A sting agonist liposome nano-preparation, a preparation method thereof and anti-tumor application

Abstract

The application discloses a STING agonist liposome nano-preparation, a preparation method thereof and anti-tumor application, relates to the technical field of nano-drug preparations, and the liposome nano-preparation comprises a liposome membrane and a STING agonist loaded in a water phase in the liposome; the liposome membrane is composed of distearoyl phosphatidylcholine, cholesterol and DSPE-PEG2000-Mal; the mass ratio of the distearoyl phosphatidylcholine, the cholesterol and the DSPE-PEG2000-Mal is 8:1:1; the surface of the liposome is not coupled with a targeting ligand or an antibody; by optimizing the specific proportion of the components of the liposome membrane, the preparation components are simplified and the system is highly stable without the aid of a targeting antibody modification, the aggregation problem caused by macromolecule coupling is effectively avoided, the drug encapsulation efficiency is improved, and the uniform nanoparticle size is maintained; the preparation can be passively enriched at a tumor site by virtue of the PEGylation long-circulating characteristics and the enhanced permeability and retention effect, plays an anti-tumor role by efficiently activating the STING pathway, and reduces systemic toxicity.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine formulation technology, and in particular to a STING agonist liposome nanoformulation, its preparation method, and its antitumor application. Background Technology

[0002] The TING (Stimulator of Interferon Genes) pathway is a key signaling pathway in the innate immune response. It activates type I interferon signaling upon recognition of cytoplasmic DNA, thereby initiating an anti-tumor immune response. STING agonists (such as di-ABZI) have shown promising anti-tumor potential in various tumor models. However, existing small-molecule STING agonists suffer from poor water solubility, short in vivo half-life, lack of tumor targeting, and systemic administration easily induces systemic inflammatory responses, severely limiting their clinical application.

[0003] Liposomes, as a mature drug delivery carrier, can effectively improve drug solubility and pharmacokinetic properties. Although targeted delivery studies for STING agonists have been conducted, developing a basic liposomal nanoformulation with simple components, good stability, high encapsulation efficiency, and long-circulation characteristics remains of great significance for the clinical translation of STING agonists. Such basic formulations can not only be used directly for tumor treatment or intratumoral injection where targeted targeting is not critical, but also serve as a universal platform, providing standardized prodrug carriers for subsequent on-demand surface functionalization modifications. Summary of the Invention

[0004] In view of the aforementioned existing problems, the present invention is proposed.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a STING agonist liposome nanoformulation, characterized in that: the liposome nanoformulation comprises a liposome membrane and a STING agonist loaded in the aqueous phase of the liposome; The liposome membrane is composed of distearate phosphatidylcholine (DSPC), cholesterol, and DSPE-PEG2000-Mal; The mass ratio of DSPC, cholesterol and DSPE-PEG2000-Mal is 8:1:1; The liposomes were not coupled with a targeting ligand or antibody on their surface.

[0006] Preferably, the STING agonist is di-ABZI.

[0007] Preferably, the liposomes were measured to have a particle size of 140-155 nm, a polydispersity index (PDI) of no more than 0.2, and a zeta potential of 5-10 mV at 25 °C using dynamic light scattering.

[0008] Preferably, the liposomes encapsulate 65%-75% of the STING agonist and have a drug loading of 7%-10%.

[0009] Secondly, the present invention provides a method for preparing the above-mentioned STING agonist liposome nanoformulation, characterized by comprising the following steps: (1) DSPC, cholesterol, DSPE-PEG2000-Mal and STING agonist were co-dissolved in an organic solvent, and the organic solvent was removed by rotary evaporation to form a lipid film; (2) After adding the hydration medium, the lipids are subjected to ultrasonic treatment to form liposomes carrying the STING agonist. (3) The obtained liposomes are purified to obtain the STING agonist liposome nanoformulation.

[0010] Preferably, the organic solvent in step (1) is a mixture of chloroform and dimethylformamide.

[0011] Preferably, the ultrasonic treatment in step (2) includes water bath ultrasound and probe ultrasound in sequence.

[0012] Preferably, the purification in step (3) uses ultrafiltration centrifugation or dialysis to remove free drug.

[0013] Thirdly, the present invention provides the application of the above-mentioned STING agonist liposome nanoformulation in the preparation of antitumor drugs.

[0014] Preferably, the antitumor drug is used to treat solid tumors.

[0015] Preferably, the solid tumor is selected from melanoma, colorectal cancer, breast cancer, or lung cancer.

