Glutathione-responsive PEGylated GQD-based nanomaterials for diagnosis and treatment of breast cancer

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Abstract

Graphene quantum dots-based nanoparticles (GQD-NPs) with reduction-responsive properties were synthesized via conjugation with two precursors, doxorubicin-disulfide-GQD that endowed the drug loading capability and polyethylene glycol-disulfide-Herceptin that enhanced the half-life and conferred active targeting ability towards HER2-overexpressed cancer. Disulfide linkages between the compartments are cleaved at a physiologically relevant concentration of glutathione in cancer cells, resulting a controlled release of drugs. In vitro analysis confirmed successful internalization of GQD-NPs in the cytoplasm and enhanced cellular uptake of DOX in HER2-positive breast cancer cells. The GQD-NPs show potential as a theranostic agent for HER2-overexpressed breast cancer cells.

Introduction

Breast cancer is one of the most fatal and common invasive cancers [1]. Chemotherapy has been used as the major treatment method as it causes remission of cancer and destroys nodular aggregates floating in the blood vessels, decreasing the chance of metastasis. However, conventional chemotherapeutic agents exert high toxicity to normal cells and cause severe side effects. Several platforms have been developed to overcome such limitations [2], [3], [4]. Although many of these platforms are effective, quantum dots (QDs) have attracted significant attention as novel drug delivery systems due to their unique intrinsic chemical and physical properties for treating and diagnosing breast cancer [5], [6], [7], [8], [9]. Initially, inorganic QDs were proposed as theranostic agents. They exhibit lower photobleaching, narrower photoluminescence spectra, and higher chemical stability [10]. However, as they are composed of inorganic materials such as cadmium, zinc, or selenium, their toxicity has been a critical barrier to the clinical translation [11]. Graphene quantum dots (GQDs), on the other hand, are suitable for cancer therapy as they are highly biocompatible while maintaining stable photoluminescence properties [12], [13]. For instance, GQDs containing 10–40% oxygen are biocompatible and show favorable optical properties for the diagnosis of cancer at low cost [14].

The targeting ability is an important consideration in the design of drug delivery systems (DDS). A tumor-targeting ligand plays an important role in recognizing receptors on the surface of tumor cells. For example, Herceptin® (HCT or trastuzumab), a clinically used antibody, can act as a targeting moiety for patients with HER2-overexpressing cancer; these patients exhibit overexpressed HER2 receptors on the breast cancer cell surface. Conjugating HCT on the GQDs can significantly enhance the active targeting efficacy and reduce side effects because the complex can distinguish between healthy cells and cancer cells. Additionally, receptor-mediated endocytosis can significantly increase the rate of internalization by breast cancer cells. This may particularly be effective for treating resistant breast cancer.

An ideal GQD-based DDS should have excellent colloidal stability in the blood vessels. Conjugation of bulky HER can significantly decrease colloidal stability in the body. As a measure to improve aqueous solubility, polyethylene glycol (PEG) can be incorporated to provide flexibility and hydrophilicity [15], [16]. In addition, the PEG moiety provides “stealth” effect to help evading the immune system [17], [18]. Therefore, hydrophobic drugs in the core of the GQDs-PEG-HER complex can be protected from degradation and rapid removal by renal filtration [19].

To achieve on-demand release of encapsulated therapeutic agents, a stimuli-responsive degradation (SRD) platform can be introduced into the GQD complex. The versatile SRD platform undergoes chemical transition upon external stimuli. GQD-based colloids with dynamic covalent linkages can be destabilized in a controlled manner [20], [21]. A few examples of SRD platforms include endogenous stimuli-responsive systems (pH [22], glutathione (GSH) [23], reactive oxygen species [24], enzyme [25]) and exogenous stimuli-responsive systems (light [26], temperature [27], magnetic field [28]). GSH-responsive systems are especially promising, as a higher concentration of GSH is found in intracellular compartments (2–10 mM) of cancer cells as opposed to extracellular compartments (<20 μM) [29], [30]. To date, few GSH-responsive GQDs have been reported [31], [32], [33]. The reported platforms have a stimuli-responsive component at a single location and demonstrated a relatively slow release rate of encapsulated drugs. Disulfide linkages can be placed at multiple locations to achieve more rapid release of therapeutics [34], but a multi-location GSH-responsive GQD system has never been reported.

Herein, novel GSH-responsive degradable GQD-based nanoparticles (GQD-NPs) were successfully synthesized for breast cancer therapy and diagnosis (Scheme 1). The GQD-NPs were designed to contain doxorubicin (DOX), GQDs, and PEGylated HCT covalently conjugated through disulfide linkages. Direct conjugation of DOX provides optimal loading of DOX. PEG moiety endows stealth effects to the GQD-NPs and HCT enables selective targeting ability for HER2-overexpressing breast cancer. The results showed that, upon exposure to physiologically relevant concentrations of GSH in cancer cells, the disulfide linkages between the blocks were cleaved. Encapsulated DOX was then rapidly released from GQD-NPs in a controlled manner. The confocal laser scanning microscopy (CLSM) and cell viability measurements revealed anticancer activity and enhanced cellular uptake of GQD-NPs in SK-BR-3 cells (HER2-positive breast cancer cells) compared to MDB-MB-231 cells (HER2-negative breast cancer cells). These results suggest that GQD-NPs have excellent potential as a theranostic agent for the diagnosis and treatment of HER2-overexpressing breast cancer.

Section snippets

Materials and instrumentation

N-doped GQDs (nGQDs) were synthesized by pyrolysis of L-glutamic acid and the detailed procedure was described in our previous report [12]. 3,3′-Dithiodipropionic acid (ss-COOH), 4-dimethylaminopyridine (DMAP), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), doxorubicin hydrochloride (DOX), polyethylene glycol (PEG, MW = 3000 g/mol), trimethylamine (Et3N), tetrahydrofuran (THF), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and sodium bicarbonate (NaHCO3) were purchased from

Synthesis and characterization of GQD-NPs

Scheme 2 illustrates the synthetic approach used to prepare GQD-based NPs (GQD-NPs) containing DOX and HCT.

First, DOX and GQD were conjugated by a disulfide linkage in a facile EDC/DMAP coupling reaction in situ, producing DOX-SS-GQD. Amine groups on the surface of the nGQD were covalently attached to SS-COOH to introduce terminal carboxyl groups. These carboxyl groups enabled conjugation to the secondary amine on DOX via a facile coupling reaction. Direct conjugation of DOX enabled increased

Conclusion

GSH-responsive degradable GQD-NPs were successfully synthesized as a promising breast cancer theranostic agent. The GQD-NPs were designed to contain covalently conjugated DOX and PEGylated HER2 antibody, with each component linked via GSH-responsive disulfide linkages. Direct conjugation of DOX provided the optimal loading level of DOX at a low concentration of GQD-NPs. The addition of PEGylated HCT conferred stealth effects by the PEG and selective targeting ability towards HER2-overexpressing

Acknowledgments

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1A6A3A11934989), Development Program of the National Research Foundation funded by the Korea Ministry of Science, ICT & Future Planning (NRF-2017M2A2A6A01070927) and Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea

Conflicts of interest

The authors declare no conflict of interest.

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