Degradable and elastic embolism microsphere with uniform particle size as well as preparation method and application of embolism microsphere

Abstract

The invention discloses degradable and elastic embolism microspheres with uniform particle size as well as a preparation method and application of the embolism microspheres, and belongs to the technical field of biomedical polymer materials. Crosslinkable micromolecules with an acrylic acid structure are connected to the macromolecules in a covalent bond mode, and the functionalized macromolecules are formed. The embolism microspheres are prepared through microfluidic and free radical polymerization technologies, and the microspheres prepared by the method have good application prospects in the field of arterial embolism treatment. According to the embolism microsphere, the embolism microsphere with the uniform particle size is prepared through the microfluidic technology, the generation operation is further simplified, the production cost is reduced, and the production process is more economical and environmentally friendly. The embolism microsphere disclosed by the invention is high in safety, convenient to prepare, good in dispersity, relatively good in elasticity and good in embolism effect, has relatively large transformation potential in the aspect of transarterial embolism treatment, and is expected to assist the development of vascular embolism agents.

Description

TECHNICAL FIELD

[0001] The present application relates to a preparation method and application of embolism microspheres with degradability, elasticity and uniform particle size, in particular to a preparation method through microfluidic and free radical polymerization technology, and application of the embolism microspheres with degradability, elasticity and uniform particle size in the field of interventional embolism. BACKGROUND

[0002] Transarterial embolization therapy is a clinical interventional therapy that occludes target blood vessels by injecting embolic materials into the target blood-supplying arteries, and has been widely used in minimally invasive interventional therapy of solid tumors, gastrointestinal bleeding, uterine fibroids and arteriovenous malformations. The principle of this therapy is to use imaging equipment to guide the catheter to the lesion site in the human body under X-ray fluoroscopy, and then deliver embolic agents through the catheter to block the blood supply of the target tissue, thereby achieving the treatment purpose (Reig, M. J. Hepatol. 2022, 76, 681-693). It is worth noting that the key to embolization therapy is to select appropriate embolic agents to block the blood supply of tumors, and the physical properties of embolic agents are crucial to the treatment effect. Embolic agents usually include mechanical occlusion devices, microspheres and liquid / gel materials. Mechanical occlusion devices can accurately embolize target sites and achieve permanent embolization, but their disadvantages include incomplete embolization, displacement in blood vessels and unavoidable damage to blood vessel walls. Liquid embolic materials such as iodized oil can flow into finer blood vessels, thereby achieving more complete embolization, but these iodized oil systems have poor stability in blood and still face limitations such as recanalization, non-specific embolization and potential toxicity. A variety of embolic microparticles such as Embosphere, CalliSpheres, DCBead and HepaSphere have the advantages of easy operation, predictable shape and calibratable size, and have been approved for embolization therapy. However, most of the currently marketed and developed (CN119613620A, CN114106252B) microspheres have a wide particle size distribution, such as 40-120 μm, 100-300 μm and 300-500 μm, and if the product has uniform particle size and good elasticity, it can form better embolization therapy effect. In addition, microspheres often do not tightly pack and passively distribute in complex vascular systems, resulting in poor embolization therapy effect (Liu, M. H. Adv. Funct. Mater. 2023, 33, 2305153). The mechanical properties of embolic microspheres, especially their viscoelastic deformation ability and vascular adaptability to hepatic tumor arterial branches, are not only crucial to achieving efficient embolization, but also can reduce surgical complications (Tao, S. J. Nat. Commun. 2023, 14, 5575). It is worth noting that most of the current embolic microspheres are based on non-degradable materials such as polyvinyl alcohol or polymethacrylic acid (CN117323294B, CN101810587A), which can hinder re-embolization aimed at inhibiting tumor recurrence and often induce foreign body reactions. Therefore, there is an urgent need to develop a new type of degradable, elastic and uniform particle size embolic microsphere, which is of great significance to achieve effective arterial embolization. SUMMARY

[0003] The application aims to provide a preparation method of embolism microspheres with degradability, elasticity and uniform particle size, so as to solve the problems of uneven particle size, non-degradability, insufficient elasticity and poor embolism effect of the existing embolism microspheres. The embolism microspheres prepared by the method have degradability, uniform particle size and excellent elasticity, and have a wide application prospect in the field of vascular embolism.

[0004] To achieve the above-mentioned application purposes, the application adopts the following technical solutions. The application provides a preparation method of embolism microspheres with degradability, elasticity and uniform particle size, comprising the following steps: polymerizing macromolecules with cross-linking groups to obtain embolism microspheres with degradability, elasticity and uniform particle size.

[0005] In the application, the macromolecules are medical macromolecules, including one or more of hyaluronic acid, dextran, chitosan, heparin, alginic acid, collagen or polyethylene glycol-polyester.

[0006] In the application, the polymerization method includes free radical polymerization, such as microfluidic free radical polymerization.

[0007] In the application, the macromolecules are medical macromolecules, including one or more of hyaluronic acid, dextran, chitosan, heparin, alginic acid, collagen or polyethylene glycol-polyester.

[0008] In the application, the macromolecules are medical macromolecules, including one or more of hyaluronic acid, dextran, chitosan, heparin, alginic acid, collagen or polyethylene glycol-polyester.

[0009] In the application, the macromolecules are medical macromolecules, including one or more of hyaluronic acid, dextran, chitosan, heparin, alginic acid, collagen or polyethylene glycol-polyester. Figure 4 In the application, the macromolecules are medical macromolecules, including one or more of hyaluronic acid, dextran, chitosan, heparin, alginic acid, collagen or polyethylene glycol-polyester.

