Polymer microspheres loaded with metal nanoparticles, preparation method therefor, and use thereof

Abstract

The present invention relates to polymer microspheres loaded with metal nanoparticles, a preparation method therefor, and use thereof. The present invention pertains to the technical field of tumor embolization therapy. The polymer microspheres loaded with metal nanoparticles are characterized by comprising a polymer microsphere framework and metal nanoparticles distributed in the polymer microsphere framework; the metal nanoparticles are manganese nanoparticles or iron nanoparticles; the particle size of the metal nanoparticles is 600 nm-1000 nm. The particle size of the polymer microspheres meets the requirements of embolization therapy. The polymer microspheres can embolize local blood vessels, achieving an embolization therapy effect. Due to the addition of the metal nanoparticles, the microspheres have a higher rigidity and a better embolization effect than traditional polymer microspheres. The preparation yield of the polymer microspheres loaded with metal nanoparticles of the present invention is significantly higher (2-3 times) than the yield of polymer microspheres loaded with active metal microparticles such as magnesium microparticles, the production efficiency and the production safety are higher, and the production cost is lower.

Description

A polymer microsphere loaded with nano-metal particles and its preparation method and application Technical Field

[0001] The present invention relates to the technical field of tumor embolization therapy, and in particular to a polymer microsphere loaded with nano-metal particles, and a preparation method and application thereof. Background Art

[0002] Transarterial embolization / chemoembolization for liver tumors is the most commonly used non-surgical treatment for liver cancer. However, during traditional embolization, the lesion is exposed to a severe hypoxic environment, where a large number of hypoxia-tolerant liver cancer cells and tumor-derived stem cells accumulate, leading to failure of transarterial tumor embolization. This excessively hypoxic microenvironment, accompanied by a weakly acidic and immunosuppressive microenvironment, also promotes the rapid proliferation of liver cancer cells. Furthermore, because traditional transarterial tumor embolization rarely results in complete tumor necrosis, residual tumor lesions often lead to recurrence or metastasis after transarterial tumor embolization.

[0003] Drug-loaded microspheres are a new type of embolic material capable of loading chemotherapy drugs. They are typically composed of polymers, and through electrostatic interactions between the polymer microspheres and the chemotherapy drugs, they achieve sustained and slow drug release, resulting in high local drug concentrations within the tumor and low concentrations in the surrounding area, enhancing the effectiveness of embolic therapy. Although these polymer-loaded microspheres are already widely used, they still cannot overcome the drug resistance and tumor escape caused by the hypoxic environment at the tumor site. Therefore, the development of new embolic microspheres is needed to improve the local microenvironment during transarterial tumor embolization and avoid the problem of poor chemotherapy efficacy caused by tumor drug resistance.

[0004] Patent CN111514368A discloses a multifunctional liquid metal embolic agent comprising liquid metal, functional nanoparticles, and a polymer material. The liquid metal is spherical and forms the core of the embolic agent. The functional nanoparticles are dispersed on the surface of the liquid metal, and the polymer material coats the core and the functional nanoparticles. The liquid metal is selected from a gallium-indium alloy, a gallium-indium-tin alloy, a gallium-indium-tin-zinc alloy, or a gallium-indium-tin-zinc-bismuth alloy. Because the metal used in this patent is a liquid alloy, it lacks the ability to improve the local acidic microenvironment, resulting in limited efficacy and safety risks.

[0005] In addition, due to the strong activity of metal particles in the existing technology, the stability of the preparation process may be poor, making it difficult to achieve industrial transformation; therefore, there is an urgent need to provide a polymer microsphere loaded with nano-metal particles that has production safety, higher yield, improved microsphere embolization ability, and prolonged embolization time.

[0006] Summary of the Invention

[0007] In response to the above problems, the present invention provides a polymer microsphere loaded with nano-metal particles, and a preparation method and application thereof. The polymer gel is wrapped or loaded with metal nanoparticles. The particle size of the polymer microspheres meets the requirements of embolization therapy and can embolize local blood vessels to achieve the effect of embolization therapy. Compared with traditional polymer microspheres, due to the addition of metal nanoparticles, the microspheres have greater rigidity and strength, and better embolization effect. The safety of the microspheres described in the present invention is more controllable, especially production safety; the yield is higher, which is convenient for reducing costs; and the stability is better, which is convenient for extending the shelf life. In addition, the metal particles can improve the embolization ability of the microspheres, prolong the embolization time, and have the function of regulating the local microenvironment of the tumor, thereby enhancing the efficacy of chemotherapy or immunotherapy.

