Development of functionalized alginate-based hydrogels to reduce fibrosis in the transplantation of microencapsulated cells

Nowadays, the development of cell therapy relies mainly on the advances made toward cell microencapsulation which allows to evade the need for immunosuppression during cell transplantation. Among the materials considered for cell encapsulation, hydrogels distinguish themselves by their exceptional properties. Due to its gelling properties in contact with divalent cations, the biopolymer sodium alginate (Na-alg), has been widely studied for the microencapsulation and transplantation of cells. However, several defects notably in permselectivity and durability in vivo is limiting the translation of alginate-based hydrogels in clinical applications. In addition, the transplantation of cells without immunosuppressive treatment have to face adverse host responses such as inflammation and fibrosis. At the moment, several approaches have been considered for the development of an ideal material for cell encapsulation; however many challenges still remain in this domain. Hence, this research project focused on the development of hydrogels in order to improve their properties toward cell encapsulation.

The first part of this research focused on the development of new types of one-component hybrid alginate-based hydrogels. These hydrogels combine the gelling properties of Na-alg with covalent crosslinking within the same polymeric structure. In order to do so, a new synthetic approach was developed allowing the functionalization on the hydroxyl position of alginate with heterobifunctional poly(ethylene glycol) (PEG) linkers through carbamate bond. This allowed to maintain the carboxyl moieties available for the formation of electrostatic interactions. Two types of one-component hybrid hydrogel were developed with this method adding either thiols or lipoyl moieties on alginate leading to an improvement of mechanical properties while preserving a good biocompatibility.

Yet, transplantation of encapsulated cells in a foreign host body have to face immune response, in particular inflammation and pericapsular fibrosis overgrowth (PFO) which ultimately leads to necrosis of the encapsulated cells and loss of graft functionality. Therefore, the second part of this research project focused on tuning the composition of the polymeric components of the hydrogels with anti-inflammatory agents which would reduce PFO in vivo. Different anti-inflammatory agents were considered for this purpose. The best candidate (ketoprofen) was then PEGylated via either ester or amide bond and grafted on the backbone of alginate to ensure a controlled release at the site of transplantation. By analyzing the fibrotic tissue around the microspheres, it was observed that the incorporation of ketopreofen on the structure of the hydrogel significantly reduced PFO 30 days after transplantation. In summary, Na-alg was functionalized with a new synthetic methodology allowing the incorporation of either functionalities reinforcing the mechanical resistance of the hydrogel or anti-inflammatory compounds improving the biocompatibility of the graft.

Multifunctional hybrid alginate-based hydrogels for cell immobilization

Luca Szabó

Cell therapy could be a solution for many diseases that currently pose a huge health burden on patients. Especially, cell therapies based on cell microencapsulation are of great interest. Immunoprotection of cells would allow to avoid the need for immunosuppression and the need for human donors, as the use of xenogeneic cell sources could be feasible. An optimal biomaterial for the microencapsulation of cells must be tailored for the type of cells and the intended application. Hydrogels are great candidates for cell immobilization, due to their high biocompatibility and extracellular matrix-like properties. Amongst them, alginate has outstanding potential with its favorable gelling properties, high biocompatibility, and relatively easy availability. However, in many aspects, it cannot fulfill the requirements of an optimal biomaterial for cell immobilization and transplantation. It lacks good mechanical properties, long-term durability and stability, and has poor shape recovery ability. In addition, severe immune reactions of the host system to the transplanted microcapsules can happen, which eventually result in cell death and early graft failure. Hence, the main focus of the present project was functionalization of alginate to develop new hydrogel materials with improved properties for cell microencapsulation. First, the thesis presents a robust, straightforward strategy and its optimization for the functionalization of alginate on the hydroxyl groups with poly(ethylene)glycol (PEG) via stable carbamate bond. The end-functionality of the heterobifunctional PEG provides a reactive group for covalent crosslinking. Hence, dual ionic-covalent microsphere preparation is achieved by extrusion of the aqueous polymer solution into a gelation bath containing ionic cross-linker and followed by concomitant covalent cross-linking. The possibility of using a variety of covalent cross-linkers was explored. Depending on the end-functionality of the heterobifunctional PEG, the chemical structure of the cross-linker is adjustable. This thesis presents thiol-thiol, thiol-acrylate, thiol-maleimide and thiol-acrylamide interactions, and compares the mechanical properties of the resulted microspheres to those of Ca-alginate. Both the mechanical resistance and the shape recovery performance of microspheres stabilized by an additional chemical cross-linking was improved. In addition, the stability of these microspheres towards non gellifying solutions was significantly enhanced. Still, biomaterials are exposed to the risk of immune responses when transplanted, such as inflammation and fibrosis. Introduction of the anti-inflammatory drug ketoprofen to alginate backbone through a PEG linker and consecutive microsphere formation provides controlled ketoprofen release. Based on this strategy, development of new types of anti-fibrotic derivatives of pirfenidone were realized. Conjugation of the most potential derivative to PEG followed by grafting on alginate resulted in a polymer capable of forming microspheres. Covalent conjugation of the drug was further compared to co-encapsulation of the drug with Ca-alginate. Eventually, the thesis presents the development of multicomponent hybrid hydrogel microspheres, combining the introduction of covalent cross-linking and anti-inflammatory compounds within the hydrogel network. The feasibility of using the newly developed biomaterials are confirmed by in vitro and in vivo assays.

