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Being a major metabolite for maintaining cellular homeostasis, as well as an important structural component in lipid membrane, cholesterol also plays critical roles in the life cycles of some viruses, including human immunodeficiency virus-1 (HIV-1). The involvement of cholesterol in HIV-1 infectivity, assembly and budding has made it an important research target. Viral protein R (Vpr) is an accessory protein of HIV-1, which is involved in many major events in the life cycle of HIV-1. In addition to its multi-functional roles in the HIV-1 life cycle, it is shown to interact with lipid membrane and form a cation-selective channel. In this work, we examined the effect of cholesterol on the interaction of Vpr and lipid membrane. Using calcein release assay, we found that the membrane permeability induced by the membrane binding of Vpr was significantly reduced in the presence of cholesterol in membrane. In addition, using solid-state NMR (ssNMR) spectroscopy, Vpr was shown to experience multiple chemical environments in lipid membrane, as indicated by the broad line shape of carbonyl 13C resonance of Cys-76 residue ranging from 165–178 ppm, which can be attributed to the existence of complex Vpr-membrane environments. We further showed that the presence of cholesterol in membrane will alter the distribution of Vpr in the complex membrane environments, which may explain the change of the Vpr induced membrane permeability in the presence of cholesterol.

In the present investigation we studied the molecular mechanisms of the monodesmosidic saponin digitonin on natural and artificial membranes. We measured the hemolytic activity of digitonin on red blood cells (RBCs). Also different lipid membrane models (large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs) in the presence and absence of cholesterol were employed. The stability and permeability of the different vesicle systems were studied by using calcein release assay, GUVs membrane permeability assay using confocal microscopy (CM) and fluorescence correlation spectroscopy (FCS) and vesicle size measurement by dynamic light scattering (DLS). The results support the essential role of cholesterol in explaining how digitonin can disintegrate biological and artificial membranes. Digitonin induces membrane permeability or causes membrane rupturing only in the presence of cholesterol in an all-or-none mechanism. This effect depends on the concentrations of both digitonin and cholesterol. At low concentrations, digitonin induces membrane permeability while keeping the membrane intact. When digitonin is combined with other drugs, a synergistic potentiation can be observed because it facilitates their uptake.

The effects of local anesthetics (LAs), namely, lidocaine (LDC), prilocaine (PLC), mepivacaine (MPV), bupivacaine (BPV), procaine (PC), and tetracaine (TTC), on the steady-state transmembrane conductance induced by the cis -side addition of the antifungal polyene macrolide antibiotic, nystatin (NYS), in planar lipid bilayers were studied. The addition of TTC to model membranes comprising DOPC and cholesterol (33 mol%) led to a nearly twenty-fold increase in the steady-state NYS-induced membrane conductance. BPV slightly enhanced the channel-forming activity of polyene. LDC, PLC, MPV, and PC did not affect the NYS-induced transmembrane current. We concluded that the effects of LAs on the channel-forming activity of NYS were in agreement with their effects on the elastic properties of model membranes. The ability of aminoamide LAs to promote calcein leakage from large unilamellar DOPC-vesicles was decreased in the following order: BPV >> LDC ≈ PLC ≈ MPV. LDC, PLC, and MPV produced a graded leakage of fluorescent marker from liposomes, up to 10–13%. A initial sharp jump in fluorescence after the introduction of BPV was attributed to the solubilization of liposomes and the formation of mixed DOPC:BPV-micelles. Differential scanning microcalorimetry (DSC) of large unilamellar DPPC-vesicles showed that the main transition temperature ( Tm ) is continuously decreased upon increasing concentrations of TTC. A sharp drop in the enthalpy of the transition at higher TTC concentrations indicated a formation of anesthetic/lipid mixed micelles. In contrast to TTC, PC slightly decreased Tm , broadened the DSC signal and did not provoke vesicle-to-micelle transition. Both the calcein leakage and DSC data together with the results of measurements of threshold voltages that are required to cause the lipid bilayer breakdown might indicate an alteration in the curvature lipid packing stress, induced by BPV and TTC. The data presented here lend support to a lipid-mediated mode of LAs action on NYS pores via an alteration in curvature stress near the trans -mouth. Similar results were obtained for several lipid pores, formed by polyene amphotericin B, lipopeptide syringomycin E, and the peptides magainin and melittin. This finding further developed the concept of non-specific regulation of lipid pores by LAs. In conclusion, the combination of nystatin with LAs could be a novel treatment for efficient therapy of superficial and mucosal candidiasis. [ABSTRACT FROM AUTHOR]

