Tunable Coassembly of Octaarginine with Thiazolyl Benzenesulfonamides Exerts Variable Antibacterial Activity.

The cationic peptide octaarginine (R8) is a prominent cell-penetrating peptide and has been extensively researched as a carrier of diverse cell-destined cargo. In this work, we describe the coassembly of R8 with small molecule thiazolyl benzenesulfonamide (TBS) derivatives. Physical complexation of R8 with three TBS derivatives across a range of weight ratios results in the formation of a distinctive set of nano- and microstructures. A detailed structural characterization of the R8:TBS-derivative coassemblies has been performed by a combination of FTIR, XRD, SEM, and DSC. The major functional groups that facilitate coassembly include sulfonamide SO2 and NH groups of the TBS derivatives, and the guanidinium of R8, via a combination of cation-π and hydrogen-bonding interactions. The R8:4F-TBS coassembly displays singular topological features compared to R8:4Br-TBS and R8:4CH3-TBS complexes. These differences are attributed to the changes in the preferred orientation of the guanidino groups of R8 with respect to the π-surface of TBS derivatives. The modulation of forces of interaction across the R8:TBS-derivative coassemblies aligns with their respective thermal stabilities. The single-crystal structure of bare 4F-TBS has been subjected to Hirshfeld and 2D fingerprinting analysis and indicates notable variations from the crystal packing of the R8:4F-TBS coassembly. The structural differences among the R8:TBS-derivative coassemblies correlate with distinctive profiles of antibacterial activity in each case. The coassembled structures exert a variable extent of bacterial membrane disruption and damage based on the unique disposition of R8 and the potency of small molecule in each case. The aqueous suspension of R8:4F-TBS displays significant outer membrane disruption and bacterial killing compared with the other complexes. This work successfully demonstrates the hitherto unreported potential for coassembly of cell-penetrating peptides with other entities. The coassembly of R8 with small molecules highlights an attractive strategy for tuning the functional properties of each component.

Reusable semi-IPN polymer networks as long-term antibacterial coatings.

The current study aimed to develop a reusable antibacterial coating that can be employed for efficient bacterial killing. We synthesized a water-soluble methacrylamide-based copolymer consisting of cationic and hydrophobic groups and coated it onto a glass surface through the formation of semi-interpenetrating polymer networks (semi-IPN) of aminopropyl triethoxysilane and glutaraldehyde. The coated surface was exposed to Gram-negative and Gram-positive bacteria, where the surface exhibited rapid bacterial killing ability within 5-15 min. The substrates displayed a minimal loss of antibacterial activity even after two water rinse cycles. The coatings were able to kill both the bacterial strains even after 5 weeks, suggesting excellent longevity. The surfaces were stable after repeated wiping cycles with 70% IPA using Kim wipes and 5 min sonication in DI water as no bactericidal activity was lost. Thus, a sustainable antibacterial copolymer coating was developed, and it is stable and reusable against bacterial contamination and could be employed as a long-term antibacterial coating.

Enhancing Bactericidal Properties of Ti6Al4V Surfaces through Micro and Nano Hierarchical Laser Texturing.

Orthopedic and dental implants made from Ti6Al4V are widely used due to their excellent mechanical properties and biocompatibility. However, the long-term performance of these implants can be compromised by bacterial infections. This study explores the development of hierarchically textured surfaces with enhanced bactericidal properties to address such challenges. Hierarchical surface structures were developed by combining microscale features produced by a microsecond laser and superimposed submicron features produced using a femtosecond laser. Microscale patterns were produced by the pulsed laser surface melting process, whereas submicrometer laser-induced periodic surface structures were created on top of them by femtosecond laser processing. Escherichia coli bacterial cells were cultured on the textured surface. After 24 h, a staining analysis was performed using SYTO9 and PI dyes to investigate the samples with a confocal microscope for live dead assays. Results showed bacterial colony formation onto the microscale surface textures with live bacterial cells, whereas the hierarchical surface textures display segregated and physically damaged bacterial cell attachments on surfaces. The hierarchical surface textures showed ∼98% dead bacterial cells due to the combined effect of its multiscale surface features and oxide formation during the laser processing steps. The efficacy of hierarchical surface textures in enhancing the antibacterial behavior of Ti6Al4V implants is evident from the conducted research. Such laser-based surface treatments can find potential applications in different industrial sectors.

