This thesis details our early efforts towards the discovery of polymeric and macromolecular platforms for the targeted delivery of sensors and actuators to specific cell types in the living brain tissue. Chapter 1 of this thesis discusses the small molecule tropane tag chosen as a homing ligand and the dopamine transporter (DAT) chosen as a cellular target, as well as the synthesis of new tropane-based molecular tags for evaluation in cultured human DAT (hDAT)-expressing cells and targeting in brain tissue. Chapter 2 discusses the results obtained from evaluation of the new tropane tags in hDAT-expressing and hNET-exressing cells, including early results from the first example of a DAT-specific voltage sensing dye. In Chapter 3, we discuss the principles governing molecular targeting of probes in the living brain tissue. Part I of Chapter 3 gives important background necessary for understanding some of the complexities involved in targeting chemical probes to specific sites in living brain tissue. Part II of Chapter 3 discusses early results obtained from targeting of our tropane tags in living brain tissue. We provide, perhaps, the first example of a binding-site barrier effect in healthy tissue and demonstrate successful delivery of a moderate-sized protein, neutravidin, to dopaminergic axons. Chapter 3 also discusses preliminary results demonstrating the behavior of our small molecule tag and tagged quantum dot construct in the living mouse brain. Studies of our tagged polymers in cultured cells and our work thus far in the brain suggest which polymers may be most effective as delivery platforms for chemical targeting to specific cell types in living brain tissue.

by Tai Fai Fok. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. 211-215). / Microfiche. Ann Arbor, Mich. UMI, 1998. 3 microfiches ; 11 x 15 cm.

Aim: Subunit vaccines have received increasing attention due to their good safety profile. However, subunit vaccines feature low immunogenicity, and soluble antigen is largely ignored by the immune system due to its lack of danger signals. To stimulate an appropriate immune response, subunit antigen vaccines require the addition of an adjuvant and multiple administrations. This study aimed to formulate biodegradable lipid implants, containing a suitable adjuvant, which delivers antigen in a sustained manner. The physico-chemical characteristics of the implants and their ability to stimulate immune responses towards a model antigen in vivo were investigated. Methods: Lipid implants were prepared from phospholipid and cholesterol. Different adjuvants were added, and their potential to induce an immune response to the model antigen ovalbumin (OVA) was investigated. The adjuvants and immunomodulators assessed were Quil-A (QA), imiquimod, and an α-Galactosylceramide (α-GalCer) analogue. Liposomal dispersions were prepared using the lipid film hydration method. These were freeze-dried, and the powder compressed into matrices (diameter of 2 mm). Physico-chemical characterisation was undertaken by transmission electron microscopy (TEM) to investigate the release of colloidal structures (liposomes, immunostimulating complexes [ISCOMs]) upon hydration with release media. Surface changes of the implant matrices were analysed using scanning electron microscopy (SEM). The release of the fluorescently-labelled antigen ovalbumin (FITC-OVA) and its entrapment into the colloidal particles was investigated using spectrofluorophotometry. Additionally, incorporation of the cationic cholesterol derivative DC-cholesterol (DCCHOL) into implants to allow for charge-charge interactions with the negatively-charged OVA, and replacement of the phospholipid with a phospholipid having a higher transition temperature to facilitate the manufacturing process, were attempted and assessed. The immune response stimulated towards OVA released from the implants was analysed in vivo using a C57Bl/6 mouse model. Expansion of CD8⁺ T cells and CD8 T cells specific for the CD8 epitope of OVA (SIINFEKL), as well as expansion of CD4⁺ T cells, were assessed. The ability of implants to stimulate T cell proliferation and interferon-γ production after in vitro restimulation with OVA was analysed. Serum samples were analysed for OVA-specific IgG antibodies. Results: Lipid implants containing Quil-A released colloidal structures upon hydration with buffer. The type of colloids observed by TEM depended on the ratio of QA:cholesterol:phospholipid. Release of OVA was sustained over ten days in implants prepared with egg yolk PC. However, the release kinetics depended strongly on the choice of phospholipid. In vivo, lipid implants containing Quil-A evoked expansion of CD8⁺ T cells. The immune response to one implant was comparable to that obtained by two equivalent injection immunisations. Therefore, the implants obviated the need for multiple immunisations in the vaccination regime tested here. Expansion of CD8⁺ T cells towards the Quil-A-containing implant was greater than that achieved by the immunomodulators imiquimod and the α-GalCer analogue. Quil-A-containing implants produced OVA-specific IgG antibodies to a greater extent than the implants containing imiquimod or α-GalCer. Incorporation of the cationic DCCHOL did not increase the entrapment efficiency of OVA into liposomes. However, the in vivo investigation of DCCHOL-containirig implants showed an adjuvant effect of DCCHOL on antibody responses, but not on cell-mediated immunity. Conclusion: Lipid implants offer great potential as sustained release vaccine delivery systems. The lipid components in the implant formulation were well-tolerated and biodegradable. Lipid implants combine the advantages of sustained release of antigen and particulate delivery by the formation of colloidal particles.

