Highly Biocompatible, fluorescence and Zwitterionic carbon dots as a

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Biological and Medical Applications of Materials and Interfaces

Highly Biocompatible, fluorescence and Zwitterionic carbon dots as a novel approach for Bioimaging applications in Cancerous Cells smriti sri, Robin Kumar, Dr. Amulya Panda, and Pratima R Solanki ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b13217 • Publication Date (Web): 11 Oct 2018 Downloaded from http://pubs.acs.org on October 14, 2018

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ACS Applied Materials & Interfaces

Highly Biocompatible, Fluorescence and Zwitterionic Carbon Dots as a Novel Approach for Bioimaging Applications in Cancerous Cells Smriti Sria#, Robin Kumarb#, Amulya K Pandab and Pratima R Solankia* aSpecial

Centre for Nanoscience, Jawaharlal Nehru University, New Delhi-110067

bNational

Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067 #authors have contributed equally

*Corresponding author: Email: [email protected]; [email protected]

Abstract Highly biocompatible, excellent photostable, nitrogen and sulphur containing novel zwitterionic carbon dots (CDs) were synthesised by microwave assisted pyrolysis. The size of CDs were of 25 nm with an average size of 2.61±0.7 nm. CDs were characterised by UV/Vis spectroscopy, fluorescence spectroscopy, zeta potential, Fourier-transform infrared spectroscopy (FTIR), XRay Diffraction (XRD) and Time-resolved fluorescence spectroscopy (TRFS). CDs were known to emit blue fluorescence, when excited at 360 nm i.e., UV region and emit in the blue region of visible spectrum i.e. at 443 nm. CDs show excitation independent photoluminescence behaviour and were highly fluorescent even at lower concentration under UV light. These CDs were highly fluorescent in nature with the quantum yield being as high as 80% which is comparable to organic dyes. The CDs were further used to image two different oral cancer cell lines namely, FaDu (Human Pharyngeal Carcinoma) and Cal-27 (Human Tongue Carcinoma). The cell viability assay shows that CDs were highly biocompatible which was further confirmed by the side scattering studies as no any change in the granularity was observed even at highest concentration of 1600 μg/mL. The generation of Reactive Oxygen Species (ROS) was also investigated and negligible formation of ROS was detected. In addition to that, the uptake phenomenon, cell cycle analysis, exocytosis, cellular uptake at 4°C and in the presence of ATP inhibitor have been studied. It was found that CDs easily cross the plasma membrane without hampering the cellular integrity. Keywords: Carbon dots, Zwitterionic particles, Microwave pyrolysis, Fluorescence, Biocompatibility, Bioimaging, Oral cancer cell lines.

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1. Introduction Carbon dots (CDs), a new member of the carbon family has received colossal interest because of its ultra-small size, having excellent photostability as well as biocompatibility with fluorescent properties. CDs are zero-dimensional, highly biocompatible as compared to metal-based quantum dots1-2. However, CDs exhibit non-blinking photoluminescence which is advantageous over organic dyes. CDs are synthesized mostly employing hydrothermal, microwave assisted pyrolysis and electrochemistry routes3-4. Of these synthesis routes, microwave synthesis is an efficient and fastest route usually taking few minutes to prepare small-sized fluorescent CDs owing to the on and off pulses of electromagnetic waves which produces heat thereby forming CDs having high yield with good quality5-6. The first microwave synthesis of CDs was done by Zhu et al. using saccharide and PEG200 as surface passivating agent7. Since then several CDs have been synthesized using microwave irradiation8.These CDs has been purified using numerous methods such as centrifugation, dialysis, high performance liquid chromatography (HPLC)9-10, polyacrylamide gel electrophoresis (PAGE)11 and capillary electrophoresis12 to get uniform size. CDs can be synthesied from a limitless substrates broadly classified into organic and greener precursors. Many research groups used green precursors due to their easy availability. Liu et al. for the first time synthesised CD using grass as the precursor molecule for carbon using hydrothermal method. Since then insanely cost-effective substrates have been explored for its potency in making CDs. The substrates being mango leaf13, cornflour14, ginko fruits15, watermelon pulp, apple juice, saffron, hair fibers, honey, meat, orange juice, egg shell membrane, coriander leaf, garlic, aloe vera, sesame oil to name a few16-17 and references therein. However, these CDs requires intensive purification steps and are also low in fluorescence when compared to CDs synthesised from organic precursors4. Therefore, organic precursors are the choice for synthesising of CDs with higher yields and purity. The main exciting feature of CDs is they exhibit excellent photoluminescence for biomedical application. These CDs can be stored at 4°C for months without losing its photoluminescence properties18-19. CDs show excitation dependent as well as excitation independent phenomenon 19.

