Name: models and methods to evaluate transport of drug delivery systems across cellular barriers
Uploaded: 2013-10-17T19:30:00
Description: Watch this Scientific Journal Video about Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers at JoVE.com

这项工作是由霍华德休斯医学研究所和美国国家科学基金会的RG，并颁发给SM卫生（批准R01-HL98416）全国学院和美国心脏协会（批准09BGIA2450014）基金的奖学金支持。

1。培养在Transwell小插入一个细胞单层<ol><li>在无菌，生物安全2级细胞培养罩，放置0.8毫米毛孔的PET Transwell小室在24孔板（4每口井条件，统计显着性），用血管钳。所有进入罩的材料应与消毒乙醇。 </li></ol> 注意：需要的过滤器的孔径，在符合被选择为在NC中使用的平均尺寸，从而使运输穿过细胞膜。另外，对于显著结果，各实验条件需要相同的实验中的最低的4个重复（孔?...

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使用上面讨论的方法中，细胞模型用于研究跨细胞屏障靶向NC的传输可以被建立，例如提供的Caco-2上皮细胞，这是有关从GI管腔评价运输到血液中的情况下的示例的口服药物传递系统。胃肠道上皮细胞单层在Transwell小室培养启用TEER的测量和紧密连接的荧光免疫染色，以确认形成的细胞渗透屏障。随后，定位和治疗药物的125単我提供了有针对性的纳米晶的快速，灵敏的定量成和整个胃肠道细...

