一种装载茶多酚与双醋瑞因的缺氧敏感型纳米凝胶的制备

近年来免疫治疗在临床上取得了巨大的成功，但由于肿瘤异质性和肿瘤免疫抑制微环境对于肿瘤生长的促进等原因，多数肿瘤的治疗效率不及预期。因此，本研究构建了一种缺氧敏感型纳米凝胶装载小分子药物茶多酚与双醋瑞因，茶多酚可以降低肿瘤细胞PD-L1的表达，双醋瑞因是一种IL-1β抑制剂，联合调控免疫检查点与肿瘤免疫微环境。通过透明质酸和羟丙基环糊精接枝双键基团，交联合成10-20 nm的纳米凝胶核心，环糊精空腔可装载药物茶多酚，聚乙二醇和双醋瑞因通过具有缺氧响应断裂特性的小分子连接在纳米凝胶核心表面，最终制备成功EGCG&DIA-P@NG-PEG药物。采用体外模拟缺氧环境，验证纳米凝胶尺寸与电荷的响应可变性，响应后粒径恢复至10-20 nm，响应之后的纳米凝胶在酸性条件下表面携带正电荷。细胞毒性实验和红细胞溶血实验验证载体的生物相容性，细胞摄取实验证明肿瘤细胞对缺氧响应后的载体有更高的摄取作用，并且细胞实验证明载体装载茶多酚后能够更多的降低肿瘤细胞表面PD-L1表达，增强免疫抗肿瘤效果。小动物活体成像实验证明合成的药物载体能够延长药物在体内的循环时间。综上所述，本论文针对肿瘤缺氧微环境制备一种缺氧响应型纳米凝胶装载两种药物，为免疫检查点阻断与肿瘤微环境调控结合的免疫疗法奠定基础，为肿瘤联合疗法提供新的思路。

Immunotherapy has achieved great success in clinical practice in recent years. However, due to tumor heterogeneity and the promotion of tumor growth by immunosuppressive microenvironment, the therapeutic efficiency of tumors is not as expected. Therefore, we constructed a hypoxia- responsive nanogel loaded with EGCG and Diacerein. EGCG can reduce PD-L1 expression on the surface of tumor cells, and Diacerein is an IL-1β inhibitor, which combines immune checkpoint blockade therapy with tumor microenvironment regulation. By grafting double bond groups with hyaluronic acid and hydroxypropyl cyclodextrin, free radicals initiate cross-linking to form a 10-20 nm nanogel. The cyclodextrin cavity could be loaded with EGCG. DIA-P and mPEG were connected on the surface of nanogel through 4,4'-azobisbenzoic acid, and EGCG&DIA-P@NG-PEG was finally prepared. In vitro simulation of anoxic environment was used to verify the response variability of the size and charge of the nanogel. After hypoxia-responsive, the size was restored to 10-20 nm, and the surface of EGCG&DIA-P@NG-PEG carried positive charge under acidic conditions. Cytotoxicity test and hemolysis test verified the biocompatibility of nanogel. Cell uptake test proved that tumor cells had a higher uptake effect on NG-PEG after hypoxia response, and EGCG@NG-PEG could reduce the expression of PD-L1 of tumor cells. In vivo imaging experiments proved that nanogel can prolong the drug circulation time in vivo. In conclusion, a hypoxia-responsive nanogel loaded with two drugs was prepared, which laid a foundation for immunotherapy combining immune checkpoint blocking and tumor microenvironment regulation, and provided a new idea for combined tumor therapy.

[1] Siegel R L, Miller K D, Fuchs H E, et al. Cancer statistics, 2022 [J]. CA Cancer J Clin, 2022, 72(1): 7-33.
[2] Morad G, Helmink B A, Sharma P, et al. Hallmarks of response, resistance, and toxicity to immune checkpoint blockade [J]. Cell, 2021, 184(21): 5309-37.
[3] He X, Xu C. Immune checkpoint signaling and cancer immunotherapy [J]. Cell Res, 2020, 30(8): 660-9.
[4] Ribas A, Wolchok J D. Cancer immunotherapy using checkpoint blockade [J]. Science, 2018, 359(6382): 1350-5.
[5] 王旭晨. 重组腺病毒表达PD-1单抗（Nivolumab）的研究 [D]; 中国科学院大学(中国科学院上海巴斯德研究所), 2019.
