Phospholipid Molar Ratio Research Articles

Self-assembled fisetin-phospholipid complex: Fisetin-integrated phytosomes for effective delivery to breast cancer

Nowadays, fisetin (FIS) is extensively studied as potent anticancer surrogate with a multitarget actions against various types of cancers including breast cancer. However, its poor aqueous solubility handicapped its clinical utility. The current work endeavored, for the first time, to develop FIS phytosomes (FIS-PHY) for improving its physicochemical properties and subsequently its anticancer activity. Optimization of FIS- phytosomes involved different preparation techniques (Thin film hydration and ethanol injection) and different FIS: phospholipid molar ratios (1:1, 1:2, and 1:3). Complex formation was confirmed by complexation efficiency, infrared spectroscopy (IR), solubility studies and transmission electron microscope. The optimized FIS-PHY of 1:1 M ratio (PHY1) exhibited a nanometric particle size of 233.01 ± 9.46 nm with homogenous distribution (PDI = 0.27), negative zeta potential of − 29.41 mV, 100% complexation efficiency and controlled drug release over 24 h. In-vitro cytotoxicity study showed 2.5-fold decrease in IC50 of PHY1 compared with free FIS. Also, pharmacodynamic studies confirmed the promoted cytotoxicity of PHY1 against breast cancer through modulating TGF-β1/MMP-9 molecular pathways of tumorigenesis. Overall, overcoming FIS drawbacks were successfully achieved through development of innovative biocompatible phytosomal system.

Investigating the mechanochemistry of membrane area asymmetry generation: A combined mass-action and micromechanical study using methyl-beta-cyclodextrin.

Membrane asymmetry is important for cell signaling and is coupled with membrane morphological remodeling protein conformational regulation, sorting, and function. With respect to the lipids composing a bilayer, membrane asymmetry is divided into two contributions including 1) area asymmetry- that is, unequal monolayer surface areas that two apposed leaflets would tend to favor were they uncoupled and 2) compositional asymmetry- that is, different lipid compositions between the leaflets. For in vitro research on membrane asymmetry, methyl-β-cyclodextrin (mbCD) has been previously applied as a lipid-exchange catalyst to tune the compositional asymmetry of synthetic membranes. Inspired by this approach, we opted to study the physical chemistry of 1-component vesicle systems where mbCD was present not as a catalyst but as a reactant. In reaction mixtures at ambient conditions containing nanoscale vesicles, we performed a fluoresence-quenching assay to see that mbCD extracts phospholipids from only the outer leaflets of vesicles and, thereby, must generate area asymmetry in such mixtures. For certain molar ratios, mbCD can fully solubilize all phospholipid and we used 90o light scattering to map this behavior on a two-dimensional phase diagram at ambient temperature. Through temperature-cycling experiments, we showed an irreversibility in mbCD-phospholipid phase behavior. This granted access to stable solutions containing no self-assembled lipid structures with larger molar ratios of phospholipid to mbCD than could be achieved by isothermal reaction conditions. By adjusting the molar ratios of these solutions, we generated both negative and positive curvature in microscopic vesicles (i.e., inward and outward bending, respectively). Tunability of the curvature is consistent with mass-action considerations relating to net flow of phospholipid out of/into vesicles. We believe our approach using mbCD pre-complexed with phospholipid will enhance experimental research on the biophysical consequences of area asymmetry.

Abstract 15: Targeting vismodegib-resistant medulloblastoma using novel hedgehog pathway inhibitor and miR-29b mimic

