Novel immunogenic cell death inducer combined with autophagy inhibitor to amplify photodynamic synergistic immunotherapy for triple-negative breast cancer

Novel immunogenic cell death inducer combined with autophagy inhibitor to amplify photodynamic synergistic immunotherapy for triple-negative breast cancer | 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 Novel immunogenic cell death inducer combined with autophagy inhibitor to amplify photodynamic synergistic immunotherapy for triple-negative breast cancer Gankun Yuan, Ruyue Yang, Wenjing Wen, Zhaoyi Wei, Meiru Song, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4950334/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 Activating immunogenic cell death (ICD) represents a promising therapeutic strategy for tumor immunotherapy. However, photodynamic therapy (PDT)-mediated ICD effects are severely limited due to the extremely short half-life and limited diffusion radius of reactive oxygen species (ROS) hinder effective endoplasmic reticulum (ER) stress induction. In addition, targeted therapy of triple-negative breast cancer (TNBC) remain hugely challenging due to the lack of expression of multiple receptors. Results Herein, we synthesized a hierarchical targeting and controllable intelligent nanodelivery material Da-CD@CET@CQ, loaded with highly efficient ER-targeted photosensitizers CET and autophagy inhibitor chloroquine (CQ). Excitingly, Da-CD@CET@CQ NPs can selectively target TNBC tumor cells and the CET was effectively released in the tumor microenvironment, enabling local accumulation of photosensitizers in the ER and in situ ROS production, which causing stronger ER stress and amplifying the ICD effect, further increasing the immune suppression of tumor growth. More importantly, CQ released by Da-CD@CET@CQ NPs can inhibit autophagy to destroy damaged cell repair, and further enhance the anti-tumor ability of PDT. Conclusions Our findings indicate that we reported a novel strategy of photosensitizing ICD inducer to amplify ICD effects and combination with autophagy inhibition, which provides a meaningful guideline for targeted PDT synergistic immunotherapy of TNBC in the future. Immunogenic cell death Photodynamic therapy Reactive oxygen species Endoplasmic reticulum stress Autophagy inhibitor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Immunotherapy as a “revolutionary”therapy for cancer treatment, which has shown breakthrough therapeutic effects on tumor recurrence and metastasis due to its unique treatment mode [ 1 – 3 ] . Immunogenic cell death (ICD) is a type of programmed cell death that can regulate the tumor immune microenvironment and activate systemic antitumor immune responses by the release of damage-associated molecular patterns (DAMPs), which is attributed to its ability to mobilize the immune system to destroy cancer cells via the“by stander effect” [ 4 – 7 ] . According to previous literature reports, sustained endoplasmic reticulum (ER) stress after reactive oxygen species (ROS) or drug stimulation can promote the induction of ICD and the release of DAMPs, such as calreticulin (CRT), high mobility group protein 1 (HMGB1), and adenosine triphosphate (ATP), from dying tumor cells [ 8 , 9 ] . Unfortunately, most of the ICD inducers that can cause ER stress are mainly toxic chemotherapeutic agents, and the number is still very small [ 10 ] . Therefore, it still has great significance that to develop a novel and effective ICD inducer. Photodynamic therapy (PDT) has become a popular cancer treatment modality due to its unique advantages, such as minimal invasiveness, good efficacy and lack of notable drug resistance [ 11 – 13 ] . In addition to direct oxidative killing of tumor cells, PDT can also induce ICD effects in the body to promote anti-tumor immunotherapy [ 14 – 18 ] . Unfortunately, the effect of this synergistic immunotherapy for tumors is still limited due to the insufficient PDT-mediated ICD effect [ 19 , 20 ] . Moreover, traditional photosensitizers (PS) such as phthalocyanines and porphyrins show insufficient tumor tissue targeting and poor biosolubility, which further hinder the anti-tumor effect of PDT. With the development and application of nanotechnology, the use of nanodelivery systems as PS carriers has provided a new platform for the development of PDT, increasing the selective enrichment of PS in tumors [ 21 , 22 ] . However, the desired PDT efficacy is undoubtedly limited to the vicinity of the generation site of ROS due to its extremely short lifespan (< 0.04 µs) and diffusion range (< 0.02 µm), which is still one of the main factors hindering the combination of nano-delivery materials and PDT for tumor therapy [ 23 – 25 ] . In addition, autophagy can repair and reverse tumor cell damage caused by PDT, resulting in incomplete cell death, which increases the tolerance of tumor cells to PDT [ 26 – 29 ] . Therefore, it is urgent to develop a strategy that can further delivery of PS to major suborganelles and inhibition of autophagy to enhance the anti-tumor effects of PDT. Endoplasmic reticulum(ER)is the main site of protein synthesis, lipid production and calcium ion storage in eukaryotic cells [ 30 ] . Therefore, targeting PS to the ER to achieve in situ ER PDT has become a promising tumor treatment strategy. Especially, PS enriched in the ER can act as an ICD inducer to induce excessive or abnormal ER stress through the continuous production of ROS, which can further amplify the PDT-mediated ICD effect. Moreover, triple-negative breast cancer (TNBC) is a subtype of breast cancer that is aggressive, easily metastasizes, result in a poor prognosis. As the expression of estrogen receptor, progesterone receptor and the proto-oncogene Her-2 is negative on the surface of TNBC tumors, very few anticancer drugs can be enriched in tumor cells, substantially diminishing their antitumor effects [ 31 – 33 ] . Excitingly, several studies have shown that the levels of SRC proteins are significantly higher in TNBC than in other breast cancer subtypes [ 34 – 36 ] . Therefore, it is essential to develop a safe and effective strategy for TNBC targeted therapy by targeting SRC protein to improve the anti-tumor effect. Herein, we successfully covalently coupled the ER-targeting ligand p-toluenesulfonyl chloride with the PS chlorin e6 (Ce6) via an ethylenediamine chain to obtain the CET. Subsequently, the CET and autophagy inhibitor CQ were mixed with poly-β-CD NPs, and the Da-Ad obtained by the reaction of adamantanamine and a tyrosine kinase inhibitor dasatinib, which can precisely target SRC family proteins [ 37 , 38 ] , can linked to the surface of the nanomaterial via host-guest interactions to obtain the cascade-targeted nanomaterial Da-CD@CET@CQ (Scheme 1 ). Given that Da is involved in SRC-mediated targeting recognition in TNBC cells, Da-CD@CET@CQ NPs achieved specific enrichment in TNBC cells and selective accumulation of CET in ER due to the acidic environment of the tumors that prompts the release of the drug from the nanomaterials. On the one hand, CET generated sufficient ROS in ER to kill tumor cells under 670 nm light irradiation; on the other hand, the in situ generation of ROS in ER led to disruption of calcium homeostasis and a sustained ER stress, which in turn amplified the ICD effect and increased the DAMPs released by dying tumor cells. Moreover, stimulation by DAMPs induced dendritic cell (DC) maturation and increased the DC antigen-presenting capacity, thereby promoting the immune response. More importantly, the autophagy inhibitor CQ released by the Da-CD@CET@CQ NPs inhibited the autophagic protection against PDT-induced damage to cells, further increasing the anti-tumor effect of PDT. Collectively, the Da-CD@CET@CQ NPs not only showed cascade targeting of TNBC tumor cells and ER, but also further sustainably increased ER stress, resulting in enhanced ICD effect, and combined with autophagy inhibitor to obtain a more robust PDT effect, ultimately realizing more effective synergistic photodynamic immunotherapy for TNBC. Methods Materials and Apparatus β-Cyclodextrin, dasatinib, amantadine, p-methylbenzene sulfonyl chloride, ethylenediamine, chlorin e6 and chloroquine purchased from Aladdin. Hoechst 33258, ER-Tracker Green, 4',6-diamidino-2-phenylindole (DAPI), DCFH-DA, Fluo-4 AM probe and methyl thiazolyl tetrazolium (MTT) were purchased from Beyotime Biotechnology. Beijing Solarbio Science&Technology Co., Ltd. offers the Calcein-AM/PI double staining kit and Annexin V-FITC apoptosis detection kit. The antibodies used in the experiment were provided by Proteintech Group, Inc. The relevant biological reagents used in the cell culture were purchased from Hyclone, Cytiva. The transmission electron microscope (TEM, Thermo Scientific FEI, USA) was applied for the morphological examinations of the nanoparticles. The hydrodynamic diameter and zeta potential of the nanoparticles were measured by Zatasizer Nano ZSE (Malvern instrument, UK) in deionized water. The ultraviolet-visible (UV-vis) near infrared spectrometer (Shimadzu, UV-3600, Japan) and the fluorospectro photometer (Cary Eclipse, Agilent) were recorded the photophysics property of the nanoparticles. The confocal laser scanning microscope (CLSM, C2+, Nikon) was used for the fluorescent cell images of the nanoparticles. The Flow cytometer (BD Accuri C6, USA) was applied for the cell uptake and apoptosis assays. In vivo tumor tissue uptake were observed by Small Animal Live Imaging System (Vilber BT100, France). Preparation of Da-CD@CET@CQ NPs The obtained Poly-β-CD (21.0 mg), CET (2.0 mg) and CQ (2.0 mg) were dissolved in a mixture of DMSO and water (1:3) at room temperature and shielded from light for 2 h. Then, Da-Ad (0.5 mg) was dissolved in a mixture of DMSO and water (1:3) and slowly added to the reaction system. At room temperature and away from light, the reaction continued for 12 h. After the reaction, the reaction mixture was transferred to a dialysis bag with MW = 3000 for dialysis for 24 h, and the water was changed every 3 h. Finally, the brown black solid powder Da-CD@CET@CQ NPs was obtained by vacuum freeze-drying. The storage stability of Da-CD@CET@CQ NPs First, Da-CD NPs (2.0 mg), CD@CET NPs and Da-CD@CET NPs solids were dissolved in 3 mL deionized water and RPMI 1640 respectively. Then, the clarified state of nanomaterials in deionized water was observed. At the designated time, the mean hydrodynamic diameter and PDI values of the solution were measured by DLS. Evaluation of CET and CQ releasing To evaluate the release efficiency of CET and CQ under physiological and acidic conditions, the Da-CD@CET@CQ NPs was dissolved in different PBS (pH = 7.4, 6.0 and 5.0), and the absorbance of CET and CQ were determined by UV-vis spectroscopy at different time points, the drug release rates at different pH were calculated, and time-dependent release curves were plotted. Cell culture Triple negative breast cancer 4T1 cells and human embryonic renal cell HEK293T cells were purchased from Institute of Basic Medical Sciences (IBMS) of the Chinese Academy of Science. The 4T1 cells were incubated in RPMI 1640 and HEK293T cells were cultured with DMEM medium, which containing 10% fetal bovine serum and 1% antibiotics (penicillin/streptomycin, 100 U mL − 1 ) at 37°C in a humidified environment of 5% CO 2 . Cellular uptake 4T1 cells and HEK293T cells were incubated in cell culture dishes for 24 h, and new medium containing CD@CET@CQ NPs or Da-CD@CET@CQ NPs (25 µg/mL) was added for 12 h, respectively. For the targeted competitive uptake assay, the 4T1 cells were preincubated with different concentrations of free dasatinib (0、5、10 and 20 nM) for 12 h. After PBS cleaning, the Da-CD@CET@CQ NPs (25 µg/mL) was added for further 12 h. The CLSM and flow cytometry were used to observe the intracellular fluorescence intensity. The ER Localization Study In the cell culture dish, the free Ce6, CET, Da-CD@CET@CQ NPs (25 µg/mL) were incubated with the 4T1 cells for 12 h, respectively. Subsequently, the PBS was used to wash the dish. The ER-Tracker Green was incubated for 20 min and washed with PBS. The fluorescence of the ER-Tracker Green was excited at 488 nm and monitored at 510–570 nm, and the photosensitizer fluorescence was excited at 633 nm and monitored at 650–750 nm by CLSM. Autophagy inhibition experiment In Vitro Autophagy marker proteins LC3 and P62 were quantified to evaluate the ability to inhibit autophagy. Cells treated with different drugs and a laser, then supernatant cells were collected and cut in a RIPA(Radio Immunoprecipitation Assay) buffer containing protease inhibitors. The protein was quantified by Coomassie leucocyanin assay. The primary antibodies were anti-LC3 (81004-1-RR) and anti-P62 (66184-1-Ig),which were purchased from Proteintech Group. Refer to western blot test for specific steps. Measurements of cytosolic Ca 2+ The cytoplasmic Ca 2+ homeostasis was examined by CLSM analysis. First, 1 mL 4T1 cells (6 x 10 4 ) were added to the confocal dish and incubated for 12 h. Subsequently, the suspension treated with different drugs was incubated with 2 µM Fluo-4-AM solution for 30 min under dark, then centrifuged and washed. The nuclei were stained with DAPI. Finally, the image was captured with CLSM. ER stress analysis Four unfolded protein responses (UPR) were quantified: proapoptotic C/EPB homologous protein, inositol requires enzyme 1α phosphorylation (p-IRE-1α), activated transcription factor 6 (ATF6), and protein kinase R-like ER kinase (PERK) to assess ER stress capacity. Different drug-treated cells were laser treated, and subsequently the supernatant cells were collected and cleaved in a buffer of RIPA (Radio-Immunoprecipitation Assay) containing protease inhibitors. The protein was quantified by the Coomassie brilliant blue protein quantitaion method. The primary antibodies used anti-p-DDIT3/CHOP (ET1703-05), anti-p-IRE-1α (Ser724), anti-ATF6 (EM1701-94), anti-p-eIF-2α (HA721510) and anti-β-Actin (AF2811) were purchased from HuaBio Biotechnology and Beyotime Institute of Biotechnology. Refer to western blot test for specific steps. Immunogenic cell death In vitro Exposure to CRT was measured using CLSM. First, 1 mL 4T1 cell (1 x 10 5 ) was added to the confocal dish and incubated for 12 h. The cells were treated with equal photosensitive unit concentration of different groups for 12 h. Then, the cells were fixed with 4% paraformaldehyde for 20 min and immersed in 1% fetal bovine serum PBS for 30 min. The primary antibody of CRT was incubated at 4℃ overnight, and the secondary antibody of 488 coupled was incubated with PBS for 1h after washing three times. Finally, the nucleus was stained with DAPI, and the fluorescence intensity of the cell surface was observed under CLSM with 405 nm and 488 nm lasers, respectively. We used CLSM fluorescence distribution assay to evaluate intracellular HMGB1 migration. Firstly, 1 mL 4T1 cell (1 × 10 5 ) was added to the confocal dish and incubated for 12 h. The cells were then treated under the same conditions as the CRT analysis. Then, the cells were fixed with 4% paraformaldehyde for 20 min and infiltrated with 0.1% Triton X-100 for 10 minutes, and incubated with 1% fetal bovine serum PBS for 30 minutes. The primary antibody of HMGB1 was incubated at 4℃ overnight, after washing three times, the secondary antibody of iFluor™ 647 was incubated with PBS for 1 h. Finally, the nucleus was stained with DAPI, and the fluorescence intensity of the cell surface was observed under CLSM with 405 nm and 633 nm lasers, respectively. The collected cells in each group were added with 0.3 mL ATP test reagent (in 2 mL EP tube) for every 2 × 10 6 cells, covered tightly and mixed evenly, inserted into a float, placed in a boiling water bath for 10 minutes, and centrifuged at 4℃ at 10000×g for 10 minutes after water cooling, and the superserum was taken. The extracellular ATP concentration in each supernatant was analyzed according to the operating instructions of the ATP chemiluminescence assay kit (Cat: E-BC-F002, Elabscience). Western Blotting The cell lysate (RIPA: protease inhibitor: phosphorylase inhibitor = 100:1:1) was added to the cell culture dish to extract the protein. The protein was quantified by Coomassie leucocyanin assay. The separation gel with appropriate concentration was selected according to the protein molecular weight and 30 µg of protein sample was added to each well. The sample was separated by SDS-PAGE gel electrophoresis and transferred to PVDF membrane, which was sealed with 5% bovine serum albumin for 1 h and incubated with primary antibody at 4℃ overnight. After TBST cleaning, the corresponding secondary antibodies were incubated at room temperature for 1 h. After TBST cleaning, the membranes were visualized using the Tanon 5200 Multi Imaging System. Intracellular ROS Generation Assay The 4T1 cells were co-cultured with the PBS, Da-CD NPs, free CQ, free CET, CD@CET@CQ, Da-CD@Ce6@CQ, Da-CD@CET and Da-CD@CET@CQ NPs (25 µg/mL) group for 12 h, respectively. Meanwhile, the Da-CD@CET@CQ NPs at different concentration (0、12.5、25 and 50 µg/mL) were incubated with 4T1 cells for 12 h. The PBS was used to wash the dish, and all groups were incubated with DCFH-DA (1:1000) probe for 30 min. A light source (λ = 670 nm, 4 mW·cm − 2 ) was applied for 5 min in all groups. Finally, the CLSM was used to measure the fluorescence of DCF excited at 488 nm. In vitro Photodynamic Cytostatic Activities The 4T1 cells were seeded per well in 96-well cell plates with different groups: PBS, Da-CD NPs, free CQ, free CET, CD@CET@CQ, Da-CD@Ce6@CQ, Da-CD@CET and Da-CD@CET@CQ NPs with various concentrations. After 12 h incubation, the medium was replaced with a new medium and the cells were irradiated with a light source for 5 min (λ = 670 nm, 4 mW·cm − 2 ), the cell viability was counted by MTT assay kit. The cell growth inhibitory effects were calculated by the following equation: Cell viability (%) = {(A treatment - A blank )/(A control - A blank )} × 100% For live/dead cells experiments, 4T1 cells with 50 µg/mL different drugs were incubated in confocal dishes for 12 h and washed repeatedly with saline. Meanwhile, the Da-CD@CET@CQ NPs at different concentration (0、6.25、12.5 and 50 µg/mL) were incubated with 4T1 cells for 12 h. All samples were irradiated with a light source for 5 min (λ = 670 nm, 4 mW·cm − 2 ). After that, a calcein AM solution (500 µL, 5 µM) and a PI solution (500 µL, 10 mM) were added to the culture medium and incubated for 20 min to stain the cells. Finally, the fluorescence images were observed by CLSM (excited at 488 nm and 546 nm, respectively). The cell apoptosis study 50 µg/mL of different drugs were incubated with 4T1 cells for 12 h and washed repeatedly with PBS. All samples were irradiated with a light source for 5 min (λ = 670 nm, 4 mW·cm − 2 ), and then incubated with FITC-Annexin V and PI for 20 min. Finally, the cell apoptosis was detected by FCM. In vivo tumor tissue targeting All animal experiments were approved by the Animal Care and Use Committee of Henan Academy of Sciences. Female BALB/c mice with 4T1 tumors were randomly divided into two groups (n = 5 mice per group) and then treated with CD@CET@CQ NPs and Da-CD@CET@CQ NPs (10 mg/kg) via tail vein injection, respectively. All mice were observed