CN111494619B

CN111494619B - Preparation method of squalene-based cationic nanostructured lipid carrier immunologic adjuvant

Info

Publication number: CN111494619B
Application number: CN202010341140.0A
Authority: CN
Inventors: 郭大伟; 蔡莹
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2020-04-26
Filing date: 2020-04-26
Publication date: 2022-03-18
Anticipated expiration: 2040-04-26
Also published as: CN111494619A

Abstract

The invention discloses a preparation method of a squalene-based cationic nanostructured lipid carrier immunologic adjuvant, which comprises the following steps: (1) heating palmitic acid, squalene, emulsifier Tween 80 and span 85 in water bath for melting, and stirring to obtain oil phase; (2) heating chitosan solution in water bath to obtain water phase; (3) under the condition of stirring, transferring the oil phase into the water phase, uniformly stirring, and shearing by using a high shear apparatus to obtain a crude dispersion; (4) and (3) processing the crude dispersion by using an ultrasonic crusher, transferring the crude dispersion into cold water for shearing to obtain primary emulsion, then carrying out ice-water bath, and solidifying to obtain a product, namely csNLCs. The immunologic adjuvant has the functions of promoting antigen presentation cells to take up antigen and enhancing the immune response of organisms, and has stable performance.

Description

Preparation method of squalene-based cationic nanostructured lipid carrier immunologic adjuvant

Technical Field

The invention relates to the technical field of veterinary drug preparations, and in particular relates to a preparation method of a squalene-based cationic nanostructured lipid carrier immunologic adjuvant.

Background

The novel vaccine needs the assistance of an immune adjuvant due to low immunogenicity so as to improve the immune response level, and the nanoparticles are widely researched as the immune adjuvant. As a new generation of lipid nanoparticles, Nanostructure Lipid Carriers (NLCs) have the advantages of high encapsulation efficiency, good stability, and capability of efficiently encapsulating antigen substances or immunostimulating substances, and can enhance the stability of antigens and protect the antigens from being degraded. And the NLCs are composed of solid lipid, liquid lipid and surfactant, the preparation method is simple, the cost is low, and the NLCs can be easily prepared into lyophilized powder for long-term storage. However, the existing carrier has low stability, and the effect of the immune adjuvant is not strong.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a preparation method of a squalene-based cationic nanostructured lipid carrier immune adjuvant.

The technical scheme is as follows: the invention provides a preparation method of a squalene-based cationic nanostructured lipid carrier immune adjuvant, which comprises the following steps:

(1) heating palmitic acid, squalene, emulsifier Tween 80 and span 85 in water bath for melting, and stirring to obtain oil phase;

(2) heating chitosan solution in water bath to obtain water phase;

(3) under the condition of stirring, transferring the oil phase into the water phase, uniformly stirring, and shearing by using a high shear apparatus to obtain a crude dispersion;

(4) and (3) processing the crude dispersion by using an ultrasonic crusher, transferring the crude dispersion into cold water for shearing to obtain primary emulsion, then carrying out ice-water bath, and solidifying to obtain a product, namely csNLCs.

Further, the mass ratio of the palmitic acid to the squalene is 1-9: 1-9.

Further, the mass ratio of the Tween 80 to the span 85 is 0.5-1: 1.

Further, the ratio of the composite emulsifier formed by the Tween 80 and the span 85 to the mixed lipid formed by mixing the palmitic acid and the squalene is 1: 0.1-0.5.

Furthermore, the chitosan solution is prepared by dissolving medium-viscosity chitosan in a glacial acetic acid solution, and the mass fraction of solute chitosan is 0.1-0.5%.

Further, the particle size of the product is 235.80 +/-5.991 nm, the Zeta potential value is 34.90 +/-6.954 mv, and the polydispersity index is 0.283 +/-0.015.

In the above technical scheme:

squalene (squalene) is an unsaturated triterpenoid aliphatic hydrocarbon. Squalene recruits immune cells to the injection site and enhances local cytokine production, resulting in an increase in the number of antigen-loaded antigen-presenting cells. MF59 adjuvant containing squalene component has been approved by FDA in the united states for marketing to various subunit vaccines, such as hepatitis b vaccine, hepatitis c vaccine, influenza vaccine, etc.

