WO2020119174A1 - 一种多功能负载维甲酸的钆参杂四氧化三铁复合纳米粒子及其制备方法 - Google Patents

一种多功能负载维甲酸的钆参杂四氧化三铁复合纳米粒子及其制备方法 Download PDF

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WO2020119174A1
WO2020119174A1 PCT/CN2019/102375 CN2019102375W WO2020119174A1 WO 2020119174 A1 WO2020119174 A1 WO 2020119174A1 CN 2019102375 W CN2019102375 W CN 2019102375W WO 2020119174 A1 WO2020119174 A1 WO 2020119174A1
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retinoic acid
loaded
gadolinium
stem cells
nanoparticles
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王冠男
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王冠男
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Priority claimed from CN201811503730.8A external-priority patent/CN109536448B/zh
Priority claimed from CN201811503738.4A external-priority patent/CN109568654B/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K49/00—Preparations for testing in vivo
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    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • 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
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    • 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
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    • 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
    • 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

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  • the invention relates to the technical field of medical biomaterials and tissue engineering, and more particularly to a material and method for nanomedicine biomaterials to induce human stem cells to differentiate into nerve cells for repair of human central nervous system damage.
  • the central nervous system refers to the main part of the nervous system, including the spinal cord in the spinal canal and the brain in the cranial cavity.
  • Diseases caused by central nervous system injury seriously affect human health, and there are many patients, mainly including spinal cord injury, traumatic brain injury, stroke and Parkinson's disease.
  • the incidence of mid-term spinal cord injury is one in a thousand.
  • spinal cord injury There are currently about 1.3 million people with spinal cord injury in my country, and it is growing at a rate of 50,000 to 70,000 per year. The number of patients is increasing year by year.
  • Spinal cord injury is recognized as the most painful group of severely disabled people. 90% of people with spinal cord injury are from trauma.
  • the high incidence is 20 to 30 years old, mostly due to traffic accidents, falls, violence (including suicide) and Caused by spinal cord disease.
  • Traumatic brain injury is also a traumatic disease. About 1.5 million traumatic brain injuries are added every year, because there are many different types of brain injury, lesions and basic damage scope, and different pathological mechanisms, The mechanism of secondary injury in different conditions, so far, there is no effective treatment for traumatic brain injury. Stroke can be divided into hemorrhagic stroke, ischemic stroke and local thrombosis. It is the second leading cause of death in the world. 6% of deaths are caused by stroke. 80% of strokes in the interim period are ischemic strokes.
  • Stem cells refer to cells with multi-directional differentiation potential and self-renewal ability, which exist in many tissues and embryos of adult individuals.
  • stem cells such as neural stem cells, bone marrow mesenchymal stem cells, etc. have the potential to differentiate into neuronal cells, and have the characteristics of sufficient sources, easy access, and autologous transplantation, which effectively avoids the use of The advantages of immune rejection and ethical controversy brought about by stem cells.
  • the rise of stem cell medicine has provided a new solution for the treatment of central nervous system injury, and has become a research hotspot.
  • stem cell transplantation faces several major problems: (1) the survival rate of stem cell transplantation is low, (2) the effective differentiation rate of stem cells is low, less than 13% of the survival neural stem cells differentiate into neurons, (3) the later evaluation method is invasive, most of the traditional methods The use of tissue sections after sacrificed animals to verify whether there is improvement in neural function or behavior after stem cell transplantation is not conducive to dynamic tracking of stem cell migration in vivo after transplantation. These problems have seriously hindered the promotion and application of stem cell therapy in scientific research and clinical practice.
  • the invention designs a multifunctional gadolinium triiron trioxide composite nanoparticle loaded with retinoic acid and a preparation method thereof.
  • the technical problem solved by the invention is that the multifunctional gadolinium triiron trioxide composite iron oxide has not been seen in the prior art.
  • Nanoparticles induce directional differentiation of human stem cells and are used for central nerve injury repair and real-time MRI imaging.
  • the present invention adopts the following solutions:
  • a multifunctional gadolinium ginseng triiron tetroxide composite nanoparticle for inducing directional differentiation of stem cells and for real-time MRI contrast in which gadolinium ginseng triiron tetroxide is an MRI contrast agent and nanocarrier, and its loaded retinoic acid As an active ingredient, it has the effect of promoting the differentiation of stem cells into neuronal cells.
  • the structural formula of the gadolinium triiron trioxide nanoparticles can be expressed as Gd:Fe 3 O 4
  • the structural formula of the multifunctional retinoic acid-loaded gadolinium triiron trioxide nanoparticles can be expressed as Gd:Fe 3 O 4 @RA, where: @ means load, RA stands for retinoic acid.
  • the Gd:Fe 3 O 4 and Gd:Fe 3 O 4 @RA are prepared by the following method, including the following steps:
  • Step 1 A single source precursor of Fe/Gd was prepared, and then added to the reaction mixture of oleylamine, oleic acid and benzyl ether, gradually heated up, and then cooled to room temperature to obtain a black/brown substance, separated, washed with ethanol and then separated , Dried to get Gd:Fe 3 O 4
  • Step 2 Surface-modify Gd:Fe 3 O 4 with an active polymer to make it into an aqueous solution, and the surface function of the active group carboxyl group, so that the retinoic acid-loaded cyclodextrin is connected to Gd:Fe through an ester bond 3 O 4 surface, that is, the o-hydroxybenzene-PEG-carboxyl terminal compound on the surface of the nanomaterial reacts with the hydroxyl group on the surface of cyclodextrin loaded with retinoic acid, and is connected by an ester bond to prepare Gd:Fe 3 O 4 @RA.
  • step 1 the Fe/Gd single source precursor is prepared by the Fe source and the Gd source, wherein the molar ratio is 5:3.
