颗粒稳定乳液的细胞亲和力促进抗原内化
1Graduate School of Bio-Applications and Systems Engineering,Tokyo University of Agriculture and Technology, 2Institute of Global Innovation Research,Tokyo University of Agriculture and Technology, 3State Key Laboratory of Biochemical Engineering, Institute of Process Engineering,Chinese Academy of Sciences, 4University of Chinese Academy of Sciences
Summary
为了合理设计高效的佐剂，我们开发了聚乳酸-乙醇酸纳米颗粒稳定的皮克林乳液（PNPE）。PNPE具有独特的柔软性和疏水界面，可实现有效的细胞接触，并提供高含量的抗原负载，改善递送系统与抗原呈递细胞的细胞亲和力，并诱导抗原的有效内化。
Abstract
微/纳米颗粒的细胞亲和力是细胞识别、细胞摄取和激活的先决条件，这对于药物递送和免疫反应至关重要。本研究源于通常考虑固体颗粒的电荷、大小和形状对细胞亲和力的影响，但我们很少意识到柔软性、动态重组现象和复杂界面相互作用在细胞亲和力中的重要作用。在这里，我们开发了聚乳酸-共乙醇酸（PLGA）纳米颗粒稳定的皮克林乳液（PNPE），克服了刚性形式的缺点，模拟了病原体的灵活性和流动性。建立了一种方法来测试PNPE对细胞表面的亲和力，并详细说明免疫细胞随后的内化。使用具有耗散监测功能的石英晶体微量天平（QCM-D）测定PNPE与仿生细胞外囊泡（bEVs）的亲和力 – 骨髓树突状细胞（BMDCs）的替代品，该天平可以实时监测细胞乳液粘附。随后，使用PNPE递送抗原（卵清蛋白，OVA），并使用共聚焦激光扫描显微镜（CLSM）观察BMDC对抗原的摄取。代表性结果表明，PNPE在遇到bEVs时立即降低频率（ΔF），表明PNPE对BMDC的粘附速度快且亲和力高，PNPE与细胞膜的结合明显强于PLGA微粒（PMPs）和AddaVax佐剂（表示为表面活性剂稳定的纳米乳液[SSE]）。此外，由于通过动态曲率变化和横向扩散增强了细胞对免疫细胞的亲和力，与PMP和SSE相比，抗原摄取随后得到增强。该协议为设计具有高细胞亲和力和高效抗原内化的新型制剂提供了见解，为开发高效疫苗提供了平台。
Introduction
为了对抗流行性、慢性和传染病，必须开发有效的佐剂用于预防和治疗性疫苗接种1，2。理想情况下，佐剂应具有优异的安全性和免疫活化3，4，5。抗原呈递细胞（APC）有效摄取和处理抗原被认为是下游信号级联和免疫应答启动的重要阶段6，7，8。因此，清晰了解免疫细胞与抗原相互作用的机制，设计佐剂以增强内化是提高疫苗效率的有效策略。
具有独特性质的微/纳米颗粒先前已被研究为抗原递送系统，以介导抗原的细胞摄取以及与病原体相关分子模式的细胞相互作用9，10。与细胞接触后，输送系统开始与细胞外基质和细胞膜相互作用，导致内化和随后的细胞反应11，12。以前的研究表明，颗粒的内部化是通过细胞膜 – 颗粒粘附13发生的，然后是细胞膜的柔性变形和受体扩散到表面膜14，15。在这种情况下，输送系统的性质取决于对APC的亲和力，这随后影响摄取量16，17。
为了深入了解改善免疫反应的输送系统的设计，已经开展了广泛的工作，研究颗粒的性质与细胞摄取之间的关系。本研究源于观察结果，即具有各种电荷，大小和形状的固体微/纳米颗粒经常从这个角度进行研究，而流动性在抗原内化中的作用很少被研究18，19。事实上，在粘附过程中，软颗粒表现出动态曲率变化和横向扩散，以增加多价相互作用的接触面积，这是固体颗粒20，21难以复制的。此外，细胞膜在摄取部位是磷脂双层（鞘脂或胆固醇），疏水物质可以改变脂质的构象熵，减少细胞摄取所需的能量22，23。因此，扩增流动性和促进递送系统的疏水性可能是加强抗原内化以增强免疫应答的有效策略。
皮克林乳液，由固体颗粒稳定地组装在两种不混溶液体之间的界面处，已广泛应用于生物领域24，25。事实上，油/水界面上的聚集颗粒决定了多级结构的形成，促进了多级递送系统-细胞相互作用，并进一步诱导药物递送中的多功能理化性质。由于其变形性和横向移动性，Pickering乳液有望与免疫细胞进行多价细胞相互作用，并被膜蛋白识别26。此外，由于皮克林乳液中的油性胶束芯没有完全被固体颗粒覆盖，皮克林乳液在油/水界面上的颗粒之间具有不同大小的间隙，从而导致更高的疏水性。因此，探索皮克林乳液与APC的亲和力并详细说明随后的内化以开发有效的佐剂至关重要。
基于这些考虑，我们设计了一种PLGA纳米颗粒稳定的皮克林乳剂（PNPE）作为流动性疫苗递送系统，这也有助于获得有关PNPE与BMDC和细胞内化的亲和力的宝贵见解。使用具有耗散监测功能的石英晶体微量天平（QCM-D）通过无标记方法 监测 仿生细胞外囊泡（bEV;BMDC的替代品）对PNPE的实时粘附进行监测。在表征PNPE对BMDC的亲和力后，使用共聚焦激光扫描显微镜（CLSM）来确定抗原摄取。结果表明，PNPE对BMDC的亲和力更高，抗原内化有效。我们预计PNPE将对APC表现出更高的亲和力，这可能更好地刺激抗原的内化以增强免疫反应。