Abstract The purpose of this study to develop targeted liposomes of MDB5 and miR-29b combination for medulloblastoma (MB) treatment. Due to high heterogeneity and diverse genetic make-up, MB treatment is a challenge. Aberrant activation of hedgehog (Hh) pathway regulates cell growth, cancer stem cell (CSC) proliferation, and tumorigenicity in sonic Hh subgroup of MB. Further, Hh signaling regulates yes-associated protein 1 (YAP1) and glutaminolysis for energy need of tumor cells. GDC-0449 a smoothened receptors (SMO) antagonist treats (SHH)-MB initially, but resistance develops after repeated administration. Cholesterol potentiates Hh pathway independent of SHH by binding directly to the SMO. On the other hand, our RNA-seq and miRNA profiling confirmed downregulation of tumor suppressor miR-29b-3p in human MB samples. miR-29b control genes of apoptosis; p53, MDM2, AKT2, and cholesterol biosynthesis including HMGCR and SREBP1. We recently synthesized a potent GDC-0449 analog MDB5 (SMO inhibitors), which binds strongly to various Smo-mutants compared to GDC-0449. We found that a combination of MDB5 and miR-29b can treat MB synergistically. When we modified miR-29b backbone with 2-O’-methoxyethyl phosphorothioate (OMe-PS-miR-29b-3p), it showed enhanced stability in 50% serum. We designed a cationic lipid MDC2 for liposomal delivery of MDB5 and miR-29b1 simultaneously. Liposomes were prepared using film hydration technique and characterized for particle size and zeta potential, drug loading and release profile, miRNA complexation, stability, and transfection efficiency. These liposomes incorporated 88% MDB5 when the drug to phospholipid molar ratio was kept at 5%. Complete complexation was observed at N/P ratio of 3:1. These liposomes when loaded with green fluorescent protein (GFP) siRNA, resulted in 70% of signal silencing into DAOY MB cells (Shh driven). Liposomal MDB5 had higher cytotoxicity in DAOY cells compared to GDC-0449. Further, MDB5 and OMe-PS-miR-29b loaded liposomes resulted in higher, cytotoxicity and % of apoptosis, G0-G1 phase arrest, and decreased target gene expression in DAOY cells. Combination of drugs to simultaneously target multiple pathways related to Hh, glutaminolysis, cholesterol biosynthesis, and apoptosis has the potential to treat MB. Successful completion of this study will provide a nanomedicine for treating SHH-MB and other brain tumors. Citation Format: Virender Kumar, Bharti Sethi, Vinod Kumar, Timothy R. Mcguire, Don W. Coulter, Ram I. Mahato. Targeting vismodegib-resistant medulloblastoma using novel hedgehog pathway inhibitor and miR-29b mimic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 15.

FORMULATION AND CHARACTERIZATION OF LONG CIRCULATING LIPOSOMES OF ANTI FUNGAL DRUG

Voriconazole is an effective antifungal drug, used as first line treatment for Invasive Aspergillosis. The present work focusses on formulation of PEGylated liposomes to achieve longer circulation in the blood, prevent the liposomes from opsonisation by mononuclear phagocytic system of RES. The higher plasma concentration of voriconazole leads to visual disturbance and dermatological side effect, which are minimized by PEGylation. Compatibility studies like FTIR (Fourier Transform IR) and DSC (Differential Scanning Calorimetry) showed that lipids or drug do not interact and are highly compatible. Liposomes were prepared by thin lipid film hydration method, applying 32 full factorial design with various molar ratio of phospholipids like DPPC /Cholesterol /DSPE mPEG2000. Vesicular size and zeta potential was found within desired range of 100-300 nm to effectively pass through intra venous circulation. Long circulating liposomes were found to have entrapment efficiency of 75-85%. TEM (Transmission Electron Microscopy) showed distinctive spherical shaped vesicles. Long circulatory effect was confirmed from biodistribution study. Stability studies of long circulating liposomes were carried out and Pegylated liposomes successfully showed long circulatory action inside the body and minimized side effect of drug with appreciable antifungal activity determined by microbiological assay (zone of inhibition).

Investigating lipids as a source of chemical exchange-induced MRI frequency shifts.