[0010] Further, the present application dissolves hyaluronic acid, dextran, chitosan, heparin, alginic acid, collagen or polyethylene glycol-polyester in water, then adds acrylate, acid anhydride or amide compound, and stirs conventionally until completely dissolved; then, adds triazine, carbodiimide or phosphonium salt coupling agent, stirs at 30-50 ℃ for 12-24 hours to obtain functional macromolecule solution, and freeze-dries to obtain functional macromolecule; dissolves the functional macromolecule in water or buffer, and in a microchannel pipeline, the water-phase material forms water-in-oil droplets under the shearing force of oil-phase material, and is crosslinked and solidified by free radical polymerization to form microspheres; after solidification, the microspheres are washed with organic solvent and water in turn, and finally obtain degradable, elastic and uniform-particle-size embolization microspheres.

[0011] In the present application, the water-phase material includes macromolecule with crosslinking group, buffer and initiator; the oil-phase material includes surfactant and oil solvent; the microchannel pipeline includes cross type, Y type, T type or flow focusing type microchannel pipeline, preferably, the microchannel depth is 50-1000 μm, such as 100-500 μm.

[0012] In the present application, the crosslinking and solidification includes photocuring, thermal curing, wherein the photocuring is preferably ultraviolet curing, and when the ultraviolet curing, the ultraviolet wavelength is 365 nm, the crosslinking and solidification time is 10-30 min, and the ultraviolet surface light source intensity is 20-50 mW / cm 2 .

[0013] In the present application, the buffer includes phosphate buffer, tris buffer solution, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid half sodium salt buffer solution or 2-morpholinoethanesulfonic acid buffer solution; the initiator is 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, 2,2'-azo(2-methyl-N-(2-hydroxyethyl)propionamide) or phenyl(2,4,6-trimethylbenzoyl) lithium phosphate salt, etc.; the organic solvent includes small molecule alcohol, such as isopropyl alcohol, n-hexane and ethanol.

[0014] Preferably, the mass ratio of macromolecule with crosslinking group (functional macromolecule), buffer and initiator is 100:2000-4000:5-15.

[0015] In the present application, the surfactant includes Span surfactant, and the oil solvent includes one or more of mineral oil, paraffin, corn oil or soybean oil.

[0016] Preferably, the mass ratio of surfactant and oil solvent is 2-20:80-98.

[0017] In the present application, the photocuring includes ultraviolet photocuring.

[0018] In the present application, the flow rate of the oil phase material is 5-100 μL / min, and the flow rate of the water phase material is 1-20 μL / min.

[0019] In the present application, the macromolecule is reacted with an acrylate, an acrylic acid anhydride or an acrylic acid amide in a solution to obtain a macromolecule with crosslinking groups. Preferably, the macromolecule is reacted with the acrylate, the acid anhydride or the amide in a solution in the presence of a catalyst to obtain the macromolecule with crosslinking groups. Preferably, the mass ratio of the macromolecule to the acrylate, the acid anhydride or the amide is 100:30-60.

[0020] The present application discloses a kind of medicine, including the above-mentioned degradable, elastic and uniform particle size embolism microsphere.

[0021] The present application discloses the application of the above-mentioned degradable, elastic and uniform particle size embolism microsphere in the preparation of medicine, preferably the application of the above-mentioned degradable, elastic and uniform particle size embolism microsphere in the preparation of vascular embolism agent;Vascular embolism agent includes renal artery embolism and liver cancer embolism etc.

[0022] The present application focuses on realizing the degradability, elasticity and uniform particle size of microspheres. The uniform particle size microspheres generally have a relatively narrow particle size distribution range, wherein the particle size of most microspheres is concentrated in a relatively small interval, for example, within an average particle size ±10% range;Degradable microspheres refer to microspheres that can gradually degrade into small molecular substances in the body environment through hydrolysis or enzymatic degradation, etc., and are ultimately absorbed or excreted by the human body;The elasticity of embolism microspheres refers to the fact that the microspheres can be compressed according to their elastic properties, so as to better adapt to the shape of blood vessels and achieve a more compact embolism effect.

[0023] In the present application, water-in-oil droplets are formed by microfluidic technology, and then free radical polymerization technology is used to obtain microspheres with smooth surfaces, regular shapes, uniform sizes, degradability and elasticity.

[0024] In the present application, the degradable, elastic and uniform particle size embolism microsphere obtained by microfluidic technology not only simplifies the production operation and reduces the production cost, but also makes the production process more economical and environmentally friendly.

[0025] In the present application, the degradable, elastic and uniform particle size embolism microsphere can achieve effective embolism effect in vivo. Under simulated experiment, the microsphere embolism agent with excellent elasticity of the present application can achieve a tight capillary embolism in vitro, and the microspheres can exhibit a shape that is adapted to the shape of blood vessels in the capillary. In particular, the embolism microsphere has a broad application prospect in the field of tumor treatment, such as liver cancer embolism treatment.

[0026] Compared with the prior art, the application has the following advantages: 1. The degradable, elastic and uniform particle size embolism microspheres are prepared by microfluidic and free radical polymerization technology, and the preparation method has the characteristics of simplicity and high efficiency, does not need complex process conditions and procedures, and has high economic benefits.

[0027] 2. The embolism microspheres disclosed by the application have degradability, excellent elasticity and uniform size, and can realize strong embolism of the kidney.

[0028] 3. The degradable, elastic and uniform particle size embolism microspheres prepared by the application have the ability of embolizing tumor blood supply arteries; in a rabbit orthotopic liver cancer model, the embolism microspheres with a size of 90 μm effectively inhibit the growth of tumors and prolong the survival time of tumor-bearing rabbits.

