WO2024017165A1 - 一种基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球、制备方法和应用 - Google Patents

一种基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球、制备方法和应用 Download PDF

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WO2024017165A1
WO2024017165A1 PCT/CN2023/107488 CN2023107488W WO2024017165A1 WO 2024017165 A1 WO2024017165 A1 WO 2024017165A1 CN 2023107488 W CN2023107488 W CN 2023107488W WO 2024017165 A1 WO2024017165 A1 WO 2024017165A1
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polyethylene glycol
microspheres
peal
glycolic acid
monomethyl ether
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PCT/CN2023/107488
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English (en)
French (fr)
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段友容
武志华
汪权
廖静涵
景宏舒
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森心(上海)科技有限公司
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Priority claimed from CN202210865881.8A external-priority patent/CN115177747A/zh
Priority claimed from CN202210873493.4A external-priority patent/CN115252877A/zh
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Publication of WO2024017165A1 publication Critical patent/WO2024017165A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/04—X-ray contrast preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/06—Macromolecular compounds, carriers being organic macromolecular compounds, i.e. organic oligomeric, polymeric, dendrimeric molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/06—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to the field of biomedical technology, and in particular relates to a developed porous microsphere based on polyethylene glycol monomethyl ether-polylactic acid glycolic acid-polylysine, preparation method and application.
  • Microspheres are spherical particles with diameters in the micron range (usually 1 ⁇ m to 1000 ⁇ m), or microparticles. According to the characteristics of free-flowing powdery particles, microspheres can be divided into protein, synthetic polymer, glass, ceramic microspheres, etc. according to different components. Among the most commonly used materials are polymers such as polylactic acid, poly(lactic-glycolic acid) (PLGA), chitosan, polycaprolactone (PCL), divinylbenzene and other polymers or copolymers.
  • PLGA poly(lactic-glycolic acid)
  • PCL polycaprolactone
  • the material When used to form microspheres, it can be combined with a pore-forming agent to form porous polymer microspheres, which greatly increases the specific surface area of the microspheres and expands its applications in the fields of chemistry, medicine, biomedical engineering and other fields.
  • a pore-forming agent to form porous polymer microspheres, which greatly increases the specific surface area of the microspheres and expands its applications in the fields of chemistry, medicine, biomedical engineering and other fields.
  • a pore-forming agent to form porous polymer microspheres, which greatly increases the specific surface area of the microspheres and expands its applications in the fields of chemistry, medicine, biomedical engineering and other fields.
  • a pore-forming agent Such as drug delivery and controlled release, 3D cell culture scaffolds, tissue repair excipients, interventional imaging microspheres and multifunctional microspheres that combine the above functions.
  • microspheres The size of microspheres is larger than that of nanoparticles, so it has been widely used in the field of vascular embolization. Compared with ordinary microspheres, porous microspheres have a larger specific surface area and are a better carrier. It has been widely studied in aspects such as controlled release of drugs, adsorption of heavy metal ions, and biomedical engineering applications, and has broad application prospects. For example, microspheres prepared into a size similar to the diameter of blood vessels can effectively cause blood vessel embolization, which can effectively inhibit tumor growth and prolong the survival of patients in tumor treatment.
  • Porous microspheres can reduce microsphere density by adjusting particle size and porosity, while achieving controlled release of drugs.
  • Porous microspheres have the structural characteristics of high porosity and large specific surface area, which not only enhances the adsorption performance, but also facilitates cell adhesion and migration.
  • the voids in the microspheres and on the surface provide space for the growth of cells, and at the same time, they contain The loaded drug molecules are easier to release to achieve controlled release, and are expected to improve the drug release stagnation period defects of solid microspheres.
  • interventional embolization is one of the scenarios where microspheres are widely used. Interventional treatments are usually performed on imaging equipment such as electronic digital subtraction angiography (DSA), electronic computed tomography (CT), and magnetic resonance imaging. With the assistance and monitoring of the patient, embolization microspheres and other implanted materials are introduced into the tumor site in the body through minimally invasive wounds through the use of puncture needles, guide wire catheters and other interventional treatment equipment, such as femoral artery puncture, to cut off the tumor. The nutrient supply starves the tumor cells to death, thereby achieving the purpose of treating tumors. Because it has its own imaging function, it does not need the assistance of other contrast agents and avoids ectopic embolism.
  • DSA electronic digital subtraction angiography
  • CT electronic computed tomography
  • magnetic resonance imaging With the assistance and monitoring of the patient, embolization microspheres and other implanted materials are introduced into the tumor site in the body through minimally invasive wounds through the use of punct
  • interventional therapy the R&D and manufacturing of interventional therapy materials have developed rapidly, which has also put forward higher requirements for interventional therapy-related materials, such as the traceability of materials and drug coating capabilities.
  • Inorganic materials that develop under X-rays are metal salt contrast agents.
  • these metal salt contrast agents affect the mechanical properties of embolization materials. In the later stages of use, they can easily spread to other parts of the body and cause toxicity to the body.
  • the embolization agents currently used clinically generally cannot be developed by themselves and need to be developed with contrast agents. Simple physical mixing will cause the separation of contrast agents and embolic materials, resulting in blurred imaging and misdiagnosis. Therefore, it is very necessary to develop a developed porous microsphere with excellent performance.
  • the present invention provides a developed porous microsphere based on polyethylene glycol monomethyl ether-polylactic acid glycolic acid-polylysine, preparation method and application.
  • the prepared developed porous microspheres use polyethylene glycol monomethyl ether-polylactic acid glycolic acid-polylysine (mPEG-PLGA-PLL, abbreviated as PEAL, patent number CN 101732723 A) cationic polymer as the skeleton.
  • PEAL has Polymer materials with excellent biocompatibility can be used to encapsulate water-soluble drugs and water-insoluble drugs.
  • the amino groups on polylysine have positive electrical properties and are suitable for adsorbing or encapsulating negatively charged drugs, nucleic acids, An ideal carrier for proteins and other substances.
  • the components produced by the decomposition of PEAL by the human body are all biocompatible molecules, especially the degradation of PLL into lysine, which is an essential amino acid for the human body.
  • PEAL is a cationic polymer. This material has good biocompatibility and drug loading capabilities. It can be modified to have multifunctional properties, such as tumor targeting, reversal of drug resistance, and medical diagnostic functions. It has been proven to be useful for loading. Organic drugs, water-soluble drugs, water-insoluble drugs or imaging agents for diagnostic purposes. In addition, PEAL is easily soluble in dichloromethane, tetrahydrofuran and other volatile compounds. organic solvents, which makes it easier to remove residual organic solvents during the preparation of PEAL-based nanoparticles, microspheres, etc.
  • the first object of the present invention is to provide a developed porous microsphere based on polyethylene glycol monomethyl ether-polylactic acid glycolic acid-polylysine, which is polyethylene glycol monomethyl ether-polylactic acid glycolic acid-
  • the molecular weight of polylysine is 1.0 ⁇ 10 3 -9.0 ⁇ 10 6
  • the molar ratio of polyethylene glycol monomethyl ether/lactic acid is 1-50:50-100
  • the molar ratio of lactic acid/glycolic acid is 1-100: 1-100
  • the molar ratio of glycolic acid/lysine is 50-100:1-50
  • the developed porous microspheres are developed and transformed from polyethylene glycol monomethyl ether-polylactic acid glycolic acid-polylysine , prepared by microfluidic method
  • the development transformation is to use barium sulfate for compounding and/or use iodine molecules for iodine reaction;
  • the particle size of the developed porous microspheres obtained is 1 ⁇ m-1000 ⁇ m.
  • the developed porous microspheres include polyethylene glycol monomethyl ether-polylactic acid glycolic acid-polylysine. 80wt%-99wt%, barium sulfate 1wt%-20wt%;
  • the pore diameter of the developed porous microspheres obtained is 10nm-1000nm.
  • polyethylene glycol monomethyl ether-polylactic acid glycolic acid-polyethylene glycol The molar ratio of amino acid and iodine molecules is 1:1-1:50; the iodine molecules are selected from 2,3,5-triiodobenzoic acid (TIBA), 3-amino-2,4,6-tris
  • TIBA 2,3,5-triiodobenzoic acid
  • ATBA 2,3,5-triiodobenzylaldehyde
  • TCA 2,3,5-triiodobenzoyl chloride
  • the barium sulfate also known as barite
  • the barium sulfate is an odorless, tasteless white amorphous powder. Stable in nature and difficult to dissolve in water, acid, alkali or organic solvents. In terms of medical applications, radiology utilizes its X-ray absorption properties, mainly as a contrast agent in the gastrointestinal tract.
  • the PEAL material itself does not have a developing function, and the prepared microspheres can be given a developing function by encapsulating a developer.
  • the developing ability of the prepared PEAL/barium sulfate developing porous microspheres is related to the ratio of barium sulfate. The higher the barium sulfate content, the stronger the developing ability of the microspheres.
  • Porous microspheres can reduce the density of microspheres to a certain extent.
  • 10nm-1000nm pores can be produced on microspheres with a particle size of 1 ⁇ m-1000 ⁇ m. While having the development function, they can also load a variety of components. point.
  • PEAL is an extremely biocompatible carrier material and has excellent resistance to inorganic materials. It has a certain physical loading capacity and does not affect its own properties. After being combined with barium sulfate, it can still contain and adsorb drugs, nucleic acids, proteins, radionuclides, etc.
  • PEAL Compared with common chitosan microspheres, PEAL does not need to be dissolved in acidic solutions and only needs to be dissolved in organic solvents, and its acid resistance is better than that of chitosan microspheres. Compared with another common sodium alginate microsphere, PEAL has better spherical properties, while sodium alginate usually requires cross-linking to form spheres. Even so, its spherical properties are still poor. Because sodium alginate displays negative electricity, it can generally only contain positively charged substances, which greatly limits its application in the biomedical field. PEAL is easily soluble in most organic reagents, most of which are volatile reagents. This makes the removal of organic reagents simple and fast, does not affect the physical and chemical properties of materials and drugs, and avoids biological toxicity caused by incomplete removal of organic reagents.
  • radiology utilizes the X-ray absorption properties of iodine to enhance image viewing.
  • Iodinated contrast agent has a higher density under degree.
  • the PEAL material itself does not have a developing function, so it needs to be iodine-modified to give it a developing function.
  • the developing ability of iodinated PEAL porous microspheres is related to the proportion of linked iodine atoms. The higher the iodine content, the stronger the developing ability of the microspheres. However, lack of pores will make the microspheres denser, making them less maneuverable.
  • PEAL can be covalently grafted with iodine-containing compounds to give it a developing function, and then the related properties can be adjusted by changing the molecular weight or the arm length of the molecular chain.
  • PEAL is a biodegradable material with good performance and good biocompatibility.
  • the iodine element contained in the chemically bonded iodine-PEAL developed porous microspheres is more stable than the iodine-containing microspheres prepared by encapsulation or mixing methods. The iodine atoms are not easily released. Compared with iodized oil exposed to air or sunlight, Zhonghui analyzed the characteristics of free iodine and increased the safety of microspheres.
  • the selected iodine molecules contain three iodine atoms on each molecule, and significant development effects can be obtained with a smaller amount of reagents. Under the same content, the four iodine PEAL developing porous microspheres show Same developing effect. In addition, through the adjustment of the production process, pores of 10nm-1000nm can be produced on microspheres with a particle size of 1 ⁇ m-1000 ⁇ m. Iodo-PEAL is easily soluble in most organic reagents. Compared with chitosan, it does not use acidic solutions, and most of them are volatile reagents.
  • the developed porous microspheres further include a drug with a mass fraction of 0.1 wt% to 30 wt% relative to the developed porous microspheres.
  • the drug is selected from one or more of common clinical drugs, anti-tumor genes, anti-tumor drugs, molecular targeted drugs and radionuclides.
  • the common clinical drug is selected from lidocaine.
  • anti-tumor gene is selected from siRNA, microRNA, piRNA, IncRNA, and circRNA;
  • the anti-tumor gene is selected from the group consisting of siRNA that downregulates NF- ⁇ Bp65 gene expression, siRNA that interferes with Gab1, siRNA that silences the protein kinase C ⁇ gene, miRNA-21, miRNA-605, miRNA-376c, miRNA-200b/c and One or more of miRNA-101 and the like.
  • the anti-tumor drug is selected from the group consisting of oxaliplatin, cisplatin, carboplatin, imiplatin, loplatin, nedaplatin, cycloplatin, paclitaxel, mitoxantrone, doxorubicin, epirubicin, Doxorubicin, mitomycin, 5-fluorouracil, raltitrexed, docetaxel, gemcitabine, bleomycin, arsenic trioxide, calcium leucovorin, deoxyfluridine, irinotecan, topotecan , hydroxycamptothecin, etoposide, vinorelbine, vincristine and methotrexate, etc. One or more.