1,

Most of the CDs emit in the blue region of visible spectrum when excited by UV light. The

PL mechanism of CDs is not clear yet and many factors are known to affect its fluorescence such 2 ACS Paragon Plus Environment

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as surface passivation, defects, and formation of fluorescent species 20.CDs based on citric acid (CA) was highly fluorescent due to the formation of fluorescent organic species of 5-oxo-1,2,3,5tetrahydroimidazo (1,2-α) pyridine-7-carboxylic acid

21-23.

It was found that CDs with nitrogen

as the functional group enhances the PL19. Recent work showed that CDs doped with nitrogen and sulfur are more fluorescent compared to only nitrogen counterparts, with quantum yield reached as high as 73%24-25. In the past, numerous CDs with nitrogen and sulfur groups have been synthesised through various synthesis routes which is shown in Table 1. CA, l-cysteine, glycerol, acrylic acid were used as the carbon precursor whereas l-cysteine, cystamine dihydrochloride were used as surface passivating agents. Researchers have reported the CDs yield as 70% maximum and used for water remediation and bio-imaging of Hela cells. In this context, there is scope to optimize the synthesis parameters to get high yield of CDs. Besides CDs, other carbon based nanomaterials including reduced graphene oxide is also shown as a biocompatible platform for biomedical applications26,27-29. It is well known that the Zwitterionic surface provides a better platform compared to their counterparts for bioconjugation with different analytes without causing any aggregation30-31. Negatively charged CDs are found to be low in their fluorescent quantum yield compared to positive or neutral particles12. Along with that, the positively charged CDs are low in their biocompatibility31. So, the zwitterionic property of the CD enhances its biocompatibility as well as fluorescent quantum yield. It is significant that interaction of CDs on the surface of plasma membrane results in adsorption of serum proteins which in turn internalizes the particle through receptor-mediated endocytosis. Zwitterionic particles less specifically adsorb proteins on their surface so are quickly taken into cytosol without the fear of their agglomeration32. Due to rich carboxylic moieties present at the surface of CDs they easily bioconjugate with various biomolecules such as antibodies, aptamers, enzymes etc33-36. Also, when zwitterionic particles interact with cancer cell microenvironment, they sheds their anionic component leaving a positive charge on their surface enhancing their entry into cells37. Chen et al., used polycation-b-polysulfobetaine block copolymer, poly[2-(dimethylamino) ethyl methacrylate]-b-poly[N-(3-(methacryloylamino)

propyl)-N,N-dimethyl-N-(3-sulfopropyl)

ammonium hydroxide] (PDMAEMA-b-PMPDSAH) for grafted luminescent carbon dots (CDs) via surface-initiated atom transfer radical polymerization (ATRP). The zwitterionic CDs yield was obtained as 41.5%.31 They have used complex system for CDs fabrications. However, Jung 3 ACS Paragon Plus Environment


