Dicationic amphiphiles bearing an amino acid head group with a long-chain hydrophobic tail for in vitro gene delivery applications.

Amino acid-based cationic lipids, which have proven their efficacy as plasmid DNA nanocarriers, were employed as dicationic forms to transfect genes into cancer and non-cancer cells in this study. Proline, methionine, and serine amino acids are involved as hydrophilic moieties and the hydrocarbon long-chain serves as a hydrophobic tail. In a multicultural investigation, cationic lipids were employed as nano-vectors in conjunction with the helper lipid DOPE. To quantify the lipid efficient size, charge, and pDNA binding, biophysical analyses such as hydrodynamic diameter, zeta potential, agarose gel electrophoresis, and serum stability were done primarily. The liposomal particle composition was examined by scanning electron microscopy (SEM). Synthesized dicationic vector lipoplex formulations with reporter genes were found to be non-toxic to the cells investigated by MTT assay, and in addition, therapeutic gene p53 transfected into oral and brain cancer cells causing cell death was examined. In vitro investigations further validated that the proline-based lipid (C14-P) has high gene knockdown efficacy than methionine-based lipid (C14-M) and serine-based lipid (C14-S) at optimal N/P ratios as measured by ß-galactosidase protein and eGFP expression. C14-P lipid shows superior cellular internalization compared to C14-M and C14-S in HEK-293 and CAL-27 cells attested by confocal study. These findings could include the proline-based lipid vector's exceptional gene delivery activity.

Cimetidine-Based Cationic Amphiphiles for In Vitro Gene Delivery Targetable to Colon Cancer.

Cimetidine, a histamine-2 (H2) receptor antagonist, has been found to have anticancer properties against a number of cancer-type cells. In this report, we have demonstrated that cimetidine can acts as a hydrophilic domain in cationic lipids and targetable to the gastric system by carrying reporter genes and therapeutic genes through in vitro transfection. Two lipids, namely, Toc-Cim and Chol-Cim consisting cimetidine as the main head group and hydrophobic moieties as alpha-tocopherol or cholesterol, respectively, were designed and synthesized. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is a well-known co-lipid employed to produce liposomes as uniform vesicles. The liposomes and lipoplexes were structurally and functionally evaluated for global surface charges and hydrodynamic diameters, and results found that both liposome and lipoplex size and surface charges are optimal to screen the transfection potentials. DNA-binding studies were analyzed as complete binding at all formulated N/P ratios. The liposomes and lipoplexes of both the lipids Toc-Cim and Chol-Cim show minimal cytotoxicity even though at higher concentrations. The results of the transfection experiments revealed that tocopherol-based cationic lipids (Toc-Cim) show finer transfection efficacy with optimized N/P ratios (2:1 and 4:1) in the colon cancer cell line. Toc-Cim lipoplexes show higher cellular uptake compare to Chol-Cim in the colon cancer cell line at 2:1 and 4:1 N/P ratios. Toc-Cim and Chol-Cim lipids showed highly compatible serum, examined up to 50% of the serum concentration. To evaluate the apoptotic cell death in CT-26 cells, exposed to Toc-Cim:p53 and Chol-Cim:p53 lipoplexes at 2:1 N/P ratios, superior results showed with Toc-Cim:p53. An effect of TP53 protein expression in CT-26 cell lines assayed by western blot, transfected with Toc-Cim:p53 and Chol-Cim:p53 lipoplexes, demonstrated the superior efficacy of Toc-Cim. All of the findings suggest that Toc-Cim lipid is relatively secure and is an effective transfection agent to colon cancer gene delivery.

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.

Phytochemical analysis of Moringa Oleifera leaves extracts by GC-MS and free radical scavenging potency for industrial applications.

Natural extracts have been of very high interest since ancient time due to their enormous medicinal use and researcher's attention have further gone up recently to explore their phytochemical compositions, properties, potential applications in the areas such as, cosmetics, foods etc. In this present study phytochemical analysis have been done on the aqueous and methanolic Moringa leaves extracts using Gas Chromatography-Mass spectrometry (GCMS) and their free radical scavenging potency (FRSP) studied using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical for further applications. GCMS analysis revealed an extraction of range of phytochemicals in aqueous and methanolic extracts. In aqueous, extract constituents found with high percent peak area are Carbonic acid, butyl 2-pentyl ester (20.64%), 2-Isopropoxyethyl propionate (16.87%), Butanedioic acid, 2-hydroxy-2-methyl-, (3.14%) (also known as Citramalic acid that has been rarely detected in plant extracts) and many other phytochemicals were detected. Similarly, fifty-four bio components detected in methanolic extract of Moringa leaves, which were relatively higher than the aqueous extract. Few major compounds found with high percent peak area are 1,3-Propanediol, 2-ethyl-2- (hydroxymethyl)- (21.19%), Propionic acid, 2-methyl-, octyl ester (15.02%), Ethanamine, N-ethyl-N-nitroso- (5.21%), and 9,12,15-Octadecatrienoic acid etc. FRSP for methanolic extract was also recorded much higher than aqueous extract. The half-maximal inhibitory concentration (IC50) of Moringa aqueous extract observed is 4.65 µl/ml and for methanolic extract 1.83 µl/ml. These extracts can act as very powerful antioxidants, anti-inflammatory ingredient for various applications in diverse field of food, cosmetics, medicine etc.

