Effect of Phosphatidylethanolamine on Pore Formation Induced by the Antimicrobial Peptide PGLa.

Most antimicrobial peptides (AMPs) induce pore formation and a burst of lipid bilayers and plasma membranes. This causes severe leakage of the internal contents and cell death. The AMP PGLa forms nanopores in giant unilamellar vesicles (GUVs) comprising dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG). We here elucidated the effect of the line tension of a prepore rim on PGLa-induced nanopore formation by investigating the interaction of PGLa with single GUVs comprising dioleoylphosphatidylethanolamine (DOPE)/DOPG (6:4) in buffer using the single GUV method. We found that PGLa forms nanopores in the GUV membrane, which evolved into a local burst and burst of GUVs. The rate of pore formation in DOPE/DOPG-GUVs was smaller than that in DOPC/DOPG-GUVs. PGLa is located only in the outer leaflet of a GUV bilayer just before a fluorescent probe AF647 leakage from the inside, indicating that this asymmetric distribution induces nanopore formation. PGLa-induced local burst and burst of GUVs were observed at 10 ms-time resolution. After nanopore formation started, dense particles and small vesicles appeared in the GUVs, followed by a decrease in the GUV diameter. The GUV was finally converted into smaller GUV or lipid membrane aggregates. We discuss the mechanisms of PGLa-induced nanopore formation and its direct evolution to a GUV burst.

Estimation of negative membrane tension in lipid bilayers and its effect on antimicrobial peptide magainin 2-induced pore formation.

Positive membrane tension in the stretched plasma membrane of cells and in the stretched lipid bilayer of vesicles has been well analyzed quantitatively, whereas there is limited quantitative information on negative membrane tension in compressed plasma membranes and lipid bilayers. Here, we examined negative membrane tension quantitatively. First, we developed a theory to describe negative membrane tension by analyzing the free energy of lipid bilayers to obtain a theoretical equation for negative membrane tension. This allowed us to obtain an equation describing the negative membrane tension (σosm) for giant unilamellar vesicles (GUVs) in hypertonic solutions due to negative osmotic pressure (Π). Then, we experimentally estimated the negative membrane tension for GUVs in hypertonic solutions by measuring the rate constant (kr) of rupture of the GUVs induced by the constant tension (σex) due to an external force as a function of σex. We found that larger σex values were required to induce the rupture of GUVs under negative Π compared with GUVs in isotonic solution and quantitatively determined the negative membrane tension induced by Π (σosm) by the difference between these σex values. At small negative Π, the experimental values of negative σosm agree with their theoretical values within experimental error, but as negative Π increases, the deviation increases. Negative tension increased the stability of GUVs because higher tensions were required for GUV rupture, and the rate constant of antimicrobial peptide magainin 2-induced pore formation decreased.

Effect of membrane tension on antimicrobial peptide PGLa-induced pore formation in lipid bilayers.

The osmotic pressure (Π) method has recently been developed to quantitatively examine the effect of membrane tension (σ) on pore formation in giant unilamellar vesicles (GUVs) induced by antimicrobial peptides (AMPs). Here, we used the Π method to reveal the effect of σ on the interaction of an AMP, PGLa, with lipid bilayers comprising dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (4/6). PGLa induced leakage of fluorescent probes from single GUVs under Π, indicating nanopore formation. Membrane tension did not transform a PGLa-induced nanopore into a micropore nor cause GUV burst up to 3.4 mN/m, which is in contrast with the effect of σ on another AMP, magainin 2-induced pore formation, where lower σ resulted in GUV burst. The fraction of leaking GUVs at a specific time increased with increasing σ, indicating that the rate of PGLa-induced pore formation increases with increasing σ. The rate of transfer of fluorescent probe-labeled PGLa across the lipid bilayer without pore formation also increased with increasing σ. PGLa-induced pore formation requires a symmetric distribution of peptides in both leaflets of the GUV bilayer, and thus we infer that the increase in the rate of PGLa transfer from the outer leaflet to the inner leaflet underlies the increase in the rate of pore formation with increasing σ. On the basis of these results, we discuss the difference between the effect of σ on nanopore formation in GUV membranes induced by PGLa and that by magainin 2.

