Paclitaxel-based supramolecular hydrogel loaded with mifepristone for the inhibition of breast cancer metastasis

Abstract

Breast cancer is the leading cause of cancer death among women and almost all of the breast cancer-caused mortality is related to metastasis. It has been reported that glucocorticoid facilitates the metastasis of breast cancer in mice, and mifepristone can antagonize the effect of glucocorticoid. Paclitaxel is one of the important drugs in the treatment of breast cancer. Mifepristone combined with paclitaxel could be an effective strategy for inhibiting breast cancer metastasis. However, their inherent defects, in terms of short blood circulation half-life and lack of tumor targeting, not only limit their effectiveness but also cause adverse reactions. Therefore, our aim is to explore a novel protocol against breast cancer metastasis, further optimize its therapeutic efficacy by a nanodelivery system, and explore its mechanism. Herein, a paclitaxel-conjugated and mifepristone-loaded hydrogel (PM-nano) was prepared by self-assembly. Its characterizations were studied. The antimetastatic effect was evaluated in vitro and in vivo and its mechanism was also explored by western blot assay. The resultant PM-nano was developed with favorable water solubility and good biocompatibility. Moreover, PM-nano displayed increased cell uptake properties and stimulated drug release in the tumor micro-acidic environment. The PM-nano was more effective in inhibiting the proliferation and metastasis of breast cancer than other groups in vitro and in vivo. The PM-nano might inhibit metastasis through glucocorticoid receptor/receptor tyrosine kinase-like orphan receptor 1 and MMPs. Taken together, PM-nano showed superior antimetastatic effects against breast cancer and excellent biocompatibility in vitro and in vivo, providing a new option for limiting metastasis.

Keywords: breast cancer; metastasis; mifepristone; paclitaxel; supramolecular hydrogel.

FIGURE 1

Characterizations of paclitaxel (PTX)‐conjugated and mifepristone (MIF)‐loaded hydrogel (PM‐nano) and PTX hydrogel (P‐nano). A, Photographs of P‐nano hydrogel, PM‐nano hydrogel, free PTX solution with the same PTX concentration as PM‐nano, and free MIF solution with the same MIF concentration as PM‐nano before (a, b, c, d) and after (a′, b′, c′, d′) the addition of equal volume of water. B, Angular frequencies of PM‐nano and P‐nano. C, Shear strains of PM‐nano and P‐nano. D, Transmission electron microscopy images of PM‐nano. E, Circular dichroism spectra of succinic anhydride (SA)‐modified PTX compound (PTX‐SA), MIF, PM‐nano, and P‐nano

FIGURE 2

Drug release in vitro and cellular uptake of paclitaxel (PTX)‐conjugated and mifepristone (MIF)‐loaded hydrogel (PM‐nano) in breast cancer cells. A, Drug release of PM‐nano in PBS with different pH values (7.4 and 5.5, simulated normal physiological conditions and tumor micro‐acidic environment, respectively). B, Fluorescence images of MDA‐MB‐231 cells treated with free FITC or FITC‐loaded PM‐nano hydrogel (FITC@Hydrogel) after incubation at an equivalent FITC concentration (10 µg/mL) for 4 h (green, FITC; blue, DAPI). C, D, Cellular uptake amounts of PTX (C) and MIF (D) in breast cancer cells evaluated by HPLC after 4 h of incubation with free MIF, free PTX, or PM‐nano. ***P < .001

FIGURE 3

Biocompatibility of paclitaxel (PTX)‐conjugated and mifepristone (MIF)‐loaded hydrogel (PM‐nano). A, Hemolysis photographs after incubation with 2% red blood cell (RBC) suspension and various materials. B, Hemolysis ratios in the PTX concentration range of 0‐0.5 mg/mL. C, Hematology analyses of mice on day 2 (D2) and D8 of treatment. ***P < .001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; Cr, creatinine; P, paclitaxel; PD, PTX + dexamethasone; HB, hemoglobin; PLT, platelets; PM, PTX + MIF; PMD, PTX + MIF + dexamethasone; P‐nano, PTX hydrogel; UA, uric acid; WBC, white blood cells

FIGURE 4

Pharmacokinetics of paclitaxel (PTX)‐conjugated and mifepristone (MIF)‐loaded hydrogel (PM‐nano). A, B, Blood clearance curves of free PTX and hydrogel‐loaded PTX (A), and free MIF and hydrogel‐loaded MIF (B). C‐F, Biodistribution of free PTX (C), hydrogel‐loaded PTX (D), free MIF (E), and hydrogel‐loaded MIF (F) at various time points after treatment.*** P < .001. PTX‐SA, succinic anhydride‐modified PTX compound

FIGURE 5

Effects on the biological functions of breast cancer cells. A‐C, Images and quantification analysis of cell scratch assays before and after 24 h of treatment with various materials. D‐F, Images and quantification analysis of invaded cells in Transwell assays. G, H, Cytotoxicity of various materials against MDA‐MB‐231 cells and 4T1 cells evaluated by MTT assays. I, Western blots of receptor tyrosine kinase like orphan receptor 1 (ROR1), MMP9, and MMP2 in different cells after various treatments. *P < .05, **P < .01, ***P < .001. P, paclitaxel; PD, paclitaxel + dexamethasone; PM, paclitaxel + mifepristone; PMD, paclitaxel + mifepristone + dexamethasone; PM‐nano, paclitaxel‐conjugated and mifepristone‐loaded supramolecular hydrogel; P‐nano, paclitaxel hydrogel

FIGURE 6

Inhibition of tumor growth in vivo. A, Tumor volumes of mice during the whole experiment. B, Body weights of 4T1 tumor‐bearing mice. C, Photographs of excised tumors on day 21 after treatment. *P < .05, **P < .01. P, paclitaxel; PD, paclitaxel + dexamethasone; PM, paclitaxel + mifepristone; PMD, paclitaxel + mifepristone + dexamethasone; PM‐nano, paclitaxel‐conjugated and mifepristone‐loaded supramolecular hydrogel; P‐nano, paclitaxel hydrogel

FIGURE 7

Antimetastatic potential in vivo. A, Bioluminescence images of 4T1‐Luc tumor‐bearing mice on day 21 after treatment. B, C, Images of organs with and without breast cancer metastasis confirmed by gross observation (B) and H&E staining (C). The control group was defined as positive control and negative control groups, including paclitaxel + mifepristone (PM), paclitaxel hydrogel (P‐nano), paclitaxel (P), paclitaxel + mifepristone + dexamethasone (PMD), paclitaxel + dexamethasone (PD), and PBS groups. D, Number of mice with metastasis in each group evaluated by pathologic analysis. E, Number of organs with metastasis in each group confirmed by pathologic analysis. F, Number of organs with metastasis in all groups detected by pathologic analysis. PM‐nano, paclitaxel‐conjugated and mifepristone‐loaded supramolecular hydrogel