GSH-responsive Nanoplatform for Intra/Extracellular Lactate Exhaustion to Enhance Antitumor Immunotherapy

GSH-responsive Nanoplatform for Intra/Extracellular Lactate Exhaustion to Enhance Antitumor Immunotherapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article GSH-responsive Nanoplatform for Intra/Extracellular Lactate Exhaustion to Enhance Antitumor Immunotherapy Yandi Tan, Ju Huang, Liang Zhang, Xinyi Tang, Chunmei Zhang, Hongwei Xiang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2168046/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Immune checkpoint blockade (ICB) therapies have reshaped tumor treatment by activating the antitumor immune response. However, the antitumor effect of ICB is seriously restricted by the immunosuppressive tumor microenvironment (ITM). A variety of strategies to alleviate the ITM have been investigated. Direct regulation of lactate metabolism in tumor microenvironment holds promise for ITM modulation. Results Glutathione (GSH) -responsive hollow mesoporous organosilicon (HMOP) was successfully fabricated, with monocarboxylate transporter 1/4 inhibitor (diclofenac, DC) and lactate oxidase (LOD) were loaded in/onto the HMOP (designed as DC-HMOP-LOD). DC-HMOP-LOD could spontaneously be biodegraded in tumor microenvironment due to disulfide bonds, and then DC/LOD could be released to exhaust intra/extracellular lactate. Consequently, lactate depletion induced an immunocompetent tumor microenvironment by activating immune-promoting cells including dendritic cells, CD4 + T cells, CD8 + T cells, and natural killer cells, and inactivating immunosuppressive cells containing tumor-associated macrophages and myeloid-derived suppressor cells, ultimately strengthening the antitumor effect of ICB therapy. Conclusion DC-HMOP-LOD effectively hindered the transmission of lactate and directly oxidized lactate, collaboratively depleting lactate in the TME. The synergetic depletion reversed the ITM and could improve the antitumor effects of aPD1-based immunotherapy. GSH-responsive hollow mesoporous organosilicon Lactate Immunosuppressive tumor microenvironment Immunotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Immune checkpoint blockade (ICB) therapies have achieved unprecedented clinical benefits. Unfortunately, only few patients could respond to the ICB therapy because of the restriction of immunosuppressive tumor microenvironment (ITM) [ 1 ]. Numerous findings point out that ITM is featured with potent immune suppressive activity including affecting the recruitment, proliferation, and functions of immune cells [ 2 , 3 ]. Therefore, reconstructing the ITM could be a promising approach for improving ICB therapy [ 4 – 6 ]. Recent nanotechnology in biomedicine has reversed ITM by advancing various modalities, such as chemotherapy [ 7 ], sonodynamic therapy [ 8 ], gene therapy [ 9 ] etc . Although these nanosystems have improved antitumor effect of ICB therapy, which are usually resulted from immunogenic death effect and the activated innate immunity, the therapeutic efficacy still is unsatisfactory due to the relatively high chemoresistance of cancer cells enabled by their reprogrammed metabolism [ 10 ]. This situation prompts us to reconsider new therapeutic modality that focuses on the basic laws of biological processes in tumor cells, and solve the most essential issues by rational metabolic regulation. Accumulating data have provided evidence supporting the functional role of lactate in constructing the ITM [ 11 – 13 ]. Concretely, the excessive accumulation of lactate (10–30 mM) in tumor microenvironment (TME) is closely related to the enhanced glycolysis in tumor cells [ 14 ]. In order to balance the rapid biosynthetic processes and a huge demand of ATP production, tumor cells are prone to adopt aerobic glycolysis to support cell proliferation. Lactate accumulation in TME influences various tumor processes, such as tissue invasion and metastasis, angiogenesis, and drug resistance [ 15 ]. Furthermore, lactate acts as a critical regulator of the ITM by influencing the differentiation and proliferation of immune cell [ 16 ]. Concretely, lactate impairs the infiltration of immune effector cells such as natural killer (NK) cells and cytotoxic T cells. Additionally, lactate can suppress the differentiation and impair the functions of dendritic cells. Meanwhile, lactate can promote the activity of immunosuppressive cells, including myeloid-derived suppressor cells (MDSCs) and M2-type tumor-associated macrophages (TAMs). It has also been shown that lactate-induced ITM is reversible by altering the lactate concentration in the TME [ 17 , 18 ]. Nowadays, various mesoporous silica nanoparticles and porous hollow silica nanoparticles have been studied as drug delivery systems due to good biocompatibility. However, some limitations restrict the potential for clinical transformation such as uncontrolled biodegradability of the silica framework, unclear retention times in vivo , and pathway. In this study, we synthesized PEGylated hollow mesoporous organosilicon (HMOP) nanocomposites. HMOP can achieve a fast bio-degradation by intracellular glutathione (GSH) stimulation because of disulfide bonds in the silica framework, enhancing its biosafety and facilitating its excretion [ 19 ]. And HMOP is a well-defined shell/core structure with desirable physical condition, pharmacokinetics, affinity, and biosafety [ 20 ]. Herein, Lactate oxidase (LOD) and diclofenac (DC) were spatially conjunct to the shell and loaded in the core of HMOP, respectively. HMOP can selectively accumulate and release LOD and DC locally by the enhanced permeability and retention (EPR) effect without the need for external energy input [ 21 ]. The released LOD and DC synergistically deplete intracellular and extracellular lactic acid. LOD is an enzyme that can directly consume a high level of intra/extracellular lactate by catalyzing the oxidation of lactate to H 2 O 2 and pyruvate [ 22 ]. To achieve a synergistic reduction in lactate in the TME, we simultaneously used DC, a classic nonsteroidal anti-inflammatory drug, to inhibit lactate transport from tumor cells into the TME by blocking lactate transporters (monocarboxylate transporters 1 (MCT1) and MCT4) on the cancer cell membrane. Lactate catalysis and lactate transport inhibition could achieve synergistic lactate reduction in the TME. The lactate exhaustion strategy could efficiently reverse the ITM by activating immune-promoting cells, inactivating immunosuppressive cells, and regulating immune cytokines. The immunocompetent TME ultimately improved the antitumor efficacy of anti-PD1 (aPD1) immunotherapy (Scheme 1 ). Materials And Methods Materials Tetraethylorthosilicate (TEOS), hexadecyltrimethylammonium chloride (CTAC), triethanolamine (TEA), bis[3-(triethoxysilyl) propyl] tetrasulfide (BTES), sodium carbonate (Na 2 CO 3 ), methanol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 3-aminopropyl triethoxysilane (APTES), ammonia solution (25-28%), and diclofenac (DC) were purchased from Macklin (China). Polyethylene glycol (PEG)-silane was purchased from Xi'an Ruixi Biological Technology Co., Ltd. (China). Lactate oxidase (LOD) was purchased from Shanghai Yingxin Laboratory Equipment Co., Ltd. (China). 1,1-Dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) and fluorescein isothiocyanate isomer (FITC) were purchased from Beyotime Biotechnology (China). Anti-PD-1 was purchased from Bio X Cell (USA). Cell counting kit-8 (CCK-8) was purchased from Dojindo (Japan). Enzyme linked immunosorbent assay (ELISA) kits were obtained from Meimian Industrial Co., Ltd. (Jiangsu, China). The Bradford Protein Assay Kit, Lactic Acid Assay Kit, and H 2 O 2 Content Assay Kit were purchased from Solarbio (Beijing, China). FITC-conjugated anti-mouse CD11c, phycoerythrin (PE)-conjugated anti-mouse CD86, allophycocyanin (APC)-conjugated anti-mouse CD80, cyanine (Cy)5.5-conjugated anti-mouse CD45, APC-conjugated anti-mouse CD49b, FITC-conjugated anti-mouse CD4, APC-conjugated CD8, APC-conjugated anti-mouse CD206, FITC-conjugated anti-mouse F4/80, Cy5.5-conjugated anti-mouse Foxp3, Cy5.5-conjugated anti-mouse CD11b, and FITC-conjugated anti-mouse Gr-1 were purchased from Thermo Fisher Scientific (USA). Cell culture and animals Male BALB/c mice (6–8 weeks old) were obtained from the Experimental Animal Center of Chongqing Medical University (CQMU, SYXK(YU)2018-0003). Animal experiments were approved and performed in accordance with the guidelines of the Institutional Animal Care and Animal Ethics Committee of CQMU. The mouse colon adenocarcinoma cell line CT26 was provided by the Cell Bank of Shanghai Institute (Chines Academy of Science). The cells were cultured in RPMI-1640 medium (M&C, Beijing, China) with 10% fetal bovine serum (FBS, Gibco, Australia), 1 % penicillin/streptomycin (37 °C and 5 % CO 2 ). Synthesis of HMOM The synthesis of HMON used a selective etching strategy. First, ethanol (74 mL), deionized water (ddH 2 O) (10 mL), and ammonia solution (3.14 mL) were stirred at room temperature for 5 minutes, and then TEOS (6 mL) was quickly added. The mixture was stirred for 1 h at room temperature, washed with ethanol 2 times, and then distributed into ddH 2 O (60 mL) to form monodispersed SiO 2 nanocomposites. Second, CTAC (2 g) and TEA (0.04 g) were added to ddH 2 O (90 mL), and the solution was stirred for 1.5 h at room temperature. Then, the above SiO 2 nanocomposites (30 mL) were added and stirred for another 1.5 h at room temperature. BTES (1.8 mL) was dropped into the mixed solution and stirred for 1 h at 80 °C. The SiO 2 @MON was washed with ethanol 2 times. Third, SiO 2 @MON was added to Na 2 CO 3 aqueous solution (0.6 M, 100 mL) and incubated at 80 °C for another 1 h to form the HMON products. Fourth, the HMON products were suspended with NaCl in methanol (1 wt %, 30 mL) for 3 h at room temperature to remove the unreacted CTAC. Fifth, HMON (50 mg) and PEG-silane (30 mg) were stirred in ethanol (50 mL) at 80 °C for 12 h to form the HMOP. Finally, the HMOP products were obtained after washing with ddH 2 O 2 times and dispersing in ddH 2 O (15 mL). Synthesis of DC-HMOP-LOD HMOP (30 mg) and DC (60 mg) were dissolved in 10 mL ethanol, stirred at room temperature for 24 h, washed with ddH 2 O 2 times and dispersed in ddH 2 O (6 mL) to collect the HMOP-DC. LOD (150 U) was amino-functionalized by stirring in a mixture of EDC (38 mg), NHS (57 mg), APTES (45 µL), and ddH 2 O (6 mL) for 8 h at room temperature. Then, HMOP-DC (20 mg) in ddH 2 O (4 mL) was added and further stirred for 24 h at room temperature. The final DC-HMOP-LOD was collected by centrifugation (10,000 rpm, 5 min), washed with ddH 2 O 2 times, and finally dispersed in ddH 2 O (5 mL). Synthesis of DC-HMOP-LOD-FITC or DC-HMOP-LOD-Cy5.5 To obtain DC-HMOP-LOD-FITC, FITC (1 mg) was amino-functionalized by stirring for 8 h at room temperature in a mixture of EDC (6.6 mg), NHS (9.6 mg), APTES (45 µL), and ddH 2 O (8 mL). Then, DC-HMOP-LOD (10 mg) in ddH 2 O (2 mL) was added and further stirred for 24 h at room temperature. The final DC-HMOP-LOD-FITC was collected by centrifugation (10,000 rpm, 5 min), washed with ddH 2 O 2 times, and finally dispersed in ddH 2 O (2 mL). To fabricate DC-HMOP-LOD-Cy5.5, the pH of the HMOP was modulated at 8.5. Then, carboxylic-functionalized Cy5.5 (20 μg) and DC-HMOP-LOD (5 mg) were mixed for 24 h at 4 °C. Characterization of DC-HMOP-LOD The morphology of DC-HMOP-LOD was visualized using scanning electron microscope (SEM) (Hitachi S-3400 N, Japan) and transmission electron microscope (TEM) (Hitachi-7500, Japan). The particle diameter was measured using a Malvern Zetasizer instrument (Nano ZS90, UK). Area-elemental mapping and EDS spectra confirmed the existence of the major elements (Si, O, S) in DC-HMOP-LOD on the FEI-Talos F200S electron microscope. The encapsulation efficiency (EE) and loading capacity (LC) of DC was calculated using a standard curve by plotting the absorbance against concentration using a UV–Vis-NIR spectrophotometer (Shimadzu, Kyoto, Japan). The EE and LC of LOD was confirmed by a Bradford Protein Assay Kit. The EE of DC or LOD in DC-HMOP-LOD was calculated according to the following formula: EE (%) = (weight of loaded DC or LOD / weight of total DC or LOD) x 100. The LC of DC or LOD in DC-HMOP-LOD was calculated according to the following formula: LC (%) = (weight of loaded DC or LOD / weight of total nanocomposites) x 100. GSH -dependent responsive degradation of DC-HMOP-LOD was measured as follows: DC-HMOP-LOD was dispersed into GSH (10 mM) and then stirred gently for 3, 7, and 14 days at 37 °C. The morphology of these samples was detected by TEM and SEM. In vivo biodistribution and pharmacokinetics assays The standard curve of DC-HMOP-LOD-FITC was obtained using a UV–Vis-NIR spectrophotometer (excitation and emission wavelengths of Ex 488 nm/Em 525 nm). Male BALB/c mice were subcutaneously injected with CT26 cells (1 ×10 6 , 50 μL in phosphate buffered saline (PBS) suspension). When the tumor volume was approximately 100 mm 3 , mice were intravenously injected with DC-HMOP-LOD-FITC (10 mg/mL, 150 μL). At predetermined time points (0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h), 10 μL of blood was collected by gently nicking the tail vein. At 24 h, tumor tissues and the main organs (heart, liver, spleen, lung, and kidney) were collected. The blood, tumor tissues, and main organs were digested with lysis solution (400 μL), and then the blood was sonicated for 10 min. Tumor tissues and main organs were ground into single-cell samples. Finally, the supernatants of blood, tumor tissues, and main organs were collected by centrifugation (8,000 rpm, 5 min). Fluorescence quantification of the supernatants was measured by a UV–Vis-NIR spectrophotometer. LOD catalysis assay in vitro The catalytic activity of LOD conjugated onto the surface of DC-HMOP-LOD was studied, including the consumption of lactate and O 2 and the production of H 2 O 2 . The lactate-PBS solution (500 μL, 3 mM) was mixed with HMOP-DC, HMOP-LOD, or DC-HMOP-LOD (500 μL, 5 mg/mL) with slight shaking at 37 °C. The solution (10 μL) was collected at different time points (0, 5, 10, 20, 40, 80, and 160 min) for the quantification of lactate by the Lactic Acid Assay Kit (KeyGEN Biotech Corp., Ltd, China, Jiangsu). The oxygen content in the mixed solution was investigated at different time points (1, 2, 3, 4, and 5 min) using a dissolved oxygen meter in real time (JPBJ-608, Shanghai INESA Scientific Instrument Co., Ltd, China). Furthermore, the different concentrations (500 μL; 0, 1, 2, 3, 4 mM) of the lactate-PBS solution were mixed with HMOP-DC, HMOP-LOD, or DC-HMOP-LOD (500 μL, 5 mg/mL) with slight shaking at 37 °C for 2 h. The solution was collected for the quantification of H 2 O 2 using an H 2 O 2 Content Assay Kit (Solarbio, Beijing). Measurement of H 2 O 2 , lactate concentration, and pH in vitro CT26 cells were incubated in T75 flasks (3×10 6 per well) for 24 h at 37 °C, and then the culture medium was replaced with fresh media containing HMOP-DC, HMOP-LOD, or DC-HMOP-LOD (200 or 400 μg/mL) for 24 h. To measure the extracellular lactate level, H 2 O 2 level, and pH values, the cell supernatant was tested with a Lactic Acid Assay Kit (KeyGEN Biotech Corp., Ltd., China, Jiangsu), H 2 O 2 Content Assay Kit, and a pH meter (Shanghai INESA Scientific Instrument Co., Ltd, China). The cells were collected to measure the intracellular lactate level using a Lactic Acid Assay Kit (Solarbio, Beijing). Measurement of intratumoral lactate concentration in vivo CT26 xenotransplanted tumor mice with a tumor volume of approximately 100 mm 3 were divided into the following 4 groups: control, HMOP-DC, HMOP-LOD, and DC-HMOP-LOD. 24 h after intravenous injection of 150 μL PBS or different nanocomposites (10 mg/mL), 100 mg of tumor tissues was collected from sacrificed mice and digested to obtain single-cell samples. After the centrifugation of tissue homogenates, the supernatants were collected to measure the extracellular lactate level, and the sediments were used to investigate the intracellular lactate level with a Lactic Acid Content Assay Kit (Solarbio, Beijing). Intracellular uptake of HMOP Intracellular endocytosis was detected by confocal laser scanning microscopy (Nikon, Japan) and flow cytometry (BD FACS Vantage SE). CT26 cells were cultured in CLSM dishes (1 × 10 5 cells per well) for 24 h, and then the medium was replaced with 200 μg/mL DC-HMOP-LOD-FITC dispersed in serum-free medium. After different incubation times (2, 4, 6, 8, and 24 h), the cells were rinsed with PBS, fixed with 4 % polyformaldehyde for 15 min, and then stained with DAPI for 15 min before confocal laser scanning microscopy (CLSM) observation. For the flow cytometry analysis, cells were seeded in 6-well plates (1 × 10 5 cells per well) for 24 h. After incubation with DC-HMOP-LOD-Cy5.5 for 2, 4, 6, 8, and 24 h, the cells were digested with trypsin, suspended in PBS, and analyzed by flow cytometry. In vitro therapeutic efficacy and safety of nanocomposites To examine the treatment efficacy of DC-HMOP-LOD in vitro , CT26 cells cultured in 96-well plates (5 × 10 3 cells per well) were divided into control, HMOP-DC, HMOP-LOD, and DC-HMOP-LOD groups. Then, the medium in the plates was replaced with fresh medium containing the corresponding nanocomposites (100, 200, 300, and 400 μg/mL). 24 h later, a CCK-8 kit was used to assess CT26 cell viability. Similarly, the cells were cultured in 6-well plates (1 × 10 5 cells per well) and then subjected to different nanocomposites (200 μg/mL) for 24h. After digestion and centrifugation, flow cytometry was conducted to evaluate apoptosis. For CLSM observation, the cells were seeded in CLSM dishes (1 × 10 5 cells per well) and subjected to different nanocomposites (200 μg/mL) for 24 h. Then, the cells were dyed with CAM (10 μL) and PI (10 μL) and finally visually observed by CLSM. In addition, the cytotoxicity of HMOP (7.5−500 μg/mL) to CT26 cells was measured using the above cell counting kit-8 steps. Immune system activation First, we evaluated the phenotypic polarization of RAW 264.7 Cells. CT26 cells were incubated in the upper chamber (1 × 10 5 per well), and RAW 264.7 cells were incubated in the lower chamber (1 × 10 5 per well) for 24 h. Then, the original medium of CT26 cells was removed, and fresh medium with different nanocomposites (200 μg/mL) was added. The medium was replaced with fresh medium containing interleukin-4 (IL)-4 (50 ng/ml). After 24 h of incubation, the RAW 264.7 cells were stained with PE-anti-CD86 and APC-anti-CD206 and then detected by flow cytometry. For the evaluation of the immune response in vivo , CT26 xenotransplanted tumor mice with a tumor volume of approximately 70 mm 3 were randomly divided into 4 groups: Control, HMOP-DC, HMOP-LOD, and DC-HMOP-LOD. Then, 150 μL PBS or different nanocomposites (10 mg/mL) was intravenously administered on days 1, 3, 6, and 9. On the 10th day, the tumor tissues were digested to obtain single-cell samples. Then, the cell samples were stained with different antibodies (FITC-anti-CD11c, PE-anti-CD86, APC-anti-CD80, Cy5.5-anti-CD45, APC-anti-CD49b, FITC-anti-CD4, APC-anti-CD8, APC-anti-CD206, FITC-anti- F4/80, Cy5.5-anti-Foxp3, Cy5.5-anti-CD11b, FITC-anti-Gr-1) and measured by flow cytometry. In addition, blood was extracted from each mouse, and plasma samples were obtained by centrifugation of the blood (8,000 rpm, 20 min). Then, the plasma samples were used to test cytokines (INF-γ, TNF-α, IL-10, and IL-12) by corresponding ELISA kits (Huyu Biotechnology, Shanghai China). In vivo synergistic therapeutic efficacy of DC-HMOP-LOD CT26 xenotransplanted tumor mice were randomly divided into six groups: Control, HMOP-DC, HMOP-LOD, DC-HMOP-LOD, aPD1, and aPD1 + DC-HMOP-LOD. The mice were intravenously injected with PBS or different nanocomposites (10 mg/mL, 150 μL) on days 1, 3, 6, and 9. Mice in the aPD1 and aPD1 + DC-HMOP-LOD groups were injected with aPD1 (25 μg/mouse) on days 2, 4, 7, and 10. Tumor volumes and mouse weights were measured, and images of the mice were captured every 3 days. The percent survival was monitored for 40 days. Tumors were dissected 24 h after treatments and then fixed with 4 % polyoxymethylene for pathological examination, including hematoxylin-eosin (H&E), proliferating cell nuclear antigen (PCNA), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The tumor inhibition rates and the growth curves were determined according to the tumor volumes. The safety of DC-HMOP-LOD was determined according to the mouse weights. Biosafety assay of DC-HMOP-LOD Twenty-five naïve BALB/c mice were treated with DC-HMOP-LOD (10 mg/mL, 150 μL). At different time points (0, 1, 7, 14, 21, and 28 d), blood samples of mice were collected for blood panel analysis and biochemistry assays. H&E staining of major organs was conducted to observe the corresponding pathological toxicity. Statistical analysis Statistical analysis in this paper was performed with SPSS 26.0 software. The quantitative data are presented as the mean ± standard deviation. The significance of the data was compared using Student’s t-test (*P＜0.05, **P＜0.01). Results And Discussion Synthesis and characterization of DC-HMOP-LOD After the success synthesis of DC-HMOP-LOD, TEM (Fig. 1a) and SEM (Fig. 1b) showed that DC-HMOP-LOD was spherical and homogeneous in size. The average diameter of DC-HMOP-LOD measured by dynamic light scattering was approximately 214.0 nm, with a polydispersity index of 0.059 (Fig. 1c), which enable the nanocomposites accumulate in tumors via the EPR effect. DC-HMOP-LOD is a shell-core structure that separately carries LOD in the shell and DC in the core. The spectrum of HMOP-DC showed a maximum absorption peak at approximately 274 nm. The EE and LC of DC was calculated to be 12.19 % and 40.65 wt %according to the linear correlation equation (Fig. S1a). The EE and LC of LOD was 16.92 % and 26.65 wt % according to the standard curve of the Bradford Protein Assay Kit (Fig. S1b). Additionally, the sediment of HMOP-DC presented milky white, whereas the sediment DC-HMOP-LOD was light yellow (Fig. S1c), indicating the successful conjugation of LOD onto the surface of HMOP-DC. Additionally, as shown in Fig. 1d, e, the area elemental mapping analysis and energy dispersive spectroscopy (EDS) spectrum revealed that the major elements (Si, O, S) were distributed peripherally and homogeneously in HMOP. Moreover, DC-HMOP-LOD could be biodegraded in the reductive TME with a high concentration of reduced GSH, because the disulfide bonds in the silica framework could be cleaved in the reductive TME. Thus, GSH-dependent responsive degradation of DC-HMOP-LOD was observed by TEM (Fig. 1f) and SEM (Fig. 1g). DC-HMOP-LOD was gradually degraded with the extension of mixture time and ultimately completely degraded, indicating the biosafety of nanocomposites that could be bio-degradable and eliminated through feces and urine [23]. Then, the pharmacokinetic behaviors of DC-HMOP-LOD-FITC were analyzed on CT26 tumor-bearing mice. The level of DC-HMOP-LOD-FITC in the blood dramatically decreased at 1-2 h (Fig. 1h). Massive accumulation in the liver, spleen, kidney and tumor (Fig. 1i) was observed at 24 h. The tumor accumulation may benefit from the EPR effect of nanocomposites. Our previous experiment found that mice injected with free LOD died within 48 h. This phenomenon was also found by Tang [24]. The quick diffusion in blood and distribution in organs of free LOD may account for the phenomenon. The design of DC-HMOP-LOD could prevent the side effect of LOD, and lay foundation for the following applications. In summary, HMOP could deliver drugs in the tumor without the need for external energy input, which may enhance the treatment effect and prevent side effects. The catalytic activity and lactate consumption of DC-HMOP-LOD After the successful fabrication and characterization of DC-HMOP-LOD, the catalytic activity of LOD was further determined by measuring the generation of O 2 and H 2 O 2 and the consumption of lactate in vitro . The lactate solution was mixed with HMOP, HMOP-DC, HMOP-LOD, and DC-HMOP-LOD, respectively. It was found that the concentrations of lactate (Fig. 2a) and O 2 (Fig. 2b) in the HMOP-LOD and DC-HMOP-LOD groups gradually decreased with time. The concentration of H 2 O 2 increased in response to the enhanced concentration of lactate (Fig. 2c) and nanocomposites (Fig. 2d), verifying that LOD retained its high selectivity and catalytic efficiency after conjugation. Afterwards, the synergistic lactate consumption of DC-HMOP-LOD was verified by measuring the concentration of intracellular and extracellular lactate in vivo and in vitro . Compared with the control group (2.51 μmol/mL), the in vitro intracellular lactate concentrations in the HMOP-LOD (0.05 μmol/mL) and DC-HMOP-LOD (0.07 μmol/mL) groups were decreased, whereas the concentration of intracellular lactate in the HMOP-DC groups (4.17 μmol/mL) was increased (Fig. 2g). The intracellular lactate showed the same tendency in vivo (Fig. 2h). The intracellular lactate concentrations in the HMOP-LOD and DC-HMOP-LOD groups were decreased, whereas the concentration in the HMOP-DC group was increased. And the effect of DC-HMOP-LOD in regulating intracellular lactate was lower than that of HMOP-LOD group because of the effect of DC. It was demonstrated that LOD could be transported into the cell by HMOP and oxidized intracellular lactate. DC increased intracellular lactate due to the inhibition of lactate efflux by blocking lactate transporters on the cancer cell membrane. Then, we measured the extracellular (i.e., TME) lactate concentrations. In comparison to the control group (15.36 μmol/mL) in vitro , the extracellular lactate concentrations in the HMOP-DC group (13.26 μmol/mL) and HMOP-LOD (8.61 μmol/mL) group were decreased, especially in the DC-HMOP-LOD group (6.41 μmol/mL) (Fig. 2e), which resulted in a