联合递送IDO抑制剂和紫杉醇用于癌症治疗

doi:10.5246/jcps.2021.01.001

中国药学(英文版) ›› 2021, Vol. 30 ›› Issue (1): 1-16.DOI: 10.5246/jcps.2021.01.001

• 【研究论文】 • 下一篇

联合递送IDO抑制剂和紫杉醇用于癌症治疗

王大宽¹, 彭博¹, 秦蒙蒙¹, 李明慧¹, 宋歌¹, 何冰¹, 张华¹, 代文兵¹, 张强¹^,³, 孟祥豹², 孟幻¹^,^*(), 王学清¹^,^*()

1. 北京大学药学院分子药剂学与新释药系统北京市重点实验室, 北京 100191
2. 北京大学药学院化学生物学系, 北京 100191
3. 北京大学药学院天然药物及仿生药物国家重点实验室, 北京 100191

收稿日期:2020-08-20 修回日期:2020-09-11 接受日期:2020-09-15 出版日期:2021-01-29 发布日期:2021-01-29
通讯作者: 孟幻, 王学清
作者简介:
+ Tel.: +86-10-82805935, E-mail: wangxq@bjmu.edu.cn
+ E-mail: menghuanpharma@163.com
基金资助:
The National Natural Science Foundation of China (Grants No. 31671017, 81872809), the National Key Research and Development Program of China (Grants No. 2017YFA0205600) and Beijing Natural Science Foundation (Grants No. 7162108).

Integrated combination delivery of IDO inhibitor and paclitaxel for cancer treatment

Dakuan Wang¹, Bo Peng¹, Mengmeng Qin¹, Minghui Li¹, Ge Song¹, Bing He¹, Hua Zhang¹, Wenbing Dai¹, Qiang Zhang¹^,³, Xiangbao Meng², Huan Meng¹^,^*(), Xueqing Wang¹^,^*()

1 Beijing Key Laboratory of Molecular Pharmaceutics, New Drug Delivery Systems, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
2 Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
3 State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China

Received:2020-08-20 Revised:2020-09-11 Accepted:2020-09-15 Online:2021-01-29 Published:2021-01-29
Contact: Huan Meng, Xueqing Wang

摘要/Abstract

摘要：

为实现化学疗法与免疫疗法的联合应用, 本研究合成了具有还原响应性的紫杉醇(Paclitaxel, PTX)前药PEG-SS-PTX, 并以此为载体包载吲哚胺2,3-双加氧酶(Indoleamine 2,3-dioxygenase, IDO)抑制剂CY-1-4制备PEG-SS-PTX/CY-1-4 NPs。通过细胞毒作用、免疫原性细胞死亡(Immunogenic cell death, ICD)诱导能力和抗肿瘤功效对PEG-SS-PTX/CY-1-4 NPs进行评估。动态光散射(Dynamic light scattering, DLS) 结果显示PEG-SS-PTX/CY-1-4 NPs粒径约为149 nm。体外实验结果表明PEG-SS-PTX/CY-1-4 NPs的细胞毒性具有浓度依赖性, 并且能够引发B16-F10细胞的ICD。体内实验结果表明PEG-SS-PTX/CY-1-4 NPs在小鼠黑色素瘤模型中能够显著抑制肿瘤生长, 降低IDO在肿瘤组织中的表达, 并且能够增加脾脏中CD8⁺ T细胞比例。综上, PEG-SS-PTX/CY-1-4 NPs达到良好抗肿瘤效果的同时降低了化疗药物的给药剂量, 是一种安全有效的联合递送平台。

关键词: 化学疗法, 免疫疗法, 联合给药, 纳米药物递送系统, IDO活性抑制, 免疫原性死亡

Abstract:

In order to realize the combination of chemotherapy and immunotherapy, a reduction-responsive paclitaxel (PTX) prodrug PEG-SS-PTX was synthesized and used as a carrier to encapsulate IDO inhibitor CY-1-4 for preparing PEG-SS-PTX/CY-1-4 NPs. PEG-SS-PTX/CY-1-4 NPs were evaluated by cytotoxicity, immunogenic cell death (ICD) induction ability and anti-tumor efficacy. Dynamic light scattering (DLS) results showed that the size of PEG-SS-PTX/CY-1-4 NPs was about 149 nm. In vitro experiments indicated that its cytotoxicity was in a concentration-dependent manner, and it induced the ICD of B16-F10 cells. In vivo studies in melanoma mouse model indicated that PEG-SS-PTX/CY-1-4 NPs significantly inhibited the tumor growth and reduced the expression of IDO in tumor tissues. Moreover, it increased the rate of CD8⁺ T cells in the spleen. In summary, PEG-SS-PTX/CY-1-4 NPs achieved good anti-tumor effects and reduced the dose of chemotherapy drugs, which was a safe and effective combined delivery system.

Key words: Chemotherapy, Immunotherapy, Combination drug delivery, Nano drug delivery system, IDO activity inhibition, Immunogenic cell death

Supporting:

Figure S1. The structure of CY-1-4. The molecular weight is 294.23 and the chemical formula is C₁₄H₆N₄O₄.

Figure S2. Quantified individual tumor volumes of different groups of mice after various treatments.

Figure S3. The routine blood test result including PLT, PCT, MO, GR, GR%, WBC, LY%, RBC, MCHC, PDW, MPV, MO%, MCV, MCH, RDW, HGB, and HCT. (^*P < 0.05, ^****P < 0.0001 vs. PBS, ^# P < 0.05, ^### P < 0.001 vs. PEG-SS-PTX/CY-1-4 NPs.)

