No description available.

<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)

No description available.

Acute liver failure is a major cause of death in the world, and effective treatments are greatly needed. Liver macrophages (Kupffer cells) play a major role in the pathology of acute liver failure, and drug delivery vehicles that can target therapeutics to Kupffer cells have great therapeutic potential for treating acute liver failure. Microparticles, formulated from biodegradable polymers, are advantageous for treating acute liver failure because they can passively target therapeutics to Kupffer cells. However, existing biomaterials are not suitable for the treatment of acute liver failure because of their slow hydrolysis and acidic degradation products. In this dissertation, I present the development of a new class of biodegradable materials, termed aliphatic polyketals, which have considerable potential as drug delivery vehicles for the treatment of acute liver failure because of their neutral degradation products and tunable hydrolysis kinetics. The anti-inflammatory enzyme, superoxide dismutase (SOD), was delivered using polyketal microparticles to the liver for treating acute liver Failure. Our results demonstrated that polyketal microparticles significantly improved the efficacy of SOD in treating LPS-induced acute liver damage in vivo, as evidenced by decreased levels of serum alanine transaminase, which corresponds to the extent of damage in the liver, and serum level of tumor necrosis factor-alpha, which corresponds to the secretion of pro-inflammatory cytokines. The completion of this thesis research demonstrates the ability of polyketal-based drug delivery systems for treating acute inflammatory diseases and creates a potential therapy for enhancing the treatment of acute liver failure.

Doctor of Philosophy / Department of Chemical Engineering / Stefan H. Bossmann / Both chemotherapy for cancer treatment and antibiotic therapy for bacterial infections require systemic applications of the drug and a systemic application is always linked to a number of disadvantages. To circumvent these a targeted drug delivery system was developed. It utilizes the ability of phagocytes from the hosts own immune system to recognize and internalize antigens. Deactivated M. luteus, a non-pathogenic gram positive bacteria was loaded with high concentrations (exceeding the IC50 at least 60 fold in local intracellular concentration) the chemotherapeutics doxorubicin or DP44mt or with the bactericidal chlorhexidine. The modified bacteria is fed to phagocytes (Monocytes/Macrophages or neutrophils) and serves as protective shell for the transporting and targeting phagocyte. The phagocyte is recruited to the tumor site or site of infection and releases the drug along with the processed M. luteus via the exosome pathway upon arrival. The chlorhexidine drug delivery system was successfully tested both in vitro and in vivo, reducing the pathogen count and preventing systemic spread of a F. necrophorum infection in a mouse model. The doxorubicin drug delivery system reduced the viability of 4T1 cancer cells to 20% over the course of four days in vitro.

New imidazotetrazinones have been synthesised that possess features in their structures to release aziridinium ions upon ring opening. Unstable 2-aminoethylisocyanates were required in this preparation, which were synthesized with BOC-protection of the amino group to counteract the reactivity of the amine towards the isocyanate group in the case of aliphatic amines; in contrast, anilinoethylisocyanates were synthesized unprotected. Substituents with a range of electron-withdrawing and electron-releasing properties were introduced at the p-position of the aniline ring. A 13C-labelled study confirmed the release of the aziridinium ion by these imidazotetrazinones in neutral pH buffer solution. Furthermore the kinetics of the hydrolysis in neutral aqueous solution of some these new tetrazines were similar to temozolomide, in addition to useful acid stability. Other imidazotetrazinones were synthesised for the purpose of releasing alcohols and phenols. Their synthesis was performed with a one-carbon linker between the imidazotetrazinone 3-position and the alcohols or phenols to be released. The release of alcohol and phenol through the hydrolysis of the intermediate diazonium ions to the unstable hemiacetals that decomposed to the alcohol and phenol was confirmed by 1H NMR. The kinetics of the hydrolysis of these tetrazines in neutral aqueous solution showed a faster reaction rate compared with temozolomide (t1/2 = 0.53 and 0.36 h compared with temozolomide 1.4 h).

