CN111632038A - 一种血小板载药微纳米马达及其制备方法和应用 - Google Patents

一种血小板载药微纳米马达及其制备方法和应用 Download PDF

Info

Publication number
CN111632038A
CN111632038A CN202010509926.9A CN202010509926A CN111632038A CN 111632038 A CN111632038 A CN 111632038A CN 202010509926 A CN202010509926 A CN 202010509926A CN 111632038 A CN111632038 A CN 111632038A
Authority
CN
China
Prior art keywords
platelet
micro
drug
nano motor
suspension
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.)
Granted
Application number
CN202010509926.9A
Other languages
English (en)
Other versions
CN111632038B (zh
Inventor
万密密
毛春
李婷
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 Normal University
Original Assignee
Nanjing Normal University
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 Normal University filed Critical Nanjing Normal University
Priority to CN202010509926.9A priority Critical patent/CN111632038B/zh
Publication of CN111632038A publication Critical patent/CN111632038A/zh
Application granted granted Critical
Publication of CN111632038B publication Critical patent/CN111632038B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • 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
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Nanotechnology (AREA)
  • Optics & Photonics (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Diabetes (AREA)
  • Dermatology (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Immunology (AREA)
  • Botany (AREA)
  • Zoology (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Molecular Biology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

本发明公开了一种血小板载药微纳米马达及其制备方法和应用，该微纳米马达包括血小板外侧包裹的聚多巴胺涂层以及与聚多巴胺结合的含胺类化合物或芳香族化合物药物分子。本发明利用多巴胺在弱碱性条件下易于自聚合的特点，在血小板表面原位聚合形成聚多巴胺涂层，再利用聚多巴胺与药物分子结合装载药物，形成血小板载药微纳米马达。本发明利用血小板本身的不对称性，在近红外光的照射下，具有光热效应的聚多巴胺涂层可形成不均匀的热泳现象，推动血小板载药微纳米马达的运动。本发明制备方法原料简单、反应条件温和、操作简便，制得的材料具有良好的生物相容性可以自主趋向炎症或伤口部位，并通过血小板细胞活化产生血小板衍生微粒，从而释放药物。

