Physical PEGylation to Prevent Insulin Fibrillation

Insulin is one of the most marketed therapeutic proteins worldwide. However, its formulation suffers from fibrillation, which affects the long-term storage limiting the development of novel devices for sustained delivery including portable infusion devices. We have investigated the effect of physical PEGylation on structural and colloidal stability of insulin by using 2 PEGylating agents terminating with polycyclic hydrophobic moieties, cholane and cholesterol: mPEG5kDa-cholane and mPEG5kDa-cholesterol, respectively. Microcalorimetric analyses showed that mPEG5kDa-cholane and mPEG5kDa-cholesterol efficiently bind insulin with binding constants (Ka) of 3.98 104 and 1.14 105 M-1, respectively. At room temperature, the 2 PEGylating agents yielded comparable structural stabilization of \u3b1-helix conformation and decreased dimerization of insulin. However, melting studies showed that mPEG5kDa-cholesterol has superior stabilizing effect of the protein conformation than mPEG5kDa-cholane. Furthermore, the fibrillation study showed that at a 1:1 and 1:5 insulin/polymer molar ratios, mPEG5kDa-cholesterol delays insulin fibrillation 40% and 26% more efficiently, respectively, as compared to mPEG5kDa-cholane which was confirmed by transmission electron microscopy imaging. Insulin was released from the mPEG5kDa-cholane and mPEG5kDa-cholesterol assemblies with comparable kinetic profiles. The physical PEGylation has a beneficial effect on the stabilization and shielding of the insulin structure into the monomeric form, which is not prone to fibrillation and aggregation

Nanosystèmes multifonctionnels pour une délivrance contrôlée des principes actifs

