脂质纳米颗粒（LNP）是mRNA肝脏递送的主流非病毒载体。经典配方由四大组分构成：可电离脂质、辅助磷脂、胆固醇和PEG脂质。过去十年，研究者的目光几乎都聚焦在可电离脂质上，而辅助磷脂常只被视为LNP填充用的膜材。此外，mRNA蛋白质替代疗法需要长期反复给药（每周、两周或三周一次），而多数关于辅助脂质的研究都基于单次给药。这种研究方式可能适用于疫苗场景的情况（1-3次给药），但不一定适合需要终身给药的代谢性疾病治疗。近日，上海交通大学医学院附属仁济医院肝外科团队和迈安纳团队在Materials Today Bio上发表题为“Elucidate the structural role of helper lipids in modulating hepatic expression following repeated intravenous administration of mRNA-LNPs”的研究，并系统回答了这个问题。

迈安纳微流控XNano系列设备及试剂盒

实验设计：六种磷脂 × 三种间隔 × 四次注射

研究者选用六种辅助磷脂，覆盖三个结构维度（图1A），给药方案：四次尾静脉注射，间隔分别为1周、2周、3周，每次检测：肝脏荧光素酶表达、抗PEG IgM/IgG、ALT/AST、IL-6/TNF-α：

Fig. 1. The types and ratios of lipids in LNPs, and the effects of different phospholipids on the characterization of LNPs. (A) The ionizable lipid, phospholipids, cholesterol, and PEG-lipid used for the preparation of mRNA-LNPs.

Fig. 2. Investigation on the effects of different phospholipids on in vitro mRNA expression, cellular uptake, and endosomal escape of the LNPs. (A-B) Luciferase expression in AML12 cells: (A) effects of phospholipid chain length, acyl unsaturation, and polar head group; (B) correlation between chain length and expression.

Fig. 3. Effects of helper phospholipids on in vivo mRNA expression, anti-PEG antibodies, liver enzymes, and cytokines under weekly intravenous administration in BALB/c mice. (A) Schematic of the experimental workflow. (B) Representative bioluminescence images at 6 and 24 h after the 1st, 2nd, 3rd, and 4th injections.

· IL-6与烷基链长呈正相关（每周、每两周）：DSPC（C18）> DPPC（C16）> DMPC（C14）（图3I）

· DSPC（临床标准配方）在每周注射中诱导最高IL-6，对需要长期、频繁给药的蛋白替代疗法而言，这可能不是理想选择

· 延长注射间隔至每三周后，链长-IL-6相关性消失（图4S-T），说明炎症反应不仅取决于脂质结构，还高度依赖于给药频率

ALT/AST波动：

· 所有检测值基本维持在Balb/c小鼠正常范围（ALT 15–84 U/L，AST 54–298 U/L）

· 每周注射时ALT在第三次注射时达峰（非首次），说明单次给药研究可能低估或误判肝酶反应

Fig. 3. Effects of helper phospholipids on in vivo mRNA expression, anti-PEG antibodies, liver enzymes, and cytokines under weekly intravenous administration in BALB/c mice. (A) Schematic of the experimental workflow. Grouped by helper lipid structural parameters: Carbon chain length (DMPC, DPPC, DSPC): effects on luciferase expression (C), anti-PEG IgM (E), ALT (G), and IL-6 (I). Headgroup and unsaturation (DOPC, DSPC, DOPE, DSPE): effects on luciferase expression (D), anti-PEG IgM (F), ALT (H), and IL-6 (J).

在所有测试条件下，DSPE表现最佳：

· 体外：DSPE在三种肝细胞系中表达均最高（图2A）

· 体内单次：DSPE是DSPC的20倍（第一次注射，图3D）

· 多次注射（每周）：前三次注射DSPE均显著优于DSPC，直到第四次优势缩小

· 更换离子化脂质仍有效：用ALC-0315时，DSPE比DSPC高1.6倍；用MC3时高3.1倍（图6A-B）

· 基因编辑：DSPE递送PCSK9 Cas9 mRNA/sgRNA，编辑效率达~60%，是DSPC的4倍（图6E），血清PCSK9降低99.4%（图6D）

· 蛋白替代：两次注射hOTC mRNA后，DSPE的OTC蛋白表达是DSPC的2.8–11.2倍（图6G-H）

Fig. 6. (A-B) Compared to the control phospholipid DSPC, the helper phospholipid DSPE enhanced the in vivo expression of Luciferase mRNA delivered by LNPs formulated with the ionizable lipids ALC0315 (A), MC3 (B). (C) Schematic of the experimental workflow for investigating the PCSK9 knockdown efficacy and gene-editing efficiency in C57BL/6 mice of PCSK9 gene editors delivered by LNPs formulated with DSPE. (D) Compared with the DSPC-based LNP, DSPE-based LNPs delivering PCSK9 gene editor exhibited greater reductions in serum PCSK9 levels at days 5, 8, and 14 postadministrations. (E) Compared with the control phospholipid DSPC, DSPE enhanced the editing efficiency of LNP-delivered PCSK9 gene editor in mouse liver. (F) Schematic of the experimental workflow for investigating the hepatic expression of human OTC following repeated administration at a two-week interval of human OTC mRNA/LNP. (G) Western blot analysis showed the expression of human OTC protein in BALB/c mice at 24 h and 13 days after two administrations. (H) Quantitative analysis of the Western blot showing the expression of human OTC protein.

第一步：更强的ApoE吸附可介导更高细胞摄取

蛋白质冠分析显示（图2F），DSPE-LNP吸附的ApoE水平最高之一（与DOPC、DOPE并列）。ApoE丰度与细胞摄取呈正相关（图2G）：更高ApoE吸附导致更多LDLR介导的内吞。

第二步：更低pKa使得内体逃逸的效率更高

DSPE-LNP的表观pKa仅6.6，是所有配方中最低的。这意味着：

在血液（pH 7.4）：几乎不带正电荷，可减少非特异性吸附和毒性

在内体（pH 5.5–6.5）：大量质子化，可与内体膜负电荷结合，触发膜融合

第三步：PE头基的“锥形”几何促进自发形成HII相

PE头基比PC头基更小，呈锥形（而非PC的圆柱形）。锥形几何天然倾向负曲率结构，即反六角相（HII），这是已知的高融合性脂质相，能有效扰动内体膜，释放mRNA。验证实验（图2J）：用巴弗洛霉素A1阻断内体酸化后，DSPE-LNP的表达降至低于DOPC-LNP的水平。这表明：DSPE的优势依赖于酸触发的内体逃逸；若非如此，阻断酸化的效果不会如此剧烈。

Fig. 2. Investigation on the effects of different phospholipids on in vitro mRNA expression, cellular uptake, and endosomal escape of the LNPs. (A-B) Luciferase expression in AML12 cells: (A) effects of phospholipid chain length, acyl unsaturation, and polar head group; (B) correlation between chain length and expression. Cellular uptake of Cy5-LNPs: (C) fluorescence microscopy images in AML12, Hep1-6, and HepG2 cells at 4 h; (D) flow cytometry quantification in AML12 cells; (E) effects of chain length, unsaturation, and head group on uptake in AML12 cells. Serum protein corona: (F) relative ApoE abundance on LNPs; (G) correlation between ApoE abundance and uptake in AML12 cells. Endosomal escape in AML12 cells: (H) colocalization analysis with lysosomes; (I) representative images at 3 h post-delivery of Cy5-mRNA. (J) Inhibition of endosomal acidification reduced the in vitro expression of Luciferase mRNA/LNP.