Improving Tumoral Acidic pH-Responsive Drug Eciency Based on GO@Au Nanocomposite

Background: to accurately deliver drugs to the lesion site and realize its timed and quantitative release is the focus of tumor therapy. In this paper, a tumor targeted near-infrared light controlled release drug delivery system integrating hyperthermia and chemotherapy was successfully constructed by combining tumor hyperthermia and chemotherapy. Results: by hydrothermal method, Au nanoparticles were rstly loaded onto the GO surface to obtain GO@Au nanocomposites, then PEG was loaded onto the GO@Au by pH sensitive hydrazone bond to obtain GO@Au-PEG, and DOX is nally loaded onto the GO surface via π-π stacking. The target controlled release drug delivery system showed good tumor targeting property, can be the ecient transfer of drug to the tumor cells, and the tumor cells in near-infrared light control of DOX release. In vivo tumor inhibition experiments in mice, the targeted drug delivery system showed obvious tumor inhibition characteristics and had good effects of hyperthermia and chemotherapy. Conclusions: therefore, this tumor targeted near-infrared controlled release drug delivery system has great potential in tumor therapy.


Background
Cancer has become one of the major diseases threatening human life [1][2][3][4] . At present, chemotherapy is still an indispensable and important means in the treatment of cancer. However, due to the poor speci city of chemotherapy drugs to tumor tissues, it has great toxic and side effects, so its therapeutic effect is not ideal [5]. Using nanomaterials as drug carriers to construct a chemotherapeutic drug controlled release system can reduce the toxic and side effects of chemotherapeutic drugs and improve their e cacy [6][7][8][9] .
The appearance of controlled release drug delivery system is expected to solve the shortcomings of poor speci city of chemotherapy and high toxic and side effects [10][11][12] . It enables drugs to be released as much as possible at the site of disease, and can maintain the effective therapeutic concentration of drugs at the site of disease, while remaining within the safe concentration range in other tissues [13] . Therefore, the toxic and side effects of chemotherapy drugs can be reduced and the curative effect can be improved.
Stimuli-responsive controlled release drug delivery system is an intelligent drug delivery system, which refers to the drug delivery system in the external environment or the human body's own environmental factors (pH, temperature, light, enzyme and ion intensity, etc.) under the stimulation of some physical or chemical changes, so as to achieve the controlled release of drug delivery system [14][15][16][17] . A wide range of stimuli-responsive controlled release drug delivery systems have been studied, including pH responsive controlled release drug delivery system, temperature responsive controlled release drug delivery system and photothermal response controlled release drug delivery system [18][19][20] .
The use of thermotherapy to treat cancer has also attracted a lot of attention. High temperatures can cause cancer cells to die, and when the temperature rises above 42℃, overheating can cause damage to the cells. Photothermal therapy has attracted wide attention in recent years and is regarded as one of the most promising tumor treatment technologies, because of its controllable, minimally invasive and e cient tumor treatment advantages [21][22][23] . But a big disadvantage of this approach is the lack of speci city. As the temperature of the tumor area rises, other surrounding tissues may also be damaged by the high temperature [24] .
Combining nanomaterials with chemotherapeutic drugs to construct a tumor targeting drug delivery system can realize the synergistic effect of chemotherapy and hyperthermia on tumors. In order to integrate various functions into a single structure to match the treatment process, many people have been working to develop hybrid nanosystems [25][26][27][28] . Despite the exciting recent progresses in the development of multi-functional drug delivery system. However, more effective tumor targeting drug delivery systems need to be developed to enable the coordinated treatment of cancer with chemotherapy and hyperthermia.
In the current study, a new doxorubicin (DOX) , one of the most effective drugs against a wide range of cancers, delivery system with pH sensitivity and photodynamic therapy was designed, synthesized, characterized and explored for its potential applications both in drug delivery and in photodynamic therapy. Firstly, graphene oxide (GO) and gold (Au) bybrid nanocomposite is synthesized. The composite is functionalized by polyethylene glycol (PEG2000). Then DOX was employed as the model drug and loaded onto GO@Au-PEG with high e cacy. The new drug delivery system is characterized by transmission electron microscopy (TEM), dynamic laser scattering (DLS), Fourier transmission infrared spectroscopy (FTIR) and UV spectrums. The tumor diagnostic and positioning ability, tumor-targeting property, photothermal therapy (PTT) and combination therapeutic e cacy of GO@Au-PEG/DOX was evaluated.

