No description available.

Synthetic CpG containing oligodeoxynucleotides (ODNs) are recognized by Toll like Receptor 9 (TLR9) and induce a strong pro-inflamatory immune response. To date, four different CpG ODN classes have been described. K-Class ODNs (also known as B-ODN) are potent B cell activators and stimulate TNF

Sulfonated porphyrins and phthalocyanines have been under consideration as microbicides, compounds which, when used in a topical formulation, can prevent transmission of the human immunodeficiency virus. Our studies have been directed toward the characterization of members of these classes. For the sulfonated phthalocyanines, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was helpful in determining the extent of sulfonation. We present the first report of spectroscopic characterization of a pentasulfonated phthalocyanine. Capillary electrophoresis data were sensitive to the concentration of the compounds (Chapter 1). Mass spectrometry was also very useful for establishing the extent of sulfonation in series of sulfonated porphyrins. Capillary electrophoresis was very useful in separating mixtures of these species. A study on sulfonation of a series of tetra(difluorophenyl)porphyrins showed that species with red-shifted Soret peaks were being formed. Data were consistent with an intramolecular sulfone bridge from the phenyl substituent to the porphyrin core. Sulfonation of the tetranaphthylporphyrins ring readily gave more than one sulfonic acid group per naphthyl side chain (Chapter 2). In cancer chemotherapy of solid tumors, it is desired to kill the tumor cells with minimal damage to the surrounding tissue. Brachytherapy seeds have been a considerable help in this regard for some tumors. In further developing approaches to selective tumor damage, we have evaluated a technique, Auger Electron Therapy (AET) in which one introduces a compound that is expected to bind to DNA, absorb the radiation, and then catalyze clustered DNA damage via release of a series of Auger electrons. We chose a series of metals (silver, indium, molybdenum, palladium, platinum, ruthenium, silver and zirconium) with appropriate energy levels to absorb an x-ray photon from the brachytherapy seed and used the tetracationic porphyrin 5,10,15,20-tetrakis(1-methylpyridinium-4-yl) porphyrin (TMPyP4) as a scaffold. The amount of clustered DNA damage was quantitated by a plasmid assay. Experiments evaluated the effect of buffer, concentration of glycerol, irradiation time, and concentration of the porphyrin. No metal studied gave significant double stranded (localized) DNA damage. Significant single stranded DNA damage was observed, however, in the order zirconium >> ruthenium > palladium > platinum > silver ~ indium (Chapter 3).

In this study / ferrocenyl and naphthalenyl substituted quinoxaline derivatives / 5,8- bis (2,3- dihydrothieno [3,4-b] [1,4] dioxin-5-yl)-2- (naphthalen-2-yl)- 3- ferrocenyl- 4a,8a-dihydroquinoxaline / 5,8- bis (2,3-dihydrothieno [3,4-b] [1,4]dioxin-5-yl) -2- (phenyl) -3-ferrocenylquinoxaline / 5,8-bis (2,3-dihydrothieno [3,4-b] [1,4]dioxin-5-yl) -2,3- di(naphthalen-2-yl)quinoxaline and trihexylthiophene and thiophene coupled benzimidazole derivatives / 4-(tert-butyl)-4,7-bis(4-hexylthiophen-2-yl)spiro[benzo[d]imidazole-2,1-cyclohexane] and 4-(tert-butyl)-4, 7-bis(thiophenyl)spiro[benzo[d]imidazole-2,1-cyclohexane] were used as photosensitizers to broaden the active area of diaryliodonium salts. Both quinoxaline and benzimidazole derivatives are expected to be efficient in cationic photopolymerization with a variety of vinyl and oxide monomers at room temperature upon long wavelength UV irradiation. Photopolymerization will be initiated by diphenyliodonium salts and monitored with Optical Pyrometry. Characterization will be completed with optical absorption, flourescence studies and photopolymerization under solar irradiation.

No description available.

Benzimidazole and benzotriazole derivatives, 4-(2,3-Dihydrothieno[3,4-b][1,4] dioxin-5-yl)-7-(2,3-dihydrothieno[3,4b][1,4]dioxin-7-yl)-2-benzyl-1H-benzo[d] imidazole (BIm-Ed), 2-benzyl-4,7-di(thiophen-2-yl)-2H-benzo[d] [1,2,3] triazole (BBTS), and 2-benzyl-4,7-bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)-2Hbenzo[ d] [1,2,3] triazole (BBTES) were employed as photosensitizers for diaryliodonium salt photoinitiators in cationic photopolymerization of various epoxide and vinyl ether monomers. Diphenyliodonium hexafluorophosphate (Ph2I+PF6&macr / ) salt was used as the photoinitiator in this study. Extended conjugation and electron-rich moieties of the photosensitizers enabled the use of long wavelength UV and visible light emitting light sources in cationic photopolymerizations. Polymerizations were achieved at room temperature and monitored by optical pyrometry. Photopolymerization of a diepoxide monomer with ambient solar irradiation was examined.

