CN109694484B

CN109694484B - Immunologic adjuvant and preparation method thereof

Info

Publication number: CN109694484B
Application number: CN201811224649.6A
Authority: CN
Inventors: 巩长旸; 吴秦洁; 何涛; 魏于全
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2017-10-20
Filing date: 2018-10-19
Publication date: 2021-08-27
Anticipated expiration: 2038-10-19
Also published as: CN109694484A

Abstract

The invention discloses an immunologic adjuvant and a preparation method thereof. The invention relates to carboxymethyl chitosan-polyaldehyde mannan hydrogel, which is synthesized by a sodium periodate oxidation method, and can react with carboxymethyl chitosan through Schiff alkali to form hydrogel OX-M/NOCC through crosslinking. The OX-M/NOCC hydrogel used as an adjuvant for in vivo antigen delivery has the advantages of injectability and low toxicity, and meanwhile, the OX-M/NOCC hydrogel has the effect of 'antigen storage reservoir', and can promote the uptake of antigens by DCs. The OX-M/NOCC hydrogel combination protein vaccine increases antigen aggregation in lymph nodes, prolongs duration of action, and elicits a strong antigen-specific humoral immune response after immunization of mice, compared to aluminum adjuvants. The results indicate that OX-M/NOCC hydrogel is a potential novel vaccine adjuvant.

Description

Immunologic adjuvant and preparation method thereof

Technical Field

The invention relates to an immunologic adjuvant and a preparation method thereof, in particular to an immunologic adjuvant based on natural polysaccharide hydrogel and a preparation method thereof.

Background

With the rapid development of life science and technology, a plurality of novel vaccines are developed, and the vaccines have good antigen specificity and higher safety, but have small antigen molecular weight and high purification degree, so that the immunogenicity is poor. The immune adjuvant is also called as a non-specific immunopotentiator, and can improve the immunogenicity of antigens and enhance the self-protection capability of organisms: including stimulating the body to produce a functionally appropriate type of immune response; increasing the production of memory cells, particularly memory T cells; increase the speed of the initial immune response and alter the breadth, specificity or affinity of the immune response, etc. In recent years, a large number of basic and clinical researches on the immunologic adjuvant suitable for the novel vaccine are carried out by scholars at home and abroad, and great progress is made. Although many new adjuvants have been studied clinically, the safety requirements for human vaccine adjuvants are very strict, and only a very few adjuvants are approved. Therefore, the development of safe and efficient immunoadjuvants remains a major challenge at present.

Aluminum salt adjuvants and freund's adjuvants are currently widely used immunological adjuvants. The Freund's adjuvant combined with protein antigen can effectively stimulate plasma cells to generate antibodies and stimulate Th1 and Th17 cellular immune responses, but mineral oil in the Freund's adjuvant cannot be metabolized by organisms and easily causes side effects such as inflammatory reaction, granuloma and ulcer of injection sites, so the adjuvant cannot be used for human vaccines. Aluminum salt adjuvants have been widely used because of their antigen depot effect, to up-regulate MHC class II molecule expression, to promote antigen presentation, to induce Th2 immune response, but aluminum salt adjuvants cannot be lyophilized, and the prepared adjuvants have large batch-to-batch variation and are difficult to control quality. Therefore, a novel immunologic adjuvant which can enhance the immune response of the organism and has lower toxic and side effects is in urgent need of development.

The hydrogel is a material widely researched at present, has the characteristics of biocompatibility, biodegradability, flexible design and the like, and has a wide selection range of basic raw materials for forming the hydrogel. Various materials (dextran, gelatin, hyaluronic acid, chitosan, polypeptides, etc.) and crosslinking techniques (Schiff base reaction, Michael addition, self-assembly, change in physical conditions, etc.) have been investigated to design hydrogels of different porosity and mechanical strength. The hydrogel is insoluble in water but can expand in an aqueous medium to form a three-dimensional network structure, and the high swelling ratio also makes the hydrogel permeable to oxygen, nutrients and metabolites.

Dendritic cells are the most potent antigen presenting cells in the body and are critical for the generation of an effective protective immune response. After uptake by DCs, the intracellular localization and processing of antigens significantly affects the strength and quality of the immune response. Studies have shown that the Mannose Receptor (MR) is highly expressed on the surface of immature DC cells, and that MR plays a crucial role in the uptake, processing, presentation of antigens by DC cells and in the initiation of downstream immune responses. After mannan and antigen are combined through chemical action, researchers have remarkably promoted the uptake of antigen by DC cells through MR-mediated endocytosis, and the MHC II restricted antigen specific T cell reaction is enhanced.

Chitosan is a natural polysaccharide cationic polymer material, and is a polymer of glucosamine and N-acetylglucosamine. Chitosan has good biocompatibility and biodegradability, low toxicity, low immunogenicity and easy modification, and has been widely used as a drug carrier. Chitosan has low solubility in water and can only be dissolved under acidic conditions. In order to overcome the low solubility characteristic, different chitosan derivatives have been synthesized for enhancing the immune effect of the vaccine, such as N-trimethyl chitosan chloride salt, carboxymethyl chitosan and trimethyl chitosan, etc. The chitosan derivatives have good solubility and can generate better absorption promoting effect under physiological conditions.

