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），和至少3个独立实验。 <ol start="2"><li>制备含有补充用4.5克/升葡萄糖，15％胎牛血清和1％青霉素链霉素的DMEM培养基的细胞培养液，并加热至37℃的水浴中。 </li><li>稀人上皮大肠腺癌（Caco-2细胞）细胞在细胞培养基和地点200-400微升的细胞溶液进入的transwell插入物的上（顶）室，在1.5×10 5个细胞/ cm 2的密度。 900微升细胞培养基 - 700填补较低（基底）室。 </li><li>培养细胞在37℃，5％CO 2和95％湿度下16-21天，更换在上部和下部腔室中的介质，每3-4天，使用在步骤1.3所表示的体积。从下腔室吸出培养基，从上部腔室吸出培养基，注入新鲜的培养基中的上腔室，并填入下部腔室有：在以下，以保持上述（而不是下面）细胞单层中的压力取代介质新鲜培养基。 </li></ol> 2。胃肠上皮通透性屏障使用跨上皮电阻（TEER）和免疫染色紧密连接的验证<ol><li>对于短期存储（不超过2个星期），淹没STX100电极在电解质溶液化（0.1-0.15米氯化钾或氯化钠）。电极电缆连接至EVOM电压电阻计电极端口，以便该系统是内部短路和电极对称性保持不变。对于长期储存，冲洗用DH 2 O和存放在干燥，避光条件。 </li><li>消毒电极，浸入在乙醇中15分钟，并允许在空气中干燥15秒。或者，所述电极可以被存储在一个紫外线罩。冲洗电极在无菌电解质溶液（磷酸盐缓冲盐水，PBS）或0.1-0.15摩尔KCl或NaCl溶液中，每个电阻测量之前。 </li><li>用伏特 - 欧姆计设定为电阻设置，垂直放置的电极在井含有的transwell插入物，具有在上部腔室的短电极和在下部腔室触及孔底部的长电极。每口井;一旦EVOM读数稳定后，记录电阻值（Ω（欧姆）给出）。 </li><li>计算电阻率（resistanCE归区域;厘米2）减去背景阻力Transwell小室无细胞（TEER）和膜表面积乘以样本Ω×。电阻率值是最经常在文献中报道。 （见讨论） </li><li>重复测量，每隔1-2天，直到TEER值上升到最高和高原，表明形成了渗透屏障，它通常需要2-3周的细胞编结的时刻。高原TEER值和必要的时间来实现屏障的完整性可以根据细胞传代次数而变化。 </li><li>以验证紧密连接的细胞单层的存在下具有高的TEER（等于或大于为势垒形成的阈值）时，通过培养用冷的2％多聚甲醛固定15分钟，固定细胞。然后，在室温下用1微克/毫升抗紧密连接蛋白用PBS洗涤细胞，并培育他们30分钟。用PBS再次洗涤细胞，并用7.5微克/毫升f孵蛋30分钟，在室温下luorescently标记的第二抗体。使用井低TEER作为阴性对照屏障的形成。 </li></ol> 注意：作为一个替代的抗紧密连接蛋白中，可以使用抗体来替代紧密连接蛋白。 <ol start="7"><li>仔细切除其上固定单层附着在过滤膜，并安装使用上或落射荧光共聚焦显微镜载玻片成像。 </li></ol> 3。评估目标的载体跨上皮转运<ol><li>标记的靶向抗体（小鼠单克隆抗ICAM-1抗体，抗ICAM，在这个例子）与125 I，如前所述7。标记抗体的使用Bradford测定法来估计蛋白质浓度和γ计数来确定该抗体的125 I含量，然后计算出在每分钟（CPM）的计数的比活性/毫克。 </li></ol> 注：为了控制特异性，重新通过标记小鼠IgG，这将被用于制备非靶向涂覆纳米晶泥炭的过程。研究的治疗性货物运输，在本例中重复该步骤标记的货物，例如，α-半乳糖苷酶（α-半乳糖）。替代方案可用于靶向剂，非特异性对照，或治疗性的货物。可供选择的方法也可以用于标记的化合物和估计的标记对应的浓度。 <ol start="2"><li>夫妇标记靶向抗体（抗ICAM在我们的例子），以纳米晶的表面。在这个例子中，孵育聚苯乙烯纳米珠（100纳米直径）为1小时，在室温下，用125 I-抗ICAM允许表面吸附，如所描述7。 </li></ol> 注：对于非特定的控制使用125 I-IgG抗体。要跟踪货物的运输，使用靶向抗体和125 I标记的货物（α-半乳糖在我们的例子）的组合。 <ol start="3"><li>离心机以13,000×g离心3分钟，并通过抽吸除去上清液中的未涂覆的同行。重悬含有用1％牛血清白蛋白（BSA）的PBS中通过移液，超声在低功率扰乱潜在的颗粒聚集体（20-30简要脉冲）涂覆的纳米晶沉淀。 </li><li>数控表征，测量尺寸，多分散性，和使用动态光散射（以下供应商的说明）涂覆颗 ​​粒的ζ-电势，和量化的数的125 I-标记的在粒子表面上靶向抗体分子（ 如抗ICAM），使用伽玛计数器。 </li></ol> 注意：这些方法可以被重复用于非特异性和治疗的同行。大约200纳米涂覆的颗粒的尺寸范围内。 <ol start="5"><li>加入125 I-NC的抗体，（ 如 125 I-抗ICAM纳米晶; 56 NCI /毫升），以上述融合的Caco-2细胞单层与跨膜电阻及上腔＃8805; 350Ω×2厘米（见讨论）在背景（16-21天后播种）。在37℃下的一个或多个所需的时间间隔。之前和之后孵育测量TEER（第2.3节），以评估NC的对渗透性屏障的作用。 </li></ol> 注意：此过程可以重复进行非特异性和治疗的同行。 <ol start="6"><li>从上，下腔收集培养基，于37℃（上院）或1毫升DH 2 O（下腔）用0.5毫升的DMEM一旦把它们洗干净。用γ计数器收集洗涤液总放射性同位素含量的测定。 </li><li>对于细胞级分进行测定，用剃刀刀片切割的边缘，并孵育在γ计数管，用1％的Triton X-100的10分钟（以释放细胞内容物），测定细胞前切除的透气性过滤器， 例如相关的总放射性。 </li><li>为了测量125我重新在运输过程中的NC或租赁由于潜在的降解，第一混合300微升的样品（从上，下，或细胞级分）与700微升PBS中的3％BSA和200微升三氯乙酸（TCA）。在室温下孵育15分钟。另外此时，测量该样品的在γ计数器中的总放射性。 </li><li>离心机TCA样品在3000×g离心5分钟，以从蛋白质降解或125 I部分（上清液）分离完整蛋白质（粒料）。量化免费125我分数的放射性和减去总放射性这个值离心前测量。这将提供标记的蛋白质的未降解的量。 </li></ol> 4。靶向纳米载体的跨上皮转运机制<ol><li>标签在3.1节中描述并预先用125 I白蛋白报7。 </li></ol> 注：白蛋白是66.5kDa的蛋白质，因此，相对大的物质。虽然较大的对象（例如200 nm的纳米晶用在这里）的被动转运有效的控制，应该学习较小的药物载体转运时被较小的惰性示踪剂分子（见讨论）。 <ol start="2"><li>使用白蛋白旁漏，培养的Caco-2细胞单层上，如上所述的transwell插入物评估细胞旁转运。 </li><li>到上面的细胞单层上腔介质，添加或125单独的I-白蛋白（阴性对照，显示泄漏的基础水平），或125 I-白蛋白和非放射性标记的抗体靶向纳米晶（如在第3.5节中所述）。在37°C为选定的时间间隔（S），这应与测试数控交通工具时检查。前测量TEER（2.3节），在此期间，和孵化后，然后收集所有组分总125我和免费125 I测量所示，第3节中说明：伴随另外的125 I-白蛋白和非放射性标记的对照IgG纳米晶可以被用作对照。 </li><li>作为阳性对照用于打开间连接中，添加含细胞介质5mM的H 2 O 2到Transwell小插入物的上部和下部腔室，并在37℃下进行30分钟。然后，测量TEER（2.3节），并加入125 I-白蛋白选定的时间间隔上院。测量TEER在整个孵化不同的时间点，以确定引起H 2 O 2诱导的开幕细胞连接的TEER值衰减。 </li><li>在平行实验，评估跨细胞运输，也被称为胞转，125 I-NC的针对性孵化融合的Caco-2细胞单层带为50微米monodansylcadaverine（MDC;抑制网格蛋白介导的内吞作用），1微克/毫升律平（抑制剂胞膜窖介导的内吞作用），0.5μM沃特曼在（抑制剂的磷脂酰肌醇3激酶PI3K]，参与巨胞饮作用），或20μM的[5 - （N-乙基-N-异丙基）阿米洛利]（EIPA，抑制剂巨胞饮作用和CAM介导的内吞作用15）。 </li></ol> 注意：125 I-IgG的纳米晶和125 I-白蛋白可作为对这些抑制剂的作用阴性对照，以及其他方法（例如siRNA的技术），可以提供更多的选择性抑制。 <ol start="6"><li>测量TEER之前（第2.3节），在此期间，与细胞抑制剂和放射性材料的温育后，作为一个额外的控制的抑制剂在单层通透性的影响。 </li></ol>

