Use of silver ion-coated particles in the preparation of a medicament for treating b-cell malignancies

Abstract

The application belongs to the tumor treatment field, and particularly relates to application of silver ion coated particles in preparation of a drug for treating B cell malignant tumors. The silver ion coated particles include one of silver-quinoline albumin nanoparticles, silver liposome nanoparticles, silver coordination polymer nanoparticles and silver-loaded ZIF-8 nanoparticles. The application has a simple preparation process, and the obtained nanoparticles can significantly improve the stability of silver ions, specifically kill B cell tumor cells, have low toxicity to normal immune cells, have excellent in-vivo anti-tumor effect, can be used for treating various B cell malignant tumors, and is easy to be mass-produced and clinically transformed.

Description

Technical Field

[0001] This invention belongs to the field of tumor treatment, specifically relating to the application of silver ion-coated particles in the preparation of drugs for treating B-cell malignant tumors. Background Technology

[0002] B-cell malignancies are common malignant tumors of the hematologic system, mainly including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), Burkitt lymphoma, chronic lymphocytic leukemia (CLL), and B-cell acute lymphoblastic leukemia, accounting for more than 80% of non-Hodgkin's lymphomas, and the incidence rate is showing an increasing trend year by year.

[0003] Currently, clinical treatment options for B-cell malignancies mainly include traditional chemotherapy, CD20 monoclonal antibody targeted therapy, antibody-drug conjugates (ADCs), and CAR-T cell therapy. These treatments have significantly improved remission rates, but still have many limitations: relapsed and refractory patients have a poor overall prognosis, and multidrug resistance is common; targeted drugs such as CD20 monoclonal antibodies have limited efficacy against some subtypes; CAR-T cell therapy is expensive, has a long preparation cycle, and carries the risk of adverse reactions such as cytokine storms, making it difficult to cover all patients.

[0004] Silver ions possess broad-spectrum biological activity, and studies have confirmed that they exert anti-tumor effects through multiple pathways, including inducing excessive production of intracellular reactive oxygen species (ROS), causing DNA damage, disrupting mitochondrial function, and arresting the cell cycle. However, free silver ions exhibit extremely poor stability in the physiological environment, readily binding to chloride ions, serum proteins, and bio-thiols in body fluids and becoming inactive, resulting in a very short half-life in vivo. Furthermore, free silver ions lack tissue selectivity, easily causing damage to normal tissues and exhibiting significant systemic toxicity, which greatly limits their clinical application in tumor treatment.

[0005] Existing research has reported the use of metals to construct nanomedicines, but there is no research on the use of silver ion-coated particles for the treatment of B-cell malignancies. Therefore, there is an urgent need to develop a stable, efficient and safe silver ion delivery system to provide a new treatment strategy for B-cell malignancies. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides an application of silver ion-coated particles in the preparation of drugs for treating B-cell malignant tumors. The silver ion-coated particles comprise one of silver-quinoline albumin nanoparticles, silver liposome nanoparticles, silver coordination polymer nanoparticles, and silver-loaded ZIF-8 nanoparticles; wherein:

[0007] The preparation method of the silver-quinoline albumin nanoparticles includes the following steps:

[0008] (1) Dissolve albumin in water to obtain an albumin aqueous solution;

[0009] (2) Add an alkaline regulator to the albumin aqueous solution to obtain an alkaline mixture;

[0010] (3) Add silver ion solution to the alkaline mixture and stir to obtain the first reaction solution;

[0011] (4) Add quinoline ligand solution to the first reaction solution and stir to obtain the second reaction solution;

[0012] (5) Dialyze the second reaction solution to remove free silver ions and free ligands to obtain a purified product;

[0013] (6) After freeze-drying the purified product, silver-quinoline albumin nanoparticles are obtained.

[0014] Preferably, in step (1), albumin is dissolved in ultrapure water to obtain an albumin aqueous solution with a concentration of 2.5-10 mg / mL;

[0015] And / or, the albumin includes one or more of bovine serum albumin (BSA), human serum albumin (HSA), and recombinant albumin.

[0016] Preferably, in step (2), an alkaline regulator is added to the albumin aqueous solution at a speed of 400-800 r / min to obtain an alkaline mixture with a pH of 9.0-11.0;

[0017] And / or, the alkalinity regulator is a sodium hydroxide solution.

[0018] Preferably, in step (3), under the conditions of avoiding light, 20-30℃ (room temperature), and 400-800r / min, silver ion solution is added to the alkaline mixture and stirred for 10-30min to obtain the first reaction solution;

[0019] And / or, the silver ion solution is an aqueous solution of silver nitrate or an aqueous solution of silver acetate;

[0020] And / or, the concentration of silver ions in the silver ion solution is 50-200 mM (dissolved in 250-1000 μL of pure water).

[0021] Preferably, in step (4), under conditions of avoiding light, 20-30℃ (room temperature), and 400-800r / min, a quinoline ligand solution is added to the first reaction solution and stirred for 20-24h to obtain the second reaction solution;

[0022] And / or, the quinoline ligand solution is an acetone solution of 8-hydroxyquinoline (8-HQ) ligand or an acetone solution of nitroquinoline (NQ) ligand;

[0023] And / or, the concentration of the quinoline ligand solution is 25-100 mM (dissolved in 1150-4600 μL of acetone).

[0024] And / or, the molar ratio of the silver ions to the quinoline ligand is 1:2 to 1:2.5.

