Design of bioactive peptide matrices: from molecular structure to biomedical applications
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Data
2024-02-16
Autores
Mello, Lucas Rodrigues de [UNIFESP]
Orientadores
Silva, Emerson Rodrigo da [UNIFESP]
Tipo
Tese de doutorado
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Introduction: Peptide materials are at the forefront of biomedical research, particularly in the development of nanotherapeutics. In addition to this application-oriented appeal, peptides are also fascinating systems from the perspective of fundamental research. In thesis, it is presented an experimental study on the self-assembly and cell interaction behaviour of nanostructured matrices based on three classes of bioactive peptides: cell-penetrating peptides (CPPs), extracellular matrix-derived related (ECM), and anti-inflammatory peptides (AIPs). Objectives: We aimed to comprehensively describe the self-assembly of representative molecules of these bioactive groups, aiming to generate knowledge to control supramolecular organisation and establish structure-activity relationships regarding their interaction with cells. In the case of CPPs, we aimed to prepare peptiplexes (i.e., peptide/DNA complexes) for DNA delivery into cells. Our focus was on the hydrophobic peptide C105Y, the cationic CPPs Tat-HIV and SV40-T, and the amphipathic CPP Transportan 10. The group of ECM peptides was analysed through the study of a designed sequence, DGRL4DGW, combining fibronectin-related motifs with a self-assembling leucine backbone, to produce hydrogelled matrices capable of sustaining cell growth. Additionally, model sequences enriched in arginine and phenylalanines were analysed to explore the impact of amphipathic layout on both supramolecular arrangement and cell response. Finally, rationalised AIPs based on the thymosin-b4 related Ac-SDKP peptide were designed to control self-assembly and investigate supramolecular organisation. In this case, we aimed to unveil the self-assembly of chimaeras prepared with either a nonpolar hexa-alanine append to generate the Ac-SDKPA6 peptide or a palmitoylated appendix to produce the lipopeptide Ac-SDKPK-palmitoyl. Methods: An advanced set of structural techniques was employed, encompassing nano-infrared spectroscopy coupled with atomic force microscopy (AFM-IR), synchrotron small-angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM), and various spectroscopies. A combination of routine molecular biology tools and sophisticated approaches was used to perform cell assays, including viability tests, fluorescence confocal microscopy imaging, and flux cell cytometry quantification. Results: We successfully produced peptiplexes between all investigated CPPs and 200 bp DNA fragments; however, the structural landscape of the different classes exhibited strong contrast. The hydrophobic C105Y CPP showed self-aggregation capacity with a propensity to form nanoscopic fibrils endowed with amyloidogenic features. This feature was preserved when this peptide was complexed with DNA, and although the resulting peptiplexes could enter cells, they suffered endosomal entrapment. The cationic Tat-HIV and SV40-T did not show self-aggregation at all but formed irregular particles when complexed with DNA. The delivery of DNA from these peptiplexes was found to be limited. In contrast, Transportan 10 was not only able to form nanoflakes on its own but exhibited intracellular delivery rates comparable to the lipofectamine standard. In the case of designed sequences related to ECM peptides, it was found that DGRL4DGW forms highly stable hydrogel scaffolds capable of inducing morphogenesis of fibroblasts into tumor-like spheroids. For this purpose, the bolaamphiphilic layout of the primary sequence emerged as a key factor to induce the formation of fibril/tape-like nanostructures that evolved to produce an interconnected self-sustained matrix. Finally, the control of the amphipathic design in AIP peptides, appending nonpolar amino acid segments or hydrocarbon palmitoyl tails, led to self-assembly of the Ac-SDKP motif into rod-like assemblies and cylinder micelles, respectively. Also, we found that these species can form hydrogels. Conclusions: It was possible to produce and apply matrices composed of bioactive peptides from different classes in living cells throughout this work. A crucial discovery from the study was that the amphipathic layout of these peptides appears to be the key factor driving self-assembly. This feature represents a viable tool for overcoming the challenge of controlling aggregation into regular assemblies. The cell response seemed to be modulated by the supramolecular structure, with nanostructures displaying higher regular morphologies and sizes exhibiting more effectiveness compared to their larger and irregular counterparts.
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Citação
MELLO, Lucas Rodrigues de. Design of bioactive peptide matrices: from molecular structure to biomedical applications. 2024. 210 f. Tese (Doutorado em Biologia Molecular) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP). São Paulo, 2024.