Asparaginase organic-inorganic hybrid nanocrystal and preparation process thereof, and coated asparaginase particles and preparation process thereof

Abstract

The invention relates to the technical field of nano material synthesis, in particular to an asparaginase organic-inorganic hybrid nanocrystal and a preparation process thereof, and a coated asparaginase particle and a preparation process thereof. The preparation process of the asparaginase organic-inorganic hybrid nanocrystal comprises the following preparation step: synthesizing the organic-inorganic hybrid nanocrystal (NCs) consisting of asparaginase and copper phosphate (Cu3 (PO4) 2.3 H2O) by adopting a coprecipitation method. The prepared nanocrystal is combined with a layer-by-layer assembly technology to prepare the core-shell structure particle which takes the nanocrystal as a core and takes a biocompatible polymer layer (chitosan / chitosan oligosaccharide and polydopamine) as a shell, so that the prepared particle not only retains the performance of enzyme specific recognition and asparagine hydrolysis, but also can be used for preparing the biocompatible polymer layer (chitosan / chitosan oligosaccharide and polydopamine). And the defects of poor stability and high immunogenicity of the free asparaginase are effectively overcome to a certain extent.

Description

Technical Field

[0001] This invention relates to the field of nanomaterial synthesis technology, and in particular to an organic-inorganic hybrid nanocrystal of asparaginase and its preparation process, and a microparticle encapsulating asparaginase and its preparation process. Background Technology

[0002] Acute lymphoblastic leukemia (ALL) is a hematologic malignancy characterized by the massive proliferation and widespread infiltration of primitive and immature lymphocytes. It can invade the bone marrow, blood, and extramedullary tissues, suppressing normal hematopoietic function and significantly affecting the central nervous system, resulting in a high mortality rate. This disease is more common in children, with the highest incidence in children aged 1-4 years. Patients with ALL often experience various clinical symptoms. Most present with fever, pallor, and bruising, early signs of bone marrow suppression or hematological abnormalities. Over 60% of patients may exhibit organ enlargement (spleen and liver), accompanied by recurrent infections, fatigue, lymphadenopathy, and skin rashes. In severe cases, lethargy, sweating, loss of appetite, nausea, and vomiting may occur. These symptoms not only significantly impact the patient's quality of life but may also affect prognosis. Current treatments for ALL include combination chemotherapy, hematopoietic stem cell transplantation (HSCT), immunotherapy, targeted drug therapy, and monoclonal antibody therapy.

[0003] Asparaginase is an indispensable drug in chemotherapy regimens for ALL (Allergic Respiratory Syndrome). Asparaginase is a crucial enzyme in the treatment of ALL, widely used clinically both domestically and internationally, and boasts the highest therapeutic index for ALL. It catalyzes the hydrolysis of asparagine, breaking it down into aspartic acid and ammonia. This depletion of plasma asparagine leads to reduced DNA, RNA, and protein synthesis in tumor cells, ultimately causing tumor cell death. However, asparaginase derived from microorganisms presents two challenges after entering the body: firstly, as an exogenous protein, it is easily degraded by various proteolytic enzymes, exhibiting poor stability and a short half-life; secondly, it readily induces immunogenicity, and direct use can cause severe side effects such as immunosuppression, chills and fever, gastrointestinal reactions, abnormal liver function, other metabolic abnormalities, and allergic reactions. These adverse reactions significantly limit the enzyme's use. Therefore, improving the stability and catalytic activity of asparaginase and reducing its immunogenicity is of paramount importance. Summary of the Invention

[0004] The main objective of this invention is to provide an organic-inorganic hybrid nanocrystal of asparaginase and its preparation process, as well as a process for preparing asparaginase-encapsulated microparticles, aiming to improve the technical problems of poor stability and short half-life of existing asparaginases when they enter the body; at the same time, it can also improve the technical problem that asparaginases are prone to causing serious side effects when they enter the body.

[0005] To achieve the above objectives, the present invention proposes a preparation process for asparaginase organic-inorganic hybrid nanocrystals, comprising the following preparation steps: adding CuSO4 aqueous solution to a phosphate buffer solution containing asparaginase and bovine serum albumin, and shaking to obtain the asparaginase organic-inorganic hybrid nanocrystals.

[0006] Preferably, the concentration ratio of the asparaginase to the bovine serum albumin is (1-3):(1-7), and the concentration ratio of the asparaginase to the CuSO4 aqueous solution is... 2+ The concentration ratio is (1.5-5):1.

[0007] Preferably, the concentration of bovine serum albumin is 0.1-0.7 mg / mL; the concentration of asparaginase is 0.1-0.3 mg / mL; and the CuSO4 aqueous solution contains Cu... 2+ The concentration is 0.5-3.5 mM.

[0008] More preferably, the concentration of bovine serum albumin is 0.2-0.35 mg / mL; the concentration of asparaginase is 0.1-0.15 mg / mL; and the CuSO4 aqueous solution contains Cu... 2+ The concentration is 1.6-2 mM.

[0009] Preferably, the oscillation time is 45-90 minutes, and the reaction temperature during oscillation is 30-40°C.

[0010] Preferably, the pH value of the phosphate buffer solution is 7-8.

[0011] In addition, this invention also proposes an asparaginase organic-inorganic hybrid nanocrystal, which is prepared by the above-mentioned preparation process of asparaginase organic-inorganic hybrid nanocrystal.

[0012] In addition, this invention also proposes a preparation process for encapsulating asparaginase microparticles, comprising the following preparation steps: S1. The above-mentioned asparaginase organic-inorganic hybrid nanocrystal precipitate is dispersed in a chitosan / chitosan oligosaccharide solution, stirred, centrifuged and washed to obtain an intermediate product; wherein, the concentration ratio of the asparaginase organic-inorganic hybrid nanocrystal to the chitosan / chitosan oligosaccharide solution is 1:(1-5). S2. The intermediate product precipitate is dispersed in a boric acid-borax / Mops mixed buffer solution, dopamine solution is added, the mixture is magnetically stirred at room temperature, and the precipitate is obtained by centrifugation and washing, which is the encapsulated asparaginase microparticle; wherein the concentration ratio of the intermediate product to the dopamine solution is (1-5):1.

[0013] Preferably, the preparation of the chitosan / chitosan oligosaccharide solution includes the following steps: S11. Add chitosan to the acetic acid solution and stir magnetically until completely dissolved to obtain a concentrated chitosan stock solution; S12. Add chitosan oligosaccharide to ultrapure water and stir to dissolve to obtain chitosan oligosaccharide solution; S13. The chitosan concentrate and chitosan oligosaccharide solution are mixed evenly in a ratio of (0.5-5):1 to obtain the chitosan / chitosan oligosaccharide solution.

[0014] Preferably, the preparation of the boric acid-borax / Mops mixed buffer solution includes the following steps: S21. Add borax to ultrapure water and stir to dissolve to obtain borax solution; add boric acid to ultrapure water and stir to dissolve to obtain boric acid solution. S22. Add Mops buffer salt to ultrapure water and stir to dissolve to obtain Mops buffer solution; S23. Mix the borax solution and the boric acid solution evenly to obtain a boric acid-borax solution; mix the boric acid-borax solution with the Mops buffer evenly to obtain the boric acid-borax / Mops mixed buffer.

[0015] Preferably, the molecular weight of the chitosan is 10,000-100,000, and the molecular weight of the chitosan oligosaccharide is 1,000-5,000.

[0016] Preferably, in step S21, the volume ratio of the borax solution and the boric acid solution when mixed is (7-10):11, and the pH value of the Mops buffer solution is 7.5-9.0; In step S23, the volume ratio of the boric acid-borax solution and the Mops buffer solution is 7:(1-5), and the pH value of the boric acid-borax / Mops mixed buffer solution is 7.5-9.5.

[0017] In addition, the present invention also proposes a microparticle encapsulated with asparaginase, which is prepared by the above-described preparation process for microparticle encapsulated with asparaginase.

[0018] Compared with the prior art, the organic-inorganic hybrid nanocrystals of asparaginase and their preparation process of the present invention, as well as the asparaginase-encapsulated microparticles and their preparation process, have the following beneficial effects: 1. This scheme utilizes immobilized enzyme technology and surface encapsulation technology to prepare core-shell structured microparticles with asparaginase nanocrystals as the core and biocompatible polymer layers (chitosan / chitooligosaccharide, dopamine) as the outer shell.

