Preparation method and application of nucleotide-loaded hydrotalcite nano aluminum adjuvant

Abstract

The invention relates to a preparation method and application of a nucleotide-loaded hydrotalcite nano aluminum adjuvant. The preparation method comprises the following steps: obtaining a magnesium salt and aluminum salt mixed aqueous solution; the method comprises the following steps: adding a magnesium salt and aluminum salt mixed aqueous solution into a sodium hydroxide solution which is in a violent stirring state and contains a certain amount of nucleotide, and continuously stirring for a certain time at a certain temperature to obtain a nucleotide-loaded precipitate; collecting a nucleotide-loaded precipitate, washing with ultrapure water, and re-suspending to a certain volume; and standing the turbid liquid under a constant temperature condition for a certain time for curing, and dispersing precipitated particles to obtain a nucleotide-loaded hydrotalcite nano aluminum adjuvant solution. The nucleotide-loaded hydrotalcite nano-aluminum adjuvant has the beneficial effects that the antigen capture / loading and delivery efficiency of the nucleotide-loaded hydrotalcite nano-aluminum adjuvant is high, rapid endocytosis escape can be realized, and the antigen is effectively released to cytoplasm of DC cells to promote antigen presentation and cross presentation.

Description

Technical Field

[0001] The invention relates to the field of aluminum adjuvant preparation, and more specifically, to a preparation method and application of a nucleotide-loaded hydrotalcite nano aluminum adjuvant. Background Art

[0002] Aluminum adjuvants, including aluminum hydroxide or aluminum phosphate, can absorb antigens and enhance the body's immune response to antigens, thereby inducing strong and lasting immune responses, and have been widely used in the field of vaccines. However, commercial aluminum adjuvants mainly induce Th2 immune responses, but the effect of inducing Th1 cell immunity needs to be improved, and there is no application of activating the cyclic guanosine monophosphate-adenosine synthetase-interferon gene stimulator (cGAS-STING) pathway.

[0003] In recent years, STING agonists such as cyclic dinucleotides can activate the immune response of anti-tumor killer T cells and promote their secretion of a large amount of type I interferon, anti-tumor cytokines, chemokines, etc., and have received extensive attention in the research and development of new anti-tumor drugs. However, the preparation process of cyclic dinucleotides (CDNs) is complicated, and there are problems such as poor stability and easy degradation. They cannot persist in the blood for a long time. In addition, CDNs themselves have certain immunogenicity, and systemic administration can induce unnecessary immune side effects in the body and cause side effects such as inflammatory cytokine storm syndrome. Therefore, the development of economically stable STING agonists has important scientific significance and clinical transformation potential. Summary of the invention

[0004] The purpose of the present invention is to address the deficiencies of the prior art and to provide a preparation method and application of a nucleotide-loaded hydrotalcite nano-aluminum adjuvant.

[0005] In a first aspect, a method for preparing a nucleotide-loaded hydrotalcite nano-aluminum adjuvant is provided, comprising:

[0006] Step 1, dissolving magnesium salt and aluminum salt in deionized water at a certain molar ratio to obtain a mixed aqueous solution of magnesium salt and aluminum salt;

[0007] Step 2, adding the mixed aqueous solution of magnesium salt and aluminum salt to a sodium hydroxide solution containing a certain amount of nucleotides in a vigorously stirred state, and continuing to stir at a certain temperature for a certain period of time to obtain a precipitate loaded with nucleotides;

[0008] Step 3, collecting the nucleotide-loaded precipitate by centrifugation;

[0009] Step 4, washing the nucleotide-loaded precipitate with ultrapure water and resuspending it to a certain volume until the pH of the suspension is between 7 and 9;

[0010] Step 5: leaving the suspension to stand for a certain period of time under constant temperature conditions to mature, so that the precipitated particles are dispersed, and a nucleotide-loaded hydrotalcite nano-aluminum adjuvant solution is obtained.

[0011] Step 6: Mix the dispersed nucleotide-loaded hydrotalcite nanoaluminum adjuvant solution and the antigen molecule solution for a period of time to obtain nucleotide-loaded hydrotalcite nanoaluminum adsorbing antigen molecules.