Protein-directed synthesis of ZIF-8 functionalized with a polymer as core-shell drug coatings with antibacterial and anti-inflammatory properties.

We developed a strategy to use lysozyme (Lys) as a template to produce mesoporous zeolitic imidazolate framework (ZIF-8) structures under physiological conditions. Thereafter, an amphiphilic triblock copolymer, PEG-PPG-PEG, was used to form protective core-shell ZIF-8 nanocomposite coatings to protect the encapsulated drug epigallocatechin-3-gallate (EGCG), to achieve notable antibacterial properties against E. coli, S. aureus and MRSA strains. Moreover, nanocomposites exhibited anti-inflammatory activity by counteracting the secretion of cytokines in THP-1 macrophages.

Histopathological Evidence of Thyroid Dermopathy and its Correlation with Thyroid-Associated Orbitopathy in Patients with Graves' Disease having Normally Appearing Pretibial Skin: A Case-Control Study.

Objective: Thyroid dermopathy (TD), reportedly encountered in less than 5% of patients with Graves' disease (GD), is supposed to coexist only with thyroid-associated orbitopathy (TAO). However, clinically inapparent TD, detected non-invasively by thermal imaging or ultrasonography, seems to be present in a larger proportion of GD. Histopathological examination (HPE), though considered as gold standard for detecting TD, has not been performed widely to identify subclinical TD in GD. Materials and Methods: In this single-centre, cross-sectional, case-control study, 50 patients with GD (cases) and normal appearing pretibial skin were compared with 45 age- and sex-matched individuals (39 healthy volunteers, 3 with toxic multinodular goitre and 3 with solitary toxic nodule) (control). All patients were evaluated clinically for presence of TAO. Punch biopsy specimens were obtained from the pretibial skin in all 95 participants. Tissue sections were examined under light microscopy for mucin deposition, splitting of collagen fibrils and perivascular lymphocytic infiltration. Results: Sixty per cent of patients with GD demonstrated at least one of the above three histological features, while 52% had any combination of two features and 46% harboured all the three features. Mucin deposition, splitting of collagen fibrils and lymphocytic infiltration were found overall in 52%, 54% and 52% of GD, respectively; 4.4-11.1% of controls also had some evidence of TD on HPE. Subclinical TD was not related to age, duration of disease and TAO in our study. Conclusions: TD, particularly in its subclinical form, Seems to be widely prevalent in GD (46-60%) and exists even in absence of TAO. HPE, though more sensitive than the various non-invasive tests, is not specific (ranges from 89% to 95%) for TD. However, HPE can accurately diagnose TD in appropriate clinical background.

Intracellular Bacterial Targeting by a Thiazolyl Benzenesulfonamide and Octaarginine Peptide Complex.

A brominated thiazolyl benzenesulfonamide (BTB) derivative is conjugated with the cell-penetrating peptide octaarginine (R8) in an effort to construct innovative antibacterial products. The noncovalent complex of BTB and R8 is characterized by Fourier transform infrared (FTIR) spectroscopy, which indicates hydrogen bonding between the two constituents. Attachment of the peptide moiety renders aqueous solubility to the hydrophobic benzenesulfonamide drug and bestows bactericidal activity. Confocal imaging in conjunction with dye probes shows successful clearance of intracellular Staphylococcus aureus bacteria by the BTB-R8 complex. Scanning electron micrographs and studies with a set of fluorescent dyes suggest active disruption of the bacterial cell membrane by the BTB-R8 complex. In contrast, the complex of BTB with octalysine (K8) fails to cause membrane damage and displays a modest antibacterial effect. A complex of BTB with the water-soluble hydrophilic polymer poly(vinylpyrrolidone) (PVP) does not display any antibacterial effect, indicating the distinctive role of the cell-penetrating peptide (CPP) R8 in the cognate complex. The leakage of the encapsulated dye from giant unilamellar vesicles upon interaction with the BTB-R8 complex further highlights the membrane activity of the complex, which cannot be accomplished by bare sulfonamide alone. This work broadens the scope of use of CPPs with respect to eliciting antibacterial activity and potentially expands the limited arsenal of membrane-targeting antibiotics.

