Sugar-based tertiary amino gemini surfactants with a vesicle-to-micelle transition in the endosomal pH range mediate efficient transfection in vitro.

Novel reduced sugar gemini amphiphiles linked through their tertiary amino head groups via alkyl spacers of 4 or 6 carbons, and with varying (unsaturated) alkyl tail lengths of 12--18, have been synthesized and tested for transfection in vitro in an adherent Chinese hamster ovary cell line (CHO-K1). Transfection efficiencies peaked at 2.7 times that of the commercial standard Lipofectamine Plus/2000 for pure solutions of the compound bearing unsaturated (oleyl) alkyl tails. For those compounds bearing saturated alkyl tails, transfection efficiency peaked at a tail length of 16, at a level similar to Lipofectamine Plus/2000. All of the amphiphiles formed bilayer vesicles at physiological pH. Some of the amino groups at the surface were protonated, and vesicles therefore bore a positive charge. Increased protonation with reduced pH resulted in greatly increased monomer solubility and a morphology change from vesicle to micelle at characteristic pH values, dependent on the tail length. For the compounds promoting high transfection efficiency, this characteristic pH was within the range found in the endosomal compartment (7.4--4.0). Formation of mixed micelles between gemini surfactant and membrane phospholipids at reduced pH may therefore provide a method of endosome rupture and subsequent escape of entrapped DNA, thus discarding the need for extra fusogenic or endosomolytic agents. The positive charge on the vesicles at physiological pH drives the colloidal association with DNA. Small angle X-ray scattering measurements indicate that lamellar aggregates are formed, which have a d spacing of 48--54 A. Preliminary differential scanning calorimetric measurements suggest that reduction of pH causes a disordering of the hydrocarbon region of the DNA-surfactant complex.     

Gemini ester quat surfactants and their biological activity

Cationic gemini surfactants are an important class of surface-active compounds that exhibit much higher surface activity than their monomeric counterparts. This type of compound architecture lends itself to the compound being easily adsorbed at interfaces and interacting with the cellular membranes of microorganisms. Conventional cationic surfactants have high chemical stability but poor chemical and biological degradability. One of the main approaches to the design of readily biodegradable and environmentally friendly surfactants involves inserting a bond with limited stability into the surfactant molecule to give a cleavable surfactant. The best-known example of such a compound is the family of ester quats, which are cationic surfactants with a labile ester bond inserted into the molecule. As part of this study, a series of gemini ester quat surfactants were synthesized and assayed for their biological activity. Their hemolytic activity and changes in the fluidity and packing order of the lipid polar heads were used as the measures of their biological activity. A clear correlation between the hemolytic activity of the tested compounds and their alkyl chain length was established. It was found that the compounds with a long hydrocarbon chain showed higher activity. Moreover, the compounds with greater spacing between their alkyl chains were more active. This proves that they incorporate more easily into the lipid bilayer of the erythrocyte membrane and affect its properties to a greater extent. A better understanding of the process of cell lysis by surfactants and of their biological activity may assist in developing surfactants with enhanced selectivity and in widening their range of application.     

Preparation and in vitro evaluation of novel lipopeptide transfection agents for efficient gene delivery

