Green Synthesis of Yellow-fluorescent Carbon Nano-dots for cancer-cell image and photocatalytic inactivation by using microplasma

: In recent years, multifunctional nanoparticles with combined diagnostic and therapeutic functions show great promise in nanomedicine. In this work, we report the environmentally friendly synthesis of fluorescent carbon nano-dots such as carbon quantum dots (CQDs) by microplasma using o-phenylenediamine. The produced CQDs exhibited a wide absorption peaks at 380-500 nm and emitted bright yellow fluorescence with a peak at 550 nm. The CQDs were rapidly taken up by HeLa cancer cells. When excited under blue light, a bright yellow fluorescence signal and intense reactive oxygen species (ROS) were efficiently produced, enabling simultaneous fluorescent cancer cell imaging and photodynamic inactivation, with a 40% decrease in relative cell viability. Furthermore, the CQDs has 98% cell viability at concentrations of 400 μg·mL -1 in the dark demonstrating excellent biocompatibility. The newly prepared CQDs are thus demonstrated to be materials that can be effectively for imaging-guided cancer therapy. irradiation for 15 min or without; (C) A simplified energy level diagram shows potential fluorescence and cell death energy transfer pathways. (S0, ground state of the fluorophore molecule; S1, first singlet excited state; Sn (n > 1); T1, first triplet excited state; Ex, excitation by photon absorption; FL, fluorescence).


Introduction
Cancer remains a leading cause of death worldwide [1]. Multifunctional nanoparticles with both diagnostic and therapeutic functions have promising applications in nanomedicine. Simultaneous image-guided therapy is a new concept in cancer treatment and shows promise with respect to the optimization of therapeutic efficiency.
It can provide useful information regarding the size and location of tumors, the optimal time window for phototherapy, and therapeutic efficacy [2][3][4]. Photodynamic therapy (PDT) is used to treat many kinds of cancer and other diseases owing to its spatiotemporal selectivity and non-invasive nature [5,6]. Ideal photosensitizers generally possess the following characteristics: (1) highly efficient generation of reactive oxygen species (ROS), (2) good biocompatibility, and (3) water solubility [7].
However, current applications of PDT are limited by the poor water solubility, instability, and sub-optimal excitation wavelengths of photosensitizers. Therefore, the generation of photosensitizer substitutes with good water-solubility and biocompatibility by environmentally friendly and low-cost methods is needed.
Carbon quantum dots (CQDs) have received tremendous attention owing to their unique beneficial properties, such as simple and environmentally friendly synthesis, low toxicity, excellent water solubility and light stability [8]. CQDs have potential uses in cellular imaging, biosensing, targeted drug delivery, and other biomedical applications [9][10][11][12][13]. There are two major approaches for the synthesis of carbon dots, bottom-up and top-down approaches. Top-down methods include electrochemical oxidation, laser ablation, chemical oxidation, and ultrasonic synthesis methods.
Bottom-up methods consist of hydrothermal treatment, microwave synthesis, and thermal decomposition [14][15][16][17]. However, the high temperature, high pressure, and strong acids required lead to substantial energy consumption, complicated processes, and unavoidable harm to the environment. Moreover, CQDs can be produced within just a few minutes using a microplasma-liquid method without high temperature condition, large energy input, and laborious procedures [18][19][20]. Microplasmas supply a unique physicochemical environment to both fundamental studies and applications involving advanced materials. The chemical and electronic environments provided by the microplasmas are highly nonequilibrium and can store energy. In this environment, a large number of electrons, ions, free radicals, and other excited, ionized, and other types of active substances can be produced [21,22]. Although o-phenylenediamine is used as raw material to synthesize carbon nano dots, it has not been used in microplasma synthesis [23][24][25].
In this study, o-phenylenediamine was used as a raw material to synthesize CQDs by micro plasma treatment. The CQDs generated by this method were uniform in size (around 3.2 nm in diameter) and exhibited an emission peak at around 550 nm. We demonstrated that the newly synthesized CQDs could produce a large amount of ROS under light conditions. In vitro, CQDs could be absorbed by HeLa tumor cells and emitted yellow light under blue wavelength excitation at 420-500 nm with low toxicity.
We also observed the inactivation of HeLa tumor cells under irradiation at 460 nm.
These results suggest that the newly prepared CQDs are promising materials for imaging-guided or targeted cancer therapy.

