从金属支架表面使用的腺病毒载体栓通过水解的交联剂血管基因转移
Summary
These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.
Abstract
支架内再狭窄呈现的广泛使用，以重新建立血液穿过冠状动脉和外周动脉的极为狭窄段流基于支架的血管成形术的主要并发症。可调谐释放基因具有抗再狭窄活性可能存在另一种战略血管内支架，以目前使用的药物洗脱支架。为了达到临床平移，基因洗脱支架必须具有支架固定化基因载体释放和血管系统的位点特异性转导的可预测的动力学，同时避免通常与用于所述载体的物理截留在聚合物涂层相关联的严重的炎症反应。本文描述了一种腺病毒基因载体coatless束缚到基于所述腺病毒颗粒的可逆结合支架的详细方法，以聚烯丙胺二膦酸盐（PABT）经由水解的交联剂（HC）改性不锈钢表面。一个家庭的双官能团（胺和硫醇反应性）的HC与中链酯水解测距5-50天的平均吨半被用于与所述支架连接的载体中。该载体的固定化过程典型地是在9小时，包括几个步骤：1）温育的金属样品在PABT（4小时）的水溶液; 2）脱保护以三（2 – 羧乙基）膦（20分钟）安装在PABT硫醇基; 3）膨胀的金属表面的巯基反应的能力通过使与聚乙烯亚胺衍生的吡啶基二硫（PDT）基团（2小时）的样品; 4）变换PDT基与硫醇与二硫苏糖醇（10分钟）; 5）修改腺病毒与HC（1小时）; 6）纯化修饰的腺病毒颗粒通过尺寸排阻柱色谱法（15分钟）和在硫醇化钢表面（1小时），巯基反应性的腺病毒颗粒7）固定。该技术具有超越支架宽的应用潜力，通过促进生物假体装置的表面工程通过基板介导的基因递送到细胞中的接口的植入外来物质，以提高其生物相容性。
Introduction
基因疗法作为治疗方法的有效性是通过基因治疗的载体1,2的差的定位能力受到阻碍。适当的定位结果中的转基因表达在目标位置的亚治疗水平的缺乏，导致载体对非靶器官3，包括那些负责安装针对两个矢量和编码的治疗产物4的免疫应答的广泛传播， 5。一电位是指，以抵消转导的滥交和促进目标是引入基因的载体在所需位置处在于，通过血液和淋巴排除其自由传播的形式。典型地，这样的努力依赖于局部注射的递送系统，包括混有纤维蛋白，胶原蛋白或透明质酸水凝胶基质6-10，其能够在注射部位短暂维持基因的载体通过物理截留第要么病毒或非病毒载体的时间在聚合网络。
用于局部基因治疗另一普遍接受的范例利用基因载体的固定化植入假体装置11,12的表面上。永久性医疗植入物（血管，支气管，泌尿系统和胃肠道支架，起搏器，人工关节，手术和妇科网等 ），每年都用在数以千万计的患者13。虽然通常是有效的，这些设备很容易出现并发症，是由当前的医疗行为14-17控制不佳的。植入假体装置提出了一个独特的机会，作为代理平台局部基因治疗。从药物动力学观点来看，医疗植入物与基因载体的结果相对低的输入剂量植入物/组织界面上实现既高局部浓度基因载体和减缓兵卫的动力学的表面衍生ř消除从这个位置。至于长期居留的后果和增强摄取目标细胞群，固定化载体的最小基因载体的传播。因而不慎接种的非靶组织中被减少。
在植入生物材料的基因的载体（也称为基底介导的基因递送或固相的基因递送）的表面束缚已在细胞培养和动物实验使用实施两个特定（抗原-抗体18-20生物素蛋白-生物素21,22）和非特异性23-26（充电，范德华力）的相互作用。共价连接的载体，以植入装置的表面之前已被视为非功能由于与表面过于强键排除向量的内化通过靶细胞。最近已证明，这种限制可以通过使用作为四面体的使用自发水解的交联剂来克服所述腺病毒载体27,28的支架和壳体蛋白的经修饰的金属表面之间存。此外，该载体释放速率和在体外和体内转基因表达的时间进程可以调制与使用可水解的交联剂呈现不同水解28的动力学。
本文件提供了腺病毒载体活化金属表面的可逆共价连接的详细协议，并引入了一个有用的实验装置来研究在体外随后传导事件在培养的平滑肌和内皮细胞和体内的支架成形术的大鼠颈动脉模型。
Protocol
1，准备Cy3标记的腺病毒为实验版 （; pH值9.3 CBB）在650微升的碳酸盐/碳酸氢盐缓冲液暂停2×10 12颗粒广告空 （约2×10 11感染单位）。 溶解的1瓶（0.2毫克）的胺反应性的荧光染料（Cy3标记（NHS）2）在1ml CBB的含量为0.2毫克/毫升的最终浓度。 加入100微升的染料溶液，以病毒悬浮液，涡旋5秒并保温1小时，在28℃振荡（100-200转）。 平衡用PBS 2…
Representative Results
矢量释放实验 腺病毒载体到植入物的表面，包括介入器械如血管内支架的束缚，近似向量到疾病部位，部分避免了缺乏矢量“物理定位。然而，为了能够实现通过靶组织的转导的治疗效果，所述载体必须释放从表面（ 图2）。使用水解的交联剂是假设为允许通过该交联剂的水解介1）〜polybisphosphonate改性的有效附着载体，硫醇 – 安装金属植入物和2）?…
Discussion
所提出的协议描述了一种用于通过腺病毒载体的可逆附着取得到coatless不锈钢表面衬底介导的基因递送的操作方法。而开发出用于血管再狭窄的支架为基础的基因疗法的特定目的，该技术具有在生物材料，生物医学植入物和基因治疗等领域的更广泛的应用。
虽然提出的研究仅使用不锈钢作为一个典型的金属基板，PABT结合其他金属的合金，如钴/铬和镍钛合金具有相当的亲?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors do not have competing financial interests to disclose.
