Anionic Vesicles Research Articles

DNA-involved thermotropic liquid crystals from catanionic vesicles

Thermotropic liquid crystals (TLCs) have created unpredictable applications ranging from sensor to electronic devices. DNA can interact with positively charged cationic and anionic (catanionic) vesicles by electrostatic interaction to form DNA-based TLCs. In the present work, the polarizing optical microscopy (POM) images clearly demonstrate the formation of DNA-involved liquid crystals, we explore the general role of DNA-involved TLC formation and the regulating factors of properties of DNA-based TLCs. We verify the effect of the chain lengths of surfactants and DNA on the formation and properties of TLCs. We found that DNA-involved TLCs constructed by the different surfactants with same chain length present the excellent properties. The position of functional group of ferrocenyl surfactants greatly affects the formation of TLCs. The bulky group closing to the headgroup of surfactant and the strong electrostatic interaction between positively charged catanionic vesicles and DNA affect the formation of TLCs. Furthermore, we found that the shorter DNA strand length, the more consistent fit better with the vesicles to enhance the thermal stability of DNA-based TLCs. The influence factors of the formation of DNA-based TLCs were explored and the importance of interactions to the architecture of DNA-based TLCs also was underscored. Our results may direct the design and synthesis of functional DNA-based TLCs and lay the foundation of applications in the field of electrochemistry and flexible electronic devices.

Design and Characterization of Myristoylated and Non-Myristoylated Peptides Effective against Candida spp. Clinical Isolates.

The increasing resistance of fungi to antibiotics is a severe challenge in public health, and newly effective drugs are required. Promising potential medications are lipopeptides, linear antimicrobial peptides (AMPs) conjugated to a lipid tail, usually at the N-terminus. In this paper, we investigated the in vitro and in vivo antifungal activity of three short myristoylated and non-myristoylated peptides derived from a mutant of the AMP Chionodracine. We determined their interaction with anionic and zwitterionic membrane-mimicking vesicles and their structure during this interaction. We then investigated their cytotoxic and hemolytic activity against mammalian cells. Lipidated peptides showed a broad spectrum of activity against a relevant panel of pathogen fungi belonging to Candida spp., including the multidrug-resistant C. auris. The antifungal activity was also observed vs. biofilms of C. albicans, C. tropicalis, and C. auris. Finally, a pilot efficacy study was conducted on the in vivo model consisting of Galleria mellonella larvae. Treatment with the most-promising myristoylated peptide was effective in counteracting the infection from C. auris and C. albicans and the death of the larvae. Therefore, this myristoylated peptide is a potential candidate to develop antifungal agents against human fungal pathogens.

Effects of small molecule inhibitors on membrane-dependent amyloid aggregation of the BAX C-terminal peptide

The pro-apoptotic Bcl-2 family protein BAX oligomerized and forms pores in the outer mitochondrial membrane in the intrinsic pathway of apoptosis. Currently, the structure of the BAX pore is unknown and various models for the initiation and propagation of membrane permeabilization have been proposed. Using FTIR and 2D IR spectroscopy, we have found that a peptide representing the C-terminal α-helix (α9p) of BAX is amyloidogenic and forms stable aggregates in solution and on the surfaces of anionic lipid vesicles.

Hemin-catalyzed oxidative oligomerization of p-aminodiphenylamine (PADPA) in the presence of aqueous sodium dodecylbenzenesulfonate (SDBS) micelles.

In a previous report on the enzymatic synthesis of the conductive emeraldine salt form of polyaniline (PANI-ES) in aqueous solution using PADPA (p-aminodiphenylamine) as monomer, horseradish peroxidase isoenzyme C (HRPC) was applied as a catalyst at pH = 4.3 with H2O2 as a terminal oxidant. In that work, anionic vesicles were added to the reaction mixture for (i) guiding the reaction to obtain poly(PADPA) products that resemble PANI-ES, and for (ii) preventing product precipitation (known as the “template effect”). In the work now presented, instead of native HRPC, only its prosthetic group ferric heme b (= hemin) was utilized as a catalyst, and micelles formed from SDBS (sodium dodecylbenzenesulfonate) served as templates. For the elaborated optimal reaction conditions, complementary UV/vis/NIR, EPR, and Raman spectroscopy measurements clearly showed that the reaction mixture obtained after completion of the reaction contained PANI-ES-like products as dominating species, very similar to the products formed with HRPC as catalyst. HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonate) was found to have a positive effect on the reaction rate as compared to dihydrogenphosphate. This work is the first on the template-assisted formation of PANI-ES type products under mild, environmentally friendly conditions using hemin as a cost-effective catalyst.

