Ana Paula Dos

Santos Matos

E D Megep

Professeur Carla Holandino Quaresma

Bruno Filipe Carmelino

Professeur Cardoso Sarmento

Rapporteur Lucas

Professeur Antonio Ferreira Miranda

Professeur Bartira Rossi Bergmann

Maria Inês Ré

Ana Paula Cajueiro

João Vitor Da Costa

Juliana Paiva

Gleyce Barbosa

Fortune Homsani

Vania Bucco

Camila Siqueira

Adriana Oliveira

Michelle Nonato

Luciana Siqueira

Priscila Elias

Fiammeta Nigro

Iris Giovanini

Mayra Leal

Raphaela Schuenck

Leide Lene

Tatiana Zanella

Prof Barbara Lorca

Prof Ana Aline Villa

Prof. Elisabete Guerra

Prof. Santos

Sylvie Del Confetto

Christine Rolland

Laurent Devriendt

Rachel Calvet

Phillipe Accart

Véronique Nallet

Séverine Patry

Céline Boachon

Fabienne Anne-Marie Fontes

Lori Gonnet

Margot Chauvet

Yi Rong

Martin Giraud

Rajesh Munirathinam

Son Phan

Razac Sane

Lina Romero

Graciela Pacheco

Chaima Chaabani

Lucia Perez

Marwa Said

Bruna Rego

Hugo Hael

Thomas Blanchard

Théo Tellouk

Diana Avram

Diana Lucia

Issa Munu

Ana Paula

Development of micro and nanostructured systems for cutaneous leishmaniasis treatment et discipline ou spécialité

Keywords: Leishmaniose Cutanée, Amphotericine B, Paromomycine, Nanoémulsion, Microparticules, Mélanges Polymériques, PLGA, PLA, Séchage

La leishmaniose est une maladie négligée causée par des parasites protozoaires du genre Leishmania et affecte des millions de personnes dans plusieurs pays. Le traitement actuel pour la leishmaniose cutanée (LC) a de nombreux effets secondaires. Ces facteurs intensifient la recherche de nouvelles formulations et l'étude de différences voies d'administration principalement pour le traitement LC, pour lesquelles les micro-et nanotechnologies pourraient représenter des solutions technologiques intéressantes capables de moduler le relargage ou la biodisponibilité des principes actifs. Un des médicaments les plus étudiés pour le traitement LC est la paromomycine (PM), un antibiotique aminoglycoside utilisé par voie intraveineuse et topique. Un autre médicament important est l'amphotéricine B (AmB), un antibiotique macrolide injectable utilisé comme traitement de deuxième choix. Cette thèse porte sur le développement d'une nanoémulsion huile-dans-l'eau (HE) contenant AmB pour une administration topique et des microparticules polymériques contenant PM obtenues par séchage par atomisation pour une administration intralésionnelle pour le traitement LC. Les formulations ont été caractérisées en termes de taille et morphologie de particules et gouttelettes d'huile, contenu d'actif, stabilité, propriétés thermiques, relargage in vitro. L'activité biologique a été évaluée par securité in vitro sur des macrophages et par activité antileshmanienne sur des promastigotes de Leishmania amazonensis. Les nanoémulsions HE contenant AmB sont caractérisées par des tailles de gouttelettes d'huile de l'ordre d'environ 50 nm, avec un indice de polydispersité inférieur à 0,5 et une teneur en AmB de 0,5 mg/mL. Les nanoémulsions ont présenté un profil de relargage lent et une faible perméation cutanée. Malgré une bonne activité antileishmanienne, ces formulations s'avèrent toxiques. Elles présentent une bonne stabilité physique jusqu'à 365 jours, mais doivent être conservées dans la chaîne du froid. Pour les microparticules polymériques, différentes compositions ont été évaluées (PLGA et mélange PLGA-PLA). Les résultats ont montré que le profil de relargage de PM était influencé par le type de polymère utilisé, avec un relargage plus lent pour le mélange PLGA-PLA. Ces microparticules polymériques contenant PM ne sont pas toxiques et présentent une bonne activité leishmanienne. Par conséquent, les deux types de formulations étudiées présentent un potentiel en tant que nouvelles alternatives pour le traitement par LC. Néanmoins, d'autres études sont nécessaires pour leur optimisation comme l'activité antileishmanienne contre les amastigotes de L. amazonensis et des études in vivo sur des souris BALB/c.

Haurie) and Jacqueline Azevedo for all support, aids, partnership and attention gave to me during the development of my formulations.

I would like to thank, in specially, Bhianca Lins, Roger Lima, Rababe Sani, Sinan Boztepe, Ercan Boztepe, Marion Boztepe, Ludovic Moulin, Augustina Ephrain and Audrey Trba for all support, care, friendship and good moments that we spent together during my stay in France.

I want to thank Laboratório de Imunofarmacologia -IBCCF -UFRJ, specially Maria Paula Borsodi, Ariane Batista, Felipe Gondim e Prof. Bartira Bergmann for the support, expertise and aid in development of in vitro studies.

I want to thank Luis from LabCQ, which help me in some IR analysis and Aline da Costa for help me in HPLC analysis.

I would like to thank Plataforma de Microscopia Eletrônica Rudolf Barth -IOC -Fiocruz for SEM and TEM analyses.

I want to thank Farmanguinhos -Fiocruz, specially Laboratório de Farmacotécnica Experimental for care, support and parterneship during all these years. I want to thank my friends Amanda Dumani, Clarice Costa, Rafael Sá, Renato Costa, Fernanda Lima, Fernanda Paschoal, Lais Monteiro, Kalline Franco, Yuka Nakamura, Marlon Heggdorne, Natália Linhares, Jéssica Barbosa for all support, care, friendship and patience for my absence during this work.

I wish to dedicate my gratefulness for IMT Mines Albi, MEGEP, School of Pharmacy -UFRJ, Prof. Ana Luisa Pallhares, Prof. Yraima Cordeiro, Prof. Cláudia Figueiredo and Secretaria Pós-graduação (Marcelo and Valeria) for all support, care and opportunities to belong of these Institutions.

I would like to thank Prof. Alexandre Pyrrho, Prof. Helena Keiko and Prof. Flavia Almada for all advices and suggestions in Qualification step.

I would like to thank the titular members of jury Prof. Bruno Sarmento, Prof. Lucas Miranda, Prof. Bartira Bergmann, Prof. Igor de Almeida and the substitute members Prof.

Elisabete dos Santos and Prof. Alexandre Pyrrho for availability and accept to be a part of this work.

I want to thank all the person that contribute direct or indirect for this work.

I would like to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the financial support of my Ph.D. project and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) for the financial support of a part of my Ph.D. Project developed in France.

RESUMO

Ana Paula dos Santos Matos. Desenvolvimento de sistemas nano e microestruturados para o tratamento da leishmaniose cutânea. Rio de Janeiro, 2018. Tese (Doutorado em Ciências Farmacêuticas), Faculdade de Farmácia da Universidade Federal do Rio de Janeiro, Brasil; (Doutorado em Engenharia de Processos e Meioambiente), IMT Mines Albi, Universidade de Toulouse, França, 2018.

A leishmaniose é uma doença negligenciada causada por parasitas protozoários do gênero Leishmania e que afeta milhões de pessoas em vários países. O tratamento atual para a leishmaniose cutânea (LC) causa muitos efeitos colaterais. Com base neste cenário, há uma busca intensiva de novas formulações e o estudo de diferentes vias de administração, principalmente para o tratamento da LC. Desta forma, micro e nanotecnologia são tecnologias importantes, que podem ser úteis para a modulação do perfil de liberação dos fármacos, melhorando sua biodisponibilidade. Um dos fármacos mais investigados para o tratamento da LC é a paromomicina (PM), um antibiótico aminoglicosídeo administrado por via intravenosa e tópica. Outro fármaco importante é a anfotericina B (AmB), um antibiótico macrolídeo, administrado de forma injetável, como tratamento de segunda escolha. Os objetivos deste estudo foram desenvolver uma nanoemulsão óleo-água (O/A) contendo AmB para administração tópica e micropartículas poliméricas secas por atomização contendo PM para administração intralesional para o tratamento da LC. As formulações foram caracterizadas principalmente em termos de tamanho e morfologia de partículas ou gotículas de óleo, propriedades térmicas, teor de fármaco, estabilidade e perfil de liberação. A atividade biológica foi avaliada através da segurança in vitro em macrófagos e atividade antileishmanial contra promastigotas de Leishmania amazonensis. As nanoemulsões O/A contendo AmB, foram caracterizadas por gotículas de óleo com tamanho de cerca de 50 nm, com índice de polidispersão menor que 0,5 e teor de fármaco de 0,5 mg / mL. As nanoemulsões mostraram uma cinética de liberação lenta e controlada de AmB e baixa permeação cutânea. No entanto, essas formulações foram citotóxicas com boa atividade antileishmanial. Além disso, a melhor formulação mostrou estabilidade até os 365 dias com necessidade de armazenamento em câmara fria. Para as micropartículas poliméricas carregadas com PM, foram investigados diferentes transportadores (apenas PLGA ou uma mistura de PLGA-PLA). Verificou-se que a cinética de liberação de PM foi influenciada pela composição carreadora, com liberação lenta para a mistura de polímeros. As formulações investigadas foram não-tóxicas e revelaram boa obtained from database 'Web of Science' using keywords as "AmB + Leishmaniasis", "AmB + drug delivery system (DDS)", "AmB + Nanoemulsions", "AmB +

LIST OF TABLES

Table 1. Current treatments of leishmaniasis (adapted from ZULFIQAR, SHELPER, [START_REF] Zulfiqar | Leishmaniasis drug discovery: recent progress and challenges in assay development[END_REF][START_REF] Lindoso | Review of the current treatments of leishmaniases[END_REF][START_REF] Menezes | Advances in development of new treatment for leishmaniasis[END_REF]..45 [START_REF] Singh | Leishmaniasis: Current status of available drugs and new potential drug targets[END_REF][START_REF] Tran | Why and how to prepare biodegradable, monodispersed, polymeric microparticles in the field of pharmacy[END_REF] and is one of the major health problems in the world. This disease has many clinical manifestations and it is divided in visceral, muco-cutaneous and cutaneous leishmaniasis. [START_REF] Hubert | In vitro leishmanicidal activity of some Cameroonian medicinal plants[END_REF][START_REF] Yahya | Effect of cantharidin on apoptosis of the Leishmania major and on parasite load in BALB/c mice[END_REF][START_REF] Alvar | Leishmaniasis worldwide and global estimates of its incidence[END_REF][START_REF] Chattopadhyay | A novel mechanism for an old drug: Amphotericin B in the treatment of visceral leishmaniasis[END_REF]. Besides, cases of Leishmania with the co-infection human immunodeficiency virus (HIV) are increasing and have been described in 35

countries [START_REF] Sundar | An update on pharmacotherapy for leishmaniasis[END_REF]PACE, 2014;[START_REF] Tiuman | Recent advances in leishmaniasis treatment[END_REF][START_REF] Soares | Leishmanicidal activity of a supercritical fluid fraction obtained from Tabaernaemontana catharinensis[END_REF].

The etiological agent of leishmaniasis is an intracellular parasite of Leishmania genus, which presents a life cycle with two morphological forms: a flagellated or promastigote form, found in the insect vector organism and amastigote form found inside the macrophages of the vertebrate host.

(MINISTÉRIO DA [START_REF] Ministério Da Saúde | Secretaria de Vigilância em Saúde. Manual de Vigilância da Leishmaniose Tegumentar Americana[END_REF][START_REF] Singh | Leishmaniasis: Current status of available drugs and new potential drug targets[END_REF][START_REF] Tiuman | Recent advances in leishmaniasis treatment[END_REF][START_REF] Reithinger | Cutaneous leishmaniasis[END_REF]. The biological cycle of leishmaniasis is showed in Figure 2. [START_REF] Bates | Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies[END_REF][START_REF] Bates | Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies[END_REF], which depends on the infective specie and the host immunological response. Some authors consider diffuse cutaneous leishmaniasis form as the forth clinical patterns [START_REF] Chappuis | Visceral leishmaniasis: what are the needs for diagnosis, treatment and control[END_REF][START_REF] Reithinger | Cutaneous leishmaniasis[END_REF][START_REF] Khatami | Treatment of acute Old World cutaneous leishmaniasis: a systematic review of the randomized controlled trials[END_REF].

The visceral leishmaniasis (VL), also known as kala-azar, is the most serious leishmaniasis form and can be fatal when not treated (WHO, 2017;NO, 2016;[START_REF] Ministério Da Saúde | Secretaria de Vigilância em Saúde. Manual de Vigilância e Controle da Leishmaniose Visceral[END_REF]. The clinical diagnosis is complex because this disease shows nonspecific symptoms. However, in general, the patients with VL present prolonged fever, weight loss, anemia, abdominal pain, hepatomegaly and splenomegaly (WHO, 2017). These last two symptoms are the most characteristic of this clinical manifestation. The Figure 3 shows one patient with hepatosplenomegaly caused by VL. VL is endemic in more than 65 countries with widespread distribution around the world [START_REF] Mansueto | Leishmaniasis in travelers: A literature review[END_REF][START_REF] Ministério Da Saúde | Secretaria de Vigilância em Saúde. Manual de Vigilância e Controle da Leishmaniose Visceral[END_REF]. The main species of Leishmania, which causes this type of leishmaniasis, are:

Leishmania infatum and Leishmania donovani (NO, 2016;[START_REF] Singh | Leishmaniasis: Current status of available drugs and new potential drug targets[END_REF]. Kala-azar presents the incidence rate, approximately, 2 million cases and 500.000 new cases each year. Around 90% of visceral leishmaniasis cases in the world occur in five countries: India, Bangladesh, Nepal, Sudan and Brazil (NO, 2016;[START_REF] Singh | Leishmaniasis: Current status of available drugs and new potential drug targets[END_REF][START_REF] Tiuman | Recent advances in leishmaniasis treatment[END_REF]. Each year, an estimated 50,000 to 90,000 new cases of VL take place worldwide (WHO, 2017) and an estimative between 20,000 and 40,000 cases of deaths happen per year [START_REF] Alvar | Leishmaniasis worldwide and global estimates of its incidence[END_REF]. The status of endemicity of VL in the world, in 2016, is showed in Figure 4. In Brazil, VL affects specially babies and small children. However, there is an increase of number of cases in adults, which were HIV-positive or were immunosuppressed [START_REF] Sundar | An update on pharmacotherapy for leishmaniasis[END_REF][START_REF] Mansueto | Leishmaniasis in travelers: A literature review[END_REF]PACE, 2014;[START_REF] Ministério Da Saúde | Secretaria de Vigilância em Saúde. Manual de Vigilância e Controle da Leishmaniose Visceral[END_REF]. The cutaneous leishmaniasis (CL) or American tegumentary leishmaniasis (ATL) is usually characterized by the presence of rounded or oval skin lesions, which arise, primarily at the insect bite site (MINISTÉRIO DA SAÚDE, 2017; [START_REF] Carneiro | Drug delivery systems for the topical treatment of cutaneous leishmaniasis[END_REF]. The first lesion is, in general, single and painless (Figure 5). After a period of 1 to 12 weeks, arise one lesion as a papule erythematosus that develops into a nodule with necrotic base and raised edges in a period of two weeks to six months [START_REF] Ministério Da Saúde | Secretaria de Vigilância em Saúde. Manual de Vigilância da Leishmaniose Tegumentar Americana[END_REF][START_REF] Reithinger | Cutaneous leishmaniasis[END_REF][START_REF] Hepburn | Cutaneous leishmaniasis[END_REF]. Sometimes, CL lesion may be leave life-long scars (WHO, 2017 CL is endemic in more than 70 countries with 90% reported cases occurred in seven countries (Afghanistan, Saudi Arabia, Algeria, Brazil, Pakistan, Peru and Syria) [START_REF] Singh | Leishmaniasis: Current status of available drugs and new potential drug targets[END_REF][START_REF] Tiuman | Recent advances in leishmaniasis treatment[END_REF][START_REF] Reithinger | Cutaneous leishmaniasis[END_REF]. The status of endemicity of CL in the world, in 2016, is showed in Figure 6. The species of Leishmania most common are: Leishmania major, Leishmania tropica, Leishmania mexicana, Leishmania guyanensis, Leishmania braziliensis, Leishmania amazonensis and Leishmania panamensis [START_REF] Carneiro | Drug delivery systems for the topical treatment of cutaneous leishmaniasis[END_REF]. According to the WHO, this type of leishmaniasis is considered one of the six infectious diseases most important because CL shows high capacity to cause deformations and high detection coefficient (MINISTÉRIO DA SAÚDE, 2017).

In Brazil, is one of the most important dermatological affection diseases with slight drop of number of cases in the last years, but with an incidence still very high being widely disseminated in brazilian territory (MINISTÉRIO DA SAÚDE, 2017). The mucocutaneous form (MCL) presents destructive lesions located in the mucous membranes of the mouth and upper respiratory tract (Figure 7). It is believed that occur a hematogenous or lymphatic spread of the parasites from a cutaneous lesion. In most cases, MCL results from a chronic evolution of cured CL lesion without adequate treatment or without treatment (MINISTÉRIO DA [START_REF] Ministério Da Saúde | Secretaria de Vigilância em Saúde. Manual de Vigilância da Leishmaniose Tegumentar Americana[END_REF]PACE, 2014;WHO, 2010). Some studies in Brazil indicated that MCL can happen months or more than 20 years after CL lesion (WHO, 2010). In severe cases, MCL lesion can lead to mutilations with partial or total loss of the nose and lips induced serious deformations and severe social problems [START_REF] Akbari | Application of nanotechnology in treatment of leishmaniasis: A review[END_REF]PACE, 2014;WHO, 2010). Moreover, secondary infections may increase the patient's death risk and my lead to death (WHO, 2010). 2017). This type of medicines has not to be use for pregnant and elderly people because they induce severe side effects such as hepatoxicity, nephrotoxicity and cardiotoxicity (AKBARI, [START_REF] Akbari | Application of nanotechnology in treatment of leishmaniasis: A review[END_REF][START_REF] Espuelas | In vitro antileishmanial activity of amphotericin B loaded in poly (εcaprolactone) nanospheres[END_REF].

Intralesional administration can cause a local reaction and systemic side effects similar as intravenous and intramuscular administration, but of mild to moderate intensity and reversible after end of treatment (MINISTÉRIO DA SAÚDE, 2017).

Besides side effects, there are some reports of parasite resistance to these drugs (SINGH, KUMAR, SINGH, 2012).

The second-choice treatment involves the administration of amphotericin B and pentamidine [START_REF] Nassif | Safety and efficacy of current alternatives in the topical treatment of cutaneous leishmaniasis: a systematic review[END_REF][START_REF] Tiuman | Recent advances in leishmaniasis treatment[END_REF].

Amphotericin B (AmB) (Figure 9) is a polyene antifungal of the macrolide Nowadays, there are 4 commercial formulations of amphotericin B available on the market for hospital use: an amphotericin B deoxycholate (Fungizone ® ), a liposomal amphotericin B (Ambisome ® ), cholesterol dispersion amphotericin B (Amphocil ® /Amphotec ® ) and lipid complex of amphotericin B (Abelcet ® ) [START_REF] Caldeira | Nanoemulsions loaded with amphotericin B: A new approach for the treatment of leishmaniasis[END_REF][START_REF] Ibrahim | Assessment of novel oral lipid-based formulations of amphotericin B using in vitro lipolysis model[END_REF][START_REF] Lindoso | Review of the current treatments of leishmaniases[END_REF] GOTO & LINDOSO, 2010).

Although its higher efficacy than antimonial pentavalent based-products, AmB, in all commercial forms available, still presents high toxicity (nephrotoxicity)

and several side effects such as fever, headache, nausea, vomiting, tremors, hypotension among others (MINISTÉRIO DA SAÚDE, 2017; FILIPPIN, SOUZA, [START_REF] Filippin | Amphotericin B associated with triglyceride-rich nanoemulsion: Stability studies and in vitro antifungal activity[END_REF][START_REF] Golenser | New formulations and derivates of amphotericin B for treatment of leishmaniasis[END_REF]. Commercial AmB lipid formulations (Ambisome ® , Abelcet ® and Amphocil ® /Amphotec ® ) are less toxic than Fungizone ® , however the high cost and difficulty of production restrict their widespread use [START_REF] Caldeira | Nanoemulsions loaded with amphotericin B: A new approach for the treatment of leishmaniasis[END_REF][START_REF] Singh | Leishmaniasis: Current status of available drugs and new potential drug targets[END_REF].

It is well known that the pharmacokinetics parameters are dependent on the composition of the AmB lipid formulations. Some examples: Ambisome ® has the lower size, negative charge and presents higher peak plasma level (Cmax) and larger area under curve (AUC) than Fungizone ® ; Abelcet ® has the largest size and the highest volume of distribution and clearance of all AmB formulations; Amphocil ® /Amphotec ® shows fast elimination from the body circulation by phagocytosis and achieves the lowest Cmax, which requires larger therapeutic doses and consequently, higher toxicity; Amphocil ® /Amphotec ® has a toxicity similar to Fungizone ® , reason why the FDA suspended its distribution in 2011 [START_REF] Hamill | Amphotericin B formulations: A comparative review of efficacy and toxicity[END_REF].

Pentamidine (Figure 10) is an aromatic diamidine, which has been The pentamidine is administered by intravenously or intramuscularly with recommended dose of 3 to 4 mg/ kg/ day. This drug can be cause different side effects such as hypoglycemia, hyperglycemia, nephrotoxicity and hepatotoxicity.

Furthermore, pentamidine may be induced insulin dependent diabetes. There are already reports of parasite resistance [START_REF] Lindoso | Review of the current treatments of leishmaniases[END_REF].

There is an alternative treatment using paromomycin, miltefosine (PACE, 2014;[START_REF] Singh | Leishmaniasis: Current status of available drugs and new potential drug targets[END_REF][START_REF] Tiuman | Recent advances in leishmaniasis treatment[END_REF] and sitamaquine (Phase II study) [START_REF] Sundar | An update on pharmacotherapy for leishmaniasis[END_REF].

Paromomycin (PM) (Figure 11) is an aminoglycoside antibiotic [START_REF] Pujol-Brugués | Development of a liquid chromatographic method for the quantification of paromomycin. Application to in vitro release and ex-vivo permeation studies[END_REF][START_REF] Singh | Leishmaniasis: Current status of available drugs and new potential drug targets[END_REF][START_REF] Tiuman | Recent advances in leishmaniasis treatment[END_REF], produced by Streptomyces rimosus [START_REF] Pujol-Brugués | Development of a liquid chromatographic method for the quantification of paromomycin. Application to in vitro release and ex-vivo permeation studies[END_REF]USP, 2012b). The mechanism of action is not yet well known, but it is believed that paromomycin acts in the inhibition of protein synthesis (inhibition of translocation and recycling of ribosome subunits) and cause mitochondria disturb (modification in mitochondrial membrane potential) [START_REF] Brugués | Developing cutaneous applications of paromomycin entrapped in stimuli-sensitive block copolymer nanogel dispersions[END_REF][START_REF] Kharaji | Drug targeting to macrophages with solid lipid nanoparticles harboring paromomycin: an in vitro evaluation against L. major and L. tropica[END_REF][START_REF] Lindoso | Review of the current treatments of leishmaniases[END_REF][START_REF] Singh | Leishmaniasis: Current status of available drugs and new potential drug targets[END_REF]. PM has been used

with alternative drug in leishmaniasis treatment at a dose of 15 mg/ kg/ day for 20 days (DAVIDSON, DEN BOER, RITMEIJER, 2009). Nowadays, there is an ointment for topical application containing 15% paromomycin with 12% methylbenzethonium chloride (Leishcutan ® ), available only in Israel market, which was developed in 1984 by El-On and colleagues. This formulation presents a local toxicity that was described as local irritancy associated with the use of methylbenzethonium chloride [START_REF] Salah | Topical paromomycin with or without gentamicin for cutaneous leishmaniasis[END_REF].

There is, also, one study involving an ointment with 15% paromomycin and 10% urea, which further studies are required in order to confirm the effectiveness [START_REF] Bryceson | Treatment of 'Old World' cutaneous leishmaniasis with aminosidine ointment: results of an open study in London[END_REF]. Another formulation was developed as a cream containing 15% of paromomycin and 0.5% gentamicin, which had the same efficacy as paromomycin cream against L. major (SALAH et al., 2009major (SALAH et al., , 2013)). In Brazil, there is one gel formulation containing 15% paromomycin under development for topical use [START_REF] Gonçalves | Activity of a paromomycin hydrophilic formulation for topical treatment of infections by Leishmania (Leishmania) amazonensis and Leishmania (Vianna) braziliensis[END_REF]. It is the first drug approved for leishmaniasis treatment that is used orally with a dose between 2 and 5 mg/ kg/ day for 28 days [START_REF] Singh | Leishmaniasis: Current status of available drugs and new potential drug targets[END_REF][START_REF] Reithinger | Cutaneous leishmaniasis[END_REF]. This drug shows several side effects such as vomiting, diarrhea, toxicity in gastrointestinal tract. Furthermore, presents a teratogenic effect and is forbidden to use in pregnant women (AKBARI, [START_REF] Akbari | Application of nanotechnology in treatment of leishmaniasis: A review[END_REF][START_REF] Menezes | Advances in development of new treatment for leishmaniasis[END_REF][START_REF] Singh | Leishmaniasis: Current status of available drugs and new potential drug targets[END_REF]. As other drugs used to treat leishmaniasis, miltefosine is already triggering resistance in parasites (AKBARI, ORYAN, HATAM, 2017; SINGH, KUMAR, SINGH, 2012).

Sitamaquine (Figure 13), an aminoquinoline, is the only drug developed for visceral leishmaniasis treatment. Its mechanism of action affects the mobility, morphology and growth of the protozoans, through an electrostatic interaction between polar ionic groups of the phospholipids present in the parasite It is a new medicine, used orally, which is in clinical studies (Phase II study in India and Kenya), but presents promising results [START_REF] Imbert | Sitamaquine resistance in Leishmania donovani affects drug accumulation and lipid metabolism[END_REF]. The worst problem of sitamaquine is the poor knowledge of the toxicity of its metabolism [START_REF] Singh | Leishmaniasis: Current status of available drugs and new potential drug targets[END_REF][START_REF] Kulshrestha | Antimony-resistant clinical isolates of Leishmania donovani are susceptible to paromomycin and sitamaquine[END_REF].

Table 1 shows the advantages and disadvantages of current leishmaniasis treatment.

In addition to these treatments, there are some drugs of other therapeutic Another alternative option to treat cutaneous leishmaniasis, already described in literature, is Heat Therapy or Thermotherapy [START_REF] Lobo | Heat therapy for cutaneous leishmaniasis elicits a systemic cytokine response similar to that of antimonial (Glucantime) therapy[END_REF].

In general, this therapy consists a local heat application, on the edge of lesion, at 50ºC for 30 seconds, varying methods and devices [START_REF] Cardona-Arias | Comprehensive economic evaluation of thermotherapy for the treatment of cutaneous leishmaniasis in Colombia[END_REF][START_REF] Aronson | A randomized controlled trial of local heat therapy versus intravenous sodium stibogluconate for the treatment of cutaneous Leishmania major infection[END_REF][START_REF] Lobo | Heat therapy for cutaneous leishmaniasis elicits a systemic cytokine response similar to that of antimonial (Glucantime) therapy[END_REF]. One of the devices, ThermoMed ® was approved by Food Drug and Administration (FDA) for CL treatment [START_REF] Lobo | Heat therapy for cutaneous leishmaniasis elicits a systemic cytokine response similar to that of antimonial (Glucantime) therapy[END_REF]. There are some studies that compared the efficacy of thermotherapy with meglumine antimoniate and showed efficacy similar than pentavalent antimonials with safety results [START_REF] Cardona-Arias | Comprehensive economic evaluation of thermotherapy for the treatment of cutaneous leishmaniasis in Colombia[END_REF][START_REF] López | An alternative for the treatment of American cutaneous leishmaniasis[END_REF][START_REF] Aronson | A randomized controlled trial of local heat therapy versus intravenous sodium stibogluconate for the treatment of cutaneous Leishmania major infection[END_REF]. However, this therapy cannot be applied in cutaneous lesions near mucosa or in lesions [START_REF] López | An alternative for the treatment of American cutaneous leishmaniasis[END_REF].

In addition to other drugs treatment and heat treatment, there are, also, [START_REF] Herrero-Vanrell | Nano and microtechnologies for ophthalmic administration, an overview[END_REF][START_REF] Mei | Pharmaceutical nanotechnology for oral delivery of anticancer drugs[END_REF] (Figure 14). Although, some marketed products can be find in countries such as India and Bangladesh in conventional pharmaceutical forms (ointment), cutaneous leishmaniasis treatment has also been approached in research studies with liposomal forms (for antimonials compounds), solid lipid nanoparticles (for AmB or paromomycin molecules), and nanoparticles (metallic or polymeric) [START_REF] Islan | Nanopharmaceuticals as a solution to neglected diseases: It is possible[END_REF].

Figure 14. Different types of drug delivery system with micro and nanotechnology (adapted from [START_REF] Wen | Nanotechnology based drug delivery systems for Alzheimer's disease management: Technical, industrial and clinical challenges[END_REF].

Whatever the route of administration for a given disease treatment, the vehicle used (drug delivery system) should be able to deliver the good drug dosage to the targeted site. Topical and intralesional are two interesting routes for CL treatment. From one side, topical administration shows some advantages as easy of administration, low cost, few side effects and can be used for local treatment [START_REF] Garnier | Topical treatment for cutaneous leishmaniasis[END_REF]. However, stratum corneum barrier difficult drug permeation across the skin and decrease the efficacy of drugs (SENGUPTA & CHATTERJEE, 2017). In addition, the high molecular weight of drugs [START_REF] Bos | The 500 Dalton rule for the skin penetration of chemical compounds and drugs[END_REF] and the location of leishmania parasites, in the dermis layer, increase the challenges of developing effective topical formulations for CL [START_REF] Singh | Challenges and new discoveries in the treatment of leishmaniasis[END_REF].

From the other side, intralesional administration presents some advantages as lower total dose, less toxic effects, local application and more adjustable schedule for drug administration (BRITO, RABELLO, COTA, 2017).

However, repeated dose injections, rapid clearance, sporadically systemic effects, risk of late mucosal complications and efficacy rate difficult the patience compliance and the implementation of this kind of administration in large scale [START_REF] Silva | Towards a standard protocol for antimony intralesional infiltration technique for cutaneous leishmaniasis treatment[END_REF][START_REF] Brito | Efficacy of pentavalent antimoniate intralesional infiltration therapy for cutaneous leishmaniasis: a systematic review[END_REF]SOUSA-BATISTA et al., 2017).

Intralesional treatment is recommended, since 2013, for patients with single lesion (not in face or joints) and with contraindications to systemic treatment by Pan American Health Organization (PAHO) (BRITO, [START_REF] Brito | Efficacy of pentavalent antimoniate intralesional infiltration therapy for cutaneous leishmaniasis: a systematic review[END_REF][START_REF] Duque | Standardization of intralesional meglumine antimoniate treatment for cutaneous leishmaniasis[END_REF]. In Brazil, intralesional administration of meglumine antimoniate has been used without a standard protocol to evaluate the efficacy of this treatment [START_REF] Silva | Towards a standard protocol for antimony intralesional infiltration technique for cutaneous leishmaniasis treatment[END_REF].

Thereby, the search of new therapies and the improvement of current therapy for CL treatment are still required. Based on this emergency situation, this thesis work focuses on polymeric microparticles and nanoemulsions, aiming to develop useful formulations respectively for intralesional use of Paromomycin and topical administration of AmB-based formulation, both for cutaneous leishmaniasis treatment.

GENERAL OBJECTIVE

This work aims to develop two different formulations for cutaneous leishmaniasis treatment, one using Amphotericin B for topical administration and other using Paromomycin for intralesional administration.

SPECIFIC OBJECTIVES

✓ [START_REF] Singh | Nanoemulsion: concepts, development and applications in drug delivery[END_REF]. They are kinetically and thermodynamically stable with a droplet size between 20-400 nm and can be considered nanoemulsion until 500 nm [START_REF] Rai | Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications[END_REF][START_REF] Singh | Nanoemulsion: concepts, development and applications in drug delivery[END_REF]. Nanoemulsions could be classified as neutral, anionic and cationic nanoemulsion based on the surface charge presents in droplets [START_REF] Rai | Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications[END_REF]. NE droplets are prepared considering the optimal hydrophilic-lipophilic relation and the greatest surfactant concentration [START_REF] Lu | Preparation, characterization and antimicrobial activity of nanoemulsions incorporating citral essential oil[END_REF], being oil phase, surfactant/cosurfactant and aqueous phase important ingredients in this type of formulation [START_REF] Jaiswal | Nanoemulsion: an advance mode of drug delivery system[END_REF].

The oil phase is used to solubilize lipophilic drugs and varies from 2% to 20% (w/w) in O/W nanoemulsions. This type of emulsion is indicated, mainly, for drugs belongs to class II and IV of biopharmaceutical classification system (BCS).

The oils most used in NE formulations are: isopropyl myristate, triacetin, soybean oil, sesame oil, coconut oil, triacyl glycerides, vitamin E [START_REF] Rai | Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications[END_REF][START_REF] Singh | Nanoemulsion: concepts, development and applications in drug delivery[END_REF] and essential oils. Essential oils can cross the cell membrane and permeate due to chemical composition (lipophilic mixture) [START_REF] Dhifi | Essential oils' chemical characterization and investigation of some biological activities: a critical review[END_REF].

