Biomimetic Photoregulation Nanoplatform for Synergetic Photodynamic therapy and Immunotherapy Guided by NIR-II Imaging

Biomimetic Photoregulation Nanoplatform for Synergetic Photodynamic therapy and Immunotherapy Guided by NIR-II Imaging | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 25 April 2025 V1 Latest version Share on Biomimetic Photoregulation Nanoplatform for Synergetic Photodynamic therapy and Immunotherapy Guided by NIR-II Imaging Authors : Yu Ji , Jing Qian , Liansheng Fan , Gaoyu Shi , Ke Liu , Guangzhao Yang , Suchen Qu , Jie Tang , Yanni Song , Liping Yin , Chao Yin , and Xin Han 0000-0002-9620-7502 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174560769.94815918/v1 236 views 107 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Uncertain molecular targets and a complex immune-suppressive tumor microenvironment (TME) present significant challenges for triple-negative breast cancer (TNBC) treatment. This study develops a light-triggered biomimetic nanodelivery system that enables remotely controlled gene editing-assisted TME remodeling and enhanced cancer immunotherapy. The nanosystem integrates a semiconductor polymer with second near-infrared (NIR-II) fluorescence imaging capabilities and CRISPR/Cas9 ribonucleoprotein (RNP) targeting programmed cell death ligand 1 (PD-L1), linked via a singlet oxygen ( 1 O 2 )-cleavable thioketal (TK) linker. Photosensitizer Ce6 in the nanoparticles generates 1 O 2 under irradiation, triggering TK linker cleavage and subsequent Cas9 RNP release. Concurrently, photodynamic therapy (PDT) induces immunogenic cell death (ICD) by promoting tumor-associated antigen release. The combination of PDT-activated ICD and PD-L1 pathway blockade synergistically amplifies immune responses and reprograms the immune-suppressive TME. Utilizing tumor cell membrane camouflage, the nanoparticles achieve homologous tumor targeting under NIR-II imaging guidance while maintaining biocompatibility and precise tumor accumulation. This integrated platform demonstrates enhanced antitumor efficacy through coordinated immune activation and TME modulation, providing a safe and effective strategy for TNBC clinical management. Biomimetic Photoregulation Nanoplatform for Synergetic Photodynamic therapy and Immunotherapy Guided by NIR-II Imaging Yu Ji [1, 2] | Jing Qian [2] | Liansheng Fan [2] | Gaoyu Shi [2] | Ke liu [1] | Guangzhao Yang [3] | Suchen Qu [2] | Jie Tang [1] | Yanni Song [4] | Liping Yin [1] | Chao Yin [3] | Xin Han [1,2] 1 The Second Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing University of Chinese Medicine, No. 138, Xianlin Rd. Nanjing 210023, China E-mail: [email protected] | 2 State Key Laboratory of Technologies for Chinese Medicine Pharmaceutical Process Control and Intelligent Manufacture, School of Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China | 3 State Key Laboratory of Organic Electronics and Information Displays & Institute of Advanced Materials (IAM), Nanjing University of Posts & Telecommunications, 9 Wenyuan Road, Nanjing 210023, China | 4 Department of Breast Surgery, Harbin Medical University Cancer Hospital, Harbin 150081, China Correspondence：Yanni Song ( [email protected] ) | Liping Yin ( [email protected] [email protected] ) | Chao Yin ( [email protected] [email protected] ) | Xin Han ( [email protected] [email protected] ) Funding: This study was funded by the National Natural Science Foundation of China (32471475); the Natural Science Foundation of Jiangsu Province (BK20210685); Yangtze River Delta joint sci-tech innovation and research projects (2023CSJZN0600); the Innovation Projects of State Key Laboratory of Technologies for Chinese Medicine Pharmaceutical Process Control and Intelligent Manufacture（NZYSKL240201); Beijing MDK Public Welfare Foundation Research Fund (MDK 2022-1001); Pandeng Fund of Harbin Medical University Cancer Hospital (PDTS 2024A-03); Postdoctoral Scientific Research Developmental Fund of Heilongjiang (LBH-Q22); Jiangsu Key Discipline Construction Fund of the 14th Five-Year Plan (Biology); National Natural Science Foundation of China (22475105) and the Project of Jiangsu Specially-Appointed Professor (RK030STP22003). Keywords： light-triggered | CRISPR/Cas9 system | biomimetic | semiconductor polymer | NIR-II imaging ABSTRACT Uncertain molecular targets and a complex immune-suppressive tumor microenvironment (TME) present significant challenges for triple-negative breast cancer (TNBC) treatment. This study develops a light-triggered biomimetic nanodelivery system that enables remotely controlled gene editing-assisted TME remodeling and enhanced cancer immunotherapy. The nanosystem integrates a semiconductor polymer with second near-infrared (NIR-II) fluorescence imaging capabilities and CRISPR/Cas9 ribonucleoprotein (RNP) targeting programmed cell death ligand 1 (PD-L1), linked via a singlet oxygen ( 1 O 2 )-cleavable thioketal (TK) linker. Photosensitizer Ce6 in the nanoparticles generates 1 O 2 under irradiation, triggering TK linker cleavage and subsequent Cas9 RNP release. Concurrently, photodynamic therapy (PDT) induces immunogenic cell death (ICD) by promoting tumor-associated antigen release. The combination of PDT-activated ICD and PD-L1 pathway blockade synergistically amplifies immune responses and reprograms the immune-suppressive TME. Utilizing tumor cell membrane camouflage, the nanoparticles achieve homologous tumor targeting under NIR-II imaging guidance while maintaining biocompatibility and precise tumor accumulation. This integrated platform demonstrates enhanced antitumor efficacy through coordinated immune activation and TME modulation, providing a safe and effective strategy for TNBC clinical management. 