One major disadvantage of nucleic acid delivery systems is the low transfection or transduction efficiency of large-sized plasmids into cells. In this communication, we demonstrate the efficient transfection of a 15.5 kb green fluorescent protein (GFP)-fused HIV-1 molecular clone with a nucleic acid delivery system prepared from the highly potent peptide-mimicking cationic lipid OH4 in a mixture with the phospholipid DOPE (co-lipid). For the transfection, liposomes were loaded using a large-sized plasmid (15.5 kb), which encodes a replication-competent HIV type 1 molecular clone that carries a Gag-internal green fluorescent protein (HIV-1 JR-FL Gag-iGFP). The particle size and charge of the generated nanocarriers with 15.5 kb were compared to those of a standardized 4.7 kb plasmid formulation. Stable, small-sized lipoplexes could be generated independently of the length of the used DNA. The transfer of fluorescently labeled pDNA-HIV1-Gag-iGFP in HEK293T cells was monitored using confocal laser scanning microscopy (cLSM). After efficient plasmid delivery, virus particles were detectable as budding structures on the plasma membrane. Moreover, we observed a randomized distribution of fluorescently labeled lipids over the plasma membrane. Obviously, a significant exchange of lipids between the drug delivery system and the cellular membranes occurs, which hints toward a fusion process. The mechanism of membrane fusion for the internalization of lipid-based drug delivery systems into cells is still a frequently discussed topic.

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)

Made available in DSpace on 2014-12-17T14:05:18Z (GMT). No. of bitstreams: 1 LourenaMF_TESE_Capa_ate_pag79.pdf: 4572586 bytes, checksum: 2630047160ff526cc964f166fb86ab33 (MD5) Previous issue date: 2011-03-14 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Gene therapy is one of the major challenges of the post-genomic research and it is based on the transfer of genetic material into a cell, tissue or organ in order to cure or improve the patient s clinical status. In general, gene therapy consists in the insertion of functional genes aiming substitute, complement or inhibit defective genes. The achievement of a foreigner DNA expression into a population of cells requires its transfer to the target. Therefore, a key issue is to create systems, vectors, able to transfer and protect the DNA until it reaches the target. The disadvantages related to the use of viral vectors have encouraged efforts to develop emulsions as non-viral vectors. In fact, they are easy to produce, present suitable stability and enable transfection. The aim of this work was to evaluate two different non-viral vectors, cationic liposomes and nanoemulsions, and the possibility of their use in gene therapy. For the two systems, cationic lipids and helper lipids were used. Nanoemulsions were prepared using sonication method and were composed of Captex? 355; Tween? 80; Spam? 80; cationic lipid, Stearylamine (SA) or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) and water (Milli-Q?). These systems were characterized by average droplet size, Polidispersion Index (PI) and Zeta Potential. The stability of the systems; as well as the DNA compaction capacity; their cytotoxicity and the cytotoxicity of the isolated components; and their transfection capacity; were also evaluated. Liposomes were made by hydration film method and were composed of DOTAP; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), containing or not Rhodaminephosphatidylethanolamine (PE- Rhodamine) and the conjugate Hyaluronic Acid DOPE (HA-DOPE). These systems were also characterized as nanoemulsions. Stability of the systems and the influence of time, size of plasmid and presence or absence of endotoxin in the formation of lipoplexes were also analyzed. Besides, the ophthalmic biodistribution of PE-Rhodamine containing liposomes was studied after intravitreal injection. The obtained results show that these systems are promising non-viral vector for further utilization in gene therapy and that this field seems to be very important in the clinical practice in this century. However, from the possibility to the practice, there is still a long way / A terapia g?nica ? um dos maiores desafios propostos pela pesquisa p?s-gen?mica e se baseia na transfer?ncia de material gen?tico a uma c?lula, tecido ou ?rg?o com o intuito de curar ou melhorar o estado cl?nico do paciente. Em sua forma mais simples, a terapia g?nica consiste na inser??o de genes funcionais em c?lulas com genes defeituosos objetivando substituir, complementar ou inibir esses genes causadores de doen?as. Para que o DNA ex?geno seja expresso em uma popula??o celular faz-se necess?ria a sua transfer?ncia at? o local de a??o. Assim, ? necess?rio criar ve?culos, que transportem e protejam o DNA at? que este chegue a uma popula??o celular alvo. Os obst?culos encontrados com a utiliza??o de vetores virais t?m proporcionado o interesse no desenvolvimento de vetores n?o-virais, por serem f?ceis de produzir, apresentarem estabilidade control?vel e facilitarem a transfec??o g?nica. O objetivo deste trabalho foi avaliar dois diferentes vetores n?o virais, lipossomas e nanoemuls?es cati?nicos, e sua poss?vel utiliza??o na terapia g?nica. Para isso, foram utilizados lip?deos cati?nicos e co-tensoativos na produ??o dos dois sistemas. As nanoemuls?es foram produzidas pelo m?todo de sonica??o e compostas por Captex? 355; Tween? 80; Spam? 80; lip?deo cati?nico, Estearilamina (EA) ou N-[1-(2,3-Dioleoiloxi)propil]-N,N,Ntrimetilamonio metilsulfato (DOTAP); e ?gua ultra-pura (Milli-Q?). Estes sistemas foram caracterizados quanto ao tamanho m?dio de got?cula, ?ndice de polidispers?o (PI) e potencial zeta. Avaliou-se ainda a estabilidade dos sistemas e suas capacidades de compacta??o do material gen?tico. Os lipossomas foram preparados a partir do m?todo de hidrata??o do filme e compostos por DOTAP, Dioleilfosfatidiletanolamina (DOPE), na presen?a ou aus?ncia de Rodaminafosfatidiletanolamina (PE-Rodamina) e do conjugado ?cido Hialur?nico DOPE (HA-DOPE). Estes sistemas foram caracterizados da mesma forma que as nanoemuls?es e tamb?m foram avaliados estabilidade, influ?ncia do tempo, tamanho de material gen?tico e presen?a ou aus?ncia de endotoxinas na forma??o dos lipoplexos. Os resultados obtidos permitem afirmar que os sistemas s?o promissores para posterior utiliza??o na terapia g?nica e que esta ?rea promete ser uma ?rea f?rtil de pesquisa cient?fica e cl?nica por muitos anos, e provavelmente se tornar? uma pr?tica cl?nica importante neste s?culo. No entanto, da possibilidade ? pr?tica existe um longo caminho a percorrer