[0019] 2. The nanocrystalline core constructed using immobilized enzyme technology effectively improves the stability and catalytic activity of free asparaginase. Combined with layer-by-layer assembly technology, a biocompatible polymer coating is introduced. This coating system enhances the conformational stability and resistance to enzymatic hydrolysis of the enzyme. Furthermore, it shields the antigenic epitopes on the enzyme molecule surface through steric hindrance, reducing immune system recognition and antibody neutralization reactions, thereby prolonging drug circulation time in vivo. This synergistic effect allows the microparticles to reduce immunogenicity while improving therapeutic efficacy. This opens up new technical pathways and theoretical basis for delivery systems of other enzyme-based biological agents with stability defects or high immunogenicity. The prepared microparticles not only retain the enzyme's ability to specifically recognize and hydrolyze asparaginase but also effectively address the shortcomings of poor stability and high immunogenicity of free asparaginase to a certain extent. Moreover, the prepared asparaginase-encapsulated microparticles have uniform particle size. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application 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 this application. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The appearance diagram of Asp / BSA-Cu3(PO4)2·3H2O; Figure 2 SEM characterization images of Asp / BSA-Cu3(PO4)2·3H2O and Cu3(PO4)2·3H2O; Figure 3 TEM characterization of organic-inorganic hybrid nanocrystals of asparaginase; Figure 4 XRD characterization of organic-inorganic hybrid nanocrystals of asparaginase; Figure 5 Fourier transform infrared spectra (A: Asp / BSA-Cu3(PO4)2·3H2O; B: Asp / BSA; C: BSA-Cu3(PO4)2·3H2O; D: Cu3(PO4)2·3H2O). Figure 6 The particle size distribution of asparaginase organic-inorganic hybrid nanocrystals; Figure 7Figure for particle size stability of organic-inorganic hybrid nanocrystals of asparaginase (n=3); Figure 8 Figure 3 shows the reusability of organic-inorganic hybrid nanocrystals of asparaginase. Figure 9 Figure 1 shows the resistance of asparaginase organic-inorganic hybrid nanocrystals to trypsin hydrolysis (n=3). Figure 10 An image showing the appearance of the intermediate product CS / COS-NCs; Figure 11 An image showing the appearance of PDA@CS / COS-NCs encapsulated asparaginase microparticles; Figure 12 The particle size distribution of the intermediate product CS / COS-NCs is shown. Figure 13 Particle size distribution of PDA@CS / COS-NCs encapsulated asparaginase microparticles; Figure 14 SEM characterization of the intermediate product CS / COS-NCs; Figure 15 SEM characterization of PDA@CS / COS-NCs encapsulated asparaginase microparticles; Figure 16 TEM characterization of PDA@CS / COS-NCs encapsulated asparaginase microparticles; Figure 17 Fourier transform infrared spectra (A: CS / COS-NCs; B: PDA-NCs; C: PDA@CS / COS-NCs); Figure 18 Figure for particle size stability of asparaginase-encapsulated microparticles (n=3); Figure 19 Figure for the reusability of encapsulated asparaginase particles (n=3); Figure 20 Figure 3 shows the plasma stability of asparaginase microparticles. Figure 21 Figure 4℃ (n=3) shows the storage stability of asparaginase microparticles. Figure 22 Figure for the storage stability study of asparaginase microparticles at -25℃ (n=3); Figure 23 Cytotoxicity assays for Asp, NCs, and PDA@CS / COS-NCs (n=4), where A 12 h; B 24 h; C 48 h; D vector; a-Asp; b-NCs; c-PDA@CS / COS-NCs; Figure 24 DNA gel electrophoresis images for apoptosis, where A is Control; B is PDA@CS / COS-NCs (0.05 U / mL); C is Asp (0.1 U / mL); D is NCs (0.1 U / mL); E is PDA@CS / COS-NCs (0.1 U / mL). Figure 25 The diagram shows the formation of colonies on plates (n=3), where A is Control; B is PDA@CS / COS-NCs (0.05 U / mL); C is Asp (0.1 U / mL); D is NCs (0.1 U / mL); and E is PDA@CS / COS-NCs (0.1 U / mL). Figure 26 Jurkat cell colony diagram; Figure 27 Flow cytometry apoptosis plots of Jurkat cells (n=3), where A is Control; B is PDA@CS / COS-NCs (0.05 U / mL); C is Asp (0.1 U / mL); D is NCs (0.1 U / mL); E is PDA@CS / COS-NCs (0.1 U / mL). Figure 28 Changes in specific IgM and IgG in the serum of mice after intravenous administration (n=6). Figure 29 Hematological parameters of mice after intravenous administration (n=6); Figure 30 The levels of AST, ALT, UREA, UA and CREA in mice after intravenous administration (n=6). Figure 31 The organ index of major organs in mice (n=6).

[0022] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0024] Furthermore, the technical solutions of the various embodiments can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.

[0025] A process for preparing asparaginase organic-inorganic hybrid nanocrystals includes the following steps: adding CuSO4 aqueous solution (120 mM) to a phosphate buffer solution (0.01 M) containing asparaginase and bovine serum albumin, and shaking on a shaker to obtain asparaginase organic-inorganic hybrid nanocrystals; wherein the concentration ratio of asparaginase to bovine serum albumin is (1-3):(1-7), and the CuSO4 aqueous solution contains asparaginase and bovine serum albumin. 2+ The concentration ratio is (1.5-5):1.

[0026] This scheme employs a co-precipitation method to synthesize organic-inorganic hybrid nanocrystals (Asp / BSA-Cu3(PO4)2·3H2O, abbreviated as NCs) composed of asparaginase (Asp) and copper phosphate crystals (Cu3(PO4)2·3H2O). Utilizing Cu... 2+ As an inorganic component, Asp and bovine serum albumin are used as organic components to prepare organic-inorganic hybrid nanocrystals. The formation mechanism of these nanocrystals is that phosphate ions combine with copper ions to form copper phosphate crystal sheets. The complex formed by the protein and copper ions can then become the nucleation sites for the primary copper phosphate crystal sheets. The interaction between the protein and copper ions promotes the growth of micron-sized particles. These particles have nanoscale characteristics and are shaped like petals. Due to their hierarchical high specific surface area nanocrystal petal structure, the catalytic activity and stability of the nanocrystals are significantly improved and enhanced.

[0027] When the concentration ratios of the raw materials in this scheme are within the above-mentioned limits, the resulting product has better long-term storage stability, maintains high catalytic activity in both acidic and alkaline environments, maintains high catalytic activity in high-temperature environments, and has a higher reusability.

[0028] Phosphate buffer solutions may be a mixture of sodium dihydrogen phosphate, disodium hydrogen phosphate and water, or a mixture of potassium dihydrogen phosphate, dipotassium hydrogen phosphate and water, or a mixture of potassium dihydrogen phosphate, disodium hydrogen phosphate and water.

[0029] Furthermore, the concentration of bovine serum albumin was 0.1-0.7 mg / mL; the concentration of asparaginase was 0.1-0.3 mg / mL; and the concentration of Cu in CuSO4 aqueous solution was... 2+ The concentration is 0.5-3.5 mM.

[0030] Nanocrystals of different sizes can be prepared by controlling the concentration of bovine serum albumin (BSAL), with particle sizes ranging from 1 to 4 μm and zeta potentials ranging from -10 to -13 mV. The preferred concentration of BSAL is 0.25 mg / mL, which yields nanocrystals with a particle size of approximately 1 μm and good stability.

[0031] Asparaginase and Cu 2+ The concentration of Cu also has a certain influence on the particle size of Asp / BSA-Cu3(PO4)2·3H2O hybrid nanocrystals. The preferred concentration of bovine serum albumin is 0.25 mg / mL. 2+ The preferred concentration of the asparaginase is 1.6 mM and the preferred concentration of the asparaginase is 0.117 mg / mL. At this concentration, the nanocrystal size is controlled within 1 μm and is relatively stable, and its zeta potential is also relatively high, which is -11.9 mV.

[0032] Furthermore, the shaking time is 45-90 min, and the reaction temperature during shaking is 30-40℃. Besides the aforementioned influencing factors, the shaking time and reaction temperature in this scheme also have a certain impact on the particle size of the Asp / BSA-Cu3(PO4)2·3H2O hybrid nanocrystals. Both excessively long and short reaction times are detrimental to nanocrystal formation. When the reaction time is too short, there are fewer nucleation sites formed by the protein and copper ions, making it difficult for copper phosphate crystals to fully attach to the limited nucleation sites, resulting in a less compact nanocrystal structure and thus a larger nanocrystal size. When the reaction time is too long, copper phosphate crystal sheets may continuously grow anisotropically at the nucleation sites, leading to the formation of nanocrystals with thicker petals, resulting in a larger particle size. The preferred shaking time is 45 min, resulting in nanocrystals with a particle size within 1 μm that are relatively stable and negatively charged. Neither excessively high nor excessively low reaction temperatures are suitable for the formation of nanocrystals. When the reaction temperature is 37℃, the nanocrystals prepared have a particle size of less than 1 μm and are relatively stable.

[0033] Furthermore, the pH of the phosphate buffer solution is 7-8. The preferred pH of the reaction system in this scheme is 7.4, resulting in nanocrystals with superior performance, a particle size within 1 μm, and good stability, while also carrying a negative charge.

[0034] In addition, this invention also proposes an asparaginase organic-inorganic hybrid nanocrystal, which is prepared by the above-mentioned preparation process of asparaginase organic-inorganic hybrid nanocrystal.