1 | Introduction Triple-negative breast cancer (TNBC) lacks clear molecular targets and exhibits significant tumor heterogeneity. [1-3] The efficacy of standard treatments, including surgery and chemotherapy, is severely limited due to significant side effects and their adverse impact on the host immune system. [4,5] Particularly, the unique immune-suppressive tumor microenvironment (TME) of TNBC further limits the effectiveness of immunotherapy. [6] The emergence of immune checkpoint blockade (ICB) strategies, such as utilizsed clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) ribonucleoprotein (RNP) targeting pro-grammed cell death ligand 1 (PD-L1) [7] to assisted immune-suppressive TME remodeling, [8-11] has brought new hope. This powerful and innovative tool holds unparalleled promise in permanently modifying target gene expression and stimulating the patient’s immune system to fight against cancer cells. [12] Therefore, exploring and developing effective strategies to prolong the duration of immunotherapy and enhance its effectiveness is of great significance in cancer treatment. Immunogenic cell death (ICD) releases damage-associated molecules, including adenosine-5’-triphosphate (ATP) and surface-exposed calreticulin (CALR), [13] which induce an immune response and play a crucial role in activating the immune-suppressive TME. [14-16] Specifically, when combined with ICB, ICD can effectively activate and significantly enhance tumor-specific immune responses. [17] The commonly used photothermal therapy (PTT) can cause irreversible damage to normal tissues and tissue edema due to the high temperatures generated during the process. [18] In contrast, photodynamic therapy (PDT) offers more controllable spatiotemporal properties and safety. [19,20] PDT shows great potential in efficiently treating tumors by inducing ICD in tumors and combining with CRISPR technology to amplify the immune response. The development of nanodelivery systems has provided an effective platform for the integration of multifunctional candidate materials. [21,22] Currently, various nanoliposomes, polymers, and self-assembled nanomaterial delivery systems have been developed. [23,24] Semiconducting polymer nanomaterials composed of polymers have become versatile NIR absorbing/emitting biomaterials, used for molecular imaging, phototherapy, and biological regulation, among other applications. [25-29] The low toxicity and high spatiotemporal resolution of light make it an ideal choice for the controlled release of drugs and RNPs, enabling on-demand release of the payload. [30-33] It is urgent to custom-develop polymer-based controlled drug delivery carriers to achieve specific light-triggered cleavage for remote activation of photosensitizer and CRISPR/Cas9 RNP. At the same time, the targeting issue of nanodelivery systems has also received significant attention. Nanoparticles coated with membranes, especially those disguised with biological cell membranes, can leverage their inherent biocompatibility and homotypic targeting properties to optimize their distribution and retention time in the body. [34-36] In this paper, we report a biomimetic nanodelivery system (OPTC-RNP@CM) based on near-infrared photoregulation semiconductor polymers (OPT) that effectively inhibits TNBC tumor growth (Figure 1). This system is first assembled by semiconductor polymers OPT and photosensitizer (Ce6) to form OPTC, and then connected to Cas9/sgRNA (PD-L1) (Cas9-RNP) via a thioketal (TK) bond (¹O₂-cleavable linker) to synthesize OPTC-RNP. Finally, the 4T1 cell membranes are wrapped around the OPTC-RNP to form a light-triggered biomimetic nanodelivery system. Meanwhile, the homologous targeting and camouflage properties of the cell membranes ensure that OPTC-RNP@CM is effectively delivered to the tumor site. It is noteworthy that, under the guidance of second near-infrared (NIR-II) fluorescence imaging, a large accumulation of biomimetic nanomaterials is observed at the tumor site. Polymer nanomaterial OPTC produce 1 O 2 under near-infrared light irradiation, and the side chains can safely release RNP through the cleavage of the linkers (sulfur-containing parts) by 1 O 2 . Subsequently, the light-induced PDT effect activates ICD and combines with Cas9-RNP to synergistically enhance the immune response, making the anti-tumor effect more pronounced. Overall, this innovative biomimetic nanodelivery system demonstrates the potential of OPTC-RNP@CM as a nanoscale drug delivery system for TNBC. Not only does it improve the targeting and efficiency of therapeutic drugs, but it also specifically activates the immune-suppressive TME through the synergistic effects of multiple therapies, providing a novel and referable approach for the efficient clinical treatment of breast cancer. [1]¿p#1 FIGURE 1 | Schematic illustration of light-triggered biomimetic OPTC-RNP@CM NPs for synergistic PDT-induced ICD and CRISPR/Cas9-mediated knockout of PD-L1 enhanced immunotherapy under NIR-II imaging guidance. 