超声协同靶向纳米粒抑制乏氧肺癌细胞增殖、迁移及侵袭

doi:10.12122/j.issn.1673-4254.2026.06.08

摘要/Abstract

摘要：

目的针对肿瘤微环境构建一种能同时负载阿霉素（DOX）与过氧化氢酶（CAT）的AS1411适配体修饰的介孔聚多巴胺纳米颗粒（AS1411-D/C-MPDA），并探讨其联合超声对Lewis肺癌（LLC）细胞的协同治疗效果。方法通过一步组装法合成负载DOX和CAT并经AS1411适配体修饰的AS1411-D/C-MPDA，检测其物理特性；在细胞水平，将LLC细胞分为游离DOX组、DOX-MPDA组及AS1411-D/C-MPDA组及对应的超声组进行处理。超声参数设置为：频率1.0 MHz，强度1.0 W/cm²，持续时间3 min。通过CCK-8法检测细胞活性，流式细胞术（Annexin V-FITC/PI染色）分析细胞凋亡，划痕实验评估细胞迁移能力，以及Transwell实验评估细胞侵袭能力。结果成功制备AS1411-D/C-MPDA纳米材料，其呈大小均匀的球形颗粒，粒径为183.36±15.99 nm、包封率DOX（91.17±0.08）%，CAT（29.59±0.2）%，具有pH响应性，能裂解并促进药物释放，DOX释药率为（81.25±1.61）%，该纳米材料有较强的产氧能力，缓解了缺氧状态。AS1411修饰的纳米材料对LLC细胞的摄取显著增强。与游离DOX和DOX-MPDA联合超声相比，AS1411-D/C-MPDA联合超声能诱导LLC细胞产生更多的活性氧，从而诱导细胞凋亡，细胞活性明显降低、划痕愈合面积减小（0.097±0.010 mm²vs 0.278±0.001 mm²，P<0.01）及侵袭细胞数量减少（24.33±12.01个vs 352±4.36个，P<0.001）。结论 AS1411-D/C-MPDA联合超声通过靶向递送抗癌药物，有望成为物理声学、药物化学、微环境调控协同治疗肺癌的新策略。

关键词: 肺癌, 声动力疗法, 多巴胺, 纳米药物, 主动靶向

Abstract:

Objective To construct AS1411 aptamer-modified mesoporous polydopamine (MPDA) nanoparticles co-loaded with doxorubicin (DOX) and catalase (AS1411-D/C-MPDA nanoparticles) targeting the tumor microenvironment and evaluate their efficacy in synergy with ultrasound for inhibiting Lewis lung carcinoma (LLC) cells. Methods AS1411-D/C-MPDA nanoparticles were synthesized using a one-step assembly method, and their physicochemical properties were characterized. Cultured LLC cells were treated with free DOX, DOX-loaded MPDA nanoparticles or AS1411-D/C-MPDA nanoparticles with or without ultrasound exposures (frequency 1.0 MHz and intensity 1.0 W/cm²) for 3 min. The changes in viability, apoptosis, and migration and invasion abilities of the cells were assessed using CCK-8 assay, flow cytometry (Annexin V-FITC/PI staining), wound healing assay, and Transwell assay, respectively. Results The prepared AS1411-D/C-MPDA nanomaterials showed a uniform spherical morphology, a mean particle size of 183.36±15.99 nm, and DOX and catalase encapsulation efficiencies of (91.17±0.08)% and (29.59±0.2)%, respectively. The nanoparticles demonstrated good pH responsiveness to enable disintegration for promoting drug release, with a cumulative DOX release rate of (81.25±1.61)%. The nanoparticles possessed strong oxygen-generating capacity to alleviate cell hypoxia. AS1411-modified nanoparticles showed significantly enhanced cellular uptake by LLC cells. Compared with free DOX and DOX-MPDA nanoparticles combined with ultrasound, AS1411-D/C-MPDA in synergy with ultrasound induced higher levels of ROS in LLC cells, resulted in a higher cell apoptosis rate, and more efficiently reduced cell viability and suppressed cell migration and invasion. Conclusion AS1411-D/C-MPDA nanoparticles combined with ultrasound allow targeted delivery of anticancer drugs and represent a promising strategy for synergistic treatment of lung cancer by integrating physical acoustics, pharmaceutical chemistry, and tumor microenvironment regulation.