  • the molecular targeted drug is selected from the group consisting of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, epidermal growth factor receptor 2 (HER-2) inhibitors, immune checkpoint inhibitors and vascular endothelial growth factor One or more of inhibitors, etc.
  • EGFR epidermal growth factor receptor
  • HER-2 epidermal growth factor receptor 2
  • immune checkpoint inhibitors vascular endothelial growth factor
  • vascular endothelial growth factor One or more of inhibitors, etc.
  • the immune checkpoint inhibitor is selected from one or more of anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, and the like.
  • the radionuclides are selected from 90 Y, 131 I, 125 I, 123 I, 32 P, 153 Sm, 186 Re, 211 At, 212 Bi, 166 Ho, 177 Lu, 188 Re, 18 F, 61 Cu , 64 Cu, 89 Zr, 66 Ga, 68 Ga, 44 Sc, 72 As, 69 Ge, 51 Mn , 52 Mn, 45 Ti, 86 Y, 55 Co, 111 In and 225 Ac, etc.
  • the second object of the present invention is to provide a method based on polyethylene glycol monomethyl ether-polylactic acid hydroxyethyl
  • the preparation method of acid-polylysine developed porous microspheres includes the following steps:
  • polyethylene glycol monomethyl ether-polylactic acid glycolic acid-polylysine reacts with iodine molecules to obtain purified iodopolyethylene glycol monomethyl ether -Polylactic acid glycolic acid-polylysine is dissolved in the organic phase solution to obtain a dispersed phase with a concentration of 1-40wt%;
  • a surfactant aqueous solution with a concentration of 1wt%-6wt% serves as the continuous phase and collection phase;
  • the preparation method of barium sulfate specifically includes the following steps: mixing water-soluble barium salt and water-soluble sulfate in a molar ratio of 1-10:1-10 Mix and stir, the stirring rate is 500rpm-2000rpm, the stirring time is 10min-30min, after the reaction, use water to wash 3 times, and then use methanol to wash 3 times, the centrifugation rate is 5000rpm-10000rpm, the centrifugation time is 5min-30min.
  • the stirring rate is 500rpm-2000rpm
  • the stirring time is 10min-30min
  • the centrifugation rate is 5000rpm-10000rpm
  • the centrifugation time is 5min-30min.
  • the washing method used in this application to prepare barium sulfate can increase its dispersion in organic solvents such as chloroform. Based on this, it can be used in the preparation When forming microspheres, barium sulfate is directly wrapped into the microspheres without subsequent adsorption and water washing.
  • the catalyst is selected from the group consisting of dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), 1-ethyl-(3- One or more of dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS).
  • DCC dicyclohexylcarbodiimide
  • DMAP 4-dimethylaminopyridine
  • EDC 1-ethyl-(3- One or more of dimethylaminopropyl)carbodiimide
  • NHS N-hydroxysuccinimide
  • iodinated PEAL imaging porous microspheres have the ability to develop under X-rays and good embolization and drug-loading capabilities. They are used in biomedicine It can have wider application value in the field of science.
  • the organic phase solution is selected from one or more of dichloromethane, chloroform, acetone, tetrahydrofuran, petroleum ether and ethyl acetate.
  • the purification method is selected from dialysis, filtration, water washing, freeze-drying or recrystallization.
  • the surfactant is selected from the group consisting of methylcellulose, carbopol, sodium alginate, polyvinyl alcohol, ammonium salt type, quaternary ammonium salt type, amphoteric Surfactant, higher fatty acid salt, sulfonate, sulfate ester salt, poloxamer, polyethylene glycol, polyethylene glycol-polylactic acid, polyethylene glycol-polylactic acid-glycolic acid copolymer, polyethylene glycol Diol - one or more of polycaprolactone and polyvinylpyrrolidone.
  • the inner diameter of the dispersed phase microchannel is 20 ⁇ m-1000 ⁇ m
  • the inner diameter of the continuous phase microchannel is 40 ⁇ m-3mm
  • the flow rate ratio of the dispersed phase to the continuous phase is 1:6-1:10.
  • step (2) also includes using deionized water to wash and develop the porous microspheres several times, freeze-drying and then use.
  • step (2) there is also a step of adding drugs to the developed porous microspheres.
  • the third object of the present invention is to provide an application of the developed porous microspheres based on polyethylene glycol monomethyl ether-polylactic acid glycolic acid-polylysine in the preparation of disease treatment drugs or disease treatment devices. .
  • the developed porous microspheres based on polyethylene glycol monomethyl ether-polylactic acid glycolic acid-polylysine are used in embolization, drug-loading subcutaneous or muscle or in situ disease treatment, oral administration and applications such as intraperitoneal drug delivery, imaging, and 3D cell culture.
  • the particle size of the developed porous microspheres based on polyethylene glycol monomethyl ether-polylactic acid glycolic acid-polylysine according to the present invention is uniform and controllable. Since the microspheres have a porous structure, it ensures that the microspheres are During the preparation process, barium sulfate can be evenly distributed in the microspheres. The porous and round structure further increases the specific surface area. This gives the microspheres multiple functions such as imaging, drug loading, and embolization, which greatly expands the application of microspheres. The method is simple to operate, has high drug loading capacity, is easy to repeat, and is stable in batches. The microsphere is expected to become an excellent carrier for drugs, genes, proteins, metal ions, and radionuclides. It is expected to be used in clinical embolization, drug loading, disease diagnosis and treatment and other fields.
  • the preparation method of the present invention realizes the process of mass production of embolization microspheres using microfluidic technology through the construction of microfluidic equipment and chip sets.
  • Figure 1 is an SEM picture of pure PEAL/barium sulfate developed porous microspheres in Example 1 of the present invention
  • Figure 2 is an SEM picture of PEAL/barium sulfate developed porous microspheres loaded with 90 Y in Example 1 of the present invention
  • Figure 3 is a development picture of a centrifuge tube of PEAL/barium sulfate developed porous microspheres in X-ray download of 90 Y in Example 1 of the present invention
  • Figure 4 is a diagram of the development effect of injecting 90 Y-loaded PEAL/barium sulfate developing porous microspheres into the left and right hind legs of mice in Example 1 of the present invention
  • Figure 5 is a DSA diagram of rabbit renal artery embolization experiment in Example 1 of the present invention.
  • Figure 6 is a CT image of the kidney of a rabbit 4 weeks after embolization in Example 1 of the present invention.
  • Figure 7 is a H&E stained tissue section of the kidney of a rabbit 4 weeks after embolization in Example 1 of the present invention.
  • Figure 8 is an SEM of pure iodinated PEAL developed porous microspheres and its developed image in Example 2 of the present invention
  • Figure 9 is an SEM of the 90 Y-loaded iodoPEAL developed porous microspheres and its developed image in Example 2 of the present invention.
  • Figure 10 is an SEM of doxorubicin-loaded iodoPEAL developed porous microspheres and its developed image in Example 2 of the present invention
  • Figure 11 shows the iodinated PEAL-developed porous microspheres loaded with miRNA-376c in Example 2 of the present invention. SEM and its developed images;
  • Figure 12 is an optical microscope picture of the iodinated PEAL-developed porous microspheres loaded with anti-PD-L1 antibodies in Example 2 of the present invention
  • Figure 13 is an SEM picture of the cell-loaded iodinated PEAL-developed porous microspheres in Example 2 of the present invention.
  • Figure 14 is a diagram of the development effect of injecting iodo-PEAL developing porous microspheres into the left and right hind legs of mice in Example 2 of the present invention.
  • Figure 15 is a digital photo of the rabbit kidney embolization experimental process in Example 2 of the present invention.
  • Figure 16 is a CT image of the rabbit kidney 4 weeks after rabbit embolization in Example 2 of the present invention.
  • Figure 17 is a H&E stained tissue section of the kidney of a rabbit 4 weeks after embolization in Example 2 of the present invention.
  • sequences of miRNA-376c used in the examples are: AACAUAGAGGAAAUUCCACGU and GGUGGAUAUUCCUUCUAUGUU.
  • Example 1 PEAL/barium sulfate developed porous microspheres
  • Preparation of PEAL/barium sulfate developed porous microspheres by microfluidic method prepare the dispersed phase, continuous phase and collection phase required for the preparation of microspheres.
  • the dispersed phase is a methylene chloride solution of PEAL containing barium sulfate, in which the molecular weight of PEAL is approximately 1 ⁇ 10 4 g/mol, the molar ratio of polyethylene glycol monomethyl ether/lactic acid is 1:20, and the molar ratio of lactic acid/glycolic acid The ratio is 1:4, the molar ratio of glycolic acid/lysine is 4:1; the PEAL mass fraction is 10wt%, the barium sulfate mass fraction is 3wt%, the continuous phase and the collection phase are 3wt% polyvinyl alcohol (PVA) aqueous solution .
  • PVA polyvinyl alcohol
  • the collected phase was continuously stirred at a stirring rate of 60 rpm.
  • a microfluidic device was built and PEAL/barium sulfate developed porous microspheres were prepared using microfluidic technology.
  • the microfluidic pipeline is constructed with polytetrafluoroethylene tubes.
  • the inner diameter of the two-phase intersection and collection channel is 500 ⁇ m.
  • the inner diameter of the dispersed phase microchannel is 210 ⁇ m.
  • the inner diameter of the continuous phase microchannel is 500 ⁇ m.
  • the inner diameter of the dispersed phase and the collection point are 500 ⁇ m.
  • Preparation of PEAL/barium sulfate developed porous microspheres by microfluidic method prepare the dispersed phase, continuous phase and collection phase required for the preparation of microspheres.
  • the dispersed phase is a methylene chloride solution of PEAL containing barium sulfate, in which the molecular weight of PEAL is approximately 1 ⁇ 10 4 g/mol, the molar ratio of polyethylene glycol monomethyl ether/lactic acid is 1:20, and the molar ratio of lactic acid/glycolic acid The ratio is 1:4, the molar ratio of glycolic acid/lysine is 4:1; the mass fraction of barium sulfate is 2wt%, and the mass fraction of PEAL is 15wt%.
  • the continuous phase and collection phase were 2 wt% PVA aqueous solution. During the preparation process, the collected phase was stirred at a stirring rate of 60 rpm.
  • a microfluidic device was built and PEAL/barium sulfate developed porous microspheres were prepared using microfluidic technology.
  • the microfluidic pipeline is constructed with polytetrafluoroethylene tubes.
  • the inner diameter of the two-phase intersection and collection channel is 500 ⁇ m.
  • the inner diameter of the dispersed phase microchannel is 210 ⁇ m.
  • the inner diameter of the continuous phase microchannel is 500 ⁇ m.
  • the inner diameter of the dispersed phase and the collection point are 500 ⁇ m.
  • Microspheres After the preparation process is completed, let it stand for 48 hours and collect the microspheres ( Figure 2). Wash 3 times with deionized water, freeze-dry and set aside for later use.
  • the PEAL/barium sulfate developed porous microspheres loaded with 90 Y have good development effects, and the signal can be clearly observed under X-ray irradiation (Figure 3).
  • the dispersed phase is a methylene chloride solution of PEAL containing barium sulfate, in which the molecular weight of PEAL is approximately 1 ⁇ 10 5 g/mol, the molar ratio of polyethylene glycol monomethyl ether/lactic acid is 1:20, and the molar ratio of lactic acid/glycolic acid The ratio is 1:4, the molar ratio of glycolic acid/lysine is 4:1; the mass fraction of barium sulfate is 3wt%, and the mass fraction of PEAL is 15wt%.
  • the continuous phase and collection phase were 1 wt% PVA aqueous solution.
  • a microfluidic device was built and PEAL/barium sulfate developed porous microspheres were prepared using microfluidic technology.
  • the microfluidic pipeline is made of polytetrafluoroethylene.
  • the inner diameter of the channel at the intersection and collection point of the two phases is 400 ⁇ m
  • the inner diameter of the dispersed phase microchannel is 210 ⁇ m
  • the inner diameter of the continuous phase microchannel is 500 ⁇ m.
  • the flow rates of the dispersed phase and continuous phase are 120 ⁇ L/min and 720 ⁇ L/min, respectively.
  • Preparation by microfluidic method prepare the dispersed phase, continuous phase, and collection phase required to prepare microspheres.
  • the dispersed phase is a chloroform solution of PEAL containing barium sulfate, in which the molecular weight of PEAL is approximately 1 ⁇ 10 5 g/mol.
  • the molar ratio of ethylene glycol monomethyl ether/lactic acid is 1:20, the molar ratio of lactic acid/glycolic acid is 1:4, the molar ratio of glycolic acid/lysine is 4:1; the mass fraction of barium sulfate is 3wt%, PEAL mass fraction is 20wt%.
