Green and Facile Synthesis of Highly Photoluminescent Multicolor

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Green and Facile Synthesis of Highly Photoluminescent Multicolor Carbon Nanocrystals for Cancer Therapy and Imaging Abolghasem Abbasi Kajani, Abdol Khalegh Bordbar, Masoud Ayatollahi Mehrgardi, Sayyed Hamid Zarkesh Esfahani, Hasan Motaghi, Mohammad Kardi, Ahmad Reza Khosropour, John Ozdemir, Mourad Benamara, and Mohammad Hassan Beyzavi ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00407 • Publication Date (Web): 18 Oct 2018 Downloaded from http://pubs.acs.org on October 21, 2018

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ACS Applied Bio Materials

Green and Facile Synthesis of Highly Photoluminescent Multicolor Carbon Nanocrystals for Cancer Therapy and Imaging

Abolghasem Abbasi Kajani,† Abdol-Khalegh Bordbar,†* Masoud Ayatollahi Mehrgardi,† Sayyed Hamid ZarkeshEsfahani,‡ Hasan Motaghi,† Mohammad Kardi,‡ Ahmad Reza Khosropour,† John Ozdemir,§ Mourad Benamara⊥, M. Hassan Beyzavi,§, ⊥

† Department

‡

§ Department

⊥ Institute

To

of Chemistry, University of Isfahan, Isfahan, 81746-73441, Iran

Department of Biology, University of Isfahan, Isfahan, 81746-73441, Iran

of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, USA

for Nano Science and Engineering, University of Arkansas, Fayetteville, Arkansas 72701, USA

whom corresponding should be addressed: Department of Chemistry, University of Isfahan, Isfahan 81746-

73441, Iran. E-mail: [email protected]; [email protected]: Fax: +983136689732; Tel: +983137934941

ABSTRACT Carbon dots (CDs), as a new generation of fluorescent nanoparticles, have greatly considered for different biomedical applications. In the present study, a one-pot hydrothermal method was developed for the synthesis of a series of carbon dots (CDs) for cancer imaging and therapy. Taxane diterpenoids were utilized as the carbon source, different diamines were used as the nitrogen source, and folic acid as a targeting agent. High quality photostable and multicolor (blue and green) carbon nanocrystals with a hexagonal shape, the narrow size distribution of less than 20 nm, and high fluorescence quantum yield of up to 50.4% were obtained from Taxanes in combination with m-phenylenediamine and folic acid to give the best results. The nanoparticles displayed potent anticancer activity with IC50 values of 31.3 ± 2.7 and 34.1 ± 1.1 µg mL-1 for the human MCF-7 and HeLa cancer cell lines, respectively, and IC50 value of 120.5 ± 3.8 µg mL-1 on the normal human fibroblast cells. The flow cytometry studies determined apoptosis mediated cell death as the main anticancer mechanism of CDs and the molecular studies revealed the

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induction of both extrinsic and intrinsic apoptosis pathways. The overall results indicated the great potential of synthesized CDs for the simultaneous cancer imaging and therapy. Keywords: carbon dots, green synthesis, cancer theranostics, fluorescence, apoptosis 1. Introduction Within the past five years carbon dot (CD) structures with the ability to detect and treat cancer have been designed.12

The latest generation of theranostic carbon dot treatments involves the integration of several imaging modalities and

therapeutic constituents onto one nanoscaled platform.3-4 Integrated nanosystems with the potential for simultaneous diagnosis and therapy of disease could also monitor biological responses in real time.5-7 As the subject of so many theranostic studies bioavailable nano-agent cancer targeting therapies with minimum side effects is the great desire of scientists. CDs are zero-dimensional nanomaterials with the size of less than 10 nm consisting of sp2/sp3 carbon and oxygen/nitrogen-based groups, with the biological applications derived from their stable emission, high water solubility, significant cell permeability and biocompatibility.1,8-9 These exceptional properties of CDs provide myriad applications especially in biomedicine such as imaging, sensing, drug delivery, disease diagnostics and theranostics.8,10-11 Moreover, unlike QDs, the production of CDs does not suffer from high toxicity, harsh synthetic conditions and high-cost issues.12,13 Fluorescent heteroatom doped CDs which exhibit high photoluminescence and long-time fluorescence while being resistant to photo-bleaching have been developed as nanostructured bio-imaging tools.14-16 CDs prepared from a variety of different carbon sources result in nanoparticles of different fluorescence properties.8,17 Surface modification and heteroatom (such as N, S, B or P) doping of CDs improve the fluorescence efficiency with nitrogen-doped CDs being successfully utilized in fluorescence sensoring.13,17-19 Among various methods using for the synthesis of CDs, the hydrothermal treatment of organic substances has been employed as a simple, scalable and green procedure.13,17,20 The present study was conducted with the aim of achieving CDs with powerful and specific therapeutic activity against cancer cells. Accordingly, a series of folic acid targeted CDs was synthesized via a one-pot hydrothermal method using Taxane diterpenoids, as a carbon source, and different diamines, as a nitrogen source. Taxanes, as a family of diterpenoids, originally extracted from Taxus species, display strong anticancer activity against a wide range


