Effect of a Ketogenic Diet on Oxidative Posttranslational Protein Modifications and Brain Homogenate Denaturation in the Kindling Model of Epilepsy in Mice.

This study focused on the ketogenic diet (KD) effects on oxidative posttranslational protein modification (PPM) as presumptive factors implicated in epileptogenesis. A 28-day of KD treatment was performed. The corneal kindling model of epileptogenesis was used. Four groups of adult male ICR mice (25-30 g) were randomized in standard rodent chow (SRC) group, KD-treatment group; SRC + kindling group; KD + kindling group (n = 10 each). Advanced oxidation protein products (AOPP) and protein carbonyl contents of brain homogenates together with differential scanning calorimetry (DSC) were evaluated. Two exothermic transitions (Exo1 and Exo2) were explored after deconvolution of the thermograms. Factor analysis was applied. The protective effect of KD in the kindling model was demonstrated with both decreased seizure score and increased seizure latency. KD significantly decreased glucose and increased ketone bodies (KB) in blood. Despite its antiseizure effect, the KD increased the AOPP level and the brain proteome's exothermic transitions, suggestive for qualitative modifications. The ratio of the two exothermic peaks (Exo2/Exo1) of the thermograms from the KD vs. SRC treated group differed more than twice (3.7 vs. 1.6). Kindling introduced the opposite effect, changing this ratio to 2.7 for the KD + kindling group. Kindling significantly increased glucose and KB in the blood whereas decreased the BW under the SRC treatment. Kindling decreased carbonyl proteins in the brain irrespectively of the diet. Further evaluations are needed to assess the nature of correspondence of calorimetric images of the brain homogenates with PPM.

The effect of prolonged intense physical exercise of special forces volunteers on their plasma protein denaturation profile examined by differential scanning calorimetry.

The human blood plasma proteome profile has been an area of intensive investigation and differential scanning calorimetry (DSC) has come forward as a novel tool in analyzing plasma heat capacity changes to monitor various physiological responses in health and disease. This study used DSC to assess potential alterations in the plasma heat capacity profile of albumin and globulins during extremely demanding physical exercise. We monitored the changes in denaturation profiles of those plasma proteins for five consecutive days of an extraordinary exercise training schedule in 14 young male Special Forces volunteers, as well as after a 30-day recovery period. The major effect of the prolonged intense exercise was the continuous upward shift of the albumin peak by 2°-3 °C on the initial days of exercise, with a tendency to plateau circa the 5th day of exercise. In addition, some redistribution of the denaturational enthalpy was observed upon exercise, where the globulins peak increased relative to the albumin peak. Noteworthy, the alterations in the plasma proteome denaturational profiles were not persistent, as virtually full recovery of the initial status was observed after 30 days of recovery. Our findings indicate that 5 days of exhaustive physical exercise of highly trained individuals enhanced the thermal stability of plasma albumin shifting its denaturational transition to higher temperatures. We surmise that these effects may be a result of increased blood oxygenation during the prolonged intense exercise and, consequently, of albumin oxidation as part of the overall adaptation mechanisms of the body to extreme physical and/or oxidative stress.

Type IV collagen conforms to the organization of polylaminin adsorbed on planar substrata.

Tissue engineering demands the development of scaffolds that mimic natural extracellular matrices (ECM). Despite the success in obtaining synthetic interstitial ECM, the production of an artificial basement membrane (BM), the specialized thin sheet of ECM that is pivotal for the functional organization of most tissues and internal organs, is still not achieved. With the long-term aim of developing a flat BM-like structure here we investigated the behavior of acid-soluble Col IV during simultaneous assembly with laminin (LM) in acidic conditions. The underlying rationale was the previously observed phenomenon of acid-triggered LM polymerization, giving rise to biomimetic polylaminin (polyLM) that can be adsorbed on the substrate. Unexpectedly, we found that Col IV (that does not polymerize in acidic conditions) readily incorporated into the polyLM layer, forming a network that mimics to a great extent the characteristic polygonal morphology of single polyLM observable at micrometric scale. Scanning calorimetry and light scattering measurements supported the notion that polyLM and Col IV could directly interact. The biological properties of the proposed artificial BM-like structure were characterized using human keratinocytes (HACAT) and umbilical vein endothelial cells (HUVEC). HACAT formed stratified cell layers on the hybrid polyLM/Col IV layer, but not on Matrigel, nor on LM or Col IV alone, while HUVEC improved cortical F-actin and tight juctions organization on polyLM/Col IV. Thus, the proposed artificial BM reproduces not only morphological but also some functional properties of the natural BM. STATEMENT OF SIGNIFICANCE: Basement membranes (BMs) are flat biological matrices separating tissue compartments in the body. Their peculiar sheet-like structure is thought to result from the association of two independent protein networks of laminin and collagen IV. While pursuing the development of an artificial BM, we found that, when mixed with acid-induced polymerized laminin, collagen IV immediately conformed to the laminin shape. This implies that the protein networks may not be independently assembled as believed so far, but instead that laminin may command the assembly of collagen IV. Our hybrid matrix was structurally more stable than the commercial BM extract Matrigel and, unlike the latter, supported in vitro formation of a stratified layer of keratinocytes that approximated the organization of the natural epidermis.

