超分子纳米疫苗用于黑色素瘤免疫治疗

doi:10.7503/cjcu20240245

摘要/Abstract

摘要：

纳米疫苗是肿瘤免疫治疗的重要组成部分，主要通过激活机体免疫系统抑制并清除肿瘤；但是肿瘤组织普遍存在的免疫抑制微环境会大大降低纳米疫苗的治疗效果. 因此，设计制备既可以逆转肿瘤组织免疫抑制微环境，又可以激活机体抗肿瘤免疫响应的纳米疫苗，对肿瘤免疫治疗的发展具有重要意义. 本文利用铁离子（Fe³⁺）和紫草素（Shik）之间的配位相互作用构筑了Fe/Shik超分子纳米结构，并通过负载鸡卵白蛋白（OVA， B16-OVA肿瘤抗原）和R837（TLR7激动剂，佐剂）制备了OVA/R837@Fe/Shik纳米疫苗. 该纳米疫苗具有良好的胶体稳定性及抗肿瘤活性，可以在肿瘤微环境中特异性拆解并释放Fe²⁺， Shik， OVA和R837，通过诱导肿瘤细胞铁死亡和程序性坏死引发免疫原性细胞死亡并释放细胞裂解物，细胞裂解物协同OVA以及R837共同诱导树突细胞成熟，促进细胞毒性T淋巴细胞的激活与浸润以及巨噬细胞向M1表型极化，从而在逆转肿瘤组织免疫抑制微环境的同时激活机体的抗肿瘤免疫反应.

关键词: 纳米疫苗, 铁死亡, 程序性坏死, 免疫疗法, 免疫原性细胞死亡

Abstract:

As an important part of tumor immunotherapy， nanovaccines eradicate tumor mainly through activating the body’s immune system. However， the immunosuppressive microenvironment can largely reduce the therapeutic effect of nanovaccines in tumor tissues. Therefore， the preparation of nanovaccines that can both reverse the immunosuppressive microenvironment of tumor tissues and activate the antitumor immune response is of great significance for the development of tumor immunotherapy. In this study， the Fe/Shik supramolecular nanostructures were constructed by utilizing the coordination interaction between Fe³⁺ and Shikonin（^{Shik）. Furthermore， OVA/R837@Fe/Shik nanovaccines were prepared by loading ovalbumin（OVA， B16-OVA tumor antigen） and R837 （TLR7 agonist， adjuvant） on the basis of Fe/Shik nanostructures. Thanks to the good colloidal stability and antitumor activity， the nanovaccines can specifically disassemble and release Fe²⁺， Shik， OVA， R837 in the tumor microenvironment， eliciting immunogenic cell death by ferroptosis and necroptosis of tumor cells. The released cell lysates cooperate with OVA and adjuvants to stimulate dendritic cells maturation， promoting the activation and infiltration of cytotoxic T-lymphocytes， repolarization of macrophage， thereby activating the antitumor immune response. The study effectively overcames the inhibitory effect of the immunosuppressive microenvironment of tumor tissues on the nanovaccines， and provide a new strategy for tumor immunotherapy.}

Key words: Nanovaccines, Ferroptosis, Necroptosis, Immunotherapy, Immunogenic cell death

中图分类号:

O625.31

TrendMD:

崔彦琪, 封文洁, 刘轶. 超分子纳米疫苗用于黑色素瘤免疫治疗. 高等学校化学学报, 2025, 46(1): 20240245.

CUI Yanqi, FENG Wenjie, LIU Yi. Supramolecular Nanovaccines for Melanoma Immunotherapy. Chem. J. Chinese Universities, 2025, 46(1): 20240245.

图/表 11

Fig.1 TEM image of OVA/R837@Fe/Shik(A), DLS hydrodynamic diameter of OVA/R837@Fe/Shik(B), UV⁃Vis absorption spectra of R837, Fe/Shik, OVA@Fe/Shik and OVA/R837@Fe/Shik(C), FTIR spectra of OVA, Shik, Fe/Shik and OVA/R837@Fe/Shik(D), UV⁃Vis absorption spectra of R837(E) and the standard absorption curve of R837(F)

Fig.2 Color changes of the supernatant(A), UV⁃Vis absorption spectra of MB solution under different treatments(B), fluorescence spectra of disodium terephthalate under different treatments(C), physiological stability of OVA/R837@Fe/Shik in H2O, PBS, and RPMI 1640 medium with or without 10% FBS before(D) and after(E) 72 h, time⁃dependent hydrated diameter of OVA/R837@Fe/Shik in H2O, PBS and RPMI 1640 medium with 10% FBS(F)Inset of （A）： color changes of the supernatant with the addition of water(a), potassium thiocyanate(b), and potassium ferricyanide(c). Inset of （B）： photographs of MB solution under different treatments， I—VI: OVA/R837@Fe/Shik+GSH+H2O2, OVA/R837@Fe/Shik+H2O2, OVA/R837@Fe/Shik+GSH, GSH+H2O2, GSH, H2O2.

