Cancer Cell Membrane Camouflaged Nanoparticles to Realize

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Cancer Cell Membrane Camouflaged Nanoparticles to Realize Starvation Therapy Together with Checkpoint Blockade for Enhancing Cancer Therapy Wei Xie, Wei-Wei Deng, Minghui Zan, Lang Rao, Guang-Tao Yu, Dao-Ming Zhu, WenTao Wu, Bei Chen, Li-Wei Ji, Liben Chen, Kan Liu, Shi-Shang Guo, Hui-Ming Huang, WenFeng Zhang, Xingzhong Zhao, Yufeng Yuan, Wenfei Dong, Zhi-Jun Sun, and Wei Liu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b03788 • Publication Date (Web): 25 Feb 2019 Downloaded from http://pubs.acs.org on February 26, 2019

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Liu, Wei; Wuhan University, Department of Physics

ACS Paragon Plus Environment

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Cancer Cell Membrane Camouflaged Nanoparticles to Realize Starvation Therapy Together with Checkpoint Blockade for Enhancing Cancer Therapy Wei Xie,1,# Wei-Wei Deng,2,3,# Minghui Zan,1 Lang Rao,1 Guang-Tao Yu,2,3 Dao-Ming Zhu,1 Wen-Tao Wu,1 Bei Chen,1 Li-Wei Ji,1 Liben Chen,4 Kan Liu,5,6 Shi-Shang Guo,1 Hui-Ming Huang,1 Wen-Feng Zhang,2,3 Xingzhong Zhao,1 Yufeng Yuan,7 Wenfei Dong,8,* Zhi-Jun Sun,2,3,* and Wei Liu1,7,* 1

Key Laboratory of Artificial Micro- and Nano-Structures of Ministry of Education, School of Physics and

Technology, Wuhan University, Wuhan 430072, China; 2

The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of

Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan 430072, China; 3 Department

of Oral Maxillofacial-Head Neck Oncology, School and Hospital of Stomatology, Wuhan University,

Wuhan 430072, China; 4

Department of Biomedical Engineering and Department of Mechanical Engineering, Johns Hopkins University,

Maryland 21218, United States; 5

School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu

610054, China; 6 College 7

of Electronic and Electrical Engineering, Wuhan Textile University, Wuhan 430200, China;

Department of Hepatobiliary and Pancreatic Surgery Zhongnan Hospital of Wuhan University Wuhan, Hubei

430071, China; 8

Key Laboratory of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology,

Chinese Academy of Sciences, Suzhou 215163, China; # These authors contributed equally to this work; * Corresponding author Email: [email protected]; [email protected]; [email protected]


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ABSTRACT Although anti-PD-1 immunotherapy is widely used to treat melanoma, its efficacy still has to be improved. In this work, we present a therapeutic method that combines immunotherapy and starvation therapy to achieve better anti-tumor efficacy. We designed the CMSN-GOx method, in which mesoporous silica nanoparticles (MSN) are loaded with glucose oxidase (GOx) and then encapsulate the surfaces of cancer cell membranes to realize starvation therapy. By functionalizing the MSN’s biomimetic surfaces, we can synthesize nanoparticles that can escape the host immune system and homologous target. These attributes enable the nanoparticles to have improved cancer targeting ability and enrichment in tumor tissues. Our synthetic CMSN-GOx complex can ablate tumors and induce dendritic cell maturity to stimulate an anti-tumor immune response. We performed an in vivo analysis of these nanoparticles and determined that our combined therapy CMSN-GOx plus PD-1, exhibits a better anti-tumor therapeutic effect than therapies using CMSN-GOx or PD-1 alone. Additionally, we used the positron emission tomography imaging to measuring the level of glucose metabolism in tumor tissues, for which investigate the effect with the cancer therapy in vivo. KEYWORDS：cancer cell membrane, immunotherapy, starvation therapy, positron emission tomography

Recently, cancer immunology treatment that utilize immunoregulatory means to treat