  • the gradual temperature increase in step 1 includes two processes, respectively heating at 100-200°C for 30 minutes and heating at 200-350°C for 30 minutes.
  • step 1 The heating process in step 1 needs to be carried out under the protection of inert gas
  • the diameter of the prepared Gd:Fe 3 O 4 in step 1 is 7-10 nm, and the smaller diameter is beneficial for its entry into cells and reducing cytotoxicity.
  • the living polymer in step 2 is an o-hydroxybenzene-PEG-carboxy terminal compound with an average molecular weight of 4000-8000.
  • step 2 After surface-modifying the active polymer in step 2, dialyzed in distilled water for 24 hours under stirring conditions to remove unconnected polymer.
  • step 2 under the conditions of acidity and heating at about 80°C, the cyclodextrin loaded with retinoic acid reacts its hydroxyl group with the active group carboxyl group on the surface of the nanoparticle through an ester bond to prepare Gd:Fe 3 O 4 @RA.
  • step 2 the prepared Gd:Fe 3 O 4 @RA needs to be purified by propylene dextran gel, and is to be used.
  • step 2 the prepared Gd:Fe 3 O 4 @RA is dissolved in an aqueous solution to obtain an aqueous solution or lyophilized and stored.
  • An experimental method for Gd:Fe 3 O 4 @RA nanoparticles to induce neural stem cells to differentiate into neuron cells includes the following steps:
  • a coverslip pre-treated with polylysine in the culture plate and inoculate primary and passaged neural stem cells derived from rat embryo brain hippocampus into the culture wells at 1 ⁇ 10 7 L -1 respectively.
  • Remove the basic fibroblast growth factor from the complete culture medium of stem cells and add 1.0ml of Gd:Fe 3 O 4 @RA composite nano ions at a concentration of 1mg/ml and fetal bovine serum with a volume fraction of 5% to promote neural stem cell differentiation
  • After culturing for 6 days continue to culture in neural stem cell culture medium containing 5% fetal bovine serum for 6 days, and take out the coverslip after the cell morphology matures;
  • each sample system randomly takes N fields of view, N is a natural number, and the fluorescent marker is positive.
  • the percentage of neural stem cells differentiated into neurons in each film is calculated according to the following formula:
  • Percentage of neuron cells (number of fluorescently labeled positive cells/total number of cells) ⁇ 100%; compare neural stem cells differentiated and cultured with Gd:Fe 3 O 4 @RA composite nano-ions with conventional adherent differentiated cultured neural stem cells.
  • a Gd:Fe 3 O 4 @RA composite nanomolecule used in the repair of spinal nerve injury and MRI imaging method includes the following steps:
  • Step 2 After intraperitoneal anesthesia, the rat is fixed under a special coil for rat magnetic resonance scanning, and the labeled neural stem cells transplanted into the body can be traced in vivo. The migration of the transplanted cells can be tracked in vivo by detecting changes in the labeled T1-MRI signal. With distribution.
  • the mechanism of the present invention is as follows: the Gd:Fe 3 O 4 @RA nanoparticles of the present invention are applied to the directional differentiation of stem cells into neuronal cells for repair of central nervous system damage, and at the same time used for real-time MRI imaging to investigate the nerves after stem cell transplantation The role of function or behavior, and dynamic tracking of migration in vivo.
  • the toxicity test proves that the Gd:Fe 3 O 4 and Gd:Fe 3 O 4 @RA of the present invention have little toxicity to neural stem cells and mesenchymal stem cells.
  • Immunofluorescence staining proved that Gd:Fe 3 O 4 @RA can promote the directional differentiation of mesenchymal stem cells and mesenchymal stem cells into neuronal cells.
  • neural stem cells labeled with Gd:Fe 3 O 4 @RA in the lesion area of spinal cord injury in rats, it can be effectively used for the repair of spinal cord injury, and real-time MRI weighted imaging of the lesion area can also be performed.
  • the state of aggregation, differentiation and migration of the injected stem cells in the body can be clearly observed in real time.
  • the present invention adopts a simple method to prepare Gd:Fe 3 O 4 @RA nanoparticles. This method has simple process, mild reaction conditions, easy operation and separation, environmental friendliness, low price, strong repeatability, and has the prospect of industrialization.
  • FIG. 1 is a transmission electron micrograph A of Gd:Fe 3 O 4 @RA nanoparticles in the present invention.
  • FIG. 2 is a transmission electron micrograph B of Gd:Fe 3 O 4 @RA nanoparticles in the present invention.
  • Fig. 3 is the MRI imaging of the rat spine on the seventh day after transplantation of Gd:Fe 3 O 4 @RA-labeled stem cells in the present invention, where the white highlights are Gd:Fe 3 O 4 @RA-labeled stem cell aggregation areas and black areas Schematic diagram of the area of spinal cord injury.
  • FIG. 4 is a schematic diagram of BBB exercise scores of spinal cord injured rats after transplanting Gd:Fe 3 O 4 @RA-labeled stem cells, unlabeled stem cells and PBS in the present invention.
  • Example 3 Multifunctional retinoic acid-loaded gadolinium triiron tetroxide composite nanoparticles induce directional differentiation of neural stem cells into neuronal cells:
  • a coverslip pre-treated with polylysine into a 24-well culture plate, and inoculate primary and passaged neural stem cells derived from rat embryo hippocampus into culture wells at 1 ⁇ 10 7 L -1 , Remove basic fibroblast growth factor from the complete culture medium of neural stem cells, and add 1.0ml, 1mg/ml of Gd:Fe 3 O 4 @RA and fetal bovine serum with a volume fraction of 5% to promote differentiation of neural stem cells, culture 6 Days later, continue to culture in neural stem cell culture medium containing 5% fetal bovine serum for 6 days, and take out the coverslip after the cell morphology matures.
