Cationic microbubbles for non-selective binding of cavitation nuclei to bacterial biofilms

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

Facile production of quercetin nanoparticles using 3D printed centrifugal flow reactors

Drug nanocrystals are a delivery system comprised of an active pharmaceutical ingredient, with small amounts of a surface stabilizer. Despite offering simplicity in formulation, their manufacture can be a challenging endeavour; this is especially true when the production is performed using microfluidic devices. Although precipitation within microchannels can lead to issues such as clogging, microfluidics is an appealing manufacturing method as it provides fine control over mixing conditions. This allows production of nanoparticles with a narrower size distribution and greater reproducibility compared to batch methods. To generate microfluidic devices cost effectively, replica moulding techniques are considered the manufacturing standard. Due to its simplicity and relatively low cost, 3D printing has become prevalent at the laboratory scale, especially during iterative development of new devices. A challenge of microfluidic-based methods is that they require specialized equipment and multi-step procedures, making them less accessible to users with no previous experience. In a recent study we developed a 3D printed flow-through reactor, referred to as reactor-in-a-centrifuge (RIAC). It is a simple device designed to fit in a 50 mL tube and actuated using a laboratory centrifuge, which removes the need for specialized instrumentation. The manufacturing capabilities of the RIAC have been already proven, by reproducible production of liposomes and silver nanoparticles. The present work demonstrates the use of RIACs with a straight- and spiral-shaped channel architecture to produce quercetin nanocrystals, with therapeutically relevant size (190–302 nm) and very low size dispersity (polydispersity index, PDI < 0.1). The work focused on evaluating how changes in operational parameters (actuation speed) and formulation components (medium viscosity and stabilizer type), impacted on nanocrystal size and PDI. Under all tested conditions the obtained nanocrystals had a smaller size and narrower size distribution, when compared to those produced with alternative methods. The obtained quercetin nanosuspensions however showed limited stability, which should be addressed in future investigations. The simplicity of the RIAC makes it an appealing technology to research groups, especially in low-resource settings and without prior expertise in microfluidics

Ultrasound-compatible 3D-printed Franz diffusion system for sonophoresis with microbubbles

Sonophoresis is a topical drug delivery approach that utilises ultrasound as a physical stimulus to enhance permeation of active pharmaceutical ingredients through the skin. Only limited research has however been conducted to evaluate the potential of ultrasound-responsive drug carriers, such as gas microbubbles, in sonophoresis. Franz diffusion cells have been extensively used for measuring drug permeation in vitro; however, traditional systems lack compatibility with ultrasound and only limited characterisation of their acoustical behaviour has been carried out in previous research. To overcome this limitation, we designed and manufactured a novel Franz cell donor compartment coupled with a conventional glass receptor, and performed a functional characterisation of the assembly for application in sonophoresis with ultrasound-responsive agents (specifically imiquimod-loaded gas microbubbles). The donor was fabricated using a photoreactive resin via 3D printing and was designed to enable integration with a therapeutically relevant ultrasound source. The assembly was capable of effectively retaining liquids during prolonged incubation and the absorption of imiquimod onto the 3D-printed material was comparable to the one of glass. Moreover, a predictable ultrasound field could be generated at a target surface without any significant spatial distortion. Finally, we demonstrated applicability of the developed assembly in sonophoresis experiments with StratM®, wherein ultrasound stimulation in the presence of microbubbles resulted in significantly enhanced drug permeation through and partitioning within the membrane (2.96 ± 0.25 μg and 3.84 ± 0.39 μg) compared to passive diffusion alone (1.74 ± 0.29 μg and 2.29 ± 0.32 μg), over 24 h

Ultrasound-mediated therapies for the treatment of biofilms in chronic wound infections