  • the continuous phase and collection phase were 4 wt% poloxamer aqueous solution.
  • a microfluidic device was built and PEAL/barium sulfate developed porous microspheres were prepared using microfluidic technology.
  • the microfluidic pipeline is made of polytetrafluoroethylene.
  • the inner diameter of the channel at the intersection and collection point of the two phases is 500 ⁇ m
  • the inner diameter of the dispersed phase microchannel is 210 ⁇ m
  • the inner diameter of the continuous phase microchannel is 500 ⁇ m.
  • the flow rates of the dispersed phase and continuous phase are 600 ⁇ L/min and 3.6mL respectively.
  • PEAL/barium sulfate developed porous microspheres with a particle size of 180 ⁇ m, a pore size of 100 nm-400 nm, and a barium sulfate mass fraction of 13%.
  • the dispersed phase is a methylene chloride solution of PEAL containing barium sulfate, in which the molecular weight of PEAL is approximately 5 ⁇ 10 4 g/mol, the molar ratio of polyethylene glycol monomethyl ether/lactic acid is 1:20, and the molar ratio of lactic acid/glycolic acid The ratio is 1:4, the molar ratio of glycolic acid/lysine is 4:1; the mass fraction of barium sulfate is 4wt%, and the mass fraction of PEAL is 25wt%.
  • the continuous phase and collection phase were 2 wt% PVA aqueous solution.
  • a microfluidic device was built and PEAL/barium sulfate developed porous microspheres were prepared using microfluidic technology.
  • the microfluidic pipeline is made of polytetrafluoroethylene.
  • the inner diameter of the channel at the intersection and collection point of the two phases is 600 ⁇ m
  • the inner diameter of the dispersed phase microchannel is 210 ⁇ m
  • the inner diameter of the continuous phase microchannel is 500 ⁇ m.
  • the flow rates of the dispersed phase and continuous phase are 600 ⁇ L/min and 3.6mL respectively.
  • the dispersed phase is a methylene chloride solution of PEAL containing barium sulfate, in which the molecular weight of PEAL is approximately 8 ⁇ 10 4 g/mol, the molar ratio of polyethylene glycol monomethyl ether/lactic acid is 1:20, and the molar ratio of lactic acid/glycolic acid The ratio is 1:4, the molar ratio of glycolic acid/lysine is 4:1; the mass fraction of barium sulfate is 7wt%, and the mass fraction of PEAL is 30wt%.
  • the continuous phase and collection phase were 2 wt% PVA aqueous solution.
  • a microfluidic device was built and PEAL/barium sulfate developed porous microspheres were prepared using microfluidic technology.
  • the microfluidic pipeline is made of polytetrafluoroethylene.
  • the inner diameter of the channel at the intersection and collection point of the two phases is 600 ⁇ m
  • the inner diameter of the dispersed phase microchannel is 100 ⁇ m
  • the inner diameter of the continuous phase microchannel is 500 ⁇ m.
  • the flow rates of the dispersed phase and continuous phase are 600 ⁇ L/min and 3.6mL respectively.
  • Preparation by microfluidic method prepare the dispersed phase, continuous phase, and collection phase required to prepare microspheres.
  • the dispersed phase is a methylene chloride solution of PEAL containing barium sulfate, in which the molecular weight of PEAL is approximately 1 ⁇ 10 6 g/mol, the molar ratio of polyethylene glycol monomethyl ether/lactic acid is 1:20, and the molar ratio of lactic acid/glycolic acid The ratio is 1:4, hydroxyethyl The molar ratio of acid/lysine is 4:1; the mass fraction of barium sulfate is 5wt%, and the mass fraction of PEAL is 20wt%.
  • the continuous phase and the collected phase were 3 wt% carbapol aqueous solution.
  • a microfluidic device was built and PEAL/barium sulfate developed porous microspheres were prepared using microfluidic technology.
  • the microfluidic pipeline is made of polytetrafluoroethylene.
  • the inner diameter of the channel at the intersection and collection point of the two phases is 500 ⁇ m
  • the inner diameter of the dispersed phase microchannel is 210 ⁇ m
  • the inner diameter of the continuous phase microchannel is 500 ⁇ m.
  • the flow rates of the dispersed phase and continuous phase are 120 ⁇ L/min and 720 ⁇ L/min, respectively.
  • Preparation by microfluidic method Prepare a methylene chloride solution of PEAL containing barium sulfate, in which the molecular weight of PEAL is approximately 9 ⁇ 10 5 g/mol, the molar ratio of polyethylene glycol monomethyl ether/lactic acid is 1:20, and the lactic acid/ The molar ratio of glycolic acid is 1:4, the molar ratio of glycolic acid/lysine is 4:1; the mass fraction of barium sulfate is 1wt%, and the mass fraction of PEAL is 15wt%.
  • the continuous phase and collection phase were 3 wt% PVA aqueous solution. During the preparation process, the collected phase was stirred at a stirring rate of 60 rpm.
  • a microfluidic device was built and PEAL/barium sulfate developed porous microspheres were prepared using microfluidic technology.
  • the microfluidic pipeline is constructed with polytetrafluoroethylene tubes.
  • the inner diameter of the two-phase intersection and collection channel is 500 ⁇ m.
  • the inner diameter of the dispersed phase microchannel is 100 ⁇ m.
  • the inner diameter of the continuous phase microchannel is 500 ⁇ m.
  • the inner diameter of the dispersed phase and the collection point are 500 ⁇ m.
  • Microspheres After the preparation process is completed, let it stand for 48 hours and collect the microspheres. Wash 3 times with deionized water, freeze-dry and set aside.
  • microspheres containing 0.2 mg lidocaine When using this microsphere, apply 100 mg PEAL/barium sulfate developing porous microspheres containing 0.2 mg lidocaine to the wound (the proportion of the drug in the microspheres is approximately 0.2%). The wound undergoing tumor resection will cause pain to the patient. Microspheres containing lidocaine slowly release the drug at the wound suture interface. While providing analgesia, the microspheres serve as a cell growth substrate and are more conducive to cell adhesion and growth, promoting cell adhesion and growth. Wound healing, lidocaine has the effect of inhibiting cell growth, and at the same time achieves the purpose of preventing tumor recurrence.
  • mice were anesthetized by intraperitoneal injection of sodium pentobarbital solution.
  • Use surgical instruments to open the mouse's left hind leg, and embed pure PEAL/barium sulfate-developed porous microspheres into the muscle space of the left hind leg using a syringe.
  • the left hind leg of the mouse was then sutured.
  • Use CT to check the development of mouse embolization microspheres on the left hind leg of mice.
  • Figure 4 pure PEAL/barium sulfate developed porous microspheres showed good development effects, stayed at the injection site for a long time, and did not cause ectopic embolism, indicating that the microspheres have good stability and development effects.
  • Adsorption experiment under acidic conditions Weigh 0.852g of yttrium chloride hexahydrate and dissolve it in 250mL of 0.001mol/L hydrochloric acid to prepare a Y 3+ stock solution. The Y 3+ concentration in the stock solution was measured using ICP-OES analysis. Pipette 3 mL of the adsorption stock solution into a centrifuge tube, then add 0.1 g of 90 Y-loaded PEAL/barium sulfate-developed porous microspheres, vortex and mix, then place on a shaker and oscillate horizontally for 1 hour.
  • Adsorption experiment under neutral conditions Weigh 0.852g of yttrium chloride hexahydrate and dissolve it in 250mL of deionized water to prepare a Y 3+ stock solution.
  • the Y 3+ concentration in the stock solution was measured using ICP-OES analysis. Pipette 3 mL of the adsorption stock solution into a centrifuge tube, then add 0.1 g of pure PEAL/barium sulfate developed porous microspheres, vortex and mix, then place on a shaker and oscillate horizontally for 1 hour. After centrifugation, remove the supernatant, then add 5 mL of deionized water to the centrifuge tube, shake horizontally for 1 hour, centrifuge, and remove the supernatant. The supernatants were diluted twice with 2% nitric acid. Finally, the Y 3+ concentration was analyzed using ICP-MS.
  • Elution experimental steps Add 5 mL of physiological saline to the adsorbed sample, vortex and mix, and let stand for 120 hours. After centrifugation, the supernatant was removed, diluted with 2% nitric acid, and analyzed for Y 3+ concentration using ICP-MS.
  • PEAL/barium sulfate developed porous microspheres have a good adsorption effect on 90 Y under both acidic and neutral conditions.
  • the elution rates of the two are only 2.4% and 2.8%.
  • ketamine hydrochloride (3.5 mg/kg) was injected into the rabbit ear vein for intravenous anesthesia, the hind legs were opened, and 2 ligation wires were introduced along the inside of the hind leg arteries, the distal end was ligated, and the proximal end was pulled upward to stop bleeding. Cut a small opening in the artery and insert a 24G intravenous indwelling hose into the renal artery. Perform DSA angiography to confirm that the renal artery and branches are visualized, and then inject 1.5 mL of pure PEAL/barium sulfate imaging porous microspheres through the catheter. Repeat angiography to see below the renal artery segment.
  • PEAL 1:10 (molar ratio) iodinated PEAL material, in which PEAL molecules The amount is about 1 ⁇ 10 4 g/mol, the molar ratio of polyethylene glycol monomethyl ether/lactic acid is 1:20, the molar ratio of lactic acid/glycolic acid is 1:4, and the molar ratio of glycolic acid/lysine is 4:1;
  • TIBA 2,3,5-Tri Dissolve iodobenzoic acid
  • Preparation of iodinated PEAL developing porous microspheres by microfluidic method prepare the dispersed phase, continuous phase and collection phase required for the preparation of microspheres.
  • the dispersed phase was a 10 wt% iodinated PEAL solution in methylene chloride, and the continuous phase and collection phase were a 3 wt% PVA aqueous solution.
  • the collected phase was continuously stirred at a stirring rate of 60 rpm/min.
  • a microfluidic device was built and microfluidic technology was used to prepare iodinated PEAL developing porous microspheres.
  • the microfluidic pipeline is constructed with polytetrafluoroethylene tubes.
  • the inner diameter of the two-phase intersection and collection channel is 500 ⁇ m.
  • the inner diameter of the dispersed phase microchannel is 150 ⁇ m.
  • the inner diameter of the continuous phase microchannel is 500 ⁇ m.
  • the inner diameter of the dispersed phase and the collection point are 500 ⁇ m.
  • PEAL 1:1 (molar ratio) iodinated PEAL material, in which the PEAL molecular weight is about 2 ⁇ 10 5 g/mol, the molar ratio of polyethylene glycol monomethyl ether/lactic acid is 1:20, and the lactic acid/ The molar ratio of glycolic acid is 1:4, and the molar ratio of glycolic acid/lysine is 4:1; weigh 0.3g PEAL, 0.002g EDC and 0.0012g NHS, add them to a 100mL flask, and drain under nitrogen protection.
  • Microfluidic preparation of iodinated PEAL developing drug-loaded microspheres Preparation of dispersed phase, continuous phase and collection phase required for preparation of microspheres.
  • the dispersed phase is 10wt% iodinated PEAL in methylene chloride solution, the continuous phase and the collection phase It is a 2wt% PVA aqueous solution.
  • a microfluidic device was built and microfluidic technology was used to prepare iodinated PEAL developing porous microspheres.
  • the microfluidic pipeline is constructed with polytetrafluoroethylene tubes.
  • the inner diameter of the two-phase intersection and collection channel is 500 ⁇ m.
  • the inner diameter of the dispersed phase microchannel is 200 ⁇ m.
  • the inner diameter of the continuous phase microchannel is 500 ⁇ m.
  • the inner diameter of the dispersed phase and the collection point are 500 ⁇ m.
  • TIBA 1:5 (molar ratio) iodinated PEAL material, in which the PEAL molecular weight is about 1 ⁇ 10 5 g/mol, the molar ratio of polyethylene glycol monomethyl ether/lactic acid is 1:20, and the lactic acid/ The molar ratio of glycolic acid is 1:4, and the molar ratio of glycolic acid/lysine is 4:1; weigh 0.3g PEAL, 0.01g EDC and 0.006g NHS, add them to a 100mL flask, and drain under nitrogen protection for 3 After several times, add it to the flask, dissolve 0.025g of 2,3,5-triiodobenzoic acid (TIBA) in dichloromethane, and react for 12 hours after evacuating nitrogen three times. The dichloromethane was removed by rotary evaporation, then 3 mL of DMF was added to dissolve, transferred to a dialysis bag for dialysis for 48 hours, and lyophilized for later use.