Rutin-loaded polymeric nanorods alleviate nephrolithiasis by inhibiting inflammation and oxidative stress in vivo and in vitro.

Polycrystalline aggregates formed in the glomerulus or other components of the urinary system represent the most critical step in kidney stone formation. The most common form of these crystals is calcium oxalate monohydrate (CaC2O4·H2O). Rutin is a potent antioxidant phytochemical, however, hydrophobicity and limited bioavailability restrain it from clinical applications. We developed a biocompatible amphiphilic triblock copolymer, PLGA-PEG-PLGA-loaded rutin nanorods, by simple and efficient self-assembly. Incorporation of polymer changed the topology of crystalline rutin into nanorods with non-Fickian sustained drug release kinetics by the Korsmeyer-Peppas model and thermodynamically non-spontaneous release of rutin. Rutin nanorods changed the growth and morphology of CaC2O4 crystals from the monohydrate to dihydrate form by increased adsorption and specific surface area from 0.8027 to 5.4233 m2 g-1, respectively. Rutin nanorods restored cell viability and oxidative stress in MDCK cells by modulating OPN expression and counteracts the proinflammatory signaling in THP-1 macrophages triggered by CaC2O4 crystals (80 µg cm-2). Rutin nanorods resulted in significant protection in serum and urinary biochemistry with reduced calcifications and increased tissue viability of kidneys without any toxicity and achieved high bioavailability. Our data provide a facile strategy for the use of rutin nanorods as a targeted drug system to treat and prevent renal stone formations.

Experimental and simulation studies reveal mechanism of action of human defensin derivatives.

Antimicrobial peptides (AMPs) are naturally occurring promising candidates which can be used as antibiotics against a wide variety of bacteria. The key component for using them as a potent antibiotic is that their mechanism of action is less prone to bacterial resistance. However, the molecular details of their mechanism of action is not yet fully understood. In this study, we try to shed light on the mode of action of AMPs, possible reason behind it, and their interaction with lipid bilayers through experimental as well as molecular dynamics (MD) simulation studies. The focal of our study was Human beta defensin 3 (hBD-3) which is a naturally occurring AMP. We chose three derivatives of hBD-3, namely CHRG01, KSR, and KLR for the detailed analysis presented in this study. These three peptides are evaluated for their antibacterial potency, secondary structure analysis and mechanism of action. The experimental results reveal that these peptides are active against gram positive as well as gram negative bacteria and kill bacteria by forming membrane pores. The MD simulation results correlate well with the antibacterial activity and shed light into the early membrane insertion dynamics. Moreover, the specific amino acids responsible for membrane disruptions are also identified from the MD simulations. Understanding the molecular level interaction of individual amino acids with the lipid bilayer will greatly help in the design of more efficient antimicrobial peptides.

Optimal Balance of Hydrophobic Content and Degree of Polymerization Results in a Potent Membrane-Targeting Antibacterial Polymer.

Globally, excessive use of antibiotics has drastically raised the resistance frequency of disease-causing microorganisms among humans, leading to a scarcity of efficient and biocompatible drugs. Antimicrobial polymers have emerged as a promising candidate to combat drug-resistance pathogens. Along with the amphiphilic balance, structural conformation and molecular weight (M n) play an indispensable role in the antimicrobial potency and cytotoxic activity of polymers. Here, we synthesize cationic and amphiphilic methacrylamide random copolymers using free-radical copolymerization. The mole fraction of the hydrophobic groups is kept constant at approximately 20%, while the molecular weight (average number of linked polymeric units) is varied and the antibacterial and cytotoxic activities are studied. The chemical composition of the copolymers is characterized by 1H NMR spectroscopy. We observe that the average number of linked units in a polymer chain (i.e., molecular weight) significantly affects the polymer activity and selectivity. The antibacterial efficacy against both of the examined bacteria, Escherichia coli and Staphylococcus aureus, increases with increasing molecular weight. The bactericidal activity of polymers is confirmed by live/dead cell viability assay. Polymers with high molecular weight display high antibacterial activity, yet are highly cytotoxic even at 1 × MIC. However, low-molecular-weight polymers are biocompatible while retaining antibacterial potency. Furthermore, no resistance acquisition is observed against the polymers in E. coli and S. aureus. A comprehensive analysis using confocal and scanning electron microscopy (SEM) techniques shows that the polymers target bacterial membranes, resulting in membrane permeabilization that leads to cell death.