Relationship between antimicrobial peptides-induced cell membrane damage and bactericidal activity.

Most antimicrobial peptides (AMPs) act by killing bacterial cells. However, there is little information regarding the required interaction time between AMPs and bacterial cells to exert the bactericidal activity. One of the causes of the bactericidal activity is considered to be cell membrane damage, although little direct evidence is available. Here, we investigated the relationship between AMP-induced cell membrane damage in Escherichia coli and AMP-induced cell death at the single-cell level. Magainin 2, lactoferricin B, and PGLa were selected as the AMPs. First, we examined the interaction time (t) of AMPs with cells required to induce cell death using the single-cell analysis. The fraction of microcolonies containing only a single cell, Psingle (t), which indicates the fraction of dead cells, increased with time to reach ∼1 in a short time (≤5 min). Then, we examined the interaction between AMPs and single cells using confocal laser scanning microscopy in the presence of membrane-impermeable SYTOX green. Within a short time interaction, the fluorescence intensity of the cells due to SYTOX green increased, indicating that AMPs induced cell membrane damage through which the dye entered the cytoplasm. The fraction of cells in which SYTOX green entered the cytoplasm among all examined cells after the interaction time (t), Pentry (t), increased with time, reaching ∼1 in a short time (≤5 min). The values of Psingle (t) and Pentry (t) were similar at t ≥ 3 min for all AMPs. The bindings of AMPs to cells were largely reversible, whereas the AMP-induced cell membrane damages were largely irreversible because SYTOX green entered the cells after dilution of AMP concentration. Based on these results, we conclude that the rapid, substantial membrane permeabilization of cytoplasmic contents after a short interaction time with AMPs and the residual damage after dilution induce cell death.

Effect of monolayer spontaneous curvature on constant tension-induced pore formation in lipid bilayers.

The methodology of constant tension-induced rupture of giant unilamellar vesicles (GUVs) has provided information on tension-induced pore formation. This method was used to investigate the effect of spontaneous curvature (H0) for a lipid monolayer on the rate constant (kr) for constant tension (σ)-induced rupture, which originates from pore formation in lipid bilayers. Lipids were incorporated with different H0 values into GUV membranes to change the overall H0 value for the GUV monolayer. The dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylethanolamine (DOPE) (4/6, molar ratio, here and elsewhere) monolayer has a negative H0, whereas the DOPG/dioleoylphosphatidylcholine (DOPC) (4/6) monolayer has an essentially zero H0. A higher tension was required to induce the rupture of DOPG/DOPE (4/6)-GUVs compared with DOPG/DOPC (4/6)-GUVs. The line tension (Γ) for a pre-pore in DOPG/DOPE (4/6)-GUVs, determined by the analysis of the tension dependence of kr, was 1.5 times larger than that in DOPG/DOPC (4/6)-GUVs. The kr values for GUVs comprising DOPG/DOPC/18:1 lysophosphatidylcholine (LPC) (40/55/10), which has a positive H0, were larger than those for DOPG/DOPC (4/6)-GUVs under the same tension. The Γ value for DOPG/DOPC/LPC (40/55/10)-GUVs was almost half that for DOPG/DOPC (4/6)-GUVs. These results indicate that Γ decreases with increasing H0, which results in an increase in kr. Based on these results, the effect of H0 on kr and Γ is discussed.

Antimicrobial peptide magainin 2-induced rupture of single giant unilamellar vesicles comprising E. coli polar lipids.