Les récentes avancées dans les domaines des nanotechnologies, sciences des matériaux et ingénierie des polymères ont permis de surmonter les obstacles associés aux formulations pharmaceutiques conventionnelles. Au sein de ce manuscrit, des systèmes de délivrance pharmaceutiques hybrides rassemblant les avantages technologiques et structurels des composants lipidiques et polymères ont été développés. La conception de la formulation a été pensée selon les propriétés spécifiques de la voie d’administration envisagée et les caractéristiques intrinsèques des classes de substances actives choisies. Dans la première partie de cette thèse, les systèmes hybrides ont été conçus pour l’administration orale de petites molécules hydrophobes afin de répondre à un besoin médical : augmenter les effets anti-inflammatoires locaux pour le traitement des maladies inflammatoires chroniques de l’intestin (MICI). Les nanocomposites macroscopiques formés par l’encapsulation de nanoémulsions (NEs) dans une matrice d’hydrogel à base de peptides ont d’abord été développés et caractérisés afin d’investiguer les propriétés mécanistiques qui pourraient permettre de les administrer oralement. Les études de biodistribution in vivo ont démontré la capacité des nanocomposites hybrides à agir comme “dépôt” dans l’estomac via la précipitation des peptides. Le comportement, dépendant du pH, des nanocomposites hybrides permet la reconstitution de l’hydrogel au niveau intestinal où ils peuvent adhérer à la surface de l'épithélium et ainsi libérer les NEs conférant alors une plus longue durée d’action. Les études d’efficacité sur des souris atteintes de colite ont démontré le potentiel de cette formulation en tant que traitement innovant pour les MICI. Ensuite, des nanocomposites hybrides sous forme de microsphères ont été préparés en incorporant les NEs au sein d’une matrice polysaccharidique. L’alginate a été utilisé pour ses propriétés bioadhésives connues pour augmenter la durée de rétention intestinale. La formulation des nanoparticules a été optimisée par un procédé microfluidique et la caractérisation in vitro a été réalisé sur des lignées cellulaires spécifiques. Les résultats obtenus soulignent l’efficacité des NEs pour le passage du principe actif à travers les muqueuses et l'épithélium intestinal au sein d’un modèle de co-culture ainsi que son activité sur des monocytes. Les études ex-vivo sur des intestins de rats excisés ont démontré les propriétés bioadhésives des nanocomposites, leur conférant une durée de rétention prolongée. La seconde partie de ce travail de thèse repose sur l’administration d’ARN messagers via la conception de systèmes hybrides polymères-lipides créant ainsi une plateforme versatile pour le traitement potentiel d’une large variété de pathologies. Des lipoplexes conventionnels ont été initialement formés à partir d’un mélange innovant de lipides et caractérisés en vue d’obtenir les propriétés physico chimiques recherchées. L’innovation de ce travail consiste en l’addition d’un élément important, une enveloppe électrostatique de polymères liée à la surface des lipoplexes, afin d’améliorer leur stabilité, apporter une charge de surface différence et permettre une fonctionnalisation pour une délivrance ciblée. Les deux systèmes ont été comparés sur leur capacité à transfecter à la fois in vitro et in vivo. Les biodistributions des systèmes ont également été comparées. Les résultats obtenus soulignent que les lipoplexes et leurs homologues enveloppés ont des comportements similaires, démontrant ainsi que la composition des lipides est le paramètre majeur affectant ces propriétés, et non leur charge de surface ni leur fonctionnalisation. Finalement, ce travail de thèse offre une étude complète des stratégies de formulation de systèmes hybrides polymères-lipides ayant pour but de franchir les barrières biologiques pour une administration précise de principes actifs.Remarkable developments in the field of nanotechnology, material science, and polymer engineering have enabled the growth of multidisciplinary approaches, aiming at overcoming the hurdles associated with conventional drug formulations. In the present manuscript this integrative modus operandi has been applied to the field of drug delivery to design hybrid multifunctional drug delivery systems merging the technological and structural advantages of two individual lipid and polymeric components. The formulation design was shaped in function of the specific properties related to the administration route and the intrinsic characteristics of the chosen active substances. In the first part of the thesis, hybrid systems were designed for the oral administration of small, hydrophobic molecules, to answer the medical need of increasing locally the anti-inflammatory effects of actives for the treatment of inflammatory bowel diseases (IBDs). Firstly, nanocomposites created by the embodiment of nanoemulsions (NEs) in a peptide-based hydrogel matrix were developed and characterized to dig on the mechanical properties that could allow their application upon oral administration. In vivo biodistribution studies showed the ability of the hybrid nanocomposite to act as “depot” in the stomach, where the peptide collapse. The pH-dependent behavior of the formulated hybrid nanocomposite, allow the hydrogel reconstitution at the intestinal level, where it can adhere to the epithelium surface slowly releasing the NEs, thus allowing a longer local drug effect. Efficacy studies on colitic mice models proved the potential of this formulation as innovative treatment for IBDs. Next, hybrid nanocomposites shaped as microspheres were prepared by incorporating NEs into a polysaccharide matrix. Alginate was used for its known bioadhesive properties to increase the system residence time by interacting with the intestinal mucosa. An optimization of nanoparticles formulation strategy by using microfluidic and an in-depth in vitro characterization of the system on relevant cell-lines, together with the design of a stable approach to create the nanocomposite, were the key facets of this work. The results obtained outlined the efficacy of the NEs to allow drug internalization through a mucus layer and intestinal epithelium on a co-culture model, as well as its activity on monocytes. Ex vivo studies on excised intestines of rats confirmed the bioadhesive properties of the nanocomposites, allowing a prolonged residence time compared to the NEs alone. The second part of the thesis aimed at designing lipid-polymer hybrid systems for the administration of mRNA, to create a versatile platform to address different diseases. Lipoplexes were prepared and characterized to ensure a product bearing define physicochemical qualities. The innovation relies in the addition of a polymeric shell electrostatically bound to their surface, to improve stability and to allow a further functionalization in sight of a targeted delivery. Both systems were studied for their ability to transfect in vitro and in vivo, and for their fate once injected in animals. As results, similar behaviors were obtained for the lipoplexes and their coated counterpart, evidencing that the lipid composition is the feature affecting the particle behavior, rather than surface charge and functionalization. Overall, this thesis provided an extensive investigation on lipid-polymer hybrid formulation strategies aimed at overcoming the biological hurdles for precise drug delivery

Alginate-pectin microparticles loaded with nanoemulsions as nanocomposites for wound healing

