A liposome for capturing cGAMP and its preparation method and use

Abstract

The present application discloses a liposome for capturing cGAMP, a preparation method and a use thereof. The liposome comprises a liposome membrane and dendritic polylysine encapsulated in the liposome membrane, and the mass ratio of the raw material of the liposome membrane to the dendritic polylysine is (1-100):1. The nanosystem of the present application can transport cGAMP in tumor cells across cells to dendritic cells, activate the STING pathway, produce anti-tumor immunity, significantly inhibit tumor growth and metastasis, and prolong animal survival.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a liposome for capturing cGAMP, and a preparation method and application thereof. Background Art

[0002] Stimulator of interferon genes (STING) is a key signaling molecule that regulates innate immunity. Activating STING in tumor tissue can enhance tumor immunogenicity and improve the response rate of immune checkpoint inhibitors, and is a hot topic in anti-tumor immunotherapy research. At present, tumor immunotherapy based on STING activation strategy generally lacks tumor targeting and is prone to induce systemic immune-related adverse events (irAEs).

[0003] A large number of agonists that can directly activate STING protein have been developed for research on tumor immunotherapy, such as natural CDN STING agonists, synthetic CDN STING agonists, and non-CDN STING agonists. Most of the currently developed CDN STING agonists and non-CDN STING agonists are still administered by intratumoral injection in clinical trials. Intratumoral administration can directly target the tumor site, reducing the exposure of the drug in normal tissues and organs. While ensuring the efficacy of STING agonists in treating tumors, it avoids the risk of STING protein being activated in normal tissues and organs, and then inducing irAEs. However, the treatment method of intratumoral administration is only applicable to a few superficial tumors in clinical practice, such as melanoma, breast tumors, head and neck tumors, etc. Most deep tumors, such as colon tumors, liver tumors, ovarian tumors, etc., cannot achieve intratumoral administration.

[0004] The latest research reveals that tumor cells themselves synthesize the natural agonist of STING protein - cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Although most tumor cells, especially those with higher malignancy, lack STING protein, they cannot use the cGAMP produced by themselves to produce anti-tumor immunity. However, some cGAMP produced by tumor cells can enter immune cells through the cell connection between tumor cells and immune cells, thereby activating the STING protein of immune cells and achieving the activation of immune cells. However, relying on endogenous cGAMP in tumor cells to activate the STING protein of immune cells to achieve anti-tumor immune function is relatively weak, which is mainly caused by the following reasons: 1) In tumor tissue, the physical space between tumor cells and dendritic cells is often far away. In addition, most tumors in the advanced stage have tumor tissues in an immunosuppressive tumor microenvironment, and there are fewer dendritic cells in the tumor tissue. 2) cGAMP secreted by tumor cells outside the cell is easily hydrolyzed by phosphodiesterase 1 (ENPP1) expressed by the tumor cells themselves to generate adenosine monophosphate (AMP) and guanosine monophosphate (GAMP). The more malignant the tumor is, the more ENPP1 it expresses. 3) Extracellular cGAMP is negatively charged and hydrophilic, so it is difficult to passively diffuse into dendritic cells. Although cGAMP-related transporters are expressed on some dendritic cells, they are affected by the tumor's immunosuppressive microenvironment, and the expression of transporters on the surface of dendritic cells and the activity of transporters are low, thereby limiting the activation of dendritic cells. These three points allow tumor cells to escape immune surveillance.

[0005] Dendritic macromolecules mainly include polyamide dendritic macromolecules (PAMAM), polypropylene imine (PPI) and dendritic polylysine (DGL). DGL is polymerized from L-lysine that can be absorbed by the human body (L-lysine is one of the essential amino acids required by the human body). It has the advantages of non-immunogenicity, extremely low cytotoxicity, and can be degraded and recycled by the body and used for protein synthesis. It is regarded as a new generation of dendritic macromolecules that can replace PAMAM and PPI. At present, antiviral drugs based on DGL (SPL7013 and Astodrimer sodium) have been used in the clinical treatment of human immunodeficiency virus and herpes simplex virus.

[0006] Studies have found that nanomedicines with a particle size of about 100nm have a longer blood circulation time. In addition, nanomedicines with a particle size of about 100nm have a strong EPR effect and good tumor targeting. However, nanomedicines of this size are difficult to diffuse into tumor tissues through blood vessels due to their large particle size. However, the dendritic macromolecule DGL has an overall particle size of less than 20nm and is easily cleared by the reticuloendothelial system in the systemic circulation.

[0007] In order to break through the physical space barrier between tumor cells and dendritic cells in tumor tissues and transport cGAMP in tumor cells across tumor cells to dendritic cells, nanomedicines need to achieve transcellular transport capabilities. In addition, in addition to considering the surface charge, the design of DGL-based nanomedicines also needs to consider the effect of the particle size of nanomedicines on targeting and toxic side effects.

[0008] Therefore, it is necessary to create a strategy to precisely activate STING at the tumor target site, which can enhance the effect of tumor immunotherapy while reducing the risk of systemic irAEs. Summary of the invention

[0009] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a liposome for capturing cGAMP and a preparation method and use thereof.

[0010] To achieve the above objectives and other related objectives, the present invention is achieved through the following technical solutions.

[0011] The first aspect of the present invention protects a liposome for capturing cGAMP, wherein the liposome comprises a liposome membrane and dendritic polylysine encapsulated in the liposome membrane, and the mass ratio of the raw material of the liposome membrane to the dendritic polylysine is (1-100):1.

[0012] In certain embodiments, the mass ratio of the raw material of the liposome membrane to the dendritic polylysine can be (1-30):1, (20-50):1, (30-60):1, (40-70):1, (50-80):1, (60-90):1, or (70-100):1. In a specific embodiment, it is 12:5.

[0013] In certain embodiments, the dendritic polylysine is selected from one or more of G1 generation dendritic polylysine (DGL-G1), G2 generation dendritic polylysine (DGL-G2), G3 generation dendritic polylysine (DGL-G3), G4 generation dendritic polylysine (DGL-G4) and G5 generation dendritic polylysine (DGL-G5). Dendritic macromolecules are highly branched and structurally precise molecules synthesized by repeated growth reactions, and each repeated cycle reaction adds a branching layer, which is called a "generation". The dendritic polylysine of the present invention can be purchased from Colcom, France; the dendritic polylysine can also be obtained by a common preparation method, as shown in reference (16) Francoia, JP; Rossi, JC; Monard, G.; Vial, L. Digitizing poly-l-lysine dendrigrafts: from experimental data to molecular dynamics simulations. J. Chem. Inf. Model. 2017, 57, 2173-2180.

[0014] In certain embodiments, the raw materials of the liposome membrane comprise phospholipids, cholesterol and long-circulating materials.

[0015] Preferably, the phospholipid is selected from one or both of dioleoylphosphatidylethanolamine (DOPE) and dipalmitoylphosphatidylcholine (DPPC).

[0016] More preferably, it is dioleoylphosphatidylethanolamine (DOPE).

[0017] Preferably, the cholesterol is selected from one or both of cholesterol monosuccinate (CHEMS) and DC-cholesterol.

[0018] More preferably, it is cholesterol monosuccinate (CHEMS). Cholesterol is a hydrophobic molecule that can combine with phospholipids and embed into the membrane, preventing phospholipids from aggregating into a crystal structure and regulating the fluidity of the membrane structure. Cholesterol, as a component of the liposome membrane, can stabilize the liposome.

[0019] Preferably, the long circulation material is selected from polyethylene glycol (PEG) and its derivatives, and the PEG derivatives are selected from one or more of polyethylene glycol-vitamin E succinate (PEG-TPGS), polyethylene glycol-cholesterol (PEG-Chol), polyethylene glycol-modified distearoylphosphatidylethanolamine (MPEG-DSPE), polyethylene glycol-modified dimyristoylphosphatidylethanolamine (MPEG-DMPE), polyethylene glycol-modified dipalmitoylphosphatidylethanolamine (MPEG-DPPE). The liposome membrane can be modified with polyethylene glycol (PEG) or its derivatives to enhance the hydrophilicity of the liposome membrane, reduce the interaction between plasma proteins and the liposome membrane, prevent the aggregation and fusion of liposomes, avoid the uptake of liposomes by reticuloendothelial system cells, and prolong the in vivo circulation time of liposomes. In some embodiments, the molecular weight of polyethylene glycol is 500 Daltons to 10,000 Daltons, for example, 1,000 Daltons to 7,000 Daltons, preferably 2,000 Daltons to 5,000 Daltons. In some preferred embodiments, the long-circulating material is distearoylphosphatidylethanolamine (DSPE-PEG2000).

