1. 核心主旨与研究背景#

胰腺癌是目前临床上最致命的恶性肿瘤之一，其五年生存率长期低于10%。导致这一现象的主要原因在于胰腺癌组织内部存在极其致密的纤维化基质以及高间质流体压力，构成了坚固的物理屏障，使得常规化疗药物或常规纳米颗粒仅能滞留在肿瘤外围，难以深入到肿瘤核心区域。此外，胰腺癌微环境呈现出高度缺氧和高抗氧化（如高谷胱甘肽）的特征，严重削弱了依赖氧气和活性氧成分的治疗效果。为了克服这些难题，研究团队设计并构建了一款名为rCeGLI@PM的仿生双驱动Janus非对称纳米机器人，通过主动运动打破物理屏障，并结合多模态协同治疗手段，实现了对胰腺癌的高效深度穿透与综合杀伤。

Scheme.1 Schematic Illustration of the Construction and Therapeutic Mechanism of rCeGLI@PM Nanorobots

2. 核心创新与结构设计#

该纳米机器人的核心创新在于其非对称Janus结构（即颗粒两侧具有不同的化学组成与功能）以及光热与气体联合驱动的自推进机制。研究人员首先制备了具有多种纳米酶活性的氧化铈（CeOx）纳米颗粒作为核心底座，并对其进行单侧表面环氧功能化改性。随后利用皮克林乳液法在熔融蜡滴界面实现非对称定位，在暴露的一侧接枝结合了L-精氨酸（L-Arg，一氧化氮前体） 与吲哚美辛/吲哚氰绿（ICG，光热与光动力试剂） 的聚酰胺-胺（PAMAM， generation 5）树枝状高分子（GLI模块）。最后，在纳米机器人表面裹上一层** panc02胰腺癌细胞膜（PM）**，赋予其同源肿瘤靶向伪装能力。

这种设计巧妙地利用了非对称结构产生的驱动力不平衡。在近红外光（808 nm）照射和肿瘤微环境化学刺激下，纳米机器人能够同时启动近红外引发的热泳作用与L-精氨酸分解产生的一氧化氮（NO）气体推进作用，使其在复杂微环境中由无规则布朗运动转变为高速定向游动，主动深入肿瘤内部。

Fig.1 (A) TEM image and (B) size distribution histogram of CeOx. (C) High-resolution XPS spectra of Ce 3d for CeOx. (D) SOD-like activities of CeOx at different concentrations. (E) CAT-like activities of CeOx at different concentrations. (F) POD-like activities of CeOx at pH 7.4, 6.5, and 5.5 with/without GSH. (G) Time-dependent GSH consumption behavior with CeOx. (H) TEM image of CeOx with GSH at different time points. (I) Schematic diagram of the multienzyme activities of CeOx. Data are shown as mean ± SD (n = 3). Fig.2 (A) 1H NMR spectrum of GL.NH2 and GLI. (B) UV–Vis spectra of CeOx, CeOx-G, ICG, GL.NH2, GLI, and rCeGLI. (C) Hydrodynamic size distribution of GL.NH2, GLI.NH2, and GLI. (D) Thermogravimetric analysis curves of CeOx, CeOx-G, and rCeGLI. (E) Hydrodynamic size and zeta potential of CeOx, CeOx-G, rCeGLI, and rCeGLI@PM. (F) SEM image of CeOx/solid wax droplet. (G) TEM image of rCeGLI. Red dashed lines outline the asymmetrically modified GLI regions on individual particles. (H) Thermal response variations of rCeGLI solutions at varying CeOx concentrations upon 808 nm laser exposure (1.0 W/cm2) during 10 min irradiation. (I) Recycling–heating curves of rCeGLI solution ([CeOx] = 1000 μg/mL) under 808 nm laser irradiation (1.0 W/cm2). Data are shown as mean ± SD (n = 3).

