Calcein Leakage Assay Research Articles

Charge and Coordination Directed Liposome Fusion onto SiO2 and TiO2 Nanoparticles.

TiO2 and SiO2 are very useful materials for building biointerfaces. A particularly interesting aspect is their interaction with lipid bilayers. Many past research efforts focused on phosphocholine (PC) lipids, which form supported lipid bilayers (SLB) on SiO2 at physiological conditions but are adsorbed as intact liposomes on TiO2. Low pH was required to form PC SLBs on TiO2. This work intends to understand the surface forces and chemistry responsible for such differences. Two charge neutral lipids: 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC) and 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl phosphate (DOCPe) and two negatively charged lipids: 1,2-dioleoyl- sn-glycero-3-phospho-l-serine (DOPS) and 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl hydrogen phosphate (DOCP) were used. Using calcein leakage assays, adsorption measurement, cryo-TEM, and washing, we concluded that charge is the dominating factor on SiO2. The two neutral lipids form SLB on SiO2 at pH 3 and 7, but the two negatively charged ones cannot form. On TiO2, both charge and coordination chemistry are important. The two anionic lipids formed SLB from pH 3 to 10. DOCP had stronger affinity than DOPS likely due to the tighter terminal phosphate binding of the former. The two neutral liposomes formed SLB only at pH 3, where phosphate interaction and van der Waals force are deemed important. The pH 3 prepared TiO2 DOPC SLBs are destabilized at neutral pH, indicating the reversible nature of the interaction. This work has provided new insights into two important materials interacting with common liposomes, which are important for reproducible biosensing, device fabrication, and drug delivery applications.

Membrane interaction of off-pathway prion oligomers and lipid-induced on-pathway intermediates during prion conversion: A clue for neurotoxicity

Soluble oligomers of prion proteins (PrP), produced during amyloid aggregation, have emerged as the primary neurotoxic species, instead of the fibrillar end-products, in transmissible spongiform encephalopathies. However, whether the membrane is among their direct targets, that mediate the downstream adverse effects, remains a question of debate. Recently, questions arise from the formation of membrane-active oligomeric species generated during the β-aggregation pathway, either in solution, or in lipid environment. In the present study, we characterized membrane interaction of off-pathway oligomers from recombinant prion protein generated along the amyloid aggregation and compared to lipid-induced intermediates produced during lipid-accelerated fibrillation. Using calcein-leakage assay, we show that the soluble prion oligomers are the most potent in producing leakage with negatively charged vesicles. Binding affinities, conformational states, mode of action of the different PrP assemblies were determined by thioflavin T binding-static light scattering experiments on DOPC/DOPS vesicles, as well as by FTIR-ATR spectroscopy and specular neutron reflectivity onto the corresponding supported lipid bilayers. Our results indicate that the off-pathway PrP oligomers interact with lipid membrane via a distinct mechanism, compared to the inserted lipid-induced intermediates. Thus, separate neurotoxic mechanisms could exist following the puzzling intermediates generated in the different cell compartments. These results not only reveal an important regulation of lipid membrane on PrP behavior but may also provide clues for designing stage-specific and prion-targeted therapy.

A Martini coarse-grained model of the calcein fluorescent dye

Calcein leakage assays are a standard experimental set-up for probing the extent of damage induced by external agents on synthetic lipid vesicles. The fluorescence signal associated with calcein release from liposomes is the signature of vesicle disruption, transient pore formation or vesicle fusion. This type of assay is widely used to test the membrane disruptive effect of biological macromolecules, such as proteins, antimicrobial peptides and RNA and is also used on synthetic nanoparticles with a polymer, metal or oxide core. Little is known about the effect that calcein and other fluorescent dyes may have on the properties of lipid bilayers, potentially altering their structure and permeability. Here we develop a coarse-grained model of calcein that is compatible with the Martini force field for lipids. We validate the model by comparing its dimerization free energy, aggregation behavior at different concentrations and interaction with a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane to those obtained at atomistic resolution. Our coarse-grained description of calcein makes it suitable for the simulation of large calcein-filled liposomes and of their interactions with external agents, allowing for a direct comparison between simulations and experimental liposome leakage assays.

