PEGylated nano-graphene oxide as a nanocarrier for delivering mixed anticancer drugs to improve anticancer activity

Due to their high specific surface area, graphene oxide and graphene oxide-base nanoparticles have great potential both in dual-drug delivery and combination chemotherapy. Herein, we developed cisplatin (Pt) and doxorubicin (DOX) dual-drug-loaded PEGylated nano-graphene oxide (pGO) to facilitate combined chemotherapy in one system. In this study, nano-sized pGO-Pt/DOX ranged around 161.50 nm was fabricated and characterized using zeta-potential, AFM, TEM, Raman, UV-Vis, and FTIR analyses. The drug delivery efficacy of Pt was enhanced through the introduction of pGO, and the final weight ratio of DOX: Pt: pGO was optimized to 0.376: 0.376: 1. In vitro studies revealed that pGO-Pt/DOX nanoparticles could be effectively delivered into tumor cells, in which they induced prominent cell apoptosis and necrosis and exhibited higher growth inhibition than the single drug delivery system or free drugs. The pGO-Pt/DOX induced the most prominent cancer cell apoptosis and necrosis rate with 18.6%, which was observed almost 2 times higher than that of pGO-Pt or pGO-DOX groups. in the apoptosis and necrotic quadrants In vivo data confirmed that the pGO-Pt/DOX dual-drug delivery system attenuated the toxicity of Pt and DOX to normal organs compared to free drugs. The tumor inhibition data, histopathology observations, and immunohistochemical staining confirmed that the dual-drug delivery system presented a better anticancer effect than free drugs. These results clearly indicated that the pGO-Pt/DOX dual-drug delivery system provided the means for combination drug delivery in cancer treatment.


Results
Synthesis, preparation and characterization. Synthesis of dual-drug delivery system (pGO-Pt/DOX) consisted of the following steps (Fig. 1A). Firstly, PEGylated nano-graphene oxide (pGO) was synthesized with PEG covalent binding to GO via amide linkage 12 . As shown in Fig. S1 and Table 1, nano-sized (146.1 nm) pGO was fabricated, and the zeta potential of GO increased from −36.8 ± 7.3 mV to −16.8 ± 0.87 mV, indicating that the 4-arm PEG-amine banded and neutralized some negatively charged carboxylic acid groups in GO 12 .
Then, cisplatin was reacted with H 2 O 2 and succinic anhydride to form Pt(NH 3 ) 2 Cl 2 (OOCCH 2 CH 2 C-OOH) (OH) (abbreviated as Pt(IV) or Pt), and the Pt-loaded pGO (pGO-Pt) nanoparticles was accomplished by a covalent reaction 19 . The coupling reaction was achieved in the presence of EDC and NHS as the activating agent for the carboxylic group at room temperature (Fig. 1A). As shown in Fig. 2 and Table 1, nanoscale pGO-Pt was achieved ( Fig. 2A) with the size slightly increased from 146.10 nm (pGO) to 154.39 nm after the loading of Pt (Table 1). Furthermore, the attachment of Pt, which could be prompted in TEM images ( Fig. 2A), because of a portion of positively charged amino groups on pGO, the Zeta potential showed a decreased trend (Table 1) 20 . Raman spectroscopy (Fig. 2B) gives a nondestructive way to characterize the graphene and its derivatives. In Fig. 2B, the G and D bonds of graphene could be found at 1580 and 1340 cm −1 , indicating that no substantial structural damage emerged during the modification procedure. A trend was observed from pGO to pGO-Pt, with a tiny difference at 3209 cm −1 , which was in good agreement with the peak of Pt. The results indicated that Pt was attached to the pGO without affecting the basic structure. In Fig. 2C, the attachment of Pt onto pGO was evident from the UV-Vis spectra of pGO-Pt solution, which showed the characteristic absorption peaks of Pt at 204 nm. FTIR spectra (Fig. 2D) of pGO, pGO-Pt, and Pt nanoconjugates were also collected. The characteristic CO-NH at 1565 and 1650 cm −1 and NH at 2920 cm −1 were detected in pGO, which may owing to the formation of amide linkage between GO and polyethylene glycol 21,22 . The spectrum of pGO-Pt was almost the same as that of pGO, however, the typical but weakened Pt absorption (1639, 1304, and 800 cm −1 ) was observed 23,24 . All these results indicated the successful incorporation of Pt into pGO.
