Invasive and non-invasive electrodes for successful drug and gene delivery in electroporation-based treatments

Electroporation is an effective physical method for irreversible or reversible permeabilization of plasma membranes of biological cells and is typically used for tissue ablation or targeted drug/DNA delivery into living cells. In the context of cancer treatment, full recovery from an electroporation-based procedure is frequently dependent on the spatial distribution/homogeneity of the electric field in the tissue; therefore, the structure of electrodes/applicators plays an important role. This review focuses on the analysis of electrodes and in silico models used for electroporation in cancer treatment and gene therapy. We have reviewed various invasive and non-invasive electrodes; analyzed the spatial electric field distribution using finite element method analysis; evaluated parametric compatibility, and the pros and cons of application; and summarized options for improvement. Additionally, this review highlights the importance of tissue bioimpedance for accurate treatment planning using numerical modeling and the effects of pulse frequency on tissue conductivity and relative permittivity values

Epidermal sensors for monitoring skin physiology

Wearable sensors are revolutionizing personalised healthcare and have continuously progressed over the years in both research and commercialization. However, most efforts on wearable sensors have been focused on tracking movement, spatial position and continuous monitoring of vital signs such as heart rate or respiration rate. Recently, there is a demand to obtain biochemical information from the body using wearables. This demand stems from an individuals’ desire for improved personal health awareness as well as the drive for doctors to continuously obtain medical information for a patients’ disease management. Epidermal sensors are a sub-class of wearable sensors that can intimately integrate with skin and have the potential for monitoring physical changes as well as detecting biomarkers within skin that can be related to human health. The holy grail for these types of sensors is to achieve continuous real-time monitoring of the state of an individual and the development of these sensors are paving the way towards personalised healthcare. However, skin is highly anisotropic which makes it challenging to keep epidermal sensors in consistent contact with skin. It is important that these sensors remain in contact with skin in order to measure its electrical properties and acquire high fidelity signals. The key objective of this thesis is to develop thin conformable, stretchable epidermal sensors for tracking changes in skin physiology. The initial iteration of the screen printed epidermal sensor comprised of a flexible silver film. Impedance spectroscopy was used to understand the electrical signals generated on skin and it was used to measure relative changes due to varying water content. However, this iteration was more suited for single use. The next chapters explore different ink formulations and adherence methodologies to enhance the epidermal sensors adherence to skin. Impedance spectroscopy was used to characterise the electrical signals from these different epidermal sensor iterations, while tensile testing and on-body assessment was used to characterise its mechanical properties. The final chapter focused on investigating the use of phenyl boronic acid (PBA) functionalized hydrogels to modify the epidermal sensor with responsive hydrogel materials to enable chemical sensing of analytes relevant to skin physiology. Impedance spectroscopy was used to characterise and understand the electrical signals generated by the binding interaction of the PBA and analytes using the sensor. Overall, the work demonstrates the challenges of developing these epidermal sensors as well as presenting their potential for continuous monitoring of human skin in the future

Advanced Electrohydrodynamic Atomisation Engineered Microneedle Coatings

Transdermal drug delivery (TDD) is an emerging field in the pharmaceutical remit compared to conventional methods (oral and parenteral). Microneedle (MN) based devices have gained significant interest as a strategy to overcome the skins formidable barrier; the stratum corneum and enhance drug delivery. The research presented here shows the successful coating of MNs with polymeric dye composites using Electrohydrodynamic atomisation (EHDA). Initially the rheological behaviours of polymeric solutions and morphological characterisation was carried out. This was followed by Quality by Design (QBD) implementation for the optimisation of the key process parameters in EHDA. Here, the electrosprayed nanoparticles and electrospun nano/micro fibres consisted of a polymeric matrix and dye. Eight formulations were assessed consisting of 5% w/v of polycaprolactone (PCL) in dichloromethane (DCM) and 5% w/v polyvinylpyrrolidone (PVP) in ethanol. A full factorial Design of Experiments (DoE) was used to assess the various parameters (applied voltage, deposition distance and flow rate). Further particle and fibre analysis was carried out using Scanning Electron Microscopy (SEM), Differential Scanning Calorimetry (DSC), Fourier Transform Infrared Spectroscopy (FTIR), particle/fibre size distribution. In addition to this, in vitro release studies were carried out using fluorescein (FL) and rhodamine B (RhB) as model dyes and in vitro permeation studies were applied. From optimising EHDA more than 52% of particles were under 500 nm and fibres were in the micron range. In vitro drug release studies showed 100% drug release after 7 days for PCL particles and fibres. It also showed 100% drug release within 120 min for PVP particles and 300 minutes for PVP fibres. The release kinetics and the permeation study showed that the MN successfully pierced the membrane and the Es and Esp MN coatings released a large amount of the loaded drug within 6 hours for all formulations. This study has demonstrated the capability of these robust MNs to encapsulate a diverse range of drugs within a polymeric matrix giving rise to the potential of developing personalised medical devices

