The effect of proteolytic enzymes on the transformation and transfection of Streptococcus lactis LM2301 protoplasts was examined in an attempt to eliminate the variability observed. By using both chymotrypsin and mutanolysin to form protoplasts followed by spread plating, consistent frequencies of 104 to 105 transformants per μg of pGB301 DNA, and 105 transfectants per μg of c2 bacteriophage DNA where achieved. Optimum transformation and transfection frequencies were obtained when 16 h cultures were treated simultaneously with 25 U/ml mutanolysin and 1.25 U/ml chymotrypsin for 15 min. Trypsin and pronase in conjunction with mutanolysin also increased transformation frequencies higher than when mutanolysin was used alone, but pronase was not as effective as chymotrypsin or trypsin. These results may explain the variability in transformation of mutanolysin-treated cells of S. lactis since commercial sources of mutanolysin contain varying amounts of proteolytic enzyme activity. Transformation of Streptococcus cremoris CS224 at low frequency (5 transformants per μg of pGB301 DNA) was achieved. Plasmid pGB301 was able to replicate and express antibiotic resistance in the resultant transformant (designated S. cremoris SW301). The presence of pGB301 in S. cremoris SW301 was confirmed by agarose gel electrophoresis.

Gene therapy is the process of delivering genetic material, such as DNA (encoding for an important protein) into a patient’s cells in order to treat a particular disease such as a genetic disorder or heart disease. This process of DNA delivery into cells is known as “transfection” and it is important that the efficiency of transfection be optimized such that a patient can obtain maximum therapeutic benefit from such a treatment. DNA is susceptible to being destroyed by harsh physiological environments prior to reaching its target. This problem can be diminished with the use of vectors that not only protect against harsh conditions but also encourage entry into cells. By mixing 1) DNA with 2) positively charged polymers, “polyplexes” form which protect DNA from degradation and increase transfection efficiency. The development of effective polyplex formulations requires optimization. In the work presented here, it was discovered that when polyplexes contained specific sequences within the DNA called “CpG”, this lowered transfection efficiencies and increased inflammatory responses compared to DNA without CpG, as measured using a mouse lungs model. Thus, DNA composition played an important role in influencing DNA transfection efficiency of polyplexes. Another aspect to take into account is the degree of positive charge of the polymer. We tested a new polymer called poly(galactaramidoamine) or PGAA. We found that this PGAA can form polyplexes with DNA and could be used in gene therapy. At the present time, mechanisms by which the polyplexes get inside and transfect the cells are still unclear. We also introduced a new system called high-content screening to the gene delivery field. This system offers automated measurements of transfection efficiency and cytotoxicity and could be used to reveal the polyplexes trafficking inside cells.

Cytosolic double stranded DNA (dsDNA) is sensed as a “danger signal” by host cells. Detection of viral and bacterial nucleic acid is emerging as a major route for cells to identify an infection by a pathogen. Recognition of cytoplasmic DNA causes death of some cells and interferon (IFN) and cytokine induction, which are appropriate anti-viral responses. Responses to cytoplasmic DNA may not only be relevant to certain retrovirus, DNA virus and bacterial infections, but could also be generated by reverse transcription of endogenous retro-elements. Introduction of DNA into the cytoplasm of bone marrow derived macrophages (BMM) causes upregulation of MHC Class I, induction of IFNβ and other cytokines and cell death. Both cytokine induction and cell death were independent of recognition of “CpG motifs” through TLR9. In order to determine whether a single receptor was likely to mediate these responses, the types of DNA eliciting these responses was compared. Both cellular activation to produce cytokines and IFNβ, as well as cell death were seen only with dsDNA but not single stranded DNA (ssDNA). Both responses increased with increasing DNA length, with little detectable effect of a double stranded 22bp oligonucleotide (ODN). The sequences of DNA leading to optimal induction of IFNβ and death were different. Although all dsDNA induced death of primary macrophages, poly(dA):(dT) was a particularly potent and rapid pro-death stimulus. In contrast, poly(dA):(dT) was a relatively poor stimulus for IFNβ, even at doses which were minimally toxic, or in cells which are resistant to DNA induced cell death. The alternating co-polymer poly(dA-dT) was the most potent inducer of IFNβ. This data suggests that separate DNA receptors mediate cell death and IFNβ induction in response to dsDNA Transfected dsDNA also rapidly activated caspase 3, a classical pro-apoptotic caspase, in BMM as early as 2½ minutes post-transfection with DNA. Caspase 3 is an effector caspase which is activated by an upstream initiator caspase. Although the apical caspase in the DNA detection system has not been defined, use of Bcl2 overexpressing BMM and caspase 2-/- BMM showed that DNA-dependent caspase 3 activation did not occur via the mitochondrial damage or the caspase 2 activation pathways. The inflammatory caspase, caspase 1 was also activated in response to DNA transfection, although whether caspase 1 is responsible for cleavage of caspase 3 has not been established. Caspase 1 activation suggests the involvement of the inflammasome, which is important for processing pro-inflammatory cytokines such as IL-1β into their biologically active forms. Furthermore, there is recent evidence suggesting that DNA-transfected cells die by a caspase 1-dependent cell death called pyroptosis. Other work in our lab identified the HIN-200 family member and candidate lupus susceptibility factor p202 as a candidate receptor for cytoplasmic dsDNA; p202 bound stably and rapidly to transfected DNA. Here, knockdown studies revealed p202 to be a regulatory protein limiting DNA-induced caspase 1 and 3 activation. Conversely, the related pyrin domain-containing HIN-200 factor AIM2 (p210), a candidate tumour suppressor, was required for caspase 1 and 3 activation by cytoplasmic dsDNA. Recently published work suggests that AIM2 multimerises along the length of the DNA leading to the formation of an inflammasome complex. The pyrin domain of AIM2 recruits the adaptor protein ASC through homotypic pyrin domain interactions. ASC subsequently recruits caspase 1, which results in its auto-activation. The inhibitory effect of p202 on caspase activation is likely to be due to its lack of a pyrin signalling domain. p202 rapidly binds to cytoplasmic DNA, and may reduce the clustering of AIM2 pyrin domains which results in caspase activation. Consistent with this proposal, DNA-dependent caspase activation correlated inversely with p202 expresssion in 3 mouse strains. This work defines HIN-200 proteins as a new class of pattern recognition receptors mediating responses to dsDNA. Work in this thesis aimed to understand the biological role and mechanism of responses to cytoplasmic DNA. Responses to cytoplasmic DNA are likely to be relevant not only to infectious disease but also to autoimmune diseases such as systemic lupus erythmatosus (SLE), where DNA appears to act as an adjuvant, and even tumour progression where there is evidence for a role for active endogenous retro-elements. In addition, responses to DNA may limit transfection efficiency and the efficacy of non-viral gene therapy.