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of cancers through inhibition of mitosis, motility, and intracellular transport, leading to the apoptotic cell death.21-22 Hence, it would be expected that using of Taxanes leads to the synthesis of functionalized CDs with therapeutic activity against cancer. The probable attachment of hydrophobic Taxanes to the surface of synthesized hydrophilic CDs would improve the bioavailability of Taxanes and has been the main goal of many recent work.23-28 To meet the needs of diagnostic and imaging applications this work also focuses on the fluorescence properties of the targeted CDs. Although, Liu et al. have recently reported the synthesis of CDs from Metronidazole with selective antibacterial activity,29 but to the best of our knowledge, this is the first report of one-pot hydrothermal synthesis of highly photoluminescent CDs with potent anticancer properties by using of medicinal plants extract.

2. Experimental Section 2.1. Materials. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), ethylenediamine (EDA), ethylenediaminetetraacetic acid (EDTA), fluorescein, folic acid (FA), m-phenylenediamine (mPDA), o-phenylenediamine (oPDA), p-phenylenediamine (pPDA), propidium iodide, and standards of baccatin III and 10-deacetyl baccatin III were purchased from Sigma (Sigma-Aldrich, Germany). Organic solvents for the extraction and HPLC analysis of Taxane compounds were purchased from Merck (Darmstadt, Germany). Standard of Taxol was purchased from Calbiochem (San Diego, CA, USA). Annexin V-FITC conjugate, annexin V binding buffer and cell staining buffer were obtained from Biolegend (San Diego, CA, USA). Roswell Park Memorial Institute medium (RPMI-1640), fetal bovine serum (FBS), and antibiotic solution (penicillin-streptomycin) were prepared from Gibco (Invitrogen, Grand Island, NY). TriPure reagent for RNA extraction was purchased from Roche (Germany). Reverta-L kit was purchased from ILS (Moscow, Russia). RealQ Plus 2x Master Mix Green for real-time PCR reaction was purchased from Ampliqon (Denmark). All of the aqueous solutions were prepared with double distilled water. 2.2. Extraction and analysis of Taxanes from T. baccata L. T. baccata needles were collected freshly from the flower garden of Isfahan, Iran. The needles were dried overnight in the oven and 0.5 gram of the dried powder was suspended in 50 mL of 80% ethanol under sonication for 30 min and shacking for 2 h. The extract was finally filtered through Whatman No.1 filter paper and stored at -20 °C before use. The reverse-phase HPLC analysis was used to confirm the efficient extraction of Taxanes, according to our previous report.24 After concentration of extract in a rotary evaporator at 75 °C, the residue was dissolved in 2 mL methanol and degreased for 4 h and finally filtered before HPLC analysis (Sykam, Eresing, Germany) using a C18 column (Kromasil, 250 mm×4.6 mm). The mobile 3 ACS Paragon Plus Environment