by small animal live imaging system at different time points. In vivo anti-tumor study Female BALB/c mice with 4T1 tumors were randomly divided into eight groups (n = 5 mice per group) include: PBS、Da-CD NPs 、free CQ、free CET、CD@CET@CQ NPs、Da-CD@Ce6@CQ NPs、Da-CD@CET NPs and Da-CD@CET@CQ NPs (10 mg/kg). After the drug was injected into the mice through the tail vein for 12 h, the tumor site was irradiated by a laser for 10 min (670 nm, 50 mW). Tumor volume and body weight were measured every other day. After 15 days of treatment, the major organs and tumor of mice were removed and weighed. The tumor volume was ascertained employing the formula below: Tumor volume (mm 3 ) = (L×W 2 ) /2 where L is the tumor length and W is the tumor width. Histological analysis The treated mice were sacrificed, and the main organs such as heart, liver, lung, spleen, kidney, and tumor were collected and fixed in 10% formalin. Paraffin sections were stained with hematoxylin and eosin (H&E) and stained with the TUNEL kit and immunohistochemistry (CD34 and Ki67). Immunity Response Study After the tissue sections were dehydrated and repaired, different primary antibodies were incubated at room temperature, and then different secondary antibodies were incubated. Antigen recovery was performed after fluorescence staining of PPD650. Different primary antibodies were used successively to culture the secondary antibodies coupled with horseradish peroxidase, and the tyramide signal was amplified. Incubated with 4'-6' -diamino-2-phenylindole (DAPI) at room temperature for 10 min, washed with TBST buffer and sterile water for 3 min, respectively. Finally, anti-hardening sealant and nail polish were added, and the stained tissue sections were observed under fluorescence microscope. Different groups of mice were killed after treatment and blood samples were collected. The blood samples were then centrifuged at 4℃ at 12000 RPM for 10 min to collect plasma for analysis of TNF-α, IFN-γ and IL-10 expression. Finally, the test kit is used to measure according to the manufacturer's instructions. Statistical Analysis All the experiments were performed for at least three independent times and data are expressed as the means ± SD. GraphPad Prism 6 software was applied for statistical analysis. The statistically significance level is * p < 0.05, ** p < 0.01, *** p < 0.001. Results and Discussion Preparation and characterization of Da-CD@CET@CQ NPs Poly-β-CD was synthesized as previously described in the literature [ 39 , 40 ] . The ER-targeted PS CET was obtained by alkylation as well as amidation reactions (Fig. S1 ). The properties of the Da-CD@CET@CQ NPs were characterized. Transmission electron microscopy (TEM) images revealed the specific morphology of the Da-CD and Da-CD@CET@CQ NPs, both of which exhibited uniform spherical structures with diameters of approximately 60 nm (Fig. 1 A and B). The results of dynamic light scattering experiments further confirmed that the average hydrodynamic diameters of the Da-CD NPs and Da-CD@CET@CQ NPs were about 58 nm and 62 nm, and the polydispersity indices (PDIs) were approximately 0.23 and 0.22, respectively, as shown in Fig. 1 C and D. The stability of materials has always been a prerequisite and key to their applicability and effectiveness, so we evaluated the stability of several nanomaterials. As shown in Fig. 1 E, the solutions of Da-CD NPs, CD@CET@CQ NPs, Da-CD@Ce6@CQ NPs and Da-CD@CET@CQ NPs did not show obvious turbidity or precipitation after 7 days in deionized water, similar to the initial state ( Fig. S2), and the PDIs of several nanomaterials were very small and remained basically unchanged within 7 days (Fig. 1 F). Moreover, the average hydrodynamic diameters of the Da-CD@CET@CQ NPs were stable and unchanged after 7 days of treatment in deionized water or RPMI 1640 medium (Fig. 1 G and H), which suggests that these nanocomplex based on poly-β-CD are stable and homogeneous in the long term. The UV-vis absorption spectra of the Da-CD@CET@CQ NPs at 330 nm showed characteristic absorbance peaks for Da, as well as for CET and CQ at 670 nm and 260 nm (Fig. 1 I), indicating that Da had been modified on the Da-CD@CET@CQ NPs surface by stronger host-guest interactions, and the successful loading of CET and CQ. In addition, the emission spectra of the Da-CD@CET@CQ NPs had nearly the same sharp fluorescence band centered at 680 nm as that of CET, further confirming the effective loading of CET (Fig. S3). The zeta potential results showed that the Da-CD NPs were positively charged, decreasing from 15.6 mV to 8.7 mV, further indicating the successful loading of CQ in the Da-CD@CET@CQ NPs (Fig. 1 J). The drug loading capacities (DLCs) of CET and CQ were 6.32 ± 0.15% and 7.04 ± 0.19%, respectively, and the drug loading efficiencies (DLEs) were 79.10 ± 0.18% and 87.50% ± 0.23%, respectively, as calculated by using UV-vis spectrophotometry and based on the drug standard curve, which demonstrated that the Da-CD NPs had good loading capacities. Poly-β-CD is in a stable state under physiological conditions, which ensures safety during drug delivery, but its structure is damaged, resulting in sparse nanoparticles, in acidic tumor regions according to previous studies [ 41 ] ; in addition, under acidic conditions, the payload drug is easily protonated, which in turn reduces its noncovalent interaction with poly-β-CD, leading to a decrease in the degree of poly-β-CD crosslinking and disintegration [ 42 – 45 ] . To verify the release mechanism and effects of the Da-CD@CET@CQ NPs, the UV-vis spectrophotometry was used to determine the release kinetics of the drug under different acidic conditions. The release rates of CET and CQ after 24 h were only 3.2% and 5.2%, respectively (pH = 7.4), which effectively prevented the premature release of the drugs during transportation (Fig. 1 K and L). Moreover, the release rate increased significantly when the pH was further reduced to 6.0 and 5.0, especially at pH = 5.0, the release rates of CET and CQ reached 89.1% and 91.5%, respectively. These results indicated that the lower pH of the tumor microenvironment could trigger the release of CET and CQ from the Da-CD@CET@CQ NPs, thereby increasing the biosafety and effectiveness of the drugs. Intracellular uptake and endoplasmic reticulum localization of Da-CD@CET@CQ NPs To verify the ability of the Da-CD@CET@CQ NPs to target TNBC cells, we used 4T1 cells with high expression of SRC proteins for in vitro cell uptake experiments. Confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) were used to determine the fluorescence intensity of the Da-CD@CET@CQ NPs in the cells to assess their ability to target 4T1 cells. Based on the time-dependent of drug uptake and the data at different time points (Fig. S4), 12 h was chosen as the appropriate time point for uptake studies. The CLSM and FCM fluorescence data showed that the Da-CD@CET@CQ NPs had stronger intracellular fluorescence after uptake for 12 h, which was approximately 5-fold greater than the cellular fluorescence of the CD@CET@CQ NPs, suggesting that they exhibited more pronounced cellular uptake (Fig. 2 A and B, Fig. S5). We hypothesize that this result may be due to the high expression of SRC in 4T1 cells and dasatinib-mediated targeting. To test this hypothesis, we preincubated 4T1 cells with different concentrations of free Da (0, 5, 10, and 20 nM) for 12 h before they were incubated with the Da-CD@CET@CQ NPs so that they could pre-bind to intracellular SRC, thereby reducing the amount of free intracellular SRC. The CLSM (Fig. S6) and FCM results (Fig. 2 C and D) showed that with prebound SRC, the intracellular fluorescence intensity of the Da-CD@CET@CQ NPs decreased with increasing concentrations of free Da. The intracellular fluorescence intensity was significantly reduced by approximately 9-fold when the preincubation concentration was 20 nM, indicating that the Da-CD@CET@CQ NPs could indeed achieve greater accumulation in 4T1 cells through Da and SRC-mediated targeting. Subsequently, to further demonstrate the tumor specificity of the Da-CD@CET@CQ NPs for TNBC, we used a human embryonic kidney cell line (HEK293T) with SRC-negative expression as the control. The CLSM (Fig. S7) and FCM (Fig. 2 E and F) results showed that the Da-CD@CET@CQ NPs had an approximately 5-fold greater fluorescence intensity in 4T1 cells than in HEK293T cells. Taken together, the cellular uptake results indicated that Da modification significantly increased the ability of Da-CD@CET@CQ NPs to specifically target TNBC cells, providing a potential strategy for targeted PDT of TNBC. Based on the exciting tumor cell targeting results, we validated the ability of the Da-CD@CET@CQ NPs to further localize PS in ER. The PS modified wit h p-toluenesulfonamide were heavily enriched in ER and in turn directly and instantaneously damaged the structure of ER and increased ER stress, thereby increasing the efficiency of apoptosis. Therefore, the ER-Tracker Green probe was used to study the ER localization of free Ce6, CET and Da-CD@CET@CQ NPs in 4T1 cells. As shown in Fig. 2 G, all the red fluorescence of free Ce6 had almost no overlap with the green fluorescence signal of the ER probe. Further qualitative analysis of the line-scanning spectra of the fluorescence intensities showed that Ce6 and the ER-Tracker Green had substantially separated signals (Fig. 2 J-I). Qualitative analysis of the line-scanning spectra of the fluorescence intensities showed a partial overlap of CET and the ER-Tracker Green, which indicated that the free CET exhibited further ER localization after passive cellular uptake (Fig. 2 H, J-II). Compared with that of free CET, the red fluorescence signal of the Da-CD@CET@CQ NPs group significantly overlapped with the green fluorescence signal of the ER probe, and the qualitative analysis of the line-scanning spectra of the fluorescence intensities showed a high degree of overlap (Fig. 2 I, J-III). Thus, these data provide strong evidence that the Da-CD@CET@CQ NPs can significantly increase the ability of PS to effectively enter ER. Intracellular ROS imaging and In vitro phototoxicity Reactive oxygen species (ROS) with superior oxidative ability play a critical role in directly causing apoptosis and necrosis of tumor cells and play a key role in continuously increasing ER stress. The ROS indicator we selected was nonfluorescent 2',7'-dichlorodihydrofluorescein (DCFH-DA), which can be oxidized by ROS to green fluorescent DCF. Herein, we used CLSM to determine the fluorescence intensity of DCF. As shown in Fig. 3 A and B, the green fluorescence intensity of the Da-CD NPs and CQ groups without PS involvement was negligible. Compared with those of the free single-targeting CET and CD@Ce6@CQ NPs groups, the green fluorescence intensities of the Da-CD@Ce6@CQ NPs, Da-CD@CET NPs and Da-CD@CET@CQ NPs groups were approximately 5 times greater. These results indicated that the nanocomplex group with surface-modified Da had a higher ROS generation efficiency, which was also consistent with the cell uptake results. In addition, our further study revealed that the intracellular ROS production increased with increasing Da-CD@CET@CQ NPs incubation concentration (Fig. S8). Thus, these results suggest that poly-β-CD surface modification of Da increases the cell-targeted uptake of Da-CD@CET@CQ NPs and further promotes ROS generation. To determine whether the notable tumor cell targeting and ER localization ability of the Da-CD@CET@CQ NPs also resulted in superior cellular photodynamic activity, we evaluated the ability of the Da-CD@CET@CQ NPs to inhibit cellular activity via the MTT assay. First, as shown in Fig. 3 C, both the poly-β-CD-based nanomaterials and PS showed no significant cytotoxicity against 4T1 cells in the absence of light, whereas free CQ and the nanomaterials encapsulating CQ (CD@CET@CQ NPs, Da-CD@Ce6@CQ NPs and Da-CD@CET@CQ NPs) exhibited weak cytotoxicity as the concentration increased but still maintained a cell survival rate above 85%. Moreover, as shown in Fig. 3 D, the cellular phototoxicities of the Da-CD@Ce6@CQ NPs, Da-CD@CET NPs and Da-CD@CET@CQ NPs with cellular targeting were significantly greater than those of free CET and the CD@CET@CQ NPs with single-targeting ability after laser irradiation (λ = 670 nm, 4 mW·cm − 2 ), which might be due to the increased intracellular uptake mediated by Da. More importantly, at limited concentrations up to 25–50 µg/mL, the live cell inhibitory abilities of the Da-CD@CET@CQ NPs (15.2% and 7.3%) were both significantly greater than those of the Da-CD@Ce6@CQ NPs (37.9% and 21.6%), which also suggested that PS with both tumor cell and ER-targeting abilities exhibit increased photodynamic activity and proapoptotic abilities. In addition, we further evaluated its cytotoxicity under light exposure by live and dead assay. As shown in Fig. 3 E and F, the Da-CD NPs and CQ did not significantly inhibit cell activity. The red-to-green ratio was significantly higher in the free CET and CD@CET@CQ NPs groups, and the ratio was close to 50%. Notably, the red fluorescence after Da-CD@CET@CQ NPs treatment was significantly stronger than that in the Da-CD@Ce6@CQ NPs and Da-CD@CET NPs groups, which further indicated that the Da-CD@CET@CQ NPs treatment induced more apoptotic cell death. Moreover, as shown in Fig. 3 G and H, the cell death increased with increasing Da-CD@CET@CQ NPs incubation concentration, which was also consistent with the MTT results. In addition, apoptotic fluorescence staining was performed by using Annexin V-FITC and PI analysis of different drug groups after light irradiation. As shown in Fig. 3 I and J, the proportions of the two regions of early apoptosis (Q2) and late apoptosis (Q3) in the Da-CD@CET@CQ NPs group (95.76%) were higher than those in the Da-CD@Ce6@CQ NP group (69.06%) and Da-CD@CET NPs group (81.36%), and late apoptosis was predominant (76.68%), which indicated that the Da-CD@CET@CQ NPs had a greater ability to induce apoptosis; this result was consistent with the above MTT assay and the live-dead cell staining results. Therefore, the Da-CD@CET@CQ NPs, which showed tumor cell targeting and further localization of PS to ER, could effectively improve the photodynamic antitumor effect. Autophagy inhibition In Vitro and ICD effect induced by ER stress Autophagy maintains cell homeostasis through digestion and degradation of damaged, denatured or senescent proteins and organelles, which significantly inhibits the ability of PDT to induce cell apoptosis. Therefore, inhibition of autophagy has become one of the key factor to improve the therapeutic effect of PDT. To evaluate the autophagic inhibitory effect of the Da-CD@CET@CQ NPs loaded with CQ, a series of studies were performed by Western blot assay and confocal microscopy. The presence of LC3 in autophagosomes and its conversion to the smaller molecular weight LC3-II are indicators of autophagic [ 46 ] . After 4T1 cells were co-incubated with Da-CD@CET NPs and irradiated by a laser, the LC3-Ⅱ protein in the cells was significantly increased compared with cells without irradiation (Fig. S9), indicating that PDT effect can induce obvious autophagy, which is consistent with previous literature reports [ 47 – 49 ] . As shown in Fig. 4 A and B, compared with other groups, the LC3-II protein content in the groups treated with free CQ or the nanocomplex encapsulated with CQ was significantly higher, which indicated that free or loaded CQ molecules did not prevent the protein conversion of LC3-I to LC3-Ⅱ in cells. In addition, P62, a bridge linking LC3 and polyubiquitinated proteins, can be degraded by proteolytic enzymes in autophagic lysosomes during autophagy. In the case of reduced or defective autophagy, the P62 protein accumulates in the cytoplasm, which is used as the indicator of autophagic inhibition [ 50 ] . As shown in Fig. 4 A and C, the abundant expression of the P62 protein was found in the presence of free CQ and CQ-encapsulated nanocomplex compared to control cells, indicating that autophagy was significantly inhibited. Moreover, LysoTracker Green staining was performed on intracellular acidic vesicles (lysosomes). The results showed that Da-CD@CET@CQ NPs exhibited more significant intracellular accumulation of autophagy vesicles (green) (Fig. 4 D). These results further indicate that CQ molecules released by Da-CD@CET@CQ NPs had no affect on the transformation of LC3-I to LC3-Ⅱ, but can significantly inhibit lysosomal degradation of autophagosome, and thus promote the accumulation of more autophagy vesicles in cells. Thus, it can be observed that Da-CD@CET@CQ NPs had a more significant anti-tumor effect of PDT in vitro . ER is a multifunctional organelle in the cell and the main reservoir of intracellular Ca 2+ . Excessive production of ROS in ER leads to dysregulation of intracellular Ca 2+ homeostasis. To determine whether the Da-CD@CET@CQ NPs disrupt Ca 2+ homeostasis after laser irradiation, we used the sensitive Ca 2+ probe Fluo-4 AM. Free Fluo-4 AM has very weak fluorescence, however, when it was sheared by intracellular esterases to form Fluo-4, which can bind with Ca 2+ to produce strong green fluorescence. CLSM revealed that the cells treated with Da-CD@CET NPs and Da-CD@CET@CQ NPs displayed stronger green fluorescence, with intensities 5-fold and 2-fold higher than those of the ER-targeted CET group and the Da-CD@Ce6@CQ NP group, respectively (Fig. 4 E and Fig. S10). Thus, these results demonstrated that the nanomaterials with cellular and ER targeting capabilities have a more significant ability to disrupt intracellular Ca 2+ homeostasis. Dysregulation of intracellular Ca 2+ homeostasis impedes protein synthesis and folding, which leads to the accumulation of unfolded or misfolded proteins in ER, inducing the ER stress. On the one hand, at the onset of ER stress, unfolded or misfolded proteins in ER lumen are highly bound to the molecular chaperone binding immunoglobulin (BIP), leading to its dissociation from ER stress sensors and promoting the activation of activating transcription factor 6 (ATF6), inositol-required enzyme 1 alpha (IRE1 alpha), and protein kinase R-like ER kinase (PERK), which further induce an unfolded protein response (UPR) to remove unfolded or misfolded proteins to maintain the homeostatic balance of the ER. On the other hand, excessive ROS continuously increase ER stress and induce apoptosis by upregulating the expression of proapoptotic C/EPB homologous protein (CHOP), which was mediated by the PERK/eIF2/ATF6/CHOP pathway [ 51 – 53 ] . To assess the extent of ER stress, we first used CLSM to analyze the expression of CHOP proteins. The CLSM revealed that the red fluorescent signal of CHOP were stronger in the Da-CD@CET NPs and Da-CD@CET@CQ NPs group, which had an approximately 2-fold greater value than that in the Da-CD@Ce6@CQ NPs group, suggesting increased CHOP expression by targeting PS to ER (Fig. 4 F and Fig. S11). In summary, these experimental results fully confirmed that the Da-CD@CET@CQ NPs can produce a large amount of ROS in ER, resulting in redox balance disorders and Ca 2+ homeostatic imbalances and activating proapoptotic pathways through continuous ER stress. Recently, a growing number of studies have demonstrated that the continuous increase in ER