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本文内容

  • 摘要
  • 摘要
  • 引言
  • 研究方案
  • 结果
  • 讨论
  • 披露声明
  • 致谢
  • 材料
  • 参考文献
  • 转载和许可

摘要

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

摘要

细胞和治疗剂的靶向递送将通过浓缩的治疗效果在目标部位，同时最小化有害的影响，以脱靶部位受益广泛的生物医学应用。磁性细胞靶向是一种高效，安全，简单的交付技术。超顺磁性氧化铁纳米颗粒（SPION）是生物可降解的，生物相容的，并且可以被内吞进入细胞，使它们响应于磁场。合成过程涉及创建磁铁矿铁 （Fe 3 O 4）纳米颗粒随后高速乳化以形成聚（乳酸-共-乙醇酸）（PLGA）的涂层。所述PLGA-磁铁矿SPIONs是大约120纳米的直径，包括约10nm直径磁铁矿核心。时，放置在培养基中，SPIONs自然被细胞内吞和细胞质内内涵体小簇存储。这些颗粒赋予足够的磁质细胞以允许磁场内定位。许多细胞分选和靶向应用是由呈现各种细胞类型响应于磁场启用。 SPIONs具有多种其它生物医学应用，以及包括用作医学成像造影剂，靶向药物或基因递送，诊断测定，并生成局部热疗的肿瘤治疗或组织焊接。

引言

Targeted delivery and capture of cells to specific sites within the body is desirable for a variety of biomedical applications. Delivery of neural stem cells to the brain by MRI-guided focused ultrasound has been proposed as a possible treatment option for neurodegenerative disease, traumatic brain injury, and stroke1. Mesenchymal stem cells are being studied for their ability to deliver anti-cancer drugs to tumors due to their natural tumor-tropic properties2,3. Cardiac stem cells have been delivered to the heart as a possible treatment for myocardial infarction4,5. Vascular stents have been developed with CD34 antibodies to capture circulating progenitor cells6. While promising, these cell targeting approaches present drawbacks including lack of cell specificity, inconsistent cell retention, and off-target cell delivery.

The overall goal of the current method is to enable magnetically directed targeting of cells for a variety of cell delivery and sorting applications. Magnetic targeting allows for controlled delivery of specific cells to a specific target site with minimal off-target effects7. The magnetic fields can be generated by implanted or external devices to safely direct the movement of magnetically-labeled cells within the body8. Numerous research efforts have focused on magnetically directed targeting of stem cells to injured tissues such as the heart9-14, retina15, lung16, skin17, spinal cord18,19, bone20, liver21, and muscle22,23 in order to improve regeneration outcomes.

Magnetic targeting of cells has also been studied extensively as a means to endothelialize implantable cardiovascular devices. A uniform and complete endothelium provides a barrier between the device and circulating blood elements to mitigate thrombosis and inflammation. Endothelial cells can be delivered to the device either prior to implantation or via the vascular system following implantation. In both cases, magnetic fields are used to capture cells to the surface of the device and retain the cells when subjected to the shear stress generated by circulating blood. Magnetic vascular stents24-27 and vascular grafts28 have both been fabricated and tested for this purpose.

Magnetic cell targeting requires a strategy for labeling cells with magnetic carrier particles. These particles can be bound to the surface of cells via antibodies or ligand/receptor pairs or they can be endocytosed into the cells. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and readily endocytosed by a variety of cell types29. These particles effectively render a cell responsive to magnetic fields and are naturally degraded over time. SPIONs provide a straightforward and safe means of magnetically labeling cells in culture for a variety of magnetic targeting and sorting applications. A method for synthesizing SPIONs with a magnetite (Fe3O4) core and poly(lactic-co-glycolic acid) (PLGA) shell is provided. In addition, a method for labeling cells in culture with SPIONs is provided.

