Supharat Inphonlek

Panya Sunintaboon

Michèle Leonard

Alain Durand
email: alain.durand@univ-lorraine.fr

Chitosan/carboxymethylcellulose-stabilized poly(lactide-co-glycolide) particles as biobased drug delivery carriers

Keywords: Chitosan, Carboxymethylcellulose, Polyelectrolyte complex, Suspension, Encapsulation, Release kinetics

Poly(lactide-co-glycolide) (PLGA) colloidal particles stabilized by complexes of two oppositely charged polysaccharides, chitosan (cationic, CS) and sodium carboxymethylcellulose (anionic, NaCMC), were fabricated. Dichloromethane containing dissolved PLGA was first emulsified in an aqueous phase containing mixtures of CS and NaCMC. Evaporation of dichloromethane from the resulting emulsion led to CS/NaCMCcovered-PLGA particles. CS and NaCMC contents affected the short-term stability of PLGA particles and also their intrinsic characteristics. The particles displayed pH-dependent characteristic. Zeta potential varied from +54 to -50 mV when pH was varied from 3 to 10. CS/NaCMC-covered-PLGA particles showed colloidal stability, over a wider pH range as compared to CS-covered-PLGA particles. Curcumin, a model hydrophobic drug, was encapsulated into the particles up to 10 wt% of PLGA. The CS/NaCMC-covered-PLGA particles loaded with curcumin showed delayed release in mildly acidic conditions and faster release in neutral and basic conditions. Cytotoxicity experiments were carried out with human colorectal carcinoma cells.

Introduction

Polymeric particles consisting of biodegradable and biocompatible polymers have received much attention for various applications, especially in biomedical and food fields. Indeed, such materials exhibited low toxicity and thus were safe when being interfaced with human tissues or organs. One important example of their usage is for preparing systems for controlled delivery of molecules. In such usage, polymeric particles can be formulated for encapsulating active ingredients or therapeutic agents and carrying them to the target tissues or organs and thus enhancing drug bioavailability. Poly(lactide-co-glycolide) (PLGA, Fig. 1), a FDA-approved biodegradable linear aliphatic polyester, has been extensively used in biomedical applications [START_REF] Liang | Bilayered PLGA/PLGA-HAp Composite Scaffold for Osteochondral Tissue Engineering and Tissue Regeneration[END_REF][START_REF] Mariano | Design of PLGA Based Nanoparticles for Imaging Guided Applications[END_REF]. PLGA is usually prepared under the form of particles, which are used as vehicles for encapsulating and delivering drugs [START_REF] De Moraes Profirio | Formulation of functionalized PLGA nanoparticles with folic acid-conjugated chitosan for carboplatin encapsulation[END_REF][START_REF] Ding | Recent advances of PLGA micro/nanoparticles for the delivery of biomacromolecular therapeutics[END_REF][START_REF] Turino | Ferritin Decorated PLGA/Paclitaxel Loaded Nanoparticles Endowed with an Enhanced Toxicity Toward MCF-7 Breast Tumor Cells[END_REF]. It can be biologically degraded by hydrolysis, providing the controlled release of those molecules for medical treatment. Polymer complexes or coacervates involving biopolymers have been showed to efficiently encapsulate and release drugs. [START_REF] Jain | Perspectives of Biodegradable Natural Polysaccharides for Site-Specific Drug Delivery to the Colon[END_REF][START_REF] Nasery | Curcumin Delivery Mediated by Bio-Based Nanoparticles: A Review[END_REF][START_REF] Situ | Resistant Starch Film-Coated Microparticles for an Oral Colon-Specific Polypeptide Delivery System and Its Release Behaviors[END_REF] Nevertheless, the use of aliphatic polyesters increased the possibilities of encapsulating hydrophobic drugs. However, because of the hydrophobicity of PLGA, its use under the form of dispersed particles raised several difficulties as keeping colloidal stability or preventing the action of immune system which would shorten their residence time.

Modification of PLGA particles was accomplished by introducing various hydrophilic polymers, among which chitosan [START_REF] Chen | Surface modification of PLGA nanoparticles with biotinylated chitosan for the sustained in vitro release and the enhanced cytotoxicity of epirubicin[END_REF][START_REF] Li | Preparation of biocompatible chitosan grafted poly(lactic acid) nanoparticles[END_REF][START_REF] Miele | Chitosan Oleate Salt as an Amphiphilic Polymer for the Surface Modification of Poly-Lactic-Glycolic Acid (PLGA) Nanoparticles. Preliminary Studies of Mucoadhesion and Cell Interaction Properties[END_REF][START_REF] Wang | Fabrication of monodisperse drugloaded poly(lactic-co-glycolic acid)-chitosan core-shell nanocomposites via Pickering emulsion[END_REF], poly(ethylene glycol) [START_REF] Devulapally | Gemcitabine and Antisense-microRNA Co-encapsulated PLGA-PEG Polymer Nanoparticles for Hepatocellular Carcinoma Therapy[END_REF][START_REF] Fernandes | PEGylated PLGA Nanoparticles As a Smart Carrier to Increase the Cellular Uptake of a Coumarin-Based Monoamine Oxidase B Inhibitor[END_REF][START_REF] Parveen | Long circulating chitosan/PEG blended PLGA nanoparticle for tumor drug delivery[END_REF][START_REF] Saneja | Gemcitabine and betulinic acid co-encapsulated PLGA-PEG polymer nanoparticles for improved efficacy of cancer chemotherapy[END_REF], or polyethyleneimine [START_REF] Park | Neoangiogenesis of human mesenchymal stem cells transfected with peptide-loaded and gene-coated PLGA nanoparticles[END_REF][START_REF] Yu | Enhanced delivery of Paclitaxel using electrostatically-conjugated Herceptin-bearing PEI/PLGA nanoparticles against HER-positive breast cancer cells[END_REF].

