Micro-nano macromolecular biological medicine and preparation method thereof

Abstract

The invention discloses a micro-nano macromolecular biological medicine and a preparation method thereof, and the process comprises the following steps: dissolving a macromolecular biological medicine and a polymer which is not adsorbed with the macromolecular biological medicine in distilled water to prepare a macromolecular biological medicine-non-adsorption polymer aqueous solution; thoroughly freezing the aqueous solution of the macromolecular biological medicine-non-adsorption polymer; freeze-drying the intermediate product in the previous step to obtain a freeze-dried product; and eluting the free non-adsorption polymer in the freeze-dried product by using a volatile organic solvent, and obtaining the micro-nano macromolecular biological medicine particles after the organic solvent is completely volatilized. The preparation method has the advantages that the preparation process is simple, the condition is mild, the controllability is high, the cost is low, the biological activity of the macromolecular biological medicine can be kept to the maximum extent, industrial production is realized, and a technical support is provided for micro-nanocrystallization of the macromolecular biological medicine; the prepared micron-sized, submicron-sized and nano-sized macromolecular biological medicine particles are beneficial to research and development of new biological medicine dosage forms, and have wide application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceutical preparation, in particular to micro-nano biopharmaceuticals. Background Art

[0002] Macromolecular biological drugs, such as recombinant protein drugs, antibody drugs, antibody-drug conjugates (ADCs), CRISPR-Cas9 gene editing drugs, etc., play an increasingly important role in the development of health science in modern society. Among them, the market size of protein drugs is about to reach 400 billion US dollars. Compared with small molecule drugs, protein drugs have higher specificity and efficacy, as well as lower side effects. Therefore, the FDA's approval time for protein drugs is relatively short.

[0003] However, protein drugs have a short half-life in the body and require frequent and high-dose injections to maintain effective drug concentrations. This mode of administration can cause complications caused by dose fluctuations and may reduce patient compliance. Therefore, innovative macromolecular biopharmaceutical preparations such as injectable preparations and long-acting sustained-release protein drug preparations are in urgent need of development. These preparations can achieve sustained and controlled release of protein drugs, thereby achieving the purpose of extending the half-life of protein drugs. The key to developing and preparing the above-mentioned innovative macromolecular biopharmaceutical preparations is to prepare micro-nanosized particles of macromolecular biopharmaceuticals by micro-nano-nano-nano-nano-nano-sized macromolecular biopharmaceuticals through micro-nano ... Summary of the invention

[0004] The purpose of the present invention is to provide a micro-nano macromolecular biopharmaceutical and a preparation method thereof, so as to provide key technical support for the development of innovative macromolecular biopharmaceutical preparations such as injectable preparations, long-acting sustained-release protein drug preparations, etc. These innovative macromolecular biopharmaceutical preparations can solve the problems of short half-life of protein drugs in existing drug preparations, as well as the complications and low patient compliance caused by them.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A micro-nano macromolecular biological drug and a preparation method thereof, the process comprising:

[0007] (1) dissolving a macromolecular biodrug and a polymer that does not adsorb to the macromolecular biodrug (nonadsorbing polymer) in distilled water to prepare a macromolecular biodrug-nonadsorbing polymer aqueous solution;

[0008] (2) completely freezing the aqueous solution of the macromolecular biopharmaceutical and non-adsorbed polymer;

[0009] (3) freeze-drying the intermediate product in the previous step to obtain a freeze-dried product;

[0010] (4) The free non-adsorbed polymer in the freeze-dried product is eluted with a volatile organic solvent, and micro-nano-sized macromolecular biopharmaceutical particles are obtained after the organic solvent is completely evaporated.

[0011] Furthermore, in step (1), the concentration of the macromolecular biodrug is 0.4 mg / ml to 500 mg / ml, the mass concentration ratio of the non-adsorbing polymer to the macromolecular biodrug is 0.5:1 to 100:1, the number average molecular weight (Mn) of the non-adsorbing polymer is 400-30000, and the molecular weight of the macromolecular biodrug is 500-900000 Da.

[0012] Furthermore, the macromolecular biopharmaceutical is a protein molecule or a modified protein molecule;

[0013] The non-adsorbing polymer is polysaccharide or polyethylene glycol, wherein the number average molecular weight (Mn) of the polyethylene glycol is 8,000.