[0012] Preferably, in step 1, the magnesium salt and the aluminum salt have the same anion, and the magnesium salt and the aluminum salt are chloride, nitrate, lactate or sulfate; and the molar ratio of the magnesium salt to the aluminum salt is 1:1-5:1.

[0013] Preferably, in step 2, the nucleotides include at least one of adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, thymine deoxyribonucleotide, adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide and uracil ribonucleotide; and the nucleotides contain 1-3 phosphate groups.

[0014] Preferably, in step 2, the reaction temperature is 0-100 degrees Celsius and the stirring time is 10-30 minutes.

[0015] Preferably, in step 4, stirring, shaking or ultrasonic dispersion is used to fully resuspend the nucleotide-loaded precipitate in ultrapure water.

[0016] Preferably, in step 5, the constant temperature is 0-100 degrees Celsius and the standing time is 2-240 hours.

[0017] Preferably, in step 6, the nucleotide-loaded hydrotalcite nanoaluminum adjuvant effectively adsorbs antigen molecules through electrostatic interaction, and the antigen molecules include protein molecules, polypeptide Trp2 and mRNA; the protein molecules include ovalbumin OVA.

[0018] In a second aspect, a nucleotide-loaded hydrotalcite nanoaluminum adjuvant prepared by any method described in the first aspect is provided.

[0019] In a third aspect, a use of a nucleotide-loaded hydrotalcite nanoaluminum adjuvant prepared by any method described in the first aspect in the preparation of a drug for inhibiting solid tumors is provided.

[0020] The beneficial effects of the present invention are:

[0021] 1. The nucleotide-loaded hydrotalcite nano-aluminum adjuvant (LDH-N) of the present invention has a simple synthesis process, clear composition and structure, and the particle size of the aluminum adjuvant particles is uniform and distributed in the range of 20-200 nm.

[0022] 2. The nucleotide-loaded hydrotalcite nanoaluminum adjuvant of the present invention has high antigen capture / loading and delivery efficiency, and can achieve rapid endosome escape, effectively releasing antigens into the cytoplasm of DC cells to promote antigen presentation and cross-presentation.

[0023] 3. The nucleotide-loaded hydrotalcite nanoaluminum adjuvant of the present invention effectively activates the STING pathway of DC cells and promotes the expression of type I interferon.

[0024] 4. The nucleotide-loaded hydrotalcite nanoaluminum adjuvant of the present invention can effectively enhance the antigen presentation and cross-presentation of DC cells after peritumoral injection, activate the immune response of T cells, and directly inhibit the growth of solid tumors, with the inhibition rate of tumor volume reaching 60%. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a transmission electron microscope image of the nucleotide-loaded hydrotalcite nanoaluminum adjuvant of the present invention;

[0026] Figure 2 It is a schematic diagram of the particle size distribution test results of the nucleotide-loaded hydrotalcite nano-aluminum adjuvant of the present invention;

[0027] Figure 3 This is a schematic diagram of the surface potential test results of the nucleotide-loaded hydrotalcite nanoaluminum adjuvant of the present invention;

[0028] Figure 4 It is a schematic diagram for identifying the nucleotide loading capacity of the nucleotide-loaded hydrotalcite nanoaluminum adjuvant of the present invention;

[0029] Figure 5 It is a schematic diagram of the in vitro cell phagocytic effect of the nucleotide-loaded hydrotalcite nano-aluminum adjuvant of the present invention;

[0030] Figure 6 This is a schematic diagram of promoting DC maturation and differentiation by the nucleotide-loaded hydrotalcite nanoaluminum adjuvant of the present invention;

[0031] Figure 7 This is a schematic diagram of the nucleotide-loaded hydrotalcite nanoaluminum adjuvant of the present invention upregulating the mRNA expression related to the STING pathway;

[0032] Figure 8 This is a schematic diagram of the nucleotide-loaded hydrotalcite nanoaluminum adjuvant of the present invention upregulating the expression of STING pathway-related proteins;

