Lipid composition influences the membrane-disrupting activity of antimicrobial methacrylate co-polymers

Amphiphilic methacrylate co-polymers recently demonstrated antimicrobial activity. To understand their activity mechanism, we prepared three homologous methacrylate co-polymers with activity ranging from inactive (MMA) over specifically active (EMA) to non-specifically active (BMA) against bacteria and human erythrocytes. Fluorescent dye leakage assays were used to characterize their membrane-disrupting activity against liposomes of different compositions. From bacterial membrane-mimicking liposomes (composed of Escherichia coli extract or 20:80 DOPG/DOPE), the two active forms, EMA and BMA, caused more dye leakage than the inactive MMA, which mirrors their antibacterial activity trend. From mammalian membrane-mimicking liposomes (composed of DOPC or 20:80 DOPG/DOPC), the highly hemolytic BMA caused significantly more leakage than MMA and EMA, which mirrors its hemolytic activity trend. Moreover, to dissect the effect of intrinsic membrane curvature from that of membrane charge, we used a ternary membrane with constant charge and tunable intrinsic curvature. Specifically, we used membranes composed of DOPG/DOPE/DOPC with constant DOPG content and varying DOPE/DOPC ratio. To significantly disrupt this model, methacrylate co-polymers with different activity profiles required a different minimum threshold DOPE content. In contrast, variation in DOPG/DOPC ratio at constant DOPE concentration did not show a similar influence on the selective membrane-disrupting activity of these co-polymers. Our results suggested that the intrinsic membrane curvature, rather than membrane charge, may play a major role in the selective membrane-disrupting activity of methacrylate co-polymers. Since more PE lipids exist in bacterial membranes than in eukaryotic membranes, our results imply that negative-intrinsic-curvature lipids such as PE may contribute to the selective antimicrobial activity.     

Electron microscopic examination of the interaction between human erythrocytes and liposomes containing DC-Cholesterol

Human erythrocytes treated with cationic liposomes containing DOPC/DC cholesterol (2∶3 mol/mol) or DOPE/DC cholesterol (2∶3 mol/mol) are studied by electron microcopy. DOPE-containing liposomes exhibit a markedly greater ability to interact with the erythrocyte surface and to initiate a mosaic structure represented by smooth and rough surfaces on freeze-fractures of the erythrocyte plasma membrane. The interaction between DOPE/DC-cholesterol-containing liposomes and the plasma membrane provides the basis for using these liposomes to deliver DNA into the cell cytoplasm. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 7, pp. 83–86, July, 1996     

Organic‐Inorganic Hybrid Membranes for Removal of Benzene from an Aqueous Solution by Pervaporation

Summary: Preparation of organic‐inorganic hybrid membranes and their pervaporation permeation and separation characteristics for an aqueous solution of 0.05 wt.‐% benzene are described. In this study, we prepared organic‐inorganic hybrid membranes by the sol‐gel reaction of tetraethoxysilane (TEOS) as an inorganic component with poly(methyl methacrylate‐co‐vinyltriethoxysilane) (P(MMA‐co‐VTES)) and poly(butyl methacrylate‐co‐vinyltriethoxysilane) (P(BMA‐co‐VTES)) as organic components. When an aqueous solution of dilute benzene (0.05 wt.‐%) was permeated through the P(MMA‐co‐VTES)/TEOS and P(BMA‐co‐VTES)/TEOS hybrid membranes, the benzene concentration in the permeate through all hybrid membranes was higher than that in the feed solution. This result demonstrates that these hybrid membranes are benzene selective for an aqueous solution containing dilute benzene. The benzene/water selectivity of the P(BMA‐co‐VTES)/TEOS hybrid membrane was about 20 times higher than that of the P(MMA‐co‐VTES)/TEOS hybrid membrane. Specifically, the P(BMA‐co‐VTES)/TEOS hybrid membrane with a TEOS content of 75 mol‐% showed the highest benzene/water selectivity. The benzene/water selectivity of the hybrid membranes depended significantly on the cross‐linked structures formed by the sol‐gel reaction of VTES and TEOS.

