Ketoprofen is a potent non-steroidal anti-inflammatory drug which is used for the treatment of rheumatoid arthritis. The oral administration of ketoprofen can cause gastric irritation and adverse renal effects. Transdermal delivery of the drug can bypass gastrointestinal disturbances and provide relatively consistent drug concentrations at the site of administration. The release of ketoprofen from proprietary gel products from three different countries was evaluated by comparing the in vitro release profiles. Twenty extemporaneously prepared ketoprofen gel formulations using Carbopol® polymers were manufactured. The effect of polymer, drug concentration, pH and solvent systems on the in vitro release of ketoprofen from these formulations were investigated. The gels were evaluated for drug content and pH. The release of the drug from all the formulations obeyed the Higuchi principle. Two static FDA approved diffusion cells, namely the modified Franz diffusion cell and the European Pharmacopoeia diffusion cell, were compared by measuring the in vitro release rate of ketoprofen from all the gel formulations through a synthetic silicone membrane. High-performance liquid chromatography and ultraviolet spectrophotometric analytical techniques were both used for the analysis of ketoprofen. The validated methods were employed for the determination of ketoprofen in the sample solutions taken from the receptor fluid. Two of the three proprietary products registered under the same manufacturing license exhibited similar results whereas the third product differed significantly. Among the variables investigated, the vehicle pH and solvent composition were found have the most significant effect on the in vitro release of ketoprofen from Carbopol® polymers. The different grades of Carbopol® polymers showed statistically significantly different release kinetics with respect to lag time. When evaluating the proprietary products, both the modified Franz diffusion cell and the European Pharmacopoeia diffusion cell were deemed adequate although higher profiles were generally obtained from the European Pharmacopoeia diffusion cells. Smoother diffusion profiles were obtained from samples analysed by high-performance liquid chromatography than by ultraviolet spectrophotometry in both diffusion cells. Sample solutions taken from Franz diffusion cells and analysed by ultraviolet spectrophotometry also produced smooth diffusion profiles. Erratic and higher diffusion profiles were observed with samples taken from the European Pharmacopoeia diffusion cell and analysed by ultraviolet spectrophotometry. The choice of diffusion cells and analytical procedure in product development must be weighed against the relatively poor reproducibility as observed with the European Pharmacopoeia diffusion cell.

The research undertaken in these studies aimed to investigate the feasibility of developing and manufacturing innovative solid lipid carriers, such as solid lipid nanoparticles (SLN) and/or nanostructured lipid carriers (NLC) using a hot high pressure homogenization method, for didanosine(DDI). In addition, studies using in vitro differential protein adsorption were undertaken to establish whether the SLN and/or NLC have the potential to deliver DDI to the central nervous system (CNS). Prior to initiating pre-formulation, formulation development and optimization studies of DDI-Ioaded SLN and/or NLC, it was necessary to develop and validate an analytical method for the in vitro quantitation and analysis of DDI. An accurate, precise and sensitive RP-HPLC method with UV detection set at 248 nm was developed, optimized and validated for the quantitative in vitro analysis of DDI in formulations. Pre-formulation studies were designed to evaluate the thermal stability of DDI and to select and characterize lipid excipients that may be used for the manufacture of the nanocarriers. It was established that DDI is thermostable at temperatures not exceeding 163°C and therefore a hot high pressure homogenization technique could be used to manufacture DDI-loaded SLN and/or NLC. Lipid screening studies revealed that DDI is poorly soluble in both solid and liquid lipids. A combination of Precirol® ATO 5 and Transcutol® HP was found to have the best solubilizing-potential for DDI of all lipids investigated. The inclusion of Transcutol® HP into Precirol® ATO 5 changed the polymorphic form of the solid lipid from the stable 13-modification to a material that exhibited the co-existence between α- and β-polymorphic forms. The relatively high solubility of DDI in Transcutol® HP compared to Precirol® ATO 5 was an indication that a solid lipid matrix prepared from a binary mixture of Precirol® ATO 5 and Transcutol® HP was likely to have a higher loading capacity and encapsulation efficiency for DDI than a matrix consisting of Precirol® ATO 5 alone. Furthermore, the potential for the solid lipid matrix to exist in the α- and/or β-modifications when Transcutol® HP was added to Precirol® ATO 5 suggested that expulsion of DDI from a solid lipid matrix during prolonged storage periods was likely to be minimal. Therefore it was considered logical to investigate the feasibility of incorporating DDI into NLC and not in