Unlocking roles of cationic and aromatic residues in peptide amphiphiles in treating drug-resistant gram-positive pathogens.

Multidrug resistance (MDR) is a rising threat to global health because the number of essential antibiotics used for treating MDR infections is increasingly compromised. In this work we report a group of new amphiphilic peptides (AMPs) derived from the well-studied G3 (G(IIKK)3I-NH2) to fight infections from Gram-positive bacteria including susceptible Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA), focusing on membrane interactions. Time-dependent killing experiments revealed that substitutions of II by WW (GWK), II by FF (GFK) and KK by RR (GIR) resulted in improved bactericidal efficiencies compared to G3 (GIK) on both S. aureus and MRSA, with the order of GWK > GIR > GFK > GIK. Electronic microscopy imaging revealed structural disruptions of AMP binding to bacterial cell walls. Fluorescence assays including AMP binding to anionic lipoteichoic acids (LTA) in cell-free and cell systems indicated concentration and time-dependent membrane destabilization associated with bacterial killing. Furthermore, AMP's binding to anionic plasma membrane via similar fluorescence assays revealed a different extent of membrane depolarization and leakage. These observations were supported by the penetration of AMPs into the LTA barrier and the subsequent structural compromise to the cytoplasmic membrane as revealed from SANS (small angle neutron scattering). Both experiments and molecular dynamics (MD) simulations revealed that GWK and GIR could make the membrane more rigid but less effective in diffusive efficiency than GIK and GFK through forming intramembrane peptide nanoaggregates associated with hydrophobic mismatch and formation of fluidic and rigid patches. The reported peptide-aggregate-induced phase-separation emerged as a crucial factor in accelerated membrane disintegration and fast bacterial killing. This work has demonstrated the importance of membrane interactions to the development of more effective AMPs and the relevance of the approaches as reported in assisting this area of research.

Effective Treatment of Helicobacter pylori Infection Using Supramolecular Antimicrobial Peptide Hydrogels.

Helicobacter pylori can cause various gastric conditions including stomach cancer in an acidic environment. Although early H. pylori infections can be treated by antibiotics, prolonged antibiotic administrations may lead to the development of antimicrobial resistance, compromising the effectiveness of the treatments. Antimicrobial peptides (AMPs) have been reported to possess unique advantages against antimicrobial-resistant bacteria due to their rapid physical membrane disruptions and anti-inflammation/immunoregulation properties. Herein, we have developed an AMP hydrogel, which can be orally administered for the treatment of H. pylori infection. The hydrogel has potent antimicrobial activity against H. pylori, achieving bacterial eradication within minutes of action. Compared with the AMP solution, the hydrogel formulation significantly reduced the cytotoxicity and enhanced proteolytic stability. In vivo experiments suggested that the hydrogel formed at pH 4 had superior therapeutic effects to those at pH 7 and 10 hydrogels, attributed to its rapid release and bactericidal action within the acidic stomach environment. Compared to conventional antibiotic treatments, the AMP hydrogel had the advantages of fast bacterial killing in the gastric juice and obviated proton pump inhibitors during the treatment. Although both the AMP hydrogel and antibiotics suppressed the expression of pro-inflammatory cytokines, the former uniquely promoted inflammation resolution. These results indicate that the AMP hydrogels with effectiveness and biosafety may be potential candidates for the clinical treatment of H. pylori infections.

Combination of a pH-responsive peptide amphiphile and a conventional antibiotic in treating Gram-negative bacteria.

