Name: comprehensive evaluation of the effectiveness and safety of placenta-targeted drug delivery using three complementary methods
Uploaded: 2018-09-10T14:30:00.000+00:00
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这项工作得到了国家自然科学基金 (81771617) 和广东省自然科学基金 (2016A030313178) 授予范的赠款的支持;深圳市基础研究基金 (JCYJ20170413165233512) 授予 x F 的补助金;和尤尼斯肯尼迪美国国立卫生研究院儿童健康 & 人类发展研究所授予编号 R01HD088549 (内容完全由作者负责, 不一定代表官方国家卫生研究院) 对 N.N. 的看法

所有鼠标实验严格遵循的协议 (SIAT-IRB-160520-YYS-FXJ-A0232) 批准的动物护理和使用委员会的深圳先进技术研究院, 中国科学院。
1. 胎盘硫酸软骨素 A 靶向脂质高分子纳米粒的合成
<ol><li>将甲氨喋呤-和微球负载的脂质高分子纳米粒子 (MNPs 和修订为期三) 和 plCSA-BP-共轭纳米粒子 (plCSA MNPs 和 plCSA 修订为期三) 进行了详细的描述, 其他18。</li>
</ol>2.体内荧光成像
<ol><li>
孕鼠的制备
	<ol>
<li>将雌性 CD-1 小鼠 (8-12 周) 与一笼中同一株 (男: female=1:2) 的肥沃雄性保持在下午, 并检查第二天早晨的阴道插头。如果观察到阴道插头, 将鼠标定义为胚胎日 0.5 (E0.5)。</li>
		<li>在没有病原体的动物室里单独饲养怀孕的老鼠, 14 小时 light/10 h 黑暗循环, 并提供免费的食物和水, 直到 E14.5。</li>
	</ol>
</li>
	<li>
静脉注射纳米微粒
	<ol>
<li>在手术前, 通过0.22 微米的注射器过滤器对纳米微粒进行消毒。在 E11.5 中对怀孕的老鼠进行称量, 以确定纳米微粒注射液的数量和体积。 注: 纳米颗粒注射液体积应小于 1% (体积/重量) 的怀孕小鼠的体重。例如, 在25克的老鼠中, 纳米粒子的注入量应该小于0.25 毫升。</li>
		<li>为了扩大尾部静脉, 用加热垫将尾部预热5-10 分钟。</li>
		<li>注射前, 将修订为期三或 plCSA 修订为期三吸入28克胰岛素注射器。</li>
		<li>将怀孕的老鼠转移到抑制老鼠的手持装置上, 同时允许进入尾部静脉。用酒精拭子清洁尾部。然后将注射器插入尾部静脉。慢慢地注射修订为期三或 plCSA-修订为期三 (5 毫克/千克当量), 甚至压力超过5-10 秒。 注: 如果尾部出现水泡, 则停止注射, 因为此结果表明针头不在静脉内。小鼠不应共享注射器, 以减少疾病传播和交叉污染。</li>
		<li>记录注射时间。同时, 对注射部位施加温和压力, 直至出血停止, 通常需要30-60 秒。</li>
	</ol>
</li>
	<li>
体内成像
	<ol>
<li>注射后30分钟, 用体内荧光成像系统对怀孕小鼠进行图像成像。</li>
		<li>麻醉妊娠小鼠, 其氧流速为1.0 升/分和异氟醚, 2-4% 在麻醉单位的相关室, 并通过缓慢和定期呼吸验证完全麻醉。然后, 将它们移动到成像室。将麻醉的怀孕小鼠放入成像室, 使动物处于仰卧位。</li>
		<li>将鼻锥放在嘴和鼻子上, 允许吸入1-2% 异氟醚, 氧气流速为1.0 升/分钟, 以维持麻醉。</li>
		<li>选择2维荧光和照相参数来图像的干扰荧光信号。设置曝光到自动和激发/发射波长到710/820 毫微米。</li>
		<li>在成像过程结束时, 关闭异氟醚的流入以停止麻醉, 并小心地将怀孕的老鼠送回笼子里。</li>
		<li>48 h 纳米微粒注射后, 麻醉与 isofluorane 的怀孕小鼠, 然后通过颈椎脱位牺牲大坝。使用 Graefe 钳、Graefe 组织钳和解剖剪刀收集胎儿和胎盘。</li>
		<li>将胎盘和胎儿放入成像室, 并使用步骤2.3.4 中描述的方法进行图像处理。</li>
	</ol>
</li>
</ol>3. HFUS 对胚胎发育的评价
<ol><li>
动物模型
	<ol>
<li>如步骤2.1 所述, 获得并准备怀孕小鼠。</li>
		<li>使用 HFUS 在 E 6.5 (3.2 和 3.3.3) 对怀孕小鼠进行图像处理。首先, 通过在日 E6.5 上可视化胚胎来确认怀孕, 然后将怀孕的小鼠随机分配到三组: 自然人流动组、plCSA-自然人流动组和磷酸盐缓冲盐 (PBS) 组。</li>
		<li>将 PBS、MNPs 或 plCSA-MNPs (1 毫克/千克甲氨蝶呤当量) 注射到怀孕小鼠的尾脉中, 每隔一天从 E6.5 开始, 如步骤2.2 所述。</li>
	</ol>
</li>
	<li>
成像准备
	<ol>
<li>24小时注射纳米微粒后, 用 HFUS 成像系统对怀孕小鼠进行图像处理。</li>
		<li>麻醉在步骤2.3.2 中描述的怀孕小鼠。打开成像平台的集成温度控制, 将平台预热至摄氏37-42 摄氏度。用胶带将怀孕小鼠放在平台的仰卧位。</li>
		<li>将鼻锥与麻醉装置连接在鼻子上。应用2% 异氟醚, 氧气流量为1.0 升/分, 以保持稳定的麻醉。</li>
		<li>用脱毛膏从腹部化学去除毛发。用浸泡过的纱布彻底擦拭残留奶油, 然后用声学耦合凝胶将腹部涂上。</li>
	</ol>
</li>
	<li>
成像过程
	<ol>
<li>将 40 MHz 传感器放在机械臂中。</li>
		<li>调整换能器位置, 获取胎儿和胎盘的纵向图像, 该区域位于焦点区域。</li>
		<li>
B 型成像与分析 注: 见电影 1。<ol>
<li>单击B 模式按钮, 并降低在腹部的传感器, 直到胎儿和胎盘进入视野。按下扫描/冻结以启动/停止成像, 按电影商店存储电影循环, 并按帧存储存储帧图像。</li>
			<li>单击 "测量" 按钮以分析妊娠囊长度 (GS)、胎冠尾部长度 (CRL)、双顶直径 (BPD)、腹围 (AC)、胎盘直径 (PD) 和胎盘厚度 (PT)。</li>
		</ol>
</li>
		<li>
多普勒成像与分析 注: 见电影 1。<ol>
<li>使用相同的扫描投影, 单击 "密码" 按钮, 将取样音量框放置在脐动脉的中心, 然后按扫描/冻结以启动成像。点击电影商店收集脐动脉图像。</li>
			<li>单击 "测量" 按钮计算脐动脉峰值速度 (UA)。</li>
		</ol>
</li>
		<li>
彩色多普勒模式成像与分析
		<ol>
<li>使用相同的扫描投影, 单击颜色按钮并调整换能器位置以获得胎儿心脏的图像。按扫描/冻结启动图像和电影商店收集图像。</li>
			<li>单击 "测量" 按钮计算胎儿心率 (HR)。</li>
		</ol>
</li>
	</ol>
</li>
</ol>4. HPLC 分析
<ol><li>
组织准备
	<ol>
<li>在妊娠晚期 (e.、E14.5) 中, 按步骤3.1.3 中所述, 向怀孕小鼠注射一剂 MNPs 或 plCSA-MNPs (1 毫克/千克甲氨蝶呤当量)。</li>
		<li>24小时后, 麻醉腹腔注射 avertin 240 微克/体重 (g)。确保没有对脚捏的反应, 以验证小鼠是完全麻醉。</li>
		<li>用75% 乙醇喷洒胸部区域。进行心脏灌注 (切开右心房和灌注通过左心室), 如前所述详细19,20与50毫升冰冷0.9% 生理盐水10分钟, 以消除未绑定纳米粒子。</li>
		<li>弄死水坝。执行剖宫产, 收集胎儿和胎盘使用 Graefe 钳, 解剖剪刀, Graefe 组织钳, 并存储在-80 °c 的组织在分析之前。</li>
		<li>准备均匀溶液 (10% 高氯酸), 并保持冰。收集大约200毫克的组织, 并添加 500 ul 均匀化溶液的每个样品。融汇的样品使用一个均质机全速三十年代, 并重复此过程两次。</li>
		<li>离心样品在1.4万×g 20 分钟在4°c。通过0.45 微米的注射器过滤器过滤上清 (约 300 ul), 并将产生的液体转移到 HPLC 瓶中。将样品瓶放入 autosampler 托盘中注射。</li>
	</ol>
</li>
	<li>
标准的编制
	<ol>
<li>为移动阶段准备以下解决方案:40 毫米磷酸钾二元 (pH 4.5) 和乙腈 (88:12, v/v)。过滤解决方案通过0.45 微米孔径注射器过滤器, 并将产生的液体转移到一个干净的 HPLC 储集瓶。 注: 用0.1 米磷酸调节 pH 值。使用超声波振动15分钟, 以德加的移动相在每次使用之前。</li>
