Ultrasonic activated liposome for regulating ATP-ADO axis to release immunosuppression and application thereof
Ultrasound-activated liposomes generate ROS in tumor cells and release CD39 inhibitors, regulating the ATP-ADO axis, solving the problem of adenosine-mediated immunosuppression, and achieving immune activation of tumor cells and lasting immunotherapy effects.
Patent Information
- Application Number
- CN202411779040.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-05
- Publication Date
- 2025-10-24
- Estimated Expiration
- 2044-12-05
AI Technical Summary
Existing immunotherapies are limited in their efficacy in tumor treatment due to adenosine-mediated immunosuppression. Single immunotherapy is only effective in about a quarter of tumor patients, and CD39 inhibitors have a short half-life in the blood circulation, making it difficult for them to accumulate to effective concentrations in tumors.
An ultrasound-activated liposome was designed, containing lipids, sonosensitizers, and reactive oxygen species-responsive CD39 inhibitor prodrugs. It was formed by self-assembly and activated by ultrasound to generate ROS, triggering tumor cell ICD. The CD39 inhibitor was then released under the action of reactive oxygen species, regulating the ATP-ADO axis and overcoming adenosine-mediated immunosuppression.
It significantly improves the effect of tumor immunotherapy, avoids off-target toxicity, achieves a lasting anti-tumor immune response, and has a dual anti-tumor immune effect.
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Figure CN119745799B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological medicine, in particular to an ultrasound-activated liposome for relieving immune suppression by regulating ATP-ADO axis and application thereof. BACKGROUND
[0002] Immune therapy, especially immune checkpoint inhibitors (ICIs), has made significant progress in the application of various tumor types. However, the efficacy of immune suppression tumors is limited due to the low immunogenicity of tumors, insufficient intratumoral infiltration of T lymphocytes, and immunosuppressive tumor microenvironment. Single immunotherapy only achieves effective treatment in about one-fourth of tumor patients.
[0003] Sonodynamic therapy (SDT) is an emerging non-invasive tumor treatment method, which 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, accurate positioning, and little damage to surrounding normal tissues. In the tumor microenvironment, endoplasmic reticulum (ER) as an important organelle, participates in protein synthesis, folding and transportation, and plays a key role in the process of tumor cells coping with 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] Immune 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, anti-cancer therapies focused on stimulating tumor cells to undergo ICD, while ignoring that ATP produced during the ICD process can be rapidly converted into immunosuppressive metabolite adenosine (ADO) through exonucleases, exacerbating the microenvironment immunosuppression. Studies have shown that high levels of adenosine are associated with increased expression of immune checkpoint molecule PD-L1 on tumor cells, thus deducing that adenosine is one of the key inducers of poor response to immune checkpoint inhibitors. Therefore, the ATP-ADO axis is considered as a regulatory pathway of immune response, which is the basis for the production of adenosine to promote tumor development and induce treatment failure of immunotherapy.
[0005] CD39 is a 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 enzyme activity of CD39, but due to its short half-life in blood circulation, it prevents them from accumulating to an effective concentration in tumors, and it is easy to produce target external toxicity.
[0006] To solve the above problems, it is crucial to prepare a drug delivery system that can not only activate ICD in tumor cells, but also overcome adenosine-mediated immune suppression to maintain a persistent anti-tumor immune effect to improve the immunotherapy effect of immunosuppressive tumors. SUMMARY
[0007] The purpose of the present application is to provide a nano delivery system that can not only cause tumor immunogenic cell death, but also overcome adenosine-mediated immune suppression to achieve a persistent anti-tumor immune effect.
[0008] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:
[0009] The present application provides an ultrasound-activated liposome for regulating ATP-ADO axis immune suppression, which is formed by self-assembly of lipids, a sonosensitizer and an active oxygen-responsive CD39 inhibitor prodrug.