Most antimicrobial peptides (AMPs) damage the cell membrane of bacterial cells and induce rapid leakage of the internal cell contents, which is a main cause of their bactericidal activity. One of the AMPs, magainin 2 (Mag), forms nanopores in giant unilamellar vesicles (GUVs) comprising phosphatidylcholine (PC) and phosphatidylglycerol (PG), inducing leakage of fluorescent probes. In this study, to elucidate the Mag-induced pore formation in lipid bilayer region in E. coli cell membrane, we examined the interaction of Mag with single GUVs comprising E. coli polar lipids (E. coli-lipid-GUVs). First, we investigated the Mag-induced leakage of a fluorescent probe AF488 from single E. coli-lipid-GUVs, and found that Mag caused rupture of GUVs, inducing rapid AF488 leakage. The rate constant of Mag-induced GUV rupture increased with the Mag concentration. Using fluorescence microscopy with a time resolution of 5 ms, we revealed the GUV rupture process: first, a small micropore was observed in the GUV membrane, then the pore radius increased within 50 ms without changing the GUV diameter, the thickness of the membrane at the pore rim concomitantly increased, and eventually membrane aggregates were formed. Mag bound to only the outer monolayer of the GUV before GUV rupture, which increased the area of the GUV bilayer. We also examined the physical properties of E. coli-lipid-GUVs themselves. We found that the rate constant of the constant tension-induced rupture of E. coli-lipid-GUVs was higher than that of PG/PC-GUVs. Based on these results, we discussed the Mag-induced rupture of E. coli-lipid-GUVs and its mechanism.

Role of interfacial hydrophobicity in antimicrobial peptide magainin 2-induced nanopore formation.

Antimicrobial peptide magainin 2 (Mag) forms nanopores in lipid bilayers and induces membrane permeation of the internal contents from vesicles. The binding of Mag to the membrane interface of a giant unilamellar vesicle (GUV) increases its fractional area change, δ, which is one of the main causes of Mag-induced nanopore formation. However, the role of its amino acid composition in the Mag-induced area increase and the following nanopore formation is not well understood. Here, to elucidate it we examined the role of interfacial hydrophobicity of Mag in its nanopore formation activity by investigating de novo-designed Mag mutants-induced nanopore formation in GUVs. Aligned amino acid residues in the α-helix of Mag were replaced to create 3 mutants: F5A-Mag, A9F-Mag, and F5,12,16A-Mag. These mutants have different interfacial hydrophobicity due to the variation of the numbers of Phe and Ala because the interfacial hydrophobicity of Phe is higher than that of Ala. The rate constant of Mag mutant-induced nanopore formation, kp, increased with increasing numbers of Phe residues at the same peptide concentration. Further, the Mag mutant-induced δ increased with increasing numbers of Phe residues at the same peptide concentration. These results indicate that kp and δ increase with increasing interfacial hydrophobicity of Mag mutants. The relationship between kp and δ in the Mag and its mutants clearly indicates that kp increases with increasing δ, irrespective of the difference in mutants. Based on these results, we can conclude that the interfacial hydrophobicity of Mag plays an important role in its nanopore formation activity.

Single-Cell Analysis of the Antimicrobial and Bactericidal Activities of the Antimicrobial Peptide Magainin 2.

Antimicrobial peptides (AMPs) inhibit the proliferation of or kill bacterial cells. To measure these activities, several methods have been used, which provide only the average value of many cells. Here, we report the development of a method to examine the antimicrobial and bactericidal activities of AMPs at the single-cell level (i.e., single-cell analysis) and apply this strategy to examine the interaction of an AMP, magainin 2 (Mag), with Escherichia coli cells. Using this method, we monitored the proliferation of single cells on agar in a microchamber and measured the distribution of the number of cells in each microcolony using optical microscopy. For method A, we incubated cells in the presence of various concentrations of AMPs for 3 h. The fraction of microcolonies containing only a single cell, Psingle, increased with the Mag concentration and reached 1 at a specific concentration, which corresponded to the MIC. For method B, after the interaction of a cell suspension with an AMP for a specific time, an aliquot was diluted to stop the interaction, and the proliferation of single cells then was monitored after a 3-h incubation; this method permits the definition of Psingle(t), the fraction of dead cells after the interaction. For the interaction of Mag with E. coli cells, Psingle(t) increased with the interaction time, reaching ~1 at 10 and 20 min for 25 and 13 µM Mag, respectively. Thus, these results indicate that a short interaction time between Mag and E. coli cells is sufficient to induce bacterial cell death. IMPORTANCE To elucidate the activity of antimicrobial peptides (AMPs) against bacterial cells, it is important to estimate the interaction time that is sufficient to induce cell death. We have developed a method to examine the antimicrobial and bactericidal activities of AMPs at the single-cell level (i.e., single-cell analysis). Using this method, we monitored the proliferation of single cells on agar in a microchamber and measured the distribution of the number of cells in each microcolony using optical microscopy. We found that during the interaction of magainin 2 (Mag) with E. coli cells, the fraction of dead cells, Psingle(t), increased with the interaction time, rapidly reaching 1 (e.g., 10 min for 25 µM Mag). This result indicates that Mag induces cell death after a short time of interaction.

