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PEGylated magnetic nanographene oxide for targeted delivery of arsenic trioxide and sec-o-glucosylhamaudol in tumor treatment with improved dual-drugs synergistic effect

PEGylated magnetic nanographene oxide for targeted delivery of arsenic trioxide and... A pH-sensitive polyethylene glycol-modified magnetic graphene oxide loaded with ATO and SOG (PEG@MGO@ ATO + SOG) was prepared for the magnetically targeted and efficient synergistic-chemo cancer therapy, which exhib - ited high specificity and good biocompatibility. oxidized form of graphene widely used in the biomedical Introduction field. Graphene and its derivatives are biologically safe at Compared with immunotherapy and radiotherapy, the cellular and organic levels, even at relatively high con- chemotherapy is more acceptable, with many alternative centrations (Ou et  al. 2016). GO has large oxygen-con- chemotherapeutic agents. Nevertheless, effective cancer taining functional groups (Allahbakhsh et al. 2013), good therapies are still unavailable. The main problem is that hydrophilicity (Tian et  al. 2019), huge surface area (Liu chemotherapeutic drugs are toxic to both cancerous and et al. 2013), and potentially low manufacturing cost (Kim normal cells (Reddy et  al. 2005). Hence, an effective and et  al. 2013). Those oxygen-containing groups in GO, like safe drug-delivery system is necessary (Mu et  al. 2020). C = O, -COOH, -OH, and -C-O-C, make it easier to be Increasing attention has been paid to the development chemically functionalized (Kazempour et  al. 2019). The of nano-drug carriers. Nano-drug carriers with diam- hydrophobic interactions and/or π–π stacking of these eters between 10 and 1000  nm work as media to trans- functional groups make drug loading possible (Xing et al. port chemotherapeutic agents (Gong et  al. 2016). This 2016; Xing et  al. 2016). The above properties facilitate novel drug-delivery system can improve drug solubility, the design of novel nano-carriers based on GO to deliver prolong the blood circulation period, and enhance drug therapeutic drugs (Priyadarsini et  al. 2018; Wang et  al. accumulation by passive or active targeting, which will 2018; Pooresmaeil et al. 2018; Abdelhamid et al. 2021). help to minimize adverse effects of clinical drugs (Geng A magnetic nanoparticle-based drug delivery system et al. 2018; Khursheed et al. 2020). can transfer drugs to a certain site under the influence Natural and synthetic polymeric materials, inorganic of an external magnetic field (Yang et al. 2018, Feng et al. materials, and lipids have been used as drug carriers 2018). Ferroferric oxide (F e O ) is an ideal choice to pre- (Karthik et  al. 2013; Truong et  al. 2013). Graphene is a 3 4 unique carbon-based nanomaterial, which looks like hon pare the magnetic drug delivery system for its paramag- netism, and there is no magnetization after removing the eycombs (Balandin 2020). Graphene oxide (GO) is the Cheng  et al. AAPS Open (2023) 9:12 Page 3 of 14 magnetizing field. Besides, reversible magnetism can pre - new combinations of ATO under the guidance of these vent the aggregation of nanoparticles, which can enhance principles. The ancient Chinese books named “YanFan - the stability of nanomedicines. Generally, the application gHuiJi” and “JiJiuBianFang” recorded that the root of the of Fe O in vivo requires surface modifications to prevent traditional Chinese medicine, Radix Saposhnikoviae, can 3 4 exocytosis and increase biocompatibility. significantly reduce ATO toxicity. In addition, modern Polyethylene glycol (PEG) is one of the most widely pharmacological research show this traditional Chinese studied superhydrophilic polymers and surface modifi - medicine can protect the liver from oxidization (Jiang ers. PEG is cheap, versatile, non-toxic, highly water-sol- et al. 2014). What is more, we find sec-O-glucosylhamau - uble, biocompatible, and can transport nanomolecules. dol (SOG), a compound extracted from Radix Saposh- Because of its appropriate pharmacokinetics and tissue nikoviae, expressing anti-cancer enhancement of ATO in distributions, the usage of PEG in pharmaceuticals is in vitro and in vivo experiments. approved by the Food and Drug Administration (FDA). We introduce a novel nano-drug, controlled-release The accumulation of nanoparticles modified with PEG nano-magnetic carrier, based on Fe O nanoparticles and 3 4 (PEGylation) in liquids decreased compared with that of GO nanosheets, which was conjugated with ATO and nanoparticles without PEG. Moreover, PEG will increase SOG to improve the therapeutic efficacy of HCC. The the internal circulation time and reduce excretion via the drug cargo was constituted of PEG-modified Fe O as 3 4 reticuloendothelial system (RES) (Tas et  al. 2021). Thus, hydrophilic corona and GO as a hydrophobic core. The PEGylated magnetic nanographene oxide (PEG@MGO) morphology, size, microstructure, and magnetic proper- is a potential nano-carrier to deliver hydrophobic drugs ties of the nanoparticles were examined. A series experi- in biological systems (Deb et al. 2018; Ma et al. 2020). ments were implemented, and the results demonstrated Arsenic trioxide (ATO) is a traditional Chinese medi- that ATO and SOG could be released in a controlled cine known as the “king of poisons” with a lethal dose manner in targeted lesions. This nano platform repre - value (LD ) of 15 mg/kg (rat, oral) (Vogt 2017). The usage sents a new approach for the treatment of HCC. of ATO was significantly reduced in the past century due to the public’s fear of its toxicity (Evens et  al. 2004). In the late twentieth century, ATO became popular again. Experimental methods It was approved by the FDA as the frontline therapy for Materials acute promyelocytic leukemia (APL) in 2000 (Hoonjan Graphene oxide (GO) was purchased from XFNANO (Nan- et al. 2018), and it was also approved for the treatment of jing, China). Ferric chloride hexahydrate (FeCl ·6H O), fer- 3 2 newly diagnosed APL by the European Medicines Agency rous chloride tetrachloride (F eCl ·4H O), trisodium citrate 2 2 (EMA) in 2016 (European Medicines Agency 2016). Sub- dihydrate (Na C H O ·2H O), arsenic trioxide (ATO), and 3 6 5 7 2 sequently, ATO was proven effective in other hemato - polyethylene glycol (PEG, average MV 400) were purchased logical malignancies, such as acute myeloid leukemia, from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, chronic myelogenous leukemia, and Hodgkin’s disease China). sec-O-Glucosylhamaudol (SOG) was bought from (Swindell et  al. 2013). With the blood clearance efficacy, Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China). ATO powder was not suitable for solid tumors therapy. All the materials mentioned were used without further Several attempts have been made to develop ATO’s anti- purification. cancer properties by increasing its bioavailability and Dulbecco’s modified Eagle’s medium (DMEM) was reducing systemic toxicity. These methods include sensi - purchased from HyCone (Logan, UT, USA). Fetal tizing carcinoma cells before ATO treatment, combining bovine serum (FBS) was obtained from Sijiqing (Hang- ATO therapy with other conventional chemotherapeutic zhou, China). A total of 0.25% trypsin-EDTA solution, agents, and developing ATO-loaded nano-drugs (Wang 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium et  al. 2012). Henceforth, ATO has become a “potential bromide (MTT), Prussian blue iron staining kit (with broad-spectrum anti-cancer drug” (Akhtar et  al. 2017). Eosin solution), dimethyl sulfoxide (DMSO), dou- Hepatocellular carcinoma (HCC) is one of the most ble antibiotic (streptomycin/penicillin), phosphate malignant cancers and has caused substantial mortal- buffer solution (PBS, pH 7.4), fluorescein isothio - ity worldwide (Bray et al. 2018, Yang et al. 2019). HCC is cyanate, and sodium dodecyl sulfate were obtained insensitive to adriamycin and platinum chemotherapeu- from Solarbio (Beijing, China). The Annexin V-FITC/ tics, so the development of ATO-loaded nano-drugs will PI Apoptosis Detection Kit was purchased from Bec- provide new treatment options for liver cancer. ton, Dickinson, and Company (San Digo, USA), and 2ʹ, Synergism and detoxication are important principles 7ʹ-dichlorodihydrofluorescein diacetate (DC-FHDA) was of traditional Chinese medicines. We try to find some bought from Sigma-Aldrich, USA. Cheng et al. AAPS Open (2023) 9:12 Page 4 of 14 Cell line and cell culture Preparation of drug‑loaded PEG‑Fe O @GO 3 4 The human hepatoma cell line (HepG2) and human In order to evaluate the adsorption process, different hepatocyte cell line (L02) were purchased from Shang- formulations were prepared. ATO and SOG co-loaded hai Cell Bank, Chinese Academy of Sciences (Shanghai, PEG@MGO (PEG@MGO@ATO + SOG) were prepared China). Both cells were cultivated in a CO incubator as follows: 10 mg PEG@MGO was added into 10 mL ethyl (Thermo Scientific, USA). HepG2 and L02 were cul - alcohol solution containing 2 mg/mL ATO and 4 mg/mL tured in a DMEM medium supplemented with fetal SOG. ATO-loaded PEG@MGO (PEG@MGO@ATO) bovine serum (10%, v/v), penicillin (100 UI/mL), and was prepared in the solution containing 2  mg/mL ATO streptomycin (100 UI/mL). When the cell confluence only, while SOG-loaded PEG@MGO (PEG@MGO@ reached nearly 80%, the cells were digested and pas- SOG) was prepared in the solution containing 4  mg/mL saged with 0.25% trypsin for subsequent experiments. SOG only. These resulting mixtures were stirred at 50 °C for 6 h, and then the nanoparticles were collected via an Nd magnet and washed with double distilled water and ethanol three times in sequence to remove unabsorbed Preparation of  Fe O and polyethylene glycol‑modified 3 4 ATO and SOG. Finally, the above drug-loaded nanopar- magnetic GO (PEG@MGO) ticles were freeze-dried at − 20 °C for 24 h. The method The preparation for PEG@MGO was according to pre - of ATO and SOG loading on PEG@MGO was optimized vious reports with some developments (Wang et  al. through preliminary experiments based on the solubility 2018; Mao et al. 2019). GO powder (20 mg) was poured of SOG and inhibition rates of HepG2. into 50 mL purified water with 2  g PEG-400 and son - After drug loading, the supernatant was collected and icated for 1  h. Then, 2.162  g FeCl · 6H O and 0.795  g 3 2 filtered via a 0.22  μm membrane filter. The concentra - FeCl · 4H O were added. The suspension was stirred 2 2 tions of ATO and SOG in the supernatant were deter- and maintained at 60  °C for 30  min with N protec- mined through an inductively coupled plasma emission tion to generate magnetic cores according to reac- spectrum (ICP 6300, Thermo Electron Corporation, tion 1. After that, sodium hydroxide (NaOH) solution USA) and high-performance liquid chromatography (1 mol/L) was dropwise added until the pH value to 11. (HPLC 1100, Shimadzu, Japan), respectively. Detailed Then, 0.247  g sodium citrate dihydrate (C H Na O ) 6 5 3 7 analytical methods were showed in supporting infor- was added under constant magnetic stirring. The tem - mation. Drug encapsulation efficiency (EE%) and drug perature of the mixture was kept at 60  °C and stirred content (DC%) of PEG@MGO@ATO + SOG and PEG@ for 2  h. The precipitation was collected by magnetic MGO@ATO were calculated according to Eqs.  (1) and separation. After being washed three times with water (2), respectively. and ethanol, the black nanoparticles were dried in the vacuum oven at 60 °C for 6 h. Amount of drug in nanoparticles EE% = × 100% Amount of drug added 2+ 3+ − (1) Fe + 2Fe + 8OH → Fe O + 4H O 3 4 2 Amount of drug in nanoparticles Reaction 1: The preparation of Fe O DC% = × 100% 3 4 Amount of nano − carrier (2) Characterization In vitro drug release Fourier transform infrared (FTIR) spectra were recorded The release behaviors of SOG and ATO on PEG@MGO on a Nicolet Ncxus 670 FTIR spectrometer (Thermo Sci - − 1 were investigated at different pH conditions. Briefly, entific, USA) in the range of 500–4000  cm by the KBr 10  mg drug-loaded nanoparticles were dispersed in 10 pellet technique. X-ray powder diffraction (XRD) data mL buffer solution with different pH values (pH 5.0, 6.8, were collected in the range of 2θ = 4 − 90° using a Rigaku and 7.4) and given to continuous shaking at 37  °C. At XRD S2 powder diffractometer (Rigaku Corporation, desired time intervals, 1 mL released solution was taken Japan). Morphological evaluation of the freeze-dried nan- from the stirring dissolution medium. Subsequently, an oparticles was recorded by a Tescan mira3 field emission equal amount of fresh buffer saline was added to the orig - scanning electron microscope (FE-SEM, Tescan, Czech inal media. The percent of released ATO and SOG was Republic). The magnetic property of nanoparticles was calculated according to the following formula: measured by the vibrating sample magnetometer (VSM) using Lakeshore 730T (Lakeshore, USA). Dynamic light W released dose Release(%) = × 100% (3) scattering (DLS) analysis was performed on a Nano-Zeta- W loaded dose Sizer ZEN3600 (Malvern, UK). Cheng  et al. AAPS Open (2023) 9:12 Page 5 of 14 where    W represents the weight of drug medium was replaced by MTT solution, and these cells released dose released into solution from the drug-loaded nanoparti- were further cultured for 4 h. DMSO (150 µL) was subse- cles; W represents the weight of drug loaded on quently added to dissolve the formazan crystals formed. loaded dose nanoparticles. The absorbance (OD) values of different groups at 570 nm were recorded by a microplate reader (Multiskan MK3, Thermo Electron Corporation, USA). The meas - Examine of stability ured OD values of the blank, control, and experimental The residual moisture content was studied for the PEG@ groups were defined as OD, OD , and OD . Cell survival MGO and PEG@MGO@ATO + SOG which were newly b c e rates were calculated according to Eq.  (4). Data are pre- prepared and stored after 30 days. The residual moisture sented as mean ± standard deviation (n = 6). content was measured by Karl Fischer titration using a Mettler DL 38 titrator (Mettler-Toledo, Switzerland). OD − OD e b 100.0  mg samples of the above nanoparticles were used Survival rate(%) = × 100% (4) OD − OD c b for the analysis and the measured moisture content was expressed in percentage. What is more, the dispersive For cell apoptosis assay, cells were seeded in 6-well capacity of PEG@MGO after 30 days’ storage over 25 °C plates at a density of 2 × 10 cells/well. After 24  h of /60RH and 40  °C /75RH was detected by dispersing incubation, nano-drugs at different concentrations were 10 mg PEG@MGO into 10mL water. added to the cell medium, and the cells were incubated for another 24 h. Then, the cells were harvested, washed Cellular uptake of nano‑drug carriers twice with cold PBS, and stained with 5 µL Annexin The cellular uptake of the nano-drug was analyzed by V-FITC and 5 µL PI for 15  min at room temperature in Prussian blue staining and a fluorescence microscope. the dark. These cells were resuspended in 200 µL binding To perform Prussian blue staining, the following steps buffer and were analyzed using flow cytometry (FACS - were made. HepG2 was seeded in 12-well plates at a den- Verse, Becton, Dickinson and Company, USA). sity of 1 × 10 cells/well and incubated for 24 h (37 °C ,5% CO ). Then the cells were treated differently. One group Combination effect of SOG and ATO was only treated with PEG@MGO at a concentration of Tumor-cell proliferation-inhibition behaviors of SOG 15  µg/mL in DMEM medium for 4  h. The other group and ATO against HepG2 were evaluated. The concentra - was treated with PEG@MGO at a concentration of 15 µg/ tions of ATO and SOG ranged from 0.5 to 64 µmol/L and mL in DMEM medium for 4  h and a small Nd perma- from 16 to 2048 µmol/L, respectively. In the combina- nent magnet was placed under each well during the first tion group, the drug concentrations are the same as the 1  h of incubation. After treatment, cells were fixed with above, the combination effects of SOG and ATO loaded 4% paraformaldehyde for 30  min and then were stained on PEG@MGO were also explored. The concentrations by freshly-prepared Prussian blue staining solution for of PEG@MGO@ATO, PEG@MGO@SOG, and PEG@ 30 min and counterstained by eosin for 1 min. MGO@ATO + SOG ranged from 2.5 to 120 µg/mL. After To perform microscopic inspection, HepG2 cells were the cells were incubated for 24  h, 48  h, and 72  h under seeded into a 6-well plate (4 × 10 cells/well). After 24  h, drug application, the cell survival rates were detected by the DMEM medium containing FITC and PEG@MGO/ the microplate reader at 570  nm by MTT assay and the FITC was added to replace the previous solution. FITC process showed above. CI was measured according to and PEG @MGO in the medium were 10  µg/mL and Chou’s method (Chou 2006). 15 µg/mL, respectively. After being incubated at different D D times, the cells were washed three times with sterilized 1 2 CI = + PBS and fixed by 75% absolute alcohol. The cells were (D ) (D ) n n 1 2 finally observed and recorded by an inverted fluorescence In the equation, where (Dn) and (Dn) represent the microscope (DMI3000B, Leica, Germany). 1 2 IC value when drug 1 or 2 works singly. D and D rep- 50 1 2 resent the concentrations of drug 1 and drug 2 when In vitro cytotoxicity and cell apoptosis analysis given simultaneously at the IC value. CI > 1 was used 50 50 The in  vitro cytotoxicity of the PEG@MGO on HepG2 to indicate antagonism between two drugs, C I = 1, the and L02 was studied using the MTT assay. These cells additive effect, and CI < 1, synergism. were seeded in 96-well plates at a density of 1 × 10 cells/well and incubated for 24  h (37  °C, 5% C O ). After removing the culture medium, 200 µL DMEM medium Cellular reactive oxygen species (ROS) measurements containing different concentrations of nanoparticles was The released intracellular ROS in different groups was added. Following 48  h incubation, the DMEM culture measured using DCFH-DA. HepG2 cells were seeded Cheng et al. AAPS Open (2023) 9:12 Page 6 of 14 in 6-well plates with a density of 4 × 10 cells/well and Results and discussion were incubated at 37  °C for 24  h. Then, the cells were Characterizations of the nanoparticles incubated with free ATO, the mixture of ATO and SOG, Figure  1a shows the FTIR spectrums of GO, PEG@ PEG@MGO@ATO, and PEG@MGO@SOG + ATO for MGO, and PEG@MGO@ATO + SOG. The peaks of the − 1 − 1 − 1 24  h. The concentration of nanoparticles was 15  µg/ml GO sample at 3650  cm, 1500  cm , and 1075  cm and the amount of ATO and SOG added are equal to are related to -OH stretching, C-O stretching vibration, − 1 the amount of drugs loaded on nanoparticles. At the epoxy, and alkoxy, respectively. The peak at 1650  cm end of the cultivation, the collected cells were resus- was attributed to C-C stretching vibration. The char - − 1 pended in a DMEM medium containing DCHF-DA (10 acteristic peak of the PEG@MGO sample at 945  cm µM) at 37  °C for 30  min. The cells were washed with was caused by the –CH groups in PEG. Additionally, − 1 serum-free culture solution three times to remove the the absorption peaks of -OH stretching at 3600  cm − 1 DCFH-DA that did not enter the cells. Then, the fluo -and 3050  cm contributed to -CH group bands, which rescence was measured by flow cytometry (excitation at confirmed the successful attachment of PEG to GO − 1 485 nm and emission at 530 nm). surface. The peaks at 530  cm related to vibrations of Fe-O show the successful modification of Fe O . The 3 4 FTIR spectrums of PEG@MGO@ATO + SOG are simi- In vivo tumor inhibition lar to those of PEG@MGO, which means the ATO and Twenty-eight 6-week-old male BALB/c nude mice were SOG loading will not affect the nano-carrier structure bought from Beijing Vital River Laboratory Animal (Farani et al. 2020; Dong et al. 2010). Technology Co., Ltd. (Beijing, China). Approximately The presence of different compositions was verified 2 × 10 HepG2 cells dispersed in 0.2 mL saline solu- with XRD analysis. Figure  1B exhibits the crystalline tion were injected subcutaneously into the right flank phases of GO, Fe O , and PEG@MGO. The peak of the 3 4 region of every mouse. When the volume of tumors PEG@MGO at 2θ = 11.29° is related to 002 diffractions approached 100 mm (about 10 days after the tumor of GO flakes. Peaks at 2θ = 30.23°, 37.23°, 41.22°, 57.15°, inoculation), the tumor-bearing mice were randomly 66.91° display the typical peaks of cubic spinel F e O 3 4 divided into four groups (7 mice /group) for different NPs. This suggests the remaining of the inner core treatments. The therapy method for groups were listed structure even after modification. DLS analysis was as follows: (1) inject saline solution via the tail vein; (2) used to evaluate the size and particle distributions of inject PEG@MGO@SOG + ATO at a dose of 