A low molecular weight protamine (LMWP)-mediated, polyrotaxane-based intracellular delivery system for anti-tumor agents.

In the development of anti-cancer drugs, it is important to maintain selective toxicity towards tumor tissues. Presented herein is a novel polyrotaxane (PR)-based delivery system that possesses the potential to achieve a highly effective yet less toxic cancer therapy. Briefly, the approach involves synthesis of the PR-anti-cancer drug (i.e. camptothecin or daunorubicin) conjugates by threading alpha-cyclodextrin (alpha-CD) monomers onto a poly(ethylene glycol) (PEG) chain, blocking both ends of the PEG thread with bulky tyrosine residues, and then coupling the anti-cancer drugs to the activated functional groups on alpha-CD via hydrolyzable ester linkages. To design a system for enhancement of therapeutic efficacy of anti-cancer drugs and reduction in their toxic effect towards tissues surrounding the tumor, a cell-penetrating peptide (i.e. low molecular weight protamine (LMWP)) is linked to the PR-drug conjugate to allow for a tumor-specific, intracellular delivery of anti-cancer drugs. In order to avoid non-selective uptake of the conjugates by normal tissues following their administration, the transmembrane function of LMWP on the conjugates is masked by binding with heparin. This system was designed in such a manner that after accumulation of the conjugates at the tumor site by passive targeting, protamine sulfate, a clinical heparin antagonist that binds heparin stronger than LMWP, is administered subsequently to unmask heparin inhibition on LMWP, permitting an effective intracellular uptake of the LMWP-PR-drug conjugates. Once inside tumor cells, anti-cancer drug molecules are released from the PR polymer chain by hydrolysis, yielding a sustained level of the drug within tumor cells. In this thesis, in vitro cell culture results were presented to demonstrate the feasibility of this drug delivery system. The LMWP-PR-drug conjugates, which yielded a linear and sustained release of anti-cancer drug over a period of greater than 4 days, were synthesized and characterized. Intracellular uptake of these LMWP-PR-drug conjugates by A2780 cells and regulation of such uptake by heparin and protamine were tested by the MTT assay and confocal/flow cytometry methods, respectively. These results suggest that this system offers the potential generic system for delivery of small anti-tumor agents possessing poor solubility or reaction selectivity.PhDHealth and Environmental SciencesPharmaceutical sciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126515/2/3253359.pd