Micro-needle platforms are the core components of many recent drug delivery and gene-editing techniques, which allow for intracellular access, controlled cell membrane stress or mechanical trapping of the nucleus. This dissertation work is devoted to the development of micro-needle platforms that offer customized fabrication and new capabilities for enhanced cell analyses. The highest degree of geometrical flexibility is achieved with 3D printed micro-needles, which enable optimizing the topographical stress environment for cells and cell populations of any size. A fabrication process for 3D-printed micro-needles has been developed as well as a metal coating technique based on standard sputter deposition. This extends the functionalities of the platforms by electrical as well as magnetic features. The micro-needles have been tested on human colon cancer cells (HCT116), showing a high degree of biocompatibility of the platform. Moreover, the capabilities of the 3D-printed micro-needles have been explored for drug delivery via the well-established electroporation technique, by coating the micro-needles with gold. Antibodies and fluorescent dyes have been delivered to HCT116 cells and human embryonic kidney cells with a very high transfection rate up to 90%. In addition, the 3D-printed electroporation platform enables delivery of molecules to suspended cells or adherent cells, with or without electroporation buffer solution, and at ultra-low voltages of 2V. In order to provide a micro-needle platform that exploits existing methods for mass fabrication a custom designed template-based process has been developed. It has been used for the production of gold, iron, nickel and poly-pyrrole micro-needles on silicon and glass substrates. A novel delivery method is introduced that activates the micro-needles by electromagnetic induction, which enables to wirelessly gain intracellular access. The method has been successfully tested on HCT116 cells in culture, where a time-dependent delivery rate has been found. The electromagnetic delivery concept is particularly promising for future in-vivo applications. Finally, the micro-needle platforms developed in this work will provide researchers with new capabilities that will help them to further advance the field of mechanobiology, drug delivery treatments, stem cells research and more. The proposed platforms are capable of applying various stimuli, analyzing cell responses in real time, drug delivery, and they also have the potential to add additional functionalities in the future.

Naloxone (NAL) is administered parenterally or intranasally for treating opioid overdose. The short duration of action of NAL calls for frequent re-dosing which may be eliminated by the development of a transdermal system. This study aimed to assess the effect of microneedles on improving the skin permeation of NAL hydrochloride. In vitro permeation of NAL across intact and microneedle-treated (Dr. Pen™ Ultima A6) porcine skin was evaluated. The effect of microneedle length and application duration, and donor concentration on NAL permeation were investigated. In-vitro in-vivo correlation of the permeation results was done to predict the plasma concentration kinetics of NAL in patients. In vitro passive permeation of NAL after 6 h was observed to be 8.25±1.06 µg/cm2. A 56- and 37-fold enhancement was observed with 500 and 250 µm needles applied for 1 min, respectively. Application of 500 µm MNs for 2 min significantly reduced the lag time to ~ 8 min and increasing the donor concentration for the same treatment group doubled the permeation (p < 0.05). Modeling simulations demonstrated the attainment of pharmacokinetic profile of NAL comparable to those obtained with the FDA-approved intramuscular and intranasal devices. Microneedle-mediated transdermal delivery holds potential for rapid and sustained NAL delivery for opioid overdose treatment.

The ability to inject DNA and other foreign particles into cells, both germ cells (e.g. to produce transgenic animals) and somatic cells (e.g. for gene therapy), is a powerful tool in genetic research. Nanoinjection is a method of DNA delivery that combines mechanical and electrical methods. It has proven to have higher cell viability than traditional microinjection, resulting in higher integration per injected embryo. The nanoinjection process can be performed on thousands of cells simultaneously using an array of microneedles that is inserted into a monolayer of cells. This thesis describes the needle array design requirements and the fabrication process used to meet them. The process uses unpassivated and passivated deep reactive ion etching (DRIE) to create needles with a constant diameter shaft and a pointed tip. The needle diameter and height are about 1 µm and 8 µm, respectively. A buckling analysis and physical testing show that the needles can withstand the force required to penetrate the cells. The chip is attached to a plastic suspension with a counter electrode and electrical connections to a voltage source. The suspension's motion is defined by two compliant orthoplanar springs that have been vertically and rotationally offset for added stability. The base of the suspension is designed to exactly fit in the bottom of a cell culture dish, where the needle array can be pushed into the cell monolayer. Injection protocol was created and followed to perform tests with needle insertion only, voltage application only, and the full nanoinjection process. The average cell viability for the full injection process was 98.2% compared to an average control viability of 99.5%. Zero volt injections with a high concentration of propidium iodide, a cell impermeable dye with two positive charges, resulted in dye uptake from diffusion, proving that the needles are penetrating the cells. Tests comparing injections with and without voltage had high variability in dye uptake. Therefore, glass cover slips were placed in the culture dishes to provide more consistent injection conditions. This reduced variation in zero voltage tests. It is recommended that this procedure be followed for performing injections with voltage.

