Improvement of DNA Vector Delivery of DOTAP Lipoplexes by Short-Chain Aminolipids

Abstract

Cellular delivery of DNA vectors for the expression of therapeutic proteins is a promising approach to treat monogenic disorders or cancer. Significant efforts in a preclinical and clinical setting have been made to develop potent nonviral gene delivery systems based on lipoplexes composed of permanently cationic lipids. However, transfection efficiency and tolerability of such systems are in most cases not satisfactory. Here, we present a one-pot combinatorial method based on double-reductive amination for the synthesis of short-chain aminolipids. These lipids can be used to maximize the DNA vector delivery when combined with the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). We incorporated various aminolipids into such lipoplexes to complex minicircle DNA and screened these systems in a human liver-derived cell line (HuH7) for gene expression and cytotoxicity. The lead aminolipid AL-A12 showed twofold enhanced gene delivery and reduced toxicity compared to the native DOTAP:cholesterol lipoplexes. Moreover, AL-A12-containing lipoplexes enabled enhanced transgene expression in vivo in the zebrafish embryo model.

Non-viral Vectors in Gene Therapy: Recent Development, Challenges, and Prospects

“…For example, Buck et al . combined short-chain aminolipids to DOTAP:chol-based lipoplexes, finding that the longer lipid tails (C 12 ) improved transfection efficiency of minicircle DNA as compared with the shorter ones (C 10 ) ( 69 ). By constructing a series of α -cyclodextrin-threaded polyrotaxanes using polyethylene glycols (PEG) with different molecular weights, Ghodke et al exhibited that the composite containing the largest PEG in the study (PEG 1500) had the least silencing efficiency on Turbo GFP siRNA as compared with smaller PEG (PEG 200, 400, and 600) ( 70 ).…”

Nanotechnology-Based Strategies to Overcome Current Barriers in Gene Delivery

“…These systems take advantage of the self-assembling properties of amphiphilic lipids, such as phospholipids, to generate carriers that protect nucleic acids. Lipoplexes are electrostatic complexes spontaneously formed when liposomes composed of cationic lipids such as DOTAP (dioleoyl-3-trimethylammonium propane) interact with negatively charged oligonucleotides [46,47]. However, liposomes are highly dynamic systems that lack stability, which can significantly impact nucleic acid encapsulation, causing the genetic material's release before its arrival to the site of action.…”

Preparation and in vitro characterization of histidine trimethyl chitosan conjugated nanocomplex incorporated into injectable thermosensitive hydrogels for localized gene delivery

“…However, nonviral vectors are easier and safer to manufacture and less immunogenic and carcinogenic than viral carriers 2 . Generally, nonviral gene delivery systems including complexation of cationic polymers or lipids with plasmids have been used as a bolus intravenous injection or local injection into a target tissue 3 . Although cationic polymers and lipids can fabricate stable nanometer complexes with DNA and siRNA to be endocytosed by cells, these complexes are rapidly inactivated during in vivo transport due to different deactivation processes, such as degradation, aggregation, or clearance from tissue hindering the efficacy of bolus delivery of these complexes 3,4 .…”

“…2 Generally, nonviral gene delivery systems including complexation of cationic polymers or lipids with plasmids have been used as a bolus intravenous injection or local injection into a target tissue. 3 Although cationic polymers and lipids can fabricate stable nanometer complexes with DNA and siRNA to be endocytosed by cells, these complexes are rapidly inactivated during in vivo transport due to different deactivation processes, such as degradation, aggregation, or clearance from tissue hindering the efficacy of bolus delivery of these complexes. 3,4 Localized delivery of DNA and siRNA using three-dimensional scaffolds would increase the efficacy of gene therapy in tissue engineering and cancer therapy, especially when patterning of gene expression is important.…”

“…3 Although cationic polymers and lipids can fabricate stable nanometer complexes with DNA and siRNA to be endocytosed by cells, these complexes are rapidly inactivated during in vivo transport due to different deactivation processes, such as degradation, aggregation, or clearance from tissue hindering the efficacy of bolus delivery of these complexes. 3,4 Localized delivery of DNA and siRNA using three-dimensional scaffolds would increase the efficacy of gene therapy in tissue engineering and cancer therapy, especially when patterning of gene expression is important. 5 For tissue regeneration, local delivery of genetic materials using biomaterial scaffolds can provide supporting medium for cell attachment, proliferation, and migration, and would promote the expression of tissue inductive factors and consequently accelerate progenitor cell responses and tissue formation.…”