Therapeutic efficacy of drugs depends on their ability to reach the treatment target. Drugs that exert their effect within cells are constrained by an inability to cross the cell membrane. Methods are being developed to overcome this barrier including biochemical and biophysical strategies. The application of ultrasound with microbubbles increases the permeability of cell membranes allowing molecules, which otherwise would be excluded, to enter the intracellular space of cells; a phenomenon known as sonoporation. This thesis describes studies aimed at improving our understanding of the mechanism underpinning sonoporation and of the exposure parameters affecting sonoporation efficiency. Cancer cells (KHT-C) in suspension were exposed to ultrasound and microbubbles – total of 97 exposure conditions. The effects on cells were assessed through uptake of cell-impermeable molecules (10 kDa to 2 MDa FITC-dextran), cell viability and microscopic observations of the plasma membrane using flow cytometry, colony assay and electron microscopy techniques. Sonoporation was a result of the interaction of ultrasound and microbubbles with the cell membrane. Disruptions (30-100 nm) were generated on the cell membrane allowing cell impermeable molecules to cross the membrane. Molecules up to 2 MDa in size were delivered at high efficiency (~70% permeabilisation). Sonoporation was short lived; cells re-established their barrier function within one minute, which allowed compounds to remain inside the cell. Following uptake, cells remained viable; ~50% of sonoporated cells proliferated. Sonoporation efficiency depended on ultrasound and microbubble exposure conditions. Microbubble disruption was a necessary but insufficient indicator of ultrasound-induced permeabilisation. The exposure conditions can be tailored to achieve a desired effect; cell permeability of ~70% with ~25% cell death versus permeability of ~35% with ~2% cell death. In addition, sonoporation depended on position in the cell cycle. Cells in later stages were more prone to being permeabilised and killed by ultrasound and microbubbles. This study indicated that sonoporation can be controlled through exposure parameters and that molecular size may not be a limiting factor. However, the transient nature may necessitate that the drug be in close vicinity to target cells in sonoporation-mediated therapies. Future work will extend the investigation into in vivo models.

<p>A new modality for preventing HIV transmission is emerging in the form of topical microbicides. Some clinical trials have shown some promising results of these methods of protection while other trials have failed to show efficacy. Due to the relatively novel nature of microbicide drug transport, a rigorous, deterministic analysis of that transport can help improve the design of microbicide vehicles and understand results from clinical trials. This type of analysis can aid microbicide product design by helping understand and organize the determinants of drug transport and the potential efficacies of candidate microbicide products.</p><p>Microbicide drug transport is modeled as a diffusion process with convection and reaction effects in appropriate compartments. This is applied here to vaginal gels and rings and a rectal enema, all delivering the microbicide drug Tenofovir. Although the focus here is on Tenofovir, the methods established in this dissertation can readily be adapted to other drugs, given knowledge of their physical and chemical properties, such as the diffusion coefficient, partition coefficient, and reaction kinetics. Other dosage forms such as tablets and fiber meshes can also be modeled using the perspective and methods developed here.</p><p>The analyses here include convective details of intravaginal flows by both ambient fluid and spreading gels with different rheological properties and applied volumes. These are input to the overall conservation equations for drug mass transport in different compartments. The results are Tenofovir concentration distributions in time and space for a variety of microbicide products and conditions. The Tenofovir concentrations in the vaginal and rectal mucosal stroma are converted, via a coupled reaction equation, to concentrations of Tenofovir diphosphate, which is the active form of the drug that functions as a reverse transcriptase inhibitor against HIV. Key model outputs are related to concentrations measured in experimental pharmacokinetic (PK) studies, e.g. concentrations in biopsies and blood. A new measure of microbicide prophylactic functionality, the Percent Protected, is calculated. This is the time dependent volume of the entire stroma (and thus fraction of host cells therein) in which Tenofovir diphosphate concentrations equal or exceed a target prophylactic value, e.g. an EC50.</p><p>Results show the prophylactic potentials of the studied microbicide vehicles against HIV infections. Key design parameters for each are addressed in application of the models. For a vaginal gel, fast spreading at small volume is more effective than slower spreading at high volume. Vaginal rings are shown to be most effective if inserted and retained as close to the fornix as possible. Because of the long half-life of Tenofovir diphosphate, temporary removal of the vaginal ring (after achieving steady state) for up to 24h does not appreciably diminish Percent Protected. However, full steady state (for the entire stromal volume) is not achieved until several days after ring insertion. Delivery of Tenofovir to the rectal mucosa by an enema is dominated by surface area of coated mucosa and whether the interiors of rectal crypts are filled with the enema fluid. For the enema 100% Percent Protected is achieved much more rapidly than for vaginal products, primarily because of the much thinner epithelial layer of the mucosa. For example, 100% Percent Protected can be achieved with a one minute enema application, and 15 minute wait time.</p><p>Results of these models have good agreement with experimental pharmacokinetic data, in animals and clinical trials. They also improve upon traditional, empirical PK modeling, and this is illustrated here. Our deterministic approach can inform design of sampling in clinical trials by indicating time periods during which significant changes in drug concentrations occur in different compartments. More fundamentally, the work here helps delineate the determinants of microbicide drug delivery. This information can be the key to improved, rational design of microbicide products and their dosage regimens.</p> / Dissertation

