Fe(III)-Shikonin supramolecular nanomedicines as immunogenic cell death stimulants and multifunctional immunoadjuvants for tumor vaccination

期刊名：Theranostics

文章地址：https://www.thno.org/v13p5266.htm

Abstract
Immunoadjuvants, as an indispensable component of tumor vaccines, can observably enhance the magnitude, breadth, and durability of antitumor immunity. However, current immunoadjuvants suffer from different issues such as weak immunogenicity, inadequate cellular internalization, poor circulation time, and mono-functional bioactivity.

Methods: Herein, we construct Fe3+-Shikonin metal-phenolic networks (FeShik) nanomedicines as immunogenic cell death (ICD) stimulants and multifunctional immunoadjuvants for tumor vaccination. The multifunctionality of FeShik nanomedicines is investigated by loading ovalbumin (OVA) as the model antigen to construct OVA@FeShik nanovaccines or 4T1 tumor cell fragment (TF) as homologous antigen to construct TF@FeShik nanovaccines. In vitro examinations including GSH responsive, •OH generation, colloid stability, cellular uptake, cytotoxicity mechanism of ferroptosis and necroptosis, ICD effect, the promotion of DC maturation and antigen cross-presentation were studied. In vivo observations including pharmacokinetics and biodistribution, antitumor effect, abscopal effect, immune memory effect, and biosafety were performed.

Results: The presence of FeShik nanomedicines can significantly prolong the blood circulation time of antigens, increasing the bioavailability of antigens. Upon phagocytosis by tumor cells, FeShik nanomedicines can disassemble into Fe2+ and Shikonin in response to tumor microenvironments, leading to ICD of tumor cells via ferroptosis and necroptosis. Consequently, ICD-released autologous tumor cell lysates and pro-inflammatory cytokines not only stimulate DC maturation and antigen cross-presentation, but also promote macrophage repolarization and cytotoxic T lymphocyte infiltration, resulting in the activation of adaptive immune responses toward solid tumors.

Conclusion: In a word, our FeShik supramolecular nanomedicines integrate bioactivities of ICD stimulants and immunoadjuvants, such as eradicating tumor cells, activating antitumor immune responses, modulating immunosuppressive tumor microenvironments, and biodegradation after immunotherapy. Encouraged by the diversity of polyphenols and metal ions, our research may provide a valuable paradigm to establish a large library for tumor vaccination.

Keywords: shikonin, metal-phenolic networks, nanomedicines, immunogenic cell death, immunoadjuvants, tumor nanovaccines

Methods
Chemicals and Reagents
Shikonin was purchased from Chengdu Gelipu Biotechnology Co., Ltd. Ovalbumin (OVA) and IR780 were obtained from Sigma-Aldrich. Iron chloride hexahydrate (FeCl3·6H2O), Glutathione (GSH), 1,10-phenanthroline, and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) were purchased from Aladdin. FITC was obtained from Solarbio. Hydrogen peroxide (H2O2) was obtained from Beijing Chemical Works. Disodium terephthalate (TPA) was bought from Alfa Aesar. Every Green Fetal bovine serum (FBS) was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. PBS, RMPI-1640 basic, and DMEM were purchased from Gibco. Cell counting kit-8 (CCK-8) was obtained from Bimake. Hoechst 33342, Lyso-Tracker Red, Hanks' Balanced Salt Solution, Reactive oxygen species assay kit, ATP assay kit, Coomassie Blue Staining Kit, and BCA protein assay kit were obtained from Beyotime. Ferrostatin-1 (Fer-1), necrostatin-1 (Nec-1), AC-DEVD-CHO (APO), and deferoxamine (DFO) were purchased from Selleck Chemicals. FerroOrange was obtained from Dojindo. GSH assay kit was bought from Nanjing Jiancheng. Anti-Glutathione Peroxidase 4 (GPX4) antibody, anti-receptor-interacting serine/threonine-protein kinase 1 (RIP1) antibody, anti-Calreticulin (CRT) antibody, and anti-HMGB1 antibody were purchased from Abcam. Anti-RIP3 antibody was purchased from Proteintech. GPX4, RIP1, RIP3, tumor necrosis factor α (TNF-α), interferon-γ (IFN-γ) ELISA assay kit were bought from Spbio. BODIPY581/591-C11 was bought from Thermo Fisher. Goat Anti-rabbit IgG H&L/FITC antibody was bought from Bioss. Annexin-V-FITC apoptosis analysis kit was purchased from Sungene Biotech. Anti-CD11c-FITC, Anti-CD80-PE, CD86-PE-Cyanine5, OVA (SIINFEKL) peptide bound to H-2Kb antibody-PE, MHC-I-PE, CD3-FITC, CD4-PE, CD8a-PE-Cyanine5, Foxp3-PE-Cyanine5, F4/80-FITC, CD206-PE, CD62L-APC, and CD44-PE were purchased from eBioscience. Interleukin-6 (IL-6) ELISA assay kit was purchased from Shanghai Tongwei Biotechnology Co., Ltd. Bouin Fixative Solution was purchased from XinFan Bio-technology Co., Ltd. 4% Paraformaldehyde (PFA) solution was purchased from Biosharp.