【ACS Nano】东北林业大学赵修华|硒桥联纳米粒突破p53突变胃癌耐药：277.2 nm粒径实现高效抑瘤与毒性翻倍降低#

Figure 1. Synthesis of dimeric prodrugs and NPs, redox-responsive drug release, and apoptotic pathway induction.#

Figure 2. Physicochemical characterization of SSCPT NPs and SSeSCPT NPs. (A) Chemical structures of the dimeric prodrugs. (B) Hydrodynamic size distribution measured by dynamic light scattering (DLS). (C) Polydispersity index (PDI) distribution. (D) Zeta potential. (E) Transmission electron microscopy (TEM) image (scale bar: 100 nm). (F) Long-term storage stability: size changes over 120 days at 4 °C. Stability in biological media: size changes after 48 h incubation at 37 °C in PBS containing 10% fetal bovine serum (FBS) (G) and in 5% glucose solution (H). In vitro cumulative release profiles in simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 6.8): (I) SSCPT NPs and (J) SSeSCPT NPs. All data are presented as mean ± standard deviation (n = 3 independent experiments).#

Figure 3. Assembly behavior and colloidal stability of SSCPT NPs and SSeSCPT NPs. (A) Schematic illustration of the self-assembly of prodrug molecules into nanoparticles from molecular dynamics simulations (0–50 ns). (B) Time evolution of van der Waals and electrostatic interaction energies, and the calculated binding free energy (Δ_G_) during assembly. (C) Hydrodynamic diameter changes of both nanoparticles over 24 h in 150 mM NaCl or 0.2% (w/v) SDS. (D, E) Time-resolved UV–vis absorption spectra of SSCPT NPs (D) and SSeSCPT NPs (E) in 0.2% SDS over 24 h. (F, G) UV–vis absorption spectra of SSCPT NPs (F) and SSeSCPT NPs (G) in NaCl solutions (0–1.0 M). (H) Absorption spectra of CPT standard in DMSO and the two nanoparticles dispersions (equivalent CPT loading). (I) Absorption spectra of CPT standards in DMSO/H2O mixtures with DMSO volume fractions from 0% to 100%. Data are mean ± SD (n = 3 independent experiments).#

Figure 4. Cumulative release of camptothecin (CPT) from SSCPT NPs (A, C) and SSeSCPT NPs (B, D) in the presence of varying concentrations of dithiothreitol (DTT; A, B) or hydrogen peroxide (H2O2; C, D). (E) Schematic illustration of the oxidative degradation pathway of the nanoparticles. Data are presented as mean ± SD (n = 3 independent experiments).#

在响应性释放方面，如图4所示，在肿瘤微环境高浓度谷胱甘肽和活性氧的双重刺激下，SSeSCPT NPs中的硒硫键发生断裂并迅速释放活性喜树碱与氧化还原活性硒物种，而在正常生理环境下则保持极低的药物泄露。如图5所示，细胞实验表明，SSeSCPT NPs对p53突变的HGC-27胃癌细胞表现出显著增强的摄取效率与选择性细胞毒性。机制研究发现，肿瘤细胞表面高表达的TLR4受体能够特异性识别纳米颗粒表面的硒基团，介导网格蛋白及网状小窝依赖性胞吞作用。这一主动靶向机制使得277.2 nm的大粒径纳米颗粒能够高效穿越细胞膜，成功克服了传统增强渗透与滞留效应对小粒径的苛刻要求。

Figure 5. Quantitative cellular uptake of CPT, SSCPT NPs, and SSeSCPT NPs in L929, GES-1, AGS, and HGC-27 cells at (A) 1 h and (B) 4 h post-treatment. (C) Cellular uptake of SSeSCPT NPs after treatment with various endocytosis inhibitors. (D) Half-maximal inhibitory concentration (IC50) values of CPT, Na2SeO3, SSCPT NPs, and SSeSCPT NPs in the same cell lines after 24 h. (E) Quantitative analysis of intracellular reactive oxygen species (ROS) levels in AGS and HGC-27 cells following the indicated treatments. (F) Changes in intracellular glutathione (GSH) levels in AGS and (G) HGC-27 cells after identical treatments. (H) Cellular uptake of nanoparticles in antibody neutralization experiments. Data are presented as mean ± SD (n = 3 independent experiments). Data are presented as mean ± SD (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.#

Figure 6. Evaluation of DNA damage by γ-H2AX immunofluorescence in (A, B) AGS and (C, D) HGC-27 cells after treatment with CPT, SSCPT NPs, SSeSCPT NPs, or a high dose of CPT (50 μM, positive control). Analysis of mitochondrial membrane potential using JC-1 fluorescence in (E, F) AGS and (G, H) HGC-27 cells after treatment with CPT, SSCPT NPs, or SSeSCPT NPs. Data are presented as mean ± SD (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.#

Figure 7. Apoptosis rates in (A) AGS and (B) HGC-27 cells following treatment with drugs or pathway inhibitors. Semiquantitative Western blot analysis of (C) Bax/Bcl-2 ratio, (D) cleaved caspase-3 (C-Caspase-3), (E) p-p53, and (F) γ-H2AX in AGS cells. Corresponding analysis in HGC-27 cells: (G) Bax/Bcl-2 ratio, (H) C-Caspase-3, (I) p-p38, (J) γ-H2AX, and (K) CHOP. Data are presented as mean ± SD (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.#

如图6和图7所示，在p53突变的HGC-27细胞中，释放的硒物种引发了剧烈的活性氧爆发，显著消耗了胞内谷胱甘肽。硒介导的氧化应激不仅诱导了严重的DNA损伤与线粒体膜电位崩溃，还激活了p38 MAPK signaling 通路与内质网应激-UPR轴。这一“ROS-p38-DNA损伤”与“ROS-内质网应激-CHOP”的双轴致死网络，绕过了缺失的p53信号通路，直接激活下游Caspase级联反应，诱导p53非依赖性的细胞凋亡。如图8所示，体内药代动力学与抑瘤实验进一步证实，口服SSeSCPT NPs能够显著延长喜树碱的血浆半衰期，并在小鼠胃癌异种移植模型中展现出强效的肿瘤生长抑制作用。同时，外周血象与脏器组织学检查表明，硒元素的协同作用有效减轻了喜树碱引起的骨髓抑制与肝肾毒性，表现出优异的安全窗口。

Figure 8. Pharmacokinetics and in vivo therapeutic responses. (A) Plasma concentration–time curves of CPT, SSCPT NPs, and SSeSCPT NPs after oral administration in mice. (B) Body weight changes and (C) tumor volume changes over 14 days in different treatment groups. (D) Survival rates of gastric cancer-bearing mice. Peripheral blood parameters on days 3, 7, and 14: (E) white blood cells (WBC), (F) red blood cells (RBC), (G) hemoglobin (HGB), (H) platelets (PLT), (I) lymphocyte percentage (Lym%), and (J) granulocyte percentage (Gran%). Serum biochemical markers: (K) alanine aminotransferase (ALT), (L) aspartate aminotransferase (AST), (M) alkaline phosphatase (ALP), (N) creatinine (CREA), and (O) uric acid (UA). Data are presented as mean ± SD (n = 6 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.#