[0008] The present invention is achieved through the following technical solutions:

[0009] The first object of the present invention is to provide a polymer microsphere loaded with metal nanoparticles, comprising a polymer microsphere skeleton and metal nanoparticles distributed inside the polymer microsphere skeleton;

[0010] The metal nanoparticles are manganese nanoparticles or iron nanoparticles;

[0011] The particle size of the metal nanoparticles is 600nm to 1000nm.

[0012] If the particle size of metal nanoparticles is too small, they will be too active and react violently with water, making it impossible to prepare polymer microspheres loaded with metal nanoparticles. If the particle size of metal nanoparticles is too large, they will be insufficiently active and have limited therapeutic effects.

[0013] In one embodiment of the present invention, the particle size of the polymer microspheres is 10 μm to 500 μm.

[0014] In one embodiment of the present invention, the particle size of the polymer microspheres is 50 μm to 500 μm.

[0015] In one embodiment of the present invention, the metal nanoparticles occupy less than or equal to 50% of the volume of the polymer microspheres to ensure sufficient contact space with the microenvironment.

[0016] A second object of the present invention is to provide a method for preparing the polymer microspheres loaded with metal nanoparticles, which is characterized by comprising the following steps:

[0017] Step 1, preparing a polymer solution from a polymer material;

[0018] Step 2: Mix the polymer solution in step 1 with metal nanoparticles, and prepare the polymer microspheres loaded with metal nanoparticles by a microsphere preparation method.

[0019] In one embodiment of the present invention, in step 1, the polymer material contains positively charged or negatively charged functional groups, so that the polymer microspheres are not electrically neutral.

[0020] In one embodiment of the present invention, in step 1, the polymer material is a raw material that can be processed into microspheres.

[0021] In one embodiment of the present invention, the polymer material is selected from one or more of gelatin, polyvinyl alcohol, chitosan, degradable polyurethane, silk fibroin, collagen, alginic acid and degradable starch.

[0022] In one embodiment of the present invention, in step 1, the polymer solution includes a polymer solution that can undergo physical polymerization, chemical polymerization, or biological polymerization.

[0023] In one embodiment of the present invention, the chemical polymerization includes cross-linking and grafting.

[0024] In one embodiment of the present invention, in step 2, the metal nanoparticles are manganese nanoparticles or iron nanoparticles;

[0025] In one embodiment of the present invention, in step 2, the particle size of the metal nanoparticles is 600 nm to 1000 nm.

[0026] In one embodiment of the present invention, in step 2, the microsphere preparation method includes a reverse microemulsion method combined with UV curing and cross-linking.

[0027] The third object of the present invention is to provide an embolic agent comprising the polymer microspheres loaded with metal nanoparticles.

[0028] In one embodiment of the present invention, the embolic agent further comprises a dispersant.

[0029] In one embodiment of the present invention, the dispersant is an aqueous solvent.

[0030] In one embodiment of the present invention, the dispersant is selected from at least one of physiological saline, isotonic neutral buffer solution, and glucose solution.

[0031] In one embodiment of the present invention, the prepared polymer microspheres loaded with metal nanoparticles are freeze-dried.

[0032] The fourth object of the present invention is to provide a tumor embolic agent, comprising the polymer microspheres loaded with metal nanoparticles and a tumor therapeutic drug embedded or adsorbed on the surface or inside of the polymer microspheres loaded with metal nanoparticles.

[0033] In one embodiment of the present invention, the tumor therapeutic drug is combined with the polymer microspheres loaded with metal nanoparticles through electrostatic interaction.

[0034] In one embodiment of the present invention, the polymer microspheres loaded with metal nanoparticles are composed of one or more polymer materials such as gelatin, polyvinyl alcohol, chitosan, degradable polyurethane, silk fibroin, collagen, alginate and degradable starch.

[0035] In one embodiment of the present invention, the polymer microspheres loaded with metal nanoparticles contain a functional group R with a positive charge or a negative charge, and the drug contains a functional group with a charge opposite to that of the functional group R.

[0036] In one embodiment of the present invention, when the functional group R is one or more of a carboxyl group, an aldehyde group, a sulfonic acid group, a cyano group and a nitroamino group, the drug is selected from any one of epirubicin, pirarubicin, irinotecan, gemcitabine and aspirin.

[0037] In one embodiment of the present invention, when the functional group R is an amino group, the drug is selected from methotrexate.