		<li>将10毫克的甲氨喋呤重分为1.5 毫升离心管。加入1毫升1米氢氧化钠。</li>
		<li>高速涡流, 直到甲氨蝶呤完全溶解。 注: 这是主要库存, 可以储存在-20 °c 几个月。</li>
		<li>要创建二甲喋呤的股票 (500 微克/毫升), 稀释 50 ul 的主要库存在 950 ul 的移动阶段。 注: 储存在冰上, 直到使用, 并准备新鲜的每日。重要的是使用移动阶段来制定标准, 以避免在注射样品后混合不同溶液产生的峰值。</li>
		<li>进一步稀释制定标准 (表 1)。把标准储存在冰上, 每天准备新鲜的。用实验样品在系列中运行标准。</li>
	</ol>
</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>数量</td>
			<td>最终浓度 (微克/毫升)</td>
			<td>500微克/毫升标准, ul</td>
			<td>移动阶段 (ul)</td>
		</tr>
<tr>
<td>1</td>
			<td>0。5</td>
			<td>1</td>
			<td>999</td>
		</tr>
<tr>
<td>2</td>
			<td>1</td>
			<td>2</td>
			<td>998</td>
		</tr>
<tr>
<td>3</td>
			<td>2。5</td>
			<td>5</td>
			<td>995</td>
		</tr>
<tr>
<td>4</td>
			<td>10</td>
			<td>20</td>
			<td>980</td>
		</tr>
<tr>
<td>5</td>
			<td>25</td>
			<td>50</td>
			<td>950</td>
		</tr>
<tr>
<td>6</td>
			<td>50</td>
			<td>100</td>
			<td>900</td>
		</tr>
<tr>
<td>7</td>
			<td>100</td>
			<td>200</td>
			<td>800</td>
		</tr>
</tbody></table>表1。制备甲氨蝶呤标准曲线.甲氨蝶呤标准溶液的最终浓度为 0.5-100 微克/毫升。
<ol start="3"><li>
高效液相色谱仪操作参数 注: 用溶剂泵、紫外分光光度计 (313 nm) 和 C18 柱 (250×4.6 毫米、5微米粒径) 的 HPLC 系统对样品进行了分析。<ol>
<li>打开高效液相色谱 degasser, 从系统中去除空气。打开该流, 用移动相平衡该列以减少基线噪音30分钟。</li>
		<li>将该列的温度设置为25摄氏度, 以1毫升/分钟的流速注入 20 ul 样品卷, 然后单击Run 方法开始分析。</li>
		<li>当运行完成后, 手动将移动相转换为 HPLC 级乙腈。运行约15分钟以保护系统。 注意: 如果在建议的运行时间之后不执行此步骤, 可能会导致列损坏。</li>
		<li>为了定量分析, 使用 HPLC 系统软件计算出标准的氨蝶呤峰值的区域。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Rodger, M. A., 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+Association+of+Factor+V+Leiden+and+Prothrombin+Gene+Mutation+and+Placenta-Mediated+Pregnancy+Complications:+A+Systematic+Review+and+Meta-analysis+of+Prospective+Cohort+Studies.">The Association of Factor V Leiden and Prothrombin Gene Mutation and Placenta-Mediated Pregnancy Complications: A Systematic Review and Meta-analysis of Prospective Cohort Studies.</a> PLOS Medicine. 7 (6), e1000292 (2010).</li><li>Rodger, M. A., 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=Inherited+thrombophilia+and+pregnancy+complications+revisited.">Inherited thrombophilia and pregnancy complications revisited.</a> Obstetrics &amp; Gynecology. 112 (2 Pt 1), 320-324 (2008).</li><li>Brenner, B., Aharon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombophilia+and+adverse+pregnancy+outcome.">Thrombophilia and adverse pregnancy outcome.</a> Clinics in Perinatology. 34 (4), 527-541 (2007).</li><li>Fisk, N. M., McKee, M., Atun, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+and+absolute+addressability+of+global+disease+burden+in+maternal+and+perinatal+health+by+investment+in+R&amp;D.">Relative and absolute addressability of global disease burden in maternal and perinatal health by investment in R&amp;D.</a> Tropical Medicine &amp; International Health. 16 (6), 662-668 (2011).</li><li>Fisk, N. M., Atun, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Market+failure+and+the+poverty+of+new+drugs+in+maternal+health.">Market failure and the poverty of new drugs in maternal health.</a> PLOS Medicine. 5 (1), e22 (2008).</li><li>Kaitu'u-Lino, T. u. J., 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=Targeted+nanoparticle+delivery+of+doxorubicin+into+placental+tissues+to+treat+ectopic+pregnancies.">Targeted nanoparticle delivery of doxorubicin into placental tissues to treat ectopic pregnancies.</a> Endocrinology. 154 (2), 911-919 (2013).</li><li>King, A., 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=Tumor-homing+peptides+as+tools+for+targeted+delivery+of+payloads+to+the+placenta.">Tumor-homing peptides as tools for targeted delivery of payloads to the placenta.</a> Science Advances. 2 (5), e1600349 (2016).</li><li>Beards, F., Jones, L. E., Charnock, J., Forbes, K., Harris, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+Homing+Peptide-microRNA+Inhibitor+Conjugates+for+Targeted+Enhancement+of+Intrinsic+Placental+Growth+Signaling.">Placental Homing Peptide-microRNA Inhibitor Conjugates for Targeted Enhancement of Intrinsic Placental Growth Signaling.