A HIFU nano-synergist and its preparation and application

Abstract

The present invention belongs to the technical field of medical nanomaterials, and specifically discloses a HIFU nano-synergist and its preparation and application. The synergist includes nanoparticles loaded with banovanthraquinone and cyclic dinucleotides, and the nanoparticles are spherical nanoparticles with a metal organic framework formed by self-assembly of metal ions and banovanthraquinone cyclic dinucleotides, and the metal ions are preferably manganese ions. The present invention utilizes the response characteristics of metal coordination bonds to acid to effectively release and synergistically treat anticancer drugs banovanthraquinone and cyclic dinucleotides in tumor tissues, thereby enhancing the anti-tumor effect; banovanthraquinone can selectively act on hypoxic cells, while overcoming hypoxia resistance to kill tumor cells without harming normal tissues; manganese ions and cyclic dinucleotides are used in combination to activate the interferon gene stimulator (STING) pathway, enhance the body's innate immunity and adaptive response, thereby reversing the immunosuppression caused by the tumor microenvironment and promoting tumor immunotherapy.

Description

Technical Field

[0001] The present invention relates to the technical field of medical nanomaterials, in particular to the technical field of nanomaterials for high-intensity focused ultrasound, and specifically to a HIFU nano synergist and its preparation and application. Background Art

[0002] High-intensity focused ultrasound (HIFU) is a minimally invasive, safe, and effective local ablation treatment method that has been used to treat a variety of solid tumors. HIFU primarily utilizes the thermal and cavitation effects of ultrasound to deposit energy in the target area, causing thermalization and coagulation necrosis of tumor cells, thereby achieving targeted tumor ablation. However, during treatment, the target energy decays exponentially with increasing tissue penetration depth, making complete ablation difficult for some deep and large tumors.

[0003] To solve the problem of residual tumors, researchers have developed nano-synergists to synergistically enhance the anti-tumor efficacy of HIFU. Shen et al. used neutrophils as carriers and PEGylated liposomal doxorubicin as a model chemotherapy drug to form a nano-drug [see "Journal of Nanobiotechnology", Vol. 19, No. 1, p. 345, (2021)]. HIFU was used to trigger the release of doxorubicin to kill cells in residual lesions. However, after HIFU irradiation, most of the blood vessels in the tumor tissue were damaged, resulting in insufficient blood supply, which aggravated the hypoxia level of the residual tumor tissue. Moreover, conventional chemotherapy drugs loaded with such nano-synergists are prone to drug resistance under hypoxic conditions. In addition, existing HIFU technology and nano-synergists mainly focus on the removal of primary tumors, while metastasis is another difficult problem faced after surgery. Therefore, solving the problem of residual tumor tissue and metastatic lesions is of great strategic significance for realizing the rise of the focused ultrasound industry.

[0004] Studies have shown that the occurrence and development of tumors are closely related to the body's immune system. Anti-tumor immune response is another powerful measure to activate the body's immune system after tumor ablation to achieve systemic tumor cell clearance and prevent metastasis. Inducing the body's immune response has become a new research hotspot in HIFU treatment of tumors in order to improve clinical prognosis. Li et al. conjugated camptothecin with the polymer C9F17-b-PAsp and wrapped it with PGA-gmPEG to form drug delivery nanodroplets. [See "Molecular Pharmaceutics", Vol. 18, No. 5, pp. 2091-2103, (2021)]. Under HIFU irradiation, the nanodroplets inhibit tumor recurrence by activating adaptive immunity, such as inducing immunogenic cell death, activating dendritic cells (DCs) maturation, and enhancing effector T cell infiltration in tumors.

[0005] Although research on HIFU enhancers has yielded promising results, existing enhancers still fail to overcome hypoxia-resistant tumor cells or only suppress tumor recurrence and metastasis by enhancing adaptive immunity, neglecting the importance of innate immunity. Consequently, they fail to effectively address the issues of residual tumors and metastasis faced by HIFU technology. In summary, research on nanoenhancers for eliminating residual hypoxia-resistant tumor cells after HIFU surgery while simultaneously enhancing the body's anti-tumor innate and adaptive immune responses has yet to be reported, and further research is needed. Summary of the Invention

[0006] In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a HIFU nano-synergist and its preparation and application, which can be used to solve the problems of the existing synergists, such as the inability to overcome the hypoxic drug resistance of tumor cells and the lack of enhancement of the innate immune response.