Chitosan (CS, Fig. 1) is a natural and non-toxic polymer with convenient properties and great potential in biomedical and food applications. CS comprises amine groups in its chemical structure, which are produced during deacetylation of chitin. According to its structure (particularly its deacetylation degree and its molar mass distribution) CS could be used as stabilizer for colloidal particles [START_REF] Caon | Chitosan-decorated polystyrene-b-poly(acrylic acid) polymersomes as novel carriers for topical delivery of finasteride[END_REF][START_REF] Inphonlek | Synthesis of poly(methyl methacrylate) core/chitosan-mixed-polyethyleneimine shell nanoparticles and their antibacterial property[END_REF]. Surface active properties of CS could be improved by covalent attachment of hydrocarbon groups [START_REF] Babak | Dynamic surface tension and dilational viscoelasticity of adsorption layers of alkylated chitosans and surfactant-chitosan complexes[END_REF]. Alternatively, the positively charged CS can interact with negatively charged components via electrostatic interaction forming noncovalent complexes which exhibit convenient stabilizing properties [START_REF] Anal | Biopolymeric-based emulsions and their effects during processing, digestibility and bioaccessibility of bioactive compounds in food systems[END_REF][START_REF] Babak | Dynamic surface tension and dilational viscoelasticity of adsorption layers of alkylated chitosans and surfactant-chitosan complexes[END_REF]. The use of electrostatic complex between cationic CS and anionic surfactant, sodium dodecyl sulphate (SDS), has been reported as efficient for stabilizing oil-in-water (O/W) emulsions [START_REF] Milinković | Interfacial and emulsifying properties of chitosan/sodium lauryl ether sulfate system[END_REF][START_REF] Pérez-Calderón | Optimal clarification of emulsified oily wastewater using a surfactant/chitosan biopolymer[END_REF][START_REF] Sunintaboon | One-step preparation of chitosan/sodium dodecyl sulfate-stabilized oil-in-water emulsion of Zingiber cassumunar Roxb. oil extract[END_REF]. Alternatively non-covalent complexes involving biological macromolecules like proteins or polysaccharides have been reported as relevant formulation alternatives [START_REF] Bouyer | Proteins, polysaccharides, and their complexes used as stabilizers for emulsions: Alternatives to synthetic surfactants in the pharmaceutical field?[END_REF].

Sodium carboxymethylcellulose (NaCMC, Fig. 1) is an anionic polysaccharide amongst the most commonly applied biopolymers in food, cosmetic or phamaceutical applications [START_REF] Cai | Impact of distribution of carboxymethyl substituents in the stabilizer of carboxymethyl cellulose on the stability of acidified milk drinks[END_REF][START_REF] Mandal | In Situ Silver Nanowire Deposited Cross-Linked Carboxymethyl Cellulose: A Potential Transdermal Anticancer Drug Carrier[END_REF][START_REF] Park | A novel pH-responsive hydrogel based on carboxymethyl cellulose/2-hydroxyethyl acrylate for transdermal delivery of naringenin[END_REF][START_REF] Rakhshaei | A potential bioactive wound dressing based on carboxymethyl cellulose/ZnO impregnated MCM-41 nanocomposite hydrogel[END_REF]. It is prepared from cellulose by covalent attachment of carboxylic groups along its backbone. The use of NaCMC as stabilizer to prevent the aggregation of iron sulfide (FeS) particles has been reported [START_REF] Gong | Immobilization of Mercury by Carboxymethyl Cellulose Stabilized Iron Sulfide Nanoparticles: Reaction Mechanisms and Effects of Stabilizer and Water Chemistry[END_REF]. The adsorption of NaCMC macromolecules onto FeS nanoparticles led to a strong negative zeta potential around -43 to -68 mV, providing the colloidal stability through electrostatic repulsion. In addition, NaCMC can form electrostatic complexes with oppositely charged proteins, such as whey protein and casein. Such complexes have been used as stabilizers and improved shelf life and stability of emulsions in food applications [START_REF] Estévez | Encapsulation of grape seed phenolic-rich extract within W/O/W emulsions stabilized with complexed biopolymers: Evaluation of their stability and release[END_REF]. Oil-in-water Pickering emulsions were prepared using chitosan hydrochloride/carboxymethylstarch complex nanogels as stabilizers [START_REF] Li | Chitosan hydrochloride/carboxymethyl starch complex nanogels as novel Pickering stabilizers: Physical stability and rheological properties[END_REF]. The nanogels formed a superficial layer onto oil droplets and the resulting emulsions exhibited long-term stability over 3 months of storage. Emulsion stability and average droplet size were closely related to pH which influenced zeta potential. CS/NaCMC complexes were also used for stabilizing paraffin oil emulsion. [START_REF] Roy | Chitosan-Carboxymethylcellulose-Based Polyelectrolyte Complexation and Microcapsule Shell Formulation[END_REF] Additionally composite beads have been prepared on the basis of electrostatic interactions between ZnO nanoparticles, NaCMC and CS, and used as drug carriers for 5-fluorouracil (5-FU) [START_REF] Sun | pHsensitive ZnO/carboxymethyl cellulose/chitosan bio-nanocomposite beads for colon-specific release of 5-fluorouracil[END_REF]. Such beads could be applied as pH-sensitive carriers for treatment of colonic cancer.

In this work, PLGA particles were prepared by a two-step procedure including emulsification and solvent evaporation. Ionic complexes between CS and NaCMC were used as stabilizers during the first step. CS/NaCMC complexes were showed to provide surface anchoring, excluded volume and more specific functionality like pH-sensitivity. The effect of CS and NaCMC contents on various characteristics of PLGA particles was studied and discussed on the basis of polymer properties. The obtained particles were evaluated regarding encapsulation and release of curcumin (CUR, Fig. 1) which was selected as a model hydrophobic drug. Preliminary cytotoxicity experiments were carried out.

Materials and methods

Materials

Chitosan (CS; molecular weight of 150,000 g/mol, and 85% deacetylation) was obtained from Seafresh Lab. Poly(lactide-co-glycolide) (PLGA; lactide:glycolide ratio of 50:50, and molecular weight of 38,000 g/mol), sodium carboxymethylcellulose (NaCMC; commercial "low viscosity" sample), and curcumin (CUR) were purchased from Sigma-Aldrich. Stock solutions of CS (5000 ppm CS solution in 1% v/v acetic acid in water) and NaCMC (2000 ppm NaCMC solution in water) were prepared. Deionized water was used throughout the study. All other chemicals were commercial products of analytical grade.

Aggregation behavior between CS and NaCMC

Conductivity measurement

Various solutions of CS and NaCMC were mixed together in a beaker before conductivity measurement. The concentration of CS solution was varied as 50, 100, 150, 300, 450 and 600 ppm. Each solution of CS was mixed with NaCMC solutions in which NaCMC concentration was varied as 50, 100, 150, 300, 500, 700, 1000, and 1500 ppm. The conductivity of each mixture was measured at room temperature using a handheld conductometer (EC100, Extech).

Potentiometric titration of CS and NaCMC

The number of amine groups of CS and carboxylic groups of NaCMC, as well as "apparent" pKa values were experimentally determined by potentiometric titration using 100 mM NaOH solution. The number of amine groups of CS and carboxylic groups of NaCMC were found to be 4.2 10 -3 mol.g -1 and 2.8 10 -3 mol.g -1 , respectively. The "apparent" pKa values of CS and NaCMC were found equal to 6.1 and 4.1, respectively (Fig. A.1). These values were consistent with previous data from literature giving "apparent" pKa values of 6.3 for CS and 4.6 for NaCMC [START_REF] Peter | Applications and Environmental Aspects of Chitin and Chitosan[END_REF][START_REF] Vleugels | Determination of the 'apparent pKa' of selected food hydrocolloids using ortho-toluidine blue[END_REF].