[0020] More preferably, the mass ratio of the phospholipid, cholesterol, long-circulating material and dendritic polylysine is (2-4.5): (1.5-2): (5.5-8): 5. In some specific embodiments, it is 4.5:2:5.5:5, 2:1.5:8:5.

[0021] In certain embodiments, the drug loading of the dendritic polylysine in the liposome is 0.3-17%, for example, 0.3-1.5%, 1-5%, 4-10%, 9-17%, and in a specific embodiment, 0.8% or 12%.

[0022] In certain embodiments, the encapsulation rate of dendritic polylysine in the liposome is 10-95%, for example, 10-32%, 25-65%, 45-65%, 60-95%, and in a specific embodiment, 15% or 90%.

[0023] In some embodiments, the particle size of the liposome is 100-200 nm, for example, 100-155 nm, 145-165 nm, 150-170 nm, or 160-200 nm, and in a specific embodiment, 150 nm.

[0024] The second aspect of the present invention protects the method for preparing liposomes as described above, wherein the raw materials of liposome membrane are dissolved in an organic solvent, and colostrum is formed with a dendritic polylysine solution by a thin film hydration method, a detergent dialysis method, a reverse evaporation method or a solvent injection method, and granulated to obtain the liposomes.

[0025] In certain embodiments, the film hydration method is: phospholipids, cholesterol, long-circulation materials and organic solvents are mixed, evaporated to obtain a film, a dendritic polylysine solution is added to the film, hydrated, and granulated to obtain the liposome. The present application removes the organic solvent by evaporation, and after the organic solvent is removed, the liposome can form a transparent film on the glass substrate, and then the dendritic polylysine solution is added to form colostrum, so that the lipid components are hydrated, spontaneously swell and detach from the glass substrate to form liposomes encapsulated with dendritic polylysine.

[0026] Preferably, the organic solvent is selected from one or more of chloroform and methanol. For example, the volume ratio of chloroform to methanol is (1-10):1, (1-5):1, (4-8):1, or (5-10):1, and in a specific embodiment is 3:1.

[0027] Preferably, the mass volume ratio of the total mass of phospholipids, cholesterol and long-circulating materials to the mass volume ratio of the organic solvent is (7-17) mg:4 mL. For example, it can be (7-9) mg:4 mL, or (8-13) mg:4 mL, or (12-17) mg:4 mL, and in a specific embodiment, it is 12 mg:4 mL.

[0028] Preferably, the evaporation temperature is 20-80° C. For example, it may be 20-60° C., 40-70° C., or 50-80° C., and in a specific embodiment, it is 50° C.

[0029] Preferably, the evaporation time is 1 to 6 hours, for example, 1 to 3 hours, 2 to 5 hours, or 4 to 6 hours, and in a specific embodiment, 2 hours.

[0030] Preferably, the dendritic poly-lysine solution is a mixture of dendritic poly-lysine and PBS solution.

[0031] Preferably, based on the volume of the dendritic polylysine solution, the concentration of the dendritic polylysine is 50-150 mg / L, for example, 50-80 mg / L, 60-120 mg / L, 100-150 mg / L, and in a specific embodiment, 100 mg / L.

[0032] Preferably, the hydration temperature is 20-80° C. For example, it may be 20-60° C., 40-70° C., or 50-80° C., and in a specific embodiment, it is 50° C.

[0033] Preferably, the hydration time is 0.2 to 0.8 h, for example, 0.2 to 0.5 h, 0.3 to 0.6 h, or 0.5 to 0.8 h, and in a specific embodiment, 0.5 h.

[0034] In certain embodiments, the reverse evaporation method is: phospholipids, cholesterol, long-circulation materials and organic solvents are mixed, dendritic polylysine solution is added to form colostrum, evaporated, granulated, and the liposomes are obtained. The reverse evaporation method of the present application is to form colostrum by mixing the raw materials of the liposomes and the dendritic polylysine solution, water bath ultrasound, to form oil-in-water reverse micelles, and to remove the organic solvent by rotary evaporation to form a transparent gel on a glass substrate. When the organic solvent is completely removed, the gel collapses to form liposomes encapsulated with dendritic polylysine. The present application found that the drug loading and encapsulation efficiency of the liposomes obtained by the reverse evaporation method are 15 times and 6 times higher than those of the thin film hydration method, respectively.

[0035] Preferably, the organic solvent is diethyl ether.

[0036] Preferably, the mass volume ratio of the total mass of phospholipids, cholesterol and long-circulating materials to the mass volume ratio of the organic solvent is (7-17) mg:3 mL. For example, it can be (7-11) mg:3 mL, or (9-15) mg:3 mL, or (12-17) mg:3 mL, and in a specific embodiment, it is 12 mg:3 mL.

[0037] Preferably, the dendritic polylysine solution is a mixture of dendritic polylysine and PBS solution.

[0038] More preferably, the mass volume ratio of the dendrimer poly-lysine to the PBS solution is (1-10) mg:2 mL.

[0039] Preferably, the heating temperature is 10-50° C. For example, it may be 10-30° C., 20-40° C., or 30-50° C., and in a specific embodiment, it is 30° C.

[0040] Preferably, the hydration time is 0.5 to 3 hours, for example, 0.5 to 1.2 hours, 0.9 to 1.8 hours, or 1.5 to 3 hours, and in a specific embodiment, 1 hour.

[0041] Preferably, the granulation is performed by a shearing machine, a homogenizer or an extrusion device.

[0042] In certain embodiments, the preparation further includes purification, wherein the purification comprises placing the liposome in a dialysis bag for dialysis.

[0043] The third aspect of the present invention protects a pharmaceutical composition, which comprises the liposome as described above, and a pharmaceutically acceptable carrier.

[0044] In certain embodiments, the liposomes are in an effective dose.

[0045] In certain embodiments, the lipid nanoparticles are used alone or in combination with other drugs, that is, the lipid nanoparticles can be used as the only active ingredient or one of the active ingredients of the drug.

[0046] In certain embodiments, the pharmaceutically acceptable carrier may be, for example, physiological saline, isotonic glucose, isotonic sucrose, Ringer's solution, and Hanks' solution. A buffer substance may be added to obtain a preferred pH for storage stability, for example, a pH between 6.0 and 8.0. In some embodiments, a pH of 6.0 to 7.5 is preferred, at which the liposome membrane has better stability and encapsulation stability for the drug.

[0047] In certain embodiments, the liposome nanoparticle or pharmaceutical composition can be administered in any medically acceptable manner, depending on the disease being treated. Possible routes of administration include injection, parenteral routes, such as intramuscular, subcutaneous, intravenous, intraarterial, intraperitoneal, intraarticular, intrameningeal, intrathecal, or other routes, such as oral, nasal, ocular, rectal, vaginal or pulmonary, such as by inhalation. For liposome drugs prepared according to the present invention are delivered to tumors of the central nervous system, liposomes are slowly and continuously directly intracranially infused into the tumor. Liposomes of the present invention can also be directly applied to tissue surfaces.

[0048] The fourth aspect of the present invention protects the use of the liposome as described above or the pharmaceutical composition as described above in the preparation of a product for preventing or treating a disease related to STING.

[0049] In certain embodiments, the STING-associated disease is a tumor.

[0050] Preferably, the tumor is selected from one or more of melanoma, breast tumor, head and neck tumor, colon tumor, liver tumor and ovarian tumor.

[0051] More preferably, it is a colon tumor, such as colon carcinoma in situ or colon subcutaneous adenoma.