SLN. However, due to the limited solubility of DDI in lipids, formulation development of DDI-loaded NLC commenced using small quantities of DDI. Formulation development and optimization studies of DDI-loaded NLC were initially aimed at selecting a surfactant system that was capable of stabilizing NLC in an aqueous environment. Solutol® HS alone or a ternary mixture consisting of Solutol® HS, Tween® 80 and Lutrol® F68 was found to stabilize the nanoparticles in terms of particle size and the polydispersity index. The use of the ternary mixture as the surfactant system was preferred to using Solutol® HS alone as Lutrol® F68 and especially Tween® 80 have been successfully used to target the delivery of API to the brain. Aqueous DDI-free and DDI-Ioaded NLC containing increasing amounts of DDI were manufactured using hot high pressure homogenization at 800 bar for three cycles. The NLC formulations were characterized in terms of particle size, polydispersity index, zeta potential, and polymorphism, degree of crystallinity, encapsulation efficiency (EE), shape and surface morphology. The mean particle size for all formulations was below 250 nm with narrow polydispersity indices, indicating that narrow particle size distribution had been achieved. The d99% values for all formulations tested, were generated using laser diffractometry, and were below 400 nm, with span values ranging from 0.84 - 1.19 also suggesting that a narrow particle size distribution had been achieved. The zeta potential values measured in double distilled water with the conductivity adjusted to 50 μS/cm ranged from -18.4 to -11.4 mV. In addition, all the formulations showed a decrease in the degree of crystallinity as compared to the bulk lipid material and WAXS shows that the formulations existed in a single β-modification form. Furthermore DDI that had been incorporated into the NLC appeared to be molecularly dispersed in the lipid matrices. These parameters remained unaffected for most formulations following storage for two months at 25°C. In addition these formulations contained a mixture of spherical and non-spherical particles irrespective of the amount of DDI that was added during the manufacture of the formulations. These studies showed that it was feasible to develop and incorporate small amounts of DDI into NLC. However in order to use these delivery systems for oral administration of DDI to paediatric patients, strategies to improve the amount of DDI that could be loaded into the particles and to achieve high encapsulation efficiencies had to be developed. The limited solubility of DDI in lipid media was identified as a major factor that affected the loading capacity and encapsulation efficiency of DDI in the NLC. Therefore, a novel strategy aimed at increasing the saturation solubility of DDI in the lipid by attempting to increase the dissolution velocity of the drug in the lipid using a particle size reduction approach, was designed and investigated. DDI was dispersed in Transcutol® HP and the particle size of DDI in the liquid lipid medium was reduced gradually using hot high pressure homogenization and the product obtained from these studies was used to manufacture DDI-loaded NLC using a cold high pressure homogenization procedure. Although the encapsulation efficiency and drug loading following use of this approach was relatively high, the particles were large and showed a tendency to grow in size leading to the formation of microparticles after storage for two months at 25°C. In addition, the degree of crystallinity of the nanoparticles increased rapidly over the same storage period which led to expulsion of DDI nanoparticles for the NLC, despite the DDI loading in NLC being unaffected. It was clearly evident that this new approach of manufacturing solid lipid nanocarriers could be used as a platform not only for enhancing the loading capacity of DDI in solid lipid nanocarriers but also for other hydrophilic drugs. Differential protein adsorption patterns of DDI-loaded NLC were generated in vitro using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) in order to establish the potential for these systems to deliver DDI to the CNS. NLC formulations containing small amounts of DDI were used as these formulations showed a better stability profile than the formulation with a higher encapsulation efficiency and drug loading capacity. Furthermore, the encapsulation efficiency and drug loading of DDI were considered sufficient for use in 2-D PAGE studies. Data obtained from 2-D PAGE analysis reveal that DDI-loaded NLC preferentially adsorb proteins in vitro that are responsible for specific brain targeting in vivo. More importantly, these studies reveal that in addition to Tween® 80 that has already been shown to have the potential to target CDDS to the brain, Solutol® HS 15 has the potential to achieve a similar objective. Consequently, DDI-loaded NLC have the potential to deliver DDI to the brain and these results may be used as a platform for conducting in vivo studies to establish whether DDI can cross the blood brain barrier and enter the CNS when administered in NLC which may in turn lead to a major breakthrough in the management of HIV/AIDS and Aids Dementia Complex (ADC).