Effect of osmotic pressure on pore formation in lipid bilayers by the antimicrobial peptide magainin 2.

Osmotic pressure (Π) induces membrane tension in cells and lipid vesicles, which may affect the activity of antimicrobial peptides (AMPs) by an unknown mechanism. We recently quantitated the membrane tension of giant unilamellar vesicles (GUVs) due to Π under physiological conditions. Here, we applied this method to examine the effect of Π on the interaction of the AMP magainin 2 (Mag) with single GUVs. Under low Π values, Mag induced the formation of nanometer-scale pores, through which water-soluble fluorescent probe AF488 permeates across the membrane. The rate constant for Mag-induced pore formation (kp) increased with increasing Π. It has been proposed that the membrane tension in the GUV inner leaflet (σin) caused by Mag binding to the outer leaflet plays a vital role in Mag-induced pore formation. During the interactions between Mag and GUVs under Π, the σin increases due to Π, thereby increasing kp. The relationship between the kp and the total σin due to Π and Mag agreed with that without Π. In contrast, Mag induced rupture of a subset of GUVs under higher Π. Using fluorescence microscopy with a high-speed camera, the GUV rupture process was revealed. First, a small micrometer-scale pore was observed in individual GUVs. Then, the pore radius increased within ∼100 ms without changing the GUV diameter and concomitantly the thickness of the membrane at the pore rim increased, and finally the GUV transformed into a membrane aggregate. Based on these results, we discussed the effect of Π on Mag-induced damage of GUV membranes.

A Single GUV Method for Revealing the Action of Cell-Penetrating Peptides in Biomembranes.

The mechanism of entry of cell-penetrating peptides (CPPs) into the cytosol of various cells has been studied by examining the interaction of CPPs with lipid bilayers and their entry into lipid vesicle lumens using various methods. Here we describe a single giant unilamellar vesicle (GUV) method to study CPPs. In this new method, we use GUVs containing small GUVs in the mother GUV lumen or GUVs containing large unilamellar vesicles (LUVs) in the GUV lumen and investigate the interaction of fluorescent probe-labeled CPPs with single GUVs in real time using confocal laser scanning microscopy. This method can detect CPPs in the GUV lumen with high sensitivity, allowing immediate measurement of the time course of entry of CPPs into the vesicle lumen. This method allows simultaneous measurement of the entry of CPPs and of CPP-induced pore formation, allowing the relationship between the two events to be determined. One can also simultaneously measure the entry of CPPs and the CPP concentration in the GUV membrane. The rate of entry of CPPs into a single GUV lumen can be estimated by obtaining the fraction of GUVs into which CPPs entered before a specific time t without pore formation among all examined GUVs (i.e., the fraction of entry) and the lumen intensity due to LUVs with bound CPPs. This method is therefore useful for elucidating the mechanism of entry of CPPs into lipid vesicles.

Sulfur-doped carbon dots@polydopamine-functionalized magnetic silver nanocubes for dual-modality detection of norovirus.