Exothermic transitions in the heat capacity profiles of human cerebrospinal fluid.

In this work, we examined by DSC protein denaturation heat capacity profiles for two body fluids, cerebrospinal fluid (CSF) and blood plasma obtained from brain tumor (mainly glioblastoma) patients and healthy volunteers. We observed large distinctions between the heat capacity profiles of CSF and blood plasma, although their protein compositions are believed to have much in common. A prominent, previously unreported CSF feature was the existence of a pre-denaturation exothermic transition peaking at ~ 50-52 °C, recorded for both control and brain tumor CSF. This appears to be the first observation of a pre-denaturation exotherm in a human body fluid. In all studied samples, the exotherms deconvoluted with high precision into a sum of two Gaussian peaks. These exotherms are apparently specific, originating from brain tissue-soluble proteins in the CSF not present in blood plasma. Malignant brain tumors (glioblastoma multiforme, Grade IV, and low-grade glioma, Grade II) reduced twofold the enthalpy of the exotherms relative to the control. These results suggest that the amount and/or conformational state of the CSF proteins (e.g., intrinsic disorder) giving rise to pre-denaturation exothermic events substantially changed upon brain tumor progression. Concomitantly, the enthalpy of the CSF endothermic peaks was partially redistributed from a lower-temperature (main) transition to a higher-temperature transition. The presented data demonstrated that the heat capacity profiles of intrinsic CSF proteins constitute a sensitive biomarker of glioblastoma and other brain malignancies.

Calorimetric monitoring data of the evolution of the lamellar-inverted hexagonal phase transition in phosphatidylethanolamine dispersions upon temperature cycling.

The data presented in this article are related to the research article entitled "Cubic phases in phosphatidylethanolamine dispersions: formation, stability and phase transitions" (Tenchov and Koynova, 2017) [1]. This article presents thermodynamic data obtained by differential scanning calorimetry following the evolution of the Lα - HII endotherm upon temperature cycling during the lamellar to cubic phase conversion.

Drug Exchange between Albumin Nanoparticles and Erythrocyte Membranes.

The effects of thioridazine (TDZ) and chlorpromazine (CPZ) and bovine serum albumin nanoparticles (BSA-NPs) on erythrocyte membranes have been investigated. Two kinds of hemolytic assays were used; hemolysis under hypotonic conditions and hemolysis in physiological conditions. Under hypotonic conditions for 50% hemolysis, both TDZ and CPZ have a biphasic effect on membranes; namely, stabilization at low concentrations and destabilization after reaching a critical concentration. In physiological conditions, there are other critical concentrations above which both drugs hemolyse the erythrocites. In each case, the critical concentrations of TDZ are lower than those of CPZ, which is consistent with the ratio of their partition coefficients. When BSA-NPs are added to the erythrocyte suspension simultaneously with the drugs, the critical concentrations increase for both drugs. The effect is due to the incorporation of a portion of drug substances into the BSA-nanoparticles, which consequently leads to the decrease of the active drug concentrations in the erythrocyte suspension medium. Similar values of the critical concentrations are found when the BSA-NPs are loaded with the drugs before their addition to the erythrocyte suspension in which case the events of the partition are: desorption of the drug from BSA-NPs, diffusion through the medium, and adsorption on erythrocyte membranes. This result suggests that the drugs are not influenced by the processes of adsorption and desorption onto and out of the BSA-NPs, and that the use of BSA-NPs as drug transporters would allow intravenous administration of higher doses of the drug without the risk of erythrocyte hemolysis.

Natural Product Formulations for the Prevention and Treatment of Alzheimer's disease: A Patent Review.