Disclosure of Invention

The invention aims to provide an immunologic adjuvant and a preparation method thereof, and particularly relates to an immunologic adjuvant based on natural polysaccharide hydrogel and a preparation method thereof.

The invention provides carboxymethyl chitosan-polyaldehyde mannan hydrogel, which is prepared by crosslinking carboxymethyl chitosan and polyaldehyde mannan.

The carboxymethyl chitosan-polyaldehyde mannan hydrogel is prepared by the following method: and uniformly mixing the carboxymethyl chitosan solution and the polyaldehyde mannan solution to obtain the carboxymethyl chitosan-polyaldehyde mannan hydrogel.

Further, the volume ratio of the carboxymethyl chitosan solution to the polyaldehyde mannan solution is 1: 3-3: 1, preferably 1: 1.

Further, the oxidation degree of the aldehyde-group mannan is 7.66-45.33%.

Preferably, the oxidation degree of the aldehyde-based mannan is 13.41-45.33%.

Further, the concentration of the carboxymethyl chitosan solution is 10-30 mg/mL; the solvent of the carboxymethyl chitosan solution is normal saline.

Preferably, the concentration of the carboxymethyl chitosan solution is 15-30 mg/mL, and preferably 20-30 mg/mL.

Further, the concentration of the multi-aldehyde mannan solution is 10-30 mg/mL, and the solvent of the multi-aldehyde mannan is normal saline.

Preferably, the concentration of the polysaccharide-based mannan solution is 15-30 mg/mL, preferably 20-30 mg/mL.

Further, the temperature of the blending is 25 ℃.

Further, the time for uniformly mixing is 0-527 s.

Preferably, the time for uniformly mixing is 100-210 s.

Further, the preparation method of the polyaldehyde mannan comprises the following steps: dissolving mannan in phosphate buffer solution to form uniform solution, and adding NaIO₄Adding into the solution, reacting for 12 hours in the dark at 25 ℃, terminating the reaction,dialyzing to obtain the product.

Further, the pH of the phosphate buffer was 6.0.

Further, the mass-to-volume ratio of the mannan to the phosphate buffer is 1: 50 g/mL.

Further, the NaIO₄The molar ratio of the mannan monomer units to the mannan monomer units is 1: 10-3: 5.

Further, the termination reaction is a termination reaction with ethylene glycol.

Further, the dialysis bag used for dialysis has a molecular weight cutoff of 3500.

The carboxymethyl chitosan-polyaldehyde mannan hydrogel is used as an immunologic adjuvant.

The invention provides an immunologic adjuvant which is prepared by taking carboxymethyl chitosan-polyaldehyde mannan hydrogel as an effective component and adding pharmaceutically acceptable auxiliary materials or auxiliary components.

The normal saline solution formed by the invention is an aqueous solution of NaCl with the mass fraction of 0.9%.

In the present invention, OX-M represents polyaldehyde mannan; NOCC stands for carboxymethyl chitosan.

The invention has the beneficial effects that: the invention prepares an immunologic adjuvant, and the effective component of the immunologic adjuvant is carboxymethyl chitosan-polyaldehyde mannan hydrogel. Polyaldehyde mannan (OX-M) was synthesized by sodium periodate oxidation, which was cross-linked with carboxymethyl chitosan (NOCC) by Schiff base reaction to form hydrogel OX-M/NOCC. The OX-M/NOCC hydrogel used as an adjuvant for in vivo antigen delivery has the advantages of injectability and low toxicity, and meanwhile, the OX-M/NOCC hydrogel has the effect of 'antigen storage reservoir', and can promote the uptake of antigens by DCs. The OX-M/NOCC hydrogel combination protein vaccine increases antigen aggregation in lymph nodes, prolongs duration of action, and elicits a strong antigen-specific humoral immune response after immunization of mice, compared to aluminum adjuvants. The results indicate that OX-M/NOCC hydrogel is a potential novel vaccine adjuvant. In addition, compared with polyaldehyde mannan (OX-M) and carboxymethyl chitosan (NOCC), the OX-M/NOCC hydrogel as an immune adjuvant has the advantages of being capable of triggering stronger antigen-specific humoral immune response, promoting the uptake of DC to antigen and the like.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is the synthesis of polyaldehyde mannan and the preparation of hydrogel, (A) the synthesis of polyaldehyde mannan; (B) schematic preparation of hydrogel;

FIG. 2 is a chart of infrared spectra and X-ray diffraction analysis of (A) Mannan (Mannan), polyaldehyde Mannan (OX-M), carboxymethyl chitosan (NOCC) and OX-M/NOCC crosslinked hydrogel; (B) NOCC, OX-M with a theoretical degree of oxidation of 20%, and XRD pattern of the formed cross-linked hydrogel; (C) XRD patterns of hydrogels formed by cross-linking mannan (2%, w/v) with different theoretical oxidation degrees with NOCC (2%, w/v);

