Designing siRNA/chitosan-methacrylate complex nanolipogel for prolonged gene silencing effects

Ye Cao,Subbu S. Venkatraman,Yee Shan Wong,Bhuthalingam Ramya,Melvin Wen Jie Liew,Muhammad Aminuddin,Jiaxin Liu,Yang Fei Tan

doi:10.1038/s41598-022-07554-0

Abstract

Despite immense revolutionary therapeutics potential, sustaining release of active small interfering RNA (siRNA) remains an arduous challenge. The development of nanoparticles with siRNA sustained release capabilities provides an avenue to enhance the therapeutic efficacy of gene-based therapy. Herein, we present a new system based on the encapsulation of siRNA/chitosan-methacrylate (CMA) complexes into liposomes to form UV crosslinkable Nanolipogels (NLGs) with sustained siRNA-release properties in vitro. We demonstrated that the CMA nanogel in NLGs can enhance the encapsulation efficiency of siRNA and provide sustained release of siRNA up to 28 days. To understand the particle mechanism of cellular entry, multiple endocytic inhibitors have been used to investigate its endocytosis pathways. The study saw positively charged NLGs entering cells via multiple endocytosis pathways, facilitating endosomal escape and slowly releasing siRNA into the cytoplasm. Transfection experiments confirmed that the crosslinked NLG delivery system provides effective transfection and prolonged silencing effect up to 14 days in cell cultures. We expect that this sustained-release siRNA NLG platform would be of interest in both fundamental biological studies and in clinical applications to extend the use of siRNA-based therapies.

Highlights

We designated covalently crosslinked nanogel core in NLGs to control the release of Small interfering RNA (siRNA), while lipid bilayers were designed to protect the chitosan-siRNA nanocomplex from dissociation at physiological pH and further facilitate the cellular internalization and intracellular trafficking
To enhance water solubility of chitosan and better control release of siRNA, we proposed the use of CMA via conjugating methacrylate groups to the chitosan chains
The chemical structure of CMA was characterized by 1H NMR spectrum (Fig. 2A), which showed the existence of vinyl protons at 5.57 and 5.78 ppm (g, 2H, C H2), methyl protons of methacrylic anhydride residues at 1.88 ppm (h, 3H, C H3)

Summary

Introduction

Nucleic acids into the cytosol (without sustained release behavior). none of the above mentioned systems have controlled release ability and have to be administered repeatedly to achieve therapeutic effect for a prolonged period. Similar to the LNPs, chitosan-methacrylate (CMA) form nano-complexes with siRNA as cores These cores are in turn encapsulated into liposomes to form UV crosslinkable NLGs to sustain release siRNA. The polydispersity index (PDI) of the particles were in a broad range from 0.24 to 0.39, and the nanoparticles released up to 60% of the drug within 180 min of release s tudy[21] In another example, Min Jiang et al reported cationic lipid-coated TPP crosslinked chitosan nanoparticles for the delivery of plasmids. Experiments inclusive of drug release, cellular entry, endocytotic pathway, toxicity, intracellular trafficking and gene silencing activity of siRNA encapsulated in NLGs using human foreskin fibroblast were performed to validate our concept of a nanocarrier incorporating siRNA that can enter cells, escape the endosome and slowly release the siRNA into the cytosol of cells over a period

Methods

Results

Discussion

Conclusion