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tumors have attracted much attention.1-4 Although PD-1 immunotherapy has shown promise, off-target side effects in metastatic melanoma remain.5-7 Moreover, lacking a co-stimulation tumor microenvironment in situations where mature dendritic cells encounter cancer cells and effecter T cells so that its efficacy is limited. 8-11 Therefore, inducing more mature dendritic cells and increasing the content of effecter T cells would greatly improve the performance of PD-1 cancer immunotherapy.12-14 In effort to develop approaches for PD-1 immunotherapy, many researchers have focused on dendritic cells. Recently, many groups have reported the methods, which use the photothermal or photodynamic therapy with nanoparticles can stimulate a vaccine-like immune response, which indicates its powerful anti-tumor efficacy and potential for cancer immunotherapy.15-21 Glucose is a crucial nutrient supplier for tumor growth. Using glucose oxidase (GOx) to transform glucose into gluconic acid and toxic hydrogen peroxide is a therapy model. This starvation therapy provides tumor-ablation effects more effectively than traditional therapies by simply stopping the energy supply to tumor cells. To increase the efficacy of starvation therapy, some groups have combined glucose starvation with other therapeutic strategies such as phototherapy.

22-27

Nanoparticles also have shown potential to improve starvation therapy outcomes in the treatment of cancers, but few clinical trials have been successful

28, 29

due to

immune escape responses and poor cancer targeting.30 Recently, a biomimetic technology has emerged, in which nanoparticles are coated with natural cell membranes.31-35


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Herein, we present a combination of starvation and immunotherapy for targeted cancer treatment, in which we utilize nanoparticles that encapsulate cancer cell membranes as well as a PD-1 immune checkpoint blockade. As shown in Figure 1, we prepared mesoporous silica nanoparticles (MSN) loaded with GOx (MSN-GOx), and then used an extrude method to encapsulate cancer cell membranes in the MSN-GOx. We found that these nanoparticles allow membrane-bound proteins to combine with cell apoptosis caused by GOx, which can be delivered to antigen presenting cells of the tumor so that efficiently enhance PD-1 immune checkpoint blockade effect. In addition, we utilized the positron emission tomography (PET) imaging to characterize the glucose metabolism in tumors.

Figure 1. Schematic illustration of anti-tumor immune response and enhanced anti-PD-1 immunotherapy induced by CMSN-GOx.

RESULTS AND DISCUSSION We characterized the physicochemical properties of the nanoparticles and verified the


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successful synthesis of CMSN-GOx. We constructed the CMSN-GOx as follows. First, we loaded GOx onto the nanoparticles by mixing them overnight with a magnetic stirrer. Next, we prepared natural cancer cell vesicles from B16-F10 cells, and extruded cancer cell membranes onto the surface of the MSN-GOx. We used transmission electron microscopy (TEM) imaging to confirm the completeness of the CMSN core-shell structure (Figure 2A). The diameters of the CMSN-GOx were about 90 nm and they showed high monodispersity in PBS. The outer membrane thicknesses of the CMSN-GOx were about 8 nm, which accords with other reports.36 These results indicated that the MSN nanoparticles were successfully coated into the cancer cell membranes. Additionally, we used dynamic light scattering (DLS) analysis to determine that the hydrodynamic diameter of the MSN nanoparticles increased by about 15 nm after the encapsulation of the cancer cell vesicle. The zeta potential results suggest a successful coating, as the surface charges of the MSN cores increased to approximately that of the membrane vesicles after being coated (Figure 2B).

