肿瘤化疗效果受肿瘤异质性、耐药性等多因素的制约，同时肿瘤微环境中的免疫抑制细胞和细胞因子促进肿瘤的生长和转移，降低了化疗的有效性。临床治疗中，已逐渐形成以化疗+免疫治疗为主的多种疗法联合应用的局面。本课题开发了一种基于番荔辛（Squamocin，Squ）及MnO₂纳米粒的化疗-免疫治疗联合方案。其中，Squ抗肿瘤活性强、耐药性低，作为化疗药物；MnO₂纳米粒可消耗肿瘤微环境中高浓度的H⁺和GSH，改善肿瘤微环境的弱酸、乏氧特性，产生的Mn²⁺可进一步激活cGAS-STING信号通路，诱导Ⅰ型IFN的产生，作为免疫治疗手段。为了克服Squ水溶性差、毒性大，以及MnO₂纳米粒生理介质中稳定性差的问题，本课题拟以具有生物相容性、生物可降解性、安全性的多糖为载体，构建Squ及MnO₂纳米粒的递送平台，以适应静脉注射及口服给药，并分别在小鼠原位结肠癌和乳腺癌皮下瘤模型中验证纳米递送平台的药效。主要内容如下：

1. Squ@IN NPs + MnO₂@IN NPs口服给药抗结肠癌研究

以结肠降解材料菊粉多糖（Inulin，IN）为载体，以维生素E（Vitamin E，VE）为稳定剂，构建了装载番荔辛的纳米粒Squ@IN NPs（Squ:VE:IN = 0.5:5:10）及装载二氧化锰的MnO₂@IN NPs（MnO₂:IN = 1:2）纳米粒，这两种纳米粒的平均粒径分别为174.4 ± 5.021 nm及155.4 ± 3.301 nm，稳定性良好。体内抗肿瘤实验表明，PTX组、Squ@IN NPs组、MnO₂@IN NPs组、Squ@IN NPs + MnO₂@IN NPs组抑瘤率分别为33.0%、31.4%、61.9%、77.7%，除此，MnO₂@IN NPs组和Squ@IN NPs + MnO₂@IN NPs组小肠肿瘤结节显著低于生理盐水组，表明MnO₂@IN NPs在抑制肿瘤生长和小肠转移上发挥重要作用，同时，Squ@IN NPs与MnO₂@IN NPs显示出一定的协同作用。但研究发现，Squ@IN NPs口服给药毒副作用较强，在静脉注射给药0.3 mg/kg时，即可引起小鼠体重急剧下降，结合前期实验结果，认为Squ能加剧肠损伤，不适合口服给药，故转向静脉注射给药研究。

2. Squ@APS-IR820 NPs + MnO₂@APS-IR820 NPs静脉注射给药抗乳腺癌研究

以KMnO₄和无水乙醇为原料在室温条件下合成了MnO₂纳米粒，通过红外光谱法、X射线能谱分析确定了其化学组成。通过席夫碱反应和酰胺化反应在黄芪多糖上接枝了具有肿瘤靶向性近红外荧光探针IR820，合成了功能载体材料黄芪多糖(Astragalus polysaccharides, APS)-IR820（APS-IR820），以油酸（Oleic acid，OA）为稳定剂包封化疗药番荔辛制备得到纳米粒Squ@APS-IR820 NPs（Squ:OA:APS-IR820 = 0.2:1:8），粒径为220.1 ± 11.16 nm，并制备了二氧化锰纳米粒MnO₂@APS-IR820 NPs（MnO₂:IN = 1:5），粒径为193.4 ± 1.701 nm；同时制备了没有IR820修饰的Squ@APS NPs和MnO₂@APS NPs作为对照。体外细胞摄取实验表明，APS-IR820的包封使4T1细胞对纳米粒的摄取增强；体内组织分布实验中，与Squ@APS相比，Squ@APS-IR820 NPs具有更强的肿瘤靶向性，相对肿瘤靶向指数为0.54，约是Squ@APS NPs的5倍。体内药效学实验表明，Squ@APS-IR820 NPs与MnO₂@APS-IR820 NPs具有协同抗肿瘤作用，体积抑瘤率为91.9%。同时，该给药方案显著促进BMDC成熟和BMDM重极化为抗肿瘤的M1型，并显著诱导脾中DC细胞成熟、CD8⁺ T细胞分化及活化，B细胞增殖，增强肿瘤中的CD8⁺ T细胞浸润，降低MDSC比例，从而激发先天性免疫及适应性免疫，解除免疫抑制。

综上所述，本课题基于多糖和纳米技术构建的Squ和 MnO₂的化疗免疫治疗纳米平台，可满足静脉注射及口服给药的要求，在极低剂量的番荔辛用量下，实现了对肿瘤的高效杀伤作用，为肿瘤治疗提供了新的选项。