corresponding pH change in the cell supernatant (Fig. 2f). Furthermore, extracellular lactate in vivo showed the same tendency (Fig. 2i). The extracellular lactate concentrations in the HMOP-DC, HMOP-LOD and DC-HMOP-LOD groups were decreased. In sum, the extracellular lactate concentrations in the HMOP-DC, HMOP-LOD, and DC-HMOP-LOD were decreased in vitro and in vivo , which showed that treatment with DC-HMOP-LOD synergistically depleted extracellular lactate, leading to a pH change in the TME. Lactate and an acidic TME both play important roles in suppressing anticancer immunity [25, 26], and thus we speculated that DC-HMOP-LOD could contribute to the restoration of anticancer immunity. Cell uptake and cytotoxicity evaluation To ensure the DC could be transported into tumor cells, we explored whether DC-HMOP-LOD could be efficiently engulfed by tumor cells. Intracellular phagocytosis was visualized by CLSM and measured by flow cytometry. As shown in Fig. 3a, the intracellular green FL gradually increased as a result of the efficient uptake of DC-HMOP-LOD with the extension of incubation time. The flow cytometry results were in accordance with the CLSM observations (Fig. 3b), demonstrating the high affinity of DC-HMOP-LOD to tumor cells. Then, the antitumor effect of DC-HMOP-LOD in vitro was measured. The cytotoxicity was evaluated by CCK-8. As shown in Fig. 3c, after various nanocomposite treatments for 24 h, the cell viabilities in the HMOP-LOD and DC-HMOP-LOD groups were approximately 33.05 % and 33.41 %, respectively. Cellular apoptosis detected by flow cytometry showed the same cytotoxicity. The cell damage rates were 80.90 % and 81.32 % (Fig. 3d) in the HMOP-LOD and DC-HMOP-LOD groups, respectively. From direct observation by CLSM (Fig. S2), massive quantities of dead cells were found in the HMOP-LOD and DC-HMOP-LOD groups, which indicated the antitumor efficacy of LOD. We verified that HMOP-LOD increased H 2 O 2 in vitro , which can cause oxidative damage and kill tumor cells. HMOP-DC had no obvious cytotoxicity, which was consistent with the results of Renner [27]. Renner found that the viabilities of tumor cells and T cells were not affected by DC for 72 h. But reduction of lactate secretion by DC boosted T-cell-mediated killing in programmed cell death-ligand 1(PD-L1) knocked down melanoma cells, while T cells were not able to kill tumor cells without the treatment of DC. Immunosuppression mitigation After the demonstration of lactate reduction of DC-HMOP-LOD, we further explored whether lactate depletion could relieve ITM and activate the immune response. Concretely, we measured the proportions of immune-promoting cells and inactivating immunosuppressive cells in tumors by flow cytometry analysis, and measured the levels of immune cytokines by ELISA. DC-HMOP-LOD significantly increased the recruitment of immune-promoting cells. Dendritic cells can activate adaptive immune responses by activating naïve T cells and inducing antigen-specific T-cell-mediated immunity [28]. Lactate suppresses this activation by impairing monocyte differentiation into dendritic cells [29]. We found that HMOP-LOD caused moderate dendritic cell maturation (10.45 %), whereas HMOP-DC generated more maturation (17.30 %). DC-HMOP-LOD markedly facilitated the maturation of dendritic cells (23.13 %), which was 2.21-fold higher than that of the control group (Fig. 4a, d). CD8 + cytotoxic T lymphocytes (CTLs) play important roles in antitumor immunity due to the selective recognition and killing of cancer cell [30]. However, lactate impairs CTLs function by influencing respiratory activity, chemotaxis, and recruitment [31]. In addition, lactate accumulation can inhibit glycolysis and decrease the motility of CD4 + helper T cells [32]. CD4 + T cells can generate proinflammatory factors, including tumor necrosis factor (TNF) -α and interferon (IFN)-γ, further activating the innate immune response. Herein, the increased proportions of both CD8 + T cells and CD4 + T cells in the HMOP-DC group and HMOP-LOD group showed superior activation for cellular immunity. Importantly, the highest proportion was found in the DC-HMOP-LOD group (CD3 + CD4 + T cells were 21.20 % and CD3 + CD8 + T cells were 31.37 %) (Fig. 4b, e, f). Additionally, NK cells, innate immune cells, exhibit an antitumor effect via effective cytolytic activity by generating granzymes, cytokines, and additional factors [33]. Lactate can inhibit NK-cell activity and reduce the expression and production of IFN-γ in NK cells [34]. Compared to untreated tumors in mice (3.17 %), NK cells recruitment was obviously induced by DC-HMOP-LOD (8.28 %). The proportions in the HMOP-DC group and the HMOP-LOD groups were 3.61 and 3.80 times higher, respectively, than the proportion in the control group (Fig. 4c, g). From the above results, it was substantiated that DC-HMOP-LOD markedly increased the activity and infiltration of immune-promoting cells and transformed immunosuppressive tumors into “hot” tumors through lactate reduction in vivo . In addition to immune stimulation, immunosuppression remission is also worthy of attention. TAMs [35] and MDSCs [36] are vital elements in the immunosuppressive TME. TAMs are sensitive to lactate, and lactate can polarize protumor M1-type TAMs to antitumor M2-type TAMs [37]. M2-type TAMs are related to tumor growth, metastasis, and immunosuppression because M2-type TAMs can not only directly target tumor cells through phagocytosis or the release of cytokines (TNF-α, etc. ) but can also indirectly recruit and activate other immune effector cells (IFN-γ, interleukin (IL) -12, etc. ) [38]. M2-type TAMs can release multiple growth factors and cytokines (IL-10, etc. ) to result in immune suppression [39]. Furthermore, M1-type TAMs exhibit decreased viability compared with M2-type TAMs at lower pH values [40]. Herein, we demonstrated that DC-HMOP-LOD could regulate macrophage phenotype polarization in vitro and in vivo . After RAW 264.7 Cells were treated with DC-HMOP-LOD (Fig. 5a, b), M2-type TAMs (3.35 %) were polarized to M1-type TAMs (76.9 %). The results in vivo showed the same trend (Fig. 5 c, d, e). The percentage of M1-type TAMs increased (58.03 %), and the competency of M2-type TAMs decreased (20.17 %) (Fig. 5f, g) in the tumor after treatment with DC-HMOP-LOD. From the above results, it was verified that DC-HMOP-LOD had an effective impact on macrophage phenotype, skewing differentiation from protumor M2-type TAMs to the antitumor M1 phenotype. MDSCs normally differentiate into TAMs, dendritic cells, and granulocytes. However, lactate impairs this differentiation, resulting in MDSCs accumulation. This accumulation can stimulate other immnosuppressive cells, inhibit lymphocyte homing, and decrease T-cell activity [41, 42]. Furthermore, MuDSCs increase PD-L1 expression in the acidic TME [43]. As shown in Fig. 6a, b, the lowest MDSCs proportion appeared in the DC-HMOP-LOD group, with a reduction of 5.02 % compared with the control group. TAMs and MDSCs levels in tumor tissues demonstrate the efficiency of DC-HMOP-LOD in recovering the immunosuppressive TME. Immune cytokines in serum also changed after the treatment of DC-HMOP-LOD. Here, we measured the levels of TNF-α (Fig. 6c), IFN-γ (Fig. 6d), IL-10 (Fig. 6e), and IL-12 (Fig. 6f) by ELISA. IFN-γ and TNF-α are both critical cytokines in cellular immunity that indicate the activation of effector T cells. Although both HMOP-DC and HMOP-LOD were able to increase the secretion of TNF-α and IFN-γ, the levels induced by DC-HMOP-LOD (IFN-γ 2.27 pg/mL and TNF-α 2.28 ng/mL) were obviously higher than the concentrations of IFN-γ (1.32 pg/mL) and TNF-α (1.37 ng/mL) in the control group. IL-10 is secreted by M2-type TAMs to suppress the immune response and promote tumor invasion and metastasis. IL-12 is secreted by M1-type TAMs to promote the immune response and inflammation [44-46]. IL-12 can also inhibit the differentiation of Tregs from naïve T cells [47]. Lactate can increase the expression of IL-10 and decrease the generation of IL-12, resulting in reduced inflammation and the promotion of immune tolerance [48]. DC-HMOP-LOD reduced the levels of IL-10 and IL-12 by consuming lactate in the TME. The secretion of IL-10 (1.46 ng/mL) was significantly decreased, whereas the secretion of IL-12 (1.81 ng/mL) was increased in the DC-HMOP-LOD group. The regulatory effects of HMOP-DC and HMOP-LOD on immune cells were unsatisfactory due to their inability to produce excessive lactic acid in the TME. The synergistic reduction of lactate by DC-HMOP-LOD had an excellent ability to relieve the ITM and activate the immune response. Antitumor capacity Based on the mitigated immunosuppression by DC-HMOP-LOD, we speculated that the efficacy of aPD1 immunotherapy could be improved by DC-HMOP-LOD. As shown in Fig. 7a, b, and Fig. S3a, the tumors in the control groups grew rapidly. DC-HMOP-LOD and aPD1 treatment exhibited an unsatisfactory tumor inhibition effect, especially after the cessation of administration. The efficiency of HMOP-LOD and HMOP-DC was even worse than that of the aPD1 group. However, the aPD1 + DC-HMOP-LOD group performed best in suppressing tumor growth. Additionally, the mice in the control group all died on the 16th day. LOD and DC could not induce a sustained antitumor response. The tumors progressed, and the animals ultimately died in the HMOP-DC and HMOP-LOD groups after cessation of injection. Compared with the mice in the HMOP-DC, HMOP-LOD, and aPD1 groups that died rapidly before the 37th day, the survival was slightly extended in the DC-HMOP-LOD group. However, much longer survival was observed in the aPD1 + DC-HMOP-LOD group (Fig. 7c). With the extension of treatment time, representative photographs of mice and tumor growth curves of every mouse are shown in Fig. S 3b. The results of H&E, PCNA, and TUNEL staining further indicated satisfactory therapeutic outcomes of synergistic therapy. To evaluate necrosis and apoptosis of the tumor cells after various treatments, H&E and TUNEL staining of tumors were performed. H&E staining (Fig. 7d) showed abundant necrosis of tumor cells in the aPD1 + DC-HMOP-LOD group, which was much more remarkable than that in the single treatment groups. The apoptosis of tumor cells revealed by TUNEL staining (Fig. 7e) showed the same trend as the H&E staining. PCNA staining (Fig. 7f) results evaluated the proliferative activities of cancer cells (stained brown) and showed a lower proliferation index after treatments, especially in the aPD1 + DC-HMOP-LOD group. MCTs have crucial roles in glycolytic cells because the glycolytic flux that MCTs provide is important for tumor cells to supply metabolic intermediates. Therefore, DC can limit glucose metabolism and impair the proliferation and survival of tumor cells [49]. This ability has been demonstrated in a clinical trial, and both Pantziarka [50] and Singer [51] showed that DC could augment the response to checkpoint inhibition by upregulating IFN-γ expression in T cells and NK cells. In addition, the reactive oxygen species level was elevated significantly due to the catalysis of LOD, which could cause cell apoptosis. However, compared with the high tumor inhibition activity in vitro , HMOP-LOD only had a moderate antitumor effect in vivo , which may result from the heterogeneous distribution of intratumoral lactate and oxygen. The moderate antitumor activity and immunosuppression mitigation also indicated the necessary combination of LOD and DC. The consumption of lactate in the TME can directly suppress tumor growth by suppressing cell migration, metastasis, and angiogenesis. Furthermore, it has been shown in clinical trials that reducing lactate levels in the TME can reduce tumor burden indirectly by promoting immunotherapeutic efficacy [52]. Herein, we also demonstrated that strategies using DC-HMOP-LOD effectively improve the antitumor activity of aPD1 treatment primarily by modulating the ITM. Safety of DC-HMOP-LOD The biosafety of nanocomposites is a key factor for ensuring the feasibility of clinical transformation. Therefore, we investigated the safety of DC-HMOP-LOD in vitro and in vivo . In vitro , over 85 % of cells remained alive after incubation with HMOP for 24 h (Fig. S4a). At the endpoint of the treatment period in vivo , negligible fluctuations were shown in the average body weights of mice from all groups (Fig. S4b). Additionally, after administration of DC-HMOP-LOD, no obvious abnormality was seen in blood panel analysis or biochemistry assays (Fig. S4c), and no detectable change was found in histopathology for major organs (Fig. S4d). These results demonstrated that DC-HMOP-LOD showed desirable biocompatibility and therapeutic safety. Conclusion In summary, we successfully designed DC-HMOP-LOD, which safely accumulated and locally released LOD and DC in the tumor. DC effectively hindered the transmission of lactate, and LOD directly oxidized lactate, collaboratively depleting lactate in the TME. The synergetic