Surface tension measurement

The surface tension of the CS/NaCMC aqueous mixtures was measured using the K20 EasyDyne tensiometer (Kruss). Before any measurement, the glass vessel was washed with sulfuric acid to eliminate the organic traces, and then rinsed with water and acetone before drying. 20 mL of each aqueous mixture of CS and NaCMC at different concentrations was added into the glass vessel and stored at 4 °C overnight. Then the sample was carefully removed without causing agitation and was allowed to return to room temperature. The measurements were carried-out at 25 °C with the Du Nouy ring method.

Preparation of chitosan/carboxymethylcellulose-stabilized poly(lactide-co-glycolide) (CS/NaCMC-covered-PLGA) particles

The CS/NaCMC-covered-PLGA particles were prepared following the emulsification-solvent evaporation procedure. Firstly, 3 mL of dichloromethane containing 100 mg of PLGA was mixed with various aqueous solutions of NaCMC (50-700 ppm or 1.25-17.50 mL) and CS (50-600 ppm or 0.50-6.00 mL) into a glass bottle. The total volume of the (biphasic) mixture was adjusted to 50 mL by adding deionized water. The mixture was ultrasonicated for 3 min at the amplitude of 50% (Bandelin Sonoplus HD2200) to form oil-in-water emulsion. An ice bath was used for heat dissipation. Dichloromethane was then evaporated under stirring at ambient temperature during 5 h to obtain CS/NaCMC-covered-PLGA particles.

Characterization

Size distribution of suspensions of particles

The size distribution of the prepared particle suspensions was determined by laser light scattering. The measurement was performed in the wet mode of particle size analyzer (Mastersizer 2000, Malvern) using distilled water as a medium. The background was run before measurement and the sample was then added into the chamber.

For all measurements, a small amount of sample was added in the container filled with 500 mL of dispersing liquid (distilled water). The particle suspension was diluted with high amount of distilled water, so the pH of the measurement was controlled by pH of water (pH around 5.8-6.0).

The values of d(0.1), d(0.5), and d(0.9), the diameters at 10%, 50%, and 90% cumulative volumes, respectively, were used for characterizing size distributions. The broadness of the size distribution was evaluated by calculating the Span value (Eq. (1)).

Span=

d 0.9 -d(0.1) d(0.5) (1)

SEM analysis

The morphology of the prepared particles was observed by using scanning electron microscope (SEM, SU8010, Hitachi) at 15 kV. A drop of diluted particle latex was dried on an aluminum sheet overnight. The dried sample was coated with platinum and palladium under vacuum using a sputter coater (E-102, Hitachi).

Colloidal stability of the particle suspensions

The colloidal stability of the particle suspensions was examined by the method called turbidimetry either in the presence of various salt concentrations or as a function of pH [START_REF] Long | The equilibrium aspects of weak flocculation[END_REF].

The concentration of NaCl was varied from 10 -4 to 4 M. The pH was varied as 4, 6, 8, and 10, in the presence of 1 mM NaCl. The particle suspensions used in these experiments were prepared by the emulsification/solvent evaporation method (section 2.3) with a given CS concentration equal to 450 ppm and different concentrations of NaCMC ranging from 50 to 300 ppm. The 600 µL of particle suspension was added to 3 mL of NaCl solution at the convenient pH, well mixed, and left for 1 h before measurement.

The absorbance of the samples was measured over the range of 400 to 700 nm, using UVvisible spectrophotometer (Lambda 35, Perkin Elmer). The colloidal stability of particle suspensions was determined by calculating the index denoted |n|, which was the absolute value of the slope of the linear variation observed when plotting logOD vs. logλ, over the previous range of wavelength. For suspensions with average particle diameters below 1 µm, the value of |n| remained around 1.5 -2. Any increase of the diameter of the dispersed objects, resulting from aggregation of individual particles, induced a decrease of |n| well below 1.

Zeta potential measurement

The zeta potential of prepared particles was determined using Zetasizer (Zetasizer 3000, Malvern Instruments). The measurements were done at 25 °C. The pH of particle dispersions in 1 mM NaCl was adjusted over the range from 3 to 10.

Drug delivery experiments 2.5.1 Miscibility between CUR and PLGA

The differential scanning calorimetry (DSC) analysis was carried out using the Q2000 apparatus (TA Instruments). The heating rate was 5 °C/min under nitrogen and temperature was varied from -20 °C to 200 °C.

The mixture of CUR and PLGA was prepared by dissolving both constituents in 2 mL dichloromethane, and then evaporating the solvent in an oven at 30 °C for 4 h. Then, the recovered sample was further dried under vacuum at 30 °C for 24 h. The miscibility of CUR and PLGA was deduced from the differential scanning calorimetry analysis of solid samples.

Particle size and encapsulation efficiency of CUR-loaded PLGA particles

CUR was encapsulated into the CS-or CS/NaCMC-covered-PLGA particles by emulsification-solvent evaporation as described above (section 2.3). Known amounts of CUR and 100 mg of PLGA were dissolved in dichloromethane. The solution was emulsified in an aqueous solution containing CS and NaCMC by sonication during 30 seconds. Subsequently, dichloromethane was evaporated. The obtained latex was centrifuged at 10,000 rpm for 15 min. The particle size of the CUR-loaded CS-or CS/NaCMC-covered-PLGA particles was characterized using laser light scattering (Mastersizer 2000, Malvern). The measurement was repeated three times at 25 °C for each sample.

Encapsulation efficiency of CUR-loaded CS-or CS/NaCMC-PLGA particles was determined by using nuclear magnetic resonance spectroscopy (NMR, Bruker AM400, 400 MHz). The (2) and ( 3)).

m encapsulated = M CUR M PLGA A 6H CUR 6A 1H PLGA (2) EE% = 100 m encapsulated m initial load (3)
In Eq. ( 2) and (3), A6H CUR was the area of the signal of 6 protons of CUR at 3.83 ppm, A1H

PLGA was the area of the signal of PLGA proton at 5.25 ppm, MPLGA was the molar mass of PLGA repeat unit (130 g.mol -1 ), and MCUR was the molar mass of CUR (368 g.mol -1 ).