[0035] In addition, this invention also proposes a preparation process for asparaginase-encapsulated microparticles, comprising the following preparation steps: S1. Dispersing the above-mentioned asparaginase organic-inorganic hybrid nanocrystal precipitate in chitosan / chitosan oligosaccharide solution (pH 6.2-7.5), stirring magnetically for 15-30 min with a constant temperature magnetic stirrer, and then washing by centrifugation with deionized water (washing 3-5 times) to obtain an intermediate product; wherein, the concentration ratio of asparaginase organic-inorganic hybrid nanocrystals to chitosan / chitosan oligosaccharide solution is 1:(1-5); S2. Dispersing the intermediate product precipitate in boric acid-borax / Mops mixed buffer solution, adding dopamine solution to make its final concentration 0.05-0.1 mg / mL, stirring magnetically at room temperature, and washing by centrifugation to obtain the precipitate, which is the asparaginase-encapsulated microparticles; wherein, the concentration ratio of intermediate product to dopamine solution is (1-5):1.

[0036] This method uses asparaginase organic-inorganic hybrid nanocrystals as the core of the polymer. First, a chitosan / chitosan oligosaccharide layer is coated on its surface. This coating can enhance the encapsulation effect of the nanocrystals and shorten the polymerization time of the dopamine layer. This allows it to be better assembled on the surface of the nanocrystals coated with chitosan / chitosan oligosaccharide layer through electrostatic adsorption. Finally, asparaginase-encapsulated microparticles (PDA@CS / COS-NCs) are prepared. The microparticles have pores on their surface, which improves the stability of the nanocrystals without affecting their hydrolysis of the substrate.

[0037] When the concentration ratio of nanocrystals to chitosan / chitosan oligosaccharide solution, and the mass of intermediate product to dopamine solution are limited to the above range, the obtained product has uniform particle size and good stability, exhibits a regular and compact flower-like structure, and has a microporous structure on the surface; plasma stability and storage stability are improved, and recyclability is stronger. Further, the preparation of chitosan / chitooligosaccharide solution includes the following steps: S11. Add chitosan to acetic acid solution and stir magnetically until completely dissolved to obtain concentrated chitosan stock solution; S12. Add chitooligosaccharide to ultrapure water and stir to dissolve to obtain chitooligosaccharide solution; S13. Mix concentrated chitosan stock solution and chitooligosaccharide solution at a concentration ratio of (0.5-5):1 until homogeneous to obtain chitosan / chitooligosaccharide solution.

[0038] Further, the preparation of the boric acid-borax / Mops mixed buffer includes the following steps: S21. Add borax to ultrapure water and stir to dissolve to obtain a borax solution; add boric acid to ultrapure water and stir to dissolve to obtain a boric acid solution; S22. Add Mops buffer salt to ultrapure water and stir to dissolve to obtain a Mops buffer; S23. Mix the borax solution and the boric acid solution evenly to obtain a boric acid-borax solution; mix the boric acid-borax solution and the Mops buffer evenly to obtain the boric acid-borax / Mops mixed buffer.

[0039] Furthermore, the molecular weight of the chitosan is 10,000-100,000, and the molecular weight of the chitosan oligosaccharide is 1,000-5,000.

[0040] Further, in step S21, the volume ratio of borax solution to boric acid solution is (7-10):11, and the pH value of Mops buffer is 7.5-9; in step S23, the volume ratio of boric acid-borax solution to Mops buffer is 7:(1-5), and the pH value of boric acid-borax / Mops mixed buffer is 7.5-9.5.

[0041] In addition, the present invention also proposes a microparticle encapsulated with asparaginase, which is prepared by the above-described preparation process for microparticle encapsulated with asparaginase.

[0042] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0043] Example 1 A process for preparing asparaginase organic-inorganic hybrid nanocrystals includes the following steps: 80 μL of molecular biological grade CuSO4 aqueous solution (120 mM) is added to 6 mL of PBS (0.01 M, pH 7.4) containing asparaginase and bovine serum albumin to adjust the concentration of asparaginase to 0.117 mg / mL and the concentrations of bovine serum albumin to 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, and 0.35 mg / mL, respectively. The mixture is then shaken on a shaker at 37°C for 45 min.

[0044] After the reaction was completed, the particles were centrifuged and washed (10,000 rpm, 6 min), dispersed in ultrapure water, and then their hydrated particle size and Zeta potential were measured using a Malvern particle size analyzer. The specific test results are shown in the table below: Effect of BSA concentration on particle size of Asp / BSA-Cu3(PO4)2·3H2O (x̅ ±s, n=3)

[0045] The results in the table above show that nanocrystals of different sizes can be prepared by controlling the concentration of BSA, with a particle size range of approximately 1–4 μm and a zeta potential of approximately -10 to -13 mV. At a BSA concentration of 0.25 mg / mL, the prepared nanocrystals have a particle size of approximately 1 μm and are relatively stable.

[0046] Example 2 A process for preparing asparaginase organic-inorganic hybrid nanocrystals includes the following steps: 80 μL of molecular biological grade CuSO4 aqueous solution (120 mM) is added to 6 mL of PBS (0.01 M, pH 7.4) containing 0.250 mg / mL bovine serum albumin and different concentrations of asparaginase, and the concentrations of asparaginase are adjusted to 0.108 mg / mL, 0.117 mg / mL, 0.125 mg / mL, 0.133 mg / mL, 0.141 mg / mL, and 0.150 mg / mL, respectively, and the mixture is shaken on a shaker at 37 °C for 45 min.

[0047] After the reaction was completed, the particles were centrifuged and washed (10,000 rpm, 6 min), dispersed in ultrapure water, and then their hydrated particle size and Zeta potential were measured using a Malvern particle size analyzer. The specific test results are shown in the table below: Effect of Asp concentration on particle size of Asp / BSA-Cu3(PO4)2·3H2O (x̅ ±s, n=3)

[0048] The test results in the table above show that at a BSA concentration of 0.25 mg / mL and Cu... 2+ When the concentration is 1.6 mM and the Asp concentration is 0.117 mg / mL, the prepared nanocrystals have a particle size of less than 1 μm and are relatively stable, and their Zeta potential is also high, at -11.9 mV.

[0049] Example 3 A process for preparing asparaginase organic-inorganic hybrid nanocrystals includes the following steps: adding different concentrations of molecular biological grade CuSO4 aqueous solution (120 mM) to 6 mL of PBS (0.01 M, pH 7.4) containing 0.117 mg / mL asparaginase and 0.250 mg / mL bovine serum albumin, adjusting the Cu... 2+ The concentrations were 1.6 mM, 1.8 mM, and 2.0 mM, and the mixtures were shaken on a shaker at 37°C for 45 min.

[0050] After the reaction was completed, the particles were centrifuged and washed (10,000 rpm, 6 min), dispersed in ultrapure water, and then their hydrated particle size and Zeta potential were measured using a Malvern particle size analyzer. The specific test results are shown in the table below: Cu 2+ Effect of concentration on particle size of Asp / BSA-Cu3(PO4)2·3H2O (x̅ ±s, n=3)

[0051] The test results in the table above show that in Cu 2+When the concentration is 1.6-1.8 mM, the prepared nanocrystals have a particle size of less than 1 μm and are relatively stable, and are negatively charged.

[0052] Example 4 A process for preparing asparaginase organic-inorganic hybrid nanocrystals includes the following steps: adding 80 μL of molecular biological grade CuSO4 aqueous solution (120 mM) to 6 mL of PBS (0.01 M, pH 7.4) containing 0.117 mg / mL asparaginase and 0.250 mg / mL bovine serum albumin, and shaking on a shaker at 37 °C for 45, 60, and 90 min.

[0053] After the reaction was completed, the particles were centrifuged and washed (10,000 rpm, 6 min), dispersed in ultrapure water, and then their hydrated particle size and Zeta potential were measured using a Malvern particle size analyzer. The specific test results are shown in the table below: Effect of shaking time on particle size of Asp / BSA-Cu3(PO4)2·3H2O (x̅ ±s, n=3)

[0054] The test results in the table above show that when the reaction time in the shaker is 45-60 min, the prepared nanocrystals have a particle size of less than 1 μm and are relatively stable, and are negatively charged.

[0055] Example 5 A process for preparing asparaginase organic-inorganic hybrid nanocrystals includes the following steps: adding 80 μL of molecular biological grade CuSO4 aqueous solution (120 mM) to 6 mL of PBS (0.01 M, pH 7.4) containing 0.117 mg / mL asparaginase and 0.250 mg / mL bovine serum albumin, and shaking on a shaker for 45 min at 30℃, 37℃, and 40℃ respectively.

[0056] After the reaction was completed, the particles were centrifuged and washed (10,000 rpm, 6 min), dispersed in ultrapure water, and then their hydrated particle size and Zeta potential were measured using a Malvern particle size analyzer. The specific test results are shown in the table below: Effect of reaction temperature on particle size of Asp / BSA-Cu3(PO4)2·3H2O (x̅ ±s, n=3)

[0057] The test results in the table above show that when the reaction temperature is 37℃, the prepared nanocrystals have a particle size of less than 1μm and are relatively stable.