Gene therapy by delivery of nonviral expression vectors is highly desirable, due to their safety, stability, and suitability for production as bulk pharmaceuticals. However, low transfection efficiency remains a limiting factor in application on nonviral gene delivery. Despite recent advances in the field, there are still major obstacles to overcome. In an attempt to construct more efficient nonviral gene delivery vectors, we have designed a series of novel lipopeptide transfection agents, consisting of an alkyl chain, one cysteine, 1 to 4 histidine and 1 to 3 lysine residues. The lipopeptides were designed to facilitate dimerization (by way of the cysteine residues), DNA binding at neutral pH (making use of charged lysine residues), and endosomal escape (by way of weakly basic histidine residues). DNA/lipopeptide complexes were evaluated for their biophysical properties and transfection efficiencies. The number and identity of amino acids incorporated in the lipopeptide construct affected their DNA/lipopeptide complex forming capacity. As the number of lysine residues in the lipopeptide increased, the DNA complexes formed became more stable, had higher zeta potential (particle surface charge), and produced smaller mean particle sizes (typically 110 nm at a charge ratio of 5.0 and 240 nm at a charge ratio of 1.0). The effect of inclusion of histidines in the lipopeptide moiety had the opposite effect on complex formation to lysine, but was necessary for high transfection efficiency. In vitro transfection studies in COS-7 cells revealed that the efficiency of gene delivery of the luciferase encoding plasmid, pCMV-Luc, mediated by all the lipopeptides, was much higher than poly(L-lysine) (PLL), which has no endosomal escape system, and in two cases was slightly higher than that of branched polyethylenimine (PEI). Lipopeptides with at least two lysine residues and at least one histidine residue produced spontaneous transfection complexes with plasmid DNA, indicating that endosomal escape was achieved by incorporation of histidine residues. These low molecular weight peptides can be readily synthesized and purified and offer new insights into the mechanism of action of transfection complexes.     

Effect of the head-group geometry of amino acid-based cationic surfactants on interaction with plasmid DNA

The interaction between DNA and different types of amino acid-based cationic surfactants was investigated. Particular attention was directed to determine the extent of influence of surfactant head-group geometry toward tuning the interaction behavior of these surfactants with DNA. An overview is obtained by gel retardation assay, isothermal titration calorimetry, fluorescence spectroscopy, and circular dichroism at different mole ratios of surfactant/DNA; also, cell viability was assessed. The studies show that the surfactants with more complex/bulkier hydrophobic head group interact more strongly with DNA but exclude ethidium bromide less efficiently; thus, the accessibility of DNA to small molecules is preserved to a certain extent. The presence of more hydrophobic groups surrounding the positive amino charge also gave rise to a significantly lower cytotoxicity. The surfactant self-assembly pattern is quite different without and with DNA, illustrating the roles of electrostatic and steric effects in determining the effective shape of a surfactant molecule.     

Biological and surface-active properties of double-chain cationic amino acid-based surfactants

Cationic amino acid-based surfactants were synthesized via solid phase peptide synthesis and terminal acylation of their α and ε positions with saturated fatty acids. Five new lipopeptides, N-α-acyl-N-ε-acyl lysine analogues, were obtained. Minimum inhibitory concentration and minimum bactericidal (fungicidal) concentration were determined on reference strains of bacteria and fungi to evaluate the antimicrobial activity of the lipopeptides. Toxicity to eukaryotic cells was examined via determination of the haemolytic activities. The surface-active properties of these compounds were evaluated by measuring the surface tension and formation of micelles as a function of concentration in aqueous solution. The cationic surfactants demonstrated diverse antibacterial activities dependent on the length of the fatty acid chain. Gram-negative bacteria and fungi showed a higher resistance than Gram-positive bacterial strains. It was found that the haemolytic activities were also chain length-dependent values. The surface-active properties showed a linear correlation between the alkyl chain length and the critical micelle concentration.     

Disintegration by surfactants of egg yolk phosphatidylcholine vesicles stabilized with carboxymethylchitin

Disintegration by surfactants of egg yolk phosphatidylcholine vesicles stabilized with carboxymethylchitin was investigated by measuring the amount released of a marker dye from the vesicles. In solutions of pH around 7, anionic and nonionic surfactants caused vesicle disintegration at very low concentrations, while cationic surfactants produced a breakdown of the vesicles at rather high concentrations. Increase in the alkyl chain-length of surfactant molecules brought about decrease in the surfactant concentration at which vesicle disintegration starts. As the length of the polyoxyethylene chain in nonionic surfactant molecules increased, the tendency of vesicle disintegration to occur decreased. Both anionic and cationic surfactants gave clear solutions above their critical micelle concentrations when they acted on the phospholipid vesicles, whereas nonionic surfactants left ghost cell-like debris consisting of carboxymethylchitin molecules in their micellar solutions. The effect of pH on vesicle disintegration was notable for ionic surfactants but not for nonionic surfactants. Thus, anionic surfactants increased the degree of disintegration as pH increased, while cationic surfactants produced an identical vesicle disintegration curve below pH 8 above which the curve started to shift toward the lower concentration region of the agents. These findings were explained in terms of surfactant penetration into phospholipid bilayers and solubilization of phospholipid molecules by surfactant micelles.     

