【ACS Nano】疏水塌缩减弱可将多肽两亲性自组装体的血脑屏障透过率提升近30微米级尺寸聚集差异#

Figure 1. Chemical structures and morphology of peptide amphiphile (PA) supramolecular assemblies. (a–c) Chemical structures of C12-VVEE (a), C14-VVEE (b), and C16-VVEE (c), composed of a common β-sheet forming amino acid sequence with charged residues and palmitoyl alkyl tails of varying lengths. (d–f) Small-angle X-ray scattering (SAXS) profiles of C12–VVEE (g), C14-VVEE (h), and C16-VVEE (i) in aqueous solutions at 5 mM concentration after annealing. Slope values are averaged over duplicates. (g–i) Representative negative-staining TEM images of C12-VVEE (d), C14-VVEE (e), and C16-VVEE (d) after annealing. Scale bars correspond to 500 nm.#

Figure 2. Internal structures and thermodynamic profiles of annealed PA assemblies. (a) Synchrotron wide-angle X-ray scattering (WAXS) profiles of C12-VVEE, C14-VVEE, and C16-VVEE assemblies in aqueous solution at a concentration of 5 mM (the baselines of PAs were offset by 0.002 for each pattern). (b) Transmission Fourier-transform infrared (FTIR) spectra of PA assemblies at 5 mM concentration (the baselines of PAs were offset by 0.3 for each spectrum). (c) Deconvoluted carboxylate peak of the CD spectra of the three PA assemblies in (b). (d) Bar graph showing the average total interaction energy (short range Lennard–Jones and Coulomb) of lipid tails in the three PA assemblies at 0, −1, and −2 charge states. Triplicate measurements were performed with the error bars representing standard error of the mean (SEM). (e) Bar graph showing the average number of hydrogen bonding interactions among peptide backbones in the three PA assemblies with 0, −1, and −2 charge states. Triplicate measurements were performed with the error bars representing SEM (f–h) Simulation boxes depicting C12-VVEE (−2) (f), C14-VVEE (−1) (g), and C16-VVEE (0) (h) assemblies with the lipid tails in transparent gray and the peptide segments as either coils or ribbons (β-sheets). The V residues are colored orange and E residues are colored red.#

在超分子内聚力与热力学特性的探究中，图2的广角X射线散射、红外光谱以及全原子分子动力学模拟结果表明，尾链变短显著降低了组装体内部的内部有序度与β-折叠含量。同时，酸碱滴定实验证实尾链缩短使谷氨酸残基的有效酸度系数从8.2降至7.0。这是因为较弱的疏水塌缩改变了纳米纤维内部的介电环境，导致负电荷密度增加，进而通过静电排斥作用弱化了氢键网络与液晶相转变温度。

Figure 3. Preparation of PA assemblies and internalization assays with mouse brain endothelial cells bEnd.3. (a) Schematic of PA supramolecular assembly preparation procedures: (74) PAs are dispersed in aqueous solutions at neutral pH and subjected to annealing at 80 °C for 1 h, followed by probe sonication. The short PA fibers formed are then diluted in media at 100 mM PA concentration and added to bEnd.3 cultures. (b) Representative negative-staining TEM images of PA assemblies after probe sonication. Scale bars correspond to 500 nm. (c) Bar graph of the measured lengths of PA short fibers in (b) with error bars representing SEM, n = 100 for each assembly. (d) Representative fluorescence confocal micrographs of bEnd.3 cells incubated with PA assemblies for 2, 6, and 24 h. PA assemblies were labeled with 5 mol % C12-VVEE-TAMRA, C14-VVEE-TAMRA, and C16-VVEE-TAMRA, respectively (red). Nuclei were labeled with Hoechst (blue). The scale bar corresponds to 100 μm. (e) Bar graph showing the quantification of PA internalization as median TAMRA fluorescence intensity using flow cytometry after 2, 6, and 24 h of treatment with error bars representing SEM n = 2 for each condition. Unpaired t-test was performed: * P < 0.05, ** P < 0.01, *** P < 0.001.#

