Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults and has a median survival of 13.4 months. Convection enhanced delivery (CED) has shown promise for the treatment of GBM by allowing intratumoral delivery of therapeutics, bypassing the blood brain barrier. A fiberoptic microneedle device (FMD) CED catheter enables simultaneous delivery of laser energy and therapeutic. The laser allows for heating of tissue in the region of infusion, called thermotherapy. Thermotherapy offers the advantages of increasing the volume of distribution (Vd) of the infusate, as well as facilitating intracellular penetration of the therapeutic. We hypothesize that heating of brain tissue will increase infusate Vd in ex vivo CED brain infusions. Methods: Formalin fixed mouse brains were infused by FMD-CED with Evans blue for 1 hour at 0.1 μl/min, at 22°C, 37°C and 42°C (n=4 brains/group). The Vd was determined and compared using one-way ANOVA. Results: FMD-CED performed at 42°C resulted in significantly higher mean Vd (4.90+2.2mm3; p =0.03) than those at 22°C (1.49+0.4 mm3), although no differences in Vd were observed between the other temperature groups. 42°C brains demonstrated interstitial and intracellular distribution, while rare intracellular distribution was noted in the other groups. Discussion: The Vd of FMD-CED infusions is facilitated by sub-lethal thermotherapy. This study indicates that thermotherapeutic enhancement of infusate Vd does not occur exclusively via vascular mechanisms. Thermotherapy facilitates advective-diffusion by decreasing interstitial fluid pressure and increasing transcellular fluid transport. These results were validated in a companion in vivo FMD-CED study in the rodent brain. / Master of Science

Infantile hemangiomas (IHs), benign vascular lesions present on the surface of the skin of children, are treated with systemic or topical beta-adrenergic antagonists (known as “beta-blockers”). However, systemic beta-blocker therapy is associated with serious adverse events in pediatric patients, and there are currently no topical formulations optimized for the skin. The objectives of this work were to 1) evaluate the local skin concentrations and drug permeation through the skin using novel beta-blocker formulations, and 2) characterize the epidermal properties and skin surface inflammatory mediators of IH skin. Skin concentrations and drug permeation through the skin from current topical treatment options were quantified in vitro; these data served as benchmarks to which other treatment paradigms in later studies were compared. Microneedle (MN)-mediated delivery of two beta-blockers, propranolol and timolol, was evaluated in vitro using solid MNs and two dissolving MN array formulations. Solid MNs increased skin concentrations of timolol compared to intact skin, while producing similar skin concentrations of propranolol. Drug permeation through the skin was increased for both drugs after MN pretreatment. Both formulations of dissolving MN arrays were ineffective at increasing local skin concentrations compared to intact skin. This was likely due to the small loading capacity of drug into the array. Drug-loaded microemulsions (ME) of varying composition were formulated and characterized. All ME formulations had solubilization properties, and water rich MEs had the greatest cumulative release through a homogenous membrane compared to surfactant rich MEs. Drug-loaded MEs did not increase local skin concentrations in vitro compared to a drug solution; however, water rich ME formulations produced greater skin-to-receiver ratio of drug concentration, indicating their potential for skin accumulation. MN pretreatment increased the skin-to-receiver ratios for surfactant rich formulations but not for water rich formulations, indicating this enhancement in skin retention after MN pretreatment is formulation dependent. These results demonstrate the potential for topical treatment of IHs upon further optimization of delivery and formulation parameters. The epidermal properties and skin surface mediators of IH skin were compared to normal, unaffected skin. Significant differences in barrier function and color, as well as chemokine and growth factor concentrations, were observed between the two sites. These results provide a greater understanding of the IH properties that have previously not been quantified. Similar changes in lesion color, which correlate to efficacy, were observed after beginning treatment with oral propranolol or topical timolol, while changes in barrier function were similar between the two treatment groups. These results indicate topical timolol may be a safe alternative for systemic treatment for superficial IHs without a loss of efficacy.

