Functionalized fluorescent carbon nanoparticles for sensitively targeted of folate-receptor-positive cancer cells

Accepted Manuscript Functionalized fluorescent carbon nanoparticles for sensitively targeted of folate-receptor-positive cancer cells

Yuan Jiao, Haiyun Sun, Yanchun Jia, Yang Liu, Yifang Gao, Ming Xian, Shaomin Shuang, Chuan Dong PII: DOI: Reference:

S0026-265X(18)31629-1 https://doi.org/10.1016/j.microc.2019.01.003 MICROC 3574

To appear in:

Microchemical Journal

Received date: Revised date: Accepted date:

16 November 2018 31 December 2018 1 January 2019

Please cite this article as: Yuan Jiao, Haiyun Sun, Yanchun Jia, Yang Liu, Yifang Gao, Ming Xian, Shaomin Shuang, Chuan Dong , Functionalized fluorescent carbon nanoparticles for sensitively targeted of folate-receptor-positive cancer cells. Microc (2018), https://doi.org/10.1016/j.microc.2019.01.003

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ACCEPTED MANUSCRIPT Functionalized fluorescent carbon nanoparticles for sensitively targeted of folate-receptor-positive cancer cells Yuan Jiao, a Haiyun Sun, a Yanchun Jia, a Yang Liu, a Yifang Gao, a Ming Xian, a,b Shaomin Shuang, a Chuan Dong, a* a

Institute of Environmental Science, and School of Chemistry and Chemical

Department of Chemistry, Washington State University, Pullman, WA, 99164, USA.

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b

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Engineering, Shanxi University, Taiyuan 030006, China

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*: Corresponding author: Chuan Dong

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Email address: [email protected] Tel: +86-351-7018613

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Fax: +86-351-7018613 Abstract

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The folic acid-functionalized fluorescent carbon dots (FA-CDs) was synthesized via the assembly of FA to the surface of CDs. A facile hydrothermal method with proline and ethylenediamine as precursors was used to fabricate CDs. The as-prepared

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CDs possessed active amino groups where the CDs could be further engineered for

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the conjugation with FA. The uptake of the as-synthesized FA-CDs by FR positive MCF-7 cells (FR++) and HepG-2 cells (FR+) via receptor-mediated endocytosis was demonstrated by confocal laser scanning microscopy, which is further verified by a comparative study with FR-negative PC-12 cells (FR-). The bright fluorescence can be observed in FR positive MCF-7 cells while negligible fluorescence was observed in PC-12 cells with low-expressed FR, demonstrating that FA-CDs could accurately identify FR-positive cancer cells from normal cells. The FA-CDs shared favorable 1

ACCEPTED MANUSCRIPT biocompatibility, excellent optical properties and ultra-low toxicity etc. Holding these superior properties, the FA-CDs was implemented as a highly effective platform for biological labeling and imaging, which may provide a innovative vision for cancer diagnosis and succeeding personalized therapy.

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Keywords: Carbon dots; Folic acid-functionalized; Targeted bioimaging; Cancer cells

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1 Introduction

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Cancer remains a major public health issue worldwide [1], which has become one of the leading cause of mortality with its high recurring and death rates. Early

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diagnosis is of great significance for precancerous lesions, prognosis, and treatment of

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carcinomas [2]. The effectiveness of the cancer therapy platform has always been focused on reducing and eliminating tumours without damaging healthy tissues [3].

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Therefore, a distinct function to target cancer cells with limited effect on healthy

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tissues is the key strategy for the success of cancer therapy. In solid tumors, cancerous cells are able to form a unique permissive milieu, so-called tumor microenvironment

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(TME), in which cancer cells overexpress certain receptors and tumor-associated

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surface antigens[4, 5]. Thus, the corresponding ligands or antibodies can be utilized to improve their active identification and targeting ability to tumors, which can decrease the side effects as expected [6]. Folate acid (FA) has been recognized as one of the most important ligands for specific labeling of cancer cells owing to the over-expression of folate receptors on the membrane of cancer cells [7, 8]. Compared with other targeting molecules, folate has many advantages including relatively small molecular weight [9, 10], 2

