Tiralizumab immune sustained-release embolization microsphere and preparation method thereof
By using polylactic acid-glycolic acid copolymer microspheres loaded with tislelizumab and coated with a modified gel material in the treatment of hepatocellular carcinoma, the problem of rapid drug release in traditional methods was solved, achieving targeted and slow drug release and improving treatment efficacy.
Patent Information
- Application Number
- CN202511498921.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-20
- Publication Date
- 2026-02-17
AI Technical Summary
In current treatments for hepatocellular carcinoma, traditional chemotherapy drugs are rapidly released into the systemic circulation, leading to reduced local efficacy and systemic adverse reactions. Furthermore, the concentration of targeted drugs is insufficient, affecting the treatment outcome.
Tislelizumab was loaded onto polylactic acid-glycolic acid copolymer microspheres and encapsulated with a modified gel material to form sustained-release embolization microspheres. Combined with a targeting ligand, this achieved targeted and slow-release of the drug.
It increased the targeted concentration of the drug, reduced systemic adverse reactions, prolonged the drug release cycle, significantly improved drug loading and sustained-release performance, and enhanced the anti-tumor effect.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical materials technology, and in particular to a tislelizumab immunosuppressive microsphere and its preparation method. Background Technology
[0002] Hepatocellular carcinoma (HCC) is a prevalent malignant tumor worldwide. The latest epidemiological studies show that its mortality rate has risen from third place in 2018 to second place globally among malignant tumor-related deaths. HCC is characterized by its insidious onset, high malignancy, and rapid progression. This results in over 75% of patients being diagnosed at an advanced stage, missing the optimal window for surgical resection, and often only being able to opt for palliative treatments such as radiotherapy, chemotherapy, and interventional therapy, leading to extremely limited clinical management strategies.
[0003] Transcatheter arterial chemoembolization (TACE)-based interventional therapy has demonstrated remarkable anti-tumor efficacy and has become an important treatment method for advanced liver cancer in clinical practice. Traditional TACE often uses a mixture of iodized oil and doxorubicin, cisplatin, or other chemotherapeutic drugs. However, this method has two main drawbacks: ① Local deposition of the iodized oil emulsion sometimes fails to achieve satisfactory results, and the cytotoxic effect of the chemotherapeutic drugs on tumor tissue decreases over time; ② Traditional drug carriers are lipids, while chemotherapeutic drugs are water-soluble. This traditional emulsion leads to the rapid release of chemotherapeutic drugs into the bloodstream, resulting in rapid entry into the systemic circulation, increasing systemic adverse reactions and reducing local efficacy. However, in the treatment of liver cancer, sustained drug release and maintenance of drug concentration within the tumor play a crucial role.
[0004] Numerous studies have confirmed the efficacy and safety of TACE combined with targeted immunotherapy for HCC. Furthermore, clinical studies have reported that the combination of lenvatinib and tislelizumab can effectively reduce tumor burden while activating the immune system by releasing tumor neoantigens and immune-related inflammatory factors, thereby enhancing the immune anti-tumor response, improving TACE-induced hypoxia, and regulating the immunosuppressive microenvironment of HCC. Currently, tislelizumab is administered intravenously, typically 200 mg every 3 weeks. Although tislelizumab has a targeted effect, there is a risk of insufficient drug concentration in the later stages, ultimately leading to a shorter drug release cycle. Summary of the Invention
[0005] In view of this, the purpose of the present invention is to provide tislelizumab immunorelief embolization microspheres and the preparation method thereof. By loading tislelizumab onto the embolization microspheres and forming a coating, the embolization microspheres can make better contact with the lesion site.
[0006] The present invention solves the above-mentioned technical problems through the following technical means:
[0007] A tislelizumab immunorestricted embolization microsphere, the embolization microsphere comprising a sustained-release microsphere and a modified gel material, wherein the sustained-release microsphere is loaded with tislelizumab, and the modified gel material is coated on the sustained-release microsphere; the sustained-release microsphere is made of polylactic acid-glycolic acid copolymer, and the sustained-release microsphere is loaded with tislelizumab inside by a low-temperature emulsification-phase separation method.
