Ultrasonic activated liposome for regulating ATP-ADO axis to relieve immunosuppression and application of ultrasonic activated liposome

Abstract

The invention discloses an ultrasonic activated liposome for regulating an ATP-ADO axis to relieve immunosuppression and application of the ultrasonic activated liposome, and belongs to the technical field of biological medicines. The liposome is formed by self-assembly of lipid, a sound-sensitive agent and an active oxygen response type CD39 inhibitor prodrug. According to the invention, the sonosensitizer capable of generating ROS and the ROS responsively released excision nucleotidase inhibitor prodrug are utilized to synthesize the ultrasonic activation type cascade drug release liposome for regulating the ATP-ADO axis to relieve immunosuppression, and only after the liposome is subjected to ultrasonic irradiation, the sonosensitizer can generate active oxygen and further activate the prodrug to release the excision nucleotidase inhibitor, so that the effect of relieving immunosuppression is achieved. According to the present invention, the tumor cell ICD is induced while the ATP level with immune activation is improved by adjusting the ATP-ADO axis, and the ADO accumulation with immunosuppression is reduced so as to achieve the dual anti-tumor immune effect; targeted external toxic and side effects caused by the excision nucleotidase inhibitor are avoided in an ultrasonic triggering mode.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to an ultrasound-activated liposome capable of regulating the ATP-ADO axis to relieve immunosuppression and an application thereof. Background Art

[0002] Immunotherapy, especially immune checkpoint inhibitors (ICIs), has made significant progress in the application of various tumor types. However, the efficacy of immunosuppressive tumors is limited by the low immunogenicity of tumors, insufficient infiltration of T lymphocytes into tumors, and immunosuppressive tumor microenvironment. Single immunotherapy is only effective in about a quarter of tumor patients.

[0003] Sonodynamic therapy (SDT) is an emerging non-invasive tumor treatment method that uses sonosensitizers to produce reactive oxygen species (ROS) under the action of ultrasound to induce immunogenic death of tumor cells. Compared with traditional photodynamic therapy, SDT has the advantages of strong penetration, precise positioning, and little damage to surrounding normal tissues. In the tumor microenvironment, the endoplasmic reticulum (ER) is an important organelle that participates in protein synthesis, folding and transport, and plays a key role in the process of tumor cells responding to internal and external stress. Sonodynamic therapy causes endoplasmic reticulum stress, which can effectively destroy the protein homeostasis of tumor cells and induce cell apoptosis and immune response.

[0004] Immunogenic cell death (ICD) refers to a specific type of cell death characterized by the release of damage-associated molecular patterns (DAMPs), including high-mobility group protein B-1 (HMGB-1), calreticulin (CRT), and ATP. In previous studies, anticancer therapy focused on stimulating ICD in tumor cells, but ignored the fact that ATP produced during ICD is rapidly converted into the immunosuppressive metabolite adenosine (ADO) by exonucleases, exacerbating microenvironmental immunosuppression. Studies have shown that high levels of adenosine are associated with increased expression of the immune checkpoint molecule PD-L1 on tumor cells, which infers that adenosine is one of the key inducers of poor response to immune checkpoint inhibitors. Therefore, the ATP-ADO axis is regarded as a regulatory pathway for the immune response, which is the basis for the production of adenosine to promote tumor development and induce the failure of immunotherapy treatment.

[0005] CD39 is the top rate-limiting enzyme that catalyzes the degradation of ATP to adenosine. FPL 67156 is a nucleotide derivative that has a good inhibitory effect on the enzymatic activity of CD39, but its short half-life in the blood circulation prevents them from accumulating to effective concentrations in tumors and is prone to off-target toxicity.

[0006] In order to solve the above problems, it is crucial to prepare a drug delivery system that can not only activate tumor cells to undergo ICD but also overcome adenosine-mediated immunosuppression to maintain a lasting anti-tumor immune effect, which is crucial to improving the immunotherapy efficacy of immunosuppressive tumors. Summary of the invention

[0007] The purpose of the present invention is to provide a nano-delivery system that can induce immunogenic cell death in tumors and overcome adenosine-mediated immunosuppression to achieve a lasting anti-tumor immune effect.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] The invention provides an ultrasound-activated liposome for regulating the ATP-ADO axis to relieve immunosuppression. The liposome is formed by self-assembly of a lipid, a sonosensitizer and an active oxygen responsive CD39 inhibitor prodrug.