20  mg/ Fe O . As shown in Fig. S1, the average particle diam- 3 4 kg via the tail vein, then fix an external magnet on eter for Fe O was 150 nm for a volume. The PDI value 3 4 the back of the tumor with glues; (3) inject free ATO was 0.179 showed a great homogeneity of this magnetic at a dose of 5  mg/kg via the tail vein; (4) inject PEG@ nanoparticle. MGO@SOG + ATO at 20  mg/kg via the tail vein. The Figure  1c and e shows the SEM images of differ - initial body weight was recorded and monitored every 3 ent nanocomposites. As shown in Fig.  1c, the GO has a days before treatment. The tumor size was determined sheet-like structure with smooth surfaces and a wrinkled and calculated by the formula V = a × b /2, where a and edge. After the modification with the Fe O and PEG400, 3 4 b were the longest and shortest diameters of the tumor, the SEM image of the nanocomposites revealed the respectively. Mice were sacrificed on the 18th day after regular spherical morphology (Fig.  1d). Figure  1e shows treatment, the tumors were excised for weighing. Then, the image of ATO- and SOG-loaded nanoparticles. The tumors and main organs (heart, liver, spleen, lung, and rough surface may be attributed to the adsorption of kidney) were fixed in 10% formalin, followed by hema - drugs on the surface of the PEG@MGO. In conclusion, toxylin and eosin (H&E) staining assay. the modified GO sheets can prevent the restacking of GO sheets and enlarge the surface area to absorb active drugs. The magnetic properties of Fe O , PEG@MGO, and Statistical analysis 3 4 PEG@MGO@ATO + SOG were studied by the mag- Data were processed using Spss.20 (SPSS Inc., Chi- netic hysteresis loop, which is shown in Fig.  2a. The cago, USA) and presented as mean ± standard devia- saturation magnetization value of Fe O , PEG@MGO, tion (SD). Statistical analysis was performed using a 3 4 and PEG@MGO@ATO + SOG was 61.1, 41.4, and 16.1 one-way analysis of variance (ANOVA). The difference emu/g, respectively. The above results demonstrate the was regarded as statistically significant when P  ≤ 0.05. good superparamagnetic ability of the nano-drug with Statistic software Graph-pad Prism 5.0 (GraphPad no coercivity or remanence (Atacan et  al. 2015; Cheng Software, California, USA) was used for all graphical et  al. 2018). The insert picture shows the good water illustrations. Cheng  et al. AAPS Open (2023) 9:12 Page 7 of 14 Fig. 1 a FT-IR spectra of GO, PEG@MGO, and PEG@MGO@ATO + SOG. b X-ray diffraction patterns of GO, Fe O , and PEG@MGO. SEM image of GO 3 4 (c), PEG@MGO (d), and PEG @ MGO@ATO + SOG (e) Fig. 2 a Magnetization curves of Fe O , PEG@MGO, and PEG@MGO@ATO + SOG, magnetic recovery of PEG@MGO@ATO + SOG from aqueous 3 4 solution (insert). b, c Release profile of ATO and SOG from PEG@MGO@ATO + SOG at different pH values dispersibility and easy magnetic separation of the PEG@ of ATO and SOG mainly depended on the electrostatic MGO@ATO + SOG. interaction between drugs and the PEG@MGO. The EE% and DC% of PEG@MGO@ATO and PEG@MGO@ Drug loading and in vitro release study ATO + SOG were listed in Table  1. These data demon - Drug loading capacity is a very important factor in evalu- strated the good drug encapsulation efficiency of the ating the therapeutic effect of nanodrugs. The loading PEG@MGO nanocomposites. This phenomenon can Cheng et al. AAPS Open (2023) 9:12 Page 8 of 14 Table 1 Drug loading ability of different nanoparticles Table 2 Residual moisture content of the nanoparticles after different storage time and determined by Karl Fischer titration PEG@MGO@ATO PEG@MGO@ATO + SOG (mean ± SD; n = 3) ATO ATO SOG Formulations Residual moisture (%) EE(%) 14.76 ± 3.2 18.03 ± 4.2 38.25 ± 3.8 Newly prepared After DC(%) 29.89 ± 2.6 36.52 ± 5.0 153.00 ± 4.2 30 days storage PEG@MGO 0.52 ± 0.01 1.01 ± 0.2 be explained as the addition of PEG on the surface of PEG@MGO@ATO + SOG 0.63 ± 0.11 1.06 ± 0.3 the dendrimer can prevent the diffusion of drugs to the solution. In addition, the high loading capacity might be related to the high surface area of the nanocomposites. In vitro cellular uptake Also, the EE% and DC% of the combination drugs are Cellular internalization is essential for nanoparticles used higher than those of only ATO-loaded nano-drug. It may as drug carriers. Prussian blue staining, which selec- 3+ be explained as positively charged PEG@MGO naturally tively stains Fe , can be used to evaluate the endocytosis absorbs the negatively charged ATO and the addition behaviors of PEG@MGO. Figure  3 shows that blue dots of SOG can produce covalent interaction between two accumulated in cells after being treated with the mag- drugs to enhance the drug loading efficiency. netic drug carrier, indicating that PEG@MGO could be The release profiles of ATO and SOG from PEG@ uptaken by tumor cells. What is more, the intracellular MGO@ATO + SOG at pH 5.0, 6.8, and 7.4 are shown in amount of PEG@MGO was significantly increased by an Fig. 2b and c. The drug release rates of ATO from PEG@ external Nd-magnet (Fig.  3c, d). These results indicated MGO@ATO + SOG with the three pH values were close that a magnetic field would enhance the endocytosis of during the initial 6 h. The results showed that the cumula - PEG@MGO. tive release rate of ATO from PEG@MGO@ATO + SOG To investigate the motion law of the nano-carrier, reached up to 77.6% ± 1.5%, 55.3.00% ± 1.9%, and 53.1% PEG@MGO was labeled with FITC (green) for subcel- ± 2.2% at pH 5.0, 6.8, and 7.4 respectively after 108  h. lular observation. Green fluorescence appeared after 4 h The releasing rule of SOG was similar to that of ATO. co-culture and was widely distributed in the cells after 24 After 108  h, the cumulative release rate of SOG from h co-culture, which is shown in Fig. 3e. This revealed that PEG@MGO@ATO + SOG was approximately 79.21% PEG@MGO exhibited a high level of cell uptake through ± 2.5%, 58.3% ± 1.2%, and 53.4% ± 2.2% at pH 5.0 6.8, endocytosis in a time-dependent manner. and 7.4, respectively. These results show that the release of ATO and SOG from nanocarrier is pH-sensitive and Cytotoxicity assay and cellular apoptosis analysis the release rate increased with the decrease of pH values. For the potential biomedical applications, it is necessary The addition PEG enhances the hydrophilic nature of to investigate the cytotoxicity of nano-carriers. Figure  4 the dendrimer thus improving its stability. Under acidic showed the results for cells treated with PEG@MGO for conditions, the hydrogen bonds are stronger than those 48 h and with drugs loaded with inhibition effect, respec - occurring at pH 7.4. Therefore, the high release of ATO tively. It should be noticed that the viability of the tumor and SOG from PEG@MGO@ATO + SOG nanocom- cells (HepG2) and liver cells (L02) were observed to be posite under acidic pH conditions indicates the poten- larger than ~ 70% even at higher concentration of 250 µg/ tial application of the proposed nanocarrier in cancer mL after 48 h (Fig. 4a, b), indicating the excellent biocom- treatment. patibility of blank nano-carriers. ATO- and SOG-loaded PEG@MGO showed cell inhibition to HepG2 cells and Stability of nanoparticles L02 cells, while the inhibition effect of L02 is lower than The residual moisture content of PEG@MGO and PEG@ that of HepG2. The results implied that the PEG@MGO MGO@ATO + SOG were showed in Table  2. It is well nanoparticles have minor toxicity and great selectivity as known that the residual moisture content plays impor- a drug delivery in cancer treatment. tant roles in determining a power’s long-term stability, Annexin V/PI staining was carried out to investigate both physically and chemically. The results showed the the influence of various concentrations of the novel nano- moisture content of the both two nanoparticles were less drug on the apoptosis rates of HepG2 cells. The apopto - than 1.2% which can prove the stability of PEG@MGO sis rates of cells treated with PEG@MGO@ATO + SOG whether it loads drug or not. Meanwhile, the dispersibil- under 10  µg/mL, 15  µg/mL, 20  µg/mL, 25  µg/mL, and ity of PEG@MGO remains nearly unchanged regardless 50  µg/mL were studied, respectively. After the incuba- the store condition (Fig. S3). tion of 24 h, the apoptosis rates were 13.5%, 16.0%,17.9%, Cheng  et al. AAPS Open (2023) 9:12 Page 9 of 14 Fig. 3 Cellular uptake of PEG@MGO. Images of HepG2 stained with Prussian blue: a cells with no treatment, b treated with 15 µg/ml PEG@MGO for 4 h, c treated with 15 µg/ml PEG@MGO under the magnetic field for the first 1 h. d Area percentage analysis after staining with Prussian blue. e Microscopy images of HepG2 incubation with FITC-labeled PEG@MGO after different time 19.2% and 21.3% in Fig.  5a. The results demonstrate that 24  h, 48  h, and 72  h incubation were 0.714, 0.83, and the inhibitory activity of the nano-drug increased with 0.964, respectively. The CI values smaller than 1 indicated the increase of its concentrations and the cellular apop- the synergistic effect of SOG and ATO. The inhibition tosis of HepG2 cells caused by PEG@MGO@ATO + SOG ratios of HepG2 at different combination concentrations was a concentration dependent manner (Fig.  5b). It can of ATO and SOG shown in Fig. S4 added evidence of be also hypothesized that PEG@MGO@ATO + SOG will the cell growth inhibition under the combination usage inhibit tumor proliferation by triggering the apoptotic of SOG and ATO. The IC values of PEG@MGO@ path way of cancer cells. ATO + SOG were smaller than those of PEG@MGO@ SOG and PEG@MGO@ATO. From the above results, we Cytotoxicity and synergism can conclude that the active targeting of PEG@MGO@ The cytotoxicity of free drugs and drug-loaded nanopar - ATO + SOG leads more drug molecules to enter tumor ticles on HepG2 cells was measured by the MTT assay. cells to inhibit tumor growth. The IC values of free drugs, co-drugs, drug-loaded PEG@MGO, and combination index (CI) values of co- Cellular ROS analysis drugs were summarized in Tables  3 and 4. The results To investigate whether the novel nano-drug causes oxi- show that the cytotoxicity of all experimental groups is dative stress in cancer cells, ROS levels of HepG2 cells dose-and time-dependent. Compared with single-drug were measured by flow cytometry after being incubated treatment, dual-drug combination treatment exhibits with different formulations (Fig.  5c). The results showed higher cytotoxicity. The CI values of SOG + ATO after that the intracellular ROS