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A cisplatin prodrug-based self-assembling ozone delivery nanosystem sensitizes radiotherapy in triple-negative breast cancer
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Tianyue Xua, Dan Zhenga, b, Meixu Chena, Linlin Songa, c, Zhihui Liua, Yan Chengb, d, Yujie Zhaoa, Liwen Huanga, Yixuan Lia, Zhankun Yange, Cong Lig, Biao Dongf, *, Jing Jinga, *, Hubing Shia, *
Acta Pharmaceutica Sinica B | 2025, 15(5) : 2703 - 2722
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Acta Pharmaceutica Sinica B | 2025, 15(5): 2703-2722
• ORIGINAL ARTICLES •
A cisplatin prodrug-based self-assembling ozone delivery nanosystem sensitizes radiotherapy in triple-negative breast cancer
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Tianyue Xua, Dan Zhenga, b, Meixu Chena, Linlin Songa, c, Zhihui Liua, Yan Chengb, d, Yujie Zhaoa, Liwen Huanga, Yixuan Lia, Zhankun Yange, Cong Lig, Biao Dongf, *, Jing Jinga, *, Hubing Shia, *
Affiliations
  • aInstitute of Breast Health Medicine, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu 610041, China
  • bCancer Center, Breast Center, West China Hospital, Sichuan University, Chengdu 610041, China
  • cDepartment of Ultrasound & Laboratory of Ultrasound Medicine, West China Hospital, Sichuan University, Chengdu 610041, China
  • dMulti-omics Laboratory of Breast Diseases, State Key Laboratory of Biotherapy, National Collaborative, Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China
  • eCollege of Chemical Engineering, Shijiazhuang University, Shijiazhuang 050035, China
  • fNational Clinical Research Center for Geriatrics and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China
  • gCore Facilities of West China Hospital, Sichuan University, Chengdu 610041, China
About Author:

E-mail addresses: (Biao Dong),

(Jing Jing),

(Hubing Shi).

†

These authors made equal contributions to this work.

Author contributions

Tianyue Xu: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Conceptualization. Dan Zheng: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Conceptualization. Meixu Chen: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis. Linlin Song: Methodology, Investigation. Zhihui Liu: Methodology, Investigation. Yan Cheng: Methodology, Investigation. Yujie Zhao: Methodology, Investigation. Liwen Huang: Investigation. Yixuan Li: Investigation. Zhankun Yang: Writing – review & editing. Cong Li: Methodology. Biao Dong: Writing – review & editing, Supervision, Project administration, Funding acquisition. Jing Jing: Writing – review & editing, Supervision, Project administration, Funding acquisition. Hubing Shi: Writing – review & editing, Writing – original draft, Supervision, Project administration, Funding acquisition, Conceptualization.

doi: 10.1016/j.apsb.2025.03.020
Outline
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Lacking therapeutic targets highlights the crucial roles of chemotherapy and radiotherapy in the clinical management of triple-negative breast cancer (TNBC). To relieve the side effects of the chemoradiotherapy combination regimen, we design and develop a self-assembled micelle nanosystem consisting of perfluorocarbon chain-modified cisplatin prodrug. By incorporating perfluorodecalin, this nanosystem can effectively carry ozone and promote irradiation-derived reactive oxygen species (ROS) production. By leveraging the perfluorocarbon sidechain, the nanosystem exhibits efficient internalization by TNBC cells and effectively escapes from lysosomal entrapment. Under X-ray irradiation, ozone-generated ROS disrupts the intracellular redox balance, thereby facilitating the release of cisplatin in a reduction-responsive manner mediated by reduced glutathione. Moreover, oxygen derived from ozone decomposition enhances the efficacy of radiotherapy by alleviating tumor hypoxia. Notably, the combination of irradiation with ozone-loaded cisplatin prodrug nano system synergistically prompts antitumor efficacy and reduces cellular/systemic toxicity in vitro and in vivo. Furthermore, the combo regimen remodels the tumor microenvironment into an immune-favored state by triggering immunogenic cell death and relieving hypoxia, which provides a promising foundation for a combination regimen of immunotherapy. In conclusion, our nanosystem presents a novel strategy for integrating chemotherapy and radiotherapy to optimize the efficacy and safety of TNBC clinical treatment.

Cisplatin  /  Prodrug  /  Radiotherapy  /  Controlled-release system  /  Ozone  /  Triple-negative breast cancer  /  Reactive oxygen species  /  Immunogenic cell death
Tianyue Xu, Dan Zheng, Meixu Chen, Linlin Song, Zhihui Liu, Yan Cheng, Yujie Zhao, Liwen Huang, Yixuan Li, Zhankun Yang, Cong Li, Biao Dong, Jing Jing, Hubing Shi. A cisplatin prodrug-based self-assembling ozone delivery nanosystem sensitizes radiotherapy in triple-negative breast cancer[J]. Acta Pharmaceutica Sinica B, 2025 , 15 (5) : 2703 -2722 . DOI: 10.1016/j.apsb.2025.03.020
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1.　Introduction
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The prognosis of breast cancer has markedly improved thanks to achievements in hormone therapy for hormonal receptor-positive subtypes and targeted therapy for HER2-positive subtypes using anti-human epidermal growth factor receptor 2 (anti-HER2) agents1. However, patients with triple-negative breast cancer (TNBC), which lacks these druggable targets, often rely on conventional treatments such as chemotherapy and radiotherapy. Chemotherapy holds significant importance in the systemic treatment of TNBC2. For example, cisplatin, one of the most commonly used chemotherapy drugs, disrupts DNA replication and eliminates rapidly dividing cells3. Based on the principle, the advantage of this chemotherapy is to clean the micronodules even if they are invisible. On the flip side, radiotherapy serves as a major local therapy approach for advanced breast cancer4,5, particularly for patients who may not be suitable candidates for surgery. Radiotherapy exerts its antitumor effects primarily by directly causing DNA strand breaks and generating hydroxyl radicals, leading to the indirect killing of tumor cells. Therefore, the advantage of radiotherapy is to locally eliminate advanced tumors with a heavy burden.
The distinct antitumor mechanism and advantages of radiotherapy and chemotherapy provide a foundation for their combination in cancer treatment. Theoretically, the combination of irradiation and chemotherapy drugs, such as cisplatin, has a synergistic effect. This synergy can be attributed to two potential mechanisms referred to as “overlap” and “complementation”6. From the perspective of “overlap”, platinum (Pt) in cisplatin boosts the generation of free radicals induced by ionizing radiation. Pt also captures free electrons liberated from irradiated DNA, causing chemical damage to DNA. From the perspective of “complementation”, Pt not only induces tumor cell G2 arrest, amplifying the radiosensitivity of tumor cells, but also inhibits the repair of radiation-induced DNA damage. In turn, irradiation promotes the cellular uptake of Pt. In practice, combining local treatment (radiotherapy) and systemic therapy (chemotherapy) is beneficial to achieving more comprehensive efficacy. Radiotherapy effectively targets localized lesions, making it an ideal option for preventing local recurrence after surgery or treating a limited number of metastatic lesions to alleviate symptoms. Systemic chemotherapy can suppress or eradicate occult cancer cells, complementing the local treatment.
Inspired by these points, clinicians have combined chemotherapy and radiotherapy in controlling several malignant diseases, including head and neck cancer, larynx cancer, lung cancer, and stage II‒III rectal tumors. For instance, in a clinical trial for patients with locally advanced larynx cancer, concomitant cisplatin/radiotherapy led to notable improvements in locoregional control and preservation of larynx compared with the induction cisplatin/fluorouracil arm or radiotherapy alone7. Furthermore, concurrent administration of chemotherapeutic drugs and irradiation has been found to be superior to sequential administration. In a Japan clinical oncology group study, concurrent chemoradiotherapy (etoposide and cisplatin) demonstrated better survival outcomes in the limited stage of small-cell lung cancer compared to sequential regimens, with a median survival time of 27.2 vs. 19.7 months8. Another clinical study on treating advanced laryngeal cancer with concurrent chemoradiotherapy reported that concurrent chemoradiotherapy with cisplatin is significantly superior to induction chemotherapy, followed by irradiation in terms of laryngeal preservation and locoregional control9. Additionally, clinical trials FFCD 92–03 and EORTC 22921 demonstrated that concurrent preoperative chemoradiotherapy remarkably improved the pathological complete response (pCR) as well as the local control rate of resectable rectal cancer10.
However, in breast cancer, there is no distinction in terms of recurrence or survival outcomes between different sequencing formats of radiotherapy and chemotherapy. Concurrent chemoradiotherapy even increases the risk of side effects such as anemia, telangiectasia, and pigmentation11. Therefore, to apply concurrent chemoradiotherapy in TNBC and improve patient outcomes, some severe problems need to be addressed. Firstly, most chemotherapeutic agents have inevitable systemic toxicities, which are further exacerbated when combined with radiotherapy. Secondly, the hypoxic tumor microenvironment limits the efficacy of chemotherapy and radiotherapy. Thirdly, drug-induced cytotoxicity compromises (neutralizes or reverses) the T-cell-mediated tumor elimination that is promoted by immunogenic cell death (ICD) caused by the combination regimens.