[0010] The lipid is a basic raw material for forming a liposome, including phospholipids, polyethylene glycol functionalized lipids and cholesterol. The phospholipid can be, but is not limited to, distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylethanolamine (DOPE), and the polyethylene glycol functionalized lipid can be, but is 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) under ultrasonic irradiation, generating immunogenic cell death (ICD). The sonosensitizer can be, but is not limited to, a porphyrin-based sonosensitizer or a phthalocyanine-based sonosensitizer.
[0012] The active oxygen-responsive CD39 inhibitor prodrug is prepared by modifying the CD39 inhibitor with a ROS-responsive group. The prodrug can release the biologically active CD39 inhibitor under the action of reactive oxygen species (ROS), which inhibits the enzyme activity of the top rate-limiting enzyme CD39 of the ATP-ADO axis.
[0013] The ultrasound-activated liposome for relieving immune suppression provided by the application has the following action mode: due to the enhanced permeability and retention of tumor tissues, the nanoscale liposome can be effectively enriched in the tumor and internalized by cancer cells. Under ultrasonic irradiation, the sonosensitizer acts to generate a large amount of intracellular ROS, a part of which causes endoplasmic reticulum stress to produce immunogenic cell death (ICD), and a part of which triggers the release of CD39 inhibitor by the active oxygen response type CD39 inhibitor prodrug, thereby regulating the ATP-ADO axis by inhibiting the enzyme activity of CD39, increasing the ATP level with immune activation, and reducing the accumulation of ADO with immune suppression, to achieve a dual anti-tumor immune effect.
[0014] Preferably, the active oxygen response type CD39 inhibitor prodrug is prepared by modifying a borate group on the ortho-phenol site of FPL 67156.
[0015] FPL 67156, as a nucleotide derivative, has good inhibitory effect on the enzyme activity of CD39. Other small molecule inhibitors modified with a borate group are also suitable for use in the application.
[0016] More preferably, the active oxygen response type CD39 inhibitor prodrug is prepared by forming a reversible covalent ester bond and positive-negative charge interaction between polybromo(2-acryloyloxy)ethyl(p-boronic benzyl)diethylamine (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 active oxygen response type CD39 inhibitor prodrug comprises: mixing B-PDEAEA and FPL 67156 in 4-hydroxyethylpiperazine ethanesulfonic acid buffer according to an N / P ratio of 9-50, and allowing B-PDEAEA and FPL 67156 to self-assemble to form B-PDEAEA / FPL 67156 nanopolymers through phenyl borate ester bond and positive-negative charge attraction and other interactions.
[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 most stable.
[0019] Preferably, the sonosensitizer is synthesized by amidation of protoporphyrin IX with 4-methylbenzenesulfonyl urea, BOP reagent and triethylamine, and the sonosensitizer material has endoplasmic reticulum targeting effect; or the sonosensitizer is protoporphyrin IX. The sonosensitizer applicable to the application is not limited to this.
[0020] As preferred, the liposome is synthesized by thin film hydration method from the active oxygen response type CD39 inhibitor prodrug and the lipid film loaded with the photosensitizer.
[0021] As preferred, the lipid film is obtained by mixing and dissolving DSPE-PEG2000, DSPE-PEG2000-cRGD, DOPE, cholesterol and photosensitizer in an organic solvent, and then rotary evaporation.
[0022] As preferred, the mass ratio of DSPE-PEG2000, DSPE-PEG2000-cRGD, DOPE, cholesterol and photosensitizer is 1:1.1:3.16:0.54:0.6.
[0023] In the preparation of the drug-loaded liposome, the dosage ratio is adjusted according to the effective concentration and the body tolerance of the CD39 inhibitor and the photosensitizer. As preferred, the mass ratio of the prodrug calculated as FPL 67156 and the lipid film calculated as photosensitizer is 1:2-3.
[0024] The application also provides the use of the ultrasound-activated liposome for preparing an antitumor drug.
[0025] The liposome is enriched in tumor tissues, and after ultrasound irradiation, it stimulates the tumor to generate ICD, and releases the CD39 inhibitor, thereby regulating the ATP-ADO axis and overcoming adenosine-mediated immunosuppression.
