CN113713093A - Novel tretinoin nanoemulsion adjuvant capable of efficiently enhancing humoral immune response and mucosal immune response and preparation method and application thereof - Google Patents

Novel tretinoin nanoemulsion adjuvant capable of efficiently enhancing humoral immune response and mucosal immune response and preparation method and application thereof Download PDF

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CN113713093A
CN113713093A CN202111016885.0A CN202111016885A CN113713093A CN 113713093 A CN113713093 A CN 113713093A CN 202111016885 A CN202111016885 A CN 202111016885A CN 113713093 A CN113713093 A CN 113713093A
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tretinoin
parts
adjuvant
immune response
nanoemulsion adjuvant
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李海波
孙红武
金喆
邹全明
冒灵
刘畅
刘婧怡
曾浩
薛若怡
冯壤
李郭成
金滋力
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Third Military Medical University TMMU
Army Medical University
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Abstract

The invention discloses a novel tretinoin nanoemulsion adjuvant for efficiently enhancing humoral immune response and mucosal immune response, and a preparation method and application thereof. The tretinoin nanoemulsion adjuvant provided by the invention can be co-delivered with various antigens (mode antigen OVA, staphylococcus aureus recombinant protein antigen and new coronavirus recombinant protein antigen) respectively, can enhance the immune response level of vaccine antigens after injection immunization, has a good immune protection effect, can induce systemic humoral immune response, can efficiently activate the immune response of mucosal parts (intestinal mucosa, vaginal mucosa, pulmonary mucosa, gastric mucosa and nasal mucosa), and has great market application value and wide application prospect.

Description

Novel tretinoin nanoemulsion adjuvant capable of efficiently enhancing humoral immune response and mucosal immune response and preparation method and application thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to a novel tretinoin nanoemulsion adjuvant for efficiently enhancing humoral immune response and mucosal immune response and a preparation method and application thereof.
Background
The mucosa is used as the first defense line for blocking pathogen invasion, is widely distributed in respiratory, digestive, urinary and reproductive systems of human bodies, and plays an irreplaceable role in the process of protecting human health. If the mucosa is not effective in blocking pathogens, its wet, fragile structure will become the ideal hotbed for the pathogen, and many vital organs will be directly exposed to the pathogen's threat. Pathogens such as vibrio cholerae, salmonella, rotavirus and the like which infect human bodies from mucosal parts have seriously threatened human health. However, a physiological barrier exists between systemic immunity and mucosal immunity, and vaccines injected through muscles can only induce systemic immune response and cannot establish an effective immune protective barrier at mucosal parts, so that the infection of pathogens is difficult to block at the mucosal parts, and the popularization and the application of the vaccines are seriously hindered. How to effectively and efficiently activate mucosal immune response has become a key scientific problem which needs to be solved urgently by vaccine researchers.
In recent years, the important role and mechanism of tretinoin (RA) in mucosal immune response has been revealed: it can induce lymphocyte to homing intestinal tract by regulating alpha 4 beta 7 integrin, cell factor CCR9, Thymic Stromal Lymphopoietin (TSLP), lactoferrin and other ways, promote the conversion of Ig class and raise the level of secretory sIgA in intestinal mucosa. However, RA has poor water solubility and poor photostability, which severely limits its use as an adjuvant. Chinese patent publication No. CN108324938A discloses that the use of all-trans retinoic acid to prepare a granular adjuvant can activate humoral immunity and intestinal mucosal immune response, but no immune response effect is seen in vaginal mucosa, pulmonary mucosa, gastric mucosa, and nasal mucosa. The nano-emulsion can greatly improve the solubility of insoluble drugs, promote drug absorption, enhance drug stability and improve bioavailability, but the tretinoin nano-emulsion adjuvant is not reported at home and abroad.
Disclosure of Invention
Nanoemulsion adjuvants are highly thermodynamically stable colloidal dispersions that can dissolve and deliver poorly water soluble drugs. Meanwhile, emulsion adjuvant and antigen are co-delivered to enhance the systemic immune protective response, such as MF59 emulsion, which has been used as a human vaccine adjuvant to be marketed for many years, and the safety and effectiveness thereof have been widely accepted clinically. According to the two characteristics of the emulsion adjuvant, on the basis of a screening experiment of a previous prescription, an MF59(MF59-like) oil-in-water type tretinoin nano-emulsion adjuvant (ATRA-NE) which takes squalene, GTCC and IPM dissolved with tretinoin as an oil phase, Tween as a surfactant and Span-80/Span-85 as a cosurfactant is designed, and the nano-emulsion adjuvant is co-delivered with a recombinant protein antigen in an intramuscular injection mode, so that the nano-emulsion adjuvant can simultaneously trigger specific systemic humoral immune response and mucosal immune response, and a new thought is provided for designing a mucosal invasion pathogen vaccine.
In view of the above, an object of the present invention is to provide a tretinoin nanoemulsion adjuvant; the second purpose of the invention is to provide a preparation method of the tretinoin nanoemulsion adjuvant; the third purpose of the invention is to provide a vaccine containing the tretinoin nanoemulsion adjuvant; the fourth purpose of the invention is to provide an application of the tretinoin nanoemulsion adjuvant as a vaccine adjuvant.
In order to achieve the purpose, the invention provides the following technical scheme:
1. tretinoin nanoemulsion adjuvant
Comprises the following components in parts by weight: 0.01-5 parts of tretinoin, 1-30 parts of surfactant, 0.1-10 parts of cosurfactant, 0.1-10 parts of oil phase and 44-97.9 parts of water.