levels were significantly Cheng et al. AAPS Open (2023) 9:12 Page 10 of 14 Fig. 4 Cytotoxicity of PEG@MGO against a HepG2 cells and b L02 cells after incubation. Cytotoxicity of ATO + SOG@ PEG@MGO against c HepG2 cells and d L02 cells after incubation increased after 24  h’s drug treatment. The intracel - In vivo synergistic anti‑cancer effect lular ROS levels in ATO + SOG and PEG@MGO@ Based on the effective therapy of the nanocomposite ATO + SOG groups were higher than those in the in  vitro, a HepG2 xenograft model was established by groups of ATO and PEG@MGO@ATO, which is due intravenous administration with different formulations to the synergistic effect. The intracellular ROS levels to study the synergistic efficacy. As shown in Fig.  6a, in PEG@MGO@ATO and PEG@MGO@ATO + SO G the volume of tumor showed significant differences groups increased 2.30-fold and 2.59-fold, respectively, among different groups. Treatment with ATO led to a as compared with those of the free ATO group. The val - slight inhibition of HepG2 tumor growth compared to ues of those two groups increased 1.46-fold and 1.59- the PBS group. The group treated with PEG@MGO@ fold compared with those of the ATO + SOG group. A ATO + SOG under a magnetic field displayed the most significant increase in ROS level was observed in the significant tumor growth inhibition, outperforming both cells when treated with a co-drug. Excessive intracel- the group of free ATO and PEG@MGO@ATO. After lular ROS may induce oxidative stress in mitochondria 18 days of observation, tumor tissues were extracted, and destruction of the integrity of the mitochondria weighed, and photographed. Tumor weights were 95.2, membrane structure and finally, induce cellular apop - 476.2, and 226.1  mg in the group of magnet + PEG@ tosis and death. PEG@MGO@ATO + SOG was more MGO@ATO + SOG, ATO, and magnet + PEG@MGO@ likely to produce ROS than other drugs, which may be ATO, respectively, as compared with 707.5 mg of the PBS ascribed to its sustained drug release manner. group. The average tumor weight of the PEG@MGO@ Cheng  et al. AAPS Open (2023) 9:12 Page 11 of 14 Fig. 5 The apoptosis rates of HepG2 cells after incubation with PEG@MGO@ATO + SOG at different concentrations. a Flow cytometry analysis via * ** *** Annexin V/PI staining and b quantitative analysis of tumor cells apoptosis. P < 0.05, P < 0.01, P < 0.001. c Eec ff t of different treatment on the ** production of intracellular ROS according to relative fluorescence intensity. Compared with ATO treated group, P < 0.01; compared with ATO + SOG ## treated group, P < 0.01 Table 3 IC and CI of SOG and ATO against HepG2 cells for different incubation time Time IC drug alone IC drug combination CI at IC 50 50 50 (95% confidence interval) (95% confidence interval) SOG(µmol/L) ATO(µmol/L) SOG(µmol/L) ATO(µmol/L) 24 h 1575.0 ± 59.5 52.0 ± 2.8 586.8 ± 33.0 17.8 ± 1.7 0.71 48 h 773.9 ± 36.6 19.9 ± 2.1 289.8 ± 27.2 9.9 ± 1.4 0.83 72 h 458.5 ± 18.5 14.2 ± 2.4 219.6 ± 16.1 6.9 ± 0.3 0.96 tumor growth curve, tumor weight (Fig. 6d), and in vitro Table 4 IC of different formulations against HepG2 cells for different incubation time experiments. The tumor growth in mice treated with PBS showed a fast and unrestrained tendency, and the Time Nanodrugs IC (µg/mL) final volume was about 11-fold of the initial size. The free 24 h PEG@MGO@SOG 87.41 ± 1.84 ATO could not prevent tumor growth might because of PEG@MGO@ATO 51.87 ± 4.73 the quick dilution of fluid flow. PEG@MGO@ATO + SOG 27.07 ± 0.86 H&E staining examinations of the tumor tissues after 48 h PEG@MGO@SOG 78.89 ± 2.55 treatment are displayed in Fig.  6e; it appeared that the PEG@MGO@ATO 41.69 ± 2.22 tumor tissue displayed a typical necrotic response after PEG@MGO@ATO + SOG 21.91 ± 1.78 treatment; the cell necrosis of PEG@MGO@ATO + SOG 72 h PEG@MGO@SOG 68.99 ± 1.78 under magnet was the most obvious. All these indicate PEG@MGO@ATO 30.89 ± 0.66 that the nano-drug PEG@MGO@ATO + SOG owns a PEG@MGO@ATO + SOG 15.62 ± 0.84 remarkable tumor inhibition effect, and the magnetic microenvironment may promote the accumulation of the anti-cancer drug in tumor cells. The possible toxicity of the formulations was also stud - ATO + SOG group was much lower than those of the ied. As shown in Fig. 6c, no significant reduction in body other groups (Fig. 6b). The pictures of the tumors among weight was observed in the different groups during the the different groups are consistent with the results of the treatment period, indicating the high biocompatibility Cheng et al. AAPS Open (2023) 9:12 Page 12 of 14 Fig. 6 In vivo tumor inhibitory effects on HepG2 xenograft tumors. a Tumor volumes were measured every three days. Compared to PBS group, ***p < 0.001; compared to magnet + PEG@MGO@ATO group, p < 0.05. b Weight of tumors of each therapeutic group after 18 days of treatments. ## ### Compared to PBS group, ***p < 0.001. **p < 0.01; compared to ATO group, p < 0.01. p < 0.001. c The body weight of mice in treatments. d The optical image of the excised tumor tissues after 18 days of treatments. e The H&E analysis for different groups (scale bar: 50 μm) CI Combination index of these PEG@MGO-based nano-drugs. H&E stained DC-FHDA 2ʹ, 7ʹ-Dichlorodihydrofluorescein diacetate images of major organs (heart, spleen, lung, and kidney) DC% Drug content shown in Fig. S5, revealing nearly no difference in patho - DMSO Dimethyl sulfoxide DMEM Dulbecco’s modified Eagle’s medium logical lesions of varied groups. These results collectively EE% Drug encapsulation efficiency indicated that the nanoparticles did not cause appreci- EMA European Medicines Agency able systemic toxicity or an inflammatory response. FBS Fetal bovine serum FDA Food and Drug Administration Fe O Ferroferric oxide 3 4 FTIR Fourier transform infrared spectra Conclusion FeCl ·6H O Ferric chloride hexahydrate 3 2 In summary, a pH-sensitive polyethylene glycol-modified HepG2 Human hepatoma cell line magnetic graphene oxide loaded with ATO and SOG HCC Hepatocellular carcinoma HPLC High-performance liquid chromatography (PEG@MGO@ATO + SOG) was first prepared for the GO Graphene oxide magnetically targeted and efficient synergistic-chemo LD50 Lethal dose value cancer therapy. This new biocompatible drug delivery L02 Human hepatocyte cell line MTT 4,5-Dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide system was prepared by coating hollow F e O nanoparti- 3 4 PEG Polyethylene glycol cles on the surface of GO sheets via electrostatic interac- PEG@MGO PEGylated magnetic nanographene oxide tion and then immobilized with hydrophilous PEG-400. RES Reticuloendothelial system ROS Reactive oxygen species The combination of ATO and SOG, the active ingredient SEM Scanning emission microscope of traditional Chinese medicines, can improve the inhi- SOG Sec-o-Glucosylhamaudol bition of HepG2. These two drugs were loaded on the VSM Vibrating sample magnetometer XRD X-ray powder diffraction nano-carrier due to the large surface area of the PEG@ MGO. The nanocomposite exhibited excellent magnetic Supplementary Information hyperthermia effect, controlled drug release, and pH sen - The online version contains supplementary material available at https:// doi. sitivity, which could be used for accurate cancer therapy. org/ 10. 1186/ s41120- 023- 00079-4. Furthermore, it showed excellent anti-cancer perfor- mance in vitro and vivo experiments. The results showed Additional file 1: Fig. S1. DLS result of Fe O nanoparticles. Fig. S2. TEM 3 4 that this ATO- and SOG-co-loaded nanodrug exhibited image of Fe O nanoparticles. Fig. S3. Photos of PEG@MGO dispersed in 3 4 water. A. PEG@MGO stored over 25℃/60%RH for 30 days; B. PEG@MGO high potential in the HCC adjuvant therapy. stored over 40℃/75% for 30 days. Fig. S4. The inhibition ratios of HepG2 at different combination concentrations of ATO and SOG after 48h co- culture. Fig S5. H&E histology images of the major organs in mice after Abbreviations administration of (A) PBS, (B) Magnet+PEG@MGO@ATO+SOG, (C) ATO, (D) APL Acute promyelocytic leukemia Magnet+PEG@MGO@ATO for 18 days. ATO Arsenic trioxide Cheng  et al. AAPS Open (2023) 9:12 Page 13 of 14 Authors’ contributions Dong H, Zhao Z, Wen H, Li Y, Guo F, Shen A, Pilger F, Lin C, Shi D (2010) Jinlai Cheng: performed laboratory experiments, analyzed, interpreted data, Poly(ethylene glycol) conjugated nano-graphene oxide for photody- and wrote the first copy of the manuscript. Kun Hong: contributed to the namic therapy. Sci China Chem 53(11):2265–2271 experiments. Jianhui Sun: co-write, revised the manuscript, and provided the Evens AM, Tallman MS, Gartenhaus RB (2004) The potential of arsenic triox- final approval of the version to publish. Hongmei Li: contributed to the design ide in the treatment of malignant disease: past, present, and future. of the work, supervised the research, and provided the final approval of the Leuk Res 28(9):891–900 version to publish and agreed to be accountable for all aspects of the work. European Medical Agency (2016) Trisenox EMEA/H/C000388/II/0058 assess- Yuqing Tan: supervised the project. Miyi Yang: put the design of the work, ment report contributed in revision, analysis, interpretation of data and provided financial Farani MR, Khadiv–Parsi P, Riazi GH, Ardestani MS, Rad HS (2020) PEGyla- support of this project. All authors discussed the results and contributed to tion of graphene/iron oxide nanocomposite: assessment of release of the final manuscript. The author(s) read and approved the final manuscript. doxorubicin, magnetically targeted drug delivery and photothermal therapy. Appl Nanosci 10(4):1205–1217 Feng L, Xie R, Wang C, Gai S, He F, Yang D, Lin J (2018) Magnetic targeting, Declarations tumor microenvironment responsive intelligent nanocatalysts for enhanced tumor ablation. ACS Nano 12:11000–11012 Availability of data and materials Geng S, Wu L, Cui H, Tan W, Chen T, Chu PK, Yu XF (2018) Synthesis of The datasets used and/or analyzed during the current study are available from lipid-black phosphorus quantum dot bilayer vesicles for near-infrared- the corresponding author on reasonable request. controlled drug release. Chem Commun 54(47):6060–6063 Gong R, Chen G (2016) Preparation and application of functionalized nano Competing interests drug carriers. Saudi Pharm J 24(3):254–257 The authors declare no conflict of interests. Hoonjan M, Jadhav V, Bhatt P (2018) Arsenic trioxide: insights into its evolu- tion to an anticancer agent. J Biol Inorg Chem 23(3):313–329 Funding Jiang C, Li W, Zheng Y (2014) Protective effect of Saposhnikovia divaricata This work was financially supported by the Fundamental Research Funds for extract on liver. J Jilin Agricultural Univ 36(3):3 the Central public welfare research institutes (ZZ13-YQ-056). Karthik S, Puvvada N, Kumar BN, Rajput S, Pathak A, Mandal M, Singh ND (2013) Photoresponsive coumarin-tethered multifunctional magnetic Acknowledgements nanoparticles for release of anticancer drug. ACS Appl Mater Interfaces Not applicable. 5(11):5232–5238 Kazempour M, Namazi H, Akbarzadeh A, Kabiri R (2019) Synthesis and Authors’ information characterization of PEG-functionalized graphene oxide as an effective Not applicable. pH-sensitive drug carrier. 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PEGylated magnetic nanographene oxide for targeted delivery of arsenic trioxide and sec-o-glucosylhamaudol in tumor treatment with improved dual-drugs synergistic effect