[0026] Specifically, the tumor is a solid tumor. The liposome accumulates in the tumor and is internalized into tumor cells, and then, in combination with ultrasound irradiation, achieves the purpose of tumor treatment. The conditions of ultrasound irradiation can be changed according to the physical condition of the subject to be treated, so that the photosensitizer is activated and then reacts to produce active oxygen.
[0027] Further, the tumor is an immunosuppressive tumor, such as triple-negative breast cancer, pancreatic cancer and other solid tumors.
[0028] The application has the following beneficial effects:
[0029] The present application utilizes a ROS-producing sonosensitizer and a ROS-responsive release exonuclease inhibitor prodrug synthesis to regulate the ultrasound-activated cascade drug release liposome of immune suppression released by ATP-ADO axis, significantly improves the immunotherapy effect, and avoids obvious toxic side effects. The liposome can only produce active oxygen after ultrasonic irradiation, and further activate the prodrug to release the exonuclease inhibitor, induce ICD of tumor cells, at the same time, regulate the ATP-ADO axis, increase the ATP level with immune activation, and reduce the accumulation of ADO with immune suppression, realize the double anti-tumor immune effect. The targeted external toxic side effects caused by the exonuclease inhibitor are avoided through the ultrasonic triggering mode. The preparation method of the present application is simple, the reaction condition is mild, and the present application has great potential in the field of tumor drug preparation and application. BRIEF DESCRIPTION OF DRAWINGS
[0030] Figure 1 It is a preparation of B-PDEAEA / FPL 67156 nanopolymer and a schematic diagram of ROS-responsive activation principle.
[0031] Figure 2 It is a particle size diagram of BFPL with N / P ratio of 9 in Example 1.
[0032] Figure 3 It is a ROS production result diagram after the cell incubates the nanodrug under the action of ultrasound in Example 1.
[0033] Figure 4 It is a survival rate result diagram after the cell incubates the nanodrug under the action of ultrasound in Example 1.
[0034] Figure 5 It is the relative content of ATP in the cell supernatant after the cell incubates the nanodrug under the action of ultrasound in Example 1.
[0035] Figure 6 It is the content of ADO in the cell supernatant after the cell incubates the nanodrug under the action of ultrasound in Example 1.
[0036] Figure 7 It is the in vivo tumor inhibition effect of the drug-loaded liposome under the action of ultrasound in the breast cancer transplantation tumor model of mice in Example 1.
[0037] Figure 8 It is the H&E staining image of the main organs (heart, liver, spleen, lung, kidney) of Balb / C mice after drug-loaded liposome drug treatment in Example 1.
[0038] Figure 9 It is a particle size diagram of BFPL with N / P ratio of 13 in Example 2.
[0039] Figure 10Relative content of ATP in cell supernatant after incubation of nanomedicine under ultrasound in Example 2.
[0040] Figure 11 Particle size diagram of BFPL with N / P ratio of 20 in Example 3.
[0041] Figure 12 Relative content of ATP in cell supernatant after incubation of nanomedicine under ultrasound in Example 3.
[0042] Figure 13 Particle size diagram of BFPL with N / P ratio of 30 in Example 4.
[0043] Figure 14 Relative content of ATP in cell supernatant after incubation of nanomedicine under ultrasound in Example 4.
[0044] Figure 15 Particle size diagram of BFPL with N / P ratio of 50 in Example 5.
[0045] Figure 16 Relative content of ATP in cell supernatant after incubation of nanomedicine under ultrasound in Example 5.
[0046] Figure 17 Relative content of ATP in cell supernatant after incubation of nanomedicine under ultrasound in Example 6. DETAILED DESCRIPTION
[0047] The application will be further described below in conjunction with specific examples. The following examples are only used to illustrate the application, and are not used to limit the application scope. Modifications or replacements of the method, steps or conditions of the application, without departing from the spirit and essence of the application, all belong to the scope of the application.