[0058] Example 6 A process for preparing asparaginase organic-inorganic hybrid nanocrystals includes the following steps: Pre-preparing PBS solutions with pH values ​​of 6.8 (0.01 M), 7.4 (0.01 M), and 8.0 (0.01 M). Adding asparaginase and bovine serum albumin to 6 mL of the above PBS solutions at different pH values, adjusting the final concentrations of Asp and BSA to 0.117 mg / mL and 0.250 mg / mL respectively, and then adding 90 μL of molecularly biological grade CuSO4 aqueous solution (120 mM), and shaking on a shaker at 37°C for 45 min.

[0059] After the reaction was completed, the particles were centrifuged and washed (10,000 rpm, 6 min), dispersed in ultrapure water, and then their hydrated particle size and Zeta potential were measured using a Malvern particle size analyzer. The specific test results are shown in the table below: Effect of the reaction system on the particle size of Asp / BSA-Cu3(PO4)2·3H2O (x̅ ±s, n=3)

[0060] The test results in the table above show that it is more suitable to prepare nanocrystals at a reaction system pH of 7.4. The prepared nanocrystals have a particle size of less than 1 μm and are relatively stable and negatively charged.

[0061] Figure 1 The image shows the appearance of the Asp / BSA-Cu3(PO4)2·3H2O hybrid nanocrystals (asparaginase organic-inorganic hybrid nanocrystals) of this scheme, as follows. Figure 1 As shown in Figure A, the prepared Asp / BSA-Cu3(PO4)2·3H2O solution is a clear, pale milky white color. After centrifugation and washing, it appears as... Figure 1 As shown in B, it is a blue precipitate.

[0062] Depend on Figure 2 As shown in SEM images A~D, the Asp / BSA-Cu3(PO4)2·3H2O hybrid nanocrystals exhibit a multi-layered petal-shaped spherical particle structure with a particle size of approximately 1 μm. Figure 2 E~F are scanning electron microscope images of Cu3(PO4)2·3H2O crystals. As can be seen from the images, without the incorporation of organic components (proteins), copper phosphate crystal sheets cannot form a complete flower-like structure and instead exhibit a loose sheet-like structure.

[0063] Figure 3 Transmission electron microscopy was used to characterize Asp / BSA-Cu3(PO4)2·3H2O crystals. As shown in the figure, the surface of a single Asp / BSA-Cu3(PO4)2·3H2O nanocrystal has multiple petal-like structures around it, which are lighter in color than the central region. The particle size is about 1 μm.

[0064] Figure 4 X-ray powder diffraction was used to analyze Asp / BSA-Cu3(PO4)2·3H2O and Cu3(PO4)2·3H2O, as shown in the figure. The blue line at the bottom represents the standard spectral peaks of Cu3(PO4)2·3H2O (JPSCD 00-022-0548), the red curve represents the spectral lines of crystalline Cu3(PO4)2·3H2O, and the black curve represents the spectral lines of hybrid nanocrystals Asp / BSA-Cu3(PO4)2·3H2O. Analysis of the figure shows that the X-ray diffraction patterns of Asp / BSA-Cu3(PO4)2·3H2O hybrid nanocrystals and Cu3(PO4)2·3H2O are in good agreement with those of Cu3(PO4)2·3H2O (JPSCD00-022-0548). This indicates that copper phosphate crystals do indeed exist in the Asp / BSA-Cu3(PO4)2·3H2O hybrid nanocrystals, which is consistent with the formation mechanism of nanocrystals.

[0065] Figure 5 Fourier transform infrared spectroscopy was used to analyze the samples (BSA / Asp, Cu3(PO4)2·3H2O, BSA-Cu3(PO4)2·3H2O, Asp / BSA-Cu3(PO4)2·3H2O), as shown in the figure. A and D are the infrared absorption spectra of BSA / Asp doped nanocrystals, BSA / Asp, Cu3(PO4)2·3H2O, and BSA doped nanocrystals, respectively. Figures A, C, and D show the infrared absorption spectra at 1090 cm⁻¹. -1 978 cm -1 The absorption at that location is PO4 3- Antisymmetric and symmetric stretching vibrations, while at 610 cm -1 567 cm -1 The absorption at that location is PO4 3- The in-plane bending vibrations indicated the presence of phosphate. In contrast, B, at 3000 cm⁻¹... -1 ~2860 cm -1 Absorption occurs at 1680 cm⁻¹ due to the stretching vibrations of -CH₂ and -CH₃. -1 ~1655 cm -1 An amide I absorption band is present at 1550 cm⁻¹. -1 ~1535 cm -1 Amide II absorption band, 1300 cm⁻¹ -1 ~1200cm -1 Amide III absorption band and 1350 cm -1 ~1250 cm -1The stretching vibrations of the secondary amine -CN, and the absorption peaks present in A and C but absent in D, demonstrate that the hybrid nanocrystals contain BSA or Asp. Furthermore, the absence of new absorption peaks or large chemical shifts in A and C indicates that the structures of Asp, BSA, and Cu3(PO4)2·3H2O remain essentially unchanged, suggesting that Asp and BSA are fixed through self-assembly rather than covalent bonding.

[0066] In addition, this scheme also investigated the particle size stability of Asp / BSA-Cu3(PO4)2·3H2O hybrid nanocrystals, according to the above-mentioned optimal process conditions (BSA concentration of 0.25 mg / mL, Cu... 2+ Asp / BSA-Cu3(PO4)2·3H2O hybrid nanocrystals were prepared by means of 1.6 mM, 0.117 mg / mL Asp concentration, 45 min of shaking reaction, 37 °C reaction temperature, and pH 7.4 reaction system. The hydrated particle size and zeta potential were measured by Malvern particle size analyzer, and the particle size stability of the nanocrystals at 0, 24, 48, and 72 h were investigated.

[0067] like Figure 6 The particle size distribution of Asp / BSA-Cu3(PO4)2·3H2O is shown, exhibiting a single peak with a particle size of approximately 760 nm. Figure 7 (A) shows the particle size stability results of Asp / BSA-Cu3(PO4)2·3H2O. The figure shows that after storage at 4℃ for 72 h, the particle size and PDI of Asp / BSA-Cu3(PO4)2·3H2O did not change significantly during the experiment, and there was no statistically significant difference (P>0.05). This protocol also calculated the protein encapsulation efficiency of the Asp / BSA-Cu3(PO4)2·3H2O hybrid nanocrystals. 1 mL of PBS (0.01 M, pH 7.4) containing 0.250 mg / mL BSA was taken, and Asp enzyme solution was added to adjust the final Asp concentration to 0.117 mg / mL. Then, 15 μL of molecular biological grade CuSO4 aqueous solution (120 mM) was added and mixed well. The mixture was shaken on a shaker at 37℃ for 45 min. After the reaction, the mixture was centrifuged (10000 rpm, 6 min), and the supernatant was collected for analysis. Add 1 mL of ultrapure water to wash the nanocrystal precipitate, centrifuge, and collect the supernatant for analysis. Repeat the measurement three times. The Asp content in the supernatant was determined using a microbial asparaginase (ASP) ELISA kit. The Asp encapsulation amount of the Asp / BSA-Cu3(PO4)2·3H2O hybrid nanocrystals = total Asp added to the reaction - Asp content in the supernatant. The Asp encapsulation efficiency was then calculated, as shown in the table below. Asp encapsulation efficiency of Asp / BSA-Cu3(PO4)2·3H2O (x̅ ±s, n=3)

[0068] The test results in the table above show that the average encapsulation efficiency of Asp / BSA-Cu3(PO4)2·3H2O hybrid nanocrystals is 78.02%, and the encapsulation amount is 36.41 U.

[0069] This study also investigates the recyclability of Asp / BSA-Cu3(PO4)2·3H2O hybrid nanocrystals: (1) Asparaginase activity determination by the Masben-Listen method Reagent Preparation: ① Mixed Buffer: Boric acid-borax buffer; Mops buffer (7:3, pH 8.6). ② Trichloroacetic acid (TCA) solution: Dissolve 0.492g of TCA in 2mL of distilled water. ③ Nessler indicator: Dissolve 16g of NaOH in 50mL of ammonia-free water and cool to room temperature. Separately dissolve 7g of KI and 10g of HgI2 in ammonia-free water, and slowly add this solution to the sodium hydroxide solution while stirring. Dilute with water to 100mL. ④ Substrate solution: Dissolve 18.75mg of asparagine in the mixed buffer and bring the volume to 100mL. ⑤ Ammonium sulfate solution: Dissolve 110.1mg of ammonium sulfate in distilled water and bring the volume to 100mL. ⑥ Enzyme solution: Dissolve the enzyme in the mixed buffer before assay to prepare several dilution solutions with concentrations between 2 and 50 U / mL.

