基于多肽-泊洛沙胺纳米颗粒载体的呼吸道黏膜mRNA疫苗研究

杨七华; 刘宇恒; 张丹丹; 李超; 龙艳碧; 陈阳; 张靖靖; 张卫军; 罗萍; 鲁东水; 程平; 肖琴; 邹全明

doi:10.16016/j.2097-0927.202303115

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基于多肽-泊洛沙胺纳米颗粒载体的呼吸道黏膜mRNA疫苗研究

基础医学 | 更新时间：2026-01-09

- 基于多肽-泊洛沙胺纳米颗粒载体的呼吸道黏膜mRNA疫苗研究
- Development of mucosal vaccine via respiratory tract based on mRNA-peptide-poloxamine nanoparticles
- 陆军军医大学学报 2023年45卷第13期页码：1377-1387
- 作者机构：
  
  1.671003 云南大理，大理大学药学院
  2.400038 重庆，陆军军医大学(第三军医大学)药学与检验医学系
- 作者简介：
  
  [ 肖琴，E-mail：XZ953179866@163.com XIAO Qin, E-mail: XZ953179866@163.com
  [ 邹全明，E-mail：qmzou2007@163.com ZOU Quanming, E-mail: qmzou2007@163.com
- 基金信息：
  
  国家自然科学基金面上项目(82173764);重庆自然科学基金面上项目(cstc2021jcyj-msxmX0136)
- DOI：10.16016/j.2097-0927.202303115
  中图分类号： R392.13;R392.7;R965.1
- 收稿：2023-03-18，
  
  修回：2023-03-30，
  
  纸质出版：2023-07-15
- 稿件说明：
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杨七华, 刘宇恒, 张丹丹, 等. 基于多肽-泊洛沙胺纳米颗粒载体的呼吸道黏膜mRNA疫苗研究[J]. 陆军军医大学学报, 2023,45(13):1377-1387.

Qihua YANG, Yuheng LIU, Dandan ZHANG, et al. Development of mucosal vaccine via respiratory tract based on mRNA-peptide-poloxamine nanoparticles[J]. Journal of Army Medical University, 2023, 45(13): 1377-1387.
杨七华, 刘宇恒, 张丹丹, 等. 基于多肽-泊洛沙胺纳米颗粒载体的呼吸道黏膜mRNA疫苗研究[J]. 陆军军医大学学报, 2023,45(13):1377-1387. DOI： 10.16016/j.2097-0927.202303115.

Qihua YANG, Yuheng LIU, Dandan ZHANG, et al. Development of mucosal vaccine via respiratory tract based on mRNA-peptide-poloxamine nanoparticles[J]. Journal of Army Medical University, 2023, 45(13): 1377-1387. DOI： 10.16016/j.2097-0927.202303115.

摘要

目的

设计并制备mRNA-多肽-泊洛沙胺纳米颗粒(mRNA peptide polymer ternary complex

mRNA-PPTC)，考察其理化特性、体外和体内呼吸道组织相关细胞递送效率、免疫后小鼠特异性抗体诱导能力。

方法

通过体外转录分别制备编码萤火虫荧光素酶(firefly luciferase

F-luc)、增强型绿色荧光蛋白(enhanced green fluorescent protein

EGFP)和新冠病毒受体结合域蛋白(receptor binding domain

RBD)的mRNA，利用动态光散射法检测mRNA-PPTC的粒径、多分散系数、Zeta电位。通过透射电镜观察纳米颗粒形态。将F-luc mRNA-PPTC及对照组制剂分别转染至人支气管上皮细胞(16 human bronchial epithelial cell

16HBE)、鼠源树突状细胞(dendritic cell 2.4

DC2.4)、鼠源巨噬细胞(mouse leukemia cells of monocyte macrophage

RAW264.7)，检测荧光素酶报告基因转染效率和细胞活性；将EGFP mRNA-PPTC及对照组制剂转染至16HBE细胞，使用荧光显微镜观察EGFP表达情况。利用活体成像技术考察F-luc mRNA-PPTC在小鼠呼吸道的转染效率。选用编码RBD蛋白的mRNA作为疫苗抗原模型，通过酶联免疫吸附实验检测免疫后小鼠血清中抗原特异性免疫球蛋白G(immunoglobulin G