phase consisted of methanol and water (70:30, v/v) with the flow rate of 1 mL min-1 and UV detection was performed at 227 nm. The concentration of 10-deacetyl baccatin III, baccatin III, and Taxol in the sample was determined based on the corresponding standards. 2.3 Synthesis of CDs. The potential use of Taxanes for the synthesis of CDs was comprehensively studied by conducting different reactions. Accordingly, the effect of reaction temperature, reaction time, type and concentration of diamines and the presence and absence of FA were evaluated. Briefly, 50 mL of as-prepared Taxane extract was first concentrated by heating at 75 °C to minimize the ethanolic portion and subsequently, the hydrothermal reaction carried out at 150 °C in the final volume of 20 mL using a Teflon-lined autoclave. The effects of different diamines including mPDA, oPDA, pPDA, EDA and EDTA on the synthesis and fluorescence properties of CDs were investigated. The resulting brown carbonized colloids were centrifuged at 6000 rpm for 15 min to remove the large particles and freeze-dried for 24 h before further characterization. 2.4. Characterization of CDs. The optical properties of CDs were characterized using UV-Vis (Lambda 265, Perkin Elmer) and fluorescence (RF-5000, Shimadzu) spectroscopy. The size, morphology and selected area electron diffraction (SAED) patterns of nanoparticles were determined using a high resolution transmission electron microscope (FEI Titan 80-300 TEM fitted with CEOS image corrector) at an accelerating voltage of 80-300 kV after drop coating of the colloidal CDs onto a hydrophilic carbon-coated copper grid and drying in a dust-protected atmosphere. The X-ray diffraction (XRD) pattern of CDs was studied using a D-8 Advance powder X-ray diffractometer (Bruker, USA). PHI 5000 VersaProbe X-ray Photoelectron Spectroscopy (XPS) was used with a monochromated Al Kα X-ray source (1,486.7 eV) at 100 W and with a beam spot size of 100 μm. The hydrodynamic size and zeta potential of CDs were analyzed using SZ-100 zeta potential analyzer (Horiba, Japan). Fourier transform infrared (FT-IR) spectroscopy carried out on a Jasco FT/IR-6300 spectrometer using KBr plate method with the wavelength range of 4000 cm−1 to 400 cm−1. The quantum yield (QY) value of CDs was calculated by comparing the emission intensity and absorbance value of CDs with those of fluorescein (QY = 95%), as a standard according to the following equation: QYx = QYstd (Ix/Istd)(Rstd/Rx)(n2x/n2std) Where I is the fluorescence intensity, R is the absorbance value at the excitation wavelength, n is the refractive index, and the subscripts x and std signify analyte and standard, respectively. To minimize the re-absorption effects, both of CDs and fluorescein were dispersed in water (refractive index, 1.33) so that their absorbance values at the excitation


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wavelength was less than 0.1. The photostability of CDs was also studied under different conditions including UV illumination, various physiological buffers, and different concentrations of NaCl. 2.5. in vitro anticancer studies. The colloidal CDs were dialyzed in ultrapure water (~2 L) using dialysis membranes (Cellu-Sep T1/Nominal MWCO: 3.5 kD, USA) for 2 h and freeze-dried before use for cell culture studies. The anticancer activity of CDs was initially evaluated on the human breast (MCF-7) and cervical (HeLa) cancer cell lines using MTT assay according to the previous report.25 The unwanted cytotoxicity potential of CDs was also studied on the normal human fibroblast (HFB) cells. For this purpose, the cells were first seeded in 96-well plates at a density of 104 cells per well and incubated overnight at 37 °C in a humidified 5% CO2 incubator before exposure to different concentrations of CDs (5, 10, 20, 50 and 100 µg mL-1) for 48 h. The medium was then removed and 100 μL MTT solution (0.5 mg mL-1 in media) was added into each well and incubated at the same conditions for 4 h. The medium was then carefully discarded and the formazan crystals were dissolved in 150 µL DMSO before the absorbance measurement at 570 nm. The cell viability was calculated as the ratio of absorbance values from each treatment and the control. Two commercial anti-cancer drugs of 5-fluorouracil (5-FU) and methotrexate were used as a control, in the same condition. The cell morphology changes after exposure to CDs were also investigated by optical microscopy. The potential application of CDs for fluorescence imaging and labeling was studied in vitro using an inverted microscope system (Nikon, Eclipse Ti-U) after 2 h incubation of MCF-7 cells with CDs. The frequency of cell apoptosis and necrosis were determined by flow cytometry after annexin V-FITC/ propidium iodide staining. To this aim, 4×105 cancer cells were seeded in 6 cm dishes and exposed to the CDs for 24 and 48 h after overnight incubation at 37 °C in a humidified 5% CO2 incubator. The cells were then harvested by trypsin, washed twice with cold staining buffer and re-suspended in 100 µL annexin V binding buffer, according to the manufacturer’s instruction (Biolegend, San Diego, CA). Subsequently, 5 µL annexin V-FITC (100 µg mL-1) and 10 µL propidium iodide solution (0.5 µg mL-1) were added to the cells and incubated for 15 min under dark condition. After addition of 400 µL binding buffer, the cells were studied using flow cytometry (BD FACS Calibur™, BD Biosciences, San Jose, CA, USA) at an excitation wavelength of 488 nm. The data were finally analyzed by Win MDI 2.8 software. The molecular mechanism of cell death was further elucidated using real-time PCR analysis of apoptosis related genes including bcl-2, bax, p53, caspase 3, and caspase 9. The specifications of each primer have been presented in Table S1. Briefly, total RNA was first extracted from the CDs exposed and control cells by TRipure reagent and