BACKGROUND: Clinical treatments ofgastric infections using antibiotics suffer from the undesired killing of commensal bacteria and emergence of antibiotic resistance. It is desirable to develop pH-responsive antimicrobial peptides (AMPs) that kill pathogenic bacteria such as H. pyloriand resistant E. coli under acidic environment with minimal impact to commensal bacteria whilst not causing antibiotic resistance. EXPERIMENTS: Using a combined approach of cell assays, molecular dynamics (MD) simulations and membrane models facilitating biophysical and biochemical measurements including small angle neutron scattering (SANS), we have characterized the pH-responsive physiochemical properties and antimicrobial performance of two amphiphilic AMPs, GIIKDIIKDIIKDI-NH2 and GIIKKIIDDIIKKI-NH2 (denoted as 3D and 2D, respectively), that were designed by selective substitutions of cationic residues of Lys (K) in the extensively studied AMP G(IIKK)3I-NH2 with anionic residue Asp (D). FINDINGS: Whilst 2D kept non-ordered coils across the entire pH range studied, 3D displayed a range of secondary structures when pH was shifted from basic to acidic, with distinct self-assembly into nanofibers in aqueous environment. Further experimental and modeling studies revealed that the AMPs interacted differently with the inner and outer membranes of Gram-negative bacteria in a pH-responsive manner and that the structural features characterized by membrane leakage and intramembrane nanoaggregates revealed from fluorescence spectroscopy and SANS were well linked to antimicrobial actions. Different antimicrobial efficacies of 2D and 3D were underlined by the interplay between their ability to bind to the outer membrane lipid LPS (lipopolysaccharide), outer membrane permeability change and inner membrane depolarization and leakage. Furthermore, AMP's binding with the inner membrane under acidic condition caused both the dissipation of membrane potential (Δψ) and the continuous dissipation of transmembrane ΔpH, with Δψ and ΔpH being the key components of the proton motive force. Combinations of antibiotic (Minocycline) with the pH-responsive AMP generated the synergistic effects against Gram-negative bacteria only under acidic condition. These features are crucial to target applications to gastric infections, anti-acne and wound healing.

How do antimicrobial peptides disrupt the lipopolysaccharide membrane leaflet of Gram-negative bacteria?

HYPOTHESIS: It is widely regarded that antimicrobial peptides (AMPs) kill bacteria by physically disrupting microbial membranes and causing cytoplasmic leakage, but it remains unclear how AMPs disrupt the outer membrane (OM) of Gram-negative bacteria (GNB) and then compromise the inner membrane. We hypothesise that different AMPs impose different structural disruptions, with direct implications to their antimicrobial efficacies. EXPERIMENTS: The antimicrobial activities of three typical AMPs, including the designed short AMP, G3, and two natural AMPs, melittin and LL37, against E. coli and their haemolytic activities were studied. Lipopolysaccharide (LPS) and anionic di-palmitoyl phosphatidyl glycerol (DPPG) monolayer models were constructed to mimic the outer membrane and inner membrane leaflets of Gram-negative bacteria. The binding and penetration of AMPs to the model lipid monolayers were systematically studied by neutron reflection via multiple H/D contrast variations. FINDING: G3 has relatively high antimicrobial activity, low cytotoxicity, and high proteolytic stability, whilst melittin has significant haemolysis and LL37 has weaker antimicrobial activity. G3 could rapidly lyse LPS and DPPG monolayers within 10-20 min. In contrast, melittin was highly active against the LPS membrane, but the dynamic process lasted up to 80 min, with excessive stacking in the OM. LL37 caused rather weak destruction to LPS and DPPG monolayers, leading to massive adsorption on the membrane surface without penetrating the lipid tail region. These findings demonstrate that the rationally designed AMP G3 was well optimised to impose most effective destruction to bacterial membranes, consistent with its highest bactericidal activity. These different interfacial structural features associated with AMP binding shed light on the future development of active and biocompatible AMPs for infection and wound treatments.

Intramembrane Nanoaggregates of Antimicrobial Peptides Play a Vital Role in Bacterial Killing.

Recent developments in antimicrobial peptides (AMPs) have focused on the rational design of short sequences with less than 20 amino acids due to their relatively low synthesis costs and ease of correlation of the structure-function relationship. However, gaps remain in the understanding of how short cationic AMPs interact with the bacterial outer and inner membranes to affect their antimicrobial efficacy and dynamic killing. The membrane-lytic actions of two designed AMPs, G(IIKK)3 I-NH2 (G3 ) and G(IIKK)4 I-NH2 (G4 ), and previously-studied controls GLLDLLKLLLKAAG-NH2 (LDKA, biomimetic) and GIGAVLKVLTTGLPALISWIKRKR-NH2 (Melittin, natural) are examined. The mechanistic processes of membrane damage and the disruption strength of the four AMPs are characterized by molecular dynamics simulations and experimental measurements including neutron reflection and scattering. The results from the combined studies are characterized with distinctly different intramembrane nanoaggregates formed upon AMP-specific binding, reflecting clear influences of AMP sequence, charge and the chemistry of the inner and outer membranes. G3 and G4 display different nanoaggregation with the outer and inner membranes, and the smaller sizes and further extent of insertion of the intramembrane nanoaggregates into bacterial membranes correlate well with their greater antimicrobial efficacy and faster dynamic killing. This work demonstrates the crucial roles of intramembrane nanoaggregates in optimizing antimicrobial efficacy and dynamic killing.