[0025] Preferably, in step (5), the second reaction solution is placed in a dialysis bag, and then the dialysis bag is placed in pure water and dialyzed at 3-5°C for 24-48 hours to remove free silver ions and free ligands, thereby obtaining the purified product.

[0026] Preferably, in step (6), the purified material is pre-frozen at -80°C and then freeze-dried at -90°C and vacuum degree ≤10Pa for 24-30 hours to obtain silver-quinoline albumin nanoparticles.

[0027] Based on the same technical concept, the present invention provides another drug for treating B-cell malignant tumors, wherein the drug is a formulation prepared by adding pharmaceutically acceptable excipients to silver ion-coated particles (silver-quinoline albumin nanoparticles, silver liposome nanoparticles, silver coordination polymer nanoparticles and / or silver-loaded ZIF-8 nanoparticles) as raw materials.

[0028] Preferably, the formulation is an oral formulation or an injectable formulation.

[0029] In addition, the preparation method of the silver liposome nanoparticles (Ag@lipo) is as follows:

[0030] (1) Weigh distearyl phosphatidylcholine (DSPC), cholesterol and distearyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) and mix them so that the molar ratio of the three is 22-26:8-10:0.8-1.2 to obtain mixed raw materials.

[0031] (2) Dissolve the mixed raw materials in a mixed solvent of chloroform and anhydrous ethanol (the volume ratio of chloroform and anhydrous ethanol is 3:7) to obtain a mixed solution.

[0032] (3) The mixture is rotary evaporated in a water bath at 35-45℃ to form a film, and then vacuum dried for 1-2 hours to obtain a dried lipid film.

[0033] (4) Dissolve the dried lipid membrane in pure water, add silver nitrate solution to hydrate it in a water bath at 65-75℃, and stop when the membrane is completely hydrated to obtain the hydrated solution.

[0034] (5) The hydrated liquid is first passed through a membrane with a large pore size (350-450nm), then through a membrane with a target pore size (150-250nm), and finally placed in a 3500Da dialysis membrane for dialysis to remove free silver ions for 24-48h, thus obtaining the silver liposome nanoparticles (Ag@lipo).

[0035] The preparation method of the silver coordination polymer nanoparticles (Ag-ICPs) is as follows:

[0036] (1) Dissolve polyvinylpyrrolidone K30 (PVP-K30) in pure water to obtain a PVP-K30 aqueous solution with a concentration of 1-3% (w / v).

[0037] (2) Dissolve silver nitrate and 4,4'-bipyridine in pure water and anhydrous ethanol respectively to obtain silver nitrate solution and 4,4'-bipyridine solution.

[0038] (3) Place the PVP-K30 aqueous solution in a round-bottom flask at a water bath of 20-30℃ and a rotation speed of 700-900r / min, and slowly add silver nitrate solution and 4,4'-bipyridine solution at the same time. Stir for 1-3 hours in the dark to obtain the reaction solution.

[0039] (4) The reaction solution is transferred into a 3500 Da dialysis bag for dialysis to remove free silver ions and 4,4'-bipyridine, thereby obtaining the silver coordination polymer nanoparticles (Ag-ICPs).

[0040] The preparation method of the silver-loaded ZIF-8 nanoparticles (Ag@ZIF-8) is as follows:

[0041] (1) Dissolve zinc nitrate hexahydrate and 2-methylimidazole in methanol respectively, and sonicate at room temperature for 5-10 min to completely dissolve them, to obtain zinc nitrate hexahydrate methanol solution and 2-methylimidazole methanol solution.

[0042] (2) Place the 2-methylimidazolium methanol solution in a round-bottom flask and stir magnetically (400-800 r / min), then quickly pour in the zinc nitrate hexahydrate methanol solution and stir at room temperature for 2 h.

[0043] (3) Add silver nitrate and continue stirring overnight at room temperature in the dark.

[0044] (4) Wash at 7000-9000 r / min for 8-12 min and discard the supernatant. Then resuspend in methanol and repeat 2-4 times to obtain silver-loaded ZIF-8 nanoparticles (Ag@ZIF-8). Finally, they can be stored in an appropriate volume of methanol.

[0045] The beneficial effects of this invention are as follows:

[0046] 1. The preparation process is simple and controllable, and easy to scale up production: The preparation process of silver ion coated particles provided by this invention is simple and easy to control: Silver-quinoline albumin nanoparticles are formed by coordination self-assembly using natural albumin as a carrier, without the need to add organic crosslinking agents, and the reaction conditions are mild; the preparation steps of silver liposomes, silver coordination polymers and silver-loaded ZIF-8 nanoparticles are few, the parameters are highly controllable, all schemes can be directly scaled up for production, and the preparation cost is low.

[0047] 2. Significantly improves the stability and bioavailability of silver ions: The silver ion-coated particles provided by this invention can fix silver ions inside the particles through coating, coordination, or pore confinement, effectively preventing silver ions from binding and becoming inactive with components such as chloride ions and thiol groups in the physiological environment, prolonging the in vivo circulating half-life, and increasing the accumulation of silver ions at the tumor site; among them, silver-quinoline albumin nanoparticles can form stable coordination complexes with silver ions through quinoline ligands, further enhancing the structural stability and controllable release of silver ions.

