Protein loaded microparticles with modified surfaces for the targeting of dendritic cells

Titelblatt und Inhaltsverzeichnis I Vorwort 1 1. Einleitung und Zielstellung 2 2. Material und Methoden 49 3. Ergebnisse und Diskussion 82 4. Zusammenfassung 165 5. Summary 167 6. Literatur 169 7. Abkürzungen 195 8. Publikationen 198 10. Danksagungen 201Im Rahmen dieser Arbeit wurden proteinbeladene, biodegradable Mikropartikel aus Poly(Lactid-co-Glycolid) [PLGA] in einem Mikromischer-basierten Doppelemulsions-Lösemittelverdampfung-Verfahren unter aseptischen Bedingungen hergestellt sowie die Partikel umfassend charakterisiert. Die Primäremulgierung des Modellproteins FITC-BSA in der Lösung des Matrixpolymers wurde unter Berücksichtigung der Ultrastruktur der Mikropartikel, der Proteinverteilung sowie des Freisetzungsverhaltens optimiert. Der Restlösungsmittelgehalt unterschritt den Grenzwert des Arzneibuches für Dichlormethan um drei Zehnerpotenzen. Die Verwendung des Modellproteins FITC- BSA ermöglichte bei Untersuchungen zur Proteinverteilung im Mikropartikel und zur Partikellokalisation im Zellexperiment zahlreiche Schlussfolgerungen, hingegen schlug die fluorimetrische Quantifizierung des FITC-BSA in Freisetzungsproben aufgrund von Dequenching fehl. Zur kationischen Oberflächenmodifizierung von PLGA-Mikropartikeln wurde erstmals DEAE-Dextran eingesetzt. Bei den DEAE-Dextran-ummantelten Teilchen konnte im Gegensatz zu Chitosan-modifizierten Partikeln auch bei pH 7 eine positive Ladung festgestellt werden. Die in der Literatur beschriebene Methode zur Oberflächenmodifizierung mit Cetyltrimethylammoniumbromid (CTAB) war nicht nachvollziehbar, da CTAB von der Partikeloberfläche desorbierte. In Zellexperimenten wurden die DEAE-Dextran-modifizierten Mikropartikel effektiver als unmodifizierte Partikel in phagozytierende Zellen aufgenommen. Die PLGA-Mikropartikel lösten bei Abwesenheit von Endotoxinen und anderen starken Antigenen keine Reifung von humanen dendritischen Zellen (DCs) oder Veränderungen in der Expression ihrer Oberflächenmarker aus und besaßen bei den verwendeten Konzentrationen keine Zytotoxizität. Damit stellen PLGA- Mikropartikel ein sicheres Trägersystem dar, um Antigene in DCs einzuschleusen. Für eine verbesserte immunologische Tumortherapie sollte die Antigenpulsung autologer DCs mit mikroverkapselten Antigenen erfolgen. Zukünftig könnten an DEAE-Dextran-modifizierte Mikropartikel Liganden von Toll-like Rezeptoren gebunden werden, die die nachgeschaltete Immunantwort verstärken und zu einer verbesserten Wirksamkeit DC-basierter Zelltherapien führen könnten.The present contribution describes the preparation of protein loaded, biodegradable microparticles from poly(lactide-co-glycolide) [PLGA] using a double emulsion solvent evaporation technique based on a micromixer under aseptic conditions as well as the comprehensive characterisation of the particles. The primary emulsification of the model protein FITC-BSA in the solution of the PLGA matrix polymer has been optimized under consideration of the ultrastructure of the microparticles, the protein distribution, as well as the release behaviour. The residual solvent in the microparticles was one thousand times smaller than required in the pharmacopoeia. The usage of FITC- BSA as a model protein allowed a number of conclusions with respect to the protein distribution in the microparticles and the localisation of the particles in the cell experiment. However, the fluorimetric quantification of FITC-BSA in samples of release studies failed because of dequenching effects. For a cationic surface modification of PLGA microparticles DEAE dextran has been used for the first time. Contrary to the surface modification with Chitosan the DEAE dextran coated particles had a positive surface charge even at pH 7. A method of cationic surface modification using cetyltrimethylammonium bromide (CTAB), as described in the literature, could not be reproduced, because CTAB showed desorption from the particle surface. In cell experiments the uptake of DEAE dextran modified particles by phagocytising cells was more efficient than that of non-modified particles. In the absence of endotoxins or other strong antigens PLGA microparticles did not initiate the maturation of human dendritic cells (DCs) or changes in the expression of their surface markers, and did not lead to cytotoxicity under the used concentration of the particles. Thus PLGA microparticles are safe carrier systems to deliver antigens to DCs. For an improved immunological cancer therapy antigen pulsing of autologous DCs should be carried out with microencapsulated antigens. In future studies ligands of Toll-like receptors could be adsorbed to DEAE dextran modified microparticles. This might amplify the immune response and lead to an improved efficiency of DC based cell therapies

