Coordination Polymers Integrating Metalloimmunology with Immune Modulation to Elicit Robust Cancer Chemoimmunotherapy

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Oct 2021Coordination Polymers Integrating Metalloimmunology with Immune Modulation to Elicit Robust Cancer Chemoimmunotherapy Longlong Tian, Yujie Zhu, Jun Xu, Ming Shao, Wenjun Zhu, Zhisheng Xiao, Qian Chen and Zhuang Liu Longlong Tian Institute of Molecular Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127 , Yujie Zhu Institute of Functional Nano and Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Soochow University, Suzhou, Jiangsu 215123 , Jun Xu Institute of Functional Nano and Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Soochow University, Suzhou, Jiangsu 215123 , Ming Shao Institute of Functional Nano and Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Soochow University, Suzhou, Jiangsu 215123 , Wenjun Zhu Institute of Functional Nano and Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Soochow University, Suzhou, Jiangsu 215123 , Zhisheng Xiao Institute of Functional Nano and Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Soochow University, Suzhou, Jiangsu 215123 , Qian Chen Institute of Functional Nano and Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Soochow University, Suzhou, Jiangsu 215123 and Zhuang Liu *Corresponding author: E-mail Address: [email protected] Institute of Functional Nano and Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Soochow University, Suzhou, Jiangsu 215123 https://doi.org/10.31635/ccschem.020.202000539 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail Nanoscale coordination polymers (NCPs) constructed by metal ions and organic ligands via metal–ligand bonds have attracted great attention for their biomedical application. Herein, a new type of NCP is constructed from Zn-ions and 4-phenylimidazole (PI), the latter of which is an inhibitor of indoleamine 2,3-dioxygenase (IDO) and acts as an organic bridging ligand via Zn–N coordination bonds. After modification with polyethylene glycol (PEG) and further loading with an immunogenic cell death (ICD) inducing therapeutic, doxorubicin (DOX), the metal-ligand coordination complex, ZnPI-PEG/DOX NCPs obtained, could be utilized as an effective cancer chemoimmunotherapy. In this system, Zn-ions cell membrane surface exposure of calreticulin (CRT) to enhance DOX-triggered ICD of tumor cells, while PI reversed the immunosuppressive tumor microenvironment (TME) by inhibiting IDO. Such synergistic action effects dramatically elicited antitumor immune responses, leading to inhibition of tumor growth, along with facilitation of long-term immune memory effect. Our work presents a smart design of NCPs as a "carrier-free" drug delivery system that enabled highly effective chemoimmunotherapy in cancer treatment. Download figure Download PowerPoint Introduction Cancer immunotherapy has achieved incredible breakthroughs in recent years and is considered a promising method for curing cancer.1,2 This approach eliminates tumors through activating host antitumor immune responses in which tumor-infiltrating T lymphocytes (TILs) play an essential role.3,4 However, the immunosuppressive tumor microenvironment (TME), on the one hand, inhibits TILs recruitment and stimulation.5,6 Thus, TILs-secreted cytokine interferon-gamma (IFN-γ) would lead to the upregulation of endogenous indoleamine 2,3-dioxygenase (IDO), an enzyme that catalyzes the main pathway of tryptophan (Trp) metabolism to kynurenine (Kyn).7–9 While Trp exhaustion would, in turn, suppress TILs' activation, Kyn would promote the intratumoral recruitment of the immunosuppressive regulatory T (Treg) cells to worsen the TME immunosuppression further.10–13 Hence, it has been reported that IDO inhibition could overcome immune suppression to enhance antitumor immune responses.14–16 Nanoscale coordination polymers (NCPs) are constructed via metal–ligand coordination bonds and have attracted significant attention in the area of nanomedicine.17–23 Taking the advantages of tunable types of metal ions and organic ligands, NCPs could act as programmable platforms.24,25 Previous studies have focused on