Finally, in Fig. 1, the doxorubicin hydrochloride (DOX) was adhered to pGO-Pt nanoparticles by a π-π stacking interaction between the large π conjugated from GO and quinone portion of DOX 25 . As shown in Fig. 3A, the dispersive pGO-Pt/DOX nanoparticles with a narrow distribution of particle sizes were achieved. The size of pGO-Pt/DOX (159.2 ± 7.0 nm) was slightly increased compared to that of pGO-Pt (151.1 nm, Table 1). Furthermore, the slightly increased zeta potential might be attributed to the addition of amino groups in DOX after the conjugation of DOX to pGO-Pt nanoparticles 26 . These could be evidence for the successful conjugation. Then, more results of the interaction between pGO-Pt and DOX was provided by UV-Vis absorption spectra (Fig. 3B,C). In Fig. 3B, the absorption peaks of DOX were located at 232, 253, 291 and 480 nm 27 . After forming the dual-drug system, the UV-Vis spectra of pGO-Pt/DOX not only confirmed the stacking of DOX onto pGO-Pt nanoparticles but also showed the red-shift due to the interaction 28 . All these results demonstrated that DOX molecules were successfully loaded onto pGO-Pt, and the nanoscale dual-drug delivery system was formed.    33 and Jianmin Shen (For DOX release) 34 . These results suggested that both Pt and DOX release kinetics from pGO-Pt/ DOX nanoparticles were pH-dependent, and such environmentally sensitive behavior could prevent drug loss during delivery and enhance drug release after reaching the tumor site.
In vitro cell viability and cytotoxicity. To investigate in vitro the anticancer effect of pGO-Pt/DOX nanoparticles, the cell viability and cytotoxicity of PBS, pGO, pGO-Pt, pGO-DOX, pGO-Pt/DOX, and Pt/DOX mixture (1-20 μg/mL) were tested in CAL-27 and MCF-7 cancer cells. The cell viability of PBS was set as 100%. In Fig. 4A-D, the empty pGO revealed a high viability (above 90%) to CAL-27 and MCF-7 cells at 1-20 μg/mL after 24 and 48 h, and even at a concentration as high as 100 μg/mL (Fig. S2), the L929 cell viability still reached 94.3%. The results indicated that the PEG-functionalized GO is a safe drug carrier 2,4,7,26 . Then, the cell viability of all drug delivery systems and pure drug samples exhibited a dose-dependent pattern after incubation for 24 and 48 h. It is encouraging that the anti-tumor activity of the dual-drug delivery system (pGO-Pt/DOX nanoparticles) was obviously higher than that of the single-drug delivery system (pGO-Pt and pGO-DOX), indicating that the anti-tumor ability might be enhanced by using the confederate delivery of two drugs. The IC 50 values of the drug delivery system and mixed drug were assessed in Table 2 and the following results were observed: First, the IC 50 value of pGO-Pt/DOX was higher than that of pGO-Pt and pGO-DOX at 24 h and 48 h for both CAL-27 and MCF-7 cells; Second, the IC 50 value of pGO-Pt/DOX was lower than that of mixed drug at 24 h for both CAL-27 and MCF-7 cells. However, pGO-Pt/DOX displayed a comparable IC 50 value to mixed drug for the CAL-27 cells after 2 days. The reason is that the dual-drug delivery system was first disassembled after being internalized by cancer cells (Fig. 1B). Once enough drug released and accumulated within the tumor cells, the anticancer activity would be improved 35 . Moreover, the amount of the drugs in mixture drug (Pt/DOX) is 2.33-fold higher than the drugs in pGO-Pt/DOX (weight ratio of DOX: Pt: pGO = 0.376: 0.376: 1), that means 2.33 times the amount of mixture drug could only obtain comparable cytotoxicity compared with pGO-Pt/DOX system. In view of the above results, we used a more sensitive cancer cell line (CAL-27) for further studies.
In vitro cellular uptake. Cellular uptake of DOX-labeled pGO-Pt/DOX and Pt/DOX mixture by CAL-27 human squamous cell carcinoma cell line was examined by fluorescence imaging (Fig. 4E). When CAL-27 cells were incubated with pGO-Pt/DOX, highlighted red fluorescence (DOX) was observed in the nucleus and cytoplasm. However, when CAL-27 cells were cultured with the Pt/DOX drug mixture, the red fluorescence mainly appeared in the cytoplasm, with obviously reducing of fluorescence intensity in the cytoplasm and nucleus. The results illustrated that the GO-based dual-drug delivery system could be effectively delivered into targeted tumor cells due to their higher efficiency of endocytosis than small molecular drugs 36 .
