Biomimetic graphene oxide quantum dots nanoparticles targeted photothermal-chemotherapy for gastric cancer

Abstract Direct use of chemotherapy drugs in the treatment of gastric cancer often leads to systemic side effects and unsatisfied therapeutic efficacy due to the lack of tumour-targeting ability. The excellent properties of nanoparticles make them good tools to provide more options for the targeted delivery of chemotherapeutic drugs. Herein, we developed a novel nanomedicine (GOQD-ICG-CS-6@HM nanoparticles, GIC@HM NPs), which employed graphene oxide quantum dots (GOQDs) to co-load photosensitizer indocyanine green (ICG) and chemotherapeutic drug gamabufotalin (CS-6) as the core and wrapped with the hybrid membrane (erythrocyte membrane and gastric cancer cell membrane, HM) on its surface. This nanomedicine possesses the functions of photothermal therapy and chemotherapy, making it a good choice for the treatment of gastric cancer. The results showed that the bionic-coated hybrid membrane not only improves the biocompatibility of the nanomedicine, and prolong its circulating half-life, but also delivers the drug to the tumour site precisely and improves the efficiency of drug utilisation. In vitro and in vivo studies further showed that GIC@HM NPs exhibited combinational effects on tumour therapy while displaying no obvious side effects on normal tissue. To sum up, the newly developed GIC@HM NPs provide a safer, more efficient, and more precise method for gastric cancer treatment.


Introduction
Gastric cancer is the fifth most frequent cancer worldwide and the third leading cause of cancer death [1]. In the current treatment guidelines, perioperative chemotherapy, or chemotherapy plus radiotherapy after surgery is listed as the preferred method [2]. Preoperative chemotherapy can increase the chances of radical resection, eliminate an early micro diffusion, evaluate in vivo response of treatment, and also improve the quality of life of patients with locally advanced, unresectable, or metastatic gastric cancer [3]. However, clinical investigations have found that chemotherapy for gastric cancer has poor prognosis and side effects [4], so there is an urgent need for a new strategy to solve these clinical problems.
In recent years, phototherapy have attracted wide attention for their selective killing of cancer cells with the least toxicity to normal tissue [5]. Moreover, the combination of phototherapy and chemotherapy can further enhance the efficacy of tumour treatment [6][7][8][9]. Among these, indocyanine green (ICG) acts as a photosensitizer because its strong absorption band allows deeper tissue penetration to cause significant heating [10][11][12][13]. In addition, gamabufotalin (CS-6), the main derivative of toad dienic acid lactone, can effectively inhibit proliferation and angiogenesis in cancers including multiple myeloma, lung cancer, breast cancer, and glioma [6,[14][15][16]. Therefore, the combination of ICG and CS-6 for gastric cancer is expected to achieve improved efficacy and reduced toxicity. However, ICG and CS-6 are tiny molecular chemicals, resulting in their inability to selectively target tumour sites. Meanwhile, due to the limited solubility and bioavailability of CS-6, higher doses are required for clinical use, leading to decreased efficacy and even toxicity during long-term in vivo application.
In recent years, multi-functional nanomaterials reported to effectively load and deliver drugs, and avoid side effects of chemotherapy drugs [17]. Among these nanomaterials, graphene oxide (GO) is widely used in material science, energy, and biomedicine [18][19][20]. Graphene oxide quantum dots (GOQDs), as a derivative of GO, are widely used in biomedicine, biological imaging, and sensor fields due to their excellent optoelectronic properties, low toxicity, high biocompatibility, and light bleaching ability [21][22][23]. While retaining the excellent characteristics of GO, GOQDs show better safety performance in biomedical applications and can be used as potential nanocarriers for ICG and CS-6 [24]. In addition, one of the important capabilities of nanocarriers is to endow the nanoparticles with the ability to evade the immune system and enhance tumour targeting functions [25]. Cell membrane encapsulations have been widely adopted to endow nanoparticles with good biocompatibility, maintenance of cellular characteristics, and adaptability to a variety of therapeutic applications. For example, erythrocyte membrane coating significantly extended the circulation half-life of nanomaterials [26]. In addition, by wrapping cancer cell membranes on the surface, nanoparticles gained good self-recognition, internalisation ability, and good immune escape ability in vivo [27]. Thus, heterogeneous membrane wrapping using fusion of erythrocyte and tumour cell membranes can confer immune escape and tumour targeting capabilities to nanoparticles.
Based on the above clues, in this work, we designed and established a nanomedicine for photothermal-chemotherapy combined therapy (Scheme 1). GOQDs effectively load ICG and CS-6 as the core through π-π stacking and hydrophobic interactions (GOQD-ICG-CS-6, GIC NPs). Then, coating with the hybrid membranes (erythrocyte membrane and gastric cancer cell membrane, HM), GIC@HM NPs possessed good biocompatibility and tumour-targeting ability. under laser irradiation, the photosensitizer of ICG performs photothermal function to improve the release and tumour-killing ability of the drug CS-6. With the combination of photo-chemotherapy, efficient gastric cancer therapy could be achieved.

Cell lines and animals
All the cell lines used in the following experiments were provided by Cell Library from Xiangya Central Laboratory (Central-South university). Dulbeccos-modified Eagle's medium (DMEM, HyClone) containing 1% streptomycin and penicillin (PS, Invitrogen) and 10% foetal bovine serum (FBS, Gibco) was used to culture the cells at 37 °C and 5% CO 2 . BALB/c nude mice (4-5 weeks old) were bought from Hunan SJA Laboratory Animal Co. Ltd.

