土贝母皂苷丙纳米乳的构建及其佐剂效应评价

WEI Jing; LIU Shulin; YE Yan; XU Mingqi; SONG Zhen; DENG Yan; SUN Hongwu; MA Lei; LI Haibo

doi:10.16016/j.2097-0927.202501034

您当前的位置：

首页 >

文章列表页 >

土贝母皂苷丙纳米乳的构建及其佐剂效应评价

Pharmacy | 更新时间：2025-04-25

- 土贝母皂苷丙纳米乳的构建及其佐剂效应评价
- Journal of Army Medical University Vol. 47, Issue 8, Pages: 784-793(2025)
- 作者机构：
  
  1.佳木斯大学临床医学院，黑龙江佳木斯
  2.陆军军医大学（第三军医大学）药学与检验医学系微生物与生化药学教研室，国家免疫生物制品工程技术研究中心，重庆
  3.重庆市沙坪坝区陈家桥医院检验科，重庆
- 作者简介：
  
  MA Lei， E-mail： maleijms@163.com
  LI Haibo， E-mail： lihaibo@tmmu.edu.cn
- 基金信息：
  
  the National Key Research and Development Program of China(2021YFC2302603);the General Program of National Natural Science Foundation of China(32270988)
- DOI：10.16016/j.2097-0927.202501034
  CLC： R282.71;R944.9;R969.2
- Received：13 January 2025，
  
  Revised：06 February 2025，
  
  Published：30 April 2025
- 稿件说明：
移动端阅览
魏静,刘姝琳,叶演,等.土贝母皂苷丙纳米乳的构建及其佐剂效应评价[J].陆军军医大学学报,2025,47(08):784-793.

WEI Jing,LIU Shulin,YE Yan,et al.Preparation of tubeimoside Ⅲ nanoemulsion and evaluation of its adjuvant effect[J].J Army Med Univ,2025,47(08):784-793.
魏静,刘姝琳,叶演,等.土贝母皂苷丙纳米乳的构建及其佐剂效应评价[J].陆军军医大学学报,2025,47(08):784-793. DOI：10.16016/j.2097-0927.202501034.

WEI Jing,LIU Shulin,YE Yan,et al.Preparation of tubeimoside Ⅲ nanoemulsion and evaluation of its adjuvant effect[J].J Army Med Univ,2025,47(08):784-793. DOI：10.16016/j.2097-0927.202501034.

摘要

目的

制备土贝母皂苷丙纳米乳（tubeimoside Ⅲ nanoemulsion，TBMⅢ-NE），并评价其疫苗佐剂效应。

方法

利用低能乳化法制备TBMⅢ-NE，采用动态光散射法表征TBMⅢ-NE的粒径和平均分散系数，透射电镜观察TBMⅢ-NE的形态，CCK-8法检测TBMⅢ-NE对骨髓来源的树突状细胞（bone marrow-derived dendritic cells，BMDCs）的细胞毒性；红细胞溶血实验评价TBMⅢ-NE的体外安全性；激光共聚焦显微镜观察TBMⅢ-NE促进DC2.4对抗原的吞噬作用；TBMⅢ-NE与BMDCs共孵育后，利用流式细胞术检测BMDCs表面CD40、CD86、MHC-Ⅰ和CCR7的表达水平，酶联免疫吸附（ELISA）法检测BMDCs上清中细胞因子的表达水平；新冠病毒抗原RBD与TBMⅢ-NE联用免疫小鼠，ELISA测定小鼠血清中特异性IgG、IgG

、IgG

抗体水平，酶联免疫斑点（ELISpot）法检测小鼠脾淋巴细胞中特异性分泌γ干扰素（IFN-γ）的细胞数量。

结果

制备的空白纳米乳（blank nanoemulsion，BNE）和TBMⅢ-NE的平均粒径分别为25.46 nm和25.89 nm，平均多分散系数分别为0.214和0.125，透射电镜观察TBMⅢ-NE呈均匀“球型”且分散性良好；当TBMⅢ-NE佐剂稀释400倍时，BMDCs存活率约为86%；与游离土贝母皂苷丙（TBMⅢ）相比，TBMⅢ-NE的溶血毒性显著降低（