[0052] In some embodiments, the product has at least one of the following functions:

[0053] D1) capture of cyclic GMP-AMP;

[0054] D2) transports cyclic GMP-AMP across the membrane;

[0055] D3) Promote dendritic cell maturation;

[0056] D4) Enhances antigen presentation by dendritic cells;

[0057] D5) Promote T lymphocyte activation and proliferation;

[0058] D6) inhibit tumor growth;

[0059] D7) Inhibit tumor metastasis;

[0060] D8) Prolong survival.

[0061] The liposome of the present application can capture cyclic guanosine monophosphate-adenylic acid (cGAMP), which means that it can capture cGAMP in tumor cells.

[0062] The liposome of the present application can transport cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) across the membrane, which means that the liposome can enter the outside world from the tumor cell.

[0063] The liposomes of the present application can promote the maturation of dendritic cells, which means that cGAMP is taken up by dendritic cells, resulting in an increase in the cGAMP content in the dendritic cells, and can promote the expression of maturation-related cell markers CD80 and CD83

[0064] The liposome in the present application can enhance the antigen presentation function of dendritic cells, which means that the liposome can promote the expression of IFNγ.

[0065] The liposomes of the present application can inhibit tumor metastasis, which means that they can inhibit the metastasis of CT26 colon tumor to distant heart, liver, spleen, lung, kidney, etc., and only a small amount of mesenteric lymph nodes have metastasis of colon tumor cells.

[0066] The liposomes of the present application can prolong the survival period, which means that they can prolong the median survival period of patients with CT26 colon tumors. In the CT26 colon tumor orthotopic mouse model, the median survival period was extended by 36 days; in the CT26 intestinal tumor subcutaneous mouse model, the median survival period was extended by 12 days.

[0067] The liposomes of the present application can increase the expression of inflammatory factors in colon tumor tissue and activate anti-tumor immunity in tumor tissue.

[0068] The fifth aspect of the present invention protects the use of the liposome as described above or the pharmaceutical composition as described above in vitro capturing cyclic GMP-AMP (cGAMP), promoting transmembrane transport of cyclic GMP-AMP or promoting maturation of dendritic cells.

[0069] The liposomes of the present application were found in a co-culture system of colon tumor cells and dendritic cells, and the DGL released by the liposomes can capture cGAMP in tumor cells and deliver it to dendritic cells, thereby promoting the maturation of dendritic cells.

[0070] On the other hand, the present invention also protects the liposome as described above or the pharmaceutical composition as described above, for activating the STING signaling pathway.

[0071] In another aspect, the present invention also protects the liposome as described above or the pharmaceutical composition as described above, for use in preventing or treating STING-related diseases.

[0072] In another aspect, the present invention also protects a method for preventing or treating a disease associated with STING, comprising administering an effective amount of the liposome as described above or the pharmaceutical composition as described above to a subject in need thereof (such as a mammal). The method may also be in vitro or non-therapeutic.

[0073] The subject or individual for therapeutic or preventive treatment is preferably a mammal, such as, but not limited to, a human, a primate, livestock (such as sheep, cattle, horses, donkeys, pigs), a pet (such as dogs, cats), a laboratory test animal (such as mice, rabbits, rats, guinea pigs, hamsters) or a captured wild animal (such as foxes, deer). The subject is preferably a primate. The subject is most preferably a human. The subject can be a patient with a tumor or an individual who expects to prevent a tumor.

[0074] The liposome or the pharmaceutical composition can be administered to a subject before, during, or after receiving anti-cancer treatment.

[0075] The liposomes used to capture cGAMP in the present application are to encapsulate DGL with liposomes with a particle size of about 150nm, so that it can realize the charge flipping function while having the ability to convert the particle size (150nm to 20nm), thereby significantly improving the tumor targeting and biosafety of dendritic polylysine. The liposomes of the present application can be administered intravenously, and a large amount of enrichment is achieved at the tumor site by the EPR effect, and the dendritic polylysine (DGL) loaded therein is released in response to the acidic environment of the tumor. After DGL is released, cGAMP in tumor cells can be captured and transported across cells to dendritic cells, activating the STING pathway in dendritic cells and producing anti-tumor immune effects.

[0076] Compared with the prior art, the present invention has the following beneficial effects:

[0077] 1) The overall particle size of the liposome used to capture cGAMP in the present application is about 150nm and carries a neutral charge. Liposomes with this particle size and surface potential are not easily cleared quickly after intravenous injection into the blood circulation, can achieve long circulation in the body, have a good EPR effect, can passively target the tumor site, and efficiently transport the loaded dendritic polylysine (DGL) to the tumor site.

[0078] 2) After the liposomes used to capture cGAMP in the present application reach the tumor site, the lipid layer (ALipo) can respond to the acidic environment of the tumor site to achieve rapid release of dendritic polylysine (DGL). The released dendritic polylysine (DGL) can use the positive charge it carries to enter the cytoplasm of tumor cells through adsorption-mediated transcytosis (AMT), capture the negatively charged cGAMP in the cytoplasm of tumor cells, and pass out of the tumor cells. The DGL that passes through the tumor cells can protect the loaded cGAMP from being hydrolyzed by the ENPP1 hydrolase expressed by the tumor cells, and carry cGAMP into the dendritic cells, increasing the cGAMP content in the dendritic cells by about 10 times.

[0079] 3) After the liposomes used to capture cGAMP in the present application capture endogenous cGAMP in tumor cells and then enter dendritic cells, the tumor endogenous cGAMP loaded by DGL can activate the STING pathway in dendritic cells, promote dendritic cell maturation, secrete multiple cytokines, enhance the ability of dendritic cells to present cross-antigens, and the infiltration of cytotoxic T lymphocytes into tumor tissues, thereby relieving the local immunosuppressive microenvironment of the tumor.

[0080] 4) After intravenous administration, the liposomes used to capture cGAMP in the present application have an inhibition rate of 74% on the growth of CT26 orthotopic colon tumors, prolonging the median survival of tumor-bearing mice by 36 days without causing systemic irAEs.

[0081] 5) The immune activation site of the liposome used to capture cGAMP in this application is limited to the local tumor, which greatly reduces the risk of systemic immune-related adverse events induced by activation of the STING pathway in normal organ tissues. On the basis of ensuring the therapeutic effect, safe systemic administration is achieved, solving the dilemma that the current nano drug delivery technology cannot achieve efficient and low-toxic activation of the local STING pathway in the tumor on the basis of systemic administration. This change in the mode of administration expands the scope of diseases treated by STING-related nano drugs, improves patient compliance, and lays the foundation for further clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 The results show the drug loading and encapsulation efficiency of the acid-responsive liposome DGL@ALipo prepared by the thin film hydration method and the reverse evaporation method in this application. DL is the drug loading, EE is the encapsulation efficiency; the results are expressed as mean±SD (n=5), ***p<0.001.

[0083] Figure 2 The particle size, Zeta potential and transmission electron microscopy characterization of the liposomes in this application are shown. (A) Characterization of the particle size and Zeta potential of dendritic polylysine DGL, acid-responsive liposome DGL@ALipo and non-acid-responsive liposome DGL@Lipo. (B) Transmission electron microscopy image of acid-responsive liposome DGL@ALipo. The results are expressed as mean±SD (n=5).

[0084] Figure 3 The results show that the acid-responsive release of dendritic polylysine (DGL) from acid-responsive liposomes DGL@ALipo and non-acid-responsive liposomes DGL@Lipo was studied using dynamic light scattering technology (Malvern Zetasizer Nano ZS) in this application.

[0085] Figure 4 The results show the efficiency of acid-responsive release of dendritic polylysine (DGL) from liposomes and the investigation of permeability in tumor stroma. (A) The efficiency of acid-responsive release of DGL by acid-responsive liposomes DGL@ALipo and non-acid-responsive liposomes DGL@Lipo within 48 hours. (B) Investigation of permeability of acid-responsive liposomes DGL@ALip (red fluorescence) and non-acid-responsive liposomes DGL@Lipo (blue fluorescence) in tumor stroma. The results are expressed as mean±SD (n=5).

