Co-Assembling Ultra-Short Ionic Complementary Peptide Emulgels for Controlled Delivery of Combination Anticancer Therapy for Glioblastoma

When and Where

Apr 27, 2023
11:15am - 11:20am

SB06-virtual

Presenter

Abdulwahhab Khedr

Mohamed Soliman

Mohamed Elsawy

Co-Author(s)

Abdulwahhab Khedr^1,2,Mohamed Soliman^1,3,Mohamed Elsawy¹

De Montfort University¹,Zagazig University²,Cairo University³

Abstract

Abdulwahhab Khedr^1,2,Mohamed Soliman^1,3,Mohamed Elsawy¹

De Montfort University¹,Zagazig University²,Cairo University³

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 co-complementary peptide sequences (5 amino acids long, each) capable of co-assembly into β-sheet nanofibers, immediately forming hydrogels upon mixing the countercharge peptide solutions. 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 biomaterials were used for emulsifying pharmaceutical oils, forming O/W emulsion hydrogels (emulgels). These dual-phase systems were used for loading both hydrophilic and hydrophobic anticancer drugs for sustained localised drug delivery against human malignant glioblastoma U87 MG cells. EXPERIMENTAL METHODS 1. Emulgel formation: Peptide solutions were mixed at different molar ratios, pH and total peptide concentrations and at different O:W ratios. 2. ATR-FTIR spectroscopy: Characterisation of peptides’ secondary structures. 3. Thioflavin T assay: Quantification of β-sheet secondary structures and for co-assembly kinetics. 4. Oscillatory rheology: Characterisation of gels viscoelastic properties. 5. Scanning electron microscopy (SEM): Characterisation of the emulgel's network and vesicular structures. 6. Transmission electron microscopy (TEM): Characterisation of nanostructures morphology. 7. In vitro drug release. 8. Cell culture and cell viability assay: Cytotoxicity assay of emulgels and drug-loaded emulgels 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. Mixing counter charge peptide solutions at pH 7.4 spontaneously formed self-supported hydrogels over a wide range of peptides molar ratios (1:9-9:1) of total peptide concentrations 10-50 mg/mL. The highest abundance of β-sheets was observed for mixtures prepared at and around the equimolar ratio with fast rate of co-assembly (equilibrium within 1 hour after mixing). Also, these peptide mixtures showed a unique surface activity, forming O/W emulsions when mixed with oily phase at 5-50 mg/mL peptide concentration and different O:W ratios (2:8-8:2). Oscillatory rheology showed that gel stiffness is controllable by fine-tuning molar ratios of peptide components. SEM showed the formation of microspheres (15-60 µm diameter) within the gel nanofibrous network for all formulations, with fibres diameter estimated using TEM. Plain gels with a net positive charge (high cationic to anionic peptide molar ratio) showed significant cytotoxicity against U87 cells. In addition, the hydrophilic anticancer drug; vincristine sulfate, loaded into the emulgel aqueous phase showed sustained drug release profiles over 96 hours with improved cytotoxicity against U87 cells. These results suggest that the physico-mechanical properties, drug loading capacity and cytotoxicity of these systems are dependent on pH, peptides molar ratio, total peptide concentration and O:W ratio, which in turn will have a direct effect on emulgel’s anticancer activity. CONCLUSIONS In conclusion, these emulgels showed that the mesoscopic and bulk mechanical properties and cytotoxicity can be tuned by molecular design, pH, peptide concentration, and molar ratio and O:W ratio control, to develop biomaterials that can satisfy the needs of combination anticancer therapy. REFERENCES [1] Wychowaniec, J et al., Biomacromolecules, 21(7):2670-2680, 2020.