Injectable click-crosslinked hydrogel containing resveratrol to improve the therapeutic effect in triple negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) patients are considered intractable, as this disease has few effective treatments and a very poor prognosis even in its early stages. Here, intratumoral therapy with resveratrol (Res), which has anticancer and metastasis inhibitory effects, was proposed for the effective treatment of TNBC. An injectable Res-loaded click-crosslinked hyaluronic acid (Res-Cx-HA) hydrogel was designed and intratumorally injected to generate a Res-Cx-HA depot inside the tumor. The Res-Cx-HA formulation exhibited good injectability into the tumor tissue, quick depot formation inside the tumor, and the depot remained inside the injected tumor for extended periods. In vivo formed Res-Cx-HA depots sustained Res inside the tumor for extended periods. More importantly, the bioavailability and therapeutic efficacy of Res remained almost exclusively within the tumor and not in other organs. Intratumoral injection of Res-Cx-HA in animal models resulted in significant negative tumor growth rates (i.e., the tumor volume decreased over time) coupled with large apoptotic cells and limited angiogenesis in tumors. Therefore, Res-Cx-HA intratumoral injection is a promising way to treat TNBC patients with high efficacy and minimal adverse effects.

Keywords: Click-crosslinking; Injectable hydrogel; Intratumoral injection; Resveratrol; Triple-negative breast cancer.

Graphical abstract

Fig. 1

Schematic image of injectable, click-crosslinkable Res-HA-Tet and Res-HA-TCO formulations for intratumoral injection of Res and effective inhibition of tumor growth.

Fig. 2

Images of (A) injectable formulations of (a1) HA and (a2) Res-HA in a single-barrel syringe, (a3) HA-Tet and HA-TCO and (a4) Res-HA-Tet and Res-HA-TCO in a dual-barrel syringe, (B) the formed depot of (b1) HA, (b2) Res-HA, (b3) Cx-HA, and (b4) Res-Cx-HA after injection through a 23-gauge needle and (C) SEM images of the formed (c1) HA and (c2) Cx-HA depot (scale bar: 100 ​μm).

Fig. 3

(A) Rheological characterization of HA and Cx-HA hydrogels with and without Res; (a) storage and loss modulus, (b) complex viscosity analysis, (c) tan ​δ value measurements (∗p ​< ​0.001). (B) Injectability of HA and Cx-HA hydrogels with and without Res; (d) force versus injection distance of HA and Res-HA in a single-barrel syringe, HA-Tet and HA-TCO with and without Res in a dual-barrel syringe, (e) force versus enlarged injection distance of each formulation, and (f) maximum force of each formulation (∗p ​< ​0.001).

Fig. 4

In vitro viability of MDA-MB-231 ​cell line in all formulations measured in this study. (a) Fluorescent images showing the morphology of MDA-MB-231 treated with all formulations after 1, 2, and 3 days (Scale bar: 200 ​μm) and (b) MTT assay results (∗p ​< ​0.005).

Fig. 5

Near-infrared (NIR) observations. (a) NIR images [(a1) black background, (a2) white background, and (a3) brightness] of NIR-HA-Tet and NIR-HA-TCO loaded into a dual-barrel syringe and the formed NIR-Cx-HA. (b) NIR image after subcutaneous injection with NIR-HA or NIR-Cx-HA for 18 days (scale bar ​= ​1 ​cm). (c) Time after injection of NIR-HA or NIR-Cx-HA versus signal-to-background ratio (SBR) determined at each time point. (d) NIR-Cx-HA image of the formed NIR-Cx-HA (indicated with a circle) after 18 days. (e) NIR images of NIR-HA in tumors of animals and the removed tumor after 1 and 6 days. (f) NIR images of NIR-Cx-HA in tumors of animals and the removed tumor after 1, 6, 12, and 18 days (scale bar ​= ​1 ​cm).

Fig. 6

(a) Changes in tumor volume (∗p ​< ​0.005, ∗∗p ​< ​0.05, Free Res, Res-HA and Res-Cx-HA versus the control at 10 and 18 days), (b) images of the removed tumor (the images were taken from mice bearing MDA-MB-231 tumor cell xenografts) and (c) tumor volume doubling time and tumor volume growth rate after injection of control, Res, Res-HA, and Res-Cx-HA solutions (^ap ​< ​0.1 and ^bp ​< ​0.05 versus control).

Fig. 7

(a) Distribution of Res in tumors after intratumoral injection of Res, Res-HA, and Res-Cx-HA on days 1, 12, and 18. Distribution of Res in tumors and organs after intratumoral injection of Res, Res-HA, and Res-Cx-HA on days (b) 1, (c) 12, and (d) 18 (∗∗p ​< ​0.05, ∗p ​< ​0.001).

Fig. 8

H&E staining of tumor sections on days 1, 12, and 18 after intratumoral injection of xenograft-bearing mice with Free Res, Res-HA, and Res-Cx-HA (Scale bar for the staining image in a white square box: 5000 ​μm; scale bar in enlarged images: 200 ​μm, Yellow and red arrows indicated blood vessels and necrosis respectively).

Fig. 9

(a) Merged images of 4′,6-diamidino-2-phenylindole (DAPI; blue, nuclei) and CD31 (green, blood vessels cells) staining (scale bar: 200 ​μm) and (b) CD31-positive cells per DAPI in tumors on days 1, 12, and 18 after intratumoral injection of xenograft-bearing mice with Res, Res-HA, and Res-Cx-HA (∗∗p ​< ​0.05, ∗p ​< ​0.001).

Fig. 10

(a) Merged images of 4′,6-diamidino-2-phenylindole (DAPI; blue, nuclei) and TUNEL (green, apoptotic cells) staining (scale bar: 200 ​μm) and (b) TUNEL positive cells per DAPI in tumors on days 1, 12, and 18 after intratumoral injection of xenograft-bearing mice with Res, Res-HA, and Res-Cx-HA (∗∗p ​< ​0.05, ∗p ​< ​0.001).

Fig. 11

(a) Merged images of 4′,6-diamidino-2-phenylindole (DAPI; blue, nuclei) and cleaved caspase-3 (CCP-3, red) staining (scale bar: 200 ​μm) and (b) CCP-3-positive cells per DAPI in tumors on days 1, 12, and 18 after intratumoral injection of xenograft-bearing mice with Res, Res-HA, and Res-Cx-HA (∗∗p ​< ​0.05, ∗p ​< ​0.001).