[0038] In one embodiment of the present invention, the polymer microspheres loaded with metal nanoparticles can also be modified with positively charged polymer molecules, and the positively charged polymer molecules include but are not limited to polyallylamine hydrochloride, polyacrylic acid polyethyleneimine, polyvinyl pyrrolidone, cellulose, carboxyl-modified cellulose and polyvinyl alcohol.

[0039] By modifying the polymer microspheres loaded with metal nanoparticles with positively charged polymer molecules, the amount of charge carried by the polymer microspheres loaded with metal nanoparticles can be increased, thereby improving the quality of the drug loaded by the polymer microspheres loaded with metal nanoparticles per unit solid phase volume.

[0040] In one embodiment of the present invention, the polymer microspheres loaded with metal nanoparticles can also be modified with negatively charged polymer molecules, and the negatively charged polymer molecules include but are not limited to polyallylamine hydrochloride, polyacrylic acid, polyethylene glycol, polylactic acid, polyglycolic acid, and polylactic acid-glycolic acid copolymer.

[0041] By modifying the polymer microspheres loaded with metal nanoparticles with negatively charged polymer molecules, the amount of charge carried by the polymer microspheres loaded with metal nanoparticles can be increased, thereby improving the quality of the drug loaded by the polymer microspheres loaded with metal nanoparticles per unit solid phase volume.

[0042] The drug is selected from

[0043] In one embodiment of the present invention, the mass of the tumor therapeutic drug loaded in the polymer microspheres loaded with metal nanoparticles per unit solid volume is 0-30 mg / mL.

[0044] In one embodiment of the present invention, the drug is selected from one or more of immune checkpoint inhibitors, cytokines and immune adjuvants.

[0045] In one embodiment of the present invention, the drug is selected from one or more of epirubicin, pirarubicin, irinotecan, gemcitabine, methotrexate and aspirin.

[0046] A fifth object of the present invention is to provide a method for preparing polymer drug-loaded microspheres, comprising the following steps:

[0047] (1) preparing a polymer solution from a polymer material;

[0048] (2) The polymer solution of step (1) is mixed with metal nanoparticles, and the polymer microspheres loaded with metal nanoparticles are prepared by a microsphere preparation method.

[0049] (3) The polymer microspheres loaded with metal nanoparticles prepared in step (2) are mixed and shaken with the drug solution to obtain the polymer drug-loaded microspheres.

[0050] In one embodiment of the present invention, in step (1), the polymer material is a raw material that can be processed into microspheres.

[0051] In one embodiment of the present invention, in step (1), the polymer material is selected from one or more of gelatin, polyvinyl alcohol, chitosan, degradable polyurethane, silk fibroin, collagen, alginic acid and degradable starch.

[0052] In one embodiment of the present invention, the polymer solution includes a polymer solution that can undergo physical polymerization, chemical polymerization, or biological polymerization.

[0053] In one embodiment of the present invention, the chemical polymerization includes cross-linking and grafting.

[0054] In one embodiment of the present invention, in step (2), the microsphere preparation method includes a reverse microemulsion method.

[0055] In one embodiment of the present invention, in step (2), the reverse phase microemulsion method is implemented by microfluidics technology, which can better control the size of the microspheres.

[0056] In one embodiment of the present invention, the process further comprises curing the obtained polymer microspheres loaded with metal nanoparticles.

[0057] In one embodiment of the present invention, the curing treatment is selected from one or more of light curing, heat curing, X-ray curing, ultrasonic curing and cryo-curing; and the light curing is ultraviolet radiation curing.

[0058] In one embodiment of the present invention, the curing treatment is achieved by adding a curing agent to the polymer solution, and the curing agent includes methacrylic anhydride, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, etc.

[0059] In one embodiment of the present invention, the polymer solution further contains polymer molecules with positive or negative charges.

[0060] The negatively charged polymer molecules are selected from one or more of polyacrylic acid, polyethylene glycol, polylactic acid, polyglycolic acid, and polylactic-co-glycolic acid.

[0061] The positively charged polymer molecules are selected from one or more of polyallylamine, polyethyleneimine, polyvinylpyrrolidone, cellulose, carboxyl-modified cellulose and polyvinyl alcohol.

[0062] By modifying the polymer material by adding positively or negatively charged polymer molecules to the polymer solution, the charge carried by the prepared polymer microspheres can be increased, and the rate and loading capacity of the polymer microspheres loading drugs carrying opposite charges through electrostatic adsorption can be improved.