O objetivo do presente estudo foi sintetizar e caracterizar um material reparador para uso endodôntico com propriedades anti-inflamatória, antimicrobiana e remineralizante. A pasta experimental tem como propósito ser um sistema carreador de fármacos para regiões de difícil acesso em Odontologia. A apresentação do material é em forma de pó:líquido. No pó se encontra α-fosfato tricálcico, tungstato de cálcio e microesferas de amoxicilina (AMX-MS), já no líquido estão contidas nanocápsulas de indometacina (IndOHNC). A pasta experimental foi testada em relação a suas características físicoquímicas e biológicas. As AMX-MS obtiveram tamanho de 1,604 μm ± 0,08, forma esférica confirmada por MEV e teor da droga foi 1,63 mg g-1. As IndOHNC obtiveram tamanho de 162 ± 7,5 nm e forma esférica confirmada por MET. O teor do fármaco foi de 1 mg mL-1 ± 0,02. O escoamento da pasta foi de 18.56 ± 0.29, a espessura de película obtida foi 33 μm e radiopacidade de 1,81 mmAl. A pasta experimental demonstrou atividade antibacteriana contra o Enterococcus faecalis. A maior concentração de pasta experimental apresentou o maior valor em relação à viabilidade celular, com 187,03% no teste SRB. A atividade da enzima fosfatase alcalina e a formação de nódulos mineralizados obtiveram um gradual aumento em função do tempo. A migração celular demonstrou fechamento da ferida, e a pasta experimental foi capaz de acelerar o processo (p<0.05). Em conclusão, a pasta experimental demonstrou propriedades físico químicas e biológicas confiáveis, podendo ser um material promissor para o reparo da região periapical. / The aim of this study was to synthesize and characterize a new reparative material with anti-inflammatory, antimicrobial and remineralizing properties. The reparative material was developed to be a drug delivery system for regions with difficult access in Dentistry. The formulation is presented in powder/liquid. The powder is composed of α-tricalcium phosphate, calcium tungstate and amoxicillin microspheres (AMX-MS). The liquid is composed of nanocapsules containing indomethacin (IndOH-NC). The physicochemical and biological properties of the experimental endodontic paste were evaluated. The AMX-MS obtained a mean size of 1.604 μm ± 0.08, spherical shape and the encapsulation capacity was 1.63 mg g-1. IndOH-NCs obtained a mean size of 162 ± 7.5 nm and spherical shape confirm by MET. The content of the encapsulated drug was 1 mg mL-1 ± 0.02. The experimental paste flow was 18.56 ± 0.29 mm, mean film thickness was 33 μm and radiopacity equivalent to 1.81 mmAl. The experimental paste showed antibacterial activity against Enterococcus faecalis. The highest concentration of experimental paste presented the highest value in cell viability (187.03% in SRB test). The activity of the phosphatase alkaline enzyme and the formation of mineralized nodules showed a gradual increase as a function of time. Cell proliferation showed continuous wound closure, and the experimental paste was able to accelerate the process (p<0.05). In conclusion, the experimental paste demonstrated reliable physicochemical and biological properties, and it could be a promising material for periapical region repair.

