Eudragit L100修饰的铝锰双金属有机框架作为猪流行性腹泻灭活疫苗口服递送载体的初步评价

doi:10.11843/j.issn.0366-6964.2025.05.027

摘要/Abstract

摘要：

本研究旨在对制备的Eudragit L100修饰的铝锰双金属有机框架纳米粒作为灭活猪流行性腹泻病毒(porcine epidemic diarrhea virus，PEDV)口服递送载体进行评价。利用金属离子Al³⁺、Mn²⁺和2-氨基对苯二甲酸有机配体之间的配位作用，将灭活PEDV原位封装Al/Mn-MOF中, 得到Al/Mn-MOF@PEDV纳米粒。通过静电相互作用，将阴离子聚合物Eudragit L100结合于Al/Mn-MOF@PEDV纳米粒表面，得到L100@Al/Mn-MOF@PEDV纳米粒。采用扫描电子显微镜考察其形态、纳米粒度电位仪测定表面电势、傅里叶红外光谱仪分析组成成分、X射线衍射仪测定晶格结构。采用ELISA和抗原检测卡表征对灭活PEDV的负载效率。配制模拟胃液考察L100@Al/Mn-MOF@PEDV纳米粒的稳定性，并用ELISA测定灭活PEDV的释放率。使用激光共聚焦观察巨噬细胞对Al/Mn-MOF@PEDV纳米粒的摄取。采用CCK8法测定不同浓度的纳米粒的细胞毒性。采用ELISA试剂盒测定Al/Mn-MOF@PEDV纳米粒对巨噬细胞分泌免疫调节因子的影响。结果表明，L100@Al/Mn-MOF@PEDV纳米粒成功制备，尺寸小于1 μm。在PEDV/金属离子/有机配体投料比为0.42 ∶1 ∶1时，对灭活PEDV的负载率可达到99.6%。L100@Al/Mn-MOF@PEDV在模拟胃液中孵育2 h仍具有高稳定性，对灭活PEDV的释放率仅为12.73%左右。在小鼠RAW264.7细胞的摄取试验中，用激光共聚焦观察到细胞对Al/Mn-MOF@PEDV摄取情况优于未经负载的灭活PEDV。且Al/Mn-MOF@PEDV能够显著增强RAW264.7细胞中Th1型因子IFN-γ和Th2型因子IL-4的表达。本研究为PED灭活疫苗口服载体的设计提供了新思路，并为后续应用于动物免疫的研究奠定基础。

关键词: 猪流行性腹泻病毒, 灭活疫苗, 纳米载体, 金属有机框架, 口服递送

Abstract:

This study aims to evaluate aluminum-manganese bimetallic organic framework nanoparticles modified with Eudragit L100 as an oral delivery vehicle for inactivated porcine epidemic diarrhea virus (PEDV). By utilizing the coordination interaction between metal ions (Al³⁺, Mn²⁺) and the organic ligand 2-aminoterephthalic acid, inactivated PEDV is encapsulated in situ within Al/Mn-MOF to form Al/Mn-MOF@PEDV nanoparticles. An anionic polymer, Eudragit L100, is then electrostatically attached to the surface of Al/Mn-MOF@PEDV nanoparticles, producing L100@Al/Mn-MOF@PEDV nanoparticles. The morphology is examined using scanning electron microscopy, the surface charge is measured by zeta potential meter, the composition is analyzed by fourier transform infrared spectroscopy, and the crystal structure is determined by X-ray Diffraction. The loading efficiency of inactivated PEDV is characterized using ELISA and antigen detection cards. The stability of L100@Al/Mn-MOF@PEDV nanoparticles in simulated gastric fluid and the release rate of inactivated PEDV are examined using ELISA. Macrophage uptake of the nanoparticles is observed using confocal laser scanning microscopy. The cytotoxicity of nanoparticles with different concentrations is determined by CCK8 method, and the effect on the secretion of immunomodulatory factors by macrophages is evaluated using ELISA kits. Results indicate successful preparation of L100@Al/Mn-MOF@PEDV nanoparticles, with sizes under 1 μm. At a PEDV/metal ion/organic ligand ratio of 0.42 ∶1 ∶1, the loading rate of inactivated PEDV reaches 99.6%. After 2 hours in simulated gastric fluid, L100@Al/Mn-MOF@PEDV nanoparticles maintained high stability with only about 12.73% release rate of inactivated PEDV. Uptake experiments in mouse RAW264.7 cells show enhanced uptake of Al/Mn-MOF@PEDV compared to unencapsulated inactivated PEDV. Additionally, Al/Mn-MOF@PEDV significantly boosts the expression of Th1-type factor IFN-γ and Th2-type factor IL-4 in RAW264.7 cells. This research offers new insights into the design of oral carriers for inactivated PED vaccines and lays the foundation for future applications in animal immunization.

Key words: PEDV, inactivated vaccine, nanoparticle carrier, metal-organic framework, oral delivery

中图分类号:

S855.3

胡米, 沈瑶歆, 范宝超, 孙敏, 周金柱, 郭容利, 李彬. Eudragit L100修饰的铝锰双金属有机框架作为猪流行性腹泻灭活疫苗口服递送载体的初步评价[J]. 畜牧兽医学报, 2025, 56(5): 2292-2230.

HU Mi, SHEN Yaoxin, FAN Baochao, SUN Min, ZHOU Jinzhu, GUO Rongli, LI Bin. Evaluation of the Characteristics of Eudragit L100-Modified Aluminum-Manganese Metal-Organic Framework as an Oral Delivery Vehicle for Inactivated Porcine Epidemic Diarrhea Vaccine[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(5): 2292-2230.

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试验方案 Protocol	灭活PEDV/金属离子/有机配体投料比 Feeding ratio of PEDV/Metal ion/Organic linker	负载效率/% Loading efficiency (LE)
1	0.42∶1.0∶1.0	56.3
2	0.21∶1.0∶1.0	85.7
3	0.14∶1.0∶1.0	99.6

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