Anti-tumour effects of ICG and CS-6
BGC-823 cells were seeded into 96-well plates. After 24 h of culture, different concentrations of CS-6 were added. After 24 h of incubation, the images of BGC-823 cells treated with different concentrations of CS-6 were photographed by an inverted microscope, and the cell survival rate of each group was detected by the MTT method.
BGC-823 cells were inoculated into 96-well plates. After 24 h of culture, different concentrations of ICG were added. After 4 h of incubation, the laser (1 W/cm 2 , 808 nm) was used to irradiate 5 min in each well. The MTT assay was used to detect the cell survival rate of each group after 20 h of incubation.

Preparation of HM
Preparation of RBCM: Fresh whole blood of BALB/c nude mice was stored in the blood vessels of heparin sodium, washed with 1 × PBS and centrifuged (4 °C, 3600 rpm), then cracked with 0.25 × PBS at 4 °C for 2 h, then washed with 0.25 × PBS and centrifuged (4 °C, 12,000 rpm) until the supernatant became colourless and transparent, and the red sediment below was collected and resuspended with PBS. Squeeze with 0.45 μm and 0.22 μm filters.
Preparation of BGC-823M: When the cultured BGC-823 cells grew to about 90%, the scraped cells were collected, the cell extract A (1 mM) was added, and the cells were lysed at 4 °C for 1 h. The cells were repetitively frozen and thawed 5 times after lysis, then the cell lysate was centrifuged at 4 °C (800 rpm, 10 min) and the supernatant was centrifuged at 4 °C (13,500 rpm, 30 min). The precipitate obtained is the BGC-823 cell membrane.
Preparation of HM: The concentration of BGC-823 cells and RBC membrane protein was determined using a BCA protein kit, and the weight of the membrane was twice the weight of the membrane protein, RBC membrane was added to the BGC-823 membrane at the Scheme 1. (a) the preparation scheme of giC nPs encapsulated by a hybrid membrane (HM) (giC@HM nPs). (B) the mechanism of tumour inhibition mediated by giC@HM nPs was proposed. membrane protein weight ratios of 1:1. RBCM and BGC-823M were re-suspended into the PBS of 1 ml, mixed and stirred at 37 °C for 2 h, then the hybrid membrane was filtered by pulsed ultrasound (80 W, 5 min) and filtered with 0.45 and 0.22 μm filter, respectively.

Synthesis of GIC@HM NPs
EDC (40 mg) and NHS (10 mg) were added to GOQDs (1 mg) aqueous solution (1 mg/mL), respectively, and stirred to activate the carboxyl group of GOQDs for 30 min. NH 2 -PEG 2000 -NH 2 (10 mg) was added and stirred for 24 h, the GOQDs modified by PEG (pGOQDs) were obtained by using 3.5 KDa dialysis membrane to dialysis for 3 days. And the PEG-modified GOQDs (pGOQDs) were obtained. 8 μL ICG aqueous solutions (25 mg/mL) and 8 μL CS-6 (25 mg/mL) were added to 1 mg pGOQDs and stirred at room temperature for 24 h. GIC NPs were adsorbing ICG and CS-6 on the surface through π − π stacking and hydrophobic interactions [20,28], and GIC nanoparticles were dialysed with a 2.5 KDa dialysis membrane for 24 h.
The hybrid membrane-coated GIC NPs (GIC@HM NPs) were obtained by stirring 1 mg GIC NPs and 200 μL hybrid membrane (HM) in the water bath at 37 °C and stirring for 2 h.

Characterisation of GIC@HM NPs
The images of GOQDs, GIC, and GIC@HM NPs were taken by transmission electron microscope (JEM-2100F, Japan). The particle size of GOQDs, GIC, and GIC@HM NPs and the potential of GOQDs, pGOQDs, GI, GIC, and GIC@HM NPs were measured by particle size analyser (Zetasizer Nano ZS, uK). GOQDs, ICG, CS-6, HM, GI, GIC, and GIC@HM NPs were placed in a uV-vis spectrophotometer to detect the characteristic uV absorption peak of each sample.
SDS-PAGE: The protein of RBCM, 823 M, HM, pGOQD@HM NPs was extracted from membrane protein extract, and the protein concentration was determined. After heating denaturation, the protein was separated on 12% gel and stained with Coomassie brilliant blue.
Membrane colocalisation experiments: The experiment of membrane colocalisation was carried out according to previous reports [29]. In brief, the erythrocyte membrane (RBCM) was labelled with DiI, and the tumour cell membrane (823 M) was labelled with DiO. The dye-labelled film was observed under a confocal laser scanning microscopy.

Loading and release behaviour of GIC@HM NPs
To evaluate the drug loading (DLC) and entrapment efficiency (EE), in the process of preparing GI and GC NPs, using a dialysis membrane (MWCO = 3.5 kDa) to collect the release medium after dialysis for 24 h. Combined with the corresponding standard curve, the drug loading of ICG and CS-6 was determined. The following formulas are used to calculate LC and EE for drugs: where M T represent the total mass of ICG or CS-6, M u represent the mass of the unencapsulated ICG or CS-6, and M P represent the quantity of pGOQDs.
2 ml GIC@HM NPs were dialysed in PBS with pH7.4 or pH5.4 of 50 ml and shaken gently in the dark at room temperature to test drug release behaviour (MWCO = 3.5 kDa). At different time points (12,24,36,48,60, and 72 h), the amount of CS-6 in the solution was detected by uV-vis analysis with 300 nm absorbance. To detect the control and release of the laser on the nano-platform, an 808 nm laser was used to irradiate 2 ml GIC@HM NPs for 30 min. The GIC@HM NPs are therefore dialysed, and the dialysate is collected at the appropriate time.