[0048] 3. Excellent biocompatibility and safety: The carrier materials of the silver ion coated particles provided by this invention all have good biodegradability and low immunogenicity: albumin is an endogenous plasma protein that can be completely biodegraded in vivo; liposomes, coordination polymers and ZIF-8 carriers are all recognized biocompatible materials in the biomedical field, and there are no toxic cross-linking agents or highly toxic reagents left in the entire preparation process. The nanoparticles have no obvious killing effect on normal immune cells, have a wide therapeutic window and low systemic toxicity.

[0049] 4. Possesses B-cell-specific antitumor activity: The silver ion-coated particles provided in this invention, including silver-quinoline albumin nanoparticles, silver liposome nanoparticles, and silver coordination polymer nanoparticles, can selectively kill B cells and B-cell-derived malignant tumor cells. They exhibit no significant toxicity to normal immune cells such as T cells, NK cells, monocytes / macrophages, and neutrophils, and show potent inhibitory effects on the proliferation of various B-cell malignant tumor cell lines. Silver-loaded ZIF-8 nanoparticles demonstrate cytotoxic effects on different cell types compared to the other three types of nanoparticles.

[0050] 5. Significant anti-tumor effect in vivo: The silver ion-coated particles provided by this invention can be administered through multiple routes such as intraperitoneal and intratumoral administration, and can effectively inhibit the in vivo growth of B-cell lymphoma. The tumor-suppressing effect is obvious, providing a new treatment option for relapsed and refractory B-cell malignant tumors.

[0051] 6. Great potential for formulation development and clinical translation: The silver ion coated particles provided by this invention are stable and can be stored for a long time after freeze-drying. They can be further prepared into commonly used clinical dosage forms such as injections, and have good clinical application prospects. Attached Figure Description

[0052] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0053] Figure 1 This graph shows the cytotoxic effects of different groups on different cells at a dose of 10 μM, based on the metal content in the nanoparticles. The cells were all derived from human peripheral blood; the x-axis represents B cells and CD4 cells, respectively. + Helper T cells, CD8 + Markers for cytotoxic T cells, NK cells, monocytes / macrophages, myeloid cells, and neutrophils.

[0054] Figure 2 This is a diagram showing the results of an in vitro cell experiment (cell proliferation) using Ag-8-HQ@BSA.

[0055] Figure 3 This is a diagram showing the results of an in vitro cell experiment (cell proliferation) with Ag@lipo.

[0056] Figure 4 This is a diagram showing the results of an in vitro cell experiment (cell proliferation) using Ag-ICPs.

[0057] Figure 5 This is a diagram showing the results of an in vitro cell experiment (cell proliferation) using Ag@ZIF-8.

[0058] Figure 6 This is the particle size distribution diagram of Ag-8-HQ@BSA.

[0059] Figure 7 This is the particle size distribution diagram of Ag@lipo.

[0060] Figure 8 This is a particle size distribution diagram of Ag-ICPs.

[0061] Figure 9 This is the particle size distribution diagram of Ag@ZIF-8.

[0062] Figure 10 This is a Zeta potential diagram.

[0063] Figure 11 This is a TEM image of Ag-8-HQ@BSA.

[0064] Figure 12 This is a TEM image of Ag@lipo.

[0065] Figure 13 This is a TEM image of Ag-ICPs.

[0066] Figure 14 This is a TEM image of Ag@ZIF-8.

[0067] Figure 15 This is a schematic diagram of mouse modeling and drug administration.

[0068] Figure 16 This is a statistical graph of mouse tumor volume. Detailed Implementation

[0069] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0070] Example 1

[0071] This embodiment provides a method for preparing silver-quinoline bovine serum albumin nanoparticles, the preparation method comprising the following steps:

[0072] (1) Weigh 50 mg of bovine serum albumin (BSA) with a purity ≥ 98%, add 10 mL of ultrapure water, and place it in a 25°C water bath and stir at low speed until completely dissolved to obtain a clear albumin aqueous solution with a concentration of 5 mg / mL.

[0073] (2) At room temperature (25℃), place the albumin aqueous solution on a magnetic stirrer and stir at a speed of 600 r / min; use a microsyringe to slowly add 0.1 mol / L sodium hydroxide solution at a speed of 50 μL / min, monitor the pH value of the system in real time until the pH stabilizes at 10.0, and obtain an alkaline mixture.

[0074] (3) Keep the stirring speed at 600 r / min, wrap the reaction vessel with aluminum foil to avoid light throughout the process, and slowly add 500 μL of 100 mM silver nitrate aqueous solution at a rate of 30 μL / min; after the addition is completed, continue to stir the reaction in the dark for 20 min to obtain the first reaction solution.

[0075] (4) Maintain a stirring speed of 600 r / min and a light-protected environment, slowly add 2200 μL of 50 mM 8-hydroxyquinoline acetone solution at a rate of 40 μL / min, and control the molar ratio of silver ions to 8-hydroxyquinoline to be 1:2.2. After the addition is complete, continue stirring at room temperature in the dark for 24 h to obtain the second reaction solution.

[0076] (5) After the reaction is complete, take a dialysis bag with a molecular weight cutoff of 30 kDa, boil it in ultrapure water for 10 minutes in advance, and cool it to room temperature before use; transfer all the second reaction solution into the dialysis bag, clamp the two ends to seal, place it in a beaker containing 1 L of ultrapure water, and place it in a 4℃ refrigerator for dialysis for 36 h. Change the ultrapure water every 8 h, and change it 3 to 4 times in total to fully remove free silver ions and free ligands, and obtain purified nanoparticle dispersion.

