DEVELOPMENT OF NEW PEPTIDE-BASED NANOMATERIALS AS POTENTIAL TOOLS FOR CRYOPRESERVATION OF TISSUES AND ORGANS
Transplant practise has improved life duration and quality of many patients. However, the worldwide need is not met yet due to the limited number of donor organs and the inefficient preservation methods, that hinder their long-term preservation. The main obstacle to achieve a successful cryopreservation is represented by the freezing and thawing processes needed to achieve cryogenic temperatures that can cause permanent and irreversible cellular damages. The solution to this issue may be found in nature, as several organisms living in extreme conditions produce specific proteins, termed antifreeze (glyco)proteins (AF(G)Ps), that are able to depress the freezing point of body fluids, control the ice crystal nucleation and inhibit their growth. In particular, the latter AFP property, the ice recrystallization inhibition (IRI), is highly desirable for new synthetic antifreeze materials as ice recrystallization during thawing is associated to cellular damage during cryopreservation. Therefore, one of the main challenges in biomedical research for organ transplantation is finding non-toxic and biocompatible antifreeze compounds that enable subzero storage and cryopreservation without causing tissue damage. As the synthesis of molecules to be used for cryopreservation represents an urgent need, in the last decades researchers have been extensively focused on it. Since the production of AF(G)Ps is expensive and presents several difficulties in its scale-up, and considering their potential cytotoxicity/immunogenicity and their tendency to denature, efforts have been focused on synthesising macromolecular cryoprotectant-polymers or minimising the AFP primary sequence through the synthesis of peptides or lastly, increasing the antifreeze activity through the exploitation of a multimeric approach, taking advantage from self-assembly processes or the simultaneous presentation of several ice binding motifs, that allows to reduce the concentration required for the effect. Although extensive efforts have been devoted to the synthesis of novel peptide or peptide-based cryoprotectants, limited results were achieved probably due to the poor understanding of the AF(G)Ps mechanism of action at molecular level and the lack of comprehensive structure-activity relationships. Considering the relevance and the global impact of that research field, finding new effective antifreeze compounds that can be translated into clinic represents an urgent need. Being inspired by the multimeric approach, the aim of this PhD project was to develop new peptide-capped materials possessing an antifreeze activity by conjugating the N-terminus of peptides derived from type I AFP HPLC6 to different moieties that are known for their self-assembling ability. The goal of decorating nanoparticles (NPs) with peptides relies on combining the ice binding capability of peptides with the size of a NP, mimicking the globular protein bulkiness. In order to reach the goal, different approaches were exploited, as i) the on-resin peptide functionalization with lauric acid, ii) the in-solution peptide functionalization with the FDA approved polymer poly(lactide-co-glycolide) (PLGA) or iii) to gold nanoparticles (AuNPs), by inserting in peptide sequence a thiol-bearing binding functionality, and lastly, iv) the on-resin peptide functionalization with Aggregation-Induced Emission (AIE) luminophores, organic molecules characterized by uniquely fluorescent properties in the aggregate or solid state. First of all, the synthesis of the precursor peptides has been optimized and their secondary structure has been characterized by circular dichroism (CD) and Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy. Considering the presence of different conformations and the lack of a preferred helical one, the secondary structure of the minimised sequence of type I AFP HPLC6 was stabilised using a click chemistry reaction, the copper(I)-catalysed azide-alkyne cycloaddition (CuAAC), by the insertion of the i, i+4 side-chain-to-side-chain triazole as conformational constraint. The conformation analyses carried out in solution revealed that the helical structure of both the obtained stapled peptides was successfully stabilised. Afterwards, the different approaches to obtain different types of nanoparticles exposing multiple copies of the synthesised antifreeze-derived peptides were described. First, soft NPs were synthesised by N-terminus functionalization of the precursor and stapled peptides with lauric acid. The secondary structure of the synthesised lipopeptides was assessed both in solution and in the solid state by CD and ATR-FTIR, respectively, whereas their tendency to self-assemble was investigated using a solvent displacement technique and characterised by Dynamic Light Scattering (DLS). Another strategy was the in-solution functionalization of the purified peptides with PLGA. As for the lipopeptides, the four obtained conjugates were characterised based on their secondary structure and their self-assembly abilities. The antifreeze peptides were also used to functionalize gold nanoparticles (AuNPs) by inserting in their sequence an appropriate binding functionality, able to stabilise the nanocolloidal systems with a set of helical, Ala-rich model peptides. Once synthesised and purified, the secondary structure of the thiol-derived antifreeze peptides was evaluated by CD and ATR-FTIR. Their conjugation to citrate-capped AuNPs via ligand exchange procedure was investigated by UV-Vis, DLS and zeta potential measurement. Besides, the peptides were conjugated to AIE luminophores. To this aim, two hybrid compounds were synthesised, combining the AIE unique properties and the surfactant features of fatty acids, to be used as N-terminus capping agents in SPPS. These molecules were found to self-assemble into different supramolecular emissive structures depending on the chemical composition and water content. Furthermore, to assess the feasibility of the synthetic strategy, a model peptide was N-terminus functionalised, leading to the obtainment of luminescent fibrillary materials that were not cytotoxic and were able to create supramolecular gels in aqueous environment. Unluckily, the use of the two hybrid compounds for the synthesis of antifreeze peptide materials resulted in the formation of highly insoluble fluorescent compounds, characterized by a low purity degree. Finally, the IRI activity of the peptides and peptide-capped based conjugates was evaluated. The ability of the different compounds to inhibit the ice crystal growth was assessed by optical microscopy, using the ‘sucrose sandwich’ assay. While the investigated peptides were not IRI active in the evaluated conditions, and the synthesised lipopeptides and PLGA peptide-based conjugates were found insoluble, the thiol-bearing peptides and the peptide-capped AuNPs showed an ice crystal growth rate lower than the sucrose solution or the naked gold nanoparticles. In particular, the gold nanoparticles conjugated with the stapled thiol-derived peptide outperformed the other systems in decelerating, although partially, the ice recrystallization.
cryopreservation, AFP, peptide-based material, CuAAC, alpha-helix, Settore CHIM/06 - Chimica Organica
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