FIG. 3 is a plot of the rheology analysis, scanning electron microscopy and optical images of OX-M/NOCC hydrogels (A) storage modulus (G ') and loss modulus (G') over time during formation of crosslinked hydrogels from OX-M (2%, w/v) and NOCC (2%, w/v). The time corresponding to the intersection point of the G 'curve and the G' curve is the time point of the formation of the mechanical gel; (B) scanning electron microscopy of OX-M/NOCC crosslinked hydrogel (100 ×); (C) OX-M (2%, w/v), NOCC (2%, w/v) and crosslinked hydrogel optical patterns formed therefrom;

FIG. 4 is a MTT cytotoxicity assay of OX-M, NOCC and OX-M/NOCC hydrogel extracts, (A) MTT assay cell viability after 48h of OX-M and NOCC treatment of 3T3 cells; (B) after OX-M and NOCC process L929 cells for 48 hours, MTT detects the cell survival rate; (C) after 3T3 and L929 cells are respectively treated by OX-M/NOCC hydrogel leaching liquor with different concentrations for 48 hours, the survival rate of the cells is detected by MTT;

FIG. 5 is a graph of the antigenic library effect of OX-M/NOCC hydrogels, in vivo fluorescence images (A) of Balb/c mice injected with OX-M/NOCC hydrogels and FITC-OVA vaccine systems, respectively, and injected with large doses of FITC-OVA, at various time points; injection site fluorescence intensity as a percentage of the initial fluorescence intensity at each time point (B), 3 Balb/c mice per group at each time point ([ p ] 0.05, [ p ] 0.01, [ p ] 0.001);

FIG. 6 is a graph showing the effect of OX-M/NOCC hydrogels on OVA antigen uptake by bone marrow-derived dendritic cells (BMDCs) incubated for 2h at 37 deg.C (A) and 4 deg.C (B) with water-soluble OVA-FITC (OF) and Hydrogel/OVA-FITC (HOF), respectively, and after 4h, the ratio of OVA-FITC positive cells was flow-detected; (C) after incubation of BMDCs with water-soluble OVA-FITC and Hydrogel/OVA-FITC for 4h at 37 ℃, antigen intracellular localization was observed by confocal laser microscopy ([ p ] 0.05);

FIG. 7 is an OX-M/NOCC hydrogel promoting the production of antigen-specific antibodies in humoral immunity. Antigen-specific antibody detection in serum as time varies after immunization of groups of mice: (A) IgG, (B) IgG1, and (C) IgG 2B. (. about.p)<0.05,**p<0.01 represents the statistical difference between the Hydrogel group and the OVA group;^#p<0.05 represents the statistical difference between the Hydrogel group and the Alum group).

FIG. 8 is a line graph of the development of antigen-specific antibody titers in the promotion of humoral immunity by NOCC and OX-M/NOCC hydrogels. (A) IgG, (B) IgG1, and (C) IgG 2B. (. p <0.05,. p <0.01 indicates statistical difference between Hydrogel group and NOCC group).

Detailed Description

The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.

EXAMPLE 1 preparation of OX-M

Separately weighing 1.0g mannan, dissolving in 50mL phosphate buffer solution with pH of 6.0, dissolving under magnetic stirring to obtain homogeneous solution, and mixing 130mg, 260mg, 520mg and 780mg NaIO₄Adding into mannan solution (adding NaIO to the solution respectively₄The molar ratio of the mannan monomer units is 1:10, 1:5, 2:5 and 3:5), and the mixture is stirred in a dark place at the temperature of 25 DEG CStirring for 12 hours; the reaction was stopped with 0.5mL of ethylene glycol, and after 1 hour the solution was transferred to a dialysis bag with a molecular weight cut-off of 3500 and dialyzed in double distilled water for three days, changing the water 3 times a day, removing unreacted NaIO₄With ethylene glycol; after dialysis, respectively transferring dialysate to a wide-mouth bottle, placing the wide-mouth bottle in a refrigerator (-20 ℃), freezing, transferring the wide-mouth bottle to a precooled freeze dryer, and freeze-drying to obtain the polyaldehyde mannan with theoretical oxidation degrees of 10%, 20%, 40% and 60%, wherein the theoretical oxidation degree is calculated by: 1mol NaIO₄Oxidizing the ortho-hydroxyl structure in 1mol of mannan monomer unit, wherein the mannan is composed of a plurality of mol of monomer unit structures, so that the total mol number of the monomer unit can be obtained according to calculation, and then the mol of NaIO required by the polyaldehyde mannan with different oxidation ratios can be obtained through calculation₄The oxidation degree is given as an example of the ratio of the number of moles of the monomer units to be oxidized to the total number of moles of the monomer units, and the theoretical oxidation degree obtained by calculation is an absolute value. And (3) using a proper amount of product for infrared spectrum characterization and X-ray diffraction analysis, and placing the rest in a refrigerator (-20 ℃) for later use.

EXAMPLE 2 preparation of OX-M/NOCC hydrogels

Equal volumes of OX-M solution (solvent normal saline) with theoretical degree of oxidation of 20% and concentration of 20mg/mL and NOCC solution (solvent normal saline) with concentration of 20mg/mL were mixed for 191s, and OX-M and NOCC were crosslinked to form hydrogel.

EXAMPLE 3 preparation of OX-M/NOCC hydrogels

Equal volumes of OX-M solution (solvent normal saline) with theoretical degree of oxidation of 10% at a concentration of 30mg/mL and NOCC solution (solvent normal saline) at a concentration of 20mg/mL were mixed for 208s, and OX-M and NOCC were crosslinked to form a hydrogel.