Quantitative structure-activity relationship (QSAR) analysis of surfactants influencing attachment of a Mycobacterium sp. to cellulose acetate and aromatic polyamide reverse osmosis membranes.

A series of 23 neutral, anionic, and zwitterionic surfactants were tested at a concentration of 0.1% wt/vol for their influence on attachment of a Mycobacterium sp. to cellulose acetate (CA) and polyamide (PA) reverse osmosis (RO) membranes. Four cell attachment bioassays were used: (1) semiconcurrent addition of surfactant and bacteria to RO coupons (standard assay); (2) surfactant pretreatment of RO membranes (membrane pretreatment assay); (3) surfactant treatment of adsorbed cells (detachment assay); and (4) surfactant pretreatment of mycobacteria (cell pretreatment assay). Seventeen surfactants inhibited attachment to PA membranes, whereas 15 inhibited attachment to CA in standard assays and, in 13 cases, the same surfactant inhibited attachment to both PA and CA. Despite greater cell attachment to PA than CA, surfactants were typically more effective in the former membrane system. More surfactants were effective in impairing cell attachment than in promoting detachment and a number enhanced attachment in membrane pretreatment assays, suggesting surface modification of RO membranes. Cell pretreatment inhibited attachment to CA membranes, suggesting the bacterial surface was also a target for detergent activity. Multivariate regression and cluster analyses indicated that critical micellar concentration (CMC) was positively correlated with Mycobacterium attachment in CA and PA standard assays. Surfactant dipole moment and octanol/water partitioning (LogP) also contributed to detergent activity in the PA system, whereas dipole moment, molecular topology (i.e., connectivity indices), and charge properties influenced activity in the CA system. Influential variables in membrane pretreatment assays included the LogP, topology indices, and charge properties, whereas CMC played a diminished role. Surfactant dipole moment was most influential in CA membrane detachment assays. Increasing system ionic strength by LiBr addition strengthened inhibition of cell attachment to CA membranes by dodecylbenzene sulfonic acid (DBSA) and promoted DBSA adsorption to CA surfaces as indicated by attenuated total reflection Fourier-transform infrared spectrometry. Results indicate that inhibition of bacterial attachment to RO membranes may be maximized by manipulating surfactant molecular structure to optimize surface adsorption behavior.     

Interaction of alpha-amylase with n-alkylammonium bromides

The interaction of alpha-amylase with n-alkylammonium bromides above and below their critical micellar concentrations (cmc) has been studied in buffer at pH 7 and 10 by UV spectrophotometry, photon correlation spectroscopy and Doppler microelectrophoresis. This interaction produces a complex that is dependent on pH of the medium. This complex appears at surfactant concentrations below the cmc, which means that individual surfactant molecules can bind tightly to native alpha-amylase. The complex maintains its aggregation state when the concentration of surfactants with a hydrocarbon chain of 16 carbons increases, but not for surfactants of 12 and 14 carbons. Measurements of zeta-potential indicate the influence of electrostatic and hydrophobic forces. When the size of the aggregate is maximal, proteins are at their point of zero charge. In such conditions, Van der Waals forces and contacts between the alkyl chain and the hydrophobic core of the protein favour the formation of a larger aggregate.     

