Stapled ß-Hairpin Antimicrobial Peptides with Improved Stability and Activity against Drug-Resistant Gram-Negative Bacteria.

Different stapling techniques have been used recently to address the subpar performance of antimicrobial peptides (AMPs) in clinical trials with ample focus on α-helical AMPs. In comparison, a systematic evaluation of such strategies on ß-hairpin AMPs is lacking. Herein, we report the design, synthesis, and evaluation of a library of all-hydrocarbon-stapled ß-hairpin AMPs with variation in key parameters intended as potent therapeutics against drug-resistant pathogens. We observed an interesting interplay between the activity, stability, and structural strength. Single-stapled peptides with a 6-carbon staple at peptide termini such as 5(c6) displayed the most potent activity against colistin-resistant clinical isolates. Using imaging techniques, we observed translocation of 5(c6) across bacterial membranes without causing extensive damage. Overall, we have engineered novel all-hydrocarbon-stapled ß-hairpin AMPs with structural and functional proficiency that can effectively combat resistant pathogens, with findings from this study a point of reference for future interests in developing novel ß-hairpin AMPs.

Manipulating turn residues on de novo designed ß-hairpin peptides for selectivity against drug-resistant bacteria.

Synthetic ß-hairpin antimicrobial peptides (AMPs) offer a useful source for the development of novel antimicrobial agents. ß-hairpin peptides generally consist of two side strands bridged by a reverse turn. In literature, most studies focused on the modifications of the side strands to manipulate the stability and activity of ß-hairpin peptides, and much less is known about the impact of the turn region. By designing a series of de novo ß-hairpin peptides with identical side strands but varied turns, we demonstrated that mutations of only 2 to 4 amino acids at the turn region could impart a wide range of antimicrobial profiles among synthetic ß-hairpin AMPs. BTT2-4 and BTT6 displayed selective potency against Gram-negative bacteria, with minimum inhibitory concentrations (MICs) of 4-8 µM. In contrast, BTT1 exhibited broad-spectrum activity, with MICs of 4-8 µM against both Gram-positive and Gram-negative strains. Additionally, BTT1 was potent against methicillin-resistant Staphylococcus aureus (MRSA) and colistin-resistant Enterobacterales. The antimicrobial potency of BTT1 persisted after 14 days of serial passage. Mechanistic studies revealed that interactions between lipopolysaccharide (LPS) and the peptides were critical to their membranolytic activity against the bacterial inner membrane. Aside from folding stability, we observed that a degree of conformational flexibility was required for disruptive membrane interactions. STATEMENT OF SIGNIFICANCE: By examining the significance of the turn region of ß-hairpin peptides, we present valuable knowledge to the design toolkit of novel antimicrobial peptides as alternative therapeutics to overcome antibiotic resistance. Our de novo designed synthetic peptides displayed selective activity against Gram-negative bacteria and potent activity against clinically relevant antibiotic-resistant strains (e.g. colistin-resistant Enterobacterales and methicillin-resistant Staphylococcus aureus). The bactericidal activity of our peptides was shown to be robust in the presence of proteolytic trypsin and saline, conditions that could suppress peptide activity. Our peptides were also determined to be non-cytotoxic against a human cell line.

Silica Nanoparticles-A Versatile Tool for the Treatment of Bacterial Infections.

The rapid emergence of drug resistance continues to outpace the development of new antibiotics in the treatment of infectious diseases. Conventional therapy is currently limited by drug access issues such as low intracellular drug accumulations, drug efflux by efflux pumps and/or enzymatic degradation. To improve access, targeted delivery using nanocarriers could provide the quantum leap in intracellular drug transport and retention. Silica nanoparticles (SiNPs) with crucial advantages such as large surface area, ease-of-functionalization, and biocompatibility, are one of the most commonly used nanoparticles in drug delivery applications. A porous variant, called the mesoporous silica nanoparticles (MSN), also confers additional amenities such as tunable pore size and volume, leading to high drug loading capacity. In the context of bacterial infections, SiNPs and its variants can act as a powerful tool for the targeted delivery of antimicrobials, potentially reducing the impact of high drug dosage and its side effects. In this review, we will provide an overview of SiNPs synthesis, its structural proficiency which is critical in loading and conjugation of antimicrobials and its role in different antimicrobial applications with emphasis on intracellular drug targeting in anti-tuberculosis therapy, nitric oxide delivery, and metal nanocomposites. The role of SiNPs in antibiofilm coatings will also be covered in the context of nosocomial infections and surgical implants.