IgG)和支气管肺泡灌洗液(bronchoalveolar lavage fluid

BALF)中抗原特异性分泌型免疫球蛋白A(secretory immunoglobulin A

sIgA)水平。

结果

成功制备粒径约100 nm近球形且均一稳定的弱阳离子mRNA-PPTC纳米颗粒；mRNA-PPTC能有效转染16HBE、DC2.4、RAW264.7细胞，与对照组比较差异有统计学意义(

0.01)，并且细胞毒性较低；mRNA-PPTC通过滴鼻免疫后，在小鼠鼻部和肺组织中检测到F-luc报告基因的有效表达，与对照组比较差异有统计学意义(

0.01)；在初免后第28天的小鼠血清和BALF样品中，能分别检测到高效价的RBD抗原特异性IgG和sIgA，与对照组比较差异有统计学意义(

0.01

0.05)。

结论

mRNA-PPTC纳米颗粒有良好的体外mRNA递送能力且细胞毒性较低；滴鼻给药后在小鼠上下呼吸道均能高效表达目的基因；小鼠滴鼻免疫后能产生高水平的抗原特异性血清IgG和支气管肺泡灌洗液sIgA。

Abstract

Objective

To design and prepare mRNA-pe

ptide-poloxamine ternary complex (mRNA-PPTC) nanoparticles and explore their physicochemical properties

in vitro

vivo

delivery efficiency

and induction of specific antibody production in immunized mice.

Methods

The mRNAs coding firefly luciferase (F-luc)

enhanced green fluorescent protein (EGFP) and SARS-CoV-2 receptor binding domain (RBD) were prepared through

in vitro

transcription. The size

polydispersity (PDI) and Zeta potential of the mRNA-PPTC nanoparticles were studied with dynamic light scattering

and their morphology was observed with transmission electron microscopy (TEM). After F-luc reporter gene was loaded in the mRNA-PPTC nanoparticles

the obtained F-luc mRNA-PPTC nanoparticles were used to transfect human bronchial epithelial cell line 16HBE

mouse dendritic cell line DC2.4 and mouse macrophage cell line RAW264.7. The transfection efficiency and cell viability were observed with luminescence reporter assay. Fluorescence microscopy was employed to observe the expression of EGFP in EGFP mRNA-PPTC nanoparticles-transfected 16HBE cells.

In vivo

imaging system was adopted to investigate the delivery efficiency through the intranasal administration in mice. Then

RBD mRNA was used as antigen for vaccine preparation

the levels of specific IgG in the serum and sIgA in bronchoalveolar lavage fluid (BALF) were detected with ELISA after the mice were immunized with RBD mRNA-PPTC nanoparticles.

Results

Our mRNA-PPTC nanoparticles were successfully assembled

as weak cationic nanoparticles in sphere shape

in a diameter of about 100 nm

and with low PDI. The obtained mRNA-PPTC could effectively transfect 16HBE

DC2.4 and RAW264.7 cells (

controls

0.01) with low cytotoxicity. The mRNA-PPTC nanoparticles effectively expressed the reporter gene F-luc in the nose and lung of mice immunized though an intranasal route (

controls

0.01). High titers of antigen-specific IgG and sIgA were found in the serum and BALF from the mice in 28 d after intranasal immunization with RBD mRNA-PPTC (

controls

0.01

0.05).

Conclusion

Our prepared mRNA-PPTC have good capacity to delivery mRNA

in vitro

without significant cytotoxicity and

in vivo

at respiratory mucosal site

and can induce high levels of specific serum IgG and BALF sIgA by intranasal inoculation.

关键词

Keywords

references

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