药物传递系统的发展旨在提高药物的疗效并降低其毒性。脂质体作为一种生物相容性好、非免疫原性的药物载体，自20世纪70年代以来便受到广泛关注。随着纳米技术的进步，纳米脂质体和脂质纳米颗粒在药物传递领域的应用日益广泛。特别是mRNA-LNP疫苗的成功应用，进一步推动了脂质基纳米载体技术的发展。尽管挤压法是当前工业生产纳米脂质体的主流方法，但其生产过程中需在脂质相变温度以上进行，且常伴随多步操作和潜在的膜材料降解风险。相比之下，微流控技术提供了一种连续、可控且低温的生产方式，有望解决传统方法中的诸多问题。Regina Bleul团队在Journal of Controlled Release上发表名为“Comparing continuous micromixing and extrusion downsizing for PEGylated nanoliposomes remotely loaded with doxorubicin or the steroid pro-drug methylprednisolone hemisuccinate”的研究。本研究通过比较微流控连续制备与挤压法制备的纳米脂质体，旨在为纳米药物的生产提供科学依据和技术支持。

Fig. 1. Schematic comparison of the conventional liposome production method using the batch process with MLV production followed by extrusion steps (top) for downsizing and the microfluidic approach presented here (bottom) using a micromixer (instead of extruder) in which the desired nanoliposomes are produced by one step and without the need to work above the lipid phase transition temperature.

01
实验结果

1.1 脂质体制备与表征：

本研究通过微流控技术和传统的挤出技术分别制备了聚乙二醇化（PEGylated）纳米脂质体，并比较了两种方法在制备远程主动加载药物产品方面的效果。实验中使用了两种不同的盐梯度（硫酸铵和醋酸钙）来远程加载药物分子，分别是阿霉素（doxorubicin，DXR）和甲基泼尼松龙琥珀酸钠（methylprednisolone hemisuccinate，MPS）。

Table 1 Comparison of the technical parameters of all tested micromixers for continuous liposome fabrication.

1.2 粒径分布与形态学特征：

通过微流控法制备的聚乙二醇化纳米脂质体展现出显著的粒径均一性。具体而言，使用split-and-recombine微混合器（CAT300和CAT600）在不同总流速（TFR）条件下制备的脂质体，其平均粒径稳定在80±10 nm范围内，多分散指数（PDI）均低于0.1，SPAN值小于0.75（表2）。这一结果显著优于传统挤压法制备的脂质体，后者在相同条件下测得的PDI和SPAN值明显较高，表明粒径分布较宽。Cryo-TEM图像进一步证实了微流控法制备脂质体的优越性。如图2所示，微流控法制备的脂质体呈现出高度均一的球形结构，膜结构清晰，内部水相均匀分布。相比之下，挤压法制备的脂质体则表现出更多的非均一性，包括不规则形状和多室结构。

Table 2 Comparison of physicochemical characteristics of nanoliposomes with ammonium sulfate gradient obtained with different micromixers at various flow rate scales

Fig. 2. Cryogenic Transmission Electron Microscopy (cryo-TEM) image of continuously manufactured liposomes with CAT300 micromixer before DXR remote loading as representative example.

1.3 药物负载效率：

在药物负载方面，微流控法制备的脂质体同样表现出色。在甲基泼尼松龙琥珀酸钠（MPS）负载实验中，微流控法制备的脂质体实现了92%的高负载效率。电镜结果显示，脂质体内存在明显的纳米沉淀，并与文献报道的挤出法结果类似（图3）。除此之外，在药物释放动力学上表现出更稳定的性能（图4）。具体而言，微流控法制备的脂质体在体外释放实验中的t1/2值为90小时，略长于挤压法制备的脂质体（t1/2值为75小时），表明其具有更好的药物控释能力。

Fig. 3. Cryogenic transmission electron microscopy images of continuous manufactured liposomes before (a) and after (b) remote loading with MPS.

Fig. 4. Comparison of release kinetics (“dissolution” assay) of MPS at 37 ◦C in human plasma from nanoliposomes produced using a microfluidic-based process and conventional extrusion.