cDNA strands were subsequently amplified using random hexamer primers and M-MLV reverse transcriptase, according to the manufacturer’s instructions. Real-time PCR was carried out using a Rotor-Gene 6000 (Corbett, Australia) for 40 cycles of 12 sec at 95 °C, 25 sec at 60 °C and 30 sec at 72 °C, after initial denaturation at 95 °C for 15 min. Each sample was assayed three times and the relative expression level of genes was calculated using ΔΔCt method while gapdh was used as internal reference gene. 2.6. Statistical analyses. The in vitro experiments were carried out in triplicate and the results were expressed as the mean value of three independent experiments ± standard deviation. The Student’s t-test (Microsoft Excel, Microsoft Corporation, USA) was used to analyze the data and P values of less than 0.05 were considered as statistically significant. 3. Results and discussion 3.1. Synthesis and characterization of CDs. Following the extraction of Taxane compounds from T. baccata needles using 80% ethanolic solution, the presence of 10-deacetylbaccatin III, baccatin III, and Taxol, as main diterpenoid Taxanes, was studied in the extract using HPLC analysis. The results (Figure S1) clearly confirmed the efficient extraction of Taxanes using 80% ethanol as a cost-effective and ecofriendly solvent appropriate for biomedical applications. Hydrothermal synthesis of CDs using Taxus extract, as the only substrate, failed. The investigation of intrinsic potential of Taxus extract for hydrothermal synthesis of CDs at different reaction conditions (150 and 200 °C for 2, 4 and 6 h) led to the colloids with no detectable fluorescence, indicating inability of pure Taxanes to synthesize CDs. Therefore, in the next stage the use of diamines as a supplementary nitrogen source to induce the synthesis of CDs and to improve their QY was comprehensively studied. Nitrogen doping has been well known as an efficient approach to drastically improve the fluorescence QY of CDs.11,13,30-31 This phenomena has been attributed to the comparable atomic size of nitrogen to carbon and to the five valence electrons of nitrogen which can bind to carbon.32 The change in electronic density of states and generation of emissive trap states caused by nitrogen doping has been suggested to modify the band-gap energy which leads to a change in the optoelectronic properties of CDs.33 Optoelectronic properties of CDs are also altered by the induction of more polyaromatic structures by incorporation and protonation of nitrogen atoms on CDs.13 Experiments using T. baccata extracts in combination with mPDA led to the successful hydrothermal synthesis of fluorescent CDs. T. baccata extracts and mPDA in 3:1 w/w ratio were heated at 150°C in the aqueous solution with


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reaction times of 2, 4, 6, 8, and 10 h. The reaction time of 6 h produced CDs with the highest QYs of 26.4% and 29% in the emission wavelengths of 500 (green) and 530 nm (light green), respectively (Figure 1). Longer reaction times led to a decrease in colloidal stability and QY of CDs which may be due to the destruction of stabilizing surface groups and subsequent aggregation of nanoparticles.32 Moreover, the fluorescence intensity of CDs produced in reaction times greater than 6 h shifted toward longer wavelengths (Figure S2) that also indicated the growth and agglomeration of CDs. Performing the reaction at 100 °C resulted in the CDs with significantly fewer QYs while the conducting reactions at 200 °C negatively affected the colloidal stability of CDs without enhancement of QY. Therefore, all of the subsequent reactions carried out at 150 °C for 6 h. The CDs displayed excitation dependent fluorescence as the emission peak red-shifted by increasing the excitation wavelength. The higher concentrations of mPDA not only reduced the QYs of CDs but also led to the less colloidal stability and substantial precipitation that represents the dependency of CDs emission on the nitrogen content. This excitation wavelength dependency of fluorescence emission could be related to different size, composition, structure, or surface oxidation degree of CDs.20,34-35 Different fluorescence properties were obtained using other diamine nitrogen sources, in the same condition (Figure 1). Among different examined diamines, pPDA was not a suitable nitrogen source as no significant fluorescence was obtained from the resulted colloids. The using of EDA, EDTA, and oPDA, led to the CDs with fluorescence QYs up to 1.4, 0.6 and 1.6% in the emission wavelengths of 490 (blue), 390 (violet) and 530 nm (yellow), respectively (Table S2).



Figure 1. Fluorescence emission spectra of the CDs synthesized using the mixtures of T. baccata extract and EDA (A), EDTA (B), oPDA (C), and mPDA (D), at different excitation wavelengths.