Lipoplexes formed from sugar-based gemini surfactants undergo a lamellar-to-micellar phase transition at acidic pH. Evidence for a non-inverted membrane-destabilizing hexagonal phase of lipoplexes

The present study aims at a better understanding of the mechanism of transfection mediated by two sugar-based gemini surfactants GS1 and GS2. Previously, these gemini surfactants have been shown to be efficient gene vectors for transfection both in vitro and in vivo. Here, using Nile Red, a solvatochromic fluorescent probe, we investigated the phase behavior of these gemini surfactants in complexes with plasmid DNA, so-called lipoplexes. We found that these lipoplexes undergo a lamellar-to-non-inverted micellar phase transition upon decreasing the pH from neutral to mildly acidic. This normal (non-inverted) phase at acidic pH is confirmed by the colloidal stability of the lipoplexes as shown by turbidity measurements. We therefore propose a normal hexagonal phase, H(I), for the gemini surfactant lipoplexes at acidic endosomal pH. Thus, we suggest that besides an inverted hexagonal (H(II)) phase as reported for several transfection-potent cationic lipid systems, another type of non-inverted non-bilayer structure, different from H(II), may destabilize the endosomal membrane, necessary for cytosolic DNA delivery and ultimately, cellular transfection.     

Novel polymerizable surfactants with pH-sensitive amphiphilicity and cell membrane disruption for efficient siRNA delivery

Small interfering RNA (siRNA) is a promising new therapeutic modality that can specifically silence disease-related genes. The main challenge for successful clinical development of therapeutic siRNA is the lack of efficient delivery systems. In this study, we have designed and synthesized a small library of novel multifunctional siRNA carriers, polymerizable surfactants with pH-sensitive amphiphilicity based on the hypothesis that pH-sensitive amphiphilicity and environmentally sensitive siRNA release can result in efficient siRNA delivery. The polymerizable surfactants comprise a protonatable amino head group, two cysteine residues, and two lipophilic tails. The surfactants demonstrated pH-sensitive amphiphilic hemolytic activity or cell membrane disruption with rat red blood cells. Most of the surfactants resulted in low hemolysis at pH 7.4 and high hemolysis at reduced pH (6.5 and 5.4). The pH-sensitive cell membrane disruption can facilitate endosomal-lysosomal escape of siRNA delivery systems at the endosomal-lysosomal pH. The surfactants formed compact nanoparticles (160-260 nm) with siRNA at N/P ratios of 8 and 10 via charge complexation with the amino head group, lipophilic condensation, and autoxidative polymerization of dithiols. The siRNA complexes with the surfactants demonstrated low cytotoxicity. The cellular siRNA delivery efficiency and RNAi activity of the surfactants correlated well with their pH-sensitive amphiphilic cell membrane disruption. The surfactants mediated 40-88% silencing of luciferase expression with 100 nM siRNA and 35-75% with 20 nM siRNA in U87-luc cells. Some of the surfactants resulted in similar or higher gene silencing efficiency than TransFast. EHCO with no hemolytic activity at pH 7.4 and 6.5 and high hemolytic activity at pH 5.4 resulted in the best siRNA delivery efficiency. The polymerizable surfactants with pH-sensitive amphiphilicity are promising for efficient siRNA delivery.     

Lipoplexes formed from sugar-based gemini surfactants undergo a lamellar-to-micellar phase transition at acidic pH. Evidence for a non-inverted membrane-destabilizing hexagonal phase of lipoplexes

The present study aims at a better understanding of the mechanism of transfection mediated by two sugar-based gemini surfactants GS1 and GS2. Previously, these gemini surfactants have been shown to be efficient gene vectors for transfection both in vitro and in vivo. Here, using Nile Red, a solvatochromic fluorescent probe, we investigated the phase behavior of these gemini surfactants in complexes with plasmid DNA, so-called lipoplexes. We found that these lipoplexes undergo a lamellar-to-non-inverted micellar phase transition upon decreasing the pH from neutral to mildly acidic. This normal (non-inverted) phase at acidic pH is confirmed by the colloidal stability of the lipoplexes as shown by turbidity measurements. We therefore propose a normal hexagonal phase, H_I, for the gemini surfactant lipoplexes at acidic endosomal pH. Thus, we suggest that besides an inverted hexagonal (H_II) phase as reported for several transfection-potent cationic lipid systems, another type of non-inverted non-bilayer structure, different from H_II, may destabilize the endosomal membrane, necessary for cytosolic DNA delivery and ultimately, cellular transfection.     