To address these challenges, we design a self-assembled micelle nanosystem consisting of fluorocarbon chain-modified cisplatin prodrug. The nano micelle is loaded with perfluorodecalin (PFD) via its sidechain, which carries and delivers ozone into the tumor microenvironment. Upon internalization, the cisplatin prodrug in nano micelle backbone is transformed into cisplatin by the high level of reduced glutathione (GSH) present in tumor cells, a process further enhanced by ozone-derived reactive oxygen species (ROS) under irradiation stimulation. The released cisplatin, in combination with radiotherapy, synergistically induces oncolysis (Fig. 1). The advantages of this nanosystem are: 1) the cisplatin prodrug specifically releases cisplatin within tumor cells, reducing systemic toxicity; 2) ozone decomposition under irradiation generates oxygen, improving the efficacy of radiotherapy by alleviating tumor microenvironmental hypoxia; 3) enhanced ICD and oxygen level create an immune-favorable microenvironment, laying the foundation for future combination with immunotherapy.
2.　Materials and methods
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2.1.　 Materials
Meryer Biochemical Technology Co., Ltd. (Shanghai, China) provided the cisplatin. PFD and perfluorooctanoic acid (PFOA) were acquired from J&K Scientific (Beijing, China). Macklin Biochemical Technology Co., Ltd. (Shanghai, China) supplied the thionyl chloride (SOCl2). Thiazolyl blue tetrazolium bromide (MTT), terephthalic acid (TA), and bovine serum albumin (BSA) were acquired through Sigma–Aldrich (St. Louis, MO, USA). Fluorescein isothiocyanate (FITC) and NileRed fluorescence probe were obtained from MedChemExpress (Shanghai, China). Vitamin C sodium salt (NaVc) was provided by Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Tween-80 was acquired through BBI Life Sciences Corporation (Shanghai, China). Invitrogen (Carlsbad, CA, USA) supplied the Alexa Fluor 488 or Alexa Fluor 594-conjugated secondary antibodies and Alexa Fluor 647-conjugated wheat germ agglutinin (WGA). Beyotime Biotechnology Co., Ltd. (Nantong, China) provided the assay kit for GSH and oxidized glutathione (GSSG), citrate and ethylenediaminetetraacetic acid (EDTA) antigen retrieval solution, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and endogenous peroxidase-blocking buffer. Ozone was generated by ionizing oxygen with an ozone generator (Chuanghuan Ozone Electrical Equipment Co., Ltd., CH-ZTW3G, Guangzhou, China). Gibco Life Technologies (Grand Island, NY, USA) provided the fetal bovine serum (FBS), collagenase, deoxyribonuclease I (DNase I), and Hanks’ balanced salt solution (HBSS). Merck Millipore (Billerica, MA, USA) provided streptomycin, penicillin, trypsin–EDTA, and radioimmunoprecipitation assay (RIPA). HyClone (Logan, UT, USA) provided the RPMI and DMEM medium. Secondary antibodies conjugated with species-specific horseradish peroxidase (HRP) and 3, 3′-diaminobenzidine (DAB) were acquired from Dako (Glostrup, Denmark).
2.2.　 Cells and mice
The SUM149 cell line was acquired from the State Key Laboratory of Biotherapy of Sichuan University (Chengdu, China). The 4T1, BT-549, and MCF-10A cell lines were acquired from the National Collection of Authenticated Cell Cultures (Shanghai, China). The cells were all cultured in a completed RPMI/DMEM medium, which was supplemented with 10% FBS, 100 μg/mL streptomycin, and 100 U/mL penicillin in a 37 °C CO2 incubator. The female BALB/c mice were obtained from Beijing HFK Bioscience Co., Ltd. (Beijing, China) and kept in specialized facilities under pathogen-free conditions. The mice received humane treatment throughout the experiments. The animal experiments followed the protocols approved by the Ethics Review Committee of Animal Experimentation of Sichuan University (20230302071, 20231221004).
2.3.　 Synthesis of Pt(IV)-PFOA
Cisplatin (100 mg) was suspended in distilled water (2.5 mL) at 55 °C under stirring. Then, 3.5 mL of hydrogen peroxide (H2O2) with a weight percentage of 30% was introduced into the mixture, which was then stirred at 55 °C for 1.5 h. Subsequently, the reaction solution was cooled to 4 °C overnight to allow complete crystallization of the resulting product, which was then collected by filtration. The collected precipitate was sequentially washed with pre-cooled distilled water (240 μL), absolute ethanol (240 μL), and absolute ether (600 μL) to obtain oxoplatin as a pale-yellow powder (yield: 80.5%).
To synthesize pentadecafluorooctanoyl chloride (PFOC), PFOA (1 g), SOCl2 (104 μL), and N,N-dimethylformamide (DMF, 50 μL) were stirred and reacted under a nitrogen atmosphere at 75 °C for 4 h. After the reaction, the remaining SOCl2 and DMF in the system were removed by rotary evaporation, yielding PFOC as a pale-yellow liquid (yield: 80.7%).
Subsequently, oxoplatin (100 mg) and excess PFOC were thoroughly mixed under nitrogen protection and reacted at 75 °C for 12 h. The product precipitate was washed with saturated sodium bicarbonate solution, followed by thorough washing with pure water. After drying the precipitate, a white powdery solid Pt(IV)-PFOA was obtained (yield: 31.4 %).
The products were dissolved in chloroform-d (CDCl3) or dimethyl sulfoxide-d6 (DMSO-d6) and analyzed by 1H-NMR and 9F-NMR using a nuclear magnetic resonance (NMR) spectrometer (Bruker, Bruker Avance 400, Rheinstetten, Germany). Analysis of Fourier-transform infrared spectroscopy (FTIR, Bruker, INVENIO R, Ettlingen, Germany) was conducted. The molecule weight was measured by ultra-high-performance liquid chromatography coupled to high-resolution Orbitrap mass spectrometry (UHPLC–Orbitrap–MS, ThermoFisher Scientific, Q Exactive Plus, Bremen, Germany).
2.4.　 Preparation of micelle PtF and PFD@PtF
PtF nanoparticles were prepared by self-assembling Pt(IV)-PFOA using nanoprecipitation. A dimethyl sulfoxide (DMSO) solution of Pt(IV)-PFOA (100 μL, 15 mg/mL) was slowly added drop by drop into distilled water (2 mL) under 35 W ultrasound (Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China) to facilitate the formation of PtF nanoparticles. After adding the organic phase, further ultrasonic treatment at 35 W (Ningbo Scientz Biotechnology Co., Ltd.) was applied to achieve a uniform particle size. The ultrasound (Ningbo Scientz Biotechnology Co., Ltd.) was programmed with a duty cycle of 5 s on and 2 s off, lasting for a total of 10 min. Finally, the resulting PtF nanoparticles were dialyzed with a 3500 D dialysis membrane to remove DMSO from the system.
To prepare the PFD@PtF, PFD (50 μL) was added to a Pt(IV)-PFOA acetone solution (1 mL, 2 mg/mL). The mixture underwent ultrasonication until emulsified. Afterward, the system received an additional 2 mL of distilled water, and sonication was maintained until a consistent emulsion was achieved. The dispersed PFD@PtF in water was obtained after removing the acetone in the system using rotary evaporation. To obtain O3_PFD@PtF, we introduced ozone into the PFD@PtF by blowing for approximately 3 min until saturation was reached. The ozone gas was generated by ionizing oxygen, and the ozone concentration was 50 mg/L.
2.5.　 Characterization
The hydrodynamic diameter was determined using ZetaView TWIN PMX-220 (Particle Metrix, Meerbusch, Germany). Transmission electron microscope (TEM) images were captured using a JEOL microscope (JEOL, 1200 EX II, Tokyo, Japan).
The amount of Pt(IV)-PFOA loaded in the PFD@PtF was quantified by high-performance liquid chromatography (HPLC, Agilent Technologies, 1260 Infinity, Santa Clara, CA, USA) analysis as follows: After centrifuging the PFD@PtF, the resulting precipitates were dissolved in acetone until a concentration of approximately 1 mg/mL of Pt(IV)-PFOA was reached. Next, 5 μL of the organic solution was injected in a Sunfire Analysis C18 column (5 μm, 150 mm × 4.6 mm, Waters Corporation, Wexford, Ireland) and eluted with methanol and water in a ratio of 80:20. The signals were detected by an ultraviolet detector (Agilent Technologies) at 210 nm.
The drug loading (DL) of PFD was determined by quantifying PFD using gas chromatography (Agilent Technologies, 7890B, Wilmington, DE, USA). Specifically, the PFD@PtF nanoparticles were subjected to centrifugation (ThermoFisher Scientific, Sorvall Legend Micro 17R, Osterode am Harz, Germany) at 13,000 rpm for 10 min in order to break the emulsion and separate the loaded compound. Subsequently, the separated PFD was dissolved in 2 mL chloroform. The sample solution was injected into a gas chromatographic column (Agilent Technologies, DB-624, Santa Clara, CA, USA) using the headspace sampling method. The temperature of the column was initially set at 40 °C for 1 min. After that, it rose at a rate of 20 °C/min to a maximum temperature of 200 °C, which it maintained steadily for 5 min. The signals were detected using a flame ionization detector (Agilent Technologies). The calculation of the DL was performed as shown in Eq. (1):
DL(%)=W1/(W2+W1)×100
In this equation, W1 represents the weight of entrapped PFD, and W2 indicates the weight of entrapped Pt(IV)-PFOA.