[0008] Preferably, the specific method for loading tislelizumab onto the embolization microspheres includes:
[0009] A1. Dissolve the polylactic acid-glycolic acid copolymer in a solvent, add a stabilizer, and form a dispersion.
[0010] A2. Dissolve tislelizumab in a buffer solution with a pH of 6.2-7.2 to form tislelizumab solution;
[0011] A3. Add tislelizumab solution to the dispersion and stir slowly at 4°C to form an emulsion;
[0012] A4. Add the emulsion dropwise to deionized water, stir at 4°C to separate the phases, and obtain the precipitate;
[0013] A5. The precipitate was washed and freeze-dried to obtain embolic microspheres loaded with tislelizumab.
[0014] Based on the above-mentioned technical means, by forming an emulsion of tislelizumab solution and dispersion of embolic microspheres, and then adding it to an aqueous solution for phase separation, embolic microspheres loaded with tislelizumab can be obtained with high drug loading and the effect of high temperature on the activity of tislelizumab can be avoided.
[0015] More preferably, in step A1, the polylactic acid-glycolic acid copolymer is dissolved in a mixed solvent of dichloromethane and ethyl acetate at a volume ratio of 4:1 to obtain an organic phase; hydrophobic silica nanoparticles with a particle size of 10-30 nanometers are added to the organic phase as a stabilizer, and the mixture is ultrasonically dispersed uniformly; the organic phase is pre-cooled to -20°C and held for 20-30 minutes to obtain a dispersion. In this technical solution, the addition of dichloromethane and ethyl acetate can moderately adjust the evaporation rate of the solvent, forming a denser microsphere structure; the deep cooling of the organic phase greatly inhibits protein denaturation caused by shear heat generation during emulsification; and the synergistic use of the hydrophobic silica nanoparticle solid stabilizer and the mixed solvent helps to form more stable primary emulsion droplets with a narrower particle size distribution, providing a basis for high drug loading.
[0016] More preferably, in step A1, the concentration of polylactic acid-glycolic acid copolymer in the dispersion is 8-20 wt%.
[0017] More preferably, in step A2, the concentration of tislelizumab in the tislelizumab solution is 0.4-0.8 mg / mL.
[0018] Based on the above-mentioned technical means, the drug loading of microspheres can be controlled by controlling the concentration of polylactic acid-glycolic acid copolymer and tislelizumab.
[0019] Preferably, in step A2, a protective agent is added to the buffer solution.
[0020] More preferably, the protective agent is one or more combinations of trehalose, sucrose, Tween 20, Tween 80, histidine, and arginine.
[0021] More preferably, the protective agent is a mixture of trehalose, Tween 20 and arginine, wherein the ratio of trehalose, Tween 20 and arginine is 1:0.1-0.3:0.2-0.5.
[0022] Based on the above-mentioned technical means, by using multiple protective agents and controlling their proportions, the aggregation of tislelizumab can be reduced and can play a synergistic role, which is beneficial to the uniformity of tislelizumab solution in emulsion.
[0023] Preferably, the coating thickness of the modified gel material is 100-300 nm.
[0024] More preferably, the coating thickness of the modified gel material is 140-230 nm.
[0025] Preferably, the modified gel material is prepared by activating the gel material and then reacting it with the targeting ligand.
[0026] Preferably, the preparation of the modified gel material specifically includes the following steps:
[0027] B1. Dissolve the natural gel in a buffer solution with a pH of 5.2-6.2, add EDC, and activate for 2-5 hours to obtain activated natural gel;
[0028] B2. Dissolve the targeting ligand in a solvent to form a ligand solution. Add the ligand solution to an activated natural gel containing a buffer solution and react for 3-6 hours. After the reaction, add a blocking agent to obtain a modified gel material.
[0029] B3. Purify the modified gel material from step B2 to obtain the purified modified gel material.
[0030] Based on the above-mentioned technical means, by loading the targeting ligand onto the natural gel through covalent binding, the targeting ligand can be made to have high stability and specificity, so that it can better bind to the lesion site when used, thereby improving the embolization effect to a certain extent. It is also beneficial to improve the targeting effect of tislelizumab loaded in the embolization microspheres.