[0063] The above technical solution of the present invention has the following advantages over the prior art:

[0064] 1. Polymer gel encapsulates or loads metal nanoparticles. The particle size of polymer microspheres meets the requirements of embolization therapy and can embolize local blood vessels to achieve the effect of embolization therapy. Compared with traditional polymer microspheres, due to the addition of metal nanoparticles, the microspheres have greater rigidity and better embolization effect.

[0065] 2. Metal nanoparticles are small in size and have the size effect of nanoparticles. They decompose in the local slightly acidic tumor microenvironment, neutralize the local pH, generate hydrogen, and further improve the immunosuppressive microenvironment at the tumor site.

[0066] 3. Manganese ions or iron ions themselves can also participate in the immune signaling pathway, enhance the anti-tumor immune response, and thus combine with immunotherapy to achieve better therapeutic effects.

[0067] 4. The polymer microspheres loaded with metal nanoparticles of the present invention can utilize the electrostatic interaction between the polymer and the drug to achieve the loading of chemotherapy drugs, and can be combined with chemotherapy for transarterial chemoembolization (TACE).

[0068] 5. The preparation yield of the polymer microspheres loaded with metal nanoparticles described in the present invention is greatly improved (2 to 3 times) compared with the yield of the polymer microspheres loaded with active metal particles such as magnesium particles in PCT / CN2024 / 085858. Since magnesium powder is flammable and explosive, it has strict temperature and humidity requirements for the storage environment. Therefore, the production efficiency, cost and production safety of the polymer microspheres loaded with nano-metal particles of the present invention are advantageous, and the industrial application prospects are better. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0070] FIG1 is an optical microscope image of polymer microspheres loaded with iron nanoparticles;

[0071] FIG2 is an optical microscope image of polymer microspheres loaded with manganese nanoparticles;

[0072] FIG3 is an optical microscope image of polymer drug-loaded microspheres prepared using polymer microspheres loaded with iron particles as raw materials in Example 2;

[0073] FIG4 is an optical microscope image of polymer drug-loaded microspheres prepared using polymer microspheres loaded with manganese particles as raw materials in Example 2;

[0074] FIG5 is a line graph of tumor volume growth in the mouse H22 subcutaneous tumor model in Example 5.1;

[0075] FIG6 is a line graph showing tumor volume growth in the mouse H22 subcutaneous tumor model in Example 5.2. DETAILED DESCRIPTION

[0076] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0077] As used in this specification and claims, unless the context clearly indicates otherwise, the terms "a," "an," "an," and / or "the" are not intended to refer to the singular and may include the plural, unless the context clearly indicates otherwise. The terms "include" and "comprising" only indicate that the elements specifically identified are included, and these elements do not constitute an exclusive list; the device may also include other elements. The term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment." Relevant definitions of other terms are provided in the following description.

[0078] As described in this specification, the metal nanoparticles are usually spherical, and may also be ellipsoidal or irregular in shape. Unless otherwise specified, the size is defined by the major diameter. The major diameter of spherical particles is equal to the particle diameter.

[0079] Description of experimental materials: Polyacrylic acid (PAA, MW1800) and lithium phenyl-2,4,6-trimethylbenzoyl phosphite were purchased from Sigma-Aldrich.

[0080] Example 1: Preparation of polymer microspheres loaded with metal particles

[0081] The preparation method of this embodiment includes mixing and stirring an oily outer phase (oil phase) with an aqueous inner phase (aqueous phase) containing polymers and metal particles. The mixing and stirring process can be carried out under the control of a microfluidic chip or added dropwise through a pipette; then, the mixed phase is solidified by ultraviolet light to obtain a polymer microsphere product loaded with metal particles. The product can be separated from the solvent phase by centrifugation or other methods.

[0082] Example 1.1: Polymer microspheres loaded with iron particles

[0083] A polymer microsphere loaded with iron particles, wherein the polymer is methacrylic anhydride-modified gelatin protein, and each polymer microsphere contains one or more iron particles. The preparation method is as follows:

[0084] First, gelatin was modified to obtain methacrylic anhydride (MA)-grafted gelatin. Specifically, 2.5 g of gelatin was dissolved in 25 mL of DPBS solution and heated to dissolve. Then, 2 mL of methacrylic anhydride was added and stirred in a 50°C water bath for 3 h. The solution was then dialyzed in a 40°C water bath and freeze-dried to obtain methacrylated gelatin (GelMA).

