2.4. Experimental section 2.4.1. Instruments 2.4.2. Synthesis of the light-degradable bis-alkoxysilane 1 2.4.3. Preparation of particles 2.4.4. 7-DH loading of MSPs, SS-MSPs and LB-MSPs 2.4.5. Light-triggered degradation study of compound 1 2.4.6. Light-triggered degradation study of LB-MSPs 2.4.7. Release of 7-DH from LB-MSPs, SS-MSPs and MSPs 2.4.8. Cell viability assay 2.4.9. DFT calculations 2.5. References Chapter 3: Novel biodegradable organosilica cages as nanocarriers for biomedical Applications

Résumé de la thèse

Chapitre 1 : Introduction L'émergence des nanotechnologies dans les années 80 a non seulement changé notre perception de la matière à petite échelle, mais a également contribué à des nouvelles découvertes dans presque tous les domaines scientifiques. En appliquant les outils et les connaissances issus des avancées nanotechnologiques à la médecine pour la détection, la prévention et le traitement des maladies, ces deux sciences ont convergé et créé la nanomédecine. Des matériaux à l'échelle nanométrique ont été étudiés pour obtenir des thérapies supérieures contre les maladies. Les objets à l'échelle nanométrique (c'est-à-dire les nanoparticules) possèdent au moins une de leurs dimensions extérieures dans la gamme de 1 à 100 nm 1,2 et présentent des propriétés physicochimiques caractéristiques qui ne sont pas observées dans le matériaux de base. Des nombreux auteurs, en particulier dans le domaine de la nanomédecine, étendent souvent le terme "nanomatériau" pour inclure les particules de plus de 100 nm (jusqu'à 1 µm), tant que de nouveaux effets biologiques ou physico-chimiques sont observés qui ne sont pas autrement obtenus avec le matériau brut. 3 Le changement des propriétés physico-chimiques des nanoparticules dans ce régime de taille peut conduire au développement de nouveaux agents d'imagerie et de systèmes d'administration de médicaments, qui peuvent en principe, surpasser les thérapies actuelles en termes d'efficacité et de rendement. Depuis l'approbation en 1995 par la FDA (U.S. Food and Drug Administration) du premier Doxil®, plus de 50 nouveaux nanomédicaments ont été mis à la disposition des cliniques. En 2019, ONPATTRO® est devenu le premier nanothérapeutique à base de siRNA/RNAi utilisé en clinique, ce qui démontre une fois de plus l'importance des nanotechnologies pour l'avenir de la médecine. 4,5 Bien que la nanomédecine soit un domaine de recherche encore relativement jeune, elle a déjà prouvé qu'elle contribuait au développement de nouveaux médicaments.Grâce à cette recherche, on obtient de nouvelles connaissances sur la manière dont les nanomatériaux sont produits, modifiés et interagissent dans un environnement biologique. Ce sont des connaissances pertinentes non seulement pour la nanomédecine, mais aussi pour d'autres branches de la science. Malgré leur succès évident, les formulations de médicaments liposomiques souffrent de plusieurs propriétés sous-optimales, telles que la stabilité à court terme, la libération prématurée du médicament ou le ciblage limité; tous ces aspects sont cruciaux pour améliorer les propriétés pharmacocinétiques optimales globales des thérapies basées sur les liposomes. [6][7][8][9] La recherche sur les nanomatériaux alternatifs pour les applications biomédicales peut non seulement améliorer les thérapies actuelles basées sur les nanomédicaments liposomiques, mais promet aussi d'améliorer l'efficacité de médicaments auparavant inadaptés, permettant ainsi la renaissance de molécules thérapeutiques abandonnées. Le taux de réussite des petites molécules atteignant les normes de la FDA pour les essais cliniques de phase I est de 9 % et cette faible valeur est principalement liée au manque d'efficacité et à la faible sécurité clinique observés. 10 L'encapsulation de ces petites molécules dans une particule porteuse de la taille du nanomètre pourrait aider à acheminer la cargaison spécifiquement vers le site malade, ce qui améliorerait considérablement l'efficacité du médicament tout en réduisant les effets secondaires indésirables. Outre les applications d'administration de médicaments, les nanoparticules sont très intéressantes pour les techniques de bio-imagerie. Par exemple, l'encapsulation de colorants fluorescents à l'intérieur de nanoparticules entraîne une amélioration de leur durée de vie en circulation, de leur stabilité chimique et de leurs propriétés photophysiques, rendant les fluorophores de pointe encore plus efficaces pour les applications d'imagerie. Des agents de contraste supérieurs peuvent être obtenus simplement par la nature même de la nanoparticule, comme cela a été démontré avec succès avec des matériaux magnétiques adaptés aux techniques d'imagerie par résonance magnétique ou par des points quantiques dans les techniques d'imagerie optique déclenchées par l'infrarouge proche. 11 Grâce à une conception intelligente des nanoparticules, il est possible de combiner l'imagerie multimodale et les propriétés d'administration de médicaments, créant ainsi des particules pour une utilisation en théranostique.

Bien que les particules de silice mésoporeuse (MSP) aient déjà fait l'objet de recherches pour des applications biomédicales, 12 les thérapies basées sur les MSP n'ont pas encore fait la transition vers des applications cliniques. La raison principale en est la faible clairance des particules observée chez les organismes vivants après injection intraveineuse, qui ne répond pas aux exigences de dégradabilité totale fixées par la FDA. 13 Bien que les MSP se dégradent en milieu (épiderme et derme), les particules organosiliciques dégradables par la lumière UV peuvent servir de systèmes de libération prolongée de médicaments hydrophobes photosensibles. D'autres travaux présentés dans cette thèse sont axés sur l'étude de l'utilisation de nouvelles particules organosiliciques de 20 nm de taille semblables à des cages comme vecteurs de médicaments pour l'administration intraveineuse. Ces particules n'ont pas été décrites auparavant dans la littérature, les propriétés physico-chimiques et le comportement biologique in vitro et in vivo de la matière ont donc été étudiés. Une attention particulière a été accordée à la caractérisation physique du matériau, comme sa morphologie, ses capacités de charge des médicaments hydrophiles ou hydrophobes et la fonctionnalisation de sa surface avec de petits peptides. Des recherches ont été menées afin de comprendre comment la taille, la morphologie et la fonctionnalisation affectent la biodistribution in vivo chez les souris saines. Les avantages et les obstacles de l'utilisation de particules organosilicielles de petite taille, semblables à des cages, sont en cours d'évaluation.

Enfin, la possibilité d'introduire des oligonucléotides d'ADN ou d'ANP dans les particules de silice a été étudiée, en vue de développer de nouveaux matériaux hybrides et biocompatibles capables de reconnaître et de réagir à la présence de biomarqueurs spécifiques dans les organismes vivants. L'accent a été mis sur la synthèse de nouveaux dérivés d'alcoxysilane d'ADN et d'ANP et leur utilisation pour la préparation de particules d'organosilice par différentes méthodologies.

Outre les stratégies de modélisation douce dans lesquelles les alcoxysilanes d'ADN/ANP sont hydrolysés et co-condensés à côté d'une source de silice commune afin de préparer des particules d'organosilice, une méthode directe, guidée par l'interaction d'oligonucléotides complémentaires, a été étudiée pour la préparation de particules hybrides. Une attention supplémentaire a été portée sur le comportement de réponse aux stimuli résultant de la présence d'oligonucléotides d'ADN ou de PNA dans le cadre de la silice.

Chapitre 2: Particules organosiliciques mésoporeuses cassables par la lumière

Les particules organosiliciques mésoporeuses représentent de futurs vecteurs de médicaments très attrayants, en raison de leur non-toxicité générale et de leurs propriétés physico-chimiques facilement réglables, telles que leur taille, leur forme, leur porosité et leur dégradabilité.

L'intégration d'un groupe fonctionnel organique dans la structure des particules permet de préparer des particules organosiliciques poreuses cassables. Diverses sources de stimulation, telles que des agents réducteurs, [18][19][20] des changements de pH 21,22 ou une activité enzymatique, 23,24 peuvent être utilisées. La lumière représente un stimulus intéressant en raison de sa nature non invasive et de son contrôle spatio-temporel simple. Parmi les matériaux dégradables par la lumière signalés précédemment, on trouve les hydrogels, 25,26 les polymères, 27 les systèmes micellaires 28 et les cristaux colloïdaux. 29 Plusieurs nanoparticules à base de silice photosensible ont été précédemment rapportés et comprennent des colloïdes photosensibles 30 et des particules de silice mésoporeuses à grille, 31,32 ainsi que des supports de médicaments photocopiés à base de silice. 33 En revanche, des particules organosiliciques mésoporeuses qui se brisent lors de l'irradiation lumineuse n'ont jamais été rapportées. Nous avons conçu un nouveau bis-alcoxysilane, portant un groupe nitrobenzyle photolabile, à utiliser comme fraction organique de pontage dans la préparation de particules organosiliciques mésoporeuses cassables par la lumière (LB-MSPs). La fraction photolabile utilisée dans ce travail est un groupe 2-nitrobenzyle éther, qui est connu pour subir une réaction Norrish de type II lors de l'irradiation par la lumière UV qui conduit à son clivage 34,35 . Afin d'intégrer par covalence un groupe fonctionnel organique photolabile dans des particules organosiliciques mésoporeuses, un bis-alcoxysilane hydrolysable, portant le groupe 2-nitrobenzyle, a été synthétisé. Ce bis-alcoxysilane 1 photolabile a été préparé (Figure 1a) et sa dégradation déclenchée par la lumière lors de l'irradiation aux UV a été caractérisée avec succès par spectroscopie d'absorption UV-Vis (Figures 1b et c) et par spectroscopie RMN 1 H. La formation du produit de photodégradation 3 a été confirmée par la comparaison de ses spectres UV-Vis caractéristiques avec les résultats rapportés dans la littérature 34 et des calculs de théorie fonctionnelle de densité dépendante du temps (TD-DFT) en utilisant la suite de calculs Gaussian09®. De l´actinométrie sur le composé 1 a montré que la réaction de photodégradation se produit avec des rendements quantiques de réaction de 30 %. Le composé 1 a été utilisé pour préparer les LB-MSPs via le protocole de synthèse Stöber modifié. 36 Des LB-MSPs sphériques d'un diamètre moyen de 303 ± 34 nm ont été obtenues.

L'intégration covalente réussie du lieur dégradable par la lumière dans les particules a été confirmée par analyse thermogravimétrique, par spectroscopie photoélectronique des rayons X et par spectroscopie d'absorption UV-Vis. La dégradation réussie de la particule sous irradiation UV a été prouvée par microscopie électronique (Figure 2 Enfin, la possibilité de charger les particules avec des molécules bioactives a été explorée. Du 7déhydrocholestérol (7-DH), un précurseur naturel de la vitamine D3, a été chargée dans des LB-MSPs poreuses (charge de 7-DH=23 % en poids). Sa libération déclenchée par la lumière, due à la dégradation de la particule, a été prouvée avec succès par spectroscopie d'absorption UV-Vis.

Comme le montre la Figure 3, une libération accrue de 7-DH par les LB-MSPs a pu être observée lors de l'irradiation aux UV, ce qui peut s'expliquer par la progression de la dégradation des LB-MSPs et donc par la libération du médicament. En l'absence de lumière, seule une faible quantité de 7-DH a été libérée, très probablement en raison de la diffusion passive de la molécule chargée dans les pores ou physioadsorbée à la surface de la particule. Dans ce travail, la préparation et la caractérisation d'un nouveau bis-alcoxysilane clivable à la lumière et son utilisation pour la préparation de particules organosiliciques mésoporeuses sphériques qui se dégradent lors de l'exposition à la lumière ont été présentées. Des tests préliminaires de cytotoxicité ont en outre montré que les particules et leurs produits de photodégradation ne provoquent pas de cytotoxicité grave à l'égard des cellules HeLa, ce qui indique une bonne cytocompatibilité de ce matériau. Dans les travaux futurs, la capacité des LB-MSPs à servir de systèmes d'administration de médicaments pour des applications topiques sera d'un grand intérêt, car les particules assurent une protection contre la lumière UV, en absorbant les rayonnements nocifs tout en libérant simultanément une molécule bioactive, utilisée pour des applications thérapeutiques.

Chapitre 3: Cages organosiliciques biodégradables comme nouveaux nanocarriers pour les applications biomédicales

En ce qui concerne les particules poreuses de silice et d'organosilice, beaucoup d'efforts ont été déployés pour réduire la taille de la particule finale par des voies de formation dirigées par les tensioactifs, tout en maintenant une bonne homogénéité de la distribution des tailles. [37][38][39] Très récemment, Wiesner et ses collaborateurs ont signalé la préparation de particules de silice inorganiques en cage de moins de 20 nm, très symétriques, qui n'avaient jamais été observées auparavant pour les particules de silice poreuse. 40 Ces particules, qui possèdent une morphologie et une taille uniques, pourraient offrir de nouvelles possibilités pour le développement de systèmes de distribution de médicaments à base de silice. Inspirés par ces derniers travaux, nous avons envisagé la préparation de particules organosiliciques en forme de cage de 20 nm, portant des ponts disulfure biodégradables dans le cadre de la silice, qui, à notre connaissance, n'ont pas encore été décrits dans la littérature. La condensation d'un disulfure contenant du bisalcoxysilane avec une source de silice commune et en présence du tensioactif CTAB a permis la préparation de cages organosiliceuses à ponts disulfure (OSCs). Comme le montre la Figure 4a, bien que la dispersion finale des OSCs obtenue après purification ait été très concentrée (c=3 mg•mL -1 ), un colloïde transparent et stable a été obtenu. Une analyse par diffusion dynamique de la lumière (DLS) a révélé que le diamètre hydrodynamique moyen de la particule était de 23 nm (figure 4d). Ces particules ont ensuite été entièrement caractérisées à l'aide de techniques standards pour les matériaux poreux, telles que la spectroscopie photoélectronique à rayons X, l'analyse thermogravimétrique, la mesure de l'adsorption de N2, la spectroscopie d'absorption UV-Vis et la spectroscopie de fluorescence. La structure en cage a été prouvée par la reconstruction 3D d'images de particules TEM individuelles à l'aide du programme d'élucidation de structure Relion® (Figure 4c). 41 L'intégration des groupes fonctionnels disulfure dans le cadre de la silice rend les OSCs dégradables en présence de thiolates en raison des réactions d'échange thiol/disulfure qui se produisent. 42 Le glutathion (GSH), un tripeptide endogène contenant un thiol, est l'un des principaux médiateurs de l'homéostasie redox cellulaire et est capable de réduire le pont disulfure au sein des OSCs. Il est connu pour être présent en concentrations relativement élevées dans l'espace intracellulaire (1-10 mM). Comme le montre la Figure 5, une dégradation rapide des particules a été observée sur les images TEM (Figure 5b), comme l'indique la formation de débris plus petits lorsque le glutathion est présent dans la dispersion de la particule. La dégradation relativement rapide qui s'est produite au cours des 2 premières heures peut probablement s'expliquer par la petite taille des particules et la structure en cage des OSCs, qui offre une grande surface et une diffusion facile du GSH. Les OSCs ont été chargées avec le colorant hydrophile HOECHST 33258 (HE) et sa libération en présence de GSH par spectroscopie de fluorescence a été utilisée pour mettre en évidence la dégradation des particules, confirmant ainsi la capacité des OSCs à servir de vecteurs de médicaments sensibles aux stimuli.

Figure 5. a) Représentation schématique de la dégradation des OSCs en présence de l'agent réducteur glutathion (GSH). Dans des conditions physiologiques, le GSH est capable de réduire les ponts disulfure de la particule, ce qui entraîne leur rupture. b) Images TEM des OSCs (c=0,1 mg•mL -1 ) à t0 et c) après 2 h, lorsqu'ils sont incubés dans du PBS à 37 °C, avec et en l'absence de GSH (10 mM). Toutes les barres d'échelle=20 nm. d) Thermogrammes enregistrés sur les OSCs, les OSCs chargées de HE (OSCs-HE) et HE. e) Expériences de libération de HE enregistrant l'émission de fluorescence (λem=492 nm, λex=350 nm), avant et après l'ajout de GSH.

Après avoir préparé les nanoparticules, la capacité à fonctionnaliser davantage les OSCs par des techniques de greffe post-synthèse avec des molécules biologiquement pertinentes, telles que des sucres et des peptides, a été démontrée. Après avoir observé l'instabilité colloïdale de la particule lors de la modification de la surface des OSCs, des procédures de greffe de surface optimisées pour ces particules, visant à conserver la stabilité colloïdale, ont été explorées. La chimie des alcoxysilanes a d'abord été sélectionnée pour fonctionnaliser les OSCs avec de la D-(+)-glucosamine (OSCs-Sucre) et la réaction de cycloaddition azide-alcyne sans cuivre (SPAAC) a été utilisée pour greffer la surface de la particule avec le tripeptide Arg-Gly-Asp (RGD). La possibilité de fonctionnaliser ces nouveaux types de particules organosiliciques en forme de cage avec une variété de petites molécules bioactives a été démontrée, tout en maintenant leur stabilité colloïdale dans les dispersions aqueuses.

La biodistribution des petites particules organosiliciques en forme de cage est encore inexplorée et une première évaluation de leur comportement in vivo est donc intéressante, puisqu'un comportement de biodistribution distinct selon la taille et la forme des nanomatériaux a été signalé. 43 Les études de biodistribution ont également porté sur des particules fonctionnalisées de glucosamine, car la présence d'une fraction desucre peut augmenter l'absorption cellulaire lors de l'interaction avec les récepteurs de surface des cellules GLUT présents sur la couche endothéliale de la barrière hémato-encéphalique (BHE), ce qui pourrait permettre une meilleure adhérence des particules sur cette barrière biologique. Les premières études de biodistribution des OSCs ont été réalisées sur un modèle de souris CD1 en bonne santé. La présence de sCy5 sur les OSCs s'est avérée idéale puisque l'imagerie se produit dans la région rouge des spectres électromagnétiques, ce qui diminue le signal d'autofluorescence des corps des souris tout en réduisant davantage l'atténuation induite par l'oxygène. Les résultats obtenus par l'imagerie optique ex vivo des particules dans divers organes sont illustrés dans la Figure 6a Les signaux fluorescents ex vivo sur les échantillons de cerveau des souris qui ont été traitées avec des CSE, n'ont révélé qu'un signal mineur provenant du cerveau, qui après 24h était comparable aux intensités mesurées pour le véhicule (Figure 7a). En outre, aucune variation statistique significative concernant le signal détecté entre les OSCs et les OSCs-Sucre n'a été observée. Pour obtenir une information plus quantitative sur la présence des particules dans le cerveau, une spectroscopie d'émission atomique à plasma inductif (ICP-AES) a été effectuée sur les échantillons de cerveau après 4h de traitement. Les résultats de l'ICP-AES obtenus sont illustrés sur la Figure 7b, qui indique qu'un nombre mineur de particules ont pu traverser la BHE, indépendamment de la présence d'une fraction de sucre à la surface des particules. Bien que les expériences in vitro précédemment rapportées utilisant des modèles de BHE aient suggéré un croisement accru de la BHE avec des nanoparticules à fonction sucre, nos expériences ont confirmé que cet effet est négligeable et presque absent dans les modèles in vivo, 44,45 ce qui est en accord avec les résultats récemment publiés par Kataoka et ses collègues. 46,47 (ssPNA). Ces molécules présentent des groupes fonctionnels organiques intéressants pour la préparation des OSP en raison de leurs propriétés physico-chimiques uniques, telles que la complémentarité programmable des paires de bases et la dégradabilité enzymatique. En outre, grâce à l'utilisation de séquences d'ADN spécifiquement conçues, telles que les aptamères, une variété de petites molécules, de métaux et même de cellules vivantes peuvent être détectées avec une grande sélectivité. 48,49 L'intégration des oligonulcéotides dans un cadre inorganique peut donc conduire à la préparation de matériaux hybrides ayant de fortes propriétés dépendantes des stimuli, allant de la dégradation sensible aux stimuli à la liaison sélective de petites molécules ou même de cellules.

Pour intégrer le groupe de pontage de l'ADN et de l'ANP dans les OSP, des dérivés de bisalcoxysilane (voir le chapitre 4 de cette thèse) ont été préparés, qui ont ensuite été hydrolysés et co-condensés à côté du tétraéthylorthosilicate (TEOS), source commune de silice, pour former des nanoparticules organosilicielles pontées par l'ADNs ou l'ANPs, marquées respectivement comme ADNs et ANPs (Figure 8). Il a été démontré que les ssDNA-OSPs sont dégradables par l'activité enzymatique de la DNase I, une endonucléase qui hydrolyse les liaisons phosphordiester de l'ADN, et la dégradation des particules a été suivie par analyse MEB (Figure 9a) et DLS (Figure 9b). Particules organosiliciques à base de PNA préparées en exploitant les interactions supramoléculaires L'utilisation d'oligonucléotides ssANP complémentaires pour préparer des assemblages supramoléculaires de bis-organoalkocysilanes a été envisagée pour produire des matériaux possédant des propriétés similaires à celles des polymères à liaisons hybrides (HBP). 50 Après avoir évalué la capacité des brins d'ANP à s'auto-assembler, leurs dérivés alcoxysilanes correspondants ont été préparés (voir le chapitre 4 pour plus de détails). Par la suite, les deux brins de dérivés d'alcoxysilane ont été mélangés, ce qui a donné lieu à la formation du bis-alcoxysilane Supra-PNA-Si assemblé et à base de ANP (Figure 12a). Le Supra-PNA-Si a été utilisé pour la préparation de particules organosiliciques, avec ou sans la présence d'orthosilicate de tétraéthyle (TEOS), source supplémentaire de silice, après son hydrolyse et sa polycondensation catalysées par une base dans un mélange H2O/EtOH, ce qui a conduit à la formation de particules organosiliciques Supra-OSPs (Figure 12b et c). En plus de l'analyse spectroscopique UV-Vis et IR, la présence du pont PNA supramoléculaire au sein des Supra-OSPs a été étudiée par l'analyse FRET (Figure 13). Une dispersion de particules de Supra-OSPs (c=0,008 mg•mL -1 ) dans du PBS (pH=5,8) a été excitée à 450 nm et la photoluminescence a été enregistrée. Comme le montre la Figure 13c,l L'hydrolyse et la condensation subséquentes du Supra-PDP-Si catalysées par une base avec et sans la source secondaire de silice TEOS, en présence du tensioactif CTAB, ont été étudiées dans un mélange H2O/EtOH. Pour suivre la formation des particules organosiliciques, des images de microscopie électronique du brut de réaction ont été analysées à différents intervalles de temps.

Les images MEB enregistrées des mélanges réactionnels à 1h, 3h et 12h de temps de réaction en l'absence de TEOS sont présentées à la Figure 15a-c. Comme le montre la Figure 15a, après 1h, des structures fibreuses de moins de 1 µm (entourées en noir) ont été observées, qui ont continué à se développer après 3h de temps de réaction pour devenir de grandes structures fibreuses, très probablement en raison d'une hydrolyse et d'une condensation retardées d'organoalcoxysilanes volumineux. Après 12h de temps de réaction, les structures fibreuses ont commencé à se dégrader en structures fibreuses plus petites, très probablement en raison de l'instabilité des interactions supramoléculaires du groupe de pontage supramoléculaire ANP-ADN-ANP. Lorsque des organosilices ont été préparées à partir de Supra-PDP-Si avec une quantité équimolaire de TEOS (Figure 15d-f), aucune structure fibreuse n'a pu être imagée après 1h de temps de réaction. Après 3h, de grandes structures fibreuses ont été observées (Figure 15e), et des structures fibreuses stables ont encore été observées à 12h de temps de réaction (Figure 15f). La stabilité accrue des structures fibreuses d'organosilice peut s'expliquer par la cocondensation du TEOS en Supra-PDP-Si et la présence du CTAB, qui a stabilisé les silicates en milieu aqueux contre la dépolymérisation.

General introduction

In this chapter, a general introduction of nanomedicine will be given, focusing in particular on the use of porous organosilica particles for biomedical applications. Although there is a wide selection of materials that are currently investigated for new nanomedicines, silica-based materials possess promising properties that are useful for the development of next generation carriers for drug-delivery and bioimaging applications. After a general overview of the current investigations on nanoparticles for medical use, the advantages of organosilica particles for drug delivery applications will be discussed while providing some insight into their preparation, physicochemical characteristics and biological properties. Finally, the aim of the work reported in this thesis will be presented.

What is "nano" in nanomedicine?

The emergence of nanotechnology in the 1980s has not only changed the way we perceive matter at the small scale but has also contributed to new discoveries in almost every field of science. By applying the tools and knowledge gained from nanotechnological advances to medicine for the detection, prevention, and treatment of diseases, these two sciences have converged and created nanomedicine. In the field of nanomedicine, nanoscale materials are being researched for the development of superior therapies against diseases. Nanoscaled objects (i.e. nanoparticles) are defined as having at least one of their external dimensions in the range of 1 to 100 nm (IUPAC and EU definition of a nanomaterial) 1,2 and feature characteristic physicochemical properties that are not observed in the bulk phase. Many authors, especially in the field of nanomedicine, often extend the term "nanoparticle" to include particles bigger than 100 nm (up to 1 µm), as long as novel biological or physicochemical properties are observed that are otherwise not obtained from the bulk (Figure 1). 3 The change in physicochemical properties of nanoparticles in this size regime can lead to the development of novel imaging agents and drug delivery systems, which hold the potential to outperform current therapies in terms of efficiency and efficacy. In fact, since the FDA approval of the first nanomedicine Doxil® in 1995, more than 50 new nanomedicines have reached clinical use. 4 In 2019, ONPATTRO® became the first siRNA/RNAi based nanotherapeutic to pass clinical testing, further demonstrating the importance of nanotechnology in the future of medicine. 5 Although nanomedicine is still a relatively young research field, it has already contributed to the development of new materials for biomedical applications and to an understanding on how nanomaterials interact with cells.

Therefore, further research into the production, modification and the interaction of nanomaterials with the biological environment will lead to a better understanding of these materials, which is not uniquely relevant to the biomedical field, but will affect also other branches of science that work with nanoparticles. Comparison of sizes for different objects. Nano-objects require that at least one of their size domains lie between 1-100 nm. This definition is not consequently applied throughout the literature, since different physiochemical characteristic of the material regarding its bulk may not strictly be limited to this definition-therefore, the borders remain somewhat undefined.

Nanoparticles for biomedical applications

The use of nanoparticles for biomedical applications has been shown to be a powerful strategy for the targeted transport of drugs or imaging agents within a living organism. Upon the precise delivery of therapeutic molecules within an organism, unwanted toxicity related to molecular diffusion of the drug in biological healthy tissues can be minimized, resulting in the development of safer and more efficient therapies. Most of the current FDA and EMA nanomedicines in clinical trials or which are already used in clinics, are based on liposomal or polymeric reformulations of previously approved drugs. 6 Despite their obvious success, liposomal drug formulations suffer from several suboptimal properties, such as short term stability, premature drug release, and/or limited possibilities of active-targeting; all aspects that are critical for optimal pharmacokinetic performance of liposomal based therapeutics. [7][8][9][10] Research into alternative nanomaterials for biomedical applications can address the shortcomings of current therapies that are based on liposomal nanomedicines, while holding promise to improve the efficacy of previously unsuitable drugs, allowing for the revival of abandoned therapeutic molecules. The success rate of smallmolecules reaching the FDA standards for phase I clinical trials lies at 9%, where the low approval rate generally results from an observed lack of drug efficacy and poor biological safety. 11 Encapsulation of such small molecules within a nanometer-sized carrier particle could help to deliver the cargo specifically to the diseased site, greatly improving drug efficacy while reducing unwanted side effects. Encapsulation of fluorescent dyes used in imaging studies within nanoparticles leads to an improvement in their circulation lifetimes, chemical stability and photophysical properties, rendering state of the art fluorophores even more efficient for imaging applications. [12][13][14] Due to their small size, nanoparticles itself can become superior contrasts agents as successfully demonstrated with magnetic materials suitable for magnetic-resonanceimaging techniques or by quantum dots in near-infrared triggered optical imaging techniques. 15- 17 Upon intelligent design of nanoparticles, multimodal imaging and drug-delivery properties can be combined, creating particles for theranostic use.

Physicochemical considerations for nanoparticles

Physiochemical properties such as size, morphology, and chemical composition are known to influence a material's therapeutic or diagnostic efficacy and biological fate (Figure 2). For example, intravenously injected nanoparticles are known to bioaccumulate based on their particle size, morphology and surface charge (Figure 3). [18][19][20][21][22][23] Relatively small particles with sizes below 5 nm are effectively cleared out from body by the kidneys, while larger particles tend to accumulate in the liver (50-200 nm) and spleen (generally bigger than 200 nm), organs commonly associated with general particle clearance. Positive charges on the nanoparticle surface have been shown to increase nanoparticle uptake by the mononuclear phagocyte system, sequestering nanomaterials preferentially in the liver. Surface functionalization has also been demonstrated to greatly influence a particle's pharmacokinetic properties. Surface functionalization with polymers such as polyethylene glycol 24,25 , or with protein-coatings that decrease the particle´s sequestration by the mononuclear phagocyte system [26][27][28] , are effectively used to enhance the particles bloodcirculation lifetime and in vivo stability. Another important concept in nanomedicine is active targeting, where surface functionalization with ligands specific to the surface receptors of the target cell leads to improved accumulation at the desired site. Actively targeting liposomal formulations, such as SGT-53 that possesses transferrin targeting antibodies on its surface, are currently in clinical studies and showed promising bioaccumulation effects due to the presence of the targeting unit. 29 Small peptides have been shown to deliver nanoparticles to tumors when they are specific antigens of cell-surface receptors. For example, the RGD (Arg-Asp-Gly) motif was successfully used in order to prove in vivo targeting of nanoparticles to tumor sites. [30][31][32][33] More recently it was observed that functionalization of nanoparticles with the tumor-homing peptide (AR) causes a selective accumulation of the material in breast tumors in mice. 34,35 Caruso and coworkers showed that upon functionalization of poly(ethylene glycol) nanoparticles (PEG) with the αvβ3-integring ligand RGD peptide causes selective accumulation of the materials in the tumor site of U-87 MG xenografted mice when the particles were intravenously administered (Figure 4). 36 When the RGD sequence was instead modified to RDG, no tumor accumulation was observed, confirming the active targeting role of cell receptor ligands on the nanoparticles.