Chitosan (CS) is the only positively charged natural polysaccharide in nature. The cationic coating is used for modifying the surface of the nanoparticle, so that the antigen can be stabilized, the antigen can be prevented from being subjected to enzymatic degradation and mechanical degradation, and the cationization of the surface of the nanoparticle can promote the antigen to enter target cells. The chitosan is used as a modified material of the controlled-release carrier, and the modified nano carrier not only has a protection effect on the nano carrier, but also can improve the stability of the nano carrier, promote the absorption of active substances and enhance the slow release of the active substances.

Has the advantages that: the method is simple and rapid, the cost is low, the prepared csNLCs are milky emulsion in appearance, the emulsion is free of layering, and the system is stable. The csNLCs prepared by the invention can be used as a carrier of an antigen or other immune enhancing substances, has the functions of promoting the antigen uptake of antigen presenting cells and enhancing the immune response of an organism, fully plays the role of an immune adjuvant, and is a potential antigen delivery system.

Drawings

FIG. 1 is a particle size distribution plot of csNLCs from example 2;

FIG. 2 is a graph of the potential distribution of csNLCs obtained in example 2;

FIG. 3 is a TEM photograph of csNLCs prepared in example 2;

FIG. 4 is a graph of the results of flow cytometry for the cellular uptake experiments performed after loading of antigen OVA with csNLCs in example 4;

FIG. 5 is a graph showing the IgG content of the mouse serum antibodies measured after the csNLCs are injected into mice after being entrapped with the OVA antigen in example 5.

Detailed Description

The optimization process of the preparation conditions of the method of the invention is as follows:

(1) determining the ratio of palmitic acid to squalene and the ratio of emulsifier to lipid mixture

Palmitic acid and squalene were compounded at 1: 9, 3: 7, 5: 5, 7: 3 and 9: 1 to give 1g of a molten mixed lipid as an oil phase to which 10%, 20%, 30%, 40% and 50% of an emulsifier (tween 80: span 85 ═ 1: 1) was added. 10mL of pure water was used as the aqueous phase, and the cold water dispersion volume was 11 mL. The hydrodynamic size, zeta potential and PDI of sNLCs and the layering after standing were examined as the screening basis (see Table 1).

Table 1 prescription screening and parameter measurements (n ═ 3, mean ± SD)

Note: -: no layering; +: there is delamination.

According to the screening in the table above, when the ratio of palmitic acid to squalene is 1: 9, no layering phenomenon occurs at each emulsifier concentration, and when the emulsifier accounts for 30% of the mixed lipid ratio, the emulsifier has the smallest PDI value at the ratio, has a moderate particle size, has an absolute potential value of more than 30mV, is stable, and considering the toxicity of the emulsifier and the problem of cost reduction, the most preferable ratio is 1: 9 of palmitic acid to squalene, and the total amount of the emulsifier accounts for 30% of the mixed lipid.

(2) Determination of the concentration of Chitosan

Based on the above-selected lipid ratio as the prescribed amount of the oil phase, chitosan solutions having concentrations of 0.1%, 0.2%, 0.3%, 0.4% and 0.5% were prepared, respectively, and 10mL was taken as the aqueous phase. The cold water dispersion volume was 11 mL. The optimal chitosan concentration was determined based on hydrodynamic size, zeta potential and PDI of the csNLCs produced and the layering after standing (see Table 2).

Table 2 prescription screening and parameter measurements (n ═ 3, mean ± SD)

According to the screening in the table, when the chitosan concentration is 0.1%, the chitosan has the smallest particle size and meets the requirements that PDI is less than 0.3, the potential absolute value is more than 30mV, and no layering phenomenon exists. Most preferably 0.1%.

Example 1

Materials: 0.1g of palmitic acid, 0.9g of squalene, 0.5g of emulsifier (Tween 80: span 85 ═ 0.5: 1), 10mL of 0.3% chitosan solution and 10mL of pure water

The preparation method comprises the following steps: weighing 0.1g of palmitic acid, 0.9g of squalene, 0.125g of tween 80 and 0.25g of span 85, placing in a penicillin bottle, heating and melting in a magnetic stirring water bath kettle at 80 ℃, and uniformly stirring to obtain an oil phase; then 10mL of 0.3% chitosan solution is weighed and placed in another penicillin bottle, and the solution is heated in a magnetic stirring water bath kettle under the same temperature condition to obtain a water phase. And (3) under the condition of stirring, transferring the oil phase into the water phase, taking out magnetons after uniform mixing, and shearing for 5min at the rotating speed of 11000rpm by using a high shear apparatus to obtain a crude dispersion. Then crushing with ultrasonic crusher at 300W amplitude for 20min to obtain colostrum, and shearing in cold water of the same volume for 1 min. And (4) carrying out ice-water bath on the obtained emulsion for 10min, and solidifying to obtain csNLCs.