Characterization of a smart pH-cleavable PEG polymer towards the development of dual pH-sensitive liposomes

To facilitate the development of PEG-cleavable pH-sensitive liposomes (CL-pPSL), this study aimed to fully characterize a new pH-sensitive polymer, PEGB-Hz-CHEMS. Polyethylene glycol (PEG) functionalised with 4-carboxybenzaldehyde (PEGB) was linked to cholesteryl hemisuccinate (CHEMS) via an acid labile hydrazide–hydrazone hybrid bond (CONHNCH) to form PEGB-Hz-CHEMS. The polymer was post-inserted into DOPE/CHEMS liposomes to form CL-pPSL. A validated stability-indicating HPLC-UV method was developed with the aid of multiple linear regression for the mobile phase. The assay was used to evaluate the pH-sensitivity, pathways of cleavage of the polymer and the PEGylation degree of CL-pPSL. The pH-sensitivity of CL-pPSL was compared with conventional PEGylated pH-sensitive (pPSL) using a calcein leakage assay. At 37 °C, PEGB-Hz-CHEMS was relatively stable at pH 7.4 with a half-life of 24 h. In comparison, at pH 5.5 and pH 6.5 PEG detachment within 1 h was determined as 80%, and 50%, respectively. PEG detachment of the polymer was through simultaneous cleavage of the hydrazine (CON) and hydrazone (NC) bonds, depending on pH, thus the polymer is more pH-sensitive than those with a hydrazine bond only. The grafting densities of PEGB-Hz-CHEMS on CL-pPSL were optimised to achieve a PEG density of 1.7% (mol). The unilamellar CL-pPSL (123 nm) were shown to be sable at least for 3 months at 4 °C and have enhanced pH-sensitivity compared with pPSL in the calcein leakage assay. Therefore, the smart cleavable PEG polymer is promising in liposome formulation to overcome the PEG dilemma.

Isothermal titration calorimetry and vesicle leakage assays highlight the differential behaviors of tau repeat segments upon interaction with anionic lipid membranes

Tau misfolding has been implicated in a variety of tauopathies, including Alzheimer's disease. The microtubule binding domain of tau consists of four repeat segments (R1-R4), and aggregation of these segments leads to the formation of neurofibrillary tangles. Previous studies indicate that misfolded tau associates with anionic phospholipid membranes, invoking structural transformations that could play a role in aggregation. Here, we investigated the role of membrane surface charge on the binding affinity of individual tau repeat segments, and whether these segments exhibit lytic activity. We quantified the thermodynamics of this process in terms of the affinity (Kd), enthalpy (ΔH), entropy (ΔS), and change in specific heat capacity (ΔCp). While neutral membranes exhibited weak interactions with each tau repeat segment, segments R2 and R3 exhibited relatively strong binding with anionic membranes with favorable ΔS and a negative value of ΔCp. Calcein leakage assays show that each repeat segment displays lytic activity, but only upon the interaction with anionic membranes. Taken together, these results distinguish the relative selectivity for anionic membranes by each repeat segment and the degree of membrane disruption that results.

AvBD1 nucleotide polymorphisms, peptide antimicrobial activities and microbial colonisation of the broiler chicken gut

BackgroundThe importance of poultry as a global source of protein underpins the chicken genome and associated SNP data as key tools in selecting and breeding healthy robust birds with improved disease resistance. SNPs affecting host peptides involved in the innate defences tend to be rare, but three non-synonymous SNPs in the avian β-defensin (AvBD1) gene encoding the variant peptides NYH, SSY and NYY were identified that segregated specifically to three lines of commercial broiler chickens Line X (LX), Line Y(LY) and Line Z. The impacts of such amino acid changes on peptide antimicrobial properties were analysed in vitro and described in relation to the caecal microbiota and gut health of LX and LY birds.ResultsTime-kill and radial immune diffusion assays indicated all three peptides to have antimicrobial properties against gram negative and positive bacteria with a hierarchy of NYH > SSY > NYY. Calcein leakage assays supported AvBD1 NYH as the most potent membrane permeabilising agent although no significant differences in secondary structure were identified to explain this. However, distinct claw regions, identified by 3D modelling and proposed to play a key role in microbial membrane attachment, and permeation, were more distinct in the NYH model. In vivo AvBD1 synthesis was detected in the bird gut epithelia. Analyses of the caecal gut microbiota of young day 4 birds suggested trends in Lactobacilli sp. colonisation at days 4 (9% LX vs × 30% LY) and 28 (20% LX vs 12% LY) respectively, but these were not statistically significant (P > 0.05).ConclusionAmino acid changes altering the killing capacity of the AvBD1 peptide were associated with two different bird lines, but such changes did not impact significantly on caecal gut microbiota.