stress generated by ROS can induce ICD [ 54 – 57 ] . Tumor cells induce ICD while producing a series of DAMPs, including CRT, which is exposed on the cell surface; HMGB1, which is secreted to the outside world; and ATP molecules, which are released by the cells [ 58 – 61 ] . Therefore, to determine whether ER stress triggered by the Da-CD@CET@CQ NPs can induce more intense ICD effects and promote DAMPs release, we conducted immunofluorescence and western blot analyses. First, we investigated the migration of HMGB1 from the nucleus to the cellular matrix. As shown in Fig. 4 G and Fig. S12, compared with those in the other groups, the overlap between HMGB1 (red) and the nucleus (blue) almost disappeared (pink) in the Da-CD@CET NPs and Da-CD@CET@CQ NPs group, which suggested that the tumor cells and ER targeting nanocomplex can increased HMGB1 migration from the nucleus. Second, we assessed the exposure of CRT on the cell surface by CLSM fluorescence intensity. Compared with those of the other groups, the Da-CD@CET NPs and Da-CD@CET@CQ NPs groups with dual-targeting ability displayed almost uniform and stronger green fluorescence, which was 3.2 times and 2.0 times higher than that of the CD@CET@CQ NPs and Da-CD@Ce6@CQ NPs groups, respectively, indicating that the nanocomplex with tumor cells and ER targeting can trigger higher CRT expression after light treatment (Fig. 4 H and I). We further evaluated ATP production in the cell culture medium. As shown in Fig. 4 J, there were more ATP secretion in the cellular supernatant of the Da-CD@CET NPs and Da-CD@CET@CQ NPs groups, which was almost 3.0 times and 2.0 times greater than that of the CD@CET@CQ NP and Da-CD@Ce6@CQ NP groups, respectively. Finally, western blotting was used to further analyze the expression of ER stress related proteins. As shown in Fig. 4 K and Fig. S13, the western blot results showed that the expression of ER stress marker, such as CHOP, PERK, ATF6, and p-IRE-1α, was significantly higher in the Da-CD@CET NPs and Da-CD@CET@CQ NPs group than that of other groups. All these results indicated that ROS continuously increased ER stress in the Da-CD@CET NPs and Da-CD@CET@CQ NPs groups, effectively induced ICD in tumor cells and further promoted antitumor effects. In vivo tumor targeting To further confirm the ability of the Da-CD@CET@CQ NPs to target TNBC tumors in vivo , we used small animal live imaging system to assess the distribution of the nanocomplex in mouse xenograft tumor model. CD@CET@CQ NPs and Da-CD@CET@CQ NPs were injected into BALB/c mice with tumors derived from 4T1 cells via the tail vein, and the fluorescence intensity of both nanocomplex at the tumor site was assessed. As shown in Fig. 5 A and B, the fluorescence intensity at the tumor site of the mice in the Da-CD@CET@CQ NPs group was significantly increased at 2 h post-injection and was approximately 3.0 times higher than that in the CD@CET@CQ NPs group, which indicated that the Da-CD@CET@CQ NPs could be enriched at the tumor site faster and more abundantly. With prolonged injection time, the enrichment of both the CD@CET@CQ NPs and the Da-CD@CET@CQ NPs at the tumor site peaked at 12 h, and the fluorescence intensity at the tumor site in the Da-CD@CET@CQ NPs group was approximately 2.5 times higher than that in the CD@CET@CQ NPs group. The fluorescence intensity at the tumor site gradually decreased over time, indicating that this type of nanocomplex could be metabolized by the organism in a timely manner. In addition, to further assess the fluorescence intensity at the tumor site and major organs, the mice were sacrificed at 12 h after administration. As shown in Fig. 5 C and D, the fluorescence intensity of the Da-CD@CET@CQ NP group at the tumor site was approximately 2.7 times higher than that of the CD@CET@CQ NP group, which was consistent with the in vivo fluorescence imaging data. Moreover, the concentration of Da-CD@CET@CQ NPs or CD@CET@CQ NPs at the tumor site was lower than that in the liver but higher than that in other major organs. Given the good biocompatibility and negligible cytotoxicity of the Da-CD@CET@CQ NPs and CD@CET@CQ NPs, the adverse effects of their accumulation in other organs in mice should be negligible. Meanwhile, fluorescence detection was performed on the tumor sections dissociated 12 h after administration, as shown in Fig. 5 E and F, Da-CD@CET@CQ NPs group showed more significant fluorescence intensity at the edge site of tumor. Moreover, fluorescence intensity 3 times higher than that of CD@CET@CQ group was also observed in the tumor core site of Da-CD@CET@CQ NPs group, which further proved that Da-CD@CET@CQ NPs can achieve abundant and more uniform accumulation of photosensitizer CET at the tumor site (Fig. 5 G and H). All these results demonstrate that Da-CD@CET@CQ NPs indeed exhibit more significant 4T1 tumor-targeting ability, which can be attributed to Da-mediated tumor specificity. In vivo antitumor activity To assess whether drug enrichment results in more significant antitumor effects, we conducted antitumor studies using BALB/c mice with a 4T1 tumor model. First, the mice were randomly divided into eight groups (n = 5 mice per group): control, Da-CD NPs, CQ, CET, CD@CET@CQ NPs, Da-CD@Ce6@CQ NPs, Da-CD@CET NPs and Da-CD@CET@CQ NPs. Based on the results of the drug targeting experiment, a laser was applied 12 h after injection (670 nm, 50 mW·cm − 2 , 10 min), and then, the tumor volume and body weight of the mice were monitored every other day (Fig. 6 A). As shown in Fig. 6 B and C, in the control group, almost no significant inhibition of tumor growth was observed after the injection of Da-CD NPs or CQ. The free CET and single-targeting groups, including the CD@CET@CQ NP and Da-CD@Ce6@CQ NP groups, only showed partial inhibition of tumor growth. Compared with the Da-CD@CET NP group, the Da-CD@CET@CQ NP group exhibited a more significant tumor inhibitory effect, and the tumors almost completely stopped growing, which may be attribute to the autophagic inhibitory ability of the Da-CD@CET@CQ NPs group, resulting in an increase in the final PDT effect. After 15 days of treatment, the mice were dissected, and the tumor tissues were removed for weighing. As shown in Fig. 6 D, the average tumor weight further confirmed that the Da-CD@CET@CQ NPs had a more significant antitumor effect. In addition, we further confirmed the antitumor effect of each group by H&E and TUNEL staining, and immunohistochemical analysis of the tumor tissues. As shown by H&E staining, more pronounced necrosis and increased vesicles, as well as nuclear shrinkage, were found in the Da-CD@CET@CQ NPs group. Moreover, a stronger green fluorescence signal was observed in the Da-CD@CET@CQ NPs group in the TUNEL experiment, which indicated that the Da-CD@CET@CQ NPs group had a greater number of apoptosis cells. Subsequently, the tumor stem cell marker CD34 and the cell proliferation marker Ki67 were selected for immunohistochemistry experiments to further assess vascular proliferation and tumor proliferation. The percentage of positive cells in Da-CD@CET@CQ NPs group was significantly lower than that in other groups, which indicated that Da-CD@CET@CQ NPs effectively inhibited vascular regeneration and cell proliferation (Fig. 6 E). Finally, we also performed H&E staining of isolated lung tissues. Compared with those in the other groups with more metastatic nodules in the lung tissues, no obvious metastatic nodules in the lung tissues were observed in the Da-CD@CET@CQ NPs group, which indicated that this treatment effectively inhibited tumor metastasis (Fig. 6 F and Fig. S14). The relative body weight of each group of mice remained almost unchanged throughout the experimental period (Fig. S15), and H&E staining analysis of isolated organs (heart, liver, spleen, lungs, and kidneys) revealed normal physiological morphology of tissues in each group (Fig. S16), indicating that all nanocomplexes have almost negligible systemic toxicity as well as side effects in vivo . These results strongly demonstrate that precise delivery of PS to ER of tumor cells and combined with autophagy inhibition has significant in vivo antitumor and metastasis inhibition effects. In vivo antitumor immunity We further explored the antitumor immune responses of the Da-CD@CET@CQ NPs by immunofluorescence staining. First, in order to evaluate ER stress and DAMPs release, we detected CHOP expression, CRT exposure and HMGB1 release (red fluorescence) in different tumor tissues by IF staining. The results showed that the red fluorescence intensity of CHOP in the Da-CD@CET NPs and Da-CD@CET@CQ NPs groups was significantly higher than that in the other groups due to the targeting of tumor cells and the presence of ER targeting, indicating the enhancement of ER stress (Fig. S17). In addition, the red fluorescence intensity of CRT and HMGB1 in the Da-CD@CET@CQ NPs group was significantly higher than that in the other groups, indicating that ER-PDT combined with autophagy inhibition could induce stronger ICD effects (Fig. S17). Second, we used a nuclear probe and four different antibodies (CD19-labeled B cells, CD8-labeled T cells, CD49B-labeled NK cells, and iNOS-labeled M1 macrophages) to determine the effect of the body's immune response in the different groups, and the expression levels of the different immunoantibodies were determined by fluorescence intensity. As shown in Fig. 7 A, the fluorescence signals of different antibodies in the dual-targeting Da-CD@CET NPs and Da-CD@CET@CQ NPs groups exhibited stronger than other groups. Meanwhile, we measured the expression levels of different antibodies based on fluorescence intensity. As shown in Fig. 7 B and C, the expression level of CD8 and CD19, compared with other groups, the Da-CD@CET NPs and Da-CD@CET@CQ NPs group show a significant increase. In addition, the expression level of CD49B and iNOS in the Da-CD@CET NPs and Da-CD@CET@CQ NPs group exhibit a obviously higher level compared to other groups (Fig. 7 D and E). These results suggest that in situ ER-PDT increased B cell, T-cell and NK cell expression and promoted macrophage polarization to M1 macrophages. More importantly, four antibodies in the Da-CD@CET@CQ NPs group exhibited a higher expression level than the Da-CD@CET NPs group, possibly because autophagic inhibition further promoted the antitumor effect of PDT, which in turn induced a stronger immune response. Moreover, peripheral blood of mice in different treatment groups were collected on the 15th day after laser irradiation for analysis of cytokine levels. The results showed that the mean levels of TNF-α and IFN-γ in Da-CD@CET@CQ NPs treatment group were significantly higher than those in other groups (Fig. 7 F and G), while the mean levels of IL-10 were lower than those in other groups (Fig. 7 H). Thus, these results suggest that the PS with tumor cells and ER targeting ability, combined with autophagic inhibition can further increase the ability of PDT to activate the body's immune response, which in turn promotes the synergistic effect of tumor treatment. Conclusion In this study, we have successfully designed and developed a smart controllable nanomaterial for the delivery of novel ICD inducer. Compared with free PS and single-target nanocomplexes, the final constructed Da-CD@CET@CQ NPs can precisely target TNBC tumor cells with SRC-overexpression, and has good tumor cell enrichment and retention ability, which may be attributed to the high affinity between Da and SRC in tumor cells. Meaningfully, the ICD inducer CET released in the tumor environment shows its remarkable ER localization ability, not only realizing in-situ ER PDT, but also enhancing the induction of ER stress to amplify the ICD effect, further promoting the maturation of DCs, and thus inducing a more drastic systemic immune response. Subsequently, autophagy inhibitor CQ released by Da-CD@CET@CQ NPs can inhibit autophagy, destroy cell homeostasis, and further promote the ability of PDT to induce apoptosis. In summary, the multifunctional nanoplatform constructed can achieve dual targeting of PS to tumor cells and ER, which can amplify the ICD effect and further enhance the anti-tumor effect of PDT by inhibiting autophagy, which is of great significance for the development and clinical application of TNBC targeted photodynamic synergistic immunotherapy in the future. Abbreviations PDT Photodynamic therapy Ce6 Chlorin e6 ICD Immunogenic cell death ROS Reactive oxygen species ER Endoplasmic reticulum TNBC Triple-negative breast cancer CQ Chloroquine DAMPs Damage-associated molecular patterns CRT Calreticulin HMGB1 High mobility group protein 1 ATP Adenosine triphosphate PS Photosensitizers DAPI 4',6-diamidino-2-phenylindole MTT Methyl thiazolyl tetrazolium TEM Transmission electron microscope UV-vis Ultraviolet-visible CLSM Confocal laser scanning microscope FCM Flow cytometer DMSO Dimethyl sulfoxide DMEM Dulbecco’s modified eagle medium ATF6 Activated transcription factor 6 CHOP Proapoptotic C/EPB homologous protein PBS Phosphate buffered saline H&E Hematoxylin and eosin TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling PDIs Polydispersity indices DLCs Drug loading capacities DLEs Drug loading efficiencies PERK Protein kinase R-like ER kinase UPR Unfolded protein responses Declarations Acknowledgments Not applicable Author contributions GY and GL conceived and designed the study, and wrote the manuscript. GY and RY performed most of the in vitro and in vivo experiments. WW and ZW participated in the in vivo experiments and analyzed some experimental data. MS and GL participating in revising the paper. LZ and KH contributed to the in vivo experimental design. Fundings This work was financially supported by the National Key Research and Development Program of China (2022YFE132800), Key R&D project of Henan Province (221111310600), and Special Foundation for Basic Research Program of Higher Education Institutions of Henan Province (22ZX005). Joint Fund of Henan Province Science and Technology R&D Program (225200810020). Postdoctoral Initiation Fund of Henan Academy of Sciences (231828050). The Scientific and Technological Research Project of Henan Province (232102311177). Data availability All data analyzed during this study are included in this published article and its supplementary information files. Ethics approval and consent to participate All animal experiments were approved by the Ethics Committee of the Animal Experimental Center of Henan Academy of Sciences (Approval Number: 2023A1017), and were carried out in compliance with all relevant ethical regulations. Consent for publication Not applicable. Competing interests The authors have declared that no competing interest exists. References Emens LA, Ascierto PA, Darcy PK, Demaria S, Eggermont AMM, Redmond WL, Seliger B, Marincola FM. Cancer immunotherapy: Opportunities and challenges in the rapidly evolving clinical landscape. Eur J Cancer. 2017;81:116–29. Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age. Nature. 2011;480:480–9. Kennedy LB, Salama AKS. A review of cancer immunotherapy toxicity. CA Cancer J Clin. 2020;70:86–104. Powles T, Kockx M, Rodriguez-Vida A, Duran I, Crabb SJ, Van Der Heijden MS, Szabados B, Pous AF, Gravis G, Herranz UA, et al. Clinical efficacy and biomarker analysis of neoadjuvant atezolizumab in operable urothelial carcinoma in the ABACUS trial. Nat Med. 2019;25:1706–14. Kroemer G, Galassi C, Zitvogel L, Galluzzi L. Immunogenic cell stress and death. Nat Immunol. 2022;23:487–500. Krysko DV, Garg AD, Kaczmarek A, Krysko O, Agostinis P, Vandenabeele P. Immunogenic cell death and DAMPs in cancer therapy. Nat Rev Cancer. 2012;12:860–75. Galluzzi L, Buqué A, Kepp O, Zitvogel L, Kroemer G. Immunogenic cell death in cancer and infectious disease. Nat Rev Immunol. 2017;17:97–111. King AP, Wilson JJ. Endoplasmic reticulum stress: an arising target for metal-based anticancer agents. Chem Soc Rev. 2020;49:8113–36. Rufo N, Garg AD, Agostinis P. The unfolded protein response in immunogenic cell death and cancer immunotherapy. Trends Cancer. 2017;3:643–58. Liu P, Zhao L, Pol J, Levesque S, Petrazzuolo A, Pfirschke C, Engblom C, Rickelt S, Yamazaki T, Iribarren K, et al. Crizotinib-induced immunogenic cell death in non-small cell lung cancer. Nat Commun. 2019;10:1486. Kuimova MK, Botchway SW, Parker AW, Balaz M, Collins HA, Anderson HL, Suhling K, Ogilby PR. Imaging intracellular viscosity of a single cell during photoinduced cell death. Nat Chem. 2009;1:69–73. Lovell JF, Liu TWB, Chen J, Zheng G. Activatable photosensitizers for imaging and therapy. Chem Rev. 2010;110:2839–57. Zhou Z, Song J, Nie L, Chen X. Reactive oxygen species generating systems meeting challenges of photodynamic cancer therapy. Chem Soc Rev. 2016;45:6597–626. Zhang S, Wang J, Kong Z, Sun X, He Z, Sun B, Luo C, Sun J. Emerging photodynamic nanotherapeutics for inducing immunogenic cell death and potentiating cancer immunotherapy. Biomaterials. 2022;282:121433. Vaes RDW, Hendriks LEL, Vooijs M, De Ruysscher D. Biomarkers of radiotherapy-induced immunogenic cell death. Cells. 2021;10:930. He L, Nie T, Xia X, Liu T, Huang Y, Wang X, Chen T. Designing bioinspired 2D MoSe2 nanosheet for efficient photothermal-triggered cancer immunotherapy with reprogramming tumor‐associated macrophages. Adv Funct Mater. 2019;29:1901240. Lan G, Ni K, Xu Z, Veroneau SS, Song Y, Lin W. Nanoscale metal-organic framework overcomes hypoxia for photodynamic therapy primed cancer immunotherapy. J Am Chem Soc. 2018;140:5670–3. Chen P, Huang P, Chen J, Shi Q, Zhu Y, Chen Y, Liu L, Zhang X. A self-delivery chimeric peptide for high efficient cell membrane-targeting low-temperature photothermal/photodynamic combinational therapy and metastasis suppression of tumor. Biomaterials. 2022;286:121593. Zhang C, Xu C, Gao X, Yao Q. Platinum-based drugs for cancer therapy and anti-tumor strategies. Theranostics. 2022;12:2115–32. Park SJ, Ye W, Xiao R, Silvin C, Padget M, Hodge JW, Van Waes C, Schmitt NC. Cisplatin and oxaliplatin induce similar immunogenic changes in preclinical models of head and neck cancer. Oral Oncol. 2019;95:127–35. Cheng Y, C Samia A, Meyers JD, Panagopoulos I, Fei B, Burda C. Highly efficient drug delivery with gold nanoparticle vectors for in vivo photodynamic therapy of cancer. J Am Chem Soc. 2008;130:10643–7. Tian B, Wang C, Zhang S, Feng L, Liu Z. Photothermally enhanced photodynamic therapy delivered by nano-graphene oxide. ACS Nano. 2011;5:7000–9. Liu C, Cao Y, Cheng Y, Wang D, Xu T, Su L, Zhang X, Dong H. An open source and reduce expenditure ROS generation strategy for chemodynamic/photodynamic synergistic therapy. Nat Commun. 2020;11:1735. Ethirajan M, Chen Y, Joshi P, Pandey RK. The role of porphyrin chemistry in tumor imaging and photodynamic therapy. Chem Soc Rev. 2011;40:340–62. Yu Z, Sun Q, Pan W, Li N, Tang B. A Near-infrared triggered nanophotosensitizer inducing domino effect on mitochondrial reactive oxygen species burst for cancer therapy. ACS Nano. 2015;9:11064–74. Yu N, Ding M, Wang F, Zhou J, Shi X, Cai R, Li J. Near-infrared photoactivatable semiconducting polymer nanocomplexes with bispecific metabolism interventions for enhanced cancer immunotherapy. Nano Today. 