  • TIBA 2,3,5
  • Microfluidic preparation of iodinated PEAL developing drug-loaded microspheres Preparation of dispersed phase, continuous phase and collection phase required for preparation of microspheres.
  • the dispersed phase was a 15 wt% iodinated PEAL solution in methylene chloride, and the continuous phase and collection phase were a 1 wt% PVA aqueous solution.
  • a microfluidic device was built and microfluidic technology was used to prepare iodinated PEAL developing porous microspheres.
  • the microfluidic pipeline is constructed with polytetrafluoroethylene tubes.
  • the inner diameter of the two-phase intersection and collection channel is 400 ⁇ m.
  • the inner diameter of the dispersed phase microchannel is 150 ⁇ m.
  • the inner diameter of the continuous phase microchannel is 400 ⁇ m.
  • the inner diameter of the dispersed phase and the collection point are 400 ⁇ m.
  • microsphere suspension When used, prepare a microsphere suspension with a concentration of 0.5g/mL, and add 0.2g of doxorubicin to 2 mL of water containing developed porous microspheres before use (the proportion of the drug in the microspheres is 20%).
  • PEAL 1:10 (molar ratio) iodinated PEAL material, in which the molecular weight of PEAL is about 3 ⁇ 10 5 g/mol, the molar ratio of polyethylene glycol monomethyl ether/lactic acid is 1:20, and the molar ratio of lactic acid/ The molar ratio of glycolic acid is 1:4, and the molar ratio of glycolic acid/lysine is 4:1; weigh 0.3g PEAL, 0.02g EDC and 0.012g NHS, add them to a 100mL flask, and drain under nitrogen protection 3 After several times, add it to the flask, dissolve 0.05g of 2,3,5-triiodobenzoic acid (TIBA) in dichloromethane, and react for 12 hours after evacuating nitrogen three times. The dichloromethane was removed by rotary evaporation, then 3 mL of DMF was added to dissolve, transferred to a dialysis bag for dialysis for 48 hours, and lyophilized for later
  • TIBA 2,3,5
  • Microfluidic preparation of iodinated PEAL developing drug-loaded microspheres Preparation of dispersed phase, continuous phase and collection phase required for preparation of microspheres.
  • the dispersed phase was a 20 wt% iodinated PEAL solution in methylene chloride, and the continuous phase and collected phase were a 4 wt% poloxamer aqueous solution.
  • a microfluidic device was built and microfluidic technology was used to prepare iodinated PEAL developing porous microspheres.
  • the microfluidic pipeline is constructed with polytetrafluoroethylene tubes.
  • the inner diameter of the two-phase intersection and collection channel is 600 ⁇ m.
  • the inner diameter of the dispersed phase microchannel is 100 ⁇ m.
  • the inner diameter of the continuous phase microchannel is 600 ⁇ m.
  • the inner diameter of the dispersed phase and the collection point are 600 ⁇ m.
  • microsphere suspension When used, prepare a microsphere suspension with a concentration of 0.5g/mL, add 50 nmol of miRNA-376c to 3 mL of water containing developed porous microspheres (the proportion of the drug in the microspheres is 0.1%).
  • TIBA 1:50 (molar ratio) iodinated PEAL material, in which the PEAL molecular weight is about 3 ⁇ 10 6 g/mol, the molar ratio of polyethylene glycol monomethyl ether/lactic acid is 1:20, and the lactic acid/ The molar ratio of glycolic acid is 1:4, and the molar ratio of glycolic acid/lysine is 4:1; weigh 0.3g PEAL, 0.04g EDC and 0.023g NHS, add them to a 100mL flask, and drain under nitrogen protection 3 After several times, add it to the flask, Dissolve 0.25g of 2,3,5-triiodobenzoic acid (TIBA) in dichloromethane, and react for 12 hours after evacuating nitrogen three times. The dichloromethane was removed by rotary evaporation, then 3 mL of DMF was added to dissolve, transferred to a dialysis bag for dialysis for 48 hours, and lyophilized for later use.
  • TIBA 2,3,5
  • Microfluidic preparation of iodinated PEAL developing drug-loaded microspheres Preparation of dispersed phase, continuous phase and collection phase required for preparation of microspheres.
  • the dispersed phase was a 20 wt% iodinated PEAL solution in methylene chloride, and the continuous phase and collection phase were a 3 wt% PVA aqueous solution.
  • a microfluidic device was built and microfluidic technology was used to prepare iodinated PEAL developing porous microspheres.
  • the microfluidic pipeline is constructed with polytetrafluoroethylene tubes.
  • the inner diameter of the two-phase intersection and collection channel is 500 ⁇ m.
  • the inner diameter of the dispersed phase microchannel is 150 ⁇ m.
  • the inner diameter of the continuous phase microchannel is 500 ⁇ m.
  • the inner diameter of the dispersed phase and the collection point are 500 ⁇ m.
  • microsphere suspension When used, prepare a microsphere suspension with a concentration of 0.5g/mL, and add 100 ⁇ g of anti-PD-L1 antibody to 2 mL of water containing developed porous microspheres before use (the proportion of the drug in the microspheres is 0.1%).
  • PEAL 1:1 (molar ratio) iodinated PEAL material, in which the molecular weight is about 3 ⁇ 10 6 g/mol, the molar ratio of polyethylene glycol monomethyl ether/lactic acid is 1:20, and the lactic acid/hydroxyl group
  • the molar ratio of acetic acid is 1:4, and the molar ratio of glycolic acid/lysine is 4:1; weigh 0.3g PEAL, 0.002g EDC and 0.0012g NHS, add them to a 100mL flask, and drain them three times under nitrogen protection.
  • Microfluidic preparation of iodinated PEAL developing drug-loaded microspheres Preparation of dispersed phase, continuous phase and collection phase required for preparation of microspheres.
  • the dispersed phase was a 15 wt% iodinated PEAL solution in methylene chloride, and the continuous phase and collected phase were a 2 wt% carbapol aqueous solution.
  • a microfluidic device was built and microfluidic technology was used to prepare iodinated PEAL developing porous microspheres.
  • the microfluidic pipeline is constructed with polytetrafluoroethylene tubes, and the two-phase intersection and collection channels are The inner diameter is 500 ⁇ m, the inner diameter of the dispersed phase microchannel is 150 ⁇ m, and the inner diameter of the continuous phase microchannel is 500 ⁇ m.
  • microsphere suspension When used, prepare a microsphere suspension with a concentration of 0.5g/mL, add 10 4 -10 5 cells to 2 mL of water containing developed porous microspheres, and culture for 48 hours before use.
  • PEAL 1:5 (molar ratio) iodinated PEAL material, in which the PEAL molecular weight is about 3 ⁇ 10 6 g/mol, the molar ratio of polyethylene glycol monomethyl ether/lactic acid is 1:20, and the lactic acid/hydroxyl group
  • the molar ratio of acetic acid is 1:4, and the molar ratio of glycolic acid/lysine is 4:1; weigh 0.3g PEAL, 0.01g EDC and 0.006g NHS, add them to a 100mL flask, and drain under nitrogen protection for 3 After several times, add it to the flask, dissolve 0.025g of 2,3,5-triiodobenzoic acid (TIBA) in dichloromethane, and react for 12 hours after evacuating nitrogen three times. The dichloromethane was removed by rotary evaporation, then 3 mL of DMF was added to dissolve, transferred to a dialysis bag for dialysis for 48 hours, and lyophilized for
  • Microfluidic preparation of iodinated PEAL developing drug-loaded microspheres Preparation of dispersed phase, continuous phase and collection phase required for preparation of microspheres.
  • the dispersed phase was a 20 wt% iodinated PEAL solution in methylene chloride, and the continuous phase and collection phase were a 1 wt% PVA aqueous solution.
  • a microfluidic device was built and microfluidic technology was used to prepare iodinated PEAL developing porous microspheres.
  • the microfluidic pipeline is constructed with polytetrafluoroethylene tubes.
  • the inner diameter of the two-phase intersection and collection channel is 500 ⁇ m.
  • the inner diameter of the dispersed phase microchannel is 210 ⁇ m.
  • the inner diameter of the continuous phase microchannel is 500 ⁇ m.
  • the inner diameter of the dispersed phase and the collection point are 500 ⁇ m.
  • microsphere suspension When used, prepare a microsphere suspension with a concentration of 0.5g/mL, and apply 2 mL of 200 mg developing porous microspheres containing lidocaine to the wound (the proportion of microspheres accounted for by the drug is 20%).
  • mice were anesthetized by intraperitoneal injection of sodium pentobarbital solution.
  • Use surgical instruments to open the left and right hind legs of the mouse, and embed pure iodinated PEAL porous microspheres into the muscle spaces of the left and right hind legs using a syringe. Then suture the left and right hind legs of the mouse.
  • Use CT to check the development of mouse embolization microspheres on the left and right hind legs of mice. The results are shown in Figure 14. From Figure 14, it can be seen that pure iodinated PEAL developed porous microspheres show a clean imaging effect. It has good stability and can be well embolized to the same position without shifting or spreading, indicating that the pure iodinated PEAL imaging porous microspheres have imaging and positioning functions.
  • ketamine hydrochloride (3.5-4 mg/kg) was injected into the rabbit ear vein for intravenous anesthesia, the hind legs were opened, and 2 ligation threads were introduced along the inside of the hind leg arteries, the distal end was ligated, and the proximal end was pulled upward to stop bleeding. Afterwards, cut a small opening in the artery and insert a 24G intravenous indwelling hose into the renal artery. Perform DSA angiography to confirm the development of the renal artery and branches, and then inject 1.5 mL of pure iodinated PEAL imaging porous microspheres through the catheter (Figure 15). Review the angiography.
  • FIG. 16 shows that the pure iodinated PEAL imaging porous microspheres can be stably fixed at the embolization site, with clear imaging and without spreading to other sites.
  • the H&E stained image in Figure 17 shows that pure iodinated PEAL-developed porous microspheres are embolized in the renal artery and cause necrosis of the renal tissue. The above results indicate that the microspheres are embolization microspheres with excellent imaging capabilities.