Kinetics of drug release

The suspension of CUR-loaded particles was placed in a dialysis bag (CelluSep T4, molecular cut-off 6-8 kDa) which was immersed in 100 mL of aqueous medium containing 3 wt% Tween 80. Tween 80 was added to the release medium due to the poor solubility of CUR in water so as to ensure sink conditions (i.e. to remain far enough from saturation of the external phase by released CUR). The release experiment was performed at three different pHs: 4.0 and 9.0 with 0.9 wt% NaCl, and 7.4 with phosphate buffered saline solution. The external medium was kept under fixed stirring condition (100 rpm) at 37 °C and it was changed once a day. Samples of particle suspension were taken out at various time intervals and washed with water for 3 times. Then, the samples were freeze-dried (Freezone plus 6, Labconco Freeze Dry System) for 24 h. The amount of CUR remaining in the particles was determined using 1 H-NMR. From the found value, the percentage of released CUR relatively to the initial load was estimated.

Cytotoxicity assay 2.6.1 Cell culture

HCT 116 human colorectal carcinoma cells, were kindly provided by collaborators. The cells were propagated in Dulbecco's Modified Eagle Medium (DMEM medium (Gibco, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin (Sigma). Cells were grown in a humidified incubator at 37 °C under 5% CO2/air and used for assays during the exponential phase of growth.

Cell Proliferation Assay by Using Tetrazolium Salt (MTT)

Cell proliferation was assessed by MTT assay (Sigma-Aldrich). Briefly, Cells (8 x 10 3 cells)

were seeded in a 96-wells flat-bottomed microplate at 100 μL overnight before treatment.

After 24 h, the media were removed and replaced with fresh complete medium. Cells were exposed to graded concentrations of tested compounds for 24 h at pH = 7.4. Cells incubated with 0.5 % DMSO (vehicle) were used as a negative control. DMSO concentration was maintained at 0.5 % for all groups. After the incubation period, each well was washed with phosphate-buffered saline (PBS) and replaced with 1 mg/mL MTT for 4 h incubation. The resulting crystals product from MTT assay was dissolved in 100 µL of 100 % DMSO and measured at 550 nm using a microplate reader (Model No; AOPUS01 and A153601; A Packard bioscience company). The experiment was run in triplicate. The percentage of cell viability was calculated (Eq. (4)).

% cell viability = average A550 of experimental group / average A550 of control group (4)

Results and discussion

Study of the aggregation behavior of CS and NaCMC in aqueous solution

The aggregation behavior between CS and NaCMC in aqueous medium was first investigated in order to find the optimal conditions for the preparation of PLGA particles. Conductometric titration has been reported as an efficient method for studying CS-NaCMC interactions in aqueous solutions [START_REF] Rosca | Interaction of chitosan with natural or synthetic anionic polyelectrolytes. 1. The chitosan-carboxymethylcellulose complex[END_REF]. Thus the complex formation between the two oppositely charged species was monitored in situ by measuring the conductivity of the aqueous mixtures as a function of the concentrations of loaded CS (50 to 600 ppm) and added NaCMC (0 to 1500 ppm). For low enough amounts of added NaCMC, the conductivity exhibited, if any, a limited variation. When exceeding a "critical value", the conductivity increased more markedly and followed an almost linear variation with NaCMC concentration (Fig. 2a). This "critical value" of NaCMC concentration was experimentally determined by the abscissa of the intersection point of linear variations (Fig. 2a,inset). This critical value of NaCMC concentration was compared to the critical aggregation concentration (CAC), i.e. the 1:1 stoichiometry of ammonium groups from CS and carboxylate groups from NaCMC calculated on the basis of loaded concentrations and chemical formulas [START_REF] Rosca | Interaction of chitosan with natural or synthetic anionic polyelectrolytes. 1. The chitosan-carboxymethylcellulose complex[END_REF]. It appeared that the "critical concentration" was close to the CAC value or slightly below (Fig. 2b). For NaCMC concentrations lower than CAC, NaCMC addition led to the formation electrostatic complexes, which released sodium chloride salt, and thus had little impact on conductivity. Above CAC, NaCMC macromolecules were in excess and remained in solution thus progressively increasing conductivity. The value of CAC increased almost linearly with loaded CS concentration (Fig. 2b).

These results showed that, for each CS concentration in the load, added amounts of NaCMC such that NaCMC concentration remained below CAC value led to complex formation with an excess of positive charges. These conditions were selected for the rest of this work.

The preparation of oil-in-water emulsions was carried-out using high-energy emulsification step to form oil droplets dispersed in water. Oil droplets could not remain stable in water phase without the accumulation of stabilizers at their surface for preventing aggregation and coalescence. The stabilizer should reduce the interfacial tension and form a physical barrier between the phases, thus providing long enough colloidal stability to the oil droplets. The surface tension of the CS/NaCMC aqueous mixtures was examined as a way to assess the ability of macromolecular species (complexes or single macromolecules in excess) to serve as stabilizers (Fig. 3). increasing CS concentration up to 600 ppm (Fig. 3a). When, the concentration of CS was fixed at 450 ppm and the concentration of NaCMC was varied from 0 to 300 ppm, the surface tension of the aqueous CS/NaCMC mixtures remained close to 59.0 mN/m (Fig. 3b). Upon further increasing the concentration of NaCMC to 500 ppm, precipitation was observed.

Therefore, the surface tension of CS/NaCMC aqueous mixtures was mainly controlled by the amount of CS while NaCMC content exhibited no significant influence in the explored showed that CS adsorption, depending on the concentration range, may last several hours [START_REF] Babak | Dynamic surface tension and dilational viscoelasticity of adsorption layers of alkylated chitosans and surfactant-chitosan complexes[END_REF]. Nevertheless, for the preparation of PLGA particles, emulsification step involved the creation of interfaces by sonication during not more than 3 min. Thus, even if our measurements proved the adsorption of macromolecular species at interfaces, the exact kinetics of this phenomenon was not evaluated. Detailed investigation of the preparation of oil-in-water emulsions, which were the precursors of PLGA particle suspensions, was carried-out.

Preparation of CS/NaCMC-stabilized PLGA particles

On the basis of previous results, the preparation of PLGA particles in the presence of CS/NaCMC mixtures and following emulsification-solvent evaporation technique was further investigated (Fig. 4). Control experiments were carried-out using either CS or NaCMC alone. No stable emulsion could be formed after sonication when using NaCMC as the sole polymer and consequently no particles were produced. This was consistent with surface tension measurements indicating the absence of any interfacial adsorption of NaCMC and thus its incapacity to stabilize initial emulsion (see section 3.1). On the contrary, suspensions of particles were obtained using CS alone in the aqueous phase as could be anticipated from surface tension data.

The visual aspect of the obtained CS/NaCMC-PLGA particles prepared by varying the concentrations of CS and NaCMC confirmed that the procedure was successful over a range of CS/NaCMC concentrations (Fig. 5). With 50 ppm of CS, milky white latex of PLGA particles dispersed in the aqueous medium was formed when NaCMC was used at 50 ppm.