Micromachined needles have been shown to successfully transport biological molecules into the body with minimal invasiveness and pain, following the insertion of needles into the skin. The aim of this research is to demonstrate that micromachined hollow polymer needle arrays fabricated using UV lithography into micromolds, a potential batch-manufacturable process, can exhibit comparable insertion and injection performance to conventional hypodermic needles for drug delivery into skin. A dual-exposure-and-single-development process flow is proposed for the above-mentioned UV lithography into micromolds approach to construct a pyramidal-tip hollow microneedle array with an integral baseplate and fluidic manifold. The developed process ultimately resulted in the ability to fabricate a 10×10 array of hollow SU-8 microneedles measuring 825 μm in height, 400 μm in width, and possessing a lumen of 120 μm in diameter. The tip diameter of the microneedles ranges from 15 μm to 25 μm. The insertion force of single needles characterized using excised porcine skin as a substrate is 2.4±1.2 N. Nevertheless, the high insertion force of 2.4 N per needle may cause a significant concern when a large number of needles are required to insert into skin for drug delivery. Conventional hypodermic needles have two key structural characteristics: a sharp beveled tip and a large side-terminated lumen. Integration of these two key characteristics of hypodermic needles into microneedle design can potentially enhance microneedle performance. To reduce the insertion force and to incorporate the two key characteristics of hypodermic needles into the design of microneedles, a new needle tip design, namely the hypodermic-needle-like design, is presented. A 6×6 array of hypodermic-needle-like microneedles of 1 mm in height, approximate 350 μm in width, and with a lumen of 150 μm in diameter is demonstrated with successful insertion of the needle array into skin and an 85% lumen openness yield. The insertion force is significantly reduced by an order of magnitude with the new needle tip design and is 0.275±0.113 N per needle, comparable to that of hypodermic needles, i.e., 0.284±0.059 N. The hypodermic-needle-like microneedles exhibit a margin of safety of 180 for successful needle insertion into skin prior to needle fracture. A successful manual fluid injection into skin using single microneedle is demonstrated. The micromachined hypodermic-needle-like polymer needle arrays presented in this dissertation are fabricated using UV lithography into micromolds, a potentially batch-manufacturable process, and exhibit comparable insertion performance to conventional hypodermic needles. Injection capability into skin is also demonstrated with a hypodermic-needle-like microneedle, illustrating the utility of these devices.

Despite the existence of effective vaccines for both diseases, measles and poliomyelitis still cause significant worldwide morbidity and mortality. The live-attenuated measles and inactivated polio vaccines are both given using a standard needle and syringe injection. This method of delivery poses many problems for large-scale vaccination campaigns. Microneedles are micron-scale needles which have the potential to overcome many of these hurdles. In the first study, we showed that the measles vaccine could be successfully incorporated into a solid, metal microneedle system which induced potent neutralizing antibody titers after administration into cotton rats. This response was statistically identical to the same dose delivered using a subcutaneous injection. The second study focused on enhancing the stability of the measles vaccine after drying and long-term storage. Using a new assay developed from a measles virus variant engineered to encode for green fluorescent protein, it was determined that a combination of sucrose and threonine provided the highest stabilizing effect. Vaccine mixed with this solution retained more than 90% of its activity after 6 months of storage at 4°C and 25°C. The third study involved the incorporation of the measles vaccine into a dissolving microneedle patch. These patches were used to vaccinate rhesus macaques and the immune response was found to be statistically identical to the same dose delivered by syringe injection. Furthermore, after creation and storage, these patches retained 100% of their infectivity after 2 months at 4°C and 25°C. The final study attempted to create a dissolving microneedle patch containing a full dose of the inactivated polio vaccine. These patches were then used to deliver a full dose of IPV into the skin of a rhesus macaque. This delivery method produced neutralizing antibody titers to IPV type 1 and 2 that were statistically identical to the same dose delivered using a needle and syringe. Overall, these studies show that the microneedle patch was a safe, simple and effective method for measles and polio vaccination. This delivery platform has the potential to overcome many of the hurdles that currently stand in the way of measles elimination and polio eradication.

