Shaojing Zhao, Li Huang, Yong Xie, Bin Wang, Feng Wang, Minhuan Lan,*

1 Hunan Provincial Key Laboratory of Micro & Nano Materials Interface Science, College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China

2 State Key Laboratory of the Discovery and Development of Novel Pesticide, Shenyang Sinochem Agrochemicals R&D Co., Ltd., Shenyang 110021, China

3 Key Laboratory of Materials Chemistry for Energy Conversion and Storage of Ministry of Education, Hubei Key Laboratory of Material Chemistry and Service Failure, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China

Keywords:Carbon dots Photothermal therapy Anti-fungus Delivery Pesticide

ABSTRACT Carbon dots (CDs) have become popular nanomaterials in biomedical and agricultural fields.Herein we synthesized multifunctional CDs which showed anti-cancer and anti-fungal activities.The low cytotoxicity, stable fluorescence and high photothermal conversion efficiency enable the CDs with imagingguided photothermal therapy.The CDs also exhibited intrinsic anti-fungal activity even at a low concentration, i.e., 40 mg·L-1 of CDs induced 20% mortality in cucumber downy mildew.Moreover, the large πconjugated nanostructure and the richness of amino and hydroxyl groups make them a powerful delivery platform for flumorph (a fungicide) with a high loading efficiency of 47.18%.Meanwhile, the heat converted from the light can accelerate the release of flumorph from CDs, and thus efficiently kill fungus.

1.Introduction

Carbon dots(CDs)have become an interesting carbon nanomaterial due to their small size, excellent photochemical stability,tunable fluorescence,low cytotoxicity,good water dispersity,easy synthesis and surface modification [1-3].CDs are widely used in catalysis, drug delivery, biosensing, bioimaging, and phototherapy[4-7].Since the firstly discovered CDs by Xu et al.[8],kinds of precursors, including organic molecules, polymers, biomass, and bulk carbon, were selected to synthesize CDs [9-12].In general, methods for preparing CDs can be divided into two types: ‘‘top-down”and ‘‘bottom-up”[13].In the ‘‘top-down” method, carbon sources,such as fullerene,carbon nanotube,and bulk carbon black were cut into CDs by using strong acid or base solution,while these solvents are harmful to the environment.Moreover,the poor water solubility and weak fluorescence of the CDs prepared by the ‘‘top-down”method are also not suitable for their biomedical applications.In contrast, in the ‘‘bottom-up” route, small molecules or polymers were used as raw materials to prepare CDs through dehydration,assembly, and carbonization processes [14-16].Among the‘‘bottom-up” methods, the hydrothermal method by using water as solvent is a simple and economical method.The as-prepared CDs also have better water solubility and fluorescence properties[17].

One of the most attractive applications of CDs is their biomedical potential, especially in bioimaging and phototherapy [18-20].Using conjugated polythiophene derivates (named PT1, PPA, and PBA) as carbon sources, we prepared three kinds of S-doped red fluorescent CDs, which exhibit high singlet oxygen generation quantum yield and capable of converting photon energy into heat.These CDs were applied in photodynamic therapy (PDT) and photothermal therapy (PTT) of cancer cells and tumor-bearing mice[21-23].Through S and Se co-doping, we further prepared near infrared fluorescent CDs with a high photothermal conversion efficiency of 58% [24].However, the synthesis of these conjugated polythiophenes is complex and expensive.Recently, we synthesized CDs by hydrothermal treatment of organic small molecules(nitropyrene),which showed a high photothermal conversion efficiency of 73.5%.Moreover,these CDs could generate singlet oxygen and hydroxyl radical under light irradiation [25,26].It should be noted that nitropyrene is highly toxic to human body [27].Due to their capacity to generate reactive oxygen spices under light excitation,CDs display promising light-triggered microbicide properties, and their anti-biotic and anti-viral efficiencies have been reported [28-31].