the physicochemical properties of the metal units in coordination polymers such as magnetic properties for magnetic resonance imaging (MRI) and Fenton reaction activity for enhanced cancer chemodynamic therapy.26–29 However, metal ions detached from the intrinsically biodegradable coordination polymers would also take part in life processes, especially those of the immune system.30–32 For instance, it has recently been reported that manganese ions (Mn+2) and Zn-ions (Zn+2) could greatly enhance the cyclic guanosine monophosphate–adenosine monophosphate synthase–stimulator of IFN genes (cyclic GMP–AMP synthase–stimulator of interferon genes) pathway and subsequently trigger multifaceted type I IFN-driven inflammatory responses to activate innate immunity.33,34 The role of metal elements in the immune system's control, namely, metalloimmunology, is one of the future directions in metal-ion-activated cancer immunotherapy.35,36 Coordination polymers inheriting metalloimmunology exhibited great potential in cancer immunotherapy. In this work, to overcome the immunosuppressive TME, a new type of coordination polymer was constructed with Zn-ions and IDO inhibitor 4-phenylimidazole (PI) via Zn–N coordination bond.37–39 Zn-ions could induce cell membrane surface exposure of calreticulin (CRT), an "eat-me" signal that promotes phagocytosis of dying tumor cells and maturation of antigen-presenting cells (APCs).40–42 Besides, nanoscale Zn–PI coordination polymers (namely ZnPI) inheriting IDO inhibition activity could inhibit Trp metabolism, and subsequently decrease intratumoral Treg cells recruitment and increase TILs infiltration, resulting in reversing the TME from immunosuppression to immunostimulatory state. Then doxorubicin (DOX), loaded efficiently in ZnPI, was used as a chemotherapeutic model drug to induce immunogenic cell death (ICD)43,44 by the entire ZnPI-PEG/DOX nanoparticles' metal-ligand coordination complex. After intravenous injection, ZnPI-PEG/DOX amplified DOX-induced ICD and dramatically elicited potent antitumor immune response duo, as follows: (1) enhanced CRT exposure by Zn-ions and (2) reversed the immunosuppressive TME after IDO inhibition. Moreover, in mice in which the tumors were eliminated after ZnPI-PEG/DOX treatment, a strong long-term immune memory effect was noted that protected the mice from tumor rechallenge. Our work, thus, involved the development of a unique coordination polymer that combined metalloimmunology with immune TME modulation to induce robust cancer immunochemotherapy (Scheme 1). Scheme 1 | Schematic representation of ZnPI integrating metalloimmunology with immune modulation to elicit robust cancer immunotherapy. ZnPI was synthesized through a solvothermal method with Zn-ions and IDO inhibitor PI. Beyond as an efficient drug delivery system, ZnPI could release Zn-ions to induce cell member surface exposure of CRT for enhancing chemotherapy-induced ICD, and PI to reverse the immunosuppressive TME via IDO inhibition. Such effects acting synergistically could dramatically elicit antitumor immune responses in inhibiting tumor growth and facilitating the long-term immune memory effect. Download figure Download PowerPoint Experimental Methods Synthesis and modification of ZnPI Precisely 0.0625 mmol/0.125 mmol/0.25 mmol Zn nitrate hexahydrate in 5 mL methanol and 0.5 mmol PI in 5 mL N,N-dimethylformamide (DMF) were added in 20 mL Teflon-sealed autoclave reactor. The well-mixed solution was kept in a 75 °C oven for 72 h. The product was collected by centrifugation (4000g for 5 min) and washed with DMF and methanol. After drying under a vacuum at 75 °C, the ZnPI product was collected and weighed. The Zn and PI contents of the as-products were measured by inductively coupled plasma optical emission spectrometer (ICP-OES) and UV–vis spectrometry, respectively. The product was further characterized by transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS). Precisely 10 mg of ZnPI was dispersed in 20 mL ethanol was added with 1 mg 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) in 1 mL chloroform. The well-mixed solution was ultrasonicated for 15 min. The DOPA-coated ZnPI was collected