One of essential oils used in nanoemulsions is clove oil, which has eugenol as the major constituent and has analgesic, anti-inflammatory, antioxidant and antimicrobial properties [START_REF] Teixeira | Synthesis and leishmanicidal activity of eugenol derivatives bearing 1,2,3trialoze functionalities[END_REF][START_REF] Choudhury | Recent uptake on nanoemulgel as topical drug delivery system[END_REF][START_REF] Sharma | Sodium caseinate stabilized clove oil nanoemulsion: Physicochemical properties[END_REF][START_REF] Shahavi | Evaluation of critical parameters for preparation of stable clove oil nanoemulsion[END_REF][START_REF] Morais | Thymol and eugenol derivatives as potential antileishmanial agents[END_REF].

Surfactants are amphiphilic molecules selected by their solubility in oil and aqueous phases, emulsification ability and lower toxicity profile. They reduce interfacial tension and avoid droplets agglomeration. Non-ionic surfactants have lower toxicity and irritability than anionic or cationic emulsifiers [START_REF] Rai | Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications[END_REF][START_REF] Singh | Nanoemulsion: concepts, development and applications in drug delivery[END_REF] [START_REF] Halnor | Nanoemulsion: A novel platform for drug delivery system[END_REF][START_REF] Rai | Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications[END_REF][START_REF] Singh | Nanoemulsion: concepts, development and applications in drug delivery[END_REF].

The aqueous phase can affect the droplet size and the stability of nanoemulsions. The pH and presence of electrolytes in this phase must be consider for good formulation development [START_REF] Halnor | Nanoemulsion: A novel platform for drug delivery system[END_REF][START_REF] Setya | Nanoemulsions: Formulation methods and stability aspects[END_REF].

The selection of ingredients, their concentration, the appropriate preparation method and the optimal process parameters are important to produce a good nanoemulsion [START_REF] Rai | Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications[END_REF]. One option to evaluate the best correlation and ingredients concentration is plot a pseudo-ternary phase diagram, which is based on surfactant, oil and water proportions [START_REF] Halnor | Nanoemulsion: A novel platform for drug delivery system[END_REF].

NE have some advantages compared to conventional emulsions, such as: high stability, increase of interfacial area, improve of drug solubility and bioavailability, increase of drug absorption through biological membranes, ease to produce and scale-up [START_REF] Rai | Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications[END_REF][START_REF] Campos | Nanoemulsions as a delivery system for lipophilic drugs[END_REF][START_REF] Date | Self-nanoemulsifying drug delivery systems: formulation insights, applications and advances[END_REF]. Despite the several advantages, NE systems have low viscosity and low spreadability, which difficult their topical use. Based on these problems, some studies suggested an increment in NE formulations through gelling agent addition. Thus, nanoemulsions can be transformed into nanoemulgel [START_REF] Choudhury | Recent uptake on nanoemulgel as topical drug delivery system[END_REF] (Figure 16). There are some gelling agents commonly used in nanoemulgel formations, such as chitosan, carbopol family, methyl cellulose and poloxamer 407. The gelling agent's incorporation in NE change formulation physical state and some modifications in pharmacokinetic properties of NE system. Nanoemulgels containing drugs have already been described [START_REF] Choudhury | Recent uptake on nanoemulgel as topical drug delivery system[END_REF]SENGUPTA & CHATTERJEE, 2017).

There are some articles describing nanoemulsions with gelling agents.

Arantes and co-workers (2017) prepared nifedipine nanoemulsions with poloxamer 407 for topical administration. Hussain and co-workers (2016a) developed AmB nanoemulsion gel or nanoemulgel (AmB nanoemulsion incorporated into carbopol gel) for topical delivery to antifungal treatment. Zheng and co-workers (2016) developed nanoemulsions gel, using carbopol 934 as gelling agent, for topical and transdermal drug delivery of terbinafine and citral.

Poloxamers properties

Poloxamers (Figure 17), also known with trademark Pluronic ® , are a non- Poloxamers have some advantages for their use, as commercial availability, wide range of molecular weights and PEO/PPO composition There are some studies using poloxamer 407 for several pharmaceutical applications. [START_REF] Ali | Oral nanoemulsions of candesartan cilexetil: formulation, characterization and in vitro drug release studies[END_REF] prepared nanoemulsions containing candesartan cilexetil for oral use using different poloxamers, including poloxamer 407. [START_REF] Chauhan | Development and in vitro characterization of nanoemulsion embedded thermosensitive in-situ ocular gel of diclofenac sodium for sustained delivery[END_REF] developed diclofenac sodium nanoemulsions dispersed in poloxamer 407 for ocular application. [START_REF] Inal | Effect of mechanical properties on the release of meloxicam from poloxamer gel bases[END_REF] evaluated the mechanical properties on meloxicam release from poloxamers (407 and 188) alone or combined. Pereira and co-workers (2013) produced poloxamer 407 gel containing polymeric microparticles and hyaluronic acid for burn wounds treatment.

Methods of nanoemulsion production

NE can be prepared by methods, which involve low energy emulsification (spontaneous emulsification, phase inversion method and emulsion inversion method) or high energy emulsification (microfluidizer, high pressure homogenizer and ultrasonication) [START_REF] Mungure | Potential application of pectin for the stabilization of nanoemulsions[END_REF][START_REF] Rai | Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications[END_REF][START_REF] Singh | Nanoemulsion: concepts, development and applications in drug delivery[END_REF][START_REF] Katouzian | Nano-encapsulation as a promising approach for targeted delivery and controlled release vitamins[END_REF][START_REF] Campos | Nanoemulsions as a delivery system for lipophilic drugs[END_REF][START_REF] Date | Self-nanoemulsifying drug delivery systems: formulation insights, applications and advances[END_REF]. The methods to prepare nanoemulsions are simple and fast when compared to other techniques to produce nanosystems [START_REF] Caldeira | Nanoemulsions loaded with amphotericin B: A new approach for the treatment of leishmaniasis[END_REF][START_REF] Campos | Nanoemulsions as a delivery system for lipophilic drugs[END_REF]. Figure 18 shows a scheme, which represents all methods used to obtain nanoemulsions. Low-energy methods utilize the internal chemical energy of the system to produce small droplets. There are two types of phase inversion method: phase inversion temperature (PIT) and phase inversion composition (PIC) [START_REF] Rai | Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications[END_REF][START_REF] Campos | Nanoemulsions as a delivery system for lipophilic drugs[END_REF]SOLANS & SOLE, 2012).

PIT is based on changes in aqueous/oil solubility of surfactants induced by temperature changes [START_REF] Rai | Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications[END_REF][START_REF] Singh | Nanoemulsion: concepts, development and applications in drug delivery[END_REF] 2012).

Spontaneous emulsification or self-emulsification method involves an addition of oil phase with surfactant/cosurfactant and drug into aqueous phase under continuous agitation. This technique is good for oral delivery hydrophobic drugs and the components can be able to oral administration as nonionic surfactants and medium chain triglycerides (oil phase) [START_REF] Singh | Nanoemulsion: concepts, development and applications in drug delivery[END_REF][START_REF] Eid | The preparation and evaluation of self-nanoemulsifying systems containing Swietenia oil and an examination of its anti-inflammatory effects[END_REF]SOLANS & SOLE, 2012). Emulsification happens immediately because of negative/positive and low free energy [START_REF] Eid | The preparation and evaluation of self-nanoemulsifying systems containing Swietenia oil and an examination of its anti-inflammatory effects[END_REF].

High energy methods depend on mechanical devices able to create high degree forces to disrupt the oil phase into small oil droplets [START_REF] Mungure | Potential application of pectin for the stabilization of nanoemulsions[END_REF][START_REF] Rai | Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications[END_REF][START_REF] Singh | Nanoemulsion: concepts, development and applications in drug delivery[END_REF]SOLANS & SOLE, 2012).

Microfluidization technique is based on emulsification through collision of two immiscible phases moving in microchannels under high pressure. Usually to obtain the desire droplet size and dispersity, NE is passed many times in microfluidizer. The correct surface device (hydrophobic or hydrophilic) is chosen considering the type of nanoemulsion required (W/O or O/W, respectively). The advantages of this technique are high scalability and zero contamination of feed material [START_REF] Rai | Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications[END_REF][START_REF] Singh | Nanoemulsion: concepts, development and applications in drug delivery[END_REF].

High pressure homogenizers technique depends on the emulsion pass through a gap between a fixed rotor and high-speed rotor. The emulsion is submitted to hydraulic shear and intense turbulence resulting in small droplets of emulsion [START_REF] Halnor | Nanoemulsion: A novel platform for drug delivery system[END_REF][START_REF] Rai | Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications[END_REF][START_REF] Singh | Nanoemulsion: concepts, development and applications in drug delivery[END_REF]. This method has high efficiency and scalable, but consumes much energy and temperature, which can be promote drug or excipients degradation (SETYA, TALEGAONKAR, RAZDAN, 2014).

The method of nanoemulsion preparation by ultrasonication is based on ultrasonic waves caused by a sonicator probe introduced in formulation. These waves promote cavitation bubbles that collapse generating high energy into the system, which break the internal phase in small droplets [START_REF] Lu | Preparation, characterization and antimicrobial activity of nanoemulsions incorporating citral essential oil[END_REF][START_REF] Rai | Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications[END_REF][START_REF] Singh | Nanoemulsion: concepts, development and applications in drug delivery[END_REF][START_REF] Jaiswal | Nanoemulsion: an advance mode of drug delivery system[END_REF][START_REF] Campos | Nanoemulsions as a delivery system for lipophilic drugs[END_REF]. Ultrasonication is the high energy method, which requires less energy. However, this technique can be induced contamination by sonicator probe. Furthermore, the scale up is not easy, but commercial homogenizers using ultrasonication have already been developed [START_REF] Singh | Nanoemulsion: concepts, development and applications in drug delivery[END_REF]. The sonication time and the ultrasonic wave amplitude affect directly the droplet size of formulation (CAMPOS, RICCI-JUNIOR, MANSUR, 2012). Figure 19 shows the relation between droplet size and sonication time to obtain nanoemulsions.

Nanoemulsions can be administered in different routes as topical, parenteral, intranasal, pulmonary, oral and ocular [START_REF] Halnor | Nanoemulsion: A novel platform for drug delivery system[END_REF][START_REF] Singh | Nanoemulsion: concepts, development and applications in drug delivery[END_REF].

Topical administration

Topical administration is an interesting non-invasive route of drugs administration, which can be used for local or systemic effects. This route has many advantages, such as: painless self-medication, improved drugs permeation avoiding hepatic first-pass effects or gastric degradation, reduction of dose frequency (constant drug delivery for a prolonged period) and hydrophilic or lipophilic drugs administration. However, the major problem of this administration is the skin barrier that difficult drugs penetration [START_REF] Rai | Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications[END_REF]SENGUPTA & CHATTERJEE, 2017;[START_REF] Montenegro | From nanoemulsions to nanostructured lipids carriers: A relevant development in dermal delivery of drugs and cosmetics[END_REF][START_REF] Bhowmik | Recent advances in novel topical drug delivery system[END_REF], mainly hydrophobic drugs [START_REF] Choudhury | Recent uptake on nanoemulgel as topical drug delivery system[END_REF].

The skin is the largest organ of the human body with a surface area around 2 m 2 and composes the natural barrier between the body and external environment. It is composed by three functional layers (Figure 20): epidermis, dermis and hypodermis. The epidermis is most superficial layer and provides protective and sensory properties. In this layer, there are keratinocytes cells divided in four different layers (stratum corneum, stratum granulosum, stratum spinousum and stratum basale). The stratum corneum represents the major barrier to drugs permeation. The dermis provides the skin mechanical properties and presents high vascularization. The hypodermis (subcutaneous layer) has the adipose tissue, nerves and blood vessels and connect the skin with deeper tissues (muscles and bones) [START_REF] Rai | Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications[END_REF][START_REF] Moronkeji | The role of subcutaneous tissue stiffness on microneedle performance in a representative in vitro model of skin[END_REF][START_REF] Montenegro | From nanoemulsions to nanostructured lipids carriers: A relevant development in dermal delivery of drugs and cosmetics[END_REF][START_REF] Ruela | Evaluation of skin absorption of drugs from topical and transdermal formulations[END_REF]. The small droplet size, low skin irritation, high permeation ability (NE can disrupt lipid bilayer) and increase drug retention at the site of action provide nanoemulsions an interesting system for topical and transdermal drug delivery [START_REF] Rai | Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications[END_REF][START_REF] Choudhury | Recent uptake on nanoemulgel as topical drug delivery system[END_REF][START_REF] Singh | Nanoemulsion: concepts, development and applications in drug delivery[END_REF][START_REF] Mou | Hydrogelthickened nanoemulsion system for topical delivery of lipophilic drugs[END_REF]. For this reason, NE is a promising alternative for skin diseases treatment [START_REF] Mattos | Nanoemulsions containing a synthetic chalcone as an alternative for treating cutaneous leishmaniasis: optimization using a full factorial design[END_REF].

One of the most important skin diseases in the world is cutaneous leishmaniasis. For CL topical treatment, the drug needs to achieve the dermis, where occurs parasites phagocytosis by macrophages [START_REF] Mattos | Nanoemulsions containing a synthetic chalcone as an alternative for treating cutaneous leishmaniasis: optimization using a full factorial design[END_REF].

NE can be used as drug delivery system for improve topical cutaneous leishmaniasis treatment, because can reduces the drugs toxicity, is easy to prepare and has low-cost production [START_REF] Caldeira | Nanoemulsions loaded with amphotericin B: A new approach for the treatment of leishmaniasis[END_REF][START_REF] Mattos | Nanoemulsions containing a synthetic chalcone as an alternative for treating cutaneous leishmaniasis: optimization using a full factorial design[END_REF]. Moreover, nanoemulsions prepared with poloxamer 407 (Pluronic ® F127)

promote wound healing and can be improve cutaneous leishmaniasis treatment.

Amphotericin B (AmB) is an effective second-line drug in CL treatment [START_REF] Caldeira | Nanoemulsions loaded with amphotericin B: A new approach for the treatment of leishmaniasis[END_REF][START_REF] Santos | Amphotericin B-loaded nanocarriers for topical treatment of cutaneous leishmaniasis: development, characterization, and in vitro skin permeation studies[END_REF] and has been chosen as model drug for this work. Besides has low solubility, AmB has a low permeability, being classified with drug belongs to class IV of biopharmaceutics classification system (BCS).

For this reason, presents low bioavailability for oral use and it is administered intravenously [START_REF] Ibrahim | Assessment of novel oral lipid-based formulations of amphotericin B using in vitro lipolysis model[END_REF]. Furthermore, this drug has high toxicity and topical administration could be a useful alternative to reduce this problem [START_REF] Santos | Amphotericin B-loaded nanocarriers for topical treatment of cutaneous leishmaniasis: development, characterization, and in vitro skin permeation studies[END_REF].

Several studies involving amphotericin B in new topical formulations, mainly lipid-based formulation, as liposomes, complexes with cholesterol, emulsions, microemulsions, nanoemulsions have been developed to improve leishmaniasis treatment [START_REF] Caldeira | Nanoemulsions loaded with amphotericin B: A new approach for the treatment of leishmaniasis[END_REF][START_REF] Santos | Amphotericin B-loaded nanocarriers for topical treatment of cutaneous leishmaniasis: development, characterization, and in vitro skin permeation studies[END_REF]. Another studies focused on AmB nanoemulgel or emulgel production to treat cutaneous leishmaniasis [START_REF] Mendonça | Poloxamer 407 (Pluronic ® F127)-based polymeric micelles for amphotericin B: In vitro biological activity, toxicity and in vivo therapeutic efficacy against murine tegumentary leishmaniasis[END_REF][START_REF] Pinheiro | Evaluation of the in vivo leishmanicidal activity of amphotericin B emulgel: An alternative for the treatment of skin leishmaniasis[END_REF] Figure 21 shows a review of number of publications, obtained in database "Web of Science", related to AmB physicochemical characterization or AmB drug delivery systems between 1997 and 2017 using keywords as "AmB + Leishmaniasis", "AmB + drug delivery system (DDS)", "AmB + Nanoemulsions", "AmB + Nanoemulsions + drug delivery systems (DDS)" and "AmB + Leishmaniasis + drug delivery systems (DDS)". database 'Web of Science' using keywords as "AmB + Leishmaniasis", "AmB + drug delivery system (DDS)", "AmB + Nanoemulsions", "AmB + Nanoemulsions + drug delivery systems (DDS)".

It can be observed a several publications about amphotericin B plus leishmaniasis with an increase of number of publications after 2011. In the last three years, there are more researches involving AmB and drug delivery systems.

Furthermore, there is an increase of number of publications after 2011 involving drug delivery systems, AmB and leishmaniasis. It can also be observed that are few studies about nanoemulsions and AmB, which began in 2012. Therefore, this work aims to develop nanoemulsions of amphotericin B prepared by ultrasonication method for topical administration for CL treatment.

OBJECTIVES

This chapter was developed in School of Pharmacy at Federal University of Rio de Janeiro (Brazil) and aims:

• Physicochemical characterization of amphotericin B;

• AmB nanoemulsions development;

• Physicochemical characterization of amphotericin B nanoemulsions;

• Stability study of AmB nanoemulsions;

• In vitro release profile of AmB nanoemulsions;

• In vitro skin permeation study of AmB nanoemulsions;

• In vitro cytotoxicity assay of AmB nanoemulsions;

• In vitro antileishmanial activity of AmB nanoemulsions.

MATERIALS AND METHODS

MATERIALS

Amphotericin B (AmB) was kindly donated by Xellia Pharmaceuticals (Denmark).

Clove oil and mint oil were purchased from Ferquima (Brazil) and were obtained from Eugenia caryophyllata flower oil and Mentha piperita leaf oil, respectively.

Poloxamer 407 (Pluronic ® F127) was purchased from Sigma-Aldrich (Brazil). Polysorbate 20 was obtained from Tedia Brazil (Brazil) and imidazolidinyl urea was obtained from All Chemistry (Brazil). All other materials were of analytical reagent grade.

METHODS

Physicochemical Characterization of Amphotericin B

The drug amphotericin B was physicochemical characterized to verify the quality of the sample and evaluate its physicochemical properties. This analysis followed amphotericin B monography of United States Pharmacopeia (USP, 2012a) with adequate potency (see Annex 1 -Xellia certificate of analysis).

Scanning Electron Microscopy (SEM)

Morphologic evaluation was performed using scanning electronic microscope (Jeol -JSM6390LV, Japan) belonging to the electron microscopy platform Rudolf Barth from Oswaldo Cruz Institute -Fiocruz. Amphotericin B sample was added on a SEM stub with a double-sided carbon adhesive tape.

After that, the sample was coated in a sputter coater equipment Denton Vacuum Desk (USA) with a gold-palladium thin layer for 8 minutes in 10 mA under Argon gas atmosphere.

Thermogravimetry Analysis (TGA)

Thermal gravimetric analysis of AmB was performed using an equipment TGA-QR500 (TA Instruments, USA) at heating rate of 10 K/min under synthetic air atmosphere. Approximately 8 mg of sample were weighed and placed into standard aluminum pan, which was hermetically sealed. The heating temperature ranged from 25 ºC to 700 ºC.

Fourier Transform Infrared Spectroscopy (FTIR)

The FTIR spectrum of the pure AmB was analyzed using a FTIR spectrophotometer (Perkin Elmer Frontier, USA) in the range of 4000 cm -1 to 600 cm -1 . The AmB sample was diluted with KBr powder at 1% and analysed as powder mixture at room temperature (adapted from [START_REF] Zu | Preparation and characterization of amorphous amphotericin B nanoparticles for oral administration through liquid antisolvent precipitation[END_REF].

Amphotericin B Nanoemulsions Development

AmB nanoemulsions were prepared by high emulsification energy method using ultrasonic processor UP 100H (Hielscher, Germany) (CAMPOS, RICCI-JUNIOR, MANSUR, 2012). Firstly, the drug was solubilized in dimethyl sulfoxide (DMSO) to obtain a 20 mg/mL solution. Then, concentrated AmB solution was diluted in oil phase (1:3 v/v ratio). Oil phase containing AmB was dispersed in aqueous phase with or without surfactant under ultrasound device for 5 minutes in ice bath [START_REF] Shahavi | Evaluation of critical parameters for preparation of stable clove oil nanoemulsion[END_REF]. Figure 22 shows the main steps of AmB nanoemulsions preparation. Twelve batches of nanoemulsions (NE) were prepared, codified from A to L, varying the essential oil used in the oil phase (clove oil or mint oil), the presence of surfactant (0 to 5% w/w), the presence of AmB and the amount of polymer used in the aqueous phase (10 and 15% w/w). Essential oils were chosen for oil phase due to their ability to increase drugs permeation. Nanoemulsions containing drug presented a theoretical final concentration of 0.5 mg/mL and the final nanoemulsions volume were 10 mL (1 mL of oil phase and 9 mL of aqueous phase). The final concentration of DMSO in nanoemulsions were lower than 2.5%. The composition of each formulation is described in Table 3.

Table 3. AmB nanoemulsions composition

Sample

Physicochemical Characterization of Amphotericin B Nanoemulsions

The following characteristics of nanoemulsions A to L were determined: mean oil droplet size and polydispersity index, pH and drug content.

Determination of Droplet Size, Polydispersity Index and pH

The droplet size and polydispersity index of developed formulations were analyzed following method previous described in the literature (CAMPOS, RICCI-JUNIOR, [START_REF] Campos | Nanoemulsions as a delivery system for lipophilic drugs[END_REF][START_REF] Campos | L-Tyrosine-loaded nanoparticles increase the antitumoral activity of direct electric current in a metastatic melanoma cell model[END_REF] in Zetasizer Nano Model S90

(Malvern Instruments, UK) equipment. The pH was verified in pH-indicator strips pH 0 -14 Universal indicator (Merck, Germany).

Drug Content

The AmB concentration in the nanoemulsions was determined by UV-vis (UV-vis model V-630, Jasco, Brazil) spectrophotometric method adapted from THE INTERNATIONAL PHARMACOPEIA, 2017a and USP, 2012a using methanol as a solvent and wavelength of 410 nm. The calibration curve (Figure 23) was prepared in methanol in the range of 0.

Stability Study of Amphotericin B Nanoemulsions

The NE that presented the best physicochemical properties were chosen and submitted to stability study. The stability study was conducted by one year.

The samples were stored under two different conditions: 4ºC ± 2ºC (refrigerator) and 28ºC ± 2°C (room temperature) in amber glass flasks hermetically sealed.

Transmission Electron Microscopy (TEM)

The morphology of AmB nanoemulsions was analyzed by transmission electron microscopy (TEM) in Jeol JEM 1011 (JEOL Inc., Peabody, USA) equipment available in Rudolf Barth Electronic Microscopy Platform (IOC -Fiocruz). The samples were diluted in distilled water at 1:4 and 1:8. Then, 5 μL of diluted NE was added through a 300-mesh copper grid with sterile 2% of Collodion (Parlodion strips) in amyl acetate coating (adapted from [START_REF] Mattos | Nanoemulsions containing a synthetic chalcone as an alternative for treating cutaneous leishmaniasis: optimization using a full factorial design[END_REF]adapted from FUKUI et al., 2003). The excess was removed with filter paper and the samples deposited on the grid were analyzed in the microscopy.

Determination of Droplet Size, Polydispersity Index and pH

The droplet size and polydispersity index of developed formulations were analyzed in Zetasizer Nano Model S90 (Malvern Instruments, UK) equipment following method described before in item 3.3.2.3.1. The pH was verified in pHindicator strips pH 0 -14 Universal indicator (Merck, Germany). The measurements were performed at 0, 7, 15, 30, 60, 90, 180, 365 days in two storage conditions (room temperature -28ºC and refrigerator -4ºC).

Drug Content

The AmB concentration in the nanoemulsions was determined by UV-vis (UV-vis model V-630, Jasco, Brazil) spectrophotometric method previously described in item 3.3.2.3.2. The AmB concentration was evaluated at 7, 30, 60, 90, 180, 365 days in two storage conditions (room temperature -28ºC and refrigerator -4ºC).

In vitro Release of Amphotericin B Nanoemulsions

AmB nanoemulsions were submitted to in vitro release study as well as free AmB. Free AmB dissolved in 1 ml of DMSO was analyzed in the same amount of AmB contained in nanoemulsions. In brief, 300 mg of NE were weighted and then inserted on cellulose acetate membrane (Sigma-Aldrich, Brazil), previously attached to a cut falcon tube. The cellulose acetate membrane was hydrated in distilled water for, at least, 12 hours before use. Therefore, the tubes with membrane and material were inserted on the surface of 20 mL of receptor medium. The receptor medium was phosphate buffer pH 7.4 (USP, 2012c) containing 2% of Tween ® 20. Experiments were carried out at 37ºC under constant stirring (300 rpm) for 48 hours. Hence, 3 mL of receptor medium was collected at 1, 2, 4, 6, 24, 30 and 48h with sample return to collected recipient.

The AmB concentration was determined by UV-visible spectrophotometry method (adapted from [START_REF] Santos | Amphotericin B-loaded nanocarriers for topical treatment of cutaneous leishmaniasis: development, characterization, and in vitro skin permeation studies[END_REF] in wavelength 410 nm. The experiments were conducted in quadruplicate. Furthermore, free AmB sample dissolved in DMSO was also evaluated in the same system used for NE release. A previous study (data not shown) showed good solubility of AmB solution in the receptor medium. Figure 24 presents a scheme of in vitro release study.

In vitro Skin Permeation Study of Amphotericin B Nanoemulsions

Skin Permeation Method

The in vitro skin permeation study of AmB nanoemulsions were evaluated in a Franz vertical cell diffusion system (Figure 26), composed of a donor compartment with diffusional area of 1.54 cm 2 and receptor compartment with 7 mL corresponding to maximum volume. The experiments were carried out following method described before by our group [START_REF] Marques | Development and characterization of a nanoemulsion containing propranolol for topical delivery[END_REF][START_REF] Arantes | Promotion of cutaneous penetration of nifedipine for nanoemulsion[END_REF] and adapted from Santos and co-workers (2012). of AmB penetrated in 1, 2, 4 and 6h (data not shown). For this reason, 1 mL of the receptor medium was collected after 6h and after 8h with refiling with 1 mL of fresh buffer solution. The aliquots were filtered through 0.20 μm pore diameter filters discs (Chromafil ® Xtra PES -20/25) and analyzed by High Performance Liquid Chromatography (HPLC).

After 8h, the permeation study was finished. The pig ear skins were removed from Franz diffusion cell and nanoemulsions excess were removed with a cotton swab dipped in distilled water. In order to evaluate the drug retained in epidermis and in dermis, the layers were separated using a scalpel to collect the epidermis following method standardized by our group. Thereafter, the epidermis and dermis (cut into small pieces) were inserted in eppendorf containing 1 mL of mobile phase (sodium acetate buffer solution pH 5.0 + acetonitrile -60:40 v/v respectively) for AmB extraction. The eppendorfs were vortexed for 60 seconds three times and centrifuged for 10 minutes in 8000 rpm. Then, all samples were filtered through 0.20 μm pore diameter filters discs (Chromafil ® Xtra PES -20/25) and analyzed by HPLC. The results were expressed as mean of triplicates.

High Performance Liquid Chromatography (HPLC) Assay

The quantification of AmB in the skin permeation study was measured by reverse phase HPLC using a Kromasil C18 column ( 250 Pig ear skin pieces without contact with nanoemulsions were cut and inserted in Eppendorf containing 1mL of mobile phase. The eppendorfs were vortexed for 1 minute three times and centrifuged for 10 minutes in 8000 rpm.

Then, all samples were filtered through 0.20 μm pore diameter filters discs (Chromafil ® Xtra PES -20/25) and analyzed by HPLC. Moreover, three different solutions containing AmB in higher concentration used for calibration curve (20 µg/mL) were placed on pig ear skin pieces and added 1 mL of mobile phase.

Then, the eppendorfs were submitted to method described for blank analyses.

These quantifications showed the retention time of AmB around 3.2 minutes after peaks corresponding to skin interferences. Figure 28 shows the blank chromatogram (skins pieces only in mobile phase) and AmB solution chromatogram (extracted from skin pieces).

BIOLOGICAL ASSAYS

Assessment of cytotoxicity of AmB nanoemulsions in BMDM cells

Bone marrow-derived macrophages (BMDM) were obtained from femurs and tibia of BALB/c mice as described by Marim and co-workers (2010) et al., 2018;[START_REF] Siqueira | Development and evaluation of zinc phthalocyanine nanoemulsions for use in photodynamic therapy for Leishmania spp[END_REF]. Moreover, the negative control was RPMI medium without treatment and positive control was Triton 100x. Triton 100x is a nonionic surfactant most used for lysing cells (KOLEY & BARD, 2010) and can be used as positive control in cytotoxicity assays. After incubation time, the cytotoxicity assay was investigated using resazurin method (RAMPERSAD, 2012). Resazurin (7-hydroxy-10-oxidophenoxazin-10-ium-3-one), also known Alamar Blue ® , is a redox indicator, which is reduced to resorufin (7-hydroxy-3Hphenoxazin-3-one) by viable cells [START_REF] Csepregi | Complex formation of resorufin and resazurin with bcyclodextrins: Can cyclodextrins interfere with a resazurin cell viability assay? Molecules[END_REF]RAMPERSAD,2012).

Figure 29 shows the reduction reaction of resazurin. 20 µL of resazurin solution were added and the plate was incubated for more 4 hours at 36ºC. Following incubation, the fluorescence was measured using microplate Fluorimeter Spectra Max M5 (Molecular Devices, USA) at 560 nm for excitation and 590 nm for emission. The results were reported as % cell viability. The 50% cytotoxicity concentration (CC50), which represents the concentration of drug or formulation that reduces 50% cell viability when compared to negative control/untreated control, were determined by logarithmic regression analysis.

Antileishmanial Activity

The et al., 2013;[START_REF] Rolón | Development of resazurin microtiter assay for drug sensibility testing of Trypanosoma cruzi epimastigotes[END_REF].

Promastigotes (10 7 parasites/mL) were seeded on 96-well microtiter plates. 180 µL of samples (AmB nanoemulsions, nanoemulsions without drug, AmB in DMSO solution and medium with FBS + antibiotics) were added on 96well microtiter plates containing promastigotes. AmB solution was tested in following concentrations: 0.05, 0.1, 0.5, 1, 5 µg/mL. AmB nanoemulsions and blank nanoemulsions were evaluated in concentrations between 0.1 and 5 µg/mL. Moreover, the essential oil was analyzed in various concentrations (0.3, 1.5, 3, 7.5 and 15 µg/mL) in order to investigate the effect of this substance alone.

DMSO was evaluated, also, in concentration used to dissolve AmB. Negative control was only medium and positive control in nanoemulsions tests was AmB solution in concentration that promotes lesser than 50% of viable promastigotes.

Thereafter, the plate was incubated for 24 hours at 28ºC. After incubation time, 20 µL of resazurin solution 0.05% were added and the plate was incubated for more 4 hours at 28ºC. Following incubation, the fluorescence was measured using microplate Fluorimeter Spectra Max M2e (Molecular Devices, USA) at 560 nm for excitation and 590 nm for emission. The results were reported as % parasite viability. The 50% inhibitory concentration (IC50), which represents the concentration of drug or formulation that promote 50% of parasite's death in comparison with negative control/untreated control, were determined by logarithmic regression analysis.

The selective index (SI) was calculated as the ratio between cytotoxicity (CC50) and activity (IC50) against Leishmania amazonensis promastigotes.

Statistical Analysis

One-Way ANOVA with Tukey Post-test, T test paired, CC50 and IC50 were calculated using GraphPad Prism 5.0 (GraphPad Software, USA). The P value < 0.05 was considered significant.

RESULTS AND DISCUSSION

PHYSICOCHEMICAL CHARACTERIZATION OF AMPHOTERICIN B 3.4.1.1. Scanning Electron Microscopy (SEM)

The SEM images of AmB raw material were evaluated in three different magnifications (220x, 650x and 1300x) and were shown in Figure 30. AmB powder showed a spherical shape with porous surface and varied sizes between 2 µm and 20 µm (measures held in Figure 30c, data not shown).

Additionally, AmB powder presented some agglomerates. [START_REF] Rodríguez | Desarrollo farmacéutico de formulaciones de poliagregados de anfotericina B[END_REF] evaluated the influence of sodium deoxycholate addition in AmB formulations and the physicochemical, kinetical and toxicological effects. In this study, AmB raw material showed scaly agglomerate particles with particle size under 15 µm and short particles adhered on surface. Zu and co-workers (2014) produced AmB nanoparticles and evaluated some physicochemical properties. It was detected irregular crystals with mean particle size around 14 µm. These two works showed raw AmB with similar morphology than observed in Figure 30, which confirms AmB raw material structure.

Thermogravimetry Analysis (TGA)

Figure 31 shows the Thermogravimetric Analysis (TGA) of AmB raw material. AmB TGA presents two lines, being the black one related to mass loss (%) and the blue the first derivation of mass loss (%/ºC).

Figure 31. TGA curve of raw AmB.

According to TGA curve, raw AmB presented two mass loss events, being the first at 177.70ºC and the second at 368.49ºC. Connors and co-workers (1986) described a gradual decomposition of AmB above 170ºC. Zu and co-workers (2014) prepared and characterized AmB nanoparticles. They evaluated thermal properties of nanoparticles comparing with raw drug and observed, in TGA curve of raw AmB, only one mass loss event at 168ºC due to TGA was held between 25ºC and 250ºC. Ghosh and co-workers ( 2017) developed and investigated some properties of mannose modified PLGA nanoparticles containing AmB. In Thermal Analysis held in the range of 30-360ºC, TGA curve of raw drug presented a mass loss event around 80ºC corresponding to drug dehydration followed by a high mass loss event around 195ºC corresponding to drug degradation.

Franzini (2010) studied AmB complexes with cyclodextrin and their incorporation in microemulsions. During the study of the thermal behavior of complexes and AmB, three mass loss events for AmB in the range of 151-245ºC, 245-303ºC and 303-542ºC were observed, also attributed to drug degradation.

