Gemini surfactants are an intriguing class of surface active agents that are comprised of two surfactant monomers chemically linked at or near the headgroups by a rigid or flexible spacer. In comparison to their corresponding monomer counterparts, gemini surfactants are more efficient at reducing surface tension, have better wetting properties, and typically have critical micelle concentration values that are one to two orders of magnitude lower. These intriguing properties characteristic of gemini surfactants make them of special interest for pharmaceutical applications. Within this work, two different projects were carried out to assess the pharmaceutical applications of gemini surfactants. The aim of the first project was to assess the applications of gemini surfactants as transfection agents for non-viral gene delivery by evaluating the physical stability characteristics of gemini surfactant-based lipoplex systems. Prior to this investigation, an evaluation of the interaction properties between gemini surfactants and DNA, and between gemini surfactants and the neutral helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine was carried out using a combination of isothermal titration calorimetry, particle size, zeta potential and surface tension measurements. Following these evaluations, the physical stability of the gemini surfactant-based DNA delivery systems was assessed by examining the particle size distribution and membrane integrity characteristics of the lipoplexes. The results from this analysis revealed that the physical stability of these systems is limited by the membrane integrity characteristics of the lipoplex structure. The second project carried out was an evaluation of the interactions between gemini surfactants and a series of Tween surfactants commonly found in pharmaceutical formulations. The results from this analysis were analyzed using Clint’s, Rubingh’s, Motomura’s and Maeda’s theories for mixed micelle formation, where it was observed that there is a general synergistic mixing interaction present between gemini and Tween surfactants. The strength of synergism was found to be dependent upon the chain length and saturation of the Tween alkyl tail.

Gemini surfactants are an intriguing class of surface active agents that are comprised of two surfactant monomers chemically linked at or near the headgroups by a rigid or flexible spacer. In comparison to their corresponding monomer counterparts, gemini surfactants are more efficient at reducing surface tension, have better wetting properties, and typically have critical micelle concentration values that are one to two orders of magnitude lower. These intriguing properties characteristic of gemini surfactants make them of special interest for pharmaceutical applications. Within this work, two different projects were carried out to assess the pharmaceutical applications of gemini surfactants. The aim of the first project was to assess the applications of gemini surfactants as transfection agents for non-viral gene delivery by evaluating the physical stability characteristics of gemini surfactant-based lipoplex systems. Prior to this investigation, an evaluation of the interaction properties between gemini surfactants and DNA, and between gemini surfactants and the neutral helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine was carried out using a combination of isothermal titration calorimetry, particle size, zeta potential and surface tension measurements. Following these evaluations, the physical stability of the gemini surfactant-based DNA delivery systems was assessed by examining the particle size distribution and membrane integrity characteristics of the lipoplexes. The results from this analysis revealed that the physical stability of these systems is limited by the membrane integrity characteristics of the lipoplex structure. The second project carried out was an evaluation of the interactions between gemini surfactants and a series of Tween surfactants commonly found in pharmaceutical formulations. The results from this analysis were analyzed using Clint’s, Rubingh’s, Motomura’s and Maeda’s theories for mixed micelle formation, where it was observed that there is a general synergistic mixing interaction present between gemini and Tween surfactants. The strength of synergism was found to be dependent upon the chain length and saturation of the Tween alkyl tail.

None

None

None

None

Non-viral delivery vectors have potential advantages over the viral systems that currently are used extensively for delivering therapeutic genes of interest. However, non-viral gene therapy has low efficiencies in vivo, in part due to the aggregation of the particles in the delivery system associated with serum proteins and other components of the blood. An effective technique for overcoming this problem to use PluronicTM block copolymers to cover the surfaces of the particles in the delivery system with polyethylene oxide, which decreases their charge density and reduces their interactions with the serum proteins. The objectives of this project were to characterize a Pluronic-gemini surfactant system to be used as non-viral vectors for gene therapy. Five Pluronics (L44, F68, F87, F108, and F127) were evaluated by studying their physiochemical properties, including particle size and zeta potential. Also, these systems were evaluated in OVCAR-3 cell culture for gene expression and cell viability. The in vitro systems showed small particle sizes (approximately 200 nm) for all Pluronics. The particle sizes in the systems were increased dramatically (up to 2000 nm) by adding dioleylphosphatidylethanolamine (DOPE) to the systems. The zeta potential of these systems shifted the negative zeta potential of DNA (-43 mV) to a positive value (+35 mV). The addition of DOPE had very little effect on zeta potential. The in vitro transfection efficiency in OVCAR-3 showed that all of the Pluronics were able to transfect OVCAR-3 at various DNA/gemini surfactant ratios. The highest transfection efficiency was obtained with Pluronics L44, F87 and F108. PluronicF127 demonstrated the lowest transfection efficiency among the five Pluronics. Adding DOPE did not improve the transfection efficiency in any of the pluronic-gemini surfactant systems. The viabilities of the cells in these systems were high, and there were greater than the positive control (Lipofectamine 2000). The greatest cell viability (about 60%) was observed when the DNA to gemini surfactant ratio was 1:2. After adding DOPE, the cell viability decreased in all of the Pluronics except for Pluronic F68. The results of this investigation indicated that Pluronic block copolymers can transfect OVCAR-3 cell cultures in vitro and that they had a low level of cytotoxicity.