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V., Lankelma, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+transepithelial+drug+transport+by+on-line+measurement:+cellular+control+of+paracellular+and+transcellular+transport.">In vitro transepithelial drug transport by on-line measurement: cellular control of paracellular and transcellular transport.</a> J. Pharm. Sci. 88, 1340-1347 (1999).</li><li>Morris, M. C., Deshayes, S., Heitz, F., Divita, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-penetrating+peptides:+from+molecular+mechanisms+to+therapeutics.">Cell-penetrating peptides: from molecular mechanisms to therapeutics.</a> Biol. Cell. 100, 201-217 (2008).</li><li>Kapus, A., Szaszi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupling+between+apical+and+paracellular+transport+processes.">Coupling between apical and paracellular transport processes.</a> Biochem. Cell Biol. 84, 870-880 (2006).</li><li>Hood, E. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidant+protection+by+PECAM-targeted+delivery+of+a+novel+NADPH-oxidase+inhibitor+to+the+endothelium+in+vitro+and+in+vivo.">Antioxidant protection by PECAM-targeted delivery of a novel NADPH-oxidase inhibitor to the endothelium in vitro and in vivo.</a> J. Control. Release. 163, 161-169 (2012).</li><li>Simone, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+targeting+of+polymeric+nanoparticles+stably+labeled+with+the+PET+imaging+radioisotope+iodine-124.">Endothelial targeting of polymeric nanoparticles stably labeled with the PET imaging radioisotope iodine-124.</a> Biomaterials. 33, 5406-5413 (2012).</li><li>Vercauteren, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+inhibitors+to+study+endocytic+pathways+of+gene+carriers:+optimization+and+pitfalls.">The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls.</a> Mol. Ther. 18, 561-569 (2010).</li></ol>