[0048] The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.
[0049] FPL 67156 trisodium used in the following examples was purchased from MCE. Protoporphyrin IX, 4-methylbenzenesulfonyl urea and BOP reagent were purchased from Macklin. DSPE-PEG2000, DSPE-PEG2000-cRGD, DOPE, cholesterol were purchased from Xi'an Ruixi Biological Technology Co., Ltd.
[0050] 4T1-Luci cells were purchased from ATCC cell library.
[0051] B-PDEAEA was synthesized by the method described in the reference (Liu, X., et al. Fusogenic Reactive Oxygen Species Triggered Charge-Reversal Vector for Effective Gene Delivery. Advanced Materials, 2015, 28(9): 1743-1752.), and the structural formula is shown in formula (I), 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 exonuclease inhibitor FPL 67156 and dilute it to 2.5 mL (the final concentration of FPL 67156 is 40 μg / mL) by 4-hydroxyethyl piperazine ethanesulfonic acid buffer (HEPES, pH 7.4, 10 mM), and mix 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, and 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 vortex immediately for 5 minutes to ensure complete crosslinking, and prepare B-PDEAEA / FPL 67156 nanopolymer. The reaction equation is shown in Figure 1 .
[0056] Preparation of ER-targeted sonosensitizer PMPS: Dissolve 4-methyl phenylsulfonyl urea (MPSU, 85.7 mg, 0.4 mmol), BOP (176.9 mg, 0.8 mmol) and triethylamine (TEA, 55.6 μL, 0.4 mmol) in 5 mL dichloromethane (DCM), and add the solution dropwise to protoporphyrin IX (56.3 mg, 0.1 mmol). Stir in 5 mL DCM at room temperature for 4 hours. The DCM solution is rotary evaporated, and the PMPS product is washed with 0.1M NaOH solution (for removing protoporphyrin IX), acetone (for removing BOP, TEA and MPSU) three times in turn.
[0057] Preparation of ER-targeted PMPS-loaded lipid membrane: 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 CHCl3. The solution was poured into a 25 mL round-bottom flask and rotary evaporated to form a lipid membrane.
[0058] Preparation of drug-loaded liposomes BFPL: B-PDEAEA / FPL 67156 liquid was transferred into the above-mentioned round-bottom flask and magnetically stirred overnight to obtain the final product BFPL drug-loaded liposomes.
[0059] In the preparation of drug-loaded liposomes, the amount of prodrug and lipid membrane is adjusted according to the target object. Specifically, at the cellular level, the effective concentration of FPL 67156 is 8 μg / mL, and the maximum dose of PMPS cannot exceed 25 μg / mL; at the animal level, such as in a mouse model, the effective dose of FPL 67156 is 5 mg / kg, and the maximum dose of PMPS is 10 mg / kg.
[0060] 2. Particle size characterization of drug-loaded liposomes
[0061] 1 mL of the prepared BFPL sample was placed in a sample pool dedicated to the particle size analyzer, and dynamic light scattering test was performed using the particle size analyzer to measure the hydration kinetic particle size and particle size distribution of the nanoparticles. The experiment was repeated three times and the average value was taken.
[0062] As shown in Figure 2 , 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. Verification of ROS generation and tumor cell death induced by drug-loaded liposomes at the cellular level.
[0064] 4,000 4T1 breast cancer cells were inoculated 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 was performed (3 MHz, 1.0 W / cm 2 , 50% duty cycle, 1 minute). ROS signal was detected by cell ROS kit.
[0065] As shown in Figure 3 , after incubation with drug-loaded liposomes and ultrasonic irradiation, the intracellular ROS fluorescence intensity increased by about 8 times, while no ROS signal was detected in cells without ultrasonic irradiation.
[0066] In 96-well plates, each well was inoculated with 5000 4T1 breast cancer cells, and the above-mentioned drugs were added. After 24 hours of incubation, ultrasonic irradiation (3MHz, 1.0W / cm 2 , 50% duty cycle, time 1 minute) was performed. Cell toxicity was detected by CCK8 kit.