Examples of nanomaterials currently studied for biomedical applications

When it comes to the choice of the nanomaterial used for biomedical applications, one can classify the particles according to their specific properties that emerge from their small size.

Magnetic particles, such as iron oxide nanoparticles, have been studied for magnetic resonance imaging (MRI), [37][38][39][40][41][42] drug delivery, [43][44][45] hypothermal treatments, 46,47 immunomodulation 48,49 and magneto-mechanical attenuation of cells. 50,51 Cheon and coworkers investigated 15 nm sized exchange-coupled CoFe2O4 core-shell MnFe2O4 particles (CoFe2O4@MnFe2O4), where the combination of two materials characterized by hard (core) and soft (shell) magnetic phases led to greater induction heat enhancement when compared to Ferrite® iron oxide nanoparticles (Figure 5). 46 The resulting enhanced heating power of CoFe2O4@MnFe2O4 in the presence of an alternating external magnetic field was used for hypothermal treatment of tumors in mice and the particles showed superior tumor reduction ability compared to Ferrite® particles or doxorubicin. Nobel-metal-based nanoparticles, such as gold nanoparticles (AuNPs) have gained attention in the biomedical field, since their morphology and surface functionalization can be easily tuned and control over their surface plasmon resonance properties can be precisely adjusted. [52][53][54] Therefore, AuNPs are extensively investigated for photothermal therapy, [55][56][57] since the particles possess strong absorption in the NIR region, and are further considered as promising contrast agents for CT imaging applications. 58,[START_REF] Xi | Gold Nanoparticles as Computerized Tomography (CT) Contrast Agents[END_REF] Zhang and coworkers prepared immunological AuNPs (AuNP@DCB16F10) derived from melanoma tumor cells (B16F10 cells), encapsulated within a dendrimer derived vesicle (DC10) that showed improved anti-cancer activity when used for photothermal treatment of melanoma in mice (Figure 6). [START_REF] Lu | A Size-Shrinkable Nanoparticle-Based Combined Anti-Tumor and Anti-Inflammatory Strategy for Enhanced Cancer Therapy[END_REF] Due to the presence of melanoma antigens on AuNP@DCB16F10, these particles were able to activate a systematic anti-cancer immune response, which in combination with the photothermal activity of the AuNPs core upon NIR-light irradiation led to the successful reduction of melanoma cancer. Colloidal semiconductor nanocrystals (QDs) are studied for the development of novel optical imaging probes for biomedical applications due to their high fluorescent quantum yields and superior photostability. The photoluminescence properties of QDs are highly dependent on size, chemical composition and shell thickness. 61,[START_REF] Han | Size Control and Photophysical Properties of Quantum Dots Prepared via a Novel Tunable Hydrothermal Route[END_REF] The majority of research on QDs is focused on their use as luminescent probes, [START_REF] Tsuboi | Immunoglobulin Binding (B1) Domain Mediated Antibody Conjugation to Quantum Dots for in Vitro and in Vivo Molecular Imaging[END_REF][START_REF] Medintz | Quantum-Dot/Dopamine Bioconjugates Function as Redox Coupled Assemblies for in Vitro and Intracellular PH Sensing[END_REF][START_REF] Jeong | Multiplexed In Vivo Imaging Using Size-Controlled Quantum Dots in the Second Near-Infrared Window[END_REF] although coverage of QDs with porous polymers or an inorganic shell has shown the possibility to perform drug release. [START_REF] Hu | Functionalized Near-Infrared Quantum Dots for Invivo Tumor Vasculature Imaging[END_REF][START_REF] Bruun | Study on Intracellular Delivery of Liposome Encapsulated Quantum Dots Using Advanced Fluorescence Microscopy[END_REF] The main disadvantages in the use of QDs is their toxicity related to the inclusion of metal ions, such as Cd 2+ , Pb 2+ or Hg 2+ . For example, Kim and coworkers prepared PbS core-shell CdS-QDs (PbS/CdS-QDs) for real-time in vivo imaging in the second near infrared region (NIR-II, 1000-1700 nm). [START_REF] Jeong | Multiplexed In Vivo Imaging Using Size-Controlled Quantum Dots in the Second Near-Infrared Window[END_REF] The authors found that the particles photophysical properties could be varied by altering the PbS core-sized of PbS/CdS-QDs, allowing for multiplex imaging upon injection of particles with different core size (λem1=1080 and λem2=1280 nm). In order to reduce the particles toxicity, the material was covered with a biocompatible polymer (PQDs) that also allowed for the folic acid functionalization of PQDs, which served as tumor targeting moiety on the surface of PQDs (Figure 7). The use of carbon quantum dots (CQDs), also called carbon dots, represents an attractive, nontoxic alternative to QDs, though the current generation of CQDs possess inferior photoluminescence quantum yields when compared to traditional QDs. [START_REF] Molaei | Carbon Quantum Dots and Their Biomedical and Therapeutic Applications: A Review[END_REF][START_REF] Du | Carbon Dots for In Vivo Bioimaging and Theranostics[END_REF] Other carbon-based materials currently investigated for biomedical applications include fullerenes, [START_REF] Liang | Metallofullerene Nanoparticles Circumvent Tumor Resistance to Cisplatin by Reactivating Endocytosis[END_REF][START_REF] Montellano | Fullerene C60 as a Multifunctional System for Drug and Gene Delivery[END_REF][72] graphene oxide [START_REF] Chung | Biomedical Applications of Graphene and Graphene Oxide[END_REF][START_REF] Liu | PEGylated Nanographene Oxide for Delivery of Water-Insoluble Cancer Drugs[END_REF][START_REF] Campbell | Graphene Oxide as a Multifunctional Platform for Intracellular Delivery[END_REF] and carbon nanotubes. Carbon nanotubes (CNTs) are especially interesting since their inner cavity can be loaded with molecules allowing for possible delivery. [START_REF] Wang | SV119-Gold Nanocage Conjugates: A New Platform for Targeting Cancer Cells via Sigma-2 Receptors[END_REF][START_REF] Hong | Filled and Glycosylated Carbon Nanotubes for in Vivo Radioemitter Localization and Imaging[END_REF][START_REF] Lamanna | Multifunctionalized Carbon Nanotubes as Advanced Multimodal Nanomaterials for Biomedical Applications[END_REF] The so-called "softmaterials", such as polymeric aggregates, DNA nanoobjects, nanogels and dendrimers are further researched for future nanomedicines and possess great potential for successful translation into clinics, as indicated on the number of polymeric nanoparticles that are currently under clinical trials for drug-delivery applications. [START_REF] Ferrari | Polymer Nanoparticles for the Intravenous Delivery of Anticancer Drugs: The Checkpoints on the Road from the Synthesis to Clinical Translation[END_REF] Monomers for the preparation of polymeric nanoparticles can be derived from natural sources or be synthetic. The latter allow for the precise tuning of the particle´s size, morphology, and chemical functionality. Upon the introduction of stimuliresponsive functional groups within the macromolecular monomer, cargo release can be triggered precisely in the presence of external stimuli, such as a change in pH, [START_REF] Gao | Responsive Nanoparticles for Drug Delivery[END_REF][START_REF] Banerjee | PH-Degradable and Thermoresponsive Water-Soluble Core Cross-Linked Polymeric Nanoparticles as Potential Drug Delivery Vehicle for Doxorubicin[END_REF] enzymatic activity, 82,83 temperature [START_REF] Soga | Thermosensitive and Biodegradable Polymeric Micelles for Paclitaxel Delivery[END_REF][START_REF] Fan | Thermoresponsive Supramolecular Chemotherapy by "V"-Shaped Armed β-Cyclodextrin Star Polymer to Overcome Drug Resistance[END_REF] or the presence of reducing agents [START_REF] Yan | Cellular Association and Cargo Release of Redox-Responsive Polymer Capsules Mediated by Exofacial Thiols[END_REF][START_REF] Lim | Redox-Responsive Polymeric Nanocomplex for Delivery of Cytotoxic Protein and Chemotherapeutics[END_REF][START_REF] Zhao | Redox-Responsive Nanocapsules for Intracellular Protein Delivery[END_REF] . Recently, Langer and coworkers reported the preparation of hybrid lipid-polymeric nanoparticles for RNAi therapy (Figure 8). [START_REF] Dahlman | In Vivo Endothelial SiRNA Delivery Using Polymeric Nanoparticles with Low Molecular Weight[END_REF] RNAi therapy aims to deliver siRNA in order to silence gene expression at the posttranscriptional level and has been shown to be a powerful strategy to treat diseases, leading to the first FDA-approved RNAi-based treatment with ONPATTRO® in 2019. The authors used a polymer-based nanoparticle that enables efficient siRNA delivery to lung endothelial cells at low doses effectively reducing lung tumor growth and metastasis. A very appealing approach for the development of soft nanoparticles for biomedical application is the use of particles which have been developed in the field of structural DNA nanotechnology. [START_REF] Hu | DNA Nanotechnology-Enabled Drug Delivery Systems[END_REF] The bottom-up assembly of synthetic oligonucleotide strands via tile-based construction strategies or DNA-origami procedures allows for the preparation of well-defined 3D nano-sized DNA-objects, in which the self-assembly of single oligonucleotide strands occurs with high precision due to Watson-Crick base pairing and π-π stacking interactions. Favorable advantages in the use of synthetic DNA are its biocompatibility and programmable assembly, which allows controlling the precise number of functional groups on the final nanoobject. For example, DNA nanotubes [START_REF] Zhao | DNA Origami Delivery System for Cancer Therapy with Tunable Release Properties[END_REF] have been studied for in vivo cancer therapy, serving as vehicles for the delivery of doxorubicin to human breast cancer cell lines. In addition, DNA-icosahedra [START_REF] Chang | Aptamer-Conjugated DNA Icosahedral Nanoparticles as a Carrier of Doxorubicin for Cancer Therapy[END_REF] have been proven able to deliver anticancer drugs to tumor cells in vitro. Despite the great control over the assembly of nanoobjects obtained by DNA nanotechnology, their biological stability and the difficultly in unambiguously tracking these nanoobjects in a biological environment represents a critical problem that must be solved in order to facilitate their successful translation for in vivo applications. [START_REF] Madhanagopal | DNA Nanocarriers: Programmed to Deliver[END_REF][START_REF] Lacroix | Uptake and Fate of Fluorescently Labeled DNA Nanostructures in Cellular Environments: A Cautionary Tale[END_REF] In this respect, Gang and coworkers were able to show that by covering octahedral DNA-origami with peptoids increases their stability against enzymatic degradation (Figure 9). [START_REF] Wang | DNA Origami Protection and Molecular Interfacing through Engineered Sequence-Defined Peptoids[END_REF] Furthermore, the potential to use octahedral DNA origami for drug-delivery application was shown by encapsulating BSA within the octahedral cavity. Moreover, by suitably functionalizing the peptoids that surround the DNA nanoobject, antibodies and fluorescent marker have been successfully covalently linked, thereby demonstrating the possibility to use these constructs for imaging and targeted drug-delivery applications. Other inorganic nanocarriers investigated for biomedical applications are silica-based particles.

Ease of preparation and functionalization as well as their good biocompatibility make them attractive platforms for imaging and drug-delivery applications. Sub-10 nm sized silica core-shell PEG nanoparticles (C´Dots), functionalized with the tumor targeting peptide RGD are currently in FDA-approved human clinical trials for tumor mapping applications via fluorescence (Phase II: ClinicalTrials.gov identifier: NCT02106598) and PET-based (Phase I: ClinicalTrials.gov identifier: NCT03465618) imaging techniques. C´-Dots, which have been developed by the group of Prof.

Ulrich Wiesner, possess high fluorescent quantum yields per particle and are easily prepared via sol-gel chemistry, allowing for further functionalization of the particles with cell-targeting peptides or contrast agents used for PET imaging techniques (Figure 10). 32,[START_REF] Ma | Control of Ultrasmall Sub-10 Nm Ligand-Functionalized Fluorescent Core-Shell Silica Nanoparticle Growth in Water[END_REF][START_REF] Chen | Target-or-Clear Zirconium-89 Labeled Silica Nanoparticles for Enhanced Cancer-Directed Uptake in Melanoma: A Comparison of Radiolabeling Strategies[END_REF] The small size of the particles ensures efficient renal clearance from the body without causing undesired bioaccumulation in non-targeted tissues. With regard to silica-based particles, the most efficient strategy to deliver therapeutic drugs is the use of porous nanoparticles, since high payloads can in principle be achieved. For instance, porous silicon nanoparticles (mSiNPs) have been used and have been approved for pharmaceutical applications by the FDA, given the biocompatibility of both mSiNPs and their degradation products. [START_REF] Croissant | Degradability and Clearance of Silicon, Organosilica, Silsesquioxane, Silica Mixed Oxide, and Mesoporous Silica Nanoparticles[END_REF] mSiNPs are produced from silicon-wafers by electrochemical etching and subsequent sonication cycles, allowing for control over the final particle's size and porosity. The tunable porosity of mSiNPs is especially advantageous for the delivery of large bioactive drugs used for example in RNAi therapy. In this regard, Sailor and coworkers have recently reported the preparation of 50 nm-sized and highly porous mSiNPs, with high siRNA loading (25 wt%) for brain delivery applications (Figure 11). [START_REF] Kang | Self-Sealing Porous Silicon-Calcium Silicate Core-Shell Nanoparticles for Targeted SiRNA Delivery to the Injured Brain[END_REF] The high loading could be retained upon capping of the loaded pores with calcium silicate precipitate. An advantageous feature of mSiNPs is that the particles are photoluminescent upon light excitation 100 and can therefore be easily followed by optical imaging techniques, allowing for their unambiguous identification in biological tissues. To achieve in vitro targeted delivery of the siRNA payload to neuronal cells, mSiNPs have been functionalized with a virus-derived peptide able to target the acetylcholine receptor of neuronal cells, and positive cell targeting ability was confirmed. An enhanced in vivo accumulation on brain injury sites was observed by optical imaging, indicating the potential for these particles to image leaky BBB regions of the brain of injured mice. The variety of materials that can be used for the preparation of novel nanomedicines is vast, although only a few reach clinical use. As mentioned before, a major obstacle for their translation remains the effective nanoparticle clearance from the body after administration, which is necessary to prevent bioaccumulation and subsequent undesired chronic toxicities. Effective renal clearance of nanomaterials is possible when the particles are generally smaller than 10 nm, which on the other hand limits their application for high payload delivery of bioactive molecules.

When bigger particles are used, they must be rendered degradable to facilitate their biological elimination. In this regard, a promising approach to solve the degradability problem of larger particles is the use of mesoporous organosilica particles. The preparation of mesoporous organosilica particles allows for the facile introduction of stimuli-responsive organic bridging groups, which enables the particle's framework to degrade in the presence of a specific external stimulus (e.g. pH change, enzymatic activity or the presence of reducing agents). In the next subchapter, the use of mesoporous organosilica particles for biomedical applications is discussed, by giving a general introduction in the preparation of this type of materials. Further recent developments in the preparation of stimuli-responsive organosilica particles and their use in biomedical applications are also presented.

Mesoporous stimuli-responsive organosilica particles for biomedical applications 1.3.1. Preparation of mesoporous silica particles

According to the IUPAC definition, mesoporous materials possess pores with sizes ranging from 2 to 50 nm. Since the discovery of ordered mesoporous silicas, such as MCM-41 aluminosilicates by Kresge et al. 101 and Yanagisawa et al. 102 in the early 1990s by surfactant-templated approach, great effort has been devoted to developing new methodologies to tune the size, shape and pore size of mesoporous silica nanoparticles. Pioneering works in the development of nanosized mesoporous silica particles were first published in the 2000s, showing that precise control over synthetic parameters during the particle formation (e.g. pH, temperature, water content and type of surfactant used) enabled the preparation of porous silica with different morphologies. 103- 105 Notably, Yano and coworkers modified the Stöber synthesis 106 by introducing a positively charged surfactant and prepared monodisperse and mesoporous silica particles with radially aligned pores. 107 As explained more in detail below, the addition of positively charged surfactants aids in the formation of mesoporous silica particles that form from the base-catalyzed hydrolysis and polycondensation of a silica source, such as tetraethyl orthosilicate (TEOS), in an aqueous or alcohol/water medium. Quaternary ammonium salts are commonly used for the preparation of mesoporous silica particles (MSPs), cetyltrimethylammonium bromide (CTAB) being one of the most used surfactants. In aqueous mixtures, CTAB forms different positively charged aggregates and phases depending on its concentration. For example, at relatively low concentrations, that is, in the range between its critical micelle concentrations (CMC) CMC 1 (in pure water that is 1.0 mM at T=25°C) and CMC 2 (in pure water that is ca. 20 mM at T=25°C), CTAB forms spherical micelles. 108 The CTAB concentration will therefore strongly affect the appearance of the final mesophase in MSPs in terms of pore-order and arrangement (depending on the CTAB concentration used, hexagonal, cubic or worm-like pore structures can be obtained). However, the MSP formation mechanism depends on a number of other variable parameters, including the nature of surfactant and silica source, the solvent mixture, the concentration of the single reaction components, pH, temperature, catalyst or the presence of salts and organic additives.

Generally, the formation of mesoporous silica can be explained by using two models: 1) the liquid crystal templated pathway 101,109 , which was first established by researchers at the now ExxonMobil company and normally provide bulk silica, and the 2) cooperative self-assembly pathway, which was proposed by Stucky and coworkers and according to which mesoporous silica nanoparticles form. 110 Figure 12 shows a graphical representation of the simplified formation mechanism of MSPs according to the cooperative self-assembly mechanism.

Hydrolysis and polycondensation of the silica source (e.g. TEOS) occur catalyzed by the presence of a base. At the same time, the polymeric silicate anions assemble with the positively charged surfactant (e.g. CTAB) micelles through electrostatic interactions, leading to nucleation and subsequent growth. This results in the formation of mesoporous particles whose pores are filled by surfactant molecules. Removal of unreacted starting materials and the surfactantcan occur through calcination, dialysis or extractions in organic solvents providing purified mesoporous particles. The hydrolysis and condensation mechanism of alkoxysilanes, such as TEOS, has been excellently described by Brinker and Scherer in their book Sol-Gel Science -The Physics and Chemistry of Sol-Gel Processing (1990 Academic Press, Inc). The base-catalyzed hydrolysis of TEOS is initialized by the nucleophilic attack of the hydroxide anion ( -OH) according to a SN-like mechanism on the silicon atom of the alkoxysilane. A negatively charged pentacoordinated hypervalent intermediate forms, which then decays through a second transition state by displacing an ethoxide anion (Figure 13a). Since the transition states accumulate either partial or net negative charges, the base catalyzed hydrolysis of TEOS experiences inductive effects from the ligands. In fact, ethoxide groups are less able to stabilize negative charges when compared to hydroxyl groups, resulting in relatively slow initial hydrolysis reaction rates for TEOS (for example when compared to hydrolysis rates of TEOS in acid-catalyzed reactions or other silica sources such as tetramethyl orthosilicate, TMOS). Upon greater ethoxy substitution with stronger electronwithdrawing hydroxyl ligands that can stabilize better the partial negative charges on the silicon atoms, hydrolysis rates increase. Condensation reactions (Figure 13b) involve the nucleophilic attack of deprotonated silanols on the silicon atom of another TEOS molecule or partially 55 hydrolyzed polyalkoxysilanes, in which a SN2 mechanism is believed to be involved. Since the hydrolysis reaction speeds up upon increased ethoxy substitution, the formation of highly branched polysilicates is promoted, which will eventually be electrostatically attracted by the positively charged CTAB micelles. The surfactant-templated approach for the preparation of mesoporous particles is a powerful methodology to prepare a variety of different particles that differ in size, shape, pore geometry and pore size. Upon variation of synthesis parameters and nature of the surfactant used, mesoporous materials are prepared, possessing typically high surface areas (up to ca. 1000 m 2 •g - Of great interest for the development of nanomedicines, is the development of sub-20 nm sized, or so-called, ultra-small mesoporous silica particles (usMSPs). The small size of usMSPs can potentially result in an accelerated hydrolysis of the silica in biological media due to the particle's increased surface area, rendering the them less persistent in biological tissues and making their use safer for biological applications when compared to that of bigger silica particles. Furthermore, smaller particles are less likely to be sequestered by macrophages of the reticuloendothelial system (RES), allowing for improved delivery efficacy of the nanomaterial to diseased sites after intravenous injection. After the pioneering work reported in small amorphous SiO2 particles, 115,116 Wiesner and coworkers successfully reported the preparation of 10 nm sized single-pore usMSPs (Figure 15a), 117 and more recently the preparation of 10-20 nm-sized highly ordered cage-like silica particles (Figure 15b), 118 a newly observed morphology for porous silica particles.

Preparation and functionalization of mesoporous organosilica particles

The principles that are valid for the surfactant-templated preparation of porous silica particles by alkoxysilane chemistry can be applied to the preparation of mesoporous organosilica particles (MONs). MONs are porous silica-based particles, which possess organic functional groups as bridging units within their silica framework (Figure 16). In order to introduce an organic bridging group within the particle structure, an organo-bis(alkoxy)silane is hydrolyzed and co-condensed with a common alkoxysilane (e.g. TEOS) in the presence of a positively charged surfactant. 119 MONs are generally purified by dialysis or extraction methods, following the removal of the unreacted species and the surfactant. As for the preparation of MSPs, upon variation of synthetic parameters (surfactant, silica source, pH, presence of additives/salts), size, shape and porosity of the final organosilica particles can be finely tuned (Figure 17). The introduction of organic functional groups provides the particles with new physicochemical properties that depend on the nature of the organic bridging molecule that was integrated within the silica framework. The role and importance of these porous organosilica particles in the biomedical field will be discussed in the next subchapter. The sol-gel based surfactant-templated preparation of MSPs and MONs represents an easy and relatively low-priced synthetic strategy to produce porous carriers for theranostic applications.

Additionally, since the particles possess hydroxyl groups on their surface, easy post-synthesis grafting with various functional groups is possible by using simple alkoxysilane chemistry. Furthermore, the functionalization can occur selectively solely on the particle surface when the surfactant has not been extracted from the pores of MSPs/MONs. As depicted in Figure 18, the outer surface of the particle can be selectively functionalized with specific functional groups when the pores are still occupied by surfactant molecules. Once the template has been removed, a selective functionalization, with different functional groups, of the pore inner surface can be achieved, thus allowing for the preparation of complex and multifunctional drug delivery carriers. Surface functionalization is in fact a crucial factor that has to be considered in order to enhance particle biocompatibility and in vivo circulation lifetime or to obtain active targeting. For example, functionalization of MONs with polyethylene glycol chains imparts enhanced in vivo circulation lifetimes due to reduced reticuloendothelial system (RES) related clearance of the particles.

When targeting peptides or antibodies are desired, the possibility to modify the particles with functional groups suitable for bioconjugation (e.g. amines, thiols, azides, carboxylic acids) reactions is essential.

Mesoporous organosilica particles for biomedical applications

Although MSPs have been previously investigated for biomedical applications, 124 MSPs-based therapies have yet to find success in clinical applications. The main reason for this issue lies in the observed poor clearance of these particles from living organisms once intravenously injected, which does not meet the requirements of total degradability set by the FDA. [START_REF] Croissant | Degradability and Clearance of Silicon, Organosilica, Silsesquioxane, Silica Mixed Oxide, and Mesoporous Silica Nanoparticles[END_REF] Although MSPs degrade in aqueous environments due to the hydrolysis of the amorphous silica framework depending on the size, porosity, preparation procedure (such as pH-value for the preparation and presence of co-solvents) and additional surface functionalities of the particles, complete dissolution is not achieved and slow dissolution is frequently observed. 125 The development of MONs with a cleavable organic bridging group in their structure holds great potential to overcome the degradability and clearance issues of silica-based nanocarriers. In fact, upon the introduction of stimuli-responsive bridging groups within the framework of MONs, precise degradation and payload release can be tuned (Figure 9) After Inagaki and coworkers reported the preparation of mesoporous organosilica for the first time, 126 the group of Prof. Coradin was able to report the synthesis of disulfide-bridged organosilicas and demonstrated their in vitro degradation within cells. 127 The authors were able to demonstrate that disulfide bond reduction occurs upon the exposure of the particles to the reducing agent dithiothreitol (DTT), causing particle disintegration, as observed through electron microscopy. In fact, the disulfide-bridged organosilica particles began degrading also after cellinternalization due to the presence of a cytosolic reducing agent, that is, the tripeptide glutathione (GSH). Since the first preparation of disulfide-bridged organosilicas, great effort has been devoted to the preparation of mesoporous disulfide-bridged organosilica particles (SS-MSPs), as their use for stimuli-responsive in vitro and in vivo drug delivery applications has been successfully proven (Figure 20). [128][129][130] Since the stimuli-responsiveness of the MONs is dictated by the chemistry of the organic bridging group, many different breakable particles for drug-delivery applications have been investigated employing different stimuli, such as pH, 131 enzymatic activity 132,133 or reactive oxygen species 134 in order to trigger the degradation of MONs. More recently Leong and coworkers prepared bioinspired diselenide-bridged MONs, which were able to degrade in cells upon the presence of the reducing agent GSH or reactive oxygen species (Figure 21). 135 Furthermore, the authors showed homologous in vivo targeting upon encapsulation of the particles in cancer-cell-derived cellular membranes, and anticancer activity was investigated upon loading the particles with a cytotoxic RNAse enzyme. Given that morphology and porosity of MONs can be easily tuned, efforts have been devoted to developing degradable particles characterized by a high cargo loading efficiency, and the capability to accommodate large molecules like proteins and enzymes. To this end, stimuliresponsive hollow mesoporous organosilica silica particles (HMONs) and organosilica capsules have been developed. In order to prepare hollow or capsule-like organosilica particles either hard-templated approaches or liquid-interface assembly methodologies have been employed. 136 Shi and coworkers prepared dual-functionalized HMONs with enhanced doxorubicin loading capacity (148 mg per gram of particles, 14.8 wt%) for in vivo antitumor studies on mice (Figure 22). 137 The hollow structure was prepared via hard-templating method by co-condensation of two organoalkoxysilanes containing benzene and tridisulfide functional groups as bridging units.

It has been shown that the integration of the benzene bridging group not only enhanced the loading capacity, due to enhanced π-π interaction with doxorubicin, but also resulted in decreased hemolytic activity of HMONs, since less Si-OH groups are present on the particle surface. Furthermore, beside the GSH triggered-degradability of HMONs the authors showed that the particles could also release their payload when triggered by the influence of high-intensity focused ultrasound; the authors attributed the enhanced drug release upon ultrasound presence to induced tridisulfide bond cleavage and ultrasonic cavitation effects.

Applying supramolecular concepts on organoalkoxysilane chemistry: New opportunities in the preparation of smart organosilica particles 1.4.1. Supramolecular polymers in biomedical research

A major hurdle for current nanomedicines to reach clinical application are safety issues caused by the limited degradation and clearance of nanoparticles from living organisms. 138,139 To solve this safety issue, degradable hard and soft nanomaterials, such as organosilica 119 and polymeric particles, 140 possessing breakable stimuli-responsive functional groups within their framework are currently investigated. Supramolecular polymers prepared by the self-assembly of natural synthetic or natural occurring monomers through noncovalent interactions, such as H-bonds, ππ stacking or electrostatic interactions, have gained much attention in the development of dynamic and smart materials in electronic devices or for biomedical applications. 141 Pioneering work in the preparation of supramolecular polymers by Lehn and coworkers used H-bonds between bifunctional diamidopyridines and uracil derivatives to form linear polymers (Figure 23a). 142 The preparation of more stable supramolecular polymers was later reported by Meijer and coworkers, upon increasing the number of H-bonding motifs present in the monomers used for the preparation of the supramolecular polymer. 143 The authors showed that by introducing a bifunctional monomer containing two 2-ureido-4-pyrimidon units, stronger interactions mediated by four hydrogen bonds, yielded higher polymerization degrees in the final polymer as compared to monomers with less H-bond moieties (Figure 23b). Beside H-bonds, π-π interactions between conjugated compounds (Figure 23c) or coordination bonds of metal complexes (Figure 23d) have been used to prepare a variety of new supramolecular materials. [144][145][146] interactions between hexa-peri-hexabenzocoronene. 144 c) Supramolecular polymer formed via metalligand coordination bond from Cu(I)-o-phenanthroline complexes. 145 Supramolecular polymers based on macromolecular building blocks represent attractive materials for biomedical applications, such as drug delivery and tissue engineering, allowing the possibility to introduce stimuli-responsive behavior. 147 For example, Langer and coworkers reported the use of a pH-responsive supramolecular gels for the development of gastric devices that were envisioned for use in oral drug delivery given that they possessed prolonged retention and safe gastrointestinal passage (Figure 24). 148 The authors show that while the supramolecular polymer prepared from poly(acryloyl 6-aminocaproic acid) and poly(methacrylic acid-co-ethyl acrylate) is stable in acidic media, it degrades at neutral pH values, allowing for safe excretion of the device. The use of macromolecular starting materials for the preparation of supramolecular polymers has been proven to yield relatively stable polymers, which can be used for biomedical applications. A drawback in the use of macromolecular precursors in the preparation of supramolecular polymers lies in the limited number of functional groups available for supramolecular assemblies, greatly limiting the preparation of multicomponent assemblies. In contrast, the use of small molecules allows the introduction of multiple unique functionalities within the final supramolecular polymer. For example, small molecule-based supramolecular polymers have been described by Dankers and coworkers as a platform for the intracellular delivery of therapeutic siRNA molecules (Figure 25). 149 The authors showed that the assembly of positively charged 1,3,5-benzenetricarboxamide derivatives which possessed several different functional groups lead to the preparation of multicomponent supramolecular polymers. The resulting fibers were able to physically entrap hydrophobic molecules within the fiber's lipophilic core, while siRNA was electrostatically adsorbed on the outer surface of the aggregate. Further in vitro experiments showed that the supramolecular assembly was internalized into cells and colocalized with the endoplasmic reticulum of the cells. Although small-molecule-based supramolecular polymers allow for the facile preparation of multicomponent assemblies, their monomer design must be optimized to enhance the polymer's mechanical stability, while maintaining its dynamic properties. In fact, compared to macromolecular based monomers, small-molecule-assemblies suffer from poor mechanical properties when biomedical applications are aimed. 147,150 A possibility to prepare small-molecule-based supramolecular aggregates with increased mechanical properties, while retaining their dynamic properties, is the preparation of so-called hybrid bonding polymers (HBP). This this type of material is constituted of molecules that possess the ability to self-assemble via supramolecular interactions and to polymerize by forming covalent bonds. 151 In this regard, Stupp and coworkers reported the preparation of HBPs formed by the covalent and noncovalent polymerization of monomers bearing aromatic dialdehyde, diamine and a peptide sequence (Figure 26). 152 Interestingly, the authors showed that a supramolecular assembly could be reversibly formed and removed on a preexisting covalently linked polymeric fiber.