The Arginines in the N-Terminus of the Porcine Circovirus 2 Virus-like Particles Are Responsible for Disrupting the Membranes at Neutral and Acidic pH

Non-enveloped viruses that are endocytosed employ numerous mechanisms to disrupt endosomal membranes for escape into the cellular cytoplasm. These include the use of amphipathic helices or sheets, hydrophobic loops, myristoylated peptides, and proteins with phospholipase activity. Some mechanisms result in immediate deterioration of the endosome, while others form pores in the membrane causing osmolysis to disrupt the endosome and allow viral escape. We describe an additional mechanism by a non-enveloped virus to disrupt endosomal membranes. Porcine circovirus 2 (PCV2) possesses a 41-amino acid arginine-rich motif (ARM) at the N-terminus of its capsid protein that appears to be in the interior of the virus-like particle (VLP). Using in vitro membrane disruption assays, we demonstrate that PCV2 VLP, unassembled capsid, and ARM peptide possess the ability to disrupt endosomal-like membranes, whereas VLP lacking the ARM sequence does not possess this capability. Membrane disruption by VLP is insensitive to pH, but unassembled capsid protein and ARM peptide exhibit diminished activity at low pH. Our liposome disruption assays, circular dichroism, and intrinsic tryptophan fluorescence assays allow us to propose a model for PCV2–endosomal membrane interaction wherein the ARM peptide externalizes from the capsid, its C-terminus (amino acids 28–40) anchors into the membrane, and the arginine-rich N-terminus (amino acids 1–27) drives membrane disruption. To our knowledge, this is the first example of a non-enveloped virus using the arginines of an ARM to disrupt membranes. Also, this is the first example of such study for the Circoviridae family of viruses.     

Polyacrylamide gel electrophoresis of several proteins in the presence of sodium oligooxyethylene dodecyl ether sulfates or a commercially available analog

The behavior of water-soluble proteins and a typical membrane protein in polyacrylamide gel electrophoresis was studied in the presence of sodium oligooxyethylene dodecyl ether sulfates with a defined number of oxyethylene units or a commercially available analog with distribution and heterogeneity for the oxyethylene chain length and alkyl group, respectively. It was concluded that most water-soluble proteins do not interact with the anionic surfactants as long as their oxyethylene chain lengths are sufficiently long; the commercially available surfactant binds exceptionally well to beta-lactoglobulin without causing denaturation and subsequent dissociation; such surfactants are expected to solubilize membrane proteins without causing denaturation as judged from the result with Na+,K+-ATPase and are promising as new solubilizing agents for membrane proteins which enable efficient electrophoretic analysis or separation after the solubilization.     

The influence of the polar head and the hydrophobic chain on the skin penetration enhancement effect of poly(ethylene glycol) derivatives

The effect of a homologue series of nonionic surfactants, namely poly(ethylene glycol) (PEG) fatty acid esters, differing in oxyethylene (PEG 8, PEG 12, and PEG 40) and fatty acid (stearate, mono and di-laurate, and mono and di-oleate) chain lengths, on in vitro skin permeability of ketoprofen (KTP) vehicled in plasters was investigated. The drug diffusion through hairless mouse skin as well as the effect of the surfactant type and strength was studied by Franz diffusion cells and ATR-FTIR spectroscopy. The use of PEG stearate series revealed that the surfactant with the largest polar head, namely PEG 40, was ineffective in enhancing the skin permeation of KTP, independently of the plaster concentrations. The effect of the hydrophobic chain was investigated only by using the shortest oxyethylene chains. The experimental results revealed that the oxyethylene chain length of surfactants appeared to be more influent than the alkyl chain. The prediction of the absorption enhancing capability of these PEG derivatives appeared related to the vehicle other than the proper combination of the number of ethylene oxide groups and alkyl groups.     