2.6.　 Measurement of Pt release
Dynamic dialysis was performed to explore the release behavior of Pt from PtF nanoparticles in the presence or absence of NaVc. Specifically, PtF nanoparticles were separated in 1 mL of Tris-buffered saline (TBS) or 5 mmol/L NaVc solution (containing 0.75 mg/mL of Pt(IV)-PFOA). These particles were placed into a 3500 D dialysis bag and immersed in 20 mL corresponding solution, then shaken at 37 °C and 100 rpm (Shanghai Zhichu Instrument Co., Ltd., ZQTY-50S, Shanghai, China). At specific intervals, 2 mL of dialysate was sampled and substituted with an identical amount of the corresponding buffer solution. The collected samples were digested overnight at 150 °C with concentrated nitric acid. Excess nitric acid was evaporated, and the remaining volume was diluted with dilute aqua regia. Platinum content was quantified using inductively coupled plasma mass spectrometry (ICP-MS, Agilent Technologies, Agilent 7900, Santa Clara, CA, USA).
2.7.　 Measurement of dissolved ozone
To assess the saturation of ozone, PFD@PtF nanoparticles, Tween-80 emulsified PFD (PFD-equivalent), and distilled water were saturated with ozone. Next, 1 mL of each sample was added to 2 mL of ozone-free distilled water. A pen-type detector (Clean Instruments Co., Ltd., CLEAN DOZ30, Xinbei, China) was used to monitor the change in ozone concentration in the ozone-free distilled water.
2.8.　 Hydroxyl radical generation of O3_PFD@PtF
The generation of hydroxyl radicals was monitored using TA as a fluorescence probe. To accomplish this, distilled water, PtF, and PFD@PtF were either exposed to 20Gy X-ray irradiation (Rad Source Technologies, RS-2000, Buford, GA, USA) or not. Then, 50 mmol/L H2O2 and TA (1 mg/mL TA, 0.8 mg/mL NaOH) solutions were sequentially added to create a system that contained 12.5 mmol/L H2O2, 0.5 mg/mL TA, and 0.4 mg/mL NaOH. Fluorescent 2-hydroxyterephthalic acid resulting from the reaction between hydroxyl radical and TA can be identified using a fluorescence spectrophotometer (Bio-Tek Instruments, SYNERGY H1, Winooski, VT, USA) at approximately 435 nm when excited with light at 315 nm.
2.9.　 Cellular uptake of PtF
In an effort to evaluate the uptake of PtF in TNBC cells, both confocal laser-scanning microscopy (CLSM, Andor, Dragonfly 200, Belfast, UK) and flow cytometry (BD Biosciences, FACSAria III, San Jose, CA, USA) were utilized. TNBC cells were plated on glass slides in 12-well plates (5 × 104 per well) for CLSM (Andor) observation. Following an overnight period of incubation, cells were exposed to FITC-loaded PtF (30 μmol/L Pt(IV)-PFOA) for a duration of 0.5 h. For cell membrane visualization, the cells were stained with Alexa Fluor 647-conjugated WGA. Following 30 min fixation with 4% paraformaldehyde, the slides were prepared with an antifading 4′,6-diamidino-2-phenylindole (DAPI) mounting medium (YEASEN, Shanghai, China) and examined using CLSM (Andor). For flow cytometry (BD Biosciences) analysis, 5 × 105 cells were incubated in 6-well plates overnight. Medium containing NileRed-labeled PtF (30 μmol/L Pt(IV)-PFOA) was introduced and left to incubate for 0.5 h. After that, the cells were then washed, resuspended, and examined with flow cytometry (BD Biosciences) for uptake study.
To elucidate the internalization mechanism of PtF, classical internalization relevant biomarkers, including clathrin, caveolin-1, and lysosomal-associated membrane protein 1 (LAMP1), were visualized by immunofluorescence (IF). In brief, cell slides were treated with NileRed-labeled PtF (30 μmol/L Pt(IV)-PFOA) for 0.5 h. Following fixation with 4% paraformaldehyde for 20 min, the slides were treated with a 3% Triton X-100-10% BSA-PBS solution for 1 h at room temperature (RT). Subsequently, the cell slides were treated with primary antibodies targeting clathrin (Cell Signaling Technology, #4796S, Danvers, MA, USA), caveolin-1 (Cell Signaling Technology, #3267S), and LAMP1 (lysosome tracker, Abcam, ab208943, Cambridge, UK, mouse use; Cell Signaling Technology, #9091T, human use) at 4 °C overnight. Subsequently, the cell slides were treated with a secondary antibody labeled with Alexa Fluor 488 for 1 h at RT. The slides were prepared using an antifading DAPI mounting medium. High-magnification images were captured using CLSM (Andor).
2.10.　 Cytotoxicity measurement
The MTT assay was performed to evaluate cell viability. TNBC cells, normal mammalian epithelial cells, and immortalized lymphocytes were placed in 96-well plates (2000 per well). After 24 h, the cells were exposed to varying concentrations of PtF nanoparticles and cisplatin, respectively, with or without 20 Gy irradiation (Rad Source Technologies). TNBC cells were also exposed to varying concentrations of O3_PFD@PtF particles with or without different doses of X-ray irradiation. After 96 h incubation, MTT solution was introduced (final concentration: 400 μg/mL). After incubating for an additional 4 h, the MTT-containing medium was substituted with 150 μL of DMSO, and the optical density (OD) at 570 nm was determined. The cell viability (%) was then calculated as shown in Eq. (2):
Cellviability(%)=(ODtest‒ODblank)/(ODcontrol‒ODblank)×100
GraphPad Prism software (GraphPad Software, 8.3.0, Boston, MA, USA) was used to determine the IC50 value at half-maximal inhibition.
2.11.　 Intracellular ROS and GSH/GSSG evaluation
The intracellular ROS generation was evaluated utilizing a DCFH-DA-dependent ROS assay kit. Firstly, TNBC cells were seeded onto cell slides in a 12-well cell culture plate (5 × 104 per well). After 24 h, TNBC cells were treated with serum-free DMEM cell–culture medium containing a diluted solution of DCFH-DA (1:1000 dilution) for 30 min. After PBS washing, cells were treated with PtF nanoparticles, O3_PFD@PtF nanoparticles, and cisplatin individually for 2 h with or without 20 Gy irradiation (Rad Source Technologies). After being rinsed, the slides were mounted with DAPI medium. Images were captured using CLSM (Andor).
In order to examine the levels of GSH and GSSG in cells, 2 × 105 TNBC cells were incubated in 6-well plates for 24 h. The cells were exposed to PtF nanoparticles, O3_PFD@PtF nanoparticles, and cisplatin at a concentration equivalent to 5 μmol/L of cisplatin, along with ozone-loaded perfluorodecalin emulsion, either with or without irradiation. After 24 h incubation, the GSH and GSSG levels in cells were determined using GSH and GSSG assay kits.
2.12.　 Intracellular Pt/DNA measurement
In order to detect cisplatin release in vitro, the Pt content in the nucleus DNA was measured. TNBC cells were placed into 6-well plates (2 × 105 per well). After 24 h, the cells were then exposed to PtF nanoparticles, O3_PFD@PtF nanoparticles, and cisplatin at a concentration equivalent to 5 μmol/L of cisplatin, with or without 20 Gy irradiation (Rad Source Technologies). Following 24 h incubation, the cells were collected. To measure Pt/DNA, the nucleus DNA was extracted utilizing the phenol/chloroform extraction method and quantitatively determined using a spectrophotometer (ThermoFisher Scientific, NanoDrop One, Waltham, MA, USA). Subsequently, concentrated nitric acid was used to digest the cell DNA, and then ICP-MS (Agilent Technologies) was applied to perform Pt content analysis.
2.13.　 Western blot
In order to conduct the western blot analysis, SUM149 cells were plated in 6-well plates (5 × 105 per well) and allowed to incubate for 24 h. Subsequently, the cells were exposed to PtF nanoparticles, O3_PFD@PtF nanoparticles, and cisplatin at a concentration of 10 μmol/L, with or without 20 Gy irradiation (Rad Source Technologies). After 24 h, cells were disrupted with RIPA solution containing phosphatase and protease inhibitors. Protein samples were electrophoresed and then moved onto polyvinylidene fluoride membranes (Merck Millipore, Carrigtwohill, Ireland). Following that, western blots analysis was conducted with antibodies for calreticulin (CRT, Abcam, ab92516), high mobility group box 1 (HMGB1, Abcam, ab79823), and β-ACTIN (ASGB-Bio, TA-09, Beijing, China).
2.14.　 Study of biodistribution and therapeutics in tumor-bearing mice
For the biodistribution study and therapeutic evaluation, female BALB/c mice (7 weeks, 18–20 g) were used to establish the TNBC tumor model. To establish the subcutaneous tumor model, 5 × 105 4T1 or luciferase-expressing 4T1 (4T1-Luc) cells were injected subcutaneously on the right or both dorsal flanks of each mouse. For the lung metastatic tumor model, each mouse was injected 2 × 105 4T1-Luc cells through the tail vein.
