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>
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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. 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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 E9.521左右开始形成。此外, 考虑到成像的分辨率,在体内成像实验应该在 E 10.5 之后进行。根据该协议, 在 E 11.5 plCSA 注射后, 在描述的条件下没有检测出荧光信号, 这可能是由于皮肤和内脏防止信号传输22。为了克服这种限制, 必须利用增加注射剂量或胎盘和胎儿的采集来进行活体成像。
HFUS 成像的一个关键步骤是使用合适的传感器来获得高质量的胚胎图像。对小鼠胚胎学成像的优化频率为40-50 兆赫。此外, 在获取图像之前保持怀孕小鼠的生理体温也很重要。最后, 观察者在早期胚胎发育过程中记录 B 型电影时应该小心 (e 6.5-e 8.5), 这更依赖于经验。在超声处理16、23、24期间, 将解剖特征与胎儿和胎盘运动的参考框架进行比较, 可以弥补测量的不确定度。通过多项测量和增加胎儿和胎盘的数量, 可以提高成像数据的准确性。
血管内未绑定的纳米微粒是评估靶向胎盘和胎儿的药物传递的有效因素。因此, 在采集胎儿和胎盘之前, 进行心脏灌注以去除未绑定的纳米颗粒。以前的研究7,8,9还指出, 在分析肽的能力, 以结合胎盘, 对老鼠进行心脏灌注是必不可少的。
HPLC 分析中的一个可能的陷阱是甲氨蝶呤与其他峰值的重叠。乙腈用于洗脱甲氨喋呤的柱。如果重叠峰发生在5分钟之前, 降低乙腈在流动阶段的浓度可能是有益的。如果在30分钟后没有峰或重叠峰发生, 增加乙腈的浓度是有用的。高效液相色谱法的一个主要局限性是, 它不能揭示纳米颗粒在胎盘中的定位。plCSA-BP 制导纳米粒子专门针对胎盘迷宫在小鼠胎盘11。因此, 胎盘的形态学分析是必要的。
这是首次采用体内成像、HFUS 和 HPLC 相结合的方式来确定胎盘靶向分娩的效率。HFUS 已成为一种先进、无创、安全、实时的成像方法, 已成功用于小鼠胚胎发育17、25、26的高分辨率成像。虽然体内荧光成像已被广泛用于可视化的肿瘤形成和转移的活鼠27,28,29, 它以前没有被用于研究胎盘药物传递。作为一种替代方法,在体内荧光成像具有明显的优势超过 HFUS, 能够直接可视化的分布, 静脉注射纳米粒子在活鼠, 但不能监测胎盘和胎儿的发展。因此, 我们结合荧光在体内成像和高分辨率 HFUS 的优势, 前者在体内的 plCSA-BP 引导修订为期三的可视化, 后者使体内监测 plCSA MNPs 对胎盘和胎儿发育和生存的影响。此外, HPLC 证实, plCSA-MNPs 是专门送到胎盘, 并没有到达胎儿。
靶向纳米医学是妊娠期疾病领域的一项新的发展, 需要采取大量新的方法, 专门向产妇器官提供药物, 以治疗30门诊的妊娠障碍。本协议所描述的三方法系统是将纳米微粒靶向的体内时间过程成像与胎盘和胎儿发育的相应影响相结合, 从而更精确的生化测量组织中药物的数量, 用以评估靶向胎盘分娩治疗胎盘介导的妊娠并发症的工具。