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Abstract

一种基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球、制备方法和应用。显影多孔微球是对聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸进行显影改造，经微流控法制备得到；所述显影改造为采用硫酸钡进行复合和/或采用碘代分子进行碘代反应；采用硫酸钡进行复合的方式进行显影改造时，得到的显影多孔微球的粒径为1μm-1000μm，采用碘代分子进行碘代反应的方式进行显影改造时，得到的显影多孔微球的孔径为10nm-1000nm。该显影多孔微球可显影、负载药物，载药量高、易于重复、批次稳定。

Description

一种基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球、制备方法和应用 技术领域
本发明属于生物医学技术领域，尤其涉及一种基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球、制备方法和应用。
背景技术
微球是直径在微米范围内(通常为1μm至1000μm)的球形颗粒，或称为微颗粒。根据微球是特征自由流动的粉末状颗粒，根据组分的不同微球可分为蛋白质、合成聚合物、玻璃、陶瓷微球等。其中最常用的材料是聚合物，如聚乳酸、聚(乳酸-乙醇酸)(PLGA)、壳聚糖、聚己内酯(PCL)、二乙烯基苯和其他聚合物或共聚物，这些聚合物在形成微球时配合成孔剂可形成多孔聚合物微球，极大地增加了微球的比表面积，拓展了其在化学、医学、生物医学工程等领域的应用。如药物递送及控制释放、3D细胞培养支架、组织修复辅料、介入显影微球及综合以上功能的多功能微球。
微球的尺寸相较于纳米级颗粒较大，因而在血管栓塞领域得到了广泛的应用，且多孔微球相比于普通微球具有更大的比表面积，是一种更为优良的载体，在药物控制释放、重金属离子的吸附、生物医学工程应用等方面被广泛的研究，具有广阔的应用前景。例如将微球制备成与血管直径相近的尺寸可以有效的引起血管的栓塞，在肿瘤治疗方面能有效抑制肿瘤的生长，延长病人的生存期。
多孔微球可以通过调节粒径和孔隙率减轻微球密度，同时实现药物的控制释放。多孔微球具有高孔隙率、比表面积大的结构特性，在增强吸附性能的同时也使得细胞的粘附和迁移更有利，微球其中及表面的空隙为细胞的生长提供了空间，同时其包载的药物分子更易于释放从而达到控释的目的，有望改善实心微球的药物释放停滞期的缺陷。
在微球的应用中，介入栓塞是微球应用较多的场景之一，介入治疗通常是在电子数字减影血管造影机(DSA)、电子计算机断层扫描(CT)、以及核磁共振等影像设备的辅助和监测下，通过使用穿刺针、导丝导管和其他一些介入治疗器材，通过股动脉穿刺等方法以微创型的伤口将栓塞微球等植入材料导入体内的肿瘤部位，以切断肿瘤的营养供给，饿死肿瘤细胞，从而达到治疗肿瘤的目的。由于其自身具有显影功能，故无需借助其他造影剂的辅助，避免了异位栓塞。
随着介入治疗的发展，介入治疗材料研发与制造发展迅速，这也对介入治疗相关材料提出了更高的要求，例如材料的示踪性以及药物包覆能力等。X射线下具有显影的无机材料为金属盐类造影剂。但这些金属盐类造影剂影响栓塞材料的机械性能，在使用后期，容易扩散至机体其它部位，并对机体产生毒性。另外当前临床使用的栓塞剂一般自身不可显影，需借助造影剂显影，单纯的物理混合会使造影剂与栓塞材料的分离，导致成像模糊和误诊。因此开发一种性能优异的显影多孔微球是非常有必要的。
发明内容
为解决上述技术问题，本发明提供了一种基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球、制备方法和应用。制备的显影多孔微球以聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸(mPEG-PLGA-PLL，简写为PEAL，专利号CN 101732723 A)阳离子聚合物为骨架，PEAL是具有优良生物相容性的高分子材料，可用于水溶性药物及水不溶性药物的包载，聚赖氨酸上的氨基集团带有正电特性，是吸附或包载具有负电性质的药物、核酸、蛋白等物质的理想载体。PEAL经人体分解所产生的组分均为生物相容性分子，特别是PLL降解为赖氨酸是人体必需氨基酸。PEAL为阳离子聚合物，该材料具有良好的生物相容性、药物包载能力，可被修饰而具有多功能特性，如肿瘤靶向、逆转耐药和医学诊断功能等，已被证明可用于负载有机药物、水溶性药物、水不溶性药物或用于诊断用的显影剂。此外，PEAL易溶于二氯甲烷、四氢呋喃等易挥发有 机溶剂，这使得以PEAL为基础的纳米颗粒、微球等在制备时更容易去除残留的有机溶剂。
本发明的第一个目的是提供一种基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球，所述聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的分子量为1.0×103-9.0×106，聚乙二醇单甲醚/乳酸的摩尔比为1-50：50-100，乳酸/羟基乙酸的摩尔比为1-100：1-100，羟基乙酸/赖氨酸的摩尔比为50-100：1-50，所述显影多孔微球是对聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸进行显影改造，经微流控法制备得到；所述显影改造为采用硫酸钡进行复合和/或采用碘代分子进行碘代反应；
采用硫酸钡进行复合的方式进行显影改造时，得到的显影多孔微球的粒径为1μm-1000μm，所述显影多孔微球包括聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸80wt％-99wt％，硫酸钡1wt％-20wt％；
采用碘代分子进行碘代反应的方式进行显影改造时，得到的显影多孔微球的孔径为10nm-1000nm，所述显影多孔微球中聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸和碘代分子的摩尔比为1：1-1：50；所述碘代分子选自2,3,5-三碘苯甲酸(TIBA)、3-氨基-2,4,6-三碘苯甲酸(ATBA)、2,3,5-三碘苯甲基醛(TBA)和2,3,5-三碘苯甲酰氯(TBC)中的一种或多种。
在本发明的一个实施例中，所述硫酸钡又名重晶石，为无臭、无味的白色无定型粉末。性质稳定，难溶于水、酸、碱或有机溶剂。在医学应用方面，放射学利用其吸收X射线特性，主要用作胃肠道内的造影剂。PEAL材料本身不具备显影功能，所制备的微球可通过包载显影剂赋予其显影功能。制备的PEAL/硫酸钡显影多孔微球的显影能力与硫酸钡的比例相关，硫酸钡含量越高，微球的显影能力越强。然而硫酸钡含量过高将使微球的密度变大，从而使其可操作性变差。多孔微球可以在一定程度上减小微球密度，通过生产工艺的调节可以在粒径为1μm-1000μm的微球上产生10nm-1000nm的孔，在具有显影功能的同时还能负载多种组分。PEAL是生物相容性极好的载体材料，且对无机材料具有 一定的物理负载能力，而且不影响其本身的性质，在与硫酸钡结合后仍可对药物、核酸、蛋白、放射性核素等进行包载、吸附。与常见的壳聚糖微球相比，PEAL不需要酸性溶液溶解，只需溶解在有机溶剂中，且耐酸性优于壳聚糖微球。与另一常见的海藻酸钠微球相比，PEAL成球性更好，而海藻酸钠通常需要交联才能成球，即使如此，其成球性仍不佳。因海藻酸钠显示负电性，一般只能包载带正电的物质，这极大限制其在生物医学领域的应用。PEAL易溶于多数有机试剂，其中多数为挥发性试剂，这使得有机试剂的清楚变得简单快捷，不影响材料和药物的理化特性，也避免了因有机试剂清除不彻底带来的生物毒性。
在本发明的一个实施例中，放射学利用碘吸收X射线特性来增强影像观察效果。碘造影剂在X下有更高的密度，增加正常与异常组织间的差异，造影剂充盈的地方就可以充分显影，使医生发现并鉴定一些早期的、小的病变，并区分病变的良恶程度。PEAL材料本身不具备显影功能，所以需要对其进行碘代改性以赋予其显影功能。碘代PEAL显影多孔微球的显影能力与链接的碘原子比例相关，碘含量越高，微球的显影能力越强。但无孔将使微球的密度变大，从而使其可操作性变差。通过改变碘代分子的比例可得到同时含有碘代分子与氨基的碘代PEAL。PEAL可通过共价键接枝含碘化合物使其具有显影功能，之后可以通过改变分子量或者分子链的臂长调节相关性能。PEAL为一种性能良好的生物可降解材料，具有良好的生物相容性。通过化学键合的碘代PEAL显影多孔微球所含碘元素相较于包载或混合方法制备的含碘微球更为稳定，碘原子不易游离出来，相较于碘化油露置空气或日光中会分解析出游离碘的特点，增加了微球的安全性。所选的碘代分子每个分子上均含有三个碘原子，在较少的试剂用量下即可获得显著的显影效果，在含量相同的情况下，四种碘代PEAL显影多孔微球表现出相同的显影效果。此外，通过生产工艺的调节可以在粒径为1μm-1000μm的微球上产生10nm-1000nm的孔。碘代PEAL易溶于多数有机试剂，与壳聚糖相比不使用酸性溶液，其中多数为挥发性试剂，这使得有机试剂的清除变得简单快捷，不影响材料和药物的理化特性，也避免了因有机试剂清 除不彻底带来的生物毒性，且该材料成球不需要交联试剂的加入，成球性好于常见的海藻酸钠。
在本发明的一个实施例中，所述显影多孔微球还包括相对于显影多孔微球的质量分数为0.1wt％-30wt％的药物。
在本发明的一个实施例中，所述药物选自临床常见药、抗肿瘤基因、抗肿瘤药物、分子靶向药物和放射性核素中的一种或多种。
进一步地，所述临床常见药选自利多卡因。
进一步地，所述抗肿瘤基因选自siRNA、microRNA、piRNA、IncRNA、circRNA；
优选地，所述抗肿瘤基因选自下调NF-κBp65基因表达的siRNA、干扰Gab1的siRNA、沉默蛋白激酶Cε基因的siRNA、miRNA-21、miRNA-605、miRNA-376c、miRNA-200b/c和miRNA-101等中的一种或多种。
进一步地，所述抗肿瘤药物选自奥沙利铂、顺铂、卡铂、米铂、洛铂、奈达铂、环铂、紫杉醇、米托蒽醌、阿霉素、表阿霉素、吡喃阿霉素、丝裂霉素、5-氟尿嘧啶、雷替曲塞、多西他赛、吉西他滨、博来霉素、三氧化二砷、亚叶酸钙、脱氧氟尿苷、伊立替康、拓扑替康、羟基喜树碱、依托泊苷、长春瑞宾、长春新碱和甲氨蝶呤等中的一种或多种。
进一步地，所述分子靶向药物选自表皮生长因子受体(EGFR)酪氨酸激酶抑制剂、表皮生长因子受体2(HER-2)抑制剂、免疫检查点抑制剂和血管内皮生长因子抑制剂等中的一种或多种。
优选地，所述免疫检查点抑制剂选自抗PD-1抗体、抗PD-L1抗体和抗CTLA-4抗体等中的一种或多种。
进一步地，所述放射性核素选择90Y、131I、125I、123I、32P、153Sm、186Re、211At、212Bi、166Ho、177Lu、188Re、18F、61Cu、64Cu、89Zr、66Ga、68Ga、44Sc、72As、69Ge、51Mn、52Mn、45Ti、86Y、55Co、111In和225Ac等中的一种或多种。
本发明的第二个目的是提供一种所述的基于聚乙二醇单甲醚-聚乳酸羟基乙 酸-聚赖氨酸的显影多孔微球的制备方法，包括以下步骤，
(1)向浓度为1wt％-40wt％的聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的有机相溶液中加入硫酸钡，得到分散相；所述分散相中硫酸钡的浓度为1wt％-20wt％；
和/或，在有催化剂和无催化剂的反应条件下，聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸和碘代分子反应，纯化后的碘代聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸溶于有机相溶液，得到浓度为1-40wt％的分散相；
浓度为1wt％-6wt％的表面活性剂水溶液作为连续相和收集相；
(2)将S1所述的分散相和连续相分别注入微流控装置的分散相微通道和连续相微通，使分散相在液滴生成管中被连续相剪切，用S1所述的收集相进行收集，得到所述的显影多孔微球。
在本发明的一个实施例中，在步骤(1)中，所述硫酸钡的制备方法具体包括以下步骤，将可水溶性钡盐与可水溶性硫酸盐按摩尔比1-10：1-10混合搅拌，搅拌速率为500rpm-2000rpm，搅拌时间为10min-30min，反应后使用水清洗3次，再使用甲醇清洗3次，离心速率为5000rpm-10000rpm，离心时间为5min-30min。后期制备含硫酸钡的微球时，采用吸附的方式往往容易导致微球的团聚，而本申请采用洗涤方式制备硫酸钡，能增加其在氯仿等有机溶剂中的分散度，基于此可在制备微球时直接将硫酸钡包裹到微球中，无需后续的吸附和水洗。