While magnetic susceptibility is a major contributor to NMR resonance frequency variations in the human brain, a substantial contribution may come from the chemical exchange of protons between water and other molecules. Exchange-induced frequency shifts fe have been measured in tissue and protein solutions, but relatively lipid-rich white matter (WM) has a larger fe than gray matter, suggesting that lipids could contribute. Galactocerebrosides (GC) are a prime candidate as they are abundant in WM and susceptible to exchange. To investigate this, fe was measured in a model of WM lipid membranes in the form of multilamellar vesicles (MLVs), consisting of a 1:2 molar ratio of GC and phospholipids (POPC), and in MLVs with POPC only. Chemical shift imaging with 15% volume fraction of dioxane, an internal reference whose protons are assumed not to undergo chemical exchange, was used to remove susceptibility-induced frequency shifts in an attempt to measure fe in MLVs at several lipid concentrations. Initial analysis of these measurements indicated a necessity to correct for small unexpected variations in dioxane concentration due to its effect on the water frequency shift. To achieve this, the actual dioxane concentration was inferred from spectral analysis and its additional contribution to fe was removed through separate experiments which showed that the water-dioxane frequency shift depended linearly on the dioxane concentration at low concentrations with a proportionality constant of -0.021 ± 0.002 ppb/mM in agreement with published experiments. Contrary to expectations and uncorrected results, for GC + POPC vesicles, the dependence of the corrected fe on GC concentration was insignificant (0.023 ± 0.037 ppb/mM; r2 = 0.085, p > 0.57), whereas for the POPC-only vesicles a small but significant linear increase with POPC concentration was found: 0.044 ± 0.008 ppb/mM (r2 = 0.877, p < 0.01). These findings suggest that the exchange-induced contribution of lipids to frequency contrast in WM may be small. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Differential immune responses to HIV-1 envelope protein induced by liposomal adjuvant formulations containing monophosphoryl lipid A with or without QS21

Liposomes have shown promise as constituents of adjuvant formulations in vaccines to parasitic and viral diseases. A particular type of liposomal construct, referred to as Army Liposome Formulation (ALF), containing neutral and anionic saturated phospholipids, cholesterol, and monophosphoryl lipid A (MPLA), has been used as an adjuvant for many years. Here we investigated the effects of physical and chemical changes of ALF liposomes on adjuvanted immune responses to CN54 gp140, a recombinant HIV-1 envelope protein. While holding the total amounts of liposomal MPLA and the gp140 antigen constant, different liposome sizes and liposomal MPLA:phospholipid molar ratios, and the effect of adding QS21 to the liposomes were compared for inducing immune responses to the gp140. For liposomes lacking QS21, higher titers of IgG binding antibodies to gp140 were induced by small unilamellar vesicle (SUV) rather than by large multilamellar vesicle (MLV) liposomes, and the highest titers were obtained with SUV having the MPLA:phospholipid ratio of 1:5.6. ALF plus QS21 (ALFQ) liposomes induced the same maximal binding antibody titers regardless of the MPLA:phospholipid ratio. ALF MLV liposomes induced mainly IgG1 and very low IgG2a antibodies, while ALF SUV liposomes induced IgG1≥IgG2a>IgG2b antibodies. Liposomes containing QS21 induced IgG1>IgG2a>IgG2b>IgG3 antibodies. ELISPOT analysis of splenocytes from immunized mice revealed that ALF liposomes induced low levels of IFN-γ, but ALFQ induced high levels. ALF and ALFQ liposomes each induced approximately equivalent high levels of IL-4. Based on antibody subtypes and cytokine secretion, we conclude that ALF liposomes predominantly stimulate Th2, while ALFQ strongly induces both Th1 and Th2 immunity. When CN54 gp140 was adjuvanted with either ALF or ALFQ liposomes, antibodies were induced that neutralized two HIV-1 tier 1 clade C strain pseudoviruses.

Preparation of transfersomes encapsulating sildenafil aimed for transdermal drug delivery: Plackett–Burman design and characterization

The aim of this work was to study the effect of different processing and formulation parameters on the preparation of sildenafil (SD) transfersomes utilizing the Plackett–Burman design. The drug to phospholipid molar ratio (X1), phospholipid to surfactant ratio (X2), hydrophilic–lipophilic balance of the surfactant (X3), hydration medium pH (X4), hydration time (X5) and the temperature of hydration (X6) were investigated to study their effect on the vesicle size (Y1) and entrapment efficiency (EE) of the drug (Y2). The preparation conditions were optimized to minimize the vesicle size and maximize the EE. The prepared transfersomes were also subjected to zeta potential measurements, morphological and physicochemical characterization. The combinations of factors that achieve the optimum desirability were identified. An optimized formulation was prepared and characterized once more for its vesicle size, EE, in vitro permeation and deformability index. The results revealed that both X3 and X6 had a pronounced effect on Y1, while X1 and X4 showed a significant effect on Y2. Morphological and physicochemical study confirmed the transfersomes spherical shape and compatibility of the formulation ingredients. The formulation with optimum desirability showed EE and vesicle size of 97.21% and 610 nm, respectively. In vitro permeation of the drug-loaded transfersome showed more than 5-fold higher permeation rate compared with drug suspension. Deformability index verified elasticity of the preparation. The significant variables could be optimized again to produce smaller vesicle size that could increase SD permeation from transdermal delivery systems loaded drug optimized transfersomes.