Cationic lipids are efficient tools to introduce nucleic acids and proteins into cells. Elucidation of the mechanism and cellular pathways associated to such a transport has been relatively slow, even though significant progress has been made in the characterization of the intracellular trafficking of cationic lipid/DNA complexes. Surprisingly, little is known about the effects of these delivery vectors on cell functioning. In the present thesis, we show that cationic lipids and cationic lipid/DNA complexes strongly increase the intracellular Ca2+ concentration. The end point of the Ca2+ increase was ~400 nM from a basal level of ~100 nM. The [Ca2+]i increase was studied using K562 and Jurkat cells cultured in vitro. This effect is weakened following addition of DNA to cationic liposomes, although remaining very large at cationic lipid/DNA ratios commonly used for cell transfection experiments. Removal of extracellular Ca2+ did not abolish this effect significantly and preincubating K562 cells with the Ca2+-ATPase inhibitor thapsigargin strongly abolished intracellular Ca2+ concentration increase, indicating that Ca2+ was released mainly from internal Ca2+ stores sensitive to thapsigargin. Pretreatment of the cells with the phospholipase C inhibitor U73122 blocked the intracellular Ca2+ concentration rise, suggesting an inositol pathway-dependent mechanism. LDH release assay indicates that in the conditions used for fluorescence measurement and in those used to transfer DNA into cells, cationic liposomes diC14-amidine and DOTAP had no massive cytotoxic effects. Cationic liposomes showed more toxicity than their corresponding complexes; this toxicity decreases in the presence of serum. The effect of cationic lipids on phosphatidylinositol-specific phospholipase C (PI-PLC) was quantitatively assessed using phosphatidylinositol (PI) and radiolabeled phosphatidylinositol ([3H]-PI). Incorporation of diC14-amidine into PC/PI vesicle activated PI-PLC and was shown to activate the hydrolysis of PI and [3H]-PI. Our data may suggest that mobilization of intracellular Ca2+ by complex could have an effect on the transfection process itself. These results indicate for the first time that cationic lipids and cationic lipid/DNA complexes are not inert and can affect the functioning of the cells by increasing their intracellular Ca2+. <p><p> / Doctorat en sciences, Spécialisation chimie / info:eu-repo/semantics/nonPublished

The thesis entitled “Novel Cationic Gemini Lipids, Click Chemistry Based Adducts and Amphiphile-Capped Silver Nanostructures: Synthesis, Aggregation and Biological Properties” elucidates the design, synthesis, aggregation and gene transfection properties of novel gemini cationic lipids based on cholesterol and pseudoglyceryl backbone, and click chemistry based adducts. This thesis also elucidates the synthesis and aggregation properties of silver nanoparticles loaded cationic liposomes and silver nanorods stabilized by micellar solutions of gemini surfactants. The work has been divided into six chapters. Chapter 1: Introduction: Membrane Formation from Cholesterol-based Cationic Lipids and their use as Non-Viral Gene Delivery Agents This chapter describes the importance of cholesterol in biological membranes, the aggregation properties of cholesterol-based cationic lipids and their interactions with phospholipid membranes. This chapter also gives a comprehensive account of the research towards the development of novel cationic cholesterol-based monomeric and gemini lipids. It also reviews the utilization of cholesterol-based cationic monomeric and gemini lipids in gene transfection properties. Chapter 2A: Effect of Hydroxyl group on the Cationic Headgroups of Cholesterol-based Gemini Lipids on their Aggregation, DNA Binding Properties and Interaction with Phospholipid Membranes This chapter describes the syntheses and aggregation properties of two series of cholesterol-based monomeric and gemini cationic lipids with and without hydroxyl functionality (Figure 1). The gemini lipids of a given series differ in their spacer polymethylene -(CH2)n- chain lengths between the cationic headgroups. Figure 1. Molecular structures of non-hydroxylated and hydroxylated cationic cholesterol-based gemini lipids and their monomeric counterparts. All monomeric and gemini lipids were found to generate stable suspensions in aqueous media. Electron microscopic studies showed that all the lipids form vesicular aggregates in aqueous media. The structures seen under TEM for the non-hydroxylated series of monomeric (C-M) and gemini lipids are of variable sizes, they appeared like separated vesicular aggregates. For the hydroxylated series of lipids, however, both the monomeric lipid aggregates (CH-M) and aggregates of their gemini counterparts were found to be ‘connected’ with each other to form elongated chain of aggregates of different length scales. XRD studies with the cast films of lipids revealed that the monomeric lipids of either series have higher bilayer width than the corresponding gemini lipids. Incorporation of the -(CH2)n- spacer units at the head group level joining the two monomeric units lowered the bilayer thicknesses of both series of the lipid aggregates. Thus the monomeric lipids (C-M and CH-M) appear to form nearly regular bilayer type arrangements whereas gemini lipids form interdigited and tilted bilayer arrangements in their aggregates. Calorimetry studies of the coaggregates showed that ~10 mol-% of most of the cholesterol gemini lipids is enough to abolish the phase transition of DPPC membranes whereas more than 10 mol-% is required in case of their monomeric counterparts. Further these thermotropic properties depend upon the length of the spacer of the gemini lipid included in the mixture. We have observed greater quenching of the thermal phase transition of DPPC membranes with 10 mol-% of C-M as compared to CH-M doped liposomes. At 10 mol-% of all the cationic lipid doped DPPC covesicles, only CG-3 doped liposomes showed an observable transition temperature. Maximum broadening of the DPPC transition peak was observed in the case of the gemini lipids, CHG-6 and CHG-12. DNA binding and release studies show that the interactions between gemini lipids and DNA depend upon the nature of the head group as well as the length of the spacer between the cationic head groups. For the non-hydroxylated cholesterol-based cationic lipid series, the monomeric liposomes of C-M facilitates the dissociation of EB from the EB-DNA complex to an extent of 93% at a maximum lipid:DNA ratio of 3.0 whereas the liposomes of CG-4 and CG-12 showed the lowest extent of maximum EB exclusion (~74%) from the EB-DNA complex at lipid:DNA ratio of 3.0. For hydroxylated cholesterol-based cationic lipid series, the monomeric liposomes of CH-M facilitate the dissociation of EB from the intercalated EB-DNA complex to an extent of 81 % whereas