Novel gallium-based immunologic adjuvant as well as preparation method and application thereof

Abstract

The invention relates to the technical field of immunologic adjuvants, in particular to a gallium-based immunologic adjuvant and a preparation method and application thereof.The gallium-based immunologic adjuvant is prepared from pure gallium, gallium ions (gallium nitrate), gallium oxide, gallium hydroxide and hydroxyl gallium oxide. The gallium-based immunologic adjuvant designed in the invention can make up for the obstacle of insufficient anticancer curative effect caused by shortage of efficient immunologic adjuvants, can realize high-yield adjuvant output at lower cost, and also can effectively avoid the problem of immunosuppression microenvironment such as insufficient immune cell infiltration. It is worth mentioning that under the same adjuvant dosage, the maturation effect of the gallium-based immunologic adjuvant on the dendritic cells is obviously higher than that of an aluminum salt control group, and it is indicated that the effect of the immunologic adjuvant is high, and the immunologic adjuvant has the possibility of replacing a traditional inorganic nano immunologic adjuvant. The preparation method of the novel gallium-based immunologic adjuvant provided by the invention provides possibility for more effectively, quickly and conveniently enhancing the immune response capability of an organism to an antigen or changing the immune response type.

Description

Technical Field

[0001] The present invention relates to the technical field of immune adjuvants, and in particular to a gallium-based immune adjuvant and a preparation method and application thereof. Background Art

[0002] Cancer remains the leading cause of death and the main obstacle to extending life expectancy worldwide. Although cancer treatment strategies such as surgery, chemotherapy, and radiotherapy have therapeutic effects on primary lesions, metastatic lesions, and subclinical metastatic lesions, they have a high recurrence rate and severe toxic side effects. As an active immunotherapy, therapeutic cancer vaccines (TCVs) have become an emerging frontier in the field of immunotherapy in recent years due to their personalization, wide range of adaptability, and low risk. However, due to problems such as tumor heterogeneity and low antigen utilization, vaccines are often used in combination with immune adjuvants. Immune adjuvants are referred to as adjuvants, that is, non-specific immunoproliferative agents, which refer to auxiliary substances that can enhance the body's immune response to antigens or change the type of immune response when injected into the body together with antigens or in advance. The immune response rate of TCVs developed by traditional methods is low, mainly limited by the lack of efficient immune adjuvants, resulting in low efficiency of antigen utilization and immune stimulation. In addition, the development of immune adjuvants has been very slow. Previous adjuvant research has focused on single aluminum salts (such as aluminum hydroxide), but aluminum adjuvants mainly induce humoral immunity rather than cell-mediated immune responses. Although aluminum adjuvants have been widely used, they are considered to be relatively "weak" vaccine adjuvants. In addition, a small number of individuals may develop granulomas or contact hypersensitivity to aluminum after injection of aluminum adjuvant vaccines. Therefore, it is urgent to develop new immune adjuvants and develop efficient cancer treatments to cope with the severe cancer development trend.

[0003] In recent years, gallium-based liquid metals have received extensive attention in the fields of antibacterial, anti-inflammatory, and anti-tumor due to their good biocompatibility, degradability, fluidity (allowing adjustable size), electrical conductivity, thermal conductivity, and other physical and chemical properties. Currently, there are fewer studies focusing on immunity. In addition, as a third-group element, gallium exhibits amphoteric chemical properties similar to aluminum. Therefore, gallium-based liquid metals have the ability to develop immune adjuvants. However, pure gallium prepared by ultrasound is often doped with a variety of gallium derivatives. Therefore, it is particularly important to explore whether gallium-based liquid metals have an immune enhancement effect equivalent to aluminum hydroxide or whether pure gallium / pure gallium derivative (pure gallium oxide, gallium ions, etc.) complexes play an adjuvant role. Summary of the invention

[0004] In order to overcome the deficiency of the current single type of immune adjuvant, the present invention proposes a gallium-based immune adjuvant with equivalent immune enhancement effect but higher safety or stronger immune stimulation effect. Through the application research of gallium-based immune adjuvant, the immunosuppressive microenvironment can be reversed (increasing the number of antigen presenting cells, etc.) and the selection of immune adjuvant types can be increased.