Liposome electrokinetic chromatography based in vitro model for early screening of the drug-induced phospholipidosis risk

Drug-induced phospholipidosis (PLD) is a storage disorder of lysosomes characterized by the excessive accumulation of phospholipids as a result of improper medical treatments. Although few evidences have supported that PLD can induce significant pathological consequences, this potential toxicity indeed can put off the drug discovery process. In this research, a high-throughput liposome electrokinetic chromatography (LEKC) method was validated to evaluate the PLD risk of drug candidates by screening drug-phospholipid interaction, which correlates to the phospholipidosis inducing risk. A statistical analysis based on the Spearman's correlation test showed that the retention factors (log k) of the tested drugs in the LEKC system and the literature reported in vivo and in vitro PLD data were highly correlated. In order to investigate the predictability of LEKC, the effect of liposome composition such as the molar ratio of phospholipids and the addition of cholesterol were also discussed in this study. The results indicated that the LEKC method could offer a fast, reliable and cost-effective screening tool for early prediction of the PLD inducing potential of drug candidates.

Mechanisms underlying the antifungal resistance in Candida albicans

Various antifungal agents have been used in the treatment of Candida albicans infection.As a common opportunistic pathogen,Candida albicans has shown an increasing antifungal resistance year by year,which are believed to result from the collaborative interaction between multiple complex mechanisms,including changes in ergosterol biosynthesis,enhancement of drug efflux,alterations in the molar ratio of phospholipid to sterol in cell membrane,glucan in cell wall and in DNA synthesis in cell nuclei,as well as biofilm formation.Active measures should be taken to prevent and control Candida albicans infection. Key words: Candida albicans; Antifungal agents; Drug resistance

Implication of BBM lipid composition and fluidity in mitigated alkaline phosphatase activity in renal cell carcinoma

Previous study has documented reduced alkaline phosphatase (ALP) activity in brush border membrane (BBM) isolated from renal cell carcinoma (RCC). Diminished activity of ALP is associated with alteration in both increased K(m) as well as decreased V(max) of enzyme suggests that there may be a change in the conformation of enzyme as well as decreased number of ALP active molecules. The present study was conducted to find out any role of BBM lipid composition and its fluidity in diminished activity of alkaline phosphatase in renal cell carcinoma. Total phospholipids and glycolipids were significantly augmented in BBM from RCC as compared to control. Fractional analysis of total phospholipids revealed significantly increased phosphatidylethanolamine. Decreased fractions of sphingomyelin and phosphatidylinositol were observed. Cholesterol-to-total phospholipid molar ratios in tumor BBM was a significantly lower in tumor BBM. A significant reduction in polarization and microviscosity was found in BBM from RCC. Therefore, we conclude that alteration in membrane lipid composition and fluidity may play a substantial role in reduced activity of ALP in RCC.

Therapeutic efficacy of a lipid-based prodrug of mitomycin C in pegylated liposomes: Studies with human gastro-entero-pancreatic ectopic tumor models