Subsequent the creation of CDs with desirable QYs and relevant emission FA, a well-known cancer targeting agent, was incorporated into the CDs. The best result was obtained by using the mixture of extract, mPDA and FA (Ex-mPDA-FA) with the maximum QYs of 23.6 and 50.4% in the emission wavelengths of 460 (blue) and 520 nm (green), respectively (Table S2). The results represent a significant enhancement of QY after addition of FA. The high QY of resulted multicolor CDs clearly indicates their high potential for the biological and analytical applications. The minor blue shift of emission peaks in the presence of FA could be attributed to the surface functionalization of resulted CDs with FA (Figure 2). Moreover, the synthesized CDs displayed high stability and monodispersity such that the colloids retained up to 88% of their initial QYs without any agglomeration and precipitation after 6 months of exposure to visible light.


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Figure 2. Fluorescence emission spectra of the CDs synthesized using the mixtures of Taxus extract, FA, and EDA (A), EDTA (B), oPDA (C), and mPDA (D), at different excitation wavelengths.

The maximum QYs of 19.6, 12.2 and 5.6% in the emission wavelengths of 450 (blue), 490 (green) and 520 nm (green) were obtained, at the same condition, for the CDs synthesized by using other nitrogen sources of EDTA, EDA, and oPDA, respectively (Table S2). Interestingly, the hydrothermal treatment of the reaction mixture containing Taxus extract and FA without using of any nitrogen source, at the same conditions, led to the synthesis of CDs with low QY (1.67% in the emission wavelength of 445 nm (Figure S3)). This result clearly proved the synergic role of both FA and nitrogen source in the QY of resulted CDs. With regards to the complex and not well-defined photoluminescence mechanism of CDs, this observation could be attributed to different factors such as quantum confinement effect, conjugated π-domains, defects and surface states, free zigzag sites, molecule state and sp2-carbon networks.36 The photostability of Ex-mPDA-FA CDs was studied following the exposure to continuous UV irradiation for different periods as well as under different physiological buffers and NaCl solutions. The results indicated high 9 ACS Paragon Plus Environment


photostability of CDs as more than 80% of fluorescence QY was maintained after 12 h exposure to UV illumination at 395 nm with 20 W power (Figure S4). The CDs also displayed high stable fluorescence in NaCl solutions with different concentrations up to 1 M (Figure S4). Moreover, no significant change in the fluorescence intensity occurred when the CDs were dispersed in the PBS buffer or RPMI culture medium. This high photostability of CDs would be very valuable in diagnostics and bioimaging applications. HR-TEM analysis of Ex-mPDA-FA synthesized CDs illustrates that the nanodots are mostly monodisperse with a hexagonal shape and 10 to 20 nm dimensions (Figure 3). HR-TEM image revealed the semi-crystalline structure of CDs, containing amorphous parts, indicating the presence of structural defects in CDs. The CDs represent a lattice spacing between 0.31 and 0.33 nm which corresponds to (002) facet of graphitic carbon. The crystalline structure of CDs was also confirmed by SAED and XRD analysis (Figure 3). The XRD pattern (Figure 3) revealed a broad diffraction peak centered at around 21°, attributed to the highly disordered carbon atoms in the graphitic structure,37 and in agreement with the result of HR-TEM lattice pattern. The nanoparticles had average hydrodynamic size of 43.2 nm and polydispersity index (PDI) of 0.241 with Zeta potential value of –47.6, respectively, indicating their narrow particle size distribution, excellent surface functionalization and high colloidal stability. The surface chemistry of purified CDs was investigated using FTIR. The FTIR spectrum of Ex-mPDA and ExmPDA-FA synthesized CDs (Figure 3) showed several strong peaks demonstrating the presence of many functional groups on the surface. The most obvious absorption peaks are 3352 cm−1, 3241 cm−1, 2925 cm−1, 1608 cm−1, 1499 cm−1, 1323 cm−1, 1050 cm−1, and 845 cm−1 corresponding to N–H stretch, sp C–H stretching of alkynes, sp3 –C–H stretching of alkanes, C=C stretching of alkenes, C–O–C stretching vibrations, C–N stretching of aromatic and aliphatic amines, C–O stretching of alcohols, and C–H stretching of aromatics. The presence of these peaks in the FTIR spectrum of freeze-dried Taxus extract clearly represents the protection of the functional groups during the hydrothermal synthesis of CDs. The shoulder peak located at 1675 cm−1 in the FTIR spectrum of Ex-mPDA-FA CDs is related to C=O stretching of carboxylic acid group of FA, indicating the presence of FA on the surface of CDs. The FTIR spectrum of other CDs contains relatively similar peaks (Figure S5). These various surface functional groups have substantially improved the colloidal stability of CDs and facilitate their further functionalization.