[0085] Next, 5 mg of polyacrylic acid (PAA), 100 mg of methacrylated gelatin, and 5 mg of lithium phenyl-2,4,6-trimethylbenzoylphosphite (LAP) were dissolved in 1 mL of deionized water and heated to 60°C for complete dissolution. 100 mg of iron particles with a particle size of approximately 800 nm were then dispersed into the solution to form an aqueous phase. Simultaneously, 10 mL of mineral oil containing 2% Span 80 was prepared as the oil phase. While the oil phase was stirring, the aqueous phase was added dropwise. After the addition was complete, stirring was continued for approximately 10 minutes. The product was then cured by UV irradiation to obtain polymer microspheres loaded with 800 nm iron particles.

[0086] Figure 1 shows an optical microscope image of polymer microspheres loaded with 800nm ​​iron particles. The results show that the particle size of the polymer microspheres is approximately 100 microns. The surface is smooth, and the iron particles are evenly loaded inside. The morphology is relatively uniform, with good dispersion and no aggregation, facilitating embolization.

[0087] Due to the ionic bond between the surface of the iron particles and the methacrylated gelatin protein, the loading is stable. After the methacrylated gelatin protein microspheres loaded with iron particles are used for tumor embolization, the iron particles react in the acidic microenvironment of the tumor and the size of the iron particles gradually decreases. Due to the existence of ionic bond forces, the iron particles are not easy to leak out of the high-molecular-weight methacrylated gelatin protein spheres. The iron particles can still stay at the embolization site under the action of the high-molecular-weight methacrylated gelatin protein shell and continue to play a role.

[0088] Example 1.2: Polymer microspheres loaded with manganese particles

[0089] A polymer microsphere loaded with manganese particles, wherein the polymer is methacrylated gelatin, and each polymer microsphere contains one or more manganese particles. Polymer microspheres loaded with manganese particles of different sizes were prepared, wherein the manganese particles were 900 nm in size and were prepared by the method described in Example 1.1.

[0090] Figure 2 shows an optical microscope image of polymer microspheres loaded with 900nm manganese particles. The average particle size of the manganese-loaded polymer microspheres is approximately 80 microns. The microspheres are smooth on the outside, with uniform manganese particles inside, a relatively uniform morphology, and good dispersion without aggregation, facilitating embolization.

[0091] Example 2: Preparation of polymer drug-loaded microspheres

[0092] The polymer microspheres loaded with metal particles prepared in Example 1 and epirubicin hydrochloride were used as raw materials to prepare polymer drug-loaded microspheres. The specific preparation method is as follows:

[0093] The polymer microspheres loaded with metal particles were added to a drug solution containing epirubicin hydrochloride (EPI), placed in an oscillator and shaken up and down for 3-5 minutes, and centrifuged again (100 rpm, 3 minutes) to remove the supernatant solution to obtain the polymer drug-loaded microspheres successfully loaded with EPI.

[0094] The product morphology was observed by optical microscopy. Figures 3 and 4 are the optical microscopy imaging results of polymer drug-loaded microspheres prepared using polymer microspheres loaded with iron particles and polymer microspheres loaded with manganese particles as raw materials. The results show that the morphology of the microspheres did not change significantly after drug loading, and the particle size changed slightly, indicating that the microspheres obtained by the above preparation method have good stability. After observation, the polymer drug-loaded microspheres prepared in this embodiment under room temperature conditions were placed in a neutral buffer solution (physiological saline) for 1 day, and their drug loading was stable, and no obvious drug release occurred. This shows that the preparation method of the polymer drug-loaded microspheres of the present invention is simple, reproducible, and the product is relatively stable.

[0095] Example 3: Preparation of tumor embolic agent

[0096] This embodiment provides a tumor embolic agent comprising the metal particle-loaded polymer microspheres described in Examples 1.1 and 1.2, or the drug-loaded polymer microspheres described in Example 2. The agent is stored in a dry, nitrogen-filled environment. Upon use, the microspheres are dispersed in an aqueous solvent, such as physiological saline, isotonic neutral buffer, glucose solution, or other biocompatible solution, and delivered to the site of embolization via an interventional catheter for embolization therapy.

[0097] Example 4: Drug loading performance test of polymer drug-loaded microspheres

[0098] Drug loading rate test of polymer drug-loaded microspheres:

[0099] Polymer drug-loaded microspheres were prepared according to the method described in Example 2. After preparation, the supernatant solution was collected and the mass of residual epirubicin hydrochloride was determined. The actual mass of loaded epirubicin hydrochloride was calculated by subtracting the known mass of the charged epirubicin hydrochloride from the mass of the residual epirubicin hydrochloride in the supernatant solution after preparation. The drug loading efficiency was calculated as follows: Drug loading efficiency = actual loaded epirubicin hydrochloride mass / charged epirubicin hydrochloride mass * 100%.