Self-assembly of organoalkoxysilanes

The self-assembly of organoalkoxysilanes has also been studied [153][154][155] and reported for the preparation of 2D surface coatings, 156,157 but to date their use has not been considered to develop of new stimuli-responsive materials for biomedical applications or for the preparation of HBPs with dynamic properties. Beside the fabrication of 2D-materials from self-assembled organoalkoxysilanes, a remarkable approach for the preparation of hybrid micellar nanocrystals was described by Fan and coworkers (Figure 27). 158 In this work, the authors self-assembled octadecyldimethyl(3-trimethoxysilylpropyl)ammonium chloride on the surface of hydrophobic gold nanoparticles mediated by hydrophobic interactions. After the assembly, the alkoysilane groups were hydrolyzed and subsequently condensed to form 3D assemblies of hybrid nanocrystals, while retaining the desired plasmon resonance properties of the gold nanoparticle core. The use of organosilica particles, which self-assemble on hydrophobic nanocrystals, represented a novel approach described by the authors, for the preparation of functionalized nanocrystals. Another example of the use of organoalkoxysilanes for the preparation of self-assembling hybrid structures was reported by Kikuchi and coworkers, which reported the preparation of hybridbonding liposomes, named cerasomes (Figure 28). 159 Cerasomes were prepared upon the selfassembly of amphiphilic organoalkoxysilanes, which formed liposomal structures in aqueous solutions. After subsequent polymerization of the liposome alkoxysilane head group, a rigid silicate layer was obtained on the liposomal structure, thus resulting in the formation of stable liposomal structures. A relatively new and attractive approach to prepare supramolecular structures is the use of oligonucleotides, such as deoxyribonucleic acids (DNAs) or peptide nucleic acids (PNAs). The use of DNA as supramolecular building blocks for the preparation of nanoobjects has been previously demonstrated, providing a power means of developing novel nanotechnologies 160,161 Oligonucleotides are one of the most programmable molecules with self-assembly behavior. DNA self-assembly is dictated by the oligonucleotide nucleobase sequence, involving H-bonding through Watson-Crick base pairs and π-π stacking interactions (Figure 29). PNA oligonucleotides possess a repeating N-(2-aminoethyl)-glycine unit linked via amide bonds as the backbone (Figure 29) and lack therefore the negatively charged phosphodiester backbone as in DNA. Since PNA lacks a negatively charged backbone, it is especially easy to work with in organic solvents, such as DMSO or DMF, and PNA assembly is strong due to the absence of electrostatic repulsions. 162 Further additional features of PNAs is their excellent stability against hydrolases, such as DNases and proteases, a good stability over a wide range of pH values and no need of divalent cations for hybridizations to occur. 163,164 The preparation of self-assembled nanostructure from PNA has been extensively reviewed by Gazit and coworkers. 165 Zhang and coworkers have reported the synthesis of PNA-based amphiphilic structures and their use for the preparation of spherical micelles by tuning both, the PNA's Watson-Crick base pairing, the π-π stacking interactions and intermolecular hydrophilic/hydrophilic interactions (Figure 30). 166 use in the immunological treatment of cancer. 167 As depicted in Figure 31, the hybrid DNA-PNAaggregate (INA) was constructed upon the self-assembly of 3 components. The final construct was built on a primary micelle formed by the self-assembly of amphiphilic lipid-DNA (U4T), able to hybridize with the adjuvant DNA, the immune activator and a PNA-labeled antigen (e.g.: pOVA). Due to good in-vitro serum stability, the DNA-PNA aggregates were able to reduce the tumor growth in mice through the delivery of immunostimulants.

Aim of the thesis

The work presented in this thesis aimed to design and prepare novel porous and stimuliresponsive organosilica particles for drug-delivery applications. In particular, novel bisalkoxysilanes have been synthesized and used in the preparation of organosilica particles through different methodologies, that is, soft-templating using surfactants or direct self-assembly of organoalkoxysilanes and their subsequent polycondensation. Furthermore, focus was put on the development of morphologically new organosilica particles for theranostic applications.

In the second chapter, the development of new light-degradable mesoporous organosilica particles for potential topical delivery is presented. A new UV-light-responsive bis-alkoxysilane was designed, synthesized, and successfully incorporated in the particle structure. The idea behind this project was to absorb harmful UV light while simultaneously delivering a hydrophobic bioactive molecule, namely provitamin D3. Pathophysiological vitamin D3 deficiencies are often observed in people having liver, renal or thyroidal dysfunctions; provitamin D3 delivery by topical application, in which the active vitamin D3 formation can occur on site, is therefore of great interest. 168 In addition, skin-related diseases, such as melanoma or inflammations, can be treated with drug-loaded nanoparticles, allowing for deeper penetration through the skin (e.g. through hair follicle canals). 169 Given that UV light is known to penetrate the upper skin layers (epidermis and dermis), UV-light-degradable organosilica particles have potential to serve as breakable drug-delivery systems for sustained release of photosensitive hydrophobic drugs.

In the third chapter, novel cage-like 20 nm sized organosilica particles were prepared and investigated as drug delivery carriers for intravenous administration. Special focus was put on the material physicochemical characterization, such as morphology, loading capacities of hydrophilic or hydrophobic drugs and surface functionalization with small peptides. To understand how size, morphology and functionalization affect the biological behavior both in vitro and in vivo studies were carried out. Biodistribution in healthy mice showed the particle ability to strongly accumulate in the endothelial layer of the liver, avoiding macrophage capture, suggesting the possibility to use them for the treatment of small liver tumors. With the research presented the advantages and hurdles in the use of small-sized cage-like organosilica particles are being assessed.

In the fourth chapter, the possibility to prepare organosilica particles with alkoxysilane derivatives of DNA or PNA oligonucleotides was investigated. The aim was to develop novel hybrid and biocompatible materials able to recognize specific biomarkers (e.g. enzymes, complementary strands) and to react in their presence. To prepare organosilica particles with DNA-or PNA-alkoxysilanes, three different preparation methodologies were investigated: 1) Stöber synthesis, 2) modified Stöber synthesis employing surfactants as structure-directing agents and 3) the possibility to form organosilicas via supramolecular interactions between the PNA-alkoxysilane molecules, inspired by the preparation of hybrid bonding polymers as discussed in section 1.4. 152 To the best of our knowledge, the preparation of oligonucleotide bridged organosilica particles has been scarcely investigated in the literature. Special focus was put on the preparation of bis-alkoxysilanes from mono-PNA-alkoxysilanes driven by supramolecular interactions, namely Watson-Crick base pairing, and their use to prepare organosilicas, which possess dynamic supramolecular moieties within their framework. The results show that, by using alkoxysilane chemistry, it is possible to develop new materials that bear site-specific binding moieties and that can react with specific oligonucleotides, which could pave the way to possible sensing applications.

Introduction

Mesoporous organosilica particles represent highly attractive drug-delivery carriers for potential biomedical applications, due to their general non-toxicity and easily tunable physicochemical properties, such as size, shape, porosity and degradability. Upon integration of stimuli-responsive organic functional groups within the silica framework, breakable porous organosilica particles can be prepared. In fact, the presence of an external trigger can mediate the particle disintegration, allowing for a controlled payload release over a defined period of time, which is essential for the development of new drug-delivery systems for optimized disease treatment.

Improved degradation leads to improved material clearance from living organisms, leading to the development of safer drug carriers. [1][2][3] A variety of stimuli sources, such as reducing agents, [4][5][6] pH changes 7,8 or enzymatic activity 9,10 , can be used; light represents a particularly attractive stimulus owing to its non-invasive nature and simple spatiotemporal control. Previously reported light-degradable materials include hydrogels 11,12 , polymers 13 , micellar systems 14 and lightresponsive colloidal crystals 15 . Several light-responsive silica-based nanoparticles have been previously reported and include light-responsive colloidosomes 16 and gated mesoporous silica particles 17,18 , as well as silica-based photocaged drug carriers 19 , but mesoporous organosilica particles that break upon light irradiation have not yet been reported. Previously, Shea and coworkers reported the preparation of photoresponsive bridged-polysilsesquioxanes, that undergo a change in morphology upon light irradiation. 20,21 Inspired by this work, we designed a new bis-alkoxysilane bearing a photolabile nitrobenzyl group to be used as the bridging organic moiety in the preparation of light-breakable mesoporous organosilica particles (LB-MSPs). The photolabile moiety used in this work is a 2-nitrobenzyl ether group, which is known to undergo a Norrish type II reaction upon UV-light irradiation that leads to its cleavage 22,23 . The choice to use the nitrobenzyl functional group as the light-responsive moiety was dictated by the fact that this group is widely used, and its photochemical properties have been therefore well studied. In fact, the 2-nitrobenzylgroup serves as the caging group for bioactive molecules [24][25][26] , as the photolabile protecting group in organic synthesis 27,28 or as the photoresponsive gatekeeper 29 for controlled drug release using mesoporous silica particles. In this chapter, the synthesis and thorough photochemical characterization of the new photodegradable bis-alkoxysilane and its use in the preparation of LB-MSPs is presented. The particles have been characterized by several techniques and their degradation behavior upon UV-light exposure has been investigated. To demonstrate the applicability of LB-MSPs as porous containers for the light-stimulated release of a cargo molecule, the particles were loaded with 7-dehydrocholesterol (7-DH), a natural previtamin D3 precursor, and the triggered release of this molecule upon UV-light exposure was investigated. The preparation of pristine mesoporous silica particles (MSPs) and disulfide-bridged mesoporous organosilica particles (ss-MSPs) to be used as model particles for comparison in the light-induce degradation and release studies has also been reported. In principle, LB-MSPs possess the potential to be used as novel organosilica carriers for topical drug-delivery applications being capable of absorbing harmful UV-light radiation while simultaneously releasing a natural precursor of a previtamin D3.

Results and discussion

Synthesis and characterization of the photolabile bis-alkoxysilane

In order to covalently integrate a photolabile organic functional group within the framework of mesoporous organosilica particles, a hydrolysable bis-alkoxysilane bearing the 2-nitrobenzyl group was synthesized. The photolabile bis-alkoxysilane 1 was prepared according to the synthetic path depicted in Scheme 1. The diallyl intermediate 2 was successfully prepared from 5-hydroxy-2-nitrobenzyl alcohol after reaction with allyl bromide, employing sodium hydride as a base. Subsequently, hydrosylilation of compound 2 with triethoxysilane, in the presence of Karstedt´s catalyst, yielded the final compound 1 in good yields ( 1 H and 13 C NMR spectra are reported in section 2. As discussed in the introduction, the photodegradable 2-nitrobenzyl ether moiety undergoes a Norrish type II reaction when irradiated with UV light, leading to its specific Obenz-C bond cleavage (Figure 1a). The occurrence of this photodegradation reaction in compound 1 was first characterized by using UV-Vis absorption spectroscopy. Therefore, a solution of 1 (0.11 mM) in a EtOH/H2O mixture (5:1, v/v) was irradiated with light at λirr=320 nm and the evolution of the spectra was recorded over time (Figure 1b andc). At t0 the UV-Vis absorption spectrum of 1

shows the characteristic absorption band of the n-π* transition centered at 312 nm. The profile evolution in time upon light irradiation shows a decrease in intensity of the band at 312 nm, which resulted to be absent after the first 5 min of irradiation, whereas a new n-π* transition band at 352 nm appeared, indicating the formation of the nitroso photodegradation product 3 (Figure 1a). Considering that the developed particles would serve as novel drug-delivery carriers for topical delivery applications, the specific solvent mixture for the UV-Vis absorption spectroscopy studies was chosen in order to contain a water fraction comparable to that present in creams and ointments. Furthermore, the high EtOH/H2O ratio used for these experiments ensured that, at a later stage when LB-MSPs would be tested, no particle degradation could occur due to hydrolysis of the amorphous silica.

95

The formation of the photodegradation product 3 was confirmed by comparing its characteristic UV-Vis spectra with results reported in the literature 22 and by time-dependent density functional theory (TD-DFT) calculations using Gaussian09® calculation suite (see section 2.4.9 for further information). DFT calculations on compound 3 revealed that the lowest and highest molecular frontier orbitals were only located on the conjugated system (Figure 2a), allowing TD-DFT calculations to be performed on the truncated structure (Figure 2b). As predicted, the calculated absorption spectra of this truncated structure matched the experimental results obtained, confirming further the formation of the nitroso benzaldehyde upon light irradiation of 1. An additional NMR analysis was used to determine the photodegradation quantum yield of 1 through a previously reported NMR-based chemical actinometry method that uses quartz NMR tubes for optimal irradiation conditions (see section 2.4.5 for additional details). 30,31 . The intensity (I0) of a 365 nm LED light source was calibrated by measuring the photodecay rate of the chemical actinometer 2-nitrobenzaldehyde (NBA) by monitoring changes in the NMR spectra.

NBA was selected as the chemical actinometer due to its well-known photochemical degradation quantum yield (φ365) of 0.5 at 365 nm. The degradation rate of the actinometer can be described by first-order kinetics, where the observed rate is equal to the product of the incident light intensity (I0), the quantum yield (φ365) at 365 nm and the fraction of light absorbed (f) by the sample (Equation 1).

𝑣 𝑁𝐵𝐴 = - 𝑑[𝑁𝐵𝐴] 𝑑𝑡 = 𝑘 0 • [𝑁𝐵𝐴] = 𝐼 0 • 𝜑 365 • 𝑓 (1)
The fraction of light absorbed by the sample can be reformulated as in Equation 2

𝑓 = 𝐼 0 -𝐼 𝐼 0 = 1 - 𝐼 𝐼 0 (2)
The term I/I0 in Equation 2 can be easily derived from the Lambert-Beer law (Equations 3 and 4)

and re-expressed as in Equation 5.

𝐴 = log ( 𝐼 0 𝐼 ) = 𝜀 • 𝑑 • [𝑁𝐵𝐴] (3) log ( 𝐼 𝐼 0 ) = -𝜀 • 𝑑 • [𝑁𝐵𝐴] ( 4
) 𝐼 𝐼 0 = 10 -𝜀•𝑑•[𝑁𝐵𝐴] ( 5
)
Combining Equations 5 and 2 in Equation 1 results in the rate law for the disappearance of the actinometer (i.e., NBA) shown in Equation 6, used for the determination of I0.

𝑣 𝑁𝐵𝐴 = - 𝑑[𝑁𝐵𝐴] 𝑑𝑡 = 𝐼 0 • 𝜑 365 • (1 -10 -𝜀•𝑑•[𝑁𝐵𝐴] ) (6)
In order to extract I0 from Equation 6, first the initial reaction rates for the photodecay of NBA was determined over a series of starting concentrations (Figure 4a). The initial reaction rate was monitored through the decay of the aldehyde peak in the NMR spectrum, where the area of the residual acetone solvent peak was used as an internal standard. At these short time intervals, the

[NBA] decays linearly with time, where the slope of these data corresponds to the initial reaction rate. The reactions rates obtained were plotted against the initial concentrations of NBA (Figure 4b) and the data was fitted with the help of equation 4 in order to extract I0, with ε=260 L•mol - Once calibrated, the LED was used in subsequent actinometry experiments performed on 1. For these experiments, CDCl3 was used as the solvent to avoid any undesired hydrolysis and condensation of 1 due to the presence of water. For these experiments, hexamethyldisilane was introduced as the internal standard. The reaction rate 𝑣(1) was monitored over time by the decrease in the peak area for the benzylic proton peak. Given the high concentrations used (> 60 mM) and the large extinction coefficient for the compound (6980 L mol-1 cm -1 ), Equation 6simplifies to Equation 7 and the reaction rate constant for the decay of 1 can be extracted from a linear fit (Figure 5). Applying equation 5 from the rate vs time plot allowed to estimate the reaction quantum yield for 1 having an average value of φ365=0.30±0.09.

𝐼 0 = 𝑣(𝟏) 𝜑 365 = 𝑘 0 𝜑 365 (7)

Preparation and characterization of light-breakable mesoporous organosilica particles

Light-breakable mesoporous silica particles (LB-MSPs) were prepared according to a modified recently reported procedure 8 that employed a modified Stöber synthesis protocol. 32 As depicted in Scheme 2, the photolabile bis-alkoxysilane 1 was hydrolyzed and co-condensed with tetraethyl orthosilicate (TEOS), with a TEOS/1 molar ratio of 2.25:1, in the presence of the surfactant cetyltrimethylammonium bromide (CTAB) and by using aqueous ammonia as the reaction catalyst. The particle preparation was performed in the dark in order to prevent the degradation of 1 due to ambient light. The particles were collected by centrifugation and the surfactant was removed by extraction in EtOH heated to reflux for 24 h (see section 2.4.3 for further details). The morphology of the particles was first analyzed by electron microscopy. SEM (Figure 6a) and TEM images (Figure 6b) of LB-MSPs revealed the presence of spherical particles with a size of 100 303±34 nm (Figure 6c). The hydrodynamic diameter (Dh) was determined to be 409±0 nm by DLS analysis (Figure 6d), which indicates a good dispersibility of the particles. TEM images of LB-MSPs taken at higher magnification (Figure 7a) indicated the absence of a highordered mesoporous phase. From the SAXS pattern (Figure 7b) of LB-MSPs, two broad peaks of low intensity centered at q=1.8 and q=2.9 nm -1 were found, confirming the absence of an ordered mesoporous phase, which is in agreement with the findings obtained from TEM. The reduced order of the mesoporous phase can be explained by the presence of the bulky organic linker in the reaction mixture during the preparation of LB-MSPs, affecting the formation of the porous phase. 33,34 N2 adsorption-desorption sorption measurements were used to characterize the porosity of LB-MSPs (Figure 8a), which confirmed the presence of mesopores with an average pore diameter of 2.4 nm, having a total pore volume of 0.26 cm 3 g -1 (Figure 8b) and a BET specific surface area of 318 m 2 g -1 . N2 adsorption-desorption measurements were used to characterize the porosity of SS-MSPs (Figure 14a), which demonstrated the presence of mesopores with an average pore diameter of 2.2 nm (Figure 14 b), having a total pore volume of 0.44 cm 3 g -1 and a BET specific surface area of 470 m 2 g -1 .

Preparation and characterization of MSPs

Pristine mesoporous silica particles (MSPs) have been prepared to compare the photoresponsive behavior of LB-MSPs with particles that do not possess an organic functional group within the inorganic structure. The particles have been prepared according to a previously reported procedure (see section 2.4.3 for details ). 8 SEM and STEM images of MSPs (Figure 16a and b) showed that spherical particles with a size of 209±20 nm (Figure 16c) were obtained and the presence of a relatively ordered porous mesophase was confirmed by the SAXS pattern recorded on MSPs (Figure 16d), which shows a broad peak centered at q=1.9 nm -1 . The porosity of MSPs was assessed by N2 adsorption-desorption experiments. As depicted in Figure 17a, the adsorption branch follows a sigmoidal type IV adsorption branch, typical for mesoporous materials. DFT pore size distributions were calculated from the adsorption branch and an average pore diameter of 2.5 nm having a total pore volume of 0.86 cm 3 g -1 was calculated for MSPs. The specific BET surface area was calculated to be 1243 m 2 g -1 . Compared to LB-MSPs and SS-MSPs, pristine MSPs possess more accessible mesopores, which explains the increased specific surface areas and pore volumes for these particles. Since the particles were prepared in the absence of a sterically hindered organic bis-alkoxysilane, the mesoporous phase of the particles was, compared to SS-MSPs and LB-MSPs, more uniformly dispersed, which reflected in the narrow pore-size distribution obtained from N2 sorption measurements (Figure 17b).

Light-triggered degradation of light-breakable mesoporous organosilica particles

The light-induced degradation of LB-MSPs was investigated by irradiating a dispersion of the particles (c=0.1 mg•mL -1 ) in an EtOH/H2O mixture (5:1 v/v) with 327 nm light and by analyzing aliquots of the dispersion by SEM and STEM. UV-Vis absorption spectra of the dispersion were also recorded over time. The specific solvent mixture was selected to have a water content similar to that of creams and ointments, considering the potential use of these particles as drug-delivery carriers for topical applications. In order to exclude the possibility that the particles degrade because of eventual hydrolysis of the amorphous silica control tests in solutions containing 20% of water were also performed. The breakability of the particles upon light-irradiation was followed by electron microscopy images to observe the light-induced change in morphology of LB-MSPs. Furthermore, UV-Vis absorption spectroscopy was used to confirm the light-triggered degradation of the light-responsive linker within LB-MSPs. As shown in the SEM images (Figure 18a), the surface of LB-MSPs became rougher after 20 min of UV-light irradiation and an amorphous material could be visualized due to the degradation of the particles. Prolonged irradiation induced an advanced structural disintegration of the particles, and after 6 h of light irradiation the complete loss of the initial morphology could be seen. The aggregates present in the SEM images could be due to a drying effect on the glass coverslip during sample preparation 110 on the small debris formed upon degradation. STEM images (Figure 18b) recorded on the same samples confirmed the degradation of the particles into small debris. Furthermore, no particles with reduced core densities could be observed suggesting that the degradation process occurs from the exterior of the particle. For further confirmation that the particles break upon light irradiation, N2 sorption experiments were performed on LB-MSPs. A LB-MSPs dispersion (0.1 mg•mL -1 in EtOH/H2O 5:1, v/v) was irradiated for 24 h with 327 nm light and subsequently dried prior to measurement. As depicted in Figure 20, after light-irradiation the amount of nitrogen adsorbed by the material decreases significantly and the calculated total pore volume and BET specific surface area decreased substantially to 0.03 cm 3 g -1 and 57 m 2 g -1 . The decrease in total pore volume and specific surface area can be explained by the light-triggered degradation of the particles and as a consequence the loss of the porous structure and spherical morphology, diminishing therefore the amount of nitrogen gas that can be adsorbed on the material. A control experiment was performed in which LB-MSPs (c=0.1 mg•mL -1 ) were stirred in an EtOH/H2O (5:1 v/v) mixture for 12 h in the dark. SEM (Figure 21a) and STEM (Figure 21b) images of the control experiment indicated that no degradation occurred after 12 h in the absence of light. Also, the UV-Vis absorption spectra of the particles remained unchanged, indicating the integrity of the light-degradable linker within LB-MSPs (Figure 21c). This control experiment confirms that the particles disintegration is only caused upon UV-light irradiation and that the solvent mixture has no effect on the particle morphology.

Light-triggered cargo release from LB-MSPs

Once the unique light-triggered degradation of LB-MSPs has been assessed, the possibility of loading the particles with a bioactive molecule and its release upon light-irradiation were investigated. For this purpose, LB-MSPs were loaded with 7-dehydrocholesterol (7-DH), a hydrophobic molecule and natural vitamin D3 precursor. The particles were suspended in a highly concentrated solution of 7-DH (20 mg•mL -1 ) in ethanol and stirred in the dark for 24 h. After solvent evaporation, the particles were washed and dried under reduced pressure (see section 2.4.4.). The final drug loading was determined to be 23 wt% by TGA (Figure 22a). For comparison, also SS-MSPs and pristine MSPs have been loaded with 7-DH using the same loading protocol as for LB-MSPs. The amount of 7-DH loaded within SS-MSPs was determined to be 28 wt% by TGA (Figure 22b), while for MSPs it resulted to be 5 wt% (Figure 22c). The cargo loading for SS-MSPs was comparable to the one obtained for LB-MSPs. The lowerloading efficacy of 7-DH within MSPs could be explained by the particle higher hydrophilicity, since no organic functional groups are present within the silica structure, rendering an adsorption of the hydrophobic 7-DH onto the pore-walls more difficult. The release of 7-DH from LB-MSPs upon light irradiation was investigated by UV-Vis absorption spectroscopy. An aqueous particle dispersion (ca. 0.8 mg•mL -1 ) was prepared and a 500 µL aliquot was placed in a 10 mm path length quartz cuvette equipped with a small stirring bar. A layer of cyclohexane (2 mL) was then added on top of the aqueous particle dispersion in order to extract the released 7-DH. Under gentle stirring, avoiding to mix the biphasic system, the particle dispersion was irradiated at 327 nm and the variation in absorbance in the cyclohexane layer at λmax=281 nm was monitored over time (see section 2.4.7).

As depicted in Figure 23a The amount of 7-DH released from LB-MSPs, with and without light-irradiation, shown in Figure 23a, was calculated by comparing the absorbance values at 281 nm obtained for t=230 min with a calibration curve made from solutions of 7-DH in cyclohexane at different concentrations (Figure 24). Considering that 0.39 mg of LB-MSPs were used and the loading of 7-DH is 23 wt%, the amount of drug loaded was 0.0897 mg (2.33•10 -7 mol) of 7-DH, which corresponds to 100% of drug present in the system. The absorption value of 7-DH after 230 min of UV-light irradiation was measured to be 0.3685, which corresponds to 7.3•10 -7 mol, or 31 wt% of 7-DH released upon light irradiation. In contrast, when no UV light was present, the absorbance measured after 230 min was 0.1540, which corresponded to 2.9•10 -8 mol, or 12 wt% of 7-DH released. Finally, in order to exclude the possibility that the observed 7-DH release from LB-MSPs may be caused by thermal effects upon light exposure, the particles were irradiated at 500 nm, a wavelength at which the photochemical degradation reaction of the nitrobenzyl moiety does not occur. As depicted in Figure 25a, upon irradiation at 500 nm, no 7-DH release was observed by monitoring its absorbance at 281 nm. The triggered release of the drug could be detected when changing the irradiation wavelength from 500 nm to 327 nm. The lower overall absorbance measured for 7-DH compared to the other release experiments here reported was since the particles had been further washed with EtOH/H2O (1:9 v/v; 1x500μL). This was done in order to reduce the amount of physisorbed material, and consequently of passive release of 7-DH, and to better visualize any variation upon light exposure. No light-induced degradation of the lightdegradable linker occurred upon light irradiation at 500 nm, indicating that the light-triggered release of 7-DH is only due to UV-light irradiation (Figure 25b).

Preliminary cytotoxicity tests

Taking into consideration that the particles could be used for biomedical applications, preliminary cytocompatibility test of the particles and their photodegradation products formed during UV-light irradiation were performed. The preliminary cytotoxicity study of LB-MSPs and their degradation products was done on HeLa cells, since this type of cell line is a standard for cytotoxicity experiments and the cells are especially easy to handle. Nevertheless, future cytotoxicity experiments on LB-MSPs will be done on healthy endothelial cell lines to simulate the particle´s toxicity when used for topical applications. HeLa cells were incubated for 12 and 24 h with different concentrations of LB-MSPs or light-breakable mesoporous organosilica particles that have been irradiated beforehand with UV-light at 320 nm for 12 h (LB-MSPs_irradiated) and the AlamarBlue® assay was used for the evaluation of the cytotoxicity (see section 2.4.8). As depicted in Figure 26, no relevant decrease in cell viability was observed for both LB-MSPs and LB-MSPs_irradiated, concluding that the particles do not show any relevant cytotoxicity at the investigated concentrations.

Conclusions

In this chapter, the preparation and characterization of a novel light-cleavable bis-alkoxysilane and its use for the preparation of spherical mesoporous organosilica particles that degrade upon light exposure has been presented. The cleavage of the nitrobenzyl-group containing bisalkoxysilane takes place upon UV-light irradiation, with a quantum yield of 0.30±0.09.

Incorporation of the linker within the silica structure of mesoporous particles allowed for the preparation of light-breakable particles and their potential as drug-delivery systems for the lighttriggered release of provitamin D3 (7-dehydrocholesterol) was demonstrated. The stability of the particles in the absence of light, while being degradable upon light irradiation, opens new possibilities in the design of novel organosilica carriers that release hydrophobic drug molecules in the presence of a non-chemical stimulus. Preliminary cytotoxicity tests further showed that the particles and their photodegradation products do not provoke any serious cytotoxicity towards HeLa cells, indicating a good cytocompatibility of this material. In future work the ability of LB-MSPs to serve as drug-delivery systems for topical applications will be of most interest, since the particles provide UV-light protection by absorbing harmful radiation, while simultaneously releasing a bioactive molecule.

Experimental section 2.4.1. Instruments

1 H, 13 C Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 400 spectrometer. The 1 H NMR chemical shifts (δ) are given in ppm and refer to residual protons of the corresponding deuterated solvent. All deuterated solvents were used as received without any further purification. Signal splittings are described as singlet (s), doublet (d), triplet (t), multiplet (m). Coupling constants (J) are given in Hertz (Hz).

