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.

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.

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.

Bactericidal Effect of Ultrasound-Responsive Microbubbles and Sub-inhibitory Gentamicin against Pseudomonas aeruginosa Biofilms on Substrates With Differing Acoustic Impedance.

The aim of this research was to explore the interaction between ultrasound-activated microbubbles (MBs) and Pseudomonas aeruginosa biofilms, specifically the effects of MB concentration, ultrasound exposure and substrate properties on bactericidal efficacy. Biofilms were grown using a Centre for Disease Control (CDC) bioreactor on polypropylene or stainless-steel coupons as acoustic analogues for soft and hard tissue, respectively. Biofilms were treated with different concentrations of phospholipid-shelled MBs (107-108 MB/mL), a sub-inhibitory concentration of gentamicin (4 µg/mL) and 1-MHz ultrasound with a continuous or pulsed (100-kHz pulse repetition frequency, 25% duty cycle, 0.5-MPa peak-to-peak pressure) wave. The effect of repeated ultrasound exposure with intervals of either 15- or 60-min was also investigated. With polypropylene coupons, the greatest bactericidal effect was achieved with 2 × 5 min of pulsed ultrasound separated by 60 min and a microbubble concentration of 5 × 107 MBs/mL. A 0.76 log (83%) additional reduction in the number of bacteria was achieved compared with the use of an antibiotic alone. With stainless-steel coupons, a 67% (0.46 log) reduction was obtained under the same exposure conditions, possibly due to enhancement of a standing wave field which inhibited MB penetration in the biofilm. These findings demonstrate the importance of treatment parameter selection in antimicrobial applications of MBs and ultrasound in different tissue environments.

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 < 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.

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.