Description

一种血小板载药微纳米马达及其制备方法和应用
技术领域
本发明属于生物医学材料，具体涉及一种血小板载药微纳米马达及其制备方法和应用。
背景技术
近年来，多功能的人工合成微纳米马达在催化降解、生物传感、毒素吸附和药物运输等领域表现出良好的应用潜能。然而，为了实现微纳米马达在生物体内的实际应用，需要设计开发完全生物兼容的智能材料，使合成的微纳米马达能够在复杂生物环境中发挥作用，同时不产生有害的影响。
部分天然细胞和微生物具有固有的趋化特性和生物功能。目前已有研究将这些生物组件与纳米材料相结合制造细胞基微纳米马达，用于生物环境的操作和执行各种生物功能。细胞基微纳米马达能够充分发挥生物组件固有的趋化特性，促进微纳米马达在复杂的生物环境中有效运动；并模仿天然细胞的生物功能，在特定位置发挥作用。细胞基微米马达由于不需要额外的燃料或动力装置，在生物医学领域具有广阔的应用前景。
尽管这类细胞基微米马达具有独特的优点，但它们在体内的应用潜力仍面临重大挑战。其中包括对运动速度与方向的控制以及微米级马达的尺寸限制。例如：巨噬细胞马达尺寸达几十微米，很难将药物有效递送至目标细胞内部，极大地限制其作为药物运输载体的功效。
发明内容
发明目的：针对现有技术存在的问题，尤其是现有大多数微纳米马达生物相容性欠佳的问题，本发明提供了一种血小板载药微纳米马达及其制备方法。本发明提供的血小板载药微纳米马达以天然细胞血小板为基材，采用温和的表面改性技术在其表面包裹聚多巴胺涂层，再利用聚多巴胺的邻苯二醌基团与含胺类药物的氨基发生席夫碱反应，或利用聚多巴胺和含氨基或者芳香族药物之间的π-π堆积负载药物。该方法制备工艺简单，材料生物相容性良好，制得的血小板载药微纳米马达可以利用血小板固有的趋化性自主趋向炎症、血栓、手术伤口等病理环境的组织和细胞，通过微纳米马达的运动可以增强细胞摄取和组织渗透，从而提高药物递送效率，在生物医学领域具有广阔的应用前景。
本发明提供一种具有刺激响应性形貌变化且生物相容性良好的血小板微纳米马达，以天然细胞血小板为基材，采用温和的表面改性技术在其表面部分包裹聚多巴胺涂层，一方面可以利用血小板天然的趋化性，自主趋向到手术伤口或炎性环境；另一方面还可以利用聚多巴胺的光热效应，在近红外激光的照射下产生不均匀的热泳现象，驱动血小板及其衍生微粒载药微纳米马达的运动。
技术方案：为了实现上述目的，如本发明所述一种血小板载药微纳米马达，包括血小板外侧包裹的聚多巴胺涂层以及物理或化学方式与聚多巴胺结合的含胺类化合物或芳香族化合物药物分子，在近红外照射下能够运动，成为一种载药微纳米马达。作为优选，所述聚多巴胺涂层的厚度为0.1-1μm。
本发明所述的血小板载药微纳米马达的制备方法，其特征在于，包括以下步骤：
(1)将提取的血小板(PLTs)，分散于PBS缓冲液，得到血小板悬浊液，以防止不必要的血小板活化，室温保存备用；
(2)将盐酸多巴胺溶解于Tris-HCl缓冲液(pH＝8.5)，并与上述血小板悬浊液按一定体积比混合后，在光照下聚合，得到含有聚多巴胺涂层的血小板微纳米马达悬浊液；离心洗涤后分散于PBS缓冲液保存备用；
(3)将含胺类化合物或芳香类化合物药物溶解于PBS缓冲液，与上述含有聚多巴胺涂层的血小板微纳米马达悬浊液在室温下混合振荡，得到负载药物的血小板载药微纳米马达悬浊液；离心洗涤后分散于PBS缓冲液保存备用。
其中，所述聚多巴胺涂层的厚度为0.1-1μm。
作为优选，所述步骤(1)提取血小板为使用梯度离心法从新鲜全血中分离提取血小板，梯度离心法先用1000-1500rpm离心15-20分钟去除红细胞，再使用2500-3000rpm离心10-15分钟富集血小板。
作为优选，步骤(2)所述盐酸多巴胺的Tris-HCl溶液浓度为0.1-2mg/mL，与血小板悬浊液的体积比为1:1-1:10，血小板悬浊液中血小板密度为1×107-8个/mL，聚合反应时间为2-6h。
其中，步骤(4)所述药物为含胺类化合物或芳香类化合物，包括如阿霉素、尼莫司汀、丝裂霉素等。
作为优选，步骤(3)所述药物浓度为0.1-2mg/mL，与含有聚多巴胺涂层的血小板微纳米马达悬浊液的体积比为1:1-5:1，其中血小板密度为1×107-8个/mL，与含有聚多巴胺涂层的血小板微纳米马达悬浊液在室温下混合2-12小时。
本发明所述的血小板微纳米马达在制备用于防止癌症的转移和复发药物中的应用。
其中，所述血小板载药微纳米马达可以自主趋向手术切除后的伤口，受到炎性环境或肿瘤微环境刺激，活化释放纳米尺寸的血小板衍生微粒，促进化疗药物如阿霉素释放。施加的近红外激光照射，可以促进血小板衍生微粒被癌细胞摄取，更好地发挥阿霉素的药效。
具体而言，本发明利用多巴胺在弱碱性条件下易于自聚合的特点，在血小板表面原位聚合形成聚多巴胺涂层，再利用聚多巴胺丰富的邻苯二醌基团与含胺类药物发生席夫碱反应，或利用聚多巴胺和含胺类化合物或芳香族药物之间的π-π堆积装载药物，形成血小板载药微纳米马达。利用血小板本身的不对称性，在近红外光的照射下，具有光热效应的聚多巴胺涂层可形成不均匀的热泳现象，由此推动血小板载药微纳米马达的运动。