Facile and efficient encapsulation of antimicrobial peptides via crosslinked DNA nanostructures and their application in wound therapy.

There is growing interest in the development of nucleic acid nanostructures as smart functional materials for applications in drug delivery. Inspired by the diverse physical interactions that exist in nature, crosslinked DNA nanostructures can serve as attractive affinity binding networks that interact with therapeutic cargos or living cells. Herein we report a strategy that addresses the challenges of topical oligopeptide therapy by exploiting high binding affinity between polyanionic DNA nanostructures and cationic antimicrobial peptides (AMPs) to fabricate hydrogels that release a model antimicrobial L12 peptide in response to pathogenic S. aureus infections. We further demonstrated controlled peptide release profiles via the DNA hydrogels that were biocompatible and delivered superior antimicrobial activity against nuclease-releasing susceptible and methicillin-resistant S. aureus infections. Single application of the L12-loaded DNA hydrogels on porcine explant S. aureus infections revealed potent efficacy after 24h. As a result of the capacity of the crosslinked DNA nanostructures to elicit a strong anti-inflammatory response, in vivo treatment of mice excision wounds translated into faster healing rates. Overall, the crosslinked DNA nanostructures reported in this study offer significant advantage as functional wound dressings and their future adaptation holds equally great promise for the delivery of cationic antimicrobials.

Phenylboronic Acid Functionalized Polycarbonate Hydrogels for Controlled Release of Polymyxin B in Pseudomonas Aeruginosa Infected Burn Wounds.

While physically crosslinked polycarbonate hydrogels are effective drug delivery platforms, their hydrophobic nature and lack of side chain functionality or affinity ligands for controlled release of hydrophilic drugs underscore the importance of their chemical compositions. This study evaluates an array of anionic hydrogel systems of phenylboronic acid functionalized triblock copolymers prepared via reversible physical interactions. Variation of key chemical functionalities while maintaining similar core structural features demonstrates the influence of the substitution position and protection of the boronic acid functionality on gel viscoelasticity and mechanical strength at physiological pH. The optimum gel systems obtained from the meta-substituted copolymers (m-PAP) are stable at physiological pH and nontoxic to mammalian dermal cells. The polymyxin B loaded m-PAP hydrogels demonstrate controlled in vitro drug release kinetics and in vitro antimicrobial activity against Pseudomonas aeruginosa over 48 h. In vivo antimicrobial efficacy of the drug loaded hydrogels further corroborates the in vitro results, demonstrating sustained antimicrobial activity against P. aeruginosa burn wound infections. The current strategy described in this study demonstrates a straightforward approach in designing physiologically relevant boronic acid hydrogel systems for controlled release of cationic antimicrobials for future clinical applications.

Studying in vitro phagocytosis of apoptotic cancer cells by recombinant GMCSF-treated RAW 264.7 macrophages.

Granulocyte macrophage colony stimulating factor (GMCSF), a therapeutically important cytokine that helps in the proliferation of macrophages, was recombinantly expressed in E. coli BL21 and purified as a GST-tagged protein. Cell viability assay demonstrated significant enhancement in proliferation of RAW 264.7 (murine macrophage) in presence of GMCSF. In vitro activation of macrophages was carried out by lipopolysaccharide (LPS) or pyrogallol and probed by the generation of reactive oxygen species (ROS). Following the induction of apoptosis in A549 lung cancer cells with anticancer drug cisplatin (at 25µM), apoptotic cancer cells were effectively phagocytosed by the recombinant GMCSF-treated and exogenously activated RAW 264.7 cells as observed in fluorescence microscopic images. The current findings attribute possible role of GMCSF as adjuvant in scavenging treated cancer cells.