[0033] Fig. 9 It is a schematic diagram for characterizing the ability of the nucleotide-loaded hydrotalcite nanoaluminum adjuvant of the present invention to adsorb OVA antigen protein;

[0034] Fig.10 This is a schematic diagram of the effect of the nucleotide-loaded hydrotalcite nano-aluminum adjuvant of the present invention on promoting DC cell cross-presentation of antigens;

[0035] Fig.11 This is a schematic diagram of the effect of the nucleotide-loaded hydrotalcite nano-aluminum adjuvant of the present invention on promoting T cell proliferation;

[0036] Fig.12 This is a schematic diagram of the effect of the nucleotide-loaded hydrotalcite nano-aluminum adjuvant of the present invention on enhancing the anti-tumor immune response of T cells;

[0037] Fig.13 This is a schematic diagram of the effect of the nucleotide-loaded hydrotalcite nano-aluminum adjuvant of the present invention on promoting T cell-specific tumor killing;

[0038] Fig.14 This is a schematic diagram of the effect of the nucleotide-loaded hydrotalcite nanoaluminum adjuvant of the present invention on inhibiting the growth of solid tumors;

[0039] Fig.15 Schematic diagram of the effect of the nucleotide-loaded hydrotalcite nanoaluminum adjuvant of the present invention on activating DC cells, T cells and macrophages in tumors. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with embodiments. The description of the following embodiments is only used to help understand the present invention. It should be noted that for ordinary persons in the art, without departing from the principle of the present invention, the present invention can also be modified in some ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0041] Embodiment 1:

[0042] In order to solve the problems of the prior art, the present invention provides a method for preparing a nucleotide-loaded hydrotalcite nano-aluminum adjuvant, which is a method for preparing a nano-aluminum adjuvant with a clear product structure, uniform particle size, low cost, high biosafety and reliable quality. After the nucleotide-loaded hydrotalcite nano-aluminum adjuvant prepared by the method enters the tumor tissue, it can effectively neutralize the tumor micro-acidic microenvironment and induce tumor cells to secrete antigens. The nucleotide-loaded hydrotalcite nano-aluminum adjuvant can also effectively adsorb and deliver tumor antigens, and promote tumor antigen presentation and cross-presentation by antigen-presenting cells such as DC cells. In addition, the Mg released when neutralizing excessive lactic acid in the tumor microenvironment 2+ It can also play a role in metal immunity, further improve immune regulation, promote the activation of anti-tumor immunity, and enhance the effect of specific anti-tumor immune response.

[0043] Specifically, the preparation method of the nucleotide-loaded hydrotalcite nano-aluminum adjuvant comprises:

[0044] Step 1: dissolving magnesium salt and aluminum salt in deionized water at a certain molar ratio to obtain a mixed aqueous solution of magnesium salt and aluminum salt.

[0045] In step 1, the magnesium salt and the aluminum salt have the same anion, and the magnesium salt and the aluminum salt are chloride, nitrate, lactate or sulfate, preferably chloride in this example; the molar ratio of the magnesium salt to the aluminum salt is 1:1-5:1, preferably 2:1-3:1 in this example.

[0046] Step 2: Add the mixed aqueous solution of magnesium salt and aluminum salt to a sodium hydroxide solution containing a certain amount of nucleotides in a vigorously stirred state, and continue stirring at a certain temperature for a certain period of time to obtain a precipitate loaded with nucleotides. In this example, it is preferably stirred in an ice water bath (0 degrees Celsius) for 10-30 minutes.

[0047] In step 2, the stirring time is 10-30 minutes, and the nucleotides include at least one of adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, thymine deoxyribonucleotide, adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, and uracil ribonucleotide; the nucleotides contain 1-3 phosphate groups. The mass, type and molar ratio of the nucleotides are arbitrary. In this example, three schemes are preferred, with a mass accounting for 5% of the mass of hydrotalcite, adenine ribonucleotide monophosphate (AMP), guanine ribonucleotide monophosphate (GMP) and a mixed nucleotide (GAMP) with a molar ratio of 1:1.