A major drawback of chemotherapy is the lack of selectively leading to damage in healthy tissue, which results in severe acute side effects to cancer patients. The use of nanoparticles as a drug delivery system has emerged as novel strategy to overcome the barriers of immunogenic response, controlled release of therapeutic, and targeting the toxicity only to cancerous cells. In this study, polymeric nanoparticles composed of transition metals and particles derived from natural biopolymers have been generated via self-assembly. For example, nanoparticles composed of cobalt crosslinked with albumin (Co-alb NPs) via Co-amine coordination chemistry of lysine residue were syntheisized in various sizes. The method to generate Co-alb NPs involves no thermal heat, organic solvent or any surfactants, which is ideal for the production of large amounts in a timely manner. The Co-alb NPs displayed exceptional stability under physiological conditions (pH 7.4) for several days with minor changes in size; however degradation could be triggered by reductant (reduced glutathione (GSH), 10 mM) with complete disappearance of particles in less than 2 hour. Numerous therapeutics that are highly effective toward cancer cells have been developed; however, many cannot be administered to patients due to poor solubility in water and pH dependent properties. We have successfully encapsulated 7-ethyl-10-hydroxycampothecin (SN-38) into Co-alb NPs with encapsulation efficiency as high as 94% and loading capacities greater than 30%. We employed an emulsion-solvent evaporation method to incorporate SN-38 into Co-alb (SN38 Co-Alb NPs). Release of the drug from SN38 Co-Alb NPs was determined for particles incubated in PBS or PBS-GSH. SN38 Co-Alb NPs were exceptionally stable under physiological condition (PBS pH 7.4), but exhibited sustained release of SN-38 over time in the presence of GSH. Uptake and toxicity of the particles were also investigated in a gastric carcinoma cell line (SNU-5) where high degrees of macropinocytic uptake were observed. The particles displayed significant toxicity making them a prime candidate for further testing in animal models.

No description available.

The potential use of polymeric, functionalized nanoparticles (NPs) as drug delivery vectors was explored. Covalent conjugation of albumin to the surface of NPs via maleimide chemistry proved problematic. However, microwave assisted synthesis of NPs was not only time efficient, but enabled the exploration of size control by changing the following parameters: temperature, microwave power, reaction time, initiator concentration, and percentage of monomer used. About 1.5 g of fluorescently-labeled, carboxylic acid-functionalized NPs (100 nm diameter) were synthesized for a total cost of less than $1. Future work will address further functionalization of the NPs for the coupling of albumin (or other targeted proteins), and tests for in vivo biodistribution.

Thesis (M. Pharm. (Pharmaceutics)) -- University of Limpopo, 2019 / Various approaches have been developed to retain dosage forms in the gastrointestinal tract. One of the commonly used approaches is the use of microspheres. Due to their intrinsic low density and small size, they are distributed throughout the gastrointestinal tract which improves drug absorption thus improving bioavailability. Ranitidine hydrochloride, an antiulcer drug is poorly absorbed from the lower gastrointestinal tract and has a short half-life of 2.5-3 hours. The aim of this study was to formulate and evaluate gastroretentive microspheres of ranitidine hydrochloride in order to extend gastric retention in the upper gastrointestinal tract, which may result in enhanced absorption and thus improved bioavailability. Pre-formulation studies were conducted to develop and validate the analytical method to identify and quantify ranitidine hydrochloride; to select the suitable polymers for further formulation development and; to determine the compatibility of the chosen polymers with ranitidine hydrochloride. The analytical method was validated and found to be sensitive, linear, precise and accurate. Preliminary formulations lead to the selection of ethyl cellulose and PEG 4000 as polymers and solvent evaporation as the method of manufacture. Compatibility studies were determined by DSC/TGA, FTIR and short-term accelerated studies and no incompatibilities were observed. Two prototype formulations of the preliminary formulations F24 and F26 were manufactured comprised of varying drug: polymer concentration. The microspheres were evaluated for morphology, particle size, flow properties, percentage yield, buoyancy and in vitro drug release. Both formulations resulted in spherical microspheres with good flow properties, high yield and buoyancy studies revealed that the microspheres would float immediately upon contact with the dissolution media and floating would continue for more than 8 hours. In vitro drug release studies revealed that polymer concentration greatly affected drug release. Dissolution kinetic studies revealed that formulation F24 and v F26 were best described by the Korsmeyer-Peppas and Higuchi kinetic models respectively. Formulation F26 was considered the best formulation, which comprised of a drug: PEG 4000 ratio of 1:2 w/w, as it yielded better in better drug encapsulation, better buoyancy results and had complete drug release.