[0007] To achieve the above objectives and other related objectives, the present invention provides a HIFU nano-synergist in a first aspect, wherein the synergist comprises nanoparticles loaded with banothramine and cyclic dinucleotide.

[0008] Furthermore, the nanoparticles are spherical nanoparticles with a metal organic framework formed by self-assembly of metal ions, anthraquinone and cyclic dinucleotide.

[0009] Furthermore, the metal ion is a divalent manganese ion (Mn 2+ ).

[0010] Furthermore, the particle size of the nanoparticles is 190±50 nm.

[0011] The second aspect of the present invention provides a method for preparing the HIFU nanoboost according to the first aspect, comprising the following steps:

[0012] (1) dissolving banovanthraquinone dihydrochloride and a soluble salt containing metal ions in water to obtain solution A and solution B; adding solution B to solution A, stirring and reacting to obtain reaction solution 1;

[0013] (2) adding an organic base to the reaction solution 1 and stirring the reaction to remove HCl to obtain a reaction solution 2;

[0014] (3) dissolving the cyclic dinucleotide in an organic solvent, adding the solution B, and stirring to react to obtain a reaction solution 3;

[0015] (4) Adding the reaction solution 2 and the reaction solution 3 into water, stirring and reacting, and after the reaction is completed, removing the uncoordinated anthraquinone and cyclic dinucleotide by dialysis to obtain the nano HIFU enhancer.

[0016] Furthermore, in step (1), the soluble salt containing metal ions is selected from manganese dichloride.

[0017] Furthermore, in the steps (1) and (3), the mass ratio of anthraquinone dihydrochloride, the soluble salt containing metal ions and the cyclic dinucleotide is (15-25):1:(0.8-1.2).

[0018] Furthermore, in step (1), the concentration of solution A is 1.5 to 2.0 mg / mL, and the concentration of solution B is 0.5 to 1.2 mg / mL.

[0019] Furthermore, the step (1) further comprises: dissolving the anthraquinone dihydrochloride in water under ultrasonication to obtain solution A; preferably, the ultrasonication time is 3 to 5 minutes.

[0020] Furthermore, in the step (1), the stirring reaction time is 3 to 5 hours.

[0021] Furthermore, in the step (1), the solution B is added to the solution A drop by drop.

[0022] Furthermore, in the step (2), the organic base is selected from triethylamine; preferably, the dosage ratio of anthraquinone dihydrochloride to triethylamine is 10:(1-1.2) (mg / mL).

[0023] Furthermore, in the step (2), the organic base is added to the reaction solution 1 by dropwise addition.

[0024] Furthermore, in the step (2), the stirring reaction time is not less than 8 hours.

[0025] Furthermore, in step (3), cyclic dinucleotides (CDNs) are dissolved in an organic solvent to prepare a solution of 0.5 to 1.0 mg / mL.

[0026] Furthermore, in step (3), the organic solvent is selected from methanol.

[0027] Furthermore, in step (3), the solution B is added dropwise.

[0028] Furthermore, the step (3) further comprises: after the solution B is added, first reacting under ultrasonic action, and then stirring the reaction to obtain a reaction solution 3; preferably, the ultrasonic reaction time is 1 to 2 minutes.

[0029] Furthermore, in step (3), the stirring reaction time is 1 to 3 hours.

[0030] Furthermore, in the step (4), the volume ratio of the reaction liquid 2, the reaction liquid 3 and water is 1:1:(4-6).

[0031] Furthermore, in step (4), the reaction solution 1 and the reaction solution 2 are added to water dropwise.

[0032] Furthermore, in step (4), the stirring reaction time is 2 to 3 hours.

[0033] Furthermore, in step (4), the dialysis times are 4 to 8 times, preferably 5 to 6 times; and the dialysis time for each time is not less than 15 minutes.

[0034] Furthermore, in steps (1) to (4), the reaction and operation are carried out at room temperature (25° C.), and the entire reaction is carried out under light-proof conditions.

[0035] The third aspect of the present invention provides use of the HIFU nanoboost according to the first aspect and / or the HIFU nanoboost prepared according to the method described in the second aspect in high-intensity focused ultrasound.