Protocol
本协议中描述的所有方法均已获得中国科学院过程工程研究所的批准。所有动物实验均严格按照《实验动物护理使用条例》和《动物伦理审查指南》（GB/T35892-2018）进行。 1. PLGA纳米粒子的制备与表征 聚血游病毒聚碳酸酯纳米颗粒的制备在90°C下将0.5g聚乙烯醇（PVA）加入120mL去离子水中，搅拌至完全溶解以制备PVA溶液。冷却至室温后将溶液储存在冰箱…
Representative Results
使用简单的一步超声检测来获得PNPE。首先，我们制备了均匀的PNP作为固体稳定剂（图1A）。通过SEM观察PNP的形态，表明它们大多是均匀和球形的（图1B）。通过DLS 检测 配方的流体动力学尺寸和zeta电位。PNP的直径为187.7 ± 3.5 nm，zeta电位为-16.4 ± 0.4 mV（图1C 和 补充表1）。为了制备PNPE，将PNP溶液（0.4%，w / v）和?…
Discussion
我们开发了PLGA纳米颗粒稳定的油/水乳液作为增强抗原内化的递送系统。制备的PNPE具有密集的表面以支持着陆点，并具有独特的柔软性和流动性，可与免疫细胞膜进行有效的细胞接触。此外，油/水界面提供了高含量的抗原负载，两亲性PLGA赋予PNPE高稳定性，用于将抗原转运到免疫细胞。PNPE可以快速粘附在细胞表面，表明PLGA纳米颗粒稳定的乳液对细胞膜具有很强的亲和力，用于细胞摄取。此外，PN…
Declarações
The authors have nothing to disclose.
Acknowledgements
本工作得到了国家重点研发计划（2021YFE020527、2021YFC2302605、2021YFC2300142）、中国科学院基础前沿科学研究计划（ZDBS-LY-SLH040）0-1原创创新项目、国家自然科学基金创新课题组基金（批准号21821005）的支持。
Materials
| AddVax | InvivoGen | Vac-adx-10 | |
| Cell Strainer | Biosharp | BS-70-CS | 70 μm |
| Confocal Laser Scanning Microscope (CLSM) | Nikon | A1 | |
| Cy3 NHS Ester | YEASEN | 40777ES03 | |
| DAPI Staining Solution | Beyotime | C1005 | |
| Fetal Bovine Serum (FBS) | Gibco | 16000-044 | |
| FITC Phalloidin | Solarbio | CA1620 | |
| Mastersizer 2000 Particle Size Analyzer | Malvern | ||
| Micro BCA protein Assay Kit | Thermo Science | 23235 | |
| Membrane emulsification equipment | Zhongke Senhui Microsphere Technology | FM0201/500M | |
| Mini-Extruder | Avanti Polar Lipids, Inc | ||
| NANO ZS | Malvern | JSM-6700F | |
| Polycarbonate membranes | Avanti Polar Lipids, Inc | ||
| Poly (lactic-co-glycolic acid) (PLGA) | Sigma-Aldrich | 26780-50-7 | Mw 7,000-17,000 |
| Poly-L-lysine Solution | Solarbio | P2100 | |
| Poly (vinyl alcohol) (PVA) | Sigma-Aldrich | 9002-89-5 | |
| QSense Silicon dioxide sensor | Biolin Scientific | QSX 303 | Surface roughness < 1 nm RMS |
| Quartz Crystal Microbalance | Biosharp | Q-SENSE E4 | |
| RPMI Medium 1640 basic | Gibco | C22400500BT | L-Glutamine, 25 mM HEPES |
| Scanning Electron Microscopy (SEM) | JEOL | JSM-6700F | |
| Squalene | Sigma-Aldrich | 111-02-4 |
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Cao, F., Ming, Y., Gao, W., Ge, J., Ogino, K. Cellular Affinity of Particle-Stabilized Emulsion to Boost Antigen Internalization. J. Vis. Exp. (187), e64406, doi:10.3791/64406 (2022).