# Research on the Targeted Treatment of Squamous Cell Carcinoma by Nanographene Drug Delivery System

Authors: Xiangyu Zhang; Liwei Guo; Zhenbang Sun; Deming Han; Lihui Zhao
Journal: Advances in Materials Science and Engineering
 (2023)

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## Abstract

In order to find an effective cure for squamous cell carcinoma, we innovatively used nano-graphene oxide as a pharmaceutical delivery system, overcoming resistance to cisplatin-targeted anti-squamous carcinoma. For this purpose, we prepared a nano-graphene oxide using an oxidation method, functionally modified with polyethylene glycol (PEG) and branched polyethyleneimine (BPEI) and loaded with the antitumor drug cisplatin (CDDP), a compound with preliminary anticancer efficacy. Then, anti-human squamous cell carcinoma monoclonal antibody was combined to construct a target graphene nanopharmaceutical system. The administration system was applied to nude mice carrying human squamous cell carcinoma. Through the detection of frozen tissue slices, the anti-squamous cell carcinoma effect and targeting of the graphene nanoloaded system were analyzed. The safety of the pharmaceutical system was confirmed through further experiments. Results showed that the NGO-PEG-BPEI-CDDP-Antibody complex had a significant antitumor effect and was able to enter the nude mice and targeted squamous cell carcinoma cell and effectively kill squamous cell carcinoma cells, thus reducing the use of clinical chemotherapeutic drugs, improving the efficacy and providing a new answer for the treatment of squamous cell carcinoma.


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## Body

## 1. Introduction

Squamous cell carcinoma is a malignant tumor occurring in the skin, appendages, or mucosa. In recent years, malignant tumors of the head and neck have become the sixth most common tumor in the world, among which squamous cell carcinoma of the head and neck is the main tumor, with 650,000 new cases each year and 350,000 deaths [1]. The incidence of squamous cell carcinoma is the second place to that of basal cell carcinoma in skin cancer [2]. It could occur in any normal tissue or in the presence of photokeratosis, mucosal leukoplakia, or burn scar [3]. There are 80,000∼100,000 new cases annually in the United States. Although the treatment of squamous cell carcinoma has made breakthroughs in the past few decades, the mortality rate is still high. Cancer can be almost cured by surgery or radiation therapy in the early stages. However, for advanced squamous cell carcinoma, it is difficult to achieve a cure effect even if the treatment combined with surgery, radiotherapy, and chemotherapy. Therefore, it is urgent to find more sensitive markers and more efficient treatment methods. Although a variety of drugs have made great progress in tumor chemotherapy, many side effects may occur due to the unsatisfactory targeting selection of chemotherapeutic drugs between normal cells and tumor cells [4]. Moreover, most antitumor drugs have poor water solubility and low bioavailability, resulting in a limited therapeutic effect on tumor tissues. In addition, chemotherapy can have serious side effects on other normal tissues and cells. It can not only damage the patient’s physical function but also lead to drug resistance, which seriously affects the efficacy.With the development of nanotechnology, antitumor drugs based on nanoparticles can achieve controlled release and targeted drug delivery by using special materials and surface modification. At the same time, nanotechnology used in tumor immunotherapy can prevent the degradation of drugs, improve the enrichment of drugs in the tumor site, change the distribution and release of drugs, and make them target the stromal cells, tumor cells, and immune cells in the tumor microenvironment for local immune regulation, so as to more effectively treat cancer and prevent systemic toxicity, overcoming the limitations of traditional antitumor drugs that have been widely used. Moreover, active modification of the surface of the carrier can also improve its biological properties [5]. Gupta et al. [6] have synthesized PLGA nanoparticles containing docetaxel, whose toxicity to human oral squamous carcinoma cells was much greater than that of free docetaxel. Wang et al. [7] have found that the uptake of cisplatin-loaded PEG and PLGA/NR7-targeted nanoparticles by oral squamous cell carcinoma cells was stronger than that of nontargeted nanoparticles, and the targeted nanoparticles had higher lethality to cancer cells. These findings suggest that the combination of nanomedicine technology and chemotherapy can significantly enhance the efficacy of drugs and reduce adverse reactions, which could be a new choice for the treatment of squamous cell carcinoma.At present, the materials used for drug carriers are very extensive, including macromolecular carrier systems, particle carrier systems, magnetic drug preparations, and multitargeting preparations. Although targeted drug delivery carriers have obvious advantages in drug therapy and have achieved some results, with the deepening of research, people gradually realize that there are still many problems in targeted drug delivery systems [8]. For example, many targeted drug delivery carriers will enter the blood, reach tissues, organs, or cells under passive, active, physical, or chemical targeting, and further penetrate into cells. In this process, targeted drug delivery carriers will encounter the removal of various physiological barriers, including the reticuloendothelial system. There are also problems such as low cell targeting and low drug treatment concentration.Graphene is a honeycomb carbon structure formed by the sp2 hybrid connection of carbon atoms, which is hexagonal honeycomb layered structure [9]. Graphene oxide (GO) is a derivative of graphene oxide, and its edge and plane contain a large number of oxygen-containing functional groups such as carboxyl, epoxy, and hydroxyl groups, which are active sites for chemical modification [10]. The existence of a large number of oxygen-containing groups makes GO have good water solubility and dispersibility [11]. The GO skeleton is an aromatic ring, which has a large specific surface area and rich functional groups. On the one hand, it can adsorb a large number of anticancer drugs and improve drug loading rate under π-π stacking [12]. On the other hand, proteins, nucleotide fragments, aptamers, and other biological macromolecules can be combined to achieve target recognition and functional modification [13, 14], which is more suitable for biomedical applications. In addition, GO is an exceptional nanomaterial that possesses multiple physical properties critical for biomedical applications. GO exhibits a pH-dependent fluorescence emission in the visible/near-infrared, providing a possibility of molecular imaging and pH-sensing. It is also water soluble and has a substantial platform for functionalization, allowing for the delivery of multiple therapeutics [15]. Wang et al. achieved the purpose of GO biological detection by detecting metals through the bimetallic labeling GO method [16]. The results showed that the thin GO (1∼2 layers) could pass through the vascular endothelium or be excreted through the glomerular filtration barrier by folding, curling, and other deformations. Cisplatin is a commonly used drug for the treatment of head and neck squamous cell carcinoma. However, cisplatin resistance significantly weakens the efficacy of chemotherapy for oral squamous cell carcinoma and even leads to the failure of treatment for oral squamous cell carcinoma [17, 18]. Cisplatin combined with functionalized graphene oxide is expected to possess a good therapeutic and killing effect on squamous cell carcinoma.In this paper, graphene oxide nanoparticles (NGOs) were prepared by the modified chemical oxidation reduction method, and functionalized with polyethylene glycol (PEG) and branched polyethyleneimine (BPEI). After loading the anticancer drug cisplatin (CDDP), a macromolecular targeted nano-drug delivery system was constructed by electrostatic adsorption combined with specific antibodies in serum, and the killing effect on tumor cells was achieved by the specific enrichment of nanomaterials. The modified nano-drug delivery system was characterized by circular dichroism and atomic force microscopy, and the pathological analysis of organs of nude mice bearing human squamous cell carcinoma after administration was carried out. Finally, the targeting property of the nano-drug-loaded composite was confirmed by frozen section, and the safety of the targeted nano-drug-loaded composite was detected by blood routine and biochemical indexes. The NGO-PEG-BPEI-CDDP-Antibody complex constructed by us has the ability to increase tumor tissue permeability and has high permeability and long retention compared to conventional chemotherapy or single drug therapy. In addition, the drug carrier constructed by nano-graphene oxide can exert the combined therapeutic effect of drugs while covalently loading a variety of drugs and safely targeting the transport of drugs CDDP and specific monoclonal antibodies to tumor cells and tumor microenvironment.


## 2. Materials and Methods

### 2.1. Chemicals and Apparatus

Graphene oxide solution (Jilin Yatai Pharmaceutical Co., Ltd.), N-hydroxysuccinimide (Xi’an Kaixin Biotechnology Co., Ltd.), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (Zhengzhou Akem Chemical Co., Ltd.), four-arm amino polyethylene glycol (Shanghai Xibao Biotechnology Co., Ltd.), TEA (0.5 mg/mL) (Shanghai Xinfan Biotechnology Co., Ltd.), branched polyethyleneimine (25 KDa) (Lianshu Biotechnology Co., Ltd.), KBr (spectral level) (Tianjin Bojun Biotechnology Co., Ltd.), RPMI-1640 (10% FBS) (Wuhan Boshide Biotechnology Co., Ltd.), cisplatin (Nanjing Biotechnology Co., Ltd.), tumor-specific antibody SCCA (8H11) (SIGMA Co., Ltd.), Trypsin (Wuhan Boschder Bioengineering Co., Ltd.), penicillin-streptomycin mixture (Wuhan Boschder Bioengineering Co., Ltd.), and 5-week-old SPF healthy Balb/c female nude mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used.The apparatus used are as follows: cell breaker (Suzhou Purification Equipment Co., Ltd.), high-speed centrifuge (Germany Eppendorf Co., Ltd.), vacuum freeze dryer (Olympus Co., Ltd.), magnetic mixer (Suzhou Indillter Instrument Technology Co., Ltd.), pH meter (Foshan Liyi Instrument Technology Co., Ltd.), Fourier transform infrared spectrometer (Tianjin Tianguang New Optical Instrument Technology Co., Ltd.), infrared compactor (American BIO-RAD Co., Ltd.), UV-3600 (Japan Shimadzu Co., Ltd.), circular dichroism spectrometer (British Applied Photophysics Co., Ltd.), and atomic force microscope (Japan Shimadzu Co., Ltd.).


### 2.2. Preparation of NGO by the Chemical Redox Method

1 g flake graphite, 0.75 g NaNO3, and 4.5 g KMnO4 were put into the beaker, and the beaker was placed in an ice water bath. Then 100 Ml concentrated H2SO4 solution was slowly added to the beaker for stirring, keeping the temperature of the reaction solution not higher than 15°C and stirring for 1 h. The heat was kept up to 60°C, stirring was done for 10 h, further heating to 95°C was done, followed by slowly dropping 100 mL distilled water into the beaker and continuing the stirring process for 1 h. Then, after cooling to 50°C, 9 mL of H2O2 with a mass fraction of 5% was poured into the filter cake. The filter cake was filtered while it was heated. Finally, the filter cake was fully washed with 5% hydrochloric acid until there was no SO42− in the filtrate (detected with BaCl2). Graphene oxide (GO) was prepared and dried to obtain solid. 1 g/L GO aqueous solution 100 mL under 570 W ultrasonic for 10 hours was added. The product was taken out every 1 hour and centrifuged at 12,000 r/min for 5 min to remove the large precipitation. After 10-hour ultrasonic processing, GO was broken into NGO and the solid NGO production was obtained by freeze-drying.


### 2.3. Preparation of PEG and BPEI Bifunctionalized GO

#### 2.3.1. The Carboxylation of GO

10 mL graphene oxide solution (mass concentration 2 mg/mL) was added with 1 g chloroacetic acid and 1.2 g sodium hydroxide, and then processed ultrasonically at 400 W for 3 h, and then stirred at room temperature for 24 h. The mixture was centrifuged at 10,000 r/min for 5 min in a high-speed centrifuge, and the supernatant was removed. The graphene precipitation was washed with a large amount of deionized water until the pH value of the washing solution reached about 7. The filtrate was washed and dried in a vacuum freeze dryer to obtain carboxylate graphene oxide.


#### 2.3.2. PEG and BPEI Loading on the GO Surface

10 mg carboxylated graphene oxide was dispersed in water at a concentration of 1 mg/mL. 150 mg NHS was added to the solution to activate the shuttle for 30 min, then 50 mg EDC was added, and 1 mg/mL NaOH was used to adjust the pH value of the solution to make pH = 5.76. Four-arm amino polyethylene glycol (4-Arm-NH2-PEG) with a mass of 100 mg was added and stirred at room temperature for 24 h. The final solution was centrifuged at 10000 g for 10 min on a high-speed centrifuge, and the supernatant was removed. A large amount of deionized water was used to slowly wash the graphene precipitation many times until the pH value of the washing solution reached 7. The solution was dried in a vacuum freeze dryer to obtain polyethylene glycol-modified graphene oxide powder.10 mg GO-PEG was diluted to 1 mg/mL in deionized water. After ultrasonic treatment for 30 min at 400 W, 54.3 mg EDC and 50.6 mg NHS were added. In addition, 5 mg of polymer polyethyleneimine (25 kDa) was added to 1 mL of TEA, which was mixed and added to GO-PEG solution. The mixture was ultrasonically treated at 400 W for 30 min, stirred at room temperature, and reacted for 24 h. Finally, the materials with complete reaction were dialyzed in a 100 kDa dialysis bag for 3 d, and the reactants with multiple reactions were removed. Then, the graphene solution was dried in a vacuum freeze dryer to obtain the polyethyleneimine-modified GO-PEG.