Mantle cell lymphoma (MCL) is an extremely difficult to treat subtype of non-Hodgkin lymphoma (NHL) with a low patient survival rate compared to most common cancers. Recently, nanoparticle delivery systems have received a great deal of interest for treating NHL. One of the more promising cargo options for these systems is short interfering RNA (siRNA). siRNA is a 18-23 nucleotide long double stranded RNA that is used to inhibit the protein expression of target mRNAs in a sequence specific manner. MCLs have several commonly overexpressed genes compared to normal cells making it an ideal candidate for siRNA therapies. For RNA interference to occur, A delivery vehicle is needed for the siRNA to reach the cytoplasm of the cell. In this thesis, ionizable lipid-like materials termed lipidoids are formulated into lipid nanoparticles (LNPs) to deliver siRNA. A new library of lipidoids is constructed to gain a better understanding of how the lipidoid tail-structure affects the silencing ability of LNPs. A novel tail precursor is identified as conferring potency to LNPs. Then, LNPs are used to silence genes within difficult to transfect MCL cells. LNPs targeting the anti-apoptotic protein Mcl-1 exhibit potent gene silencing and cause an increase in the fraction of cells undergoing apoptosis. This is important because there is no therapeutic that is FDA approved that targets this commonly overexpressed protein. Because of this LNP’s potency, siRNAs targeting multiple genes can be encapsulated into LNPs without causing unwanted toxicity. LNPs targeting several genes in multiple pathways cause a larger fraction of MCL cells to undergo apoptosis compared to cells treated with LNPs targeting only one gene. A major issue in cancer therapeutics is that the majority of nanoparticles accumulate in the liver. In an effort to improve the delivery of LNPs to target cells, changes to their formulations and administration methods are investigated as a means to improve LNP circulation time, biodistribution, and silencing ability. Overall, this work identifies lipidoid nanoparticles as potent siRNA delivery systems to treat MCL and investigates key properties for further improvement in LNP siRNA delivery to target cells.

In the field of cancer research and diagnostics it is crucial to have reliable methods for detecting molecules involved in the disease. New and better methods for diagnostics, prognostics and drug delivery therefore remain a permanent aim. In this thesis applications of the in situ proximity ligation assay (in situ PLA) were developed for diagnostics and research. Two new methods were developed, one more cost effective proximity assay without the use of enzymes and one method for loading pharmaceuticals in lipid rafts made from detergent resistant membranes (DRMs) to be used as a drug delivery platform. In Paper I the aim was to develop a flow cytometric detection method of the fusion protein BCR-ABL that is the hallmark of chronic myeloid leukemia (CML). By using in situ PLA the malignant cells carrying the fusion protein could be detected in patients in a convenient workflow. Paper II describes an application of multiplex in situ PLA, where extracellular vesicles (EVs) are detected and identified using flow cytometry. Up to five different antigens are targeted on the EVs, reflected in three different colors during detection in the flow cytometer. By using antibodies targeting proteins specific for prostasomes a population of prostasomes could be identified in human blood plasma. In Paper III a new method is described for using lipid raft for drug delivery. In this method, lipid rafts, derived from prostasomes or erythrocytes, are loaded with pharmaceuticals. The vehicles were loaded with doxorubicin, added to cells and counted. Cells that received the vehicle with doxorubicin stopped proliferating and died, while controls that received the lipid raft vehicle without doxorubicin were not affected, suggesting that the vehicles are effectively loaded with the drug and that they are safe. This lipid raft vehicle could provide a safe drug delivery system.      Paper IV investigates the crosstalk between the two major signal pathways Hippo and Wnt, and how these are affected in gastric cancer. When looking at different colon cancer tumor stages, we found that the cellular localization of TAZ/β-catenin interactions were different. We also found that protein complexes involved in the crosstalk increased in sparsely growing cells compared to more densely growing cells. On the basis of these results the protein E-cadherin, involved in maintenance of the epithelial integrity, was investigated and was found to have a probable role in regulating the crosstalk between Hippo and Wnt.     A new method for localized protein detection is described in paper V. Here a proximity assay, based on the hybridization chain reaction (HCR), was developed. This assay, proxHCR, is more cost effective than in situ PLA because no enzymes are required. ProxHCR successfully detects protein interactions and can be used together with both fluorescence microscopy and flow cytometry.