【JACS】华南理工大学pH 6.8精准爆破！JACS发表新型电离脂质纳米颗粒：通过TTCN参数实现肿瘤选择性溶瘤治疗#

文章标题：Tumor Acidity-Activatable Ionizable Lipid Nanoparticles for Selective Oncolytic Therapy

通讯作者：（Menghua Xiong）

文章链接：https://doi.org/10.1021/jacs.6c07591

文章概要#

本研究报道了一种基于电离脂质的创新型肿瘤酸性激活溶瘤纳米颗粒（aoLNPs）。研究团队通过对脂质分子的烷基链长度和数量进行程序化调节，首次发现了总尾部碳数（TTCN） 与脂质纳米颗粒在不同pH环境下细胞毒性之间的明确相关性。通过对脂质库的筛选，鉴定出的最优候选者aoLNP_E14A6-2能够特异性地在pH 6.8（模拟肿瘤微环境）下诱导肿瘤细胞死亡，而在正常生理pH值下保持稳定。该研究不仅揭示了通过物理化学参数精确控制溶瘤活性的机制，也为溶瘤治疗开辟了除溶瘤病毒之外的新路径。

引言#

肿瘤微环境的酸性特征是恶性肿瘤的重要标志之一，这主要源于肿瘤细胞活跃的无氧糖脂代谢。长期以来，科学家们一直试图利用这种天然的pH梯度来设计靶向药物，但如何实现高度的选择性依然是巨大的科学挑战。电离脂质通常被认为是核酸药物递送的“搬运工”，但在特定酸性条件下，它们展现出的膜破坏能力引起了研究者的关注。如果能将这种膜破坏活性锁定在酸性肿瘤组织中，这些脂质就能从“配角”变为直接杀伤肿瘤的“致命武器”。

Figure 1. Construction and screening of aoLNPs. (a) Synthesis route of ionizable lipids via a ring-opening reaction between 1,2-epoxyalkanes and an amine, followed by the acetylation or butyrylation of the hydroxyl groups. (b) Schematic illustration of aoLNPs preparation and screening. (c) Apparent p_K_a values of aoLNPs with different ionizable lipids determined via TNS assay. (d–f) CV7.4 (d), CV6.8 (e), and RCT (f) of aoLNPs (100 μg/mL of ionizable lipids) as a function of TTCN against Panc02 cells after 24 h of incubation. (g) Chemical structure of ionizable lipids with three tails in the second library. (h) RCT of aoLNPs (100 μg/mL of ionizable lipids) with different ionizable lipids in (g) against Panc02 cells after 24 h of incubation. (i) Hemolysis rate of aoLNPs (400 μg/mL of ionizable lipids) after incubation with red blood cells for 1 h and MTD of aoLNPs against ICR mice following i.v. administration, aoLNPs containing ionizable lipids with TTCN = 28 or 30 were used. (j) Chemical structure of E14A6-2. (k) The pH- and concentration-dependent cytotoxicity of aoLNPE14A6-2 against a range of cancer cells after 24 h of incubation. (l) Average diameter and zeta potential of aoLNPE14A6-2 under different pH values. (m) Cryo-transmission electron microscope (TEM) images of aoLNPE14A6-2 at pH 7.4 and pH 6.8. Scale bars, 50 nm. (n) Dye leakage from cancer cell membrane-mimicking liposomes incubated with aoLNPE14A6-2 (200 μg/mL of E14A6-2) for 2 h at different pH conditions. In c–f, h, i, k, l, and n, n = 3. In d–f, h, l, and n, data are presented as mean ± standard deviation (S.D.).#

主要实验及结论#

研究人员首先构建了一个包含多种结构变体的电离脂质库，其核心逻辑在于系统性地改变脂质疏水尾部的链长和链数。如图1中的模式图所示，脂质分子的头部负责对pH值的响应，而尾部则决定了其与细胞膜相互作用的强度。通过高通量筛选，团队观察到一个引人注目的现象：细胞毒性并非随着疏水性线性增加，而是存在一个最佳的TTCN数值范围。当TTCN处于最优区间时，脂质纳米颗粒展现出最强的pH依赖性细胞毒性。