Acyl chain length tuning improves antimicrobial potency and biocompatibility of short designed lipopeptides.

HYPOTHESIS: Designed antimicrobial lipopeptides (ALPs) offer the attractive benefits of short peptide sequences and flexible tuning of amphiphilicity by altering the acyl chain length. These lipopeptides kill microbes by forming intriguing in-membrane nanostructures and causing the leakage of internal contents. However, how subtle differences in the molecular structures of the lipopeptides affect their antimicrobial efficacy and biocompatibility to host cells is still under-investigated. EXPERIMENTS: This work focuses on assessing changes in the acyl chain length of CH3(CH2)n-2CO-KKKIII-NH2 (n = 10, 12 and 14, K = lysine, I = isoleucine, denoted as CnKI3) on the antimicrobial potency and cytotoxicity by combining biological assays with physical measurements. Aggregation properties were characterized by changes in critical aggregation concentration (CAC) from surface tension measurements. Antimicrobial susceptibility tests, cytotoxic MTT assays, haemolytic tests, and dynamic bactericidal experiments were employed to reveal their bioactive potency toward different types of cells. To further investigate lipopeptides' underlying antimicrobial and cytotoxic mechanisms, lipid monolayer and lipid small unilamellar vesicle (SUV) models were established and biophysically characterized. FINDINGS: An increase in n led to the decrease in the CAC of CnKI3, showing a rising membrane-lytic power. Subsequent bioactive measurements revealed the optimal performance of C12KI3 from this series of lipopeptides. The selective membrane binding behaviour was well supported by neutron reflection data from charged lipid monolayer models, revealing membrane-supported nanostructures of ALPs. However, increased membrane-lytic actions in C14KI3 led to notably increased toxicity and reduced selectivity. On the other hand, C14KI3 can impose faster dynamic killing than natural lipopeptide polymyxin B, showing the distinct impact of the amphiphilic balance from the designed lipopeptide. In contrast, the distinctly weaker binding to zwitterionic membrane models (monolayers and SUVs) provided direct nanoscale structural evidence to the mildness of the designed ALPs on host cells. This work demonstrates the high selectivity and fast killing of rationally designed short ALPs to microbes via in-membrane nanostructuring.

Correction: Coordination-assembled myricetin nanoarchitectonics for sustainably scavenging free radicals.

[This corrects the article DOI: 10.3762/bjnano.13.23.].

Orally administered covalently-assembled antioxidative peptide nanoparticles for inflammatory bowel disease therapy.

HYPOTHESIS: Inflammatory bowel disease (IBD) is a chronic inflammation disease and still faces many therapeutic challenges, such as ineffective treatments, antibiotic resistance, and systematic toxicity. In order to improve the therapeutic efficacy of IBD, it is thus urgent to develop efficient, non-toxic and conveniently-administrated nanoagents to replace the currently used medicines. Casein phosphopeptide (CPP) has been found capable of chelating transition metal ions to suppress reactive oxygen species (ROS) generation, showing the potential for the treatment of IBD. However, CPP easily suffers from hydrolysis and enzymatic degradation, which limits its further clinical application. Covalent assembly of CPP to form nanoparticles (GCPP NPs) may be an efficient way to enhance the CPP stability in physiological environment and finally improve its capability of in vivo antioxidation and IBD treatment. EXPERIMENTS: We synthesized GCPP NPs through covalent assembly of Genipin and CPP, followed by characterizing their physicochemical properties. GCPP NPs were incubated under different physiological conditions including phosphate buffered saline, cell culture media, simulated gastrointestinal fluid for evaluation of stability. Cytotoxicity and antioxidation activities of GCPP NPs were tested in vitro under the 3T3 cell line using the ABTS and MTT assays, respectively. Finally, a DSS-induced mouse colitis model was established to assess specific accumulation and good therapeutic efficacy of GCPP NPs via an oral administration strategy. FINDINGS: GCPP NPs are robust and stable to overcome easy degradation of CPP even under the harsh gastrointestinal environments, which are adapted for oral administration. As-prepared GCPP NPs show benign antioxidant activity to scavenge ROS. Meanwhile, nanoscale GCPP NPs can passively accumulate and maintain at inflamed sites. The body weight and colon length of DSS-induced colitis mice treated by GCPP NPs perform a rehabilitation trend. These results indicate that GCPP NPs, as peptide-based therapeutic nanoagents, have great potential in the anti-inflammatory treatment of IBD by oral administration.