研究方案

1.合成磁铁矿凝胶

  1. 通过用浓盐酸，随后通过离子交换水接着乙醇清洗所有玻璃器皿。晾干O / N，优选在干燥炉中。
    注意！盐酸是有害的 - 佩戴个人防护装备和工作在通风橱;酒精是有害的 - 佩戴个人防护装备。
  2. 使用Dreschel瓶中脱气500ml的去离子H 2 O的通过轻轻鼓泡N 2气30分钟。
  3. 建立的化学通风橱内的磁铁矿合成装置。
    1. 放置的500毫升三颈圆底烧瓶中的电热套加热器内并使用夹子固定中心颈和站立。
    2. 安装橡胶隔片进入圆底烧瓶的侧颈之一和回流冷凝器用橡胶隔片进入的剩余侧颈部。不断通过回流冷凝器运行冷水。
    3. 穿刺个Ë圆底烧瓶的橡胶隔片与连接到N 2气体管线针和穿刺回流冷凝器的橡胶隔片与连接到运行到鼓泡的气体线的针（即，烧瓶与水）以可视化气体流出。
    4. 经由桨适配器安装刀片桨到圆底烧瓶的中心颈。附加叶片桨的轴，顶部搅拌器安装到一个支架上。
  4. 吹扫的圆底烧瓶用N 2气和离开N 2气流入以低，但检测率。
  5. 从圆底烧瓶中去除回流冷凝器和添加1.000克铁（III），氯化0.6125克铁（Ⅱ）四水合氯化和50脱气H 2 O的溶液
    注意！氯化铁和氯化亚铁四是有害的 - 穿戴个人防护装备。
  6. 替换回流冷凝器和搅拌以1,000rpm，同时加热到50C。在这些条件下搅拌产生10纳米直径磁铁矿纳米颗粒。
  7. 一旦在50℃下，通过在橡胶隔膜的圆底烧瓶注入，同时仍然搅拌加入10毫升28％的氢氧化铵溶液。
    注意！氢氧化铵是有害的 - 佩戴个人防护装备。
    注:氢氧化铵溶液用于以沉淀磁铁矿和溶液应变黑。
  8. 取下橡胶隔膜和 N 2气体管线从圆底烧瓶中，并加热到90℃，蒸出掉氨气，同时仍然搅拌。
    注意:这是可选的通过刺穿回流冷凝器的橡胶隔片保持N 2流入圆底烧瓶中，然而，磁铁矿到磁赤铁矿的氧化作用是在此步骤中可忽略不计。
  9. 一旦达到90℃，加入1 ml的油酸向圆底烧瓶，同时仍然搅拌。油酸是用于涂覆磁铁矿呐noparticles形成磁铁矿凝胶。
    注意！油酸是有害的 - 佩戴个人防护装备。
  10. 更换橡胶隔膜和 N 2气体管线到圆底烧瓶中并取出回流冷凝器。
  11. 熄火和搅拌在500rpm下2小时。
  12. 从电热套加热器除去圆底烧瓶中，并在使用保持抵靠烧瓶保留磁铁矿凝胶底部强磁铁倒出任何剩余的液体。
    注意！处理强力磁铁格外小心，以免损坏或人身伤害。
  13. 允许磁铁矿凝胶风干O / N（可选）。

2.净化磁铁矿凝胶

  1. 加入40毫升己烷进圆底烧瓶以溶解磁铁矿凝胶
    注意！正己烷是有害的 - 佩戴个人防护装备和工作在通风橱。
  2. 使用分液漏斗用40ml脱气H 2 O的，以去除残留的H 2 O的往复米磁铁矿的解决方案。
    1. 慢慢倒入分液漏斗内的磁铁矿溶液到的 H 2 O和轻轻摇动两相液5分钟。
    2. 排出并丢弃下部水分数。
    3. 慢慢加入40毫升去脱气的 H 2 O至分液漏斗中使得其沉降的磁铁矿溶液下方并轻轻地旋转并如前排水。
    4. 重复洗第三次。
  3. 转移磁铁矿溶液至锥形瓶中，加几铲值得无水硫酸钠，并乱舞从磁铁矿溶液中除去任何剩余的残留H 2 O的。
  4. 过滤通过1微米的滤纸的磁铁矿溶液在过滤漏斗以除去硫酸钠和残余H 2 O
    注:建议使用真空辅助。
  5. 磁铁矿溶液转移至50毫升烧瓶中蒸发，并使用旋转蒸发器以蒸发己烷为在以下条件下2小时:中等旋转速度，施加真空，在50℃的水浴蒸发烧瓶中，并通过冷凝器24℃的循环水。
    注:可选，存储涂覆PLGA之前磁铁矿凝胶。

