【Adv.Mater.】国科大温州研究院沈建良|传统声动力治疗遭遇“免疫逃逸”？新一代自调节纳米增敏剂实现“一箭三雕”，肿瘤消退率超85%#

SCHEME 1 Schematic illustration of porphyrin-biguanide-loaded albumin nanoparticles (POR-BG@Alb)-mediated synergistic sonodynamic immunotherapy. To address the immune escape faced by some sonodynamic therapy (SDT) sensitizer-assisted SDT, a porphyrin-biguanide-loaded albumin nanoparticles POR-BG@Alb nanosystem was developed. Apart from enhancing reactive oxygen species (ROS) generation capacity of SDT by reversing tumor hypoxia and decreasing energy level, POR-BG@Alb nanosystem also avoided innate and adaptive immune resistance via affecting the mitochondria/AMPK/c-MYC axis. By doing this, POR-BG@Alb enhanced T-cell cytotoxicity and macrophage phagocytosis. In preclinical models, this nanosystem suppressed the growth of primary tumor and abscopal tumor growth, as well as slowed tumor metastasis and induced long-term immune memory for durable antitumor efficacy.#

FIGURE 1 Rational design of the novel self-oxygen regulation and self-immune regulation sonosensitizer POR-BG. (A) Schematic structures and functional comparison of conventional organic sonosensitizers (ICG, PCN, and POR) and the highly efficient sonosensitizer POR-BG. (B) Schematic singlet/triplet energy level structures and corresponding transition pathways of POR and POR-BG. (C) UV–Vis absorption spectra of POR and POR-BG. (D) Electron cloud distributions of frontier molecular orbitals (HOMO/LUMO) and schematic of corresponding energy levels for POR and POR-BG, with the HOMO-LUMO energy gap labeled. (E) Comparative analysis of dipole moment change (Δµ) and charge transfer index (D index) of POR and POR-BG based on DFT calculations. (F) Electron-hole distributions and calculated excited-state parameter (Hct) of POR and POR-BG, showing changes in charge transfer capacity after BG modification. (G) Time-dependent curves of singlet oxygen (1O2) probe signals at 529 nm for different treatment groups (PBS, POR, POR-BG) under ultrasound irradiation. (H) Time-dependent absorption curves of superoxide anion (O2•−) probe at 420 nm for different treatment groups under ultrasound irradiation. (I) Western blot analysis of HIF-1α and GLUT-1 protein expression levels in cells after treatment with different concentrations of POR-BG. (J) Western blot analysis of AMPK phosphorylation level (p-AMPK) and total AMPK protein expression in cells after treatment with different concentrations of POR-BG. (K) Effects of different treatment groups on the expression of CD47, PD-L1, and c-MYC in the presence or absence of the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC). (L) Flow cytometry analysis of intracellular ROS levels induced by different sonosensitizers under ultrasound stimulation. (M) Regulatory effects of POR-BG on the protein expression of CD47, PD-L1, and c-MYC by using AMPKα1/α2 knockdown tumor cells. (N) Western blot analysis of the regulatory effects of POR-BG on the expression of CD47, PD-L1, and c-MYC by using c-MYC overexpression tumor cells. (O) Schematic illustrating that POR-BG integrates oxygen self-regulation capability, high quantum yield, and immune self-regulation function compared with conventional sonosensitizers.#

在细胞层面的机制探究中，团队发现该纳米系统展现出了独特的亚细胞定位特性。共聚焦显微成像显示该系统能特异性靶向细胞内的线粒体，其皮尔逊相关系数高达0.961。通过蛋白质组学测序、蛋白印迹及透射电镜观察，超声激活的纳米系统引发了显著的线粒体肿胀及嵴断裂，不仅破坏了其膜电位，更抑制了线粒体呼吸作用使胞内ATP产量骤降。这种对线粒体能量代谢的抑制能够被动减少细胞耗氧，从而在固有的缺氧环境中实现自供氧调节。最核心的免疫重塑实验表明，该系统通过激活线粒体/AMPK/c-MYC信号通路，在生成大量ROS触发免疫原性细胞死的同时，逆转了传统声动力导致的免疫耐受，显著下调了肿瘤表面的PD-L1与CD47表达。在共培养体系中，巨噬细胞对肿瘤细胞的吞噬效率从4.43%飙升至20.03%，同时显着增强了T细胞介导的细胞毒性杀伤效应。

FIGURE 2 Preparation and characterization of POR-BG@Alb nanosystem. (A) Schematic of the fabrication process for POR-BG@Alb nanosystem. (B) Schematic of molecular docking results and corresponding binding energy between POR-BG and albumin. (C) Transmission electron microscope (TEM) image of POR-BG@Alb nanoparticles. (D) Fluorescence emission spectra of POR-BG under different methanol volume fractions. (E) Fluorescence changes of POR-BG at different Alb concentrations. (F) Changes in fluorescence intensity of POR-BG@Alb after co-incubation with various Alb-binding inhibitors to analyze its binding mode with Alb. (G) SDS-PAGE analysis results of POR-BG@Alb to demonstrate the formation of a stable complex with Alb Sudlow’s Site I. (H) Two-dimensional principal component analysis (PCA) and Gibbs free energy distribution of POR-BG and Alb to evaluate conformational stability. (I) UV–Vis absorption spectra of POR-BG and POR-BG@Alb. (J) Hydrodynamic diameter distribution of POR-BG@Alb. (K) Zeta potential of POR-BG@Alb and POR@Alb. (L) Stability of POR-BG@Alb under different media and temperature conditions. (M) Release behavior of POR-BG under different pH conditions. (N) Absorption spectral changes of ultrasound-triggered ROS generation. (O) Kinetic analysis of ultrasound-induced ROS generation under different treatment conditions. (P) Optical stability of POR-BG@Alb after multiple ultrasound cycles. (Q) Particle size distribution of POR-BG@Alb. (R) Effects of different concentrations of POR-BG@Alb on the hemolysis rate of red blood cells (RBCs). (S) In vivo fluorescence imaging and major organ distribution of POR-BG@Alb in mice.#