### 2.4. Raman Analysis

The freeze-dried powder of 0.5 mg graphene oxide was put on the sample pad to make the sample in a dark environment. The wavelength generated by the semiconductor laser is 514.5 nm, the microscopic size range is ≤1μm, and the spectral resolution is 1 cm−1. The backscattering method was used at room temperature. The scanning range of detector was 100–2000 cm−1, the step length was 1 cm−1, and the slit width of the monochromator was 24 μm. The phase of nanosized graphite oxide powder was determined, and its average particle size was estimated.


### 2.5. SEM Characterization of Nano-Graphene Oxide

SEM sample preparation: GO and NGO were ultrasonically dissolved in ultrapure water to form solution, dripped on hydrophilic silicon wafer, and dried naturally. The surface morphology and size of nano-graphene oxide were observed under an electron microscope.


### 2.6. FTIR Characterization of Graphene Oxide Nanoparticles

NGO, NGO-PEG, and NGO-PEG-PEI powder 1 mg were mixed with KBr powder 50 mg after drying. After stirring and shaking, the mixture was placed in an agate bowl and continued to grind. The mixture was dried with infrared light. The mixed powder was placed in an infrared tabletting mold and pressed into a transparent sheet structure by an infrared tabletting machine at 5 × 107 Pa. The slice structure was put into the sample holder and detected and analyzed by infrared spectroscopy.


### 2.7. Solubility Analysis of Graphene Oxide Nanoparticles

NGO, NGO-PEG, and NGO-PEG-BPEI (0.5 mg each) were in deionized water, normal saline, and RPMI-1640 (10% FBS) solution, respectively. The samples were centrifuged at 1200 rpm for 10 min at room temperature, and the morphology of NGO, NGO-PEG, and NGO-PEG-BPEI after centrifugation was recorded.


### 2.8. Loading and Load Rate Calculation of Cisplatin

#### 2.8.1. Drawing Standard Curve of Cisplatin

Different concentrations of CDDP solution were prepared with 0.9% saline solution. The absorption peak at 301 nm was measured by UV-3600, and the standard curve was drawn.


#### 2.8.2. Loading and Characterization of Cisplatin

5 mg nGO-PEG-BPEI was dispersed in deionized water according to a concentration of 1 mg/mL, and then 5 mL CDDP (5 mg/mL) was slowly put into it. The mixture was stirred in the dark at 37°C for 24 h, and centrifuged at 12000 r/min for 8 min. The precipitate was nGO-PEG-BPEI-CDDP. 1 mg nGO-PEG-BPEI-CDDP powder was mixed with 50 mg dry KBr powder and analyzed by infrared spectroscopy.


#### 2.8.3. Load Rate Calculation

The supernatant after centrifugation was taken, and the absorbance was measured at 301 nm. According to the following formula, the loading rate of nGO-PEG-BPEI on CDDP was calculated.(1)W1%=ρ0V0−ρ1V1ρ0V0×100%.In formula (1), W1 is the drug loading rate, ρ0 is the initial concentration of CDDP (mg/mL), V0 is the initial volume of CDDP (mL), ρ1 is the concentration of CDDP in the supernatant obtained by centrifugation after loading (mg/mL), and V1 is the volume of the supernatant obtained by centrifugation after loading (mL).


### 2.9. NGO-PEG-BPEI-CDDP Binding to Tumor-Specific Antibodies

NGO-PEG-BPEI-CDDP powder was mixed with 0.9% saline to prepare NGO-PEG-BPEI-CDDP solutions with concentrations of 1.5μg/mL, 1 μg/mL, and 0.5 μg/mL. 1 mL of each solution was added to 10 μL tumor-specific antibody SCCA (8H11) and incubated at room temperature for 2 h.


### 2.10. Characterization of NGO-PEG-BPEI-CDDP-Antibody Circular Dichroism Chromatography

The stereo structure of tumor-specific antibody SCCA (8H11) was determined by circular dichroism (CD). 10μL of 200 μg/mL SCCA (8H11) protein and 90 μL of 1.5 μg/mL NGO-PEG-BPEI-CDDP nanoparticles were incubated at 37°C for 2 h, and then the weak nitrogen was used to blow dry hexafluoro-2-propanol as the experimental group. SCCA (8H11) and NGO-PEG-BPEI-CDDP nanoparticles were used as control group, respectively. The effect of NGO-PEG-BPEI-CDDP nanoparticles on the conformational changes of the tumor-specific antibody SCCA (8H11) was investigated at 200–250 nm.


### 2.11. Cell Culture and Establishment of Tumor-Bearing Nude Mice Model

#### 2.11.1. Cell Culture

KB cell lines were routinely cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/L streptomycin and cultured in a 5% CO2 incubator at 37°C. Cell culture medium was replaced every 24 hours and subculture cells every 72 hours. Cells in the logarithmic growth phase were taken for experiment.


#### 2.11.2. Establishment and Administration of Tumor-Bearing Nude Mice Model

After the cell viability was higher than 90%, about 1 × 107 KB cells were resuspended by 50 μL serum-free RPMI 1640 medium. The centrifuge tube of the cell suspension was sealed and stored in the ice box for a short time to maintain the lowest metabolic level. Then, the cells were sent to the hands of the operating table to cover the hot ice for 1 min. When it approached 37°C, it was absorbed and injected into the left armpit of 5-week-old female nude mice subcutaneously. The tumor size of nude mice was observed daily, and the weight of nude mice was measured until the tumor was about 0.5 cm in diameter.Nude mice bearing tumor were divided into the experimental group and the control group. Nude mice were intravenously injected with 200μL sterile saline and 200 μL sterile graphene nanocarriers at different concentrations. The mice were weighed daily and the tumor diameter was measured after the end of administration (take medicine on time every day for seven days).


### 2.12. Collection of Tumor-Bearing Nude Mice Model Specimens

After 7 days of administration, 0.2 mL whole blood was obtained from each nude mouse by the tail-broken blood collection method, stored in a blood routine tube containing EDTA, and sent to the animal hospital for routine blood and serum biochemical tests. The mice were dissected with a scalpel, and the heart, liver, kidney, and part of tumors (15 × 15 × 2 mm) of nude mice were taken for paraffin sections. The sections were cut into 3-4μm for staining for staining. The morphological changes of the tissue cells in the sections were observed for pathological analysis.


### 2.13. Immunohistochemistry

Some tumors were taken for a frozen section, and immunohistochemistry was performed. The sheep anti-mouse secondary antibody labeled with horseradish peroxidase was used for incubation. The horseradish peroxidase-DAB method was used to detect the monoclonal antibody SCCA1 (note: SCCA1 is a mouse monoclonal antibody against recombinant human SCCA1).


## 2.1. Chemicals and Apparatus

Graphene oxide solution (Jilin Yatai Pharmaceutical Co., Ltd.), N-hydroxysuccinimide (Xi’an Kaixin Biotechnology Co., Ltd.), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (Zhengzhou Akem Chemical Co., Ltd.), four-arm amino polyethylene glycol (Shanghai Xibao Biotechnology Co., Ltd.), TEA (0.5 mg/mL) (Shanghai Xinfan Biotechnology Co., Ltd.), branched polyethyleneimine (25 KDa) (Lianshu Biotechnology Co., Ltd.), KBr (spectral level) (Tianjin Bojun Biotechnology Co., Ltd.), RPMI-1640 (10% FBS) (Wuhan Boshide Biotechnology Co., Ltd.), cisplatin (Nanjing Biotechnology Co., Ltd.), tumor-specific antibody SCCA (8H11) (SIGMA Co., Ltd.), Trypsin (Wuhan Boschder Bioengineering Co., Ltd.), penicillin-streptomycin mixture (Wuhan Boschder Bioengineering Co., Ltd.), and 5-week-old SPF healthy Balb/c female nude mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used.The apparatus used are as follows: cell breaker (Suzhou Purification Equipment Co., Ltd.), high-speed centrifuge (Germany Eppendorf Co., Ltd.), vacuum freeze dryer (Olympus Co., Ltd.), magnetic mixer (Suzhou Indillter Instrument Technology Co., Ltd.), pH meter (Foshan Liyi Instrument Technology Co., Ltd.), Fourier transform infrared spectrometer (Tianjin Tianguang New Optical Instrument Technology Co., Ltd.), infrared compactor (American BIO-RAD Co., Ltd.), UV-3600 (Japan Shimadzu Co., Ltd.), circular dichroism spectrometer (British Applied Photophysics Co., Ltd.), and atomic force microscope (Japan Shimadzu Co., Ltd.).


## 2.2. Preparation of NGO by the Chemical Redox Method

1 g flake graphite, 0.75 g NaNO3, and 4.5 g KMnO4 were put into the beaker, and the beaker was placed in an ice water bath. Then 100 Ml concentrated H2SO4 solution was slowly added to the beaker for stirring, keeping the temperature of the reaction solution not higher than 15°C and stirring for 1 h. The heat was kept up to 60°C, stirring was done for 10 h, further heating to 95°C was done, followed by slowly dropping 100 mL distilled water into the beaker and continuing the stirring process for 1 h. Then, after cooling to 50°C, 9 mL of H2O2 with a mass fraction of 5% was poured into the filter cake. The filter cake was filtered while it was heated. Finally, the filter cake was fully washed with 5% hydrochloric acid until there was no SO42− in the filtrate (detected with BaCl2). Graphene oxide (GO) was prepared and dried to obtain solid. 1 g/L GO aqueous solution 100 mL under 570 W ultrasonic for 10 hours was added. The product was taken out every 1 hour and centrifuged at 12,000 r/min for 5 min to remove the large precipitation. After 10-hour ultrasonic processing, GO was broken into NGO and the solid NGO production was obtained by freeze-drying.


## 2.3. Preparation of PEG and BPEI Bifunctionalized GO

### 2.3.1. The Carboxylation of GO

10 mL graphene oxide solution (mass concentration 2 mg/mL) was added with 1 g chloroacetic acid and 1.2 g sodium hydroxide, and then processed ultrasonically at 400 W for 3 h, and then stirred at room temperature for 24 h. The mixture was centrifuged at 10,000 r/min for 5 min in a high-speed centrifuge, and the supernatant was removed. The graphene precipitation was washed with a large amount of deionized water until the pH value of the washing solution reached about 7. The filtrate was washed and dried in a vacuum freeze dryer to obtain carboxylate graphene oxide.


### 2.3.2. PEG and BPEI Loading on the GO Surface

10 mg carboxylated graphene oxide was dispersed in water at a concentration of 1 mg/mL. 150 mg NHS was added to the solution to activate the shuttle for 30 min, then 50 mg EDC was added, and 1 mg/mL NaOH was used to adjust the pH value of the solution to make pH = 5.76. Four-arm amino polyethylene glycol (4-Arm-NH2-PEG) with a mass of 100 mg was added and stirred at room temperature for 24 h. The final solution was centrifuged at 10000 g for 10 min on a high-speed centrifuge, and the supernatant was removed. A large amount of deionized water was used to slowly wash the graphene precipitation many times until the pH value of the washing solution reached 7. The solution was dried in a vacuum freeze dryer to obtain polyethylene glycol-modified graphene oxide powder.10 mg GO-PEG was diluted to 1 mg/mL in deionized water. After ultrasonic treatment for 30 min at 400 W, 54.3 mg EDC and 50.6 mg NHS were added. In addition, 5 mg of polymer polyethyleneimine (25 kDa) was added to 1 mL of TEA, which was mixed and added to GO-PEG solution. The mixture was ultrasonically treated at 400 W for 30 min, stirred at room temperature, and reacted for 24 h. Finally, the materials with complete reaction were dialyzed in a 100 kDa dialysis bag for 3 d, and the reactants with multiple reactions were removed. Then, the graphene solution was dried in a vacuum freeze dryer to obtain the polyethyleneimine-modified GO-PEG.


## 2.3.1. The Carboxylation of GO

10 mL graphene oxide solution (mass concentration 2 mg/mL) was added with 1 g chloroacetic acid and 1.2 g sodium hydroxide, and then processed ultrasonically at 400 W for 3 h, and then stirred at room temperature for 24 h. The mixture was centrifuged at 10,000 r/min for 5 min in a high-speed centrifuge, and the supernatant was removed. The graphene precipitation was washed with a large amount of deionized water until the pH value of the washing solution reached about 7. The filtrate was washed and dried in a vacuum freeze dryer to obtain carboxylate graphene oxide.