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 border="0" cellpadding="0" cellspacing="0" style="width:785px;" width="786">
	<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 &#176;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 &#956;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)。
然后, 我们用 HFUS 来监测注射纳米微粒后胚胎发育的情况。生物统计学测量包括妊娠囊长度 (GS), 胎儿冠尾长度 (CRL), 双顶直径 (桶), 腹部周长 (AC), 胎盘直径 (PD), 胎盘厚度 (PT), 脐动脉峰值速度 (UA), 和胎儿心脏率 (HR) (电影 1)。表 2列出了不同胎龄的形态学参数。在 plCSA, 相对于 PBS 组, 平均胎儿腹围和脐动脉峰值速度在 E12.5 (图 2A和2H) 显著降低, 冠臀长度和胎盘直径在 E10.5 显著减少 (图 2B和2F)。从 E9.5 开始, 妊娠囊长度也显著降低 (图 2C), 双顶直径、胎盘厚度和胎儿心率在 E 11.5 上开始显著下降, 相对于 PBS 组 (图2D、2E和2G)。这些发现共同表明, plCSA-MNPs 对胎儿和胎盘发育有很强的细胞毒性作用。有趣的是, MNPs 的治疗也轻微地损害了胎儿和胎盘发育 (图 2 2 h), 表明纳米粒子可能通过增强的通透性和保留 (EPR) 效应改善甲氨喋呤对胎盘的传递。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>孕龄</td>
			<td>组</td>
			<td>蜕膜 (mm)</td>
			<td>GS (mm)</td>
			<td>CRL (mm)</td>
			<td>桶 (mm)</td>
			<td>AC (mm)</td>
			<td>PD (mm)</td>
			<td>PT (mm)</td>
			<td>HR (bpm)</td>
			<td>UA (毫米/秒)</td>
		</tr>
<tr>
<td>E6。5</td>
			<td></td>
			<td>0.92±0.23</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
		</tr>
<tr>
<td rowspan="3">E7。5</td>
			<td>Pbs</td>
			<td>/</td>
			<td>0.82±0.24</td>
			<td>0.72±0.18</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
		</tr>
<tr>
<td>MNPs</td>
			<td>/</td>
			<td>0.83±0.14</td>
			<td>0.83±0.14</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
		</tr>
<tr>
<td>plCSA-MNPs</td>
			<td>/</td>
			<td>0.65±0.23</td>
			<td>0.65±0.23</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
		</tr>
<tr>
<td rowspan="3">E8。5</td>
			<td>Pbs</td>
			<td>/</td>
			<td>2.02±0.54</td>
			<td>1.88±0.40</td>
			<td>0.93±0.23</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
		</tr>
<tr>
<td>MNPs</td>
			<td>/</td>
			<td>1.49±0.50</td>
			<td>1.49±0.50</td>
			<td>0.82±0.20</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
		</tr>
<tr>
<td>plCSA-MNPs</td>
			<td>/</td>
			<td>1.14±0.46</td>
			<td>1.02±0.42</td>
			<td>0.83±0.18</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
		</tr>
<tr>
<td rowspan="3">E9。5</td>
			<td>Pbs</td>
			<td>/</td>
			<td>3.31±0.62</td>
			<td>3.49±0.65</td>
			<td>1.39±0.54</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
		</tr>
<tr>
<td>MNPs</td>
			<td>/</td>
			<td>2.34±0.68</td>
			<td>2.23±0.49</td>
			<td>0.98±0.34</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
		</tr>
<tr>
<td>plCSA-MNPs</td>
			<td>/</td>
			<td>1.83±0.42</td>
			<td>1.59±0.59</td>
			<td>0.94±0.25</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
		</tr>
<tr>
<td rowspan="3">E10。5</td>
			<td>Pbs</td>
			<td>/</td>
			<td>4.43±0.67</td>
			<td>4.97±0.80</td>
			<td>2.10±0.61</td>
			<td>4.83±1.40</td>
			<td>2.91±0.23</td>
			<td>2.24±0.24</td>
			<td>100±30</td>
			<td>30.16±9.40</td>
		</tr>
<tr>
<td>MNPs</td>
			<td>/</td>
			<td>3.28±0.64</td>
			<td>2.91±0.83</td>
			<td>1.46±0.54</td>
			<td>3.95±1.28</td>
			<td>2.66±0.33</td>
			<td>2.17±0.19</td>
			<td>87±21</td>
			<td>24.63±7.35</td>
		</tr>
<tr>
<td>plCSA-MNPs</td>
			<td>/</td>
