Deirdre Fitzgerald-Hughes

As antibiotic resistant infections become ever more prevalent, the WHO has designated the antimicrobial resistance (AMR) crisis as a silent epidemic. With over 1.1 million deaths directly caused by AMR in 2021 and a further 4.7 million associated deaths annually, there has never been a more pertinent time to investigate novel drug modalities and methods to tackle these challenging infections. Herein, we report a series of antimicrobial photocatalysts ( 1 – 4 ), which exhibit low-nanomolar activity against a panel of pathogenic reference strains, including all six ESKAPE pathogens and minimal off-target toxicity towards mammalian cells in-vitro. These catalysts have been extensively screened for their photoredox and biological properties, where we have used a series of spectroscopic techniques in combination with cellular assays, chemoproteomics, and super-resolution microscopy to uncover the mechanistic actions of these agents. They act primarily through intracellular NADH depletion, and induce biomolecular damage - located primarily at protein histidine residues. Furthermore, given the excellent antibacterial activity of this class of complexes we developed a targeted bioconjugate of our lead, 1 which shows augmented activity against ESKAPE pathogens. This work provides further evidence toward the utility of rationally designed photocatalysts in the fight against AMR.

In this study, we synthesised and characterised a copper(II) naphthalenediimide-phenanthroline (NDI-Phen) compound and the corresponding copper complex. The photophysical properties of both the copper complex, Cu(NDI-Phen)Cl₂ (Cu1) and the N-octyl-N-[1,10]phenanthroline-1,4,5,8-naphthalenetetracarboxylic-diimide (NDI-Phen, L1) were probed using laser flash photolysis on the nano to microsecond timescales. The biological properties of the Cu1 complex and L1 were evaluated based on their interactions with DNA, bovine serum albumin (BSA), and their antimicrobial properties against clinically important pathogens were determined. The binding affinity of the Cu1 complex with calf thymus DNA (CT DNA) was evaluated using UV-Vis absorption titrations, and fluorescence quenching measurements. The results indicate a stronger intercalative binding mode for the copper complex, compared to L1, suggesting enhanced stabilisation with the DNA helix. The interaction with BSA was also studied for the Cu1 complex, which exhibited a higher quenching and binding constant compared to L1. This suggests a stronger and more stable interaction with the protein, which may improve its bioavailability and resistance to degradation under physiological conditions. The Cu1 complex demonstrated superior antibacterial activity when compared to L1. These findings suggest that complexation of the NDI-Phen unit with a copper centre significantly improves its potential as an antibacterial agent.

This work presents the synthesis, characterisation, photophysical properties, time-resolved spectroscopic behaviour, and biological evaluation of two structurally distinct heavy-atom-free BODIPY-anthracene dyads (BDP-1) and the newly designed 2,6-bis[1-(tert-butyl) 4-(prop-2-yn-1-yl) piperazine-1,4-dicarboxylate] BODIPY-anthracene (BDP-2), incorporating 2,6-alkynyl-piperazine substituents for potential application in antimicrobial photodynamic therapy. BDP-1 exhibits absorption and emission maxima at 507 nm and 516 nm, respectively, with a Stokes shift of 344 cm⁻¹ in dichloromethane (DCM), characteristic of unsubstituted BODIPYs. In contrast, BDP-2 undergoes a red-shift in the absorption maximum to 552 nm (Stokes shift of 633 cm⁻¹), which is attributed to the extended conjugation from the introduction of the alkyne groups. Time-resolved infrared spectroscopy confirmed efficient spin-orbit charge transfer intersystem crossing, and nanosecond transient absorption studies confirmed the formation of a long-lived triplet state for BDP-2 (up to 138 µs in MeCN). A binding constant (K_b) of 9.6 × 10⁴ M⁻¹ was obtained for BDP-2 when titrated with bovine serum albumin (BSA), which is higher than comparable BODIPY derivatives. BDP-2 displayed improved hemocompatibility compared to BDP-1 (−1). Antimicrobial activity of BDP-1 and BDP-2 was most potent when irradiated at 370 nm compared to the other wavelengths employed. However, BDP-2 did not retain the potent (6 log) and rapid (within 15 min) eradication of Staphylococcus aureus achieved by BDP-1 under irradiation at 370 nm. These findings demonstrate the rational design of BDP-2 as a biocompatible, and heavy-atom-free BODIPY offering promise for targeted antimicrobial photodynamic therapeutic applications.