[0048] In addition, since the mixed aqueous solution of the magnesium salt and the aluminum salt can produce the nucleotide-carrying hydrotalcite nano-aluminum adjuvant precipitation when it contacts the sodium hydroxide solution containing nucleotides, there is no special requirement for the dosage of the mixed aqueous solution of the magnesium salt and the aluminum salt and the sodium hydroxide solution containing nucleotides, as long as the mixed aqueous solution of the magnesium salt and the aluminum salt contacts the sodium hydroxide solution containing nucleotides to produce the nucleotide-carrying hydrotalcite aluminum adjuvant precipitation. In order to make full use of the raw materials, preferably, (Mg 2+, ... 2+ +Al 3+ ) and OH - The metal ions in the mixed aqueous solution of magnesium salt and aluminum salt react with OH in the sodium hydroxide solution. - The molar ratio is 1:2. In addition, the concentration and volume of the mixed aqueous solution of magnesium salt and aluminum salt and the sodium hydroxide solution containing nucleotides can also be arbitrary. In order to make full use of the raw materials and facilitate operation, preferably, the concentration of magnesium salt is 0.6 mol / L, the concentration of aluminum salt is 0.3 mol / L, the total volume is 10 ml, and the concentration of sodium hydroxide solution containing nucleotides is 0.45 mol / L, and the volume is 40 ml.

[0049] Step 3: Collect the nucleotide-loaded precipitate by centrifugation.

[0050] Step 4: Wash the nucleotide-loaded precipitate with ultrapure water and resuspend it to a certain volume until the pH of the suspension is between 7 and 9.

[0051] In step 4, the precipitate loaded with nucleotides is fully resuspended in ultrapure water by stirring, shaking or ultrasonic dispersion. There is no clear limit on the volume of ultrapure water. For ease of operation, preferably, 40 ml of ultrapure water is used to fully resuspend the precipitate. The product obtained by mixing the mixed aqueous solution of magnesium salt and aluminum salt with the alkaline aqueous solution containing nucleotides spontaneously generates nanosheets under a neutral or weakly alkaline environment.

[0052] Step 5: The suspension is allowed to stand for a certain period of time under a constant temperature condition (0-40° C.) to mature, so that the precipitated particles are dispersed, and a nucleotide-loaded hydrotalcite nanoaluminum adjuvant suspension is obtained.

[0053] In step 5, the standing time is 2-240 hours. For example, the standing time is 16 hours at room temperature 25°C.

[0054] The nano aluminum adjuvant obtained above effectively adsorbs antigen molecules through electrostatic interaction, including protein molecules (such as ovalbumin, OVA), polypeptide Trp2 and mRNA, etc. In this example, OVA is selected as the model antigen.

[0055] In the present invention, in order to deliver antigen molecules more efficiently, it is necessary to effectively adsorb the protein, polypeptide or nucleic acid antigen to the surface of the nano-aluminum adjuvant to achieve efficient load and release balance. There is no specific requirement for the molecular weight of the protein, polypeptide or nucleic acid antigen, and the antigen type covers most antigens involved in the treatment of diseases such as preventive vaccines, therapeutic vaccines, gene therapy, tumor therapy, inflammatory and chronic diseases, so the application prospects are very broad and are not limited to the field of tumor immunotherapy.

[0056] The above reactor can be any reactor known in the art, such as glassware, reaction kettle, etc.

[0057] The present invention also provides an example of anti-tumor immunotherapy based on the above-mentioned nucleotide-loaded hydrotalcite nanoaluminum adjuvant. By local injection of the above-mentioned nucleotide-loaded hydrotalcite nanoaluminum adjuvant, the tumor immune microenvironment is improved, the infiltration of mature DC cells presenting antigens is enhanced, and the killer T cells in the tumor immune microenvironment are activated, thereby effectively inhibiting the growth of solid tumors.

[0058] The nucleotide-loaded hydrotalcite nanoaluminum adjuvant prepared by the present invention is suitable for various solid tumors, that is, tumor tissues with clear entities, including melanoma, colon cancer, liver cancer, breast cancer, otolaryngology cancer, etc. The local injection methods include intratumoral injection and peritumoral injection.

