CN114469875A - 一种冷冻多孔载药载细胞微球的制备方法及其在制备治疗急性肝衰竭药物中的应用 - Google Patents

一种冷冻多孔载药载细胞微球的制备方法及其在制备治疗急性肝衰竭药物中的应用 Download PDF

Info

Publication number
CN114469875A
CN114469875A CN202210218796.2A CN202210218796A CN114469875A CN 114469875 A CN114469875 A CN 114469875A CN 202210218796 A CN202210218796 A CN 202210218796A CN 114469875 A CN114469875 A CN 114469875A
Authority
CN
China
Prior art keywords
loaded
porous drug
cell
preparation
frozen porous
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210218796.2A
Other languages
English (en)
Inventor
赵远锦
王经琳
任昊桢
商珞然
张大淦
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing Drum Tower Hospital
Original Assignee
Nanjing Drum Tower Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanjing Drum Tower Hospital filed Critical Nanjing Drum Tower Hospital
Priority to CN202210218796.2A priority Critical patent/CN114469875A/zh
Publication of CN114469875A publication Critical patent/CN114469875A/zh
Pending legal-status Critical Current

Links

Images

Classifications

    • 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
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1635—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/739—Lipopolysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0042—Photocleavage of drugs in vivo, e.g. cleavage of photolabile linkers in vivo by UV radiation for releasing the pharmacologically-active agent from the administered agent; photothrombosis or photoocclusion
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
    • 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
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1617—Organic compounds, e.g. phospholipids, fats
    • 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
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1658—Proteins, e.g. albumin, gelatin
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/08—Carbon ; Graphite
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/16—Macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/222—Gelatin
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/23—Carbohydrates
    • A61L2300/232—Monosaccharides, disaccharides, polysaccharides, lipopolysaccharides
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/30—Compounds of undetermined constitution extracted from natural sources, e.g. Aloe Vera
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/41—Anti-inflammatory agents, e.g. NSAIDs
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/62—Encapsulated active agents, e.g. emulsified droplets
    • A61L2300/622—Microcapsules

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Dermatology (AREA)
  • Transplantation (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Virology (AREA)
  • Botany (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Inorganic Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Medicinal Preparation (AREA)

Abstract

本发明提供一种冷冻多孔载药载细胞微球的制备方法，包括以下步骤：S1、将脂多糖、光热响应性材料以及水凝胶材料添加到去离子水中，配置得到混合水凝胶溶液；以混合水凝胶溶液为内相，硅油为外相，通过微流控装置制备得到油包水型溶液；S2、将S1制备得到的油包水型溶液置于‑20℃环境中进行预冷冻，再置于液氮中进行冷冻处理，然后使用紫外线照射固化，使用乙醇清洗后得到光热响应性冷冻多孔载药微球；S3、将S2得到的光热响应性冷冻多孔载药微球浸泡在含有间充质干细胞的培养基中，得到具有光热响应的冷冻多孔载药载细胞微球。