In the present investigation we studied the molecular mechanisms of the monodesmosidic saponin digitonin on natural and artificial membranes. We measured the hemolytic activity of digitonin on red blood cells (RBCs). Also different lipid membrane models (large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs) in the presence and absence of cholesterol were employed. The stability and permeability of the different vesicle systems were studied by using calcein release assay, GUVs membrane permeability assay using confocal microscopy (CM) and fluorescence correlation spectroscopy (FCS) and vesicle size measurement by dynamic light scattering (DLS). The results support the essential role of cholesterol in explaining how digitonin can disintegrate biological and artificial membranes. Digitonin induces membrane permeability or causes membrane rupturing only in the presence of cholesterol in an all-or-none mechanism. This effect depends on the concentrations of both digitonin and cholesterol. At low concentrations, digitonin induces membrane permeability while keeping the membrane intact. When digitonin is combined with other drugs, a synergistic potentiation can be observed because it facilitates their uptake. [ABSTRACT FROM AUTHOR]

Pseudomonas syringae pv. syringae produces two groups of cyclic lipodepsipeptides (LDPs): the nona-peptides syringomycins, syringostatins, and syringotoxin (ST), and the more complex syringopeptins composed of either 22 or 25 amino acid residues (SP22 and SP25). Both classes of peptides significantly contribute to bacterial pathogenesis and their primary target of action seems to be the plasma membrane. We studied and compared the activity of some members of these two classes of LDPs on red blood cells and on model membranes (monolayers and unilamellar vesicles). All peptides induced red blood cell hemolysis. The mechanism was apparently that of a colloid-osmotic shock caused by the formation of pores, as it could be prevented by osmoticants of adequate size. Application of the Renkin equation indicated a radius of approximately 1 nm for the lesions formed by syringopeptins SP22A and SP25A, whereas those formed by syringomycin E (SRE) had a variable, dose-dependent size ranging from 0.7 up to 1.7 nm. All tested LDPs displayed surface activity, forming peptide monolayers with average molecular areas of 1.2 nm2 (SRE), 1.5 nm2 (SP22A), and 1.3 nm2 (SP25A). They also partitioned into preformed lipid monolayers occupying molecular areas that ranged from 0.6 to 1.7 nm2 depending on the peptide and the lipid composition of the film. These LDPs formed channels in lipid vesicles as indicated by the release of an entrapped fluorescent dye (calcein). The extent of permeabilization was dependent on the concentration of the peptide and the composition of the lipid vesicles, with a preference for those containing a sterol. From the dose dependence of the permeabilization it was inferred that LDPs increased membrane permeability by forming oligomeric channels containing from four to seven monomers. On average, syringopeptin oligomers were smaller than SRE and ST oligomers.

Crossing the cellular membrane and delivering active pharmaceuticals or biologicals into the cytosol of cells is an essential step in the development of nanomedicines. One of the most important intracellular processes regarding the cellular uptake of biologicals is the endolysosomal pathway. Sophisticated nanocarriers are developed to overcome a major hurdle, the endosomal entrapment, and delivering their cargo to the required site of action. In parallel, in vitro assays are established analyzing the performance of these nanocarriers. Among them, the release of the membrane-impermeable dye calcein has become a popular and straightforward method. It is accessible for most researchers worldwide, allows for rapid conclusions about the release potential, and enables the study of release mechanisms. This review is intended to provide an overview and guidance for scientists applying the calcein release assay. It comprises a survey of several applications in the study of endosomal escape, considerations of potential pitfalls, challenges, and limitations of the assay, and a brief summary of complementary methods. Based on this review, it is hoped to encourage further research groups to take advantage of the calcein release assay for their own purposes and help to create a database for more efficient cross-correlations between nanocarriers., (© 2022 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH.)