In vitro cell apoptosis assay. The result of further cell apoptosis evaluation are shown in Fig. 5. After incubating with the drug carrier for 4 h, the pGO showed a negligible effect on cell apoptosis and necrosis, revealing the excellent biocompatibility. And pGO-Pt and pGO-DOX nanoparticles induced 1.8 and 1.9 times higher cell apoptosis and necrosis than that of the pGO, respectively. Furthermore, pGO-Pt/DOX showed the highest cell apoptosis and necrosis compared with any other groups, which achieved 18.6% from data analysis (Fig. 5E) 37 .
Although the mixed drug induced less cell apoptosis and necrosis, no significant differences were found between the pGO-Pt/DOX and Pt/DOX mixtures. According to the apoptosis assays, the pGO-Pt/DOX showed markedly apoptotic and necrotic effects, which verified the conclusion of CCK-8 assay (Fig. 4A,B) and fluorescence imaging (Fig. 4E). www.nature.com/scientificreports www.nature.com/scientificreports/ In vivo antitumor efficacy. Our in vitro studies suggest pGO-Pt/DOX nanoparticles might has better anti-cancer efficacy. The CAL-27 tumor models were generated by subcutaneous injection of CAL-27 cells into nude mice. After the tumor volumes reached 50-100 mm 3 , mice were subjected to intravenous administration with drugs every 3 days, for a total of seven injections.   www.nature.com/scientificreports www.nature.com/scientificreports/ The effective tumor targeting of pGO-Pt/DOX was performed by a quantification analysis of both Pt and DOX amounts in tumor after 12 h post-injection. As shown in Fig. 6A, the concentrations of Pt and DOX in tumor for Pt/DOX mixture administration group were 3.06 and 3.45 μg/mL, respectively, while the concentrations reached 4.87 and 4.43 μg/mL for Pt and DOX dual-drug delivery system, indicating that the amounts of Pt and DOX accumulated in the tumor for pGO-Pt/DOX were slightly higher than that for the Pt/DOX mixture. It should be noted that the amount of the drugs in the Pt/DOX mixture is 2.33-fold higher than that in pGO-Pt/DOX. Therefore, the above results suggested that dual-drug delivery system could accumulate in the tumor cells more efficiently.
The change in body weight was monitored to investigate the drug safety throughout the therapeutic period 20 , and the results were depicted in Fig. 6B. The body weights in the pGO-DOX/Pt and pGO groups showed a slow and sustained increase with no indication of toxicity versus the control group. However, mice receiving Pt/DOX mixture showed obvious weight loss, implying the side effects were produced by the mixture drugs. The typical H&E staining slices of organs were also monitored after the treatment (Fig. 6C). No significate histopathological changes were found in the main organs from the pGO-DOX/Pt, pGO and control groups (PBS). However, a cardiac section from Pt/DOX-treated sample showed marked damage changes including myocardial congestion and cardiomyocyte hypertrophy, indicating a considerable amount of DOX accumulated in heart tissue and caused evident cardiac toxicity 38 . Moreover, the obvious histopathological changes of the spleen were found in Pt/DOX-treated mice, suggesting the accumulation of Pt 20,39 . Additionally, blood chemistry was also introduced to analyze the www.nature.com/scientificreports www.nature.com/scientificreports/ hepatic function, nephrotoxicity, and cardiac damage of tumor-bearing nude mice that were treated with PBS, pGO, pGO-Pt/DOX and Pt/DOX. The data were collected in Table 3. Mice treated with pGO and pGO-Pt/DOX showed no significant differences in all these parameters compared with controls, indicating no obvious toxicity to the liver, kidney and heart, which was consistent with H&E staining results. As suggested by H&E staining, the cardiac damage of Pt/DOX-treated mice agreed with the significant changes of CK and LDH (Table 3). Although no obvious histopathological changes in kidney were observed, the BUN levels of Pt/DOX mixture group were 3-fold higher than that of control group (PBS), and this increase in BUN might indicate the beginning of nephrotoxicity and was consistent with previous study 40 . The above results indicated that the dual-drug delivery system substantially attenuated the toxicity of Pt and DOX to normal organs compared to free drugs.
