Conventional drugs used for antibacterial therapy display several limitations. This is not due to antibiotics being ineffective, but rather due to their low bioavailability, limited penetration to sites of infection and the rise of drug-resistant bacteria. Although new delivery systems (e.g., nanoparticles) that are loaded with antibacterial drugs have been designed to overcome these limitations, therapeutic efficacy does not seem to have improved. Against this backdrop, stimuli-responsive antibiotic-loaded nanoparticles and materials with antimicrobial properties (nanoantibiotics) present the ability to enhance therapeutic efficacy, while also reducing drug resistance and side effects. These stimuli can either be exogenous (e.g., light, ultrasound) or endogenous (e.g., pH, variation in redox gradient, enzymes). This promising therapeutic approach relies on advances in materials science and increased knowledge of microorganism growth and biofilm formation. This review provides an overview in the field of antibacterial drug-delivery systems and nanoantibiotics that benefit from a response to specific triggers, and also presents a number of future prospects.

The paralogous multifunctional adaptor proteins YAP and TAZ are the nuclear effectors of the Hippo pathway, a central mechanism of organ size control and stem cell self-renewal. WW domains, mediators of protein-protein interactions, are essential for YAP and TAZ function, enabling interactions with PPxY motifs of numerous partner proteins. YAP has single and double WW domain isoforms (YAP1 and YAP2) whereas only a single WW domain isoform of TAZ has been described to date. Here we identify the first example of a double WW domain isoform of TAZ. Using NMR, we have characterized conformational features and peptide binding of YAP and TAZ tandem WW domains (WW1-WW2). The solution structure of YAP WW2 confirms that it has a canonical three-stranded antiparallel β-sheet WW domain fold. While chemical shift-based analysis indicates that the WW domains in the tandem WW pairs retain the characteristic WW domain fold, 15N relaxation data show that, within the respective WW pairs, YAP WW1 and both WW1 and WW2 of TAZ undergo conformational exchange. 15N relaxation data also indicate that the linker between the WW domains is flexible in both YAP and TAZ. Within both YAP and TAZ tandem WW pairs, WW1 and WW2 bind single PPxY-containing peptide ligand concurrently and noncooperatively with sub-mM affinity. YAP and TAZ WW1-WW2 bind a dual PPxY-containing peptide with approximately 6-fold higher affinity. Our results indicate that both WW domains in YAP and TAZ are functional and capable of enhanced affinity binding to multi-PPxY partner proteins such as LATS1, ErbB4, and AMOT.

The association of photosensitisers to peptides and proteins is a recognised and successful method for enhancing the selectivity and efficacy of photodynamic treatment. The covalent attachment of porphyrins and related macrocycles to peptides and proteins can generate new phototoxic species that allow the concentration of the oxidative damage to the target area, thanks to their enhanced cellular uptake, favourable sub-cellular distribution, and ability to target receptors or enzymes over-expressed by a given tissue or cell. The need to exert control over the regioselectivity of the conjugation led to the exploration of a variety of chemistries; in some cases based on bioorthogonal ligations, in others exploring the reactivity of naturally occurring functional groups. In this review we place a major emphasis on the synthetic strategies adopted to generate such conjugates. All such strategies will be surveyed, together with the methods used to introduce or unmask the appropriate reactive functionalities both on the peptide moiety and the photosensitiser.

Given that cell-penetrating peptides (CPP) are cationic and often amphipathic, similar to membrane-active antimicrobial peptides, it may be possible to use CPP conjugation to improve the delivery of photosensitisers for antimicrobial photodynamic therapy (antimicrobial PDT). We investigated the possibility of using a Tat peptide to deliver the photosensitiser, tetrakis(phenyl)porphyrin (TPP) and kill bacteria. The Tat peptide is a positively-charged mammalian cell-penetrating peptide with potent antimicrobial activity but no haemolytic activity. Fluorescence spectroscopy revealed that the bioconjugate can bind to and/or be incorporated into all bacterial species tested. All species were susceptible to the Tat-porphyrin, with the bactericidal effect being dependent on both the concentration and the light dose. Using the highest light dose, treatment with the Tat-porphyrin achieved reductions of 6.6 log(10) and 6.37 log(10) in the viable counts of Staphylococcus aureus and Streptococcus pyogenes, and reductions of 5.74 log(10) and 6.6 log(10) in the viable counts of Pseudomonas aeruginosa and Escherichia coli. Moreover, the Tat moiety appears to confer antimicrobial properties to the conjugate, particularly for the Gram positive strains, based on the observation of dark toxicity using 1 μM of Tat-porphyrin. Finally, the conjugate induced membrane destabilization by synergistic action of the peptide and PDT, resulting in carboxyfluorescein leakage from bacterial membrane-mimicking liposomes. These findings demonstrate that the use of CPP to deliver a photosensitiser is an effective way of improving the uptake and the treatment efficacy of antimicrobial PDT.