EXAMPLE 4 preparation of OX-M/NOCC hydrogels

Equal volumes of OX-M solution (solvent normal saline) with a theoretical degree of oxidation of 40% and a concentration of 20mg/mL and NOCC solution (solvent normal saline) with a concentration of 10mg/mL were mixed for 136s, and OX-M and NOCC were crosslinked to form a hydrogel.

EXAMPLE 5 preparation of OX-M/NOCC hydrogels

Equal volumes of OX-M solution (solvent is normal saline) with theoretical degree of oxidation of 60% and concentration of 10mg/mL and NOCC solution (solvent is normal saline) with concentration of 20mg/mL are mixed for 110s, and OX-M and NOCC are crosslinked to form hydrogel.

The beneficial effects of the invention are illustrated by way of experimental examples as follows:

experimental example 1 measurement of actual degree of oxidation of OX-M

Drying hydroxylamine hydrochloride to constant weight, weighing 4.35g, and dissolving in 40mL of distilled water to form a uniform solution; adding 1.5mL of 0.05% methyl orange aqueous solution into hydroxylamine hydrochloride solution, uniformly mixing, transferring into a 250mL volumetric flask, and adding water to dilute to a scale for later use; taking 100mg of freeze-dried OX-M with each theoretical oxidation degree, adding the OX-M into 25mL of the prepared hydroxylamine hydrochloride-methyl orange solution, and fully stirring for 12 hours; measuring the amount of HCI generated in the solution by adopting a potentiometric titration method by taking 0.1mol/L NaOH solution as a titrant, and observing the color change of the solution; the titration was stopped when the final solution became yellow (pH 5), and the aldehyde group concentration and the actual degree of oxidation of OX-M were calculated from the consumption of NaOH solution.

Calculation of the actual degree of oxidation of OX-M:

Mannan-(CHO)_n+nH₂N-OH-HCI＝Mannan-(CH＝N-OH)_n+nH2O+nHCI (1)

HCI+NaOH＝NaCI+H₂O (2)

△V×0.001×n_NaOH＝n_CHO (3)

OD＝(n_CHO/2)/(w_mannan/162) (4)

Wherein Δ V is the volume of NaOH consumed at the time of titration, in mL; n is_NaOHIs the molar concentration of NaOH, and the unit is mol/L; n is_CHOIs the number of moles of aldehyde groups on mannan; w is a_MannanIs the mass of mannan in g; 162 is the molar mass of mannan monomer units; OD represents the actual degree of oxidation, which is the molar mass of mannan.

We prepared polyaldehyde mannans (OX-M) with theoretical oxidation degrees of 10%, 20%, 40% and 60% by sodium periodate oxidation method, respectively (see FIG. 1A), and measured the actual oxidation degree of OX-M by hydroxylamine hydrochloride method (see Table 1). Furthermore, we characterized Mannan and polyaldehyde Mannan by FTIR, as shown in fig. 2A, the infrared spectra of Mannan and OX-M are very similar, and no distinct aldehyde group absorption peak appears, which may be due to the fact that the aldehyde group obtained by oxidation forms a hemiacetal structure with the adjacent hydroxyl group, which makes it difficult to detect the presence of aldehyde group in the polymer chain. In experiments we also found that OX-M obtained by oxidation with sodium periodate was soluble in aqueous solution, but the solubility was significantly lower than mannan at 25 ℃ and required heating to 37 ℃ for dissolution. It is also possible that this temporary cross-linked network (hemiacetal structure) reduces the solubility of OX-M.

TABLE 1 preparation of polyaldehyde mannan

Material	Theoretical degree of oxidation	Actual degree of oxidation
			OX-M-1	10％	7.66±2.20
OX-M-2	20％	13.41±1.20
			OX-M-3	40％	31.25±1.24
OX-M-4	60％	45.53±2.19

After equal volumes of NOCC (20mg/mL) and OX-M (20mg/mL) at 20% theoretical oxidation were mixed together, the OX-M crosslinked with NOCC by Schiff base reaction to form a hydrogel (FIG. 1B). We analyzed the structure of the hydrogel by FTIR and XRD, respectively. FIG. 2A clearly shows that the IR spectrum of the hydrogel contains the characteristic absorption peak of NOCC (symmetrical and asymmetrical carboxylate anion stretching vibration absorption peak: 1411 cm)^-1And 1319cm^-1(ii) a N-H stretching vibration: 3379cm^-1) Furthermore, the absorption peak of the hemiacetal structure in OX-M of 814cm was not observed in the hydrogel pattern^-1Indicating that the aldehyde group of OX-M has Schiff base reaction with the amino group of NOCC.