[0014] Furthermore, the macromolecular biological drug is selected from bovine serum albumin, horseradish peroxidase, lysozyme, genetically recombinant protein drugs, antibody drugs, antibody-conjugated drugs, and CRISPR-Cas9 gene editing drugs.

[0015] Furthermore, in the step (2), the aqueous solution of the macromolecular biopharmaceutical-non-adsorbing polymer is completely frozen at -20 to -196°C.

[0016] Furthermore, the freeze-drying temperature in step (3) is -20 to -80°C.

[0017] Furthermore, the organic solvent in step (4) is selected from dichloromethane, chloroform, alcohol, ketone, glyceride, and aromatic hydrocarbon.

[0018] Furthermore, in step (4), the organic solvent is naturally evaporated or vacuum dried.

[0019] Furthermore, the size range of the micro-nanosized macromolecular biopharmaceutical particles is 20 nm to 10 μm.

[0020] The present invention also provides a micro-nano macromolecular biological medicine obtained by the preparation method.

[0021] The advantages of the present invention are:

[0022] (1) This method only requires freeze-drying of PEG-macromolecule biological drug aqueous solution and elution of PEG with organic solvent to obtain micron, submicron, and nanometer-sized macromolecule biological drug particles. The preparation process is simple, the conditions are mild, the controllability is strong, the cost is low, and the biological activity of macromolecule biological drugs can be maintained to the maximum extent. Industrial production can be achieved, providing technical support for the micro-nanoization of macromolecule biological drugs.

[0023] (2) The present invention is applicable to a variety of macromolecular biopharmaceutical molecules with different molecular weights and charges, and is a universal method for preparing micron, submicron, and nanometer-sized macromolecular biopharmaceutical particles;

[0024] (3) The process of the present invention can control the particle size of macromolecular biopharmaceutical particles from micron, submicron, to nanometer sizes;

[0025] (4) The prepared micron, submicron, and nanometer-sized macromolecular biopharmaceutical particles are conducive to the development of new dosage forms of biopharmaceuticals, such as long-acting sustained-release preparations, injectable preparations, and biological preparations, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The drawings described herein are used to provide a further understanding of the present invention, constitute a part of the present application, and do not constitute an improper limitation of the present invention. In the drawings:

[0027] Figure 1 a is the zeta potential result of bovine serum albumin (BSA) molecules in a pH 5.3, 10 mM NaCl aqueous solution;

[0028] Figure 1 b is a scanning electron micrograph of BSA particles prepared under the conditions of bovine serum albumin (BSA): polyethylene glycol (PEG) (w / w%) = 1:5 and a freezing temperature of -20°C;

[0029] Figure 1 c is a particle size distribution diagram of BSA particles prepared under the conditions of bovine serum albumin (BSA): polyethylene glycol (PEG) (w / w%) = 1:5 and a freezing temperature of -20°C;

[0030] Figure 2 a is a scanning electron micrograph of BSA particles prepared under the conditions of bovine serum albumin (BSA): polyethylene glycol (PEG) (w / w%) = 1:5 and a freezing temperature of -70°C;

[0031] Figure 2 b is a particle size distribution diagram of BSA particles prepared under the conditions of bovine serum albumin (BSA): polyethylene glycol (PEG) (w / w%) = 1:5 and a freezing temperature of -70°C;

[0032] Figure 3 a is a scanning electron micrograph of BSA particles prepared under the conditions of bovine serum albumin (BSA): polyethylene glycol (PEG) (w / w%) = 1:5 and a freezing temperature of -195°C;

[0033] Figure 3b is a particle size distribution diagram of BSA particles prepared under the conditions of bovine serum albumin (BSA): polyethylene glycol (PEG) (w / w%) = 1:5 and a freezing temperature of -195°C;

[0034] Figure 4 a is a scanning electron micrograph of BSA particles prepared under the conditions of bovine serum albumin (BSA): polyethylene glycol (PEG) (w / w%) = 1:10 and a freezing temperature of -20°C;

[0035] Figure 4 b is a particle size distribution diagram of BSA particles prepared under the conditions of bovine serum albumin (BSA): polyethylene glycol (PEG) (w / w%) = 1:10 and a freezing temperature of -20°C;

[0036] Figure 5 a is a scanning electron micrograph of BSA particles prepared under the conditions of bovine serum albumin (BSA): polyethylene glycol (PEG) (w / w%) = 1:2 and a freezing temperature of -20°C;