This work combines natural polymers with nanoemulsions (NEs) to formulate nanocomposites as an innovative wound dressing. Spray-drying has been used to produce alginate-pectin in situ gelling powders as carriers for NEs loaded with curcumin (CCM), a model antimicrobial drug. The influence of NEs encapsulation in polymer-based microparticles was studied in terms of particle size distribution, morphology, and stability after spray-drying. NEs loading did not affect the size of microparticles which was around 3.5 µm, while the shape and surface morphology analyzed using scanning electron microscope (SEM) changed from irregular to spherical. Nanocomposites as dried powders were able to form a gel in less than 5 min when in contact with simulated wound fluid (SWF), while the value of moisture transmission of the in situ formed hydrogels allowed to promote good wound transpiration. Moreover, rheologic analyses showed that in situ formed gels loaded with NEs appeared more elastic than blank formulations. The in situ formed gel allowed the prolonged release of CCM-loaded NEs in the wound bed, reaching 100% in 24 h. Finally, powders cytocompatibility was confirmed by incubation with keratinocyte cells (HaCaT), proving that such nanocomposites can be considered a potential candidate for wound dressings

Nanoemulsions Embedded in Alginate Beads as Bioadhesive Nanocomposites for Intestinal Delivery of the Anti-Inflammatory Drug Tofacitinib

International audienceOral administration of nanoparticles (NPs) is a promising strategy to overcome solubility and stability issues of many active compounds. However, this route faces major obstacles related to the hostile gastrointestinal (GI) environment, which impairs the efficacy of orally administered nanomedicines. Here, we propose nanocomposites as a promising approach to increase the retention time of NPs in the intestinal tract by using bio-and mucoadhesive matrixes able to protect the cargo until it reaches the targeted area. A microfluidic-based approach has been applied for the production of tailored nanoemulsions (NEs) of about 110 nm, used for the encapsulation of small hydrophobic drugs such as the antiinflammatory JAK-inhibitor tofacitinib. These NEs proved to be efficiently internalized into a mucus-secreting human intestinal monolayer of Caco-2/ HT29-MTX cells and to deliver tofacitinib to subepithelial human THP-1 macrophage-like cells, reducing their inflammatory response. NEs were then successfully encapsulated into alginate hydrogel microbeads of around 300 μm, which were characterized by rheological experiments and dried to create a long-term stable system for pharmaceutical applications. Finally, ex vivo experiments on excised segments of rats′ intestine proved the bioadhesive ability of NEs embedded in alginate hydrogels compared to free NEs, showing the advantage that this hybrid system can offer for the treatment of intestinal pathologies

Monomethyl Auristatin E Grafted-Liposomes to Target Prostate Tumor Cell Lines

Novel nanomedicines have been engineered to deliver molecules with therapeutic potentials, overcoming drawbacks such as poor solubility, toxicity or short half-life. Lipid-based carriers such as liposomes represent one of the most advanced classes of drug delivery systems. A Monomethyl Auristatin E (MMAE) warhead was grafted on a lipid derivative and integrated in fusogenic liposomes, following the model of antibody drug conjugates. By modulating the liposome composition, we designed a set of particles characterized by different membrane fluidities as a key parameter to obtain selective uptake from fibroblast or prostate tumor cells. Only the fluid liposomes made of palmitoyl-oleoyl-phosphatidylcholine and dioleoyl-phosphatidylethanolamine, integrating the MMAE-lipid derivative, showed an effect on prostate tumor PC-3 and LNCaP cell viability. On the other hand, they exhibited negligible effects on the fibroblast NIH-3T3 cells, which only interacted with rigid liposomes. Therefore, fluid liposomes grafted with MMAE represent an interesting example of drug carriers, as they can be easily engineered to promote liposome fusion with the target membrane and ensure drug selectivity

Supersaturable self-microemulsifying delivery systems: an approach to enhance oral bioavailability of benzimidazole anticancer drugs

