Mechanism of action and cellular responses of HEK293 cells on challenge with zwitterionic carbon dots

https://doi.org/10.1016/j.colsurfb.2021.111698Get rights and content

Highlights

  • Synthesis and characterization of carbon dots (CDs).

  • Evaluation of cell material interactions using different methods.

  • Determination of Intracellular uptake of CDs and ROS generation.

  • Cell-CDs mediated gene expression using RT-qPCR.

Abstract

Carbon, an extremely versatile element has great demand in the field of nanoscience. Carbon-based nanostructures are exponentially increased due to its wide range of applications in biotechnological and environmental approaches; hence, its safety assessment is of greater concern. In the present study, high quantum yielding zwitterionic carbon dots were synthesized, characterized and its safety assessment at different concentration ranges (50–1600 μgmL−1) on HEK 293 cells was carried out. Cellular, mitochondrial, lysosomal integrity and ROS generation were assessed using specific fluorochromes.The key cellular event apoptosis was assessed by annexinpropidium iodide staining using imaging flow cytometry. Moreover, the mRNA levels of the apoptotic genes were determined by real-time PCR. The results revealed that the cell viability assays (MTT, NR) and mitochondrial membrane potential were altered on exposure to a higher concentration of zwitterionic CDs for 24 h. Also, annexinpropidiumiodidestaining exhibited an increased percentage of apoptotic cells upon exposure to zwitterionic CDs at higher concentrations. Further, apoptosis was confirmed by significantlyincreased expression of pro-apoptotic gene (Bax) together with decreased expression of Bcl-2/Bax ratio. Collectively, this study suggests that zwitterionic CDs induce apoptosis in HEK 293 at higher concentration and the safe range for its intended application is found to be 50−200 μg/mL.

Introduction

Carbon dots (CDs) are an emerging subclass of nanomaterials, defined by characteristic sizes of <10 nm. CDs possess a carbon core that is functionalized by various groups at the surface. Carbon dots are considered as an epitome for various environmental, biological and energy-related applications. CDs with varying chemical structures have been actively produced through the optimization of synthesis, doping/surface modification and purification conditions.Increased permeability of CDs toward the cell membrane and even nuclear membrane during the long incubation make it potent reliable candidates for biomedical applications [1]. The key application of CDs includes bio-imaging, biosensing, therapeutic vehicles, and photocatalysis [2]. Bio-imaging of both cancerous and non-cancerous cell lines are well explored in different cell lines. The highly luminescent CDs are a potential class of biolabels due to their biocompatibility, low toxicity and can be mass-produced by a simple preparation method [[3], [4], [5], [6]].

A great number of studies have been performed to synthesize CDs using different precursors; it may vary from green to inorganic precursors. Currently, a wide variety of materials have been explored for the synthesis of CDs and it ranges from organic compounds to the greener substrates. Initially, the synthesis was limited to carbonaceous materials. However, now many greener precursors ranging from food waste, peels, juices and even human residues have been explored for their potential in CD synthesis. These precursors are broadly classified into fruit source, food and beverages, animal derivatives, vegetables, waste materials, leaves and human derivative [7]. However, the CDs synthesized from the green source are generally excitation dependent with non-uniform size distribution. This could be because the greener source varies in composition widely and the surface functionalization depends on the type of greener precursor used. Also, the carbonization degree, which further depends on the type of synthesis method used, also affects the CDs synthesis and in turn its quantum yield. However, in contrast, according to the literature, high quantum yield CDs have been synthesized using organic compounds or organic precursors as it does not have many impurities and thus carbonization is also rapid [2,8].

The synthesis of CDs is relatively simple, low cost, and applicable to large scales. The existing approaches for synthesizing CDs include dry methods [9,10], laser ablation [11], solution methods combustion/thermal [12,13], electrochemical oxidation [14], organic synthesis [15], and microwave methods [[16], [17], [18]]. The microwave-assisted synthesis is a unique and simple technique which offers fast and efficient processing of materials with higher reproducibility. Also, it involves rapid, uniform heating and high purity over other conventional techniques [19].

It has been documented that most CDs are localized to the cell cytoplasm, including lysosomes, mitochondria, Golgi apparatus, and endoplasmic reticulum [[20], [21], [22]]. Size and surface charge are known to be the main determinants of cytoplasmic and nuclear uptake of nanoparticles [[23], [24], [25]]. Recent studies report that positively charged nanoparticles are mainly internalized by cells, whereas negatively charged ones after internalization interact with nuclei [[26], [27], [28]]. Besides, studies also report negatively charged CDs are found to be low in their fluorescent quantum yield compared to positive or neutral particles [29]. Along with that, the positively charged CDs are low in their biocompatibility [30].