Ultrasound is used to trigger the cytotoxicity of chemical compounds, known as sonosensitisers, in an approach called sonodynamic therapy (SDT), which is under investigation herein. The generation of reactive oxygen species (ROS) has been proposed as the main biological occurrence that leads to the cytotoxic effects, which are achieved via the synergistic action of two components: the energy-absorbing sonosensitiser and ultrasound (US), which are both harmless per se. Despite some promising results, a lack of investigation into the mechanisms behind US sonosensitiser-mediated ROS generation has prevented SDT from reaching its full potential. The aim of this work is to investigate the US-responsiveness of a variety of metal-porphyrin complexes, free-base porphyrin and Fe(III), Zn(II) and Pd(II) porphyrin, by analyzing their ROS generation under US exposure and related bio-effects. All experiments were also carried out under light exposure and the results were used as references. Our results show that porphyrin ultrasound-responsiveness depends on the metal ion present, with Zn(II) and Pd(II) porphyrin being the most efficient in generating singlet oxygen and hydroxyl radicals. ROS production efficiency is lower after ultrasound exposure than after light exposure, because of the various physico-chemical mechanisms involved in sensitiser activation. US and porphyrin-mediated ROS generation is oxygen-dependent and the activation of porphyrin by US appears to be more compatible with sonoluminescence-based photo-activation rather than a radical path process that occurs via the homolytic bond rupture of water. Notably, the cytotoxicity results reported herein, which are mirrored by ex-cellulo data, confirm that the type of ROS generation achieved by the US activation of intracellular porphyrins is pivotal to the effectiveness of cancer cell killing.

Photochemical internalisation (PCI) is a site-specific technique for improving cellular delivery of macromolecular drugs. In this study, a cell penetrating peptide, containing the core HIV-1 Tat 48-57 sequence, conjugated with a porphyrin photosensitiser has been shown to be effective for PCI. Herein we report an investigation of the photophysical and photobiological properties of a water soluble bioconjugate of the cationic Tat peptide with a hydrophobic tetraphenylporphyrin derivative. The cellular uptake and localisation of the amphiphilic bioconjugate was examined in the HN5 human head and neck squamous cell carcinoma cell line. Efficient cellular uptake and localisation in endo/lysosomal vesicles was found using fluorescence detection, and light-induced, rupture of the vesicles resulting in a more diffuse intracellular fluorescence distribution was observed. Conjugation of the Tat sequence with a hydrophobic porphyrin thus enables cellular delivery of an amphiphilic photosensitiser which can then localise in endo/lysosomal membranes, as required for effective PCI treatment. PCI efficacy was tested in combination with a protein toxin, saporin, and a significant reduction in cell viability was measured versus saporin or photosensitiser treatment alone. This study demonstrates that the cell penetrating peptide-photosensitiser bioconjugation strategy is a promising and versatile approach for enhancing the therapeutic potential of bioactive agents through photochemical internalisation.

Amphiphilic cell-penetrating peptide–porphyrin conjugates have been developed for application in light-based therapeutic techniques.

Sonodynamic therapy (SDT) relies on the ability of ultrasound to activate sonosensitisers and trigger the generation of reactive oxygen species (ROS) to achieve cell death. SDT was explored as an anticancer approach until 6 years ago, when its potential application as an antimicrobial strategy was pointed out and the term "sonoantimicrobial chemotherapy" (SACT) was coined. The excellent penetration of ultrasound in liquid media make SACT particularly promising approach for the noninvasive treatment of deep-seated infections, and for the reduction of bacterial load in turbid water. In this review we provide an account of the brief history of SACT, from its molecular bases to the current state of the art and perspective applications. Graphical abstract