Le glioblastome (GB), tumeur primitive du cerveau, la plus agressive, et la plus fréquente chez l’adulte, présente une prolifération vasculaire importante. Des agents thérapeutiques innovants ciblant à la fois l'angiogenèse et les cellules tumorales sont recherchés, ainsi que des systèmes pour augmenter leur délivrance dans la tumeur cérébrale. Un de ces agents est le sorafénib (SFN), un inhibiteur de tyrosine kinase. Sa mauvaise solubilité aqueuse et ses effets secondaires indésirables limitent son utilisation. Le premier objectif de cette thèse était d'encapsuler cet agent dans des nanocapsules lipidiques (NCL) pour contrer ces inconvénients. Nous avons développé des NCL avec une haute efficacité d'encapsulation du SFN qui inhibaient in vitro l'angiogenèse et la viabilité de la lignée de GB humain U87MG. La délivrance intratumorale de SFN-NCL chez des souris porteuses d’une tumeur intracérébrale U87MG induit une normalisation vasculaire tumorale précoce qui pourrait améliorer l'efficacité de la chimiothérapie et de la radiothérapie. Le second objectif était de définir si la délivrance intranasale de NCL pouvait constituer une voie non-invasive alternative. Nous avons étudié via le transfert d'énergie par résonance de type Förster, le devenir des NCL chargées d’un fluorochrome à travers des monocouches de cellules Calu-3, un modèle de l'épithélium nasal. L'utilisation de NCL augmente le passage du fluorochrome à travers les cellules Calu-3, mais les particules sont rapidement dégradées après leur capture. Ces données mettent en évidence que les NCL sont appropriées pour la délivrance locale du SFN mais doivent être modifiées pour une délivrance intranasale. / Glioblastoma (GB), the most aggressive, and the most frequent primary tumor of the brain in adults, present a prominent vascular proliferation. Innovative therapeutic agents targeting both angiogenesis and tumor cells are urgently required, along with competent systems for their delivery to the brain tumor. One such agent is sorafenib (SFN), a tyrosine kinase inhibitor. However, poor aqueoussolubility and undesirable side effects limit its clinical application. The first objective of this thesis was to encapsulate this drug inside lipid nanocapsules(LNCs) to overcome these drawbacks. We developed LNCs with a high SFN encapsulation efficiency (>90%) that inhibited in vitro angiogenesis and the viability of the human U87MG GB cell line. Intratumoral delivery of SFN-LNCs in mice bearing intracerebral U87MG tumors induced early tumor vascular normalization which could be used to improve the efficacy of chemotherapy and radiotherapy in the treatment of GB. The second objective was to define whether intranasal delivery of LNCs could be an alternative non-invasive route. In this regard, we investigated through Förster resonance energy transfer, the fate of dye-loaded LNCs across Calu-3 cell monolayers, a model of the nasal mucosa. We showed that employment of LNCs dramatically increased the delivery of the dye acrossCalu-3 cell monolayer but they were rapidly degraded after their uptake. These data highlight that LNCs are suitable nanocarriers for the local delivery of SFN but must be redesigned for enhancing their nose-to-brain delivery.