[0100] The examples are grouped as follows:

[0101] Control Example 4.1: Blank polymer microspheres were prepared according to the method described in Example 1.1, except that no metal particles and polyacrylic acid (PAA) were added to the aqueous phase;

[0102] Comparative Example 4.2: Iron-loaded polymer microspheres were prepared according to the method described in Example 1.1. The iron particles were approximately 1 μm in size, except that polyacrylic acid (PAA) was not added to the aqueous phase.

[0103] Comparative Example 4.3: Polymer microspheres loaded with manganese particles were prepared according to the method described in Example 1.2. The size of the manganese particles was about 1 μm, except that polyacrylic acid (PAA) was not added to the aqueous phase.

[0104] Control Example 4.4: Blank polymer microspheres were prepared as described in Example 1.1, except that no metal particles were added to the aqueous phase;

[0105] Example 4.5: Polymer microspheres loaded with iron particles prepared according to the method described in Example 1.1, the iron particles having a size of about 1 μm;

[0106] Example 4.6: Polymer microspheres loaded with manganese particles were prepared according to the method described in Example 1.2, and the size of the manganese particles was about 1 micron.

[0107] The test results of the drug loading rate of the microspheres involved in this embodiment are shown in Table 1.

[0108] Table 1: Drug loading rate test of polymer drug-loaded microspheres

[0109] The test results in Table 1 show that the drug loading rates in Examples 4.5-4.6 were significantly higher than those in Control Examples 4.2-4.3, indicating that the introduction of PAA can significantly improve the loading efficiency of the microspheres for cationic drugs. In addition, the polymer microspheres containing metal particles have a higher drug loading rate within the same drug loading time. This is because the introduction of metal particles increases the specific surface area of ​​the polymer microspheres, resulting in a higher drug loading rate. At this EPI concentration, the polymer drug-loaded microspheres can quickly load most of the epirubicin in a short period of time, which is more conducive to practical drug loading.

[0110] Maximum drug loading capacity test of polymer drug-loaded microspheres:

[0111] The above test results show that the drug loading of the polymer drug-loaded microspheres has not reached saturation at this EPI concentration; the maximum drug loading capacity of the microspheres per unit solid phase volume was further tested: a certain solid phase volume (0.5 mL) of GelMA-PAA-Mg microspheres prepared according to the method described in Example 9.6 was mixed with 2 mL of high-concentration epirubicin hydrochloride and placed in an oscillator and shaken up and down for 10 minutes. After completion, centrifugation was performed again (100 r / min, 3 minutes), the supernatant solution was collected and the mass of the residual epirubicin hydrochloride therein was detected to evaluate the maximum drug loading capacity of the microspheres per unit solid phase volume. The calculation formula is as follows: Maximum drug loading capacity of microspheres per unit solid phase volume = maximum drug loading capacity / microsphere solid phase volume

[0112] The drug loading ranges of different microspheres were tested according to the above method, and the test results are shown in Table 2.

[0113] Table 2: Maximum drug loading capacity test of polymer drug-loaded microspheres

[0114] Testing has shown that when the total amount of epirubicin hydrochloride added exceeds 16 mg, the drug loading of the metal particle-loaded polymer microspheres with a solid phase volume of 0.5 mL gradually reaches saturation. The maximum drug loading of the polymer drug-loaded microspheres in Examples 4.5-4.6 is significantly improved compared to that of Control Examples 4.2-4.3, further demonstrating the effectiveness of polymer modification in increasing the charge carried by the microspheres. The drug loading of these polymer drug-loaded microspheres is high, meeting the needs of clinical use, and combined with the functionality of the metal particles, is expected to achieve even better therapeutic effects.

[0115] Example 5: Study on the Tumor Inhibition Effect of Polymer Drug-Loaded Microspheres in H22 Subcutaneous Tumors

[0116] Female Balb / c mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. and maintained according to protocols approved by the Experimental Animal Center of our university. The tumor inhibitory effect of polymer-loaded drug-loaded microspheres was verified in a mouse hepatocyte subcutaneous tumor model (H22 subcutaneous tumor). The experimental methods are as follows:

[0117] H22 tumor cells were inoculated on the lower right side of the back of BALB / c mice to establish a subcutaneous H22 liver cancer model. When tumors reached 100 mm³, the mice were randomly divided into four groups of six and treated with intratumoral microsphere injections. Day 0 was designated as treatment, and tumor growth was monitored every two days.