ACCEPTED MANUSCRIPT non-immunogenicity [11-14], better stability [15], higher affinity to receptors [16] and easier modification of functional groups [17, 18] etc. Recently, numerous nanomaterials have been modified with FA for cancer’s targeting imaging, diagnosis, and therapy purposes. The commonly used nanomaterials include gold nanoparticles

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[19-21], silica nanoparticles [22, 23], quantum dots [24, 25], silver nanoparticles

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[26-28] and magnetic-fluorescent nanoparticles [29] etc. However, some of these

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functionalized nanomaterials are expensive and with large diameters [30] or potentially toxic effects [31] etc. Thus, the development of non-cytotoxic,

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cost-effective and excellent luminescence nanomaterials for targeted fluorescence

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imaging of cancer cells are still of important concern.

As a new type of photoluminescent (PL) nanomaterials, carbon dots (CDs) have

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been utilized advantageously in fluorescent probes, bioimaging [32] and drug delivery

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[33] due to their excellent optical properties [34, 35], high fluorescent stability [36], good biocompatibility [37] and high quantum yield [38, 39] etc. Among these

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applications, CDs have been extensively employed as fluorescent markers for

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intracellular imaging. However, the construction of carbon dots for targeted imaging is rarely reported. Ma et al. presented the first report about FA-conjugated luminescence CDs for targeting and detecting cancer cells, which open a new avenues for targeted fluorescence imaging of cancer cells [30]. Consecutively, Lei et al. developed a turn-on fluorescent nanoprobe through non-covalent conjugation of folic acid to polyethyleneimine modified CDs and demonstrated their utilization in targeted imaging of cancer cells [31]. In addition, there are some other literatures reported the 3

ACCEPTED MANUSCRIPT constructed FA functionalized CDs as a high-performance platform for accurately recognizing special cancer cells [40, 41]. Significant efforts of these pioneers have had a positive impact on the development of CDs as a diagnostic platform for cancer imaging. However, the quantum yields of fluorescent CDs immobilization with folate

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are relatively low. Thus, the development of targeted fluorescence CDs with excellent

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luminescence property becomes extremely critical to extend the utilization of CDs in

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targeted bioimaging.

Herein, we fabricated blue fluorescent CDs through facile one-step hydrothermal

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approach using proline and ethylenediamine as the carbon source and nitrogen source,

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respectively. The prepared CDs present a high quantum yield of 17.3% and rich in active amino groups on the surface which governed covalent conjugation with FA.

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The FA-CDs shared some exceptional properties such as favorable biocompatibility,

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excellent reversibility, and non-immunogenicity. Holding these superior properties, the FA-CDs can recognize the FR-positive MCF-7 cells, which is further confirmed

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by a comparative study with low-expressed FR PC-12 cells. The as-prepared FA-CDs

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can implement as a bioimaging platform for efficient identification of FR-positive cancer cells from normal cells, which may provide a new perspective for cancer diagnosis and therapy. 2 Experimental 2.1 Preparation of fluorescent CDs The fluorescent CDs were prepared via one-step hydrothermal strategy using proline as carbon source and ethylenediamine as nitrogen source，respectively. Firstly, 4

ACCEPTED MANUSCRIPT 0.1151 g of proline was dissolved in 10 mL of distilled water, 34 μL of EDA was added continuously for the functionalization of the carbon materials. The mixture solution was transferred to a 50 mL Teflon-lined stainless autoclave and heated at 180℃ for 5 h. The mixture was automatically cooled to room temperature. The

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obtained pale-yellow suspensions were freeze-dried to afford a powder.

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2.2 Conjugation of CDs with FA

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The FA-CDs was synthesized based on the known procedure [30]. In brief, 0.0100 g FA was dissolved in 6 mL PBS buffer (0.01M, pH=7.4). Then, 0.0130 g EDC and

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0.0078 g NHS were successively added, then the solution was stirred at room

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temperature overnight. 1 mL of CDs (22 mg·mL-1) dispersion was added to the resulting solution and continue the reaction for 12 h. The obtained solution was

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of 500–1000 Da for 48 hours.