本发明利用了血小板天然特征，生理条件下的血小板处于静息态，呈圆盘状，在血管壁附近循环。当受到激活信号刺激时，血小板发生黏附、激活和聚集。激活态血小板伸出伪足，呈树突状，同时释放血小板衍生微粒(血小板的质膜)，直径约0.1-1微米。本发明从全血中提取血小板，并在其膜表面修饰聚多巴胺涂层(静息态)，然后添加凝血酶体外激活修饰后的血小板，得到激活态血小板及血小板衍生微粒，这两种产物都来源于原来的血小板，因此它们表面也覆盖有聚多巴胺涂层。而与聚多巴胺结合的药物分子也存在于激活态血小板和血小板衍生微粒表面，形成纳米级的血小板衍生微粒携带药物，比微米级的细胞更有利于药物的组织渗透。
有益效果：与现有技术相比，本发明具有以下优点：
本发明选用天然细胞血小板和生物相容性良好的多巴胺为原料制备血小板载药微纳米马达。该方法原料简单、反应条件温和、操作简便，制得的材料具有良好的生物相容性，可以自主趋向炎症或伤口部位，并通过血小板细胞活化产生血小板衍生微粒，从而释放药物。本发明制备的血小板载药微纳米马达与现有的细胞基微纳米马达相比，本发明提供的血小板载药微纳米马达具有刺激响应性形貌变化，激活后可以产生纳米级衍生微粒，与现有的活细胞给药体系相比(如：巨噬细胞、***、细菌等)克服了当前生物类微米马达给药体系本身尺寸较大的不足，更有利于药物在组织内的渗透，同时利用血小板本身的不对称性，在近红外光的照射下，具有光热效应的聚多巴胺涂层可形成不均匀的热泳现象，由此推动血小板载药微纳米马达的运动，微纳米马达的运动性能可以增强细胞摄取和组织渗透，从而提高药物递送效率。
本发明制得的血小板载药微纳米马达可以利用血小板固有的趋化性自主趋向炎症、血栓、术后创口等病理环境的组织和细胞，再通过微纳米马达的运动可以增强细胞摄取和组织渗透，从而提高药物递送效率，可以有效用于制备用于防止癌症的转移和复发药物中，在生物医学领域具有广阔的应用前景。
附图说明
图1为血小板的透射电镜图；
图2为血小板微纳米马达的透射电镜图；
图3为负载阿霉素的血小板载药微纳米马达的免疫荧光图像；
图4为近红外激光(808nm，2.5W/cm2)照射下负载阿霉素的血小板载药微纳米马达运动轨迹(5s)；
图5为不同材料与MCF-7细胞共孵育24小时后的细胞存活率。
具体实施方式
下面通过具体实施例对本发明所述的技术方案给予进一步详细的说明，但有必要提出，以下实施例只用于对发明内容的描述，并不构成对本发明保护范围的限制。
实施例1
一种血小板载药微纳米马达的制备方法，具体包括以下步骤：
(1)血小板微纳米马达的制备：
采集新鲜兔全血，1500rpm离心15分钟，小心吸取收集上层富血小板血浆；3000rpm离心10分钟，弃去上层血浆，将沉淀血小板缓慢重悬于PBS(pH＝7.4，下同)缓冲溶液，得到血小板悬浊液。血小板的透射电镜图如图1所示，从图1可以看出血小板呈圆盘状。称取10mg盐酸多巴胺，溶于10mL，10mM Tris-HCl缓冲液(pH＝8.5)。通过细胞计数法调节血小板悬浊液中血小板密度为1×108个/mL，取前述盐酸多巴胺溶液1mL与1mL(1×108个)血小板悬浊液混合，置于静音混合器，室温下聚合2小时，得到含有聚多巴胺涂层的血小板微纳米马达悬浊液，3000rpm离心10min取下层沉淀，加入PBS缓冲液混匀，离心洗涤后分散于PBS缓冲液备用。图2为血小板微纳米马达的透射电镜图，从图2可以看出聚多巴胺包裹在圆盘状血小板外周，其厚度约为0.3-1μm，其外圈颜色较浅的为聚多巴胺涂层厚度约为0.1-1μm。
(2)血小板载药微纳米马达的制备：
称取10mg盐酸阿霉素，超声溶解于10mL PBS缓冲液(pH＝7.4，下同)。通过细胞计数法调节(1)中含有聚多巴胺涂层的血小板微纳米马达悬浊液中血小板的密度为1×108个/mL。取前述盐酸阿霉素溶液4mL与1mL含有聚多巴胺涂层的血小板微纳米马达悬浊液(1×108个)混合，置于静音混合器，室温下避光反应2小时，得到负载阿霉素的血小板载药微纳米马达悬浊液，3000rpm离心10min取下层沉淀，加入PBS缓冲液混匀，离心洗涤后分散于PBS缓冲液备用。将所得负载阿霉素的血小板载药微纳米马达与DiO染料共孵育，标记血小板膜并置于共聚焦显微镜下观察，其免疫荧光图像如图3所示。从图3可以看出红色荧光(阿霉素DOX，中图)与绿色荧光(血小板PLTs，左图)几乎重合(右图)，证明了药物的成功负载。将所得的负载阿霉素的血小板载药微纳米马达在近红外激光(808nm，2.5W/cm2)照射下的运动视频截图如图4所示，黑色细线代表血小板载药微纳米马达在5s内的运动轨迹，证明血小板载药微纳米马达在近红外激光的照射下可以运动。