[0070] (2) Measurement procedure ① Add 0.2 mL of mixed buffer and 1.7 mL of substrate solution to each of several centrifuge tubes, place them in a 37°C water bath, add 0.1 mL of enzyme solution, and incubate for 10 min. After the reaction, add 0.1 mL of trichloroacetic acid solution to terminate the reaction. Centrifuge at 3500 rpm for 5 min to separate the test solution. Take 0.5 mL of the supernatant and add it to 7 mL of distilled water, then add 1 mL of Nessler indicator. Let it stand at room temperature for 10 min, and then measure the absorbance using a spectrophotometer (480 nm) with a blank value as a reference. The blank value determination method is the same as the main value determination method, except that 0.1 mL of trichloroacetic acid solution is added before adding the enzyme solution. ② Plot the ammonium sulfate standard curve according to the above method. The amount of each reagent used is shown in the table below:

[0071] The corresponding ammonia content can be calculated from the standard curve, and then the relative enzyme activity (%) can be calculated. Determination of the recyclability of hybrid nanocrystals: Take the hybrid nanocrystal sample solution and examine the recycling of nanocrystals according to the enzyme activity determination method described above. After one round of reaction, determine the corresponding amount of enzyme produced according to the method. Immediately centrifuge the remaining reaction solution at 10,000 rpm for 5 min, discard the supernatant, wash the precipitate with mixed buffer, centrifuge, and then perform the next catalytic cycle. Using the ammonia content determined in the first cycle as 100%, calculate the relative enzyme activity (%) after each of the other cycles.

[0072] Free enzymes have the disadvantages of being non-recyclable and non-reusable. Immobilizing free enzymes to make them reusable is one of the advantages of enzyme immobilization technology. For example... Figure 8 The figure shown is a graph of the reusability of Asp / BSA-Cu3(PO4)2·3H2O hybrid nanocrystals (NCs). After three cycles of use, the activity of the nanocrystals is still about 47%, indicating that the prepared nanocrystals have reusable catalytic properties.

[0073] Example 7 In this embodiment, all preparation steps and parameters are the same as in Example 1, except that the concentration ratios of the raw materials for preparing asparaginase organic-inorganic hybrid nanocrystals are different, as shown in the table below:

[0074] The asparaginase organic-inorganic hybrid nanocrystals prepared in Example 7 were subjected to the following performance tests to detect enzyme activity. The test results are shown in the table below:

[0075] As shown in the table above, the detection data for each group in Example 7 indicate that the concentration ratio of each raw material in the preparation of asparaginase organic-inorganic hybrid nanocrystals has a significant impact on the performance of the product. The preferred concentration ratio of asparaginase to bovine serum albumin in this scheme is (1-3):(1-7), and the concentration ratio of asparaginase to CuSO4 aqueous solution is... 2+ The preferred concentration ratio is (1.5-5):1; the optimal concentration ratio of asparaginase to bovine serum albumin is 1:(1-3), and the concentration ratio of asparaginase to CuSO4 aqueous solution is... 2+ The optimal concentration ratio is (1.5-2.5):1. Within this concentration ratio range, the product exhibits better long-term storage stability, higher catalytic activity, and higher reusability.

[0076] This protocol also investigated the anti-trypsin hydrolysis ability of Asp / BSA-Cu3(PO4)2·3H2O hybrid nanocrystals: Free Asp and NCs (Asp / BSA-Cu3(PO4)2·3H2O hybrid nanocrystals) were mixed with 0.25% trypsin and incubated in a 37℃ water bath for 0, 10, 20, 30, 40, 50, and 60 min. Enzyme samples were taken at the corresponding time points, and the activities of free Asp and NCs were determined according to the previously described method (Maschben-Leiston method). The relative activity of Asp at each incubation time point was calculated with the initial activity as 100%. Figure 9 As shown, free Asp was rapidly hydrolyzed by trypsin, and its activity decreased sharply. After 30 min of incubation with trypsin, only about 4% of the free Asp activity remained, while the Asp activity in Asp / BSA-Cu3(PO4)2·3H2O hybrid nanocrystals (NCs) was still about 74%. At 60 min, free Asp was almost completely inactivated, while the Asp activity in NCs was still about 49%. Under the experimental conditions, the Asp activity in NCs was higher than that in free Asp, and the difference was statistically significant (P < 0.05; 0 min), suggesting that the immobilized Asp has better resistance to enzymatic hydrolysis.

[0077] Example 8 A process for preparing asparaginase-encapsulated microparticles includes the following steps: 1. Add 15 μL of molecular biological grade CuSO4 aqueous solution (120 mM) to 1 mL of PBS (0.01 M, pH 7.4) containing asparaginase and bovine serum albumin to adjust the final concentration of asparaginase to 0.117 mg / mL and the final concentration of bovine serum albumin to 0.25 mg / mL. Shake the mixture at 37 °C for 45 min. After the reaction is complete, centrifuge (10000 rpm, 6 min) and wash to obtain the precipitate, which is the asparaginase organic-inorganic hybrid nanocrystals (NCs).

[0078] 2. The above-mentioned nanocrystal (NCs) precipitate was dispersed in a chitosan / chitosan oligosaccharide (CS / COS) solution (pH 6.2), stirred on a constant temperature magnetic stirrer for 15 min, and washed three times with deionized water by centrifugation (10000 rpm, 5 min) to obtain the intermediate product -CS / COS-NCs.

[0079] 3. Disperse the above CS / COS-NCs precipitate in a boric acid-borax / Mops mixed buffer, add dopamine solution to make the final concentration 0.075 mg / mL, stir magnetically at room temperature for 40 min, centrifuge (10000 rpm, 6 min) and wash to obtain the precipitate, which is the asparaginase-encapsulated microparticle.

[0080] To prepare a CS solution, weigh 0.02875 g of chitosan (CS) and add it to 100 mL of 1% acetic acid solution. Stir the solution on a magnetic stirrer until completely dissolved to obtain a CS stock solution with a concentration of 0.2875 mg / mL. Weigh 0.02875 g of chitosan oligosaccharide (COS) and add it to 100 mL of ultrapure water. Stir to dissolve the COS solution to obtain a COS solution. Mix the prepared CS stock solution and COS solution in a 1:1 ratio and adjust the pH of the solution to 6.2 to obtain a CS / COS mixture.

[0081] Preparation of 0.05 mol / L borax solution: Weigh 1.907 g borax, add 100 mL ultrapure water, and stir to dissolve. Preparation of 0.2 mol / L boric acid solution: Weigh 1.237 g boric acid, add 100 mL ultrapure water, and stir to dissolve. Preparation of boric acid-borax buffer solution: Mix the prepared borax solution and boric acid solution in a 9:11 ratio until homogeneous. Preparation of Mops buffer solution (0.01 M, pH 8.0): Weigh 0.2093 g Mops buffer salt solid, add 100 mL ultrapure water, stir to dissolve, and adjust the pH to 8.0. Preparation of boric acid-borax / Mops mixed buffer solution: Mix the prepared boric acid-boric acid solution and Mops buffer solution in a 7:3 ratio until homogeneous, and adjust the pH to 8.5.

[0082] Preparation of 1 mg / mL dopamine (DA) concentrate: Weigh 10 mg of DA powder and dissolve it in 10 mL of ultrapure water.

[0083] Figure 10 Here is an appearance diagram of the CS / COS-NCs solution, such as... Figure 10 As shown in Figure A, the prepared CS / COS-NCs solution is a clear milky white color; after centrifugation and washing, it appears as follows: Figure 10 As shown in (B), it is a blue precipitate. Figure 11 This is an appearance diagram of the PDA@CS / COS-NCs solution, as shown below. Figure 11 As shown in Figure A, the prepared PDA@CS / COS-NCs solution is a clear, dark brown color; after centrifugation and washing, it appears as follows: Figure 11 As shown in (B), it is a black precipitate.

[0084] As shown in the table below, the nanoflowers (CS / COS-NCs) coated with chitosan / chitooligosaccharide composite coating have a particle size of about 1 μm, PDI < 0.3, relatively uniform particle size distribution, and are positively charged. Figure 12 The particle size distribution of CS / COS-NCs is shown as a single peak, with a particle size of approximately 1 μm.

[0085] Particle size and Zeta potential (x̅ ±s, n=3) of CS / COS-NCs

[0086] As shown in the table below, the asparaginase-encapsulated microparticles (PDA@CS / COS-NCs) have a particle size of about 1 μm, a PDI of <0.3, a relatively uniform particle size distribution, and a negative charge. Figure 13 The particle size distribution of PDA@CS / COS-NCs is shown as a single peak, with a particle size of approximately 900 nm. Experiments revealed that the particle size of PDA@CS / COS-NCs is slightly smaller than that of CS / COS-NCs. This may be because the polydopamine layer (formed by the oxidative polymerization of dopamine under alkaline conditions) has strong adhesive properties. Coating CS / COS-NCs with polydopamine makes its flower-like structure more compact, thus altering the particle size.

[0087] Particle size and Zeta potential (x̅ ±s, n=3) of PDA@CS / COS-NCs

[0088] The CS / COS-NCs were characterized using scanning electron microscopy (SEM), and the results are shown in Figure 14. The particles exhibit a multi-layered, petal-like spherical structure, similar in morphology to NCs, but their spherical structure is flatter and the petals are more loosely packed and thicker, with a particle size of approximately 1 μm. The PDA@CS / COS-NCs were also characterized using SEM, and the results are shown in Figure 14. Figure 15 As shown, the microparticles have a flower-like spherical structure, similar to the morphology of CS / COS-NCs, but their petal-like layered structure is not obvious due to the covering of polydopamine, and the structure is more compact with a particle size of about 1 μm.