2 | Results and Discussion 2.1 | Synthesis and Characterization of OPTC-RNP@CM We constructed a biomimetic tumor membrane-enclosed light-responsive nanodelivery platform (OPTC-RNP@CM). The synthesis rout of OPT is shown in Figure 2. First, monomer A and monomer B were synthesized according to previous reports [37] (Supporting Information: Figure S1 and S2). Monomer B was employed to synthesize OSP (Supporting Information: Figures S3 and S4), which was then reacted with NH 2 -PEG-NH 2 to yield water-soluble OSP-PEG. The resulting OSP-PEG exhibited characteristic peaks corresponding to the -CH 2 CH 2 O- moiety (3.6 ppm) from NH 2 -PEG-NH 2 in the 1 H NMR spectrum (Supporting Information: Figure S5,). Next, a ROS-sensitive thioketal (TK) linker was introduced to obtain OSP-PEG-TK (OPT). The successful attachment of the TK linker was confirmed by the appearance of characteristic peaks (-SCCH 3 CH 3 S-, 1.6 ppm) in the 1 H NMR spectrum (Supporting Information: Figure S6,). OPT and Ce6 were co-assembled to form OSP-PEG-TK-Ce6 (OPTC), and the successful encapsulation of Ce6 was confirmed through UV-visible absorption spectrum. Transmission electron microscopy (TEM) revealed that OPTC self-assembled into spherical particles with an average diameter of 112 nm (Figure 3B), a finding further supported by dynamic light scattering (DLS) analysis (Figure 3E). Next, the carboxyl group of TK was conjugated with Cas 9 RNP targeting PD-L1, forming OPTC-RNP. To optimize the loading efficiency of Cas9 RNP, OPTC-RNP nanoparticles with varying mass ratios of OPTC to Cas9 RNP were synthesized. The Cas9 RNP content in the OPTC-RNP nanoparticles was assessed via agarose gel electrophoresis, which identified the optimal OPTC to Cas9-RNP ratio as 50 (Supporting Information: Figure S7,). Finally, based on previous reports, [38,39] cancer cell-mimicking vesicles (CMs) were prepared using 4T1 breast cancer cells and used to coat OPTC-RNP, forming OPTC-RNP@CM. This modification enabled selective targeting of homologous tumor cells. [1]¿p#1 FIGURE 2 | Synthesis route of the 1 O 2 -responsive semiconducting polymer OPT. To evaluate the stability of OPTC-RNP@CM, we simulated complex vascular environments and conducted dynamic light scattering (DLS) measurements in phosphate-buffered saline (PBS, pH 7.4), Dulbecco’s Modified Eagle Medium (DMEM) with 50% fetal bovine serum (FBS), and water at various time points. The hydrodynamic diameter of OPTC-RNP@CM remained largely unchanged across the different media, indicating that these membrane-coated, self-assembled nanoparticles exhibit good stability (Supporting Information: Figure S8). Transmission electron microscopy (TEM) images clearly reveal that the OPTC-RNP is encapsulated within a low-contrast bio-membrane structure, likely derived from the 4T1 cell membrane. To verify the retention of membrane proteins on OPTC-RNP@CM, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. Notably, the same protein profiles were observed in both the 4T1 cell membrane group and the OPTC-RNP@CM group, confirming the presence of adhesion molecules that mediate homotypic cell-cell adhesion (Figure 3C). This encapsulation was further confirmed by DLS, which showed an increase in size from 126 ± 0.34 nm for OPTC-RNP to 213 ± 0.64 nm for OPTC-RNP@CM (Figure 3E). Additionally, zeta potential measurements indicated a significant change in surface charge from −14.80 mV to −22.43 mV, which is attributed to the negatively charged nature of the 4T1 cell membrane (Figure 3D). Additionally, the UV-Vis absorption spectrum and fluorescence emission spectrum demonstrated that OPTC-RNP@CM exhibits strong near-infrared absorption and second region emission characteristics, providing an advantage in penetrating deep tissues, making it suitable for in vivo light modulation and tracking (Figure 3F, G). 