In-Membrane Nanostructuring of Cationic Amphiphiles Affects Their Antimicrobial Efficacy and Cytotoxicity: A Comparison Study between a De Novo Antimicrobial Lipopeptide and Traditional Biocides.

Cationic biocides have been widely used as active ingredients in personal care and healthcare products for infection control and wound treatment for a long time, but there are concerns over their cytotoxicity and antimicrobial resistance. Designed lipopeptides are potential candidates for alleviating these issues because of their mildness to mammalian host cells and their high efficacy against pathogenic microbial membranes. In this study, antimicrobial and cytotoxic properties of a de novo designed lipopeptide, CH3(CH2)12CO-Lys-Lys-Gly-Gly-Ile-Ile-NH2 (C14KKGGII), were assessed against that of two traditional cationic biocides CnTAB (n = 12 and 14), with different critical aggregation concentrations (CACs). C14KKGGII was shown to be more potent against both bacteria and fungi but milder to fibroblast host cells than the two biocides. Biophysical measurements mimicking the main features of microbial and host cell membranes were obtained for both lipid monolayer models using neutron reflection and small unilamellar vesicles (SUVs) using fluorescein leakage and zeta potential changes. The results revealed selective binding to anionic lipid membranes from the lipopeptide and in-membrane nanostructuring that is distinctly different from the co-assembly of the conventional CnTAB. Furthermore, CnTAB binding to the model membranes showed low selectivity, and its high cytotoxicity could be attributed to both membrane lysis and chemical toxicity. This work demonstrates the advantages of the lipopeptides and their potential for further development toward clinical application.

Contrasting impacts of mixed nonionic surfactant micelles on plant growth in the delivery of fungicide and herbicide.

HYPOTHESIS: Nonionic alkyl ethoxylate surfactants are widely used in agrochemicals to facilitate the permeation of systemic herbicides and fungicides across the plant waxy film. Industrial grade surfactants are often highly mixed and how the mixing affects their interactions with pesticides and wax films remains largely unexplored. A better understanding could enable design of mixed nonionic surfactants for herbicides and fungicides to maximize their efficiency and reduce wastage whilst controlling their impact on plant wax films. EXPERIMENT: In this study, nonionic surfactants with general structure n-oxyethylene glycol monododecyl ether (C12En) were used to form surfactant mixtures with the same average ethoxylate numbers but different hydrophilic-lipophilic balance (HLB) values. Their mixed micellar systems were then used to solubilize a herbicide diuron (DN) and a fungicide cyprodinil (CP), followed by plant wax solubilization upon contact with wax films. These processes were monitored by 1H NMR and SANS. FINDING: Pesticide solubilization made surfactant micelles effectively more hydrophobic but subsequent wax dissolution caused pesticide release and the restoration of the micellar amphiphilicity. Nonionic surfactants with lower HLBs form larger nanoaggregates, show enhanced wettability, and have better ability to solubilize and permeate pesticides across the wax film, but may cause significant damage to plant growth. These observations help explain why herbicides applied on weeds would benefit from surfactants with lower HLB values while fungicides require surfactants with HLBs to balance between delivery efficiency and potential phytotoxicity risks.

Coordination-assembled myricetin nanoarchitectonics for sustainably scavenging free radicals.