[0086] Figure 5 The results show the investigation of liposome stability in this application. (A) 24-hour serum stability of acid-responsive liposome DGL@ALipo. (B) 7-day storage stability of acid-responsive liposome DGL@ALipo. (C) 24-hour DGL leakage rate of acid-responsive liposome DGL@ALipo in serum environment. (D) 7-day DGL leakage rate of acid-responsive liposome DGL@ALipo in storage environment. The results are expressed as mean±SD (n=5).

[0087] Figure 6The results show the investigation of the ability of dendritic polylysine DGL to capture cGAMP and transport cGAMP across cells in this application. (A) The ability of DGL to capture cGAMP was investigated by agarose electrophoresis. (B) The ability of DGL to cross mouse CT26 colon tumor cells was investigated by fluorescence quantification. (C) The ability of DGL to cross human COLO 205 colon tumor cells was investigated by fluorescence quantification. (D) The uptake of DGL by mouse bone marrow-derived dendritic cells (BMDC) after crossing mouse CT26 colon tumor cells was investigated by laser confocal microscopy. Scale bar: 10 μm. (E) The uptake of DGL by human peripheral blood monocyte-derived dendritic cells (MoDC) after crossing human COLO 205 colon tumor cells was investigated by laser confocal microscopy. Scale bar: 10 μm. (F) Mass spectrometry curve of cGAMP standard. (G) Changes in cGAMP content in mouse BMDC after DGL transported cGAMP across cells. (H) Changes in cGAMP content in human MoDCs after DGL transcellular transport of cGAMP. Results are expressed as mean±SD (n=5), ***p<0.001.

[0088] Figure 7 The statistics of the bioluminescent signal intensity of the acid-responsive liposome DGL@ALipo and the non-acid-responsive liposome DGL@Lipo in this application in inhibiting the growth of CT26-Luc colon tumors are shown. The results are expressed as mean±SD (n=5), *p<0.05, ***p<0.001.

[0089] Figure 8 The results are shown in the study of the effects of acid-responsive liposomes DGL@ALipo and non-acid-responsive liposomes DGL@Lipo on the survival of BALB / c tumor-bearing mice in this application. The results are expressed as mean±SD (n=5). DETAILED DESCRIPTION

[0090] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0091] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0092] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0093] Dipalmitoylphosphatidylcholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) was purchased from Avanti, USA;

[0094] DC-cholesterol (3β-[N-(N,N-dimethylaminoethane)carbamoyl]cholesterolhydrochloride, DC-cholesterol) was purchased from Avanti, USA;

[0095] Fluorescein isothiocyanate and n-hydroxysuccinimide ester (FITC-NHS) was purchased from Thermo Fisher, USA.

[0096] Cy3-NHS (Cy3 labeled N-hydroxysuccinimide ester) was purchased from Sigma-Aldrich, USA;

[0097] Cy5-NHS (Cy5 labeled N-hydroxysuccinimide ester) was purchased from Sigma-Aldrich, USA;

[0098] Cy7-NHS (Cy7-labeled N-hydroxysuccinimide ester) was purchased from Sigma-Aldrich, USA;

[0099] In the present application, dendritic macromolecule polylysine G1-DGL, G2-DGL, G3-DGL, G4-DGL and G5-DGL of different generations are purchased from Colcom, France, and their weight average molecular weights are 1450 g / mol, 8600 g / mol, 22000 g / mol, 65300 g / mol and 172300 g / mol, respectively. In the present application, dendritic macromolecule polylysine G-DGL of different generations are the same as DGL-G1, such as G1-DGL and DGL-G1 are the same substance; in the following embodiments, DGL refers to G5-DGL unless otherwise specified.

[0100] Example 1 Preparation of liposomes for capturing cGAMP

[0101] In this example, for subsequent research, fluorescently labeled dendritic polylysine (DGL) was used, and then liposomes for capturing cGAMP were prepared with the raw materials of liposome membrane, including acid-responsive liposomes (labeled as DGL@ALipo) and non-acid-responsive liposomes (labeled as DGL@Lipo). The following steps are included:

[0102] 1.1. Preparation of FITC fluorescently labeled dendritic polylysine

[0103] Dendritic polylysine (DGL) is labeled with FITC by using the chemical reaction principle of NHS and the amino group of lysine to specifically form an amide bond. The specific preparation method is as follows:

[0104] Take 10 mg DGL and 3 μg FITC-NHS, dissolve them in 5 mL solution of pH 8.0, and stir overnight at room temperature in the dark. After the reaction is completed, the reaction product is concentrated by ultrafiltration (ultrafiltration tube specification: 10 kDa, 50 mL; ultrafiltration conditions: 1000 rpm, 30 min, room temperature). After the ultrafiltration is completed, add 2 mL PBS solution to re-dissolve, and the resulting liquid is added to 2 EP tubes of 1.5 mL (1 mL per tube) and freeze-dried.

[0105] 1.2 Preparation of acid-responsive liposomes DGL@ALipo by thin film hydration method

[0106] 1) The raw materials of the lipid layer include: 4.5 mg of dioleoylphosphatidylethanolamine (DOPE), 2 mg of cholesterol hemisuccinate (CHEMS) and 5.5 mg of distearoylphosphatidylethanolamine (DSPE-PEG2000), and the molar ratio is 3:2:1. The raw materials of the lipid layer are mixed with 4 mL of an organic solvent to form a mixed solution A. The organic solvent is composed of chloroform and methanol, and the volume ratio of chloroform to methanol is 3:1.

[0107] 2) Place the mixed solution A in a round-bottom flask and connect the round-bottom flask to a rotary evaporator. Set the water bath temperature of the rotary evaporator to 50°C, the rotation speed to 50 rpm, and the rotary evaporation time to 2 hours. After the rotary evaporation is completed, remove the round-bottom flask from the rotary evaporator and observe the bottom of the flask. If a uniform, transparent, dry film is visible, the hydration operation can be carried out.

[0108] 3) The specific steps of the hydration operation are as follows: 5 mL of dendritic polylysine solution is added to the round-bottom flask. Subsequently, the mouth of the round-bottom flask is sealed with a sealing film, and 4-5 ventilation holes are pierced on the sealing film with a needle, and the round-bottom flask is placed in a shaker, and the temperature in the shaker is set to 50°C, the shaker speed is set to 250rpm, and the shaker working time is set to 0.5h for hydration. Among them, the dendritic polylysine solution is composed of dendritic polylysine and PBS solution; based on the total volume of the dendritic polylysine solution, the concentration of dendritic polylysine (DGL) is 1 mg / mL.

[0109] 4) After the film is hydrated, transfer the milky white liquid in the round-bottom flask to a 15 mL centrifuge tube for later use. Take a polycarbonate membrane with a pore size of 200 nm and assemble it in a liposome extrusion device. Set the temperature of the liposome extrusion device to 50°C and the number of extrusions to 20. Then, add the hydrated liposome solution (i.e., milky white liquid) into the liposome extrusion device, and obtain the DGL@ALipo solution after extrusion.

[0110] 5) Dialysis purification: Add the obtained DGL@ALipo solution to a dialysis bag (dialysis bag specification: 1000 kDa), seal the open end of the dialysis bag with a dialysis clip, and place the dialysis bag in a beaker filled with PBS solution. Subsequently, place the beaker on a magnetic stirrer, set the speed of the magnetic stirrer to 300 rpm, the stirring time to 24 hours, the ambient temperature to 4°C, and replace the PBS solution in the beaker every 6 hours during the dialysis period to obtain the purified acid-responsive liposome DGL@ALipo.

[0111] 1.3. Preparation of acid-responsive liposomes DGL@ALipo by reverse evaporation method

[0112] 1) Take 4.5 mg DOPE, 2 mg CHEMS and 5.5 mg DSPE-PEG2000 and dissolve them in 3 mL ether to form an organic phase.

[0113] Meanwhile, 5 mg DGL and 2 mL PBS were mixed to form the aqueous phase.