Fourier transform infrared (FTIR) transmittance spectra were measured with a Shimadzu

IRAffinity-1 spectrometer. The spectrum of 1 was recorded in CHCl3 (ca. 1 mg mL -1 , 0.1 mm path length NaCl window cell). The attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra of the particle samples were collected using a spectral resolution of Δ = 4 cm -1 , accumulating 64 scans and the ATR-FTIR spectra were ATR corrected (penetration depth mode).

High resolution mass spectrometry (HR-MS) experiments were performed on a Bruker Daltonics microToF spectrometer (Bruker Daltonik GmgH, Bremen, Germany) with the electrospray ionization (ESI) interface operating in a positive ion mode.

Absorption spectra were measured on a Shimadzu UV-3600 spectrophotometer double-beam UV-VIS-NIR spectrometer and baseline corrected. Quartz cuvettes with path length of 10.0 mm were used for the recording of UV-Vis absorption spectra.

Light-breakability experiments on LB-MSPs and the photolabile linker 1 (but not for the quantum yield determination for 1) were performed with a Horiba Jobin-Yvon IBH FL-322 Fluorolog 3 spectrometer equipped with a 450 W xenon arc lamp, double grating excitation, emission monochromators (2.1 nm/mm of dispersion; 1200 grooves/mm), and a TBX-04 detector. The emission slit opening were set to 14 mm. Quartz cuvettes with 10.0 mm path length were used.

Scanning electron microscopy (SEM) and scanning transmission electron microscopy (STEM)

images were recorded with a FEI Quanta FEG 250 instrument (FEI corporate, Hillsboro, S3 Oregon,

4-(allyloxy)-2-((allyloxy)methyl)-1-nitrobenzene (2):

NaH (311 mg, 12.98 mmol) was added portion-wise to a stirring solution of 5-hydroxy-2-nitrobenzyl alcohol (1.00 g, 5.91 mmol) in dry DMF (10 mL) at 0 °C. After the completed addition of NaH, the reaction mixture was further stirred for 10 min at 0 °C. Subsequently, allyl bromide (1.17 mL, 13.52 mmol) was added dropwise to the stirring reaction mixture at 0 °C. The reaction mixture was stirred for 1 h at 0°C and for a further 1 h at room temperature and was then quenched with water (5 mL). The aqueous layer was extracted with ethyl acetate (3×, 20 mL), and the combined organic layers were washed with a saturated Na2CO3 solution and brine, and finally dried over anhydrous Na2SO4. Subsequently, the solvent was evaporated under reduced pressure and the crude oil was purified by column chromatography (silica gel, EtOAc/cyclohexane=1:2), affording the diallyl intermediate 2 as a yellowish oil (1.05 g, 71%).

7-DH loading of MSPs, SS-MSPs and LB-MSPs

A suspension of particles (4 mg) and 7-dehydrocholesterol (7-DH; 4 mg) in EtOH (200 μL) was stirred for 24 h at room temperature. Subsequently, the organic solvent was evaporated, the particles were washed with a mixture EtOH/H2O=1:9 (v/v; 2×500μL) and dried overnight under reduced pressure.

Light-triggered degradation study of compound 1

Spectroscopic studies: A solution of 1 (0.11 mM in EtOH/H2O=5:1 v/v) in a quartz cuvette was irradiated with UV light at λirr=320 nm using a Horiba Jobin-Yvon IBH FL-322 Fluorolog 3 spectrometer equipped with a 450 W xenon arc lamp. The emission slit opening were set to 14 mm. UV-Vis absorption spectra were recorded at different time intervals. When the degradation of 1 was investigated using NMR spectroscopy, a solution of 1 (2 mg in 0.6 mL of deuterated chloroform) placed in a non-quartz NMR tube was irradiated and spectra were recorded.

Actinometry: In a typical experiment, 1 and the actinometer (NBA=2-nitrobenzoic acid, Sigma-Aldrich 98%) were dissolved each in ca. 0.5 mL of the appropriate (deuterated acetone or CDCl3) deuterated solvent and then added to a quartz NMR tube. The tube was then irradiated with a 365 nm LED (ThorLabs) driven at 550 mA, where the distance between the sample and the light source was kept fixed. Changes in the NMR spectra were monitored as function of irradiation time. Given that NMR integration-based quantitation techniques can be inaccurate if the system is not allowed to fully relax between pulses, a 30-degree pulse with a 4 second delay between pulses was employed to ensure that full relaxation is achieved. Experiments were either performed in a darkened room, or with the sample wrapped in aluminum foil when not in use to reduce stray light exposure. The rate of the light driving decomposition reactions can be described by Equation 6(section 2.2.1). The LED intensity was calibrated through the quantification of the initial reaction rate for 2-nitrobenzaldehyde performed over a series of starting concentrations prepared in deuterated acetone. The initial reaction rate was monitored through the decay of the aldehyde peak in the NMR spectrum, where the area of the acetone solvent peak was used as an internal standard. The rate was then abstracted through a first-order kinetic fit to these data, Figure 4a. A plot of initial reaction rate vs initial concentration was subsequently modeled by using Equation 6, where ε365 = 260 M -1 cm -1 and, Φ365=0.5 (Figure 4b).

Once calibrated, the LED was used in subsequent actinometry experiments performed on 1.

These experiments were performed in CDCl3, where hexamethyldisilane was introduced as an internal standard. The reaction rate was monitored by the decrease in the peak area for the benzylic proton peaks. Given the high concentrations used (> 60 mM) and the large extinction coefficient for the compound (6980 M -1 cm -1 ), Equation 6simplifies to Equation 7 (section 2.2.1).

The initial reaction rate abstracted from a first order kinetic fit (Figure 5, section 2.2.1) were then used to calculate the reaction quantum yield. An average value for Φ365= 0.30±0.09 (mean value±standard deviation, N=3).

Light-triggered degradation study of LB-MSPs

A dispersion of LB-MSPs (0.1 mg•mL -1 ) in EtOH/H2O (5:1, v/v) was placed in a quartz cuvette equipped with a magnetic stirrer. The dispersion was kept under stirring and irradiated at λirr=327 nm using a Horiba Jobin-Yvon IBH FL-322 Fluorolog 3 spectrometer equipped with a 450 W xenon arc lamp. The emission slit opening were set to 14 mm. The UV-Vis absorption spectra were recorded on the dispersion of particles. To follow the degradation by electron microscopy, a dispersion of LB-MSPs (0.1 mg•mL -1 ) in EtOH/H2O (5:1 v/v) was placed in a quartz cuvette equipped with a magnetic stirrer. The particle dispersion was irradiated at λirr=327 nm under stirring and aliquots were taken for SEM and STEM investigations at different time intervals.

Release of 7-DH from LB-MSPs, SS-MSPs and MSPs

Release of 7-DH upon UV-light irradiation from LB-MSPs: Cyclohexane (2 mL) was slowly added to a dispersion of 7-DH loaded particles (0.39 mg) in H2O (500 μL) contained in a quartz cuvette equipped with a magnetic stir bar. Under stirring, to avoid the mixing of the two separate phases, the aqueous layer was irradiated with light at 327 nm using a Horiba Jobin-Yvon IBH FL-322

Fluorolog 3 spectrometer equipped with a 450 W xenon arc lamp (the emission slit opening were set to 14 mm) and over time the UV-Vis absorption spectra were recorded on the cyclohexane phase.

Release of 7-DH upon UV-light irradiation from SS-MSPs, MSPs and LB-MSPs:

Cyclohexane (2 mL) was slowly added to a dispersion of 7-DH loaded particles (0.8 mg) in H2O (500 μL) contained in a quartz cuvette equipped with a magnetic stir bar. Under stirring, avoiding the mixing of the two separate phases, the aqueous layer was irradiated with light at 327 nm using a Horiba Jobin-Yvon IBH FL-322 Fluorolog 3 spectrometer equipped with a 450 W xenon arc lamp (the emission slit opening were set to 14 mm) and over time the UV-Vis absorption spectra were recorded on the cyclohexane phase.

Cell viability assay

The cytotoxicity evaluation of LB-MSPs and their degradation products after 12 h of UV-light irradiation (LB-MSPs_irradiated) was performed by using HeLa cell line and the AlamarBlue® assay. LB-MSPs_irradiated were prepared by irradiating a suspension of LB-MSPs (3.2 mg•mL -1 )

in EtOH/H2O (5:1, v/v) at 327 nm for 12 h. The suspension was lyophilized prior to the biological evaluation to remove residual EtOH that was detrimental for the cell growth. The HeLa cells were seeded in a 96-well plate (1.5×104 cells per well) and incubated at 37 °C (5% CO2) overnight to allow them to attach on the plate surface. The culture medium (CM; Dulbecco's Modified Eagle Medium) was then replaced by fresh CM (100 μL) containing different concentrations of nanoparticles (20, 100, 200 μg•mL -1 ). After 12-and 24-h incubation, AlamarBlue® (10 μL) was added and the cells were incubated for additional 2 h. A solution of DMSO in CM (10%, v/v) and CM were used as the positive and negative control, respectively. The percentage of living cells was determined by monitoring the absorbance at 570 and 600 nm by using a microplate reader (Perkin Elmer, VICTOR™ X5).

DFT calculations

DFT calculations were performed using the Gaussian09 calculation suite. Ground state structure optimizations were performed at the B3LYP/6-31+G(d,p) level of theory, using a self-consistent reaction field (SCRF) PCM to model the influence of the surrounding solvent. Optimization into a global minimum was confirmed by the lack of negative vibration modes. Time-dependent (TD)-DFT calculations were performed to calculate electronic excitation energies using the random phase approximation at the B3LYP/6-311+G(2d,p) level of theory. The cartesian coordinates for the truncated nitroso-model were as followed: C -1.23112600 -0.33193900 -0.26303900 C -0.16254500 -

Chapter 3

Novel biodegradable organosilica cages as nanocarriers for biomedical applications

In this chapter, the preparation and characterization of 20 nm sized cage-like organosilica nanoparticles is presented. The particles possess stimuli-responsive disulfide functional groups within the silica framework, which renders this material degradable upon the presence of endogenous reducing agents, such as the tripeptide glutathione. The possibility to load the particles with hydrophilic and hydrophobic drugs is shown along with the stimuli-triggered cargo release, demonstrating their potential for drug-delivery applications. Moreover, grafting procedures for easy and efficient functionalization of the particles with various biomolecules employing alkoxysilane chemistry are reported that allow ensuring a good particle colloidal stability. Since little is known about the biological fate of cage-like particles intravenous administration in healthy mice models was done and their biological feature were investigated.

In-vivo results suggested that the particles do not accumulate preferentially in liver macrophages after occurred injection but are taken up by endothelial cells of the liver. Due to the particles ability to escape the uptake by liver macrophages, their use to treat effectively hepatocellular carcinoma can be envisioned, which will be further investigated in vivo in future experiments.

Introduction

Although tremendous research efforts have been made to develop nanomaterials for future applications in nanomedicine, only few have met the rigorous standards required for clinical implementation. Degradation represents a major hurdle for hard nanomaterials, where poor degradation leads to prolonged bioaccumulation, which in turn leads to an undesired chronic toxicity in living organisms. 1 Of many degradable nanoparticles currently under study for biomedical applications, porous organosilicas have attracted great interest in recent years due to several attractive physicochemical properties, including the facile tunability of properties, such as size, 2,3 morphology 4-6 and biodegradability 7 . Size represents an important factor in the particle´s biodistribution 8 , and small nanoparticles ("small" meaning a diameter below 50 nm)

are considered to possess favorable biodistribution properties due to accelerated elimination of these small particles from the body. 9 Regarding porous silica and organosilica particles, much effort has been devoted to decrease the final particle size via surfactant-directed formation pathways, while still maintaining a high degree of dispersibility and colloidal stability. [10][11][12] Over the past years, many reports have been published on the effect of different nanoparticle shapes on the biodistribution and bioaccumulation, [13][14][15] nevertheless little is known about the biological behavior of the recently discovered cage-like silica nanoparticles reported by Wiesner and coworkers. 16 These nanoparticles represent an attractive system to be explored for biomedical applications, owing to their small size and unique morphology characterized by a relatively large empty cavity. Inspired by Wiesner's work, we envisioned the preparation of 20 nm sized cagelike organosilica particles bearing biodegradable disulfide bridges within the silica framework, which to the best of our knowledge have not yet been described in the literature. Cocondensation of a disulfide containing bis-alkoxysilane with a common silica source in the presence of the surfactant CTAB and subsequent polyethylene glycol (PEG) and sulfo-cyanine 5 (sCy5) surface-grafting, allowed for the preparation of disulfide-bridged organosilica cages (OSCs). These particles have been fully characterized with standard characterization techniques for porous materials and the cage-like structure has been proven by 3D reconstruction of singleparticle TEM images. Further it was shown that the cavity of the particles can be loaded with hydrophilic and hydrophobic molecules, proving their applicability for drug delivery applications.

Due to the presence silanol groups on the surface of the particles, fluorescent dyes and bioactive molecules for cellular recognition were successfully grafted on the particle surface using simple alkoxysilane chemistry. Considering the particle´s unique morphology and non-toxicity, preliminary biodistribution studies were performed on OSCs in healthy rat models to assess their potential use in biomedical applications, such as drug delivery or imaging. The obtained biodistributions results for OSCs in various organs, especially the liver, revealed the potential of these particles for anti-cancer applications in the latter organ, since histological analysis suggested that the particles are not sequestered by liver macrophages. Finally, the particles ability to cross stiff biological barriers, such as the blood-brain barrier, was investigated and future strategies to increase the ability of these particles to accumulate in the brain will be discussed.

Results and discussion

Preparation and characterization of cage-like organosilica particles

Disulfide-bridged organosilica cages (OSCs) were prepared by modifying previously reported procedure for the preparation of 20 nm sized mesoporous organosilica particles. 17 As depicted in Figure 1, the final organosilica cages were obtained through a two-step bottom-up procedure, according to which a disulfide-containing bis-alkoxysilane, namely bis(triethoxysilylpropyl) disulfide (BTDS), was hydrolyzed and co-condensed with tetraethyl orthosilicate (TEOS) in the presence of the surfactant cetylammonium bromide (CTAB) and of the aqueous NH3 as the basic catalyst. The co-condensation of BTDS ensures the covalent linkage of the disulfide functional groups within the particle framework, thereby rendering the particles degradable in a reducing environment, such as the intracellular space given its high glutathione concentrations (2-10 mM). 18,19 In a second step (Figure 1), after the initial particle formation, the surface functionalization was obtained via alkoxysilane chemistry; polyethylene glycol silane (mean PEG chain length of 6-9 ethyleneoxy groups) and the sulfo-cyanine5-silane (sCy5-silane) have been added to the colloidal particle dispersion, obtaining a covalent grafting of these molecules onto the particle surface. The purification of the particles from unreacted species and the surfactant was obtained via dialysis (see section 3.4.4.) Transmission electron microscopy images (TEM) of OSCs (Figure 2a) revealed the cage-like structure of the particles possessing an average diameter of 20 nm (Figure 2b) as seen from the characteristic high and low contrast areas observed (Figure 2a inset). In order to reconstruct the 3D structure of OSCs, 17.946 single-particle TEM images were recorded and analyzed using the structure elucidation program Relion®. 20,21 As depicted in Figure 3b, the 3D-reconstruction confirmed the cage-like structure of OSCs, revealing the presence of a relatively large inner cavity of around 5 nm in diameter. The presence of an inner cavity within OSCs was further confirmed by N2 adsorption-desorption measurements. Sorption isotherms (Figure 3a) show a characteristic hysteresis indicating the presence of a porous material, and a pore size distribution with an average size of 8 nm (Figure 3b) and an average total pore volume of 0.577 cm3•g -1 were calculated. The calculated specific BET surface area of was measured to be 437.0 m 2 •g -1 . The results obtained were comparable with the nitrogen-adsorption data reported by Wiesner and co-workers for cage-like inorganic silica particles. 16 Although the final OSCs dispersion obtained after purification was highly concentrated (c=3 mg•mL -1 ), a transparent and stable colloid was obtained (Figure 4a) and DLS analysis (Figure 4b) revealed the particle mean hydrodynamic diameter to be a 23 nm. The presence of short PEGchains (6-9 units) on the particle surface was crucial for the colloidal stability of OSCs, since intense aggregation of the particles was observed when prepared without, as confirmed by DLS (Figure 4c). The enhanced colloidal stability upon PEGylation can be explained by steric repulsion between single particles created by the coiled polymers chains. 22,23 In order to investigate the biological features of OSCs, the red-emissive dye sulfo-cyanine5 (sCy5) was grafted onto the particles (see section 3.4.2. and 3.4.4. for details). As depicted in Figure 4d, upon sCy5 grafting of the particles surface, OSCs showed the characteristic absorption (λmax=650 nm) and emission (λmax=670 nm, λex=600 nm) spectra of sCy5, possessing high fluorescent quantum yield of 52%. The organic content of OSCs was first investigated by TGA on dried samples (Figure 5a). The overall organic content was determined to be roughly 50 wt%. The amount of disulfide-linker integrated within the silica framework was determined to be 11 wt% by comparing the thermogram of non-functionalized OSCs with that of particles prepared analogously but not containing the disulfide bridge. Consequently, the amount of PEG chains grafted onto OSCs was calculated to be around 32 wt%, whereas the amount of dye could not be discriminated by TGA, due to the low amount used to functionalize OSCs. TGA analysis also confirmed the complete removal of the surfactant upon dialysis. ATR-FTIR spectra (Figure 5b) recorded on dried OSCs The OSCs were loaded with the hydrophilic dye HOECHST 33258 (HE) and its release in the presence of GSH was studied by fluorescence spectroscopy to further prove particle degradation.

OSCs non functionalized with PEG were loaded by impregnation method (see section 3.4.6. for details) with a highly concentrated HE solution and subsequently PEGylated to entrap the dye within the internal cavity (OSCs-HE). The particles were subsequently washed to ensure the removal of physiosorbed HE on the particle surface (see section 3.4.6. for details). OSCs-HE dispersions (c=0.1 mg•mL -1 ) in PBS buffer solutions were prepared with and without GSH (10 mM) and the cumulative cargo-release of HE was followed by fluorescence spectroscopy in the supernatant of the particle dispersions (λem=492 nm, λex=350 nm). As depicted in Figure 10a, the cargo loading was estimated to be 10 wt% upon comparison of particles that have been prepared

as OSCs-HE, but not loaded with the dye. Ϛ-potential analysis of OSCs-HE showed that the surface potential became more positive, likely due to charge compensation of loaded HE, since the cargo molecule possesses three positive charges per molecule. Release experiments (Figure 10c)

showed that a boosted HE-release was present when OSCS-HE particles were dispersed in PBS containing GSH, indicating the particles degradation and subsequent HE-release. The stimulitriggered release was further confirmed by stirring an OSCs-HE dispersion and measuring the HE emission at different time intervals before and after GSH addition and as depicted in Figure 10d.

Only upon the addition of GSH to the particle dispersion a boosted HE release was observed, further confirming the exclusive degradation of the particles in the presence of an external reducing agent. Having assessed the particles stimuli-triggered degradation and cargo release of a model drug molecule, the possibility to load OSCs with the hydrophobic anti-cancer drug docetaxel (DT) was investigated. DT, a member of the taxane class of anti-cancer drugs, is widely applicated for the treatment of cancers such as breast, stomach and prostate. Due to its hydrophobic nature, nanoformulations have been developed to increase the efficacy of the drug, resulting in the successful preparation of liposome-based delivery systems. The use of liposomes as carrier vehicle still faces challenges, such as liposome instability, short blood-circulation lifetimes and high productions costs. 27,28 The use of porous organosilicas, such as the here presented OSCs would instead provide small (20 nm) delivery system characterized by high stability and a stimuli responsive degradation behavior. Therefore, the particles have been loaded with DT in a two-step procedure. First, the drug was loaded into the particles cavity by stirring non-PEG functionalized

OSCs in an ethanolic DT solution (1/1 wt/wt) over night. Subsequently, the particles were functionalized with PEG, ensuring DT entrapment within OSCs, and the particles were washed by dispersion-centrifugation cycles to remove any physisorbed drug on the surface, yielding DT loaded OSCs (OSCs-DT, see section 3.4.6. for details). The removed supernatants were analyzed by UV-Vis absorption spectroscopy, which revealed that the physiosorbed drug was successfully removed from the particles surface, since no absorption of DT was measured in the washing solutions (Figure 11a). TGA allowed to determine a DT loading of 13 wt% (Figure 11b) upon comparison with the thermogram recorded for OCSs. After particle loading, the colloidal stability was not preserved, and massive aggregation was observed. Prolonged sonication and subsequent filtration of OSCs-DT dispersion (1 mg•mL -1 ) with a nylon syringe filter (cut-off 0.2 µm) helped to obtain a colloidal dispersion with a relatively homogeneous size distribution of OSCs as confirmed by DLS on purified OSCs-DT (Figure 12). The main drawback of the purification method was the high dilution of the purified OSCs-DT obtained, unpractical for in vivo experiments when the injection volume should be minimized.

Future work will further focus on establishing a robust and efficient methodology to load hydrophobic drugs within OSCs, while persevering their colloidal stability. The encapsulation of 150 hydrophobic drugs will be of great interest since its encapsulation in hydrophilic drug carriers, while obtaining high loadings, is still hardly achieved.

Surface functionalization of cage-like organosilica particles

As already discussed in the general introduction, the functionalization of nanoparticles with biological active molecules, such as cell-targeting peptides or sugar molecules, has the potential to increase the particle´s therapeutic efficacy. To functionalize silica nanoparticles with biological relevant biomolecules, such as sugars or peptides, alkoxysilane chemistry offers an opportune strategy for covalent surface grafting. 29 We aimed therefore to find a working protocol to graft the surface of OSCs with the tripeptide Arg-Gly-Asp (RGD) or the sugar D-(+)-glucosamine, aiming for mild functionalization reaction, which retain good colloidal stability of the particles and do not require laborious purification steps. Alkoxysilane chemistry was first selected to functionalize OSCs with D-(+)-glucosamine, and copper-free azide-alkyne cycloaddition reaction (SPAAC) was used to graft the particle surface with RGD. After sugar functionalization, the colloidal stability of the particles was investigated by DLS. As depicted in Figure 13, the colloidal stability of Sugar-OSCs was confirmed as the hydrodynamic diameter was 28±7 nm (Figure 14a). The Ϛ-potential measured for Sugar-OSCs was -23±1 mV, a slightly more positive mean value when compared to that for OSCs. The particles morphology after surface functionalization was analyzed by TEM, which indicated that particle morphology was retained after surface functionalization (Figure 13b). The amount of sugar-silane used for the functionalization was 3.5 mg per 100 mg of non-functionalized OSCs. Higher amounts of sugarsilane led to the formation of unstable colloidal dispersions of Sugar-OSCs, most likely due to the increased presence of H-bonding units, mediating interparticle attraction forces. 31 154

In order to functionalize the particles with the alkyne-RGD peptide, OSCs were first functionalized with azide groups, upon reaction of OSCs with 3-azidopropyltrimethoxysilane (AzTMS, see section 3.4.2. for its preparation), and the particles purification occurred by dialysis against an EtOH/H2O (1:1 v/v) mixture for 12 h (Scheme 4). The amount of AzTMS was chosen to be 5 mol% in respect to TEOS used for the preparation of OSCs, which did not affect the final colloidal stability of N3-OSCs as confirmed by DLS, which revealed an average hydrodynamic of 26±4 nm. The presence of azide groups on the particle surface was determined by ATR-FTIR (Figure 15), which indicated the presence of an intense band at 2100 cm -1 corresponding to the characteristic asymmetric -N=N=N stretching vibration, 32 and was in agreement with spectra recorded on neat AzTMS. Finally, the peptide was conjugated onto the particle surface upon the addition of Alkyne-RGD to a stirring N3-OSCs particle dispersion, allowing to react overnight. The particle dispersion was then purified by means of dialysis in water for 48 h, yielding RGD-OSCs particles (see experimental section). The amount of peptide conjugated onto the particle´s surface was estimated via fluorimetric analysis using a previously reported fluorescence quantification method (see experimental section). 33 For this purpose an aliquot of RGD-OSCs was allowed to react with 9,10-phenantrenequinone (PQ), a reagent which specifically reacts with arginine in order to give a fluorescent reaction product after the addition of acid (Scheme 5). The amount of RGD covalently linked onto the particle surface was estimated upon measuring the fluorescence intensity of RGD-COS (Figure 16a) that have been derivatized with PQ (c=0.0107 mg/mL) at the wavelength of emission λem=424 nm (λex=312 nm). Upon comparison of the measured emission with a previously established calibration curve based on the reaction of RGD with PQ at different concentrations (Figure 16b), a total amount of 25 µg per mg of particles was calculated (see section 3.4.5.). Herein, the possibility to functionalize these novel types of cage-like organosilica particles with a variety of bioactive small-molecules was proven, while maintaining their colloidal stability in aqueous dispersions. Future work will focus on the functionalization of the particles with specific cell surface receptors to evaluate the possibility to quantitatively cross the blood-brain-barrier (BBB), one of the major challenges for the development of nanomedicines for brain delivery applications.

Preliminary biological evaluation of cage-like organosilica particles

Once the physicochemical properties of OSCs have been assessed and their biodegradability successfully proven, the particle cytotoxicity was investigated. The preliminary biological evaluation was carried out by the group of Prof. Bigini (Mario Negri Institute). Although previously reported in vitro cytotoxicity studies on disulfide-bridged porous silica particles have shown good biocompatibility towards different cell-lines, the toxicity of OSCs were investigated to ensure safety during in vivo applications. Therefore, glioma C6 cells were incubated with OSCs at different concentrations (50, 100 and 200 µg•mL -1 ) and the cell viability was evaluated using the trypan blue test, a dye-exclusion procedure that stains dead cells. As depicted in Figure 17, OSCs showed no relevant cytotoxicity even at high concentrations after 24 and 48 h of incubation time, confirming the non-toxicity of disulfide-based organosilica particles. With the positive cell-viability tests obtained the biodistribution of OSCs and Sugar-OSCs in healthy rodents was investigated. The biodistribution of small cage-like organosilica particles is still unexplored and a first evaluation of their in vivo behavior is therefore of interest, since distinct biodistribution behavior according to the size and shape nanomaterials has been reported. 15 The biodistribution studies included also the glucosamine functionalized particles, since the presence of the sugar moiety may enhance cellular uptake upon the interaction with GLUT-cell surface receptors present on the endothelial layer of the BBB, therefore having the potential to enable enhanced particle adherence on this biological barrier. Glucosamine was chosen considering that the C-2 position of the sugar can be modified without loss of receptor activity for the GLUT1-cell surface receptor. 35 In addition, glucosamine would provide a reactive amine group that could be easily used for OSC functionalization through alkoxysilane coupling.

First biodistribution studies on OSCs were performed on healthy CD1 mice model, injecting OSCs via one-time intravenous tail injection (dose=7 mg particles per kg of mouse) and analyzing the animals after different time points (30 min, 1 h, 4 h and 24), using 6 animals for each experimental group (see section 3.4.9. for details). The presence of sCy5 on OSCs resulted to be ideal since the imaging occurs in the red region of the electromagnetic spectra, diminishing the autofluorescence signal from the mice bodies while further reducing the oxygen-induced attenuation. The results obtained from ex vivo optical imaging of the particles in various organs are shown in Figure 18; the fluorescent signals are normalized with respect to the mouse to which only a saline solution has been administered (vehicle). The particles were found to accumulate in various organs well known for the elimination of nanoparticles. Interestingly, OSCs showed a 13-fold increased signal in the lung compared to Sugar-OSCs, which decreased by about 3 times after 24 h (Figure 18a). This behavior may be explained by the fact that OSCs possess a longer blood circulation lifetime compared to Sugar-OSCs, leading to their accumulation in the lungs. Similar results were previously reported for spherical nanoparticles of 20-30 nm that were shown to accumulate in the lungs within the first 30 min after intravenous administration. 18 This can be explained most probably by the fact that the PEG-chains protect the particles in the blood-stream from instability, while the presence of a sugar may decrease this ability of PEG stabilization in the blood-stream. To evaluate the uptake in the lung, histological analysis was done on lung sections (Figure 19). OSCs were only found in the lung parenchyma, whereas no signal related to the presence of Sugar-OSCs was observed. In the liver, a comparison between the accumulation of OSCs and Sugar-OSCs did not show a statistical difference, indicating that the presence of the sugar on the particle surface did not affect the kinetics or liver uptake (Figure 18b). Most interestingly, immunohistology of the liver indicated that OSCs and Sugar-OSCs did not accumulate in hepatic macrophages (Kupffer cells) usually involved in the clearance of nanoparticles from the liver. As depicted in Figure 20, no colocalization of the particles (red) with macrophages (green), which had been stained with fluorescein-labeled CD68 antibodies, was observed. This differentiated accumulation indicated the possibility that the particles are taken up by other cells of the liver, most likely epithelial cells.

These findings suggest that the particles could serve for the development of drug-delivery carriers for the treatment of liver cancers. In the kidneys, the fluorescence signal of OSCs and Sugar-OSCs was relatively low, excluding a renal clearance of the particles (Figure 18c). It is known that renal clearance of nanoparticles is very strong for particles smaller than 5 nm, and the particles subjected in this study were too large to be excreted by the kidneys. 36 The particles were found to accumulate to a greater extend in the spleen than the liver and kidneys, which can be explained by the fact that the spleen is the main organ particle excretion (Figure 18d). Compared to OSCs, Sugar-OSCs seemed to be eliminated more slowly from this organ, which could be explained by the fact that sugarfunctionalized particles form bigger aggregates in the blood that persist longer in the spleen.