Over the last six decades, the field of drug delivery has advanced considerably, from sustained oral release technology to pH-responsive polymers. Innovation in the space has progressed alongside the development of new categories of drugs, as well as improvements in electronics and material science which have enabled new modalities of external stimulation. Nevertheless, the traditional challenges of drug delivery persist, including the need to reduce off-target toxicity, minimize invasiveness of administration, and bypass biological barriers; these challenges are particularly apparent for drug delivery applications in difficult-to-reach areas of the body, such as tumors or areas beyond the blood-brain barrier. Furthermore, as therapeutics become more targeted, the need for corresponding delivery methods becomes even more vital to ensure treatment effectiveness with minimal side effects. In this dissertation, we aim to demonstrate a new strategy for on-demand and localized drug delivery which is easy to fabricate and delivers a large payload relative to device size, is responsive to external stimulation for triggered release, and can be integrated into a system for real-time actuation during a physiological process. In Aim 1, we developed a microfluidic fabrication technique for making biphasic microcapsules loaded with model drug. This method relied on microfluidic droplet methods, with sufficient interfacial tension between two on-chip phases to cause droplet formation. Typically, these systems rely on an aqueous-oil interface for sufficient interfacial tension; to fabricate a biocompatible microcapsule, we formed biphasic microcapsules composed of an aqueous-based inner and outer phase, without an oil intermediate phase, with aqueous two-phase system properties. Additionally, we incorporated on-chip photopolymerization, designing the microfluidic chip and light source to minimize refracted ultraviolet exposure. The resulting drug-loaded microcapsules were stable, with minimal background leakage. This fabrication technique can produce a high-throughput supply of monodisperse microcapsules, which can be modified for a variety of therapeutic payloads and easily injected in targeted region in the body. In Aim 2, we adapted these drug-loaded microcapsules for ultrasound-triggered release. Focused ultrasound (FUS) is a minimally-invasive method of stimulating release from a device, which can penetrate deep within the body and is compatible with a variety of materials; when applied at sufficient intensity and duration, it can induce heating, cavitation, or both. We tuned the applied ultrasound parameters to minimize temperature increases in surrounding tissue phantoms, while inducing step-like release profiles from the microcapsules over the course of multiple cycles of pulsed FUS. Under these applied conditions, we detected acoustic signatures consistent with inertial cavitation and visually observed structural breakdown of the microcapsules corresponding to cavitation-related effects. This release strategy is highly targeted, inducing drug release from microcapsules within a narrow focal area with minimal risk to surrounding tissue. Finally, in Aim 3, we performed in vitro demonstrations of drug-loaded actuators, as initial demonstrations towards a system of integrated sensors, actuators, and adaptive learning algorithms for closed-loop control over physiological processes involved in wound healing. We experimented with both the aforementioned microcapsules and with a liposome-loaded scaffold as drug-loaded actuators, and tested both actuators with three ultrasound transducers which offered a range of portability, intensity ranges, and imaging capacities. Next, we developed in vitro testing setups incorporating the actuators with either a cell monolayer or a three-dimensional cell construct, mimicking a wound site, and validated ultrasound-triggered drug-release with minimal cell damage. To demonstrate cell uptake of the released therapeutic agents, we modified the microcapsules’ payload, performed the in vitro release experiments, and then observed correlating cell response over the following week of culturing. These demonstrations have provided guidance towards a more integrated system, which will validate the impact of the localized actuators in stimulating enhancing wound healing rates. More broadly, the eventual integrated system, incorporating both sensors and the adaptive algorithm, will be able to sense and respond to physiological changes within a wound in real-time. This work explores how wireless, deep-tissue devices coupled with external control modalities will facilitate interventions with high spatiotemporal accuracy; when combined with sensing and regulating algorithms, it will empower real-time monitoring and interventions in physiological processes. Aim 1 focused on the fabrication of such implantable microcapsule devices and Aim 2 demonstrated a method for triggering the devices using an external control modality. In Aim 3, we investigated a use case for these microcapsules to promote rapid wound healing, alongside flexible electronics, sensors, and additional actuators. To provide additional context on implantable microdevices and biocompatibility, we provide a framework for designing medical microrobotics in Appendix I and an application of a thermally-responsive hydrogel coating in Appendix II. Overall, the sum of this work illustrates the potential impact of soft microdevices for localized and on-demand applications, towards a future of spatiotemporally-targeted biological interventions.