[0114] 2) Mix the organic phase with the aqueous phase and place the mixed solution in a round-bottom flask. Then place the round-bottom flask in a water bath sonicator, set the frequency of the water bath sonicator to 42kHz, the power of the water bath sonicator to 100W, the water bath temperature to 25°C, and the water bath sonication time to 10min. After the water bath sonication is completed, let the resulting solution stand for 0.5h and observe whether the milky white liquid in the round-bottom flask becomes turbid or precipitated. If not, it means that the oil-in-water emulsion is successfully prepared and the solution system is relatively stable, and the next step can be performed.

[0115] 3) Connect the round-bottom flask containing the water-in-oil emulsion to the rotary evaporator, set the rotary evaporation temperature to 30°C, the rotary evaporation speed to 40 rpm, and the rotary evaporation time to 1 hour. After the rotary evaporation is completed, the organic phase in the water-in-oil emulsion can be completely removed. At this time, add PBS solution into the round-bottom flask to make the total volume of the solution 5 mL.

[0116] 4) The obtained solution was then added into a liposome extruder equipped with a polycarbonate membrane (pore size of 200 nm) and extruded repeatedly 20 times to obtain a DGL@ALipo solution.

[0117] 5) Dialysis purification: Add the obtained DGL@ALipo solution to a dialysis bag (dialysis bag specification: 1000 kDa), seal the open end of the dialysis bag with a dialysis clip, and place the dialysis bag in a beaker filled with PBS solution. Subsequently, place the beaker on a magnetic stirrer, set the speed of the magnetic stirrer to 300 rpm, the stirring time to 24 hours, the ambient temperature to 4°C, and replace the PBS solution in the beaker every 6 hours during the dialysis period to obtain the purified acid-responsive liposome DGL@ALipo.

[0118] At the same time, a control group was set up, and the non-acid-responsive liposomes constructed in the control group were labeled DGL@Lipo.

[0119] The difference between the control group and the acid-responsive liposomes in step 1.3 is that dipalmitoylphosphatidylcholine (DPPC) is used instead of dioleoylphosphatidylethanolamine, DC-cholesterol (DC-cholesterol) is used instead of cholesterol succinate monoester (CHEMS), the molar ratio of dipalmitoylphosphatidylcholine, DC-cholesterol and DSPE-PEG2000 is 20:1:2, and the rest is the same as step 1.3 in this embodiment, and the non-acid-responsive liposomes DGL@Lipo of the control group are obtained for subsequent experimental studies.

[0120] Example 2 Determination of drug loading and encapsulation efficiency of liposomes

[0121] In this Example 2, Cy3-labeled liposomes were used as the object, and the drug loading (Drug loading, DL%) and encapsulation efficiency (Encapsulation efficiency, EE%) of dendritic polylysine (DGL) were investigated by fluorescence quantitative analysis.

[0122] The specific method is as follows:

[0123] 2.1、 Cy3 Preparation of DGL@ALipo

[0124] 1) First, dendritic polylysine (DGL) was labeled with Cy3 fluorescent dye to obtain Cy3-labeled DGL (abbreviated as Cy3 DGL), and then the thin film hydration method of step 1.2 of Example 1 and the reverse evaporation method of step 1.3 were used to load Cy3 DGL gets Cy3 The DGL@ALipo solution was then dialyzed and purified to obtain the purified Cy3-labeled liposomes (abbreviated as Cy3 DGL@ALipo).

[0125] Cy3 The preparation method of DGL is as follows: using the chemical reaction principle of NHS and the amino group of lysine to specifically form an amide bond, the dendritic polylysine (DGL) is labeled with Cy3. The specific preparation method is as follows: take 1 mg of DGL and 1 μg of Cy3-NHS, dissolve them in a solution with a pH of 8.0 (the volume of the solution is 5 mL), and stir overnight in the dark at room temperature. After the reaction is completed, the reaction product is concentrated by ultrafiltration (ultrafiltration tube specifications: 10 kDa, 50 mL; ultrafiltration conditions: 1000 rpm, 30 min, room temperature). After the ultrafiltration is completed, add 2 mL of PBS solution to dissolve it, and the resulting liquid is freeze-dried to obtain Cy3 DGL lyophilized powder.

[0126] 2) Cy3 Determination of DGL mass concentration-fluorescence intensity standard curve

[0127] Pick Cy3 DGL freeze-dried powder was dissolved in deionized water to prepare a 1 g / L standard solution. Cy3 The DGL standard solution was diluted in series. Cy3 The concentration gradient of DGL solution was set to: 1, 2, 5, 10, 20, 50, 100 μg / mL.

[0128] Take 200 μL of the serially diluted Cy3The fluorescence intensity of DGL standard solution can be obtained by reading it with a multifunctional microplate reader. Cy3 DGL mass concentration-fluorescence intensity standard curve.

[0129] 3) Concentration and freeze drying

[0130] Use a 30 kDa ultrafiltration tube to filter the Cy3 DGL@ALipo was concentrated by ultrafiltration (ultrafiltration conditions: 5000 rpm, 30 min, 25°C) and freeze-dried to obtain Cy3 DGL@ALipo lyophilized powder.

[0131] 2.2. Drug loading (DL) determination

[0132] The principle is: ultrasonic destruction Cy3 The outer acid-sensitive phospholipid bilayer (ALipo) of DGL@ALipo makes the Cy3 DGL leaked into the external solution, and the fluorescence intensity of the solution was read using a multifunctional microplate reader to calculate Cy3 Drug loading capacity of DGL@ALipo.

[0133] Weigh 1 mg of each of the Cy3 DGL@ALipo freeze-dried powder was dispersed in 2 mL of deionized water. Then a probe sonicator was used and the sonication time was 10 min at a power of 600 W. 200 μL of the sonicated solution was placed in a black 96-well plate and the fluorescence intensity of the solution in the well plate was read using a multifunctional microplate reader. The fluorescence intensity of the sample was taken into Cy3 DGL concentration-fluorescence intensity standard curve can be calculated to obtain the Cy3 The quality of DGL. The results are shown in Figure 1 .

[0134] in, Cy3 The preparation of DGL concentration-fluorescence intensity standard curve is as follows: Cy3 DGL freeze-dried powder was dissolved in deionized water to prepare a 1 g / L standard solution. Cy3 The DGL standard solution was diluted in a gradient manner as follows: 1, 2, 5, 10, 20, 50, 100 μg / mL, and 200 μL of each solution was taken to obtain the fluorescence intensity and obtain the standard curve.

[0135] Drug loading DL (%) = Cy3 DGL quality / Cy3 DGL@ALipo total mass × 100%

[0136] from Figure 1It can be seen that the drug loading of DGL in the thin film hydration method is about 0.8%, and the drug loading of DGL in the reverse evaporation method is about 12%.

[0137] 2.3. Encapsulation efficiency (EE) determination

[0138] 1) Weigh 1 mg of the Cy3 DGL@ALipo lyophilized powder (not dialyzed and purified before lyophilization to remove free Cy3 DGL) lyophilized powder was dispersed in 1 mL of deionized water to obtain a solution.

[0139] 2) Then, the obtained solution was injected into a G-50 polysaccharide gel separation column for separation, and the free Cy3 DGL solution. In order to accurately collect the free Cy3 DGL solution should be collected in sections when the separation liquid is collected, and a multifunctional ELISA reader should be used for detection and determination.

[0140] 3) The measured Cy3 Substitute the fluorescence intensity of DGL solution into the standard curve and calculate the free Cy3 At the same time, take the same Cy3 DGL@ALipo deionized water solution, using probe ultrasound, Cy3 Loaded in DGL@ALipo Cy3 DGL completely leaked into the solution and Cy3 The total amount of DGL was determined. Figure 1 .

[0141] Encapsulation efficiency EE (%) = (1-free Cy3 DGL quality / Cy3 DGL total mass) × 100%

[0142] from Figure 1 It can be seen that the encapsulation rate of DGL in the thin film hydration method is about 15%, and the encapsulation rate of DGL in the reverse evaporation method is about 90%.

[0143] Example 3 Morphology and structural characterization of liposomes

[0144] In this Example 3, the acid-responsive liposome DGL@ALipo obtained in steps 1.2 and 1.3 of Example 1 was used to characterize the morphology and structure as well as the acid-responsive function.