			<td>2.64±0.66</td>
			<td>2.17±0.85</td>
			<td>1.12±0.33</td>
			<td>3.82±1.13</td>
			<td>2.13±0.35</td>
			<td>1.94±0.15</td>
			<td>83±22</td>
			<td>15.37±5.70</td>
		</tr>
<tr>
<td rowspan="3">E11。5</td>
			<td>Pbs</td>
			<td>/</td>
			<td>5.68±0.73</td>
			<td>6.45±0.90</td>
			<td>3.08±0.70</td>
			<td>8.67±2.08</td>
			<td>4.16±0.39</td>
			<td>2.75±0.26</td>
			<td>124±28</td>
			<td>31.62±7.76</td>
		</tr>
<tr>
<td>MNPs</td>
			<td>/</td>
			<td>4.36±0.39</td>
			<td>3.74±1。2</td>
			<td>2.31±0.53</td>
			<td>6.69±1.85</td>
			<td>3.56±0.40</td>
			<td>2.39±0.23</td>
			<td>106±22</td>
			<td>25.20±6.18</td>
		</tr>
<tr>
<td>plCSA-MNPs</td>
			<td>/</td>
			<td>3.42±0.76</td>
			<td>2.61±0.84</td>
			<td>1.51±0.54</td>
			<td>4.59±1.57</td>
			<td>2.54±0.49</td>
			<td>2.09±0.27</td>
			<td>79±20</td>
			<td>16.66±5.69</td>
		</tr>
<tr>
<td rowspan="3">E12。5</td>
			<td>Pbs</td>
			<td>/</td>
			<td>/</td>
			<td>8.12±1.29</td>
			<td>3.90±0.65</td>
			<td>12.43±2.48</td>
			<td>5.37±0.42</td>
			<td>3.14±0.24</td>
			<td>141±26</td>
			<td>40.62±10.89</td>
		</tr>
<tr>
<td>MNPs</td>
			<td>/</td>
			<td>/</td>
			<td>4.87±1.29</td>
			<td>2.87±0.62</td>
			<td>8.29±1.78</td>
			<td>4.25±0.67</td>
			<td>2.65±0.26</td>
			<td>119±18</td>
			<td>27.76±7.52</td>
		</tr>
<tr>
<td>plCSA-MNPs</td>
			<td>/</td>
			<td>/</td>
			<td>3.2±1.28</td>
			<td>1.75±0.60</td>
			<td>5.47±1.39</td>
			<td>3.05±0.50</td>
			<td>2.28±0.26</td>
			<td>72±22</td>
			<td>18.76±7.20</td>
		</tr>
<tr>
<td rowspan="3">E13。5</td>
			<td>Pbs</td>
			<td>/</td>
			<td>/</td>
			<td>10.04±1。2</td>
			<td>4.67±0.65</td>
			<td>15.64±2.33</td>
			<td>6.03±0.60</td>
			<td>3.49±0.23</td>
			<td>157±28</td>
			<td>54.62±12.37</td>
		</tr>
<tr>
<td>MNPs</td>
			<td>/</td>
			<td>/</td>
			<td>6.17±1.29</td>
			<td>3.37±0.55</td>
			<td>9.39±1.88</td>
			<td>4.77±0.69</td>
			<td>2.92±0.43</td>
			<td>109±22</td>
			<td>35.84±9.49</td>
		</tr>
<tr>
<td>plCSA-MNPs</td>
			<td>/</td>
			<td>/</td>
			<td>3.57±1.71</td>
			<td>1.87±0.73</td>
			<td>6.25±1.41</td>
			<td>3.42±0.63</td>
			<td>2.37±0.34</td>
			<td>60±23</td>
			<td>20.02±11.20</td>
		</tr>
<tr>
<td rowspan="3">E14。5</td>
			<td>Pbs</td>
			<td>/</td>
			<td>/</td>
			<td>12.35±1。6</td>
			<td>5.36±0.71</td>
			<td>18.38±2.53</td>
			<td>6.70±0.64</td>
			<td>3.75±0.35</td>
			<td>167±27</td>
			<td>71.48±10.72</td>
		</tr>
<tr>
<td>MNPs</td>
			<td>/</td>
			<td>/</td>
			<td>7.6±1.56</td>
			<td>3.90±0.70</td>
			<td>10.31±2.31</td>
			<td>5.23±0.76</td>
			<td>3.10±0.39</td>
			<td>99±23</td>
			<td>45.80±13.07</td>
		</tr>
<tr>
<td>plCSA-MNPs</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
			<td>/</td>
		</tr>
</tbody></table>表2。测量每个胎龄的形态学参数.GS: 妊娠囊长度;CRL: 皇冠尾部长度;桶: 双顶直径;AC: 腹部周长;胎盘直径;铂: 胎盘厚度;HR: 胎儿心率;脐动脉峰值速度;/: 无法测量。
我们接下来用 HPLC 测定胎盘和胎儿的甲氨喋呤浓度。使用以上所述的 HPLC 操作参数, 甲氨蝶呤保留时间确定为7分钟, 在 plCSA-自然人流动组的胎盘中检测到甲氨喋呤 (图 3)。用甲氨蝶呤标准曲线测定胎盘和胎儿的甲氨喋呤浓度 (图 4)。24小时注射后, 自然人流动组胎盘氨甲喋呤水平明显低于 plCSA-自然人流动组, plCSA-自然人流动组的胎儿未发现甲氨喋呤。在 plCSA-流动注射后48小时内仍可检测到甲氨蝶呤 (图 5)。这些结果表明, plCSA-MNPs 不能跨越胎盘, 从而减少潜在的不利影响胎儿。