  • the coverslips carrying cells of conventional adherent differentiation culture and induced differentiation culture were divided into two groups respectively, and immunohistochemistry of ⁇ -tubulin (TUJ1) labeled with neuronal markers was respectively divided. Randomly select 5 films as samples, each sample system randomly selects 5 fields of view, with fluorescent markers as positive, and calculate the percentage of neural stem cells differentiated into neuronal cells according to the following formula:
  • Neuronal cell percentage (number of fluorescently labeled positive cells/total number of cells) ⁇ 100%;
  • Example 4 Multifunctional retinoic acid-loaded gadolinium triiron trioxide composite nanoparticles induce directional differentiation of neural stem cells into neuronal cells for repair of spinal cord injury and MRI imaging:
  • a rat spinal cord injury model was made, and the rats were anesthetized with 10% chloral hydrate. Fix the prone position of the rat on the rat board, perform laminectomy at the T9-T10 segment, take the self-made striking device (weight 109, diameter 2.5mm), and freely fall vertically from the height of 25mm to hit the T9 segment spinal cord ( The dura mater should not be torn after the blow), and the muscles and skin are sutured in turn. Gentamicin (8 mg/kg/day) was intraperitoneally injected into rats 3 days after surgery to prevent urinary tract infection. After the operation, artificial urination was assisted twice a day until the rats recovered their voluntary urination function.
  • T1-MRI signal refers to the T1-weighted MRI signal, which is the signal that is brightened first. Commonly used in clinical MRI signal.
  • the T1 high signal in situ of the injury can be clearly seen under 3T MRI, distributed around the injection point and the injury area; after 14 days, the T1 signal intensity around the injection point has decreased, and the injection point and the center of the injury area The area between them has diffused high-density shadows.
  • the recovery of rat motor function after transplantation of Gd:Fe 3 O 4 @RA-labeled neural stem cells and unlabeled neural stem cells was evaluated. From 14 days after spinal cord injury, the BBB scores of the two groups after stem cell transplantation were significantly higher than those of the PBS control group (P ⁇ 0.05). The BBB scores of rats transplanted with Gd:Fe 3 O 4 @RA labeled neural stem cells and those transplanted with unlabeled neural stem cells were also significantly different (P ⁇ 0.05). This shows that Gd:Fe 3 O 4 @RA can effectively induce the directional differentiation of neural stem cells, can significantly improve the motor function of injured rats, and complete the repair of spinal cord injury.