在本发明的一个实施例中，在步骤(1)中，所述催化剂选自二环己基碳二亚胺(DCC)、4-二甲氨基吡啶(DMAP)、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDC)和N-羟基丁二酰亚胺(NHS)中的一种或多种。PEAL在催化剂作用下将碘代分子(显影小分子)接到聚赖氨酸上，合成了具有X射线自显影能力的碘代PEAL，极大降低了碘造影剂引起的诸多不良反应，并进一步使用微流控法制备了碘代PEAL显影多孔微球。小鼠和兔的动物实验表明碘代PEAL显影多孔微球具有在X射线下的显影能力和较好的栓塞及载药能力，在生物医 学领域能够有更为广泛的应用价值。
在本发明的一个实施例中，在步骤(1)中，所述有机相溶液选自二氯甲烷、三氯甲烷、丙酮、四氢呋喃、石油醚和乙酸乙酯中的一种或多种。
在本发明的一个实施例中，在步骤(1)中，所述纯化的方法选自透析、过滤、水洗、冷冻干燥或重结晶。
在本发明的一个实施例中，在步骤(1)中，所述表面活性剂选自甲基纤维素、卡巴浦尔、藻酸钠、聚乙烯醇、铵盐型、季铵盐型、两性表面活性剂、高级脂肪酸盐、磺酸盐、硫酸酯盐、泊洛沙姆、聚乙二醇、聚乙二醇-聚乳酸、聚乙二醇-聚乳酸-羟基乙酸共聚物、聚乙二醇-聚己内酯和聚乙烯吡咯烷酮中的一种或多种。
在本发明的一个实施例中，在步骤(2)中，所述分散相微通道的内径为20μm-1000μm，所述连续相微通道的内径为40μm-3mm，分散相与连续相的流速比为1：6-1：10。
在本发明的一个实施例中，在步骤(2)中，还包括使用去离子水清洗显影多孔微球数次，冷冻干燥后备用。
在本发明的一个实施例中，在步骤(2)结束后，还包括向显影多孔微球中加入药物的步骤。
本发明的第三个目的是提供一种所述的基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球在制备疾病治疗药物或者治疗疾病的器件中的应用。
在本发明的一个实施例中，所述的基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球在栓塞、载药皮下或者肌肉或者原位疾病治疗、口服和腹腔给药、显影、细胞3D培养等应用。
本发明的技术方案相比现有技术具有以下优点：
(1)本发明所述的基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球粒径均一可控，由于微球具有多孔的结构，保证了在微球制备过程中，硫酸钡能够均匀的分布于微球中。多孔、圆形的结构使其比表面积进一步增大， 这赋予了微球显影、负载药物、栓塞等多重功能，在很大程度上拓展了微球的应用。本发明操作简单、载药量高、易于重复、批次稳定。该微球有望成为药物、基因、蛋白、金属离子、放射性核素的优良载体。有望用于临床栓塞、药物负载、疾病的诊断与治疗等领域。
(2)本发明所述的制备方法与现有技术相比，微流控设备与芯片组的搭建实现了使用微流控技术大批量生产栓塞微球的工艺。
附图说明
为了使本发明的内容更容易被清楚地理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明，其中：
图1为本发明实施例1中的纯PEAL/硫酸钡显影多孔微球的SEM图片；
图2为本发明实施例1中的载90Y的PEAL/硫酸钡显影多孔微球的SEM图片；
图3为本发明实施例1在X射线下载90Y的PEAL/硫酸钡显影多孔微球的离心管的显影图片；
图4为本发明实施例1中将载90Y的PEAL/硫酸钡显影多孔微球注入到老鼠左右后腿中的显影效果图；
图5为本发明实施例1中兔肾动脉栓塞实验DSA图；
图6为本发明实施例1中兔栓塞4周后肾部CT图像；
图7为本发明实施例1中兔栓塞4周后肾部H&E染色组织切片图；
图8为本发明实施例2中的纯碘代PEAL显影多孔微球的SEM及其显影图像；
图9为本发明实施例2中的载90Y的碘代PEAL显影多孔微球的SEM及其显影图像；
图10为本发明实施例2中的载阿霉素的碘代PEAL显影多孔微球的SEM及其显影图像；
图11为本发明实施例2中的载miRNA-376c的碘代PEAL显影多孔微球的 SEM及其显影图像；
图12为本发明实施例2中的载抗PD-L1抗体的碘代PEAL显影多孔微球的光学显微镜图片；
图13为本发明实施例2中的载细胞的碘代PEAL显影多孔微球的SEM图片；
图14为本发明实施例2中将碘代PEAL显影多孔微球注入到老鼠左右后腿中的显影效果图；
图15为本发明实施例2中兔子肾部栓塞实验过程数码照片；
图16为本发明实施例2中兔栓塞4周后兔肾部CT图像；
图17为本发明实施例2中兔栓塞4周后肾部H&E染色组织切片图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
在本发明中，除非另有说明，实施例中采用的miRNA-376c的序列为：AACAUAGAGGAAAUUCCACGU和GGUGGAUAUUCCUUCUAUGUU。
实施例1 PEAL/硫酸钡显影多孔微球
(1)纯PEAL/硫酸钡显影多孔微球的制备
分别配制0.05mol/L的氯化钡溶液与0.05mol/L的硫酸钠水溶液，将等体积的硫酸钠水溶液缓慢滴加到氯化钡水溶液中，并在1500rpm下高速搅拌30min。反应结束后，使用去离子水离心清洗3次，再使用甲醇清洗3次，5000rpm离心30min后备用。
微流控法制备PEAL/硫酸钡显影多孔微球：配制制备微球所需的分散相、连续相、收集相。分散相为含有硫酸钡的PEAL的二氯甲烷溶液，其中PEAL分子量约为1×104g/mol，聚乙二醇单甲醚/乳酸的摩尔比为1：20，乳酸/羟基乙酸的摩尔比为1：4，羟基乙酸/赖氨酸的摩尔比为4：1；PEAL质量分数为10wt％，硫酸钡质量分数为3wt％，连续相和收集相为3wt％聚乙烯醇(PVA)水溶液。 制备过程中，不停搅拌收集相，搅拌速率为60rpm。搭建微流控装置，使用微流控技术制备PEAL/硫酸钡显影多孔微球。微流控管道选择聚四氟乙烯管搭建，两相交汇处及收集处通道内径为500μm，分散相微通道内径为210μm，连续相微通道内径为500μm，使用单个微流控芯片时分散相与连续相流速分别为40μL/min与240μL/min(分散相：连续相＝1：6)，得到粒径为200μm，孔径为100nm-200nm的纯PEAL/硫酸钡显影多孔微球。制备过程结束后，静置48h，收集微球(图1)。使用去离子水清洗3次，冷冻干燥后备用。
(2)载90Y的PEAL/硫酸钡显影多孔微球的制备
分别配制0.05mol/L的氯化钡溶液与0.05mol/L的硫酸钠水溶液，将等体积的硫酸钠水溶液缓慢滴加到氯化钡水溶液中，并在1500rpm下高速搅拌30min。反应结束后，使用去离子水离心清洗3次，再使用甲醇清洗3次，5000rpm离心30min后备用。
微流控法制备PEAL/硫酸钡显影多孔微球：配制制备微球所需的分散相、连续相、收集相。分散相为含有硫酸钡的PEAL的二氯甲烷溶液，其中PEAL分子量约为1×104g/mol，聚乙二醇单甲醚/乳酸的摩尔比为1：20，乳酸/羟基乙酸的摩尔比为1：4，羟基乙酸/赖氨酸的摩尔比为4：1；硫酸钡质量分数为2wt％，PEAL质量分数为15wt％。连续相和收集相为2wt％的PVA水溶液。制备过程中，搅拌收集相，搅拌速率为60rpm。搭建微流控装置，使用微流控技术制备PEAL/硫酸钡显影多孔微球。微流控管道选择聚四氟乙烯管搭建，两相交汇处及收集处通道内径为500μm，分散相微通道内径为210μm，连续相微通道内径为500μm，使用单个微流控芯片时分散相与连续相流速分别为40μL/min与240μL/min(分散相：连续相＝1：6)，得到粒径为200μm，孔径为100nm-500nm，硫酸钡质量分数为11％的PEAL/硫酸钡显影多孔微球。制备过程结束后，静置48h，收集微球(图2)。使用去离子水清洗3次，冷冻干燥后备用，载90Y的PEAL/硫酸钡显影多孔微球具有良好的显影效果，在X光照射下能清晰观察到信号(图3)。
使用时，向3mL的含有1g显影多孔微球的水中加入0.1g的90Y(药物所占 微球比重为10％)。
(3)载阿霉素的PEAL/硫酸钡显影多孔微球的制备
分别配制0.2mol/L的氯化钡溶液与0.2mol/L的硫酸钠溶液，将等体积的硫酸钠溶液缓慢滴加到氯化钡溶液中，并在1200rpm下高速搅拌。反应30min，结束后，使用去离子水离心清洗3次，再使用甲醇清洗3次，5000rpm离心30min后备用。
微流控法制备：配制制备微球所需的分散相、连续相、收集相。分散相为含有硫酸钡的PEAL的二氯甲烷溶液，其中PEAL分子量约为1×105g/mol，聚乙二醇单甲醚/乳酸的摩尔比为1：20，乳酸/羟基乙酸的摩尔比为1：4，羟基乙酸/赖氨酸的摩尔比为4：1；硫酸钡质量分数为3wt％，PEAL质量分数为15wt％。连续相和收集相为1wt％的PVA水溶液。搭建微流控装置，使用微流控技术制备PEAL/硫酸钡显影多孔微球。微流控管道选择聚四氟乙烯材质。两相交汇处及收集处通道内径为400μm，分散相微通道内径为210μm，连续相微通道内径为500μm，使用3个微流控芯片时分散相与连续相流速分别为120μL/min与720μL/min(分散相：连续相＝1：6)，得到粒径为250μm，孔径为200nm-600nm，硫酸钡质量分数为16％的PEAL/硫酸钡显影多孔微球。制备过程结束后，静置48h，收集微球。使用去离子水清洗3次，冷冻干燥后备用。
使用时，向3mL的含有1g显影多孔微球的水中加入0.3g的阿霉素(药物所占微球比重为30％)。
(4)载131I的PEAL/硫酸钡显影多孔微球的制备
分别配制0.3mol/L的氯化钡溶液与0.3mol/L的硫酸钠溶液，将等体积的硫酸钠溶液缓慢滴加到氯化钡溶液中，并在1000rpm下高速搅拌。反应30min，结束后，使用去离子水离心清洗3次，再使用甲醇清洗3次，5000rpm离心30min后备用。
微流控法制备：配制制备微球所需的分散相、连续相、收集相。分散相为含有硫酸钡的PEAL的三氯甲烷溶液，其中PEAL分子量约为1×105g/mol，聚 乙二醇单甲醚/乳酸的摩尔比为1：20，乳酸/羟基乙酸的摩尔比为1：4，羟基乙酸/赖氨酸的摩尔比为4：1；硫酸钡质量分数为3wt％，PEAL质量分数为20wt％。连续相和收集相为4wt％的泊洛沙姆水溶液。搭建微流控装置，使用微流控技术制备PEAL/硫酸钡显影多孔微球。微流控管道选择聚四氟乙烯材质。两相交汇处及收集处通道内径为500μm，分散相微通道内径为210μm，连续相微通道内径为500μm，使用5个微流控芯片时分散相与连续相流速分别为600μL/min与3.6mL/min(分散相：连续相＝1：6)，得到粒径为180μm，孔径为100nm-400nm，硫酸钡质量分数为13％的PEAL/硫酸钡显影多孔微球。制备过程结束后，静置48h，收集微球。使用去离子水清洗3次，冷冻干燥后备用。
使用时，向3mL的含有1g显影多孔微球的水中加入150mg的131I核素(药物所占微球比重为15％)。
(5)载miRNA-376c的PEAL/硫酸钡显影多孔微球的制备
分别配制0.4mol/L的氯化钡溶液与硫酸钠溶液，将等体积的硫酸钠溶液缓慢滴加到氯化钡溶液中，并在1000rpm下高速搅拌。反应30min，结束后，使用去离子水离心清洗3次，再使用甲醇清洗3次，5000rpm离心30min后备用。
微流控法制备：配制制备微球所需的分散相、连续相、收集相。分散相为含有硫酸钡的PEAL的二氯甲烷溶液，其中PEAL分子量约为5×104g/mol，聚乙二醇单甲醚/乳酸的摩尔比为1：20，乳酸/羟基乙酸的摩尔比为1：4，羟基乙酸/赖氨酸的摩尔比为4：1；硫酸钡质量分数为4wt％，PEAL质量分数为25wt％。连续相和收集相为2wt％的PVA水溶液。搭建微流控装置，使用微流控技术制备PEAL/硫酸钡显影多孔微球。微流控管道选择聚四氟乙烯材质。两相交汇处及收集处通道内径为600μm，分散相微通道内径为210μm，连续相微通道内径为500μm，使用5个微流控芯片时分散相与连续相流速分别为600μL/min与3.6mL/min(分散相：连续相＝1：6)，得到粒径为180μm，孔径为200-600nm，硫酸钡质量分数为14％的PEAL/硫酸钡显影多孔微球。制备过程结束后，静置48h，收集微球。使用去离子水清洗3次，冷冻干燥后备用。
使用时，向3mL的含有1g显影多孔微球的水中加入50nmol的miRNA-376c(药物所占微球比重为0.1％)。
(6)载抗PD-L1抗体的PEAL显影多孔微球的制备
分别配制0.4mol/L的氯化钡溶液与硫酸钠溶液，将等体积的硫酸钠溶液缓慢滴加到氯化钡溶液中，并在1500rpm下高速搅拌。反应30min，结束后，使用去离子水离心清洗3次，再使用甲醇清洗3次，5000rpm离心30min后备用。