by centrifugation, washed with ethanol, and redispersed in chloroform. Then 1,2-Dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (DSPE-PEG 5000) were added with vigorous stirring overnight. After rotary evaporation, deionized water was added to obtain well-dispersed ZnPI-PEG. Zn-ions-induced CRT exposure Cancer cells (colon carcinoma CT26 or human breast carcinoma 4T1 cell lines) were incubated with 100 μM of each of the following reagents for 24 h: CaCl2, FeCl2, MnCl2, NiCl2, CuCl2, or ZnCl2. Oxaliplatin (OXA; 5 μM) was used as a positive control. Then cells were washed three times with fresh phosphate-buffered saline (PBS) and stained with Alexa Fluor 488-labeled CRT antibody (ab196158; Abcam, Shanghai, China) for flow cytometry analysis. In vitro IDO inhibition CT26 cells or 4T1 cells were seeded at a density of 5 × 103 cells per well in a 96-well plate and grew overnight. About 100 μL 100 ng/mL IFN-γ was added to each well. Meanwhile, 100 μL ZnPI-PEG or free PI at varying concentrations was added to each set of wells. After 48 h of incubation, 100 μL of cell culture supernatant was transferred from each well into a new 96-well plate. About 100 μL of 70% trichloroacetic acid (TCA) was added to each well, and the mixture was incubated at 50 °C for 4 h to hydrolyze N-formylkynurenine to Kyn. The Kyn concentration was measured at 490 nm after adding an equal volume of Ehrlich reagent (2% p-dimethylamino-benzaldehyde w/v in acetic acid). In vivo IDO inhibition Female Balb/c mice were purchased from Nanjing Peng Sheng Biological Technology Co., Ltd (Nanjing, China) and used under protocols approved by Soochow University Laboratory Animal Center (Jiangsu, China). Mice bearing subcutaneous CT26 tumors (about 100 mm3) were randomized into four groups (n = 5), as follows: PBS vehicle group, the ZIF-8 group, the free PI group, and ZnPI-PEG group. Then the mice received intravenous (i.v.) injection of the substances (10 mg/kg, except the PBS group) indicated in each group on day 0 and 3 (two times). Two days after the second treatment (day 5), the mice were sacrificed by cervical dislocation, and the tumors were harvested, along with serum collection from each group. Tumor homogenates for each mice group were prepared. Either the sera or tumor homogenates were mixed with water/TCA/acetonitrile (62%/30%/8%) solution and incubated at 50 °C for 4 h to fully hydrolyze N-formylkynurenine to Kyn. After centrifugation, the Kyn/Trp ratios in the supernatants were examined by high-performance liquid chromatography with an ultraviolet (HPLC-UV) detection method (360 nm for Kyn, 254 nm Ex/404 nm Em for Trp). Single-cell suspensions were prepared and stained for flow cytometry analysis of Treg cells (CD3, CD4, and Foxp3) and T cells (CD3, CD4, and CD8). Fresh tumors were embedded in tissue freezing medium (opti-mum cutting temperature compound [OCT]) at −80 °C for frozen sectioning (8 μm). The tumor slices were stained with CD8a or Foxp3 antibody, and confocal images were gathered using Zeiss confocal fluorescence microscopy (Carl Zeiss Shanghai Co. Ltd., Shanghai, China). ZnPI complex drug loading and release ZnPI-PEG solution was mixed with varying concentrations of DOX hydrochloride in saline. After incubation with stirring in the dark for 24 h, ZnPI-PEG/DOX was collected by ultrafiltration to remove free DOX. The free DOX was detected at 490 nm. The loading capacity was calculated using the formula (DOXadd − DOXfree)/ZnPIadd. The ZnPI-PEG was dissolved in PBS at varying pH (7.4, 6.8, and 5.5); the released PI was collected by ultrafiltration and measured at 260 nm. Further, the ZnPI-PEG/DOX was dissolved in PBS at varying pH (7.4, 6.8, and 5), and the released DOX was collected by ultrafiltration and measured at 490 nm. Cellular uptake of ZnPI-PEG/DOX CT26 cells were seeded at a density of 20,000 cells per well in a 24-well plate and grew overnight. Adhered CT26 cells were incubated with ZnPI-PEG/DOX or free DOX for 0, 4, and 12 h, and then with fresh lysosome staining solution. Next, the cells were washed three times with a fresh PBS buffer, fixed with formaldehyde, and stained with 4′,6-diamidino-2-phenylindole (DAPI). After protected