细胞屏障在体内充当外部环境和内部隔室之间的网关。这是上皮衬里分离胃肠（GI）道和血液1-3的外部暴露表面上的情况。细胞屏障也代表了血液和软组织及组织和器官的细胞成分之间的界面。这是用于在血管，如血肺屏障，血-脑屏障等在内的内皮衬里的情况下1遍历这些细胞屏障在身体的能力对于有效递送治疗剂和诊断剂的关键为那些需要干预的循环和组织/器官。 以改善递送治疗或诊断剂，这些化合物可以被加载到亚微米的纳米载体（NCS）。这些药物递送载体可以与各种被配制化学和结构优化药物的溶解度，保护，药代动力学，释放和代谢4,5。纳米晶也可官能化亲和或靶向部分（ 如抗体，肽糖，核酸适体等 ），以促进粘附到那里的治疗作用是必需的2,6身体的区域。 NC的靶向表达的细胞屏障的表面上的决定可以进一步便于运输进入和/或在这些衬片2,6。 两个环境之间选择性地运送物质的作用需要细胞间层的某些独特的功能。一个这样的功能是细胞极性，从 ​​而面临着腔的内腔的顶膜从基底外侧膜面向组织间质的变化时，相对于膜的形态和脂质，转运蛋白，受体和2的组合物。另一个特点涉及到细胞间junctioNS连接相邻细胞。形成紧密连接，特别是连接粘附分子（卡纸），occludins和claudins蛋白的调节，调节的屏障功能，以选择性地允许或没有的细胞，称为细胞旁转运之间的物质运输，允许材料从内腔通道到基底外侧空间3。许多天然的和合成的元素（白细胞，分子，粒子和药物递送系统），以在体内细胞屏障的结合可诱导细胞结开口，这可能是短暂的，相对无毒或更长时间的，因此，与不安全的访问跨越障碍2,5,7-9不需要的物质。因此，该途径可通过测量跨上皮电阻（TEER）和分子的被动旁细胞扩散（在本文中称为旁泄漏）进行评估，从而阻力下降到的电流或INE的增加细胞旁漏RT复合成的基底空间指示开幕细胞交界处，分别为5,10,11。为了配合这些方法，任何上面列出的紧密连接蛋白的可染色，以评估其诚信，染色的地方应该会出现集中在细胞与细胞边框四周的细胞外围5,10,12。 可替换地，药物递送系统针对特定的细胞表面决定簇，如那些相关联的网格蛋白小窝或烧瓶形膜内陷称为小窝，可能引发水泡性口摄取到细胞通过内吞作用，提供了一个途径药物递送至细胞内区室5， 13。此外，细胞内吞作用可能导致拐卖整个细胞体囊泡释放的基底侧，这种现象称为transyctosis，或跨细胞运输14。因此，内吞作用的动力学和机制的知识可能被用来利用细胞内的一个ND跨细胞给药，它提供了一个相对安全和控制下交付模式相比，旁路线。内吞作用的机制可能与经典途径（网格蛋白和小窝蛋白介导的内吞作用和巨胞饮作用）或者非经典途径的调节剂进行评估（如细胞粘附分子的情况下（CAM） -介导的胞吞作用）5,13,15 。 而细胞内运输往往是研究标准井或盖玻片，没有一个基底室的排除细胞极化，研究运输跨越细胞层的能力。为了克服这个障碍，跨细胞单层转运已久使用Transwell小室10,11,16,17，它由一个上（顶）腔室，一多孔可渗透膜，其中细胞附着和形成紧密的单层和下研究（基底）室（ 图1）。在这种结构中，运输可以在被测量心尖​​至基底外侧通过施用治疗入上腔体，通过细胞单层，并在下面的多孔膜下面的运输，并最终收集培养基中的下腔室用于输送物料的定量方向。运输在基底外侧至顶端的方向也可以通过最初给药测定从上部腔室5,10,12,16的下部腔和随后的集合。各种技术存在以验证渗透性屏障的上转孔，包括TEER和细胞旁转运测定法形成的，如上面所述。此外，可渗透的过滤器上的细胞进行培养可用于成像分析（ 例如，通过荧光，激光共聚焦，电子显微镜）被除去，因为该细胞单层模型的进一步验证，以及传输的机制。选择膜类型，它可在不同的孔尺寸，材料和表面区域的，​​取决于各种实际RS诸如物质的大小或者被运送的物体，细胞类型和成像方法16,18-20。 Transwell小室也便于控制和准确的运输量化比较复杂的哺乳动物系统中，作为商会和细胞表面积体积是已知常数。而参与体内输送许多因素被消除，包括肠粘液的存在，剪切应力，消化酶，免疫细胞等 ，这种小规模的体外模型提供有关运输有用的初步信息。 作为一个例子来说明这些方法的适应性研究数控运输跨越细胞屏障10,11,16,17，我们在这里描述的地方为整个胃肠道上皮细胞的NC迁移的潜力为蓝本通过评估模型药物输送通道的情况下系统通过人上皮结肠直肠腺癌（Caco-2细胞）的细胞单层。为了这个目的，C细胞l培养于Transwell小室，在0.8微米孔径的聚对苯二甲酸乙二醇酯（PET）的过滤器（6.4毫米直径），它是透明的，并且可以用于显微镜成像。渗透性屏障的状态，通过测量TEER，控制物质，白蛋白，并且紧密连接，occludin的蛋白中的一个元素的荧光显微镜目测的心尖至基底外侧运输进行了验证。有针对性的聚合物数控的模型时，由100纳米，生物降解聚苯乙烯纳米颗粒。纳米晶是由表面吸附有单独的靶向抗体或靶向抗体的结合和治疗的货物，其中任一组分可以被标记的125 I放射性同位素示踪包衣。在选定实施例中，所述抗体识别细胞间粘附分子-1（ICAM-1），蛋白质胃肠上皮的表面上表达（和其他）细胞，其已经显示出促进细胞内和跨细胞转运ö<html> f药物载体及其货物21。货物是α-半乳糖苷酶（α-半乳糖），用于治疗法布里病，一种遗传性溶酶体贮积症22的治疗酶。 该涂层NC的大小约为200纳米，被添加到心尖部室对细胞单层培养在37℃期限不同的时间，在这之后125我对NC的可检测相关的细胞单层和/或输送到下面的细胞基底外侧腔室。额外的决心免费125我的允许涂层数控运输的退化分数和估计的减法。传输的机制是通过检查变化有关的细胞旁路线的通透性屏障，通过上面描述的参数，而跨细胞运输是通过调节内吞和胞转的通路时，检查改变传输确定进一步评估。 ontent“&gt;这些方法提供关于蜂窝障碍模型的有价值的信息，药物递送系统的运输的程度和速率，并且这样传输的机制，完全允许跨细胞屏障的药物递送的潜力的评价。