Based on this information, the two events shown in Figure 31 are attributed to drug degradation in high temperatures.

Fourier Transform Infrared Spectroscopy (FTIR)

Figure 32 presents the FTIR spectrum of raw AmB.

Figure 32. FTIR spectrum of raw AmB.

AmB spectrum showed characteristics peaks at 3358 cm -1 corresponding to hydroxyl group (OH), at 1690 cm -1 (C=O stretching), at 1553 cm -1 (C=C stretching), at 1068 cm -1 (C-O asymmetric stretching) and at 1007 cm -1 (C-H bending). Chhonker and co-workers (2015) developed and characterized lecithin/chitosan nanoparticles of AmB. It was observed strong characteristics peaks at 3401 cm -1 corresponding to hydroxyl group (OH) and at 1645 cm -1 corresponding to C=O stretching in AmB FTIR spectrum. Ghosh and co-workers (2017) identified some AmB characteristics peaks, in FTIR analysis, at 3416-3382 cm -1 (N-H stretching), at 2936-2916 cm -1 (long alkane chains asymmetric stretching), at 1680 cm -1 (C=C non-conjugated stretching) and one peak near 1100 cm -1 (C-O stretching). Zia and co-workers (2017) investigated the physicochemical properties and fungal activity of nanoparticles of AmB produced using Aloe vera leaf extract. Raw AmB spectrum presented some peaks as at 3440 cm -1 (OH stretching), at 2936 cm -1 (C-H stretching), at 1657 cm -1 (C=O stretching), at 1579 cm -1 (C=C stretching), at 1095 cm -1 (C-O asymmetric stretching) and at 994 cm -1 (C-H bending). FTIR spectrum of AmB used in this study, showed in Figure 32, presents the same characteristic peaks reported in these studies for AmB.

AMPHOTERICIN B NANOEMULSIONS DEVELOPMENT

The AmB nanoemulsions prepared were stored in amber glass flasks hermetically sealed at room temperature (Figure 33) in order to protect from light. From a visual analysis of Figure 33, it can be seen that nanoemulsions A, B, J and K presented different physical aspects, from liquid to gelled formulations.

More specifically, A-NE, B-NE and J-NE samples showed yellow color, stable and limpid aspect, while K-NE exhibited yellow color with cloudy aspect. There are no literature reports about AmB nanoemulsions using essential oils, even if AmB nanoemulsions have already been described before, prepared by different methods and, with the oil phase generally composed by triglycerides.

As examples, Filippin and co-workers (2008) Mendonça and colleagues (2016) prepared AmB polymeric micelles using Pluronic ® F127 and evaluated in vitro and in vivo response against murine tegumentary leishmaniasis. The polymeric micelles were produced under vigorous magnetic agitation of AmB solution (AmB dissolved in dichloromethane) addition in Pluronic ® F127 solution. Then, dichloromethane was evaporated under rotary evaporation and, after dispersed in water. They used the same polymer selected for this thesis. However, they used more steps and organic solvent to produce AmB polymeric micelles, while here we are using ultrasonic processor (one step formulation) with an organic solvent of Class 3 (DMSO)

instead Class 2 (dichloromethane). Solvents in Class 2 (ICH Guidelines) should be limited in pharmaceutical products because of their inherent toxicity. Ideally, less toxic solvents (Class 3) should be used where practical (ICH, 2016).

PHYSICOCHEMICAL CHARACTERIZATION OF AMPHOTERICIN B NANOEMULSIONS

Determination of Droplet Size, Polydispersity Index and pH

The physicochemical characterization of twelve batches of AmB nanoemulsions is shown in Table 4. However, formulation G showed elevated PdI (0.432 ± 0.002), suggesting an instable system. The batches H-NE and L-NE, which had not AmB, exhibited average droplet size lower than 100 nm and lower than respectively formulations produced with AmB (F and B). The PdI of these nanoemulsions were lower than PdI values of respectively nanoemulsions containing drug. Formulation K presented an average droplet size higher than 100 nm and a moderate value of PdI.

The achievement of nanoemulsions containing droplet size lower than 100 nm allows their characterization in nanoscale formulations, since nanoscale concept is controversial in literature (CAMPOS, RICCI-JUNIOR, MANSUR, 2012). However, in general, nanoemulsions are defined as oil-water emulsions with droplet size below 500 nm [START_REF] Rai | Nanoemulsion as pharmaceutical carrier for dermal and transdermal drug delivery: Formulation development, stability issues, basic considerations and applications[END_REF]. Thus, the twelve batches prepared can be consider in nanoscale.

The small droplets size observed in nanoemulsions prepared with clove oil (Table 4) was related to the ultrasonication time used (5 minutes). It is known that the ultrasonication time interferes in average droplets size. For example, Shahavi and co-workers (2015) evaluated physicochemical parameters for clove oil nanoemulsions. They prepared nanoemulsions containing between 2.5% and 15% of clove oil and 5% of Tween ® 80/Span ® 80, under ultrasonication.

Ultrasonication times higher than 2.5 minutes allowed average droplets sizes lower than 80 nm.

In relation to pH values of nanoemulsions, the values remained 4.5 and 5.0, which correspond to a compatible pH with human skin pH. The pH of human skin has a value in the range of 4.0 to 6.0 [START_REF] Ali | Skin pH: From basic science to basic skin care[END_REF][START_REF] Lambers | Natural skin surface pH is on average below 5, which is beneficial for its resident flora[END_REF]. Furthermore, the values of pH allow topical administration of AmB nanoemulsions, which it is the objective of this development.

Drug Content

Prior to drug content assay of nanoemulsions, the AmB ultraviolet spectrum was identified in the range of 200 to 800 nm (Figure 34). Four peaks can be seen respectively at 333 nm, 360 nm, 389 nm and 415 nm.

According to European Pharmacopeia (2009), AmB UV spectrum is characterized by three peaks at 362 nm, 381 nm and 405 nm. Some studies have related these peaks to the presence of AmB aggregates or AmB monomers: AmB aggregates at 328 nm followed by three peaks at 365 nm, 384 nm and 408 nm [START_REF] Rodrigues | Amphotericin B-loaded poly(lactide)-poly (ethylene glycol)-blend nanoparticles: Characterization and in vitro efficacy and toxicity[END_REF]; AmB aggregates at 340 nm, followed by three peaks at 365 nm, 388 nm and at 410 nm corresponding to AmB monomers [START_REF] Caldeira | Nanoemulsions loaded with amphotericin B: A new approach for the treatment of leishmaniasis[END_REF]. Based on these data, the AmB UV spectrum obtained in our study are considered to be related to the self-association of AmB (peak at 333 nm) and the presence of AmB monomers (peaks at 360 nm, 389 nm and 415 nm).

Figure 34. AmB UV-spectrum.

In the following, UV spectra of two nanoemulsions were obtained in order to evaluate the best wavelength for drug content assay. The nanoemulsions selected were F-NE (nanoemulsion with AmB) and H-NE (blank nanoemulsion).

Figure 35 shows the UV spectra of these formulations in the range of 220 to 500 nm. This range of wavelength was chosen due to previous identification of AmB absorption peaks between 300 and 420 nm (Figure 34). It could be observed that F-NE, which contains AmB, presented four peaks in the range of 340-420 nm, corresponding to AmB peaks. H-NE (formulation without drug) presented no peaks in the range of 340-420 nm, which confirmed the absence of AmB in this sample. However, some absorption between 300 and 400 nm was also detected in H-NE spectrum, suggesting that other material used to produce nanoemulsions had some absorption in the same region as AmB. This interference allowed the choice of 410 nm as the wavelength for drug content assay. In this wavelength, the absorbance value was related to AmB presence in the formulations. Moreover, in the UV spectra of two other samples, it could be identified highest absorbance values in the range of 220-300 nm, which are probable related to other constituents as essential oil or polymer in the formulations.

Table 5 presents the drug content of twelve batches of nanoemulsions.

These batches were stored at room temperature for seven days before the drug content analysis. The theoretical concentration of AmB was 0.5 mg/mL. Referring to the literature, it can be confirmed that others AmB nanoemulsions have reached higher loads of AmB. For example, Santos and coworkers (2012) developed AmB nanoemulsions with triglycerides and stearylamine and obtained 95% of drug content in their formulations for topical application. Santos and co-workers (2017) prepared AmB nanoemulsions, for intravenous administration, with triglycerides, Tween ® 80 and glycerol and with a higher drug content than the best values observed for our AmB nanoemulsions (around 77%), probably due to the differences in the formulations (presence of stearylamine formed ion pairing with drug), affecting, among other things, the stability of AmB nanoemulsions. Another possibility for lower drug content values observed in this work is the storage time at room temperature (7 days) before the analysis indicating a possible drug degradation.

To summarize, the AmB nanoemulsions selected for further studies (stability and in vitro analyses) showed, on average, 77% of drug content.

STABILITY STUDY OF AMPHOTERICIN B NANOEMULSIONS

New batches (B-NE, E-NE and F-NE, H-NE and L-NE) were prepared for stability study. These new nanoemulsions were prepared with 0.3% of antimicrobial preservative (imidazolidinyl urea) to protect the formulations. E-NE was discarded from the study due to its visual changes (became more fluid and cloudier) in seven days of storage. Then, B-NE and F-NE (with AmB) and H-NE and L-NE (without drug) were submitted for one year of storage in two different temperature conditions: at room temperature (28ºC) and in refrigerator (4ºC), in amber flasks hermetically sealed.

Transmission Electron Microscopy (TEM)

Prior to stability study, the recent nanoemulsions were evaluated by TEM to investigate the morphology of droplets. Figure 36 TEM images of four nanoemulsions showed spherical shaped droplets.

Moreover, droplets lower than 100 nm in size were observed, according to the average droplets size determined by dynamic light scattering analysis. This is the range of droplet size of dispersed phase frequently reported in the literature: AmB microemulsion comprising various lipids, Span ® and Tween ® as surfactants with an average droplet size around 120 nm were described by Silva and co-workers (2013); AmB nanoemulsions with variable compositions of surfactant and co-surfactant with an average droplet size lower than 100 nm were developed by Hussain andco-workers (2013 and2016), while AmB nanoemulsions prepared with various lipids were characterized by monodispersed oil droplets with spherical shape and sizes lower than 150 nm [START_REF] Sosa | Amphotericin B releasing topical nanoemulsion for the treatment of candidiasis and aspergillosis[END_REF]. These studies have described similar morphology than that observed in our work. Moreover, they supported the use of TEM analysis to investigate the morphology and average size of nanoemulsions confirmed by dynamic light scattering analysis.

Determination of Droplet Size, Polydispersity Index and pH

The AmB nanoemulsions, stored at 0, 7, 15, 30, 60, 90, 180, 365 days under two storage conditions (room temperature -RT and refrigerator -R) and placed into amber glass flasks in order to protect to light due to AmB is photo sensible (EUROPEAN PHARMACOPEIA, 2009), were evaluated in terms of their average droplet size over time.

The results were given in Figure 37 for B-NE and its blank L-NE and in Figure 38 for others two AmB nanoemulsions, F-NE and its blank H-NE.

In Figure 37, B-NE and L-NE presented droplets size, on average, lower than 60 nm. Moreover, B-NE (which have AmB) showed higher droplets size (around 40 nm) than L-NE, which was prepared without drug and had droplets size around 30 nm. It was possible to observe an increase of droplets size at 90 days of storage, when samples were storage under refrigeration. This can be explained by droplets agglomeration and/or no efficacy of droplets dispersion in distilled water for DLS measurements. The two nanoemulsions were considered physically stable until 365 days, even if an enhancement of droplet sizes was detected (B-NE -around 200 nm and L-NE 130 nm).

However, L-NE stored in refrigerator (4ºC) remained more stable than sample stored at room temperature in the same period (365 days). Based on these results, it seems that AmB nanoemulsion (B-NE) and blank nanoemulsion (L-NE) can be produced, stored in room temperature or under refrigeration (4ºC), and used until 365 days from their manufacturing data. In Figure 38, F-NE and H-NE showed droplets size, on average, lower than 60 nm as observed to B-NE and L-NE as well.

F-NE (with AmB) presented droplets size around 43 nm, while H-NE (blank) were around 33 nm. The same trend was observed with B-NE (with AmB)

and L-NE (without AmB). Therefore, the presence of drug in nanoemulsion formulation increased the droplets size when compared to blank formulations.

As for B-NE and L-NE, F-NE and H-NE presented a good stability and remained stable until 365 days of storage. Furthermore, when samples were stored in refrigerator, it was possible to detect:

-An increase of droplets size in 90 days of storage. A possible explanation, as already mentioned, could be some droplets agglomeration and/or no homogeneous dispersion in distilled water to DLS measurements;

-An enhancement of droplets size in F-NE (droplet size around 120 nm)

in the last storage time;

-A slight increase of droplets size in H-NE (droplets around 55 nm).

Results from the literature have shown that AmB nanoemulsions may represent stable systems, even varying the components and the preparation method. Taking some examples:

Santos and co-workers (2012) developed AmB nanoemulsions using triglycerides, Tween ® 80, Span ® 80 and stearylamine and investigated the stability of formulations for 15 and 30 days. The formulations were stored into amber glass containers. They obtained nanoemulsions (with or without AmB) with droplet size around 200 nm and stable for 30 days. However, the storage condition of these formulations was not described. Singh and co-workers (2017) developed AmB nanoemulsions using Pluronic ® F127 and chitosan. The formulations were submitted to a stability study during 6 months at 4ºC. They obtained stable nanoemulsions with droplets size around 110 nm. Shahavi and co-workers (2015) evaluated physicochemical parameters and the stability of clove oil nanoemulsions (containing between 2.5% and 15% of clove oil and 5%

of Tween ® 80/Span ® 80) for 180 days. They observed a better stabilization in nanoemulsions with higher concentrations of oil. Moreover, droplets size increased during storage.

It is important to emphasize here that our study evaluated two different storage temperature conditions over longer period than those reported in the literature.

In addition to droplets size evaluation, the polydispersity index (PdI) of four NE were determined and are presented in Figure 39 There are some studies that described the stability of AmB nanoemulsions evaluating the PdI. Santos and co-workers (2012) investigated the stability of AmB nanoemulsions for 30 days. They observed that blank nanoemulsions and AmB nanoemulsions showed values of PdI very similar varying between 0.2 and 0.3 and concluded that these systems were stable and homogeneous. Singh and co-workers (2017) evaluated the stability of AmB nanoemulsions with or without chitosan for 180 days. The nanoemulsions produced without chitosan showed values of PdI between 0.120 and 0.200 while nanoemulsions with chitosan presented values of PdI varying from 0.100 to 0.300. However, it was not statistically different and all nanoemulsions were considered stable for 180 days.

In addition to droplets size and PdI values, the pH of the four nanoemulsions (B-NE, F-NE, H-NE and L-NE) were evaluated in stability study.

The values of pH during all storage period under two storage conditions for all four samples were maintained 5.0 without any variation. This value of pH is in agreement of desired for topical formulations due to belongs the range of human skin pH, which is between 4.0 and 6.0 [START_REF] Ali | Skin pH: From basic science to basic skin care[END_REF][START_REF] Lambers | Natural skin surface pH is on average below 5, which is beneficial for its resident flora[END_REF].

One study evaluated the pH values during stability of AmB nanoemulsions for topical administration. Santos and co-workers (2012) observed that nanoemulsions, with and without AmB, presented no significant differences of pH values, which were in the range of 7.3 to 7.7. These values were higher than consider optimal pH for topical application and higher than values obtained in this work. This difference is related to the formulation composition, which is different between this work and work of Santos and colleagues (2012).

Other studies investigated the pH values for intravenous administration.

Drug Content

Besides monitoring droplet size, PdI and pH during storage (365 days under two different temperature conditions), the drug content was investigated in B-NE and F-NE. Figure 41 shows the results of drug content in these nanoemulsions. B-NE and F-NE stored at room temperature (Figure 41a and41c, respectively) showed a considerable reduction of AmB concentration during storage. Furthermore, B-NE samples were statistically different among them as observed for F-NE samples. F-NE presented higher value of AmB concentration in sample stored for 30 days than that for sample stored for 7 days. An error of analysis can explain this difference. B-NE showed a drug content around 95% for sample stored 7 days at room temperature, while sample stored for 365 days in the same condition presented only 20% of drug content.

F-NE showed 71% of drug content in sample stored 7 days, while sample stored for 365 days had only 17% of AmB content. This reduction of AmB concentration in samples storage at room temperature can be related to AmB degradation. AmB exhibits poor stability already described in literature when exposed to light, heat, ultraviolet irradiation, extremes of pH and suffers oxidation in presence of oxygen [START_REF] Bonner | Stability studies with amphotericin B and amphotericin B methyl ester[END_REF].

Moreover, there are, also, reports about AmB degradation when incorporated in oil-water systems [START_REF] Walker | Amphotericin B in lipid emulsion: stability, compatibility, and in vitro antifungal activity[END_REF]. The possible AmB degradation observed in nanoemulsions stored at room temperature will be investigate through HPLC in a complementary study.

B-NE and F-NE stored in refrigerator (4ºC) (Figure 41b and41d, respectively) presented unchanged AmB concentration over whole storage period, presenting only a statistically significant reduction at the end of the experiment (365 days). However, the reduction was lower than reduction observed in samples stored at room temperature. B-NE presented 95% of drug content in 7 days of storage and maintained this value until 365 days, in which AmB concentration was reduced to 82%. F-NE had 100% of drug content in 7 days of storage while the last analysis (365 days) showed 83% of drug content.

According to these results, it is advisable to store AmB nanoemulsions at 4ºC to maintain their stability until 365 days. The nanoemulsions prepared for in vitro studies were then maintained at 4ºC.

Filippin and co-workers (2008) evaluated the AmB content during 180 days of storage at 25ºC and 4ºC. They observed that drug content was 98% in the beginning of stability study and was maintained around 90% AmB content during stability tests. It was, also, verified that nanoemulsions stored in both conditions remained stable chemically and physically, which was explained by the high purity of soybean phosphatidylcholine.

Santos and co-workers (2012) analyzed the stability of AmB nanoemulsions prepared with or without stearylamine for 90 days of storage. The AmB concentration remained constant during stability study for nanoemulsion prepared with stearylamine while a significant reduction of AmB content after 90 days was observed for formulation without stearylamine. The presence of stearylamine promotes ion pairing formation between this amine and carboxylic group of AmB, which stabilized oil-water interface and increase the nanoemulsions stability. The same group, in 2015, evaluated the stability of AmB nanoemulsions (with or without stearylamine) for 180 days stored at 4ºC using cholesterol instead of sorbitan monooleate used before. It was observed that NE with or without stearylamine presented a constant AmB content after 180 days, suggesting that both formulations were stable [START_REF] Caldeira | Nanoemulsions loaded with amphotericin B: A new approach for the treatment of leishmaniasis[END_REF].

Singh and co-workers (2017) investigated the stability of AmB nanoemulsions containing chitosan or not during 180 days at 4ºC. The encapsulation efficiency remained around 60% for nanoemulsions with or without chitosan and during whole storage period, suggesting stable formulations.

It is possible to observe that, in general, AmB nanoemulsions described in literature were stable and submitted to stability studies corresponding to a period of 180 days at 4ºC. These results support the results obtained in this work that AmB nanoemulsions remained stable until 365 days and storage under refrigeration is advisable.

Comparing our formulation with AmB formulations presented on the market, our nanoemulsions are more stable than Ambisome ® and Anforicin ® B, formulations for intravenous administration. These market formulations have stability for 24 hours after reconstitution under refrigeration (2ºC -8ºC), while our formulation remains stable for 365 days under same temperature (4ºC).

IN VITRO RELEASE OF AMPHOTERICIN B NANOEMULSIONS

New AmB nanoemulsions (B-NE and F-NE) were prepared and stored at 4ºC for in vitro studies. These formulations presented oil droplets size around 45 nm and AmB content around 98%wt. Free AmB (AmB dissolved in DMSO), B-NE and F-NE in vitro release profiles are shown in Figure 42. showed faster release than nanoemulsions (around 15% of drug released in 30h, against 0.2% from B-NE and F-NE formulations). However, after 30 hours, free AmB presented a decrease of release profile, possible due to some drug degradation. In fact, AmB degradation in phosphate buffer solution maintained at 37ºC has already report by Connors and co-workers (1986).

AmB toxicity is one of the challenges for conventional therapy to leishmaniasis treatment. A slow release of AmB nanoemulsions should be interesting for a controlled drug release, decreasing the drug toxicity and protecting it from degradation. Due to the very slow AmB release from our nanoemulsions (lower than 1%wt), these systems were not submitted to a mathematical kinetic analysis.

Santos and co-workers (2012) evaluated and compared the in vitro release profile, for 8 hours, of three formulations containing AmB: AmB in solution (methanol/DMSO), Fungizone ® and AmB nanoemulsion developed by the authors. They verified that AmB solution presented the highest percentage of drug release after 8 hours (around 69%), while AmB nanoemulsion presented the lowest percentage (only 8%). The low release profile of nanoemulsion may be explained by the strong absorption of AmB at the oil-water interface and this property allows a reduction of AmB toxicity in comparison to Fungizone ® toxicity.

Sosa and co-workers (2017) developed AmB nanoemulsion for topical application for antifungal treatment. The physicochemical properties, drug release, permeation and antifungal activity were evaluated. The release of AmB from AmB nanoemulsions (prepared with triglycerides) was evaluated for 75 hours and compared with AmB solution (in DMSO). AmB nanoemulsions released 100% of drug after 75 hours, while AmB solution released only 10%.

The percentage of AmB released from nanoemulsions increased over time until 100%. Therefore, AmB nanoemulsion presented a higher and extended drug release than AmB solution.

These results indicated that nanoemulsions is an interesting formulation for topical delivery of AmB. Despite two different formulations, this work and the work of Santos and colleagues (2012) suggested a slow release of AmB from this kind of formulation. A controlled release was also demonstrated by the nanoemulsion formulation of Sosa and colleagues (2017), even if with higher drug release rate.

Furthermore, there are some more recent studies, which described in vitro release profiles from AmB nanoparticles. Al-Quadeib and co-workers (2015)

developed PEG-PLGA nanoparticles of AmB for oral delivery and evaluated the in vitro release profile of different batches prepared for 24 hours. They observed a biphasic release profile with a rapid release in the first 6 hours (varying 21-51% of drug released) and a slow release after 6 hours until 24 hours with 11% of drug released. This release profile was explained, in the first 6 hours (fast release) by the dissolution and diffusion of AmB not entrapped, followed by a continuous and slow release attributed to a drug diffusion from the particle core.

Chhonker and co-workers (2015) developed a lecithin AmB nanoparticles with or without chitosan for ocular application and analyzed the in vitro release profile for 10 hours. They observed a biphasic release with a burst release in the first hours followed by a continuous release. The initial fast release was related to desorption and diffusion of AmB adhered to nanoparticles surface. The continuous release was explained by drug diffusion from nanoparticles matrix.

However, the amount of AmB released after 10 hours was higher than 80%.

It can be verified that AmB release is dependent on formulation types.

Nanoparticles presented a biphasic release while nanoemulsions promoted a slow and continuous release. The two type of formulations are interesting for prolonged release of drugs.

IN VITRO SKIN PERMEATION STUDY OF AMPHOTERICIN B NANOEMULSIONS

In vitro skin permeation study evaluated the amount of AmB permeated across the pig ear skin and presented in the receptor chamber after 6 and 8 hours of experiment (Figure 43). The amount of AmB permeated from the two nanoemulsions was lower than 2 µg/cm 2 (or lower than 0.2%) after 8 hours. A higher amount of AmB was permeated from F-NE than from B-NE. The lower values of AmB permeated after 8 hours can be related to the in vitro release profile of these formulations, which showed very slow release. Furthermore, the low amount of AmB permeated could suggest a possible low systemic action of these formulations, decreasing the toxicity and side effects of AmB and supporting the local action desired for these formulations.

Santos and co-workers (2012) evaluated in vitro skin permeation study for different formulations containing AmB (AmB in solution, Fungizone ® , Amphocil ® , AmB nanoemulsion and Fungizone ® -Lipofundin ® mixture) and investigated the amount of drug permeated after 24 hours. It was observed no amount of AmB (concentration lower than 0.02 µg/mL) detectable in receptor chamber for all formulations. This might probably be explained by the drug hydrophobicity and high molecular weight, which difficult the drug passage through dermis hydrophilic barrier. This article confirmed the results observed in Figure 43 that AmB nanoemulsions presented a low permeation across skin.

There are some articles that use albino rat skin as skin model instead of pig ear skin. Hussain and co-workers (2013) investigated the in vitro permeation study of three oil-water AmB nanoemulsions, Fungisome ® (liposomal AmB gel)

and drug solution for 24 hours. It was collected aliquots in various times (1, 2, 4, 6, 8, 10, 12, 16, 20 and 24 hours) to evaluate the amount of AmB presented in receptor chamber. Nanoemulsions prepared with polyethylene glycol 400 (PEG-400) as co-surfactant showed a higher cumulative amount of AmB (higher than 950 µg) after 24 hours than nanoemulsion prepared with propylene glycol, drug solution and Fungisome ® . The presence of PEG-400 allowed the increase of drug penetration due to increases the drug solubility in stratum corneum layer. Butani and co-workers (2014) developed AmB microemulsions and evaluated the in vitro skin permeation of these formulations. Microemulsions showed higher amount of AmB permeated (higher than 95 µg/cm 2 ) than drug solution (around 50 µg/cm 2 ) after 24 hours. The microemulsions increased the drug permeability. This finding was explained by the authors by the water content in microemulsions, which allowed the drug permeation due to stratum corneum hydration promoting the enlargement of channels in keratin layer.

In addition to evaluation the amount of AmB presented in the receptor chamber, it was investigated the amount of AmB presented in epidermis and in dermis after 8 hours of experiment. Figure 44 shows the amounts of AmB retained in each skin layer (epidermis and dermis). B-NE and F-NE showed some amount of AmB retained in epidermis and dermis. However, epidermis presented more AmB retained than dermis, which can be explained by the stratum corneum layer presence in the epidermis that is the major barrier to drug permeation [START_REF] Ruela | Evaluation of skin absorption of drugs from topical and transdermal formulations[END_REF]. The two formulations presented AmB retention higher than 0.7 µg/cm 2 in epidermis and between 0.16-0.36 µg/cm 2 in dermis. Nanoemulsions showed no statistically differences (p>0.05) between then conforming to T test in relation to AmB retention in epidermis and in dermis. Moreover, no statistically differences (p>0.05) was observed comparing AmB retention in epidermis and dermis for each NE based on T test. Although these results suggested low permeation of AmB from nanoemulsions, there is some amount of drug retained in dermis that can be promote the CL treatment as the parasites are located. Thus, these results were compared with in vitro antileishmanial activity in order to evaluate if this low amount of drug retained in dermis can be able to treat CL.

Despite this experiment was conducted in intact skin, which has skin composition different from skin with cutaneous leishmaniasis lesions, it is not possible investigate in vitro skin permeation with skin containing lesion.

Therefore, skin permeation study allows have an idea of formulations behavior on skin.

Our results demonstrated low penetration across the skin with some AmB retention in dermis and epidermis. A low AmB penetration across the skin was already described in literature, as well as high amounts of drug retained into skin layers. Among them:

Santos and co-workers (2012) evaluated the amount of AmB penetrated into dermal membranes from various formulations (AmB in solution, Fungizone ® , Amphocil ® , AmB nanoemulsion and Fungizone ® -Lipofundin ® mixture). These experiments were investigated in pig ear skin without epidermis. It was observed that AmB solution showed the highest value of penetration into dermal membranes, around 18 µg/cm 2 , while nanoemulsion and Fungizone ® -Lipofundin ® mixture presented the lowest values (1.1 µg/cm 2 and 1.7 µg/cm 2, respectively). This may be justified by the water-oil interface of these formulations, which allows slow release of drug. These results showed higher penetration into dermal membranes than those observed in Figure 44(b).

However, in our work, the penetration study was evaluated in intact skin with epidermis while Santos and co-workers evaluated in skin without epidermis.

Some studies evaluated in vitro skin permeation of AmB formulations in different skin models. Butani and co-workers (2014) evaluated the amount of AmB retained in the rat skin from four AmB microemulsions and AmB solution after 24 hours. It was investigated the drug retained in the skin (epidermis + dermis). They verified that microemulsions had high drug retained in the skin (higher than 80 µg) than drug solution (lower than 40 µg). Perez and co-workers (2016) evaluated the amount of AmB retained in the stratum corneum and viable epidermis (epidermis without stratum corneum) of two liposomal formulations (Ambisome ® and AmB-ultradeformable liposome) after 1 hour in excised human skin for topical administration. The AmB-ultradeformable liposome presented higher drug retention in the stratum corneum (around 33%) and in the viable epidermis (around 7%) than Ambisome ® , suggesting an improvement in ultradeformable liposome development for increase AmB target delivery to viable epidermis and dermis.

Figure 45 shows a dose-dependent relation to cytotoxicity, especially for free AmB. Free AmB showed high toxicity in concentrations higher than 0.2 µg/mL while AmB nanoemulsions and blank nanoemulsion presented toxicity in all concentrations tested.

L-NE showed toxicity similar than B-NE and F-NE, indicating that NE formulations had some ingredients which promote toxicity. H-NE (blank formulation of F-NE) was not evaluated in this experiment because all NE had elevated toxicity. The two higher concentrations (0.8 µg/mL and 3.2 µg/mL) of nanoemulsions presented no statistically differences (P > 0.05) to positive control while free AmB presented only the highest concentration (3.2 µg/mL) without statistically differences (P > 0.05) to positive control. The two-lower concentration (0.05 µg/mL and 0.2 µg/mL) of free AmB and nanoemulsions presented no statistically differences (P > 0.05) between them. However, all concentrations are statistically different (P < 0.05) in comparison with Negative Control.

The ingredient in all NE that can be the cause of high toxicity is clove oil.

The high cytotoxicity of clove oil has already described in literature. Prashar and co-workers (2006) evaluated the cytotoxicity of clove oil in human fibroblasts and endothelial cells. It was observed that at concentration of 0.03%, clove oil promotes more than 90% of cell death. Furthermore, it was suggested that clove oil cytotoxicity can be attributed to eugenol, which is the major constituent of this oil. Kouidhi and co-workers (2010) investigated clove oil cytotoxicity in different cells type (macrophages RAW 264.7, adenocarcinoma cells -A549 and HT-29, human epidermoid cancer Hep-2, human fibroblast-like fetal lung cell MRC-5).

They found higher oil toxicity in cells MRC-5, HT29 and RAW 264.7 (IC50 values lower than 30 µg/mL) than Hep-2 and A549 cells (IC50 500 and 112 µg/mL, respectively). The decrease of oil droplet size of nanoemulsions may increase the cytotoxicity of this ingredient, by enlarging the surface area of contact of macrophages cells.

Caldeira and co-workers (2015) developed AmB nanoemulsions and investigated the cytotoxicity in macrophages J774. AmB nanoemulsion and blank nanoemulsion with two concentration of stearylamine (0.1% or 0.2%) were used in these studies. The results were compared with conventional AmB formulation (AmB deoxycholate). It was found that nanoemulsion with and without AmB presented high cytotoxicity and similar values of CC50. Moreover, nanoemulsions prepared with 0.2% stearylamine showed higher toxicity than nanoemulsions with 0.1%. AmB presence in formulation promoted low influence in cytotoxicity values.

However, when the concentration of stearylamine in nanoemulsions was increased, the cytotoxicity was also increased. This was attributed to electrostatic interactions between stearylamine and anionic components of the cell membrane. Despite high toxicity of nanoemulsions, conventional AmB presented higher toxicity, suggesting that nanoemulsions decreased the drug toxicity.

Mendonça and co-workers (2016) produced AmB polymeric micelles formulations using Pluronic ® F127 and investigated the cytotoxicity of this formulations in murine peritoneal macrophages. It was verified that blank formulation and AmB formulation presented similar values of CC50 (around 120 µM) and lesser toxicity than free AmB. These formulations presented no essential oil as nanoemulsions produced in this work. For this reason, the formulations presented more safety than nanoemulsions. Furthermore, it was possible to identify the most cytotoxicity component in nanoemulsions, which was clove oil.

Silva and co-workers (2013) studied the cytotoxicity of microemulsions (with and without AmB) and free AmB in macrophages J774. They observed a dose-dependent relation for toxicity as well as observed in Figure 45. Moreover, microemulsion with or without drug showed similar results and lesser toxicity than free AmB when AmB concentration was lower than 10 µg/mL. However, when AmB concentration was 25 µg/mL, microemulsions presented toxicity similar than free drug.

Antileishmanial Activity

The antileishmanial activity of AmB nanoemulsions was investigated against L. amazonensis promastigotes as well as free AmB and free clove oil.

Figure 46 shows the results of antileishmanial activity of free AmB. Free AmB showed an antileishmanial activity against promastigotes in concentrations higher than 0.1 µg/mL. The lower concentrations of AmB analyzed in this study (0.05 µg/mL and 0.1 µg/mL) presented no statistically significant differences (P > 0.05) in comparison with DMSO solution (amount higher than used to dissolved AmB) and with control (without treatment).

Based on these results, it was possible to observe no interference of DMSO in parasites viability then the antileishmanial activity was related only to drug effects. Furthermore, the two higher concentrations of AmB (1 µg/mL and 5 µg/mL) promoted more the 70% of parasites death and presented no statistically significant differences (P > 0.05) between then.