2.2 | In Vitro Light-responsive Drug Release Assessment To verify whether OPTC-RNP@CM nanoparticles exhibit ideal PDT effects, we evaluated their ability to generate 1 O 2 in vitro by measuring 1,3-diphenylisobenzofuran (DPBF). As the irradiation time with a 660 nm laser increased, the absorbance of the characteristic peak of DPBF at 420 nm significantly decreased, indicating that OPTC-RNP@CM can efficiently generate 1 O 2 under 660 nm laser irradiation (Supporting Information: Figure 3H and Figure S9). This is a prerequisite for achieving controlled release of CRISPR RNP through ROS-triggered cleavage of the thioether bond in OPTC-RNP@CM. The thioether bond in the molecule modified with TK can be easily cleaved by 1 O 2 , thereby enabling controlled release of Cas9-RNP triggered by near-infrared light irradiation. Considering the high efficiency of 1 O 2 generation by OPTC-RNP@CM, we evaluated the in vitro release behavior of Cas9-RNP from OPTC-RNP@CM triggered by near-infrared light. The OPTC-RNP@CM nanoparticles were irradiated with NIR at different time points, and the residual amount of Cas9-RNP in OPTC-RNP@CM was detected using SDS-PAGE. Compared to the group without near-infrared irradiation, the residual amount of Cas9-RNP in OPTC-RNP@CM rapidly decreased with prolonged incubation time. Approximately 70% of Cas9-RNP was released 8 hours after near-infrared irradiation (Figure 3I, J). This reflects that the Cas9-RNP can be effectively released from OPTC-RNP@CM nanoparticles through 1 O 2 -mediated activation upon NIR irradiation. FIGURE 3 | Preparation and characterization of OPTC-RNP@CM nanomaterials. A) Representative TEM images of OPTC and OPTC-RNP@CM. Scale bar: 200 nm. B) SDS-PAGE analysis of (I) marker, (II) 4T1cell lysate, (III) 4T1 CM, (IV) OPTC-RNP@CM, (V) RNP and (VI) marker. C) Zeta potential of OP, OPT, OPTC, OPTC-RNP, and OPTC-RNP@CM. D) DLS of OPTC, OPTC-RNP, and OPTC-RNP@CM. E) UV-Visible absorption spectra of OP, OPT, OPTC, Ce6, and TK. F) Fluorescence emission spectrum of OPT, OPTC, OPTC-RNP, and OPTC-RNP@CM. G) Relative absorbance of DPBF after adding OPTC-RNP@CM with or without NIR irradiation. H) Residual amount of RNP in OPTC-RNP@CM after NIR irradiation for different durations, determined by SDS-PAGE. I) RNP release from OPTC-RNP@CM corresponding to different durations of NIR irradiation. 2.3 | In Vitro Cellular Uptake and Gene Editing Effects We investigated the intracellular delivery capability of this nanoplatfrom and assessed its gene-editing efficiency in 4T1 cells. Before conducting in vitro therapeutic studies, it is essential to confirm whether the designed OPTC-RNP@CM nanoparticles can be effectively endocytosed by cancer cells. First, we co-incubated OPTC-RNP@CM with 4T1 cells for 6 hours, where the RNP in OPTC-RNP@CM was labeled with EGFP. Subsequently, the cells were fixed and the nuclei were stained. Using confocal laser scanning microscopy (CLSM), we observed a clear green ERFR fluorescent signal of Cas9-RNP in the near-infrared light irradiation group, indicating that the OPTC-RNP@CM nanoparticles were successfully taken up by the cells and delivered into the nucleus (Figure 4A). Subsequent targeted gene editing can only proceed after the release of Cas9 RNP from the nanoparticles. The 1 O 2 generated by OPTC-RNP@CM under near-infrared light irradiation can cleave the thioether bonds, thereby promoting the release of Cas9-RNPs from the nanoparticles and efficiently delivering them to the cell nucleus. Therefore, the OPTC-RNP@CM nanoparticles we designed hold great potential for near-infrared light-activated genome editing in deep tissues. Next, to evaluate the gene-editing capability of this therapeutic platform in vitro, we divided the four T1 cells into different treatment groups (I: PBS/NIR, II: OPTC, III: OPTC/NIR, IV: OPTC-RNP@CM, V: OPTC-RNP@CM/NIR) and verified the gene-editing effects in vitro. First, the mutation frequency was quantified using the T7 endonuclease 1 (T7E1) assay. The mutation frequency of PD-L1 detected in the OPTC-RNP@CM/NIR group was 26.22% (Figure 4E). To further verify, we used Western blot (WB) analysis to detect the expression of PD-L1 protein in 4T1 cells after different treatments. The expression level of PD-L1 protein in the OPTC-RNP@CM/NIR group was lower than that in the control group (Figure 4F). The experimental results were consistent with the T7E1 assay results, confirming the precise deletion of the PD-L1 gene in 4T1 cells. Finally, to further demonstrate that OPTC-RNP@CM can accomplish gene editing of PD-L1, we performed DNA sequencing of the PD-L1 gene in the OPTC-RNP@CM/NIR group. The target gene showed three representative deletions and insertions near the PAM sequence, further confirming the CRISPR/Cas9-mediated knockout of PD-L1 (Figure 4G). 2.4 | In Vitro ROS Generation Evaluation and Cytotoxicity To confirm that OPTC-RNP@CM can generate ROS under NIR light after entering cells, we used the 2′,7′-dichlorofluorescin diacetate (DCFH-DA) probe to assess the intracellular total free radical levels. This probe is hydrolyzed and oxidized into the green fluorescent product dichlorofluorescein (DCF) under high oxidative stress. No intracellular green fluorescence signal was observed in 4T1 cells treated with OPTC or OPTC-RNP@CM without NIR light exposure. In contrast, intracellular green fluorescence was observed in the OPTC/NIR and OPTC-RNP@CM/NIR groups (Figure 4B). Since the surface encapsulation of cancer cell membranes can help nanoparticles target homologous cells, the accumulation of more nanoparticles within the cells can generate more ROS under near-infrared light excitation. Therefore, the fluorescence signal intensity in the OPTC-RNP@CM/NIR group is higher than that in the OPTC-RNP@CM group. The OPTC-RNP@CM nanoparticles produce a large amount of 1 