Structure-activity relationship of serotonin derived tocopherol lipids.

Tocopherol-based lipids are widely used for nucleic acid delivery. Using tocopherol molecules, we designed and synthesized 5-HT functionalized lipids by tethering 5-hydroxytryptamine (5-HT), a small molecule ligand as the head group to a natural amphiphilic molecule namely α-tocopherol (Vitamin E). This is with the aim of delivering nucleic acids specifically into cells expressing the serotonin receptors (5-hydroxytryptamine[5-HT]) which are abundant in the central nervous system. In order to achieve target recognition, we adopted an approach wherein two structurally different lipid molecules having serotonin as the head group was conjugated to tocopherol via different linkers thus generating lipids with either free -NH2 or -OH moiety. The corresponding lipids designated as Lipid A (Tocopheryl carbonate serotonin-NH2) and Lipid B (Tocopheryl 2-hydroxy propyl ammonium serotonin-OH), were formulated with co-lipids 1,2-dioleoyl-sn-glycero-3-phosphatidyl-ethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-sn-3-phosphatidylcholine (DOPC) and evaluated for their ability to deliver plasmid DNA through reporter gene expression assays in vitro. Furthermore, the physicochemical characteristics and cellular interactions of the formulations were examined using serotonin-receptor enriched cells in order to distinguish the structural and functional attributes of both lipids. Cell-based gene expression studies reveal that in comparison to Lipid A, a formulation of Lipid B prepared with DOPE as the co-lipid, contributes to efficient uptake leading to significant enhancement in transfection. Specific interactions explored by molecular docking studies suggests the role of the hydroxyl moiety and the enantiospecific significance of serotonin- conjugated tocopherol lipids in recognizing these receptors thus signifying a promising lipid-based approach to target the serotonin receptors in the central nervous system.

α-Tocopherol-ascorbic acid hybrid antioxidant based cationic amphiphile for gene delivery: Design, synthesis and transfection.

Natural antioxidants and vitamins have potential to protect biological systems from peroxidative damage induced by peroxyl radicals, α-tocopherol (Vitamin E, lipid soluble) and ascorbic acid (vitamin C, water soluble), well known natural antioxidant molecules. In the present study we described the synthesis and biological evaluation of hybrid of these two natural antioxidants with each other via ammonium di-ethylether linker, Toc-As in gene delivery. Two control cationic lipids N14-As and Toc-NOH are designed in such a way that one is with ascorbic acid moiety and no tocopherol moiety; another is with tocopherol moiety and no ascorbic acid moiety respectively. All the three cationic lipids can form self-assembled aggregates. The antioxidant efficiencies of the three lipids were compared with free ascorbic acid. The cationic lipids (Toc-As, N14-As and Toc-NOH) were formulated individually with a well-known fusogenic co-lipid DOPE and characterization studies such as DNA binding, heparin displacement, size, charge, circular dichroism were performed. The biological characterization studies such as cell viability assay and in vitro transfection studies were carried out with the above formulations in HepG2, Neuro-2a, CHO andHEK-293T cell lines. The three formulations showed their transfection efficiencies with highest in Toc-As, moderate inN14-As and least in Toc-NOH. Interestingly, the transfection efficiency observed with the antioxidant based conjugated lipid Toc-As is found to be approximately two and half fold higher than the commercially available lipofectamine 2000 at 4:1 charge ratio in Hep G2 cell lines. In the other cell lines studied the efficiency of Toc-As is found to be either higher or similarly active compared to lipofectamine 2000. The physicochemical characterization results show that Toc-As lipid is showing maximum antioxidant potency, strong binding with pDNA, least size and optimal zeta potential. It is also found to be least toxic in all the cell lines studied especially in Neuro-2a cell lines when compared to other two lipids. In summary, the designed antioxidant lipid can be exploited as a delivering system for treating ROS related diseases such as malignancy, brain stroke, etc.

Hepatocellular targeted α-tocopherol based pH sensitive galactosylated lipids: design, synthesis and transfection studies.

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 via 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, i.e. HepG2 and SK-Hep-1, respectively.

α-Tocopherol-based cationic amphiphiles with a novel pH sensitive hybrid linker for gene delivery.

Endosomal escape is one of the barriers for the efficient liposomal gene delivery. To address this and based on earlier encouraging results using tocopherol cationic lipids, we elaborated chemical modifications on tocopherol cationic lipids by introducing a novel hybrid pH sensitive linker "ether-ß-hydroxy-triazole" between tocopherol, the anchoring moiety and the basic tris(2-hydroxy ethyl)quaternary ammonium head group (Lp2). As control lipids we designed two lipids (Lp1 and Lp3), one is with only the ether-ß-hydroxy linker in between α-tocopherol and quaternary tris(2-hydroxyethyl)ammonium (Lp1) and the other is with the same novel hybrid linker i.e. "ether-ß-hydroxy-triazole" between the α-tocopherol linked and quaternary tris-ethyl ammonium head group (Lp3). Liposomes were formulated with a combination of a well-known co-lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and biophysical characteristics such as DNA binding, hydrodynamic diameters and global surface charges for liposomes and lipoplexes of respective lipids were evaluated. Cell viability assay and in vitro transfection studies were carried out in NIH3 T3, B16F10, HEK-293, and HepG2 cell lines. In vitro transfection data for the liposomes of lipids (Lp1, Lp2 and Lp3) revealed that the Lp2 lipid with a novel hybrid pH sensitive linker showed superior transfection efficiency when compared with the remaining two analogues. More importantly, Lp2 has shown a similar pattern of transfection efficiency in HepG2 and HEK-293 cell lines when compared with commercially available Lipofectamine 3000.