Is Structural Precision of Platonic Micelles Controlled Solely by Close-Packed Head Groups or Also by Hydrophobic Tail Packing? A DFT Exploration.

Platonic micelles have been defined as structurally precise amphiphilic aggregates with discrete aggregation numbers corresponding to the close packing of spherical caps (representing head groups) on a sphere (representing hydrophobic core), analogous to the Tammes problem in geometry. Here, we use DFT to explore how an actual molecule behaves compared to the idealized picture based on the Tammes problem by also considering the packing of the tails. We modeled micelles of aggregation numbers 4 to 8 generated from the calix[4]arene amphiphile, PACaL3, with the tails forming a close-packed configuration while the headgroups are arranged as in Platonic solids. The DFT calculations reveal that tail packing overwhelmingly influences the equilibrium aggregation number. While the DFT prediction of a PACaL3 micelle of aggregation number 6 agrees with the scattering experiments of the Sakurai group, DFT calculations also suggest small concentrations of micelles of aggregation number 7. More interestingly, DFT calculations reveal that PACaL3 micelle formation occurs even though less than 20% of the hydrophobic tail surface is removed from contact with water, in contrast to the roughly 80% removal observed for classical surfactant micelles. While the close-packed head groups model predicts higher coverage of the hydrophobic surface for aggregation numbers 4 and 6 compared to 5 and 7, the DFT calculations also accounting for tail packing show that the surface coverage for aggregation numbers 5 and 7 is practically no different than that for aggregation number 4. Finally, although both the close-packed head groups model and the DFT calculations agree that the exposed hydrophobic surface area controls the equilibrium micelle aggregation number, the DFT calculations demonstrate how this exposed hydrophobic area is overwhelmingly determined by the tail group packing and not just by the close packing of head groups.

Enhancing enzyme stability by construction of polymer-enzyme conjugate micelles for decontamination of organophosphate agents.

Enhancing the stability of enzymes under different working environments is essential if the potential of enzyme-based applications is to be realized for nanomedicine, sensing and molecular diagnostics, and chemical and biological decontamination. In this study, we focus on the enzyme, organophosphorus hydrolase (OPH), which has shown great promise for the nontoxic and noncorrosive decontamination of organophosphate agents (OPs) as well as for therapeutics as a catalytic bioscavanger against nerve gas poisoning. We describe a facile approach to stabilize OPH using covalent conjugation with the amphiphilic block copolymer, Pluronic F127, leading to the formation of F127-OPH conjugate micelles, with the OPH on the micelle corona. SDS-PAGE and MALDI-TOF confirmed the successful conjugation, and transmission electron microscopy (TEM) and dynamic light scattering (DLS) revealed ∼100 nm size micelles. The conjugates showed significantly enhanced stability and higher activity compared to the unconjugated OPH when tested (i) in aqueous solutions at room temperature, (ii) in aqueous solutions at higher temperatures, (iii) after multiple freeze/thaw treatments, (iv) after lyophilization, and (v) in the presence of organic solvents. The F127-OPH conjugates also decontaminated paraoxon (introduced as a chemical agent simulant) on a polystyrene film surface and on a CARC (Chemical Agent Resistant Coating) test panel more rapidly and to a larger extent compared to free OPH. We speculate that, in the F127-OPH conjugates (both in the micellar form as well as in the unaggregated conjugate), the polypropylene oxide block of the copolymer interacts with the surface of the OPH and this confinement of the OPH reduces the potential for enzyme denaturation and provides robustness to OPH at different working environments. The use of such polymer-enzyme conjugate micelles with improved enzyme stability opens up new opportunities for numerous civilian and Warfighter applications.

Effect of interaction with coesite silica on the conformation of Cecropin P1 using explicit solvent molecular dynamics simulation.