## 2.3.2. PEG and BPEI Loading on the GO Surface

10 mg carboxylated graphene oxide was dispersed in water at a concentration of 1 mg/mL. 150 mg NHS was added to the solution to activate the shuttle for 30 min, then 50 mg EDC was added, and 1 mg/mL NaOH was used to adjust the pH value of the solution to make pH = 5.76. Four-arm amino polyethylene glycol (4-Arm-NH2-PEG) with a mass of 100 mg was added and stirred at room temperature for 24 h. The final solution was centrifuged at 10000 g for 10 min on a high-speed centrifuge, and the supernatant was removed. A large amount of deionized water was used to slowly wash the graphene precipitation many times until the pH value of the washing solution reached 7. The solution was dried in a vacuum freeze dryer to obtain polyethylene glycol-modified graphene oxide powder.10 mg GO-PEG was diluted to 1 mg/mL in deionized water. After ultrasonic treatment for 30 min at 400 W, 54.3 mg EDC and 50.6 mg NHS were added. In addition, 5 mg of polymer polyethyleneimine (25 kDa) was added to 1 mL of TEA, which was mixed and added to GO-PEG solution. The mixture was ultrasonically treated at 400 W for 30 min, stirred at room temperature, and reacted for 24 h. Finally, the materials with complete reaction were dialyzed in a 100 kDa dialysis bag for 3 d, and the reactants with multiple reactions were removed. Then, the graphene solution was dried in a vacuum freeze dryer to obtain the polyethyleneimine-modified GO-PEG.


## 2.4. Raman Analysis

The freeze-dried powder of 0.5 mg graphene oxide was put on the sample pad to make the sample in a dark environment. The wavelength generated by the semiconductor laser is 514.5 nm, the microscopic size range is ≤1μm, and the spectral resolution is 1 cm−1. The backscattering method was used at room temperature. The scanning range of detector was 100–2000 cm−1, the step length was 1 cm−1, and the slit width of the monochromator was 24 μm. The phase of nanosized graphite oxide powder was determined, and its average particle size was estimated.


## 2.5. SEM Characterization of Nano-Graphene Oxide

SEM sample preparation: GO and NGO were ultrasonically dissolved in ultrapure water to form solution, dripped on hydrophilic silicon wafer, and dried naturally. The surface morphology and size of nano-graphene oxide were observed under an electron microscope.


## 2.6. FTIR Characterization of Graphene Oxide Nanoparticles

NGO, NGO-PEG, and NGO-PEG-PEI powder 1 mg were mixed with KBr powder 50 mg after drying. After stirring and shaking, the mixture was placed in an agate bowl and continued to grind. The mixture was dried with infrared light. The mixed powder was placed in an infrared tabletting mold and pressed into a transparent sheet structure by an infrared tabletting machine at 5 × 107 Pa. The slice structure was put into the sample holder and detected and analyzed by infrared spectroscopy.


## 2.7. Solubility Analysis of Graphene Oxide Nanoparticles

NGO, NGO-PEG, and NGO-PEG-BPEI (0.5 mg each) were in deionized water, normal saline, and RPMI-1640 (10% FBS) solution, respectively. The samples were centrifuged at 1200 rpm for 10 min at room temperature, and the morphology of NGO, NGO-PEG, and NGO-PEG-BPEI after centrifugation was recorded.


## 2.8. Loading and Load Rate Calculation of Cisplatin

### 2.8.1. Drawing Standard Curve of Cisplatin

Different concentrations of CDDP solution were prepared with 0.9% saline solution. The absorption peak at 301 nm was measured by UV-3600, and the standard curve was drawn.


### 2.8.2. Loading and Characterization of Cisplatin

5 mg nGO-PEG-BPEI was dispersed in deionized water according to a concentration of 1 mg/mL, and then 5 mL CDDP (5 mg/mL) was slowly put into it. The mixture was stirred in the dark at 37°C for 24 h, and centrifuged at 12000 r/min for 8 min. The precipitate was nGO-PEG-BPEI-CDDP. 1 mg nGO-PEG-BPEI-CDDP powder was mixed with 50 mg dry KBr powder and analyzed by infrared spectroscopy.


### 2.8.3. Load Rate Calculation

The supernatant after centrifugation was taken, and the absorbance was measured at 301 nm. According to the following formula, the loading rate of nGO-PEG-BPEI on CDDP was calculated.(1)W1%=ρ0V0−ρ1V1ρ0V0×100%.In formula (1), W1 is the drug loading rate, ρ0 is the initial concentration of CDDP (mg/mL), V0 is the initial volume of CDDP (mL), ρ1 is the concentration of CDDP in the supernatant obtained by centrifugation after loading (mg/mL), and V1 is the volume of the supernatant obtained by centrifugation after loading (mL).


## 2.8.1. Drawing Standard Curve of Cisplatin

Different concentrations of CDDP solution were prepared with 0.9% saline solution. The absorption peak at 301 nm was measured by UV-3600, and the standard curve was drawn.


## 2.8.2. Loading and Characterization of Cisplatin

5 mg nGO-PEG-BPEI was dispersed in deionized water according to a concentration of 1 mg/mL, and then 5 mL CDDP (5 mg/mL) was slowly put into it. The mixture was stirred in the dark at 37°C for 24 h, and centrifuged at 12000 r/min for 8 min. The precipitate was nGO-PEG-BPEI-CDDP. 1 mg nGO-PEG-BPEI-CDDP powder was mixed with 50 mg dry KBr powder and analyzed by infrared spectroscopy.


## 2.8.3. Load Rate Calculation

The supernatant after centrifugation was taken, and the absorbance was measured at 301 nm. According to the following formula, the loading rate of nGO-PEG-BPEI on CDDP was calculated.(1)W1%=ρ0V0−ρ1V1ρ0V0×100%.In formula (1), W1 is the drug loading rate, ρ0 is the initial concentration of CDDP (mg/mL), V0 is the initial volume of CDDP (mL), ρ1 is the concentration of CDDP in the supernatant obtained by centrifugation after loading (mg/mL), and V1 is the volume of the supernatant obtained by centrifugation after loading (mL).


## 2.9. NGO-PEG-BPEI-CDDP Binding to Tumor-Specific Antibodies

NGO-PEG-BPEI-CDDP powder was mixed with 0.9% saline to prepare NGO-PEG-BPEI-CDDP solutions with concentrations of 1.5μg/mL, 1 μg/mL, and 0.5 μg/mL. 1 mL of each solution was added to 10 μL tumor-specific antibody SCCA (8H11) and incubated at room temperature for 2 h.


## 2.10. Characterization of NGO-PEG-BPEI-CDDP-Antibody Circular Dichroism Chromatography

The stereo structure of tumor-specific antibody SCCA (8H11) was determined by circular dichroism (CD). 10μL of 200 μg/mL SCCA (8H11) protein and 90 μL of 1.5 μg/mL NGO-PEG-BPEI-CDDP nanoparticles were incubated at 37°C for 2 h, and then the weak nitrogen was used to blow dry hexafluoro-2-propanol as the experimental group. SCCA (8H11) and NGO-PEG-BPEI-CDDP nanoparticles were used as control group, respectively. The effect of NGO-PEG-BPEI-CDDP nanoparticles on the conformational changes of the tumor-specific antibody SCCA (8H11) was investigated at 200–250 nm.


## 2.11. Cell Culture and Establishment of Tumor-Bearing Nude Mice Model

### 2.11.1. Cell Culture

KB cell lines were routinely cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/L streptomycin and cultured in a 5% CO2 incubator at 37°C. Cell culture medium was replaced every 24 hours and subculture cells every 72 hours. Cells in the logarithmic growth phase were taken for experiment.


### 2.11.2. Establishment and Administration of Tumor-Bearing Nude Mice Model

After the cell viability was higher than 90%, about 1 × 107 KB cells were resuspended by 50 μL serum-free RPMI 1640 medium. The centrifuge tube of the cell suspension was sealed and stored in the ice box for a short time to maintain the lowest metabolic level. Then, the cells were sent to the hands of the operating table to cover the hot ice for 1 min. When it approached 37°C, it was absorbed and injected into the left armpit of 5-week-old female nude mice subcutaneously. The tumor size of nude mice was observed daily, and the weight of nude mice was measured until the tumor was about 0.5 cm in diameter.Nude mice bearing tumor were divided into the experimental group and the control group. Nude mice were intravenously injected with 200μL sterile saline and 200 μL sterile graphene nanocarriers at different concentrations. The mice were weighed daily and the tumor diameter was measured after the end of administration (take medicine on time every day for seven days).


## 2.11.1. Cell Culture

KB cell lines were routinely cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/L streptomycin and cultured in a 5% CO2 incubator at 37°C. Cell culture medium was replaced every 24 hours and subculture cells every 72 hours. Cells in the logarithmic growth phase were taken for experiment.


## 2.11.2. Establishment and Administration of Tumor-Bearing Nude Mice Model

After the cell viability was higher than 90%, about 1 × 107 KB cells were resuspended by 50 μL serum-free RPMI 1640 medium. The centrifuge tube of the cell suspension was sealed and stored in the ice box for a short time to maintain the lowest metabolic level. Then, the cells were sent to the hands of the operating table to cover the hot ice for 1 min. When it approached 37°C, it was absorbed and injected into the left armpit of 5-week-old female nude mice subcutaneously. The tumor size of nude mice was observed daily, and the weight of nude mice was measured until the tumor was about 0.5 cm in diameter.Nude mice bearing tumor were divided into the experimental group and the control group. Nude mice were intravenously injected with 200μL sterile saline and 200 μL sterile graphene nanocarriers at different concentrations. The mice were weighed daily and the tumor diameter was measured after the end of administration (take medicine on time every day for seven days).


## 2.12. Collection of Tumor-Bearing Nude Mice Model Specimens

After 7 days of administration, 0.2 mL whole blood was obtained from each nude mouse by the tail-broken blood collection method, stored in a blood routine tube containing EDTA, and sent to the animal hospital for routine blood and serum biochemical tests. The mice were dissected with a scalpel, and the heart, liver, kidney, and part of tumors (15 × 15 × 2 mm) of nude mice were taken for paraffin sections. The sections were cut into 3-4μm for staining for staining. The morphological changes of the tissue cells in the sections were observed for pathological analysis.


## 2.13. Immunohistochemistry

Some tumors were taken for a frozen section, and immunohistochemistry was performed. The sheep anti-mouse secondary antibody labeled with horseradish peroxidase was used for incubation. The horseradish peroxidase-DAB method was used to detect the monoclonal antibody SCCA1 (note: SCCA1 is a mouse monoclonal antibody against recombinant human SCCA1).


## 3. Results Analysis

### 3.1. Characterization of Graphene Oxide

The prepared NGO was characterized by Raman spectroscopy to confirm that it reached nanometer level. In order to further analyze the thickness, particle size, and surface oxidation degree of graphene oxide and verify that the graphene oxide reaches nanometer level, the GO obtained by ultrasound was tested and analyzed by SEM, as shown in Figure1.Figure 1
Characterization of graphene oxide. (a) Raman spectra of graphene oxide before centrifugation (left) and after centrifugation (right); (b) (A) and (B) are the SEM images of graphene oxide before ultrasound, and (C) and (D) are the SEM images of graphene oxide after ultrasound.
(a)(b)It is one of the common methods for rapid characterization of carbon materials. The typical NGO has two obvious characteristic peaks in Raman spectra, which are 1338 cm−1 (called D peak) and 1580 cm−1 (called G peak). G peak, caused by sp2 hybrid carbon hexagonal network structure, is the characteristic peak of C group elements; D peak is caused by the disorder of sp2 hybrid carbon hexagonal network structure defects and oxidation. GO contains oxygen-containing functional groups and other defects, which is a unique peak of GO. Therefore, the more obvious the D peak is relative to the G peak, the closer the sample is to NGO, and the less graphene oxide at other levels there is, the purer the sample is.Figure1(a) is the sample before centrifugation (left chart) atlas appeared at 1580 cm−1 at the G peak and at 1338 cm−1 at the D peak, but the D peak is not obvious relative to the G peak, which shows that the sample is nano, micron, and other levels of GO mixing, and that sample purity is not enough. After centrifugation (right chart), the D peak in the two peaks was more obvious, and the D peak and G peak were significantly widened, which was caused by the nanosize effect of nano-GO. Compared with the samples before centrifugation, it can be preliminarily explained that most of the prepared samples are nano-graphene oxide (NGO).In order to further analyze the thickness, particle size, and surface oxidation degree of graphene oxide and verify whether the graphene oxide reaches the nanometer level, the GO obtained by ultrasonic was tested and analyzed by SEM. It can be seen from Figure1(b) that the particle size of graphene oxide before ultrasound is about 10 μm in Figure 1(b) (A and B). After 8 hours of ultrasound, the flake structure of graphene oxide is obvious and uniform, and the particle size of graphene oxide is about 150 nm in Figure 1(b) (C and D). The nanosheet structure increases the space for drug delivery. The experimental results confirmed that the graphene oxide lamellar structure prepared by ultrasound reached the nanometer level.