On the other hand, pesticides can efficiently control fungi and are widely used in agriculture to increase crop yield[32,33].However, more than 90% of pesticides are quickly fallen off from the crop pages and thus wasted due to their poor water solubility[34].Although several nano-carriers have been used to load these hydrophobic pesticides, the development of multifunctional carriers with intrinsic anti-fungal activity and high drug loading capacity are still commendable [35-37].

Here we report the green synthesis of multifunctional CDs for anti-cancer and anti-fungal applications.The nanostructure and chemical composition of the CDs were studied,and the biocompatibility, photo- and chemical stability were investigated in detail.Results demonstrated that these CDs have good cell-permeability and high photothermal conversion efficiency of 42.4%.The application of these CDs in multi-color fluorescence imaging and cancer photothermal therapy was investigated (Scheme 1).Further, the CDs can efficiently kill fungus even at low concentration(40 mg·L-1).To further improve CDs anti-fungal properties, the commercial pesticide (flumorph) was loaded on the CDs with a loading efficiency of 47.18%.Meanwhile, the heat converted from the sunlight can accelerate the release of flumorph from CDs, and thus efficiently kill fungus.

Scheme 1.The applications of CDs in multi-color imaging, photothermal therapy,loading pesticide and anti-fungus.

2.Experimental

2.1.Preparation and characterization of CDs

A specific amount(0.6 g)of dried seaweed were resuspended in 30 mL water and autoclaved under hydrothermal treatment for 4 h at 200 °C in a hydrothermal reactor (K50, Shanghai LABE Instrument CO.,Ltd).The solution was cooled to room temperature,centrifuged at 16,000 r·min-1for 15 min,and then filtrated to exclude large particles.The supernatant was dialyzed using 3500 MW dialysis bag.The purified CDs (～30 mg) were dispersed in de-ionized water.

Transmission electron microscopy (TEM) observations were made with a JEOL JEM-2100F electron microscope.Atomic Force Microscope (AFM) images were collected by the Nano-ScopeIIIaMulti-Mode microscope.X-ray photoelectron spectroscopy (XPS)spectra were recorded on a VG ESCALAB 220i-XL surface analysis system.Fourier transform infrared (FTIR) spectrum was obtained with an IR spectrophotometer (IFS 66 V/S, Bruker, Germany).The Raman spectrum was obtained using an Invia-Reflex Raman system.The UV-vis absorption and fluorescence spectra were recorded on UV-2600 spectrometer and RF-6000 spectrofluorometer (Shimadzu), respectively.

2.2.Cell imaging and in vitro PTT

HeLa cells were seeded in a 6-well plate for 24 h,and then incubated with CDs(40 μg·ml-1)at 37°C for additional 4 h.After,cells were washed with PBS three times and imaged by the Nikon fluorescent microscopy under different excitation wavelength.To study the cytotoxicity of CDs, HeLa cells were seeded into a 96-well plate for 12 h, and then incubated with different concentrations of CDs for 24 h.To investigate the phototoxicity of CDs,L929 or HeLa cells in the 96-well plate were incubated with different concentration of CDs for 4 h, and then exposed to a 635 nm laser(2 W·cm-2) irradiating for 10 min.CDs cytotoxicity and phototoxicity were assessed by measuring cell viability by standard MTT assay.

2.3.Pesticide loading

The flumorph-loaded CDs were prepared as following:2 ml flumorph (99.3% technical material, 1 mg·ml-1in THF) was added into 10 ml of CDs aqueous solution (0.15 mg·ml-1) under ultrasound condition.The mixture solution was further subjected to ultrasounds for another 30 min, purified by dialysis with a MW 3500 dialysis bag to remove THF, and then filtered by a 0.45 μm filter membrane.The solution was freeze dried for further use.

The loading efficiency of flumorph into CDs was calculated by the following equation [38,39]:

The amount of flumorph in the supernatant was calculated to be ～1.34 mg, and the loading efficiency was thus calculated to be～47.18%.