Opioid addiction is a serious national crisis impacting public health. Naloxone is a potent opioid antagonist administered to reverse the effects of opioid overdose. It is currently administered as an intravenous, intramuscular, subcutaneous injection and intranasal spray. The short duration of action of naloxone results in requirement of frequent re-dosing, especially in cases of larger overdoses, which may impact successful outcomes, especially when drug administration is provided by non-medical personnel as in case of intranasal sprays. These weaknesses necessitate the development of a non-injectable dosage form that has a rapid onset and extended duration of action. Delivery of drugs via skin is an attractive alternative that provides these benefits. Our study aimed to assess the effect of microneedles on the amount and lag time of permeation of naloxone across skin. In vitro permeation studies were performed to assess the delivery of naloxone through dermatomed porcine ear skin using Franz Diffusion cells. The donor and receptor chamber of the cells contained the drug solution and phosphate buffered saline, respectively. The receptor was sampled until 6 h and analyzed using HPLC. The permeation of naloxone across intact (passive) and microneedle-treated (Dr. Pen™ Ultima A6) skin was evaluated. Two microporation conditions with donor concentration of 10 mg/mL were investigated: needle lengths (500 µm and 250 µm) for 1 minute and 500 µm needle length for different durations (1 and 2 minutes). Further, the effect of application of different naloxone concentrations (10 and 20 mg/mL) on skin treated with 500 µm microneedles for 2 minutes was also tested. One-way ANOVA was applied to ascertain statistical difference between the different test groups. The amount of passive permeation after 6 h and lag time for naloxone was observed to be 8.251.06 µg/cm2 and88.58 ± 3.05 min, respectively. One minute treatment with 500 µm needles significantly enhanced the permeation to 463.24 ± 30.21 µg/cm2 and reduced the lag time to 15.90 ± 1.63 min (p0.05). Microneedles were found to enhance the permeation of naloxone across skin. The observation of quick onset of drug permeation in the in vitro settings is very encouraging and future studies would focus on developing a microneedle patch for quick onset and extended drug release.

Highly integrated and customizable systems have been a principal focus of development for parenteral and oral drug administration. Extensive work has been done to optimize drug efficacy via localized delivery and dosage control providing new ways for accomplishing targeted therapeutic effects. However, many challenges and opportunities for advancement remain. One promising research path is introducing novel microfabrication methods or engineering discoveries in concept realization, making devices more versatile and effective. Firstly, this dissertation focuses on designing and fabricating a miniaturized, 3D printed, wirelessly powered drug delivery system for biomedical applications. The drug delivery system is composed of an electrolytic micropump integrated into a 3D printed reservoir equipped with hollow microneedles. The electrolytic pump is composed of interdigitated electrodes and a bellows membrane. A simple and customizable manufacturing process is developed to fabricate miniaturized bellows membranes. To improve the integration of microneedles in microelectromechanical devices, a high-resolution 3D printing technique is implemented to produce a reservoir equipped with an array of hollow microneedles. Penetration tests of microneedles into a skin-like material confirm sufficient stability of microneedles. Furthermore, the microneedle arrays are used to pierce and deliver into mouse skin successfully. The assembled system (electrolytic micropump integrated into the 3D printed reservoir equipped with hollow microneedles) is actuated using inductive wireless powering. Secondly, this dissertation tackles one of the most challenging diseases, Coronary Artery Disease. Delivering a therapeutic agent directly to the inner wall of affected blood vessels can be a transformative step toward a better treatment option. To open the door for such an approach, a catheter delivery system is developed based on a conventional balloon catheter where a fluidic channel and microneedles are integrated on top of it. This enables precise and localized delivery of therapeutics directly into vessel walls. Ex vivo tests on rabbit aorta confirm the microneedles-upgraded balloon catheter’s performance on real tissue. This study shows that microneedles-upgraded balloon catheter is capable of localized and targeted drug delivery into artery walls. The fabrication process ensures a highly customizable solution that can be tailored to patient-specific requirements.