[0010] The lipid is a basic raw material for forming liposomes, including phospholipids, polyethylene glycol functionalized lipids, and cholesterol. The phospholipids may be, but are not limited to, distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylethanolamine (DOPE), and the polyethylene glycol functionalized lipid may be, but are not limited to, distearoylphosphatidylcholine-polyethylene glycol 2000 (DSPC-PEG2000) or distearoylphosphatidylcholine-polyethylene glycol 2000-cyclic RGD peptide (DSPC-PEG2000-cRGD).

[0011] The sonosensitizer is an organic small molecule that can generate reactive oxygen species (ROS) upon ultrasound irradiation, thereby causing immunogenic cell death (ICD). The sonosensitizer may be, but is not limited to, a porphyrin sonosensitizer or a phthalocyanine sonosensitizer.

[0012] The reactive oxygen species responsive CD39 inhibitor prodrug is prepared by modifying the CD39 inhibitor with a ROS responsive group. The prodrug can release a CD39 inhibitor with biological activity under the action of reactive oxygen species (ROS) to inhibit the enzyme activity of CD39, the apical rate-limiting enzyme of the ATP-ADO axis.

[0013] The ultrasound-activated liposomes provided by the present invention that regulate the ATP-ADO axis to relieve immunosuppression act in the following manner: due to the enhanced permeability and retention of tumor tissues, nano-sized liposomes can be effectively enriched in tumors and internalized by cancer cells. Under ultrasound irradiation, the sonosensitizer produces a large amount of intracellular ROS, a portion of which causes endoplasmic reticulum stress and produces immunogenic cell death (ICD), and a portion of which triggers the release of CD39 inhibitors by reactive oxygen species-responsive CD39 inhibitor prodrugs, thereby regulating the ATP-ADO axis, increasing the level of ATP with immunoactivation, and reducing the accumulation of ADO with immunosuppression, thereby achieving a dual anti-tumor immune effect.

[0014] Preferably, the reactive oxygen species-responsive CD39 inhibitor prodrug is prepared by modifying the catechol site of FPL 67156 with a boronate group.

[0015] As a nucleotide derivative, FPL 67156 has a good inhibitory effect on the enzymatic activity of CD39. Other small molecule inhibitors that can be modified with boronate groups are also suitable for the present invention.

[0016] More preferably, the reactive oxygen species responsive CD39 inhibitor prodrug is prepared by forming a reversible covalent ester bond and positive and negative charge interactions between polybromo(2-acryloyloxy)ethyl(p-boronic acid benzyl)diethylammonium (B-PDEAEA) and FPL 67156. B-PDEAEA forms a reversible covalent ester with 1,2- or 1,3-cis diol on FPL 67156.

[0017] Preferably, the preparation method of the reactive oxygen species responsive CD39 inhibitor prodrug comprises: mixing B-PDEAEA and FPL 67156 in 4-hydroxyethylpiperazineethanesulfonic acid buffer at an N / P ratio of 9-50, and self-assembling B-PDEAEA and FPL 67156 to form a B-PDEAEA / FPL67156 nanopolymer through interactions such as phenyl borate bonds and positive and negative charge attraction.

[0018] The N / P ratio refers to the molar ratio of B-PDEAEA in terms of N to FPL 67156 in terms of P. Studies have shown that when N / P=9, the size and distribution of the formed nanopolymers are the most stable.

[0019] Preferably, the sonosensitizer is synthesized from protoporphyrin IX, 4-methylbenzenesulfonylurea, BOP reagent and triethylamine amide, and the sonosensitizer material has endoplasmic reticulum targeting effect; or the sonosensitizer is protoporphyrin IX. The sonosensitizer applicable to the present invention is not limited thereto.

[0020] Preferably, the liposome is synthesized by thin film hydration method from the reactive oxygen species responsive CD39 inhibitor prodrug and the lipid membrane loaded with sonosensitizer. The method applicable to the present invention is not limited thereto.

[0021] Preferably, the lipid film is obtained by mixing and dissolving DSPE-PEG2000, DSPE-PEG2000-cRGD, DOPE, cholesterol and sonosensitizer in an organic solvent, and then rotary evaporating.

[0022] Preferably, the mass ratio of DSPE-PEG2000, DSPE-PEG2000-cRGD, DOPE, cholesterol and sonosensitizer is 1:1.1:3.16:0.54:0.6.