[0059] The nucleotide-loaded hydrotalcite nanoaluminum adjuvant prepared by the present invention is suitable for various cells, including various human or mouse tumor cells such as mouse colon cancer cell line CT26, breast cancer cell line 4T1, melanoma cell line B16F10, human cervical cancer cell line Hela, etc., as well as immune cells such as DC, macrophages, etc.

[0060] The nucleotide-loaded hydrotalcite nanoaluminum adjuvant of the present invention is used to induce DC cell maturation by activating the STING pathway, thereby promoting the expression of type I interferon secretion, which is beneficial to further activate the anti-tumor immune response of T cells.

[0061] Embodiment 2:

[0062] Weigh a certain amount of MgCl2·6H2O and AlCl3·6H2O, dissolve them in ultrapure water, and prepare 10 ml of a mixed aqueous solution with magnesium ion and aluminum ion concentrations of 0.6 mol / L and 0.3 mol / L, respectively; weigh a certain amount of NaOH, dissolve it in distilled water, and add 10 micromoles of adenine monophosphate ribonucleotide (GMP), guanine monophosphate ribonucleotide (AMP) or an equimolar mixture of the two (GAMP) to 40 ml of 0.45 mol / L NaOH aqueous solution. Pre-place 40 ml of sodium hydroxide solution containing nucleotides in a flask and stir at high speed using a magnetic stirrer, then quickly add MgCl2·6H2O and AlCl3·6H2O solutions to the sodium hydroxide solution containing nucleotides, and stir in an ice-water bath for 20 minutes. Collect the suspension, collect the precipitate using a centrifugal force of 4750g, and wash it twice with ultrapure water. Disperse the precipitate in 40 ml of ultrapure water, stand and mature at room temperature for 16 hours to obtain a nano-aluminum adjuvant loaded with nucleotides. The particle morphology of the samples was observed using a transmission electron microscope. Figure 1 .from Figure 1 It can be seen that the nano aluminum adjuvant obtained by the present invention is a hexagonal particle with uniform size and uniform dispersion of nucleotide-loaded hydrotalcite nano aluminum adjuvant (LDH-GMP, LDH-AMP and LDH-GAMP; collectively referred to as LDH-N). The particle size distribution and surface charge of the sample particles were measured using a nanoparticle size and Zeta potential analyzer. The particle size of the nucleotide-loaded hydrotalcite nano aluminum adjuvant obtained by the present invention is uniformly distributed in the range of 20-200nm, with an average particle size of about 40nm and a Zeta surface potential of about +40mV. The results are shown in FIG. Figure 2 and Figure 3 .

[0063] Embodiment 3:

[0064] The above-mentioned nucleotide-loaded hydrotalcite nano-aluminum adjuvant was centrifuged at high speed and washed to remove free nucleotides, and then fully dissolved with hydrochloric acid solution to release the loaded nucleotides. The concentration of nucleotides released from the above-mentioned nucleotide-loaded hydrotalcite nano-aluminum adjuvant was detected using an ultraviolet spectrophotometer to determine the nucleotide molecular loading amount. The results are shown in Table 1. Figure 4 .from Figure 4 It can be seen that the nucleotide-loaded hydrotalcite nano-aluminum adjuvant obtained in the present invention efficiently loads nucleotide molecules. Through calculation and correction, the nucleotide loading rate exceeds 90%.