[6] Gaikwad S, Agrawal M Y, Kaushik I, et al. Immune checkpoint proteins: Signaling mechanisms and molecular interactions in cancer immunotherapy [J]. Seminars in Cancer Biology, 2022,
[7] Lui Y, Davis S J. LAG-3: a very singular immune checkpoint [J]. Nature Immunology, 2018, 19(12): 1278-9.
[8] Abril-Rodriguez G, Ribas A. SnapShot: Immune Checkpoint Inhibitors [J]. Cancer Cell, 2017, 31(6): 848- e1.
[9] Motzer R J, Tannir N M, Mcdermott D F, et al. Nivolumab plus Ipilimumab versus Sunitinib in Advanced Renal-Cell Carcinoma [J]. N Engl J Med, 2018, 378(14): 1277-90.
[10] Upadhaya S, Neftelino S T, Hodge J P, et al. Combinations take centre stage in PD1/PDL1 inhibitor clinical trials [J]. Nat Rev Drug Discov, 2021, 20(3): 168-9.
[11] Wang D Y, Salem J E, Cohen J V, et al. Fatal Toxic Effects Associated With Immune Checkpoint Inhibitors: A Systematic Review and Meta-analysis [J]. JAMA Oncol, 2018, 4(12): 1721-8.
[12] Long G V, Atkinson V, Lo S, et al. Combination nivolumab and ipilimumab or nivolumab alone in melanoma brain metastases: a multicentre randomised phase 2 study [J]. Lancet Oncol, 2018, 19(5): 672-81.
[13] Hellmann M D, Ciuleanu T E, Pluzanski A, et al. Nivolumab plus Ipilimumab in Lung Cancer with a High Tumor Mutational Burden [J]. N Engl J Med, 2018, 378(22): 2093-104.
[14] Chiloiro S, Bianchi A, Giampietro A, et al. The changing clinical spectrum of endocrine adverse events in cancer immunotherapy [J]. Trends Endocrinol Metab, 2022, 33(2): 87-104.
[15] Pillai R N, Behera M, Owonikoko T K, et al. Comparison of the toxicity profile of PD-1 versus PD-L1 inhibitors in non-small cell lung cancer: A systematic analysis of the literature [J]. Cancer, 2018, 124(2): 271-7.
[16] Callahan M K, Postow M A, Wolchok J D. CTLA-4 and PD-1 Pathway Blockade: Combinations in the Clinic [J]. Front Oncol, 2014, 4(385.
[17] Greten F R, Grivennikov S I. Inflammation and Cancer: Triggers, Mechanisms, and Consequences [J]. Immunity, 2019, 51(1): 27-41.
[18] Binnewies M, Roberts E W, Kersten K, et al. Understanding the tumor immune microenvironment (TIME) for effective therapy [J]. Nat Med, 2018, 24(5): 541-50.
[19] Cao R, Ji H, Feng N, et al. Collaborative interplay between FGF-2 and VEGF-C promotes lymphangiogenesis and metastasis [J]. Proc Natl Acad Sci U S A, 2012, 109(39): 15894-9.
[20] Patel A, Sant S. Hypoxic tumor microenvironment: Opportunities to develop targeted therapies [J]. Biotechnol Adv, 2016, 34(5): 803-12.
[21] Span P N, Bussink J. Biology of hypoxia [J]. Semin Nucl Med, 2015, 45(2): 101-9.
[22] Vaupel P, Schlenger K, Knoop C, et al. Oxygenation of human tumors: evaluation of tissue oxygen distribution in breast cancers by computerized O2 tension measurements [J]. Cancer Res, 1991, 51(12): 3316-22.
[23] Helmlinger G, Yuan F, Dellian M, et al. Interstitial pH and pO2 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation [J]. Nat Med, 1997, 3(2): 177-82.
[24] Lee P, Chandel N S, Simon M C. Cellular adaptation to hypoxia through hypoxia inducible factors and beyond [J]. Nat Rev Mol Cell Biol, 2020, 21(5): 268-83.
[25] Al Tameemi W, Dale T P, Al-Jumaily R M K, et al. Hypoxia-Modified Cancer Cell Metabolism [J]. Front Cell Dev Biol, 2019, 7(4.
[26] Martinez-Reyes I, Chandel N S. Cancer metabolism: looking forward [J]. Nat Rev Cancer, 2021, 21(10): 669-80.