Preferably, the surfactant is any one or combination of tween-20, tween-60, tween-80, tween-85, polyoxyethylene castor oil or polyoxyethylene hydrogenated castor oil.
Preferably, the cosurfactant is span-80, span-85, absolute ethyl alcohol, propylene glycol, glycerol and 1, 3-butanol, or any one or more of the combination thereof.
Preferably, the oil phase is any one or combination of ethyl acetate, squalane, squalene or ethyl oleate.
Preferably, the composition comprises the following components in parts by weight: 6 parts of tretinoin, 5 parts of squalene, 8030 parts of tween-8030 parts of span-8015 parts of water and 44 parts of water.
Preferably, the particle size distribution of the tretinoin nanoemulsion adjuvant is 1-100nm, the dispersion index is less than 0.3, and the Zeta potential absolute value is less than 30 mV.
2. Preparation method of tretinoin nanoemulsion adjuvant
Comprises the following steps: weighing tretinoin, surfactant, cosurfactant, oil phase and water according to a proportion; dissolving the tretinoin in the oil phase fully, then dripping the surfactant and the cosurfactant, and finally stirring and slowly dripping the sterilized pure water to obtain the tretinoin nanoemulsion adjuvant. Vaccine containing tretinoin nanoemulsion adjuvant
3. Vaccine containing tretinoin nanoemulsion adjuvant
The vaccine antigens are a model antigen OVA, a staphylococcus aureus recombinant protein antigen HI and a neocoronavirus vaccine recombinant protein antigen RBD.
4. The tretinoin nanoemulsion adjuvant is applied to preparation of a vaccine adjuvant, and the adjuvant is an adjuvant for activating systemic humoral immune response or mucosal immune adjuvant.
Preferably, the mucosa is intestinal mucosa, vaginal mucosa, pulmonary mucosa, gastric mucosa, nasal mucosa.
The invention has the beneficial effects that:
the invention discloses a tretinoin nanoemulsion adjuvant for efficiently enhancing humoral immune response and mucosal immune response, and a preparation method and application thereof. The structure is complete, stable and good in uniformity, the particle size distribution of the preparation is 10-100 nm, and the size of the preparation is favorable for the preparation to gather to lymph nodes in a short time, so that the preparation is favorable for recognition and presentation by APC (antigen-plasma) and enhances immune response.
The tretinoin nanoemulsion adjuvant provided by the invention can be co-delivered with various antigens (mode antigen OVA, staphylococcus aureus recombinant protein antigen and new coronavirus recombinant protein antigen) respectively, the immune response level of the vaccine antigen can be enhanced after injection immunization, the good immune protection effect is achieved, and after intramuscular injection administration, the high expression of the IL-6 and the IL-15 at the injection part is beneficial to the generation of immune response.
The result of serum IgG shows that the atRA-NE induces the IgG level equivalent to that of the aluminum adjuvant, and the sIgA level of the mucosa parts such as intestinal mucosa, vaginal mucosa, lung mucosa, gastric mucosa, nasal mucosa and the like is obviously higher than that of the aluminum adjuvant group, so that the serum IgG has the characteristics of obvious mucosal adjuvant while effectively activating the systemic humoral immune response, and has great market application value and wide application prospect.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a diagram showing the identification of the physicochemical properties of atRA-NE (a: particle size distribution diagram; b: Zeta potential distribution diagram; c: scanning electron microscopy diagram; d: transmission electron microscopy diagram);
FIG. 2 shows the stability test of nanoemulsion (a: before centrifugation and b: after centrifugation);
figure 3 is flow cytometry measured cell surface marker data (a) and CCR9 gene expression levels (b) (") indicates P <0.05 versus blank," × "indicates P <0.01 versus blank;
FIG. 4 shows secretion of IL-6(a) and IL-15(b) at the injection sites after immunization ("-" indicates that P < 0.05; "-" indicates that P < 0.01; ". indicates that P < 0.001;" indicates that P < 0.001. relative to OVA);
FIG. 5 is a graph of ATRA-NE induced serum OVA antigen-specific IgG and its subtypes (a: IgG; b: IgG 1; c: IgG2 a; d: IgG1/IgG2 a; ". indicates P < 0.01;" indicates P < 0.001. relative to the PBS group);
FIG. 6 is the induction of intestinal mucosal OVA-specific sIgA by atRA-NE (a: OD450nm after 2-fold dilution; b: antibody titer; ". indicates that P <0.001 relative to PBS group);
figure 7 is a graph of the induction of vaginal mucosal OVA-specific sIgA by atRA-NE ("×" indicates P <0.001 relative to PBS group);
figure 8 is the case where atRA-NE induced pulmonary mucosal OVA specific sIgA ("×" indicates P <0.001 relative to PBS group);
figure 9 is a graph of the induction of gastric mucosal OVA-specific sIgA by atRA-NE ("×" indicates P <0.001 relative to PBS group);
FIG. 10 shows cytokine secretion by splenic lymphocyte culture supernatants of immunized mice (a: IL-4 content; b: IL-17; "+" indicates P <0.01 relative to PBS group; and "+" indicates P <0.001 relative to PBS group);
FIG. 11 shows IFN-. gamma.secretion from splenic lymphocyte culture supernatants (a: OVA257-264 epitope peptide as stimulator; b: OVA323-339 epitope peptide as stimulator; c: representative ELISPOT results; ". indicates P < 0.01;" indicates P < 0.001. relative to PBS group);
figure 12 is a graph of the serum HI-specific IgG profile induced by atRA-NE ("×" indicates P <0.001 relative to the PBS group);
figure 13 is the induction of vaginal HI-specific sIgA profiles by atRA-NE ("×" indicates P <0.001 relative to PBS group);
figure 14 is a graph of (a) ("x") indicates P <0.001 relative to the RBD group for atRA-NE induced serum RBD-specific IgG and (b) ("x" indicates P <0.001 relative to the RBD group for atRA-NE induced serum RBD-specific IgA);
figure 15 is a graph of the induction of vaginal RBD-specific IgA by atRA-NE ("×" indicates P <0.001 relative to the RBD group);
figure 16 is a graph of the induction of lung mucosal RBD specific sIgA by atRA-NE ("×" indicates P <0.001 relative to the RBD group);
figure 17 is a graph of the induction of nasal mucosal RBD specific sIgA by atRA-NE ("×" indicates P <0.001 relative to the RBD group);
figure 18 is a graph of the induction of RBD-specific IgG secretion by splenic lymphocytes by atRA-NE ("×" indicates that P <0.001 relative to the RBD group);
fig. 19 shows the expression of atRA-NE inducing spleen lymphocytes to secrete RBD-specific IgAASC and IgG ASC ("×" indicates P <0.001 relative to the RBD group).