Microfabricated needles have the potential for inexpensive drug delivery without pain. The ability to deliver medication painlessly to patients will someday be not just hoped for but expected by the general public. The commercialization of this technology will also lead to other valuable technologies, such as systems that continually monitor and control insulin or other drugs in diabetic patients. This research presents fabrication procedures developed to produce pyramidal-shaped microneedles with microchannels that will allow for fluid delivery. The microchannels are etched into the substrate surface of a [100] silicon wafer using inductively coupled plasma etching. After the channel etch a layer of silicon nitride is deposited onto the inner walls of the microchannels and on the surface of the substrate. The nitride on the substrate surface provides the hard mask necessary to etch the microneedles, which are wet etched in a bath of potassium hydroxide (KOH). The selectivity of the KOH on [100] silicon is such that octagonal shaped pyramids are etched into the surface of the wafer. The pyramids are aligned with the previously etched microchannels to allow for needles with channels running through them. This research presents the first needles demonstrated with drug delivery channels running through the robust pyramidal needle shape. In addition to the microchannel/microneedle fabrication procedure, microchannels were developed with inner structures as a method of creating hydrophobic surfaces on the inner walls of the channels. It was found that the channels developed had far too much variability in the diameter to accurately create a measurable reduction in flow; however, a loss coefficient was calculated showing increased flow rates in hydrophobically coated microchannels when hydrophobic structures are incorporated into the channel design. It was also discovered that a hydrophobic coating, typically used to increase flow rates through a channel, can impede flow rate. There was no evidence found to suggest that hydrophobically coated microchannels of this size, with or without structures, will yield higher flow rates than non-coated microchannels.

This dissertation describes the development of the fiberoptic microneedle device (FMD), a microneedle technology platform for fluid and light delivery, from general engineering characterization to specific applications in treating bladder and brain cancers. The central concept of the FMD is physical modification of silica fiberoptics and capillary tubes into sharp microneedles capable of penetrating a tissue's surface, enabling light and fluid delivery into the interstitial spaces. Initial studies sought to characterize the mechanical penetration and optical delivery of multimode fiberoptics and capillary tubes modified through a custom, CO2 laser melt-drawing technique. Additional work with multimode fibers investigated using an elastomeric lateral support medium to ensure robust penetration of small diameter fibers. These early experiments laid an engineering foundation for understanding the FMD technology. Subsequent studies focused on developing the FMD to treat specific diseases. The first such investigation sought to leverage the high aspect ratio nature of FMDs made from long capillary tubes as a therapy delivery device deployable through the instrument channel of a urological cystoscope. The therapeutic strategy was to infuse single-walled carbon nanohorns (SWNHs), a carbon-based nanoparticle allowing surface modification and drug encapsulation, into the infiltrating front of later stage bladder tumors. The SWNHs primarily serve as exogenous chromophores, enabling a fluid-based control of photothermal heat generation created when the SWNHs interacted with laser energy from an interstitial FMD or a light-emitting fiber in the bladder's interior. The study described here primarily sought to characterize the dispersal of the infused SWNHs and the photothermal response of the particles when heated with a 1064 nm laser. The FMD was also developed as a platform capable of conducting convection-enhanced delivery (CED), a therapeutic approach to treat invasive tumors of the central nervous system such as malignant glioma (MG). Intracranial CED involves the placement of small catheters local to the tumor site and slow infusion of a chemotherapeutic over long timeframes (12-72 hours). A primary challenge of this treatment approach is infused chemotherapeutics not dispersing sufficiently to reach the infiltrating cells in the tumor's margins. The hypothetical improvement provided by the FMD technology is using sub-lethal photothermal heating to sufficiently increase the diffusive and convective transport of an infusate to reach infiltrative cells in the tumor's periphery. Initial experiments sought to demonstrate and characterize a heat-mediated increase of volumetric dispersal in Agarose tissue phantoms and ex vivo tissue. Subsequent studies with in vivo rodent models determined the best laser parameters to achieve the desired levels of diffuse, sub-lethal heat generation and then demonstrated the hypothesis of increasing the rate of volumetric dispersal though concurrent local hyperthermia. This research was the first demonstration of photothermal augmentation of an interstitially infused fluid's dispersal rate, which may have uses outside of the CED approach to brain cancer exhibited here. Taken in sum, this manuscript describes the potency and versatility of the FMD technology platform through its development in various biomedical applications. / Ph. D.