Abstract: Background: Microbial antibiotic resistance is a challenging medical problem nowadays. Two scorpion peptides displaying antibiotic activity: hadrurin and vejovine were taken as models for the design of novel shorter peptides with similar activity. Methods: Using the standard Fmoc-based solid phase synthesis technique of Merrifield twelve peptides (18 to 29 amino acids long) were synthesized, purified and assayed against a variety of multi-drug resistant Gram-negative bacteria from clinical isolates. Hemolytic and antiparasitic activities of the peptides and their possible interactions with eukaryotic cells were verified. Release of the fluorophore calcein from liposomes treated with these peptides was measured. Results: A peptide with sequence GILKTIKSIASKVANTVQKLKRKAKNAVA), and three analogs: Δ(Α29), Δ(K12-Q18; Ν26−Α29), and K4N Δ(K12-Q18; Ν26−Α29) were shown to inhibit the growth of Gram-negative (E. coli ATCC25922) and Gram-positive bacteria (S. aureus), as well as multi-drug resistant (MDR) clinical isolated. The antibacterial and antiparasitic activities were found with peptides at 0.78 to 25μM and 5 to 25μM concentration, respectively. These peptides have low cytotoxic and hemolytic activities at concentrations significantly exceeding their minimum inhibitory concentrations (MICs), showing values between 40 and 900μM for their EC50, compared to the parent peptides vejovine and hadrurin that at the same concentration of their MICs lysed more than 50% of human erythrocytes cells. Conclusions: These peptides promise to be good candidates to combat infections caused by Gram-negative bacteria from nosocomial infections. General significance: Our results confirm that well designed synthetic peptides can be an alternative for solving the lack of effective antibiotics to control bacterial infections. [Copyright &y& Elsevier]

Abstract: eLiposomes are liposomes encapsulating emulsions and therapeutics for targeted delivery. By applying ultrasound to eLiposomes, emulsion droplets can transform from liquid to gas and rupture the lipid bilayer of the eLiposome to release a drug or plasmid. In this study, perfluoropentane (PFC5) emulsions were encapsulated inside folated eLiposomes carrying a model drug (calcein) or a model GFP plasmid to examine the effects of a folate ligand, PFC5 emulsion and various ultrasonic acoustic parameters in drug delivery and gene transfection into HeLa cells. Confocal microscopy was used to quantify drug delivery and the level of plasmid transfection into HeLa cells. The results showed that drug delivery or transfection was minimal without incorporation of internal PFC5 emulsions and folate ligand on the eLiposome surface. It was also shown that application of ultrasound greatly enhanced the drug delivery and plasmid transfection. Delivery of these therapeutics appears to be to the cytosol, indicating that the expansion of the emulsion droplets disrupted both the eLiposomes and the endosomes. [Copyright &y& Elsevier]

Abstract: Bax is a cytosolic protein that responds to various apoptotic signals by binding to the outer mitochondrial membrane, resulting in membrane permeabilization, release of cytochrome c, and caspase-mediated cell death. Currently discussed mechanisms of membrane perforation include formation of hetero-oligomeric complexes of Bax with other pro-apoptotic proteins such as Bak, or membrane insertion of multiple hydrophobic helices of Bax, or formation of lipidic pores physically aided by mitochondrial membrane-inserted proteins. There is compelling evidence provided by our and other groups indicating that the C-terminal “helix 9” of Bax mediates membrane binding and pore formation, yet the mechanism of pore forming capability of Bax C-terminus remains unclear. Here we show that a 20-amino acid peptide corresponding to Bax C-terminus (VTIFVAGVLTASLTIWKKMG) and two mutants where the two lysines are replaced with glutamate or leucine have potent membrane pore forming activities in zwitterionic and anionic phospholipid membranes. Analysis of the kinetics of calcein release from lipid vesicles allows determination of rate constants of pore formation, peptide–peptide affinities within the membrane, the oligomeric state of transmembrane pores, and the importance of the lysine residues. These data provide insight into the molecular details of membrane pore formation by a Bax-derived peptide and open new opportunities for design of peptide-based cytotoxic agents. [Copyright &y& Elsevier]