[0023] When preparing the drug-loaded liposomes, the dosage ratio of the CD39 inhibitor and the sonosensitizer is adjusted according to the effective concentration and body tolerance. Preferably, the mass ratio of the prodrug in terms of FPL 67156 to the lipid membrane in terms of sonosensitizer is 1:2-3.

[0024] The present invention also provides the use of the ultrasound-activated liposome for regulating the ATP-ADO axis to relieve immunosuppression in the preparation of anti-tumor drugs.

[0025] The liposomes are enriched in tumor tissues, and stimulate the tumor to generate ICD after ultrasound irradiation, while releasing CD39 inhibitors, thereby overcoming adenosine-mediated immunosuppression by regulating the ATP-ADO axis.

[0026] Specifically, the tumor is a solid tumor. After the liposomes accumulate in the tumor and are internalized into the tumor cells, they are combined with ultrasound irradiation to achieve the purpose of tumor treatment. The conditions of the ultrasound irradiation can be changed according to the physical condition of the subject to be treated, so as to achieve the activation of the sonosensitizer and then react to produce reactive oxygen.

[0027] Furthermore, the tumor is an immunosuppressive tumor, such as triple-negative breast cancer, pancreatic cancer and other solid tumors.

[0028] The present invention has the following beneficial effects:

[0029] The present invention utilizes a sonosensitizer that can generate ROS and an exonucleotidase inhibitor prodrug that is released in response to ROS to synthesize an ultrasound-activated cascade drug-releasing liposome that regulates the ATP-ADO axis to relieve immunosuppression, thereby significantly improving the immunotherapy effect and avoiding obvious toxic side effects. Only after the liposome is irradiated with ultrasound, the sonosensitizer can generate reactive oxygen and further activate the prodrug, releasing the exonucleotidase inhibitor, inducing the ICD of tumor cells while regulating the ATP-ADO axis to increase the level of ATP with immune activation and reduce the accumulation of ADO with immunosuppression, thereby achieving a dual anti-tumor immune effect. The targeted external toxic side effects caused by the exonucleotidase inhibitor are avoided by ultrasound triggering. The preparation method of the present invention is simple, the reaction conditions are mild, and it has great potential in the field of tumor drug preparation and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the preparation of B-PDEAEA / FPL 67156 nanopolymers and the principle of ROS-responsive activation.

[0031] Figure 2 This is the particle size diagram of BFPL with an N / P ratio of 9 in Example 1.

[0032] Figure 3 This is a graph showing the generation of ROS after cells are incubated with nanomedicine under ultrasound in Example 1.

[0033] Figure 4 This is a graph showing the survival rate of cells after incubation with nanomedicine under ultrasound in Example 1.

[0034] Figure 5 It is the relative content of ATP in the cell supernatant after the cells are incubated with the nanodrug under ultrasound in Example 1.

[0035] Figure 6 It is the content of ADO in the cell supernatant after the cells are incubated with nano-drugs under ultrasound in Example 1.

[0036] Figure 7 The in vivo tumor inhibition effect of the ultrasound-loaded drug-loaded liposomes in Example 1 in a mouse breast cancer transplant tumor model.

[0037] Figure 8 These are H&E staining images of the main organs (heart, liver, spleen, lung, and kidney) of Balb / C mice after treatment with drug-loaded liposome drugs in Example 1.

[0038] Fig. 9 This is the particle size diagram of BFPL with an N / P ratio of 13 in Example 2.

[0039] Fig.10It is the relative content of ATP in the cell supernatant after the cells are incubated with nanomedicine under ultrasound in Example 2.

[0040] Fig.11 This is the particle size diagram of BFPL with an N / P ratio of 20 in Example 3.

[0041] Fig.12 It is the relative content of ATP in the cell supernatant after the cells are incubated with nanomedicine under ultrasound in Example 3.

[0042] Fig.13 This is the particle size diagram of BFPL with an N / P ratio of 30 in Example 4.

[0043] Fig.14 It is the relative content of ATP in the cell supernatant after the cells are incubated with nanomedicine under ultrasound in Example 4.

[0044] Fig.15 This is the particle size diagram of BFPL with an N / P ratio of 50 in Example 5.

[0045] Fig.16 It is the relative content of ATP in the cell supernatant after the cells are incubated with nanomedicine under ultrasound in Example 5.