  • the BBB (Basso, Beattie & Bresnahan locomotor rating) scale which is the scoring standard for rat spinal cord injury, was established based on the observation of three stages of recovery of spinal cord injured rats.
  • Late stage includes fine movements, such as dragging toes and tail, unstable torso and alternating rotation of paws.
  • the 21 scores are synchronized with the progress of recovery.
  • the first part assesses the recovery of early hindlimb joint movement.
  • the second part assesses mid-restoration gait and coordinated movements.
  • the sole of the foot is only in the weight-bearing position, or occasionally/frequently/continuously walking on the back of the foot with no weight.

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Abstract

一种多功能负载维甲酸的钆掺杂四氧化三铁复合纳米粒子及其制备方法，其用于诱导干细胞定向分化及用于实时MRI造影。钆掺杂四氧化三铁为MRI造影剂及纳米载体，其所负载的维甲酸为活性成分，维甲酸具有促进干细胞分化为神经元细胞的作用。钆掺杂四氧化三铁纳米粒子结构式可表述为Gd:Fe 3O 4，多功能负载维甲酸的钆掺杂四氧化三铁纳米粒子的结构式表述为Gd:Fe 3O 4@RA，其中：@为负载的意思，RA代表维甲酸。该Gd:Fe 3O 4@RA纳米粒子用于将干细胞分化为神经元，并用于中枢神经损伤的修复及MRI无侵实时造影。

Description

一种多功能负载维甲酸的钆参杂四氧化三铁复合纳米粒子及其制备方法 技术领域
本发明涉及涉及医用生物材料与组织工程技术领域，更具体地涉及一种纳米医用生物材料诱导人干细胞定向分化为神经细胞用于人中枢神经损伤修复的材料和方法。
背景技术
中枢神经系统是指神经系统的主要部分，包括位于椎管内的脊髓和位于颅腔内的脑。中枢神经损伤所引发的疾病严重影响着人类的健康，且患者众多，主要包括脊髓损伤，创伤性脑损伤，脑中风及帕金森病。期中脊髓损伤的发生率为千分之一，我国目前有约130万脊髓损伤者，且以每年5至7万的速度增长，患者数量呈逐年上升的趋势。脊髓损伤是公认的重度肢体残障群体中最痛苦的一个群体,90％的脊髓损伤者是源自于外伤，高发年龄为20至30岁,多因交通事故、跌落、暴力行为(含自杀)及脊髓疾病而发病。脊髓损伤是一种严重的中枢神经系统损伤,致双下肢或四肢瘫痪,伴随数十种并发症。其治疗一直是世界性难题，目前尚无有效治疗方式。创伤性脑损伤同样是一种外伤性疾病，每年新增大约150万的外伤性脑损伤，因为脑损伤具有多种不同的明确的类型，病灶和基本的损伤范围，而且不同的病理学机制、不同条件二次损伤机制，故到目前为止，外伤性脑损伤还没有针对性的有效治疗手段。脑中风可以分为出血性脑中风，缺血性脑中风和局部血栓等，是世界上第二致死病因，6％的死亡是由于脑中风引起的。期中80％的脑中风为缺血性脑中风，到目前为止缺血性脑中风仍然没有行之有效的治疗方案，目前的治疗方案仅限于血栓溶解和神经保护， 但研究表明这些方案只是针对极少的患者才会具有一点效果。帕金森是一类神经退行性疾病，是65岁以上老年人常见疾病。通常帕金森患者运动平衡和思考认知能力均受到严重影响，目前治疗办法为口服可以在体内转化生成多巴胺的L-3,4-二羟基苯丙氨酸进行治疗，而且存在运动障碍的副作用。由于中枢神经难以再生，到目前为止中枢神经损伤仍未找到合理的解决办法，因此中枢神经再生引起了世界学者们的广泛关注，多年来受到研究学者的高度重视。
干细胞是指有多向分化潜能和自我更新能力的细胞，这种细胞存在于成年个体的许多组织和胚胎中。近年来，研究证实多种干细胞如神经干细胞，骨髓间充质干细细胞等，具有向神经元细胞分化的潜能，并具有来源充足、获取方便、可进行自体移植的特点，这有效避免了使用干细胞所带来的免疫排斥反应和伦理学争议等优势。目前干细胞医学的兴起，为治疗中枢神经损伤提供了新的解决方案，已成为一个研究热点。但干细胞移植面临几大难题：(1)干细胞移植成活率低，(2)干细胞有效分化率低，存活的神经干细胞不足13％分化成神经元，(3)后期评价方式具有侵入性，传统大多采用处死动物后进行组织切片加以验证干细胞移植后对神经功能或行为是否有改善，不利于对移植后干细胞在活体内的迁移的动态示踪。这些问题严重阻碍了干细胞治疗在科研及临床得推广和应用。