[0029] 4. The precursor material of the degradable, elastic and uniform particle size embolism microspheres disclosed by the application has good biocompatibility and degradability, low cost and wide source; the microsphere preparation process is controllable, has good repeatability, and is suitable for and beneficial to industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 It is a process flow chart of the degradable, elastic and uniform particle size embolism microspheres prepared by the application.

[0031] Figure 2 It is a nuclear magnetic resonance hydrogen spectrum diagram of hyaluronic acid methacrylate derivative (HA-AMA) in Example 1.

[0032] Figure 3 It is a nuclear magnetic resonance hydrogen spectrum diagram of chitosan methacrylate derivative (CS-MA) in Example 2.

[0033] Figure 4 It is a structural schematic diagram of a microfluidic chip in Example 3.

[0034] Figure 5 It is a physicochemical performance characterization diagram of hyaluronic acid embolism microspheres (3Asphere) in Example 3, wherein a is the influence of polymer concentration and chip depth on the size of the microspheres, b is the influence of the water-oil phase flow rate ratio on the size of the hyaluronic acid embolism microspheres, c (60 μm), d (90 μm) and e (150 μm) are the morphology and appearance characterization of three sizes of hyaluronic acid embolism microspheres, and the scale is 100 μm.

[0035] Figure 6 It is the high temperature and high pressure sterilization stability of 3Asphere in Example 3, and the scale is 100 μm.

[0036] Figure 7is the long-term storage stability of 3Asphere in Example Three, scale: 100 μm.

[0037] Figure 8 is the physicochemical property characterization of chitosan embolization microspheres in Example Four.

[0038] Figure 9 is the cell compatibility characterization of 3Asphere in Example Five, where a is L929 cells, b is three hepatoma cells (Hepa1-6, SMMC-7721, HepG2), and c is VX2 cells.

[0039] Figure 10 is the blood compatibility characterization of 3Asphere in Example Five, where a is hemolysis rate, and b is a representative picture.

[0040] Figure 11 is the characterization of 3Asphere binding to CD44 in Example Six, where a is the binding image of microspheres with HUVEC cells, and b is the number of microspheres bound to different cells, scale: 100 μm.

[0041] Figure 12 is the mechanical property characterization of microspheres in Example Seven, where a is the viscosity of microspheres, b is the storage modulus and loss model of microspheres, and c is the compression deformation curve of microspheres.

[0042] Figure 13 is the stability of microspheres before and after delivery through a microcatheter in Example Seven, scale: 100 μm.

[0043] Figure 14 is the capillary embolization characterization of microspheres in Example Eight, where a is the confocal image of microspheres in a capillary, b is an enlarged view of the confocal image of 3Asphere, c is the ratio of the embolization volume (V em ) in the capillary glass tube to the total theoretical volume (V th ) of 3Asphere or Embosphere, d is the porosity of the capillary after embolization, e is the ratio of the long diameter to the short diameter of microspheres in the capillary, scale: 100 μm.

[0044] Figure 15 is the renal artery embolization characterization of 3Asphere in Example Nine, where a is the DSA image of the rabbit kidney, b is the enhanced CT image of the rabbit kidney after embolization, and c is the histological section image of the rabbit renal artery after embolization.

[0045] Figure 16 is the H&E staining image of the kidney embolized by 3Asphere in Example Nine.

[0046] Figure 17Figure 1 is the characterization of 3Asphere in different sizes for transarterial embolization of VX2 tumor-bearing rabbits in Example 10, where a is the DSA images of rabbits before and after embolization, b is the representative enhanced CT images, scale: 1 cm, c is the relative tumor growth volume, d is the MRI images, scale: 1 cm, e is the apparent diffusion coefficient (ADC) condition, f is the survival of tumor-bearing rabbits, g is the weight change of rabbits.

[0047] Figure 18 Figure 2 is the H&E staining images of heart, liver, spleen, lung and kidney of each treatment group in Example 10.

[0048] Figure 19 Figure 3 is the effect of each treatment group on liver and kidney function of rabbits before and after embolization in Example 10.

[0049] Figure 20 Figure 4 is the distribution characterization of 3Asphere in rabbit liver, tumor and blood vessels in Example 11, where a is the IVIS image of rabbit liver in vitro, b is the accumulation of Cy5-3Asphere in the tumor site.

[0050] Figure 21 Figure 5 is the histological characterization of the distribution of different microspheres in tumor blood vessels of different sizes in Example 11, scale: 100 μm.

[0051] Figure 22 Figure 6 is the distribution curve of different microspheres in tumor blood vessels of different sizes in Example 11. DETAILED DESCRIPTION

[0052] In the prior art, commercially available Embosphere and CalliSpheres microspheres are currently considered to be the two products with good performance in clinical application, but the elasticity and particle size uniformity of both still need to be improved. The present application discloses a preparation method of degradable, elastic and particle size uniform embolization microspheres, which comprises the following steps: covalently connecting a cross-linkable small molecule with an acrylic structure to hyaluronic acid, dextran, chitosan, heparin, alginic acid, collagen or polyethylene glycol-polyester macromolecule to form a functional macromolecule; and obtaining the cross-linked polymeric degradable, elastic and particle size uniform embolization microspheres through microfluidic and free radical polymerization technology, see Figure 1 .