BackgroundA mitomycin-C lipid-based prodrug (MLP) formulated in pegylated liposomes (PL-MLP) was previously reported to have significant antitumor activity and reduced toxicity in mouse tumor models (Clin Cancer Res 12:1913–20, 2006). MLP is activated by thiolysis releasing mitomycin-C (MMC) which rapidly dissociates from liposomes. The purpose of this study was to examine the plasma stability, pharmacokinetics, and antitumor activity of PL-MLP in mouse models of human gastroentero-pancreatic tumors. MethodsMLP was incorporated with almost 100% efficiency in pegylated liposomes composed of hydrogenated phosphatidylcholine, with or without cholesterol (Chol). Mean vesicle size was 45–65nm for liposome preparations downsized by homogenization, and 80–100nm when downsized by extrusion, the latter displaying narrower polydispersity. MLP to phospholipid mole ratio was 5% (~20μg MMC-equivalents/μmol). Therapeutic studies were carried out in the N87 gastric carcinoma (Ca), HCT15 colon Ca, and Panc-1 pancreatic Ca models implanted s.c. in CD1 nude mice. Treatment was administered i.v. in mice with established tumors. ResultsPL-MLP was very stable when incubated in plasma, and whole blood with a maximum of 5% release and activation to free MMC after 24h. In the presence of a strong reducing agent (dithiotreitol), MLP was almost entirely activated to free MMC. Pharmacokinetic studies revealed major differences in plasma clearance between free MMC and PL-MLP. The longest half-lives were observed for extruded and Chol-containing preparations. Using a liposome radiolabel, it was found that the plasma levels of liposomes and prodrug were nearly superimposable confirming the absence of drug leakage in circulation. In vivo prodrug activation was significantly increased by co-injection of a large dose of a biocompatible reducing agent, N-acetylcysteine. PL-MLP was significantly more effective in delaying tumor growth and resulted in more tumor regressions than irinotecan in the N87 and HCT15 models, and than gemcitabine in the Panc-1 model. PL-MLP was ~3-fold less toxic than free MMC at MMC-equivalent doses, and displayed mild myelosuppression at therapeutic doses. ConclusionsDelivery of MLP in pegylated liposomes is more effective than conventional chemotherapy in the treatment of gastroentero-pancreatic ectopic tumor models, and may represent an effective tool for treatment of these malignancies in the clinical setting with improved safety over free MMC. Reducing agents offer a tool for controlling in vivo prodrug release.

Abstract B65: Molecular basis of mitigated alkaline phosphatase activity in renal cell carcinoma

Abstract The present study was conducted to find out the molecular basis of aberrant expression/activity of ALP in BBM from RCC in comparison to normal renal BBM. Thirty histopathologically confirmed cases of RCC were included in the present investigation. A significant decrease was observed in the activity of alkaline phosphatase in BBM isolated from RCC as compared to BBM isolated from normal renal parenchyma. Kinetic studies revealed that diminished activity of alkaline phosphatase in BBM isolated from RCC was fraternized with decrease in maximal velocity (Vmax) and increase in affinity constant (Km) of the enzyme. SDS-PAGE analysis illustrated the less expression of higher molecular weight BBM proteins in RCC. Incubation of native polyacrylamide gel with ALP specific BCIP/NBT dye showed reduced expression of ALP in BBM from RCC as compared to BBM from normal kidney tissue. In addition, Western blot analysis using anti-ALP antibody also confirmed the reduced expression of ALP protein in BBM of RCC. This finding was corroborated by immunofluorescence and immunohistochemical study. Further, semiquantitative mRNA expressions of ALP demonstrated a markedly reduced expression of ALP gene in RCC as compared to normal kidney tissue. No mutation in C-terminal end of ALP gene was detected. Therefore the C-terminal end of the ALP gene might not play a role in decreased activity of ALP in RCC. Further, to elucidate the role of secondary messengers viz. cAMP and intracellular Ca2+ in the reduction of ALP activity/expression. cAMP and intracellular Ca2+ levels were measured in RCC. The cAMP level was found to be significantly increased while the intracellular Ca2+ was significantly decreased in RCC as comparison to normal renal tissue indicating their possible role in reduced activity of ALP in RCC. Antecedent studies have suggested that ALP is directly correlated with membrane lipid composition and fluidity. In view of this, we determined the role of membrane lipid composition and fluidity of BBM in decreased activity of ALP in BBM from RCC. The fluorescence anisotropy (rDPH) and polarization (pDPH) of diphenyl hexatriene were significantly decreased. These parameters are inversely proportional to membrane fluidity. Therefore, this finding was suggestive of increased membrane fluidity in BBM isolated from RCC. Cholesterol has condensing effect on phospholipid and significantly reduced membrane fluidity. The molar ratio of cholesterol to phospholipid in BBM was of utmost importance to determine and compare the membrane fluidity. BBM lipid composition analysis revealed that phospholipids and glycolipids were increased whereas, cholesterol was decreased in BBM of RCC as compared to that in normal renal BBM. Cholesterol to phospholipid ratio was found to be decreased in BBM from RCC. The differential membrane phospholipid was also found altered in BBM from RCC. Therefore, the alteration in membrane lipids, cholesterol to phospholipid molar ratio and membrane fluidity might be playing an important role in decreased activity of ALP in RCC as compared to normal renal tissue. Results from the present study revealed those molecular events that might play a collaborative role in decreased activity of ALP in RCC. Our data suggest that the reduced activity of ALP in BBM isolated from RCC is due to decreased expression of ALP gene at transcription level. Besides this membrane fluidity and lipid composition might also contribute to the decrease activity of ALP. In the above mechanisms secondary messengers such as cAMP and intracellular Ca2+ may be having contributory role. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B65.