In vitro photothermal characteristics and stability
The 808 nm laser (1 W/cm 2 , 5 min) was used to irradiate GI, GI@ HM NPs solution (10 µg/mL), and PBS. The temperature was recorded once per 1 min with a thermal infra-red image (Flir C2, uSA). The aqueous solutions of ICG (10 µg/mL) and GI@HM NPs (10 µg/mL) were irradiated with laser (808 nm, 1 W/cm 2 , 5 min) and then cooled to room temperature for 5 times to compare the photothermal stability. An ultraviolet spectrophotometer was used to measure the final absorption spectra of GI@HM NPs and ICG.

Detection of ROS in vitro
BGC-823 cells were planted in 12-well plates and incubated for 24 h at 37 °C. PBS, ICG, CS-6, GIC, and GIC@HM NPs (the concentrations of ICG and CS-6 were 2 µg/mL and 100 nM) were added to the well. Another group was treated with the lasers (808 nm, 1 W/cm 2 , 5 min) after 4 h of incubation. BGC-823 cells were rinsed with serum-free medium after 20 h of growth, stained with DCFH-DA for 20 min at 37 °C, and photographed under a confocal microscope.
1 ml each of ICG, GI, and GIC@HM NPs were taken and then irradiated with laser for 5 min and then 10 μL TEMP trapping agent was added. Finally, the signal of single-linear state oxygen was detected by ESR.

Biocompatibility analysis
Cell cytotoxicity assay: BGC-823, MDA-MB-231, SMC, and HuVEC cells were inoculated into 96-well plates for 24 h. Then, GOQDs, pGOQDs, and pGOQDs@HM NPs were added to each kind of cell. After 24 h of incubation, the survival rate of each group of cells was detected by MTT method.
Immune evasion assay: The macrophages were placed in a 12-well plate. After 24 h of culture, GRB, GRB@Lip, and GRB@HM NPs (the concentration of RB was 5 µg/mL) were added to the well. The macrophages were fixed and stained with DAPI after 4 h of incubation. Finally, using the CLSM to capture the results.
Haemolysis assay and coagulation assay: 4% red blood cell suspension was mixed with different concentrations of CS-6, GOQDs, GI, and GI@HM NPs and incubated at 37 °C for 4 h. The red blood cells dissolved in pure water and PBS was used as positive control and negative control, respectively. The absorbance of the supernatant at 562 nm was measured after centrifugation at 3500 rpm for 10 min at 4 °C and then estimated the haemolysis rate.
Platelet-rich plasma (PRP) was prepared from the whole blood of allogeneic BALB/c nude mice with anticoagulants. The absorbance of the above components at 650 nm was measured by a microplate instrument. The samples treated with thrombin and PBS were used as positive control and negative control, respectively.

Cell uptake and intracellular distribution of GIC@HM NPs
Homotypic targeting analysis of hybrid membrane (HM): To make the GOQDs produce a red glow, they were initially loaded with rhodamine B (RB). The GRB NPs were first loaded with RB to emit red colour. Plates were seeded with BGC-823 cells, 4T1 cells, and SMC cells were incubated in 12-well plates (1 × 10 4 cells per well), and cultured for 24 h at 37 °C, after that, three different types of cells were given fresh culture media containing the RB, GRB, GRB@RBCM and GRB@HM NPs (5 μg/mL, RB). The nucleus was coloured with DAPI after 4 h of cultivation, and the imaging was done under CLSM.
The time of drug uptake was investigated. BGC-823 cells were inoculated in a 12-well plate at a density of 1 × 10 4 per well and cultured in an incubator at 37 °C for 24 h. The cells were treated with RB, GRB, GRB@HM NPs (the concentration of RB was 5 µg/mL) at 1, 2, and 4 h. 4% paraformaldehyde-fixed cells, and the cells were stained with DAPI, and the images of the cells were collected by CLSM.
The mechanism of GRB@HM NPs uptake was investigated by treating cells with three inhibitors, methyl-β-cyclodextrin (caveolin-mediated endocytosis inhibitor), chloroquine (grid-mediated endocytosis inhibitor), colchicine (micropinocytosis inhibitor), and low temperature (4 °C, inhibition of energy channel). BGC-823 cells were inoculated in a 12-well plate and cultured in 37 °C incubators for 24 h. After treating BGC-823 cells with each of the three inhibitors mentioned above for 1 h, GRB@HM NPs were added and incubated for 4 h at 37 °C. Then, cells were fixed with 4% paraformaldehyde and stained with DAPI, and finally CLSM images were acquired.
To study the localisation and uptake efficiency of nanoparticles in cells, GOQDs were initially loaded with rhodamine B (RB) to give them a red glow. BGC-823 cells were incubated in a 12-well plate for 24 h, then cultured with GRB and GRB@HM NPs (5 μg/mL, RB) for 4 h, followed by the addition of 1 μL lysosome probe (1 μM) and incubation for 30 min for lysosome staining. At the same time, 4% paraformaldehyde-fixed cells and stained the nucleus with DAPI.