Detailed Description
The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
Example 1 formulation screening of tretinoin nanoemulsion adjuvant (RA-NE)
Formula 1: tretinoin nanoemulsion adjuvant (100 g): 1g of tretinoin, 802 g of tween-802 g, 2g of propylene glycol and 95g of distilled water.
And (2) formula: tretinoin nanoemulsion adjuvant (100 g): 0.1g of tretinoin, 8030 g of Tween-8030 g, 10g of glycerol, 10g of caprylic/capric Glyceride (GTCC) and 49.9g of distilled water.
And (3) formula: tretinoin nanoemulsion adjuvant (100 g): 2g of tretinoin, 205 g of tween-205 g, 0.1g of propylene glycol, 0.1g of ethyl acetate and 92.8g of distilled water.
And (4) formula: tretinoin nanoemulsion adjuvant (100 g): 5g of tretinoin, 6020 g of tween-6020 g, 4g of propylene glycol, 6g of medicinal paraffin oil and 65g of distilled water.
And (5) formula: tretinoin nanoemulsion adjuvant (100 g): 2g of tretinoin, 8530 g of Tween-8530 g of 1, 3-butanediol, 15g of GTCC and 43g of distilled water.
And (6) formula: tretinoin nanoemulsion adjuvant (100 g): 1g of tretinoin, 802 g of tween-802 g, 0.2g of propylene glycol, 0.1g of glycerol, 0.1g of GTCC, 0.1g of IPM and 96.5g of distilled water.
And (3) formula 7: tretinoin nanoemulsion adjuvant (100 g): 2g of tretinoin, 851 g of tween-851 g, 1g of ethanol, 0.1g of paraffin oil and 95.9g of distilled water.
And (4) formula 8: tretinoin nanoemulsion adjuvant (100 g): 2g of tretinoin, 601 g of tween, 802 g of tween-802 g, 600.1 g of span-600, 0.1g of isopropyl myristate (IPM) and 94.8g of distilled water.
Formula 9: tretinoin nanoemulsion adjuvant (100 g): 5g of tretinoin, 0.5g of RH403.5g of ethanol, 0.5g of GTCC and 90.5g of distilled water.
Formula 10: tretinoin nanoemulsion adjuvant (100 g): 1g of tretinoin, 3530 g of EL, 0.5g of 1, 3-butanediol, 10g of ethyl acetate and 58.5g of distilled water.
Formula 11: tretinoin nanoemulsion adjuvant (100 g): 2g of tretinoin, 10g of EL 4030 g, 10g of ethanol, 10g of ethyl oleate and 48g of distilled water.
Formula 12: tretinoin nanoemulsion adjuvant (100 g): tretinoin 5g, EL 401 g, 1,3 propylene glycol 0.1g, paraffin oil 0.5g, and distilled water 93.4 g.
Formula 13: tretinoin nanoemulsion adjuvant (100 g): 10g of tretinoin, 601 g of tween-601, 800.1 g of span, 0.1g of squalene and 88.8g of distilled water.
Formula 14: tretinoin nanoemulsion adjuvant (100 g): 5g of tretinoin, 805 g of tween-805 g, 850.1 g of span-5 g, 0.5g of squalene and 89.4g of distilled water.
Formula 15: tretinoin nanoemulsion adjuvant (100 g): tretinoin 0.1g, Tween-8025 g, span-6010 g, squalane-10 g and distilled water 54.9 g.
Formula 16: tretinoin nanoemulsion adjuvant (100 g): 10g of tretinoin, 855 g of tween-855 g, 801 g of span-801 g, 0.1g of squalane, 0.5g of squalene and 83.4g of normal saline.
Formula 17: tretinoin nanoemulsion adjuvant (100 g): 2g of tretinoin, 8525 g of Tween-8525 g of span-8515 g of squalene and 48g of distilled water.
Formula 18: tretinoin nanoemulsion adjuvant (100 g): 5g of tretinoin, 10g of polyoxyethylene castor oil (EL35), 800.1 g of span-800, 0.1g of squalene and 84.8g of distilled water.
Formulation 19: tretinoin nanoemulsion adjuvant (100 g): 6g of tretinoin, 8030 g of Tween, 8015 g of span, 5g of squalene and 44g of distilled water.