2.4.Anti-fungal activity

The anti-fungal activity of the CDs, flumorph, and flumorphloaded CDs was assessed by fungicidal assays in greenhouse.In detail,CDs(25.0 mg),flumorph(25.2 mg,99.3%technical material),and flumorph-loaded CDs(53.0 mg,containing 25.0 mg flumorph)were each dissolved in 5 ml of acetone/methanol (1:1, volume ratio) solution, and then mixed with 5 ml of water containing 0.1%Tween 80(stock solutions).Test solutions with concentration between 10 and 640 mg·L-1were prepared by dilution of the stock solutions.The fungicidal activity of each compound against cucumber downy mildew in vivo were measured as follows:

Seeds (cucumber: Cucumis sativus L.) were grown to the oneleaf stage.Then, test solutions were sprayed on the host plants.After 24 h,the leaves of the host plants were inoculated with sporangial suspensions of cucumber downy mildew(Pseudoperonospora cubensis, cultured by Shenyang Sinochem Agrochemicals R&D Co.Ltd.Shenyang, China) at a concentration of 5 × 105spores per ml using a PS289 Procon Boy WA double action 0.3 mm airbrush(GSI,Tokyo,Japan).The cucumber plants were stored in a humidity chamber ((24 ± 1) °C, Relative Humidity ＞95%, dark) and then transferred to a greenhouse (18-30 °C, Relative Humidity ＞(50-60)%) 24 h after infection.Three independent experiments were performed.The anti-fungal activity was studied by visual inspection after 7 days.The inhibitory activity was calculated as following:

The EC50values of flumorph and flumorph-loaded CDs were calculated by Duncan’s new multiple-range test (DMRT) using DPS version 14.5.

3.Results and Discussion

3.1.Characterization of CDs

Fig.1.CDs characterization.(a) TEM image and (b) The size distribution of CDs.The inset in (a) shows the HR-TEM of one CD.(c) AFM image and height profile of CDs.(d)Raman spectrum of CDs.

TEM and AFM images were collected to study CDs morphology.Fig.1(a) and (b) revealed that the prepared CDs had a monodisperse spherical morphology with an average size distribution of 2.2 nm.The lattice space of 0.23 nm was observed from the HR-TEM image of one particle (inset in Fig.1(a)), revealing the crystal structure of the CDs.The AFM analysis (Fig.1(c)) revealed a CDs height lower than 2 nm,which is agreed with the TEM observation.Furthermore, the Raman spectrum (Fig.1(d)) showed two peaks at 1326 cm-1and 1577 cm-1, which are attributed to the D and G band of graphitic.

The XPS spectra presented in Fig.2 provide information regarding the elemental CDs composition.The three peaks at the binding energy of ～285, 400, and 531 eV, which is corresponding to C1s,N1s and O 1s, were observed from the XPS survey spectrum(Fig.2(a)).The atomic ratios of C, N, and O were calculated to be～74.0%, 6.3%, and 19.7% respectively (Fig.2(a), insets).The deconvoluted high-resolution C1s XPS spectrum shows four peaks:284.3 eV for C-C, 285.4 eV for C-N, 286.5 eV for C-O, and 288.4 eV for C=O.The deconvolute N1s spectrum in Fig.2(c)revealed that the CDs have pyridinic N,pyrrolic N and N-H.Moreover,the FTIR spectrum of CDs in Fig.2(d)shows several significant absorption bands: 1700, 1650, 1400 and 1050 cm-1, which is attributed to the C=O, C=C, C-N and C-O bonds, and the peaks at ～2950 and 3400 cm-1are originated from C-H and O-H/N-H, respectively.Furthermore, a negative Zeta-potential of-17.1 mV demonstrated that the CDs rich in -OH and -COOH groups on the surface.

3.2.Optical properties of CDs

The optical properties of the prepared CDs were studied by UVvis absorption and FL spectra.As shown in Fig.3 (black line), the CDs have the absorbance ranging from 250 to 750 nm.The CDs exhibit blue emission under 365 nm light irradiation, with a fluorescence quantum yield of ～1.5%.Fig.3(b) shows the fluorescence spectra of the CDs under different excitation wavelengths, revealing a typical excitation wavelength-dependent feature, i.e., when excited from 300 to 360 nm, the fluorescence intensity progressively increased.At higher wavelengths,the fluorescence intensity gradually decreased.The highest fluorescence intensity at 455 nm was obtained under 360 nm excitation.The emission wavelength was red-shifted with increasing excitation wavelength.This is agreed with the excitation spectrum of the CDs(Fig.1(a),red line),showing the highest intensity of ～360 nm.