FIG. 2B shows XRD patterns of OX-M, NOCC, and OX-M/NOCC hydrogels. As shown, the OX-M/NOCC hydrogel detected two distinct diffraction peaks at 2 θ of 31.9 ° and 45.6 °, respectively. This indicates that the polymer chains are altered to form a new crystal structure after the aldehyde group of OX-M is combined with the amino group of NOCC to form a Schiff base. Furthermore, we prepared hydrogels by mixing NOCC and OX-M of different theoretical oxidation degrees (10%, 20%, 40%, 60%) in equal proportions and examined the change in crystalline form by XRD. As shown in fig. 2C, hydrogels prepared from four different theoretical degrees of oxidation OX-M and NOCC all observed distinct diffraction peaks at 31.9 ° and 45.6 ° 2 θ, except that the higher the degree of oxidation, the weaker the peak intensity. The hydrogen bond of amino group with hydroxyl group is an important factor for stabilizing the crystal structure, which indicates that the more aldehyde groups in OX-M, the more amino groups in NOCC are consumed by Schiff base, and the reduction of hydrogen bond results in the crystal structure of hydrogel becoming more and more unstable.

EXAMPLE 2 rheological analysis of OX-M/NOCC hydrogels

The time to gel formation at 37 ℃ and gel strength of different degrees of oxidation OX-M and NOCC were analyzed by rheometry and orthogonal methods to screen combinations of material concentrations suitable for in vivo applications. Firstly, opening a rheometer and preheating, and selecting a 37 ℃ oscillation mode; respectively dissolving NOCC and OX-M with different oxidation degrees in physiological saline according to the concentration of 10mg/mL, 20mg/mL, 40mg/mL and 60mg/mL, and stirring to form uniform solution; 200 mu L of NOCC and OX-M are added on a parallel plate of the rheometer by adopting a double injector, so that bubbles are prevented from being generated; the gap between the mixed solution and the rotor was maintained at 1mm, the change in storage modulus (G ') and loss modulus (G') over time was recorded over 20min, and the time to hydrogel formation of OX-M with NOCC at different concentrations and degrees of oxidation was analyzed by Origin mapping.

We examined the gel formation time of 36 hydrogel samples at 37 ℃ using a rheometer and recorded the storage modulus G' values at 10 and 20 minutes (see Table 2). The gelation time was longer when the theoretical degree of oxidation of OX-M was 10%, the gelation time was shorter and larger as the degree of oxidation and the concentration increased, and the gelation time was less than 160 seconds when the theoretical degree of oxidation of OX-M was 60%, and the gelation time could not be detected after the concentration of OX-M reached 20 mg/mL. The gelation time of the crosslinked hydrogel that can be used in vivo cannot be too short because the semisolid hydrogel cannot be used for subcutaneous injection, and additional damage to the body can be caused by subcutaneous implantation. In addition, the storage modulus of the hydrogel should not be too high or too low, since too high would be detrimental to the degradation of the hydrogel in vivo, and too low would be detrimental to the sustained release of the antigen in vivo. For these reasons, hydrogels prepared with OX-M and NOCC at concentrations of 20mg/mL, both theoretical degrees of oxidation being 20%, are preferred, as are hydrogels prepared under this condition for subsequent experiments.

TABLE 2 preparation of different OX-M/NOCC hydrogels

As shown in FIG. 3A, when OX-M (20mg/mL) was mixed with NOCC (20mg/mL) at equal ratio, the loss modulus G "was small and tended to increase slowly, and the storage modulus G' just started to be lower than 0.1pa and increased rapidly with time. When t is 191 seconds, G '═ G ", and then G' > G". After freeze-drying of the OX-M/NOCC hydrogel, we observed the cross-sectional morphology of the hydrogel by scanning electron microscopy, as shown in FIG. 3B, which clearly shows the porous and highly connected internal structure of the hydrogel, which may have high permeability to protein antigens and support infiltration of cells. OX-M, NOCC, appeared as a flowable solution and non-flowable gel after cross-linking (FIG. 3C).

EXAMPLE 3 in vitro cytotoxicity assay

Taking L929 and 3T3 cells in a logarithmic growth phase, carrying out trypsinization and centrifugation, counting by using a cell counting plate, and carrying out heavy suspension by using a fresh culture medium; 100. mu.L of L929 and 3T3 cell suspensions were added to different 96-well plates, respectively, with cell densities set at 4X 10³A/hole and 2.5X 10³Per well; the cells were cultured overnight for adherent growth, and 100 μ L of each well was added with a medium containing different concentrations of OX-M and NOCC (0mg/mL to 2mg/mL) and different amounts of OX-M/NOCC hydrogel leaching solution prepared in example 2 (1mL of hydrogel plus 4mL of medium, and the leaching solution was prepared by placing in a shaker at 37 ℃ for 24h, followed by dilution of the leaching solution with the medium to make the stock solution content 0%, 25%, 50% and 100% >), respectively, with 6 duplicate wells for each material concentration; after continuing culturing for 48h, adding 20 mu L of sterile MTT solution (5mg/mL) into each well, and placing the mixture in an incubator for continuing culturing for 4 h; discarding the culture solution, and adding 150 mu L of DMSO into each well to dissolve the generated formazan; and placing the 96-well plate on an enzyme labeling instrument, oscillating for 1min, measuring the absorbance of 570nm wavelength, taking an untreated group as a control, and calculating the survival rate of cells under the materials with different concentrations according to the average value of the absorbance.