[0053] Further, first, hyaluronic acid, dextran, chitosan, heparin, alginic acid, collagen or polyethylene glycol-polyester is dissolved in water, and then an acrylate, anhydride or amide compound is mixed conventionally; then a triazine, carbodiimide or phosphonium salt coupling agent is added as a catalyst, and the reaction is carried out under stirring at 30 ~ 50 ℃ for 12 ~ 24 hours to obtain a functional macromolecule solution, which is freeze-dried to obtain the functional macromolecule.

[0054] Functionalized macromolecules are dissolved in water or buffer solution. Within a microchannel, the aqueous phase is sheared into water-in-oil droplets by the shear force of the oil phase, and then cross-linked and solidified through free radical polymerization to form microspheres. After solidification, the microspheres are washed sequentially with an organic solvent and water, ultimately yielding biodegradable, elastic, and uniformly sized embolic microspheres.

[0055] Furthermore, the buffer solution includes phosphate buffer, tris(hydroxymethyl)aminomethane buffer, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonate hemisodium salt buffer, or 2-morpholine ethanesulfonate buffer; the organic solvent includes isopropanol, n-hexane, and ethanol.

[0056] Furthermore, the aqueous phase material is composed of functionalized macromolecules, buffer solution and initiator; preferably, the mass ratio of the functionalized macromolecules, buffer solution and initiator is 100:2000-2500:4-6.

[0057] Furthermore, the oil phase material is composed of Span surfactants and oils (including mineral oil, paraffin wax, corn oil, soybean oil, etc.); the mass ratio of the Span surfactant to the oil is 2-10:90-98, preferably, the mass ratio of the Span surfactant to the oil is 3-8:97-92.

[0058] Furthermore, the microchannel tubing is a cross-shaped, Y-shaped, T-shaped, or flow-focusing type microchannel tubing, with a microchannel depth of 50-500 μm.

[0059] Furthermore, the wavelength of the ultraviolet light is 365 nm, the cross-linking curing time is 10-30 min, and the intensity of the ultraviolet light source is 20-50 mW / cm². 2 .

[0060] Furthermore, the oil phase flow rate is 5-100 μL / min, and the water phase flow rate is 1-20 μL / min.

[0061] Furthermore, the initiator includes 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone; 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylpropanone; 2,2'-azo (2-methyl-N-(2-hydroxyethyl)propionamide); and lithium phenyl (2,4,6-trimethylbenzoyl)phosphate, etc.

[0062] This invention discloses a method for preparing biodegradable, elastic, and uniformly sized embolic microspheres. The invention will be further described below with reference to the accompanying drawings and embodiments. The raw materials used are all existing products, and the specific preparation operations and performance tests are all conventional techniques.

[0063] Example 1 Synthesis of hyaluronic acid methacrylic acid derivative (HA-AMA) Under the protection of nitrogen, 100 mL two-necked flask was added hyaluronic acid (1.0 g), deionized water 40 mL, dimethyl sulfoxide 10 mL, and stirred until dissolved, then 2-aminoethyl methacrylate (AMA, 65 mg, 130 mg, 220 mg, 400 mg) was added to the solution, stirred for 10 minutes, then 4-(4, 6-dimethoxytriazin-2-yl)-4-methyl morpholine hydrochloride (DMTMM, 160 mg, 320 mg, 550 mg, 650 mg) was added, and reacted at 35 ℃ oil bath for 12 hours. After the reaction was completed, dialysis was performed with deionized water, and finally freeze-drying was performed to obtain the hyaluronic acid derivative, with a yield of 85.7%; the nuclear magnetic resonance characterization of HA-AMA (with a degree of substitution of 22.5) is shown in the appendix Figure 2 , 1 H NMR (D2O / DMSO-d6): HA: δ 1.98, 3.15-4.05, 4.38-4.65; AMA: δ 1.90, 5.72, 6.12. The degree of substitution of the methacrylate group (the number of methacrylate groups in 100 sugar units) was calculated by the peak area of the signal peak, which was 10, 16.5, 22.5, and 25, respectively.

[0064] Example 2 Synthesis of chitosan methacrylic acid derivative (CS-MA) Under the protection of nitrogen, 100 mL round-bottom flask was added 300 mg chitosan (CS), 30 mL deionized water, and stirred at 1000 rpm until the chitosan was dissolved to obtain a 10 mg / mL CS solution at 25 ℃. Subsequently, at 60 ℃, methacrylic anhydride (MA, 200, 400, 600 mg) was added dropwise into the CS solution, and stirred at 1000 rpm for 12 hours. After the reaction was completed, NaHCO3 was added to the flask to adjust the pH of the solution to neutral, and then dialyzed in deionized water for 72 hours. Finally, freeze-drying was performed to obtain the chitosan methacrylic acid derivative, with a yield of 85.3%; (with a degree of substitution of 15.0) the nuclear magnetic resonance characterization of CS-MA is shown in the appendix Figure 3 , 1 HNMR (D2O): CS: δ 3.44-4.00; MA: δ 1.95, 5.53, 5.76. The degree of substitution of the methacrylic acid group was calculated by the peak area of the signal peak, which was 10.3, 15.0, and 18.9, respectively.