Oxidative stress can lead to permanent and irreversible damage to cellular components and even cause cancer and other diseases. Therefore, the development of antioxidative reagents is an important strategy to alleviate chronic diseases and maintain the redox balance in cells. Small-molecule bioactive compounds have exhibited huge therapeutic potential as antioxidants and anti-inflammatory agents. Myricetin (Myr), a well-known natural flavonoid, has drawn wide attention because of its high antioxidant, anti-inflammatory, antimicrobial, and anticancer efficacy. Especially regarding antioxidation, Myr is capable of not only chelating intracellular transition metal ions for removing reactive oxygen species, but also of activating antioxidant enzymes and related signal pathways and, thus, of sustainably scavenging radicals. However, Myr is poorly soluble in water, which limits its bioavailability for biomedical applications, and even its clinical therapeutic potential. The antioxidant peptide glutathione (GSH) plays a role as antioxidant in cells and possesses good hydrophilicity and biocompatibility. However, it is easily metabolized by enzymes. To take advantages of their antioxidation activity and to overcome the abovementioned limitations, GSH, Zn2+, and Myr were selected to co-assemble into Myr-Zn2+-GSH nanoparticles or nanoarchitectonics. This study offers a new design to harness stable, sustainable antioxidant nanoparticles with high loading capacity, high bioavailability, and good biocompatibility as antioxidants.

How do terminal modifications of short designed IIKK peptide amphiphiles affect their antifungal activity and biocompatibility?

HYPOTHESIS: The widespread and prolonged use of antifungal antibiotics has led to the rapid emergence of multidrug resistant Candida species that compromise current treatments. Natural and synthetic antimicrobial peptides (AMPs) offer potential alternatives but require further development to overcome some of their current drawbacks. AMPs kill pathogenic fungi by permeabilising their membranes but it remains unclear how AMPs can be designed to maximise their antifungal potency whilst minimising their toxicity to host cells. EXPERIMENTS: We have designed a group of short (IIKK)3 AMPs via selective terminal modifications ending up with different amphiphilicities. Their antifungal performance was assessed by minimum inhibition concentration (MICs) and dynamic killing to 4 Candida strains and Cryptococcus neoformans, and the minimum biofilm-eradicating concentrations to kill 95% of the C. albicans biofilms (BEC95). Different antifungal actions were interpreted on the basis of structural disruptions of the AMPs to small unilamellar vesicles from fluorescence leakage, Zeta potential, small angle neutron scattering (SANS) and molecular dynamics simulations (MD). FINDING: AMPs possess high antifungal activities against the Candida species and Cryptococcus neoformans; some of them displayed faster dynamic killing than antibiotics like amphotericin B. G(IIKK)3I-NH2 and (IIKK)3II-NH2 were particularly potent against not only planktonic microbes but also fungal biofilms with low cytotoxicity to host cells. It was found that their high selectivity and fast action were well correlated to their fast membrane lysis, evident from data measured from Zeta potential measurements, SANS and MD, and also consistent with the previously observed antibacterial and anticancer performance. These studies demonstrate the important role of colloid and interface science in further developing short, potent and biocompatible AMPs towards clinical treatments via structure design and optimization.

Structural Disruptions of the Outer Membranes of Gram-Negative Bacteria by Rationally Designed Amphiphilic Antimicrobial Peptides.

Gram-negative bacteria are covered by both an inner cytoplasmic membrane (IM) and an outer membrane (OM). Antimicrobial peptides (AMPs) must first permeate through the OM and cell wall before attacking the IM to cause cytoplasmic leakage and kill the bacteria. The bacterial OM is an asymmetric bilayer with the outer leaflet primarily composed of lipopolysaccharides (LPSs) and the inner leaflet composed of phospholipids (PLs). Two cationic α-helical AMPs were designed to target Gram-negative bacteria, a full peptide G(IIKK)3I-NH2 (G3), and a hydrophobic lipopeptide C8-G(IIKK)2I-NH2 (C8G2, with C8 denoting the octanoyl chain). LPS dominates OM functions as the first line of defense against antibiotics, thereby reducing drug susceptibility. This work explores how the two AMPs interact with LPS through several carefully chosen OM models that facilitated measurements from solid-state nuclear magnetic resonance (ss-NMR), small-angle neutron scattering (SANS), and neutron reflectivity (NR). The results revealed that G3 molecules bound preferably to the LPS head region and functioned as bridge molecules to reassemble the dislocated lipids into bilayer stacks. In contrast, C8G2 lipopeptides could quickly penetrate into the central region of the OM to cause direct removal of some membrane lipids. Different structural disruptions implicated different antimicrobial efficacies from these AMPs. The demonstration of the structural features underlying different susceptibilities of the OM to AMPs offers a useful route for the future development of strain-specific AMPs against antimicrobial-resistant pathogens.