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dialyzed in PBS buffer (0.01M, pH=7.4) through a dialysis membrane with MWCO

3 Results and discussion

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3.1 Preparation and characterization of fluorescent CDs

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A variety of factors affecting the preparation of CDs need to be optimized. The molar ratio of the initial raw materials, the reaction time and the heating temperature were studied in detail. As shown in Fig.S.1a and Fig.S.1c, when the material molar ratio of proline to ethylenediamine is 1:2 and the reaction temperature is 180 °C, the synthesized CDs holding exceptional fluorescence properties. The influence of reaction time on the fluorescence of CDs was demonstrated in Fig.S.1b, the fluorescence intensity of CDs enhanced gradually with the increase of reaction time. 5

ACCEPTED MANUSCRIPT When the reaction time reaches 5 hours, the fluorescence intensity has no significantly increased. Meanwhile, as the reaction time increases, the carbon powder looked much darker. Prolonged continuous heating may lead to excessive carbonization and the formation of by-products. Therefore, we choose 5h as the

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optimum time.

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The morphology of the as-prepared CDs was investigated by transmission electron

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microscopy (TEM). As illustrated in Fig.1a and Fig.1b, the CDs are nearly spherical and well dispersed with an average diameter of 3.13±0.34 nm. The inset of HRTEM

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in Fig.1a depict that the lattice spacing of CDs was 0.21 nm, which is in line with the

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facet (100) of graphene.

The surface functional states and components of the CDs were investigated by

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X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR). The

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survey spectrum in Fig. 1c reveals three prominent peaks of C1s at 284 eV, N1s at 400 eV and O1s at 531 eV [42-44]. The typical C1s spectrum (Fig. 1d) exhibits four distinct

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peaks located at 283.9, 285.0, 286.9 and 288.0 eV, which correspond to C- C/C-H,

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C-N, C-O and C=O groups, respectively. The N1s spectrum (Fig.1e) can be deconvoluted into three components at 398.2, 400.1 and 402 eV, attributed to C-N, N-H and pyrrolic N, respectively. The O1s spectrum (Fig.S.2) exhibits two peaks at 530.0 and 531.2eV, representing -C=O and C-OH/C-O-C. The FTIR spectrum was shown in Fig.1f, the absorption bands 3440 and 2995 cm-1 represent the typical stretching vibrations of O-H/N-H and C-H, respectively. Simultaneously, the absorption band at 1626 cm-1 is attributed to the stretching vibration of C=O. Several 6

ACCEPTED MANUSCRIPT sharp peaks at 1511, 1411 and 1031 cm-1 correspond to the vibrations of N-H, C=C and C-N, respectively. The analytical results of XPS and FTIR demonstrate that there existed effective -NH2 and -COOH groups on the surface of as-synthesized CDs, which can be directly conjugated with FA without further modification.

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As shown in fig.2a, the CDs exhibit maximum emission peak centered at 405 nm

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when excitation at 320 nm. The inset of Fig.2a demonstrates that the pale-yellow CDs

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solution emitted intense blue luminescence under UV light at 365 nm. The excitation-wavelength-dependent PL behavior of CDs was described in Fig.2b. The

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PL spectra are batho-chromically red-shift with the increase of excitation wavelengths

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in the range of 280 to 420 nm. Meanwhile, the intensity of the emission peak first increases and then gradually decreases. It reaches a maximum at an excitation of

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nm,

indicating

that

the

prepared

CDs

has

an

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wavelength

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excitation-wavelength-dependent PL behavior. Furthermore, using quinine sulfate as a reference (QY = 58% in 0.1M H2SO4, λex= 350 nm), the relative quantum yield (QY)

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of the fluorescent CDs was measured to be 17.3% at λex/λem of 350/370–680nm.