[0065] Example 3 Preparation and characterization of degradable, elastic and uniform particle size hyaluronic acid embolism microspheres (3Asphere) Hyaluronic acid microspheres were prepared by microfluidic and free radical polymerization techniques as follows: 30 mg, 40 mg or 45 mg of hyaluronic acid derivative (HA-AMA, degree of substitution 22.5 in Example 1) and 6 mg of photoinitiator I2959 were dissolved in 1 mL of phosphate buffer (PB, pH 7.4, 10 mM) as the aqueous phase material in the aqueous phase flask. At the same time, mineral oil containing 10 wt.% Span 80 was placed in the oil phase flask as the oil phase material. Subsequently, the materials in the aqueous and oil phase flasks were respectively flowed into the microfluidic chip (chip structure see Figure 4 , flow focusing microchannel pipeline, the microchannel pipeline is a rectangular structure, the depth is 70 μm, 100 μm, 150 μm, and the corresponding width is 150 μm, 210 μm, 310 μm); after the two-phase liquid enters the chip, the flow rate ratio of the aqueous phase and the oil phase is adjusted to 1:5, the aqueous phase flow rate is 1 μL / min, and the aqueous phase forms uniform droplets under the continuous laminar shear action of the oil phase. Under the irradiation of a UV lamp (365 nm, 30 mM / cm 2 ), the cured microspheres 3Asphere were formed by free radical polymerization reaction; the cured microspheres were washed with isopropanol, n-hexane and water in turn to obtain the purified 3Asphere solution. Inverted fluorescence microscope and scanning electron microscope were used to measure the size and surface morphology of 3Asphere, and the particle size is shown in Figure 5 a.

[0066] Referring to the above method, 40 mg of hyaluronic acid derivative (HA-AMA) was used, the flow rate ratio of the aqueous phase and the oil phase was adjusted, the aqueous phase flow rate was 1 μL / min, and the particle size of 3Asphere prepared at different microchannel pipeline depths is shown in Figure 5 b.

[0067] By adjusting the polymer concentration, the depth of the microfluidic chip and the flow rate of the aqueous and oil phases, the size of 3Asphere can be accurately controlled in the range of 40-180 μm Figure 5 a, b). The microspheres with diameters of 60, 90 and 150 μm were named 3Asphere-60, 3Asphere-90 and 3Asphere-150 respectively to study the related properties. The three kinds of 3Asphere microspheres have spherical shape and the coefficient of variation (CV) is less than 2.0 %, see Figure 5 c-e, wherein Figure 5 c condition: using a chip with a depth of 70 microns, the concentration of HA-AMA is 40 mg / mL, and the flow rate ratio of the aqueous and oil phases is 1:5; Figure 5 d condition: using a chip with a depth of 100 microns, the concentration of HA-AMA is 40 mg / mL, and the flow rate ratio of the aqueous and oil phases is 1:4;Figure 5 e provided that the chip depth is 150 microns, the HA-AMA concentration is 40 mg / mL, and the water to oil flow rate ratio is 1 :4. In addition, 3Asphere exhibits significant stability under sterilization at 120 °C, 0.1 MPa and long-term (>12 months) storage with little change in size Figure 6 , 7). As common knowledge, according to the technical solution disclosed in the present application, the microdroplet size can be conveniently controlled by adjusting the chip depth, macromolecule concentration and flow rate ratio, thus 3Asphere with a size of 20-1100 μm can be conveniently prepared.

[0068] Cy3-3Asphere was prepared by amidation reaction of carboxyl (-COOH) on 3Asphere with Sulfo-Cy5-NH2 under the action of coupling agent DMTMM. The specific steps are as follows: first, 3Asphere (10 mg, 25 μmol -COOH) was dissolved in 0.75 mL PB. Then, DMTMM (0.61 mg, 3.75 μmol) was added and shaken on a 900 rpm shaker for 30 minutes. Then, Sulfo-Cy3-NH2 (0.54 mg, 0.75 μmol) was added and the reaction was continued to shake at 50 °C for 24 hours. After the reaction was completed, the microspheres were washed with PBS to obtain the purified Cy3-3Asphere solution.

[0069] Preparation and characterization of degradable, elastic and uniform size chitosan embolization microspheres Chitosan microspheres were prepared by microfluidic technology and free radical polymerization technology. The specific steps are as follows: 50 mg of chitosan derivative (CS-MA, degree of substitution 15.0 in Example 2) and 6 mg of photoinitiator I2959 were dissolved in 1 mL of PB (pH 6.5, 10 mM) solution as water phase material in the water phase flask. At the same time, mineral oil containing 10 wt.% Span 80 was used as oil phase material in the oil phase flask. Then, the materials in the water phase and oil phase flasks were made to flow into the microfluidic chip through the conduit by constant pressure pump. After the two-phase liquid entered the chip, the flow rates of the water phase and the oil phase were adjusted, and uniform droplets were formed under the continuous laminar shear of the oil phase. These droplets were irradiated under a UV lamp (365 nm, 30 mM / cm 2 ) to form chitosan microspheres through free radical polymerization. Finally, the solidified microspheres were washed with isopropanol, n-hexane and water in turn to obtain the purified chitosan microsphere solution. The size and surface morphology of the chitosan microspheres were measured by inverted fluorescence microscope. It can be observed that the size of the microspheres is about 90 μm, which has a spherical shape and the coefficient of variation can be controlled within 5% ( Figure 8 ).Figure 8 The preparation conditions of the microspheres are as follows: using a chip with a depth of 100 microns, the concentration of CS-MA is 50 mg / mL, the flow rate ratio of water phase to oil phase is 1:4, and the flow rate of water phase is 1 μL / min.