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Abstract

A pH-sensitive polyethylene glycol-modified magnetic graphene oxide loaded with ATO and SOG (PEG@MGO@ ATO + SOG) was prepared for the magnetically targeted and efficient synergistic-chemo cancer therapy, which exhib - ited high specificity and good biocompatibility. oxidized form of graphene widely used in the biomedical Introduction field. Graphene and its derivatives are biologically safe at Compared with immunotherapy and radiotherapy, the cellular and organic levels, even at relatively high con- chemotherapy is more acceptable, with many alternative centrations (Ou et  al. 2016). GO has large oxygen-con- chemotherapeutic agents. Nevertheless, effective cancer taining functional groups (Allahbakhsh et al. 2013), good therapies are still unavailable. The main problem is that hydrophilicity (Tian et  al. 2019), huge surface area (Liu chemotherapeutic drugs are toxic to both cancerous and et al. 2013), and potentially low manufacturing cost (Kim normal cells (Reddy et  al. 2005). Hence, an effective and et  al. 2013). Those oxygen-containing groups in GO, like safe drug-delivery system is necessary (Mu et  al. 2020). C = O, -COOH, -OH, and -C-O-C, make it easier to be Increasing attention has been paid to the development chemically functionalized (Kazempour et  al. 2019). The of nano-drug carriers. Nano-drug carriers with diam- hydrophobic interactions and/or π–π stacking of these eters between 10 and 1000  nm work as media to trans- functional groups make drug loading possible (Xing et al. port chemotherapeutic agents (Gong et  al. 2016). This 2016; Xing et  al. 2016). The above properties facilitate novel drug-delivery system can improve drug solubility, the design of novel nano-carriers based on GO to deliver prolong the blood circulation period, and enhance drug therapeutic drugs (Priyadarsini et  al. 2018; Wang et  al. accumulation by passive or active targeting, which will 2018; Pooresmaeil et al. 2018; Abdelhamid et al. 2021). help to minimize adverse effects of clinical drugs (Geng A magnetic nanoparticle-based drug delivery system et al. 2018; Khursheed et al. 2020). can transfer drugs to a certain site under the influence Natural and synthetic polymeric materials, inorganic of an external magnetic field (Yang et al. 2018, Feng et al. materials, and lipids have been used as drug carriers 2018). Ferroferric oxide (F e O ) is an ideal choice to pre- (Karthik et  al. 2013; Truong et  al. 2013). Graphene is a 3 4 unique carbon-based nanomaterial, which looks like hon pare the magnetic drug delivery system for its paramag- netism, and there is no magnetization after removing the eycombs (Balandin 2020). Graphene oxide (GO) is the Cheng  et al. AAPS Open (2023) 9:12 Page 3 of 14 magnetizing field. Besides, reversible magnetism can pre - new combinations of ATO under the guidance of these vent the aggregation of nanoparticles, which can enhance principles. The ancient Chinese books named “YanFan - the stability of nanomedicines. Generally, the application gHuiJi” and “JiJiuBianFang” recorded that the root of the of Fe O in vivo requires surface modifications to prevent traditional Chinese medicine, Radix Saposhnikoviae, can 3 4 exocytosis and increase biocompatibility. significantly reduce ATO toxicity. In addition, modern Polyethylene glycol (PEG) is one of the most widely pharmacological research show this traditional Chinese studied superhydrophilic polymers and surface modifi - medicine can protect the liver from oxidization (Jiang ers. PEG is cheap, versatile, non-toxic, highly water-sol- et al. 2014). What is more, we find sec-O-glucosylhamau - uble, biocompatible, and can transport nanomolecules. dol (SOG), a compound extracted from Radix Saposh- Because of its appropriate pharmacokinetics and tissue nikoviae, expressing anti-cancer enhancement of ATO in distributions, the usage of PEG in pharmaceuticals is in vitro and in vivo experiments. approved by the Food and Drug Administration (FDA). We introduce a novel nano-drug, controlled-release The accumulation of nanoparticles modified with PEG nano-magnetic carrier, based on Fe O nanoparticles and 3 4 (PEGylation) in liquids decreased compared with that of GO nanosheets, which was conjugated with ATO and nanoparticles without PEG. Moreover, PEG will increase SOG to improve the therapeutic efficacy of HCC. The the internal circulation time and reduce excretion via the drug cargo was constituted of PEG-modified Fe O as 3 4 reticuloendothelial system (RES) (Tas et  al. 2021). Thus, hydrophilic corona and GO as a hydrophobic core. The PEGylated magnetic nanographene oxide (PEG@MGO) morphology, size, microstructure, and magnetic proper- is a potential nano-carrier to deliver hydrophobic drugs ties of the nanoparticles were examined. A series experi- in biological systems (Deb et al. 2018; Ma et al. 2020). ments were implemented, and the results demonstrated Arsenic trioxide (ATO) is a traditional Chinese medi- that ATO and SOG could be released in a controlled cine known as the “king of poisons” with a lethal dose manner in targeted lesions. This nano platform repre - value (LD ) of 15 mg/kg (rat, oral) (Vogt 2017). The usage sents a new approach for the treatment of HCC. of ATO was significantly reduced in the past century due to the public’s fear of its toxicity (Evens et  al. 2004). In the late twentieth century, ATO became popular again. Experimental methods It was approved by the FDA as the frontline therapy for Materials acute promyelocytic leukemia (APL) in 2000 (Hoonjan Graphene oxide (GO) was purchased from XFNANO (Nan- et al. 2018), and it was also approved for the treatment of jing, China). Ferric chloride hexahydrate (FeCl ·6H O), fer- 3 2 newly diagnosed APL by the European Medicines Agency rous chloride tetrachloride (F eCl ·4H O), trisodium citrate 2 2 (EMA) in 2016 (European Medicines Agency 2016). Sub- dihydrate (Na C H O ·2H O), arsenic trioxide (ATO), and 3 6 5 7 2 sequently, ATO was proven effective in other hemato - polyethylene glycol (PEG, average MV 400) were purchased logical malignancies, such as acute myeloid leukemia, from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, chronic myelogenous leukemia, and Hodgkin’s disease China). sec-O-Glucosylhamaudol (SOG) was bought from (Swindell et  al. 2013). With the blood clearance efficacy, Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China). ATO powder was not suitable for solid tumors therapy. All the materials mentioned were used without further Several attempts have been made to develop ATO’s anti- purification. cancer properties by increasing its bioavailability and Dulbecco’s modified Eagle’s medium (DMEM) was reducing systemic toxicity. These methods include sensi - purchased from HyCone (Logan, UT, USA). Fetal tizing carcinoma cells before ATO treatment, combining bovine serum (FBS) was obtained from Sijiqing (Hang- ATO therapy with other conventional chemotherapeutic zhou, China). A total of 0.25% trypsin-EDTA solution, agents, and developing ATO-loaded nano-drugs (Wang 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium et  al. 2012). Henceforth, ATO has become a “potential bromide (MTT), Prussian blue iron staining kit (with broad-spectrum anti-cancer drug” (Akhtar et  al. 2017). Eosin solution), dimethyl sulfoxide (DMSO), dou- Hepatocellular carcinoma (HCC) is one of the most ble antibiotic (streptomycin/penicillin), phosphate malignant cancers and has caused substantial mortal- buffer solution (PBS, pH 7.4), fluorescein isothio - ity worldwide (Bray et al. 2018, Yang et al. 2019). HCC is cyanate, and sodium dodecyl sulfate were obtained insensitive to adriamycin and platinum chemotherapeu- from Solarbio (Beijing, China). The Annexin V-FITC/ tics, so the development of ATO-loaded nano-drugs will PI Apoptosis Detection Kit was purchased from Bec- provide new treatment options for liver cancer. ton, Dickinson, and Company (San Digo, USA), and 2ʹ, Synergism and detoxication are important principles 7ʹ-dichlorodihydrofluorescein diacetate (DC-FHDA) was of traditional Chinese medicines. We try to find some bought from Sigma-Aldrich, USA. Cheng et al. AAPS Open (2023) 9:12 Page 4 of 14 Cell line and cell culture Preparation of drug‑loaded PEG‑Fe O @GO 3 4 The human hepatoma cell line (HepG2) and human In order to evaluate the adsorption process, different hepatocyte cell line (L02) were purchased from Shang- formulations were prepared. ATO and SOG co-loaded hai Cell Bank, Chinese Academy of Sciences (Shanghai, PEG@MGO (PEG@MGO@ATO + SOG) were prepared China). Both cells were cultivated in a CO incubator as follows: 10 mg PEG@MGO was added into 10 mL ethyl (Thermo Scientific, USA). HepG2 and L02 were cul - alcohol solution containing 2 mg/mL ATO and 4 mg/mL tured in a DMEM medium supplemented with fetal SOG. ATO-loaded PEG@MGO (PEG@MGO@ATO) bovine serum (10%, v/v), penicillin (100 UI/mL), and was prepared in the solution containing 2  mg/mL ATO streptomycin (100 UI/mL). When the cell confluence only, while SOG-loaded PEG@MGO (PEG@MGO@ reached nearly 80%, the cells were digested and pas- SOG) was prepared in the solution containing 4  mg/mL saged with 0.25% trypsin for subsequent experiments. SOG only. These resulting mixtures were stirred at 50 °C for 6 h, and then the nanoparticles were collected via an Nd magnet and washed with double distilled water and ethanol three times in sequence to remove unabsorbed Preparation of  Fe O and polyethylene