Structural elucidation upon binding of antimicrobial peptides into binary mixed lipid monolayers mimicking bacterial membranes.

HYPOTHESIS: Antimicrobial peptides (AMPs) kill microorganisms by causing structural damage to bacterial membranes. Different microorganisms often require a different type and concentration of an AMP to achieve full microbial killing. We hypothesise that the difference is caused by different membrane structure and composition. EXPERIMENTS: Given the complexities of bacterial membranes, we have used monolayers of the binary DPPG/TMCL mixture to mimic the cytoplasmic membrane of Gram-positive bacteria and the binary DPPG/DPPE mixture to mimic the cytoplasmic membrane of Gram-negative bacteria, where DPPG, TMCL and DPPE stand for 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1',3'-bis[1,2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, respectively. A Langmuir trough was specially designed to control the spread lipid monolayers and facilitate neutron reflectivity measurements. FINDINGS: Surface pressure-area isotherm analysis revealed that all binary lipid systems mix non-ideally, but mixing is thermodynamically favoured. An increase in the surface pressure encourages demixing, resulting in phase separation and formation of clusters. Neutron reflectivity measurements were undertaken to study the binding of an antimicrobial peptide G(IIKK)4-I-NH2 (G4) to the binary DPPG/TMCL and DPPG/DPPE monolayer mixtures at the molar ratios of 6/4 and 3/7, respectively. The results revealed stronger binding and penetration of G4 to the DPPG/TMCL monolayer, indicating greater affinity of the antimicrobial peptide due to the electrostatic interaction and more extensive penetration into the more loosely packed lipid film. This work helps explain how AMPs attack different bacterial membranes, and the results are discussed in the context of other lipid models and antibacterial studies.

Surface adsorption and solution aggregation of a novel lauroyl-l-carnitine surfactant.

HYPOTHESIS: l-carnitine plays a crucial role in the cellular production of energy by transporting fatty acids into mitochondria. Acylated l-carnitines are amphiphilic and if appropriate physical properties were demonstrated, they could replace many currently used surfactants with improved biocompatibility and health benefits. EXPERIMENTS: This work evaluated the surface adsorption of lauroyl-l-carnitine (C12LC) and its aggregation behavior. The size and shape of the aggregates of C12LC surfactant were studied at different temperatures, concentrations, pH and ionic strength by dynamic light scattering (DLS) and small-angle neutron scattering (SANS). Surface tension measurements were carried out to determine the critical micellar concentration (CMC) of C12LC. Combining with the Gibbs equation, the surface excess at different concentrations could be determined. Neutron reflection (NR) was used to determine the structure of the adsorbed layer at the air/water interface with the help of isotopic contrast variations. FINDINGS: At pH 7, the limiting area per molecule (ACMC) of the zwitterionic C12LC adsorbed layer at the air/water interface was found to be 46 Å2 from surface tension and neutron reflection, smaller than the values of C12PC, C12E5, DTAB, C12C4betaine and C12C8betaine but close to that of SDS. A pronounced surface tension minimum at pH 2 at the low ionic strength was linked to a minimum value of area per molecule of about 30 Å2, indicating the competitive adsorption from traces of lauric acid produced by hydrolysis of C12LC. As the concentration increased, area per molecule reached a plateau of 37-39 Å2, indicating the dissolution of the more surface-active lauric acid into the micelles of C12LC. DLS and SANS showed that the size and shape of micelles had little response to temperature, concentration, ionic strength or pH. The SANS profiles measured under 3 isotopic contrasts could be well fitted by the core-shell model, giving a spherical core radius of 15.7 Å and a shell thickness of 10.5 Å. The decrease of pH led to more protonated carboxyl groups and more positively charged micelles, but the micellar structures remained unchanged, in spite of their stronger interaction. These features make C12LC potentially attractive as a solubilizing agent.