将0.1mL细胞密度为5×104个/mL的人乳腺癌细胞(MCF-7)接种于96孔板，置于37℃、5％CO2培养箱中培养12h。然后吸出培养基，实验组每孔分别加入0.2mL(1×108个/mL)血小板(PLTs)或上述血小板载药微纳米马达或新鲜培养基，加入新鲜培养基的组别每孔再用近红外激光器(808nm，2.5W/cm2)照射10min，空白组(Control)每孔加入等量新鲜细胞培养基作对照，孵育24h后采用MTT法测试细胞存活率。结果如图5所示，实验组细胞存活率均大于90％，表明本研究制备的血小板微纳米马达具有良好的生物相容性。
实施例2
一种血小板载药微纳米马达的制备方法，具体包括以下步骤：
(1)血小板微纳米马达的制备：
按照实施例1所述梯度离心法分离提取血小板，得血小板悬浊液。通过细胞计数法调节血小板悬浊液中血小板密度为1×107个/mL。称取20mg盐酸多巴胺，溶于10mL，10mMTris-HCl缓冲液(pH＝8.5)，取出1mL与10mL(1×107个)血小板悬浊液混合，置于静音混合器，室温下聚合2小时，得到血小板微纳米马达悬浊液，3000rpm离心10min取下层沉淀，加入PBS缓冲液混匀，离心洗涤后分散于PBS缓冲液备用。
(2)血小板载药微纳米马达的制备：
称取10mg盐酸阿霉素，超声溶解于10mL PBS缓冲液。通过细胞计数法调节(1)中含有聚多巴胺涂层的血小板微纳米马达悬浊液中血小板的密度为1×107个/mL。移取前述盐酸阿霉素溶液1mL与1mL含有聚多巴胺涂层的血小板微纳米马达悬浊液混合(1×107个)，置于静音混合器，室温下避光反应2小时，得到负载阿霉素的血小板载药微纳米马达悬浊液，3000rpm离心10min取下层沉淀，加入PBS缓冲液混匀，离心洗涤后分散于PBS缓冲液备用。
实施例3
一种血小板载药微纳米马达的制备方法，具体包括以下步骤：
(1)血小板微纳米马达的制备：
采集新鲜兔全血，1000rpm离心20分钟，小心吸取收集上层富血小板血浆；2500rpm离心15分钟，弃去上层血浆，将沉淀血小板缓慢重悬于PBS缓冲溶液，得到血小板悬浊液。通过细胞计数法调节血小板悬浊液中血小板密度为1×108个/mL。称取1mg盐酸多巴胺，溶于10mL，10mM Tris-HCl缓冲液(pH＝8.5)，取出1mL与1mL(1×108个)血小板悬浊液混合，置于静音混合器，室温下聚合6小时，得到血小板微纳米马达悬浊液，3000rpm离心10min取下层沉淀，加入PBS缓冲液混匀，离心洗涤后分散于PBS缓冲液备用。
(2)血小板载药微纳米马达的制备：
称取4mg盐酸尼莫司汀，溶解于4mL PBS缓冲液。通过细胞计数法调节(1)中含有聚多巴胺涂层的血小板微纳米马达悬浊液中血小板的密度为1×108个/mL，取1mL与前述4mL盐酸尼莫司汀溶液混合，室温下避光反应2小时，得负载尼莫司汀的血小板载药马达悬浊液，3000rpm离心10min取下层沉淀，加入PBS缓冲液混匀，离心洗涤后分散于PBS缓冲液保存备用。
实施例4
一种血小板载药微纳米马达的制备方法，具体包括以下步骤：
(1)血小板微纳米马达的制备：
按照实施例1所述梯度离心法分离提取血小板，得血小板悬浊液。通过细胞计数法调节血小板悬浊液中血小板密度为1×108个/mL。称取5mg盐酸多巴胺，溶于10mL，10mMTris-HCl缓冲液(pH＝8.5)，取出1mL与1mL(1×108个)血小板悬浊液混合，置于静音混合器，室温下聚合2小时，得到血小板微纳米马达悬浊液，3000rpm离心10min取下层沉淀，加入PBS缓冲液混匀，离心洗涤后分散于PBS缓冲液备用。
(2)血小板载药微纳米马达的制备：
称取2mg丝裂霉素，溶解于2mL PBS缓冲液。通过细胞计数法调节(1)中含有聚多巴胺涂层的血小板微纳米马达悬浊液中血小板的密度为1×108个/mL，取1mL与前述2mL丝裂霉素溶液混合，室温下避光反应2小时，得负载丝裂霉素的血小板载药马达悬浊液，3000rpm离心10min取下层沉淀，加入PBS缓冲液混匀，离心洗涤后分散于PBS缓冲液保存备用。
实施例5
实施例5与实施例1制备方法相同，不同之处在于，盐酸阿霉素药物浓度为0.1mg/mL，与含有聚多巴胺涂层的血小板微纳米马达悬浊液的体积比为4:1，其中血小板密度为1×107个/mL，在室温下混合4小时。
实施例6
实施例6与实施例1制备方法相同，不同之处在于，盐酸阿霉素药物浓度为2mg/mL，与含有聚多巴胺涂层的血小板微纳米马达悬浊液的体积比为5:1，其中血小板密度为1×107个/mL，在室温下混合12小时。
本发明实施例制备的血小板微纳米马达在制备用于防止癌症的转移和复发药物中，血小板载药微纳米马达可以自主趋向手术切除后的伤口，受到炎性环境或肿瘤微环境刺激，活化释放纳米尺寸的血小板衍生微粒，促进化疗药物如阿霉素释放。本发明通过施加的近红外激光照射，利用血小板本身的不对称性，在近红外光的照射下，具有光热效应的聚多巴胺涂层可形成不均匀的热泳现象，由此推动血小板载药微纳米马达的运动，可以促进血小板衍生微粒被癌细胞摄取，更好地发挥如阿霉素、尼莫司汀或丝裂霉素的药效。