We used the MTT assay to study cytotoxicity of OX-M and NOCC and OX-M/NOCC hydrogel extracts on two mouse fibroblast cell lines 3T3 and L929. As shown in fig. 4, NOCC-treated cells all had an average survival rate of over 80%. For OX-M treated cells, 3T3 cell viability was maintained at 68.9% and L929 cell viability was over 80% at concentrations of 2mg/mL (FIG. 4). Hydrogel extracts at different concentrations did not show significant cytotoxicity (figure 4C). As analyzed from MTT cytotoxicity results, OX-M, NOCC and the formed hydrogel thereof are low in cytotoxicity and are safe biological materials.

Experimental example 4 in vivo Release test

OX-M/NOCC and fluorescently-labeled ovalbumin OVA-RBITC can be combined through non-covalent bonds to form hydrogel in situ under the skin of a mouse, and the rate of releasing OVA by the OX-M/NOCC is observed and quantitatively calculated by a living body imager. Preparing 8 Balb/c, dividing into PBS group and OX-M/NOCC hydrogel group prepared in example 2, 4 in each group, anesthetizing the mice with chloral hydrate (10%), and removing the injection sites and surrounding hairs on the back of the mice by using a hair shaving machine; respectively preparing PBS solution and hydrogel containing OVA-RBITC with concentration of 1mg/mL, and respectively injecting the solution to the subcutaneous back of mice with 200 μ L per mouse; after 10min of injection, the mice are placed in a black box of a living body imaging instrument for fluorescence imaging, the mice are marked and the corresponding fluorescence intensity is recorded; thereafter, fluorescence imaging was performed on each mouse at 1d, 3d, 6d, 10d, and 18d, respectively, and the fluorescence intensity corresponding to the mouse was recorded, and the in vivo release rate of the antigen was calculated using the fluorescence intensity obtained by imaging the injected mouse for 10min as the total amount.

We used Rhodamine (RBITC) labeled OVA for in vivo imaging to detect the rate of antigen release from OX-M/NOCC hydrogels in vivo by quantitative analysis. FIG. 5A shows a representative in vivo fluorescence image of each group of mice at various time points. The OVA-RBITC group injected alone showed a rapid decay in antigen fluorescence intensity within 1 day, leaving only 33.31% of the initial fluorescence intensity. This then continued to decrease and almost no fluorescence signal was detected by day 18. The OVA-RBITC fluorescence intensity of the hydrogel group slowly decreases, and the fluorescence signal is still detected by 18 days and reaches 15.26 percent of the initial fluorescence intensity. These results indicate that aqueous OVA solutions are rapidly broken down in vivo, while OX-M/NOCC hydrogels are able to slowly release antigen as a "antigen pool".

Experimental example 5 DC cell uptake experiment

OX-M/NOCC hydraulic: example 2 preparation.

Killing a C57 mouse by cervical dislocation, taking out a femur and a tibia under a sterile state, soaking in 75% alcohol, and transferring to RPMI1640 double-culture medium after 5 min; sucking RPMI1640 double non-culture with 1ml syringePuncturing a bone marrow cavity from one end of a backbone, flushing bone marrow into a sterile culture dish, repeating for 4-6 times for each bone, collecting bone marrow cell suspension in the culture dish, and centrifuging at 1500rpm for 5 min; discarding the supernatant, adding 5ml of sterile erythrocyte lysate to resuspend the cells, standing for 5 minutes at room temperature, dissolving the erythrocytes, centrifuging at 1500rpm for 5 minutes again, and discarding the supernatant; washing cells with RPMI1640 double-no culture solution, re-suspending with X-vivo15 culture medium, counting, adjusting cell density to 1 × 10⁶Dividing into 6-well culture plates, adding 4mL of cell-containing culture medium into each well, and adding rmGM-CSF to a final concentration of 20ng/mL and rmIL-4 to a final concentration of 10 ng/mL; the cell culture plate was placed at 37 ℃ and 5% CO₂Culturing in the incubator for 48-72 hours; after the cells are lightly blown, the suspended cells are sucked together with the culture solution, and only the adherent cells are reserved; adding fresh X-vivo15 culture medium and the same concentration of rmGM-CSF and rmIL-4, and continuing to culture for 5 days; half the amount of the solution is changed, rmGM-CSF is replenished, and suspension cells are kept as much as possible; the culture was continued until day 7, and all the suspension cells were collected by gently pipetting.

The cells were plated in 24-well plates at 500. mu.L/well and divided into a blank control group, a PBS/OVA-FITC group and an OX-M/NOCC/OVA-FITC group at an OVA-FITC concentration of 2.5. mu.g/mL; after incubation for 4h in the incubator in the dark, the cells were washed three times with PBS, resuspended in 300. mu.L of PBS, and tested on a flow-type machine.

The cells were plated in a polylysine-pretreated confocal 8-well chamber at 100. mu.L/well into a blank control group, a PBS/OVA-FITC group and an OX-M/NOCC/OVA-FITC group at a concentration of 2.5. mu.g/mL; incubating in an incubator for 4h in the dark, discarding culture supernatant, washing with precooled PBS for three times, and staining lysosome for 20min in the dark by Lyso-Tracker Red (1: 1000); removing lysosome staining solution, washing with precooled PBS for three times, and staining Hoechst (1:100) for 5min to mark cell nucleus; the cell nuclear staining solution is discarded, precooled PBS is washed for three times, then fixed for 10min by 4 percent paraformaldehyde, and observed by a laser confocal scanning microscope.