[0077] (6) The dialysis nanoparticle dispersion was transferred to a freeze-drying bottle and pre-frozen in a -80℃ freezer for 4 hours; then it was placed in a freeze dryer and freeze-dried for 24 hours at -90℃ and a vacuum degree ≤10Pa to obtain silver-8-hydroxyquinoline bovine serum albumin nanoparticles (denoted as: Ag-8-HQ@BSA).

[0078] Example 2

[0079] This embodiment provides a method for preparing silver-quinoline human serum albumin nanoparticles, the preparation method comprising the following steps:

[0080] (1) Weigh 75 mg of human serum albumin (HSA) with a purity ≥ 99%, add 10 mL of ultrapure water, and place it in a 25°C water bath and stir at low speed until completely dissolved to obtain a clear albumin aqueous solution with a concentration of 7.5 mg / mL.

[0081] (2) At room temperature (25℃), place the albumin aqueous solution on a magnetic stirrer and stir at a speed of 500 r / min. Use a microsyringe to slowly add 0.1 mol / L sodium hydroxide solution at a speed of 50 μL / min, and monitor the pH value of the system in real time until the pH stabilizes at 9.5 to obtain an alkaline mixture.

[0082] (3) Keep the stirring speed at 500 r / min, wrap the reaction vessel with aluminum foil to avoid light throughout the process, and slowly add 400 μL of 150 mM silver nitrate aqueous solution at a rate of 25 μL / min; after the addition is completed, continue to stir the reaction in the dark for 15 min to obtain the first reaction solution.

[0083] (4) Maintain a stirring speed of 500 r / min and a light-protected environment, slowly add 1600 μL of 75 mM 8-hydroxyquinoline acetone solution at a rate of 35 μL / min, and control the molar ratio of silver ions to 8-hydroxyquinoline to be 1:2.0. After the addition is complete, continue stirring at room temperature in the dark for 24 h to obtain the second reaction solution.

[0084] (5) After the reaction is completed, take a dialysis bag with a molecular weight cutoff of 30 kDa, boil it in ultrapure water for 10 minutes in advance, and cool it to room temperature before use; transfer all the second reaction solution into the dialysis bag, clamp the two ends to seal, place it in a beaker containing 1 L of ultrapure water, and place it in a 4℃ refrigerator for dialysis for 24 hours. Change the ultrapure water every 8 hours for a total of 3 times to remove free silver ions and free ligands, and obtain purified nanoparticle dispersion.

[0085] (6) The dialysis nanoparticle dispersion was transferred to a freeze-drying bottle and pre-frozen in a -80℃ freezer for 4 hours; then it was placed in a freeze dryer and freeze-dried for 24 hours at -90℃ and a vacuum degree ≤10Pa to obtain silver-quinoline human serum albumin nanoparticles.

[0086] Example 3

[0087] This embodiment provides a method for preparing silver-quinoline bovine serum albumin nanoparticles, the preparation method comprising the following steps:

[0088] (1) Weigh 25 mg of bovine serum albumin (BSA) with a purity ≥ 98%, add 10 mL of ultrapure water, and place it in a 25°C water bath and stir at low speed until completely dissolved to obtain a clear albumin aqueous solution with a concentration of 2.5 mg / mL.

[0089] (2) At room temperature (25℃), place the albumin aqueous solution on a magnetic stirrer and stir at a speed of 700 r / min. Use a microsyringe to slowly add 0.1 mol / L sodium hydroxide solution at a speed of 50 μL / min, and monitor the pH value of the system in real time until the pH stabilizes at 11.0 to obtain an alkaline mixture.

[0090] (3) Keep the stirring speed at 700 r / min, wrap the reaction vessel with aluminum foil to avoid light throughout the process, and slowly add 1000 μL of 50 mM silver acetate aqueous solution at a rate of 40 μL / min; after the addition is completed, continue to stir the reaction in the dark for 30 min to obtain the first reaction solution.

[0091] (4) Maintain a stirring speed of 700 r / min and a light-protected environment, slowly add 4500 μL of 25 mM 8-hydroxyquinoline acetone solution at a rate of 50 μL / min, and control the molar ratio of silver ions to 8-hydroxyquinoline to be 1:2.25. After the addition is complete, continue stirring at room temperature in the dark for 24 h to obtain the second reaction solution.

[0092] (5) After the reaction is completed, take a dialysis bag with a molecular weight cutoff of 30 kDa, boil it in ultrapure water for 10 minutes in advance, and cool it to room temperature before use; transfer all the second reaction solution into the dialysis bag, clamp the two ends to seal, place it in a beaker containing 1.5 L of ultrapure water, and place it in a 4℃ refrigerator for dialysis for 48 h. Change the ultrapure water every 8 h for a total of 5 times to completely remove free silver ions and free ligands, and obtain purified nanoparticle dispersion.

[0093] (6) The dialysis nanoparticle dispersion was transferred to a freeze-drying bottle and pre-frozen in a -80℃ freezer for 6 hours; then it was placed in a freeze dryer and freeze-dried for 30 hours at -90℃ and a vacuum degree ≤10Pa to obtain silver-quinoline bovine serum albumin nanoparticles.