Synthesis of GO@Au
Weigh 50 mg GO, place it in a small beaker, add 150 ml ultra-pure water, and ultrasound makes it completely dispersed. In the dark condition, 10 ml HAuCl 4 (0.025 M) solution was added while shaking, and then 10 ml sodium citrate solution (0.1 M) was added to the mixed solution. After ultrasonic mixing for 10 min, the solution was transferred to the reactor and reacted at 120 ℃ for 10 h. At the end of the reaction, the reaction products were naturally cooled to room temperature, centrifuged at 12000 rpm for 10 min, then the precipitation was washed for 3 times with ultrapure water and anhydrous ethanol, and dried in a vacuum at 60 ℃ for 12 h to obtain GO@Au nanocomposites.

Synthesis of GO@Au-PEG/DOX
Put 50 mg GO@Au powder in a three-mouth ask, and an appropriate amount of DMF was added, followed by ultrasonic cleaning until the powder was completely dispersed, and then 2 ml pyridine, 20 mg DMAP and 400 mg P-nitrophenyl chloroformate were added. The powder was then reacted in a water bath at 0℃ under the protection of nitrogen, and stirred for 48h ( ultrasonic treatment1 h per 8 h). At the end of the reaction, diethyl ether was added to the reaction system to precipitate the black substance. The reaction product was repeatedly washed step by step with diethyl ether, dichloromethane and isopropanol. Put the product into vacuum drying at 60 ℃ for 12h to obtain activated GO@Au. 50 mg activated GO@Au were dispersed in DMF and treated with ultrasonic under nitrogen protection for 30 min to disperse them. Then 10 ml ethylenediamine and 2 ml diisopropyl ethylamine were added and stirred at room temperature for 24h. Then it was cleaned with methanol and pure water and dried at 60℃ for 12h to obtain GO@Au-NH 2 . GO@Au-PEG (50 mg) and DOX (100 ml) were added to DMSO (50 ml) stirred at room temperature in the dark for 24 h. Then the nanosuspension was centrifuged to remove free DOX. After freeze-drying, the solid products GO@Au-PEG/DOX were dried in vacuum at 30 ℃ for 24 h and stored at 4℃ until use. Figure 1 shows the schematic of the synthetic steps from GO to GO@Au-PEG/DOX.

Results And Discussion
3.1 Synthesis of GO@Au-PEG/DOX Figure 1 shows the synthesis scheme of GO@Au-PEG/DOX. During the synthesis of GO@Au, mild sodium citrate was selected as the reducing agent in the experiment, and gold nanoparticles were loaded on the surface of GO by hydrothermal method to form GO@Au nanocomposite. In GO@Au-PEG synthesis process, chloroformic acid p-nitrophenyl ester was used to activate the hydroxyl group on the GO surface, to get GO@Au of nitro phenyl acetic derivatives, then connect ethylenediamine chemical by ester bond on the GO@Au nanocomposites, to form the GO@Au-NH 2 , then introduced with hydrazine base HBA, add CHO-PEG2000, modi ed PEG by pH sensitive hydrazone bond on GO@Au nanocomposites. DOX is e ciently loaded onto the GO@Au-PEG surface through a strong π-π stacking. Figure 2a and b shows the TEM image of GO and GO@Au. It can be seen that GO is a typical strati ed structure and Au is a sphere with a diameter of 10~20 nm. Au nanoparticles were uniformly distributed on the GO sheets, indicating that Au was successfully loaded on the GO@Au nanocomposite. Figure 2c shwos the UV spectrum of the GO and GO@Au. The the UV spectrum of GO@Au showed a strong absorption at 527 nm, showing that the gold nanoparticles are indeed loaded on the GO surface.

