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

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.

Targeted Liposomes: A Nonviral Gene Delivery System for Cancer Therapy

“…In addition, octaarginine-loaded liposomes produced by this method showed higher transfection capacity than unmodified liposomes. This study demonstrated the potential of the CPP to enhance the incorporation of siRNA in liposomes and improve the transfection ability, which plays an important role in medical applications [147].…”

“…The cellular uptake mechanism of CPPs is still understood. It is not yet known if the cellular uptake o CCPs are mediated or not by specific cellular receptors [144][145][146][147]. By contrast, tumor-targeting/homing peptides (e.g., RGD, angiopep-2, Lyp-1, and GE11 peptides) can be recognized by specific receptors in the tumor cells or the tumor microenvironment, being able to differentiate tumor and normal cells [145].…”

A Multifunctional Hybrid Nanocarrier for Non-Invasive siRNA Delivery to the Retina

“…STR-CH2R4H2C can modify the surface of liposomes through the hydrophobic interactions between the stearyl group of STR-CH2R4H2C and the fatty acid ester residues of the lipid bilayer of the liposome [ 43 , 44 ]. We determined the optimized composition of the hybrid nanocarriers to be 2.0 mol% STR-CH2R4H2C and cationic DOTAP/DOPC/CHO liposomes composed of 0.25 µmol DOTAP (per 3 µmol of total lipid).…”

Biomaterials for Drug Delivery and Human Applications

“…The versatility, biocompatibility, and tunable properties of these carriers make them invaluable tools for the pharmaceutical industry. As research in drug delivery continues to advance, lipid-based nanocarriers will undoubtedly remain at the forefront, offering innovative solutions to improve the treatment of various diseases and enhance the overall patient experience [206].…”