RÉSUMÉ en françaisLes systèmes de délivrance de médicaments sont fortement investigués dans le but d’obtenir la meilleure efficacité thérapeutique et la plus faible toxicité possible. De nombreux systèmes existent à l’heure actuelle qu’ils soient mécaniques ou chimiques. Dans ce travail, nous nous sommes tout d’abord intéressés à une nouvelle approche basée sur la macropinocytose pour augmenter l’internalisation d’agents thérapeutiques aussi bien dans des cellules cibles de médicaments que dans des cellules souches mésenchymateuses d’origine musculaire (mdMSCs) actuellement de plus en plus étudiées comme nouveaux systèmes de délivrance. La macropinocytose est en effet une voie d’endocytose clathrine-indépendante permettant l’internalisation non sélective de fluide extracellulaire. Ce processus semble contribuer à l’agressivité des cancers en favorisant leur apport en nutriments, le recyclage de la membrane plasmatique et de ses récepteurs ainsi que l’internalisation d’exosomes. La macropinocytose est déclenchée notamment par l’activation des récepteurs epidermal growth factor receptor (EGFR) et platelet-derived growth factor receptor (PDGFR) qui sont des marqueurs connus d’agressivité des gliomes. Au cours de ce travail, nous nous sommes demandé si la macropinocytose ne pourrait pas être utilisée pour améliorer l’internalisation d’agents chimiothérapeutiques dans les cellules de glioblastomes. Nous avons tout d’abord montré que l’expression des acteurs clés de la macropinocytose au niveau de leur ARNm est fortement altérée en fonction du grade de la tumeur en utilisant des bases de données publiques d’échantillons de tumeurs gliales humaines. Une « signature » basée sur l’expression de 38 gènes liés à la macropinocytose permet la discrimination des échantillons de glioblastomes, c’est-à-dire des tumeurs les plus agressives. Nous avons ensuite comparé les effets induits par le MOMIPP et le Vacquinol-1, deux inducteurs connus de macropinocytose, à ceux de l’Honokiol qui induit également une vacuolisation sévère au sein de cellules de gliomes. Malgré de fortes similarités morphologiques, l’Honokiol se distingue par l’induction de vacuoles provenant à la fois d’endocytose mais aussi du réticulum endoplasmique. Chacun des trois composés induit néanmoins une nette augmentation de l’internalisation de dextrans de 10 kDa. Le marquage à l’acridine orange suggère pourtant des différences dans le devenir des vacuoles induites par ces trois molécules. Il a récemment été démontré que le MOMIPP agit en empêchant la maturation des macropinosomes ce qui concorde avec notre observation d’une fusion avec les lysosomes qui serait très limitée. Si les trois molécules permettent bien d’augmenter également la concentration de témozolomide (traitement de référence) au sein des cellules de gliomes, leur impact sur l’internalisation de la doxorubicine est plus faible. L’augmentation de l’internalisation des médicaments par l’induction de macropinocytose ne semble donc pas toujours efficace et pourrait dépendre de leurs propriétés physico-chimiques ainsi que de leur voie d’internalisation basale. Étant donné que l’induction soutenue de macropinocytose peut également déclencher la mort cellulaire tant de cellules de glioblastome chimio-sensibles que chimio-résistantes, nous proposons d’envisager la macropinocytose dans des approches combinées pour bénéficier d’une augmentation de l’internalisation de médicaments et d’effets additifs/ synergiques. Comme les cellules souches mésenchymateuses sont intensément étudiées pour leur utilisation en tant que transporteur de médicaments en plus de leur potentiel intrinsèque pour la médecine régénérative, les effets de ces inducteurs de vacuolisation ont également été étudiés sur des mdMSCs. Ces dernières sont caractérisées par un niveau basal de macropinocytose plus élevé que les modèles de gliomes utilisés et elles semblent mieux répondre au Vacquinol-1 qui permet d’ailleurs d’augmenter plus efficacement la concentration intracellulaire en doxorubicine que le MOMIPP, avec une internalisation environ 2 fois plus importante qu’en absence de stimulation. Ces cellules pouvant être utilisées comme véhicule pour le transport de molécules ayant une faible biodisponibilité afin d’améliorer leur usage en clinique, nous avons ensuite évalué l’impact du chargement de mdMSCs avec de la curcumine, un polyphénol naturel bien connu pour ses propriétés antioxydantes et anti-inflammatoires dont le potentiel clinique reste limité notamment par sa faible solubilité. Pour cette raison, l’utilisation du NDS27, un complexe de lysinate