The thrust of this thesis is to study oral solid dosage formulation using hot melt coating method and to use pharmacokinetic modeling and simulation (PK M&S) as a tool that can help to predict pharmacokinetics of a drug in human and the probability of passing various bioequivalence criteria of the formulation based on the PK of the drug. Hot-melt coating using a new method, direct blending, was performed to create immediate and sustained release formulations (IR and SR). This new method was introduced to offer another choice to produce IR and SR drug delivery formulations using single and double coating layer of waxes onto sugar beads and/or drug loaded pellets. Twelve waxes were applied to coat sugar cores. The harder the wax the slower the drug was released from single coated beads. The wax coating can be deposited up to 28% of the weight of the core bead with 58% drug loading efficiency in the coating The cores were coated with single or double wax layers containing acetaminophen. Carnauba wax coated beads dissolved in approximately 6 hrs releasing 80% of loaded drug. However, when covered with another layer, the drug loaded beads released drug for over 20 hrs. When drug loaded pellets were used as cores, 33-58% drug loading was achieved. Double coated pellets exhibited a near zero order drug release for up to 16 hrs. Hot melt coating by direct blending using waxes is a simple process compared to conventional hot melt coating using coating pan or fluid bed coating machines. It offers an alternative way of making immediate, sustained drug release (IR, SR) and modified release (IR+SR) oral dosage forms of drugs which are stable at high temperature (100°C). The pellet-containing-drug coated formulations provide options when higher drug loading is warranted. It is required by the US Food and Drug Administration (FDA) that a new modified –release (MR) product or identical generic product be regarded as bioequivalent (BE) to the originators reference drug product. However, there are concerns that current regulatory criteria are not sufficient when evaluating bioequivalence (BE) for many MR products, and additional metrics for BE assessment of the products should be applied to ensure therapeutic equivalence. This study used pharmacokinetic modeling and simulation (M&S) to investigate 1) the probability of BE occurring between the MR test and reference products 2) the rates of false positive and true negative of the BE test; and 3) the estimation of the sample size in pivotal BE studies; all of which when partial area under the curves (pAUCs) were applied as additional BE criteria. Reference data of two MR forms of methylphenydate HCl (MPH) were simulated and obtained from literature (formulation Q and Metadate CD, respectively). Monte Carlo simulations were performed to simulate the test drug concentration profiles and BE assessment was carried out utilizing the mean (method 1) and individual concentration time curves (method 2). For formulation Q, adding pAUC₀₋[subscript Tmax] to current BE criteria reduced the possibility of passing BE from approximately 98% to 85%, with a true negative rate of 5%. The earlier the time points used to determine for pAUC before Tmax, the lower the chance of passing BE for the test product. The possibility of passing BE varied and depended on the coefficient of variations (CV) of T[subscript lag], K[subscript a] and K[subscript e] and that considerable variability in the parameters affected the earlier segments of the drug concentration profile curves more. Similar drug concentration time profiles between the test and reference products is recommended to ensure bioequivalence occurs with a reasonable subject sample size. A similar scenario was seen when Metadate CD was used as the reference product. PK M&S can help provide appropriate additional metrics to assure the BE test is a better tool ensuring therapeutic equivalence for MR products with little negative impact to generic manufacturers. Predictions can also be made about the required sample size and the chances of passing BE with any addition to the conventional three criteria for the test product. PK M&S was also used to predict drug concentrations of levofloxacin in tissue. Levofloxacin has been widely used in clinical practice as an effective broad-spectrum antimicrobial, however tendonitis and tendon rupture have been reported with increasing use of this agent. Here, these incidents will be assessed by investigating pharmacokinetic behavior of the compound to see if they are related to drug's tissue disposition. The PK model for levofloxacin was established. Mean concentration time profiles of single or multiple dosing of 500 mg levofloxacin following oral and IV infusion administration were simulated. Monte Carlo simulation was used to simulate the drug concentration time profiles in plasma (compartment 1) and tissue (compartment 2) after seven dosing regimens while varying the drug's elimination and distribution rates to see the effect of changing those rates have on the drug accumulation in tissue. Monte Carlo Simulation shows that low elimination rates affect the drug concentration in plasma and tissue significantly with the level in plasma rising up to 35 μg/mL at day 7. A normal elimination rate together with escalation of distribution rates from plasma to tissue could increase the tissue concentration after 7 doses to 9.5 µg/mL, a value that is more than twice that of normal. PK M&S can be used as an effective tool to evaluate drug concentration in different compartments (plasma and tissues, for example). The unexpectedly high concentration values in some cases may explain, at least in part, the reason of tendinopathy occurs in the clinical setting. / Graduation date: 2012 / Access restricted to the OSU Community at author's request from June 7, 2012 - June 7, 2014