[0065] Embodiment 4:

[0066] The nucleotide-loaded hydrotalcite nanoaluminum adjuvant was labeled with FITC green fluorescence and incubated with DC2.4 cells for 1 or 4 hours. Its cellular uptake and endosomal escape were observed using a confocal microscope. Figure 5 .from Figure 5 It can be seen that after the nucleotide-loaded hydrotalcite nanoaluminum adjuvant was co-incubated with DC2.4 cells for 1 hour, a significant green fluorescence signal appeared in the cytoplasm, that is, the above-mentioned nanoaluminum adjuvant was phagocytosed by the cells. At the same time, we observed that the red fluorescence signal of the endosome / lysosome in the cytoplasm of DC2.4 cells was weakened. This is because the weakly alkaline LDH-N neutralized the acidity of the endosome / lysosome, resulting in the inability of the Lysotracker probe to emit red fluorescence. After 4 hours of co-incubation, the green fluorescence signal in the cytoplasm was stronger and the red signal was weaker. In addition, we can observe that the green fluorescence-labeled LDH-N and the red fluorescence-stained lysosomes have obvious non-overlapping areas, proving that the nucleotide-loaded hydrotalcite nanoaluminum adjuvant can quickly escape from the endosome into the cytoplasm.

[0067] Embodiment 5:

[0068] The above-mentioned nucleotide-loaded hydrotalcite nanoaluminum adjuvant was incubated with DC cells for 24 hours, and the expression of DC cell maturation and differentiation markers CD80, CD86, and CD40 was detected by flow cytometry. The results are shown in Figure 6 .from Figure 6 It can be seen that free nucleotides alone had little effect on the expression of CD80, CD86, and CD40 in DC cells, while the CD80, CD86, and CD40 in the blank LDH treatment group increased to a certain extent. In contrast, LDH-N can more significantly increase the expression of CD80, CD86, and CD40 in DC cells. These results indicate that LDH-N can efficiently induce the maturation and differentiation of DC cells.

[0069] Embodiment 6:

[0070] The above-mentioned nucleotide-loaded hydrotalcite nanoaluminum adjuvant was co-incubated with DC cells for 24 hours, and the expression of isg56 and ifn-β mRNA related to the STING pathway was detected by qT-PCR. The results are shown in Figure 7 .from Figure 7 It can be seen that compared with the stimulation of free nucleotides or blank hydrotalcite alone, LDH-N can significantly increase the expression of DC cell interferon stimulating factor gene isg56 and type I interferon ifn-β, indicating that LDH-N has the ability to activate the STING pathway of DC cells.

[0071] Embodiment 7:

[0072] The above-mentioned nucleotide-loaded hydrotalcite nanoaluminum adjuvant was co-incubated with DC cells for 24 hours, and the expression of STING, IRF3, TBK1 proteins and their phosphorylated proteins related to the STING pathway and the expression level of downstream IFN-β cytokines were detected by Western Blot. The results are shown in Figure 8 .from Figure 8 It can be seen that the LDH-N treatment group significantly increased the expression of STING protein, IRF3 protein, TBK1 protein and its phosphorylated protein related to the STING pathway of DC cells and the expression level of downstream IFN-β cytokine, further proving that LDH-N can efficiently activate the STING pathway of DC cells and is a potential STING agonist or its analogue.

[0073] Embodiment 8:

[0074] The above-mentioned nucleotide-loaded hydrotalcite nanoaluminum adjuvant was incubated with different concentrations of OVA antigen protein solutions for 15 minutes, and the supernatant was centrifuged at 15,000 rpm to determine the free protein concentration. The Langmuir isotherm adsorption model Qe=QmCe / (1 / K+Ce) was used to fit and analyze the adsorption capacity of the above-mentioned nucleotide-loaded hydrotalcite nanoaluminum adjuvant for antigen protein. The results are shown in Fig. 9 .from Fig. 9 It can be seen that the maximum adsorption capacity of antigen protein per mg of LDH, LDH-GMP, LDH-AMP and LDH-GAMP is 0.8260, 0.8820, 0.8486 and 0.8396 mg of OVA protein, which proves that the above-mentioned nucleotide-loaded hydrotalcite nanoaluminum adjuvant can efficiently adsorb antigen protein, which is beneficial to deliver antigen protein into cells and promote antigen presentation.