</a> Theranostics. 7 (11), 2940-2955 (2017).</li><li>Cureton, N., 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=Selective+Targeting+of+a+Novel+Vasodilator+to+the+Uterine+Vasculature+to+Treat+Impaired+Uteroplacental+Perfusion+in+Pregnancy.">Selective Targeting of a Novel Vasodilator to the Uterine Vasculature to Treat Impaired Uteroplacental Perfusion in Pregnancy.</a> Theranostics. 7 (15), 3715-3731 (2017).</li><li>Paul, J. W., 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=Drug+delivery+to+the+human+and+mouse+uterus+using+immunoliposomes+targeted+to+the+oxytocin+receptor.">Drug delivery to the human and mouse uterus using immunoliposomes targeted to the oxytocin receptor.</a> American Journal of Obstetrics and Gynecology. 216 (3), e281-e283 (2017).</li><li>Zhang, B., 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=Placenta-specific+drug+delivery+by+trophoblast-targeted+nanoparticles+in+mice.">Placenta-specific drug delivery by trophoblast-targeted nanoparticles in mice.</a> Theranostics. 8 (10), 2765-2781 (2018).</li><li>Fan, X., 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=Noninvasive+monitoring+of+placenta-specific+transgene+expression+by+bioluminescence+imaging.">Noninvasive monitoring of placenta-specific transgene expression by bioluminescence imaging.</a> PloS One. 6 (1), e16348 (2011).</li><li>Murata, M., Tahara, K., Takeuchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+in+vivo+imaging+of+surface-modified+liposomes+to+evaluate+their+behavior+after+pulmonary+administration.">Real-time in vivo imaging of surface-modified liposomes to evaluate their behavior after pulmonary administration.</a> European Journal of Pharmaceutics and Biopharmaceutics. 86 (1), 115-119 (2014).</li><li>Ito, A., 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=New+whole-body+multimodality+imaging+of+gastric+cancer+peritoneal+metastasis+combining+fluorescence+imaging+with+ICG-labeled+antibody+and+MRI+in+mice.">New whole-body multimodality imaging of gastric cancer peritoneal metastasis combining fluorescence imaging with ICG-labeled antibody and MRI in mice.</a> Gastric Cancer. 17 (3), 497-507 (2014).</li><li>Mazza, M., 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=Liposome-Indocyanine+Green+Nanoprobes+for+Optical+Labeling+and+Tracking+of+Human+Mesenchymal+Stem+Cells+Post-Transplantation+In+Vivo.">Liposome-Indocyanine Green Nanoprobes for Optical Labeling and Tracking of Human Mesenchymal Stem Cells Post-Transplantation In Vivo.</a> Advanced Healthcare Materials. 6 (21), (2017).</li><li>Greco, A., 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=High+frequency+ultrasound+for+in+vivo+pregnancy+diagnosis+and+staging+of+placental+and+fetal+development+in+mice.">High frequency ultrasound for in vivo pregnancy diagnosis and staging of placental and fetal development in mice.</a> PloS One. 8 (10), e77205 (2013).</li><li>Spurney, C. F., Leatherbury, L., Lo, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+ultrasound+database+profiling+growth,+development,+and+cardiovascular+function+in+C57BL/6J+mouse+fetuses.">High-frequency ultrasound database profiling growth, development, and cardiovascular function in C57BL/6J mouse fetuses.</a> Journal of the American Society of Echocardiography. 17 (8), 893-900 (2004).</li><li>Zhang, B., 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=Synthesis+and+characterization+of+placental+chondroitin+sulfate+A+(plCSA)+-targeting+lipid-polymer+nanoparticles.">Synthesis and characterization of placental chondroitin sulfate A (plCSA) -targeting lipid-polymer nanoparticles.</a> Journal of Visualized Experiments. , (2018).