The thesis entitled “Design, Synthesis, Aggregation and Gene Transfection Properties of Novel Gemini Cationic Lipids and Lipopolymers” elucidates the design, synthesis, aggregation and gene transfection properties of novel gemini cationic lipids based on pseudoglyceryl, aromatic and cholesterol/thiocholesterol backbone, and PEI-cholesterol based lipopolymers . The work has been divided into five chapters. Chapter 1: Introduction to Gene Delivery This chapter presents an overview of the general area of gene delivery and also gives a comprehensive account of the research towards the development of novel cationic lipids and PEI derived polymers. Utilization of these non-viral vectors for gene delivery and their aggregation studies has also been reviewed. Chapter 2 deals with the Design, Synthesis, Membrane-Forming and Gene Transfection Properties of Pseudoglyceryl Gemini Lipids and has been divided into four parts. Part 2A: Synthesis of Pseudoglyceryl Gemini Lipids Possessing Polymethylene and Oxyethylene Spacers We have synthesized pseudoglyceryl gemini cationic lipids possessing polymethylene [-(CH2)m-] or oxyethylene [-CH2-(CH2-O-CH2)m-CH2-] spacers between the cationic ammonium headgroups. We have varied the length and nature of the spacer between the headgroups, from hydrophobic polymethylene [-(CH2)m-] to hydrophilic oxyethylene [-CH2-(CH2-O-CH2)m-CH2-] units (Figure 1). In these two series, we have also varied the hydrocarbon chain lengths from tetradecyl (n-C14H29) to hexadecyl (nC16H33) chains. Ether functionality has been introduced between the pseudoglyceryl backbone and the hydrocarbon chains. Figure 1(Refer PDF File) Part 2B: Thermotropic and Hydration Studies of Membranes Formed from Pseudoglyceryl Gemini Lipids Possessing Polymethylene spacers In this part, the aggregation, thermotropic and hydration properties of pseudoglyceryl gemini lipids possessing polymethylene [-(CH2)m-] spacers (Figure 1) have been discussed using transmission electron microscopy (TEM), high sensitivity differential scanning calorimetry (DSC) and Paldan fluorescence studies. Electron microscopic studies revealed the vesicular nature of all the lipid aggregates. Thermotropic studies showed that the incorporation of a -(CH2)3- (lipid (16)2-3-(16)2) spacer between cationic ammonium headgroups dramatically increased the phase transition temperature (Tm) for gemini lipid aggregates irrespective of the hydrocarbon chain lengths. Further increase in the number of polymethylene units brought about decreases in the Tm. Hydration studies indicate that gemini lipid aggregates bearing hexadecyl (n-C16H33) chains sense greater hydration at membrane interfaces and among them, aggregates of lipid (16)2-12-(16)2 were found to be most hydrated in the gel state. Part 2C: Membrane-Forming Properties of Pseudoglyceryl Gemini Lipids Possessing Oxyethylene Spacers Here, we report the membrane-forming properties of glycerol backbone based gemini cationic lipids with two pairs of hexadecyl (n-C16H33) chains and with a hydrophilic, flexible oxyethylene [-CH2-(CH2-O-CH2)m-CH2-] spacer of variable length and hydration properties between headgroups (Figure 1). Their membrane-forming properties have been studied by transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements, X-Ray diffraction (XRD), differential scanning calorimetry (DSC), Paldan fluorescence studies. The aggregates of lipid (16)2-1ox-(16)2 possess the highest phase transition temperature (Tm), lowest zeta potential and are highly hydrated, whereas that of gemini lipid (16)2-5ox-(16)2 aggregates are smallest in size, have highest zeta potential and greater bilayer width in the series examined, but possess comparable Tm as that of monomeric lipid (16)2. Part 2D: Gene Transfection Properties of Pseudoglyceryl Gemini Lipids Possessing Polymethylene and Oxyethylene Spacers We undertook a chemical-biology investigation on gene delivery efficacies of pseudoglyceryl gemini lipids (Figure 1). These gemini lipid formulations showed a significant enhancement in the gene transfection activities as compared to that of Lipofectin, which is a monomeric, structurally related to the present set of gemini lipids and commercially available reagent based on 1:1(w/w) ratio of DOTMA:DOPE formulation. The transfection efficacies depend on the hydrocarbon chains lengths and the spacer between the cationic ammonium headgroups as shown in Figure 2. The present set of gemini lipids were found to be serum compatible and even the presence of serum caused enhancement of the gene transfection activities of some of the lipid formulations. Lipid (16)2-3ox-(16)2/DOPE formulation was able to