DISSERTATION (PH.D.)--THE UNIVERSITY OF MICHIGAN

Thesis (Ph. D.)--University OF MICHIGAN.

This is a study of drug delivery problems from hydrogels and it is mainly focused on the effects of the hydrogel's geometry on the behaviour of the release profile. Studying drug delivery also offers one the opportunity to delve into the physical mechanisms involved in the release process to better understand its theoretical implications. We can apply the well-developed theory of diffusion in order to understand many aspects of drug delivery. Thus, this thesis starts with a presentation of concepts and theories used in the following two articles (chapters). We go over the basic concept of drug delivery, followed by an introduction on diffusion. We then explain the ideas governing drug release and how we have modeled it. The tools used to generate our drug release platforms are presented followed by a discussion on characterizing the resulting drug release profiles. This thesis is presented as a series of two articles that have been submitted to peer-reviewed scientific journals. The first article presents a novel combinatorial technique used to obtain controlled drug delivery profiles. We use an exact enumeration diffusion model in order to obtain our drug release profiles and test its validity by comparing these results with analytical theory and widely used empirical tools. By using a genetic algorithm, we then show that it is possible to tailor the drug delivery platform in order to get a specific functional form of the release profile. The second article consists in testing two widely used empirical functions that are used in the literature to characterize drug release profiles. Several claims have been made regarding the interpretation of these functions and we have used our exact enumeration data to argue that although the functions fit the data relatively well on certain time scales, they do not necessarily convey reliable information about the mechanism of release. Finally, we conclude with preliminary work that was done on a second optimization technique to be used in controlling drug delivery profiles. The Metropolis simulated annealing was used to further optimize the design of the drug release platform and was shown to be quite effective, albeit being computationally demanding.

The human ovarian carcinoma cell line, A2780s, and its variant, A2780cp, provide an isogenic tumour model for examining factors which determine intrinsic radiosensitivity. A2780s demonstrates sensitivity to ionization radiation while A2780cp is relatively radioresistant (surviving fraction following a 2 Gy dose is 0.083 and 0.43, respectively). The increased radioresistance of A2780cp can not be explained by greater cellular recovery, neither from sublethal nor potentially lethal damage. Moreover, A2780s demonstrates significantly greater (P < 0.05) sublethal and potentially lethal damage recovery than A2780cp. The induction of double strand breaks (dsb), as measured by asymmetric field inversion gel electrophoresis (AFIGE), is identical in these cell lines. Dsb rejoining efficiency is similar between A2780s and A2780cp. Initially, rejoining kinetic experiments reveal that A2780s is deficient in the slow phase of rejoining, while A2780cp is not (80% of dsb remain for A2780s vs 29% A2780cp) (P < 0.01). This deficiency however, is not reproduced in residual dsb experiments and further analysis reveals that it may be an artifact of the normalization process. When the level of residual dsb as a function of dose is examined, the slopes are similar for both cell lines and the only significant difference is seen for the highest dose used, 150 Gy (50% for A2780s and 33% for A2780cp) (P < 0.05). Finally, the cell-free fidelity assay demonstrates that for blunt-cut plasmid, no differences exist in the rejoining efficiency of nuclear extracts. The ligation infidelity of blunt-cut plasmid however, is significantly less (P < 0.05) in A2780cp extracts (0.021 for A2780s vs 0.0030 for A2780cp). Therefore, neither the induction of dsb, nor the efficiency of dsb repair can explain observed differences in radiosensitivity. The fidelity of rejoining however, is significantly greater in the resistant variant and may, in part, explain these differences.

Damage to the immune system of exposed individuals renders the host susceptible to opportunistic infection and disease. The purpose of this investigation was to contribute to the understanding of mechanisms underlying depression of host immune responses following radiation exposure. Peripheral blood mononuclear cells (PBMC) of mice irradiated with 0-700 rad $\gamma$-whole body irradiation (WBI) were analyzed by flow cytometry (FCA). Natural killer (NK) cells and CD4+ T lymphocytes were selectively enhanced following radiation exposure, demonstrating radioresistance of these cell types over other PBMC, while B lymphocytes were dramatically radiosensitive. Dextran sulfate mobilization of mononuclear cells (MNC) from lymphoid tissues into the blood revealed that the same pattern of MNC loss had occurred throughout the lymphoid tissues. PBMC alterations reflected similar changes occurring in previous studies of splenic mononuclear cells $\{$1$\}$, and may promote immune dysregulation. The role of apoptosis in radiation-induced injury to the immune system in the low to intermediate dose range (0 to 400 rad) was investigated in PBMC. 25 rad induced apoptosis in PBMC above the unirradiated control within 2 hours post-irradiation; apoptosis induction increased with higher doses (100-400 rad). Additionally, the impact of ionizing radiation on NK cell function was assessed. 24 hours following radiation exposure, NK cytotoxicity against YAC-1 target cells was depressed by doses of 25 or 50 rad, with little change in the 100 to 400 rad range. By day 7, NK cytolytic potential was reduced or unaffected by doses lower than 200 rad, while a single exposure of 400 rad enhanced cytotoxicity. The results of this investigation have furthered our understanding of factors which may be important in the impairment of immune responses post-irradiation.

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