【Angew.Chem.】中科大刘世勇、邓正玉、胡进明等|首次实现！自组装突破热力学限制：可见光触发“无迹交叉联结”构筑5种仿生盘状高分子囊泡#

Schematics of hierarchical self-assembly of amphiphilic BCPs containing photolabile thiocoumarin moieties and concurrent traceless crosslinking and permeabilizing of discocytes triggered by visible light for controlled drug delivery inside live cells. (a, b) BCP amphiphiles self-assemble into five distinct nanostructures, including uncommon PDs. (c, d) After cellular uptake of PDs by live cells, visible light irradiation leads to the efficient cleavage of thiocoumarin groups, followed by decarboxylation and cascade eliminations (m = 1) to liberate primary amines. These newly generated primary amines with suppressed p_K_a undergo extensive amidation reactions with neighboring ester linkages, resulting in traceless covalent crosslinking and permeabilization of bilayer membranes, which can be utilized to construct drug delivery nanocarriers.#

Hierarchical nanostructures fabricated from photoresponsive BCPs. Representative (a) TEM and (b) SEM images were recorded for nanostructures formed by adding 9 mL deionized water into separate BCP solutions (1.0 g/L, 1 mL) in 1,4-dioxane, in which the water addition rate was varied (36 or 2 mL/h). (c) Microstructural changes of nanostructures with varying water contents during the formation of PDs. TEM images were recorded at varying intermediate water contents when deionized water was added into T2 solution in 1,4-dioxane (1.0 g/L) at a rate of 36 mL/h. All dispersions were subsequently diluted with water to a polymer concentration of 0.1 g/L before TEM sample preparation and imaging.#

为了验证这些纳米结构的光响应行为及材料的化学变化，研究团队利用460纳米的蓝色可见光对盘状高分子囊泡进行了照射。如图2所示，紫外-可见吸收光谱中硫代香豆素特征吸收峰的减弱和新峰的出现，证实了光引发的裂解反应极为高效。Confined双层膜环境中的强$\pi-\pi$堆积作用使得其吸收峰产生了显著红移。光解反应在 hydrophobic 微环境中原位释放出具有高亲核性的伯胺基团，并自发与邻近的酯键发生主链间酰胺化反应（无迹共价交叉联结）。红外光谱和核磁共振波谱的数据明确证实了酯键向酰胺键的转化。这一独特的机理使得囊泡在流体动力学直径仅微幅增加的情况下，不仅通过共价强化锁定了盘状形貌，还同时促使双层膜发生疏水向亲水的转变，显著提升了膜的渗透性。

(a) Proposed mechanisms for concurrent amidation-actuated crosslinking and hydrophobic-to-hydrophilic transition within hydrophobic domains. Upon visible light irradiation, photoactive thiocoumarin moieties are removed, producing primary amine functionalities within the hydrophobic domains of hierarchical nanostructures. Prominent interchain amidation reactions then occur, leading to covalent stabilization of polymeric assemblies, which is associated with significant hydrophobic-to-hydrophilic transformation. (b) Irradiation time-dependent UV-Vis absorbance spectra recorded for aqueous dispersion of PDs of T2 (0.1 g/L). (c) Intensity-average hydrodynamic diameters, <_D_h>, and (d) normalized scattering intensities recorded for aqueous dispersion of PVs of T1, PDs of T2, and FVs of T3 before and after photoirradiation with 460 nm LED light for 15 min. Data are presented as mean ± SD (n = 3). (e) FTIR and (f) 1H NMR spectra recorded for PDs of T2 before and after photoirradiation with 460 nm LED light for 15 min. The irradiated PD dispersion was then subjected to dialysis and lyophilization before running FTIR and 1H NMR. (g) Representative SEM images (scale bar: 200 nm) of an aqueous dispersion of PVs of T1, PDs of T2, and FVs of T3 at a concentration of 0.1 g/L before and after photoirradiation with 460 nm LED light for 15 min.#