de curcumine et d’hydroxy-propyl-bêta-cyclodextrine qui présente une bien meilleure solubilité dans l’eau, a été préférée. Nous avons observé que l’internalisation de la curcumine, provenant du NDS27, dans les mdMSCs est concentration-dépendante et non temps-dépendante. De plus, elle ne peut pas être augmentée par l’utilisation d’inducteurs de macropinocytose. De plus amples investigations des effets de l’internalisation de la curcumine à partir du NDS27 en fonction de la concentration sur leur viabilité, prolifération, propriétés mésenchymateuses et leur fonction mitochondriale nous ont permis de montrer qu’un chargement de 2 h avec 7 µM de NDS27 était acceptable en vue de préserver la possibilité de bénéficier des mdMSCs en tant qu’agent de thérapie cellulaire régénérative en sus de leur aspect de délivrance. L’investigation du potentiel thérapeutique de telles mdMSCs chargées en curcumine pour traiter l’arthrose chez le cheval est toujours en cours. / SUMMARYDrug delivery systems are highly investigated with the aim to promote the best therapeutic efficacy and the lowest possible toxicity. Nowadays, several systems either mechanical or chemical exist. In this work, we were first interested in studying a new approach based on macropinocytosis to increase the internalisation of therapeutic agents in both target cells of drugs and skeletal muscle-derived mesenchymal stem cells (mdMSCs) currently more and more studied as new deliverance systems. Macropinocytosis is indeed a clathrin-independent endocytosis pathway allowing the nonselective internalisation of extracellular fluid. This process seems to contribute to cancer aggressiveness by favouriting nutrient supply, recycling of plasma membrane and its receptors, and exosome internalisation. Macropinocytosis may notably be triggered by epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR), two well-known markers of glioma aggressiveness. During this work, we wondered whether macropinocytosis could be used to improve the internalisation of chemotherapeutic agents into glioblastoma cells. We first showed that the mRNA expression levels of key actors of macropinocytosis are strongly altered according to the grade of the tumour using public datasets of human glioma tumour samples. A “signature” based on the expression of 38 macropinocytosis-related gene allows the discrimination of the glioblastoma samples, i.e. the most aggressive tumours. We next compared the effects induced by MOMIPP and Vacquinol-1, two well-known macropinocytosis inducers to those of Honokiol which also induces a severe vacuolisation within glioma cells. Despite high phase-contrast morphological similarities, Honokiol appeared different from the others by the induction of vacuoles from both endocytosis and endoplasmic reticulum origins. Each of the three compounds nevertheless induces a marked increase in 10 kDa dextran internalisation. Acridine orange staining however suggested differences in the fate of vacuoles induced by those three molecules. MOMIPP has recently been shown to prevent the macropinosomes maturation which is consistent with our observation ofthe lack of acridine orange staining of the vacuoles that may be attributed to a limited fusion with lysosomes. Although each of the three compounds allows nevertheless a marked increase of temozolomide (i.e. first-line treatment) concentration into glioma cells, their impact on the internalisation of doxorubicin is poor. The increased internalisation of drugs by inducing macropinocytosis does not seem to be always efficient and could depend on their physicochemical properties and their basal way of internalisation. Considering that sustained macropinocytosis induction may also trigger cell death of both chemo-sensitive and chemo-resistant glioblastoma cells, we proposefor future perspectives to consider macropinocytosis in combination approaches to benefit from an increased drug uptake and additive/synergistic effects. As mesenchymal stromal / stem cells are increasingly studied for their use as drug-carrier in addition to their intrinsic potential for regenerative medicine, the effects of these vacuolisation inducers were also studied on mdMSCs. These lasts are characterised by a higher basal level of macropinocytosis than the glioma models used in this work. They seem to better respond to Vacquinol-1 which allows to increase more efficiently the intracellular concentration of doxorubicin than MOMIPP, with an uptake approximatively 2-fold higher than without stimulation. Given that these cells can be used as vehicle to transport molecules with a