INTRODUCTION: Although considerable efforts have been made to develop effective therapeutic agents for Alzheimer's Disease (AD), neither a consensus concerning the pathogenesis of the disease nor a successful therapy for its treatment is yet available. The natural product chemistry brings tremendous diversity and abundant resource for medical needs. OBJECTIVES: The present review summarizes recent patents on natural extracts and derived drugs as agents for the prevention and treatment of AD. It also sums up the suggested mechanisms of action of the formulated natural remedies. CONCLUSION: It is now becoming well accepted that multiple factors contribute to the progression of AD. The pathogenesis of the disease involves amyloid-ß cascade, tau hyperphosphorylation, oxidative stress, inflammation, mitochondrial dysfunction, protein misfolding, gene mutation, etc. It has been suggested that the multifactorial nature of AD pathogenesis requires the design of medicines with a wide spectrum of activity. Medicinal herbs are known to consist of multiple compounds and may implicate multiple mechanisms, thus being advantageous over the simple single-target drugs in the treatment of complex diseases. Indeed, natural products attract increased attention. In the last decades, they have become a major focus in the quest for AD remedies and may represent a real promise for curing the disease.

Cubic phases in phosphatidylethanolamine dispersions: Formation, stability and phase transitions.

The non-lamellar phases formed by membrane lipids in diluted aqueous dispersions are mainly represented by the inverted hexagonal phase, HII, and phases of cubic symmetry, among them the bicontinuous cubic phases Pn3m (Q224), Im3m (Q229) and Ia3d (Q230). Here we report X-ray diffraction data on phosphatidylethanolamine (PE) dispersions forming highly stable Im3m and Pn3m cubic phases at ambient temperature as a result of a temperature cycling through the Lα - HII transition and complement the structural characterization of the PE phase transitions with thermodynamic data obtained by differential scanning calorimetry and differential scanning densitometry. All studied PEs displayed irreversible two-state Im3m → Pn3m phase transitions in the range ∼75-85°C with enthalpy of ∼100cal/mol. By contrast with the Lα - HII transition, the Im3m → Pn3m transition was not accompanied by a stepwise change of the specific volume. The cubic phases induced in dipalmitoleoyl PE dispersions are of particular interest because of their facile formation, especially in the presence of small amounts of charged lipid admixtures, and long-term stability at physiologically relevant conditions in a broad temperature range around room temperature.

A novel DSC approach for evaluating protectant drugs efficacy against dementia.

Differential scanning calorimetry was applied to evaluate the efficacy of preventive treatments with biologically active compounds of plant origin against neurodegenerative disorder in mice. As we reported recently, large differences exist between the heat capacity profiles of water-soluble brain proteome fractions from healthy animals and from animals with scopolamine-induced dementia: the profiles for healthy animals displayed well expressed exothermic event peaking at 40-45°C, by few degrees above body temperature, but still preceding in temperature the proteome endothermic denaturational transitions; the low-temperature exotherm was completely abolished by the scopolamine treatment. Here we explored this signature difference in the heat capacity profiles to assess the efficacy of preventive treatments with protectant drugs anticipated to slow down or block progression of dementia (myrtenal, ellagic acid, lipoic acid and their combinations, including also ascorbic acid). We found that these neuroprotectants counteract the scopolamine effect and partially or completely preserve the 'healthy' thermogram, and specifically the low-temperature exotherm. These results well correlate with the changes in the cognitive functions of the animals assessed using the Step Through Test for learning and memory. The exothermic event is deemed to be associated with a reversible process of fibrillization and/or aggregation of specific water-soluble brain protein fractions preceding their denaturation. Most importantly, the results demonstrate that the effect of scopolamine and its prevention by protectant substances are clearly displayed in the heat capacity profiles of the brain proteome, thus identifying DSC as a powerful method in drug testing and discovery.

Identification of Specific Effect of Chloride on the Spectral Properties and Structural Stability of Multiple Extracellular Glutamic Acid Mutants of Bacteriorhodopsin.

