Summary: Bacterial biofilms are an ever‐growing concern for public health, featuring both inherited genetic resistance and a conferred innate tolerance to traditional antibiotic therapies. Consequently, there is a growing interest in novel methods of drug delivery, in order to increase the efficacy of antimicrobial agents. One such method is the use of acoustically activated microbubbles, which undergo volumetric oscillations and collapse upon exposure to an ultrasound field. This facilitates physical perturbation of the biofilm and provides the means to control drug delivery both temporally and spatially. In line with current literature in this area, this review offers a rounded argument for why ultrasound‐responsive agents could be an integral part of advancing wound care. To achieve this, we will outline the development and clinical significance of biofilms in the context of chronic infections. We will then discuss current practices used in combating biofilms in chronic wounds and then critically evaluate the use of acoustically activated gas microbubbles as an emerging treatment modality. Moreover, we will introduce the novel concept of microbubbles carrying biologically active gases that may facilitate biofilm dispersal.

Obstructions of the ureter lumen can originate from intrinsic or extrinsic factors, such as kidney stones, tumours, or strictures. These can affect the physiological flow of urine from the kidneys to the bladder, potentially causing infection, pain, and kidney failure. To overcome these complications, ureteral stents are often deployed clinically in order to temporarily re-establish urinary flow. Despite their clinical benefits, stents are prone to encrustation and biofilm formation that lead to reduced quality of life for patients; however, the mechanisms underlying the formation of crystalline biofilms in stents are not yet fully understood. In this study, we developed microfluidic-based devices replicating the urodynamic field within different configurations of an occluded and stented ureter. We employed computational fluid dynamic simulations to characterise the flow dynamic field within these models and investigated bacterial attachment (Pseudomonas fluorescens) by means of crystal violet staining and fluorescence microscopy. We identified the presence of hydrodynamic cavities in the vicinity of a ureteric occlusion, which were characterised by low levels of wall shear stress (WSS < 40 mPa), and observed that initiation of bacterial attachment occurred in these specific regions of the stented ureter. Notably, the bacterial coverage area was directly proportional to the number of cavities present in the model. Fluorescence microscopy confirmed that the number density of bacteria was greater within cavities (3 bacteria·mm⁻²) when compared to side-holes of the stent (1 bacterium·mm⁻²) or its luminal surface (0.12 bacteria·mm⁻²). These findings informed the design of a novel technological solution against bacterial attachment, which reduces the extent of cavity flow and increases wall shear stress over the stent’s surface.

Nanoscale liposomes have been extensively researched and employed clinically for the delivery of biologically active compounds, including chemotherapy drugs and vaccines, offering improved pharmacokinetic behaviour and therapeutic outcomes. Traditional laboratory-scale production methods often suffer from limited control over liposome properties (e.g., size and lamellarity) and rely on laborious multistep procedures, which may limit pre-clinical research developments and innovation in this area. The widespread adoption of alternative, more controllable microfluidic-based methods is often hindered by complexities and costs associated with device manufacturing and operation, as well as the short device lifetime and the relatively low liposome production rates in some cases. In this study, we demonstrated the production of liposomes comprising therapeutically relevant lipid formulations, using a cost-effective 3D-printed reactor-in-a-centrifuge (RIAC) device. By adjusting formulation- and production-related parameters, including the concentration of polyethylene glycol (PEG), temperature, centrifugation time and speed, and lipid concentration, the mean size of the produced liposomes could be tuned in the range of 140 to 200 nm. By combining selected experimental parameters, the method was capable of producing liposomes with a therapeutically relevant mean size of ~174 nm with narrow size distribution (polydispersity index, PDI ~0.1) at a production rate of >8 mg/min. The flow-through method proposed in this study has potential to become an effective and versatile laboratory-scale approach to simplify the synthesis of therapeutic liposomal formulations.