[0031] More preferably, in step B1, the natural gel is one or more combinations of agarose gel, chitosan gel, sodium alginate gel, and hyaluronic acid gel.
[0032] Further preferred, the natural gel is a hyaluronic acid gel, and the hyaluronic acid is low molecular weight hyaluronic acid, that is, hyaluronic acid with a molecular weight of 50,000-150,000 Daltons.
[0033] More preferably, in step B1, the buffer solution is a citrate-sodium citrate buffer solution.
[0034] More preferably, in step B1, the concentration of EDC is 0.1-0.5 mg / mL.
[0035] More preferably, in step B2, the targeting ligand is a polypeptide.
[0036] More preferably, the polypeptide is an RGD polypeptide.
[0037] More preferably, in step B2, the blocking agent is bovine serum albumin.
[0038] Based on the above technical means, by covalently binding RGD peptides to the surface of hyaluronic acid gel and blocking unreacted active groups with bovine serum albumin to reduce non-specific binding, and then removing unbound peptides and EDC through purification, it is beneficial to reduce the possibility of triggering immune responses or toxicity.
[0039] This application discloses a method for preparing tislelizumab immunorelief microspheres, comprising the following steps:
[0040] The modified gel material was dissolved in a buffer solution with a pH of 4.2-5.2, and the sustained-release microspheres were slowly added while stirring. Sodium chloride was added during the stirring process, and stirring was continued for 1-5 hours to obtain a precipitate. The precipitate was then washed and freeze-dried to obtain embolization microspheres.
[0041] Based on the above technical means, by dispersing sustained-release microspheres in a modified gel solution and performing electrostatic adsorption self-assembly, the modified gel is coated onto the sustained-release microspheres. Sodium chloride is added to adjust the ionic strength, and stirring helps the microspheres coated with the gel to precipitate. Furthermore, by controlling the stirring speed, it is beneficial to achieve uniform coating of the gel and enable the embolization microspheres to continuously and stably release tislelizumab, thus prolonging the drug release cycle.
[0042] Preferably, the concentration of sodium chloride is 0.1-0.3 mM, and the stirring speed is 60-200 r / min.
[0043] Based on the above-mentioned technical means, controlling the concentration of sodium chloride and the stirring speed helps to control the uniformity and thickness of the gel coating.
[0044] The present application, employing the above-described scheme, has the following beneficial effects:
[0045] 1. This invention utilizes a gel loaded with a targeting ligand, which is then coated onto polylactic-co-glycolic acid copolymer microspheres loaded with tislelizumab. This allows the gel to bind specifically to the lesion site during use, thereby increasing the targeting concentration and embolization effect of tislelizumab and reducing the possibility of adverse reactions.
[0046] 2. The embolization microspheres of this invention have a drug loading capacity of up to 28.2%, and a cumulative release rate of only 48.2% after 300 minutes, which is far lower than the rapid release of existing technologies, demonstrating their excellent sustained-release performance. Therefore, this invention significantly improves the stability and drug loading of tislelizumab through the synergistic effect of a specific combination of protective agents (trehalose, Tween 20, and arginine) and a modified gel coating process, and achieves long-acting sustained release, with unexpected effects.
[0047] 3. The addition of dichloromethane and ethyl acetate in this invention can moderately adjust the evaporation rate of the solvent, forming a denser microsphere structure; the deep cooling of the organic phase greatly inhibits protein denaturation caused by shear heat during emulsification; and the synergistic use of hydrophobic silica nanoparticle solid stabilizers and mixed solvents helps to form more stable primary emulsion droplets with a narrower particle size distribution, providing a basis for high drug loading; finally, by forming an emulsion from tislelizumab solution and the dispersion of embolic microspheres, and then adding it to an aqueous solution for phase separation, embolic microspheres loaded with tislelizumab can be obtained with a high drug loading, and the phase separation loading can reduce the impact on the activity of tislelizumab.