[0037] Figure 5 b is a particle size distribution diagram of BSA particles prepared under the conditions of bovine serum albumin (BSA): polyethylene glycol (PEG) (w / w%) = 1:2 and a freezing temperature of -20°C;

[0038] Figure 6 a is a scanning electron micrograph of BSA particles prepared under the conditions of bovine serum albumin (BSA): polyethylene glycol (PEG) (w / w%) = 1:1 and a freezing temperature of -20°C;

[0039] Figure 6 b is a particle size distribution diagram of BSA particles prepared under the conditions of bovine serum albumin (BSA): polyethylene glycol (PEG) (w / w%) = 1:1 and a freezing temperature of -20°C;

[0040] Figure 7 1 is the circular dichroism spectra of bovine serum albumin (BSA) raw material (1 red line) and BSA particles prepared using the raw material (2 black line);

[0041] Figure 8 a is the zeta potential result of horseradish peroxidase (HRP) molecules in an aqueous solution of pH 5.3 and 10 mM NaCl;

[0042] Figure 8 b is a scanning electron micrograph of HRP particles prepared at a ratio of horseradish peroxidase (HRP) to polyethylene glycol (PEG) (w / w%) = 1:5 and a freezing temperature of -20°C;

[0043] Figure 8 c is a particle size distribution diagram of HRP particles prepared at a ratio of horseradish peroxidase (HRP): polyethylene glycol (PEG) (w / w%) = 1:5 and a freezing temperature of -20°C;

[0044] Fig. 9 a is a scanning electron micrograph of HRP particles prepared under the conditions of horseradish peroxidase (HRP): polyethylene glycol (PEG) (w / w%) = 1:5 and a freezing temperature of -70°C;

[0045] Fig. 9 b is a particle size distribution diagram of HRP particles prepared under the conditions of horseradish peroxidase (HRP): polyethylene glycol (PEG) (w / w%) = 1:5 and a freezing temperature of -70°C;

[0046] Fig.10 a is a scanning electron micrograph of HRP particles prepared under the conditions of horseradish peroxidase (HRP): polyethylene glycol (PEG) (w / w%) = 1:5 and a freezing temperature of -195°C;

[0047] Fig.10 b is a particle size distribution diagram of HRP particles prepared under the conditions of horseradish peroxidase (HRP): polyethylene glycol (PEG) (w / w%) = 1:5 and a freezing temperature of -195°C;

[0048] Fig.11 a is the zeta potential result of lysozyme molecules in an aqueous solution of pH 5.3 and 10 mM NaCl;

[0049] Fig.11 b is a scanning electron micrograph of Lysozyme particles prepared at a lysozyme:polyethylene glycol (PEG) (w / w%) ratio of 1:5 and a freezing temperature of -20°C;

[0050] Fig.11 c is a particle size distribution diagram of Lysozyme particles prepared under the conditions of lysozyme (Lysozyme): polyethylene glycol (PEG) (w / w%) = 1:5 and a freezing temperature of -20°C;

[0051] Fig.12 a is a scanning electron micrograph of Lysozyme particles prepared at a lysozyme:polyethylene glycol (PEG) (w / w%) ratio of 1:5 and a freezing temperature of -70°C;

[0052] Fig.12b is a particle size distribution diagram of Lysozyme particles prepared under the conditions of lysozyme (Lysozyme): polyethylene glycol (PEG) (w / w%) = 1:5 and a freezing temperature of -70°C;

[0053] Fig.13 a is a scanning electron micrograph of Lysozyme particles prepared under the conditions of lysozyme (Lysozyme): polyethylene glycol (PEG) (w / w%) = 1:5 and a freezing temperature of -195°C;

[0054] Fig.13 b is the particle size distribution diagram of Lysozyme particles prepared under the conditions of lysozyme (Lysozyme): polyethylene glycol (PEG) (w / w%) = 1:5 and a freezing temperature of -195°C. DETAILED DESCRIPTION

[0055] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. The exemplary embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0056] Example 1

[0057] Bovine serum albumin (BSA) with a molecular weight of 66.5 kDa was dissolved in an aqueous solution with a pH of 5.3 and an ion concentration of 10 mM NaCl. The zeta potential of the BSA molecule under this condition was measured using a zeta potential meter to be -7.51 ± 0.55 mV, indicating that it is negatively charged. Figure 1 As shown in a.