And (3) detection: the prepared csNLCs have the particle size of 247.3 +/-4.115 nm, the zeta potential of 35.93 +/-1.457 mV and the PDI of 0.356 +/-0.051.

Example 2

Materials: 0.1g of palmitic acid, 0.9g of squalene, 0.3g of emulsifier (Tween 80: span 85: 1), 10mL of 0.1% chitosan solution and 10mL of pure water

The preparation method comprises the following steps: weighing 0.1g of palmitic acid, 0.9g of squalene, 0.15g of tween 80 and 0.15g of span 85, placing in a penicillin bottle, heating and melting in a magnetic stirring water bath kettle at 80 ℃, and uniformly stirring to obtain an oil phase; then 10mL of 0.1% chitosan solution is weighed and placed in another penicillin bottle, and the solution is heated in a magnetic stirring water bath kettle under the same temperature condition to obtain a water phase. And (3) under the condition of stirring, transferring the oil phase into the water phase, taking out magnetons after uniform mixing, and shearing for 5min at the rotating speed of 11000rpm by using a high shear apparatus to obtain a crude dispersion. Then crushing with ultrasonic crusher at 300W amplitude for 20min to obtain colostrum, and shearing in cold water of the same volume for 1 min. And (4) carrying out ice-water bath on the obtained emulsion for 10min, and solidifying to obtain csNLCs.

The particle size distribution diagram of csNLCs prepared in this example is shown in FIG. 1, and the average particle size of the micelles obtained by measuring the particle size of the micelles by using a Malvern laser particle sizer is 235.80 + -5.991 nm. The potential distribution diagram is shown in figure 2, potential measurement is carried out on the obtained micelle by using a Malvern laser particle size analyzer, and the Zeta potential value is measured to be 4.90 +/-6.954 mV, which indicates that the system is stable; the Polydispersity (PDI) was 0.283. + -. 0.015, indicating a uniform particle size. The transmission electron microscope photograph is shown in fig. 3, the micelle is in a nearly spherical shape and uniform in size when observed under an electron microscope, and corona wrapped by chitosan can be seen.

Example 3

Materials: 0.9g of palmitic acid, 0.1g of squalene, 0.3g of emulsifier (Tween 80: span 85: 1), 10mL of 0.5% chitosan solution and 10mL of pure water

The preparation method comprises the following steps: weighing 0.9g of palmitic acid, 0.1g of squalene, 0.15g of tween 80 and 0.15g of span 85, placing in a penicillin bottle, heating and melting in a magnetic stirring water bath kettle at 80 ℃, and uniformly stirring to obtain an oil phase; then 10mL of 0.5% chitosan solution is weighed and placed in another penicillin bottle, and the solution is heated in a magnetic stirring water bath kettle under the same temperature condition to obtain a water phase. And (3) under the condition of stirring, transferring the oil phase into the water phase, taking out magnetons after uniform mixing, and shearing for 5min at the rotating speed of 11000rpm by using a high shear apparatus to obtain a crude dispersion. Then crushing with ultrasonic crusher at 300W amplitude for 20min to obtain colostrum, and shearing in cold water of the same volume for 1 min. And (4) carrying out ice-water bath on the obtained emulsion for 10min, and solidifying to obtain csNLCs.

And (3) detection: the prepared csNLCs have the particle size of 1196.00 +/-6.120 nm, the zeta potential of 39.30 +/-1.587 mV and the PDI of 0.843 +/-0.151.