Characterization of CQDs
The yellow-emissive CQDs could be prepared in a facile and environmentally friendly manner by a micro plasma method using o-phenylenediamine as the carbon precursor. Figure 1A shows transmission electron microscope (TEM) images of the CQD particles. The particles produced by micro plasma were cyclo-sharp or oval-shaped with an average diameter of 3.2 nm. As shown in the high-resolution image Figure 1A (inset), the lattice distance in the CQDs is 0.21 nm, belonging to the (1,1,0) plane of graphite.
The Raman spectrum shown in Figure 1E, as a result of sp3 and sp2-hybridization of carbon, D mode, named as disorder induced mode, located around 1342 cm-1 and G mode centers around 1507 cm -1 respectively. It is known that the intensity of D mode compared with G mode depends on the size of the graphite micro-crystals in the sample.
The higher disorder of the sample leads to the higher intensity ratio of ID/IG and the smaller the graphite micro-crystal. The D and G mode of CQD powders can be viewed as small graphite flakes with a relatively high intensity ratio ID/IG (0.77).
FTIR and XPS are powerful tools to characterize the chemical composition and structure of carbon-based materials. FTIR data for CQDs were recorded in the range of 400-4000 cm -1 , as shown in Figure 1F. The FTIR spectrum revealed that the CQDs mainly contain amine (3052 and 3324 cm -1 ), OH (3200 cm -1 ), C=O (1595 cm -1 ), C-N/C-O (1200 cm -1 ), C=C (1500 cm -1 ), and CH (748 cm -1 ) functional groups or chemical bonds [26,27]. The surface components of the CQDs, as determined by XPS, were consistent with the FTIR results. The full spectrum presented in Figure 2A showed three typical peaks: C 1s (285 eV), N 1s (400 eV), and O 1s (531 eV). Figure  The O 1s band contained peaks at 531.6 and 533.1 eV for C-O and C=O, respectively [28,29]. Furthermore, the optical properties of CQDs were investigated using fluorescence spectroscopy and UV-Vis absorption. The fluorescence emission spectra of CQDs are shown in Figure 1C. The prepared CQDs exhibit an excitation-dependent fluorescence emission behavior. When excited at wavelengths from 400 to 500 nm, the maximum fluorescence emission peak red-shifted from 473 to 519 nm and the fluorescence intensity decreased sharply [30]. As shown in Figure 1D, the UV-vis spectra of CQD features a strong absorption peak in the wavelength range of 400-490 nm. The CQDs exhibited two characteristic absorption peaks at 280 and 420 nm, which can be assigned to π-π* (aromatic C=C) and n-π* (carboxyl and/or C-N) transitions, respectively [31,32]. Therefore, These optical properties of CQDs provide feasibility for simultaneous biological imaging and photodynamic inactivation.

Bioimaging and cytotoxicity of CQDs
To assess the capability of CQDs for bioimaging and cell labeling, in vitro cellular imaging using CQDs was investigated on HeLa cells by CLSM and illustrated in Figure   3A, HeLa cells showed bright yellow fluorescence evenly distributed throughout the cell. It should be noted that low CQDs concentration of 200 μg·mL −1 was required for these cell imaging experiments for the bio-fluorescence labeling of HeLa cancer cells.
The as-prepared CQDs are accordingly a potential bio-labelling reagent. For biomedical applications, materials must be highly biocompatible at recommended dosages. To examine cytotoxicity, HeLa cells were treated with CQDs at final concentrations ranging from 0 to 400 μg·mL -1 for 24 h. As shown in Figure 3B, over 95% of cells survived, as determined by MTT assays. These data suggest that the newly generated CQDs have low cytotoxicity and high biocompatibility.