近年来，随着纳米技术的迅速发展，其应用领域也越来越广，包括医用生物材料与组织工程技术领域。高生物适应性的多功能纳米材料的构建可有效地提高干细胞移植的成活率；其负载的药物和生物分子可提高干细胞定向分化为神经元细胞；其构建的无机元素可有效地提供磁靶向传递以及提供无侵入性的实时动态造影。目前，尚未见经多功能钆参杂四氧化三铁复合纳米粒子诱导人干细胞定向分化用于中枢神经损伤修复及实时MRI造影的方法报道。
发明内容
本发明设计了一种多功能负载维甲酸的钆参杂四氧化三铁复合纳 米粒子及其制备方法，其解决的技术问题是现有技术中尚未见经多功能钆参杂四氧化三铁复合纳米粒子诱导人干细胞定向分化用于中枢神经损伤修复及实时MRI造影的方法。
为了解决上述存在的技术问题，本发明采用了以下方案：
一种用于诱导干细胞定向分化及用于实时MRI造影的多功能钆参杂四氧化三铁复合纳米粒子，其中，钆参杂四氧化三铁为MRI造影剂及纳米载体，其负载的维甲酸为活性成分，其具有促进干细胞分化为神经元细胞的作用。
本发明中，所述的钆参杂四氧化三铁纳米粒子结构式可表述为Gd:Fe 3O 4,所述的多功能负载维甲酸的钆参杂四氧化三铁纳米粒子的结构式可表述为Gd:Fe 3O 4@RA,其中：@为负载的意思，RA代表着维甲酸。
本发明中，所述的Gd:Fe 3O 4及Gd:Fe 3O 4@RA是由以下方法制备，包括以下步骤：
步骤1、制备了Fe/Gd单源前驱体，然后加入到油胺、油酸和苄醚的反应混合物中，逐步升温加热，后冷却至室温，得到黑色/棕色物质，分离、乙醇洗涤再分离，干燥得到Gd:Fe 3O 4
步骤2、用活性聚合物对Gd:Fe 3O 4进行表面改性，使其成为水溶液，且表面功能上活性基团羧基，令通过酯键将负载维甲酸的环糊精连在Gd:Fe 3O 4表面，即：通过纳米材料表面的邻羟基苯-PEG-羧基端化合物与载有维甲酸的环糊精表面的羟基进行反应，通过酯键连接，从而制备成Gd:Fe 3O 4@RA。
步骤1中Fe/Gd单源前驱体通过Fe源和Gd源制备，其中摩尔比为5：3。
步骤1中的逐步升温包括两个进程，分别为100-200℃加热30分钟，200-350℃加热30分钟。
步骤1中升温加热过程需要在惰性气体保护下进行
步骤1中的制备的Gd:Fe 3O 4的直径为7-10nm,较小的直径有益于其进入细胞和减少细胞毒性。
步骤2中活性聚合物为邻羟基苯-PEG-羧基端化合物，平均分子量为4000-8000。
步骤2中表面修饰活性聚合物后，需要搅拌条件下，在蒸馏水中透析24小时除去未连接的聚合物。
步骤2中，在酸性和加热约80℃的条件下，将载有维甲酸的环糊精通过酯键将其羟基与纳米粒子表面的活性基团羧基进行反应，从而制备Gd:Fe 3O 4@RA。
步骤2中，制备的Gd:Fe 3O 4@RA需要通过丙烯葡聚糖凝胶进行提纯，待用。
步骤2中，制备的Gd:Fe 3O 4@RA溶于水溶液中得到水溶液或冻干保存。
一种Gd:Fe 3O 4@RA纳米粒子诱导神经干细胞定向分化为神经元细胞的实验方法，包括以下步骤：
在培养板中放入预先用多聚赖氨酸处理的盖玻片，将来源于大鼠胚胎脑海马的原代和传代神经干细胞以1×10 7L -1分别接种到培养孔中，神经干细胞完全培养液中去除碱性成纤维细胞生长因子，并添加1.0ml,浓度为1mg/ml的Gd:Fe 3O 4@RA复合纳米离子和体积分数为5％的胎牛血清促进神经干细胞分化，培养6天后，继续在含有体积分数为5％胎牛血清的神经干细胞培养基中培养6天，待细胞形态发育成熟后取出盖玻片；
将载有常规贴壁分化培养和Gd:Fe 3O 4@RA复合纳米离子诱导分化培养的细胞的盖玻片各分为2组，分别神经元标志物标记的β-微管蛋白(TUJ1)的免疫组化；
随机选取N张片子作为样本，每个样本系统随机取N个视野，N为自然数，以荧光标记为阳性，按照下列公式计算出各个片子神经干细胞分化为神经元细胞的百分率：
神经元细胞百分率＝(荧光标记阳性细胞数/细胞总数)×100％；将Gd:Fe 3O 4@RA复合纳米离子分化培养的神经干细胞与常规贴壁分化培 养的神经干细胞比较。
一种Gd:Fe 3O 4@RA复合纳米分子用于脊髓神经损伤的修复及MRI造影方法，包括以下步骤：
步骤1、大鼠脊髓损伤造模后第7天，对大鼠进行10％水合氯醛腹腔麻醉、固定及损伤处再暴露，将含有1×10 6个Gd:Fe 3O 4@RA标记的神经干细胞、未标记的神经干细胞悬液10μl或PBS(“PBS”为磷酸盐平衡生理盐水，pH＝7.4)，分别用微量注射器，进针方向朝向损伤处中心区，进针角度30度，进针深度约2mm,注入损伤处头端的边缘区，距损伤中心点约5mm，注射速率为2.5μl/分钟，注射完成后针头留在注射点2分钟利于细胞向周围扩散；
步骤2、大鼠腹腔麻醉后，固定于大鼠磁共振扫描专用线圈下，对移植入体内标记的神经干细胞进行活体示踪可以通过检测标记T1-MRI信号上的改变在体内追踪移植细胞的迁移与分布。
本发明的机理为：本发明的Gd:Fe 3O 4@RA纳米粒子应用于干细胞的定向分化为神经原细胞用于中枢神经损伤的修复，同时用于实时的MRI造影考察干细胞移植后对神经功能或行为的作用，和在活体内的迁移的动态示踪。毒性检测证明本发明的Gd:Fe 3O 4和Gd:Fe 3O 4@RA对神经干细胞及间充质干细胞等几乎没有毒性。免疫荧光染色证明了Gd:Fe 3O 4@RA具有促进经干细胞及间充质干细胞等定向分化为神经元细胞。同时，通过在大鼠脊髓损伤病灶区注射标记有Gd:Fe 3O 4@RA的神经干细胞，可有效用于用于脊髓损伤的修复，并可对病灶区进行实时的MRI加权造影，同时也可清楚实时观测到注射的干细胞在体内的聚集，分化和迁移状态。
