【Adv.Mater.】酯化胆固醇偶联物实现mRNA向血脑屏障的高效靶向递送：转染效率超传统配方且完整保留屏障功能#

文章标题：Esterified Cholesterol Conjugates Enable LNP‐Mediated mRNA Delivery to the Blood–Brain Barrier Following Intravenous Administration 文章作者：Zeru Tian, Xu Wang, Alexis Pacheco Benitez, Erick Guerrero, Yaou Duan, Jingwen Wei, Lukas Farbiak, Daniel J. Siegwart 文章链接：https://doi.org/10.1002/adma.74762

一、核心主旨与研究突破#

这项由美国德克萨斯大学西南医学中心团队发表的研究，开发了一种全新且无需额外连接靶向配体的脂质纳米颗粒（LNP）递送平台。常规脂质纳米颗粒在静脉注射后绝大部分会被肝脏截留，极难到达脑部；而该研究通过模块化合成策略设计了一类带有酯化修饰的胆固醇偶联脂质，在完全去除游离胆固醇的前提下构建了四组分新型LNP，在小鼠静脉注射后实现了对脑血管内皮细胞的高效、高选择性mRNA递送与蛋白表达。更为关键的是，这种靶向递送过程完全不破坏血脑屏障（BBB）原有的紧密结构与生理屏障功能，为治疗神经炎症、脑卒中及阿尔茨海默病等与脑血管病变相关的中枢神经系统疾病提供了全新的非侵入式基因治疗策略。

Fig.1 A three-dimensional (3D) combinatorial synthesis approach was used to develop and evaluate lipids for mRNA delivery to the BBB. (a) Schematic representation of the 3D combinatorial synthesis strategy employed to identify BBB-targeting lipids. (b) Chemical structures of the lipid library components, including amine head groups (C1–C6), alkyl groups (O1 and O2), and cholesterol tails (B1, B2, and B3). (c) Schematic illustration of mRNA-encapsulating lipid nanoparticles (LNPs). (d) Heatmap showing the quantification of total luciferase activity in the brain following systemic administration of BBB-targeting LNPs (n = 3 biologically independent samples). (e) Total brain luciferase activity mediated by BBB-targeting LNPs formulated with cholesterol-conjugated lipids (synthesized core structures are shown), and benchmark MC3 and SM-102 LNPs formulated with unmodified ionizable lipids. (f) Impact of piperazine incorporation in the lipid core on BBB-targeting efficacy, demonstrating significantly enhanced activity in piperazine-containing formulations (****p < 0.0001).#

二、研究背景与传统递送痛点#

血脑屏障是中枢神经系统与外周血液循环之间的关键屏障，主要由脑微血管内皮细胞通过紧密连接构成，严格限制着离子、大分子物质及免疫细胞的进出。在过去，绝大多数中枢神经系统递送研究都专注于“如何跨越BBB进入脑实质（神经元与神经胶质细胞）”，常用的手段包括受体介导的转胞吞作用、聚焦超声（FUS）物理破坏屏障等。然而，强行跨越或物理破坏血脑屏障往往伴随着极低的递送效率、脱靶毒性以及脑出血和组织损伤的巨大风险。

脑微血管内皮细胞本身不仅是屏障，更是调节脑血流、神经免疫通讯的重要靶标，在脑部肿瘤转移、神经退行性疾病及脑缺血中均起着关键作用。许多疾病仅需在脑血管表面表达治疗蛋白或抑制内皮炎症即可达到治疗目的，无需让药物侵入深部脑实质。然而，目前直接靶向脑血管内皮的系统通常依赖昂贵且易引起免疫排斥的抗体或配体修饰。因此，开发一种完全不依赖外源配体、通过静脉注射即可靶向脑血管内皮且不损伤屏障完整性的新型LNP系统，是领域内亟待解决的核心难题。

三、化学设计创新与高通量配方筛选#

传统的LNP通常由四种成分组成：可电离脂质、辅助磷脂、游离胆固醇和聚乙二醇化脂质（PEG-脂质）。其中，胆固醇负责稳定双分子层膜并促进膜融合。研究团队打破常规，不再将胆固醇作为单独的添加剂，而是将其共价偶联到氨基脂质核心上，通过一步两阶段的三组分模块化合成反应，高效构建了包含51种全新胆固醇偶联脂质的化合物库，总反应收率均超过80%。