O 2 under near-infrared light irradiation, causing DNA damage to tumor cells, leading to apoptosis and activating immunogenic cell death. Near-infrared-controlled genome editing enhances the effectiveness of immunotherapy by knocking out the PD-L1 gene, thereby improving the immune system’s ability to recognize and kill tumor cells. To validate the anti-tumor potential of OPTC-RNP@CM nanoparticles under near-infrared light irradiation, we first performed a standard CCK-8 assay to assess the viability of 4T1 cells after co-incubation with different concentrations (0, 10, 20, 40, 80 µg/mL) of OPTC and OPTC-RNP@CM without near-infrared light exposure. The cell survival rates were all above 90%, indicating no significant cytotoxicity. FIGURE 4 | Synergistic cancer therapy with PDT and gene editing of OPTC-RNP@CM in vitro. A) CLSM analysis of intracellular delivery of OPTC-RNP@CM carrying FITC-labeled Cas9 (green) to 4T1 cells under different conditions. Blue: DAPI; Red: CM-Dil. Scale bars: 10 μm. B) Representative fluorescent images of intracellular ROS in 4T1 cells stained with DCFH-DA after different treatments (Scale bar: 300 μm). C) The representative fluorescent images of 4T1cells after different treatments. Green, calcein-AM; Red, PI. Scale bar: 300 μm. D) The apoptosis of 4T1 cells under different treatments was analyzed by flow cytometry. E) T7EI assay for indels frequency analysis of 4T1 cells of PD-L1 loci after transfection with different treatments. F) Western blotting for PD-L1 expression levels in 4T1 cells with different treatments. G) The representative DNA sequence of PD-L1 was detected in the mutant colonies treated with OPTC-RNP@CM/NIR. I: PBS/NIR, II: OPTC, III: OPTC/NIR, IV: OPTC-RNP@CM, V: OPTC-RNP@CM/NIR. When 4T1 cells were exposed to near-infrared light at different power levels, even at a power density of 1 W·cm⁻², there was no significant decrease in cell viability (Figure S10, Supporting Information). These results indicate that OPTC-RNP@CM, as a CRISPR-Cas9 delivery system, has good biocompatibility. After co-incubating OPTC-RNP@CM with 4T1 cells, near-infrared light was applied to evaluate the efficacy of PDT in vitro. To further validate the enhanced PDT of the genomic editing nanoplatform OPTC-RNP@CM, we evaluated the in vitro therapeutic effects of OPTC-RNP@CM at different concentrations (0, 10, 20, 40, 80 µg/mL) under 660 nm near-infrared light irradiation. OPTC-RNP@CM demonstrated higher therapeutic efficacy compared to OPTC at the same incubation concentration. This indicates that OPTC-RNP@CM enhances PDT through genomic editing, showing promising therapeutic effects under near-infrared light irradiation. Next, the 4T1 cells were stained with both Calcein-AM and Propidium Iodide (PI). The killing effect was evaluated by detecting live cells (green fluorescence signal) and dead cells (red fluorescence signal) under a fluorescence microscope. For the PBS/NIR, OPTC, and OPTC-RNP@CM groups, almost all cells exhibited strong green fluorescence signals, indicating that the damage to tumor cells was negligible. The OPTC-RNP@CM group showed both red and green fluorescence signals. However, in the OPTC-RNP@CM/NIR group, bright red PI fluorescence was distinctly observed, indicating the strongest 4T1 cell killing capability (Figure 4C). Finally, we performed flow cytometry with Annexin-V FITC and PI dual staining to detect cell apoptosis, which also supports this trend (Figure 4D). We demonstrated that OPTC-RNP@CM/NIR exhibits good in vitro antitumor effects. This preliminary evidence confirms the feasibility of our CRISPR gene editing-enhanced near-infrared light therapy strategy and provides a basis for further exploration of its in vivo antitumor effects. 2.5 | Biodistribution of OPTC-RNP@CM In Vivo NIR-II imaging can used for intelligent real-time monitoring and evaluation of breast cancer progression after treatment, as well as metabolic detection, making it highly valuable for prognosis. [40] To evaluate the in vivo combined therapeutic effect of OPTC-RNP@CM nanodrugs, we subcutaneously implanted 4T1 cells into the mammary glands of female BALB/c mice, establishing an in situ mammary cancer mouse model. First, to assess the targeting potential of OPTC-RNP@CM nanodrugs to tumors in vivo, we used a NIR-II in vivo imaging system to capture OPTC-RNP@CM distribution in tumor-bearing mice at different time points following tail vein injection (Figure 5A). Over time, the signal at the tumor site gradually increased, peaking at 24 hours and remaining detectable even after 72 hours (Figure 5B). This indicates that the OPTC-RNP@CM nanomedicine can accurately target the tumor site and remain there long enough for subsequent treatment. Furthermore, we euthanized the mice 48 hours after tail vein injection of OPTC-RNP@CM nanomedicine and performed ex vivo organ imaging to observe the fluorescence in the tumor and major organs. The fluorescence was found to be primarily distributed in the liver, tumor, and spleen (Figure 5C). 