transfect nearly 35% of the cells in 50% FBS conditions. The simplicity of the use of pseudoglyceryl backbone, their high chemostability and shelf-life make these formulations particularly attractive. Figure 2(Refer PDF File) Chapter 3 deals with Design, Synthesis, Membrane-Forming and Gene Transfection Properties of Cationic Gemini Lipids based on Aromatic Backbone and have been divided into four parts. Part 3A: Synthesis of Gemini Lipids Possessing Aromatic backbone between the Hydrocarbon chains and the Cationic Headgroup In this chapter, we report the synthesis of new gemini cationic lipids based on an aromatic backbone that differ in the hydrocarbon chain lengths. We have also varied the length and nature of the spacer segment from hydrophobic polymethylene [-(CH2)m-] to hydrophilic oxyethylene [-CH2-(CH2-O-CH2)m-CH2-] units between the cationic headgroups .(Fig3) Figure 3(Refer PDF FILE) Part 3B: Membrane-Forming Properties of Aromatic derived Gemini Lipids Possessing Polymethylene Spacers The membrane-forming properties of lipids (12)2Bz and (12)2Bz-(CH2)m-Bz(12)2 (Figure 3) have been studied in detail by transmission electron microscopy (TEM), dynamic light scattering (DLS), X-ray diffraction (XRD), high sensitivity differential scanning calorimetry (DSC), Paldan fluorescence studies and UV-vis absorption spectroscopy. The vesicle sizes, morphologies and thermotropic phase transition properties of the lipid aggregates depend on the length of the spacer chain. Paldan fluorescence studies indicate that the gemini lipid aggregates are less hydrated as compared to that of their monomeric counterpart in their solid-gel state. In contrast in their fluid liquid-crystalline phase, the hydration was found to depend strongly on the length of the spacer. UV-vis absorption studies suggest an H-type aggregate formation in the gemini lipid membranes in the gel states. In fluid state of the lipid membranes, H-aggregate formation was found to be enhanced depending on the length of the spacer. Part 3C: Gene Transfection Properties of Aromatic derived Gemini Lipids Possessing Polymethylene Spacers Gene transfection properties of novel aromatic derived gemini possessing polymethylene [-(CH2)m-] spacers and three monomeric cationic lipids (Figure 3) that differ in the hydrocarbon chain lengths have been reported in this chapter. We investigated their gene transfection properties in detail in HeLa cells in the absence and presence of serum conditions. The lipids bearing n-C14H29 hydrocarbon chain lengths have been found to be the best transfecting agents as compared to their analogues with n-C12H25 and n-C16H33 hydrocarbon chains (Figure 4). Formulation of lipid (14)2Bz-5-Bz(14)2, possessing tetradecyl hydrocarbon chains and pentamethylene [-(CH2)5-] spacer showed highest gene transfection efficacy in this series. Lipid (14)2Bz-5-Bz(14)2 formulation is also able to deliver genes in the presence of high percentages of serum. Figure 4(Refer PDF File) Part 3D: Gene Transfection Properties of Aromatic derived Gemini Lipids Possessing Oxyethylene Spacers In this part, the transfection properties of six novel gemini cationic lipids based on aromatic backbone possessing n-C14H29 or n-C16H33 hydrocarbon chains (Figure 3) have been reported. We have varied the length of oxyethylene type spacers [(-CH2-CH2-O-CH2-CH2-)m] between the headgroups, where m varies from 1 to 3. Transfection studies showed that among lipids bearing n-C14H29 chains, transfection efficacies decrease with increase in the length of the spacer, whereas in case of lipids bearing n-C16H33 chains, transfection efficacies increase with increase in the length of the spacer. Lipid ((16)2Bz-3ox-Bz(16)2) bearing n-C16H33 hydrocarbon chains with [-(CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2)-] spacer was found to be highly serum compatible even in the presence of 50% serum conditions. Chapter 4 deals with the Design, Synthesis and Gene Transfection Properties of Gemini Cationic Lipids based on Cholesterol/Thiocholesterol backbone and have been divided into three parts. Part 4A: Design, Synthesis and Gene Transfection Properties of Cholesterol based Gemini Cationic Lipids Possessing Polymethylene Spacers Here we represent the synthesis and gene transfection properties of five cholesterol based gemini cationic lipids, which differ in the length of the polymethylene [-(CH2)m-] spacer between cationic ammonium headgroups (Figure 5). Transfection studies showed that with the increase in spacer chain length from propanediyl [-(CH2)3-] to pentanediyl [-(CH2)5-], transfection efficiency increased both in the absence and presence of serum (Figure 