[0048] 4. In this application, by loading the targeting ligand onto the natural gel through covalent binding, the targeting ligand can be made to have high stability and specificity, so that it can better bind to the lesion site when used, thereby improving the embolization effect to a certain extent. It is also beneficial to improve the targeting effect of tislelizumab loaded in the embolization microspheres, so that the embolization microspheres can continuously and stably release tislelizumab. Detailed Implementation
[0049] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0050] This invention discloses a tislelizumab immunorelief microsphere comprising polylactic-co-glycolic acid copolymer (PLGA) sustained-release microspheres and a modified hyaluronic acid gel material. The sustained-release microspheres are loaded with tislelizumab, and the modified hyaluronic acid gel material coats the PLGA sustained-release microspheres. Unless otherwise specified, each example and comparative example uses three parallel samples (n=3). Experimental data are expressed as mean ± standard deviation to ensure the reliability and statistical validity of the results. The specific preparation process is as follows:
[0051] Example 1
[0052] Preparation of tislelizumab loaded onto embolized microspheres
[0053] A1. Dissolve 10 mg of polylactic acid-glycolic acid copolymer in 91 mL of solvent, add 1 mg of stabilizer to form a dispersion; specifically, dissolve the polylactic acid-glycolic acid copolymer in a mixed solvent of dichloromethane and ethyl acetate at a volume ratio of 4:1 to obtain an organic phase; add Aerosil R972 hydrophobic silica nanoparticles with a particle size of 10-30 nm as a stabilizer to the organic phase, and ultrasonically disperse until uniform; pre-cool the organic phase to -20°C and maintain for 20 minutes to obtain a dispersion.
[0054] A2. At 4°C, 0.4 mg of tislelizumab was dissolved in 9.5 mL of buffer solution with a pH of 6.5, and then 0.5 mL of a protective agent was added. The protective agent consisted of trehalose, Tween 20 and arginine, with a mass ratio of trehalose, Tween 20 and arginine of 1:0.2:0.3, to form the tislelizumab solution.
[0055] A3. Add tislelizumab solution to the dispersion and stir at 60 r / min for 5 min at 4°C to form an emulsion;
[0056] A4. Add the emulsion dropwise to deionized water and stir at 30 r / min at 4°C to separate the phases and obtain the precipitate;
[0057] A5. The precipitate was washed three times with deionized water and then freeze-dried to obtain embolic microspheres loaded with tislelizumab.
[0058] Preparation of modified gel materials:
[0059] B1. Dissolve 10g of low molecular weight hyaluronic acid gel in 100mL of citrate-sodium citrate buffer solution with pH 6.2, add 3mL of 0.2mg / mL EDC, and activate for 3h to obtain activated hyaluronic acid gel;
[0060] B2. Dissolve 5g of RGD peptide in 20mL of deionized water to form a ligand solution. Add the ligand solution to 100mL of activated natural gel containing citrate-sodium citrate buffer and react for 3h. After the reaction, add 10mL of bovine serum albumin to obtain the modified gel material.
[0061] B3. Dialyze the modified gel material from step B2 for 3 days to obtain the purified modified gel material.
[0062] Preparation of tislelizumab immunorelief microspheres
[0063] At 4°C, 2.0 mg of modified gel material was dissolved in 100 mL of citrate-sodium citrate buffer solution with pH 4.8. 1.0 mg of embolic microspheres loaded with tislelizumab was slowly added and stirred at 80 r / min. During stirring, 3 mL of 0.2 mM sodium chloride was added and stirring was continued for 3 h to obtain a precipitate. The precipitate was then washed three times with deionized water and freeze-dried to obtain embolic microspheres.
[0064] The thickness of the modified gel on the embolized microspheres was measured to be 140 nm.
[0065] Aseptic and low-temperature operation is required when preparing the tislelizumab immunorestricted embolization microspheres of the present invention. In use, inject 30 mL of 0.9 wt% sodium chloride solution into a sealed vial, mix thoroughly, draw it out with a syringe, and gently shake the syringe to prevent precipitation of the tislelizumab immunorestricted embolization microspheres. Using the Seldinger method, insert a catheter through the femoral artery and slowly inject the tislelizumab immunorestricted embolization microspheres into the common hepatic artery or other blood vessels at the site of liver cancer lesion.
