Development of mucosal vaccine <italic style="font-style: italic">via</italic> respiratory tract based on mRNA-peptide-poloxamine nanoparticles

Qihua YANG; Yuheng LIU; Dandan ZHANG; Chao LI; Yanbi LONG; Yang CHEN; Jingjing ZHANG; Weijun ZHANG; Ping LUO; Dongshui LU; Ping CHENG; Qin XIAO; Quanming ZOU

doi:10.16016/j.2097-0927.202303115

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Development of mucosal vaccine via respiratory tract based on mRNA-peptide-poloxamine nanoparticles

Basic Medicine | 更新时间：2026-01-09

- Development of mucosal vaccine via respiratory tract based on mRNA-peptide-poloxamine nanoparticles
- Journal of Army Medical University Vol. 45, Issue 13, Pages: 1377-1387(2023)
- 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：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(82173764);the General Program of Natural Science Foundation of Chongqing(cstc2021jcyj-msxmX0136)
- DOI：10.16016/j.2097-0927.202303115
  CLC： R392.13;R392.7;R965.1
- Received：18 March 2023，
  
  Revised：2023-03-30，
  
  Published：15 July 2023
- 稿件说明：
移动端阅览
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：

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

HOGAN M J, PARDI N. mRNA vaccines in the COVID-19 pandemic and beyond[J]. Annu Rev Med, 2022, 73: 17-39. DOI: 10.1146/annurev-med-042420-112725.

FANG E Y, LIU X H, LI M, et al . Advances in COVID-19 mRNA vaccine development[J ] . Signal Transduct Target Ther, 2022, 7(1): 94. DOI: 10.1038/s41392-022-00950-y.

ROHNER E, YANG R, FOO K S, et al . Unlocking the promise of mRNA therapeutics[J ] . Nat Biotechnol, 2022, 40(11): 1586-1600. DOI: 10.1038/s41587-022-01491-z.

WITTRUP A, AI A, LIU X, et al . Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown[J ] . Nat Biotechnol, 2015, 33(8): 870-876. DOI: 10.1038/nbt.3298.

TANG J, CAI L, XU C F, et al . Nanotechnologies in delivery of DNA and mRNA vaccines to the nasal and pulmonary mucosa[J ] . Nanomaterials (Basel), 2022, 12(2): 226. DOI: 10.3390/nano12020226.

SUN L, KALLOLIMATH S, PALT R, et al . Increased in vitro neutralizing activity of SARS-C oV-2 IgA1 dimers compared to monomers and IgG[J ] . Proc Natl Acad Sci USA, 2021, 118(44): e2107148118. DOI: 10.1073/pnas.2107148118..

WANG Z J, LORENZI J C C, MUECKSCH F, et al . Enhanced SARS-CoV-2 neutralization by dimeric IgA[J ] . Sci Transl Med, 2021, 13(577): eabf1555. DOI: 10.1126/scitranslmed.abf1555.

HAMEED S A, PAUL S, DELLOSA G K Y, et al . Towards the future exploration of mucosal mRNA vaccines against emerging viral diseases; lessons from existing next-generation mucosal vaccine strategies[J ] . NPJ Vaccines, 2022, 7(1): 71. DOI: 10.1038/s41541-022-00485-x.

LAMBIASE A, SACCHETTI M, MALLONE F, et al . Evaluation of the effectiveness of BNT162b2 primary vaccination and booster dose to SARS-CoV-2 in eliciting stable mucosal immunity[J ] . Biomedicines, 2022,10(10): 2430. DOI: 10.3390/biomedicines10102430.

SHEIKH-MOHAMED S, ISHO B, CHAO G Y C, et al . Systemic and mucosal IgA responses are variably induced in response to SARS-CoV-2 mRNA vaccination and are associated with protection against subsequent infection[J ] . Mucosal Immunol, 2022, 15(5): 799-808. DOI: 10.1038/s41385-022-00511-0.

GUERRIERI M, FRANCAVILLA B, FIORELLI D, et al . Nasal and salivary mucosal humoral immune response elicited by mRNA BNT162b2 COVID-19 vaccine compared to SARS-CoV-2 natural infection[J ] . Vaccines (Basel), 2021, 9(12): 1499. DOI: 10.3390/vaccines9121499.

CHAN R W Y, CHAN K C C, LUI G C Y, et al . Mucosal antibody response to SARS-CoV-2 in paediatric and adult patients: a longitudinal study[J ] . Pathogens, 2022, 11(4): 397. DOI: 10.3390/pathogens11040397.

THOMAS A C, OLIVER E, BAUM H E, et al . Evaluation of isotype specific salivary antibody assays for detecting previous SARS-CoV-2 infection in children and adults[J ] . medRxiv, 2022. DOI: 10.1101/2022.04.11.22273690.

FRÖBERG J, GILLARD J, PHILIPSEN R, et al . SARS-CoV-2 mucosal antibody development and persistence and their relation to viral load and COVID-19 symptoms[J ] . Nat Commun, 2021, 12(1): 5621. DOI: 10.1038/s41467-021-25949-x.

GUAN S, MUNDER A, HEDTFELD S, et al . Self-assembled peptide-poloxamine nanoparticles enable in vitro and in vivo genome restoration for cystic fibrosis[J ] . Nat Nanotechnol, 2019, 14(3): 287-297. DOI: 10.1038/s41565-018-0358-x..