该多功能负载维甲酸的钆参杂四氧化三铁复合纳米粒子具有以下有益效果：
(1)本发明采用简单的方式制备Gd:Fe 3O 4@RA纳米粒子，此法工艺简单，反应条件温和，易于操作分离，环境友好，价格低廉，重复性强，具有产业化实施的前景
(2)本发明提出的Gd:Fe 3O 4@RA纳米粒子在干细胞的神经元分化中的应用，并提出用于中枢神经损伤的修复及MRI无侵实时造影。
附图说明
图1是本发明中Gd:Fe 3O 4@RA纳米粒子的透射电镜图A。
图2是本发明中Gd:Fe 3O 4@RA纳米粒子的透射电镜图B。
图3是本发明中移植Gd:Fe 3O 4@RA标记的干细胞后第七天，大鼠脊柱的MRI成像，其中白色亮点为Gd:Fe 3O 4@RA标记的干细胞聚集区域，黑色区域为脊髓损伤的区域示意图。
图4是本发明中移植Gd:Fe 3O 4@RA标记的干细胞，未标记的干细胞和PBS后脊髓损伤大鼠的BBB运动评分示意图。
具体实施方式
下面结合图1至图4，对本发明做进一步说明：
实施例1：钆参杂四氧化三铁复合纳米粒子的制备：
首先，将1.88g苯甲酸钠溶液加入100ml蒸馏水中，然后加入含有0.5g FeCl 2.4H 2O和0.46gGdCl 3.2H 2O的水溶液中，将25毫升乙腈加入到该溶液中；反应混合物在室温下搅拌至少12小时，得到浅棕色沉淀物；该浅棕色沉淀物为Fe/Gd单源前驱体,经过滤后用乙醚洗涤,真空泵干燥，获得Fe/Gd干燥前驱体。
接下来将0.2g制备的Fe/Gd干燥前驱体加入含有油胺、油酸和苄醚的反应混合物中，比例为体积比为4.5ml∶3ml∶2.5ml，然后在氮气流下加热到110℃保持30分钟，然后温度缓慢加热到350℃保持30分钟得到黑色/棕色物质；冷却至室温后分离，加入至少10ml的乙醇，通过洗涤和再分离，得到的黑色/棕色的Gd:Fe 3O 4纳米粒子，最后在真空干燥，将得到直径为7-10纳米的Gd:Fe 3O 4纳米粒子，该纳米粒子能在室温下稳定存在，容易保存。通过透射电子显微镜观察，结果如图1所示。
实施例2：多功能负载维甲酸的钆参杂四氧化三铁复合纳米粒子的制备：
将0.02克Gd:Fe 3O 4纳米粒子加入到含有0.1g活性聚合物邻羟基苯-PEG-羧基端化合物(平均分子量为4000)的10毫升CHCl 3中；然后将混合物在室温下在黑暗中搅拌12小时；加入20ml正己烷，离心沉淀得到表面功能化的Gd掺杂四氧化三铁纳米粒子Gd:Fe 3O 4@DIB-PEG-COOH(邻羟基苯-PEG-羧基端化合物)，经10ml CHCl 3和正己烷(1:5v/v)洗涤后，分散于5ml蒸馏水中，用蒸馏水透析24小时，除去未反应的有机分子，并储存于黑暗中。
其次，利用酸催化的酯键制备将载有维甲酸的环糊精与纳米粒子表面的羧基进行相连，从而完成制备Gd:Fe 3O 4@RA：将1ml的HCl溶液(浓度为：1mol/L)与1mlGd:Fe 3O 4@DIB-PEG-COOH(邻羟基苯-PEG-羧基端化合物)溶液混合搅拌30分钟，然后加入0.05g维甲酸-β-环糊精包合物(分子比维甲酸：β-环糊精＝6:1),搅拌均匀后，混合物保持在黑暗中以及80℃，并在旋转摇动器上以15-30rpm/min速度搅拌，并反应至少12小时。最后，通过丙烯葡聚糖凝胶色谱柱进行分离，从而得到负载维甲酸的钆参杂四氧化三铁复合纳米粒子Gd:Fe 3O 4@RA，冻干保存。通过透射电子显微镜观察，制备的Gd:Fe 3O 4@RA尺寸约为15-30nm,结果如图2所示。
实施例3：多功能负载维甲酸的钆参杂四氧化三铁复合纳米粒子诱导神经干细胞定向分化为神经元细胞：
在24孔培养板中放入预先用多聚赖氨酸处理的盖玻片，将来源于大鼠胚胎脑海马的原代和传代神经干细胞以1×10 7L -1分别接种到培养孔中，神经干细胞完全培养液中去除碱性成纤维细胞生长因子，并添加1.0ml,1mg/ml的Gd:Fe 3O 4@RA和体积分数为5％的胎牛血清促进神经干细胞分化，培养6天后，继续在含有体积分数为5％胎牛血清的神经干细胞培养基中培养6d，待细胞形态发育成熟后取出盖玻片。将载有常规贴壁分化培养和诱导分化培养的细胞的盖玻片各分为2组，分别神经元标志物标记的β-微管蛋白(TUJ1)的免疫组化。随机选取5张片子作为样本，每个样本系统随机取5个视野，以荧光标记为阳性， 按照下列公式计算处神经干细胞分化为神经元细胞的百分率：
神经元细胞百分率＝(荧光标记阳性细胞数/细胞总数)×100％；
结果显示，与常规贴壁分化培养的神经干细胞比较，Gd:Fe 3O 4@RA诱导分化培养后，神经干细胞向神经元分化率明显升高，分化率从19.73±1.32提高到38.06±2.78。
实施例4：多功能负载维甲酸的钆参杂四氧化三铁复合纳米粒子诱导神经干细胞定向分化为神经元细胞用于脊髓神经损伤的修复及MRI造影:
制作大鼠脊髓损伤模型，大鼠用10％水合氯醛腹腔麻醉。将大鼠俯卧位固定于鼠板上，在T9-T10节段行椎板切除术，取自制打击器(重109，直径2.5mm)，自25mm高处自由落体垂直打击T9节段脊髓(打击后硬脊膜不应撕破)，依次缝合肌肉和皮肤。术后3天给予大鼠腹腔注射庆大霉素(8mg/kg/天)以预防泌尿道感染。术后每天两次人工协助排尿直至大鼠恢复自主排尿功能。
大鼠脊髓损伤造模后第7天，对大鼠进行10％水合氯醛腹腔麻醉、固定及损伤处再暴露。将含有1×10 6个Gd:Fe 3O 4@RA标记的神经干细胞、未标记的神经干细胞悬液10μl或PBS(“PBS”为磷酸盐平衡生理盐水，pH＝7.4)，用微量注射器(进针方向朝向损伤处中心区，进针角度30度，进针深度约2mm,注入损伤处头端的边缘区(距损伤中心点约5mm)。注射速率为2.5μl/分钟，注射完成后针头留在注射点2分钟(利于细胞向周围扩散)。