Materials
| 316 stainless steel mesh disks | Electon Microscopy Sciences | E200-SS | |
| Generic 304-grade stainless steel stents | Laserage | custom order | |
| AdeGFP | University of Pennsylvania Vector Core | AD-5-PV0504 | |
| AdLuc | University of Pennsylvania Vector Core | AD-5-PV1028 | |
| AdEMPTY | University of Pennsylvania Vector Core | A858 | |
| Cy3(NHS)2 | GE Healthcare | PA23000 | |
| Sepharose 6B | Sigma-Aldrich | 6B100-500ML | |
| UV 96-well plates | Costar | 3635 | |
| Fluorometry 96-well plates | Costar | 3915 | |
| Cell culture 96-well plates | Falcon | 353072 | |
| Tris(2-carboxyethyl)phosphine hydrochloride (TCEP ) | Pierce Thermo Scientific | 20490 | |
| dithiothreitol (DTT) | Pierce Thermo Scientific | 20290 | |
| sulfo-LC-SPDP | Pierce Thermo Scientific | 21650 | |
| Spectrophotometer | Molecular Devices | SpectraMax 190 | |
| Spectrofluorometer | Molecular Devices | SpectraMax Gemini EM | |
| Orbital shaker incubator | VWR | 1575R | |
| Horizontal airflow oven | Shel Lab | 1350 FM | |
| Centra-CL2 centrifuge | International Equipment Company | 426 | |
| Digital vortex mixerer | Fisher Thermo Scientific | 02-215-370 | |
| Eclipse TE300 fluorescence microscope | Nikon | TE300 | |
| DC 500 CCD camera | Leica | DC-500 | |
| 7500 Real-Time PCR system | Applied Biosystems | not available | |
| IVIS Spectrum bioluminescence station | Perkins-Elmer | not available | |
| EDTA dipotassium salt | Sigma-Aldrich | ED2P | |
| Bovine serum albumin fraction V (BSA) | Fisher Thermo Scientific | BP1600-100 | |
| Tween-20 | Sigma-Aldrich | P1379 | |
| Dumont forceps | Fine Science Tools | 11255-20 | |
| A10 cell line | ATCC | CRL-1476 | |
| Bovine aortic endothelial cells | Lonza | BW-6002 | |
| Luciferin, potassium salt | Gold Biotechnology | LUCK-1Ge | |
| Pluronic F-127 | Sigma-Aldrich | P2443-250G | |
| PBS without calcium and magnesium | Gibco | 14190-136 | |
| Fetal bovine serum | Gemini Bio-Products | 100-106 | |
| Penicillin/Streptomycin solution | Gibco | 11540-122 | |
| DMEM, high glucose | Corning cellgro | 10-013-CV | |
| 0.25% Trypsin/EDTA | Gibco | 25200-056 | |
| QIAamp DNA micro kit | Qiagen | 56304 | |
| Power Sybr Green PCR Master Mix | Applied Biosystems | 4367659 | |
| MicroAmp Optical 96-well Reaction Plate | Applied Biosystems | N8010560 | |
| MicroAmp Optical Adhesive Film | Applied Biosystems | 4360954 | |
| Cephazolin | Apotex | not available | |
| Loxicom (Meloxicam) | Norbrook | not available | |
| Heparin sodium | APP Pharmaceuticals | not available | |
| Ketavet (Ketamine) | VEDCO | not available | |
| Anased (Xylazine) | Lloid | not available | |
| Forane (Isoflurane) | Baxter | not available | |
| Curved Moria iris forceps | Fine Science tools | 11370-31 | |
| Curved extra-fine Graefe forceps | Fine Science Tools | 11152-10 | |
| Dumont #5 forceps | Fine Science Tools | 11252-20 | |
| Vannas spring scissors | Fine Science Tools | 15018-10 | |
| Fine scissors – ToughCut | Fine Science Tools | 14058-09 | |
| Surgical scissors | Fine Science Tools | 14101-14 | |
| Vicryl suture (5-0) | Ethicon | J385 | |
| Suture thread (4/0 silk) | Fine Science Tools | 18020-40 | |
| Michel suture clips | Fine Science Tools | 12040-02 | |
| Wound dilator (Lancaster eye specula) | KLS Martin | 34-149-07 | |
| Hot bead sterilizer | Fine Science Tools | 18000-45 | |
| Michel suture clip applicator | Fine Science Tools | 112028-12 | |
| Insyte Autoguard 24G IV catheter | Beckton-Dickinson | 381412 | |
| 2F Fogarty catheter | Edwards Lifesciences | 120602F | |
| Teflon tubing | Vention | 041100BST | |
| PTA catheter | NuMed | custom order | |
| Gauze pads | Kendall Healthcare | 9024 | |
| Cotton applicators | Solon Manufacturing | WOD1003 | |
| Saline | Baxter | 281321 | |
| 10 ml syringe (Luer-Lok) | Beckton-Dickinson | 309604 | |
| 1 ml syringe (Luer-Lok) | Beckton-Dickinson | 309628 | |
| Clippers with #40 blade | Oster | 78005-314 | |
| Transpore surgical tape | 3M | MM 15271 | |
| Puralube vet ointment | Pharmaderm | not available |
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Cite This Article
Fishbein, I., Forbes, S. P., Adamo, R. F., Chorny, M., Levy, R. J., Alferiev, I. S. Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers. J. Vis. Exp. (90), e51653, doi:10.3791/51653 (2014).