Key words: lung cancer, sonodynamic therapy, mesoporous polydopamine, nanodrug, active targeting

董俊余, 郭佳俊, 李岱容, 杜永洪. 超声协同靶向纳米粒抑制乏氧肺癌细胞增殖、迁移及侵袭[J]. 南方医科大学学报, 2026, 46(6): 1278-1289.

Junyu DONG, Jiajun GUO, Dairong LI, Yonghong DU. Ultrasound-synergized targeted nanoparticles suppress proliferation, migration and invasion of hypoxic lung cancer cells in vitro[J]. Journal of Southern Medical University, 2026, 46(6): 1278-1289.

图/表 11

图1 AS1411-DOX/CAT-MPDA的制备示意图

Fig.1 Schematic diagram of the preparation of AS1411-DOX/CAT-MPDA nanoparticles.

图2 透射电镜图及DOX、CAT和AS1411-D/C-MPDA的紫外光谱图

Fig.2 Transmission electron microscopy and UV-Vis spectra of DOX, CAT, and AS1411-D/C-MPDA. A: Transmission electron microscopy of AS1411-D/C-MPDA nanoparticles (Original magnification: ×18 500, scale bar=200 nm). B: Transmission electron microscopy of AS1411-D/C-MPDA nanoparticles at pH5.5 (×6800, scale bar=600 nm). C: UV-Vis spectra of DOX, CAT, and AS1411-D/C-MPDA nanoparticles (n=3).

图3 AS1411-D/C-MPDA具有良好稳定性和pH响应性

Fig.3 AS1411-D/C-MPDA exhibits excellent stability and pH responsiveness. A: Average hydrodynamic size of AS1411-D/C-MPDA nanoparticles. B: Size changes of AS1411-D/C-MPDA nanoparticles after treatment with PBS (pH7.4), PBS (pH5.5), or 10% FBS. C: Average zeta potential of AS1411-D/C-MPDA nanoparticles (n=3).

图5 CLSM显示AS1411-D/C-MPDA的体外肿瘤靶向性

Fig.5 CLSM images showing in vitro tumor-targeting capability of AS1411-D/C-MPDA nanoparticles (×60).

图6 CLSM显示LLC细胞摄取AS1411-D/C-MPDA

Fig.6 CLSM images showing the cellular uptake of AS1411-D/C-MPDA nanoparticles by LLC cells (×60).

图7 不同方式处理24、48、72 h后16HBE的相对细胞活性

Fig.7 Relative viability of 16HBE cells after treatment with different formulations for 24, 48 and 72 h (n=3). **P<0.01, ***P<0.001.

图8 CCK-8测定LLC细胞活性、流式检测LLC细胞周期分布

Fig.8 Assessment of viability and cell cycle distribution of LLC cells using CCK-8 assay and flow cytometry. A: Relative viability of LLC cells after 24 h of treatment with different formulations. B: Cell cycle distribution of LLC cells after 24 h of treatment with different formulations. *P<0.05, **P<0.01, ***P<0.001 (n=3).

图9 CLSM分析AS1411-D/C-MPDA联合超声对LLC细胞活死状态的影响,流式细胞术检测各组LLC细胞的凋亡率

Fig.9 CLSM images showing effect of AS1411-D/C-MPDA nanoparticles combined with ultrasound on viability and apoptosis of LLC cells. A: Viability of LLC cells (×20). B: Apoptosis rates of LLC cells in different treatment groups detected by flow cytometry. C: Statistical results of apoptosis rates in each treatment group (n=3). *P<0.05, ***P<0.001.

图10 CLSM显示各组LLC细胞内活性氧水平

Fig.10 CLSM images showing intracellular ROS levels in LLC cells in each group (×20).