[0075] Embodiment 9:

[0076] The model antigen protein OVA was added to the DC cell culture medium and incubated for 4 hours, and then the above-mentioned nucleotide-loaded hydrotalcite nanoaluminum adjuvant was added and incubated for another 20 hours. The expression of H-2Kb-bound SIINFEKL (MHC I) and IA / IE (MHC II) presented by DC cells was detected by flow cytometry. The results are shown in Fig.10 .from Fig.10 It can be seen that the expression of H-2Kb-bound SIINFEKL (MHC I) in DC cells treated with PBS or GMP / AMP nucleotides did not increase significantly, but the expression of IA / IE (MHC II) increased, which may be caused by the degradation of OVA entering the lysosome through endocytosis. In contrast, LDH-N can significantly upregulate the expression of H-2Kb-bound SIINFEKL (MHC I) in DC cells, but will not significantly increase the expression of IA / IE (MHC II), indicating that LDH-N can efficiently deliver antigens to DC cells and escape from lysosomes, which is beneficial to the ability of DC cells to process OVA protein into cross-presented antigen peptides.

[0077] Embodiment 10:

[0078] DC cells activated by nucleotide-loaded hydrotalcite nanoaluminum adjuvant in Example 8 were co-incubated with OT-I T cells for 72 hours, and the green fluorescence attenuation of CFSE-labeled OT-I T cells was detected by flow cytometry to characterize the proliferation of T cells. The results are shown in Fig.11 .from Fig.11 It can be seen that DC cells activated by LDH / OVA promoted the proliferation and activation of OT-IT cells to a certain extent. In contrast, LDH-N / OVA significantly increased the proliferation rate of OT-I T cells by significantly enhancing the antigen presentation of DC cells. This shows that after LDH-N delivers OVA antigen to activate DC cells, it can enhance cross-antigen presentation and promote the proliferation of cytotoxic T cells.

[0079] Embodiment 11:

[0080] DC cells activated by nucleotide-loaded hydrotalcite nanoaluminum adjuvant in Example 8 were compared with CD8 + T cells were co-incubated for 24 hours and CD8 + The expression of interferon (IFN-γ), granzyme B and perforin in T cells was used to characterize the activation of T cells. The results are shown in Fig.12 .from Fig.12It can be seen that compared with DC cells treated with nucleotides and LDH / OVA, N-LDH / OVA treatment significantly improved the antigen cross-presentation ability of DC cells and significantly increased the CD8 + The expression of interferon (IFN-γ), granzyme B and perforin secreted by T cells indicates that CD8 + The effector activity of T cells was significantly enhanced.

[0081] Embodiment 12:

[0082] Take the CD8 activated in Example 10 + T cells were co-incubated with B16-OVA cells for 24 h, and CD8 + The specific killing ability of T cells on tumor cells is shown in Fig.13 .from Fig.13 It can be seen that the nucleotide-loaded hydrotalcite nanoaluminum adjuvant in the embodiment of the present invention can effectively enhance CD8 + The specific killing ability of T cells on tumor cells.

[0083] Embodiment 13:

[0084] 1 mg of the nucleotide-loaded hydrotalcite nanoaluminum adjuvant prepared by the present invention was injected into the tumor with a volume of about 100 mm by peritumoral injection. 3 4T1 tumors were treated with injections every 3 days for a total of three injections. Tumor size was measured with a vernier caliper every three days and tumor volume was calculated (volume = 0.5 × length × width 2 ), and the tumor growth inhibition effect was detected. The results are shown in Fig.14 .from Fig.14 It can be seen that the nucleotide-loaded hydrotalcite nanoaluminum adjuvant prepared by the present invention can effectively inhibit the growth of solid 4T1 triple-negative breast cancer, and on the 18th day, the tumor inhibition rate of the nucleotide-loaded hydrotalcite nanoaluminum adjuvant of the present invention reaches 60%.

[0085] Embodiment 14:

[0086] The tumor tissue in Example 12 was digested into a single cell suspension and then analyzed by flow cytometry for the composition ratio of DC cells, T cells and macrophages in the tumor. The results are shown in Fig.15 .from Fig.15 It can be seen that compared with free nucleotides or LDH alone, the LDH-N prepared by the present invention can more significantly increase the number of mature DC cells and cytotoxic CD8 +It can also reduce the infiltration of T cells, reduce the proportion of regulatory T cells, and enhance the development of tumor-associated macrophages from the cancer-promoting M2 type to the cancer-suppressing M1 type, indicating that LDH-N can significantly improve the tumor immunosuppressive microenvironment.