Methacrylamide based antibiotic polymers with no detectable bacterial resistance.

The growing number of multidrug-resistant pathogens is a major healthcare concern. In search of alternatives to antibiotics, synthetic mimics of antimicrobial peptides (SMAMPs) in the form of antimicrobial polymers have gained tremendous attention. Here, we report the synthesis of a set of 7 amphiphilic water-soluble cationic copolymers using aminopropyl methacrylamide and benzyl methacrylamide repeat units that show significant antibacterial activity. The antibacterial activity was evaluated using a broth microdilution assay against S. aureus and E. coli, while toxicity to mammalian cells was quantified by hemolysis assay with human red blood cells (RBCs). We find that the antibacterial activity and selectivity of the polymers depends on the mole fraction of aromatic benzyl units (fbenzyl) and the average molecular weight (Mn). Polymers with fbenzyl of 0.10 and 0.19, named AB-10 and AB-19 respectively, exhibited the highest antibacterial efficacy without inducing hemolysis and were chosen for further study. Liposome dye leakage study and observations from confocal and scanning electron microscopy indicate that the AB polymers killed bacterial cells primarily by disrupting the cytoplasmic membrane. No resistant mutants of E. coli and S. aureus were obtained with AB-19 in a 30 day serial passage study.

Emergent antibacterial activity of N-(thiazol-2-yl)benzenesulfonamides in conjunction with cell-penetrating octaarginine.

Hybrid antimicrobials that combine the effect of two or more agents represent a promising antibacterial therapeutic strategy. In this work, we have synthesized N-(4-(4-(methylsulfonyl)phenyl)-5-phenylthiazol-2-yl)benzenesulfonamide derivatives that combine thiazole and sulfonamide, groups with known antibacterial activity. These molecules are investigated for their antibacterial activity, in isolation and in complex with the cell-penetrating peptide octaarginine. Several of the synthesized compounds display potent antibacterial activity against both Gram-negative and Gram-positive bacteria. Compounds with 4-tert-butyl and 4-isopropyl substitutions exhibit attractive antibacterial activity against multiple strains. The isopropyl substituted derivative displays low MIC of 3.9 µg mL-1 against S. aureus and A. xylosoxidans. The comparative antibacterial behaviour of drug-peptide complex, drug alone and peptide alone indicates a distinctive mode of action of the drug-peptide complex, that is not the simple sum total of its constituent components. Specificity of the drug-peptide complex is evident from comparison of antibacterial behaviour with a synthetic intermediate-peptide complex. The octaarginine-drug complex displays faster killing-kinetics towards bacterial cells, creates pores in the bacterial cell membranes and shows negligible haemolytic activity towards human RBCs. Our results demonstrate that mere attachment of a hydrophobic moiety to a cell penetrating peptide does not impart antibacterial activity to the resultant complex. Conversely, the work suggests distinctive modes of antibiotic activity of small molecules when used in conjunction with a cell penetrating peptide.

Enhancing Aqueous Solubility and Antibacterial Activity of Curcumin by Complexing with Cell-Penetrating Octaarginine.

Bacterial resistance to antimicrobial drugs is one of the biggest threats to human health and novel drugs, and strategies are needed to obviate this resistance crisis. An innovative strategy for designing novel antimicrobial drugs is based on the hybridization of an antimicrobial agent with a second functional entity. Here, we use a cell-penetrating peptide-octaarginine (R8) as the second functional entity and develop a complex or hybrid of R8 and curcumin that possibly targets the bacterial cell membrane. Minimum inhibitory concentration assays show that the antibacterial activity of the complex is enhanced in a synergistic manner and rapid killing kinetics are obtained, emphasizing a bactericidal mode of action. In addition, electron microscopy images reveal bacterial membrane disruption by the complex. The R8-curcumin complex also displays activity against HeLa cells.