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3.2 Characterization of FA-CDs The CDs possessed active amino groups where the CDs could be covalently bound to FA via a covalent bond reaction. As shown in Fig.3a, the average diameter of FA-CDs is 3.38 ± 0.49 nm, which slightly larger than CDs after conjugated with folic acid. The Zeta potential of CDs and FA-CDs were -11.1 and -22.6 mV (Fig.3b), suggesting the surface of the carbon dot has been successfully covered by FA with negatively charged. According to UV–vis absorption spectra, the blue line in Fig.3c 7

ACCEPTED MANUSCRIPT existed two dominant absorbance peaks located at 261 and 307 nm, we propose that the strong absorption peak of CDs at 261nm correspond to the π-π* transition of the aromatic sp2. The absorption peak at 307 nm is attributed to the n→π* transition of the C-N bond. As shown in black line in Fig.3c, the characteristic absorption peaks of

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FA at 282 nm and 361 nm is emerged in the UV–vis absorption spectra of FA-CDs,

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demonstrating that FA has been connected to the surface of CDs successfully. This

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result is further confirmed by FTIR (Fig.3d), the absorption peak at 1691 cm-1 is attributed to C=O stretching of –COOH on folic acid. Meanwhile, other absorption

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bands at 1604 and 1480 cm-1 are attributed to the phenyl and pterin rings. All these

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characteristic peaks of FA are reflected in the FA-CDs, demonstrating FA has fixed on the surface of the CDs successfully.

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3.3 Photostabilities of FA-CDs

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The photostability of FA-CDs was verified by measuring the fluorescent intensity under continuous irradiation with a xenon arc lamp for 60 min (Fig.S3), the

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fluorescence intensity was continuously monitored and recorded at λex/λem

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=320/405nm. The PL intensity of FA-CDs has no obvious change in comparison with CDs, which demonstrated that the FA-CDs possess excellent photostability and can be utilized to bioimaging applications. Fig.S4 shows that the CDs and FA-CDs maintains a nearly constant fluorescence intensity in the pH region of 3 to 11. The effects of different concentration of NaCl on the PL stability of CDs and FA-CDs were described in Fig.S5. The PL intensity and spectral feature of FA-CDs do not change much at different concentrations of NaCl, 8

ACCEPTED MANUSCRIPT which is beneficial to the practical use of FA-CDs under physical salt conditions. Moreover, the influence of commonly cell substances on the fluorescence of CDs and FA-CDs was described in Fig.S6. These substances show no significant interference on the fluorescence intensity of FA-CDs, which certificate that FA-CDs can be a good

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candidate for the biological imaging.

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3.4 Targeted imaging of FA-CDs to FR-positive cancer cells

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The cytotoxicity of FA-CDs was determined by a standard MTT assay with FR+ HepG-2 cells. As shown in Fig.S7, the cell viability remained at approximately 87%

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after incubation of HepG-2 with FA-CDs at concentrations ranging from 0 to 200

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μg·mL-1, demonstrating low toxicity of the FA-CDs and inferring their potential use in the biological system.

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To assess the feasibility of the prepared FA-CDs for recognition cancer cells,

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FR-positive MCF-7 cells (FR++), HepG2 cells (FR+) and FR-negative PC-12 cells (FR-) were selected for comparison experiment of cell targeting imaging. As

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demonstrated in Fig.4, It is obvious that MCF-7 cells display bright blue fluorescence

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after incubation with the FA-CDs for 2h. However, there only weak fluorescence is shown in PC-12 cells with low-expressed FR. The fluorescence intensity of HepG2 cells is in an intermediate order among the three cell lines. All these results indicate that FA-CDs can discrimination of FR-positive cancer cells from normal cells via receptor-mediated endocytosis, which demonstrated that FA-CDs are a good candidate for targeted biological imaging of cancer cells. Demonstration results have been described in some representative work[45-47], which conjugated magnetic 9

ACCEPTED MANUSCRIPT nanoparticles (MNPs) with folic acid (FA) and loaded anticancer chemotherapeutics for targeted imaging and therapy of cancer. Consecutively, some developed researches have concentrated on designing targeted substance-conjugated metal quantum dots [48, 49] or chitosan micellar nanoparticles[50] for multifunctional fluorescence

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targeted imaging and cancer diagnosis. All these reports showed a significant

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instrument for developing new targeted material and further demonstrating our works

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reliability for constructed folic acid-functionalized fluorescent carbon dots as a highly effective platform for biological labeling and imaging.