2022;46:101600. Sun Q, Hou X, Yang J, Zhang M, Yang Y, Liu Y, Shen W, Yin D. Heparin-coated photosensitive metal-organic frameworks as drug delivery nanoplatforms of autophagy inhibitors for sensitized photodynamic therapy against breast cancer. ACS Appl Mater Interfaces. 2021;13:55577–90. Zhu B, Li S, Yu L, Hu W, Sheng D, Hou J, Zhao N, Hou X, Wu Y, Han Z, et al. Inhibition of autophagy with chloroquine enhanced sinoporphyrin sodium mediated photodynamic therapy-induced apoptosis in human colorectal cancer cells. Int J Biol Sci. 2019;15:12–23. Xie L, Wang L, Li L, Liu C, Guo L, Liao Y, Zhou S, Wu W, Duo Y, Shi L, et al. Novel carrier-free nanodrug enhances photodynamic effects by blocking the autophagy pathway and synergistically triggers immunogenic cell death for the efficient treatment of breast cancer. ACS Appl Mater Interfaces. 2024;16:5683–95. Schwarz DS, Blower MD. The endoplasmic reticulum: structure, function and response to cellular signaling. Cell Mol Life Sci. 2016;73:79–94. Zhu Y, Zhu X, Tang C, Guan X, Zhang W. Progress and challenges of immunotherapy in triple-negative breast cancer. Biochim Biophys Acta Rev Cancer. 2021;1876:188593. Gupta GK, Collier AL, Lee D, Hoefer RA, Zheleva V, van Siewertsz LL, Tang-Tan AM, Guye ML, Chang DZ, Winston JS, et al. Perspectives on triple-negative breast cancer: current treatment strategies, unmet needs, and potential targets for future therapies. Cancers. 2020;12:2392. Leon-Ferre RA, Goetz MP. Advances in systemic therapies for triple negative breast cancer. BMJ. 2023;381:e071674. Hiscox S, Nicholson RI. Src inhibitors in breast cancer therapy. Expert Opin Ther Targets. 2008;12:757–67. Duffy MJ, McGowan PM, Crown J. Targeted therapy for triple-negative breast cancer: where are we? Int J Cancer. 2012;131:2471–7. Finn RS, Dering J, Ginther C, Wilson CA, Glaspy P, Tchekmedyian N, Slamon DJ. Dasatinib, an orally active small molecule inhibitor of both the src and abl kinases, selectively inhibits growth of basal-type/triple-negative breast cancer cell lines growing in vitro. Breast Cancer Res Treat. 2007;105:319–26. Bayraktar S, Glück S. Molecularly targeted therapies for metastatic triple-negative breast cancer. Breast Cancer Res Treat. 2013;138:21–35. Araujo J, Logothetis C. Dasatinib: a potent SRC inhibitor in clinical development for the treatment of solid tumors. Cancer Treat Rev. 2010;36:492–500. Koopmans C, Ritter H. Formation of physical hydrogels via host-guest interactions of β-cyclodextrin polymers and copolymers bearing adamantyl groups. Macromolecules. 2008;41:7418–22. Yuan G, Wang Q, You Z, Chen X, Xue J, Jia X, Chen J. A novel hierarchical targeting and controllable smart nanoparticles for enhanced in situ nuclear photodynamic therapy. Nano Res. 2022;15:4212–23. Huang L, Tonelli AE. Polymer inclusion compounds. Taylor Francis. 1998;38:781–837. Fan W, Xu Y, Li Z, Li Q. Folic acid-modified β-cyclodextrin nanoparticles as drug delivery to load DOX for liver cancer therapeutics. Soft Mater. 2019;17:437–47. Ang CY, Tan SY, Teh C, Lee JM, Wong MFE, Qu Q, Poh LQ, Li M, Zhang Y, Korzh V, et al. Redox and pH dual responsive polymer based nanoparticles for in vivo drug delivery. Small. 2017;13:1602379. Xu X, Zeng Z, Chen J, Huang B, Guan Z, Huang Y, Huang Z, Zhao C. Tumor-targeted supramolecular catalytic nanoreactor for synergistic chemo/chemodynamic therapy via oxidative stress amplification and cascaded Fenton reaction. Chem Eng J. 2020;390:124628. Wankar J, Kotla NG, Gera S, Rasala S, Pandit A, Rochev YA. Recent advances in host-guest self-assembled cyclodextrin carriers: implications for responsive drug delivery and biomedical engineering. Adv Funct Mater. 2020;30:1909049. Tanida I, Ueno T, Kominami E. LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol. 2004;36:2503–18. Chen T, Cen D, Ren Z, Wang Y, Cai X, Huang J, Di Silvio L, Li X, Han G. Bismuth embedded silica nanoparticles loaded with autophagy suppressant to promote photothermal therapy. Biomaterials. 2019;221:119419. Dai H, Yan H, Dong F, Zhang L, Du N, Sun L, Li N, Yu G, Yang Z, Wang Y, et al. Tumor-targeted biomimetic nanoplatform precisely integrates photodynamic therapy and autophagy inhibition for collaborative treatment of oral cancer. Biomater Sci. 2022;10:1456–69. Shi M, Liu Y, Huang J, Chen Z, Ni C, Lu J, Zhang Y, Liu Z, Bai J. Multifunctional theranostic nanoplatform loaded with autophagy inhibitor for enhanced photothermal cancer therapy under mild near-infrared irradiation. Biomaterials Adv. 2022;138:212919. Islam MA, Sooro MA, Zhang P. Autophagic regulation of p62 is critical for cancer therapy. Int J Mol Sci. 2018;19:1405. Senft D, Ronai ZA. UPR, autophagy, and mitochondria crosstalk underlies the ER stress response. Trends Biochem Sci. 2015;40:141–8. Qi Z, Chen L. Endoplasmic reticulum stress and autophagy. Adv Exp Med Biol. 2019;1206:167–77. Sudsaward S, Khunchai S, Thepmalee C, Othman A, Limjindaporn T, Yenchitsomanus P-T, Mutti L, Krstic-Demonacos M, Demonacos C. Endoplasmic reticulum stress, unfolded protein response and autophagy contribute to resistance to glucocorticoid treatment in human acute lymphoblastic leukaemia cells. Int J Oncol. 2020;57:835–44. Li W, Yang J, Luo L, Jiang M, Qin B, Yin H, Zhu C, Yuan X, Zhang J, Luo Z, et al. Targeting photodynamic and photothermal therapy to the endoplasmic reticulum enhances immunogenic cancer cell death. Nat Commun. 2019;10:3349. Wan J, Zhang X, Li Z, Mo F, Tang D, Xiao H, Wang J, Rong G, Liu T. Oxidative stress amplifiers as immunogenic cell death nanoinducers disrupting mitochondrial redox homeostasis for cancer immunotherapy. Adv Healthc Mater. 2023;12:2202710. Miao Z, Li J, Zeng S, Lv Y, Jia S, Ding D, Li W, Liu Q. Endoplasmic reticulum-targeting AIE photosensitizers to boost immunogenic cell death for immunotherapy of bladder carcinoma. ACS Appl Mater Interfaces. 2024;16:245–60. Zhang X, Wan J, Mo F, Tang D, Xiao H, Li Z, Jia J, Liu T. Targeting bone tumor and subcellular endoplasmic reticulum via near infrared II fluorescent polymer for photodynamic-immunotherapy to break the step‐reduction delivery dilemma. Adv Sci. 2022;9:e2201819. Fucikova J, Kepp O, Kasikova L, Petroni G, Yamazaki T, Liu P, Zhao L, Spisek R, Kroemer G, Galluzzi L. Detection of immunogenic cell death and its relevance for cancer therapy. Cell Death Dis. 2020;11:1013. Wang L, Guan R, Xie L, Liao X, Xiong K, Rees TW, Chen Y, Ji L, Chao H. An ER-targeting iridium(III) complex that induces immunogenic cell death in non-small-cell lung cancer. Angew Chem Int Ed Engl. 2021;60:4657–65. Zhang X, Tang J, Li C, Lu Y, Cheng L, Liu J. A targeting black phosphorus nanoparticle based immune cells nano-regulator for photodynamic/photothermal and photo-immunotherapy. Bioact Mater. 2021;6:472–89. Li Z, Lai X, Fu S, Ren L, Cai H, Zhang H, Gu Z, Ma X, Luo K. Immunogenic cell death activates the tumor immune microenvironment to boost the immunotherapy efficiency. Adv Sci. 2022;9:e2201734. Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.docx 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4950334","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":355954332,"identity":"9d854e4f-ef5c-4b91-a897-5de9e33a825d","order_by":0,"name":"Gankun Yuan","email":"","orcid":"","institution":"Henan Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Gankun","middleName":"","lastName":"Yuan","suffix":""},{"id":355954333,"identity":"47e35a79-0ac7-4594-b845-b76fb2852f22","order_by":1,"name":"Ruyue Yang","email":"","orcid":"","institution":"Henan University of Science \u0026 Technology","correspondingAuthor":false,"prefix":"","firstName":"Ruyue","middleName":"","lastName":"Yang","suffix":""},{"id":355954334,"identity":"2d2344c3-ffd7-4e48-8aff-28a5c2bbc487","order_by":2,"name":"Wenjing Wen","email":"","orcid":"","institution":"Henan University of Science \u0026 Technology","correspondingAuthor":false,"prefix":"","firstName":"Wenjing","middleName":"","lastName":"Wen","suffix":""},{"id":355954335,"identity":"ec737dba-7351-4fad-a902-c142dcb89d11","order_by":3,"name":"Zhaoyi Wei","email":"","orcid":"","institution":"Henan University of Science \u0026 Technology","correspondingAuthor":false,"prefix":"","firstName":"Zhaoyi","middleName":"","lastName":"Wei","suffix":""},{"id":355954336,"identity":"1172ecc7-209b-4e94-8cfa-18b373a77be2","order_by":4,"name":"Meiru Song","email":"","orcid":"","institution":"Henan Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Meiru","middleName":"","lastName":"Song","suffix":""},{"id":355954337,"identity":"6104442c-9f28-464d-91d3-afd182833816","order_by":5,"name":"Lingyang Zhang","email":"","orcid":"","institution":"Henan Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lingyang","middleName":"","lastName":"Zhang","suffix":""},{"id":355954338,"identity":"5b240261-b36d-4ba0-98c4-546ad6c61160","order_by":6,"name":"Kun Hou","email":"","orcid":"","institution":"Henan Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Kun","middleName":"","lastName":"Hou","suffix":""},{"id":355954339,"identity":"a2145c4a-736f-410d-81f4-08f321b87087","order_by":7,"name":"Gaofeng Liang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYBACPgbGB0DKRo6N4UDigw8GNnYEtbAxMBsAqTRjfsYDjw1nFKQlE6vlcOLM5oPPpHk+HGJsIKiFfzHj44JfhxM3HDucbGxjcICZgf3w0Q14tUg8Zjae2ZduvOHMscTHOQZ3+Bh40tJu4Ndy/pg0b4+17IYbZ5KNcwyeMTNI8JgR0HKY/TdvDzPjhvvvv0lbGBxmbCCohb+ZjZnnh7PizIYDadIMRGmRYGaW5m0ABjLDgWTDHoO0ZDZCfuHnP8z4mecPNCp//LGx42c/fAyvFgaJBAYGxjZke/EqB1tzAEj8IahsFIyCUTAKRjIAALFcT1Pmo2K5AAAAAElFTkSuQmCC","orcid":"","institution":"Henan Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Gaofeng","middleName":"","lastName":"Liang","suffix":""}],"badges":[],"createdAt":"2024-08-21 09:21:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4950334/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4950334/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65296001,"identity":"9161f4cb-fc25-4751-90e1-e9c744259d3c","added_by":"auto","created_at":"2024-09-25 19:15:09","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":643356,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of Da-CD@CET@CQ NPs. The transmission electron microscope (TEM) images of A) Da-CD NPs and B) Da-CD@CET@CQ NPs. The diameter of C) Da-CD NPs and D) Da-CD@CET@CQ NPs by dynamic light scattering. E) The state and F) PDI change of Da-CD NPs, CD@CET@CQ NPs, Da-CD@Ce6@CQ NPs and Da-CD@CET@CQ NPs in deionized water for 7 days. The stability of G) Da-CD NPs and H) Da-CD@CET@CQ NPs in deionized water and RPMI 1640 medium for 7days. I) UV-vis absorption spectra of free CET, CQ, Da-CD NPs, Da-CD@CET NPs and Da-CD@CET@CQ NPs in DMF. J)Zeta potential of CD, CQ, CET, Da-CD NPs and Da-CD@CET@CQ NPs in deionized water. K) Release ratio curve of CET and L) CQ at different pH values. Data were shown as mean ± SD (n = 3). Scale bar: 200 nm.\u003c/p\u003e","description":"","filename":"Figure.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4950334/v1/29329f20239996b455ec223d.jpg"},{"id":65296477,"identity":"885d6190-7f29-46bb-9f80-a96f7361af3f","added_by":"auto","created_at":"2024-09-25 19:31:09","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":926011,"visible":true,"origin":"","legend":"\u003cp\u003eA) Intracellular uptake and B) the quantitative study ofCD@CET@CQ NPs and Da-CD@CET@CQ NPs incubated with 4T1 cells by FCM. C) Intracellular uptake and D) the quantitative study of Da-CD@CET@CQ NPs incubated with 4T1 cells pretreated with free Da (0, 5, 10, 20 nM) by FCM. E) Intracellular uptake and F) the quantitative study of Da-CD@CET@CQ NPs incubated with 4T1 cells and 293T cells by FCM. G) ER targeting of Ce6, H) CET and I) Da-CD@CET@CQ NPs in 4T1 cells. J) Qualitative analysis of the line-scanning spectra of lines I, II and III in (G), (H) and (I), the green and red curves represent the fluorescence intensity of ER Tracker and PS, respectively. Scale bar: 20 μm. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4950334/v1/0e5352787e31aeedc4f05615.jpg"},{"id":65296225,"identity":"05491d68-ce87-4400-9441-74f192452b9c","added_by":"auto","created_at":"2024-09-25 19:23:09","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2217136,"visible":true,"origin":"","legend":"\u003cp\u003eA) ROS production capacity of different groups of 4T1 cells after light irradiation by DCFH-DA staining assay (670 nm, 4 mW·cm\u003csup\u003e-2\u003c/sup\u003e). Scale bare: 50 μm. B) The quantitative study of DCF in different groups. C) Cell viability of 4T1 cells with different treatments by MTT assay without irradiation and D) under light illumination (670 nm, 4 mW·cm\u003csup\u003e-2\u003c/sup\u003e). E) Live/dead co-staining assay of 4T1 cells with different treatments and F) Quantitative data indicating the percentage of live and dead cells. G）Live/dead co-staining assay of 4T1 cells with different concentrations of Da-CD@CET@CQ NPs and H) Quantitative data indicating the percentage of live and dead cells. Scale bar: 100 μm. I) and J) Cell apoptosis of 4T1 cells with different treatments by using Annexin V-FITC and PI after light irradiation . Data are expressed as the mean ± SD (n = 3). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4950334/v1/08d0d15435aca6d78863f885.jpg"},{"id":65296230,"identity":"067e3cba-b0a6-4c92-9389-1784abd5b362","added_by":"auto","created_at":"2024-09-25 19:23:09","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":846845,"visible":true,"origin":"","legend":"\u003cp\u003eA) Western blot analysis for the autophagy-related proteins LC3-II and P62 in 4T1 cells after various treatments. B) Expression of LC3-II relative to β-actin. C）Expression of P62 relative to β-actin. D) CLSM images of 4T1 cells stained by DAPI and LysoTracker Green. E) CLSM images of the cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e level in cells with different treatments. F) CLSM images of CHOP-stained cells with different treatments. G) CLSM images of the migration of HMGB1 in 4T1 cells with different treatments. H) CLSM images of the exposure of CRT in 4T1 cells with different treatments. I) The quantitative study of the exposure of CRT by CLSM. G) The relative ATP release of 4T1 cells with different treatments. K) Western blot results of the ER stress proteins with different treatments. Scale bar: 50 μm. Data are shown as mean ± SD (n = 3). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4950334/v1/42893cf1172f24fb89ac6f64.jpg"},{"id":65296005,"identity":"1bb2a90f-33a2-4681-9e15-dd912b9448c7","added_by":"auto","created_at":"2024-09-25 19:15:09","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1323094,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e targeting study in BALB/c mice. A) \u003cem\u003eIn vivo\u003c/em\u003e tumor imaging after administration of both nanocomplex in 0-24 h (n = 5 mice per group). B) The relative mean tumor content of both nanocomplex over time. C) Ex vivo fluorescence images of the major organs and tumor regions of the mice sacrificed at 12 h after administration of both nanocomplex. H: Heart; Li: Liver; S: Spleen; Lu: Lung; K: Kidney; T: Tumor. D) The quantitative study of the fluorescence intensities of the major organs and tumors. E）The CLSM image of the edge site of the tumor section and F) the average fluorescence intensity of CET. G) The CLSM image of the core site of the tumor section and H) the average fluorescence intensity of CET. Blue channel: DAPI of nucleus; Red channel:CET. Data are presented as mean ± SD (n = 3). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4950334/v1/5a10b95307308371dc8a865b.jpg"},{"id":65296231,"identity":"2a51fda3-627c-48bb-b9f5-8d57c5337e2f","added_by":"auto","created_at":"2024-09-25 19:23:09","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2180825,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e PDT efficiency of Da-CD@CET@CQ NPs in BABL/c mice. A) Establishment of an orthotopic 4T1 tumor model for \u003cem\u003ein vivo\u003c/em\u003e therapeutic (670 nm，50 mW·cm\u003csup\u003e-2\u003c/sup\u003e, 10 min). B) Tumor photographs of stripped from mice with different treatments at day 15. C) Tumor growth curves of mice with different treatments at day 15. D) Average tumor weight with different treatments at day 15. E) H\u0026amp;E, TUNEL staining and immunohistochemical analysis of tumor tissues in different groups. F) Lung metastatic nodule numbers and H\u0026amp;E staining images of lung tissue from 4T1 tumor-bearing mice after treatments 15 days under different conditions. Data are presented as mean ± SD (n = 5). Scale bar: 50 μm. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01,***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4950334/v1/779015ab0d6e7b1d97c1ffa7.jpg"},{"id":65296007,"identity":"c0e5feba-b375-4e98-9e24-aba3bf06d6ca","added_by":"auto","created_at":"2024-09-25 19:15:09","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1899317,"visible":true,"origin":"","legend":"\u003cp\u003eCascade targeting Da-CD@CET@CQ NPs enhanced the antitumor immunity \u003cem\u003ein vivo\u003c/em\u003e. A) Staining of different antibodies in the different group: DAPI (Blue), CD8(Cyan) CD49B (Yellow), CD19 (Red), iNOS (Green). B) CD8, C) CD19, D) CD49B, E) iNOS antibodies’expression levels in the different groups. F) Levels of TNF-α, G) IFN-γ and H) IL-10 in tumor tissues of different treatment groups measured by ELISA. Data were shown as the mean ± SD (n = 3). Scale bar: 20 μm. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4950334/v1/8d62969375c90234fe69de20.jpg"},{"id":65993853,"identity":"5a2b03b0-519a-43fc-be0b-c5b7c3915d16","added_by":"auto","created_at":"2024-10-05 22:01:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10820704,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4950334/v1/1ee654e0-ebec-49ff-bb82-541d0a1e7d7e.pdf"},{"id":65296040,"identity":"ab9ba7b2-2914-4953-90d1-077ad3907530","added_by":"auto","created_at":"2024-09-25 19:15:09","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":3829983,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4950334/v1/26d6e365e7385926673f0374.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Novel immunogenic cell death inducer combined with autophagy inhibitor to amplify photodynamic synergistic immunotherapy for triple-negative breast cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eImmunotherapy as a \u0026ldquo;revolutionary\u0026rdquo;therapy for cancer treatment, which has shown breakthrough therapeutic effects on tumor recurrence and metastasis due to its unique treatment mode\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Immunogenic cell death (ICD) is a type of programmed cell death that can regulate the tumor immune microenvironment and activate systemic antitumor immune responses by the release of damage-associated molecular patterns (DAMPs), which is attributed to its ability to mobilize the immune system to destroy cancer cells via the\u0026ldquo;by stander effect\u0026rdquo;\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. According to previous literature reports, sustained endoplasmic reticulum (ER) stress after reactive oxygen species (ROS) or drug stimulation can promote the induction of ICD and the release of DAMPs, such as calreticulin (CRT), high mobility group protein 1 (HMGB1), and adenosine triphosphate (ATP), from dying tumor cells\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Unfortunately, most of the ICD inducers that can cause ER stress are mainly toxic chemotherapeutic agents, and the number is still very small\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Therefore, it still has great significance that to develop a novel and effective ICD inducer.