Thereafter free AmB antileishmanial activity, free clove oil was submitted to antileishmanial assay. Figure 47 presents the results of clove oil antileishmanial activity against L. amazonensis promastigotes. Clove oil showed an antileishmanial activity against promastigotes in concentrations higher than 3 µg/mL. The antileishmanial activity of clove oil was previously described in literature, which correlate the effect to eugenol, the major constituent of clove oil [START_REF] Morais | Thymol and eugenol derivatives as potential antileishmanial agents[END_REF]. Lower concentrations (0.3 µg/mL, 1.5 µg/mL and 3 µg/mL) presented no statistically significant differences (P > 0.05) in comparison with control. However, the highest concentrations of clove oil (7.5 µg/mL and 15 µg/mL) were statistically significant differences (P < 0.05) compared with other concentrations, control and between them. The highest concentration of clove oil analyzed was lower than oil concentration in nanoemulsions (around 78 µg/mL). Nonetheless, it was observed a high amount of oil in wells that contained 15 µg/mL. For this reason, it was not investigated higher concentrations than 15 µg/mL and not analyzed the real oil concentration present in nanoemulsions.

The antileishmanial activity of all nanoemulsions (B, F, H and L) is illustrated in Figure 48. The positive control (C1) used in these experiments was free AmB in concentration (1 µg/mL) that promotes higher than 50% of parasites death based on assay showed in Figure 45.

According to Figure 48(a), it was possible to observe that antileishmanial activity of negative control (C2) was statistically significant different (P < 0.05) than positive control (C1) and nanoemulsions in all concentrations.

B-NE presented high leishmanicidal effect in all four concentrations analyzed with higher than 70% of parasite death. The four concentrations of B-NE showed no statistically significant differences (P > 0.05) among them, positive control and L-NE in two higher concentrations (1 µg/mL and 5 µg/mL). These results showed a greater leishmanicidal effect on B-NE than free AmB, which can be explained by the presence of clove oil, besides the low droplet size of nanoemulsions. Moreover, lower concentrations of L-NE (0.1 µg/mL and 0.5 µg/mL) had no statistically significant differences (P > 0.05) between them.

In relation to Figure 48 Based on cytotoxicity assay and anti-promastigotes activity, it was calculated the CC50, IC50 and the selective index of free AmB, free clove oil and of the four nanoemulsions. The values of CC50, IC50 and selective index are shown in Table 6. Table 6 shows lower CC50 values for B-NE, F-NE and L-NE than free AmB.

The nanoemulsions had lower CC50 than CC50 described in literature for clove oil.

This suggests that nanoemulsions had higher toxicity due to their small oil droplets size, which increases the interaction between formulation and cells membrane.

In relation to IC50 values, free AmB showed value higher than observed for NE containing drug (B-NE = 0.03 µg/mL and F-NE = 0.14 µg/mL) and lower than values for blank NE (H-NE = 0.84 µg/mL). However, L-NE showed higher activity than free drug, which may be explained by the presence of Tween ® 20 in formulation. Moreover, clove oil showed IC50 around 5.26 µg/mL suggesting that free oil had a good leishmanicidal effect, which was already described in the literature. It was possible to notice an increase of antileishmanial activity of AmB nanoemulsion in comparison with free drug. This can be explained by the small size of oil droplets, which facilitates the uptake from parasites as well as observed in the cytotoxicity study. Furthermore, the presence of clove oil and AmB combined in nanoemulsions increased the leishmanicidal activity. Although blank NE presented IC50 higher than AmB nanoemulsions, these values showed interesting antileishmanial activity. Nevertheless, the problem of clove oil nanoemulsions is its high cytotoxicity.

In order to investigate the selectivity of these formulations, the selective index (SI) was calculated. Free AmB and F-NE showed values of 1.03 and 1.25 respectively, indicating low selectivity with cytotoxicity dose close to the effective dose to promote parasite death.

L-NE presented SI=0.79, which suggests that effective dose against L.

amazonensis promastigotes is higher than the cytotoxicity dose. SI for free clove oil and B-NE are respectively 3.58 and 6.33, which means that effective dose against promastigotes is lower than cytotoxicity dose.

As already discussed, formulations B-NE and F-NE had retained in dermis 0.25 µg/mL and 0.56 µg/mL, respectively, which are higher than their IC50 (0.03 µg/mL and 0.13 µg/mL, respectively). It should be noted that dermis retention was evaluated after 8 hours of experiment, that could represent an extended period of exposition for topical application. For this reason and considering the slow drug release from these formulations, more studies are required to investigate their cytotoxicity nature.

Comparing with SI of AmB nanoemulsions from other studies: Caldeira and co-workers (2015) developed AmB nanoemulsions with and without stearylamine and investigated CC50 in macrophages J774, IC50 (in L.

amazonensis amastigotes) and SI of nanoemulsions and compared with conventional AmB (AmB deoxycholate). The CC50 of nanoemulsions with and without stearylamine (6.3 µg/mL and > 200 µg/mL, respectively) were higher than CC50 of conventional AmB (1 µg/mL), showing that stearylamine increased the cytotoxicity of nanoemulsion. IC50 of the two nanoemulsions were 0.11 µg/mL and 0.21 µg/mL, close to IC50 of conventional AmB (0.06 µg/mL). The SI index were: 57 (AmB nanoemulsion with stearylamine), 952 (AmB nanoemulsion without stearylamine) and 16 (conventional AmB). All SI index were higher than 10 for all formulations, showing that nanoemulsions with or without stearylamine were selective for Leishmania and more safe than conventional AmB.

Mendonça and co-workers (2016) analyzed in vitro antileishmanial activity of free AmB and AmB in polymeric micelles system against L. amazonensis promastigotes and amastigotes. Moreover, the cytotoxicity of these formulations was investigated in peritoneal macrophages. The CC50 values were 9.5 µM, 119.5 µM and 134.7 µM for free AmB, AmB-polymeric micelles and blank-formulation, respectively. These results showed that blank and AmB formulation had lower toxicity than the free drug. The values of IC50 detected for free AmB was 1.2 µM, for AmB micelles was 1.8 µM and blank micelles was 22.1 µM against promastigotes. The presence of AmB increases the antileishmanial effect of polymeric micelles. The SI index corresponded to 61.5 for AmB polymeric micelles, in comparison with blank formulation (SI = 5.4) and free drug (SI = 7.9).

From these results, the AmB polymeric micelles were considered a safe formulation and with low toxicity than the free drug.

Our results (see Table 6) and the previous studies reported here [START_REF] Mendonça | Poloxamer 407 (Pluronic ® F127)-based polymeric micelles for amphotericin B: In vitro biological activity, toxicity and in vivo therapeutic efficacy against murine tegumentary leishmaniasis[END_REF][START_REF] Caldeira | Nanoemulsions loaded with amphotericin B: A new approach for the treatment of leishmaniasis[END_REF] agreed on the toxicity and high antileishmanial activity of free AmB. However, the two mentioned studies obtained nanoemulsions with lower toxicity than the free drug and safe (SI higher than 10), while our AmB nanoemulsions are more cytotoxic than free AmB and need more studies and perhaps optimization (in particular B-NE), to be considered safe (at present, their SI are lower than 10).

There are some studies described in literature, which developed nanoemulsions using clove oil as oil phase. Siqueira and co-workers (2017) investigated in vitro antileishmanial activity of free AmB and different nanoemulsions against L. amazonensis. The cytotoxicity of free drug and formulations were evaluated in macrophages RAW 264.7. The blank nanoemulsion contained 5% of clove oil and Pluronic ® F127. Blank nanoemulsion and free AmB presented IC50 of 4000 µg/mL and 0.55 µg/mL, respectively.

Furthermore, CC50 of free AmB was 0.99 µg/mL, while blank nanoemulsion showed no activity in macrophages. These results indicated lower toxicity and lower antileishmanial activity of blank nanoemulsion in comparison with free drug.

The results obtained for blank nanoemulsion were different than those obtained in our work (see Table 6). The differences in cytotoxicity assay can be explained by the different macrophage cells used and the method to quantify toxicity.

Cardoso and co-workers (2018) produced different sulphonamide nanoemulsions with clove oil and Pluronic ® F127 using the same method described in this work. It was evaluated the antileishmanial activity against L.

amazonensis promastigotes and L. infantum promastigotes as the cytotoxicity in macrophages RAW 264.7 of sulphonamide nanoemulsions and compared with free AmB. Six nanoemulsions presented CC50 between 2.24 and 18.26 µg/mL and IC50 between 3.21 and 8.13 µg/mL. Free AmB showed CC50 of 1.07 µg/mL and IC50 of 0.61 µg/mL. The SI values of free AmB and sulphonamide nanoemulsions were lower than 2.5, then nanoemulsions presented cytotoxicity similar to free AmB. The in vitro response for blank nanoemulsion (that could be compared to the cytotoxicity of clove oil given in Table 6) was not investigated.

Furthermore, the cytotoxicity and antileishmanial activity of clove oil were studied in previous works. Kouidhi and co-workers (2010)

CONCLUSION

In this chapter, AmB nanoemulsion using clove oil and Pluronic ® F127

were proposed for topical cutaneous leishmaniasis treatment. The development of AmB nanoemulsions was already described in the literature, but the formulation (essential oil, polymer and AmB) proposed in this work was not held before.

The choice of clove oil as oil phase is based on its several pharmacology activities already described including antileishmanial activity. Moreover, the use of Pluronic ® F127 is related to the thermoreversible property of this compound and its gelling ability in high concentrations favoring a simple formulation for topical administration.

After a first characterization study of the raw AmB (SEM images, FTIR analysis), the development of the AmB nanoemulsions started with their preparation, during which several parameters were varied: -the presence of surfactant (Tween ® 20); -the amount of polymer used in aqueous phase (10% w/w or 15% w/w); -the essential oil (mint oil or clove oil). The nanoemulsions were characterized in terms of physicochemical properties, release behavior, skin permeation and biological assays.

It could be demonstrated that the type of essential oil influenced the nanoemulsion stability, with nanoemulsions produced with clove oil being more stable than those produced with mint oil. Furthermore, the pH for all formulations was in the ideal range for topical application. Based on some preliminary results, two nanoemulsions containing AmB (B-NE -with Tween ® 20 as surfactant and F-NE-without surfactant) and respectively blank-formulation (L-NE -with Tween ® 20 as surfactant and H-NE-without surfactant) were chosen for further studies.

The nanoemulsions submitted to a stability study presented droplets size lower than 60 nm and polydispersity index lower than 0.400 until 365 days of storage. The drug content in formulations stored at room temperature was reduced over storage time, probably related to the AmB degradation. Then, these formulations need to be stored under refrigeration (approximately 4ºC).

In vitro release tests showed a very slow release profile (lower than 1% released in 48 hours). Furthermore, in skin permeation study, the nanoemulsions showed low values of drug permeated after 8 hours, which confirms the slow release profile. The slow drug permeation could be an indicative that nanoemulsions could promote a local treatment without a systemic effect.

The cytotoxicity of nanoemulsions were investigated in bone marrowderived macrophages cells and compared with antileishmanial activity against Leishmania amazonensis promastigotes. The results revealed a higher cytotoxicity but a higher antileishmanial activity of nanoemulsions in comparison to the free drug. The increased antileishmanial activity of AmB nanoemulsions related to free drug was attributed to the small oil droplets of nanoemulsions that could increase the contact of the drug with the macrophage cells.

The selective index was calculated, and the values were lower than 10, which correlated free drug, clove oil and nanoemulsions with or without AmB as cytotoxicity products with low selectivity for leishmanicidal effect.

Biodegradable Polymeric Depots

Over the past few years, drug delivery systems development received considerable attention, specially depot formulations. Depot formulations offer some advantages, such as localized delivery, ease of application, drug-controlled One the most drug carrier study for depot formulation is biodegradable polymeric particles, which have been used for biomedical applications. These particles require polymers, which present several properties as biodegradability, in vivo toxicological safety with ease elimination of degradations products by the metabolic pathways [START_REF] Swider | Customizing poly(lactic-co-glycolic acid) particles for biomedical applications[END_REF].

Poly (lactic acid) (PLA), poly(glycolic acid) (PGA), and their copolymer poly(lactic-co-glycolic acid) (PLGA) are appropriate polymers to prepare depot deliveries due to their favorable biocompatibility and biodegradability (SWIPER et al., 2018;[START_REF] Fredenberg | The mechanism of drug release in poly(lactic-co-glycolic acid)-based drug delivery systems -A review[END_REF]. They are the most commonly polymers investigated for controlled or sustained release and some have been approved

by Food Drug Administration (FDA) [START_REF] Wang | Design of PLGA-based depot delivery systems for biopharmaceuticals prepared by spray drying[END_REF][START_REF] Kapoor | PLGA: a unique polymer for drug delivery[END_REF][START_REF] Ansary | Biodegradable poly (D, L-lacticco-glycolic acid) based micro/nanoparticles for sustained release of protein drugs -A review[END_REF][START_REF] Wang | PLGA microspheres as a delivery vehicle for sustained release of tetracycline: biodistribution in mice after subcutaneous administration[END_REF][START_REF] Re | Formulating drug delivery systems by Spray Drying[END_REF] and by European Medicines Agency (EMA) for pharmaceutical products development and can be administered orally or parenterally and as implant without surgery [START_REF] Han | Bioerodable PLGA-based microparticles for production sustained-release drug formulations and strategies for improving drug loading[END_REF][START_REF] Wang | Design of PLGA-based depot delivery systems for biopharmaceuticals prepared by spray drying[END_REF]. Since the 1970s, PLGA is used as a biomaterial, when it was developed as biodegradable sutures and artificial prostheses, followed by advances use in devices production as implants and therapeutic devices [START_REF] Swider | Customizing poly(lactic-co-glycolic acid) particles for biomedical applications[END_REF]KAPPOR et al., 2015).

Physicochemical Properties of PLGA

PLGA is a synthetic linear copolymer composed of lactic and glycolic acid monomers, which can be organized as a block-copolymer or statistical copolymer. This copolymer can be synthesized by several methods, which the most used methods are ring opening polymerization and polycondensation reactions. The conditions of reactions and process parameters strongly affect the physicochemical properties of final product (PLGA) (MIR, AHMED, [START_REF] Mir | Recent applications of PLGA based nanostructures in drug delivery[END_REF][START_REF] Sharma | PLGA-based nanoparticles : A new paradigm in biomedical applications[END_REF][START_REF] Kapoor | PLGA: a unique polymer for drug delivery[END_REF][START_REF] Lü | Current advances in research and clinical applications of PLGA-based nanotechnology[END_REF]. Figure 49 shows its molecular structure, besides those of PGA and PLA. Generally, a range of common organic solvents can dissolve PLGA, depending on its composition. Chlorinated solvents, like dichloromethane or chloroform, and water-miscible solvents, like acetone or tetrahydrofuran, can dissolved PLGA with a higher amount of lactic acid. However, PLGA with a higher amount of glycolic acid is dissolved by fluorinated solvents, like hexafluoroisopropanol [START_REF] Swider | Customizing poly(lactic-co-glycolic acid) particles for biomedical applications[END_REF][START_REF] Makadia | Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier[END_REF]. It is also possible organic solvent combinations in order to regulate the polymer solubility (SOSNIK & SEREMETA, 2015).

In addition, the glass transition temperature of PLGA can also be explored.

Usually, PLGA has glass transition above body temperature, between 42 and 55°C. The glass transition temperature increases with an increase of PLA content and an increase of molecular weight (see Table 7).

Preparation Methods of PLGA Microparticles

According to the internal structure, PLGA microparticles are classified into two types: microcapsules (one drug core or various drug core in a polymer layer)

and microspheres (drug dispersed in a polymeric matrix) (Figure 50) [START_REF] Bravo-Osuna | Pharmaceutical microscale and nanoscale approaches for efficient treatment of ocular diseases[END_REF][START_REF] Herrero-Vanrell | Nano and microtechnologies for ophthalmic administration, an overview[END_REF][START_REF] Singh | Microencapsulation: A promising technique for controlled drug delivery[END_REF]. PLGA microspheres are the most commonly particles type produced due to their ease development. Unfortunately, the structure of microspheres is, mainly, adequate for hydrophobic drugs encapsulation. This particle type presents some difficulties for peptides, proteins and water-soluble drugs encapsulation, such as our drug of interest, paromomycin, due to low encapsulation [START_REF] Abulateefeh | Synthesis and characterization of PLGA shell microcapsules containing aqueous core prepared by internal phase separation[END_REF]. Prior and co-workers (2000) developed gentamicin (watersoluble drug) PLGA microspheres by two different techniques, solvent evaporation and spray drying, and obtained low encapsulation efficiency and drug content.

In fact, production of aqueous core-PLGA shell microcapsules is an evident challenge and few studies have addressed this point in the literature. Han and co-workers (2016) reported some strategies to improve water-soluble drugs in PLGA microparticles as drug conversion into hydrophobic form (complexation with ionic surfactant), solid dispersion in polymer solution or use of doubleemulsion method. [START_REF] Abulateefeh | Synthesis and characterization of PLGA shell microcapsules containing aqueous core prepared by internal phase separation[END_REF] synthesized and characterized core-PLGA shell microcapsules containing risedronate (watersoluble drug) prepared with internal phase separation and obtained higher encapsulation efficiency in comparison with conventional PLGA microspheres.

Gasparini and co-workers ( 2010) investigated the encapsulation of model watersoluble drug, blue-dextran, using double-emulsion method and PLGA as polymer.

It was observed that encapsulation efficiency can be increased varying production parameters as polymer concentration. Lin and co-workers (2005) evaluated the doxorubicin, water-soluble drug, PLGA/PLLA microparticles prepared by spray-dried varying organic solvent and polymer composition and obtained encapsulation efficiency lower than 30%.

There are several methods of particle preparation from PLGA copolymers [START_REF] Herrero-Vanrell | Nano and microtechnologies for ophthalmic administration, an overview[END_REF][START_REF] Tomaro-Duchesneau | Microencapsulation for the therapeutic delivery of drugs, live mammalian and bacterial cells, and other biopharmaceutics: Current status and future directions[END_REF][START_REF] Jyothi | Microencapsulation techniques, factors influencing encapsulation efficiency[END_REF][START_REF] Singh | Microencapsulation: A promising technique for controlled drug delivery[END_REF][START_REF] Re | Microencapsulation by Spray Drying[END_REF]. One the most conventional methods refers to single and double emulsion-solvent evaporation methods [START_REF] Ramazani | Strategies for encapsulation of small hydrophilic and amphiphilic drugs in PLGA microspheres: state-of-the-art and challenges[END_REF][START_REF] Sharma | PLGA-based nanoparticles : A new paradigm in biomedical applications[END_REF]. Single-emulsion method consists of an organic phase containing PLGA and the lipophilic agent (e.g. drug) in a volatile organic solvent, and an aqueous phase with a surfactant, usually poly vinyl alcohol (PVA). The organic phase is added, under homogenization or sonication, to the aqueous phase with surfactant. Then, organic solvent is removed by evaporation and particles are collected after washing (to remove the surfactant), separation generally by centrifugation and, finally, freeze-drying as last step [START_REF] Ding | Recent advances of PLGA micro/nanoparticles for the delivery of biomacromolecular therapeutics[END_REF][START_REF] Swider | Customizing poly(lactic-co-glycolic acid) particles for biomedical applications[END_REF][START_REF] Ramazani | Strategies for encapsulation of small hydrophilic and amphiphilic drugs in PLGA microspheres: state-of-the-art and challenges[END_REF][START_REF] Sharma | PLGA-based nanoparticles : A new paradigm in biomedical applications[END_REF][START_REF] Wischke | Principles of encapsulating hydrophobic drugs in PLA/PLGA microparticles[END_REF]. Double-emulsion method, commonly W1/O/W2, consists in a water-soluble drug dissolved in a first water phase (W1), which is dispersed in an oil phase O (polymer dissolved in an organic solvent), then dispersed in a second water phase (W2). The W1 phase is added under stirring into the organic phase, forming the first W1/O emulsion. Then, W1/O emulsion is emulsified in the W2 phase, followed by extraction/evaporation of the organic solvent and collection of polymeric hardened particles [START_REF] Ding | Recent advances of PLGA micro/nanoparticles for the delivery of biomacromolecular therapeutics[END_REF][START_REF] Swider | Customizing poly(lactic-co-glycolic acid) particles for biomedical applications[END_REF][START_REF] Sharma | PLGA-based nanoparticles : A new paradigm in biomedical applications[END_REF].

To prepare particles with controlled properties, such as size or degradation rate, it is important to choose not only the right materials but also the right synthesis parameters. For example, the polymer concentration and the stirring speed, time of stirring and temperature (sonication, homogenization) can affected particles size [START_REF] Swider | Customizing poly(lactic-co-glycolic acid) particles for biomedical applications[END_REF][START_REF] Ramazani | Strategies for encapsulation of small hydrophilic and amphiphilic drugs in PLGA microspheres: state-of-the-art and challenges[END_REF][START_REF] Sharma | PLGA-based nanoparticles : A new paradigm in biomedical applications[END_REF].

The single and double emulsion technique can be scaled up by adjusting process parameters, such as the amount of polymer, type of solvents, type of surfactant, speed and temperature of sonication or homogenization [START_REF] Sharma | PLGA-based nanoparticles : A new paradigm in biomedical applications[END_REF]. Single-emulsion method is applicable for hydrophobic drugs encapsulation, while double-emulsion method is used to the encapsulation of more hydrophilic drugs [START_REF] Swider | Customizing poly(lactic-co-glycolic acid) particles for biomedical applications[END_REF][START_REF] Ramazani | Strategies for encapsulation of small hydrophilic and amphiphilic drugs in PLGA microspheres: state-of-the-art and challenges[END_REF][START_REF] Makadia | Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier[END_REF][START_REF] Lü | Current advances in research and clinical applications of PLGA-based nanotechnology[END_REF][START_REF] Wischke | Principles of encapsulating hydrophobic drugs in PLA/PLGA microparticles[END_REF].

Spray drying is a rapid, well-established, reproducible, scalable and onestep process, which can be used in a closed system (organic solvents using inert gas) or an open system (aqueous mode using compressed air) [START_REF] Patel | Revealing facts behind spray dried solid dispersion technology used for solubility enhancement[END_REF][START_REF] Sosnik | Advantages and challenges of spray-drying technology for the production of pure drug particles and drug-loaded polymeric carries[END_REF]WAN et al., 2014a). Moreover, spray-drying can be used to encapsulate both hydrophilic and lipophilic drugs, peptides and proteins into a biodegradable polymer like PLGA [START_REF] Das | Microencapsulation techniques and its practice[END_REF].

Drug-loaded particles are prepared by spraying a solid-in-oil (S/O) dispersion or W/O emulsion in a hot stream gas [START_REF] Ding | Recent advances of PLGA micro/nanoparticles for the delivery of biomacromolecular therapeutics[END_REF][START_REF] Swider | Customizing poly(lactic-co-glycolic acid) particles for biomedical applications[END_REF][START_REF] Wang | Design of PLGA-based depot delivery systems for biopharmaceuticals prepared by spray drying[END_REF][START_REF] Patel | Revealing facts behind spray dried solid dispersion technology used for solubility enhancement[END_REF][START_REF] Sosnik | Advantages and challenges of spray-drying technology for the production of pure drug particles and drug-loaded polymeric carries[END_REF]KAŠPAR, JAKUBEC, ŠTĚPÁNEK, 2013a;[START_REF] Re | Formulating drug delivery systems by Spray Drying[END_REF] as shown in Figure 52. This method may induce particles agglomeration (JAMEKHORSHID, SADRAMELI, FARID, 2014). Furthermore, encapsulation by spray drying depends on solubility and diffusion coefficient of materials (KONDO, NIWA, DANJO, 2014). The characteristics of spray-dried microparticles can be influenced by different parameters related to the process, the formulation and the equipment design (SINGH & [START_REF] Singh | Spray drying formulation of amorphous solid dispersions[END_REF][START_REF] Patel | Revealing facts behind spray dried solid dispersion technology used for solubility enhancement[END_REF][START_REF] Sosnik | Advantages and challenges of spray-drying technology for the production of pure drug particles and drug-loaded polymeric carries[END_REF]. The choice of the solvent is influenced by the type of drug (hydrophilic or hydrophobic) to be encapsulated [START_REF] Ding | Recent advances of PLGA micro/nanoparticles for the delivery of biomacromolecular therapeutics[END_REF]. Table 8 presents the different adjustable variables in a spray drying process.

that encounters the liquid feed transferring the energy for its disintegration in liquid droplets. Generally, this configuration produces microspheres (drug dispersed in a polymer matrix) as shown in Figure 54a (SUNDERLAND, KELLY, RAMTOOLA, 2015; KONDO, NIWA, DANJO, 2014).

In three-fluid nozzles, two liquid feeds are pumped separately through two liquid passages and the gas is flowing through a third passage (Figure 54b). This

Release from PLGA microparticles

In aqueous medium, PLGA copolymer suffers degradation/biodegradation by hydrolysis through breakage of polymer backbone ester linkages into oligomers, which increase polymer hydrophilicity, and then are cleavage into monomers (lactic and glycolic acids). These monomers are breakage into energy, carbon dioxide and water [START_REF] Ding | Recent advances of PLGA micro/nanoparticles for the delivery of biomacromolecular therapeutics[END_REF][START_REF] Kapoor | PLGA: a unique polymer for drug delivery[END_REF][START_REF] Makadia | Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier[END_REF]. PLGA is consider a bulk-eroding polymer and during the polymer erosion, the water uptake into matrix is higher than polymer degradation rate (hydrolysis) [START_REF] Han | Bioerodable PLGA-based microparticles for production sustained-release drug formulations and strategies for improving drug loading[END_REF][START_REF] Kapoor | PLGA: a unique polymer for drug delivery[END_REF][START_REF] Makadia | Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier[END_REF].

PLGA particles follow some of three degradation mechanism: erosion of polymer surface with drug physically release, splitting of drug-polymer bonding with drug diffusion release and/or drug release by diffusion followed by polymer erosion [START_REF] Han | Bioerodable PLGA-based microparticles for production sustained-release drug formulations and strategies for improving drug loading[END_REF][START_REF] Kapoor | PLGA: a unique polymer for drug delivery[END_REF]. The degradation rate of PLGA can regulate the drug release [START_REF] Swider | Customizing poly(lactic-co-glycolic acid) particles for biomedical applications[END_REF][START_REF] Fredenberg | The mechanism of drug release in poly(lactic-co-glycolic acid)-based drug delivery systems -A review[END_REF].

Figure 55 gives a schematically representation of possible degradation mechanisms for PLGA particles. Generally, PLGA particles show a biphasic release profile. In phase I, occurs the water penetration into polymer matrix forming pores corresponding to an initial burst drug release; the polymer degradation that follows corresponds to a slower release (phase II) [START_REF] Han | Bioerodable PLGA-based microparticles for production sustained-release drug formulations and strategies for improving drug loading[END_REF][START_REF] Sharma | PLGA-based nanoparticles : A new paradigm in biomedical applications[END_REF][START_REF] Makadia | Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier[END_REF]. Formulations properties affecting PLGA particles degradation, include size and shape of matrix (large particles have small initial burst release with slow release than smaller particles) and drug load (low drug content allows slow burst release). The presence of enzymes and pH (alkaline or acid) can, also, affect PLGA particles release [START_REF] Ding | Recent advances of PLGA micro/nanoparticles for the delivery of biomacromolecular therapeutics[END_REF][START_REF] Swider | Customizing poly(lactic-co-glycolic acid) particles for biomedical applications[END_REF][START_REF] Sharma | PLGA-based nanoparticles : A new paradigm in biomedical applications[END_REF][START_REF] Kapoor | PLGA: a unique polymer for drug delivery[END_REF][START_REF] Makadia | Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier[END_REF].

PLGA microparticles for depot delivery

There are several published reviews focusing on the PLGA particles formulation due to the polymer broad use and acceptance. [START_REF] Ding | Recent advances of PLGA micro/nanoparticles for the delivery of biomacromolecular therapeutics[END_REF] discussed the recent advances of PLGA micro and nanoparticles for bio macromolecular molecules delivery, as preparation methods, drug release and macromolecular compounds deliveries. Swiper and co-workers (2018) focused on PLGA particles preparation, novels particles design and how to improve effects on treatments using these particles. [START_REF] Wang | Design of PLGA-based depot delivery systems for biopharmaceuticals prepared by spray drying[END_REF][START_REF] Wang | PLGA microspheres as a delivery vehicle for sustained release of tetracycline: biodistribution in mice after subcutaneous administration[END_REF]. There are already some marketed formulations using PLGA, especially for cancer treatment, as: Lupron Depot ® , Suprecur ® MP, Somatuline ® LA, Trelstar ™ Depot and others (ANSARY, AWANG, RAHMAN, 2014). Moreover, the preparation method fully influences in the properties of PLGA particles. Particles, used in specific function such as therapeutic delivery or cell imaging/targeting, require accurate physicochemical properties [START_REF] Swider | Customizing poly(lactic-co-glycolic acid) particles for biomedical applications[END_REF]KAPPOR et al., 2015).

One of alternative administration route, which has been studied is intralesional administration. This alternative route has some advantages as decrease of drug dose and/or the administration frequency, reduction of side effects, improvement of treatment efficiency [START_REF] Pinero | New administration model of trans-chalcone biodegradable polymers for the treatment of experimental leishmaniasis[END_REF]. Moreover, it can be a potential route for treat complex diseases [START_REF] Angamuthu | Controlled-release injectable containing terbinafine/PLGA microspheres for Onychomycosis treatment[END_REF].

A positive example is the formulation containing PLGA and terbinafine for intralesional administration to treat fungal nail infections, which had good results [START_REF] Angamuthu | Controlled-release injectable containing terbinafine/PLGA microspheres for Onychomycosis treatment[END_REF]. A poorer result was obtained with one formulation called OncoGel TM (PLGA-PEG-PLGA triblock copolymer with paclitaxel) for local tumor treatment, which failed in phase IIb [START_REF] Jain | Injectable formulations of poly (lactic acid) and its copolymers in clinical use[END_REF].

Intralesional administration has already been described in literature for leishmaniasis treatment [START_REF] Batista | Depot subcutaneous injection with chalcone CH8-loaded poly (lactic-co-glycolic acid) microspheres as a single dose treatment of cutaneous leishmaniasis[END_REF]. In 2013, it was included in leishmaniasis guidelines intralesional treatment using pentavalent antimonials to decrease antimonials dose and decrease side effects in comparison with traditional treatment using intramuscular or intravenous routes (BRITO, RABELLO, COTA, 2017). Moreover, there are studies involving PLGA formulations with chalcones (natural product with a broad spectrum of biological activities including antileishmanial) for this type of administration, which showed interesting results [START_REF] Batista | Depot subcutaneous injection with chalcone CH8-loaded poly (lactic-co-glycolic acid) microspheres as a single dose treatment of cutaneous leishmaniasis[END_REF][START_REF] Pinero | New administration model of trans-chalcone biodegradable polymers for the treatment of experimental leishmaniasis[END_REF].

The present thesis aims to develop PLGA microspheres and PLGA coreshell microcapsules to encapsulate paromomycin, a water-soluble drug to be tested in leishmaniasis treatment by intralesional administration. The process of choice is spray drying.

The production of PLGA microparticles by spray drying seems represent a promising way for depot deliveries development, including with water-soluble drugs [START_REF] Re | Formulating drug delivery systems by Spray Drying[END_REF].

Paromomycin

Paromomycin (PM) is a white or slightly yellow powder, which is in salt form The efficacy of PM formulations against Leishmania species from 'New World' still controversial and several clinical studies are required to evaluate their performance [START_REF] Gonçalves | Activity of a paromomycin hydrophilic formulation for topical treatment of infections by Leishmania (Leishmania) amazonensis and Leishmania (Vianna) braziliensis[END_REF]. The controversy is generated by less efficacy showed by these studies compared to parenteral meglumine antimoniate [START_REF] Kim | Is paromomycin an effective and safe treatment against cutaneous leishmaniasis? A meta-analysis of 14 randomized controlled trials[END_REF]. In Brazil, paromomycin has not been yet approved to use in leishmaniasis treatment (DNDi, 2018), probably related to PM low efficacy reported against Leishmania "New World" species. Furthermore, PM current topical formulations present low permeability because this drug belongs to class III of BCS. Moreover, the parasite resistance of paromomycin current formulations has been reported and it is related to decrease of PM uptake by Leishmania [START_REF] Jhingran | Paromomycin: uptake and resistance in Leishmania donovani[END_REF].

The parasite resistance, the few studies of PM efficacy in New World, the few numbers of clinical studies, the toxicity of formulations presented on the market and the poor permeability of this drug are motivating the development of new PM formulations for leishmaniasis treatment.

However, there are still few studies described in literature about PM physicochemical characterization or paromomycin drug delivery systems. Figure 56 shows the number of publications, obtained in database "Web of Science", related to PM physicochemical characterization or paromomycin drug delivery systems between 1997 and 2017 using keywords as "Paromomycin + Leishmaniasis", "Paromomycin + Characterization", "Paromomycin + Drug delivery system (DDS)", "Paromomycin + PLGA" and "Paromomycin + Leishmaniasis + Drug delivery systems (DDS)". Table 9 shows PM drug delivery systems described in literature, including information about the preparation methods and some product characteristics. It can be observed that studies on PM drug delivery systems for leishmaniasis were not numerous before 2009. However, an increasing interest on this topic has been observed from 2010, mainly in the last three years. There are, also, few studies about PM formulations for subcutaneous administration, which is one of objectives of this work.

Ionic complexation

Topical

Ion pairing NMR, thermal analysis, solubility [START_REF] Nogueira | characterization, and topical delivery of paromomycin ion pairing[END_REF].

PM Solid lipid nanoparticles

-Microemulsion and solvent diffusion

Particle size, encapsulation efficiency, drug release, DSC [START_REF] Ghadiri | Loading hydrophilic drug in solid lipid media as nanoparticles: Statistical modeling of entrapment efficiency and particle size[END_REF].

Microspheres of albumin containing PM

Parenteral

Spray drying Stability study [START_REF] Khan | Paromomycinloaded albumin microspheres: efficacy and stability studies[END_REF].