[0070] Example Five Biocompatibility of 3Asphere The hyaluronic acid microspheres prepared in Example Three are taken as an example to test the cell compatibility of the microspheres. Fibroblasts (L929), hepatoma cells (SMMC-7721, HepG2 and Hepa1-6) and rabbit anaplastic epidermoid squamous carcinoma cells (VX2) are respectively spread on a 96-well cell culture plate, about 3000 cells per well, and cultured at 37 ℃, 5% carbon dioxide for 12 hours. Then, 10 μL of different concentrations of 3Asphere is added to each well. For L929 cells, the cell compatibility of 3Asphere-60, 3Asphere-90 and 3Asphere-150 is detected; for SMMC-7721, HepG2, Hepa1-6 and VX2 cells, 3Asphere-90 is detected. After incubation at 37 ℃ for 48 hours, 10 μL of CCK8 solution is added to each well and incubated for another 2 hours. Finally, the ultraviolet absorption of each well at 450 nm is determined by using an enzyme marker. The relative survival rate of cells is obtained by comparing the absorption at 450 nm with the control well with only blank cells, and each group of experiments is repeated for 5 times on average. It can be seen from the experimental results that the survival rate of cells after co-incubation with microspheres for 48 hours reaches more than 95%, indicating that the microspheres have good biocompatibility. Figure 9 ).

[0071] The blood of healthy rabbits is collected into an anticoagulant blood collection tube, and then centrifuged at 4 ℃, 1500 rpm for 10 minutes to remove the supernatant. Then the bottom cells are washed with physiological saline until the supernatant is colorless. Then the red blood cells are diluted with physiological saline to 5% (v / v). Then, 3Asphere-90 is incubated with an equal volume of 5% red blood cells at a concentration of 200 μg / mL and 1000 μg / mL at 37 ℃ for 20 minutes. After incubation, the sample is centrifuged, and the supernatant is used to determine the absorbance at 545 nm. Among them, physiological saline is the negative control group and deionized water is the positive control group. The hemolysis rate is calculated by the following formula: HR (%) = (OD s −OD n ) / (OD p – OD n ) × 100. OD s , OD n and OD pUV absorbance of samples, negative control and positive control. It was found that the hemolysis rate was less than 1% for both 200 μg / mL and 1000 μg / mL of microspheres. Figure 10 These results show that the microspheres have good biocompatibility.

[0072] Example Six Binding of 3Asphere to CD44 5 x 10 4 Hepal-6, human umbilical vein endothelial cells (HUVEC) and L929 cells were cultured in 48-well plates. After 12 hours, 100 Cy3-labeled microspheres (3Asphere-90 and Embosphere) were added to each well. After incubation at 37 °C for 4 hours, each well was washed twice with PBS, and the cells were fixed with 4% paraformaldehyde for 10 minutes and then stained with DAPI. Finally, the microspheres and cells in each well were photographed using an inverted fluorescence microscope, and the number of microspheres in each well was counted. As can be seen from the fluorescence micrographs, 3Asphere can actively bind to HUVEC endothelial cells and Hepal-6 liver cancer cells, while the interaction of the three types of cells with commercial microspheres Embosphere (a well-performing product on the market) is weak. Figure 11

[0073] Example Seven Elasticity Characterization of 3Asphere All rheological tests were performed using an RS6000 rheometer. For viscosity tests, different sizes of 3Asphere (60, 90 and 150 μm) and Embosphere (500-700 μm) were added to the parallel plates of the rheometer, and the change in microsphere viscosity was tested at 37 °C, 0.1-100 s -1 of shear rate. In addition, the storage modulus (G') and loss modulus (G'') of different microspheres were tested at 37 °C, 0.001-100 % of shear strain. It was found that 3Asphere has a lower viscosity (20-1400 mPa∙s) than Embosphere, and both microspheres have shear thinning behavior Figure 12 a). In addition, the storage modulus of 3Asphere is only 90-100 Pa, while the storage modulus of Embosphere is 1200 Pa Figure 12 b).

[0074] ​The elasticity of 3Asphere was evaluated using a texture analyzer with a 6.0 mm cylindrical probe and a 5.0 g sensor. Here, a 500 μm deep chip was used, with an HA-AMA concentration of 40 mg / mL, a water-to-oil flow rate ratio of 1:1.5, and an aqueous phase velocity of 10 μL / min to prepare 3Aspheres with a size of 580 μm. For compression testing, monolayers of 3Asphere (580 μm) and Embosphere (500–700 μm) were placed in a 35 mm diameter petri dish. The initial contact force was set to 0.01 N, and the probe was lowered at a speed of 0.01 mm / s until the microspheres achieved 50% deformation. The results showed that both 3Asphere and Embosphere exhibited quasi-elastic deformation behavior, achieving 50% compressive deformation under compressive forces of approximately 0.8 N and 1.8 N, respectively, indicating that 3Asphere has better elasticity. Figure 12 c). Furthermore, after injection via microcatheter, the 3Asphere maintained its intact structure and morphology, without any fragmentation. Figure 13 This indicates the feasibility of delivery via catheter.

[0075] Example 8: Characterization of the extracorporeal embolization performance of 3Asphere A semi-enclosed glass capillary with an inner diameter of 0.7 mm was used to simulate in vivo embolism. Simply put, 25 μL of 3Asphere-90 and Embosphere were injected into the capillary at a rate of 0.2 mL / min using a syringe pump. Finally, the morphology of 3Asphere-90 and Embosphere within the capillary was observed using laser confocal microscopy (CLSM) to evaluate the embolic effect and deformability of 3Asphere in the capillary. V em V is the volume of the plug inside the capillary glass tube. th This represents the total 3Asphere-90 or Embosphere theoretical volume. V em V is the cross-sectional area of ​​the capillary multiplied by the plug height. th It is the number of microspheres multiplied by the volume of one microsphere.