We

used

sodium

dodecyl

sulfate-polyacrylamide

gel

electrophoresis

(SDS-PAGE) for determine that the cell membrane’s surface proteins weather can be keeping on the CMSN. As shown in Figure 2C, the CMSN and cell membranes showed similar protein profiles, indicating that the proteins had been fully retained in the CMSN. After verifying the successful loading of the GOx and membrane proteins, we conducted a GOx-catalyzed decomposition reaction to characterize the catalytic properties of CMSN-GOx. As shown in Figure 2D, the pH of the MSN-GOx solution


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stays close to 7.4 in no glucose culture conditions. In contrast, a significant pH decline from 7.4 to 3.6 occurs after treatment with glucose due to the glucose decomposition caused by the GOx in the MSN or CMSN. Glucose can promote the cancer cells’ proliferation. To better understand the process of glucose metabolism, we tested the hydrogen peroxide content. The results showed that the H2O2 can be effectively released regardless of whether the nanoparticles are coated or not cancer cell membranes (Figure S1, Supporting Information). Compared with conventional starvation therapy methods that only block the glucose supply,25 our method also produces toxic H2O2 by the GOx-triggered decomposition of glucose, and thereby demonstrates stronger anti-tumor effects. (Figure 2E).37 In our experiments, we tested B16-F10 cancer cell lines to determine the anti-tumor efficacy of our nanoparticles. We used Calcein-AM to stain live cells and observed green fluorescence by fluorescence microscopy, and we used PI to stain dead cells and observed red fluorescence by fluorescence microscopy. When treated with CMSN-GOx, the B16-F10 cells shown strong fluorescence signal as displayed the CLSM images (Figure S2, Supporting Information). These above results indicate that a combined therapy is recommended to achieve an optimal therapeutic effect in vitro. Next, we tested B16-F10 cancer cells by incubating them with CMSN. We labeled the MSN and cancer membranes with Cy5 and fluorescein isothiocyanate (FITC), respectively. Under a confocal laser scanning microscope (CLSM), we observed fully overlapped Cy5 (red) and FITC (green) signals surrounding the nuclei, which indicates the core-shell structure of the CMSN and their ability to target B16-F10 cells (Figure 2F).


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Furthermore, we conducted cellular uptake experiments to verify the nanoparticles' immune evasion capability, at the same time, verify the nanoparticles' cancer targeting capability. To do so, we incubated MSN, red blood cell (RBC) membrane coated MSN (RMSN), and CMSN nanoparticles with RAW 264.7 cells or B16-F10 cancer cells, and quantified their intracellular Si using inductively coupled plasma mass spectrometry (ICP-MS). We found CMSN has the lowest macrophage uptake. As the same time, it has highest cancer cell uptake (Figure S3, Supporting Information), which demonstrates CMSN’s capabilities with respect to immune evasion and cancer targeting. In addition, we found the CMSN retain the protein of CD47, which is a membrane protein that helps cancer cells escape from phagocytosis (Figure 2G). To further verify the cancer targeting ability of the CMSN, we incubated the CMSN with B16-F10 and 4T1 cells, respectively. The results indicated that the CMSN preferred to accumulate in the B16-F10 cells rather than the 4T1 cells (Figure S4, Supporting Information). Furthermore, to analyze the tumor target ability of CMSN, we used ICP-MS to quantitatively determine the Si contents in tumor sites and major organs by intravenous injection of MSN, RMSN and CMSN. The accumulation of CMSN at the tumor site had significantly increase (Figure 2H), which indicates that CMSN have a good targeting ability in vivo. We also worth note that the accumulations of liver and spleen in both CMSN and RMSN are lower than those in the MSN, which reveals that nanoparticles coated by cell membranes have an excellent immune escape ability in vivo.


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Figure 2. Physicochemical characterizations of CMSN. (A)The samples TEM images of MSN and CMSN. We used phosphotungstic acid to negatively stain the samples. (B) Mean diameter and zeta potential of MSN, CC Me mbranes, and CMSN. (C) The protein analysis of MSN, CC Membranes, and CMSN by SDS-PAGE. (D) The pH changes of MSN and CMSN solutions at the lack and exist of glucose. (E) B16-F10 cell viability after different treatments at the lack and exist of glucose (F) Confocal laser scanning microscopy images of single B16-F10 cell by cultured with CMSN. DAPI (blue) used for stain cell nucleus, Cy5 (red) used for stain MSN, and FITC (green) used for stain CC-Membranes, respectively. Scale bar is 5 μm. (G) The analysis of CD47 with western blot in MSN, cancer cell lysate, CC Membranes, and CMSN. (H) The nanoparticles' biodistribution in mice after injecting 48 h. Five samples in each group. ( * ) P < 0.05, ( ** ) P < 0.01 and ( *** ) P < 0.001, respectively, as compared with the MSN group.