Multiple sclerosis (MS) is a chronic neurological disease where immune cells from the periphery enter the central nervous system and attack the myelin sheaths resulting in damage to the axons. It is an unpredictable disease that affects each person differently. One of the symptoms of MS includes muscle spasticity and depending on the severity it can cause gait and mobility issues. Oral baclofen is the first-line recommendation to treat spasticity in people with MS whose treatment goals include improving mobility or easing pain and discomfort. Baclofen works pre- and post-synaptically as a gamma aminobutyric acid-B agonist at the spinal cord to reduce the amount of excitatory neurotransmitters that are responsible for muscle contractions. Oral baclofen has been used to treat spasticity, however its short half-life of 2-6 hours warrants its need for three times a day dosing schedule. The multiple daily dosing can be a burden to the patient taking it and for the caregiver who will be giving the medication which can negatively affect adherence and acceptability. The goal of the current study was to design and assess baclofen loaded dissolving microneedle (MN) patch on the amount permeated and sustained delivery of baclofen in the management of spasticity. A 1.5 mg/mL MN patch was initially made by dissolving baclofen in polyvinylpyrrolidone (PVP) and fabricated using the mold casting technique. The permeation profile of fabricated baclofen loaded MNs over a predetermined time was assessed in vitro through dermatomed porcine ear skin using Franz Diffusion cells. Employing the use of MNs significantly increased the flux from 2.10 ± 0.35 µg/sq.cm/h to 5.92 ± 4.17 µg/sq.cm/h and the average cumulative amount permeated from 92.67 ± 11.25 µg/sq.cm to 457.11 ± 111.47 µg/sq.cm over 72 h (p<0.05). However, because of baclofen’s hydrophilic nature, drug permeation across skin is limited, so a nanosuspension with reduced particle size was formulated to be used for MN fabrication to increase drug loading and permeation. Baclofen was formulated into a suspension through wet media milling followed by mechanical homogenization using PVP K30 and sodium dodecyl sulfate as stabilizers. The suspension was successfully loaded in dissolving PVP-based MNs. The donor chamber of the cell contained the MN patch mounted onto the porcine skin and the receptor chamber contained phosphate buffered saline. The receptor was sampled over 72 h and analyzed using HPLC. The baclofen suspension loaded MNs produced an average drug flux of 26.51 ± 4.17 µg/sq.cm/h and an average cumulative amount of 1718.96 ± 217.12 µg/sq.cm over 72 h. The use of suspension loaded MNs was found to enhance the permeation and produce a sustained delivery of baclofen across skin and depicts the applicability of fabricated needles for sustained delivery in the management of muscle spasticity in multiple sclerosis.

No description available.

Skin is the largest immune organ and an ideal site to administer vaccines. However, by nature, skin is not permeable to antigens, which are macromolecules. The major hurdle in skin permeation is the outermost stratum corneum layer. Microneedles have proven feasible to create micron-sized channels in the epidermis of the skin, through which protein and plasmid DNA antigens can penetrate into the viable skin epidermis and dermis. However, the immune responses induced by microneedle-mediated transcutaneous immunization with protein or plasmid DNA alone are generally weak, and a vaccine adjuvant is often required to induce strong immune responses. Data from numerous previous studies have shown that nanoparticles as a vaccine carrier can significantly enhance the immunogenicity of antigens, but the feasibility of utilizing nanoparticles as a vaccine carrier to enhance the immune responses induced by microneedle-mediated transcutaneous immunization has rarely been studied. In this dissertation, using protein antigen (OVA) chemically conjugated onto the surface of solid-lipid nanoparticles and plasmid DNA (pCMV-beta, pVax/opt-BoNT/C-Hc50, and pCI-neo-sOVA) physically coated on the surface of cationic polymeric nanoparticles, we showed that the immune responses induced by microneedle-mediated transcutaneous immunization with protein antigens or plasmid DNA vaccines are significantly enhanced by delivering the proteins and plasmid DNA with nanoparticles. Importantly, microneedle-mediated transcutaneous immunization with proteins or plasmid DNA induces not only systemic immune responses, but also mucosal immune responses. In addition, it is generally believed that microneedles are safe. However, it remained unclear whether the micropores created by microneedles on the skin will also facilitate the permeation of microbes such as bacteria into the skin. In this dissertation, we also designed an unique ex vivo model to evaluate the permeation of live bacteria through mouse skin pretreated with microneedles. The results demonstrated that the risk of potential bacterial infection associated with microneedle treatment is not greater than that associated with a hypodermic needle injection. / text