[0094] Example 4

[0095] This embodiment provides a method for preparing silver liposome nanoparticles (Ag@lipo), the preparation method comprising the following steps:

[0096] (1) Weigh 22.34 mg distearylphosphatidylcholine (DSPC), 4.28 mg cholesterol and 3.38 mg distearylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) and mix them so that the molar ratio of the three is about 24:9:1 to obtain the mixed raw materials.

[0097] (2) Dissolve the mixed raw materials in a mixed solvent of chloroform and anhydrous ethanol (3 mL of chloroform and 7 mL of anhydrous ethanol) to obtain a mixed solution.

[0098] (3) The mixture was rotary evaporated in a water bath at 40°C to form a film, and then vacuum dried for 2 hours to obtain a dried lipid film.

[0099] (4) Dissolve 42.5 mg of silver nitrate in 10 mL of pure water, add 10 mL of silver nitrate solution to the above-mentioned dried lipid membrane in a 70°C water bath to hydrate it, and stop when the membrane is completely hydrated to obtain the hydrated solution.

[0100] (5) The hydrated liquid is first passed through a membrane with a larger pore size (400 nm), then through a membrane with a target pore size (200 nm), and finally placed in a 3500 Da dialysis membrane for dialysis to remove free silver ions for 36 h, thus obtaining the silver liposome nanoparticles (Ag@lipo).

[0101] Example 5

[0102] This embodiment provides a method for preparing silver coordination polymer nanoparticles (Ag-ICPs), the preparation method comprising the following steps:

[0103] (1) Dissolve 0.5g of polyvinylpyrrolidone K30 (PVP-K30) in 21mL of pure water to obtain an aqueous solution of PVP-K30.

[0104] (2) Dissolve 42.5 mg of silver nitrate and 39.0 mg of 4,4'-bipyridine in 2 mL of pure water and 2 mL of anhydrous ethanol respectively to obtain silver nitrate solution and 4,4'-bipyridine solution.

[0105] (3) In a 25°C water bath at 800 r / min, PVP-K30 aqueous solution was placed in a round-bottom flask, and silver nitrate solution and 4,4'-bipyridine solution were added slowly at a rate of 0.5 mL / min and stirred in the dark for 2 h to obtain the reaction solution.

[0106] (4) The reaction solution is transferred into a 3500 Da dialysis bag for dialysis to remove free silver ions and 4,4'-bipyridine, thereby obtaining the silver coordination polymer nanoparticles (Ag-ICPs).

[0107] Example 6

[0108] This embodiment provides a method for preparing silver-loaded ZIF-8 nanoparticles (Ag@ZIF-8), the preparation method comprising the following steps:

[0109] (1) Dissolve 0.366g of zinc nitrate hexahydrate and 0.811g of 2-methylimidazole in 10mL of methanol respectively, and sonicate at room temperature for 8min to completely dissolve them (ultrasonic power is 50W, each ultrasonic cycle is 2s on and 3s off) to obtain zinc nitrate hexahydrate methanol solution and 2-methylimidazole methanol solution.

[0110] (2) Place the 2-methylimidazolium methanol solution in a round-bottom flask and stir magnetically (600 r / min), then quickly pour in the zinc nitrate hexahydrate methanol solution and stir at room temperature for 2 h.

[0111] (3) Add 42.5 mg of silver nitrate and continue stirring overnight at room temperature in the dark.

[0112] (4) Wash at 8000 r / min for 10 min and discard the supernatant. Then resuspend in methanol and repeat 3 times to obtain silver-loaded ZIF-8 nanoparticles (Ag@ZIF-8). Finally, they can be stored in an appropriate volume of methanol.

[0113] Comparative Example 1

[0114] The preparation method of this comparative example is basically the same as that of Example 1, except that the silver nitrate aqueous solution in step (3) is replaced with an equimolar amount of manganese chloride aqueous solution, and the other process parameters remain unchanged. Finally, Mn-8-HQ@BSA is prepared.

[0115] Comparative Example 2

[0116] The preparation method of this comparative example is basically the same as that of Example 1, except that the silver nitrate aqueous solution in step (3) is replaced with an equimolar amount of cobalt chloride aqueous solution, and the other process parameters remain unchanged. Co-8-HQ@BSA is finally prepared.

[0117] Comparative Example 3

[0118] The preparation method of this comparative example is basically the same as that of Example 1, except that the silver nitrate aqueous solution in step (3) is replaced with an equimolar amount of nickel chloride aqueous solution, and the other process parameters remain unchanged. Finally, Ni-8-HQ@BSA is prepared.

[0119] Comparative Example 4

[0120] The preparation method of this comparative example is basically the same as that of Example 1, except that the silver nitrate aqueous solution in step (3) is replaced with an equimolar amount of copper chloride aqueous solution, and the other process parameters remain unchanged. Finally, Cu-8-HQ@BSA is prepared.

[0121] Comparative Example 5

[0122] The preparation method of this comparative example is basically the same as that of Example 1, except that the silver nitrate aqueous solution in step (3) is replaced with an equimolar amount of zinc chloride aqueous solution, and the other process parameters remain unchanged. Finally, Zn-8-HQ@BSA is prepared.

[0123] Comparative Example 6

[0124] The preparation method of this comparative example is basically the same as that of Example 1, except that the silver nitrate aqueous solution in step (3) is replaced with an equimolar amount of aluminum chloride aqueous solution, and the other process parameters remain unchanged. Finally, Al-8-HQ@BSA is prepared.