Synergistic dual-mode optical platforms are up-and-coming detection tools in the diagnosis and management of infectious diseases. Here, novel dual-modality fluorescence (FL) and surface-enhanced Raman scattering (SERS) techniques have been integrated into a single probe for the rapid and ultrasensitive detection of norovirus (NoV). The developed FL-SER-based biosensor relies on the dual-signal enhancements of newly synthesized sulfur-doped agar-derived carbon dots (S-agCDs). The antigen-antibody immunoreaction results in forming a core-satellite immunocomplex between anti-NoV antibody-conjugated S-agCDs and polydopamine-functionalized magnetic silver nanocubes [poly (dop)-MNPs-Ag NCs]. By deploying an immunomagnetic enrichment protocol and performing the SERS modality on a single-layer graphene substrate, norovirus-like particles (NoV-LPs) were detected across a wide range of 1 fg mL-1 - 10 ng mL-1 with an excellent limit of detection of 0.1 fg mL-1. The combined advantage of the dual-signaling properties of the biosensor was demonstrated using FL confocal imaging for "hotspots" tracking prior to SERS detection of clinical NoV in fecal specimen down to ⁓10 RNA copies mL-1. The proposed dual-modality biosensor's performance increases the prospect of a rapid and low-cost sensitive NoV detection and surveillance option for public health.

Translocation of the nonlabeled antimicrobial peptide PGLa across lipid bilayers and its entry into vesicle lumens without pore formation.

Fluorescent-probe-labeled peptides are used to study the interactions of peptides with cells and lipid vesicles but labeling peptides with fluorescent probes can significantly change these interactions. We recently developed a new method to detect the entry of nonlabeled peptides into the lumen of single giant unilamellar vesicles (GUVs). Here we applied this method to examine the interaction of the antimicrobial peptide PGLa with single GUVs to elucidate whether PGLa can enter the GUV lumen without pore formation. First, we examined the interaction of nonlabeled PGLa with single GUVs comprising dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (4/6) whose lumens contain the fluorescent probe AF647 and DOPG/DOPC (8/2)-large unilamellar vesicles encapsulating a high concentration of calcein. After a large lag period from starting the interaction with PGLa, the fluorescence intensity of the GUV lumen due to calcein (Icalcein) increased gradually without leakage of AF647, indicating that PGLa enters the GUV lumen without pore formation in the GUV membrane. The fraction of entry of PGLa increased with increasing PGLa concentration. Simultaneous measurement of the fractional area change of the GUV membrane (δ) and PGLa-induced increase in Icalcein showed that the entry of PGLa occurs only during the second increase in δ, indicating that PGLa enters the lumen during its translocation from the outer leaflet to the inner leaflet. The fraction of entry of PGLa without pore formation increased with increasing membrane tension. Based on these results, we discuss the elementary processes and the mechanism of the entry of PGLa into the GUV lumen.

Effect of membrane potential on entry of lactoferricin B-derived 6-residue antimicrobial peptide into single Escherichia coli cells and lipid vesicles.