glycol‑modified 3 4 ATO and SOG. Finally, the above drug-loaded nanopar- magnetic GO (PEG@MGO) ticles were freeze-dried at − 20 °C for 24 h. The method The preparation for PEG@MGO was according to pre - of ATO and SOG loading on PEG@MGO was optimized vious reports with some developments (Wang et  al. through preliminary experiments based on the solubility 2018; Mao et al. 2019). GO powder (20 mg) was poured of SOG and inhibition rates of HepG2. into 50 mL purified water with 2  g PEG-400 and son - After drug loading, the supernatant was collected and icated for 1  h. Then, 2.162  g FeCl · 6H O and 0.795  g 3 2 filtered via a 0.22  μm membrane filter. The concentra - FeCl · 4H O were added. The suspension was stirred 2 2 tions of ATO and SOG in the supernatant were deter- and maintained at 60  °C for 30  min with N protec- mined through an inductively coupled plasma emission tion to generate magnetic cores according to reac- spectrum (ICP 6300, Thermo Electron Corporation, tion 1. After that, sodium hydroxide (NaOH) solution USA) and high-performance liquid chromatography (1 mol/L) was dropwise added until the pH value to 11. (HPLC 1100, Shimadzu, Japan), respectively. Detailed Then, 0.247  g sodium citrate dihydrate (C H Na O ) 6 5 3 7 analytical methods were showed in supporting infor- was added under constant magnetic stirring. The tem - mation. Drug encapsulation efficiency (EE%) and drug perature of the mixture was kept at 60  °C and stirred content (DC%) of PEG@MGO@ATO + SOG and PEG@ for 2  h. The precipitation was collected by magnetic MGO@ATO were calculated according to Eqs.  (1) and separation. After being washed three times with water (2), respectively. and ethanol, the black nanoparticles were dried in the vacuum oven at 60 °C for 6 h. Amount of drug in nanoparticles EE% = × 100% Amount of drug added 2+ 3+ − (1) Fe + 2Fe + 8OH → Fe O + 4H O 3 4 2 Amount of drug in nanoparticles Reaction 1: The preparation of Fe O DC% = × 100% 3 4 Amount of nano − carrier (2) Characterization In vitro drug release Fourier transform infrared (FTIR) spectra were recorded The release behaviors of SOG and ATO on PEG@MGO on a Nicolet Ncxus 670 FTIR spectrometer (Thermo Sci - − 1 were investigated at different pH conditions. Briefly, entific, USA) in the range of 500–4000  cm by the KBr 10  mg drug-loaded nanoparticles were dispersed in 10 pellet technique. X-ray powder diffraction (XRD) data mL buffer solution with different pH values (pH 5.0, 6.8, were collected in the range of 2θ = 4 − 90° using a Rigaku and 7.4) and given to continuous shaking at 37  °C. At XRD S2 powder diffractometer (Rigaku Corporation, desired time intervals, 1 mL released solution was taken Japan). Morphological evaluation of the freeze-dried nan- from the stirring dissolution medium. Subsequently, an oparticles was recorded by a Tescan mira3 field emission equal amount of fresh buffer saline was added to the orig - scanning electron microscope (FE-SEM, Tescan, Czech inal media. The percent of released ATO and SOG was Republic). The magnetic property of nanoparticles was calculated according to the following formula: measured by the vibrating sample magnetometer (VSM) using Lakeshore 730T (Lakeshore, USA). Dynamic light W released dose Release(%) = × 100% (3) scattering (DLS) analysis was performed on a Nano-Zeta- W loaded dose Sizer ZEN3600 (Malvern, UK). Cheng  et al. AAPS Open (2023) 9:12 Page 5 of 14 where    W represents the weight of drug medium was replaced by MTT solution, and these cells released dose released into solution from the drug-loaded nanoparti- were further cultured for 4 h. DMSO (150 µL) was subse- cles; W represents the weight of drug loaded on quently added to dissolve the formazan crystals formed. loaded dose nanoparticles. The absorbance (OD) values of different groups at 570 nm were recorded by a microplate reader (Multiskan MK3, Thermo Electron Corporation, USA). The meas - Examine of stability ured OD values of the blank, control, and experimental The residual moisture content was studied for the PEG@ groups were defined as OD, OD , and OD . Cell survival MGO and PEG@MGO@ATO + SOG which were newly b c e rates were calculated according to Eq.  (4). Data are pre- prepared and stored after 30 days. The residual moisture sented as mean ± standard deviation (n = 6). content was measured by Karl Fischer titration using a Mettler DL 38 titrator (Mettler-Toledo, Switzerland). OD − OD e b 100.0  mg samples of the above nanoparticles were used Survival rate(%) = × 100% (4) OD − OD c b for the analysis and the measured moisture content was expressed in percentage. What is more, the dispersive For cell apoptosis assay, cells were seeded in 6-well capacity of PEG@MGO after 30 days’ storage over 25 °C plates at a density of 2 × 10 cells/well. After 24  h of /60RH and 40  °C /75RH was detected by dispersing incubation, nano-drugs at different concentrations were 10 mg PEG@MGO into 10mL water. added to the cell medium, and the cells were incubated for another 24 h. Then, the cells were harvested, washed Cellular uptake of nano‑drug carriers twice with cold PBS, and stained with 5 µL Annexin The cellular uptake of the nano-drug was analyzed by V-FITC and 5 µL PI for 15  min at room temperature in Prussian blue staining and a fluorescence microscope. the dark. These cells were resuspended in 200 µL binding To perform Prussian blue staining, the following steps buffer and were analyzed using flow cytometry (FACS - were made. HepG2 was seeded in 12-well plates at a den- Verse, Becton, Dickinson and Company, USA). sity of 1 × 10 cells/well and incubated for 24 h (37 °C ,5% CO ). Then the cells were treated differently. One group Combination effect of SOG and ATO was only treated with PEG@MGO at a concentration of Tumor-cell proliferation-inhibition behaviors of SOG 15  µg/mL in DMEM medium for 4  h. The other group and ATO against HepG2 were evaluated. The concentra - was treated with PEG@MGO at a concentration of 15 µg/ tions of ATO and SOG ranged from 0.5 to 64 µmol/L and mL in DMEM medium for 4  h and a small Nd perma- from 16 to 2048 µmol/L, respectively. In the combina- nent magnet was placed under each well during the first tion group, the drug concentrations are the same as the 1  h of incubation. After treatment, cells were fixed with above, the combination effects of SOG and ATO loaded 4% paraformaldehyde for 30  min and then were stained on PEG@MGO were also explored. The concentrations by freshly-prepared Prussian blue staining solution for of PEG@MGO@ATO, PEG@MGO@SOG, and PEG@ 30 min and counterstained by eosin for 1 min. MGO@ATO + SOG ranged from 2.5 to 120 µg/mL. After To perform microscopic inspection, HepG2 cells were the cells were incubated for 24  h, 48  h, and 72  h under seeded into a 6-well plate (4 × 10 cells/well). After 24  h, drug application, the cell survival rates were detected by the DMEM medium containing FITC and PEG@MGO/ the microplate reader at 570  nm by MTT assay and the FITC was added to replace the previous solution. FITC process showed above. CI was measured according to and PEG @MGO in the medium were 10  µg/mL and Chou’s method (Chou 2006). 15 µg/mL, respectively. After being incubated at different D D times, the cells were washed three times with sterilized 1 2 CI = + PBS and fixed by 75% absolute alcohol. The cells were (D ) (D ) n n 1 2 finally observed and recorded by an inverted fluorescence In the equation, where (Dn) and (Dn) represent the microscope (DMI3000B, Leica, Germany). 1 2 IC value when drug 1 or 2 works singly. D and D rep- 50 1 2 resent the concentrations of drug 1 and drug 2 when In vitro cytotoxicity and cell apoptosis analysis given simultaneously at the IC value. CI > 1 was used 50 50 The in  vitro cytotoxicity of the PEG@MGO on HepG2 to indicate antagonism between two drugs, C I = 1, the and L02 was studied using the MTT assay. These cells additive effect, and CI < 1, synergism. were seeded in 96-well plates at a density of 1 × 10 cells/well and incubated for 24  h (37  °C, 5% C O ). After removing the culture medium, 200 µL DMEM medium Cellular reactive oxygen species (ROS) measurements containing different concentrations of nanoparticles was The released intracellular ROS in different groups was added. Following 48  h incubation, the DMEM culture measured using DCFH-DA. HepG2 cells were seeded Cheng et al. AAPS Open (2023) 9:12 Page 6 of 14 in 6-well plates with a density of 4 × 10 cells/well and Results and discussion were incubated at 37  °C for 24  h. Then, the cells were Characterizations of the nanoparticles incubated with free ATO, the mixture of ATO and SOG, Figure  1a shows the FTIR spectrums of GO, PEG@ PEG@MGO@ATO, and PEG@MGO@SOG + ATO for MGO, and PEG@MGO@ATO + SOG. The peaks of the − 1 − 1 − 1 24  h. The concentration of nanoparticles was 15  µg/ml GO sample at 3650  cm, 1500  cm , and 1075  cm and the amount of ATO and SOG added are equal to are related to -OH stretching, C-O stretching vibration, − 1 the amount of drugs loaded on nanoparticles. At the epoxy, and alkoxy, respectively. The peak at 1650  cm end of the cultivation, the collected cells were resus- was attributed to C-C stretching vibration. The char - − 1 pended in a DMEM medium containing DCHF-DA (10 acteristic peak of the PEG@MGO sample at 945  cm µM) at 37  °C for 30  min. The cells were washed with was caused by the –CH groups in PEG. Additionally, − 1 serum-free culture solution three times to remove the the absorption peaks of -OH stretching at 3600  cm − 1 DCFH-DA that did not enter the cells. Then, the fluo -and 3050  cm contributed to -CH group bands, which rescence was measured by flow cytometry (excitation at confirmed the successful attachment of PEG to GO − 1 485 nm and emission at 530 nm). surface. The peaks at 530  cm related to vibrations of Fe-O show the successful modification of Fe O . The 3 4 FTIR spectrums of PEG@MGO@ATO + SOG are simi- In vivo tumor inhibition lar to those of PEG@MGO, which means the ATO and Twenty-eight 6-week-old male BALB/c nude mice were SOG loading will not affect the nano-carrier structure bought from Beijing Vital River Laboratory Animal (Farani et al. 2020; Dong et al. 2010). Technology Co., Ltd. (Beijing, China). Approximately The presence of different compositions was verified 2 × 10 HepG2 cells dispersed in 0.2 mL saline solu- with XRD analysis. Figure  1B exhibits the crystalline tion were injected subcutaneously into the right flank phases of