et al., 2015 fabricated the zwitterionic functional groups of the CDs introduced by a simple onestep synthesis methd using β-alanine and citric acid. This is simple method as compared to previous one but CDs yield obtained as 21.9 %. In this context, there is a possibility to improve the quantum yield of zwitterionic CDs30. In this context, we have proposed a one-step simple synthesis method using citric acid and L-cysteine which is cost effective and providing high yield (80%) of zwitterionic CDs. Thus, our synthesis method of CDs is superior with high yield and cost effective as compared to CDs synthesised by Chen et al and Jung et al.30,31. Without a shadow of doubt, cancer is the deadliest disease that humans encounter in their lives. Diagnosis in early stage remains the major bottleneck for its prognosis. A plethora of study is dedicated to diagnose in its early stage and to target using anti-cancerous drugs. Compared to 1977 the survival of cancer patients is stipulated to increase by 400%, because of a large amount of research in cancer. Oral cancer is the 6th most common type of cancer38. Oral cancer is more prevalent in Asian countries especially south Asia because of more consumption of oral tobacco in the form of beetel, quid, cigarette and smoking39. More than 90% of oral cancer arise from the epithelial lining of the oral cavity and is called Oral Squamous Cell Carcinoma (OSCC). Even though OSCC can be screened by naked eye but it is diagnosed in advanced stages which limits the survival rate of patients. There is a need for early diagnosis to increase the prognosis of OSCC. Imaging intracellular compartments, cells and tissues enable more accurate diagnosis and treatment of disease. Fluorescent imaging will be useful to detect real time monitoring of cancer cells in patients tissue samples. Owing to its small size CDs are known to permeate the cell membrane and even nuclear membrane when incubated for longer duration. Since its serendipitous discovery, CD have mainly been used for bioimaging, biosensing, therapeutic vehicle and photocatalysis4. CDs have been extensively explored for bioimaging in different cell lines (cancerous as well as noncancerous) and bacteria40. Bioimaging of non-cancerous cell lines such as L92919, 41-42,Vero43, MC3T3, RAW 264.7 COS-7 were explored with CDs. Further, cervical cancer cell line such as Hela, breast cancer MCF-7 and MDA-MB-2340, 44, Liver cancer HepG245 and Huh-746, receptor negative cells NIH-3T347, lung carcinoma A54940, neural cell line PC12 and RSC9648 were also studied. According to the best of my knowledge no work has been devoted towards the bioimaging of oral cancer cells using CDs.


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In the present work, novel zwitterionic CDs were synthesized by microwave pyrolysis using citric acid as the precursor and L-cysteine as the surface passivating agent, synthesised first time as per our knowledge. The quantum yield obtained 80% using microwave synthesis method. These CDs were characterized using various spectroscopic techniques such as UV/Vis spectroscopy, fluorescence spectroscopy, FTIR, Raman Spectroscopy and time resolved Fluorescence spectroscopy. Zeta potential and XRD were done to investigate the ionic nature and the crystalline behaviour of CDs respectively. These CDs having N and S group, enhanced the biocompatibility of two different oral cancer cell lines namely FaDu and Cal-27, upto 80% viability with highest concentration (400 µg/mL) of CDs. Biocompatibility of CDs were confirmed by the side scattering studies and found no any change in the granularity of cells even at higher concentration (1600 µg/mL). Beside this, reactive oxygen species (ROS) were determined and found no significant ROS generation. It was also observed that there is no any change in cell division at different incubation time (12 and 24 h). Exocytosis studies confirmed the CDs removed easily from the cell in a span of 48h. Also, ATP depletion and 4°C incubation were studied which shows that CD mostly diffuse passively across plasma membrane. 2.0 Materials and Methods 2.1 Materials Citric acid (CA) was purchased from Sd fine chemicals limited (Cat no. 77-92-9), India. Lcysteine (W326305), sodium azide (S2002), cell counting kit 8 (CCK 8) (Cat no. 96992) and 2',7' –dichlorofluorescin diacetate (DCFDA) (Cat. no. 287810) were purchased from Sigma Aldrich. Oral Cancer Cell lines namely FaDu (Human Pharyngeal Carcinoma) (Cat. no. ATCC® HTB43™) and Cal-27 (Human Tongue Carcinoma) (ATCC® CRL-2095™) were purchased from American Type Culture Collection (ATCC). Cell culture media namely Dulbecco’s Modified Eagle’s Medium (DMEM) (Cat no. AL001A), Fetal Bovine Serum (FBS) (Cat no. RM1112), Trypsin (Cat no. TCL070) and phosphate buffer saline (PBS) pH 7.4 (Cat no. TL1006) were purchased from Himedia. Cell culture Flasks (Cat no. CLS430720, CLS3599, CLS3289), 24 well plates were procured from corning (Cat no.CLS3527), cover slips from blue star. 2.2 Carbon dots synthesis 0.52 M, CA and 20.8 mM L-cysteine was dissolved in 10 mL ultrapure water. The transparent solution was then transferred to domestic microwave for 3 min at 700W. The colourless solution changed to yellow foamy solid. After cooling at room temperature, 5 mL ultrapure water was 5 ACS Paragon Plus Environment