3.涂层的磁性纳米颗粒与PLGA壳牌

  1. 溶解3.60克的PLGA（75/25共混物）于240ml乙酸乙酯中以创建一个1.5％（M / V）溶液。注意:醋酸乙酯是有害的 - 佩戴个人防护装备和工作在通风橱。
  2. 用磁力搅拌器以创建一个5.0％（M / V）溶液溶解25.00克尼克F-127的在500毫升脱气H 2 O的。
    注:尼克F-127是充当一个生物相容的表面活性剂的非离子两亲嵌段共聚物。它有助于稳定在步骤3.3.2油包水乳液。
  3. 使用microspatula，收集磁铁矿凝胶成加权玻璃瓶中在六0.040克等分。 PERFORM下面的涂层和洗涤过程中的每个等份。
    注:等份是必要的，以确保有效的处理和磁倾析，这将最大化纯度和收率，同时最小化在步骤4中降解前，冷冻干燥。
    1. 添加磁铁矿凝胶的0.040克等分试样和40毫升的PLGA溶液至塑料烧杯中，并声处理的在超声波清洗10分钟。
    2. 添加80毫升的Pluronic溶液到塑料烧杯中，并立即用实验室混合器乳化在最高设定为7分钟，以在磁铁矿纳米颗粒为油包水乳液中的PLGA涂层。
    3. 立即稀释SPION溶液在1升去离子H 2 O和超声的1小时在化学通风橱中以蒸发乙酸乙酯。
    4. 一个强有力的磁铁旁SPION解决方案，并轻轻搅拌，收集棕SPIONs的磁铁。
      注意:这可能是必要的，以间歇性地搅拌几个小时该溶液s之前变成发白，表明大多数SPIONs的已收集。
    5. 滗水溶液，同时保留SPIONs与磁铁的烧杯中。
    6. 如下洗SPIONs三次。
      1. 暂停SPIONs于1L去离子H 2 O的
      2. 超声清洗20分钟的SPION解决方案。
      3. 一个强有力的磁铁旁SPION解决方案，并轻轻搅拌，收集棕SPIONs的磁铁。它可能需要间歇地搅拌几小时前的溶液变清，表明大多数SPIONs的已收集。
      4. 滗水溶液，同时保留SPIONs与磁铁的烧杯中。
  4. 收集来自六个磁铁矿凝胶等分的合成为一个单一的加权玻璃小瓶作为含水悬浮液的SPIONs。根据需要可选择倒出多余的水磁。

4.冻结SPIONs的-drying

  1. 冻结SPION解决方案。
  2. 冷冻干燥SPION解决方案O / N在冻干机。
  3. 称重冻干SPIONs。冻干SPIONs可以储存在-20℃，直到用于细胞标记。
    注:储存在-20℃下显着降低降解动力学，增加的货架寿命。

5.标记细胞SPIONs的

  1. 暂停在磷酸盐缓冲盐水（PBS）SPIONs的等分试样以40mg / ml的浓度，并超声处理30分钟。
  2. 添加SPION溶液到细胞的几乎汇合烧瓶中的细胞培养基的5微升/毫升的浓度。确保均匀分布轻摇烧瓶中。
  3. 孵育细胞16小时，在37℃。
  4. 轻轻吸培养基并用PBS洗涤细胞两次。
  5. 收集磁标记的细胞和用于实验。
  6. 未使用的SPION溶液可以储存在4℃，并应我们在短短几个月内编。超声清洗每次使用前30分钟。

结果

磁铁矿纳米颗粒是大约10的直径为在50℃下，以1,000rpm（图1）搅拌铁（III），氯化铁（Ⅱ）四水合氯化的水溶液的结果纳米。这些结果表明成功合成磁铁矿纳米粒子。它来验证从批次的一个小样本试图合成首次时采取磁铁矿纳米粒子的大小和形状是重要的。透射电子显微镜（TEM）是用于可视化这些粒子的首选方法。将批料应该被丢弃，合成应再次尝试，如果磁铁矿纳米颗粒不约10nm的直...

讨论

正如任何纳米颗粒合成的协议中，反应物的化学物质的纯度是实现高质量SPIONs，将具有最小的细胞毒性作用是至关重要的。它购买很纯试剂，包括油酸（≥99％），铁（II），氯化四水合物（≥99.99％），铁（III），氯化（≥99.99％），乙酸乙酯因此重要（HPLC级，≥99.9％ ），己烷（HPLC级，≥97.0％），氢氧化铵（≥99.99％）和硫酸钠（≥99.0％）。它是特别重要的，购买很纯和高品质的PLGA，其可以?...