Since its discovery 15 years ago there has been an explosion of research in the field of DNA immunisation. Unfortunately despite early promises that DNA immunisation had the potential to cure almost any infectious disease, autoimmune disease or even cancer, progress towards clinical trials has been slow. This has been due in part to the huge range of permutations possible in delivering the DNA. One approach is to deliver the DNA by gene gun. Gene gun delivery is a very efficient way of transfecting cells however also has a number of possible disadvantages. These drawbacks include a weak immunogenicity in larger animals as well as the tendency to bias towards the development of a strong type 2 response. In an effort to enhance antigen-specific immune responses and counter the type 2 polarisation of gene gun delivery, a series of DNA vaccines were created where the extracellular portion of the hemagglutinin (HA) gene from influenza A/PR8/34 virus was genetically fused the type 1 cytokines IFNγ, IL-12 and IL-23. Interleukin-23 has been recently discovered and even though both IL-12 and IL-23 contain the p40 subunit they seem to have dissimilar functions. The vaccine constructs were first tested in cellular assays in vitro to ensure correct production and biological activity of the attached cytokines. They were then delivered in various combinations to groups of BALB/c mice to test development of immune responses and the effect of different delivery regimes. Finally mice were immunised then challenged with live influenza virus to determine the different DNA vaccines� protective efficacy. DNA vaccines containing the HA gene alone (pHA) or fused to IFNγ (pIFNγHA), IL-12 (pIL-12HA) or IL-23 (pIL-23HA) were successfully constructed. The fusion of the HA gene to the genes for IFNγ, IL-12 or IL-23 did not significantly disturb the structure of the antigen or prevent the biological actions of the cytokines. Mice immunised three times with pHA had high titres of serum IgG1 antibody and their splenocytes produced approximately equal amounts of IFNγ and IL-5. Co-delivery of IFNγ was unable to alter immune responses regardless of whether it was delivered at the first, last or during all immunisations. Surprisingly co-delivery of IL-12 acted to suppress both antibody and cellular immune responses, possibly through an IFNγ/nitric oxide feedback loop. On the other hand co-delivery of IL-23 tended to enhanced immune responses and, while it did not significantly alter the type 1 to type 2 balance, it was able to increase the ability of mice to clear live influenza virus from their lungs when they were challenged 26 weeks after immunisation. This protection was associated with increased levels of neutralising antibody in the serum of pIL-23HA immunised mice. This research has illuminated several of the pitfalls in the development of DNA vaccines and the use of cytokine as adjuvants. However it has also broadened our understanding of IL-23 and implies that IL-23 could be effectively used to increase the development of longterm immunity after immunisation.