the liposomes of CHG-3 showed the minimum binding to DNA. Thus the two monomeric liposomes C-M and CH-M were the more efficient formulations that allow dissociation of DNA from the corresponding lipoplexes. These findings have important being in their gene transfection activity compared their respective gemini lipid counterparts. Chapter 2B: Novel Cholesterol-based Cationic Gemini Lipids possessing Hydroxyethyl group on the Headgroup: Transfection Efficacy and Cell Toxicity Properties This chapter describes the transfection efficacy and cell toxicity properties of five cholesterol based gemini cationic lipids possessing hydroxyethyl functionality on each head group, which differ in the length of the polymethylene spacer [-(CH2)n-] chain (Figure 2). These gemini lipids are important to gene delivery processes as they possess pre-optimized molecular features, e.g., cholesterol backbone, ether linkage and a variable spacer chain between both the headgroups of the gemini lipids. Cationic liposomes were prepared from each of these lipids individually and as a mixture of individual cationic gemini lipid and 1,2-dioleoylphosphatidylethanolamine (DOPE). The gemini lipid with a hydroxyethylated headgroup and a -(CH2)5- spacer, CHG-5 showed the highest transfection activity at N/P (lipid/DNA) ratio of 0.5 and lipid:DOPE molar ratio of 2. Upon comparison of the relevant parameters, e.g., % transfected cells, the amount of DNA transfected to each cell and % cell viability all together against LipofectAMINE 2000, one of the most potent commercially available transfecting agents, the optimized lipid formulation based on CHG-5/DOPE was found to be comparable. In terms of its ability to induce gene-transfer in presence of serum and shelf-life CHG-5/DOPE liposome was found to be better than its commercial counterpart. Recording of confocal images confirmed that in presence of 10% serum using 1.2 µg DNA per well and lipid:DOPE ratio of 1:4 and N/P charge ratio of 0.75, CHG-5 is better than LipofectAMINE 2000. These properties render them to be reagents of practical value for various gene delivery applications. Figure 2. Molecular structures of cholesterol-based cationic monomeric lipid and gemini lipids possessing hydroxyethyl group on the headgroup synthesized. Chapter 3: Bilayer Membrane and Stable Monolayer Forming Properties of Cationic Pseudoglyceryl Gemini Lipids having Polymethylene Spacers and Oxyethylene Linkages This chapter describes the synthesis of five new cationic pseudoglyceryl gemini lipid versions of their monomeric counterpart (Figure 3). Each cationic lipid aggregate in aqueous media was found to form vesicular structures as evidenced from the negatively stained TEM experiments and DLS measurements. XRD experiments with their cast films of aqueous dispersions revealed that introduction of the polymethylene -(CH2)n-spacer chain joining the two monomers decreased the bilayer widths of the gemini lipid aggregates. The inter-lipidic packing and the hydration of the lipid vesicles were examined using fluorescence anisotropy and generalized polarization measurements using membrane-soluble probes, DPH and Paldan respectively. Fluorescence anisotropy measurements showed that the aggregates of lipid 2c with -(CH2)5- spacer chain were highly packed and ordered in the vesicular aggregates than that of the other cationic lipid aggregates in the series. Paldan hydration studies showed that incorporation of the polymethylene -(CH2)n- spacer chains joining two monomeric units lowered the hydration of the gemini lipid aggregates in the solid gel state. Each of these cationic lipid aggregates showed sharp transition temperatures (Tm) as observed from differential scanning calorimetric studies. DSC studies further revealed that the incorporation of oxyethylene group at the linker region of cationic pseudoglyceryl gemini lipid 2a with (CH2)3- spacer chain length lowered the thermotropic phase transition temperature (Tm) of the aggregates in aqueous media when compared with the corresponding gemini analogue without oxyethylene linkages. Langmuir film balance studies showed that each cationic gemini lipid and their monomeric counterpart were able to form stable monolayers at the air-water interphase. We observed that the mean molecular area (collapse area) of each of the cationic lipid obtained from the Langmuir monolayer studies increased with increase in the spacer chain lengths. Figure 3. Molecular structures of the cationic pseudoglyceryl gemini lipids and their monomeric counterpart. Chapter 4: Vesicle and Stable Monolayer Formation from Simple ‘Click’ Chemistry Adducts in Water This chapter describes successful use of Cu(I) catalyzed “Click Chemistry” for the syntheses of a series of hitherto unknown amphiphilic adducts (M1, M2, D1 and T1) which on dispersal in water afforded vesicular aggregates as evidenced from dye entrapment, TEM, SEM, AFM and DLS studies (Figure 4). Figure 4. Molecular structures of triazole based adducts. XRD experiments with their cast films of aqueous suspensions indicate the formation of a tilted bilayer arrangement for the aggregates of M1 whereas regular bilayer structures are predominant for the aggregates derived from M2, D1 and T1. Measurement of pKa values using UV-Vis spectroscopy showed that the aggregates of monomeric click adducts (M1 and M2) possess less pKa value than that of the aggregates of dimeric (D1) and tetrameric (T1) analogues and the values lie within the range of 2.8-3.2. The hydrodynamic diameter of the aggregates of each click adduct increased with decrease in the pH of the media. Thus, the protonation of the triazole groups in the aggregates of each click adduct increased the hydrodynamic diameter. Dye entrapment studies showed that each click chemistry based adduct formed closed vesicular aggregates with inner aqueous compartment in aqueous media. The temperature induced order-to-disorder transitions of the aggregates and the accompanying changes in hydration were examined using high sensitive DSC, fluorescence anisotropy and generalized polarization measurements using a membranesoluble probe, DPH and Paldan respectively. In the solid state, M1 remains as the most hydrated species whereas in the fluidized phase, D1 maintains as the most hydrated aggregate. Clearly simple variation in the adduct molecular architecture bring about significant changes in their packing in aggregates and also the hydration of the resulting vesicles. Langmuir monolayer studies confirmed that these click adducts do form stable monolayers as well on water subphase at the air-water interface. We also calculated the mean molecular areas from the Langmuir monolayer studies and as perhaps expected the adduct T1 has the highest head group area. Thus click chemistry based simple