It is well established that the zwitterionic surface provides a prospective platform for bioconjugation with different analytes devoid of any aggregation [31,32]. Hence, zwitterionic CDs with both positively and negatively charged functional groups can facilitate cytoplasmic uptake and subsequent nuclear translocation. A study reported that zwitterionic CDs with a quantum yield of 21.9 % are synthesized by a simple one-step synthesis method using β-alanine and citric acid [33]. In contrast, highly fluorescent, biocompatible zwitterionic CDs intended to monitor cancer in early stages have been synthesized with 80 % quantum yield [1]. Even though, numerous studies on the potential applications of CDs in the biomedical field, toxicity studies have been limited. CDs can cross the various biological barriers and may reach the most sensitive organs [34]. Similar to nanomaterial, most CDs are localized to the cell cytoplasm, including the lysosomes, mitochondria, Golgi apparatus, and endoplasmic reticulum [35]. Studies have also documented on the subcellular targeting features of surface-regulated CDs [36]. Hence, a detailed understanding of cellular interactions and intracellular distributions of CDs is essential.

In the present study, novel highly fluorescent zwitterionic CDs were synthesized by microwave pyrolysis and its toxicity assessment was carried out in HEK 293 cells.These CDs are intended to be used for drug delivery and bioimaging applications. The advantage of zwitterionic CDs in cancerous cell environment has been reported by sriet al., but its cellular fate and interaction on normal cells remain elusive. Zwitterionic carbon dots due to its small size it may reach potentially sensitive targets through blood and lymph circulation. The kidney is considered as the major secondary target organs in which nanoparticle toxicity should be thoroughly evaluated. The human embryonic kidney, HEK293 cells are specific cell lines commonly used in cell biology and toxicology studies [37]. Hence, in this study, the cellular response and interaction of synthesized zwitterion CDs on HEK 293 cells were assessed. An attempt also made to assess the cellular mechanism involved in response to zwitterionic CDs

Section snippets

Materials and methods

All the chemicals used in the study were of analytical grade. Dulbecco's modified eagle's medium (DMEM), Trypsin-EDTA was obtained from Gibco (Grand Island, NY, USA). 2,7dichlorofluorescein acetate (DCFH-DA) and JC1 were procured from Invitrogen (Carlsbad, CA, USA). DAPI, 4, 6-diamidino-2-phenylindole and acridine orange was purchased from Hi-media Pvt. Ltd. (Mumbai, India). RhodaminePhalloidin obtained from Abcam.Neutral Red stain(SRL, Mumbai, India). RNA isolation kit wasprocured from Qiagen,

Characterization of zwitterionic CDs

Various techniques have been used to characterize the size, crystal structure, elemental composition and various other physical properties of nanoparticles. In the present study, UV–vis spectrophotometry, zeta potential analysis, Raman spectra, FTIR, TEM, AFM and LCsingle bondMS analysis were used to characterize the microwave-assisted synthesized zwitterionic CDs. The optical properties of CDs were determined by UV visible spectroscopy. Fig. 1(a), depicts the UV visible spectra of the synthesized

Conclusion

Among various nanoparticles, small carbon nanoparticles (CDs) are gaining importance due to its application in biomedical research and targeted drug delivery. Toxicological behavior of nanomaterial’s and its cellular fate is of major concern for its safety assessment. In this study, novel highly fluorescent, cost-effective zwitterionic CDs was synthesized and a systematic approach has been made to assess the cellular response and interaction of zwitterionic CDs on HEK 293 cells. In conclusion,

CRediT authorship contribution statement

P.V. Mohanan: Design, literature survey, drafting, discussion, submission. V.P. Sangeetha, Sri Smriti, Pratima R. Solanki contributed equally: literature survey, drafting.

Funding

No funds received from other sources.

Data availability

The authors declared that the research data referred to correctly cited in the manuscript’s reference section.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

VPS, PVM wish to express their thanks to the Director and Head, Biomedical Technology Wing, SreeChitraTirunal Institute for Medical Sciences and Technology (Govt. of India), Trivandrum, Kerala, India for their support and providing the infrastructure to carry out this work. SS, SPR express their thanks to the Vice-Chancellor, Jawaharlal Nehru University, New Delhi for providing the infrastructure to carry out this work.

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      The utilization of a three-electrode system was done where silver-silver chloride (Ag/AgCl), platinum (Pt), and modified ITO coated glass substrate acted as a reference, counter, and working electrode, respectively. The characterization of CDs was discussed in detail by Sri et al., in the previously published paper [4,47]. Here, TEM images and average size calculations are shown in Fig. 2.

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