Claims (8)

1.一种血小板载药微纳米马达，其特征在于，包括血小板外侧包裹的聚多巴胺涂层以及与聚多巴胺结合的含胺类化合物或芳香族化合物药物分子，形成为一种血小板载药微纳米马达。
2.根据权利要求1所述的血小板载药微纳米马达，其特征在于，所述聚多巴胺涂层的厚度为0.1-1μm。
3.一种血小板载药微纳米马达的制备方法，其特征在于，包括以下步骤：
(1)将提取的血小板(PLTs)分散于PBS缓冲液得到血小板悬浊液，室温保存备用；
(2)将盐酸多巴胺溶解于缓冲液，与上述血小板悬浊液混合后在光照下聚合，得到含有聚多巴胺涂层的血小板微纳米马达悬浊液，离心洗涤后分散于PBS缓冲液保存备用；
(3)将所需含胺类化合物或芳香族化合物药物溶解于PBS缓冲液，与上述含有聚多巴胺涂层的血小板微纳米马达悬浊液在室温下振荡混合，得到负载药物的血小板载药微纳米马达悬浊液，即为血小板载药微纳米马达。
4.根据权利要求3所述的制备方法，其特征在于，所述步骤(1)提取血小板优选为使用梯度离心法从新鲜全血中分离提取血小板，梯度离心法先用1000-1500rpm离心15-20分钟去除红细胞，再使用2500-3000rpm离心10-15分钟富集血小板。
5.根据权利要求3所述的制备方法，其特征在于，步骤(2)所述盐酸多巴胺的Tris-HCl缓冲液的浓度为0.1-2mg/mL，与血小板悬浊液的体积比为1:1-1:10，血小板悬浊液中血小板密度为1×107-8个/mL；所述聚合反应时间为2-6h。
6.根据权利要求3所述的制备方法，其特征在于，步骤(3)所述药物为含胺类化合物或芳香族化合物包括阿霉素、尼莫司汀或丝裂霉素。
7.根据权利要求3所述的制备方法，其特征在于，步骤(3)所述药物浓度为0.1-2mg/mL，与含有聚多巴胺涂层的血小板微纳米马达悬浊液的体积比为1:1-5:1，与含有聚多巴胺涂层的血小板微纳米马达悬浊液在室温下混合2-12小时。
8.一种权利要求1所述的血小板载药微纳米马达在制备用于防止癌症的转移和复发药物中的应用。
CN202010509926.9A 2020-06-05 2020-06-05 一种血小板载药微纳米马达及其制备方法和应用 Active CN111632038B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010509926.9A CN111632038B (zh) 2020-06-05 2020-06-05 一种血小板载药微纳米马达及其制备方法和应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010509926.9A CN111632038B (zh) 2020-06-05 2020-06-05 一种血小板载药微纳米马达及其制备方法和应用