6). However, with further increase in the length from pentanediyl [-(CH2)5-] to dodecanediyl [-(CH2)12-] spacer transfection efficiency decreases. Transfection efficiencies of all the gemini lipids except lipid chol-3-chol were maintained even when the serum was present during the transfection conditions as compared to the monomeric lipid M, with which a dramatic decrease in transfection efficiency was observed(figure6) Figure 5 and 6(Refer PDF File) . Part 4B: Synthesis and Gene Transfection Properties of Cholesterol based Gemini Cationic Lipids Possessing Oxyethylene type Spacers Four novel cholesterol based gemini cationic lipids differing in the length of oxyethylene [(-CH2-CH2-O-CH2-CH2-)m] type spacers between each ammonium headgroups have been synthesized (Figure 7) and studied for gene transfection properties. All the cholesterol based gemini lipids induced better transfection activity than their monomeric counterpart M. Major characteristic feature of these oxyethylene spacer based cholesterol gemini lipids was that 10% serum conditions does not inhibit the transfection activity of these gemini lipids, whereas the transfection activity of their monomeric counterpart decreased drastically in the presence of serum. One of cholesterol based gemini lipid chol-1ox-chol possessing -CH2-CH2-O-CH2-CH2- spacer showed highest transfection activity. Figure 7(Refer PDF File) Part 4C: Effect of the Nature of the Spacer on Gene Transfection Properties of Novel Thiocholesterol derived Gemini Cationic Lipids In this chapter, we present the synthesis and gene transfection properties of three thiocholesterol derived gemini cationic lipids possessing biodegradable disulfide linkages between the cationic ammonium headgroup and thiocholesterol backbone (Figure 8). We varied the nature of the spacer between cationic headgroups from hydrophobic flexible -(CH2)5- (Lipid TC-5) to hydrophobic rigid (-C6H4-) (Lipid TC-px) to hydrophilic flexible (-CH2-CH2-O-CH2-CH2-) (Lipid TC-1-ox) spacer, to examine the effect of the nature of the spacer on gene transfection properties in different cell lines. Gene transfection properties of these gemini lipids were found to depend upon the nature of the spacer and the cell line. Cytotoxic studies confirmed the nontoxic nature of these lipid:DNA complexes at different N/P ratios used for transfection studies. Figure 8(Refer PDF File) Chapter 5 deals with the Synthesis and Gene Transfection Properties of PEI-Cholesterol based Lipopolymers, and Their Interactions with L-α-dipalmitoyl phosphatidylcholine (DPPC) membranes and has been divided into two parts Part 5A: Synthesis and Gene Transfection Properties of PEI-Cholesterol based Lipopolymers Nine lipopolymers based on low molecular weight Polyethyleneimines (PEI) and cholesterol via an ether linkage between the polymer amine and the cholesterol backbone have been synthesized (Figure 9). Different percentage of cholesterol moieties had been grafted on three types of PEI of molecular weights 800 (Mw), 1200 (Mn), 2000 (Mw). These lipopolymers were studied for gene transfection activities in HeLa cells. All lipopolymer formulations are better transfecting agents and highly serum compatible than commercially available PEI-25KDa. Transfection efficacies and serum compatibility of lipopolymer formulations depend upon the M.W. of PEI used for lipopolymers’ synthesis and percentage of cholesterol grafting on lipopolymers. Cell viability assay showed that PEI-25KDa is highly toxic as compared to all the lipopolymers. Figure 9(Refer PDF File) Part 5B: Thermotropic and Fluorescence studies of the Interactions of PEI-Cholesterol based Lipopolymers with L-α-dipalmitoyl phosphatidylcholine (DPPC) membranes The interactions of PEI-cholesterol based lipopolymers (Figure 9) with L-α-dipalmitoyl phosphatidylcholine (DPPC) membranes had been examined using fluorescence anisotropy and differential scanning calorimetry (DSC). These lipopolymers were found to quench the chain motion of the acyl chains of DPPC, when incorporated in membranes. Detailed analysis of the fluorescence anisotropy and DSC data indicates that the nature of perturbation induced by lipopolymers is dependent upon the molecular weight of the PEI used and the % of cholesterol grafting on PEI.