3. 驱动机制与深度穿透能力#

在运动分析测试中，传统的氧化铈纳米颗粒在溶液中仅表现出被动的布朗运动，而具有非对称结构的rCeGLI纳米机器人在微环境介质中展现出显著的自主定向运动能力。在存在双重刺激条件时，其自主推进速度可达到12.5 μm/s。这种高速自推进力使其能够突破致密细胞外基质的阻碍，深入肿瘤组织的坏死核心。

此外，光动力疗法产生的活性氧自由基与光热引发产生的一氧化氮在局部结合，可原位生成强氧化性的过硝酸盐（ONOO⁻）。这种活性氮成分能够有效降解肿瘤区域的细胞外基质，破坏其坚固的物理屏障，从而建立起“基质降解-渗透深化-积累增加”的正反馈循环，极大地提升了纳米颗粒在实体肿瘤内部的渗透深度与蓄积总量。

Fig.3 (A) Evaluation of motion performance of various nanorobots under different conditions by calculating MSD. (B) Motion trajectories of CeOx in water. (C) Motion trajectories of rCeGLI in 100 μM H2O2 solution. (D) Motion trajectories of rCeGI in water upon laser irradiation. (E) Motion trajectories of CeGLI in 100 μM H2O2 solution upon laser irradiation. (F) Motion trajectories of rCeGLI in 100 μM H2O2 solution upon laser irradiation. (G) SDS-PAGE protein analysis of Panc02 cell membrane (lane 1), rCeGLI@PM (lane 2), rCeGLI (lane 3), and protein marker (lane 4). (H) TEM image of rCeGLI@PM. (I) Hemolysis percentage and representative images of blood samples incubated with water (positive control), PBS (negative control), and rCeGLI@PM at different concentrations. Data are shown as mean ± SD (n = 3).

4. 级联催化与微环境调控机制#

除了物理层面的主动穿透，该纳米机器人还具备强大的肿瘤微环境调控与多模态协同治疗能力：

多重纳米酶级联催化：氧化铈（CeOx）表面兼具三价与四价铈离子，赋予其类似超氧化物歧化酶（SOD）、过氧化氢酶（CAT）以及过氧化物酶（POD）的多重活性。在肿瘤酸性微环境中，它能首先将超氧阴离子转化为过氧化氢，再将过氧化氢分解生成氧气，有效缓解肿瘤内部的严重缺氧状态；同时，在酸性环境下进一步催化产生强毒性的羟基自由基（•OH），引发肿瘤细胞氧化应激损伤。

谷胱甘肽消耗与抗氧化防御削弱：氧化铈的氧化还原循环能够高效消耗肿瘤细胞内高浓度的谷胱甘肽（GSH），削弱肿瘤自身的抗氧化防御体系，从而放大了氧化应激杀伤效果。

光热与光动力协同增效：封装的ICG在808 nm近红外光照射下表现出优异的光热转换效率（η = 38.6%），局部产生的温度升高不仅能直接进行光热治疗，还能显著增强氧化铈纳米酶的催化活性。与此同时，催化产生的氧气又充当了光动力疗法的原料，源源不断地生成单线态氧，实现了光热、光动力、气体与纳米酶催化治疗的高效互补与自我放大。

Fig.4 (A) Cell viability of Panc02 cells after incubation with rCeGLI, rCeGLI + NIR irradiation, rCeGLI@PM, and rCeGLI@PM + NIR irradiation at serial CeOx concentrations (0, 12.5, 25, 50, 100, 200 μg/mL) for 24 h. (B) Flow cytometric quantitative detection of cellular uptake and (C) statistical analysis of the corresponding mean fluorescence intensity in Panc02 cells incubated with rCeGLI@PM (CeOx = 100 μg/mL) for 2, 4, 8, and 12 h. (D) CLSM uptake images of Panc02 cells incubated with different formulations (CeOx = 100 μg/mL) for 8 h. (E) Lysosome co-localization CLSM of rCeGLI@PM (CeOx = 100 μg/mL) in Panc02 cells at 0.5 h, 1 h, 2, 4, and 8 h post-incubation. (F) Z-stack CLSM images of Panc02 and NIH-3T3 MCSs after 8 h of treatment under different conditions at a CeOx concentration of 100 μg/mL. Data are shown as mean ± SD (n = 3). In (A), *** p < 0.001.