added and the yellow precipitate was dissolved. Finally, we obtained the yellow solution having the desired CDs. In order to remove the impurities or unreacted substance from CDs, it was dialysed against pure water through a dialysis membrane (MWCO 1000 Da) for 3 days. Finally, the solution containing CDs were oven dried at 80°C and CDs collected of 0.157g (the yield of the reaction was about 16%). 1 mg CD was weighed and dispersed in 1 mL ultrapure water and further used for cellular and characterization studies. The quantum yield (QY) of CDs was determined by the slope method taking quinine sulfate as the reference (Figure S5B). The slope of both the fluorescent molecules were calculated by fitting the graph and fed into the equation for QY calculation. Φx= Φst(Kx/Kst) (ηx/ηst)2......................................................................(Eq. 1) Where ϕ is the fluorescence quantum yield, x and st represents test and standard, respectively, K is the gradient from the plot of integrated fluorescence intensity vs absorbance, and η is the refractive index of the solvent. 2.3 Cellular Studies of CDs 2.3.1 Cytotoxicity Cytotoxicity of CDs was measured by cell counting kit 8 (CCK 8). FaDu and Cal 27 cells were seeded in 96 well plates at a density of 1x 104 cells per well. The cells were incubated for 24h at 37° C and 5% CO2 incubator for attachment. Different concentrations of CDs (400, 200, 100, 50, 25 μg/mL) were added and incubated for another 24h. Next 10 μL of CCK-8 was added to the wells and incubated for another 2h. The absorbance was measured at 450 nm with reference of 630 nm. 2.3.2 Cell cycle analysis 2.5x106 cells of FaDu and Cal 27 were seeded in 24 well plate and incubated for 24h at 37° C and 5% CO2 incubator. Further, CDs were added for different time points (0h, 12h, 24h). Cells were trypsinized and harvested in PBS. Cells pellet was dislodged and fixed with chilled 70% ethanol. To avoid clumping, cells were vortexed properly and then kept at 4° C for 1h. After that, cells were centrifuged and ethanol was removed. Cells pellet was dislodged again and washed twice with PBS. Cells were resuspended and treated with 200 µg mL-1 of RNase A for 15 min to degrade RNA and then DNA was stained with 50 µg mL-1 of propidium iodide for 20 min at 4°C in dark. Cells were collected in tubes for Fluorescence-activated cell sorting (FACS) analysis. 6 ACS Paragon Plus Environment

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For FACS analysis, 10,000 events were collected. Untreated cells were taken as a control and results were compared with untreated cells for analyzing effect of CDs on mammalian cell cycle. 2.3.3 Uptake of CDs Time-dependent as well as concentration-dependent uptake studies of CDs were performed. For both types of the experiments, 2.5 x 105 cells of FaDu and Cal 27 were seeded in 24 well plate and incubated for 24h at 37° C and 5% CO2 incubator. Different concentration of CDs (400, 200, 100, 50, 25 μg/mL) were added and incubated for 2h and visualised by confocal laser scanning microscope (CLSM). Similarly, for time-dependent uptake studies of CDs, 200 μg/mL were added for different time points (0h, 1h, 2h, 4h) and visualised by CLSM with 60X objective excited by a 405 nm laser. 2.3.4 Exocytosis studies of internalised CDs 1x105 cells of FaDu and Cal 27 were seeded in 24 well plate and incubated for 24h at 37° C and 5% CO2 incubator. Cells were treated with 200 μg/mL CDs and incubated for 3h. Cells were further washed thrice with PBS. Fresh media without CDs were added to the wells for different time (48, 24, 12, 6, 3, and 1h). Cells were fixed with 4% paraformaldehyde and visualised using CLSM with 60x objective excited by 405 nm laser. 2.3.5 Internalisation of CDs based on side scattering 1x105 cells of FaDu and Cal 27 were seeded in 24 well plate and incubated for 24h at 37° C and 5% CO2 incubator. Cells were treated with different concentrations (1600, 800, 400, 200, 100 μg/mL) of CDs and incubated for 24h. Further, cells were trypsinized and centrifuged to collect the pellet. Pellet was resuspended in PBS and studied by Flow cytometry. 2.3.6 Reactive oxygen species (ROS) detection 1x105 cells of FaDu and Cal 27 were seeded in 24 well plate and incubated for 24h at 37° C and 5% CO2 incubator. Cells were treated with different concentrations (200, 100 μg/mL) of CDs and incubated for 24h. Next day the cells were trypsinized and centrifuged to collect cell pellets. 200 μL of 10 μM DCFDA dye was added to the cells and incubated for another 30 min in dark. After completion of incubation time cells were analysed with Flow cytometry. 2.4 Characterizations Size and crystallinity of CDs were characterized by High Resolution Transmission Electron Microscopy (HR-TEM, JEOL JEM-2200 FS, JAPAN). Absorbance was measured on T90+ UV/VIS spectrometer. Fluorescence spectra were measured on Cary Eclipse Fluorescence 7 ACS Paragon Plus Environment