[0145] 3.1 Characterization of morphology and structure

[0146] 3.1.1 Characterization of particle size and potential

[0147] The particle size and Zeta potential of acid-responsive liposomes DGL@ALipo were characterized using the Malvern Zetasizer Nano ZS laser particle size analyzer. Figure 2 Middle A.

[0148] from Figure 2 As shown in Figure A, the particle size of DGL is about 17nm and the Zeta potential is +59mV. It is generally believed that nanoparticles with small particle size (<50nm) and a large amount of positive charge are easily cleared by the reticuloendothelial system after intravenous injection and are not easy to be enriched in large quantities at the tumor site; the particle size of the non-acid-responsive liposome DGL@Lipo is 150nm and the Zeta potential is +12mV; the particle size of the acid-responsive liposome DGL@ALipo is about 150nm, and the Zeta potential is +8mV, which is a neutral potential. This shows that at this particle size and potential, the acid-responsive liposome DGL@ALipo has a good enhanced permeation and retention effect (EPR) in vivo, can better penetrate into tumor tissues, and retain for a long time.

[0149] 3.1.2 Characterization of morphology

[0150] Transmission electron microscopy was used to characterize the morphology of the acid-responsive liposome DGL@ALipo. Figure 2 Middle B.

[0151] from Figure 2 As shown in Figure B, the acid-responsive liposome DGL@ALipo is a white spherical structure. The particle size of the spherical structure is about 150nm, which is consistent with the particle size data of the acid-responsive liposome DGL@ALipo measured by the Malvern Zetasizer Nano ZS laser particle size analyzer.

[0152] 3.2 Characterization of acid response function

[0153] 3.2.1 Characterization of acid-responsive DGL release from liposomes

[0154] The particle size distribution of acid-responsive liposome DGL@ALipo was studied by dynamic light scattering technology (Malvern Zetasizer Nano ZS laser particle size analyzer) in a simulated tumor acidic environment of pH 6.5 and a normal physiological environment of pH 7.4. At the same time, non-acid-responsive liposome DGL@Lipo was used as a control.

[0155] like Figure 3As shown in the figure, when the acid-responsive liposome DGL@ALipo is in a normal physiological environment of pH 7.4, its particle size is about 150nm, and the particle size distribution presents a single-peak image that is approximately normal distribution; when the acid-responsive liposome DGL@ALipo is in a simulated tumor acidic environment of pH 6.5, it can be seen that in addition to the main peak at 150nm, another peak appears at 20nm, indicating the presence of free DGL.

[0156] At the same time, when the non-acid-responsive liposome DGL@Lipo was placed in a simulated tumor acidic environment of pH 6.5 and a normal physiological environment of pH 7.4, its particle size distribution remained basically unchanged, both of which were a single-peak image at 150nm, which was approximately normal distribution.

[0157] The above results show that in the simulated tumor acidic environment of pH 6.5, the acid-responsive liposome DGL@ALipo can achieve acid-responsive release of DGL.

[0158] 3.2.2 Characterization of acid-responsive DGL release from liposomes

[0159] 1) Prepared Cy3 fluorescent dye labeled Cy3 DGL@ALipo.

[0160] 2) Then, Cy3 DGL@ALipo was placed in PBS solutions of pH 6.5 and pH 7.4 to simulate the acidic environment of tumors, and the released Cy3 DGL was subjected to fluorescence quantitative analysis and the release rate of DGL was calculated. Figure 4 .

[0161] Cy3 DGL release rate (%) = free Cy3 Fluorescence intensity of DGL / total Cy3 Fluorescence intensity of DGL × 100%

[0162] from Figure 4 As shown in Figure A, in the first 12 hours, the acid response Cy3 DGL@ALipo can responsively release nearly 80% of Cy3 DGL, not acid response Cy3 DGL@Lipo can only release less than 10% Cy3 DGL.

[0163] 3.2.3. After the acid-responsive release of DGL from acid-responsive liposomes DGL@ALipo, the penetration of DGL in tumor tissue was investigated

[0164] To mimic in vivo tumor tissue in vitro, a capillary Matrigel diffusion device was designed.

[0165] Figure 4 As shown in the schematic diagram of B, the upper part of the capillary is filled with a physiological saline solution of pH 6.5 to simulate the acidic environment of the tumor; the lower part of the capillary is filled with matrix gel to simulate the environment of the tumor tissue matrix.

[0166] Cy3 fluorescent dye labeled acid response Cy3 DGL@ALipo was added to the capillary matrix gel diffusion device and then observed using a fluorescence microscope at 0, 4, 8, 12, and 24 h. Cy3 The release and diffusion of DGL. Cy3 DGL@Lipo served as the control group.

[0167] from Figure 4 As shown in Figure B, compared with the non-acid-responsive liposome DGL@Lipo, the acid-responsive liposome DGL@ALipo is permeable in the simulated tumor acidic environment at pH 6.5, indicating that DGL@ALipo can easily penetrate into the tumor matrix.

[0168] Example 4 Stability study of liposomes

[0169] In this Example 4, the stability of the acid-responsive liposome DGL@ALipo was studied, including 24h serum stability and 7-day storage stability. The stability evaluation indicators mainly included particle size change, dispersibility change and leakage of the loaded DGL.

[0170] 4.1. Investigation of serum stability

[0171] The acid-responsive liposome DGL@ALipo lyophilized powder was dissolved in a solution containing 10% fetal bovine serum (FBS) to simulate the in vivo blood environment, and incubated on a constant temperature shaker at 37°C for 24 hours. 2 mL of the solution was taken at 0, 1, 2, 4, 8, 12, and 24 hours to measure the particle size, dispersibility, and DGL leakage rate. Figure 6 .

[0172] from Figure 5 As shown in Figure A, in the simulated in vivo blood environment, the particle size of the acid-responsive liposome DGL@ALipo increased slightly (less than 10 nm) within 24 hours, and the dispersion index PDI remained between 0.1 and 0.2, indicating that the acid-responsive liposome DGL@ALipo did not undergo obvious aggregation and had good dispersion.

[0173] from Figure 5 As shown in Figure C, in the simulated in vivo blood environment, the leakage of DGL loaded in the acid-responsive liposome DGL@ALipo was less than 10%.

[0174] 4.2. Investigation of storage environment stability

[0175] The acid-responsive liposome DGL@ALipo freeze-dried powder was dissolved in PBS solution to simulate the storage environment, and the concentration of the acid-responsive liposome DGL@ALipo was 10 g / L. It was stored at 4°C for 7 days, and 1 mL of the solution was taken at 0, 1, 2, 3, 4, 5, 6, and 7 days to measure the particle size, dispersibility, and DGL leakage rate. The results are shown in Figure 5 .

[0176] from Figure 5 As shown in Figure B, in a simulated storage environment, the particle size of the acid-responsive liposome DGL@ALipo increased by less than 10 nm after being stored in PBS for 7 days, and its PDI remained between 0.1 and 0.2, indicating good dispersibility.

[0177] from Figure 5 As shown in Figure 5D, in the simulated storage environment, the leakage of DGL loaded in the acid-responsive liposome DGL@ALipo was also less than 10%.

[0178] In summary, the acid-responsive liposome DGL@ALipo of the present application has good structural stability in a serum environment or a storage environment. The nanostructure of the acid-responsive liposome DGL@ALipo can remain intact and uniform in a normal environment, and the DGL leakage rate of the acid-responsive liposome DGL@ALipo does not exceed 10%.

[0179] Example 5 Investigation of the ability of liposomes to transport cGAMP across cells

[0180] In this Example 5, the co-culture system of colon tumor cells and dendritic cells was used to investigate the ability of the acid-responsive liposome DGL@ALipo to transport cGAMP across cells and its ability to be taken up by dendritic cells.

[0181] 5.1. Investigation of the ability of dendrimer polylysine to capture cGAMP

[0182] Agarose gel electrophoresis migration assay was used to investigate the cGAMP capture ability of liposomes formed by the first-generation dendritic polylysine (G1-DGL, abbreviated as G1), the second-generation dendritic polylysine (G2-DGL, abbreviated as G2), the third-generation dendritic polylysine (G3-DGL, abbreviated as G3), the fourth-generation dendritic polylysine (G4-DGL, abbreviated as G4), and the fifth-generation dendritic polylysine (G5-DGL, abbreviated as G5) respectively encapsulated in the lipid layer of dioleoylphosphatidylethanolamine, cholesterol succinate monoester and distearoylphosphatidylethanolamine.