[0087] In summary, the present invention obtains a nano-aluminum adjuvant loaded with nucleotides having a clear lamellar crystal structure and a particle size in the range of 20-200nm by coprecipitating a mixed aqueous solution of magnesium salt and aluminum salt in a sodium hydroxide solution containing nucleotides, washing and dispersing in deionized water, and standing at room temperature. The nano-aluminum adjuvant can activate the STING pathway to induce DC cell maturation. At the same time, the nano-aluminum adjuvant loaded with nucleotides can effectively adsorb and deliver antigens to antigen-presenting cells such as DC, promote antigen presentation and cross-presentation, thereby enhancing the specific tumor killing function of T cells, and has great potential for improving tumor immunotherapy.

Claims

1. A method for preparing a nucleotide-loaded hydrotalcite nano-aluminum adjuvant, characterized in that: include: Step 1, dissolving magnesium salt and aluminum salt in deionized water at a certain molar ratio to obtain a mixed aqueous solution of magnesium salt and aluminum salt; Step 2, adding the mixed aqueous solution of magnesium salt and aluminum salt to a sodium hydroxide solution containing a certain amount of nucleotides in a vigorously stirred state, and continuing to stir at a certain temperature for a certain period of time to obtain a precipitate loaded with nucleotides; Step 3, collecting the precipitate loaded with nucleotides by centrifugation; Step 4, washing the nucleotide-loaded precipitate with ultrapure water and resuspending it to a certain volume until the pH of the suspension is between 7 and 9; Step 5: leaving the suspension to stand for a certain period of time under constant temperature conditions to mature, so that the precipitated particles are dispersed, and a nucleotide-loaded hydrotalcite nano-aluminum adjuvant solution is obtained. Step 6: Mix the dispersed nucleotide-loaded hydrotalcite nanoaluminum adjuvant solution and the antigen molecule solution for a period of time to obtain nucleotide-loaded hydrotalcite nanoaluminum adsorbing antigen molecules.

2. The method for preparing the nucleotide-carrying hydrotalcite nano-aluminum adjuvant according to claim 1, characterized in that: In step 1, the magnesium salt and the aluminum salt have the same anion, and the magnesium salt and the aluminum salt are chloride, nitrate, lactate or sulfate; and the molar ratio of the magnesium salt to the aluminum salt is 1:1-5:

1.

3. The method for preparing the nucleotide-carrying hydrotalcite nano-aluminum adjuvant according to claim 2, characterized in that: In step 2, the nucleotides include at least one of adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, thymine deoxyribonucleotide, adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, and uracil ribonucleotide; and the nucleotides contain 1-3 phosphate groups.

4. The method for preparing the nucleotide-carrying hydrotalcite nano-aluminum adjuvant according to claim 3, characterized in that: In step 4, the mixed precipitate loaded with nucleotides is fully resuspended in ultrapure water by stirring, shaking or ultrasonic dispersion.

5. The method for preparing the nucleotide-carrying hydrotalcite nano-aluminum adjuvant according to claim 5, characterized in that: In step 2, the stirring time is 10-30 minutes; in step 5, the standing time is 2-240 hours.

6. The nucleotide-carrying hydrotalcite nano-aluminum adjuvant according to claim 5 can adsorb antigen molecules, characterized in that: In step 6, the nucleotide-loaded hydrotalcite nanoaluminum adjuvant effectively adsorbs antigen molecules through electrostatic interaction, and the antigen molecules include protein molecules, polypeptide Trp2 and mRNA; the protein molecules include ovalbumin OVA.

7. A nucleotide-loaded hydrotalcite nanoaluminum adjuvant prepared by the method according to any one of claims 1 to 6.

8. Use of the nucleotide-loaded hydrotalcite nanoaluminum adjuvant prepared by the method of any one of claims 1 to 6 in the preparation of drugs for inhibiting solid tumors.

Images