triazole adducts, which can be very easily prepared, are good candidates for further investigations involving syntheses of novel self-assembling structures. Chapter 5: Lipid Mediated Synthesis of Silver Nanoparticles, their Physical Characterizations and DNA Binding Abilities In this chapter, work on the Ag-NP (silver nanoparticle) loaded liposomes preparation using four cationic lipids (1-4) in which the Ag-NPs were entrapped within lipid bilayer has been described. A novel method was developed to synthesize the Ag-NPs where the lipid itself capped and stabilized the Ag-NPs. Consequently there was no need of inclusion of any other capping agents like citrate. Confocal microscopy confirmed that these Ag-nanoparticles are fluorescent in character. It was also demonstrated that silver nanoparticles are indeed entrapped in lipid bilayer with transmission electron microscopy (TEM). DLS experiments provided information about the hydrodynamic diameter of the lipid vesicles which increased with the increase in Ag concentrations. This could be due to the ‘loosening’ of the lipid packing in vesicles. Zeta potential measurements showed that the zeta potential value decreased with the increase in the concentration of Ag-NPs in the cationic lipid vesicles. XRD studies with the cast films of the lipid or Ag-NP loaded lipid suspensions revealed that when the Ag-NPs get entrapped into the bilayer of the multilamellar vesicles of the lipid in the aqueous media, the unit bilayer thickness of the aggregates increased. Paldan experiments showed that with the incorporation of Ag-NPs in the lipid vesicles, the hydration of the lipid vesicles increased to a significant extent but the phase transition temperatures remained practically unaltered for all the lipids. Fluorescence anisotropy experiments revealed that the hydrocarbon chain packing of the lipid vesicles ‘loosens’ with the incorporation of Ag-NPs. Ag-NP loaded liposomes showed enhanced DNA binding ability and also the presence of Ag-NPs in cationic liposomes induced the release of DNA from silver nanoparticle-loaded lipoplexes more effectively. Figure 5. Molecular structures of the cationic lipids mentioned in the present chapter. Chapter 6: Dependence of Spacer Chain Lengths in the Synthesis of Ag-Nanorods in Gemini Cationic Surfactant Micelles Figure 6. Chemical structures of cationic gemini surfactants. This chapter describes the synthesis of Ag-nanospecies by seed-mediated wet synthesis method using four gemini surfactants (16-2-16, 16-4-16, 16-5-16 and 16-1216) as the capping agents (Figure 6). For this, we first synthesized Ag-nanoseeds of diameter ~7 nm stabilized by trisodium citrate (as capping agent). Then the solution containing Ag-nanoseeds was used to synthesize Ag-nanorods of different aspect ratios. It was that with decreasing Ag-nanoseed concentration, the aspect ratios of Agnanorods stabilized by gemini surfactants (16-2-16 and 16-4-16) increased gradually as evidenced from TEM images. These Ag-nanoseeds and Ag-nanorods were further characterized using UV-Vis spectroscopy (to know the surface plasmon bands), transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDAX) and X-ray diffraction (XRD). It was observed that when gemini surfactant 164-16 was used to stabilize Ag-nanorods, the λmax of the longitudinal band shifted more towards the red region (red-shift) as observed by UV-Vis spectroscopy when compared to that of gemini surfactant with shortest spacer, 16-2-16. Thus the gemini surfactants with shorter -(CH2)2- and -(CH2)4- spacer chains promoted the growth of Ag-nanorods in their micellar solutions whereas -(CH2)5- and -(CH2)12- spacer chains of gemini surfactants did not. So, the growth of Ag-nanorods in micellar solutions is found to be highly spacer-chain length specific. TEM micrographs revealed that the aspect ratios of Ag-nanorods stabilized by gemini surfactants 16-4-16 are larger than those compared to the Ag-nanorods stabilized by gemini surfactants 16-2-16 at a particular amount of Agnanoseed solution. TEM images of the samples containing micellar solutions of gemini surfactants 16-5-16 and 16-12-16 showed that the formation of only Ag-nanoparticles of larger sizes (compared to Ag-nanoseeds stabilized by trisodium citrate) and Agnanoprisms irrespective of the amount of Ag-nanoseed solution added. No Ag-nanorod formation in the micellar solutions of gemini surfactants 16-5-16 and 16-12-16 was observed. Gemini surfactants (16-2-16 and 16-4-16) formed bilayer arrangements to facilitate the growth and stabilization of Ag-nanorods in aqueous media where the inner layer is attached to the Ag-nanorod surface through the gemini surfactant ammonium headgroups. X-ray diffraction (XRD) studies showed that these Ag-nanorods stabilized by gemini surfactants 16-2-16 and 16-4-16 crystallized in the aqueous media via (111), (220) and (222) lattice faces. Thus this study demonstrated the way one can control structures and shapes of the silver nanoobjects using gemini surfactant micelles. (For structural formula pl refer the thesis)

La grande majorité des essais cliniques de transfert de gènes in vivo utilise des vecteurs viraux. Si ces derniers sont efficaces, ils présentent des risques immunogènes, toxiques, voire mutagènes avérés. Les vecteurs synthétiques (non viraux), par leur grande modularité et leur faible toxicité représentent une alternative très prometteuse. Le principal frein à leur utilisation est leur manque d’efficacité. L’objectif majeur de ce travail de thèse a été de comprendre le mécanisme de transfert de gènes associé à différents complexes vecteurs synthétiques/ADN plasmidique, ce qui est indispensable pour une conception rationnelle de nouveaux vecteurs. Nous avons étudié, sur cellules en culture, le mécanisme de transfert de gènes associé à deux lipides cationiques ; le BGTC (bis(guanidinium)-tren-cholesterol) et la DOSP (DiOleylamine A-Succinyl-Paromomycine) qui sont connus pour être des vecteurs efficaces in vitro. Nous avons ainsi pu visualiser par microscopie électronique leurs voies d’entrée, leurs remaniements structuraux ainsi que leur échappement endosomal qui représente une étape clé du processus de transfert de gènes. L’identification non ambigüe des lipoplexes tout au long de leur trafic intracellulaire a été rendue possible grâce au marquage de l’ADN par des nanoparticules de silice dotées d’un cœur de maghémite (Fe2O3) dense aux électrons. Cette stratégie de marquage a également été appliquée à l’étude du mécanisme d’action d’un autre vecteur synthétique de type polymère, le copolymère à blocs non ionique P188 ou Lutrol. Contrairement à la plupart des vecteurs synthétiques, celui-ci présente une efficacité de transfection in vivo chez la souris par injection in situ pour le tissu musculaire ou en intra