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Efficacy of nitric oxide donors and EDTA against Pseudomonas aeruginosa biofilms: Implications for antimicrobial therapy in chronic wounds.
Opportunistic pathogen Pseudomonas aeruginosa plays a crucial role in chronic wound biofilms, increasing infection's morbidity and mortality. In recent years, the signalling molecule nitric oxide (NO) and chelating agent tetrasodium EDTA (T-EDTA) have been applied therapeutically owing to their multifactorial effects including bacterial killing, biofilm dispersal, and wound healing. However, previous studies assessing NO's antibiofilm efficacy have not considered the variable pH and temperature of the wound environment. Here, pH-dependent NO donors N-diazeniumdiolates (NONOates), PAPA NONOate (PA-NO) and Spermine NONOate (SP-NO), and T-EDTA were applied in wound-relevant pH environments (pH 5.5-8.5) and temperatures (32 °C and 37 °C) to P. aeruginosa PAO1 biofilms grown for either 24 or 48 h. At 32 °C and pH 7.5, 250 μM PA-NO reduced 24-h biofilm biomass by 35 %. At 37 °C, 250 μM PA-NO and 4 % w/v T-EDTA caused 21 % and 57 % biomass reduction in 24-h biofilms, respectively. In 48-h biofilms, NONOates did not induce significant biomass reduction, while T-EDTA maintained its efficacy with a 64 % reduction. A subsequent experiment investigated the impact of NONOates and T-EDTA as pre-treatments before exposure to ciprofloxacin. Unexpectedly, NONOate pre-treatment decreased ciprofloxacin's effectiveness, resulting in approximately 1-log increase in viable planktonic and biofilm-residing cells compared to ciprofloxacin alone. It was hypothesized that this protective effect might stem from NO-induced decreased cellular respiration, which inhibits reactive oxygen species (ROS)-mediated bactericidal mechanisms. These findings highlight both the potential and complexities of developing effective antimicrobial strategies for chronic wound infections, emphasizing the need for further research to optimize treatment approaches.
Sonothrombolysis with Magnetically Targeted Microbubbles.
Microbubble-enhanced sonothrombolysis is a promising approach to increasing the tolerability and efficacy of current pharmacological treatments for ischemic stroke. Maintaining therapeutic concentrations of microbubbles and drugs at the clot site, however, poses a challenge. The objective of this study was to investigate the effect of magnetic microbubble targeting upon clot lysis rates in vitro. Retracted whole porcine blood clots were placed in a flow phantom of a partially occluded middle cerebral artery. The clots were treated with a combination of tissue plasminogen activator (0.75 µg/mL), magnetic microbubbles (∼107 microbubbles/mL) and ultrasound (0.5 MHz, 630-kPa peak rarefactional pressure, 0.2-Hz pulse repetition frequency, 2% duty cycle). Magnetic targeting was achieved using a single permanent magnet (0.08-0.38 T and 12-140 T/m in the region of the clot). The change in clot diameter was measured optically over the course of the experiment. Magnetic targeting produced a threefold average increase in lysis rates, and linear correlation was observed between lysis rate and total energy of acoustic emissions.
Temperature-controlled acoustofluidic production of microbubble contrast agents with enhanced stability and monodispersity
In this paper, we propose a simple acoustofluidic method for production of microbubble contrast agents in disposable microfluidic chips by the use of a low-frequency ultrasound transducer and a temperature controller unit. The technique is used to increase the lifetime and to narrow down the size distribution of the microbubbles as compared to traditional microfluidic-based methods.
pH-sensitive release of nitric oxide gas using peptide-graphene co-assembled hybrid nanosheets.