When the concentration of NaCMC was increased up to 100 ppm, unstable latex was obtained.

Unstable latex products were obtained for conditions close to CAC point, as determined by the conductivity measurement (section 3.1). However, when further increasing the concentration of NaCMC from 150 to 700 ppm, milky white latexes were obtained again. For CS concentrations equal to 100, 150, 300, 450, and 600 ppm, similar behaviors were observed. CS/NaCMC-stabilized PLGA particles prepared from the conditions corresponding to an excess of positive charges from CS were selected for further characterization.

CS

Characterization of CS/NaCMC-stabilized PLGA particles

Particle size distribution of prepared suspensions

The size distributions of the suspensions exhibiting sufficient colloidal stability after solvent evaporation were further characterized using laser granulometry (Table 1). For all the explored conditions, aggregate-free suspensions of particles could be prepared with CS/NaCMC mixtures provided that the composition was far enough from CAC. Indeed, close to CAC, most of ionic groups of CS and NaCMC were involved into 1:1 electrostatic interactions. As a result, there were not enough free polymeric sequences for generating electrostatic or steric repulsions between particles, which resulted in the production of high amounts of aggregates. However, keeping NaCMC at 50 ppm, and increasing CS from 150 to 20 600 ppm, allowed producing stable PLGA particles ( The data obtained by laser granulometry were confirmed by SEM observations (Fig. 6). The suspensions used for SEM observations were prepared by keeping the concentration of CS at 450 ppm and increasing the concentration of NaCMC from 50 to 700 ppm (Table 1, lines 5 and 7-11). These conditions were selected because, according to laser granulometry, they led to particle suspensions that could be divided into two groups, one exhibiting rather narrow size distribution (from 50 to 150 ppm NaCMC) and the other containing significant amounts of micrometric aggregates. Such differences should be detected by SEM observations.

For the lower concentrations of NaCMC (50-150 ppm), no aggregates were observed and particles with spherical shape were evidenced (Fig. 6A-C). Formation of aggregates was detected on SEM micrographs for the highest NaCMC concentrations (Fig. 6E-F In what follows, the majority of the experiments used CS450/NaCMC100 load composition for particle preparation as it led to PLGA particles with rather well-defined size distribution and the 450 ppm concentration of CS allowed preparing PLGA particles with CS alone, which was very useful for comparison and interpretation of results.

Surface coverage of particles

The change of zeta potential of the particles upon varying pH value was investigated to study the surface coverage of the particles (Fig. 7a). The zeta potentials of the CS450/NaCMC100-PLGA particles at different pHs were examined and compared to CS450-PLGA particles (i.e.

PLGA particles prepared using 450 ppm of CS as the sole stabilizer in the aqueous phase). In order to discard any artifact caused by non-adsorbed polymers present in the aqueous phase, freshly prepared PLGA particles were centrifuged, washed and redispersed in aqueous media with varying pHs. For the CS450-PLGA particles, positive surface charge was measured in acidic conditions (the maximum positive zeta potential was around +58 mV at pH 3.5). The zeta potential gradually decreased upon increasing the pH, and then reached slightly negative values (-11 mV at pH 10). For pH values between 7 and 8, zeta potential was close to 0. These observations were fully consistent with the assumption that CS macromolecules were covering PLGA particles and with the "apparent" pKa value of 6.3 that was found by potentiometric titration. In acidic environment, the particles exhibited positive surface potentials because the amine groups of chitosan on particle surface were protonated.

Increasing the pH values, the zeta-potential decreased because of the neutralization of ammonium groups. The slightly negative value measured at pH 10 (-11 mV) was attributed to carboxylic end groups of PLGA accumulated at the surface of particles. In the case of CS450/NaCMC100-PLGA particles, the trend was similar that observed with CS450-PLGA particles. Positive values were measured in acidic medium, with the maximum positive zeta potential of +54 mV at pH 3.5. This was due to the protonation of excess amine groups of chitosan adsorbed on the PLGA particles. However, the positive values were lower than that of CS-PLGA particles in the 5-6 pH range, which could be explained by charge cancellation triggered by partial electrostatic complexation. Upon increasing the pH above 6.2, the zeta potential was decreased down to -50 mV at pH 10. These highly negative values could only result from the presence of NaCMC at the surface of particles. More precisely, our assumption was that, upon increasing pH, deprotonation of ammonium groups led to partial dissociation of ions pairs and thus to the release of anionic carboxylic groups in dangling NaCMC sequences at the surface of the particles. These results demonstrated that electrostatic complexes between CS and NaCMC were located onto the surface of PLGA particles. CS sequences were responsible from the adsorption onto hydrophobic PLGA surface while electrostatic complexation between ammonium and carboxylate groups allowed the anchoring of NaCMC macromolecules as an outer layer onto oil droplets. Such ion pairs remained even at pH 10 despite deprotonation of a majority of ammonium groups from CS.

Thus the use of CS/NaCMC complexes appeared as an efficient strategy to adjust surface characteristics of PLGA particles. In addition, zeta potential measurements demonstrated that, in the presence of CS/NaCMC mixtures, electrostatic complexes were adsorbed at the surface of particles (while surface tension measurements did not allow identification of species adsorbing at air/water interface, see section 3.1).

Colloidal stability of suspensions vs. pH and ionic strength

The colloidal stability of particle suspensions was examined using turbidimetry as a function of pH and ionic strength.

First, CS450-PLGA and CS450/NaCMC100-PLGA particle suspensions were compared as a function of pH (Fig. 7b). Both suspensions exhibited good colloidal stability in acidic media, owing to electrostatic repulsions from CS at the particle surface. However, the CS450-PLGA particles were not stable for pH above 8. This was explained by the neutralization of ammonium groups leading to insufficient electrostatic repulsions between particles. On the contrary, PLGA particles covered with CS/NaCMC complex exhibited colloidal stability in neutral and basic conditions (close to pH 10). This was explained by negative charges of NaCMC sequences which were predominant at the surface of particles above pH 6. This confirmed that the biopolymer CS/NaCMC complex enhanced colloidal stability of PLGA particles at high pH values.

In order to utilize these particles as delivery carriers, the influence of ionic strength on their colloidal stability is an important factor because salts are generally found in the body fluid as well as in blood. PLGA particles were prepared from CS at 450 ppm and NaCMC from 50 to 300 ppm. The colloidal stability of suspensions was examined in the presence of NaCl concentrations ranging between 10 -4 and 4 mol.L -1 (Fig. 8).

Fig. 8 Colloidal stability of the CS/NaCMC-PLGA particles as indicated by the variation of |n| index with NaCl concentration ranging from 10 -4 M to 4 mol.L -1 (for detail see section

2.4.3).