\u003c/p\u003e\n\u003cp\u003ePhotodynamic therapy (PDT) has become a popular cancer treatment modality due to its unique advantages, such as minimal invasiveness, good efficacy and lack of notable drug resistance\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. In addition to direct oxidative killing of tumor cells, PDT can also induce ICD effects in the body to promote anti-tumor immunotherapy\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Unfortunately, the effect of this synergistic immunotherapy for tumors is still limited due to the insufficient PDT-mediated ICD effect\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Moreover, traditional photosensitizers (PS) such as phthalocyanines and porphyrins show insufficient tumor tissue targeting and poor biosolubility, which further hinder the anti-tumor effect of PDT. With the development and application of nanotechnology, the use of nanodelivery systems as PS carriers has provided a new platform for the development of PDT, increasing the selective enrichment of PS in tumors\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. However, the desired PDT efficacy is undoubtedly limited to the vicinity of the generation site of ROS due to its extremely short lifespan (\u0026lt;\u0026thinsp;0.04 \u0026micro;s) and diffusion range (\u0026lt;\u0026thinsp;0.02 \u0026micro;m), which is still one of the main factors hindering the combination of nano-delivery materials and PDT for tumor therapy\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. In addition, autophagy can repair and reverse tumor cell damage caused by PDT, resulting in incomplete cell death, which increases the tolerance of tumor cells to PDT\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is urgent to develop a strategy that can further delivery of PS to major suborganelles and inhibition of autophagy to enhance the anti-tumor effects of PDT.\u003c/p\u003e\n\u003cp\u003eEndoplasmic reticulum(ER)is the main site of protein synthesis, lipid production and calcium ion storage in eukaryotic cells\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Therefore, targeting PS to the ER to achieve in situ ER PDT has become a promising tumor treatment strategy. Especially, PS enriched in the ER can act as an ICD inducer to induce excessive or abnormal ER stress through the continuous production of ROS, which can further amplify the PDT-mediated ICD effect. Moreover, triple-negative breast cancer (TNBC) is a subtype of breast cancer that is aggressive, easily metastasizes, result in a poor prognosis. As the expression of estrogen receptor, progesterone receptor and the proto-oncogene Her-2 is negative on the surface of TNBC tumors, very few anticancer drugs can be enriched in tumor cells, substantially diminishing their antitumor effects\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Excitingly, several studies have shown that the levels of SRC proteins are significantly higher in TNBC than in other breast cancer subtypes\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is essential to develop a safe and effective strategy for TNBC targeted therapy by targeting SRC protein to improve the anti-tumor effect.\u003c/p\u003e\n\u003cp\u003eHerein, we successfully covalently coupled the ER-targeting ligand p-toluenesulfonyl chloride with the PS chlorin e6 (Ce6) via an ethylenediamine chain to obtain the CET. Subsequently, the CET and autophagy inhibitor CQ were mixed with poly-\u0026beta;-CD NPs, and the Da-Ad obtained by the reaction of adamantanamine and a tyrosine kinase inhibitor dasatinib, which can precisely target SRC family proteins\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e, can linked to the surface of the nanomaterial via host-guest interactions to obtain the cascade-targeted nanomaterial Da-CD@CET@CQ (Scheme \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Given that Da is involved in SRC-mediated targeting recognition in TNBC cells, Da-CD@CET@CQ NPs achieved specific enrichment in TNBC cells and selective accumulation of CET in ER due to the acidic environment of the tumors that prompts the release of the drug from the nanomaterials. On the one hand, CET generated sufficient ROS in ER to kill tumor cells under 670 nm light irradiation; on the other hand, the in situ generation of ROS in ER led to disruption of calcium homeostasis and a sustained ER stress, which in turn amplified the ICD effect and increased the DAMPs released by dying tumor cells. Moreover, stimulation by DAMPs induced dendritic cell (DC) maturation and increased the DC antigen-presenting capacity, thereby promoting the immune response. More importantly, the autophagy inhibitor CQ released by the Da-CD@CET@CQ NPs inhibited the autophagic protection against PDT-induced damage to cells, further increasing the anti-tumor effect of PDT. Collectively, the Da-CD@CET@CQ NPs not only showed cascade targeting of TNBC tumor cells and ER, but also further sustainably increased ER stress, resulting in enhanced ICD effect, and combined with autophagy inhibitor to obtain a more robust PDT effect, ultimately realizing more effective synergistic photodynamic immunotherapy for TNBC.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials and Apparatus\u003c/h2\u003e \u003cp\u003eβ-Cyclodextrin, dasatinib, amantadine, p-methylbenzene sulfonyl chloride, ethylenediamine, chlorin e6 and chloroquine purchased from Aladdin. Hoechst 33258, ER-Tracker Green, 4',6-diamidino-2-phenylindole (DAPI), DCFH-DA, Fluo-4 AM probe and methyl thiazolyl tetrazolium (MTT) were purchased from Beyotime Biotechnology. Beijing Solarbio Science\u0026amp;Technology Co., Ltd. offers the Calcein-AM/PI double staining kit and Annexin V-FITC apoptosis detection kit. The antibodies used in the experiment were provided by Proteintech Group, Inc. The relevant biological reagents used in the cell culture were purchased from Hyclone, Cytiva. The transmission electron microscope (TEM, Thermo Scientific FEI, USA) was applied for the morphological examinations of the nanoparticles. The hydrodynamic diameter and zeta potential of the nanoparticles were measured by Zatasizer Nano ZSE (Malvern instrument, UK) in deionized water. The ultraviolet-visible (UV-vis) near infrared spectrometer (Shimadzu, UV-3600, Japan) and the fluorospectro photometer (Cary Eclipse, Agilent) were recorded the photophysics property of the nanoparticles. The confocal laser scanning microscope (CLSM, C2+, Nikon) was used for the fluorescent cell images of the nanoparticles. The Flow cytometer (BD Accuri C6, USA) was applied for the cell uptake and apoptosis assays. \u003cem\u003eIn vivo\u003c/em\u003e tumor tissue uptake were observed by Small Animal Live Imaging System (Vilber BT100, France).\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003ePreparation of Da-CD@CET@CQ NPs\u003c/h2\u003e \u003cp\u003eThe obtained Poly-β-CD (21.0 mg), CET (2.0 mg) and CQ (2.0 mg) were dissolved in a mixture of DMSO and water (1:3) at room temperature and shielded from light for 2 h. Then, Da-Ad (0.5 mg) was dissolved in a mixture of DMSO and water (1:3) and slowly added to the reaction system. At room temperature and away from light, the reaction continued for 12 h. After the reaction, the reaction mixture was transferred to a dialysis bag with MW\u0026thinsp;=\u0026thinsp;3000 for dialysis for 24 h, and the water was changed every 3 h. Finally, the brown black solid powder Da-CD@CET@CQ NPs was obtained by vacuum freeze-drying.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eThe storage stability of Da-CD@CET@CQ NPs\u003c/h2\u003e \u003cp\u003eFirst, Da-CD NPs (2.0 mg), CD@CET NPs and Da-CD@CET NPs solids were dissolved in 3 mL deionized water and RPMI 1640 respectively. Then, the clarified state of nanomaterials in deionized water was observed. At the designated time, the mean hydrodynamic diameter and PDI values of the solution were measured by DLS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of CET and CQ releasing\u003c/h2\u003e \u003cp\u003eTo evaluate the release efficiency of CET and CQ under physiological and acidic conditions, the Da-CD@CET@CQ NPs was dissolved in different PBS (pH\u0026thinsp;=\u0026thinsp;7.4, 6.0 and 5.0), and the absorbance of CET and CQ were determined by UV-vis spectroscopy at different time points, the drug release rates at different pH were calculated, and time-dependent release curves were plotted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eTriple negative breast cancer 4T1 cells and human embryonic renal cell HEK293T cells were purchased from Institute of Basic Medical Sciences (IBMS) of the Chinese Academy of Science. The 4T1 cells were incubated in RPMI 1640 and HEK293T cells were cultured with DMEM medium, which containing 10% fetal bovine serum and 1% antibiotics (penicillin/streptomycin, 100 U mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at 37\u0026deg;C in a humidified environment of 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCellular uptake\u003c/h2\u003e \u003cp\u003e4T1 cells and HEK293T cells were incubated in cell culture dishes for 24 h, and new medium containing CD@CET@CQ NPs or Da-CD@CET@CQ NPs (25 \u0026micro;g/mL) was added for 12 h, respectively. For the targeted competitive uptake assay, the 4T1 cells were preincubated with different concentrations of free dasatinib (0、5、10 and 20 nM) for 12 h. After PBS cleaning, the Da-CD@CET@CQ NPs (25 \u0026micro;g/mL) was added for further 12 h. The CLSM and flow cytometry were used to observe the intracellular fluorescence intensity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eThe ER Localization Study\u003c/h2\u003e \u003cp\u003eIn the cell culture dish, the free Ce6, CET, Da-CD@CET@CQ NPs (25 \u0026micro;g/mL) were incubated with the 4T1 cells for 12 h, respectively. Subsequently, the PBS was used to wash the dish. The ER-Tracker Green was incubated for 20 min and washed with PBS. The fluorescence of the ER-Tracker Green was excited at 488 nm and monitored at 510\u0026ndash;570 nm, and the photosensitizer fluorescence was excited at 633 nm and monitored at 650\u0026ndash;750 nm by CLSM.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAutophagy inhibition experiment\u003c/b\u003e \u003cb\u003eIn Vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAutophagy marker proteins LC3 and P62 were quantified to evaluate the ability to inhibit autophagy. Cells treated with different drugs and a laser, then supernatant cells were collected and cut in a RIPA(Radio Immunoprecipitation Assay) buffer containing protease inhibitors. The protein was quantified by Coomassie leucocyanin assay. The primary antibodies were anti-LC3 (81004-1-RR) and anti-P62 (66184-1-Ig),which were purchased from Proteintech Group. Refer to western blot test for specific steps.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMeasurements of cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e\u003c/h2\u003e \u003cp\u003eThe cytoplasmic Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis was examined by CLSM analysis. First, 1 mL 4T1 cells (6 x 10\u003csup\u003e4\u003c/sup\u003e) were added to the confocal dish and incubated for 12 h. Subsequently, the suspension treated with different drugs was incubated with 2 \u0026micro;M Fluo-4-AM solution for 30 min under dark, then centrifuged and washed. The nuclei were stained with DAPI. Finally, the image was captured with CLSM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eER stress analysis\u003c/h2\u003e \u003cp\u003eFour unfolded protein responses (UPR) were quantified: proapoptotic C/EPB homologous protein, inositol requires enzyme 1α phosphorylation (p-IRE-1α), activated transcription factor 6 (ATF6), and protein kinase R-like ER kinase (PERK) to assess ER stress capacity. Different drug-treated cells were laser treated, and subsequently the supernatant cells were collected and cleaved in a buffer of RIPA (Radio-Immunoprecipitation Assay) containing protease inhibitors. The protein was quantified by the Coomassie brilliant blue protein quantitaion method. The primary antibodies used anti-p-DDIT3/CHOP (ET1703-05), anti-p-IRE-1α (Ser724), anti-ATF6 (EM1701-94), anti-p-eIF-2α (HA721510) and anti-β-Actin (AF2811) were purchased from HuaBio Biotechnology and Beyotime Institute of Biotechnology. Refer to western blot test for specific steps.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunogenic cell death\u003c/b\u003e \u003cb\u003eIn vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eExposure to CRT was measured using CLSM. First, 1 mL 4T1 cell (1 x 10\u003csup\u003e5\u003c/sup\u003e) was added to the confocal dish and incubated for 12 h. The cells were treated with equal photosensitive unit concentration of different groups for 12 h. Then, the cells were fixed with 4% paraformaldehyde for 20 min and immersed in 1% fetal bovine serum PBS for 30 min. The primary antibody of CRT was incubated at 4℃ overnight, and the secondary antibody of 488 coupled was incubated with PBS for 1h after washing three times. Finally, the nucleus was stained with DAPI, and the fluorescence intensity of the cell surface was observed under CLSM with 405 nm and 488 nm lasers, respectively.\u003c/p\u003e \u003cp\u003eWe used CLSM fluorescence distribution assay to evaluate intracellular HMGB1 migration. Firstly, 1 mL 4T1 cell (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) was added to the confocal dish and incubated for 12 h. The cells were then treated under the same conditions as the CRT analysis. Then, the cells were fixed with 4% paraformaldehyde for 20 min and infiltrated with 0.1% Triton X-100 for 10 minutes, and incubated with 1% fetal bovine serum PBS for 30 minutes. The primary antibody of HMGB1 was incubated at 4℃ overnight, after washing three times, the secondary antibody of iFluor\u0026trade; 647 was incubated with PBS for 1 h. Finally, the nucleus was stained with DAPI, and the fluorescence intensity of the cell surface was observed under CLSM with 405 nm and 633 nm lasers, respectively.\u003c/p\u003e \u003cp\u003eThe collected cells in each group were added with 0.3 mL ATP test reagent (in 2 mL EP tube) for every 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells, covered tightly and mixed evenly, inserted into a float, placed in a boiling water bath for 10 minutes, and centrifuged at 4℃ at 10000\u0026times;g for 10 minutes after water cooling, and the superserum was taken. The extracellular ATP concentration in each supernatant was analyzed according to the operating instructions of the ATP chemiluminescence assay kit (Cat: E-BC-F002, Elabscience).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blotting\u003c/h2\u003e \u003cp\u003eThe cell lysate (RIPA: protease inhibitor: phosphorylase inhibitor\u0026thinsp;=\u0026thinsp;100:1:1) was added to the cell culture dish to extract the protein. The protein was quantified by Coomassie leucocyanin assay. The separation gel with appropriate concentration was selected according to the protein molecular weight and 30 \u0026micro;g of protein sample was added to each well. The sample was separated by SDS-PAGE gel electrophoresis and transferred to PVDF membrane, which was sealed with 5% bovine serum albumin for 1 h and incubated with primary antibody at 4℃ overnight. After TBST cleaning, the corresponding secondary antibodies were incubated at room temperature for 1 h. After TBST cleaning, the membranes were visualized using the Tanon 5200 Multi Imaging System.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular ROS Generation Assay\u003c/h2\u003e \u003cp\u003eThe 4T1 cells were co-cultured with the PBS, Da-CD NPs, free CQ, free CET, CD@CET@CQ, Da-CD@Ce6@CQ, Da-CD@CET and Da-CD@CET@CQ NPs (25 \u0026micro;g/mL) group for 12 h, respectively. Meanwhile, the Da-CD@CET@CQ NPs at different concentration (0、12.5、25 and 50 \u0026micro;g/mL) were incubated with 4T1 cells for 12 h. The PBS was used to wash the dish, and all groups were incubated with DCFH-DA (1:1000) probe for 30 min. A light source (λ\u0026thinsp;=\u0026thinsp;670 nm, 4 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) was applied for 5 min in all groups. Finally, the CLSM was used to measure the fluorescence of DCF excited at 488 nm.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003ePhotodynamic Cytostatic Activities\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe 4T1 cells were seeded per well in 96-well cell plates with different groups: PBS, Da-CD NPs, free CQ, free CET, CD@CET@CQ, Da-CD@Ce6@CQ, Da-CD@CET and Da-CD@CET@CQ NPs with various concentrations. After 12 h incubation, the medium was replaced with a new medium and the cells were irradiated with a light source for 5 min (λ\u0026thinsp;=\u0026thinsp;670 nm, 4 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e), the cell viability was counted by MTT assay kit. The cell growth inhibitory effects were calculated by the following equation:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCell viability (%) = {(A\u003csub\u003e\u003cem\u003etreatment\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-\u003c/em\u003eA\u003csub\u003e\u003cem\u003eblank\u003c/em\u003e\u003c/sub\u003e)/(A\u003csub\u003e\u003cem\u003econtrol\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e-\u003c/em\u003eA\u003csub\u003e\u003cem\u003eblank\u003c/em\u003e\u003c/sub\u003e)} \u0026times; 100%\u003c/h2\u003e \u003cp\u003eFor live/dead cells experiments, 4T1 cells with 50 \u0026micro;g/mL different drugs were incubated in confocal dishes for 12 h and washed repeatedly with saline. Meanwhile, the Da-CD@CET@CQ NPs at different concentration (0、6.25、12.5 and 50 \u0026micro;g/mL) were incubated with 4T1 cells for 12 h. All samples were irradiated with a light source for 5 min (λ\u0026thinsp;=\u0026thinsp;670 nm, 4 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e). After that, a calcein AM solution (500 \u0026micro;L, 5 \u0026micro;M) and a PI solution (500 \u0026micro;L, 10 mM) were added to the culture medium and incubated for 20 min to stain the cells. Finally, the fluorescence images were observed by CLSM (excited at 488 nm and 546 nm, respectively).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eThe cell apoptosis study\u003c/h2\u003e \u003cp\u003e50 \u0026micro;g/mL of different drugs were incubated with 4T1 cells for 12 h and washed repeatedly with PBS. All samples were irradiated with a light source for 5 min (λ\u0026thinsp;=\u0026thinsp;670 nm, 4 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e), and then incubated with FITC-Annexin V and PI for 20 min. Finally, the cell apoptosis was detected by FCM.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003etumor tissue targeting\u003c/b\u003e\u003c/p\u003e \u003cp\u003e All animal experiments were approved by the Animal Care and Use Committee of Henan Academy of Sciences. Female BALB/c mice with 4T1 tumors were randomly divided into two groups (n\u0026thinsp;=\u0026thinsp;5 mice per group) and then treated with CD@CET@CQ NPs and Da-CD@CET@CQ NPs (10 mg/kg) via tail vein injection, respectively. All mice were observed by small animal live imaging system at different time points.