Effects of the TEOS content on the sorption selectivity (○) and the diffusion selectivity (?) for an aqueous solution of 0.05 wt.‐% benzene through P(MMA‐co‐VTES)/TEOS and P(BMA‐co‐VTES)/TEOS hybrid membranes at 40 °C.     

Inhibition of Sendai virus fusion with phospholipid vesicles and human erythrocyte membranes by hydrophobic peptides.

Hydrophobic di- and tripeptides which are capable of inhibiting enveloped virus infection of cells are also capable of inhibiting at least three different types of membrane fusion events. Large unilamellar vesicles (LUV) of N-methyl dioleoylphosphatidylethanolamine (N-methyl DOPE), containing encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and/or p-xylene bis(pyridinium bromide) (DPX), were formed by extrusion. Vesicle fusion (contents mixing) and leakage were then monitored with the ANTS/DPX fluorescence assay. Sendai virus fusion with lipid vesicles and Sendai virus fusion with human erythrocyte membranes were measured by following the relief of fluorescence quenching of virus labeled with octadecylrhodamine B chloride (R18), a lipid mixing assay for fusion. This study found that the effectiveness of the peptides carbobenzoxy-L-Phe-L-Phe (Z-L-Phe-L-Phe), Z-L-Phe, Z-D-Phe, and Z-Gly-L-Phe-L-Phe in inhibiting N-methyl DOPE LUV fusion or fusion of virus with N-methyl DOPE LUV also paralleled their reported ability to block viral infectivity. Furthermore, Z-D-Phe-L-PheGly and Z-Gly-L-Phe inhibited Sendai virus fusion with human erythrocyte membranes with the same relative potency with which they inhibited vesicle-vesicle and virus-vesicle fusion. The evidence suggests a mechanism by which these peptides exert their inhibition of plaque formation by enveloped viruses. This class of inhibitors apparently acts by inhibiting fusion of the viral envelope with the target cell membrane, thereby preventing viral infection. The physical pathway by which these peptides inhibit membrane fusion was investigated. 31P nuclear magnetic resonance (NMR) of proposed intermediates in the pathway for membrane fusion in LUV revealed that the potent fusion inhibitor Z-D-Phe-L-PheGly selectively altered the structure (or dynamics) of the hypothesized fusion intermediates and that the poor inhibitor Z-Gly-L-Phe did not. One possible interpretation of these 31P NMR results was that the inhibitory peptide stabilized a membrane structure with a large radius of curvature, when the fusion pathway demanded a membrane defect with a small radius of curvature. This hypothesis was tested by determining the influence of an inhibitory and a noninhibitory peptide on the formation of membraneous structures with small radii of curvature, through ultrasonic irradiation of phospholipid dispersions. The inhibitory peptide prevented the formation of membrane structures with small radii of curvature, while the noninhibitory peptide did not prevent the formation of such structures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Novel Polymer Blends for the Preparation of Membranes for Biohybrid Liver Systems

It was found previously that membranes based on co-polymers of acrylonitrile (AN) and 2-acrylamido-2-methyl-propansulfonic acid (AMPS) greatly stimulated the functionality and survival of primary hepatocytes. In those studies, however, the pure AN–AMPS co-polymer had poor membrane-forming properties, resulting in quite dense rubber-like membranes. Hence, membranes with required permeability and optimal biocompatibility were obtained by blending the AN–AMPS co-polymer with poly(acrylonitrile) homopolymer (PAN). The amount of PAN (P) and AN–AMPS (A) in the blend was varied from pure PAN (P/A-100/0) over P/A-75/25 and P/A-50/50 to pure AN–AMPS co-polymer (P/A-0/100). A gradual decrease of molecular cut-off of membranes with increase of AMPS concentration was found, which allows tailoring membrane permeability as necessary. C3A hepatoblastoma cells were applied as a widely accepted cellular model for assessment of hepatocyte behaviour by attachment, viability, growth and metabolic activity. It was found that the blend P/A-50/50, which possessed an optimal permeability for biohybrid liver systems, supported also the attachment, growth and function of C3A cells in terms of fibronectin synthesis and P-450 isoenzyme activity. Hence, blend membranes based on a one to one mixture of PAN and AN–AMPS combine sufficient permeability with the desired cellular compatibility for application in bioreactors for liver replacement.     