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.

In recent years there has been increasing interest in novel forms of surfactants. Of particular interest are gemini surfactants, which consist of two conventional surfactants joined by a spacer at the head groups, as they exhibit lower critical micelle concentrations than can be achieved by conventional surfactants. In this work, the self-assembly behaviour of several nonionic gemini surfactants with polyethylene oxide head groups (GemnEm, where n (= 20) is the number of carbons per tail and m (= 10, 15, 20 and 30) is the number of ethylene oxides per head group) were investigated. The Critical Micelle Concentrations (CMCs) were measured using a fluorescence probe technique. The CMCs are all ~2 x 10?7 M, with almost no variation with m. The CMCs are several orders of magnitude lower than conventional C12Em nonionic surfactants. The mixing behaviour of the gemini surfactants with conventional surfactants was also studied. They obeyed ideal mixing behaviour with both ionic and nonionic surfactants. Micelle morphologies were studied using Small Angle Neutron Scattering. The gemini surfactants with the larger head groups (i.e. Gem20E20 and Gem20E30) formed spherical micelles. Gem20E15 showed strong scattering at low Q, characteristic of elongated micelles. As the temperature was increased towards the cloud point, the scattering approached the Q-1 dependence predicted for infinite, straight rods. The existence of anisotropic micelles was supported by the viscosity of Gem20E15, which increases by several orders of magnitude on heating towards its cloud point. Phase behaviour was determined using Diffusive Interfacial Transport coupled to near-infrared spectroscopy. Much of the behaviour of these systems is similar to conventional nonionic surfactants. For example, Gem20E10 forms a dilute liquid isotropic phase (W) coexisting with a concentrated lamellar phase (La) at around room temperature and forms a sponge phase at higher temperatures. This is similar to the behaviour of C12E3 and C12E4. The other surfactants studied are all quite soluble in water and form liquid isotropic and hexagonal phases from room temperature. At higher concentrations Gem20E15 formed a cubic and then a lamellar phase while Gem20E20 formed a cubic phase and then an intermediate phase. This is also comparable to the phase behaviour of conventional nonionic surfactants except the intermediate phase, which is often only observed for surfactant systems with long alkyl tails.

Cationic gemini surfactants have been used for delivery of DNA into cells. These cationic surfactants are known to strongly bind to DNA to form a complex. In the dilute regimen, when the gemini-DNA complexes are mixed with helper neutral lipids, they undergo spontaneous assembly to form particles that are able to transfect DNA into the cells. In this study, the structure of several gemini surfactants, gemini-DNA complexes and gemini-DNA-neutral lipids complexes were systematically examined by small angle x-ray scattering (SAXS). The gemini surfactants were found to form micelles of varying shapes and arrangements modulated by the nature of spacer region and tail lengths. This includes ellipsoidal and worm-like micelles (as in the case of the 12- s-12 series) and disk-shaped hexagonally packed micelles (as in the case of 16-3-16). In addition to the study of the gemini surfactants, the effect of varying the DNA: gemini charge ratio on the DNA-gemini assembly was studied. The scattering pattern has shown that in the presence of excess gemini surfactants, free unbound surfactants exist in the solution. Upon the addition of neutral lipids, DNA-gemini-neutral lipid complexes are formed. The scattering patterns of the latter showed evidence of a strong interaction of the neutral lipids with the free gemini surfactants and the overcharged DNA-gemini complexes. Effectively, overcharging DNA-gemini complexes seem to aid in its incorporation into the neutral lipid matrix. These findings shed the light on the structure of DNA-gemini-neutral lipid systems and provide insights into the factors that influence the spontaneity of the self-assembly process. More importantly, the presented work provides a general strategy that can be applied to the study of similar systems using small angle x-ray scattering. A helium and vacuum chambers were made to enable testing the feasibility of the technique at the Canadian Light Source. Further, a pipeline was written to automate the reduction and analysis of SAXS data.