This study presents the development and characterization of a novel porphyrin-Jeffamine polymer conjugate designed to function as a photosensitizer prodrug for antimicrobial photodynamic therapy (aPDT). The conjugate features a photosensitive porphyrin unit covalently attached to a biocompatible polymer backbone, with enhanced solubility, stability, and bioavailability compared to those of the free porphyrin derivatives. The photophysical properties were studied using transient absorption spectroscopy spanning the fs-μs time scales in addition to emission studies. The production of reactive oxygen species upon photoactivation enabled effective bacterial cell killing. Spectroscopic studies confirmed strong binding of the conjugate to DNA through intercalation, likely disrupting DNA replication and transcription. Interaction studies with bovine serum albumin demonstrated substantial serum protein binding, which may positively impact the pharmacokinetics and biodistribution. Overall, this porphyrin-polymer conjugate offers a multifunctional theranostic platform, combining antimicrobial action with DNA and protein binding potential, positioning it as a promising candidate for aPDT and bioimaging applications.

Hydrogels with low toxicity, antimicrobial potency and shear-thinning behavior are promising materials to combat the modern challenges of increased infections. Here, we report on 8-arm star block copolypeptides based on poly(L-lysine), poly(L-tyrosine) and poly(S-benzyl-L-cysteine) blocks. Three star block copolypeptides were synthesized with poly(S-benzyl-L-cysteine) always forming the outer block. The inner block comprised either two individual blocks of poly(L-lysine) and poly(L-tyrosine) or a statistical block copolypeptide from both amino acids. The star block copolypeptides were synthesized by the Ring Opening Polymerization (ROP) of the protected amino acid N-carboxyanhydrides (NCAs), keeping the overall ratio of monomers constant. All star block copolypeptides formed hydrogels and Scanning Electron Microscopy (SEM) confirmed a porous morphology. The investigation of their viscoelastic characteristics, water uptake and syringe extrudability revealed superior properties of the star polypeptide with a statistical inner block of L-lysine and L-tyrosine. Further testing of this sample confirmed no cytotoxicity and demonstrated antimicrobial activity of 1.5-log and 2.6-log reduction in colony-forming units, CFU/mL, against colony-forming reference laboratory strains of Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus, respectively. The results underline the importance of controlling structural arrangements in polypeptides to optimize their physical and biological properties.

Introduction. As growing numbers of patients are at higher risk of infection, novel topical broad-spectrum antimicrobials are urgently required for wound infection management. Robust pre-clinical studies should support the development of such novel antimicrobials. Gap statement. To date, evidence of robust investigation of the cytotoxicity and antimicrobial spectrum of activity of antimicrobial peptides (AMP)s is lacking in published literature. Using a more clinical lens, we address this gap in experimental approach, building on our experience with poly-l-lysine (PLL)-based AMP polymers. Aim. To evaluate the in vitro bactericidal activity and cytotoxicity of a PLL-based 16-armed star AMP polymer, designated 16-PLL10, as a novel candidate antimicrobial. Methods. Antimicrobial susceptibilities of clinical isolates and reference strains of ESKAPE (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) pathogens, to 16-PLL10 were investigated. Human erythrocyte haemolysis and keratinocyte viability assays were used to assess toxicity. Modifications were made to 16-PLL10 and re-evaluated for improvement. Results. Minimum bactericidal concentration of 16-PLL10 ranged from 1.25 µM to ≥25 µM. At 2.5 µM, 16-PLL10 was broadly bactericidal against ESKAPE strains/wound isolates. Log-reduction in colony forming units (c.f.u.) per millilitre after 1 h, ranged from 0.3 (E. cloacae) to 5.6 (K. pneumoniae). At bactericidal concentrations, 16-PLL10 was toxic to human keratinocyte and erythrocytes. Conjugates of 16-PLL10, Trifluoroacetylated (TFA)−16-PLL10, and Poly-ethylene glycol (PEG)ylated 16-PLL10, synthesised to address toxicity, only moderately reduced cytotoxicity and haemolysis. Conclusions. Due to poor selectivity indices, further development of 16-PLL10 is unlikely warranted. However, considering the unmet need for novel topical antimicrobials, the ease of AMP polymer synthesises/modification is attractive. To support more rational development, prioritising clinically relevant pathogens and human cells, to establish selective toxicity profiles in vitro, is critical. Further characterisation and discovery utilising artificial intelligence and computational screening approaches can accelerate future AMP nanomaterial development.

Ciprofloxacin has been used to replace tryptophan at positions 3 and 6 in the antimicrobial peptide (AMP) Bac8c. Bac8c(Cip^3,6) showed comparable antimicrobial activity but increased selectivity toward some Gram-negative bacteria with MIC values of ≤6.25 μM. Bac8c(Cip^3,6) was also non-cytotoxic to cultured HepG2 cells at antimicrobial concentrations, with a CC₅₀ >300 μM.