[0125] Comparative Example 7

[0126] The preparation method of this comparative example is basically the same as that of Example 1, except that the silver nitrate aqueous solution in step (3) is replaced with an equimolar amount of chromium chloride aqueous solution, and the other process parameters remain unchanged. Finally, Cr-8-HQ@BSA is prepared.

[0127] Comparative Example 8

[0128] The preparation method of this comparative example is basically the same as that of Example 1, except that the silver nitrate aqueous solution in step (3) is replaced with an equimolar amount of ferric chloride aqueous solution, and the other process parameters remain unchanged. Finally, Fe-8-HQ@BSA is prepared.

[0129] Comparative Example 9

[0130] The preparation method of this comparative example is basically the same as that of Example 1, except that the silver nitrate aqueous solution in step (3) is replaced with an equimolar amount of gallium chloride aqueous solution, and the other process parameters remain unchanged. Finally, Ga-8-HQ@BSA is prepared.

[0131] Comparative Example 10

[0132] The preparation method of this comparative example is basically the same as that of Example 1, except that the silver nitrate aqueous solution in step (3) is replaced with an equimolar amount of lanthanum chloride aqueous solution, and the other process parameters remain unchanged. Finally, La-8-HQ@BSA is prepared.

[0133] Verification Example

[0134] (I) Evaluation of in vitro antitumor activity

[0135] 1. Specific killing assay of normal immune cells (flow cytometry)

[0136] Peripheral blood was collected from healthy volunteers, and peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation. The cells were resuspended in RPMI 1640 medium containing 10% fetal bovine serum and the cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 6 The nanoparticles were seeded at a concentration of 1 mL / well in a 24-well plate. Nanoparticles prepared in Example 1 and Comparative Examples 1-10 were added to each well, with the final concentration of metal ions in the nanoparticles set at 10 μM. A blank control group without any added drugs was also set up. Each group had 3 replicates.

[0137] After incubating the cells in a 37°C, 5% CO2 saturated humidity incubator for 24 h, all cells were collected and washed twice with pre-cooled PBS. Fluorescently labeled antibodies corresponding to CD20, CD4, CD8, CD56, CD14, CD11b, CD16, and CD66b were added, mixed thoroughly, and incubated at 4°C in the dark for 30 min. Unbound antibodies were removed by washing with PBS and the cells were resuspended. Before flow cytometry, antibodies to determine cell viability were added, and the survival rate of different cell subpopulations was detected. The relative survival rate of the drug-treated groups was calculated with the cell viability of the blank control group being 100%.

[0138] The results are as follows Figure 1 As shown: At a dosage concentration of 10 μM, Ag-8-HQ@BSA can significantly kill CD20. + B cells showed a cell survival rate dropping below 30%; while for CD4 cells... + Helper T cells, CD8 + Cytotoxic T cells, CD56 + NK cells, CD14 + Monocytes / macrophages, CD11b + Myeloid cells, CD16 + CD66b +Neutrophils and other normal immune cells maintained a survival rate of over 85% with no significant killing effect. Nanoparticles prepared from other metal ions showed weak B-cell killing activity or non-specific toxicity to various normal cells, resulting in a narrow therapeutic window. The results confirmed that Ag-8-HQ@BSA has a specific killing effect on B cells, possessing a broad therapeutic window and good safety.

[0139] 2. Tumor cell proliferation inhibition assay (MTT method)

[0140] Tumor cell lines in logarithmic growth phase were used: EG.7-OVA mouse T-cell lymphoma cells, 4T1 mouse breast cancer cells, BT549 human breast cancer cells, A20 mouse B-cell lymphoma cells, Jeko-1 human mantle cell lymphoma cells, MB-1 mouse B-cell lymphoma cells, NALM-6 human B-cell acute lymphoblastic leukemia cells, Ramos human Burkitt lymphoma cells, and SU-DHL-6 human diffuse large B-cell lymphoma cells. Adherent cells were seeded into 96-well plates 12 hours in advance, while suspension cells were directly seeded. The cell density per well was adjusted to an appropriate level, and 50 μL of cell suspension was added to each well. It should be emphasized that the above 9 cell lines were only used for the detection of proliferation inhibition of Ag-8-HQ@BSA nanoparticles; for Ag@lipo, Ag-ICPs and Ag@ZIF-8 nanoparticles, only 7 cell lines were selected for the experiment, and Jeko-1 human mantle cell lymphoma cells and NALM-6 human B-cell acute lymphoblastic leukemia cells were not included.

[0141] After the cells stabilized, serially diluted Ag-8-HQ@BSA nanoparticles were added. Seven concentration gradients (0, 3.13, 6.25, 12.5, 25, 50, and 100 μM) were set up using a silver ion final concentration meter, with five replicates per group. A blank well containing only culture medium was also included for zeroing. The 96-well plate was incubated at 37°C in a 5% CO2 incubator for 48 hours. Then, 10 μL of 5 mg / mL MTT solution was added to each well, and incubation continued for another 4 hours. After centrifugation, the supernatant was discarded, and 100 μL of DMSO was added to each well. The plate was then incubated at 37°C in a 5% CO2 incubator for 4-6 hours to allow the formazan crystals to dissolve completely. The absorbance (OD) value of each well was measured at 570 nm using a microplate reader.

