近红外生物正交激活的研究背景#

在复杂生物系统中进行人工化学反应的生物正交化学，为原位生成治疗药物提供了强大工具。利用外部光源触发药物的前体释放，能够获得极高的时空控制精度。然而，传统光敏化学保护基团大多依赖紫外光或高能量可见光激发。这类短波长光线在生物组织中的穿透深度非常有限，且易对正常组织造成光毒性损伤，严重阻碍了临床转化。

为了克服这一瓶颈，科研人员尝试使用组织穿透力强且损伤小的近红外光（700–1100 nm）作为激发源。但近红外光子能量较低，传统光敏前药对双光子吸收截面极小，往往需要极高的激光功率才能驱动化学键断裂，这极易引发剧烈的热效应。因此，如何在低功率近红外光照射下高效激活前体药物，成为光控药物递送领域的核心难题。

Fig.1 a) AuNSt-mediated prodrug (proDox1) photoactivation by NIR light. Schematic illustration of the process: Upon irradiation with NIR light, AuNSt induce the photo-cleavage of proDox1 with the subsequent release of Dox in a spatiotemporally controlled manner by the generation of an intense EM field in their tips. b) Representative TEM image of AuNSt. Scale bar: 500 nm Inset: High magnification TEM image of AuNSt. Scale bar: 50 nm c) Dox release from aqueous solutions containing only proDox1 as a control (black) and both proDox1 and AuNSt (red), after selected irradiation times using an 808 nm laser (150 mW cm−2). Normalized fluorescence at 560 nm (λex = 490 nm). Data represent mean ± SD, n = 3. d) Photoactivation of AuNSt@proDox2 by NIR light. Schematic illustration of the process: Upon irradiation with NIR light, AuNSt induce the photo-cleavage of the attached proDox2 with the subsequent release of Dox. e) Optical extinction spectra of AuNSt (black) and AuNSt@proDox2 (red) in water. f) Dox release from AuNSt@proDox2 dispersion in water in non-irradiated control samples (black) and irradiated samples (red) after selected irradiation times using an 808 nm laser (150 mW cm−2). Normalized fluorescence at 560 nm (λex = 490 nm). Data represent mean ± SD, n = 3.

核心实验过程与发现#

研究团队设计并合成了两种具有不同金表面亲和力的阿霉素前体药物。第一种前药proDox 1直接通过4,5-二甲氧基-2-硝基苄基氨基甲酸酯封闭阿霉素的氨基活性位点。第二种前药proDox 2则引入了含二硫键的2-硝基苄基二醇连接锁，能够直接通过金-硫键共价锚定在金纳米星（AuNSt） 表面，如图1所示。通过高效液相色谱、质谱及核磁共振光谱等手段，研究人员证实了两种前药的高纯度与结构稳定性。实验所采用的金纳米星平均粒径为120 nm，在830 nm处展现出强烈的局域表面等离子体共振吸收峰，如图1所示。

在近红外光激活实验中，金纳米星尖端产生的强烈局域电磁场显著增强了相邻前药分子的多光子吸收过程，如图1所示。当使用功率密度仅为150 mW cm⁻²的808 nm CW激光照射时，混合有金纳米星的proDox 1溶液释放出大量游离阿霉素，荧光强度大幅上升，如图1所示。相比之下，无金纳米星参与的纯前药组在相同照射下几乎不释放药物，证明了局域场增强效应的关键作用，如图1所示。

Fig.2 SERS spectra of AuNSt aggregates on a glass slide in contact with drug in solution and Raman spectra of the drugs in powder form. Dox, proDox1 and proDox2 were dissolved in ethanol and then added to a dispersion of AuNSt in water, with final concentrations of 10 and 500 µM of the drug and 0.58 mM (100 µg mL−1) estimated Au in the AuNSt colloid in 50% (vol/vol) ethanol/water. SERS line scan average of at least 100 points were acquired focusing the 785 nm laser on the top layer of the AuNSt aggregates. In parallel, the different drug powders were placed on top of a glass slide and Raman spectra were acquired. a) SERS spectra of the AuNSt aggregates on the bottom of the slide with drugs in solution (size of the field 760 µm x 560 µm). b) Raman spectra of the drug samples powder. Red bars highlight SERS peaks matching the characteristic Raman spectra of the powder drugs.

Fig.3 AuNSt mediated photoactivation of proDox in cancer cells. a) Confocal laser scanning microscopy (CLSM) images of Sk-Mel-103 cells (blue: cell nuclei, red: Dox). a-i) From top to bottom: untreated cells (Control), cells treated with proDox1+AuNSt yet not irradiated (proDox1 + AuNSt), and cells treated with proDox1 + AuNSt and NIR irradiation (AuNSt+proDox1+NIR). a-ii) From top to bottom: untreated cells (Control), cells treated with AuNSt@proDox2 yet not irradiated (AuNSt@proDox2), and cells treated with AuNSt@proDox2 and NIR irradiation (AuNSt@proDox2+NIR). Scale bars: 50 µm. NIR irradiation for 15 min at 300 mW cm−2. From left to right: DNA marker (Hoechst 33342), doxorubicin, and combined (merge) fluorescence channels b) Quantification of the corresponding fluorescence intensity of Dox: (i) AuNSt+proDox1, (ii) AuNSt@proDox2. Error bars represent standard deviation, n = 6. c) CLSM images of spatiotemporal control in AuNSt-mediated prodrug photoactivation. c-i) Intracellular photorelease of Dox in HeLa cells treated with AuNSt+proDox1 at different NIR irradiation times. HeLa cells were irradiated for 90 and 300 s and CLSM micrographs were immediately collected. From top to bottom: DNA marker (Hoechst 33342), doxorubicin, and combined (merge) fluorescence channels. From left to right: time of exposure to NIR light (0, 90 and 300 s). Scale bars: 10 µm. c-ii) TEM images of HeLa cells after incubation with AuNSt (left) and zoomed area (right). Scale bars: 10 µm (a), 200 nm (b). d) Cell viability assays: d-i) Scheme of the experiment. d-ii) Cell viability of HeLa cells incubated with proDox1 (10 µM) and AuNSt (100 µg mL−1) or with AuNSt@proDox2 (100 µg mL−1) upon 808 nm laser irradiation at 300 mW cm−2 for 15 min (black = non-irradiated controls; red = NIR irradiated samples). Cell viability experiments were performed in triplicate. Error bars represent standard deviations. GraphPad Prism 9 was used for statistical analysis. Two-way ANOVA followed by Tukey’s post-test were used to compare the findings, (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

