该研究由资助号资助：NCI P30 CA023108和NCI U54 CA151662。

达特茅斯学院动物护理和使用计划由美国实验动物护理认证协会（iAAALAC）认可，并遵守所有UDSA和NIH（实验动物福利办公室）指南和法规。所有体内研究均获得达特茅斯学院机构动物护理和使用委员会（IACUC）的批准。安乐死程序遵守 2020 年 AVMA 动物 安乐死指南。
1. 系统的仪表/设计
<ol><li>将定制的AMF天线（线圈）设计为闭环，选择形状以创建所需的磁场。使用发电机选择中的电感公式和特性来设计兼容的线圈以产生所需的磁场。使用不同的设计进行体外和体内实验。</li>
	<li>确保AMF天线电感在发电机的可接受范围内。添加或减去电容器以使天线与发电机匹配（调谐）。</li>
	<li>对于体外实验，设计一个 14 圈螺旋线圈，内径 2 cm，长度 14 cm，可以包含 1.5 mL 管，允许同时处理多个样品。用乙烯基聚合物绝缘线圈，并使用聚苯乙烯垫片将线圈与管子分开。设计规范和考虑因素的详细信息见 补充文件 1。</li>
	<li>对于体内实验，请从具有专有设计信息的制造商处购买定制的全身螺旋线圈。使用8毫米方形管（因为它在线圈孔内产生更均匀的磁场），并在目标治疗区域使用浓缩器。使浓缩器长 5.0 厘米，总共转 5 圈，产生 3.6 厘米内径、5.2 厘米外径，并将其定位在目标治疗区域。用聚碳酸酯外壳包围线圈。</li>
	<li>使用功率和频率可调的AMF发电机作为电源，额定功率为10kW或更高。电感将电源和天线/线圈匹配在 0.62 至 1.18 μH 的范围内，允许频率范围在 30-300 kHz 之间。通过离心增压泵使用循环水冷却发电机，压力调节至 50 psi。</li>
	<li>用 5.6 吨制冷量冷却器冷却盘管，该冷水机泵送用水稀释的 25% 乙二醇基传热流体通过 AMF 天线。将冷却器的温度设置为天线不会加热或冷却样品。</li>
	<li>对于动物收容，构造一个管状支架，该支架可以悬挂在线圈的中心，支架和线圈表面之间有 0.5 厘米的气隙。连接一个可调节的调节气泵，该气泵使空气通过线圈周围的外壳循环，并将其设置为保持正常的动物核心温度。将麻醉机连接到动物头部附近的管状动物支架上，以确保麻醉的正确输送。</li>
	<li>对于细胞遏制，创建一个装置，使水浴中的水通过放置管子的垫片循环。设置此水浴的温度，使管子被水包围在37°C。</li>
	<li>使用光纤探针监测肿瘤、动物核心和动物环境的温度，或进行体外研究，监测细胞沉淀的温度和管周围的水。</li>
	<li>在所有实验中使用尺寸为 100 nm 的磁性氧化铁纳米颗粒。 注意：浓度和比吸收率（SAR）是选择纳米颗粒时必须考虑的两个特性，因为它们直接影响可能的加热和热剂量18。</li>
</ol>2. 体外热疗
<ol><li>在含有 10% FBS 和 1% Pen/链球菌的 RPMI 培养基中培养 B16F10 鼠黑色素瘤细胞。在 6 孔板中板 150， 000 个细胞/孔，用 2 mL 完全培养基。</li>
	<li>确定每个孔的适当处理，即没有mNP和没有AMF的细胞，有mNP和没有AMF的细胞，没有mNP和AMF的细胞，有mNP和AMF的细胞。 注意：此外，如果将热疗与另一种疗法相结合，请确保有适当的控制措施。AMF在标准研究台实验室中进行，该实验室经过改造，具有所需的电源和冷却能力。</li>
	<li>电镀后24小时，按照上一步中的确定将mNP添加到适当的孔中。添加 mNP 至浓度为 3 毫克铁/毫升。通过创建储备培养基/mNP溶液（去除旧培养基，添加此溶液）或直接添加mNP并轻轻旋转板以实现均匀分布，确保mNP分布在整个孔中。</li>
	<li>加入mNPs后48小时开始处理，当孔~80%汇合时，通过去除培养基并用新鲜培养基洗涤孔。取出介质。</li>
	<li>向每个接受治疗的孔中加入 0.5 mL 胰蛋白酶，然后轻轻旋转。使用显微镜检查细胞是否分离。</li>
	<li>向每个孔中加入 1 mL 培养基，将细胞收集到 1.5 mL 管中。从孔中收集所有细胞（~1 x 106 个细胞）。为每个孔使用明确标记的单独管。</li>
	<li>以60× g 离心管2-3分钟以使细胞沉淀。将颗粒保留在培养基中。</li>
	<li>将管子放在盘管内装满水的垫片中。设置水浴的温度，使培养基和细胞沉淀保持在37°C。 使用单独的光纤温度探头监测管内和水浴内的温度。</li>