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.

The present thesis titled “Molecular Modulation of Material Properties: Stud- ies on Nanoparticles, Nanoassemblies and Low Molecular Mass Gelator” deals with the preparation, characterization, and investigations into the properties of gold nanoparticles coated with novel thiols. The coverage of nanoparticle surfaces with these thiols renders them with special characteristics that will be of interest in biological and sensor applications. Also, a novel low molecular mass tetrameric sugar-based hydrogelator was synthesized and its gelation properties were studied in detail. Chapter 1 gives a general introduction and an overview about Nanomaterials, with emphasis towards nanoparticles of gold, which form the basis of this work. It delves with the history of research in noble metal nanoparticles, their interesting electronic and optical properties, the present methods of synthesis of high quality nanoparticles of noble metals, numerous potential applications of these novel materials, as well as the challenges in their real-life applications, and ends with the future outlook of this field of research. Chapter 2 describes the synthesis and characterization of three cationic lipid-like disulfides whose molecular structures are shown in Fig. 2.1. Gold nanoparticles capped with these molecules were then synthesized in small size dispersion by a simple one-phase protocol. These particles exhibited remarkably different solubility properties that were dictated by the molecular structure of the capping agent. The nanoparticles were characterized by a variety of techniques like UV-visible spec- troscopy, Transmission Electron Microscopy (TEM), proton Nuclear Magnetic Resonance (1H NMR), Fourier Transform Infra-red (FTIR) spectroscopy, and Zeta Potential measurements. These nanoparticles were then examined for their interactions (structural formula) Figure 1: Chemical Structures of the cationic lipid-like thiols used for nanoparticle preparation with dipalmitoyl phosphatidyl choline (DPPC) vesicles as model biological membranes. TEM, UV-vis, and Differential Scanning Calorimetry (DSC) were employed to probe the interactions. It was found that the capping agent of the nanoparticle had a strong bearing upon the interactions of the nanoparticles with DPPC vesicles. Chapter 3 describes the assembly of hydrophilic cationic nanoparticles upon elec- trostatic interaction with a variety of anionic surfactants. The chemical structures of some of the anions employed in the study, as well as a schematic of cationic nanopar- ticle are shown in Fig. 2. Upon ion pairing with long-chain anionic surfactants, the hydrophilic cationic nanoparticles were completely hydrophobized. They could then be phase-transferred to organic layer. TEM showed that nanoparticles assemble in to a variety of mesostructures upon ion-pairing with anions. The aggregate formation was found to depend critically upon length of the hydrophobic alkyl chain as well as the head-group of the anion. Isothermal Titration Calorimetry (ITC) was employed to probe the interactions of these nanoparticles with anions. It was found that the anions that resulted in nanoparticle precipitation displayed exothermic interactions with the nanoparticle. Chapter 4 deals with the synthesis of -thiolated metal chelator derivatives whose structures are shown in Fig. 3. The molecules are based on well-known chelators viz. iminodiacetic acid and bis-(2-pyridylmethyl)amine. While the first one is carboxylic acid-based chelator, the second one is pyridine-based. Nanoparticles coated with these chelators were synthesized in a size-controlled manner. These nanoparticles exhibited pH-controlled reversible assembly. However, while S-IDA based nanoparticles aggregated at low pH values, the S-BPA based nanoparticles aggregated in high pH regimes. Mixed monolayer protected gold nanoparticles were synthesized by employing S-BPA and C12H25SH as capping agents. It resulted in the formation of nanoparticles in low size-dispersion. These nanoparticles were characterized by 1H NMR spectroscopy to infer the ratio of the two capping agents on the nanoparticle surface. These nanoparticles demonstrated metal-ion induced aggregation. It was found that the nanoparticles could differentiate Cu2+ ions from other ions, and immediately formed aggregates in presence of Cu2+ ions. Chapter 5 describes the synthesis of novel mono-thiolated “Gemini” surfactants for nanoparticle synthesis. Gemini surfactants with different spacers were prepared. These surfactants had a 12-n-12 kind of molecular structure as shown in the Fig. 4. Upon preparation of nanoparticles with these thiols, the resulting material was soluble in water in the case of rigid thiols like D2S and DBPS Chapter 6 deals with the synthesis and hydrogelation properties of a low molecular mass hydrogelator based on an azobenzene based tetrameric sugar derivative (Fig. 5). The pKa of carboxylic acids in the molecule were determined using 13C NMR. The trans-to-cis isomerization of the compound was probed by time-dependent UV-vis studies. The sugar derivative exhibited pronounced hydrogelation capacity, gelling water at micromolar concentration. The gel formed was characterized extensively (structural formula) Figure 2: Schematic of cationic nanoparticles and molecular structures of the anions employed for nanoparticle assembly (structural formula) Figure 3: Chemical structures of metal-chelator containing thiols employed for the pH-controlled and metal-ion mediated nanoparticle assembly (structural formula) Figure 4: Schematic of cationic nanoparticles and molecular structures of the anions employed for nanoparticle assembly (structural formula) Figure 5: Chemical Structure of azobenzene-based tetrameric sugar derivative exhibit- ing pronounced hydrogelation using melting temperature analysis, UV-vis, FT-IR, circular dichroism spectroscopy and scanning electron microscopy. The resultant gel exhibited impressive tolerance to the pH variation of the aqueous phase and gelated water in the pH range of 4 to 10. While UV-vis and CD spectroscopy indicated that pronounced aggregation of the azobenzene chromophores in the gelator was responsible for gelation, FT-IR studies showed that hydrogen bonding is also a contributing factor in the gelation process. The melting of gel was found to depend upon the pH of the aqueous medium in which gel was formed. The gel showed considerable photostability to UV irradiation indicating tight intermolecular packing inside gelated state that render azobenzene groups in the resultant aggregate refractory to photoisomerization. The electron micrographs of the aqueous gels thus formed showed the existence of spongy globular aggregates in such gelated materials. Addition of salts to the aqueous medium led to a delay in the gelation process and also caused remarkable morphological changes in the microstructure of the gel. Appendix A describes the employment of ligand-free palladium nanoparticles towards efficient catalysis of Heck and Suzuki reactions in aqueous medium. Hexadecyl trimethylammonium bromide was employed as the surfactant to achieve solubilization of organic compounds in aqueous medium. UV-vis and TEM investigations into the formation of nanoparticles in the reaction media were undertaken. These studies indicate that the nanoparticles were formed by reduction of potassium tetrachloropalladinate by methyl acrylate used as one of the reactants. TEM investigation indicated the formation of nanoparticle assemblies upon solvent drying. Efficient and catalytic synthesis of a number of organic compounds could be achieved in high yield.