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Figure 3. High resolution TEM image (A), SAED pattern (B) XRD pattern (C) and FTIR spectra (D) of Ex-mPDAFA synthesized CDs. The elemental composition of Ex-mPDA-FA synthesized CDs was analyzed using PHI 5000 VersaProbe X-ray Photoelectron Spectroscopy (XPS) with a monochromated Al Kα X-ray source (1,486.7 eV) at 100 W and with a beam spot size of 100 μm. As can clearly be seen in Figure 4 and Figure S6, O 1s, N 1s, and C 1s were observed which are consistent with the elemental composition of the Ex-mPDA-FA synthesized CDs.

Figure 4. The study of the elemental composition of Ex-mPDA-FA synthesized CDs using PHI 5000 VersaProbe Xray Photoelectron Spectroscopy (XPS).



The synthesis of yellow-green-emissive CDs with QYs of 27.7 and 28.6% at 515 and 520 nm, respectively, using solvothermal treatment of m-aminophenol has been reported recently; however, the QY of CDs prepared in the present study is significantly higher than those previously reported, representing a more potent use of these CDs for bioimaging applications.38 Additionally, the present synthetic method was carried out with renewable natural precursors with only ethanol and water as solvents under more mild conditions than the previous work making its capacity for large scale production of high QY CDs for bio-imaging and treatment more viable.

3.2. in vitro studies. The anticancer potential of synthesized CDs was comprehensively studied using several cellular and molecular methods including MTT assay, microscopic observation of cell morphology, flow cytometry and realtime PCR. The results of MTT experiments were presented in Table 1 based on IC50 values of the synthesized CDs after 48 h exposure with two major human cancer cell lines of the breast (MCF-7) and cervical (HeLa) as well as normal human fibroblast (HFB) cells. The overall results showed the considerable cytotoxicity of synthesized CDs using Taxane compounds and different diamines. As the previously reported CDs were biocompatible without the toxic and anticancer activity, the observed activity could be attributed to the carbon source of Taxanes. According to Table 1, a diverse range of obtained IC50 values could be related to the role of reaction composition on the biological activity of CDs. Interestingly, FA targeting considerably enhanced the anticancer activity of CDs. The highest anticancer effects were observed for Ex-EDA-FA and Ex-mPDA-FA synthesized CDs with IC50 values of 38.2 ± 2.2 and 34.1 ± 1.1 µg mL-1 for HeLa and 35.1 ± 1.9 and 31.3 ± 2.7 µg mL-1 for MCF-7 cancer cells, respectively, representing up to 400% enhancement on the anticancer activity of CDs due to the FA targeting. As no significant difference was observed in the toxicity of targeted and non-targeted CDs on HFB cells (for instance, the IC50 values of Ex-mPDA and Ex-mPDAFA are 125.6 ± 2.0 and 120.5 ± 3.8 µg mL-1, respectively), the enhanced anticancer activity could be attributed to the overexpression of folate receptors on the surface of cancer cells leading to the increased delivery of CDs into cancer cells.39 The comparison of anticancer activity and cytotoxicity effects of 5-fluorourdacil and methotrexate, as the wellknown commercial anticancer drugs, with those of the CDs (Table 1), emphasized the comparable anticancer activity and reduced toxicity of CDs. To the best of our knowledge, the obtained anticancer activity in the present study is considerably higher than those reported until now for CDs. Moreover, the QY of CDs is higher than the other


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synthesized CDs with anticancer activity.20 The low toxicity of aqueous phase synthesized CDs using FA, as the precursor, has also been reported recently,39-40 indicating the importance of precursor in the biological effects of obtained CDs.

Table 1. IC50 values (µg mL-1) of different CDs, T. baccata extract, 5-fluorouracil, and methotrexate on HFB, HeLa, and MCF-7 cell lines. cell line Sample

HFB (µg mL-1)

HeLa (µg mL-1)

MCF-7 (µg mL-1)