[0183] The specific experimental method is as follows:

[0184] 1) Take cGAMP labeled with DY547 fluorescent dye ( DY547 cGAMP), mixed with G1-DGL, G2-DGL, G3-DGL, G4-DGL and G5-DGL, and adjusted the mixed solution DY547 The mass concentration ratios of cGAMP and DGL were 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, and 1:10, respectively.

[0185] 2) Then vortex for 10 min in a vortex shaker to DY547 cGAMP and DGL of different generations are fully contacted to obtain a sample solution.

[0186] 3) Place the 7% agarose gel in a horizontal electrophoresis tank, add TBE electrophoresis buffer solution, add 30 μL sample solution, connect the electrophoresis instrument, and perform electrophoresis for 15 minutes at a voltage of 80 mV. After the electrophoresis is completed, remove the agarose gel from the electrophoresis tank and place it in the Odyssey two-color infrared imaging system for imaging. The results are shown in Figure 6 .

[0187] The preparation method of 7% agarose gel is as follows: weigh 4.9g agarose gel powder, disperse it in 70mL TBE electrophoresis buffer solution, heat it to completely melt the agarose gel powder and form a transparent colloid. Then, pour the agarose gel solution into the gel making tank to form a uniform gel layer, and cool and solidify it at room temperature. DY547 cGAMP is inherently fluorescent, so there is no need to add ethidium bromide dye during the preparation of agarose gels.

[0188] from Figure 6 From A, we can see that DY547 When cGAMP is mixed with DGL of different generations, DY547 cGAMP can be captured by DGL; when DY547 When cGAMP was mixed with G1-DGL, G2-DGL, G3-DGL, G4-DGL and G5-DGL at a mass ratio of 1:10, 1:5, 1:2, 1:1 and 2:1, respectively, DY547 cGAMP is completely captured by DGL.

[0189] 5.2. Investigation of the ability of dendrimer poly-lysine to cross colon cancer cells

[0190] DGL labeled with Cy3 fluorescent dye ( Cy3DGL was co-incubated with mouse CT26 colon tumor cells and human COLO205 colon tumor cells for 4 h, and then the culture medium was replaced with fresh medium. At 1, 2, 4, 8, 12, 24, and 48 h, the free Cy3 DGL was quantified and its ability to cross colon tumor cells was investigated.

[0191] from Figure 6 As shown in Figure B, within 48 hours, the contents of G1-DGL, G2-DGL, G3-DGL, G4-DGL and G5-DGL entering the external culture medium from mouse CT26 colon tumor cells were 10%, 20%, 30%, 50% and 70%, respectively.

[0192] from Figure 6 As shown in Figure C, within 48 hours, the contents of G1-DGL, G2-DGL, G3-DGL, G4-DGL and G5-DGL entering the external culture medium from human COLO 205 colon tumor cells were 8%, 12%, 20%, 45% and 60%, respectively.

[0193] 5.3. Investigation of dendritic cell uptake of dendritic poly-lysine

[0194] After investigating the ability of acid-responsive liposome DGL@ALipo to cross colon tumor cells, we further studied the uptake of DGL@ALipo by dendritic cells after crossing colon tumor cells using a co-culture system of mouse CT26 colon tumor cells and mouse bone marrow-derived dendritic cells (BMDC) and a co-culture system of human COLO 205 colon tumor cells and human peripheral blood monocyte-derived dendritic cells (MoDC). Laser confocal microscopy was used for observation.

[0195] from Figure 6 As shown in Figure D, the acid-responsive liposome DGL@ALipo was taken up by mouse bone marrow-derived dendritic cells after crossing over mouse CT26 colon tumor cells.

[0196] from Figure 6 As shown in Figure E, the same cellular uptake phenomenon occurs in human peripheral blood monocyte-derived dendritic cells (MoDC), that is, after crossing human COLO 205 colon tumor cells, G5-DGL loaded by the acid-responsive liposome DGL@ALipo is taken up by human MoDC.

[0197] 5.4. Determination of cGAMP content in dendritic cells

[0198] Using ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry, we further investigated the changes in cGAMP content in dendritic cells after different generations of DGL transported cGAMP across cells.

[0199] from Figure 6 As shown in Figure 1, the cGAMP content in normal mouse BMDC is about 31fg. After treating tumor cells with liposomes formed by G1-DGL, G2-DGL, G3-DGL, G4-DGL and G5-DGL, the cGAMP in the cells were 34fg, 47fg, 66fg, 90fg and 120fg, respectively. In summary, except for G1-DGL, the rest of the generations of DGL will increase the cGAMP content in mouse BMDC after transcellular transport of cGAMP, which can reach 4 times the cGAMP content in normal mouse BMDC.

[0200] Figure 6 It can be seen from Figure 3 that the content of cGAMP in normal human MoDC is about 3fg. After the liposomes formed by G1-DGL, G2-DGL, G3-DGL, G4-DGL and G5-DGL were used to treat tumor cells, the cGAMP in the cells were 3fg, 43fg, 57fg, 73fg and 100fg, respectively, which can reach 33 times the cGAMP content in normal human MoDC.

[0201] In summary, after different generations of DGL transport cGAMP across cells, the cGAMP content in human and mouse dendritic cells will increase, and as the DGL generation increases (the number of grafted lysine increases), its ability to transport cGAMP across cells into dendritic cells becomes stronger.

[0202] Example 6 In vivo anti-tumor study of liposomes

[0203] In this example, an orthotopic animal model of CT26-Luc colon tumor was constructed and used as a research subject. Liposomes were injected into the tumor of the research subject to investigate the inhibitory effect of liposomes on CT26 colon cancer.

[0204] 6.1. Construction of CT26-Luc colon tumor orthotopic animal model

[0205] 1) Obtain CT26 colon tumor cells labeled with luciferase (luciferase-labeled CT26 colon tumor cells are labeled as CT26-Luc colon tumor cells) through market channels.

[0206] 2) 4-5 week old BALB / c mice were anesthetized and inoculated subcutaneously in the groin with 50 μL of CT26-Luc colon tumor cell suspension. The CT26-Luc colon tumor cell suspension was 2×10 7CT26-Luc colon tumor cells were dispersed in 1 mL of a mixed solution of Matrigel and serum-free 1640 culture medium (the volume ratio of Matrigel to serum-free 1640 culture medium was 4:1).

[0207] 3) One week after inoculation, the bioluminescent signals of the tumors in the tumor-bearing mice were detected using the IVIS Spectrum CT small animal imaging system at intervals of 5 days.

[0208] 4) 14 days after inoculation of CT26-Luc colon tumor cells, the bioluminescence signal intensity was selected at 3×10 8 BALB / c tumor-bearing mice with a photons / s were entered into the in vivo pharmacodynamic evaluation experiment.

[0209] 5) Subsequently, the selected BALB / c tumor-bearing mice were randomly divided into 5 groups, 5 mice in each group, namely: ① saline treatment group; ② Lipo liposome treatment group; ③ ALipo liposome treatment group; ④ non-acid responsive liposome DGL@Lipo treatment group; ⑤ acid responsive liposome DGL@ALipo treatment group. The dosage was 30 mg / kg (measured by the mass of the nanosystem freeze-dried powder), the number of administrations was 3 times, and the dosing interval was 2 days.

[0210] Lipo liposome treatment group: DGL was not loaded, that is, DGL was not added in the preparation of the non-acid-responsive liposome DGL@Lipo in Example 1, and an aqueous phase was added to the organic phase formed by DPPC, DC-cholesterol and DSPE-PEG2000, and then evaporated.

[0211] ALipo liposome treatment group: DGL was not loaded, that is, DGL was not added in the reverse evaporation method in step 1.3 of Example 1, and the aqueous phase was added to the organic phase formed by DOPE, CHEMS and DSPE-PEG2000, and then evaporated.