Modifying Polydiacetylene Vesicle Compositions to Reduce Non-Specific Interactions.

Polydiacetylene (PDA) vesicles provide useful stimuli-responsive behavior as well as by the modular structure afford a means for the design of sensing and delivery systems with tunable target specificity. To reduce inherent non-specific interaction with either anionic or cationic formulations of polydiacetylene vesicles, we explored the use of various lengths of poly(ethylene glycol) (PEG) amphiphiles for integration and polymerization within PDA vesicles. Our results established that as little as 1% of polyethylene glycol amphiphile integration into anionic vesicles was sufficient to significantly reduce non-specific association with mammalian cells. Similarly integrating a low percent of PEG amphiphile content within cationic vesicles could also significantly reduce non-specific cell association, and moreover reduced cytotoxicity. These results may be prove useful in augmenting PDA vesicles formulations for reduced non-specific interaction which is of particularly interest to enhancing selectivity in vesicles designed with integrated targeting moieties for sensing and drug delivery applications.

Synthetic Peptides Derived From Lycosa Erythrognatha Venom: Interaction With Phospholipid Membranes and Activity Against Resistant Bacteria.

Superbugs are a public health problem, increasing the need of new drugs and strategies to combat them. Our group has previously identified LyeTxI, an antimicrobial peptide isolated from Lycosa erythrognatha spider venom. From LyeTxI, we synthesized and characterized a derived peptide named LyeTxI-b, which has shown significant in vitro and in vivo activity. In this work, we elucidate the interaction of LyeTxI-b with artificial membranes as well as its effects on resistant strains of bacteria in planktonic conditions or biofilms. Isothermal titration calorimetry revealed that LyeTxI-b interacts more rapidly and with higher intensity with artificial vesicles, showing higher affinity to anionic vesicles, when compared to synthetic LyeTxI. In calcein experiments, LyeTxI-b caused greater levels of vesicle cleavage. Both peptides showed antibacterial activity at concentrations of μmol L−1 against 12 different clinically isolated strains, in planktonic conditions, in a concentration-dependent manner. Furthermore, both peptides elicited a dose-dependent production of reactive oxygen species in methicillin-resistant Staphylococcus aureus. In S. aureus biofilm assay, LyeTxI-b was more potent than LyeTxI. However, none of these peptides reduced Escherichia coli biofilms. Our results show LyeTxI-b as a promising drug against clinically resistant strains, being a template for developing new antibiotics.

Modulatory Effects of Acidic pH and Membrane Potential on the Adsorption of pH-Sensitive Peptides to Anionic Lipid Membrane.

Anionic lipid membrane electrostatic potential and solution pH can influence cationic peptide adsorption to these bilayers, especially those containing simultaneously acid and basic residues. Here, we investigate the effects of the pH solution on MP1 (IDWKKLLDAAKQIL-NH2) adsorption to anionic (7POPC:3POPG) lipid vesicles in comparison to its analog H-MP1, with histidines substituting lysines. We used the association of adsorption isotherms and constant pH molecular dynamic simulations (CpHMD) to explore the effects of membrane potential and pH on peptides’ adsorption on this lipid membrane. We analyzed the fluorescence and zeta potential adsorption isotherms using the Gouy–Chapman theory. In CpHMD simulations for the peptides in solution and adsorbed on the lipid bilayer, we used the conformations obtained by conventional MD simulations at a μs timescale. Non-equilibrium Monte Carlo simulations provided the protonation states of acidic and basic residues. CpHMD showed average pKa shifts of two to three units, resulting in a higher net charge for the analog than for MP1, strongly modulating the peptide adsorption. The fractions of the protonation of acidic and basic residues and the peptides’ net charges obtained from the analysis of the adsorption isotherms were in reasonable agreement with those from CpHMD. MP1 adsorption was almost insensitive to solution pH. H-MP1 was much more sensitive to partitioning, at acidic pH, with an affinity ten times higher than in neutral ones.