[0016] The beneficial effects of this invention are as follows: by optimizing the specific ratio of liposome membrane components, the formulation components are simplified and the system is highly stable without relying on targeted antibody modification. This effectively avoids aggregation problems that may be caused by macromolecular coupling, improves drug encapsulation efficiency and maintains uniform nanoparticle size. The formulation, with its PEGylated long-cycle characteristics, can be passively enriched at the tumor site by enhancing the permeation and retention effects. While efficiently activating the STING pathway to exert anti-tumor effects, it reduces systemic toxicity. Moreover, the pre-reserved active sites on the surface make it a universal platform carrier, providing a standardized precursor for subsequent functional modification as needed. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a flowchart of the preparation method of the STING agonist liposome nanoformulation in Example 1. Detailed Implementation

[0019] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0020] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0021] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0022] Example 1, the first embodiment of the present invention, provides a method for preparing a high-encapsulation-efficiency STING agonist liposome nanoformulation and its physicochemical characterization, including: Accurately weigh 80 mg of distearate phosphatidylcholine (DSPC), 10 mg of cholesterol, 10 mg of DSPE-PEG2000-Mal, and 6 mg of the STING agonist di-ABZI, and dissolve them together in 10 mL of a mixed organic solvent of chloroform and dimethylformamide (9:1 volume ratio). Transfer the mixture to a round-bottom flask and remove the organic solvent by rotary evaporation at 45°C and 100 rpm, forming a uniform lipid film on the flask wall. Then, place the flask in a vacuum drying oven overnight to completely remove residual solvent. Add 10 mL of ultrapure water as a hydration medium and sonicate in a water bath at 50°C for 30 minutes to detach the film. Then, use a probe sonicator to sonicate at 20% power for 10 minutes in an ice-water bath to promote the full self-assembly of lipids. The resulting suspension was filtered sequentially through 0.45 μm and 0.22 μm aqueous membranes, and finally centrifuged for 20 minutes at 4000 g using a 10 kDa ultrafiltration centrifuge tube to remove free drug. The retentate was collected to obtain the STING agonist liposome nanoformulation. Dynamic light scattering (DLS) at 25 °C showed that the average particle size of the formulation was 148.3 nm, the polydispersity index (PDI) was 0.13, and the zeta potential was +7.8 mV. Ultraviolet spectrophotometry determined that the encapsulation efficiency of di-ABZI was 68.5%, and the drug loading was 8.2%.

[0023] Example 2, the second embodiment of the present invention, provides a method for studying the in vitro serum stability and storage stability of a STING agonist liposome nanoformulation, comprising: STING agonist liposome nanoformulations were prepared according to the method described in Example 1. To investigate serum stability, the prepared liposomes were mixed with fetal bovine serum (FBS) at a volume ratio of 1:4 and incubated in a 37°C constant-temperature shaker. Samples were taken at 0, 2, 4, 8, 12, and 24 hours, and particle size changes were monitored using DLS. The results showed that after 24 hours of incubation, the liposome particle size only slightly increased from 148 nm to 155 nm, and the PDI remained below 0.2, with no obvious protein adsorption and aggregation. This demonstrates that the PEGylated hydration layer formed by DSPE-PEG2000-Mal endows the formulation with excellent anti-protein adsorption ability. To investigate storage stability, the formulation was stored in a 4°C refrigerator, and samples were taken and analyzed on days 0, 7, 14, and 30. The results showed that after 30 days of storage, the liposome particle size did not change significantly, the encapsulation efficiency decreased by only about 2.5%, and no precipitation occurred, confirming that the lipid membrane with this specific ratio (8:1:1) has extremely high bilayer rigidity and thermodynamic stability, meeting the storage requirements of clinical formulations.

[0024] Example 3, the third embodiment of the present invention, provides a passive targeted pharmacodynamic validation of a STING agonist liposome nanoformulation in a melanoma model, including: A subcutaneous melanoma xenograft model was constructed in B16-F10 mice. When the tumor volume grew to approximately 80 mm³, tumor-bearing C57BL / 6 mice were randomly divided into a PBS control group, a free di-ABZI group, and a liposome group prepared in this embodiment (n=8 per group). Both the free and liposome groups were administered di-ABZI at a dose of 2 mg / kg via tail vein every 3 days for a total of 4 administrations. Tumor volume was measured and mouse weight was recorded every other day. The results showed that the free di-ABZI group exhibited a tumor inhibition rate of only 35% due to rapid drug clearance and systemic toxicity, and mice showed significant weight loss and brittle hair, indicating an inflammatory response. In contrast, the liposome group, thanks to the long-cycle properties of PEG and the EPR effect (enhanced penetration and retention), effectively accumulated at the tumor site, achieving a tumor inhibition rate of 72%, while maintaining normal mouse weight and showing no significant pathological damage in organ H&E staining. These results fully demonstrate that this invention achieves highly efficient and safe anti-tumor therapy without the need for conjugation with a targeting antibody, relying solely on the optimized basic liposome structure.

[0025] Comparative Example 1 The difference from Example 1 is that the ratio of liposome membrane components was adjusted, and the mass ratio of DSPC, cholesterol and DSPE-PEG2000-Mal was adjusted to 6:3:1 (increasing the proportion of cholesterol and decreasing the proportion of DSPC). The remaining preparation steps and drug dosages are the same as in Example 1.

[0026] Characterization results showed that the average particle size of the obtained liposomes increased to 185 nm, and the PDI increased to 0.28, indicating a wider particle size distribution and decreased uniformity. More importantly, due to the excessively high cholesterol ratio leading to excessive lipid membrane stiffness and difficulty in hydration, the encapsulation efficiency of di-ABZI plummeted to 42.1%. This comparative example demonstrates that the 8:1:1 ratio specified in this invention is the optimal solution for balancing membrane fluidity and encapsulation efficiency. Deviating from this ratio will lead to a significant deterioration in formulation quality and is not a conventional choice in the art.