Then  www.nature.com/scientificreports www.nature.com/scientificreports/ (PBS) and pGO groups exhibited non-uniform dense cellularity and huge cell nuclei, indicating the tumors from these two groups have vigorous proliferative capability 20 . Compared with the PBS-treated group, both therapeutic groups showed uniform dense cellularity and less huge cell nuclei, which indicated that treating tumor with Pt and DOX resulted in tumor cells inhibition 34 . It was noteworthy that fewer tumor cells, smaller cell nuclei, and the formation of tumor nests were observed for the mice treated with the dual-drug delivery system. Furthermore, to evaluate the effect of the dual-drug delivery system on tumor proliferation, immunohistochemical staining  Table 3. Biochemical assay in serum (Data presented as mean ± SD, n = 6). www.nature.com/scientificreports www.nature.com/scientificreports/ Ki-67 was performed. As shown in Fig. 7B,C, the percentage of Ki-67 active cells was significant lower in pGO-Pt/ DOX group (1.03 ± 0.76%) than that in PBS (12.27 ± 2.15%), pGO (11.89 ± 3.01%), and Pt/DOX mixture (2.57 ± 0.61%) groups. These results indicated that pGO-Pt/DOX could suppress tumor cell proliferation.

Discussion
Here, we successfully synthesized a dual-drug delivery system using PEG-functionalized graphene oxide (pGO) and anticancer drugs (Pt and DOX). We demonstrated that pGO nanosheets had enhanced biocompatibility and could be used as a safe nanocarrier. Moreover, the surface modified dual-drug system (pGO-Pt/DOX) showed high DLE, controlled release and high cellular uptake properties, and could enhance antitumor effect both in vitro and in vivo.
peG functioned Go is a safe drug delivery carrier. As graphene oxide (GO) has functional groups for better bonding, is excellently dispensability within water, and is easy to manufacture 3 , it is considered highly interesting in biomedical fields including drug delivery 4 . In this study, we introduced GO nanosheets as the carrier of anticancer drugs to fabricate a dual-drug delivery system (Fig. S1). However, nanomaterials might present side effects sometimes 41,42 , and the biological toxicity of GO remains controversial. For example, Matthew and colleagues 43 directly administered the solution of GO into the lungs of mice could cause lung injury. On the other hand, Yanli and colleagues 44 explored the toxicity of graphene oxide (GO) by analyzing the influences of GO on morphology, viability and membrane integrity, and reported that GO had no obvious cytotoxicity for A549 cells. Moreover, Liu and colleagues 45 indicated that PEGylated GO showed distinctive in vivo behaviors, such as reduced reticuloendothelial system accumulation and improved tumor passive targeting effect.
Interestingly, the results in Fig. 4 showed that PEG-functionalized GO (pGO) revealed a high viability to both CAL-27 and MCF-7 cells at 1-20 μg/mL after 24 and 48 h. The viability of L929 cells was still as high as 94.3% when the concentration reached 100 μg/mL (Fig. S2). The pGO also showed a negligible effect on cell apoptosis and necrosis in cell apoptosis assay (Fig. 5). Furthermore, in mice treated with pGO, there was little difference in body weights (Fig. 6B), blood chemistry (Table 3) and hematoxylin-eosin (HE) staining for the main organ ( Fig. 6C) compared to that of PBS group. These results collectively indicated that pGO was highly biocompatible and could be used as the nanocarrier of dual-drug delivery system, which is consistent with the pGO drug delivery studies 2,4,7,26,36,45 . The possible reasons of the highly biocompatibility of pGO were suggested as follows: (1) Surface modification could enhance biocompatibility. Unlike Matthew and colleagues, who used nonfunctional GO in their research, many studies have indicated that PEG modification could lower the cytotoxicity of GO. For instance, it has been shown that pGO was not toxic against Hela or MCF-7 cells 46 and had no effect on cell viability even at concentrations up to 100 mg/L 6 , which is consistent with our results. Liu and colleagues 45 also found that pGO (20 mg/kg) intravenously administered into mice did not cause any organ damage. (2) The nanoscale size of GO was crucial for biological response. In our study, the diameter of all the particles were approximately 140 nm (Table 1), which is considered nanoscale 47 . Su and colleagues 48 showed that both nanoscale (350 nm) and microscale (2 μm) GO could be taken up by macrophages, but only microscale GO initiated a severe inflammatory responses. Based on the above discussion, we suggested that pGO could be used as a safe and effective nanocarrier for dual-drug delivery system. Dual-drug delivery system possesses high DLE, superior cell uptake and controlled release property. The discovery and application of pGO as a carrier for drug loading extending the chemotherapeutic filed 49 . Anticancer drugs, both hydrophobic (Pt) and hydrophilic (DOX), were effectively loaded and protected by the dual-drug delivery system in this study (Figs. 2 and 3). Previous studies showed that DOX has been loaded onto GO by