Fig. 3A shows the design of our animal experiment, in which we challenged mice with either CMSN-GOx or PD-1 antibody (10 mg/kg per mouse for each injection) on days 9, 12, and 15, and then measured their body weights and tumor sizes. As shown in Figure 3B, the results indicate that the CMSN-GOx plus PD-1 treatment more


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significantly inhibited tumor development than the GOx, PD-1, or CMSN-GOx treatment alone. To further test the combined treatment effect, we conducted a long-term close observation of the mice after various treatments, and found that their survival time after the CMSN-GOx plus PD-1 treatment was significantly extended (Figure 3C). We measured the mice’ body weight, as we expected, experimental and control mice were not shown obvious difference in body weight, and there were not shown abnormality in the mice during the entire treatment period. Therefore, we determined that we used materials had not obvious toxicity to the mice and had potential in clinical practice (Figure S5, Supporting Information). In addition, we performed a histological assay to characterize the cancer cell proliferation. As shown in Figures 3D-3E, the results of hematoxylin and eosin (H&E) stain validated the tumor tissue was disappeared though treatment with the CMSN-GOx plus PD-1 and the tissue cells had shrunk and the nuclei had disappeared.38 The Ki-67 staining results revealed that the CMSN-GOx plus PD-1 treatment more significantly inhibited the proliferation of tumor cells than the other treatments. All these data confirm that the combination therapy demonstrated an excellent capability of inhibiting tumor growth.


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Figure 3. (A) Protocol of CMSN+GOx and anti-PD-1 combination therapy. (B) Tumor volume in the mice after different treatments. (n= 5 in per group). (C) Survival curves of the mice after different treatments. (n= 5 in per group). ** indicate P < 0.01, the PBS group as contrast. (D-E) H&E and Ki-67 stained tumor slice images of groups through different treated.

To further investigate the efficacy of our nanoparticles in cancer therapy, we used the

positron

emission

tomography

scanning

technique

to

detect

[18F]

2-fluoro-2deoxy-d-glucose (18F-FDG) levels in a mouse model.39 Unlike other imaging techniques, PET imaging can provide glucose metabolism data, and hence has been widely used in disease diagnosis and therapy.40, 41 We analyzed the glucose metabolism in typical coronal, transverse, and sagittal sites (Figure 4). The PET imaging results reveals that the tumor tissue had minimum glucose metabolism in vivo


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after treatment with CMSN-GOx+PD-1. These results indicate that the combined CMSN-GOx and PD-1 therapy can achieve an optimal induced tumor ablation effect. By looking at the values of Mean SUV, we found that the combination therapy can achieve the strongest anti-tumor effect.

Figure 4. PET images and Mean SUV of B16-F10 tumor-bearing mice taken with different treatments.

Next, we used flow cytometry to investigate the state of dendritic cells (DCs) in vivo after treatment with CMSN-GOx+PD-1. DCs is the essential types of antigen-presenting cells (APC), which play a significant part in initiating, regulating and adaptive immunities.42 Once the tumor antigens are exposed, immature DCs will capture and digest them into peptides as they migrate to the lymph nodes.43 These immature

DCs

will

then

mature

and

present

major

histocompatibility

complex-peptides to T-cell receptors upon their arrival at lymph nodes. Therefore, inducing more DCs maturation is of great significance for enhanced cancer immunotherapy effect. Cancer cell apoptosis can induce an immune response in vivo, so we used the Tunel staining protocol to characterize the tumor issue after different