</li><li>Devraj, K., Guerit, S., Macas, J., Reiss, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+In+Vivo+Blood-brain+Barrier+Permeability+Assay+in+Mice+Using+Fluorescently+Labeled+Tracers.">An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers.</a> Journal of Visualized Experiments. (132), (2018).</li><li>Beeton, C., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mononuclear+cells+from+the+central+nervous+system+of+rats+with+EAE.">Isolation of mononuclear cells from the central nervous system of rats with EAE.</a> Journal of Visualized Experiments. (10), 527 (2007).</li><li>Watson, E. D., Cross, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+structures+and+transport+functions+in+the+mouse+placenta.">Development of structures and transport functions in the mouse placenta.</a> Physiology. 20 (3), 180-193 (2005).</li><li>Frangioni, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+near-infrared+fluorescence+imaging.">In vivo near-infrared fluorescence imaging.</a> Current Opinion in Chemical Biology. 7 (5), 626-634 (2003).</li><li>Flores, L. E., Hildebrandt, T. B., Kuhl, A. A., Drews, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+detection+and+staging+of+spontaneous+embryo+resorption+by+ultrasound+biomicroscopy+in+murine+pregnancy.">Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy.</a> Reproductive Biology and Endocrinology. 12, 38 (2014).</li><li>Khankin, E. V., Hacker, M. R., Zelop, C. M., Karumanchi, S. A., Rana, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+high-frequency+ultrasonography+to+evaluate+cardiovascular+and+uteroplacental+blood+flow+in+mouse+pregnancy.">Intravital high-frequency ultrasonography to evaluate cardiovascular and uteroplacental blood flow in mouse pregnancy.</a> Pregnancy Hypertension. 2 (2), 84-92 (2012).</li><li>Phoon, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tools+for+the+developmental+biologist:+ultrasound+biomicroscopy+of+mouse+embryonic+development.">Imaging tools for the developmental biologist: ultrasound biomicroscopy of mouse embryonic development.</a> Pediatric Research. 60 (1), 14-21 (2006).</li><li>Pallares, P., Gonzalez-Bulnes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+ultrasonographic+characterization+of+phenotypic+changes+during+embryo+development+in+non-anesthetized+mice+of+different+genotypes.">Non-invasive ultrasonographic characterization of phenotypic changes during embryo development in non-anesthetized mice of different genotypes.</a> Theriogenology. 70 (1), 44-52 (2008).</li><li>Parvani, J. G., Gujrati, M. D., Mack, M. A., Schiemann, W. P., Lu, Z. -. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+&#946;3+integrin+by+targeted+ECO/siRNA+nanoparticles+inhibits+EMT+and+metastasis+of+triple-negative+breast+cancer.">Silencing &#946;3 integrin by targeted ECO/siRNA nanoparticles inhibits EMT and metastasis of triple-negative breast cancer.</a> Cancer Research. 75 (11), 2316-2325 (2015).</li><li>Zhang, B., 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=Targeted+delivery+of+doxorubicin+by+CSA-binding+nanoparticles+for+choriocarcinoma+treatment.">Targeted delivery of doxorubicin by CSA-binding nanoparticles for choriocarcinoma treatment.</a> Drug Delivery. 25 (1), 461-471 (2018).</li><li>Jenkins, D. 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=Bioluminescent+imaging+(BLI)+to+improve+and+refine+traditional+murine+models+of+tumor+growth+and+metastasis.">Bioluminescent imaging (BLI) to improve and refine traditional murine models of tumor growth and metastasis.</a> Clinical &amp; Experimental Metastasis. 20 (8), 733-744 (2003).</li><li>Keelan, J. A., Leong, J. W., Ho, D., Iyer, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+and+safety+considerations+of+nanoparticle-mediated+drug+delivery+in+pregnancy.">Therapeutic and safety considerations of nanoparticle-mediated drug delivery in pregnancy.</a> Nanomedicine. 10 (14), 2229-2247 (2015).</li></ol>