0.01）；TBMⅢ-NE可促进DC2.4对抗原的吞噬，并显著提高BMDCs表面CCR7的表达水平（

0.05），具有促进树突状细胞向淋巴结迁移的潜力；TBMⅢ-NE还可促进BMDCs培养上清中IL-6和IL-1β的表达（

0.05）；与RBD联用，TBMⅢ-NE可显著提升小鼠血清中特异性IgG、IgG2a、IgG1抗体的水平（

01），并促进脾淋巴细胞中特异性IFN-γ的分泌（

0.01），提示TBMⅢ-NE可以增强特异性T细胞免疫应答。

结论

成功构建出质量稳定、具有高效诱导体液和细胞免疫应答的TBMⅢ-NE。

Abstract

Objective‍　‍To prepare tubeimoside Ⅲ nanoemulsion (TBMⅢ-NE) and evaluate its adjuvant effect in vaccines. Methods‍　‍TBMⅢ-NE was prepared using low-energy emulsification. Dynamic light scattering was used to characterize the particle size and polydispersity index of the obtained TBMⅢ-NE

and transmission electron microscopy (TEM) was employed to observe the morphology. CCK-8 assay was utilized to determine the cytotoxicity of TBMⅢ-NE on bone marrow-derived dendritic cells (BMDCs). The

in vitro

safety of TBMⅢ-NE was evaluated using a hemolysis assay. The ability of TBMⅢ-NE to promote the phagocytosis of antigens by DC2.4 cells was observed using confocal laser microscopy. After co-incubation of TBMⅢ-NE with BMDCs

the expression levels of CD40

CD86

MHC-Ⅰ

and CCR7 on the surface of BMDCs were detected using flow cytometry

and the levels of cytokines in the supernatant of BMDCs were measured using enzyme-linked immunosorbent assay (ELISA). After female BALB/c mice were immunized with the SARS-CoV-2 antigen RBD in combination with TBMⅢ-NE

ELISA was conducted to determine the serum levels of specific IgG

IgG2a

and IgG1 antibodies. The number of specific IFN-γ-secreting cells in mouse splenocytes was detected using enzyme-linked immunospot (ELISpot) assay. Results‍　‍The prepared blank nanoemulsion (BNE) and TBMⅢ-NE were in a particle size of 25.46 and 25.89 nm

and a polydispersity index of 0.214 and 0.125

respectively. TEM displayed that TBMⅢ-NE was in uniform sphere and well dispersed. When the TBMⅢ-NE adjuvant was diluted by 400-fold

the survival rate of BMDCs was approximately 86%. Compared with free TBMⅢ

the hemolytic toxicity of TBMⅢ-NE was significantly reduced (

0.01). TBMⅢ-NE promoted the phagocytosis of antigens by DC2.4 cells and significa

ntly increased the expression of CCR7 on the surface of BMDCs (

0.05)

indicating its potential to promote more dendritic cells to effectively migrate to lymph nodes. TBMⅢ-NE also promoted the expression of IL-6 and IL-1β in the supernatant of BMDCs (

0.05). When combined with RBD

TBMⅢ-NE significantly increased the levels of specific IgG

IgG2a

and IgG1 antibodies in mouse serum (

0.01) and promoted the secretion of specific IFN-γ in splenocytes (

0.01)

indicating that TBMⅢ-NE could enhance specific cellular immune responses. Conclusion‍　‍A stable and highly effective TBMⅢ-NE that can induce humoral and cellular immune responses is successfully prepared.

关键词

Keywords

references

ZHAO T M ， CAI Y L ， JIANG Y J ， et al . Vaccine adjuvants： mechanisms and platforms ［J］. Signal Transduct Target Ther ， 2023 ， 8 （ 1 ）： 283 . DOI： 10.1038/s41392-023-01557-7 http://dx.doi.org/10.1038/s41392-023-01557-7 .

IWASAKI A ， OMER S B . Why and how vaccines work ［J］. Cell ， 2020 ， 183 （ 2 ）： 290 - 295 . DOI： 10.1016/j.cell. 2020. 09.040 http://dx.doi.org/10.1016/j.cell.2020.09.040 .

SILVA M ， KATO Y ， MELO M B ， et al . A particulate saponin/TLR agonist vaccine adjuvant alters lymph flow and modulates adaptive immunity ［J］. Sci Immunol ， 2021 ， 6 （ 66 ）： eabf1152 . DOI： 10.1126/sciimmunol.abf1152 http://dx.doi.org/10.1126/sciimmunol.abf1152 .