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.

Most gram-negative organisms require lipopolysaccharide and its membrane anchor, lipid A, for growth and survival. Also known as endotoxin, lipid A is synthesized via a nine-step enzymatic process, culminating in a conserved hexa-acylated, bis-phosphorylated disaccharide of glucosamine. This framework is often altered by condition- or species-specific lipid A modifications, which change the biochemical properties of the molecule in response to and to defend against environmental stress signals. Here, we expound on two stories in different gram-negative organisms, both involving novel or unanticipated biochemistry that impacts lipid A structure. First, the missing acyltransferase in the Epsilonproteobacterium Helicobacter pylori lipid A biosynthesis pathway is identified. This enzyme transfers a secondary acyl chain to the 3'-linked primary acyl chain of lipid A like E. coli LpxM, but shares almost no sequence similarity with the E. coli acyltransferase. It is reannotated as LpxJ and demonstrated to possess an unprecedented ability to act before the 2'-secondary acyltransferase, LpxL, as well as the 3-deoxy-D-manno-octulosonic acid transferase, KdtA. LpxJ is one member of a large class of acyltransferases found in a diverse range of organisms that lack an E. coli LpxM homolog, suggesting that LpxJ participates in lipid A biosynthesis in place of an LpxM homolog. The second story focuses on regulation of modifications to endotoxin structure that occur after the conserved biosynthesis pathway. E. coli pmrD is shown to be required for PmrAB-dependent lipid A modifications in conditions that exclusively activate PhoPQ; this result proves that PmrD connects PhoPQ and PmrAB despite previous reports that it is an inactive connector in this organism. Further, RNA sequencing and polymyxin B survival assays solidify the role of E. coli pmrD in influencing expression of pmrA and its target genes and promoting survival during exposure to cationic antimicrobial peptides. Notably, the presence of an unknown factor or system capable of activating pmrD to promote lipid A modification in the absence of the PhoPQ system is also revealed. In all, the findings presented here expand our understanding of alternative approaches to lipid A biosynthesis and the complex systems that regulate modifications of this dynamic molecule.