Environmentally Benign Nanoantibiotics with a Built-in Deactivation Switch Responsive to Natural Habitats.

The massive use of antibiotics in healthcare and agriculture has led to their artificial accumulation in natural habitats, which risks the structure and function of the microbial communities in ecosystems, threatens food and water security, and accelerates the development of resistome. Ideally, antibiotics should remain fully active in clinical services while becoming deactivated rapidly once released into the environment, but none of the current antibiotics meet this criterion. Here, we show a nanoantibiotic design that epitomizes the concept of carrying a built-in "OFF" switch responsive to natural stimuli. The environmentally benign nanoantibiotics consist of cellulose backbones covalently grafted with hydrophilic polymer brushes that by themselves are antimicrobially inactive. In their nanostructured forms in services, these cellulose-based polymer molecular brushes are potent killers for both Gram-positive and Gram-negative bacteria, including clinical multidrug-resistant strains; after services and being discharged into the environment, they are shredded into antimicrobially inactive pieces by cellulases that do not exist in the human body but are abundant in natural habitats. This study illuminates a new concept of mitigating the environmental footprints of antibiotics with rationally designed nanoantibiotics that can be dismantled and disabled by bioorthogonal chemistry occurring exclusively in natural habitats.

Influenza virus A M2 protein generates negative Gaussian membrane curvature necessary for budding and scission.

The M2 protein is a multifunctional protein, which plays several roles in the replication cycle of the influenza A virus. Here we focus on its ability to promote budding of the mature virus from the cell surface. Using high-resolution small-angle X-ray scattering we show that M2 can restructure lipid membranes into bicontinuous cubic phases which are rich in negative Gaussian curvature (NGC). The active generation of negative Gaussian membrane curvature by M2 is essential to influenza virus budding. M2 has been observed to colocalize with the region of high NGC at the neck of a bud. The structural requirements for scission are even more stringent than those for budding, as the neck must be considerably smaller than the virus during 'pinch off'. Consistent with this, the amount of NGC in the induced cubic phases suggests that M2 proteins can generate high curvatures comparable to those on a neck with size 10× smaller than a spherical influenza virus. Similar experiments on variant proteins containing different M2 domains show that the cytoplasmic amphipathic helix is necessary and sufficient for NGC generation. Mutations to the helix which reduce its amphiphilicity and are known to diminish budding attenuated NGC generation. An M2 construct comprising the membrane interactive domains, the transmembrane helix and the cytoplasmic helix, displayed enhanced ability to generate NGC, suggesting that other domains cooperatively promote membrane curvature. These studies establish the importance of M2-induced NGC during budding and suggest that antagonizing this curvature is a viable anti-influenza strategy.

Arginine in α-defensins: differential effects on bactericidal activity correspond to geometry of membrane curvature generation and peptide-lipid phase behavior.

The conserved tridisulfide array of the α-defensin family imposes a common triple-stranded ß-sheet topology on peptides that may have highly diverse primary structures, resulting in differential outcomes after targeted mutagenesis. In mouse cryptdin-4 (Crp4) and rhesus myeloid α-defensin-4 (RMAD4), complete substitutions of Arg with Lys affect bactericidal peptide activity very differently. Lys-for-Arg mutagenesis attenuates Crp4, but RMAD4 activity remains mostly unchanged. Here, we show that the differential biological effect of Lys-for-Arg replacements can be understood by the distinct phase behavior of the experimental peptide-lipid system. In Crp4, small-angle x-ray scattering analyses showed that Arg-to-Lys replacements shifted the induced nanoporous phases to a different range of lipid compositions compared with the Arg-rich native peptide, consistent with the attenuation of bactericidal activity by Lys-for-Arg mutations. In contrast, such phases generated by RMAD4 were largely unchanged. The concordance between small-angle x-ray scattering measurements and biological activity provides evidence that specific types of α-defensin-induced membrane curvature-generating tendencies correspond directly to bactericidal activity via membrane destabilization.

Translocation of HIV TAT peptide and analogues induced by multiplexed membrane and cytoskeletal interactions.