Anticancer activity of GIC@HM NPs in vitro
Cell viability: BGC-823 cells inoculated in 96-well plates were separated into two groups. After the fusion degree of the cells reached 85%, PBS, ICG, CS-6, GIC, and GIC@HM NPs (the concentration of ICG and CS-6 was 2 µg/mL and 100 nM, respectively) were added and incubated for 4 h. The laser treatment group was exposed to lasers (808 nm, 1 W/cm 2 , 5 min). The cytotoxicity was detected by MTT assay after additional incubation for 20 h.
Flow Cytometry analysis: BGC-823 cells were inoculated in a 6-well plate, and after the cell fusion degree reached around 70%, the cells were treated according to the grouping Control, ICG + Laser, CS-6, GIC + Laser, and GIC@HM + Laser. The cells of each component were collected and the apoptosis rate of BGC-823 cells was determined using an Alexa Fluor 488ANNEXIN V/PI apoptosis kit after 24 h of incubation.
Live/dead staining assay of Cells: BGC-823 cells were inoculated into 12-well plates and divided into two groups. After 24 h of culture, PBS, ICG, CS-6, GIC, and GIC@HM NPs (the concentration of ICG and CS-6 were 2 µg/mL and 100 nM) were added. 4 h later, the laser group was treated with laser for 5 min (1 W/cm 2 , 808 nm). After 20 h of culture, the cells were treated with Calcein AM/Propidium Iodide (PI) for 10 min, and the cell images were captured by CLSM photography.
Western blotting: BGC-823 cells were seeded into a 6-well plate and cultured for 24 h. After the cells were treated according to the five groups of Control, ICG + Laser, CS-6, GIC + Laser, and GIC@HM + Laser, the protein of each well was extracted, the protein concentration was detected, and the expression of related proteins (Caspase-3, PARP, Bcl-2, Bax, and Cyclin D1) was detected by western blotting.

Pharmacokinetics in vivo
After the mice were treated with GIC and GCI@M NPs (150 µL, 5 mg/kg of ICG) respectively, the orbital blood of mice was collected at 1, 2, 3, 4, 6, 8, 12, and 24 h for fluorescence visualisation imaging, and analysed by IVIS kinetic optical system (PerkinElmer, CA) to determine the fluorescence intensity of ICG.

Targeted analysis and biodistribution in vivo
BGC-823 cells in the logarithmic growth phase were subcutaneously implanted into the back of female BALB/c nude mice. After the tumour size reached 100 mm 3 , the tumour-bearing mice were divided into three groups injected with 150 µL GIC and GCI@M NPs (ICG concentration was 5 mg/kg). Kodak multimode imaging system (Ex/Em = 740/820 nm) was used to collect fluorescence images in vivo at 0, 0.5, 1, 2, 4, 8, 12, 24, and 48 h. After 48 h, the tumours, lung, spleen, liver, kidney, and heart were collected and captured under Kodak multimode imaging system.

In vivo photothermal effect of GIC@HM NPs
The tumour-bearing nude mice were infused with 150 μL GIC and GCI@M NPs (ICG concentration was 5 mg/kg) into the caudal vein for 24 h, then the tumour area was irradiated with laser (808 nm, 1 W/cm 2 ) for 5 min, and then the nude mice were photographed with an infra-red camera and the images were collected.

Antitumor activity assay in vivo
The tumour-bearing nude mice were grouped into 5 groups (n = 3) randomly: PBS, IC + Laser, GI@HM + Laser, GC@HM + Laser, GIC@HM, and GIC@HM + Laser, and the concentrations of ICG and CS-6 were 5 and 2 mg/kg, respectively (Group IC + Laser is free ICG and CS-6 plus laser irradiation; Group GI@HM + Laser is GOQDs loaded ICG, wrapped in a layer of HM, plus laser irradiation; Group GC@HM is GOQDs loaded CS-6, wrapped in a layer of HM; GIC@HM is GOQDs loaded ICG and CS-6, wrapped in a layer of HM; while GIC@HM + Laser is GOQDs loaded ICG and CS-6, wrapped in a layer of HM, plus laser irradiation). After the tumour volume reached 100 mm 3 , the drug was injected into the tail vein of nude mice every other day. After 24 h, the nude mice were treated with 808 nm laser (1 W/cm 2 , 5 min), and the drug was given 6 times in the whole treatment cycle. During this period, the body weight and tumour size of nude mice were recorded, and the length (L) and width (W) of the tumour were measured with a Vernier calliper. The formula for calculating the volume of the tumour is V = (L × W 2 )/2. The tumours of each group of nude mice were collected and fixed in formalin. The tumours were fixed in paraffin and then stained with H&E and TuNEL.

Safety evaluation of GIC@HM NPs
After 12 days of treatment, the nude mice were dissected, and the heart, liver, spleen, lung, and kidney of each group were collected and fixed in formalin. The above organs were paraffin-fixed and coloured with H&E. The liver and kidney function and blood count of nude mice was tested.

Statistical analysis
The above-mentioned related experiments were repeated 3 times. SPSS19.0 statistical software was used for analysis. Metrological data of normal distribution were expressed as x s. T-test was used to compare the mean between the two groups. The homogeneity test of variance was performed first and then single-factor analysis of variance was used to make comparisons between the means of the two groups. In terms of * p < 0.05, * * p < 0.01, and * * * p < 0.001, the difference was statistically significant.