图11 划痕实验评价各组LLC细胞的迁移能力及其数据定量分析

Fig.11 Wound healing assay for evaluating migration ability of LLC cells in different treatment groups (×20). ***P<0.001 (n=3).

图12 Transwell小室实验评价各组LLC细胞的侵袭能力

Fig.12 Transwell chamber assay for evaluating invasion ability of LLC cells in different treatment groups (×20). ***P<0.001 vs DOX (n=3).

参考文献 30

[1]	Chen J, Luo HL, Liu Y, et al. Oxygen-self-produced nanoplatform for relieving hypoxia and breaking resistance to sonodynamic treatment of pancreatic cancer[J]. ACS Nano, 2017, 11(12): 12849-62. doi：10.1021/acsnano.7b08225
[2]	Siegel RL, Kratzer TB, Giaquinto AN, et al. Cancer statistics, 2025[J]. CA Cancer J Clin, 2025, 75(1): 10-45. doi：10.3322/caac.21871
[3]	Kuerban K, Gao XW, Zhang H, et al. Doxorubicin-loaded bacterial outer-membrane vesicles exert enhanced anti-tumor efficacy in non-small-cell lung cancer[J]. Acta Pharm Sin B, 2020, 10(8): 1534-48. doi：10.1016/j.apsb.2020.02.002
[4]	Dai ZD, Song XZ, Cao JK, et al. Dual-stimuli-responsive TiOx/DOX nanodrug system for lung cancer synergistic therapy[J]. RSC Adv, 2018, 8(39): 21975-84. doi：10.1039/c8ra02899k
[5]	Bhatia SN, Chen XY, Dobrovolskaia MA, et al. Cancer nanomedicine[J]. Nat Rev Cancer, 2022, 22(10): 550-6. doi：10.1038/s41568-022-00496-9
[6]	赵雅洁. 几种用于化学动力学及其相关联合疗法抗肿瘤纳米催化剂的设计和应用[D].合肥:中国科学技术大学, 2023.
[7]	Liang S, Deng XR, Ma PA, et al. Recent advances in nanomaterial-assisted combinational sonodynamic cancer therapy[J]. Adv Mater, 2020, 32(47): 2003214. doi：10.1002/adma.202003214
[8]	Jia L, Li X, Liu H, et al. Ultrasound-enhanced precision tumor theranostics using cell membrane-coated and pH-responsive nanoclusters assembled from ultrasmall iron oxide nanoparticles[J]. Nano Today, 2021, 36: 101022. doi：10.1016/j.nantod.2020.101022
[9]	Wang JJ, Wang XY, Lu SY, et al. Integration of cascade delivery and tumor hypoxia modulating capacities in core-releasable satellite nanovehicles to enhance tumor chemotherapy[J]. Biomaterials, 2019, 223: 119465. doi：10.1016/j.biomaterials.2019.119465
[10]	何雨初.刺激响应性纳米药物系统强化体内传递与抗肿瘤研究[D]. 河北: 燕山大学, 2021.
[11]	He YC, Cong C, Liu ZW, et al. Stealth surface driven accumulation of "Trojan Horse" for tumor hypoxia relief in combination with targeted cancer therapy[J]. Chem Eng J, 2019, 378: 122252. doi：10.1016/j.cej.2019.122252
[12]	Mei L, Zhu GZ, Qiu LP, et al. Self-assembled multifunctional DNA nanoflowers for the circumvention of multidrug resistance in targeted anticancer drug delivery[J]. Nano Res, 2015, 8(11): 3447-60. doi：10.1007/s12274-015-0841-8
[13]	Peng L, Hung CT, Wang SW, et al. Versatile nanoemulsion assembly approach to synthesize functional mesoporous carbon nanospheres with tunable pore sizes and architectures[J]. J Am Chem Soc, 2019, 141(17): 7073-80. doi：10.1021/jacs.9b02091
[14]	Rizvi SAA, Saleh AM. Applications of nanoparticle systems in drug delivery technology[J]. Saudi Pharm J, 2018, 26(1): 64-70. doi：10.1016/j.jsps.2017.10.012