Electrostatic Interactions between Tau Peptides and Model Biological Membranes: A Folding-Upon-Binding Mechanism

Found in neuronal cells of the central nervous system, tau proteins primarily function in cytoskeletal stability by associating with microtubules. During Alzheimer's disease and other tauopathies, misfolded tau proteins form neurofibrillary tangles (NFTs), thus impairing neuronal function and causing various neurodegenerative effects. The microtubule binding domain of tau consists of four repeat segments (R1-R4), and aggregation of these segments has previously been observed within NFTs. Previous research has also found misfolded tau components associated with the Golgi membrane, but not the plasma membrane. Here, we have used circular dicroism (CD) and isothermal titration calorimetry (ITC) to examine interactions between repeat segments R1-R4 and lipid bilayers of varying charge (DOPC and DOPC/DOPG 80:20). Our data indicate that significant structural changes are induced by charged membrane mimics and an entropy-driven binding process occurs during this interaction. Indeed, binding of tau occurs concomitantly with a classical hydrophobic effect. Thus, we propose a folding-upon-binding event occurring between negatively-charged membranes and the more promiscuous tau repeat segments. Results from calcein leakage assays and membrane fusion FRET measurements will also be presented to further probe the mechanism of bilayer disruption. These results will help explain a possible mechanism of tau misfolding during Alzheimer's disease, and are potentially beneficial to design strategies that prevent or reverse the aggregation process.

Profiling Metal Oxides with Lipids: Magnetic Liposomal Nanoparticles Displaying DNA and Proteins.

Metal oxides include many important materials with various surface properties. For biomedical and analytical applications, it is desirable to engineer their biocompatible interfaces. Herein, a phosphocholine liposome (DOPC) and its headgroup dipole flipped counterpart (DOCP) were mixed with ten common oxides. Using the calcein leakage assay, cryo-TEM, and ζ-potential measurement, these oxides were grouped into three types. The type 1 oxides (Fe3 O4 , TiO2 , ZrO2 , Y2 O3 , ITO, In2 O3 , and Mn2 O3 ) form supported bilayers only with DOCP. Type 2 (SiO2 ) forms supported bilayers only with DOPC; type 3 (ZnO and NiO) are cationic and damage lipid membranes. Magnetic Fe3 O4 nanoparticles were further studied for conjugation of fluorophores, proteins, and DNA to the supported DOCP bilayers via lipid headgroup labeling, covalent linking, or lipid insertion. Delivery of the conjugates to cells and selective DNA hybridization were demonstrated. This work provides a general solution for coating the type 1 oxides with a simple mixing in water, facilitating applications in biosensing, separation, and nanomedicine.

Profiling Metal Oxides with Lipids: Magnetic Liposomal Nanoparticles Displaying DNA and Proteins

AbstractMetal oxides include many important materials with various surface properties. For biomedical and analytical applications, it is desirable to engineer their biocompatible interfaces. Herein, a phosphocholine liposome (DOPC) and its headgroup dipole flipped counterpart (DOCP) were mixed with ten common oxides. Using the calcein leakage assay, cryo‐TEM, and ζ‐potential measurement, these oxides were grouped into three types. The type 1 oxides (Fe3O4, TiO2, ZrO2, Y2O3, ITO, In2O3, and Mn2O3) form supported bilayers only with DOCP. Type 2 (SiO2) forms supported bilayers only with DOPC; type 3 (ZnO and NiO) are cationic and damage lipid membranes. Magnetic Fe3O4 nanoparticles were further studied for conjugation of fluorophores, proteins, and DNA to the supported DOCP bilayers via lipid headgroup labeling, covalent linking, or lipid insertion. Delivery of the conjugates to cells and selective DNA hybridization were demonstrated. This work provides a general solution for coating the type 1 oxides with a simple mixing in water, facilitating applications in biosensing, separation, and nanomedicine.