La recherche de vecteurs non viraux pour la thérapie génique est un domaine très développé. Les systèmes cationiques montrent une forte efficacité de transfection in vitro, mais cette activité est considérablement diminuée in vivo car les complexes ADN/vecteur, du fait de leur charge globale positive, sont rapidement éliminés de la circulation sanguine. Les lipopolythiourées sont des systèmes non cationiques qui représentent une alternative aux lipides cationiques: ils permettent de réduire de moitié l'élimination précoce après une injection intraveineuse. Dans ce mémoire est décrite la synthèse d'une famille de seize lipopolythiourées dont la structure repose sur une tête polaire branchée à deux motifs thiourée. Ces lipides présentent une grande diversité au niveau de l'ancre hydrophobe, de l'espaceur, du répartiteur et des terminaisons. L'évaluation de cette famille a été réalisée de façon systématique et la recherche du mécanisme d'action a été entreprise. Ces études ont permis la mise au point de lipopolythiourées d'une formulation facile et possédant un pouvoir transfectant du même ordre de grandeur que celui des lipides cationiques.

Les microsporidies, parasites intracellulaires obligatoires, forment des spores délimitées par une paroi épaisse, qui renferment un appareil invasif original, le tube polaire à l'origine du transfert du matériel infectieux dans une cellule hôte. PTP1 et PTP2, protéines du tube polaire ont été décrites au sein du genre Encephalitozoon. Pour améliorer nos connaissances sur la composition du tube polaire, de nouvelles PTP ont été recherchées. L'identification des protéines PTP1 et PTP2 chez Antonospora locustae a été facilitée par la conservation de l'organisation des gènes (synténie) entre E. cuniculi et A. locustae. L'analyse du protéome d'E. cuniculi a permis d'identifier deux nouvelles PTP conservées chez A. locustae. Des études de co-expression chez E. coli ont montré des interactions entre PTP1 et PTP2. Chez E. hellem, les séquences complètes de 2 protéines localisées au niveau de l'exospore ont été caractérisées. Enfin, des essais de transfection d'E. cuniculi ont été réalisés