A Liposomal Formulation Able to Incorporate a High Content of Paclitaxel and Exert Promising Anticancer Effect

A liposome formulation for paclitaxel was developed in this study. The liposomes, composed of naturally unsaturated and hydrogenated phosphatidylcholines, with significant phase transition temperature difference, were prepared and characterized. The liposomes exhibited a high content of paclitaxel, which was incorporated within the segregated microdomains coexisting on phospholipid bilayer of liposomes. As much as 15% paclitaxel to phospholipid molar ratio were attained without precipitates observed during preparation. In addition, the liposomes remained stable in liquid form at 4°C for at least 6 months. The special composition of liposomal membrane which could reduce paclitaxel aggregation could account for such a capacity and stability. The cytotoxicity of prepared paclitaxel liposomes on the colon cancer C-26 cell culture was comparable to Taxol. Acute toxicity test revealed that LD50 for intravenous bolus injection in mice exceeded by 40 mg/kg. In antitumor efficacy study, the prepared liposomal paclitaxel demonstrated the increase in the efficacy against human cancer in animal model. Taken together, the novel formulated liposomes can incorporate high content of paclitaxel, remaining stable for long-term storage. These animal data also demonstrate that the liposomal paclitaxel is promising for further clinical use.

家族性lecithin:cholesterol acyltransferase欠損症の一例

Lecithin: cholesterol acyltransferase (LCAT) is an enzyme that catalyzes the esterifying reaction of cholesterol in plasma high density lipoprotein (HDL). Deficiency of LCAT is a rare hereditary disease characterized by several clinical symptoms such as proteinuria, corneal opacity, and anemia due to a shortened life span of erythrocytes. In this communication, we report a case of 40 year-old female patient of LCAT deficiency. She visited a hospital for work-up of proteinuria, corneal opacity and anemia. Activity of her serum LCAT was found to be extremely low, and characteristic changes in plasma lipids due to deficiency of LCAT was observed: those were marked decreases in HDL-cholesterol, degree of esterification in serum cholesterol, and apoprotein A-I, A-II, B and C-II levels. The diagnosis of LCAT deficiency was finally made. We studied about histopathological changes in the patient's kidney, and erythrocyte membrane lipid composition and fluidity. Histopathological findings in renal biopsy were follows: a) Light microscopy showed spherical deposits stained with periodic acid-Schiff in mesangial matrix and adjacent capillary loops, and hyaline deposits in arterioles, b) Electron microscopy showed vacuoles in mesangial matrix and along the glomerular basement membranes. In erythrocyte membrane lipids, increase of cholesterol to phospholipid molar ratio was evident, being accompanied by changes in phospholipid fractions: increase of phosphatidylcholine, and decreases of phosphatidylethanolamine, sphingomyelin and lysophosphatidylcholine. In phospholipid acyl chains, increase of C18:2 and decreased of C18:1 were evident in the patient. Erythrocyte membrane fluidity was found to be decreased in the patient in a measurement by pyrene, probably being related to the changes in membrane lipid composition.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved therapeutic index of a mitomycin-C (MMC) prodrug formulated in pegylated liposomes: Studies in human xenograft tumor models of gastrointestinal (GI) origin.

