Effect of Methylation of the Hydrophilic Domain of Tocopheryl Ammonium-Based Lipids on their Nucleic Acid Delivery Properties.

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.

Influence of Hydrophobicity in the Hydrophilic Region of Cationic Lipids on Enhancing Nucleic Acid Delivery and Gene Editing.

Intracellular delivery of biomolecules using non-viral vectors critically depends on the vectors' ability to allow the escape and release of the contents from the endosomes. Prior findings demonstrated that aromatic/hydrophobic group-containing amino acids such as phenylalanine (F) and tryptophan (W) destabilize cellular membranes by forming pores in the lipid bilayer. Taking cues from these findings, we have developed four α-tocopherol-based cationic amphiphiles by varying the aromatic/hydrophobic amino acids such as glycine (G), proline (P), phenylalanine (F), and tryptophan (W) as head groups and triazole in the linker region to study their impact on endosomal escape for the enhanced transfection efficacy. The lipids tocopherol-triazole-phenylalanine (TTF) and tocopherol-triazole-tryptophan (TTW) exhibited similar potential to commercial transfecting reagents, Lipofectamine (LF) 3000 and Lipofectamine Messenger Max (LFMM), respectively, in transfecting plasmid DNA and messenger RNA in multiple cultured cell lines. The TTW liposome was also found to be effective in delivering Cas9 mRNA and demonstrated equal efficiency of gene editing AAVS1 locus compared to LFMM in CHO, Neuro-2a, and EA.HY926 cell lines. In this current investigation, it is shown that the synthesized cationic lipids with aromatic hydrophobic R group-containing amino acids are safe, economic, and actually more efficient in nucleic acid delivery and genome-editing applications. These findings can be further explored in the genome-editing approach for treating genetic disorders.

α-Tocopherol-anchored gemini lipids with delocalizable cationic head groups: the effect of spacer length on DNA compaction and transfection properties.

Understanding the role of structural units in cationic lipids used for gene delivery is essential in designing efficient gene delivery vehicles. Herein, we report a systematic structure-activity investigation on the influence of the spacer length on the DNA compaction ability and the transfection properties of gemini lipids with delocalizable cationic head groups. We have synthesized a series of dimeric cationic lipids varying in spacer length. The DNA binding interactions of liposomal formulations were characterized by gel electrophoresis and ethidium bromide (EtBr) exclusion assays. Condensation potentials were optimized and the best results were observed with cationic lipids possessing a 6 methylene spacer (TIM 6). We found that the size of the lipid/DNA complex decreased with the increase in spacer chain length up to a 6 methylene spacer TIM 6 and increased further. We have optimized the dimeric lipid/DOPE molar formulation using the ß-galactosidase activity assay and found that the molar ratio of 1 : 1.5 (gemini lipid/DOPE) showed the maximum transfection among all molar ratios. The cellular uptake and co-localization of lipoplexes were observed by cell analysis and imaging using confocal microscopy. The results confirm that the lipoplex derived from lipid TIM 6 and pCMV-bgal/DNA internalizes via cellular endocytosis. The cytotoxicity studies using the MTT assay revealed that all formulations show comparable cell viability to the commercial standard even at higher charge ratios. Overall, the data suggest that the DNA compaction ability of these lipid dimers depends on the spacer chain length and the gemini lipid containing a six methylene aliphatic spacer has the maximum potential to deliver genes.

Evolution of New "Bolaliposomes" using Novel α-Tocopheryl Succinate Based Cationic Lipid and 1,12-Disubstituted Dodecane-Based Bolaamphiphile for Efficient Gene Delivery.

Nonviral lipid-based vectors are promising transporting systems for the intracellular delivery of therapeutic gene sequences and directly influence the success of gene delivery. However, the associated drawbacks like lower transfection, toxicity, and targetability require further improvement. Thus, herein, we report a novel lipid formulation by the mixing of two distinct cationic surfactants such as tocopheryl succinate based cationic lipid and 1,12 dodecane based bolaamphiphile and prove it to be a good transfection reagent with its competing potential with the "golden standard", Lipofectamine 3000 (L3K). These interesting aggregations were named "Bolaliposome" and showed adequate unilamellar vesicle morphology under transmission electron microscopy, having a size of around 100 nm and could transfect efficiently different varieties of cell lines. Moreover, the generated complexes from bolaliposome and DNA (bolalipoplex) were characterized in terms of surface potential, hydrodynamic size, and gel electrophoresis. Various pharmacological inhibitors were also used in reporter gene expression to prove that the complexes followed the clathrin-mediated endocytosis. Finally, these findings would be helpful in the making of new aggregates and the development of better cytofectins. This was developed by optimizing the formulation based on the efficiency of reporter gene expression performed using the pEGFP-N3 plasmid.

Novel 1,2,3-triazolium-based dicationic amphiphiles synthesized using click-chemistry approach for efficient plasmid delivery.

Herein, we report the synthesis, characterization and evaluation of the transfection efficiencies of a series of dicationic amphiphiles designed to construct quaternary ammonium ion-based cationic lipids varying in chain length of the hydrophobic back bone connected individually through head group to a 1,2,3-triazolium cation consisting of 2-hydroxy ethyl chain as substitution. Accordingly, three dicationic amphiphiles were synthesized by "click chemistry" approach and formulated to bilayered vesicles using DOPE as a co-lipid. The transfection efficacies of these novel lipid formulations were measured and correlated with the results obtained from various physicochemical techniques. Importantly, the observed gradient in the activity profile, where the transfection potential increased with decreasing chain length of the lipid hydrophobic back bone, highlights the synergistic interplay of the lipid alkyl chain length in coordination with charge delocalization in modulating the transfection potency of these 1,2,3-triazolium-based lipids.