[27] Loscalzo J. Adaptions to Hypoxia and Redox Stress: Essential Concepts Confounded by Misleading Terminology [J]. Circ Res, 2016, 119(4): 511-3.
[28] Murciano-Goroff Y R, Warner A B, Wolchok J D. The future of cancer immunotherapy: microenvironment-targeting combinations [J]. Cell Research, 2020, 30(6): 507-19.
[29] Mantovani A, Barajon I, Garlanda C. IL-1 and IL-1 regulatory pathways in cancer progression and therapy [J]. Immunol Rev, 2018, 281(1): 57-61.
[30] Garlanda C, Mantovani A. Interleukin-1 in tumor progression, therapy, and prevention [J]. Cancer Cell, 2021, 39(8): 1023-7.
[31] Kaplanov I, Carmi Y, Kornetsky R, et al. Blocking IL-1beta reverses the immunosuppression in mouse breast cancer and synergizes with anti-PD-1 for tumor abrogation [J]. Proc Natl Acad Sci U S A, 2019, 116(4): 1361-9.
[32] Song X, Krelin Y, Dvorkin T, et al. CD11b+/Gr-1+ immature myeloid cells mediate suppression of T cells in mice bearing tumors of IL-1beta-secreting cells [J]. J Immunol, 2005, 175(12): 8200-8.
[33] Huang B, Lei Z, Zhao J, et al. CCL2/CCR2 pathway mediates recruitment of myeloid suppressor cells to cancers [J]. Cancer Lett, 2007, 252(1): 86-92.
[34] De Vlaeminck Y, Gonzalez-Rascon A, Goyvaerts C, et al. Cancer-Associated Myeloid Regulatory Cells [J]. Front Immunol, 2016, 7(113.
[35] De Henau O, Rausch M, Winkler D, et al. Overcoming resistance to checkpoint blockade therapy by targeting PI3Kgamma in myeloid cells [J]. Nature, 2016, 539(7629): 443-7.
[36] Qian B Z, Pollard J W. Macrophage diversity enhances tumor progression and metastasis [J]. Cell, 2010, 141(1): 39-51.
[37] Guerriero J L. Macrophages: The Road Less Traveled, Changing Anticancer Therapy [J]. Trends Mol Med, 2018, 24(5): 472-89.
[38] Koul H, Huh J S, Rove K O, et al. Molecular aspects of renal cell carcinoma: a review [J]. Am J Cancer Res, 2011, 1(2): 240-54.
[39] Bingle L, Brown N J, Lewis C E. The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies [J]. J Pathol, 2002, 196(3): 254-65.
[40] Obradovic A, Chowdhury N, Haake S M, et al. Single-cell protein activity analysis identifies recurrence-associated renal tumor macrophages [J]. Cell, 2021, 184(11): 2988-3005 e16.
[41] Anderson N R, Minutolo N G, Gill S, et al. Macrophage-Based Approaches for Cancer Immunotherapy [J]. Cancer Res, 2021, 81(5): 1201-8.
[42] Hinshaw D C, Shevde L A. The Tumor Microenvironment Innately Modulates Cancer Progression [J]. Cancer Res, 2019, 79(18): 4557-66.
[43] Anduran E, Dubois L J, Lambin P, et al. Hypoxia-activated prodrug derivatives of anti-cancer drugs: a patent review 2006 - 2021 [J]. Expert Opin Ther Pat, 2022, 32(1): 1-12.
[44] Wilson W R, Hay M P. Targeting hypoxia in cancer therapy [J]. Nat Rev Cancer, 2011, 11(6): 393-410.
[45] Wang Y, Xiao D, Li J, et al. From prodrug to pro-prodrug: hypoxia-sensitive antibody-drug conjugates [J]. Signal Transduct Target Ther, 2022, 7(1): 20.
[46] Cheng M H Y, Mo Y, Zheng G. Nano versus Molecular: Optical Imaging Approaches to Detect and Monitor Tumor Hypoxia [J]. Adv Healthc Mater, 2021, 10(2): e2001549.
[47] Sharma A, Arambula J F, Koo S, et al. Hypoxia-targeted drug delivery [J]. Chem Soc Rev, 2019, 48(3): 771-813.
[48] Guo X, Liu F, Deng J, et al. Electron-Accepting Micelles Deplete Reduced Nicotinamide Adenine Dinucleotide Phosphate and Impair Two Antioxidant Cascades for Ferroptosis-Induced Tumor Eradication [J]. ACS Nano, 2020, 14(11): 14715-30.