Exogenous antigens are first taken up by antigen presenting cells, such as DC cells, macrophages or B cells, after entering the human body. This process has an important role in the initiation of downstream immune responses. As shown in fig. 6A, the uptake of DC cells in the Hydrogel group was significantly increased (p-values of 0.0225 and 0.0138, respectively), whereas the DC antigen uptake was significantly lower than 37 ℃ in 2h or 4h of co-incubation at 4 ℃ (fig. 6B), indicating that the DC taken up antigen mainly by endocytosis. Confocal fluorescence imaging results next showed that the Hydrogel group antigens fluoresced more strongly than the water-soluble OVA-FITC, which also indicates that Hydrogel promotes antigen uptake by DCs.

EXAMPLE 6 ELISA detection of antigen-specific antibody production

OX-M/NOCC hydraulic: example 2 preparation.

C57 mice 6 to 8 weeks old were randomly divided into 4 groups: saline group, OVA group, Alum/OVA group and OX-M/NOCC/OVA group, each group consisting of 6 mice; immunizing each group of mice for three times (0d, 14d and 21d), injecting 100 mu L of corresponding solution into the back subcutaneous tissue at two points each time, wherein the concentration of OVA is 0.2mg/mL, the mass ratio of Alum to OVA-FITC is 5:1, and the concentration of OX-M/NOCC hydrogel is 20 mg/mL; performing orbital bleeding on each group of mice at 21d, 28d, 35d and 42d respectively, placing in a refrigerator at 4 ℃ overnight, centrifuging at 3500rpm at 4 ℃ for 15min, separating serum, subpackaging, and storing in a refrigerator at-80 ℃ for later use; adding carbonate buffer solution containing 10 mu g/mL OVA into an ELISA plate, placing the ELISA plate at 100 mu L/hole, and placing the ELISA plate in a refrigerator at 4 ℃ overnight for pre-coating; taking the coated ELISA plate, washing for 3 times by PBST, and drying on filter paper each time; adding 150 mu L of confining liquid into each hole, and incubating for 1h in a constant-temperature incubator at 37 ℃; PBST was washed 5 times, each time with filter paper and dried; adding 100 μ L diluted serum into each well, incubating in a constant temperature incubator at 37 deg.C for 1.5h, washing with PBST for 5 times, and drying on filter paper each time; adding 100 mu L of diluted HRP-labeled secondary antibody, incubating for 1h in a constant-temperature incubator at 37 ℃, washing for 5 times by PBST, and drying on filter paper each time; adding 100 mu L of TMB single-component color development liquid into each hole, and developing for 20min in a constant-temperature incubator at 37 ℃ in a dark place; after 50 μ L of stop solution was directly added to each well, the absorbance value at 450nm was read on a microplate reader. The lowest dilution corresponding to a test well absorbance value 2.1-fold greater than the control well is the antibody titer corresponding to that group of sera.

The slow release of the OX-M/NOCC hydrogels towards the antigen OVA and the promotion of antigen aggregation in lymph nodes prompted us to investigate further whether they induced a stronger protective immune response in vivo. As can be seen in fig. 7A, from the last immunization, the water-soluble OVA group produced lower antigen-specific IgG antibody titers than the Hydrogel and Alum groups, the Hydrogel group produced IgG antibody titers that were significantly higher at each time point than the OVA and Alum groups, and reached the highest value at day 28 after the last booster immunization, and the Hydrogel group was also significantly higher than the Alum and OVA groups for IgG-typed antibodies (fig. 7B and C). These results indicate that OX-M/NOCC hydrogels are able to enhance the immune response of the body in vivo.

EXAMPLE 7 ELISA detection of the Generation of antigen-antibody titers of OX-M/NOCC hydrogel group and NOCC group

OX-M/NOCC hydraulic: example 2 preparation.

C57 mice 6 to 8 weeks old were randomly divided into 4 groups: saline, OVA, NOCC and OX-M/NOCC/OVA groups, 6 mice per group; three times of immunization (0d, 14d and 21d) are carried out on each group of mice, 100 mu L of corresponding solution is injected into the back subcutaneous tissue at two points each time, wherein the concentration of OVA is 0.2mg/mL, the concentration of NOCC is 20mg/mL, and the concentration of OX-M/NOCC hydrogel is 20 mg/mL; performing orbital bleeding on each group of mice at 21d, 28d and 35d respectively, placing in a refrigerator at 4 ℃ overnight, centrifuging at 3500rpm at 4 ℃ for 15min, separating serum, subpackaging, and storing in a refrigerator at-80 ℃ for later use; adding carbonate buffer solution containing 10 mu g/mL OVA into an ELISA plate, placing the ELISA plate at 100 mu L/hole, and placing the ELISA plate in a refrigerator at 4 ℃ overnight for pre-coating; taking the coated ELISA plate, washing for 3 times by PBST, and drying on filter paper each time; adding 150 mu L of confining liquid into each hole, and incubating for 1h in a constant-temperature incubator at 37 ℃; PBST was washed 5 times, each time with filter paper and dried; adding 100 μ L diluted serum into each well, incubating in a constant temperature incubator at 37 deg.C for 1.5h, washing with PBST for 5 times, and drying on filter paper each time; adding 100 mu L of diluted HRP-labeled secondary antibody, incubating in a constant-temperature incubator at 37 ℃ for 1h PBST, washing for 5 times, and drying on filter paper each time; adding 100 mu L of TMB single-component color development liquid into each hole, and developing for 20min in a constant-temperature incubator at 37 ℃ in a dark place; after 50 μ L of stop solution was directly added to each well, the absorbance value at 450nm was read on a microplate reader. When the absorbance value of the experimental hole is 2.1 times larger than that of the control hole (the serum of the normal saline control group mouse), the corresponding lowest dilution factor is the antibody titer corresponding to the group of the serum.