PM liposomes and PM+miltefosine liposomes

Subcutaneous Double emulsion and lyophilization

Particle size, zeta potential, encapsulation efficiency, drug loading Momeni et al., 2013.

PM nanogel

OBJECTIVES

The chapter presents the part of this thesis developed at RAPSODEE Research Centre (France) related to:

• The development of new formulations of PM loaded in biodegradable polymeric microparticles, intending intralesional administration;

• The physicochemical characterization of pure PM and PM-loaded polymeric microparticles;

• The study of their performance behavior under the following in vitro studies:

-In vitro PM release profile;

-In vitro cytotoxicity assay;

-In vitro antileishmanial activity.

MATERIAL AND METHODS

MATERIAL

Paromomycin sulfate (PM) was obtained from Biovet (Bulgaria).

Polymer poly lactic co-glycolic acid (PLGA) (Purasorb ® PDLG 5004 -ester terminated) was purchased from Corbion (Netherlands). PLGA has 0.4 dl/g of inherent viscosity midpoint, 44 kg/mol of molecular weight and 50:50 lactide/glycolide ratio.

Poly lactic acid (PLA) (Resomer ® L206S -ester terminated) was purchased from Sigma-Aldrich (France).

All other materials were of analytical reagent grade.

( KHAN & KUMAR, 2011a,b;[START_REF] Kalimouttou | Evaluation of the paromomycin loading characteristics in nanoprecipitated PLGA nanospheres[END_REF]. The DSC was calibrated with indium for temperature and enthalpy calibration and with sapphire for heat capacity calibration. The sample was submitted to two heating cycles.

X-ray Scattering Diffraction (XRD)

The solid state of PM powders was evaluated in an X-ray Diffraction equipment Philips X'Pert (Philips, USA). The measurements were made with Cu Kα radiation at scanning speed of 0.02º/sec, in 2θ, from 5 to 80º. Furthermore, the analyses were performed at 40 KV and 40 mA (adapted from KANG et al., 2008).

Fourier Transform Infrared Spectroscopy (FTIR)

The FTIR spectra of the raw and reprocessed PM were analyzed using a

Raman Spectroscopy

Raman mapping of PM powders was performed with a Raman microscopy (Alpha 300R Raman-AFM spectrophotometer, WITEC GmbH, Germany). The measurements were obtained at room temperature with 50X magnification using a confocal laser wavelength of 532 nm. The PM Raman spectra were analyzed four times, collected for 1s of integration using ten accumulations. When using a two-fluid nozzle (Figure 57a), only one liquid formulation was spray dried in contact with the drying/carrying gas. This formulation contains both compounds, PM and polymer. Firstly, an organic solution was prepared by dissolving the polymer in a selected solvent (1.5% w/w). The drug (PM powder)

PAROMOMYCIN POLYMERIC MICROPARTICLES

was then mixed to the organic solution (6.8 mg/mL).

With this nozzle design, the nozzle orifice diameter was varied in the study (0.7 mm, 1.4 mm and 2.0 mm).

When three-fluid nozzle was used (Figure 53b), the outer solution was the organic solution of polymer. In turn, the inner feed was constituted by an aqueous solution of PM in a concentration of 20.2 mg/mL. The nozzle orifice diameter was fixed (1.4 mm). In this configuration, two solutions (PM in water and PLGA in organic solvent) or one solution (PLGA in organic solvent) and one dispersion (PM dispersed in organic solvent) were pumped separately and joined at the nozzle tip, where they are mixed and atomized by pressurized gas provided by the third channel. Subsequently, the solvents evaporate from the droplets by a stream of hot gas and the particles are formed.

The ratio of flow ratio desired was the proportion 1:4 (core: shell), corresponding to a flow rate of 2.6 mL/min for inner (core) and 10.4 mL/min for out (shell).

Figure 58 shows the different nozzles used in this work.

b) Type of Organic Solvent

Two organic solvents were used to dissolve the polymeric carrier, PLGA alone or a mixture of PLGA and PLA. The solvents used were ethyl acetate or acetone.

Inlet gas temperature used in all batches varied from 60 to 72 ºC and the outlet temperature varied from 60 to 66 ºC when ethyl acetate was chosen as organic solvent, and from 35 to 50 ºC, when acetone was chosen.

c) Type of Polymer

Most of formulations were generated with a single polymer, PLGA.

However, the use of a mixture of PLGA and PLA were also tested in different mass proportions in relation to the total polymer amount: PLGA 95%-PLA 5%;

PLGA 90%-PLA 10%; 60%-PLA 40%; PLA 100%.

d) PM Mass Concentration

Different PM mass percentage in the binary mixtures constituted of PM and polymer: 30% and 70% wt. for two-fluid batches and 15%, 30%, 45% and 60% wt. for three-fluid batches.

e) The Physical State of PM formulation

PM-loaded polymeric microparticles were generated from different physical states of the drug: dispersed in water (with two-fluid nozzle) or dissolved in water (with the three-fluid nozzle). Blank particles were also generated. They will be presented and discussed in detail in the next sections.

PHYSICOCHEMICAL CHARACTERIZATION OF PAROMOMYCIN POLYMERIC MICROPARTICLES

PM-loaded polymeric microparticles produced by the two-fluid nozzle will be named 'PM-MS', while those produced by the three-fluid nozzle will be named 'PM-MC' from this point of the manuscript.

The PM-MS and PM-MC batches were characterized with respect to some physical characteristics (particle size and morphology), thermal properties, FT-IR and Raman spectra as described below. In short, 100 mg of PM-MS or PM-MC samples were weighed and 1% of micronized Pluronic (dispersing agent) were mixed. Then, 5 mL of dichloromethane was added and homogenized using vortex mixer during approximately 2 minutes. After homogenization, 5 mL of distilled water was added and homogenized for another 2 minutes using vortex mixer. Subsequently, the suspension was added into separatory funnel for a liquid-liquid extraction. The material was left until complete phase's separation. The organic phase was discarded and the aqueous phase, containing PM extracted from PM-MS samples, was collected and submitted to derivatization reaction with DNFB (adapted from RYAN,1984). The absorbance values were evaluated at 415 nm.

The drug content, DL (%), in PM-MS and PM-MC products was then calculated by Equation 1:

The encapsulation efficiency, EE (%), was defined by Equation 2:

Equation 2

Equ ation 17

Equation 4 Equation 1Eq uation 1 Equation 4Eq uation 2 Equation 4The yield of batches (%) was calculated following the Equation 3:

Fourier Transform Infrared Spectroscopy (FTIR)

The FTIR spectra of PM-MS/PM-MC samples were analyzed using a FTIR spectrophotometer (Thermo Scientific Nicolet iS10, Thermo Fisher Scientific, France) following method described previously in item 4.3.2.1.4.

Particle Size

The particle size and polydispersity index of PM microparticles were investigated in equipment Mastersizer 3000 (Malvern Instruments, United Kingdom). The microparticles were dispersed in distilled water containing 0.01% (w/v) of Tween ® 80 and sonicating 1 min in a water bath to prevent aggregation phenomena at room temperature [START_REF] Wang | Comparative studies on the properties of glycyrrhetinic acid-loaded PLGA microparticles prepared by emulsion and template methods[END_REF]adapted

IN VITRO RELEASE OF PAROMOMYCIN POLYMERIC

MICROPARTICLES

In vitro release assay has been used for investigating the drug release profile from micro and nano formulations. Membrane diffusion method, also known as dialysis method, is an appropriate method for drug release determination and several adaptations for this method is already described in literature (JUG et al., 2018). In this work, to evaluate the in vitro release profile of PM-MS and PM-MC batches, it was chosen an adapted dialysis method, which have been adopted by the Brazilian research group for similar in vitro release studies.

In brief, 10 mg of PM-MS or PM-MC samples were weighed and mixed to 1 mg of micronized Pluronic (dispersing agent). These powders were inserted on a cellulose acetate membrane (Sigma-Aldrich, Brazil), previously attached to a cut falcon tube. The cellulose acetate membrane was hydrated in distilled water for, at least, 12 hours before use. Thereafter, 0.5 mL of distilled water was added inside each tube containing powder. Therefore, the tubes with membrane and material were inserted on the surface of 10 mL of receptor medium. The receptor medium was phosphate buffer pH 7.4 (USP, 2012c) containing 2% of Tween ® 20.

Experiments were carried out at room temperature under constant stirring (100 rpm) for 4 hours. Hence, 1 mL of receptor medium was collected at 1, 2, and 4 hours with refiling with 1 mL of fresh buffer solution.

Furthermore, two other samples were also evaluated:

1. A physical mixture of PM and PLGA in the same mass proportion of the PM-MS/PM-MC particles; 2. A free PM sample in the same amount of PM contained in PM-MS/PM-MC particles. Figure 61 shows a scheme of the in vitro release assay used in this study. The PM concentration was determined by UV-visible spectrophotometry method (adapted from [START_REF] Ryan | Colorimetric Determination of Gentamicin, Kanamycin, Tobramycin and Amikacin Aminoglycosides with 2,4-dinitrofluorobenzene[END_REF] in wavelength 415 nm. The experiments were conducted in triplicate for samples (free PM, physical mixture and PM-MS or PM-MC batches). The calibration curve (Figure 62) was prepared in PBS with PM derivatized in the range of 25 to 500 µg/mL and a regression equation (y= 0.0028x) was obtained (linearity r 2 = 0.9941).

Methods used to compare drug release profiles from PMloaded spray dried polymeric microparticles

Drug release profiles were analyzed using independent [dissolution efficiency (DE)] and a model-dependent (curve-fitting) approach, the Korsmeyer-Peppas model. 10.

Korsmeyer-Peppas model is a semi-empirical model, relating exponentially the drug release to the elapsed time. It is used to analyze the release of pharmaceutical polymeric dosage forms, when the release mechanism is not well known or when more than one type of release phenomenon could be involved [START_REF] Bruschi | Mathematical Models of Drug Release[END_REF][START_REF] Ramteke | Mathematical models of drug dissolution: a review[END_REF][START_REF] Costa | Avaliação in vitro da lioequivalência de formulações farmacêuticas[END_REF][START_REF] Costa | Modeling and comparison of dissolution profiles[END_REF]. Nonlinear regressions were applied for cumulative dissolved drug.

The coefficient of determination (R 2 ), root-mean-square error (RMSE, Equation 2, Table 10) and Akaike's information criterion (AIC, Equation 3, Table 10) values were determined using the same software Kinet DS 3.0 rev. 2010.

RMSE 𝑅𝑀𝑆𝐸 = √ ∑ (𝑦𝑖𝑜𝑏𝑠 -𝑦𝑖𝑝𝑟𝑒𝑑) 2 𝑛 𝑖=1 𝑛 (2)
AIC 𝐴𝐼𝐶 = 2𝑘 + 𝑛[ln(∑ (𝑦𝑖𝑜𝑏𝑠 -𝑦𝑖𝑝𝑟𝑒𝑑) 𝑛 𝑖=1
2 )] ( 3)

Independent Dissolution Efficiency (DE) DE = ( ∫ 𝑄𝑡𝑑𝑡 𝑡 0 𝑄𝑡𝑚𝑎𝑥 𝑡 ) 𝑥 100 (4)
Legend: Q t -amount of drug released in time t; Q tmax -maximal amount of drug released (=100%); Q∞ -amount of drug released at an infinite time;

Q0 -initial amount of drug in dosage form; KK -release rate constant; n -diffusional exponent.

SPATIAL DISTRIBUTION OF CONSTITUENTS IN THE

MICROPARTICLES

In order to investigate the spatial localization of the constituents, polymer and drug in the microparticles, different techniques were associated. They are:

Raman mapping -following method described in item 4.3.2.1.5. Each spectral scan was collected for 0.05 s of integration using ten accumulations and each sample was analyzed three times. 2D (surface) and 3D (depth) analyses were performed on the samples.

SEM observation -following method described previously in item 4.3.2.1.1.

PM-loaded polymer microparticles were observed just after manufacturing. Some microparticles were then added to water and exposed to the contact with this medium during 24h (to solubilize and remove PM not protected by PLGA), after which they were centrifuged in a microcentrifuge (HMAC CT15RE, Hitachi, Japan) for 15 minutes (12000 rpm, 21ºC). The water was discarded, the powder was dried at room temperature for 48 hours in desiccator and observed by SEM.

BIOLOGICAL ASSAYS

Assessment of cytotoxicity of PM-loaded microparticles in BMDM cells

Bone marrow-derived macrophages (BMDM) were obtained from femurs and tibia of BALB/c mice as described by Marim and co-workers (2010). Mice were anaesthetized with 5% of isoflurane and euthanized as approved by Ethics Committee of Animal Use (CEUA) of the Instituto de Biofísica Carlos Chagas Filho (IBCCF) under number IBCCF-118. The protocol to obtain BMDM was described in chapter 3, item 3.3.2.7.1.

BMDM (1x10 5 cells/100 µL) were seeded on 96-well microtiter plate and incubated with different concentrations of free PM, two PM-MS batches and respective blank microparticles (812, 1625, 3250, 7500 µg/mL) for 48 hours at 36ºC in a 5% CO2 atmosphere. The range of PM concentration selected for cytotoxicity was based on previous studies described in literature [START_REF] Brugués | Developing cutaneous applications of paromomycin entrapped in stimuli-sensitive block copolymer nanogel dispersions[END_REF][START_REF] Kharaji | Drug targeting to macrophages with solid lipid nanoparticles harboring paromomycin: an in vitro evaluation against L. major and L. tropica[END_REF]. Moreover, the negative control was RPMI medium without treatment and positive control was Triton 100X. After incubation time, the cytotoxicity assay was investigated using resazurin method [START_REF] Rampersad | Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays[END_REF]. 20 µL of resazurin solution were added and the plate was incubated for more 4 hours at 36ºC. After 4 hours, the fluorescence was measured using microplate Fluorimeter Spectra Max M5 (Molecular Devices, USA) at 560 nm for excitation and 590 nm for emission. The results were reported as % cell viability.

The 50% cytotoxicity concentration (CC50) values were determined by logarithmic regression analysis as previously described in item 3.3.2.7.1.

Antileishmanial Activity

The promastigotes metacyclic form of Leishmania L. amazonensis (WHOM/BR/75/JOSEFA) were maintained in M-199 medium with 10% of inactivated FBS and 1% of antibiotics (penicillin + streptomycin). Promastigotes were grown at 26ºC in climatic chamber (adapted from [START_REF] Torres-Santos | Altered sterol profile induced in Leishmania amazonensis by a natural dihydroxymethoxylated chalcone[END_REF]. The antileishmanial activity of free PM and best PM-MS was investigated by resazurin reduction method [START_REF] Kulshrestha | Validation of a simple resazurin-based promastigote assay for the routine monitoring of miltefosine susceptibility in clinical isolates of Leishmania donovani[END_REF][START_REF] Rolón | Development of resazurin microtiter assay for drug sensibility testing of Trypanosoma cruzi epimastigotes[END_REF].

Promastigotes (5x10 5 parasites/ 200µL) were seeded on 96-well microtiter plate. Different concentrations of free PM and PM-MS batches (40, 120, 361, 1083 and 3250 µg/mL) were evaluated and the plate was incubated for 48 hours at 26ºC. The negative control was M-199 medium without treatment and positive control was free PM in microparticles analysis.

After incubation time, 20 µL of resazurin solution were added and the plate was incubated for more 4 hours at 26ºC. After that, the fluorescence was measured using microplate Fluorimeter Spectra Max M5 (Molecular Devices, USA) at 560 nm for excitation and 590 nm for emission. The results were reported as % parasite viability. The 50% inhibitory concentration (IC50) values were determined by logarithmic regression analysis and the selectivity index (SI) was calculated as the ratio CC50/IC50, as previously described in item 3.3.2.7.2.

STATISTICAL ANALYSIS

Statistical analysis, CC50 and IC50 were calculated using GraphPad Prism 5.0 (GraphPad Software, USA). The P value < 0.05 was considered significant.

RESULTS AND DISCUSSION

PHYSICOCHEMICAL CHARACTERIZATION OF PAROMOMYCIN

Scanning Electron Microscopy (SEM)

SEM images of raw PM are shown in two different magnifications (800x and 3200x) in Figure 63. PM powder presents a smooth shriveled spherical morphology. This morphology suggested that PM powder was probably produced by spray drying.

As spray drying was our process of choice to produce PM-loaded polymeric microparticles, the raw PM powder was also reprocessed alone by in the bench scale spray dryer used in our study. The reprocessed spray-dried PM powder can be visualized as SEM images in Figure 64. It can be observed that spray-dried PM particles has the shape (predominantly spherical and shriveled morphology) already showed by the raw PM powder. However, reprocessed spray-dried PM particles are smaller in size with an apparently large particle size distribution, which could be explained by different scales of production (industrial versus bench scale).

Differential Scanning Calorimetry (DSC)

The MDSC curve of raw and spray-dried PM was shown in Figure 65.

The thermogram of raw and spray-dried PM present no endothermic melting peak through rev Heat Flow and Heat Flow analyses. The large endotherm event (between 25ºC and 175ºC) showed in non-reversible heat flow (nonrev Heat Flow) and Heat Flow analyses in both samples of PM probably corresponds to the loss of moisture of the drug, which is very hygroscope. The spray-dried PM showed a higher loss of moisture than raw PM, what could be due to their smaller particle size (and then greater surface area) favoring increased water uptake. The MDSC analysis stopped at 200ºC, because PM suffers degradation and the powder changes the color without melt as shown in Figure 66.

Little information on thermal properties of PM is found in the literature.

However, we can mention few works that have observed a broad peak corresponding to loss of moisture between 30ºC to 100ºC during thermal analyses (DSC and TGA) of pure PM (KHAN & KUMAR, 2011a, b). A broad peak corresponding to loss of moisture between 30ºC and 185°C was also measured for PM by Brugués and co-workers (2015).

In turn, some works have confirmed PM degradation above 210°C (KHAN & KUMAR, 2011a, b) or 230ºC [START_REF] Brugués | Developing cutaneous applications of paromomycin entrapped in stimuli-sensitive block copolymer nanogel dispersions[END_REF], attributed to PM pyrolysis.

At our knowledge, only one paper published in 2008 [START_REF] Kalimouttou | Evaluation of the paromomycin loading characteristics in nanoprecipitated PLGA nanospheres[END_REF] have identified a melting point (Tm) for a crystalline sample of PM corresponding to an endothermic peak at 179.64ºC. However, no data could be found related to the glass transition temperature (Tg) of the amorphous form of PM. Generally, the Tg of materials can be found in the interval between ⅔ and ⁴⁄5

of Tm (JÓJÁRT-LACZKOVICH & SZABÓ-RÉVÉSZ, 2010). From this statement, Tg of PM could be expected to be between 28ºC (⅔ Tm) and 89ºC (⁴⁄5 Tm).

Nevertheless, DSC analyses did not detect any Tg on both, raw and reprocessed PM powders. A supplementary analysis was done increasing the interval of temperature from -90ºC to 200ºC (data not shown) in an attempt to investigate the presence of Tg, but none was identified in this temperature range, either.

X-ray Scattering Diffraction (XRD)

X-ray powder scattering diffraction patterns of raw and reprocessed PM powders are shown in Figure 67. It can be seen that the diffraction patterns did not show sharp Bragg peaks, which is characteristic of amorphous materials.

Fourier Transform Infrared Spectroscopy (FTIR)

Figure 68 displays the FTIR spectra for raw PM powder. The PM spectrum

shows characteristic peaks at 3408 cm -1 (NH2 amine group), at 1632 cm -1 (N-H bending coupled with C-N stretch), at 1534 cm -1 (CH2 bending) and at 1027 cm -1 (C-O-C stretch). The reprocessed PM sample presented a similar FTIR spectrum.

Khan and Kumar (2011a, b) described PM characteristic peaks at 1629 cm -1 (N-H bending coupled with C-N stretch), 1535 cm -1 (CH2 bending) and at 1091 cm -1 (C-O-C stretch). Moreover, they observed two bands at 2928 cm -1 and at 3377 cm -1 corresponding to hydroxyl and NH2 amine groups. Therefore, the PM FTIR spectrum presented in Figure 68 revealed similar PM characteristics peaks to those described in literature.

Raman Spectroscopy

The Raman spectrum of PM is shown in Figure 69. It can be seen one peak around 1000 cm -1 and one peak between 2900 cm -1 and 3100 cm -1 . Also, it was observed that PM presents fluorescence. Raman and infrared are complementary techniques which identify the molecular structure. For this reason, the Raman spectrum and FTIR spectrum are linked [START_REF] Larkan | Infrared and Raman Spectroscopy: Principles and Spectral Interpretation[END_REF].

According to this information, the characteristic peak around 1000 cm -1 can be related to C-O-C stretch and peak around 3000 cm -1 can be related to NH2 because hydroxyls have weak signal in Raman spectra [START_REF] Larkan | Infrared and Raman Spectroscopy: Principles and Spectral Interpretation[END_REF]. The two spectra (FTIR and Raman) provide the molecular structure "fingerprint" of PM. However, Raman spectrum of PM was not found in the literature.

PAROMOMYCIN POLYMERIC MICROSPHERES DEVELOPMENT

Development of PM-loaded PLGA microspheres

The first series of tests aimed, on one hand, to select the organic solvent for the polymeric carrier, and on the other hand, to investigate the ability of the spray drying process to produce PM-loaded polymeric microspheres.

Solvent Choice

The most common solvent used to produce PLGA microspheres by the conventional emulsification-evaporation process is dichloromethane because it has low boiling point around 40ºC, which allows fast extraction and evaporation under controlled conditions of temperature and pressure (SINGH & [START_REF] Singh | Spray drying formulation of amorphous solid dispersions[END_REF][START_REF] Re | Formulating drug delivery systems by Spray Drying[END_REF][START_REF] Wang | Etanidazole-loaded microspheres fabricated by spraydrying different poly(lactide/glycolide) polymers: effects on microspheres properties[END_REF][START_REF] Wang | Sustained release of etanidazole from spray dried microspheres prepared by non-halogenated solvents[END_REF].

However, the use of halogenated solvents (dichloromethane or chloroform) have regulatory restriction because they belong to the class II solvents (solvents to be limited) of ICH classification [START_REF] Ich | International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use. ICH Harmonized Guideline. Impurities: Guideline for Residual Solvents Q3C(R6)[END_REF]. In this work, two solvents belonging to the class III of ICH classification (solvents with low toxic potential) were selected for the first tests: ethyl acetate and acetone.

Samples were then prepared with ethyl acetate (PM-MS01, PM-MS02 and PM-MS03 samples) or acetone (PM-MS06) to solubilize the polymeric carrier.

Table 11 shows the formulation and process parameters fixed: PM was dispersed in the organic polymeric solution (dispersion type), the initial PM concentration in the formulation was fixed at 30%wt in relation to the total content of solids in the liquid formulation, the polymeric carrier was PLGA 50:50 and a two-fluid nozzle with a nozzle orifice diameter of 1.4 mm was used. The spray-dried products were characterized and compared in terms of particle shape (Figures 70 and71) and yield (Table 11).

Table 11. Formulation conditions, process and product characteristics of PM-loaded microspheres: solvent effect. et al., 2013a). If a spray-dried powder is exposed to high temperatures, it can move to a rubbery state, adhering to the spray drying chamber or cyclone walls and be hardly recovered, which explains the low yield of our batches (Table 11).

The production of PM-loaded PLGA microspheres employing ethyl acetate as solvent requires higher temperatures than acetone, since their boiling points are 77°C and 56°C, respectively. The outlet temperatures were kept in the range 65-67ºC to evaporate ethyl acetate and in the range 42-46ºC to evaporate acetone. The latter condition is more favorable to improve the yield production of PLGA microspheres by spray drying.

Indeed, using literature as basis, some works can be mentioned. Silva-Junior and colleagues (2009) prepared triamcinolone PLGA spray-dried microparticles using acetone as solvent with gas inlet temperature of 70ºC and outlet temperature of 45ºC. They obtained smooth spherical agglomerated microparticles similarly to those shown in Figure 71 (a andb). Oliveira and coworkers (2013) evaluated the physicochemical properties of methotrexate PLGA spray-dried microparticles prepared with different proportions drug/polymer using acetone as solvent. They observed that methotrexate microparticles had spherical smooth shape with some agglomerates and suggested that the Referring to literature data: drug-loaded PLGA microspheres have been generated from two-fluid nozzles with different orifice diameters. For example, [START_REF] Mok | Water-free microencapsulation of proteins within PLGA microparticles by spray drying using PEG-assisted protein solubilization technique in organic solvent[END_REF] developed spray-dried human growth hormone-loaded PLGA microspheres using 0.5 mm nozzle orifice diameter. Spray-dried rifampicinloaded PLGA microspheres were also produced from 0.5 mm nozzle orifice diameter [START_REF] Son | Preparation of sustained release rifampicin microparticles for inhalation[END_REF]. In both studies the aim was to produce fine particles and they used the smaller orifice diameter existing for two-fluid nozzles operating with conventional bench spray dryers. Oliveira and co-workers (2013) used 0.7 mm nozzle orifice diameter to prepare methotrexate-loaded PLGA microspheres, while Nakashima and co-workers (2017) used 1.4 mm nozzle orifice diameter to produce spray-dried aripiprazole-loaded PLGA microparticles for injectable sustained release.

In this study, Table 12 shows that yield, drug loading and encapsulation efficiency increased with the increase of two-fluid nozzle orifice diameter from 0.7 to 2.0 mm. For the next steps of this work, the orifice diameter of 1.4 mm was choses even if higher EE, drug loading and yield could be reached with the larger (2.0 mm) diameter orifice. The apparent larger particle size distribution obtained with the largest orifice diameter could impact the drug release from these microspheres as the release rate depends, among others, of the surface area exposed to the release medium.

Paromomycin Concentration

Other parameter evaluated during the development of PM-loaded PLGA microspheres was the PM concentration, as indicated in Table 13. SEM images of PM-loaded PLGA microspheres generated from two-fluid nozzles with different PM concentrations are shown in Figure 73. It is possible to observe that the particles are similar in shape and degree of agglomeration.

However, some differences can be verified in Table 13: 1. Reduced encapsulation efficiency and yield when increasing the PM concentration in the initial liquid formulation.

2. PM-MS07 batch (30% PM) showed higher yield and encapsulation efficiency than PM-MS08 batch (70% PM).

There are some studies described in literature with a similar correlation between drug content and encapsulation efficiency. Lin and co-workers (2005) evaluated the production of doxorubicin-loaded PLGA or PLLA/PLGA microparticles obtained by spray-drying. They observed that the encapsulation efficiency and yield decreased when increasing the drug content in the microparticles. It was explained by the drug insolubility in the organic solvent used. Prior and co-workers (2000) evaluated two different microencapsulation techniques (solvent evaporation and spray-drying) to produce gentamicin PLGA microparticles. Two batches were prepared from each technique, varying the gentamicin concentration. They also attributed the lower encapsulation efficiency for more concentrated batches to gentamicin insolubility in the organic solvent.

However, in our case, PM is dispersed and not dissolved in the organic solvent and the lower encapsulation efficiency and yield are not related to the recrystallization of PM in acetone but more probably to some loss of PM powder during the atomization step (segregation in the feeding pump).

3. Increased PM concentration in the initial liquid formulation leads to increased drug loading in the PM-MS batches.

Despite the losses, PM-MS08 batch has a drug loading higher than PM-MS07 batch. This trend has been reported with other drugs encapsulated in PLGA microspheres (WAN et al., 2014a;[START_REF] Oliveira | Structural and thermal properties of spray-dried methotrexateloaded biodegradable microparticles[END_REF].

Development of PM-loaded core-shell PLGA microcapsules

In a second study, core-shell PLGA microcapsules were developed to encapsulate PM, thanks to a three-fluid nozzle. They are named PM-MC samples from this point of manuscript.

Table 14 presents all batches produced. The studied adjustable parameters were the initial PM concentration in the initial core formulation (from 15 to 60% PM); the shell composition (PLGA or a mixture of PLGA and PLA with varying PLA mass proportions); the physical state of PM in the initial core formulation (solubilized in water or dispersed in the organic solvent).

From these results, it could be concluded that:

-An increase in the PM concentration in the initial core formulation led to an increase the drug loading in the microcapsules (see PM-MC04, PM-MC12 and PM-MC13, then PM-MC14 and PM-MC15). The same effect could be observed in encapsulation efficiency (EE) for the first series MC04, MC12, MC13 but not for MC14 and MC15. The yield was less influenced by this parameter.

-PM, introduced in a solubilized form in the core formulation, allowed higher drug loading in the microcapsules compared to the powder form (see PM-MC15, PM-MC16). EE and yield seemed to be less influenced by this parameter.

-The change in the polymeric matrix from PLGA 50:50 to PLA (see PM-MC12 x PM-MC22) led to a reduction in drug loading in the microcapsules and EE, despite the yield increase.

-The use of a PLGA/PLA blend as polymeric carrier influenced the drug loading, EE and yield, being favored by the increase of the PLA proportion in the matrix (see PM-MC19 to PM-MC21). In general, and in quantitative terms, A Tg of 40.8ºC was identified for PLGA through rev Heat Flow in the first heating cycle (Figure 74a), moving to 42.2°C in the second heating cycle. These values are close to values of Tg reported in the literature for PLGA 50:50 [START_REF] Erbetta | Synthesis and characterization of poly (D, L-lactide-co-glycolide) copolymer[END_REF][START_REF] Park | Predictors of glass transition in the biodegradable poly-lactide and poly-lactide-co-glycolide polymers[END_REF].

When analyzing the microparticles produced by spray drying the first cycle of heating reveals the thermal history from the process used to generate them.

DSC thermograms for PM-MS06 revealed only a single Tg of 39.79ºC in the first heating cycle and of 41.33ºC in the second cycle, which is close to the Tg of pure PLGA.

PM-MC12 sample has a higher Tg of 44.08ºC in the first heating cycle that moved to 45.68ºC in the second cycle. No other thermal events were observed.

According to these results, no differences in thermal properties were found between the MDSC thermograms of PM-loaded PLGA microspheres (PM-MS06) compared to PM-loaded core-shell PLGA microcapsules (PM-MC12).

Comparing to some published data, it can be seen that these values are close to the Tg measured during the preparation of PLGA microspheres loaded with another drug (aripiprazole). A Tg of 46.7ºC was identified for pure PLGA with a displacement to 50.2ºC in presence of the drug [START_REF] Nakashima | Design of highly dispersible PLGA microparticles in aqueous fluid for the development of longacting release injectables[END_REF]. In our case, for PM-MC12, it is difficult to attribute the displacement of Tg to the PM presence, because the Tg of PM was not identified by our DSC analysis, as already shown in previous section of this chapter. The thermogram of PLA (Figure 75a) showed a single Tg at 59ºC in rev Heat Flow analysis. In the nonrev Heat Flow and Heat Flow analyses can be observed an exothermic peak related to PLA crystallization at 73ºC followed by an endothermic peak at 170ºC, which corresponds to the melting of the polymer.

PM-MC20 and MC24 presented MSDC curves very similar: Tg of polymers around 45ºC (rev Heat Flow) and one endothermic event at 169ºC corresponding to the melting point of PLA (Heat Flow). 16, the Tg of the blends (PM-MC219 to PM-MC21) appears to be more or less proportional to the composition of the blend, since the value of the single Tg detected increases by increasing the proportion of PLA (individual component with a higher Tg). A single Tg could be an indicative of miscibility of the amorphous phase in in the blends. However, DSC curves showed that PLA is partially crystalline (Tm) in these samples.

The thermal properties of PLA and PLGA have been reported in the literature. For example, [START_REF] Lu | Poly (lactic acid) In: Polymer Data Handbook[END_REF] described that PLA has two enantiomers and can be found in three forms: PDLA (stereoisomer D), PLLA (stereoisomer L) and PDLLA (racemic mixture). PLLA has a Tg between 52.85-63.85ºC and a melting point between 144.85-185.85ºC. [START_REF] Kim | Comparative study on sustained release of human growth hormone from semi-crystalline poly (L-lactic acid) and amorphous poly (D, Llactic-co-glycolic acid) microspheres: morphological effect on protein release[END_REF] investigated the sustained release of human growth hormone encapsulated in PLA, PLGA and PLGA-PLA blend prepared by solvent evaporation method. They identified a Tg of PLA at 32ºC with melting point at 145ºC and a Tg of PLGA at 28ºC. However, they did not characterize the thermal properties of the PLGA-PLA blends.

Farah and co-workers (2016) described a Tg for PLLA at 55-65ºC and the melting point between 170-200ºC. Our data (Tg and Tm for PLA) shown in Table 16 are falling within this range.

In contrast, the study of [START_REF] Park | Predictors of glass transition in the biodegradable poly-lactide and poly-lactide-co-glycolide polymers[END_REF] showed Tg values of PLA (41.9ºC in first heating cycle and 45.3ºC in second heating cycle) lower than those given in Table 16. This difference could be related to intrinsic viscosity of the PLA used (0.39 dl/g for Park's study and between 0.8 dl/g and 1.2 dl/g for PM-MC batches).

Scanning Electron Microscopy (SEM)

Figure 76 shows SEM images of PM-loaded PLGA microspheres (PM-MS06) compared to PM-loaded core-shell PLGA microcapsules (PM-MC12 and PM-MC16). The difference between PM-MC12 and PM-MC16, both produced with the three-fluid nozzle, was the PM solid state in the feed solution: PM-MC12

was produced from PM dissolved in water in the core feed, while PM-MC16 was produced from a liquid core containing PM dispersed in acetone. PM-C18 contains a higher load of PM (28.9%wt) than PM-MS06 (6.9%).

SEM images show particles very similar to raw PM particles (see Figure 63) in both MC and MS samples prepared from raw PM particles dispersed in acetone. They are mixed to smaller particles, which sometimes are adhered to their surface (see higher magnifications).