[0046] Fig.17 It is the relative content of ATP in the cell supernatant after the cells are incubated with nanomedicine under ultrasound in Example 6. DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0048] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0049] The FPL 67156 trisodium used in the following examples was purchased from MCE. Protoporphyrin IX, 4-methylbenzenesulfonylurea and BOP reagent were purchased from Macklin. DSPE-PEG2000, DSPE-PEG2000-cRGD, DOPE and cholesterol were purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0050] 4T1-Luci cells were purchased from ATCC cell bank.

[0051] B-PDEAEA was synthesized according to the method described in the literature (Liu, X., et al. Fusogenic Reactive Oxygen Species Triggered Charge-Reversal Vector for Effective Gene Delivery. Advanced Materials, 2015, 28(9): 1743-1752.), and its structural formula is shown in formula (I), where n represents the degree of polymerization, n=27, 39, 60, 150;

[0052]

[0053] Example 1

[0054] 1. Preparation method of drug-loaded liposome BFPL:

[0055] Preparation of exonuclease inhibitor prodrug: Take the exonuclease inhibitor FPL 67156 and dilute it to 2.5 mL (the final concentration of FPL 67156 is 40 μg / mL) with 4-hydroxyethylpiperazineethanesulfonic acid buffer (HEPES, pH 7.4, 10 mM), and mix it with 2.5 mL of B-PDEAEA working solution (the final concentration of B-PDEAEA is 248.7 μg / mL, the degree of polymerization n is 27, then the N / P molar ratio is 9, N corresponds to the nitrogen element in B-PDEAEA, and P corresponds to the phosphorus element in FPL 67156). Then transfer the mixture to a centrifuge tube and immediately vortex for 5 minutes to ensure complete cross-linking, and prepare B-PDEAEA / FPL 67156 nanopolymer. The reaction equation is as follows: Figure 1 shown.

[0056] Preparation of ER-targeted sonosensitizer PMPS: 4-Methylbenzenesulfonylurea (MPSU, 85.7 mg, 0.4 mmol), BOP (176.9 mg, 0.8 mmol) and triethylamine (TEA, 55.6 μL, 0.4 mmol) were dissolved in 5 mL of dichloromethane (DCM) and the solution was added dropwise to protoporphyrin IX (56.3 mg, 0.1 mmol). Stir at room temperature for 4 hours in 5 mL of DCM. The DCM solution was rotary evaporated and the PMPS product was washed three times with 0.1 M NaOH solution (for removing protoporphyrin IX) and acetone (for removing BOP, TEA and MPSU).

[0057] Preparation of lipid membrane loaded with ER-targeted sonosensitizer PMPS: lipid compounds DSPE-PEG2000, DSPE-PEG2000-cRGD, DOPE, cholesterol (1 mg, 1.1 mg, 3.16 mg, and 0.54 mg, respectively) and PMPS (0.6 mg) were mixed and dissolved in 5 mL CHCl 3 The solution was poured into a 25 mL round-bottom flask and rotary evaporated to form a lipid film.

[0058] Preparation of drug-loaded liposomes BFPL: Transfer the B-PDEAEA / FPL 67156 liquid to the above round-bottom flask and stir it magnetically overnight to obtain the final product BFPL drug-loaded liposomes.

[0059] When preparing drug-loaded liposomes, the dosage of prodrug and lipid membrane is adjusted according to the target of action. Specifically, at the cellular level, the effective concentration of FPL 67156 is 8μg / mL, and the maximum dose of PMPS cannot exceed 25μg / mL; at the animal level, such as in the mouse model, the effective dose of FPL 67156 is 5mg / kg, and the maximum dose of PMPS is 10mg / kg.

[0060] 2. Particle size characterization of drug-loaded liposomes

[0061] Take 1 mL of the prepared BFPL sample and place it in a special sample cell of the particle size analyzer. Use the particle size analyzer to perform a dynamic light scattering test to measure the hydration kinetic particle size and particle size distribution of the nanoparticles. The experiment is repeated three times to take the average value.

[0062] like Figure 2 As shown, when N / P=9, the size and distribution of B-PDEAEA / FPL 67156 nanopolymers are most stable, and the average particle size of the prepared BFPL is about 113.6 nm.

[0063] 3. Verify the effect of drug-loaded liposomes in producing ROS and inducing tumor cell death at the cellular level.

[0064] 40,000 4T1 breast cancer cells were seeded in each well of a 24-well plate, and BFPL was added. The concentration of FPL 67156 in the reaction system reached 8 μg / mL, and the concentration of PMPS was 24 μg / mL. After incubation for 24 hours, ultrasonic irradiation (3 MHz, 1.0 W / cm 2 , 50% duty cycle, time 1 min). ROS signal was detected by cell ROS kit.