A more quantitative understanding of peptide loading and release from polyethylene oxide (PEO) brush layers will provide direction for development of new strategies for drug storage and delivery. The antimicrobial peptide nisin shows potent activity against Gram-positive bacteria including the most prevalent implant-associated pathogens, its mechanism of action minimizes the opportunity for the rise of resistant bacteria and it does not appear to be toxic to humans, suggesting good potential for its use in antibacterial coatings for selected medical devices. In this work, optical waveguide lightmode spectroscopy was used to record changes in adsorbed mass during cyclic adsorption-elution experiments with nisin, at uncoated and PEO-coated surfaces. PEO layers were prepared by radiolytic grafting of Pluronic® surfactant F108 or F68 to silanized silica surfaces, producing long- or short-chain PEO layers, respectively. Kinetic patterns were interpreted with reference to a model accounting for history-dependent adsorption, in order to evaluate rate constants for nisin adsorption and desorption, as well as the effect of pendant PEO on the lateral clustering behavior of nisin. Lateral rearrangement and clustering of adsorbed nisin was apparent on uncoated and F68-coated surfaces, but not on F108-coated surfaces. In addition, nisin showed greater resistance to elution by peptide-free buffer from uncoated and F68-coated surfaces. These results are consistent with shorter PEO chains allowing for peptide adsorption to the base substrate in the case of F68-coated surfaces, while adsorption to the F108-coated surfaces is apparently governed by the presence of a hydrophobic core within the brush layer itself. Further, these results suggest that while peptide location within the hydrophobic core provides stability against lateral rearrangement, the pendant PEO chains themselves provide no steric barrier to nisin rearrangement within the brush layer. / Graduation date: 2012

This dissertation describes two different projects. The first is the development of an oral DNA vaccine delivery system for fish. A novel oral DNA vaccine delivery system was developed for Rainbow Trout by combining non-viral vectors (polycationic liposomes or polycationic polymer) to facilitate the DNA vaccine's uptake by cell membranes along with enteric-coated protection of the DNA embedded in microparticles to prevent DNA degradation in the gastrointestinal tract. Spray drying and spray coating bead techniques were employed in the preparation of the DNA vaccine microparticles. The spray drying technique allowed production of spherical shape enteric-coated microparticles with a particle size range of 0.18 to 20 ��m. Larger particle sizes of 40-50 mesh were obtained from the spray-coated bead technique. The resultant DNA vaccine microparticles were granulated with regular fish feed and given to fish to investigate the efficacy of the delivery system in providing protection against IHNV, and to demonstrate the ease of administration in fish. An in vivo fish trial experiment showed improvement in fish survival rate when fish were immunized with larger particle size DNA vaccine microparticles. Further research to find effective vector carriers for the DNA vaccine delivery system and to seek modifications of the delivery system that will still prevent the denaturation of plasmid DNA that will also facilitate membrane uptake of the DNA vaccine is needed in order to develop a safe, effective, and commercially viable vaccine to control the outbreak of IHNV. The second project of the dissertation is prediction of in vitro drug release profiles from a novel matrix tablet spray-coated with a barrier membrane using mathematical and statistical models. Tablets were prepared by direct compression followed by spray coating. The relationship of the amount of hydrophilic materials in the core tablets and barrier thickness on drug release mechanism was investigated using factorial design and regression analysis. Drug release characteristics were influenced and can be controlled by modifying the amount of hydrophilic materials in the core tablet and the barrier thickness. Mathematical equation generated from regression analysis of n-value, lag time, and percent drug release as a function of the amount of hydrophilic material and the amount of coating material applied can now be used as a tool for predicting and optimizing in vitro drug release from matrix tablets spray-coated with a barrier membrane. / Graduation date: 2003

The assembly of poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAm-co-AAc) microgel thin films into disordered and ordered arrays was investigated. Disordered pNIPAm-co-AAc microgel arrays were assembled based on electrostatic attractions between polyanionic pNIPAm-co-AAc microgels and polycationic poly(allylamine hydrochloride) (PAH). These interactions were studied in solution and subsequently used to assemble thin films following a Layer-by-Layer assembly protocol. Thin films were assembled as a function of pNIPAm-co-AAc microgel solution temperature and the resultant film thermoresponsivity characterized as a function of microgel layer number and pH. The response of assembled thin films to pH 3.0 and 6.5 exposure was then characterized by quartz crystal impedance and surface plasmon resonance spectroscopy, which showed that the thin film solvation was highly dependent on the pH of the solution it was in. Assembled thin films were also shown to be useful as controlled drug delivery platforms, where it was found that small molecules could be released from the films in a temperature regulated fashion. Microgel thin films also exhibited unique optical properties and were used as microlens arrays, which were able to focus pattern in air as well as in solution and had focal lengths that could be tuned in response to pH and temperature changes. Ordered microgel arrays were assembled following a thermal annealing process, in order to make light diffracting materials. These ordered arrays were photopolymerized and exhibited temperature dependent Bragg diffraction properties.