范和 B.Z. 是 SIAT 提出的专利申请 PCT/CN2017/108646 的发明者, 涵盖了胎盘特异的药物传递方法及其应用。所有其他作者声明他们没有竞争的兴趣。

在这篇手稿中, 我们概述了一个三方法系统, 以确定是否 plCSA-BP 制导纳米粒子是一个有效的工具, 以提供药物到胎盘。利用活体成像技术监测红外荧光干扰信号, 证实了 plCSA 的胎盘靶向特异性. 采用 HFUS 和 HPLC 法, 证明了 plCSA-bp 共轭纳米粒子能有效地将氨蝶呤输送到胎盘细胞, 而不是胎儿。
在活体荧光成像实验中, 孕鼠的妊娠年龄是重要的。胎盘在 E9.5<sup class="xref"...

胎盘介导的妊娠并发症, 包括先兆子痫、妊娠丢失、胎盘早剥和小胎龄 (SGA), 是常见的, 导致大量的胎儿和产妇发病率和死亡率1,2, 3, 很少有药物被证明是有效的治疗妊娠疾病4,5。在怀孕期间, 制定更有选择性和更安全的胎盘靶向药物的策略在现代药物治疗中仍然具有挑战性。
近年来, 几份报告着重于以胎盘为靶的工具, 用多肽或抗体包衣纳米微粒, 将药物子宫胎盘组织定向交付。这些包括抗表皮生长因子受体 (EGFR)6抗体, 肿瘤自导肽 (CGKRK 和 iRGD)7, 胎盘靶向肽8, 胎盘血管靶向肽9和抗体对催产素受体10。
在这里, 我们证明, 合成胎盘硫酸软骨素 a 结合肽 (plCSA BP) 可用于靶向交付纳米微粒及其药物有效载荷的胎盘11。plCSA-BP 制导纳米粒子与报告的子宫胎盘靶向方法是互补的, 因为它们靶向胎盘滋养层。
作为一种非侵入性的方法,在体内成像已被用来监测12小鼠胎盘特异基因的表达, 吲哚菁绿 () 已被广泛应用于用荧光成像系统跟踪纳米粒子13, 14,15。因此, 我们静脉注射注射 plCSA-BP-共轭纳米粒子负载的 plCSA-修订为期三), 以可视化的 plCSA, 在怀孕小鼠与荧光成像仪的分布。然后, 我们静脉注射注射甲氨蝶呤 (MTX) 加载 plCSA 核动力源到怀孕的小鼠。高频超声 (HFUS), 另一种无创, 实时成像工具16,17被用来监测胎儿和胎盘发育的小鼠。最后, 我们用高效液相色谱 (HPLC) 定量分析了甲氨蝶呤在胎盘和胎儿中的分布情况。
在本议定书中, 我们详细描述了用 plCSA-BP 引导 nanocarriers 评估胎盘靶向药物传递效率的三方法系统。