The CS/NaCMC-PLGA particle suspensions prepared with NaCMC loaded concentration between 50 and 150 ppm exhibited colloidal stability up to 1 mol.L -1 NaCl. The reduction in colloidal stability was explained by the screening of the electrostatic interactions by salt, leading to insufficient repulsions between particles [START_REF] Ding | Impact of pH, ionic strength and chitosan charge density on chitosan/casein complexation and phase behavior[END_REF].

When the CS/NaCMC-PLGA particles were prepared with NaCMC concentration equal to 300 ppm, the colloidal stability of the resulting suspension was maintained only up to 0.1 mol.L -1 NaCl. For the mixture containing 450 ppm CS and 300 ppm NaCMC, the charge ratio between ammonium groups and carboxylate groups was close to unity. Thus, the amount of excess cationic charges present at the surface of particles was significantly decreased which limited the stabilizing capacity against electrostatic screening by salt.

Drug delivery application

The results described in section 3.3 demonstrated the production of PLGA particles combining a hydrophobic polyester core with a surface covered by polyelectrolyte complexes. No chemical modification of stabilizing polymers was required; the composition of the mixture was the relevant parameter. Such suspensions of particles were further evaluated regarding their possible use for encapsulation and controlled release of hydrophobic molecules. Curcumin (CUR) was selected as a model hydrophobic molecule for a first evaluation of the performances of CS/NaCMC-PLGA particles.

Miscibility between curcumin and PLGA

The miscibility of CUR and PLGA in the solid state was evaluated through DSC measurements (Fig. A.5). Indeed, pure PLGA, as an amorphous polymer, exhibited a single glass transition around 47 °C as expected from literature. [START_REF] Blasi | Plasticizing effect of water on poly(lactideco-glycolide)[END_REF] Neat CUR exhibited one single peak corresponding to a melting temperature around 176 °C. This value was consistent with literature data. [START_REF] Jamali | Evaluation of targeted curcumin (CUR) loaded PLGA nanoparticles for in vitro photodynamic therapy on human glioblastoma cell line[END_REF][START_REF] Mukerjee | Formulation, Characterization and Evaluation of Curcuminloaded PLGA Nanospheres for Cancer Therapy[END_REF][START_REF] Yallapu | Fabrication of curcumin encapsulated PLGA nanoparticles for improved therapeutic effects in metastatic cancer cells[END_REF] For CUR/PLGA solid mixtures, the absence of any peak corresponding to the melting of CUR was an indication of molecularly dispersed CUR, i.e. total miscibility of CUR and PLGA. On the contrary, the detection of a melting peak in the vicinity of 176 °C would be the signature of partial phase separation, i.e. partial miscibility.

For CUR:PLGA weight ratios up to 0.1:1, no melting peak was detected, which showed that both compounds were fully miscible in solid state. At CUR:PLGA weight ratio equal to 0.2:1 and for higher amounts of CUR, a melting phenomenon was detected between 160 and 170 °C, meaning that within that range of composition, CUR and PLGA were only partly miscible. [START_REF] Jamali | Evaluation of targeted curcumin (CUR) loaded PLGA nanoparticles for in vitro photodynamic therapy on human glioblastoma cell line[END_REF] Over the whole composition range, glass transition temperature remained close to that of pure PLGA, indicating a negligible plasticizing effect of CUR, which was consistent with its rather high melting temperature and also consistent with previous report. [START_REF] Jamali | Evaluation of targeted curcumin (CUR) loaded PLGA nanoparticles for in vitro photodynamic therapy on human glioblastoma cell line[END_REF]

Encapsulation of curcumin in CS/NaCMC-PLGA particles

The particle size distribution and encapsulation efficiency (EE%) of CS450/NaCMC100-PLGA particles loaded with different CUR contents were determined (Table 2).

Particle diameter increased when CUR was added into the organic phase, with weight ratio up to 0.2:1. This variation could be explained by the increased amount of matter in the organic phase, everything else being equal. Moreover, it was shown that CUR was successfully encapsulated in the CS/NaCMC-PLGA particles with encapsulation efficiencies higher than 90 %.

The size distribution of the particles became significantly broader when CUR:PLGA weight ratio was increased from 0.1:1 to 0.2:1 and overall precipitation was observed at 0.3:1 CUR:PLGA weight ratio. A similar effect of the initial load of active molecule on final particle size distribution has been reported previously. [START_REF] Chebil | Nanoparticulate delivery systems for alkyl gallates: Influence of the elaboration process on particle characteristics, drug encapsulation and in-vitro release[END_REF]) These results led to the conclusion that CUR could be efficiently encapsulated in CS/NaCMC-PLGA particles provided that the loaded amount of CUR did not exceed the limit of solid state miscibility. The 10 % load of CUR in particles was comparable to previously reported performances with polyester particles. [START_REF] Arzani | Preparation of curcumin-loaded PLGA nanoparticles and investigation of its cytotoxicity effects on human glioblastoma U87MG cells[END_REF][START_REF] Jamali | Evaluation of targeted curcumin (CUR) loaded PLGA nanoparticles for in vitro photodynamic therapy on human glioblastoma cell line[END_REF][START_REF] Nagy | Highly efficient encapsulation of curcumin into and pH-controlled drug release from poly(ε-caprolactone) nanoparticles stabilized with a novel amphiphilic hyperbranched polyglycerol[END_REF]. The high encapsulation efficiency was consistent with other reports about encapsulation of CUR in polymers. [START_REF] Arzani | Preparation of curcumin-loaded PLGA nanoparticles and investigation of its cytotoxicity effects on human glioblastoma U87MG cells[END_REF][START_REF] Umerska | Polymeric Nanoparticles for Increasing Oral Bioavailability of Curcumin[END_REF]

Drug release

Apart from the efficiency of encapsulation, the kinetics of drug release out of particles was also a key performance parameter for potential applications. Thus, the release of curcumin out of CUR-loaded CS/NaCMC-PLGA particles was determined at pH 4.0, 7.4 and 9 after 24 h in dialysis bags. For comparison, free CUR and CUR-loaded CS-PLGA particles, were also investigated (Fig. 9a). Our aim was to reveal the main aspects of pH sensitivity of release kinetics. Nevertheless, according to the conditions of application and the chemical structure of the drug, several questions related to pH may appear. For a specific application, other pH conditions as well as other CS/NaCMC combinations may be explored.