e13628 Background: A MMC lipid-based prodrug (MLP) formulated in pegylated liposomes (PL-MLP) was previously reported to have significant antitumor activity and reduced toxicity in mouse tumor models (Clin Cancer Res 12:1913-20, 2006). MLP is activated by thiolysis releasing MMC which rapidly dissociates from liposomes. The purpose of this study was to examine the plasma stability, pharmacokinetics, in vivo toxicity, and antitumor activity of PL-MLP in human xenograft models of GI-derived tumors. Methods: MLP was incorporated with nearly 100% efficiency in pegylated liposomes with hydrogenated phosphatidylcholine as the main component with or without cholesterol (Ch). Mean vesicle size was 45-65 nm for liposome preparations downsized by homogenization, and 80-100 nm for those downsized by extrusion, the latter displaying narrower polydispersity. MLP to phospholipid molar ratio was 5% (∼20 μg MMC-equivalents/μmol). Therapeutic studies were carried out in 3 tumor models (HCT15 colon Ca, N87 gastric Ca, and Panc-1 pancreatic Ca) implanted s.c. in CD1 nude mice. Treatment was administered i.v. into mice with established tumors. Results: PL-MLP was very stable when incubated in plasma, and whole blood with a maximum of 5% of MLP undergoing activation to free MMC. In the presence of a strong reducing agent (dithiothreitol, 0.5 mM), MLP was massively activated to free MMC. Pharmacokinetic studies in mice revealed huge differences in key parameters (t, Cl, Vss) between free MMC and PL-MLP. The longest t were observed for extruded and/or Ch-containing preparations. PL-MLP was ∼3-fold less toxic than free MMC at MMC-equivalent doses. PL-MLP was significantly more effective in delaying tumor growth and resulted in more tumor regressions than irinotecan in the HCT15 and N87 models, and than gemcitabine in the Panc-1 model. Conclusions: Delivery of MLP in pegylated liposomes is more effective than conventional chemotherapy used in the treatment of GI-derived tumor models, and may represent an effective tool for treatment of these malignancies in the clinical setting with improved safety over free MMC. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Teva Teva

Use of Cholesterol in Sperm Cryopreservation: Present Moment and Perspectives to Future

Sperm cryosurvival rates are not optimal for most species. Therefore, new cryopreservation strategies are needed with the objective of increasing the number of surviving sperm and the quality of those sperm after thawing. Cholesterol plays important roles in many sperm functions, including effects on membrane properties. One of these effects is to stabilize membranes at low temperatures. Thus, species that produce sperm which possess high membrane cholesterol : phospholipid ratios are more resistant to cold shock than sperm with low cholesterol : phospholipid ratios. Therefore, increasing the cholesterol content of sperm membranes may be a strategy that can improve sperm quality after freeze-thawing. In this review, information is presented related to using cyclodextrins pre-loaded with cholesterol for cryopreserving sperm from different species. The topics discussed include both in vitro and in vivo assessments of sperm quality after cryopreservation, as well as how increasing sperm cholesterol content affects other sperm functions.

Development of simple thiol-reactive liposome formulations, one-step analysis and physicochemical characterization

The aim of this study was to synthesize simple thiol-reactive conjugates from maleimide and lipoamines (stearylamine or oleylamine) and to develop a simple, fast and low-cost method for the preparation of lyophilized general-purpose thiol-reactive liposomes. A formulation of egg phosphatidylcholine-dipalmitoylphoshatidylglycerol (9:0.1 molar ratio) was developed and characterized. Freeze-drying methodology was established to produce a stock of liposomes and the physicochemical characteristics of the reconstituted liposomes were compared with those of the initial preparation. The physicochemical properties (size and zeta potential) of the new liposomal formulations were studied. High-performance thin-layer chromatography coupled to a flame ionization detector was applied for one-step analysis of the liposomal components and for determining the maleimide-lipoamine conjugates phospholipid molar ratio. The differences concerning the incorporation efficiency of the synthetic conjugates into liposomes were discussed on the basis of their conformational properties. The small difference in structure between the two thiol-reactive conjugates (i.e., the C18 alkyl chain double bond) causes a considerable difference in phospholipids packing of the resulting lipidic bilayers of the liposomes; the conformational bending of conjugate maleimide-oleylamine may contribute to the final architecture of liposomes.

