Toxicity of carbon dots – Effect of surface functionalization on the cell viability, reactive oxygen species generation and cell cycle

Abstract

Carbon dots (CDs) are fluorescent nanoprobes offering a great potential in biological and medical applications due to their superior biocompatibility compared to metal chalcogenide quantum dots (e.g., CdSe). Key factors determining their cytotoxicity and cellular/intracellular tracking involve chemical nature and charge of surface functional groups. For the first time, we present a comprehensive cytotoxic study including cell cycle analysis of carbon dots differing in surface functionalization, namely pristine CDs (CDs-Pri) with negative charge due to carboxylic groups, polyethyleneglycol modified dots with neutral charge (CDs-PEG), and polyethylenimine coated dots with a positive charge (CDs-PEI). The CDs in vitro toxicity was studied on standard mouse fibroblasts (NIH/3T3). The results suggest that neutral CDs-PEG are the most promising for biological applications as they do not induce any abnormalities in cell morphology, intracellular trafficking, and cell cycle up to concentrations of 300 μg mL⁻¹. Negatively charged CDs-Pri arrested the G2/M phase of the cell cycle, stimulated proliferation and led to higher oxidative stress, however they did not enter the cell nucleus. In contrast, positively charged CDs-PEI are the most cytotoxic, entering into the cell nucleus and inducing the largest changes in G0/G1 phase of cell cycle, even at concentrations of around 100 μg mL⁻¹.

Introduction

Carbon dots (CDs) have been demonstrated as a new class of fluorescent nanomaterials, already competitive in many respects to conventional metal chalcogenide based quantum dots (QDs) [1], [2], [3]. Unlike conventional semiconducting QDs containing cadmium or other heavy metals, carbon quantum dots do not cause any serious health or environmental concerns [3], [4], [5], [6]. Due to their fluorescence brightness, high photostability and good biocompatibility, CDs have been developed as fluorescent nanoprobes for optical bioimaging applications [5], [7], [8], [9], [10] and shown promise for clinical translation [11].

One of the most important properties affecting biological applications of nanomaterials is their surface functionalization [12], [13] as it can determine the pathway of cellular uptake, intracellular trafficking and cytotoxicity [13]. Polyethylene glycol (PEG) is biocompatible and biodegradable polymer [6], [14], [15], mainly used for passivation of nanomaterials to obtain neutral surface charge which prevents nonspecific protein absorption, immunological reaction and accumulation in the reticuloendothelial system [16]. Polyethylene imine (PEI) is a cationic macromolecule commonly used in gene transfer therapy because provides high transfection efficiency [17]. PEI molecules interact with negatively charged proteins in cytoskeleton (such as actin and beta-tubulin) [18] and modify membrane integrity. From this reason, PEI is often used for surface functionalization of nanomaterials to obtain positive charge and improve permeabilization of plasma membrane. PEI destabilizes endosomal membranes [17], is able to escape from endosomes via the so-called “proton sponge” mechanism and binds to DNA [19], [20]. Hence, CDs attached to PEI may exhibit thermo- and pH-responsive properties particularly suited for stimuli drug release, as previously reported [21].

Many cytotoxicity studies have demonstrated that CDs (with or without surface passivation) exhibit very low toxicity and can be easily internalized into cells for imaging [22], [23]. The cytotoxicity effects (viability, mortality, proliferation) of CDs have been tested on various types of cell line, at various concentrations and with different surface coverages. A brief overview of the parameters determining the cytotoxicity is given in Table 1. In almost all of the cytotoxicity studies performed to date, CDs have been demonstrated to cause negligible loss in cell viability at concentrations sufficient for cell labeling (approximately 10–100 μg mL⁻¹) [24].

In this study, for the first time, we examined differences in the cell cycle, ROS generation and cytotoxicity for three most commonly used CDs differing in surface coverages. As a result, we identified principal differences in toxicity of CDs on the cellular level depending on their surface chemistry. The presented study offers a new view on the toxicity of CDs, which cannot be evaluated simply on cell viability measurements but other phenomena including proliferation, entering the cell nucleus, and changes in the cell cycle should also be taken into account. Thus, this study could help improve our understanding of the carbon dot toxic profile.