Characterization of GO@Au-PEG
The FT-IR spectrum of GO, GO@Au, activated GO@Au, GO@Au-NH 2 , GO@Au-HBA and GO@Au-PEG are show in Figure 2d. GO shows an obvious absorption peak at 1710 cm -1 , which is the absorption peak of -COOH carbonyl, and a strong absorption peak at 3400 cm -1 , which is the characteristic peak of -OH.
GO@Au shows an obvious absorption peak at 3400 cm -1 , which means there remains a lot of -OH that can react with -OH on GO. When nitrophenyl was introduced and activated GO-Au was obtained, the -OH absorption peak was signi cantly weakened and N-O absorption peak appeared (1382 cm -1 ), indicating that nitrophenyl was successfully attached to GO@Au. Then, after ethylenediamine is conjugated on the activated GO@Au by an ester bond, the characteristic absorption peak of C-N appears at 1140 cm -1 , the absorption peak of N-H bond appears at 1624 cm -1 and 3400 cm -1 , and the absorption peak of N-O bond at 1382 cm -1 weakens, indicating the successful synthesis of GO@AU-NH 2 . When -NH 2 is introduced into GO@Au, the HBA links to GO@Au through the reaction between -COOH and -NH 2 to form amide bonds.
Compared with GO@Au-NH 2 , GO@Au-HBA has absorption peaks at 1661 cm -1 and 1388 cm -1 . Meanwhile, the absorption peaks of -NH 2 at 1600 cm -1 and 992 cm -1 prove the existence of N-N bonds, indicating the successful synthesis of GO@Au-HBA. Finally, CHO-PEG2000 generates pH-sensitive hydrazone by reaction of aldehyde group with hydrazine group on HBA, and absorption peaks appear at 1716 cm -1 ,1241 cm -1 and 1600 cm -1 , indicating successful preparation of GO@Au-PEG.

Characterization of GO@Au-PEG/DOX
As shown in Figure 3a, DOX has strong absorption peaks at 252 nm and 480 nm, GO@Au-PEG has a strong absorption peak at 527 nm, GO@Au-PEG/DOX has strong absorption peaks at 252, 480 and 527 nm. The mean Zeta potential of GO@Au-PEG/DOX was 35.5±2.6 mV, indicating that the Zeta potential of GO@Au-PEG/DOX was stable.
GO@Au, GO@Au-PEG and GO@Au-PEG/DOX are all well dispersed in water. After one week of placement, the GO@Au sample had partial precipitation, while the Go@Au-PEG sample had no aggregation precipitation. This indicates that PEG improves the water solubility of GO@Au. In addition, GO@Au-PEG and GO@Au-PEG/DOX was stable in normal saline, medium, fetal bovine serum and PBS solution, and will not accumulate for several weeks.

Photothermal effect and pH sensitivity
The heating condition of GO@Au under 808 nm laser is shown in Figure 4a. When the water is irradiated by 808 nm laser, the temperature is almost unchanged and there is no obvious thermal effect. The solution temperature of GO and GO@Au increased signi cantly under the irradiation of 808 nm laser, and the heating e ciency of GO@Au solution was much higher than that of GO solution. This indicates that Au nanoparticles loaded on the GO surface signi cantly improve the heating e ciency.
The pH sensitivity of PEG in GO@Au-PEG is caused by the break of hydrazone bond in acidic environment, and its pH sensitivity in vitro is shown in Figure 4b. Photostimulation is an effective stimulus to achieve controlled drug release. In this study, 808 nm laser irradiation of GO@Au was used to generate near-infrared light and release DOX from go@Au-PEG /DO drug delivery system. As shown in Figure 5, under laser irradiation, the DOX release rate from GO@Au-PEG/DOX is very fast. When there is no laser irradiation, the release rate of DOX slows down signi cantly.
At the same time, in the presence of laser irradiation, the DOX release rate of pH 5.5 group was signi cantly faster than that of pH 7.4 group. This indicates that laser irradiation under acidic conditions is conducive to the rapid release of DOX, which can release 80.1%.

