GE11 peptide modified and reduction-responsive hyaluronic acid-based nanoparticles induced higher efficacy of doxorubicin for breast carcinoma therapy

Abstract

Novel breast carcinoma dual-targeted redox-responsive nanoparticles (NPs) based on cholesteryl-hyaluronic acid conjugates were designed for intracellular delivery of the antitumor drug doxorubicin (DOX). A series of reduction-responsive hyaluronic acid derivatives grafted with hydrophobic cholesteryl moiety (HA-ss-Chol) and GE11 peptide conjugated HA-ss-Chol (GE11–HA-ss-Chol) were synthesized. The obtained conjugates showed attractive self-assembly characteristics and high drug loading capacity. GE11–HA-ss-Chol NPs were highly stable under conditions mimicking normal physiological conditions, while showing a fast degradation of the vehicle’s structure and accelerating the drug release dramatically in the presence of intracellular reductive environment. Furthermore, the cellular uptake assay confirmed GE11–HA-ss-Chol NPs were taken up by MDA-MB-231 cells through CD44- and epidermal growth factor receptor-mediated endocytosis. The internalization pathways of GE11–HA-ss-Chol NPs might involve clathrin-mediated endocytosis and macropinocytosis. The intracellular distribution of DOX in GE11–HA-ss-Chol NPs showed a faster release and more efficient nuclear delivery than the insensitive control. Enhanced in vitro cytotoxicity of GE11–HA-ss-Chol DOX-NPs further confirmed the superiority of their dual-targeting and redox-responsive capacity. Moreover, in vivo imaging investigation in MDA-MB-231 tumor-bearing mice confirmed that GE11–HA-ss-Chol NPs labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide, a near-infrared fluorescence dye, possessed a preferable tumor accumulation ability as compared to the single-targeting counterpart (HA-ss-Chol NPs). The antitumor efficacy showed an improved therapy efficacy and lower systemic side effect. These results suggest GE11–HA-ss-Chol NPs provide a good potential platform for antitumor drugs.

Keywords:

• self-assembled nanoparticles
• redox responsive
• dual-ligand targeting
• hyaluronic acid
• cancer therapy

Supplementary materials

Figure S1 ¹H-NMR spectra of cholesteryl chloroformate (A), Chol–cys (B), and Chol–eda (C).

Notes: (a): δ (ppm) 5.36 (s, 1H, alkenyl); (b): 4.48 (m, 1H, oxycyclohexyl); (c): 5.05 (s, 1H, CONH); (d): 3.48 (m, 2H, CH₂NH); (e): 3.00 (m, 2H, CH₂N(H₂)); (f): 2.76–2.79 (m, 4H, 2CH₂S); (g): 4.99 (s, 1H, CONH); (h) 2.82–2.84 (m, 2H, CONCH₂); (i): 3.21–3.23 (m, 2H, N(H₂)CH₂).

Abbreviations: Chol–cys, cholesteryl–cystamine; Chol–eda, cholesteryl–ethylenediamine; NMR, nuclear magnetic resonance.

Figure S2 Synthesis procedures of GE11–HA-Chol.

Notes: (i) TEA/DCM, (ii) EDC/HOBT, (iii) EDC/sulfo-NHS.

Abbreviations: Chol–eda, cholesteryl–ethylenediamine; DCM, dichloromethane; EDC, 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydro chloride; HOBT, 1-hydroxybenzotriazole monohydrate; sulfo-NHS, N-hydroxysulfosuccinimide; TEA, triethylamine; HA, hyaluronic acid; HA-Chol₇, reduction-nonresponsive hyaluronic acid derivatives grafted with hydrophobic cholesteryl moiety with feed ratio of hydrophobic cholestryl equalling to 0.07; GE11–HA-Chol₇, GE11 peptide conjugated HA-Chol₇.

Figure S3 HPLC assessment of GE11 peptide conjugated to the distal end of HA-ss-Chol by monitoring absorbance at 220 nm.

Notes: (a) HA-ss-Chol conjugate, (b) GE11 peptide before reaction, (c) GE11 peptide after reaction.

Abbreviations: HA-ss-Chol, reduction-responsive hyaluronic acid derivatives grafted with hydrophobic cholesteryl moiety; HPLC, high-performance liquid chromatography.

Figure S4 Influence of various endocytosis inhibitors on the uptake efficiency of GE11–HA-ss-Chol₇ C6-NPs in MDA-MB-231 and MCF-7 cells.

Notes: Data represented as the mean ± SD (n=3). *P<0.05 versus control group.

Abbreviations: CPZ, chloropromazine; HA-ss-Chol₇, reduction-responsive hyaluronic acid derivatives grafted with hydrophobic cholesteryl moiety with feed ratio of hydrophobic cholestryl equalling to 0.07; GE11–HA-ss-Chol₇, GE11 peptide conjugated HA-ss-Chol₇; NPs, nanoparticles; SD, standard deviation.

Figure S5 Cytotoxicity of blank NPs against MCF-7 (A) and MDA-MB-231 (B) cells (mean ± SD, n=5).

Abbreviations: HA-ss-Chol₇, reduction-responsive hyaluronic acid derivatives grafted with hydrophobic cholesteryl moiety with feed ratio of hydrophobic cholestryl equalling to 0.07; GE11–HA-ss-Chol₇, GE11 peptide conjugated HA-ss-Chol₇; HA-Chol₇, reduction-responsive hyaluronic acid derivatives grafted with hydrophobic cholesteryl moiety with feed ratio of hydrophobic cholestryl equalling to 0.07; GE11–HA-Chol₇, GE11 peptide conjugated HA-Chol₇; NPs, nanoparticles; SD, standard deviation.

Table S1 Relative tissue exposures of DiR-NPs compared with DiR solution

Table S2 Tumor-targeting efficiency and relative tumor-targeting efficiency of DiR-NPs

Acknowledgments

This work was financially supported by the Program of the 333 High-level Personnel Training Project (number BRA2015392) of the Young Leaders in Science and Technology of Jiangsu Province. The authors express their gratitude to Jiangsu Province Public Technology Service Center of Nanodrug Preparation and Evaluation and Jiangsu Key Laboratory of Carcinogenesis and Intervention (China Pharmaceutical University) for assistance with the cell and animal experiments.

Disclosure

The authors report no conflicts of interest in this work.