[0005] Another object of the present invention is to provide a method for preparing the novel gallium-based immune adjuvant.

[0006] Another object of the present invention is to propose the application of the novel gallium-based immune adjuvant.

[0007] The first aspect of the present invention provides a novel gallium-based immune adjuvant, and the preparation raw materials include: a gallium-based immune adjuvant and a polymer material; wherein the polymer material is coated on the surface of the gallium-based immune adjuvant.

[0008] The gallium-based immune adjuvant includes pure gallium, gallium nitrate, gallium oxide, gallium hydroxide, and gallium oxyhydroxide.

[0009] In some preferred embodiments, the weight ratio of pure gallium, gallium nitrate, gallium oxide, gallium hydroxide and gallium oxyhydroxide is 3:(1-2):(1-2):(1-2):(1-2).

[0010] Preferably, the weight ratio of pure gallium, gallium nitrate, gallium oxide, gallium hydroxide and gallium oxyhydroxide is 3:1:2:1:1.

[0011] The polymer material is one or more of gelatin, chitosan, polyvinyl alcohol, polyethylene glycol, polylactic acid, hyaluronic acid, and dopamine.

[0012] The second aspect of the present invention provides a method for preparing a gallium-based immune adjuvant, comprising:

[0013] (1) preparing gallium-based immune adjuvant nanoparticles; the preparation method includes one or more of mechanical pulverization, ultrasound, fluid jet, and microfluidic focusing;

[0014] (2) screening gallium-based immune adjuvant nanoparticle solutions; the screening methods include one or more of natural precipitation and centrifugation;

[0015] (3) drying and weighing the gallium-based immune adjuvant nanoparticles to obtain powdered gallium-based immune adjuvant nanoparticles;

[0016] (4) placing powdered gallium-based immune adjuvant nanoparticles into a solution containing a polymer material, and ultrasonically treating for 10-30 minutes; the concentration of the solution containing the polymer material is 1-10wt%, and the solvent of the solution is any one of ethanol, acetone, ethylene glycol, and trichloroethane;

[0017] (5) placing the gallium-based immune adjuvant nanoparticles after ultrasonic treatment in step (4) in a washing solution, washing 2-5 times and then drying; the washing solution includes one or more of pure water, anhydrous ethanol, acetone, PBS buffer, and PB buffer.

[0018] Among them, in step (1), the preparation method is specifically: placing the gallium-based liquid metal in a dispersant, ultrasonically treating it for 0.5-3h, and the ultrasonic power is 70%-90%; the dispersant includes one or more of a neutral buffer, deionized water, and anhydrous ethanol, and the ultrasonic method is preferred.

[0019] Wherein, in step (2), the screening method includes natural sedimentation, centrifugation or a combination thereof, preferably a combination of natural sedimentation and centrifugation.

[0020] Wherein, in step (4), the mass ratio of gallium-based immune adjuvant nanoparticles to polymer-containing materials is 1:(0.5-2).

[0021] In step (1), the preparation method is specifically as follows: placing gallium-based liquid metal in a dispersant, ultrasonically treating for 0.5-3h, with an ultrasonic power of 70%-90%; the dispersant includes one or more of a neutral buffer, deionized water, and anhydrous ethanol.

[0022] In step (2), the screening method is specifically: centrifuging the gallium-based immune adjuvant nanoparticle solution 1-3 times, with a centrifugal speed of 200-4000 rpm and a time of 2-5 minutes.

[0023] In step (4), the concentration of the polymer surface material solution may be in the range of 1-10 wt %, preferably 1-2 wt %.

