Receptor mediated gene delivery to the liver offers advantages in treating genetic disorders such as hemophilia and hereditary tyrosinemia type I (HTI). Prior findings demonstrated that tethering the d-galactose head group to cationic lipids directs genes to the liver asialoglycoprotein receptors (ASGPRs). In our continued efforts to develop safer and efficient lipofectins, we demonstrated that cationic lipids bearing α-tocopherol, an antioxidant, as a hydrophobic domain could deliver genes efficiently with high safety profiles in multiple cell lines. Towards developing ASGPR targeted pH sensitive cationic lipids, we have designed a galactosylated cationic lipid (Toc-Gal) with α-tocopherol as the hydrophobic core covalently connected with a pH responsive triazole moiety and a non-targeting control lipid (Toc-OH) without the galactose head group. In this study, we present the design and synthesis of a pH sensitive galactosylated cationic lipid (Toc-Gal), its comparative transfection biology, cellular uptake studies, serum stability and cytotoxicity profiles in both ASGPR positive and negative liver cells, HepG2 and SK-Hep-1, respectively.
Cationic
lipid-guided nucleic acid delivery holds great promise
in gene therapy and genome-editing applications for treating genetic
diseases. However, the major challenge lies in achieving therapeutically
relevant efficiencies. Prior findings, including our own, demonstrated
that asymmetry in the hydrophobic core of cationic lipids imparted
superior transfection efficiencies. To this end, we have developed
a lipid nanocarrier system with an asymmetric hydrophobic core (PS-Lips) derived from a mixture of fatty acids of food-grade
palmstearin and compared its efficiency with symmetric palmitic acid-based
nanocarrier system (P-Lip). PS-Lips exhibited
superior transfection efficiencies with both plasmid DNA (pDNA) and
mRNA in multiple cultured cells than the control P-Lip. More importantly, PS-Lips exhibited 2-fold superior
transfections with linear nucleic acid, green fluorescent protein
(GFP) mRNA in hematopoietic cells, when compared with the commercial
control lipofectamine RNAiMAX. PS-Lips was also found
to be effective in delivering genome-editing tools (CRISPR/Cas9, sgRNA
encoded pDNA with a reporter GFP construct) than P-Lip in HEK-293 cells. In the present study, we report that cationic
liposomes derivatized from natural food-grade fat palmstearin with
a natural hydrophobic core asymmetry are efficient in delivering both
linear and circular nucleic acids. In particular, PS-Lips is efficient in delivering mRNA to hematopoietic cells. These findings
can be further exploited in the genome-editing approach for treating
β-globinopathies.
Intracellular delivery of nucleic acids is one of the critical steps in the transfections. Prior findings demonstrated various strategies including membrane fusion, endosomal escape for the efficient cytoplasmic delivery. In our continuing efforts to improve the nucleic acids transfections, we harnessed cell permeable properties of Tomatidine (T), a steroidal alkaloid abundantly found in green tomatoes for maximizing intracellular delivery of lipoplexes. We doped Tomatidine into liposomes of cationic lipid with amide linker (A) from our lipid library. Six liposomal formulations (AT) of Lipid A (1 mM) with varying concentrations of Tomatidine (0-1 mM) were prepared and evaluated for their transfection efficacies. Owing to its signature characteristic of cell membrane permeability, Tomatidine modulated endocytosis process, enhanced the intracellular delivery of the lipoplexes, and in turn increased the transfection efficacy of cationic liposomes. Our findings provide 'proof of concept' for enhancing transfections in gene delivery applications with Tomatidine in cationic liposomal formulations. These findings can be further applied in lipid mediated gene therapy and drug delivery applications.
The CRISPR/Cas9 system holds great promise in treating genetic diseases, owing to its safe and precise genome editing. However, the major challenges to implementing the technology in clinics lie in transiently limiting the expression of genome editing factors and achieving therapeutically relevant frequencies with fidelity. Recent findings revealed that non-viral vectors could be a potential alternative delivery system to overcome these limitations. In our previous research, we demonstrated that liposomal formulations with amide linker-based cationic lipids and cholesterol were found to be effective in delivering a variety of nucleic acids. In the current study, we screened steroidal sapogenins as an alternative co-lipid to cholesterol in cationic liposomal formulations and found that liposomes with diosgenin (AD, Amide lipid: Diosgenin) further improved nucleic acid delivery efficacy, in particular, delivering Cas9 pDNA and mRNA for efficient genome editing at multiple loci, including AAVS1 and HBB, when compared to amide cholesterol. Mechanistic insights into the endocytosis of lipoplexes revealed that diosgenin facilitated the lipoplexes’ cholesterol-independent and clathrin-mediated endocytosis, which in turn leads to increased intracellular delivery. Our study identifies diosgenin-doped liposomes as an efficient tool to deliver CRISPR/Cas9 system.
Lipid-enabled nucleic
acid delivery has garnered tremendous attention
in recent times. Tocopherol among the cationic lipids, 3b-[
N
-(
N
′,
N
′-dimethylamino-ethane)carbamoyl]-cholesterol
hydrochloride (DC-Chol) with a headgroup of dimethylammonium, and
cholesterol as a hydrophobic moiety are found to be some of the most
successful lipids and are being used in clinical trials. However,
limited efficacy is a major limitation for their broader therapeutic
application. In our prior studies, we demonstrated tocopherol to be
a potential alternative hydrophobic moiety having additional antioxidant
properties to develop efficient and safer liposomal formulations.
Inspired by DC-Chol applications and taking cues from our own prior
findings, herein, we report the design and synthesis of four alpha-tocopherol-based
cationic derivatives with varying degrees of methylation, AC-Toc (no
methylation), MC-Toc (monomethylation derivative), DC-Toc (dimethylation
derivative), and TC-Toc (trimethylation derivative) and the evaluation
of their gene delivery properties. The transfection studies showed
that AC-Toc liposomes exhibited superior transfection compared to
MC-Toc, DC-Toc, TC-Toc, and control DC-Chol, indicating that methylation
in the hydrophilic moiety of Toc-lipids reduced their transfection
properties. Cellular internalization studies in the presence of different
endocytosis blockers revealed that all four tocopherol lipids were
internalized through clathrin-mediated endocytosis, whereas control
DC-Chol was found to be internalized through both macropinocytosis
and clathrin-mediated endocytosis. These novel Toc-lipids exhibited
higher antioxidant properties than DC-Chol by generating less reactive
oxygen species, indicating lower cytotoxicity. Our present findings
suggest that AC-Toc may be considered as an alternative to DC-Chol
in liposomal transfections.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.