Activity of tick antimicrobial peptide from Ixodes persulcatus (persulcatusin) against cell membranes of drug-resistant Staphylococcus aureus

Persulcatusin (IP), which is an antimicrobial peptide found in Ixodes persulcatus midgut, is active against Gram-positive bacteria such as Staphylococcus aureus. Multidrug-resistant bacteria in particular methicillin-resistant S. aureus (MRSA), vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) are a worldwide clinical concern. In the present study, to explore the potential of IP as a new agent against multidrug-resistant S. aureus infections, we evaluated the antimicrobial activity of IP against multidrug-resistant S. aureus strains by MIC90, morphological observation with scanning electron microscope (SEM), and the calcein leakage assay of membrane integrity. Among the six antimicrobial peptides used in this study, IP exhibited the lowest MIC90 values for both vancomycin-susceptible and -resistant S. aureus strains. The IP MIC90 against a VISA strain was equivalent to vancomycin, while the MIC90 against VRSA was relatively low. SEM observations indicated that bacterial cells exposed to IP were crumpled and showed prominent structural changes. Moreover, IP influenced the cell membranes of both MRSA and VRSA in a mere 5 min, leading to leakage of the preloaded calcein. Although a VISA strain was resistant to the action of IP on cell membrane, the MIC90 of IP was lower than that of Nisin, suggesting that IP had another bactericidal mechanism in addition to cell membrane attack. Our results indicate that the synthetic tick antimicrobial peptide, IP exhibits strong antibacterial activity against multidrug-resistant S. aureus strains, including VRSA, via both cell membrane attack and another unknown mechanism. IP represents a promising candidate for a new anti-VRSA therapy.

Enantiomeric CopA3 dimer peptide suppresses cell viability and tumor xenograft growth of human gastric cancer cells.

The CopA3 dimer peptide is a coprisin analog that has an anticancer effect against human cancer cells in vitro. In this study, we investigated the anticancer activity of the enantiomeric CopA3 dimer peptide in human gastric cancer cell lines as well as in an in vivo tumor xenograft model. Enantiomeric CopA3 reduced gastric cancer cell viability and exhibited cytotoxicity against cancer cells. Enantiomeric CopA3-induced cell death was mediated by specific interactions with phosphatidylserine and phosphatidylcholine, membrane components that are enriched in cancer cells, in a calcein leakage assay. Moreover, acridine orange/ethidium bromide staining, flow cytometric analysis, and Western blot analysis showed that enantiomeric CopA3 induced apoptotic and necrotic gastric cancer cell death. The antitumor effect was also observed in a mouse tumor xenograft model in which intratumoral inoculation of the peptide resulted in a significant decrease in the SNU-668 gastric cancer tumor volume. In addition, periodic acid-Schiff and hematoxylin staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay revealed apoptotic and necrotic cell death in tumor masses treated with greater than 150μg CopA3. Collectively, these results indicate that the enantiomeric CopA3 dimer peptide induces apoptosis and necrosis of gastric cancer cells in vitro and in vivo, indicating that the peptide is a potential candidate for the treatment of gastric cancer, which is a common cause of cancer and cancer deaths worldwide.

A novel antimicrobial peptide, scolopendin, from Scolopendra subspinipes mutilans and its microbicidal mechanism.