Antineoplastic effect of CS-6 and ICG
To achieve an ideal treatment effect for gastric cancer, we first optimised the effective concentration of CS-6 and ICG to kill BGC-823 cells. As shown in Figure S1(A), when BGC-823 cells were treated with CS-6 for 24 h, the IC 50 value was about 180 nM, which is a very low dose compared to the clinical chemotherapy drug 5-fluorouracil [30]. Meanwhile, we observed the morphological changes of BCC-823 cells treated with different concentrations of CS-6 and found that with the increase of drug concentration, the number of live BGC-823 cells gradually decreased, and many cells gradually shrunk and became round ( Figure S1(B)), which indicated the possible effect of CS-6 on the growth and apoptosis of BGC-823 cells. In addition, with the help of laser irradiation, IGC can induce stronger tumour-killing effect against BGC-823 cells and the IC 50 value was about 2.3 μg/mL ( Figure S1(C)). It means that CS-6 combined with photothermal treatment has the potential to enhance the killing of BGC-823 cells.

Characterisation of the GIC@HM NPs
To address the shortcomings in drug delivery, we use nanotechnology to improve the bioavailability. GOQDs is an ideal nano-carrier for the construction of drug delivery system because of its ultra-small size, high safety, and modifiability [24]. We characterised the shape and size of GOQDs, GIC, and GIC@HM NPs using transmission electron microscope (Figure 1(A-C)) and particle size analyser (Figure 1(D-F)). The results of TEM and DLS showed that the particle size of GOQDs was about 5 nm ( Figure  1(A) and 1(D)). After being modified by PEG and loaded with ICG and CS-6, the particle size of GIC NPs increased to about 16 nm (Figure 1(B) and 1(E)), and when coated with a hybrid membrane, the particle size of GIC@HM NPs increased to about 120 nm, which may be due to the presence of multiple GIC NPs clustered in cell membrane vesicles (Figure 1(C) and 1(F)). Then, we detected the zeta potential of different materials. The potential of GOQDs, pGOQDs, GI, GIC NPs was −26.5 mV, −8.26 mV, −10.2 mV, and −18.9 mV, respectively, and the potential of GIC@HM NPs changes to −10.5 mV (Figure 1(G)). In addition, we detected the ultraviolet absorption peak of each component (Figure 1(H)). The characteristic ultraviolet absorption peak of GOQDs, ICG, CS-6, and HM was at 360, 780, 300, and 400 nm, respectively, while the final material of GIC@HM NPs indicated these characteristic absorption peaks. These results indirectly proved the successful synthesis of GIC@HM NPs.
Moreover, we carried out the membrane fusion experiment to prove the successful synthesis of the hybrid membrane (Figure 1(I)). The erythrocyte membranes and BGC-823 cell membranes were labelled with red and green fluorescent dyes, respectively, and the result showed that the red and green fluorescent overlapped and appeared as yellow, which proved the successful synthesis of the hybrid membrane. Figure 1(J) showed the ultraviolet absorption peaks of the erythrocyte membrane, tumour cell membrane, and hybrid membrane. Erythrocyte membrane and hybrid membranes showed similar characteristic absorption peaks. In addition, the membrane proteins of HM and pGOQD@HM NPs inherited the characteristic proteins of both RBCM and BGC-823M (Figure 1(K)), which proves the successful synthesis of hybrid membrane.
Next, we studied the drug loading capacity of nanoparticles. As shown in Figure S2(A) and S2(B), when the mass ratio of ICG/ pGOQDs was 0.2, 0.4, and 0.6, the loading rate of ICG was 10%, 35%, and 44%, respectively, and when the mass ratio of CS-6/ pGOQDs was 0.2, 0.4 and 0.6, the loading rate of CS-6 was 1.7%, 12%, and 26%, respectively. Next, we studied the loading rate and entrapment efficiency of GI (ICG/pGOQDs) mass ratio 0.2, 0.4, and 0.6) to CS-6. As shown in Figure S2(C), the loading rate of CS-6 was 7%, 14%, and 20%, respectively, corresponding to the CS-6/ GI mass ratio of 0.2, 0.4, and 0.6. With the increase in the mass ratio, the loading rate did not increase significantly, and the entrapment efficiency was unchanged, so the drug was synthesised according to the mass ratio of 0.2. Finally, when the ratio of ICG and CS6 to pGOQDs was 0.2, the loading rates of ICG and CS-6 were 13% and 7%, respectively ( Figure S2(D)). These results demonstrate that pGOQDs can improve the load rate of CS-6 after loading with ICG.