Figure 2. aoLNPE14A6-2 induced lysosomal vacuolation and permeabilization under tumor acidity. (a) Representative images of Panc02 cells after incubation with Cy5aoLNPE14A6-2 (200 μg/mL of E14A6-2) at pH 6.8 for 2 h. Lysosomes and cell nuclei were stained with Lyso-Tracker Green and Hoechst 33342, respectively. Scale bars, 10 μm. (b) Representative differential interference contrast microscopy images of Panc02 cell treated with aoLNPE14A6-2 (200 μg/mL of E14A6-2) at pH 7.4 or pH 6.8 for varying durations. Scale bars, 40 μm. (c) Representative images of Panc02 cells after being treated with Cy5aoLNPE14A6-2 (200 μg/mL of E14A6-2) at pH 6.8 for varying durations. Lysosomes and cell nuclei were stained with Lyso-Tracker Red and Hoechst 33342, respectively. Scale bars, 10 μm. (d) Size of lysosomes in Panc02 cells after being treated with Cy5aoLNPE14A6-2 (200 μg/mL of E14A6-2) at pH 6.8 for varying durations. (e) Representative TEM images of Panc02 cells treated with or without aoLNPE14A6-2 (200 μg/mL of E14A6-2) at pH 6.8 for 6 h. Red arrows indicate vacuolated lysosomes. Scale bars for TEM images and magnified TEM images are 2.5 and 1 μm, respectively. (f) Representative images of Panc02Gal3-GFP cells treated with aoLNPE14A6-2 (200 μg/mL of E14A6-2) at pH 6.8 for varying durations. White arrows indicate Gal3-GFP puncta. Scale bars, 20 μm. (g) Representative lysosome images of Panc02 cells after incubation with aoLNPE14A6-2 (200 μg/mL of E14A6-2) in the absence or presence of BafA (20 nM) or CMA (20 nM) at pH 6.8 for 8 h. Lysosomes and cell nuclei were stained with Lyso-Tracker Red and Hoechst 33342, respectively. Scale bars, 10 μm. (h) Diameter of lysosomes in Panc02 cells after incubation with aoLNPE14A6-2 (200 μg/mL of E14A6-2) in the absence or presence of BafA (20 nM) or CMA (20 nM) at pH 6.8 for 8 h. (i) Cell viability of Panc02 cells treated with aoLNPE14A6-2 (200 μg/mL of E14A6-2) in the absence or presence of BafA (20 nM) or CMA (20 nM) at pH 6.8 for 14 h. Representative images are shown in a–c, e, f, and g (at least 3 images were taken for each sample). At least 804 (d) or 693 (h) lysosomes were measured, and n = 3 (i). In d, h, and i, data are presented as mean ± S.D., and P values were determined using a one-way ANOVA followed by Tukey’s multiple comparisons test.#

为了验证这一规律，研究人员对比了多种化合物在pH 7.4和pH 6.8条件下的表现。实验结果证明，aoLNP_E14A6-2在模拟肿瘤酸性环境下具有极强的杀伤力，而在模拟正常生理环境的pH 7.4条件下几乎没有毒性。这种高度的灵敏度确保了药物在循环系统中是安全的。在机制探究中，研究发现这种特异性杀伤并非源于简单的坏死，而是由于电离脂质在酸性环境下发生质子化，随后引发了剧烈的溶酶体空泡化和内质网应激，最终导致细胞质膜的彻底破裂。

在随后的体内实验中，研究者通过静脉注射aoLNP_E14A6-2观察其在荷瘤小鼠模型中的表现。结果显示，该纳米颗粒能够显著抑制多种实体瘤的生长，且表现出优异的生物安全性。生化分析和组织学切片均证实，受试动物的肝、肾等主要脏器未出现结构损伤或慢性毒性反应。这一发现极大地拓宽了电离脂质的应用边界，证明了通过分子水平的参数调控，可以赋予药物递送系统直接溶瘤的生物学功能。

Figure 4. aoLNPE14A6-2 induced membranolytic cell death under tumor acidity. (a) Representative time-lapse imaging showing the change of cell morphology of Panc02GFP/mCherry cells after treatment with aoLNPE14A6-2 (200 μg/mL of E14A6-2) at pH 7.4 or pH 6.8. White arrows indicate the bubble-like herniations on plasma membranes. Scale bar: 10 μm. (b) Relative mean fluorescence intensity (MFI) of mCherry in Panc02GFP/mCherry cells treated with aoLNPE14A6-2 (200 μg/mL of E14A6-2) at pH 7.4 or pH 6.8. (c) LDH release from Panc02 cells treated with different concentrations of aoLNPE14A6-2 at pH 7.4 or pH 6.8 for 24 h. (d) TEM images of Panc02 cells treated with or without aoLNPE14A6-2 (200 μg/mL of E14A6-2) at pH 6.8 for 12 h. White arrows and red arrows indicate the intact and ruptured plasma membrane. Scale bars for TEM images and magnified TEM images are 2 and 1 μm, respectively. (e) MFI of FITC in Panc02 cells treated with FITCaoLNPE14A6-2 at 4 or 37 °C and pH 6.8 for 4 h. (f) Cell viability of Panc02 cells treated with aoLNPE14A6-2 at 4 or 37 °C and pH 6.8 for 24 h. (g) Cell viability of Panc02 cells treated with aoLNPE14A6-2 (100 μg/mL of E14A6-2) and different inhibitors (NaN3 (10 mM), 2-DG (20 mM), MβCD (50 μM), Chl (50 μM), Wor (10 nM)) for 24 h. (h) Concentration-dependent cytotoxicity of aoLNPE14A6-2 against Panc02 cells at pH 6.8 after pretreatment with VX-765 (a caspase 1/4 inhibitor, 40 μM), Z-VAD-FMK (a pan-caspase inhibitor, 40 μM), ferrostatin-1 (Fer-1, a ferroptosis inhibitor, 1 μM), or Necrostatin-1 (Nec-1, a necrosis inhibitor, 50 μM) for 24 h. In b, at least 24 cells were measured and data are presented as mean ± S.D. In c, e–h, n = 3 and data are presented as mean ± S.D. P values in g were determined using a one-way ANOVA followed by Tukey’s multiple comparisons test.#

总结及展望#

本研究成功开发了一种通过TTCN参数驱动的酸性激活型溶瘤脂质纳米颗粒。该系统不仅克服了传统溶瘤药物选择性不足的难题，更利用了电离脂质在低pH值下特有的物理化学特性实现了对肿瘤细胞的“物理爆破”。未来，该技术有望与免疫检查点抑制剂联合使用，通过溶瘤作用释放肿瘤抗原，激活原位免疫反应。尽管该研究仍处于实验阶段，但其展示的分子结构-生物活性相关性为下一代响应性生物材料的设计提供了极具价值的理论支持。