A Recombinant Biopolymeric Platform for Reliable Evaluation of the Activity of pH-Responsive Amphiphile Fusogenic Peptides

Abstract

Over the past couple of decades, the sequences of several cationic and anionic pH-responsive amphiphile fusogenic peptides (FPs) have been reported in the literature. Due to their endosome membrane disrupting activity, these peptides have been routinely used for enhancing the efficacy of drug/gene delivery systems. However, no accurate comparative study has been performed to establish the precise correlation between FP sequence and its impact on enhancing drug/gene delivery efficiency. Therefore, there has been no clear rationale for selecting one FP over another in the past, and it is still unclear which FP is the most suitable and efficient construct for use in drug/gene delivery system design. To address this shortcoming, we examined the use of a recombinant biopolymeric platform as a tool to assess the pH-dependent membrane disruption activity, cell toxicity and impact on gene transfer efficiency of the five most widely used cationic and anionic pH-responsive FPs, INF7, GALA, KALA, H5WYG, and RALA. We first developed specific expression methods for the production of five identical recombinant biopolymers that were different only in FP sequence in their structures. Through the use of physicochemical and biological assays, the biopolymers were characterized and compared in terms of DNA condensation ability, cell toxicity, pH-dependent cell membrane disruption activity, and gene transfer efficiency. Overall, our data suggests that, among the tested constructs, GALA is the most suitable pH-responsive FP for enhancing the efficiency of gene delivery systems due mostly to its efficient endosomolytic activity and negligible cell toxicity. Most importantly, this study demonstrates the application of an effective biopolymeric tool that facilitates reliable evaluation of the physicochemical and biological activities of any pH-responsive FP independent of its charge. Therefore, whether artificially designed or inspired by nature, the FPs can be screened for their efficacy with a higher degree of accuracy in the future.

RALA-Mediated Delivery of FKBPL Nucleic Acid Therapeutics

“…Hatefi's group have created numerous multifunctional nonviral delivery systems with discrete interchangeable motifs, each one of which is designed to overcome a specific biological barrier [10,11]. While such systems are excellent examples of what can be achieved, the large-scale production of such recombinant proteins can be time-consuming and therefore peptide-mediated gene delivery presents a highly attractive option.…”

Taming Cell Penetrating Peptides: Never Too Old To Teach Old Dogs New Tricks

“…Whether GALA should be labeled as fusogenic peptide or cell penetrating peptide remained in disputation, however, its marvelous drug delivery capacity should never be ignored; GALA and GALA-related peptide (such as cationic KALA and RALA, etc.) could escape endosome entrapment and improve gene transfection [120][121][122][123][124][125].Harashima et al…”

“…It was worth noticing that the penetrating and fusion property was concealed until these peptides reached acidic environment such as endosomes, minimizing its cytotoxicity to normal tissues. Among them, GALA peptide should be counted as the most classic one since it showed high endosomal activity and low cytotoxicity [125]. Recently, Harashima et al discovered that GALA peptide could recognize the sialic acid-terminated sugar chains on pulmonary endothelium, which could aid in the cellular uptake of GALA-functionalized MEND by lung endothelial cells, and the nucleic acid delivery could be further improved by the endosomal membrane fusion by GALA peptide [126].…”

A fusogenic peptide from a sea urchin fertilization protein promotes intracellular delivery of biomacromolecules by facilitating endosomal escape

“…Although improving the efficiency of endosomal escape by CPPs alone is difficult, the ability to facilitate endosomal escape has been discovered in fusogenic peptides (FPs), and some studies have utilized FPs for intracellular delivery [3,[15][16][17]. For example, Wadia et al combined the TAT peptide and fusogenic HA2 peptide derived from hemagglutinin from influenza virus and demonstrated that dTAT-HA2 efficiently promoted the escape from macropinosomes [16].…”

“…FPs have membrane-disrupting activities under acidic conditions, through pH-dependent conformational changes [18], and consequently, they are expected to facilitate endosomal escape at acidic pH. The pH-dependence of FPs is an important characteristic for their application in drug delivery systems (DDS) with cell targeting because it reduces non-specific effects [15]. However, the FPs studied so far are not adequately efficient in facilitating endosomal escape; thus, novel FPs with higher activity are required.…”