Photothermal properties and drug release of GIC@HM NPs
We tested the photothermal properties of ICG and nanomaterials. Figure 2(A) showed the dose-dependent temperature increase of ICG under laser irradiation (808 nm, 1 W/cm 2 ). After GOQDs loaded ICG, the temperature of GI NPs and GI@HM NPs was higher than that of ICG under the irradiation of laser (Figure 2(B)), which indicated that GOQDs could improve the photothermal properties of ICG [31]. Meanwhile, the photothermal curve of GI@HM NPs was similar to that of GI NPs. This result demonstrated the negligible effect of hybrid membrane coating on the photothermal property of GI@HM NPs. The photothermal map also showed that the materials wrapped by the hybrid membranes were relatively concentrated in the high-temperature area without significant change (Figure 2(C)). In addition, using ICG as the control, we detected the photothermal stability of GI@HM NPs. As shown in Figure 2(D), after five cycles of laser irradiation, the maximum temperature of ICG gradually decreased, while the maximum temperature of GI@ HM NPs remained stable, which illustrated that this nano-platform improved the photothermal stability of free ICG.
The controlled release of nanomedicines can greatly increase the therapeutic efficacy and decrease the side effects. Since the tumour microenvironment is acidic [32], we researched the release of CS-6 at different pH values and observed that at a pH7.4, CS-6 could only be released about 40% at 72 h. In contrast, at pH5.4, CS-6 was able to release about 60% at 72 h (Figure 2(E)). In addition, the release rate of CS-6 in GIC@HM NPs pH5.4 can reach 80% under laser irradiation. However, it was increased about 20% under pH7.4 condition (Figure 2(F)). These findings indicated that drug release from GIC@HM NPs could be accelerated by the acidic microenvironment and laser, and this property of it could prevent too much dose of CS-6 from being released too early in the blood circulation, achieving the effect of reducing side effects, enriching the drug at the tumour location and improving the therapeutic effect.

Biocompatibility of GIC@HM NPs in vitro
Biocompatibility refers to the properties that nanoparticles can produce appropriate host reactions in drug delivery applications, generally referring to cytotoxicity and blood compatibility [33]. First of all, we treated BGC-823, MDA-MB-231, SMC, and HuVEC cells with GOQDs, pGOQDs, and pGOQD@HM NPs. The results showed that GOQDs had almost no toxicity to these investigated cells. However, PEG modification reduced the survival rate of cells with the increase of pGOQDs concentration. On contrary, the hybrid membrane coating decreased the toxicity of pGOQDs and the cell survival rate was higher than 85% even with treatment with high concentration, which indicated the good biocompatibility of pGOQD@HM NPs ( Figure S3(A-D)).
Haemolysis is another indicator of biocompatibility. Figure 3(A) indicated that the haemolysis rate was only 2% in the presence of 10 μg/mL CS-6, demonstrating the safety of CS-6 for haemolysis. Meanwhile, Figure 3(B,C) showed the effects of different nanomaterials on red blood cells. GOQDs were extremely safe even at high concentrations. While at the concentration of 100 μg/mL, the supernatant became slightly reddish, and the haemolysis rate was 7% in the GI NPs group. In contrast, the haemolysis rate was reduced to 2% in the GI@HM NPs group due to the hybrid membrane coating. These results indicated that the HM can improve the biosafety of nanomaterials. Figure S4 showed the microscopic morphology of red blood cells treated with different materials. From this figure, it can be found that these treatments did not cause morphological change in red blood cells. Next, we investigated the effect of GI@HM NPs on blood coagulation. In Figure  3(D), compared with the positive control group, GI@HM NPs did not cause blood clotting. This finding is compatible with the previous report [34], which noted the improvement of biomimetic membranes on the biosafety and biocompatibility of GI NPs.
Moreover, we investigated the immune escape ability of GI@HM NPs by using macrophages to mimic the immune environment in vitro. As shown in Figure 3(E,F), compared with GRB NPs without membrane coating and GRB@Lipo NPs groups, the uptake efficiency of GRB@HM NPs by macrophages was dramatically decreased (***p < 0.001). This result demonstrated that the hybrid membranes encapsulation can improve the immune escape ability of GRB@HM NPs, the ability of which can improve the effective utilisation of drugs by reducing phagocytosis during blood circulation.

Tumour targeting and uptake of GIC@HM NPs in vitro
To verify the function of the hybrid membrane on targeting ability in vitro, we compared the uptake efficacy of different tumour cell lines to GI@HM NPs (Figure 4(A,B)). In this experiment, GRB with red fluorescence was used to locate the position of GOQDs. As we expected, the red fluorescence intensity of rhodamine B increased approximately 13.17-fold and 11.07-fold in BGC-823 cells treated with GRB@HM NPs compared with 4T1 and SMC cells, correspondingly. This finding supported that the hybrid membranes can endow the GIC@HM NPs with good homomorphic targeting ability.
To monitor the uptake efficiency of nanomaterials by BGC-823 cells at different time points, a rhodamine B (RB) fluorescence reagent was used to label nanomaterials. As shown in Figure 4(C,D), we found that the red fluorescence intensity of GRB@HM NPs was 8.61-fold stronger than that of GRB NPs, which proved the improvement of the uptake rate of nanomaterials coating with hybrid membranes. Then we explored the cellular uptake mechanism of nanomaterials by investigating the effect of different inhibitors. Figure 4(E,F) showed that methyl-β-cyclodextrin could significantly inhibit the uptake of nanomaterials which indicated that GRB@HM NPs entered cells mainly through caveolin-mediated endocytosis. In addition, by exploring the relationship between nanomaterials and lysosomes, we found that nanomaterials could co-locate with lysosomes after cells were processed with different components of nanomaterials for 4 h (Figure 4(G)), indicating that nanomaterials can enter lysosomes and then be released into the cytoplasm.

