Graphene quantum dots‐based targeted nanoprobes detecting drug delivery, imaging, and enhanced chemotherapy of nasopharyngeal carcinoma

Abstract One of the main clinical treatments for advanced nasopharyngeal carcinoma is chemotherapy, but systemic administration can cause serious adverse reactions. New type of nanomaterial which can actively targeting, imaging, and treating nasopharyngeal carcinoma at the same time to enhance the effect of chemotherapy, meanwhile monitoring the intracellular drug release process at the level of single cancer cell was urgently needed. GE11, an EGFR antagonist peptide, was used to target nasopharyngeal carcinoma which has positive expression of EGFR on its nucleus. GE11‐modified graphene quantum dots (GQDs@GE11) were used as drug carriers for clinical chemotherapeutics cisplatin (CDDP) and doxorubicin (DOX). The emission spectrum of GQDs (460 nm) and the excitation spectrum of DOX (470 nm) have a good overlap, thus the transfer and release process of DOX can be sensitively detected by the fluorescence resonance energy transfer (FRET). CDDP was used to enhance the chemotherapy effect of nanoprobe, and the loading amount of DOX and CDDP on GQDs@GE11 nanoprobe were up to 67 and 50 mg/g, respectively. In vivo experiments have confirmed that GQDs@GE11/CDDP/DOX nanoprobe can be enriched to tumor site through specific targeting effect, and significantly inhibit tumor cell proliferation. This new type of targeted therapy fluorescent probe provides new ideas for the study of drug release process and the treatment of nasopharyngeal carcinoma.


| INTRODUCTION
Nasopharyngeal carcinoma is a common malignant tumor of the head and neck, [1][2][3] it has a deep focus and the clinical manifestations of nasopharyngeal carcinoma are not obvious. Therefore, once diagnosed, nasopharyngeal carcinoma usually reaches an intermediate or advanced stage. High-intensity radiotherapy and chemotherapy can effectively control and kill tumor cells, 2,4 but due to lack of selectivity, normal tissues are also severely damaged, leading to many complications, which greatly affect the treatment effect and plan. 5,6 Therefore, there is an urgent need to develop a new treatment method with minimal side effects at low doses and the best antitumor effect for the treatment of nasopharyngeal carcinoma. 7 Drug delivery platforms based on nano-drugs have been widely studied and applied in the treatment of nasopharyngeal carcinoma. 1,8,9 Among inorganic nanocarriers, graphene derivatives such as graphene oxide (GO) and graphene quantum dots (GQDs) are less toxic compared with other inorganic nanomaterials containing heavy metal ions, 10 and have been used for tumor therapy research for many years. 11,12 The high drug loading capacity, excellent physiological stability and biocompatibility, strong photoluminescence, and ease of use of GQDs have also been proven, which makes them a promising nanocarrier for the delivery of anticancer drugs. [13][14][15][16] The importance of targeting delivery has been emphasized due to the systemic toxicity caused by nonspecific administration. Specific targeting to cancer tissues or cancer cells can significantly reduce the side effects of chemotherapy drugs. 17 Nasopharyngeal carcinoma has positive expression of EGFR on the tumor cells nucleus. 18 GE11, an EGFR antagonist peptide, is a potential targeted modification peptide for nasopharyngeal carcinoma. 19 Fluorescence resonance energy transfer (FRET) is an interesting technique which can transmit photo-excitation energy from a donor fluorophore to an acceptor fluorophore. 20 FRET-based fluorescence quenching and reproduction can be used for biological detection or monitoring of some dynamic processes, such as the delivery process of drugs. 21 In recent years, people have studied the potential applications of GQDs in bioimaging and drug delivery. [21][22][23] GQDs have been applied in biosensing owing to its incredible fluorescence characteristics (such as high brightness, long fluorescence lifetime, and photostability). 20 It can be excited by a short-wavelength, like ultra-violent, and the emission wavelength is 460 nm, which is very close to the excitation wavelength of doxorubicin (DOX, 450 nm), thus making it possible to construct fluorescent probes based on FRET.
At the same time, DOX is also one of the most famous anticancer drugs in clinical practice. GQDs were also reported to have a high drug loading rate for DOX. 17,18 However, single-drug loading platforms usually require large doses of drugs to achieve the desired therapeutic effect, and excessive drugs can also cause systemic side effects. Therefore, the development of intelligent therapeutic nanoplatform with multiple synergistic antitumor drugs will be of great significance. 24,25 Cisplatin (CDDP) is a cell cycle nonspecific drug and has therapeutic effects on many tumors, including nasopharyngeal carcinoma. 26 Nanoplatform combine DOX and CDDP will have enhanced therapeutic effects and low side effects at the same time.
Here, we proposed a noncytotoxic, targeted, GQD-based nanoprobe with dual functions of drug delivery and cell imaging.
GQDs were modified with the targeting polypeptide GE11 to target EGFR which highly expressed on the nucleus of nasopharyngeal carcinoma. 17,[27][28][29] GQDs nanoprobe had good biocompatibility and high drug loading capacity that would be a potential drug carrier for cancer treatment. DOX and CDDP, the two most widely used chemotherapeutics in clinical practice, 30,31 were loaded in the GQDs@GE11 nanoprobe to construct the anticancer nanoprobe GQDs@GE11/DOX/CDDP. Due to the inherent fluorescence imaging capabilities of GQDs and DOX, we also built a FRET system based on GQDs and DOX to study the cellular delivery and release of drugs. The co-delivery system showed excellent DOX and CDDP delivery capabilities to CNE-2 cells and tumors, and its combined antitumor effect was much better than using only DOX or CDDP treatment, indicating that it had potential applications in nasopharyngeal carcinoma therapy and visualization of intracellular drug uptake behavior (Scheme 1).