随后，研究人员将退火后的纳米纤维经超声破碎为150至250纳米的短纤维，并与脑微血管内皮细胞共孵育。如图3的激光共聚焦显微镜与流式细胞术结果所示，十六碳组装体在细胞内展现出最强的荧光累积，而十二碳组装体的胞内信号相对最弱。内吞抑制剂实验表明，所有组装体均主要通过 ATP 依赖的脂质花生微区及网格蛋白介导的内吞作用进入细胞，同时伴随部分被动扩散。

Figure 4. Experimental setup of an in vitro BBB-mimetic transwell assay with bEnd.3 to quantify PA passage across cell layers of brain endothelial cells with tight junctions. (a) Representative fluorescence confocal micrographs of bEnd.3 cells cultured on the transwell membrane to form tight junctions (nuclei are labeled with Hoechst (blue), tight junction protein marker ZO-1 is labeled with Alex Fluor 488-conjugated anti ZO-1 (green), and JAM-A is labeled with Alex Fluor 568-conjugated anti JAM-A (red)). The scale bar corresponds to 100 μm. (b) Schematic of the transwell assay, PA treatment, and quantification of PA passage using TAMRA fluorescence and high-performance liquid chromatography (HPLC). (74) (c) Quantification of PA passage across the transwell using TAMRA fluorescence intensity at the bottom chamber over 72 h of treatment, with error bars representing SEM and n = 2. (d) Quantification of PA passage across the transwell using quantitative HPLC analysis of aliquots from the bottom chamber over 72 h with error bars representing SEM and n = 2. One-way ANOVA with the Tukey test was performed. ** P < 0.01, *** P < 0.001. Unpaired t-test was performed: # P < 0.05, ## P < 0.01.#

然而，在建立的血脑屏障双室转运模型中，图4的高效液相色谱及质谱定量分析给出了完全相反的结论。在长达72小时的监测中，跨膜透过效率最高的恰恰是胞内累积最少的十二碳组装体，而胞内累积最高的十六碳组装体在下室中的浓度极低。这一现象打破了以往“胞内摄取量高即代表跨膜递送能力强”的传统假设。

Figure 5. Localization of PAs in live bEnd.3 cells and the aggregation profiles of PA assemblies over a range of decreasing pH values. (a) Representative fluorescence confocal micrographs of live bEnd.3 cells after 6 h of PA treatment. Nuclei are labeled with Hoechst (blue); PA assemblies are labeled with 5 mol % C12-VVEE-TAMRA, C14-VVEE-TAMRA, and C16-VVEE-TAMRA, respectively (red); lysosomes are labeled with DND-26-conjugated LysoTracker (green). The scale bar corresponds to 50 μm. (b) Representative fluorescence confocal micrographs of PA assemblies diluted in phosphate buffer at pH 7.4, phosphate buffer at pH 6.5, acetate buffer at pH 5.5, and acetate buffer at pH 4.5, respectively, and incubated for 24 h (2 mM Nile Red is used to visualize PA filaments and bundles). Scale bars correspond to 100 μm. (c) Representative negative-staining TEM of PA assemblies diluted in acetate buffer at pH 4.5 and incubated for 24 h. Scale bars correspond to 1 μm. (d–f) SAXS profiles of % C12-VVEE (d), C14-VVEE (e), and C16-VVEE (f) incubated in phosphate buffer at pH 7.4, phosphate buffer at pH 6.5, acetate buffer at pH 5.5, and acetate buffer at pH 4.5, respectively. The baselines of PA tracess were offset by a multiplying factor of 100 for each pattern. (g) Bar graph showing the slope of scattering curves in the Guinier region of the SAXS profiles in (d–f). Error bars correspond to standard deviation derived from linear fitting.#

为了解开这一矛盾，图5的活细胞共聚焦成像揭示了关键的胞内命运差异。所有组装体在内吞后均进入了溶酶体，但十六碳组装体由于其较高的酸度系数，在酸性的溶酶体环境（pH 4.5）中极易完全去质子化并失去静电排斥，从而诱发了尺寸高达20微米级的巨型纤维束沉淀与过度聚集，被永久滞留在溶酶体内部。相反，十二碳组装体因保持较低的酸度系数而维持了部分静电排斥，微观尺寸仅出现轻微增加，这使其能够成功逃逸溶酶体滞留并高效执行胞吐过程，最终实现高水平的跨细胞转运。