[0067] As shown in Figure 4 , after incubation of nanomedicine and ultrasonic irradiation, the cell survival rate was only 34%, indicating that a large number of tumor cells died.
[0068] The above results show that the drug-loaded liposomes can produce a large amount of ROS after ultrasonic irradiation and cause tumor cell death.
[0069] 4. Verify the content of extracellular ATP and ADO at the cellular level
[0070] In 12-well plates, each well was inoculated with 80,000 4T1 breast cancer cells, and the above-mentioned drugs were added. After 24 hours of incubation, ultrasonic irradiation (3MHz, 1.0W / cm 2 , 50% duty cycle, time 1 minute) was performed. The content of ATP in the cell supernatant was detected by ATP enhancement test kit.
[0071] As shown in Figure 5 , after incubation of nanomedicine and ultrasonic irradiation, the extracellular ATP content was significantly higher than that of the control group.
[0072] As described above, the content of ADO in the cell supernatant was detected by adenosine assay kit.
[0073] As shown in Figure 6 , after incubation of nanomedicine and ultrasonic irradiation, the extracellular ADO content was significantly lower than that of the control group.
[0074] 5. Verify the anti-tumor effect of liposomes at the experimental animal level
[0075] 4T1 tumor cells (100 million tumor cells per mouse) were inoculated into Balb / C mice in situ, and when the tumor volume reached 70mm 3 , breast cancer orthotopic tumor model mice were obtained. The mice were divided into 3 groups, 5 mice in each group, and were injected with PBS, FPL 67156 (dose 5mg / kg) and drug-loaded liposomes BFPL (FPL 67156 dose 5mg / kg, maximum dose of PMPS 10mg / kg) via tail vein, respectively. After 6 hours, ultrasonic irradiation (frequency 3MHz, duty cycle 50%, intensity 2.0W / cm 2 , time 10min) was performed, and the tumor volume of mice was measured every two days.
[0076] AsFigure 7 As shown in Figure 6, compared with the control groups (PBS and FPL 67156 group), the tumor growth of BAPL+US experimental group was significantly inhibited, indicating that the nanomaterial can improve the tumor treatment effect through ultrasound triggered mode.
[0077] 6. Safety evaluation
[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), and after three days, the mice were reasonably sacrificed according to the animal ethics, and the main organs (heart, liver, spleen, lung, kidney) were obtained by dissection, and the biological safety was characterized by H&E staining.
[0079] As shown in Figure 6, compared with the control groups (PBS and FPL 67156 group), the tumor growth of BAPL+US experimental group was significantly inhibited, indicating that the nanomaterial can improve the tumor treatment effect through ultrasound triggered mode. Figure 8 As shown in Figure 6, compared with the control groups (PBS and FPL 67156 group), the tumor growth of BAPL+US experimental group was significantly inhibited, indicating that the nanomaterial can improve the tumor treatment effect through ultrasound triggered mode.
[0080] Example 2
[0081] 1. Preparation method of drug-loaded liposome BFPL:
[0082] The exonuclease inhibitor FPL 67156 was diluted to 2.5 mL (FPL 67156 final concentration 40 μg / mL) by HEPES (pH 7.4, 10 mM), and mixed with 2.5 mL B-PDEAEA working solution (359.2 μg / mL, polymerization degree n is 39, N / P=13). Then the mixture was transferred to a centrifuge tube and vortexed immediately for 5 minutes to ensure complete crosslinking, and the B-PDEAEA / FPL 67156 nanopolymer was prepared.
[0083] The preparation method of the lipid membrane loaded with ER-targeting sonosensitizer PMPS is the same as that of Example 1.
[0084] The B-PDEAEA / FPL 67156 liquid was transferred to the lipid membrane and magnetically stirred overnight to obtain the final product BFPL drug-loaded liposome.