[0027] Comparative Example 2 The difference from Example 1 is that DSPE-PEG2000-Mal is replaced with an equal mass of conventional long-circulating lipid material DSPE-PEG2000-OH (hydroxyl-terminated, without maleimide active groups), while the rest of the preparation steps and proportions are the same as in Example 1.

[0028] Characterization and stability results showed that although the initial particle size was similar to that of Example 1, after 14 days of storage at 4°C, this comparative formulation exhibited significant flocculation and precipitation, with a polydispersity index exceeding 0.35 and a decrease in the absolute value of the Zeta potential. Furthermore, due to the lack of polar contribution from the maleimide group, its encapsulation efficiency was only 55%. This comparative example demonstrates that even without antibody conjugation, the maleimide group in DSPE-PEG2000-Mal plays an irreplaceable role in maintaining the specific surface charge, hydration layer structure, and long-term physical stability of liposomes; replacing it with ordinary PEG lipids cannot achieve the technical effects of this invention.

[0029] Comparative Example 3 The difference from Example 1 is that the probe ultrasonic step is omitted in the preparation process, and only water bath ultrasonication is used for hydration and dispersion. The remaining components and proportions are the same as in Example 1.

[0030] Characterization results showed that the obtained product was a mixture of multilayer vesicles (MLVs) and giant monolayer vesicles (GUVs), with an average particle size of up to 450 nm and a PDI greater than 0.5. Due to the large particle size, the encapsulated drug was readily and rapidly cleared by the reticuloendothelial system (RES) in vivo. When used in the melanoma model of Example 3, its tumor inhibition rate was only 28%, which was not significantly different from that of the free drug. This comparative example demonstrates that the specific process steps defined in the claims of this invention, including water bath ultrasound and probe ultrasound, are necessary conditions for obtaining 140-155 nm uniform monolayer vesicles (SUV / LUVs). Omitting this step would prevent the passive targeted pharmacodynamics of this invention from being achieved.

[0031] In summary, this invention achieves a simplified formulation and high system stability by optimizing the specific ratio of liposome membrane components without relying on targeted antibody modification. It effectively avoids aggregation problems that may be caused by macromolecular coupling, improves drug encapsulation efficiency, and maintains uniform nanoparticle size. The formulation, with its PEGylated long-cycle characteristics, can passively accumulate at the tumor site through enhanced permeation and retention effects. It can exert anti-tumor effects by efficiently activating the STING pathway while reducing systemic toxicity. Furthermore, the pre-reserved active sites on the surface make it a universal platform carrier, providing a standardized precursor for subsequent functional modification as needed.

[0032] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A liposome nanoformulation of a STING agonist, characterized in that: The liposome nanoformulation includes a liposome membrane and a STING agonist encapsulated in the aqueous phase of the liposome. The liposome membrane is composed of distearate phosphatidylcholine, cholesterol, and DSPE-PEG2000-Mal. The mass ratio of distearate phosphatidylcholine, cholesterol and DSPE-PEG2000-Mal is 8:1:1; The liposomes were not coupled with a targeting ligand or antibody on their surface.

2. The STING agonist liposome nanoformulation according to claim 1, characterized in that: The STING agonist is di-ABZI.

3. The STING agonist liposome nanoformulation according to claim 1 or 2, characterized in that: The particle size of the liposomes was measured to be 140-155 nm, the polydispersity index was not greater than 0.2, and the zeta potential was 5-10 mV at 25 °C using dynamic light scattering.

4. The STING agonist liposome nanoformulation according to any one of claims 1-3, characterized in that: The liposomes encapsulate 65%-75% of the STING agonist and have a drug loading of 7%-10%.

5. A method for preparing a STING agonist liposome nanoformulation as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) Distearate phosphatidylcholine, cholesterol, DSPE-PEG2000-Mal and STING agonist are co-dissolved in an organic solvent, and the organic solvent is removed by rotary evaporation to form a lipid film. (2) After adding the hydration medium, the lipids are subjected to ultrasonic treatment to form liposomes carrying the STING agonist. (3) The obtained liposomes are purified to remove free drug, thus obtaining the STING agonist liposome nanoformulation.

6. The preparation method according to claim 5, characterized in that: The organic solvent mentioned in step (1) is a mixture of chloroform and dimethylformamide.

7. The preparation method according to claim 5, characterized in that: The ultrasonic treatment in step (2) includes water bath ultrasound and probe ultrasound in sequence.

8. The preparation method according to claim 5, characterized in that: The purification described in step (3) is performed using ultrafiltration centrifugation or dialysis.

9. The use of the STING agonist liposome nanoformulation as described in any one of claims 1-4 in the preparation of antitumor drugs.

10. The application according to claim 9, characterized in that: The antitumor drug is used to treat solid tumors; the solid tumors are selected from melanoma, colorectal cancer, breast cancer, or lung cancer.

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