[0058] 10 g of polyethylene glycol (PEG) with a number average molecular weight (Mn) of 8000 and 2 g of BSA with a molecular weight of 66.5 kDa were dissolved in 500 ml of distilled water to form a mixed solution of PEG-BSA. The mixed solution was then frozen at -20°C and freeze-dried at -40°C for 48 hours. 300 ml of dichloromethane was added to the lyophilized sample, and the PEG in the sample was washed away by centrifugation. After repeated washing three times, the obtained BSA sample was placed in a fume hood to evaporate the dichloromethane to obtain BSA particles.

[0059] The prepared BSA particles were loaded onto the conductive glue and sprayed with gold. The sample morphology was observed using a scanning electron microscope. The morphology of the micron-sized BSA particles is shown in Figure 1 As shown in b; the prepared BSA particles were dispersed in acetone, and the particle size and particle size distribution of the sample were detected using a laser light scattering instrument. The hydrodynamic diameter of the BSA particles was 2.15±0.62μm. Figure 1 As shown in c.

[0060] Example 2

[0061] 10 g of polyethylene glycol (PEG) with a number average molecular weight (Mn) of 8000 and 2 g of BSA with a molecular weight of 66.5 kDa were dissolved in 500 ml of distilled water to form a PEG-BSA mixed solution. The mixed solution was then frozen at -70°C and freeze-dried at -40°C for 48 hours. 300 ml of dichloromethane was added to the lyophilized sample, and the PEG in the sample was washed away by centrifugation. After repeated washing three times, the obtained BSA sample was placed in a fume hood to evaporate the dichloromethane to obtain BSA particles.

[0062] The prepared BSA particles were loaded onto the conductive glue and sprayed with gold. The sample morphology was observed using a scanning electron microscope. The morphology of the micron-sized BSA particles is shown in Figure 2 As shown in a; the prepared BSA particles were dispersed in acetone, and the particle size and particle size distribution of the sample were detected using a laser light scattering instrument. The hydrodynamic diameter of the BSA particles was 0.94±0.27μm. Figure 2 As shown in b.

[0063] Example 3

[0064] 10 g of polyethylene glycol (PEG) with a number average molecular weight (Mn) of 8000 and 2 g of BSA with a molecular weight of 66.5 kDa were dissolved in 500 ml of distilled water to form a mixed solution of PEG-BSA. The mixed solution was then frozen at -195°C and freeze-dried at -40°C for 48 hours. 300 ml of dichloromethane was added to the lyophilized sample, and the PEG in the sample was washed away by centrifugation. After repeated washing three times, the obtained BSA sample was placed in a fume hood to evaporate the dichloromethane to obtain BSA particles.

[0065] The prepared BSA particles were loaded onto the conductive glue and sprayed with gold. The sample morphology was observed using a scanning electron microscope. The morphology of the nanosized BSA particles is shown in Figure 3 As shown in a; the prepared BSA particles were dispersed in acetone, and the particle size and particle size distribution of the sample were detected using a laser light scattering instrument. The hydrodynamic diameter of the BSA particles was 0.28±0.08μm. Figure 3 As shown in b.

[0066] Example 4

[0067] 20 g of polyethylene glycol (PEG) with a number average molecular weight (Mn) of 8000 and 2 g of BSA with a molecular weight of 66.5 kDa were dissolved in 500 ml of distilled water to form a mixed solution of PEG-BSA. The mixed solution was then frozen at -20°C and freeze-dried at -40°C for 48 hours. 300 ml of dichloromethane was added to the lyophilized sample, and the PEG in the sample was washed away by centrifugation. After repeated washing three times, the obtained BSA sample was placed in a fume hood to evaporate the dichloromethane to obtain BSA particles.

[0068] The prepared BSA particles were loaded onto the conductive glue and sprayed with gold. The sample morphology was observed using a scanning electron microscope. The morphology of the micron-sized BSA particles is shown in Figure 4 As shown in a; the prepared BSA particles were dispersed in acetone, and the particle size and particle size distribution of the sample were detected using a laser light scattering instrument. The hydrodynamic diameter of the BSA particles was 2.17±0.56μm. Figure 4 As shown in b.