[49] Zhang T X, Zhang Z Z, Yue Y X, et al. A General Hypoxia-Responsive Molecular Container for Tumor-Targeted Therapy [J]. Adv Mater, 2020, 32(28): e1908435.
[50] Hubbell J A, Chilkoti A. Chemistry. Nanomaterials for drug delivery [J]. Science, 2012, 337(6092): 303-5.
[51] Owen S C, Chan D P Y, Shoichet M S. Polymeric micelle stability [J]. Nano Today, 2012, 7(1): 53-65.
[52] Kang N, Perron M E, Prud'homme R E, et al. Stereocomplex block copolymer micelles: core-shell nanostructures with enhanced stability [J]. Nano Lett, 2005, 5(2): 315-9.
[53] Pedrosa S S, Goncalves C, David L, et al. A novel crosslinked hyaluronic acid nanogel for drug delivery [J]. Macromol Biosci, 2014, 14(11): 1556-68.
[54] Song Q, Zhang G, Wang B, et al. Reinforcing the Combinational Immuno-Oncotherapy of Switching "Cold" Tumor to "Hot" by Responsive Penetrating Nanogels [J]. ACS Appl Mater Interfaces, 2021, 13(31): 36824-38.
[55] Zhou Z, Ma X, Jin E, et al. Linear-dendritic drug conjugates forming long-circulating nanorods for cancer-drug delivery [J]. Biomaterials, 2013, 34(22): 5722-35.
[56] Xu P, Wang L, Zhang X, et al. High-Performance Smart Hydrogels with Redox-Responsive Properties Inspired by Scallop Byssus [J]. ACS Appl Mater Interfaces, 2022, 14(1): 214-24.
[57] Niland S, Eble J A. Hold on or Cut? Integrin- and MMP-Mediated Cell-Matrix Interactions in the Tumor Microenvironment [J]. Int J Mol Sci, 2020, 22(1):
[58] Tanaka A, Fukuoka Y, Morimoto Y, et al. Cancer cell death induced by the intracellular self-assembly of an enzyme-responsive supramolecular gelator [J]. J Am Chem Soc, 2015, 137(2): 770-5.
[59] Chen M, Tan Y, Hu J, et al. Injectable Immunotherapeutic Thermogel for Enhanced Immunotherapy Post Tumor Radiofrequency Ablation [J]. Small, 2021, 17(52): e2104773.
[60] Yan X, Sun T, Song Y, et al. In situ Thermal-Responsive Magnetic Hydrogel for Multidisciplinary Therapy of Hepatocellular Carcinoma [J]. Nano Lett, 2022, 22(6): 2251-60.
[61] Guedes G, Wang S, Fontana F, et al. Dual-Crosslinked Dynamic Hydrogel Incorporating {Mo154 } with pH and NIR Responsiveness for Chemo-Photothermal Therapy [J]. Adv Mater, 2021, 33(40): e2007761.
[62] Komatsu S, Tago M, Ando Y, et al. Facile preparation of multi-stimuli-responsive degradable hydrogels for protein loading and release [J]. J Control Release, 2021, 331(1-6.
[63] Wang S, Zheng H, Zhou L, et al. Injectable redox and light responsive MnO2 hybrid hydrogel for simultaneous melanoma therapy and multidrug-resistant bacteria-infected wound healing [J]. Biomaterials, 2020, 260(120314.
[64] Tang M L, Zhou M L, Huang Y A, et al. Dual-sensitive and biodegradable core-crosslinked HPMA copolymer-doxorubicin conjugate-based nanoparticles for cancer therapy [J]. Polym Chem-Uk, 2017, 8(15): 2370-80.
[65] Wibowo D, Hui Y, Middelberg A P, et al. Interfacial engineering for silica nanocapsules [J]. Adv Colloid Interface Sci, 2016, 236(83-100.
[66] Bedard P L, Hyman D M, Davids M S, et al. Small molecules, big impact: 20 years of targeted therapy in oncology [J]. Lancet, 2020, 395(10229): 1078-88.
[67] Islam M T. Diterpenes and Their Derivatives as Potential Anticancer Agents [J]. Phytother Res, 2017, 31(5): 691-712.
[68] Pascolutti M, Quinn R J. Natural products as lead structures: chemical transformations to create lead-like libraries [J]. Drug Discov Today, 2014, 19(3): 215-21.