physisorption or chemical conjugation with a high DLE 22 . However, for the delivery of cisplatin, although many drug delivery system [50][51][52][53] have been investigated, the DLE was relatively low. Therefore, building a superior carrier for Pt with high DLE has been the primary challenge. Xueqiong 11 prepared doxorubicin and platinum-base compounds delivery system using carboxymethyl chitosan nanoparticles. Their result showed anticancer effect with the drug loading efficacy (DLE) relatively low. In addition, the size of chitosan nanoparticles (274 nm) was bigger that the pGO (159 nm) in this study, making it less susceptible to achieve endocytosis which may reduce chitosan nanoparticles' anticancer effect. On the other hand, the mesoporous silica nanoparticles (MSN) also had been used to build a doxorubicin and platinum-base compounds delivery system 13 . However, using pGO to transport DOX and Pt could achieve a loading rate of 0.367 mg/mg, which is significantly higher than that of MSN. Moreover, the pGO-Pt/DOX system showed effective antitumor ability in vivo, while the above tow studies only showed enhanced in vitro anticancer properties. In our study, Pt was first covalently bound with pGO followed by the non-covalent loading of DOX (Fig. 1). The results (Fig. 3C) showed that the pGO-based nanoparticles could load Pt with higher DLE (0.376 mg/mg), compared with carbon nanotubes (0.21 mg/mg) 30 and gelatin hydrogels (0.1 to 0.3 mg/mg) 31 . The high DLE could be attributed to the large and negatively charged surface of pGO 4 ( Table 1).
A controlled drug release profile is essential to prolong the plasma half-life of the drug 54 . It is well known that the pH in tumor cell lysosomes, endosomes and tumor microenvironment is acidic 26,55 . Therefore, it is also critical for the drug delivery system to respond to PH. Our study (Fig. 3D) showed that the Pt and DOX release rates were significantly higher at pH 5.3 versus pH 7.4, which suggested that both Pt and DOX release kinetics from dual-drug delivery system were pH-dependent. This pH-dependent release behavior could enhance the permeability and retention (EPR) effect of solid tumor 56 . The EPR effect could be achieved by the following reasons: first, there is a leaking vasculature in the tumor, and the holes in the tumor vessel wall range from 200 nm to 2 μm with an average of about 400 nm 57 . Many studies suggest that 100-200 nm diameter nanoparticles are easier to supply blood to tumors 24,57 . Secondly, the negative charge (Table 1) and PEG segments (Fig. 1) of the drug delivery system prevented themselves from being recognized and prolonged their circulation time in blood, (2020) 10:2717 | https://doi.org/10.1038/s41598-020-59624-w www.nature.com/scientificreports www.nature.com/scientificreports/ which in turn enhanced the EPR effect 58 . Therefore, in this study, the dual-drug delivery system (pGO-Pt/DOX) was PEG-functionalized and the size was suitable for the EPR effect. The EPR effect was then indicated by the in vitro cell uptake assay (Fig. 4E), and this in turn gave a comparable IC 50 value between the pGO-Pt/DOX group and the 3-fold higher amount of the Pt/DOX group (Table 2). These results revealed that the dual-drug delivery system could satisfy the harsh request of the long circulation time in the plasma, maintain their antitumor effect. the superiority of the dual drugs for the cancer cell. In recent years, combination chemotherapy has been adopted as a standard clinical strategy against many types of cancer 14 . In this study, Pt and DOX were chosen to demonstrate the possibility of combination chemotherapy simultaneously on graphene. The carboxylic acid group at the edge of GO allowed amide interactions with Pt, and the basal plane of GO, which was mainly composed of polyaromatic networks, allowed π-π stacking of DOX (Fig. 1). These two drugs are widely used in clinic for combination chemotherapy with a synergistic effect 11,59 . Figure 4A-D demonstrated that the anticancer activity could be improved by using the simultaneous delivery ability. Figure 5 showed that this dual-drug delivery system induced not only apoptosis but also necrosis of cancer cells. Furthermore, in vivo Ki67 staining (Fig. 7) indicated that the cancer cell proliferation has been inhibited by the use of a dual-drug delivery system. The mechanisms might be explained as follows (Fig. 1B): First, the anticancer effects of Pt could be to generate DNA lesions, then inducing cell senescence or apoptosis. Pt was also shown to trigger cell necrosis by arousing cytotoxic effects from both nuclear and cytoplasmic signaling pathway 60 . On the other hand, the anticancer effect of DOX could be to prevent DNA replication, inhibiting cell proliferation activity. DOX was also shown to damage mitochondrial DNA (mtDNA) and led to lower tumor energy supply 38 . Therefore, although the molecular mechanisms underlying the cytotoxic potential of Pt and DOX remain poorly understood, pGO-Pt/DOX dual-drug delivery system has presented a more excellent anticancer effect than either single drug or free drugs.