trachéale dans le poumon. En revanche, il est totalement inefficace in vitro. Nous avons montré que le Lutrol permet une augmentation de l’internalisation d’ADN par les cellules mais n’induit pas son échappement endosomal, ce qui expliquerait son absence d’efficacité in vitro. D’autres voies d’entrée sont alors à envisager in vivo pour comprendre son mécanisme d’action. / The vast majority of clinical trials of gene transfer in vivo use viral vectors. Although they are effective, they induce immunogenic, toxic or mutagenic risks. Due to their high modularity and low toxicity, synthetic vectors (non viral), represent a promising alternative despite their lack of effectiveness. The major objective of this work was to understand the mechanism of gene transfer using two prototypic synthetic vectors, in the context of a rational design of new vectors. We studied on cultured cells, the mechanism of action of two cationic lipids; BGTC (bis(guanidinium)-tren-cholesterol) and DOSP (DiOleylamine A-Succinyl-Paromomycine) formulated with plasmid DNA (lipoplexes) which are in vitro efficient vectors. We have been able to visualize by electron microscopy, their intracellular pathways, their structural alterations and their endosomal escape, the latter being a key step in the process of gene transfer. The unambiguous identification of lipoplexes throughout their intracellular trafficking has been made possible thanks to the labelling of DNA by core-shell silica nanoparticles with an electron dense maghemite core (Fe2O3). The labeling strategy has also been applied to study the mechanism of action of a nonionic block copolymer (P188 or Lutrol). Interestingly, these synthetic vectors have an in vivo transfection efficiency in mice lung and muscle tissue while they are totally inefficient in vitro. We have shown that Lutrol induces an increase of DNA internalization into cells and fails to trigger endosomal escape, which would explain the lack of in vitro efficacy. These findings suggest that the in vivo mechanism of action of Lutrol would involve other internalization pathways.

Bei der Hornhauttransplantation (Keratoplastik) handelt es sich um die häufigste Transplantation humanen Gewebes. Das Hornhautendothel, eine empfindliche Zellschicht auf der Innenseite der Kornea, verfügt nicht über die Fähigkeit, Zellverluste durch Proliferation auszugleichen. Zwei grundsätzliche Probleme ergeben sich aus dieser Eigenschaft des kornealen Endothels für die Keratoplastik: ein Endothelzellverlust tritt zum einen während der Hornhautkonservierung vor Keratoplastik auf und führt zu einem Ausschluss vieler Transplantate, zum anderen ist er oftmals im Zuge von immunvermittelten Abstoßungsreaktionen oder in Folge chronischer Vorgänge nach Keratoplastik zu beobachten. Aus diesem Grund ist eine möglichst hohe Endothelzelldichte auf kornealen Transplantaten eine Voraussetzung für den Erfolg einer Keratoplastik. Das erste Ziel dieser Arbeit war deshalb die Übertragung des Gens für den aziden FGF (aFGF) in korneale Endothelzellen mit Hilfe des nicht-viralen Gentransfers. Dazu wurden verschiedene Lipid-Formulationen für den Gentransfer in humane korneale Endothelzellen in vitro optimiert. Der Einsatz von DAC-30 und Lipofectin für den aFGF-Gentransfer führte zu einer deutlichen Proliferationssteigerung (um ca. 50 %) der Zellen, womit der Einsatz dieses Wachstumsfaktors eine gute Möglichkeit darzustellen scheint, die prä-operative Ausgangssituation kornealer Transplantate zu verbessern. Der zweite Teil dieser Arbeit befasste sich mit Untersuchungen zur Immunmodulation nach Keratoplastik. Eine Hauptrolle bei der Abstoßung kornealer Transplantate spielen CD4+ T- Lymphozyten. In anderen Transplantationsmodellen konnte durch die lokale Überexpression immunmodulatorischer Zytokine die Entstehung und Aktivierung dieser Zellen gehemmt und eine verlängerte Transplantatüberlebenszeit erzielt werden. Mit Hilfe gentherapeutischer Vektoren (Adenoviren, Liposomen) wurden die immunmodulatorischen Zytokine vIL-10 und rIL-4 ex vivo in korneale Transplantate eingebracht. Nach erfolgreichen in vitro- Untersuchungen zur Genexpression wurden die transduzierten/transfizierten Hornhäute in einem starken Abstoßungsmodell der Ratte transplantiert, was jedoch zu keiner signifikanten Verlängerung der Transplantatüberlebenszeit führte. Diese Arbeit liefert Hinweise darauf, dass der lokale Gentransfer von vIL-10 in der Keratoplastik nicht für eine Immunmodulation geeignet ist, und dass sowohl die Zytokindosis, als auch der Zeitpunkt und der Ort der Vektorapplikation eine entscheidende Rolle für den Transplantationserfolg spielen. / Keratoplasty is the most common transplantation of human tissue. The corneal endothelium constitutes a damagable cell layer on the inner surface of the cornea, unable to proliferate. From this, two general problems arise for the outcome of keratoplasty: loss of corneal endothelial cells occurs on the one hand during corneal long time storage before keratoplasty and enforces the lack of donor tissue, on the other hand it is often correlated with immune mediated rejections as well as with chronic processes after keratoplasty. Therefore an endothelial cell number as high as possible on corneal grafts displays a requirement for successful keratoplasties. The first aim of this study was non-viral gene transfer of the aFGF (acidic FGF) gene in corneal endothelial cells. Different lipid formulations were optimized for gene transfer in human corneal endothelial cells in vitro. Application of DAC-30 and Lipofectin for aFGF gene transfer clearly showed a stimulating effect on cell proliferation (approximately 50 %). Thus, the use of aFGF seems to be a good possibility for improving the pre-operative situation of corneal allografts. The second part of this study deals with the immune modulation after keratoplasty. CD4+ T- lymphocytes play a key role in rejection processes after keratoplasty. Local over expression of immunomodulatory cytokines in different transplantation models could inhibit the development and activation of these cells and was able to prolong the allograft survival time. Using different gene therapeutic vectors (adenoviruses, liposomes) the immunomodulatory cytokines vIL-10 and rIL-4 were transferred ex vivo in corneal allografts. After successfully determined gene expression in vitro, the transduced/transfected corneal allografts were transplanted in a strong rejection model of the rat. However, this was not sufficient in prolonging the graft survival time significantly. This study provides indications, that local gene transfer of vIL-10 in keratoplasty is not suitable for an immune modulation, and that both cytokine dose as well as time point and site of vector application play an important role for successful transplantation.