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treatments. Our results showed that the CMSN-GOx plus PD-1 treatment group to have the most powerful Tunel fluorescence signal of all the treatments (Figure 5A). Because the co-stimulatory molecules CD80 and CD86 are widely used as biomarkers for DC maturation, we analyzed the CD80 and CD86 percentage in vivo to assess the level of DCs maturation. In this experiment, we collected drained inguinal lymph nodes and stained them with CD11c, then used flow cytometry to investigate their immunoregulation effect on DCs after the CMSN-GOx treatment to calculate the upregulation rate of CD80 and CD86. The percentage of CD80 and CD86 increases from 21.48% to 54.70% after the PBS and CMSN-GOx+PD-1 treatments (Figure 5B). The DCs maturation results indicated that the combined CMSN-GOx plus PD-1 therapy generated strong immune responses, which shows its potential for improving immunotherapy outcomes.

Figure 5. (A) Tumor slice images of different treatment groups were stained by Tunel. Scale bars represent 50 μm


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in Tunel stained slice images. (B) DCs maturation induced after different treatments of mice-bearing B16-F10 tumors. Cells in the tumor-draining lymph nodes were obtained and co-stained with CD80 and CD86 by flow cytometry after 72-hour treatments. Five mice were used in each group in 5B. ** indicate P < 0.01, PBS group as a contrast.

To better understand the checkpoint blockade process of our combined CMSN-GOx plus PD-1 immunotherapy, we further analyzed the tumor-infiltrating lymphocytes profiles of each tumor with flow cytometry. CD8+ was known as cytotoxic T cells and CD4+ was helper T cells in adaptive immunity, the increase of CD8+ and CD4+ content can promote the cancer immunotherapy.16 We observed that the CD8+ CTL infiltration values and the percentage of effective T cells in those treated with CMSN-GOx+PD-1 were higher than in the other groups (Figure 6A-B). In contrast, regulatory T cells (Tregs) (CD4+Foxp3+) were known to suppress anti-tumor immune responses. We collected T cells from tumor tissues and analyzed them using flow cytometry after they had been co-stained with CD3, CD4, and Foxp3. Although the mice after CMSN-GOx treatment experienced vaccine-like immune responses, the anti-tumor effect was not satisfactory due to the large number of Tregs in the tumor issues. As shown in Figure 6C-D, the CMSN-GOx+PD-1 combination therapy significantly decreased the proportion of Tregs (CD4+Foxp3+) in vivo. Furthermore, the CMSN-GOx+PD-1 treatment significantly improved the CD8+ Teff/Treg and CD4+ Teff/Treg ratios (Figure 6E). In addition, by comparing the PD-1 and CMSN-GOx+PD-1 treatment (Figure 6), we found that the combination therapy can increase the effective T cell ratio (CD4+ T cells, CD8+ T cells, CD8+ Teff/Treg ratio, and CD4+ Teff/Tregs ratio) and lower the Tregs rate. This may be because by


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these nanoparticles possess immune evasion and cancer targeting capabilities. Furthermore, we know that cytokine secretion plays an important part at the immune response. Therefore, in the cellular immunity experiment, we obtained from B16-F10 tumor mice which had been treated differently to determine the immunofluorescence intensities of TNF-α. As shown in the Figure 6F, mice treated with CMSN-GOx+PD-1 showed the strongest fluorescence, which indicates that the combination therapy can cause a strong anti-tumor immune response.

Figure 6. Checkpoint blockade immunotherapeutic effect of our nanoparticles. (A) Flow cytometry of various


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treatments of T cells in tumors. B16-F10 was analyzed for T-cell infiltration. (B) Portions of tumor-infiltrating CD4+ and CD8+ T cells corresponding to the data in A. (C) Flow cytometry plots of Tregs by different treatments. (D) Proportions of tumor-infiltrating CD4+ FoxP3+ regulatory T cells corresponding to the data in C. (E) CD8+ CTL: Treg ratios and CD4+ effecter T cells: Treg ratios in the tumors with different treatments. (F) The immunofluorescence of TNF-α in mouse after various treatments. Scale bars represent 50 μm in TNF-α stained slice images. (n = 5), (NS) no statistical difference, (*) P < 0.05, and (**) P < 0.01.