大鼠腹腔麻醉后，固定于大鼠磁共振扫描专用线圈下，对移植入体内标记的神经干细胞进行活体示踪可以通过检测标记T1-MRI信号上的改变在体内追踪移植细胞的迁移与分布。“T1-MRI信号”指的是T1加权的MRI信号，为信号增强，先明亮的信号。临床上常用的MRI信号的方式。
移植7天后，3T MRI下可以清晰的见到损伤原位的T1高信号，分布于注射点及损伤区域周围；14天后注射点周围的T1信号强度有所降 低，而注射点和损伤区中心之间的区域有弥散的高密度影。
如图4所示，对Gd:Fe 3O 4@RA标记的神经干细胞和未标记的神经干细胞移植后的大鼠运动功能恢复进行评价。从脊髓损伤后14天开始，两组经干细胞移植组的BBB评分与PBS对照组的评分相比有显著性的增高(P<0.05)。并且移植Gd:Fe 3O 4@RA标记的神经干细胞的大鼠和移植未标记神经干细胞的大鼠的BBB评分也有显著性差异(P<0.05)。这说明Gd:Fe 3O 4@RA可有效地诱导神经干细胞的定向分化，能够明显改善损伤大鼠的运动功能，完成脊髓损伤的修复。
BBB(Basso,Beattie&Bresnahan locomotor rating scale)评分,即大鼠脊髓损伤评分标准，是根据观察脊髓损伤大鼠经过三个阶段的恢复而建立的。
1、早期：以无或极少的后肢关节运动为特征。
2、中期：包括几次共济失调步态。
3、晚期：包括精细运动，如拖着脚趾和尾巴，躯干不稳定以及爪子交替轮转。
大体上，21种评分表与恢复的进展是同步的。
第一部分，0-7类，评估恢复早期的后肢关节运动。
0.未见后肢运动。
1.一个或两个关节的轻微运动，通常是髋关节和/或膝关节。
2.一个关节的广泛运动或一个关节的广泛运动加上其它关节的轻微运动。
3.两个关节的广泛运动。
4.后肢三个关节的轻微运动(髋关节，膝关节和踝关节)。
5.两个关节的轻微运动和另一个关节的广泛运动。
6.两个关节的广泛运动和另一个关节的轻微运动。
7.后肢三个关节的广泛运动。
第二部分，8-13类，评估恢复中期的步态和协调运动。
8.无负重拖动或足置于无负重位。
9.足底仅位于负重位，或偶尔/频繁/持续的足背负重步行，无足底步行。
10.偶尔负重步行，无前后肢协调运动。
11.频繁到持续的负重步行，无前后肢协调运动。
12.频繁到持续的负重步行，偶有前后肢协调运动。
13.持续的负重步行，频繁的前后肢协调运动。
第三部分，14-21类，评估运动时爪子的精细运动。
14.持续协调步态，持续前后肢运动协调；运动时优势爪旋转或频繁足底步行，持续前后肢运动协调和偶尔的足背步行。
15.持续协调步态；当前肢前进时无或偶有伸趾；优势爪刚触地时与身体平行。
16.持续协调步态；频繁伸趾；优势爪触地时与身体平行，提起时旋转。
17.持续协调步态；频繁伸趾；优势爪在触地及提起时均与身体平行。
18.持续协调步态；持续伸趾；优势爪在触地与身体平行，提起时旋转。
19.持续协调步态；持续伸趾；优势爪在触地及提起时均与身体平行。
20.持续协调步态；持续伸趾；优势爪在触地及提起时均与身体平行；但躯体不稳定，尾巴持续上翘。
21.持续协调步态；持续伸趾；优势爪在触地及提起时均与身体平行；躯体稳定，尾巴持续上翘。
上面结合附图对本发明进行了示例性的描述，显然本发明的实现并不受上述方式的限制，只要采用了本发明的方法构思和技术方案进行的各种改进，或未经改进将本发明的构思和技术方案直接应用于其它场合的，均在本发明的保护范围内。

Claims (9)

  1. 一种多功能负载维甲酸的钆参杂四氧化三铁复合纳米粒子，其特征在于：其用于用于诱导干细胞定向分化及用于实时MRI造影；所述钆参杂四氧化三铁为MRI造影剂及纳米载体，其负载的维甲酸为活性成分，其具有促进干细胞分化为神经元细胞的作用；
    所述的钆参杂四氧化三铁纳米粒子结构式可表述为Gd:Fe 3O 4,所述的多功能负载维甲酸的钆参杂四氧化三铁纳米粒子的结构式可表述为Gd:Fe 3O 4@RA,其中：@为负载的意思，RA代表着维甲酸。
  2. 根据权利要求1所述多功能负载维甲酸的钆参杂四氧化三铁复合纳米粒子，其特征在于：所述的钆参杂四氧化三铁纳米粒子制备方法如下：
    制备了Fe/Gd单源前驱体，然后加入到油胺、油酸和苄醚的反应混合物中，逐步升温加热，后冷却至室温，得到黑色/棕色物质，分离、乙醇洗涤再分离，干燥得到Gd:Fe 3O 4。
  3. 根据权利要求1和2所述多功能负载维甲酸的钆参杂四氧化三铁复合纳米粒子，其特征在于：所述的多功能负载维甲酸的钆参杂四氧化三铁纳米粒子制备方法如下：
    用活性聚合物对Gd:Fe 3O 4进行表面改性，使其成为水溶液，且表面功能上活性基团羧基，令通过酯键将负载维甲酸的环糊精连在Gd:Fe 3O 4表面，即：通过纳米材料表面的邻羟基苯-PEG-羧基端化合物与载有维甲酸的环糊精表面的羟基进行反应，通过酯键连接，从而制备成Gd:Fe 3O 4@RA。
  4. 一种多功能负载维甲酸的钆参杂四氧化三铁复合纳米粒子的制 备方法，包括以下步骤：
    步骤1、制备了Fe/Gd单源前驱体，然后加入到油胺、油酸和苄醚的反应混合物中，逐步升温加热，后冷却至室温，得到黑色/棕色物质，分离、乙醇洗涤再分离，干燥得到Gd:Fe 3O 4；
    步骤2、用活性聚合物对Gd:Fe 3O 4进行表面改性，使其成为水溶液，且表面功能上活性基团羧基，令通过酯键将负载维甲酸的环糊精连在Gd:Fe 3O 4表面，即：通过纳米材料表面的邻羟基苯-PEG-羧基端化合物与载有维甲酸的环糊精表面的羟基进行反应，通过酯键连接，从而制备成Gd:Fe 3O 4@RA。