3D printed Microneedles for Transdermal Drug Delivery

3D printing is a revolutionary manufacturing and prototyping technology that has altered the outlooks of numerous industrial and scientific fields since its introduction. Recently, it has attracted attention for its potential as a manufacturing tool for transdermal microneedles for drug delivery. In the present thesis, the 3D printability of solid and hollow microneedles via photopolymerisation-based 3D printing was investigated, aiming at establishing robust manufacturing strategies for reproducible, mechanically strong and versatile microneedles. The developed microneedles were employed as drug delivery systems for the treatment of diabetes via insulin administration. Solid microneedles featuring different geometries were designed and 3D printed. It was demonstrated that the printing and post-printing parameters affected the printed quality, a finding that was employed to optimise the manufacturing strategy. Microneedle geometry was also found to have an impact on the piercing and fracture behaviour; however all microneedle designs were found to be mechanically safe upon application. The solid microneedles were subsequently coated with insulin-polymer films, using a 2D inkjet printing technology. The coating process achieved spatial control of the drug deposition, with quantitative accuracy. The microneedle geometry was shown to influence the morphology of the coating film, an effect that was pronounced during in the in vitro delivery studies of insulin to porcine skin. Furthermore, hollow microneedles were designed and 3D printed, featuring different heights. Two photopolymerisation-based technologies were studied, and their performance was compared. The key influential parameters of the printing outcome and microneedle quality were identified to be the printing angle and the size of the microneedle opening. The hollow microneedles were found to be effective in piercing porcine skin without structural damaging. The hollow microneedles were incorporated into complex patches with internal microfluidic structures for the provision and distribution of drug-containing solutions. The developed complex hollow microneedle patches were coupled with a microelectromechanical system to create a novel platform device for controlled, personalised transdermal drug delivery. Advanced imaging techniques revealed that the device achieved distribution of the liquid within porcine skin tissue without the creation of depots that would delay absorption. The device was evaluated for its efficacy to transdermally deliver a model dye and insulin in vitro. In vivo trials were also conducted using diabetic rodents, with the device achieving faster onset of insulin action and sustained glycemic control, in comparison to subcutaneous injections. Overall, the findings of the present research are anticipated to elucidate key problematic areas associated with the application of 3D printing for microneedle manufacturing and propose feasible solutions. The outermost goal of this work is to contribute to the advancement of knowledge in the field of 3D printed transdermal drug delivery systems, in order to bring them one step closer to their adoption in the clinical setting

Real-time bioimpedance measurements of stem cellbased disease models-on-a-chip

In vitro disease models are powerful platforms for the development of drugs and novel therapies. Stem-cell based approaches have emerged as cutting-edge tools in disease modelling, allowing for deeper insights into previously unknown disease mechanisms. Hence the significant role of these disease-in-a-dish methods in therapeutics and translational medicine. Impedance sensing is a non-invasive, quantitative technique that can monitor changes in cellular behaviour and morphology in real-time. Bioimpedance measurements can be used to characterize and evaluate the establishment of a valid disease model, without the need for invasive end-point biochemical assays. In this work, two stem cell-based disease models-on-a-chip are proposed for acute liver failure (ALF) and age-related macular degeneration (AMD). The ALF disease model-on-a-chip integrates impedance sensing with the highly-differentiated HepaRG cell line to monitor in real-time quantitative and dynamic response to various hepatotoxins. Bioimpedance analysis and modelling has revealed an unknown mechanism of paracetamol hepatotoxicity; a temporal, dose-dependent disruption of tight junctions (TJs) and cell-substrate adhesion. This disruption has been validated using ultrastructural imaging and immunostaining of the TJ-associated protein ZO-1. Age-related macular degeneration (AMD) is the leading cause of blindness in the developed world with a need for disease models for its currently incurable forms. Human induced pluripotent stem cells (hiPSCs) technology offers a novel approach for disease modelling, with the potential to impact translational retinal research and therapy. Recent developments enable the generation of Retinal Pigment Epithelial cells from patients (hiPSC-RPE), thus allowing for human retinal disease in vitro studies with great clinical and physiological relevance. In the current study, the development of a tissue-on- a-chip AMD disease model has been established using RPE generated from a patient with an inherited macular degeneration (case cell line) and from a healthy sibling (control cell line). A reproducible Electric Cell-substrate Impedance Sensing (ECIS) electrical wounding assay was conducted to mimic RPE damage in AMD. First, a robust and reproducible real-time quantitative monitoring over a 25-day period demonstrated the establishment and maturation of RPE layers on microelectrodes. A spatially-controlled RPE layer damage that mimicked cell loss in AMD was then initiated. Post recovery, significant differences in migration rates were found between case and control cell lines. Data analysis and modelling suggested this was due to the lower cell-substrate adhesion of the control cell line. These findings were confirmed using cell adhesion biochemical assays. Moreover, different-sized, individually-addressed square microelectrode arrays with high spatial resolution were designed and fabricated in-house. ECIS wounding assays were performed on these chips to study immortalized RPE migration. Migration rates comparable to those obtained with ECIS circular microelectrodes were determined. The two proposed disease-models-on-a-chip were then used to explore the therapeutic potential of the antioxidant N-Acetyl-Cysteine (NAC) on hiPSC-RPE and HepaRG cell recovery. Addition of 10 mM NAC at the end of a 24h paracetamol challenge caused a slight increase in the measured impedance, suggesting partial cell recovery. On the other hand, no effect on case hiPSC-RPE migration has been observed. More experiments are needed to examine the effect of different NAC concentrations and incubation periods. The therapeutic potential of electrical stimulation has also been explored. A preliminary study to evaluate the effect of electrical stimulation on RPE migration has been conducted. An externally applied direct current electric field (DC EF) of 300 mV/mm was found to direct the migration of the immortalized RPE cell line (hTERT-RPE1) perpendicular to the EF. The cells were also observed to elongate and to realign their long axes perpendicular to the applied EF. The proposed tissue-on-a-chip disease models are powerful platforms for translational studies. The potential of such platforms has been demonstrated through revealing unknown effects of acetaminophen on the liver as well as providing deeper insights into the underlying mechanisms of macular degeneration. Combining stem cell technology with impedance sensing provides a high throughput platform for studying patient-specific diseases and evaluating potential therapies