spectrometer. Zeta potential measurements were performed using a zetasizer Nano-ZS (Malvern Instruments). Decay time was calculated by Time Resolved Fluorescence Spectrometer (TRFS Edinburgh FL920 Fluorescence Life Time Spectrometer). Confocal Microscopy studies were done on Nikon Real Time Laser Scanning Confocal Microscope Model A1R. Flow cytometry studies were done on BD FACS Verse. 3.0 Results and Discussion 3.1 Characterization studies CDs have been synthesised using nitrogen and sulfur which was zwitterionic in nature as confirmed by the zeta potential results [Scheme 1a]. These CDs were found highly fluorescent (QY 80%) when quinine sulfate was taken as the reference which is explained later. CDs were found to emit blue fluorescence (emission at 443 nm) when excited in the UV region 360 nm [Figure1A (a)]. Figure 1A (a) inset shows the CDs under visible light and the right side image shows CDs excited in the UV region. The absorption peak at 360 nm is due to the n-π* transition of C=O bonds, which further show that the n-π* transition in carbons are responsible for the PL mechanism. The CDs were excited with different wavelengths ranging from 300-400 nm [Figure1A (b)]. The highest emission was observed when CDs were excited with 360 nm and emit in the visible region i.e. 443 nm. There is no any shift in the emission spectra on increasing the excitation wavelength. This behaviour of CDs may be due to different emissive sites on CDs surface49. The fluorescence spectra of CDs shows excitation independent PL behaviour which is unlikely for most of the CDs, but is obvious for CDs prepared from same substrates24. Excitation independent PL behaviour is because of either difference in size or different surface characteristics such as more amine groups on the surface50. Figure 1A (c) shows the FTIR spectrum of CDs. The FTIR spectra of CDs show hump at 3340 cm-1 which was attributed to the stretching vibration of NH2 of L-cysteine. However, bending vibration of N-H stretching was observed at 1652, 1570,1558 and 1542 cm-1 assigned to L-cysteine. Strong absorption IR peaks were observed at 1716, 1731 and 1635 cm-1 was assigned to the C=O stretch. Absorption bands at 1223 cm-1 was due to C-O-C stretch. The presence of sulfur in CDs was confirmed by the absorption peak at 2600 and 635 cm-1. The FTIR spectra confirmed that the CDs contain of thearomatic ring of carbon and carboxylic acid, primary amine and sulfur as the functional groups correspond to CA and L-cysteine.


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The excitation independent PL behaviour was correlated with the TEM images [Figure1B (a)]. Figure 1B (a) clearly shows that the CDs were monodispersed in aqueous solution. Figure 1B (b) shows a narrow size distribution of CDs varying from 1-5 nm and were non-uniform in nature with an average size of 2.61±0.7 nm. It suggests that the excitation independent PL behaviour may be attributed to surface morphology which indeed should be uniform in nature to minimise the surface defects and hence the PL mechanism. Figure 1B(c) shows the magnified image of CDs and inset shows the HRTEM image of CDs also possess crystallinity, the lattice spacing for CDs was found to be 0.206 nm which is the characteristics of graphitic carbon (100 facet) in CDs51. However, SAED pattern [Figure 1B (d)] significantly indicate that the CDs were mostly amorphous in nature, only rare particles possessing well-resolved lattice fringes19. The ring pattern corresponds to the (002) plane of graphite. The CDs were found to be zwitterion in nature [Supplementary data; Figure S1 (A)]. The overall charges on CDs were found to be -0.0147 mV which is almost zero. But three different charge species were present, the highest number of particles (83%) possess negative charge of -8.06 mV. Some of the CDs (17%) possess a positive charge of 35.7 mV. Only a few CDs were (only 0.3%) positively charged with a charge of 146 mV [Supplementary data; Figure SIB]. The high QY of zwitterionic CDs is apparent from earlier studies where it was shown that neutral species are more fluorescent12. The zwitterionic surface of CDs provide an anti-fouling surface i.e. they have minimal electrostatic or nonspecific adsorption resulted in easy bioconjugation with different analytes without their aggregation52. Also on perspective of cellular environment they can easily invade plasma membrane and even can be translocated to nucleus by surpassing the nuclear core complex. The XRD spectra of CDs [Supplementary data; Figure S2], clearly shows the broad single hump near 23° (2θ) which corresponds to the graphite lattice (002). These results indicated that the most of the particles are amorphous in nature. However, some of the particle showed crystallinity according to HRTEM results53-54. The Raman spectra of CDs [Supplementary data; Figure S3] shows two prominent D and G bands at 1343 and 1590 cm-1, respectively. The intensity of D band was more compared to G band which corresponds to more defects in the structure of CDs and may contribute towards fluorescence55-56. The fluorescence decay time of CDs was experimentally done using TRFS by exciting the particles with a laser of 375nm and a bandpass filter of 390 nm was used [Supplementary data; Figure S4]. The FL decay time of CDs was 9 ACS Paragon Plus Environment