### 3.2. FTIR Spectra Analysis of Functionally Modified Graphene Oxide Nanoparticles

In order to increase the biocompatibility and loading rate, the prepared NGO was modified with PEG, and the NGO-PEG complex was analyzed by FTIR. The FTIR spectroscopy can reveal the fine structural changes between the nanosized NGO and the carrier NGO-PEG.As shown in Figure2 (left), the absorption peaks of NGO at 3377 cm−1 and 1730 cm−1 are due to the stretching vibration of hydroxyl (-OH) and carbonyl (C = O), respectively. In the FTIR spectra of NGO-PEG, the absorption peak at 3375 cm−1 is the stretching vibration of hydroxyl (-OH), the absorption peaks at 1653 cm−1 and 2874 cm−1 are respectively from the stretching vibration peaks of -CO-NH- and -NH- bonds, and the absorption peak at 1099 cm−1 is due to the stretching of -C-O-C- bonds, which can be attributed to the conjugation of PEG chains, indicating that PEG is successfully connected with NGO to form PEG-modified NGO (NGO-PEG).Figure 2
FTIR spectra of NGO and NGO-PEG (left), and FTIR spectra of NGO-PEG and NGO-PEG-BPEI (right). (a) The absorption peaks of NGO are 3377 cm−1 and 1730 cm−1, and the absorption peaks of NGO-PEG are 3375 cm−1, 1653 cm−1, 2874 cm−1, and 1099 cm−1; (b) the absorption peaks of NGO-PEG are 3375 cm−1, 1653 cm−1, 2874 cm−1, and 1099 cm−1, and the absorption peaks of NGO-PEG-BPEI are 3370 cm−1, 3248 cm−1, 2927 cm−1, 1623 cm−1, and 1095 cm−1.
(a)(b)NGO-PEG and branched polyethyleneimine (BPEI) formed a double-modified complex (NGO-PEG-BPEI) throughπ-π conjugation, which was a good carrier of drugs. FTIR spectroscopy was used to characterize and analyze the complex, and whether BPEI was grafted onto NGO-PEG to form NGO-PEG-BPEI was analyzed. As shown in Figure 2 (right), in the FTIR spectra of NGO-PEG, the absorption peak at 3375 cm−1 is the stretching vibration characteristic peak of hydroxyl (-OH), the absorption peaks at 1653 cm−1 and 2874 cm−1 are respectively from the stretching vibration characteristic peaks of -CO-NH- and -NH- bonds, and the absorption peak at 1099 cm−1 is due to the stretching of -C-O-C- bonds, which can be attributed to the conjugation of PEG chains.In the FTIR spectra of NGO-PEG-BPEI, a broad absorption peak at 3370 cm−1 is attributed to the stretching vibration of hydroxyl (-OH), the absorption peak at 3248 cm−1 is due to the stretching vibration of -NH-bond in polyethyleneimine BPEI, the absorption peak at 2927 cm−1 is due to the stretching vibration of methylene (-CH2) in BPEI, and the absorption peak at 1623 cm−1 is associated with -CO-NH-bond, indicating that NGO-PEG can form an amide bond with BPEI. The band of NGO-PEG-BPEI at 1095 cm−1 is due to the stretching vibration absorption characteristic peak of -C-O-C- bond, which indicates the successful connection between BPEI and GO-PEG.Only the stable structure of prepared NGO-PEG-BPEI under physiological conditions allows it to be used as a carrier to perform drug-loading function. Therefore, the prepared NGO, NGO-PEG, and NGO-PEG-BPEI were dissolved in deionized water, normal saline, and RPMI (10% FBS) solution, respectively, to detect and analyze their stability. The experimental results show that the solubility of NGO, NGO-PEG, and NGO-PEG-BPEI in water is very high, while the solubility of NGO in normal saline and RPMI (10% FBS) solution is significantly reduced. Black NGO aggregates precipitate after centrifugation for 10 min. The results further confirmed that the NGO grafted with PEG and BPEI (NGO-PEG and NGO-PEG-BPEI) showed good dispersion in deionized water, saline, and RPMI (10% FBS) solution, and the solution was uniform without precipitation, indicating that the NGO grafted with PEG and BPEI had excellent solubility and stability in physiological solutions.


### 3.3. Characterization of Drug-Loaded Graphene Nanocomplex

Graphene nanocomplex NGO-PEG-BPEI loaded anticancer drug cisplatin (CDDP) via the covalent bond. The NGO-PEG-BPEI-CDDP was characterized by FTIR spectroscopy to verify whether cisplatin (CDDP) was loaded on graphene oxide (NGO) nanosheets.As shown in Figure3, in the FTIR spectra of NGO-PEG-BPEI, a wide absorption peak at 3372 cm−1 was due to the stretching vibration of hydroxyl (−OH), and the absorption peak at 3240 cm−1 was due to the stretching vibration of -NH- bond in polyethyleneimine BPEI. The absorption peak at 2932 cm−1 was due to the stretching vibration of methylene (−CH2) in BPEI. The absorption peak at 1619 cm−1 was associated with −CO-NH- bond, indicating that amide bond was formed between BPEI and NGO-PEG. The band of NGO-PEG-BPEI at 1097 cm−1 is due to the stretching vibration of -C-O-C- bond. In the FTIR spectra of NGO-PEG-BPEI-CDDP, a broad absorption peak at 3358 cm−1 was attributed to the stretching vibration of hydroxyl (-OH), the absorption peak at 2880 cm−1 was attributed to the stretching vibration of methylene (-CH2) in BPEI, the absorption peaks at 1629 cm−1 and 1105 cm−1 were attributed to the stretching vibration of -CO-NH- bond and the stretching of -C-O-C- bond, and the absorption peak at 1250 cm−1 was attributed to the stretching of GO carbonyl (-C = O-) bond. Typical but weak CDDP absorption peaks (831 cm−1) were observed, indicating that NGO-PEG-BPEI successfully loaded CDDP.Figure 3
FTIR spectra of NGO-PEG-BPEI and NGO-PEG-BPEI-CDDP. The absorption peaks of NGO-PEG-BPEI are 3372 cm−1, 3240 cm−1, 2932 cm−1, 1619 cm−1, and 1097 cm−1; the absorption peaks of NGO-PEG-BPEI-CDDP are 3358 cm−1, 2880 cm−1, 1629 cm−1, 1250 cm−1, 1105 cm−1, and 831 cm−1.The covalent reaction between CDDP and the carboxylic acid (-COOH) group of graphene oxide attached CDDP to NGO-PEG-BPEI. The absorbance of the supernatant of the NGO-PEG-CDDP nano-drug delivery system solution at 301 nm was measured to be 1.574 (Figure2). According to the concentration-absorption standard curve (y = 0.4649x + 0.0858, R2 = 0.9988), the drug concentration corresponding to this absorbance was 3.20 mg/mL, and the loading rate of NGO-PEG-BPEI on CDDP was calculated by the formula W1/%=ρ0V0−ρ1V1/ρ0V0×100%. 31% of CDDP was bound to NGO-PEG-BPEI; according to the formula, the encapsulation efficiency ER% was calculated, and finally the encapsulation efficiency of CDDP encapsulated by NGO-PEG-BPEI was 61.02%. According to the formula ER%=W−ρ1V1/W×100% (W is the total mass of NGO-PEG-BPEI and CDDP added to the system), the encapsulation efficiency ER% was calculated and the encapsulation efficiency of NGO-PEG-BPEI was 61.02%.


### 3.4. Characterization of Graphene Oxide Nanocomposite by Circular Dichroism Spectroscopy

CDDP was loaded on graphene oxide (NGO) nanosheets to form a graphene nano drug-loading complex (NGO-PEG-BPEI-CDDP), which was combined with specific antibodies against squamous cell carcinoma to form a targeted graphene nano drug-loading system. The adsorption of the squamous cell carcinoma-specific antibody on graphene nanodrug delivery system was detected by circular dichroism spectroscopy. Figure4 shows the circular dichroism spectra of the stereostructure of antibody protein.Figure 4
CD spectra of Antibody, NGO-PEG-BPEI-CDDP, and NGO-PEG-BPEI-CDDP@Antibody. The CD spectra of free squamous cell carcinoma antibody, NGO-PEG-BPEI-CDDP, and NGO-PEG-BPEI-CDDP@Antibody in aqueous solution.Figure4 shows the CD spectra of free squamous cell carcinoma antibody, NGO-PEG-BPEI-CDDP and NGO-PEG-BPEI-CDDP@Antibody in aqueous solution, respectively. Obviously, NGO-PEG-BPEI-CDDP did not display spirals and layers. The secondary structures of free squamous cell carcinoma antibody and immobilized NGO-PEG-BPEI-CDDP@Antibody have similar characteristics, showing the minimum value at −215 nm, corresponding to the obvious structure of β-sheet. The peak of NGO-PEG-BPEI-CDDP@Antibody is not very sharp, which is due to the combination of NGO-PEG-BPEI-CDDP surface, thus masking the secondary structure. The above results showed that NGO-PEG-BPEI-CDDP nanocomplex could play an essential role in maintaining the stability of protein conformation.To further confirm whether squamous cell carcinoma-specific antibody is adsorbed on graphene nanocarriers, the structure of NGO-PEG-BPEI-CDDP-Antibody was observed by atomic force microscopy.Figure5(a) shows that the typical AFM two-dimensional image of NGO-PEG-BPEI-CDDP presents a smooth sheet (surface roughness analysis Ra∼0.06 nm) with some wrinkles and burrs and an average thickness of about 1.8 nm corresponding to a layer of NGO-PEG-BPEI-CDDP. Two-dimensional AFM images of 1.5 μg/mL NGO-PEG-BPEI-CDDP@Antibody showed that the antibody protein molecules were evenly and densely distributed over the entire graphene nano-drug delivery system, as shown in Figure 5(b). After NGO-PEG-BPEI-CDDP adsorbed antibody protein molecules, the thickness (3.7 nm) and roughness (Ra∼0.96 nm) were further increased. So, AFM image visualization confirmed the binding of NGO-PEG-BPEI-CDDP surface with antibody protein.Figure 5
AFM images of NGO-PEG-BPEI-CDDP and NGO-PEG-BPEI-CDDP@Antibody. (a) The typical AFM two-dimensional image of NGO-PEG-BPEI-CDDP shows a smooth sheet with some wrinkles and burrs (left); an average thickness of about 1.8 nm (right); (b) the typical AFM two-dimensional image of NGO-PEG-BPEI-CDDP@Antibody (left); the thickness (3.7 nm) and roughness (Ra∼0.96 nm) were further increased (right).
(a)(b)