Nitric oxide (NO) donating drugs such as organic nitrates have been used to treat cardiovascular diseases for more than a century. These donors primarily produce NO systemically. It is however sometimes desirable to control the amount, location, and time of NO delivery. We present the design of a novel pH-sensitive NO release system that is achieved by the synthesis of dipeptide diphenylalanine (FF) and graphene oxide (GO) co-assembled hybrid nanosheets (termed as FF@GO) through weak molecular interactions. These hybrid nanosheets were characterised by using X-ray diffraction, Raman spectroscopy, Fourier transform infrared spectroscopy, zeta potential measurements, X-ray photoelectron spectroscopy, scanning and transmission electron microscopies. The weak molecular interactions, which include electrostatic, hydrogen bonding and π-π stacking, are pH sensitive due to the presence of carboxylic acid and amine functionalities on GO and the dipeptide building blocks. Herein, we demonstrate that this formulation can be loaded with NO gas with the dipeptide acting as an arresting agent to inhibit NO burst release at neutral pH; however, at acidic pH it is capable of releasing NO at the rate of up to 0.6 μM per minute, comparable to the amount of NO produced by healthy endothelium. In conclusion, the innovative conjugation of dipeptide with graphene can store and release NO gas under physiologically relevant concentrations in a pH-responsive manner. pH responsive NO-releasing organic-inorganic nanohybrids may prove useful for the treatment of cardiovascular diseases and other pathologies.
An investigation into the cytotoxic effects of microbubbles and their constituents on osteosarcoma and bone marrow stromal cells.
BACKGROUND: Ultrasound-responsive microbubbles offer a means of achieving minimally invasive, localised drug delivery in applications including regenerative medicine. To facilitate their use, however, it is important to determine any cytotoxic effects they or their constituents may have. The aim of this study was to test the hypothesis that phospholipid-shelled microbubbles are non-toxic to human bone-derived cells at biologically-relevant concentrations. METHODS: Microbubbles were fabricated using combinations of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC), polyoxyethylene(40) stearate (PEG40S) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene-glycol)-2000] (DSPE-PEG2000). Microbubble size and concentration were measured as a function of time and temperature by optical microscopy. Effects on MG63 osteosarcoma and human bone marrow stromal cells (BMSCs) were measured for up to 72 h by assay for viability, metabolic activity and proliferation. RESULTS: DBPC:DSPE-PEG2000 microbubbles were significantly more stable than DSPC:PEG40S microbubbles under all conditions tested. Serum-containing medium had no detrimental effect on microbubble stability, but storage at 37 °C compared to at 4 °C reduced stability for both preparations, with almost complete dissolution of microbubbles at times ≥24 h. DSPC:PEG40S microbubbles had greater inhibitory effects on cell metabolism and growth than DBPC:DSPE-PEG2000 microbubbles, with PEG40S found to be the principle inhibitory component. These effects were only evident at high microbubble concentrations (≥20% (v/v)) or with prolonged culture (≥24 h). Increasing cell-microbubble contact by inversion culture in a custom-built device had no inhibitory effect on metabolism. CONCLUSIONS: These data indicate that, over a broad range of concentrations and incubation times, DBPC:DSPE-PEG2000 and DSPC:PEG40S microbubbles have little effect on osteoblastic cell viability and growth, and that PEG40S is the principle inhibitory component in the formulations investigated.
An Acoustic Device for Ultra High-Speed Quantification of Cell Strain During Cell-Microbubble Interaction.
Microbubbles utilize high-frequency oscillations under ultrasound stimulation to induce a range of therapeutic effects in cells, often through mechanical stimulation and permeabilization of cells. One of the largest challenges remaining in the field is the characterization of interactions between cells and microbubbles at therapeutically relevant frequencies. Technical limitations, such as employing sufficient frame rates and obtaining sufficient image resolution, restrict the quantification of the cell's mechanical response to oscillating microbubbles. Here, a novel methodology was developed to address many of these limitations and improve the image resolution of cell-microbubble interactions at high frame rates. A compact acoustic device was designed to house cells and microbubbles as well as a therapeutically relevant acoustic field while being compatible with a Shimadzu HPV-X camera. Cell viability tests confirmed the successful culture and proliferation of cells, and the attachment of DSPC- and cationic DSEPC-microbubbles to osteosarcoma cells was quantified. Microbubble oscillation was observed within the device at a frame rate of 5 million FPS, confirming suitable acoustic field generation and ultra high-speed image capture. High spatial resolution in these images revealed observable deformation in cells following microbubble oscillation and supported the first use of digital image correlation for strain quantification in a single cell. The novel acoustic device provided a simple, effective method for improving the spatial resolution of cell-microbubble interaction images, presenting the opportunity to develop an understanding of the mechanisms driving the therapeutic effects of oscillating microbubbles upon ultrasound exposure.