Fig.3 Relative cell viability of B16⁃OVA cells after 24 h incubation with different concentrations of OVA(A), Fe/Shik(B), OVA@Fe/Shik(C) and OVA/R837@Fe/Shik(D)Data are shown as mean±SD. n represents the number of biologically independent samples. Statistical significance was calculated by Student’s t⁃test and designated as *P<0.05， **P<0.01， ***P<0.001， ****P<0.0001.

Fig.4 CLSM images of ROS, Fe2+ and LPO levels in B16⁃OVA cells after different treatmentsGroup: I(control), II(OVA), III(Shik), IV(Fe/Shik), V(OVA@Fe/Shik), VI(OVA/R837@Fe/Shik).

Fig.5 Quantification of ROS(A), Fe2+(B) and LPO(C) levels by flow cytometry in B16⁃OVA cells after different treatments, GSH content of B16⁃OVA cells after different treatments by GSH assay kid(D), the expression of RIPK1(E) and RIPK3(F) analyzed by ELISA assay kit after different treatmentsGroup: I(OVA), II(OVA), III(Shik), IV(Fe/Shik), V(OVA@Fe/Shik), VI(OVA/R837@Fe/Shik). Data are shown as mean±SD. n represents the number of biologically independent samples. Statistical significance was calculated by Student's t⁃test and designated as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig.6 CLSM images of CRT and HMGB1 expression in B16⁃OVA cells after different treatments(A), the expression of HMGB1(B) and ATP(C) analyzed by ELISA assay kit after different treatments, corresponding quantification DCs maturation(CD11c+CD80+CD86+) by flow cytometry(D)Group: I(control), II(OVA), III(Shik), IV(Fe/Shik), V(OVA@Fe/Shik), VI(OVA/R837@Fe/Shik). Data are shown as mean±SD. n represents the number of biologically independent samples. Statistical significance was calculated by Student's t⁃test and designated as *P<0.05， **P<0.01， ***P<0.001， ****P<0.0001.

Fig.7 Average tumor growth curves(A), average body weight of mice(B) and average tumor weight(C) with various treatments, H&E⁃stained images of tumor and immunofluorescence staining images(GPX4, RIPK1, RIPK3, CRT and HMGB1) of tumor tissue slices after different treatments(D)Group: I(control), II(OVA), III(Shik), IV(Fe/Shik), V(OVA@Fe/Shik), VI(OVA/R837@Fe/Shik). Data are shown as mean±SD. n represents the number of biologically independent samples. Statistical significance was calculated by Student’s t⁃test and designated as *P<0.05， **P<0.01， ***P<0.001， ****P<0.0001.

Fig.8 Corresponding quantification DCs maturation(CD11c+CD80+CD86+) by flow cytometry in spleen(A), flow cytometric statistical analysis of the percentages of T helper cells(CD3+CD4+)(B), cytotoxic T lymphocytes(CD3+CD8+)(C), M1 macrophage(D), M2 macrophage(E) and Tregs(F) in tumors after different treatments, fluorescence intensity measured by Image J(G, H) and corresponding immunofluorescence staining images(I) of CD4+ and CD8+ T cells in tumor tissue slices after different treatmentsGroup: I(control), II(OVA), III(Shik), IV(Fe/Shik), V(OVA@Fe/Shik), VI(OVA/R837@Fe/Shik). Data are shown as mean±SD. n represents the number of biologically independent samples. Statistical significance was calculated by Student’s t⁃test and designated as *P<0.05， **P<0.01， ***P<0.001， ****P<0.0001.

Fig.9 Representative images(A) and H&E⁃stained images(B) of lungs of mice after different treatments

Fig.10 Main indexes of liver and renal functions tests of mice in each group(A) Total bile acid(TBA); (B) total bilirubin(TBIL); (C) direct bilirubin(DBIL); (D) creatinine(CREA); (E) uric acid(UA); (F) alanine aminotransferase(ALT); (G) aspartate transaminase(AST); (H) alkaline phosphatase(ALP); (I) γ⁃glutamyl transpeptidase; (J) albumin(ALB); (K) urea. Group: I(OVA)， II(OVA)， III(Shik)， IV(Fe/Shik)， V(OVA@Fe/Shik)， VI(OVA/R837@Fe/Shik). Data are shown as mean±SD. n represents the number of biologically independent samples. Statistical significance was calculated by Student’s t⁃test and designated as *P<0.05， **P<0.01， ***P<0.001， ****P<0.0001.

Fig.11 H&E⁃stained splanchnic slices of heart, liver, spleen, lungs and kidneys of mice after different treatmentsGroup: I(OVA), II(OVA), III(Shik), IV(Fe/Shik), V(OVA@Fe/Shik), VI(OVA/R837@Fe/Shik).

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