The major hurdle in the development of transdermal route as a versatile drug delivery method is the formidable transport barrier provided by the stratum corneum. Despite decades of research to overcome the stratum corneum barrier, limited success has been achieved. The objectives of this research were to develop and characterize two different strategies to overcome the stratum corneum barrier for transdermal delivery of biopharmaceuticals and vaccines. In the first strategy, coated microneedles (sharp-tipped, micron-sized structures) were developed to enable delivery of drugs directly into the skin by bypassing the stratum corneum barrier. In the second strategy, instead of bypassing the barrier, microdermabrasion was used to selectively abrade stratum corneum with sharp microparticles for topical drug application. Coated microneedles For developing painless microneedles, the first detailed study was performed to characterize the effect of microneedle geometry on pain caused by microneedle insertions in human volunteers. This study demonstrated that microneedles are significantly less painful than a 26-gage hypodermic needle and that decreasing microneedle length and numbers reduces pain. Next, the first in-depth study of microneedle coating methods and formulations was performed to (i) develop a novel micron-scale dip-coating process, (ii) test the breadth of compounds that can be coated onto microneedles, and (iii) develop a rational basis to design novel coating formulations based on the physics of dip-coating. Finally, a plasmid DNA-vaccine was coated onto microneedles to immunize mice, to provide the first evidence that microneedle-based skin immunization can generate a robust in vivo antigen-specific cytotoxic-T-lymphocyte response using similar, or lower, DNA doses on microneedles as when using the gene gun or intramuscular injection. Microdermabrasion We demonstrated for the first time that microdermabrasion in monkeys and humans can selectively, yet completely remove the stratum corneum layer. Using a mobile mode of microdermabrasion, an increase in the number of treatment passes led to greater tissue removal. Furthermore, topical application of Modified Vaccinia Ankara virus after microdermabrasion induced virus-specific antibodies in monkeys. In conclusion, both coated microneedles and microdermabrasion were developed to enable delivery of biomolecules into the skin, indicating their potential for transdermal delivery of a wide range of biopharmaceuticals and vaccines.

Flexible and stretchable wearable biomedical devices provide a platform for continues long-term monitoring of biological signals during neutral body movements thus enabling early intervention and diagnostics of various diseases. This thesis evaluates novel flexible and stretchable bio interfacing medical devices based on microneedle patches and split ring resonator for healthcare diagnostics. Flexible and stretchable microneedle patches were realized by integrating a soft polymer substrate with sharp stainless steel microneedles. This was realized using a magnetic assembly technique. Investigations have shown that the flexible microneedle patch can provide conformal and reliable contact with wrinkles and deformations of the skin. In addition, transdermal monitoring of potassium ions using the proposed flexible microneedle patch have been demonstrated by coating the microneedles with a potassium sensing membrane. Ex-vivo test on the microneedle potassium sensor performed on chicken and porcine skin was able to detect change in potassium concentration in the skin. Furthermore, a novel flexible bio-interface spilt ring resonator (SRR) for the monitoring of intera cranial pressure (ICP) is demonstrated. The sensor was fabricated by depositing a 500 nm gold film on a thermoset thiolene epoxy polymer substrate. The flexible sensor was able to clearly detect the pressure variation that might be an indication of increased ICP in the skull. The proposed methodology of heterogeneous integration of hard materials on a soft and flexible substrate demonstrates a first proof of concept of flexible wearable bio-interfacing devices with vastly different material properties with the potential for continuous and real-time health monitoring. / <p>QC 20190306</p>