Membrane-dependent amyloid aggregation of human BAX α9 (173-192).

Mitochondrial outer membrane permeabilization, which is a critical step in apoptosis, is initiated upon transmembrane insertion of the C-terminal α-helix (α9) of the proapoptotic Bcl-2 family protein BAX. The isolated α9 fragment (residues 173-192) is also competent to disrupt model membranes, and the structures of its membrane-associated oligomers are of interest in understanding the potential roles of this sequence in apoptosis. Here, we used ultrafast two-dimensional infrared (2D IR) spectroscopy, thioflavin T binding, and transmission electron microscopy to show that the synthetic BAX α9 peptide (α9p) forms amyloid aggregates in aqueous environments and on the surfaces of anionic small unilamellar vesicles. Its inherent amyloidogenicity was predicted by sequence analysis, and 2D IR spectra reveal that vesicles modulate the β-sheet structures of insoluble aggregates, motivating further examination of the formation or suppression of BAX amyloids in apoptosis.

Oral delivery of all-trans retinoic acid mediated by liposome carriers

All-trans retinoic acid (ATRA) is a molecule that finds wide applications in medicine. Connection between cancer cell proliferation and ATRA is a well-established item. Driven by the potential applications of liposomes in stabilizing and protecting therapeutic compounds thus enabling effective delivery of encapsulated compounds, recent research efforts have been directed to understanding mechanisms of oral delivery through the gastrointestinal tract. The surface charge of the liposome bilayers can modify the interactions between the aggregates and the gastrointestinal fluids.Here, we investigated the ability of cationic and anionic liposomes to encapsulate, protect and deliver ATRA in an in-vitro digestion process as a different oral administration route.Stability and encapsulation efficiency of ATRA in negatively and positively charged liposomes enriched with α-tocopherol were investigated by means of UV–vis spectroscopy, dynamic light scattering and ζ-potential. The applicability of the carriers was tested by means of an in-vitro digestion procedure allowing for the measurement of the bioavailability of ATRA.From this study evidence was provided that the water insoluble molecules, ATRA and α-tocopherol are intercalated in liposome membranes regardless of the surface charge of the vesicle bilayers. Comparisons between cationic and anionic liposomes incorporating retinoic acid show differences in bioavailability. The cationic vesicles are preferable for a larger amount of ATRA bioavailability, which can be understood from electrostatic interactions. Thus ATRA is ionized in a wide range of pHs but protonated in anionic vesicles.

Adsorption Kinetics of Oppositely Charged Hard and Soft Nanoparticles with Phospholipid Membranes.

Nanoparticles (NPs) have great potential for biological applications as typically they exhibit strongly size-dependent properties. Specifically, the interaction of NPs with phospholipid membranes is significantly relevant to nanomedicine and the related field of nanotoxicology. Therefore, the investigation of interactions of NPs with model membranes is not only fundamentally important but also practically valuable to understand interactions of NPs with more complex cell membranes. Here, we report on the interaction of anionic vesicles of different charge densities and cationic SiO2 NPs, either covered by a bare surface functionalized with amino moieties (-NH2) or covered by poly[2-(dimethylamino) ethyl methacrylate]. We studied the kinetics of binding of NPs to the vesicle surface by time-resolved scattering experiments. A key result of the study is that binding is favored in the presence of electrostatic attraction, but the polymer layer decreases the binding rate drastically.

How Insertion of a Single Tryptophan in the N-Terminus of a Cecropin A-Melittin Hybrid Peptide Changes Its Antimicrobial and Biophysical Profile.

In the era of antibiotic resistance, there is an urgent need for efficient antibiotic therapies to fight bacterial infections. Cationic antimicrobial peptides (CAMP) are promising lead compounds given their membrane-targeted mechanism of action, and high affinity towards the anionic composition of bacterial membranes. We present a new CAMP, W-BP100, derived from the highly active BP100, holding an additional tryptophan at the N-terminus. W-BP100 showed a broader antibacterial activity, demonstrating a potent activity against Gram-positive strains. Revealing a high partition constant towards anionic over zwitterionic large unilamellar vesicles and inducing membrane saturation at a high peptide/lipid ratio, W-BP100 has a preferential location for hydrophobic environments. Contrary to BP100, almost no aggregation of anionic vesicles is observed around saturation conditions and at higher concentrations no aggregation is observed. With these results, it is possible to state that with the incorporation of a single tryptophan to the N-terminus, a highly active peptide was obtained due to the π–electron system of tryptophan, resulting in negatively charged clouds, that participate in cation–π interactions with lysine residues. Furthermore, we propose that W-BP100 action can be achieved by electrostatic interactions followed by peptide translocation.

