Redox-Responsive Heparin-Chlorambucil Conjugate Polymeric Prodrug for Improved Anti-Tumor Activity

Abstract

Polymeric prodrug-based delivery systems have been extensively studied to find a better solution for the limitations of a single drug and to improve the therapeutic and pharmacodynamics properties of chemotherapeutic agents, which can lead to efficient therapy. In this study, redox-responsive disulfide bond-containing amphiphilic heparin-chlorambucil conjugated polymeric prodrugs were designed and synthesized to enhance anti-tumor activities of chlorambucil. The conjugated prodrug could be self-assembled to form spherical vesicles with 61.33% chlorambucil grafting efficiency. The cell viability test results showed that the prodrug was biocompatible with normal cells (HaCaT) and that it selectively killed tumor cells (HeLa cells). The uptake of prodrugs by HeLa cells increased with time. Therefore, the designed prodrugs can be a better alternative as delivery vehicles for the chlorambucil controlled release in cancer cells.

Keywords: chlorambucil; combination chemotherapy; heparin; prodrug; self-assembly.

Scheme 1

Schematic illustration of the Hep–Chl prodrug self-assembly and Chl intracellular drug release.

Figure 1

¹H NMR spectra of Hep (a), cystamine (Cys) (b), Hep-Cys (c), Chl (d), and Hep–Chl (e) conjugate. CDCl₃ is used as a solvent for Chl and D₂O for others.

Figure 2

Raman spectra of heparin (Hep), chlorambucil (Chl), cystamine dihydrochloride (Cy), and the conjugate Hep–Cy–Chl.

Figure 3

(a) UV-vis spectra of Hep, Hep-Cys, Chl, and Hep–Chl. (b) The concentration of Chl absorbance for the calibration curve at 304 nm.

Figure 4

Particle morphology and size: (a) AFM images, (b) SEM images, and (c) particle size distribution Hep–Chl-1, Hep–Chl-2, and Hep–Chl-3 self-assembled prodrugs.

Figure 4

Particle morphology and size: (a) AFM images, (b) SEM images, and (c) particle size distribution Hep–Chl-1, Hep–Chl-2, and Hep–Chl-3 self-assembled prodrugs.

Figure 5

Redox-responsive drug release profile of Hep–Chl self-assembled prodrug nanoparticles in PBS only, 2 mg/mL GSH/PBS, and 0.1% H₂O₂ PBS.

Figure 6

Cell viability of HeLa (red) and HaCaT (black) cells treated with Hep–Chl for 24 h. Cell viability was measured using the standard MTT assay. Data are presented as mean ± standard deviation.

Figure 7

Cell viability. Fluorescence images of the HeLa cells stained with calcein-acetoxymethyl ester (AM)/propidium iodide (PI) with Hep–Chl, Chl, and PBS (control).

Figure 8

Cellular uptake of prodrug nanoparticles. The fluorescence intensity measuring Rhodamine B-labeled Hep–Chl uptake after 3, 6, and 12 h of treatment of HeLa cells.