Explicit solvent molecular dynamics (MD) simulation was carried out for the antimicrobial peptides (i) Cecropin P1 and C-terminus cysteine modified Cecropin P1 (Cecropin P1 C) in solution, (ii) Cecropin P1 and Cecropin P1 C adsorbed onto coesite -Si - O - and Si - O - H surfaces, and (iii) Cecropin P1 C tethered to coesite -Si - O - surface with either (PEO)(3) or (PEO)(6) linker. Low energy structures for Cecropin P1 and Cecropin P1 C in solution consists of two regions of high α helix probability with a sharp bend, consistent with the available structures of other antimicrobial peptides. The structure of Cecropin P1 C at low ionic strength of 0.02 M exhibits two regions of high α helix probability (residues AKKLEN and EGI) whereas at higher ionic strength of 0.12 M, the molecule was more compact and had three regions of higher α helix probability (residues TAKKLENSA, ISE, and AIQG) with an increase in α helical content from 15.6% to 18.7% as a result of shielding of electrostatic interactions. In the presence of Cecropin P1 C in the vicinity of -Si - O - surface, there is a shift in the location of two peaks in H - O - H density profile to larger distances (2.95 Å and 7.38 Å compared to 2.82 Å and 4.88 Å in the absence of peptide) with attenuated peak intensity. This attenuation is found to be more pronounced for the first peak. H-bond density profile in the vicinity of -Si - O - surface exhibited a single peak in the presence of Cecropin P1 C (at 2.9 Å) which was only slightly different from the profile in the absence of polypeptide (2.82 Å) thus indicating that Cecropin P1 C is not able to break the H-bond formed by the silica surface. The α helix probability for different residues of adsorbed Cecropin P1 C on -Si - O - surface is not significantly different from that of Cecropin P1 C in solution at low ionic strength of 0.02 M whereas there is a decrease in the probability in the second (residues ISE) and third (residues AIQG) α helical regions at higher ionic strength of 0.12 M. Though the total α helical content of adsorbed and tethered Cecropin P1 C was lower for hydrophilic Si - O - H surface compared to hydrophobic -Si - O -, hydrophobicity of the surface did not significantly affect the α helix probability of different residues. The conformation of Cecropin P1 C in solution is closer to that of tethered to -Si - O - with (PEO)(6) than that tethered with (PEO)(3) as a result of less surface interaction of tethered polypeptide with a longer linker. At low ionic strength of 0.02 M, tethered Cecropin P1 C to -Si - O - is found to exhibit lower α helix (13.0%) compared to adsorbed (15.6%) for (PEO)(3) linker with this difference being insignificant for larger (PEO)(6) linker molecule. Experimental values of % α helix inferred from circular dichroism spectra of Cecropin P1 in solution as well as in adsorbed state on silica surface compared well with the corresponding values obtained from MD simulation thereby validating the simulation procedure.

Formation of a Fully Anionic Supported Lipid Bilayer to Model Bacterial Inner Membrane for QCM-D Studies.

Supported lipid bilayers (SLBs) on quartz crystals are employed as versatile model systems for studying cell membrane behavior with the use of the highly sensitive technique of quartz crystal microbalance with dissipation monitoring (QCM-D). Since the lipids constituting cell membranes vary from predominantly zwitterionic lipids in mammalian cells to predominantly anionic lipids in the inner membrane of Gram-positive bacteria, the ability to create SLBs of different lipid compositions is essential for representing different cell membranes. While methods to generate stable zwitterionic SLBs and zwitterionic-dominant mixed zwitterionic-anionic SLBs on quartz crystals have been well established, there are no reports of being able to form predominantly or fully anionic SLBs. We describe here a method for forming entirely anionic SLBs by treating the quartz crystal with cationic (3-aminopropyl) trimethoxysilane (APTMS). The formation of the anionic SLB was tracked using QCM-D by monitoring the adsorption of anionic lipid vesicles to a quartz surface and subsequent bilayer formation. Anionic egg L-α-phosphatidylglycerol (PG) vesicles adsorbed on the surface-treated quartz crystal, but did not undergo the vesicle-to-bilayer transition to create an SLB. However, when PG was mixed with 10-40 mole% 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) (LPG), the mixed vesicles led to the formation of stable SLBs. The dynamics of SLB formation monitored by QCM-D showed that while SLB formation by zwitterionic lipids followed a two-step process of vesicle adsorption followed by the breakdown of the adsorbed vesicles (which in turn is a result of multiple events) to create the SLB, the PG/LPG mixed vesicles ruptured immediately on contacting the quartz surface resulting in a one-step process of SLB formation. The QCM-D data also enabled the quantitative characterization of the SLB by allowing estimation of the lipid surface density as well as the thickness of the hydrophobic region of the SLB. These fully anionic SLBs are valuable model systems to conduct QCM-D studies of the interactions of extraneous substances such as antimicrobial peptides and nanoparticles with Gram-positive bacterial membranes.