We performed hematology tests, blood biochemistry, and histology analysis to verify the biocompatibility of CMSN-GOx+PD-1 in vivo. As shown in FigureS3-S4, comparing to control groups, we found no obvious differences in the treatment groups (i.e., alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), and creatinine (CRE)). Furthermore, H&E-staining verified no major organ damage (Figure S6-S7, Supporting Information). The above results indicate that CMSN-GOx+PD-1 have high biocompatibility and great potential for clinical applications. CONCLUSIONS In a word, we have developed a therapeutic method that combines CMSN-GOx starvation therapy and anti-PD-1 immunotherapy to enhance anti-tumor efficacy. By functionalizing the nanoparticles’ surfaces, CMSN-GOx can more readily escape immune clearance and target tumor tissue. This homotypic targeting approach can significantly enhance the capacity to localize CMSN-GOx in tumor sites. Our CMSN-GOx cuts off the glucose source to inhibit tumor growth and induce immune responses. We also demonstrated that a combined starvation and immunotherapy treatment can provide more effective cancer ablation and better stimulation of adaptive immune responses than single therapies. Moreover, we introduced the use of


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PET imaging, which enables the clear and upright imaging of tumor tissues, to characterize glucose metabolism. Our combined CMSN-GOx starvation therapy and anti-PD-1 immunotherapy shows great potential for clinical applications.

EXPERIMENTAL SECTION Preparation of CC-Membranes: To prepare cancer cell membranes, we used a culture dishes with 10 cm in diameter to culture B16-F10 cells at 37 ℃ , 5% CO2, then used a cell scraper to detach the cells. Next, isolating the cells through a centrifugation treated at 720 g for 6 min. Resuspending the collected cells into pre-cooled PBS buffer (pH=7.4), and centrifuging again at 600 g for 6 min. The collected cells pellets were resuspended in a hypotonic lysing buffer, including membrane protein extraction reagent and phenylmethanesulfonyl fluoride (PMSF) (Beyotine Institute of Biotechnology). Then, the sample above was incubated in ice bath for 10-15 min. Next, we used a freeze-thaw method repeatedly to break the cells in the above solution, and then make a centrifugation at 700 g, 10 min, 4 oC. To collect the cell membrane fragments, the supernatant was subjected to further centrifugation at 14,000 g for 30 min. Subsequently, we used an Avanti mini extruder to extrude 11 times. Finally, centrifuge to remove the redundant CC-vesicles. Preparation and Characterization of CC-MSN: To make the MSN were coated into the cancer cell membranes. The mixture of 50 μg MSN and prepared CC-vesicles were mixed and re-suspend into 1 mL PBS. Next, we used an Avanti mini extruder to extrude 11 times, and centrifuge to remove the redundant CC-vesicles. At the last, the


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preparation CC-MSN at 4 ℃ in 1 × PBS overnight for further use.

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We used DLS to

evaluate the performance of the nanoparticle. 50 μg MSN or CC-MSN were suspended in 1 mL 1 × PBS. Finally, we used TEM (JEM-2010HT, Japan) to characterize the morphology of CC-MSN at room temperature. The way of preparing the TEM samples successful is contacted the suspension of CC-MSN with copper grids for 60 s, which were negatively stained 30 s by using phosphotungstic acid and dried in environmental conditions before characterization. Stability Study of CC-MSN: We used Cy5 to label MSN through mixed MSN with DSPE-PEG-Cy5. We used FITC to label the CC-vesicles. The nanoparticles (100 μg) and B16-F10 cells were co-cultured for 4 h, 37 °C. We used PBS wash the cells for 3 times at 25 °C, and used PFA to fix the cells for 30 min. Finally, we used DAPI to stain the cells, and then CLSM (IX81, Olympus, Japan) was used for imaging. FITC, DAPI, and Cy5 channels obtained images of green, blue, and red fluorescence, respectively. In Vitro Immune Escape study: The cells of B16-F10 were seeded in 12-well plates as same as RAW 264.7 cells, and then cultured cells in 12 h, 37 °C. As a control, erythrocyte (RBC) membranes coated MSN (RBC-MSN) was obtained according to our previous report.31 We added the MSN, RBC-MSN, and CC-MSN (100 μg Si mL−1) to medium. At the same time, cell growth without any particles being as a contrast. We used the PBS to wash the cells three times. The cells were cultured in an incubator for 4 h. Finally, we washed the cells three times with PBS. To assess Si content, the cells were lysed by adding 0.5 mL 1% Tween 80 to each well. Heat the