  5. 根据权利要求4所述多功能负载维甲酸的钆参杂四氧化三铁复合纳米粒子的制备方法，其特征在于：
    步骤1中Fe/Gd单源前驱体通过Fe源和Gd源制备，其中两者的摩尔比为5：3；
    步骤1中的逐步升温加热包括两个进程，分别为100-200℃加热30分钟，200-350℃加热30分钟，步骤1中逐步升温加热需要在惰性气体保护下进行；
    步骤1中的制备的Gd:Fe 3O 4的直径为7-10nm,该较小的直径有益于其进入细胞和减少细胞毒性。
  6. 根据权利要求4所述多功能负载维甲酸的钆参杂四氧化三铁复合纳米粒子的制备方法，其特征在于：
    步骤2中活性聚合物为邻羟基苯-PEG-羧基端化合物，平均分子量为4000-8000；
    步骤2中表面修饰活性聚合物后，需要搅拌条件下，在蒸馏水中透析24小时除去未连接的聚合物；步骤2中，在酸性和加热约80℃的条件下，将载有维甲酸的环糊精通过酯键将其羟基与纳米粒子表面的活性基团羧基进行反应，从而制备Gd:Fe 3O 4@RA。
  7. 根据权利要求4所述多功能负载维甲酸的钆参杂四氧化三铁复合纳米粒子的制备方法，其特征在于：
    首先、将1.88g苯甲酸钠溶液加入100ml蒸馏水中，然后加入含有0.5g FeCl 2.4H 2O和0.46gGdCl 3.2H 2O的水溶液中，将25毫升乙腈加入到该溶液中；反应混合物在室温下搅拌至少12小时，得到浅棕色沉淀物；该浅棕色沉淀物为Fe/Gd单源前驱体,经过滤后用乙醚洗涤,真空泵干燥，获得Fe/Gd干燥前驱体；
    其次、接下来将0.2g制备的Fe/Gd干燥前驱体加入含有油胺、油酸和苄醚的反应混合物中，比例为体积比为4.5ml∶3ml∶2.5ml，然后在氮气流下加热到110℃保持30分钟，然后温度缓慢加热到350℃保持30分钟得到黑色/棕色物质；冷却至室温后分离，加入至少10ml的乙醇，通过洗涤和再分离，得到的黑色/棕色的Gd:Fe 3O 4纳米粒子，最后在真空干燥，将得到直径为7-10纳米的Gd:Fe 3O 4纳米粒子；
    再次、将0.02克Gd:Fe 3O 4纳米粒子加入到含有0.1g活性聚合物邻羟基苯-PEG-羧基端化合物(平均分子量为4000)的10毫升CHCl 3中；然后将混合物在室温下在黑暗中搅拌12小时；加入20ml正己烷，离心沉淀得到表面功能化的Gd掺杂四氧化三铁纳米粒子Gd:Fe 3O 4@DIB-PEG-COOH(邻羟基苯-PEG-羧基端化合物)，经10ml CHCl 3和正己烷(1:5v/v)洗涤后，分散于5ml蒸馏水中，用蒸馏水透析24小时，除去未反应的有机分子，并储存于黑暗中；
    从次、利用酸催化的酯键制备将载有维甲酸的环糊精与纳米粒子表面的羧基进行相连，从而完成制备Gd:Fe 3O 4@RA：将1ml的HCl溶液(浓度为：1mol/L)与1mlGd:Fe 3O 4@DIB-PEG-COOH(邻羟基苯-PEG-羧基端化合物)溶液混合搅拌30分钟，然后加入0.05g维甲酸-β-环糊精包合物(分子比维甲酸：β-环糊精＝6:1),搅拌均匀后，混合物保持在黑暗中以及80℃，并在旋转摇动器上以15-30rpm/min速度搅拌，并反应至少12小时；
    最后，通过丙烯葡聚糖凝胶色谱柱进行分离，从而得到负载维甲 酸的钆参杂四氧化三铁复合纳米粒子Gd:Fe 3O 4@RA，冻干保存。
  8. 一种Gd:Fe 3O 4@RA纳米粒子诱导神经干细胞定向分化为神经元细胞的实验方法，包括以下步骤：
    在培养板中放入预先用多聚赖氨酸处理的盖玻片，将来源于大鼠胚胎脑海马的原代和传代神经干细胞以1×10 7L -1分别接种到培养孔中，神经干细胞完全培养液中去除碱性成纤维细胞生长因子，并添加1.0ml,浓度为1mg/ml的Gd:Fe 3O 4@RA复合纳米离子和体积分数为5％的胎牛血清促进神经干细胞分化，培养6天后，继续在含有体积分数为5％胎牛血清的神经干细胞培养基中培养6天，待细胞形态发育成熟后取出盖玻片；
    将载有常规贴壁分化培养和Gd:Fe 3O 4@RA复合纳米离子诱导分化培养的细胞的盖玻片各分为2组，分别神经元标志物标记的β-微管蛋白(TUJ1)的免疫组化；
    随机选取N张片子作为样本，每个样本系统随机取N个视野，N为自然数，以荧光标记为阳性，按照下列公式计算出各个片子神经干细胞分化为神经元细胞的百分率：
    神经元细胞百分率＝(荧光标记阳性细胞数/细胞总数)×100％；将Gd:Fe 3O 4@RA复合纳米离子分化培养的神经干细胞与常规贴壁分化培养的神经干细胞比较。
  9. 一种Gd:Fe 3O 4@RA复合纳米分子用于脊髓神经损伤的修复及MRI造影方法，包括以下步骤：
    步骤1、大鼠脊髓损伤造模后第7天，对大鼠进行10％水合氯醛腹腔麻醉、固定及损伤处再暴露，将含有1×10 6个Gd:Fe 3O 4@RA标记的神经干细胞、未标记的神经干细胞悬液10μl或PBS(“PBS”为磷酸盐平衡生理盐水，pH＝7.4)，分别用微量注射器，进针方向朝向损伤处中心区，进针角度30度，进针深度约2mm,注入损伤处头端的边缘区， 距损伤中心点约5mm，注射速率为2.5μl/分钟，注射完成后针头留在注射点2分钟利于细胞向周围扩散；
    步骤2、大鼠腹腔麻醉后，固定于大鼠磁共振扫描专用线圈下，对移植入体内标记的神经干细胞进行活体示踪可以通过检测标记T1-MRI信号上的改变在体内追踪移植细胞的迁移与分布。
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