### 3.5. Study on Antitumor Effect of Graphene Nano-Drug-Loaded Complex

The KB cells were cultured to 5 × 106/mL and 5 × 107/mL by the cell culture method, and 200 μL (1.0 × 106/mouse) and 200 μL (1.0 × 107/mouse) cell suspensions were extracted with a liquid gun and inoculated subcutaneously in the right armpit of nude mice. No subcutaneous mass was found in 1.0 × 106/nude mice. Small subcutaneous mass was found in 1.0 × 107/nude mice about 2 days. The tumor was touchable, nodular, and slightly soft. The tumor volume increased gradually, and the texture became hard. Until the 20th day, the tumor volume reached 120 mm3. The tumor volume-time growth curve of nude mice was established in Figure 6.Figure 6
The tumor volume-time growth curve of nude mice. (a) Tumor growth curve in nude mice; (b) weight change in nude mice; (c) tumor volume change after administration. Group I: control; group II: CDDP; group III: NGO-PEG-BPEI-CDDP-Antibody (100μg/one); group IV: NGO-PEG-BPEI-CDDP-Antibody (200 μg/one); group V: NGO-PEG-BPEI-CDDP-Antibody (300 μg/one).
(a)(b)(c)The experimental results showed that the nude mice were in good condition on the first day of inoculation and had no effect on the normal life of nude mice. With the extension of time, the tumor volume increased gradually, and the tumor volume reached more than 120 mm3 on the 20th day. The subcutaneous inoculation rate was 100%. There was infiltration of surrounding tissues, but no metastasis. In addition, the body weight of tumor-bearing nude mice in each experimental group was not significantly changed compared with that before the tumor. During the experiment, no deaths were found in the tumor-bearing nude mice in each group, indicating that the tumor-bearing method is feasible. Subcutaneous inoculation of KB cells into tumors can be used as an animal model for drug treatment of anti-squamous cell carcinoma in vivo.The administration experiment was carried out on the nude mice bearing tumor. Three administration concentrations (about 20 g for each nude mouse) of graphene nano-drug-loaded complex (100μg/mouse, 200 μg/mouse, and 300 μg/mouse) were set for a total of 7 days. The observation of each experimental group showed that the nude mice had free movement, a normal diet, normal urine and feces, good drinking, eating, and development, and no abnormal weight loss. The weight changes of tumor-bearing nude mice in each experimental group were recorded (see Figure 6(a)). By comparing the weight change of tumor-bearing nude mice before and after administration, it was found that the weight of tumor-bearing nude mice in the control group and the experimental group did not change significantly compared with that before administration, and no deaths occurred in each group during the whole administration.The experimental results showed that the tumor volume of mice in group II, group III, group IV, and group V gradually decreased with the increase of treatment time under the treatment of graphene nano-drug-loading complex. Compared with the control group I, the tumor volume was significantly reduced, and the tumor volume of a high-dose group V decreased significantly with the increase of the treatment time. From the effect of tumor treatment, the effect of the graphene nano-drug-loaded composite treatment group is more obvious than that of CDDP alone, indicating that the effect of graphene nano-drug-loaded composite treatment group is better than that of the CDDP group. The tumor volume in the CDDP group decreased with varying degrees as compared with that in the control group I, indicating that this dose of CDDP drugs had therapeutic effect on tumor growth. The above results showed that the inhibitory effect of graphene nano-drug-loaded composite on the tumor of nude mice bearing squamous cell carcinoma was better than that of CDDP alone and that it had a synergistic therapeutic effect.Tumor quality detection and tumor inhibition rate calculation: on the 7th day, the nude mice in the tumor-bearing group were dissected after eyeball bloodletting and neck transection. The nude mice were fixed on the foam board with a large scalp needle, and then the tumor was weighed and the tumor inhibition rate was calculated. Tumor inhibition rate = (tumor mass of negative control group-tumor mass of drug group)/tumor mass of negative control group × 100%. The average tumor weight of normal saline group was 0.0270 ± 0.0102 g. After administration, the final tumor weight of nude mice in different administration groups was reduced compared with the negative control group. Different groups of tumor inhibition rate are different; the specific results are as shown in Table1.Table 1
Effects of different experimental groups on tumor growth.
GroupDose (μg/rat)Number of miceAverage tumor mass (g)Tumor inhibition rate (%)Negative control group (saline)20080.0270 ± 0.0102—Positive control group (cisplatin)20080.0126 ± 0.007153.33Low-dose group (graphene nanocomposite)10080.0151 ± 0.008244.07Medium-dose group (graphene nanocomposite)20080.0111 ± 0.004558.89High-dose group (graphene nanocomposite)30080.0089 ± 0.003367.03The tumor inhibition rate of nude mice in middle- and high-dose groups of graphene nanodrug complexes was significantly increased. Compared with the negative dose group and the positive control group, the difference was statistically significant (P<0.05).The pathological examination and analysis of tumor tissue sections in tumor-bearing mice showed that the HE staining tumor tissue taken under the microscope was basically consistent with the measured volume during the administration of squamous cell carcinoma. The cells in the control group were normal, with normal cell morphology and a complete nucleus. The tumor cells treated with CDDP chemotherapy were the most severely damaged, and the tumor cells under the microscope were all inactive tumor cells. After the treatment of graphene nano drug-loaded composite, the low dose group showed nest-like growth of implanted tumor cells and small focal necrosis. There was more necrosis and tumor cell degeneration in the middle dose group, and tumor cell nucleus division was inhibited. The tumor tissue in the high-dose group decreased until disappeared, and the tumor cells showed degeneration with few mitotic phases and a large number of tumor cells dying (Figure7).Figure 7
HE staining of nude mice tumor in different treatment groups. (a) Nude mice tumors in the control group (200μg/saline, ×200), (b) nude mice tumors in the CDDP group (200 μg/CDDP, ×200), (c) nude mice tumors in the experimental group (100 μg/graphene carrier complex, ×200); (d) nude mice tumors in the experimental group (200 μg/graphene carrier complex, ×200), (e) nude mice tumors in the experimental group (300 μg/graphene carrier complex, ×200).
(a)(b)(c)(d)(e)Anticancer drugs used in clinical practice usually have no selectivity and have a killing effect on cancer cells, but they also cause damage to other cells and tissues of the human body. If the chemotherapy takes too long or too much, the drug concentration in the blood will increase and cause acute toxic reactions in normal tissues. Targeted therapy can directly kill tumor cells, reduce the damage of anticancer drugs to normal cells and tissues, and increase the antitumor effect. After treatment, the heart, liver, kidney, and tumor of tumor-bearing mice were frozen sectioned, and the horseradish peroxidase-DAB method was used to detect whether there was monoclonal antibody SCCA1 on tumor sections.The experimental results showed that there were many positive substances in the tumor nest of nude mice, which were distributed in the intercellular space of the tumor cell nest as fissures or star-shaped, and the distribution of positive substances in necrotic cells in the central part of the cell nest was obvious (Figure8(a)). At the same time, the OCT sections of the heart, liver, and kidney of the same tumor-bearing nude mice were observed. It was found that the positive substances were expressed in the cardiac cell stroma, hepatic sinusoid, and renal tubulointerstitium (Figures 8(b)–8(d)) but did not cause the death and injury of myocardial cells, hepatocytes, and renal cells. The experiment further shows that the antibody binding between graphene nano drug-loaded complexes and squamous cell carcinoma mainly acts on tumor cells through blood circulation, killing or inhibiting tumor cells.Figure 8
Immunohistochemical detection of tumor and its organs (heart, liver, kidney) in nude mice. (a) Nude mice tumors in the experimental group (300μg/graphene carrier complex, ×200); (b) heart of the nude mice (300 μg/graphene carrier complex, ×200); (c) liver of the nude mice (300 μg/graphene carrier complex, ×200), (d) kidney of the nude mice (300 μg/graphene carrier complex, ×200).
(a)(b)(c)(d)


### 3.6. Safety Evaluation of Graphene Nano-Drug-Loaded Complex

In order to further clarify the toxicity of graphene nano-drug-loading complex treatment, the liver, kidney, heart, and other organs of tumor-bearing nude mice were taken for pathology analysis to test the effects of the graphene nano-drug-loading complex treatment or the CDDP treatment alone [19]. The results of pathology analysis in the CDDP administration group showed the uneven staining of myocardium, myocardial interstitial edema, the changing of myocardial ischemia, and the gathering of chromatin nuclear membrane in hepatocytes. Intrarenal proximal tubular degeneration is a drug metabolic reaction. The heart, liver, and kidney of the low-dose group and the middle-dose group were basically normal, while the heart of the high-dose group had a few degenerative myocardial cells and the other tissues were basically normal. The hepatocyte was slightly loose, and the proximal convoluted tubules were degenerated. The kidney was slightly swollen, and the other kidney tissues were significantly changed. These results show that graphene nano-drug-loaded composites can achieve a good therapeutic effect at a relatively low dose and can reduce the toxicity to normal tissues caused by CDDP alone (Figure 9).Figure 9
HE staining of major organs of mice in different treatment groups after treatment group. (a) Control group (200μg/saline solution, heart, liver, kidneys, ×200); (b) experimental group (200 μg/CDDP, heart, liver, kidneys, ×200); (c) experimental group (200 μg/graphene carrier complex, heart, liver, kidney, ×200).
(a)(b)(c)After administration for 7 consecutive days, 1 mL of the whole blood of each tumor-bearing nude mice was collected and about 0.1 mL blood was used for routine blood test. Table2 is the blood routine test results of mice in each group after administration.Table 2
Blood routine examination results of nude mice in each group.
Group 1Group 2Group 3Group 4Group 5Group 6Total white blood cell (WBC), 109/L4.13.36.81.93.84.5Total number of lymphocytes (LYM), 109/L3.50.70.70.92.20.8Total neutrophil count (GRAN), 109/L0.52.35.80.81.13.5RBC, 1012/L9.118.8311.299.019.038.53The main physiological indexes were analyzed with the number of mice in the abscissa and the relative values of each test result in the ordinate, as shown in Figure10. Group 1 was the blood test of nontumor-bearing nude mice, which was an intravenous injection of 200 μg/saline; group 2 was the blood test of tumor-bearing nude mice, which was an intravenous injection of 200 μg/saline; group 3 was tumor-bearing nude mice were given 200 μg/CDDP; four, five, and six groups of tumor-bearing nude mice were given graphene nano-drug-loading complex, with concentrations of 100 μg/CDDP, 200 μg/CDDP, and 300 μg/CDDP, respectively. The physiological function indexes of the blood test in each group tended to be normal, and the experimental results showed no toxic tendency.Figure 10
Analysis of blood routine examination in different groups of nude mice. WBC = white blood cell; LYM = lymphocyte amount; GRAN = granulocyte; RBC = red blood cell.The results of the blood routine examination in nude mice showed that, compared with the negative control group, the total number of white blood cells and lymphocytes in the CDDP-treated group were normal, and the total number of neutrophils and red blood cells increased. The total number of white blood cells, lymphocytes, neutrophils, and red blood cells in the group 4 (100μg/graphene-loaded composite) and the group 5 (200 μg/graphene-loaded composite) of the nano-drug-loaded composite were normal. The total number of white blood cells, lymphocytes, and red blood cells in the group 6 (300 μg/graphene-loaded composite) was normal while the total number of neutrophils increased. The experiment confirmed that, compared with the impact from CDDP, the nano-drug-loaded complex did not affect the physiology of nude mice. Similarly, about 1 mL whole blood was obtained from each tumor-bearing nude mouse, and about 0.1 mL blood was used for biochemical detection. Table 3 is the biochemical test results of nude mice after administration.Table 3
Biochemical results of nude mice in each group.
Group 1Group 2Group 3Group 4Group 5Group 6Albumin (ALB), g/L23.425.320.625.428.820.3Total protein (TP), g/L38.546.6128.6152.363.6547.05Globulin (GLOB), g/L15.121.3826.934.926.7Alanine aminotransferase (ALT), U/L4581148484784Creatinine (crea), umol/L65.760.9859.317.51822Urea (UREA), mmol/L7.186.5429.156.987.258.62Glucose (GLU), mmol/L6.015.030.86.77.344.78The main biochemical indexes were analyzed with the number of mice in the abscissa and the relative values of each test result in the ordinate, as shown in Figure11.Figure 11
Analysis of biochemical test results in different groups of nude mice. ALB = albumin; TP = total protein; GLOB = globulin; ALT = alanine transaminase; Crea = creatinine; UREA = urea; GLU = glucose.Group 1 was the blood routine test value of nontumor-bearing nude mice, which was intravenous injection of 200μg/saline, and group 2 was the blood test of tumor-bearing nude mice, i.e., 200 μg/mouse normal saline intravenous injection, group 3 was the blood test of tumor-bearing nude mice with 200 μg CDDP by intravenous injection. Group 4, 5, and 6 were injected with graphene nano-drug-compound for 100 μg, 200 μg, and 300 μg, respectively. The biochemical test results showed that the albumin, total protein, and glucose in the CDDP administration group were decreased, and the alanine aminotransferase (ALT) and urea nitrogen were increased. The biochemical indexes of the low-dose group (Group 4) and the middle-dose group (Group 5) were not affected, and the albumin and glucose in the high dose group (Group 6) were lower than the normal value (Figure 11).Albumin was decreased because about 90% of albumin in the blood is synthesized by the liver, and alanine aminotransferase (ALT) increased. This enzyme exists in the liver and striated muscle, increased to consider two factors. Need to grasp the mental state of nude mice, with or without ascites, jaundice, diarrhea, etc. Urea nitrogen is the nitrogen in urea, the final product of mammalian protein catabolism in the liver, which is discharged from the kidney. Combined with a normal creatinine value, this sample should consider gastrointestinal bleeding, effective blood volume reduction, heart failure, etc. If chronic liver injury with abnormal liver function is considered, this value should be reduced. Some drugs can change or increase the ability of kidney concentration. The decrease of blood glucose per hour will be normal due to the timely test time and without considering the problem of artificial sample processing. Considering both pancreatic tumors, severe struggles, intestinal diseases, etc., and the liver glycogen storage capacity of juvenile animals, the appropriate dose of targeted graphene nano-drug-loading complex has little effect on the tumor-bearing nude mice, and the same dose of CDDP has a very serious impact on the physiology of tumor-bearing nude mice.