Cancer cell inactivation
As shown in Figure 3C, viability did not differ between HeLa cells treated with CQDs or blue light alone. Simultaneous treatment with CQDs and blue light markedly reduced cell viability, depending on the duration of photo-exposure. After irradiation at 460 nm for 15 min, the CQDs displayed remarkable antitumor activity; the viability of HeLa cells decreased by about 40% at a concentration of 200 μg·mL −1 . These results indicate that the CQDs were as effective as some clinical anticancer drugs, like Photofrin [33].

ROS generation of CQDs
During PDT, cancer cells can be killed by cytotoxic ROS generated by the endocytosed photosensitizer under appropriate irradiation conditions [34,35]. ROS can inactivate target cells by apoptosis or necrosis with little side effects via PDT in several diseases [36][37][38][39]. Inspection of Figure 4A shows that ROS reagent emits red fluorescence, indicating the generation of ROS, which is well overlapped with the fluorescence of CQDs. As shown in Figure 4B, compared with the control and no laser irradiation groups, the experimental groups under 460 nm laser irradiation for 15 min showed obvious ROS generation. Our results indicating that CQDs could significantly promote the production of the intracellular ROS under the irradiated of 460 nm laser and have great potential for application in PDT. As shown in Figure 4C, the CQDs can be excited from the ground state (S0 in Figure 4C) to an excited state (Sn in Figure   4C), and the efficiency of this process is determined by the intensity of the light source and the extinction coefficient. After solvent-mediated relaxation, CQDs remain at the lowest vibration level of the first singlet excited state. Due to the rapid vibrational relaxation following excitation, the energy of the photon emitted from the first singlet excited state (S1 in Figure 4C) is lower than the energy of the excitation photon, resulting in a wavelength increase. Fluorescence imaging utilizes the CQDs transition from S0 to Sn to S1 [40]. The CQDs were ingested by HeLa tumor cells and emit fluorescence when illuminated by a suitable wavelength source, thereby allowing the cells to be labeled. S1 can return to the S0 state by fluorescence or by intersystem crossing to a non-fluorescent triplet excited state (T1 in Figure 4C) [41,42]. The fluorescent group of T1 is especially active in electron transfer reactions, generating superoxide free radicals and subsequently resulting in fluorescent group degradation.
The energy from T1 transferred to molecular oxygen would produce an excited singlet oxygen oxidizing agent that is stronger than ground state molecular oxygen. The superoxide radicals and singlet oxygen, as well as other ROS, including OH and H2O2, react with nearby biological molecules to exert phototoxicity, leading to cell death.

Synthesis of carbon quantum dots
The micro plasma processing system is summarized in Figure S1. During micro plasma treatment, argon (Ar) gas flowed through the pipe at a flow rate of 60 sccm, and the DC current was kept at 17 mA. After 10 min of plasma treatment, the brownish-black product was dialyzed using a dialysis membrane (molecular weight cut-off, 500 Da) against 2 L of deionized water for 12 h and subsequently filtered through a 0.22 μm ultrafiltration membrane. Finally, pure CQDs were obtained by freeze drying.

Characterization of the structure, composition, and optical properties of CQDs
The size and morphology of the CQDs were characterized by TEM using a JEM-2100F system (JEOL, Tokyo, Japan). Fluorescence spectroscopy was performed using a Perkin Elmer LS 55 Luminescence Spectrometer (Waltham, MA, USA). The UV/Vis absorption spectra were measured using the Varian Cary 50 UV-VIS spectrophotometer (Palo Alto, CA, USA). FTIR spectra were obtained using a Nicolet 6700 spectroscope (Thermo Scientific, Waltham, MA, USA), and Raman spectroscopy was performed using the 800 UV micro-Raman spectrometer (Invia-reflex, UK). XPS experiments were performed using an Axis Ultra DLD system (Shimadzu/Kratos Analytical Ltd., Kyoto, Japan).

Availability of data and materials
All data and materials of the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This work was financially supported by the National Natural Science Foundation of