This study explored the design of supersaturable self-microemulsifying drug delivery systems (S-SMEDDS) to address poor solubility and oral bioavailability of a novel benzimidazole derivative anticancer drug (BI). Firstly, self-microemulsifying drug delivery systems SMEDDS made of Miglyol (R) 812, Kolliphor (R) RH40, Transcutol (R) HP, and ethanol were prepared and loaded with the BI drug. Upon dispersion, the systems formed neutrally charged droplets of around 20 nm. However, drug precipitation was observed following incubation with simulated gastric fluid (pH 1.2). Aiming at reducing this precipitation and enhancing drug payload, supersaturable systems were then prepared by adding 1% hydroxypropyl cellulose as precipitation inhibitor. Supersaturable systems maintained a higher amount of drug in a supersaturated state in gastric medium compared with conventional formulations and were stable in simulated intestinal medium ( pH 6.8). In vitro cell studies using Caco-2 cell line showed that these formulations reduced in a transient manner the transepithelial electrical resistance of the monolayers without toxicity. Accordingly, confocal images revealed that the systems accumulated at tight junctions after a 2 h exposure. In vivo pharmacokinetic studies carried out following oral administration of BI-loaded S-SMEDDS, SMEDDS, and free drug to healthy mice showed that supersaturable systems promoted drug absorption compared with the other formulations. Overall, these data highlight the potential of using the supersaturable approach as an alternative to conventional SMEDDS for improving oral systemic absorption of lipophilic drugs.National Research Agency (ANR), HyDNano projectFrench National Research Agency (ANR) [ANR-18-CE18-0025-01]; ANRFrench National Research Agency (ANR) [42306YB]; Fundacao para a Ciencia e Tecnologia (FCT)Portuguese Foundation for Science and TechnologyEuropean Commission [42306YB]; FCTPortuguese Foundation for Science and TechnologyEuropean Commission [UID/Multi/04326/2019

Nanocomposite sponges for enhancing intestinal residence time following oral administration

In this work, nanocomposites that combine mucopenetrating and mucoadhesive properties in a single system are proposed as innovative strategy to increase drug residence time in the intestine following oral administration. To this aim, novel mucoadhesive chitosan (CH) sponges loaded with mucopenetrating nanoemulsions (NE) were developed via freeze-casting technique. The NE mucopenetration ability was determined studying the surface affinity and thermodynamic binding of the nanosystem with mucins. The ability of nanoparticles to penetrate across a preformed mucins layer was validated by 3D-time laps Confocal Laser Scanning Microscopy imaging. Microscopy observations (Scanning Electron Microscopy and Optical Microscopy) showed that NE participated in the structure of the sponge affecting its stability and in vitro release kinetics. When incubated with HCT 116 and Caco-2 cell lines, the NE proved to be cytocompatible over a wide concentration range. Finally, the in vivo biodistribution of the nanocomposite was evaluated after oral gavage in healthy mice. The intestinal retention of NE was highly enhanced when loaded in the sponge compared to the NE suspension. Overall, our results demonstrated that the developed nanocomposite sponge is a promising system for sustained drug intestinal delivery.Portuguese Foundati on for Science and Technology and European Commission: 42306YB; Portuguese Foundation for Science and Technology: UID/Multi/04326/2020info:eu-repo/semantics/publishedVersio

Hybrid core-shell particles for mRNA systemic delivery

International audiencemRNA based infectious disease vaccines have opened the venue for development of novel nucleic acids-based therapeutics. For all mRNA therapeutics dedicated delivery systems are required, where different functionalities and targeting abilities need to be optimized for the respective applications. One option for advanced formulations with tailored properties are lipid-polymer hybrid nanoparticles with complex nanostructure, which allow to combine features of several already well described nucleic acid delivery systems. Here, we explored hyaluronic acid (HA) as coating of liposome-mRNA complexes (LRCs) to investigate effects of the coating on surface charge, physicochemical characteristics and biological activity. HA was electrostatically attached to positively charged complexes, forming hybrid LRCs (HLRCs). At different N/P ratios, physico-chemical characterization of the two sets of particles showed similarity in size (around 200 nm) and mRNA binding abilities, while the presence of the HA shell conferred a negative surface charge to otherwise positive complexes. High transfection efficiency of LRCs and HLRCs in vitro has been obtained in THP-1 and human monocytes derived from PBMC, an interesting target cell population for cancer and immune related pathologies. In mice, quantitative biodistribution of radiolabeled LRC and HLRC particles, coupled with bioluminescence studies to detect the protein translation sites, hinted towards both particles' accumulation in the hepatic reticuloendothelial system (RES). mRNA translated proteins though was found mainly in the spleen, a major source for immune cells, with preference for expression in macrophages. The results showed that surface modifications of liposome-mRNA complexes can be used to fine-tune nanoparticle physico-chemical characteristics. This provides a tool for assembly of stable and optimized nanoparticles, which are prerequisite for future therapeutic interventions using mRNA-based nanomedicines