In the present work we combine spectroscopic, DSC and computational approaches to examine the multiple extracellular Glu mutants E204Q/E194Q, E204Q/E194Q/E9Q and E204Q/E194Q/E9Q/E74Q of bacteriorhodopsin by varying solvent ionic strength and composition. Absorption spectroscopy data reveal that the absorption maxima of multiple EC Glu mutants can be tuned by the chloride concentration in the solution. Visible Circular dichroism spectra imply that the specific binding of Cl- can modulate weakened exciton chromophore coupling and reestablish wild type-like bilobe spectral features of the mutants. The DSC data display reappearance of the reversible thermal transition, higher Tm of denaturation and an increase in the enthalpy of unfolding of the mutants in 1 M KCl solutions. Molecular dynamics simulations indicate high affinity binding of Cl- to Arg82 and to Gln204 and Gln194 residues in the mutants. Analysis of the experimental data suggests that simultaneous elimination of the negatively charged side chain of Glu194 and Glu204 is the major cause for mutants' alterations. Specific Cl- binding efficiently coordinates distorted hydrogen bonding interactions of the EC region and reconstitutes the conformation and structure stability of mutated bR in WT-like fashion.

Recent Progress in Liposome Production, Relevance to Drug Delivery and Nanomedicine.

From their discovery half a century ago and the subsequent appreciation of their clinical utility, liposomes currently hold a recognized position in the mainstream of drug delivery systems. Conventional techniques for liposome preparation and size reduction are simple to implement and do not require sophisticated equipment. However, most of them are not easy to scale-up for industrial liposome production. With several liposomal formulations already on the market, and more in final clinical trials, the industrial scale production of liposomes has become reality, and so the range of liposome preparation methods has been extended by a number of techniques which are increasingly attractive, such as microfluidic hydrodynamic focusing, supercritical fluid processing, freeze-drying and spray-drying. Some of these new techniques generally represent advancements of the conventional methods allowing for scale-up, better reproducibility and process control. This review summarizes patents in the last decade offering new techniques for production of liposomes as related to their application in drug delivery.

Nonlamellar Phases in Cationic Phospholipids, Relevance to Drug and Gene Delivery.

Lipid aggregates have been used as drug carriers for several decades. Recently, nonlamellar liquid crystalline lipid systems have attracted attention as possible drug-delivery vehicles because of their unique nanostructure and physicochemical properties. Here we summarize data on the nonlamellar phase-forming propensity of the cationic phosphatidylcholines (cationic PCs). The class of cationic PCs has been specifically designed and explored for the purpose of nonviral gene delivery. These lipids were found to comprise an attractive cationic lipid class because they are biodegradable, have low toxicities, and in a number of cases, display high transfection activity. Lipids of this class form a variety of polymorphic and mesomorphic phases-lamellar and nonlamellar, depending on the structure of their hydrocarbon chains and especially on the third hydrocarbon chain used to alkylate the PC phosphate group and convert the zwitterionic PC headgroup into a cation. Here we characterize the phase behavior and transfection activity of eight cationic PCs that have been identified as forming nonlamellar phases-inverted hexagonal and cubic. We then demonstrate that those cationic PCs that also form nonlamellar lipoplexes are notably less efficient gene nanocarriers in comparison with the cationic PCs forming lamellar phase lipoplexes.

Enhancing nucleic acid delivery, insights from the cationic phospholipid carriers.

The development of nucleic acid-based drugs has attracted considerable interest in the past two decades as a new category of biologics. A key challenge in successfully achieving the full potential of nucleotide therapeutics is their efficient delivery. Synthetic cationic lipids are currently the most extensively used non-viral nucleotide carriers because of their ability to form complexes with the nucleic acids. Here we examine the properties of oligonucleotide lipoplexes with a particularly noteworthy cationic lipid class, the cationic phosphatidylcholines (PCs) which exhibit low toxicity and good nucleotide delivery efficacy. Studies on a set of cationic PCs reveal the existence of a strong, systematic dependence of their carrier efficiency on the lipid hydrocarbon chain structure. Their activity rises with the increase in chain unsaturation and declines with the increase in chain length. Maximum transfection is detected for ethyl-PC (ePC) with monounsaturated 14:1 chains. The same lipid exhibits maximum activity also in intracellular delivery of siRNA. As the lipid phase behavior is known to depend substantially on the hydrocarbon chain structure, the above relationships validate a view that cationic PC phase properties are an important factor for their activity. Indeed, time-resolved X-ray diffraction studies showed that the rate of the nucleotide release from the lipoplexes, as well as their transfection activity, correlate with the non-lamellar phase progressions detected in mixtures of cationic PCs with biomembrane lipids. These findings emphasize the role of the non-lamellar lipid mesophases in the nucleic acid transport across the cellular membranes and their intracellular release.

Recent patents on nonlamellar liquid crystalline lipid phases in drug delivery.