微流控法制备：配制制备微球所需的分散相、连续相、收集相。分散相为含有硫酸钡的PEAL的二氯甲烷溶液，其中PEAL分子量约为8×104g/mol，聚乙二醇单甲醚/乳酸的摩尔比为1：20，乳酸/羟基乙酸的摩尔比为1：4，羟基乙酸/赖氨酸的摩尔比为4：1；硫酸钡质量分数为7wt％，PEAL质量分数为30wt％。连续相和收集相为2wt％的PVA水溶液。搭建微流控装置，使用微流控技术制备PEAL/硫酸钡显影多孔微球。微流控管道选择聚四氟乙烯材质。两相交汇处及收集处通道内径为600μm，分散相微通道内径为100μm，连续相微通道内径为500μm，使用5个微流控芯片时分散相与连续相流速分别为600μL/min与3.6mL/min(分散相：连续相＝1：6)，得到粒径为100μm，孔径为250nm-600nm，硫酸钡质量分数为18.9％的PEAL/硫酸钡显影多孔微球。制备过程结束后，静置48h，收集微球。使用去离子水清洗3次，冷冻干燥后备用。
使用时，向3mL的含有1g显影多孔微球的水中加入100μg抗PD-L1抗体(药物所占微球比重为0.1％)。
(7)PEAL/硫酸钡显影多孔微球在细胞3D培养中的应用
分别配制0.2mol/L的氯化钡溶液与硫酸钠溶液，将等体积的硫酸钠溶液缓慢滴加到氯化钡溶液中，并在1200rpm下高速搅拌。反应30min，结束后，使用去离子水离心清洗3次，再使用甲醇清洗3次，5000rpm离心30min后备用。
微流控法制备：配制制备微球所需的分散相、连续相、收集相。分散相为含有硫酸钡的PEAL的二氯甲烷溶液，其中PEAL分子量约为1×106g/mol，聚乙二醇单甲醚/乳酸的摩尔比为1：20，乳酸/羟基乙酸的摩尔比为1：4，羟基乙 酸/赖氨酸的摩尔比为4：1；硫酸钡质量分数为5wt％，PEAL质量分数为20wt％。连续相和收集相为3wt％的卡巴浦尔水溶液。搭建微流控装置，使用微流控技术制备PEAL/硫酸钡显影多孔微球。微流控管道选择聚四氟乙烯材质。两相交汇处及收集处通道内径为500μm，分散相微通道内径为210μm，连续相微通道内径为500μm，使用3个微流控芯片时分散相与连续相流速分别为120μL/min与720μL/min(分散相：连续相＝1：6)，得到粒径为200μm，孔径为250nm-550nm，硫酸钡质量分数为20％的PEAL/硫酸钡显影多孔微球。制备过程结束后，静置48h，收集微球。使用去离子水清洗3次，冷冻干燥后备用。
使用时，向含有100mg的PEAL/硫酸钡显影多孔微球的培养基中加入104-105个细胞，培养48h，可以观察到细胞在微球表面生长成团，表明该微球可作为细胞生长基底。
(8)PEAL/硫酸钡显影多孔微球在伤口愈合的应用
分别配制0.05mol/L的氯化钡溶液与硫酸钠溶液，将等体积的硫酸钠溶液缓慢滴加到氯化钡溶液中，并在1500rpm下高速搅拌。反应30min，结束后，使用去离子水离心清洗3次，再使用甲醇清洗3次，5000rpm离心30min后备用。
微流控法制备：配制含有硫酸钡的PEAL的二氯甲烷溶液，其中PEAL分子量约为9×105g/mol，聚乙二醇单甲醚/乳酸的摩尔比为1：20，乳酸/羟基乙酸的摩尔比为1：4，羟基乙酸/赖氨酸的摩尔比为4：1；硫酸钡质量分数为1wt％，PEAL质量分数为15wt％。连续相和收集相为3wt％的PVA水溶液。制备过程中，搅拌收集相，搅拌速率为60rpm。搭建微流控装置，使用微流控技术制备PEAL/硫酸钡显影多孔微球。微流控管道选择聚四氟乙烯管搭建，两相交汇处及收集处通道内径为500μm，分散相微通道内径为100μm，连续相微通道内径为500μm，使用单个微流控芯片时分散相与连续相流速分别为40μL/min与240μL/min(分散相：连续相＝1：6)，得到粒径为100μm，孔径为100nm-500nm，硫酸钡质量分数为6.25％的PEAL/硫酸钡显影多孔微球。制备过程结束后，静置48h，收集微球。使用去离子水清洗3次，冷冻干燥后备用。
使用该微球时，向伤口上涂覆载有0.2mg利多卡因的100mg PEAL/硫酸钡显影多孔微球(药物所占微球比重约为0.2％)。行肿瘤切除术的伤口将给患者带了疼痛感，载有利多卡因的微球在伤口缝合界面缓慢释药，镇痛的同时微球作为细胞生长基底更有利于细胞的粘附生长，促进伤口愈合，利多卡因具有抑制在细胞生长的作用，同时到达了防止肿瘤复发的目的。
(9)小鼠体内栓塞显影多孔微球成像实验
常规准备下，在小鼠腹腔注射戊巴比妥钠溶液，麻醉小鼠。使用手术器械，开小鼠左后腿部位，将纯PEAL/硫酸钡显影多孔微球使用注射器包埋到左后腿肌肉间隙中。然后缝合小鼠左后腿。使用CT查看小鼠显影栓塞微球在小鼠左后腿部位的显影情况，结果如图4所示。从图4可以看出，纯PEAL/硫酸钡显影多孔微球表现出良好的显影效果，在注射部位停留时间长，未发生异位栓塞的现象，表明该微球具有良好的稳定性以及显影效果。从图5的肾栓塞实验可以看出，纯PEAL/硫酸钡显影多孔微球成功地阻断了肾动脉的营养供给，肾表现出坏死的情况，表明其可作为栓塞剂使用。
(10)Y3+吸附及洗脱实验
酸性条件下的吸附实验：称取0.852g六水氯化钇溶于250mL 0.001mol/L盐酸中，配制Y3+储备液。采用ICP-OES分析测得储备液中Y3+浓度。移取3mL吸附原液至离心管中，后加入0.1g载90Y的PEAL/硫酸钡显影多孔微球，漩涡振荡混匀后置于摇床，水平振荡吸附1h。后离心分离，移出上清液，然后再在离心管中加入5mL去离子水，水平振荡1h，离心分离，移出上清液。将两次上清液经2％硝酸稀释后，利用ICP-MS分析其中Y3+浓度。
中性条件下吸附实验：称取0.852g六水氯化钇溶于250mL去离子水中，配制Y3+储备液。采用ICP-OES分析测得储备液中Y3+浓度。移取3mL吸附原液至离心管中，后加入0.1g纯PEAL/硫酸钡显影多孔微球，漩涡振荡混匀后置于摇床，水平振荡吸附1h。后离心分离，移出上清液，然后再在离心管中加入5mL去离子水，水平振荡1h，离心分离，移出上清液。将两次上清液经2％硝酸稀释 后，利用ICP-MS分析其中Y3+浓度。
洗脱实验步骤：在吸附完的样品中加入5mL生理盐水，漩涡振荡混匀后，静置120h。离心后移出上清液，经2％硝酸稀释后，利用ICP-MS分析其中Y3+浓度。
表1所示为最终测得的相关数据：
表1 PEAL/硫酸钡显影多孔微球对抗肿瘤药物90Y的吸附与洗脱实验数据。
从表1可以看出，PEAL/硫酸钡显影多孔微球在酸性和中性条件下对90Y均具有较好的吸附效果，吸附率分别达到了92％和95％，且吸附牢固，不易游离出来，二者的洗脱率仅为2.4％和2.8％。这些结果表明，吸附90Y的PEAL/硫酸钡显影多孔微球具有很好的稳定性，能很好地控制90Y的分布，减少了潜在的毒性问题。
(11)健康试验兔肾动脉栓塞
常规准备下，兔耳缘静脉注射盐酸氯胺酮(3.5mg/kg)行静脉麻醉后，开后腿，沿后腿动脉内侧引入2根套扎线，远端结扎，近端向上牵拉止血后，将动脉剪一小口后插入24G静脉留置软管进入肾动脉内，行DSA造影确定肾动脉及分支显影后再经导管注射纯PEAL/硫酸钡显影多孔微球1.5mL，复查造影见肾动脉段以下细小分支完全性栓塞后，拔管、包扎缝合伤口，结束手术。术后4周分别进行CT复查，如图6所示可以看出栓塞微球仍存在于肾动脉处，保持较好的显影功能，微球未见扩散。术后4周取肾部，HE染色观察，如图7所示，肾组织出现明显的坏死，表明纯PEAL/硫酸钡显影多孔微球成功对肾动脉进行了栓塞。
实施例2碘代PEAL显影多孔微球
(1)纯碘代PEAL显影多孔微球的制备
制备PEAL：TIBA＝1：10(摩尔比)的碘代PEAL材料，其中PEAL分子 量约为1×104g/mol，聚乙二醇单甲醚/乳酸的摩尔比为1：20，乳酸/羟基乙酸的摩尔比为1：4，羟基乙酸/赖氨酸的摩尔比为4：1；称取0.3g的PEAL，0.02g EDC及0.012g NHS，加入到100mL烧瓶中，在氮气保护下抽排3次后，加入烧瓶中，将0.05g的2,3,5-三碘苯甲酸(TIBA)溶解二氯甲烷中，在氮气抽排3次后反应12h。旋蒸除去二氯甲烷，之后加3mL DMF溶解，转移到透析袋透析48h，冷冻干燥后备用。
微流控法制备碘代PEAL显影多孔微球：配制制备微球所需的分散相、连续相、收集相。分散相为10wt％的碘代PEAL的二氯甲烷溶液，连续相和收集相为3wt％的PVA水溶液。制备过程中，不停搅拌收集相，搅拌速率为60rpm/min。搭建微流控装置，使用微流控技术制备碘代PEAL显影多孔微球。微流控管道选择聚四氟乙烯管搭建，两相交汇处及收集处通道内径为500μm，分散相微通道内径为150μm，连续相微通道内径为500μm，使用单个微流控芯片时分散相与连续相流速分别为40μL/min与240μL/min(分散相：连续相＝1：6)，得到粒径为180μm，孔径为100nm-300nm的纯碘代PEAL显影多孔微球。制备过程结束后，静置48h，收集微球(图8)，可以看出所得微球为多孔微球。使用去离子水清洗3次，冷冻干燥后备用。
(2)载90Y的碘代PEAL显影多孔微球的制备
制备PEAL：TIBA＝1：1(摩尔比)的碘代PEAL材料，其中PEAL分子量约为2×105g/mol，聚乙二醇单甲醚/乳酸的摩尔比为1：20，乳酸/羟基乙酸的摩尔比为1：4，羟基乙酸/赖氨酸的摩尔比为4：1；称取0.3g的PEAL，0.002g EDC及0.0012gNHS，加入到100mL烧瓶中，在氮气保护下抽排3次后，加入烧瓶中，将0.005g的2,3,5-三碘苯甲酸(TIBA)溶解二氯甲烷中，在氮气抽排3次后反应12h。旋蒸除去二氯甲烷，之后加3mL DMF溶解，转移到透析袋透析48h，冻干后备用。
微流控制备碘代PEAL显影载药微球：配制制备微球所需的分散相、连续相、收集相。分散相为10wt％的碘代PEAL的二氯甲烷溶液，连续相和收集相 为2wt％的PVA水溶液。搭建微流控装置，使用微流控技术制备碘代PEAL显影多孔微球。微流控管道选择聚四氟乙烯管搭建，两相交汇处及收集处通道内径为500μm，分散相微通道内径为200μm，连续相微通道内径为500μm，使用单个微流控芯片时分散相与连续相流速分别为40μL/min与240μL/min(分散相：连续相＝1：6)，得到粒径为210μm，孔径为50nm-500nm的碘代PEAL显影多孔微球。制备过程结束后，静置48h，收集微球(图9)，得到具有较大孔径的微球。使用去离子水清洗3次，冷冻干燥后备用。
使用时向3mL含有1g的显影多孔微球的水中加入0.3g的90Y后使用(药物所占微球比重为30％)。
(3)载阿霉素的碘代PEAL显影多孔微球的制备
制备PEAL：TIBA＝1：5(摩尔比)的碘代PEAL材料，其中PEAL分子量约为1×105g/mol，聚乙二醇单甲醚/乳酸的摩尔比为1：20，乳酸/羟基乙酸的摩尔比为1：4，羟基乙酸/赖氨酸的摩尔比为4：1；称取0.3g的PEAL，0.01gEDC及0.006gNHS，加入到100mL烧瓶中，在氮气保护下抽排3次后，加入烧瓶中，将0.025g的2,3,5-三碘苯甲酸(TIBA)溶解二氯甲烷中，在氮气抽排3次后反应12h。旋蒸除去二氯甲烷，之后加3mL DMF溶解，转移到透析袋透析48h，冻干后备用。
微流控制备碘代PEAL显影载药微球：配制制备微球所需的分散相、连续相、收集相。分散相为15wt％的碘代PEAL的二氯甲烷溶液，连续相和收集相为1wt％的PVA水溶液。搭建微流控装置，使用微流控技术制备碘代PEAL显影多孔微球。微流控管道选择聚四氟乙烯管搭建，两相交汇处及收集处通道内径为400μm，分散相微通道内径为150μm，连续相微通道内径为400μm，使用单个微流控芯片时分散相与连续相流速分别为50μL/min与350μL/min(分散相：连续相＝1：7)，得到粒径为180μm，孔径为50nm-500nm的碘代PEAL显影多孔微球。制备过程结束后，静置48h，收集微球(图10)，可以看出所得微球为多孔微球。使用去离子水清洗3次，冷冻干燥后备用。
使用时，配制成浓度为0.5g/mL的微球悬液，并向2mL的含有显影多孔微球的水中加入0.2g的阿霉素后使用(药物所占微球比重为20％)。
(4)载miRNA-376c的碘代PEAL显影多孔微球的制备
制备PEAL：TIBA＝1：10(摩尔比)的碘代PEAL材料，其中PEAL分子量约为3×105g/mol，聚乙二醇单甲醚/乳酸的摩尔比为1：20，乳酸/羟基乙酸的摩尔比为1：4，羟基乙酸/赖氨酸的摩尔比为4：1；称取0.3g的PEAL，0.02gEDC及0.012gNHS，加入到100mL烧瓶中，在氮气保护下抽排3次后，加入烧瓶中，将0.05g的2,3,5-三碘苯甲酸(TIBA)溶解二氯甲烷中，在氮气抽排3次后反应12h。旋蒸除去二氯甲烷，之后加3mL DMF溶解，转移到透析袋透析48h，冻干后备用。