[0085] 2. Particle size characterization of drug-loaded liposome
[0086] The test method is the same as that of Example 1, and the results are shown in Figure 9 As shown in Figure 6, compared with the control groups (PBS and FPL 67156 group), the tumor growth of BAPL+US experimental group was significantly inhibited, indicating that the nanomaterial can improve the tumor treatment effect through ultrasound triggered mode.
[0087] 3. Verification of extracellular ATP content at the cellular level
[0088] The test method is the same as that of Example 1, and the results are shown in Figure 10As shown, after incubation of the nanomedicine and ultrasound irradiation, the extracellular ATP content was significantly higher than that of 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 by HEPES (pH 7.4, 10 mM) (the final concentration of FPL 67156 was 40 μg / mL) and mixed with 2.5 mL of B-PDEAEA working solution (552.7 μg / mL, degree of polymerization n was 60, N / P = 20). Then the mixture was transferred to a centrifuge tube and immediately vortexed for 5 minutes to ensure complete crosslinking, and B-PDEAEA / FPL 67156 nanopolymers were prepared.
[0092] The preparation method of the lipid membrane loaded with ER-targeting photosensitizer PMPS is the same as that of Example 1.
[0093] The B-PDEAEA / FPL 67156 liquid was transferred to the above lipid membrane and magnetically stirred overnight to obtain the final product BFPL drug-loaded liposome.
[0094] 2. Particle size characterization of drug-loaded liposome
[0095] The test method is the same as that of Example 1, and the results are shown in Figure 11 The average particle size of the nanomedicine prepared in this embodiment is about 183.0 nm.
[0096] 3. Verification of extracellular ATP content at the cellular level
[0097] The test method is the same as that of Example 1, and the results are shown in Figure 12 After incubation of the nanomedicine and ultrasound irradiation, the extracellular ATP content was significantly higher than that of 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 by HEPES (pH 7.4, 10 mM) (the final concentration of FPL 67156 was 40 μg / mL) and mixed with 2.5 mL of B-PDEAEA working solution (552.7 μg / mL, degree of polymerization n was 60, N / P = 20). Then the mixture was transferred to a centrifuge tube and immediately vortexed for 5 minutes to ensure complete crosslinking, and B-PDEAEA / FPL 67156 nanopolymers were prepared.
[0101] The preparation method of the lipid film loaded with the ER-targeting sonosensitizer PMPS is the same as that in Embodiment 1.
[0102] The B-PDEAEA / FPL 67156 liquid is transferred into the above lipid film, and magnetic stirring is performed overnight to obtain the final product BFPL drug-loaded liposome.
[0103] 2. Particle size characterization of the drug-loaded liposome
[0104] The test method is the same as that in Embodiment 1, and the results are shown in Table 1. Figure 13 The average particle size of the nano-drug prepared in this embodiment is about 164.7 nm.
[0105] 3. Verification of the extracellular ATP content at the cellular level
[0106] The test method is the same as that in Embodiment 1, and the results are shown in Table 1. Figure 14 After incubation of the nano-drug and ultrasonic irradiation, the extracellular ATP content is significantly higher than that of the control group.
[0107] Embodiment 5
[0108] 1. Preparation method of the drug-loaded liposome BFPL:
[0109] The exonuclease inhibitor FPL 67156 is diluted to 2.5 mL (the final concentration of FPL 67156 is 40 μg / mL) through HEPES (pH 7.4, 10 mM), and is mixed with 2.5 mL of B-PDEAEA working solution (1381.7 μg / mL, the degree of polymerization n is 150, N / P=50). Then the mixture is transferred into a centrifuge tube and vortexed immediately for 5 minutes to ensure complete crosslinking, to prepare the B-PDEAEA / FPL 67156 nanopolymer.
[0110] The preparation method of the lipid film loaded with the ER-targeting sonosensitizer PMPS is the same as that in Embodiment 1.
[0111] The B-PDEAEA / FPL 67156 liquid is transferred into the above lipid film, and magnetic stirring is performed overnight to obtain the final product BFPL drug-loaded liposome.