For biodistribution analysis of PtF, NileRed-labeled PtF nanoparticles were injected into the mice intravenously once the tumor size reached around 200 mm3. At the specified time points (1, 6, 12, and 24 h) post-injection, the mice were humanely euthanized. Tumors and major organs (including the heart, liver, spleen, lung, and kidney) were collected and analyzed with fluorescence imaging (excitation: 500 nm, emission: 600 nm) utilizing the small animal fluorescence imaging system (PerkinElmer, IVIS Spectrum, Waltham, MA, USA).
The therapeutic efficacy of O3_PFD@PtF under X-ray irradiation was evaluated in mice bearing subcutaneous 4T1 tumors. Upon the subcutaneous tumor volume reaching approximately 100 mm3, the mice were randomly divided into various treatment groups (n = 6): (1) PBS (Blank), (2) PtF, (3) cisplatin, (4) O3_PFD@PtF, (5) PBS plus X-ray irradiation, (6) PtF plus X-ray irradiation, (7) cisplatin plus X-ray irradiation, and (8) O3_PFD@PtF plus X-ray irradiation. Each reagent was administered intravenously every 3 days at a cisplatin-equivalent dose of 0.8 mg/kg of body weight. The injection was administered 3 times with or without localized X-ray irradiation (6 Gy/fraction, Rad Source Technologies) on the right dorsal flank 6 h after injection. Tumor diameters were assessed with a vernier caliper every 3 days before treatment and every other day after treatment. The tumor volume was computed as shown in Eq. (3):
Tumorvolume=(Tumorlength)×(Tumorwidth)2/2
Additionally, the mice's body weight was recorded every 2 days following the treatment. The mice were euthanized on Day 26 after tumor injection, and both the tumors and major organs were excised.
In the 4T1 lung metastatic mice model, the antitumor efficacy of O3_PFD@PtF combined with X-ray irradiation was also examined. On Day 6 post-inoculation, the mice were assigned to various treatment groups (n = 6), as mentioned above. Each reagent was administered intravenously at a cisplatin-equivalent dosage of 0.8 mg/kg body weight every 2 days. This injection was repeated 3 times in the presence or in the absence of whole-body X-ray irradiation (2 Gy/fraction, Rad Source Technologies) at 6 h after injection. The growth of lung metastatic tumors was measured using bioluminescence imaging (Spectral Instruments Imaging, KINO, Tucson, AZ, USA) starting from Day 5 after inoculation and then every 2 days. On Day 13 post-inoculation, the mice were euthanized in a human manner, and their lungs were dissected for additional analysis.
2.15.　 Immunohistochemistry and immunofluorescence staining
The samples of tumors and major organs were fixed using a solution of 10% formalin, followed by embedding in paraffin and cutting into 4 μm-thick slides. These slides were then placed onto glass slides and subjected to staining with hematoxylin–eosin (H&E). For immunohistochemical (IHC) and IF staining, the obtained tumor sections were first deparaffinized and then exposed to 3% H2O2. Antigen retrieval was carried out by subjecting the samples to microwave irradiation at 95 °C for 16 min using either EDTA (pH = 9.0) or citrate (pH = 6.0) buffer.
For IHC, the tumor slides were sequentially treated with an endogenous peroxidase-blocking buffer in the dark for 15 min and 5 % BSA for 1 h at RT. Following a rinse with PBS, the slides were incubated overnight at 4 °C with primary antibodies including BAX (Cell Signaling Technology, #14796), Cleaved caspase-3 (Cell Signaling Technology, #9664), Ki67 (Cell Signaling Technology, #12202), CRT (Abcam, ab92516), HMGB1 (Abcam, ab79823), and heat shock protein70 (HSP70, Abcam, ab194360). Afterward, the tumor samples were washed with PBS and exposed to secondary antibodies conjugated with species-specific HRP at RT for 40 min. After another round of PBS washing, the slides were visualized using DAB. The slides were subsequently processed by water washing, hematoxylin counterstaining, ethanol dehydrating, and media mounting.
For IF staining, the tumor slides were incubated with 5 % BSA at RT for 1 h, followed by a PBS rinse. Following this, the slides underwent treatment with either CD4 antibody (Abcam, ab183685) or CD8α antibody (Abcam, ab217344) for an overnight incubation at 4 °C. After two PBS rinses, the slides were exposed to Alexa Fluor 488 or Alexa Fluor 594-conjugated secondary antibodies in 5% BSA at 37 °C for 40 min. Finally, the slides were mounted using an antifading DAPI mounting medium for fluorescence imaging.
2.16.　 Flow cytometry analysis
In order to assess the tumor-infiltrating lymphocyte of each treatment group, single-cell suspensions were prepared following the method below: tumor samples were dissected into small pieces using scissors in MACS Tissue Storage Solution (Miltenyi Biotec, Bergisch Gladbach, Germany). Tissue pieces were broken down using a combination of collagenase type I and type IV (1 mg/mL) along with DNase I (50 μg/mL) at 37 °C for a duration of 10 min. Then, digested tissues were passed through a cell strainer to obtain single-cell suspensions. After rinsing the suspensions twice with the HBSS buffer, they were subsequently resuspended in the same HBSS buffer.
Different fluorophore-conjugated anti-mouse antibodies were used to co-stain the cells: CD45-APC-Cyanine7 (BioLegend, 103116, San Diego, CA, USA), CD3-PE (BioLegend, 100205), CD4-FITC (BioLegend, 100510) and CD8-Brilliant Violet 510 (BioLegend, 100752). Multiparameter staining was used to differentiate the specific cell groups: tumor-infiltrating CD3+ T-cells (CD45+CD3+), tumor-infiltrating CD8+ T-cells (CD45+CD3+CD8+), and tumor-infiltrating CD4+ T-cells (CD45+CD3+CD4+). Following the staining process, flow cytometry (BD Biosciences) was used to analyze the cells, and FlowJo_v10.6.2 software (BD Biosciences, San Jose, CA, USA) was utilized to process the data. Nonviable cells and doublets were eliminated using forward and side scatter characteristics.
2.17.　 Statistical analysis
GraphPad Prism 8 (8.3.0) was utilized for the statistical analysis. The results were presented in the form of mean ± standard deviation (SD). Two-way ANOVA analysis was performed for multiple comparisons in Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9, J, L, M and Supporting Information Figs. S8B and S8C. The statistical parameters, encompassing the F and P values for factors and interaction, were included in the Supporting Information Table S1‒S13.
3.　Results
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3.1.　 Synthesis of cisplatin prodrug Pt(IV)-PFOA
To constitute the self-assemble small-molecule prodrug nanoparticles, a platinum(IV) prodrug (Pt(IV)-PFOA) tethering with two PFOA molecules is synthesized according to the route illustrated in Fig. 2A. Firstly, oxoplatin is obtained by oxidization of cisplatin with H2O2. Secondly, PFOC is synthesized by an acylating chlorination reaction with PFOA and SOCl2. Finally, the Pt(IV)-PFOA is obtained by esterification between PFOC and oxoplatin, as indicated in Fig. 2A. The appearance of the final product Pt(IV)-PFOA, raw compound, and intermediate products are shown in Fig. 2B.
The structure of Pt(IV)-PFOA and the synthesis process are quality controlled by FTIR, 1H-NMR, and 19F-NMR (Fig. 2C and D; Supporting Information Figs. S1 and S2). The FTIR spectrum of oxoplatin exhibits the stretching vibration peaks of –OH and Pt–OH at 3458 and 532 cm−1, respectively, indicating the correct synthesis of oxoplatin. Comparing the FTIR spectra of PFOC and PFOA, it is observed that the –OH stretching vibration peak at 3551 cm−1 and the C–O stretching vibration peak at 1450 cm−1 disappears, proving the production of PFOC. Meanwhile, the stretching vibration peak of CO is red-shifted from 1694 to 1778 cm−1, which confirms the proper transformation of acyl chloride from carboxylic acid. In the FTIR spectrum of Pt(IV)-PFOA, the CO stretching vibration peak at 1711 cm−1 and the C–O stretching vibration peak at 1335 cm−1 indicate the formation of the ester bond. The peaks of C–F stretching vibration at 1200 and 1143 cm−1 are attributed to fluoroalkyl groups derived from PFOA. In addition, no peak between 3300 and 3600 cm−1 is observed, suggesting the absence of the –OH group. It indicates that both hydroxyl groups in oxoplatin have reacted with PFOC and PFOA in synthesized Pt(IV)-PFOA are covalently connected as axial ligands.
In the 1H-NMR spectrum of Pt(IV)-PFOA, the amino H appearing at 6.5 ppm (a) is attributed to the ammonia ligands of platinum(IV). In the 19F-NMR spectra, the peaks that appeared at −80.18 ppm (b, –CF3),–125.72 ppm (c, –CF2–CF3), −121.25 to −122.45 ppm (d, e, f, g, –CF2–), and −114.99 ppm (h, –O–CO–CF2–) confirmed the presence of fluoroalkyl groups in Pt(IV)-PFOA. In summary, the successful synthesis of Pt(IV)-PFOA is confirmed by analyzing the results obtained from FTIR, 1H-NMR, and 19F-NMR.
Furthermore, the molecular weight of Pt(IV)-PFOA is determined by UHPLC-Orbitrap-MS analysis (Supporting Information Fig. S3). In the mass spectrum of Pt(IV)-PFOA, the exact molecular ion mass of [M + H]+, [M + NH4]+ and [M + Na]+ are observed at m/z 1125.89, 1142.92, and 1147.87, respectively, which are in excellent agreement with the corresponding theoretical simulation results simulated by Thermo Xcalibur Qual Browser software (ThermoFisher Scientific, version 4.3, Waltham, MA, USA). These data pinpoint the molecular weight of Pt(IV)-PFOA to be 1126.17 g/mol.