[69] Ghanbari-Movahed M, Jackson G, Farzaei M H, et al. A Systematic Review of the Preventive and Therapeutic Effects of Naringin Against Human Malignancies [J]. Front Pharmacol, 2021, 12(639840.
[70] Hanahan D, Weinberg R A. Hallmarks of cancer: the next generation [J]. Cell, 2011, 144(5): 646-74.
[71] Lu J M, Yao Q, Chen C. Ginseng compounds: an update on their molecular mechanisms and medical applications [J]. Curr Vasc Pharmacol, 2009, 7(3): 293-302.
[72] Zhang H, Park S, Huang H, et al. Anticancer effects and potential mechanisms of ginsenoside Rh2 in various cancer types (Review) [J]. Oncol Rep, 2021, 45(4):
[73] Wani M C, Taylor H L, Wall M E, et al. Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia [J]. J Am Chem Soc, 1971, 93(9): 2325-7.
[74] Scribano C M, Wan J, Esbona K, et al. Chromosomal instability sensitizes patient breast tumors to multipolar divisions induced by paclitaxel [J]. Sci Transl Med, 2021, 13(610): eabd4811.
[75] Zhu L, Chen L. Progress in research on paclitaxel and tumor immunotherapy [J]. Cell Mol Biol Lett, 2019, 24(40.
[76] Sharifi-Rad J, Quispe C, Patra J K, et al. Paclitaxel: Application in Modern Oncology and Nanomedicine-Based Cancer Therapy [J]. Oxid Med Cell Longev, 2021, 2021(3687700.
[77] Holton R A, Somoza C, Kim H B, et al. First Total Synthesis of Taxol .1. Functionalization of the B-Ring [J]. Journal of the American Chemical Society, 1994, 116(4): 1597-8.
[78] Ravindran Menon D, Li Y, Yamauchi T, et al. EGCG Inhibits Tumor Growth in Melanoma by Targeting JAK-STAT Signaling and Its Downstream PD-L1/PD-L2-PD1 Axis in Tumors and Enhancing Cytotoxic T-Cell Responses [J]. Pharmaceuticals (Basel), 2021, 14(11):
[79] Gao Y, Chen X, Fang L, et al. Rhein exerts pro- and anti-inflammatory actions by targeting IKKbeta inhibition in LPS-activated macrophages [J]. Free Radic Biol Med, 2014, 72(104-12.
[80] Maeda H, Nakamura H, Fang J. The EPR effect for macromolecular drug delivery to solid tumors: Improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo [J]. Adv Drug Deliv Rev, 2013, 65(1): 71-9.
[81] Ding Y X, Xu Y J, Yang W Z, et al. Investigating the EPR effect of nanomedicines in human renal tumors via ex vivo perfusion strategy [J]. Nano Today, 2020, 35(
[82] Gong G, Pan J, He Y, et al. Self-assembly of nanomicelles with rationally designed multifunctional building blocks for synergistic chemo-photodynamic therapy [J]. Theranostics, 2022, 12(5): 2028-40.
[83] Hui L, Chen Y. Tumor microenvironment: Sanctuary of the devil [J]. Cancer Lett, 2015, 368(1): 7-13.
[84] Patel R D, Raval M K, Pethani T M. Application of a Validated RP-HPLC Method in Solubility and Dissolution Testing for Simultaneous Estimation of Diacerein and Its Active Metabolite Rhein in Presence of Coformers in the Eutectic Tablet Formulation [J]. J Chromatogr Sci, 2021, 59(8): 697-705.
[85] Kianfar S, Keshtkar A R, Zarenezhad B. Graft polymerization of acrylonitrile onto cross-linked (alginate/polyvinyl alcohol) beads initiated by potassium persulfate: synthesis and artificial neural network modeling [J]. Polymer Bulletin, 2021, 78(1): 295-311.
[86] Yang G, Phua S Z F, Lim W Q, et al. A Hypoxia-Responsive Albumin-Based Nanosystem for Deep Tumor Penetration and Excellent Therapeutic Efficacy [J]. Adv Mater, 2019, 31(25): e1901513.
[87] Kraft S, Fernandez-Figueras M T, Richarz N A, et al. PDL1 expression in desmoplastic melanoma is associated with tumor aggressiveness and progression [J]. J Am Acad Dermatol, 2017, 77(3): 534-42.