found to be 9.7 ns which are comparable to organic dyes. QY is defined as the ratio of number of photons emitted to that of number of photons absorbed. The QY was determined by the slope method and taking qunine sulfate as the reference [Supplementary data; Figure S5].The slope for both the fluorescent molecule was calculate by fitting the graph and fed into the equation No 1. The QY was found to be 80% which means that on exciting the colloidal solution of CDs with 360 nm wavelength out of hundred particles eighty particles emits photons, which is quite high for the CDs synthesized till present. It is well known that the surface passivation increases the fluorescence57-59. The high QY of the prepared CDs shows that CDs mainly consist of carbogenic core with COOH, OH and NH2 SH as the surface passivating agent60. This increase in fluorescence compared to other methods may be due to capping with N and S as well as due to formation of fluorescent organic species of 5-oxo-1,2,3,5-tetrahydroimidazo (1,2-α) pyridine-7carboxylic acid (IPCA)21, 24. Moreover, microwave synthesized CDs create more surface defects resulted in increased fluorescence.

3.2 Cellular Studies 3.2.1 Cytotoxicity Cytotoxicity assay was done on two oral cancer cell lines namely FaDu and Cal-27 [Figure 2]. Cells were incubated for 24h with different CDs concentrations (25-400 μg/mL). On lower concentration upto 50 μg/mL more than 90% of the cells were viable for FaDu. However, on higher concentration of 400 μg/mL the cellular viability decreases a bit marginally but remains as high as 80% while the similar behaviour was observed in Cal-27. The low cytotoxicity of zwitterionic CDs is similar to other zwitterionic CDs prepared from different substrates30-31, 61. The biocompatibility makes CDs a promising candidate for use in various applications for in vitro as well as in vivo studies. 3.2.1. (A) Concentration dependent FaDu and Cal-27 cells were incubated with different concentrations of CDs (50-200 μg/mL) for 2h and CLSM was done to study the fluorescence behaviour of CDs [Figure 3A]. The study shows that the uptake phenomenon depends on the concentration. Visible fluorescence were observed in the cells treated with CDs concentration as low as 50 μg/mL and no saturation