Description

一种冷冻多孔载药载细胞微球的制备方法及其在制备治疗急 性肝衰竭药物中的应用
技术领域
本发明涉及生物材料领域，具体涉及一种冷冻多孔载药载细胞微球的制备方法及其在制备治疗急性肝衰竭药物中的应用。
背景技术
急性肝衰竭(ALF)是一种由药物毒性或病毒性肝炎引起的高发病率和死亡率的临床综合征。各种治疗策略已被用于治疗ALF，其中间充质干细胞(MSCs)移植显示出良好的疗效。MSCs因其来源广泛，容易获得，具有免疫调节作用，并对肝脏修复有促进作用在再生医学领域被广泛研究。然而，由于移植后的宿主免疫细胞攻击和异物排斥，传统的细胞植入往往具有细胞存活率低和定植效果差的特点。为了防止植入的细胞受到免疫系统的攻击，并使细胞直接定位到目标区域，人们开始用各种凝胶和微载体来封装细胞。
微载体释放系统的应用解决了间充质干细胞的传输问题，但这些传输系统大多仍具有不稳定的化学特性、较差的生物相容性和潜在的细胞毒性，这极大地阻碍了细胞的生存能力，限制了治疗效果。此外，由于未经训练的MSCs很难保持抗炎和免疫调节能力，封装的MSCs严重影响了应用效果。因此，改进间充质干细胞输送系统，使其具有长期的生物功能，低免疫反应和高生物相容性，仍有望用于ALF治疗。基于上述几点，亟需一种冷冻多孔载药载细胞微球，用于制备治疗急性肝衰竭的药物。
发明内容
本发明针对现有技术中的不足，提供一种冷冻多孔载药载细胞微球的制备方法。
为实现上述目的，本发明采用以下技术方案：
一种冷冻多孔载药载细胞微球的制备方法，包括以下步骤：
S1、将脂多糖(LPS)、光热响应性材料以及水凝胶材料添加到去离子水中，配置得到混合水凝胶溶液；以混合水凝胶溶液为内相，硅油为外相，通过微流控装置制备得到油包水型溶液；使用微流控装置可以获得形态大小均一的油包水型溶液，进而促使后续制备得到的冷冻多孔载药微球形态大小均一；
S2、将S1制备得到的油包水型溶液置于-20℃环境中进行预冷冻，再置于液氮中进行冷冻处理，然后使用紫外线照射固化，使用乙醇清洗后得到光热响应性冷冻多孔载药微球；使用梯度冷冻技术有利于使获得的冷冻多孔载药微球具有多孔结构；多孔结构的存在可以使冷冻多孔载药微球在后续装载药物的过程中吸附更多的细胞并在定植后促进细胞的粘附；
S3、将S2得到的光热响应性冷冻多孔载药微球浸泡在含有间充质干细胞(MSCs)的培养基中，得到具有光热响应的冷冻多孔载药载细胞微球。
冷冻多孔载药载细胞微球在微球上实现了载药和载细胞的结合，冷冻多孔载药载细胞微球收缩时LPS的释放可激活MSCs，促进MSCs在肝脏的定植并可以避免免疫系统的攻击，相较于传统的单纯载药或载细胞方式，定植效果更好。
为优化上述技术方案，采取的具体措施还包括：
进一步地，步骤S1中，脂多糖的添加量为1mg/mL。
进一步地，步骤S1中，所述光热响应性材料为具有将光能转化为热能特性的氧化石墨烯(GO)和具有良好热敏性的聚(N-异丙基丙烯酰胺)(PNIPAM)。
进一步地，所述氧化石墨烯的添加量为0.2wt％，聚(N-异丙基丙烯酰胺)的添加量为10wt％。
进一步地，步骤S1中，所述水凝胶材料为对细胞粘附和定植具有良好的生物相容性的甲基丙烯酸明胶(GelMA)、甲叉丙烯酰胺(BIS)和α-羟基异丁酰苯(HMPP)。
进一步地，所述甲基丙烯酸明胶的添加量为2.5wt％，甲叉丙烯酰胺的添加量为0.34wt％，α-羟基异丁酰苯的浓度为1v/v％。
进一步地，步骤S2中，所述预冷冻的时间为0-60分钟，通过控制冷冻时间可以获得不同孔径大小的冷冻多孔载药微球。
进一步地，步骤S3中，所述含有间充质干细胞的培养基的浓度为1g/L。
一种如上述任一项制备方法所制备的冷冻多孔载药载细胞微球用于制备治疗急性肝衰竭的药物；具体的，结合3D打印技术，将冷冻多孔载药载细胞微球打印成肝脏补片，将该肝脏补片移植到大鼠的肝脏区域以治疗急性肝衰竭大鼠。
本发明的有益效果是：