GO, Fe O , and PEG@MGO. The peak of the 3 4 region of every mouse. When the volume of tumors PEG@MGO at 2θ = 11.29° is related to 002 diffractions approached 100 mm (about 10 days after the tumor of GO flakes. Peaks at 2θ = 30.23°, 37.23°, 41.22°, 57.15°, inoculation), the tumor-bearing mice were randomly 66.91° display the typical peaks of cubic spinel F e O 3 4 divided into four groups (7 mice /group) for different NPs. This suggests the remaining of the inner core treatments. The therapy method for groups were listed structure even after modification. DLS analysis was as follows: (1) inject saline solution via the tail vein; (2) used to evaluate the size and particle distributions of inject PEG@MGO@SOG + ATO at a dose of 20  mg/ Fe O . As shown in Fig. S1, the average particle diam- 3 4 kg via the tail vein, then fix an external magnet on eter for Fe O was 150 nm for a volume. The PDI value 3 4 the back of the tumor with glues; (3) inject free ATO was 0.179 showed a great homogeneity of this magnetic at a dose of 5  mg/kg via the tail vein; (4) inject PEG@ nanoparticle. MGO@SOG + ATO at 20  mg/kg via the tail vein. The Figure  1c and e shows the SEM images of differ - initial body weight was recorded and monitored every 3 ent nanocomposites. As shown in Fig.  1c, the GO has a days before treatment. The tumor size was determined sheet-like structure with smooth surfaces and a wrinkled and calculated by the formula V = a × b /2, where a and edge. After the modification with the Fe O and PEG400, 3 4 b were the longest and shortest diameters of the tumor, the SEM image of the nanocomposites revealed the respectively. Mice were sacrificed on the 18th day after regular spherical morphology (Fig.  1d). Figure  1e shows treatment, the tumors were excised for weighing. Then, the image of ATO- and SOG-loaded nanoparticles. The tumors and main organs (heart, liver, spleen, lung, and rough surface may be attributed to the adsorption of kidney) were fixed in 10% formalin, followed by hema - drugs on the surface of the PEG@MGO. In conclusion, toxylin and eosin (H&E) staining assay. the modified GO sheets can prevent the restacking of GO sheets and enlarge the surface area to absorb active drugs. The magnetic properties of Fe O , PEG@MGO, and Statistical analysis 3 4 PEG@MGO@ATO + SOG were studied by the mag- Data were processed using Spss.20 (SPSS Inc., Chi- netic hysteresis loop, which is shown in Fig.  2a. The cago, USA) and presented as mean ± standard devia- saturation magnetization value of Fe O , PEG@MGO, tion (SD). Statistical analysis was performed using a 3 4 and PEG@MGO@ATO + SOG was 61.1, 41.4, and 16.1 one-way analysis of variance (ANOVA). The difference emu/g, respectively. The above results demonstrate the was regarded as statistically significant when P  ≤ 0.05. good superparamagnetic ability of the nano-drug with Statistic software Graph-pad Prism 5.0 (GraphPad no coercivity or remanence (Atacan et  al. 2015; Cheng Software, California, USA) was used for all graphical et  al. 2018). The insert picture shows the good water illustrations. Cheng  et al. AAPS Open (2023) 9:12 Page 7 of 14 Fig. 1 a FT-IR spectra of GO, PEG@MGO, and PEG@MGO@ATO + SOG. b X-ray diffraction patterns of GO, Fe O , and PEG@MGO. SEM image of GO 3 4 (c), PEG@MGO (d), and PEG @ MGO@ATO + SOG (e) Fig. 2 a Magnetization curves of Fe O , PEG@MGO, and PEG@MGO@ATO + SOG, magnetic recovery of PEG@MGO@ATO + SOG from aqueous 3 4 solution (insert). b, c Release profile of ATO and SOG from PEG@MGO@ATO + SOG at different pH values dispersibility and easy magnetic separation of the PEG@ of ATO and SOG mainly depended on the electrostatic MGO@ATO + SOG. interaction between drugs and the PEG@MGO. The EE% and DC% of PEG@MGO@ATO and PEG@MGO@ Drug loading and in vitro release study ATO + SOG were listed in Table  1. These data demon - Drug loading capacity is a very important factor in evalu- strated the good drug encapsulation efficiency of the ating the therapeutic effect of nanodrugs. The loading PEG@MGO nanocomposites. This phenomenon can Cheng et al. AAPS Open (2023) 9:12 Page 8 of 14 Table 1 Drug loading ability of different nanoparticles Table 2 Residual moisture content of the nanoparticles after different storage time and determined by Karl Fischer titration PEG@MGO@ATO PEG@MGO@ATO + SOG (mean ± SD; n = 3) ATO ATO SOG Formulations Residual moisture (%) EE(%) 14.76 ± 3.2 18.03 ± 4.2 38.25 ± 3.8 Newly prepared After DC(%) 29.89 ± 2.6 36.52 ± 5.0 153.00 ± 4.2 30 days storage PEG@MGO 0.52 ± 0.01 1.01 ± 0.2 be explained as the addition of PEG on the surface of PEG@MGO@ATO + SOG 0.63 ± 0.11 1.06 ± 0.3 the dendrimer can prevent the diffusion of drugs to the solution. In addition, the high loading capacity might be related to the high surface area of the nanocomposites. In vitro cellular uptake Also, the EE% and DC% of the combination drugs are Cellular internalization is essential for nanoparticles used higher than those of only ATO-loaded nano-drug. It may as drug carriers. Prussian blue staining, which selec- 3+ be explained as positively charged PEG@MGO naturally tively stains Fe , can be used to evaluate the endocytosis absorbs the negatively charged ATO and the addition behaviors of PEG@MGO. Figure  3 shows that blue dots of SOG can produce covalent interaction between two accumulated in cells after being treated with the mag- drugs to enhance the drug loading efficiency. netic drug carrier, indicating that PEG@MGO could be The release profiles of ATO and SOG from PEG@ uptaken by tumor cells. What is more, the intracellular MGO@ATO + SOG at pH 5.0, 6.8, and 7.4 are shown in amount of PEG@MGO was significantly increased by an Fig. 2b and c. The drug release rates of ATO from PEG@ external Nd-magnet (Fig.  3c, d). These results indicated MGO@ATO + SOG with the three pH values were close that a magnetic field would enhance the endocytosis of during the initial 6 h. The results showed that the cumula - PEG@MGO. tive release rate of ATO from PEG@MGO@ATO + SOG To investigate the motion law of the nano-carrier, reached up to 77.6% ± 1.5%, 55.3.00% ± 1.9%, and 53.1% PEG@MGO was labeled with FITC (green) for subcel- ± 2.2% at pH 5.0, 6.8, and 7.4 respectively after 108  h. lular observation. Green fluorescence appeared after 4 h The releasing rule of SOG was similar to that of ATO. co-culture and was widely distributed in the cells after 24 After 108  h, the cumulative release rate of SOG from h co-culture, which is shown in Fig. 3e. This revealed that PEG@MGO@ATO + SOG was approximately 79.21% PEG@MGO exhibited a high level of cell uptake through ± 2.5%, 58.3% ± 1.2%, and 53.4% ± 2.2% at pH 5.0 6.8, endocytosis in a time-dependent manner. and 7.4, respectively. These results show that the release of ATO and SOG from nanocarrier is pH-sensitive and Cytotoxicity assay and cellular apoptosis analysis the release rate increased with the decrease of pH values. For the potential biomedical applications, it is necessary The addition PEG enhances the hydrophilic nature of to investigate the cytotoxicity of nano-carriers. Figure  4 the dendrimer thus improving its stability. Under acidic showed the results for cells treated with PEG@MGO for conditions, the hydrogen bonds are stronger than those 48 h and with drugs loaded with inhibition effect, respec - occurring at pH 7.4. Therefore, the high release of ATO tively. It should be noticed that the viability of the tumor and SOG from PEG@MGO@ATO + SOG nanocom- cells (HepG2) and liver cells (L02) were observed to be posite under acidic pH conditions indicates the poten- larger than ~ 70% even at higher concentration of 250 µg/ tial application of the proposed nanocarrier in cancer mL after 48 h (Fig. 4a, b), indicating the excellent biocom- treatment. patibility of blank nano-carriers. ATO- and SOG-loaded PEG@MGO showed cell inhibition to HepG2 cells and Stability of nanoparticles L02 cells, while the inhibition effect of L02 is lower than The residual moisture content of PEG@MGO and PEG@ that of HepG2. The results implied that the PEG@MGO MGO@ATO + SOG were showed in Table  2. It is well nanoparticles have minor toxicity and great selectivity as known that the residual moisture content plays impor- a drug delivery in cancer treatment. tant roles in determining a power’s long-term stability, Annexin V/PI staining was carried out to investigate both physically and chemically. The results showed the the influence of various concentrations of the novel nano- moisture content of the both two nanoparticles were less drug on the apoptosis rates of HepG2 cells. The apopto - than 1.2% which can prove the stability of PEG@MGO sis rates of cells treated with PEG@MGO@ATO + SOG whether it loads drug or not. Meanwhile, the dispersibil- under 10  µg/mL, 15  µg/mL, 20  µg/mL, 25  µg/mL, and ity of PEG@MGO remains nearly unchanged regardless 50  µg/mL were studied, respectively. After the incuba- the store condition (Fig. S3). tion of 24 h, the apoptosis rates were 13.5%, 16.0%,17.9%, Cheng  et al. AAPS Open (2023) 9:12 Page 9 of 14 Fig. 3 Cellular uptake of PEG@MGO. Images of HepG2 stained with Prussian blue: a cells with no treatment, b treated with 15 µg/ml PEG@MGO for 4 h, c treated with 15 µg/ml PEG@MGO under the magnetic field for the first 1 h. d Area percentage analysis after staining with Prussian blue. e Microscopy images of HepG2 incubation with FITC-labeled PEG@MGO after different time 19.2% and 21.3% in Fig.  5a. The results demonstrate that 24  h, 48  h, and 72  h incubation were 0.714, 0.83, and the inhibitory activity of the nano-drug increased with 0.964, respectively. The CI values smaller than 1 indicated the increase of its concentrations and the cellular apop- the synergistic effect of SOG and ATO. The inhibition tosis of HepG2 cells caused by PEG@MGO@ATO + SOG ratios of HepG2 at different combination concentrations was a concentration dependent manner (Fig.  5b). It can of ATO and SOG shown in Fig. S4 added evidence of be also hypothesized that PEG@MGO@ATO + SOG will the cell growth inhibition under the combination usage inhibit tumor proliferation by triggering the apoptotic of SOG and ATO. The IC values of PEG@MGO@ path way of cancer cells. ATO + SOG were smaller than those of PEG@MGO@ SOG and PEG@MGO@ATO. From the above results, we Cytotoxicity and synergism can conclude that the active targeting of PEG@MGO@ The cytotoxicity of free drugs and drug-loaded nanopar - ATO + SOG leads more drug molecules to enter