3.2.　 Preparation and characterization of micelle PtF and PFD@PtF
The synthesized prodrug Pt(IV)-PFOA can form nano-scaled particles either alone (PtF) or with PFD (PFD@PtF) through self-assembly based on the hydrophobic interaction between fluorine residues (Fig. 3A). The self-assembled PtF nanoparticles are prepared using the nanoprecipitation method by adding a DMSO solution of Pt(IV)-PFOA dropwise into an aqueous solution under ultrasound. The PtF particle presents uniform spherical morphology revealed by TEM (Fig. 3C) with a hydrated diameter of around 130 nm (Fig. 3B). To efficiently load ozone, PFD, an excellent ozone solvent, is incorporated into PtF nanoparticles based on the similarity-intermiscibility theory, resulting in PFD@PtF nanoparticles with a hydrated diameter of around 170 nm (Fig. 3B). Our results demonstrate a high maximal incorporation rate of 91.24% (PFD/PFD@PtF), ensuring effective ozone loading and delivery. To evaluate the stability of PtF and PFD@PtF nanoparticles, we measure their hydrated diameter at indicated temperatures and pH values. The results indicate that both PtF and PFD@PtF particles remain stable for up to 72 h under acidic, alkaline, and neutral conditions at 4 °C, RT, and even 37 °C, respectively. This excellent stability fulfills the criteria for in vitro as well as in vivo uses (Fig. 3D).
According to our design strategy, cisplatin can be released from the Pt(IV)-PFOA prodrug in a reductive environment, such as the cytosol of tumor cells. In order to verify this, PtF nanoparticles are incubated with a buffer containing NaVc at 37 °C. ICP-MS results demonstrate that the reductive environment remarkably promotes the release of cisplatin and the disintegration of PtF nanoparticles. After 24 h incubation in 5 mmol/L NaVc, as much as 77.0% of cisplatin is released from PtF particles (Fig. 3E). The strong electron-withdrawing effect by fluorine atoms facilitates the reduction of platinum(IV) to platinum(II) and consequently boosts the release of cisplatin.
To measure the ozone loading capacity of PFD@PtF particles, ozone release curves are profiled with ozone-saturated water solution (negative control), Tween-80 emulsified PFD (positive control), and PFD@PtF particles (Fig. 3F). The results reveal that the ozone loading capacity of PFD@PtF vehicles is approximately 20-fold higher than that of water solution. Furthermore, the release curves indicate that PFD@PtF nanoparticles exhibit a slow and sustained ozone-releasing capacity for up to 24 h. Upon X-ray irradiation, the ozone-loaded particles decompose into ROS and oxygen, providing a foundation for improving radiotherapy. The results of the TA assay show that ozone-loaded PFD@PtF particles (O3_PFD@PtF) efficiently generate ROS under X-ray ionizing conditions (Fig. 3G). The amount of ROS is positively correlated with the amount of O3_PFD@PtF.
3.3.　 Biodistribution and cytotoxicity of PtF and PFD@PtF in vitro
We investigate the subcellular distribution of FITC-labeled PtF nanoparticles in three TNBC cell lines, including one murine (4T1) and two human cell lines (SUM149 and BT-549). After a 0.5 h incubation period, an intensive fluorescence signal of FITC is detected within the cytosol of all three cell lines, indicating that PtF nanoparticles can be promptly and efficiently uptake by TNBC cells (Fig. 4A and Supporting Information Fig. S4A). The excellent cellular uptake efficiency of PtF nanoparticles is also clearly confirmed by flow cytometry (Fig. 4B and Fig. S4B). To gain further insights into the internalization mechanism, we co-visualize the NileRed-labeled PtF with two canonical endocytosis-associated proteins (clathrin and caveolin-1) and a lysosome tracker (LAMP1) in three TNBC cell lines (SUM149, 4T1, and BT-549) (Fig. 4C and Supporting Information Fig. S5). The results show that caveolin- and clathrin-mediated endocytosis pathways are engaged in tested TNBC cell lines. It is in line with previous reports that tumor cells may internalize particles through multiple endocytosis pathways12-14. In addition, the obvious separation between NileRed-labeled PtF and the lysosome tracker indicates the effective escape of PtF from lysosomal entrapment, which may be potentially due to the amphiphilic features of perfluorocarbon15-17.
To assess the cytotoxicity of synthesized PtF particles, we determine the cell viability under indicated treatment conditions with two TNBC cell lines (SUM149 and 4T1), a normal mammalian epithelial cell line (MCF-10A), and two immortalized lymphocytes (EG7 and Jurkat) using a 96 h MTT assay (Fig. 4D‒H and Supporting Information Table S1). Overall, both cisplatin and PtF show tumor inhibition compared to the control group. Specifically, with or without irradiation, the prodrug PtF particles consistently show better tumor cell inhibition than cisplatin at the equimolar equivalent in TNBC cell lines. A potential explanation for the better efficacy of PtF is that the perfluorocarbon sidechains of prodrug improve the bioavailability of cisplatin by enhancing the efficiency of platinum internalization18. Importantly, like other prodrugs of modified cisplatin, PtF shows less toxicity in normal mammalian cells and lymphocytes. This may be attributed to the fact that the level of GSH (which triggers the release of cisplatin from prodrug) in normal cells is significantly lower compared to tumor cells (see Fig. 5D‒F below). The IC50 of each condition above is calculated, and similar results are obtained (Fig. 4I‒M and Supporting Information Table S2). The specificity and selectivity of cisplatin and PtF are evaluated by calculating the tumor-selective index (SI). Both SI on normal mammalian epithelial vs. tumor and SI on lymphocytes vs. tumor show high selectivity and tumor cell-specific toxicity (Fig. 4N). All these data suggest that PtF shows higher tumor inhibitory efficacy and lower cytotoxicity than cisplatin.
3.4.　 O3_PFD@PtF and irradiation combination promote Pt releasing in vitro
We then evaluate the pharmacodynamics of ozone-loaded prodrug particles (O3_PFD@PtF) and their combination with irradiation. Cell toxicity is evaluated using a 96 h MTT assay with indicated treatment (Fig. 5A and Supporting Information Table S3). Consistently, cisplatin and PtF show tumor inhibition in two TNBC cell lines. Compared with cisplatin or PtF, ozone-loaded PtF particle (O3_PFD@PtF) significantly inhibits tumor cell growth. In the presence of irradiation, the efficacy of O3_PFD@PtF is further enhanced. We then assess the synergistic effect between O3_PFD@PtF and X-ray irradiation by figuring the combination index (CI) with the assistance of Calcusyn software (REACHSOFT, version 2.1, Beijing, China). The results show that O3_PFD@PtF and irradiation present considerable synergistic effects in terms of cell growth inhibition across all four doses (Supporting Information Fig. S6). According to previous reports19,20, we hypothesize that the growth inhibition induced by O3_PFD@PtF under irradiation may be attributed to the synergistic impact of hydroxyl radicals and cisplatin. On the flip side, a large number of hydroxyl radicals generated by ozone under irradiation directly kill tumor cells. On the other hand, hydroxyl radicals indirectly promote the release of cisplatin from prodrug nanoparticles by impacting the balance of GSH-GSSG in the cytosol. Specifically, hydroxyl free radicals disrupt the intracellular redox balance and promote the generation of reducing mediators such as GSH21. The generated GSH subsequently releases cisplatin from prodrug PtF by reducing Pt(IV) to Pt(II).
To confirm this speculation, we visualize the ROS in three TNBC cell lines (SUM149, 4T1, and BT-549) treated with indicated conditions using a DCFH-DA fluorescence probe (Fig. 5B and Supporting Information Fig. S7A). The fluorescence signals are observed in cells treated with ozone-loaded particles in the presence or absence of X-ray irradiation. The quantification of the fluorescence signal indicates that ROS levels in cells treated with PtF and cisplatin are barely detectable, regardless of X-ray exposure (Fig. 5C and Fig. S7B). On top of O3_PFD@PtF particles treatment, X-ray irradiation remarkably improved the ROS generation by 3.1-fold (in SUM149), 2.1-fold (in 4T1), and 3.3-fold (in BT-549). The results of intracellular ROS detection are in line with those of the extracellular hydroxyl radical generation experiment in Fig. 3G.
Then, we evaluate the impact of ROS on the intracellular balance of GSH/GSSG in tumor cells. SUM149 cells are treated with ozone-loaded perfluorodecalin emulsion (O3_PFD emulsion) with or without irradiation. After intensive washing, the intracellular GSH and GSSG levels are measured using the DTNB (5,5′-dithiobis-(2-nitrobenzoic acid)) reduction reaction. As shown in Fig. 5D, O3_PFD emulsion, X-ray irradiation, and their combo remarkably increase the intracellular GSH/GSSG ratio (Supporting Information Table S4). On top of this assay, we incorporate the prodrug PtF, which will release cisplatin by consuming the generated GSH (Fig. 5E). The results show that PtF, O3_PFD@PtF, and PtF plus irradiation slightly down-regulate the GSH/GSSG ratio. On the contrary, O3_PFD@PtF combined with irradiation dramatically upregulates the GSH/GSSG ratio. This suggests that the combination of ozone and X-ray irradiation, rather than each single treatment, maintains a high level of GSH. As a result, the released cisplatin will incorporate into chromosome DNA and form a Pt–DNA complex. We then extracted the nucleus DNA and measured the DNA-bond platinum. Not surprisingly, the amount of DNA-bound platinum is consistent with the ratio of GSH/GSSG (Fig. 5F and Supporting Information Table S5).