depletion reversed the ITM by activating antitumor immune responses and inhibiting negative regulators of innate as well as adaptive immune cells. Therefore, DC-HMOP-LOD induced necrosis and apoptosis, decreased the proliferative activities of the tumor cells, suppressed tumor growth, and extended the survival of mice, indicating that DC-HMOP-LOD could improve the antitumor effects of aPD1. Thus, the intra/extracellular lactate exhaustion by DC-HMOP-LOD may overcome the existing restrictions of ICB therapy to some extent. Abbreviations Abbreviations: ICB, Immune checkpoint blockade; ITM, immunosuppressive tumor microenvironment; MCTs, monocarboxylate transporters; HMOP, PEGylated hollow mesoporous organosilicon; DC, diclofenac; LOD, lactate oxidase; TME, tumor microenvironment; NK, natural killer; MDSCs, myeloid-derived suppressor cells; TAMs, tumor-associated macrophages; Tregs, regulatory T cells; EPR, enhanced permeability and retention; aPD1, anti-PD1; TEM, transmission electron microscope; SEM, scanning electron microscope; EE, encapsulation efficiency; LC, loading capacity ; EDS, energy dispersive spectroscopy; GSH, glutathione; CLSM, confocal laser scanning microscopy; CCK-8, cell counting kit-8; PD-L1, programmed cell death-ligand 1; ELISA, enzyme linked immunosorbent assay; CTLs, cytotoxic T lymphocytes; TNF-α, tumor necrosis factor-α; interferon-γ, IFN -γ; H&E, hematoxylin-eosin; PCNA, proliferating cell nuclear antigen; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. Declarations Ethics approval and consent to participate Animal experiments were approved and performed in accordance with the guidelines of the Institutional Animal Care and Animal Ethics Committee of Chongqing Medical University. Consent for publication Not applicable. Availability of data and materials The data supporting the findings of this study are available from the corresponding author upon reasonable request Competing interests The authors declare no competing financial and non-financial interests. Funding This study was supported by the Research Incubation Project of the Third Affiliated Hospital of Chongqing Medical University (KY08025), Basic Research and Frontier Exploration Project of Yuzhong District of Chongqing (20190107), and Chongqing Talent Plan (cstc2021ycjh-bgzxm0077). Authors’ contributions YDT JH, and RL conceived and designed experiments; YDT, CMZ and HWX conducted the experiments and obtained the results; XJL and JZ provided guidance for data analysis and instrumental support; YDT and XYT analyzed data; YDT JH and LZ wrote the manuscript; BS contributed to manuscript revision; RL offered financial support and provided final approval of the manuscript. Acknowledgements Not applicable. Author details 1 Department of Ultrasonography, The Third Affiliated Hospital of Chongqing Medical University, Chongqing 401120, People’s Republic of China. 2 Ultrasound Department, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400010, People’s Republic of China. 3 Chongqing Key Laboratory of Ultrasound Molecular Imaging & Institute of Ultrasound Imaging, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400016, People’s Republic of China. References O'Donnell JS, Long GV, Scolyer RA, Teng MW, Smyth MJ. 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Supplementary Files Additionalfiles.docx Scheme1.tif Scheme 1 DC-HMOP-LOD gradually accumulated and locally released LOD and DC in the tumor. Then, LOD directly catalyzed lactate oxidation to consume lactate in the TME and tumor cells. Meanwhile, DC blocked lactate transporters to inhibit lactate efflux and thus decreased lactate in the TME. Thus, DC-HMOP-LOD could synergistically deplete lactate in the TME, restoring an immunocompetent TME by activating immune-promoting cells (dendritic cells, CD4 + T cells, CD8 + T cells, and NK cells), inactivating immunosuppressive cells (M2-TAM and MDSCs), and ultimately increasing antitumor efficiency by increasing the antitumor effect of aPD1 immunotherapy. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Li","email":"data:image/png;base64,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","orcid":"","institution":"The Third Affiliated Hospital of Chongqing Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2022-10-15 03:14:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2168046/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2168046/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":27993747,"identity":"bcaf6eeb-ab2c-48fd-b715-1d6b7e121433","added_by":"auto","created_at":"2022-10-19 14:28:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21814422,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of DC-HMOP-LOD. \u003cstrong\u003ea\u003c/strong\u003e TEM image and (\u003cstrong\u003eb\u003c/strong\u003e) SEM image of DC-HMOP-LOD (scale bar = 200 nm). \u003cstrong\u003ec\u003c/strong\u003eThe hydrodynamic diameter of DC-HMOP-LOD. \u003cstrong\u003ed\u003c/strong\u003e Energy-dispersive X-ray spectroscopy spectrum of DC-HMOP-LOD. \u003cstrong\u003ee\u003c/strong\u003e Elemental mapping of DC-HMOP-LOD (scale bar = 500 nm). \u003cstrong\u003ef\u003c/strong\u003e TEM images and (\u003cstrong\u003eg\u003c/strong\u003e) SEM images of DC-HMOP-LOD in 10 mM GSH solutions at 3, 7, and 14 days (scale bar = 200 nm).\u003cstrong\u003eh-i \u003c/strong\u003ePharmacokinetic study of DC-HMOP-LOD-FITC in (\u003cstrong\u003eh\u003c/strong\u003e) blood circulation and (\u003cstrong\u003ei\u003c/strong\u003e) major organs.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2168046/v1/c00859c4bdc28a922d574749.png"},{"id":27993739,"identity":"15f6d079-c1c2-4629-b925-cd5f851693b0","added_by":"auto","created_at":"2022-10-19 14:28:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1657920,"visible":true,"origin":"","legend":"\u003cp\u003eThe catalytic activity and lactate consumption of DC-HMOP-LOD. \u003cstrong\u003ea\u003c/strong\u003e Lactate and (\u003cstrong\u003eb\u003c/strong\u003e) O\u003csub\u003e2\u003c/sub\u003e consumption effects of nanocomposites in lactate solution at different time points. \u003cstrong\u003ec-d\u003c/strong\u003e The generated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003econcentration arises from the reaction between nanocomposites and (\u003cstrong\u003ec\u003c/strong\u003e) different concentrations of lactate and also from (\u003cstrong\u003ed\u003c/strong\u003e) cells. \u003cstrong\u003ee\u003c/strong\u003e Extracellular lactate consumption effect and (\u003cstrong\u003ef\u003c/strong\u003e) corresponding pH values at the cellular level. \u003cstrong\u003eg\u003c/strong\u003e Intracellular lactate consumption at the cellular level. \u003cstrong\u003eh\u003c/strong\u003e Extracellular and (\u003cstrong\u003ei\u003c/strong\u003e) intracellular lactate consumption effects in CT26 tumors 24 h after injection of nanocomposites. (*P＜0.05, **P＜0.01)\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2168046/v1/7dafdc698a7b5e5d4aa39cfa.png"},{"id":27994816,"identity":"82a4aad4-dba6-40b1-9b91-5d609b11736b","added_by":"auto","created_at":"2022-10-19 14:38:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9416087,"visible":true,"origin":"","legend":"\u003cp\u003eIntracellular phagocytosis and therapeutic performance of nanocomposites in vitro. \u003cstrong\u003ea\u003c/strong\u003e CLSM images (scale bar = 50 μm) and (\u003cstrong\u003eb\u003c/strong\u003e) flow cytometry of CT26 cells after treatment with DC-HMOP-LOD for 2, 4, 6, 8, and 24 h. \u003cstrong\u003ec-d\u003c/strong\u003eThe therapeutic effects of various nanocomposites were detected by (\u003cstrong\u003ec\u003c/strong\u003e) CCK-8 and (\u003cstrong\u003ed\u003c/strong\u003e) flow cytometry. (**P＜0.01)\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2168046/v1/d2571f5ee01fee41188900e8.png"},{"id":27994357,"identity":"1ed55d8d-f75e-4ff8-a7c9-61d1a85cb2b3","added_by":"auto","created_at":"2022-10-19 14:33:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5910605,"visible":true,"origin":"","legend":"\u003cp\u003eImmune promoting stimulation. \u003cstrong\u003ea-c\u003c/strong\u003e Representative fluorescence activated cell sorting (FACS) plots of tumor infiltration by (\u003cstrong\u003ea\u003c/strong\u003e) dendritic cells, (\u003cstrong\u003eb\u003c/strong\u003e) T cells, and (\u003cstrong\u003ec\u003c/strong\u003e) NK cells. \u003cstrong\u003ed–g\u003c/strong\u003e Quantitative analyses of (\u003cstrong\u003ed\u003c/strong\u003e) dendritic cells, (\u003cstrong\u003ee\u003c/strong\u003e) CD4\u003csup\u003e+\u003c/sup\u003e T cells, (\u003cstrong\u003ef\u003c/strong\u003e) CD8\u003csup\u003e+\u003c/sup\u003e T cells, and (\u003cstrong\u003eg\u003c/strong\u003e) NK cells. (*P＜0.05, **P＜0.01)\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2168046/v1/f51daabd98f75356ff33afb7.png"},{"id":27994356,"identity":"e9ee2d8a-dcea-4d69-97e8-d5961ea752cc","added_by":"auto","created_at":"2022-10-19 14:33:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4658903,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of lactate on macrophage polarization. \u003cstrong\u003ea, b\u003c/strong\u003e Flow cytometry analysis of the expression of (\u003cstrong\u003ea\u003c/strong\u003e) CD86 and (\u003cstrong\u003eb\u003c/strong\u003e) CD206 in RAW264.7 cells after coculturing with CT26 cells. \u003cstrong\u003ec, d\u003c/strong\u003e Representative FACS plots of (\u003cstrong\u003ec\u003c/strong\u003e) M1-type TAMs and (\u003cstrong\u003ed\u003c/strong\u003e) M2-type TAMs. \u003cstrong\u003ee-g\u003c/strong\u003e Quantitative analysis of (\u003cstrong\u003ee\u003c/strong\u003e) M1-type TAMs, (\u003cstrong\u003ef\u003c/strong\u003e) M2-type TAMs and (\u003cstrong\u003eg\u003c/strong\u003e) the ratio between M1-type TAMs and M2-type TAMs. (*P＜0.05, **P＜0.01)\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2168046/v1/03f6e9bb16cbbbbd49da3e40.png"},{"id":27993745,"identity":"e514aa57-6bc3-448a-94e6-6fcc903932d7","added_by":"auto","created_at":"2022-10-19 14:28:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2421961,"visible":true,"origin":"","legend":"\u003cp\u003eImmunosuppression remission and cytokine regulation. \u003cstrong\u003ea\u003c/strong\u003e Representative FACS plots of tumor-infiltrating MDSCs. \u003cstrong\u003eb\u003c/strong\u003eQuantitative analyses of MDSCs. \u003cstrong\u003ec-f\u003c/strong\u003e Cytokine levels of (\u003cstrong\u003ec\u003c/strong\u003e) TNF-α, (\u003cstrong\u003ed\u003c/strong\u003e) IFN-γ, (\u003cstrong\u003ee\u003c/strong\u003e) IL-10 and (\u003cstrong\u003ef\u003c/strong\u003e) IL-12 in serum. (*P＜0.05, **P＜0.01)\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-2168046/v1/1e184eea7e7ab2634743efd3.png"},{"id":27994359,"identity":"cfbaffbf-9375-4f43-b337-8e4e345493fa","added_by":"auto","created_at":"2022-10-19 14:33:20","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":28138664,"visible":true,"origin":"","legend":"\u003cp\u003eTherapeutic performance of nanocomposites \u003cem\u003ein vivo\u003c/em\u003e. After various treatments with nanocomposites, (\u003cstrong\u003ea\u003c/strong\u003e) tumor volume curves, (\u003cstrong\u003eb\u003c/strong\u003e) tumor inhibition rates and (\u003cstrong\u003ec\u003c/strong\u003e) morbidity-free survival of CT26-bearing mice in six groups were assessed. (\u003cstrong\u003ed\u003c/strong\u003e) H\u0026amp;E, (\u003cstrong\u003ee\u003c/strong\u003e) TUNEL and (\u003cstrong\u003ef\u003c/strong\u003e) PCNA staining of tumor sections were observed (scale bar = 200 μm). (*P＜0.05, **P＜0.01)\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-2168046/v1/3273887265e20e0748fcf187.png"},{"id":29619771,"identity":"beda8ffe-7a0a-4ebf-88c7-b3eb165c81ed","added_by":"auto","created_at":"2022-11-28 22:29:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4636658,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2168046/v1/f6ce9205-3e1b-44e4-ae88-0b33b016a493.pdf"},{"id":27993740,"identity":"e627f782-250f-45c6-947e-ae37bcde9674","added_by":"auto","created_at":"2022-10-19 14:28:20","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2976422,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-2168046/v1/cd81e9802ce77e5578167407.docx"},{"id":27993744,"identity":"04220dc4-73cb-4b3c-8203-f35275a65e15","added_by":"auto","created_at":"2022-10-19 14:28:20","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":9997268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e\u0026nbsp; DC-HMOP-LOD gradually accumulated and locally released LOD and DC in the tumor. Then, LOD directly catalyzed lactate oxidation to consume lactate in the TME and tumor cells. Meanwhile, DC blocked lactate transporters to inhibit lactate efflux and thus decreased lactate in the TME. Thus, DC-HMOP-LOD could synergistically deplete lactate in the TME, restoring an immunocompetent TME by activating immune-promoting cells (dendritic cells, CD4\u003csup\u003e+\u003c/sup\u003e T cells, CD8\u003csup\u003e+\u003c/sup\u003e T cells, and NK cells), inactivating immunosuppressive cells (M2-TAM and MDSCs), and ultimately increasing antitumor efficiency by increasing the antitumor effect of aPD1 immunotherapy.