Example 4

The csNLCs prepared in the example 2 are coated with fluorescein labeled model antigen chicken ovalbumin (FITC-OVA) to prepare FITC-OVA-csNLCs, the cold water dispersion in the preparation step (4) is replaced by FITC-OVA solution with a certain concentration, and the rest is unchanged. Resuspending cells using fresh media to obtain 5X 10⁶And (3) inoculating 1mL of cell suspension per well of a 24-well plate, culturing in a cell culture box for 12h, removing culture medium in the well, adding 1mL of culture medium containing FITC-OVA or FITC-OVA-csNLCs, wherein the concentration gradient is 100, 200, 400, 500 and 800 mu g/mL (based on the concentration of csNLCs), and diluting the FITC-OVA until the concentration of the FITC-OVA is the same as that of the FITC-OVA in the FITC-OVA-csNLCs. 3 replicates were set for each concentration and a negative control was set. Culturing in a cell culture box for 4h, and discarding the culture medium containing FITC-OVA or FITC-OVA-csNLCs in each well. The wells were washed with cold PBS, then trypsinized and the digestion was stopped with serum-containing medium, and the cells were collected in a centrifuge tube protected from light. Centrifuging at 3000rpm for 3min, discarding the supernatant, resuspending with 1mL PBS, and analyzing fluorescence of each group of cells by flow cytometryLight intensity.

As shown in FIG. 4, it was found that the fluorescence intensity of cells did not change significantly as the FITC-OVA treatment concentration increased; with the increase of the treatment concentration of FITC-OVA-csNLCs, the fluorescence of the cells is gradually increased. When the concentration of the csNLCs is higher than 400 mu g/mL, the fluorescence intensity is continuously increased along with the increase of the concentration of the csNLCs, and the amount of the taken antigen is also continuously increased. When the concentration of csNLCs is not lower than 400 mu g/mL, the difference of the fluorescence intensity of cells is obvious compared with that of the FITC-OVA group with the same concentration. Indicating that csNLCs can promote the uptake of FITC-OVA by RAW264.7 cells.

Example 5

Animal experiments are used for further evaluating the immune adjuvant effect of the csNLCs, and Balb/c SPF mice are selected as animal models for in vivo experiments. 40 female Balb/c mice of 6-8 weeks old were statically maintained for one week before the experiment. Randomly divided into 4 groups, which are: (1) physiological saline; (2) OVA; (3) csNLCs and (4) OVA-csNLCs, wherein the saline group is a blank control.

On

day

0, 200. mu.L of each sample was injected subcutaneously into the back, and 20. mu.g of OVA was contained in both the (2) and (4) samples, and the csNLCs content in the (3) group was the same as that in the (4) group. The same injection amount was used for the booster immunization on day 14, and blood serum was collected on day 21. The content of the antibody in the serum of each group of mice was measured using a mouse IgG (OVA-sIgG) elisa kit, and the results are shown in FIG. 5.

The results show that compared with a blank group only injected with physiological saline, the contents of OVA specific antibody IgG in the serum of three groups of mice injected with OVA, csNLCs and OVA-csNLCs are all increased, particularly the difference between the OVA-csNLCs group and the blank control group injected with single antigen is very obvious, and the prepared csNLCs can enhance the humoral immune response level after being coated with the antigen.

The particle size of the cationic nanostructured lipid carrier based on squalene prepared by the invention is 235.80 +/-5.991 nm. The zeta potential value is 4.90 +/-6.954 mV, the system is stable, the polydispersity index (PDI) is 0.283 +/-0.015, and the particle size is uniform. The micelle is in a nearly spherical shape under the observation of an electron microscope, and no agglomeration phenomenon exists, so that corona wrapped by chitosan can be seen. The antigen carrier can promote the antigen presenting cell to phagocytize antigen and raise the humoral immune response level of mouse.

Claims

1. A preparation method of a squalene-based cationic nanostructured lipid carrier immune adjuvant is characterized by comprising the following steps: the method comprises the following steps:

(2) heating chitosan solution in water bath to obtain water phase;

(4) processing the coarse dispersion by using an ultrasonic crusher, transferring the processed coarse dispersion into cold water for shearing to obtain primary emulsion, then carrying out ice-water bath, and solidifying to obtain a product, and recording csNLCs;

the mass ratio of the palmitic acid to the squalene is 1-9: 1-9;

the mass ratio of the Tween 80 to the span 85 is 0.5-1: 1;

the ratio of the composite emulsifier formed by the Tween 80 and the span 85 to the mixed lipid formed by mixing the palmitic acid and the squalene is 1: 0.1 to 0.5;

the chitosan solution is prepared by dissolving medium-viscosity chitosan in a glacial acetic acid solution, wherein the mass fraction of solute chitosan is 0.1-0.5%.

2. The method for preparing the squalene-based cationic nanostructured lipid carrier immunoadjuvant of claim 1, wherein: the particle size of the product is 235.80 +/-5.991 nm, the Zeta potential value is 34.90 +/-6.954 mv, and the polydispersity index is 0.283 +/-0.015.