<p>T lymphocytes can be rendered specific against a wide range of antigens by the genetic transfer of a chimeric receptor, a fusion between the antigen-binding domain of an antibody and the signalling domain of a T cell receptor. The use of such chimeric T lymphocytes has shown promising results for cancer therapy. Previous experiments in our laboratory have shown low rates of gene transfer using retroviral vectors. In this study, investigations have been done to increase the number of genetically modified cells. Different enhancers such as PLL and polybrene have previously been used in combination with retroviral transduction. The optimal retroviral protocol in this study showed to be the use of retrovectors produced with twice the normal concentration of the plasmids encoding env and gag-pol rather than the use of the enhancers. A 6-day pre stimulation of T lymphocytes prior transduction together with a centrifugation step increased the rate of modified cells even further. Alternative approaches of gene transfer were also investigated, including plasmid transfection and adenoviral transduction. While transfection protocols yielded low numbers of modified cells, adenoviral vectors showed the highest rate of gene transfer.</p> / <p>Cancer är den sjukdom som idag, efter hjärt-kärl-sjukdomar, kräver flest dödsfall i i-länder. Som en alternativ behandlingsmetod mot cancer pågår just nu forskning om genetiskt förbättrade immunceller, s.k. chimära T lymfocyter, skulle kunna användas för att döda tumörceller. De chimära cellerna är utrustade med en konstgjord receptor som är en fusion av en antikropp och en signalkedja. Det gör att cellerna kan riktas mot ett brett urval av cancertyper. Att få cellerna att ta upp generna som behövs för den konstgjorda receptorn har visats sig vara problematiskt. Den här studien har därför som mål att förbättra cellernas förmåga att ta upp gener. För detta har vi använt oss av retrovirus- och adenovirus-system tillsammans med försök att få cellerna att spontant ta upp generna, sk. plasmid-transfektion. Studien har visat att de båda virussystemen ger högst antal modifierade celler. Olika substanser som tidigare har visat sig förhöja graden av gentillförsel har testats, men vår studie har visat att tillverkningen av virusvektorerna har större påverkan på resultaten än någon av de olika hjälpmedlen.</p>

Gene expression knockdown using RNA interference has dramatically altered the ability to silence target genes without the need for a creation of a genetic knockout. The pitfalls surrounding successful siRNA gene expression knockdown fall in the broad category of delivery. This work focuses on the use of N-isopropyl-acrylamide conjugated polyglycerol (PGNIPAM) as a novel cationic vector of in vitro and possible in vivo delivery of siRNA. The hyper-branched structure of the PGNIPAM molecule bears a biocompatible core with cationic subunits on the surface, providing a less toxic alternative to other cationic polymers used in the past. Further PGNIPAM shows excellent binding and release characteristics over other comparable molecules and systems. Activity of the siRNA requires access to the cell cytoplasm, which in turn requires passage of the siRNA through the cell membrane and release into the internal environment with no degradation. PGNIPAM has shown the ability to traverse the endocytic pathway and release the siRNA directly into the cytoplasm where it can interact with cellular machinery. Knockdown of known oncogene survivin was observed in vitro both through mRNA expression reduction as well as through protein reduction in MDA-MB-231 human breast cancer cells. Additionally, early stage animal work with a human breast cancer model shows positive results for coupled treatment of tumors using siRNA against survivin and doxorubicin, an anticancer drug. PGNIPAM offers a safer alternative to other cationic delivery systems and has shown improvement over standard modes of knockdown from commercial products.

We used megahertz pulsed ultrasound and studied gene transfection with a human prostate cancer cell line. We first studied the compromise of cell viability and uptake efficiency and found out that increasing sonication temperature or changing US contrast agents could improve drug/gene delivery mediated by US exposure. We also found that accounting for cell debris after sonication was important to correctly determine cell viability. Next, we verified the capability of US to deliver DNA into the cell nuclei, which is necessary for successful gene transfection. Under the optimal sonication conditions, ~ 30% of cells showed DNA uptake right after US exposure and most had a portion of DNA already localized in the cell nuclei. The maximum transfection efficiency was ~ 12% at 8 h post US exposure. From the DNA perspective, ~ 30% of DNA was localized in the cell nuclei immediately after US exposure and ~ 30% was in the autophagosomes/ autophagolysosomes with the rest ¡°free¡± in the cytoplasm. At later time up to 24 h, DNA continued to be distributed ~ 30% in the nuclei and most or all of the rest in autophagosomes/autophagolysosomes. Our results showed that US was able to deliver DNA into the cell nuclei shortly after the treatment and that the rest of DNA was mostly cleared by autophagosomes/autophagolysosomes. To further increase transfection efficiency, we then studied the differences between live cells with DNA uptake and those with successful gene transfection post US exposure using cell sorting, cell cycle and microarray analysis. Cells with gene transfection were found to accumulate at the G1 phase of cell cycle and associate with the up-regulation of 32 genes (e.g., GADD45¦Á) and the down-regulation of 46 genes (e.g., TOP2¦Á). Drugs that regulate the expression levels of GADD45¦Á and TOP2¦Á were found to further enhance the transfection mediated by US. A maximun increase of ~ 2 fold in transfection efficiency was observed when cells were sonicated with 0.6 mg/mL ethyl methanesulfonate to up-regulate GADD45¦Á. These results suggestted that using drugs that regulate certain introcellular processes could further enhance US-mediated gene transfection. Over a broad range of US conditions, the integrity of three common gene delivery vectors, plasmid DNA, siRNA and adeno-associated virus, were not affected by US exposure. This thesis verified that US was able to delivery DNA into the cell nuclei to facilitate rapid gene transfection, and provided a proof of princible that by modulating certain intracellular processes, the efficiency of US-mediated gene transfection could be further increased. US could potentially be a safe and efficient method for gene therapy.

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.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 152-169).