## 3.1. Characterization of Graphene Oxide

The prepared NGO was characterized by Raman spectroscopy to confirm that it reached nanometer level. In order to further analyze the thickness, particle size, and surface oxidation degree of graphene oxide and verify that the graphene oxide reaches nanometer level, the GO obtained by ultrasound was tested and analyzed by SEM, as shown in Figure1.Figure 1
Characterization of graphene oxide. (a) Raman spectra of graphene oxide before centrifugation (left) and after centrifugation (right); (b) (A) and (B) are the SEM images of graphene oxide before ultrasound, and (C) and (D) are the SEM images of graphene oxide after ultrasound.
(a)(b)It is one of the common methods for rapid characterization of carbon materials. The typical NGO has two obvious characteristic peaks in Raman spectra, which are 1338 cm−1 (called D peak) and 1580 cm−1 (called G peak). G peak, caused by sp2 hybrid carbon hexagonal network structure, is the characteristic peak of C group elements; D peak is caused by the disorder of sp2 hybrid carbon hexagonal network structure defects and oxidation. GO contains oxygen-containing functional groups and other defects, which is a unique peak of GO. Therefore, the more obvious the D peak is relative to the G peak, the closer the sample is to NGO, and the less graphene oxide at other levels there is, the purer the sample is.Figure1(a) is the sample before centrifugation (left chart) atlas appeared at 1580 cm−1 at the G peak and at 1338 cm−1 at the D peak, but the D peak is not obvious relative to the G peak, which shows that the sample is nano, micron, and other levels of GO mixing, and that sample purity is not enough. After centrifugation (right chart), the D peak in the two peaks was more obvious, and the D peak and G peak were significantly widened, which was caused by the nanosize effect of nano-GO. Compared with the samples before centrifugation, it can be preliminarily explained that most of the prepared samples are nano-graphene oxide (NGO).In order to further analyze the thickness, particle size, and surface oxidation degree of graphene oxide and verify whether the graphene oxide reaches the nanometer level, the GO obtained by ultrasonic was tested and analyzed by SEM. It can be seen from Figure1(b) that the particle size of graphene oxide before ultrasound is about 10 μm in Figure 1(b) (A and B). After 8 hours of ultrasound, the flake structure of graphene oxide is obvious and uniform, and the particle size of graphene oxide is about 150 nm in Figure 1(b) (C and D). The nanosheet structure increases the space for drug delivery. The experimental results confirmed that the graphene oxide lamellar structure prepared by ultrasound reached the nanometer level.


## 3.2. FTIR Spectra Analysis of Functionally Modified Graphene Oxide Nanoparticles

In order to increase the biocompatibility and loading rate, the prepared NGO was modified with PEG, and the NGO-PEG complex was analyzed by FTIR. The FTIR spectroscopy can reveal the fine structural changes between the nanosized NGO and the carrier NGO-PEG.As shown in Figure2 (left), the absorption peaks of NGO at 3377 cm−1 and 1730 cm−1 are due to the stretching vibration of hydroxyl (-OH) and carbonyl (C = O), respectively. In the FTIR spectra of NGO-PEG, the absorption peak at 3375 cm−1 is the stretching vibration of hydroxyl (-OH), the absorption peaks at 1653 cm−1 and 2874 cm−1 are respectively from the stretching vibration peaks of -CO-NH- and -NH- bonds, and the absorption peak at 1099 cm−1 is due to the stretching of -C-O-C- bonds, which can be attributed to the conjugation of PEG chains, indicating that PEG is successfully connected with NGO to form PEG-modified NGO (NGO-PEG).Figure 2
FTIR spectra of NGO and NGO-PEG (left), and FTIR spectra of NGO-PEG and NGO-PEG-BPEI (right). (a) The absorption peaks of NGO are 3377 cm−1 and 1730 cm−1, and the absorption peaks of NGO-PEG are 3375 cm−1, 1653 cm−1, 2874 cm−1, and 1099 cm−1; (b) the absorption peaks of NGO-PEG are 3375 cm−1, 1653 cm−1, 2874 cm−1, and 1099 cm−1, and the absorption peaks of NGO-PEG-BPEI are 3370 cm−1, 3248 cm−1, 2927 cm−1, 1623 cm−1, and 1095 cm−1.
(a)(b)NGO-PEG and branched polyethyleneimine (BPEI) formed a double-modified complex (NGO-PEG-BPEI) throughπ-π conjugation, which was a good carrier of drugs. FTIR spectroscopy was used to characterize and analyze the complex, and whether BPEI was grafted onto NGO-PEG to form NGO-PEG-BPEI was analyzed. As shown in Figure 2 (right), in the FTIR spectra of NGO-PEG, the absorption peak at 3375 cm−1 is the stretching vibration characteristic peak of hydroxyl (-OH), the absorption peaks at 1653 cm−1 and 2874 cm−1 are respectively from the stretching vibration characteristic peaks of -CO-NH- and -NH- bonds, and the absorption peak at 1099 cm−1 is due to the stretching of -C-O-C- bonds, which can be attributed to the conjugation of PEG chains.In the FTIR spectra of NGO-PEG-BPEI, a broad absorption peak at 3370 cm−1 is attributed to the stretching vibration of hydroxyl (-OH), the absorption peak at 3248 cm−1 is due to the stretching vibration of -NH-bond in polyethyleneimine BPEI, the absorption peak at 2927 cm−1 is due to the stretching vibration of methylene (-CH2) in BPEI, and the absorption peak at 1623 cm−1 is associated with -CO-NH-bond, indicating that NGO-PEG can form an amide bond with BPEI. The band of NGO-PEG-BPEI at 1095 cm−1 is due to the stretching vibration absorption characteristic peak of -C-O-C- bond, which indicates the successful connection between BPEI and GO-PEG.Only the stable structure of prepared NGO-PEG-BPEI under physiological conditions allows it to be used as a carrier to perform drug-loading function. Therefore, the prepared NGO, NGO-PEG, and NGO-PEG-BPEI were dissolved in deionized water, normal saline, and RPMI (10% FBS) solution, respectively, to detect and analyze their stability. The experimental results show that the solubility of NGO, NGO-PEG, and NGO-PEG-BPEI in water is very high, while the solubility of NGO in normal saline and RPMI (10% FBS) solution is significantly reduced. Black NGO aggregates precipitate after centrifugation for 10 min. The results further confirmed that the NGO grafted with PEG and BPEI (NGO-PEG and NGO-PEG-BPEI) showed good dispersion in deionized water, saline, and RPMI (10% FBS) solution, and the solution was uniform without precipitation, indicating that the NGO grafted with PEG and BPEI had excellent solubility and stability in physiological solutions.


## 3.3. Characterization of Drug-Loaded Graphene Nanocomplex

Graphene nanocomplex NGO-PEG-BPEI loaded anticancer drug cisplatin (CDDP) via the covalent bond. The NGO-PEG-BPEI-CDDP was characterized by FTIR spectroscopy to verify whether cisplatin (CDDP) was loaded on graphene oxide (NGO) nanosheets.As shown in Figure3, in the FTIR spectra of NGO-PEG-BPEI, a wide absorption peak at 3372 cm−1 was due to the stretching vibration of hydroxyl (−OH), and the absorption peak at 3240 cm−1 was due to the stretching vibration of -NH- bond in polyethyleneimine BPEI. The absorption peak at 2932 cm−1 was due to the stretching vibration of methylene (−CH2) in BPEI. The absorption peak at 1619 cm−1 was associated with −CO-NH- bond, indicating that amide bond was formed between BPEI and NGO-PEG. The band of NGO-PEG-BPEI at 1097 cm−1 is due to the stretching vibration of -C-O-C- bond. In the FTIR spectra of NGO-PEG-BPEI-CDDP, a broad absorption peak at 3358 cm−1 was attributed to the stretching vibration of hydroxyl (-OH), the absorption peak at 2880 cm−1 was attributed to the stretching vibration of methylene (-CH2) in BPEI, the absorption peaks at 1629 cm−1 and 1105 cm−1 were attributed to the stretching vibration of -CO-NH- bond and the stretching of -C-O-C- bond, and the absorption peak at 1250 cm−1 was attributed to the stretching of GO carbonyl (-C = O-) bond. Typical but weak CDDP absorption peaks (831 cm−1) were observed, indicating that NGO-PEG-BPEI successfully loaded CDDP.Figure 3
FTIR spectra of NGO-PEG-BPEI and NGO-PEG-BPEI-CDDP. The absorption peaks of NGO-PEG-BPEI are 3372 cm−1, 3240 cm−1, 2932 cm−1, 1619 cm−1, and 1097 cm−1; the absorption peaks of NGO-PEG-BPEI-CDDP are 3358 cm−1, 2880 cm−1, 1629 cm−1, 1250 cm−1, 1105 cm−1, and 831 cm−1.The covalent reaction between CDDP and the carboxylic acid (-COOH) group of graphene oxide attached CDDP to NGO-PEG-BPEI. The absorbance of the supernatant of the NGO-PEG-CDDP nano-drug delivery system solution at 301 nm was measured to be 1.574 (Figure2). According to the concentration-absorption standard curve (y = 0.4649x + 0.0858, R2 = 0.9988), the drug concentration corresponding to this absorbance was 3.20 mg/mL, and the loading rate of NGO-PEG-BPEI on CDDP was calculated by the formula W1/%=ρ0V0−ρ1V1/ρ0V0×100%. 31% of CDDP was bound to NGO-PEG-BPEI; according to the formula, the encapsulation efficiency ER% was calculated, and finally the encapsulation efficiency of CDDP encapsulated by NGO-PEG-BPEI was 61.02%. According to the formula ER%=W−ρ1V1/W×100% (W is the total mass of NGO-PEG-BPEI and CDDP added to the system), the encapsulation efficiency ER% was calculated and the encapsulation efficiency of NGO-PEG-BPEI was 61.02%.


## 3.4. Characterization of Graphene Oxide Nanocomposite by Circular Dichroism Spectroscopy

CDDP was loaded on graphene oxide (NGO) nanosheets to form a graphene nano drug-loading complex (NGO-PEG-BPEI-CDDP), which was combined with specific antibodies against squamous cell carcinoma to form a targeted graphene nano drug-loading system. The adsorption of the squamous cell carcinoma-specific antibody on graphene nanodrug delivery system was detected by circular dichroism spectroscopy. Figure4 shows the circular dichroism spectra of the stereostructure of antibody protein.Figure 4
CD spectra of Antibody, NGO-PEG-BPEI-CDDP, and NGO-PEG-BPEI-CDDP@Antibody. The CD spectra of free squamous cell carcinoma antibody, NGO-PEG-BPEI-CDDP, and NGO-PEG-BPEI-CDDP@Antibody in aqueous solution.Figure4 shows the CD spectra of free squamous cell carcinoma antibody, NGO-PEG-BPEI-CDDP and NGO-PEG-BPEI-CDDP@Antibody in aqueous solution, respectively. Obviously, NGO-PEG-BPEI-CDDP did not display spirals and layers. The secondary structures of free squamous cell carcinoma antibody and immobilized NGO-PEG-BPEI-CDDP@Antibody have similar characteristics, showing the minimum value at −215 nm, corresponding to the obvious structure of β-sheet. The peak of NGO-PEG-BPEI-CDDP@Antibody is not very sharp, which is due to the combination of NGO-PEG-BPEI-CDDP surface, thus masking the secondary structure. The above results showed that NGO-PEG-BPEI-CDDP nanocomplex could play an essential role in maintaining the stability of protein conformation.To further confirm whether squamous cell carcinoma-specific antibody is adsorbed on graphene nanocarriers, the structure of NGO-PEG-BPEI-CDDP-Antibody was observed by atomic force microscopy.Figure5(a) shows that the typical AFM two-dimensional image of NGO-PEG-BPEI-CDDP presents a smooth sheet (surface roughness analysis Ra∼0.06 nm) with some wrinkles and burrs and an average thickness of about 1.8 nm corresponding to a layer of NGO-PEG-BPEI-CDDP. Two-dimensional AFM images of 1.5 μg/mL NGO-PEG-BPEI-CDDP@Antibody showed that the antibody protein molecules were evenly and densely distributed over the entire graphene nano-drug delivery system, as shown in Figure 5(b). After NGO-PEG-BPEI-CDDP adsorbed antibody protein molecules, the thickness (3.7 nm) and roughness (Ra∼0.96 nm) were further increased. So, AFM image visualization confirmed the binding of NGO-PEG-BPEI-CDDP surface with antibody protein.Figure 5
AFM images of NGO-PEG-BPEI-CDDP and NGO-PEG-BPEI-CDDP@Antibody. (a) The typical AFM two-dimensional image of NGO-PEG-BPEI-CDDP shows a smooth sheet with some wrinkles and burrs (left); an average thickness of about 1.8 nm (right); (b) the typical AFM two-dimensional image of NGO-PEG-BPEI-CDDP@Antibody (left); the thickness (3.7 nm) and roughness (Ra∼0.96 nm) were further increased (right).
(a)(b)