[0036] As described above, the HIFU nano-synergist of the present invention and its preparation and application have the following beneficial effects:

[0037] 1. The HIFU nano-synergist of the present invention utilizes the acid-responsive properties of metal coordination bonds to effectively release the anticancer drugs, anthraquinone and cyclic dinucleotide, within tumor tissues for synergistic treatment, thereby enhancing the anti-tumor effect.

[0038] 2. The anthraquinone drug in the HIFU nano-synergist of the present invention can selectively act on hypoxic cells, effectively overcoming hypoxia resistance and killing tumor cells without harming normal tissues.

[0039] 3. The manganese ions in the HIFU nanoboost of the present invention, when used in combination with cyclic dinucleotides, can activate the stimulator of interferon genes (STING) pathway, while simultaneously enhancing the body's innate immune and adaptive responses, thereby reversing the immunosuppression caused by the tumor microenvironment and promoting tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Shown is the particle size distribution diagram of the HIFU nano-boosting agent in Example 1 of the present invention.

[0041] Figure 2 Shown is a CAM / PI double-stained laser confocal image of cells treated with the HIFU nanosynergist loaded with anthraquinone / cyclic dinucleotide (abbreviated as AMC) in Example 1 of the present invention.

[0042] Figure 3Shown is the interferon beta (IFN-β) level in mice after treatment with the HIFU nanosynergist (AMC) loaded with anthraquinone / cyclic dinucleotide in Example 1. DETAILED DESCRIPTION

[0043] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0044] The present invention provides a HIFU nano-synergist, such as Figure 1 As shown, the HIFU nano-synergist comprises Banoxantrone dihydrochloride (AQ4N), divalent manganese ions (Mn 2+ ) and cyclic dinucleotides (CDNs) self-assembled into spherical nanoparticles with a metal organic framework (abbreviated as AMC), the particle size of the nanoparticles is 190±50nm. Among them, AQ4N can be activated into cytotoxic AQ4 under tumor hypoxia conditions. CDNs can reprogram tumor-associated immune cells into anti-tumor active immune cells. Manganese ions (Mn 2+ ) can enhance the activity of STING agonists and induce anti-tumor T cell responses with long-term memory. Therefore, the present invention enables the nano-synergist to decompose and release drugs in the tumor microenvironment. AQ4N can overcome hypoxia resistance and kill hypoxic tumor cells. At the same time, Mn 2+ The combination of AQ4N, Mn, and MgCl2 can enhance the activation of the STING pathway, reprogram tumor-associated immune cells into anti-tumor active immune cells, reverse the immunosuppression caused by the tumor microenvironment, and enhance the activation of the body's anti-tumor innate immunity and adaptive immune response. 2+ The combined effect with CDNs can effectively eliminate the residual hypoxia-resistant tumor cells after HIFU surgery, rebuild the tumor immune microenvironment, and improve the therapeutic effect of HIFU.

[0045] One embodiment of the present application provides a method for preparing the HIFU nano-synergist as described above, comprising the following steps:

[0046] (1) dissolving anthraquinone dihydrochloride (AQ4N·HCl) and a soluble salt containing manganese ions in water to obtain solution A and solution B; adding solution B to solution A, stirring and reacting to obtain reaction solution 1;

[0047] (2) adding an organic base to the reaction solution 1 and stirring the reaction to remove HCl to obtain a reaction solution 2;

[0048] (3) dissolving cyclic dinucleotides (CDNs) in an organic solvent, adding the solution B, and stirring to react to obtain a reaction solution 3;

[0049] (4) Adding the reaction solution 2 and the reaction solution 3 into water, stirring and reacting, and after the reaction is completed, removing the uncoordinated anthraquinone and cyclic dinucleotide by dialysis to obtain the nano HIFU enhancer.

[0050] In another embodiment of the present application, in step (1), the soluble salt containing manganese ions is selected from manganese dichloride.

[0051] In another embodiment of the present application, in steps (1) and (3), the mass ratio of anthraquinone dihydrochloride, the soluble salt containing manganese ions and the cyclic dinucleotide is (15-25):1:(0.8-1.2); the concentration of the solution A is 1.5-2.0 mg / mL, and the concentration of the solution B is 0.5-1.2 mg / mL.

[0052] In another embodiment of the present application, the step (1) further comprises: dissolving the anthraquinone dihydrochloride in water under ultrasonication to obtain solution A; preferably, the ultrasonication time is 3 to 5 minutes.