[0069] Example 5

[0070] 4 g of polyethylene glycol (PEG) with a number average molecular weight (Mn) of 8000 and 2 g of BSA with a molecular weight of 66.5 kDa were dissolved in 500 ml of distilled water to form a mixed solution of PEG-BSA. The mixed solution was then frozen at -20°C and freeze-dried at -40°C for 48 hours. 300 ml of dichloromethane was added to the lyophilized sample, and the PEG in the sample was washed away by centrifugation. After repeated washing three times, the obtained BSA sample was placed in a fume hood to evaporate the dichloromethane to obtain BSA particles.

[0071] The prepared BSA particles were loaded onto the conductive glue and sprayed with gold. The sample morphology was observed using a scanning electron microscope. The morphology of the micron-sized BSA particles is shown in Figure 5 As shown in a; the prepared BSA particles were dispersed in acetone, and the particle size and particle size distribution of the sample were detected using a laser light scattering instrument. The hydrodynamic diameter of the BSA particles was 2.93±0.56μm. Figure 5 As shown in b.

[0072] Example 6

[0073] 2 g of polyethylene glycol (PEG) with a number average molecular weight (Mn) of 8000 and 2 g of BSA with a molecular weight of 66.5 kDa were dissolved in 500 ml of distilled water to form a mixed solution of PEG-BSA. The mixed solution was then frozen at -20°C and freeze-dried at -40°C for 48 hours. 300 ml of dichloromethane was added to the lyophilized sample, and the PEG in the sample was washed away by centrifugation. After repeated washing three times, the obtained BSA sample was placed in a fume hood to evaporate the dichloromethane to obtain BSA particles.

[0074] The prepared BSA particles were loaded onto the conductive glue and sprayed with gold. The sample morphology was observed using a scanning electron microscope. The morphology of the micron-sized BSA particles is shown in Figure 6 As shown in a; the prepared BSA particles were dispersed in acetone, and the particle size and particle size distribution of the sample were detected using a laser light scattering instrument. The hydrodynamic diameter of the BSA particles was 4.19±0.86μm. Figure 6 As shown in b.

[0075] Example 7

[0076] The BSA raw material and the BSA particles prepared in Example 1 were dissolved in phosphate buffered saline (PBS) respectively, and the secondary structure of BSA was determined by circular dichroism. The results are as follows: Figure 7 As shown. The BSA raw material and the prepared BSA particles have the same absorption peaks at 209 and 222nm wavelengths, which are characteristic signals of the α-helical conformation of BSA. This result shows that the secondary structure of the sample protein is intact during the preparation of micro-nanoparticles using this technology.

[0077] Example 8

[0078] Horseradish peroxidase (HRP) with a molecular weight of 40 kDa was dissolved in an aqueous solution with a pH of 5.3 and an ion concentration of 10 mM NaCl. The zeta potential of the HRP molecule under this condition was measured using a zeta potential meter to be -4.24 ± 0.33 mV, which is negatively charged. Figure 8 As shown in a.

[0079] 10g of polyethylene glycol (PEG) with a number average molecular weight (Mn) of 8000 and 2g of HRP with a molecular weight of 40kDa were dissolved in 500ml of distilled water to form a mixed solution of PEG-HRP. The mixed solution was then frozen at -20°C and freeze-dried at -40°C for 48 hours. 300ml of dichloromethane was added to the lyophilized sample, and the PEG in the sample was washed away by centrifugation. After repeated washing three times, the obtained HRP sample was placed in a fume hood to evaporate the dichloromethane to obtain HRP particles.

[0080] The prepared HRP particles were loaded onto the conductive glue and sprayed with gold. The sample morphology was observed using a scanning electron microscope. The morphology of the micron-sized HRP particles is shown in Figure 8 b; the prepared HRP particles were dispersed in acetone, and the particle size and particle size distribution of the sample were detected using a laser light scattering instrument. The hydrodynamic diameter of the HRP particles was 2.67±0.68μm. Figure 8 As shown in c.

[0081] Example 9

[0082] 10g of polyethylene glycol (PEG) with a number average molecular weight (Mn) of 8000 and 2g of HRP with a molecular weight of 40kDa were dissolved in 500ml of distilled water to form a mixed solution of PEG-HRP. The mixed solution was then frozen at -70°C and freeze-dried at -40°C for 48 hours. 300ml of dichloromethane was added to the lyophilized sample, and the PEG in the sample was washed away by centrifugation. After repeated washing three times, the obtained HRP sample was placed in a fume hood to evaporate the dichloromethane to obtain HRP particles.