Atomic Force Microscopy to Characterize Antimicrobial Peptide-Induced Defects in Model Supported Lipid Bilayers.

Antimicrobial peptides (AMPs) interact with bacterial cell membranes through a variety of mechanisms, causing changes extending from nanopore formation to microscale membrane lysis, eventually leading to cell death. Several AMPs also disrupt mammalian cell membranes, despite their significantly different lipid composition and such collateral hemolytic damage hinders the potential therapeutic applicability of the AMP as an anti-microbial. Elucidating the mechanisms underlying the AMP-membrane interactions is challenging due to the variations in the chemical and structural features of the AMPs, the complex compositional variations of cell membranes and the inadequacy of any single experimental technique to comprehensively probe them. (1) Background: Atomic Force Microscopy (AFM) imaging can be used in combination with other techniques to help understand how AMPs alter the orientation and structural organization of the molecules within cell membranes exposed to AMPs. The structure, size, net charge, hydrophobicity and amphipathicity of the AMPs affect how they interact with cell membranes of differing lipid compositions. (2) Methods: Our study examined two different types of AMPs, a 20-amino acid, neutral, α-helical (amphipathic) peptide, alamethicin, and a 13-amino acid, non-α-helical cationic peptide, indolicidin (which intramolecularly folds, creating a hydrophobic core), for their interactions with supported lipid bilayers (SLBs). Robust SLB model membranes on quartz supports, incorporating predominantly anionic lipids representative of bacterial cells, are currently not available and remain to be developed. Therefore, the SLBs of zwitterionic egg phosphatidylcholine (PC), which represents the composition of a mammalian cell membrane, was utilized as the model membrane. This also allows for a comparison with the results obtained from the Quartz Crystal Microbalance with Dissipation (QCM-D) experiments conducted for these peptides interacting with the same zwitterionic SLBs. Further, in the case of alamethicin, because of its neutrality, the lipid charge may be less relevant for understanding its membrane interactions. (3) Results: Using AFM imaging and roughness analysis, we found that alamethicin produced large, unstable defects in the membrane at 5 µM concentrations, and completely removed the bilayer at 10 µM. Indolicidin produced smaller holes in the bilayer at 5 and 10 µM, although they were able to fill in over time. The root-mean-square (RMS) roughness values for the images showed that the surface roughness caused by visible defects peaked after peptide injection and gradually decreased over time. (4) Conclusions: AFM is useful for helping to uncover the dynamic interactions between different AMPs and cell membranes, which can facilitate the selection and design of more efficient AMPs for use in therapeutics and antimicrobial applications.

Effects of L-alanine and inosine germinants on the elasticity of Bacillus anthracis spores.