[0066] Example 2
[0067] Preparation of tislelizumab loaded onto embolized microspheres
[0068] A1. Dissolve 15 mg of polylactic acid-glycolic acid copolymer in 94 mL of solvent, add 1 mg of stabilizer to form a dispersion; specifically, dissolve the polylactic acid-glycolic acid copolymer in a mixed solvent of dichloromethane and ethyl acetate at a volume ratio of 4:1 to obtain an organic phase; add Aerosil R972 hydrophobic silica nanoparticles with a particle size of 10-30 nm as a stabilizer to the organic phase, and ultrasonically disperse until uniform; pre-cool the organic phase to -20°C and maintain for 25 minutes to obtain a dispersion.
[0069] A2. At 4°C, 0.6 mg of tislelizumab was dissolved in 9.5 mL of buffer solution with a pH of 6.2, and then 0.5 mL of a protective agent was added. The protective agent consisted of trehalose, Tween 20 and arginine, with a mass ratio of trehalose, Tween 20 and arginine of 1:0.2:0.3, to form the tislelizumab solution.
[0070] A3. Add tislelizumab solution to the dispersion and stir at 50 r / min for 10 min at 4°C to form an emulsion;
[0071] A4. Add the emulsion dropwise to deionized water and stir at 40 r / min for 15 min at 4°C to separate the phases and obtain the precipitate;
[0072] A5. The precipitate was washed three times with deionized water and then freeze-dried to obtain embolic microspheres loaded with tislelizumab.
[0073] Preparation of modified gel materials:
[0074] B1. Dissolve 15g of low molecular weight hyaluronic acid gel in 100mL of citrate-sodium citrate buffer solution with pH 5.8, add 5mL of 0.2mg / mL EDC, and activate for 2h to obtain activated hyaluronic acid gel;
[0075] B2. Dissolve 6.5g of RGD peptide in 20mL of deionized water to form a ligand solution. Add the ligand solution to 100mL of activated natural gel containing citrate-sodium citrate buffer and react for 4h. After the reaction, add 12mL of bovine serum albumin to obtain the modified gel material.
[0076] B3. Dialyze the modified gel material from step B2 for 3 days to obtain the purified modified gel material.
[0077] Preparation of tislelizumab immunorelief microspheres
[0078] At 4°C, 1.5 mg of modified gel material was dissolved in 100 mL of citrate-sodium citrate buffer solution with pH 5.2. 1.2 mg of embolic microspheres loaded with tislelizumab was slowly added and stirred at 60 r / min. During stirring, 4 mL of 0.2 mM sodium chloride was added and stirring was continued for 3 h to obtain a precipitate. The precipitate was then washed three times with deionized water and freeze-dried to obtain embolic microspheres.
[0079] The thickness of the modified gel on the embolized microspheres was measured to be 165 nm.
[0080] Aseptic and low-temperature operation is required when preparing the tislelizumab immunorestricted embolization microspheres of the present invention. In use, inject 30 mL of 0.9 wt% sodium chloride solution into a sealed vial, mix thoroughly, draw it out with a syringe, and gently shake the syringe to prevent precipitation of the tislelizumab immunorestricted embolization microspheres. Using the Seldinger method, insert a catheter through the femoral artery and slowly inject the tislelizumab immunorestricted embolization microspheres into the common hepatic artery or other blood vessels at the site of liver cancer lesion.