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Moreover, in order to investigate that whether FA-CDs entrance into the cells is

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via receptor-mediated endocytosis, a competition experiment was conducted where MCF-7 cells were saturated with an excess of free FA firstly and then incubated with

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FA-CDs under the same culture conditions. The result shows that there is no

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fluorescence signal in the MCF-7 cells (Fig.S8), which may attribute to the specific binding between FA and folic acid receptor of cancer cells. Meanwhile, the result also

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endocytosis.

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proved that FA-CDs can target recognition of cancer cells via receptor-mediated

4 Conclusion

In summary, we developed an efficient approach for targeted bioimaging of cancer cells through covalent modification of FA on the surface of CDs. The prepared FA-CDs shared excellent optical properties, favorable biocompatibility, and ultra-low toxicity etc. Holding these exceptional properties, the FA-CDs can be implemented as a good candidate for a biological system. The comparative experiments of MCF-7 10

ACCEPTED MANUSCRIPT cells (FR++), HepG2 cells (FR+) and (FR-) PC-12 cells demonstrated that FA-CDs can accurately identify FR-positive cancer

cells

from

normal

cells

via

receptor-mediated endocytosis. The FA-CDs can serve as a promising nanomaterial to construct a high-performance platform for biological imaging of cancer cells, which

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have great potential significance for cancer diagnostic studies.

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Conflicts of interest

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There are no conflicts to declare. Acknowledgments

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This work was supported by the National Natural Science Foundation of China

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(No.21705101, 21475080 and 21575084) and the Hundred Talent Programme of

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Notes and references

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Shanxi Province.

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ACCEPTED MANUSCRIPT Figure captions Scheme.1 Illustration of the synthetic of the FA-CDs and the FA-CDs based fluorescence bioimaging platform for the targeted imaging of FR+ cancer cells. Fig.1 (a) TEM image and HRTEM image (top inset) of as-synthesised CDs; (b)

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Particle size distribution of CDs; (c) XPS survey scan of CDs; (d) C1s XPS and (e) N1s

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XPS and of CDs; (f) FTIR spectrum of CDs.

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Fig.2 (a) Fluorescence spectra of CDs; Inset: Photographs of CDs under visible (left) and UV light (right) at 365 nm; (b) Excitation–emission matrix for CDs at different

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excitation wavelengths.

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Fig.3 (a) TEM image and particle size distribution (top inset) of FA-CDs; (b) Zeta potential distributions of the CDs (top) and FA-CDs (down); (c) UV-vis absorption

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CDs and (c) FA.

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spectra of (a) FA-CDs, (b) CDs and (c) FA; (d) FTIR spectrum of (a) FA-CDs, (b)

Fig.4 Confocal images of the MCF-7 cell, HepG 2 cell, and PC-12 cell after incubated

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with FA-CDs.

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Scheme.1

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Fig. 1

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ACCEPTED MANUSCRIPT Highlights 1. The fluorescent CDs were facilely prepared by hydrothermal method and present a high quantum yield of 17.3%, which is advantageous in bioimaging applications.

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2. The fluorescent CDs possessed active amino groups where the CDs could be

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engineered for the conjugation with FA directly without further modification.

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3. Outstanding performance of the FA-CDs with favorable biocompatibility, high photostability and ultra-low toxicity, which can implemented as a bioimaging

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platform for efficient identification of FR-positive cancer cells from normal cells.

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4. The FA-CDs can implemented as a highly effective platform for biological labeling and imaging, which may provide a new tool for cancer diagnosis and

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succeeding personalized therapy.

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