The thesis entitled “The Role of Liposomal Hybrids and Gold Nanoparticles in the Efficacious Transport of Nucleic Acids and Small Molecular Drugs for Cancer Nanomedicine” elucidates the preparation of various liposomal formulations of cationic monomeric and gemini lipids where hydrophobic domains were consisted of tocopherol, cholesterol and pseudoglyceryl backbone for the cellular transport of nucleic acids. The thesis continues while elucidating the role of various pH sensitive molecules and gold nanoparticles in liposomes to improve the delivery efficacy levels. This thesis also elucidates the role of gold nanoparticles stabilized with natural pH sensitive molecules for efficacious drug delivery applications. Additionally, the role of such pH sensitive gold nanoparticles in association with liposomes for the co-delivery of drug and gene has been discussed. The work has been divided into six chapters. Chapter 1A: Dimeric Lipids Derived from α-Tocopherol as Efficient Gene Transfection Agents. Mechanistic Insights into Lipoplex Internalization and Therapeutic Induction of Apoptotic Activity In this chapter, we present cationic dimeric (gemini) lipids for significant plasmid DNA (pDNA) delivery to different cell lines without any marked toxicity in the presence of serum. The six gemini lipids possess α-tocopherol as their hydrophobic backbone and differ from each other in terms of their spacer chain lengths. Each of these gemini lipids mixed with a helper lipid 1, 2-dioleoyl phosphatidyl ethanolamine (DOPE), was capable of forming stable aqueous suspensions. These co-liposomal systems were examined for their potential to transfect pEGFP-C3 plasmid DNA in to nine cell lines of different origins. The transfection efficacies noticed in terms of EGFP expression levels using flow cytometry were well corroborated using independent fluorescence microscopy studies. Significant EGFP expression levels were reported using the gemini co-liposomes which counted significantly better than one well known commercial formulation lipofectamine 2000 (L2K). Transfection efficacies were also analyzed in terms of the degree of intracellular delivery of labeled plasmid DNA (pDNA) using confocal microscopy which revealed an efficient internalization in the presence of serum. The cell viability assays performed using optimized formulations demonstrated no significant toxicity towards any of the cell lines used in the study. We also had a look at the lipoplex internalization pathway to profile the uptake characteristics. A caveolae/lipid raft route was attributed to their excellent gene transfection capabilities. The study was further advanced by using a therapeutic p53-EGFP-C3 plasmid and the apoptotic activity was observed using FACS and growth inhibition assay. Figure 1. The co-liposomes of tocopheryl gemini lipids and DOPE for efficient delivery of p53-EGFP-C3 plasmid DNA that induces significant apoptotic response. Chapter 1B: Efficacious Gene Silencing in Serum and Significant Apoptotic Activity Induction by Survivin Downregulation Mediated by Cationic Gemini Tocopheryl Lipids Non-viral gene delivery offers cationic liposomes as promising instruments for the delivery of double-stranded RNA (ds RNA) molecules for successful sequence-specific gene silencing (RNA interference). The efficient delivery of siRNA (small interfering RNA) to cells while avoiding the unexpected side effects is an important prerequisite for the exploitation of the power of this excellent tool. We discuss in this chapter about six tocopherol based cationic gemini lipids, which induce substantial gene knockdown without any obvious cytotoxicity. All the efficient co-liposomal formulations derived from each of these geminis and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE) were well characterized using physical methods such as atomic force microscopy (AFM) and dynamic light scattering (DLS). Zeta potential measurements were conducted to estimate the surface charge of these formulations. Flow cytometric analysis showed that the optimized co-liposomal formulations could transfect anti-GFP siRNA efficiently in three different GFP expressing cell lines, viz. HEK 293T, HeLa and Caco-2 significantly better than a potent commercial standard Lipofectamine 2000 (L2K) both in the absence and presence of serum (FBS). Notably, the knockdown activity of co-liposomes of gemini lipids was not affected even in the presence of serum (10% and 50% FBS) while it dropped down for L2K significantly. Observations under a fluorescence microscope, RT-PCR and western blot analysis substantiated the flow cytometry results. The efficient cellular entry of labeled siRNA in GFP expressing cells as evidenced from confocal microscopy put forward these gemini lipids among the potent lipidic carriers for siRNA. The efficient transfection capabilities were also profiled in a more relevant fashion while performing siRNA transfections against survivin (an anti-apoptotic protein) which induced substantial apoptosis. Furthermore, the survivin downregulation improved the therapeutic efficacy levels of an anticancer drug, doxorubicin significantly. In short, the new tocopherol based gemini lipids appear to be highly promising for achieving siRNA mediated gene knockdown in various cell lines. Figure 2. The co-liposomes of tocopheryl gemini lipids and DOPE for efficient delivery of siRNA against survivin that induces significant apoptotic response. Chapter 2: Efficacious in Vitro EGFP Expression and Silencing in Serum by Cationic Pseudoglyceryl Gemini Lipids To elicit the desirable efficacy levels in cationic liposome mediated nucleic acid therapeutics has been part of extensive scientific efforts. This chapter describes three cationic gemini lipids and application of their co-liposomes with DOPE as potent pDNA (plasmid DNA) and siRNA (small interfering RNA) cytofectins for remarkably advanced efficacy levels in numerous cell lines in the presence of serum. The hydrophobic structural lineament of cationic gemini lipids is made up of pseudoglyceryl backbone linked to the hydrocarbon chains via oligo-oxyethylene units. The stable aqueous co-liposomal suspensions of gemini lipids showed an efficient binding to pDNA or siRNA and their significant intracellular delivery in various cell lines. The transfection capabilities of different co-liposomal