| Synthesis of GO
GO was synthesized through modified Hummer's method. 32 The graphite powder was pre-oxidated by the following process:

| Synthesis of GQDs
GQDs was obtained by ultrasonic peeling. 33 GO was dispersed in N, N-dimethylformamide (DMF) at a concentration of 20 mg/ml, then the mixture was sonicated for 30 min (120 W, 100 kHz). After that, the mixed solution was transferred to a 40 ml autoclave lined with Teflon and heated at 200 C for 4 h. Then the container was cooled to 25 C with water and the black precipitate was collected, washed with water, and resuspended in PBS for later use.

| Synthesis of GQDs@GE11
The coupling of GQDs and GE11 was achieved by amide reaction.
Specifically, 24 mg of EDC and 24 mg of NHS were added to 3 ml of GQDs solution (1 mg/ml) and stirred for 4 h. Then, 10 mg of GE11 was added dropwise to the above solution and reacted at room temperature overnight. Finally, the mixture was dialyzed against deionized water (MWCO = 500 Da) for 72 h to remove impurities to obtain GQDs@GE11 solution.

| Loading of CDDP and DOX on GQDs@GE11
For the preparation of DOX and CDDP co-loaded complexes, DOX was firstly added to GQDs solution, and then CDDP was mixed. In short, 5 mg of DOXÁHCl and 10 mg of GQDs@GE11 were dissolved in distilled water, and then 5 μl of triethylamine solution was added to the mixture and the reaction was stirred for 2 h to neutralize hydrochloric acid. Finally, 2 ml of cisplatin (2 mg/ml, DMSO) solution was added to the mixed system, and the reaction was stirred overnight. After that, the solution was dialyzed and lyophilized to obtain the GQDs@GE11/CDDP/DOX complex. The loads of DOX and CDDP in the GQDs@GE11/CDDP/DOX complex were determined by ultraviolet-visible spectrophotometry and high-performance liquid chromatography, respectively.

| Characterization of nanoprobe
The shape, size, and morphology of the synthesized nanoprobe were

| Cytotoxicity of GQDs@GE11
The cytotoxicity of GQDs and GQDs@GE11 was measured on CNE-2 cells using the CCK-8 assay. Briefly, the CNE-2 cells were seeded in 96-well plates with cell density of 5 Â 10 3 cells per well and cultured for 12 h. The cells were then incubated with fresh cell medium containing GQDs or GQDs@GE11 with concentrations ranging from 0 to 100 μg/ml and incubated for 24 h. After treatment, cells were washed with PBS and added with fresh cell medium containing 10% CCK-8 to all wells. Finally, the absorbance at 450 nm was tested by a microplate reader (Thermo Scientific, USA). CNE-2 cells incubated with RPMI 1640 medium were used as control groups.

