Rapidly Dissolving Polymeric Microneedle Skin Patch of Naloxone for Opioid Overdose Treatment Tijani Akeemat1, Maria J. Peláez2, Prashant Dogra2,3, Ashana Puri1 1 Department of Pharmaceutical Sciences, Bill Gatton College of Pharmacy, East Tennessee State University, Johnson City, TN 37614. 2 Mathematics in Medicine Program, Houston Methodist Research Institute, Houston, TX 77030, USA 3 Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY 10065, USA Worldwide opioid abuse affects over 16 million people. A major cause of death in abusers is overdosing. Naloxone (NAL) is an opioid inhibitor that reverses its respiratory depressing effect. The use of this drug is limited mostly to invasive delivery: intravenous (IV), intramuscular (IM) and subcutaneous (SC) due to its significant hepatic clearance and poor oral bioavailability (2%). These routes are painful and worse still is the need for frequent injections for patient stabilization due to the short half-life of NAL. Non-invasive intranasal forms exist but this is fraught with a couple of limitations such as nasal damage and epistaxis. The need for alternatives without these limitations is thus evident. The feasibility of the use of metal microneedles (MNs) for the transdermal delivery of NAL was demonstrated in-vitro and through in-vitro in-vivo correlation modeling in our lab. The goal of the current study was to design a rapidly dissolving polymeric MN patch with delivery and pharmacokinetic (PK) properties comparable to that seen with the commercially available NAL products, eliminating their highlighted limitations. NAL loaded rapidly dissolving polyvinyl pyrrolidone-based MN arrays (500 µm, 100 needles) were fabricated by the mold casting technique. The permeation profile of fabricated MNs over a predetermined time were assessed via an in-vitro permeation set up using porcine ear skin. Samples were analyzed via HPLC. To improve on drug flux and amount permeated, the effect of increasing MN length and density (no. of needles/unit area) were assessed by fabricating MNs 300 µm longer and those with density double that of the initial array. Factors such as drug load and polymer strength influenced the needle fabrication. Compared to passive permeation, a reduced lag time of about 15 min was observed with a significant drug flux of 15.09 ± 7.68 g/cm2/h seen in the first 1 h (pin-vitro in-vivocorrelation we were able to predict an optimized design of the patch that can reproduce the clinical PK of NAL obtained with commercial devices. Increasing needle density and/or patch area was found to be of greater significance. Overall, drug flux seen over 1 h depicts the applicability of fabricated needles in opioid overdose emergencies with delivery properties comparable to that with IM and IN delivery.

The use of naloxone (NAL) for opioid overdose treatment is limited mostly to parenteral (intravenous, intramuscular, and subcutaneous) or intranasal route due to significant first-pass metabolism associated with oral delivery. Injectables are painful and frequent administrations by the existing routes for patient stabilization due to the short half-life of NAL are needed. Alternative delivery systems would be beneficial if they provide a balance between sustained release properties and a comparable rapid release as is achievable with the available parenteral forms. Thus, the goal of our study is to design a clinically viable polymeric microneedle (MN) patch for NAL. MNs of varying geometric dimensions were fabricated. In vitro skin permeation data for the best-performing patch was mathematically modeled and predictions on geometric parameters for a MN patch of comparable pharmacokinetic properties to parenteral and intranasal NAL as seen in the market were determined. From these evaluations, the need to devise ways to improve flux and amount of drug released from a patch per time was identified. We explored the influence of iontophoretically driving ionized drug content in MN patches on cumulative permeation of NAL from the best-performing MN patch. To optimize the iontophoresis parameters, the influence of citrate phosphate buffer strength on drug release profile was evaluated. Also, the impact of combining iontophoresis and higher drug loading was evaluated. A reduced lag time of about 5-15 min was observed with fabricated polymeric MN patches. From the polymeric MN patch P1 loaded with 50 mg/mL of NAL, a significant drug flux of 15.09 ± 7.68 ��g/cm2/h was observed in the first 1 h (p.Increasing MN length and density (P2 and P3) made a significant difference in the amount permeated and flux (pin-vitrorelease from the best-performing patch (P3) revealed the significance of needle base diameter and needle count in improving systemic pharmacokinetics of NAL from the MN patches. With this approach, an optimized design of the patch that can reproduce the clinical pharmacokinetics of NAL obtained with commercial devices was predicted. Investigation on the influence of iontophoresis in improving flux from the P3 patch shows about a 2-fold (p