微流控制备碘代PEAL显影载药微球：配制制备微球所需的分散相、连续相、收集相。分散相为20wt％的碘代PEAL的二氯甲烷溶液，连续相和收集相为4wt％的泊洛沙姆水溶液。搭建微流控装置，使用微流控技术制备碘代PEAL显影多孔微球。微流控管道选择聚四氟乙烯管搭建，两相交汇处及收集处通道内径为600μm，分散相微通道内径为100μm，连续相微通道内径为600μm，使用单个微流控芯片时分散相与连续相流速分别为60μL/min与360μL/min(分散相：连续相＝1：6)，得到粒径为150μm，孔径为50nm-400nm的碘代PEAL显影多孔微球。制备过程结束后，静置48h，收集微球(图11)。使用去离子水清洗3次，冷冻干燥后备用。
使用时，配制成浓度为0.5g/mL的微球悬液，向3mL的含有显影多孔微球的水中加入50nmol的miRNA-376c后使用(药物所占微球比重为0.1％)。
(5)载抗PD-L1抗体的碘代PEAL显影多孔微球的制备
制备PEAL：TIBA＝1：50(摩尔比)的碘代PEAL材料，其中PEAL分子量约为3×106g/mol，聚乙二醇单甲醚/乳酸的摩尔比为1：20，乳酸/羟基乙酸的摩尔比为1：4，羟基乙酸/赖氨酸的摩尔比为4：1；称取0.3g的PEAL，0.04gEDC及0.023gNHS，加入到100mL烧瓶中，在氮气保护下抽排3次后，加入烧瓶中， 将0.25g的2,3,5-三碘苯甲酸(TIBA)溶解二氯甲烷中，在氮气抽排3次后反应12h。旋蒸除去二氯甲烷，之后加3mL DMF溶解，转移到透析袋透析48h，冻干后备用。
微流控制备碘代PEAL显影载药微球：配制制备微球所需的分散相、连续相、收集相。分散相为20wt％的碘代PEAL的二氯甲烷溶液，连续相和收集相为3wt％的PVA水溶液。搭建微流控装置，使用微流控技术制备碘代PEAL显影多孔微球。微流控管道选择聚四氟乙烯管搭建，两相交汇处及收集处通道内径为500μm，分散相微通道内径为150μm，连续相微通道内径为500μm，使用单个微流控芯片时分散相与连续相流速分别为70μL/min与560μL/min(分散相：连续相＝1：8)，得到粒径为150μm，孔径为80nm-450nm的碘代PEAL显影多孔微球。制备过程结束后，静置48h，收集微球(图12)。使用去离子水清洗3次，冷冻干燥后备用。
使用时，配制成浓度为0.5g/mL的微球悬液，并向2mL的含有显影多孔微球的水中加入100μg抗PD-L1抗体后使用(药物所占微球比重为0.1％)。
(6)载细胞的碘代PEAL显影多孔微球的制备
制备PEAL：TIBA＝1：1(摩尔比)的碘代PEAL材料，其中分子量约为3×106g/mol，聚乙二醇单甲醚/乳酸的摩尔比为1：20，乳酸/羟基乙酸的摩尔比为1：4，羟基乙酸/赖氨酸的摩尔比为4：1；称取0.3g的PEAL，0.002gEDC及0.0012gNHS，加入到100mL烧瓶中，在氮气保护下抽排3次后，加入烧瓶中，将0.005g的2,3,5-三碘苯甲酸(TIBA)溶解二氯甲烷中，在氮气抽排3次后反应12h。旋蒸除去二氯甲烷，之后加3mL DMF溶解，转移到透析袋透析48h，冻干后备用。
微流控制备碘代PEAL显影载药微球：配制制备微球所需的分散相、连续相、收集相。分散相为15wt％的碘代PEAL的二氯甲烷溶液，连续相和收集相为2wt％的卡巴浦尔水溶液。搭建微流控装置，使用微流控技术制备碘代PEAL显影多孔微球。微流控管道选择聚四氟乙烯管搭建，两相交汇处及收集处通道 内径为500μm，分散相微通道内径为150μm，连续相微通道内径为500μm，使用单个微流控芯片时分散相与连续相流速分别为50μL/min与500μL/min(分散相：连续相＝1：10)，得到粒径为150μm，孔径为80nm-400nm的碘代PEAL显影多孔微球。制备过程结束后，静置48h，收集微球(图13)。使用去离子水清洗3次，冷冻干燥后备用。
使用时，配制成浓度为0.5g/mL的微球悬液，并向2mL的含有显影多孔微球的水中加入104-105个细胞，培养48h后使用。
(7)载利多卡因的碘代PEAL显影多孔微球的制备
制备PEAL：TIBA＝1：5(摩尔比)的碘代PEAL材料其中PEAL分子量约为3×106g/mol，聚乙二醇单甲醚/乳酸的摩尔比为1：20，乳酸/羟基乙酸的摩尔比为1：4，羟基乙酸/赖氨酸的摩尔比为4：1；称取0.3g的PEAL，0.01g EDC及0.006gNHS，加入到100mL烧瓶中，在氮气保护下抽排3次后，加入烧瓶中，将0.025g的2,3,5-三碘苯甲酸(TIBA)溶解二氯甲烷中，在氮气抽排3次后反应12h。旋蒸除去二氯甲烷，之后加3mL DMF溶解，转移到透析袋透析48h，冻干后备用。
微流控制备碘代PEAL显影载药微球：配制制备微球所需的分散相、连续相、收集相。分散相为20wt％的碘代PEAL的二氯甲烷溶液，连续相和收集相为1wt％的PVA水溶液。搭建微流控装置，使用微流控技术制备碘代PEAL显影多孔微球。微流控管道选择聚四氟乙烯管搭建，两相交汇处及收集处通道内径为500μm，分散相微通道内径为210μm，连续相微通道内径为500μm，使用单个微流控芯片时分散相与连续相流速分别为60μL/min与480μL/min(分散相：连续相＝1：8)，得到粒径为200μm，孔径为50nm-450nm的碘代PEAL显影多孔微球。制备过程结束后，静置48h，收集微球。使用去离子水清洗3次，冷冻干燥后备用。
使用时，配制成浓度为0.5g/mL的微球悬液，向伤口上涂覆2mL载有利多卡因的200mg显影多孔微球(药物所占微球比重为20％)。
(8)小鼠体内栓塞显影多孔微球成像实验
常规准备下，在小鼠腹腔注射戊巴比妥钠溶液，麻醉小鼠。使用手术器械，开小鼠左右后腿部位，将纯碘代PEAL显影多孔微球使用注射器包埋到左右后腿肌肉间隙中。然后缝合小鼠左右后腿。使用CT查看小鼠显影栓塞微球在小鼠左右后腿部位的显影情况，结果如图14所示，从图14可以看出，纯碘代PEAL显影多孔微球表现出清洗的成像效果，稳定性好，能较好地栓塞到同一位置，不发生移位、散开等现象，说明该纯碘代PEAL显影多孔微球具有成像和定位的功能。
(9)健康试验兔肾动脉栓塞
常规准备下，兔耳缘静脉注射注射盐酸氯胺酮(3.5-4mg/kg)行静脉麻醉后，开后腿，沿后腿动脉内侧引入2根套扎线，远端结扎，近端向上牵拉止血后，将动脉剪一小口后插入24G静脉留置软管进入肾动脉内，行DSA造影确定肾动脉及分支显影后再经导管注射纯碘代PEAL显影多孔微球1.5mL(图15)，复查造影见肾动脉段以下细小分支完全性栓塞后，拔管、包扎缝合伤口，结束手术。术后4周再次成像(图16)并取肾部H&E染色(图17)观察。图16表明该纯碘代PEAL显影多孔微球可稳定地固定于栓塞部位，成像清晰，未扩散到其他部位。图17的H&E染色图像显示纯碘代PEAL显影多孔微球栓塞在肾动脉内并导致肾组织的坏死。以上结果表明该微球是具有出色成像功能的栓塞微球。
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (10)

  1. 一种基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球，所述聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的分子量为1.0×103-9.0×106，聚乙二醇单甲醚/乳酸的摩尔比为1-50：50-100，乳酸/羟基乙酸的摩尔比为1-100：1-100，羟基乙酸/赖氨酸的摩尔比为50-100：1-50，其特征在于，所述显影多孔微球是对聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸进行显影改造，经微流控法制备得到；所述显影改造为采用硫酸钡进行复合和/或采用碘代分子进行碘代反应；
    采用硫酸钡进行复合的方式进行显影改造时，得到的显影多孔微球的粒径为1μm-1000μm，所述显影多孔微球包括聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸80wt％-99wt％，硫酸钡1wt％-20wt％；
    采用碘代分子进行碘代反应的方式进行显影改造时，得到的显影多孔微球的孔径为10nm-1000nm，所述显影多孔微球中聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸和碘代分子的摩尔比为1：1-1：50；所述碘代分子选自2,3,5-三碘苯甲酸、3-氨基-2,4,6-三碘苯甲酸、2,3,5-三碘苯甲基醛和2,3,5-三碘苯甲酰氯中的一种或多种。
  2. 根据权利要求1所述的基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球，其特征在于，所述显影多孔微球还包括相对于显影多孔微球的质量分数为0.1wt％-30wt％的药物。
  3. 根据权利要求2所述的基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球，其特征在于，所述药物选自临床常见药、抗肿瘤基因、抗肿瘤药物、分子靶向药物和放射性核素中的一种或多种。
  4. 权利要求1-3任一项所述的基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球的制备方法，其特征在于，包括以下步骤，
    (1)向浓度为1wt％-40wt％的聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的 有机相溶液中加入硫酸钡，得到分散相；所述分散相中硫酸钡的浓度为1wt％-20wt％；
    和/或，在有催化剂和无催化剂的反应条件下，聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸和碘代分子反应，纯化后的碘代聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸溶于有机相溶液，得到浓度为1-40wt％的分散相；
    浓度为1wt％-6wt％的表面活性剂水溶液作为连续相和收集相；
    (2)将S1所述的分散相和连续相分别注入微流控装置的分散相微通道和连续相微通，使分散相在液滴生成管中被连续相剪切，用S1所述的收集相进行收集，得到所述的显影多孔微球。
  5. 根据权利要求4所述的基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球的制备方法，其特征在于，在步骤(1)中，所述催化剂选自二环己基碳二亚胺、4-二甲氨基吡啶、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺和N-羟基丁二酰亚胺中的一种或多种。
  6. 根据权利要求4所述的基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球的制备方法，其特征在于，在步骤(1)中，所述有机相溶液选自二氯甲烷、三氯甲烷、丙酮、四氢呋喃、石油醚和乙酸乙酯中的一种或多种。
  7. 根据权利要求4所述的基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球的制备方法，其特征在于，在步骤(1)中，所述表面活性剂选自甲基纤维素、卡巴浦尔、藻酸钠、聚乙烯醇、铵盐型、季铵盐型、两性表面活性剂、高级脂肪酸盐、磺酸盐、硫酸酯盐、泊洛沙姆、聚乙二醇、聚乙二醇-聚乳酸、聚乙二醇-聚乳酸-羟基乙酸共聚物、聚乙二醇-聚己内酯和聚乙烯吡咯烷酮中的一种或多种。
  8. 根据权利要求4所述的基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球的制备方法，其特征在于，在步骤(2)中，所述分散相微通道的内径为20μm-1000μm，所述连续相微通道的内径为40μm-3mm，分散相与连续相的流速比为1：6-1：10。
  9. 根据权利要求4所述的基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球的制备方法，其特征在于，在步骤(2)结束后，还包括向显影多孔微球中加入药物的步骤。
  10. 权利要求1-3任一项所述的基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球在制备疾病治疗药物或者治疗疾病的器件中的应用。
PCT/CN2023/107488 2022-07-22 2023-07-14 一种基于聚乙二醇单甲醚-聚乳酸羟基乙酸-聚赖氨酸的显影多孔微球、制备方法和应用 WO2024017165A1 (zh)

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