abstract: In the United States, 12% of women are typically diagnosed with breast cancer, where 20-30% of these cases are identified as Triple Negative Breast Cancer (TNBC). In the state of Arizona, 810 deaths occur due to breast cancer and more than 4,600 cases are diagnosed every year (American Cancer Society). The lack of estrogen, progesterone, and HER2 receptors in TNBC makes discovery of targeted therapies further challenging. To tackle this issue, a novel multi-component drug vehicle is presented. Previously, we have shown that mitoxantrone, a DNA damaging drug, can sensitize TNBC cells to TRAIL, which is a protein that can selectively kill cancer cells. In this current study, we have formulated aminoglycoside-derived nanoparticles (liposomes) loaded with mitoxantrone, PARP inhibitors, for delivery to cancer cells. PARP inhibitors are helpful in preventing cancer cells from repairing their DNA following damage with other drugs (e.g. mitoxantrone). Various treatment liposome groups, consisting of lipid-containing polymers (lipopolymers) synthesized in our laboratory, were formulated and characterized for their size, surface charge, and stability. PARP inhibitors and treatment of cells for in-vitro and in-vivo experiments with these liposomes resulted in synergistic death of cancer cells. Finally, studies to evaluate the pre-clinical efficacy of these approaches using immuno-deficient mouse models of TNBC disease have been initiated. / Dissertation/Thesis / Masters Thesis Chemical Engineering 2018

The thesis entitled “Novel Redox Responsive Cationic Lipids, Lipopolymers, Glycolipids and Phospholipid-Cationic Lipid Mixtures: Syntheses, Aggregation and Gene Transfection Properties” elucidates the design, synthesis, aggregation and gene transfection properties of novel cholesterol based cationic lipids with ferrocene as the redox moiety, polyethylenimine based ferrocenylated lipopolymers and cholesterol based non-ionic glycolipids. The thesis also discusses the cationic phospholipid-cationic lipid mixtures as superior gene transfection agents. The work has been divided into six chapters. Chapter 1. Introduction Part A. Various Cholesterol based Systems for Applications as Biomaterials Liposomes composed of cationic lipids have become popular gene delivery vehicles. A great deal of research is being pursued to make efficient vectors by varying their molecular architecture. Cholesterol being ubiquitous component in most of the animal cell membranes is increasingly being used as a hydrophobic segment of synthetic cationic lipids. In this chapter we describe various cholesterol based cationic lipids and focus on the effect of modifying various structural segments like linker and the headgroup of the cationic lipids on gene transfection efficiency with a special emphasis on the importance of ether linkage between cholesteryl backbone and the polar headgroup. Interaction of cationic cholesteryl lipids with dipalmitylphosphatidycholine membranes is also discussed here. Apart from cholesterol being an attractive scaffold in the drug/gene delivery vehicles, certain cholesteryl derivatives have also been shown to be attractive room temperature liquid-crystalline materials. Part B. Diverse Applications of Ferrocene Derivatives This chapter gives a brief overview of ferrocene chemistry followed by description of major applications of ferrocenyl derivatives in a variety of fields like catalysis, materials chemistry, electrochemical sensors, medicinal chemistry etc. We discuss the use of ferrocene as an electrochemical and redox active switch to achieve control over supramolecular aggregation. It also reviews ferrocene based amphiphiles including surfactants, lipids and polymers with an emphasis on the role of ferrocene over aggregate formation and their utilization in biological applications. Chapter 2: Optimization of Redox Active Alkyl-Ferrocene Modified Polyethylenimines for Efficacious Gene Delivery in Serum 1a-c, n = 6, P8-C6-F1, P8-C6-F2, P8-C6-F3 2a-c, n = 11, P8-C11-F1 P8-C11-F2, P8-C11-F3 % ferrocene grafting, F1 = 15%, F2 = 25% and F3 = 50% Figure 1. Structure of the alkyl-ferrocene modified 800 Da Branched Polyethylenimine. In this chapter we present six new lipopolymers based on low molecular weight polyethylenimines (BPEI 800 Da) which are hydrophobically modified using ferrocene terminated alkyl tails of variable lengths. The effects of degree of grafting, spacer length and redox state of ferrocene in the lipopolymer on the self assembly properties were investigated in detail by transmission electron microscopy (TEM), atomic force microscopy (AFM), dynamic light scattering (DLS) and zeta potential measurements. The assemblies displayed a redox induced increase in the size of the aggregates. The coliposomes comprising of the lipopolymer and a helper lipid 1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) showed excellent gene delivery capability in serum containing environment in two cancer cell lines (HeLa, U251 cells). Optimized formulations showed remarkably