Diblock copolymers with tunable pH transitions for gene delivery

A series of diblock copolymers containing an endosomal-releasing segment composed of diethylaminoethyl methacrylate (DEAEMA) and butyl methacrylate (BMA) were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. The materials were designed to condense plasmid DNA (pDNA) through electrostatic interactions with a cationic poly(N,N-dimethylaminoethyl methacrylate) (DMAEMA) first block. The pDMAEMA was employed as a macro chain transfer agent (macroCTA) for the synthesis of a series in which the relative feed ratios of DEAEMA and BMA were systematically varied from 20% to 70% BMA. The resultant diblock copolymers exhibited low polydispersity (PDI ≤ 1.06) with similar molecular weights (M_n = 19.3-23.1 kDa). Dynamic light scattering (DLS) measurements in combination with ¹H NMR D₂O studies demonstrated that the free copolymers assemble into core-shell micelles at physiological pH. Reduction of the solution pH to values representative of endosomal/lysosomal compartments induced an increase in the net cationic charge of the core through protonation of the DEAEMA residues. This protonation promotes micelle destabilization and exposure of the hydrophobic BMA residues that destabilize biological membranes. The pH value at which this micelle-to-unimer transition occurred was dependent on the hydrophobic content of the copolymer, with higher BMA-containing copolymer compositions exhibiting pH-induced transitions to the membrane-destabilizing state at successively lower pH values. The ability of the diblock copolymers to deliver pDNA was subsequently investigated using a GFP expression vector in two monocyte cell lines. High levels of DNA transfection were observed for the copolymer compositions exhibiting the sharpest pH transitions and membrane destabilizing activities, demonstrating the importance of tuning the endosomal-releasing segment composition.     

Immobilization of DNA on Microporous PVDF Membranes by Plasma Polymerization

Microporous poly(vinylidene fluoride) (PVDF) membranes with different porous surface morphologies were prepared by immersion-precipitation from coagulation baths of different strengths. On these membranes single-strand deoxyribonucleic acid (ss-DNA) was covalently immobilized by a dual-step procedure. First, poly(glycidyl methacrylate) (PGMA) was grafted on PVDF membranes by plasma-induced free radical polymerization. Then, ss-DNA was bonded to PGMA through ring-opening reactions of epoxies with amine to form amino alcohols. The highest attainable graft yield of PGMA on PVDF membrane was 0.3 mg/cm², which was the case when a highly porous PVDF membrane was employed as the substrate. For immobilization of ss-DNA, the yield was found to depend significantly on the reaction temperature and pH. The maximal value was 48.5 μg/cm². Furthermore, adsorption tests of anti-DNA antibody were carried out on membranes with and without immobilized ss-DNA using serum obtained from systemic lupus erythematosus patients. The results indicated that the immobilized DNA could effectively adsorb the antibody in the serum.     