[0083] The prepared HRP particles were loaded onto the conductive glue and sprayed with gold. The sample morphology was observed using a scanning electron microscope. The morphology of the micron-sized HRP particles is shown in Fig. 9 As shown in a; the prepared HRP particles were dispersed in acetone, and the particle size and particle size distribution of the sample were detected using a laser light scattering instrument. The hydrodynamic diameter of the HRP particles was 1.69±0.90μm. Fig. 9 As shown in b.

[0084] Example 10

[0085] 10g of polyethylene glycol (PEG) with a number average molecular weight (Mn) of 8000 and 2g of HRP with a molecular weight of 40kDa were dissolved in 500ml of distilled water to form a mixed solution of PEG-HRP. The mixed solution was then frozen at -195°C and freeze-dried at -40°C for 48 hours. 300ml of dichloromethane was added to the lyophilized sample, and the PEG in the sample was washed away by centrifugation. After repeated washing three times, the obtained HRP sample was placed in a fume hood to evaporate the dichloromethane to obtain HRP particles.

[0086] The prepared HRP particles were loaded onto the conductive glue and sprayed with gold. The sample morphology was observed using a scanning electron microscope. The morphology of the nano-sized HRP particles is shown in Fig.10 As shown in a; the prepared HRP particles were dispersed in acetone, and the particle size and particle size distribution of the sample were detected using a laser light scattering instrument. The hydrodynamic diameter of the HRP particles was 0.76±0.41μm. Fig.10 As shown in b.

[0087] Embodiment 11

[0088] Lysozyme with a molecular weight of 14.3 kDa was dissolved in an aqueous solution with a pH of 5.3 and an ion concentration of 10 mM NaCl. The zeta potential of the Lysozyme molecule under this condition was measured using a zeta potential meter to be 1.39 ± 0.53 mV, indicating a positive charge. Fig.11 a.

[0089] 10 g of polyethylene glycol (PEG) with a number average molecular weight (Mn) of 8000 and 2 g of Lysozyme with a molecular weight of 14.3 kDa were dissolved in 500 ml of distilled water to form a PEG-Lysozyme mixed solution. The mixed solution was then frozen at -20°C and freeze-dried at -40°C for 48 hours. 300 ml of dichloromethane was added to the freeze-dried sample, and the PEG in the sample was washed away by centrifugation. After repeated washing three times, the obtained Lysozyme sample was placed in a fume hood to evaporate the dichloromethane to obtain Lysozyme particles.

[0090] The prepared Lysozyme particles were loaded onto the conductive glue and sprayed with gold. The sample morphology was observed using a scanning electron microscope. The morphology of the micrometer-sized Lysozyme particles is shown in Figure 2. Fig.11 As shown in b; the prepared Lysozyme particles were dispersed in ethanol, and the particle size and particle size distribution of the sample were detected using a laser light scattering instrument. The hydrodynamic diameter of the Lysozyme particles was 2.12±0.29μm. Fig.11 As shown in c.

[0091] Example 12

[0092] 10 g of polyethylene glycol (PEG) with a number average molecular weight (Mn) of 8000 and 2 g of Lysozyme with a molecular weight of 14.3 kDa were dissolved in 500 ml of distilled water to form a PEG-Lysozyme mixed solution. The mixed solution was then frozen at -70°C and freeze-dried at -40°C for 48 hours. 300 ml of dichloromethane was added to the freeze-dried sample, and the PEG in the sample was washed away by centrifugation. After repeated washing three times, the obtained Lysozyme sample was placed in a fume hood to evaporate the dichloromethane to obtain Lysozyme particles.

[0093] The prepared Lysozyme particles were loaded onto the conductive glue and sprayed with gold. The sample morphology was observed using a scanning electron microscope. The morphology of the micrometer-sized Lysozyme particles is shown in Figure 2. Fig.12As shown in a; the prepared Lysozyme particles were dispersed in ethanol, and the particle size and particle size distribution of the sample were detected using a laser light scattering instrument. The hydrodynamic diameter of the Lysozyme particles was 1.32±0.15μm. Fig.12 As shown in b.