Nanomedicine applications mediated by electromagnetic fields

Recently, the introduction of nanotechnologies into medical applications has become more frequent due to the growing of several diseases originating from alteration of biological processes at molecular and nanoscale level (e.g. mutated genes, cell malfunction due to viruses or bacteria). The nanomedicine combines the innovation of the nanotechnology materials (shape and size of nm scale) to health care, providing new promising techniques for the diagnosis, the prevention, the tissue regeneration and therapeutic fields. Disorders like cancer, Alzheimer’s, Parkinson’s disease, cardiovascular problems or inflammatory diseases are serious challenges to be dealt with. For this reason researches are focusing their attention to the nanomaterials unique properties [Murty et al., 2013, Xia et al., 2009]. The progress in nanomedicine ranges from nanoparticles for molecular diagnostics, imaging and therapy to integrated medical nanosystems [Nune et al., 2009, Shi, 2009] to act at the cellular level inside the body. For a recent review on challenges, opportunities, and clinical applications in nanomedicine an interesting review is the one of Wicki et al. [Wicki et al., 2015]. Despite the concerns raised by the authors in their review, the expert opinion on clinical opportunities finds a generalized consensus on stimuli-responsive systems for targeting the compound (drug, gene, biomolecule) at the site of interest and on the use of lipid based nanosystems for the biocompatible platform to be used in clinical trials. In this scenario is placed the main activity of this Ph.D. thesis whose aim is to provide a multiscale and multidisciplinary approach to demonstrate the capability to activate lipid-based nanosystems by means of electromagnetic fields (EMFs). Specifically, the attention will be focused, on a first part, on the liposome-based systems mediated by EMF to provide a proof-of-concept of EMF stimuli-response systems for applications of drug delivery. This aspect will be approached both form a theoretic, technological and experimental point of view. Moreover, because proteins are considered a fundamental pattern as bio-sensors for signaling cell processes, a molecular dynamics simulation approach will be provided to study the interaction mechanisms between EMFs and proteins structures for potential protein activation

Minimally invasive clinical monitoring and data transference in cardiac patients

'Wet' electrodes used in electrocardiography (ECG), are applied to the surface of the skin to record cardiac activity. Over time, water-based electrolytic gels between the electrodes and skin dehydrate, reducing signal quality. Microneedle-electrodes negate the need for conductive gels and potentially improve signal fidelity by circumventing the stratum corneum and contacting the underlying conductive epidermal layers. This thesis aimed to assess the wearability and functionality of microneedle-electrodes in cardiac signal acquisition. Epoxy, 500μm-length microneedles were applied to excised skin models to assess insertion performance. Increasing downward application force increased microneedle penetration efficiency from 79%±8.20 (5N) to 87%±13.32 (15N). The microneedle application technique also had an impact on penetration efficiency, with impact insertion (93%±5.75) proving more effective than manual downward force (71%±22.01). Metallised versions of the epoxy microneedles were integrated into a commercial electrode and compared to conventional wet electrodes in human volunteers. Wet electrodes recorded higher quality signals than microneedle-electrodes in healthy human participants (1.6dB difference between the electrode types). This clinical data informed the development of an in vitro laboratory skin model to assess the influence of microneedle-electrode parameters on a simulated ECG signal. Increasing microneedle length from 500μm (25.2dB±3.25) to 600μm (24.3dB±2.31) did not result in a sustained improvement in signal quality (p>0.05). Bespoke second-generation microneedle-electrodes were manufactured allowing an improved signal quality to be maintained over the recording period (17.3dB±2.11 compared to 15.0dB±1.97 for wet electrodes; p>0.05) in the laboratory model. Human participant studies assessed their wearability and functionality. At rest, the metallised epoxy (23.2dB±5.79) and bespoke (22.5dB±7.57) microneedle-electrode performance was comparable to wet electrodes (24.9dB±6.44) (p>0.05). Under active conditions, the signal-to-noise ratio declined for all electrodes and ECG traces highlighted increased motion artifacts. Participants preferred wet electrodes and highlighted seven key wearability themes. Further optimisation of microneedle-electrodes for ECG monitoring is therefore, warranted