The antimicrobial peptide (AMP) derived from lactoferricin B, LfcinB (4-9) (RRWQWR), and lissamine rhodamine B red-labeled peptide (Rh-LfcinB (4-9)) exhibit strong antimicrobial activities, and they can enter Escherichia coli cells without damaging the cell membranes. Thus, these peptides are cell-penetrating peptide (CPP) -type AMPs. In this study, to elucidate the effect of the membrane potential (Δφ) on the action of the CPP-type AMP, Rh-LfcinB (4-9), we investigated the interactions of Rh-LfcinB (4-9) with single E. coli cells and spheroplasts containing calcein in the cytosol using confocal laser scanning microscopy. At low peptide concentrations, Rh-LfcinB (4-9) entered the cytosol of single E. coli cells and spheroplasts without damaging the cell membranes, and the H+-ionophore carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) suppressed its entry. The studies using the time-kill method indicate that these low concentrations of peptide exhibit antimicrobial activity but CCCP inhibits this activity. Next, we investigated the effect of Δφ on the interaction of Rh-LfcinB (4-9) with single giant unilamellar vesicles (GUVs) comprising E. coli polar lipid extracts and containing a fluorescent probe, Alexa Fluor 647 hydrazide. At low concentrations (0.2-0.5 µM), Rh-LfcinB (4-9) showed significant entry to the single GUV lumen without pore formation in the presence of Δφ. The fraction of entry of peptide increased with increasing negative membrane potential, indicating that the rate of peptide entry into the GUV lumen increased with increasing negative membrane potential. These results indicate that Δφ enhances the entry of Rh-LfcinB (4-9) into single E. coli cells, spheroplasts, and GUVs and its antimicrobial activity.IMPORTANCE: Bacterial cells have a membrane potential (Δφ), but the effect of Δφ on action of cell-penetrating peptide-type antimicrobial peptides (AMPs) is not clear. Here, we investigated the effect of Δφ on the action of fluorescent probe-labeled AMP derived from lactoferricin B, Rh-LfcinB (4-9). At low peptide concentrations, Rh-LfcinB (4-9) enters the cytosol of Escherichia coli cells and spheroplasts without damaging their cell membrane, but a protonophore suppresses this entry and its antimicrobial activity. The rate of entry of Rh-LfcinB (4-9) into the giant unilamellar vesicles (GUVs) comprising E. coli lipids without pore formation increases with increasing Δφ. These results indicate that Δφ enhances the antimicrobial activity of Rh-LfcinB (4-9) and hence LfcinB (4-9) by increasing the rate of their entry into the cytosol.

Fluorescent and electrochemical dual-mode detection of Chikungunya virus E1 protein using fluorophore-embedded and redox probe-encapsulated liposomes.

The critical goal of sensitive virus detection should apply in the early stage of infection, which may increase the probable survival rate. To achieve the low detection limit for the early stage where a small number of viruses are present in the sample, proper amplified signals from a sensor can make readable and reliable detection. In this work, a new model of fluorescent and electrochemical dual-mode detection system has been developed to detect virus, taking recombinant Chikungunya virus E1 protein (CHIK-VP) as an example. The hydrophobic quantum dots (QDs) embedded in the lipid bilayer of liposome and methylene blue (MB) encapsulated in the inner core of liposomes played a role of dual-signaling modulator. After CHIK-VP addition, the nanocomposites and APTES-coated Fe3O4 nanoparticles (Fe3O4 NPs) were conjugated with antibodies to form a sandwich structure and separated from the medium magnetically. The nanoconjugates have been burst out by chloroform as surfactant, and both the QDs and MB are released from the liposome and were then monitored through changes in the fluorescence and electrochemical signals, respectively. These two fluorometric and electrochemical signals alteration quantified the CHIK-VP in the range of femtogram to nanogram per milliliter level with a LOD of 32 fg mL-1, making this liposomal system a potential matrix in a virus detection platform. Graphical abstract.

Membrane Tension in Negatively Charged Lipid Bilayers in a Buffer under Osmotic Pressure.

Osmotic pressure (Π) induces membrane tension in cell membranes and the lipid bilayers of vesicles and plays an important role in the functions and physical properties of these membranes. We recently developed a method to determine quantitatively the membrane tension of giant unilamellar vesicles (GUVs) under Π and applied it to GUVs comprising electrically neutral dioleoylphosphatidylcholine (DOPC). Here, we examined the effect of Π on GUVs composed of DOPC and negatively charged dioleoylphosphatidylglycerol (DOPG) in a buffer containing a physiological concentration of ions. First, we examined the rate constant, kr, for constant tension (σex)-induced rupture of DOPG/DOPC (4/6)-GUVs under Π and obtained the dependence of kr on σex in GUVs for various values of Π. Comparing this dependence in the absence of Π provided values for membrane tension due to Π, σosm, which agree with the theoretical values within the experimental error. The values of σosm for DOPG/DOPC-GUVs were smaller than those for DOPC-GUVs under the same Π. Two factors, that is, the solute concentration in a GUV suspension and the elastic modulus of the GUV membrane, can reasonably explain this difference based on the theory of σosm. We also examined the effect of Π on the rate constant, kFF, for the transbilayer movement of lipid molecules in single GUVs. The values of kFF increased with increasing Π, indicating that kFF increased with σosm. This result supports the existence of prepores in stretched lipid bilayers. Based on these results, we discuss the membrane tension of DOPG/DOPC-GUVs under Π.