The anti-tumor efficacy of chemotherapy is limited by multiple factors, such as tumor heterogeneity and drug resistance. Meanwhile, immune suppressive cells and cytokines in the tumor microenvironment promote tumor growth and metastasis, which attenuate the effectiveness of chemotherapy. In clinical practice, a combination of chemotherapy and immunotherapy has gradually become the mainstream. This study has developed a combined chemo-immunotherapy based on squamocin (Squ) and MnO₂ nanoparticles. Squ has strong anti-tumor activity and low drug resistance, used as a chemotherapy agent. MnO₂ nanoparticles can respond to high concentrations of H⁺, GSH, and H₂O₂ in tumor microenvironment, improving the weakly acidic and hypoxic conditions of the tumor microenvironment. The generated Mn²⁺ can further activate the cGAS-STING signaling pathway and induce the production of type I IFN, serving as an immunotherapy approach. To overcome the poor water solubility and high toxicity of Squ, as well as the poor stability of MnO₂ nanoparticles in physiological media, polysaccharides were applied as carriers to deliver Squ and MnO₂ for its biocompatible, biodegradable, and safe properties, which is suitable for intravenous injection and oral administration. The anti-tumor efficacy of the delivery platform was verified in subcutaneous breast cancer mouse models and orthotopic colon cancer models. The main contents are as follows:

1. Synergistic anti-colorectal cancer effects of the oral administration of Squ@IN NPs+ MnO₂@IN NPs

MnO₂ nanoparticles were synthesized using KMnO₄ and anhydrous ethanol at room temperature. The chemical composition of MnO₂ was verified by infrared spectroscopy and X-ray energy spectrum analysis. Inulin, an enzyme triggered colon-specific material, was applied as a carrier to prepare Squ@IN NPs (Squ:VE:IN = 0.5:5:10) with VE as a stabilizer, and MnO₂@IN NPs (MnO₂:IN = 1:2) were also prepared. The average particle sizes of these two nanoparticles were 174.4 ± 5.021 nm and 155.4 ± 3.301 nm, respectively, with good stability. Anti-tumor efficacy was studied in orthotopic colon cancer mouse model. The tumor inhibition rates of the PTX group, Squ@IN group, MnO₂@IN group, and Squ@IN NPs+ MnO₂@IN NPs group were 33.0%, 31.4%, 61.9%, and 77.7%, respectively. Meanwhile, the number of intestinal tumor nodules in the MnO₂@IN NPs group and Squ@IN NPs + MnO₂@IN NPs group was significantly lower than that in the Saline group, indicating that MnO₂@IN plays an important role in inhibiting tumor growth and intestinal metastasis. At the same time, Squ@IN NPs and MnO₂@IN NPs showed a certain synergistic anti-tumor effect. However, oral administration of Squ@IN NPs exhibited strong toxic side effects, causing a sharp decrease in body weight at 0.3 mg/kg (i.v.) dose. Based on previous experimental results, it was concluded that Squ could aggravate intestinal damage and was not suitable for oral administration, so attention was turned to intravenous injection administration.

2. Synergistic anti-breast cancer effects of intravenous administration of Squ@APS-IR820 NPs +MnO₂@APS-IR820 NPs

APS-IR820, a functional carrier material, was synthesized via grafting tumor-targeting near-infrared fluorescent probe IR820 onto Astragalus polysaccharides (APS) through a two-step including Schiff base reaction and amidation reaction. APS-IR820 was used as a carrier to prepare Squ@APS-IR820 NPs (Squ: OA: APS-IR820 = 0.2:1:8, Size: 220.1 ± 11.16 nm) and MnO₂@APS-IR820 NPs (MnO₂:APS-IR820 = 1:5, Size: 193.4 ± 1.701 nm). Notably, oleic acid (OA) acted as a stabilizer in Squ@APS-IR820 NPs (Squ:OA:APS-IR820 = 0.2:1:8). At the same time, Squ@APS NPs and MnO₂@APS NPs were prepared as controls. In vitro cellular uptake experiments demonstrated that the encapsulation of APS-IR820 enhanced the uptake of nanoparticles by 4T1 cells. The stronger tumor targeting capability of Squ@APS-IR820 NPs, compared to Squ@APS NPs, was confirmed through in vivo distribution experiments, with a relative tumor targeting index of 0.54, approximately five times higher than that of Squ@APS NPs. A synergistic anti-tumor effect of Squ@APS-IR820 NPs and MnO₂@APS-IR820 NPs was demonstrated by in vivo pharmacodynamic experiments, with a tumor inhibition rate of 91.9%. Additionally, this drug delivery regimen significantly promoted the maturation of bone marrow-derived dendritic cells (BMDCs) and the re-polarization of (bone marrow-derived macrophages) BMDMs to the anti-tumor M1 type, and significantly induced the maturation of dendritic cells (DC) in the spleen, the differentiation and activation of CD8⁺ T cells, and the proliferation of B cells. It also enhanced the infiltration of CD8⁺ T cells in tumors and reduced the proportion of myeloid-derived suppressor cells (MDSCs), thereby stimulating innate and adaptive immunity and relieving immune suppression.

In summary, the chemo-immunotherapy nanoplatform for Squ and MnO₂ based on polysaccharides and nanotechnology developed in this study achieved highly efficient tumor killing at an extremely low dose of Squ, providing a new option for tumor treatment.