with Fluoromount (Sigma-Aldrich, Shanghai, China), the samples were imaged by a Zeiss microscopy. DOX-induced ICD CT26 cells were seeded at a density of 2000 cells per well in a 24-well plate and incubated separately with blank medium or free DOX or ZnPI-PEG or ZnPI-PEG/DOX for 24 h. Adenosine triphosphate (ATP) concentrations in supernatants of medium were tested using ATP assay kit (Beyotime, Beijing, China), per the manufacturer's instructions. CT26 cells were washed three times with a fresh PBS buffer, fixed with formaldehyde, and stained with CRT antibody, followed by DAPI. For immunohistochemical staining of high mobility group box protein 1 (HMGB1) as ICD marker, cells were pretreated with Triton X-100 to permeabilize the membrane. The slides were mounted with the aqueous medium Fluoromount (Sigma-Aldrich) for preservation, and the samples were imaged with a Zeiss microscopy. Blood circulation and biodistribution of the ZnPI complex drug Healthy mice received i.v. injection with ZnPI-PEG/DOX or free DOX (5 mg/kg). At different time points postinjection, blood samples were drawn from the eyes, and the red blood cells (RBCs) were hemolyzed with the RBC lysis buffer. DOX concentration was measured by its fluorescence emission. Further, mice bearing subcutaneous CT26 tumors (about 100 mm3) received i.v. injection with ZnPI-PEG/DOX or free DOX (5 mg/kg). At 24 h postinjection, mice were sacrificed to remove the main organs (heart, liver, kidneys, lungs, and stomach) and the tumors were collected and weighed. After homogenation with lysis buffer, the DOX concentration was measured by fluorescence emission, as described earlier. Meanwhile, half of each tumor was embedded in tissue freezing medium (OCT) at −80 °C for frozen section preparation. Subsequently, the tumor slices were stained with anti-CD31 and DAPI. The localization of the blood vessels and DOX were visualized by Zeiss confocal microscopy. In vivo cancer therapy Mice bearing subcutaneous CT26 tumors (∼100 mm3) were randomly divided into four groups and treated as follows: Groups (1) PBS, (2) Free DOX, (3) ZnPI-PEG, or (4) ZnPI-PEG/DOX, on day 0, 3, and 6. Tumor volumes were measured with a caliper and calculated using the formula: length2 × width/2. Mice body weights were measured with a balance. According to animal ethics, mice were euthanized when their tumor volumes reached 1500 mm3. On the ninth day, four mice in each group were sacrificed, and the main organs were collected for hematoxylin and eosin (H&E) staining. Kyn/Trp ratios were measured using HPLC-UV, as indicated above. Single-cell tumor suspensions were prepared and stained for flow cytometry analysis of CRT+ cells, Treg cells [CD3-fluorescein isothiocyanate (FITC), CD4-PE, and Foxp3-APC], and T cells (CD3-FITC, CD4-PE, and CD8-APC). Half of each tumor size was embedded in tissue freezing medium (OCT) at −80 °C for frozen section preparation. Tumor slices were stained with HMGB1 and DAPI, and confocal microscopy images were gathered. Immunological memory effect The cured mice from the ZnPI-PEG/DOX treated group were gathered on the 39th day after initial therapy, and blood was collected from the mice eyes. Then the white blood cells (WBCs) or memory T cells were measured in the samples after RBC lysis using an RBC lysis buffer and staining with anti-CD3-FITC, -CD8-PerCP, -CD62L-APC, and -CD44-PE antibodies for flow cytometry analysis. On the 40th day after the initial therapy, mice received the second tumor inoculation. Tumor volumes were measured with a caliper. On day 7, after the second tumor inoculation, peripheral blood was collected from mice eyes, and sera were collected by centrifugation for enzyme-linked immune-sorbent assay (ELISA). Results and Discussion Synthesis of ZnPI In our work, ZnPI was synthesized via the solvothermal method (Figure 1a). To optimize ZnPI, different PI to Zn mole ratios (8, 4, and 2) were added during synthesis. The TEM image showed that the higher PI/Zn mole ratios during synthesis resulted in the formation of ZnPI with smaller sizes (Figure 1b). The Zn and PI contents in products were measured by ICP-OES and UV–vis spectrometer, respectively. In the obtained