The research reports that the chitosan has the function of stimulating the body immunity, but the influence of the water-soluble derivative on the body humoral immunity is not reported. As shown in fig. 8, the NOCC group alone stimulated mice to produce antigen-specific IgG antibody titers that were comparable to the water-soluble OVA group, all significantly lower than the OX-M/NOCC hydrogel group (fig. 8A, table 3). Furthermore, we found the same results for IgG classification antibodies IgG1 and IgG2B (fig. 8B and C, table 3). These results indicate that NOCC does not have the sole effect of stimulating humoral immunity in the body.

Experimental results show that the effect of NOCC on stimulating the humoral immunity of an organism independently is small, the stimulation effect can be greatly improved by adopting a mode of cross-linking OX-M and NOCC, the immunostimulation effect of OX-M is very weak, and the effect of the OX-M and NOCC in a synergistic mode is proved to be played.

TABLE 3

In conclusion, we prepared an injectable, low toxicity OX-M/NOCC hydrogel adjuvant for in vivo antigen delivery. The OX-M/NOCC hydrogel has an "antigen depot" effect, facilitating antigen uptake by DCs. The OX-M/NOCC hydrogel combination protein vaccine increases antigen aggregation in lymph nodes, prolongs duration of action, and elicits a strong antigen-specific humoral immune response after immunization of mice, compared to aluminum adjuvants. The results indicate that OX-M/NOCC hydrogel is a potential novel vaccine adjuvant. In addition, compared with polyaldehyde mannan (OX-M) and carboxymethyl chitosan (NOCC), the OX-M/NOCC hydrogel as an immune adjuvant has the advantages of being capable of triggering stronger antigen-specific humoral immune response, promoting the uptake of DC to antigen and the like.

Claims

1. Use of a carboxymethyl chitosan-polyaldehyde mannan hydrogel for the preparation of an immunoadjuvant, characterized in that: the carboxymethyl chitosan-polyaldehyde mannan hydrogel is prepared by the following method: uniformly mixing the carboxymethyl chitosan solution and the polyaldehyde mannan solution to obtain carboxymethyl chitosan-polyaldehyde mannan hydrogel; the volume ratio of the carboxymethyl chitosan solution to the polyaldehyde mannan solution is (1: 3) - (3): 1; the concentration of the carboxymethyl chitosan solution is 10-30 mg/mL; the concentration of the polyaldehyde mannan solution is 10-30 mg/mL; the oxidation degree of the polyaldehyde mannan is 13.41-45.33%; the solvent of the carboxymethyl chitosan solution is normal saline; the solvent of the polyaldehyde mannan solution is normal saline; the immune adjuvant is prepared by taking carboxymethyl chitosan-polyaldehyde mannan hydrogel as an effective component and adding pharmaceutically acceptable auxiliary materials or auxiliary components.

2. Use according to claim 1, characterized in that: the volume ratio of the carboxymethyl chitosan solution to the polyaldehyde mannan solution is 1: 1.

3. Use according to claim 1, characterized in that: the concentration of the carboxymethyl chitosan solution is 15-30 mg/mL; and/or the concentration of the multi-aldehyde mannan solution is 15-30 mg/mL.

4. Use according to claim 1, characterized in that: the concentration of the carboxymethyl chitosan solution is 20-30 mg/mL; and/or the concentration of the multi-aldehyde mannan solution is 20-30 mg/mL.

5. Use according to claim 1, characterized in that: the temperature for uniformly mixing is 25 ℃; and/or the time for hydrogel formation after mixing is less than 527 s.

6. Use according to claim 5, characterized in that: the time for uniformly mixing is 100-210 s.

7. Use according to any one of claims 1 to 6, characterized in that: the preparation method of the polyaldehyde mannan comprises the following steps: dissolving mannan in phosphate buffer solution to form uniform solution, and adding NaIO₄Adding into the solution, reacting at 25 deg.C in dark for 12 hr, terminating reaction, dialyzing, and lyophilizing.

8. Use according to claim 7, characterized in that: the pH of the phosphate buffer = 6.0; and/or the mass-to-volume ratio of the mannan to the phosphate buffer is 1: 50 g/mL; and/or, the NaIO₄The molar ratio of the mannan monomer units to the mannan monomer units is 1: 10-3: 5; and/or, the termination reaction is a termination reaction with ethylene glycol; and/or the dialysis bag used for dialysis has a molecular weight cutoff of 3500.