[0076] CLSM images show that 3Aspheres exhibit tight aggregation with shapes adapted to blood vessels, such as spherical, ellipsoidal, dumbbell-shaped, string-like, and bifurcated, while Embospheres show relatively loose filling, predominantly spherical. Figure 14 a, b). After injecting spheres of the same theoretical volume into the capillary, the embolization volume (V) of the 3Asphere... em ) and theoretical volume (V th The ratio of () to Embosphere is approximately 1.3, significantly smaller than that of Embosphere (~1.6).Figure 14 c), indicating that 3Aspheres packed more tightly. According to the CLSM images, the void fractions of Embospheres and 3Aspheres after embolization were about 65% and 28%, respectively Figure 14 d). In addition, the aspect ratios of Embospheres and 3Aspheres were 1.09 and 1.17, respectively Figure 14 e). These results indicated that 3Aspheres had better viscoelasticity and stackability compared to the microspheres used in clinic, which contributed to effective vascular embolization.

[0077] Example Nine In vivo renal artery embolization study of 3Aspheres New Zealand white rabbits were anesthetized by intramuscular injection of tiletamine (0.2 mL / kg) after fasting for 12 hours, and the rabbits were fixed on the animal operating table after anesthesia. Then the femoral artery was dissected in the groin, and the micro guide wire and catheter sheath were introduced into the femoral artery, and then the micro guide wire and micro catheter were introduced into the catheter sheath. Under the guidance of digital subtraction angiography (DSA), 3 mL of iodixanol solution was injected to observe the abdominal vascular distribution. Then under the guidance of DSA, the catheter was slowly introduced into the right renal artery, and angiography was performed again to determine the distribution of the renal artery. After the catheter reached the designated artery, 1.0 mL (about 3 x 10 5 microspheres) of 3Asphere-90 mixed with iodixanol (50%, v / v) was injected into the renal artery at a speed of 1.0 mL / min. After the microsphere injection was completed, angiography was performed again to confirm the effect of renal artery embolization. Then the microcatheter was slowly taken out, the wound was sutured and sterilized, and antibiotics were injected intramuscularly for 3 days after the operation to prevent postoperative infection of the rabbits. After renal artery embolization, the rabbits were injected with contrast agent for contrast-enhanced computed tomography (CTA) at 3, 7, 14, and 28 days to check the embolization effect. At the same time, one rabbit was euthanized, and all the kidneys were fixed in a 4.0% paraformaldehyde solution, then sectioned and analyzed by H&E staining.

[0078] The DSA image results clearly showed that the right renal artery was completely occluded Figure 15 a). Then the CTA results at 3, 7, 14, and 28 days showed that no obvious angiographic signals were observed in the right kidney within 28 days after embolization, indicating that 3Aspheres achieved long-term and robust occlusion of the kidney Figure 15 b). H&E staining further confirmed that 3Aspheres effectively filled the renal vessels Figure 15 c), and caused obvious damage to the glomerular and tubular structures and tissue necrosis in the embolized right kidney Figure 16 .

[0079] Example Ten In vivo hepatic artery embolization study of 3Aspheres Under CT imaging, VX2 (rabbit anaplastic epidermoid squamous carcinoma strain) tumor tissue was transplanted into rabbit liver lobes to establish a rabbit VX2 orthotopic liver cancer model. After 10 days, CT imaging was performed to determine whether the tumor was formed and the size of the volume. Then the VX2 tumor-bearing rabbits with a tumor maximum diameter of 0.8-1.3 cm were randomly divided into 5 groups (n = 3), and the following treatments were performed respectively: (1) Control; (2) 3Asphere-60; (3) 3Asphere-90; (4) 3Asphere-150; (5) Embosphere: 40-120 μm. Under the guidance of DSA, one microcatheter with a diameter of 2.4-F (outer diameter: 0.8 mm) was placed into the rabbit hepatic proper artery, and then angiography was performed to determine the distribution of the hepatic artery and the location of the liver tumor. Subsequently, a mixture of 0.2 mL of 3Asphere and iodixanol (70%, v / v) was slowly injected into the hepatic artery through the microcatheter. After injection, DSA imaging was performed to confirm the tumor vessel occlusion. On the 7th and 14th day after surgery, CT scanning was performed by intravenous injection of iodixanol contrast agent to evaluate the tumor treatment effect. At the same time, 3.0 T magnetic resonance imaging (MRI) was performed before embolization treatment and 14 days after treatment to perform T1 weighted imaging (T1), T2 weighted imaging (T2), diffusion weighted imaging (DWI) and Gd-DTPA enhanced T1 weighted imaging (T1+) sequence scanning. The tumor volume was calculated according to the maximum (a) and minimum (b) diameters of the tumor, and the calculation formula was as follows: tumor volume (mm 3 ) = (a x b 2 ) / 2. In addition, rabbit blood (about 1.0 mL per rabbit) was taken at time points (before TAE and 1, 3 and 7 days after TAE) into a coagulation tube and centrifuged to obtain serum, and then a biochemical analyzer was used to determine liver and kidney function indicators such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (ALB), total bilirubin (TBIL), urea (UREA) and creatinine (CREA).