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mixture in 1:1:1 H2O/HF/HNO3 to dissolve the cells. Mixture was stilling for overnight, and then heating for 80 °C, 6 h. And then, we used 1 mL DI water to re-suspend the sample, and then used ICP-MS to determine the Si content of each sample. Cell Viability Assay: To investigate the cytotoxicity of nanoparticles to B16-F10, we arranged the CCK-8 assay. Seeded the cells and cultured for 12 h. In the case of without glucose or glucose, we added the MSN-GOx and CMSN-GOx (50 μg mL−1) to the cell culture medium. At the same time, didn't add any particles to the cells as a contrast. Washing three times with PBS, followed by incubated, and used PBS washed again. Next, we added CCK-8 solution into the plate at the end of the incubation, which keeping incubate for another 4 h. Finally, we used a microplate reader (Emax Precision, USA) to determine the cell viability. To observe the ability of CMSN-GOx to kill cancer cells through the starvation therapy effect. We used Calcein-AM and PI to co-stain B16-F10 cells with the CMSN-GOx treatment, and live cell emission green fluorescence and the dead cells emission red fluorescence. SDS-PAGE Protein Analysis: The MSN and CC-MSN were prepared in SDS sample buffer. Heating the samples to 95 °C and staying for 5 min. And then each well that contained 10% SDS polyacrylamide gel loaded 20 μl of the sample. Finally, we used Commassie blue to stain the resulting polyacrylamide gel, and washed overnight. Catalytic activity measurements of GOx: Firstly, we measured the change of GOx induced pH at the absence or presence of glucose. Next, preparing the solution


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samples, which is mixed MSN-GOx, CMSN-GOx (50 μg/mL) with glucose (1 mg/mL) together, another is dispersed MSN-GOx, CMSN-GOx (50 μg/mL) in distilled water. We used pH meter to measure the pH values of the solutions in time. Measurement of H2O2 concentration: B16-F10 cells were seeded into a 96-well plate and then cultured at 37 °C for 24 h. Then MSN, CMSN, CMSN-GOx were added into each well. After co-incubation 6 h, the cells were harvested and use H2O2 Kit (TIANDZ, China). And then test the absorbance at A560 using a UV-Vis spectrophotometer. Finally, the concentration of H2O2 was determined according to the drawn standard curve. In Vivo Biodistribution: All the mice were euthanized, and then collected their major organs after injected 48 h to determine the biological distribution of the particles. As mentioned above, the Si content was measured using ICP-MS. Samples with high Si content (e.g., livers, spleens and lungs) were diluted to insure measurement accuracy. The Si content in the major organs was expressed in the unit of the percentage of tissue injected dose (% ID/g). In Vivo PET image. In vivo PET imaging was performed using PET system (Discovery VCT 64, USA), we injected the materials into the subcutaneous in mice directly, and we used PET imaging with an 18F-labelled glucose analogue, 18F-FDG, to quantify the therapy effect of the materials. This provided a quantitative measure to determine how much is reduced of the tumor metabolism in treated areas after an hour after treatment. In Vivo therapy Evaluation: Female C57B6 mice were purchased from Hubei