The formula 20 is as follows: tretinoin nanoemulsion adjuvant (100 g): tretinoin 2g, EL 4020 g, propylene glycol 1g, ethyl oleate 0.5g, and physiological saline 76.5 g.
For Surfactants (SF), such as: screening Tween-80, Tween-85, Tween 60, Tween 20, polyoxyethylene castor oil (EL40, EL35), polyoxyethylene hydrogenated castor oil, etc., and detecting according to high speed centrifugation stabilization experiment, particle size and potential, etc., finding that the formula and the range can form tretinoin nanoemulsion adjuvant.
Diluting the formula by 100 times, and detecting the granularity and the dispersity by using a nano potential analyzer; and (3) observing whether the phenomena of instability such as layering, flocculation, drug precipitation, emulsion breaking and the like occur before and after centrifugation at 13000rpm for 30 min.
The detection results are shown in table 1:
TABLE 1 results of different formulations
Formulation of Particle size (nm) Dispersibility (pdI) Layered state Flocculation conditions Drug precipitation Condition of emulsion breaking
1 24.5 0.132 Layering Flocculation Does not precipitate Without breaking emulsion
2 22.0 0.22 Not delaminating Non-flocculated Does not precipitate Without breaking emulsion
3 101 0.13 Not delaminating Non-flocculated Does not precipitate Without breaking emulsion
4 67 0.23 Not delaminating Flocculation Does not precipitate Without breaking emulsion
5 25.3 0.14 Not delaminating Non-flocculated Does not precipitate Without breaking emulsion
6 102 0.54 Not delaminating Non-flocculated Precipitation out of Without breaking emulsion
7 35.9 0.25 Not delaminating Non-flocculated Does not precipitate Without breaking emulsion
8 81.8 0.22 Not delaminating Non-flocculated Does not precipitate Demulsification
9 25.4 0.13 Not delaminating Non-flocculated Does not precipitate Without breaking emulsion
10 45.9 0.142 Not delaminating Non-flocculated Does not precipitate Without breaking emulsion
11 60.8 0.211 Layering Flocculation Precipitation out of Demulsification
12 71.0 0.42 Not delaminating Non-flocculated Does not precipitate Without breaking emulsion
13 62.9 0.113 Not delaminating Non-flocculated Does not precipitate Without breaking emulsion
14 27.3 0.22 Not delaminating Non-flocculated Does not precipitate Without breaking emulsion
15 50.0 0.222 Not delaminating Non-flocculated Does not precipitate Without breaking emulsion
16 24.9 0.124 Not delaminating Non-flocculated Does not precipitate Without breaking emulsion
17 140.1 0.141 Not delaminating Non-flocculated Does not precipitate Without breaking emulsion
18 24.0 0.424 Not delaminating Non-flocculated Does not precipitate Without breaking emulsion
19 27.0 0.191 Not delaminating Non-flocculated Does not precipitate Without breaking emulsion
20 58.0 0.234 Not delaminating Non-flocculated Does not precipitate Without breaking emulsion
The nano-emulsion adjuvant of the formula is subjected to granularity and dispersity detection by a nano-potential analyzer with the granularity of 100 times; and (3) observing whether the phenomena of layering, flocculation, drug precipitation, emulsion breaking and other instability occur before and after centrifugation at 13000rpm for 30min, wherein the particle size is 1-100nm, the dispersion index is less than 0.3, and the qualified nanoemulsion adjuvant formula is obtained after no layering, no flocculation, no drug precipitation and no emulsion breaking after centrifugation. In the experiment, the qualified formula is selected, and the formula No. 19 is randomly selected as a research formula. And the prepared tretinoin nanoemulsion adjuvant has the same result in the subsequent examples as long as the particle size range is within 100 nm.
Example 2 preparation and characterization of preferred atRA-NE
1. Preparation of atRA-NE
Precisely weighing 10mg of tretinoin (atRA) in a 50mL clean dry beaker, dripping oil phase, surfactant and cosurfactant, stirring until the oil phase, the surfactant and the cosurfactant are fully dissolved, adopting a low-energy emulsification method, namely slowly dripping sterile pure water while stirring until the volume of the system is 10mL, preparing the tretinoin nanoemulsion adjuvant (atRA-NE), standing at room temperature and storing in a dark place. Blank nanoemulsion adjuvant (BNE), retinoic acid does not need to be added into the prescription.
2. Particle size and Zeta potential detection
The prepared nanoemulsion adjuvant was diluted 100 times with ultrapure water, and the particle size and Zeta potential were measured in a Nano ZA dynamic light scattering particle size potentiostat.
3. Transmission Electron Microscopy (TEM) detection
Diluting the prepared nano-emulsion adjuvant by 100 times with ultrapure water, taking 5 mu L of the diluted nano-emulsion adjuvant on a copper net of an electron microscope, standing for 10 minutes, carefully sucking excess water by using filter paper, dropwise adding 10 mu L of 1% phosphotungstic acid for dyeing for 1 minute, then sucking excess water by using the filter paper again, and observing and acquiring images under a TEVNAI10 transmission electron microscope.
4. Scanning Electron Microscope (SEM) detection
The nano-emulsion adjuvant is sent to an analysis testing center of army and military medical university for sample preparation and detection in a scanning electron microscope room.
5. Encapsulation efficiency and drug loading measurements
0.8mL of absolute ethyl alcohol is added into 0.4mL of nano-emulsion adjuvant for demulsification or 0.8mL of pure water is added, the mixture is centrifuged at 13000rpm for 30 minutes, and the supernatant and the precipitate are taken to respectively measure the concentration of the all-trans retinoic acid in the mixture.