The surface of dormant Bacillus anthracis spores consists of a multilayer of protein coats and a thick peptidoglycan layer that allow the cells to resist chemical and environmental insults. During germination, the spore coat is degraded, making the spore susceptible to chemical inactivation by antisporal agents as well as to mechanical inactivation by high-pressure or mechanical abrasion processes. While chemical changes during germination, especially the release of the germination marker, dipicolinic acid (DPA), have been extensively studied, there is as yet no investigation of the corresponding changes in the mechanical properties of the spore. In this work, we use atomic force microscopy (AFM) to characterize the mechanical properties of the surface of Bacillus anthracis spores during germination. The Hertz model of continuum mechanics of contact was used to evaluate the Young's moduli of the spores before and after germination by applying the model to load-indentation curves. The highest modulus was observed for dormant spores, with average elasticity values of 197 +/- 81 MPa. The elasticity decreased significantly after incubation of the spores with the germinants L-alanine or inosine (47.5 +/- 41.7 and 35.4 +/- 15.8 MPa, respectively). Exposure of B. anthracis spores to a mixture of both germinants resulted in a synergistic effect with even lower elasticity, with a Young's modulus of 23.5 +/- 14.8 MPa. The elasticity of the vegetative B. anthracis cells was nearly 15 times lower than that of the dormant spores (12.4 +/- 6.3 MPa vs 197.0 +/- 80.5 MPa, respectively). Indeed from a mechanical strength point of view, the germinated spores were closer to the vegetative cells than to the dormant spores. Further, the decrease in the elasticity of the cells was accompanied by increasing AFM tip indentation depths on the cell surfaces. Indentation depths of up to 246.2 nm were observed for vegetative B. anthracis compared to 20.5 nm for the dormant spores. These results provide quantitative information on how the mechanical properties of the cell wall change during germination, which may explain how spores become susceptible to inactivation processes based on mechanical forces during germination and outgrowth. The study of spore elasticity may be a valuable tool in the design of improved antisporal treatments.

Atomic force microscopy study of germination and killing of Bacillus atrophaeus spores.

Bacterial spores such as Bacillus atrophaeus are one of the most resistant life forms known and are extremely resistant to chemical and environmental factors in the dormant state. During germination, as bacterial spores progress towards the vegetative state, they become susceptible to anti-sporal agents. B. atrophaeus spores were exposed to the non-nutritive germinant dodecylamine (DDA), a cationic surfactant that can also be used as a killing agent, for up to 60 min, or to the nutrient germinant L-alanine. In kinetic studies, 99% of the spores were killed within 5 min of exposure to DDA. Atomic force microscopy (AFM) can be used as a sensitive tool to assess how the structure of the spore coat changes upon exposure to germinants or killing agents. Changes in cell height and roughness over time of exposure to DDA were examined using AFM. DDA caused the spore height to decrease by >50%, which may have been due to a partial breakdown of the spore coat. Treatment of B. atrophaeus with the nutrient germinant resulted in a decrease in height of spores after 2 h of incubation, from 0.7 +/- 0.1 microm to 0.3 +/- 0.2 microm. However, treatment with L-alanine did not change the surface roughness of the spores, indicating that the changes that occur during germination take place underneath the spore coat. We propose that exposure to DDA at high concentrations causes pores to form in the coat layer, killing B. atrophaeus without the need to fully germinate spores.

Constructing a molecular theory of self-assembly: Interplay of ideas from surfactants and block copolymers.

Low molecular weight surfactants and high molecular weight block copolymers display analogous self-assembly behavior in solutions and at interfaces, generating nanoscale structures of different shapes. Understanding the link between the molecular structure of these amphiphiles and their self-assembly behavior has been the goal of theoretical studies. Despite the analogies between surfactants and block copolymers, models predicting their self-assembly behavior have evolved independent of one another, each overlooking the molecular feature considered critical to the other. In this review, we focus on the interplay of ideas pertaining to surfactants and block copolymers in three areas of self-assembly. First, we show how improved free energy models have evolved by applying ideas from surfactants to block copolymers and vice versa, giving rise to a unitary theoretical framework and better predictive capabilities for both classes of amphiphiles. Second we show that even though molecular packing arguments are often used to explain aggregate shape transitions resulting from self-assembly, the molecular packing considerations are more relevant in the case of surfactants whereas free energy criteria are relevant for block copolymers. Third, we show that even though the surfactant and block copolymer aggregates are small nanostructures, the size differences between them is significant enough to make the interfacial effects control the solubilization of molecules in surfactant micelles while the bulk interactions control the solubilization in block copolymer micelles. Finally, we conclude by identifying recent theoretical progress in adapting the micelle model to a wide variety of self-assembly phenomena and the challenges to modeling posed by emerging novel classes of amphiphiles with complex biological, inorganic or nanoparticle moieties.

Nanoarmoring of Proteins by Conjugation to Block Copolymer Micelles.