[0081] Example 3
[0082] Preparation of tislelizumab loaded onto embolized microspheres
[0083] A1. Dissolve 20 mg of polylactic acid-glycolic acid copolymer in 99 mL of solvent, add 1 mg of stabilizer to form a dispersion; specifically, dissolve the polylactic acid-glycolic acid copolymer in a mixed solvent of dichloromethane and ethyl acetate at a volume ratio of 4:1 to obtain an organic phase; add Aerosil R972 hydrophobic silica nanoparticles with a particle size of 10-30 nm as a stabilizer to the organic phase, and ultrasonically disperse until uniform; pre-cool the organic phase to -20 °C and maintain for 30 minutes to obtain a dispersion. A2. At 4°C, 0.8 mg of tislelizumab was dissolved in 9.5 mL of buffer solution with a pH of 7.2, and then 0.5 mL of a protective agent was added. The protective agent consisted of trehalose, Tween 20 and arginine, with a mass ratio of trehalose, Tween 20 and arginine of 1:0.2:0.3, to form the tislelizumab solution.
[0084] A3. Add tislelizumab solution to the dispersion and stir at 40 r / min for 20 min at 4°C to form an emulsion;
[0085] A4. Add the emulsion dropwise to deionized water and stir at 35 r / min at 4°C to separate the phases and obtain the precipitate;
[0086] A5. The precipitate was washed three times with deionized water and then freeze-dried to obtain embolic microspheres loaded with tislelizumab.
[0087] Preparation of modified gel materials:
[0088] B1. Dissolve 20g of low molecular weight hyaluronic acid gel in 100mL of citrate-sodium citrate buffer solution with pH 5.2, add 6mL of 0.2mg / mL EDC, and activate for 2h to obtain activated hyaluronic acid gel;
[0089] B2. Dissolve 7g of RGD peptide in 20mL of deionized water to form a ligand solution. Add the ligand solution to 100mL of activated natural gel containing citrate-sodium citrate buffer and react for 5h. After the reaction, add 12mL of bovine serum albumin to obtain the modified gel material.
[0090] B3. Dialyze the modified gel material from step B2 for 3 days to obtain the purified modified gel material.
[0091] Preparation of tislelizumab immunorelief microspheres
[0092] At 4°C, 2.0 mg of modified gel material was dissolved in 100 mL of citrate-sodium citrate buffer solution with pH 4.2. 1.5 mg of sustained-release microspheres were slowly added and stirred at 100 r / min. During stirring, 5 mL of 0.2 mM sodium chloride was added and stirring was continued for 3 h to obtain a precipitate. The precipitate was then washed three times with deionized water and freeze-dried to obtain embolic microspheres.
[0093] The thickness of the modified gel on the embolized microspheres was measured to be 210 nm.
[0094] Aseptic and low-temperature operation is required when preparing the tislelizumab immunorestricted embolization microspheres of the present invention. In use, inject 30 mL of 0.9 wt% sodium chloride solution into a sealed vial, mix thoroughly, draw it out with a syringe, and gently shake the syringe to prevent precipitation of the tislelizumab immunorestricted embolization microspheres. Using the Seldinger method, insert a catheter through the femoral artery and slowly inject the tislelizumab immunorestricted embolization microspheres into the common hepatic artery or other blood vessels at the site of liver cancer lesion.
[0095] Example 4
[0096] In this embodiment, the preparation methods of tislelizumab loaded on embolization microspheres, the preparation methods of modified gel materials, and the preparation methods of tislelizumab immunorelease embolization microspheres are the same as in Example 1, except that: A3. The tislelizumab solution is added to the dispersion and stirred at 60 r / min for 30 min at 4°C to form an emulsion.
[0097] Example 5
[0098] In this embodiment, the preparation methods of tislelizumab loaded on embolization microspheres, the preparation methods of modified gel materials, and the preparation methods of tislelizumab immunorelease embolization microspheres are the same as in Example 2, except that: A3. The tislelizumab solution is added to the dispersion and stirred at 50 r / min for 60 min at 4°C to form an emulsion.
[0099] Example 6
[0100] In this embodiment, the preparation methods of tislelizumab loaded on embolization microspheres, the preparation methods of modified gel materials, and the preparation methods of tislelizumab immunorelease embolization microspheres are the same as in Example 3, except that: A3. The tislelizumab solution is added to the dispersion and stirred at 40 r / min for 90 min at 4°C to form an emulsion.
[0101] Example 7
[0102] In this embodiment, the process is the same as in Example 3, except that: A1. 20 mg of polylactic acid-glycolic acid copolymer is dissolved in 99 mL of solvent, and 1 mg of Tween 80 is added to form a dispersion.