EPFL2020

Solution behavior of peptide and peptide-hybrid materials

Harald Nuhn

Peptide based and peptide hybrid materials represent two interesting and challenging classes of biomaterials. Both classes consist of and contain, respectively, one or more polypeptide blocks, endowing the resulting polymers with enhanced self-assembly properties, biocompatibility or novel characteristics. Peptide based materials, solely comprised of amino acids, benefit from their advanced self-assembly properties. In the case of peptide hybrid materials, being conjugates of peptides and synthetic polymers, either the polypeptide or the synthetic polymer block can drive the self-assembly process. Furthermore, the latter class benefits from combining the advantages of peptides and synthetic polymers, thus overcoming limitations related to the use of the individual components. The application of these materials is still challenging because it is not possible to predict their final self-assembly behavior based on their design. In order to facilitate a target-oriented design of novel materials for application such as drug delivery, tissue engineering, or self-healing materials, a detailed understanding of the complex interactions within and between these molecules is essential. The work presented in this thesis addresses these fundamental questions. Over the last decades, a plethora of peptide and peptide hybrid materials has been reported. Chapter 1 gives an overview of selected articles describing different synthesis routes for peptide as well as peptide hybrid materials, showing examples of molecules and their self-assembly into superstructures, and highlighting recent applications. Chapter 2 describes the synthesis and characterization of different peptide hybrid diblock oligomers composed of two classes of peptide sequences covalently attached to a poly(ethylene glycol) (PEG) block. The first class of peptide sequences contains the intrinsic propensity to form coiled coils, whereas the second class is composed of "switch" peptides and is able to adopt both amphiphilic α-helical and β-strand conformations. Upon dissolution, the first class yielded superstructures comprised of dimeric and tetrameric coiled coil aggregates, whereas the second class formed fibrillar assemblies. In both cases, the self-assembly properties were driven by the peptide block, and the final superstructure consisted of a peptidic core wrapped by the synthetic polymer block. Chapter 3 presents a detailed study on the aqueous solution self-assembly of two poly(styrene)n-b-poly(L-lysine)m peptide hybrid oligomers. The aim was to clarify whether the observed rod-like micelles, obtained by direct dissolution of the diblock oligomers in aqueous solution, reflect intrinsic self-assembly properties of the hybrid diblock oligomers or whether they present structures that are energetically trapped due to the glassy nature of the polystyrene block. To address this question, the influence of a variety of sample preparation techniques, including the use of organic solvents, dialysis from an organic solvent and several thermal treatments, was investigated. All treatments resulted in solution aggregates that differed in size and shape from samples prepared by direct dissolution in aqueous solution. Thus, it seems likely that direct dissolution of polystyrene-b-poly(L-lysine) diblock oligomers in aqueous solution results in kinetically trapped, rod-like aggregates, which can be transformed into non-spherical micelles by the appropriate treatment. Chapter 4 contains a comprehensive study on peptide-based materials, investigating four different series of short elastin-like peptides (ELPs) based on the GVGVP motif. The objective was to test whether and how defined changes in the primary structure of short ELPs affect the secondary structure and the lower critical solution temperature (LCST) behavior. Therefore, the number of repeats was altered, and within one peptide of constant length, all valines were successively substituted by the more hydrophobic amino acids isoleucine, leucine and phenylalanine, respectively. In parallel, a theoretical approach based on the partition coefficient log P was used to predict the shift of the LCST as function of chain length and composition. For short ELPs, the LCST is strongly affected by changes in the primary structure. This has also been predicted by calculating log P. An increase in the hydrophobicity resulted in a shift of the LCST towards lower temperatures. Furthermore, it was shown for the first time that the sensitivity of short ELPs towards external factors, such as salt concentration, is determined by the intrinsic propensity of the incorporated amino acids to form either α-helices or β-strands. In summary, the obtained results on peptide based and peptide-hybrid materials provides deeper insights into their self-assembly characteristics and highlights the potential of these materials to be used for technical and medical applications.

