A novel polyrotaxane-based intracellular delivery system for camptothecin:In vitro feasibility evaluation

Camptothecin (CPT) is a naturally occurring alkaloid that shows promise in antitumor activity in vitro against various tumor cell lines. Its potential clinical uses, however, are hindered by a lack of reaction selectivity and poor water solubility. Presented herein is a novel polyrotaxane (PR)-based delivery system that could potentially lead to a highly effective yet less toxic CPT therapy. The approach involves the synthesis of the PR-CPT conjugates via hydrolyzable linkages. To enhance the therapeutic efficacy of CPT, a cell-penetrating peptide, LMWP, is linked to the conjugate to allow specific, intratumoral delivery of CPT. To avoid nonselective uptake of the conjugates by normal tissues following administration, the cell-penetrating function of LMWP on the conjugates is masked by heparin binding. This system was designed such that after accumulation at the tumor via the enhanced permeability and retention (EPR) effect, protamine can be subsequently administered to unmask heparin inhibition on LMWP, permitting intracellular uptake of the LMWP-PR-CPT conjugates. Once inside the tumor, CPT molecules are detached from the PR chain by hydrolysis, yielding a sustained concentration of CPT within tumor cells. In this paper, we demonstrated the in vitro feasibility of this delivery system. The LMWP-PR-CPT conjugates yielded a sevenfold increase in the overall CPT solubility, as well as a sustained release of CPT over a period of more than 7 days. Intracellular uptake of these conjugates by A2780 human ovarian cancer cells and regulation of such uptake by heparin and protamine were tested by MTT assay and confocal/flow cytometric methods, respectively.