<table><tbody><tr><td>CD-1 mice</td><td>Beijing Vital River</td><td>201</td><td>Female (8-12 week)</td></tr><tr><td>Insulin syringe</td><td>BD</td><td>328421</td><td>for IV injection</td></tr><tr><td>Ethanol absolute</td><td>Sinopharm Chemical</td><td>10009218</td><td>for nanoparticles synthesis</td></tr><tr><td>Soybean lecithin</td><td>Avanti Polar Lipids</td><td>441601</td><td>for nanoparticles synthesis</td></tr><tr><td>DSPE-PEG-COOH</td><td>Avanti Polar Lipids</td><td>880125</td><td>for nanoparticles synthesis</td></tr><tr><td>PLGA</td><td>Sigma-Aldrich</td><td>719897</td><td>for nanoparticles synthesis</td></tr><tr><td>Ultrasonic processor</td><td>Sonics</td><td>VCX130</td><td>for nanoparticles synthesis</td></tr><tr><td>Methotrexate (MTX)</td><td>Sigma-Aldrich</td><td>V900324</td><td>for nanoparticles synthesis</td></tr><tr><td>Indocyanine green (ICG)</td><td>Sigma-Aldrich</td><td>1340009</td><td>for in vivo imaging</td></tr><tr><td>phosphate-buffered saline (PBS)</td><td>Hyclone</td><td>SH30028.01</td><td></td></tr><tr><td>IVIS spectrum instrument</td><td>Perkin Elmer</td><td></td><td>for in vivo imaging</td></tr><tr><td>Ultrasound transmission gel</td><td>Guanggong</td><td>ZC4252418</td><td>for ultrasound imaging</td></tr><tr><td>Isoflurane</td><td>Lunan Pharmaceutical</td><td>I0040</td><td>for maintain the anesthesia</td></tr><tr><td>Depilatory cream</td><td>Nair</td><td>TMG001</td><td>for removing fur</td></tr><tr><td>40 MHz transducer</td><td>VisualSonics</td><td>MS550S</td><td>for ultrasound imaging</td></tr><tr><td>High-frequency ultrasound imaging system</td><td>VisualSonics</td><td>Vevo2100</td><td>for ultrasound imaging</td></tr><tr><td>Avertin</td><td>Sigma-Aldrich</td><td>T48402</td><td>for anesthesia</td></tr><tr><td>Syringe pump</td><td>Mindray</td><td>SK-500III</td><td>forcardiac perfusion</td></tr><tr><td>0.9% saline solution</td><td>Meilunbio</td><td>MA0083</td><td>forcardiac perfusion</td></tr><tr><td>1.5 mL Polypropylene tubes</td><td>AXYGEN</td><td>MCT-150-C</td><td></td></tr><tr><td>-80 &amp;deg;C freezer</td><td>Thermo Fisher Scientific</td><td>88600V</td><td></td></tr><tr><td>Centriguge</td><td>Cence</td><td>H1650R</td><td></td></tr><tr><td>Perchloric acid</td><td>Sigma-Aldrich</td><td>311421</td><td>for precipitating protein</td></tr><tr><td>Homogenizer</td><td>SCIENTZ</td><td>SCIENTZ-48</td><td>for homogenizing tissue</td></tr><tr><td>Syringe filter (0.45 &amp;mu;m)</td><td>Millipore</td><td>SLHV033RS01</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sinopharm Chemical</td><td>10019763</td><td>for solving MTX</td></tr><tr><td>HPLC vials</td><td>Waters</td><td>670650620</td><td>for HPLC</td></tr><tr><td>Potassium phosphate dibasic</td><td>Sinopharm Chemical</td><td>20032117</td><td>for HPLC</td></tr><tr><td>Acetonitrile</td><td>JKchemical</td><td>932537</td><td>for HPLC</td></tr><tr><td>C18 column</td><td>Waters</td><td>186003966</td><td>for HPLC</td></tr><tr><td>HPLC system</td><td>Shimadzu</td><td></td><td>for HPLC</td></tr></tbody></table>

comprehensive evaluation of the effectiveness and safety of placenta-targeted drug delivery using three complementary methods

目前尚无与胎盘相关的妊娠并发症的有效治疗方法, 并制定了靶向胎盘提供药物的战略, 同时尽量减少胎儿和产妇的副作用仍然具有挑战性。靶向纳米微粒载体为治疗胎盘疾病提供了新的机会。我们最近证明, 合成胎盘硫酸软骨素 a 结合肽 (plCSA BP) 可以用来指导纳米粒子向胎盘提供药物。在本议定书中, 我们详细描述了一个系统, 以评估药物交付胎盘由 plCSA BP, 采用三种不同的方法结合使用:在体内成像, 高频超声 (HFUS), 和高性能液相色谱法 (HPLC)。plCSA-bp 制导纳米粒子在活体动物胎盘中的应用, HFUS 和 HPLC 表明, plCSA-bp-共轭纳米粒子有效地将甲氨蝶呤送到胎盘。因此, 这些方法的组合可以作为一个有效的工具, 为靶向胎盘提供药物和制定新的治疗策略, 一些妊娠并发症。

在这篇手稿中, plCSA-BP-共轭纳米粒子负载甲氨蝶呤 (plCSA MNPs) 或一组 (plCSA 修订为期三) 被静脉注射到怀孕小鼠。体外成像显示, 在 plCSA 注射30分钟后, 子宫区域内有较强的干扰信号。修订为期三主要定位于肝脾区 (图 1A)。在 plCSA 注射后48小时, 怀孕小鼠被牺牲, 只在胎盘中暴露了干扰信号, 而胎儿没有信号可检测 (图 1B)。...