poor bioavailability in order to improve their clinical usage, we evaluated the impact of the loading of mdMSCs with curcumin, a natural polyphenol well-known for its antioxidant and anti-inflammatory properties whose clinical potential remains limited notably by reason of its poor solubility. Therefore, the use of NDS27, a complex of curcumin lysinate and hydroxypropyl-beta-cyclodextrin displaying an increased solubility in aqueous solution, was preferred. We observed that the uptake of curcumin from NDS27 into mdMSCs is concentration-dependent and not time-dependent. Moreover, it cannot be improve using macropinocytosis inducers. Further investigations of the effects of curcumin internalisation from NDS27 depending on the concentration used on their viability, proliferation, mesenchymal properties and their mitochondrial function allowed us to show that a loading of 2 h with NDS27 at 7 µM seems acceptable to preserve the possibility to benefit from mdMSCs as a cellular therapy agent in addition to the delivery aspect. The investigation of the double potential therapeutic benefits of such curcumin-loaded mdMSCs to treat osteoarthritis in horses is still ongoing. / Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie) / info:eu-repo/semantics/nonPublished

This thesis presents the design and optimization of a dendrimer-based cationic nanoparticle system tailored for versatile applications, ranging from anti-inflammatory scavenging to targeted pain relief through endosomal delivery. By harnessing the unique attributes of this platform, various strategies were devised to overcome hurdles in drug delivery, offering promising avenues for nanomedicine in anti-inflammatory and nociceptive treatments. In our scavenging screening project, we rigorously screened various materials to find the best universal anti-inflammatory carrier. We started by exploring the potential of dendrimer-based materials as scavengers of inflammatory signals and studied how they could be used to develop therapeutic carriers. With intrinsic therapeutic properties and the ability to create tunable nanocarriers, dendrimer-based delivery systems are powerful multimodal delivery systems. The dendrimer base of our delivery system, cationic PAMAM Generation 3 dendrimer (PAMAM-G3), was selected due to its efficient scavenging ability and low biotoxicity. Conjugation with cholesterol facilitated the formation of polymeric micelles, exhibiting a cationic and hydrophilic exterior coupled with a hydrophobic interior, resulting in a high drug-loading capacity. Among the developed scavengers, PAMAM-Cholesterol (PAMAM-Chol) nanoparticles demonstrated a potent reduction in toll-like receptor activation with minimal toxicity and extended endosomal retention. We then exploited the endosomal retention of PAMAM-Chol nanoparticles to target the activated PAR2 receptor within endosomes of relevant cancer cells, aiming to alleviate oral cancer-induced nociception. Extensive characterization confirmed the platform's stability, physical attributes, and ability to encapsulate PAR2 inhibitor, AZ3451. The platform exhibited high drug loading capacity and sustained release profiles across various pHs. Cellular uptake studies demonstrated efficient endosomal targeting, with subsequent modulation of PAR2 signaling pathways. Preclinical studies in oral cancer pain models revealed a significant and prolonged reduction in nociception for over 24 hours, surpassing the efficacy of free drugs. Further diversification of the PAMAM-Chol platform explored its potential as a "Push" chemotherapy carrier and a "Pull" cfDNA scavenger against chemotherapy-induced neurological and neuropathic side effects. Evaluation in wild-type mice demonstrated the platform's effectiveness in mitigating chemobrain and chemotherapy-induced peripheral neuropathy, highlighting its translational potential for multimodal cancer therapy. We found that NPs loaded with chemotherapy significantly reduced the painful effects of chemotherapy-induced peripheral neuropathy and decreased recovery times. Collectively, this body of work underscores the potential of PAMAM-Chol as a versatile tool in drug delivery and endosome-localized pain therapeutics. It contributes to the evolving landscape of precision medicine through tailored therapeutic approaches for minimizing side effects and enhancing patient well-being. The innate therapeutic properties coupled with efficient and sustained drug delivery mechanisms position the PAMAM-Chol platform as a foundational element for the development and delivery of next-generation therapeutics.