[0142] Cell viability was calculated using the following formula: Cell viability (%) = (OD value of drug-treated group - OD value of zeroing well) / (OD value of control group - OD value of zeroing well) × 100%

[0143] The results are as follows Figure 2 As shown: Ag-8-HQ@BSA significantly inhibited the proliferation of six B-cell-derived tumor cell lines in a concentration-dependent manner, with a half-maximal inhibitory concentration (IC50) of 1,500%. 50The inhibitory effect was lower than 15 μM for all B cell-derived EG.7-OVA mouse T lymphoma cells, 4T1 mouse breast cancer cells, and BT549 human breast cancer cells; however, its inhibitory effect on proliferation was weaker, with an IC50 value of less than 15 μM. 50 All values ​​were above 50 μM. The results indicate that Ag-8-HQ@BSA possesses specific killing activity against B-cell malignant tumor cells.

[0144] Based on this, the silver ion-coated particles obtained in Examples 4-6 (Ag@lipo, Ag-ICPs, and Ag@ZIF-8, respectively) were further tested, and the results are as follows: Figures 3-5 As shown, the results indicate that Ag@lipo and Ag-ICPs exhibited results consistent with Ag-8-HQ@BSA, while Ag@ZIF-8 showed weaker killing activity against B-cell derived tumor cell lines than the other three silver ion-coated nanoparticles. Furthermore, Ag@ZIF-8 showed similar IC50 activity against non-B-cell derived tumor cell lines. 50 near.

[0145] (II) Characterization of the physicochemical properties of nanoparticles

[0146] The silver ion-coated particles prepared in Examples 1, 4-6 were tested, and the methods and results are as follows:

[0147] 1. Particle size and zeta potential detection

[0148] Take an appropriate amount of lyophilized nanoparticle powder and resuspend it in ultrapure water to a suitable concentration (the instrument shows approximately 200 particles). Use a nanoparticle tracking analyzer to detect the particle size distribution and Zeta potential at 25℃. Each group was measured in triplicate, and the average value was taken. The results are as follows: Figures 6-10 As shown. Wherein:

[0149] Figure 6 The results show that the Ag-8-HQ@BSA nanoparticles exhibit a single-peak distribution with an average particle size of 102 nm, falling within the range of 20–200 nm, and the particle size distribution is uniform.

[0150] Figure 7 The results show that the Ag@lipo nanoparticles exhibit a single-peak distribution with an average particle size of 193 nm, falling within the 100-200 nm range, and the particle size distribution is uniform.

[0151] Figure 8 The results show that the Ag-ICPs nanoparticles exhibit a unimodal distribution with an average particle size of 155 nm, falling within the 100-200 nm range, and the particle size distribution is uniform.

[0152] Figure 9The results show that the Ag@ZIF-8 nanoparticles exhibit a single-peak distribution with an average particle size of 228 nm, falling within the range of 100-250 nm, and the particle size distribution is uniform.

[0153] Figure 10 The results show that the Zeta potential of Ag-8-HQ@BSA nanoparticles is -19.63±1.03mV, the particle surface is negatively charged, the colloidal stability is good, and they are not prone to aggregation; the Zeta potential of Ag@lipo nanoparticles is -13.84±1.14mV, the particle surface is negatively charged, the colloidal stability is good, and they are not prone to aggregation; the Zeta potential of Ag-ICPs nanoparticles is -9.33±0.86mV, the particle surface is negatively charged, the colloidal stability is good, and they are not prone to aggregation; the Zeta potential of Ag@ZIF-8 nanoparticles is -26.81±0.85mV, the particle surface is negatively charged, the colloidal stability is good, and they are not prone to aggregation.

[0154] 2. Morphological observation by transmission electron microscopy (TEM)

[0155] Ag-8-HQ@BSA aqueous dispersion was diluted to a silver ion concentration of 5 μM. 10 μL was added to the surface of a copper mesh supported on a carbon substrate. After adsorption at room temperature for 5 min, excess liquid was absorbed along the edge of the copper mesh using filter paper. 10 μL of 2% phosphotungstic acid solution was added for negative staining. After staining for 2 min, excess stain was absorbed. The sample was then air-dried at room temperature, and the particle morphology was observed and photographed using a transmission electron microscope. The results are as follows: Figures 11-14 As shown. Wherein:

[0156] Figure 11 The results show that the Ag-8-HQ@BSA nanoparticles are regular spherical or near-spherical in shape, with good dispersibility and no obvious agglomeration. The particle size is consistent with the DLS detection results.

[0157] Figure 12 The results show that the Ag@lipo nanoparticles are regular spherical or near-spherical in shape, with good dispersibility and no obvious agglomeration. The particle size is consistent with the DLS detection results.

[0158] Figure 13 The results showed that the Ag-ICPs nanoparticles were regular spherical or near-spherical in shape, well dispersed, and without obvious agglomeration. The particle size was consistent with the DLS detection results.

[0159] Figure 14 The results show that the Ag@ZIF-8 nanoparticles are rhombic dodecahedral in shape, well dispersed, and without obvious agglomeration. The particle size is consistent with the DLS detection results.

[0160] (III) Evaluation of in vivo antitumor activity

[0161] Six-week-old female BALB / c mice, weighing 18-20g, were selected and acclimatized for 3 days before tumor modeling was initiated. B-cell lymphoma cells from A20 mice in the logarithmic growth phase were collected and resuspended in sterile PBS to adjust the cell concentration to 5 × 10⁻⁶ cells / mL. 7 Cells / mL, 0.1 mL of cell suspension (i.e., 5 × 10⁶ cells / mL) was subcutaneously injected into the left flank of each mouse. 6 (1 cell / animal) to construct a subcutaneous B-cell lymphoma model.