The encapsulation efficiency is the actual tretinoin loading/theoretical tretinoin loading multiplied by 100 percent;
the drug loading is (the content of the retinoic acid in the demulsification supernatant plus the content of the retinoic acid in the demulsification precipitate)/the total mass of the sample is multiplied by 100%.
As shown in FIG. 1, the particle size and zeta potential distribution were concentrated, and the average particle size was 27.23nm (FIG. 1, a). The dispersibility was 0.181 and the zeta potential was-25.8 mV (FIG. 1, b). The encapsulation efficiency is 99.26% and the drug loading is 0.998 mg/mL. The spherical shape of the atRA-NE, good dispersibility, no aggregation and good quality characteristics are observed by a scanning electron microscope (figure 1, c) and a transmission electron microscope (figure 1, d).
6. Stability detection
And (3) observing whether the phenomena of layering, flocculation, medicament precipitation, emulsion breaking and other instability occur before and after centrifugation at 13000rpm for 30min, wherein the particle size is 1-100nm, the dispersity index is less than 0.3, and the phenomena of layering, flocculation, medicament precipitation and emulsion breaking are not generated after the centrifugation, so that the nano-emulsion adjuvant formula is qualified. 1.5ml of the nanoemulsion adjuvant was centrifuged at 13000rpm for 30min, and then the results were observed as shown in fig. 2. The result shows that the nano-emulsion adjuvant prepared by the invention has good stability.
Example 3 ATRA-NE ability to stimulate mouse lymphocyte maturation
1. ability of ATRA-NE to promote BMDC cell maturation
Killing mouse, taking femur, sucking 1640 culture medium containing 10% FBS with 1mL syringe, washing bone marrow 5 times, collecting bone marrow cell washing liquid, centrifuging at 1500rpm for 15 min, seeding collected cells in cell culture plate, changing liquid half every other day, centrifuging on day 6, collecting cells to obtain mouse bone marrow-derived dendritic cells (BMDCs), adjusting cell concentration to 5 × 105one/mL, 2 mL/well in 6-well plates. Then 20 μ L of medium, 20 μ L1: Blank-NE at 100 dilution, 20 μ g atRA, or 20 μ L1: 100 diluted atRA-NE, which is blown and mixed evenly, is placed in a cell culture box for incubation for 24 hours. Centrifuge and wash cells with PBS, wash with stabilizing buffer at 1: 50 dilution APC marked mouse CD11c antibody, PE marked mouse CD40 antibody, PerCP/Cy7 marked mouse CD86 antibody mixed dilution, each tube of cells 100 u L staining solution heavy suspension, 4 degrees C photophobic staining for 30 minutes. The supernatant was discarded by centrifugation, resuspended in 100. mu.L of stabilizing buffer and examined by FACSVersese flow cytometer.
2. ATRA-NE promotes expression of lymphocyte surface CCR9
Grinding lymph node of mouse intestinal tract in 200 mesh screen, taking grinding fluid 1500rpm, centrifuging for 10min, adding complete culture medium, resuspending and adjusting cell concentration to 5 × 105one/mL in a 12-well plateMedium 2 mL/well, after which 20 μ L of medium, 20 μ L1: Blank-NE at 100 dilution, 20 μ g atRA, or 20 μ L1: 100 diluted atRA-NE is evenly blown and placed in a cell incubator for incubation for 48 hours, cells are collected by centrifugation, RNA is extracted by using a cell RNA extraction kit, and then reverse transcription is carried out to obtain DNA; and (3) PCR system: adding 7 mu L of enzyme-free water, CCR9 primer and 10 mu L of SYBR Green into 2 mu L of DNA, and carrying out 40 cycles of 95 ℃ for 30 seconds, (95 ℃ for 5 seconds, 58 ℃ for 20 seconds and 72 ℃ for 10 seconds), 95 ℃ for 10 seconds, (65 ℃ is increased to 95 ℃ for 5 seconds, each time is increased by 0.5 ℃) according to the program, and detecting the expression condition of the CCR9 gene by a Realtime-PCR instrument.
The proportion of CD40+/CD11c +, CD86+/CD11c + cells, the atRA-NE group was significantly higher than the remaining control group (FIG. 3, a), showing that atRA-NE has a greater ability to stimulate DC cell maturation. The expression level of CCR9 gene of cells co-incubated with atRA-NE was significantly higher than that of other control groups (FIG. 3, b), indicating that atRA-NE has stronger ability to stimulate CCR9 expression on lymphocyte surface, and the ligand CCL25 of CCR9 is highly expressed on intestinal tract surface, so that the atRA-NE improves the homing tendency of lymphocytes to intestinal tract.
Example 4 Retinoic acid nanoemulsion adjuvant enhanced immune response Using OVA-model antigen as model
1. Grouping and immunization of animals
The experiment was divided into 5 groups, in turn PBS, OVA + aluminium hydroxide, OVA + blank nanoemulsion adjuvant (BNE) and OVA + atRA-NE, with 8 BALB/c mice per group (6-8 weeks old, female, purchased from beijing vindeli). Three immunizations were administered intramuscularly at 0, 14 and 28 days. Antigen OVA is 10 mug/piece, aluminum hydroxide is 100 mug/piece, blank and retinoic acid nanoemulsion adjuvant are 100 mug/piece.