对于阿霉素（DXR）负载的脂质体，药物负载效率达到95%以上，与挤压法制备的脂质体相当（表3）。与市售的挤出法制备的制剂相比（图6），特别是Doxil®类似物，微流控法制备的脂质体在轴向比（elongatedness）和形状分布上表现出更高的均一性（表4）。

Table 3 Physicochemical characterization of liposome samples with calcium acetate gradient directly after formulation, after diafiltration and after remote drug loading with MPS. Size, size distribution and Zeta potential were measured using a Zetasizer nanoZS. 

Fig. 6. CryoTEM images of Doxil®-like liposomes prepared by a) Fraunhofer IMM using microfluidics, further processed, and loaded with Doxorubicin by Ayana Pharma; b) Caelyx® and c) Ayana Pharma starting with stepwise extrusion downsizing. Scalebar 200 nm. 

Table 4 Cryo-TEM comparison of Doxil®-like liposomes prepared with either microfluidics or extrusion.Analysis was done by measurement of multiple images with a total of N > 1000 particles as representative sample according to reference

1.4 热力学性质：

差示扫描量热法（DSC）测量结果显示，微流控法制备的脂质体在热力学性质上与传统挤压法制备的脂质体存在显著差异（表5，图5）。具体而言，微流控法制备的脂质体具有更窄的相变峰（ΔT1/2 = 10.15°C）和更低的焓变（ΔH = 1.63 kcal·mol⁻¹），表明其膜结构更为均匀和有序。相比之下，挤压法制备的脂质体相变峰较宽（ΔT1/2 = 14.78°C），且焓变较高（ΔH = 2.00 kcal·mol⁻¹），反映出其膜结构的不均一性。

Table 5 Thermodynamic parameters and size distribution of nanoliposomes with calcium acetate transmembrane gradient produced using a microfluidic based process and conventional extrusion.

Fig. 5. DSC thermograms of nanoliposomes with calcium acetate transmembrane gradient produced using a microfluidic based process and conventional extrusion

1.5 稳定性测试：

长期稳定性测试表明，微流控法制备的脂质体在储存过程中表现出优异的稳定性。无论是含有高残留乙醇含量（<380 ppm）还是低残留乙醇含量（<96 ppm）的脂质体，在24个月的储存期内，其粒径分布和药物负载效率均未发生显著变化（图7和图8）。特别是Doxil®类似物的稳定性测试中，微流控法制备的脂质体在42个月的观察期内，其粒径和药物含量均保持稳定，进一步证实了微流控法制备脂质体的长期稳定性。此外，通过监测不同时间点的导电性，确认了脂质体在储存过程中外水相离子浓度的稳定性，从而间接反映了脂质体膜结构的完整性。这些结果共同表明，微流控法制备的脂质体在长期储存和释放过程中能够保持其原有的稳定性和释放效果。

Fig. 7. a) Independence of liposome size distribution from the ethanol content in the range from below 96 ppm to up to 380 ppm. These Parameters were followed up for 24 months at 5◦ ± 3 ◦C. b) Independence of extent of doxorubicin encapsulation from ethanol content in the range from below 96 ppm to up to 380 ppm. These Parameters were followed up for 24 months at 5◦ ± 3 ◦C.

Fig. 8. Long-term stability of microfluidically prepared liposomes (three individual production runs) that comply with FDA guidelines for ethanol content of <100 ppm. DLS-Data was analyzed using One way ANOVA showing no significance regarding changes in size and PDI during the entire observation period.

02
总结

本研究通过详细的实验比较了微流控技术和传统挤出技术在制备PEGylated纳米脂质体方面的性能。结果表明，微流控技术不仅能够制备出粒径分布更窄、形态更均匀的纳米脂质体，而且在药物加载效率、热力学稳定性和长期稳定性方面均表现出色。此外，微流控技术还具有生产规模灵活可调、工艺控制精确等优点，为纳米脂质体的工业化生产提供了新的解决方案。

参考文献：Comparing continuous micromixing and extrusion downsizing for PEGylated nanoliposomes remotely loaded with doxorubicin or the steroid pro-drug methylprednisolone hemisuccinate. https://doi.org/10.1016/j.jconrel.2025.113707