该类新型脂质分子在结构上具备三大创新特性：

季铵与叔胺复合结构：在单个脂质分子中同时引入叔胺与季铵结构，精细微调脂质的电离度，从而显著增强其在内涵体酸性微环境下的缓冲能力与逃逸效率。
可降解酯键链接：在烷基链与氨基之间嵌入可水解的酯键，大幅提升体内生物相容性与降解安全性。
可调控碳链间距：在胆固醇基团与胺核心之间设置了不同长度（3、5或7个碳）的柔性碳链连接子，赋予胆固醇构象更大的自适应灵活性。

为了找到最优组分配比，研究团队针对具有代表性的分子设计了涵盖不同辅助磷脂（如DSPC、DOPE、DOPC等）及不同摩尔比例的高通量筛选库，共计合成了486种LNP配方。体外荧光素酶转染筛选（以转染难度适中的IGROV-1细胞为模型）显示，哌嗪（Piperazine）环核心（尤其是代号为C6的胺核心）表现出远超其他结构的靶向与转染能力。最终，团队筛选出综合性能最优的先导配方DSPC-3——由新型胆固醇脂质C6O2B2、可电离脂质DODAP、辅助磷脂DSPC以及DMG-PEG2000以35:50:30<2>的摩尔比例组成。

Fig.2 High-throughput screening and in vivo validation of BBB-targeting LNP formulations. (a) Chemical structure of the top-performing BBB-targeting lipid C6O2B2. (b) Schematic of the screening pipeline. A total of 486 LNP formulations with varying helper lipids and component ratios were systematically evaluated for in vitro transfection efficiency. The top-performing candidates were then assessed in vivo via intravenous (I.V.) administration of luciferase mRNA-loaded LNPs (0.1 mg/kg, 6 h) in C57BL/6 mice. (c) High-throughput screening of LNP transfection efficiency in IGROV-1 cells. Luciferase mRNA (mLuc)-loaded LNPs were prepared by pipette mixing and used to transfect IGROV-1 cells (20 ng mRNA per well, 24 h; n = 3 biologically independent samples). Heat map displays relative luciferase expression, with relative light units (RLU) >1 000 000 considered as highly efficient. (d–f) Relative hit rate of BBB-targeting LNPs at different molar ratios of (d) C6O2B2, (e) DMG-PEG, and (f) DODAP. (g) Hit rate of LNPs incorporating different helper lipids. (h) Performance classification of screened LNPs based on luminescence: >1 000 000 RLU (highly efficient), 100 000–1 000 000 RLU (moderate efficiency). (i–k) Bioluminescence imaging (BLI) was performed 6 h post-intravenous administration of mLuc-loaded LNPs (0.1 mg/kg), and total brain luminescence flux was quantified (n = 3). (i) Representative bioluminescence images of the brain for the top 14 LNP formulations. The images were selected from mice with signal intensities consistent with the group average and represent the overall trends observed across all animals. (j) Molar composition of the top 14 formulations. (k) Quantification of brain luminescence signals across the top-performing formulations. Each column was compared with DSPC-3 formulation using one-way ANOVA with Tukey’s multiple comparisons test. Statistical significance is indicated above each column. Data are presented as mean ± SEM. p < 0.05; p < 0.01; p < 0.001; ****p < 0.0001; ns, not significant, p > 0.05.#

四、核心实验表征与体内靶向验证#

在纳米物理化学表征方面，优化后的C6O2B2 LNP展现出优异的均匀度与包裹性能：其水动力学粒径约为138 nm，多分散系数（PDI）低至0.14，表面呈微弱的正电荷至近中性，mRNA包封率（Encapsulation Efficiency）高达95%。

在动物体内转染验证实验中，研究团队取得了以下突破性数据：

卓越的脑部转染能力：在小鼠静脉注射装载荧光素酶mRNA的LNP（剂量0.1 mg/kg）后，活体生物发光成像显示，新型LNP在脑部产生了极强的发光信号，而传统的MC3配方与SM-102配方在脑部的表达量微乎其微。
精准靶向脑血管内皮细胞：研究人员利用Ai14转基因小鼠（Cre-LoxP系统，仅在发生Cre重组后发出tdTomato红色荧光）进一步评估细胞特异性。高分辨率连续双光子层析成像（STPT）展示了全脑3D血管树的强劲荧光表达；脑切片共聚焦免疫荧光染色进一步证实，tdTomato荧光信号与血管内皮标志物CD31高度共定位，而在神经元标志物（NeuN）和微胶质细胞标志物（IBA1）中完全未检测到脱靶转染信号。