The world-wide transdermal drug delivery market is quite large, but only a small number of agents have FDA approval. The primary reason for such limited development is the difficulty in permeating the stratum corneum layer of human skin. In our study, we developed a novel percutaneous delivery enhancing approach. Magainin peptide was previously shown to disrupt vesicles from stratum corneum lipid components and this ability of magainin allows us to propose that magainin can increase skin permeability. Therefore, we tested the hypothesis that magainin, a pore-forming peptide, can increase skin permeability by disrupting stratum corneum lipid structure and that magainin¡¯s enhancement requires co-administration of a surfactant chemical enhancer to increase magainin penetration into the skin. In support of these hypotheses, synergistic enhancement of transdermal permeation can be observed with magainin peptide in combination of N-lauroyl sarcosine (NLS) in 50% ethanol-PBS solution. The exposure to NLS in 50% ethanol solution increased in vitro skin permeability to fluorescein 15 fold and the addition of magainin synergistically increased skin permeability 47 fold. In contrast, skin permeability was unaffected by exposure to magainin without co-enhancement by NLS-ethanol. To elucidate the mechanism of this synergistic effect, several characterization methods such as differential scanning calorimetry, Fourier transform infrared spectroscopy, and X-ray diffraction were applied. These analyses showed that NLS-ethanol disrupted stratum corneum lipid structure and that the combination of magainin and NLS-ethanol disrupted stratum corneum lipids even further. Furthermore, confocal microscopy showed that magainin in the presence of NLS-ethanol penetrated deeply and extensively into stratum corneum, whereas magainin alone penetrated poorly into the skin. Together, these data suggest that NLS-ethanol increased magainin penetration into stratum corneum, which further increased stratum corneum lipid disruption and skin permeability. Finally, skin permeability was enhanced by changing the charge of magainin peptide via pH change. We modulated pH from 5 to 11 to change the magainin charge from positive to neutral, which decreased skin permeability to a negatively charged fluorescein and increased skin permeability to a positively charged granisetron. This suggests that an attractive interaction between the drug and magainin peptide improves transdermal flux.

The use of various biodegradable polymers for the improvement of different controlled and long-lasting drug release systems is an active research area in recent years. The application of different metal prostheses, especially titanium based ones, to the human body is also very common. A most important disadvantage of these prostheses is the risk of infection at the application areas that necessitates the removing of the prosthesis with a second surgical operation and reapplication of it after recovery. One of the best ways to solve this problem is to render metal prostheses infection free with controlled and sustainable drug (antibiotic) release systems. The long term sustained release of relevant antibiotics from the various biodegradable polymer coated metal implants is studied in this thesis. Virtual fatigue analysis and drug loading capacities of titanium and stainless steel samples with different surface pattern and modifications were studied. Various biodegradable polymer and drug combinations were examined and used for coating of metal prosthesis. The aim is to design polymer-drug coated metal implants that are capable of releasing a feasible amount of drug up to a period of at least 1 month. Various coating techniques and surface modifications were also employed to improve the adhesional properties of the drug containing polymers. Their adhesion abilities on the metal substrates were tested by Lap-shear and T-peel tests. Polymer degradation kinetics was followed by viscosity studies. Calibration lines for different drugs were obtained and drug releases on different systems were followed by using UV spectroscopy and microbial antibiotic sensitivity tests. Among the techniques applied to prevent fast release of drugs initially, the coatings of Vancomycin absorbed &amp / #946 / -TCP (&amp / #946 / -tricalcium phosphate) homogeneously distributed in poly(D,L-lactide-co-glycolide) solution in chloroform followed by an inert coating with poly(L-lactide) system proved to be feasible. By this technique, initial burst release was minimized and drug release from implants lasted nearly 2 months. Multiple coatings on polymer plus drug coating layer also gave promising results. In vivo studies on dorsal muscles of native rabbits with antibiotic loaded implants gave no negative effect on the surrounding tissues with high compatibility free of infection.

In this presentation it is demonstrated that the unique magnetic properties of superparamagnetic cobalt-spinel ferrite nanoparticles can be employed in several novel applications. A method to selectively capture and remove pathogens from infected organisms to improve longevity is presented. Evidence is provided to show that automated methods using modified forms of hemofiltration or peritoneal dialysis could be used to eliminate the particle/pathogen or particle/infected cell conjugates from the organism postoperatively. It is shown that disparately functionalized nanoparticles can be used in concert as drug carrier and release mechanisms. Lastly, we provide preliminary evidence to support the use of magnetic nanoparticles for controlling reaction kinetics.