What happens when pesticides are solubilised in binary ionic/zwitterionic-nonionic mixed micelles?

HYPOTHESIS: Surfactants have been widely used as adjuvants in agri-sprays to enhance the solubility of pesticides in foliar spray deposits and their mobility through leaf cuticles. Previously, we have characterised pesticide solubilisation in nonionic surfactant micelles, but what happens when pesticides become solubilised in anionic, cationic and zwitterionic and their mixtures with nonionic surfactants remain poorly characterised. EXPERIMENTS: To facilitate characterisations by SANS and NMR, we used nonionic surfactant hexaethylene glycol monododecyl ether (C12E6), anionic sodium dodecylsulphate (SDS), cationic dodecyltrimethylammonium bromide (DTAB) and zwitterionic dodecylphosphocholine (C12PC) as model adjuvant systems to solubilise 3 pesticides, Cyprodinil (CP), Azoxystrobin (AZ) and Difenoconazole (DF), representing different structural features. The investigation focused on the influence of solubilisates in driving changes to the micellar nanostructures in the absence or presence of electrolytes. NMR and NOESY were applied to investigate the solubility and location of each pesticide in the micelles. SANS was used to reveal subtle changes to the micellar structures due to pesticide solubilisation with and without electrolytes. FINDINGS: Unlike nonionic surfactants, the ionic and zwitterionic surfactant micellar structures remain unchanged upon pesticide solubilisation. Electrolytes slightly elongate the ionic surfactant micelles but have no effect on nonionic and zwitterionic surfactants. Pesticide solubilisation could alter the structures of the binary mixtures of ionic/zwitterionic and ionic/nonionic micelles by causing elongation, shell shrinkage and dehydration, with the exact alteration being determined by the molar ratio in the mixture.

How do Self-Assembling Antimicrobial Lipopeptides Kill Bacteria?

Antimicrobial peptides are promising alternatives to traditional antibiotics. A group of self-assembling lipopeptides was formed by attaching an acyl chain to the N-terminus of α-helix-forming peptides with the sequence Cx-G(IIKK)yI-NH2 (CxGy, x = 4-12 and y = 2). CxGy self-assemble into nanofibers above their critical aggregation concentrations (CACs). With increasing x, the CACs decrease and the hydrophobic interactions increase, promoting secondary structure transitions within the nanofibers. Antimicrobial activity, determined by the minimum inhibition concentration (MIC), also decreases with increasing x, but the MICs are significantly smaller than the CACs, suggesting effective bacterial membrane-disrupting power. Unlike conventional antibiotics, both C8G2 and C12G2 can kill Staphylococcus aureus and Escherichia coli after only minutes of exposure under the concentrations studied. C12G2 nanofibers have considerably faster killing dynamics and lower cytotoxicity than their nonaggregated monomers. Antimicrobial activity of peptide aggregates has, to date, been underexploited, and it is found to be a very promising mechanism for peptide design. Detailed evidence for the molecular mechanisms involved is provided, based on superresolution fluorescence microscopy, solid-state nuclear magnetic resonance, atomic force microscopy, neutron scattering/reflectivity, circular dichroism, and Brewster angle microscopy.

Aggregated Amphiphilic Antimicrobial Peptides Embedded in Bacterial Membranes.

Molecular dynamics (MD) simulations, stochastic optical reconstruction microscopy (STORM), and neutron reflection (NR) were combined to explore how antimicrobial peptides (AMPs) can be designed to promote the formation of nanoaggregates in bacterial membranes and impose effective bactericidal actions. Changes in the hydrophobicity of the designed AMPs were found to have a strong influence on their bactericidal potency and cytotoxicity. G(IIKK)3I-NH2 (G3) achieved low minimum inhibition concentrations (MICs) and effective dynamic kills against both antibiotic-resistant and -susceptible bacteria. However, a G3 derivative with weaker hydrophobicity, KI(KKII)2I-NH2 (KI), exhibited considerably lower membrane-lytic activity. In contrast, the more hydrophobic G(ILKK)3L-NH2 (GL) peptide achieved MICs similar to those observed for G3 but with worsened hemolysis. Both the model membranes studied by Brewster angle microscopy, zeta potential measurements, and NR and the real bacterial membranes examined with direct STORM contained membrane-inserted peptide aggregates upon AMP exposure. These structural features were well supported by MD simulations. By revealing how AMPs self-assemble in microbial membranes, this work provides important insights into AMP mechanistic actions and allows further fine-tuning of antimicrobial potency and cytotoxicity.