[0053] In another embodiment of the present application, in step (1), the solution B is added to the solution A dropwise, and the stirring reaction time is 3 to 5 hours.

[0054] In another embodiment of the present application, in the step (1).

[0055] In another embodiment of the present application, in step (2), the organic base is selected from triethylamine; preferably, the usage ratio of anthraquinone dihydrochloride to triethylamine is 10:(1-1.2) (mg / mL).

[0056] In another embodiment of the present application, in step (2), the organic base is added to the reaction solution 1 by dropwise addition, and the stirring reaction time is not less than 8 hours.

[0057] In another embodiment of the present application, in step (3), cyclic dinucleotides (CDNs) are dissolved in an organic solvent to prepare a solution of 0.5 to 1.0 mg / mL.

[0058] In another embodiment of the present application, in step (3), the organic solvent is selected from methanol.

[0059] In another embodiment of the present application, in step (3), the solution B is added dropwise, and the stirring reaction time is 1 to 3 hours.

[0060] In another embodiment of the present application, the step (3) further comprises: after the solution B is added, first reacting under ultrasound, and then stirring the reaction to obtain a reaction solution 3; preferably, the ultrasonic reaction time is 1 to 2 minutes.

[0061] In another embodiment of the present application, in step (4), the volume ratio of the reaction liquid 2, the reaction liquid 3 and water is 1:1:(4-6).

[0062] In another embodiment of the present application, in step (4), the reaction solution 1 and the reaction solution 2 are added to water dropwise, and the stirring reaction time is 2 to 3 hours.

[0063] In another embodiment of the present application, in step (4), the dialysis times are 4 to 8 times, preferably 5 to 6 times; and the dialysis time for each time is not less than 15 minutes.

[0064] In addition, it should be noted that in the preparation method provided in the above embodiment of the present application, the water used is selected from deionized water and ultrapure water; the dialysis bag used during dialysis is MD44 (3500Da) from Solarbio; in the steps (1) to (4), the reactions and operations involved are all carried out at room temperature (25°C), and the entire reaction is carried out under light-proof conditions; the role of ultrasound is to dissolve or mix the reactants evenly.

[0065] The following examples are further given to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention all fall within the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make selections within a suitable range based on the description herein and are not limited to the specific values ​​exemplified below.

[0066] Example 1

[0067] The preparation process of the HIFU nanosynergist (AMC) loaded with anthraquinone / cyclic dinucleotide in this embodiment is as follows:

[0068] (1) Dissolve 20 mg of anthraquinone dihydrochloride (AQ4N·HCl) in 10 mL of deionized water and sonicate for 3 to 5 minutes.

[0069] (2) Add to a brown glass bottle and add 1 mL of 1 mg / mL manganese dichloride (MnCl2) solution dropwise. After magnetic stirring for 4 h, add 2 mL of triethylamine dropwise. After the addition is complete, continue stirring overnight to remove HCl.

[0070] (3) Dissolve cyclic dinucleotides (CDNs) in methanol to prepare a 1 mg / mL solution. Add manganese dichloride solution to the CDNs solution at a ratio of 10:1 under vigorous stirring.

[0071] (4) Slowly dropwise add the reaction solution obtained in step (2) and step (3) into a round-bottom flask containing 20 mL of deionized water, and perform magnetic stirring for 2 h.

[0072] (5) Finally, the reaction solution obtained in step (4) was dialyzed using a dialysis bag, with the water changed every 15 minutes, for 5 times to remove the uncoordinated anthraquinone and cyclic dinucleotide, thereby obtaining an HIFU nanosynergist (AMC) loaded with anthraquinone / cyclic dinucleotide.

[0073] Take 1 mL of the dialyzed product (AMC nanoparticles) obtained in step 5 and use a Maven laser particle size analyzer to measure the average particle size and distribution of the dialyzed AMC nanoparticles. The results are as follows: Figure 2 shown.

[0074] from Figure 2 From the particle size distribution diagram, we can see that the particle size of AMC nanoparticles is 190.6nm.

[0075] Example 2

[0076] The preparation process of the HIFU nanosynergist (AMC) loaded with anthraquinone / cyclic dinucleotide in this embodiment is as follows:

[0077] (1) Dissolve 10 mg of anthraquinone dihydrochloride (AQ4N·HCl) in 5 mL of deionized water and sonicate for 2 to 3 minutes.