[0103] Comparative Example 1
[0104] Preparation of tislelizumab loaded onto embolized microspheres
[0105] A1. At 4°C, 0.4 mg of tislelizumab was dissolved in 9.5 mL of buffer solution with a pH of 6.5, and then 0.5 mL of a protective agent was added. The protective agent consisted of trehalose, Tween 20 and arginine, with a mass ratio of trehalose, Tween 20 and arginine of 1:0.2:0.3, to form the tislelizumab solution.
[0106] A2. Mix tislelizumab solution with 2 mg of purchased PLGA drug-loaded microspheres, stir at 60 r / min for 10 min at 4°C, wash three times with deionized water, and then freeze-dry to obtain PLGA drug-loaded microspheres loaded with tislelizumab.
[0107] The embolic microspheres loaded with tislelizumab prepared in Examples 1-7 and Comparative Example 1 were used. The concentration of tislelizumab was determined by high performance liquid chromatography (HPLC), and the amount of tislelizumab loaded on the microspheres was calculated. The test results are shown in Table 1.
[0108] Table 1
[0109]
[0110] According to the data in Table 1, the drug loading increases with increasing tislelizumab dosage and stirring time. However, as shown in Examples 3 and 6, when the dosage is the same, the increase in drug loading with increasing stirring time is smaller. That is, under the conditions in Example 3, the drug loading can reach 28.2, which can better balance dosage, drug loading time, and drug loading. Compared with direct drug loading, the method in this application can effectively improve the drug loading. A comparison between Examples 3 and 7 shows that the present invention, through deep cooling of the organic phase, greatly inhibits protein denaturation caused by shear heat during emulsification. Furthermore, the synergistic use of hydrophobic silica nanoparticle solid stabilizers and mixed solvents helps to form more stable primary emulsion droplets with a narrower particle size distribution, thus improving the drug loading. In this example, the drug loading represents the number of micrograms of tislelizumab loaded per milligram of dried embolic microspheres.
[0111] The tislelizumab immunorestricted embolization microspheres prepared in Examples 1-7 and Comparative Example 1 were added to dialysis bags, which were then placed in beakers. 50 mL of physiological saline was added to the beakers, and the bags were sealed. Samples were taken at 25°C every 30 min, 60 min, 120 min, 180 min, and 300 min to determine the drug concentration in the solution and calculate the cumulative release rate of the drug-loaded embolization microspheres. The test results are shown in Table 2.
[0112] Table 2
[0113]
[0114] According to the data in Table 2, in Examples 1-7, the average cumulative drug release rate of the embolization microspheres was 15.3-16.8% at 30 min; 26.1-27.9% at 60 min; 36.8-39.9% at 120 min; 40.1-44.2% at 180 min; and 42.4-48.2% at 300 min. The average cumulative drug release rate in Comparative Example 1 was much higher than that in this application, indicating that the tislelizumab immunorelief embolization microspheres prepared by the method of this application have good sustained-release properties. This is mainly due to the effect of the gel loaded with the targeting ligand on the microspheres, which avoids the rapid release caused by direct loading of tislelizumab.
[0115] Animal experiments were conducted on the embolic microspheres obtained in Examples 1-7 and Comparative Example 1. The experimental design is shown in Table 3, and the experimental data are shown in Table 4.
[0116] Table 3
[0117]
[0118] Table 4
[0119]
[0120] Regarding tumor volume inhibition rate (TGI), Examples 1-7 were significantly superior to each comparative example 1, indicating that the embolization microspheres of the present invention have stronger antitumor efficacy in terms of embolization and controlled release. Regarding the AFP (alpha-fetoprotein) index, AFP is a sensitive biomarker in rabbit liver cancer models; Examples 1-7 showed a large decrease, indicating the best tumor control effect. Regarding CD8... + Regarding T-cell infiltration rate, the embolic microspheres of this invention can enhance the immunoaffinity between the microspheres and liver tissue, promote immune cell infiltration, and enhance anti-tumor immunity. Regarding survival rate, all participants in Examples 1-7 survived, and pathological examination revealed no distant metastasis.