Predictive Shapes of Ellipsoid PPDL-PTHF Copolymer Particles Prepared by the Phantom Stretching Technique

Ellipsoidal polymer particles can be prepared from spheres by unidirectional stretching at elevated temperatures, while the particles’ aspect ratios (AR) that result from this phantom stretching methodology are often not precisely predictable. Here, an elastic deformation model was exemplarily evaluated for ~50 µm spherical microparticles from PPDL-PTHF block copolymers. The prolate ellipsoidal particles, obtained by stretching in polyvinyl alcohol phantoms, differed in dimensions at identical relative phantoms elongations up to 150%, depending on the relative polymer composition and their systematically altered mechanical properties. Importantly, the resulting particle shapes within the studied range of AR up to ~4 matched the predictions of the elastic deformation model, which includes information of the elastic moduli of phantom and particle materials. These data suggest that the model may be applicable to predict the conditions needed to precisely prepare ellipsoids of desired AR and may be applicable to various deformable particle materials

Opportunities and Challenges of Switchable Materials for Pharmaceutical Use

Switchable polymeric materials, which can respond to triggering signals through changes in their properties, have become a major research focus for parenteral controlled delivery systems. They may enable externally induced drug release or delivery that is adaptive to in vivo stimuli. Despite the promise of new functionalities using switchable materials, several of these concepts may need to face challenges associated with clinical use. Accordingly, this review provides an overview of various types of switchable polymers responsive to different types of stimuli and addresses opportunities and challenges that may arise from their application in biomedicine

Design of Reservoirs Enabling Stress-Induced Sequential Release Systems

Mechanical stress is recognized as a principle for opening enclosed compartments through compression, stretching, or shear, eventually resulting in the onset of a diffusion-controlled release. Here, we hypothesized that the geometrical design of cavities (cut-outs) introduced as containers in elastic polymer substrates and sealed with a brittle coating layer would enable a pre-defined release of different compounds by stress concentration phenomena. Design criteria such as cut-out shapes, orientations, and depths were initially assessed for suitably different stress concentrations in computational models. In substrates fabricated from polydimethylsiloxane by photolithographic techniques, the local strains at horizontal rectangular, circular, and vertical rhombus-shaped cut-outs systematically increased under horizontal stretching as proposed. When filled with model compounds and coated with poly(n-butyl cyanoacrylate), a pre-defined induced breakage of the coating and compound release was confirmed upon continuous uniaxial stretching. This proof of concept demonstrates how device design and functions interlink and may motivate further exploration in technology and medicine for deformation-induced on-demand dosage applications

Characteristics and challenges of Poly(ethylene-co-vinyl acetate) solution electrospinning

Poly(ethylene-co-vinyl acetate) (PEVA) is a versatile elastic, durable and biocompatible copolymer, which can be processed by melt extrusion or solvent casting, while electrospinning has been reported as challenging. Here, a spinnability window should be identified using a total of 10 different PEVA materials with increasing vinyl acetate content (~12 – 40 wt.%) and molecular weights (~60-130 kDa). Based on solubility predictions by calculating Hansen solubility parameters, candidate solvents were experimentally evaluated. Spinning experiments with systematic alteration of solution composition and processing parameters revealed the causes of material deposition at the spraying nozzle and multi-jet spinning characteristics. By introducing a spinnability score that accounts for product characteristics and reproducibility, the spinnability of PEVA could be rationalized. Overall, it was demonstrated that PEVA solutions with an apparent viscosity of 920-3500 mPa·s can be spun to bead-free fibers of ~10 µm. This size may allow suspension electrospinning to composite fibers in the future

Design of decorin-based peptides that bind to collagen I and their potential as adhesion moieties in biomaterials