[0065] like Figure 3 As shown, after incubation with drug-loaded liposomes and ultrasound irradiation, the intracellular ROS fluorescence intensity increased by about 8 times, while no ROS signal was detected in cells without ultrasound irradiation.

[0066] 5000 4T1 breast cancer cells were inoculated in each well of a 96-well plate, and the above drugs were added. After incubation for 24 hours, ultrasound irradiation (3 MHz, 1.0 W / cm 2 , 50% duty cycle, time 1 min). Cytotoxicity was detected by CCK8 kit.

[0067] like Figure 4 As shown, after incubation with nanomedicine and ultrasound irradiation, the cell survival rate was only 34%, indicating that a large number of tumor cells died.

[0068] The above results indicate that drug-loaded liposomes can produce a large amount of ROS and cause tumor cell death after ultrasound irradiation.

[0069] 4. Verify the content of extracellular ATP and ADO at the cellular level

[0070] 80,000 4T1 breast cancer cells were seeded in each well of a 12-well plate, and the above drugs were added. After incubation for 24 hours, the cells were irradiated with ultrasound (3 MHz, 1.0 W / cm 2 , 50% duty cycle, time 1 min). The ATP content in the cell supernatant was detected by an ATP enhancement test kit.

[0071] like Figure 5 As shown, after incubation with nanodrugs and ultrasound irradiation, the extracellular ATP content was significantly higher than that in the control group.

[0072] The same experimental steps as above were followed to detect the ADO content in the cell supernatant using an adenosine assay kit.

[0073] like Figure 6 As shown, after incubation with nanodrugs and ultrasound irradiation, the extracellular ADO content was significantly lower than that in the control group.

[0074] 5. Verification of the anti-tumor efficacy of liposomes at the experimental animal level

[0075] 4T1 tumor cells (1 million tumor cells per mouse) were inoculated into Balb / C mice orthotopically until the tumor volume reached 70 mm 3 The mice were divided into 3 groups, 5 mice in each group, and injected with PBS, FPL 67156 (effective dose 5 mg / kg) and drug-loaded liposome BFPL (FPL 67156 effective dose 5 mg / kg, PMPS maximum dose 10 mg / kg) through the tail vein. After 6 hours, they were irradiated with ultrasound (frequency 3 MHz, duty cycle 50%, intensity 2.0 W / cm 2 , time 10min), and the tumor volume of mice was measured every two days.

[0076] like Figure 7 As shown, compared with the control group (PBS and FPL 67156 group), the tumor growth of the BAPL+US experimental group was significantly inhibited, indicating that the nanomaterial can improve the tumor treatment effect by ultrasound triggering.

[0077] 6. Safety Assessment

[0078] The drug-loaded liposome BFPL was injected into healthy ICR mice through the tail vein (FPL 67156 5 mg / kg, PMPS 10 mg / kg). Three days later, the mice were killed in accordance with animal ethics, and the main organs (heart, liver, spleen, lung, and kidney) were dissected and characterized by biosafety using H&E staining.

[0079] like Figure 8 As shown, after the mice were injected with nanomaterials via the tail vein, no obvious tissue damage was found in their major organs (heart, liver, spleen, lungs, and kidneys), proving the biosafety of drug-loaded liposomes to normal tissues.

[0080] Example 2

[0081] 1. Preparation method of drug-loaded liposome BFPL:

[0082] The exonuclease inhibitor FPL 67156 was diluted to 2.5 mL (FPL67156 final concentration was 40 μg / mL) by HEPES (pH 7.4, 10 mM) and mixed with 2.5 mL B-PDEAEA working solution (359.2 μg / mL, degree of polymerization n was 39, N / P=13). The mixture was then transferred to a centrifuge tube and immediately vortexed for 5 minutes to ensure complete cross-linking, to obtain B-PDEAEA / FPL 67156 nanopolymers.

[0083] The preparation method of the lipid membrane loaded with the ER targeting sonosensitizer PMPS is the same as that in Example 1.

[0084] The B-PDEAEA / FPL 67156 liquid was transferred into the lipid membrane and magnetically stirred overnight to obtain the final product, BFPL drug-loaded liposomes.