FIGURE 3 Effects of POR-BG@Alb-assisted SDT in vitro. (A) GO biological process enrichment analysis of differentially expressed genes identified by proteomics in the control and POR-BG@Alb groups. (B–E) GSEA analysis of the changes in pathways in the control and POR-BG@Alb groups. (F) Fluorescence imaging of intracellular JC-1 under different treatments. (G) TEM images of ultrastructural changes in cells after POR-BG@Alb treatment. (H) Schematic of the regulatory mechanism underlying mitochondrial function and oxygen metabolism. (I) Co-localization analysis of POR-BG@Alb with mitochondria in cells. (J) Quantitative analysis of intracellular ATP levels after different treatments. (K) Fluorescence imaging of ROS in MB49 and 4T1 cells under US irradiation. (L) Analysis of singlet oxygen generation using the SOSG probe. (M) Detection of superoxide anion generated by the DHE probe. (N) Annexin V/PI staining for the analysis of the proportion of apoptotic cells. (O) Colony formation assay to evaluate long-term cell proliferative capacity. (P) CCK-8 curves of MB49 cells treated with POR-BG@Alb assisted SDT. (Q) γH2AX immunofluorescence staining for analyzing DNA damage levels.#

随后在小鼠体内进行的治疗靶向性及多模型评估进一步验证了其临床转化潜力。体内荧光成像证实，借助白蛋白的优异靶向性，药物在注射12小时后高效富集于肿瘤部位。在原位膀胱癌模型中，该疗法展现出最浅的肿瘤浸润深度和最小的膀胱肿块重量，且完全避免了临床联用双特异性抗体带来的系统性溶血等血液毒性。在双侧肿瘤模型及肿瘤再挑战实验中，该纳米系统介导的声动力治疗不仅高效清除了原发灶，更诱导了强效的远端效应，使未受超声辐照的远端肿瘤受到显着抑制，远端肿瘤抑制率超过85%。在高度恶性的三阴性乳腺癌模型中，治疗同样显著逆转了肿瘤的表皮-间质转化进程，大幅减少了肺部转移结节的数量。生存期分析显示，该方案成功激活了长效的免疫记忆，将小鼠的中位生存期从对照组的17天大幅延长至44天，有效防止了肿瘤的复发与扩散。

FIGURE 4 POR-BG@Alb co-inhibited PD-L1/CD47 to avoid immune resistance. (A) Detection of the mRNA expression levels of PD-L1 and CD47 after different treatments. (B) Western blot analysis of the AMPK signaling pathway and PD-L1/CD47 protein expression. (C, D) Flow cytometry analysis of the surface expression of PD-L1 and CD47. (E,F) Heatmap analysis of inflammatory cytokines and immune-related gene expression in T cells and macrophages. (G) Imaging of phagocytic behavior in the macrophage-tumor cell co-culture system. (H) Flow cytometric analysis of macrophage phagocytic efficiency under different treatment conditions. (I) Analysis of T cell cytotoxic capacity in the T24 cell-T cell co-culture system. (J) Schematic of the mechanism underlying PD-L1/CD47-mediated immune escape and its reversal. (K) Western blot analysis of PD-L1/CD47 protein expression in different tissues. (L) Quantitative analysis of PD-L1/CD47 expression in tissues.#

FIGURE 5 Therapeutic efficacy of POR-BG@Alb-mediated sonodynamic immunotherapy in orthotopic bladder cancer and subcutaneous tumor-bearing models. (A) Schematic of the establishment and treatment protocol for the orthotopic bladder cancer model. (B) Intraoperative tumor images, three-dimensional reconstruction, ultrasound imaging, and H&E staining after different treatments. (C) Tumor volume curves after different treatments. (D) Quantitative analysis of bladder weight. (E) Western blot analysis of PD-L1 and CD47 protein expression in tumor tissues. (F) Immunofluorescence staining of CD3+, CD4+, and CD8+ T cells in tumor tissues. (G) Schematic of the tumor re-challenge experiment protocol. (H) Photographs of primary MB49 tumors. (I,J) Primary tumor volume and weight changes. (K,L) Flow cytometric analysis of infiltrating T cell subsets in primary tumor tissues. (M–O) Photographs, volume, and weight changes of tumors in the re-challenge model. (P,Q) Tumor growth in the abscopal tumor model. (R,S) Survival analysis.#

FIGURE 6 POR-BG@Alb induced systemic anti-tumor immunity and distant metastasis. (A) Schematic of the 4T1 tumor model and treatment protocol. (B,C) Protein expression of CD47, PD-L1, Ki67, and Bcl-2 in tumor tissues from different groups. (D,E) Flow cytometric analysis of tumor-infiltrating T cell subsets. (F-H) Quantification of CD3+, CD4+, and CD8+ T cell proportions. (I,J) Immunohistochemical and apoptosis staining analysis of tumor tissues. (K–M) Primary tumor volume, weight, and photographs. (N) Analysis of EMT-related protein expression. (O,P) Photographs and quantitative analysis of lung metastatic nodules. (Q–S) Evaluation of anti-tumor efficacy in the tumor re-challenge model. (T,U) Evaluation of antitumor efficacy in the distant tumor model.#