Tumour cell-killing ability of GIC@HM NPs in vitro
To test the killing effect of nanomaterials on gastric cancer cells, the cells were divided into 10 groups: Control (PBS), ICG, CS-6, GIC, and GIC@HM NPs normal group and PBS, ICG, CS-6, GIC, GIC@HM NPs with laser group (1 W/cm 2 , 808 nm, 5 min). By comparing the viability of BGC-823 cells with different treatments for 24 h, we found that the cell survival rate of normal groups of ICG, GIC, and GIC@HM NPs was significantly different from that of the laser groups (****p < 0.0001, ***p < 0.001) ( Figure 5(A)). Meanwhile, it was found that the photothermal effect of ICG has a certain effect on tumour cells. Compared with the single ICG (95.1%) and CS-6 (73.6%) group, the cell survival rate of the GIC NPs group was 47.5%, which proved the better therapeutic effect of GIC NPs on tumour cells. Moreover, the laser irradiation further enhanced the tumour-killing effect of GIC NPs, which was reflected by the cell viability of 22.4%. However, the cell survival rate of the final material group GIC@HM NPs without laser irradiation was 63.5%, higher than that of the GIC NPs group without laser irradiation (47.5%), which can be contributed to the sustained release effect of drugs. On the contrary, after laser irradiation, the cell survival rate of the GIC@HM NPs group was only 15.7% ( **** p < 0.0001), which was lower than that of GIC NPs (22.4%) irradiated by laser. Inducing apoptosis is one of the main mechanisms of killing tumour cells. Next, we studied the effects of different treatments on tumour cell apoptosis. FACS assay to detect the apoptosis rate of BGC-823 cells, it was found that the proportion of apoptosis was 5.21% and 18.10%, respectively, after treatment with CS-6 and ICG. However, the apoptosis rate of GIC NPs and GIC@HM NPs reached 90% and 94%, respectively after combination with laser irradiation (Figure 5(B)). In addition, to intuitively observe the death of cells caused by the action of nanomaterials, we performed live-death staining experiment. BGC-823 cells with different treatments for 24 h were stained with fluorescent dyes. Green fluorescence represents living cells and red fluorescence represents dead cells. Figure 5(C,D) showed that under irradiation of laser (1 W/cm 2 , 808 nm, 5 min), compared with the five components without laser irradiation, the proportion of red fluorescence increased. The consistent results of cell staining and MTT assay further proved the strong tumour cell-killing function of GIC@HM NPs with laser irradiation.
In addition, to prove the singlet oxygen induction of this nano-platform under laser irradiation, we carried out an ESR experiment. As shown in Figure S5, GI and GIC@HM NPs produced more singlet oxygen than ICG. It is reported that reactive oxygen species (ROS) induced by ICG is an important factor in eradication of cancer cells [35]. Then, using the ROS probe, we detected the ROS change of tumour cells caused by GIC@HM NPs. As we expected that the GIC@HM NPs treated cells showed the strongest green fluorescence signal among these investigated groups ( Figure  5(E,F)). This result demonstrated that the combination of photo-chemo therapy can efficiently kill tumour cells by inducing strong ROS. These findings strongly proved that GIC@HM NPs plus laser could contribute to cell apoptosis by causing the production of ROS. Subsequently, we explored the protein expression change of gastric cancer cells under different conditions. To ensure the reliability of FACs assay, the levels of two key pro-apoptotic proteins PARP and Caspase-3 in BGC-823 cells were detected under different treatments. As we expected that P-Caspase3 and P-PARP gradually decreased, while the trend of C-Caspase3 and C-PARP was the opposite ( **** p < 0.0001), which is consistent with the FACs assay. Because Bax/Bcl-2 signalling pathway was reported to be involved in CS-6-induced apoptosis of tumour cells [36], we detected the expression of major mitochondrial apoptosis signal-related proteins Bcl-2 and Bax, the results of Western blotting showed that could significantly reduce the expression level of Bcl-2 and the ratio of anti-apoptosis Bcl-2/Bax (****p < 0.0001). In addition, we also found the expression of cycle-related protein CyclinD1 decreased ( **** p < 0.0001) ( Figure 5(G,H)). From the expression pattern changes of these proteins, it could be indicated that GIC@HM NPs can kill tumour cells by affecting the apoptosis and cycle of BGC-823 cells.

Tumour targeting and long circulation of GIC@HM in vivo
Blood samples of female BALB/c nude mice via intravenous administration were collected to examine the pharmacokinetics of GIC@ HM NPs through fluorescence imaging. In Figure 6(A,B), the half-life of GIC@HM NPs reached 4 h, which was 1.73-fold longer than that of GIC NPs, probably because the hybrid membrane layer could prevent effective clearance by the mononuclear phagocyte system with the help of CD47 [37]. Next, we photographed the fluorescence distribution of GIC@HM NPs after being intravenously injected into tumour-bearing nude mice. As shown in Figure 6(C), the targeting effect of GIC@HM NPs was stronger than GIC NPs. Moreover, the fluorescence intensity of the GIC@HM NPs group tumour was significantly stronger 1.37-fold than GIC NPs ( Figure  6(D,E)) ( **** p < 0.0001). All these results demonstrate that GIC@HM NPs had a good tumour-targeting effect. In addition, the distribution results of fluorescence in various organs also illustrated that GIC NPs were mainly metabolised by the liver ( **** p < 0.0001), while GIC@HM NPs were mainly metabolised by the kidney ( **** p < 0.0001). Some of the GIC NPs were being detained in the spleen, while GIC@HM NPs did not stagnate in the spleen ( **** p < 0.0001), because the hybrid membrane layer could prevent effective clearance by the mononuclear phagocyte system, and effectively prolong the half-life. These results proved that GIC@ HM NPs possessed higher tumour targeting ability and prolonged circulation time in vivo than the GIC NPs group without membrane coating, which can reduce the side effects of drugs on normal cells.