\u003c/p\u003e","description":"","filename":"Scheme1.tif","url":"https://assets-eu.researchsquare.com/files/rs-2168046/v1/a17060239dffc136467c8d82.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"GSH-responsive Nanoplatform for Intra/Extracellular Lactate Exhaustion to Enhance Antitumor Immunotherapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eImmune checkpoint blockade (ICB) therapies have achieved unprecedented clinical benefits. Unfortunately, only few patients could respond to the ICB therapy because of the restriction of immunosuppressive tumor microenvironment (ITM) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Numerous findings point out that ITM is featured with potent immune suppressive activity including affecting the recruitment, proliferation, and functions of immune cells [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, reconstructing the ITM could be a promising approach for improving ICB therapy [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent nanotechnology in biomedicine has reversed ITM by advancing various modalities, such as chemotherapy [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], sonodynamic therapy [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], gene therapy [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] \u003cem\u003eetc\u003c/em\u003e. Although these nanosystems have improved antitumor effect of ICB therapy, which are usually resulted from immunogenic death effect and the activated innate immunity, the therapeutic efficacy still is unsatisfactory due to the relatively high chemoresistance of cancer cells enabled by their reprogrammed metabolism [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This situation prompts us to reconsider new therapeutic modality that focuses on the basic laws of biological processes in tumor cells, and solve the most essential issues by rational metabolic regulation. Accumulating data have provided evidence supporting the functional role of lactate in constructing the ITM [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Concretely, the excessive accumulation of lactate (10\u0026ndash;30 mM) in tumor microenvironment (TME) is closely related to the enhanced glycolysis in tumor cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In order to balance the rapid biosynthetic processes and a huge demand of ATP production, tumor cells are prone to adopt aerobic glycolysis to support cell proliferation. Lactate accumulation in TME influences various tumor processes, such as tissue invasion and metastasis, angiogenesis, and drug resistance [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Furthermore, lactate acts as a critical regulator of the ITM by influencing the differentiation and proliferation of immune cell [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Concretely, lactate impairs the infiltration of immune effector cells such as natural killer (NK) cells and cytotoxic T cells. Additionally, lactate can suppress the differentiation and impair the functions of dendritic cells. Meanwhile, lactate can promote the activity of immunosuppressive cells, including myeloid-derived suppressor cells (MDSCs) and M2-type tumor-associated macrophages (TAMs). It has also been shown that lactate-induced ITM is reversible by altering the lactate concentration in the TME [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNowadays, various mesoporous silica nanoparticles and porous hollow silica nanoparticles have been studied as drug delivery systems due to good biocompatibility. However, some limitations restrict the potential for clinical transformation such as uncontrolled biodegradability of the silica framework, unclear retention times \u003cem\u003ein vivo\u003c/em\u003e, and pathway. In this study, we synthesized PEGylated hollow mesoporous organosilicon (HMOP) nanocomposites. HMOP can achieve a fast bio-degradation by intracellular glutathione (GSH) stimulation because of disulfide bonds in the silica framework, enhancing its biosafety and facilitating its excretion [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. And HMOP is a well-defined shell/core structure with desirable physical condition, pharmacokinetics, affinity, and biosafety [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Herein, Lactate oxidase (LOD) and diclofenac (DC) were spatially conjunct to the shell and loaded in the core of HMOP, respectively. HMOP can selectively accumulate and release LOD and DC locally by the enhanced permeability and retention (EPR) effect without the need for external energy input [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The released LOD and DC synergistically deplete intracellular and extracellular lactic acid. LOD is an enzyme that can directly consume a high level of intra/extracellular lactate by catalyzing the oxidation of lactate to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and pyruvate [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. To achieve a synergistic reduction in lactate in the TME, we simultaneously used DC, a classic nonsteroidal anti-inflammatory drug, to inhibit lactate transport from tumor cells into the TME by blocking lactate transporters (monocarboxylate transporters 1 (MCT1) and MCT4) on the cancer cell membrane. Lactate catalysis and lactate transport inhibition could achieve synergistic lactate reduction in the TME. The lactate exhaustion strategy could efficiently reverse the ITM by activating immune-promoting cells, inactivating immunosuppressive cells, and regulating immune cytokines. The immunocompetent TME ultimately improved the antitumor efficacy of anti-PD1 (aPD1) immunotherapy (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003ch2\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eTetraethylorthosilicate (TEOS), hexadecyltrimethylammonium chloride (CTAC),\u0026nbsp;triethanolamine (TEA), bis[3-(triethoxysilyl) propyl] tetrasulfide (BTES), sodium carbonate (Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e), methanol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 3-aminopropyl triethoxysilane (APTES),\u0026nbsp;ammonia solution (25-28%), and diclofenac (DC) were purchased from Macklin (China). Polyethylene glycol (PEG)-silane was purchased from Xi\u0026apos;an Ruixi Biological Technology Co., Ltd. (China). Lactate oxidase (LOD) was purchased from Shanghai Yingxin Laboratory Equipment Co., Ltd. (China). 1,1-Dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) and fluorescein isothiocyanate isomer (FITC)\u0026nbsp;were\u0026nbsp;purchased from Beyotime Biotechnology (China). Anti-PD-1 was purchased from Bio X Cell (USA). Cell counting kit-8 (CCK-8) was purchased from Dojindo (Japan). Enzyme linked immunosorbent assay (ELISA) kits were obtained from Meimian Industrial Co., Ltd. (Jiangsu, China). The\u0026nbsp;Bradford Protein Assay Kit, Lactic Acid Assay Kit, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e Content Assay Kit were\u0026nbsp;purchased from\u0026nbsp;Solarbio (Beijing, China). FITC-conjugated anti-mouse CD11c, phycoerythrin (PE)-conjugated anti-mouse CD86, allophycocyanin (APC)-conjugated anti-mouse CD80, cyanine (Cy)5.5-conjugated anti-mouse CD45, APC-conjugated anti-mouse CD49b, FITC-conjugated anti-mouse CD4, APC-conjugated CD8, APC-conjugated anti-mouse CD206, FITC-conjugated anti-mouse F4/80, Cy5.5-conjugated anti-mouse Foxp3, Cy5.5-conjugated anti-mouse CD11b, and FITC-conjugated anti-mouse Gr-1 were purchased from Thermo Fisher Scientific (USA).\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eCell culture and animals\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eMale BALB/c mice (6\u0026ndash;8 weeks old) were obtained from the Experimental Animal Center of Chongqing Medical University\u0026nbsp;(CQMU, SYXK(YU)2018-0003). Animal experiments were approved and\u0026nbsp;performed in accordance with the guidelines of the Institutional Animal Care and Animal Ethics Committee of CQMU.\u0026nbsp;The mouse colon adenocarcinoma cell line CT26 was provided by the Cell Bank of Shanghai Institute (Chines Academy of\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eScience). The cells were cultured in RPMI-1640 medium (M\u0026amp;C, Beijing, China) with 10% fetal bovine serum (FBS, Gibco, Australia), 1 % penicillin/streptomycin (37\u0026nbsp;\u0026deg;C\u0026nbsp;and 5 % CO\u003csub\u003e2\u003c/sub\u003e). \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eSynthesis of HMOM\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe synthesis of HMON used a selective etching strategy. First, ethanol (74 mL), deionized water (ddH\u003csub\u003e2\u003c/sub\u003eO) (10 mL), and ammonia solution (3.14 mL) were stirred at room temperature for 5 minutes, and then TEOS (6 mL) was quickly added. The mixture was stirred for 1 h at room temperature, washed with ethanol 2 times, and then distributed into ddH\u003csub\u003e2\u003c/sub\u003eO (60 mL) to form monodispersed SiO\u003csub\u003e2\u003c/sub\u003e nanocomposites. Second, CTAC (2 g) and TEA (0.04 g) were added to ddH\u003csub\u003e2\u003c/sub\u003eO (90 mL), and the solution was stirred for 1.5 h at room temperature. Then, the above SiO\u003csub\u003e2\u003c/sub\u003e nanocomposites (30 mL) were added and stirred for another 1.5 h at room temperature. BTES (1.8 mL) was dropped into the mixed solution and stirred for 1 h at 80 \u0026deg;C. The SiO\u003csub\u003e2\u003c/sub\u003e@MON was washed with ethanol 2 times.\u0026nbsp;Third, SiO\u003csub\u003e2\u003c/sub\u003e@MON was added to Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e aqueous solution (0.6 M, 100 mL) and incubated at 80 \u0026deg;C for another 1 h to form the HMON products. Fourth, the HMON products were suspended with NaCl in methanol (1 wt %, 30 mL) for 3 h at room temperature to remove the unreacted CTAC.\u0026nbsp;Fifth,\u0026nbsp;HMON\u0026nbsp;(50 mg) and PEG-silane (30 mg) were stirred in ethanol (50\u0026nbsp;mL)\u0026nbsp;at 80 \u0026deg;C\u0026nbsp;for 12 h\u0026nbsp;to form the HMOP.\u0026nbsp;Finally, the HMOP products were obtained after washing with ddH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e times and dispersing in ddH\u003csub\u003e2\u003c/sub\u003eO (15 mL).\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eSynthesis of DC-HMOP-LOD\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eHMOP (30 mg) and DC (60 mg) were dissolved in 10 mL ethanol, stirred at room temperature for 24 h, washed with ddH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e times and dispersed in ddH\u003csub\u003e2\u003c/sub\u003eO (6 mL) to collect the HMOP-DC. LOD (150 U) was amino-functionalized by stirring in a mixture of EDC (38 mg), NHS (57 mg), APTES (45 \u0026micro;L), and ddH\u003csub\u003e2\u003c/sub\u003eO (6 mL) for 8 h at room temperature. Then, HMOP-DC (20 mg) in ddH\u003csub\u003e2\u003c/sub\u003eO (4 mL) was added and further stirred for 24 h at room temperature. The final DC-HMOP-LOD was collected by centrifugation (10,000 rpm, 5 min), washed with ddH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e times, and finally dispersed in ddH\u003csub\u003e2\u003c/sub\u003eO (5 mL).\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eSynthesis of DC-HMOP-LOD-FITC or DC-HMOP-LOD-Cy5.5\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eTo obtain DC-HMOP-LOD-FITC,\u0026nbsp;FITC (1 mg) was amino-functionalized by stirring for 8 h at room temperature in a mixture of EDC (6.6 mg), NHS (9.6 mg), APTES (45 \u0026micro;L), and ddH\u003csub\u003e2\u003c/sub\u003eO (8 mL). Then, DC-HMOP-LOD (10 mg) in ddH\u003csub\u003e2\u003c/sub\u003eO (2 mL) was added and further stirred for 24 h at room temperature. The final DC-HMOP-LOD-FITC was collected by centrifugation (10,000 rpm, 5 min), washed with ddH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e times, and finally dispersed in ddH\u003csub\u003e2\u003c/sub\u003eO (2 mL). To fabricate DC-HMOP-LOD-Cy5.5, the pH of the HMOP was modulated at 8.5. Then, carboxylic-functionalized Cy5.5 (20 \u0026mu;g) and DC-HMOP-LOD (5 mg) were\u0026nbsp;mixed\u0026nbsp;for 24 h at 4 \u0026deg;C.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eCharacterization of DC-HMOP-LOD\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe morphology of DC-HMOP-LOD was\u0026nbsp;visualized using scanning electron microscope (SEM) (Hitachi S-3400 N, Japan) and transmission electron microscope (TEM) (Hitachi-7500, Japan).