## 3.5. Study on Antitumor Effect of Graphene Nano-Drug-Loaded Complex

The KB cells were cultured to 5 × 106/mL and 5 × 107/mL by the cell culture method, and 200 μL (1.0 × 106/mouse) and 200 μL (1.0 × 107/mouse) cell suspensions were extracted with a liquid gun and inoculated subcutaneously in the right armpit of nude mice. No subcutaneous mass was found in 1.0 × 106/nude mice. Small subcutaneous mass was found in 1.0 × 107/nude mice about 2 days. The tumor was touchable, nodular, and slightly soft. The tumor volume increased gradually, and the texture became hard. Until the 20th day, the tumor volume reached 120 mm3. The tumor volume-time growth curve of nude mice was established in Figure 6.Figure 6
The tumor volume-time growth curve of nude mice. (a) Tumor growth curve in nude mice; (b) weight change in nude mice; (c) tumor volume change after administration. Group I: control; group II: CDDP; group III: NGO-PEG-BPEI-CDDP-Antibody (100μg/one); group IV: NGO-PEG-BPEI-CDDP-Antibody (200 μg/one); group V: NGO-PEG-BPEI-CDDP-Antibody (300 μg/one).
(a)(b)(c)The experimental results showed that the nude mice were in good condition on the first day of inoculation and had no effect on the normal life of nude mice. With the extension of time, the tumor volume increased gradually, and the tumor volume reached more than 120 mm3 on the 20th day. The subcutaneous inoculation rate was 100%. There was infiltration of surrounding tissues, but no metastasis. In addition, the body weight of tumor-bearing nude mice in each experimental group was not significantly changed compared with that before the tumor. During the experiment, no deaths were found in the tumor-bearing nude mice in each group, indicating that the tumor-bearing method is feasible. Subcutaneous inoculation of KB cells into tumors can be used as an animal model for drug treatment of anti-squamous cell carcinoma in vivo.The administration experiment was carried out on the nude mice bearing tumor. Three administration concentrations (about 20 g for each nude mouse) of graphene nano-drug-loaded complex (100μg/mouse, 200 μg/mouse, and 300 μg/mouse) were set for a total of 7 days. The observation of each experimental group showed that the nude mice had free movement, a normal diet, normal urine and feces, good drinking, eating, and development, and no abnormal weight loss. The weight changes of tumor-bearing nude mice in each experimental group were recorded (see Figure 6(a)). By comparing the weight change of tumor-bearing nude mice before and after administration, it was found that the weight of tumor-bearing nude mice in the control group and the experimental group did not change significantly compared with that before administration, and no deaths occurred in each group during the whole administration.The experimental results showed that the tumor volume of mice in group II, group III, group IV, and group V gradually decreased with the increase of treatment time under the treatment of graphene nano-drug-loading complex. Compared with the control group I, the tumor volume was significantly reduced, and the tumor volume of a high-dose group V decreased significantly with the increase of the treatment time. From the effect of tumor treatment, the effect of the graphene nano-drug-loaded composite treatment group is more obvious than that of CDDP alone, indicating that the effect of graphene nano-drug-loaded composite treatment group is better than that of the CDDP group. The tumor volume in the CDDP group decreased with varying degrees as compared with that in the control group I, indicating that this dose of CDDP drugs had therapeutic effect on tumor growth. The above results showed that the inhibitory effect of graphene nano-drug-loaded composite on the tumor of nude mice bearing squamous cell carcinoma was better than that of CDDP alone and that it had a synergistic therapeutic effect.Tumor quality detection and tumor inhibition rate calculation: on the 7th day, the nude mice in the tumor-bearing group were dissected after eyeball bloodletting and neck transection. The nude mice were fixed on the foam board with a large scalp needle, and then the tumor was weighed and the tumor inhibition rate was calculated. Tumor inhibition rate = (tumor mass of negative control group-tumor mass of drug group)/tumor mass of negative control group × 100%. The average tumor weight of normal saline group was 0.0270 ± 0.0102 g. After administration, the final tumor weight of nude mice in different administration groups was reduced compared with the negative control group. Different groups of tumor inhibition rate are different; the specific results are as shown in Table1.Table 1
Effects of different experimental groups on tumor growth.
GroupDose (μg/rat)Number of miceAverage tumor mass (g)Tumor inhibition rate (%)Negative control group (saline)20080.0270 ± 0.0102—Positive control group (cisplatin)20080.0126 ± 0.007153.33Low-dose group (graphene nanocomposite)10080.0151 ± 0.008244.07Medium-dose group (graphene nanocomposite)20080.0111 ± 0.004558.89High-dose group (graphene nanocomposite)30080.0089 ± 0.003367.03The tumor inhibition rate of nude mice in middle- and high-dose groups of graphene nanodrug complexes was significantly increased. Compared with the negative dose group and the positive control group, the difference was statistically significant (P<0.05).The pathological examination and analysis of tumor tissue sections in tumor-bearing mice showed that the HE staining tumor tissue taken under the microscope was basically consistent with the measured volume during the administration of squamous cell carcinoma. The cells in the control group were normal, with normal cell morphology and a complete nucleus. The tumor cells treated with CDDP chemotherapy were the most severely damaged, and the tumor cells under the microscope were all inactive tumor cells. After the treatment of graphene nano drug-loaded composite, the low dose group showed nest-like growth of implanted tumor cells and small focal necrosis. There was more necrosis and tumor cell degeneration in the middle dose group, and tumor cell nucleus division was inhibited. The tumor tissue in the high-dose group decreased until disappeared, and the tumor cells showed degeneration with few mitotic phases and a large number of tumor cells dying (Figure7).Figure 7
HE staining of nude mice tumor in different treatment groups. (a) Nude mice tumors in the control group (200μg/saline, ×200), (b) nude mice tumors in the CDDP group (200 μg/CDDP, ×200), (c) nude mice tumors in the experimental group (100 μg/graphene carrier complex, ×200); (d) nude mice tumors in the experimental group (200 μg/graphene carrier complex, ×200), (e) nude mice tumors in the experimental group (300 μg/graphene carrier complex, ×200).
(a)(b)(c)(d)(e)Anticancer drugs used in clinical practice usually have no selectivity and have a killing effect on cancer cells, but they also cause damage to other cells and tissues of the human body. If the chemotherapy takes too long or too much, the drug concentration in the blood will increase and cause acute toxic reactions in normal tissues. Targeted therapy can directly kill tumor cells, reduce the damage of anticancer drugs to normal cells and tissues, and increase the antitumor effect. After treatment, the heart, liver, kidney, and tumor of tumor-bearing mice were frozen sectioned, and the horseradish peroxidase-DAB method was used to detect whether there was monoclonal antibody SCCA1 on tumor sections.The experimental results showed that there were many positive substances in the tumor nest of nude mice, which were distributed in the intercellular space of the tumor cell nest as fissures or star-shaped, and the distribution of positive substances in necrotic cells in the central part of the cell nest was obvious (Figure8(a)). At the same time, the OCT sections of the heart, liver, and kidney of the same tumor-bearing nude mice were observed. It was found that the positive substances were expressed in the cardiac cell stroma, hepatic sinusoid, and renal tubulointerstitium (Figures 8(b)–8(d)) but did not cause the death and injury of myocardial cells, hepatocytes, and renal cells. The experiment further shows that the antibody binding between graphene nano drug-loaded complexes and squamous cell carcinoma mainly acts on tumor cells through blood circulation, killing or inhibiting tumor cells.Figure 8
Immunohistochemical detection of tumor and its organs (heart, liver, kidney) in nude mice. (a) Nude mice tumors in the experimental group (300μg/graphene carrier complex, ×200); (b) heart of the nude mice (300 μg/graphene carrier complex, ×200); (c) liver of the nude mice (300 μg/graphene carrier complex, ×200), (d) kidney of the nude mice (300 μg/graphene carrier complex, ×200).
(a)(b)(c)(d)


## 3.6. Safety Evaluation of Graphene Nano-Drug-Loaded Complex

In order to further clarify the toxicity of graphene nano-drug-loading complex treatment, the liver, kidney, heart, and other organs of tumor-bearing nude mice were taken for pathology analysis to test the effects of the graphene nano-drug-loading complex treatment or the CDDP treatment alone [19]. The results of pathology analysis in the CDDP administration group showed the uneven staining of myocardium, myocardial interstitial edema, the changing of myocardial ischemia, and the gathering of chromatin nuclear membrane in hepatocytes. Intrarenal proximal tubular degeneration is a drug metabolic reaction. The heart, liver, and kidney of the low-dose group and the middle-dose group were basically normal, while the heart of the high-dose group had a few degenerative myocardial cells and the other tissues were basically normal. The hepatocyte was slightly loose, and the proximal convoluted tubules were degenerated. The kidney was slightly swollen, and the other kidney tissues were significantly changed. These results show that graphene nano-drug-loaded composites can achieve a good therapeutic effect at a relatively low dose and can reduce the toxicity to normal tissues caused by CDDP alone (Figure 9).Figure 9
HE staining of major organs of mice in different treatment groups after treatment group. (a) Control group (200μg/saline solution, heart, liver, kidneys, ×200); (b) experimental group (200 μg/CDDP, heart, liver, kidneys, ×200); (c) experimental group (200 μg/graphene carrier complex, heart, liver, kidney, ×200).
(a)(b)(c)After administration for 7 consecutive days, 1 mL of the whole blood of each tumor-bearing nude mice was collected and about 0.1 mL blood was used for routine blood test. Table2 is the blood routine test results of mice in each group after administration.Table 2
Blood routine examination results of nude mice in each group.
Group 1Group 2Group 3Group 4Group 5Group 6Total white blood cell (WBC), 109/L4.13.36.81.93.84.5Total number of lymphocytes (LYM), 109/L3.50.70.70.92.20.8Total neutrophil count (GRAN), 109/L0.52.35.80.81.13.5RBC, 1012/L9.118.8311.299.019.038.53The main physiological indexes were analyzed with the number of mice in the abscissa and the relative values of each test result in the ordinate, as shown in Figure10. Group 1 was the blood test of nontumor-bearing nude mice, which was an intravenous injection of 200 μg/saline; group 2 was the blood test of tumor-bearing nude mice, which was an intravenous injection of 200 μg/saline; group 3 was tumor-bearing nude mice were given 200 μg/CDDP; four, five, and six groups of tumor-bearing nude mice were given graphene nano-drug-loading complex, with concentrations of 100 μg/CDDP, 200 μg/CDDP, and 300 μg/CDDP, respectively. The physiological function indexes of the blood test in each group tended to be normal, and the experimental results showed no toxic tendency.Figure 10
Analysis of blood routine examination in different groups of nude mice. WBC = white blood cell; LYM = lymphocyte amount; GRAN = granulocyte; RBC = red blood cell.The results of the blood routine examination in nude mice showed that, compared with the negative control group, the total number of white blood cells and lymphocytes in the CDDP-treated group were normal, and the total number of neutrophils and red blood cells increased. The total number of white blood cells, lymphocytes, neutrophils, and red blood cells in the group 4 (100μg/graphene-loaded composite) and the group 5 (200 μg/graphene-loaded composite) of the nano-drug-loaded composite were normal. The total number of white blood cells, lymphocytes, and red blood cells in the group 6 (300 μg/graphene-loaded composite) was normal while the total number of neutrophils increased. The experiment confirmed that, compared with the impact from CDDP, the nano-drug-loaded complex did not affect the physiology of nude mice. Similarly, about 1 mL whole blood was obtained from each tumor-bearing nude mouse, and about 0.1 mL blood was used for biochemical detection. Table 3 is the biochemical test results of nude mice after administration.Table 3
Biochemical results of nude mice in each group.
Group 1Group 2Group 3Group 4Group 5Group 6Albumin (ALB), g/L23.425.320.625.428.820.3Total protein (TP), g/L38.546.6128.6152.363.6547.05Globulin (GLOB), g/L15.121.3826.934.926.7Alanine aminotransferase (ALT), U/L4581148484784Creatinine (crea), umol/L65.760.9859.317.51822Urea (UREA), mmol/L7.186.5429.156.987.258.62Glucose (GLU), mmol/L6.015.030.86.77.344.78The main biochemical indexes were analyzed with the number of mice in the abscissa and the relative values of each test result in the ordinate, as shown in Figure11.Figure 11
Analysis of biochemical test results in different groups of nude mice. ALB = albumin; TP = total protein; GLOB = globulin; ALT = alanine transaminase; Crea = creatinine; UREA = urea; GLU = glucose.Group 1 was the blood routine test value of nontumor-bearing nude mice, which was intravenous injection of 200μg/saline, and group 2 was the blood test of tumor-bearing nude mice, i.e., 200 μg/mouse normal saline intravenous injection, group 3 was the blood test of tumor-bearing nude mice with 200 μg CDDP by intravenous injection. Group 4, 5, and 6 were injected with graphene nano-drug-compound for 100 μg, 200 μg, and 300 μg, respectively. The biochemical test results showed that the albumin, total protein, and glucose in the CDDP administration group were decreased, and the alanine aminotransferase (ALT) and urea nitrogen were increased. The biochemical indexes of the low-dose group (Group 4) and the middle-dose group (Group 5) were not affected, and the albumin and glucose in the high dose group (Group 6) were lower than the normal value (Figure 11).Albumin was decreased because about 90% of albumin in the blood is synthesized by the liver, and alanine aminotransferase (ALT) increased. This enzyme exists in the liver and striated muscle, increased to consider two factors. Need to grasp the mental state of nude mice, with or without ascites, jaundice, diarrhea, etc. Urea nitrogen is the nitrogen in urea, the final product of mammalian protein catabolism in the liver, which is discharged from the kidney. Combined with a normal creatinine value, this sample should consider gastrointestinal bleeding, effective blood volume reduction, heart failure, etc. If chronic liver injury with abnormal liver function is considered, this value should be reduced. Some drugs can change or increase the ability of kidney concentration. The decrease of blood glucose per hour will be normal due to the timely test time and without considering the problem of artificial sample processing. Considering both pancreatic tumors, severe struggles, intestinal diseases, etc., and the liver glycogen storage capacity of juvenile animals, the appropriate dose of targeted graphene nano-drug-loading complex has little effect on the tumor-bearing nude mice, and the same dose of CDDP has a very serious impact on the physiology of tumor-bearing nude mice.