2. Characterization of acute inflammation at the site of injection
IL-6 and IL-15 are important cytokines involved in immune regulation, can enhance the lysis function of natural killer cells, can promote B cell precursors to become plasma cells for generating antibodies, and other reports show that the secretion of IL-15 has a positive effect on activating ATRA receptor RAR. 48 hours after single immunization, taking the quadriceps femoris muscle of the mouse (12 +/-1 mm multiplied by 5 +/-1 mm, 0.18 +/-0.02 g) and placing the muscle in a tissue crushing tube, adding 1mL of PBS and 5 zirconium oxide grinding balls with the particle size of 3mm, and homogenizing the mixture for 15s multiplied by 10 times on a homogenizer; the homogenate was centrifuged at 14800rpm at 4 ℃ for 20 minutes to collect the supernatant, and the concentrations of IL-6 and IL-15 in the homogenate supernatants of the respective samples were quantitatively determined according to the instructions of the mouse IL-6ELISA kit and IL-15ELISA kit.
The results show that 48h after single immunization, the nanoemulsion adjuvant induces IL-6 (figure 4, a) and IL-15 (figure 4, b) with the levels equivalent to those of the aluminum adjuvant at the injection site, and an inflammation microenvironment beneficial to improving the intestinal mucosa tropism of the lymphocyte is formed.
3. Detection of OVA-specific IgG, IgG1, IgG2a in serum
14 days after 3 times of immunization, blood is taken from eye sockets, eyeball blood is kept still at room temperature until obvious layering is achieved, centrifugation is carried out at 6000rpm for 10 minutes to separate serum, and the serum is diluted 1000 times by using antibody diluent; taking an ELISA 96-well plate, adding coating buffer solution containing 10 mu g/mL OVA into 100 mu L/well, and washing the plate after overnight at 4 ℃; adding PBST containing 1% BSA at 250 μ L/well, incubating at 37 ℃ for 2 hours, and washing the plate; adding 1: diluting the serum with 1000 times of the antibody diluent, incubating at 37 ℃ for 1 hour, and washing the plate; adding 1: 10000 diluted goat anti-mouse IgG, goat anti-mouse IgG1 and goat anti-mouse IgG2a, incubating for 40min, and washing the plate; adding TMB into 100 mu L/hole for developing for 15 minutes, then adding stop solution into 100 mu L/hole to stop the color development reaction, and reading the light absorption value with the wavelength of 450nm by using an enzyme-linked immunosorbent assay; the antibody titer was calculated as a cutoff value, which was 2.1 times the average value of the PBS group mice OD450 nm.
The results show that atRA-NE elicits high levels of OVA-specific IgG in serum comparable to that of aluminum adjuvant (fig. 5, a), demonstrating that it is effective in activating systemic humoral immune responses. Their IgG subtypes were analyzed and, compared to the aluminum adjuvant, atRA-NE induced more specific IgG1 (fig. 5, b) and IgG2a (fig. 5, c), suggesting that its ability to mediate CD8+ and CD4+ T cell responses may be stronger than that of the aluminum adjuvant; and levels of IgG1 were higher than IgG2a (fig. 5, d), suggesting that atRA-NE may induce a Th2 biased cellular immune response.
4. Detection of OVA-specific sIgA in intestinal lavage fluid
Preparing PBS solution containing 0.1mg/mL of trypsin inhibitor, 50mM of EDTA-2Na and 1mM of PMSF to be used as intestinal lavage fluid; cutting the small intestine along the intestinal canal, immersing the small intestine into the intestinal lavage fluid, performing vortex oscillation in a refrigeration house at 4 ℃ for 30 minutes to fully dissolve the content of the small intestine, centrifuging the small intestine for 20 minutes at 4000rpm, taking the supernatant, centrifuging the supernatant for 10 minutes at 14800rpm, and taking the supernatant to obtain the intestinal lavage fluid sample. The ELISA plate coating step is the same as the previous step; adding PBST containing 20% FBS into 250 mu L/well after washing the plate, incubating and sealing for 2 hours, then washing the plate, adding 100 mu L PBST containing 20% FBS into each well, then adding 100 mu L small intestinal lavage fluid into each sample well, after dilution by multiple times, incubating for 1 hour at 37 ℃, and then washing the plate; 100 μ L/well 1: 10000 diluted goat anti-mouse IgA, and washing the plate after incubation for 40 minutes; color development, plate reading and detection of antibody levels.
The results show that the intramuscular injection of pure antigen and the combination with aluminum adjuvant can not induce specific sIgA in intestinal mucosa, and the atRA-NE can obviously improve the level of OVA specific sIgA in intestinal mucosa (figure 6), which indicates that the atRA-NE has obvious intestinal mucosa adjuvant effect.
5. Detection of OVA-specific sIgA antibody levels in vaginal lavage fluid
Vaginal douche solution: the vagina of OVA antigen treated mice was washed 4 times with 75. mu.l PBST to obtain 300. mu.l vaginal lavage solution, which was centrifuged at 14800rpm at 4 ℃ for 10min and the antibody content in the supernatant was measured by ELISA. The results are shown in FIG. 7, which indicates that atRA-NE can significantly enhance secretion of OVA-specific antibody IgA of vaginal mucosa, and indicates that atRA-NE can effectively induce the immune effect of vaginal mucosa.