| In vitro cellular fluorescence imaging
The cellular uptake of GQDs@GE11/DOX/CDDP by CNE-2 cells was quantified using CLSM measurements. In detail, CNE-2 cells were seeded in 2 cm confocal microscopy dish at a density of 2 Â

| In vitro cell proliferation inhibition
In order to measure the effect of co-administration of GQDs@GE11/ DOX/CDDP on CNE-2 cell survival rate, CCK-8 assay was performed.
Briefly, CNE-2 cells were planted into 96-well plate at a density of 1 Â 10 4 cell per well. After overnight, the cells were exposed to GQDs@GE11/DOX/CDDP with drug concentrations for 24 h. Then, 100 μl of CCK-8 solution (10%) was added to each well and incubated at 37 C in 5% CO 2 atmosphere for 1 h. Next, absorbance of each sample was measured at 450 nm using a microplate reader.  Each experimental group included three mice and injected via tail vein every 2 days. The tumor volume was tested with an electronic caliper and calculated as: 1/2 Â shortest diameter 2 Â longest diameter. At the same time, the weight of the mice was recorded. After seven treatments, the tumors were collected and weighed.

| Histologic and immunohistochemical analysis
The mice of all groups were killed after the treatment. The tumors were removed, fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and cryosectioned into 4 μm slices. Then, the sections were stained with hematoxylin and eosin (H&E). For immunohistochemical analysis, the level of tumor apoptosis was evaluated by the terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assay. In addition, the expression of Ki67 in tumor sections was also detected.

| In vivo fluorescence imaging
In order to explore GQDs@GE11/DOX/CDDP in vivo fluorescence imaging and tumor enrichment. The 100 μl GQDs@GE11/DOX/CDDP (DOX:2 mg/kg) was intravenously injected, and the fluorescence images of the mice were recorded at 0.5, 1, 3, 5, and 8 h intervals using the in vivo FX Pro device (Brooker, USA). After 8 h, the mice were sacrificed and the fluorescence images of the main organs (heart, liver, spleen, lung, and kidney) and tumor tissue were captured. Take GQDs/ DOX/CDDP complex without GE11 targeting peptide as control.

| In vitro hemolysis assay
The hemolysis rate was measured according to the previously reported method. In short, 2 ml of GQDs@GE11solution (0.01, 0.1, and 0.2 mg/ml) was added to 0.2 ml of 16% red blood cell (RBC) suspension and incubated for different time. After that, the hemolysis rate at different time points was tested at 540 nm by a microplate reader. The distilled water and PBS were used as positive control group and negative control group, respectively. Hemolysis ratio was calculated according to the following formula: where M 0 is the absorbance of the test sample, M 1 is the absorbance of the negative control, and M 2 is the absorbance of the positive control.

| Histological analysis
After the mice were euthanized, the heart, liver, spleen, lung, kidney, and other major organs were taken and immersed in tissue fixative.
The sections were stained with H&E and imaged with fluorescence microscope.

| Blood chemistry assay
After 14 days treatment, the mouse blood was collected, centrifuged at 3000 rpm for 5 min, and the serum in the supernatant was collected. Then detect activated partial thromboplastin time (APTT) and prothrombin time (PT).

| Statistical analysis
All data were represented as mean ± SD and each experiment was  To further explore the energy resonance transfer system, a specific concentration of GQDs@GE11 (1 mg/ml) was hatched with DOX at a series of increasing molar ratios. When DOX was loaded onto the GQD surface through pi-pi superposition interaction, the fluorescence signal of GQDs was quenched due to the energy transfer from GQDs to DOX. [35][36][37] It was observed that the fluorescence signal of GQDs decreased successively, and the fluorescence signal of DOX continued to increase, indicating that the amount of DOX molecules adsorbed on the GQDs surface was increasing (Figure 1h). In contrast, the DOX emission peak at 580 nm was observed due to the FRET effect.