3.3.Stability and cell penetration capacity of CDs

The stability is a key parameter that determines the potential application in biomedicine of the CDs.Fig.4(a) and (b) show the time-dependent absorption and fluorescence spectra of the CDs aqueous solution under the Xenon light irradiation for 60 min.No significant spectral variation was observed.In contrast, the absorption and fluorescence spectra of the fluorescein sodium were significantly decreased to ～30% of their original value after the light irradiation(Fig.4(c)and(d)).These results demonstrated that the CDs have excellent photostability.

The pH effects on the stability of the CDs fluorescence was further studied.As shown in Fig.5(a), the normalized FL intensity at 455 nm under different pH values is stable between pH 3 and pH 10, and decreased with the increase of the pH up to 13.Fig.5(b)shows the normalized FL intensity of the CDs aqueous solution with different concentrations of NaCl.We observed that the FL intensity is nearly stable, even in a NaCl concentration of 1000 mmol·L-1.The fluorescence intensity of CDs was also maintained in the presence of different amino acids, as shown in Fig.5(c).The above results demonstrated that the CDs have excellent fluorescence stability.

Fig.2.CDs characterization.(a) XPS survey spectrum, (b) the deconvolution of C1s and (c) N1s spectra.(d) FT-IR spectrum.

Fig.3.Optical properties of CDs (a) UV-vis absorption (black line) and excitation (red line) spectra of CDs in aqueous solution.(b) FL spectra of the CDs aqueous solution obtained by different excitation.

To investigate the biomedical imaging capability,CDs incubated HeLa cells were imaged by fluorescent microscopy.As shown in Fig.5(d), HeLa cells showed blue, green and red fluorescent colors when the excitation wavelength is about 330-385 nm, 450-480 nm, and 510-550 nm, respectively, suggesting the good cytomembrane penetrate capability of CDs.While the cells in the absence of CDs shows no obvious fluorescent signal (Fig.S1).The good cell membrane permeability of the CDs maybe ascribed to the small size, the negative charged surface groups of CDs, such as -OH and -COOH groups [40].

3.4.Photothermal analysis and photothermal therapy of cancer

The photothermal conversion capabilityof the CDs wasstudied by monitoring temperature variations of the CDs aqueous solutions under a 635 nm laser irradiation.As revealed in Fig.6,the temperature of water(in the absence of CDs)increased from 25°C to 30°C within 10 min of laser irradiation.However,the temperature of the CDs solution (360 μg·ml-1) was increased to 53 °C after receiving laser irradiation for 10 min.The curve of temperature gradients versus concentration of CDs shown in Fig.6(b)suggests that higher concentration of CDs solution reach a higher temperature upon irradiation,indicating that the temperature increase of the CDs aqueous solution is primarily caused by the photothermal effect of CDs,in a concentration-dependent manner.The photothermal conversion efficiency of the CDs was measured to be ～42.4% (Fig.S2).Fig.6(c)shows the temperature variation under pulses of laser irradiation.Results shows that after 10 cycles,the temperature of the CDs solution remains at ～52°C after the laser treatment.However,no obvious ROS signal was observed under the 635 nm laser irradiation(Fig.S3).

Fig.4.Time-dependent(a)absorption spectra and(b)fluorescence spectra of CDs.Time-dependent(c)absorption spectra and(d)fluorescence spectra of fluorescein sodium.

Fig.5.Effect of(a)pH,(b)NaCl,and(c)Amino acids(10 μmol·L-1)on the FL intensity at 455 nm.(d)Bright field and multi-color fluorescent imaging of CDs-incubated HeLa cells from fluorescent microscope at different excitation wavelength.Scale bar: 50 μm.