Molecular dynamics simulation of the atomistic monolayer structures of N-acyl amino acid-based surfactants

Abstract

Atomic molecular dynamics simulations have been performed on the monolayer systems of N-acyl amino acid-based surfactants. The role of intermolecular hydrogen bonds and ionic side chain length of dicarboxylate surfactants were investigated through radial and spatial distribution functions. It was found that the hydrogen bonding capability between surfactants was the major factor determining the surface area each surfactant could occupy. Tighter packing of surfactants would lead to a weaker interaction with water molecule, and the protonation of carboxylate groups resulted in stronger inter-surfactant interactions. The hydrogen bonds with water molecules were found to prevail between the carboxylate groups, and regular cage-like water distributions surrounding the surfactant headgroups were seen. The introduction of divalent ions leads to a significant increase of counterion binding, and their intramolecular and intermolecular bindings of calcium ions were also well characterised. The intramolecular chelation of calcium ions was found impossible between the carboxylate groups for N-acyl glutamate due to its flexible side chain.     

Fluorescence stopped-flow study of the interaction of alkylpyridinium salts and pyrene-substituted fatty acids with lecithin vesicles

The migration of alkylpyridinium surfactants and pyrene-substituted fatty acids between vesicles has been studied. The results are in accord with the assumption of diffusion-controlled processes. Changes in relaxation time with charge type, degree of ionization, pH, and alkyl chain length of the migrating species are investigated and discussed.The presence of a slower relaxation type, apparently due to a transmembrane transport was established in the case of the pyridinium surfactants, but could not be found for pyrene-substituted fatty acids. Fluorescence-quenching studies indicate that these are present at both vesicle interfaces.     

Transport of basic amino acids by membrane vesicles of Lactococcus lactis.

The uptake of the basic amino acids arginine, ornithine, and lysine was studied in membrane vesicles derived from cells of Lactococcus lactis which were fused with liposomes in which beef heart mitochondrial cytochrome c oxidase was incorporated as a proton motive force (PMF)-generating system. In the presence of ascorbate N,N,N'N'-tetramethylphenylenediamine-cytochrome c as the electron donor, these fused membranes accumulated lysine but not ornithine or arginine under aerobic conditions. The mechanism of energy coupling to lysine transport was examined in membrane vesicles of L. lactis subsp. cremoris upon imposition of an artificial electrical potential (delta psi) or pH gradient or both and in fused membranes of these vesicles with cytochrome c oxidase liposomes in which the delta psi and delta pH were manipulated with ionophores. Lysine uptake was shown to be coupled to the PMF and especially to the delta psi, suggesting a proton symport mechanism. The lysine carrier appeared to be specific for L and D isomers of amino acids with a guanidine or NH2 group at the C6 position of the side chain. Uptake of lysine was blocked by p-chloromercuribenzene sulfonic acid but not by maleimides. Counterflow of lysine could not be detected in L. lactis subsp. cremoris, but in the arginine-ornithine antiporter-containing L. lactis subsp. lactis, rapid counterflow occurred. Homologous exchange of lysine and heterologous exchange of arginine and lysine were mediated by this antiporter. PMF-driven lysine transport in these membranes was noncompetitively inhibited by arginine, whereas the uptake of arginine was enhanced by lysine. These observations are compatible with a model in which circulation of lysine via the lysine carrier and the arginine-ornithine antiporter leads to accumulation of arginine.     