[15]	Ren DD, Williams GR, Zhang YY, et al. Mesoporous doxorubicin-loaded polydopamine nanoparticles coated with a platelet membrane suppress tumor growth in a murine model of human breast cancer[J]. ACS Appl Bio Mater, 2022, 5(1): 123-33. doi：10.1021/acsabm.1c00926
[16]	Li L, Chen YS, Chen WJ, et al. Photodynamic therapy based on organic small molecular fluorescent dyes[J]. Chin Chemical Lett, 2019, 30(10): 1689-703. doi：10.1016/j.cclet.2019.04.017
[17]	Gao YT, Zhou H, Liu GG, et al. Tumor microenvironment: lactic acid promotes tumor development[J]. J Immunol Res, 2022, 2022: 3119375. doi：10.1155/2022/3119375
[18]	王朝阳. 基于介孔聚多巴胺的肿瘤微环境响应型纳米平台构建及增强的肿瘤治疗[D].保定: 河北大学, 2023.
[19]	Bie LH, Wang Y, Jiang FZ, et al. Insights into the binding mode of AS1411 aptamer to nucleolin[J]. Front Mol Biosci, 2022, 9: 1025313. doi：10.3389/fmolb.2022.1025313
[20]	Keizer HG, Pinedo HM, Schuurhuis GJ, et al. Doxorubicin (adriamycin): a critical review of free radical-dependent mechanisms of cytotoxicity[J]. Pharmacol Ther, 1990, 47(2): 219-31. doi：10.1016/0163-7258(90)90088-J
[21]	Cheng JJ, Zhu Y, Xing X, et al. Manganese-deposited iron oxide promotes tumor-responsive ferroptosis that synergizes the apoptosis of cisplatin[J]. Theranostics, 2021, 11(11): 5418-29. doi：10.7150/thno.53346
[22]	He YX, Liu SH, Yin J, et al. Sonodynamic and chemodynamic therapy based on organic/organometallic sensitizers[J]. Coord Chem Rev, 2021, 429: 213610. doi：10.1016/j.ccr.2020.213610
[23]	Cetindere T, Nambiar S, Santourlidis S, et al. Induction of indoleamine 2, 3-dioxygenase by death receptor activation contributes to apoptosis of melanoma cells via mitochondrial damage-dependent ROS accumulation[J]. Cell Signal, 2010, 22(2): 197-211. doi：10.1016/j.cellsig.2009.09.013
[24]	Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release[J]. Physiol Rev, 2014, 94(3): 909-50. doi：10.1152/physrev.00026.2013
[25]	Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro [J]. Nat Protoc, 2007, 2(2): 329-33. doi：10.1038/nprot.2007.30
[26]	Justus CR, Leffler N, Ruiz-Echevarria M, et al. In vitro cell migration and invasion assays[J]. JoVE, 2014(88): 51046. doi：10.3791/51046
[27]	Zhou YF. Application of acoustic droplet vaporization in ultrasound therapy[J]. J Ther Ultrasound, 2015, 3(1): 20. doi：10.1186/s40349-015-0041-8
[28]	Skandalis SS, Karalis TT, Chatzopoulos A, et al. Hyaluronan-CD44 axis orchestrates cancer stem cell functions[J]. Cell Signal, 2019, 63: 109377. doi：10.1016/j.cellsig.2019.109377
[29]	Cao YC, Shen SF, Xiang JH, et al. Sonodynamic therapy-based DNA nanocarriers with hypoxia-inducible factor-1α silencing activation for precision lung cancer therapy[J]. Biomater Res, 2025, 29: 230. doi：10.34133/bmr.0230
[30]	Pulley JM, Rhoads JP, Jerome RN, et al. Using what we already have: uncovering new drug repurposing strategies in existing omics data[J]. Annu Rev Pharmacol Toxicol, 2020, 60: 333-52. doi：10.1146/annurev-pharmtox-010919-023537