products, the PI to Zn mole ratios was calculated to be 3.19, 2.06, and 1.97 (Figure 1c), respectively. The yields based on PI were estimated to be 13.9%, 43.8%, and 67.2% (Figure 1d), respectively. Owning the proper size ∼80 nm ( Supporting Information Figure S1), as well as the yield obtained, the PI to Zn mole ratio of four was chosen during synthesis for further experiments. The energy-dispersive X-ray spectrometry (EDXS) mapping revealed ZnPI complex was composed of Zn, C, and N elements (Figure 1e), which was confirmed further by XPS (Figure 1f). Figure 1 | Material design, synthesis, and characterization of ZnPI-PEG. (a) Schematic illustration of material construction and surface modification. (b) TEM images of Zn–PI coordination polymer synthesized at varying PI/Zn stoichiometries. (c) PI/Zn mole ratios in products of different PI/Zn stoichiometries. (d) Zn–PI coordination polymer products yield based on PI at different PI/Zn stoichiometries. (e) EDXS-mapping images and (f) XPS spectrum of ZnPI. (g) Dynamic light scattering of ZnPI-PEG in saline. (h) PI release from ZnPI-PEG under different pH values. (i and j) TEM images of ZnPI-PEG after 3 h incubation within weak acidic buffers (pH 6.8 and 5.5). Download figure Download PowerPoint After surface modification with PEG,45 ZnPI-PEG exhibited well-dispersion in saline with a hydrodynamic diameter at ∼90 nm (Figure 1g). The pH-sensitive property of ZnPI-PEG was tested in PBS at varying pH values. Under physiological conditions (pH 7.4), ZnPI-PEG appeared to be stable without PI release (Figure 1h). On the contrary, in acidic buffers, ZnPI-PEG decomposed gradually with the release of PI (Figures 1i and 1j). Considering that the pKa of imidazole in PI was ∼6.953, the protonation process of nitrogen in the acidic environment would weaken the coordination interaction between the Zn-ions and PI, leading to the decomposition of the nanoparticles. Metalloimmunology property and IDO inhibition activity of ZnPI-PEG It has been reported that Zn-ions can induce CRT exposure on the cancer cell surface,46,47 an "eat-me" signal that could promote phagocytosis and maturation of APCs. To further confirm that Zn-ions could induce CRT exposure, cancer cells were incubated with different metal-ion-containing compounds (CaCl2, FeCl2, MnCl2, NiCl2, CuCl2, or ZnCl2), each at a concentration of 100 μM and analyzed by flow cytometry. At our test concentration, only Zn-ions could induce intense CRT exposure at the cell surface, similar to the level of induction by a classical ICD-inducer, OXA (Figures 2a and 2b). Notably, such behavior appeared to be unique for Zn-ions. Figure 2 | The inherent metalloimmunology and IDO inhibition properties of ZnPI-PEG. (a) Flow cytometry analyzing CRT exposure for cancer cells after incubation with various metal ions at a concentration of 100 μM. OXA was a positive control. (b) Statistics of CRT+ cell percentage in (a). (c) Relative cell viability after incubation with ZnPI-PEG at various concentrations. (d) Flow cytometry analyzing CRT exposure of cancer cells after incubation with PI, ZnCl2, ZIF-8, or ZnPI-PEG. The concentration of Zn-ions was 50 μM, corresponding to 100 μM of PI. (e) Statistics of CRT+ cell percentages in (f) images of CT26 cells stained with antibody and after ZnPI-PEG treatment. (g) Schematic illustration of IDO inhibition with PI to suppress the metabolism and Kyn inhibition of ZnPI-PEG and free PI for CT26 cells (h) and 4T1 cells Download figure Download PowerPoint The potential of ZnPI-PEG to CT26 carcinoma was examined using cell viability ZnPI-PEG showed at our test concentrations (Figure the of the nanoparticles. To ZnPI-PEG the metalloimmunology properties of cells were incubated with free PI Zn-ions or ZnPI-PEG μM of Flow cytometry analysis (Figures and showed that Zn-ions and ZnPI-PEG could induce CRT cell surface exposure the PBS control. The strong CT26 cell surface fluorescence (Figure confirmed further that ZnPI-PEG the metalloimmunology properties of Zn-ions. Next, the in vitro IDO inhibition activity of ZnPI-PEG was by the of Trp to Kyn (Figure IFN-γ was used to induce IDO in CT26 and 4T1 cells, and the Kyn released in the