The sample PM-MC12, prepared from an aqueous solution of PM fed into the 3-fluid nozzle to generate the particle core, seems be constituted by smaller particles, very agglomerated.

PM-loaded microcapsules were also generated using a blend of PLGA and PLA instead PLGA alone. Three different compositions were tested, the PLA content varying from 5% to 40% in the polymeric mixture. Microcapsules prepared with PLGA-PLA blends are shown in Figure 77. It can be observed that they are characterized by smaller particles with smooth surface, also very agglomerated and the shape seemed be influenced by the PLA content.

PM-MC22 was generated with PLA alone. The sample showed a distinct morphology (Figure 78), with irregular wrinkled shape and porous surface, and agglomerates. Similar morphologies were already found for others spray-dried PLA microparticles encapsulating sodium monofluorophosphate [START_REF] Zuo | Characteristics and release property of polylactic acid/sodium monofluorophosphate microcapsules prepared by spray drying[END_REF], methotrexate [START_REF] Oliveira | Monitoring thermal, structural properties, methotrexate release and biological activity from biocompatible spray-dried microparticles[END_REF] or ketotifen [START_REF] Guerrero | Ketotifen-loaded microspheres prepared by spray-drying poly (D, Llactide) and poly (D, L-lactide-co-glycolide) polymers: Characterization and in vivo evaluation[END_REF].

These batches were investigated subsequently by other characterization techniques and the results will be discussed later in this chapter.

Fourier Transform Infrared Spectroscopy (FTIR)

Figure 79 shows the FTIR spectra of PLGA pure, PM pure, physical mixture PM+PLGA, PM-MS06 (produced by two-fluid nozzle), PM-MC12 (threefluid nozzle, core fed by PM solubilized in water and shell fed by the organic solution) and PM-MC18 (three-fluid nozzle, core fed by PM dispersed in acetone and shell fed by the organic solution). These samples were chosen for comparison of PM-MS microspheres with PM-MC microcapsules.

The PLGA spectrum showed peaks at 2997 cm -1 and 2917 cm -1 (CH2 stretch), 1750 cm -1 (C=O stretch), 1380 cm -1 (CH2-CH wagging vibration) and at 1180 cm -1 and 1098 cm -1 (C-O stretch, ester group), similarly to characteristic peaks reported in the literature [START_REF] Erbetta | Synthesis and characterization of poly (D, L-lactide-co-glycolide) copolymer[END_REF][START_REF] Matos | Thermal behavior and stability of biodegradable spray-dried microparticles containing triamcinolone[END_REF]. The PM spectrum showed characteristic peaks at 3408 cm -1 (NH2 amine group), at 1632 cm -1 (N-H bending coupled with C-N stretch), at 1534 cm -1 (CH2 bending) and at 1027 cm -1 (C-O-C stretch), also in line with characteristic peaks previously reported for PM (KHAN & KUMAR, 2011 a,b). Some characteristic peaks of both PM and PLGA can be seen in the PM-PLGA physical mixture.

PM-MS06 presented peaks at 3380 cm -1 (NH2 amine group), 1080 cm -1 (C-O-C stretch) corresponding to PM peaks and at 2990 cm -1 , 2940 cm -1 (CH2 stretch), 1750 cm -1 (C=O stretch) corresponding to PLGA peaks ((b) in Figure 79). By its turn, PM-MC12 and PM-MC18 presented similar spectra when compare to PM-MS06 differing only in peaks intensity at 2990 cm -1 and 2940 cm -1 (CH2 stretch, corresponding to PLGA peaks) ((a) in Figure 79) and in the range of 1380 cm -1 to 1040 cm -1 ((c) in Figure 79). These ranges presented peaks corresponding to PM and PLGA, in which PM-MS06 showed broader band than PM-MC12 and PM-MC18.

To conclude, the absence of new IR peaks and the maintenance of characteristics peaks of drug and polymer suggest no chemical interactions between PM and PLGA. The effect of PLGA/PLA blends was also investigated by FT-IR analysis.

Figure 80 shows the FTIR spectra of PLA pure, physical mixture PLA+PLGA, physical mixture PM+PLA+PLGA (30:5:65), PM-MC19 (produced with 5% of PLA) and MC23 (blank microparticles). These samples were chosen to represent the results found in PM-MS batches prepared with PLGA/PLA blend. PLA spectrum showed characteristics peaks at 2989 cm -1 and 2939 cm -1 (CH2 stretch), 1750 cm -1 (C=O stretch), 1448 cm -1 and 1369 cm -1 (CH2-CH wagging vibration) and at 1175 cm -1 and 1077 cm -1 (C-O stretch, ester group), in accordance with literature [START_REF] Oliveira | Monitoring thermal, structural properties, methotrexate release and biological activity from biocompatible spray-dried microparticles[END_REF][START_REF] Lu | Poly (lactic acid) In: Polymer Data Handbook[END_REF].

Physical mixtures (PLA+PLGA and PLA+PLGA+PM), PM-MC19 and MC23 showed FTIR spectra similar to PLA, varying only in intensity (especially at 1750 cm -1 corresponding to C=O). However, it is not possibly to relate the peaks observed in samples to PLA or PLGA, because the polymers presented similar chemical structures and similar IR spectra as already reported by Kang and co-workers (2008). The results also suggested no chemical interactions between PLGA/PLA blends and PM.

Particle Size

The particle size of some batches was measured by laser granulometry as described in 4.3.2.3.5. All PM-MS/PM-MC batches presented small individual particles (lower than 10 µm), however, very agglomerated. Table 17 shows particle size distribution and span of some PM-MC batches prepared with threefluid nozzle. As particles are hardly separated, the mean particle size should be viewed cautiously. The percentage of PM released for each sample per hour of released test is given in Table 19. Dissolution efficiencies at the end of the experimental test (DE4h) are also given in Table 19.

PM-MS06, PM-MC15 and PM-MC18 showed fast release with higher than 66% of drug released in the first hour, corresponding to DE higher than 70% (see Table 19). The same behavior was observed for free PM and the physical mixture.

Nonetheless, PM-MC20 displayed a slower PM release rate reaching approximately 34% of drug release in 4h (Table 19) and DE of 27.5%.

For comparative purposes, Khan and Kumar (2011a) verified a biphasic release profile of PM from albumin microspheres with an initial burst release of 50-60% of drug released in 2 h. To investigate the mechanism of drug release, the % cumulative PM from these different structures (MS and MC) were fitted in Korsmeyer-Peppas model.

Nonlinear regressions were applied for cumulative dissolved PM. The coefficient of determination (R 2 ), root-mean-square error (RMSE) and Akaik's information criterion (AIC) values were determined using Kinet DS 3.0 rev. 2010 software.

The data obtained from Korsmeyer-Peppas for all dissolution experiments are listed in Table 20. or lower than 0.5, the release mechanism follows Fickian diffusion. If n value is between 0.5 and 1, the release mechanism follows a non-Fickian model named anomalous transport. If n value is equal to 1, the release mechanism is Case-II transport, while n value higher than 1 corresponds to super Case-II transport [START_REF] Carmelo | Factors influencing the erosion rate and the drug release kinetics from organogels designed as matrices for oral controlled release of hydrophobic drug[END_REF][START_REF] Dash | Kinetic modeling on drug release from controlled drug delivery systems[END_REF][START_REF] Costa | Avaliação in vitro da lioequivalência de formulações farmacêuticas[END_REF][START_REF] Costa | Modeling and comparison of dissolution profiles[END_REF].

The four samples showed "n" values lower than 0.5, which indicates that PM release mechanism follows Fickian diffusion model [START_REF] Carmelo | Factors influencing the erosion rate and the drug release kinetics from organogels designed as matrices for oral controlled release of hydrophobic drug[END_REF][START_REF] Dash | Kinetic modeling on drug release from controlled drug delivery systems[END_REF][START_REF] Costa | Avaliação in vitro da lioequivalência de formulações farmacêuticas[END_REF]. Based on this model, the solvent diffusion into polymeric matrix is faster than polymer relaxation and drug get out of particles by diffusion [START_REF] Bruschi | Mathematical Models of Drug Release[END_REF]. As introduced earlier in this chapter, PLGA is a bulkeroding polymer and during the polymer erosion, the water uptake into the matrix is higher than polymer degradation rate (hydrolysis). Consequently, water uptake promotes drug diffusion from outside the particles [START_REF] Han | Bioerodable PLGA-based microparticles for production sustained-release drug formulations and strategies for improving drug loading[END_REF][START_REF] Kapoor | PLGA: a unique polymer for drug delivery[END_REF][START_REF] Makadia | Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier[END_REF]. In addition, formulations properties affecting release from PLGA microparticles include their size (large particles have small initial burst release with slow release than smaller particles) and drug load (low drug content allows slow burst release), besides the solubility of the drug in the release medium. A small individual particle size (in the order of a few microns) and a drug load of 18-24% could be in part responsible for the rapid release.

Moreover, PM is a very water-soluble drug and its release will be fast if the drug is not protected by a polymeric matrix.

In an effort to explain the unexpected fast release from PLGA PM-MS06, PM-MC15 and PM-MC18 samples, additional characterizations were performed and are presented and discussed in the next section.

SPATIAL DISTRIBUTION OF CONSTITUENTS IN PM-MS AND PM-MC MICROPARTICLES

In order to investigate the spatial localization of polymer and drug in the microparticles, different techniques described in item 4.3.2.5 were associated:

Raman mapping and SEM analyses after contact with water during 24h, aiming to remove PM not encapsulated. Figure 81 shows some particles with more porous structure or voids that could be created by the release of PM. In Figures 82 and 83 PLGA Raman spectrum presented some characteristics peaks in the range of 800-1200 cm -1 , around 1400 cm -1 , between 1700-1800 cm -1 and strong peak between 2800-3100 cm -1 , corresponding to C-O stretch, C-H bend, C=O stretch, C-H stretch, respectively. These peaks can be related to peaks observed in PLGA IR spectrum. However, the strong peak in Raman is linked to C-H bond because OH -has weak signal in Raman spectra (LARKAN, 2011).

Sanna and co-workers (2012) developed PLGA nanoparticles of resveratrol and analyzed the Raman spectra of components. The PLGA spectrum presented peaks at 2882 cm -1 , 2946 cm -1 and 3000 cm -1 related to C-H stretching, at 1766 cm -1 related to C=O bond stretching, peak at 1130 cm -1 and 1044 cm -1 corresponding to C-O stretching and one peak at 1453 cm -1 corresponding to C-H bend. Yan and co-workers (2015) investigated PLGA nanoparticles with hydrophobic drugs. The Raman spectrum of polymer showed peaks at 2930 cm - 1 , 2960 cm -1 corresponding to CH3 stretch of lactic acid, at 2884 cm -1 related to C-H stretch of lactic acid, at 2850 cm -1 related to CH2 stretch of glycolic acid. It was also observed: -a peak at 1635 cm -1 corresponding to C=O stretch; -peaks between 1400-1500 cm -1 related to C-H bend; -peak at 1296 cm -1 related to C-H stretch and peaks between 1130-1000 cm -1 corresponding to C-O-C stretch.

Wang and co-workers (2016) evaluated microspheres of PLGA or Pluronic ® F127 + PEG containing exenatide and prepared by double emulsification and solvent evaporation. They evaluated the Raman spectra of pure substances and of the microparticles and observed that PLGA spectrum had a strong peak in the range of 2500-3000 cm -1 and six small peaks between 800 and 1500 cm -1 . These studies have identified the same Raman spectrum of PLGA showed in Figure 84. One of the theories proposed to describe the particle formation process via spray drying with a 3-fluid nozzle is that two liquids would form an 'emulsion mixture' upon atomization (WAN et al., 2014a). When a miscible solvent like acetone is applied in the 3-fluid nozzle, the two feeds (inner and outer feed) mix intimately to make a 'transient film' which then break up into micro-droplets.

According to this theory, particle formation is initiated by the formation of a transient liquid PLGA film at the interface between the aqueous (inner feed) and the organic solution (outer feed), which is a result of mass transfer between the organic solvent and water, leading to a decrease in the solubility of the polymer in the solvents. In the case that the transitional liquid PLGA film appears instantaneously once the organic PLGA solution comes into contact with the aqueous solution, it forms irregular PLGA aggregates in the subsequent drying process. The use of organic solvent with a low surface tension and a certain degree of miscibility with water causes the instant phase separation of the PLGA film (WAN et al., 2014a), which could explain the formation of a structure like that illustrated in Figure 85c. In other words, use of acetone as organic solvent produces more irregular PLGA aggregates resulted from lower surface tension between acetone and water, which promotes fast spreading rate of solvent on the aqueous droplets surface (WAN et al., 2014a). Then, the particles produced with acetone from 3-fluid nozzles and PLGA solution (PM-MC15 and PM-MC18) were more porous. The pores provided PM passage for receptor chamber that is composed of water medium (PBS) being favorable for drug dissolution (PM is highly hydrophilic).

PM-MC20 was prepared with a polymer blend comprising PLGA (90%)

and PLA (10%) and had a core-shell structure, possibly close to that illustrated in Figure 85b. The slow release of PM from this sample could be due to a slow diffusion through a polymeric barrier. Two factors have probably contributed to this effect:

1. The modification of the composition of the organic solution to a binary solvent mixture.

To solubilize PLA, dichloromethane was mixed to acetone leading to a mass ratio around 1:7 w/w in organic solution. Dichloromethane is immiscible with water and better solvent for PLA and PLGA and could prevent the formation of aggregates from the polymeric blend. Aggregates were unlikely to form on PLGA films when this solvent was used as observed previously (WAN et al., 2014a). A more uniform polymeric membrane can promote an increase of the resistance of polymer matrix in release processes (WAN et al., 2014a), which could explain the slow release of this batch (PM-MC20).

The polymeric composition

The release from a PLGA: PLA blend was influenced by its degradation rate, delayed by the presence of PLA. PLA increased hydrophobicity of the polymer blend, reducing water uptake, polymer hydrolysis and providing slow release.

BIOLOGICAL ASSAYS

The capacity of PM-loaded microparticles to induce cytotoxicity in BMDM cells and their antileishmanial activity against L. amazonensis promastigotes were investigated. Samples chosen for these tests were: PM-MC12 (PM in solution, microcapsules), PM-MC20 (PM in solution, PLGA/PLA mixture (90:10) microcapsules) and MC24 (blank batch for PM-MC20). Free drug was also submitted to the same assays.

Assessment of cytotoxicity of PM-loaded microparticles in BMDM cells

The cytotoxicity was evaluated in BMDM cells and the results was shown in Figure 86.

Firstly, Free PM (Figure 86a) showed a dose-dependent cytotoxicity and presented lower toxicity than PM-MC batches.

MC24, blank formulation, showed no cytotoxicity with no statistically significant differences (P > 0.05) among three concentrations (812 µg/mL, 1625 µg/mL, 3250 µg/mL) and Negative Control, suggesting that the cytotoxicity observed in microparticles batches with PM was due to the drug present in concentration between 26% wt. (PM-MC12) and 18% wt. (PM-MC20).

PM-MC12 presented no statistically significant differences (P > 0.05) among the three concentrations (1625 µg/mL, 3250 µg/mL, 7500 µg/mL) and the Positive Control (Triton). Moreover, the lower concentration of PM-MC12 analyzed (812 µg/mL) also demonstrated cytotoxicity.

PM-MC20 as well as PM-MC12 showed cytotoxicity in all concentrations in comparison to the Negative Control. However, only the highest concentration (7500 µg/mL) had no statistically significant differences (P > 0.05) in relation to Khan and co-workers (2013) developed PM albumin microspheres and investigated their cytotoxicity and PM in solution in peritoneal macrophages. The concentration ranged between 0.5-150 µg/mL and it was observed that PM solution and PM microspheres promoted no cytotoxicity, suggesting that higher concentrations than 150 µg/mL of PM solution and PM formulation should be tested to conclude about the cytotoxicity of this formulation. Our tests were performed with very higher loads of PM (812 to 7500 µg/mL) compared with the concentrations tested for PM albumin microspheres.

Brugués and co-workers (2015) developed PM nanogel and evaluated its cytotoxicity in macrophages RAW 264.7, in comparison to the nanogel without PM (blank formulation) and an aqueous solution of PM (PM solution). The cytotoxicity was investigated in the concentration range of 0.8-25000 µg/mL. The results showed that PM nanogel presented higher cytotoxicity than the PM solution, while blank nanogel showed lower cytotoxicity than PM solution or nanogel. A similar trend was observed in our results. Kharaji and co-workers (2015) developed solid lipid nanoparticles containing PM and evaluated the cytotoxicity of these formulations, blank nanoparticles and PM solution in human monocytic cell line (THP-1). The results showed no toxicity for blank nanoparticles and PM solution even in the highest concentration analyzed (6400 µg/mL). On the other hand, the nanoparticle size had some influence on cytotoxicity: the bigger PM solid lipid nanoparticles (980 nm and 1500 nm) have shown higher cytotoxicity than the smaller ones (120 nm and 240 nm).

In short, the results reported here with others different PM delivery systems (albumin microspheres, nanogel, solid lipid nanoparticles) confirmed that cytotoxicity depends on the range of PM concentration (µg/mL) tested and may be revealed for more concentrated formulations. Moreover, physical characteristics of these systems such as particle size may also influence the results. The higher cytotoxicity of PM-loaded microparticles (Figure 86 b andc) in relation to the free PM (Figure 86a) could be due to the polymer used, however it was not confirmed with the blank polymeric microparticles (Figure 86d). More studies are need to a better understanding of this biological behavior. However, compared to the literature, the PM-MC samples tested are in the same category of cytotoxicity found with other formulations aiming leishmaniasis treatment.

In order to compare the cytotoxicity and antileishmanial activities, the following parameters were calculated: CC50 (for cytotoxicity); IC50 (for antileishmanial activity) and Selective Index (CC50/IC50.) for free PM, PM-MC12, PM-MC20 and MC24. The results are given in Table 21.

Table 21 shows that free PM and MC24 presented higher values of CC50 than PM-MC batches. MC24 showed no antileishmanial activity with high value of IC50 (> 3250 µg/mL). PM-MS20 had IC50 value of 11.8 µg/mL, slightly higher than the similar value of IC50 (7.5 µg/mL) of PM-MC12 and free PM. A key part of the development of PM-MS or PM-MC formulations is the characterization and optimization of their safety and efficacy and to identify which have an appropriately balanced safety-efficacy profile for intralesional or subcutaneous leishmaniasis treatment. The selectivity index (SI) which is usually considered as the highest drug's exposure that results in no toxicity to the exposure that produces the desired efficacy -is an important parameter in efforts to achieve this balance.

In the present study, the selectivity of the free PM and its formulation was expressed in terms of selectivity index (SI). To be a safe drug, SI should be greater than 10 ( [START_REF] Katsuno | Hit and lead criteria in drug discovery for infectious diseases of the developing world[END_REF]NWAKA & HUDSON, 2006). Compared to free PM, PM-MC20 and PMC-12 had a decrease in the selectivity index, however, the SI values for both samples were approximately 50 and 41, respectively, indicating that they presented promising activity, which is higher than its cytotoxicity.

Taking the literature as reference, some results are cited here: Two different structures were expected: microspheres (matrix) and microcapsules (core-shell). The samples obtained were characterized by characteristics quite different in terms of drug loading (between 1.4% and 33.8%), EE (between 9.3% and 96.4%) and yield of production (between 6.4% and 80.5%).

Physicochemical characterizations (DSC, DRX, FT-IR analysis) revealed no interactions between the drug and polymeric carrier, which could be expected in the matrix structure (from two-fluid nozzle). In vitro release showed unexpected behaviors such as fast release from microspheres and PLGA microcapsules.

These undesirable performances were attributed to the solid structure of the microparticles. A better understanding of the mechanism of particle formation from 3-fluid nozzles helps to identify ways to better control the physical core-shell structures formed. One way could be the mixture of polymers, PLGA and PLA as demonstrated by some of our results shown in this chapter (PM controlled release).

All spray-dried samples tested in biological assays, microspheres and microcapsules, presented safety with low toxicity detected in bone marrowderived macrophages. Their Selective index was higher than 10, which confirms that these formulations were safe, with antileishmanial activity higher than cytotoxicity.

The treatment for cutaneous leishmaniasis is a challenge since it is difficult to overcome problems such as the several side effects provoked by several drugs with antileishmanial activity. For this raison, formulation strategies and alternative administration routes have been increasingly studied looking for innovative treatments, with more efficacy and safety.

The aim of this study was to provide, for the treatment of cutaneous leishmaniasis, novel formulations for two drugs with well-known antileishmanial activity, amphotericin B and paromomycin.

Amphotericin B (AmB) is the most potent drug. However, its marketed formulations present high cytotoxicity, several side effects, high cost (for Ambisome ® ) and are available only for injectable use in hospital environment.

There are several studies aiming novel formulations containing AmB and focusing in different routes of administration. Nonetheless, besides high toxicity, this drug presents low solubility and low permeability, which difficult the development of novel formulations.

Nanoemulsions were chosen as formulation strategy for AmB due to the presence of an oil phase, which in turn, should be able to entrap the drug and favor its permeation through the skin in topical application. Moreover, the use of topical formulation is a suitable alternative for decrease the toxicity, side effects and for self-application, increasing the patient's adhesion. In addition, nanoemulsions can generally be produced by simple manufacturing processes, which could be verified in this study. The formulations proposed here presented another advantage that is the use of a less toxic solvent (Class 3 ICH Guidelines)

to solubilize AmB in the oil phase, DMSO, instead the most used in AmB nanoemulsions (dichloromethane), which is Class 2. Solvents in Class 2 need to be limited in pharmaceutical products because of their inherent toxicity.

The AmB nanoemulsions developed here have demonstrated relevant characteristics like stability during 365 days under refrigeration (4ºC), very slow release profile and slow drug permeation during in vitro tests. They also revealed good antileishmanial activity, however high cytotoxicity. The most promising formulation is AmB nanoemulsion prepared with surfactant Tween ® 20 (Nanoemulsion B), which showed the lowest cytotoxicity with selective index around 6. To be safe, formulations require SI values higher than 10. However, B-NE formulation has perhaps some potential in terms of composition to further improve its selectivity, which could be seen here as recommendations for future improvements: reduction of the amount of drug, novel formulations using others poloxamers, reduction of the amount or replacement of clove oil. Furthermore, the addition of an additional compound for accelerating the drug release could be another modification to improve nanoemulsion B performance.

In follow-up to this work, complementary studies for nanoemulsion B are required and are listed here:

rheological properties, spreadability;

antileishmanial activity against L. amazonensis amastigotes, -antileishmanial activity against other Leishmania species that cause cutaneous leishmaniasis;

in vivo studies in BALB/c mice model.

Paromomycin (PM) is another drug for which the parasite resistance, the few studies of PM efficacy in New World, the few numbers of clinical studies and the toxicity of formulations presented on the market are motivating the development of new PM formulations for leishmaniasis treatment. This drug has some physicochemical characteristics as high hydrophilicity and poor permeability that difficult the efficacy of topical formulations as already reported, besides susceptibility to degradation. Another route of administration not yet studied for paromomycin is the intralesional route. In addition to avoid problems of poor permeability through skin, the potential advantages of intralesional administration could be the use of lower total doses of the drug (and thus fewer toxic effects) and a more flexible schedule without the requirement of daily drug administration.

PLGA-based formulations were chosen for PM because the use of biocompatible polymer allows injectable application. In addition, the manufacturing process chosen was spray drying, a rapid and one-step process that can be used for hydrophilic drug encapsulation.

The development of paromomycin microparticles involved the study of parameters influencing in physicochemical properties of formulations and the structure of the delivery systems, microspheres or microcapsules. Different structures could be developed with PLGA or a mixture of PLGA and PLA as polymeric carriers. Microcapsules prepared with PLGA/PLA blend (PM-MC20) had the most interesting properties as core-shell structure and slow and controlled release. In addition, this batch presented low cytotoxicity with good antileishmanial activity and high selectivity for leishmanicidal effect.

Several other possibilities can be explored to modify the release profile of all PLGA-based microparticles generated in this study. Strategies for this aim in further studies could be:

-A better control of PLGA shell formation by modifying the interfacial tension at the interface core (water)-shell (organic solvent) with the use of other solvents for example.

-The investigation of other PLGA/PLA blend compositions.

-Moreover, some analyses are required to confirm the promising formulation (PM-MC20), such as: antileishmanial activity against L.

Figure 3 .Figure 4 .Figure 5 .Figure 6 .

 3456 Figure 1. Thesis Structure .............................................................................. 30

Figure 42 .

 42 Figure 22. Main steps of the AmB nanoemulsions preparation method. ......... 68

  Cutaneous leishmaniasis is characterized by lesions on the skin, which appears, primarily, in the local of insect bite (MINISTÉRIO DA SAÚDE, 2017). In Brazil, is one of the most important dermatological illnesses and disseminate in the whole country. The actual treatment for leishmaniasis, and particularly for cutaneous leishmaniasis, presents several side effects, which are motivating the development of new pharmaceutical formulations, as the study of different routes of administration. In this context, microtechnology and nanotechnology are interesting for modulate drug release profile and bioavailability providing more treatment effectiveness and low toxicity. This PhD thesis was focused on the development of two new products aiming cutaneous leishmaniasis treatment: an oil-water nanoemulsion containing amphotericin B (one of the most drug studied for this disease) and polymeric microparticles containing paromomycin (drug that have been studied for topical administration) for intralesional administration for CL treatment. The work was undertaken under the framework of an international joint supervision between School of Pharmacy -Federal University of Rio de Janeiro in Brazil, and Mines Telecom Institute (IMT Mines Albi) in France. This manuscript is divided in five chapters as shown in Figure 1. Chapter 1 introduces a literature review on Leishmaniasis diseases, presenting types of clinical manifestations, treatment available and search of new formulations, focusing on micro and nanotechnologies. Chapter 2 describes the general and specifics objectives of this work. Chapter 3 deals with amphotericin B (AmB) nanoemulsions. It starts with a literature review about the chosen drug followed by a review on nanoemulsions and more specifically, AmB nanoemulsions. In a second part, it describes all material and methods used to produce AmB nanoemulsions and, finally the obtained results are presented and discussed. Chapter 4 reports the development of PLGA-based microparticles loaded with paromomycin (PM), the second selected drug. Following the same report outline of Chapter 3, it presents paromomycin and then a review on polymeric depot deliveries, focusing on PLGA-based systems. In a second part, material and methods used to generate PLGA-based microparticles matrix type and coreshell structures are described. Results are presented and discussed in a third and last part of this chapter. The concluding Chapter 5 summarizes the major findings of this thesis and gives suggestions for further studies.

Figure

  Figure 1. Thesis Structure

Figure 2 .

 2 Figure 2. Biological cycle of Leishmania sp (REITHINGER et al., 2007).

Figure 3 .

 3 Figure 3. Patient with hepatosplenomegaly caused by VL (MINISTÉRIO DA SAÚDE, 2006).

Figure 4 .

 4 Figure 4. Status of endemicity of visceral leishmaniasis in the world in 2016 (http://apps.who.int/neglected_diseases/ntddata/leishmaniasis/leishmaniasis.html).

Figure 5 .

 5 Figure 5. First lesion of cutaneous leishmaniasis (MINISTÉRIO DA SAÚDE, 2017).

Figure 6 .

 6 Figure 6. Status of endemicity of cutaneous leishmaniasis in the world in 2016 (http://apps.who.int/neglected_diseases/ntddata/leishmaniasis/leishmaniasis.html).

Figure 7 .

 7 Figure 7. Nasal mucosa edema characteristic of mucocutaneous leishmaniasis (MINISTÉRIO DA SAÚDE, 2017).

Figure 8 .

 8 Figure 8. Chemical structure of pentavalent antimonials salts (a) N-methyl glucamine or Glucantime®, (b) Sodium stibogluconate or Pentostam® (CHEMID PLUS, 2016).

Figure 9 .

 9 Figure 9. Chemical structure of amphotericin B (CHEMID PLUS, 2016).

  marketed in the form of two salts: isethionate (di-b-hydroxyethane sulfonate) and mesylate (di-b-hydroxymethyl sulfonate) (MINISTÉRIO DA SAÚDE, 2017; SINGH, KUMAR, SINGH, 2012;). The mechanism of action may be related to a decrease of mitochondrial membrane potential by drug accumulation in the mitochondria (LINDOSO et al., 2012; SINGH, KUMAR, SINGH, 2012; GOTO & LINDOSO, 2010).

Figure 10 .

 10 Figure 10. Chemical structure of pentamidine (CHEMID PLUS, 2016).

Figure 11 .

 11 Figure 11. Chemical structure of paromomycin (CHEMID PLUS, 2016).

  , which shows interesting results in leishmaniasis treatment and has been considered a progress in the research of new treatment of this disease. Its mechanism of action is based on drug intracellular accumulation in parasites through transporters[START_REF] Lindoso | Review of the current treatments of leishmaniases[END_REF][START_REF] Singh | Leishmaniasis: Current status of available drugs and new potential drug targets[END_REF].

Figure 12 .

 12 Figure 12. Chemical structure of miltefosine (CHEMID PLUS, 2016).

  membrane and positive charge of the drug (IMBERT et al., 2014; SINGH, KUMAR, SINGH, 2012).

Figure 13 .

 13 Figure 13. Chemical structure of sitamaquine (CHEMID PLUS, 2016).

Figure 15 .

 15 Figure 15. Different types of nanoemulsions (adapted from KATOUZIAN & JAFARI, 2016).

Figure 16 .

 16 Figure 16. Scheme for nanoemulgel preparation (CHOUDHURY et al., 2017).

Figure 17 .

 17 Figure 17. Poloxamers chemical general structure (a,b) ethylene oxide and propylene oxide units (ROWE, SHESKEY, QUINN, 2009).

Figure 18 .

 18 Figure 18. Scheme of methods of preparation of nanoemulsions (RAI et al., 2018).

Figure 19 .

 19 Figure 19. -Relation between droplet size and sonication time to obtain nanoemulsions (CAMPOS, RICCI-JUNIOR, MANSUR, 2012)

Figure 20 .

 20 Figure 20. Human skin composition (Source: http://www.naturallyhealthyskin.org/anatomy-ofthe-skin/the-dermis/dermis-anatomy-of-the-skin/).

  PHARMACOPEIA 7.0, 2009), that presents molecular formula C47H73NO17 and molecular weight of 924.08 g (CHEMID PLUS, 2016; THE INTERNATIONAL PHARMACOPEIA, 2017a; USP, 2012a; EUROPEAN PHARMACOPEIA 7.0, 2009). It has very low solubility in water around 0.75 µg/mL (CHEMID PLUS, 2016), soluble in dimethyl sulfoxide (DMSO) and propylene glycol, insoluble in ethanol and slightly soluble in methanol and dimethylformamide (THE INTERNATIONAL PHARMACOPEIA, 2017a; EUROPEAN PHARMACOPEIA 7.0, 2009, GOLENSER & DOMB, 2006). This drug is sensitive to light and must be stored in places protected from light (THE INTERNATIONAL PHARMACOPEIA, 2017a; EUROPEAN PHARMACOPEIA 7.0, 2009; CONNORS, AMIDON, STELLA, 1986) and in low temperatures (between 2°C -8°C) (THE INTERNATIONAL PHARMACOPEIA, 2017a).

Figure 21 .

 21 Figure 21. Number of publications of amphotericin B (AmB) among 1997-2017 obtained from

Figure 22 .

 22 Figure 22. Main steps of the AmB nanoemulsions preparation method.

  5 to 8 µg/mL, corresponding to a regression equation (y= 0.1063x + 0.0118) with linearity r 2 = 0.9989. A preweighed amount of each nanoemulsion was diluted in methanol (1:40 v/v) and evaluated in duplicate.

Figure 23 .

 23 Figure 23. Calibration curve of AmB in methanol.

Figure 24 .

 24 Figure 24. Scheme of AmB nanoemulsions in vitro release study.

Figure 25 .

 25 Figure 25. Calibration curve of AmB in PBS for in vitro release study.

Figure 26 .

 26 Figure 26. Franz Vertical Cell Diffusion System.

  mm X 4.6 mm; 5 μm) using method adapted fromSantos and co-workers (2012). HPLC equipment was Flexar TM Conventional LC Manual Injection HPLC System (Perkin Elmer, USA) with Flexar LC manual injection valve, Flexar LC UV/VIS detector, Flexar isocratic LC pump and software system Chromera CDS. The system was maintained at room temperature (25ºC). Moreover, the mobile phase was a mixture of sodium acetate buffer solution pH 5.0 (USP, 2012c) and acetonitrile (60:40 v/v). The flow rate was 1.0 mL/min, with 20 µL as injection volume and UV detection at 410 nm. The calibration curve (Figure27) was prepared in mobile phase in the range of 0.01 to 20 µg/mL and was obtained a regression equation (y= 141692x) and linearity (r 2 = 0.9992) with retention time of AmB around 3.2 minutes.

Figure 27 .

 27 Figure 27. Calibration curve of AmB in mobile phase for skin permeation study.

Figure 28 .

 28 Figure 28. Chromatograms of (a) skin piece in mobile phase and (b) AmB solution extracted from skin piece.

Figure 29 .

 29 Figure 29. Reduction reaction of resazurin (Alamar Blue®) (adapted from CSEPREGI et al., 2018).

Figure 30 .

 30 Figure 30. SEM images of raw AmB in magnifications (a) 220x, (b) 650x, (c) 1300x.

Figure 33 .

 33 Figure 33. AmB nanoemulsions prepared in this work.

  Yellow color may be related to AmB color. C-NE and I-NE samples, prepared with mint oil, exhibited polydisperse system with dispersed cloudy yellow particles indicating instable system. D-NE, E-NE, F-NE and G-NE were liquid, while A-NE, B-NE, J-NE and K-NE were gel-like. D-NE, E-NE, F-NE and G-NE presented stable aspect and yellow color. H-NE and L-NE samples, prepared without drug, showed whiter color than NE containing AmB, visually stable and gel-like aspect.