MARTIN L B B ， KIKUCHI S ， REJZEK M ， et al . Complete biosynthesis of the potent vaccine adjuvant QS-21 ［J］. Nat Chem Biol ， 2024 ， 20 （ 4 ）： 493 - 502 . DOI： 10.1038/s41589-023-01538-5 http://dx.doi.org/10.1038/s41589-023-01538-5 .

REED J ， ORME A ， EL-DEMERDASH A ， et al . Elucidation of the pathway for biosynthesis of saponin adjuvants from the soapbark tree ［J］. Science ， 2023 ， 379 （ 6638 ）： 1252 - 1264 . DOI： 10.1126/science.adf3727 http://dx.doi.org/10.1126/science.adf3727 .

HAN Z Y ， JIN J J ， CHEN X F ， et al . Adjuvant activity of tubeimosides by mediating the local immune microenvironment ［J］. Front Immunol ， 2023 ， 14 ： 1108244 . DOI： 10.3389/fimmu.2023.1108244 http://dx.doi.org/10.3389/fimmu.2023.1108244 .

TONG Y N ， LU G X ， WANG Z G ， et al . Tubeimuside I improves the efficacy of a therapeutic Fusobacterium nucleatum dendritic cell-based vaccine against colorectal cancer ［J］. Front Immunol ， 2023 ， 14 ： 1154818 . DOI： 10.3389/fimmu.2023.1154818 http://dx.doi.org/10.3389/fimmu.2023.1154818 .

LIM S B ， BANERJEE A ， ÖNYÜKSEL H . Improvement of drug safety by the use of lipid-based nanocarriers ［J］. J Control Release ， 2012 ， 163 （ 1 ）： 34 - 45 . DOI： 10.1016/j.jconrel.2012.06.002 http://dx.doi.org/10.1016/j.jconrel.2012.06.002 .

PULENDRAN B ， AHMED R . Immunological mechanisms of vaccination ［J］. Nat Immunol ， 2011 ， 12 （ 6 ）： 509 - 517 . DOI： 10.1038/ni.2039 http://dx.doi.org/10.1038/ni.2039 .

GOWTHAMAN U ， CHEN J S ， ZHANG B Y ， et al . Identification of a T follicular helper cell subset that drives anaphylactic IgE ［J］. Science ， 2019 ， 365 （ 6456 ）： eaaw6433 . DOI： 10.1126/science.aaw6433 http://dx.doi.org/10.1126/science.aaw6433 .

PARK G ， NA W ， LIM J W ， et al . Self-assembled nanostructures presenting repetitive arrays of subunit antigens for enhanced immune response ［J］. ACS Nano ， 2024 ， 18 （ 6 ）： 4847 - 4861 . DOI： 10.1021/acsnano.3c09672 http://dx.doi.org/10.1021/acsnano.3c09672 .

O’HAGAN D T ， OTT G S ， NEST G V ， et al . The history of MF59（®） adjuvant： a phoenix that arose from the ashes ［J］. Expert Rev Vaccines ， 2013 ， 12 （ 1 ）： 13 - 30 . DOI： 10.1586/erv.12.140 http://dx.doi.org/10.1586/erv.12.140 .

O’HAGAN D T ， OTT G S ， DE GREGORIO E ， et al . The mechanism of action of MF59—An innately attractive adjuvant formulation ［J］. Vaccine ， 2012 ， 30 （ 29 ）： 4341 - 4348 . DOI： 10.1016/j.vaccine.2011.09.061 http://dx.doi.org/10.1016/j.vaccine.2011.09.061 .

GUY B . The perfect mix： recent progress in adjuvant research ［J］. Nat Rev Microbiol ， 2007 ， 5 （ 7 ）： 505 - 517 . DOI： 10.1038/nrmicro1681 http://dx.doi.org/10.1038/nrmicro1681 .

REINKE S ， PANTAZI E ， CHAPPELL G R ， et al . Emulsion and liposome-based adjuvanted R21 vaccine formulations mediate protection against malaria through distinct immune mechanisms ［J］. Cell Rep Med ， 2023 ， 4 （ 11 ）： 101245 . DOI： 10.1016/j.xcrm.2023.101245 http://dx.doi.org/10.1016/j.xcrm.2023.101245 .

