Advances in the Formulation and Assembly of Non-Cationic Lipid Nanoparticles for the Medical Application of Gene Therapeutics.

Richard K Fisher,Michael B Freeman,Joshua D Arnold,Oscar H Grandas,Michael R Buckley,Michael M Mcnally,Michael D Best,Deidra J H Mountain,Raymond A Dieter,Samuel I Mattern-Schain,Stacy S Kirkpatrick,Phillip C West

doi:10.3390/nano11030825

Richard K Fisher, Michael B Freeman + Show 10 more

Open Access

https://doi.org/10.3390/nano11030825

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Abstract

Lipid nanoparticles have become increasingly popular delivery platforms in the field of gene therapy, but bench-to-bedside success has been limited. Many liposomal gene vectors are comprised of synthetic cationic lipids, which are associated with lipid-induced cytotoxicity and immunogenicity. Natural, non-cationic PEGylated liposomes (PLPs) demonstrate favorable biocompatibility profiles but are not considered viable gene delivery vehicles due to inefficient nucleic acid loading and reduced cellular uptake. PLPs can be modified with cell-penetrating peptides (CPPs) to enhance the intracellular delivery of liposomal cargo but encapsulate leakage upon CPP-PLP assembly is problematic. Here, we aimed to identify parameters that overcome these performance barriers by incorporating nucleic acid condensers during CPP-PLP assembly and screening variable ethanol injection parameters for optimization. CPP-PLPs were formed with R8-amphiphiles via pre-insertion, post-insertion and post-conjugation techniques and liposomes were characterized for size, surface charge, homogeneity, siRNA encapsulation efficiency and retention and cell associative properties. Herein we demonstrate that pre-insertion of stearylated R8 into PLPs is an efficient method to produce non-cationic CPP-PLPs and we provide additional assembly parameter specifications for a modified ethanol injection technique that is optimized for siRNA encapsulation/retention and enhanced cell association. This assembly technique could provide improved clinical translation of liposomal based gene therapy applications.

Highlights

Ribonucleic acid interference (RNAi) is a biological process that inhibits the posttranscriptional expression of mRNA in the cytoplasm of eukaryotic cells
Incorporation of R8-polyethylene glycol (PEG) by both preinsertion and post-conjugation resulted in increased nanoparticle size and/or aggregation and increased Polydispersity Index (PDI) (Table 3)
We aimed to establish a noncationic PEGylated liposomes (PLPs) formulation with natural lipid constituents and surface-conjugated Cell-penetrating peptides (CPPs) as an improved transfection agent compared to traditional cationic lipids in liposomal formulations (CLPs) formulations that often fail in downstream gene therapy applications

Summary

Introduction

Ribonucleic acid interference (RNAi) is a biological process that inhibits the posttranscriptional expression of mRNA in the cytoplasm of eukaryotic cells. Many multifunctional modification strategies aimed at improving the clinical efficacy of liposome-mediated gene therapy have been elucidated [2]. This includes the incorporation of polyethylene glycol (PEG) on the surface, resulting in PEGylated liposomes (PLPs) with increased stability and half-life of siRNA cargo in vivo (2nd generation liposomes) [3,4] and the development of ligand-conjugated liposomes for enhanced cell uptake and targeted drug delivery (3rd generation liposomes) [5,6]. The use of cationic lipids in liposomal formulations (CLPs) has helped improve encapsulation of negatively charged nucleic acid cargo through electrostatic-mediated “lipoplexes” [7]. Most of the techniques used to assemble CPP-modified PLPs with siRNA or other nucleic acid cargo are inefficient and costly

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