Purpose: The aim of this thesis was to investigate a suitable formulation for the topical delivery of growth factors to chronic wounds, and then to determine the concentrations reached within an animal wound model. A secondary aim was to determine if the chosen growth factor was present at levels able to stimulate the production of other cytokines, specifically IL-1β and MCP-1. Methods: An in vitro testing apparatus was designed and made and the release of model actives [bromophenol blue (BPB) and horseradish peroxidase (HRP)] from gels and films of hydroxylpropylmethylcellulose (HPMC) (E4M CR, K4M CR and E10M CR) was determined. In this study, Fibroblast Growth Factor -2 (FGF-2) (0.3 [mu]g) was incorporated into three formulations (solution, gel and dried gel film on Melolin[TM] backing) and together with a control formulation were administered to punch biopsy wounds in rats. The in vivo release was followed over three time periods (two, five and eight hours) and the amount of FGF-2 at various wound depths was quantified by ELISA. Two biological markers IL-1β and MCP-1 were quantified using ELISA. The FGF-2 was additionally tagged with a fluorescent dye so that visualisation of the penetration could be obtained via confocal microsopy. Results: For the HPMC gels, the more viscous gel (E10M) provided a greater diffusional barrier and slowed the release of BPB (12 � 3.5 [mu]g/min compared with 16 � 1.7 [mu]g/min and 18 � 1.4 [mu]g/min for K4M and E4M respectively). However, when HPMC was formulated as a dried film a burst release was seen and release of BPB was slowest from the more rapidly hydrating K4M. With the larger model active HRP, there was a slower diffusion through the gel barrier formed upon film hydration, such that time of 100% release was up to 300 minutes compared to approximately 60 minutes for BPB. When the film was dried onto a supportive backing, the initial burst release was minimised as the film did not break apart on contact with the wound, and hence film integrity was maintained and release prolonged. The in vivo studies showed that, two hours after application, the highest concentration of FGF-2 was seen in the surface granulation tissue of rats that received the solution formulation (2280 � 790 pg/g). The concentration decreased with increasing tissue depth but was significantly greater than the saline control in the surface granulation and subcutaneous fat layers (p<0.05). Tissue concentrations following application of the gel and film formulations were only marginally greater than control in the surface granulation layer. After eight hours, rats that received the solution retained elevated surface tissue concentrations (surface granulation and subcutaneous fat) of FGF-2. Repeated measures ANOVA using a general linear model showed statistically significant differences in the mean FGF-2 level with respect to formulation and length of time of application of the formulation (p<0.05). In terms of other cytokines, there was a release of both IL-1β and MCP-1 in all groups, immediately post-wounding, probably in response to cellular damage. After eight hours, the film formulation appeared to elevate IL-1β levels which may be useful to drive wound healing. Confocal microscopy images showed diffuse distribution of FGF-2 eight hours after application of the solution formulation after eight hours and that with the gel formulation FGF-2 initially aggregated at the wound surface. Conclusion: In vitro experiments investigating the effect of hydration rate and viscosity of HPLC polymers allowed a formulation to be chosen for further in vivo study. Elevated FGF-2 could be measured in superficial wound tissues up to eight hours after application of a solution. However, application of a comparable amount of FGF-2 in HPMC gels or films did not produce appreciable elevations in FGF-2 in wound tissues, although confocal microscopy indicated the penetration of FGF-2 into the wound for up to eight hours.

This thesis describes evaluation of a gastric retention device (GRD) developed at Oregon State University. The device was originally fabricated from Xanthan gum and Locust bean gum. A modified gastric retention device containing other additives was developed and investigated in this work. The modified device was evaluated in vitro for swelling and dissolution properties using riboflavin as a model drug. Different shapes and sizes of GRDs were tested in dogs to study the gastric retention potential of these devices. The effect of the device on food emptying from the stomach in dogs was also investigated. Endoscopic studies in dogs also showed that the device swells rapidly and considerably in gastric fluid. The bioavailability of riboflavin from three different size GRDs was determined in six fasted human volunteers and compared to an immediate release formulation. The biostudy indicated that the bioavailability of riboflavin from a large size GRD was more than triple that measured after administration of the immediate release formulation. Deconvolution was used to determine gastric residence time of the different size GRDs. A new colonic delivery system made of acetaminophen loaded beads produced by extrusion and spheronization and coated with different ratios of pectin and ethylcellulose coating solutions in a spray coating apparatus was also developed in this work. Such beads release their drug content in the colon due to susceptibility of pectin in the outer coat to enzymatic action of colonic bacteria. The new delivery system was evaluated in vitro by conducting release studies in different dissolution media to mimic transit times, pH and enzyme conditions in the gastrointestinal tract. The gastrointestinal transit behavior of drug beads was also assessed by conducting gamma-scintigraphic studies in dogs. The bioavailability and pharmacokinetic parameters of acetaminophen from several colonic delivery system formulations were determined in human volunteers and compared to the immediate release commercial product Tylenol®. A selected pectin-ethylcellulose coat formulation in the ratio 1:3 was further evaluated in six volunteers under both fed and fasting conditions and was found to be effective and to provide sustained drug release in the colon over a period of 12 hours. / Graduation date: 2003