As reported previously, cisplatin alone is not effective in triggering ICD because it cannot translocate the antigen-carried CRT to the surface of the cell. However, ROS promotes CRT translocation. Thus, in our design rationale, the ozone-derived ROS may serve as an enhancer for ICD induced by cisplatin. To validate this principle, we examine the levels of two ICD markers, HMGB1 and CRT, in SUM149 cells exposed to PtF, cisplatin, and O3_PFD@PtF with or without irradiation (Fig. 5G). Upon irradiation, cisplatin leads to an upregulated CRT expression, likely attributed to the generation of ROS by X-ray. Furthermore, O3_PFD@PtF treatment in combination with irradiation exhibits even higher CRT exposure compared to cisplatin plus irradiation. This may be attributed to the differential efficiency of ROS production with or without ozone in the presence of irradiation. Similarly, O3_PFD@PtF administration significantly increases the expression levels of HMGB1, particularly when combined with irradiation.
3.5.　 O3_PFD@PtF and irradiation combination inhibit tumor growth
Encouraged by the remarkable efficacy and selectivity observed in vitro, we proceed with evaluating the in vivo antitumor efficiency of the prodrug nanosystem. We characterize the biodistribution of PtF nanoparticles by intravenously injecting NileRed-labeled PtF nanoparticles into 4T1-bearing mice. The fluorescence signals of NileRed in dissected major organs and tumors are recorded at four indicated time points (1, 6, 12, and 24 h) utilizing IVIS Spectrum. The PtF particles are observed in tumor mass as early as 1 h after injection, and they continue to accumulate for at least 24 h (Fig. 6A and B), indicating that PtF is a tumor-targeted carrier with long-lasting accumulation properties. Additionally, strong fluorescence signals are observed in the liver and kidneys at 1 h, followed by a significant decrease within 24 h post-injection. This suggests that PtF nanoparticles primarily undergo hepatic and renal metabolic pathways, followed by a quick bio-elimination.
We then evaluate the antitumor efficacy of indicated regimens on a BALB/c mice model with unilateral subcutaneous 4T1 xenografts (Fig. 6C). Briefly, tumor-bearing mice are randomly divided into 8 groups and administered with indicated regimens respectively, including PBS (Blank), PtF, cisplatin, O3_PFD@PtF, PBS plus X-ray irradiation (Irradiation), PtF plus X-ray irradiation (R + PtF), cisplatin plus X-ray irradiation (R + cisplatin), and O3_PFD@PtF plus X-ray irradiation (R + O3_PFD@PtF). Tumor volumes and body weight are monitored throughout the experiment to profile tumor growth curves and alterations of body weight, respectively (Fig. 6D and E). After 3 dosages of treatment, tumors from each experimental group are excised, photographed, and weighed (Fig. 6F and G; Supporting Information Table S6) on Day 26 upon inoculation. The results of the tumor growth curve and tumor weight show that each single treatment (PtF, cisplatin, and O3_PFD@PtF) shows effective tumor inhibition compared with the blank group. The inhibition can be further improved by the combination of X-ray irradiation. Remarkably, the combination of O3_PFD@PtF and X-ray irradiation shows the most powerful inhibition of tumor growth among all groups.
These results are further confirmed on an independent bilateral subcutaneous 4T1-Luc xenografted mice model. Tumor-bearing mice are treated with indicated regimens, and X-ray irradiation is performed on the primary tumors (right side) as needed. At the endpoint of the experiment, tumors are visualized in situ using bioluminescent in vivo imaging (Supporting Information Fig. S8A). Not surprisingly, similar results of tumor inhibition in the unilateral model are obtained on the primary tumor from the bilateral model. In addition, comparable tumor inhibition is observed between the primary and abscopal tumors treated with indicated regimens except for tumors treated with irradiation alone (Figs. S8B and C; Supporting Information Table S7). Primary tumor rather than abscopal tumor responses to the irradiation. The results suggest that irradiation alone cannot effectively elicit an abscopal effect.
Despite its high efficacy, the prodrug nano system offers the advantage of reducing the systemic toxicity associated with cisplatin and radiotherapy, as proven in vitro (Fig. 4D‒N). Here, this advantage is further confirmed in vivo. As shown in Fig. 6E, mice treated with cisplatin experience body weight loss, regardless of whether it is combined with X-ray irradiation or not. To specify the systemic toxicity, we profile the body weight at the endpoint of the experiment (Fig. 6H and Supporting Information Table S8). Compared to free cisplatin, the prodrug PtF significantly alleviates chemo-induced toxicity, regardless of X-ray irradiation. At the end of the experiment, important organs like the heart, liver, spleen, lung, and kidney are collected, along with peripheral blood samples for hematological and pathological assessment. No visible histological damage is observed in the tissue slides derived from each group except for the kidney slides from the cisplatin-treated group (Supporting Information Fig. S9). The kidneys from the cisplatin-treated group exhibit obvious glomerular fusion and atrophy, which are typical signs of kidney damage. Remarkably, these pathological morphologies are not observed in the kidneys of mice receiving prodrug nanoparticle treatment. The cisplatin-related toxicities are further confirmed by the hematologic and biochemical analysis. Consistent with the H&E staining results, abnormally high levels of urea (UREAL) are observed in mice treated with cisplatin and cisplatin plus irradiation (Supporting Information Fig. S10). These findings collectively indicate that the prodrug nano system PtF effectively avoids systemic toxicity induced by cisplatin. Meanwhile, the combination regimen of O3_PFD@PtF and X-ray irradiation exhibits both efficient tumor inhibition and commendable biosafety in animal experiments.
To elucidate the molecular mechanisms behind tumor growth inhibition, we evaluate the histopathology of tumors treated with indicated regimens using H&E staining (Fig. 7A). We observe similar degrees of tumor tissue damages, as indicated by karyopyknosis and disruption of intercellular junctions, in tumors treated with cisplatin and prodrug PtF. Loading with ozone, O3_PFD@PtF clearly enhances the tumor damage. These damages are further improved by X-ray irradiation. The most severe histological damage is observed in the tumors treated with O3_PFD@PtF plus irradiation. Consistently, the index of cell apoptosis, indicated by markers BAX and Cleaved caspase-3, also shows similar trends as that in the histological analysis (Fig. 7B, C, E, F and Supporting Information Table S9). Remarkably, the combination of O3_PFD@PtF and X-ray irradiation results in very high levels of apoptotic signals, implying a severe ICD. Moreover, the number of proliferative cells, indicated by Ki67 expression, shows an opposite pattern (Fig. 7D, G and Table S9). Treatment with cisplatin, PtF, and O3_PFD@PtF significantly reduces the Ki67-positive cell number compared to the control group. Tumor cell proliferation is further attenuated by the combination of X-ray irradiation. Almost no Ki67-positive cells are observed in tumors treated with the combo regimen of O3_PFD@PtF and X-ray irradiation.
Encouraged by the excellent inhibitory effect of this combo regimen on the subcutaneous xenograft tumor model, we further validate the antitumor efficacy on a 4T1-Luc lung metastatic mice model (Fig. 8A). The lung metastatic tumor is monitored by bioluminescence imaging, and the tumor growth curves are profiled accordingly (Fig. 8B and C). After 3 dosages of treatment, the mice are euthanized, and the lungs are harvested on Day 13 upon inoculation. The number of lung metastatic lesions is evaluated with H&E staining of tumor sections (Fig. 8D and E and Supporting Information Table S10). The results indicate that the mono-drug treatment (Cisplatin, PtF, or O3_PFD@PtF) shows moderate efficacy in inhibiting lung metastasis compared to the blank group. And the tumor inhibitory efficacy is further boosted by irradiation. The combo of O3_PFD@PtF and X-ray irradiation exhibits the highest efficacy in suppressing lung metastasis among the tested regimens.
3.6.　 Combo of O3_PFD@PtF and irradiation improved immune-related cell death
As programmed cell death is usually correlated with ICD, we then investigate the intensity of ICD in tumors that are treated with indicated regimens. The ICD levels are evaluated by examining canonical biomarkers, including CRT, HMGB1, and HSP70 (Fig. 9A‒F and Supporting Information Table S11). In comparison with the control group, the results from IHC staining and integrated optical density/area quantification demonstrate that cisplatin, PtF, and O3_PFD@PtF induce a slight increase in ICD. The ICD is further enhanced by the combination of X-ray irradiation. The most severe ICD is observed in tumors treated with irradiation plus O3_PFD@PtF. Theoretically, ICD can improve neoantigen exposure and T lymphocyte infiltration into the tumors. As a result, we evaluate the levels of CD4+ and CD8+ T lymphocytes in tumors by IF staining and flow cytometry (Fig. 9G‒M, Supporting Information Tables S12 and S13). The results show similar trends to those observed in ICD marker measurements. Treatment with cisplatin, PtF, and O3_PFD@PtF significantly increases the densities of tumor-infiltrated lymphocytes, which are further improved by the combination of X-ray irradiation. Taken together, the combination of irradiation with an ozone-loaded prodrug nanosystem can significantly suppress tumor proliferation by inducing apoptotic-related ICD, which not only efficiently inhibits tumor growth but also provides a promising fundament for combination with immunotherapy.