The phase behaviour and phase stability of mixed liposomes prepared from diether polar lipids isolated from thermophilic Archaea and from conventional diester dipalymitoyl- l-α-phosphatidylcholine (DPPC) at different molar ratios were investigated by differential scanning calorimetry. The permeability of these mixed liposomes was investigated as a function of temperature, by measuring the fluorescence emission intensity for the release of calcein in the temperature range from 20 to 98 °C. Our data show that diether polar lipids isolated from hyperthermophilic Archaea can form mixed liposomes with synthetic α-DPPC. Liposomes prepared from pure archaeal lipids do not show the characteristic gel-to-liquid crystalline phase transition characteristics of DPPC liposomes in the temperature range from 0 to 100 °C. In the presence of 5 mol% archaeal lipids in mixed α-DPPC liposomes, the temperature of the phase transition decreases and the broadness of the peak increases, although the enthalpy change (Δ H) of the phase transition is not significantly influenced. At molar ratios of archaeal lipids greater than 50 mol% in these mixed liposomes, the typical gel-to-liquid crystalline phase transition disappears, which indicates that the lipids are predominantly in the liquid crystalline state. At ratios of archaeal lipids greater than 50 mol% in these mixed liposomes, their permeability for anionic calcein is the same as for liposomes made from 100 mol% archaeal lipids, across the whole temperature range. [ABSTRACT FROM AUTHOR]

Abstract: Temperature and pH effects were studied for stability, structural organization, fluidity and permeability of vesicles from a polar lipid methanol fraction isolated from the Aeropyrum pernix. We determined the permeability of C25,25 liposomes using fluorescence intensity of released calcein. At pH 7.0 and 9.0, and from 85°C to 98°C, only 10% of entrapped calcein was released. After 10h at 90°C, calcein release reached 27%, independent of pH. Fluorescence anisotropy measurements of hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene revealed gradual changes up to 60°C. At higher temperatures, the anisotropy did not change significantly. Fluorescence alone did not provide detailed and direct structural information about these C25,25 liposomes, so we used electron paramagnetic resonance spectroscopy (EPR) and differential scanning calorimetry (DSC). From EPR spectra, mean membrane fluidity determined according to maximal hyperfine splitting and empirical correlation times showed continuous increases with temperature. Computer simulation of EPR spectra showed heterogeneous membranes of these C25,25 liposomes: at low temperatures, they showed three types of membrane regions characterized by different motional modes. Above 65°C, the membrane becomes homogeneous with only one fluid-like region. DSC thermograms of C25,25 liposomes reveal a very broad and endothermic transition in the temperature range from 0°C to 40°C. [Copyright &y& Elsevier]

Abstract: Oritavancin, a lipoglycopeptide with marked bactericidal activity against vancomycin-resistant Staphylococcus aureus and enterococci, induces calcein release from CL:POPE and POPG:POPE liposomes, an effect enhanced by an increase in POPG:POPE ratio, and decreased when replacing POPG by DPPG (Domenech et al., Biochim Biophys Acta 2009; 1788:1832–40). Using vesicles prepared from lipids extracted from S. aureus, we showed that oritavancin induces holes, erosion of the edges, and decrease of the thickness of the supported lipid bilayers (atomic force microscopy; AFM). Oritavancin also induced an increase of membrane permeability (calcein release) on a time- and dose-dependent manner. These effects were probably related to the ability of the drug to bind to lipid bilayers as shown by 8-anilino-1- naphthalene sulfonic acid (ANS) assay. Interaction of oritavancin with phospholipids at the level of their glycerol backbone and hydrophobic domain was studied by monitoring changes of Laurdan excitation generalized polarization (GP ex ) and 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy upon temperature increase. Oritavancin increased GP ex values and the transition temperature, indicating a more ordered structure at the level of the glycerol backbone. Oritavancin slightly decreased DPH fluorescence depolarization intensities, suggesting an increase in fluidity at the level of acyl chains. Together, our data confirm the interaction of oritavancin with lipids and the potential role of a rigidifying effect at the level of glycerol backbone for membrane permeabilization. This work shows how AFM and biophysical methods may help in characterizing drug–membrane interactions, and sheds further light on the mode of action of oritavancin. [ABSTRACT FROM AUTHOR]