This thesis describes 1) development of a gastric retention device (GRD) to increase gastric retention time of certain drugs, 2) product formulations of an immediate release itraconazole and controlled-release ketoprofen. GRD was fabricated from crosslinked carbohydrate polymers. Rate and extent of hydration of the film in water and in simulated gastric fluid, compressibility of film, shape of the film, and in vivo gastric transit time in the stomach of dog were used as tools to evaluate gastric retention properties. Hydration studies were carried out at 37��C. Evaluation of the device containing radio-opaque agents, in dogs for gastric retention was carried out with the help of X-rays. The device was found to stay in the stomach of dogs for at least 10 hours. GRD containing amoxicillin trihydrate caplets were evaluated in a human. The area under the excretion rate curve was found to increase by 30% when compared to without the device. A successful development of a formulation of water insoluble itraconazole, without the use of organic solvents, was achieved with modifications from eutectic mixture techniques. Solubilization of the drug was achieved in polyethylene glycol of higher molecular weight. A series of formulations made by varying the amounts ingredients therein, were evaluated for dissolution profile in comparison with the reference, Sporanox��. Effect of molecular weights of PEG and types of PEG were evaluated for desired drug dissolution. Preliminary study from 6 subjects under the conditions of fasting and fed indicated that bioavailability from the new formulation was increased slightly when compared to the reference. This may be correlated to difference in the rate of in vitro dissolution, where the new formulation has initial faster dissolution. A controlled-release formulation of ketoprofen was also developed using a diffusion-controlled polymer, which was coated onto the drug beads. Release of drugs from such beads is controlled by the thickness of the coat. Thickness of the coat was evaluated by SEM and was correlated to the desired in vitro drug release in comparison to the reference Oruvail��. A three-way cross over study involving the new formulation and two marketed products in 12 subjects under fasting conditions indicated that there was a significant difference between the new product and marketed products, so as to be considered non-bioequivalent. Use of In Vitro-In Vivo Correlations and Convolution- Deconvolution relations predicted desired in vitro drug dissolution in a subsequent modification of the formulation. / Graduation date: 1999

Thesis (Ph. D.)--University of Rhode Island, 2006. / Typescript. Includes bibliographical references (leaves 210-235).

Mesoporous silica particles offer great potential benefits as vehicles for drug delivery and in other biomedical applications. They present a high loading capacity due their ordered and size-tuneable pores that allow molecules to be loaded and released. In addition, they offer the possibility to enhance oral bioavailability of drugs with limited aqueous solubility and to protect pH sensitive drugs from the acidic conditions in the stomach on their way to the intestine. The aim of this thesis was to evaluate the biocompatibility and effects of mesoporous silica particles on immunocompetent cells. Subsequently, two potential life sciences applications were investigated: as adjuvants and as weight reduction agents. Adjuvants are used in vaccines in order to enhance the immunological response towards attenuated and poorly immunogenic antigens. Their function can be mediated through dendritic cells which have a central role in the control of adaptive immunity including immunological memory. Our results show that different types of mesoporous silica particles were able to tune the development of T cells both in human cell cultures and in mice. In contrast to the approved adjuvant alum (aluminium salts) which is a specific inducer of Th2-type immune responses, the particles induced more Th1-like responses, which may be desired in vaccines against allergy and intracellular pathogens such as viruses. Particle exposure to macrophages did not affect their cell function which is crucial for tissue homeostasis, wound repair and in prevention of autoimmune responses. Likewise, the cytokine secretion was not affected, which suggest that macrophages would not modulate the immune response towards the particles. Furthermore, mesoporous silica particles were highly tolerated at daily oral administrations of up to 2000 mg/kg doses for some of the materials prepared. Large pore mesoporous silica particles were shown to act as weight and body fat reduction agents without other observable pathological signs when administered in the diet of obese mice. Together; those results are promising for the development of mesoporous silica as drug delivery systems and adjuvants for oral administration of drugs or vaccines. Additionally, large pore mesoporous silica materials are potential agents for the treatment of obesity.

This dissertation focuses on utilization of poly(N-isopropylacylamide) (pNIPAm) based mirogels for regulated macromolecule drug delivery applications. There is particular emphasis on incorporation of stimuli responsive materials into multi-layer thin film constructs with the main goal being fabrication of highly functional materials with tunable release characteristics. Chapter 1 gives a broad overview of hydrogel and microgel materials focusing on fundamental properties of pNIPAm derived materials. Chapter 2 illustrates the progression of controlled macromolecule release from hydrogel and microgel materials and sets up the scope of this thesis work. Chapter 3 details studies on thermally modulated insulin release from microgel thin films where extended pulsatile release capabilities are shown. Chapters 4 and 5 focus on more fundamental synthesis and characterization studies of PEG and acrylic acid modified pNIPAm microgels that could ultimately lead to the design of protein loaded microgel films with tunable release characteristics. Chapter 6 illustrates fundamental macromolecule loading strategies, which could also prove useful in future protein drug delivery design using stimuli responsive networks. Chapter 7 concentrates on direct insulin release studies that probe the interaction between entrapped and freely diffusing protein and microgels. These model experiments could prove useful in design of tunable macromolecule drug release from functionally modified microgels and could aid in the tailored design of peptide-loaded microgel thin films. Chapter 8 discusses the future outlook of controlled macromolecule release from microgel based materials.