The release percentages of CUR from both types of particles were lower than that observed with free CUR for all pH conditions. So, the encapsulation of CUR in the particles provided sustained release of CUR in aqueous medium over a wide range of pH conditions. The amount of released CUR from PLGA particles covered either by CS or by CS/NaCMC complex varied with pH over the range 4.0 to 9.0. In acidic condition (pH = 4.0), the release percentage of CUR from CS/NaCMC-PLGA particles was 28%, i.e. lower than that observed with CS-PLGA particles (higher than 40 %). We assumed that, in the case of CS/NaCMC-PLGA particles, NaCMC in the complex formed physical cross-links within adsorbed physical layer and slowed down the diffusion of CUR out of the particles. On the other hand, the amount of CUR released from CS/NaCMC-PLGA particles seemed to increase compared to CS-PLGA particles at neutral and basic conditions. At pH 7.4 and 9.0, the release percentages of CUR from CS/NaCMC-PLGA particles were 36% and 52%, while they were 26% and 39% from CS-PLGA particles, respectively. Increasing the pH led to deprotonation of ammonium groups of CS and thus to partial dissociation of electrostatic cross-links. So, the drug release percentage for CS/NaCMC-PLGA particles was increased when increasing pH.

The release profiles of CUR from the CUR-loaded CS/NaCMC-PLGA particles at pH 4.0 and 7.4 were compared over 7 days (Fig. 9b). At pH 7.4, the kinetics of release was much faster than that observed at pH 4.0. Because of the lack of monitoring at short times, we could not decide whether there was an initial burst. Nevertheless, when considering the experimental curves, we could conclude that, even if some burst effect occurred, it should account for less than 5 % release whatever the pH. When the pH was 7.4, the released amount of CUR reached 62 % after 48 h, and exhibited only incremental variation for longer times, then reaching 69 % after 168 h. At pH 4.0 the kinetics of release was much smoother and gradual, with a continuous increase of the amount of released CUR up to 43 % after 168 h. From such characteristics, the particles could be suitable for treatment in female genital tract [START_REF] Deák | Evaluation of pH-responsive poly(styrene-co-maleic acid) copolymer nanoparticles for the encapsulation and pHdependent release of ketoprofen and tocopherol model drugs[END_REF]. Normally, the genital tract system has a weak acidity of about 3.8 to 4.5. The bacteria in vagina area can produce lactic acid to control weak acidic condition in order to prevent fungal disease. Acid-base imbalance from menstrual blood, semen, and hormone fluctuation results in the increase of pH, leading to vaginal infection. The kinetics of CUR release at pH 4.0 could be nicely fitted by a power law (Eq.( 5)).

M t M ∞ = kt α (5)
In Eq. ( 5), Mt and M∞ were the masses (g) of drug released at instant t and after infinite time, respectively. k was a constant and α was the diffusional exponent, which was characteristic of the release mechanism. We found a rather satisfactory agreement between experimental and calculated curves with α = 0.43 (Fig. 9b), which had been shown to characterize release out of isometric spheres through Fickian diffusion [START_REF] Ritger | Simple equation for description of solute release I. Fickian and non-Fickian release from non-swellable devices in the form of slabs, spheres, cylinders or discs[END_REF]. These results

were consistent with previous characterizations revealing spherical particles with reasonably narrow size distributions.

Moreover, the results demonstrated that the CS/NaCMC-PLGA particles exhibited pHdependent properties. In this case, the CS/NaCMC-PLGA particles could be used as pHsensitive carriers. In addition, the components of the particles were bio-based polymers, especially PLGA as its hydrolysis products were lactic acid and glycolic acid, which could help increase acidity in this area to control the weak acidic pH balance. Loaded and non-loaded particles as well as solubilized CUR in equivalent amount were compared (Fig. 10). No significant cytotoxicity could be detected with non-loaded particles. Free CUR exhibited cytotoxicity to HCT 116 in a dose-dependent manner, consistently with previous reports. [START_REF] Sahu | Structure activity relationship, cytotoxicity and evaluation of antioxidant activity of curcumin derivatives[END_REF][START_REF] Watson | Curcumin induces apoptosis in HCT-116 human colon cancer cells in a p21-independent manner[END_REF] Loaded particles exhibited cytotoxicity in a dose-dependent manner. Nevertheless, the cell viability remained higher than that observed in the presence of an equivalent amount of free CUR, which could be explained by its encapsulation into the core of particles. This result was consistent with release experiments (see section 3.4.3).

These experiments confirmed the suitability of CS/NaCMC covered particles for drug delivery experiments.

Conclusion

PLGA particles stabilized by CS/NaCMC complex were successfully prepared by emulsification-solvent evaporation. The composition of the CS/NaCMC mixture was selected so as to have an excess of positive charges, which was appropriate for the preparation of stable PLGA particles, with diameters in the range of 484 to 705 nm. Unstable particles were obtained for compositions close to charge stoichiometry. Suspensions exhibited good colloidal stability over a wide pH range. Curcumin could be encapsulated in the core of the particles with the maximum loading of 10 % by weight of PLGA, and encapsulation efficiency percentage was 98.8 % of initial load. The CS/NaCMC covered on particle surface exhibited pH-dependent charge characteristics, which affected the CUR release behavior. The particles showed slower CUR release in weakly acidic conditions and faster release in neutral and basic conditions, which could be applied as pH-sensitive carriers in biomedical field.

Cytotoxicity experiments confirmed the applicability of CS/NaCMC covered particles. The concentration of free curcumin was related to the amount encapsulated in the particles. It was not possible to dissolve free curcumin at 250 µg/mL.

Figure captions

Fig. 1

 1 Fig. 1 Chemical structures of main compounds used in this work.

  signals from NMR spectra of CUR, PLGA, and CUR-loaded CS/NaCMC-covered-PLGA particles were used (Fig. A.2). Characteristic signals of CUR and PLGA were assigned on the basis of spectra of pure compounds and it was verified that all signals were found in the spectra of loaded particles. Assignments were also compared to literature. (Waghela, Sharma, Dhumale, Pandey, & Pathak, 2015) The weight ratio of CUR to PLGA after encapsulation (mencapsulated) was determined directly from the 1 H-NMR results. The maximum loading was calculated similarly but on the basis of the composition of the mixture used for preparing particles. By comparison to the initial load, the encapsulation efficiency (EE%) could be calculated (Eq.

Fig. 2

 2 Fig. 2 (a): Conductivity of mixtures of CS and NaCMC at different concentrations. Inset: Determination of "critical value" of NaCMC concentration for 150 ppm CS. (b): Variation of

Fig. 3

 3 Fig. 3 Surface tension of CS/NaCMC aqueous mixtures differing either by CS or by NaCMC content. Mixtures of CS (0, 50, 100, 150, 300, and 450 ppm) and NaCMC (50 ppm) (a), and mixtures of CS (450 ppm) and NaCMC (0, 50, 100, 150, and 300 ppm) (b).