Model Liposomal Delivery System for Drugs and Vaccines

Background Liposomes have been used for drug delivery since their discovery 60-years-ago. The advantages they provide as carriers have been recognised and exploited to improve the delivery of numerous drugs and eliminate harmful side-effects. Liposomal delivery has been tested for anticancer drugs, anti-tuberculosis drugs, variety of vaccines, just to list a few. Methods We developed a series of liposomal formulations with the addition of cholesterol and polyethylene glycol. The uptake of these formulations by human epithelial prostate cancer (PC-3) cells and mouse macrophages was examined and analysed by flow cytometry and confocal microscopy. Results Among the liposomes tested, small anionic liposome vesicles (≤200 nm) prepared with egg phosphatidylglycerol as the main lipid were most effectively taken up by PC-3 cells and macrophages. Conclusion We produced a liposome formulation that can be used as a model system for the delivery of drugs and vaccines.

An investigation into the critical tension of electroporation in anionic lipid vesicles.

Irreversible electroporation (IRE) is a technique for the disruption of localized cells or vesicles by a series of short and high-frequency electric pulses which has been used for tissue ablation and treatment in certain diseases. It is well reported that IRE induces lateral tension in the membranes of giant unilamellar vesicles (GUVs). The GUVs are prepared by a mixture of anionic lipid dioleoylphosphatidylglycerol (DOPG) and neutral lipid dioleoylphosphatidylcholine (DOPC) using the natural swelling method. Here the influence of DOPG mole fraction, XDOPG, on the critical tension of electroporation in GUVs has been investigated in sodium chloride-containing PIPES buffer. The critical tension decreases from 9.0 ± 0.3 to 6.0 ± 0.2 mN/m with the increase of XDOPG from 0.0 to 0.60 in the membranes of GUVs. Hence an increase in XDOPG greatly decreases the mechanical stability of membranes. We develop a theoretical equation that fits the XDOPG dependent normalized critical tension, and obtain a binding constant for the lipid-ion interaction of 0.75M-1. The decrease in the energy barrier for formation of the nano-size nascent or prepore state, due to the increase in XDOPG, is the main factor explaining the decrease in critical tension of electroporation in vesicles.

N-terminal acetylation of a mastoparan-like peptide enhances PE/PG segregation in model membranes

The synthetic peptides L1A and its acetylated analog (acL1A) display potent Gram-negative bactericidal activities without being hemolytic. We have gathered evidence that the N-terminal acetylation of L1A enhances the lytic activity in anionic vesicles with high capability to insert into and disturb lipid packing of model membranes. Here, the impact of L1A and acL1A was evaluated on a model membrane that mimics the cytoplasmic membrane of Gram-negative bacteria, which is rich in phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), using 3:1 mixture of POPE/DOPG and a variety of techniques. We followed peptide adsorption and penetration by zeta potential determination of large unilamellar vesicles, accessibility of tryptophan residue to acrylamide by quenching assays, and Gibbs isotherms. The secondary structure of the peptide on the membranes was assessed using circular dichroism. Peptide mixing ability with the lipids and phase segregation was assessed by the observation of Langmuir monolayers with fluorescence microscopy, as well as with differential scanning calorimetry thermograms of multilamellar vesicles. All in all, the results indicate that both peptides adsorb and penetrate POPE/DOPG membranes with similar affinities, decreasing the surface charge, and adopting alpha structures. Both peptides mix with DOPG and demix from POPE, and consequently, persist at the interface to larger surface pressures in the presence of PG than in pure PE monolayers. This selective degree of mixing of the peptides with PE and PG leads to peptide-induced segregation of PG from PE, being the less charged peptide, acL1A, able to segregate the lipids more efficiently.