<table border="1">
	<tbody>
		<tr>
			<td>Transwell inserts</td>
			<td>BD Falcon</td>
			<td>353095</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), 1x</td>
			<td>Cellgro</td>
			<td>10-013-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Cellgro</td>
			<td>35-015-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>Human epithelial colorectal adenocarcinoma (Caco-2) cells</td>
			<td>ATCC</td>
			<td>HTB-37TM</td>
			<td></td>
		</tr>
		<tr>
			<td>125Iodine</td>
			<td>Perkin Elmer</td>
			<td>NEZ033H002MC</td>
			<td>Radioactive hazard</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS)</td>
			<td>Gibco</td>
			<td>14190-235</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Equitech Bio</td>
			<td>BAH-66</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (16%)</td>
			<td>Fisher Scientific</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Immunoglobulin G (IgG)</td>
			<td>Jackson ImmunoResearch</td>
			<td>015-000-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse monoclonal antibodies to human ICAM-1 (anti-ICAM)</td>
			<td>Marlin 1987</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-Galactosidase, from green coffee beans</td>
			<td>Sigma</td>
			<td>G8507-25UN</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC latex beads, 100 nm</td>
			<td>Polysciences, Inc.</td>
			<td>17150</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>234729-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic acid (TCA)</td>
			<td>Fisher Scientific</td>
			<td>SA433-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Occludin antibody (Y-12), goat polyclonal anti-human</td>
			<td>Santa Cruz Biotechnology</td>
			<td>Sc-27151</td>
			<td></td>
		</tr>
		<tr>
			<td>Monodansylcadaverine (MDC)</td>
			<td>Sigma</td>
			<td>D4008</td>
			<td></td>
		</tr>
		<tr>
			<td>Filipin</td>
			<td>Sigma</td>
			<td>F9765</td>
			<td></td>
		</tr>
		<tr>
			<td>5-(N-ethyl-N-isopropyl) amiloride (EIPA)</td>
			<td>Sigma</td>
			<td>A3085</td>
			<td></td>
		</tr>
		<tr>
			<td>Wortmannin</td>
			<td>Sigma</td>
			<td>W1628</td>
			<td></td>
		</tr>
		<tr>
			<td>Gamma counter</td>
			<td>Perkin Elmer</td>
			<td>Wizard2</td>
			<td></td>
		</tr>
		<tr>
			<td>Volt-ohm meter</td>
			<td>World Precision Instruments</td>
			<td>EVOM2</td>
			<td></td>
		</tr>
		<tr>
			<td>TEER electrodes</td>
			<td>World Precision Instruments</td>
			<td>STX100</td>
			<td>Electrodes available for different well-plates</td>
		</tr>
		<tr>
			<td>Dynamic Light Scattering (DLS)</td>
			<td>Malvern</td>
			<td>Nano-ZS90</td>
			<td></td>
		</tr>
	</tbody>
</table>

models and methods to evaluate transport of drug delivery systems across cellular barriers

亚微米载体（纳米载体; NCS）通过改善溶解性，稳定性，循环时间，靶向，和释放增强药物疗效。此外，穿越在体内细胞屏障是为口服给药治疗的NC进入流通和运输从血液进入组织，在需要干预至关重要。 （i）该细胞旁路线通过该互锁相邻细胞，或（ii）在跨细胞途径，其中材料是通过细胞内吞作用内在化，在整个细胞体运送，而分泌的结的瞬态干扰，：跨细胞屏障转运的NC被实现在相对的细胞表面（transyctosis）。跨细胞屏障递送可以通过偶联治疗剂或它们的载体与特异性结合到参与运输的细胞表面标志物的靶向剂来促进。这里，我们提供的方法来测量的程度和NC传输的机制跨越模型细胞屏障，WHI通道由胃肠道的单层（GI）的上皮细胞生长在位于一个transwell小室插入一个多孔膜。的渗透性屏障的形成是通过测量跨上皮电阻（TEER），控制物质的跨上皮转运和紧密连接的免疫染色证实。作为一个例子，〜200 nm的聚合物纳米晶的使用，它携带一种治疗货物和涂有靶向的细胞表面决定簇的抗体。所述抗体或治疗性的货物被标记的125 I放射性同位素示踪标记和纳米晶加入到上室在细胞单层的时间期限不同。相关的细胞和/或输送到底层室的NC可以被检测到。免费125我的测量允许的退化分数的减法。该旁路线是通过确定所造成的NC输送到上述的势垒参数的电位变化进行评估。跨细胞运输我s由寻址调节内吞作用和胞转作用途径的作用决定的。

作为我们的细胞模型，研究对象NC的跨上皮转运的验证， 图2示出的Caco-2细胞单层镀以1.5×10 5个细胞/ cm 2的密度达到汇合〜第12天，并保持单层完整性达18天，通过跨膜电阻（ 图2A）表示。这是由occludin的阳性紧密连接的细胞单层用高TEER（390Ω×cm 2时，第14天）的存在（ 图2B）进行验证，相比于低TEER差的紧密连接标记（17Ω×cm 2时...

Watch this Scientific Journal Video about Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers at JoVE.com

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

许多治疗应用需要药物载体及其货物横跨在体内细胞屏障的安全，高效的运输。本文介绍的方法，建立一个适应，以评估药物纳米载体（NCS）跨细胞屏障，如胃肠道（GI）上皮细胞的转运速率和机制。

模型和方法来评价药物输送系统在整个细胞屏障的运输

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

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Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

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Self-reporting Scaffolds for 3-Dimensional Cell Culture

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

纳米丝绸制造和药物输送应用

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

实验和有限元议定书调查中性和带电溶质的跨关节软骨运输

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

化学共轭法在戊型肝炎病毒纳米粒子表面功能化中的应用

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

用三种互补方法综合评价胎盘靶向药物的有效性和安全性

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

用化学表观修饰因子重定向细胞机械抑制基因转录

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

趋化梯度多细胞响应的三维分析

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

使用微流体混合平台为基因传递制订和定性脂质纳米粒子

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...

Injectable Supramolecular Polymer-Nanoparticle Hydrogels for Cell and Drug Delivery Applications

用于细胞和药物输送应用的可注射超分子-纳米粒子水凝胶

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are ...