Cellure uptake
The uptake of MCF-7 cells into the GO@Au-PEG/DOX controlled release system and the near-infrared controlled release of DOX by the system were studied using the red uorescence DOX as the marker. As shown in Figure 6, GO@Au-PEG/DOX group and GO@Au-PEG*/DOX group after 4 h incubation in the complete medium pH7.4, only very weak burning red light signals, shows that only a tiny amount of DOX into MCF-7 cell. The GO@Au after PEG modi cation, big space steric of PEG seriously hindered the MCF-7 cell uptake of drug delivery system. Compared with the GO@Au-PEG/DOX (pH7.4) group, the uptake of MCF-7 cells was much faster in the GO@Au-PEG/DOX (pH5.5) group, because PEG was removed due to the pH5.5 rupture of pH-sensitive hydrazone bonds in the weakly acidic environment (pH5.5), and the uptake of GO@Au-PEG/DOX by McF-7 cells was accelerated. GO@Au-PEG*/DOX (without pH-sensitive hydrazone bonds ) did not have this sensitivity, and uptake by MCF-7 cells did not differ signi cantly between pH 7.4 and pH 5.5. Once inside the cell, DOX is quickly transferred to the nucleus, where it inserts into the DNA and interferes with its synthesis, killing tumor cells. According to Figure 6, the red and yellow light in the GO@Au-PEG/DOX (pH 5.5) group after incubation for 4 h was mainly concentrated in the cytoplasm, indicating that a large amount of DOX was not released from the GO@Au-PEG/DOX drug delivery system. However, when the GO@Au-PEG/DOX (pH 5.5) group entered the cells at 4 h and was irradiated with 808 nm laser for 10 min, strong red light appeared in the nucleus, because the 808 nm laser radiation accelerated the release of a large amount of DOX from the GO@Au-PEG/DOX and the released DOX was transferred to the nucleus quickly. The above results indicated that the weakly acidic environment was conducive to the uptake of GO@Au-PEG/DOX by MCF-7 cells, and the controlled release of DOX by GO@Au-PEG/DOX drug delivery system could be realized by 808 nm laser irradiation group after incubation for 4 h was mainly concentrated in the cytoplasm, indicating that a large amount of DOX was not released from the GO@Au-PEG/DOX drug delivery system. However, when the GO@Au-PEG/DOX (pH5.5) group entered the cells at 4 h and was irradiated with 808 nm laser for 10 min, strong red light appeared in the nucleus, because the 808 nm laser radiation released a large amount of DOX from the Go@Au-PEG/DOX controlled release drug delivery system and was transferred to the nucleus quickly. The above results indicated that the weakly acidic environment was conducive to the uptake of GO@Au-PEG/DOX by MCF-7 cells, and the controlled release of DOX by GO@Au-PEG/DOX drug delivery system could be realized by 808 nm laser irradiation.

Cytotoxicity of GO@Au-PEG
The effects of GO, GO@Au and Go@Au-PEG at different concentrations on McF-7 cells after 24 h were shown in Figure 7a. As can be seen from the gure, with the increase of GO, go@Au and Go@Au-PEG concentration, the inhibition effect on MCF-7 cells was gradually presented to a certain extent. However, when the concentration of GO, GO@Au and GO@Au-PEG was 100 μg/ml, the inhibition rate of the cells was still less than 15%, indicating that GO, GO@Au and GO@Au-PEG had no obvious toxicity on MCF-7 cells. Figure 7b shows the effects of GO and G0@AU-PEG at different concentrations on MCF-7 cell growth without and with laser irradiation. In the absence of laser irradiation, the two drugs had little inhibitory effect on cell growth. Under laser irradiation, the growth inhibition of MCF-7 cells in GO group and GO@Au-PEG group increased with the increase of concentration. In addition, at the same concentration, the GO@Au-PEG group had more signi cant inhibition on cell growth than the GO group, indicating that GO@Au-PEG had stronger photohyperthermia effect under laser irradiation and had a good hyperthermia killing effect. Meanwhile, in the absence of laser irradiation, the growth inhibition effect of DOX at all concentrations on MCF-7 was higher than that of GO@Au-PEG/DOX, which was caused by the slow release of DOX from GO@Au-PEG/DOX. However, under laser irradiation, the growth inhibition of MCF-7 cells by high concentration of GO@Au-PEG/DOX was signi cantly enhanced, but the effect was not obvious at low concentration, because the heat generated by GO@Au at low concentration was less and could not kill the tumor cells. When DOX concentration was 10 μg/ml, the survival rate of MCF-7 cells in the GO@Au-PEG/DOX group under laser irradiation was only about 10%. This is because both hyperthermia and chemotherapy enhance the therapeutic effect at the same time, the laser promotes the release of DOX, again enhancing the therapeutic effect.