Gemini Lipopeptide Bearing an Ultrashort Peptide for Enhanced Transfection Efficiency and Cancer-Cell-Specific Cytotoxicity.

Cationic gemini lipopeptides are a relatively new class of amphiphilic compounds to be used for gene delivery. Through the possibility of incorporating short peptides with cell-penetrating functionalities, these lipopeptides may be advantageous over traditional cationic lipids. Herein, we report the design, synthesis, and application of a novel cationic gemini lipopeptide for gene delivery. An ultrashort peptide, containing four amino acids, arginine-cysteine-cysteine-arginine, serves as a cationic head group, and two α-tocopherol moieties act as hydrophobic anchoring groups. The new lipopeptide (ATTA) is incorporated into the conventional liposomes, containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), at different molar ratios. The formulated liposomes are characterized and screened for better transfection efficiency. Transfection activity in multiple human cell lines from cancerous and noncancerous origins indicates that the inclusion of an optimal ratio of ATTA in the liposomes substantially enhances the transfection efficiency, superior to that of a traditional liposome, DOTAP-DOPE. Cytotoxicity of ATTA-containing formulations against multiple cell lines indicates potentially distinct activity between cancer and noncancer cell lines. Furthermore, lipoplexes of the ATTA-containing formulations with anticancer therapeutic gene, plasmid encoding tumor necrosis factor-related apoptosis-inducing ligand (pTRAIL), induce obviously more cytotoxicity than conventional formulations. The results indicate that arginine-rich cationic lipopeptide appears to be a promising ingredient in gene delivery vector formulations to enhance transfection efficiency and cell-selective cytotoxicity.

Arginine-tocopherol bioconjugated lipid vesicles for selective pTRAIL delivery and subsequent apoptosis induction in glioblastoma cells.

The incorporation of specific therapeutic gene into glioblastoma offers potent therapeutic strategy to treat the disease. Non-viral gene delivery vectors are of particular interest due to their tuneable transfection efficiency and easy scale-up. Herein, we demonstrate successful delivery of plasmid encoding tumor necrosis factor (TNF)-related apoptosis-inducing ligand (pTRAIL) using arginine-conjugated tocopherol lipid (AT) nanovesicles into glioblastoma cell lines. Another cationic lipid, glycine-conjugated tocopherol lipid (GT) having glycine in the head group region is also synthesized as a control lipid. Both lipid-derived liposomes effectively condensed the pDNA and the corresponding biomacromolecular assemblies (lipoplexes) are efficiently transfected into different cell lines. AT-based liposomes exhibit higher transfection efficacy in various cell lines, particularly selective in glioma cell lines. At an optimized N/P ratio, both the liposomal formulations show low cytotoxicity. AT-based lipoplexes have superior cellular uptake in U87 than the control lipid GT. The expression of TRAIL protein regulated death receptor and apoptosis signaling pathway is assayed by western blot using transfection of AT-based/pTRAIL into U87 cell lines. Induction of apoptosis in U87 cells exposed to AT-based/pTRAIL plasmid is evaluated by MTT assay as well as Annexin V-propidium iodide dual-staining assay. All results indicate that the developed AT-based/pTRAIL system offers a potentially safe and efficient therapeutic strategy for glioblastoma gene therapy.

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.

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.

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.

Effects of heterocyclic-based head group modifications on the structure-activity relationship of tocopherol-based lipids for non-viral gene delivery.

Gene therapy, a promising strategy for the delivery of therapeutic nucleic acids, is greatly dependent on the development of efficient vectors. In this study, we designed and synthesized several tocopherol-based lipids varying in the head group region. Here, we present the structure-activity relationship of stable aqueous suspensions of lipids that were synthetically prepared and formulated with 1,2-dioleoyl phosphatidyl ethanolamine (DOPE) as the co-lipid. The physicochemical properties such as the hydrodynamic size, zeta potential, stability and morphology of these formulations were investigated. Interaction with plasmid DNA was clearly demonstrated through gel binding and EtBr displacement assays. Further, the transfection potential was examined in mouse neuroblastoma Neuro-2a, hepatocarcinoma HepG2, human embryonic kidney and Chinese hamster ovarian cell lines, all of different origins. Cell-uptake assays with N-methylpiperidinium, N-methylmorpholinium, N-methylimidazolium and N,N-dimethylaminopyridinium head group containing formulations evidently depicted efficient cell uptake as observed by particulate cytoplasmic fluorescence. Trafficking of lipoplexes using an endocytic marker and rhodamine-labeled phospholipid DHPE indicated that the lipoplexes were not sequestered in the lysosomes. Importantly, lipoplexes were non-toxic and mediated good transfection efficiency as analyzed by ß-Gal and GFP reporter gene expression assays which established the superior activity of lipids whose structures correlate strongly with the transfection efficiency.