Abstract: Antibiotics acting on bacterial membranes are receiving increasing attention because of widespread resistance to agents acting on other targets and of potentially improved bactericidal effects. Oritavancin is a amphiphilic derivative of vancomycin showing fast and extensive killing activities against multi-resistant (including vancomycin insusceptible) Gram-positive organisms with no marked toxicity towards eukaryotic cells. We have undertaken to characterize the interactions of oritavancin with phospholipid bilayers, using liposomes (LUV) and supported bilayers made of cardiolipin (CL) or phosphatidylglycerol (POPG) and phosphatidylethanolamine (POPE), all abundant in Gram-positive organisms. Changes in membrane permeability were followed by the release of calcein entrapped in liposomes at self-quenching concentrations, and changes in nanoscale lipid organization examined by Atomic Force Microscopy (AFM). Oritavancin caused a fast (<5 min) and complete (>95%) release of calcein from CL:POPE liposomes, and a slower but still substantial (50% in 60 min) release from POPG:POPE liposomes, which was (i) concentration-dependent (0–600 nM; [microbiologically meaningful concentrations]); (ii) enhanced by an increase in POPG:POPE ratio, and decreased when replacing POPG by DPPG. AFM of CL:POPE supported bilayers showed that oritavancin (84 nM) caused a remodeling of the lipid domains combined with a redisposition of the drug and degradation of the borders. In all the above studies, vancomycin was without a significant effect at 5.5 μM. Electrostatic interactions, together with lipid curvature, lipid polymorphism as well of fluidity play a critical role for the permeabilization of lipid bilayer and changes in lipid organization induced by oritavancin. [Copyright &y& Elsevier]

[Display omitted] • Molecular electrotransfer can be controlled by intracellular Ca2+ concentration. • These effects are related with an enhanced membrane repair mechanism. • Simultaneous molecule electrotransfer and intracellular Ca2+ influx was investigated. • EP-mediated delivery of excess of Ca2+ leads to loss of Ca2+ homeostasis. • Our findings might lead to improved therapeutic outcomes. The combination of Ca2+ ions and electroporation has gained attention as potential alternative to electrochemotherapy. Ca2+ is an important component of the cell membrane repair system and its presence directly influences the dynamics of the pore cycle after electroporation which can be exploited for cancer therapies. Here, the influence of Ca2+ concentration is investigated on small molecule electrotransfer and release of Calcein from 4T1, MX-1, B16F10, U87 cancer cells after cell exposure to microsecond electric pulses. Moreover, we investigated simultaneous molecule electrotransfer and intracellular calcium ion influx when media was supplemented with different Ca2+ concentrations. Results show that increased concentrations of calcium ions reduce the electrotransfer of small molecules to different lines of cancer cells as well as the release of Calcein. These effects are related with an enhanced membrane repair mechanism. Overall, we show that the efficiency of molecular electrotransfer can be controlled by regulating Ca2+ concentration in the electroporation medium. For the first time, the cause of cancer cell death in vitro from 1 mM CaCl 2 concentrations is related to the irreversible loss of Ca2+ homeostasis after cell electroporation. Our findings provide fundamental insight on the mechanisms of Ca2+ electroporation that might lead to improved therapeutic outcomes. [ABSTRACT FROM AUTHOR]

Abstract: The ability of newly synthesized genistein benzyl and glycosylated derivatives to permeabilize the liposome membrane was studied by calcein-leakage method. All studied derivatives appeared to be more effective than their parent compound — genistein. Comparing the experimental results with theoretical calculations we found that in the case of benzyl derivatives the dipole moment of added benzene ring (with its substitutions) might be important for the strength of flavonoids–lipid interactions. This conclusion may have some implications for QSAR studies in which mostly the dipole moments of entire molecules are considered. [Copyright &y& Elsevier]