Gemini Surfactants Based on Bis-Imidazolium Alkoxy Derivatives as Effective Agents for Delivery of Nucleic Acids: A Structural and Spectroscopic Study

The success rate of gene therapy depends on the efficient transfection of genetic material into cells. The golden mean between harmlessness and high effectiveness can be provided by synthetic lipid-like molecules that are similar to the components of biological membranes. Cationic gemini surfactants are one such moiety and because of their favourable physicochemical properties (double positive electric charge, reduced toxicity, low values of critical micelle concentration), they show great potential as delivery system components for genetic material in gene therapy. The aim of this study was to investigate the process of the complexation of cationic gemini surfactants with nucleic acids: double-stranded DNA of different sizes (21 bp, ~185 bp, ~20 kbp) and siRNA (21 bp). The tested series of dicationic surfactants consists of bis-imidazolium quaternary salts with varying lengths of hydrophobic side chains (m = 5, 6, 7, 8, 9, 11, 12, 14, 16). On the basis of the data obtained by circular dichroism spectroscopy and electrophoresis, we concluded that the studied gemini surfactants with long side chains effectively bind nucleic acids at low concentrations, which leads to the formation of stable lipoplexes. Images obtained by atomic force microscopy also confirmed the formation of vesicular structures, i.e., complexes between DNA and surfactants. The cytotoxicity of selected surfactants was also tested on HeLa cells. The surfactant toxicity significantly depends on surfactant geometry (the length of hydrophobic chain).     

Functional characterization of an endosome-disruptive peptide and its application in cytosolic delivery of immunoliposome-entrapped proteins

Antibody-directed liposomes (immunoliposomes) are frequently used for targeted drug delivery. However, delivery of large biotherapeutic molecules (i.e. peptides, proteins, or nucleic acids) with immunoliposomes is often hampered by an inefficient cytosolic release of entrapped macromolecules after target cell binding and subsequent endocytosis of immunoliposomes. To enhance cytosolic drug delivery from immunoliposomes present inside endosomes, a pH-dependent fusogenic peptide (diINF-7) resembling the NH(2)-terminal domain of influenza virus hemagglutinin HA-2 subunit was used. Functional characterization of this dimeric peptide showed its ability to induce fusion between liposome membranes and leakage of liposome-entrapped compounds when exposed to low pH. In a second series of experiments, diINF-7 peptides were encapsulated in immunoliposomes to enhance the endosomal escape of diphtheria toxin A chain (DTA), which inhibits protein synthesis when delivered into the cytosol of target cells. Immunoliposomes targeted to the internalizing epidermal growth factor receptor on the surface of ovarian carcinoma cells (OVCAR-3) and containing encapsulated DTA did not show any cytotoxicity toward OVCAR-3 cells. Cytotoxicity was only observed when diINF-7 peptides and DTA were co-encapsulated in the immunoliposomes. Thus, diINF-7 peptides entrapped inside liposomes can greatly enhance cytosolic delivery of liposomal macromolecules by pH-dependent destabilization of endosomal membranes after cellular uptake of liposomes.     

Cationic surfactant mediated fibrillogenesis in bovine liver catalase: a biophysical approach

Protein aggregation into oligomers and mature fibrils are associated with more than 20 diseases in humans. The interactions between cationic surfactants dodecyltrimethylammonium bromide (DTAB) and tetradecyltrimethylammonium bromide (TTAB) with varying alkyl chain lengths and bovine liver catalase (BLC) were examined by various biophysical approaches. The delicate coordination of electrostatic and hydrophobic interactions with protein, play imperative role in aggregation. In this article, we have reconnoitered the relation between charge, hydrophobicity and cationic surfactants DTAB and TTAB on BLC at pH 7.4 and 9.4 which are two and four units above pI, respectively. We have used techniques like turbidity, Rayleigh light scattering, far-UV CD, ThT, ANS, Congo red binding assay, DLS, and transmission electron microscopy. The low concentration ranges of DTAB (0–600 μM) and TTAB (0–250 μM) were observed to increase aggregation at pH 9.4. Nevertheless, at pH 7.4 only TTAB was capable of inducing aggregate. DTAB did not produce any significant change in secondary structure at pH 7.4 suggestive of the role of respective charges on surfactants and protein according to the pI and alkyl chain length. The morphology of aggregates was further determined by TEM, which proved the existence of a fibrillar structure. The surfactants interaction with BLC was primarily electrostatic as examined by ITC. Our work demystifies the critical role of charge as well as hydrophobicity in amyloid formation.