[0118] Example 5.1: Study on the Anti-tumor Effect of Polymer Drug-Loaded Microspheres (Loaded with Iron Particles)

[0119] Iron-loaded polymer microspheres and blank polymer microspheres were prepared according to the method described in Example 1, except that no metal particles were added to the aqueous phase. Furthermore, using the above-mentioned polymer microspheres and epirubicin hydrochloride as raw materials, polymer drug-loaded microspheres were prepared according to the method described in Example 2, and their anti-tumor effects were studied.

[0120] The specific experimental groups and treatment doses are as follows:

[0121] Control Example 5.1.1: Intratumoral injection of blank polymer microspheres;

[0122] Control Example 5.1.2: Intratumoral injection of polymer microspheres loaded with iron particles, the injection dose is 1.5 mg / animal based on the weight of iron;

[0123] Control Example 5.1.3: Intratumoral injection of polymer drug-loaded microspheres prepared with blank polymer microspheres and epirubicin hydrochloride as raw materials, the injection dose is 50 μg / mouse based on EPI mass;

[0124] Example 5.1.4: Intratumoral injection of polymer drug-loaded microspheres prepared with iron-loaded polymer microspheres and epirubicin hydrochloride as raw materials, with an injection dose of 1.5 mg / mouse based on the mass of iron and 50 μg / mouse based on the mass of epirubicin;

[0125] The statistical results of the tumor volume of tumor-bearing mice after administration are shown in Figure 5. The tumor volume of the mice in Example 5.1.2 was significantly smaller than that in Control Example 5.1.1, indicating that the iron particles loaded in the polymer microspheres can continuously produce hydrogen and hydroxide after being injected into the tumor site, exerting a significant anti-tumor effect. The tumor volume of the mice in Example 5.1.4 was significantly smaller than that in the other three groups, indicating that the therapeutic effect of intratumoral injection of polymer drug-loaded microspheres loaded with iron particles was the best, achieving the combination of tumor microenvironment regulation, hydrogen immunotherapy and chemotherapy, and effectively inhibiting the growth of mouse liver cancer cells.

[0126] Example 5.2: Study on the anti-tumor effect of polymer drug-loaded microspheres (loaded with manganese particles)

[0127] Polymer microspheres loaded with manganese particles and blank polymer microspheres were prepared according to the method described in Example 1, except that no metal particles were added to the aqueous phase. Furthermore, using the above-mentioned polymer microspheres and epirubicin hydrochloride as raw materials, polymer drug-loaded microspheres were prepared according to the method described in Example 2, and their anti-tumor effects were studied.

[0128] The specific experimental groups and treatment doses are as follows:

[0129] Control Example 5.2.1: Intratumoral injection of blank polymer microspheres;

[0130] Control Example 5.2.2: Intratumoral injection of polymer microspheres loaded with manganese particles, the injection dose is 1 mg / animal based on the mass of manganese;

[0131] Control Example 5.2.3: Intratumoral injection of polymer drug-loaded microspheres prepared with blank polymer microspheres and epirubicin hydrochloride as raw materials, the injection dose is 50 μg / mouse based on EPI mass;

[0132] Example 5.2.4: Intratumoral injection of polymer drug-loaded microspheres prepared with polymer microspheres loaded with manganese particles and epirubicin hydrochloride as raw materials, with an injection dose of 1 mg / mouse based on the mass of manganese and 50 μg / mouse based on the mass of epirubicin;

[0133] The statistical results of the tumor volume of tumor-bearing mice after administration are shown in Figure 6. The tumor volume of the mice in Example 5.2.2 was significantly smaller than that in Control Example 5.2.1, indicating that the manganese particles loaded in the polymer microspheres can exert an anti-tumor effect similar to that of iron particles after being injected into the tumor site. The tumor volume of the mice in Example 5.2.4 was significantly smaller than that in the other three groups, indicating that the therapeutic effect of intratumoral injection of polymer drug-loaded microspheres loaded with manganese particles was the best, and the therapeutic effect was more significant compared with single chemical drug treatment.

[0134] The test results of Example 5 collectively illustrate that by regulating the size of metal particles, their reactivity can be regulated. For iron / manganese particles, when their particle size is reduced to the nanoscale (100-1000 nm), they still have strong reactivity after being wrapped in a polymer material. After reaching the tumor site through intratumoral injection or interventional surgery, they can produce sufficient hydrogen to achieve anti-tumor therapeutic efficacy. At the same time, the hydroxide produced can effectively improve tumor microacidity, inhibit the generation of drug-resistant cells, and amplify the efficacy of chemotherapy. It has broad application prospects in many solid tumors.