## 4. Discussion

Nanomedicines have the characteristics of small particles, a large specific surface area, high surface reactivity, many active centers, and a strong adsorption capacity. The use of nanomaterials as drug carriers can improve the absorption and utilization of drugs, achieve efficient targeted delivery, prolong the half-life of drug consumption, and reduce harmful side effects on normal tissues [20]. However, when the simple nano-drug carrier circulates in the body, it will be quickly engulfed by RES macrophages in the liver and spleen, and lose efficacy. In order to reduce the phagocytosis of RES on the carrier, hinder the adsorption of nanoparticles by plasma proteins, and prolong the circulation time of nanoparticles in the blood, so as to further improve the targeting effect, the modification with hydrophilic polymer materials can make the drug delivery carrier recyclable. The hydrophilic polymer polyethylene glycol (PEG) is the most widely used modification material and has been approved by the FDA. Studies have shown that the particles surface covered by the hydrophilic polymer PEG system show the characteristics of enhanced blood circulation time, increasing the accumulation of drugs at the infection site [21].The nanosized NGO-PEG-BPEI-CDDP drug-loading complex was combined with the tumor-specific monoclonal antibody of squamous cell carcinoma to form the NGO-PEG-BPEI-CDDP-Antibody complex targeting tumor cells and reducing the damage to normal tissue cells. NGO-PEG-BPEI-CDDP-Antibody complex was injected into nude mice via vein to target squamous cancer cells, which can effectively kill tumor cells and reduce the dose of clinical chemotherapeutic drugs. In cancer treatment, molecular targeted drugs targeting proliferation-related molecules produced by cancer cells are often used [22]. Targeted therapy is mainly aimed at some specific biological markers or important proteins in signal transduction pathways in tumor cells to block the development of tumor [23]. These drugs have the characteristics of targeting and noncytotoxicity and mainly act on tumor cells. Therefore, they are very different from cytotoxic drugs in terms of the mechanism of action and the performance of toxic and side effects. In order to distinguish them from traditional cytotoxic drugs, these drugs are named cell proliferation inhibitors. Targeted therapy has not only made breakthroughs in some relatively rare drug-resistant tumors but also made remarkable progress in the treatment of common tumors, which has improved the efficacy of some tumors on the basis of traditional chemotherapy [24]. Tumor targeting therapy will play an increasingly important role in tumor therapy with its specificity and targeting. At present, the main targeted therapy is antibody therapy, which is often used in the treatment of hematological tumors and has achieved some promising results. However, there are still problems in solid tumors. It is difficult for the treated antibodies to enter the solid tumors, so the curative effect of treating large solid tumors is still not very ideal. In addition, for gene therapy and viral therapy, the vector cannot specifically target tumor cells, and the expression efficiency of therapeutic genes in tumor cells is low, which is not enough to kill tumors. Targeted tumor stem cells have been confirmed in leukemia, breast cancer, and glioma [25–27]. Although cancer stem cells are rare in tumors, they are highly likely to be the source of tumorigenesis, drug resistance, recurrence, and metastasis. Scientists hope to find the unique antigen expression of cancer stem cells in order to design monoclonal antibodies. At present, it is more likely that the signal transduction in tumor stem cells is different from that in normal stem cells, which can be blocked by small molecule drugs. One of the most important molecules of signal transduction is protein tyrosine kinase, and the development of targeted drugs has become one of the research hotspots of anticancer drugs. Currently, small molecules approved by the FDA can specifically kill tumor cells. Various targeted drugs are being actively explored in the trial of different tumors, combination with different chemotherapy regimens, in maintenance therapy, in sequential therapy, and in combined targeted therapy. In addition to the familiar types, various drugs with new mechanisms of action are under study. Most monoclonal antibody-based targeted therapy drugs have low efficiency when used alone, and even effective patients often have secondary drug resistance. This is partly because of the complexity and heterogeneity of the pathophysiological mechanism of tumors. The same disease is the result of the interaction of multiple factors. Also, efficacy generated is varied among different patients and different disease stages of the same patient.Therefore, monoclonal antibody-targeted therapy drugs are often used in combination with cytotoxic drugs or with other targeted drugs. Tumors often have fewer molecular biological abnormalities when the tumor load is small in the early stage of disease. It is a direction of clinical research in recent years to promote targeted therapy drugs to adjuvant therapy, which requires accurate understanding of the early molecular events of the disease. In addition, exploring effective prognostic indicators and selecting appropriate patients for targeted therapy can also relatively improve the effect of targeted therapy.Squamous-cell carcinoma antigen (SCCA) is a tumor cell-related antigen, which can be used as a serological and histological marker for many squamous cell carcinomas. SCCA is one of the ovalbumin families and has a serine protease inhibitor effect. It has been found that the level of SCCA protein in blood has an obvious correlation with some clinically advanced squamous cell carcinoma (including cervical cancer, lung cancer, esophageal cancer, and cervical inverted papilloma) and it is involved in tumor growth. Thus, SCCA can be used as a biological marker and can be used as a biological marker. In addition, it has been reported that SCCA proteins (SCCA1, SCCA2, and SCCA-PD) were abnormally expressed in the liver of 85% of patients with hepatocellular carcinoma (HCC) by immunohistochemistry. The clinical study on cervical squamous cell carcinoma showed that the proportion of patients with an increased free SCCA level during tumor occurrence from 12% in Stage 0 to 90% in Stage IV. The level of free SCCA decreases after tumor resection, while it rises again in 90% of patients with relapse. This study reveals that the detection of SCCA levels after treatment could help monitor the therapeutic effect of patients and predict tumor recurrence and metastasis. The level of SCCA is related to tumor load and tumor cell activity. Continuous dynamic monitoring is helpful to monitor the therapeutic effect, especially the effect of surgery, and can also be used as an important reference index for the follow-up after treatment. SCCA determination has high specificity for the diagnosis of various squamous cell carcinomas, especially at the cervical, esophageal, head, and neck. The antibody-based targeted drug delivery system has become a research hotspot because of its small side effects. Studies have confirmed that the coupling of platinum drugs with tumor-targeting groups avoids the shortcoming of its instability in the blood and irreversible binding of plasma proteins, and the renal clearance rate is fast. The functionalized graphene oxide carrier (NGO-PEG-BPEI) was used to load the anticancer drug CDDP and bind to the specific squamous cell carcinoma monoclonal antibody (SCCA1) of squamous cell carcinoma to produce the targeted graphene nano-drug-loading complex (NGO-PEG-BPEI-CDDP-Antibody). The prepared NGO-PEG-BPEI-CDDP-Antibody complex was injected into nude mice bearing human squamous cell carcinoma by the tail vein. The experimental results showed that a certain dose of NGO-PEG-BPEI-CDDP-Antibody could inhibit the growth of tumors to a variable extent and had a good therapeutic effect on tumors. The therapeutic effect of the NGO-PEG-BPEI-CDDP-Antibody complex was better than that of CDDP alone, and it had better antitumor effect. Through frozen section, it was found that NGO-PEG-BPEI-CDDP-Antibody complex gathered around the squamous cell carcinoma tissue. The experiment further indicated that the combination of graphene nano-drug-loading complex and the antibody from squamous cell carcinoma mainly acted on tumor cells through blood circulation, killing or inhibiting tumor cells. After the treatment, the blood routine indexes and biochemical indexes of the tumor-bearing nude mice were detected in the normal range. The pathological analysis of the normal tissues from the heart, liver, and kidney showed that an appropriate amount of NGO-PEG-BPEI-CDDP-Antibody did not have toxic and side effects on the normal tissues of nude mice. Therefore, the experimental results could provide certain theoretical data reference and clinical evidence for the immune targeting therapy of NGO-PEG-BPEI-CDDP-Antibody nano-Antibody nanocarrier complex. In the presence of surfactants and additional agents such as water, the drug can be directly pulverized into nanosuspensions and administered intravenously. The drug-loading system based on nano-graphene oxide particles can target and transport drugs through passive transport and active transport. Passive targeting, mainly through the penetration and retention effect (EPR), enables the drug to be swallowed by macrophages as a foreign body immediately after entering the human body, thereby reducing the nonspecific binding to nontarget sites and reaching the target site for selective binding. Specific monoclonal antibodies can guide drug delivery systems to detect and bind squamous cell carcinoma antigens or receptors that are overexpressed on the surface of tumor cells for cell imaging and targeted delivery of antitumor drugs. Due to the limitation of time, whether the NGO-PEG-BPEI-CDDP-Antibody complex has the same biological effect in other types of tumor cells needs to be further studied. In addition, there is no systematic and quantitative study on the pharmacokinetics, specific distribution in vivo and long-term toxicity of CDDP after the NGO-PEG-BPEI-CDDP-Antibody complex enters the body, which are planned in the subsequent experiments.In summary, the NGO-PEG-BPEI-CDDP-Antibody complex has a significant antitumor effect. NGO-PEG-BPEI-CDDP-Antibody complex can encapsulate 61.02% cisplatin into nude mice and target at squamous cancer cells; the effective killing effect could greatly reduce the dose of clinical chemotherapeutic drugs and improve the clinical therapy against squamous cancer. In particular, the positive expression of tumor-specific antibody (8H11) in squamous cell carcinoma cells is feasible. These results show that when squamous cell carcinoma cells express a tumor-specific antibody (8H11) receptor, the NGO-PEG-BPEI-CDDP-Antibody complex will target tumor cells. Hence, the NGO-PEG-BPEI-CDDP-Antibody complex is expected to improve the squamous cell carcinoma treatment based on CDDP chemotherapy.


## 5. Conclusions

We conclude this work with some outlooks. The nano-graphene oxide successfully loaded with CDDP and a human squamous cell carcinoma monoclonal antibody has the ability to target squamous cancer cells, can effectively kill cancer cells while reducing the toxicity to human cells, and can reduce the use of clinical chemotherapy drugs to improve the clinical therapeutic effect of squamous cell carcinoma. The difficulty of nano-drug-loading systems is whether targeted drugs can be successfully loaded. In order to test this problem, we carried out detailed testing experiments to prove that the method described in this paper can prepare drug-loading systems with high encapsulation efficiency. Also, in the process of simulating drug administration in the nude mouse tumor-bearing model, the safety and efficacy of the drug-loading system were confirmed by biochemical tests such as tissue sections, and it has clinical value. The nano-graphene oxide drug-loading system may provide new answers for the cure of squamous cell carcinoma.



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*Source: 6336569-2023-02-02.xml*