Functional Characterization of Temporin-SHe, a New Broad-Spectrum Antibacterial and Leishmanicidal Temporin-SH Paralog from the Sahara Frog (Pelophylax saharicus).

Amphibian skin is a promising natural resource for antimicrobial peptides (AMPs), key effectors of innate immunity with attractive therapeutic potential to fight antibiotic-resistant pathogens. Our previous studies showed that the skin of the Sahara Frog (Pelophylax saharicus) contains broad-spectrum AMPs of the temporin family, named temporins-SH. Here, we focused our study on temporin-SHe, a temporin-SHd paralog that we have previously identified in this frog but was never structurally and functionally characterized. We synthesized and determined the structure of temporin-SHe. This non-amphipathic α-helical peptide was demonstrated to strongly destabilize the lipid chain packing of anionic multilamellar vesicles mimicking bacterial membranes. Investigation of the antimicrobial activity revealed that temporin-SHe targets Gram-negative and Gram-positive bacteria, including clinical isolates of multi-resistant Staphylococcus aureus strains. Temporin-SHe exhibited also antiparasitic activity toward different Leishmania species responsible for visceral leishmaniasis, as well as cutaneous and mucocutaneous forms. Functional assays revealed that temporin-SHe exerts bactericidal effects with membrane depolarization and permeabilization, via a membranolytic mechanism observed by scanning electron microscopy. Temporin-SHe represents a new member of the very limited group of antiparasitic temporins/AMPs. Despite its cytotoxicity, it is nevertheless an interesting tool to study the AMP antiparasitic mechanism and design new antibacterial/antiparasitic agents.

Development and Characterization of Ultrasound Activated Lipopolyplexes for Enhanced Transfection by Low Frequency Ultrasound in In Vitro Tumor Model.

This work focuses on the development of ultrasound contrast vesicles for ultrasound-mediated enhanced transfection of nucleic acids in the cancer cells and projects its application as a tool for diagnostic imaging. The ultrasound contrast vesicles are stable, anionic, nanoscaled vesicles with ultrasound contrast equivalent to the commercially available SonoVue. These anionic lipid vesicles establish electrostatic interaction with cationic polyplexes based on linear polyethylenimine (22kDa)forming lipopolyplexes with ultrasound contrast. The lipopolyplexes are characterized regarding shape, size, and zeta potential. When exposed to low frequency ultrasound, these carriers show elevated transfection efficiency and reduced cytotoxicity. The effect of post-transfection ultrasound on cellular uptake of lipopolyplexes is also evaluated. An analogous transfection is also observed in the tumor mimicking multicellular 3D spheroid culture of ovarian cancer cells. The emergence of tumor imaging and enhanced gene delivery by medical ultrasound, a noninvasive imaging modality, is considered paving the way for efficient theranostic gene therapy.

Membrane interactions of the anuran antimicrobial peptide HSP1-NH2: Different aspects of the association to anionic and zwitterionic biomimetic systems

Studies have suggested that antimicrobial peptides act by different mechanisms, such as micellisation, self-assembly of nanostructures and pore formation on the membrane surface. This work presents an extensive investigation of the membrane interactions of the 14 amino-acid antimicrobial peptide hylaseptin P1-NH2 (HSP1-NH2), derived from the tree-frog Hyla punctata, which has stronger antifungal than antibacterial potential. Biophysical and structural analyses were performed and the correlated results were used to describe in detail the interactions of HSP1-NH2 with zwitterionic and anionic detergent micelles and phospholipid vesicles. HSP1-NH2 presents similar well-defined helical conformations in both zwitterionic and anionic micelles, although NMR spectroscopy revealed important structural differences in the peptide N-terminus. 2H exchange experiments of HSP1-NH2 indicated the insertion of the most N-terminal residues (1–3) in the DPC-d38 micelles. A higher enthalpic contribution was verified for the interaction of the peptide with anionic vesicles in comparison with zwitterionic vesicles. The pore formation ability of HSP1-NH2 (examined by dye release assays) and its effect on the size and surface charge as well as on the lipid acyl chain ordering (evaluated by Fourier-transform infrared spectroscopy) of anionic phospholipid vesicles showed membrane disruption even at low peptide-to-phospholipid ratios, and the effect increases proportionately to the peptide concentration. On the other hand, these biophysical investigations showed that a critical peptide-to-phospholipid ratio around 0.6 is essential for promoting disruption of zwitterionic membranes. In conclusion, this study demonstrates that the binding process of the antimicrobial HSP1-NH2 peptide depends on the membrane composition and peptide concentration.