tumor ticles on HepG2 cells was measured by the MTT assay. cells to inhibit tumor growth. The IC values of free drugs, co-drugs, drug-loaded PEG@MGO, and combination index (CI) values of co- Cellular ROS analysis drugs were summarized in Tables  3 and 4. The results To investigate whether the novel nano-drug causes oxi- show that the cytotoxicity of all experimental groups is dative stress in cancer cells, ROS levels of HepG2 cells dose-and time-dependent. Compared with single-drug were measured by flow cytometry after being incubated treatment, dual-drug combination treatment exhibits with different formulations (Fig.  5c). The results showed higher cytotoxicity. The CI values of SOG + ATO after that the intracellular ROS levels were significantly Cheng et al. AAPS Open (2023) 9:12 Page 10 of 14 Fig. 4 Cytotoxicity of PEG@MGO against a HepG2 cells and b L02 cells after incubation. Cytotoxicity of ATO + SOG@ PEG@MGO against c HepG2 cells and d L02 cells after incubation increased after 24  h’s drug treatment. The intracel - In vivo synergistic anti‑cancer effect lular ROS levels in ATO + SOG and PEG@MGO@ Based on the effective therapy of the nanocomposite ATO + SOG groups were higher than those in the in  vitro, a HepG2 xenograft model was established by groups of ATO and PEG@MGO@ATO, which is due intravenous administration with different formulations to the synergistic effect. The intracellular ROS levels to study the synergistic efficacy. As shown in Fig.  6a, in PEG@MGO@ATO and PEG@MGO@ATO + SO G the volume of tumor showed significant differences groups increased 2.30-fold and 2.59-fold, respectively, among different groups. Treatment with ATO led to a as compared with those of the free ATO group. The val - slight inhibition of HepG2 tumor growth compared to ues of those two groups increased 1.46-fold and 1.59- the PBS group. The group treated with PEG@MGO@ fold compared with those of the ATO + SOG group. A ATO + SOG under a magnetic field displayed the most significant increase in ROS level was observed in the significant tumor growth inhibition, outperforming both cells when treated with a co-drug. Excessive intracel- the group of free ATO and PEG@MGO@ATO. After lular ROS may induce oxidative stress in mitochondria 18 days of observation, tumor tissues were extracted, and destruction of the integrity of the mitochondria weighed, and photographed. Tumor weights were 95.2, membrane structure and finally, induce cellular apop - 476.2, and 226.1  mg in the group of magnet + PEG@ tosis and death. PEG@MGO@ATO + SOG was more MGO@ATO + SOG, ATO, and magnet + PEG@MGO@ likely to produce ROS than other drugs, which may be ATO, respectively, as compared with 707.5 mg of the PBS ascribed to its sustained drug release manner. group. The average tumor weight of the PEG@MGO@ Cheng  et al. AAPS Open (2023) 9:12 Page 11 of 14 Fig. 5 The apoptosis rates of HepG2 cells after incubation with PEG@MGO@ATO + SOG at different concentrations. a Flow cytometry analysis via * ** *** Annexin V/PI staining and b quantitative analysis of tumor cells apoptosis. P < 0.05, P < 0.01, P < 0.001. c Eec ff t of different treatment on the ** production of intracellular ROS according to relative fluorescence intensity. Compared with ATO treated group, P < 0.01; compared with ATO + SOG ## treated group, P < 0.01 Table 3 IC and CI of SOG and ATO against HepG2 cells for different incubation time Time IC drug alone IC drug combination CI at IC 50 50 50 (95% confidence interval) (95% confidence interval) SOG(µmol/L) ATO(µmol/L) SOG(µmol/L) ATO(µmol/L) 24 h 1575.0 ± 59.5 52.0 ± 2.8 586.8 ± 33.0 17.8 ± 1.7 0.71 48 h 773.9 ± 36.6 19.9 ± 2.1 289.8 ± 27.2 9.9 ± 1.4 0.83 72 h 458.5 ± 18.5 14.2 ± 2.4 219.6 ± 16.1 6.9 ± 0.3 0.96 tumor growth curve, tumor weight (Fig. 6d), and in vitro Table 4 IC of different formulations against HepG2 cells for different incubation time experiments. The tumor growth in mice treated with PBS showed a fast and unrestrained tendency, and the Time Nanodrugs IC (µg/mL) final volume was about 11-fold of the initial size. The free 24 h PEG@MGO@SOG 87.41 ± 1.84 ATO could not prevent tumor growth might because of PEG@MGO@ATO 51.87 ± 4.73 the quick dilution of fluid flow. PEG@MGO@ATO + SOG 27.07 ± 0.86 H&E staining examinations of the tumor tissues after 48 h PEG@MGO@SOG 78.89 ± 2.55 treatment are displayed in Fig.  6e; it appeared that the PEG@MGO@ATO 41.69 ± 2.22 tumor tissue displayed a typical necrotic response after PEG@MGO@ATO + SOG 21.91 ± 1.78 treatment; the cell necrosis of PEG@MGO@ATO + SOG 72 h PEG@MGO@SOG 68.99 ± 1.78 under magnet was the most obvious. All these indicate PEG@MGO@ATO 30.89 ± 0.66 that the nano-drug PEG@MGO@ATO + SOG owns a PEG@MGO@ATO + SOG 15.62 ± 0.84 remarkable tumor inhibition effect, and the magnetic microenvironment may promote the accumulation of the anti-cancer drug in tumor cells. The possible toxicity of the formulations was also stud - ATO + SOG group was much lower than those of the ied. As shown in Fig. 6c, no significant reduction in body other groups (Fig. 6b). The pictures of the tumors among weight was observed in the different groups during the the different groups are consistent with the results of the treatment period, indicating the high biocompatibility Cheng et al. AAPS Open (2023) 9:12 Page 12 of 14 Fig. 6 In vivo tumor inhibitory effects on HepG2 xenograft tumors. a Tumor volumes were measured every three days. Compared to PBS group, ***p < 0.001; compared to magnet + PEG@MGO@ATO group, p < 0.05. b Weight of tumors of each therapeutic group after 18 days of treatments. ## ### Compared to PBS group, ***p < 0.001. **p < 0.01; compared to ATO group, p < 0.01. p < 0.001. c The body weight of mice in treatments. d The optical image of the excised tumor tissues after 18 days of treatments. e The H&E analysis for different groups (scale bar: 50 μm) CI Combination index of these PEG@MGO-based nano-drugs. H&E stained DC-FHDA 2ʹ, 7ʹ-Dichlorodihydrofluorescein diacetate images of major organs (heart, spleen, lung, and kidney) DC% Drug content shown in Fig. S5, revealing nearly no difference in patho - DMSO Dimethyl sulfoxide DMEM Dulbecco’s modified Eagle’s medium logical lesions of varied groups. These results collectively EE% Drug encapsulation efficiency indicated that the nanoparticles did not cause appreci- EMA European Medicines Agency able systemic toxicity or an inflammatory response. FBS Fetal bovine serum FDA Food and Drug Administration Fe O Ferroferric oxide 3 4 FTIR Fourier transform infrared spectra Conclusion FeCl ·6H O Ferric chloride hexahydrate 3 2 In summary, a pH-sensitive polyethylene glycol-modified HepG2 Human hepatoma cell line magnetic graphene oxide loaded with ATO and SOG HCC Hepatocellular carcinoma HPLC High-performance liquid chromatography (PEG@MGO@ATO + SOG) was first prepared for the GO Graphene oxide magnetically targeted and efficient synergistic-chemo LD50 Lethal dose value cancer therapy. This new biocompatible drug delivery L02 Human hepatocyte cell line MTT 4,5-Dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide system was prepared by coating hollow F e O nanoparti- 3 4 PEG Polyethylene glycol cles on the surface of GO sheets via electrostatic interac- PEG@MGO PEGylated magnetic nanographene oxide tion and then immobilized with hydrophilous PEG-400. RES Reticuloendothelial system ROS Reactive oxygen species The combination of ATO and SOG, the active ingredient SEM Scanning emission microscope of traditional Chinese medicines, can improve the inhi- SOG Sec-o-Glucosylhamaudol bition of HepG2. These two drugs were loaded on the VSM Vibrating sample magnetometer XRD X-ray powder diffraction nano-carrier due to the large surface area of the PEG@ MGO. The nanocomposite exhibited excellent magnetic Supplementary Information hyperthermia effect, controlled drug release, and pH sen - The online version contains supplementary material available at https:// doi. sitivity, which could be used for accurate cancer therapy. org/ 10. 1186/ s41120- 023- 00079-4. Furthermore, it showed excellent anti-cancer perfor- mance in vitro and vivo experiments. The results showed Additional file 1: Fig. S1. DLS result of Fe O nanoparticles. Fig. S2. TEM 3 4 that this ATO- and SOG-co-loaded nanodrug exhibited image of Fe O nanoparticles. Fig. S3. Photos of PEG@MGO dispersed in 3 4 water. A. PEG@MGO stored over 25℃/60%RH for 30 days; B. PEG@MGO high potential in the HCC adjuvant therapy. stored over 40℃/75% for 30 days. Fig. S4. The inhibition ratios of HepG2 at different combination concentrations of ATO and SOG after 48h co- culture. Fig S5. H&E histology images of the major organs in mice after Abbreviations administration of (A) PBS, (B) Magnet+PEG@MGO@ATO+SOG, (C) ATO, (D) APL Acute promyelocytic leukemia Magnet+PEG@MGO@ATO for 18 days. ATO Arsenic trioxide Cheng  et al. AAPS Open (2023) 9:12 Page 13 of 14 Authors’ contributions Dong H, Zhao Z, Wen H, Li Y, Guo F, Shen A, Pilger F, Lin C, Shi D (2010) Jinlai Cheng: performed laboratory experiments, analyzed, interpreted data, Poly(ethylene glycol) conjugated nano-graphene oxide for photody- and wrote the first copy of the manuscript. Kun Hong: contributed to the namic therapy. Sci China Chem 53(11):2265–2271 experiments. Jianhui Sun: co-write, revised the manuscript, and provided the Evens AM, Tallman MS, Gartenhaus RB (2004) The potential of arsenic triox- final approval of the version to publish. Hongmei Li: contributed to the design ide in the treatment of malignant disease: past, present, and future. of the work, supervised the research, and provided the final approval of the Leuk Res 28(9):891–900 version to publish and agreed to be accountable for all aspects of the work. European Medical Agency (2016) Trisenox EMEA/H/C000388/II/0058 assess- Yuqing Tan: supervised the project. Miyi Yang: put the design of the work, ment report contributed in revision, analysis, interpretation of data and provided financial Farani MR, Khadiv–Parsi P, Riazi GH, Ardestani MS, Rad HS (2020) PEGyla- support of this project. All authors discussed the results and contributed to tion of graphene/iron oxide nanocomposite: assessment of release of the final manuscript. 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Journal

AAPS OpenSpringer Journals

Published: Jun 5, 2023

Keywords: Synergistic effect; Arsenic trioxide; Sec-o-Glucosylhamaudol; Magnetic graphene oxide; Drug-delivery

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