culture medium supernatant was by a The of PI and ZnPI-PEG were calculated to be and μM with CT26 cells, and and μM with 4T1 cells, respectively. ZnPI-PEG the IDO inhibition of PI for CT26 and 4T1 cells (Figure and Next, in vivo were to the in vivo IDO inhibition. Mice bearing CT26 tumors were randomly into four group received one of the following by i.v. PBS, the modification method used as ZnPI-PEG, as blank free PI, or ZnPI-PEG, at the PI of 10 Mice were sacrificed on day 2 second injection to sera and tumors for further analysis (Figure that with the Kyn/Trp the of IDO activity in sera and tumors was after ZnPI-PEG treatment (Figure an effective in vivo IDO inhibition of ZnPI-PEG. Figure 3 | In vivo IDO inhibition and tumor immune microenvironment modification with ZnPI-PEG. (a) of the Balb/c mice bearing CT26 tumors were times on day 0 and 3 with PBS, ZIF-8, free PI, or ZnPI-PEG. Mice were sacrificed on the day for further analysis. (b) Kyn/Trp ratios in tumors and sera after PBS, ZIF-8, free PI, or ZnPI-PEG treatment measured on the Flow cytometry analysis of (c) Treg cells in cells and (d) T cells in cells in tumors of mice measured on the (e) images of stained tumor slices of mice after various Tumor slices were stained with cell and Treg T (f) The of T cells and Treg cells per image in the of the were calculated by analysis of Download figure Download PowerPoint IDO is an thus, the immune TME was after ZnPI-PEG treatment by analyzing Treg and T cells of the tumor using flow cytometry and staining. on the flow cytometry analysis of the tumor cell a decrease of Treg cells (Figure and Supporting Information Figure and an increase in T cells (Figure and Supporting Information Figure were after ZnPI-PEG treatment, which was further confirmed by confocal microscopy images of the tumor slices (Figures and that the immunosuppressive TME was reversed after ZnPI-PEG treatment, leading to the suppression of the Trp metabolism pathway via IDO inhibition. ZnPI-PEG DOX-induced ICD were as to have elicited an immune response in recent DOX, as an antitumor could ICD via of immune In our work, that DOX could be efficiently loaded in ZnPI-PEG ZnPI-PEG/DOX (Figure and Supporting Information Figure that and between DOX and PI be for the of ZnPI-PEG/DOX nanoparticles. The DOX release from ZnPI-PEG/DOX at varying pH conditions were (Figure Under physiological conditions (pH 7.4), ZnPI-PEG/DOX was However, acidic conditions (pH 6.8 and were DOX was released from the nanoparticles. Figure 4 | ZnPI-PEG (a) of ZnPI-PEG. DOX concentration was fixed at 0.5 in the following experiments. (b) DOX release from (c) Cancer cell of ZnPI-PEG/DOX and DOX to CT26 (d) uptake of The ZnPI-PEG/DOX was in after with CT26 cells for 4 h, then in cell after 12 h. (e) images of CT26 cells stained with after with PBS, free DOX, ZnPI-PEG, or (f) ATP concentrations in cell culture supernatants after with PBS, free DOX, ZnPI-PEG, or (g) Schematic illustration of ZnPI-PEG DOX-induced were calculated by and Download figure Download PowerPoint CT26 cells were incubated with ZnPI-PEG/DOX and free DOX to via the cell viability free DOX and ZnPI-PEG/DOX showed effects on cancer cell growth (Figure images of CT26 cells after incubation with ZnPI-PEG/DOX showed that cancer cells could ZnPI-PEG/DOX in a (Figure At 4 h after incubation, free DOX was in the ( Supporting Information Figure while ZnPI-PEG/DOX fluorescence with a lysosome the uptake of ZnPI-PEG/DOX through the At 12 h after incubation, of the ZnPI-PEG/DOX fluorescence was in the to the decomposition of ZnPI to promote lysosome of DOX. DOX is a type I ICD that on the and ICD via ZnPI-PEG/DOX could induce ICD and ATP from cancer cells after treatment images (Figure of stained cancer cells showed that ZnPI-PEG/DOX enhanced cells surface exposure of CRT free ZnPI-PEG/DOX and DOX group showed HMGB1 fluorescence in the that of the HMGB1 was released after treatment. ATP concentrations in the cell culture medium supernatants were measured using the ATP assay indicated (Figure The ZnPI-PEG/DOX treatment resulted in a higher ATP with that in the free DOX group. that the

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