[0085] 2. Particle size characterization of drug-loaded liposomes

[0086] The test method is the same as in Example 1, and the results are as follows Fig. 9 As shown, the average particle size of the nano drug prepared in this example is about 182.4 nm.

[0087] 3. Verify the extracellular ATP content at the cellular level

[0088] The test method is the same as in Example 1, and the results are as follows Fig.10As shown, after incubation with nanodrugs and ultrasound irradiation, the extracellular ATP content was significantly higher than that in the control group.

[0089] Example 3

[0090] 1. Preparation method of drug-loaded liposome BFPL:

[0091] The exonuclease inhibitor FPL 67156 was diluted to 2.5 mL (FPL67156 final concentration was 40 μg / mL) by HEPES (pH 7.4, 10 mM) and mixed with 2.5 mL B-PDEAEA working solution (552.7 μg / mL, degree of polymerization n was 60, N / P = 20). The mixture was then transferred to a centrifuge tube and immediately vortexed for 5 minutes to ensure complete cross-linking, to obtain B-PDEAEA / FPL 67156 nanopolymers.

[0092] The preparation method of the lipid membrane loaded with the ER targeting sonosensitizer PMPS is the same as that in Example 1.

[0093] The B-PDEAEA / FPL 67156 liquid was transferred into the above lipid membrane and magnetically stirred overnight to obtain the final product, BFPL drug-loaded liposomes.

[0094] 2. Particle size characterization of drug-loaded liposomes

[0095] The test method is the same as in Example 1, and the results are as follows Fig.11 As shown, the average particle size of the nano drug prepared in this example is about 183.0 nm.

[0096] 3. Verify the extracellular ATP content at the cellular level

[0097] The test method is the same as in Example 1, and the results are as follows Fig.12 As shown, after incubation with nanodrugs and ultrasound irradiation, the extracellular ATP content was significantly higher than that in the control group.

[0098] Example 4

[0099] 1. Preparation method of drug-loaded liposome BFPL:

[0100] The exonuclease inhibitor FPL 67156 was diluted to 2.5 mL (FPL67156 final concentration was 40 μg / mL) by HEPES (pH 7.4, 10 mM) and mixed with 2.5 mL B-PDEAEA working solution (829.0 μg / mL, degree of polymerization n was 90, N / P=30). The mixture was then transferred to a centrifuge tube and immediately vortexed for 5 minutes to ensure complete cross-linking, to obtain B-PDEAEA / FPL 67156 nanopolymers.

[0101] The method for preparing the lipid membrane loaded with the ER targeting sonosensitizer PMPS is the same as that in Example 1.

[0102] The B-PDEAEA / FPL 67156 liquid was transferred into the above lipid membrane and magnetically stirred overnight to obtain the final product, BFPL drug-loaded liposomes.

[0103] 2. Particle size characterization of drug-loaded liposomes

[0104] The test method is the same as in Example 1, and the results are as follows Fig.13 As shown, the average particle size of the nano drug prepared in this example is about 164.7 nm.

[0105] 3. Verify the extracellular ATP content at the cellular level

[0106] The test method is the same as in Example 1, and the results are as follows Fig.14 As shown, after incubation with nanodrugs and ultrasound irradiation, the extracellular ATP content was significantly higher than that in the control group.

[0107] Example 5

[0108] 1. Preparation method of drug-loaded liposome BFPL:

[0109] The exonuclease inhibitor FPL 67156 was diluted to 2.5 mL (the final concentration of FPL67156 was 40 μg / mL) by HEPES (pH 7.4, 10 mM) and mixed with 2.5 mL of B-PDEAEA working solution (1381.7 μg / mL, degree of polymerization n was 150, N / P=50). The mixture was then transferred to a centrifuge tube and immediately vortexed for 5 minutes to ensure complete cross-linking, and B-PDEAEA / FPL 67156 nanopolymers were prepared.

[0110] The method for preparing the lipid membrane loaded with the ER targeting sonosensitizer PMPS is the same as that in Example 1.

[0111] The B-PDEAEA / FPL 67156 liquid was transferred into the above lipid membrane and magnetically stirred overnight to obtain the final product, BFPL drug-loaded liposomes.

[0112] 2. Particle size characterization of drug-loaded liposomes

[0113] The test method is the same as in Example 1, and the results are as follows Fig.15 As shown, the average particle size of the nano drug prepared in this example is about 152.7 nm.