A novel antimicrobial peptide (AMP) was identified from the centipede Scolopendra subspinipes mutilans by RNA sequencing, and the amino acid sequences predicted from the sequenced mRNAs were compared with those of known AMPs. We named this peptide scolopendin, according to its origin, and investigated the molecular mechanisms underlying its antimicrobial activity. Our findings showed that scolopendin had antimicrobial activity against several pathogenic microorganisms, but did not produce hemolysis of human erythrocytes. In addition, disturbances in the cell membrane potential, induction of potassium release from the cytosol, and increased membrane permeability of the microbes Candida albicans and Escherichia coli O157 were detected by the use of 3,3'-dipropylthiacarbocyanine iodide [DiSC3(5)] dye, potassium leakage assay, and propidium iodide influx assay, respectively, following scolopendin treatment. Further evidence to support the membrane-targeted action of scolopendin was obtained using artificial liposomes as models of the cell membrane. Use of calcein and FITC-labeled dextran leakage assays from scolopendin-treated giant unilamellar vesicles and large unilamellar vesicles showed that scolopendin has a pore-forming action on microbial membrane, with an estimated pore radius of 2.3-3.3nm. In conclusion, scolopendin is a novel and potent AMP with a membrane-targeted mechanism of action.

Fungicidal mechanisms of the antimicrobial peptide Bac8c

Bac8c (RIWVIWRR-NH2) is an analogue peptide derived through complete substitution analysis of the linear bovine host defense peptide variant Bac2A. In the present study, the antifungal mechanism of Bac8c against pathogenic fungi was investigated, with a particular focus on the effects of Bac8c on the cytoplasmic membrane. We used bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] staining and 3,3’-dipropylthiacarbocyanine iodide [DiSC3(5)] assays to show that Bac8c induced disturbances in the membrane potential of Candida albicans. An increase in membrane permeability and suppression of cell wall regeneration were also observed in Bac8c-treated C. albicans. We studied the effects of Bac8c treatment on model membranes to elucidate its antifungal mechanism. Using calcein and FITC-labeled dextran leakage assays from Bac8c-treated large unilamellar vesicles (LUVs) and giant unilamellar vesicles (GUVs), we found that Bac8c has a pore-forming action on fungal membranes, with an estimated pore radius of between 2.3 and 3.3nm. A membrane-targeted mechanism of action was also supported by the observation of potassium release from the cytosol of Bac8c-treated C. albicans. These results indicate that Bac8c is considered as a potential candidate to develop a novel antimicrobial agent because of its low-cost production characteristics and high antimicrobial activity via its ability to induce membrane perturbations in fungi.

An antifungal mechanism of curcumin lies in membrane-targeted action within Candida albicans.

The aim of this study is to investigate the antifungal mechanism of curcumin. This polyphenolic compound has been used traditionally in Asia for medicinal, culinary, and other purposes. Although antifungal effect of curcumin has been reported, this is the first study for its mode of action underlying disruption of plasma membrane in Candida albicans. The leakage of potassium ion from the fungal cytosol and dissipation in membrane potential was detected by bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4 ] staining. We also investigated an increase in membrane permeability in curcumin-treated C. albicans with influx of propidium iodide assay. Fluorescence analysis with 1,6-diphenyl-1,3,5-hexatriene supported the membrane-targeted mechanism of action indicating membrane disruption. On the basis of these results, we studied the effects of curcumin treatment on model membrane to elucidate its antifungal mechanism. Using calcein leakage assays from curcumin-treated large unilamellar vesicles and giant unilamellar vesicles, we found that curcumin has membrane-active mechanism inducing leakage of intracellular component through the flappy membrane. Therefore, this study suggests that curcumin exerts antifungal activity via inducing disruption of fungal plasma membrane.

The influence of pathological mutations and proline substitutions in TDP-43 glycine-rich peptides on its amyloid properties and cellular toxicity.

TAR DNA-binding protein (TDP-43) was identified as the major ubiquitinated component deposited in the inclusion bodies in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) in 2006. Later on, numerous ALS-related mutations were found in either the glycine or glutamine/asparagine-rich region on the TDP-43 C-terminus, which hinted on the importance of mutations on the disease pathogenesis. However, how the structural conversion was influenced by the mutations and the biological significance of these peptides remains unclear. In this work, various peptides bearing pathogenic or de novo designed mutations were synthesized and displayed their ability to form twisted amyloid fibers, cause liposome leakage, and mediate cellular toxicity as confirmed by transmission electron microscopy (TEM), circular dichroism (CD), Thioflavin T (ThT) assay, Raman spectroscopy, calcein leakage assay, and cell viability assay. We have also shown that replacing glycines with prolines, known to obstruct β-sheet formation, at the different positions in these peptides may influence the amyloidogenesis process and neurotoxicity. In these cases, GGG308PPP mutant was not able to form beta-amyloid, cause liposome leakage, nor jeopardized cell survival, which hinted on the importance of the glycines (308–310) during amyloidogenesis.

