Surface modification of PVDF using non-mammalian sources of collagen for enhancement of endothelial cell functionality.

Although polyvinylidene fluoride (PVDF) is non-toxic and stable in vivo, its hydrophobic surface has limited its bio-applications due to poor cell-material interaction and thrombus formation when used in blood contacting devices. In this study, surface modification of PVDF using naturally derived non-mammalian collagen was accomplished via direct surface-initiated atom transfer radical polymerisation (SI-ATRP) to enhance its cytocompatibility and hemocompatibility. Results showed that Type I collagen was successfully extracted from fish scales and bullfrog skin. The covalent immobilisation of fish scale-derived collagen (FSCOL) and bullfrog skin-derived collagen (BFCOL) onto the PVDF surface improves the attachment and proliferation of human umbilical vein endothelial cells (HUVECs). Furthermore, both FSCOL and BFCOL had comparable anti-thrombogenic profiles to that of commercially available bovine collagen (BVCOL). Also, cell surface expression of the leukocyte adhesion molecule was lower on HUVECs cultured on non-mammalian collagen surfaces than on BVCOL, which is an indication of lower pro-inflammatory response. Overall, results from this study demonstrated that non-mammalian sources of collagen could be used to confer bioactivity to PVDF, with comparable cell-material interactions and hemocompatibility to BVCOL. Additionally, higher expression levels of Type IV collagen in HUVECs cultured on FSCOL and BFCOL were observed as compared to BVCOL, which is an indication that the non-mammalian sources of collagen led to a better pro-angiogenic properties, thus making them suitable for blood contacting applications.

Modular micro-PCR system for the onsite rapid diagnosis of COVID-19.

Effective containment of the COVID-19 pandemic requires rapid and accurate detection of the pathogen. Polymerase chain reaction (PCR) remains the gold standard for COVID-19 confirmation. In this article, we report the performance of a cost-effective modular microfluidic reverse transcription (RT)-PCR and RT-loop mediated isothermal amplification (RT-LAMP) platform, Epidax®, for the point-of-care testing and confirmation of SARS-CoV-2. This platform is versatile and can be reconfigured either for screening using endpoint RT-PCR or RT-LAMP tests or for confirmatory tests using real-time RT-PCR. Epidax® is highly sensitive and detects as little as 1 RNA copy per µL for real-time and endpoint RT-PCR, while using only half of the reagents. We achieved comparable results with those of a commercial platform when detecting SARS-CoV-2 viruses from 81 clinical RNA extracts. Epidax® can also detect SARS-CoV-2 from 44 nasopharyngeal samples without RNA extraction by using a direct RT-PCR assay, which shortens the sample-to-answer time to an hour with minimal user steps. Furthermore, we validated the technology using an RT-LAMP assay on 54 clinical RNA extracts. Overall, our platform provides a sensitive, cost-effective, and accurate diagnostic solution for low-resource settings.

Layer-by-layer coated nanoliposomes for oral delivery of insulin.

Crossing the intestinal epithelial cell barrier safely and reaching the blood with therapeutic levels of bioactive insulin have been the ultimate goal of oral insulin delivery. The optimum way to overcome the barrier lies in the design of an efficient high drug loading carrier, that can protect insulin from the harsh Gastrointestinal (GI) environment and enhance its uptake and transport by epithelial cells. In the present study, we developed a multi-layered insulin loading strategy on an anionic nanoliposome surface based on electrostatic interaction with chitosan. The layer-by-layer (LbL) coated nanoliposomes achieved high insulin loading (10.7% by weight) and offered superior protection with limited release in simulated gastric fluid (SGF) (about 6% in 1 h), simulated intestinal fluid (SIF) (2% in two weeks), and phosphate buffered saline (PBS) (5% in two weeks). Intracellular imaging revealed that the LbL coated liposomes were internalized and intracellularly trafficked towards the basolateral side of the Caco-2 monolayer. Transported insulin demonstrated retention of bioactivity while crossing the epithelial barrier in the glucose uptake study in 3T3 L1-MBX adipocytes. In rat studies, oral administration of the formulation resulted in rapid absorption with a peak in plasma insulin levels 0.5 h post oral gavaging. This technology thus serves as a promising platform for potential oral insulin applications.

Adventitial injection delivery of nano-encapsulated sirolimus (Nanolimus) to injury-induced porcine femoral vessels to reduce luminal restenosis.