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eanti-tumor study\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFemale BALB/c mice with 4T1 tumors were randomly divided into eight groups (n\u0026thinsp;=\u0026thinsp;5 mice per group) include: PBS、Da-CD NPs 、free CQ、free CET、CD@CET@CQ NPs、Da-CD@Ce6@CQ NPs、Da-CD@CET NPs and Da-CD@CET@CQ NPs (10 mg/kg). After the drug was injected into the mice through the tail vein for 12 h, the tumor site was irradiated by a laser for 10 min (670 nm, 50 mW). Tumor volume and body weight were measured every other day. After 15 days of treatment, the major organs and tumor of mice were removed and weighed. The tumor volume was ascertained employing the formula below:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eTumor volume (mm\u003csup\u003e3\u003c/sup\u003e ) = (L\u0026times;W\u003csup\u003e2\u003c/sup\u003e ) /2\u003c/h2\u003e \u003cp\u003ewhere L is the tumor length and W is the tumor width.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis\u003c/h2\u003e \u003cp\u003eThe treated mice were sacrificed, and the main organs such as heart, liver, lung, spleen, kidney, and tumor were collected and fixed in 10% formalin. Paraffin sections were stained with hematoxylin and eosin (H\u0026amp;E) and stained with the TUNEL kit and immunohistochemistry (CD34 and Ki67).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eImmunity Response Study\u003c/h2\u003e \u003cp\u003eAfter the tissue sections were dehydrated and repaired, different primary antibodies were incubated at room temperature, and then different secondary antibodies were incubated. Antigen recovery was performed after fluorescence staining of PPD650. Different primary antibodies were used successively to culture the secondary antibodies coupled with horseradish peroxidase, and the tyramide signal was amplified. Incubated with 4'-6' -diamino-2-phenylindole (DAPI) at room temperature for 10 min, washed with TBST buffer and sterile water for 3 min, respectively. Finally, anti-hardening sealant and nail polish were added, and the stained tissue sections were observed under fluorescence microscope.\u003c/p\u003e \u003cp\u003eDifferent groups of mice were killed after treatment and blood samples were collected. The blood samples were then centrifuged at 4℃ at 12000 RPM for 10 min to collect plasma for analysis of TNF-α, IFN-γ and IL-10 expression. Finally, the test kit is used to measure according to the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll the experiments were performed for at least three independent times and data are expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. GraphPad Prism 6 software was applied for statistical analysis. The statistically significance level is *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec21\"\u003e\n \u003ch2\u003ePreparation and characterization of Da-CD@CET@CQ NPs\u003c/h2\u003e\n \u003cp\u003ePoly-\u0026beta;-CD was synthesized as previously described in the literature\u003csup\u003e[\u003cspan\u003e39\u003c/span\u003e, \u003cspan\u003e40\u003c/span\u003e]\u003c/sup\u003e. The ER-targeted PS CET was obtained by alkylation as well as amidation reactions (Fig. \u003cspan\u003eS1\u003c/span\u003e). The properties of the Da-CD@CET@CQ NPs were characterized. Transmission electron microscopy (TEM) images revealed the specific morphology of the Da-CD and Da-CD@CET@CQ NPs, both of which exhibited uniform spherical structures with diameters of approximately 60 nm (Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eA and B). The results of dynamic light scattering experiments further confirmed that the average hydrodynamic diameters of the Da-CD NPs and Da-CD@CET@CQ NPs were about 58 nm and 62 nm, and the polydispersity indices (PDIs) were approximately 0.23 and 0.22, respectively, as shown in Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eC and D. The stability of materials has always been a prerequisite and key to their applicability and effectiveness, so we evaluated the stability of several nanomaterials. As shown in Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eE, the solutions of Da-CD NPs, CD@CET@CQ NPs, Da-CD@Ce6@CQ NPs and Da-CD@CET@CQ NPs did not show obvious turbidity or precipitation after 7 days in deionized water, similar to the initial state ( Fig. S2), and the PDIs of several nanomaterials were very small and remained basically unchanged within 7 days (Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eF). Moreover, the average hydrodynamic diameters of the Da-CD@CET@CQ NPs were stable and unchanged after 7 days of treatment in deionized water or RPMI 1640 medium (Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eG and H), which suggests that these nanocomplex based on poly-\u0026beta;-CD are stable and homogeneous in the long term. The UV-vis absorption spectra of the Da-CD@CET@CQ NPs at 330 nm showed characteristic absorbance peaks for Da, as well as for CET and CQ at 670 nm and 260 nm (Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eI), indicating that Da had been modified on the Da-CD@CET@CQ NPs surface by stronger host-guest interactions, and the successful loading of CET and CQ. In addition, the emission spectra of the Da-CD@CET@CQ NPs had nearly the same sharp fluorescence band centered at 680 nm as that of CET, further confirming the effective loading of CET (Fig. S3). The zeta potential results showed that the Da-CD NPs were positively charged, decreasing from 15.6 mV to 8.7 mV, further indicating the successful loading of CQ in the Da-CD@CET@CQ NPs (Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eJ). The drug loading capacities (DLCs) of CET and CQ were 6.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15% and 7.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19%, respectively, and the drug loading efficiencies (DLEs) were 79.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18% and 87.50% \u0026plusmn; 0.23%, respectively, as calculated by using UV-vis spectrophotometry and based on the drug standard curve, which demonstrated that the Da-CD NPs had good loading capacities.\u003c/p\u003e\n \u003cp\u003ePoly-\u0026beta;-CD is in a stable state under physiological conditions, which ensures safety during drug delivery, but its structure is damaged, resulting in sparse nanoparticles, in acidic tumor regions according to previous studies\u003csup\u003e[\u003cspan\u003e41\u003c/span\u003e]\u003c/sup\u003e; in addition, under acidic conditions, the payload drug is easily protonated, which in turn reduces its noncovalent interaction with poly-\u0026beta;-CD, leading to a decrease in the degree of poly-\u0026beta;-CD crosslinking and disintegration\u003csup\u003e[\u003cspan\u003e42\u003c/span\u003e\u0026ndash;\u003cspan\u003e45\u003c/span\u003e]\u003c/sup\u003e. To verify the release mechanism and effects of the Da-CD@CET@CQ NPs, the UV-vis spectrophotometry was used to determine the release kinetics of the drug under different acidic conditions. The release rates of CET and CQ after 24 h were only 3.2% and 5.2%, respectively (pH\u0026thinsp;=\u0026thinsp;7.4), which effectively prevented the premature release of the drugs during transportation (Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eK and L). Moreover, the release rate increased significantly when the pH was further reduced to 6.0 and 5.0, especially at pH\u0026thinsp;=\u0026thinsp;5.0, the release rates of CET and CQ reached 89.1% and 91.5%, respectively. These results indicated that the lower pH of the tumor microenvironment could trigger the release of CET and CQ from the Da-CD@CET@CQ NPs, thereby increasing the biosafety and effectiveness of the drugs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\"\u003e\n \u003ch2\u003eIntracellular uptake and endoplasmic reticulum localization of Da-CD@CET@CQ NPs\u003c/h2\u003e\n \u003cp\u003eTo verify the ability of the Da-CD@CET@CQ NPs to target TNBC cells, we used 4T1 cells with high expression of SRC proteins for \u003cem\u003ein vitro\u003c/em\u003e cell uptake experiments. Confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) were used to determine the fluorescence intensity of the Da-CD@CET@CQ NPs in the cells to assess their ability to target 4T1 cells. Based on the time-dependent of drug uptake and the data at different time points (Fig. S4), 12 h was chosen as the appropriate time point for uptake studies. The CLSM and FCM fluorescence data showed that the Da-CD@CET@CQ NPs had stronger intracellular fluorescence after uptake for 12 h, which was approximately 5-fold greater than the cellular fluorescence of the CD@CET@CQ NPs, suggesting that they exhibited more pronounced cellular uptake (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eA and B, Fig. S5). We hypothesize that this result may be due to the high expression of SRC in 4T1 cells and dasatinib-mediated targeting. To test this hypothesis, we preincubated 4T1 cells with different concentrations of free Da (0, 5, 10, and 20 nM) for 12 h before they were incubated with the Da-CD@CET@CQ NPs so that they could pre-bind to intracellular SRC, thereby reducing the amount of free intracellular SRC. The CLSM (Fig. S6) and FCM results (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eC and D) showed that with prebound SRC, the intracellular fluorescence intensity of the Da-CD@CET@CQ NPs decreased with increasing concentrations of free Da. The intracellular fluorescence intensity was significantly reduced by approximately 9-fold when the preincubation concentration was 20 nM, indicating that the Da-CD@CET@CQ NPs could indeed achieve greater accumulation in 4T1 cells through Da and SRC-mediated targeting. Subsequently, to further demonstrate the tumor specificity of the Da-CD@CET@CQ NPs for TNBC, we used a human embryonic kidney cell line (HEK293T) with SRC-negative expression as the control. The CLSM (Fig. S7) and FCM (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eE and F) results showed that the Da-CD@CET@CQ NPs had an approximately 5-fold greater fluorescence intensity in 4T1 cells than in HEK293T cells. Taken together, the cellular uptake results indicated that Da modification significantly increased the ability of Da-CD@CET@CQ NPs to specifically target TNBC cells, providing a potential strategy for targeted PDT of TNBC.\u003c/p\u003e\n \u003cp\u003eBased on the exciting tumor cell targeting results, we validated the ability of the Da-CD@CET@CQ NPs to further localize PS in ER. The PS modified wit\u003cspan type=\"Underline\" name=\"Emphasis\"\u003eh\u003c/span\u003e p-toluenesulfonamide were heavily enriched in ER and in turn directly and instantaneously damaged the structure of ER and increased ER stress, thereby increasing the efficiency of apoptosis. Therefore, the ER-Tracker Green probe was used to study the ER localization of free Ce6, CET and Da-CD@CET@CQ NPs in 4T1 cells. As shown in Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eG, all the red fluorescence of free Ce6 had almost no overlap with the green fluorescence signal of the ER probe. Further qualitative analysis of the line-scanning spectra of the fluorescence intensities showed that Ce6 and the ER-Tracker Green had substantially separated signals (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eJ-I). Qualitative analysis of the line-scanning spectra of the fluorescence intensities showed a partial overlap of CET and the ER-Tracker Green, which indicated that the free CET exhibited further ER localization after passive cellular uptake (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eH, J-II). Compared with that of free CET, the red fluorescence signal of the Da-CD@CET@CQ NPs group significantly overlapped with the green fluorescence signal of the ER probe, and the qualitative analysis of the line-scanning spectra of the fluorescence intensities showed a high degree of overlap (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eI, J-III). Thus, these data provide strong evidence that the Da-CD@CET@CQ NPs can significantly increase the ability of PS to effectively enter ER.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eIntracellular ROS imaging and\u003c/strong\u003e \u003cstrong\u003eIn vitro\u003c/strong\u003e \u003cstrong\u003ephototoxicity\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eReactive oxygen species (ROS) with superior oxidative ability play a critical role in directly causing apoptosis and necrosis of tumor cells and play a key role in continuously increasing ER stress. The ROS indicator we selected was nonfluorescent 2\u0026apos;,7\u0026apos;-dichlorodihydrofluorescein (DCFH-DA), which can be oxidized by ROS to green fluorescent DCF. Herein, we used CLSM to determine the fluorescence intensity of DCF. As shown in Fig. \u003cspan\u003e3\u003c/span\u003eA and B, the green fluorescence intensity of the Da-CD NPs and CQ groups without PS involvement was negligible. Compared with those of the free single-targeting CET and CD@Ce6@CQ NPs groups, the green fluorescence intensities of the Da-CD@Ce6@CQ NPs, Da-CD@CET NPs and Da-CD@CET@CQ NPs groups were approximately 5 times greater. These results indicated that the nanocomplex group with surface-modified Da had a higher ROS generation efficiency, which was also consistent with the cell uptake results. In addition, our further study revealed that the intracellular ROS production increased with increasing Da-CD@CET@CQ NPs incubation concentration (Fig. S8). Thus, these results suggest that poly-\u0026beta;-CD surface modification of Da increases the cell-targeted uptake of Da-CD@CET@CQ NPs and further promotes ROS generation.\u003c/p\u003e\n \u003cp\u003eTo determine whether the notable tumor cell targeting and ER localization ability of the Da-CD@CET@CQ NPs also resulted in superior cellular photodynamic activity, we evaluated the ability of the Da-CD@CET@CQ NPs to inhibit cellular activity via the MTT assay. First, as shown in Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003eC, both the poly-\u0026beta;-CD-based nanomaterials and PS showed no significant cytotoxicity against 4T1 cells in the absence of light, whereas free CQ and the nanomaterials encapsulating CQ (CD@CET@CQ NPs, Da-CD@Ce6@CQ NPs and Da-CD@CET@CQ NPs) exhibited weak cytotoxicity as the concentration increased but still maintained a cell survival rate above 85%. Moreover, as shown in Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003eD, the cellular phototoxicities of the Da-CD@Ce6@CQ NPs, Da-CD@CET NPs and Da-CD@CET@CQ NPs with cellular targeting were significantly greater than those of free CET and the CD@CET@CQ NPs with single-targeting ability after laser irradiation (\u0026lambda;\u0026thinsp;=\u0026thinsp;670 nm, 4 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e), which might be due to the increased intracellular uptake mediated by Da. More importantly, at limited concentrations up to 25\u0026ndash;50 \u0026micro;g/mL, the live cell inhibitory abilities of the Da-CD@CET@CQ NPs (15.2% and 7.3%) were both significantly greater than those of the Da-CD@Ce6@CQ NPs (37.9% and 21.6%), which also suggested that PS with both tumor cell and ER-targeting abilities exhibit increased photodynamic activity and proapoptotic abilities. In addition, we further evaluated its cytotoxicity under light exposure by live and dead assay. As shown in Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003eE and F, the Da-CD NPs and CQ did not significantly inhibit cell activity. The red-to-green ratio was significantly higher in the free CET and CD@CET@CQ NPs groups, and the ratio was close to 50%. Notably, the red fluorescence after Da-CD@CET@CQ NPs treatment was significantly stronger than that in the Da-CD@Ce6@CQ NPs and Da-CD@CET NPs groups, which further indicated that the Da-CD@CET@CQ NPs treatment induced more apoptotic cell death. Moreover, as shown in Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003eG and H, the cell death increased with increasing Da-CD@CET@CQ NPs incubation concentration, which was also consistent with the MTT results. In addition, apoptotic fluorescence staining was performed by using Annexin V-FITC and PI analysis of different drug groups after light irradiation. As shown in Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003eI and J, the proportions of the two regions of early apoptosis (Q2) and late apoptosis (Q3) in the Da-CD@CET@CQ NPs group (95.76%) were higher than those in the Da-CD@Ce6@CQ NP group (69.06%) and Da-CD@CET NPs group (81.36%), and late apoptosis was predominant (76.68%), which indicated that the Da-CD@CET@CQ NPs had a greater ability to induce apoptosis; this result was consistent with the above MTT assay and the live-dead cell staining results. Therefore, the Da-CD@CET@CQ NPs, which showed tumor cell targeting and further localization of PS to ER, could effectively improve the photodynamic antitumor effect.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eAutophagy inhibition\u003c/strong\u003e \u003cstrong\u003eIn Vitro\u003c/strong\u003e \u003cstrong\u003eand ICD effect induced by ER stress\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAutophagy maintains cell homeostasis through digestion and degradation of damaged, denatured or senescent proteins and organelles, which significantly inhibits the ability of PDT to induce cell apoptosis. Therefore, inhibition of autophagy has become one of the key factor to improve the therapeutic effect of PDT. To evaluate the autophagic inhibitory effect of the Da-CD@CET@CQ NPs loaded with CQ, a series of studies were performed by Western blot assay and confocal microscopy. The presence of LC3 in autophagosomes and its conversion to the smaller molecular weight LC3-II are indicators of autophagic\u003csup\u003e[\u003cspan\u003e46\u003c/span\u003e]\u003c/sup\u003e. After 4T1 cells were co-incubated with Da-CD@CET NPs and irradiated by a laser, the LC3-Ⅱ protein in the cells was significantly increased compared with cells without irradiation (Fig. S9), indicating that PDT effect can induce obvious autophagy, which is consistent with previous literature reports\u003csup\u003e[\u003cspan\u003e47\u003c/span\u003e\u0026ndash;\u003cspan\u003e49\u003c/span\u003e]\u003c/sup\u003e. As shown in Fig. \u003cspan\u003e4\u003c/span\u003eA and B, compared with other groups, the LC3-II protein content in the groups treated with free CQ or the nanocomplex encapsulated with CQ was significantly higher, which indicated that free or loaded CQ molecules did not prevent the protein conversion of LC3-I to LC3-Ⅱ in cells. In addition, P62, a bridge linking LC3 and polyubiquitinated proteins, can be degraded by proteolytic enzymes in autophagic lysosomes during autophagy. In the case of reduced or defective autophagy, the P62 protein accumulates in the cytoplasm, which is used as the indicator of autophagic inhibition\u003csup\u003e[\u003cspan\u003e50\u003c/span\u003e]\u003c/sup\u003e. As shown in Fig. \u003cspan\u003e4\u003c/span\u003eA and C, the abundant expression of the P62 protein was found in the presence of free CQ and CQ-encapsulated nanocomplex compared to control cells, indicating that autophagy was significantly inhibited. Moreover, LysoTracker Green staining was performed on intracellular acidic vesicles (lysosomes). The results showed that Da-CD@CET@CQ NPs exhibited more significant intracellular accumulation of autophagy vesicles (green) (Fig. \u003cspan\u003e4\u003c/span\u003eD). These results further indicate that CQ molecules released by Da-CD@CET@CQ NPs had no affect on the transformation of LC3-I to LC3-Ⅱ, but can significantly inhibit lysosomal degradation of autophagosome, and thus promote the accumulation of more autophagy vesicles in cells. Thus, it can be observed that Da-CD@CET@CQ NPs had a more significant anti-tumor effect of PDT \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eER is a multifunctional organelle in the cell and the main reservoir of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e. Excessive production of ROS in ER leads to dysregulation of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis. To determine whether the Da-CD@CET@CQ NPs disrupt Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis after laser irradiation, we used the sensitive Ca\u003csup\u003e2+\u003c/sup\u003e probe Fluo-4 AM. Free Fluo-4 AM has very weak fluorescence, however, when it was sheared by intracellular esterases to form Fluo-4, which can bind with Ca\u003csup\u003e2+\u003c/sup\u003e to produce strong green fluorescence. CLSM revealed that the cells treated with Da-CD@CET NPs and Da-CD@CET@CQ NPs displayed stronger green fluorescence, with intensities 5-fold and 2-fold higher than those of the ER-targeted CET group and the Da-CD@Ce6@CQ NP group, respectively (Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eE and Fig. S10). Thus, these results demonstrated that the nanomaterials with cellular and ER targeting capabilities have a more significant ability to disrupt intracellular Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis.\u003c/p\u003e\n \u003cp\u003eDysregulation of intracellular Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis impedes protein synthesis and folding, which leads to the accumulation of unfolded or misfolded proteins in ER, inducing the ER stress. On the one hand, at the onset of ER stress, unfolded or misfolded proteins in ER lumen are highly bound to the molecular chaperone binding immunoglobulin (BIP), leading to its dissociation from ER stress sensors and promoting the activation of activating transcription factor 6 (ATF6), inositol-required enzyme 1 alpha (IRE1 alpha), and protein kinase R-like ER kinase (PERK), which further induce an unfolded protein response (UPR) to remove unfolded or misfolded proteins to maintain the homeostatic balance of the ER. On the other hand, excessive ROS continuously increase ER stress and induce apoptosis by upregulating the expression of proapoptotic C/EPB homologous protein (CHOP), which was mediated by the PERK/eIF2/ATF6/CHOP pathway\u003csup\u003e[\u003cspan\u003e51\u003c/span\u003e\u0026ndash;\u003cspan\u003e53\u003c/span\u003e]\u003c/sup\u003e. To assess the extent of ER stress, we first used CLSM to analyze the expression of CHOP proteins. The CLSM revealed that the red fluorescent signal of CHOP were stronger in the Da-CD@CET NPs and Da-CD@CET@CQ NPs group, which had an approximately 2-fold greater value than that in the Da-CD@Ce6@CQ NPs group, suggesting increased CHOP expression by targeting PS to ER (Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eF and Fig. S11). In summary, these experimental results fully confirmed that the Da-CD@CET@CQ NPs can produce a large amount of ROS in ER, resulting in redox balance disorders and Ca\u003csup\u003e2+\u003c/sup\u003e homeostatic imbalances and activating proapoptotic pathways through continuous ER stress.\u003c/p\u003e\n \u003cp\u003eRecently, a growing number of studies have demonstrated that the continuous increase in ER stress generated by ROS can induce ICD\u003csup\u003e[\u003cspan\u003e54\u003c/span\u003e\u0026ndash;\u003cspan\u003e57\u003c/span\u003e]\u003c/sup\u003e. Tumor cells induce ICD while producing a series of DAMPs, including CRT, which is exposed on the cell surface; HMGB1, which is secreted to the outside world; and ATP molecules, which are released by the cells\u003csup\u003e[\u003cspan\u003e58\u003c/span\u003e\u0026ndash;\u003cspan\u003e61\u003c/span\u003e]\u003c/sup\u003e. Therefore, to determine whether ER stress triggered by the Da-CD@CET@CQ NPs can induce more intense ICD effects and promote DAMPs release, we conducted immunofluorescence and western blot analyses. First, we investigated the migration of HMGB1 from the nucleus to the cellular matrix. As shown in Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eG and Fig. S12, compared with those in the other groups, the overlap between HMGB1 (red) and the nucleus (blue) almost disappeared (pink) in the Da-CD@CET NPs and Da-CD@CET@CQ NPs group, which suggested that the tumor cells and ER targeting nanocomplex can increased HMGB1 migration from the nucleus. Second, we assessed the exposure of CRT on the cell surface by CLSM fluorescence intensity. Compared with those of the other groups, the Da-CD@CET NPs and Da-CD@CET@CQ NPs groups with dual-targeting ability displayed almost uniform and stronger green fluorescence, which was 3.2 times and 2.0 times higher than that of the CD@CET@CQ NPs and Da-CD@Ce6@CQ NPs groups, respectively, indicating that the nanocomplex with tumor cells and ER targeting can trigger higher CRT expression after light treatment (Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eH and I). We further evaluated ATP production in the cell culture medium. As shown in Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eJ, there were more ATP secretion in the cellular supernatant of the Da-CD@CET NPs and Da-CD@CET@CQ NPs groups, which was almost 3.0 times and 2.0 times greater than that of the CD@CET@CQ NP and Da-CD@Ce6@CQ NP groups, respectively. Finally, western blotting was used to further analyze the expression of ER stress related proteins. As shown in Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eK and Fig. S13, the western blot results showed that the expression of ER stress marker, such as CHOP, PERK, ATF6, and p-IRE-1\u0026alpha;, was significantly higher in the Da-CD@CET NPs and Da-CD@CET@CQ NPs group than that of other groups. All these results indicated that ROS continuously increased ER stress in the Da-CD@CET NPs and Da-CD@CET@CQ NPs groups, effectively induced ICD in tumor cells and further promoted antitumor effects.\u003c/p\u003e\n \u003cdiv id=\"Sec23\"\u003e\n \u003ch2\u003e\u003cem\u003eIn vivo\u003c/em\u003e tumor targeting\u003c/h2\u003e\n \u003cp\u003eTo further confirm the ability of the Da-CD@CET@CQ NPs to target TNBC tumors \u003cem\u003ein vivo\u003c/em\u003e, we used small animal live imaging system to assess the distribution of the nanocomplex in mouse xenograft tumor model. CD@CET@CQ NPs and Da-CD@CET@CQ NPs were injected into BALB/c mice with tumors derived from 4T1 cells via the tail vein, and the fluorescence intensity of both nanocomplex at the tumor site was assessed. As shown in Fig.\u0026nbsp;\u003cspan\u003e5\u003c/span\u003eA and B, the fluorescence intensity at the tumor site of the mice in the Da-CD@CET@CQ NPs group was significantly increased at 2 h post-injection and was approximately 3.0 times higher than that in the CD@CET@CQ NPs group, which indicated that the Da-CD@CET@CQ NPs could be enriched at the tumor site faster and more abundantly. With prolonged injection time, the enrichment of both the CD@CET@CQ NPs and the Da-CD@CET@CQ NPs at the tumor site peaked at 12 h, and the fluorescence intensity at the tumor site in the Da-CD@CET@CQ NPs group was approximately 2.5 times higher than that in the CD@CET@CQ NPs group. The fluorescence intensity at the tumor site gradually decreased over time, indicating that this type of nanocomplex could be metabolized by the organism in a timely manner. In addition, to further assess the fluorescence intensity at the tumor site and major organs, the mice were sacrificed at 12 h after administration. As shown in Fig.\u0026nbsp;\u003cspan\u003e5\u003c/span\u003eC and D, the fluorescence intensity of the Da-CD@CET@CQ NP group at the tumor site was approximately 2.7 times higher than that of the CD@CET@CQ NP group, which was consistent with the \u003cem\u003ein vivo\u003c/em\u003e fluorescence imaging data. Moreover, the concentration of Da-CD@CET@CQ NPs or CD@CET@CQ NPs at the tumor site was lower than that in the liver but higher than that in other major organs. Given the good biocompatibility and negligible cytotoxicity of the Da-CD@CET@CQ NPs and CD@CET@CQ NPs, the adverse effects of their accumulation in other organs in mice should be negligible. Meanwhile, fluorescence detection was performed on the tumor sections dissociated 12 h after administration, as shown in Fig.\u0026nbsp;\u003cspan\u003e5\u003c/span\u003eE and F, Da-CD@CET@CQ NPs group showed more significant fluorescence intensity at the edge site of tumor. Moreover, fluorescence intensity 3 times higher than that of CD@CET@CQ group was also observed in the tumor core site of Da-CD@CET@CQ NPs group, which further proved that Da-CD@CET@CQ NPs can achieve abundant and more uniform accumulation of photosensitizer CET at the tumor site (Fig.\u0026nbsp;\u003cspan\u003e5\u003c/span\u003eG and H). All these results demonstrate that Da-CD@CET@CQ NPs indeed exhibit more significant 4T1 tumor-targeting ability, which can be attributed to Da-mediated tumor specificity.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e \u003cstrong\u003eantitumor activity\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTo assess whether drug enrichment results in more significant antitumor effects, we conducted antitumor studies using BALB/c mice with a 4T1 tumor model. First, the mice were randomly divided into eight groups (n\u0026thinsp;=\u0026thinsp;5 mice per group): control, Da-CD NPs, CQ, CET, CD@CET@CQ NPs, Da-CD@Ce6@CQ NPs, Da-CD@CET NPs and Da-CD@CET@CQ NPs. Based on the results of the drug targeting experiment, a laser was applied 12 h after injection (670 nm, 50 mW\u0026middot;cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 10 min), and then, the tumor volume and body weight of the mice were monitored every other day (Fig. \u003cspan\u003e6\u003c/span\u003eA). As shown in Fig. \u003cspan\u003e6\u003c/span\u003eB and C, in the control group, almost no significant inhibition of tumor growth was observed after the injection of Da-CD NPs or CQ. The free CET and single-targeting groups, including the CD@CET@CQ NP and Da-CD@Ce6@CQ NP groups, only showed partial inhibition of tumor growth. Compared with the Da-CD@CET NP group, the Da-CD@CET@CQ NP group exhibited a more significant tumor inhibitory effect, and the tumors almost completely stopped growing, which may be attribute to the autophagic inhibitory ability of the Da-CD@CET@CQ NPs group, resulting in an increase in the final PDT effect. After 15 days of treatment, the mice were dissected, and the tumor tissues were removed for weighing. As shown in Fig. \u003cspan\u003e6\u003c/span\u003eD, the average tumor weight further confirmed that the Da-CD@CET@CQ NPs had a more significant antitumor effect. In addition, we further confirmed the antitumor effect of each group by H\u0026amp;E and TUNEL staining, and immunohistochemical analysis of the tumor tissues. As shown by H\u0026amp;E staining, more pronounced necrosis and increased vesicles, as well as nuclear shrinkage, were found in the Da-CD@CET@CQ NPs group. Moreover, a stronger green fluorescence signal was observed in the Da-CD@CET@CQ NPs group in the TUNEL experiment, which indicated that the Da-CD@CET@CQ NPs group had a greater number of apoptosis cells. Subsequently, the tumor stem cell marker CD34 and the cell proliferation marker Ki67 were selected for immunohistochemistry experiments to further assess vascular proliferation and tumor proliferation. The percentage of positive cells in Da-CD@CET@CQ NPs group was significantly lower than that in other groups, which indicated that Da-CD@CET@CQ NPs effectively inhibited vascular regeneration and cell proliferation (Fig. \u003cspan\u003e6\u003c/span\u003eE). Finally, we also performed H\u0026amp;E staining of isolated lung tissues. Compared with those in the other groups with more metastatic nodules in the lung tissues, no obvious metastatic nodules in the lung tissues were observed in the Da-CD@CET@CQ NPs group, which indicated that this treatment effectively inhibited tumor metastasis (Fig. \u003cspan\u003e6\u003c/span\u003eF and Fig. S14). The relative body weight of each group of mice remained almost unchanged throughout the experimental period (Fig. S15), and H\u0026amp;E staining analysis of isolated organs (heart, liver, spleen, lungs, and kidneys) revealed normal physiological morphology of tissues in each group (Fig. S16), indicating that all nanocomplexes have almost negligible systemic toxicity as well as side effects \u003cem\u003ein vivo\u003c/em\u003e. These results strongly demonstrate that precise delivery of PS to ER of tumor cells and combined with autophagy inhibition has significant \u003cem\u003ein vivo\u003c/em\u003e antitumor and metastasis inhibition effects.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e \u003cstrong\u003eantitumor immunity\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eWe further explored the antitumor immune responses of the Da-CD@CET@CQ NPs by immunofluorescence staining. First, in order to evaluate ER stress and DAMPs release, we detected CHOP expression, CRT exposure and HMGB1 release (red fluorescence) in different tumor tissues by IF staining. The results showed that the red fluorescence intensity of CHOP in the Da-CD@CET NPs and Da-CD@CET@CQ NPs groups was significantly higher than that in the other groups due to the targeting of tumor cells and the presence of ER targeting, indicating the enhancement of ER stress (Fig. S17). In addition, the red fluorescence intensity of CRT and HMGB1 in the Da-CD@CET@CQ NPs group was significantly higher than that in the other groups, indicating that ER-PDT combined with autophagy inhibition could induce stronger ICD effects (Fig. S17). Second, we used a nuclear probe and four different antibodies (CD19-labeled B cells, CD8-labeled T cells, CD49B-labeled NK cells, and iNOS-labeled M1 macrophages) to determine the effect of the body\u0026apos;s immune response in the different groups, and the expression levels of the different immunoantibodies were determined by fluorescence intensity. As shown in Fig. \u003cspan\u003e7\u003c/span\u003eA, the fluorescence signals of different antibodies in the dual-targeting Da-CD@CET NPs and Da-CD@CET@CQ NPs groups exhibited stronger than other groups. Meanwhile, we measured the expression levels of different antibodies based on fluorescence intensity. As shown in Fig. \u003cspan\u003e7\u003c/span\u003eB and C, the expression level of CD8 and CD19, compared with other groups, the Da-CD@CET NPs and Da-CD@CET@CQ NPs group show a significant increase. In addition, the expression level of CD49B and iNOS in the Da-CD@CET NPs and Da-CD@CET@CQ NPs group exhibit a obviously higher level compared to other groups (Fig. \u003cspan\u003e7\u003c/span\u003eD and E). These results suggest that in situ ER-PDT increased B cell, T-cell and NK cell expression and promoted macrophage polarization to M1 macrophages. More importantly, four antibodies in the Da-CD@CET@CQ NPs group exhibited a higher expression level than the Da-CD@CET NPs group, possibly because autophagic inhibition further promoted the antitumor effect of PDT, which in turn induced a stronger immune response. Moreover, peripheral blood of mice in different treatment groups were collected on the 15th day after laser irradiation for analysis of cytokine levels. The results showed that the mean levels of TNF-\u0026alpha; and IFN-\u0026gamma; in Da-CD@CET@CQ NPs treatment group were significantly higher than those in other groups (Fig. \u003cspan\u003e7\u003c/span\u003eF and G), while the mean levels of IL-10 were lower than those in other groups (Fig. \u003cspan\u003e7\u003c/span\u003eH). Thus, these results suggest that the PS with tumor cells and ER targeting ability, combined with autophagic inhibition can further increase the ability of PDT to activate the body\u0026apos;s immune response, which in turn promotes the synergistic effect of tumor treatment.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we have successfully designed and developed a smart controllable nanomaterial for the delivery of novel ICD inducer. Compared with free PS and single-target nanocomplexes, the final constructed Da-CD@CET@CQ NPs can precisely target TNBC tumor cells with SRC-overexpression, and has good tumor cell enrichment and retention ability, which may be attributed to the high affinity between Da and SRC in tumor cells. Meaningfully, the ICD inducer CET released in the tumor environment shows its remarkable ER localization ability, not only realizing in-situ ER PDT, but also enhancing the induction of ER stress to amplify the ICD effect, further promoting the maturation of DCs, and thus inducing a more drastic systemic immune response. Subsequently, autophagy inhibitor CQ released by Da-CD@CET@CQ NPs can inhibit autophagy, destroy cell homeostasis, and further promote the ability of PDT to induce apoptosis. In summary, the multifunctional nanoplatform constructed can achieve dual targeting of PS to tumor cells and ER, which can amplify the ICD effect and further enhance the anti-tumor effect of PDT by inhibiting autophagy, which is of great significance for the development and clinical application of TNBC targeted photodynamic synergistic immunotherapy in the future.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePDT \u0026nbsp; \u0026nbsp; \u0026nbsp;Photodynamic therapy\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCe6 \u0026nbsp; \u0026nbsp; \u0026nbsp;Chlorin e6\u003c/p\u003e\n\u003cp\u003eICD \u0026nbsp; \u0026nbsp; \u0026nbsp;Immunogenic cell death\u003c/p\u003e\n\u003cp\u003eROS \u0026nbsp; \u0026nbsp; \u0026nbsp;Reactive oxygen species\u003c/p\u003e\n\u003cp\u003eER \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Endoplasmic reticulum\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTNBC \u0026nbsp; \u0026nbsp;Triple-negative breast cancer\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCQ \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Chloroquine\u003c/p\u003e\n\u003cp\u003eDAMPs \u0026nbsp; \u0026nbsp; \u0026nbsp;Damage-associated molecular patterns\u003c/p\u003e\n\u003cp\u003eCRT \u0026nbsp; \u0026nbsp; \u0026nbsp;Calreticulin\u003c/p\u003e\n\u003cp\u003eHMGB1 \u0026nbsp; High mobility group protein 1\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eATP \u0026nbsp; \u0026nbsp; \u0026nbsp; Adenosine triphosphate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Photosensitizers\u003c/p\u003e\n\u003cp\u003eDAPI \u0026nbsp; \u0026nbsp; 4\u0026apos;,6-diamidino-2-phenylindole\u003c/p\u003e\n\u003cp\u003eMTT \u0026nbsp; \u0026nbsp; Methyl thiazolyl tetrazolium\u003c/p\u003e\n\u003cp\u003eTEM \u0026nbsp; \u0026nbsp; Transmission electron microscope\u003c/p\u003e\n\u003cp\u003eUV-vis \u0026nbsp; Ultraviolet-visible\u003c/p\u003e\n\u003cp\u003eCLSM \u0026nbsp; \u0026nbsp;Confocal laser scanning microscope\u003c/p\u003e\n\u003cp\u003eFCM \u0026nbsp; \u0026nbsp; Flow cytometer\u003c/p\u003e\n\u003cp\u003eDMSO \u0026nbsp; \u0026nbsp;Dimethyl sulfoxide\u003c/p\u003e\n\u003cp\u003eDMEM \u0026nbsp; Dulbecco\u0026rsquo;s modified eagle medium\u003c/p\u003e\n\u003cp\u003eATF6 \u0026nbsp; \u0026nbsp;Activated transcription factor 6\u003c/p\u003e\n\u003cp\u003eCHOP \u0026nbsp;\u0026nbsp;Proapoptotic C/EPB homologous protein\u003c/p\u003e\n\u003cp\u003ePBS \u0026nbsp; \u0026nbsp; Phosphate buffered saline\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E \u0026nbsp; \u0026nbsp;Hematoxylin and eosin\u003c/p\u003e\n\u003cp\u003eTUNEL \u0026nbsp;Terminal deoxynucleotidyl transferase dUTP nick end labeling\u003c/p\u003e\n\u003cp\u003ePDIs \u0026nbsp; \u0026nbsp;\u0026nbsp;Polydispersity\u0026nbsp;indices\u003c/p\u003e\n\u003cp\u003eDLCs \u0026nbsp; \u0026nbsp;Drug loading\u0026nbsp;capacities\u003c/p\u003e\n\u003cp\u003eDLEs \u0026nbsp; \u0026nbsp;Drug loading\u0026nbsp;efficiencies\u003c/p\u003e\n\u003cp\u003ePERK \u0026nbsp; \u0026nbsp;Protein kinase R-like ER kinase\u003c/p\u003e\n\u003cp\u003eUPR \u0026nbsp; \u0026nbsp; Unfolded protein responses\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGY\u0026nbsp;and GL conceived and designed the study, and wrote the manuscript. GY and RY performed most of the in vitro and in vivo experiments. WW and ZW participated in the in vivo experiments and analyzed some experimental data.