Dual Receptor-Targeted and Redox-Sensitive Polymeric Micelles Self-Assembled from a Folic Acid-Hyaluronic Acid-SS-Vitamin E Succinate Polymer for Precise Cancer Therapy

Abstract

Purpose: Poor site-specific delivery and insufficient intracellular drug release in tumors are inherent disadvantages to successful chemotherapy. In this study, an extraordinary polymeric micelle nanoplatform was designed for the efficient delivery of paclitaxel (PTX) by combining dual receptor-mediated active targeting and stimuli response to intracellular reduction potential.

Methods: The dual-targeted redox-sensitive polymer, folic acid-hyaluronic acid-SS-vitamin E succinate (FHSV), was synthesized via an amidation reaction and characterized by ¹H-NMR. Then, PTX-loaded FHSV micelles (PTX/FHSV) were prepared by a dialysis method. The physiochemical properties of the micelles were explored. Moreover, in vitro cytological experiments and in vivo animal studies were carried out to evaluate the antitumor efficacy of polymeric micelles.

Results: The PTX/FHSV micelles exhibited a uniform, near-spherical morphology (148.8 ± 1.4 nm) and a high drug loading capacity (11.28% ± 0.25). Triggered by the high concentration of glutathione, PTX/FHSV micelles could quickly release their loaded drug into the release medium. The in vitro cytological evaluations showed that, compared with Taxol or single receptor-targeted micelles, FHSV micelles yielded higher cellular uptake by the dual receptor-mediated endocytosis pathway, thus leading to significantly superior cytotoxicity and apoptosis in tumor cells but less cytotoxicity in normal cells. More importantly, in the in vivo antitumor experiments, PTX/FHSV micelles exhibited enhanced tumor accumulation and produced remarkable tumor growth inhibition with minimal systemic toxicity.

Conclusion: Our results suggest that this well-designed FHSV polymer has promising potential for use as a vehicle of chemotherapeutic drugs for precise cancer therapy.

Keywords: antitumor; cytotoxicity; dual-targeted; micelles; paclitaxel; redox-sensitive.

Figure 1

Illustration of self-assembly, tumor accumulation and intracellular release of PTX/FHSV micelles.

Figure 2

(A) Chemical structures of HCV, HSV and FHSV polymers. (B) ¹H-NMR spectra of HA in D₂O, HCV, HSV and FHSV polymers in DMSO-d₆/D₂O (v/v, 1:1). (C) The CMC of HCV, HSV and FHSV polymers.

Figure 3

Size distribution of blank micelles after incubation (A) in PBS (10 mM, pH 7.4) for 0 h and 24 h (B) in PBS (10 mM, pH 7.4) with 10 μM, 10 mM and 20 mM GSH for 24 h.

Figure 4

(A) Size distribution and TEM images of PTX-loaded micelles. Scale bar represents 50 μm. (B) In vitro drug release of Taxol and PTX-loaded micelles in PBS (10 mM, pH 7.4) with or without 10 mM GSH. Data are presented as mean ± SD (n = 3).

Figure 5

Cell viability of (A) NIH3T3 cells and (B) MCF-7 cells treated for 48 h with Taxol and PTX-loaded micelles. In the competitive inhibition experiment, the MCF-7 cells were precultured either with free HA (10 mg/mL) or with both free HA (10 mg/mL) and free FA (1 mM) for 1 h prior to treatment with PTX/FHSV micelles. (C) The IC₅₀ values of PTX-loaded formulations against MCF-7 cells. Data are presented as mean ± SD (n=6). *p < 0.05; **p < 0.01; ***p < 0.001; ns, p > 0.05. (D) Apoptotisis of MCF-7 cells treated for 12 h with Taxol and PTX-loaded micelles.

Figure 6

Time-dependent cellular uptake of C6-loaded formulations in MCF-7 cells. (A) Flow cytometry profiles and (B) quantification of MFI of MCF-7 cells following 1 h and 4 h incubation with C6-loaded formulations. Data are presented as mean ± SD. (n=3). *p < 0.05; **p < 0.01; ***p < 0.001; ns, p > 0.05. CLSM images of MCF-7 cells incubated for (C) 1 h and (D) 4 h with C6-loaded formulations. The competitive inhibition experiments were performed by treating MCF-7 cells either with free HA (10 mg/mL) or with free HA (10 mg/mL) and FA (1 mM) before adding C6/FHSV micelles.

Figure 7

Bio-distribution of DiR solution and DiR-loaded micelles. (A) In vivo imaging of DiR solution and DiR-loaded micelles in S180 tumor-bearing mice at 1 h, 6 h, 12 h and 24 h postinjection, respectively. (B) Ex vivo fluorescence imaging of excised organs and tumors at 24 h postinjection. (C) Quantitative results of major organs and tumors accumulation at 24 h post injection (n=3). ***p < 0.001.

Figure 8

In vivo antitumor efficacy against S180 tumor xenograft model. (A) Tumor growth curves of mice treated with saline, Taxol and PTX-loaded micelles. (B) Body weight changes of mice. (C) Tumor weight and TIR of different groups after the last treatments (n=5).*p < 0.05, **p < 0.01, ns, p > 0.05. (D) H&E, TUNEL and CD31 analyses of excised tumors after the last treatments.

Figure 9

H&E staining of major organs at the end of treatments.