\u0026nbsp;The particle diameter\u0026nbsp;was\u0026nbsp;measured using a Malvern Zetasizer instrument (Nano ZS90, UK). Area-elemental mapping and EDS spectra confirmed the existence of the major elements (Si, O, S) in DC-HMOP-LOD on the FEI-Talos F200S electron microscope. The encapsulation efficiency (EE) and loading capacity (LC) of DC was calculated using a standard curve by plotting the absorbance against concentration using a UV\u0026ndash;Vis-NIR spectrophotometer (Shimadzu, Kyoto, Japan). The EE and LC of LOD was confirmed by a Bradford Protein Assay Kit. The EE of DC or LOD in DC-HMOP-LOD was calculated according to the following formula: EE (%) = (weight of loaded DC or LOD / weight of total DC or LOD) x 100. The LC of DC or LOD in DC-HMOP-LOD was calculated according to the following formula: LC (%) = (weight of loaded DC or LOD / weight of total nanocomposites) x 100. GSH -dependent responsive degradation of DC-HMOP-LOD was measured as follows: DC-HMOP-LOD\u0026nbsp;was\u0026nbsp;dispersed into GSH (10 mM) and then stirred gently for 3, 7, and 14\u0026nbsp;days\u0026nbsp;at 37 \u0026deg;C. The morphology of these samples was detected by TEM and SEM.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e\u003cem\u003eIn vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;biodistribution and pharmacokinetics assays\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe standard curve of DC-HMOP-LOD-FITC was obtained using a UV\u0026ndash;Vis-NIR spectrophotometer (excitation and emission wavelengths of Ex 488 nm/Em 525 nm). Male BALB/c mice were subcutaneously injected with CT26 cells (1 \u0026times;10\u003csup\u003e6\u003c/sup\u003e, 50 \u0026mu;L in phosphate buffered saline (PBS) suspension). When the tumor volume was approximately 100 mm\u003csup\u003e3\u003c/sup\u003e, mice were intravenously injected with DC-HMOP-LOD-FITC (10 mg/mL, 150 \u0026mu;L). At\u0026nbsp;predetermined time points\u0026nbsp;(0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h), 10 \u0026mu;L of blood was collected by gently nicking the tail vein. At 24 h, tumor tissues and the main organs (heart, liver, spleen, lung, and kidney) were collected. The blood, tumor tissues, and main organs were digested with\u0026nbsp;lysis solution (400 \u0026mu;L), and then the blood was sonicated for 10 min. Tumor tissues and main organs were\u0026nbsp;ground into single-cell samples.\u0026nbsp;Finally,\u0026nbsp;the supernatants of\u0026nbsp;blood, tumor tissues, and main organs\u0026nbsp;were collected by centrifugation (8,000 rpm, 5 min). Fluorescence quantification of the supernatants was measured by a UV\u0026ndash;Vis-NIR spectrophotometer.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eLOD catalysis assay \u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe catalytic activity of LOD conjugated onto the surface of DC-HMOP-LOD was studied, including the consumption of lactate and O\u003csub\u003e2\u003c/sub\u003e and the production of\u0026nbsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The\u0026nbsp;lactate-PBS\u0026nbsp;solution\u0026nbsp;(500 \u0026mu;L, 3 mM)\u0026nbsp;was mixed with HMOP-DC, HMOP-LOD, or DC-HMOP-LOD (500 \u0026mu;L, 5 mg/mL) with slight shaking at 37 \u0026deg;C. The solution (10 \u0026mu;L) was collected\u0026nbsp;at different time points (0, 5, 10, 20, 40, 80, and 160 min)\u0026nbsp;for the quantification of lactate by the Lactic Acid Assay Kit\u0026nbsp;(KeyGEN Biotech Corp., Ltd, China, Jiangsu). The oxygen content in the mixed solution was investigated\u0026nbsp;at different time points (1, 2, 3, 4, and 5 min)\u0026nbsp;using a dissolved oxygen meter in real time (JPBJ-608, Shanghai INESA Scientific Instrument Co., Ltd, China). Furthermore, the different concentrations\u0026nbsp;(500 \u0026mu;L;\u0026nbsp;0, 1, 2, 3, 4 mM)\u0026nbsp;of the lactate-PBS\u0026nbsp;solution\u0026nbsp;were mixed with HMOP-DC, HMOP-LOD, or DC-HMOP-LOD (500 \u0026mu;L, 5 mg/mL) with slight shaking at 37 \u0026deg;C\u0026nbsp;for 2 h. The solution was collected\u0026nbsp;for the quantification of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e using an H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e Content Assay Kit (Solarbio, Beijing).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eMeasurement of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, lactate concentration, and pH \u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eCT26 cells were incubated in T75 flasks (3\u0026times;10\u003csup\u003e6\u003c/sup\u003e per well) for 24 h at 37 \u0026deg;C, and then the culture medium was replaced with fresh media containing HMOP-DC, HMOP-LOD, or DC-HMOP-LOD (200 or 400 \u0026mu;g/mL) for 24 h. To measure the extracellular lactate level,\u0026nbsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e level, and pH values, the cell supernatant was tested with a Lactic Acid Assay Kit (KeyGEN Biotech Corp., Ltd., China, Jiangsu),\u0026nbsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eContent Assay Kit, and a pH meter (Shanghai INESA Scientific Instrument Co., Ltd, China). The cells\u0026nbsp;were\u0026nbsp;collected to measure the intracellular lactate level using a Lactic Acid Assay Kit (Solarbio, Beijing).\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eMeasurement of intratumoral lactate concentration \u003cem\u003ein vivo\u003c/em\u003e\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eCT26 xenotransplanted\u0026nbsp;tumor mice with\u0026nbsp;a\u0026nbsp;tumor volume of approximately 100 mm\u003csup\u003e3\u003c/sup\u003e were divided into the following 4 groups:\u0026nbsp;control, HMOP-DC, HMOP-LOD, and DC-HMOP-LOD. 24 h after intravenous injection of 150 \u0026mu;L\u0026nbsp;PBS or different nanocomposites (10 mg/mL), 100 mg of tumor tissues was collected from sacrificed mice and digested to obtain single-cell samples. After the centrifugation of tissue homogenates, the supernatants were collected to measure the extracellular lactate level, and the sediments\u0026nbsp;were\u0026nbsp;used to investigate the intracellular lactate level with a Lactic Acid Content Assay Kit (Solarbio, Beijing).\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eIntracellular uptake of HMOP\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eIntracellular endocytosis was detected by confocal laser scanning microscopy (Nikon, Japan) and flow cytometry (BD FACS Vantage SE). CT26 cells were cultured in CLSM dishes (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well) for 24 h, and then the medium was replaced with 200 \u0026mu;g/mL DC-HMOP-LOD-FITC dispersed in serum-free medium.\u0026nbsp;After different incubation\u0026nbsp;times\u0026nbsp;(2, 4, 6, 8, and 24 h), the cells were rinsed with PBS, fixed with 4 % polyformaldehyde for 15 min, and then stained with DAPI for 15 min before confocal laser scanning microscopy (CLSM) observation. For the flow cytometry analysis, cells were seeded in 6-well plates (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well) for 24 h. After incubation with DC-HMOP-LOD-Cy5.5 for 2, 4, 6, 8, and 24 h,\u0026nbsp;the cells were digested with trypsin, suspended in PBS, and analyzed by flow cytometry.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;therapeutic efficacy and safety of nanocomposites\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eTo examine the treatment efficacy of DC-HMOP-LOD \u003cem\u003ein vitro\u003c/em\u003e, CT26 cells cultured in 96-well plates (5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per well) were divided into control, HMOP-DC, HMOP-LOD, and DC-HMOP-LOD groups. Then, the medium in the plates was replaced with fresh medium containing the\u0026nbsp;corresponding nanocomposites (100, 200, 300, and 400 \u0026mu;g/mL). 24 h later, a CCK-8 kit was used to assess CT26 cell viability. Similarly, the cells were cultured in 6-well plates (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well) and then subjected to different nanocomposites (200 \u0026mu;g/mL) for 24h. After digestion and centrifugation, flow cytometry was conducted to evaluate apoptosis. For CLSM observation, the cells were seeded in CLSM dishes (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well) and subjected to different nanocomposites (200 \u0026mu;g/mL) for 24 h. Then, the cells were dyed with CAM (10 \u0026mu;L) and PI (10 \u0026mu;L) and finally visually observed by CLSM.\u0026nbsp;In addition, the cytotoxicity of HMOP (7.5\u0026minus;500 \u0026mu;g/mL) to CT26 cells was measured using the above cell counting kit-8 steps.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eImmune system activation\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eFirst, we evaluated the phenotypic polarization of RAW 264.7 Cells. CT26 cells were incubated in the upper chamber (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e per well), and RAW 264.7 cells were incubated in the lower chamber (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e per well) for 24 h. Then, the original medium of CT26 cells was removed, and fresh medium with different nanocomposites (200 \u0026mu;g/mL) was added. The medium was replaced with fresh medium containing interleukin-4 (IL)-4 (50 ng/ml). After 24 h of incubation, the RAW 264.7 cells were stained with PE-anti-CD86 and APC-anti-CD206 and then detected by\u0026nbsp;flow cytometry. For the evaluation of the immune response \u003cem\u003ein vivo\u003c/em\u003e, CT26 xenotransplanted tumor mice with a tumor volume of approximately 70 mm\u003csup\u003e3\u003c/sup\u003e were randomly divided into 4 groups: Control, HMOP-DC, HMOP-LOD, and DC-HMOP-LOD. Then, 150 \u0026mu;L\u0026nbsp;PBS or\u0026nbsp;different nanocomposites (10 mg/mL) was intravenously\u0026nbsp;administered on days 1, 3, 6, and 9. On the 10th day, the tumor tissues were digested to obtain single-cell samples. Then, the cell samples were stained with different antibodies (FITC-anti-CD11c, PE-anti-CD86, APC-anti-CD80, Cy5.5-anti-CD45, APC-anti-CD49b, FITC-anti-CD4, APC-anti-CD8, APC-anti-CD206, FITC-anti-\u0026nbsp;F4/80, Cy5.5-anti-Foxp3, Cy5.5-anti-CD11b, FITC-anti-Gr-1) and measured by\u0026nbsp;flow cytometry. In addition, blood was extracted from each mouse,\u0026nbsp;and plasma samples were obtained by centrifugation of the blood (8,000 rpm, 20 min). Then, the plasma samples were used to test cytokines (INF-\u0026gamma;, TNF-\u0026alpha;, IL-10, and IL-12) by corresponding ELISA kits (Huyu Biotechnology, Shanghai China).\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e\u003cem\u003eIn vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;synergistic therapeutic efficacy of DC-HMOP-LOD\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eCT26 xenotransplanted tumor mice were randomly divided into six groups: Control, HMOP-DC, HMOP-LOD, DC-HMOP-LOD, aPD1, and aPD1 + DC-HMOP-LOD. The mice were intravenously injected with PBS or different nanocomposites (10 mg/mL, 150 \u0026mu;L)\u0026nbsp;on days 1, 3, 6, and 9. Mice in the\u0026nbsp;aPD1 and aPD1 + DC-HMOP-LOD\u0026nbsp;groups were injected with aPD1 (25 \u0026mu;g/mouse) on days 2, 4, 7, and 10. Tumor volumes and mouse weights were measured, and\u0026nbsp;images of the mice were captured every 3 days. The percent survival was monitored for 40 days. Tumors were dissected 24 h after treatments\u0026nbsp;and then fixed with 4 % polyoxymethylene for pathological examination, including hematoxylin-eosin (H\u0026amp;E), proliferating cell nuclear antigen (PCNA), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The tumor inhibition rates\u0026nbsp;and the growth curves\u0026nbsp;were determined according to the tumor volumes. The safety of DC-HMOP-LOD was determined according to the mouse weights.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eBiosafety assay of DC-HMOP-LOD\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eTwenty-five na\u0026iuml;ve BALB/c mice were treated with DC-HMOP-LOD (10 mg/mL,\u0026nbsp;150 \u0026mu;L). At different time points (0, 1, 7, 14, 21, and 28 d), blood samples of mice were collected for blood panel analysis and biochemistry assays. H\u0026amp;E staining of major organs was conducted to observe the corresponding pathological toxicity.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eStatistical analysis in this paper was performed with SPSS 26.0 software. The quantitative data are presented as the mean \u0026plusmn; standard deviation. The significance of the data was compared using Student\u0026rsquo;s t-test (*P＜0.05, **P＜0.01).\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003ch2\u003e\u003cstrong\u003eSynthesis and characterization of DC-HMOP-LOD\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eAfter the success synthesis of DC-HMOP-LOD, TEM (Fig. 1a) and SEM (Fig. 1b) showed that DC-HMOP-LOD was spherical and homogeneous in size. The average diameter of DC-HMOP-LOD measured by dynamic light scattering was approximately 214.0 nm, with a polydispersity index of 0.059 (Fig. 1c), which enable the nanocomposites accumulate in tumors via the EPR effect. DC-HMOP-LOD is a shell-core structure that separately carries LOD in the shell and DC in the core. The spectrum of HMOP-DC showed a maximum absorption peak at approximately 274 nm. The EE and LC of DC was calculated to be 12.19 % and 40.65 wt %according to the linear correlation equation (Fig. S1a). The EE and LC of LOD was 16.92 % and 26.65 wt % according to the standard curve of the Bradford Protein Assay Kit (Fig. S1b). Additionally, the sediment of HMOP-DC presented milky white, whereas the sediment DC-HMOP-LOD was light yellow (Fig. S1c), indicating the successful conjugation of LOD onto the surface of HMOP-DC. Additionally, as shown in Fig. 1d, e, the area elemental mapping analysis and energy dispersive spectroscopy (EDS) spectrum revealed that the major elements (Si, O, S) were distributed peripherally and homogeneously in HMOP. Moreover, DC-HMOP-LOD could be biodegraded in the reductive TME with a high concentration of reduced GSH, because the disulfide bonds in the silica framework could be cleaved in the reductive TME. Thus, GSH-dependent responsive degradation of DC-HMOP-LOD was observed by TEM (Fig. 1f) and SEM (Fig. 1g). DC-HMOP-LOD was gradually degraded with the extension of mixture time and ultimately completely degraded, indicating the biosafety of nanocomposites that could be bio-degradable and eliminated through feces and urine\u0026nbsp;[23].