<p>Sight-threatening diseases, such as age-related macular degeneration (AMD), affect the tissues of the posterior segment of the eye. Though modern classes of biomolecular based drugs are therapeutically useful, drug targeting for prolonged bioavailability to pathological sites within the eye is challenging. Current delivery approaches are invasive and lack control over drug release rates and tissue-specific localization. In this thesis, a device using microneedles embedded in a flexible platform was developed that could potentially overcome these challenges.</p> <p>New methods for microneedle fabrication were developed by co-opting simple chemical etch methods commonly used for optical probe fabrication as an alternative to current complex and expensive photolithographic technologies to produce out-of-plane, high aspect ratio microneedles which are often constrained materially to silicon and metal. Microneedles with repeatable tip and taper sizes were obtained using hydrofluoric acid, an organic phase and fused-silica capillary tubing. Microneedles with 10 um tips were made using single and batch mode methods and were then integrated into poly (dimethylsiloxane) (PDMS) for alignment using low cost micromolding approaches offering the same degree of accuracy provided by conventional photolithography<strong>. </strong></p> <p>Single microneedle-based devices successfully delivered rhodamine intrasclerally, intravitreally, suprachoroidally and to the retina. This is the first demonstration of active delivery to specific spatial regions within the posterior eye at controllable rates using a non-implantable, biocompatible device – with minimal fabrication facilities, equipment and cost. The fabricated device demonstrated a new hybrid approach of coupling a rigid microneedle with a soft and pliable substrate that could conform to biological tissues.</p> / Doctor of Philosophy (PhD)

The emergence of microneedle (MN) technologies offers a route for a pain free, straightforward and efficient way of transdermal drug delivery, but technological barriers still exist which pose significant challenges for manufacture of MN systems with high volume outputs at low cost. The main aim of this research was to develop new ways for MN manufacture primarily using micro-injection moulding processes with high performance engineering thermoplastics. During the moulding process these polymeric melts will be subjected to extreme stress and temperature gradients and detailed material characterisation combined with in-line monitoring is desirable to optimise the moulding parameters and will help in achieving sharp microneedles with acceptable quality. Hence high shear rheology of these selected materials was performed at wall shear rates carried out in excess of 107 s-1 over a range of temperatures to predict the flow behaviour of polymer melts at such high shear strain rates. This information was fed into injection moulding simulation software tools (Moldflow) to assist the MN production process design. The optimal design was then used to produce a full 3D solid model of the injection mould and mould insert. Furthermore various design of experiments were conducted considering input parameters such as injection pressure, injection speed, melt temperature, filling time and mould cavity temperature. Response variables including product quality and data acquired from the cavity pressure and temperature transducers were used to optimise the manufacturing process. The moulded MNs were geometrically assessed using a range of characterisation techniques such as atomic force microscopy, confocal microscopy and scanning electron microscopy. An attempt to make hollow MNs was performed and encountered many challenges like partial cavity filling and part ejection during processing. Studies were carried out to understand the problem and identified the major problem was in tool design and improvements to the moulding tool design were recommended. Plasma treatment and mechanical abrasion were employed to increase the surface energy of the moulded polymer surfaces with the aim of enhancing protein adsorption. Sample surface structures before and after treatment were studied using AFM and surface energies have been obtained using contact angle measurement and calculated using Owens-Wendt theory. Adsorption performance of bovine serum albumin and release kinetics for each sample set was assessed using a Franz diffusion cell. Results indicate that plasma treatment significantly increases the surface energy and roughness resulting in better adsorption and release of BSA. To assist design-optimisation and to assess performance, a greater understanding of MN penetration behaviour is required. Contact stiffness, failure strength and creep behaviour were measured during compression tests of MN against a steel surface, and in-vitro penetration of MNs into porcine skin. The MN penetration process into porcine skin was imaged using optical coherence tomography. Finally, a finite element model of skin was established to understand the effect of tip geometry on penetration. The output of findings from this research will provide proof of concept level development and understanding of mechanisms of MN penetration and failure, facilitating design improvements for micro-injection moulded polymeric MNs.