[0078] (2) Add to a brown glass bottle and add 0.5 mL of 1 mg / mL manganese dichloride (MnCl2) solution dropwise. After magnetic stirring for 4 h, add 1 mL of triethylamine dropwise. After the addition is complete, continue stirring overnight to remove HCl.

[0079] (3) Dissolve cyclic dinucleotides (CDNs) in methanol to prepare a 0.5 mg / mL solution. Add manganese dichloride solution to the CDNs solution at a ratio of 10:1 under vigorous stirring.

[0080] (4) Slowly dropwise add the reaction solution obtained in step (2) and step (3) into a round-bottom flask containing 20 mL of deionized water, and perform magnetic stirring for 2 h.

[0081] (5) Finally, the reaction solution obtained in step (4) was dialyzed using a dialysis bag, with the water changed every 15 minutes, for 6 times to remove the uncoordinated anthraquinone and cyclic dinucleotide, thereby obtaining an HIFU nanosynergist (AMC) loaded with anthraquinone / cyclic dinucleotide.

[0082] Example 3

[0083] The preparation process of the HIFU nanosynergist (AMC) loaded with anthraquinone / cyclic dinucleotide in this embodiment is as follows:

[0084] (1) Dissolve 8 mg of anthraquinone dihydrochloride (AQ4N·HCl) in 5 mL of deionized water and sonicate for 2 to 3 minutes.

[0085] (2) Add to a brown glass bottle and add 0.5 mL of 1 mg / mL manganese dichloride (MnCl2) solution dropwise. After magnetic stirring for 3 h, add 0.8 mL of triethylamine dropwise. After the addition is complete, continue stirring overnight to remove HCl.

[0086] (3) Dissolve cyclic dinucleotides (CDNs) in methanol to prepare a 0.5 mg / mL solution. Add manganese dichloride solution to the CDNs solution at a ratio of 10:1 under vigorous stirring.

[0087] (4) Slowly add the reaction solution obtained in step (2) and step (3) dropwise into a round-bottom flask containing 10 mL of deionized water, and stir magnetically for 1.5 h.

[0088] (5) Finally, the reaction solution obtained in step (4) was dialyzed using a dialysis bag, with the water changed every 15 minutes, for 5 times to remove the uncoordinated anthraquinone and cyclic dinucleotide, thereby obtaining an HIFU nanosynergist (AMC) loaded with anthraquinone / cyclic dinucleotide.

[0089] Example 4

[0090] The CAM / PI double staining method was used to study the in vitro anti-tumor effect of HIFU nanosynergist (AMC). 5) were seeded into confocal microplates, with 1.5 ml of cell suspension added to each dish and incubated in a 37°C incubator for 24 hours. After 24 hours, the culture medium in each dish was replaced with fresh culture medium containing free AQ4N and AMC (equivalent AQ4N dose: 50 μg / ml), respectively, and the cells were co-cultured for 48 hours under normoxic or hypoxic conditions. After 48 hours, the old culture medium was aspirated, and the cells were gently washed twice with PBS. Subsequently, 2 μL of calcein-AM and 4 μL of propidium iodide (PI) were added for staining for 15 minutes, followed by three washes with PBS. Cells were then observed using confocal laser scanning, where live cells were indicated by green fluorescence and dead cells by red fluorescence.

[0091] Figure 2 This is a laser confocal micrograph showing the killing effect of the HIFU nanosynergist (AMC) loaded with anthraquinone / cyclic dinucleotide in Example 1 on 4T1 tumor cells in vitro. Figure 2 As can be seen, cells treated with AQ4N under hypoxic conditions exhibited a higher proportion of green fluorescence and a lower proportion of red fluorescence compared to the normoxic group, indicating that hypoxia can effectively activate the cytotoxicity of AQ4N. In the AMC group under hypoxic conditions, almost all cells displayed strong red fluorescence, with dead cells accounting for the largest proportion. Only a small amount of green fluorescence was observed in a punctate distribution. This result demonstrates that the nanosynergist (AMC) has a higher cell-killing effect than free AQ4N and can effectively eliminate residual hypoxic heterogeneous tumor cells.