[0212] 6.2 Effects on Tumor Growth

[0213] Tumor growth detection: The time of the first administration was recorded as day 0, and the bioluminescent signal intensity of the CT26-Luc colon tumor was detected every 5 days. The termination time of the CT26-Luc colon tumor growth monitoring experiment was the time when the first BALB / c tumor-bearing mouse in all treatment groups died (i.e., day 36).

[0214] from Figure 7It can be seen that compared with the Saline treatment group, the colon tumor growth rate of the acid-responsive liposome DGL@ALipo treatment group was significantly inhibited; the non-acid-responsive liposome DGL@Lipo treatment group can suppress the growth of colon tumors to a certain extent, but the effect is far weaker than the acid-responsive liposome DGL@ALipo treatment group; the colon tumor growth rate of the non-acid-sensitive Lipo empty liposome treatment group and the acid-sensitive ALipo empty liposome treatment group is almost the same. This means that ALipo and Lipo in the non-acid-responsive liposome DGL@Lipo and acid-responsive liposome DGL@ALipo have no inhibitory effect on the growth of CT26-Luc colon tumors, but the addition of DGL can significantly inhibit the growth of CT26-Luc colon tumors.

[0215] 6.3 Survival monitoring of BALB / c tumor-bearing mice

[0216] Survival monitoring of BALB / c tumor-bearing mice: The time of the first administration was recorded as day 0. The termination time of the survival monitoring experiment of BALB / c tumor-bearing mice was the 90th day of the experiment.

[0217] from Figure 8 It can be seen that the median survival time of tumor-bearing mice in the acid-responsive liposome DGL@ALipo treatment group was 78 days; the median survival time of the Saline treatment group was only 42 days; the median survival time of tumor-bearing mice in the non-acid-responsive liposome DGL@Lipo treatment group was 52 days; the median survival time of BALB / c tumor-bearing mice in the Lipo treatment group and the ALipo treatment group were 44 days and 41 days, respectively.

[0218] In summary, the liposomes of the present application can effectively inhibit the growth of mouse colon tumor cells, inhibit the metastasis of mouse colon tumor cells, and prolong the survival of mice.

[0219] The acid-responsive liposome DGL@ALipo of the present invention has a particle size of about 150nm and a neutral potential, can penetrate into tumor tissue in vivo, and remain for a long time; it has acid response and is easy to penetrate in the tumor matrix, can release 80% of DGL under acid response in the first 12 hours, and has good structural stability when stored in a serum environment or a storage environment for 7 days

[0220] The present invention also investigates the ability of the acid-responsive liposome DGL@ALipo to transport cGAMP and be taken up by dendritic cells in mouse co-culture systems and human co-culture systems, respectively, and finds that cGAMP can be completely captured by DGL of different generations; and as the generation increases, the ability to cross the membrane from colon tumor cells is enhanced; and after crossing the membrane of colon tumor cells, it can be taken up by mouse or human dendritic cells, and the cGAMP content in dendritic cells increases with the increase in DGL generation, the cGAMP content in human dendritic cells can be as high as 33 times higher than normal, and the cGAMP content in mouse dendritic cells can be as high as 4 times higher than normal.

[0221] The present application found that there was no difference in the growth of CT26 in situ colon tumors between the non-acid-sensitive Lipo empty liposome treatment group and the acid-sensitive ALipo empty liposome treatment group, while the colon tumor growth rate of the acid-responsive DGL@ALipo treatment group was significantly inhibited and due to the non-acid-responsive DGL@Lipo treatment group; it was also found that only a small number of mesenteric lymph nodes in the acid-responsive DGL@ALipo treatment group had metastasis of CT26-Luc colon tumor cells, while no metastasis occurred to distant major organs such as the heart, liver, spleen, lungs, and kidneys; in addition, compared with the Saline treatment group, the median survival time of the acid-responsive DGL@ALipo treatment group was extended from 42 days to 78 days.

[0222] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limitations of the present invention. In addition, the various modifications listed herein and the variations of methods and compositions in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications obvious to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A liposome for capturing cGAMP, characterized in that: The liposome comprises a liposome membrane and dendritic polylysine encapsulated in the liposome membrane, and the mass ratio of the raw material of the liposome membrane to the dendritic polylysine is (1-100):

1.

2. The liposome according to claim 1, wherein The dendritic polylysine is selected from one or more of the following: G1 generation dendritic polylysine, G2 generation dendritic polylysine, G3 generation dendritic polylysine, G4 generation dendritic polylysine and G5 generation dendritic polylysine; and / or, the raw materials of the liposome membrane include phospholipids, cholesterol and long-circulating materials; Preferably, the phospholipid is selected from one or both of dioleoylphosphatidylethanolamine and dipalmitoylphosphatidylcholine; Preferably, the cholesterol is selected from one or both of cholesterol succinate and DC-cholesterol; Preferably, the long-circulating material is selected from polyethylene glycol and its derivatives, and the PEG derivative is selected from one or more of polyethylene glycol-vitamin E succinate, polyethylene glycol-cholesterol, polyethylene glycol-modified distearoylphosphatidylethanolamine, polyethylene glycol-modified dimyristoylphosphatidylethanolamine, and polyethylene glycol-modified dipalmitoylphosphatidylethanolamine.

3. The liposome according to claim 2, wherein The mass ratio of the phospholipid, cholesterol, long-circulation material and dendritic polylysine is (2-4.5):(1.5-2):(5.5-8):

5.

4. The liposome according to claim 1, wherein The drug loading amount of the dendritic polylysine in the liposome is 0.3-17%; and / or, the encapsulation efficiency of the dendritic polylysine in the liposome is 10 to 95%; And / or, the particle size of the liposome is 100-200 nm.

5. The method for preparing liposomes according to any one of claims 1 to 4, characterized in that: The raw material of liposome membrane is dissolved in organic solvent, and colostrum is formed with dendritic polylysine solution by thin film hydration method, detergent dialysis method, reverse evaporation method or solvent injection method, and granulated to obtain the liposome.

6. The preparation method according to claim 5, characterized in that: The film hydration method comprises the following steps: phospholipid, cholesterol, long-circulation material and organic solvent are mixed, evaporated to obtain a film, a dendritic polylysine solution is added to the film, hydrated and granulated to obtain the liposome.

7. The preparation method according to claim 6, characterized in that: Includes one or more of the following features: B1) the organic solvent is selected from one or more of chloroform, methanol, dichloromethane, acetonitrile, anhydrous ethanol and acetone; B2) the mass volume ratio of the total mass of phospholipids, cholesterol and long-circulating materials to the mass volume of the organic solvent is (7-17) mg: 4 mL; B3) the dendritic poly-lysine solution is a mixture of dendritic poly-lysine and PBS solution; B4) based on the volume of the dendritic poly-lysine solution, the concentration of the dendritic poly-lysine is 50 to 150 mg / L; B5) the evaporation temperature is 40 to 80°C; B6) the hydration temperature is 50-80°C; B7) The hydration time is 0.2 to 0.8 h.

8. The preparation method according to claim 5, characterized in that: The reverse evaporation method comprises the following steps: mixing phospholipids, cholesterol, long-circulation materials and organic solvents, adding dendritic polylysine solution to form colostrum, evaporating, granulating and obtaining the liposomes.

9. The preparation method according to claim 8, characterized in that: Includes one or more of the following: C1) the organic solvent is ether; C2) the mass volume ratio of the total mass of phospholipids, cholesterol and long-circulating materials to the mass volume ratio of the organic solvent is (7-17) mg: 3 mL; C3) the dendritic polylysine solution is a mixture of dendritic polylysine and PBS solution; preferably, the mass volume ratio of the dendritic polylysine and PBS solution is (1-10) mg: 2 mL; C4) The evaporation temperature is 20-80°C.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the liposome according to any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

11. Use of the liposome according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 10 in preparing a product for preventing or treating a disease associated with STING.

12. Use of the liposome according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 10 for capturing cyclic GMP-AMP in vitro, promoting transmembrane transport of cyclic GMP-AMP or promoting maturation of dendritic cells.

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