披露声明

The authors declare that they have no competing financial interests.

致谢

The authors wish to acknowledge funding from the European Regional Development Fund – FNUSA-ICRC (no. CZ.1.05/ 1.1.00/ 02.0123), the American Heart Association Scientist Development Grant (AHA #06-35185N), and the National Institutes of Health (NIH #T32HL007111).

材料

NameCompanyCatalog NumberComments
Ammonium Hydroxide solution, 28% NH3 in H2O, ≥99.99% trace metal basisSigma-Aldrich338818-100ML Harmful reagent - wear personal protective equipment
Dreschel bottle, 500 mlAce Glass5516-16
Ethyl Acetate, CHROMASOLVR Plus, for HPLC, 99.9% Sigma-Aldrich650528-1LHarmful reagent - wear personal protective equipment & work in fume hood
Ethyl alcoholSigma-AldrichE7023Harmful reagent - wear personal protective equipment
Evaporating flask, 50 ml, 24/40 jointSigma-AldrichZ515558For use with rotoevaporator
Filter paper, 3 cm dia, grade 1Fisher09-805PFor use with glass filter funnel
Glass beakers, 1 LFisherFB-101-1000For washing SPIONs
Glass filter funnel, vacuum hose adapter, fits 24/40, 30 mLFisherK954100-0344 
Glass vial capsFisher03-391-46For use with glass vials
Glass vials, 2 mlFisher03-391-44For collecting magnetite gel & SPIONs
Hexane, CHROMASOLVR, for HPLC, ≥97.0% (GC)Sigma-Aldrich34859-1L Harmful reagent - wear personal protective equipment & work in fume hood
Hydrochloric acidSigma-AldrichH1758Harmful reagent - wear personal protective equipment & work in fume hood
Iron(II) chloride tetrahydrate, ≥99.99% trace metals basis Sigma-Aldrich380024-5GHarmful reagent - wear personal protective equipment
Iron(III) chloride anhydrous, powder, ≥99.99% trace metals basisSigma-Aldrich451649-1GHarmful reagent - wear personal protective equipment
Isomantle heater, 500 mLVoight GlobalEM0500/CEX1
Laboratory mixerSilversonL5M-A
LyophilizerLabconco7670520
MicrospatulasFisher21-401-25AFor transfering magnetite gel
NdFeB magnet, 1 in x 1 in x 1 inAmazing MagnetsC1000H-MVery strong magnet, handle with care
Oleic acid, ≥99% (GC)Sigma-AldrichO1008-5G Store in freezer; Harmful reagent - wear personal protective equipment
Overhead stirrerIKA2572201
Overhead stirrer clampIKA2664000For use with overhead stirrer
Overhead stirrer H-standIKA1412000For use with overhead stirrer
Phosphate buffered salineLife Technologies10010-023
Plastic beakers, 250 mlFisher02-591-28
PLGA PURASORB PDLG (75/25 blend)PuracPDLG 7502PDLG 7502A may be used as well; Store in freezer
Pluronic F-127 powder, BioReagent, suitable for cell cultureSigma-AldrichP2443-250G 
PTFE expandable blade paddle, 8 mm diaSciQuipSP4018
PTFE vessel adapter, fits 24/40, 8 mm dia paddleMonmouth ScientificPTFE Vessel Adaptor A480For use with PTFE expandable blade paddle
Recirculating chillerClarkson696613For use with rotoevaporator
Reflux condenser, fits 24/40, 250 mmAce Glass5997-133
RotoevaporatorClarkson216949
Rubber septa, fits 24/40Ace Glass9096-56
Separatory funnel with stopper, 250 mlFisher10-438E
Sodium sulfate ACS reagent, ≥99.0%, anhydrous, granularSigma-Aldrich239313-500G 
Three neck round bottom flask, angled, 24/40 joints, 500 mlAce Glass6948-16
Ultrasonic cleaner perforated panFisher15-335-20AFor use with ultrasonic cleaner
Ultrasonic cleaner, 2.8 LFisher15-335-20
Vacuum controllerClarkson216639For use with rotoevaporator (optional)
Vacuum pumpClarkson219959For use with rotoevaporator

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