[0162] After modeling, the mice were observed daily until the tumor volume reached approximately 100 mm. 3 Then, the mice were randomly divided into 3 groups:

[0163] Control group: Intraperitoneal injection of an equal volume of sterile saline;

[0164] Intratumoral administration group: Ag-8-HQ@BSA nanoparticle dispersion was injected intratumorally at a dose of 0.72 mg / kg (based on silver ion content).

[0165] The medication is administered once every 3 days, for a total of 6 doses.

[0166] Intraperitoneal administration group: Ag-8-HQ@BSA nanoparticle dispersion was injected intraperitoneally at a dose of 0.72 mg / kg (based on silver ion content).

[0167] The medication was administered once daily for a total of 16 doses.

[0168] Mouse modeling and drug administration regimens, such as Figure 15 As shown in the figure, the tumor volume change curves of each group of mice are as follows: Figure 16 As shown in the figure. Results showed that, compared with the control group, intraperitoneal and intratumoral administration of Ag-8-HQ@BSA significantly inhibited tumor growth in mice. During the experiment, the mice in each group did not experience significant weight loss, and their mental state, food intake, and water consumption remained normal, indicating that the nanoparticles have good in vivo safety. These results confirm that Ag-8-HQ@BSA possesses excellent anti-B-cell lymphoma activity in vivo, and both administration routes demonstrate clear therapeutic effects.

[0169] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The application of silver ion-coated particles in the preparation of drugs for treating B-cell malignant tumors, characterized in that, The silver ion-coated particles include one of silver-quinoline albumin nanoparticles, silver liposome nanoparticles, silver coordination polymer nanoparticles, and silver-loaded ZIF-8 nanoparticles; wherein: The preparation method of the silver-quinoline albumin nanoparticles includes the following steps: (1) Dissolve albumin in water to obtain an albumin aqueous solution; (2) Add an alkaline regulator to the albumin aqueous solution to obtain an alkaline mixture; (3) Add silver ion solution to the alkaline mixture and stir to obtain the first reaction solution; (4) Add quinoline ligand solution to the first reaction solution and stir to obtain the second reaction solution; (5) Dialyze the second reaction solution to remove free silver ions and free ligands to obtain a purified product; (6) After freeze-drying the purified product, silver-quinoline albumin nanoparticles are obtained.

2. The application of the silver ion-coated particles according to claim 1 in the preparation of drugs for treating B-cell malignant tumors, characterized in that, In step (1), albumin is dissolved in ultrapure water to obtain an albumin aqueous solution with a concentration of 2.5-10 mg / mL; And / or, the albumin includes one or more of bovine serum albumin, human serum albumin, and recombinant albumin.

3. The application of the silver ion-coated particles according to claim 1 in the preparation of drugs for treating B-cell malignant tumors, characterized in that, In step (2), an alkaline regulator is added to the albumin aqueous solution at a speed of 400-800 r / min to obtain an alkaline mixture with a pH of 9.0-11.0; And / or, the alkalinity regulator is a sodium hydroxide solution.

4. The application of the silver ion-coated particles according to claim 1 in the preparation of drugs for treating B-cell malignant tumors, characterized in that, In step (3), under the conditions of avoiding light, 20-30℃, and 400-800r / min, silver ion solution is added to the alkaline mixture and stirred for 10-30min to obtain the first reaction solution; And / or, the silver ion solution is an aqueous solution of silver nitrate or an aqueous solution of silver acetate; And / or, the concentration of silver ions in the silver ion solution is 50-200 mM.

5. The application of the silver ion-coated particles according to claim 1 in the preparation of drugs for treating B-cell malignant tumors, characterized in that, In step (4), under the conditions of avoiding light, 20-30℃, and 400-800r / min, a quinoline ligand solution is added to the first reaction solution and stirred for 20-24h to obtain the second reaction solution; And / or, the quinoline ligand solution is an 8-hydroxyquinoline ligand acetone solution or a nitroquinoline ligand acetone solution; And / or, the concentration of the quinoline ligand solution is 25-100 mM; And / or, the molar ratio of the silver ions to the quinoline ligand is 1:2 to 1:2.

5.

6. The application of the silver ion-coated particles according to claim 1 in the preparation of drugs for treating B-cell malignant tumors, characterized in that, In step (5), the second reaction solution is placed in a dialysis bag, and then the dialysis bag is placed in pure water and dialyzed at 3-5℃ for 24-48h to remove free silver ions and free ligands, and the purified product is obtained.

7. The application of the silver ion-coated particles according to claim 1 in the preparation of a drug for treating B-cell malignant tumors, characterized in that, In step (6), the purified material is pre-frozen at -80℃ and then freeze-dried at -90℃ and vacuum degree ≤10Pa for 24-30h to obtain silver-quinoline albumin nanoparticles.

8. A drug for treating B-cell malignant tumors, characterized in that, The drug is a formulation prepared by using the silver ion-coated particles described in claim 1 as raw material and adding pharmaceutically acceptable excipients.

9. The drug for treating B-cell malignant tumors according to claim 8, characterized in that, The preparation is an oral or injectable preparation.

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