(1)本发明以脂多糖(LPS)、光热响应性材料以及水凝胶材料配置得到的混合水凝胶溶液为内相，以硅油为外相，通过微流控装置制备得到形态大小均一的油包水型溶液，通过冷冻处理和固化得到光热响应性冷冻多孔载药微球；使用的光热响应性材料为可以将光能转化为热能特性的氧化石墨烯(GO)和具有良好热敏性的聚(N-异丙基丙烯酰胺)(PNIPAM)；使用的水凝胶材料为甲基丙烯酸明胶(GelMA)、甲叉丙烯酰胺(BIS)和α-羟基异丁酰苯(HMPP)，对细胞粘附和定植具有良好的生物相容性；使用微流控技术可以使获得的冷冻多孔载药微球形态大小均一；使用梯度冷冻技术并控制冷冻时间便于获得孔径大小不同的冷冻多孔载药微球，保证药物和细胞的粘附；进一步将光热响应性冷冻多孔载药微球浸泡在含有间充质干细胞(MSCs)的培养基中，得到具有光热响应的冷冻多孔载药载细胞微球；该冷冻多孔载药载细胞微球在近红外照射下可释放其内部的LPS，促进MSCs在肝脏的定植并且可以激活MSCs获得免疫记忆效应以避免免疫系统的攻击；在组织炎症发生时，LPS激活的MSCs能以更持久和更有效的方式作出反应。
(2)本发明使用的水凝胶材料具有良好的生物相容性和稳定的化学特性，且不具有细胞毒性，极大的提高了细胞的生存能力以及治疗效果；与现有材料相比，本发明制备的冷冻多孔载药载细胞微球具有更快的响应速度，并且相应变化特征明显。
(3)本发明提供了一种冷冻多孔载药载细胞微球的制备方法，具有成本低廉，操作简单，尺寸可控，可大规模量产的优势。
附图说明
图1为微流控装置制备出的冷冻多孔载药载细胞微球图；
图2为实施例2中的药物释放图；
图3为冷冻多孔载药载细胞微球的电镜图；
图4为实施例4中的大鼠活体成像图；
图5为冷冻多孔载药载细胞微球治疗急性肝衰竭大鼠后大鼠生存情况以及肝脏炎症的评估图。
具体实施方式
实施例1：冷冻多孔载药微球的制备
将1mg/mL的脂多糖(LPS)、0.2wt％的氧化石墨烯(GO)、10wt％的聚(N-异丙基丙烯酰胺)(PNIPAM)、2.5wt％的甲基丙烯酸明胶(GelMA)、0.34wt％的甲叉丙烯酰胺(BIS)以及1v/v％的α-羟基异丁酰苯(HMPP)的混合水溶液作为内相，使用硅油作为外相，内相和外相液体分别通过内、外毛细管流入微流控装置；在剪切力和表面张力的作用下，内相液体被夹断成单分散的液滴，制备得到油包水型溶液。
将获得的油包水型溶液置于-20℃环境中冷冻一定时间(0、30、60分钟)后，转移至-196℃(液氮)环境中。通过紫外线照射使油包水型溶液固化后，用乙醇抽滤清洗，得到光热响应性冷冻多孔载药微球，如图1所示。
实施例2：药物的释放
对实施例1中制备得到的光热响应性冷冻多孔载药微球进行近红外照射，测试每10分钟由近红外照射1分钟触发的释放特性。累积释放量由LPS ELISA试剂盒检测，测试结果如图2所示。
实施例3：冷冻多孔载药微球装载细胞
对冷冻多孔载药微球紫外线照射消毒24小时后，使用PBS清洗3次，然后将冷冻多孔载药微球浸泡在含有4x106个MSCs的DMEM(含10％血清)培养基中。每天更换DMEM培养基，培养3天后制备得到冷冻多孔载药载细胞微球，孔径大小在7-20μm，如图3所示。
实施例4：冷冻多孔载药载细胞微球在制备治疗急性肝衰竭药物中的应用：
对4-6周龄的SD大鼠进行腹腔注射D-氨基半乳糖(D-Gal)以诱导急性肝衰竭大鼠模型(D-Gal的剂量为0.6g/只)。使用基质胶(Matrigel)作为生物墨水，构建3D打印肝脏补片，该肝脏补片中含有冷冻多孔载药载细胞微球。将该肝脏补片移植到大鼠的肝脏区域，体外给予近红外照射使LPS和MSCs释放。如图4所示，大鼠活体成像证明了该支架(肝脏补片)可以有效定植在大鼠肝脏区域。对各组大鼠进行生存率分析以及肝脏炎症评估，如图5所示，采用肝脏补片治疗后可以将急性肝衰竭小鼠的七天生存率从20％提高到70％，并且肝功能指标ALT、AST能够及时恢复，冷冻多孔载药载细胞微球对治疗急性肝衰竭有显著效果，该策略可以广泛用于细胞治疗和组织再生领域。
以上仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，应视为本发明的保护范围。