Gene gun assisted micro-particle delivery system is an excellent method for the delivery of DNA into target tissue so as to carry out gene transfection in the target cells. The gene gun is primarily a particle accelerator which accelerates DNA-coated micro-particles to sufficient velocities to breach the target layer enabling the micro-particles to penetrate to a desired depth and target the cells of interest to achieve gene transfer. However, an inevitable problem in this process is the tissue/cell damage due to the impaction of the pressurized gas and micro-particles on the target. The purpose of this research is developing a new conceptual system which improves the penetration depth of micro-particles at less imposed pressure and particle injection velocity. This is achieved by applying a microneedle array and ground slide in the gene gun system, thus a study involving microneedle assisted micro-particle delivery is conducted in this work. Microneedle array is used to create holes in the target which allows a number of micro-particles to penetrate through the skin which enhances the penetration depth inside target. The ground slide is used to load a pellet of the micro-particles and prevent the pressurized gas to avoid the impaction on the target. The operation principle is that the pellet is attached to ground slide which is accelerated to a sufficient velocity by the pressurized gas. The pellet is released from the ground slide which separates into individual micro-particles by a mesh and penetrates to a desired depth inside the target. An experimental rig to study various aspects of microneedle assisted micro-particle delivery is designed in this PhD research. The passage percentage of the micro-particles and size of the separated micro-particles are analysed in relation to the operating pressure, mesh pore size and Polyvinylpyrrolidone (PVP) concentration to verify the applicability of this system for the micro-particle delivery. The results have shown that the passage percentage increases from an increase in the mesh pore size and operating pressure and a decrease in PVP concentration. A mesh pore size of 178 μm and pellet PVP concentration of 40 mg/ml were used for the bulk of the experiments in this study as these seem to provide higher passage percentage and the narrow size distribution of the separated micro-particles. In addition, the velocity of the ground slide is detected by the photoelectric sensor and shown that it increases from an increase in operating pressure and reaches 148 m/s at 6 bar pressure, A further analysis in the penetration depths of the micro-particles to determine whether they achieve enhanced penetration depths inside the target after using microneedles is carried out. A skin mimicked agarose gel is obtained from comparing the viscoelastic properties of various concentration of agarose gel in comparison with the porcine skin, which is assumed to mimic the human skin. These experiments are used to relate the micro-particle penetration depth with the operating pressure, microneedle length and particle size. In addition, a theoretical model is developed based on the experimental data to simulate the microneedle assisted micro-particle delivery which provide further understanding of the microneedle assisted micro-particle delivery. The developed model was used to analyse the penetration depth of micro-particles in relation to the operation pressure, target properties, microneedle length and particle size and density. The modelling results were compared with the experimental results to verify the feasibility of the microneedle assisted micro-particle delivery for micro-particles delivery. As expected, both experimental and theoretical results show that the micro-particles achieve an enhanced penetration depth inside target. The maximum penetration depth of micro-particles is increased from an increase in operating pressure, microneedle length, particle size and density.

The present study was divided between two different drug delivery goals, each involving naltrexone (NTX) or its active metabolite, 6-β-naltrexol (NTXOL). First, amino acid esters of NTX and NTXOL were prepared in order to test their candidacy for microneedle-enhanced transdermal delivery. Second, a 3-O-(-)-cytisine-naltrexone (CYT-NTX) codrug was prepared for screening as a potential oral delivery form of NTX and (-)-cytisine (CYT). The amino acid prodrugs were intended for the treatment of alcohol abuse, while the codrug was designed as a single agent for the treatment of alcoholism and tobacco-dependency co-morbidities. One hypothesis of this work was that prodrugs of NTX or NTXOL can be designed that possess superior skin transport properties through microneedle-treated skin compared to parent NTX or NTXOL. Nine amino acid ester prodrugs were prepared, and only three 6-O amino acid ester prodrugs of NTXOL were stable enough at skin pH (pH 5.0) to move forward to studies in 50% human plasma. 6-O-β-Ala-NTXOL, the lead compound, exhibited the most rapid bioconversion to NTXOL in human plasma (t1/2 = 2.2 ± 0.1 h); however, this in vitro stability value indicates that the prodrug may require hepatic enzyme-mediated hydrolysis for sufficiently rapid bioconversion to NTXOL in vivo. A second hypothesis of this work was that a CYT-NTX codrug could be designed with appropriate stability characteristics for oral delivery. CYT-NTX was found to be stable over the time course of 24 h in buffer systems of pH 1.5, 5.0, 7.4 and 9.0, and in 80% rat plasma, 80% human plasma, simulated gastric fluid and simulated intestinal fluid. Six (3 rats/group) Sprague-Dawley male rats were dosed i.v. with 1 mg/kg CYT-NTX codrug, or 10 mg/kg, p.o. Oral administration of a 10 mg/kg dose of CYT-NTX codrug resulted in rapid absorption and distribution (5 min) of CYT-NTX codrug, and NTX was released from codrug with a peak plasma concentration of 6.8 ± 0.9 nmol/L reached within 65 minutes. Plasma CYT was not detected; however, NTX delivery was achieved with a fraction absorbed value of 13%. Thus, CYT-NTX may hold promise as a potential oral codrug for further optimization and development.