Materials and Methods
Materials. All chemical reagents used for this study were of analytical grade or above. Graphene oxide Six-week-old female athymic nude mice were obtained from the Laboratory Animal Centre of Sichuan University (Chengdu, China), and were housed in a temperature-controlled environment with 12 h light/dark cycles. All animal procedures were approved by the Institutional Animal Care and Ethics Committee of Sichuan University (Chengdu, China), in accordance with the ARRIVE guidelines. Efforts were taken to ensure that the guiding principles of the three R's were followed.
Characterizations. Transmission electron microscopic (TEM) images were performed using a FEI Tecnai G2 F20 S-TWIN high-resolution transmission electron microscope operating at 200 kV. Atomic force microscopy (AFM, SPM-9600, Shi-madzu, Japan) images were obtained on a Si substrate. The Zeta potential were determined by a Malvern Zeta Sizer (Malvern, NanoZS, UK). Fourier transform infrared (FTIR) spectra were recorded on a Nicolet 6700 FTIR spectrometer (Thermo Fisher Scientific, USA). Raman spectra were taken with a LabRAM HR-800 micro spectrometer system equipped with a 514.5 nm Arþ laser. UV-Vis spectra were performed by a UV-Vis spectrometer (Lambda 35, PerkineElmer, USA). The platinum contents were measured by inductively coupled plasma-optical emission spectroscopy (ICP-OES, Arcos, Germany).

Synthesis and characterization of PEGylated nano-graphene oxide (pGO). PEGylated
nano-graphene oxide (pGO) was first synthesized following a previously published protocol 21,61 . Briefly, nano-sized graphene oxide (NGO) were firstly obtained by soaking 10 mg GO into 5 mL deionized water and sonicating at the power of 570 W for 30 min, followed by adding the sodium hydroxide (NaOH) solution (1.2 g) and chloroacetic acid (CH 2 ClCOOH)(1.0 g). The NGO was then collected, re-dispersed in deionized water and sonicated again at 570 W for 2 h to give a clear solution in an ice bath 61 .
The NGO dispersion (1 mg/mL, 10 mL) was mixed with 10 mg of PEG in a flask by sonicator for 30 min. Then 4 mg of EDC and 6 mg of NHS were added and the mixture was stirred overnight. The PEGylated nano-graphene oxide (pGO) was filtered with MWCO of 100 kDa Millipore filters (Millipore, Bedford, MA, USA) and dried under vacuum. Eventually, the obtained pGO solution was acquired and monitored by zeta-potential analysis, TEM image, AFM image and UV-Vis spectra (Fig. SI).
oxidized with H 2 O 2 then reacted with succinic anhydride to form c,c,t-Pt(NH 3 ) 2 Cl 2 (OOCCH 2 CH 2 COOH)(OH) (abbreviated as Pt(IV) or Pt) followed the protocol reported from previous study 19 . To synthesize the pGO-Pt nanoparticles, EDC and NHS were firstly added into 0.1 mg/mL pGO (m(EDC):m(NHS):m(pGO) = 0.4:0.6:1), followed by the addition of Pt, then the mixture was bath sonicated for 30 min and stirred vigorously overnight at room temperature. The as-prepared pGO-Pt was purified by dialysis (MW cutoff of 3500) for 48 h and repeated ultrafiltration, and dried under vacuum. All the supernatant and the washing liquor after dialysis and ultrafiltration were collected. Afterwards, a series of evaluations (Zeta-potential, AFM, TEM, Raman spectrometry, UV-Vis spectra, and FTIR spectroscopy) were conducted to characterize the resultant pGO-Pt nanoparticles. All experiments based on Pt were protected from light to avoid its decomposition.