[0121] The foregoing provides a detailed description of tislelizumab immunorestricted embolization microspheres and their preparation method. The specific embodiments are provided only to aid in understanding the method and core concepts of this invention. It should be noted that those skilled in the art can make various improvements and modifications to this invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims.
[0122] It should be noted that: for experimental steps or conditions not specified in the examples, the procedures and conditions described in conventional experimental procedures in the literature of this art can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.
[0123] The above examples are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by anyone under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.
Claims
1. A tiragulimab immunocochleare embolization microsphere, characterized in that, The modified gel material is prepared by activating the gel material and then reacting with the targeting ligand.
2. The ramipiribumab immunocochleare embolization microspheres according to claim 1, characterized in that, The specific method for loading the embolization microspheres with the tirzepatamab includes: A1. Dissolving the polylactic acid-glycolic acid copolymer in a solvent, adding a stabilizer to form a dispersion liquid, A2. Dissolving the tirzepatamab in a buffer solution with a pH of 6.2-7.2 to form a tirzepatamab solution; A3. Adding the tirzepatamab solution to the dispersion liquid and slowly stirring at 4°C to form an emulsion; A4. Dropping the emulsion into deionized water and stirring at 4°C to perform phase separation to obtain a precipitate; A5. Washing and freeze-drying the precipitate to obtain the embolization microspheres loaded with the tirzepatamab.
3. The ramipiribumab immunocochleare embolization microspheres according to claim 2, characterized in that, In step A1, the polylactic acid-glycolic acid copolymer is dissolved in a mixed solvent of dichloromethane and ethyl acetate with a volume ratio of 4:1 to obtain an organic phase; hydrophobic silica nanoparticles with a particle size of 10-30 nanometers are added to the organic phase as a stabilizer and are uniformly dispersed by ultrasonic; the organic phase is pre-cooled to -20°C for 20-30 minutes to obtain a dispersion liquid.
4. The ramipiribumab immunocochleare embolization microspheres according to claim 3, characterized in that, In step A1, the concentration of the polylactic acid-glycolic acid copolymer in the dispersion liquid is 8-20 wt%; In step A2, the concentration of the tirzepatamab in the tirzepatamab solution is 0.4-0.8 mg / mL.
5. The ravulizumab immune-release embolization microspheres of claim 3, wherein, In step A2, a protective agent is added to the buffer solution, and the protective agent is one or a combination of trehalose, sucrose, Tween 20, Tween 80, histidine, and arginine.
6. The tiragulimab immunosuppressant-impregnated embolization microspheres of any one of claims 1-5, wherein, The coating thickness of the modified gel material is 100-300 nm.
7. The ramipiribumab immunocochleare embolization microspheres according to claim 6, characterized in that, The modified gel material is prepared by activating the gel material and then reacting with the targeting ligand.
8. The ramipiribumab immunocochleare embolization microspheres according to claim 7, characterized in that, The preparation of the modified gel material specifically includes the following steps: B1. Dissolving the natural gel in a buffer solution with a pH of 5.2-6.2 and adding EDC to activate for 2-5 hours to obtain an activated natural gel; B2. Dissolving the targeting ligand in a solvent to form a ligand solution, adding the ligand solution to the activated natural gel containing the buffer solution, and reacting for 3-6 hours; after the reaction, a blocking agent is added to obtain a modified gel material; B3. Purifying the modified gel material in step B2 to obtain a purified modified gel material.
9. A method of preparing the tiragolumab immunocryotherapeutic embolization microspheres according to any one of claims 1-8, characterized in that, The modified gel material is dissolved in a buffer solution with a pH of 4.2-5.2, the slow-release microspheres are added and stirred, sodium chloride is added during the stirring process, and the stirring is continued for 1-5 hours to obtain a precipitate, which is then washed and freeze-dried to obtain the embolization microspheres. The concentration of the sodium chloride is 0.1-0.3 mM, and the stirring speed is 60-200 r / min.
10. The method of claim 9, wherein,