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concentration was observed. On increasing the concentration of CDs the fluorescence intensity was also increasing linearly. 3.2.1 (B)Time dependent For time dependent cellular uptake study, FaDu and Cal-27 cells were incubated with 200 μg/mL for different time (1-4h). CLSM was used to quantify the fluorescence in cells [Figure 3B]. The fluorescence starts to appear as early, within 30 min in cells. These results indicated that the CDs are very small in size so they can easily cross the plasma membrane. Because, the uptake of CDs mainly depends upon their size. The uptake of CDs depends on the cancer microenvironment. Since the cancer microenvironment is devoid of oxygen in order to make up the energy need, cells starts to move to anaerobic mode of reproduction thereby producing lactic acid. As a result of lactic acid production, the pH of cancer microenvironment drops and become acidic (pH 6.6) in nature. When zwitterionic CDs come in the contact of acidic cancer microenvironment they switch there negative charge into positive charge (Zwitterions behave as cation in acidic pH). These positively charged CDs are easily taken up by negatively charged plasma membrane of cells lines resulting in enhanced their uptake62. This uptake phenomenon is described in the Scheme 1 (b) . This property of zwitterionic nature of CDs found a suitable candidate for bio-imaging and cellular uptake study in human oral cancer cell lines i.e. FaDu and Cal-27. 3.2.2 Cell cycle analysis Cell cycle of FaDu and Cal 27 cells were studied after the treatment with CDs. In this study, both cell lines were treated with 200 μg/mL of CDs. The experimental results are shown in term of frequency of cell cycle in G0-G1, S and G2-M phases [Figure 4]. The results on both the cells shows that major population of the cells are in the G0-G, followed by G2-M phases and S phase, respectively. No any change in the cell cycle was observed on treatment of cells with CDs. 3.2.3 Exocytosis FaDu and Cal 27 were grown for 24h and treated with 200 μg/mL of CD solution for 3 h. After that the CDs containing media was replaced by fresh media after washing with PBS twice. Exocytosis from cells was studied through CLSM [Figure 5A]. 0h cells were taken as control and the exocytosis phenomenon was studied for a period of 48h. The CDs start to disappear from the cells in as quick as 0.5h and gradually decreases with increasing time. A very little fluorescence 11 ACS Paragon Plus Environment


was observed at 48h which signifies that most of the CDs either degraded by the lysosomes in the cellular compartments or have been effluxed by the transport system of the cells. 3.2.4 4°C Incubation and ATP depletion studies For internalisation inside cells, either CDs have been taken up by energy-dependent as well as energy-independent mechanisms. To check whether the CDs follow either of the two process, cells were grown in medium devoid of ATP by either directly blocking the ATP through sodium azide or passively by growing cells at 4°C [Figure 5B]. At 4°C, most of the cellular function restricts to such a lower temperature. FaDu and Cal 27 cells were grown at 37°C for 24h. For 4°C incubation, cells were precooled at 4°C for 1h and to study ATP depletion in cells, both the cells were treated with sodium azide for 45min. After that CDs were added for 2h and 4h and intracellular fluorescence intensity was measured using CLSM. It is evident from the images that most of the CDs use energy-independent transport process i.e. passive diffusion for internalisation inside cells. With the noticeable fluorescence from cells grown at 4°C confirms that most of the CDs uses passive diffusion mode of transport inside cells. 3.2.5 Internalisation of CDs based on side scattering In order to investigate further the biocompatibility of CDs, cells were incubated with different concentration of CDs (100-1600 μg/mL). And the internalised CDs were investigated through side scattering by flow cytometry [Figure 6]. No any change was observed after incubation even at a higher concentration of 1600 μg/mL which signifies that even on higher concentration of CDs there is no change in the granularity of the cells which confirms the biocompatible study done using CCK-8 assay. 3.2.6 ROS detection Generation of ROS at different concentration (100 and 200 μg/mL) was detected using DCFDA dye and results were interpreted by flow cytometry [Figure 7]. The control signifies the cells without DCFDA dye whereas cells only signify cells incubated with dye. While there is no formation of ROS in cal-27 cell lines, negligible ROS formation was detected in FaDu at a concentration of 200 μg/mL. The results signifies that CDs have no any impact on the cells and does not contribute to ROS generation. 4.0 Conclusions Zwitterionic CDs were synthesised by microwave radiations within a short duration of 3 minute. The CD was excited in the UV region (360 nm). CDs were amorphous in nature, only some of 12 ACS Paragon Plus Environment

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the CDs were showing crystalline nature as evident from TEM and XRD analysis. The FTIR analysis shows that CDs are rich in carboxyl moieties, hydroxyl groups, sulfur groups and amine groups. Zeta potential studies suggest that the CDs are having both the charges i.e. positive and negative and were zwitterionic in nature. Imaging cancer cells (cells with altered markers or cancer stem cells) can lead to the early diagnosis of the infected area and will improve the survival rate in patients. Also imaging the cellular compartments can give a real time picture of the infected area for the physician to remove the tumour from infected area without removing healthy cells. The CCK-8 assay directly signifies 80% cells are viable at highest concentration. Uptake based on SSC shows there is no change in the granularity of cells and no significant ROS generation have been observed. Also, the CDs do not affect the cell cycle at any stage. These experiments suggest that the CDs are highly biocompatible and do not need any further modification to be used on animal models. The fluorescence of the synthesised CDs is excellent and non-blinking, the QY being 80%. The uptake studies by CLSM show that the fluorescence began to appear in the cytoplasm in as less as 0.5 h owing to the small size of CDs. The exocytosis phenomenon shows that the CDs is not retained within the cells and is flushed out within 48h from cellular system due to very small size of CDs. Further, CD incubation at 4°C and ATP depleted environment shows the passive diffusion for the transportation in cell. In summary, a novel, highly fluorescent, biocompatible CD having sulphur, carboxyl and amine group have been synthesised and found to passively diffuse very quickly into and out of the cytoplasm that provide a reliable and quick assay to monitor cancer in early stages.