[0112] 2. Particle size characterization of the drug-loaded liposome
[0113] The test method is the same as that in Embodiment 1, and the results are shown in Table 1. Figure 15 The average particle size of the nano-drug prepared in this embodiment is about 152.7 nm.
[0114] 3. Verification of the extracellular ATP content at the cellular level
[0115] The test method is the same as that in Embodiment 1, and the results are shown in Table 1. Figure 16As shown in the table, after incubation of the nano-drug and ultrasound irradiation, the extracellular ATP content was significantly higher than that of the control group.
[0116] Example 6
[0117] 1. Preparation method of drug-loaded liposome BFPL
[0118] The preparation method of B-PDEAEA / FPL 67156 nano-polymer 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 CHCl3. 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 into the above-mentioned round-bottom flask and magnetically stirred overnight to obtain the final product BFPL drug-loaded liposome.
[0121] 2. Particle size characterization of drug-loaded liposome
[0122] The test method is the same as that in Example 1, and the average particle size of the nano-drug prepared in this example is about 112.7 nm.
[0123] 3. Verification of extracellular ATP relative content at cell level
[0124] The test method is the same as that in Example 1, and the results are shown in the table. Figure 17 As shown in the table, after incubation of the nano-drug and ultrasound irradiation, the extracellular ATP content was significantly higher than that of the control group.
[0125] The above examples are only for illustrating the technical concept and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and to implement it, and it cannot limit the protection scope of the present application. Any equivalent changes or modifications made in accordance with the spirit and essence of the present application shall be covered within the protection scope of the present application.
Claims
1. An ultrasound-activated liposome modulating the ATP-ADO axis to relieve immunosuppression, characterized in that, The liposome is formed by self-assembly of lipids, a sonosensitizer, and an active oxygen-responsive CD39 inhibitor prodrug; The method for preparing the active oxygen response type CD39 inhibitor prodrug comprises: mixing polybromo(2-acryloyloxy)ethyl ammonium (PDEAEA) and FPL 67156 in 4-hydroxyethylpiperazine ethanesulfonic acid buffer according to an N / P ratio of 9-50 to self-assemble to form B-PDEAEA / FPL 67156 nanopolymers. p - mixing benzyl diethylammonium borate and FPL 67156 in 4-hydroxyethylpiperazine ethanesulfonic acid buffer to self-assemble to form B-PDEAEA / FPL 67156 nanopolymers.
2. The ultrasound-activated liposome modulating the ATP-ADO axis deimmunosuppression of claim 1, wherein, The sonosensitizer is synthesized by amidation of protoporphyrin IX with 4-methylbenzenesulfonylurea, BOP reagent, and triethylamine; or the sonosensitizer is protoporphyrin IX.
3. The ultrasound-activated liposome modulating the ATP-ADO axis deimmunosuppression according to claim 1 or 2, characterized in that, The liposome is synthesized by a thin film hydration method from an active oxygen-responsive CD39 inhibitor prodrug and a lipid membrane loaded with a sonosensitizer.
4. The ultrasound-activated liposome modulating the ATP-ADO axis deimmunosuppression of claim 3, wherein, The lipid membrane is obtained by mixing and dissolving DSPE-PEG2000, DSPE-PEG2000-cRGD, DOPE, cholesterol, and a sonosensitizer in an organic solvent, and then rotary evaporation.
5. The ultrasound-activated liposome modulating the ATP-ADO axis deimmunosuppression of claim 4, wherein, The mass ratio of DSPE-PEG2000, DSPE-PEG2000-cRGD, DOPE, cholesterol, and a sonosensitizer is 1:1.1:3.16:0.54:0.
6.
6. Use of the ultrasound-activated liposomes modulating the ATP-ADO axis deimmunosuppression according to any one of claims 1 to 5 for the preparation of an antitumor medicament, characterized in that, The tumor is an immunosuppressive tumor.
7. Use according to claim 6, wherein The tumor is triple-negative breast cancer.