Anticancer co-assembling ultra-short ionic complementary peptide hydrogels for localised therapy of malignant glioblastoma Abdulwahhab Khedr , Mohamed Soliman, Mohamed A. Elsawy De Montfort University, UK INTRODUCTION Assembling peptides represent a versatile chemical toolbox for the development of soft shear thinning nanomaterials exploited for a variety of biomedical applications. Herein, we developed two de novo ultra- short ionic-complementary peptide sequences (5 amino acids long, each) capable of co-assembly into β-sheet nanofibers, immediately forming hydrogels upon mixing. These peptides were designed based on the tetrapeptide Phg4. 1 These are the cationic peptide KPhg4 and the anionic counterpart E(Phg4) Rev , in which the charged residues are alternating with phenylglycine (Phg) and distributed in a pattern conferring a charge co-complementarity between both sequences. Both peptides were characterized for molecular assembly and hydrogelation at the physiological pH 7.4, both individually and in combination where co-assembly into nanofibres is induced through counter charge interaction. Also, these hydrogels were used for loading and sustaining the release of the anticancer drug; vincristine sulfate and tested for cytotoxicity against human malignant glioblastoma U87 MG cells. EXPERIMENTAL METHODS Hydrogel formation ‘mix & gel’:Peptide solutions were mixed at different molar ratios, pH and total peptide concentrations. ATR-FTIR spectroscopy: Characterisation of peptides’ secondary structures. Thioflavin T assay: Quantification of β-sheet secondary structures and kinetics of counter charge peptides’ co-assembly. Oscillatory rheology: Characterisation of hydrogels viscoelastic properties. Scanning electron microscopy (SEM): Characterisation of hydrogel network structure. Transmission electron microscopy (TEM): Characterisation of nanostructures morphology. Cell culture and cell viability assay (MTT): Cytotoxicity assay of hydrogels and drug- loaded hydrogels against U87 cells. RESULTS & DISCUSSION Individual peptides neither self-assembled into β-sheet structures nor formed hydrogels at the physiological pH over a wide range of peptide concentrations (FTIR and inverted vial test). Mixing counter charge peptide solutions at pH 7.4 spontaneously formed self-supported hydrogels at equimolar ratios of total peptide concentrations 10-50 mg/mL. A wide range of peptides molar ratios (1:9-9:1) were tested for binary mixes, where the highest abundance of β-sheets was observed for mixtures prepared at and around the equimolar ratio (FTIR, Thioflavin T) with very fast co-assembly kinetics reaching equilibrium within 1 hour after mixing. Oscillatory rheology results revealed that hydrogel stiffness can be controlled by fine-tuning molar ratios of peptide components. SEM showed the formation of nanofibrous networks for all formulated hydrogels, with fibres diameter estimated using TEM. These results suggest that the stability of molecular co-assembly and fibre formation is dependent on pH, molar ratio and total peptide concentration, which in turn will have a direct effect on hydrogel physico-mechanical properties. Hydrogels with a net positive charge showed significant cytotoxicity against U87 cells. In addition, vincristine-loaded hydrogels showed sustained release profiles over 96 hours. CONCLUSIONS In conclusion, the new ‘Mix &Gel’ strategy showed that the mesoscopic and bulk mechanical properties of the formed gels can be tuned by molecular design, pH, peptide concentration and molar ratios control, which can be manipulated to develop biomaterials that can satisfy the needs of anticancer applications. References 1. Wychowaniec, J et al., Biomacromolecules, 21(7):2670-2680, 2020.
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