In vitro assessment of a novel polyrotaxane-based drug delivery system integrated with a cell-penetrating peptide

Abstract

In the development of anti-cancer drugs, it is important to yield selective cytotoxicity primarily against tumor tissues. To achieve this goal, the use of a polymer-drug conjugate appears to be appealing, simply because it can take the advantage of the so-called enhanced permeability and retention (EPR) effect due to vascular leak in tumors. Among various types of polymers, polyrotaxane (PR) is an interesting candidate and warrants further consideration. It is a self-assembled polymer made entirely of biocompatible components, by threading alpha-cyclodextrin (alpha-CD) molecules with the poly(ethylene glycol) (PEG) chain. The abundance in functional -OH groups on the CD residues renders PR the capability of carrying a large dose of small anti-tumor agents for delivery. Herein, we presented a novel PR-based delivery system using doxorubicin (DOX) as the model anti-cancer drug. Daunorubicin (DNR) was conjugated to the PR polymer via hydrolysable linkages, and upon hydrolysis, doxorubicin was released as the cytotoxic drug. To facilitate an intracellular uptake by the tumor cells of the PR-DOX conjugates, a cell-penetrating low molecular weight protamine (LMWP) peptide was further attached to the two termini of the PR chain. Using an innovative principle established in our laboratory, such as via the inhibition of the cell-penetrating activity by binding with heparin and reversal of this inhibition by subsequent addition of protamine, cellular uptake of the polymer-drug conjugates could be readily regulated. In this paper, we performed in vitro studies to demonstrate the feasibility of this delivery system. The LMWP-PR-DOX conjugates, which yielded a sustained release of DOX over a period of greater than 4 days, were successfully synthesized. Intracellular uptake of these conjugates by A2780 human ovarian cancer cells and regulation of such uptake by heparin and protamine were confirmed by using the MTT assay and also the confocal microscopy method.

Figure 1

Schematic illustration of the polyrotaxane-doxorubicin conjugates.

Figure 2

Schematic illustration of the synthesis of polyrotaxane-doxorubicin conjugates.

Figure 3

NMR spectrum of the synthesized polyrotaxane-doxorubicin conjugates.

Figure 4

Doxorubicin release from polyrotaxane-doxorubicin conjugate in PBS (pH 7.4). (n=3)

Figure 5

Cytotoxicity of doxorubicin-polymer conjugates. Free DOX (◆), polyrotaxane-DOX (■), and LMWP-polyrotaxane-DOX (▲) conjugates against A2780 human ovarian cancer cells in log-phase culture. Values represented as mean ± SD. Each experiment was performed in triplicate.

Figure 6

Cellular localization of Lissamine Rhodamine B ethylenediamine-labeled LMWP-drug conjugates in A2780 human ovarian carcinoma cells. Free DOX, Lissamine Rhodamine B ethylenediamine(RHO)-labeled polyrotaxane-DOX, Rho-labeled LMWP-polyrotaxane-DOX, Rho-labeled LMWP-polyrotaxane-DOX and heparin mixture, and Rho-labeled LMWP-polyrotaxane-DOX, heparin, and protamine mixture were overlaid onto cultured A2780 cells in the presence of 10% FBS. Cellular localization was monitored by confocal microscopy. (1) Free DOX; (2) Rho-labeled polyrotaxane-DOX; (3) Rho-labeled LMWP-polyrotaxane-DOX; (4) Rho-labeled LMWP-polyrotaxane-DOX and heparin mixture; (5) Rho-labeled LMWP-polyrotaxane-DOX, heparin, and protamine mixture. (A) 488 nm (green) detection; (B) 560 nm (red) detection; (C) overlaid (A+B) Bar is 20 μm.