higher transfection activity than BPEI 25 KDa and even better than commercial Lipofectamine 2000 as evidenced from luciferase activity and EGFP expression analysis. Oxidation of ferrocene in lipopolymers led to reduced levels of gene transfection which was also followed by cellular internalization of fluorescently labeled pDNA using confocal microscopy. Cytotoxicity assay revealed no obvious toxicity for the lipopolyplexes in the range of optimized transfection levels. Overall, we have exploited the redox activity of ferrocene in PEI based polymeric gene carriers for trenchant control over gene transfection potential. RLU/mg protein HeLa Cells Figure 2. Maximum transfection efficacies of optimized redox lipopolymer/DOPE formulations by (A) Luciferase Assay and (B) Flow cytometry (GFP expression). Chapter 3. Membranes derived from Redox-active Cholesterol based Cationic Lipids and their Interactions with DNA and Phospholipid Membranes Figure 3. Molecular structures of the electroactive cholesterol based monomeric and gemini lipids. This chapter describes the synthesis and aggregation properties of two series of redox-active ferrocene containing monomeric and gemini cationic lipids with cholesterol as a hydrophobic domain. These cationic lipids are modified at their headgroup region using ferrocene terminated alkyl chains of differing length. All the four cationic lipids formed stable suspensions in water. Aggregation behavior of these cationic lipids in aqueous suspensions in their unoxidized and oxidized state was studied using TEM, DLS, zeta potential measurements and XRD studies. Cationic lipids with ferrocene in natural, reduced state were found form bigger sized vesicles which upon oxidation became smaller aggregates with increased zeta potential. XRD results indicate the existence of nice lamellar arrangements of the lipid bilayers. Thermotropic phase transition behavior of DPPC membranes incorporated with cationic ferrocene lipids was also studied using differential scanning calorimetry. Finally, we assayed pDNA (plasmid DNA) binding ability of all the four cationic lipids using ethidium bromide intercalation assay where all the cationic lipid formulations showed excellent DNA binding capability. In the experiments involving SDS-induced release of DNA, we observed that redox-active monomeric lipids (3a-b) were found to be more efficient in facilitating the release of DNA from the liposome-DNA complex in the presence of negatively charged SDS micelles than their gemini counterparts (4a-b). Chapter 4. Redox-responsive Gene Delivery by Ferrocene containing Cationic Cholesteryl Lipids in Serum This chapter describes the transfection efficacy of redox-active monomeric and gemini cationic lipids with cholesterol backbone. The transfection efficiency of all the lipids could be tuned by changing the oxidation state of the ferrocene moiety. Gene transfection capability was assayed in terms of EGFP expression using pEGFP-C3 plasmid DNA in three cancer cell lines of different origin, namely Caco-2, HEK293T and HeLa in the presence of serum. Figure 4. Effect of oxidation state of ferrocene on maximum transfection efficacies of monomeric and gemini lipids in three different cell lines (Caco-2, HEK 293T and HeLa). Cationic liposomal formulations with ferrocene in its reduced state were observed to be potent transfectants reaching the EGFP expression levels even better than commercial lipofectamine 2000 in the presence of serum as evidenced by flow cytometry. EGFP expression was further substantiated using fluorescence microscopy studies. All liposomal formulations containing oxidized ferrocene displayed diminished levels of gene expression and interestingly, these results were consistent for each formulation in all the three cell lines. Assessment of EGFP expression mediated by both reduced and oxidized ferrocene containing formulations was also undertaken following cellular internalization of labelled pDNA using confocal microscopy and flow cytometry. Lipoplexes derived from different liposomal formulations with reduced and oxidized ferrocene were characterised using TEM, AFM, zeta potential and DLS measurements. Overall, we demonstrate here controlled gene transfection levels using redox driven, transfection efficient cationic monomeric and gemini lipids. Chapter 5: Synthesis of ‘Click Chemistry’ Mediated Glycolipids: Their Aggregation Properties and Interaction with DPPC Membranes This chapter describes the synthesis and aggregation properties of cholesterol based glycolipids along with their interaction with a model phosphatidylcholine membranes. Three series of non-ionic glycolipids with