Amyotrophic lateral sclerosis (ALS) is a devastating disease. Currently, there is no cure for this disease, and effective treatment strategies are greatly needed. Calpain activation plays a major role in the motor neuron degeneration that causes ALS. Therefore, therapeutic strategies can inhibit calpain activity in the central nervous system (CNS) have great clinical potential. The calpain inhibitors AK295 and MDL-28170 have been demonstrated to be neuroprotective in animal models of neurological injury, and should have great potential to treat ALS; however delivery problems have hindered their clinical success. Therefore, development of a new strategy that can locally deliver the calpain inhibitors to the central nervous system could significantly improve the treatment of ALS. The objectives of my thesis research were (1) to develop high molecular weight polyketals that provide sustained release properties for hydrophobic molecules, (2) to formulate calpain inhibitor-encapsulated polyketal microparticles which have a release half life of one month in vitro, (3) and to evaluate the performance of polyketal microparticles for delivering calpain inhibitors to the spinal cord in vivo. In completing these specific aims, we have developed biodegradable polymeric microparticles for the delivery of calpain inhibitors, AK295 and MDL-28170 to treat ALS. The results of calpain assays showed that both AK-PKMs and MDL-PKMs maintained most of their inhibitory activities even after the robust emulsion process. The in vitro release profile of MDL-28170 in MDL-PKMs showed that 50 % of the drug was released in the first 30 days. Experiments using dye-encapsulated microparticles showed that polyketal microparticles (1-2 ìm) are not easily cleared in the neutral physiological environment and can have potential to continuously release drug from the injection sites in the spinal cord. The efficacy of calpain inhibitor-encapsulated PKMs were studied by evaluation the behavior and survival of SOD1G93A rats, a genetic rat model for ALS. We observed the trend toward improvements in grip strength and rotarod performance in the first two months from the AK-PKMs treated group, however, further improvements are needed to enhance their in vivo efficacy.

Existing methods of treating glioma are not effective for eradicating the disease. Therefore, new and innovative methods of treatment alone or in combination with existing therapies are necessary. Delivery of therapeutic agents through delivery carriers such as liposomes diminishes the harmful effects of the agent in healthy tissues and allows increased accumulation in the tumor. In addition, targeted chemotherapy using liposomes provides the opportunity for further increase in drug accumulation in tumor. However, the current targeting strategies suffer accelerated plasma clearance and are not advantageous in improving efficacy. The search for new tumor targets, novel ligands, new strategies for targeting, and particle stabilization will advance our ability to improve delivery at the tumor level while decreasing toxicity to normal tissues. The global objective of this thesis was to improve the status of current liposomal therapy to achieve higher efficacy in tumors. Here, we show a novel mechanism to increase targeting to tumor while uncompromising on the long circulation of stealth liposomes. Long circulation is essential for passive accumulation of the nanocarriers due to EPR effect, in order to see benefits of targeting. Using phage display technique, a variety of tumor specific peptides were identified for use as targeting moieties. One potential advantage of the approach proposed here is the rapid identification of patient tumor specific peptide that evades the RES. This could lead to the development of a nanocarrier system with high avidity and selectivity for tumors. Therefore, tumor accumulation of the targeted formulations will be higher than that of non‐targeted liposomes due to increased drug retention at the tumor site and uncompromised blood residence time.In addition, it has been shown that the distribution of nanocarriers, spatially within the tumor, is limited that might further hinder the distribution of the encapsulated drug, thereby limiting efficacy. It is necessary to release the drug from within the nanocarrier to promote increased efficacy. Here, we were able to address the problem of drug diffusion within the tumor interstitium using a combination therapy employing a remotely triggered thermosensitive liposomal chemotherapeutic. We fabricated a thermosensitive liposomal nanocarrier that maintained its stability at physiological temperature to minimize toxicity to healthy cells. We, then, showed a remote triggering mechanism mediated by gold nanorods heated via NIR can help in achieving precise control over the desired site for drug release. These strategies enabled increased drug availability at the tumor site and contributed to tumor retardation. Additionally, we show that the synergistic therapy employing gold nanorods and thermosensitive liposomes may have great potential to be translated to the clinic.