The creation of polymer nanoparticles with protein functionality is of great interest to many applications such as targeted drug or gene delivery, diagnostic imaging, cancer theranostics, delivery of protein therapeutics, sensing chemical and biomolecular analytes in complex environments, and design of protective clothing resembling a second skin. Many approaches to achieving this goal are being explored in the current literature. In this chapter, we describe a relatively simple and flexible approach of conjugating the protein to an amphiphilic block copolymer and creating polymer nanoparticles with protein functionality by taking advantage of the intrinsic self-assembly behavior of the amphiphilic block copolymer. The commercially available and biocompatible polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer is used as the polymer building block. For demonstrative purposes, bovine serum albumin was chosen as the protein. We determine the molecular weight of the protein-polymer conjugate and thereby the degree of conjugation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry measurements. Retention of protein secondary structure in the conjugate was determined by circular dichroism spectroscopy, and the biological activity of the protein in the conjugated state has been evaluated by kinetic assay involving hydrolysis of an organophosphate compound. Dynamic light scattering and zeta potential measurements were used to characterize the size and charge of the protein-polymer conjugate micelle. Precise control of the size of the micelle and surface number density of the proteins on the micelle surface by coassembling with a second block copolymer have been demonstrated. These studies document a rational approach to armor the protein by conjugation with a block copolymer micelle, as a general approach.

"Non-equilibrium" block copolymer micelles with glassy cores: a predictive approach based on theory of equilibrium micelles.

Micelles generated in water from most amphiphilic block copolymers are widely recognized to be non-equilibrium structures. Typically, the micelles are prepared by a kinetic process, first allowing molecular scale dissolution of the block copolymer in a common solvent that likes both the blocks and then gradually replacing the common solvent by water to promote the hydrophobic blocks to aggregate and create the micelles. The non-equilibrium nature of the micelle originates from the fact that dynamic exchange between the block copolymer molecules in the micelle and the singly dispersed block copolymer molecules in water is suppressed, because of the glassy nature of the core forming polymer block and/or its very large hydrophobicity. Although most amphiphilic block copolymers generate such non-equilibrium micelles, no theoretical approach to a priori predict the micelle characteristics currently exists. In this work, we propose a predictive approach for non-equilibrium micelles with glassy cores by applying the equilibrium theory of micelles in two steps. In the first, we calculate the properties of micelles formed in the mixed solvent while true equilibrium prevails, until the micelle core becomes glassy. In the second step, we freeze the micelle aggregation number at this glassy state and calculate the corona dimension from the equilibrium theory of micelles. The condition when the micelle core becomes glassy is independently determined from a statistical thermodynamic treatment of diluent effect on polymer glass transition temperature. The predictions based on this "non-equilibrium" model compare reasonably well with experimental data for polystyrene-polyethylene oxide diblock copolymer, which is the most extensively studied system in the literature. In contrast, the application of the equilibrium model to describe such a system significantly overpredicts the micelle core and corona dimensions and the aggregation number. The non-equilibrium model suggests ways to obtain different micelle sizes for the same block copolymer, by the choices we can make of the common solvent and the mode of solvent substitution.

Differentiating antimicrobial peptides interacting with lipid bilayer: Molecular signatures derived from quartz crystal microbalance with dissipation monitoring.

Many antimicrobial peptides (AMPs) kill bacteria by disrupting the lipid bilayer structure of their inner membrane. However, there is only limited quantitative information in the literature to differentiate between AMPs of differing molecular properties, in terms of how they interact with the membrane. In this study, we have used quartz crystal microbalance with dissipation monitoring (QCM-D) to probe the interactions between a supported bilayer membrane of egg phosphatidylcholine (egg PC) and four structurally different AMPs: alamethicin, chrysophsin-3, indolicidin, and sheep myeloid antimicrobial peptide (SMAP-29). Multiple signatures from the QCM-D measurements were extracted, differentiating the AMPs, that provide information on peptide addition to and lipid removal from the membrane, the dynamics of peptide-membrane interactions and the rates at which the peptide actions are initiated. The mechanistic variations in peptide action were related to the fundamental structural properties of the peptides including the hydrophobicity, hydrophobic moment, and the probability of α-helical secondary structures.