Effect of membrane potential on pore formation by the antimicrobial peptide magainin 2 in lipid bilayers.

The effect of membrane potential on plasma membrane damage generated by antimicrobial peptides (AMPs) is an important, yet poorly characterized, process. Here, we studied the effect of membrane potential (φm) on pore formation by magainin 2 (Mag) in single giant unilamellar vesicles (GUVs) composed of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes. Various membrane potentials in GUVs containing gramicidin A were generated as a result of K+ concentration gradients. First, we examined Mag-generated membrane permeation of the water-soluble fluorescent probe calcein in single DOPG/DOPC-GUVs in the presence of membrane potential. The results indicate that the rate constant (kp) of Mag-induced pore formation increased with increasing negative membrane potentials. Analysis of the rim intensity of single GUVs interacting with low concentrations of a fluorescent probe, carboxyfluorescein-labeled Mag (CF-Mag), using confocal laser scanning microscopy (CLSM) shows that the concentration of CF-Mag in the membrane greatly increased with negative membrane potentials. This indicates that the binding constant of CF-Mag to the membrane increased with more negative membrane potentials. To elucidate the location of Mag in a GUV with φm during Mag-induced pore formation, we examined the interaction of Mag and a low concentration of a CF-Mag mixture with single GUVs containing the water-soluble fluorescent probe AF647 using CLSM. The data indicate that CF-Mag locates in the external leaflet of single GUVs until just before pore formation. Based on these data, we conclude that the increase in the surface concentration of Mag is one of the primary causes of the increase in kp with negative membrane potential.

Detection of the Entry of Nonlabeled Transportan 10 into Single Vesicles.

The entry of cell-penetrating peptides (CPPs) into live cells and lipid vesicles has been monitored using probe (e.g., fluorescent dye)-labeled CPPs. However, probe labeling may alter the interaction of CPPs with membranes. We have developed a new method to detect the entry of nonlabeled CPPs into the lumens of single giant unilamellar vesicles (GUVs) without pore formation in the GUV membrane. The GUVs contain large unilamellar vesicles (LUVs) whose lumens contain a high (self-quenching) concentration of the fluorescent dye calcein. If the CPPs enter the GUV lumen and interact with these LUVs to induce calcein leakage, the fluorescence intensity (FI) due to calcein in the GUV lumen increases. The lipid compositions of the LUVs and GUVs allow leakage from LUVs but not from the GUVs. We applied this method to detect the entry of transportan 10 (TP10) into single GUVs comprising dioleoylphosphatidylglycerol and dioleoylphosphatidylcholine and examined the interaction of low concentrations of nonlabeled TP10 with single GUVs whose lumens contain Alexa Fluor 647 hydrazide (AF647) and the LUVs mentioned above. The FI of the GUV lumen due to calcein increased continuously with time without leakage of AF647, indicating that TP10 entered the GUV without pore formation in the GUV membrane. The lumen intensity due to calcein increased with TP10 concentration, indicating that the rate of entry of TP10 into the GUV lumen increased. We estimated the minimum TP10 concentration in a GUV lumen detected by this method. We discuss the entry of nonlabeled TP10 and the characteristics of this method.

Action of antimicrobial peptides and cell-penetrating peptides on membrane potential revealed by the single GUV method.