Lipid aggregates, mainly liposomes, have been used as drug carriers for quite some time. Recently, nonlamellar liquid crystalline lipid systems, such as inverted bicontinuous cubic, hexagonal and sponge mesophases, attract attention as possible drug-delivery vehicles because of their unique microstructure and physicochemical properties. Various bioactive molecules can be solubilized and protected from hydrolysis or oxidation in either the aqueous or the oil lipid phase. Nonlamellar liquid crystalline lipid aggregates have demonstrated high encapsulating capacity and ability for controlled release of drugs. Valuable properties such as a wide solubilization spectrum, high drug payloads, effective encapsulation, stabilization and protection of sensitive drug substances, define the nonlamellar lipid aggregates as promising pharmaceutical carriers. This review summarizes the recent patents on new formulations based on nonlamellar liquid crystalline lipid phases and their efficiency as drug carriers.

Fusion peptides promote formation of bilayer cubic phases in lipid dispersions. An x-ray diffraction study.

Small angle x-ray diffraction revealed a strong influence of the N-terminal influenza hemagglutinin fusion peptide on the formation of nonlamellar lipid phases. Comparative measurements were made on a series of three peptides, a 20-residue wild-type X-31 influenza virus fusion peptide, GLFGAIAGFIENGWEGMIDG, and its two point-mutant, fusion-incompetent peptides G1E and G13L, in mixtures with hydrated phospholipids, either dipalmitoleoylphosphatidylethanolamine (DPoPE), or monomethylated dioleoyl phosphatidylethanolamine (DOPE-Me), at lipid/peptide molar ratios of 200:1 and 50:1. All three peptides suppressed the HII phase and shifted the L(α)-H(II) transition to higher temperatures, simultaneously promoting formation of inverted bicontinuous cubic phases, Q(II), which becomes inserted between the L(α) and H(II) phases on the temperature scale. Peptide-induced Q(II) had strongly reduced lattice constants in comparison to the Q(II) phases that form in pure lipids. Q(II) formation was favored at the expense of both L(α) and H(II) phases. The wild-type fusion peptide, WT-20, was distinguished from G1E and G13L by the markedly greater magnitude of its effect. WT-20 disordered the L(α) phase and completely abolished the HII phase in DOPE-Me/WT-20 50:1 dispersions, converted the Q(II) phase type from Im3m to Pn3m and reduced the unit cell size from ∼38 nm for the Im3m phase of DOPE-Me dispersions to ∼15 nm for the Pn3m phase in DOPE-Me/WT-20 peptide mixtures. The strong reduction of the cubic phase lattice parameter suggests that the fusion-promoting WT-20 peptide may function by favoring bilayer states of more negative gaussian curvature and promoting fusion along pathways involving Pn3m phase-like fusion pore intermediates rather than pathways involving H(II) phase-like intermediates.

Preparation, biodistribution and neurotoxicity of liposomal cisplatin following convection enhanced delivery in normal and F98 glioma bearing rats.

The purpose of this study was to evaluate two novel liposomal formulations of cisplatin as potential therapeutic agents for treatment of the F98 rat glioma. The first was a commercially produced agent, Lipoplatin™, which currently is in a Phase III clinical trial for treatment of non-small cell lung cancer (NSCLC). The second, produced in our laboratory, was based on the ability of cisplatin to form coordination complexes with lipid cholesteryl hemisuccinate (CHEMS). The in vitro tumoricidal activity of the former previously has been described in detail by other investigators. The CHEMS liposomal formulation had a Pt loading efficiency of 25% and showed more potent in vitro cytotoxicity against F98 glioma cells than free cisplatin at 24 h. In vivo CHEMS liposomes showed high retention at 24 h after intracerebral (i.c.) convection enhanced delivery (CED) to F98 glioma bearing rats. Neurotoxicologic studies were carried out in non-tumor bearing Fischer rats following i.c. CED of Lipoplatin™ or CHEMS liposomes or their "hollow" counterparts. Unexpectedly, Lipoplatin™ was highly neurotoxic when given i.c. by CED and resulted in death immediately following or within a few days after administration. Similarly "hollow" Lipoplatin™ liposomes showed similar neurotoxicity indicating that this was due to the liposomes themselves rather than the cisplatin. This was particularly surprising since Lipoplatin™ has been well tolerated when administered intravenously. In contrast, CHEMS liposomes and their "hollow" counterparts were clinically well tolerated. However, a variety of dose dependent neuropathologic changes from none to severe were seen at either 10 or 14 d following their administration. These findings suggest that further refinements in the design and formulation of cisplatin containing liposomes will be required before they can be administered i.c. by CED for the treatment of brain tumors and that a formulation that may be safe when given systemically may be highly neurotoxic when administered directly into the brain.