总之, 这个由体内荧光成像、HFUS 和 HPLC 组成的三方法系统, 可以用来确定药物运载工具的靶向 nanocarriers 和向胎盘提供药物的程度。使用这些方法, 我们已经证明, plCSA-BP 制导纳米粒子是一种有效的工具, 以提供药物到胎盘。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58219/58219fig1.jpg"> 图 1.体内荧光成像.(A) E11.5 的怀孕小鼠 (n=5) 注射修订为期三或 plCSA 修订为期三 (5 毫克/千克),通过尾部静脉。30分钟后, 小鼠用荧光成像系统进行成像。(B) 在注射修订为期三或 plCSA 修订为期三后48小时, 采集胎儿 (F、n=2 每只老鼠) 和胎盘 (P、n=2 每只老鼠), 并用荧光成像系统进行成像。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58219/58219fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58219/58219fig2.jpg"> 图 2.HFUS 胚胎生长的量化.(A) 腹部周长 (n = 30-51 胚胎/天), (B) 冠臀长 (n = 30-51 胚/日), (C) 妊娠囊长度 (n = 10-30 胚胎/天), (D) 双顶直径 (n = 30-51 胚胎/天), (E) 胎盘厚度 (n = 30-51 胚胎/天), (F)胎盘直径 (n = 30-51 胚胎/天), (G) 胎儿心率 (n = 20-33 胚胎/天), 和 (H) 脐动脉峰值速度 (n = 12-36 胚胎/天) 测量非侵入超声在体内.所有试验均采用2尾配对t检验, 对 p < 0.05 进行统计学意义上的比较。价值被表示作为手段 @ p < 0.05, ** p < 0.01, *** p < 0.001 与 PBS 小组比较。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58219/58219fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58219/58219fig3.jpg"> 图 3.有代表性的 HPLC 图谱胎盘标本.妊娠小鼠 (n=5) 静脉注射 PBS 或 plCSA-MNPs, 其胎盘 (n=15 每组) 收集24小时后的 HPLC。采用标准溶液的甲氨蝶呤与紫外线检测在 313 nm, 保留时间被确定为7分钟.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58219/58219fig3large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58219/58219fig4.jpg"> 图 4.甲氨蝶呤标准曲线.甲氨蝶呤的浓度从0.5 微克/毫升到100微克/毫升不等。数据代表 n=3 的平均±SD。某些数据的误差线小于菱形符号。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58219/58219fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58219/58219fig5.jpg"> 图 5.高效液相色谱法测定胎盘和胎儿 biodistributions 中纳米颗粒的含量.妊娠期 E13.5 对妊娠小鼠进行单 MNPs 或 plCSA MNPs (1 毫克/千克甲氨蝶呤当量) 注射。在24小时和48小时后, 用 HPLC 测定胎盘 (n = 15) 和胎儿 (n = 15) 中甲氨喋呤的浓度。值被表示为 means±SD. 使用未配对学生的t检验 (** p < 0.001) 分析了 plCSA 和自然人流动组之间的氨甲喋呤浓度差异;未检测到。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58219/58219fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Movie 1" src="/files/ftp_upload/58219/58219Movie1.jpg"> 电影 1.HFUS 胎儿和胎盘的图像, 说明生物特征测量位置。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58219/Movie1.mkv" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>

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

Nanyang Technological University

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

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 ...

科研

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. ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

适应静电过程中提供三个独特的环境为一个三层次<em&gt;在体外</em&gt;的气道壁模型

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

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 ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

人工程心脏组织的收缩自动分析心脏药物安全筛选

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

高通量自动 Microbioreactor 系统在 CHO 细胞模型 IgG1 生产中的应用

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

使用自动微生物反应器系统从中国仓鼠卵巢细胞中纯化和分析单克隆抗体

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

二维和三维 i 型胶原蛋白基质角质形成细胞增殖的评价

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

人体神经细胞在体外热的化学和分子分析的生物粒子微阵列

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

使用无细胞细菌提取物、脂质体和乳液转移制备蛋白质生成合成细胞

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...