Exerting Spatial Control During Nanoparticle Occlusion within Calcite Crystals.

In principle, nanoparticle occlusion within crystals provides a straightforward and efficient route to make new nanocomposite materials. However, developing a deeper understanding of the design rules underpinning this strategy is highly desirable. In particular, controlling the spatial distribution of the guest nanoparticles within the host crystalline matrix remains a formidable challenge. Herein, we show that the surface chemistry of the guest nanoparticles and the [Ca2+ ] concentration play critical roles in determining the precise spatial location of the nanoparticles within calcite crystals. Moreover, in situ studies provide important mechanistic insights regarding surface-confined nanoparticle occlusion. Overall, this study not only provides useful guidelines for efficient nanoparticle occlusion, but also enables the rational design of patterned calcite crystals using model anionic block copolymer vesicles.

Interaction of Alpha-Synuclein and Its Mutants with Rigid Lipid Vesicle Mimics of Varying Surface Curvature.

Abnormal aggregation of alpha-synuclein (α-syn), an intrinsically disordered neuronal protein, is strongly implicated in the development of Parkinson's disease. Efforts to better understand α-syn's native function and its pathogenic role in neurodegeneration have revealed that the protein interacts with anionic lipid vesicles via adoption of an amphipathic α-helical structure; however, the ability of α-syn to remodel lipid membranes has made it difficult to decipher the role of vesicle surface curvature in protein binding behavior. In this study, sodium dodecyl sulfate (SDS)-coated gold nanoparticles (AuNPs), which mimic bilayer vesicle architecture, were synthesized in order to conduct a systematic investigation into the binding interaction of α-syn and two of its mutants (A30P and E46K) with rigid lipid vesicle mimics of defined surface curvature. By incorporating a rigid AuNP core (∼10-100 nm), the ability of α-syn to remodel the vesicle mimics was removed and their surface curvature could be fixed. Proteomics studies showed that, upon binding of free α-syn to the surface of SDS-AuNPs, the N-terminus of α-syn became less solvent accessible, whereas its C-terminus became more accessible. Interestingly, α-syn's non-amyloid-β component (NAC) region also exhibited increased solvent accessibility, suggesting that α-syn bound to rigid vesicle-like structures could possess heightened aggregation propensity and therefore pathogenicity. Additionally, both the A30P and E46K mutations were found to adopt distinct binding modes on the mimics' surface. In contrast with previous reports, similar binding affinities were observed for WT, A30P, and E46K α-syn toward SDS-AuNPs of all sizes, indicating the potential importance of vesicle deformability in determining α-syn binding behavior.

Photothermal Nano-antibiotic for Effective Treatment of Multidrug-Resistant Bacterial Infection.

Bacteria's antibiotic resistance is one of the major challenges in the treatment of infectious diseases. With increasing difficulty in discovering antibiotics, there is an urgent need to develop antibiotic-free therapeutic strategies to address this grand challenge. In this report, we developed a polypyrrole (PPy)-based photothermal nano-antibiotic (PTNA) for effective treatment of multidrug-resistant (MDR) bacterial infection. PTNA was fabricated by polymerizing pyrrole onto an anionic vesicle to integrate the cationic and photothermal properties of PPy for combinatory killing against bacteria. PTNA exhibits a strong photothermal effect in the NIR-II (1064 nm) biowindow, thus it is feasible for in vivo therapy due to deeper tissue penetration. Our in vitro experiments revealed that PTNA can significantly inhibit the growth of MDR bacteria (Salmonella typhimurium), alleviate inflammatory response of infected cells, and prevent biofilm formation. More importantly, PTNA showed a significant therapeutic effect in an S. typhimurium-infected animal disease model of acute systemic infection by inhibiting bacterial growth, lowering inflammatory responses and pathological changes, and eventually improving the survival rate of mice. Finally, PTNA had safe profiles in vitro and in vivo with no visible toxicity detected. Therefore, we believe PTNA may serve as a promising antibiotic-free antimicrobial material for the effective treatment of MDR bacterial infection.