[0094] Embodiment 13

[0095] 10 g of polyethylene glycol (PEG) with a number average molecular weight (Mn) of 8000 and 2 g of Lysozyme with a molecular weight of 14.3 kDa were dissolved in 500 ml of distilled water to form a PEG-Lysozyme mixed solution. The mixed solution was then frozen at -195°C and freeze-dried at -40°C for 48 hours. 300 ml of dichloromethane was added to the freeze-dried sample, and the PEG in the sample was washed away by centrifugation. After repeated washing three times, the obtained Lysozyme sample was placed in a fume hood to evaporate the dichloromethane to obtain Lysozyme particles.

[0096] The prepared Lysozyme particles were loaded onto conductive glue and sprayed with gold. The sample morphology was observed using a scanning electron microscope. The morphology of the nano-sized Lysozyme particles is shown in Figure 2. Fig.13 As shown in a; the prepared Lysozyme particles were dispersed in ethanol, and the particle size and particle size distribution of the sample were detected using a laser light scattering instrument. The hydrodynamic diameter of the Lysozyme particles was 0.37±0.10μm. Fig.13 As shown in b.

[0097] The technical solutions provided by the embodiments of the present invention are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present invention. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, according to the embodiments of the present invention, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A micro-nano macromolecular biopharmaceutical and a preparation method thereof, characterized in that: The process includes: (1) dissolving a macromolecular biodrug and a polymer that does not adsorb to the macromolecular biodrug in distilled water to prepare a macromolecular biodrug-non-adsorbable polymer aqueous solution; (2) completely freezing the aqueous solution of the macromolecular biopharmaceutical and non-adsorbed polymer; (3) freeze-drying the intermediate product in the previous step to obtain a freeze-dried product; (4) The free non-adsorbed polymer in the freeze-dried product is eluted with a volatile organic solvent, and micro-nano-sized macromolecular biopharmaceutical particles are obtained after the organic solvent is completely evaporated.

2. A micro-nano macromolecular biopharmaceutical and a preparation method thereof according to claim 1, characterized in that: In the step (1), the concentration of the macromolecular biopharmaceutical is 0.4 mg / ml to 500 mg / ml, the mass concentration ratio of the non-adsorbing polymer to the macromolecular biopharmaceutical is 0.5:1 to 100:1, the number average molecular weight (Mn) of the non-adsorbing polymer is 400-30000, and the molecular weight of the macromolecular biopharmaceutical is 500-900000 Da.

3. A micro-nano macromolecular biopharmaceutical and a preparation method thereof according to claim 2, characterized in that: The macromolecular biopharmaceutical is a protein molecule or a modified protein molecule; The non-adsorbing polymer is polysaccharide or polyethylene glycol, wherein the number average molecular weight (Mn) of the polyethylene glycol is 8,000.

4. A micro-nano macromolecular biopharmaceutical and a preparation method thereof according to claim 3, characterized in that: The macromolecular biological drug is selected from bovine serum albumin, horseradish peroxidase, lysozyme, genetically recombinant protein drugs, antibody drugs, antibody-coupled drugs, and CRISPR-Cas9 gene editing drugs.

5. A micro-nano macromolecular biopharmaceutical and a preparation method thereof according to claim 1 or 4, characterized in that: In the step (2), the aqueous solution of the macromolecular biopharmaceutical-non-adsorbing polymer is placed at -20 to -196°C and completely frozen.

6. The micro-nano macromolecular biopharmaceutical and the preparation method thereof according to claim 5, characterized in that: The freeze-drying temperature in step (3) is -20 to -80°C.

7. The micro-nano macromolecular biopharmaceutical and the preparation method thereof according to claim 6, characterized in that: The organic solvent in step (4) is selected from dichloromethane, chloroform, alcohol, ketone, glyceride, and aromatic hydrocarbon.

8. The micro-nano macromolecular biopharmaceutical and the preparation method thereof according to claim 7, characterized in that: In the step (4), the organic solvent is naturally evaporated or vacuum dried.

9. The micro-nano macromolecular biopharmaceutical and the preparation method thereof according to claim 7, characterized in that: The size range of the micro-nano macromolecular biopharmaceutical particles is 20 nm to 10 μm.

10. A micro-nanosized macromolecular biopharmaceutical obtained by the preparation method according to any one of claims 1 to 9.

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