formulations were profiled based on EGFP expression (pEGFP-C3 pDNA transfection) and EGFP knockdown (anti-GFP siRNA transfections) in EGFP expressing cell lines. The cellular EGFP expression levels and intracellular delivery of labeled nucleic acids were thoroughly studied using flow cytometry (FACS), fluorescence and confocal microscopy. The MTT based cell viability assay revealed no loss in cell viabilities for all of the transfection optimized lipoplexes of siRNA or pDNA. The transfection profile of gemini co-liposomes was noted to be significantly much better than a commercial lipofection reagent, Lipofectamine 2000 used for pDNA and siRNA applications in each of the cell lines studied. The co-liposomes and their transfection optimized lipoplexes were physiochemically characterized extensively by means of zeta potential, dynamic light scattering (DLS) and atomic force microscopy (AFM). In brief, these new gemini co-liposomal formulations seem to offer a great opportunity for successful nucleic acid (DNA and siRNA) delivery in a practical scenario. Figure 3. Efficacious EGFP expression (pDNA transfection) and EGFP silencing (anti GFP siRNA transfection) mediated by co-liposomes of pseudoglyceryl gemini lipids and DOPE. Chapter 3: Efficient Elicitation of Liposomal Nucleic acid delivery through the Eminence of Gold Nanoparticles Stabilized with pH Responsive Short Tripeptide Derived from Tyrosine Kinase NGF Receptors The prerequisite in the area of gene therapy today is to serve transfection efficient formulations nullifying the enduring key issues. To this end, we discuss in this chapter, the role of hybrid liposomal formulations derived from structurally distinct cationic lipids, a neutral lipid (DOPE) and pH responsive short tripeptide (KFG, Lys-Phe-Gly) capped gold nanoparticles (PAuNPs). The hybrid liposomes are presented to be efficient enough to transfect pDNA leading to remarkably high gene expression levels in various cell lines of different origins in the presence of serum (FBS). Hybrid liposomes could deliver pDNA more effectively than the native liposomes and commercial standard lipofectamine 2000 (L2K) across the entire range of N/P ratios studied under the influence of intracellular pH response and gold nanoparticles prominence. The gene transfection capabilities are profiled based on transfections performed using two different plasmids (pGL3, luciferase activity and p-EGFP-C3, green fluorescent protein expression). pDNA cellular internalization and subsequent gene expression levels are studied using flow cytometry, fluorescence microscopy and confocal microscopic studies. The extensive physiochemical characterization of hybrid liposomal formulation and their complexes with pDNA in comparison with respective native liposomes was performed using AFM, TEM, Zeta, DLS, gel retardation assay, U.V. and fluorescence emission measurements. The hybrid liposomes are shown to possess significantly higher fusion activity at lowered pH of intracellular compartments. These hybrid liposomes are fairly biocompatible across the concentration range used in transfection experiments. Precisely, introduction of these pH responsive tripeptide capped gold nanoparticles in to liposomal formulations straightforwardly must be more advantageous for a practical application in biomedical scenario to achieve therapeutic levels. Figure 4. The hybrid of liposomes and tri-peptide capped gold nanoparticles for significantly improved gene expression levels. Chapter 4: RNA Aptamer Decorated pH Sensitive Liposomes for Active Transport of Nucleic Acids in Specific Cancer Cells This chapter describes the target specific transport of pH sensitive liposomes loaded with a RNA aptamer for promising nucleic acid therapeutics. The pH sensitive liposomes are constructed from a cationic cholesteryl gemini lipid (CGL), neutral helper lipid (DOPE) and gemini analog of a pH sensitive lipid, palmitoyl homocysteine (GPHC). The liposomes are shown to be significantly fusogenic that deliver the cargoes upon lowerin the pH (6.0). The fusogenic behaviour of the liposomes was thoroughly studied by means of dynamic light scattering (DLS), zeta potential, lipid mixing, calcein dequenching and atomic force microscopy (AFM). The facile integration of cholesterol conjugated RNA aptamer in liposomes derived from cholesteryl gemini lipids was exploited for their delivery to specific cancer cells. The RNA aptamer specifically binds to epithelial cell adhesion molecule (EpCAM) with high affinity which is a cell surface marker in various solid cancers such as colorectal and breast carcinoma. These aptamer decorated pH sensitive liposomes could efficiently enter the EpCAM expressing COLO-205, Caco-2, MCF-7 and MDA-MB-231 cell lines while no such noticeable liposome transport was observed in EpCAM negative HEK 293T cells as evidenced by flow cytometry and confocal microscopy. Additionally, the liposomes are shown to be actively transported inside the cells, i.e., receptor mediated endocytosis. These liposomes could complex the nucleic acids (pDNA) in an efficient manner. The MTT based cell viability assay accounted no noticeable loss in cell viabilities for liposome treatments. Concisely, we have formulated RNA aptamer loaded pH sensitive liposomes that would certainly be promising tool in target based cancer nanomedicine. Figure 5. (A) Cellular internalization of DY-647 labeled aptamer loaded pH sensitive liposomes. (B) The liposomes were actively internalized through receptor mediated endocytosis. Each panel (A and B) represents (from left to right) bright field image, aptamer fluorescence, DAPI stained nuclei and merge of previous three impressions. Chapter 5: Natural Tri-peptide Capped Gold Nanoparticles for Efficacious Doxorubicin Delivery in Vitro and in Vivo Nanotechnology has gained ever increasing interest for the successful implementation of chemotherapy based treatment of cancer. This chapter describes the role of gold nanoparticles (AuNPs) capped with a natural pH responsive short tri-peptide (Lys-Phe–Gly or KFG) for significant intracellular delivery of an anti-cancer drug, doxorubicin (DOX). A significantly increased apoptotic response was noted for DOX treatments mediated by KFG-AuNPs in comparison with drug alone treatments in various cell lines (BT-474, HeLa, HEK 293T and U251) in vitro. Furthermore, KFG-AuNPs