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.

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.

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.

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.

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.