Anti-Trop2 antibody-conjugated bioreducible nanoparticles for targeted triple negative breast cancer therapy

Abstract

Trop2, a transmembrane glycoprotein, has emerged as a biomarker for targeted cancer therapy since it is overexpressed in 80% of triple negative breast cancer (TNBC) patients. For the site-specific delivery of the anticancer drug into TNBC, anti-Trop2 antibody-conjugated nanoparticles (ST-NPs) were prepared as the potential nanocarrier, composed of carboxymethyl dextran (CMD) derivatives with bioreducible disulfide bonds. Owing to its amphiphilicity, the CMD derivatives were self-assembled into nano-sized particles in an aqueous condition. Doxorubicin (DOX), chosen as a model anticancer drug, was effectively encapsulated into the nanoparticles. DOX-loaded ST-NPs (DOX-ST-NPs) rapidly released DOX in the presence of 10 mM glutathione (GSH), whereas the DOX release is significantly retarded in the physiological condition (PBS, pH 7.4). Confocal microscopic images and flow cytometry analysis demonstrated that DOX-ST-NPs were selectively taken up by MDA-MB-231 as the representative Trop2-expressing TNBC cells. Consequently, DOX-ST-NPs exhibited higher toxicity to Trop2-positive MDA-MB-231 cancer cells, compared to DOX-loaded control nanoparticles without the disulfide bond or anti-Trop2 antibody. Overall, ST-NPs might be a promising carrier of DOX for targeted TNBC therapy.

Introduction

Breast cancer has become the most common cancer as well as the second leading cause of death in women worldwide [1]. Among the subtypes of the breast cancer, triple negative breast cancer (TNBC) is the most aggressive one, characterized by lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) [2], [3], [4], [5], [6]. Therefore, TNBC is not effectively treated by the conventional chemotherapy, primarily ascribed to lack of the appropriate target. Although limited success was demonstrated by the combination of multiple agents [7] and epidermal growth factor receptor-targeted antibody [8], there is an urgent need to develop the carrier which can effectively deliver the drug to the site of action in TNBC.

Over the past two decades, numerous nanocarriers have been developed for targeted drug delivery for cancer therapy in forms of liposomes, dendrimers, micelles, and polymer-drug conjugates [9], [10], [11], [12]. Owing to the enhanced permeability and retention effect by the leaky vasculature and lack of lymphatic drainage, nanocarriers have exhibited preferential accumulation into the tumor tissue after their systemic administration, implying their potential for targeted delivery of the drug [13], [14]. To facilitate the site-specific release of anticancer drugs into tumor tissues, several stimuli-responsive nanocarriers have been extensively investigated [15], [16], [17], [18], [19]. They are designed to exhibit physicochemical alterations by responding to internal or external stimuli such as pH, hypoxia, glutathione, and temperature [20], [21], [22], [23]. In particular, glutathione (GSH), a thiol-containing tripeptide capable of reducing disulfide bonds, has received increasing attention to developing the nanocarriers for the intracellular drug delivery since it is abundant in the cytoplasm of the cell (1–10 mM) [24], [25]. Moreover, the GSH concentrations in cancer cells of pathological sites have been demonstrated to be at least 4-fold higher than that of normal tissues [26]. This has encouraged the development of bioreducible nanoparticles for the GSH-triggered drug release [27].

For the effective therapy of TNBC, it is obviously required to develop the nanocarriers which specifically recognize the tumor cells. Trop2, a 46-kD transmembrane glycoprotein involving numerous intracellular signaling pathways, has emerged as a biomarker for targeted cancer therapy since it is overexpressed in several cancers compared to normal tissue [28], [29], [30].

Of note, Trop2 is expressed in 80% of TNBC patients, indicating that Trop2 is an attractive target for TNBC therapy. In this study, we developed anti-Trop2 antibody-conjugated bioreducible nanoparticles (ST-NPs) for active targeting TNBC, allowing for rapid drug release at the intracellular compartment via the reduction of the disulfide bond (Fig. 1). As controls, the GSH-sensitive nanoparticles without anti-Trop2 antibody (SS-NPs), GSH-insensitive nanoparticles without anti-Trop2 antibody (CA-NPs), and GSH-insensitive nanoparticles with anti-Trop2 antibody (CT-NPs) were also prepared to investigate the potential of anti-Trop2 antibody and bioreducible bond for TNBC-targeted therapy. The physicochemical properties of ST-NPs were characterized using various instruments including ¹H NMR, transmission electron microscopy (TEM), and dynamic light scattering (DLS). The anticancer drug, doxorubicin (DOX), was physically encapsulated into nanoparticles by the emulsion method, and its GSH-dependent release behavior was assessed as a function of time. Also, Trop2-mediated endocytosis by TNBC cells was evaluated using flow cytometry analysis.