[0024] The third aspect of the present invention provides an application of a novel gallium-based immune adjuvant. The gallium-based immune adjuvant of the present invention is applied to the field of immune stimulation of cancer vaccines, aiming to avoid the inhibitory nature of the tumor microenvironment and further improve the tumor treatment effect.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention explores the immune enhancement effect produced by gallium-based immune adjuvants with different proportions of pure gallium, gallium nitrate, gallium oxide, gallium hydroxide, and gallium oxyhydroxy, and develops a new gallium-based immune adjuvant with immunostimulatory properties. The immune adjuvant effect mediated by gallium-based immune adjuvants can reduce cancer recurrence and metastasis, and further improve the effect of tumor treatment. In addition, gallium-based immune adjuvants can also be used as tumor antigen delivery carriers. The antigens produced by tumor cells after in vitro treatment can retain a variety of tumor antigens and damage-associated molecular patterns (DAMPs), which are encapsulated in gallium-based immune adjuvants to generate "nano vaccines", thereby artificially amplifying the antigen and adjuvant properties. Therefore, the ability of immune stimulation, tumor targeting, and antigen release can be achieved by loading tumor antigens with gallium-based immune adjuvants.

[0027] 2. The development of immune adjuvants has been relatively slow, and it will take about 3-10 years to develop a human vaccine immune adjuvant. The gallium-based immune adjuvant designed in the present invention can alleviate the obstacle of the shortage of high-efficiency immune adjuvants, broaden the use of liquid metals, and achieve high-yield adjuvant output at a relatively low cost. It is worth mentioning that, at the same adjuvant dosage, the gallium-based immune adjuvant has a significantly higher effect on the maturation of dendritic cells than the aluminum salt control group, indicating that the immune adjuvant has a stronger effect and is replaceable. The method for preparing the immune adjuvant proposed in the present invention provides a possibility for enhancing immune function more effectively, quickly and conveniently. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the effect of the immune adjuvants of Examples and Comparative Examples on the maturation of dendritic cells.

[0029] Figure 2 It is a schematic diagram comparing the mean fluorescence intensity of the immune adjuvants of the embodiment and the comparative example on the maturation of dendritic cells. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The sources of the raw materials and instruments used in the examples are not particularly limited and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0032] The gallium-based immune adjuvant nanoparticles in the raw materials are prepared according to the following technical scheme:

[0033] (a) Weighed pure gallium (90 mg), gallium nitrate (30 mg), gallium oxide (30 mg), gallium hydroxide (30 mg), and gallium oxyhydroxide (30 mg) are evenly dispersed at the bottom of five beakers each containing anhydrous ethanol;

[0034] (b) crushing the raw material powder by ultrasonic method (ultrasonic pulverizer, power 90%, ultrasonic 50min);

[0035] (c) After the solution is naturally precipitated, the supernatant is taken and centrifuged at 10,000 rpm for 5 min and the precipitate is taken;

[0036] (d) freeze-drying the prepared nanoparticle precipitate for later use.

[0037] Example 1

[0038] This embodiment is a method for preparing a novel gallium-based immune adjuvant, which specifically includes the following steps:

[0039] (1) 150 mg of gallium-based immune adjuvant (pure gallium, gallium nitrate, gallium oxide, gallium hydroxide, gallium oxyhydroxide, weight ratio of 3:1:1:1:1) was placed in a mortar and further crushed by mechanical crushing;

[0040] (2) The nanoparticles in (1) were placed in an anhydrous ethanol solution (2 wt %) of polyethylene glycol (purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; S30195; polyethylene glycol 20000; 250 g), and the gallium-based immune adjuvant micro-nanoparticles coated with polyethylene glycol were prepared by ultrasonic method (ultrasonication crusher, ultrasonic power 90%, time 40 min);

[0041] (3) After being fully coated, the solution was centrifuged twice at a speed of 1500 rpm for 2 min, and the supernatant was collected;

[0042] (4) Transferring the novel immune adjuvant nanoparticles into anhydrous ethanol and washing them repeatedly for 4 times;

[0043] (5) After washing, the material was centrifuged at 10,000 rpm for 5 min, and the precipitate was freeze-dried to complete the preparation of the material.