Watch this Scientific Journal Video about Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods at JoVE.com

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

我们描述一个系统, 利用三方法来评估胎盘靶向药物的安全性和有效性:在体内成像监测纳米微粒积累, 高频超声监测胎盘和胎儿发育, 并以 HPLC 量化药物对组织的传递。

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 ...

comprehensive-evaluation-effectiveness-safety-placenta-targeted-drug

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.6K 次观看.  Chinese Academy of Sciences. 我们描述一个系统, 利用三方法来评估胎盘靶向药物的安全性和有效性:在体内成像监测纳米微粒积累, 高频超声监测胎盘和胎儿发育, 并以 HPLC 量化药物对组织的传递。

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

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

科研

遗传学

Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC twin pregnancies develop complications arising from the shared placenta and associated vascular connections1. Severe twin-to-twin syndrome (TTTS) is reported to account for approximately 20% of these complications2,3. Inter-twin vascular connections occur in almost all MC placentas and are related to the prognosis and outcome of these high-risk twin pregnancies. The number, size and type of connections have been implicated in the development of TTTS and other MC twin conditions. Three types of inter-twin vascular connections occur: 1) artery to vein connections (AVs) in which a branch artery carrying deoxygenated blood from one twin courses along the fetal surface of the placenta and dives into a placental cotyledon. Blood flows via a deep intraparenchymal capillary network into a draining vein that emerges at the fetal surface of the placenta and brings oxygenated blood toward the other twin. There is unidirectional flow from the twin supplying the afferent artery toward the twin receiving the efferent vein; 2) artery to artery connections (AAs) in which a branch artery from each twin meets directly on the superficial placental surface resulting in a vessel with pulsatile bidirectional flow, and 3) vein to vein connections (VVs) in which a branch vein from each twin meets directly on the superficial placental surface allowing low pressure bidirectional flow. In utero obstetric sonography with targeted Doppler interrogation has been used to identify the presence of AV and AA connections4. Prenatally detected AAs that have been confirmed by postnatal placental injection studies have been shown to be associated with an improved prognosis for both twins5. Furthermore, fetoscopic laser ablation of inter-twin vascular connections on the fetal surface of the shared placenta is now the preferred treatment for early, severe TTTS. 
Postnatal placental injection studies provide a valuable method to confirm the accuracy of prenatal Doppler ultrasound findings and the efficacy of fetal laser therapy6. Using colored dyes separately hand-injected into the arterial and venous circulations of each twin, the technique highlights and delineates AVs, AAs, and VVs. This definitive demonstration of MC placental vascular anatomy may then be correlated with Doppler ultrasound findings and neonatal outcome to enhance our understanding of the pathophysiology of MC twinning and its sequelae. Here we demonstrate our placental injection technique.

Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins&#x2019; circulations. 
Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.

导丝辅助导管和彩色染料注射的双人单绒毛膜胎盘血管映射

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC ...

医学

Accurate and Simple Evaluation of Vascular Anastomoses in Monochorionic Placenta using Colored Dye

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia polycythemia sequence (TAPS)1,2. Injection studies of twin placentas have shown that such anastomoses are almost invariably present in monochorionic twins and extremely rare in dichorionic twins1. Three types of anastomoses have been documented: from artery to artery, from vein to vein and from artery to vein. Arterio-venous (AV) anastomoses are unidirectional and are referred to as "deep" anastomoses since they proceed through a shared placental cotyledon, whereas arterio-arterial (AA) and veno-venous (VV) anastomoses are bi-directional and are referred to as "superficial" since they lie on the chorionic plate. Both TTTS and TAPS are caused by net imbalance of blood flow between the twins due to AV anastomoses. Blood from one twin (the donor) is pumped through an artery into the shared placental cotyledon and then drained through a vein into the circulation of the other twin (the recipient). Unless blood is pumped back from the recipient to the donor through oppositely directed deep AV anastomoses or through superficial anastomoses, an imbalance of blood volumes occurs, gradually leading to the development of TTTS or TAPS. The presence of an AA anastomosis has been shown to protect against the development of TTTS and TAPS by compensating for the circulatory imbalance caused by the uni-directional AV anastomoses1,2. 
Injection of monochorionic placentas soon after birth is a useful mean to understand the etiology of various (hematological) complications in monochorionic twins and is a required test to reach the diagnosis of TAPS2. In addition, injection of TTTS placentas treated with fetoscopic laser surgery allows identification of possible residual anastomoses3-5. This additional information is of paramount importance for all perinatologists involved in the management and care of monochorionic twins with TTTS or TAPS. Several placental injection techniques are currently being used. We provide a simple protocol to accurately evaluate the presence of (residual) vascular anastomoses using colored dye injection.

Twin-to-twin transfusion syndrome and twin anemia polycythemia sequence are two potentially devastating problems in perinatal medicine. Both disorders occur only in monochorionic twins and result from unbalanced blood flow through placental vascular anastomoses. We provide a simple protocol to accurately evaluate the presence of vascular anastomoses using colored dye injection of placental vessels after birth.

在单绒毛膜胎盘血管吻合用有色染料的精确和简单的评价

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia ...