Ex-EDA

151.2 ± 3.7

161.6 ± 4.6

156.3 ± 2.3

Ex-EDTA

> 200.0

149.4 ± 3.7

141.3 ± 1.9

Ex-oPDA

120.8 ± 3.1

125.1 ± 4.4

112.5 ± 4.3

Ex-mPDA

125.6 ± 2.0

115.2 ± 4.6

82.1 ± 4.0

Ex-FA

> 200.0

166.3 ± 3.6

153.9 ± 2.1

Ex-EDTA-FA

> 200.0

160.2 ± 4.5

151.7 ± 2.6

Ex-EDA-FA

125.8 ± 3.2

38.2 ± 2.2

35.1 ± 1.9

Ex-mPDA-FA

120.5 ± 3.8

34.1 ± 1.1

31.3 ± 2.7

Ex-oPDA-FA

147.9 ± 3.3

91.5 ± 1.1

88.1 ± 2.0

Extract

98.6 ± 2.0

100.4 ± 1.8

103.6 ± 1.6

5-fluorouracil

51.4 ± 2.6

47.2 ± 2.1

44.3 ± 2.4

Methotrexate

36.6 ± 2.3

21.8 ± 1.7

27.4 ± 2.1

The changes in cell morphology following the exposure to CDs were monitored using the optical microscope to further investigate the anticancer activity of CDs. In accordance with the MTT results, the morphology of cells showed different levels of changes based on the type of CD. The maximum change was observed after exposure of cancer cells to Ex-EDA-FA and Ex-mPDA-FA synthesized CDs (Figure 5). However, a considerable change was also observed following the exposure to other CDs (Figure S7-S10). Moreover, some morphology changes are also visible in the human fibroblast cells, indicating the non-specific cytotoxicity of CDs. The initial changes include suppression



of cell proliferation following by the cellular shrinkage, rounding, and clumping. In the serve condition, the plasma membrane rupture and cell fragmentation could be clearly observed.

Figure 5. Morphology of cells before exposure to CDs (A-C) and after 48 h exposure to 50 µg mL-1 Ex-EDA-FA (D-F) and Ex-mPDA-FA CDs (G-I). Left to right: HFB, HeLa, and MCF-7 cells.

The imaging potential of CDs was evaluated by fluorescence microscopy after 2 h incubation with MCF-7 cells. The simultaneous green and red photoluminescence of CDs exposed MCF-7 cancer cells without the background light interference was observed under the blue and green light excitation, respectively (Figure 6). The results clearly indicate


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the rapid and efficient delivery of CDs to the cancer cells. However, CDs also efficiently stained the normal human fibroblast cells, at the same conditions (Figure S11), which could be due to their very small size.

Figure 6. Fluorescence images of MCF-7 cells after 2 h incubation with Ex-mPDA-FA CDs under the excitation of blue (A) and green (B) light excitation.

Flow cytometry analysis of cancer cells after staining with annexin V-FITC and propidium iodide was used to investigate the cell death mode of CD treated cells. Annexin V-FITC is commonly used for detection of apoptotic cells due to the high binding affinity of Annexin V protein to the specific apoptotic cells expressing molecules of phosphatidylserine. On the other hand, the ability of propidium iodide in the differential penetration and binding to the nuclear DNA of late apoptotic and necrotic cells was used to distinguish the CD initiated cell death from unrelated apoptosis.41 The results (Figure 7) clearly indicate the induction of apoptosis in the MCF-7 cells following the exposure to CDs. The late apoptosis was the main form of cell death which represented a time dependent increase reaching 61.78 and 93.13% after 48 h exposure to Ex-mPDA-FA and Ex-EDA-FA CDs, respectively. The overall results, consistent with the findings of MTT assays, show more than 71 and 93% cell mortality after 48 h exposure with 50 µg mL-1 of Ex-mPDA-FA and Ex-EDA-FA CDs, respectively.



Figure 7. The frequency of apoptosis and necrosis in MCF-7 cells after 24 and 48 h treatment with Ex-mPDA-FA and Ex-EDA-FA CDs (A), and the mRNA expression level of apoptotic markers (p53, bax, bcl-2, caspase 3 and caspase 9) in Ex-mPDA-FA treated MCF-7 cells (for 4 and 24 h) in comparison with untreated cells (B). To further elucidate the probable apoptosis pathway(s) involved in the CD mediated cell death, the mRNA expression levels of five important apoptotic genes of p53, bax, bcl-2, caspase-3, and caspase-9 were quantitatively investigated by real-time PCR. Caspase-3, as the most important executioner caspase which is activated by the initiator caspases of 8, 9, and 10, induces apoptosis through endonuclease-mediated degradation of chromosomal DNA and disintegration of cells into the apoptotic bodies via cytoskeletal reorganization. In contrast, bcl-2 has been recognized as an important apoptosis inhibitor by controlling the activation of caspases.42 The results showed that the MCF-7 cells exposure to CDs led to the up-regulation of cell cycle checkpoint protein p53 and apoptotic members (bax, caspase-3, and caspase-9) while the expression of the anti-apoptotic bcl-2 gene was down-regulated (Figure 7). Based on the results, it could be concluded that the exposure of cancer cells to CDs results in the up-regulation of p53 which subsequently activates the expression of pro-apoptotic bax gene. These events lead to the enhanced permeability of the outer mitochondrial membrane and subsequent release of cell death factors such as cytochrome c into the cytosol. The cell death factors eventually induce the apoptosis and destruction of cellular components due to the activation of caspases.43-44 Interestingly, Morales-Cano et al. also reported a relatively similar pathway of apoptosis induction including reductions of p53, changes in Bax and Bcl-2 and activation of caspases 3 and 9 following the exposure of acute promyelocytic human leukemia NB4 cells with paclitaxel.45 Li et al. reported the anticancer activity of tender ginger juice synthesized CDs in human hepatocellular carcinoma cells via upregulation of p53 protein and subsequent induction of apoptosis.20 This result indicates different mechanism of anticancer action based on the source of CDs.