研究人员利用表面增强拉曼光谱（SERS）深入探究了前药与金纳米星表面的距离对光解效率的影响，如图2所示。光谱分析表明，proDox 2由于二硫键的共价锚定，其特征拉曼峰在极低浓度下依然非常显著，如图2所示。相比游离状态的proDox 1，共价结合的AuNSt@proDox 2表现出更快的药物释放动力学，进一步证实紧密靠近金纳米星表面能最大化利用等离子体近场增强效应，如图1所示。温度监测表明，在150 mW cm⁻²的低功率密度下，溶液温度维持在安全范围，排除了热降解导致药物释放的可能性。

细胞与动物水平的生物学评估证实了该体系的优异疗效。在黑色素瘤细胞及小鼠皮下肿瘤模型中，光照组表现出显著的亚毫米级区域选择性药物释放。在避开高功率光热损伤的前提下，金纳米星介导的光激活前药成功诱导了肿瘤细胞凋亡，显著抑制了肿瘤生长，证明了该近红外光控生物正交体系在体内应用的安全性和高效性。

Fig.4 In vivo evaluation of the therapeutic efficacy of AuNSt@proDox2 photoactivation in an in vivo mouse model of human melanoma. a) Athymic nude mice were orthotopically injected with 5 × 105 Sk-Mel-103 cells subcutaneously on both dorsolateral flanks. When tumor volume reached 50 mm3, treatments started (days 0 and 3). Nanoparticles were administered via intratumoral injection. Treatments consisted of: a control group only irradiated with NIR (blue), AuNSt and NIR irradiation (yellow), AuNSt@proDox2 (black), and AuNSt@proDox2 with NIR irradiation (red), n = 8 tumors per group. NIR irradiation was applied at 150 mW cm−2 for 10 min and the nanoparticles (AuNSt and AuNSt@proDox2) concentration was 1 mg mL−1 (volume of injection: 50 µL; dose: 2 mg kg−1). Tumor growth was monitored for 5 days until tumor volume of control group reached values stipulated by the endpoint criteria. b) Antitumor effect of the different treatments. b-i) Images of representative tumors extracted at the end of the experiment. b-ii) Fold change of tumor volume (mm3) for different treatments. Data represent the mean ± SEM. b-iii) Kaplan-Meier graph of mice for each treatment group during the period of the study. c) Changes in temperature produced by the different treatments (control+NIR, AuNSt+NIR, and AuNSt@proDox2+NIR). c-i) Thermal images (Testo 875) recorded after NIR irradiation. c-ii) Mean temperature in tumors for different treatments. Data represent the mean ± SEM. GraphPad Prism 9 was used for statistical analysis. Two-way ANOVA followed by Tukey’s post-test were used to compare the findings, (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Fig.5 Ex vivo evaluation in tumors after treatment with AuNSt@proDox2. a) TEM images of tumor sections from animals subjected to different treatments (control+NIR, AuNSt+NIR, AuNSt@proDox2 and AuNSt@proDox2+NIR), showing the internalization of AuNSt in cancer cells. Scale bars: 2 µm (first row), 500 nm (second row), and 200 nm (last image of second row). b) Assessment of NIR-triggered Dox delivery in tumor sections upon different treatments (control+NIR, AuNSt@proDox2 and AuNSt@proDox2+NIR). b-i) CLSM images (blue: cell nuclei, red: Dox). Scale bars: 200 µm. b-ii) Corresponding quantification of Dox-related fluorescence intensity values (arbitrary units). c) Evaluation of apoptosis in tumor sections by TUNEL assay from animals subjected to different treatments (control+NIR, AuNSt+NIR, AuNSt@proDox2, and AuNSt@proDox2+NIR). c-i) CLSM images (blue: cell nuclei, green: apoptotic cells). Scale bars: 50 µm. c-ii) Corresponding quantification of TUNEL-positive cells fluorescence (arbitrary units). Data represent the mean ± SEM and statistical significance was assessed by one-way ANOVA followed by Tukey’s post-test (* p < 0.05, ** p < 0.01, *** p < 0.001) using GraphPad Prism 8 (n ≥ 5).

总结与展望#

本项工作成功构建了一种基于金纳米星与近红外光协同作用的生物正交药物释放平台。该技术巧妙利用等离子体纳米结构的近场增强效应，克服了近红外光激活光敏保护基团效率低下的难题，实现了低功率密度下前体药物的高效光解。这一策略不仅为阿霉素等毒性较强的化疗药物提供了精准的时空释放手段，也为未来开发高穿透深度、低组织损伤的远程光控靶向治疗方案奠定了重要基础。