Size dependence of gold nanoparticle interactions with a supported lipid bilayer: A QCM-D study.

Knowledge of nanoparticle (NP)-membrane interactions is important to advances in nanomedicine as well as for determining the safety of NPs to humans and the ecosystem. This study focuses on a unique mechanism of cytotoxicity, cell membrane destabilization, which is principally dependent on the nanoparticle nature of the material rather than on its molecular properties. We investigated the interactions of 2, 5, 10, and 40nm gold NPs with supported lipid bilayer (SLB) of L-α-phosphatidylcholine using quartz crystal microbalance with dissipation monitoring (QCM-D). Gold NPs were tested both in the absence of and in the presence of polymethacrylic acid (PMAA), used to simulate the natural organic matter (NOM) in the environment. In the absence of PMAA, for all NP sizes, we observed only small mass losses (1 to 6ng) from the membrane. This small lipid removal may be a free energy lowering mechanism to relieve stresses induced by the adsorption of NPs, with the changes too small to affect the membrane integrity. In the presence of PMAA, we observed a net mass increase in the case of smaller NPs. We suggest that the increased adhesion between the NP and the bilayer, promoted by PMAA, causes sufficient NP adsorption on the bilayer to overcompensate for any loss of lipid. The most remarkable observation is the significant mass loss (60ng) for the case of 40nm NPs. We attribute this to the lipid bilayer engulfing the NP and leaving the crystal surface. We propose a simple phenomenological model to describe the competition between the particle-bilayer adhesion energy, the bilayer bending energy, and the interfacial energy at bilayer defect edges. The model shows that the larger NPs, which become more adhesive because of the polymer adsorption, are engulfed by the bilayer and leave the crystal surface, causing large mass loss and membrane disruption. The QCM-D measurements thus offer direct evidence that even if NPs are intrinsically not cytotoxic, they can become cytotoxic in the presence of environmental organic matter which modulates the adhesive interactions between the nanoparticle and the membrane.

Antimicrobial peptide alamethicin insertion into lipid bilayer: a QCM-D exploration.

Alamethicin is a 20-amino-acid, α-helical antimicrobial peptide that is believed to kill bacteria through pore formation in the inner membranes. We used quartz crystal microbalance with dissipation monitoring (QCM-D) to explore the interactions of alamethicin with a supported lipid bilayer. Changes in frequency (Δf) and dissipation (ΔD) measured at different overtones as a function of peptide concentration were used to infer peptide-induced changes in the mass and rigidity of the membrane as well as the orientation of the peptide in the bilayer. The measured Δf were positive, corresponding to a net mass loss from the bilayer, with substantial mass losses at 5 µM and 10 µM alamethicin. The measured Δf at various overtones were equal, indicating that the mass change in the membrane was homogeneous at all depths consistent with a vertical peptide insertion. Such an orientation coupled to the net mass loss was in agreement with cylindrical pore formation and the negligibly small ΔD suggested that the peptide walls of the pores stabilized the surrounding lipid organization. Dynamics of the interactions examined through Δf vs. ΔD plots suggested that the peptides initially inserted into the membrane and caused disordering of the lipids. Subsequently, lipids were removed from the bilayer to create pores and alamethicin caused the remaining lipids to reorder and stabilize within the membrane. Based on model calculations, we concluded that the QCM-D data cannot confirm or rule out whether peptide clusters coexist with pores in the bilayer. We have also proposed a way to calculate the peptide-to-lipid ratio (P/L) in the bilayer from QCM-D data and found the calculated P/L as a function of the peptide concentration to be similar to the literature data for vesicle membranes.

Enhanced activity and stability of organophosphorus hydrolase via interaction with an amphiphilic polymer.

A simple approach to enhancing the activity and stability of organophosphorus hydrolase (OPH) is developed based on interactions between the hydrophobic poly(propylene oxide) (PPO) block of amphiphilic Pluronics and the enzyme. This strategy provides an efficient route to new formulations for decontaminating organophosphate neurotoxins.

Role of single-walled carbon nanotubes on ester hydrolysis and topography of electrospun bovine serum albumin/poly(vinyl alcohol) membranes.