Fig. 4

 4 Fig. 4 Formation of CS/NaCMC-stabilized PLGA particles by emulsification-solvent evaporation.

Fig. 5

 5 Fig. 5 Visual aspects of the obtained latexes observed after sonication and solvent evaporation with various initial concentrations of CS and NaCMC in the aqueous phase.

  1), d(0.5), and d(0.9) are the diameters at 10 %, 50 %, and 90 % cumulative volumes, respectively.b Unstable particles for concentrations close to CAC point.

Fig. 6

 6 Fig. 6 SEM images of CS/NaCMC-PLGA particles prepared with 450 ppm CS and different concentrations of NaCMC: 50 ppm (A), 100 ppm (B), 150 ppm (C), 300 ppm (D), 500 ppm (E), and 700 (F).

Fig. 7 :

 7 Fig. 7: Zeta potential (a), and colloidal stability (b) of the CS/NaCMC-PLGA particles compared to CS-PLGA particles at different pH values.

Fig. 9

 9 Fig. 9 Release behaviors of free CUR, 0.10CUR-loaded CS450-PLGA particles, and 0.10CUR-loaded CS450/NaCMC100-PLGA particles under different pH conditions from 4.0 to 9.0 for 24 h (a), and release profiles of CUR from 0.10CUR-CS450/NaCMC100-PLGA particles at pH 4.0 and 7.4 (b). Inset is the plot of the released fraction at pH = 4.0 versus time in logarithmic scale and dotted line is curve fitting with power law setting exponent at 0.43 (see section 3.4.3).

Fig. 10

 10 Fig. 10 In vitro cytotoxicity of CS450/NaCMC100-PLGA particles, 0.10CUR-loaded CS450/NaCMC100-PLGA particles and free CUR to HCT 116 with different concentrations. The concentration of free curcumin was related to the amount encapsulated in the particles. It was not possible to dissolve free curcumin at 250 µg/mL.

Fig. 1

 1 Fig. 1 Chemical structures of main compounds used in this work.

Fig. 2 Fig. 3

 23 Fig. 2 (a): Conductivity of mixtures of CS and NaCMC at different concentrations. Inset: Determination of "critical value" of NaCMC concentration for 150 ppm CS. (b): Variation of "critical value" of NaCMC concentration with CS concentration. Continuous line is guide for the eye. Dotted line represents NaCMC concentration corresponding to 1:1 stoichiometry.

Fig. 4 Fig. 5

 45 Fig. 4 Formation of CS/NaCMC-stabilized PLGA particles by emulsification-solvent evaporation.

Fig. 6

 6 Fig. 6 SEM images of CS/NaCMC-PLGA particles prepared with 450 ppm CS and different concentrations of NaCMC: 50 ppm (A), 100 ppm (B), 150 ppm (C), 300 ppm (D), 500 ppm (E), and 700 (F).

Fig. 7

 7 Fig. 7 Zeta potential (a), and colloidal stability (b) of the CS/NaCMC-PLGA particles compared to CS-PLGA particles at different pH values.

Fig. 8

 8 Fig. 8 Colloidal stability of the CS/NaCMC-PLGA particles as indicated by the variation of |n| index with NaCl concentration ranging from 10 -4 M to 4 mol.L -1 (for detail see section 2.4.3).

Fig. 9

 9 Fig. 9 Release behaviors of free CUR, 0.10CUR-loaded CS450-PLGA particles, and 0.10CUR-loaded CS450/NaCMC100-PLGA particles under different pH conditions from 4.0 to 9.0 for 24 h (a), and release profiles of CUR from 0.10CUR-CS450/NaCMC100-PLGA particles at pH 4.0 and 7.4 (b). Inset is the plot of the released fraction at pH = 4.0 versus time in logarithmic scale and dotted line is curve fitting with power law setting exponent at 0.43 (see section 3.4.3).

Fig. 10

 10 Fig. 10In vitro cytotoxicity of CS450/NaCMC100-PLGA particles, 0.10CUR-loaded CS450/NaCMC100-PLGA particles and free CUR to HCT 116 with different concentrations.

Table 1

 1 Particle size and distribution of prepared CS/NaCMC-PLGA particles.

		Particles	CS	NaCMC	d(0.1) a	d(0.5) a	d(0.9) a	Span
	Exp.
			(ppm)	(ppm)	(µm)	(µm)	(µm)
	1	CS50/NaCMC50-PLGA	50	50	b	b	b	b
	2	CS100/NaCMC50-PLGA	100	50	0.205	0.703	6.256	8.6
	3	CS150/NaCMC50-PLGA	150	50	0.207	0.705	1.498	1.8
	4	CS300/NaCMC50-PLGA	300	50	0.197	0.611	1.389	1.9
	5	CS450/NaCMC50-PLGA	450	50	0.186	0.600	1.289	1.8
	6	CS600/NaCMC50-PLGA	600	50	0.183	0.605	1.275	1.8
	7	CS450/NaCMC100-PLGA	450	100	0.184	0.520	1.312	2.1
	8	CS450/NaCMC150-PLGA	450	150	0.191	0.484	1.309	2.3
	9	CS450/NaCMC300-PLGA	450	300	0.209	0.694	28.241	40.4
	10 CS450/NaCMC500-PLGA	450	500

Table 1

 1

	, lines 3-6). Moreover, when the

9-11).

Except for the two compositions that were close to CAC (Table

1

, lines 2 and 9), most of particles had diameters lower than 1.5 µm and Span values of suspensions were lower than 2.3.

Table 2

 2 Particle size and encapsulation efficiency (EE%) of CUR-loaded PLGA particles with different amounts of loaded CUR and stabilized either by CS or by CS/NaCMC mixtures.

			Particle size
	Particles	d(0.1) a	d(0.5) a	d(0.9) a	Span	EE%
		(µm)	(µm)	(µm)
	CS450-PLGA	0.235	0.627	1.158	1.5	-
	0.10CUR-loaded CS450-PLGA	0.226	0.521	1.146	1.8	97.5±1.5
	CS450/NaCMC100-PLGA	0.184	0.520	1.312	2.1	-
	0.10CUR-loaded CS450/NaCMC100-PLGA 0.227	0.647	1.285	1.6	98.8±1.6
	0.20CUR-loaded CS450/NaCMC100-PLGA 0.174	0.697	4.222	6.6	91.5±2.4
	0.30CUR-loaded CS450/NaCMC100-PLGA		Aggregates		-

a For meaning see footnote a of Table

1

.

Acknowledgements

This work was financially supported by (a) the Royal Golden Jubilee Ph.D. Program, Thailand Research Fund Joint Funding with Rajamangala University of Technology Suvarnabhumi [Grant PHD/0184/2556], (b) Mahidol University, (c) Université de Lorraine, and (d) CNRS.