Supporting Information: Zeta potential, XRD, Raman spectra, Time Resolved Fluorescence spectra (TRFS) and Quantum yield of CDs. Acknowledgement: Authors are thankful to AIRF, JNU for facilitating with the instrumentation facility, NII for conducting the experiments. One of the authors, Smriti Sri is thankful to UGC for providing the financial support through fellowship. This work was supported by a grant from the UPE-II-58, Department of Science and Technology [Nanomission Project; No. SR/NM/NS1144/2013

(G)],

DST-Purse,

BT/PR10638/PFN/20/826/2013

Department and

Indo

of Russia

Biotechnology

(DBT),Project

No.

(DBT/IC-2/Indo-Rusia/2017-19/02),

Government of India, , India.



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Figures

Scheme 1. (a) Synthesis of CDs and its bio-imaging in oral cancer cell lines; (b) Mechanism underlying uptake of zwitterionic CDs.



Figure 1A. (a) UV-Vis spectra (inset shows CDs under visible light and right shows CDs excited in UV light); (b) PL emission spectra of CDs; (c) FTIR spectrum of CDs


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Figure 1B. (a) TEM images shows the distribution of CDs ; scale bar 5 nm (b) Histogram showing size distribution of CDs. (c) HRTEM images of CDs inset shows the lattice fringes with lattice spacing of 0.206 nm and (d) SAED pattern of CDs.



(b)

% cell viability

(a)

% cell viability

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Concentration (μg/ml)

Concentration (μg/ml)

Figure 2. (a) CCK-8 assay of CDs on Cal 27 and (b) FaDu cell lines.


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Figure 3A. Concentration dependent cellular uptake of CDs in (a) Cal 27 cells (b) FaDu cells

Figure 3B. Time dependent cellular uptake of CDs in (a) Cal 27 (b) FaDu cells 23 ACS Paragon Plus Environment



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(a)

(b)

Figure 4. Cell cycle analysis on (a) Cal 27 (b) FaDu cell lines.



Figure 5A. Exocytosis of CDs in (a) Cal-27 cells (b) Fadu cells

Figure 5B. 4°C and ATP depletion in (a) Cal-27 cells (b) Fadu cells


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(a)

(b)

Figure 6. SSC uptake (a) Cal-27 cells (b) Fadu cells

(a)

(b)

Figure 7. ROS detection (a) Cal-27 cells (b) Fadu cells



Page 28 of 29

Table 1. Comparison of quantum yield of different N, S-CDs

Precursor

Method

Citric acid and Thermal L-cysteine evaporation followed by hydrothermal treatment (200°C for 3h) L-cysteine and 200°C for 3min diphosphorus pentoxide Citric acid and Hydrothermal L-cysteine treatment 200° for 5h Citric acid and Hydrothermal cystamine 160°C for 6 h. dihydrochloride L-cysteine and glycerol Methionine and acrylic acid Citric acid, urea and Lcysteine Citric acid and L-cysteine

Quantum yield (%) 73.00

Application

References

Bio imaging (HeLa cells)

[24]

18.10

Metal ion detection

[63]

54.00

Investigation of photoluminescence mechanism dichromate ion detection and multicolor bioimaging Fluazinam detection Fe3+ and temperature sensor Hg2+ detection in living sytem

[25]

Targeting of oral cancer cells (FaDu and Cal-27)

This work

39.70

hydrothermal 180°C for 12 h. hydrothermal 200 ◦C for 12 h Microwave synthesis

15.20.

Microwave synthesis 700W for 3 min

80.00

10.55 25.00


[64]

[65] [66] [67]

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Table of Contents (TOC) graphic


Highly Biocompatible, fluorescence and Zwitterionic carbon dots as a

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