Cell-penetrating peptides (CPPs), such as the HIV TAT peptide, are able to translocate across cellular membranes efficiently. A number of mechanisms, from direct entry to various endocytotic mechanisms (both receptor independent and receptor dependent), have been observed but how these specific amino acid sequences accomplish these effects is unknown. We show how CPP sequences can multiplex interactions with the membrane, the actin cytoskeleton, and cell-surface receptors to facilitate different translocation pathways under different conditions. Using "nunchuck" CPPs, we demonstrate that CPPs permeabilize membranes by generating topologically active saddle-splay ("negative Gaussian") membrane curvature through multidentate hydrogen bonding of lipid head groups. This requirement for negative Gaussian curvature constrains but underdetermines the amino acid content of CPPs. We observe that in most CPP sequences decreasing arginine content is offset by a simultaneous increase in lysine and hydrophobic content. Moreover, by densely organizing cationic residues while satisfying the above constraint, TAT peptide is able to combine cytoskeletal remodeling activity with membrane translocation activity. We show that the TAT peptide can induce structural changes reminiscent of macropinocytosis in actin-encapsulated giant vesicles without receptors.

Squalamine as a broad-spectrum systemic antiviral agent with therapeutic potential.

Antiviral compounds that increase the resistance of host tissues represent an attractive class of therapeutic. Here, we show that squalamine, a compound previously isolated from the tissues of the dogfish shark (Squalus acanthias) and the sea lamprey (Petromyzon marinus), exhibits broad-spectrum antiviral activity against human pathogens, which were studied in vitro as well as in vivo. Both RNA- and DNA-enveloped viruses are shown to be susceptible. The proposed mechanism involves the capacity of squalamine, a cationic amphipathic sterol, to neutralize the negative electrostatic surface charge of intracellular membranes in a way that renders the cell less effective in supporting viral replication. Because squalamine can be readily synthesized and has a known safety profile in man, we believe its potential as a broad-spectrum human antiviral agent should be explored.

Criterion for amino acid composition of defensins and antimicrobial peptides based on geometry of membrane destabilization.

Defensins comprise a potent class of membrane disruptive antimicrobial peptides (AMPs) with well-characterized broad spectrum and selective microbicidal effects. By using high-resolution synchrotron small-angle X-ray scattering to investigate interactions between heterogeneous membranes and members of the defensin subfamilies, α-defensins (Crp-4), ß-defensins (HBD-2, HBD-3), and θ-defensins (RTD-1, BTD-7), we show how these peptides all permeabilize model bacterial membranes but not model eukaryotic membranes: defensins selectively generate saddle-splay ("negative Gaussian") membrane curvature in model membranes rich in negative curvature lipids such as those with phosphoethanolamine (PE) headgroups. These results are shown to be consistent with vesicle leakage assays. A mechanism of action based on saddle-splay membrane curvature generation is broadly enabling, because it is a necessary condition for processes such as pore formation, blebbing, budding, and vesicularization, all of which destabilize the barrier function of cell membranes. Importantly, saddle-splay membrane curvature generation places constraints on the amino acid composition of membrane disruptive peptides. For example, we show that the requirement for generating saddle-splay curvature implies that a decrease in arginine content in an AMP can be offset by an increase in both lysine and hydrophobic content. This "design rule" is consistent with the amino acid compositions of 1080 known cationic AMPs.

Small-angle X-ray scattering studies of peptide-lipid interactions using the mouse paneth cell α-defensin cryptdin-4.

In the presence of specialized proteins or peptides, a biological membrane can spontaneously restructure itself to allow communication between the intracellular and the extracellular sides. Examples of these proteins include cell-penetrating peptides and antimicrobial peptides (AMPs), which interact with cell membranes in complex ways. We briefly review cell-penetrating peptides and AMPs, and describe in detail how recombinant AMPs are made and their activity evaluated, using α-defensins as a specific example. We also review X-ray scattering methods used in studying peptide-membrane interactions, focusing on the procedures for small-angle X-ray scattering experiments on peptide-membrane interactions at realistic solution conditions, using both laboratory and synchrotron sources.