Immobilization of L-Lysine on Microporous PVDF Membranes for Neuron Culture

Microporous poly(vinylidene fluoride) (PVDF) membranes with dense or porous surface were prepared by immersion precipitation of PVDF/TEP solutions in coagulation baths containing different amounts of water. Onto the membrane surface, poly(glycidyl methacrylate) (PGMA) was grafted by plasma-induced free radical polymerization. Then, L-lysine was covalently bonded to the as-grafted PGMA through ring-opening reactions between epoxide and amine to form amino alcohol. The highest attainable graft density of PGMA on a PVDF membrane was 0.293 mg/cm². This was obtained when the reaction was carried out on a porous surface under an optimized reaction condition. For immobilization of L-lysine, the yield was found to depend on the reaction temperature and L-lysine concentration. The maximal yield was 0.226 mg/cm², a value considerably higher than reported in the literature using other immobilization methods. Furthermore, neurons were cultured on L-lysine-immobilized PVDF membranes. The results indicated that these membrane surfaces were suited to the growth of neurons, with a MTT value higher than that of the standard culture dish.     

The compressive deformation of multicomponent microcapsules: Influence of size,membrane thickness,and compression speed

The clinical application of microcapsules for the immunoisolation of living tissue requires knowledge about the mechanical stability of polymer membranes. Microcapsules of 400-1000 μm in diameter were formed through the gelation of sodium alginate/sodium cellulose sulfate droplets through calcium chloride, with the membrane produced via complex coacervation between polyanions and poly(methylene-co-guanidine) hydrochloride. The deformation behavior of these multicomponent microcapsules was investigated by uniaxial compression experiments. Specifically, the influence of the deformation speed, capsule diameter, and membrane thickness on the mechanical properties was evaluated. The bursting force was found to be dependent on the deformation speed. Therefore, the measurement of the bursting work, a speed-independent value of the resistance to high stresses and deformations, was recommended as the most valid for capsule mechanical resistance. Furthermore, the bursting force was positively correlated with membrane thickness only for membrane-radius ratios up to 20%. For thicker membranes, the bursting event occurred because the opposite membranes touched each other, and not, because of insufficient strength. Indeed, the resistance to smaller deformations was positively correlated to the membrane thickness over the whole range of membrane-radius ratios. Moreover, the forces for constant deformation were linearly correlated to the total membrane volume, independently of capsule size and membrane thickness.     

Fabrication and Biocompatibility of an Antimicrobial Composite Membrane with an Asymmetric Porous Structure

A composite slurry from silver ion-substituted nano-hydroxyapatite, titania nano-particles and polyamide 66 (Ag-nHA/TiO₂/PA66) was prepared and used to fabricate a novel antimicrobial membrane with a gradient porous structure for guided bone regeneration (GBR). Subsequently, assays were performed to determine the cytocompatibility, as well as the bone biocompatibility of the prepared membranes. To investigate the cytocompatibility of the Ag-nHA/TiO₂/PA66 membrane, in vitro studies were done with osteoblast-like cells (MG63) and the viability, alkaline phosphatase activity (ALP) and morphology of cells cultured on the membrane were determined. The bone biocompatibility of the membranes was finally assessed in animal experiments, in which nano-hydroxyapatite/polyamide 66 (nHA/PA66) and pure polyamide 66 (PA66) membranes were compared. The in vitro cell-culture experiments showed that Ag-nHA/TiO₂/PA66 antimicrobial membrane evoked good cell affinity and cytocompatibility. The in vivo study showed that Ag-nHA/TiO₂/PA66 asymmetric porous barrier membrane resulted in complete closure of 5-mm bone defects as created in the skull of rats after 8 weeks of implantation. In conclusion, the Ag-nHA/TiO₂/PA66 membrane has the potential to be applied in GBR, especially in infected tissue or areas with high bacteria concentration.     