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.
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.
Engineering solutions to ureteral stents: material, coating and design.
INTRODUCTION: An ideal stent would offer simple insertion and removal with no discomfort and/or migration, it would have no biofilm formation or encrustation and would also maintain the patient's quality of life. MATERIAL AND METHODS: In this mini-review, we outlined the engineering developments related to stent material, design and coating. RESULTS: There have been a wide variety of in-vitro, model-based, animal-based and clinical studies using a range of commercial and non-commercial stents. Ureteric stents have evolved since their first usage with a wider range of stent design, material and coating available for laboratory and clinical use. CONCLUSIONS: While engineering innovations have led to the evolution of stents, more work needs to be done to address the issues relating to stent encrustation and biofilm formation.
High throughput imaging cytometer with acoustic focussing.
We demonstrate an imaging flow cytometer that uses acoustic levitation to assemble cells and other particles into a sheet structure. This technique enables a high resolution, low noise CMOS camera to capture images of thousands of cells with each frame. While ultrasonic focussing has previously been demonstrated for 1D cytometry systems, extending the technology to a planar, much higher throughput format and integrating imaging is non-trivial, and represents a significant jump forward in capability, leading to diagnostic possibilities not achievable with current systems. A galvo mirror is used to track the images of the moving cells permitting exposure times of 10 ms at frame rates of 50 fps with motion blur of only a few pixels. At 80 fps, we demonstrate a throughput of 208 000 beads per second. We investigate the factors affecting motion blur and throughput, and demonstrate the system with fluorescent beads, leukaemia cells and a chondrocyte cell line. Cells require more time to reach the acoustic focus than beads, resulting in lower throughputs; however a longer device would remove this constraint.
The effect of ultrasound-related stimuli on cell viability in microfluidic channels.
BACKGROUND: In ultrasonic micro-devices, contrast agent micro-bubbles are known to initiate cavitation and streaming local to cells, potentially compromising cell viability. Here we investigate the effects of US alone by omitting contrast agent and monitoring cell viability under moderate-to-extreme ultrasound-related stimuli. RESULTS: Suspended H9c2 cardiac myoblasts were exposed to ultrasonic fields within a glass micro-capillary and their viability monitored under different US-related stimuli. An optimal injection flow rate of 2.6 mL/h was identified in which, high viability was maintained (~95%) and no mechanical stress towards cells was evident. This flow rate also allowed sufficient exposure of cells to US in order to induce bioeffects (~5 sec), whilst providing economical sample collection and processing times. Although the transducer temperature increased from ambient 23°C to 54°C at the maximum experimental voltage (29 Vpp), computational fluid dynamic simulations and controls (absence of US) revealed that the cell medium temperature did not exceed 34°C in the pressure nodal plane. Cells exposed to US amplitudes ranging from 0-29 Vpp, at a fixed frequency sweep period (tsw = 0.05 sec), revealed that viability was minimally affected up to ~15 Vpp. There was a ~17% reduction in viability at 21 Vpp, corresponding to the onset of Rayleigh-like streaming and a ~60% reduction at 29 Vpp, corresponding to increased streaming velocity or the potential onset of cavitation. At a fixed amplitude (29 Vpp) but with varying frequency sweep period (tsw = 0.02-0.50 sec), cell viability remained relatively constant at tsw ≥ 0.08 sec, whilst viability reduced at tsw < 0.08 sec and minimum viability recorded at tsw = 0.05 sec. CONCLUSION: The absence of CA has enabled us to investigate the effect of US alone on cell viability. Moderate-to-extreme US-related stimuli of cells have allowed us to discriminate between stimuli that maintain high viability and stimuli that significantly reduce cell viability. Results from this study may be of potential interest to researchers in the field of US-induced intracellular drug delivery and ultrasonic manipulation of biological cells.
Optimised production of multifunctional microfibres by microfluidic chip technology for tissue engineering applications.