4.　Discussion
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Although the combination of chemotherapy and radiotherapy shows promising outcomes in clinics, several defects, including intolerable systematic toxicity, as well as hypoxic and unfavorable immune microenvironments, severely dampen its application22,23. Therefore, enhancing therapeutic effects and reducing systematic toxicity are two critical issues in cancer therapy. To address these challenges, researchers have focused on improving the efficacy of combined therapy for cancer treatment by alleviating hypoxia24 and sensitizing chemotherapy25. With the advancement of antitumor strategies, various gases have been identified to possess outstanding antitumor effects while maintaining superior safety profiles26. In this manuscript, we present a self-assembling ozone delivery nanosystem consisting of fluorocarbon chain-modified cisplatin prodrug. The advantages of this system are 1) reducing toxicity, 2) improving the efficacy of chemo plus radiotherapy, and 3) providing an immune-favored niche.
Cisplatin is commonly used as a cornerstone chemotherapeutic drug for TNBC in clinical practice27. However, its broad application is hindered by severe dose-limiting toxicities, such as nephrotoxicity and ototoxicity28. Multiple nano-based platinum(IV) prodrugs have been developed29-31 to alleviate the side effects of cisplatin. In our nanosystem, the platinum is modified as a reduction-responsive Pt(IV) prodrug, which can be specifically released by GSH. GSH is a natural byproduct in mammalian cells and is essential for antioxidant defense, proliferation, cell differentiation, and apoptosis32. Compared with extracellular matrix or blood circulation, cytosolic GSH concentration is around 100–1000 times higher33. To neutralize severe oxidative stress, tumor cells typically generate more GSH (2–10 mmol/L, approximately 4 times higher) than normal cells do, which fortunately serves as a tumor-specific stimulus for cisplatin release from prodrug PtF34. The up-regulation of GSH in tumor cells is further augmented by the ozone loaded into the particle35. Thus, the prodrug shows less systematic toxicity in both in vitro experiments and in vivo experiments.
Our prodrug nano system potently improves the efficacy of chemo plus radiotherapy. Firstly, the fluorocarbon chains of prodrug PtF enhance bioavailability by facilitating the drug transmembrane36,37. Secondly, the release efficiency of cisplatin from PtF prodrug can be augmented by combining ozone and X-ray irradiation. Specifically, the loaded ozone in PFD@PtF can generate ROS upon exposure to X-ray irradiation19,38. The elevated level of ROS induces oxidative stress in tumor cells, resulting in the upregulation of the intracellular GSH/GSSG ratio21. As a result, the generated GSH facilitates the reduction-responsive release of cisplatin from the prodrug. Thirdly, oxygen produced by ozone under irradiation can alleviate the hypoxic conditions in the tumor microenvironment, enhancing the effectiveness of radiotherapy39,40.
The promising outcomes of immunotherapy in treating solid tumors have drawn large attention to its application in TNBC. Previous research has confirmed that certain nanomaterials can induce ICD and enhance the immune response against tumor cells41. In our study, we observe that the Pt(IV) prodrug nanosystem also provides a fundament for combination with immunotherapy in the future. The combo regimen is promising for enhancing antitumor immune response because it potently induces ICD and promotes the proliferation of lymphocytes. Firstly, radiotherapy induces ICD by producing ROS and increasing neoantigen generation42. On this basis, ozone plus X-ray irradiation generates even more ROS in tumors, greatly eliciting a burst of ICD by inducing endoplasmic reticulum stress (ERS) and CRT exposure in tumor cells43. Secondly, although certain chemo drugs also have the ability to induce ICD44,45, cisplatin has been reported to fail in inducing ICD because of inadequate CRT exposure46. Since both ATP and HMGB1 release can be enhanced by cisplatin, combining a CRT exposure inducer with cisplatin holds promise in achieving full-blown ICD46,47. Therefore, the combination of ozone and cisplatin released by Pt(IV) prodrug shows synergistic induction of ICD under X-ray irradiation. Finally, as a side product, the oxygen derived from ozone contributes to the creation of an immune-favored microenvironment by alleviating hypoxia, which supports lymphocyte proliferation48,49.
In this manuscript, we attempt to utilize perfluorooctanoate as the axial ligand of platinum(IV) and employ the cisplatin prodrug to construct a carrier-free ozone delivery nanosystem. In this system, the prodrug Pt(IV)-PFOA itself serves as a unit of the vehicle. By incorporating the fluorocarbon chain, the Pt(IV) prodrug has the capability to self-assemble into nanoparticles via hydrophobic interactions, significantly reducing the need for excipients and mitigating polymer-related toxicity50. The carrier-free nature of the Pt(IV) prodrug nanoparticle endows the system with an exceptionally high drug-loading capacity. As summarized by several reviews, the simplified self-assembling of micelle nanoparticles with prodrug potentially addresses the limitation of conventional nano-formulations in terms of mass production and quality control51-53, which indicates expected clinic-translational potency in the future.
5.　Conclusions
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In conclusion, we successfully developed a smart self-assembled nano micelle consisting of carrier-free cisplatin prodrug to deliver a gaseous drug into tumor masses. By loading ozone, the nanosystem efficiently suppresses tumor growth in an on-demand cisplatin release pattern upon X-ray irradiation. This prodrug nano system offers a promising approach to synchronize radiotherapy and chemotherapy applications in TNBC without significant systemic toxicity.
References
Less
1.
McDonald ES, Clark AS, Tchou J, Zhang P, Freedman GM. Clinical diagnosis and management of breast cancer. J Nucl Med 2016;57(Suppl 1):9S—16S.
2.
Bianchini G, De Angelis C, Licata L, Gianni L. Treatment landscape of triple-negative breast cancer—expanded options, evolving needs. Nat Rev Clin Oncol 2022;19:91—113.
3.
Pabla N, Dong Z. Cisplatin nephrotoxicity: mechanisms and renoprotective strategies. Kidney Int 2008;73:994—1007.
4.
Kerr AJ, Dodwell D, McGale P, Holt F, Duane F, Mannu G, et al. Adjuvant and neoadjuvant breast cancer treatments: a systematic review of their effects on mortality. Cancer Treat Rev 2022;105:102375.
5.
Speers C, Pierce LJ. Postoperative radiotherapy after breast-conserving surgery for early-stage breast cancer: a review. JAMA Oncol 2016;2:1075—82.
6.
Nagasawa S, Takahashi J, Suzuki G, Hideya Y, Yamada K. Why concurrent CDDP and radiotherapy has synergistic antitumor effects: a review of in vitro experimental and clinical-based studies. Int J Mol Sci 2021;22:3140.
7.
Forastiere AA, Zhang Q, Weber RS, Maor MH, Goepfert H, Pajak TF, et al. Long-term results of RTOG 91-11: a comparison of three nonsurgical treatment strategies to preserve the larynx in patients with locally advanced larynx cancer. J Clin Oncol 2013;31:845—52.
8.
Takada M, Fukuoka M, Kawahara M, Sugiura T, Yokoyama A, Yokota S, et al. Phase III study of concurrent versus sequential thoracic radiotherapy in combination with cisplatin and etoposide for limited-stage small-cell lung cancer: results of the Japan Clinical Oncology Group Study 9104. J Clin Oncol 2002;20:3054—60.
9.
Forastiere AA, Goepfert H, Maor M, Pajak TF, Weber R, Morrison W, et al. Concurrent chemotherapy and radiotherapy for organ preservation in advanced laryngeal cancer. N Engl J Med 2003;349:2091—8.
10.
Li Y, Wang J, Ma XW, Tan L, Yan YL, Xue CF, et al. A review of neoadjuvant chemoradiotherapy for locally advanced rectal cancer. Int J Biol Sci 2016;12:1022—31.
11.
Hickey BE, Francis DP, Lehman M. Sequencing of chemotherapy and radiotherapy for early breast cancer. Cochrane Database Syst Rev 2013;30:CD005212.
12.
Rejman J, Bragonzi A, Conese M. Role of clathrin-and caveolae-mediated endocytosis in gene transfer mediated by lipo-and polyplexes. Mol Ther 2005;12:468—74.
13.
Mathew B, Ravindran S, Liu XR, Torres L, Chennakesavalu M, Huang CC, et al. Mesenchymal stem cell-derived extracellular vesicles and retinal ischemia-reperfusion. Biomaterials 2019;197:146—60.
14.
Liu Y, Luo JS, Liu YJ, Liu W, Yu GT, Huang YT, et al. Brain-targeted biomimetic aanodecoys with neuroprotective effects for precise therapy of Parkinson’s disease. ACS Cent Sci 2022;8:1336—49.
15.
Lv J, Wang H, Rong GY, Cheng YY. Fluorination promotes the cytosolic delivery of genes, proteins, and peptides. Acc Chem Res 2022;55:722—33.
16.
Wei YS, Li XD, Lin JH, Zhou Y, Yang JD, Hou MY, et al. Oral delivery of siRNA using fluorinated, small-sized nanocapsules toward anti-inflammation treatment. Adv Mater 2023;35:2206821.