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; Then, the pharmacokinetic behaviors of DC-HMOP-LOD-FITC were analyzed on CT26 tumor-bearing mice. The level of DC-HMOP-LOD-FITC in the blood dramatically decreased at 1-2 h (Fig. 1h). Massive accumulation in the liver, spleen, kidney and tumor (Fig. 1i) was observed at 24 h. The tumor accumulation may benefit from the EPR effect of nanocomposites. Our previous experiment found that mice injected with free LOD died within 48 h. This phenomenon was also found by Tang\u0026nbsp;[24]. The quick diffusion in blood and distribution in organs of free LOD may account for the phenomenon. The design of DC-HMOP-LOD could prevent the side effect of LOD, and lay foundation for the following applications. In summary, HMOP could deliver drugs in the tumor without the need for external energy input, which may enhance the treatment effect and prevent side effects.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eThe catalytic activity and lactate consumption of DC-HMOP-LOD\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eAfter the successful fabrication and characterization of DC-HMOP-LOD, the catalytic activity of LOD was further determined by measuring the generation of O\u003csub\u003e2\u003c/sub\u003e and\u0026nbsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and the consumption of lactate \u003cem\u003ein vitro\u003c/em\u003e. The lactate solution was mixed with HMOP, HMOP-DC, HMOP-LOD, and DC-HMOP-LOD, respectively. It was found that the concentrations of lactate (Fig. 2a) and O\u003csub\u003e2\u003c/sub\u003e (Fig. 2b) in the HMOP-LOD and DC-HMOP-LOD groups gradually decreased with time. The concentration of\u0026nbsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e increased in response to the enhanced concentration of lactate (Fig. 2c) and nanocomposites (Fig. 2d), verifying that LOD retained its high selectivity and catalytic efficiency after conjugation.\u003c/p\u003e\n\u003cp\u003eAfterwards, the synergistic lactate consumption of DC-HMOP-LOD was verified by measuring the concentration of intracellular and extracellular lactate \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. Compared with the control group (2.51 \u0026mu;mol/mL), the \u003cem\u003ein vitro\u003c/em\u003e intracellular lactate concentrations in the HMOP-LOD (0.05 \u0026mu;mol/mL) and DC-HMOP-LOD (0.07 \u0026mu;mol/mL) groups were decreased, whereas the concentration of intracellular lactate in the HMOP-DC groups (4.17 \u0026mu;mol/mL) was increased (Fig. 2g). The intracellular lactate showed the same tendency \u003cem\u003ein vivo\u003c/em\u003e (Fig. 2h). The intracellular lactate concentrations in the HMOP-LOD and DC-HMOP-LOD groups were decreased, whereas the concentration in the HMOP-DC group was increased. And the effect of DC-HMOP-LOD in regulating intracellular lactate was lower than that of HMOP-LOD group because of the effect of DC. It was demonstrated that LOD could be transported into the cell by HMOP and oxidized intracellular lactate. DC increased intracellular lactate due to the inhibition of lactate efflux by blocking lactate transporters on the cancer cell membrane.\u003c/p\u003e\n\u003cp\u003eThen, we measured the extracellular (i.e., TME) lactate concentrations. In comparison to the control group (15.36 \u0026mu;mol/mL)\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e, the extracellular lactate concentrations in the HMOP-DC group (13.26 \u0026mu;mol/mL) and HMOP-LOD (8.61 \u0026mu;mol/mL) group were decreased, especially in the DC-HMOP-LOD group (6.41 \u0026mu;mol/mL) (Fig. 2e), which resulted in a corresponding pH change in the cell supernatant (Fig. 2f). Furthermore, extracellular lactate \u003cem\u003ein vivo\u003c/em\u003e showed the same tendency (Fig. 2i). The extracellular lactate concentrations in the HMOP-DC, HMOP-LOD and DC-HMOP-LOD groups were decreased. In sum, the extracellular lactate concentrations in the HMOP-DC, HMOP-LOD, and DC-HMOP-LOD were decreased \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, which showed that treatment with DC-HMOP-LOD synergistically depleted extracellular lactate, leading to a pH change in the TME. Lactate and an acidic TME both play important roles in suppressing anticancer immunity\u0026nbsp;[25, 26], and thus we speculated that DC-HMOP-LOD could contribute to the restoration of anticancer immunity.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eCell uptake and cytotoxicity evaluation\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eTo ensure the DC could be transported into tumor cells, we explored whether DC-HMOP-LOD could be efficiently engulfed by tumor cells. Intracellular phagocytosis was visualized by CLSM and measured by flow cytometry. As shown in Fig. 3a, the intracellular green FL gradually increased as a result of the efficient uptake of DC-HMOP-LOD with the extension of incubation time. The flow cytometry results were in accordance with the CLSM observations (Fig. 3b), demonstrating the high affinity of DC-HMOP-LOD to tumor cells.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; Then, the antitumor effect of DC-HMOP-LOD \u003cem\u003ein vitro\u003c/em\u003e was measured. The cytotoxicity was evaluated by CCK-8. As shown in Fig. 3c, after various nanocomposite treatments for 24 h, the cell viabilities in the HMOP-LOD and DC-HMOP-LOD groups were approximately 33.05 % and 33.41 %, respectively. Cellular apoptosis detected by flow cytometry showed the same cytotoxicity. The cell damage rates were 80.90 % and 81.32 % (Fig. 3d) in the HMOP-LOD and DC-HMOP-LOD groups, respectively. From direct observation by CLSM (Fig. S2), massive quantities of dead cells were found in the HMOP-LOD and DC-HMOP-LOD groups, which indicated the antitumor efficacy of LOD. We verified that HMOP-LOD increased H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e \u003cem\u003ein vitro\u003c/em\u003e, which can cause oxidative damage and kill tumor cells. HMOP-DC had no obvious cytotoxicity, which was consistent with the results of Renner\u0026nbsp;[27]. Renner found that the viabilities of tumor cells and T cells were not affected by DC for 72 h. But reduction of lactate secretion by DC boosted T-cell-mediated killing in programmed cell death-ligand 1(PD-L1) knocked down melanoma cells, while T cells were not able to kill tumor cells without the treatment of DC.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eImmunosuppression mitigation\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eAfter the demonstration of lactate reduction of DC-HMOP-LOD, we further explored whether lactate depletion could relieve ITM and activate the immune response. Concretely, we measured the proportions of immune-promoting cells and inactivating immunosuppressive cells in tumors by flow cytometry analysis, and measured the levels of immune cytokines by ELISA.\u003c/p\u003e\n\u003cp\u003eDC-HMOP-LOD significantly increased the recruitment of immune-promoting cells. Dendritic cells can activate adaptive immune responses by activating na\u0026iuml;ve T cells and inducing antigen-specific T-cell-mediated immunity\u0026nbsp;[28]. Lactate suppresses this activation by impairing monocyte differentiation into dendritic cells\u0026nbsp;[29]. We found that HMOP-LOD caused moderate dendritic cell maturation (10.45 %), whereas HMOP-DC generated more maturation (17.30 %). DC-HMOP-LOD markedly facilitated the maturation of dendritic cells (23.13 %), which was 2.21-fold higher than that of the control group (Fig. 4a, d). CD8\u003csup\u003e+\u003c/sup\u003e cytotoxic T lymphocytes (CTLs) play important roles in antitumor immunity due to the selective recognition and killing of cancer cell\u0026nbsp;[30]. However, lactate impairs CTLs function by influencing respiratory activity, chemotaxis, and recruitment\u0026nbsp;[31]. In addition, lactate accumulation can inhibit glycolysis and decrease the motility of CD4\u003csup\u003e+\u003c/sup\u003e helper T cells\u0026nbsp;[32]. CD4\u003csup\u003e+\u003c/sup\u003e T cells can generate proinflammatory factors, including tumor necrosis factor (TNF) -\u0026alpha; and interferon (IFN)-\u0026gamma;, further activating the innate immune response. Herein, the increased proportions of both CD8\u003csup\u003e+\u003c/sup\u003e T cells and CD4\u003csup\u003e+\u003c/sup\u003e T cells in the HMOP-DC group and HMOP-LOD group showed superior activation for cellular immunity. Importantly, the highest proportion was found in the DC-HMOP-LOD group (CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells were 21.20 % and CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells were 31.37 %) (Fig. 4b, e, f). Additionally, NK cells, innate immune cells, exhibit an antitumor effect via effective cytolytic activity by generating granzymes, cytokines, and additional factors\u0026nbsp;[33]. Lactate can inhibit NK-cell activity and reduce the expression and production of IFN-\u0026gamma; in NK cells\u0026nbsp;[34]. Compared to untreated tumors in mice (3.17 %), NK cells recruitment was obviously induced by DC-HMOP-LOD (8.28 %). The proportions in the HMOP-DC group and the HMOP-LOD groups were 3.61 and 3.80 times higher, respectively, than the proportion in the control group (Fig. 4c, g). From the above results, it was substantiated that DC-HMOP-LOD markedly increased the activity and infiltration of immune-promoting cells and transformed immunosuppressive tumors into \u0026ldquo;hot\u0026rdquo; tumors through lactate reduction \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIn addition to immune stimulation, immunosuppression remission is also worthy of attention. TAMs\u0026nbsp;[35]\u0026nbsp;and MDSCs\u0026nbsp;[36]\u0026nbsp;are vital elements in the immunosuppressive TME. TAMs are sensitive to lactate, and lactate can polarize protumor M1-type TAMs to antitumor M2-type TAMs\u0026nbsp;[37]. M2-type TAMs are related to tumor growth, metastasis, and immunosuppression because M2-type TAMs can not only directly target tumor cells through phagocytosis or the release of cytokines (TNF-\u0026alpha;, \u003cem\u003eetc.\u003c/em\u003e) but can also indirectly recruit and activate other immune effector cells (IFN-\u0026gamma;, interleukin (IL) -12, \u003cem\u003eetc.\u003c/em\u003e)\u0026nbsp;[38]. M2-type TAMs can release multiple growth factors and cytokines (IL-10, \u003cem\u003eetc.\u003c/em\u003e) to result in immune suppression\u0026nbsp;[39]. Furthermore, M1-type TAMs exhibit decreased viability compared with M2-type TAMs at lower pH values\u0026nbsp;[40]. Herein, we demonstrated that DC-HMOP-LOD could regulate macrophage phenotype polarization \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. After RAW 264.7 Cells were treated with DC-HMOP-LOD (Fig. 5a, b), M2-type TAMs (3.35 %) were polarized to M1-type TAMs (76.9 %). The results \u003cem\u003ein vivo\u003c/em\u003e showed the same trend (Fig. 5 c, d, e). The percentage of M1-type TAMs increased (58.03 %), and the competency of M2-type TAMs decreased (20.17 %) (Fig. 5f, g) in the tumor after treatment with DC-HMOP-LOD. From the above results, it was verified that DC-HMOP-LOD had an effective impact on macrophage phenotype, skewing differentiation from protumor M2-type TAMs to the antitumor M1 phenotype.\u003c/p\u003e\n\u003cp\u003eMDSCs normally differentiate into TAMs, dendritic cells, and granulocytes. However, lactate impairs this differentiation, resulting in MDSCs accumulation. This accumulation can stimulate other immnosuppressive cells, inhibit lymphocyte homing, and decrease T-cell activity\u0026nbsp;[41, 42]. Furthermore, MuDSCs increase PD-L1 expression in the acidic TME\u0026nbsp;[43]. As shown in Fig. 6a, b, the lowest MDSCs proportion appeared in the DC-HMOP-LOD group, with a reduction of 5.02 % compared with the control group. TAMs and MDSCs levels in tumor tissues demonstrate the efficiency of DC-HMOP-LOD in recovering the immunosuppressive TME.