hydrophobic cholesterol backbone and various monosaccharide and disaccharide sugars as the hydrophilic polar domain have been synthesized. These were conjugated to the cholesteryl backbone via oligooxyethylene spacers of different lengths (n = 1, 3 and 4) using Cu (I) catalyzed Huisgen [3+2] cycloaddition, which is popularly known as „Click Chemistry‟. All the synthetic glycolipids (5a-d, 6a-d and 7a-d) formed vesicular aggregates in aqueous medium as confirmed by TEM and DLS. XRD studies with the cast films of lipids revealed that the bilayer width increased with increase in the length of oligoethylene spacer unit that has been incorporated between the hydrophobic and hydrophilic domains. Also, within the same series containing a particular oligoethylene unit, bilayer widths were found to be more for the lipids containing disaccharides as their headgroup than monosaccharides. Figure 5. Molecular structures of various cholesterol-based glycolipids. Calorimetry studies of the coaggregates containing naturally occurring 1, 2-dipalmitoylphosphatidylcholine (DPPC) and various mol-% of each of the glycolipids revealed that more than 30 mol-% of glycolipids are required to completely abolish the phase transition of DPPC membranes. These results were further supported by fluorescence anisotropy measurements of the co-aggregates using 1, 6-diphenylhexatriene (DPH) as a probe. Fluorescence anisotropy of the neat vesicles revealed that 9a and 9c were more rigid than DPPC vesicles in the solid-like gel phase, while the glycolipids with longer oxyethylene spacers (n = 3 and 4) were less rigid than the DPPC vesicles. Chapter 6. Hydrophobic Moiety Decides the Synergistic Increase in Transfection Efficiency in Cationic Phospholipid/Cationic Lipid mixtures This chapter describes the effect of inclusion of cationic lipid/cationic gemini lipids into the membranes of a cationic phospholipid on the gene delivery efficiency across HeLa and HEK293T cell lines. Although all the three cationic lipids have the same quaternary ammonium moiety as their headgroup, they differ from each other in terms of their hydrophobic moiety and in the number of cationic headgroups. Chol-N is a cholesterol based monocationic lipid, while 2C14-N and 2C14N-5-N2C14N are monomeric and gemini cationic lipids respectively with pseudoglycerol backbone consisting of tetradecyl (n-C14H29) chains. Each of the three cationic lipids under the current investigation, namely, Chol-N, 2C14-N and 2C14N-5-N2C14N were added in different ratios to EtDMoPC and the resultant mixed membranes were studied for the biophysical characterization and gene delivery efficacies. Figure 6. Molecular structures of cationic lipids used in this study. All the formulations were characterized using dynamic light scattering and zeta potential measurements to obtain their hydrodynamic diameters and surface charge properties respectively. Their DNA binding ability was also studied by measuring changes in zeta potential and gel electrophoresis of the lipoplexes formed by the coliposomal formulations and pDNA at different Lipid/DNA weight ratios. The gene delivery efficacies of various formulations were studied in terms of EGFP expression using pEGFP-C3 plasmid DNA in two different cell lines, namely HeLa and HEK293T. In the absence of serum we found that the formulation (EtDMoPC+2C14N-5-N2C14N) showed better transfection efficiency than the individual lipids. However, in the case of others, i.e., (EtDMoPC+Chol-N) and (EtDMoPC+2C14-N) formulations, there was a slight decrease in transfection efficiency compared to the individual lipids. In the presence of serum, the formulations (EtDMoPC+2C14-N) and (EtDMoPC+2C14N-5-N2C14N) showed significantly higher transfection efficacies compared to their individual lipids. Fusion assay using labelled cationic lipid formulations and unlabelled anionic liposomes revealed that lipoplexes prepared from EtDMoPC+ 2C14-N and EtDMoPC+ 2C14N-5-N2C14 exhibited much higher fusogenicity as compared to the lipoplexes prepared using EtDMoPC+Chol-N as well as the individual lipids. Thus, the liposome formulations which showed better transfection activity fused more readily with the anionic liposomes than did the formulations with poorer activity. Overall, we found that the hydrophobic domain of the cationic lipid/cationic gemini lipid that is added to cationic phospholipid has an important role on the transfection efficiency of the mixed formulations. Additionally the cytotoxicity studies revealed that each of these formulations was not significantly toxic making them viable for applications in vivo. (For structural formula pl see the abstract pdf file)