Figure S4. H & E staining for hearts, livers, spleens, lungs and kidneys. Organs were harvested 2 days after last treatment. Nuclei and cytoplasm were stained by hematoxylin and eosin, respectively.

王大宽, 彭博, 秦蒙蒙, 李明慧, 宋歌, 何冰, 张华, 代文兵, 张强, 孟祥豹, 孟幻, 王学清. 联合递送IDO抑制剂和紫杉醇用于癌症治疗[J]. 中国药学(英文版), 2021, 30(1): 1-16.

Dakuan Wang, Bo Peng, Mengmeng Qin, Minghui Li, Ge Song, Bing He, Hua Zhang, Wenbing Dai, Qiang Zhang, Xiangbao Meng, Huan Meng, Xueqing Wang. Integrated combination delivery of IDO inhibitor and paclitaxel for cancer treatment[J]. Journal of Chinese Pharmaceutical Sciences, 2021, 30(1): 1-16.

图/表 9

Figure 1. Schematic representation of PEG-SS-PTX/CY-1-4 NPs combining PTX with IDO inhibitor CY-1-4 to exert anti-tumor effects.

Table 1. Basic characterization of NPs (n = 3, means ± SD).

Figure 2. Particle size distribution determined by DLS and TEM images of NPs. (A) PEG-SS-PTX NPs; (B) PEG-SS-PTX/CY-1-4 NPs; (C) CY-1-4 NPs.

Figure 3. In vitro biological characterizations of NPs. (A) Cell viability when treated with PEG-SS-PTX NPs, PEG-SS-PTX/CY-1-4 NPs and CY-1-4 NPs in B16-F10 cells in CY-1-4 concentrations from 0.4 to 0.9 μM (n = 3, mean ± SD); (B) Histogram showed the ratio of late apoptosis and early apoptosis treated with different formulations at the concentration of CY-1-4 0.8 μM (n = 3, mean ± SD); (C) Representative dot plot showed the cell apoptosis detected by flow cytometry.

Figure 4. NPs induced ICD. (A) Confocal microscopy was used to observe the release of HMGB1 from B16-F10 cell nucleus after treated with 0.75 μM NPs. HMGB1 proteins were marked by Alexa Flour 488 (green) and nucleus were marked by Hochest 33258 (blue), Scale bar, 25 μm. (B) Confocal microscopy was used to observe the CRT expression in B16-F10 cells after treated with 0.75 μM NPs. CRT was marked by Alexa Flour 488 (green) and nucleus were marked by Hochest 33258 (blue), Scale bar, 10 μm. (C) Histogram showed the percentage of B16-F10 which exposed CRT on the cell surface after the treatments of NPs by flow cytometry analysis (n = 3, *P < 0.05, ****P < 0.0001 vs. the negative control, ####P < 0.0001 vs. PEG-SS-PTX/CY-1-4 NPs). (D) Representative graph of cell CRT exposure by flow cytometry after treatments of NPs.

Figure 5. NPs stimulate DC 2.4 cells maturation and activation. (A) & (C) Flow cytometry dot plot for CD80, CD86 and CD11c in treated DC 2.4 cells. B16-F10 cells were treated with 0.75 μM NPs for 24 h in 6-well plate, and then the culture solution and debris in each plate was added to another 6-well plate full with DC 2.4 cells to co-culture for 24 h, respectively. Finally, the DC 2.4 cells were sent to analyze the change of CD80, CD86 and CD11c. (B) Histogram for statistic analysis shows the percentage of CD11c+ and CD86+ in each group. The data are expressed as mean ± SD from independent experiments. (n = 3, *P < 0.05, **P < 0.01 vs. the negative control, #P < 0.05 vs. PEG-SS-PTX/CY-1-4 NPs). (D) Histogram for statistic analysis shows the percentage of CD11c+ and CD80+ in each group. The data are expressed as mean ± SD from independent experiments. (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 vs. the negative control, ##P < 0.01 vs. PEG-SS-PTX/CY-1-4 NPs).

Figure 6. In vivo antitumor activity treated with PBS, NPs at dosage of 300 μg/kg CY-1-4, 1-MT in 200 mg/kg and Taxol at dosage of 7 mg/kg PTX. (A) Tumor photographs harvested from tumor-bearing mice in each group. 1-MT as a classical control. (B) Tumor growth sizes of different treatment groups (n = 6, *P < 0.05 vs. PBS group, #P<0.05 vs. PEG-SS-PTX/CY-1-4 NPs, mean ± SEM). (C) Average body weight of tumor-bearing mice in different treatment groups (n = 6, *P < 0.05, **P < 0.01, ****P < 0.0001, mean ± SD).

Figure 7. In vivo IDO expression in a subcutaneous mouse model of B16-F10 cells treated with NPs. Fluorescence microscopy images of the tumor tissue sections at 2 d after last treatment. IDO protein was marked by Alexa Flour 488-conjugated secondary antibodies (green) and the nucleus were marked by Hochest 33258 (blue), Scale bar, 25 μm.

Figure 8. Flow cytometry analysis of immune cells subpopulation in spleens after the mice were treated with PBS, PEG-SS-PTX NPs, PEG-SS-PTX/CY-1-4 NPs, CY-1-4 NPs at dosage of 300 μg/kg, and 1-MT in 200 mg/kg. Percentage of (A) CD3+ T cells, (B) CD8+ T cells, (C) CD4+ T cells. (D) The rate of CD4+ cells: CD8+ cells. The bars represent means ± SD (*P < 0.05, **P < 0.01, n = 3).

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联合递送IDO抑制剂和紫杉醇用于癌症治疗

Integrated combination delivery of IDO inhibitor and paclitaxel for cancer treatment

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