Mimicking the binding epitopes of protein-protein interactions by using small peptides is important for generating modular biomimetic systems. A strategy is described for the design of such bioactive peptides without accessible structural data for the targeted interaction, and the effect of incorporating such adhesion peptides in complex biomaterial systems is demonstrated. The highly repetitive structure of decorin was analyzed to identify peptides that are representative of the inner and outer surface, and it was shown that only peptides based on the inner surface of decorin bind to collagen. The peptide with the highest binding affinity for collagen I, LHERHLNNN, served to slow down the diffusion of a conjugated dye in a collagen gel, while its dimer could physically crosslink collagen, thereby enhancing the elastic modulus of the gel by one order of magnitude. These results show the potential of the identified peptides for the design of biomaterials for applications in regenerative medicine.status: publishe

Multivariate Analysis of Cellular Uptake Characteristics for a (Co)polymer Particle Library

Controlling cellular responses to nanoparticles so far is predominantly empirical, typically requiring multiple rounds of optimization of particulate carriers. In this study, a systematic model-assisted approach should lead to the identification of key parameters that account for particle properties and their cellular recognition. A copolymer particle library was synthesized by a combinatorial approach in soap free emulsion copolymerization of styrene and methyl methacrylate, leading to a broad compositional as well as constitutional spectrum. The proposed structure–property relationships could be elucidated by multivariate analysis of the obtained experimental data, including physicochemical characteristics such as molar composition, molecular weight, particle diameter, and particle charge as well as the cellular uptake pattern of nanoparticles. It was found that the main contributors for particle size were the polymers’ molecular weight and the zeta potential, while particle uptake is mainly directed by the particles’ composition. This knowledge and the reported model-assisted procedure to identify relevant parameters affecting particle engulfment of particulate carriers by nonphagocytic and phagocytic cells can be of high relevance for the rational design of pharmaceutical nanocarriers and assessment of biodistribution and nanotoxicity, respectively

Design of Controlled Release PLGA Microspheres for Hydrophobic Fenretinide

Fenretinide, a chemotherapeutic agent for cancer, is water-insoluble and has a very low oral bioavailability. Hence, the objective was to deliver it as an injectable depot and improve the drug solubility and release behavior from poly­(lactide-<i>co</i>-glycolide) (PLGA) microspheres by incorporating nonionic surfactants with fenretinide. Enhancement of drug solubilization was observed with Brij 35 or 98, Tween 20, and Pluronic F127, but not Pluronic F68. Co-incorporation of Brij 98 with fenretinide significantly changed the microsphere morphology and improved the fenretinide release profile. The most optimal microsphere formulation, with 20% Brij 98 as excipient, showed an initial in vitro burst around 20% and a sustained release over 28 days in a solubilizing release medium at 37 °C. The effect of addition of MgCO₃, drug loading, and polymer blending on the release of fenretinide from PLGA microspheres was also investigated and observed to enhance the drug release. Two sustained release formulations, one incorporating 20% Brij 98 and the other incorporating 3% MgCO₃ in the oil phase, were selected for dosing in Sprague–Dawley rats and compared to a single injection of an equivalent dose of fenretinide drug suspension. These two formulations were chosen due to their high encapsulation efficiency, high cumulative release, and desirable in vitro release profile. The drug suspension resulted in a higher initial release in rats compared to the polymeric formulations, however, sustained release was also observed beyond 2 weeks, which may be attributed to the physiological disposition of the drug in vivo. The two PLGA based test formulations provided the desired low initial burst of fenretinide followed by 4 weeks of in vivo sustained release

Multivariate Analysis of Cellular Uptake Characteristics for a (Co)polymer Particle Library

Controlling cellular responses to nanoparticles so far is predominantly empirical, typically requiring multiple rounds of optimization of particulate carriers. In this study, a systematic model-assisted approach should lead to the identification of key parameters that account for particle properties and their cellular recognition. A copolymer particle library was synthesized by a combinatorial approach in soap free emulsion copolymerization of styrene and methyl methacrylate, leading to a broad compositional as well as constitutional spectrum. The proposed structure–property relationships could be elucidated by multivariate analysis of the obtained experimental data, including physicochemical characteristics such as molar composition, molecular weight, particle diameter, and particle charge as well as the cellular uptake pattern of nanoparticles. It was found that the main contributors for particle size were the polymers’ molecular weight and the zeta potential, while particle uptake is mainly directed by the particles’ composition. This knowledge and the reported model-assisted procedure to identify relevant parameters affecting particle engulfment of particulate carriers by nonphagocytic and phagocytic cells can be of high relevance for the rational design of pharmaceutical nanocarriers and assessment of biodistribution and nanotoxicity, respectively