[0089] PDA@CS / COS-NCs were characterized and observed using transmission electron microscopy, and the results are as follows: Figure 16 As shown, the surface of PDA@CS / COS-NCs has a layered light-colored structure. Compared with NCs, no obvious needle-like petal crystal structure was observed in the central region. The color is darker and the structure is more compact, with a particle size of about 1 μm.

[0090] The following samples were analyzed using Fourier transform infrared spectroscopy, and the results are as follows: Figure 17 As shown, A and C represent the infrared absorption spectra of CS-NCs, PDA@NCs, and PDA@CS-NCs, respectively. The absorption spectra are in the range of 1100–1050 cm⁻¹. -1 1000~970 cm -1 The strong absorption at that point is PO4. 3- Antisymmetric and symmetric stretching vibrations, while at 650–610 cm -1 580~540 cm -1 The weak absorption at that point is PO4.3- The antisymmetric and symmetric stretching vibrations suggest the presence of phosphate groups. Chitosan molecules contain hydroxyl and amino groups, as do polydopamine. CS / COS-NCs (A) encapsulated with chitosan / chitosan oligosaccharides and PDA-NCs (B) encapsulated with a polydopamine film polymerized from dopamine share the same functional groups in their chemical bonds, with no significant functional group shifts between them. At 3500 cm⁻¹ -1 ~3300 cm -1 Absorption occurs at the -OH stretching vibration at 3000 cm⁻¹. -1 ~2850 cm -1 Absorption occurs at 1680 cm⁻¹ due to the stretching vibrations of -CH₂ and -CH₃. -1 ~1655 cm -1 An amide I absorption band is present at 1550 cm⁻¹. -1 ~1535 cm -1 Amide II absorption band, 1300 cm⁻¹ -1 ~1200 cm -1 Amide III absorption band and 1350 cm -1 ~1250 cm -1 Secondary amine-CN stretching vibration. Furthermore, PDA@CS / COS-NCs obtained by first encapsulating with chitosan / chitosan oligosaccharide and then polymerizing with dopamine did not exhibit any new absorption peaks or large chemical shifts, demonstrating that the structures of chitosan / chitosan oligosaccharide, polydopamine, and nanocrystals remained essentially unchanged.

[0091] The PDA@CS / COS-NCs prepared above were centrifuged (10000 rpm, 6 min), the supernatant was discarded and the precipitate was obtained. The precipitate was washed three times by centrifugation with deionized water. The precipitate was dispersed in deionized water and its hydrated particle size and zeta potential were measured using a Malvern particle size analyzer at 0, 24, 48 and 72 h to investigate the particle size stability of PDA@CS / COS-NCs.

[0092] like Figure 18 The figure shows the results of the particle size stability study of PDA@CS / COS-NCs. As can be seen from the figure, PDA@CS / COS-NCs were stored at 4℃ for 72 h. During the experiment, there was no significant change in particle size and PDI, and no statistically significant difference was found (P>0.05). Figure 18 -B is the result of the zeta potential stability study of PDA@CS / COS-NC. As shown in the figure, the zeta potential of PDA@CS / COS-NC did not change significantly during the experiment (0~72 h), and there was no statistically significant difference (P>0.05).

[0093] The CS / COS-NCs obtained by the above preparation method were centrifuged (10000 rpm, 6 min), and the supernatant was collected for analysis. 1 mL of ultrapure water was added to wash the CS / COS-NCs precipitate, and the supernatant was collected after centrifugation. This cycle was repeated three times. The encapsulation efficiency was determined using a microbial asparaginase (ASP) ELISA kit, as shown in the table below: Encapsulation efficiency of CS / COS-NCs (x̅ ±s, n=3)

[0094] The test results in the table above show that the average encapsulation efficiency of CS / COS-NCs is 89.34%, and the encapsulation amount is 32.53 U.

[0095] The PDA@CS / COS-NCs obtained by the above preparation method were centrifuged (10000 rpm, 6 min), and the supernatant was collected for analysis. 1 mL of ultrapure water was added to wash the PDA@CS / COS-NCs precipitate, and the supernatant was collected again for analysis. This cycle was repeated 3 times. The corresponding encapsulation efficiency was determined using a microbial asparaginase (ASP) ELISA kit, as shown in the table below: Encapsulation efficiency of PDA@CS / COS-NCs (x̅ ±s, n=3)

[0096] The test results in the table above show that the average encapsulation rate of PDA@CS / COS-NCs is 90.77%, and the final encapsulation amount is 29.53 U.

[0097] Take an appropriate amount of PDA@CS / COS-NCs sample solution and determine the recovery and utilization of PDA@CS / COS-NCs according to the Masben-Liston method. After one round of reaction, determine the corresponding amount of enzyme produced according to the method. Immediately centrifuge the remaining reaction solution at 10,000 rpm for 5 min, discard the supernatant, wash the precipitate with mixed buffer, centrifuge, and then perform the next catalytic cycle. Using the ammonia content determined in the first cycle as 100%, calculate the relative Asp activity (%) in each of the other cycles.

[0098] Coating nanocrystals with a biopolymer coating can further improve the reusability of enzymes, such as... Figure 19 As shown, after 8 cycles, PDA@CS / COS-NCs still had about 28% activity, indicating that the coating of the polymer layer can further improve the reusability of the nanocrystals for catalysis.

[0099] Take PDA@CS / COS-NCs and free Asp solutions, add 5 volumes of blank plasma respectively, mix well, and incubate at 37℃ for 0, 1, 2, 4, 8, 12, 24, 48, 60, 72, and 96 hours. Then, determine the Asp activity of each group according to the previously described Masben-Leiston method, and calculate the relative activity of each group with the highest enzyme activity as 100%. Figure 20 As shown, within 24–96 h, the Asp activity in PDA@CS / COS-NCs was consistently higher than that of free Asp, and the difference was statistically significant (P < 0.05). Under the experimental conditions, the activity of free Asp decreased more rapidly, with approximately 80% loss by 48 h, while the Asp activity in PDA@CS / COS-NCs only decreased by 40%. These results suggest that Asp in PDA@CS / COS-NCs exhibits better plasma stability than free Asp. This result is mainly attributed to the immobilized enzyme technology and the coating of the biopolymer layer.

[0100] Free Asp and PDA@CS / COS-NCs solutions were stored at 4℃ and 25℃, respectively. Enzyme activity was measured at 0, 7, 14, 21, and 28 days. The highest enzyme activity was considered as 100%, and the relative activity of each group was calculated. Figure 21 As shown, after storage at 4°C for 14, 21, and 28 days, the Asp activity in PDA@CS / COS-NCs was higher than that of free Asp. After 28 days of storage, the Asp activity in PDA@CS / COS-NCs was still approximately 84%, while that of free Asp was only about 67%. Figure 22 As shown, after storage at 25℃ for 21 and 28 days, the Asp activity in PDA@CS / COS-NCs was higher than that of free Asp. In summary, the results indicate that, regardless of whether stored at 4℃ or 25℃, the Asp in PDA@CS / COS-NCs exhibits better storage stability than free Asp.

[0101] Example 9 In this embodiment, all preparation steps and parameters are the same as in Example 8, except that the concentration ratios of the raw materials used to prepare the asparaginase-encapsulated microparticles are different, as shown in the table below:

[0102] The asparaginase-encapsulated microparticles prepared in Example 9 were subjected to the following performance tests, and the results are shown in the table below:

[0103] As shown in the test data of each group in Example 9 above, the concentration ratio of each raw material used to prepare asparaginase-encapsulated microparticles has a significant impact on the performance of the product. In this scheme, the preferred concentration ratio of nanocrystals to chitosan / chitosan oligosaccharide solution is 1:(1-5), the preferred concentration ratio of intermediate product to dopamine solution is (1-5):1, the optimal concentration ratio of nanocrystals to chitosan / chitosan oligosaccharide solution is 1:(1-3), and the optimal concentration ratio of intermediate product to dopamine solution is (1-3):1. Within the above concentration ratio ranges, the obtained product has uniform particle size and good stability, improved plasma stability and storage stability, and stronger recyclability.

[0104] Example 10 In this embodiment, all preparation steps and parameters are the same as in Example 8, except that the molecular weights of chitosan and chitosan oligosaccharides are different, as shown in the table below:

[0105] The asparaginase-encapsulated microparticles prepared in Example 10 were subjected to the following performance tests, and the results are shown in the table below:

[0106] As shown in the test data of each group in Example 10 above, the molecular weight of chitosan and chitosan oligosaccharide has a significant impact on the particle size and long-term storage stability of the product. In this scheme, the molecular weight of chitosan is preferably 30,000-50,000, and the molecular weight of chitosan oligosaccharide is preferably 1,000-2,000. Under the above range, the obtained product has better long-term storage stability and higher reusability.