Interactions between oxicams and membrane bilayers: an explanation for their different COX selectivity

Meloxicam was launched as a major new NSAID for the treatment of arthritis following extensive published research confirming its selectivity for COX-2. Several studies proposed possible explanations for its effectiveness and superior safety profile. The proposed theories included chemical structural relationships between meloxicam and other effective NSAIDs with low gastrointestinal toxic effects. However, other oxicams have similar chemical groups, but despite this, are not considered COX-2 selective drugs and exhibit less gastric tolerance. Hence, the aim of this work was to investigate the interactions between oxicams and biomembrane models as it could influence their resorption from the upper gastrointestinal tract and may affect their local gastromucosal tolerability. The partition of oxicams within membranes was determined by calculating their partition coefficients between liposomes and water. Moreover, their location within the bilayer was determined by fluorescence quenching. Finally, zeta-potential measurements were made to complete the information about the binding behaviour of the oxicams and steady-state anisotropy measurements were made to determine their induced perturbation in membrane structure. These studies proved that, in spite of structural similarities, oxicams present different interactions with membranes making possible a virtual division of the class in two groups. Tenoxicam and piroxicam known as COX-1 inhibitors demonstrated higher partition capacity in liposomes/water systems together with a smaller ability to change the membrane fluidity and surface potential. In contrast lornoxicam and meloxicam, which demonstrated activity against COX-2, have revealed smaller partition capacity in liposomes/water systems together with a higher ability to change the membrane fluidity and surface potential.     

Suramin inhibits macrophage activation by human group IIA phospholipase A2, but does not affect bactericidal activity of the enzyme

Objective:  Suramin is a polysulphonated napthylurea antiprotozoal and anthelminitic drug, which also presents inhibitory activity against a broad range of enzymes. Here we evaluate the effect of suramin on the hydrolytic and biological activities of secreted human group IIA phospholipase A₂ (hsPLA₂GIIA). Materials and Methods:  The hsPLA₂GIIA was expressed in E. coli, and refolded from inclusion bodies. The hydrolytic activity of the recombinant enzyme was measured using mixed dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) liposomes. The activation of macrophage cell line RAW 264.7 by hsPLA₂ GIIA was monitored by NO release, and bactericidal activity against Micrococcus luteus was evaluated by colony counting and by flow cytometry using the fluorescent probe Sytox Green. Results:  The hydrolytic activity of the hsPLA₂ GIIA was inhibited by a concentration of 100 nM suramin and the activation of macrophages by hsPLA₂ GIIA was abolished at protein/suramin molar ratios where the hydrolytic activity of the enzyme was inhibited. In contrast, both the bactericidal activity of hsPLA₂ GIIA against Micrococcus luteus and permeabilization of the bacterial inner membrane were unaffected by suramin concentrations up to 50 μM. Conclusions:  These results demonstrate that suramin selectively inhibits the activity of the hsPLA₂ GIIA against macrophages, whilst leaving the anti-bacterial function unchanged. Received 26 June 2008; returned for revision 8 August 2008; received from final revision 11 September 2008; accepted by J.Skotnicki 8 October 2008     

Influence of red cell surface charge on red cell membrane curvature

The wellknown biconcave shape of resting RBC reflects an intrinsic overall negative spontaneous membrane curvature. It depends to a high degree on the integrity of the spectrin-actin-ankyrin-Band-3 hetero-complex. Alterations of this complex have previously been shown to be associated with shape transitions, which have been abolished by treatment with enzymes reducing the surface charge of the RBC. In this report we show that treatment of erythrocytes with enzymes reducing the surface charge (e.g. neuraminidase, trypsin, chymotrypsin and pronase), consistently exerts a stomatocytogenic effect, i.e. it reduces mean mean curvature. Also the time dependency for the charge reduction and for the correlated decrease of mean mean curvature is shown. So-called stomatocytogenic agents (e.g. clorpromazine, tetracain and triton X100) and so-called echinocytogenic agents (e.g. dinitrophenol and Na-salicylate) are known to change membrane curvature in a dose dependent manner. It is further shown that by prior reduction of surface charge by various enzymes the dose response curves of all stomatocytogenic and echinocytogenic agents tested is shifted towards higher degrees of stomatocytosis or lesser degrees of echinocytosis. The data show, that in RBC pronounced curvature influences are produced by the surface charge located on the sialic acid residues in the glycocalix.Supported by SFB 109 of Deutsche Forschungsgemeinschaft (künstliche Organe)     