Surfactants, Salt, and pH Alter Nanoparticle-Model Cell Membrane Interactions

Due to their small size, nanoparticles have the ability to penetrate pulmonary and vascular tissue, and as a result, are classified as potential human carcinogens. On the other hand, nanoparticle insertion into targeted cells can play a key role in drug delivery and gene therapy applications, prompting a need to more thoroughly characterize nanoparticle/membrane interactions. Polystyrene nanoparticles with modifications in surface functionalization and detergent conditions remain monodisperse in a variety of aqueous solutions as measured by dynamic light scattering, but tend to aggregate in phosphate buffered saline at neutral pH potentially due to electrostatic screening effects. Calcein leakage assays with small unilamellar egg phosphatidylcholine vesicles were run to measure their interactions with nanoparticles and determine applicability of previous experiments performed with lipid monolayers at the air-water interface that modeled the outer leaflet of a cell membrane. In pure water, adding surfactant to detergent-free nanoparticle solutions increased the magnitude of membrane permeabilization compared to nanoparticles alone. In a buffered solution, the opposite was true; addition of surfactant decreased the nanoparticle induced membrane permeabilization. To better understand nanoparticle-detergent-membrane interactions, either nanoparticles or detergents were individually introduced to the vesicles, and following a time lapse, the other component was introduced. A model of detergent sequestration by the polystyrene nanoparticles explains these results and provides insight to the mechanism of vesicle leakage caused by surfactant-nanoparticle mixtures.

Development and In Vitro Proof-of-Concept of Interstitially Targeted Zinc- Phthalocyanine Liposomes for Photodynamic Therapy

Photodynamic therapy (PDT) has been successfully used to treat various solid tumors. However, some cancer types respond poorly to PDT, including urothelial carcinomas, nasopharyngeal carcinomas, and extrahepatic cholangiocarcinomas. The therapeutic recalcitrance is in part due to the use of photosensitizers with suboptimal optical/ photochemical properties and unfavorable pharmacokinetics. To circumvent these drawbacks, a second-generation photosensitizer with improved optical/photochemical properties, zinc phthalocyanine (ZnPC), was encapsulated in interstitially targeted, polyethylene glycol-coated liposomes (ITLs) intended for systemic administration. The ZnPC-ITLs were examined for reactive oxygen species (ROS) generation and oxidation capacity and validated for tumoricidal efficacy in human extrahepatic cholangiocarcinoma (Sk-Cha1) cells. ZnPC-ITL uptake and the mechanism and mode of PDT-induced cell death were studied. The ITL formulation was optimized on the basis of fluorescence spectroscopy and photon correlation spectroscopy. The extent of ROS generation, protein oxidation, and membrane oxidation were determined by the 2',7'-dichlorodihydrofluorescein, tryptophan oxidation, and calcein leakage assays, respectively. PDT efficacy was evaluated by measuring mitochondrial activity and apoptosis-/necrosis-specific staining in combination with flow cytometry. The uptake of fluorescently labeled ITLs was assayed by confocal microscopy, flow cytometry, and fluorescence spectroscopy. ZnPC-ITLs exhibited maximum ROS-generating and oxidation potential at a ZnPC:lipid molar ratio of 0.003. PDT of Sk-Cha1 cells incubated with ZnPC-ITLs induced cell death in a lipid concentration- dependent manner. The mode of PDT-induced cell death comprised both apoptosis and necrosis, with necrotic cell death predominating. Post-PDT cell death was attributable to pre-PDT ZnPC-ITL uptake by cancer cells, which was more efficient at smaller ITL diameters and a more positive surface charge. ZnPC-ITLs constitute a nanoparticulate photosensitizer delivery system capable of inducing apoptosis and necrosis in cultured extrahepatic cholangiocarcinoma cells by PDT-mediated oxidative processes. PDT-induced cell death is dependent on the extent of ITL uptake, which in turn relies on ITL size and zeta potential.