Yes / Industry-standard quality management systems such as Six Sigma and emerging Industry 4.0 compliant production processes demonstrate the importance of in-line condition monitoring of manufacturing methods for achieving the highest levels of product quality. Measurement data collected as the process is running can inform the operator about unexpected changes in machine operation or raw materials that could negatively impact production; and offer an opportunity for a process control intervention to stabilise production. However, micro-manufacturing production lines can pose a challenging environment for deploying such systems, since processing events can occur extremely rapidly and in harsh environments. Moreover, the small scale of micro-nano featured components can make sensor installation even more problematic. Recently, ultrasonic micromoulding has drawn attention in niche markets due to its unique advantages for processing thermoplastics as a new micro-manufacturing technology. The process differs from conventional moulding significantly by eliminating the need for a plasticising screw and using direct application of ultrasonic energy to melt the polymer. This offers numerous benefits such as decrease in energy usage, moulding at lower pressures, easier cleaning, and reduced material residence times, the latter which could be beneficial for pharma-grade polymers or polymers with active ingredients. However, very little work has been reported attempting to monitor the process using in-line measurements. This work aims to evaluate the characteristics of the ultrasonic micromoulding process for microinjection moulding of a microneedle array using a range of sensor technologies including: data recorded by the machine controller; a high-speed thermal camera and a cavity pressure transducer. The data has captured the highly dynamic process environment with a high degree of accuracy. The relationship between the process data and dimensional quality of the ultrasonically micromoulded products has been quantified and subsequently implemented as a cost-effective in-line quality assurance method. / Horizon 2020, the EU Framework Programme for Research and Innovation (Project ID: 674801). This research has also received funding and support from two other Horizon 2020 projects: HIMALAIA (Grant agreement No. 766871) and Laser4Fun (GA no. 675063)

Biopharmaceuticals, including proteins, DNA and vaccines, are one of the fastest growing segments of the overall pharmaceutical market. While the hypodermic injection, the most common delivery method for these molecules, is effective, it also has limitations, including low patient compliance, need for medically trained personnel and biohazardous sharps after delivery. The overall goal of this thesis was to develop a new delivery system for biopharmaceuticals, based on dissolving polymer microneedles, which is effective and more patient compliant than the hypodermic needle. Microneedles are microscopic needles that are large enough to insert into the skin to deliver drugs effectively, while being short enough to avoid the pain causing nerves deep in the skin. An additional benefit of polymer microneedles is that the needles completely dissolve in the skin, leaving behind no biohazardous sharps. There are significant material and fabrication issues that must be overcome in the development of this new device. The first part of this thesis focused on the development of a new fabrication process, based on in situ photopolymerization, for the creation of polymer microneedles. These microneedles were shown to successfully insert into the skin, dissolving within a minute to deliver the encapsulated cargo, and retain full activity of encapsulated proteins. Next, we applied the microneedle technology to the delivery of the influenza virus. We found that the reformulation process required to encapsulate the influenza virus in polymer microneedles did not affect the antigenicity or immunogenicity of the virus. In addition, we used coated metal microneedles to successfully immunize mice with the influenza virus, verifying the delivery capabilities of a microneedle system. Finally, we used the dissolving polymer microneedles to successfully immunize mice with the influenza virus, resulting in full protection against lethal challenge after one immunization. This immune response was equivalent to the control intramuscular injection. In conclusion, we have developed dissolving polymer microneedles as an effective and patient compliant delivery system for biopharmaceuticals. This system could be especially applicable to mass immunization efforts or home use, since it can be self-administered and allows for easy disposal with no biohazardous sharps.

Microneedles have been developed as a minimally invasive alternative to painful hypodermic needles to deliver modern biotherapeutics. Previously, several in-vitro and in-vivo animal studies have been conducted to show that microneedles increase skin permeability to a wide range of molecules that cannot cross the skin using conventional transdermal patches due to the skin's stratum corneum barrier. However, only a limited number of studies have been performed to study microneedle-based drug delivery in human subjects. Therefore, the objective of this study was to perform the first-in-humans microneedle studies to: a) characterize skin repair responses to solid microneedle insertion to determine the extent of increased skin permeability coupled with predictions of pharmacokinetics of drug delivered through premeabilized skin, b) determine the effect of hollow microneedle-based infusion parameters on flow conductivity of skin and pain and thereby identify barriers to fluid flow into the skin from hollow microneedles, c) assess the safety and efficacy of systemic therapeutic effects through measurement of pharmacokinetic parameters, pain and irritation for microneedle-based insulin delivery in type 1 diabetes subjects, and d) assess the safety and efficacy of local therapeutic effects though delivery of lidocaine to the skin. Results showed for the first time that solid microneedle-treated skin reseals rapidly (< 2 h) in the absence of occlusion whereas occluded skin reseals slowly (3-40 h) depending on microneedle geometry as determined by skin impedance measurements. Increased microneedle length, number, and cross-sectional area led to slower recovery kinetics in the presence of occlusion. This thesis also demonstrated that the flow conductivity of skin decreased as fluid was infused to the dermis through hollow microneedles due to the dense structure of the dermis. Microneedle retraction, low flow rates, and the addition of hyaluronidase helped increase flow conductivity. Microneedles were able to deliver 800 µl of saline to the dermis without causing significant pain. Further, microneedle-based insulin delivery in type 1 diabetes subjects revealed that microneedles provided faster pharmacokinetics and improved glycaemic control than conventional subcutaneous catheters. Lastly, microneedle-based lidocaine injection demonstrated that microneedles were less painful, as effective, and more preferred than hypodermic needles in anesthetizing clinically relevant areas.