Synthesis and characterization of pGO-Pt/DOX. To synthesize the dual-drug delivery system, DOX (10 mg) was dissolved into 10 mL deionized water solution. Then the solution of DOX was added to the solution of pGO-Pt, and the pH value was adjusted to 8 with NaOH solution 61 . The mixture was stirred at room temperature under light-sealed conditions for 24 h. After the reaction was completed, the products were purified by dialysis for 48 h and repeated ultrafiltration, then dried under vacuum. All the supernatant and the washing liquor after dialysis and ultrafiltration were collected. Finally, the pGO-Pt/DOX dual-drug delivery system was evaluated by zeta-potential analysis, AFM, TEM, and UV-Vis spectra. All experiments were protected from light to avoid drugs decomposition.

Drug loading efficacy (DLE) of Pt and DOX.
To determine the DLE, each series of experiments was carried out in triplicate under the corresponding test conditions (Supporting Information, SI). According to the DLE curve, we optimized the initial amount of DOX to make the finally weight ratio of DOX: Pt: pGO = 0.376: 0.376: 1 (Fig. 3C).
In vitro dual-drug release from the pGO-Pt/DOX nanoparticles. The pGO-Pt/DOX nanoparticles (6 mg) were dispersed in 6 mL of phosphate buffer solution (PBS, pH = 7.4) and the dispersion was divided into two equal aliquots. The pGO-Pt/DOX samples used for release study were transferred into a dialysis bag, which were kept in 200 mL of aqueous solution under constant stirring with pH 7.4 and 5.3, respectively. At certain time intervals, a portion of release medium (2 mL) was taken out for characterization and then fresh buffer (2 mL) was added to keep the volume constant. The amount of released Pt was determined by ICP-OES and the amount of released DOX was measured by UV-vis spectroscopy at 480 nm.

cell viability and cytotoxicity in vitro. Cell viability of pGO.
To investigate cell viability of the drug carrier material (pGO), the murine fibroblasts cell line (L929) was seeded into 96-well plate with a concentration of 1 × 10 4 cells/well and incubated for 24 h. After replacing the medium, different concentration of pGO was added into the plates at concentrations ranged from 0 to 100 μg/mL. The cells were incubated for 24, 48, and 72 h. Then, cell viability was assessed by cell counting kit-8 assay (CCK-8) according to the protocol suggested by the manufacturer. The optical density (OD) of the samples at a wavelength of 450 nm was measured by microplate reader (Thermo, Varioskan Flash). A triplicate analysis was induced from three independent experiments (n = 3).
Cytotoxicity of pGO-Pt/DOX nanoparticles. To assess the cytotoxicity and tumor targeting of pGO-Pt/DOX nanoparticles, CAL-27 and MCF-7 cell lines were chosen to incubate with PBS, pGO, pGO-Pt, pGO-DOX, pGO-Pt/DOX, and free drugs (Pt/DOX mixture, m(Pt):m(DOX) = 1:1) in vitro. In generally, CAL-27 and MCF-7 cells were firstly seeded and incubated at 37 °C for 24 h. Then PBS, pGO, pGO-Pt, pGO-DOX, pGO-Pt/DOX, and Pt/DOX were administrated at five final concentrations of 1, 2, 5, 10, and 20 μg/mL. After incubated for 24 and 48 h, the cell cytotoxicity was determined by CCK-8 assay according to the manufacturer suggested procedures. A triplicate analysis was induced from three independent experiments (n = 3). After the determination of half maximal inhibitory concentration (IC 50 ), we chose CAL-27 cell line in the following study.
In vitro cellular uptake. To investigate the cellular uptake of the dual-drug delivery system by means of fluorescence microscopy, CAL-27 cells were seeded into 24-well plates at 1 × 10 5 cell/mL. Twenty-four hours later, each well was incubated with 5 μg/mL DOX-labeled pGO-Pt/DOX and Pt/DOX mixture for 24 h at 37 °C. At the end of the incubation period, the solutions were removed, and the cells were stained with DAPI and rinsed three times with cold PBS. Then, the cells were observed under a fluorescence microscope (Leica, Wetzlar, Germany).
Cell apoptosis assay. The quantitative analysis of cell apoptosis induced by dual-drug delivery system was performed by Annexin V-FITC/PI double staining. CAL-27 cells were seeded in 6-well plates at a density of 5 × 10 5 cell/mL. After 24 h incubation, cells were treated for 4 h with PBS, pGO, pGO-Pt, pGO-DOX, pGO-Pt/DOX, and Pt/DOX mixture, the final concentration of the drugs or PBS was 5 μg/mL. Then, treated cells were harvested, washed with cold PBS, suspended in 400 μL of binding buffer, and stained with 5 μL of Annexin V-FITC for 15 min and 5 μL of PI for 5 min at 4 °C in the dark. The stained cells were then analyzed by a flow cytometer (Beckman Coulter, Fullerton, CA, USA).