Spectroscopic and thermodynamic evidence for antimicrobial peptide membrane selectivity

In our laboratory we developed a series of antimicrobial peptides that exhibit selectivity and potency for prokaryotic over eukaryotic cells ( Hicks et al., 2007). Circular dichroism (CD), isothermal calorimetry (ITC) and calcein leakage assays were conducted to determine the mechanism of lipid binding of a representative peptide 1 (Ac-GF-Tic-Oic-GK-Tic-Oic-GF-Tic-Oic-GK-Tic-KKKK-CONH 2) to model membranes. POPC liposomes were used as a simple model for eukaryotic membranes and 4:1 POPC:POPG liposomes were used as a simple model for prokaryotic membranes. CD, ITC and calcein leakage data clearly indicate that compound 1 interacts via very different mechanisms with the two different liposome membranes. Compound 1 exhibits weaker binding and induces less calcein leakage in POPC liposomes than POPC:POPG (4:1 mole ratio) liposomes. The predominant binding mechanism to POPC appears to be limited to surface interactions while the mechanism of binding to 4:1 POPC:POPG most likely involves some type of pore formation.

Effect of L- to D-Peptide Isomerisation on the Activity of Antimicrobial Peptide Anoplin

Isolated from the venom sac of solitary spider wasp, Anoplius samariensis, Anoplin is the smallest linear α-helical antimicrobial peptide found naturally up to date. It has broad spectrum activity against both Gram-positive and Gram-negative bacteria, and little hemolytic activity toward human erythrocytes (1,2). Previous studies showed that substitution of all amino acids in the peptide with D-isomers can increase the bioavailability and reduce peptide degradation without affecting the antimicrobial properties since the net charge and the hydrophobicity are retained (3). In the present work, two stereoisomers of Anoplin were studied using UV resonance Raman spectroscopy, Langmuir Blodgett, atomic force microscopy, calcein leakage assay and antimicrobial assay. UV resonance Raman data indicate that the two forms of the peptide adopt similar conformations in aqueous buffer and in membrane mimicking solutions. Monolayer isotherms show that D-Anoplin has a lightly greater area per molecule than L-Anoplin. Finally, membrane rupturing ability of both stereoisomers was found to depend strongly on membrane composition.(1) Konno, K., Hisada, M., Fontana, R., Lorenzi, C.C.B., Naoki, H., Itagaki, Y., Miwa, A., Kawai, N., Nakata, Y., Yasuhara, T., Neto, J.R., de Azevedo Jr., W.F., Palma, M.S. and Nakajima, T. (2001) Biochim. Biophys. Acta 1550: 70-80.(2) Ifrah, D., Doisy, X., Ryge, R.S. and Hansen, P.R. (2005) J. Pept. Sci. 11: 113-121.(3) Wade, D., Boman, A., Wahlin, B., Drain, C.M., Andreu, D., Boman, H.G. and Merrifield, R.B. (1990) Proc. Nat. Acad. Sci. 87: 4761-4765.

Interactions of Antimicrobial Peptide Latarcin With Model Cell Membrane

Latarcins are linear antimicrobial peptides purified from the venom of the Lachesana tarabaevi spider. They are highly active against Gram-positive and Gram-negative bacteria with minimum inhibitory concentrations (MIC) at the micromolar level and low hemolytic activity (1, 2). In the present work, a 26 residue peptide Latarcin 2a that adopts a helix-hinge-helix conformation in a membrane mimetic environment (1, 2) was studied as well as a derivative obtained by replacing the Guanine 11 by with Alanine. The interaction of the peptides with phospholipid mono and bilayers were investigated using Langmuir-Blodgett monolayer technique, Atomic Force Microscopy (AFM), calcein leakage assay and UV resonance Raman spectroscopy. Effect of small changes in the primary structure of the peptide on the membrane rupturing activity is discussed.