The emergence of microneedle (MN) technologies offers a route for a pain free, straightforward and efficient way of transdermal drug delivery, but technological barriers still exist which pose significant challenges for manufacture of MN systems with high volume outputs at low cost. The main aim of this research was to develop new ways for MN manufacture primarily using micro-injection moulding processes with high performance engineering thermoplastics. During the moulding process these polymeric melts will be subjected to extreme stress and temperature gradients and detailed material characterisation combined with in-line monitoring is desirable to optimise the moulding parameters and will help in achieving sharp microneedles with acceptable quality. Hence high shear rheology of these selected materials was performed at wall shear rates carried out in excess of 107 s-1 over a range of temperatures to predict the flow behaviour of polymer melts at such high shear strain rates. This information was fed into injection moulding simulation software tools (Moldflow) to assist the MN production process design. The optimal design was then used to produce a full 3D solid model of the injection mould and mould insert. Furthermore various design of experiments were conducted considering input parameters such as injection pressure, injection speed, melt temperature, filling time and mould cavity temperature. Response variables including product quality and data acquired from the cavity pressure and temperature transducers were used to optimise the manufacturing process. The moulded MNs were geometrically assessed using a range of characterisation techniques such as atomic force microscopy, confocal microscopy and scanning electron microscopy. An attempt to make hollow MNs was performed and encountered many challenges like partial cavity filling and part ejection during processing. Studies were carried out to understand the problem and identified the major problem was in tool design and improvements to the moulding tool design were recommended. Plasma treatment and mechanical abrasion were employed to increase the surface energy of the moulded polymer surfaces with the aim of enhancing protein adsorption. Sample surface structures before and after treatment were studied using AFM and surface energies have been obtained using contact angle measurement and calculated using Owens-Wendt theory. Adsorption performance of bovine serum albumin and release kinetics for each sample set was assessed using a Franz diffusion cell. Results indicate that plasma treatment significantly increases the surface energy and roughness resulting in better adsorption and release of BSA. To assist design-optimisation and to assess performance, a greater understanding of MN penetration behaviour is required. Contact stiffness, failure strength and creep behaviour were measured during compression tests of MN against a steel surface, and in-vitro penetration of MNs into porcine skin. The MN penetration process into porcine skin was imaged using optical coherence tomography. Finally, a finite element model of skin was established to understand the effect of tip geometry on penetration. The output of findings from this research will provide proof of concept level development and understanding of mechanisms of MN penetration and failure, facilitating design improvements for micro-injection moulded polymeric MNs.

Kidney failure results in fluid and toxin accumulation within body fluid compartments, contributing to the excess mortality seen in this condition. Such uremic toxins have been measured in plasma, with levels assumed to reflect extraplasmatic concentrations such as in interstitial fluid (ISF). ISF is separated from plasma by nanometre-order microvascular pores; toxins may not circulate “freely” between the two compartments. This work set out to characterise the ISF in uremic subjects, with the hypothesis that there may be differences with plasma. Any such difference may be clinically relevant, owing to the much larger size of the ISF compartment, its proximity to cell metabolic processes, and its expansion in renal impairment.We used a modified microdialysis technique to successfully sample subcorneal ISF of some the uremic toxins (urea, creatinine, urate, phosphate). Reverse iontophoresis (RI) was also used as a non-invasive technique to sample epidermal ISF of urea. Hollow microneedles were developed and their ability to extract ISF tested in CKD patients and controls. The mechanical properties (pressure, volume, permeability) and biochemical composition (proteomic and metabolomic profiles) of the interstitial compartment were also investigated.Microdialysis and RI performed very well as interstitial uremic toxin sampling techniques. Small differences were seen in steady states between ISF and plasma urea, creatinine, phosphate and urate, with slightly lower ISF levels. Dialysis seemed to enhance this difference, with a lag in the clearance of ISF toxins seen in some patients, most remarkable with phosphate. Metabolomic analysis identified several uremic toxins in ISF, whilst proteomics found some significant differences between the two compartments, with toxins like beta-2 microglobulin occurring in ISF only. Microneedle arrays successfully extracted ISF in 68.8% of patients with oedema. Successful extraction of ISF with microneedles occurred mainly in oedematous patients, who were found to have raised interstitial pressures (ISP) and volumes. ISP correlated significantly with body fluid volumes and seemed time-dependent, lower in more chronic oedema. ISP and volumes also correlated with the oedema depitting time (after thumb pressure), a potential novel parameter that probably relates to tissue hydraulic conductivity and hence volume status and fluid mobility within the interstitium.This study demonstrates that interstitial fluid may need to be considered as a separate active compartment in patients with renal dysfunction, with a different “uremic" composition and unique pathophysiological characteristics that cannot be explained by blood compartment based measurements alone. There is a need for more studies, to further characterise this compartment and elucidate its importance.