[0092] 4T1 bilateral tumor-bearing Balb / c mice (from the Animal Center of Chongqing Medical University) were used to conduct in vivo innate immune activation studies. When the tumor diameter was 5-6 mm, the mice were randomly divided into four groups, one group was a PBS group, one group was a free CDNs group, one group was an AMC group, and one group was an AMC+HIFU group. The HIFU nanoboost (AMC) in Example 1 was injected through the tail vein (the relative injection amount of CDNs was 50 μg / kg), once every 5 days, and the treatment cycle was 15 days. After the treatment cycle, blood was collected from the orbital cavity and the IFN-β content in the plasma was detected using a mouse IFN-β kit. The results are as follows: Figure 3 shown.

[0093] Figure 3 The figure shows the IFN-β levels in the plasma of mice treated in each group at the end of the treatment cycle. IFN-β levels are an important indicator for evaluating the level of innate immunity in the body. Figure 3 It can be seen that compared with free CDNs, the HIFU nanoboost AMC+HIFU group can significantly increase the secretion of IFN-β, indicating that AMC can effectively enhance the anti-tumor innate immune response of mice after HIFU surgery.

[0094] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A HIFU nano-synergist, characterized in that: The synergist comprises nanoparticles loaded with banothraxanthraquinone and cyclic dinucleotide, wherein the nanoparticles are spherical nanoparticles with a metal organic framework formed by self-assembly of metal ions, banothraxanthraquinone and cyclic dinucleotide, and the metal ions are divalent manganese ions Mn 2+ .

2. The HIFU nano-synergist according to claim 1, characterized in that: The particle size of the nanoparticles is 190±50 nm.

3. The method for preparing the HIFU nano synergist according to any one of claims 1 to 2, characterized in that: The steps include: (1) Dissolving banothramine dihydrochloride and a soluble salt containing metal ions in water respectively to obtain solution A and solution B; adding solution B to solution A, stirring and reacting to obtain reaction solution 1; (2) adding an organic base to the reaction solution 1 and stirring the reaction to remove HCl to obtain a reaction solution 2; (3) dissolving the cyclic dinucleotide in an organic solvent, adding the solution B, and stirring to react to obtain reaction solution 3; (4) Adding the reaction solution 2 and the reaction solution 3 into water, stirring and reacting, and after the reaction is completed, removing the uncoordinated anthraquinone and cyclic dinucleotide by dialysis to obtain the HIFU nano-synergist.

4. The preparation method according to claim 3, wherein: In the step (1), the soluble salt containing metal ions is selected from manganese dichloride; And / or, in steps (1) and (3), the mass ratio of anthraquinone dihydrochloride, the soluble salt containing metal ions, and the cyclic dinucleotide is (15-25):1:(0.8-1.2); And / or, in step (1), the concentration of solution A is 1.5-2.0 mg / mL, and the concentration of solution B is 0.5-1.2 mg / mL; And / or, the step (1) further comprises: dissolving the anthraquinone dihydrochloride in water under ultrasonication to obtain a solution A; And / or, in step (1), the stirring reaction time is 3 to 5 hours; And / or, in step (1), the solution B is added to the solution A drop by drop.

5. The preparation method according to claim 3, wherein: In the step (2), the organic base is selected from triethylamine; And / or, in step (2), the organic base is added to the reaction solution 1 by dropwise addition; And / or, in step (2), the stirring reaction time is not less than 8 hours.

6. The preparation method according to claim 3, wherein: In the step (3), the cyclic dinucleotide CDNs are dissolved in an organic solvent to prepare a solution of 0.5-1.0 mg / mL; And / or, in step (3), the organic solvent is selected from methanol; And / or, in step (3), the solution B is added dropwise; And / or, the step (3) further comprises: after the solution B is added, first reacting under ultrasound, and then stirring the reaction to obtain a reaction solution 3; And / or, in step (3), the stirring reaction time is 1 to 3 hours.

7. The preparation method according to claim 3, wherein: In the step (4), the volume ratio of the reaction liquid 2, the reaction liquid 3 and water is 1:1:(4-6); And / or, in step (4), the reaction solution 2 and the reaction solution 3 are added to water dropwise; And / or, in step (4), the stirring reaction time is 2 to 3 hours; And / or, in step (4), the dialysis frequency is 4 to 8 times; each dialysis time is not less than 15 minutes; And / or, in the steps (1) to (4), the reaction and operation are carried out at room temperature of 25° C., and the entire reaction is carried out under light-proof conditions.

8. The preparation method according to claim 7, characterized in that: In the step (4), the number of dialysis is 5 to 6 times.

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