Abstract: The proposed toxicity mechanism of the Bacillus thuringiensis Cry insecticidal proteins involves membrane penetration and lytic pore formation of the α4–α5 hairpins in the target larval midgut cell membranes. In this study, alanine substitutions of selected polar residues (Tyr178, Gln180, Asn183, Asn185, and Asn195) in the hydrophobic helix-α5 of the Cry4Ba mosquito-larvicidal protein were initially conducted via PCR-based directed mutagenesis. Upon IPTG induction, all the 130-kDa mutant protoxins were highly expressed in Escherichia coli as cytoplasmic inclusions, with yields similar to the wild-type protoxin. When E. coli cells expressing each mutant toxin were tested against Stegomyia aegypti mosquito larvae, the larvicidal activity of the N183A mutant was almost completely abolished whereas the four other mutants showed only a small reduction in toxicity. Additionally, replacements of this critical residue with various amino acids revealed that the uncharged polar residue at position 183 in α5 is crucial for larvicidal activity. Further characterisation of the N183K bio-inactive mutant revealed that the 65-kDa activated toxin was unable to form oligomers in lipid vesicles and its ability to induce the release of entrapped calcein from liposomes was much weaker than that of the wild-type toxin. These results suggest that the highly conserved Asn183 located in the middle of the transmembrane α5 of Cry4Ba plays a crucial role in toxicity and toxin oligomerisation in the lipid membranes. [Copyright &y& Elsevier]

The high larvicidal effect of Bacillus sphaericus (Bs), a mosquito control agent, originates from the presence of a binary toxin (Bs Bin) composed of two proteins (BinA and BinB) that work together to lyse gut cells of susceptible larvae. We demonstrate for the first time that the binary toxin and its individual components permeabilize receptor-free large unilamellar phospholipid vesicles (LUVs) and planar lipid bilayers (PLBs) by a mechanism of pore formation. Calcein-release experiments showed that LUV permeabilization was optimally achieved at alkaline pH and in the presence of acidic lipids. BinA was more efficient than BinB, BinB facilitated the BinA effect, and their stoichiometric mixture was more effective than the full Bin toxin. In PLBs, BinA formed voltage-dependent channels of approximately 100-200 pS with long open times and a high open probability. Larger channels (> or =400 pS) were also observed. BinB, which inserted less easily, formed smaller channels (< or =100 pS) with shorter mean open times. Channels observed after sequential addition of the two components, or formed by their 1:1 mixture (w/w), displayed BinA-like activity. Bs Bin toxin was less efficient at forming channels than the BinA/BinB mixture, with channels displaying the BinA channel behavior. Our data support the concept of BinA being principally responsible for pore formation in lipid membranes with BinB, the binding component of the toxin, playing a role in promoting channel activity. [ABSTRACT FROM AUTHOR]

In its mature form, the VacA toxin of Helicobacter pylori is a 95-kDa protein which is released from the bacteria as a low-activity complex. This complex can be activated by low-pH treatment that parallels the activity of the toxin on target cells. VacA has been previously shown to insert itself into lipid membranes and to induce anion-selective channels in planar lipid bilayers. Binding of VacA to lipid vesicles and its ability to induce calcein release from these vesicles were systematically compared as a function of pH. These two phenomena show a different pH-dependence, suggesting that the association with the lipid membrane may be a two-step mechanism. The secondary and tertiary structure of VacA as a function of pH and the presence of lipid vesicles were investigated by Fourier-transform infrared spectroscopy. The secondary structure of VacA is identical whatever the pH and the presence of a lipid membrane, but the tertiary structure in the presence of a lipid membrane is dependent on pH, as evidenced by H/D exchange. [ABSTRACT FROM AUTHOR]