5. 治疗效果与应用价值#

在小鼠皮下胰腺癌及原位胰腺癌模型的综合评估中，rCeGLI@PM纳米机器人展现出了卓越的抗肿瘤疗效。与传统的被动扩散纳米颗粒相比，该系统依靠同源细胞膜靶向与双驱动活性穿透，在肿瘤部位实现了数倍的蓄积提升。多模态联合治疗显著抑制了原位胰腺癌的生长与转移，且未观察到明显的系统性毒性，显示出极高的生物安全性与临床转化潜力。

这项研究不仅为突破胰腺癌致密基质屏障提供了全新的物理-化学联合驱动范式，也为设计具有微环境自我调节与多模态协同治疗功能的高性能纳米机器人提供了重要的理论基础与实验依据。

Fig.5 (A) Representative CLSM images and (B,C) flow cytometric results of intracellular ROS levels in Panc02 cells after different treatments. (D) Relative intracellular GSH levels in Panc02 cells following various treatments. Representative CLSM images of intracellular (E) O2 levels and (F) ONOO– levels in Panc02 cells after different treatments. All cell groups were incubated with different formulations at an equivalent CeOx concentration of 100 μg/mL for 8 h before detection. Data are shown as mean ± SD (n = 3). In (C, D), * p < 0.05, *** p < 0.001. Fig.6 (A,B) Flow cytometric analysis of MMP in Panc02 cells treated with different treatments. (C) Representative Calcein-AM/PI double-staining images (Live/Dead assay) of Panc02 cells after various treatments. (D) Flow cytometric analysis and (E) corresponding quantification of the apoptosis rate in Panc02 cells incubated with different treatments. (F) Schematic diagram illustrating the proposed mechanism of rCeGLI@PM-induced cell death. All formulations were applied at a CeOx equivalent concentration of 100 μg/mL with 24 h incubation before measurement. Data are shown as mean ± SD (n = 3). In (B, E), *** p < 0.001. Fig.7 (A,B) In vivo FLI of subcutaneous Panc02 tumor-bearing mice after intravenous injection of rCeGLI or rCeGLI@PM and the corresponding quantitative analysis of tumor fluorescence intensity over time. (C) Ex vivo FLI and (D) quantitative fluorescence intensity of major organs in Panc02 tumor-bearing mice after various treatments. (E) Infrared thermographic images of tumor regions under 808 nm laser irradiation after various treatments. Data are shown as mean ± SD (n = 3). In (B, D), *** p < 0.001. Fig.8 (A) Schematic of the treatment regimen in the subcutaneous Panc02 tumor model. (B) Tumor growth curves following different treatments. (C) Representative photographs and (D) quantitative weights of excised tumors from each group at the end point. (E) Representative images of H&E staining, TUNEL assay, and ki67 staining in tumors from different treatment groups. Immunohistochemical staining for (F) collagen I and (G) HIF-1α in tumors from different treatment groups. Data are shown as mean ± SD (n = 6). In (D), *** p < 0.001. Fig.9 (A,B) In vivo FLI of orthotopic tumor-bearing mice and quantitative analysis of the tumor region fluorescence intensity at different time points post-injection. (C) Experimental treatment process of orthotopic Panc02-luc tumor-bearing mice. (D) In vivo bioluminescence imaging analysis of Panc02-luc orthotopic PC-bearing mice on day 0, 7, and 14. (E) Changes in body weight after different treatments. (F,G) Tumor weights and photographs of resected tumors for each group.

6. 研究局限与未来展望#

尽管该纳米机器人在动物模型中展现出了出色的深度穿透与协同治疗效果，但仍存在一定的局限性。首先，目前的研究主要基于小鼠肿瘤模型，其肿瘤微环境与人体真实复杂病灶在基质密度、免疫微环境及血流动力学上仍存在差异，其在人体大体积肿瘤中的穿透深度与驱动效率仍需进一步验证；其次，利用近红外一区光（808 nm）作为驱动与治疗光源，其组织穿透深度相对有限，对于人体深部器官的临床应用可能受到一定限制。

未来研究可聚焦于开发组织穿透能力更强的近红外二区（NIR-II）光驱动体系，或者结合超声、磁场等穿透力更强的外部物理场作为辅助驱动源。同时，深入优化细胞膜伪装工艺的标准化与规模化制备，推动该类智能自推进纳米药物从实验室研究向临床应用的转化。