Fig.3 Characterization of BBB-targeting LNPs and cell type selectivity evaluation of BBB-targeting. (a) Physicochemical characterization of C6O2B2 LNPs formulated in 10 mM citrate buffer (pH 4.0) (n = 3 biologically independent samples). (b) Hydrodynamic diameter distribution of the C6O2B2 LNP. (c) Schematic illustrating Cre mRNA delivery, leading to tdTomato expression in transgenic mice through Cre-mediated deletion of the stop cassette. (d) Ex vivo fluorescence imaging of brains from tdTomato mice following intravenous injection of C6O2 and C6O2B2 LNPs loaded with Cre mRNA (0.3 mg/kg, two doses, 48 h post-final injection). (e) C6O2 and C6O2B2 LNPs induced tdTomato expression in the brain following Cre mRNA administration (0.3 mg/kg, two doses). The C6O2B2 LNP formulation exhibited significantly higher tdTomato expression compared to PBS controls. Data are presented as mean ± s.e.m. (n = 3 biologically independent animals). (f) The fold increase in brain fluorescence for C6O2 and C6O2B2 LNPs compared to PBS-treated mice. (g) Representative tissuecyte image of a whole brain from an LNP-Cre–treated mouse. (h) Confocal images of brain sections showing co-localization of tdTomato with CD31, Iba1, and NeuN, indicating expression in endothelial cells, microglia, and neurons, respectively.#

五、血脑屏障功能与安全性评估#

靶向脑血管内皮的关键前提是不能破坏屏障本身的安全结构。研究团队从三个独立维度系统验证了该系统的安全性：

多重颗粒穿透测试：在注射先导LNP后，分别追加注射针对肺或肝脏的SORT LNP，发现脑部并未出现其他非脑靶向纳米颗粒的非特异性漏入。
伊文思蓝（Evans Blue）渗漏检测：伊文思蓝染料渗漏是衡量血脑屏障通透性的金标准，实验显示给药组小鼠脑内几乎未见任何染料渗漏，屏障物理致密性完好。
7T高场小动物磁共振成像（MRI）：在注射LNP 6小时后向小鼠静脉注射钆对比剂（Gadobutrol），动态T1加权弛豫时间测量显示，给药前后脑实质内无任何造影剂渗入信号，在活体功能影像层面确认血脑屏障生理功能未受任何干扰。

在治疗应用验证中，团队在LPS诱导的急性脑损伤小鼠模型中注射装载抑炎因子IL-10 mRNA的C6O2B2 LNP，结果显著降低了脑血管渗漏，并强力下调了促炎细胞因子（TNF-α、IL-1β和IL-6）的表达水平，证实了其在血管抗炎与脑保护方面的实质疗效。

Fig.4 Evaluation of BBB integrity after treatment with BBB-targeting LNPs. (a) Schematic representation of the experimental setup used to assess the effect of BBB-targeting lipids on BBB permeability. C57BL/6 mice were intravenously injected with Cre mRNA-loaded BBB-targeting LNPs (0.3 mg/kg mRNA). At 24 or 48 h post-injection, luciferase mRNA-loaded LNPs (0.1 mg/kg) were administered intravenously (I.V.) and analyzed after 6 h. (b–d) Representative bioluminescence images of the brain following administration of (b) lung-targeting, (c) liver-targeting, and (d) BBB-targeting LNPs in C57BL/6 mice. (e) Quantification of total luciferase activity in the brain after intravenous injection of lung-, liver-, and BBB-targeting LNPs. (f) Schematic of BBB integrity assessment using Evans blue dye. C57BL/6 mice were intravenously injected with mLuc-loaded C6O2 and C6O2B2 LNPs (0.3 mg/kg mRNA). Evans blue was administered at 1, 3, 6, or 24 h post-injection, followed by (g) brain collection and imaging and (h) homogenization to quantify Evans blue penetration. (i) Schematic representation of BBB functional evaluation using magnetic resonance imaging (MRI). (j) Representative T1-weighted images of the brain before and after administration of Gadobutrol (1.0 mmol/kg). (k) Absolute change in T1 values before and after Gadobutrol administration.#