This paper describes a method for the production of alginate microfibres using glass-based microfluidic chips fabricated by a photolithography-wet etching procedure. The main focus of the work is the fabrication of a cell containing multifunctional microfibres which have great potential for applications in drug release formulations and tissue engineering scaffolds (to guide the regeneration of tissues in predefined sizes and shapes) providing cell structural support and immunoisolation. The key parameters, which critically influence the formation of microfibres and their geometries, were identified by a classical intuitive approach COST (Changing One Separate factor a Time). In particular, their effects on the microfibre diameter were investigated, which are directly associated with their functionalities relating to the implantation site, the nutrient availability and diffusion/transport of oxygen, essential nutrients, growth factors, metabolic waste and secretory products. The interplay between the alginate solution concentration, pumping rate and gelling bath concentration in controlling the diameter of the produced microfibres was investigated with a statistical approach by means of a "design of the experiments" (DoEs) optimization and screening. Finally, the processing impacts on cell viability, the cellular effect of wall thickness consistency and the spatial distribution of cells within the alginate microfibre were examined. We provide an approach for the production of alginate microfibres with controlled shape and content, which could be further developed for scaling up and working towards FDA approval.
Easy-to-perform and cost-effective fabrication of continuous-flow reactors and their application for nanomaterials synthesis.
The translation of continuous-flow microreactor technology to the industrial environment has been limited by cost and complexity of the fabrication procedures and the requirement for specialised infrastructure. In the present study, we have developed a significantly more cost-effective and easy-to-perform fabrication method for the generation of optically transparent, continuous-flow reactors. The method combines 3D printing of master moulds with sealing of the PDMS channels' replica using a pressure-sensitive adhesive tape. Morphological characterisation of the 3D printed moulds was performed and reactors were fabricated with an approximately square-shaped cross-section of 1 mm2. Notably, they were tested for operation over a wide range of volumetric flow rates, up to 20 ml/min. Moreover, the fabrication time (i.e., from design to the finished product) was <1 day, at an average material cost of ∼£5. The flow reactors have been applied to the production of both inorganic nanoparticles (silver nanospheres) and organic vesicular systems (liposomes), and their performance compared with reactors produced using more laborious fabrication methods. Numerical simulations were performed to characterise the transport of fluids and chemical species within the devices. The developed fabrication method is suitable for scaled-up fabrication of continuous-flow reactors, with potential for application in biotechnology and nanomedicine.
Contrast agent-free sonoporation: The use of an ultrasonic standing wave microfluidic system for the delivery of pharmaceutical agents.
Sonoporation is a useful biophysical mechanism for facilitating the transmembrane delivery of therapeutic agents from the extracellular to the intracellular milieu. Conventionally, sonoporation is carried out in the presence of ultrasound contrast agents, which are known to greatly enhance transient poration of biological cell membranes. However, in vivo contrast agents have been observed to induce capillary rupture and haemorrhage due to endothelial cell damage and to greatly increase the potential for cell lysis in vitro. Here, we demonstrate sonoporation of cardiac myoblasts in the absence of contrast agent (CA-free sonoporation) using a low-cost ultrasound-microfluidic device. Within this device an ultrasonic standing wave was generated, allowing control over the position of the cells and the strength of the acoustic radiation forces. Real-time single-cell analysis and retrospective post-sonication analysis of insonated cardiac myoblasts showed that CA-free sonoporation induced transmembrane transfer of fluorescent probes (CMFDA and FITC-dextran) and that different mechanisms potentially contribute to membrane poration in the presence of an ultrasonic wave. Additionally, to the best of our knowledge, we have shown for the first time that sonoporation induces increased cell cytotoxicity as a consequence of CA-free ultrasound-facilitated uptake of pharmaceutical agents (doxorubicin, luteolin, and apigenin). The US-microfluidic device designed here provides an in vitro alternative to expensive and controversial in vivo models used for early stage drug discovery, and drug delivery programs and toxicity measurements.
Fluid mechanical modeling of the upper urinary tract.