Chemo/photothermal therapy of GIC@HM NPs in vivo
Thermal imaging was used to evaluate the photothermal effect of GIC@HM NPs at the tumour site of tumour-bearing nude mice. As shown in Figure 7(B), the temperature of the tumour site with laser irradiation was not significantly increased in the ICG group as the single ICG was easily metabolised without accumulation in the tumour site. GIC NPs could be partially accumulated in the tumour part due to the EPR effect of nanomaterials. Thus, the temperature of the tumour site of the GIC NPs team increased after with laser irradiation. However, the temperature of the tumour site of the GIC@HM NPs group was the highest as hybrid membranes coated can not only slow down its metabolism but also can enhance the accumulation in the tumour site to exert strong photothermal effect.
Based on the outstanding targeting ability and strong photothermal effect in vivo, we then investigated the therapeutic effects of GIC@HM NPs against gastric cancer according to the schematic diagram in Figure 7(A). These nude mice were randomly divided into 6 groups: Control, IC + Laser, GI@HM + Laser, GC@HM + Laser, GIC@HM, and GIC@HM + Laser. Figure 7(B) showed the drug administration model. The treatment time was 12 days including administration and laser irradiation, a total of 6 cycles. As shown in Figure 7(C), the tumour size of nude mice with GIC@HM NPs administration was dramatically less than that of other teams (***p < 0.001), which suggested their strongest inhibitory effect on tumour growth. Consistent with this result, the images of tumour in Figure 7(E) visually showed the difference in tumour size of each group at the end of the experiment. The size of the tumour of the final material GIC@HM NPs was much smaller than that of other components. In addition, the body weight assay in Figure 7(D) showed that there was no significant change in the GIC@HM NPs group, indicating the ultra-low toxicity of nanomaterials to nude mice. Figure 7(F) showed the H&E staining of each group's tumour tissue. It can be seen that the tumour cells in the GIC@HM NPs administration group showed the highest apoptosis and necrosis rate among these investigated groups, which proved the strong killing effect of GIC@HM NPs on tumours at the tissue level. Similarly, the TuNEL staining assay visually demonstrated the strongest green fluorescence in the GIC@HM NPs group, compared with other groups (Figure 7(F)), which further explained the reason for GIC@HM NPs for efficient tumour therapy.

Toxicity evaluation in vivo
Although nanomaterials showed high anti-tumour efficiency, their safety was related to whether they can be effectively used [38], so we tested the biosafety of GIC@HM NPs. First of all, at the end of the administration, we collected the whole blood of nude mice to perform the liver function, kidney function assay and blood routine assay. In Figure 8(A), no obvious damage was found in the main organs of the heart, liver, spleen, and kidney in the treatment group nude mice compared to normal healthy mice. Biochemical tests showed that the blood routine (RBC, WBC, PLT, GRAN, RDW and MCV) of nude mice were all in the normal range in various treatment teams (Figure 8(B)). In addition, through the analysis of the important indexes of liver and kidney function, it was discovered that free CS-6 and ICG (IC group) could induce an elevation of AST (1.41-fold than normal), ALT (1.59-fold than normal), and CREA (1.55-fold than normal) (Figure 8(C)). Considering the low toxicity and short half-life (plasmatic half-life of 2-4 min) of ICG [39,40], the liver and kidney function of nude mice increased after treatment with free CS-6, it was consistent with the previous report [6]. However, the values of AST, ALT and CREA in nude mice recovered to the normal range in the GIC@HM NPs group. These results confirmed the good biosafety of GIC@HM NPs in vivo.

Conclusion
In this study, we successfully constructed a novel hybrid membrane-encapsulated nano complex (GIC@HM NPs), which can  1 cm). (F) The tumour tissue sections were stained with H&e staining (scale is 100 μm) and the tumour tissue was stained with Tunel (scale is 50 μm). ***p < 0.001, **p < 0.01, *p < 0.05. be applied against gastric cancer using combinational photothermal-chemo therapy. Due to the combinational therapy, the relative reduction of the effective dose of CS-6, coupled with the photothermal therapy effect of ICG under the irradiation of the laser, can enhance the anti-tumour effect of the nano-platform. The biomimetic nano-platform camouflaged showed the advantages of immune escape, prolonging half-life, and tumour targeting, so that CS-6 and ICG can gather in the tumour site, greatly improving the utilisation of drugs, reducing the side effects of drugs, and further enhancing the anti-tumour effect. In vivo and in vitro experiments showed a significantly stronger anti-tumour effect of the nano-platform, compared with the control group. Overall, these complexes with high safety showed that the efficient nano-drug loading platform had potential clinical significance for the therapy of gastric cancer.

Disclosure statement
No potential conflict of interest was reported by the author(s). Figure 8. In vivo safety of giC@HM nPs. (A) H&e stained images obtained from the major organs (heart, liver, spleen, and kidney) of mice with different treatments (scale is 100 μm). (B) Complete blood panel analysis of nude mice treated with different groups. rBC: red blood cell, WBC: white blood cell, PlT: platelets, gran: granulocyte, rDW: red blood cell distribution width, MCV: mean corpuscular volume. (C) Blood biochemistry analysis of nude mice treated with different groups. liver function markers: alT: alanine aminotransferase and aST: aspartate aminotransferase. Kidney function markers: Crea: creatinine and Bun: blood urea-nitrogen. ****p < 0.0001, **p < 0.01, *p < 0.05.