Being a major metabolite for maintaining cellular homeostasis, as well as an important structural component in lipid membrane, cholesterol also plays critical roles in the life cycles of some viruses, including human immunodeficiency virus-1 (HIV-1). The involvement of cholesterol in HIV-1 infectivity, assembly and budding has made it an important research target. Viral protein R (Vpr) is an accessory protein of HIV-1, which is involved in many major events in the life cycle of HIV-1. In addition to its multi-functional roles in the HIV-1 life cycle, it is shown to interact with lipid membrane and form a cation-selective channel. In this work, we examined the effect of cholesterol on the interaction of Vpr and lipid membrane. Using calcein release assay, we found that the membrane permeability induced by the membrane binding of Vpr was significantly reduced in the presence of cholesterol in membrane. In addition, using solid-state NMR (ssNMR) spectroscopy, Vpr was shown to experience multiple chemical environments in lipid membrane, as indicated by the broad line shape of carbonyl 13C resonance of Cys-76 residue ranging from 165–178 ppm, which can be attributed to the existence of complex Vpr-membrane environments. We further showed that the presence of cholesterol in membrane will alter the distribution of Vpr in the complex membrane environments, which may explain the change of the Vpr induced membrane permeability in the presence of cholesterol. [ABSTRACT FROM AUTHOR]

Responsive polymers, which become protonated at decreasing pH, are considered a milestone in the development of synthetic cell entry vectors. Exact correlations between their properties and their ability to escape the endosome, however, often remain elusive due to hydrophobic interactions or limitations in the design of water-soluble materials with suitable basicity. Here, we present a series of well-defined, hydrophilic polypiperazines, where systematic variation of the amino moiety facilitates an unprecedented fine-tuning of the basicity or p K a value within the physiologically relevant range (pH 6-7.4). Coincubation of HEK 293T cells with various probes, including small fluorophores or functioning proteins, revealed a rapid increase of endosomal release for polymers with p K a values above 6.5 or 7 in serum-free or serum-containing media, respectively. Similarly, cytotoxic effects became severe at increased p K a values (>7). Although the window for effective transport appears narrow, the discovered correlations offer a principal guideline for the design of effective polymers for endosomal escape.

This study was focused on the molecular mechanisms of action of saponins and related compounds (sapogenins and alkaloids) on model lipid membranes. Steroids and triterpenes were tested. A systematic analysis of the effects of these chemicals on the physicochemical properties of the lipid bilayers and on the formation and functionality of the reconstituted ion channels induced by antimicrobial agents was performed. It was found that digitonin, tribulosin, and dioscin substantially reduced the boundary potential of the phosphatidylcholine membranes. We concluded that saponins might affect the membrane boundary potential by restructuring the membrane hydration layer. Moreover, an increase in the conductance and lifetime of gramicidin A channels in the presence of tribulosin was due to an alteration in the membrane dipole potential. Differential scanning microcalorimetry data indicated the key role of the sapogenin core structure (steroid or triterpenic) in affecting lipid melting and disordering. We showed that an alteration in pore forming activity of syringomycin E by dioscin might be due to amendments in the lipid packing. We also found that the ability of saponins to disengage the fluorescent marker calcein from lipid vesicles might be also determined by their ability to induce a positive curvature stress.

Lipid membranes are involved in regulating biochemical and biological processes and in modulating the selective permeability of cells, organelles, and vesicles. Membrane composition, charge, curvature, and fluidity all have concerted effects on cellular signaling and homeostasis. The ability to prepare artificial lipid assemblies that mimic biological membranes has enabled investigators to obtain considerable insight into biomolecule-membrane interactions. Lipid nanoscale assemblies can vary greatly in size and composition and can consist of a single lipid monolayer, a bilayer, or other more complex assemblies. This structural diversity makes liposomes suitable for a wide variety of biochemical and clinical applications. Here, we describe a calcein dye leakage assay that we have developed to monitor phospholipid vesicle disruption by alpha-synuclein (αSyn), a presynaptic protein that plays a central role in Parkinson's disease (PD). We present data showing the effect of adenylylation of αSyn on αSyn-mediated vesicle disruption as an example. This assay can be used to study the effect of mutations or post-translational modifications on αSyn-membrane interactions, to identify protein binding partners or chemical entities that perturb these interactions, and to study the effects of different lipids on the permeabilization activity of αSyn or any other protein., Competing Interests: Competing interests The authors report no conflicts of interest.