Highly efficient cationic ethylphosphatidylcholine siRNA carrier for GFP suppression in modified breast cancer cells.

AIM: Cationic ethylphosphatidylcholines (ePCs) were evaluated for the delivery of siRNA in modified breast cancer cells. MATERIALS AND METHODS: Dimyristoleoyl-ePC (C14), dioleoyl-ePC (C18), and dilauroyl-ePC (C12) nanoparticles were complexed with siRNA for green fluorescent protein (GFP) suppression in modified MCF-7 breast cancer cells. The kinetics of GFP suppression were followed over the course of 72 hours. RESULTS: C14, which has been previously found to be particularly effective in gene transfection into primary human umbilical artery endothelial cells, was also remarkably effective as siRNA carrier, with an efficacy exceeding that of Lipofectamine RNAiMAX. The C14 toxicity remained comparable to that of RNAiMAX. The efficacy of the other tested cationic ePC formulations was less than that of C14 and RNAiMAX. CONCLUSION: The cationic lipid C14 is a highly efficient siRNA carrier that could be used for the development of new formulations for siRNA delivery into cancer cells. A valuable advantage of the C14 formulations is the fact that they are simple, and do not require adjuvants or complex preparation procedures.

Cubic phases in membrane lipids.

On the basis of data obtained by time-resolved X-ray diffraction, we consider in the present article the occurrence and formation pathways of inverted bicontinuous cubic phases, or bilayer cubic phases, Q (II)(B) , in diluted dispersions of lipids representing major biomembrane lipid classes [phosphatidylethanolamines (PEs), mixtures of PEs and phosphatidylcholines (PCs) with other lipids, glycolipids]. We show that Q (II)(B) formation proceeds much more easily upon cooling from the H(II) phase than upon heating or isothermal conversion from the L(α) phase, thus identifying an indirect but faster route for Q (II)(B) phase induction in lipids. The data collected consistently show that the ability to convert into cubic phase upon temperature cycling appears to be a general property of all lipids exhibiting an L(α) ↔ H(II) phase transition. Admixtures of charged phospholipids, both anionic and cationic, strongly facilitate Q (II)(B) formation in PEs. Their effect may be attributed to increased electrostatic repulsion between the lipid bilayers that reduces the unbinding energy and facilitates the dissipation of the L(α) phase required for its conversion into bilayer cubic phase.

Recent patents in cationic lipid carriers for delivery of nucleic acids.

Gene therapy is a medical technique intended for treatment of disorders caused by defective, missing, or overexpressing genes. Efficient delivery vectors are necessary in order to transport genetic material to the target cells. Such vectors include viral and non-viral carriers. Viral vectors transfect cells efficiently, however risks associated with their use have limited their clinical applications. Nonviral delivery systems are safer, easier to prepare, more versatile and cost effective. However, their transfection efficiency still falls behind that of the viral vectors. Considerable research into nonviral gene delivery has been conducted in the last two decades on synthetic soft materials such as cationic lipids, polymers, surfactants, and dendrimers as prospective nucleotide carriers for gene delivery. So far, cationic lipids are the most widely used constituents of nonviral gene carriers, with multiple strategies employed to improve their in vitro and in vivo transfection. Efforts in synthesizing new cationic lipids were not fully successful in closing the gap between the efficiency of the viral vectors and that of binary cationic lipid/DNA complexes. Current efforts for improving lipofection efficiency are focused on the development of multicomponent carriers including cationic lipids as key constituents. This review summarizes the recent patents on new cationic lipids as well as on multicomponent formulations enhancing their efficiency as nucleotide carriers.

Quantitation of cholesterol incorporation into extruded lipid bilayers.

Cholesterol incorporation into lipid bilayers, in the form of multilamellar vesicles or extruded large unilamellar vesicles, has been quantitated. To this aim, the cholesterol contents of bilayers prepared from phospholipid:cholesterol mixtures 33-75 mol% cholesterol have been measured and compared with the original mixture before lipid hydration. There is a great diversity of cases, but under most conditions the actual cholesterol proportion present in the extruded bilayers is much lower than predicted. A quantitative analysis of the vesicles is thus required before any experimental study is undertaken.