\u0026nbsp;MS and GL participating in revising the paper. LZ and KH contributed to the in vivo experimental design.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by\u0026nbsp;the National Key Research and Development Program of China (2022YFE132800),\u0026nbsp;Key R\u0026amp;D project of Henan Province\u0026nbsp;(221111310600), and Special Foundation for Basic Research Program of Higher Education Institutions of Henan Province (22ZX005). Joint Fund of Henan Province Science and Technology R\u0026amp;D Program (225200810020). Postdoctoral Initiation Fund of Henan Academy of Sciences (231828050). The Scientific and Technological Research Project of Henan Province (232102311177).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Ethics Committee of the Animal Experimental Center of Henan Academy of Sciences (Approval Number: 2023A1017), and were carried out in compliance with all relevant ethical regulations.\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\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no competing interest exists.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEmens LA, Ascierto PA, Darcy PK, Demaria S, Eggermont AMM, Redmond WL, Seliger B, Marincola FM. Cancer immunotherapy: Opportunities and challenges in the rapidly evolving clinical landscape. Eur J Cancer. 2017;81:116\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age. Nature. 2011;480:480\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKennedy LB, Salama AKS. A review of cancer immunotherapy toxicity. CA Cancer J Clin. 2020;70:86\u0026ndash;104.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePowles T, Kockx M, Rodriguez-Vida A, Duran I, Crabb SJ, Van Der Heijden MS, Szabados B, Pous AF, Gravis G, Herranz UA, et al. Clinical efficacy and biomarker analysis of neoadjuvant atezolizumab in operable urothelial carcinoma in the ABACUS trial. Nat Med. 2019;25:1706\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKroemer G, Galassi C, Zitvogel L, Galluzzi L. Immunogenic cell stress and death. Nat Immunol. 2022;23:487\u0026ndash;500.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrysko DV, Garg AD, Kaczmarek A, Krysko O, Agostinis P, Vandenabeele P. Immunogenic cell death and DAMPs in cancer therapy. Nat Rev Cancer. 2012;12:860\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGalluzzi L, Buqu\u0026eacute; A, Kepp O, Zitvogel L, Kroemer G. Immunogenic cell death in cancer and infectious disease. Nat Rev Immunol. 2017;17:97\u0026ndash;111.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKing AP, Wilson JJ. Endoplasmic reticulum stress: an arising target for metal-based anticancer agents. Chem Soc Rev. 2020;49:8113\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRufo N, Garg AD, Agostinis P. The unfolded protein response in immunogenic cell death and cancer immunotherapy. Trends Cancer. 2017;3:643\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu P, Zhao L, Pol J, Levesque S, Petrazzuolo A, Pfirschke C, Engblom C, Rickelt S, Yamazaki T, Iribarren K, et al. Crizotinib-induced immunogenic cell death in non-small cell lung cancer. Nat Commun. 2019;10:1486.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuimova MK, Botchway SW, Parker AW, Balaz M, Collins HA, Anderson HL, Suhling K, Ogilby PR. Imaging intracellular viscosity of a single cell during photoinduced cell death. Nat Chem. 2009;1:69\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLovell JF, Liu TWB, Chen J, Zheng G. Activatable photosensitizers for imaging and therapy. Chem Rev. 2010;110:2839\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou Z, Song J, Nie L, Chen X. Reactive oxygen species generating systems meeting challenges of photodynamic cancer therapy. Chem Soc Rev. 2016;45:6597\u0026ndash;626.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang S, Wang J, Kong Z, Sun X, He Z, Sun B, Luo C, Sun J. Emerging photodynamic nanotherapeutics for inducing immunogenic cell death and potentiating cancer immunotherapy. Biomaterials. 2022;282:121433.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVaes RDW, Hendriks LEL, Vooijs M, De Ruysscher D. Biomarkers of radiotherapy-induced immunogenic cell death. Cells. 2021;10:930.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe L, Nie T, Xia X, Liu T, Huang Y, Wang X, Chen T. Designing bioinspired 2D MoSe2 nanosheet for efficient photothermal-triggered cancer immunotherapy with reprogramming tumor‐associated macrophages. Adv Funct Mater. 2019;29:1901240.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLan G, Ni K, Xu Z, Veroneau SS, Song Y, Lin W. Nanoscale metal-organic framework overcomes hypoxia for photodynamic therapy primed cancer immunotherapy. J Am Chem Soc. 2018;140:5670\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen P, Huang P, Chen J, Shi Q, Zhu Y, Chen Y, Liu L, Zhang X. A self-delivery chimeric peptide for high efficient cell membrane-targeting low-temperature photothermal/photodynamic combinational therapy and metastasis suppression of tumor. Biomaterials. 2022;286:121593.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang C, Xu C, Gao X, Yao Q. Platinum-based drugs for cancer therapy and anti-tumor strategies. Theranostics. 2022;12:2115\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark SJ, Ye W, Xiao R, Silvin C, Padget M, Hodge JW, Van Waes C, Schmitt NC. Cisplatin and oxaliplatin induce similar immunogenic changes in preclinical models of head and neck cancer. Oral Oncol. 2019;95:127\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng Y, C Samia A, Meyers JD, Panagopoulos I, Fei B, Burda C. Highly efficient drug delivery with gold nanoparticle vectors for in vivo photodynamic therapy of cancer. J Am Chem Soc. 2008;130:10643\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian B, Wang C, Zhang S, Feng L, Liu Z. Photothermally enhanced photodynamic therapy delivered by nano-graphene oxide. ACS Nano. 2011;5:7000\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu C, Cao Y, Cheng Y, Wang D, Xu T, Su L, Zhang X, Dong H. An open source and reduce expenditure ROS generation strategy for chemodynamic/photodynamic synergistic therapy. Nat Commun. 2020;11:1735.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEthirajan M, Chen Y, Joshi P, Pandey RK. The role of porphyrin chemistry in tumor imaging and photodynamic therapy. Chem Soc Rev. 2011;40:340\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu Z, Sun Q, Pan W, Li N, Tang B. A Near-infrared triggered nanophotosensitizer inducing domino effect on mitochondrial reactive oxygen species burst for cancer therapy. ACS Nano. 2015;9:11064\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu N, Ding M, Wang F, Zhou J, Shi X, Cai R, Li J. Near-infrared photoactivatable semiconducting polymer nanocomplexes with bispecific metabolism interventions for enhanced cancer immunotherapy. Nano Today. 2022;46:101600.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Q, Hou X, Yang J, Zhang M, Yang Y, Liu Y, Shen W, Yin D. Heparin-coated photosensitive metal-organic frameworks as drug delivery nanoplatforms of autophagy inhibitors for sensitized photodynamic therapy against breast cancer. ACS Appl Mater Interfaces. 2021;13:55577\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu B, Li S, Yu L, Hu W, Sheng D, Hou J, Zhao N, Hou X, Wu Y, Han Z, et al. Inhibition of autophagy with chloroquine enhanced sinoporphyrin sodium mediated photodynamic therapy-induced apoptosis in human colorectal cancer cells. Int J Biol Sci. 2019;15:12\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie L, Wang L, Li L, Liu C, Guo L, Liao Y, Zhou S, Wu W, Duo Y, Shi L, et al. Novel carrier-free nanodrug enhances photodynamic effects by blocking the autophagy pathway and synergistically triggers immunogenic cell death for the efficient treatment of breast cancer. ACS Appl Mater Interfaces. 2024;16:5683\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchwarz DS, Blower MD. The endoplasmic reticulum: structure, function and response to cellular signaling. Cell Mol Life Sci. 2016;73:79\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu Y, Zhu X, Tang C, Guan X, Zhang W. Progress and challenges of immunotherapy in triple-negative breast cancer. Biochim Biophys Acta Rev Cancer. 2021;1876:188593.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta GK, Collier AL, Lee D, Hoefer RA, Zheleva V, van Siewertsz LL, Tang-Tan AM, Guye ML, Chang DZ, Winston JS, et al. Perspectives on triple-negative breast cancer: current treatment strategies, unmet needs, and potential targets for future therapies. Cancers. 2020;12:2392.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeon-Ferre RA, Goetz MP. Advances in systemic therapies for triple negative breast cancer. BMJ. 2023;381:e071674.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHiscox S, Nicholson RI. Src inhibitors in breast cancer therapy. Expert Opin Ther Targets. 2008;12:757\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuffy MJ, McGowan PM, Crown J. Targeted therapy for triple-negative breast cancer: where are we? Int J Cancer. 2012;131:2471\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFinn RS, Dering J, Ginther C, Wilson CA, Glaspy P, Tchekmedyian N, Slamon DJ. Dasatinib, an orally active small molecule inhibitor of both the src and abl kinases, selectively inhibits growth of basal-type/triple-negative breast cancer cell lines growing in vitro. Breast Cancer Res Treat. 2007;105:319\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBayraktar S, Gl\u0026uuml;ck S. Molecularly targeted therapies for metastatic triple-negative breast cancer. Breast Cancer Res Treat. 2013;138:21\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAraujo J, Logothetis C. Dasatinib: a potent SRC inhibitor in clinical development for the treatment of solid tumors. Cancer Treat Rev. 2010;36:492\u0026ndash;500.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoopmans C, Ritter H. Formation of physical hydrogels via host-guest interactions of β-cyclodextrin polymers and copolymers bearing adamantyl groups. Macromolecules. 2008;41:7418\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan G, Wang Q, You Z, Chen X, Xue J, Jia X, Chen J. A novel hierarchical targeting and controllable smart nanoparticles for enhanced in situ nuclear photodynamic therapy. Nano Res. 2022;15:4212\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang L, Tonelli AE. Polymer inclusion compounds. Taylor Francis. 1998;38:781\u0026ndash;837.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan W, Xu Y, Li Z, Li Q. Folic acid-modified β-cyclodextrin nanoparticles as drug delivery to load DOX for liver cancer therapeutics. Soft Mater. 2019;17:437\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAng CY, Tan SY, Teh C, Lee JM, Wong MFE, Qu Q, Poh LQ, Li M, Zhang Y, Korzh V, et al. Redox and pH dual responsive polymer based nanoparticles for in vivo drug delivery. Small. 2017;13:1602379.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu X, Zeng Z, Chen J, Huang B, Guan Z, Huang Y, Huang Z, Zhao C. Tumor-targeted supramolecular catalytic nanoreactor for synergistic chemo/chemodynamic therapy via oxidative stress amplification and cascaded Fenton reaction. Chem Eng J. 2020;390:124628.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWankar J, Kotla NG, Gera S, Rasala S, Pandit A, Rochev YA. Recent advances in host-guest self-assembled cyclodextrin carriers: implications for responsive drug delivery and biomedical engineering. Adv Funct Mater. 2020;30:1909049.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanida I, Ueno T, Kominami E. LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol. 2004;36:2503\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen T, Cen D, Ren Z, Wang Y, Cai X, Huang J, Di Silvio L, Li X, Han G. Bismuth embedded silica nanoparticles loaded with autophagy suppressant to promote photothermal therapy. Biomaterials. 2019;221:119419.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai H, Yan H, Dong F, Zhang L, Du N, Sun L, Li N, Yu G, Yang Z, Wang Y, et al. Tumor-targeted biomimetic nanoplatform precisely integrates photodynamic therapy and autophagy inhibition for collaborative treatment of oral cancer. Biomater Sci. 2022;10:1456\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi M, Liu Y, Huang J, Chen Z, Ni C, Lu J, Zhang Y, Liu Z, Bai J. Multifunctional theranostic nanoplatform loaded with autophagy inhibitor for enhanced photothermal cancer therapy under mild near-infrared irradiation. Biomaterials Adv. 2022;138:212919.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIslam MA, Sooro MA, Zhang P. Autophagic regulation of p62 is critical for cancer therapy. Int J Mol Sci. 2018;19:1405.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSenft D, Ronai ZA. UPR, autophagy, and mitochondria crosstalk underlies the ER stress response. Trends Biochem Sci. 2015;40:141\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQi Z, Chen L. Endoplasmic reticulum stress and autophagy. Adv Exp Med Biol. 2019;1206:167\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSudsaward S, Khunchai S, Thepmalee C, Othman A, Limjindaporn T, Yenchitsomanus P-T, Mutti L, Krstic-Demonacos M, Demonacos C. Endoplasmic reticulum stress, unfolded protein response and autophagy contribute to resistance to glucocorticoid treatment in human acute lymphoblastic leukaemia cells. Int J Oncol. 2020;57:835\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi W, Yang J, Luo L, Jiang M, Qin B, Yin H, Zhu C, Yuan X, Zhang J, Luo Z, et al. Targeting photodynamic and photothermal therapy to the endoplasmic reticulum enhances immunogenic cancer cell death. Nat Commun. 2019;10:3349.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan J, Zhang X, Li Z, Mo F, Tang D, Xiao H, Wang J, Rong G, Liu T. Oxidative stress amplifiers as immunogenic cell death nanoinducers disrupting mitochondrial redox homeostasis for cancer immunotherapy. Adv Healthc Mater. 2023;12:2202710.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiao Z, Li J, Zeng S, Lv Y, Jia S, Ding D, Li W, Liu Q. Endoplasmic reticulum-targeting AIE photosensitizers to boost immunogenic cell death for immunotherapy of bladder carcinoma. ACS Appl Mater Interfaces. 2024;16:245\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Wan J, Mo F, Tang D, Xiao H, Li Z, Jia J, Liu T. Targeting bone tumor and subcellular endoplasmic reticulum via near infrared II fluorescent polymer for photodynamic-immunotherapy to break the step‐reduction delivery dilemma. Adv Sci. 2022;9:e2201819.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFucikova J, Kepp O, Kasikova L, Petroni G, Yamazaki T, Liu P, Zhao L, Spisek R, Kroemer G, Galluzzi L. Detection of immunogenic cell death and its relevance for cancer therapy. Cell Death Dis. 2020;11:1013.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Guan R, Xie L, Liao X, Xiong K, Rees TW, Chen Y, Ji L, Chao H. An ER-targeting iridium(III) complex that induces immunogenic cell death in non-small-cell lung cancer. Angew Chem Int Ed Engl. 2021;60:4657\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Tang J, Li C, Lu Y, Cheng L, Liu J. A targeting black phosphorus nanoparticle based immune cells nano-regulator for photodynamic/photothermal and photo-immunotherapy. Bioact Mater. 2021;6:472\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Z, Lai X, Fu S, Ren L, Cai H, Zhang H, Gu Z, Ma X, Luo K. Immunogenic cell death activates the tumor immune microenvironment to boost the immunotherapy efficiency. Adv Sci. 2022;9:e2201734.\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"Immunogenic cell death, Photodynamic therapy, Reactive oxygen species, Endoplasmic reticulum stress, Autophagy inhibitor","lastPublishedDoi":"10.21203/rs.3.rs-4950334/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4950334/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e Activating immunogenic cell death (ICD) represents a promising therapeutic strategy for tumor immunotherapy. However, photodynamic therapy (PDT)-mediated ICD effects are severely limited due to the extremely short half-life and limited diffusion radius of reactive oxygen species (ROS) hinder effective endoplasmic reticulum (ER) stress induction. In addition, targeted therapy of triple-negative breast cancer (TNBC) remain hugely challenging due to the lack of expression of multiple receptors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e Herein, we synthesized a hierarchical targeting and controllable intelligent nanodelivery material Da-CD@CET@CQ, loaded with highly efficient ER-targeted photosensitizers CET and autophagy inhibitor chloroquine (CQ). Excitingly, Da-CD@CET@CQ NPs can selectively target TNBC tumor cells and the CET was effectively released in the tumor microenvironment, enabling local accumulation of photosensitizers in the ER and in situ ROS production, which causing stronger ER stress and amplifying the ICD effect, further increasing the immune suppression of tumor growth. More importantly, CQ released by Da-CD@CET@CQ NPs can inhibit autophagy to destroy damaged cell repair, and further enhance the anti-tumor ability of PDT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e Our findings indicate that we reported a novel strategy of photosensitizing ICD inducer to amplify ICD effects and combination with autophagy inhibition, which provides a meaningful guideline for targeted PDT synergistic immunotherapy of TNBC in the future.\u003c/p\u003e","manuscriptTitle":"Novel immunogenic cell death inducer combined with autophagy inhibitor to amplify photodynamic synergistic immunotherapy for triple-negative breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-25 19:15:04","doi":"10.21203/rs.3.rs-4950334/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":"630fc8e0-fbf3-42ab-a120-1215beb85d68","owner":[],"postedDate":"September 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-05T21:53:09+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-25 19:15:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4950334","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4950334","identity":"rs-4950334","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}