Histological analysis of spleen samples after 1 h and 4 h of the treatment indicated that the particles accumulated in the red pulp (RP), a region related to clearance of common antigens (Figure 21). Interestingly, no migration of the particles in other parts of the spleen, such as the marginal zone (MZ) or the white-pulp (WP), was observed. The particle´s unique morphology, small size, and the presence of a sugar functionality may enhance the particle adherence on the epithelial layer of the BBB, possibility leading to enhanced BBB crossing. In Figure 22a, ex vivo fluorescent signals on the brain samples of mice treated with OSCs, revealed only a minor signal derived from the brain, which after 24 h was comparable to the intensities measured for the vehicle. Additionally, no significant statistical variation regarding the detected signal between OSCs and Sugar-OSCs was observed. To obtain a more quantitative information on the presence of the particles in the brain, inductively coupled plasma atomic emission spectroscopy (ICP-AES) was performed on the brain samples after 4 h of treatment. The ICP-AES results are shown in Figure 21b and indicate that a minor number of particles were able to cross the BBB, indifferently from the presence of a sugar moiety on the particles surface. Similar results were obtained based on histological analysis of the brain after 4 h of treatment with Sugar-OSCs (Figure 23). The particles could be mainly detected in in the region of lateral ventricles of the brain and no inner penetration of brain areas, such as the caudate putamen (cPu) 163

These results show that BBB crossing of OSCs and Sugar-OSCs is less than 1 % with respect to the total amount of injected particles (ID), that is, 0.2 % ID for OSCs and 0.3 % ID for Sugar-OSCs (Figure 22b). Although previously reported in-vitro experiments using BBB-models suggested an enhanced BBB crossing of sugar-functionalized gold and polymeric nanoparticles, 37,38 our experiments confirmed that this effect is negligible and almost absent in in vivo models, which is in agreement with recently results published by Kataoka and coworkers. 39,40 In fact, although it has been reported that the brain uptake of nanoparticles in healthy mice is somewhat size and shape dependent and that smaller particles are preferentially accumulated in brain regions, the amount that reaches the brain is generally low and below 1% ID. 39,41,42 Therefore, for future application in which brain accumulation of OSCs is desired, the particles must be functionalized with adequate cell targeting units to enhance the particles BBB crossing via transcytosis.

Interesting cell-surface targeting ligands to be used for OSCs functionalization could be ganglioside targeting peptides such as the recently reported G23 peptide by Zuhorn and coworkers 43 or glycopeptide G7 reported by Tosi and coworkers. 44

Conclusions

In this chapter, the preparation and characterization of novel porous cage-like organosilica particles was presented. The particles were found to be biodegradable due to the presence of a reducible disulfide moiety embedded within the silica framework and showed rapid rates of degradation due to their small size and highly accessible inner cavity. It has been also reported that the particles can be loaded with hydrophilic and hydrophobic molecules, demonstrating the stimuli-triggered cargo release upon the presence of a reducing agent, such as the endogenous tripeptide glutathione. Furthermore, facile and mild OSCs functionalization procedures for have been established, proving the successful covalent conjugation of bioactive molecules to the particle surface. Preliminary biological experiments indicated that the particles are nontoxic and the OSCs bioaccumulation was investigated upon intravenous injection in healthy mice.

Interestingly, in the liver OSCs did not co-localized with macrophages, rendering these particles interesting for the development of drug-delivery carriers against liver-cancers. Strong accumulation of the particles in the lungs of mice have been observed when no sugar surface functionalization was present, which was explained by the fact that the particles possess good blood-serum circulation lifetimes. To enhance the BBB crossing of the particles, future experiments will focus on the ad hoc functionalization of OCSs with peptide-based ligands of cell surface receptors by using the functionalization procedures developed in this work.

Experimental section

Instruments

1 H, 13 C Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 400 spectrometer. The 1 H NMR chemical shifts (δ) are given in ppm and refer to residual protons on the corresponding deuterated solvent. All deuterated solvents were used as received without any further purification. All coupling constants (J) are given in Hertz (Hz).

The attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectrum and the Fourier transform infrared (FTIR) spectrum was measured with a Shimadzu IRAffinity-1 spectrometer.

The transmittance spectrum was collected using a spectral resolution of Δ=1 cm -1 , accumulating 64 scans and the ATR-FTIR spectra were ATR corrected (penetration depth mode).

Absorption spectra were measured on a Shimadzu UV-3600 spectrophotometer double-beam UV-VIS-NIR spectrometer and baseline corrected. Quartz cuvettes with path length of 10 mm were used for the recording of UV-Vis absorption spectra.

Emission spectra were measured on a Horiba Jobin-Yvon IBH FL-322 Fluorolog 3 spectrometer equipped with a 450 W xenon arc lamp, double grating excitation, emission monochromators

Docetaxel (DT) loading of OSCs.

To load non-functionalized OSCs with the hydrophobic drug docetaxel (DT), OSCs were prepared but no surface functionalization occurred. After CTAB removal via dialysis (as described above) to an aliquot of the particle dispersion (784 μL, 4 mg of particles), EtOH (600 μL) and Doxotaxel (2mg) were added. The resulting suspension was briefly sonicated and subsequently allowed to stir over night at room temperature. The particles were then functionalized with PEG to reestablish the good colloidal stability and to physically entrap the drug into the organosilica cavity. Therefore, PEG-silane (6.4 μL) was added to the drugparticle dispersion. After 30 min, water (500 μL) was added and the particle dispersion was stirred over night at 50 o C to allow the PEG-to hydrolyze and condense on the particle surface. The particles were then collected by centrifugation (5 min, 14.8 krpm) and washed with an EtOH/H2O

(1:5 v/v) mixture by dispersing and centrifuging the particles, until no Docetaxel could be observed in the washing solutions. Therefore, the supernatant of the washing solution was collected, dried under reduced pressure, and the dried sample was solubilized in EtOH (100 μL)

to record the UV-Vis absorption spectra. To evaluate the loading, an aliquot of the particles was taken (so that around 2 mg of loaded particles could be analyzed) and the sample was dried before the thermogravimetric analysis.

Cell viability experiments

5x10 4 Glioma C6 cells were seeded in two 24-well plates (culture media: DMEM supplemented with 10% FBS and 1% penicillin). After 5 hours incubation at 37°C 5% CO2, the spent cell culture media was discarded. After 20 minutes sonication, a certain volume of an aqueous stock solution of nanoparticles (1 mg•mL -1 ) was withdrawn and added to the new complete culture media in order to have three different NPs concentrations, that is, 50, 100 and 200 μg•mL -1 . The positive control was represented by a solution of 10% DMSO in complete culture media, while simple complete culture media was used as negative control. The cells were incubated at 37°C for 24 and 48 h. In each plate, each test was performed in three replicates, in order to increase the statistical validity of the results. After 24 and 48 h the Trypan Blue exclusion test was performed.

The spent cell culture media was removed from each well and stored separately in different 2 mL Eppendorf tubes. The cells were washed using 100 µL of PBS in order to collect all the dead cells.

The washing solution was added in the 2 mL Eppendorf tubes with the old media. The cells were detached using 100 µL of trypsin and incubated for 5 min. After trypsinization, 100 µL of prewarmed complete growth medium were added and transferred in the 2 mL Eppendorf tubes with the old media and the PBS, the tubes were centrifuged at 18.000 rpm for 3 min. After discarding the supernatant, 100 µL of the new media were added and the cells resuspended. 50 µL of the cell suspensions were added to 50 µL of the Trypan Blue solution, and 10 µL from the above solution were placed in a cell counting slide. The cell viability was determined using a Countess automated cell counter. The whole experiment was performed twice,to increase the statistical validity of the results.

Biodistribution studies

Animal treatment: CD1 mice, ranging from 20 to 25 grams of weight, were recruited for biodistribution experiments. All studies were carried out with healthy, specific pathogen free and immunocompetent animals in compliance with welfare laws and according to the approval of the Italian ministerialresearch project number "669/2015-PR". For the first study, a single dose of NP (7 mg Silica/ kg of body weight) was administered by intravenous injection from the caudal vein (the same dose but in a volume of 5 µL). After NP administration, the mice did not show any evidence of stress and discomfort for the whole duration of the experiment. The study was carried out including at least 6 mice for each experimental group. Mice were randomly divided and received 200 µL of the following solutions: i) PBS alone; ii) PBS+OSCs; iii) PBS+Sugar-OSCs.

Animals were sacrificed at different time-points (30 min, 1 h, 4 h, 24 h and 120 h) after the treatments with an overdose of anesthetic. Brain, lungs, spleen, liver and kidneys were removed and stored before ex vivo studies.

Optical imaging:

The presence of a stable link connecting OSCs with a fluorescent dye enabled us to determine the distribution of OSCs in the body with a high sensibility and specificity. Briefly, living animals were anesthetized at different time-points and were positioned in prone position in a room connected with a source of light with a peak of emission at 630 nm (corresponding to the peak of excitation of the fluorophore attached to NPs). The instrument Lumina XRS was used for all scanning. Since the brain is anatomically well distinguishable from the other organs, it was possible to obtain reliable results using the images directly obtained from living mice. Opposite the scanning of toraco-abdominal region does not allow to discriminate among the different organs that overlaps among them. For this reason, the in vivo imaging is not reliable, and we decided to measure the fluorescence of lungs, liver, spleen and kidneys after sacrifice and organ removal. To avoid the background contribution associated with the autofluorescence of the tissues, spectral unmixing process was performed during the scanning. Spectral unmixing permits to distinguish between the dye and the background through the kinetics of extinction of the two different signals in the whole area of analysis. Both ex vivo and in vivo analyses are quantitative, and are expressed as relative radiant efficacy normalizing the average of signal originating from untreated mice to 1.

Ex vivo analysis.

To determine the amount of silicon in brain and other organs samples were mineralized and processed by ICP-AES. Samples (1 mL) were diluted to a total volume of 10 mL using aqueous 2% nitric acid. All solutions were freshly prepared from UV-sterilized deionized

Introduction

Organosilica particles (OSPs) are hybrid organic-inorganic materials that find application in various fields such as catalysis, 1-3 photovoltaics, 4,5 separation sciences 6,7 and pollutant remediation [8][9][10] . Many different organic functional groups can be integrated within the silica structure of OSPs via sol-gel chemistry using organo-bisalkoxysilanes, thereby yielding particles endowed with specific physiochemical properties. 11 As mentioned in previous chapters, porous and stimuli-responsive OSPs that degrade upon exposure to external stimuli are attractive carriers for the development of future nanomedicines with improved biological safety. Examples of unexplored molecules for the preparation of OSPs include single-stranded deoxyribonucleic acid (ssDNA) and single-stranded peptide nucleic acid (ssPNA) based oligonucleotides. These molecules contain attractive organic functional groups for the preparation of OSPs that can ensure unique physicochemical properties, such as programmable base-pair complementarity and enzymatic degradability. Furthermore, by using specifically designed DNA sequences, such as aptamers, a variety of small molecules, metals, and even living cells can be sensed with high selectivity. 12,13 The integration of oligonucleotides within an inorganic framework can therefore lead to the preparation of hybrid materials with strong stimuli-dependent properties, ranging from stimuli-responsive degradation to selective binding of small molecules or even cells. Due to the precise programmability of DNA and PNA oligonucleotides regarding their hybridization with complementary strands, they were previously used in the preparation of self-assembled nanoparticles. [14][15][16][17][18][19] Aptamer-functionalized nanoparticles have been previously reported for either the detection of biomolecules, 20,21 cells 22,23 or for drug-delivery applications. 24,25 Porous silica particles have been previously reported as carriers for the delivery therapeutic DNA, [26][27][28] RNA [29][30][31] and PNA 32 oligonucleotides. Furthermore, the development of DNA-gated delivery systems 33,34 for biomedical applications was successfully reported, but the preparation of DNA or PNA-bridged organosilica particles has not yet been described in the literature. Integration of ssDNAs, such as aptamers, or ssPNAs would provide organosilicas with the ability to selectively bind biomolecules allowing for molecular recognition, accumulation and detection of diseaserelated biomarkers. [35][36][37] In order to prepare porous OSPs, in which the organic molecule serves as bridging group with the silica framework, typically organo bis-alkoxysilanes are hydrolyzed and condensed, beside a common silica source (e.g. tetraethyl orthosilicate), in the presence of a surfactant (e.g. cetyltrimethylammonium bromide). 11 Organoalkoxysilanes can also be thought to be used as monomers for the preparation of self-assembled structures, which can then be further hydrolyzed and condensed to form particles, consisting of supramolecular, that is noncovalent and covalent bonds. This approach would form materials which can be categorized as hybrid bonding polymers. 38 In this respect, the self-assembly of organoalkoxysilanes has been previously reported [39][40][41][42][43] and functionalized gold nanocrystals 44 as well as functionalized liposomes 45 have been reported by exploiting the self-assembly of such molecules. Nevertheless, the use of PNA oligonucleotides to form supramolecular assembled bis-alkoxysilanes, which can be further polymerized to prepare organosilica particles, has not been yet described.

In this chapter, the possibility to use various alkoxysilane derivatives of single-stranded DNA and PNA oligonucleotides to serve as novel stimuli-responsive building blocks for the preparation of stimuli-responsive organsilica particles was investigated. To this end, novel DNA and PNA bisalkoxysilanes were synthesized and used to form organosilica particles by using a modified Stöber synthesis protocol. The ability of the particles to bind complementary ssDNA oligonucleotides and their degradation upon enzymatic activity was studied. Furthermore, inspired by the recent work reported on hybrid bonding polymers, 46 the possibility to self-assemble complementary PNA-based alkoxysilanes to prepare organosilicas was investigated. To this purpose, singlestranded complementary PNAs, which have been synthesized by Corradini's group (University of Parma), were derivatized to alkoxysilane derivatives and allowed to self-assemble to form supramolecular PNA-based bis-alkoxysilanes. These assembled alkoxysilanes were directly hydrolyzed and condensed to form organosilicas bearing a supramolecular bridging group within a covalent network of condensed alkoxysilanes. The presence of the supramolecular assembly within the particles was successfully characterized, indicating the possibility to form new types of stimuli-responsive organosilicas, which resemble some of the properties of hybrid bonding polymers.

Results and Discussion

Preparation of single-stranded DNA and PNA organosilica particles

As mentioned in the previous chapters, covalent integration of an organic functional group within a silica framework requires organo bis-alkoxysilanes to be hydrolyzed and condensed together with a common silica source, such as TEOS, in aqueous media. To integrate ssDNA within the framework of organosilica particles, ssDNA oligonucleotides were first prepared and subsequently derivatized to their corresponding bis-alkoxysilanes. ssDNA oligonucleotides and their backbone-modified analogues were prepared with a DNA synthesizer using a standard phosphoramidite protocol, yielding the diamino-modified and cyanine 3 (Cy3) labeled The use of a 10-mer oligonucleotide with its specific base-pair sequence was chosen because its hybridization with complementary DNA strands can occur at room temperature. To investigate the influence of the oligonucleotide length on the final particle morphology, a shorter 4-mer ssDNA bis-alkoxysilane was prepared analogously as described for the synthesis of 2 (see section 4.4.3.). Before using 2 to prepare organosilica particles, a protocol for the preparation of pristine mesoporous silica particles (MSPs) under slightly basic (pH=8.5) and low EtOH content conditions was optimized, to ensure the chemical stability of ssDNA during the particle preparation. By applying a modified Stöber synthesis and varying the H2O/EtOH, the synthesis of MSPs was optimized to obtain 100 nm MSPs (see section 4.4.4.). TEM images (Figure 1), showed that the particle size distribution narrowed as the water content in the reaction mixture was increased, while the concentration of TEOS, CTAB and NH3 concentrations were kept constant. The formation of smaller particles in reaction mixtures with a higher water content could be explained by the fact that the contact of water molecules with TEOS increased in such mixtures, leading faster to hydrolysis and nucleation of the silica source. The reactions were carried out at pH=8.5 where condensation rates are known to outcompete hydrolysis rates for alkoxysilanes, which further explains the fast formation of nucleation sites in reaction mixtures, possessing greater amounts of water. 47 Furthermore, TEM analysis suggested an increased porosity upon formation of smaller MSPs. A solvent ratio of 1:0.3 (v/v) was selected to prepare ssDNAorganosilica particles based on the formation of a narrow distribution of small (101±7 nm) MSPs (Figure 1b andd). This loss in porosity can be explained by the high negative charge density and greater steric hindrance of the ssDNA, which interfere with the cooperative self-assembly mechanism of silanols and CTAB micelles, leading to a highly disordered pore arrangement. This hypothesis is supported by previously published work on porous organosilica particles in which a decrease in porosity and pore-order was observed when organoalkoxysilanes with greater structural flexibility were used to form OSPs. The use of a shorter ssDNA-silane composed of 4 nucleobases (GCGC) generated particles with increased porosity (Figure 3), confirming the hypothesis that negative charge density and sterically hindrance of the ssDNA organoalkoxysilanes can influence particle porosity. As depicted in Figure 4b, the SAXS pattern obtained from ssDNA-OSPs showed the presence of a broad signal at q=1.3 nm -1 , indicating the presence of an ordered structure within the particles.

Analysis of the TEM images could not determine whether the SAXS pattern was produced by the presence of mesopores or a different ordered structure existing within ssDNA-OSPs.

Interestingly, SEM images of ssDNA-OSPs at higher magnification (Figure 4a) show that the particles possess a rough surface and seem to be constituted from smaller spherical particles.

Therefore, it cannot be excluded the possibility that the observed SAXS pattern was produced by this smaller-particles, which were arranged in either a hexagonally or face-centered closed packing. Analysis regarding the approximate size of the scatterer from the SAXS pattern observed, its size was calculated to be ca. 5 nm (see section 4.4.8. for calculation). This calculated value is in good agreement with the size of the small particles observed in Figure 4a, considering that the gold sputtering during the SEM sample preparation produced an additional gold-layer with thickness of ca. 8 nm. Further, when at a later stage the degradability of the particles in the presence of a hydrolase was assesses via SEM images (Figure 7b), it was evident that ssDNA-OSPs degraded into small spherical debris, suggesting further that the initial particle was composed of smaller silica particles. The presence of the ssDNA moiety within the framework of ssDNA-OSPs was investigated by UV-Vis absorption spectroscopy, XPS analysis and ATR-FTIR spectroscopy. As depicted in Figure 5a, the UV-Vis absorption spectra of ssDNA-OSPs in H2O showed a characteristic absorption band of compound 1 (Figure 5b) as indicated by the absorption band maxima centered at λ=260 nm and λ=550 nm, which can be attributed to purine and pyrimidine DNA nucleobases and the Cy3 moiety, respectively. The ATR-FTIR spectra indicated the presence of the organic functional group within ssDNA-OSPs (Figure 5c), showing the characteristic transmissions bands at 1474 cm -1 and 1649 cm -1 , both attributable to the presence of DNA. 51 The transmission bands occurring from Once the presence of ssDNA within the particles structure was confirmed, the stimuli-responsive properties of ssDNA-OSPs upon the presence of external stimuli was investigated. First, the degradability of the particles structure upon activity of DNAse enzymes was investigated. A ssDNA-OSPs dispersion in Tris-buffer (c=0.04 mg•mL -1 , pH=7.6), DNase I enzyme was added to a particle dispersion and the particles were incubated for 1 h at 37 °C (see section 4.4.6.). DNase I enzymes are endonucleases able to hydrolyze single-stranded DNA oligonucleotides independently from the oligonucleotides sequence. The degradation of the particles was followed by SEM and DLS. As depicted in Figure 7, SEM images confirm the DNase-triggered degradation of ssDNA-OSPs, indicated by the loss of the initial particle morphology and the formation of small debris, most probable due to the particle disintegration. Particles that have been stirred in in the same conditions but in the absence of the enzyme did not indicated the formation of small degradation debris, confirming the role of the enzyme in the particle degradation. Degradation experiments suggested that the particles were composed of smaller silica particles (Figure 7b, SEM image with DNase). It can be therefore hypothesized that the ssDNA-silane would bridge the small spherical objects within ssDNA-OSPs and when hydrolyzed in the presence of DNase, the structure subsequently breaks apart and forms small debris. Additional confirmation of the particle degradation upon enzymatic activity was obtained by DLS.

As shown in Figure 8a, the hydrodynamic diameter of ssDNS-OSPs incubated with DNase for 1 h in Tris-buffer solution (see section 4.4.6) decreased significantly, while in the absence of DNase, no drastic particle degradation was observed. DLS analysis on ssDNA-OSPs showed that when the particles were stirred at 37 °C for 1 h in the absence of DNase, only a relatively small decrease of the hydrodynamic diameter was observed, most probably due to slow silica dissolution in the buffer at this temperature (Figure 8b). As previously mentioned, electron microscopy images of ssDNA-OSPs suggested that the presence of the DNA during the particles synthesis alters the formation pathway of the particles.

The altered final morphology of ssDNA-OSPs, compared to model MSPs obtained with the same protocol, could be explained by the highly negatively charged DNA that strongly interacts with other charged species in during the Stöber synthesis protocol, while the role of the DNA length was confirmed to impact particle porosity. To prepare oligonucleotide-bridged organosilica particles with a uniform size distribution, the use of uncharged single-strand peptide nucleic acid

PNA-based and supramolecularly assembled organoalkoxysilanes for the preparation of organosilicas

As mentioned in the introduction, the use of complementary ssPNA oligonucleotides to prepare supramolecular assemblies of bis-organoalkoxysilanes has been envisioned to produce novel hybrid bonding polymers (HBPs). The 10-mer ssPNA strands (F-PNA and T-PNA) used were designed to be complementary and to hybridize easily at room temperature in organic solvents, such as DMSO (Figure 11). The use of a 10-mer ssPNA was selected because it can form a helical structure already at rom temperatures, similar to that of DNA. 54 Having assessed the ability of the PNA strands to self-assemble, their alkoxysilane derivatives were prepared. To this purpose, F-PNA and T-PNA were allowed to react with 1.1 eq. of ). Subsequently, both strands were mixed, resulting in the formation of the supramolecular PNA bis-alkoxysilane (Supra-PNA-Si; Figure 13).

representing an elongated hydrolysable alkoxysilane. Therefore, the formation of the structures observed in Figure 15 could be explained by the polymerization of Supra-PNA-Si, the subsequent formation of smaller grain-like structures which then assemble to form the final μm-sized structures through hydrolysis and condensation of uncondensed silanol groups (Figure 15c). The yield of these structures observed at 3 h of reaction time was low (less than 1 mg) and no quantitative amounts of the particles were recovered by centrifugation. to form new and more stable organosilicas. Incomplete condensation degrees in organosilicas can be explained by the fact that hydrolysis and condensation rates of organosilanes are structure dependent, experiencing inductive and steric effects from the substituents. 57 In Supra-PNA-Si the Si-atoms possess one alkyl substituent, having a positive inductive effect on the Si atom.

Therefore, the alkyl group in Supra-PNA-Si renders the Si atom less electrophilic compared to tetra alkoxy silanes, resulting in decreased hydrolysis rates and condensation degrees in basic solutions. The small particles observed for Supra-OSPs are hypothesized to represent degradation debris derived from previously formed grain-like structures. In this case, the formation of grain structures would represent a metastable morphology observed for Supra-OSPs. To further investigate whether the particles degrade upon prolonged reaction time and to test the possibility to stabilize the grain-like structure of particles which were observed in the supernatant at 3 h of reaction time, hydrolysis and condensation of Supra-PNA-Si in the presence of an equimolar amount of tetraethyl orthosilicate (TEOS) was performed. A secondary silica source would increase the amount of highly condensed inorganic -Si-O-Si-bonds in the resulting organosilica, thus yielding more stable rice-grain-like structures. To follow the structural evolution of organosilica particles formed in the presence of TEOS, aliquots of the reaction mixture were analyzed at different time intervals by SEM. When TEOS was co-condensed to Supra-PNA-Si, the presence of several μm-long fiber-like structures were observed after 1 h (Figure 17a,b and c), and they continued to grow when the reaction was allowed to proceed for 3 h (Figure 17d,e). Also in this case, the fibers were primarily composed of smaller, sub-μm structures, suggesting a similar formation pathway as it was previously hypothesized for the formation of rice-grain-like structures (Figure 15c). SEM images recorded on an aliquot of the crude reaction at 12 h indicated the absence of fibrous particles. Instead, the presence of smaller spherical-like particulate was imaged, suggesting again the instability of these fiber-like structures over prolonged reaction times (Figure 17f). Indeed, some of the very few remaining fiber-like structures present after 12 h confirmed the hypothesis of the material degradation. As depicted in Figure 18, these remaining fibers showed strong fragmentation and degradation, leading to the formation of sub-µm sized degradation products, which could be imaged (Figure 18). Therefore, the preparation of more resistant fiber-like structures by co-condensation of an equimolar amount of TEOS and Supra-PNA-Si, was not effective. In conclusion, co-condensation of an equimolar amounts of TEOS and Supra-PNA-Si did not increased the stability of fiber-like structures, suggesting that increasing the number of -Si-O-Si-bonds alone is not sufficient to obtain stable fibrous structures. Ultimately, the degradation of the organosilica materials could be explained by the dissolution of silicates in aqueous basic media, considering that no positively charged surfactants, which are known to stabilize the silicates through electrostatic interactions against hydrolysis, 58 were used for the preparation of Supra-PNA-Si. Future experiments will investigate the formation of organosilica particles from Supra-PNA-Si in the presence of the surfactant CTAB. Next, the presence of the supramolecular PNA-bridge within Supra-OSPs was investigated via FRET analysis (Figure 20, see experimental section). A Supra-OSPs particle dispersion (c=0.008 mg•mL -1 ) in PBS (pH=5.8) was excited at 450 nm and the photoluminescence was recorded. As depicted in Figure 20c, excitation of the FRET-donor moiety at λex=450 nm (F-PNA moiety) saw the quenching of the donor emission (λem,F-PNA=517 nm), while emission from the FRET-acceptor (T-PNA moiety) λem,T-PNA=590 nm was observed, indicating the PNAs hybridization within Supra-OSPs. In addition, the possibility to dissociate the PNA hybridization within Supra-OSPs upon heating was also investigated by FRET-analysis. As depicted in Figure 20a, elevated temperatures can disrupt the supramolecular interactions between PNA strands in Supra-OSPs, and consequently disrupt FRET. A Supra-OSPs dispersion was heated at 60°C for 10 min and the particle dispersion was subsequently excited with light of 450 nm while monitoring the emission.

As depicted in Figure 20d, heating was found to interrupt FRET between the donor and acceptor moieties, as indicated by the increased emission intensity observed for the donor strand (F-PNA).

These experiments indicate that upon hydrolysis and condensation of Supra-PNA-Si, organosilica particles are formed that possess hybridized PNAs as bridging groups within their framework, which can be dissociated upon the presence of an external stimulus, for example, heat.

Organosilicas prepared through hybrid DNA-PNA supramolecular interactions

The successful preparation of PNA-based organosilicas prompted the preparation of stimuliresponsive organosilicas. Therefore, the possibility to use the self-assembly of a PNA-DNA-PNA assembly as stimuli-responsive bridging group for the preparation of organosilica particles has been investigated. The choice to combine of DNA and PNA interactions was due to the fact that the DNA moiety allows for precise interaction, depending on the DNA base-sequence, with biomolecules. As depicted in Figure 21, a supramolecular PNA-DNA-PNA bis-alkoxysilane Supra-PDP-Si is obtained by mixing of two single-stranded PNA-alkoxysilanes (PNA-Si1 and PNA-Si2), which were designed to be complementary to 10-mer regions at the 5´and 3´end of a 45-mer single-stranded DNA (ssDNA). The 45-mer ssDNA was designed not only to hybridize with PNAsilanes, but also to possess an ATP binding sequence, as reported by Ricci and coworkers. 13 For this purpose the group of Prof. Ricci provided us the ATP-responsive ATP aptamer. The structure of fibers obtained from the polymerization reactions in which solely Supra-PDP-Si was used, varied from the fibers obtained when Supra-PDP-Si was polymerized in the presence of TEOS. Fibrous structures obtained from the polymerization of only Supra-PDP-Si at 3 h when possess rather smooth surfaces (Figure 23a), while the fibers obtained by co-condensing TEOS appeared to possess more layered internal structure (Figure 23b). The results obtained for the use of Supra-PDP-Si to prepare organosilicas are preliminary and future experiments will focus the thoroughly characterization of Supra-PDP-Si. Although these preliminary results show the presence of fiber/like structures, their composition must be further investigated to confirm their organosilica-based nature. Future experiments will investigate the stimuli-responsive behavior of the obtained fibers when ATP is present, since the DNA behaves as an ATP-aptamer, yielding to disassembly of the particles.

Conclusions

In this chapter, the possibility to use DNA and PNA-based building blocks to prepare stimuliresponsive organosilica particles was investigated. The synthesis of DNA and PNA organo bisalkoxysilanes and their subsequent use to prepare organo-bridged silica particles was reported.

DNA-bridged organosilica particles were prepared that possessed the ability to recognize complementary DNA molecules and to degrade in the presence of specific enzymes, such as hydrolyses. The advantage of using PNA bis-alkoxysilanes lies in the formation of more uniform particles. Moreover, the possibility to self-assemble complementary PNA alkoxysilanes to form a supramolecular PNA-based organo bis-alkoxysilane to be used for the preparation of hybrid-used to remove DMT protecting group on the DNA synthesizer. After the synthesis was completed, CPG was treated with 28% aqueous ammonium hydroxide solution for 16-18 hours at 60°C. The crude mixture then was concentrated under reduced pressure at 60°C, dispersed in MilliQ water and subsequently filtered by 0.22μm centrifugal filter before purifying by RP-HPLC using CH3CN/TEAA-buffer (3-50 % CH3CN, pH = 8) as eluent mixture and freeze dried. Pure compound 1 was obtained as a pink solid (0.2 μmol, yield 20% compared to the initial CPG loading). The shorter 4-mer oligonucleotide was analogously prepared as the procedure described above, but not labeled with any fluorescent dye. Preparation of 2. To a dispersion of 1 (2 mg, 0.5 µmol) in dry DMSO (50 µL), ICPTES (0.27 µL of a 4.04 M ICPTES solution in DMOS, 1.1 µmol ,2.2 eq) and TEA (0.5 µL) was added. The reaction mixture was stirred for 1 h at room temperature. The resulting reaction crude was used for the and 4 µM, whereas the final concentration of F-PNA was 1.143 µM in all three samples. After mixing the PNA solutions, the system was let to stand for 30 min under static conditions at room temperature. Subsequently, the emission spectra of the sample solution was recorded upon light excitation at λex=450 nm.