Ce mémoire de maîtrise porte sur le développement de microaiguilles hydrogels pour la capture et la détection précoce de biomarqueurs protéiques spécifiques du liquide interstitiel cutané. Le diagnostic précoce d’une maladie et le suivi préventif des paramètres biologiques peuvent effectivement améliorer les traitements et auront un rôle plus important dans les années à venir. Cependant, des obstacles considérables à cette approche persistent, en particulier la nature hautement invasive et perturbatrice des analyses biologiques. Se rendre dans une clinique et subir un prélèvement invasif de sang (ou de liquide biologique) sont des défis considérables par rapport aux traitements courants, qui consistent souvent en des médicaments qui peuvent être pris sans douleur à la maison. Une solution à ces problèmes peut être trouvée dans l'invention de méthodes peu invasives pour le diagnostic et l'analyse des soins de santé, idéalement celles qui peuvent être utilisées à domicile sans nécessiter de personnel formé. À cet égard, les micro-aiguilles (MNs) démontrent un énorme potentiel car leur petite taille garantit qu’elles sont relativement simples et presque indolores. De plus, leur nature simple et à usage unique permet potentiellement une administration à domicile par le patient. Les micro-aiguilles d'hydrogel présentent des caractéristiques bénéfiques à des fins de diagnostic compte tenu de leurs propriétés de gonflement qui permettent d'absorber les fluides corporels tels que le liquide interstitiel (ISF) et de capturer les biomarqueurs. Ces caractéristiques remarquables ont poussé les scientifiques à utiliser des micro-aiguilles d'hydrogel pour des applications de diagnostic. Afin de fournir un contexte pour le développement de cette technologie, cette thèse commence par un examen des principes et des avancées récentes dans le domaine des applications diagnostiques des MN (Chapitre 1). Par la suite, des sections expérimentales, de résultats et de discussion seront présentes sur la fonctionnalisation de l'hydrogel avec des anticorps pour la détection de biomarqueurs spécifiques (Chapitre 2). Le dernier chapitre aborde la conclusion générale et les perspectives d'avenir de cette approche (Chapitre 3). / This master’s thesis focuses on the development of hydrogel microneedles (HMNs) for capture and early detection of specific protein biomarkers form the skin interstitial fluid. Early disease diagnosis and preventative monitoring of biological parameters can effectively improve medical results and anticipate playing a more important part in the forthcoming years. However, considerable barriers to this approach persist, specifically the highly invasive and disruptive nature of biological analyses. Visiting clinics and undergoing invasive blood (or biological fluid) sampling are considerable challenges in comparison with common treatments, which often consist of drugs that may be taken painlessly at home. A solution to these concerns can be found in the invention of minimally invasive methods for diagnostics and healthcare analyzing, ideally ones that may be utilized at home without the requirement for trained staff. In this regard, microneedles (MNs) demonstrate tremendous potential as their small size ensures that they are relatively straightforward and almost painless. Also, their simple and single-use nature potentially permits at-home administration by the patient. HMNs demonstrate beneficial features for the diagnosis purposes considering the swelling properties of them which give the chance of absorbing body fluids such as ISF and capture of the biomarkers. These remarkable features have driven scientists to employ HMNs for diagnostic applications. To provide background for the development of this technology, this thesis begins with a review of the principles and recent advances in the field of diagnostic applications of MNs (Chapter 1). Subsequently, experimental, result, and discussion sections will be present about the functionalization of hydrogel with a model antibody for specific biomarkers detection (Chapter 2). The last chapter discusses the general conclusion and future prospects of this approach (Chapter 3).