Asymmetrically porous PLGA/Pluronic F127 membrane for effective guided bone regeneration

Porous guided bone regeneration (GBR) membranes with selective permeability, hydrophilicity and adhesiveness to bone were prepared with PLGA and Pluronic F127 using an immersion precipitation method. The porous PLGA/Pluronic F127 membranes were fabricated by immersing the PLGA/Pluronic F127 mixture solution (in tetraglycol) in a mold into water. The PLGA/Pluronic F127 mixture was precipitated in water by the diffusion of water into PLGA/Pluronic F127 mixture solution. It was observed that the membrane has an asymmetric column-shape porous structure. The top surface of the membrane (water contact side) had nano-size pores (approx. 50 nm) which can effectively prevent from fibrous connective tissue invasion but permeate nutrients, while the bottom surface (mold contact size) had micro-size pores (approx. 40 μm) which can improve adhesiveness with bone. From the investigations of mechanical property, water absorbability, model nutrient permeability and preliminary in vivo bone regeneration, the hydrophilized porous PLGA/F127 (5 wt%) membrane seems to be a good candidate as a GBR membrane for the effective permeation of nutrients and osteoconductivity, as well as good mechanical strength to maintain a secluded space for bone regeneration.     

Alteration of lipid order profile and permeability of plasma membranes from Trypanosoma cruzi epimastigotes grown in the presence of ketoconazole

Highly purified preparations of plasma membranes from control and ketoconazole-treated (1 microM, 120 h) epimastigotes of Trypanosoma cruzi have been obtained by cell disruption using abrasion with glass beads, differential centrifugation and isopycnic centrifugation in continuous, self-generating Percoll gradients. The purity of the preparation was ascertained by the specific activity 125I bound to the membranes obtained from enzymatically radiolabeled epimastigotes and by the alpha-methyl-mannoside sensitive binding of 125I-concanavalin A. The membranes form closed vesicles of 0.2-0.4 micron in diameter which display Mg2+ ATPase and acid phosphatase activities, but are devoid of 5'-nucleotidase and succinate-cytochrome c oxidoreductase; these vesicles can be strongly agglutinated by concanavalin A. The lipid order profiles of membranes from control and treated cells were compared with that present in egg phosphatidylcholine/ergosterol liposomes (84:16, mol/mol) by electron spin resonance spectroscopy of doxylstearic acid probes with the nitroxide group bound to carbon 5, 10, 12 and 16 of the stearic acid chain. Membranes from treated epimastigotes have a lipid order profile which resembles that of control plasma membranes near the polar surface (positions 5 and 10) but there is an abrupt decrease of order at position 12 and from there to the center of bilayer is highly disordered, even more than in pure lipid membranes. Consistent with these results, the leakage of L-[14C]glucose from membrane vesicles of ketoconazole-treated cells is much faster than that observed in vesicles obtained from control cells. These results indicate a strong alteration of the plasma membrane physical and biological properties due to the incubation of the parasite with the drug; this alteration is consistent with the accumulation of methylated precursors of ergosterol, which affects both lipid-lipid and lipid-protein interactions in the membrane.     

Cyclosporin A inhibits T cell receptor-induced interleukin-2 synthesis of human T lymphocytes by selectively preventing a transmembrane signal transduction pathway leading to sustained activation of a protein kinase C isoenzyme,protein kinase C-β