Claims (9)

1.一种冷冻多孔载药载细胞微球的制备方法，其特征在于，包括以下步骤：
S1、将脂多糖、光热响应性材料以及水凝胶材料添加到去离子水中，配置得到混合水凝胶溶液；以混合水凝胶溶液为内相，硅油为外相，通过微流控装置制备得到油包水型溶液；
S2、将S1制备得到的油包水型溶液置于-20℃环境中进行预冷冻，再置于液氮中进行冷冻处理，然后使用紫外线照射固化，使用乙醇清洗后得到光热响应性冷冻多孔载药微球；
S3、将S2得到的光热响应性冷冻多孔载药微球浸泡在含有间充质干细胞的培养基中，得到具有光热响应的冷冻多孔载药载细胞微球。
2.根据权利要求1所述的一种冷冻多孔载药载细胞微球的制备方法，其特征在于，
步骤S1中，脂多糖的添加量为1mg/mL。
3.根据权利要求1所述的一种冷冻多孔载药载细胞微球的制备方法，其特征在于，
步骤S1中，所述光热响应性材料为氧化石墨烯和聚(N-异丙基丙烯酰胺)。
4.根据权利要求3所述的一种冷冻多孔载药载细胞微球的制备方法，其特征在于，
所述氧化石墨烯的添加量为0.2wt％，聚(N-异丙基丙烯酰胺)的添加量为10wt％。
5.根据权利要求1所述的一种冷冻多孔载药载细胞微球的制备方法，其特征在于，
步骤S1中，所述水凝胶材料为甲基丙烯酸明胶、甲叉丙烯酰胺和α-羟基异丁酰苯。
6.根据权利要求5所述的一种冷冻多孔载药载细胞微球的制备方法，其特征在于，
所述甲基丙烯酸明胶的添加量为2.5wt％，甲叉丙烯酰胺的添加量为0.34wt％，α-羟基异丁酰苯的浓度为1v/v％。
7.根据权利要求1所述的一种冷冻多孔载药载细胞微球的制备方法，其特征在于，
步骤S2中，所述预冷冻的时间为0-60分钟。
8.根据权利要求1所述的一种冷冻多孔载药载细胞微球的制备方法，其特征在于，
步骤S3中，所述含有间充质干细胞的培养基的浓度为1g/L。
9.一种如权利要求1-8任一项所述的冷冻多孔载药载细胞微球在制备治疗急性肝衰竭药物中的应用。
CN202210218796.2A 2022-03-04 2022-03-04 一种冷冻多孔载药载细胞微球的制备方法及其在制备治疗急性肝衰竭药物中的应用 Pending CN114469875A (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210218796.2A CN114469875A (zh) 2022-03-04 2022-03-04 一种冷冻多孔载药载细胞微球的制备方法及其在制备治疗急性肝衰竭药物中的应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210218796.2A CN114469875A (zh) 2022-03-04 2022-03-04 一种冷冻多孔载药载细胞微球的制备方法及其在制备治疗急性肝衰竭药物中的应用

Publications (1)

Publication Number Publication Date
CN114469875A true CN114469875A (zh) 2022-05-13

Family

ID=81486576

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210218796.2A Pending CN114469875A (zh) 2022-03-04 2022-03-04 一种冷冻多孔载药载细胞微球的制备方法及其在制备治疗急性肝衰竭药物中的应用

Country Status (1)