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Although there is no significant report about the synthesis of CDs with intrinsic anticancer activity but specific cancer cell imaging using CDs based labeling agents has attracted great attention in cancer research owing to facilitating the cancer cell biology studies and cell response monitoring to cancer therapy.39 In this context, some CDs based theranostic nano-platform have been reported, recently, for simultaneous cancer cell imaging and delivery of anticancer drugs such as doxorubicin,6-9,46-48 methotrexate,1 cisplatin,49 epirubicin,4 as well as multiple siRNAs.5 Beyond the attractive optical properties of CDs, some interesting biological effects such as the antiviral activity of ascorbic acid synthesized CDs,10 promoting the differentiation of rat bone marrow mesenchymal stem cells to osteoblasts using citric acid-based CDs 50 and reversing the resistance to hypoxia-triggered photodynamic therapy via light-driven water splitting of carbon nitride based multifunctional nanocomposite 51 have been reported recently. However, to the best of our knowledge, this is the first report regarding the development of highly photoluminescent CDs with powerful and specific anticancer activity. 4. Conclusions Development of a simple, efficient and straightforward approach to fabricate the multifunctional nanoplatforms toward cancer theranostics is an emerging and cutting-edge field of nanomedicine and is the essential aspect of advancing cancer researches. In this regard, fluorescent CDs have attracted much attention due to their fascinating characteristics especially high QY which is the main prerequisite for development of new bioimaging, biosensing, and theranostics tools with high sensitivity and accuracy. Herein, we present a green, simple and reproducible method suitable for large-scale production of fluorescent CDs with physico-chemical properties such as high colloidal stability, monodispersity, high and stable QY, multifunctionality, multicolor emission working towards selective and potent anticancer activity. The synthetic parameters that examined, including the type and concentration of diamines, temperature and reaction time. The using of FA also led to the synthesis of CDs with significantly superior fluorescence QY and higher anticancer activity. Unlike most amorphous CDs reported by previous methods, semicrystalline and hexagonal CDs containing various functional groups were obtained by the present method that is appropriate for further surface modification and different applications. A wide range of fluorescence from violet to orange with different QYs and various biological activity was obtained by changing the reaction conditions. The innovative use of anticancer Taxane compounds, as the carbon source, led to the synthesis of new generation of CDs with potent anticancer activity. This result emphasized the importance of carbon source in the final biological effects of synthesized CDs. The present approach not only led to the creation of fluorescent nanoparticles but also was a 17 ACS Paragon Plus Environment


breakthrough in overcoming the obstacle of hydrophobicity in anticancer Taxanes. Unlike most of the previously reported approaches which entail multi-step synthetic process and assembly of different counterparts for development of theranostic nanostructures, we report a one-pot synthesis method for the synthesis of a single nanoparticle with the simultaneous ability of imaging and cancer therapy. In conclusion, the present approach provides a promising way to develop high-quality multifunctional nanoparticles for cancer theranostics. Associated Content Supporting Information Table S1, Figure S1, Figure S2, Table S2, Figure S3, Figure S4, Figure S5, Figure S6, Figure S7, Figure S8, Figure S9, Figure S10, Figure S11. Author Information Corresponding Author *

Email: [email protected]

Funding Sources Iran National Elites Foundation and Research Council of University of Isfahan. Notes The authors declare no competing financial interest.

Acknowledgments This work was supported by Iran National Elites Foundation and Research Council of University of Isfahan.


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Graphical Abstract




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Green and Facile Synthesis of Highly Photoluminescent Multicolor

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