CHEN Z J ， HAO X Y ， WANG H R ， et al . Smart combination of aluminum hydroxide and MF59 to induce strong cellular immune responses ［J］. J Control Release ， 2022 ， 349 ： 699 - 711 . DOI： 10.1016/j.jconrel.2022.07.032 http://dx.doi.org/10.1016/j.jconrel.2022.07.032 .

HAUSER M A ， LEGLER D F . Common and biased signaling pathways of the chemokine receptor CCR7 elicited by its ligands CCL19 and CCL21 in leukocytes ［J］. J Leukoc Biol ， 2016 ， 99 （ 6 ）： 869 - 882 . DOI： 10.1189/jlb.2MR0815-380R http://dx.doi.org/10.1189/jlb.2MR0815-380R .

ITANO A A ， MCSORLEY S J ， REINHARDT R L ， et al . Distinct dendritic cell populations sequentially present antigen to CD4 T cells and stimulate different aspects of cell-mediated immunity ［J］. Immunity ， 2003 ， 19 （ 1 ）： 47 - 57 . DOI： 10.1016/S1074-7613（03）00175-4 http://dx.doi.org/10.1016/S1074-7613（03）00175-4 .

SU J P ， BRUNNER L ， ATES OZ E ， et al . Activation of CD4 T cells during prime immunization determines the success of a therapeutic hepatitis B vaccine in HBV-carrier mouse models ［J］. J Hepatol ， 2023 ， 78 （ 4 ）： 717 - 730 . DOI： 10.1016/j.jhep.2022.12.013 http://dx.doi.org/10.1016/j.jhep.2022.12.013 .

ZIMMERMANN P ， CURTIS N . Factors that influence the immune response to vaccination ［J］. Clin Microbiol Rev ， 2019 ， 32 （ 2 ）： e00084-18 . DOI： 10.1128/CMR.00084-18 http://dx.doi.org/10.1128/CMR.00084-18 .

MOGA E ， LYNTON-PONS E ， DOMINGO P . The robustness of cellular immunity determines the fate of SARS-CoV-2 infection ［J］. Front Immunol ， 2022 ， 13 ： 904686 . DOI： 10.3389/fimmu.2022.904686 http://dx.doi.org/10.3389/fimmu.2022.904686 .

YIN Q ， LUO W ， MALLAJOSYULA V ， et al . A TLR7-nanoparticle adjuvant promotes a broad immune response against heterologous strains of influenza and SARS-CoV-2 ［J］. Nat Mater ， 2023 ， 22 （ 3 ）： 380 - 390 . DOI： 10.1038/s41563-022-01464-2 http://dx.doi.org/10.1038/s41563-022-01464-2 .

LI D P ， MARTINEZ D R ， SCHÄFER A ， et al . Breadth of SARS-CoV-2 neutralization and protection induced by a nanoparticle vaccine ［J］. Nat Commun ， 2022 ， 13 （ 1 ）： 6309 . DOI： 10.1038/s41467-022-33985-4 http://dx.doi.org/10.1038/s41467-022-33985-4 .

XIONG H L ， WU Y T ， CAO J L ， et al . Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus （VSV） pseudovirus and ACE2-overexpressing BHK21 cells ［J］. Emerg Microbes Infect ， 2020 ， 9 （ 1 ）： 2105 - 2113 . DOI： 10.1080/22221751.2020.1815589 http://dx.doi.org/10.1080/22221751.2020.1815589 .

刘姝琳，魏静，朱宝行，等 . 植物源性免疫刺激分子麦冬皂苷的免疫增强效应及初步机制［J］. 陆军军医大学学报， 2025 ， 47 （ 4 ）： 350 - 359 . DOI： 10. 16016/j. 2097-0927. 202412021 http://dx.doi.org/10.16016/j.2097-0927.202412021 .

LIU S L ， WEI J ， ZHU B H ， et al . Immune-enhancing effect and mechanism of natural plant-derived immunostimulatory molecule ophiopogonin ［J］. J Army Med Univ ， 2025 ， 47 （ 4 ）： 350 - 359 . DOI： 10. 16016/j. 2097-0927. 202412021 http://dx.doi.org/10.16016/j.2097-0927.202412021 .

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

No data

Related Author

No data

Related Institution

No data

⁰