Publications (2)

Publication Number Publication Date
CN111632038A true CN111632038A (zh) 2020-09-08
CN111632038B CN111632038B (zh) 2022-02-11

Family

ID=72322792

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010509926.9A Active CN111632038B (zh) 2020-06-05 2020-06-05 一种血小板载药微纳米马达及其制备方法和应用

Country Status (1)

Country Link
CN (1) CN111632038B (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113633788A (zh) * 2021-08-02 2021-11-12 郑州大学第一附属医院 一种用于神经阻滞可显影的高黏附药物缓释体的制备方法
CN114425043A (zh) * 2021-12-08 2022-05-03 深圳先进技术研究院 一种基于巨噬细胞的活细胞载药***及其制备方法与应用
CN117982987A (zh) * 2024-04-03 2024-05-07 四川厚浦生物科技有限公司 一种白细胞过滤材料及其制备方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107233302A (zh) * 2017-07-25 2017-10-10 中国科学院青岛生物能源与过程研究所 一种纳米纤维素/聚多巴胺复合智能凝胶药物缓释材料的制备方法
US20180087116A1 (en) * 2017-02-11 2018-03-29 Mahmoud Amouzadeh Tabrizi Methods and biosensors for tumor detection
CN108042802A (zh) * 2017-12-11 2018-05-18 哈尔滨工业大学 一种人造红细胞马达及其制备方法
CN109091673A (zh) * 2018-09-11 2018-12-28 浙江理工大学 一种集靶向、光热于一体的红细胞仿生型纳米粒的制备方法
CN110251479A (zh) * 2019-06-06 2019-09-20 南京师范大学 一种红细胞膜包裹仿生型血液六价铬还原去除剂/磁性纳米马达及其制备方法和应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180087116A1 (en) * 2017-02-11 2018-03-29 Mahmoud Amouzadeh Tabrizi Methods and biosensors for tumor detection
CN107233302A (zh) * 2017-07-25 2017-10-10 中国科学院青岛生物能源与过程研究所 一种纳米纤维素/聚多巴胺复合智能凝胶药物缓释材料的制备方法
CN108042802A (zh) * 2017-12-11 2018-05-18 哈尔滨工业大学 一种人造红细胞马达及其制备方法
CN109091673A (zh) * 2018-09-11 2018-12-28 浙江理工大学 一种集靶向、光热于一体的红细胞仿生型纳米粒的制备方法
CN110251479A (zh) * 2019-06-06 2019-09-20 南京师范大学 一种红细胞膜包裹仿生型血液六价铬还原去除剂/磁性纳米马达及其制备方法和应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
MIMI WAN ET AL.: "Platelet-derived porous nanomotor for thrombus therapy", 《SCIENCE ADVANCES》 *
MIMI WAN ET AL.: "Systematic Research and Evaluation Models of Nanomotors for Cancer Combined Therapy", 《ANGEWANDTE》 *
吴志光等: "红细胞伪装微纳米马达", 《中国化学会第十五届胶体与界面化学会议论文集（第二分会）》 *
孔磊等: "自驱动微纳米马达的设计原理与结构简化方法", 《科学通报》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113633788A (zh) * 2021-08-02 2021-11-12 郑州大学第一附属医院 一种用于神经阻滞可显影的高黏附药物缓释体的制备方法
CN114425043A (zh) * 2021-12-08 2022-05-03 深圳先进技术研究院 一种基于巨噬细胞的活细胞载药***及其制备方法与应用
CN117982987A (zh) * 2024-04-03 2024-05-07 四川厚浦生物科技有限公司 一种白细胞过滤材料及其制备方法