Membrane potential plays various key roles in live bacterial and eukaryotic cells. So far, the effects of membrane potential on action of antimicrobial peptides (AMPs) and cell-penetrating peptides (CPPs) have been examined using cells and small lipid vesicles. However, due to the technical drawbacks of these experiments, the effect of membrane potential on the actions of AMPs and CPPs and the elementary processes of interactions of these peptides with cell membranes and vesicle membranes are not well understood. In this short review, we summarize the results of the effect of membrane potential on the action of an AMP, lactoferricin B (LfcinB), and a CPP, transportan 10 (TP10), in vesicle membranes revealed by the single giant unilamellar vesicle (GUV) method. Parts of the actions and their elementary steps of AMPs and CPPs interacting vesicle membranes under membrane potential are clearly revealed using the single GUV method. The experimental methods and their analysis described here can be used to elucidate the effects of membrane potential on various activities of peptides such as AMPs, CPPs, and proteins. Moreover, GUVs with membrane potential are more suitable as a model of cells or artificial cells, as well as GUVs containing small vesicles.

Use of Target-Specific Liposome and Magnetic Nanoparticle Conjugation for the Amplified Detection of Norovirus.

Viral diseases are one of the most life-threatening diseases as they can erupt unpredictably and spread rapidly in any medium with a very small number of particles. Therefore, the key for lethal virus detection should be highly sensitive in the early-stage detection, which can help increase the chance of survival. Amplification of the detecting signal is one of the most promising mechanisms for the detection of low-concentration analytes. A proper amplification can develop such a kind of system where a small number of particles can produce intense signals for a prominent detection. Keeping this in mind, in this report, we have presented a fluorometric method to detect norovirus (NoV) by a newly developed fluorophore-labeled liposome and a magnetically modified Fe3O4 combined system. Homogeneously distributed amine-functionalized liposomes have been constructed filled with a strong fluorophore of calcein. Simultaneously, (3-aminopropyl)-triethoxysilane (APTES)-functionalized Fe3O4 nanoparticles are also synthesized by the standard silanization process, and these two separately synthesized nanoparticles were functionalized with an antibody to achieve specificity. The Fe3O4 and calcein-liposome system has been applied for NoV detection, which was magnetically separated from the analyte medium and then externally burst to release the fluorophores from the core of the liposome. The easiness, rapidity, and sensitivity in a wide linear range can offer a huge potential of this method in point-of-care diagnostics.

Role of Membrane Potential on Entry of Cell-Penetrating Peptide Transportan 10 into Single Vesicles.

Cell-penetrating peptides (CPPs) can translocate across plasma membranes to enter the cytosol of eukaryotic cells without decreasing cell viability. We revealed the mechanism underlying this translocation by examining the effect of membrane potential, φm, on the entry of a CPP, transportan 10 (TP10), into the lumen of single giant unilamellar vesicles (GUVs). For this purpose, we used the single GUV method to detect the entry of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) into the lumen of single GUVs. First, we used various K+ concentration differences to apply different negative membrane potentials on single GUVs containing gramicidin A in their membrane and confirmed these potentials using the φm-sensitive fluorescent probe 3,3'-dihexyloxacarbocyanine iodine. The fluorescence intensity of the GUV membranes (i.e., the rim intensity) due to 3,3'-dihexyloxacarbocyanine iodine increased with |φm| up to 118 mV, and its dependence on |φm| less than 28 mV agreed with a theoretical estimation (i.e., the dye concentration in the inner leaflet of a GUV is larger than that in the outer leaflet according to the Boltzmann distribution). We then examined the effect of φm on the entry of CF-TP10 into GUVs using single GUVs containing small GUVs or large unilamellar vesicles inside the mother GUV lumen. We found that CF-TP10 entered the GUV lumen without pore formation and the rate of entry of CF-TP10 into the GUV lumen, Ventry, increased with an increase in |φm|. The rim intensity due to CF-TP10 increased with an increase in |φm|, indicating that the CF-TP10 concentration in the inner leaflet of the GUV increased with |φm|. These results indicate that the φm-induced elevation in Ventry can be explained by the increase in CF-TP10 concentration in the inner leaflet with |φm|. We discuss the mechanism underlying this effect of membrane potential based on the pre-pore model of the translocation of CF-TP10 across a GUV membrane.