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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

In this protocol, we developed a cationic nanoemulsion-encapsulated retinoic acid (RA) to be used as an adjuvant to promote antigen-specific systemic and mucosal responses. By adding the FDA-approved RA to the nanoemulsion, antigen-specific sIgA was promoted in the vagina and small intestine after intramuscular injection of the nanoemulsion.

Abstract

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen uptake and then promotes antigen-specific immune responses. In recent years, retinoic acid (RA) has received increasing attention due to its effect in activating the mucosal immune response; however, in order to use RA as a mucosal adjuvant, it is necessary to solve the problem of its dissolution, loading, and delivery. Here, we describe a cationic nanoemulsion-encapsulated retinoic acid (CNE-RA) delivery system composed of the cationic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOTAP), retinoic acid, squalene as the oil phase, polysorbate 80 as surfactant, and sorbitan trioleate 85 as co-surfactant. Its physical and chemical properties were characterized using dynamic light scattering and a spectrophotometer. Immunization of mice with the mixture of antigen (ovalbumin, OVA) and CNE-RA significantly elevated the levels of anti-OVA secretory immunoglobulin A (sIgA) in vaginal lavage fluid and the small intestinal lavage fluid of mice compared with OVA alone. This protocol describes a detailed method for the preparation, characterization, and evaluation of the adjuvant effect of CNE-RA.

Introduction

Adjuvants are often used to enhance the efficacy of a vaccine by stimulating the immune system to respond more strongly to the vaccine, thereby increasing immunity to a particular pathogen1. Nanoemulsion (NE) adjuvant refers to a colloidal dispersion system with thermodynamic stability by emulsifying a certain proportion of oil phase and aqueous phase to produce an emulsion in the form of water-in-oil (W/O) or oil-in-water (O/W)2. O/W nanoemulsion adjuvant can produce cytokines and chemokines at the injection site, induce the rapid aggregation and proliferation of important immune cells such as monocytes, neutrophils, an....

Protocol

The animal experiments were performed in accordance with the Guide to the Use and Care of Laboratory Animals and approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University.

1. Preparation of nanoemulsions (NEs)

  1. For aqueous phase preparation, dissolve 0.15 g of polysorbate 80 in 28.2 mL of phosphate buffered saline (PBS) while stirring at 40 °C.
  2. For oil phase preparation, use the oil phase formulation of th.......

Representative Results

In total, four nanoemulsion formulations were prepared and characterized by their particle size (Figure 1), their zeta potential and their encapsulation efficiency as presented in Table 2. The particle size was concentrated around 160-190nm and the addition of DOTAP reversed the Zeta potential of nanoemulsion. OVA-specific serum IgG and its subgroup antibody level in serum were detected 2 weeks post third immunization. The nanoemulsion adjuvant vaccine significantly increase.......

Discussion

In this protocol, we developed a cationic nanoemulsion-encapsulated retinoic acid to be used as an adjuvant to promote antigen-specific systemic and mucosal responses. Compared to traditional NE adjuvants, it has the following two advantages. First, in general, the surface of O/W NEs has a high negative charge, which makes it difficult to directly load antigens. Cationic NEs can effectively adsorb peptide or protein antigens and enhance the specific immunogenicity. Secondly, experience in traditional vaccine research has.......

Acknowledgements

This study was funded by Key Program of Chongqing Natural Science Foundation (No. cstc2020jcyj-zdxmX0027) and Chinese National Natural Science Foundation Project (No. 32270988).

....

Materials

NameCompanyCatalog NumberComments
1640 mediumGIBCO, USAC11875500BT
450 nm Stop Solution for TMB SubstrateAbcamab171529-1000 mL
Automated Cell CounterCountstar, ChinaIC1000
BSASigma-Aldrich, USAB2064-100G
Centrifuge 5810 REppendorf, Germany5811000398
Danamic Light ScatteringMalvernZetasizer Nano S90
DOTAPCordenPharma, SwitzerlandO02002
ELISpot Plus: Mouse IFN-gamma (ALP)mabtechab205719
Fetal Bovine SerumGIBCO, USA10099141C
Full-function Microplate ReaderThermo Fisher Scientific, USAVL0000D2
Goat Anti-Mouse IgG1(HRP)Abcamab97240-1mg
Goat Anti-Mouse IgA alpha chain (HRP)Abcamab97235-1mg
Goat Anti-Mouse IgG H&L (HRP)AbcamAb205720-500ug
Goat Anti-Mouse IgG2a heavy chain (HRP)Abcamab97245-1mg
High pressure homogenizerATS
MONTANE 85 PPISEPPIC, FranceL12910
MONTANOX 80 PPISEPPIC, France36372K
OVA257–264Shanghai Botai Biotechnology Co., Ltd.NA
OVA323-339Shanghai Botai Biotechnology Co., Ltd.NA
Phosphate buffer salineZSGB-bioZLI-9061
Red Blood Cell Lysis BufferSolarbio, ChinaR1010
retinoic acidTCI, JapanTCI-R0064-5G
SqualeneSigma, USAS3626
T10 basic Ultra-TurraxIKA, Germany
TMB ELISA SubstrateAbcamab171523-1000ml
trypsin inhibitorDiamondA003570-0100
Tween-20Macklin, China9005-64-5
Ultraviolet spectrophotometerHitachiU-3900

References

  1. Pulendran, B., Arunachalam, P. S., O'Hagan, D. T. Emerging concepts in the science of vaccine adjuvants. Nat Rev Drug Discov. 20 (6), 454-475 (2021).
  2. Pandey, P., Gulati, N., Makhija, M., Purohit, D., Dureja, H. Nanoemul....

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Cationic NanoemulsionRetinoic AcidAdjuvantOvalbuminMucosal Immune ResponseSystemic Immune ResponseDendritic CellAntigen Delivery SystemSIgA

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