Cationic Nanoemulsion-Encapsulated Retinoic Acid as an Adjuvant to Promote OVA-Specific Systemic and Mucosal Responses

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.......

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.......

Materials

Name	Company	Catalog Number	Comments
1640 medium	GIBCO, USA	C11875500BT
450 nm Stop Solution for TMB Substrate	Abcam	ab171529-1000 mL
Automated Cell Counter	Countstar, China	IC1000
BSA	Sigma-Aldrich, USA	B2064-100G
Centrifuge 5810 R	Eppendorf, Germany	5811000398
Danamic Light Scattering	Malvern	Zetasizer Nano S90
DOTAP	CordenPharma, Switzerland	O02002
ELISpot Plus: Mouse IFN-gamma (ALP)	mabtech	ab205719
Fetal Bovine Serum	GIBCO, USA	10099141C
Full-function Microplate Reader	Thermo Fisher Scientific, USA	VL0000D2
Goat Anti-Mouse IgG1(HRP)	Abcam	ab97240-1mg
Goat Anti-Mouse IgA alpha chain (HRP)	Abcam	ab97235-1mg
Goat Anti-Mouse IgG H&L (HRP)	Abcam	Ab205720-500ug
Goat Anti-Mouse IgG2a heavy chain (HRP)	Abcam	ab97245-1mg
High pressure homogenizer	ATS
MONTANE 85 PPI	SEPPIC, France	L12910
MONTANOX 80 PPI	SEPPIC, France	36372K
OVA257–264	Shanghai Botai Biotechnology Co., Ltd.	NA
OVA323-339	Shanghai Botai Biotechnology Co., Ltd.	NA
Phosphate buffer saline	ZSGB-bio	ZLI-9061
Red Blood Cell Lysis Buffer	Solarbio, China	R1010
retinoic acid	TCI, Japan	TCI-R0064-5G
Squalene	Sigma, USA	S3626
T10 basic Ultra-Turrax	IKA, Germany
TMB ELISA Substrate	Abcam	ab171523-1000ml
trypsin inhibitor	Diamond	A003570-0100
Tween-20	Macklin, China	9005-64-5
Ultraviolet spectrophotometer	Hitachi	U-3900