\u003c/p\u003e\n\u003cp\u003eImmune cytokines in serum also changed after the treatment of DC-HMOP-LOD. Here, we measured the levels of TNF-\u0026alpha; (Fig. 6c), IFN-\u0026gamma; (Fig. 6d), IL-10 (Fig. 6e), and IL-12 (Fig. 6f) by ELISA. IFN-\u0026gamma; and TNF-\u0026alpha; are both critical cytokines in cellular immunity that indicate the activation of effector T cells. Although both HMOP-DC and HMOP-LOD were able to increase the secretion of TNF-\u0026alpha; and IFN-\u0026gamma;, the levels induced by DC-HMOP-LOD (IFN-\u0026gamma; 2.27 pg/mL and TNF-\u0026alpha; 2.28 ng/mL) were obviously higher than the concentrations of IFN-\u0026gamma; (1.32 pg/mL) and TNF-\u0026alpha; (1.37 ng/mL) in the control group. IL-10 is secreted by M2-type TAMs to suppress the immune response and promote tumor invasion and metastasis. IL-12 is secreted by M1-type TAMs to promote the immune response and inflammation\u0026nbsp;[44-46]. IL-12 can also inhibit the differentiation of Tregs from na\u0026iuml;ve T cells\u0026nbsp;[47]. Lactate can increase the expression of IL-10 and decrease the generation of IL-12, resulting in reduced inflammation and the promotion of immune tolerance\u0026nbsp;[48]. DC-HMOP-LOD reduced the levels of IL-10 and IL-12 by consuming lactate in the TME. The secretion of IL-10 (1.46 ng/mL) was significantly decreased, whereas the secretion of IL-12 (1.81 ng/mL) was increased in the DC-HMOP-LOD group. The regulatory effects of HMOP-DC and HMOP-LOD on immune cells were unsatisfactory due to their inability to produce excessive lactic acid in the TME. The synergistic reduction of lactate by DC-HMOP-LOD had an excellent ability to relieve the ITM and activate the immune response.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eAntitumor capacity\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eBased on the mitigated immunosuppression by DC-HMOP-LOD, we speculated that the efficacy of aPD1 immunotherapy could be improved by\u0026nbsp;DC-HMOP-LOD. As shown in\u0026nbsp;Fig.\u0026nbsp;7a,\u0026nbsp;b, and\u0026nbsp;Fig.\u0026nbsp;S3a, the tumors in the\u0026nbsp;control groups grew rapidly.\u0026nbsp;DC-HMOP-LOD and aPD1 treatment exhibited an unsatisfactory tumor inhibition effect, especially after the cessation of administration. The efficiency of\u0026nbsp;HMOP-LOD and HMOP-DC\u0026nbsp;was even worse than that of the aPD1 group. However,\u0026nbsp;the aPD1 + DC-HMOP-LOD group performed best in suppressing tumor growth. Additionally, the mice in the control group all died on the 16th day.\u0026nbsp;LOD and DC could not induce a sustained antitumor response. The tumors progressed, and the animals ultimately died in the HMOP-DC and HMOP-LOD groups after cessation of injection.\u0026nbsp;Compared with the mice\u0026nbsp;in\u0026nbsp;the HMOP-DC, HMOP-LOD,\u0026nbsp;and\u0026nbsp;aPD1 groups that died rapidly before the 37th day, the survival was slightly extended in the DC-HMOP-LOD group. However, much longer survival\u0026nbsp;was observed in the\u0026nbsp;aPD1 +\u0026nbsp;DC-HMOP-LOD group\u0026nbsp;(Fig.\u0026nbsp;7c).\u0026nbsp;With the extension of treatment time,\u0026nbsp;representative photographs of mice and tumor growth curves of every mouse are shown in\u0026nbsp;Fig.\u0026nbsp;S 3b.\u003c/p\u003e\n\u003cp\u003eThe results of H\u0026amp;E, PCNA, and TUNEL staining further indicated satisfactory therapeutic outcomes of synergistic therapy. To evaluate necrosis and apoptosis of the tumor cells after various treatments, H\u0026amp;E and TUNEL staining of tumors were\u0026nbsp;performed. H\u0026amp;E staining\u0026nbsp;(Fig.\u0026nbsp;7d)\u0026nbsp;showed\u0026nbsp;abundant necrosis of tumor cells in the aPD1 + DC-HMOP-LOD group, which was much more remarkable than that in the single treatment groups.\u0026nbsp;The\u0026nbsp;apoptosis\u0026nbsp;of tumor cells\u0026nbsp;revealed by\u0026nbsp;TUNEL staining (Fig.\u0026nbsp;7e)\u0026nbsp;showed the same trend as the\u0026nbsp;H\u0026amp;E\u0026nbsp;staining.\u0026nbsp;PCNA staining (Fig.\u0026nbsp;7f) results evaluated the\u0026nbsp;proliferative activities of cancer cells (stained brown)\u0026nbsp;and\u0026nbsp;showed a lower proliferation index after treatments, especially in the aPD1 + DC-HMOP-LOD group.\u003c/p\u003e\n\u003cp\u003eMCTs have crucial roles in glycolytic cells because the glycolytic flux that MCTs provide is important for tumor cells to supply metabolic intermediates. Therefore, DC can limit glucose metabolism and impair the proliferation and survival of tumor cells\u0026nbsp;[49]. This ability has been demonstrated in a clinical trial, and both Pantziarka\u0026nbsp;[50]\u0026nbsp;and Singer\u0026nbsp;[51]\u0026nbsp;showed that DC could augment the response to checkpoint inhibition by upregulating IFN-\u0026gamma; expression in T cells and NK cells.\u0026nbsp;In addition, the reactive oxygen species level was elevated significantly due to the catalysis of LOD, which could cause cell apoptosis. However, compared with the high tumor inhibition activity \u003cem\u003ein vitro\u003c/em\u003e, HMOP-LOD only had a moderate antitumor effect \u003cem\u003ein vivo\u003c/em\u003e, which may result from the heterogeneous distribution of intratumoral lactate and oxygen. The moderate antitumor activity and immunosuppression mitigation also indicated the necessary combination of LOD and DC.\u0026nbsp;The consumption of lactate in the TME can directly suppress tumor growth by suppressing cell migration, metastasis, and angiogenesis.\u0026nbsp;Furthermore, it has been shown in clinical trials that reducing lactate levels in the TME\u0026nbsp;can reduce tumor burden indirectly by promoting\u0026nbsp;immunotherapeutic efficacy\u0026nbsp;[52].\u0026nbsp;Herein, we also demonstrated that strategies using\u0026nbsp;DC-HMOP-LOD\u0026nbsp;effectively improve the antitumor activity of\u0026nbsp;aPD1\u0026nbsp;treatment primarily by modulating the ITM.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eSafety\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;of DC-HMOP-LOD\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe biosafety of nanocomposites is a key factor for ensuring the feasibility of clinical transformation. Therefore, we investigated the\u0026nbsp;safety of DC-HMOP-LOD \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eIn vitro\u003c/em\u003e, over 85 % of cells remained alive after incubation with HMOP for 24 h (Fig.\u0026nbsp;S4a).\u0026nbsp;At the endpoint of the treatment period \u003cem\u003ein vivo\u003c/em\u003e, negligible fluctuations were shown in the average body weights of mice from all groups (Fig. S4b). Additionally, after administration of DC-HMOP-LOD, no obvious abnormality was seen in blood panel analysis or biochemistry assays (Fig. S4c), and no detectable change was found in histopathology for major organs (Fig. S4d). These results demonstrated that DC-HMOP-LOD showed desirable biocompatibility and therapeutic safety.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we successfully designed DC-HMOP-LOD, which safely accumulated and locally released LOD and DC in the tumor. DC effectively hindered the transmission of lactate, and LOD directly oxidized lactate, collaboratively depleting lactate in the TME. The synergetic depletion reversed the ITM by activating antitumor immune responses and inhibiting negative regulators of innate as well as adaptive immune cells. Therefore, DC-HMOP-LOD induced necrosis and apoptosis, decreased the proliferative activities of the tumor cells, suppressed tumor growth, and extended the survival of mice, indicating that DC-HMOP-LOD could improve the antitumor effects of aPD1. Thus, the intra/extracellular lactate exhaustion by DC-HMOP-LOD may overcome the existing restrictions of ICB therapy to some extent.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAbbreviations: ICB, Immune checkpoint blockade; ITM, immunosuppressive tumor microenvironment; MCTs, monocarboxylate transporters; HMOP, PEGylated hollow mesoporous organosilicon; DC, diclofenac; LOD, lactate oxidase; TME, tumor microenvironment; NK, natural killer; MDSCs, myeloid-derived suppressor cells; TAMs, tumor-associated macrophages; Tregs, regulatory T cells; EPR, enhanced permeability and retention; aPD1, anti-PD1; TEM, transmission electron microscope; SEM, scanning electron microscope; EE, encapsulation efficiency; LC, loading capacity ; EDS, energy dispersive spectroscopy; GSH, glutathione; CLSM, confocal laser scanning microscopy; CCK-8, cell counting kit-8; PD-L1, programmed cell death-ligand 1; ELISA, enzyme linked immunosorbent assay; CTLs, cytotoxic T lymphocytes; TNF-\u0026alpha;, tumor necrosis factor-\u0026alpha;; interferon-\u0026gamma;, IFN -\u0026gamma;; H\u0026amp;E, hematoxylin-eosin; PCNA, proliferating cell nuclear antigen; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal experiments were approved and performed in accordance with the guidelines of the Institutional Animal Care and Animal Ethics Committee of Chongqing Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial and non-financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Research Incubation Project of the Third Affiliated Hospital of Chongqing Medical University (KY08025), Basic Research and Frontier Exploration Project of Yuzhong District of Chongqing (20190107), and Chongqing Talent Plan (cstc2021ycjh-bgzxm0077).\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYDT JH, and RL conceived and designed experiments; YDT, CMZ and HWX conducted the experiments and obtained the results; XJL and JZ provided guidance for data analysis and instrumental support; YDT and XYT analyzed data; YDT JH and LZ wrote the manuscript; BS contributed to manuscript revision; RL offered financial support and provided final approval of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u0026nbsp;\u003c/sup\u003eDepartment of Ultrasonography, The Third Affiliated Hospital of Chongqing Medical University, Chongqing 401120, People\u0026rsquo;s Republic of China. \u003csup\u003e2\u0026nbsp;\u003c/sup\u003eUltrasound Department, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400010, People\u0026rsquo;s Republic of China. \u003csup\u003e3\u003c/sup\u003eChongqing Key Laboratory of Ultrasound Molecular Imaging \u0026amp; Institute of Ultrasound Imaging, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400016, People\u0026rsquo;s Republic of China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eO'Donnell JS, Long GV, Scolyer RA, Teng MW, Smyth MJ. 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Cell Metab. 2018;27(5):977\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"GSH-responsive hollow mesoporous organosilicon, Lactate, Immunosuppressive tumor microenvironment, Immunotherapy","lastPublishedDoi":"10.21203/rs.3.rs-2168046/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2168046/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eImmune checkpoint blockade (ICB) therapies have reshaped tumor treatment by activating the antitumor immune response. However, the antitumor effect of ICB is seriously restricted by the immunosuppressive tumor microenvironment (ITM). A variety of strategies to alleviate the ITM have been investigated. Direct regulation of lactate metabolism in tumor microenvironment holds promise for ITM modulation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eGlutathione (GSH) -responsive hollow mesoporous organosilicon (HMOP) was successfully fabricated, with monocarboxylate transporter 1/4 inhibitor (diclofenac, DC) and lactate oxidase (LOD) were loaded in/onto the HMOP (designed as DC-HMOP-LOD). DC-HMOP-LOD could spontaneously be biodegraded in tumor microenvironment due to disulfide bonds, and then DC/LOD could be released to exhaust intra/extracellular lactate. Consequently, lactate depletion induced an immunocompetent tumor microenvironment by activating immune-promoting cells including dendritic cells, CD4\u003csup\u003e+\u003c/sup\u003e T cells, CD8\u003csup\u003e+\u003c/sup\u003e T cells, and natural killer cells, and inactivating immunosuppressive cells containing tumor-associated macrophages and myeloid-derived suppressor cells, ultimately strengthening the antitumor effect of ICB therapy.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eDC-HMOP-LOD effectively hindered the transmission of lactate and directly oxidized lactate, collaboratively depleting lactate in the TME. The synergetic depletion reversed the ITM and could improve the antitumor effects of aPD1-based immunotherapy.\u003c/p\u003e","manuscriptTitle":"GSH-responsive Nanoplatform for Intra/Extracellular Lactate Exhaustion to Enhance Antitumor Immunotherapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-10-19 14:28:17","doi":"10.21203/rs.3.rs-2168046/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"70b7cc9c-39c1-432e-b650-1465b304477d","owner":[],"postedDate":"October 19th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-11-28T22:29:12+00:00","versionOfRecord":[],"versionCreatedAt":"2022-10-19 14:28:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2168046","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2168046","identity":"rs-2168046","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}