Figure 35 .

 35 Figure 35. UV spectrum of nanoemulsions F and H in methanol.

  presents the TEM images of nanoemulsions using 100K for B-NE and L-NE as magnification and 80K magnification for F-NE and H-NE.

Figure 36 .

 36 Figure 36. TEM images of nanoemulsions with AmB (a) B-NE, (b) F-NE and respectively nanoemulsions without drug (c) L-NE and (d) H-NE.

Figure 37 .

 37 Figure 37. Droplets size in stability study of (a) B-NE and (b) L-NE. Legend: RTroom temperature (28ºC), Rrefrigerator (4ºC). * -No statistically significant differences (P > 0.05); ** -No statistically significant differences (P > 0.05); *** -No statistically significant differences (P > 0.05) according to One-Way ANOVA with Tukey post-test.

Figure 38 .

 38 Figure 38. Droplets size in stability study of (a) F-NE and (b) H-NE. Legend: RTroom temperature (28ºC), Rrefrigerator (4ºC). * -No statistically significant differences (P > 0.05); ** -No statistically significant differences (P > 0.05); *** -No statistically significant differences (P > 0.05) according to One-way ANOVA with Tukey post-test.

  for B-NE and L-NE. B-NE presented, during the stability study, 0.337 of PdI on average. The sample stored for 180 days at room temperature showed the lowest value of PdI (around 0.113). This value, statistically different of all others, could be explained by an error of analysis, probably related to non-representative aliquot or high sample dilution. The samples stored for 60 days (RT), 90 days (R) and 365 days (RT and R) presented higher values of PdI. The high values observed in samples stored for 365 days (PdI > 0.390) are probably due to an instability of the nanoemulsion, in according to the increase of droplets size observed in the same conditions. Thus, B-NE showed stability until 365 days of storage. L-NE, without drug, showed a PdI value of 0.183 (on average) during the stability study. As observed in stability of B-NE, samples stored for 90 days (R) and 365 days (RT and R) presented higher values of PdI. The high values observed in samples stored for 365 days (PdI > 0.440) suggested an instability of nanoemulsion as described for B-NE and in accordance with the enhancement of droplets size in this storage period. As for B-NE, L-NE was stable until 365 days. However, the values of PdI, on average, obtained for L-NE were lower than those found for B-NE. This can be explained by the drug presence in system, which increased the droplets size and the polydispersity. PdI values close to 0 indicates monodisperse systems and values near 1 indicates large distribution (TANG et al., 2012). Nonetheless, the two nanoemulsions prepared (B-NE and L-NE) presented PdI values closer to zero than one, indicating stable systems.

Figure 39 .

 39 Figure 39. Polydispersity index (PdI) in stability study of (a) B-NE and (b) L-NE. Legend: RTroom temperature (28ºC), Rrefrigerator (4ºC). * -No statistically significant differences (P > 0.05); ** -No statistically significant differences (P > 0.05); *** -No statistically significant differences (P > 0.05); **** -Statistically significant different (P < 0.05) according to One-way

Filippin

  and co-workers (2008) developed AmB nanoemulsions using triglycerides and evaluated the stability of formulations for 180 days stored in two conditions (4ºC and 25ºC) protected from light. It was observed no differences in pH values, maintained between 6.2 and 6.6, during stability study in the two conditions of storage.Caldeira and co-workers (2015) developed nanoemulsions of AmB using triglycerides, cholesterol and with or without stearylamine. They investigated the stability of these formulations for 180 days and analyzed pH one day after preparation and after 180 days. It was observed that pH of nanoemulsions (with or without stearylamine) was between 7.2 and 7.4, while after 180 days the pH decreased to 6.2-6.4. This reduction in pH values can be explained by fatty acids formation with triglycerides hydrolysis.From these studies, the control of pH at values desired for topical application is formulation-dependent. The NE formulations developed here seemed satisfied this criterion.

Figure 40 .

 40 Figure 40. Polydispersity index (PdI) in stability study of (a) F-NE and (b) H-NE. Legend: RTroom temperature (28ºC), Rrefrigerator (4ºC). * -No statistically significant differences (P > 0.05); ** -No statistically significant differences (P > 0.05); *** -Statistically significant differences (P < 0.05); **** -Statistically significant different (P < 0.05) according to One-way ANOVA with Tukey post-test.

Figure 41 .

 41 Figure 41. Drug loading content in stability study of (a) B-NE at room temperature, (b) B-NE at refrigerator, (c) F-NE at room temperature and (d) F-NE at refrigerator. Legend: RTroom temperature (28ºC), Rrefrigerator (4ºC). * -Statistically significant different (P < 0.05) according to One-way ANOVA with Tukey post-test.

Figure 42 .

 42 Figure 42. In vitro release profile of (a) B-NE (blue) and F-NE (red) and (b) free AmB in solution (black) (n=4).

Figure 42

 42 Figure 42 shows similar and very slow release profile of AmB from B-NE and F-NE formulations. Moreover, it can be observed (Fig 4b) that free AmB

Figure 43 .

 43 Figure 43. Amount of AmB (a) AmB permeated/area and (b) % AmB permeated present in receptor chamber (n=5).

Figure 44 .

 44 Figure 44. Amount of AmB retained in (a) epidermis and (b) dermis after 8 hours of in vitro permeation study.

Figure 46 .

 46 Figure 46. Antileishmanial activity of free AmB against promastigotes of L. amazonensis. Legend: Control -untreated parasites, only with Schneider's medium supplemented with FBS; DMSOamount of DMSO (5%) higher than used to dissolved AmB. * -No statistically significant differences (P > 0.05); ** -No statistically significant differences (P > 0.05) according to One-way ANOVA with Tukey post-test. Means ± SD (n=3).

Figure 47 .

 47 Figure 47. Antileishmanial activity of clove oil against promastigotes of L. amazonensis. Legend: Control -untreated parasites, only with Schneider's medium supplemented with FBS. * -Statistically significant difference (P < 0.05); **-Statistically significant difference (P < 0.05) according to One-way ANOVA with Tukey post-test. Means ± SD (n=3).

  Figure 48(a). The lowest concentration of F-NE (0.1 µg/mL) and two concentrations of H-NE (0.1 µg/mL and 0.5 µg/mL) presented no statistically significant differences (P > 0.05) among them. Furthermore, three concentrations of F-NE (0.5 µg/mL, 1 µg/mL and 5 µg/mL), the highest concentration of H-NE (5 µg/mL) and positive control presented no statistically significant differences (P > 0.05) among them.

Figure 48 .

 48 Figure 48. Antileishmanial activity of AmB nanoemulsions (a) B-NE and L-NE, (b) F-NE and H-NE against promastigotes of L. amazonensis. Legend: B-NE -NE with AmB and Tween ® 20; L-NE -NE without AmB with Tween ® 20; F-NE -NE with AmB; H-NE -NE without AmB; C1-Positive Control -AmB solution (1 µg/mL); C2-Negative Control -untreated parasites, only with Schneider's medium supplemented with FBS. * -No statistically significant differences (P > 0.05); **-No statistically significant differences (P > 0.05); ***-Statistically significant difference (P < 0.05); ****-Statistically significant difference (P < 0.05) according to One-way ANOVA with Tukey post-test. Means ± SD (n=3).

Figure 49 .

 49 Figure 49. Chemical structures of biodegradables polymers (PLA, PGA and PLGA) (LARRAÑETA et al., 2016).

Figure 50 .

 50 Figure 50. Types of microparticles (a) microcapsules with one core, (b) microcapsules with various core and (b) microspheres.

  and can be divided in chemical methods (emulsion solvent extraction/evaporation, polymerization), physical methods (pan coating, extrusion, fluid-bed, spray drying) and physicochemical methods (coacervation, gelation, supercritical fluid) (DESHMUKH, WAGH, NAIK, 2016; HERRERO-

  Single and double emulsification methods are based on the preparation of an oil-in-water (O/W) or water-in-oil-in-water (W/O/W) emulsion, respectively, as schematically illustrated in Figure 51. These techniques are suitable to encapsulate different types of lipophilic and hydrophilic drugs (SWIDER et al., 2018).

Figure 51 .

 51 Figure 51. Emulsification methods based on the preparation of (a) an oil-in-water (O/W) or (b) water-in-oil-in-water (W/O/W) emulsion (HAN et al., 2016).

Figure 52 .

 52 Figure 52. Droplet formation and drying during spray drying process.

Figure 53

 53 Figure53shows a scheme of a typical spray-drying process and the different steps: liquid feed into the atomizing nozzle, droplets formation (atomization), gas-droplets contact in the drying chamber and solvent evaporation, gas-dried particles separation in a cyclone.

Figure 53 .

 53 Figure 53. Typical spray drying process (SOSNIK & SEREMETA, 2015).

  configuration allows immiscible liquids encapsulation because the two liquids come into contact only at the nozzle tip. Therefore, three-fluid nozzles can produce microcapsules (core-shell system) (SUNDERLAND, KELLY, RAMTOOLA, 2015; KONDO, NIWA, DANJO, 2014).

Figure 54 .

 54 Figure 54. Types of spray drying nozzles (a) two-fluid and (b) three-fluid (adapted from KAŠPAR et al., 2013b).

Figure 55 .

 55 Figure 55. Scheme of possible PLGA particle degradation mechanism (a) initial particle, (b) water uptake into polymer matrix, (c) polymer degradation (adapted from YAO et al., 2016).

(

  paromomycin sulfate) with molecular formula C23H45N5O14 xH2SO4 and molecular weight of 615.6 g of PM base and 896.86 g in sulfate form salt (CHEMID PLUS, 2016; THE INTERNATIONAL PHARMACOPEIA, 2017b; USP, 2012b; DAVIDSON, DEN BOER, RITMEIJER, 2009). PM presents high solubility in water and low solubility in ethanol (THE INTERNATIONAL PHARMACOPEIA, 2017b). PM is hygroscopic and undergo degradation in moist environment and degrades rapidly when exposed to high temperatures (THE INTERNATIONAL PHARMACOPEIA, 2017b).

Figure 56 .

 56 Figure 56. Number of publications of paromomycin (PM) among 1997-2017 obtained from database 'Web of Science' using keywords as "Paromomycin + Leishmaniasis", "Paromomycin + Characterization", "Paromomycin + Drug delivery system (DDS)", "Paromomycin + PLGA", and "Paromomycin + Leishmaniasis + Drug delivery systems (DDS)".

FTIR

  spectrophotometer (Thermo Scientific Nicolet iS10, Thermo Fisher Scientific, France) in the range of 4000 cm -1 to 450 cm -1 . The PM samples were diluted with KBr powder using conventional KBr discs method (KHAN & KUMAR, 2011a, b). The peaks identification was based on published data describing characteristic infrared bands (SILVERSTEIN, WEBSTER, KIEMLE, 2005; LARKAN, 2011).

DEVELOPMENT

  Figure 57. The three-fluid nozzle have some advantages in comparison with twofluid nozzle, such as the use of two solvents immiscible and solutions with high concentrations ([START_REF] Kauppinen | Efficient production of solid dispersions by spray drying solutions of high solid content using a 3-fluid nozzle[END_REF].

Figure 57 .

 57 Figure 57. Nozzle types used in this work (a) two-fluid nozzle and (b) three-fluid nozzle (in situ coating).

Figure 58 .

 58 Figure 58. Nozzles design and diameters used (a) three diameters of two-fluid nozzle, (b) threefluid nozzle 1.4 mm.

Figure 60 .

 60 Figure 60. Calibration curve of derivatized PM in distilled water.

  from BONIATTI, PITALUGA, SEICEIRA, 2011). The particle size distribution, exhibited as volume weighted mean diameter (Dv), was showed based on values of d10, d50 and d90, which represent the accumulated diameters of 10, 50 and 90% of the particles, respectively. Furthermore, it was calculated polydispersity index (Span) following equation below:Where d10, d50 and d90 were the diameters at 10%, 50% and 90% cumulative volumes, respectively.

Figure 61 .

 61 Figure 61. Scheme of in vitro release assay of PM-MS/PM-MC samples. Legend: RTroom temperature.

Figure 62 .

 62 Figure 62. Calibration curve of derivatized PM in PBS for in vitro release study.

  The DE was calculated by the free open source software Kinet DS 3.0 rev. 2010 software (Krakow, Poland) according to Equation4in Table

Figure 63 .

 63 Figure 63. SEM images of raw PM in magnifications (a) 800x, (b) 3200x.

Figure 65 .

 65 Figure 65. MDSC of (a) raw PM and (b) spray-dried PM with two heating cycles.

Figure 66 .

 66 Figure 66. Color change of free PM: (a) light yellow powder before DSC analysis at room temperature, (b) dark brown powder after submitted to 200ºC in DSC analysis.

Figure 67 .

 67 Figure 67. XR Diffractograms of PM powders.

Figure 68 .

 68 Figure 68. FTIR spectrum of PM.

Figure 69 .

 69 Figure 69. Raman spectrum of PM.

Figure 70

 70 Figure70shows some SEM images of samples prepared with ethyl acetate and Figure71shows some SEM images of samples prepared with acetone, where, in both figures, raw PM was dispersed in the organic solution

Figure 70 .

 70 Figure 70. SEM Images of some batches prepared with ETHYL ACETATE in two different magnifications: (a) PM-MS01 800x, (b) PM-MS01 1600x, (c) PM-MS02 800x and (d) PM-MS02 1600x.

Figure 71 .

 71 Figure 71. SEM Images of some batches prepared with ACETONE in two different magnifications: (a) PM-MS06 800x, (b) PM-MS06 1600x.

  PM-loaded PLGA microspheres generated from two-fluid nozzles with different orifice diameters are shown in Figure 72. It is possible to verify that all particles are very similar in shape and degree of agglomeration. PM-MS05 batch (0.7 mm nozzle orifice diameter) has smaller particles in comparison to PM-MS06 and PM-MS07 batches. PM-MS07 batch (2.0 mm nozzle orifice diameter) seems to be constituted by particles of different sizes, which corresponds to a wider particle size distribution. In fact, bigger particles might be expected from the bigger nozzle orifice diameter, since it produces bigger liquid droplets during atomization. Bigger liquid droplets normally produce bigger dry particles (SOSNIK & SEREMETA, 2015).

Figure 72 .

 72 Figure 72. SEM Images of some batches prepared with acetone in two different magnifications varying the nozzle diameter size: (a) PM-MS05 1600x 0.7 mm, (b) PM-MS05 3200x 0.7 mm, (c) PM-MS06 1600x 1.4 mm, (d) PM-MS06 3200x 1.4 mm, (e) PM-MS07 1600x 2.0 mm and (f) PM-MS07 3200x 2.0 mm.

Figure 73 .

 73 Figure 73. SEM Images of some batches prepared with acetone in two different magnifications varying the PM concentration in the initial formulation: (a) PM-MS07 1600x, (b) PM-MS07 3200x, (c) PM-MS08 1600x, (d) PM-MS08 3200x.

Legend: 1

 1 PM-MS06 was produced with two-fluid nozzle and PM-MC12 with three-fluid nozzle; PLGA 50:50 is the polymeric matrix in both samples; an aqueous solution of PM was introduced in the core liquid formulation.

Figure 75

 75 Figure 75 shows the MDSC thermograms of PM-loaded core-shell PLGA/PLA microcapsules and corresponding blank formulation. The thermogram for pure PLA is also displayed in this figure.

Figure 74 .

 74 Figure 74. MDSC curves of (a) pure PLGA, (b) PM-loaded PLGA microspheres (PM-MS06) and (c) PM-loaded core-shell PLGA microcapsules (PM-MC12).

  Some PLGA blends have been reported in the literature: Lin and coworkers (2012) evaluated a novel risperidone-loaded PLGA-sucrose acetate isobutyrate for sustained release injection depot. A polymer mixture composed ofPDLLA, PGA and PCL (40:40:20) was used in another study. The experimental value of Tg found for this blend was 21.8 ºC[START_REF] Liu | Synthesis, properties, and in vitro hydrolytic degradation of poly(d,l-lactide-co-glycolide-co-ε-caprolactone)[END_REF].

Figure 75 .

 75 Figure 75. MDSC curves of (a) pure PLA, (b) PM-loaded core-shell PLGA/PLA microcapsules (PM-MC20) and (c) PLGA/PLA blend microparticles (MC24).

Figure 76 .

 76 Figure 76. SEM Images of some batches prepared with acetone in two different magnifications varying the nozzle type: two-fluid PM-MS06 and three-fluid (PM-MC12 and PM-MC16).

Figure 77 .

 77 Figure 77. SEM images of some batches prepared from an aqueous of PM, three-fluid, with different compositions of PLGA/PLA blends: PM-MC19 (95:5), PM-MC20 (90:10) and PM-MC21 (60:40).

Figure 78 .

 78 Figure 78. SEM Images of PM-MC22 prepared with dichloromethane: acetone in two different magnifications: (a) 1600x, (b) 3200x.

Figure 79 .

 79 Figure 79. FTIR spectra of pure components (PLGA and PM), physical mixture PM+PLGA (30:70) and microparticles samples (PM-MS06, PM-MC12 and PM-MC18).

Figure 80 .

 80 Figure 80. FTIR spectra of pure PLA, physical mixture PLGA+PLA (95:5), physical mixture PM+PLA+PLGA (30:5:65), PM microparticles (PM-MC19) and blank microparticles (MC23).

Legend: 1

 1 Physical Mixture PM:PLGA (30:70); PM-MS06 (raw PM powder fed into the 2fluid nozzle); PM-MC15 (PM-MC12 replicated, PM in solution fed to the 3-fluid nozzle, PLGA), PM-MC18 (raw PM powder fed to the 3-fluid nozzle) and PM-MC20 (PM in solution fed to the 3-fluid nozzle, PLGA:PLA mixture 90:10) (n=3).

Figures

  Figures 81 to 83 display respectively SEM images of PM-M06 microspheres, PM-MC15 and PM-MC18 microcapsules, before and after contact with water during 24h. This test aimed to release PM not protected by PLGA and then allow the observation of the solid structure after the possible PM release.

  , in the samples exposed to water, some bigger particles like the ones visible in Figure82(top left), seem to have disappeared.

Figure 81 .

 81 Figure 81. PM-MS06 microspheres, after contact with water for 24h.

Figure 84

 84 Figure 84 shows the spatial distribution of PM and polymer mapped by Raman spectroscopy in studied samples, PM-MS06, PM-MC12 (replicate of PM-MC15), PM-MC16 (replicate of PM-MC18) and PM-MC20. The Raman spectra of PLGA and PM are also presented in this figure.

Figure 84

 84 Figure 84 allows the analysis of the spatial distribution of PLGA and PM in the microparticles prepared using two-fluid and three-fluid nozzle. The PM-loaded PLGA microspheres (MS06) (a) showed PM (in red) partially surrounded by the polymer (in blue). On the other hand, the batches of core-shell microcapsules

Figure 84 .

 84 Figure 84. Raman images of spatial distribution of some PM-MS/PM-MC batches (a) PM-MS06, (b) PM-MC12, (c) PM-MC16 and (d) PM-MC20; (e) Raman spectra of Paromomycin (red) and PLGA (blue) with correspondent colors to identify polymer and drug in spatial distribution.

Figure 85 .

 85 Figure 85. Possible structure of PM-MS/PM-MC batches based on Raman analysis. Legend: redcorresponding PM and bluecorresponding polymer.

Figure 86 .

 86 Figure 86. Cytotoxicity assay of (a) free PM, (b) PM-MC12, (c) PM-MC20 and (d) MC24 in BMDM cells. Legend: 0 -Negative Control without treatment, Triton -Positive Control (4% Triton 100x). * -No statistically significant differences (P > 0.05); **-No statistically significant differences (P > 0.05); ***-No statistically significant differences (P > 0.05) according to Oneway ANOVA with Tukey post-test.

  , PLGA-based microparticles were proposed as systems for subcutaneous delivery of Paromomycin (PM) for leishmaniasis treatment. Spray drying was the manufacturing process chosen to this end. The raw PM used in this study was commercially produced by spray drying. There are very few scientific publications on physicochemical properties of this molecule. DRX analysis confirmed the amorphous state of the sample, however its glass transition temperature (Tg) could not be determined by DSC analysis in the interval -90°C to 200 ºC. PLGA 50:50 is the polymer of first choice for subcutaneous delivery due to its biodegradability and biocompatibility. However, the development of drugloaded PLGA microparticles containing hydrophilic drug like PM represents a challenge due to low drug loadings often reached, low encapsulation efficiencies and initial burst release. Different parameters were varied during the development of the spraydried drug-loaded microparticles, among them: the type of organic solvent, drug concentration in the initial formulations, formulation and nozzle design (two-fluid and three-fluid nozzles, nozzle diameter orifice of the two-fluid nozzle) and composition of the polymeric carrier (PLGA or PLGA:PLA binary mixture). The spray-dried powders were characterized in terms of physicochemical properties, release behavior and biological assays.

  amazonensis amastigotes, stability study, antileishmanial activity against other Leishmania species that cause cutaneous leishmaniasis and in vivo studies in BALB/c mice model. To conclude, the two formulations proposed in this work could represent novel promising possibilities for cutaneous leishmaniasis treatment, one via topical administration of amphotericin B, and the other via intralesional administration of paromomycin. L'AmB est l'un des médicaments les plus étudiés pour le traitement de la leishmaniose. Elle est utilisée dans différents systèmes d'administration pour améliorer leur activité leishmanicide et réduire leurs effets secondaires. Comme l'AmB est très toxique, les formulations topiques contenant ce médicament peuvent être une option intéressante pour réduire sa toxicité. Un des médicaments alternatifs étudiés pour le traitement de la LC est la paromomycine (PM), un antibiotique aminoglycoside utilisé par voie intraveineuse et topique. La PM est fortement soluble dans l'eau, mais présente une faible perméabilité, ce qui permet d'insérer ce médicament dans la classe III du système de classification biopharmaceutique (SCB). En raison de la faible perméabilité du médicament, les formulations topiques de PM ont un usage controversé, en particulier contre les espèces de Leishmania de «Nouveau Monde». Il existe peu d'études portant sur la caractérisation physicochimique de la PM ou sur les systèmes de libération de la PM. De plus, ce médicament n'a pas encore été approuvé pour le traitement de la leishmaniose dans des pays comme le Brésil, où cette maladie est présente. Cette thèse porte sur le développement d'une nanoémulsion d'huile-dansl'eau (HE) contenant AmB pour une administration topique et des microparticules polymériques contenant PM obtenues par séchage par atomisation pour une administration intralésionnelle pour le traitement de la LC. Les formulations ont été caractérisées en termes de taille et morphologie de particules et gouttelettes d'huile, contenu d'actif, stabilité, propriétés thermiques, relargage in vitro.L'activité biologique a été évaluée in vitro sur des macrophages et l'activité antileshmanienne sur des promastigotes de Leishmania amazonensis.Les nanoémulsions HE contenant Amphotéricine B (AmB)Les nanoémulsions HE contenant AmB ont été préparées par un procédé d'émulsification sous ultrasons. Les huiles essentielles (huile de girofle et huile de menthe) ont été sélectionnées pour la phase huileuse en raison de leur capacité à augmenter la perméation des médicaments. Douze lots ont été préparés en modifiant leur composition comme l'huile essentielle utilisée, la présence de tensioactif (0 à 5% de Tween ® 20 en masse), la quantité de polymère utilisée en phase aqueuse (10 à 15% de Pluronic ® F127 en masse) et la présence d'AmB (0,5 mg/mL). Les étapes de préparation des nanoémulsions peuvent être visualisées sur la Figure1. Les lots ont été caractérisés par la taille des gouttelettes, l'indice de polydispersité, le pH et la teneur d'AmB.

Figure 1 -

 1 Figure 1 -Représentation schématique de la préparation des nanoémulsions. (DMSOdiméthylsulfoxyde et HE -huile-dans-l'eau).

Figure 2 - 3 )

 23 Figure 2 -Buses bi-fluide et tri-fluide utilisées lors de la préparation des microparticules de PM par séchage par atomisation

Table 2 .

 2 Physicochemical properties of some poloxamers used in pharmaceutical formulations (adapted from BODRATTI & ALEXANDRIDIS, 2018; adapted from DEVI, SANDHYA, HARI, 2013; adapted from ROWE, SHESKEY, QUINN, 2009 ). ............................................................................................... 57

Table 3 .

 3 AmB nanoemulsions composition ............................................ 69

Table 4 .

 4 Physicochemical characterization of AmB nanoemulsions....... 86

Table 5 .

 5 Drug Content of Nanoemulsions .............................................. 90

Table 6 .

 6 CC50, IC50 values and Selective Index of free AmB, free clove oil and

nanoemulsions. .............................................................................................. 119

Table 7. Physical properties of some PLGAs (adapted from KAPOOR et al., 2015; ANSARY et al., 2014). ..........................................................................128 Table 8. Adjustable variables in a spray drying process (adapted from PATEL et al., 2015; SOSNIK & SEREMETA, 2015; adapted from PAUDEL et al., 2013).134 Table 9. Drug delivery systems of paromomycin formulations. .............142 Table 10. Dependent and independent kinetic models for study release profile of PM delivery systems (adapted from MEDEIROS et al., 2017; adapted from COSTA, 2002)..............................................................................................................155

Table 11 .

 11 Formulation conditions, process and product characteristics of PMloaded microspheres: solvent effect. ..............................................................166

Table 12 .

 12 Formulation conditions, process and product characteristics of PMloaded microspheres: nozzle orifice diameter effect. ......................................170

Table 13 .

 13 Formulation conditions, process and product characteristics of PM-

loaded microspheres: effect of PM concentration in the initial liquid formulation.173 Table 14. Formulation conditions, process and product characteristics of PMloaded core-shell microcapsules. ...................................................................176

Table 15. Thermal properties of pure PLGA, PM-MS06 and PM-MC12.179

Table 16 .

 16 Thermal properties of PM-MC and MC (blank) batches prepared with PLGA/PLA as polymeric matrix. .....................................................................181

Table 17 .

 17 Particle size distribution and Span of some PM-MC batches.191

Table 18 .

 18 Composition of PM-MS/PM-MC batches selected and their characterization ..............................................................................................193 Table 19. % PM released per hour of released test and dissolution efficiency after 4 hours (DE4h) ........................................................................................194

Table 20 .

 20 Kinetic parameters of PM-MS/PM-MC batches using Korsmeyer-Peppas model. ...............................................................................................195

Table 21

 21 Equation 1. Drug Content (DL %) ........................................................150 Equation 2. Encapsulation Efficiency (EE %) .......................................150 Equation 3. Yield of batches ...............................................................151 Equation 4. Polydispersity Index (Span) ..............................................151

	HIV -Human Immunodeficiency Virus
	HLB -Hydrophilic Lipophilic Balance
	HPLC -High Performance Liquid Chromatography
	IBCCF -Instituto de Biofísica Carlos Chagas Filho
	ICH -International Conference on Harmonization of Technical Requirements
	for Registration of Pharmaceuticals for Human Use
	IC50 -Half Maximal Inhibitory Concentration
	IOC -Instituto Oswaldo Cruz / Oswald Cruz Foundation
	Leishcutan -Ointment containing 15% Paromomycin + 12%

. In vitro antileishmanial activity and cytotoxicity results for PM-MC formulations (CC50, IC50 values and Selective Index of free PM and PM-MC batches) against L. amazonensis promastigotes ..........................................................209

LIST OF EQUATIONS

266 ANNEX 2 -PM release fitting described by mathematical Korsmeyer-Peppas model

  Nanoemulsions ........................................................................................53 In vitro Skin Permeation Study of Amphotericin B Nanoemulsions ..........73 3.3.2.6.1. Skin Permeation Method .......................................................................73 3.3.2.6.2. High Performance Liquid Chromatography (HPLC) Assay ....................75 3.3.2.7. BIOLOGICAL ASSAYS ............................................................................77 3.3.2.7.1. Assessment of cytotoxicity of AmB nanoemulsions in BMDM cells .......77 3.3.2.7.2. Antileishmanial Activity ..........................................................................79 3.3.2.8. Statistical Analysis ...................................................................................80 Physicochemical Properties of PLGA..................................................... 4.1.3. Preparation Methods of PLGA Microparticles ........................................ 4.1.4. Challenges in improving drug loading of PLGA microparticles with acceptable control over release rate profiles ...................................................................................... 4.1.5. Release from PLGA microparticles ........................................................ 4.1.6. PLGA microparticles for depot delivery ..................................................... 4.1.7. Paromomycin ............................................................................................ .3.1. MATERIAL .............................................................................................

	3.4. 4.2.

CONTENTS GENERAL INTRODUCTION .......................................................................... 28 CHAPTER 1 DISEASE LITERATURE REVIEW ............................................. 31 CHAPTER 2 OBJECTIVES ............................................................................ 50 2.1. GENERAL OBJECTIVE ..............................................................................51 2.2. SPECIFIC OBJECTIVES .............................................................................51 CHAPTER 3 NANOEMULSIONS OF AMPHOTERICIN B .............................. 52 3.1. INTRODUCTION ..........................................................................................53 3.1.1.

3.1.2. Poloxamers properties .............................................................................55 3.1.3. Methods of nanoemulsion production ......................................................58 3.1.4. Topical administration .................................................................................62 3.1.5. Amphotericin B ............................................................................................64 3.2. OBJECTIVES...............................................................................................66 3.3. MATERIALS AND METHODS .....................................................................66 3.3.1. MATERIALS .............................................................................................66 3.3.2. METHODS ...............................................................................................67 3.3.2.1. Physicochemical Characterization of Amphotericin B ..............................67 3.3.2.1.1. Scanning Electron Microscopy (SEM) ..................................................67 3.3.2.1.2. Thermogravimetry Analysis (TGA) ........................................................67 3.3.2.1.3. Fourier Transform Infrared Spectroscopy (FTIR) ..................................67 3.3.2.2. Amphotericin B Nanoemulsions Development .........................................68 3.3.2.3. Physicochemical Characterization of Amphotericin B Nanoemulsions .....69 3.3.2.3.1. Determination of Droplet Size, Polydispersity Index and pH .................69 3.3.2.3.2. Drug Content .........................................................................................70 3.3.2.4. Stability Study of Amphotericin B Nanoemulsions ....................................70 3.3.2.4.1. Transmission Electron Microscopy (TEM) .............................................71 3.3.2.4.2. Determination of Droplet Size, Polydispersity Index and pH .................71 3.3.2.4.3. Drug Content .........................................................................................71 3.3.2.5. In vitro Release of Amphotericin B Nanoemulsions .................................71 3.3.2.6. RESULTS AND DISCUSSION.....................................................................80 3.4.1. PHYSICOCHEMICAL CHARACTERIZATION OF AMPHOTERICIN B ...80 3.4.1.1. Scanning Electron Microscopy (SEM) ......................................................80 3.4.1.2. Thermogravimetry Analysis (TGA) ...........................................................81 3.4.1.3. Fourier Transform Infrared Spectroscopy (FTIR) .....................................82 3.4.2. AMPHOTERICIN B NANOEMULSIONS DEVELOPMENT ......................83 3.4.3. PHYSICOCHEMICAL CHARACTERIZATION OF AMPHOTERICIN B NANOEMULSIONS ...........................................................................................................85 3.4.3.1. Determination of Droplet Size, Polydispersity Index and pH ....................85 3.4.3.2. Drug Content ............................................................................................87 3.4.4. STABILITY STUDY OF AMPHOTERICIN B NANOEMULSIONS ............91 3.4.4.1. Transmission Electron Microscopy (TEM) ................................................92 3.4.4.2. Determination of Droplet Size, Polydispersity Index and pH ....................93 3.4.4.3. Drug Content .......................................................................................... 102 3.4.5. IN VITRO RELEASE OF AMPHOTERICIN B NANOEMULSIONS ........ 104 3.4.6. IN VITRO SKIN PERMEATION STUDY OF AMPHOTERICIN B NANOEMULSIONS ......................................................................................................... 107 3.4.7. BIOLOGICAL ASSAYS .......................................................................... 112 3.4.7.1. Assessment of cytotoxicity of AmB nanoemulsions in BMDM cells ....... 112 3.4.7.2. Antileishmanial Activity ........................................................................... 114 3.5. CONCLUSION ........................................................................................... 123 CHAPTER 4 POLYMERIC MICROPARTICLES OF PAROMOMYCIN...........125 4.1. INTRODUCTION ........................................................................................ 126 4.1.1. Biodegradable Polymeric Depots ........................................................... 126 4.1.2. OBJECTIVES............................................................................................. 4.3. MATERIAL AND METHODS ..................................................................... 44.3.2. METHODS ............................................................................................. 4.3.2.1. PHYSICOCHEMICAL CHARACTERIZATION OF PAROMOMYCIN ..... 4.3.2.1.1. Scanning Electron Microscopy (SEM) ................................................. 4.3.2.1.2. Differential Scanning Calorimetry (DSC) ............................................. 4.3.2.1.3. X-ray Scattering Diffraction (XRD) ...................................................... 4.3.2.1.4. Fourier Transform Infrared Spectroscopy (FTIR) ................................ 4.3.2.1.5. Raman Spectroscopy .......................................................................... 4.3.2.2. PAROMOMYCIN POLYMERIC MICROPARTICLES DEVELOPMENT . a) Nozzle Design ........................................................................................ b) Type of Organic Solvent ......................................................................... c) Type of Polymer ..................................................................................... d) PM Mass Concentration ......................................................................... e) The Physical State of PM formulation .................................................... 4.3.2.3. PHYSICOCHEMICAL CHARACTERIZATION OF PAROMOMYCIN POLYMERIC MICROPARTICLES ................................................................................... 4.3.2.3.1. Scanning Electron Microscopy (SEM) ................................................. 4.3.2.3.2. Differential Scanning Calorimetry (DSC) ............................................. 4.3.2.3.3. Drug Content, Encapsulation Efficiency and Process Yield ................ 4.3.2.3.4. Fourier Transform Infrared Spectroscopy (FTIR) ................................ 4.3.2.3.5. Particle Size ........................................................................................ 4.3.2.4. IN VITRO RELEASE OF PAROMOMYCIN POLYMERIC MICROPARTICLES

CHAPTER 5 GENERAL CONCLUSIONS AND PERSPECTIVES .................214 CHAPTER 6 BIBLIOGRAPHY ......................................................................218 CHAPTER 7 RÉSUMÉ LONG EN FRANÇAIS ..............................................254 CHAPTER 8 ANNEXES ................................................................................265 ANNEX 1 -Certificate of Analysis ........................................................................ ........................... ........................................................................................................ 268 CHAPTER 9 PUBLICATIONS RELATED TO THIS WORK ...........................270 ABSTRACT ...................................................................................................272

GENERAL INTRODUCTION

Leishmaniasis belongs of the group of neglected infectious diseases caused by protozoan parasites of the genus Leishmania (SINGH, KUMAR,

Table 1 . Current treatments of leishmaniasis (adapted from ZULFIQAR, SHELPER, AVERY, 2017; LINDOSO et al., 2012 and MENEZES et al., 2015). Drugs Administration route Dosage Advantages Disadvantages Resistance Amphotericin B

 1

	classes that are used as alternative treatments such as ketoconazole, imiquimod,
	itraconazole, allopurinol among others (HANDLER et al., 2015; MITROPOULOS,
	KONIDAS, DURKIN-KONIDAS, 2010).
	Imidazoles (ketoconazole) and triazoles (fluconazole, itraconazole) are
	antifungal compounds with same mechanism of action. These drugs act binding
	to the ergosterol molecules in the cytoplasmic membrane of parasites increasing
	membrane permeability. These compounds can be administered orally and
	present lower toxic side effects than pentavalent antimonials (MITROPOULOS,
	KONIDAS, DURKIN-KONIDAS, 2010). Furthermore, triazoles are less toxic,
	interfere less with human synthesis of cholesterol and are slower metabolized
	than imidazoles (LINDOSO, 2012; GOTO & LINDOSO, 2010; MITROPOULOS,
	KONIDAS, DURKIN-KONIDAS, 2010).
	Pentoxifylline is a methyl-xanthine derivate drug belongs to peripheral
	vasodilator class with hemorheological effects (MINISTÉRIO DA SAÚDE, 2017;
	HASSAN, DORJAY, ANWAR, 2014). Some researchers have been demonstrated
	successful using pentoxifylline as adjunct drug as immunomodulate in cutaneous
	leishmaniasis reducing treatment time. This drug is available in extended release
	tablets and is recommended one tablet of 400 mg, three times a day for 30 days
	(MINISTÉRIO DA SAÚDE, 2017).