mediated DOX treatment significantly decreased cell proliferation and tumor growth in BT-474 cell xenograft model in nude mice. In addition, KFG-AuNPs showed efficacious drug delivery in DOX-resistant HeLa cells (HeLa-DOXR) in comparison with drug alone treatments. Figure 6. Representative images of excised tumors after doxorubicin treatment mediated by pH responsive tri-peptide capped gold nanoparticles (DOX-KFG-AuNPs) (C) in comparison with doxorubicin alone treatments (B) and untreated tumors (A). Extensive cell death as observed under Hematoxylin/eosin (H&E) (D) and TUNEL (E) staining of DOX-KFG-AuNPs treated tumor sections. Chapter 6: Significant Apoptotic Activity Induction by Efficacious Co-delivery of p53 Gene and Doxorubicin Mediated by the Combination of Co-liposomes of Cationic Gemini lipid and pH Responsive Tri-peptide Combining chemotherapy with gene therapy has appeared as an efficient tool to treat complex biological disorder like cancer. Herein, we show efficient co-delivery of DNA and an anti-cancer drug, doxorubicin (DOX) by means of gemini cationic liposome (GCL) based lipoplex nanoaggregates that are coated with DOX encapsulated pH responsive tripeptide nanovesicles. The lipoplex, tripeptide vesicles and their association was thoroughly studied using dynamic light scattering (DLS), zeta potential, atomic force microscopy (AFM). Flow cytometry, fluorescence and confocal microscopic analysis revealed that the GCL-tripeptide association could significantly co-deliver the p53 expression plasmid (p53-EGFP-C3) and DOX in HeLa and HEK 293T cells in the presence of serum. A synergistic increase in gene expression level and DOX internalization was observed in co-delivery which was even substantially higher than individual lipoplex transfection and DOX treatment. The apoptosis induced due to p53 expression and DOX was profiled with the help of annexin-V positivity analysis under flow cytometry and nuclear damage analysis by DAPI nuclei counterstaining under confocal microscopy which noted to be significantly higher in cells during co-delivery. The MTT based cell viability assay revealed a significantly increased loss in cell viability counts for co-delivery treatments. Such a system delivering synergistically increased significant efficacy levels in combinatorial drug and nucleic acid therapeutics would be certainly advantageous for practical biomedical applications. Figure 7. The co-delivery of pDNA and drug (doxorubicin) mediated by GCL-tripeptide association as observed under (A) confocal microscopy (pDNA; green and doxorubicin; red) and (B) flow cytometry.

The biggest hurdle in success of gene and drug therapy is designing and preparation of suitable bio-nanomaterials to carry the desired nucleic acid and drug to the targeted site. The work described in the present thesis encompasses two different approaches for the delivery of bioactive oligo/polynucleotides and drugs in vitro and in vivo using either cationic lipids or their nanocomposites with different carbon nanomaterials. The idea of using carriers for oligo/polynucleotides and drugs came into existence because of numerous physiological barriers in pathway of delivery of naked oligo/polynucleotides or drugs which reduces the overall activity of these bioactives in biological systems. These barriers trigger scientific research toward the preparation of appropriate biomaterials which can overcome the physiological barriers and improve the activity of bioactive oligo/polynucleotides and drugs in cellular systems. Toward this end, the design and synthesis of different cationic lipids and carbon nanomaterials were undertaken as described in seven chapters of the thesis. A series of novel cationic lipids with structural variability was prepared and used for gene delivery in vitro. They were further tuned chemically to sustain delivery efficiency in high serum percentage during in vitro transfection. These serum compatible lipids were used to perform transfection of reporter gene plasmid and found to be more efficient compared to the some well known commercial products for the same purpose. Another series of novel lipids were synthesized for the targeted gene delivery in vitro. These tryptophan based cholesteryl lipids were used to prepare mixed liposomes. These mixed liposomes were highly efficient in targeting sigma receptor rich HEK293T over sigma receptor negative HeLa cells. Mixed liposomes were also prepared for selective targeting of αvβ3 and αvβ5 integrins in gene transfection protocol using a palmitoyl-RAFT-RGD4 template. A mixed liposomal formulation was developed to carry out anti-sense siRNA mediated knockdown of Smad-2 protein with better efficiency compared to some of the best known commercial products for the same purpose. These mixed liposomes were also highly efficient for regression via induction of p53 mediated apoptosis in xenograft tumors developed in nude mice. Carbon nanomaterials have been extensively explored as nanoscale gene/drug carriers for potential applications. But the challenge is to solubilize these highly hydrophobic materials in aqueous medium for use in biological systems. Although there are reports for covalent modifications of such nanomaterials but it could be done only with the loss of some beneficial features of these materials. Herein a non-covalent technique has been efficiently used to suspend single walled carbon nanotubes in water using biocompatible cationic lipids. These nanosuspensions were used to complex plasmid DNA and transfect them in vitro. They proved to be highly serum compatible DNA carriers which did not drop the efficiency even in very high percentage of serum. Similarly exfoliated graphene was modified with cationic lipid and serum components to improve IC50 of Tamoxifen citrate and Methotrexate to a considerable extent in vitro. The improved Methotrexate formulations were highly efficient for regression in size of xenograft tumors developed in nude mice. Thus, the present thesis entails generation of cationic lipids and carbon nanomaterials based nanocomposites which were not only highly biocompatible themselves but their efficiency was found many fold better compare to some of the best commercial delivery agents. These were useful for the delivery of various bioactive oligo/polynucleotides and drugs in vitro and in vivo.