鉴于封闭的内腔具有装载亲水和疏水货物的潜力，研究团队进一步考察了不同形貌囊泡的细胞生物学行为。如图3所示，利用近红外荧光染料Cy7标记的共聚物进行细胞共培养实验表明，盘状高分子囊泡展现出了显著优于花状和穿孔囊泡的细胞内吞效率。这种形貌依赖性的摄取优势得益于盘状结构优异的表面积与体积比以及其特有的内在可变形性。液体原子发射显微镜的纳米力学测试也印证了高弹性对细胞膜易穿透性的贡献。生物化学抑制剂实验进一步揭示，该盘状囊泡主要通过巨胞饮作用和动力蛋白依赖性内吞作用等多条途径协同进入活细胞内部。

(a) Chemical structure of fluorescent Cy7-labeled BCPs. (b) Representative time-dependent CLSM images (scale bar: 10 µm) of HepG2 cells upon incubation with PDs and PVs of T4-Cy7 as well as FVs of T5-Cy7, respectively. (c) Variations of normalized fluorescence intensities within HepG2 cells as quantified from CLSM observations. Data are presented as mean ± SD (n = 3). Statistical significance was assessed by one-way ANOVA followed by Tukey’s multiple-comparison test. ***p < 0.001.#

最后，研究团队在活细胞和体内水平展示了这种光控纳米载体的空间精准递药能力。如图4和图5所示，团队利用笼状醌氰素（QCy）和四甲基罗丹明（TMR）双荧光报告系统，在 HepG2 细胞内部成功可视化了光触发的酰胺化释放过程。在将抗癌药物阿霉素（DOX）以及不同分子量的荧光染料共同包裹进盘状囊泡后，共聚焦显微镜观察表明，在无光照的暗处条件下药物几乎不发生泄漏；而一旦施加空间精确定位的可见光微区照射，囊泡膜的渗透性被瞬间打开，小分子的阿霉素和DAPI迅速释放并富集于细胞核中，而大分子量的右旋糖酐因尺寸效应仍被完美截留在囊泡内部。这种在维持囊泡结构完整性的同时实现小分子药物按需释放的特质，显著提高了对癌细胞的光控细胞毒性。小鼠体内实验也证实，这些囊泡在完成肝脏等免疫器官的靶向富集后能够被渐进式代谢清除，展现出优异的生物相容性。

(a) Schematics of photo-triggered traceless crosslinking and permeability switching of membranes of fluorescent PDs coloaded with nucleus-staining small molecule DAPI dye (277 Da) and TR-Dextran (MW ≈ 10 kDa) inside live cells. Visible light irradiation triggers DAPI release from PDs due to synchronized bilayer crosslinking and permeabilizing, whereas TR-Dextran is retained within the discocytes owing to its large size. (b) Representative CLSM images (scale bar: 10 µm) recorded for HepG2 cells upon incubation with DAPI and TR-Dextran coloaded PDs of T4-Cy7 at 37°C. Upon 12 h incubation, the DAPI and TR-Dextran coloaded PDs were removed, followed by washing and replacing with fresh culture medium. After image acquisition (upper panel), a single cell inside yellow box was exposed to irradiation using embedded 514 nm laser in CLSM for 2 min, followed by additional incubation for 30 min (lower panel).#

(a) Schematics of self-assembly and drug encapsulation to prepare DOX-loaded discocytes for photo-triggered crosslinking and drug release. (b) In vitro DOX release profiles from drug-loaded PDs in the absence or presence of irradiation with 460 nm LED light for 15 min. Data are presented as mean ± SD (n = 3). (c) Representative CLSM images (scale bar: 10 µm) recorded for HepG2 cells upon incubation with DOX-encapsulated PDs of T4-Cy7 at 37°C. After 12 h incubation, DOX-loaded PDs were removed, followed by washing and replacing with fresh culture medium. Following image acquisition (upper panel), cells were exposed to irradiation using embedded 514 nm laser in CLSM for 2 min, followed by additional incubation for 10 min (middle panel) and 30 min (lower panel). (d) Relative viability of HepG2 cells after treatment with DOX-loaded PDs of T2 or free DOX. After incubation for 24 h, DOX-loaded PDs and free DOX were removed, followed by washing and replacing with fresh culture medium. For photoirradiation, HepG2 cells treated with DOX-loaded PDs were exposed to 460 nm LED light for 15 min. All samples were then incubated for an additional 48 h prior to the metabolic assay. Data are reported as mean ± SD (n = 3).#