FRET experiments with Supra-OSPs. In a quartz cuvette a particle dispersion of c(Supra-OSPs)=0.008 mg•mL -1 in PBS (pH = 5.8) was placed and the emission spectra was recorded upon light excitation at λex=450 nm. To evaluate the strand melting at elevated temperatures, the same quartz cuvette with the particle dispersion was placed in a water bath and kept at 60 °C for 10 min. Subsequently the emission spectra of the warm particle dispersion were measured upon light excitation at λex=450 nm.

SAXS-based analysis of ssDNA-OSPs

Assuming that an ordered hexagonally arranged structure composed of small silica particles exists within ssDNA-OSPs and that the observed SAXS pattern centered at q=1.3 nm -1 corresponds the (100) diffraction plane of a hexagonally arranged phase, then the mean size of the scatterers can be calculated as:

𝑑 = 2𝜋 𝑞 =

Résumé en anglais

The work presented in this thesis focused on the preparation and characterization of novel stimuli-responsive organosilica particles. In this respect, a series of organoalkoxysilanes have been synthesized and used to prepare organosilicas, which possess either covalently or supramolecularly linked organic bridging-groups within their silica framework. It has been shown that porous and light-degradable and organosilicas can be prepared which are able to quantitatively release hydrophobic molecules upon lightirradiation and that oligonucleotides, such as DNA and PNA can be integrated into the particles framework in order to prepare materials, which can interact with biological molecules such as enzyme or complementary DNAs. Finally, the possibility to prepare redox-responsive cage-like organosilica particles, a novel type of morphology for organosilicas, was shown and their potential use for the development of novel nanomedicines was evaluated and discussed.

Figure 1 .

 1 Figure 1. a) Réaction de photodégradation Norrish de type II de 1 induite par l'irradiation par la lumière UV. b) Evolution des spectres d'absorption UV-Vis par l'irradiation par la lumière UV de 1 (0,11mM dans EtOH/H2O 1:1 v/v). c) Différence des spectres d'absorption signalés pour visualiser l'évolution spectrale.

  ), spectroscopie d'absorption UV-Vis et porosimétrie. Aucune dégradation des LB-MSPs n'a été observée en l'absence d'irradiation lumineuse, comme le confirment les images de microscopie électronique et la spectroscopie d'absorption UV-Vis.

Figure 2 .

 2 Figure 2. Dégradation des LB-MSP déclenchée par la lumière. a) images MEB et b) images STEM des LB-MSP à t0 et après 20 min et 6 h d'irradiation par la lumière UV à 327 nm, barres d'échelle = 500 nm.

Figure 3 .

 3 Figure 3. a) Libération de 7-DH contrôlée à 281 nm des LB-MSPs avec et sans irradiation lumineuse à λirr=327 nm. b) Libération de 7-DH des LB-MSPs comparée à celle des SS-MSPs et des MSPs vierges sous irradiation lumineuse à λirr=327 nm.

Figure 4 .

 4 Figure 4. a) Images de la dispersion colloïdale des OSCs (Fiole 2). Lorsque les particules sont irradiées par une lumière laser, l'effet Tyndall peut être observé. b) Images TEM de OSCs (barre d'échelle=100 nm) et encart montrant une image TEM de OSCs à un grossissement plus élevé (barre d'échelle=20 nm). b) Modèle 3D obtenu à partir de la reconstruction des images TEM de OSCs, en utilisant Relion®. d) Diamètre hydrodynamique et Ϛ-potentiel de OSCs.

  . Les signaux fluorescents représentés sont normalisés pour la souris à laquelle seule une solution saline a été administrée (véhicule). Il est intéressant de noter que, dans le foie, l'accumulation entre les OSCs et les OSCs-Sucre n'a pas montré de différence statistique, ce qui indique que la présence du sucre à la surface de la particule n'a pas affecté la cinétique ou l'absorption hépatique. Plus intéressant encore, l'immunohistologie du foie a indiqué que les OSCs et les OSCs-Sucre ne s'accumulaient pas dans les macrophages hépatiques (cellules de Kupffer) habituellement impliqués dans l'élimination des nanoparticules du foie. Comme le montrent les Figures 6b et c, aucune colocalisation des particules (rouge) avec les macrophages (vert), qui ont été colorés avec des anticorps CD68 marqués à la fluorescéine, n'a été observée. Cette accumulation différenciée a indiqué la possibilité que les particules soient absorbées par d'autres cellules du foie, très probablement des cellules épithéliales. Ces résultats suggèrent que les particules pourraient servir au développement de vecteurs de médicaments pour le traitement des cancers du foie.

Figure 6 .

 6 Figure 6. a) Biodistribution ex vivo des OSCs (barres rouges) et des OSCs-Sucre (barres bleues) à 1h, 4h et 24h. Les efficacités radiantes relatives des OSCs et des OSCs-Sucre sont indiquées dans le poumon, le foie, le rein et la rate. Efficacité radiante où toutes les valeurs sont normalisées au groupe de véhicules (injection de solution saline)=1. b) et c) Immunohistologie du foie après 4h du traitement par spectroscopie de fluorescence. Bleu : noyau coloré au DAPI, rouge : OSCs et OSCs-Sucre et vert : cellules de Kupffer. Échelle des barres=50 µm.]

Figure 7 .Chapitre 4 :

 74 Figure 7. a) Analyse des émissions fluorescentes ex vivo d'échantillons de cerveaux de souris de OSCs (barres rouges) et de OSCs-Sucre (barres bleues) à 1h, 4h et 24h après le traitement. Efficacité radiante où toutes les valeurs sont normalisées par rapport au groupe du véhicule (injection de solution saline)=1. b) Résultats ICP-AES obtenus à partir d'échantillons de cerveau après 4 h de traitement ; OSCs (barres rouges) et OSCs-Sucre (barres bleues).

Figure 8 .

 8 Figure 8. a) Représentation schématique des particules organosiliciques pontées par l'ADN et l'ARN. b) Image MEB de l'ADN ssDNA-OSPs. c) Images MEB de l'ANP ssPNA-OSPs.

Figure 9 .

 9 Figure 9. Étude de la dégradation par MEB des ssDNA-OSPs en présence ou en absence de DNase I. a) Image MEB des ssDNA-OSPs à t0. b) Images MEB des ssDNA-OSPs après 1h sans (image de gauche) ou lorsqu'il est incubé avec la DNase I. c) Mesure DLS sur des ssDNA-OSPs qui ont été agités pendant 1h avec et sans la présence de DNase. d) Mesure DLS sur des ssDNA-OSPs à t0 et qui ont été agités pendant 1h en l'absence de DNase.

Figure 10 .

 10 Figure 10. a) Représentation schématique du processus FRET se produisant lors de l'hybridation des brins d'ADN marqués Cy3 (donneur) et Cy5 (accepteur). b) Spectres d'absorption et d'émission normalisés de l'ADN marqué Cy3 et l´accepteur FRET dans le PBS ; λex (l'ADN marqué Cy3) = 520 nm et λex (accepteur FRET) = 650 nm. c) Expérience FRET avec des ssDNA-OSPs (c=0,96 µg•mL -1 ) et diverses concentrations d'accepteur FRET (1,76 µM et 3,52 µM).

Figure 11 .

 11 Figure 11. a) et b) Images SEM des ssPNA-OSPs. c) Distribution de taille des ssPNA-OSPs calculée à partir des images MEB.

Figure 12 .

 12 Figure 12. Représentation schématique de l'hydrolyse et de la condensation de Supra-PNA-Si pour préparer les Supra-OSPs.

  photoluminescence a été enregistrée. Comme le montre la Figure 13c, l'excitation de la fraction du donneur FRET à λex = 450 nm (fraction F-PNA) a permis d'éteindre l'émission du donneur (λem,F-PNA = 517 nm) tandis que l'émission de l'accepteur FRET (fraction T-PNA) λem,T-PNA = 590 nm a été observée, indiquant l'hybridation des PNAs dans les Supra-OSPs. Ensuite, la possibilité de dissocier l'hybridation des ANP au sein des Supra-OSPs lors du chauffage a été étudiée par analyse FRET. Comme le montre la Figure 13a, des températures élevées peuvent perturber les

Figure 13 .

 13 Figure 13. a) Représentation schématique de la perturbation du processus FRET lors du chauffage d'une dispersion de Supra-OSPs due à la dissociation des brins de ANP. b) Spectres d'absorption et d'émission normalisés des brins de F-et T-PNA dans le tampon PBS à pH=5,8. c) Expérience FRET sur une dispersion de Supra-OSPs (c=0,008 mg•mL -1 ) dans du PBS (pH=5,8) après excitation à λex=450 nm. d) Expériences FRET dépendantes de la température sur une dispersion de Supra-OSPs (c=0,008 mg-mL -1 ) dans du PBS (pH=5,8) après excitation à λex = 450 nm.

Figure 14 .

 14 Figure 14. Représentation schématique dans l'utilisation des APN-silanes, qui se lient aux extrémités de l'ADNs pour former le bis-organoalcoxysilane Supra-PDP-Si assemblé au niveau supramoléculaire.

Figure 15 .

 15 Figure 15. Images MEB des mélanges réactionnels bruts pour la formation d'organosilices à partir de Supra-PDP-Si. a-c) Images MEB du brut réactionnel à différents intervalles de temps lorsqu'aucune source de silice supplémentaire n'a été ajoutée dans la réaction d'hydrolyse et de condensation de Supra-PDP-Si. d-f) Images MEB du brut réactionnel à différents intervalles de temps lorsqu'une quantité équimolaire de TEOS a été ajoutée à la réaction.

Figure 1 .

 1 Figure 1.Comparison of sizes for different objects. Nano-objects require that at least one of their size domains lie between 1-100 nm. This definition is not consequently applied throughout the literature, since different physiochemical characteristic of the material regarding its bulk may not strictly be limited to this definition-therefore, the borders remain somewhat undefined.

Figure 2 .

 2 Figure 2. Nanoparticles researched for biomedical applications with different physicochemical properties. Copyright 2014, John Wiley and Sons. Reproduced with permission from Ref 18.

Figure 3 .

 3 Figure 3. Biodistribution of nanoparticles according to size, morphology, and surface charge. Reproduced with permission from Ref 23. Copyright 2015, Springer Nature.

Figure 4 .

 4 Figure 4. a) Schematic representation of RGD mediated uptake by αvβ3-integring expressing cells. RDG functionalized particles are not able to interact with αvβ3-integrings du to the receptors sequence-specific recognition of the RGD motive. b) TEM images of PEG particles. c) In vivo fluorescent images with dye labelled PEG, PEG-RGD and PEG-RDG nanoparticles. Only RGD functionalized particles accumulate in U-87 MG tumor cells within mice. Adapted with permission from Ref 36. Copyright 2019, American Chemical Society.

Figure 5 .

 5 Figure 5. a) Schematic depiction of 15 nm sized CoFe2O4 core-shell MnFe2O4 nanoparticles (CoFe2O4@MnFe2O4) and TEM image of CoFe2O4@MnFe2O4. b) Schematic representation of the in vivo hypothermal treatment setup done with tumor-bearing mice after intravenous particle injection. Images of tumor bearing mice before hypothermal treatment (top row, dot circle indicates tumor location) and after treatment (18 days, bottom row). c) Tumor volume versus days after treatment using CoFe2O4@MnFe2O4 particles with and without (untreated) hypothermia, Ferrite® particles combined with hypothermia, and doxorubicin. Adapted with permission from Ref 46. Copyright 2011, Springer Nature.

Figure 6 .

 6 Figure 6. a) TEM images of AuNP@DCB16F10. TEM images at higher magnification (right) indicate the presence of the dendritic cell derived vesicle with a size of ca. 5 nm. Scale bars 500 and 50 nm, respectively. b) Photothermal effect of AuNPs and immunological AuNPs. Pristine AuNPs showed decreased photothermal activity due to a lack of absorbance in the NIR region. c) Melanoma tumor growth measured with different treatments with or without laser irradiation (2 W•cm -2 ). Adapted with permission from Ref 60. Copyright 2019, American Chemical Society.

Figure 7 .

 7 Figure 7. a) TEM image of PbS/CdS-QDs with 4.2 nm core size. b) TEM image of PbS/CdS-QDs with 5.1 nm core size. c) Normalized florescence spectra of PbS/CdS-QDs with 4.2 nm core size (λem=1080 nm) and 5.1 core-size (λem=1280 nm) in water. d) In vivo NIR-II multiplexed imaging in tumor xenografted rodents with two polymer-coated and folic acid functionalized PbS/CdS-QDs probes of different core size. Adapted with permission from Ref 65. Copyright 2018, John Wiley and Sons.

Figure 8 .

 8 Figure 8. a) Structure of 7C1 lipid used in combination with PEG polymer for the preparation of lipidpolymer hybrid nanoparticles. B) Schematic representation of the lipid-polymer siRNA loaded carrier. c) Cryo-TEM images of lipid-polymer siRNA loaded carrier. d) Primary Lewis lung carcinoma (LLC) growth following three 1.0 mg•kg -1 treatments with siRNA loaded lipid-polymer nanoparticles (siRNAs used to silence protein expressions: siLuciferase in siCntrol formulation; siVascular endothelial growth factor receptor-1 in siVEGFR-1 silencing formulation; siDelta-like ligand 4 in siDll4 silencing formulation). Adapted with permission from Ref 89. Copyright 2014, Springer Nature.

Figure 9 .

 9 Figure 9. a) TEM image of octahedral DNA origami nanostructures. Scale bar=50 nm. b) Schematic representation of peptoid-protected octahedral DNA-origami. c) Schematic representation of the use of peptoids for selective antibody and/or fluorescent marker functionalization of the nanostructure. AF=Azide-Fluor 488 dye; Tz=Trastuzumab antibody. Adapted from Gang and coworkers Ref 95. Copyright 2020 the Author´s CC BY-NC-ND 4.0, published by National Academy of Sciences.

Figure 10 .

 10 Figure 10. a) Schematic representation of the general structure of sub 10 nm C´-Dots. Within an amorphous silica-core, the red-emissive dye cyanine 5 is covalently incorporated, and the surface of the particle is functionalized with PEG-chains and the tumor homing peptide cRGD. The layered core structure indicates that, according to the synthetic procedure applied, the diameter of C´-Dots can be varied upon further silica growth. b) TEM images of 4.5 nm big C´-Dots. Adapted with permission from Ref 96. Copyright 2015, American Chemical Society. c) Human trials with 124 I-labeled and cRGD functionalized C´-Dots for tumor imaging. d) Coronal CT of a patient demonstrates a hypo dense left hepatic lobe metastasis (arrowhead). e) Whole-body PET imaging after 24 h of 124 I-labeled C´-Dots. Hepatic metastasis is indicated by the yellow arrow. Adapted with permission from Ref 32. Copyright 2014, The American Association for the Advancement of Science.

Figure 11 .

 11 Figure 11. a) Schematic representation of siRNA loading and subsequent calcium silicate capping of nSiNPs. b) TEM images of mSiNPs. c) Ex vivo fluorescence images of brain samples treated with PBS (1), calcium silicate capped, and siRNA loaded mSiNPs functionalized with PEG chains (2) and calcium silicate capped, and siRNA loaded mSiNPs functionalized with the acetylcholine targeting peptide. siPPIB=siRNA for silencing the gene for peptidylprolyl isomerase B expression; DPNC=dual peptide nanocomplex composed of a rabies virus glycoprotein sequence and the transportan-based peptide. Adapted with permission from Ref 99. Copyright 2016, John Wiley and Sons.

Figure 12 .

 12 Figure 12. Schematic representation of the cooperative self-assembly pathway for the formation of MSPs.

Figure 13 .

 13 Figure 13. a) Base-catalyzed hydrolysis of TEOS. b) Base-catalyzed condensation of TEOS.

Figure 14 .

 14 Figure 14. TEM images of MSPs with different size, shape, and pore arrangement. a) Spherical particles of a) 170 nm and b) 50 nm in dimeter. Adapted with permission from Ref 111, Copyright 2013, Royal Society of Chemistry (Great Britain). Particles with c) oval and d) toroidal shape. Adapted from Ref 112. MSPs with e) highly hexagonally ordered mesopores (adapted with permission from Ref 113, Copyright 2012, Elsevier) and f) MSPs with concentrically aligned pores (adapted with permission from Ref 114, Copyright 2013, American Chemical Society). All scale bars = 100 nm.

Figure 15 .

 15 Figure 15. a) Schematic representation of single pore usMSPs and b) TEM images of usMSPs (scale bars = 100 nm and 50 nm for inset). c) 3D reconstructed representation of one cage-like silica particle class obtained from cryo-TEM images. d) TEM and (scale bar = 20 nm) e) Cryo-TEM images of cage-like silica particles (scale bars = 5 nm). Images reproduced with permission: Copyright 2012, American Chemical Society. Images a) and b) adapted with permission from Ref 117; Copyright 2018, Springer nature. Images c-e) adapted with permission from Ref 118.

Figure 16 .

 16 Figure 16. Schematic representation of the preparation for porous organosilica particles. As for the preparation of porous silica particles, porous organosilicas are prepared upon a surfactant templated hydrolysis of alkoxysilanes under acid or base catalysis.

Figure 17 .

 17 Figure 17. TEM images of organosilica particles with different size, morphology and porosity. a) Spherical particles with radially oriented large pores. Adapted with permission from Ref 120. Copyright 2013, Royal Society of Chemistry (Great Britain). b) Spherical and hollow organosilica particles. Adapted with permission from Ref 121. Copyright 2013, John Wiley and Sons. c) Rod-shaped mesoporous organosilica particles. Adapted with permission from Ref 122. Copyright 2017, John Wiley and Sons. d) Multiple-shelled mesoporous organosilica particles. Adapted with permission from Ref 123. Copyright 2012, American Chemical Society.

Figure 18 .

 18 Figure 18. Selective surface and pore functionalization of MSPs or MONs using alkoxysilane chemistry.

Figure 19 .

 19 Figure 19. a) The use of breakable MONs allows the delivery of nanoparticles on-site of the disease and their stimuli-triggered degradation, therefore promoting the efficient clearance of small debris from a living organism. b) Stimuli responsive bridging groups integrated within the silica framework allow the stimuli triggered degradation of MONs into small degradation products. C) Stimuli-responsive bridging groups that can be integrated MONs, rendering them breakable in biological environments are shown. The various functional groups can be redox-responsive (e.g. disulfide bonds), sensible to enzymatic hydrolysis (e.g. ester and amide bonds) or sensible to acidic pH-values (e.g. acetal or imine bonds).

Figure 20 .

 20 Figure 20. a) TEM images of stimuli responsive disulfide-bridged mesoporous organosilica particles which degrade upon the presence of the reducing agent glutathione (GSH). Adapted with permission from Ref 129. Copyright 2016, Royal Society of Chemistry (Great Britain). b) GSH reduces the disulfide bond and leads to its cleavage, hence triggering the degradation of the particles.

Figure 21 .

 21 Figure 21. a) Schematic representation of antitumor activity of RNAse A loaded and cell membrane (CM) encapsulated degradable MONs. When intravenously injected the particles actively target the tumor due to CM mediated recognition and upon cellular uptake the particles are degraded and release the cytotoxic cargo. b) TEM image of diselenide-bridged organosilica particles. c) In vivo anti-tumor activity of RNAse A loaded diselenide-bridged nanoparticles. Adapted with permission from Ref 135. Copyright 2018, John Wiley and Sons.

Figure 22 .

 22 Figure 22. a) Schematic representation of trisulfide and benzene bridged HMONs. b) TEM images of HMONs. c) GSH triggered doxorubicin release form degradable HMONs. d) Doxorubicin release from HMONs upon application of intensity focused ultrasounds. Adapted with permission from Ref 137. Copyright 2014, American Chemical Society.

Figure 23 .

 23 Figure 23. Non-covalent interactions which have been used in the preparation of supramolecular polymers. a) Supramolecular polymer formed between diamidopyridines and uracil derivatives and b) 2ureido-4-pyrimidon units, via H-bonding interactions. b) Supramolecular polymer formed by π-π

Figure 24 .

 24 Figure 24. a) Schematic representation in the use of a supramolecular based gastric device in a pig animal model. After oral administration of the encapsulated gastric device within a gelatin capsule, it is retained in the pig's stomach after the dissolution of the gelatin capsule. When translocated to the small and large intestine, the gastric device is degraded, facilitating its safe excretion. b) Chemical structure of poly(acryloyl 6-aminocaproic acid) and poly(methacrylic acid-co-ethyl acrylate)polymer and their supramolecular interactions via H-bonding. c) Degradation of the gastric device at neutral pH-values over time. Adapted with permission from Ref 148.Copyright 2015, Springer Nature.

Figure 25 .

 25 Figure 25. a) Schematic representation in the use of 1,3,5-benzenetricarboxamide derivatives for the preparation of supramolecular polymers. b) Supramolecular assemblies bearing the hydrophobic guest molecule nile red and negatively charged siRNA. Adapted with permission from Ref 149. Copyright 2016, American Chemical Society.

Figure 26 .

 26 Figure 26. a) Chemical structures of the monomers used for the preparation of HBPs. b) Schematic representation of the formation of the fiber-like HBPs upon supramolecular aggregation and covalent polymerization between monomer 1 and 2, while monomer 3 co-assembles driven only by non-covalent interactions. Adapted with permission from Ref 152. Copyright 2016, American Association for the Advancement of Science.

Figure 27 .

 27 Figure 27. a) Schematic representation in the organoalkoxysilane self-assembly process onto hydrophobic nanocrystals and subsequent crosslinking of the hydrolysable alkoxy-groups in acidic aqueous solution to form hexagonally or cubic arranged hybrid nanocrystals. b) UV-Vis absorption spectra of hybrid nanocrystals, in which the characteristic absorption of the surface resonance band is still visible, even after functionalization. The inset represents a TEM image of hybrid nanocrystals. c) X-ray diffraction pattern of 3D ordered hybrid nanocrystals. Adapted with permission form Ref 158. Copyright 2005, American Chemical Society.

Figure 28 .

 28 Figure 28. a) Schematic representation of the preparation of cerasomes. SEM image of b) a cerasome particle and c) of a cerasome particle coated with Pt. Adapted with permission from Ref 159. Copyright 2003, Elsevier.

Figure 29 .

 29 Figure 29. Structure of DNA and PNA oligonucleotides.

Figure 30 .

 30 Figure 30. a) Schematic representation of PNA-based monomers to PNA-based micelles. b) TEM images of PNA-based spherical micelles. c) Hydrodynamic diameter measured for PNA-based micelles. Adapted with permission from Ref 166. Copyright 2004, American Chemical Society.

Figure 31 .

 31 Figure 31. a) Schematic representation in the self-assembly driven formation of an immunostimulant based on DNA-PNA hybrid micelle (INA), formed by mixing of two DNA and a PNA component. b) TEM images of INAs. c) Hydrodynamic radius of INAs. Adapted with permission from Ref 167. Copyright 2019, Elsevier.

4 . 2 ) 1 .

 421 Scheme 1. Synthesis of photolabile bis-alkoxysilane 1. Reaction conditions: (i) NaH, DMF, 0°C; (ii) allylbromide, DMF, 0°C to r.t.; (iii) Karstedt's catalyst, triethoxysilane, toluene, 50°C.

Figure 1 .

 1 Figure 1. a) Norrish type II photodegradation reaction of 1 induced by UV-light irradiation. b) UV-Vis absorption spectra evolution upon UV-Light irradiation of 1 (0.11mM in EtOH/H2O 1:1 v/v). c) Difference absorbance spectra reported to better visualize the spectral evolution.

Figure 2 .

 2 Figure 2. a) Computed frontier molecular orbitals for compound 3. b) Comparison of the absorption spectra of 3 with the computed absorption spectra of the model nitroso-compound shown in red.

Figure 3 .

 3 Figure 3. NMR spectra recorded over time upon light-irradiation of 1. a) Intensity change of the benzylic methylene resonance signal upon light irradiation. b) Intensity change of the aldehyde resonance signal upon light irradiation.

Figure 4 .

 4 Figure 4. a) Concentration change of NBA over a series of initial different concentrations. b) Reaction rates as a function of initial NBA concentrations.

Figure 5 .

 5 Figure 5. Concentration change of 1 over light irradiation at 365 nm.

Scheme 2 .

 2 Scheme 2. Schematic representation of the preparation of LB-MSPs.

Figure 6 .

 6 Figure 6. a) SEM and b) TEM images of LB-MSPs. c) Size distribution of LB-MSPS calculated from TEM images (N = 100). d) DLS measurements on LB-MSPs in water.

Figure 7 .

 7 Figure 7. a) TEM images of LB-MSPs at higher magnification. b) SAXS pattern on LB-MSPs.

Figure 8 .

 8 Figure 8. a) N2 adsorption and desorption isotherms measured on LB-MSPs. b) DFT pore size distribution calculated from adsorption branch of the isothermal curve of LB-MSPs.

Figure 9 .

 9 Figure 9. a) TGA analysis showing the characteristic weight losses of LB-MSPs, Pristine MSPs, 1 and CTAB. b) ATR-FTIR recorded on LB-MSPs and Pristine MSPs.

Figure 10 .

 10 Figure 10. a) XPS survey spectrum on LB-MSPs. b) Deconvoluted XPS HR scan of N(1s) level recorded on LB-MSPs. c) Deconvoluted XPS HR scan of C(1s) level recorded on LB-MSPs.

Figure 11 .

 11 Figure 11. a) UV-Vis absorption spectra of 1 in EtOH/H2O (5:1 v/v); c(1)=0.1 mM. b) UV-Vis absorption spectra of LB-MSPs in EtOH/H2O (5:1 v/v); c(LB-MSPs)=0.1 mg•mL -1 .

Figure 12 .

 12 Figure 12. a) SEM and b) STEM images of SS-MSPs. c) Calculated size distribution of SS-MSPs using STEM images (N=100). d) SAXS pattern obtained from SS-MSPs.

Figure 14 .

 14 Figure 14. a) N2 adsorption and desorption isotherms measured on SS-MSPs. b) DFT pore size distribution calculated from adsorption branch of the isothermal curve of SS-MSPs.

Figure 15 .

 15 Figure 15. a) Thermogram recorded on MSPs, SS-MSPs and the surfactant CTAB. b) XPS survey spectrum recorded on SS-MSPs. c) Deconvoluted XPS HR scan of S(2p) level recorded on SS-MSPs. c) Deconvoluted XPS HR scan of C(1s) level recorded on SS-MSPs.

Figure 16 .

 16 Figure 16. a) SEM and b) STEM images of MSPs. c) Calculated size distribution of MSPs using STEM images (N=100). d) SAXS pattern recorded on MSPs.

Figure 17 .

 17 Figure 17. a) N2 adsorption and desorption isotherms measured on MSPs. b) DFT pore size distribution calculated from adsorption branch of the isothermal curve of MSPs.

Figure 18 .

 18 Figure 18. a) SEM and b) STEM images of LB-MSPs at t0 and after 20 min and 6 h of UV-light irradiation at 327 nm, scale bars = 500 nm.

Figure 19 .

 19 Figure 19. UV-vis absorption spectra recorded over time on a dispersion of LB-MSPs (0.1 mg•mL -1 ) in EtOH/H2O (5:1, v/v) upon light irradiation at λirr=327 nm.

Figure 20 .

 20 Figure 20. Comparison between the N2 adsorption-desorption isotherms obtained for LB-MSPs (black) before and after UV-light irradiation (blue).

Figure 21 .

 21 Figure 21. a) SEM and b) STEM images of LB-MSPs which have been stirred in the dark. c) UV-Vis absorption spectra of LB-MSPs stirred in the dark at t0 and after 12 h.

Figure 22 .

 22 Figure 22. a) Thermogram of LB-MSPs and 7-DH loaded LB-MSPs. b) Thermogram of SS-MSPs and 7-DH loaded SS-MSPs. c)Thermogram of MSPs and 7-DH loaded MSPs.

, a boosted release of 7 -

 7 DH from LB-MSPs could be observed upon UVlight irradiation, which can be explained by the progressing degradation of LB-MSPs and therefore occurring release of the drug. In the absence of light, only a low amount of 7-DH was released most probably due to the passive diffusion of the molecule loaded in the pores or physisorbed onto the particle surface (Figure23a). To further confirm that the release of 7-DH is triggered by light, the same release experiments were performed by using MSPs and SS-MSPs as references (See experimental section 2.4.7.). SS-MSPs particles possessed comparable particle size, shape and organic content to LB-MSPs but had the disulfide linker within their structure, which does not react upon UV-light irradiation. The release experiments showed that, besides the fast passive diffusion of 7-DH from SS-MSPs and MSPs, no boosted release of the drug due to light irradiation was observed, demonstrating the unique light-triggered cargo release from LB-MSPs (Figure23b).

Figure 23 .

 23 Figure 23. a) 7-DH release monitored at 281 nm from LB-MSPs (c=0.1 mg•mL -1 ) with and without light irradiation at λirr = 327 nm. b) 7-DH release from LB-MSPs compared to SS-MSPs and pristine MSPs upon light irradiation at λirr = 327 nm; c(particles)=0.78 mg•mL -1 .

Figure 24 .

 24 Figure 24. Left: UV-Vis absorption spectra of 7-DH in cyclohexane recorded at different concentrations. Right: Calibration curve obtained for 7-DH in order to quantify the amount of cargo release upon and without UV-light irradiation of LB-MSPs.