Tumor growth inhibition in vivo.
In order to investigate the anti-tumor activity of GO@Au-PEG/DOX in vivo, S180 tumor-bearing mice were used as the animal model in this chapter to investigate the tissue distribution characteristics and pharmacodynamic characteristics of the mice.
Several successful tumor-bearing mice were given free water and fasted for 12 hours. Their main organs (heart, liver, spleen, lung, kidney and tumor) and blood were collected separately and weighed, then saline was added to each sample (saline weight: organ weight = 1:1) and homogenized. CO@Au-PEG/DOX (5 mg) was dispersed in 1 ml of water and placed in a dialysis bag. The dialysis bag was then immersed in 10 ml of the homogenates of the different organs and kept in a horizontal laboratory shaker. After 48 h, 500 μl of homogenate was removed, added to a chloroform-methanol mixture (chloroform:methanol = 4:1, 2 ml). After centrifugation at 4000 rpm for 20 min, the chloroform layers were collected and the concentrations of DOX were determined by high-performance liquid chromatography.
As shown in Figure 9, the DOX release in blood, heart, liver, spleen, lung and kidney is very slow, with less than 24% of DOX released after 48 h. The DOX release in Tumor is very fast, with more than 55%of DOX released after 48 h. This indicates that DOX in tumor can be released by the hydrolysis of GO@Au-PEG/DOX on hydrazone bond.
The weight change curve of mice in each group during treatment was shown in Figure 10. Only DOX group mice lost weight, while the weight of other mice did not. The weight loss of mice in DOX group was due to the low selectivity of chemotherapy drug DOX in treatment, which not only killed tumor cells but also caused damage to normal cells in the body, thus causing weight loss. The mice in the GO@Au-PEG/DOX+NIR group lost a little weight (still larger than the initial weight) at the end of treatment, which may be due to the effect of reduced tumor size on weight. At the later stage of treatment, the DOX group and the blank control group showed reduced activity and dull hair color, while the remaining 5 groups showed normal behavior and hair color. The results showed that the GO@Au-PEG prepared in the experiment had low toxicity and could be applied in vivo as a drug transport carrier, which could also improve the selectivity of chemotherapy drug DOX and reduce its toxic and side effects.
Tumor volume changes in each group during administration were shown in Figure 11. During the treatment, tumors in mice of normal saline group, saline + NIR group, GO@Au-PEG group were increased rapidly. At the end of the treatment, tumor volume in the saline group was about three times that at the beginning. No signi cant changes were observed in saline + NIR group and GO@Au-PEG group compared with the control group. This suggests that NIR irradiation alone could not effectively inhibit tumor growth and GO@Au-PEG alone was not effective in inhibiting tumor growth in the absence of NIR. The tumor volume in the GO@Au-PEG + NIR group at the end of administration was about 2.1 times that at the beginning of administration, indicating that GO@Au-PEG + NIR had a certain hyperthermia effect. Tumor growth was slow in the DOX group, and tumor volume at the end of administration was about 1.9 times that at the beginning of administration, indicating that chemotherapy alone could not effectively treat tumors. The tumor volume in the GO@Au-PEG/DOX group at the end of administration was about 1.1 times that at the beginning of administration, indicating that the GO@Au-PEG/DOX controlled release drug delivery system could deliver DOX to the tumor site with certain tumor targeting. The tumor volume of the GO@Au-PEG/DOX + NIR group at the end of administration was about 52% of that at the beginning of administration, and some of the tumors in mice had been completely eliminated, showing a signi cant tumor suppressive effect.