[0044] Example 2

[0045] The only difference between this example and Example 1 is that the polyethylene glycol in step (2) of Example 1 is replaced by chitosan, which is purchased from Shanghai yuanye Bio-Technology Co., Ltd.; S11064; chitosan (chitosan); 100 g.

[0046] This example prepares a novel gallium-based immune adjuvant coated with chitosan. The chitosan shell enhances the stability of the nanoparticles and can also be further targeted and modified to promote mucosal adhesion.

[0047] Example 3

[0048] The only difference between this example and Example 1 is that the polyethylene glycol in step (2) of Example 1 is replaced by polylactic acid-co-glycolic acid copolymer, purchased from Shanghai yuanye Bio-Technology Co., Ltd; S24436; polylactic acid-co-glycolic acid copolymer, 1 g.

[0049] This example prepares a novel gallium-based immunoadjuvant coated with poly(lactic acid-co-glycolic acid) copolymer. The shell coating of poly(lactic acid-co-glycolic acid) copolymer makes the degradation rate of the nanoparticles controllable and enhances the antigen stability and solubility.

[0050] Example 4

[0051] The only difference between this embodiment and embodiment 1 is that the polyethylene glycol in step (2) of embodiment 1 is replaced by hyaluronic acid.

[0052] This example prepares a novel gallium-based immune adjuvant coated with hyaluronic acid. The outer shell of hyaluronic acid enhances the cell adhesion and migration ability of the nanoparticles, and can be modified.

[0053] Example 5

[0054] The only difference between this example and Example 1 is that the polyethylene glycol in step (2) of Example 1 is replaced by dopamine purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; D103111; dopamine hydrochloride; 5 g.

[0055] This example prepares a novel gallium-based immune adjuvant coated with dopamine. The hyaluronic acid shell coating enables the nanoparticles to enhance the immune effect while also gathering heat from external stimulation, and can be used for immune stimulation in the field of photothermal therapy.

[0056] Example 6

[0057] The only difference between this embodiment and embodiment 1 is that the anhydrous ethanol solution in step (2) of embodiment 1 is replaced by alkaline (PH=13) deionized water.

[0058] Example 7

[0059] The only difference between this embodiment and embodiment 1 is that the anhydrous ethanol solution in step (2) of embodiment 1 is replaced by a neutral PBS buffer solution.

[0060] Comparative Example 1

[0061] The specific implementation of this comparative example is the same as that of Example 1, except that the weight ratio of the corresponding components in the novel gallium-based immune adjuvant - pure gallium, gallium nitrate, gallium oxide, gallium hydroxide, and gallium oxyhydroxide - is 3:2:1:1:1.

[0062] Comparative Example 2

[0063] The specific implementation of this comparative example is the same as that of Example 1, except that the weight ratio of the corresponding components in the novel gallium-based immune adjuvant - pure gallium, gallium nitrate, gallium oxide, gallium hydroxide, and gallium oxyhydroxide - is 3:1:2:1:1.

[0064] Comparative Example 3

[0065] The specific implementation of this comparative example is the same as that of Example 1, except that the weight ratio of the corresponding components in the novel gallium-based immune adjuvant - pure gallium, gallium nitrate, gallium oxide, gallium hydroxide, and gallium oxyhydroxide - is 3:1:1:2:1.

[0066] Comparative Example 4

[0067] The specific implementation of this comparative example is the same as that of Example 1, except that the weight ratio of the corresponding components in the novel gallium-based immune adjuvant - pure gallium, gallium nitrate, gallium oxide, gallium hydroxide, and gallium oxyhydroxide - is 3:1:1:1:2.

