Supermolecule-Drug Conjugates Based on Acid-Degradable Polyrotaxanes for pH-Dependent Intracellular Release of Doxorubicin

Abstract

Doxorubicin (DOX)-conjugated acid-degradable polyrotaxanes (PRXs) were designed as supramolecular drug carriers capable of releasing drugs in acidic cellular environments. Acid-degradable PRXs composed of α-cyclodextrin (α-CD) as a cyclic molecule, poly(ethylene glycol) (PEG) as a polymer axis, and N-triphenylmethyl (N-Trt) groups as an acid-labile stopper molecules were synthesized and DOX was conjugated with the threaded α-CDs in the PRXs. Because the acid-induced cleavage of N-Trt groups in PRXs leads to PRX dissociation, the DOX-modified α-CDs were released under acidic conditions (pH 5.0). The cytotoxicity of DOX-conjugated PRXs in colon-26 cells revealed significant cell death for DOX-conjugated PRXs after 48 h of treatment. Confocal laser scanning microscopy (CLSM) analysis revealed that the fluorescence signals derived from DOX-conjugated PRXs were observed in cellular nuclei after 48 h, suggesting that the DOX-modified α-CDs were released and accumulated in cellular nuclei. These results confirmed that acid-degradable PRXs can be utilized as drug carriers capable of releasing drug-modified α-CDs in acidic lysosomes and eliciting cytotoxicity. Overall, acid-degradable PRXs represent a promising supramolecular framework for the delivery and intracellular release of drug-modified α-CDs, and PRX-drug conjugates are expected to contribute to the development of pH-responsive drug carriers for cancer therapy.

Keywords: cyclodextrin; doxorubicin; drug delivery system; polyrotaxane.

Figure 1

Schematic illustration of acid-degradable polyrotaxane (PRX)–drug conjugates and their acid-induced dissociation.

Figure 2

Synthetic scheme for doxorubicin (DOX)-conjugated acid-degradable PRXs.

Figure 3

(A) SEC charts of α-CD, axle PEG, PRX, HPR, and DOX-HPR in dimethyl sulfoxide (DMSO) monitored with refractive index (RI) and UV (500 nm) detectors. (B) ¹H NMR spectrum of DOX-PRX in DMSO-d₆.

Figure 4

(A) Fluorescence spectra of DOX (1 μM) and DOX-HPR (1 μM DOX) in PBS. (B) Number-averaged size distribution of HPR (3 mg/mL) and DOX-HPR (3 mg/mL) in PBS.

Figure 5

(A) Schematic showing the cleavage of N-Trt groups in acid-degradable PRXs under acidic condition. (B) SEC charts of DOX-HPR after incubation at pH 5.0–8.0 at 37 °C for 24 h. SEC measurements were performed using DMSO as an eluent and the peaks were detected at 500 nm. (C) Relationship between pH and release profiles of DOX-modified α-CDs from DOX-HPR after incubation at 37 °C for 24 h, (D) SEC charts of DOX-HPR after incubation at pH 7.4 and 5.0 at 37 °C for 0–24 h. (E) Release profiles of DOX-modified α-CDs from DOX-HPR with time after incubation at pH 7.4 and 5.0 at 37 °C.

Figure 6

(A) Viability of colon-26 cells treated with DOX and DOX-HPR for (A) 24 h and (B) 48 h. Data are expressed as mean ± standard deviation (n = 6). * p < 0.05, ** p < 0.0001 vs. untreated cells (100% cell viability).

Figure 7

CLSM images of colon-26 cells treated with DOX (2 μM) and DOX-HPR (50 μM DOX) for 24 h and 48 h (scale bars: 20 μm). Cellular nuclei and lysosomes were stained with Hoechst 33258 and LysoTracker Green, respectively. The histograms depict the fluorescence intensity line profiles for the yellow lines in the merged images. Red, green, and blue lines denote DOX, LysoTracker Green, and Hoechst 33258, respectively.