Conclusion
1. PEG can be successfully removed from the GO@Au-PEG system in a weak acidic environment, with good pH sensitivity. At the same time, under laser irradiation, the heating e ciency of GO@Au was signi cantly higher than that of GO alone, thus con rming that Go@Au-PEG has a good photothermal effect and can be used in the hyperthermia of tumors.
2. Under laser irradiation, GO@Au-PEG/DOX can release more DOX, and under certain conditions, DOX release is related to laser irradiation power, laser irradiation time, and the pH of the medium.
3. Go@Au-PEG system has little toxicity in vitro in the absence of laser irradiation, so it can be used as a drug carrier. However, in the absence of laser irradiation, the toxicity of GO@Au-PEG system on MCF-7 cells is signi cantly increased, showing a good photothermal treatment effect. The growth inhibition of MCF-7 in the GO@Au-PEG/DOX group was signi cantly increased after laser irradiation.
4. Go@Au-PEG was more likely to aggregate at tumor sites in mice, showing signi cant targeting. Under laser irradiation, GO@Au-PEG/DOX tumor targeted and controlled release drug delivery system had an obvious tumor suppressive effect. By the end of treatment, more than half of the tumors in mice had been completely eliminated, and tumor volume had decreased by about 52% on average, showing good effects of hyperthermia and chemotherapy.

Methods
The particle size, zeta potential and morphology of GO@Au-PEG/DOX were TEM were characterized by DLS and TEM. A UV-Vis spectrometer was used to characterize the optical properties of GO@Au-PEG and GO@Au-PEG/DOX. . Fourier transform infrared (FTIR) spectra were recorded with spectrophotometer. Au in GO@Au was measured with an inductively coupled plasma emission spectrometer (ICP). The photothermal e cacy of GO@Au-PEG/DOX was measured using a thermal infrared imager. An appropriate amount of sample powder was put into the platinum crucible, and the minimum temperature was set as 25℃, the maximum temperature as 800℃, and the temperature rising speed was set as 20℃/min. Under the protection of nitrogen, the samples were measured and recorded by thermal gravimetric analysis (TGA), and the PEG content in each sample was calculated.
To evaluate pH sensitivity, 10 mg samples were placed in phosphate buffer solutions with pH values of 7.4 and 5.5 respectively, centrifuged at room temperature for 4 h, and then dried at 60 ℃ for 24 h, and then the PEG content was measured by TGA.
The concentrations of DOX were measured at 480 nm by UV-Vis spectrometer. GO@Au-PEG/DOX was sealed in dialysis membranes (MW=8000~14000). The dialysis bags were incubated in 10 ml PBS buffer at 37 ℃ with a stirring rate of 100 r/min. A 200 μl portion of the aliquot was collected from the incubation medium at predetermined time intervals, and the released DOX was quanti ed by absorption spectroscopy recorded on UV Vis spectrophotometer at 490 nm. The dose released was quanti ed by collecting 200 μl portion of the aliquot from the culture medium at predetermined intervals and recording them with an UV-Vis spectrophotometer at 480nm. The DOX release studies were performed in triplicate for each of the samples. irradiated with 808 nm laser (2 W/cm 2 ) for 10 min as a control experiment.

Cytotoxicity Assay
The cytotoxicity of DOX, GO@Au-PEG and GO@Au-PEG/DOX against MCF-7 cells was assessed by using the standard SRB assay. The MCF-7 cells were cultured and lifted as described above before seeding (6×10 3 ) into 96-well plates (5% CO 2 , 37 ℃) and incubating for 24 h. When the cells were monolayer full of cell plate hole bottom, the original culture medium was taken out, and the medium containing different concentrations of drugs was added. 6 compound Wells were set at each concentration, and blank controls were set. The 96-well plates were taken out and the medium was moved out of the wells. Each well was washed three times with 100 L PBS buffer. Then 200 L precooled TCA solution with a nal concentration of 10% was added to the wells. After xing, wash with distilled water and dry naturally. Then, 0.4% SRB acetic acid solution of 100 L was added to each well for staining. The solution was allowed to stand at room temperature in darkness for 20min and then washed with 1% acetic acid.After drying, 10 mol/L Tris base solution of 150 L was added to each well. The cells were placed in an oscillator and oscillated at 37 ℃ for 10 min to dissolve the protein-bound SRB.Finally, the OD value of each well was measured at 515 nm with a microplate reader, so as to calculate the survival rate and inhibition rate of cells.

Statistical Analysis
Quantitative data are expressed as mean ± SD and analyzed by use of Student's t test. When P values < 0.05 were considered statistically signi cant.

Declarations
Ethics approval and consent to participate: Written informed consent was obtained from individual or guardian participants.

Consent for publication: Not applicable
Competing interests: The authors declare no interests con icts.          Changes in tumor relative volume.