Molecularly designed self-assembling matrices for applications in regenerative medicine

Abstract

The exquisite field of molecular self-assembly (SA) offers numerous opportunities to fabricate biomaterials with increased level of precision and complexity. Peptide molecules have been widely used as building blocks in biomaterial SA as they provide the possibility to form nanofibers that resemble the filamentous structure of the natural extracellular matrix (ECM). Peptide amphiphiles (PAs) were used throughout the work described in this thesis to develop new biomaterials (membranes and capsules) by SA. To develop bioactive membranes recapitulating some features of the skin ECM, positively charged PAs were combined with hyaluronan (HA). Membranes were obtained by SA and presented a nanofibrillar morphology resembling the ECM architecture. The cell-adhesive sequence (RGDS) was then integrated into the PA structure and was shown to increase the adhesion of human dermal fibroblasts (hDFbs). To further expand the versatility of these membranes, and recreate some of the aspects of the ECM remodeling, a cleavable sequence (GPQGIWGQ, octapeptide) sensitive to matrix metalloproteinase-1 (MMP-1) was incorporated into the PA structure. Enzymatic degradation studies with exogenous enzymes (hyaluronidase and MMP-1) revealed that membranes containing the MMP-1 substrate exhibited enhanced enzymatic degradation, being completely degraded after 7 days. Cell culture studies using hDFbs showed that the presence of MMP-1 cleavable sequence stimulated the secretion of MMP-1 by hDFbs and interfered with matrix deposition, particularly the deposition of collagen. A notable observation from this work was the spontaneous formation of well-defined micro-groove-like patterns on the membrane surface during the SA process. The micro-pattern formation was then investigated by varying the conditions (experimental set up, incubation time, concentration of the building blocks, HA molecular weight and nature of polyelectrolyte) during SA. These studies revealed that the presence of an air-liquid interface at the PA side and the nature of the underneath polyelectrolyte played a key role on the formation of the micro-grooved patterns. hDFbs cultured on the patterned membranes were shown to align in a parallel direction with the micro-grooves, suggesting the possibility of using the self-patterned membranes for guided-tissue regeneration. The role of the octapeptide domain on the membrane topography was also investigated using a peptide library containing 15 PAs with variations on the central octapeptide segment, in terms of length and amino acid composition. Membranes with very distinct surface morphologies, from welldefined micro-grooves to micro-sized aggregates, were obtained by simply manipulating the PA structure. The results from site-direct mutations suggest that the size of the central domain, including the presence of isoleucine in the middle, and existence of β-sheet structures are determinant for the formation of micro-groove patterns. In a distinct work, electrostatic self-assembly of PAs of opposite charge was combined with microfluidics to develop soft peptide-based capsules for cell encapsulation. The resulting capsules exhibited regular shape and size, and a gelled core made of a dense nanofibrillar network. Their properties (nanofibrillar density, permeability) were able to be modulated by changing the PAs concentration. These properties were also shown to influence the morphology of hDFbs encapsulated within the microcapsules. On less dense capsules, hDFbs exhibited their typical elongated morphology and organized cytoskeleton. In addition, the developed microcapsules allowed the co-culture of two cell types (hDFbs and keratinocytes). The research work described in this thesis highlights the use of molecular engineering strategies to design peptides, integrating SA ability and specific biochemical functionalities, for the bottom-up fabrication of novel biomaterials. The developed biomaterials exhibited intrinsic bioactivity and hierarchical structure, enabling their application as artificial matrices for probing cell behavior in defined microenvironments that recapitulate critical aspects of native tissues and thus develop novel therapeutic strategies for tissue regeneration.

Avanços na área da química supramolecular oferecem inúmeras oportunidades para o desenvolvimento de novos biomateriais com elevado nível de precisão e complexidade. Péptidos têm sido amplamente utilizados para o desenvolvimento de biomateriais por processos de auto-organização, uma vez que permitem a formação de estruturas fibrilares semelhantes às existentes na matriz extracelular. Na presente tese foram utilizados péptidos anfifílicos (PAs) para desenvolver novos biomateriais (membranas e microcápsulas) por processos de auto-organização. Para desenvolver membranas bioactivas que mimetizem alguns aspectos da matriz extracelular da pele, PAs com carga positiva foram combinados com um polímero de carga oposta (ácido hialurónico). As membranas foram obtidas por processos de auto-organização, apresentando uma estrutura fibrilar. A sequência peptídica de adesão celular (RGDS) foi integrada na estrutura do PA, para promover a adesão de fibroblastos nas membranas. Para recriar outras funcionalidades da matriz extracelular, nomeadamente o processo de remodelação, a sequência peptídica (GPQGIWGQ, octapéptido) sensível à degradação pela enzima metaloproteinase-1 (MMP-1) foi incorporada na estrutura do PA. Ensaios de degradação enzimática, na presença de hialuronidase e MMP-1, mostraram que as membranas contendo o substrato da MMP-1 apresentaram sinais evidentes de degradação, tendo sido completamente degradadas após 7 dias. Estudos celulares com fibroblastos mostraram que a presença do substrato da MMP-1 nas membranas estimulou a secreção de MMP-1 pelas células e afectou a deposição de matriz extracelular, particularmente a deposição de colagénio. Uma observação notável resultante deste trabalho, foi a formação espontânea de topografias organizadas à microescala na forma de canais. Análises de microscopia revelaram um alinhamento das células (fibroblastos) ao longo dos microcanais quando cultivadas nas membranas. Através de um dispositivo de microfluidicos, uma solução de péptido foi emulsionada num óleo a fim de gerar gotas esféricas. Estas gotas foram posteriormente gelificados quando em contacto com uma outra solução de péptido de carga oposta, formando capsulas de forma e tamanho regular, e interior gelificado. De forma a controlar as propriedades das capsulas (densidade fibrilar, permeabilidade) variou-se a concentração dos péptidos usados. Estudos celulares feitos com fibroblastos no interior das cápsulas revelaram que as células mantiveram-se viáveis. No entanto, a sua morfologia mostrou depender da densidade nanofibrillar das cápsulas. Além disso, este sistema permitiu também a co-cultura de dois tipos de células, fibroblastos e queratinócitos. Os estudos descritos nesta tese realçam a vantagem de manipular a estrutura de biomateriais à escala molecular. Os biomateriais desenvolvidos (membranas e microcápsulas) apresentam estruturas hierarquicamente organizadas e bioactividade intrínseca, permitindo a sua aplicação no desenvolvimento de novas estratégias terapêuticas para a regeneração de tecidos.