作者宣称，他们有没有竞争的财务权益。

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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46.0K 次观看.  University of Maryland. 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。 

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

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

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

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

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

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 ...

科研

生物工程

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

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 ...

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs through the blood-brain barrier (BBB), causing difficulties for effective pharmacological therapies. In recent years, different strategies for promoting brain targeting have aimed to improve drug delivery and activity at this site, including innovative nanosystems for drug delivery across the BBB. In this context, an in vitro approach based on a simplified cellular model of the BBB provides a useful tool to investigate the effect of nanoformulations on the trans-BBB permeation of molecules. This study describes the development of a double-layer BBB, consisting of co-cultured commercially available primary rat brain microvascular endothelial cells and astrocytes. A multiparametric approach for the validation of the model, based on the measurement of the transendothelial electrical resistance and the apparent permeability of a high molecular weight dextran, is also described. As proof of concept for the employment of this BBB model to study the effect of different nanoformulations on the translocation of fluorescent molecules across the barrier, we describe the use of fluorescein isothiocyanate (FITC), loaded into ferritin nanoparticles. The ability of ferritins to improve the trans-BBB permeation of FITC was demonstrated by flux measurements and confocal microscopy analyses. The results suggest this is a useful system for validating nanosystems for delivery of drugs across the BBB.

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules.

跨越大鼠血脑屏障FITC加载铁蛋白体外渗透：一个模型来研究Nanoformulated分子的交付

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

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

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

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 ...

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal microscopy, the scientific community has unprecedented access to large datasets at sub-micrometer resolution that characterize the architectural remodeling that accompanies changes in cardiomyocyte function in health and disease. However, these datasets have been under-utilized for&#160;investigating the role of cellular architecture remodeling in cardiomyocyte function. The purpose of this protocol is to outline how to create an accurate finite element model of a cardiomyocyte using high resolution electron microscopy and confocal microscopy images. A detailed and accurate model of cellular architecture has significant potential to provide new insights into cardiomyocyte biology, more than experiments alone can garner. The power of this method lies in its ability to computationally fuse information from two disparate imaging modalities of cardiomyocyte ultrastructure to develop one unified and detailed model of the cardiomyocyte. This protocol outlines steps to integrate electron tomography and confocal microscopy images of adult male Wistar (name for a specific breed of albino rat) rat cardiomyocytes to develop a half-sarcomere finite element model of the cardiomyocyte. The procedure generates a 3D finite element model that contains an accurate, high-resolution depiction (on the order of ~35 nm) of the distribution of mitochondria, myofibrils and ryanodine receptor clusters that release the necessary calcium for cardiomyocyte contraction from the sarcoplasmic reticular network (SR) into the myofibril and cytosolic compartment. The model generated here as an illustration does not incorporate details of the transverse-tubule architecture or the sarcoplasmic reticular network and is therefore a minimal model of the cardiomyocyte. Nevertheless, the model can already be applied in simulation-based investigations into the role of cell structure in calcium signaling and mitochondrial bioenergetics, which is illustrated and discussed using two case studies that are presented following the detailed protocol.

This protocol outlines a novel method to create a spatially detailed finite element model of the intracellular architecture of cardiomyocytes from electron microscopy and confocal microscopy images. The power of this spatially detailed model is demonstrated using case studies in calcium signaling and bioenergetics.

建立一种结构逼真的心肌细胞的有限元几何模型研究蜂窝结构在心肌系统生物学中的作用

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

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

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

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

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

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

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

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 ...