Figure 25 .

 25 Figure 25. a) 7-DH release from LB-MSPs (c=0.1 mg•mL -1 ) monitored at 281 nm by UV-Vis absorption spectroscopy at two different irradiation wavelengths. b) UV-Vis absorption spectra of 1 irradiated with light of 500 nm at to and after 30 min.

Figure 26 .

 26 Figure 26. ALamarBlue® cell viability test results of LB-MSPs and LB-MSPs_irradiated on HeLa cells.

Figure 1 .

 1 Figure 1. Schematic representation of the preparation and surface functionalization of disulfide-bridged organosilica cages (OSCs).

Figure 2 .

 2 Figure 2. a) TEM images of OSCs (scale bar = 100 nm) and inset showing OSCs at higher magnification (scale bar=20 nm). b) 3D-model obtained from the reconstruction of OSCs TEM images, using Relion®.c) OSCs size-distribution calculated from TEM images (N=100).

Figure 3 .

 3 Figure 3. a) N2 adsorption-desorption isotherms recorded on OSCs. b) Pore size distribution calculated on OSCs.

Figure 4 .

 4 Figure 4. a) Left: images of colloidal dispersions of OSCs (c=3 mg•mL -1 ), showing the characteristic Tyndall effect of colloids when irradiated with a laser. Right: colloidal dispersion of OSCs (c=3 mg•mL -1 ) which were functionalized with the dye sCy5. b) DLS analysis on OSCs. c) DLS analysis on OSCs which have not been functionalized with PEG-chains. d) UV-Vis absorption (black) end emission (red) spectra of an aqueous OSCs dispersion that were functionalized with sCy5.

Figure 6 .

 6 Figure 6. a) XPS survey spectrum recorded on OSCs. b) Deconvoluted XPS HR scan for C(1s) level recorded on OSCs. c) Deconvoluted XPS HR scan for S(2p) level recorded on OSCs.

Figure 7 .

 7 Figure 7.a) UV-Vis absorption spectra of OSCs (c=0.15 mg•mL -1 ) recorded in water. b) Calibration curve obtained from measuring the absorbance of sCy5 at different concentrations.

Figure 8 .

 8 Figure 8. Left: GSH-triggered reduction of disulfide groups within OSCs, leading to particle degradation.Right: TEM images of OSCs (c=0.1 mg•mL -1 ) at t0 and after 2 h, when incubated in PBS at 37 °C, with and without GSH (10 mM). All scale bars=20 nm.

Figure 9 .

 9 Figure 9. STEM images of OSCs (c=0.1 mg•mL -1 ) dispersed in PBS/GSH (10 mM) solution at t0 and after 12 h.

Figure 10 .

 10 Figure 10. a) Thermograms recorded on OSCs, OSCs-HE and HE. b) Ϛ-potential measurements on OSCs and OSCs-HE. c) Cumulative HE-release experiments following the HE fluorescence emission (λem=492 nm, λex=350 nm) from OSCs-HE dispersions (c=0.1 mg•mL -1 ), with and without GSH (10 mM). d) HE-release experiments recording the fluorescence emission (λem=492 nm, λex=350 nm), before and after addition of GSH.

Figure 11 .

 11 Figure 11. a) UV-absorption of the supernatant washing solutions recorded at λ=320 nm. b) Thermograms recorded on OSCs and OSCs-DT.

Figure 12 .

 12 Figure 12. DLS analysis of a filtered OSCs-DT particle dispersion in water.

Figure 13 .

 13 Figure 13. a) Fluorescence emission spectra of Sugar-OSCs (c=0.6 mg•mL -1 ) which have been incubated with 2-cyanoacetamide in borate buffer (λex=331 nm).b) Fluorescence emission calibration curve obtained from the reaction of glucosamine at different concentrations (λex=331 nm, λem=383 nm).

Figure 14 .Scheme 3 .

 143 Figure 14. a) DLS analysis of Sugar-OSCs and b) TEM images of Sugar-OSCs.

Scheme 4 .

 4 Scheme 4. Preparation of N3-OSCs.

Figure 15 .

 15 Figure 15. a) DLS size distribution of N3-OSCs b) ATR-FTIR spectra recorded on N3-OSCs.

Scheme 5 .

 5 Scheme 5. Reaction of the guanidine group of arginine present on RGD-OSCs, with PQ, leads to the formation of a fluorescent addition product, used for the quantification of RGD onto the particles surface.

Figure 16 .

 16 Figure 16. a) Fluorescence emission spectra of RGD-OSCs (c=0.0107 mg•mL -1 ) which have been incubated with PQ (λex=312 nm). b) Fluorescence emission calibration curve obtained from the reaction of RGD at different concentrations with PG (λex=312 nm, λem=424 nm).

Figure 17 .

 17 Figure 17. Trypan blue cell-viability results on glioma C6 cells obtained upon incubation with OSCs at different concentrations after 24 and 48 h of incubation.

Figure 18 .

 18 Figure 18. Ex vivo biodistribution of OSCs (red bars) and Sugar-OSCs (blue bars) at 1, 4 and 24 h. Relative radiant efficiencies for OSCs and Sugar-OSCs are shown in a) lung, b) liver c) kidney and d) spleen. Radiant Efficiency where all values are normalized to vehicle (saline solution injection) group=1.

Figure 19 .

 19 Figure 19. Histological analysis of OSCs and sugar-OSCs in lung sections 1 h and 4 h after the treatment obtained by fluorescent microscopy. The particles are visualized in red (sCy5 signal) and nuclei in blue (Hoechst 33258 staining). Scale bars=100 µm.

Figure 20 .

 20 Figure 20. Immunohistology of the liver after 4 h of the treatment by fluorescence spectroscopy. Blue: nucleus stained with DAPI; red: OSCs and Sugar-OSCs; green: Kupffer cells. Scale bars=50 µm.

Figure 21 .

 21 Figure 21. Histological analysis of OSCs and Sugar-OSCs in spleen sections at 1 and 4 h after the treatment obtained by fluorescent microscopy. NPs are visualized in red (sCy5 signal) and nuclei in blue (Hoechst 33258 staining). RP=red-pulp, MZ=marginal zone, WP=white-pulp. Scale bars = 100 µm.

Figure 22 .

 22 Figure 22. a) Ex vivo fluorescent emission analysis of mouse brain samples of OSCs (red bars) and Sugar-OSCs (blue bars) at 1, 4 and 24 h after treatment. Radiant Efficiency where all values are normalized to vehicle (injection of saline solution) group=1. b) ICP-AES results obtained from brain samples after 4 h of treatment; OSCs (red bars) and Sugar-OSCs (blue bars).

Figure 23 .

 23 Figure 23. a) Sagittal sections of mouse brain. Dashed squares represent the field of view examined by histology. b) Representative images of brain sections of Sugar-OSCs treated mice at 4h obtained by fluorescent microscopy. NPs are visualized in red (Cy5 signal) and nuclei in blue (Hoechst 33258 staining). CC=corpus callosum, CPu=caudate putamen, LV=lateral ventricle. Scale bar=100 µm.

( 2 . 3 . 4 . 2 . 3 . 4 . 3 . 25 . 3 . 4 . 4 .

 234234325344 1 nm mm -1 of dispersion; 1200 grooves mm -1 ) and a TBX-04 single photocounting detector was used. Quartz cuvettes with path length of 10 mm were used for all recorded spectra. coated with Au (Emitech K575X peltier cooled) for 3 min at 60 mA. The Advantage software was used for background subtraction (Shirley-type), and deconvolution of peaks. Thermogravimetric analysis (TGA) was conducted on a Netzsch model STA 449 fi Jupiter instrument. The samples (0.5-2 mg) were kept at 100 °C for 30 mi for stabilization, then heated from 25 to 800 °C at a speed of 10 °C/min, before being held at this temperature for further 30 minutes, and finally cooled. The analyses were performed under a 20 mL•min -1 air flow. ICP-AES analyses were performed with a Thermo Scientific instrument, ICAP 6500. Samples (1 mL) were diluted to a total volume of 10 mL using aqueous 2% nitric acid. All solutions were freshly prepared from UV sterilized deionized (about 18 MΩ) water and the samples were always handled in plastic falcon tubes. Standard Si solutions (0.01 ppm, 0.1 ppm, 1 ppm) were used for calibration. Reported measurements in each sample represent the average of 3 measurements and are back-calculated to offset the initial dilution. The individual measurements are typically obtained with a relative standard deviation of approximately 0.01 ppm per sample. Preparation of alkoxysilanes for the functionalization of cage-like silica particles General information. All solvents were purchased from Merck and used as received. Sulfo-cyanine5-NHS ester and sulfo-cyanine3-NHS ester were purchased from Lumiprobe. 3-(aminopropyl)triethoxy silane (APTES, 99%), 3-(triethoxysilyl)propyl isocyanate (ICPTES, 95%), triethylamine (TEA, >99%), (3-chloropropyl)trimethoxysilane (Cl-TMS, 95%), sodium azide (NaN3, synthesis grade), D-(+)-glucosamine hydrochloride, tetrabutylammonium bromide (>99%) were purchased from Sigma-Aldrich Preparation of sCy5-silane. A solution of sulfo-Cy5-NHS ester (0.5 mg, 0.643 μmol) in dry DMSO (200 μL) was prepared in in a 2 mL Eppendorf microtube . APTES (4.5 μL, 0.142 M in dry DMSO) was added and the reaction mixture was allowed to stir for 30 min in the dark. The reaction mixture was used for post-synthesis grafting of OCSs without further purification. Preparation of sugar-silane. A solution of D-(+)-glucosamine hydrochloride (1.8 mg, 8.35 μmol) in DMSO (200 μL) was prepared in a 2 mL Eppendrof microtube. TEA (1.2 μL, 11.86 μmol) was filtrate under reduced pressure. The product was obtained as transparent, yellowish liquid (930 mg, 90%). 1 H-NMR (CDCl3, 400 MHz): δ 3.57 (s, 9H), 3.26 (t, 2H, J=7.2 Hz), 1.76-1.70 (m, 2H), 0.75-0.63 (m, 2H) ppm. FTIR ṽ max [cm -1 ]: 2941, 2843, 2092, 1463, 1190, 1078, 817, 775. Preparation of alkyne-RGD General information. The reaction was carried out under inert conditions and in the dark, using dry solvents. Dry DMF was purchased from Merck and used as received. Arg-Gly-Asp peptide (RGD, >97%), (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate ester (BCN-NHS) and triethylamine (TEA, >99%) were from Merck.Synthesis of Alkyne-RGD.To a RGD peptide solution (10mg; 28,88 µmol, 1eq) in DMF (3mL), placed in a 10 mL round-bottom flask, TEA (9.2µL, 68.1 µmol) and BCN-NHS (1.1 eq; 9.2 mg, 31.6 µmol) were added. The reaction mixture was stirred at room temperature, while kept in the dark, for 24 h. Subsequently, the reaction crude was dried under a constant nitrogen-flow. The product was finally washed with Et2O (2 mL), centrifuged and dried under reduced pressure. After drying, the product was obtained ad waxy solid. HR-MS (ESI-TOF): m/z calcd for C25H35N6O8: 523.25 found 523.Preparation of particles and their surface-functionalization General information. All solvents were used as received. Hexadecyltrimethylammonium bromide (CTAB, 99+%) was purchased from ACROS ORGANICS. Bis(triethoxysilyl-propyl)disulfide (BTDS, 95%) was purchased from fluorochem. Methoxy-(poly-ethyleneoxy)propyltrimethoxysilane (PEG-silane 6-9 units, 90%) was purchased from ABCR. Aqueous ammonia (NH3, 28 wt.%) was purchased from VWR Chemicals. Tetraethyl orthosilicate (TEOS, ≥99.0 %, GC) was purchased from Sigma-Aldrich. Acetic acid (glacial, extra pure), hydrochloric acid (37%) and sodium carbonate were purchased from Fisher scientific. SnakeSkin dialysis tubing was purchased from ThermoScientific (MWCO 10.000, 22 mm). All reagents were used without further purification. Preparation of non-surface-functionalized OSCs, OSCs and Sugar-OSCs. In a 100 mL roundbottom flask, equipped with a magnetic stirrer (Teflon; cylindrical geometry; 1 cm length), CTAB (408 mg, 1.12 mmol) was dissolved in freshly deionized water (50 mL) at 50 o C, while stirring the solution for 30 min at 250 rpm. Aqueous NH3 (12.5 μL, 28 wt% in water) was added and the stirring speed was increased to 750 rpm. Subsequently a mixture of TEOS (448.1 μL, 2 µmol) and BTDS (102.8 μL, 0.223 μmol) was added and the mixture was stirred for 20 h at 50 o C, while stirring the reaction mixture at 750 rpm. Subsequently, PEG-silane (818 μL) was added to the stirring solution and the reaction mixture was stirred for 14 h at 50 °C. In the case were the dye and Sugar functionalization was desired, the sCy5-Silane solution and Sugar-silane solutions were added immediately after the addition of the PEG-silane and the mixture was stirred for further 14 h at 50 o C. The particle dispersion was purified by dialysis against EtOH/H2O/AcOH (1:1:0.007 v/v/v) mixture for 24 h and finally in an EtOH/H2O (1:1 v/v) mixture for another 24 h, whereby dispersion was lyophilized. Subsequently, 10 mg of particles were suspended via sonication (15 min, 30 W) in a solution of the dye (5 mg) in DMSO (300 μL). The suspension was briefly sonicated and allowed to stir for 24 h in the dark. Subsequently the organic solvent was removed by evaporation and the particles were washed once with a H2O/EtOH mixture (500 μL, 1:1 v/v) and collected by centrifugation at 14.8 krpm for 30 min. Subsequently the particles were dispersed in EtOH (500 μL), TEA (1 μL) and finally PEG-Silane (64 μL) was added. The mixture was briefly sonicated and subsequently stirred for 12 h in the dark. The recovered particles were washed three times by dispersing the particles in a EtOH/H2O (1:1 v/v) mixture (500 μL) and subsequent centrifugation and were finally dried under reduced pressure.

3. 4 . 7 .

 47 Degradability studies and HE release experimentsDegradability experiments of OSCs: To evaluate the degradability of OSCs upon the presence of GSH, particles (0.05 mg) were dispersed in 500 μL of a GSH solution in PBS (10mM, pH=7.4) and stirred at 37 o C. Separately, OSCs (0.05 mg) were dispersed in 500 μL of PBS (pH=7.4) and stirred at 37 o C. At different time intervals, an aliquot (6 μL) of each particle dispersion was taken and diluted with EtOH (1000 μL; the EtOH was filtered before use with a 25mm syringe filter with 0.2 μm nylon). The diluted samples were analyzed by STEM and TEM.HOECHST 33258 release from OSC-He: 0.1 mg of OSCs-He were dispersed in 500 μL PBS buffer solution containing GSH (10mM, pH = 7.4) or solely in PBS (pH = 7.4) and the particle dispersion was allowed to stir at 37 o C. At different time point the particle dispersion was centrifuged (20 min, 14.5 krpm) and aliquots (20 μL) of supernatant were collected for subsequent, fluorescence measurements and a fresh aliquot of solution was added to the particle dispersion. The fluorescence measurement of the first sampled aliquot was performed by placing it in a quartz cuvette, adding subsequently PBS (2.48 mL) and measuring its fluorescence (λex=350 nm). For each subsequent measurement, an aliquot from the cuvette was replaced by an aliquot of new supernatant solution from the centrifuged particle dispersion, obtaining therefore a cumulative release curve for HE. All The fluorescent emission spectra were recorded from 385-600 nm.

(about 18

 18 MΩ) water and the samples were always handled in plastic falcon tubes. Standard Si solutions (0.01ppm, 0.1ppm, 1 ppm) were used for calibration. Reported measurements in each sample represent the average of 3 measurements and were back-calculated to offset the initial dilution. In addition, micrometric sections from brain, spleen, liver, kidneys, and lungs were obtained from frozen samples of OSCs-treated and untreated mice. Hoechst 33258 (fluorescent nuclear dye with an excitation peak at 405 nm) was utilized to visualize the anatomy of tissues and to determine the localization cellular localization OSCs (detectable by a direct excitation at 635 nm) by confocal microscope.

oligonucleotide 1 (Scheme 1 .

 11 Scheme 1. Reaction scheme for the preparation of ssDNA alkoxysilane derivative 2.

Figure 1 .

 1 Figure 1. MSPs prepared at pH=8.5 with different H2O/EtOH ratios: a) 1:0.6, b) 1/0.3 and c) 1.02. d) Calculated size distribution for MSPs prepared at different H2O/EtOH ratios.

Figure 2 .

 2 Figure 2. a) SEM and b) TEM images of ssDNA-OSPs. c) Size distribution of ssDNA-MSPs calculated from TEM images.

Figure 3 .

 3 Figure 3. a) TEM image of organosilica particles prepared with a short ssDNA alkoxysilane (GCGC). b) TEM image of organosilica particles prepared with a short ssDNA alkoxysilane at higher magnification.

  It was not possible to obtain amounts of ssDNA-OSPs large enough to analyze the particle porosity by N2-adsorption-desorption measurements. The formation path of ssDNA-OSPs could be investigated via electron microscopy and SAXS measurements of the reaction mixture at different time intervals during particle formation. The way in which ssDNA-OSPs are formed could be significantly altered from the expected cooperative hydrolysis and assembly mechanism and rather follow altered formation pathways. Such an altered pathway could consist in the initial formation of silica particles of ca. 5 nm due to hydrolysis and condensation of TEOS and subsequent interparticle aggregation and further condensation with unreacted TEOS and 2, leading to the formation of ssDNA-OSPs. Slower hydrolysis and condensation rates for 2 can be explained by the fact that slower reaction rates for sterically demanding organoalkoxysilanes were previously reported.50

Figure 4 .

 4 Figure 4. a) SEM image of ssDNA-OSPs. b) SAXS pattern obtained from ssDNA-OSPs.

  2848-2928 cm -1 can be attributed to Csp2-H and Csp3-H stretching vibrations of ssDNA and residual CTAB within ssDNA-MSPs. The survey XPS spectra recorded on ssDNA-MSPs indicated the presence of C(1s) and N(1s) signals at 285 and 403 eV, respectively (Figure5d), confirming the presence of the organic functional group within ssDNA-OSPs. Although XPS analysis indicated the presence of residual CTAB within ssDNA-OSPs, ζ-potential measurements showed that the particles possessed an overall negative surface charge of -27±2 mV when dispersed in deionized water at pH=7.4. The latter result is important to exclude the possibility that the electrostatic interaction between positively charged particles and negatively charged DNA would result in false positive results when hybridization with a complementary ssDNA strand would be analyzed.

Figure 5 .

 5 Figure 5. a) UV-Vis absorption spectra of ssDNA-OSPs in H2O (c=0.02 mg•mL -1 ). b) UV-Vis absorption spectra of 1 in H2O (c=2.1 µM). c) ATR-FTIR spectra recorded on ssDNA-OSPs. d) XPS survey spectra recorded on ssDNA-OSPs.

Figure 6 .

 6 Figure 6. Deconvoluted XPS HR scan of C(1s) level recorded on ssDNA-OSPs. Deconvoluted XPS HR scan of N(1s) level recorded on ssDNA-OSPs. Deconvoluted XPS HR scan of P(2p) level recorded on ssDNA-OSPs

Figure 7 .

 7 Figure 7. Degradation study via SEM of ssDNA-OSPs in the presence or absence of DNase I. a) SEM image of ssDNA-MSPs at t0. b) SEM images of ssDNA-OSPs after 1 h without in the absence of DNase (left) or after being incubated 1 h with DNase I (right). Scale bars=500 nm.

Figure 8 .

 8 Figure 8. a) DLS intensity size distributions of ssDNA-OSPs in Tris buffer (pH=7.6) that have been stirred for 1 h with and without DNase. b) DLS intensity size distributions of ssDNA-OSPs at t0 and that have been stirred for 1 h in the absence of DNase.

Figure 9 .

 9 Figure 9. a) Schematic representation of the FRET process occurring upon hybridization of Cy3 (donor) and Cy5 (acceptor) labeled DNA strands. b) Normalized absorption and emission spectra of 1 and 3 in PBS; λex(1)=520 nm and λex(3)=650 nm. c) FRET experiment with ssDNA-OSPs (cssDNA-OSPs=0.96 µg•mL -1 ) and various concentrations of 3 (1.76 µM and 3.52 µM).

(Scheme 2 .

 2 Scheme 2. Reaction scheme for the preparation of ssPNA alkoxysilane derivative 5.

Figure 10 . 4 . 2 . 2 .

 10422 Figure 10. a) and b) SEM images of ssPNA-OSPs. c) Size distribution of ssPNA-OSPs calculated from SEM images.

Figure 11 . 56 Figure 12 .

 115612 Figure 11. a) Structure of the 5-carboxyfluorescein labelled F-PNA strand. b) Structure of the carboxytetramethylrhodamine labelled T-PNA.

(Figure 13 ,

 13 Figure 13, see section 4.4.3.). Subsequently, both strands were mixed, resulting in the formation

Figure 15 .

 15 Figure 15. a) SEM images and b) SEM images taken at higher magnification of an aliquot taken from the reaction crude for the preparation of organosilica after 3 h of reaction time. c) Hypothesized formation mechanism of observed rice-grain-like structures observed; I) Hydrolysis and polycondensation of Supra-PNA-Si helix occurs; II) upon further condensation the organosilane may form sub-μm sized particles, which due to the presence of uncondensed silanol groups could further assemble to form III) μm sized grain-like structures.

Figure 16 .

 16 Figure 16. a) and b) SEM images recorded on Supra-OSPs after 12 h of reaction time.

Figure 18 .

 18 Figure 18. a) SEM image of degrading fibers observed at 3 h of reaction time. b) SEM image of degrading fiber at 12 h of reaction time.

Figure 19 .

 19 Figure 19. a) UV-Vis absorption spectra recorded on a Supra-OSPs dispersion in water (c=0.075 mg•mL -1 ). b) ATR-FTIR spectra recorded on Supra-OSPs.

Figure 20 . 1 )

 201 Figure 20. a) Schematic representation in the disruption of the FRET process upon heating a dispersion of Supra-OSPs due to PNA strand dissociation. b) Normalized absorption and emission spectra of F-and T-PNA strands in PBS buffer at pH=5.8. c) FRET experiment of on a dispersion of Supra-OSPs (c=0.008 mg•mL - 1 ) in PBS (pH=5.8) upon excitation at λex=450 nm. d) Temperature dependent FRET experiments on dispersion of Supra-OSPs (c=0.008 mg•mL -1 ) in PBS (pH=5.8) upon excitation at λex=450 nm.

Figure 22 .

 22 Figure 22. SEM images of the crude reaction mixtures for the formation of organosilicas from Supra-PDP-Si. a-c) SEM images of the reaction crude at different time intervals when no additional silica source was added in the hydrolysis and condensation reaction of Supra-PDP-Si. d-f) SEM images of the reaction crude at different time intervals when an equimolar amount of TEOS was added to the reaction.

Figure X.

  Figure X. a) SEM images of fibers obtained upon hydrolysis and condensation of Supra-PDP-Si after 2 h. b) SEM images of fibers obtained upon hydrolysis and condensation of Supra-PDP-Si with TEOS after 2 h.

1 : 19 4. 4 . 3 .

 11943 Am-CAATGTAGCT-Cy3-Am MS (ESI-TOF negative mode): m/z [M-3H] 3-calcd for C139H183N43O72P12, 1326.00; found, 1326.06 Complementary strand to 1 = Cy5-Strand = Cy5-AGCTACATTG MS (ESI-TOF negative mode): m/z [M-3H] 3-calcd for C141H185N43O72P12, 1332.63; found, 1332.65 Short 4mer diamino oligonucleotide: Am-GCGC-Am MS (ESI-TOF negative mode): m/z [M-2H] 2-calcd for C50H73N20O32P5, 810.1694; found, 810.Preparation of ssDNA and ssPNA alkoxysilanes General information. All solvents were purchased from Merck and used as received. DMSO (analytical reagent grade) was purchased from Fisher Chemical and dried over Na2SO4 prior to use. 3-(triethoxysilyl)propyl isocyanate (ICPTES, 95%), triethylamine (TEA, >99%), were purchased from Merck.

Table of contents Résumé de these List of abbreviations Chapter 1: General introduction

	1.1. What is "nano" in nanomedicine?
	1.2. Nanoparticles for biomedical applications
	1.2.1. Physicochemical considerations for nanoparticles
	1.2.2. Examples of nanomaterials currently studied for biomedical
	applications
	1.3. Mesoporous stimuli-responsive organosilica particles for biomedical
	applications
	1.3.1. Preparation of mesoporous silica particles
	1.3.2. Preparation and functionalization of mesoporous organosilica particles
	1.3.3. Mesoporous organosilica particles for biomedical applications
	1.4. Applying supramolecular concepts on organoalkoxysilane chemistry:
	New opportunities in the preparation of smart organosilica particles
	1.4.1. Supramolecular polymers in biomedical research
	1.4.2. Self-assembly of organoalkoxysilanes
	1.5. Aim of the thesis
	1.6. References

Chapter 2: Light-breakable mesoporous organosilica particles

	4.2.2. Supramolecular assembled organoalkoxysilanes and their use for
	the preparation of organosilicas
	4.2.2.1. PNA-based and supramolecularly assembled organoalkoxysilanes
	for the preparation of organosilicas
	4.2.2.2. Organosilicas prepared through hybrid DNA-PNA
	supramolecular interactions
	4.3. Conclusions
	4.4. Experimental section
	4.4.1. Instruments
	4.4.2. Preparation of ssDNA oligonucleotides
	4.4.3. Preparation of ssDNA and ssPNA alkoxysilanes
	4.4.4. Preparation of model mesoporous silica particles
	4.4.5. Preparation of organosilica particles
	4.4.6. DNase I-triggered degradation of ssDNA-OSPs
	3.1. Introduction 4.4.7. FRET experiments
	3.2. Results and discussion 4.4.8. SAXS-based analysis of ssDNA-OSPs
	3.particles 4.5. References
	3.3. Conclusions
	3.4. Experimental section
	3.4.1. Instruments
	3.4.2. Preparation of alkoxysilanes for the functionalization of cage-like
	silica particles
	3.4.3. Preparation of alkyne-RGD
	2.1. Introduction 3.4.4. Preparation of particles and their surface functionalization
	2.2. Results and discussion 3.4.5. Quantification of surface functionalization
	2.2.1. Synthesis and characterization of the photolabile bis-alkoxysilane 3.4.6. Loading protocols for OSCs
	2.2.2. Preparation and characterization of light-breakable mesoporous 3.4.7. Degradability studies and HE release experiment
	organosilica particles 3.4.8. Cell viability experiments
	2.2.3. Preparation and characterization of SS-MSPs 3.4.9. Biodistribution studies
	2.2.4. Preparation and characterization of MSPs 3.5. References
	2.2.5. Light-triggered degradation of light-breakable mesoporous Chapter 4: Building organosilicas with oligonucleotides
	organosilica particles 4.1. Introduction 2.2.6. Light-triggered cargo release from LB-MSPs 4.2. Results and discussion 2.2.7. Preliminary cytotoxicity tests 2.3. Conclusions 4.2.1. Preparation of single-stranded DNA and PNA organosilica particles

2.1. Preparation and characterization of cage-like organosilica particles 3.2.2. Degradation studies and stimuli-triggered cargo release 3.2.3. Surface functionalization of cage-like organosilica particles 3.2.4. Preliminary biological evaluation of cage-like organosilica

  at 20000 ×g (15 min). Particles were thoroughly washed by sonication and centrifugation with distilled H2O/EtOH (1:1, v/v; 2x) and EtOH (2x). The organic template was removed by refluxing the particles in EtOH for 12 h. The material was recovered by centrifugation, re-dispersed in EtOH, sonicated for 2 min, and centrifuged again. The particles were re-dispersed in EtOH, the dispersion heated to reflux for additional 12 h and finally centrifuged. The particle were washed with EtOH (3×) and dried overnight under reduced pressure.

	2.4.3. Preparation of particles
	General information: All the reactions were carried out in a 100 mL round-bottom flask equipped
	with a magnetic stirring bar (1 cm Teflon-coated cylindrical bar). EtOH from Carlo Erba (ACS
	reagent), hexadecyltrimethylammonium bromide (CTAB, Acros Organics, 99+%), ammonia
	solution (VWR, 28%), tetraethyl orthosilicate (TEOS, Sigma-Aldrich, ≥99% GC), and bis[3-
	(triethoxysilyl)propyl]disulfide (fluorochem, tech grade) were used for the preparation of the
	particles. Preparation of mesoporous silica particles (MSPs): CTAB (50 mg, 0.137 mmol) was added to an
	Preparation of light-breakable mesoporous organosilica particles (LB-MSPs). CTAB (50 mg, EtOH/H2O mixture (15:30, v/v) and stirred at 250 rpm until its complete dissolution.
	Subsequently, NH3 (28 wt%, 350 μL) was added to the stirring solution and the stirring speed was
	37 (m, 1H), 6.87
	(m, 1H), 6.09-5.94 (m, 2H), 5.47-5.23 (m, 4H), 4.94 (s, 2H), 4.66-4.64 (m, 2H), 4.17-4.15 ppm (m,
	2H). 250.1079; found, 250.1073.
	Preparation of disulfide-bridged mesoporous organosilica particles (SS-MSPs). CTAB (50 mg,
	0.137 mmol) was dissolved in an EtOH/H2O mixture (14.5/30, v/v) and stirred at 250 rpm until its
	complete dissolution. Subsequently, NH3 (28 wt.%, 350 μL) was added to the stirring solution and
	the stirring speed was increased to 1000 rpm. A mixture of TEOS (87.3 μL, 0.39 mmol) and
	compound bis[3-(triethoxysilyl)propyl]disulfide (79.75μL, 0.173 mmol) in EtOH (500 μL) were
	123 126

1

H NMR (CDCl3, 400 MHz): δ 8.16 (d, 1H, J=9.1 Hz), 7.