In vivo antitumor efficacy. Animal preparation. All animal procedures were approved by the Institutional Animal Care and Ethics Committee of Sichuan University (Chengdu, China), in accordance with the ARRIVE guidelines. The CAL-27 tumor models were generated by the subcutaneous injection of 1 × 10 6 cells into the dorsal right side of six-week-old female nude mice. When the tumor volume reached 50-100 mm 3 62,63 , the mice were (2020) 10:2717 | https://doi.org/10.1038/s41598-020-59624-w www.nature.com/scientificreports www.nature.com/scientificreports/ divided into four groups (five mice in each group), minimizing the differences in weights and tumor sizes in each group. The mice were subjected to intravenous administration with (a) PBS (200 μL), (b) pGO (10 mg/kg, 200 μL), (c) pGO-Pt/DOX (10 mg/kg, 200 μL) and (d) Pt/DOX mixture (m(Pt):m(DOX) = 1:1, 10 mg/kg, 200 μL) every 3 days, for a total of seven injections. Tumors were measured and mice were weighed every two days over a period of 3 weeks. The volume of the tumor (V) was calculated using the following formula 64 : where V is the tumor volume, L is the tumor length, and W is the tumor width. In addition, the drug accumulation of pGO-Pt/DOX and Pt/DOX mixture were performed by a quantification analysis of Pt and DOX amounts in tumor tissue 12 h post-injection.
Analysis of tumor and blood chemistry. Three weeks later, the mice were anesthetized and blood samples were collected from the vein of the fundus oculi before being euthanatized. Liver function was determined by the alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP); nephrotoxicity was evaluated with blood urea nitrogen (BUN); cardiac damage was assayed by lactate dehydrogenase (LDH) and creatinine (CK) using a Biochemical Autoanalyzer (Type AU680, Beckman Coulter, USA) 65,66 .
Major organs including tumor, heart, liver, spleen, lung, and kidney were harvested and fixed in 4% paraformaldehyde overnight at 4 °C. These samples were then dehydrated through an ascending ethanol series prior to paraffin embedding. Eight micrometer sections were cut and collected on Superfrost-plus slides. Tissue sections prepared for histology and immunohistochemistry were performed by one individual and then were quantified by a blinded individual. Hematoxylin and eosin (H&E) staining for the major organs and immunohistochemistry for tumors were performed as previously described 67 , and a rabbit anti-mouse monoclonal antibody Ki67 was used as the primary antibody. To quantify Ki-67 expression, areas in each tumor sample were randomly selected and the Ki-67 labeling index was calculated as the number of Ki-67-positive cells/total number of cells. All images were acquired by using a Nikon microscope (Eclipse 80i, Japan).

conclusion
In this study, we developed a dual-drug delivery system using PEG-functionalized graphene oxide (pGO) and antitumor drugs (Pt and DOX). We employed zeta-potential, TEM, and Raman, UV-Vis, and FTIR investigations to characterize the as-prepared pGO-Pt/DOX, and the results exhibited that nano-sized pGO-Pt/DOX was successfully fabricated. The drug delivery efficacy of Pt was enhanced through the introduction of pGO, and the final weight ratio of DOX: Pt: pGO was optimized to 0.376: 0.376: 1. Drug release results indicated that both Pt and DOX release kinetics from pGO-Pt/DOX nanoparticles were pH-dependent. In vitro studies suggested that pGO-Pt/DOX nanoparticles were effectively delivered into tumor cells, indicated the most prominent cell apoptosis and necrosis, and then exhibited a higher growth inhibition property than the single drug delivery system or free drugs. In vivo data confirmed that pGO-Pt/DOX dual-drug delivery system attenuated the toxicity of Pt and DOX to normal organ compared to free drugs. The tumor inhibition data, histopathology observations, and immunohistochemical staining have confirmed that the dual-drug delivery system presented more excellent anticancer effect than free drugs. This study demonstrated that the combination of Pt and DOX onto PEG-functionalized nano-sized GO afforded numerous advantages for tumor therapy such as minimizing systemic toxicity, controlling drug release under acid environment of tumor, and enhancing therapeutic efficacy, implying this dual-drug delivery system has great potential for clinical applications.