How does substrate hydrophobicity affect the morphological features of reconstituted wax films and their interactions with nonionic surfactant and pesticide?

HYPOTHESIS: Surfactants are widely used in agri-sprays to improve pesticide efficiency, but the mechanism underlying their interactions with the surface wax film on plants remains poorly understood. To facilitate physical characterisations, we have reconstituted wheat cuticular wax films onto an optically flat silicon substrate with and without octadecyltrimethoxysilane modification to control surface hydrophobicity. EXPERIMENTS: Imaging techniques including scanning electron microscopy (SEM) unravelled morphological features of the reconstituted wax films similar to those on leaves, showing little impact from the different substrates used. Neutron reflection (NR) established that reconstituted wax films were comprised of an underlying wax film decorated with top surface wax protrusions, a common feature irrespective of substrate hydrophobicity and highly consistent with what was observed from natural wax films. NR measurements, with the help of isotopic H/D substitutions to modify the scattering contributions of the wax and solvent, revealed different wax regimes within the wax films, illustrating the impact of surface hydrophilicity on the nanostructures within the wax films. FINDINGS: It was observed from both spectroscopic ellipsometry and NR measurements that wax films formed on the hydrophobic substrate were more robust and durable against attack by nonionic surfactant C12E6 solubilised with pesticide Cyprodinil (CP) than films coated on the bare hydrophilic silica. Thus, the former could be a more feasible model for studying the wax-surfactant-pesticide interactions.

Effects of Conventional Surfactants on the Activity of Designed Antimicrobial Peptide.

In this article, the interaction between a designed antimicrobial peptide (AMP) G(IIKK)3I-NH2 (G3) and four typical conventional surfactants (sodium dodecyl sulfonate (SDS), hexadecyl trimethyl ammonium bromide (C16TAB), polyoxyethylene (23) lauryl ether (C12EO23), and tetradecyldimethylamine oxide (C14DMAO)) has been studied through surface tension measurement and circular dichroism (CD) spectroscopy. The antimicrobial activities of AMP/surfactant mixtures have also been studied with Gram-negative Escherichia coli, Gram-positive Staphylococcus aureus, and the fungus Candida albicans. The cytotoxicity of the AMP/surfactant mixtures has also been assessed with NIH 3T3 and human skin fibroblast (HSF) cells. The surface tension data showed that the AMP/SDS mixture was much more surface-active than SDS alone. CD results showed that G3 conformation changed from random coil, to ß-sheet, and then to α-helix with increasing SDS concentration, showing a range of structural transformation driven by the different interactions with SDS. The antimicrobial activity of G3 to Gram-negative and Gram-positive bacteria decreased in the presence of SDS due to the strong interaction of electrostatic attraction between the peptide and the surfactant. The interactions between G3 and C16TAB, C12EO23, and C14DMAO were much weaker than SDS. As a result, the surface tension of surfactants with G3 did not change much, neither did the secondary structures of G3. The antimicrobial activities of G3 were little affected in the presence of C12EO23, slightly improved by C14DMAO, and clearly enhanced by cationic surfactant C16TAB due to its strong cationic and antimicrobial nature, consistent with their surface physical activities as binary mixtures. Although AMP G3 did not show activity to fungus, the mixtures of AMP/C16TAB and AMP/C14DMAO could kill C. albicans at high surfactant concentrations. The mixtures had rather high cytotoxicity to NIH 3T3 and HSF cells although G3 is nontoxic to cells. Cationic AMPs can be formulated with nonionic, cationic, and zwitterionic surfactants during product development, but care must be taken when AMPs are formulated with anionic surfactants, as the strong electrostatic interaction may undermine their antimicrobial activity.