6. Detection of OVA-specific sIgA antibody levels in alveolar lavage fluid
Mice were sacrificed and lungs from OVA antigen treated mice were lavaged 3 times with 500 μ l of sterile PBS to obtain alveolar lavage fluid (BALF) from which the antibody content was measured by ELISA. The results are shown in FIG. 8, which indicates that atRA-NE can remarkably enhance the secretion of lung mucosa OVA specific antibody sIgA, and indicates that atRA-NE can effectively induce the immune effect of lung mucosa.
7. Detection of OVA-specific sIgA antibody levels in gastric homogenates
The whole stomach of the mice treated by the OVA antigen is taken, 1ml of normal saline is added, homogenate is carried out, 14800rpm is carried out, the temperature is kept at 4 ℃, and the supernatant is centrifuged for 10min to measure the content of the antibody. The results are shown in FIG. 9, which indicates that atRA-NE can significantly enhance the secretion of the gastric mucosal OVA-specific antibody IgA, and indicates that atRA-NE can effectively induce the immune effect of the gastric mucosa.
8. Detection of IL-4, IL-17 in spleen lymphocyte culture supernatant
Taking out mouse spleen, grinding in 3mL erythrocyte lysate to obtain spleen cell suspension, centrifuging, discarding supernatant, resuspending cells with complete culture medium, and adjusting spleen lymphocyte concentration to 1 × 1071 mL/mL, added in a 24-well plate; preparing complete culture medium solution containing 1mg/mL OVA, filtering and sterilizing by a 0.22 mu m filter, adding 10 mu L of cell suspension into each hole, and incubating for 48 hours in a cell culture box at 37 ℃; centrifuging at 1500rpm for 10min, and taking supernatant; the concentration of each cytokine in the cell culture supernatant was determined as described in the IL-4, IL-17 assay kit instructions.
The results show that ELISpot assay is OVA-tested257-264Or OVA323-339In spleen lymphocyte culture supernatant after epitope peptide stimulation, the level of IL-4 in the aluminum adjuvant group is obviously higher than that in the nanoemulsion adjuvant group (figure 10, a), the level of IL-17A in the nanoemulsion adjuvant group is obviously higher than that in the aluminum adjuvant group (figure 10, b), which indicates that the aluminum adjuvant mainly stimulates Th2 type cellular immune response, and the atRA-NE contributes to Th1 and Th17 type immune responses.
9. ELISPOT measures the level of IFN-gamma secretion from splenic lymphocytes
Adding 100 mu L/hole of lymphocyte suspension with adjusted concentration in reference 3.3 into an ELISPOT plate pre-coated with IFN-gamma antibody; then 100. mu.L of blank complete medium, or 20. mu.g/mL OVA was added257-264Or OVA323-339The complete medium solution of (a), incubating for 72 hours in a cell incubator; the in-plate cells were subtracted and the plate washed with PBS, 100 μ L added 1: 1000 diluted detection antibody (BVD6-24G 2-biotin), standing at room temperature for 2 hours, and washing the plate; 100 μ L of 1: standing for 1 hour at room temperature for washing plates by using 1000-diluted streptavidin-horseradish peroxidase; after 100. mu.L of TMB was added and developed for 15 minutes, the plate was washed with pure water, dried and read using a plate reader and spots were counted.
ELISpot assay by OVA257-264Or OVA323-339IFN-gamma water secretion from splenic lymphocytes after epitope peptide stimulationIn parallel, the atRA-NE group was significantly higher than the aluminum adjuvant group, and the aluminum adjuvant group was significantly higher than the blank nanoemulsion adjuvant group (fig. 11, a, b, c), suggesting that atRA-NE can induce a more significant cellular immune response compared to the aluminum adjuvant.
Example 5 Funginic acid nanoemulsion adjuvant with the antigen HI of Staphylococcus aureus as a model to enhance immune response
1. Grouping and immunization of animals
The experiment was divided into 5 groups, in turn PBS, HI + aluminium hydroxide, HI + blank nanoemulsion adjuvant (BNE), HI + atRA and HI + atRA-NE, with 8 BALB/c mice per group (6-8 weeks old, female, purchased from beijing witnessel, inc). Three immunizations were administered intramuscularly at 0, 7 and 14 days. Antigen HI 10 μ g/mouse; 100 mu g of aluminum hydroxide per unit, and 100 mu g of blank and tretinoin nano-emulsion adjuvant per unit.
2. Detection of HI-specific IgG in serum
14 days after the last immunization, the HI antigen-treated mice were sacrificed, blood was taken from the orbit, serum was allowed to stand at room temperature until stratification, diluted to a desired fold with an antibody diluent, and the antibody content was measured by ELISA. Results as shown in figure 12, atRA-NE elicited high levels of HI-specific IgG in serum comparable to aluminum adjuvant, demonstrating that it was effective in activating systemic humoral immune responses.
3. Detection of HI-specific sIgA antibody levels in vaginal lavage fluid
14 days after the last immunization, the vagina of HI antigen-treated mice was washed 4 times with 75. mu.l PBST to obtain 300. mu.l vaginal lavage fluid, centrifuged at 14800rpm at 4 ℃ for 10min, and the antibody content in the supernatant was measured by ELISA. The results are shown in FIG. 13, indicating that atRA-NE significantly enhances the secretion of the vaginal HI-specific antibody IgA.
Example 6 Retinoic acid nanoemulsion adjuvant enhances immune response using novel coronavirus recombinant protein antigen RBD as model
1. Grouping and immunization of animals
The experiment was divided into 5 groups, in turn PBS, RBD + blank nanoemulsion adjuvant (BNE) and RBD + atRA-NE, with 8 BALB/c mice per group (6-8 weeks old, female, purchased from beijing vintonia). Three immunizations were administered intramuscularly at 0, 14 and 28 days. The RBD of the antigen is 20 mu g/mouse; 100 mu g of aluminum hydroxide per unit, and 100 mu g of blank and tretinoin nano-emulsion adjuvant per unit.