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 report offers a rounded argument and research evidence to outline why ultrasound-responsive agents could be an integral part of advancing wound care. The fabrication of a stable ultrasound-responsive carrier of nitric oxide was an important first step in this research, this was achieved by exploring two primary lipid microbubble shell constituents: 1,2-distearoylphosphatidylcholine (DSPC) and 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC). It was successfully demonstrated that DBPC shelled microbubbles exhibited the greatest stability in ambient temperature over 60 minutes. Targeting biofilms to increase the efficacy of antimicrobial delivery and optimised local concentrations has proved challenging, due to the complex and heterogeneous nature of the biofilm extracellular matrix. This research assessed the utility of cationic microbubbles, as a means of non-selective electrostatic targeting of the anionic biofilm structure. Cationic microbubbles demonstrated the ability enhance microbubble-biofilm contact by up to 37%, with a binding strength comparable to that seen in specific ligand-receptor targeting of biofilm extracellular matrix constituents. The anti-biofilm and bactericidal efficacy of neutral and cationic airand nitric oxide-loaded microbubbles, were assessed both in terms of their passive nitric oxidemediated action and active response to ultrasound stimulation. To facilitate growth of biofilms with morphological and phenotypic characteristics of biofilms found in chronic wounds, a wound constituent media that featured key pathophysiological components of the in vivo wound environment was developed. It was successfully demonstrated that insonified DBPC nitric oxide microbubbles could elicit a clinically significant 99.9% (3-log reduction) in viable cells recovered from treated biofilms, by enhancing the efficacy of sub-inhibitory (4 µg/mL) concentrations of the antibiotic gentamicin. Moreover, the combination of the nitric oxide and the ultrasound mediated physical perturbation of biofilms by microbubbles, was also shown to elicit a significant 99.9% reduction in surface area covered by P. aeruginosa biofilms

Ultrasound mediated therapies for the treatment of biofilms in chronic wounds: a review of present knowledge

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

Rapid Production of Nanoscale Liposomes Using a 3D-Printed Reactor-In-A-Centrifuge: Formulation, Characterisation, and Super-Resolution Imaging

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

Quantitative evaluation of anti-biofilm cavitation activity seeded from microbubbles or protein cavitation nuclei by passive acoustic mapping

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 Staphylococcus aureus and Pseudomonas 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 centre 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 Passive Acoustic Mapping for real-time monitoring of antimicrobial processes

A microfluidic-based investigation of bacterial attachment in ureteral stents

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 &lt; 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. </p

Bactericidal and anti-biofilm effects of uncharged and cationic ultrasound-responsive nitric oxide microbubbles on Pseudomonas aeruginosa biofilms

Bacterial biofilms are a major and ongoing concern for public health, featuring both inherited genetic resistance traits and a conferred innate tolerance to traditional antibiotic therapies. Consequently, there is a growing need for novel methods of drug delivery, to increase the efficacy of antimicrobial agents. This research evaluated the anti-biofilm and bactericidal effects of ultrasound responsive gas-microbubbles (MBs) of either air or nitric oxide, using an in vitro Pseudomonas aeruginosa biofilm model grown in artificial wound medium. The four lipid-based MB formulations evaluated were room-air MBs (RAMBs) and nitric oxide MBs (NOMBs) with no electrical charge, as well as cationic (+) RAMBs+ and NOMBs+. Two principal treatment conditions were used: i) ultrasound stimulated MBs only, and ii) ultrasound stimulated MBs with a sub-inhibitory concentration (4 µg/mL) of the antibiotic gentamicin. The total treatment time was divided into a 60 second passive MB interaction period prior to 40 second ultrasound exposure; each MB formulation was tested in triplicate. Ultrasound stimulated RAMBs and NOMBs without antibiotic achieved reductions in biofilm biomass of 93.3% and 94.0%, respectively. Their bactericidal efficacy however was limited, with a reduction in culturable cells of 26.9% and 65.3%, respectively. NOMBs with sub-inhibitory antibiotic produced the most significant reduction in biofilm biomass, corresponding to a 99.9% (SD ± 5.21%); and a 99.9% (SD ± 0.07%) (3-log) reduction in culturable bacterial cells. Cationic MBs were initially manufactured to promote binding of MBs to negatively charged biofilms, but these formulations also demonstrated intrinsic bactericidal properties. In the absence of antibiotic, the bactericidal efficacy of RAMB+ and NOMB+ was greater that of uncharged counterparts, reducing culturable cells by 84.7% and 86.1% respectively; increasing to 99.8% when combined with antibiotic. This study thus demonstrates the anti-biofilm and bactericidal utility of ultrasound stimulated MBs, and specifically is the first to demonstrate the efficacy of a NOMB for the dispersal and potentiation of antibiotics against bacterial biofilms in vitro. Importantly the biofilm system and complex growth-medium were selected to recapitulate key morphological features of in vivo biofilms. The results us offer new insight for the development of new clinical treatments, for example, in chronic wounds.</p