-Sitamaquine is in phase II study for leishmaniasis treatment.

Table 2 . Physicochemical properties of some poloxamers used in pharmaceutical formulations (adapted from BODRATTI & ALEXANDRIDIS, 2018; adapted from DEVI, SANDHYA, HARI, 2013; adapted from ALVAREZ-LORENZO, SOSNIK, CONCHEIRO, 2011; adapted from ROWE, SHESKEY, QUINN, 2009). Poloxamer Pluronic ® Notation Physical Form Molecular Weight Melting Point (°C) Ethylene Oxide Units* Propylene Oxide Units* CMC ** (mM)

 2

	(BODRATTI & ALEXANDRIDIS, 2018; DEVI, SANDHYA, HARI, 2013). These
	copolymers have an interesting thermoreversible property, in which at low
	temperatures (or below sol-gel transition temperature) remain as solution and at
	high temperatures (or above sol-gel transition temperature) become gel (DEVI,
	SANDHYA, HARI, 2013; PATEL, PATEL, PATEL, 2009). Moreover, some
	poloxamers have been approval for pharmaceutical formulations by Food and
	Drug Administration (FDA) as Poloxamer 407 and Poloxamer 188 (FAKHARI,
	CORCORAN, SCHWARZ, 2017). Table 2 presents some Poloxamers, used in
	pharmaceutical formulations, with some physicochemical properties.

Legend: * -based on average molecular weight; ** -CMC -Critical micellar concentration at 37ºC.

	drug introduction into aqueous solution (DEVI, SANDHYA, HARI, 2013). The
	addition of drugs, salts, excipients or polar organic solvents into poloxamers
	solution can affect the micellization process (BODRATTI & ALEXANDRIDIS,
	2018; FAKHARI, CORCORAN, SCHWARZ, 2017).
	There are some methods for prepare drug-loaded poloxamers
	formulations as direct solubilization (simplest method), thin film hydration,
	temperature-induced emulsification, solvent displacement and kinetically frozen
	micelles (BODRATTI & ALEXANDRIDIS, 2018).
	One of the most studied poloxamer is Poloxamer 407 (Pluronic
	In aqueous solution, poloxamers exhibit micelles structures. The
	micellization process starts when the concentration of copolymer in solution
	achieve critical micellar concentration (CMC) at certain temperature or when
	increases temperature to achieve critical micellar temperature (CMT) at fixed
	polymer concentration. Then, occurs the reduction of PPO chains solubility
	resulting into micelles formations (BODRATTI & ALEXANDRIDIS, 2018; DEVI,
	SANDHYA, HARI, 2013). The micellization is a spontaneous process with
	spherical micelles are core-shell structure, in which micelles core have
	hydrophobic characteristic and micelles shell hydrophilic characteristic
	(BODRATTI & ALEXANDRIDIS, 2018). Micelles formation allow hydrophobic

® F127) due to this copolymer can form hydrogels with low polymer concentration in water, around 20% w/w (BODRATTI & ALEXANDRIDIS, 2018). Poloxamer 407 formulations can be used for drug, small molecules, peptides, biological molecules deliveries, especially for controlled release (FAKHARI, CORCORAN, SCHWARZ, 2017). Moreover, some studies have been described that poloxamer 407 gel has properties to promote wound healing

[START_REF] Patel | Poloxamers: A pharmaceutical excipients with therapeutic behavior[END_REF]

.

Table 4 . Physicochemical characterization of AmB nanoemulsions

 4

	Samples	Mean droplet size	Polydispersity index (PdI)	pH
		(nm) ± SD	± SD
	A-NE	32.63 ± 1.81	0.19 ± 0.06	5.0
	B-NE	43.50 ± 3.14	0.26 ± 0.01	5.0
	C-NE	294.27 ± 2.03	0.45 ± 0.03	4.5
	D-NE	85.88 ± 1.82	0.37 ± 0.05	4.5
	E-NE	46.83 ± 0.44	0.37 ± 0.00	4.5
	F-NE	93.91 ± 2.63	0.36 ± 0.04	5.0
	G-NE	51.27 ± 0.51	0.43 ± 0.00	4.5
	H-NE	78.50 ± 1.20	0.33 ± 0.04	5.0
	I-NE	371.40 ± 8.77	0.63 ± 0.04	4.5
	J-NE	56.65 ± 3.42	0.61 ± 0.01	5.0
	K-NE	112.10 ± 0.40	0.31 ± 0.01	5.0
	L-NE	31.43 ± 0.43	0.16 ± 0.03	5.0
	Legend: SD -standard deviation
	C-NE and I-NE, prepared with mint oil and showed the highest mean
	droplet size (294.27 ± 2.03 nm and 371.40 ± 8.77 nm, respectively) and elevated
	values of PdI (0.454 ± 0.030 and 0.633 ± 0.041, respectively). The presented a
	visual instable aspect (Figure 33) and these data confirmed an unstable behavior.
	J-NE, also prepared with mint oil, presented lower mean droplet size (56.65 ±
	3.42 nm) than formulations C and I, but presented elevated PdI (0.609 ± 0.014)
	as formulations C and I.

NE A showed the lowest mean particle droplet size and the lowest PdI of formulations prepared with drug suggesting a more stable formulation. B-NE, D-NE, E-NE, F-NE and G-NE exhibited mean droplet size lower than 100 nm.

Table 5 . Drug Content of Nanoemulsions

 5

	Batch	Concentration of	% DC 2
		AmB (mg/mL) ± SD 1
	A-NE	0.26 ± 0.00	51.2
	B-NE	0.38 ± 0.00	76.8
	C-NE	0.02 ± 0.00	4.2
	D-NE	0.30 ± 0.00	60.2
	E-NE	0.39 ± 0.00	78.2
	F-NE	0.39 ± 0.00	77.2
	G-NE	0.36 ± 0.00	72.0
	H-NE	-	-
	I-NE	0.01 ± 0.00	1.2
	J-NE	0.02 ± 0.01	3.2
	K-NE	0.28 ± 0.00	55.4
	L-NE	-	-

Legend:

1 Standard deviation; 2 Drug content in percentage

  Finally, B-NE, E-NE and F-NE showed the best %DC compared with the other batches. Furthermore, they presented satisfactory droplet sizes and PdI.

	encapsulation supporting the instability of these systems observed in visual
	analysis and through high PdI probably related to phase separation. Thus,
	samples prepared with mint oil were discarded from the AmB nanoemulsions
	development.

H-NE and L-NE are blank without drug. C-NE, I-NE and J-NE, prepared with mint oil, had the lowest values of %DC, which suggested no drug A-NE, D-NE and K-NE presented lower values of %DC than B-NE, E-NE, F-NE and G-NE being also discarded from this study. It is worth pointing out K-NE showed high droplet size as well. Although %DC of G-NE was good (72%), this formulation was discarded due to PdI higher than 0.4.

Table 6 . CC50, IC50 values and Selective Index of free AmB, free clove oil and nanoemulsions.

 6

	Formulation	CC50 *	IC50 **	Selective Index
				(SI) ***
	AmB	0.50 µg/mL	0.48 µg/mL	1.04
	Clove oil	18.80 µg/mL #	5.26 µg/mL	3.57
	B-NE	0.19 µg/mL	0.03 µg/mL	6.33
	L-NE	0.11 µg/mL	0.14 µg/mL	0.79
	F-NE	0.17 µg/mL	0.14 µg/mL	1.21
	H-NE	-	0.84 µg/mL	-

Legend: *-

concentration in µg/mL cytotoxicity for 50% of BMDM; ** -concentration in µg/mL which reduce 50% of viability of L. amazonensis promastigotes; *** -Selective Index: CC50/IC50; # -literature data obtained from macrophages RAW 264.7 (KOUIDHI, ZMANTAR, BAKHROUF, 2010).

Table 7

 7

	shows some physical properties of some PLGAs. It can be seen
	that the biodegradation rate of PLGA depends on several factors, among them,
	the composition of the polymer. The degradation of PLGA is faster for polymers

with the increased amount of glycolic acid units. Polymers with a 50:50 ratio of lactic and glycolic acids have the fastest degradation rate. PLGA with the 50:50 ratio has the degradation rate of around two months in vivo and has the fastest release rate when compared to other ratios

[START_REF] Ding | Recent advances of PLGA micro/nanoparticles for the delivery of biomacromolecular therapeutics[END_REF][START_REF] Kapoor | PLGA: a unique polymer for drug delivery[END_REF]

.

Table 7 . Physical properties of some PLGAs (adapted from KAPOOR et al., 2015; ANSARY et al., 2014).

 7

	Polymer	Molecular	Glass	Physical	Degradation
	(lactide/glycolide	Weight range	Transition	state	time
	ratio)		(Tg)		(months)
			temperature
			(°C)
	PLGA (50:50)	7,000-54,000	42-50	Amorphous	1-2
	PLGA (65:35)	24,000-38,000	45-50	Amorphous	3-4
	PLGA (75:25)	4,000-116,000	42-50	Amorphous	4-5
	PLGA (85:15)	190,000-	50-55	Amorphous	5-6
		240,000

  Mir and co-workers (2017) discussed the recent application of PLGA particles in drug delivery for different diseases treatment. Moreover, they introduced the products prepared with PLGA in clinical

	trials and on market. Han ad co-workers (2016), described some examples of
	PLGA microparticles for sustained release design and some strategies to improve
	drug loading in particles. Sharma and co-workers (2016) focused on helping to
	explain the preparation methods to obtain PLGA-based nanoparticles for
	biomedical applications. They also discussed the mechanisms of drug release,
	factors affecting particles degradation and targets strategies for efficient drug
	delivery. Wan and Yang (2016) reported PLGA-based depot delivery systems
	prepared by spray drying and discussed some aspects of the spray drying
	technology as the critical solvent properties, novel types of nozzles for
	atomization and their applications. Kappor and co-workers (2015) addressed the
	synthesis, physicochemical properties and biodegradation of PLGA,
	microencapsulation methods, problems with drug delivery and PLGA
	formulations in drug delivery systems.
	PLGA microparticles can be administered by different routes such as
	pulmonary, ocular, parenteral, oral, intramuscular, subcutaneous, nasal and
	topical (WANG

Table 9 . Drug delivery systems of paromomycin formulations.

 9

	Formulation	Administration	Method	Physicochemical	Reference
		route			characterization
	Nanoparticles	-	Nanoprecipitation	Particle size, encapsulation	Kalimouttou et
	of PM + PLGA				efficiency, DSC	al., 2008.
	PM liposomes	Topical	Fusion and	Particle size	Jaafari et al.,
			homogenization		2009.
	PM liposomes	Topical	Reverse	phase	Particle size, zeta potential,	Carneiro et al.,
			evaporation		encapsulation efficiency	2010.
	Microspheres	Parenteral	Spray drying	Particle size, encapsulation	Khan et al.,
	of albumin				efficiency, drug release,	2011a.
	containing PM				SEM, DSC, TGA, FTIR,
					contact angle

Table 10 . Dependent and independent kinetic models for study release profile of PM delivery systems (adapted from MEDEIROS et al., 2017; adapted from COSTA, 2002).

 10

	Approach	Model	Mathematical Equation
	Dependent	Korsmeyer-	𝑄𝑡 𝑄∞	= 𝐾𝑘 𝑡 n	(1)
		Peppas

Table 12 . Formulation conditions, process and product characteristics of PM-loaded microspheres: nozzle orifice diameter effect.

 12

Table 13 . Formulation conditions, process and product characteristics of PM-loaded microspheres: effect of PM concentration in the initial liquid formulation.

 13

	Batch 1	Initial	Polymeric	PM in the	Yield	Drug loading	Encapsulation
		Drug	carrier	formulation	(%)	in the	Efficiency
		content 2				microspheres	(%)
		(%wt.)				(%)
	PM-MS07	30	PLGA 50:50	Dispersed 3	44.5	18.3	61.1
	PM-MS08	70	PLGA 50:50	Dispersed 4	38.8	33.8	48.3
	Legend: 1

process conditions: acetone is the solvent for the polymer; 2.0mm two-fluid nozzle; 2 Drug percentage in relation to total of solid contents; 3 Raw PM dispersed in the polymeric solution (0.65g PM in 99.35g organic solution containing 1.5%wt PLGA); 4 Raw PM dispersed in the polymeric solution (3.50g PM in 96.50g organic solvent containing 1.5%wt PLGA).

Table 14 . Formulation conditions, process and product characteristics of PM-loaded core-shell microcapsules.

 14

	Batch 1	Initial	Core	Shell	Yield	Drug loading	Encapsulation
		drug			(%)	in the	Efficiency
		content 2				microcapsules	(%)
		(%)				(%)
	PM-MC04	45	PM in water	PLGA	48.8	30.1	66.9
			(solution)
	PM-MC12	30	PM in water	PLGA	54.8±8.3	24.3±2.5	87.1±8.3
	PM-MC15		(solution)
	PM-MC13	15	PM in water	PLGA	40.8	7.0	46.7
			(solution)
	PM-MC14	60	PM in water	PLGA	80.3	30.7	51.1
			(solution)
	PM-MC16	30	Raw PM	PLGA	31.7±12.0	25.4±4.9	80.6±22.2
	PM-MC18		powder
	PM-MC17	15	Raw PM	PLGA	45.1	1.4	28.8
			powder
	PM-MC19	30	PM in water	PLGA	42.2	2.8	9.4
			(solution)	(95%)-PLA
				(5%)
	PM-MC20	30	PM in water	PLGA	32.1	18.1	60.3
			(solution)	(90%)-PLA
				(10%)
	PM-MC21	30	PM in water	PLGA	53.4	28.0	93.4
			(solution)	(60%)-PLA
				(40%)
	PM-MC22	30	PM in water	PLA	70.9	9.2	30.5
			(solution)
	MC23	-	Water	PLGA		_	_
				(95%)-PLA
				(5%)
	MC24	-	Water	PLGA		_	_
				(90%)-PLA
				(10%)
	MC25	-	Water	PLGA		_	_
				(60%)-PLA
				(40%)
	Legend: 1

Process conditions: acetone as organic solvent; three-fluid nozzle (orifice diameter 1.4 mm); 2 Drug percentage in relation to total solid content.

  Table 14 shows that the yield of PM-

	batches ranged from 1.4% to 30.7%. EE was also highly variable, from 9.4% up batches. EE of PM-loaded liposomes batches ranged from 58 to 97%, depending
	to 93.4%. on the lipid composition.
	In comparison with PM-MS06, produced with same nozzle orifice size (1.4 The PM-MS batches produced in our study showed average values of EE
	mm), PM-microcapsules presented higher values of drug loading and EE. PM-in comparison of PM encapsulation efficiency described in literature using various
	MC16 showed the highest EE (93.4%), however, this batch changed of powder encapsulation systems.
	color (from white to light-yellow).
	Some published data reflect the difficulty to reach high drug loadings in 4.4.2.2.1. Differential Scanning Calorimetry (DSC)
	PLGA microspheres: Prior and co-workers (2004) developed spray-dried
	gentamicin-loaded PLGA microspheres with a low drug loading of 20 µg Figure 74 displays the MDSC thermograms of PM-loaded PLGA
	gentamicin/mg microspheres, compared to the expected loading of 100 µg microspheres (PM-MS06) compared to PM-loaded core-shell PLGA
	gentamicin/mg microspheres. On the other hand, Guerrero and co-workers microcapsules (PM-MC12). The thermogram for pure PLGA is also displayed in
	(2008) prepared spray-dried ketotifen-loaded microspheres using PLA or PLGA this figure.
	and obtained high values of EE and drug loadings: EE of 82% and a drug loading
	of 91% with PLGA and EE of 75% and drug loading of 83% with PLA.
	Furthermore, others micro and nano systems have been described aiming
	PM encapsulation. These systems presented different EE values:
	Kalimouttou and colleagues (2008) prepared PM-loaded PLGA
	nanoparticles by nanoprecipitation method and evaluated EE varying polymer
	and solvent amounts. The very low EE measured, lower than 4%, led the authors
	to conclude that was not possible to encapsulate PM in the nanoparticles.
	Ghadiri and co-workers (2011) produced solid lipid nanoparticles
	containing PM with EE of 39%.
	In another study performed by Jaafari and co-workers (2009), PM-loaded
	liposomes were produced by fusion method. The EE, measured by two different
	methods (direct and indirect), remained around 60%.
	Khan and Kumar (2011a) prepared spray-dried PM-loaded Albumin
	microparticles with 1:9 drug/polymer ratio and a very high EE of approximately
	100%.
	Gaspar and colleagues (2015) prepared different PM-loaded liposomes by
	dehydration and rehydration technique, varying the lipid composition among
	microcapsules varied between 32.1% and 80.3%. The drug loading in all PM-MC

Table 15

 15

	presents the thermal properties of pure PLGA and PM-loaded
	PLGA microspheres (PM-MS06) and microcapsules (PM-MC12).

Table 15 . Thermal properties of pure PLGA, PM-MS06 and PM-MC12.

 15

	Batch 1	1st Heating Cycle	2nd Heating Cycle
		Tg (°C)	ΔCp J/g. °C	Tg (°C)	ΔCp J/g. °C
	PLGA	40.80	0.4726	42.24	0.5390
	PM-MS06	39.79	0.4974	41.33	0.4533
	PM-MC12	44.08	0.4925	45.68	0.4044

Table 16

 16 shows the thermal properties of PM-MC and MC (blank) batches prepared with PLGA/PLA blends. PM-MC22 prepared only with PLA showed the highest Tg (58.89ºC in the first cycle) close to the Tg of pure PLA (59.81ºC).

	PLGA-PLA microparticles showed higher Tg than PLGA microcapsules, which
	could represent an improvement of physical stability under storage conditions.

Table 16 . Thermal properties of PM-MC and MC (blank) batches prepared with PLGA/PLA as polymeric matrix.

 16

	Batch	1st Heating Cycle	2nd Heating Cycle	T cryst	ΔH cryst	T	ΔH
		Tg (°C)	ΔCp J/g.	Tg (°C)	ΔCp J/g.	(°C)	J/g. °C	fusion	fusion
			°C		°C			(°C)	J/g. °C
	PLGA	40.80	0.47	42.24	0.54	-	-	-	-
	PLA	59.81	0.45	61.90	0.22	68.54	18.36	170.88	58.02
	PM-MC19	43.84	0.59	44.14	0.56	-	-	170.83	2.08
	PM-MC20	45.07	0.57	44.56	0.56	-	-	169.96	5.26
	PM-MC21	45.88	0.34	43.56	0.39	74.73	1.523	170.23	16.99
	PM-MC22*	58.89	0.34	67.51	0.12	69.14	11.22	170.64	37.65
	MC23	41.92	1.16	43.19	0.62	-	-	169.16	3.05
	MC24	42.93	0.51	42.81	0.55	-	-	169.02	6.83
	MC25	46.01	0.42	44.91	0.35	66.74	8.110	169.99	24.76

Legend: * Batch prepared only with PLA

	Miscibility between any two polymers in the amorphous state is detected
	by the presence of a single glass-transition temperature (Tg) intermediate
	between those of the two component polymers. Immiscibility of two polymers is
	demonstrated by the retention of the Tg values of both individual components. In
	Figure 75, the PLGA-PLA blend exhibit a single Tg, as all studied blends. As
	shown in Table

Table 17 . Particle size distribution and Span of some PM-MC batches.

 17

	Batch	d10 (µm)	d50 (µm)	d90 (µm)	Span*
	PM-MC12	2.24 ± 0.06	5.77 ± 0.17	19.5 ± 1.13	3.00 ± 0.10
	PM-MC13	2.59 ± 0.01	7.26 ± 0.03	21.7 ± 0.21	2.62 ± 0.05
	PM-MC14	1.76 ± 0.00 3.38 ± 0.01	6.53 ± 0.02	1.41 ± 0.00
	PM-MC15	2.17 ± 0.01	5.58 ± 0.02	20.4 ± 0.64	3.30 ± 0.09
	PM-MC16	2.95 ± 0.03	12.6 ± 0.28	44.6 ± 2.55	3.29 ± 0.12
	PM-MC17	4.19 ± 0.02	23.7 ± 0.28	67.2 ± 0.85	2.66 ± 0.00
	PM-MC18	3.78 ± 0.03	20.9 ± 0.14	62.7 ± 0.64	2.82 ± 0.06

Legend: * -Span = (d90 -d10)/d50

Table 17

 17

	shows that batches prepared with PM dissolved in water (PM-
	MC12, PM-MC13, PM-MC14 and PM-MC15) had smaller mean particles size (d50
	< 7.3 µm) than batches prepared with PM dispersed in acetone (PM-MC16, PM-
	MC17 and PM-MC18) (d50 > 12 µm).
	PM-MC12 presented results very similar to PM-MC15, its replicate.
	However, comparing PM-MC16 and its replicate (PM-MC18), it was possible to
	observe different results. The trouble of particles agglomeration and difficulty of
	particles dispersion probably explain the difficulty to reproduce particle size and
	span results. This finding indicates that PM dissolved in water provides smaller
	microcapsules than PM dispersed in organic solvent.

Table 18 . Composition of PM-MS/PM-MC batches selected and their characterization Batch 1 Composition of batches Characterization

 18

		Inner solution	Outer solution
		PM (g) Solvent/	Polymer	Solvent/	Yield	EE 2	DL 3
			Volume	(g)	Volume	(%)	(%)	(%)
			(g)		(g)
	PM-MS06	0.65	-	1.5 g	97.85 g	35.30 23.01 6.90
				PLGA	ACE 4
	PM-MC15	1.03	49.97 g	2.4 g	157.60 g	60.65 75.27 22.58
			Water	PLGA	ACE 4
	PM-MC18	0.77	29.23 g	1.8 g	118.20 g	40.32 64.81 21.94
			ACE 4	PLGA	ACE 4
	PM-MC20	1.03	49.97 g	2.16 g	137.60 g	32.11 60.29 18.09
			Water	PLGA /	ACE 4 /
				0.24 g	20.00 g
				PLA	DCM 5

Table 19 . % PM released per hour of released test and dissolution efficiency after 4 hours (DE4h)

 19

	Batch 1		Time (h)		DE4h
		1	2	4
	Free PM	76.62 ± 4.75	90.33 ± 1.86	90.60 ± 2.46	75.68
	PM:	71.77 ± 9.58	94.72 ± 10.06	98.35 ± 4.89	78.05
	PLGA
	physical
	mixture
	PM-MS06	70.30 ± 2.37	83.91 ± 1.82	84.14 ± 3.50	70.08
	PM-MC15	66.13 ± 1.98	98.68 ± 2.87	100.00 ± 1.58	78.54
	PM-MC18 78.34 ± 12.24	98.88 ± 2.87	100.00 ± 1.58	81.66
	PM-MC20	30.32 ± 0.35	30.11 ± 0.33	34.35 ± 0.42	27.46

Table 20 . Kinetic parameters of PM-MS/PM-MC batches using Korsmeyer-Peppas model.

 20

	Batch	Korsmeyer-Peppas model
		K (min -1 )	R 2	n	AIC 1	RMSE 2
	PM-MS06	41.19	0.9987	0.14	18.44	3.04
	PM-MC15	25.02	0.9992	0.26	25.11	7.00
	PM-MC18	41.15	0.9989	0.17	21.10	4.24
	PM-MC20	17.37	0.9986	0.12	10.38	1.11

Legend:

1 Akaike Information Criterion calculated from Equation 3, Table 10 (item 4.3.2.4.1); 2 Root Mean Squared Error calculated from Equation 2, Table 10 (item 4

.3.2.4.1).

Table 21 . In vitro antileishmanial activity and cytotoxicity results for PM- MC formulations (CC50, IC50 values and Selective Index of free PM and PM-MC batches) against L. amazonensis promastigotes

 21

	Formulation	CC50 *	IC50 **	Selective Index
				(SI) ***
	PM	2653 µg/mL	7.5 µg/mL	353.73
	PM-MC12	308 µg/mL	7.5 µg/mL	41.06
	PM-MC20	597 µg/mL	11.8 µg/mL	50.59
	MC24	5437 µg/mL	>3250 µg/mL	<1.67

Legend:

*-concentration in µg/mL cytotoxicity for 50% of BMDM; ** -concentration in µg/mL which reduces 50% of viability of L. amazonensis promastigotes; *** -Selective Index: CC50/IC50.

Micropartículas, Misturas Poliméricas, PLGA, PLA, Spray Drying

Figure 82. PM-MC15 microcapsules, before and after contact with water for 24h.

Figure 83. PM-MC18 microcapsules, before and after contact with water for 24h.

ACKNOWLEDGEMENTS

First, I would like to thank God for give me the opportunity to develop this project, for guide me during all these years and for give me health and strength to make this project real.

I wish to dedicate my gratefulness to my parents Ana Cristina and Paulo Roberto,

EE -Encapsulation Efficiency

EMA -European Medicines Agency

FBS -Fetal Bovine Serum

FDA -Food and Drug Administration

Fiocruz -Fundação Oswaldo Cruz

BIOLOGICAL ASSAYS

The free AmB, free clove oil and four AmB nanoemulsions (B, F, H and L) were submitted to cytotoxicity and antileishmanial study.

Assessment of cytotoxicity of AmB nanoemulsions in BMDM cells

The cytotoxicity results of free AmB and AmB nanoemulsions are shown in Figure 45.

% Aspiration

There are some types of nozzles, which can be used for spray drying, such as rotary atomizer, pressure nozzle, ultrasonic nozzle and pneumatic nozzle [START_REF] Patel | Revealing facts behind spray dried solid dispersion technology used for solubility enhancement[END_REF][START_REF] Sunderland | Application of a novel 3-fluid nozzle spray drying process for the microencapsulation of therapeutic agents using incompatible drug-polymer solutions[END_REF]KAŠPAR, JAKUBEC, ŠTĚPÁNEK, 2013a;[START_REF] Paudel | Manufacturing of solid dispersions of poorly water-soluble drugs by spray-drying: Formulation and process considerations[END_REF]CAL & SOLLOHUB, 2010).

Challenges in improving drug loading of PLGA microparticles with acceptable control over release rate profiles

The encapsulation of hydrophilic drugs in PLGA microparticles has some challenges as low encapsulation, high initial burst drug release [START_REF] Han | Bioerodable PLGA-based microparticles for production sustained-release drug formulations and strategies for improving drug loading[END_REF].

Most studies came mainly in an attempt to overcome drawbacks related to the conventional W1/O/W2 double-emulsion method in conventional emulsificationevaporation methods (ABULATEEFEH & ALKILANY, 2016).

When using a spray drying process, different nozzles can be employed for droplets formation. In two-fluid nozzles, only one liquid feed (containing drug and polymer) is atomized through the nozzle orifice. The name 'two-fluid' is due to the use of a second fluid (gas) in the nozzle flowing through a second passage and

Differential Scanning Calorimetry (DSC)

DSC analysis of PM powders were performed using an equipment DSC-Q200 (TA Instruments, USA) in modulated mode (MDSC) at heating rate of 2 ºC/min under dry nitrogen atmosphere (50 mL/min). Approximately 7 mg of sample were weighed and placed into standard non-hermetic aluminum pan. The heating temperature ranged from 25 ºC to 200 ºC following adapted method

Scanning Electron Microscopy (SEM)

The PM-MS/PM-MC samples images were performed using scanning electronic microscope (Philips XL30 ESEM-FEG, Netherlands) following method described previously in item 4.3.2.1.1.

Differential Scanning Calorimetry (DSC)

DSC analysis of PM-MS/PM-MC samples were performed using an equipment DSC-Q200 (TA Instruments, USA) in modulated mode (MDSC)

following method already described previously in item 4.3.2.1.2.

Drug Content, Encapsulation Efficiency and Process Yield

The PM concentration in the microparticles was determined by UV-vis (UVvis model V-630, Jasco, Brazil) spectrophotometric method using PM derivatization with 1-fluoro-2,4dinitrobenzene (DNFB) (Figure 59) in distilled water as a solvent at 415 nm (adapted from RYAN,1984) particles agglomeration is related to low Tg of the polymer.

Another option is a solvent mixture as proposed by Wan and colleagues (2013a, b). They tested different proportions of two organic solvents (acetone and methanol) to produce celecoxib PLGA spray-dried microparticles using gas inlet and outlet temperature of 45ºC and 30ºC, respectively. The SEM images showed microparticles with spherical and smooth surface.

Ethyl acetate has been used as solvent to produce Etanidazole-PLGA spray-dried particles with lower temperatures than that used in our tests. The authors kept the dryer outlet temperature in the range 40-46ºC and also reported that the dry product stuck on the cyclone wall leading to low yields [START_REF] Wang | Sustained release of etanidazole from spray dried microspheres prepared by non-halogenated solvents[END_REF].

Therefore, based on the particles shape and yield of PM-MS batches with acetone and ethyl acetate, acetone was selected as solvent for PLGA-based polymers used in this work to produce PM-loaded polymeric microparticles.

Nozzle Orifice Diameter Effect

Other parameter evaluated during the development of PM-loaded PLGA microspheres was the nozzle orifice diameter, as indicated in Table 12. Generally, in a two-fluid nozzle, droplet size is a function of liquid formulation type, liquid flow rate, atomization gas flow rate and liquid orifice diameter (RÉ, MESSIAS, [START_REF] Re | The influence of the liquid properties and the atomizing conditions on the physical characteristics of the spray-dried ferrous sulfate microparticles[END_REF].

The literature gives some information about particle size of spray-dried PLGA microparticles. Lin and co-workers (2005) evaluated the particle size distribution of spray-dried doxorubicin-loaded PLGA microparticles using a similar laser diffraction method adopted to PM-MS batches. They analyzed particles dispersed in deionized water under sonication and obtained particles in the range of 2-5 µm. Wan and co-workers (2014b) investigated the particle size distribution of spray-dried bovine serum albumin-loaded PLGA microparticles using a laser diffraction method. The particles were dispersed in water under sonication before measurement and got particles with d50 between 5 and 11 µm and span in the range 1-2.

Wang and co-workers (2015) evaluated the particle size distribution of gycyrrhetinic acid PLGA microparticles obtained by oil-water emulsion. The particles were dispersed in water containing 0.5% of PVA and 0.1% of Tween ® 80 under sonication and analyzed by laser diffraction method. They produced particles with mean size around 40 µm, which are bigger than microparticles obtained by spray-drying. Spray drying is seen as a technique available to reduce and control particle size compared to techniques such as emulsification-solvent evaporation also used to produce drug-loaded PLGA microparticles (SOLLOHUB & CAL, 2010).

IN VITRO RELEASE OF PAROMOMYCIN POLYMERIC

MICROPARTICLES

Based on the characteristics of the batches produced in this study, the choice of PM-MS/PM-MC formulations was made for in vitro release study.

The batches selected were PM-MC15 (PM-MC12 replicated), PM-MC20, PM-MC18 and PM-MS06. Free drug and a physical mixture of PM and PLGA (30:70) were also subjected to the same in vitro test. For remembering, Table 18 summarizes the initial composition of PM-MS/PM-MC batches selected and some characteristics of the products.

Antileishmanial Activity

The antileishmanial activity against L. amazonensis promastigotes was evaluated with the same PM-MC samples tested in cytotoxicity assay and the results are shown in Figure 87.