The presence of multi-drug resistant biofilms in chronic, persistent infections is a major barrier to successful clinical outcomes of therapy. The production of an extracellular matrix is a characteristic of the biofilm phenotype, intrinsically linked to antimicrobial tolerance. The heterogeneity of the extracellular matrix makes it highly dynamic, with substantial differences in composition between biofilms, even in the same species. This variability poses a major challenge in targeting drug delivery systems to biofilms, as there are few elements both suitably conserved and widely expressed across multiple species. However, the presence of extracellular DNA within the extracellular matrix is ubiquitous across species, which alongside bacterial cell components, gives the biofilm its net negative charge. This research aims to develop a means of targeting biofilms to enhance drug delivery by developing a cationic gas-filled microbubble that non-selectively targets the negatively charged biofilm. Cationic and uncharged microbubbles loaded with different gases were formulated and tested to determine their stability, ability to bind to negatively charged artificial substrates, binding strength, and, subsequently, their ability to adhere to biofilms. It was shown that compared to their uncharged counterparts, cationic microbubbles facilitated a significant increase in the number of microbubbles that could both bind and sustain their interaction with biofilms. This work is the first to demonstrate the utility of charged microbubbles for the non-selective targeting of bacterial biofilms, which could be used to significantly enhance stimuli-mediated drug delivery to the bacterial biofilm.

Objective. Bacterial biofilms represent a major challenge for effective antibiotic therapy as they confer physical and functional changes that protect bacteria from their surrounding environment. In this work, focused ultrasound in combination with cavitation nuclei was used to disrupt biofilms of S. aureus and P. aeruginosa, both of which are on the World Health Organization’s priority list for new antimicrobial research. Approach. Single species biofilms were exposed to ultrasound (0.5 MHz center frequency, 0.5–1.5 MPa peak rarefactional pressure, 200 cycle pulses, 5 Hz repetition frequency, 30 s duration), in the presence of two different types of cavitation nuclei. Quantitative passive acoustic mapping (PAM) was used to monitor cavitation emissions during treatment using a calibrated linear array. Main Results. It was observed that the cumulative energy of acoustic emissions during treatment was positively correlated with biofilm disruption, with differences between bacterial species attributed to differences in biofilm morphology. PCaN provided increased biofilm reduction compared to microbubbles due in large part to their persistence over the duration of ultrasound exposure. There was also good correlation between the spatial distribution of cavitation as characterized by PAM and the extent of biofilm disruption observed with microscopy. Significance. Collectively, the results from this work indicate the potential broad applicability of cavitation for eliminating biofilms of priority pathogens and the opportunity presented by PAM for real-time monitoring of antimicrobial processes.

Urinary tract infections (UTI) remain a major global health burden, with high recurrence despite antibiotic treatment. The escalating prevalence of antimicrobial resistance further compromises therapeutic efficacy, contributing to an estimated 260,000 deaths annually. Conventional in vitro susceptibility assays often fail to predict clinical outcomes, underscoring the urgent need for physiologically relevant infection models. Here, we examined how microenvironmental complexity shapes uropathogenic Escherichia coli (UPEC) responses to antibiotics and bacteriophages using: human urine, a three-dimensional urothelial microtissue model (3D-UHU), and a novel mesofluidic system (P-FLO) that introduces physiologically relevant flow dynamics to the 3D-UHU. P-FLO was engineered from cost-effective 3D-printed components compatible with standard Transwell systems. Among the antibiotics tested, nitrofurantoin exhibited the greatest potency in minimum inhibitory concentration assays, but it failed to fully eradicate infection within the more physiological 3D-UHU model. A bacteriophage cocktail (LCPR1) showed markedly reduced activity in urine compared with nutrient-rich media, highlighting the influence of infection-site conditions. In contrast, in 3D-UHU, LCRP1 modulated host responses without reducing bacterial burden. Combination therapy (nitrofurantoin + LCPR1) eliminated planktonic bacteria under static conditions but offered no added benefit against adherent or intracellular populations relative to antibiotic monotherapy. Incorporating flow revealed additional layers of complexity, where shear stress induced bacterial elongation and attachment and altered drug performance, diminishing the efficacy of nitrofurantoin and combination therapy against planktonic populations despite increased drug exposure. Together, these findings demonstrate that the bladder microenvironment and its mechanical forces modulate host-pathogen interactions and profoundly influence UPEC infection dynamics and therapeutic outcomes, emphasizing the need for advanced, physiologically informed models to guide treatment strategies in the post-antibiotic era.