[0107] This protocol also evaluated the in vitro anti-leukemia efficacy as follows: CCK8 cytotoxicity assay: To evaluate the cytotoxic effects of different concentrations of Asp, NCs, and PDA@CS / COS-NCs on Jurkat cells, the CCK8 assay was used to determine cell viability in each group 12 h, 24, and 48 h after drug administration. Figure 23 As shown in (A~C), compared with the Asp group, the NCs group and the PDA@CS / COS-NCs group had significant toxic effects on Jurkat cells, reduced cell survival rate, and showed a relatively obvious time-concentration-dependent inhibitory effect. Figure 23(A) Survival rate of Jurkat cells in each group 12 h after drug administration. At concentrations of 0.025 U / mL to 0.1 U / mL, there was no significant difference in cell viability among the three groups (P > 0.05). At a concentration of 0.2 U / mL, cell viability in the PDA@CS / COS-NCs group was lower than that in the Asp group (P < 0.05). At a concentration of 0.4 U / mL, cell viability in both the NCs group and the PDA@CS / COS-NCs group was significantly lower than that in the Asp group (P < 0.01), but there was no significant difference in cell viability between the NCs group and the PDA@CS / COS-NCs group (P > 0.05). Figure 23 (B~C) represent the survival rates of Jurkat cells 24 and 48 hours after drug administration, respectively. Within the concentration range of 0.025 U / mL to 0.4 U / mL, the cytotoxic effects on Jurkat cells varied among different drug administration groups. Compared with the Asp group, the NCs group and the PDA@CS / COS-NCs group showed stronger cytotoxic effects on Jurkat cells, with a significant difference in inhibitory effect (P < 0.01); while compared with the NCs group, the PDA@CS / COS-NCs group showed stronger cytotoxic effects on Jurkat cells, with a significant difference (P < 0.05). A comprehensive analysis suggests that the NCs prepared using immobilized enzyme technology may have improved the stability and catalytic activity of free Asp to some extent, leading to stronger cytotoxic effects on Jurkat cells. Furthermore, the presence of a biopolymer encapsulation layer in PDA@CS / COS-NCs may have protected the NCs from direct exposure to the complex external environment, potentially further enhancing their stability and thus exhibiting a more significant cytotoxic effect, which becomes more pronounced over time. Figure 23 (D) represents the cell survival rate of Jurkat cells after 12, 24, and 48 h of intervention with the vector PDA@CS / COS-NCs (without incorporating enzyme Asp). As shown in the figure, the vector has no obvious toxic effect on Jurkat cells, and there is no statistically significant difference (P>0.05), indicating good biocompatibility.

[0108] DNA-Ladder Detection of Apoptosis: DNA was extracted from Jurkat cells (both untreated and treated with Asp, NCs, and PDA@CS / COS-NCs for 24 h) and DNA fragments were separated by gel electrophoresis. Figure 24As shown, untreated Jurkat cells did not form a DNA ladder pattern, indicating that no obvious apoptosis occurred. However, in the drug-treated groups treated with Asp, NCs, and PDA@CS / COS-NCs for 24 h, Jurkat cells all showed DNA fragments produced due to apoptosis, forming a DNA ladder pattern. In summary, the results suggest that drug-induced Jurkat cell death may involve an apoptotic pathway, but there was no significant difference in the width and brightness of the DNA ladder among the groups.

[0109] DNA Ladder Detection Method for Apoptosis: Soft Agar Plate Cloning Assay: This assay was used to evaluate the effects of Asp, NCs, and PDA@CS / COS-NCs on the proliferation of Jurkat cells. Figure 25 As shown, compared with the Control group, the Asp group, NCs group, and PDA@CS / COS-NCs group all significantly inhibited the proliferation and clonal capacity of Jurkat cells (P < 0.05). Compared with the Asp group and NCs group, the PDA@CS / COS-NCs group with a drug concentration of 0.1 U / mL showed stronger ability to inhibit Jurkat cell colony formation, and the number of cell colonies formed was significantly lower than that of the other two groups (P < 0.05). Furthermore, the number of colonies formed was negatively correlated with the drug concentration. Figure 26 This is a diagram of the colonies formed by the proliferation of Jurkat cells, showing the cells growing densely in single clusters.

[0110] Flow cytometry detection of apoptosis: Flow cytometry was used to observe the staining of FITC and PI after drug-induced apoptosis. Figure 27 As shown, Q1 in the upper left quadrant represents necrotic or mechanically damaged cells, Q2 in the upper right quadrant represents late-stage apoptotic cells, Q3 in the lower right quadrant represents early-stage apoptotic cells, and Q4 in the lower left quadrant represents viable cells. The apoptosis rate is usually calculated using Q2 + Q3. Figure 27As shown, compared with the Control group, the apoptosis rates of Asp, NCs, and PDA@CS / COS-NCs groups were significantly increased (P < 0.01); the apoptosis rates of NCs and PDA@CS / COS-NCs groups were higher than those of the Asp group (P < 0.05), suggesting that NCs and PDA@CS / COS-NCs have better pro-apoptotic effects; the apoptosis rate of the PDA@CS / COS-NCs group was higher than that of the NCs group, but the difference was not significant (P > 0.05). Compared with free Asp, the stronger pro-apoptotic effects of NCs and PDA@CS / COS-NCs are mainly attributed to two aspects: firstly, the stability and activity of immobilized Asp are improved; secondly, the encapsulation of the biopolymer layer further enhances the stability of Asp, allowing it to maintain good catalytic activity for a longer period of time.

[0111] In summary, the apoptosis-DNA ladder experiment verified that the killing effect of the drug Asp on Jurkat cells involves the apoptotic pathway. In vitro cytotoxicity assays, soft agar plate cloning assays, and flow cytometry assays for apoptosis detection showed that PDA@CS / COS-NCs had a significant inhibitory effect on Jurkat cells, and this effect was much stronger than that of free Asp. This result is attributed to two factors: firstly, the immobilization of the enzyme improved the stability and catalytic activity of Asp; secondly, the encapsulation of the polymer layer further enhanced the stability of Asp, allowing it to remain stable in complex external environments and prolonging the catalytic hydrolysis time.

[0112] This protocol also evaluated in vivo immunogenicity as follows: Detection of specific IgM and IgG: The in vivo immunogenicity of Asp and PDA@CS / COS-NCs was examined by measuring the relative changes in the levels of specific IgM and IgG in mouse serum after drug administration. During the primary immune response, IgM, as the earliest produced antibody, plays an important role in early immune defense. Figure 28 As shown, compared with the Control group, after the initial immune stimulation, specific anti-Asp IgM was present in the serum of mice in both the Asp group and the PDA@CS / COS-NCs group. Compared with the Control group, the IgM levels in both groups were significantly increased (P < 0.05), indicating an immune response after drug administration. Compared with the PDA@CS / COS-NCs group, the Asp group showed significantly higher IgM secretion levels, suggesting a stronger immune stimulation from free Asp. After re-stimulation and the third drug administration, as... Figure 28As shown in (BC), the increase in IgM was significantly less than that of the primary immunization. This is because the content and types of specific antibodies in the body fluids change in the later stages of immunization, with the antibody type gradually shifting from predominantly IgM to predominantly IgG. After the third immunization, compared with the Control group and the PDA@CS / COS-NCs group, the IgM secretion level in the Asp group was significantly higher (P < 0.001), while the IgM secretion level in the PDA@CS / COS-NCs group was not significantly different from that in the Control group after the third immunization (P > 0.05), suggesting that free Asp provides a stronger immune stimulus to mice than PDA@CS / COS-NCs. During the primary immunization response, because the specific antibody type in the body fluids is predominantly IgM, the production of IgG occurs relatively late, resulting in relatively low IgG secretion at this stage. Figure 28 As shown in (D), compared with the Control group, the IgG levels in the Asp group and the PDA@CS / COS-NCs group were significantly increased (P < 0.01), and the increase in IgG secretion level in the Asp group was more significant compared with the PDA@CS / COS-NCs group. Upon re-stimulation and the third dose stimulation, as... Figure 28 As shown in (EF), the increase in IgG was significantly stronger than that of the primary immunization. This is because specific IgG can be produced in large quantities and dominate the immune response in the later stages of immunization. Compared with the Control group, the IgG secretion levels in the Asp group and the PDA@CS / COS-NCs group were significantly increased (P < 0.001), while the IgG secretion level in the PDA@CS / COS-NCs group was significantly decreased compared with the Asp group, suggesting that free Asp has a stronger immune stimulation on mice than PDA@CS / COS-NCs, and that PDA@CS / COS-NCs has lower immunogenicity. The lower immunogenicity of PDA@CS / COS-NCs compared with free Asp is mainly attributed to the biocompatible polymer (chitosan / chitooligosaccharide, polydopamine) coating on the surface of PDA@CS / COS-NCs. This coating can improve the performance of nanocrystals and can shield the antigenic epitopes on the Asp surface through steric hindrance, reducing the recognition of the immune system and the production of neutralizing antibodies.