The upper urinary tract (UUT) consists of kidneys and ureters, and is an integral part of the human urogenital system. Yet malfunctioning and complications of the UUT can happen at all stages of life, attributed to reasons such as congenital anomalies, urinary tract infections, urolithiasis and urothelial cancers, all of which require urological interventions and significantly compromise patients' quality of life. Therefore, many models have been developed to address the relevant scientific and clinical challenges of the UUT. Of all approaches, fluid mechanical modeling serves a pivotal role and various methods have been employed to develop physiologically meaningful models. In this article, we provide an overview on the historical evolution of fluid mechanical models of UUT that utilize theoretical, computational, and experimental approaches. Descriptions of the physiological functionality of each component are also given and the mechanical characterizations associated with the UUT are provided. As such, it is our aim to offer a brief summary of the current knowledge of the subject, and provide a comprehensive introduction for engineers, scientists, and clinicians who are interested in the field of fluid mechanical modeling of UUT. This article is categorized under: Cancer > Biomedical Engineering Infectious Diseases > Biomedical Engineering Reproductive System Diseases > Biomedical Engineering.
The role of clinically-relevant parameters on the cohesiveness of sclerosing foams in a biomimetic vein model.
We have recently reported on the development of a biomimetic vein model to measure the performance of sclerosing foams. In this study we employed the model to compare the commercially-available Varithena(®) (polidocanol injectable foam) 1% varicose vein treatment (referred to as polidocanol endovenous microfoam, or PEM) with physician compounded foams (PCFs) made using different foam generation methods (Double Syringe System and Tessari methods) and different foam formulations [liquid to gas ratios of 1:3 or 1:7; gas mixtures composed of 100% CO2, various CO2:O2 mixtures and room air (RA)]. PCFs produced using the DSS method had longer dwell times (DTs) (range 0.54-2.21 s/cm in the 4 mm diameter vein model) than those of the corresponding PCFs produced by the Tessari technique (range 0.29-0.94 s/cm). PEM had the longest DT indicating the best cohesive stability of any of the foams produced (2.92 s/cm). Other biomimetic model variables investigated included effect of vessel size, delayed injection and rate of plug formation (injection speed). When comparing the 4 and 10 mm vessel diameters, the DTs seen in the 10 mm vessel were higher than those observed for the 4 mm vessel, as the vein angle had been reduced to 5° to allow for foam plug formation. PCF foam performance was in the order RA > CO2:O2 (35:65) ≅ CO2:O2 (65:35) > CO2; PEM had a longer DT than all PCFs (22.10 s/cm) except that for RA made by DSS which was similar but more variable. The effect of delayed injection was also investigated and the DT for PEM remained the longest of all foams with the lowest percentage deviation with respect to the mean values, indicating a consistent foam performance. When considering rate of plug formation, PEM consistently produced the longest DTs and this was possible even at low plug expansion rates (mean 29.5 mm/s, minimum 20.9 mm/s). The developed vein model has therefore demonstrated that PEM consistently displays higher foam stability and cohesiveness when compared to PCFs, over a range of clinically-relevant operational variables.
Microfluidic and lab-on-a-chip preparation routes for organic nanoparticles and vesicular systems for nanomedicine applications.
In recent years, advancements in the fields of microfluidic and lab-on-a-chip technologies have provided unique opportunities for the implementation of nanomaterial production processes owing to the miniaturisation of the fluidic environment. It has been demonstrated that microfluidic reactors offer a range of advantages compared to conventional batch reactors, including improved controllability and uniformity of nanomaterial characteristics. In addition, the fast mixing achieved within microchannels, and the predictability of the laminar flow conditions, can be leveraged to investigate the nanomaterial formation dynamics. In this article recent developments in the field of microfluidic production of nanomaterials for drug delivery applications are reviewed. The features that make microfluidic reactors a suitable technological platform are discussed in terms of controllability of nanomaterials production. An overview of the various strategies developed for the production of organic nanoparticles and colloidal assemblies is presented, focusing on those nanomaterials that could have an impact on nanomedicine field such as drug nanoparticles, polymeric micelles, liposomes, polymersomes, polyplexes and hybrid nanoparticles. The effect of microfluidic environment on nanomaterials formation dynamics, as well as the use of microdevices as tools for nanomaterial investigation is also discussed.