Endovascular therapy in peripheral intervention has grown exponentially in the past decade, but the issue of high restenosis rates in lower extremity arteries still persist. While drug-coated balloons (DCB) have been the device of choice, recent controversary regarding the long-term safety of paclitaxel have raised concern over current DCBs. In our study, we proposed that the direct injection of a sirolimus nanoliposomal formulation (Nanolimus) using a infusion catheter can attenuate inflammation response in injured vessels. In vitro characterization showed retention of the nanoliposomes size and detectable drug amount up to 336 days in storage. For in vivo study, four female, mixed breed swines were subjected to balloon injury of the femoral arteries before treatment with either injection of saline (n = 4) or Nanolimus (n = 12) using the Bullfrog catheter. Pharmacokinetic analysis demonstrated sustained sirolimus release in the arteries and undetectable systemic drug level at 28 days. Arteries treated with Nanolimus showed significant reduction in neointima area (0.2 ± 0.3 mm2 vs 2.0 ± 1.2 mm2, p < 0.01) and luminal stenosis (14.2 ± 7.2% vs. 67.7 ± 24.8%, p < 0.01) compared to controls. In summary, adventitial delivery of sirolimus using an infusion catheter is a feasible and safe method to reduce vascular restenosis.

Controlled-release nanotherapeutics: State of translation.

This is a review of nanotherapeutic systems, specifically those that exhibit controlled release of the encapsulated bioactive compound. The survey includes the delivery of a range of bioactive compounds, from lipophilic small molecules to hydrophilic proteins and siRNA molecules. The research into enabling sustained delivery of these compounds from nanocarriers has been prolific, but clinical success has been harder to achieve. This is partly because achieving true sustained duration of action over several days is difficult when the carrier dimensions become less than about 400 nm, due to the much shorter diffusion path length compared to micron-sized carrier systems. Other options must be sought to control the efflux of incorporated bioactives, particularly when these bioactives have moderate to high hydrophilicity. A few of these options are discussed critically in this review. We also answer the question: is controlled release needed for nanotherapies? We present the case for controlled release in specific conditions, with two examples from our own work: one for treatment of glaucoma, and the second for inhibition of fibrosis following surgery. The former is sustaining the release of a small-molecule lipophilic drug, while the latter focusses on sustained siRNA delivery.

A bilayer swellable drug-eluting ureteric stent: Localized drug delivery to treat urothelial diseases.

A bilayer swellable drug-eluting ureteric stent (BSDEUS) is engineered and implemented, as a sustained drug delivery platform technology that enhances localized drug delivery to the highly impermeable urothelium, for the treatment of urothelial diseases such as strictures and carcinomas. On deployment, the device swells to co-apt with the ureteric wall and ensure drug availability to these tissues. BSDEUS consists of a stent spray-coated with a polymeric drug containing polylactic acid-co-caprolactone (PLC) layer which is overlaid by a swellable polyethylene glycol diacrylate (PEGDA) based hydrogel. In-vitro quantification of released drug demonstrated a tunable time-profile, indicating sustained delivery over 1-month. The PEGDA hydrogel overlayer enhanced drug release and transport into explanted porcine ureteric tissues ex-vivo, under a simulated dynamic fluid flow. A preliminary pilot in-vivo feasibility study, in a porcine model, demonstrated that the swollen hydrogel co-apts with the urothelium and thus enables localized drug delivery to the target tissue section. Kidney functions remained unaffected and device did not result in either hydronephrosis or systemic toxicity. This successful engineering of a bilayer coated stent prototype, demonstrates its feasibility, thus offering a unique solution for drug-based urological therapy.

Materials technology in drug eluting balloons: Current and future perspectives.

The coating material technology is important for the delivery of anti-proliferative drugs from the surface of drug-eluting balloons (DEBs), which are emerging as alternatives to drug-eluting stents (DES) in the field of interventional cardiology. Currently, several shortcomings limit their competition with DES, including low drug transfer efficiency to the arterial tissues and undesirable particulate generation from the coating matrix. In this review, we provide a survey of the materials used in existing DEBs, and discussed the mechanisms of actions of both the drugs and coating materials. The type of drug and the influence of the coating material characteristics on the drug uptake, distribution and retention in arterial tissues are described. We also summarize the novel coating excipients under development and provide our perspective on the possible use of nano-scale carriers to address the shortcomings of current coating technology. The scope of this review includes only materials that have been approved for biomedical applications or are generally recognized as safe (GRAS) for drug delivery.