13

C{ 1 H} NMR (CDCl3, 100 MHz): δ 163.1, 139.8, 139.0, 134.2, 132.0, 127.6, 118.6, 117.3, 113.5, 113.3, 71.9, 69.3, 69.0 ppm. ATR-FTIR ṽmax [cm -1 ]: 3861, 2854, 1577, 1508, 1336, 1321, 1282, 1228, 1066, 993, 923, 842, 754. HR-MS (ESI-TOF): m/z [M+H] + calcd for C13H16NO4, 0.137 mmol) was added to an EtOH/H2O mixture (14.5:30, v/v) and stirred at 250 rpm until its complete dissolution. Subsequently, NH3 (28 wt%, 350 μL) was added to the mixture under stirring, and the stirring speed was increased to 1000 rpm. A mixture of TEOS (87.3 μL, 0.39 mmol) and compound 1 (100 mg, 0.173 mmol) in EtOH (500 μL) was added, and the mixture was stirred (1000 rpm) at r.t. overnight in the dark. The solution was decanted, leaving behind any material which was adhered to the walls, and the particles were recovered by centrifugation at 20000 ×g (15 min). Subsequent purification and extraction steps were carried out under the exclusion of light. Particles were thoroughly washed by sonication and centrifugation with distilled H2O/EtOH (1:1, v/v; 2×) and EtOH (2×). The organic template was removed by heating the particle dispersion in EtOH to reflux for 12 h. The material was recovered by centrifugation, re-dispersed in EtOH, sonicated for 2 min, and centrifuged again. The particles were re-dispersed in EtOH, the dispersion heated to reflux for additional 12 h and finally centrifuged. The particle were washed with EtOH (3×) and dried overnight under reduced pressure. added and the mixture was stirred (1000 rpm) at r.t. overnight. The solution was decanted, leaving behind any material, which was adhered to the walls, and the particles were recovered by centrifugation increased to 1000 rpm. A mixture of TEOS (125 μL, 0.56 mmol) were added and the mixture was stirred (1000 rpm) at r.t. overnight. The solution was decanted, leaving behind any material, which was adhered to the walls, and the particles were recovered by centrifugation at 20000 ×g (15 min). Particles were thoroughly washed by sonication and centrifugation with distilled H2O/EtOH (1:1, v/v; 2×) and EtOH (2×). The organic template was removed by heating a particle dispersion in EtOH to reflux for 12 h. The material was recovered by centrifugation, re-dispersed in EtOH, sonicated for 2 min, and centrifuged again. The particles were re-dispersed in EtOH, the dispersion heated to reflux for additional 12 h and finally centrifuged. The particle were washed with EtOH (3×) and dried overnight under reduced pressure.

  7,34

  48,49

Pierre PICCHETTI Design and Preparation of Degradable Organosilica Particles for Biomedical Applications Résumé

  4.8 𝑛𝑚Les travaux présentés dans cette thèse se sont concentrés sur la préparation et la caractérisation de nouvelles particules d'organosilice sensibles aux stimuli. À cet égard, une série d'organoalkoxysilanes ont été synthétisés et utilisés pour préparer des organosilices possédant des groupes fonctionnels organiques liés de manière covalente ou supramoléculaire dans leur structure de silice. Il a été démontré qu'il est possible de préparer des organosilices poreuses et photodégradables qui sont capables, sous irradiation lumineuse, de libérer quantitativement des molécules hydrophobes. De plus, certains oligonucléotides, tels que l'ADN et l'ANP, peuvent être intégrés dans la structure des particules afin de préparer des matériaux pouvant interagir avec des molécules biologiques telles que des enzymes ou des ADN complémentaires. Enfin, la possibilité de préparer des particules d'organosilice sous forme de cage et sensibles à l'oxydoréduction a été mis en évidence et leur utilisation potentielle pour le développement de nouvelles techniques de nanomédecine a été évaluée et discutée.

aqueux en raison de l'hydrolyse de la structure de silice amorphe en fonction de la taille, de la porosité, de la procédure de préparation (comme la valeur du pH pour la préparation et la présence de co-solvants) et des fonctionnalités de surface supplémentaires des particules, la dissolution complète n'est pas obtenue et la plupart du temps la dissolution reste lente.14 Le développement de particules organosiliciques mésoporeuses (MON), avec un groupe de pontage organique cassable dans leur structure, possède un grand potentiel pour surmonter le problème de dégradation et d'élimination des nanocarriers à base de silice. L'introduction de groupes de pontage réagissant aux stimuli dans le cadre des MON permet de régler avec précision la dégradation et la libération de la charge utile.15 La présence d'un déclencheur externe devrait faciliter la désintégration des particules, permettant une libération contrôlée de la charge utile sur une période définie, essentielle pour le développement de nouveaux systèmes d'administration de médicaments pour un traitement optimisé des maladies. Une meilleure dégradation entraîne une meilleure élimination des matières des organismes vivants, ce qui permet de développer des vecteurs de médicaments plus sûrs.Les travaux présentés dans cette thèse portent sur la conception et la préparation de particules organosiliciques poreuses et sensibles aux stimuli pour des applications d'administration de médicaments. L'accent a été mis sur la possibilité d'utiliser la lumière UV pour déclencher la dégradation des particules organosiliciques mésoporeuses, où la lumière UV est un bon stimulant dans les systèmes d'administration de médicaments pour l'administration topique. L'absorption de la lumière UV nocive, tout en permettant l'administration de molécules bioactives hydrophobes, telles que la provitamine D3, était le principal objectif visé en ce qui concerne le développement de particules organosiliciques mésoporeuses dégradables par la lumière. Des carences physiopathologiques en vitamine D3 sont souvent observées chez les personnes souffrant de dysfonctionnements hépatiques, rénaux ou thyroïdiens, et la distribution de provitamine D3 par des applications topiques, dans lesquelles la formation active de la vitamine D3 peut se produire sur place, est intéressante.16 En outre, les maladies de la peau telles que le mélanome ou les inflammations peuvent être traitées avec des nanoparticules chargées de médicaments, permettant une pénétration plus profonde à travers la peau (comme les canaux des follicules pileux).17 Comme la lumière UV pénètre dans les parties supérieures de la peau

), tunable pore sizes (2-30 nm) and high total pore volumes (up to 1 cm 3 •g -1 ). In Figure14examples of MSPs with different size, morphology and mesopore arrangement obtained by modifying synthetic parameters are shown.

•L -1 .

Guillermo Moreno Alcantar, Simone Silvestrini and Alessandro Aliprandi are thanked for their precious scientific advice, support and readiness for occasional experiments -True to the

Acknowledgements Short Summary

Acknowledgements

First of all, I would like to thank Prof. Luisa De Cola for having given me the possibility to conduct research in her working group. During this time, I was able learn and grow as a chemist in many aspects. I am especially grateful for the possibility to have worked independently on research problems and never experiencing any financial or instrumental related shortcomingsessentially to thrive in research labs and enjoying experimentation. I will remember the vivid and inspiring scientific discussions we had in the lab.

I would like to thank Leana Travaglini and Brian DiMarco for their adamant support and helpful discussions, contributing to the successful conclusions of the work presented in chapter 2. Thanks also to Etienne Piantanida and Simone Silvestrini for their support regarding XPS analysis and Yang Zhang for in-vitro experimentation presented in chapter 2.

I would like to thank collaborators of the Mario Negri Institute, especially Dr. Laura Talamini, Prof. Paolo Bigini and Dr. Giovanni Sitia, which helped with in vitro and in vivo experiments presented in chapter 3. I would like to thank Adrien Mourgout, which as part of his Master's internship, worked excellently under my supervision on the functionalization of cage-like silica particles. I thank Alexander Frechat, Corinne Crucifix and Prof. Patrick Schultz for their help during the 3D structure reconstruction of cage-like particles using Relion® and assistance during long TEM sessions at the IGBMC. I would like to thank also Prof. Hanadi Sleiman for her enthusiastic support and motivating discussions we had during my placement at the McGill University. Big thanks to Michael Dore, Tuan Trinh, Aurélie Lacroix, Donatien de Rochambeau and Nicole Avakyan for the time we spend together in Montréal and for fantastic assistance to any research-related problem/discussion. Further members of the Sleiman lab and faculty members that I want to thank are: Dr. Johans Fakhoury, Xin Luo, Christophe Lachance-Brais, Daniel Saliba, Dr. Alexander Whaba and Dr. Violetta Toader. Many thanks also to Dr. Alessandro Porchetta, Dr. Marianna Rossetti, Prof Roberto Corradini and Dr. Stefano Volpi for their helpful discussions regarding the work presented in chapter 4 and for the preparation of the PNA strands.

Thanks also to Prof Marta Cerruti and Prof. Fiorenzo Vetrone for their participation and support of scientific project which originated in discussion with Vivienne Tam, during my stay at the McGill University. I would like to thank Vivienne for her enthusiastic and hard work related to this collaboration.

Chapter 2

Light-breakable mesoporous organosilica particles

In this chapter, the preparation and characterization of a novel light-responsive bis-alkoxysilane and its use for the preparation of light-breakable mesoporous organosilica particles is presented.

The use of light to trigger the particle degradation is particuarly advantageous, due to its facile spatiotemporal control and non-invasive properties. The light-breakable organosilica particles can be loaded with bioactive molecules and the payload release can be triggered upon lightirradiation. These particles hold potential as cutaneous drug delivery systems, protecting the skin from harmful UV-light radiation, while simultaneously releasing bioactive molecules, such as provitamin D3.

Preparation and characterization of SS-MSPs

Disulfide-bridged mesoporous organosilica particles (SS-MSPs) were prepared that possess similar physicochemical properties compared with LB-MSPs to used them as model particles for comparison when studying the unique light-triggered degradation and drug release from LB-MSPs. SS-MSPs were prepared by adapting the synthesis of LB-MSPs and replacing the photolabile bis-alkoxysilane with bis [3-(triethoxysilyl)propyl]disulfide (BTPS, see section 2.4.3 for details). As depicted in Scheme 3, BTPS was hydrolyzed and co-condensed with TEOS, with a TEOS/BTPS molar ratio of 2.25:1, in the presence of CTAB and using aqueous ammonia as the basic catalyst. SEM images of SS-MSPs showed the spherical morphology of the particles, with a size of 242±17 nm (Figure 12a). STEM images of SS-MSPs revealed the absence of a highly ordered mesoporous phase within the particles, which was confirmed by the SAXS pattern obtained from SS-MSPs, where no sharp peak could be observed, but only a shoulder centered around q=1.6 nm -1 . USA) with an acceleration voltage of 20 kV. The SEM sample were prepared by drop-casting the dispersion of particles in EtOH onto a glass cover slip, subsequently sputter coated with Au (Emitech K575X Peltier cooled) for 60 s at 60 mA prior to fixation on an Al support.

The STEM were prepared drop casting dispersions of particles in EtOH onto Formvar-coated Cu grids (400 mesh) and allowed to dry overnight prior visualization.

Transmission electron microscopy (TEM) samples were analyzed on a FEI/PHILIPS CM120 system operating at 100 kV. Samples were prepared drop casting dispersions of particles in EtOH (ca. 0.1 mg/mL) onto Formvar-coated Cu grids (400 mesh) and allowed to dry overnight prior visualization.

Dynamic light scattering (DLS): the measurements were conducted on a Delsa Nano C Particle Analyzer (Beckman Coulter, operative wavelength 655 nm). The DLS measurements were performed on dispersions of the particles in EtOH (ca. 0.1 mg/mL).

Nitrogen physisorption isotherms were obtained with a Micromeritics ASAP-2020 physisorption instrument. The particles were degassed at 100 °C for 12h. The measurement was performed at -196 °C. The surface areas were calculated by Brunauer-Emmett-Teller (BET) method in the relative pressure range of the adsorption branch at p/p0 0.06-0.3. The pore size distributions and pore volumes were calculated by density functional theory (DFT) method on the adsorption branch. The adsorption data were analyzed by using the SAIEUS software (model used: N2@77K, cylindrical pores in an oxide surface),8 provided by Micrometrics. The total pore volume was calculated at p/p0=0.99.

Small-angle X-ray scattering (SAXS) set-up comprised the SAXSess mc2 instrument from Anton Paar GmbH, containing a slit collimation system, and the PW3830 laboratory X-ray generator (40 kV, 50 mA) with a long-fine focus sealed X-ray tube (CuKα wavelength of λ = 0.1542 nm) from PANalytical. Detection was performed with the 2D imaging-plate reader Cyclone® by Perkin Elmer. The measurement was performed on a powder sample for 5 min and the data collected up to a scattering vector q value of 7 nm -1 , where q = (4πsinθ)/λ and 2θ is the scattering angle.

The 2D data were converted to 1D data and background-corrected by using SAXSQuant software (Anton Paar GmbH, Austria). X-Ray photoelectron spectroscopy (XPS) analysis was performed using a K-Alpha™+ X-ray Photoelectron Spectrometer System (Thermo Scientific). Monochromatic Al K alpha X-rays were used (15 keV, 72 W, 200 mm spot diameter). Spectra were measured using pass energy of 200 eV for survey spectra and 50 eV for core level spectra. The samples were analysed as powders.

The Advantage software was used for background subtraction (smart-mode), and deconvolution of peaks.

Thermogravimetric analysis (TGA) was conducted on a Netzsch model STA 449 fi Jupiter instrument. The samples (ca. 2 mg) were kept at 100°C for 30 min for stabilization, then heated from 25 to 800 °C at a speed of 10°C/min, before being held at this temperature for further 30 min, and finally cooled. The analyses were performed under a 20 mL/min air flow.

Synthesis of the light-degradable bis-alkoxy silane 1

General information: Compound 1 was prepared according to the reactions shown in Scheme 1.

All reactions were carried out under argon atmosphere. The round-bottom flasks used in the reactions were oven-dried at 110 °C prior to use. All reactions were performed in anhydrous solvents as purchased from Sigma-Aldrich. The reaction progress was monitored by thin-layer chromatography (TLC) with silica gel 60 F254 coated on aluminum sheets (Merck). Flash column chromatography was carried out using 60-200 μm silica gel (VWR Chemicals). The solvents used were reagent grade or higher, unless otherwise noted. The following compounds were purchased from the listed supplies at the given purity and were used without further purification: sodium hydride (NaH, Sigma-Aldrich, 90%), 5-hydroxy-2-nitrobenzyl alcohol (Sigma-Aldrich, 97%), N,Ndimethylformamide (DMF, Sigma-Aldrich, anhydrous, 99.8%), triethoxysilane (Sigma-Aldrich, 95%), platinum(0)-1,3-diviynl-1,1,3,3-tetramethyldisiloxane complex solution (Karstedt's catalyst, Pt∼2 wt% in xylene, Sigma-Aldrich), sodium sulfate (anhydrous, Sigma-Aldrich, ACS reagent), and sodium hydrogen carbonate (Sigma-Aldrich, ACS reagent, >99.7%).

showed characteristic transmission bands for the -CH2-wagging and deformation vibration at 1456 and 1354 cm -1 , respectively, which could be attributed to the presence of PEG chains. The transmission bands in the 2927-2868 cm -1 range could be assigned to the Csp3-H stretching vibration derived from the presence of PEG chains and the disulfide linker within the silica framework of OSCs. The covalent nature of the linkage of the disulfide moieties within the OSC silica framework was proven by XPS analysis. The survey spectrum recorded on OSCs (Figure 6a) indicates the presence of C(1s) and S(2p) signals at 287 eV and 164 eV, respectively. The deconvoluted HR scan for the C(1s) peak (Figure 6b) shows the presence of two components C-1 at 284.8 eV and C-2 at 286.4 eV, which can be attributed to the presence of C-C and C-O-C species, respectively. The analysis of the HR scan for the S(2p) peak (Figure 6c) shows the presence of the characteristic spin-orbit components of sulfur S-1 at 163.5 eV and S-2 at 164.6 eV, respectively, which are attributable to the disulfide bond present in OSCs. 24

Degradation studies and stimuli-triggered cargo release

As discussed in the introduction, the integration of disulfide functional groups within the silica framework renders OSCs degradable in the presence of thiolates due to occurring thiol-disulfide exchange reactions in the intracellular environment, caused by high concentrations (1-10 mM) of glutathione (GSH), 25 a thiol-containing endogenous tripeptide. 26 GSH is one of the main mediators for cellular redox homeostasis and is able to reduce the disulfide bridge within OSCs.

In order to investigate the GSH-triggered degradation of OSCs, particle dispersions (c=0.1 mg•mL - 1 ) in PBS buffer with and without GSH (10 mM) were prepared and incubated at 37°C. Aliquots of these dispersions were taken at different time points were analyzed by TEM and STEM. As depicted in Figure 8, the formation of small debris was observed, indicating a fast particle degradation. The relatively fast degradation that occurred within the first 2 h ca be explained by the small particle size and the cage-like structure of OSCs, which provide a high surface area and therefore facile GSH diffusion. Therefore, the disulfide reduction most likely occurred simultaneously on the inner and outer regions of the particles. STEM images of OSCs dispersed in PBS/GSH (10 mM) solution at 37°C indicated the complete degradation of the particles after 12 h (Figure 9) of incubation as visualized by the formation of low-contrast and amorphous degradation products. The amount of glucosamine covalently grafted onto Sugar-OSCs was quantified by using a previously reported fluorimetric analysis, in which the reaction of reducing sugars with cyanoacetamide in borate buffer results in the formation of luminescent reaction product. 30 The amount of glucosamine was estimated by measuring the fluorescence intensity of Sugar-OSCs (Figure 13) that have been derivatized with 2-cyanoacetamide in borate buffer (c=0.6 mg•mL -1 ) at the wavelength of emission λem=383 nm (λex=331 nm) and resulted to be 0.89 µg of glucosamine per mg of Sugar-OSCs (see section 3.4.5.).

Quantum yield measurements were performed with an absolute photoluminescence quantum yield spectrometer Quantaurus C11347 (Hamamatsu, Japan) exciting the sample at λexc=600 nm.

For the measurement of colloidal dispersions, water was used as dispersion medium.

Fluorescence anisotropy measurements were performed on a PicoHarp300 or the Multi Channel Scaling (MCS) electronics NanoHarp 250 of the PicoQuant FluoroTime 300 (PicoQuant GmbH, Germany), equipped with a PDL 820 laser pulse driver. A pulsed laser diode LDH-P-C-510 (λexc=505 nm) was used to excite the sample and mounted directly on the sample chamber at 90°. The photons were collected by a PMA-C192 photomultiplier (PMT) single-photon-counting detector.

The data were acquired by using the commercially available software EasyTau (PicoQuant GmbH, Germany), while data analysis was performed using the commercially available software FluoFit (PicoQuant GmbH, Germany).

Transmission electron microscopy (TEM) samples were analyzed on a FEI/PHILIPS CM120 system operating at 100 kV. Samples were prepared drop casting dispersions of particles in EtOH (0.1 mg•mL -1 ) onto Formvar-coated Cu grids (400 mesh) and allowed to dry overnight prior visualization.

Nitrogen physisorption isotherms were obtained with a Micromeritics ASAP-2020 physisorption instrument. The particles sample was degassed at 100 °C for 12h. The N2 adsorption-desorption measurement was performed at -196 °C. The surface areas were calculated by Brunauer-Emmett-Teller (BET) method in the relative pressure range p/p0 0.06-0.3. The pore size distributions and pore volumes were calculated by classical method on the adsorption branch using a cylindrical model. The total pore volume was estimated at p/p0 0.99. X-Ray photoelectron spectroscopy (XPS) analysis was performed using a K-Alpha™+ X-ray Photoelectron Spectrometer (XPS) System (Thermo Scientific). Monochromatic Al K alpha X-rays were used (15 keV, 72 W, 200 mm spot diameter). Spectra were measured using pass energy of 200 eV for survey spectra and 50 eV for core level spectra. The analyzed samples were prepared by drop-casting an ethanolic dispersion (0.1 mg•mL -1 ) of the particles onto a glass cover slip pre-added, followed by ICPTES (2.07 μL, 8.35 μmol), and the reaction mixture was allowed to react under stirring for 1 h. The reaction mixture was used for the post-synthetic grafting of OCSs without further purification. 1 Preparation of AzTMS. To a stirring dispersion of NaN3 (390 mg, 6 mmol) and tetrabutylammonium bromide (278 mg, 0.8 mmol) in acetonitrile (15 mL), Cl-TMS (964 µL, 4 mmol) was added dropwise and the reaction was subsequently allowed to stir for 24 at 80 °C under reflux conditions. The reaction mixture was allowed to cool down and subsequently Et2O (5 mL) was added, which induced the formation of a white precipitate(NaBr). The reaction mixture was filtered from the precipitate and the organic solvents were evaporated from the the dialysis solution was replaced every 2 h, when possible. Before conducting in-vitro and invivo experiments, the particle suspension was dialyzed against distilled water for 24 h.

Preparation of N3-OSCs.

To a stirring solution of surface extracted OSCs (5 mL, c=3mg mL -1 ) which have been dialyzed against EtOH/H2O (1:1 v/v), Az-Silane was added (11.1µL, 4.16µmol) and the mixture was allowed to stir for 12 h at 50 °C. Subsequently the particles were purified by means of dialysis against EtOH/H2O (1:1 v/v) for 12 h.

Preparation of RGD-OSCS.

To the purified Az-OSCs (2.5 mL, 3.94 mg•mL -1 ) dispersion RGD-Alkyne (1 mg, 1.92 µmol) was added and the reaction mixture was stirred for 16 h at room temperature.

Subsequently, the particle dispersion was dialyzed against water for 48 h.

Quantification of surface functionalization

Glucosamine quantification. To a dispersion of Sugar-OSCs (500 µL, c=2.3 mg/mL), 2cyanoacetamide aqueous solution (500 µL, c=0.5 M) and borate-phosphate buffer (1 mL, composed of 0.3 M sodium borate and 0.3 M potassium dihydrogen phosphate, pH = 8) was added and the reaction mixture was heated for 30 min in a boiling water bath. The samples were allowed to cool down at room temperature and the fluorescence intensity was measured upon excitation at λex=331 nm. RGD quantification. To aqueous dispersion of RGD-OSCs, an ethanolic 9,10-phenantrenequinone solution (150 µL, 150 µM) and aqueous NaOH solution (25 µL, 2 M) was added. Subsequently the reaction mixture was heated at 60 °C for 3 h. Aqueous HCl (40 µL, 1.2 M) was added and the reaction mixture was kept at room temperature for 1 h before measuring its fluorescence (λex=312 nm).

Loading protocols for OSCs

HOECHST 33258 (HE) loading of OSCs. In order to load non-functionalized OSCs with the hydrophilic dye Hoechst 33258, organosilica particles were prepared but no surface functionalization occurred. After CTAB removal via dialysis (as described above) the particle

Chapter 4

Building organosilicas with oligonucleotides In this chapter, the preparation of organosilica particles by using DNA and PNA oligonucleotides is described. To this purpose, novel DNA-and PNA-based bis-alkoxysilanes were synthesized and their hydrolysis and condensation by using sol-gel chemistry were investigated. In this regard, the influence of synthetic parameters, such as the length of the oligonucleotide, the presence of a secondary silica source or a surfactant serving as a structure-templating agent was assessed. The Supra-PNA-Si was used for the preparation of organosilica particles, with or without the presence of the additional silica source tetraethyl orthosilicate (TEOS), upon its base-catalyzed hydrolysis and polycondensation in an H2O/EtOH (1:0.3, v/v mixture), leading to the formation of organosilica particles Supra-OSPs (Figure 14, see section 4.4.5. for preparation details). The morphological evolution during the formation of Supra-PNA-Si in the absence of an additional silica source was monitored via electron microscopy analysis by taking aliquots of the crude reaction mixture at 2 different time intervals. SEM images of the crude reaction at 3 h (Figure 15a andb) indicate the presence of silica structures, which appeared to be composed of smaller grain-like structures (Figure 15 b). The formation of grain-like particles may be explained by the structure of the Supra-PNA-Si itself, which can form a linear helical assembly, 54 thus To prepare Supra-PDP-Si, the amino bearing PNA-strands were functionalized with ICPTES in the presence of the base TEA, forming their mono alkoxysilane derivatives (PNA-S 1 and PNA-Si 2, see section 4.4.3.). Subsequently, an equimolar amount of PNA-Si 1, PNA-Si 2 and ssDNA (see section 4.4.5 for its structure) were mixed to promote their hybridization, forming Supra-PDP-Si.

Subsequent base-catalyzed hydrolysis and condensation of Supra-PDP-Si with and without the secondary silica source TEOS, in the presence of the surfactant CTAB was investigated in a H2O/EtOH mixture (see section 4.4.5.). To follow the formation of organosilica particles, electron microscopy images were taken on aliquots of the reaction crude at different time intervals.

Recorded SEM images of the reaction mixtures at 1 h, 3 h and 12 h of reaction time in the absence

of TEOS are shown in Figure 22a-c. As depicted in Figure 22a, after 1 h sub-µm fiber-like structures (encircled in black) were observed, which further grew after 3 h of reaction time to form big fibrous structures, most probably due to a delayed hydrolysis and condensation of the bulky organoalkoxysilanes. After 12 h of reaction time, the fibrous structures started to degrade into smaller fibrous structures, most probably due to instability of the supramolecular interactions of the PNA-DNA-PNA supramolecular bridging groups. When organosilicas were prepared from Supra-PDP-Si with an equimolar amount of TEOS (Figure 22d-f), no fibrous structures could be imaged after 1 h of reaction time. After 3 h large fibrous structures were observed (Figure 22e), and stable fibrous structures were still observed at 12 h of reaction time (Figure 22f). The increased stability of the fibrous organosilica structures may be explained by cocondensation of TEOS to Supra-PDP-Si and the presence of CTAB, which stabilized the silicates in aqueous environments against depolymerization.

bonded polymers was shown. The successful integration of supramolecular bonds within a rigid silica-based structure was successfully proven via Förster resonance energy transfer experiments, showing the feasibility to prepare organosilicas possessing supramolecular bonds within their structure. Finally, the use of a PNA-DNA-PNA based organoalkoxysilane, held together by supramolecular interactions, to prepare organosilicas was investigated. The successful formation of fiber-like structures was confirmed, and future experiments will provide further insights into the physicochemical properties of PNA-DNA-PNA based organosilicas.

Experimental section 4.4.1. Instruments

Fourier transform infrared (FTIR) transmittance spectra were measured with a Shimadzu IRAffinity-1 spectrometer. The spectrum of 1 was recorded in CHCl3 (ca. 1 mg mL -1 , 0.1 mm path length NaCl window cell). The attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra of the particle samples were collected using a spectral resolution of Δ=4 cm -1 , accumulating 64 scans and the ATR-FTIR spectra were ATR corrected (penetration depth mode).

Mass spectrometry (MS) experiments were performed on a Bruker Daltonics microToF spectrometer (Bruker Daltonik GmgH, Bremen, Germany) with the electrospray ionization (ESI) interface operating in a positive ion (for PNA) or negative mode (for DNA).

Absorption spectra were measured on a Shimadzu UV-3600 spectrophotometer double-beam UV-VIS-NIR spectrometer and baseline corrected. Quartz cuvettes with path length of 10.0 mm were used for the recording of UV-Vis absorption spectra.

Scanning electron microscopy (SEM) and scanning transmission electron microscopy (STEM)

images were recorded with a FEI Quanta FEG 250 instrument (FEI corporate, Hillsboro, S3 Oregon, USA) with an acceleration voltage of 20 kV. The SEM sample were prepared by drop-casting the dispersion of particles in EtOH onto a glass cover slip, subsequently sputter coated with Au (Emitech K575X Peltier cooled) for 60 s at 60 mA prior to fixation on an Al support.

ζ-potential measurements were conducted on a DelsaNano C Particle Analyzer (Beckman Coulter, operative wavelength 655 nm). Measurements were performed on dispersions of the particles in EtOH (ca. 0.01 mg/mL).

Small-angle X-ray scattering (SAXS) set-up comprised the SAXSess mc2 instrument from Anton Paar GmbH, containing a slit collimation system, and the PW3830 laboratory X-ray generator (40 kV, 50 mA) with a long-fine focus sealed X-ray tube (CuKα wavelength of λ = 0.1542 nm) from PANalytical. Detection was performed with the 2D imaging-plate reader Cyclone® by Perkin Elmer. The measurement was performed on a powder sample for 5 min and the data collected up to a scattering vector q value of 7 nm -1 , where q=(4πsinθ)/λ and 2θ is the scattering angle.

The 2D data were converted to 1D data and background-corrected by using SAXSQuant software (Anton Paar GmbH, Austria). X-ray photoelectron spectroscopy (XPS) analysis was performed using a K-Alpha™+ X-ray Photoelectron Spectrometer System (Thermo Scientific). Monochromatic Al K alpha X-rays were used (15 keV, 72 W, 200 mm spot diameter). Spectra were measured using pass energy of 200 eV for survey spectra and 50 eV for core level spectra. The samples were analyzed as powders.

The Advantage software was used for background subtraction (smart mode), and deconvolution of peaks.

Preparation of ssDNA oligonucleotides

The oligonucleotides were synthesized on solid support using a BioAutomation Mer Made MM6 DNA synthesizer using a standard phosphoramidite protocol. Amino functionalities were introduced using the serinol aminomodifier (Am, GlenTech) and the fluorescent marker was introduced using Cyanine-3-phosphoramidite (Cy3, GlenTech) and Cyanine-5-phosphoramidite (Cy5, GlenTech). To this purpose, the phosphor amidite monomer modifier were dissolved in anhydrous CH3CN to achieve a final concentration of 0.1 M and the coupling occurred on the resin in a glove box with inert atmosphere upon activating the phosphonamidites with 0. Financial support was obtained from: