In Situ Forming, Enzyme-Responsive Peptoid-Peptide Hydrogels: An Advanced Long-Acting Injectable Drug Delivery System.

Long-acting drug delivery systems are promising platforms to improve patient adherence to medication by delivering drugs over sustained periods and removing the need for patients to comply with oral regimens. This research paper provides a proof-of-concept for the development of a new optimized in situ forming injectable depot based on a tetrabenzylamine-tetraglycine-d-lysine-O-phospho-d-tyrosine peptoid-D-peptide formulation ((NPhe)4GGGGk(AZT)y(p)-OH). The chemical versatility of the peptoid-peptide motif allows low-molecular-weight drugs to be precisely and covalently conjugated. After subcutaneous injection, a hydrogel depot forms from the solubilized peptoid-peptide-drug formulation in response to phosphatase enzymes present within the skin space. This system is able to deliver clinically relevant concentrations of a model drug, the antiretroviral zidovudine (AZT), for 35 days in Sprague-Dawley rats. Oscillatory rheology demonstrated that hydrogel formation began within ∼30 s, an important characteristic of in situ systems for reducing initial drug bursts. Gel formation continued for up to ∼90 min. Small-angle neutron scattering data reveal narrow-radius fibers (∼0.78-1.8 nm) that closely fit formation via a flexible cylinder elliptical model. The inclusion of non-native peptoid monomers and D-variant amino acids confers protease resistance, enabling enhanced biostability to be demonstrated in vitro. Drug release proceeds via hydrolysis of an ester linkage under physiological conditions, releasing the drug in an unmodified form and further reducing the initial drug burst. Subcutaneous administration of (NPhe)4GGGGk(AZT)y(p)-OH to Sprague-Dawley rats resulted in zidovudine blood plasma concentrations within the 90% maximal inhibitory concentration (IC90) range (30-130 ng mL-1) for 35 days.

Metal Cross-Linked Supramolecular Gel Noodles: Structural Insights and Antibacterial Assessment.

Achieving precise control over gelator alignment and morphology is crucial for crafting tailored materials and supramolecular structures with distinct properties. We successfully aligned the self-assembled micelles formed by a functionalized dipeptide 2NapFF into long 1-D "gel noodles" by cross-linking with divalent metal chlorides. We identify the most effective cross-linker for alignment, enhancing mechanical stability, and imparting functional properties. Our study shows that Group 2 metal ions are particularly suited for creating mechanically robust yet flexible gel noodles because of their ionic and nondirectional bonding with carboxylate groups. In contrast, the covalent nature and high directional bonds of d-block metal ions with carboxylates tend to disrupt the self-assembly of 2NapFF. Furthermore, the 2NapFF-Cu noodles demonstrated selective antibacterial activity, indicating that the potent antibacterial property of the copper(II) ion is preserved within the cross-linked system. By merging insights into molecular alignment, gel extrusion processing, and integrating specific functionalities, we illustrate how the versatility of dipeptide-based gels can be utilized in creating next-generation soft materials.

Unravelling the antimicrobial activity of peptide hydrogel systems: current and future perspectives.

The use of hydrogels has garnered significant interest as biomaterial and drug delivery platforms for anti-infective applications. For decades antimicrobial peptides have been heralded as a much needed new class of antimicrobial drugs. Self-assembling peptide hydrogels with inherent antimicrobial ability have recently come to the fore. However, their fundamental antimicrobial properties, selectivity and mechanism of action are relatively undefined. This review attempts to establish a link between antimicrobial efficacy; the self-assembly process; peptide-membrane interactions and mechanical properties by studying several reported peptide systems: ß-hairpin/ß-loop peptides; multidomain peptides; amphiphilic surfactant-like peptides and ultrashort/low molecular weight peptides. We also explore their role in the formation of amyloid plaques and the potential for an infection etiology in diseases such as Alzheimer's. We look briefly at innovative methods of gel characterization. These may provide useful tools for future studies within this increasingly important field.

Ultrashort self-assembling Fmoc-peptide gelators for anti-infective biomaterial applications.

Biomaterial-related infections have a significant impact on society and are a major contributor to the growing threat of antimicrobial resistance. Current licensed antibiotic classes struggle to breakdown or penetrate the exopolysaccharide biofilm barrier, resulting in sub-therapeutic concentrations of antibiotic at the surface of the biomaterial, treatment failure and increased spread of resistant isolates. This paper focuses for the first time on the ability of ultrashort Fmoc-peptide gelators to eradicate established bacterial biofilms implicated in a variety of medical device infections (Gram-positive: Staphylococcus aureus, Staphylococcus epidermidis and Gram-negative Escherichia coli, Pseudomonas aeruginosa). The effect of increasing the cationicity of FmocFF via addition of di-lysine and di-orntithine was also studied with regard to antibacterial activity. Our studies demonstrated that Fmoc-peptides (FmocFF, FmocFFKK, FmocFFFKK, FmocFFOO) formed surfactant-like soft gels at concentrations of 1% w/v and above using a method of glucono-δ-Lactone pH induction. The majority of Fmoc-peptides (0.5-2% w/v) demonstrated selective action against established (grown for 24 h) biofilms of Gram-positive and Gram-negative pathogens. These results are likely to increase the clinical translation of short-peptide gelator platforms within the area of anti-infective biomaterials including as wound dressings and coatings for prostheses, catheters, heart valves and surgical tubes. In the long term, this will lead to wider treatment choices for clinicians and patients involved in the management of medical device infections and reduce the burden of antimicrobial resistance. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Anti-biofilm activity of ultrashort cinnamic acid peptide derivatives against medical device-related pathogens.

The threat of antimicrobial resistance has placed increasing emphasis on the development of innovative approaches to eradicate multidrug-resistant pathogens. Biofilm-forming microorganisms, for example, Staphylococcus epidermidis and Staphylococcus aureus, are responsible for increased incidence of biomaterial infection, extended hospital stays and patient morbidity and mortality. This paper highlights the potential of ultrashort tetra-peptide conjugated to hydrophobic cinnamic acid derivatives. These peptidomimetic molecules demonstrate selective and highly potent activity against resistant biofilm forms of Gram-positive medical device-related pathogens. 3-(4-Hydroxyphenyl)propionic)-Orn-Orn-Trp-Trp-NH2 displays particular promise with minimum biofilm eradication concentration (MBEC) values of 125 µg/ml against methicillin sensitive (ATCC 29213) and resistant (ATCC 43300) S. aureus and activity shown against biofilm forms of Escherichia coli (MBEC: 1000 µg/ml). Kill kinetics confirms complete eradication of established 24-h biofilms at MBEC with 6-h exposure. Reduced cell cytotoxicity, relative to Gram-positive pathogens, was proven via tissue culture (HaCaT) and haemolysis assays (equine erythrocytes). Existing in nature as part of the immune response, antimicrobial peptides display great promise for exploitation by the pharmaceutical industry in order to increase the library of available therapeutic molecules. Ultrashort variants are particularly promising for translation as clinical therapeutics as they are more cost-effective, easier to synthesise and can be tailored to specific functional requirements based on the primary sequence allowing factors such as spectrum of activity to be varied.

Ultrashort cationic naphthalene-derived self-assembled peptides as antimicrobial nanomaterials.

Self-assembling dipeptides conjugated to naphthalene show considerable promise as nanomaterial structures, biomaterials, and drug delivery devices. Biomaterial infections are responsible for high rates of patient mortality and morbidity. The presence of biofilm bacteria, which thrive on implant surfaces, are a huge burden on healthcare budgets, as they are highly resistant to current therapeutic strategies. Ultrashort cationic self-assembled peptides represent a highly innovative and cost-effective strategy to form antibacterial nanomaterials. Lysine conjugated variants display the greatest potency with 2% w/v NapFFKK hydrogels significantly reducing the viable Staphylococcus epidermidis biofilm by 94%. Reducing the size of the R-group methylene chain on cationic moieties resulted in reduction of antibiofilm activity. The primary amine of the protruding R-group tail may not be as readily available to interact with negatively charged bacterial membranes. Cryo-SEM, FTIR, CD spectroscopy, and oscillatory rheology provided evidence of supramolecular hydrogel formation at physiological pH (pH 7.4). Cytotoxicity assays against murine fibroblast (NCTC 929) cell lines confirmed the gels possessed reduced cytotoxicity relative to bacterial cells, with limited hemolysis upon exposure to equine erythrocytes. The results presented in this paper highlight the significant potential of ultrashort cationic naphthalene peptides as future biomaterials.

Two into one does go: Formulation development of a multipurpose combination vaginal ring releasing dapivirine and metronidazole for prevention of HIV infection and treatment of bacterial vaginosis.

Bacterial vaginosis (BV) is a common but often asymptomatic dysbiosis of the human vagina characterized by an imbalance in the normal vaginal microbiota due to loss of lactobacilli and an overgrowth of certain anaerobic bacteria. While BV itself is not a sexually transmitted infection, it is associated with an increased risk in women of various sexually acquired infections, including human immunodeficiency virus (HIV) infection. There is, therefore, a strong rationale for pursuing new multipurpose products that seek to treat or prevent BV alongside preventing HIV infection. With the dapivirine-releasing vaginal ring for HIV prevention now approved in several African countries, here we report formulation development of a next-generation ring product that releases both dapivirine (DPV) and the antibiotic drug metronidazole (MET). Following thermal analysis studies to characterize the phase behaviour of DPV-MET mixtures and rheological analysis to assess the cure characteristics of the active silicone elastomer mixes, matrix-type rings were manufactured containing 25 or 200 mg DPV in combination with 100, 250, 500, 1000 or 2000 mg MET. The results for drug content, in vitro release, mechanical testing, and Gardnerella vaginalis time-kill experiments demonstrate the feasibility of incorporating both DPV and MET in a matrix-type ring formulation and indicate that clinically effective release rates may be possible.

Enzyme-Triggered l-α/d-Peptide Hydrogels as a Long-Acting Injectable Platform for Systemic Delivery of HIV/AIDS Drugs.

Eradicating HIV/AIDS by 2030 is a central goal of the World Health Organization. Patient adherence to complicated dosage regimens remains a key barrier. There is a need for convenient long-acting formulations that deliver drugs over sustained periods. This paper presents an alternative platform, an injectable in situ forming hydrogel implant to deliver a model antiretroviral drug (zidovudine [AZT]) over 28 days. The formulation is a self-assembling ultrashort d or l-α peptide hydrogelator, namely phosphorylated (naphthalene-2-ly)-acetyl-diphenylalanine-lysine-tyrosine-OH (NapFFKY[p]-OH), covalently conjugated to zidovudine via an ester linkage. Rheological analysis demonstrates phosphatase enzyme instructed self-assembly, with hydrogels forming within minutes. Small angle neutron scattering data suggest hydrogels form narrow radius (≈2 nm), large length fibers closely fitting the flexible cylinder elliptical model. d-Peptides are particularly promising for long-acting delivery, displaying protease resistance for 28 days. Drug release, via hydrolysis of the ester linkage, progress under physiological conditions (37 °C, pH 7.4, H2 O). Subcutaneous administration of Napffk(AZT)Y[p]G-OH in Sprague Dawley rats demonstrate zidovudine blood plasma concentrations within the half maximal inhibitory concentration (IC50 ) range (30-130 ng mL-1 ) for 35 days. This work is a proof-of-concept for the development of a long-acting combined injectable in situ forming peptide hydrogel implant. These products are imperative given their potential impact on society.

Rapidly dissolving microneedle patch of amphotericin B for intracorneal fungal infections.

Chronic fungal infection of the cornea could lead to blindness if not treated properly. Topical amphotericin B (AMP-B) is considered the first treatment of choice for ocular fungal infection. However, factors related to its poor solubility and penetration through intact cornea lead to poor bioavailability. Microneedles (MNs) are emerging as a minimally invasive method to enhance ocular drug delivery. This study aims to investigate the potential use of biodegradable poly(vinylpyrrolidone) (PVP) and hyaluronic acid (HA)-based rapidly dissolving MNs for delivery of AMP-B to treat fungal infection. The data obtained illustrates PVP/HA MN arrays' reproducibility, good mechanical strength, and faster dissolution with 100% drug recovery. Multiphoton microscopic results revealed that MNs successfully penetrate the corneal tissue and enhance AMP-B permeation through corneal layers. Furthermore, PVP/HA MN arrays showed high solubility. Both PVP and HA successfully decreased AMP-B cytotoxicity when compared to free drug. More interestingly, the biocompatible MN formulations preserved the antifungal activity of AMP-B, as demonstrated by significant inhibition of fungal growth. Therefore, this study shows the feasibility of ocular delivery of the poorly soluble AMP-B using a fast-dissolving MN patch.

Injectable Depot Forming Thermoresponsive Hydrogel for Sustained Intrascleral Delivery of Sunitinib Using Hollow Microneedles.

Purpose: Age-related macular degeneration is a vision-threatening disorder affecting the posterior segment of the eye. Drug delivery to the posterior segment is challenging owing to the complex anatomical and physiological structure, necessitating monthly injections of antivascular endothelial growth factors. Thermoresponsive hydrogels provide sustained drug delivery and ease of injection, due to their sol-gel transition. Poly (N-isopropyl acrylamide) (PNIPAAm) is a widely researched thermoresponsive hydrogel; however, insufficient wet strength and a wide mesh network make it inept for the entrapment of small molecules. Methods: A novel approach of grafting PNIPAAm with chitosan is exploited. A chitosan concentration altered in 10%, 30%, and 50% compared to PNIPAAm is investigated for entrapment of a small-molecular weight, hydrophilic drug, sunitinib (SUN), a multiple tyrosine kinase receptor inhibitor. Furthermore, these hydrogels were characterized using 1H-NMR, FTIR, differential scanning calorimetry (DSC), and thermogravimetric analysis for chemical characterization and viscosity, swellability, syringeability, degradation, and In-vitro permeation using Franz-diffusion cell. Results: In-vitro drug release kinetics suggested that the release of SUN could be controlled with the percentage of chitosan grafting; however, gel strength (3%-5% w/v) of 30% Cs-g-PNIPAAm did not significantly affect percentage drug release. Sustained release of SUN was observed for 1 month. In-vitro permeation studies on porcine sclera suggested that a thermoresponsive gel of chitosan grafted PNIPAAm (Cs-g-PNIPAAm) was able to sustain the drug release by 40%, compared to SUN solution. Conclusions: The study indicates that the synthesized Cs-g-NIPAAm hydrogel has the potential to serve as a tailorable injectable platform for intrascleral drug delivery applications.

The potential of antimicrobial peptides as biocides.

Antimicrobial peptides constitute a diverse class of naturally occurring antimicrobial molecules which have activity against a wide range of pathogenic microorganisms. Antimicrobial peptides are exciting leads in the development of novel biocidal agents at a time when classical antibiotics are under intense pressure from emerging resistance, and the global industry in antibiotic research and development stagnates. This review will examine the potential of antimicrobial peptides, both natural and synthetic, as novel biocidal agents in the battle against multi-drug resistant pathogen infections.

Ultrashort Peptide Hydrogels Display Antimicrobial Activity and Enhance Angiogenic Growth Factor Release by Dental Pulp Stem/Stromal Cells.

Recent studies on peptide hydrogels have shown that ultrashort peptides (<8 amino acids) can self-assemble into hydrogels. Ultrashort peptides can be designed to incorporate antimicrobial motifs, such as positively charged lysine residues, so that the peptides have inherent antimicrobial characteristics. Antimicrobial hydrogels represent a step change in tissue engineering and merit further investigation, particularly in applications where microbial infection could compromise healing. Herein, we studied the biocompatibility of dental pulp stem/stromal cells (DPSCs) with an ultrashort peptide hydrogel, (naphthalene-2-ly)-acetyl-diphenylalanine-dilysine-OH (NapFFεKεK-OH), where the epsilon (ε) amino group forms part of the peptide bond rather than the standard amino grouping. We tested the antimicrobial properties of NapFFεKεK-OH in both solution and hydrogel form against Staphylococcus aureus, Enterococcus faecalis and Fusobacterium nucleatum and investigated the DPSC secretome in hydrogel culture. Our results showed NapFFεKεK-OH hydrogels were biocompatible with DPSCs. Peptides in solution form were efficacious against biofilms of S. aureus and E. faecalis, whereas hydrogels demonstrated antimicrobial activity against E. faecalis and F. nucleatum. Using an angiogenic array we showed that DPSCs encapsulated within NapFFεKεK-OH hydrogels produced an angiogenic secretome. These results suggest that NapFFεKεK-OH hydrogels have potential to serve as novel hydrogels in tissue engineering for cell-based pulp regeneration.

Multicomponent Peptide Hydrogels as an Innovative Platform for Cell-Based Tissue Engineering in the Dental Pulp.

In light of the increasing levels of antibiotic resistance, nanomaterials and novel biologics are urgently required to manage bacterial infections. To date, commercially available self-assembling peptide hydrogels have not been studied extensively for their ability to inhibit micro-organisms relevant to tissue engineering sites such as dental root canals. In this work, we assess the biocompatibility of dental pulp stem/stromal cells with commercially available multicomponent peptide hydrogels. We also determine the effects of dental pulp stem/stromal cell (DPSC) culture in hydrogels on growth factor/cytokine expression. Furthermore, to investigate novel aspects of self-assembling peptide hydrogels, we determine their antimicrobial activity against the oral pathogens Staphylococcus aureus, Enterococcus faecalis, and Fusobacterium nucleatum. We show that self-assembling peptide hydrogels and hydrogels functionalized with the adhesion motif Arg-Gly-Asp (RGD) are biocompatible with DPSCs, and that cells grown in 3D hydrogel cultures produce a discrete secretome compared with 2D-cultured cells. Furthermore, we show that soluble peptides and assembled hydrogels have antimicrobial effects against oral pathogens. Given their antibacterial activity against oral pathogens, biocompatibility with dental pulp stem/stromal cells and enhancement of an angiogenic secretome, multicomponent peptide hydrogels hold promise for translational use.

Peptide Nanomaterials for Drug Delivery Applications.

Self-assembled peptides have been shown to form well-defined nanostructures which display outstanding characteristics for many biomedical applications and especially in controlled drug delivery. Such biomaterials are becoming increasingly popular due to routine, standardized methods of synthesis, high biocompatibility, biodegradability and ease of upscale. Moreover, one can modify the structure at the molecular level to form various nanostructures with a wide range of applications in the field of medicine. Through environmental modifications such as changes in pH and ionic strength and the introduction of enzymes or light, it is possible to trigger self-assembly and design a host of different self-assembled nanostructures. The resulting nanostructures include nanotubes, nanofibers, hydrogels and nanovesicles which all display a diverse range of physico-chemical and mechanical properties. Depending on their design, peptide self-assembling nanostructures can be manufactured with improved biocompatibility and in vivo stability and the ability to encapsulate drugs with the capacity for sustained drug delivery. These molecules can act as carriers for drug molecules to ferry cargo intracellularly and respond to stimuli changes for both hydrophilic and hydrophobic drugs. This review explores the types of self-assembling nanostructures, the effects of external stimuli on and the mechanisms behind the assembly process, and applications for such technology in drug delivery.

Pharmaceutical Formulation and Characterization of Dipeptide Nanotubes for Drug Delivery Applications.

Peptide nanotubes are promising materials for a variety of biomedical applications with ultrashort (≤7 amino acids) forms providing particular promise for clinical translation. The manufacture of peptide nanotubes has, however, been associated with toxic organic solvents restricting clinical use. The purpose of this work is to formulate dipeptide nanotubes using mild techniques easily translated to industrial upscale and to characterize their physiochemical and biological properties. Phenylalanine-phenylalanine variants can be successfully formulated using distilled water as demonstrated here. Formulations are homogenous in shape (tubular), with apparent size (50-260 nm) and a zeta potential of up to +30 mV. L-(H2 N-FF-COOH), and D-enantiomers (H2 N-ff-COOH) demonstrate no toxicity against glioblastoma cells and are explored for ability to deliver a model hydrophilic molecule, sodium fluorescein, at pH 5.5 (tumor) and 7.4 (physiological). Peptide nanotubes loaded with >85% sodium fluorescein, demonstrate burst release characteristics, fitting the Weibull model of drug release. This research provides important data contributing to the pharmaceutical formulation of peptide nanotubes as drug delivery platforms for hydrophilic drugs.

Tuning the antimicrobial activity of low molecular weight hydrogels using dopamine autoxidation.

We present a method to trigger the formation of dipeptide-based hydrogels by the simple addition of dopamine. Dopamine undergoes oxidation in air, reducing the pH to induce gelation. The production of polydopamine and release of reactive oxygen species such as hydrogen peroxide confers antimicrobial activity. Gel stiffness can be controlled by modulating the initial starting pH of the gelator solution. We can use this method to tune the antimicrobial activity of the gels, with gels that are less stiff demonstrating increased bactericidal efficacy against Gram-positive bacteria.

High-throughput toxicity screening of novel azepanium and 3-methylpiperidinium ionic liquids.

Ionic liquids (ILs) have been employed as potentially environmentally friendly replacements for harmful organic solvents, but have also been studied for their use in bioelectrochemical applications, such as in microbial electrochemistry for bioenergy production, or in industrial biocatalysis. For these processes, low microbial toxicity is important and there is a growing need for microbial toxicology studies for novel ILs. In this study, we report initial toxicity data for novel ILs, based on azepanium and 3-methylpiperidinium cations. Agar disc diffusion assays are used, along with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determinations, to obtain rapid and inexpensive initial toxicity data for these novel ILs against Escherichia coli and Staphylococcus epidermidis. Many of the novel ILs characterised possess low microbial toxicity relative to well-studied ILs, highlighting their potential for further study in applications where this is a desirable property.

Development of a teaching model to advance skills in industrial pharmaceutical formulation and regulatory aspects.

BACKGROUND AND PURPOSE: To design and critically evaluate a laboratory-scale pharmaceutical formulation practical that enables pharmaceutical science students to develop work-based skills relating to industrial pharmacy such as problem solving, pharmaceutical calculations, research, legal checking, communication, practical aptitude, handling of medicinal products, record keeping, and ability to interpret, analyze and report data. EDUCATIONAL ACTIVITY AND SETTING: Nine laboratory practical sessions were designed whereby students formulated a range of dosage forms and conducted corrective and preventative actions (CAPAs) exercises. A master batch formula outlined the specifications for each product and a practical sheet was provided for the students to record their activities. Student evaluation of the exercise was performed via a self-administered 17-item questionnaire in the final week. FINDINGS: Twenty-four students completed the workshops over two year groups (2015 year group n = 11, 2016 year group n = 13). The mean score across both groups was 15.6 out of 20. DISCUSSION: The questionnaire had a 100% response rate and the majority of students agreed that the classes were a useful teaching method and that they fostered key skills required for pharmaceutical formulation and regulation. SUMMARY: Laboratory classes effectively delivered course content relating to industrial-based pharmaceutical formulation and helped to develop relevant skills.

Self-assembling diphenylalanine peptide nanotubes selectively eradicate bacterial biofilm infection.

Biofilms present a major problem to industry and healthcare worldwide. Composed of a population of surface-attached microbial cells surrounded by a protective extracellular polysaccharide matrix, they are responsible for increased tolerance to antibiotics, treatment failure and a resulting rise in antimicrobial resistance. Here we demonstrate that self-assembled peptide nanostructures composed of a diphenylalanine motif provide sufficient antibacterial activity to eradicate mature biofilm forms of bacteria widely implicated in hospital infections. Modification of terminal functional groups to amino (-NH2), carboxylic acid (-COOH) or both modalities, and switch to d-isomers, resulted in changes in antibacterial selectivity and mammalian cell toxicity profiles. Of the three peptide nanotubes structures studied (NH2-FF-COOH, NH2-ff-COOH and NH2-FF-NH2), NH2-FF-COOH demonstrated the most potent activity against both planktonic (liquid, free-floating) and biofilm forms of bacteria, possessing minimal mammalian cell toxicity. NH2-FF-COOH resulted in greater than 3 Log10 CFU/mL viable biofilm reduction (>99.9%) at 5 mg/mL and total biofilm kill at 10 mg/mL against Staphylococcus aureus after 24 h exposure. Scanning electron microscopy proved that antibiofilm activity was primarily due to the formation of ion channels and/or surfactant-like action, with NH2-FF-COOH and NH2-ff-COOH capable of degrading the biofilm matrix and disrupting cell membranes, leading to cell death in Gram-positive bacterial isolates. Peptide-based nanotubes are an exciting platform for drug delivery and engineering applications. This is the first report of using peptide nanotubes to eradicate bacterial biofilms and provides evidence of a new platform that may alleviate their negative impact throughout society. STATEMENT OF SIGNIFICANCE: We outline, for the first time, the antibiofilm activity of diphenylalanine (FF) peptide nanotubes. Biofilm bacteria exhibit high tolerance to antimicrobials 10-10,000 times that of free-flowing planktonic forms. Biofilm infections are difficult to treat using conventional antimicrobial agents, leading to a rise in antimicrobial resistance. We discovered nanotubes composed of NH2-FF-COOH demonstrated potent activity against staphylococcal biofilms implicated in hospital infections, resulting in complete kill at concentrations of 10 mg/mL. Carboxylic acid terminated FF nanotubes were able to destroy the exopolysaccharide architecture of staphylococcal biofilms expressing minimal toxicity, highlighting their potential for use in patients. Amidated (NH2-FF-NH2) forms demonstrated reduced antibiofilm efficacy and significant toxicity. These results contribute significantly to the development of innovative antibacterial technologies and peptide nanomaterials.

Ultrashort Self-Assembling Peptide Hydrogel for the Treatment of Fungal Infections.

The threat of antimicrobial resistance to society is compounded by a relative lack of new clinically effective licensed therapies reaching patients over the past three decades. This has been particularly problematic within antifungal drug development, leading to a rise in fungal infection rates and associated mortality. This paper highlights the potential of an ultrashort peptide, (naphthalene-2-ly)-acetyl-diphenylalanine-dilysine-OH (NapFFKK-OH), encompassing hydrogel-forming and antifungal properties within a single peptide motif, thus overcoming formulation (e.g., solubility, drug loading) issues associated with many currently employed highly hydrophobic antifungals. A range of fungal susceptibility (colony counts) and cell cytotoxicity (MTS cell viability, LIVE/DEAD staining® with fluorescent microscopy, haemolysis) assays were employed. Scanning electron microscopy confirmed the nanofibrous architecture of our self-assembling peptide, existing as a hydrogel at concentrations of 1% w/v and above. Broad-spectrum activity was demonstrated against a range of fungi clinically relevant to infection (Aspergillus niger, Candida glabrata, Candida albicans, Candida parapsilosis and Candida dubliniensis) with greater than 4 log10 CFU/mL reduction at concentrations of 0.5% w/v and above. We hypothesise antifungal activity is due to targeting of anionic components present within fungal cell membranes resulting in membrane disruption and cell lysis. NapFFKK-OH demonstrated reduced toxicity against mammalian cells (NCTC 929, ARPE-19) suggesting increased selectivity for fungal cells. However, further studies relating to safety for systemic administration is required, given the challenges toxicity has presented in the wider context of antimicrobial peptide drug development. Overall this study highlights the promise of NapFFKK-OH hydrogels, particularly as a topical formulation for the treatment of fungal infections relating to the skin and eyes, or as a hydrogel coating for the prevention of biomaterial related infection.