WRITER M J, MARSHALL B, PILKINGTON-MIKSA M A, et al . Targeted gene delivery to human airway epithelial cells with synthetic vectors incorporating novel targeting peptides selected by phage display[J ] . J Drug Target, 2004, 12(4): 185-193. DOI: 10.1080/10611860410001724459.

ALVAREZ-LORENZO C, REY-RICO A, SOSNIK A, et al . Poloxamine-based nanomaterials for drug delivery[J ] . Front Biosci (Elite Ed), 2010, 2(2): 424-440. DOI: 10.2741/e102.

REY-RICO A, CUCCHIARINI M. PEO-PPO-PEO tri-block copolymers for gene delivery applications in human regenerative medicine—an overview[J]. Int J Mol Sci, 2018, 19(3): 775. DOI: 10.3390/ijms19030775.

MCKAY P F, HU K, BLAKNEY A K, et al . Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine candidate induces high neutralizing antibody titers in mice[J ] . Nat Commun, 2020, 11(1): 3523. DOI: 10.1038/s41467-020-17409-9.

POLLOCK K M, CHEESEMAN H M, SZUBERT A J, et al . Safety and immunogenicity of a self-amplifying RNA vaccine against COVID-19: COVAC1, a phase I, dose-ranging trial[J ] . EClinicalMedicine, 2022, 44: 101262. DOI: 10.1016/j.eclinm.2021.101262.

BEISSERT T, PERKOVIC M, VOGEL A, et al . A trans-amplifying RNA vaccine strategy for induction of potent protective immunity[J ] . Mol Ther, 2020, 28(1): 119-128. DOI: 10.1016/j.ymthe.2019.09.009.

QU L, YI Z Y, SHEN Y, et al . Circular RNA vaccines against SARS-CoV-2 and emerging variants[J ] . Cell, 2022,185(10): 1728-1744. e16. DOI: 10.1016/j.cell.2022.03.044.

LAVELLE E C, WARD R W. Mucosal vaccines—fortifying the frontiers[J]. Nat Rev Immunol, 2022, 22(4): 236-250. DOI: 10.1038/s41577-021-00583-2.

BAROUCH D H. Covid-19 vaccines-immunity, variants, boosters[J]. N Engl J Med, 2022, 387(11): 1011-1020. DOI: 10.1056/NEJMra2206573.

SUN S, LI E T, ZHAO G, et al . Respiratory mucosal vaccination of peptide-poloxamine-DNA nanoparticles provides complete protection against lethal SARS-CoV-2 challenge[J ] . Biomaterials, 2023, 292: 121907. DOI: 10.1016/j.biomaterials.2022.121907.

HASSETT K J, HIGGINS J, WOODS A, et al . Impact of lipid nanoparticle size on mRNA vaccine immunogenicity[J ] . J Control Release, 2021, 335: 237-246. DOI: 10.1016/j.jconrel.2021.05.021.

LV H T, ZHANG S B, WANG B, et al . Toxicity of cationic lipids and cationic polymers in gene delivery[J ] . J Control Release, 2006, 114(1): 100-109. DOI: 10.1016/j.jconrel.2006.04.014.

CLOGSTON J D, PATRI A K. Zeta potential measurement[J ] . Methods Mol Biol, 2011, 697: 63-70. DOI: 10.1007/978-1-60327-198-1_6.

DANAEI M, DEHGHANKHOLD M, ATAEI S, et al . Impact of particle size and polydispersity index on the clinical applications of lipidic nanocarrier systems[J ] . Pharmaceutics, 2018, 10(2): 57. DOI: 10.3390/pharmaceutics10020057.

SULTANA S, ALZAHRANI N, ALZAHRANI R, et al . Stability issues and approaches to stabilised nanoparticles based drug delivery system[J ] . J Drug Target, 2020, 28(5): 468-486. DOI: 10.1080/1061186X.2020.1722137.

RUSSELL M W, MESTECKY J. Mucosal immunity: the missing link in comprehending SARS-CoV-2 infection and transmission[J]. Front Immunol, 2022, 13: 957107. DOI: 10.3389/fimmu.2022.957107.

METTELMAN R C, ALLEN E K, THOMAS P G. Mucosal immune responses to infection and vaccination in the respiratory tract[J]. Immunity, 2022, 55(5): 749-780. DOI: 10.1016/j.immuni.2022.04.013.

BUS T, TRAEGER A, SCHUBERT U S. The great escape: how cationic polyplexes overcome the endosomal barrier[J]. J Mater Chem B, 2018, 6(43): 6904-6918. DOI: 10.1039/C8TB00967H.

SMITH S A, SELBY L I, JOHNSTON A P R, et al . The endosomal escape of nanoparticles: toward more efficient cellular delivery[J ] . Bioconjug Chem, 2019, 30(2): 263-272. DOI: 10.1021/acs.bioconjchem.8b00732.

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Development of mucosal vaccine via respiratory tract based on mRNA-peptide-poloxamine nanoparticles

DOI：10.16016/j.2097-0927.202303115

摘要

Abstract

关键词

Keywords

references