Also Published As

Publication number Publication date
CN111632038B (zh) 2022-02-11

Similar Documents

Publication Publication Date Title
CN111632038B (zh) 一种血小板载药微纳米马达及其制备方法和应用
Hoseinpour et al. Applications of zeolitic imidazolate framework-8 (ZIF-8) in bone tissue engineering: A review
Li et al. Engineering of cascade-responsive nanoplatform to inhibit lactate efflux for enhanced tumor chemo-immunotherapy
CN106806343B (zh) 一种叶酸和聚多巴胺修饰的肿瘤靶向介孔二氧化硅纳米粒及制备方法与应用
Lu et al. Medical applications based on supramolecular self-assembled materials from tannic acid
Rahimi et al. Biocompatible magnetic tris (2-aminoethyl) amine functionalized nanocrystalline cellulose as a novel nanocarrier for anticancer drug delivery of methotrexate
Zhu et al. Responsive hydrogels based on triggered click reactions for liver cancer
CN110215438B (zh) 双载蒽环类药物及光敏剂介孔硅纳米粒的制备方法与应用
CN113289030B (zh) 一种光热协同化疗的靶向长循环纳米药物载体的制备方法
CN106806344A (zh) 聚多巴胺和聚乙二醇维生素e琥珀酸酯修饰的介孔二氧化硅纳米粒及其制备方法与应用
Qu et al. Photothermal scaffolds/surfaces for regulation of cell behaviors
CN112121029A (zh) 一种仿生多巴胺聚合载药纳米递释***及其制备方法
CN111558051B (zh) 一种具有快速粘液渗透作用的复合纳米微球及其制备方法和应用
CN110404070B (zh) Pvp修饰的海藻酸钠/聚多巴胺复合纳米材料及制备和应用
CN114259477B (zh) 一种促渗透、缓解肿瘤缺氧并能靶向肿瘤细胞的纳米递送体系及其制备方法和应用
Kim et al. Macromolecular Engineering of Thermoresponsive Metal–Phenolic Networks
CN111870579B (zh) 一种肿瘤靶向纳米胶束、制备方法及作为药物载体的应用
CN111035625A (zh) 阿司匹林在血小板靶向载药***制备中的用途
CN112294776B (zh) 一种包覆细胞膜的还原响应型碳点载药纳米团簇及其制备和应用
CN113599368A (zh) 一种细胞膜拮抗联合纳米酶的仿生载药纳米***、制备方法和用途
CN113633625A (zh) 一种杂膜负载氧化磷酸化抑制剂的纳米药物及其制备方法
Zhang et al. “Motile-targeting” drug delivery platforms based on micro/nanorobots for tumor therapy
Nagesetti et al. Polyethylene glycol modified ORMOSIL theranostic nanoparticles for triggered doxorubicin release and deep drug delivery into ovarian cancer spheroids
Li et al. Temperature-and pH-responsive injectable chitosan hydrogels loaded with doxorubicin and curcumin as long-lasting release platforms for the treatment of solid tumors
CN110368501B (zh) 一种rgd肽修饰的硼载药体系及其制备和应用

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
GR01 Patent grant
GR01 Patent grant