Country Link
CN (1) CN114469875A (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104353123A (zh) * 2014-11-11 2015-02-18 四川大学 一种非匀质水凝胶及其制备方法和应用
CN110003408A (zh) * 2019-03-13 2019-07-12 上海大学 生物相容性多孔冷冻凝胶微球及其制备方法
CN111330072A (zh) * 2020-03-03 2020-06-26 南京鼓楼医院 一种仿生多孔MSCs微球的制备方法及其应用
CN112220758A (zh) * 2020-10-16 2021-01-15 温州医科大学附属第二医院（温州医科大学附属育英儿童医院） 一种具有光热响应性的可融化水凝胶载药微球的制备及其在细胞扩增中的应用
CN112245658A (zh) * 2020-10-09 2021-01-22 北京大学 一种可注射晶胶微球细胞扩增载体及其制备方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104353123A (zh) * 2014-11-11 2015-02-18 四川大学 一种非匀质水凝胶及其制备方法和应用
CN110003408A (zh) * 2019-03-13 2019-07-12 上海大学 生物相容性多孔冷冻凝胶微球及其制备方法
CN111330072A (zh) * 2020-03-03 2020-06-26 南京鼓楼医院 一种仿生多孔MSCs微球的制备方法及其应用
CN112245658A (zh) * 2020-10-09 2021-01-22 北京大学 一种可注射晶胶微球细胞扩增载体及其制备方法
CN112220758A (zh) * 2020-10-16 2021-01-15 温州医科大学附属第二医院（温州医科大学附属育英儿童医院） 一种具有光热响应性的可融化水凝胶载药微球的制备及其在细胞扩增中的应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
王洁等: ""智能响应性氧化石墨烯水凝胶微载体用于细胞的捕获与释放"", 《SCIENCE CHINA MATERIALS》 *

Similar Documents

Publication Publication Date Title
Ju et al. Extracellular vesicle-loaded hydrogels for tissue repair and regeneration
CN101891962B (zh) 丝素蛋白多孔三维材料的制备方法
Liu et al. Microcryogels as injectable 3-D cellular microniches for site-directed and augmented cell delivery
Ravichandran et al. Cardiogenic differentiation of mesenchymal stem cells on elastomeric poly (glycerol sebacate)/collagen core/shell fibers
ES2405774T3 (es) Métodos para preparar andamio poroso para la ingeniería de tejidos
CN104894062A (zh) 一种干细胞外泌体补片及其制备方法与应用
Huang et al. Solid freeform-fabricated scaffolds designed to carry multicellular mesenchymal stem cell spheroids for cartilage regeneration
Liang et al. Micronized acellular dermal matrix as an efficient expansion substrate and delivery vehicle of adipose‐derived stem cells for vocal fold regeneration
CN102218160A (zh) 神经组织基质源性组织工程支架材料的制备及其应用
CN110897993A (zh) 一种外泌体冻干粉的制备方法及其应用
CN106421902B (zh) 一种快速凝胶化丝素蛋白溶液及其制备方法
Jang et al. A MSCs-laden polycaprolactone/collagen scaffold for bone tissue regeneration
CN108822335B (zh) 一种复合膜及其制备方法和应用
CN104587531B (zh) 一种修复关节软骨损伤的凝胶支架的制备方法
CN107149702A (zh) 一种聚多巴胺改性多孔支架的制备
Yao et al. Fabrication of water-stable silk fibroin scaffolds through self-assembly of proteins
Chen et al. Mechanically active small intestinal submucosa hydrogel for accelerating chronic wound healing
Jiang et al. Dissolvable microgel-templated macroporous hydrogels for controlled cell assembly
CN113577381A (zh) 基于微凝胶支架材料构建的可注射软骨及其应用
CN114469875A (zh) 一种冷冻多孔载药载细胞微球的制备方法及其在制备治疗急性肝衰竭药物中的应用
Higuchi et al. Temperature-dependent cell detachment on Pluronic gels
TWI741263B (zh) 片狀細胞培養物的製造方法
Cai et al. Surface modification of three-dimensional poly (d, l-lactic acid) scaffolds with baicalin: a histological study
Sun et al. Cell-Incorporated Bioactive Tissue Engineering Scaffolds made by Concurrent Cell Electrospinning and Emulsion Electrospinning
Ota et al. Fabrication of scaffold-free mesenchyme tissue bands by cell self-aggregation technique for potential use in tissue regeneration

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20220513