<p>Several studies have reported beneficial effects of C-peptide supplementation in diabetic patients and animal models of insulinopenic diabetes. However, it is also established that good glycemic control is essential to minimize the risk of diabetes-induced complications. This thesis investigates potential mechanisms for the beneficial effect of C-peptide on glomerular hyperfiltration, and a novel, painless route of insulin administration.</p><p>The results demonstrate that both C-peptide and its C-terminal penta-peptide sequence reduce the diabetes-induced glomerular hyperfiltration within an hour. The results also indicate that C-peptide possibly reduces diabetes-induced hyperfiltration via three different mechanisms: 1. Constriction of the afferent arteriole was demonstrated on isolated vessels from diabetic mice. 2. A net dilation of the efferent arteriole was evident <i>in vivo</i>. 3. Inhibition of the Na⁺/K⁺-ATPase was demonstrated <i>in vivo</i> in diabetic rats as well as <i>in vitro</i> on isolated proximal tubular cells from diabetic rats. All these mechanisms are known regulators of the net glomerular filtration pressure.</p><p>The last part of this thesis demonstrates that intradermal administration with a newly developed patch-like microneedle device results in similar insulin concentration compared to standard subcutaneous delivery. </p><p>These findings provide an insight for the beneficial effects of C-peptide on diabetic kidney function, and shows that this effect can be achieved by infusion of the C-terminal penta-peptide sequence alone. This thesis also presents a novel, painless alternative to insulin injections that is controllable, requires minimal training, and therefore presents several advantages compared to current standard therapy.</p>

Patch-based transdermal drug delivery offers a convenient way to administer drugs without the drawbacks of standard hypodermic injections relating to issues such as patient acceptability and injection safety. However, conventional transdermal drug delivery is limited to therapeutics where the drug can diffuse across the skin barrier. By using miniaturized needles, a pathway into the human body can be established which allow transport of macromolecular drugs such as insulins or vaccines. These microneedles only penetrate the outermost skin layers, superficial enough not to reach the nerve receptors of the lower skin. Thus, microneedle insertions are perceived as painless. The thesis presents research in the field of microneedle-based drug delivery with the specific aim of investigating a microneedle-based transdermal patch concept. To enable controllable drug infusion and still maintain an unobtrusive and easy-to-use, patch-like design, the system includes a small active dispenser mechanism. The dispenser is based on a novel thermal actuator consisting of highly expandable microspheres. When actuated, the microspheres expand into a liquid reservoir and, subsequently, dispense stored liquid through outlet holes. The microneedles are fabricated in monocrystalline silicon by Deep Reactive Ion Etching. The needles are organized in arrays situated on a chip. To allow active delivery, the microneedles are hollow with the needle bore-opening located on the side of the needle. This way, the needle can have a sharp and well-defined needle tip. A sharp needle is a further requirement to achieve microneedle insertion into skin by hand. The thesis presents fabrication and evaluation of both the microneedle structure and the transdermal patch as such. Issues such as penetration reliability, liquid delivery into the skin and microneedle packaging are discussed. The microneedle patch was also tested and studied in vivo for insulin delivery. Results show that intradermal administration with microneedles give rise to similar insulin concentration as standard subcutaneous delivery with the same dose rate. / QC 20100623