[0135] Example 6

[0136] In this example, microspheres loaded with metal particles of different particle sizes were prepared by referring to the method described in Example 1.1, and the products were collected. Empty microspheres and aggregated particles were removed to obtain a relatively uniform product. The specific parameters and results are shown in Table 3:

[0137] Table 3

[0138] As shown in Table 3, loading manganese and iron particles with a particle size of approximately 600 to 1000 nm resulted in a higher effective yield, while loading larger metal particles significantly reduced the effective yield of the final polymer microspheres. Furthermore, when using smaller metal particles as raw materials to prepare polymer microspheres, the size effect of the nanoparticles leads to higher metal particle reactivity, making the production process uncontrollable and the effective yield fluctuating significantly, making it unsuitable for scaled-up production.

[0139] It can be seen that the size (long diameter) of the metal particles affects their reactivity and hydrogen generation efficiency. The smaller the metal particle size, the smaller the particle size of the polymer microspheres loaded with the metal particles, the stronger the reactivity, and the faster the hydrogen generation rate. Experimental verification shows that when the iron and manganese particles are less than 1000nm in size, the polymer-coated iron / manganese particles have better reactivity and can produce obvious bubbles in the buffer solution, indicating that the hydrogen generation rate is sufficient to exert anti-tumor activity. The polymer microspheres formed by loading metal particles of this size are ideally sized, with an average particle size of approximately 50-100 microns, which is conducive to subsequent drug loading.

[0140] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A polymer microsphere loaded with metal nanoparticles, characterized in that: It includes a polymer microsphere skeleton and metal nanoparticles distributed inside the polymer microsphere skeleton; The metal nanoparticles are manganese nanoparticles or iron nanoparticles; The particle size of the metal nanoparticles is 600nm to 1000nm.

2. The polymer microspheres according to claim 1, characterized in that The particle size of the polymer microspheres is 10 μm to 500 μm.

3. The polymer microspheres according to claim 1, characterized in that The metal nanoparticles account for less than or equal to 50% of the volume of the polymer microspheres.

4. A method for preparing polymer microspheres loaded with metal nanoparticles according to any one of claims 1 to 3, characterized in that: The following steps are involved: Step 1, preparing a polymer solution from a polymer material; Step 2: Mix the polymer solution in step 1 with metal nanoparticles, and prepare the polymer microspheres loaded with metal nanoparticles by a microsphere preparation method.

5. The preparation method according to claim 4, characterized in that In step 1, the polymer material contains positively charged or negatively charged functional groups, so that the polymer microspheres are not electrically neutral.

6. The preparation method according to claim 4 or 5, characterized in that The polymer material is selected from one or more of gelatin, polyvinyl alcohol, chitosan, degradable polyurethane, silk fibroin, collagen, alginic acid and degradable starch.

7. The preparation method according to claim 4, characterized in that In step 1, the polymer solution includes a polymer solution capable of physical polymerization, chemical polymerization or biological polymerization.

8. The preparation method according to claim 4, characterized in that In step 2, the metal nanoparticles are manganese nanoparticles or iron nanoparticles.

9. The preparation method according to claim 4, characterized in that In step 2, the particle size of the metal nanoparticles is 600 nm to 1000 nm.

10. An embolic agent, characterized in that: The invention comprises the polymer microspheres loaded with metal nanoparticles as claimed in claim 1.

11. A tumor embolic agent, characterized in that: The invention comprises the polymer microspheres loaded with metal nanoparticles according to any one of claims 1 to 3 and tumor therapeutic drugs embedded or adsorbed on the surface or inside of the polymer microspheres loaded with metal nanoparticles.

12. The tumor embolic agent according to claim 11, characterized in that The tumor therapeutic drug is combined with the polymer microspheres loaded with metal nanoparticles through electrostatic interaction.

13. The tumor embolic agent according to claim 11, characterized in that The polymer microspheres loaded with metal nanoparticles contain a functional group R with a positive charge or a negative charge, and the drug contains a functional group with a charge opposite to that of the functional group R.

14. The tumor embolic agent according to claim 11, characterized in that The mass of the tumor therapeutic drug loaded in the polymer microspheres loaded with metal nanoparticles per unit solid phase volume is 0-30 mg / mL.

15. The tumor embolic agent according to claim 11, characterized in that The drug is selected from one or more of immune checkpoint inhibitors, cytokines, and immune adjuvants.

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