17.
Liu Y, Zhang J, Tu Y, Zhu L. Potential-independent intracellular drug delivery and mitochondrial targeting. ACS Nano 2021;16:1409—20.
18.
Riess JG. Highly fluorinated amphiphilic molecules and self-assemblies with biomedical potential. Curr Opin Colloid Interf Sci 2009;14:294—304.
19.
Zheng D, Li YC, Song LL, Xu TY, Jiang X, Yin XM, et al. Improvement of radiotherapy with an ozone-carried liposome nanosystem for synergizing cancer immune checkpoint blockade. Nano Today 2022;47:101675.
20.
Song LL, Zheng D, Xu JS, Xu TY, Liu ZH, Zhang H, et al. Improvement of TNBC immune checkpoint blockade with a microwave-controlled ozone release nanosystem. J Control Release 2022;351:954—69.
21.
Ju EG, Dong K, Chen ZW, Liu Z, Liu CQ, Huang YY, et al. Copper(II)—graphitic carbon nitride triggered synergy: improved ROS generation and reduced glutathione levels for enhanced photodynamic therapy. Angew Chem Int Ed Engl 2016;55:11467—71.
22.
Xie JN, Gong LJ, Zhu S, Yong YA, Gu ZJ, Zhao YL. Emerging strategies of nanomaterial-mediated tumor radiosensitization. Adv Mater 2019;31:1802244.
23.
Li Y, Zhang HJ, Merkher Y, Chen L, Liu N, Leonov S, et al. Recent advances in therapeutic strategies for triple-negative breast cancer. J Hematol Oncol 2022;15:121.
24.
Gou SQ, Chen NX, Wu XA, Zu MH, Yi SX, Ying BW, et al. Multi-responsive nanotheranostics with enhanced tumor penetration and oxygen self-producing capacities for multimodal synergistic cancer therapy. Acta Pharm Sin B 2022;12:406—23.
25.
Yang Y, Hu DR, Lu Y, Chu BY, He XL, Chen Y, et al. Tumor-targeted/reduction-triggered composite multifunctional nanoparticles for breast cancer chemo-photothermal combinational therapy. Acta Pharm Sin B 2022;12:2710—30.
26.
Lin CC, Huang CY, Shi ZQ, Ou MT, Sun SJ, Yu M, et al. Biodegradable calcium sulfide-based nanomodulators for H2S-boosted Ca2+-involved synergistic cascade cancer therapy. Acta Pharm Sin B 2022;12:4472—85.
27.
Telli ML, Gradishar WJ, Ward JH. NCCN guidelines updates: breast cancer. J Natl Compr Canc Netw 2019;17:552—5.
28.
Trendowski MR, El Charif O, Dinh Jr PC, Travis LB, Dolan ME. Genetic and modifiable risk factors contributing to cisplatin-induced toxicities. Clin Cancer Res 2019;25:1147—55.
29.
Ding KK, Wang LR, Zhu JM, He D, Huang YH, Zhang WJ, et al. Photo-enhanced chemotherapy performance in bladder cancer treatment via albumin coated AIE aggregates. ACS Nano 2022;16:7535—46.
30.
Wen XD, Zhang R, Hu YX, Wu LY, Bai H, Song DF, et al. Controlled sequential in situ self-assembly and disassembly of a fluorogenic cisplatin prodrug for cancer theranostics. Nat Commun 2023;14:800.
31.
Rottenberg S, Disler C, Perego P. The rediscovery of platinum-based cancer therapy. Nat Rev Cancer 2021;21:37—50.
32.
Cook JA, Gius D, Wink DA, Krishna MC, Russo A, Mitchell JB. Oxidative stress, redox, and the tumor microenvironment. Semin Radiat Oncol 2004;14:259—66.
33.
Chaiswing L, Oberley TD. Extracellular/microenvironmental redox state. Antioxid Redox Signal 2010;13:449—65.
34.
Dong X, Brahma RK, Fang C, Yao SQ. Stimulus-responsive self-assembled prodrugs in cancer therapy. Chem Sci 2022;13:4239—69.
35.
Bai XP, Ni J, Beretov J, Graham P, Li Y. Triple-negative breast cancer therapeutic resistance: where is the Achilles’ heel?. Cancer Lett 2021;497:100—11.
36.
Wang LH, Wu DC, Xu HX, You YZ. High DNA-binding affinity and gene-transfection efficacy of bioreducible cationic nanomicelles with a fluorinated core. Angew Chem Int Ed Engl 2016;55:755—9.
37.
Zhang ZJ, Shen WW, Ling J, Yan Y, Hu JJ, Cheng YY. The fluorination effect of fluoroamphiphiles in cytosolic protein delivery. Nat Commun 2018;9:1377.
38.
Sun WH, Chen LJ, Zhang YM, Wang JL. Synergistic effect of ozonation and ionizing radiation for PVA decomposition. J Environ Sci (China) 2015;34:63—7.
39.
Wu QY, Yang ZW, Du Y, Ouyang WY, Wang WL. The promotions on radical formation and micropollutant degradation by the synergies between ozone and chemical reagents (synergistic ozonation): a review. J Hazard Mater 2021;418:126327.
40.
Garoma T, Gurol MD. Degradation of tert-butyl alcohol in dilute aqueous solution by an O3/UV process. Environ Sci Technol 2004;38:5246—52.
41.
Li QQ, Shi ZQ, Zhang F, Zeng WW, Zhu DW, Mei L. Symphony of nanomaterials and immunotherapy based on the cancer—immunity cycle. Acta Pharm Sin B 2022;12:107—34.
42.
Arina A, Gutiontov SI, Weichselbaum RR. Radiotherapy and immunotherapy for cancer: from “systemic” to “multisite”. Clin Cancer Res 2020;26:2777—82.
43.
Ma S, Song WT, Xu YD, Si XH, Lv SX, Zhang Y, et al. Rationally designed polymer conjugate for tumor-specific amplification of oxidative stress and boosting antitumor immunity. Nano Lett 2020;20:2514—21.
44.
Monteran L, Ershaid N, Doron H, Zait Y, Ye Scharff, Ben-Yosef S, et al. Chemotherapy-induced complement signaling modulates immunosuppression and metastatic relapse in breast cancer. Nat Commun 2022;13:5797.
45.
Kroemer G, Galluzzi L, Kepp O, Zitvogel L. Immunogenic cell death in cancer therapy. Annu Rev Immunol 2013;31:51—72.
46.
Hato SV, Khong A, de Vries IJM, Lesterhuis WJ. Molecular pathways: the immunogenic effects of platinum-based chemotherapeutics. Clin Cancer Res 2014;20:2831—7.
47.
Martins I, Kepp O, Schlemmer F, Adjemian S, Tailler M, Shen S, et al. Restoration of the immunogenicity of cisplatin-induced cancer cell death by endoplasmic reticulum stress. Oncogene 2011;30:1147—58.
48.
Wu JC, Wang X, Chen L, Wang JN, Zhang JL, Tang JH, et al. Oxygen microcapsules improve immune checkpoint blockade by ameliorating hypoxia condition in pancreatic ductal adenocarcinoma. Bioact Mater 2023;20:259—70.
49.
Yang ZB, Luo Y, Yu HZ, Liang KC, Wang M, Wang QG, et al. Reshaping tumor immune microenvironment based on light activated nano-platform for efficient cancer therapy. Adv Mater 2022;34:2108908.
50.
Wu PK, Zhang HT, Sun ML, Mao S, He QF, Shi YZ, et al. Manipulating offense and defense signaling to fight cold tumors with carrier-free nanoassembly of fluorinated prodrug and siRNA. Adv Mater 2022;34:2203019.
51.
Li GT, Sun BJ, Li YQ, Luo C, He ZG, Sun J. Small-molecule prodrug nanoassemblies: an emerging nanoplatform for anticancer drug delivery. Small 2021;17:2101460.
52.
Li HN, Zang WL, Mi Z, Li JY, Wang LJ, Xie D, et al. Tailoring carrier-free nanocombo of small-molecule prodrug for combinational cancer therapy. J Control Release 2022;352:256—75.
53.
Han H-H, Wang H-M, Jangili P, Li M, Wu L, Zang Y, et al. The design of small-molecule prodrugs and activatable phototherapeutics for cancer therapy. Chem Soc Rev 2023;52:879—920.
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Year 2025 volume 15 Issue 5
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doi: 10.1016/j.apsb.2025.03.020
  • Receive Date：2024-09-22
  • Online Date：2026-09-17
Article Data
Affiliations
History
  • Received：2024-09-22
  • Revised：2024-12-28
  • Accepted：2025-01-15
Affiliations
    aInstitute of Breast Health Medicine, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu 610041, China
    bCancer Center, Breast Center, West China Hospital, Sichuan University, Chengdu 610041, China
    cDepartment of Ultrasound & Laboratory of Ultrasound Medicine, West China Hospital, Sichuan University, Chengdu 610041, China
    dMulti-omics Laboratory of Breast Diseases, State Key Laboratory of Biotherapy, National Collaborative, Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China
    eCollege of Chemical Engineering, Shijiazhuang University, Shijiazhuang 050035, China
    fNational Clinical Research Center for Geriatrics and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China
    gCore Facilities of West China Hospital, Sichuan University, Chengdu 610041, China

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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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