六、作用机制深度拆解#

该LNP为何能够在不加外源配体的情况下实现脑血管靶向？研究揭示了其背后协同工作的双重机制：

结构驱动的高效内涵体逃逸：基于FRET（荧光共振能量转移）的模拟膜融合实验表明，共价结合的胆固醇具有锥形几何构型并紧密锚定在双层膜中，与内涵体模拟膜的融合速率和彻底程度显著优于普通游离胆固醇配方，赋予了其极强酸性环境下的膜破裂与细胞质mRNA释放能力。
载脂蛋白A-I（ApoA-I）介导的内源性蛋白冠靶向：质谱蛋白质组学分析表明，该LNP进入血浆后，其表面吸附形成的“蛋白冠”中富集了显著高水平的载脂蛋白A-I（ApoA-I）。细胞内吞通路阻断实验证实，破坏脂筏结构（Mβ-CD处理）会显著抑制其摄取，这表明富含ApoA-I的蛋白冠利用脂筏介导的内吞通路，有效引导纳米颗粒富集于脑内皮细胞表面。此外，巨噬细胞清除实验与转铁蛋白受体1（TfR1）抗体阻断实验证明，该过程并不依赖游走免疫细胞的转运或TfR1介导的传统转胞吞途径。

Fig.5 Mechanistic insights into BBB-targeting LNP delivery. (a) Chemical structure and molecular dynamics simulation of the top-performing BBB-targeting lipid C6O2B2, showing side and top views of its predicted conformation. (b) Schematic representation of the study design for assessing the fusogenic properties of BBB-targeting LNPs using fluorescence resonance energy transfer (FRET). (c) Lipid fusion and membrane destabilization properties of C6O2 and C6O2B2 LNPs, determined by FRET at pH 5.5. (d,e) Quantification of C6O2B2 LNP dissociation via FRET upon mixing with endosome-mimicking anionic liposomes for 30 min (d) or 120 min (e) at pH 5.5, demonstrating superior lipid fusion efficiency compared to C6O2 LNPs. (f) Evaluation of BBB-targeting LNP delivery in macrophage-depleted mice. Mice were intraperitoneally injected with clodronate liposomes (200 µL per mouse) to deplete macrophages, followed by intravenous administration of luciferase mRNA-loaded LNPs (0.1 mg/kg) 2 days later. Ex vivo bioluminescence imaging and quantification of total brain flux were performed 6 h post-treatment. (g) Assessment of BBB-targeting LNP delivery in exosome-depleted mice. Mice received daily intraperitoneal injections of GW4869 (4 mg/kg) for 5 days to inhibit exosome production, followed by intravenous administration of luciferase mRNA-loaded LNPs (0.1 mg/kg) on Day 6. Ex vivo bioluminescence imaging and total brain flux quantification were performed 6 h post-treatment. (h) Evaluation of transferrin receptor 1 (TfR1)-mediated BBB-targeting LNP delivery. Mice were intravenously injected with an anti-TfR1 antibody (4 mg/kg) to block TfR1, followed by administration of luciferase mRNA-loaded LNPs (0.1 mg/kg) 2 h later. Ex vivo bioluminescence imaging and total brain flux quantification were conducted 6 h post-treatment. (i) Comparative analysis of protein corona composition among mDLNPs, Lung SORT LNPs, and BBB-targeting LNPs. (j) Functional classification of the most abundant proteins identified in LNP protein coronas. (k) Quantification of apolipoprotein A-I enrichment in the protein coronas of BBB-targeting LNPs compared to C6O2 LNPs and Lung SORT LNPs, highlighting their potential role in facilitating BBB interaction and transport. (l) Inhibition of C6O2B2 LNP uptake by endocytic pathway inhibitors (n = 3 biologically independent samples). Uptake of C6O2B2 LNPs was assessed in the presence of specific endocytic inhibitors: chlorpromazine (CMZ), a clathrin-mediated endocytosis inhibitor; genistein (GEN), a caveolae-mediated endocytosis inhibitor; amiloride (AMI), a macropinocytosis inhibitor; and methyl-β-cyclodextrin (Mβ-CD), an inhibitor of lipid raft-mediated endocytosis. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean ± SEM.#

七、研究局限与未来展望#

尽管这项工作在材料设计与递送靶向上取得了重要成果，但文中也指出了当前体系的客观局限性：

靶向特异性仍有提升空间：虽然实现了对脑部的高效偏好性富集，但由于全身血管内皮的共有特征，在心脏等外周器官的血管内皮中仍存在少量非特异性摄取与微弱转染。
体内机制细节有待进一步深化：ApoA-I蛋白冠与脑微血管特异受体（如清道夫受体SR-B1）之间的具体结合动力学及活体动态互作细节仍需更为详尽的生化解析。

作者指出，未来的研究方向将聚焦于进一步精细调控脂质化学结构以最大限度降低外周组织摄取，同时将该递送平台推向中风、脑外伤及神经退行性疾病等更多样化、更复杂的疾病模型中开展临床前转化验证。

【Adv.Mater.】酯化胆固醇偶联物实现mRNA向血脑屏障的高效靶向递送：转染效率超传统配方且完整保留屏障功能