[0080] The DSA image showed that after injecting 3Asphere into the hepatic proper artery through the microcatheter, there was almost no angiographic signal at the tumor site, indicating successful occlusion of the tumor vessels by the microspheres ( Figure 17 a). Notably, 3Asphere showed a size-dependent inhibition of VX2 tumor growth, with smaller sizes of 3Asphere generally having better tumor inhibition effect ( Figure 17 b, c). After embolization for 14 days, smaller 3Asphere (60 and 90 μm) showed better tumor inhibition effect than the commercial Embosphere group. At the same time, the treatment effect was further evaluated by MRI on day 0 and day 14 ( Figure 17d). T1, T2, and T1+ showed that 3Asphere had a similar size-dependent inhibitory effect on tumor growth. Furthermore, DWI, which assesses tumor cell necrosis by measuring the microscopic mobility of free water molecules, could be quantified by the apparent diffusion coefficient (ADC). After 14 days of TAE treatment, the ADC values ​​of VX2 tumors in each group were as follows: 3Asphere-60 > 3Asphere-90 > 3Asphere-150 ≈ Embosphere ( Figure 17 e). Although 3Asphere-60 showed the best tumor suppression effect, its survival time was slightly increased, with a median survival time (MST) of 30 days. Figure 17 f). Conversely, the MST in rabbits treated with 3Asphere-90 was significantly prolonged by 47 days, demonstrating the importance of precise control of physicochemical properties for microparticle embolization. Importantly, there was little change in body weight among the groups after embolization (f). Figure 17 g), H&E staining analysis of major organs (heart, liver, spleen, lungs and kidneys) Figure 18 No significant damage was observed. Furthermore, although the concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the rabbit serum increased after embolization, they rapidly returned to normal levels by day 7. Figure 19 Furthermore, the changes in albumin (ALB), total bilirubin (TBIL), creatinine (CERA), and urea (UERA) caused by 3Asphere embolization during the experiment were minimal, indicating that 3Asphere did not cause liver or kidney damage.

[0081] Example 11: Distribution of Asphere vascular embolization in the liver, tumor tissue, and blood vessels in vivo. To observe the in vivo hepatic vascular distribution of 3Aspheres of different sizes, nine rabbits with VX2 tumors were divided into three groups (n = 3): (1) Cy5-3Asphere-60; (2) Cy5-3Asphere-90; (3) Cy5-3Asphere-150. Three days after embolization, the rabbits were euthanized, and their livers were harvested for observation of 3Asphere distribution using a near-infrared fluorescence imaging system (IVIS). Normal liver tissue and liver tumors were then taken, fixed with 4% paraformaldehyde solution, sectioned, and stained with H&E to analyze the distribution of microspheres in vessels of various sizes.

[0082] The results showed that, consistent with size-dependent tumor suppression, smaller 3Aspheres were more enriched in tumors. Figure 20 a), where the tumor enrichment of 3Asphere-60 (45.4%) was 1.3 times and 4 times higher than that of 3Asphere-90 (33.3%) and 3Asphere-150 (10.7%), respectively.Figure 20 b). Histological analysis was used to investigate the distribution of microspheres in tumor vessels. H&E images of tumor vessels 3 days after embolization showed that the deformability of 3Asphere enabled it to adapt to different levels of vessels and could penetrate distally, effectively embolizing fine vessels of 20-60 μm in diameter (Fig. 4B). Figure 21 In contrast to the rare distribution of 3Asphere-150 in tumor vessels, 3Asphere-60 and 3Asphere-90 showed better distribution in vessels with diameters of 50 μm and 70 μm, respectively (Fig. 4B). Figure 22 However, Embosphere, which has higher stiffness, mainly showed a spherical morphology in tumor vessels and was distributed in vessels with diameters close to the microspheres (40-120 μm).

Claims

1. A method for preparing embolizing microspheres which are degradable, elastic and uniform in particle size, characterized by, The method comprises the following steps: The macromolecule with cross-linking groups is polymerized to obtain embolization microspheres with degradability, elasticity and uniform particle size.

2. The method of claim 1, wherein the biodegradable, elastic and uniform particle size embolism microspheres are prepared by the steps of: The macromolecule is a medical macromolecule; and the polymerization comprises radical polymerization. ​ 3. The method for preparing biodegradable, elastic, and uniformly sized embolic microspheres according to claim 1, characterized in that, The embolization microspheres with degradability, elasticity and uniform particle size are prepared by microfluidic technology; and the particle size of the embolization microspheres with degradability, elasticity and uniform particle size is 20-1100 μm.

4. The method for preparing biodegradable, elastic, and uniformly sized embolic microspheres according to claim 2, characterized in that, The macromolecule comprises one or more of hyaluronic acid, dextran, chitosan, heparin, alginic acid, collagen or polyethylene glycol-polyester; the water-phase material containing the macromolecule with cross-linking groups is subjected to radical polymerization cross-linking solidification with the oil-phase material through a micro-channel pipeline to obtain the embolization microspheres with degradability, elasticity and uniform particle size.

5. The method for preparing the biodegradable, elastic, and uniformly sized embolic microspheres according to claim 3, characterized in that, The water-phase material comprises the macromolecule with cross-linking groups, buffer and initiator; the oil-phase material comprises surfactant and oil solvent; the concentration of the macromolecule with cross-linking groups is 10-400 mg / mL; the micro-channel pipeline comprises a cross-shaped, Y-shaped, T-shaped or flow-focusing micro-channel pipeline; and the solidification comprises ultraviolet light solidification and thermal solidification.

6. The method for preparing biodegradable, elastic, and uniformly sized embolic microspheres according to claim 1, characterized in that, The macromolecule is reacted with acrylate, acid anhydride or amide in a solution to obtain the macromolecule with cross-linking groups, and the degree of substitution is 5-50%.

7. The embolization microspheres with degradability, elasticity and uniform particle size prepared by the method of claim 1.

8. A medicament, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The embolization microspheres with degradability, elasticity and uniform particle size of claim 7.

9. The embolization microspheres with degradability, elasticity and uniform particle size of claim 7 for use in the preparation of a medicament.

10. The embolization microspheres with degradability, elasticity and uniform particle size of claim 7 for use in the preparation of a vascular embolization agent.

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