[0113] Hematological parameters: The in vivo immunogenicity of Asp and PDA@CS / COS-NCs was evaluated by detecting changes in blood cells and hemoglobin in Blab / c mice. Figure 29The hematological parameters of mice on day 21 are shown below: WBC (white blood cell count), RBC (red blood cell count), HGB (hemoglobin), and PLT (platelet count). The results showed that compared with the Control group, there were no significant differences in any of the hematological parameters in the PDA@CS / COS-NCs group (P>0.05), while the Asp group showed statistically significant differences (P<0.05). Free Asp affects the number of blood cells and platelets, as well as hemoglobin expression in mice, and to some extent reduces the number of WBCs, RBCs, PLTs, and the expression of HGB. This toxic side effect may be attributed to immune-related toxicity following three injections of highly immunogenic Asp. This toxic side effect was still observed in the Asp group mice one week after drug withdrawal, while the same phenomenon was not observed in the PDA@CS / COS-NCs group mice. This suggests that Asp has certain toxic side effects on normal mice, while PDA@CS / COS-NCs has no significant effect on the hematological parameters of normal mice, or that it has a small effect on the hematological parameters of mice, and the mice can recover to normal quickly after drug withdrawal.

[0114] Serum biochemical assays: The effects of Asp and PDA@CS / COS-NCs on the function of major organs in mice were evaluated by measuring serum biochemical indicators. Liver and kidney function indicators in experimental mice were as follows: Figure 30 As shown, the liver function parameters were ALT (alanine aminotransferase) and AST (aspartate aminotransferase); the kidney function parameters were UEA (urea), UA (uric acid), and CREA (creatinine). The results showed that there were no significant differences in liver and kidney function parameters between the PDA@CS / COS-NCs group and the Control group (P>0.05), suggesting that PDA@CS / COS-NCs did not significantly damage liver and kidney function in mice. The ALT, AST, and UEA values ​​in the Asp group were all higher than those in the Control group (P<0.05), suggesting that free Asp may cause some damage to liver and kidney function in mice. This difference in damage is mainly attributed to the biocompatible polymer (chitosan / chitooligosaccharide, polydopamine) coating on the surface of PDA@CS / COS-NCs. This coating can mask the active sites of Asp, leading to a decrease in the immunogenicity of PDA@CS / COS-NCs. In contrast, the antigenic epitopes of free Asp are directly exposed to body fluids, making them more easily recognized and attacked by the body's immune system, thus resulting in higher immunogenicity.

[0115] Mouse organ index assessment: By comparing the organ indices of experimental mice, the effects of drugs on organs can be reflected to some extent. For example... Figure 31As shown, compared with the Control group, there were no significant differences in organ indices of the heart, liver, spleen, lungs, kidneys, and thymus in the PDA@CS / COS-NCs group (P>0.05), suggesting that PDA@CS / COS-NCs had little effect on organ mass in mice. However, the liver and thymus in the Asp group were significantly larger than those in the Control group (P<0.05), suggesting that free Asp may have some effect on the liver of normal mice, and its relatively high immunogenicity may also stimulate the thymus, leading to its enlargement. In summary, the results indicate that Asp may have potential toxic side effects in mice, which may stimulate the liver and thymus, leading to increased mass. This phenomenon was not observed in the PDA@CS / COS-NCs group, suggesting that PDA@CS / COS-NCs may have higher safety and lower immunogenicity.

[0116] In summary, the immunogenicity of free Asp and PDA@CS / COS-NCs in vivo was assessed using normal BALB / c mice. Dynamic monitoring of specific antibody levels in mice revealed that PDA@CS / COS-NCs induced lower levels of specific antibody secretion in vivo compared to free Asp. Monitoring of complete blood count, blood biochemistry, organ indices, and pathological sections in mice showed that PDA@CS / COS-NCs had no significant effect on major organs, blood cells, or liver and kidney function, while free Asp caused some damage to certain organs, blood cells, and liver and kidney function. This suggests that PDA@CS / COS-NCs induces lower immunogenicity in vivo compared to free Asp. This result is mainly attributed to the protective effect of the biopolymer layer on the nanocrystals. This coating layer reduces the exposure of enzyme antigen epitopes to a certain extent without affecting the enzyme's hydrolytic activity, thus lowering its immunogenicity in vivo.

[0117] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the patent. The asparaginase organic-inorganic hybrid nanocrystals or asparaginase-encapsulated microparticles mentioned in the present invention are not limited to the above-mentioned types. Therefore, the above embodiments should not be regarded as a limitation on the scope of protection of the present invention. All equivalent structural transformations made using the contents of the present invention under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the scope of patent protection of the present invention.

Claims

1. A preparation process for asparaginase organic-inorganic hybrid nanocrystals, characterized in that, The preparation process includes the following steps: CuSO4 aqueous solution is added to a phosphate buffer solution containing asparaginase and bovine serum albumin and shaken to obtain the asparaginase organic-inorganic hybrid nanocrystals; The concentration ratio of the asparaginase to the bovine serum albumin is (1-3):(1-7), and the concentration ratio of the asparaginase to Cu in the CuSO4 aqueous solution is (1.5-5):

1. 2+ The concentration ratio of the asparaginase to the bovine serum albumin is (1-3):(1-7), and the concentration ratio of the asparaginase to Cu in the CuSO4 aqueous solution is (1.5-5):

1.

2. The preparation process of asparaginase organic-inorganic hybrid nanocrystals according to claim 1, characterized in that, The concentration of bovine serum albumin is 0.1-0.7 mg / mL; the concentration of asparaginase is 0.1-0.3 mg / mL; and the CuSO4 aqueous solution contains Cu... 2+ The concentration is 0.5-3.5 mM.

3. The preparation process of asparaginase organic-inorganic hybrid nanocrystals according to claim 1, characterized in that, The oscillation time is 45-90 minutes, and the reaction temperature during oscillation is 30-40℃.

4. The preparation process of asparaginase organic-inorganic hybrid nanocrystals according to claim 1, characterized in that, The pH value of the phosphate buffer solution is 7-8.

5. An asparaginase organic-inorganic hybrid nanocrystal, characterized in that, It is prepared by the preparation process of asparaginase organic-inorganic hybrid nanocrystals according to any one of claims 1-4.

6. A preparation process for encapsulating asparaginase microparticles, characterized in that, The preparation steps include the following: S1. The asparaginase organic-inorganic hybrid nanocrystal precipitate of claim 5 is dispersed in a chitosan / chitosan oligosaccharide solution, stirred, centrifuged and washed to obtain an intermediate product; wherein the concentration ratio of the asparaginase organic-inorganic hybrid nanocrystal to the chitosan / chitosan oligosaccharide solution is 1:(1-5). S2. The intermediate product precipitate is dispersed in a boric acid-borax / Mops mixed buffer solution, dopamine solution is added, the mixture is magnetically stirred at room temperature, and the precipitate is obtained by centrifugation and washing, which is the encapsulated asparaginase microparticle; wherein the concentration ratio of the intermediate product to the dopamine solution is (1-5):

1.

7. The preparation process of asparaginase-encapsulated microparticles according to claim 6, characterized in that, The preparation of the chitosan / chitosan oligosaccharide solution includes the following steps: S11. Add chitosan to the acetic acid solution and stir magnetically until completely dissolved to obtain a concentrated chitosan stock solution; S12. Add chitosan oligosaccharide to ultrapure water and stir to dissolve to obtain chitosan oligosaccharide solution; S13. The chitosan concentrate and chitosan oligosaccharide solution are mixed evenly at a concentration ratio of (0.5-5):1 to obtain the chitosan / chitosan oligosaccharide solution; The preparation of the boric acid-borax / Mops mixed buffer solution includes the following steps: S21. Add borax to ultrapure water and stir to dissolve to obtain borax solution; add boric acid to ultrapure water and stir to dissolve to obtain boric acid solution. S22. Add Mops buffer salt to ultrapure water and stir to dissolve to obtain Mops buffer solution; S23. Mix the borax solution and the boric acid solution evenly to obtain a boric acid-borax solution; mix the boric acid-borax solution with the Mops buffer evenly to obtain the boric acid-borax / Mops mixed buffer.

8. The preparation process of asparaginase-encapsulated microparticles according to claim 6, characterized in that, The molecular weight of the chitosan is 10,000-100,000, and the molecular weight of the chitosan oligosaccharide is 1,000-5,000.

9. The preparation process of asparaginase-encapsulated microparticles according to claim 8, characterized in that, In step S21, the volume ratio of the borax solution and the boric acid solution when mixed is (7-10):11, and the pH value of the Mops buffer is 7.5-9; In step S23, the volume ratio of the boric acid-borax solution and the Mops buffer solution is 7:(1-5), and the pH value of the boric acid-borax / Mops mixed buffer solution is 7.5-9.

5.

10. A microparticle encapsulating asparaginase, characterized in that, It is prepared by the preparation process of encapsulated asparaginase microparticles as described in any one of claims 6-9.

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