[0068] The experimental results of the immunostimulatory effect of the gallium-based immune adjuvants prepared in the above examples and comparative examples on dendritic cells after co-incubation are shown in Figure 1-2 As can be seen from the figure, when the weight ratio of pure gallium, gallium nitrate, gallium oxide, gallium hydroxide, and gallium oxyhydroxide is 3:1:2:1:1, the maturation effect of the gallium-based immune adjuvant on dendritic cells (DCs) is most obvious. Figure 1 In the figure, DAPI (blue) shows the number and location of DCs, FITC-CD80 (green) shows mature DCs, and the preferred gallium-based immune adjuvant (the ratio of pure gallium, gallium nitrate, gallium oxide, gallium hydroxide, and gallium oxyhydroxy is 3:1:2:1:1) has the brightest and most abundant FITC-CD80 fluorescence intensity, indicating that DCs are the most mature and the gallium-based immune adjuvant has the strongest immunostimulatory effect. Figure 2 Among them, the preferred gallium-based immune adjuvant had a higher effect on DCs maturation than other groups, and the significant difference was at four stars, which verified that the gallium-based immune adjuvant had a significant immune enhancement effect.

[0069] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A novel gallium-based immune adjuvant, characterized in that: The preparation raw materials include: a gallium-based immune adjuvant and a polymer material; wherein the polymer material is coated on the surface of the gallium-based immune adjuvant.

2. The gallium-based immune adjuvant according to claim 1, characterized in that The gallium-based immune adjuvant includes pure gallium, gallium nitrate, gallium oxide, gallium hydroxide, and gallium oxyhydroxide.

3. The gallium-based immune adjuvant according to claim 2, characterized in that: The weight ratio of pure gallium, gallium nitrate, gallium oxide, gallium hydroxide and gallium oxyhydroxide is 3:(1-2):(1-2):(1-2):(1-2).

4. The gallium-based immune adjuvant according to claim 1, characterized in that The polymer material is one or more of gelatin, chitosan, polyvinyl alcohol, polyethylene glycol, polylactic acid, hyaluronic acid, and dopamine.

5. A method for preparing a gallium-based immune adjuvant according to claim 1, characterized in that: Includes steps: (1) preparing gallium-based immune adjuvant nanoparticles; the preparation method includes one or more of mechanical pulverization, ultrasound, fluid jet, and microfluidic focusing; (2) screening gallium-based immune adjuvant nanoparticle solutions; the screening methods include one or more of natural precipitation and centrifugation; (3) drying and weighing the gallium-based immune adjuvant nanoparticles to obtain powdered gallium-based immune adjuvant nanoparticles; (4) placing powdered gallium-based immune adjuvant nanoparticles into a solution containing a polymer material, and ultrasonically treating for 10-30 minutes; the concentration of the solution containing the polymer material is 1-10wt%, and the solvent of the solution is any one of ethanol, acetone, ethylene glycol, and trichloroethane; (5) placing the gallium-based immune adjuvant nanoparticles after ultrasonic treatment in step (4) in a washing solution, washing 2-5 times and then drying; the washing solution includes one or more of pure water, anhydrous ethanol, acetone, PBS buffer, and PB buffer.

6. The gallium-based immune adjuvant according to claim 5, characterized in that In step (1), the preparation method is specifically as follows: placing gallium-based liquid metal in a dispersant, ultrasonically treating for 0.5-3h, with an ultrasonic power of 70%-90%; the dispersant includes one or more of a neutral buffer, deionized water, and anhydrous ethanol.

7. The gallium-based immune adjuvant according to claim 5, characterized in that: In step (2), the screening method is specifically: centrifuging the gallium-based immune adjuvant nanoparticle solution 1-3 times, with a centrifugal speed of 200-4000 rpm and a time of 2-5 minutes.

8. The gallium-based immune adjuvant according to claim 5, characterized in that: In step (4), the mass ratio of gallium-based immune adjuvant nanoparticles to polymer-containing materials is 1:(0.5-2).

9. A use of the gallium-based immune adjuvant according to claim 1, characterized in that: The gallium-based immune adjuvant is applied in the field of immune stimulation of cancer vaccines.

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