2.6 | Antitumor Effects of OPTC-RNP@CM In Vivo Based on the excellent tumor-targeting capability of the OPTC-RNP@CM nanodrug in vivo, we proceeded with in vivo antitumor experiments using a 4T1 tumor-bearing BALB/c mouse model. The tumor-bearing mice were randomly divided into five groups (n=5): (I: PBS/NIR, II: OPTC, III: OPTC/NIR, IV: OPTC-RNP@CM, V: OPTC-RNP@CM/NIR). After 24 hours of intravenous injection, the mice were irradiated with a 660 nm laser (1 W·cm⁻², 5 min). After 72 hours, the nanodrug was injected into the mice again. This treatment cycle was repeated four times, with the mice being euthanized on the 12th day (Figure 6A). We recorded the body weight and tumor volume and weight of mice in all experimental groups. Compared to the PBS/NIR group, the other four groups all exhibited inhibitory effects on tumor growth. The most significant tumor inhibition was observed in the OPTC-RNP@CM/NIR group, indicating that the combination of PDT and PD-L1-targeted gene editing immunotherapy has a strong therapeutic effect (Figure 6B-E). [1]¿p#1 FIGURE 5 | The distribution of OPTC-RNP@CM in vivo after intravenous injection in breast cancer-bearing mice. A) Representative in vivo NIR-II fluorescence imaging of nude mice bearing 4T1 breast cancer in BALB/C mice following intravenous injection of OPTC-RNP@CM. B) NIR-II fluorescence signals of tumor, liver was tracked at different time points. C) After 48 hours of drug administration, the mice were euthanized, and the main organs and tumor tissues were collected for two-region imaging analysis. Hereafter, to confirm whether effective knockout of PD-L1 occurred in vivo, we collected tumor tissues from mice in different treatment groups and conducted WB experiments to detect PD-L1 protein expression in the tumor tissues. In the OPTC-RNP@CM/NIR group, the PD-L1 protein level was significantly reduced, indicating that the PD-L1 gene was successfully edited in vivo (Figure 6F). To further confirm the effectiveness of PD-L1 gene editing, we performed immunohistochemical staining on tumor tissues. The results also confirmed a decrease in PD-L1 protein expression in the OPTC-RNP@CM/NIR group, consistent with the findings from WB. Subsequently, the tumor tissues were stained using Hematoxylin and Eosin (H&E) and TUNEL. The results of both staining methods confirmed that the OPTC-RNP@CM/NIR group induced tumor cell damage and apoptosis, showing a strong inhibitory effect on tumor growth (Figure 6G). Additionally, we evaluated the potential toxic side effects in vivo under near-infrared light-regulated OPTC-RNP@CM combined immunotherapy. H&E staining of major organs (including the liver, heart, lungs, spleen, and kidneys) showed no significant organ damage (Supporting Information: Figure S11). Finally, we conducted a hemolysis test on the OPTC-RNP@CM nanocomposite material. Compared to the positive control group (ddH 2 O), the hemolysis rate of the nanocomposite material at different concentrations was low, indicating good blood compatibility (Supporting Information: Fig S12). Therefore, the OPTC-RNP@CM nanocomposite material not only demonstrates strong antitumor immunotherapeutic effects in vivo but also possesses high biosafety. FIGURE 6 | Synergistic TNBC immunotherapy with gene editing and PDT based on OPTC-RNP@CM in vivo. A) Schematic illustration of the treatment process at different times of the vivo experiments with female BALB/C mice. B-D) The tumor weights, tumor volumes and mouse body weights change after different treatments. E) The physical images of tumors with different treatments. F) The analysis of PD-L1 expression in tumor tissues under different treatment methods using Western blot. G) H&E, PD-L1, TUNEL staining of tumor sections from different treatments. Scale bars: 20 µm (H&E, PD-L1) and 50 µm (TUNEL). I: PBS/NIR, II: OPTC, III: OPTC/NIR, IV: OPTC-RNP@CM, V: OPTC-RNP@CM/NIR. (*P < 0.05, **P < 0.01, ***P < 0.001). 2.7 | In Vivo Immune Response In order to investigate the synergistic activation of tumor immunity by OPTC-RNP@CM nanoparticles, we evaluated the major immune cell populations within the tumor microenvironment. The infiltration of cytotoxic T lymphocytes (CD4 + CD3 + CD8 + T cells) at the tumor site is a key indicator of the effectiveness of immunotherapy, and flow cytometry was used to analyze the different groups. The results showed that the proportion of CD3 + CD8 + T cells in tumor tissues significantly increased in the other treatment groups compared to the control group (PBS/NIR). Notably, the OPTC-RNP@CM/NIR group induced the highest proportion of CD3 + CD8 + T cell infiltration in tumor tissues, indicating that PDT-induced ICD combined with PD-L1 gene editing significantly enhanced the anti-tumor immunity in mice (Figure 7A). In contrast, for immune-suppressive regulatory T cells (Tregs), their proportion was lower in the treatment groups compared to the control group (PBS/NIR). Furthermore, the OPTC-RNP@CM/NIR group had the lowest Tregs content, suggesting a substantial improvement in the immune system of these mice (Figure 7B). Then, we investigated whether OPTC-RNP@CM/NIR could promote the maturation of dendritic cells (DCs) (CD45 + CD11c + CD80 + CD86 + ) in tumor tissues compared to the control group. The results showed that the OPTC-RNP@CM/NIR group had a significantly higher content of mature DCs in the tumor tissues compared to the control group and other experimental groups. This indicates that the OPTC-RNP@CM/NIR treatment effectively recruited a large number of DCs to the tumor tissues and stimulated their maturation, significantly enhancing the antigen-presenting capability (Figure 7C). Finally, to better demonstrate the immunotherapy effect of PDT combined with targeted PD-L1, we measured the expression levels of related inflammatory cytokines in mouse serum using ELISA. The experimental results were consistent with the trends observed in the aforementioned flow cytometry results (Figure 7D). In summary, tail vein injection of OPTC-RNP@CM nanoparticles, which synergistically induce ICD and target PD-L1 gene editing, greatly enhanced the tumor immune response in mice. FIGURE 7 | Synergistic activating T cell-mediated anti-tumor immunity by targeting PD-L1 and PDT. A) Representative flow cytometry plots and the quantitative analysis of CD8 + T cells (gated on CD3 + T cells) in tumor tissues of mice from different treatment groups. B) Representative flow cytometry plots and the quantitative analysis of Tregs in tumor tissues from mice in different treatment groups. C) Representative flow cytometry plots and the quantitative analysis of matured DCs in primary tumor (CD80 + CD86 + gated on CD11c + cells) after different treatments. D) The expression levels of IFN-γ, TNF-α and IL-6 in serum analyzed by ELISA. Group I：PBS/NIR、II：OPTC、III：OPTC/NIR、IV：OPTC-RNP@CM、V：OPTC-RNP@CM/NIR。(*P < 0.05, **P < 0.01, ***P < 0.001). 3 | Conclusion In summary, we have rationally designed and established a biomimetic targeted and synergistic treatment strategy by ingeniously combining the multifunctional polymer OPT, photosensitizer Ce6, CRISPR/Cas9 PD-L1 , and 4T1 tumor cells to form light-remote-controlled OPTC-RNP@CM nanoparticles. Under NIR light irradiation, OPTC generated 1 O 2 , which cleaved the 1 O 2 -cleavable linker, enabling the safe and efficient release of the gene carrier RNP. This nanoparticle, under NIR-II imaging-guided NIR light remote control, induced PDT-induced ICD and combined with PD-L1 to not only prevent immune escape but also synergistically amplify the immune response, ultimately activating key cytokines IFN-γ, TNF-α, and IL-6, resulting in significant antitumor immunotherapy. This multimodal approach simultaneously targets tumors and enhances the overall antitumor immune response, providing a promising multifunctional platform for targeted tumor therapy in clinical settings. [1]¿p#1 Author Contributions Yu Ji and Jing Qian contribute equally to this work. Acknowledgment This study was funded by the National Natural Science Foundation of China (32471475); the Natural Science Foundation of Jiangsu Province (BK20210685); Yangtze River Delta joint sci-tech innovation and research projects (2023CSJZN0600); the Innovation Projects of State Key Laboratory of Technologies for Chinese Medicine Pharmaceutical Process Control and Intelligent Manufacture（NZYSKL240201); Beijing MDK Public Welfare Foundation Research Fund (MDK 2022-1001); Pandeng Fund of Harbin Medical University Cancer Hospital (PDTS 2024A-03); Postdoctoral Scientific Research Developmental Fund of Heilongjiang (LBH-Q22); Jiangsu Key Discipline Construction Fund of the 14th Five-Year Plan (Biology); National Natural Science Foundation of China (22475105) and the Project of Jiangsu Specially-Appointed Professor (RK030STP22003). Conflict of Interest The authors declare no conflict of interest. Data Availability Statement The data that support the findings of this study are available in the supplementary material of this article. [1]¿p#1 References [1] N. Simon, A. Antignani, R. Sarnovsky, S.M. Hewitt, D. FitzGerald, JNCI: Journal of the National Cancer Institute 2016 , 108, djw028. [2] R.A. Leon-Ferre, M.P. Goetz, BMJ , 381 (2023) e071674. [3] F. Derakhshan, J.S. Reis-Filho, Annu Rev Pathol , 17 (2022) 181-204. [4] F. Nasiri, M. Kazemi, S.M.J. Mirarefin, M. Mahboubi Kancha, M. Ahmadi Najafabadi, F. Salem, S. Dashti Shokoohi, S. Evazi Bakhshi, P. Safarzadeh Kozani, P. Safarzadeh Kozani, Front Immunol , 13 (2022) 1018786. [5] M. Guha, S. Srinivasan, P. Raman, Y. Jiang, B.A. Kaufman, D. Taylor, D. Dong, R. Chakrabarti, M. Picard, R.P. Carstens, Y. Kijima, M. Feldman, N.G. Avadhani, Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease , 1864 (2018) 1060-1071. [6] Y. Zhang, Z. Zhang, Cellular & Molecular Immunology , 17 (2020) 807-821. [7] L. Fan, S. Qu, J. Qian, G. Shi, Q. Huang, Y. Song, Y. Ji, Q. Fan, X. Han, Chemical Engineering Journal , 493 (2024) 152754. [8] X. Wang, C. Tokheim, S.S. Gu, B. Wang, Q. Tang, Y. Li, N. Traugh, Z. Zeng, Y. Zhang, Z. Li, B. Zhang, J. Fu, T. Xiao, W. Li, C.A. Meyer, J. Chu, P. Jiang, P. Cejas, K. Lim, H. Long, M. Brown, X.S. Liu, Cell , 184 (2021) 5357-5374.e5322. [9] L. Zhao, D. Li, Y. Zhang, Q. Huang, Z. Zhang, C. Chen, C.