Development of a miniaturized stimulation device for electrical stimulation of cells.

BACKGROUND: Directing cell behaviour using controllable, on-demand non-biochemical methods, such as electrical stimulation is an attractive area of research. While there exists much potential in exploring different modes of electrical stimulation and investigating a wider range of cellular phenomena that can arise from electrical stimulation, progress in this field has been slow. The reasons for this are that the stimulation techniques and customized setups utilized in past studies have not been standardized, and that current approaches to study such phenomena rely on low throughput platforms with restricted variability of waveform outputs. RESULTS: Here, we first demonstrated how a variety of cellular responses can be elicited using different modes of DC and square waveform stimulation. Intracellular calcium levels were found to be elevated in the neuroblast cell line SH-SY5Y during stimulation with 5 V square waves and, stimulation with 150 mV/mm DC fields and 1.5 mA DC current resulted in polarization of protein kinase Akt in keratinocytes and elongation of endothelial cells, respectively. Next, a miniaturized stimulation device was developed with an integrated cell chamber array to output multiple discrete stimulation channels. A frequency dividing circuit implemented on the device provides a robust system to systematically study the effects of multiple output frequencies from a single input channel. CONCLUSION: We have shown the feasibility of directing cellular responses using various stimulation waveforms, and developed a modular stimulation device that allows for the investigation of multiple stimulation parameters, which previously had to be conducted with different discrete equipment or output channels. Such a device can potentially spur the development of other high throughput platforms for thorough investigation of electrical stimulation parameters on cellular responses.

Polymer-Enriched 3D Graphene Foams for Biomedical Applications.

Graphene foams (GFs) are versatile nanoplatforms for biomedical applications because of their excellent physical, chemical, and mechanical properties. However, the brittleness and inflexibility of pristine GF (pGF) are some of the important factors restricting their widespread application. Here, a chemical-vapor-deposition-assisted method was used to synthesize 3D GFs, which were subsequently spin-coated with polymer to produce polymer-enriched 3D GFs with high conductivity and flexibility. Compared to pGF, both poly(vinylidene fluoride)-enriched GF (PVDF/GF) and polycaprolactone-enriched GF (PCL/GF) scaffolds showed improved flexibility and handleability. Despite the presence of the polymers, the polymer-enriched 3D GF scaffolds retained high levels of electrical conductivity because of the presence of microcracks that allowed for the flow of electrons through the material. In addition, polymer enrichment of GF led to an enhancement in the formation of calcium phosphate (Ca-P) compounds when the scaffolds were exposed to simulated body fluid. Between the two polymers tested, PCL enrichment of GF resulted in a higher in vitro mineralization nucleation rate because the oxygen-containing functional group of PCL had a higher affinity for Ca-P deposition and formation compared to the polar carbon-fluorine (C-F) bond in PVDF. Taken together, our current findings are a stepping stone toward future applications of polymer-enriched 3D GFs in the treatment of bone defects as well as other biomedical applications.

Endothelial cell thrombogenicity is reduced by ATRP-mediated grafting of gelatin onto PCL surfaces.

Reducing the thrombogenicity of a tissue-engineered vascular graft prior to implantation is important for improving graft patency. As functionalization of synthetic materials with cell-adhesive proteins is routinely utilized as a means to promote endothelial cell (EC) growth, we conducted detailed investigation on the proliferation and thrombogenicity of ECs on such functionalized surfaces. We observed that polycaprolactone (PCL) surfaces functionalized with poly(glycidyl methacrylate) [(P(GMA)] brushes via atom transfer radical polymerization (ATRP) alone resulted in the enhancement of an activated EC profile characterized by low production of nitric oxide (NO), platelet activation and elevated expression levels of von Willebrand factor (vWF) and matrix metalloproteinase-2 (MMP-2). When gelatin was conjugated onto the PCL-g-P(GMA) surfaces, not only were EC proliferation and endothelial coverage significantly improved, but an anti-thrombogenic profile was also observed. We demonstrated that PCL can be successfully functionalized by a controllable surface-initiated polymerization method and importantly, the thrombogenic profile of the endothelial cells can be influenced by material surface chemistry (e.g. the presence of polymer graft chains). Our findings emphasize the importance of a careful consideration of materials for vascular graft applications, as well as differential endothelial cell physiology on surfaces with different material chemistry.