Stimulation of human peripheral blood lymphocytes via T cell receptor/CD3 complex resulted in a bimodal activation of protein kinase(s) C (PKC). Within 10 min of stimulation PKC-α was translocated to, and thus activated in, the plasma membranes of human lymphocytes, followed by a fast dissociation of this isotype from the plasma membrane. This short term activation and translocation PKC-α proved to be cyclosporin A (CsA) insensitive. After 90 min of stimulation PKC-β was translocated to and remained bound to the plasma membranes for up to 4 h. Preincubation of human lymphocytes with 200 ng/ml CsA specifically and completely abolished the sustained activation of PKC-β. Neither the phorbol ester-induced direct activation of PKC nor the specific activity of the plasma membrane-bound enzyme was influenced by CsA, suggesting that a signal transduction pathway leading to sustained activation of PKC-β was influenced by the immunosuppressive agent. In fact, CsA inhibited, in a concentration-dependent manner, the activation of lysophosphatid acyltransferase-catalyzed elevated incorporation of cis-polyunsaturated fatty acids into plasma membrane phospholipids. While interleukin-2 (IL-2) synthesis and cellular proliferation were completely inhibited by 200 ng/ml CsA in BMA 030- or BMA 031-stimulated cells, expression of high-affinity IL-2 receptors was not influenced by the immunosuppressive drug. These results suggest that synthesis and expression of high-affinity IL-2 receptors might be regulated by a signal-transducing pathway involving activation and translocation of PKC-α. Lysophosphatid acyltransferase-catalyzed incorporation of cis-polyunsaturated fatty acids might represent another mechanism of signal transduction implicated in the activation and translocation of PKC-β, which is specifically inhibited by CsA. Neutralization of PKC-β by introducing anti-PKC-β antibodies prevented IL-2 synthesis and proliferation in stimulated human lymphocytes. The results suggest a possible link between activation of PKC-β and regulation of IL-2 synthesis in activated human lymphocytes. Thus, inhibition of the activation and translocation of PKC-β by CsA may result in inhibition of IL-2 gene expression in human lymphocytes.     

Overcoming drug-resistant lung cancer by paclitaxel loaded dual-functional liposomes with mitochondria targeting and pH-response

Mitochondrion-orientated transportation of smart liposomes has been developed as a promising strategy to deliver anticancer drugs directly to tumor sites, and these have a tremendous potential for killing cancer cells, especially those with multidrug resistance (MDR). Herein we report a novel dual-functional liposome system possessing both extracellular pH response and mitochondrial targeting properties to enhance drug accumulation in mitochondria and trigger apoptosis of drug-resistant cancer cells. Briefly, peptide _D[KLAKLAK]₂ (KLA) was modified with 2, 3-dimethylmaleic anhydride (DMA) and combined with 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) to yield a DSPE-KLA-DMA (DKD) lipid. This dual-functional DKD was then mixed with other commercially available lipids to fabricate liposomes. In vitro anticancer efficacy of this liposome system was evaluated in human lung cancer A549 cells and drug-resistant lung cancer A549/Taxol cells. At tumor extracellular pH (∼6.8), liposomes could reverse their surface charge (negative to positive), facilitating liposome internalization. After cellular uptake, KLA peptide directed delivery-enabled selective accumulation of these liposomes into mitochondria and favored release of their cargo paclitaxel (PTX) into desired sites. Specifically, enhanced apoptosis of MDR cancer cells through mitochondrial signaling pathways was evidenced by release of cytochrome c and increased activity of caspase-9 and -3. These dual-functional liposomes had the greatest efficacy for treating A549 cells and A549/Taxol cells in vitro, and in treating drug-resistant lung cancer A549/Taxol cells xenografted onto nude mice (tumor growth inhibition 86.7%). In conclusion, dual-functional liposomes provide a novel and versatile approach for overcoming MDR in cancer treatment.     