Benzothiazole head group based cationic lipids: synthesis and application for gene delivery.

A series of benzothiazole based lipids (1-10) containing different derivatives of benzothiazole in the head group region were synthesized to determine the structure-activity relationship for gene delivery. The liposomes formulated were mixed with plasmid DNA encoding green fluorescent protein (α5GFP) or ß-galactosidase (pCMV-SPORT-ß-gal) and transfected into B16F10 (Human melanoma cancer cells), CHO (Chinese hamster ovary), A-549 (Human lung carcinoma cells) and MCF-7 (Human breast carcinoma cells) types of cell lines. The efficiencies of lipids 9 and 10 in particular, were found to be comparable and even more when compared to that of LipofectAmine-2000. The transfection profiles of the efficient lipids are proved to be maintained even in the presence of serum. Thus, the benzothiazole head group based lipids developed have the potential to be used as transfection reagents in vitro and in vivo.

Synthesis and gene transfer activities of novel serum compatible reducible tocopherol-based cationic lipids.

The molecular structure of the cationic lipids greatly influences their transfection efficiency. High transfection efficiencies of tocopherol-based simple monocationic transfection lipids with hydroxylethyl headgroups were recently reported by us (Kedika, B., et al. J. Med. Chem.2011, 54 (2), 548-561). Toward enhancing the transfection efficiency of tocopherol-based lipids, we have synthesized two tocopherol-based dicationic lipids (1 and 2) using simple cystine in the headgroup region. The efficiency of tocopherol-based lipids (1 and 2) were compared with nontocopherol-based lipids (3 and 4) with cystine in the headgroup region. We report also a comprehensive structure-activity relationship study that identified tocopherol-based gemini cationic lipid 1 is a better transfecting agent than its monomeric lipid counterpart 2 and two other nontocopherol-based gemini cationic lipids (3 and 4). The transfection efficiency of lipid 1 was also greater than that of commercial formulation in HepG2 cell lines. A major characteristic feature of this investigation is that serum does not inhibit the transfection activity of tocopherol-based lipids (1 and 2) in general and in particular lipid 1 which is found to be highly serum-compatible even at higher concentrations of serum when compared to its monomeric counterpart lipid 2 and the other two control lipid analogues 3 and 4.

Influence of minor backbone structural variations in modulating the in vitro gene transfer efficacies of α-tocopherol based cationic transfection lipids.

Toward probing the influence of backbone structural variation in cationic lipid mediated gene delivery of α-tocopherol based lipids, two novel α-tocopherol based lipids 1 and 2 have been designed and synthesized. The only structural difference between the cationic amphiphiles 1 and 2 is the backbone structure, where lipid 1 has a non-glycerol backbone and lipid 2 has a glycerol backbone. The lipids 1 and 2 showed contrasting transfection efficiencies: lipid 1 showed high gene transfer efficacy in multiple cultured animals cell lines, whereas lipid 2 is transfection incompetent. In summary, the present findings demonstrate that in the case of α-tocopherol based lipids even minor structural variations like backbone can profoundly influence size, DNA binding characteristics, cellular uptake, and consequently gene delivery efficacies.

Design, synthesis, and in vitro transfection biology of novel tocopherol based monocationic lipids: a structure-activity investigation.

Herein, we report on the design, synthesis, and in vitro gene delivery efficacies of five novel tocopherol based cationic lipids (1-5) in transfecting CHO, B16F10, A-549, and HepG2 cells. The in vitro gene transfer efficiencies of lipids (1-5) were evaluated by both ß-galactosidase reporter gene expression and inverted fluorescent microscopic experiments. The results of the present structure-activity investigation convincingly demonstrate that the tocopherol based lipid with three hydroxyl groups in its headgroup region showed 4-fold better transfection efficiency than the commercial formulation. The results also demonstrate that these tocopherol based lipids may be targeted to liver. Transfection efficiency of all the relevant lipids was maintained even when the serum was present during the transfection conditions. The results indicated that the designed systems are quite capable of transferring the DNA into all four types of cells studied with low or no toxicity.