[0114] 3. Verify the extracellular ATP content at the cellular level

[0115] The test method is the same as in Example 1, and the results are as follows Fig.16As shown, after incubation with nanodrugs and ultrasound irradiation, the extracellular ATP content was significantly higher than that in the control group.

[0116] Example 6

[0117] 1. Preparation method of drug-loaded liposome BFPL:

[0118] The preparation method of B-PDEAEA / FPL 67156 nanopolymer is the same as that in Example 1.

[0119] The lipid compounds DSPE-PEG2000, DSPE-PEG2000-cRGD, DOPE, cholesterol (1 mg, 1.1 mg, 3.16 mg and 0.54 mg, respectively) and PPIX (0.6 mg) were mixed and dissolved in 5 mL CHCl 3 The solution was poured into a 25 mL round-bottom flask and rotary evaporated to form a lipid film.

[0120] The B-PDEAEA / FPL 67156 liquid was transferred to the above round-bottom flask and magnetically stirred overnight to obtain the final product, BFPL drug-loaded liposomes.

[0121] 2. Particle size characterization of drug-loaded liposomes

[0122] The testing method is the same as that in Example 1. The average particle size of the nano drug prepared in this example is about 112.7 nm.

[0123] 3. Verify the relative content of extracellular ATP at the cellular level

[0124] The test method is the same as in Example 1, and the results are as follows Fig.17 As shown, after incubation with nanodrugs and ultrasound irradiation, the extracellular ATP content was significantly higher than that in the control group.

[0125] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. An ultrasound-activated liposome that regulates the ATP-ADO axis to relieve immunosuppression, characterized in that: The liposome is formed by self-assembly of lipids, sonosensitizer and active oxygen responsive CD39 inhibitor prodrug.

2. The ultrasound-activated liposome for regulating the ATP-ADO axis to relieve immunosuppression according to claim 1, characterized in that: The reactive oxygen species responsive CD39 inhibitor prodrug is prepared by modifying the catechol site of FPL 67156 with a boronate group.

3. The ultrasound-activated liposome for regulating the ATP-ADO axis to relieve immunosuppression according to claim 2, characterized in that: The active oxygen responsive CD39 inhibitor prodrug is prepared by forming a reversible covalent ester bond and positive and negative charge interactions between polybromo(2-acryloyloxy)ethyl(p-boronic acid benzyl)diethylammonium and FPL 67156.

4. The ultrasound-activated liposome for regulating the ATP-ADO axis to relieve immunosuppression according to claim 3, characterized in that: The preparation method of the active oxygen responsive CD39 inhibitor prodrug comprises: mixing polybromo(2-acryloyloxy)ethyl(p-boronic acid benzyl)diethylammonium and FPL 67156 in a 4-hydroxyethylpiperazineethanesulfonic acid buffer according to an N / P ratio of 9-50, and self-assembling to form a B-PDEAEA / FPL67156 nanopolymer.

5. The ultrasound-activated liposome for regulating ATP-ADO axis and relieving immunosuppression according to claim 1, characterized in that: The sonosensitizer is synthesized by amidation of protoporphyrin IX, 4-methylbenzenesulfonylurea, BOP reagent and triethylamine; or the sonosensitizer is protoporphyrin IX.

6. The ultrasound-activated liposome for regulating ATP-ADO axis and relieving immunosuppression according to any one of claims 1 to 5, characterized in that: The liposome is synthesized by a thin film hydration method using a reactive oxygen species-responsive CD39 inhibitor prodrug and a lipid membrane loaded with a sonosensitizer.

7. The ultrasound-activated liposome for regulating the ATP-ADO axis to relieve immunosuppression according to claim 6, characterized in that: The lipid film is obtained by mixing and dissolving DSPE-PEG2000, DSPE-PEG2000-cRGD, DOPE, cholesterol and a sonosensitizer in an organic solvent, and then performing rotary evaporation.

8. The ultrasound-activated liposome for regulating the ATP-ADO axis to relieve immunosuppression according to claim 7, characterized in that: The mass ratio of DSPE-PEG2000, DSPE-PEG2000-cRGD, DOPE, cholesterol and sonosensitizer is 1:1.1:3.16:0.54:0.

6.

9. Use of the ultrasound-activated liposome for regulating ATP-ADO axis and relieving immunosuppression according to any one of claims 1 to 8 in the preparation of anti-tumor drugs, characterized in that: The tumor is an immunosuppressive tumor.

10. The use according to claim 9, characterized in that The tumor is triple-negative breast cancer.

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