-F. Xu, X. Chu, Y. Zhang, X. Yang, ACS Nano , 16 (2022) 13821-13833. [10] X. Liu, D. Wang, P. Zhang, Y. Li, Nano Today , 29 (2019) 100801. [11] L. Zhao, Y. Luo, Q. Huang, Z. Cao, X. Yang, Small , 16 (2020) 2004879. [12] L. Li, Z. Yang, S. Zhu, L. He, W. Fan, W. Tang, J. Zou, Z. Shen, M. Zhang, L. Tang, Y. Dai, G. Niu, S. Hu, X. Chen, Advanced Materials , 31 (2019) 1901187. [13] K. Hayashi, F. Nikolos, Y.C. Lee, A. Jain, E. Tsouko, H. Gao, A. Kasabyan, H.E. Leung, A. Osipov, S.Y. Jung, A.V. Kurtova, K.S. Chan, Nature Communications , 11 (2020) 6299. [14] H. Aria, M. Rezaei, Biomedicine & Pharmacotherapy , 161 (2023) 114503. [15] Z. Li, X. Lai, S. Fu, L. Ren, H. Cai, H. Zhang, Z. Gu, X. Ma, K. Luo, Advanced Science , 9 (2022) 2201734. [16] L. Minute, A. Teijeira, A.R. Sanchez-Paulete, M.C. Ochoa, M. Alvarez, I. Otano, I. Etxeberrria, E. Bolaños, A. Azpilikueta, S. Garasa, N. Casares, J. Luis Perez Gracia, M.E. Rodriguez-Ruiz, P. Berraondo, I. Melero, Journal for ImmunoTherapy of Cancer , 8 (2020) e000325. [17] W. Xiong, Z. Cheng, H. Chen, H. Liang, M. Wang, Y. Chen, J. Ying, Y. Cai, J. Chai, K. Dou, W. Zheng, S. Zheng, L. Zhao, Advanced Functional Materials , n/a (2024) 2410841. [18] X. Dai, D. Liu, P. Pan, G. Liang, X. Wang, W. Chen, J ournal of Colloid and Interface Science , 661 (2024) 930-942. [19] G. Lan, K. Ni, Z. Xu, S.S. Veroneau, Y. Song, W. Lin, J ournal of the American Chemical Society , 140 (2018) 5670-5673. [20] M. Overchuk, R.A. Weersink, B.C. Wilson, G. Zheng, ACS Nano , 17 (2023) 7979-8003. [21] Y. Ji, L. Fan, S. Qu, X. Han, Journal of Materials Chemistry B , 10 (2022) 7694-7707. [22] Q. Deng, A. Hua, S. Li, Z. Zhang, X. Chen, Q. Wang, X. Wang, Z. Chu, X. Yang, Z. Li, Exploration , 0 (2025) e20240080. [23] S. Rahaiee, E. Assadpour, A. Faridi Esfanjani, A.S. Silva, S.M. Jafari, Advances in Colloid and Interface Science , 279 (2020) 102153. [24] Y. Lyu, C. Yang, X. Lyu, K. Pu, Small , 17 (2021) 2005222. [25] Y. Lyu, S. He, J. Li, Y. Jiang, H. Sun, Y. Miao, K. Pu, Angewandte Chemie International Edition , 58 (2019) 18197-18201. [26] Q. Miao, C. Xie, X. Zhen, Y. Lyu, H. Duan, X. Liu, J.V. Jokerst, K. Pu, Nature Biotechnology , 35 (2017) 1102-1110. [27] Y. Jiang, K. Pu, Accounts of Chemical Research , 51 (2018) 1840-1849. [28] J. Li, J. Huang, Y. Lyu, J. Huang, Y. Jiang, C. Xie, K. Pu, Journal of the American Chemical Society , 141 (2019) 4073-4079. [29] J. Li, K. Pu, Chem Soc Rev, 48 (2019) 38-71. [30] L.R. Polstein, C.A. Gersbach, Nature Chemical Biology , 11 (2015) 198-200. [31] Y. Pan, J. Yang, X. Luan, X. Liu, X. Li, J. Yang, T. Huang, L. Sun, Y. Wang, Y. Lin, Y. Song, Science Advances , 5 eaav7199. [32] A. Ju, S.W. Lee, Y.E. Lee, K.-C. Han, J.-C. Kim, S.C. Shin, H.J. Park, E. EunKyeong Kim, S. Hong, M. Jang, Biomaterials , 217 (2019) 119298. [33] X. Xu, C. Liu, Y. Wang, O. Koivisto, J. Zhou, Y. Shu, H. Zhang, Advanced Drug Delivery Reviews , 176 (2021) 113891. [34] Z. Chen, Q. Hu, Z. Gu, Accounts of Chemical Research , 51 (2018) 668-677. [35] R.H. Fang, Y. Jiang, J.C. Fang, L. Zhang, Biomaterials , 128 (2017) 69-83. [36] Y. Miao, Y. Yang, L. Guo, M. Chen, X. Zhou, Y. Zhao, D. Nie, Y. Gan, X. Zhang, ACS Nano , 16 (2022) 6527-6540. [37] J. Li, C. Tian, Y. Yuan, Z. Yang, C. Yin, R. Jiang, W. Song, X. Li, X. Lu, L. Zhang, Q. Fan, W. Huang, Macromolecules , 48 (2015) 1017-1025. [38] W. Xiong, Z. Cheng, H. Chen, H. Liang, M. Wang, Y. Chen, J. Ying, Y. Cai, J. Chai, K. Dou, W. Zheng, S. Zheng, L. Zhao, Advanced Functional Materials , (2024). [39] R.H. Fang, W. Gao, L. Zhang, Nature Reviews Clinical Oncology , 20 (2023) 33-48. [40] S. Liang, D. Hu, G. Li, D. Gao, F. Li, H. Zheng, M. Pan, Z. Sheng, Sci Bull (Beijing), 67 (2022) 2316-2326. Information & Authors Information Version history V1 Version 1 25 April 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords crispr nir-ii imaging semiconductor polymer Authors Affiliations Yu Ji Nanjing University of Chinese Medicine View all articles by this author Jing Qian Nanjing University of Chinese Medicine View all articles by this author Liansheng Fan Nanjing University of Chinese Medicine View all articles by this author Gaoyu Shi Nanjing University of Chinese Medicine View all articles by this author Ke Liu Nanjing University of Chinese Medicine View all articles by this author Guangzhao Yang Nanjing University of Posts and Telecommunications View all articles by this author Suchen Qu Nanjing University of Chinese Medicine View all articles by this author Jie Tang Nanjing University of Chinese Medicine View all articles by this author Yanni Song Harbin Medical University Cancer Hospital View all articles by this author Liping Yin Nanjing University of Chinese Medicine View all articles by this author Chao Yin Nanjing University of Posts and Telecommunications View all articles by this author Xin Han 0000-0002-9620-7502 [email protected] Nanjing University of Chinese Medicine View all articles by this author Metrics & Citations Metrics Article Usage 236 views 107 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Yu Ji, Jing Qian, Liansheng Fan, et al. 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