Square wave pulse analysis of cellular and paracellular conductance pathways in necturus gallbladder epithelium

In search for a rapid and reliable method to identify and quantitatively determine cell membrane resistances and paracellular shunt resistances in epithelia we have developed appropriate techniques to measure transepithelial and intracellular potential transients in response to transepithelially applied square wave constant current pulses. Model considerations indicate that in a unilayered, homogeneous epithelium with open lateral spaces the transient potential response across each cell membrane should obey a single exponential function in case the tight junction resistance is high, as in a tight epithelium, whereas in a leaky epithelium it should consist of a superposition of two exponentials with equal sign at the membrane with the higher intrinsic time constant and of two exponentials of different sign (overshoot with recline) at the membrane with the lower intrinsic time constant. The latter predictions were experimentally verified in a study on Necturus gallbladder epithelium and equivalent circuit parameters for the cell membrane resistances and capacitances as well as for the resistance of the shunt path were calculated from the data by curve fitting procedures. The resistances of the apical and basal cell membrane and of the shunt path averaged 1220, 201 and 91 cm² respectively while the apical and basal cell membrane capacitances were 8.0 and 26.3 F/cm² respectively. The fact that the resistance values are 4–15 times lower than estimates derived previously from 2D-cable analysis relates to a better preservation of the transport function under the present incubation conditions as verified by a new series of cable analysis data. The capacitances agree well with estimates of the surface amplification of the cell membranes from electronmicrographs, thus confirming the validity of the interpretation of the observed voltage transients.     

Effect of cholesterol on membrane Na,K-dependent adenosine triphosphatase activity

By feeding rats on a high-cholesterol diet or by treating rat brain membranes with cholesterol oxidase, preparations of membrane Na,K-ATPase differing in their cholesterol content were obtained. The cholesterol/protein ratio varied from 0.034 to 0.251 (normally 0.152). An increase in this ratio was shown to straighten Arrhenius' plots for Na,K-ATPase versus temperature, whereas a decrease increases their curvature. A change in the temperature dependence on enzyme activity takes place simultaneously with the corresponding change in water repellency in experiments with the spin-label analog of androstane. It is concluded that the cholesterol level in the brain membranes controls Na,K-ATPase activity.Department of Biochemistry, M. V. Lomonosov Moscow State University. (Presented by Academician of the Academy of Medical Sciences of the USSR S. E. Severin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 12, pp. 672–675, December, 1977.     

Biomimetic hydrogels gate transport of calcium ions across cell culture inserts

Control of the in vitro spatiotemporal availability of calcium ions is one means by which the microenvironments of hematopoietic stem cells grown in culture may be reproduced. The effects of cross-linking density on the diffusivity of calcium ions through cell culture compatible poly(2-hydroxyethyl methacrylate) [poly(HEMA)]-based bioactive hydrogels possessing 1.0 mol% 2-methacryloyloxyethyl phosphorylcholine (MPC), 5 mol% N,N-(dimethylamino)ethylmethacrylate (DMAEMA) and ca. 17 mol% n-butyl acrylate (n-BA) have been investigated to determine if varying cross-link density is a viable approach to controlling transport of calcium across hydrogel membranes. Cross-linking density was varied by changing the composition of cross-linker, tetraethyleneglycol diacrylate (TEGDA). The hydrogel membranes were formed by sandwich casting onto the external surface of track-etched polycarbonate membranes (T?=?10 μm, φ?=?0.4 μm pores) of cell culture inserts, polymerized in place by UV light irradiation and immersed in buffered (0.025 HEPES, pH 7.4) 0.10 M calcium chloride solution. The transport of calcium ions across the hydrogel membrane was monitored using a calcium ion selective electrode set within the insert. Degree of hydration (21.6?±?1.0%) and void fraction were found to be constant across all cross-linking densities. Diffusion coefficients, determined using time-lag analysis, were shown to be strongly dependent on and to exponentially decrease with increasing cross-linking density. Compared to that found in buffer (2.0-2.5?×?10?? cm2/s), diffusion coefficients ranged from 1.40?×?10?? cm2/s to 1.80?×?10?? cm2/s and tortuosity values ranged from 1.7 to 10.0 for the 1 and 12 mol% TEGDA cross-linked hydrogels respectively. Changes in tortuosity arising from variations in cross-link density were found to be the primary modality for controlling diffusivity through novel n-BA containing poly(HEMA)-based bioactive hydrogels.