Effects of acceptor composition and mechanism of ABCG1-mediated cellular free cholesterol efflux

Among the known mechanisms of reverse cholesterol transport (RCT), ATP binding cassette transporter G1 (ABCG1)-mediated free cholesterol (FC) transport is the most recent and least studied. Here, we have characterized the efficiencies of different acceptors using baby hamster kidney (BHK) cells transfected with human ABCG1 cDNA, which is inducible upon treatment with mifepristone. When normalized on particle number and particle surface area, the acceptor efficiency for FC efflux was as follows: small unilamellar vesicles (SUV)>LDL>reconstituted HDL>HDL2 = HDL3. Based on phospholipid content, the order was reversed. ABCG1 also mediated phospholipid efflux to human serum and HDL3. ABCG1-mediated FC efflux correlated significantly with a number of HDL subfractions and components in serum collected from 25 normolipidemic individuals: apolipoprotein A-II (apoA-II) (r2 = 0.7), apolipoprotein A-I (apoA-I) (r2 = 0.5), HDL-C (r2 = 0.4), HDL-PL (r2 = 0.4), α-2 HDL (r2 = 0.4), and preβ HDL (r2 = 0.2). ABCG1 did not enhance influx of FC or cholesteryl oleyl ether (COE) when cells were incubated with radiolabeled HDL3. ABCG1 expression did not increase the association of HDL3 with cells. Compared with control cells, ABCG1 expression significantly increased the FC pool available for efflux and the rate constant for efflux. In conclusion, composition and particle size determine the acceptor efficiency for ABCG1-mediated efflux. ABCG1 increases cell membrane FC pools and changes its rate of desorption into the aqueous phase without enhancing the association with the acceptor.

Solid-State NMR Study of the Human Peripheral Cannabinoid Receptor CB2 in Lipid Bilayers

Human peripheral cannabinoid receptor CB2 expressed in E. coli. has been purified, and successfully reconstituted in functional form into lipid bilayers composed of phosphatidylcholine, phosphatidylserine, and cholesteryl hemisuccinate (CHS). The reconstitution was carried out by detergent removal from the CB2-lipid-detergent mixed micelles on an adsorbent column, or by rapid dilution below the critical micelle concentration followed by washing on a concentrator. Proteoliposomes prepared at the CB2:phospholipid molar ratio of 1:600 showed the following basic physical properties: Free of detergents (as analyzed by high-resolution 1H NMR), homogeneity of the CB2-to-lipid ratio over the proteoliposome particles (by sucrose gradient centrifugation), unilamellarity with a mean diameter of ∼150-200 nm (by dynamic light scattering), and functional integrity (by G-protein activation assay). Using the proteoliposomes, CB2-lipid interactions were investigated by solid-state NMR. Order parameters of the phospholipid acyl chains measured by 2H quadrupolar splittings indicated that CHS, a detergent-soluble analog of cholesterol, preferentially interacts with the 1-palmitoyl-2-oleoyl-sn-glycero-3-phospholipids over CB2. By probing 1H NMR saturation transfer, evidence for CB2-lipid interactions at the lipid acyl chains and less significant interactions at the glycerol backbone and the headgroups were observed. 1H spin-lattice relaxation rates decreased notably at the acyl-chains upon CB2 incorporation, indicating reduced motion on the nanosecond timescale corresponding to the restriction of phospholipid wobbling about the bilayer normal. Structure-function relationships in view of the role of interactions between CB2 and anionic phosphatidylserine in activation of G-protein will also be discussed.

Use of high‐pressure freeze fixation and freeze fracture electron microscopy to study the influence of the phospholipid molar ratio in the morphology and alignment of bicelles

The high-pressure freeze fixation and freeze fracture electron microscopy techniques were combined with the (31)P nuclear magnetic resonance to study the morphological transitions of two different dimyristoyl-phosphatidilcholine/dihexanoyl-phosphocholine aggregates by the effect of temperature. Through these techniques, the relationship between magnetic alignment and the morphology of alignable and non-alignable aggregates was evaluated. The micrographs related to the non-alignable dimyristoyl-phosphatidilcholine/dihexanoyl-phosphocholine sample presented rounded objects at a temperature below the dimyristoyl-phosphatidilcholine phase transition (T(m)) and, above this temperature an increase of viscosity was followed by the appearance of large elongated aggregates. The micrographs related to the alignable dimyristoyl-phosphatidilcholine/dihexanoyl-phosphocholine sample presented discoidal objects below T(m). Above T(m), when the best alignment was achieved, the images showed large areas of lamellar stacked bilayers and the presence of some multilamellar vesicles. Our results reveal that the composition of the aggregates is a key factor determining the morphological transitions of the bicellar systems. Understanding of the rules governing these transitions is crucial to modulate characteristics of these systems and to adequate them for different applications.