| Drug release from GQDs@GE11
Due to the layered structure of GQDs, hydrophobic drugs DOX and CDDP could be encapsulated in their layers, resulting in high loading amount of DOX and CDDP as high as 67 and 50 mg/g, respectively.
The GQDs has pH-sensitive drug release property, 38  pH-sensitive drug release property, which is consistent with previous reports. 38

| GE11 targeting ability assay
Efficient uptake by in vivo cells was the key to achieve the biological performance of material design. 39 The synthetic 12-amino acid peptide GE11 (amino acid sequence: Y-H-W-Y-G-Y-T-P-Q-N-V-I) has been proved to be an effective EGFR targeting peptide in vitro and in vivo 40 ; therefore, it is one of the best choices for EGFR-targeted diagnosis and drug delivery system design. 41  FRET channel) was reduced, indicating that most DOX molecules were released from the carrier and accumulate in the nucleus. The results showed that the nanoprobe could sensitively detect the intracellular drug release process at the level of a single cancer cell, which may be an effective tool to detect the drug control release process.

| Cytotoxicity of GQDs@GE11
Low cytotoxicity is the basic requirement for nanoprobe's in vivo application. Here, we conducted an in vitro cytotoxicity assay by coculture CNE-2 cells with different concentration (0, 10, 20, 50, and 100 μg/ml) of GQD and GQD@GE11 nanoprobes, and the cell viability was tested by CCK-8 assay. As shown in Figure 3a, the cell viability of all groups exceeded 90%, indicating that the GQD and GQD@GE11 nanoprobes at all concentrations almost shown no toxicity to CNE-2 cells. These results showed that GQDs and GQDs@GE11 nanoprobes had good cell compatibility.

| Inhibition of cell proliferation
The  Figure 3b). In addition, the cytotoxicity of GQDs@GE11/DOX/CDDP nanoprobe was significantly higher than that of GQDs@GE11/DOX and GQDs@GE11/CDDP nanoprobes, which could be explained by the effect of combination therapy over single drug therapy.
As previous studies reported, the antitumor effects of DOX and CDDP both depend on their ability to interact with DNA. 43

| In vitro apoptosis assessments
Apoptosis was also conducted to evaluate the killing effect of considered to be a key factor for reflecting the safety and side effects of the single formula used. As shown in Figure 4d, the body weight of mice in each group increased, which means GQDs@GE11/CDDP/DOX nanoprobe was safe for mice in the process of tumor treatment in vivo.

| Histologic and immunohistochemical analysis
Through the histopathological analysis of H&E-stained CNE-2 tumor sections, the antitumor efficacy of co-delivery GQDs@GE11/CDDP/ DOX nanoprobe was further evaluated. As shown in Figure 4e, the results highly support the tumor suppression data.

| In vivo fluorescent imaging
To further evaluate the optical imaging and tumor enrichment of GQDs in mice, GQDs@GE11/DOX/CDDP and GQDs/DOX/CDDP were injected into Balb/c nude mice through the tail vein. Figure 5a showed the time-dependent optical image in mice after injection of the material (GQDs is 10 mg/kg dose). After intravenous injection of

| Biocompatibility evaluation
Biocompatibility was a necessary condition for the safe application of materials in nanomedicine. Therefore, we studied the in vivo toxicity of different treatment groups to the main organs and blood of mice.
H&E images of the main organs of the mice after 14 days of different treatments are shown in Figure 6a. There was no obvious damage to the tissue morphology and structure of the organs in each treatment group. Blood chemistry indexes of each group were also detected.
Compared with the PBS control group, the blood parameters of the mice in the treatment group did not change significantly (Figure 6b, c).
To sum up, GQDs@GE11 nanoprobe is safety and necessity for the treatment of nasopharyngeal carcinoma.

| Hemolysis assay in vitro
The interaction between cells and materials first occurs on the cell membrane, 44 thus affecting its structure and function in many ways.
Hemolysis refers to the release of hemoglobin from RBCs, indicating that the integrity of the RBC membrane is disturbed. Therefore, it reflects the interaction of biological materials with RBC membranes. Figure 7a showed the percentage of hemolysis of RBC exposed to different concentrations of GQDs@GE11 and different incubation times. The GQDs@GE11 had no significant hemolysis at the highest 0.2 mg/ml, indicating that the RBC membrane had no detectable interference.

| Morphology of RBCs
In humans, mature RBCs are elastic biconcave disks, lacking a nucleus and most organelles; they are very sensitive to membrane active substances. In this study, the aggregation and morphological changes of RBC by GQDs@GE11 was used to examine by scanning electron