EPFL2008

Synthesis, structural characterization and applications of hyperbranched polymers based on L-lysine

Markus Scholl

L-lysine is an essential a-amino acid and a necessary building block for all proteins in the body. As an essential amino acid, L-lysine is not synthesized by humans or animals. Industrially produced L-lysine has therefore a big market as food additive, e.g. in pig food. In the body, L-lysine plays a major role in calcium absorption, building muscle proteins, recovering from surgery or sports injuries, and the body's production of hormones, enzymes, and antibodies.1 From a chemical point of view, L-lysine is an AB2 building block that contains two amino groups and one carboxylic acid group, which makes it an interesting monomer for the synthesis of dendrimers and hyperbranched polymers. L-lysine indeed has been extensively used to synthesize dendrimers2-4 or conjugates of dendrimers and linear polymers.5,6 These dendrimers and linear-dendritic hybrid architectures have attracted interest for various medical applications, including gene delivery,3,6-8 as drug carriers,9 for the development of multiple antigen peptide systems,10,11 and as magnetic resonance imaging agents.12 The drawback of these dendrimers and linear-dendritic hybrid architectures is their time consuming synthesis and their expensive large scale production. This thesis evaluates the feasibility of L-lysine as a monomer to synthesize hyperbranched polymers and investigates the potential of these materials as encapsulation agents and for various biomedical applications. As the hyperbranched polylysines are prepared in a single step and can be produced conveniently at a large scale, they may offer an interesting alternative to their widely used dendritic analogues. Including this introduction, this thesis consists of 8 chapters, which are briefly described below. Chapter 2 gives a general overview of dendritic and hyperbranched polymers build up of amide bonds. Their synthesis and applications in medicine and catalysis as well as their self-assembling and encapsulation properties will be discussed. Chapter 3 reports on the synthesis of hyperbranched polylysines via the thermal polymerization of L-lysine hydrochloride.13 Different catalysts were investigated to improve the reaction kinetics. The structure of the polymers was determined by 1H-NMR spectroscopy, and the degree of branching and the average number of branches were calculated. Chapter 4 will discuss the feasibility of different approaches to control polymer architecture during the thermal hyperbranched polymerization of L-lysine hydrochloride.14 The reactivity of the more reactive ε-NH2 group was modulated by introducing temporary protective groups. The distribution of structural units was analyzed by 1H-NMR spectroscopy and the degree of branching and the average number of branches were calculated. Chapter 5 will describe the dilute solution and solid state structure of hyperbranched polylysines and polyelectrolyte complexes generated from hyperbranched polylysine and various anionic, sodium alkyl sulfate surfactants.15 Structural models for the obtained liquid crystalline phases will be proposed. Chapter 6 will show some preliminary results of the end group modification of hyperbranched polylysine with different hydrophilic and hydrophobic agents. The feasibility of these hydrophobically modified hyperbranched polylysines to encapsulate hydrophilic dyes and transfer them from aqueous to organic media has been investigated. Chapter 7 provides a first insight into the biological properties of hyperbranched polylysine. In this chapter the results from preliminary cytotoxicity and cellular uptake experiments will be discussed. Chapter 8 will give a short summary of the work done in this thesis and an outlook. References Nelson, D. L.; Cox, M. M. "Lehninger, Principles of Biochemistry" 3rd Ed. Worth Publishing: New York, 2000. ISBN 1-57259-153-6. Denkewalter, R. G.; Kolc, J. F.; Lukasavage, W. J. In U.S. Pat. 4289872; Allied Corporation, 1981; Chem. Abstr. 1981, 102, 79324. Ohsaki, M.; Okuda, T.; Wada, A.; Hirayama, T.; Niidome, T.; Aoyagi, H. Bioconjugate Chem. 2002, 13, 510-517. Driffield, M.; Goodall, D. M.; Smith, D. K. Org. Biomol. Chem. 2003, 1, 2612-2620. Lübbert, A.; Nguyen, T. Q.; Sun, F.; Sheiko, S. S.; Klok, H.-A. Macromolecules 2005, 38, 2064-2071. Choi, J. S.; Joo, D. K.; Kim, C. H.; Kim, K.; Park, J. S. J. Am. Chem. Soc. 2000, 122, 474-480. Vlasov, G. P.; Korol'kov, V. I.; Pankova, G. A.; Tarasenko, I. I.; Baranov, A. N.; Glazkov, P. B.; Kiselev, A. V.; Ostapenko, O. V.; Lesina, E. A.; Baranov, V. S. Russ. J. Bioorg. Chem. 2004, 30, 12-20. Choi, J. S.; Lee, E. J.; Choi, Y. H.; Jeong, Y. J.; Park, J. S. Bioconjugate Chem. 1999, 10, 62-65. Sakthivel, T.; Toth, I.; Florence, A. T. Pharm. Res. 1998, 15, 776-782. Tam, J. P. Proc. Natl. Acad. Sci. U. S. A. 1988, 85, 5409-5413. Pessi, A.; Bianchi, E.; Bonelli, F.; Chiappinelli, L. J. Chem. Soc., Chem. Commun. 1990, 8-9. Nicolle, G. M.; Tóth, E.; Schmitt-Willich, H.; Radüchel, B.; Merbach, A. E. Chem. Eur. J. 2002, 8, 1040-1048. Scholl, M.; Nguyen T. Q.; Bruchmann, B.; Klok, H.-A. J. Polym. Sci. Part A Polym. Chem. 2007, 45, 5494-5508. Scholl, M.; Nguyen T. Q.; Bruchmann, B.; Klok, H.-A. Macromolecules 2007, 40, 5726-5734. Canilho, N.; Scholl, M.; Mezzenga, R.; Klok, H.-A. Macromolecules 2007, 40, 8374-8383.

EPFL2007