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising approach to cancer therapy. The glycosaminoglycan placental chondroitin sulfate A (plCSA) is expressed on a wide range of cancer cells and placental trophoblasts, and malarial protein VAR2CSA can specifically bind to plCSA. A reported placental chondroitin sulfate A binding peptide (plCSA-BP), derived from malarial protein VAR2CSA, can also specifically bind to plCSA on cancer cells and placental trophoblasts. Hence, plCSA-BP-conjugated nanoparticles could be used as a tool for targeted drug delivery to human cancers and placental trophoblasts. In this protocol, we describe a method to synthesize plCSA-BP-conjugated lipid-polymer nanoparticles loaded with doxorubicin (plCSA-DNPs); the method consists of a single sonication step and bioconjugate techniques. In addition, several methods for characterizing plCSA-DNPs, including determining their physicochemical properties and cellular uptake by placental choriocarcinoma (JEG3) cells, are described.

Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles

Here, we present a protocol for the synthesis of placental chondroitin sulfate A binding peptide (plCSA-BP)-conjugated lipid-polymer nanoparticles via single-step sonication and bioconjugate techniques. These particles constitute a novel tool for the targeted delivery of therapeutics to most human tumors and placental trophoblasts to treat cancers and placental disorders.

胎盘硫酸软骨素 A (plCSA) 靶向脂质高分子纳米粒的合成与表征

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising ...

癌症研究

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 ...

生物工程

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

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

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. ...

Mouse In Vivo Placental Targeted CRISPR Manipulation

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients and waste between the mother and fetus and the production and delivery of growth factors and hormones. Placental genetic manipulations in mice are critical for understanding the placenta's specific role in prenatal development. Placental-specific Cre-expressing transgenic mice have varying effectiveness, and other methods for placental gene manipulation can be useful alternatives. This paper describes a technique to directly alter placental gene expression using CRISPR gene manipulation, which can be used to modify the expression of targeted genes. Using a relatively advanced surgical approach, pregnant dams undergo a laparotomy on embryonic day 12.5 (E12.5), and a CRISPR plasmid is delivered by a glass micropipette into the individual placentas. The plasmid is immediately electroporated after each injection. After dam recovery, the placentas and embryos can continue development until assessment at a later time point. The evaluation of the placenta and offspring after the use of this technique can determine the role of time-specific placental function in development. This type of manipulation will allow for a better understanding of how placental genetics and function impact fetal growth and development in multiple disease contexts.

Mouse In Vivo Placental Targeted CRISPR Manipulation

Here we describe a time-specific method to effectively manipulate critical developmental pathways in the mouse placenta in vivo. This is performed through the injection and electroporation of CRISPR plasmids into the placentas of pregnant dams on embryonic day 12.5.

小鼠 体内 胎盘靶向CRISPR操作

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients ...

Ex Vivo Perfusion of the Rodent Placenta

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An assay is described here to perfuse an isolated rat placenta and evaluate the maternal-to-fetal translocation of xenobiotics ex vivo. In addition, the evaluation of physiological processes such as fluid flow to the fetus and placental metabolism may be conducted with this methodology. This technique is suitable for evaluating maternal-to-fetal kinetics of pharmaceutical candidates or environmental contaminants. In contrast to current alternative approaches, this methodology allows the evaluation of the isolated maternal-fetal vasculature, with the systemic neural or immune involvement removed, allowing any observed changes in physiological function to be attributable to local factors within the isolated tissue.

Here is presented a protocol of ex vivo maternal-fetal vascular perfusion to enable the administration of a test article into maternal vasculature and to evaluate placental transfer of xenobiotic particles or pharmacological agents in addition to alterations in placental physiology.

啮齿类动物胎盘的体外灌注

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An ...

发育生物学

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

疏水货物封装有针对性的质膜交付在液晶纳米颗粒载体

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

人体胎盘生理学综合研究的4血管取样方法<em&gt;体内</em

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...

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

摘要

探索更多视频

此视频中的章节

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

导丝辅助导管和彩色染料注射的双人单绒毛膜胎盘血管映射

在单绒毛膜胎盘血管吻合用有色染料的精确和简单的评价

胎盘硫酸软骨素 A (plCSA) 靶向脂质高分子纳米粒的合成与表征

通过胎盘的外来物质和纳米材料的运输率测定

模型和方法来评价药物输送系统在整个细胞屏障的运输

小鼠体内胎盘靶向CRISPR操作

啮齿类动物胎盘的体外灌注

疏水货物封装有针对性的质膜交付在液晶纳米颗粒载体

人体胎盘生理学综合研究的4血管取样方法

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

摘要

探索更多视频

此视频中的章节

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

导丝辅助导管和彩色染料注射的双人单绒毛膜胎盘血管映射

在单绒毛膜胎盘血管吻合用有色染料的精确和简单的评价

胎盘硫酸软骨素 A (plCSA) 靶向脂质高分子纳米粒的合成与表征

通过胎盘的外来物质和纳米材料的运输率测定

模型和方法来评价药物输送系统在整个细胞屏障的运输

小鼠 体内 胎盘靶向CRISPR操作

啮齿类动物胎盘的体外灌注

疏水货物封装有针对性的质膜交付在液晶纳米颗粒载体

人体胎盘生理学综合研究的4血管取样方法

小鼠体内胎盘靶向CRISPR操作