2. Detection of RBD-specific antibodies in serum
The RBD antigen treated mice were sacrificed 14 days after the last immunization, blood was taken from the orbit, serum was allowed to stand at room temperature until stratified, diluted to a desired fold with antibody diluent, and the antibody content was measured by ELISA. atRA-NE elicited high levels of RBD-specific IgG (fig. 14, a), IgA (fig. 14, B) in serum, demonstrating that it was effective in activating systemic humoral immune responses.
3. Detection of RBD-specific sIgA antibody levels in vaginal lavage fluid
14 days after the last immunization, the vagina of RBD antigen-treated mice was washed 4 times with 75. mu.l PBST to obtain 300. mu.l vaginal lavage fluid, centrifuged at 14800rpm at 4 ℃ for 10min, and the antibody content in the supernatant was measured by ELISA. The results are shown in FIG. 15, indicating that atRA-NE significantly enhances the secretion of the vaginal RBD-specific antibody IgA.
4. Detection of RBD-specific sIgA antibody levels in alveolar lavage fluid
Mice were sacrificed 14 days after the last immunization, and the RBD antigen-treated mice lungs were perfused 3 times with 500. mu.l of sterile PBS to obtain alveolar lavage fluid (BALF), from which the antibody content was measured by ELISA. The results are shown in FIG. 16, which indicates that atRA-NE can significantly enhance the secretion of the lung mucosa RBD specific antibody sIgA.
5. Detection of RBD-specific sIgA antibody levels in nasal lavage fluid
The RBD antigen-treated mice were sacrificed 14 days after the last immunization, and the upper respiratory tract and nasal cavity of the mice were lavaged with 500. mu.l of sterile PBS to obtain a lavage solution for nasal cavity, and the antibody content was measured by ELISA. The results are shown in FIG. 17, which indicates that atRA-NE can significantly enhance the secretion of the nasal mucosa RBD specific antibody IgA, and indicates that atRA-NE can effectively induce the immune effect of the nasal mucosa.
6. Detection of spleen lymphocyte IgA ASC and IgG ASC level by ELISPOT
Taking mouse spleen, grinding, filtering to obtain mouse spleen lymphocyte, adjusting fine cell with culture mediumCell concentration to 106one/mL. Incubate on RBD-coated ELISPOT plates for 24h, followed by development as per ELISPOT kit (Mabtech) instructions.
As shown in the figure, the results show that the atRA-NE can remarkably enhance the secretion of RBD specific antibodies IgG ASC (figure 18) and IgA ASC (figure 19) of spleen lymphocytes, and the atRA-NE can effectively induce the expression of antibody secreting cells in the spleen lymphocytes.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims (10)

1. The tretinoin nanoemulsion adjuvant is characterized by comprising the following components in parts by weight: 0.01-5 parts of tretinoin, 1-30 parts of surfactant, 0.1-10 parts of cosurfactant, 0.1-10 parts of oil phase and 44-97.9 parts of water phase.
2. The tretinoin nanoemulsion adjuvant according to claim 1, characterized in that: the surfactant is any one or combination of tween-20, tween-60, tween-80, tween-85, polyoxyethylene hydrogenated castor oil or polyoxyethylene castor oil.
3. The tretinoin nanoemulsion adjuvant according to claim 1, characterized in that: the cosurfactant is span-80, span-85, absolute ethyl alcohol, propylene glycol, glycerol or 1, 3-butanol or the combination of a plurality of the same.
4. The tretinoin nanoemulsion adjuvant according to claim 1, characterized in that: the oil phase is any one or combination of ethyl acetate, squalane, squalene and ethyl oleate.
5. The tretinoin nanoemulsion adjuvant according to any one of claims 1 to 4, characterized by comprising the following components in parts by weight: 6 parts of tretinoin, 5 parts of squalene, 8030 parts of tween, 8015 parts of span-8015 parts and 44 parts of water.
6. The tretinoin nanoemulsion adjuvant according to any one of claims 1 to 5, characterized in that: the particle size distribution of the tretinoin nanoemulsion adjuvant is 1-100nm, the dispersion index is less than 0.3, and the Zeta potential absolute value is less than 30 mV.
7. The preparation method of tretinoin nanoemulsion adjuvant according to any one of claims 1 to 6, characterized by comprising the following steps: weighing tretinoin, surfactant, cosurfactant, oil phase and water according to a proportion; fully dissolving the tretinoin in the oil phase, then dripping the surfactant and the cosurfactant, and finally slowly dripping the sterilized water while stirring to obtain the tretinoin nanoemulsion adjuvant.
8. Vaccine comprising the tretinoin nanoemulsion adjuvant according to any one of claims 1 to 6, characterized in that: the vaccine antigens are a model antigen OVA, a staphylococcus aureus recombinant protein antigen and a new coronavirus vaccine recombinant protein antigen.
9. The tretinoin nanoemulsion adjuvant of any one of claims 1 to 6, for use in the preparation of a vaccine adjuvant, characterized in that: the adjuvant is an adjuvant which can effectively activate systemic humoral immune response and mucosal immune response.
10. Use according to claim 9, characterized in that: the mucosa is one or more of intestinal mucosa, vaginal mucosa, pulmonary mucosa, gastric mucosa or nasal mucosa.
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