Electrospun membranes were studied for the chemical deactivation of threat agents by means of enzymatic proteins. Protein loading and the surface chemistry of hybrid nanofibers influenced the efficacy by which embedded enzymes could digest the substrate of interest. Bovine serum albumin (BSA), selected as a model protein, was electrospun into biologically active fibers of poly(vinyl alcohol), PVA. Single-walled carbon nanotubes (SWNTs) were blended within these mixtures to promote protein assembly during the process of electrospinning and subsequently the ester hydrolysis of the substrates. The SWNT incorporation was shown to influence the topography of PVA/BSA nanofibers and enzymatic activity against paraoxon, a simulant for organophosphate agents and a phosphorus analogue of p-nitrophenyl acetate (PNA). The esterase activity of BSA against PNA was uncompromised upon its inclusion within nanofibrous membranes because similar amounts of PNA were hydrolyzed by BSA in solution and the electrospun BSA. However, the availability of BSA along the fiber surface was shown to affect the ester hydrolysis of paraoxon. Atomic force microscopy images of nanofibers implicated the surface migration of BSA during the electrospinning of SWNT filled dispersions, especially as greater weight fractions of protein were added to the spinning mixtures. In turn, the PVA/SWNT/BSA nanofibers outperformed the nanotube free PVA/BSA membranes in terms of paraoxon digestion. The results support the development of electrospun polymer nanofiber platforms, modulated by SWNTs for enzyme catalytic applications relevant to soldier protective ensembles.

Interactions of antimicrobial peptide chrysophsin-3 with Bacillus anthracis in sporulated, germinated, and vegetative states.

Bacillus anthracis spores contain on their surface multilayered protein coats that provide barrier properties, mechanical strength, and elasticity that aid in protecting the sporulated state and preventing germination, outgrowth, and transition into the virulent vegetative bacterial state. In this work, the antimicrobial peptide (AMP) chrysophsin-3 was tested against B. anthracis in each of the three distinct metabolic states (sporulated, germinated, and vegetative) for its bacteria-killing activity and its ability to modify the surface nanomechanical properties. Our results provide the first demonstration that chrysophsin-3 killed B. anthracis even in its sporulated state while more killing was observed for germinated and vegetative states. The elasticity of vegetative B. anthracis increased from 12 ± 6 to 84 ± 17 MPa after exposure to 0.22 mM chrysophsin-3. An increase in cellular spring constant was also observed for chrysophsin-3-treated vegetative B. anthracis. Atomic force microscopy images suggested that the changes in mechanical properties of vegetative B. anthracis after chrysophsin-3 treatment are due to loss of water content and cellular material from the cell, possibly caused by the disruption of the cell membrane by the AMP. In contrast, sporulated and germinated B. anthracis retained their innate mechanical properties. Our data indicate that chrysophsin-3 can penetrate the spore coat of B. anthracis spores and kill them without causing any significant mechanical changes on the spore surface. These results reveal a yet unrecognized role for chrysophsin-3 in the killing of B. anthracis spores without the need for complete germination or release of spore coats.

Characterization of supported lipid bilayer disruption by chrysophsin-3 using QCM-D.

Antimicrobial peptides (AMPs) are naturally occurring polymers that can kill bacteria by destabilizing their membranes. A quartz crystal microbalance with dissipation monitoring (QCM-D) was used to better understand the action of the AMP chrysophsin-3 on supported lipid bilayers (SLB) of phosphatidylcholine. Interaction of the SLB with chrysophsin-3 at 0.05 µM demonstrated changes in frequency (Δf) and energy dissipation (ΔD) that were near zero, indicating little change in the membrane. At higher concentrations of chyrsophsin-3 (0.25-4 µM), decreases in Δf of up to 7 Hz were measured. These negative frequency changes suggest that mass was being added to the SLB, possibly due to peptide insertion into the membrane. At a chrysophsin-3 concentration of 10 µM, there was a net mass loss, which was attributed to pore formation in the membrane. QCM-D can be used to describe a mechanistic relationship between AMP concentration and interaction with a model cell membrane.