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provincial center for disease control and prevention. Before the mice were randomly divided into 5 groups, which including PBS control, anti-PD-1, MSN-GOx, CMSN-GOx, and CMSN-GOx+anti-PD-1, B16-F10 tumor cells (5× 105 cells in 100μl volume) were transplanted into the back of the C57B6 mice. Since day 8, the MSN-GOx and CMSN-GOx were intravenously infused via tail vein every three days for 4 times. Since day 9, the anti-PD-1 antibody was intraperitoneally injected into mice every three days for 3 times. The mice were weighed every three days since day 6. The tumor volume was calculated as (long diameter x short diameter2)/2. At day 19, the experiment was terminated, and then the mice were euthanized. Finally, we collected the tumors and used hematoxylin-eosin, Ki-67, TUNEL, and TNF-α, and then analyzed with flow cytometry. Flow Cytometry Analysis: The tumors were harvested and digested using collagenase/hyaluronidase and DNase to analyze the immune cells in the tumor. The resulting cells were lysed with Red Blood Cell Lysis Buffer (Beyotime Institute of Biotechnology) and filtered through nylon mesh filters (70 μm). The single cell suspensions were re-suspended in PBS with 2% FBS. To analyze the T cells, the prepared cells were label with CD3 (17A2, BD), CD4 (GK1.5, Invitrogen) and CD8 (53-6.7, BD). We used CD4 (GK1.5, Invitrogen) and Foxp3 (FJK-16s, Invitrogen) to stain the single cells for analysis of regulatory T cell. The resulting cells were incubated with CD11c (N418, Invitrogen), CD80 (16-10A1, Invitrogen) and CD86 (GL1, Invitrogen) to evaluate the dendritic cells. Gating strategy for flow cytometry (Figure S8, Supporting Information).


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In Vivo Toxicity Evaluation: After the treatment, all mice were euthanized. We collected their blood samples and major organs (i.e., hearts, kidneys, spleens, livers, and lungs). Two indicators for kidney functions (i.e., CRE and BUN) were measured by using a blood biochemical auto-analyzer (7080, HITACHI, Japan), and three important hepatic indicators (i.e., AST, ALT, and ALP) were measured as described above. The complete blood panel data were tested, and the data we obtained from control mice and treated mice. Finally, we used H&E to stained the part of mice' organs. ASSOCIATED CONTENT Supporting Information Supplementary experimental section and Figures S1−S8 are included in the Supporting Information. This material is available free of charge via the internet at http://pubs.acs.org. Supplementary experimental section: Materials and Reagents. Figure S1: The change of H2O2 level. Figure S2: Representative CLSM images of B16-F10 cells after different treatments. Figure S3: RAW 264.7 macrophage-like cells and B16-F10 cancer cells with various incubation time periods. Figure S4: CLSM images of B16-F10 cells and 4T1 after incubation with CMSN over time. Figure S5: Body weight changes in 18 days after various treatments. Figure S6: Representative H&E stained slices of major organs.


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Figure S7: Blood biochemistry data including liver function markers and kidney function markers. Figure S8: Gating strategy for flow cytometry.

AUTHOR INFORMATION ORCID Wei Xie :0000-0001-7003-6349 ACKNOWLEDGMENTS The authors gratefully acknowledge support for this research from National Natural Science Foundation of China (Grant No. 61474084, Grant No.81672668), National Natural Science Foundation for Outstanding Youth Foundation (Grant No. 61722405), National Research and Development Program for Major Research Instruments (Grant No.81527801), Fundamental Research Funds for the Central Universities of China 2042017kf0171 (Outstanding Young Scholars), and National Key Research and Development Program (Grant No.2016YFC1000700). REFERENCES 1.

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TOC graphic

In this paper, we present a therapeutic method that combines immunotherapy and starvation therapy to achieve better anti-tumor efficacy. Our synthetic CMSN-GOx complex can ablate tumors and induce dendritic cell maturity to stimulate an anti-tumor immune response. We performed an in vivo analysis of these nanoparticles and determined that our combined therapy CMSN-GOx plus PD-1, exhibits a better anti-tumor therapeutic effect than therapies using CMSN-GOx or PD-1 alone. Additionally, we introduce the use of positron emission tomography imaging to evaluate the efficacy of the cancer therapy in vivo by measuring the level of glucose metabolism in tumor tissues.


Cancer Cell Membrane Camouflaged Nanoparticles to Realize

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