Next, methyl thiazolyl tetrazolium (MTT) assay was used to investigate CDs in vitro cytotoxicity to cancer cells (HeLa cells)and normal cells (L929 cells).As shown in Fig.6(d), the cell viabilities of HeLa and L929 cells was decreased with increasing CDs concentration and exposing 635 nm laser irradiation for 10 min.However, in the absence of laser, the cell viabilities were large than 80% even in a high concentration of 360 μg·mL-1of CDs,confirming the PTT-related properties of CDs.The results obtained here prompted us to study the in vivo performance of CDs.

Fig.6.(a)Temperature variation of CDs aqueous solution at different concentrations vs the irradiation time.(b)Temperature variation of CDs aqueous solution after 10 min laser irradiation vs the concentrations.(c)Temperature variation of CDs(360 μg·mL-1)over 10 laser ON/OFF cycles.(d)Cytotoxicity and phototoxicity of the CDs to L929 or HeLa cells under a 635 nm laser irradiation 2 W·cm-2 for 10 min.

3.5.Pesticide loading and fungicidal assays

Taking advantage of the large π-conjugated structure and enrichment in-NH2and-OH groups,our CDs can host hydrophobic pesticides through hydrophobic and hydrogen bonding interaction.Here flumorph was loaded on the surface of CDs with a loading efficiency of ～47.18%.The anti-fungal activity of flumorph-loaded CDs against cucumber downy mildew was determined by fungicidal assays[44].As a control,the anti-fungal activity of CDs and flumorph alone was also tested.As shown in Table 1,our CDs exhibit anti-fungal activity with a percentage of inhibition of 10%at low concentration of 10 mg·ml-1,the inhibition efficiency increased to 20% in at of 40 mg·L-1CDs, while increasing the concentration of CDs, induced no further inhibition efficiency.The antifungal ability of CDs maybe due to the oxygen-containing groups which can adsorb on the cell walls of bacteria and fungi,and then diffuse into the cell walls.These CDs destroy the integrity of cell membrane and lead to cytoplasmic leakage [45].Moreover,CDs can bind to DNA and RNA in bacteria and fungi through non covalent bonds, change the structure of DNA and RNA, and then affect the genetic process of bacteria and fungi [46].

Table 1 Anti-fungal activity of CDs, flumorph, and flumorph-loaded CDs against cucumber downy mildew

In contrast, the inhibition efficiency of flumorph gradually increased with increased concentration.By comparison,flumorph-loaded CDs show an improved antifungal activity, with the inhibition efficiency of flumorph,and flumorph-loaded CDs calculated as 65%,and 75%,respectively,in the presence of 160 mg·L-1flumorph.The EC50values of flumorph and flumorph-loaded CDs were 40.2 and 27.8 mg·L-1, respectively, suggesting that our CDs may function as an antifungal agent, and as a pesticide carrier.

4.Conclusions

We have prepared multifunctional CDs which show stable fluorescence, excellent cell permeability capacity and strong photothermal conversion capability with a high efficiency of 42.4%.The application of CDs in PTT of cancer cells was demonstrated.Moreover, the as-prepared CDs show intrinsic anti-fungal activityand high pesticide loading efficiency.The pesticide-loaded CDs exhibit improved anti-fungal activity and a lower EC50value, as compared with the pesticide.With this study we generated an efficient and green photothermal and antifungal agent for biomedical and agricultural applications.

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.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (61805287), Natural Science Foundation of Hunan Province, China (2018JJ3632 and 2019JJ50824), Fundamental Research Funds for State Key Laboratory of the Discovery and Development of Novel Pesticide, Shenyang Sinochem Agrochemicals Research and Development Co., Ltd.(2018NYRD02).

Supplementary Material

Bright field and fluorescent images of HeLa cells in the absence of CDs.Photothermal conversion efficiency measurements of the CDs.reactive oxygen species detection Supplementary data to this article can be found online at https://doi.org/10.1016/j.cjche.2021.03.008.