	<li>打开冷却器，检查冷却液是否流过盘管。打开电源并将最大值的百分比调整为所需的字段。以 165 kHz 和 23.87 kA/m （300 Oe） 的频率运行由 10 kW 发电机供电的 14 匝电磁线圈。</li>
	<li>将单独的光纤温度探头放入其中一个管中。处理细胞直到先前确定的方案热剂量。一个例子是在43°C下30分钟（CEM43的30）。</li>
	<li>将细胞重悬于试管中的培养基中，然后将细胞重新铺板到新的 6 孔板中。清楚地标记新板。目标是重新铺板所有收集的细胞（~1 x 106 个细胞）。 注意：应使用新的6孔板以确保培养的细胞接受处理。如果使用旧板，则平板上仍可能残留未成功胰蛋白酶消化的细胞。</li>
	<li>如有必要，对于下一个实验程序，裂解细胞以进行RNA或蛋白质表达分析。</li>
</ol>3.体内热疗
<ol><li>细胞培养和接种<ol><li>在含有 10% FBS 和 1% Pen/链球菌的 RPMI 培养基中培养 B16F10 鼠黑色素瘤细胞。使用能够提供足够细胞接种所需数量动物的盘子/培养皿。例如，10， 100 mm培养皿，接种在100， 000个细胞上，将在48小时内与足够的细胞汇合以进行20只小鼠注射。</li>
		<li>胰蛋白酶消化细胞并使用纯RPMI培养基（无FBS或笔/链球菌）收集。</li>
		<li>计数细胞并根据接种体积和小鼠数量为所需浓度的细胞创建溶液。</li>
		<li>使用汽化异氟醚和氧气麻醉6周龄雌性C57Bl / 6小鼠。将动物放入含有5%异氟醚和95%氧气的有机玻璃盒中，直至诱导。诱导后，取出动物并使用2%异氟醚的面锥完成步骤3.1.5-3.1.7和3.3.3-3.3.6。 注意：对于治疗期间的麻醉，请使用内置麻醉容器。遵循小鼠麻醉的标准机构方案。在动物实验之前，请确保适当的IACUC批准。麻醉后，将动物放回笼子并监测恢复情况，以确保没有并发症。</li>
		<li>检查是否对矫正反射缺乏反应。</li>
		<li>使用电动剃须刀剃除右胁。</li>
		<li>用酒精湿巾清洁注射区域。使用带有28G针头的100μL玻璃注射器注射1-2 x 106 细胞，在麻醉的剃光右胁皮内分散在50μL培养基中。</li>
	</ol></li>
	<li>肿瘤生长/纳米颗粒注射<ol><li>使用卡尺（长度、宽度和深度）测量 3 维肿瘤，并通过（长 x 宽 x 深度 x π）/6 计算体积。</li>
		<li>当肿瘤体积达到120mm 3（+/- 20mm3）时，将动物置于研究中。设计研究，确保有适当的对照和治疗组，包括联合治疗队列（即对照、mNPH、放疗和联合治疗）。</li>
		<li>麻醉将接受mNP的小鼠，如3.1.4中所述。</li>
		<li>用酒精湿巾清洁该区域。在AMF治疗前3小时将mNPs注射到肿瘤中。注射体积，使剂量为7.5mg铁/cm 3 肿瘤。 注意：实验室未发表的数据表明最大mNP摄取发生在3-6小时。</li>
	</ol></li>
	<li>AMF治疗<ol><li>麻醉鼠标并放在加热垫上以保持核心温度。</li>
		<li>检查是否对矫正反射缺乏反应。取下鼠标上的耳标或任何其他金属物体。</li>
		<li>轻轻地将润滑的光纤温度探头放入鼠标的直肠中。</li>
		<li>将导管插入肿瘤，取出针头。切开导管，使其不会过多地从肿瘤中伸出。 注意：在小鼠全身麻醉时放置和移除光纤温度导管，即导管仅在肿瘤加热过程中就位。在手术时给予小鼠单次皮下剂量的NSAID镇痛药物酮洛芬（5mg / kg）。我们没有观察到与导管放置相关的短期或长期不适或发病率。</li>
		<li>将 3 传感器光纤温度探头插入导管。导管保护光纤温度探头传感器。</li>
		<li>将直肠和肿瘤内探针粘在动物的尾巴上，以确保它们保持在原位。</li>
		<li>将鼠标放入 50 mL 管中，头部到底部。管子应该在头部附近有一个孔，麻醉将连接和输送。</li>
		<li>将管子放在设置好的线圈内，然后重新连接麻醉。</li>
		<li>将光纤温度探头松散地放入管中以测量环境温度。</li>
		<li>打开冷却器，并确保冷却液正在循环。</li>
		<li>检查并确保计算机软件显示各种温度并开始记录，以便实时显示CEM43计算。所需的CEM43是先前确定的剂量。 注意：在打开磁铁之前，请确保没有金属物品附着在动物身上，因为它们会迅速加热。此外，确保房间里的每个人都没有心脏起搏器，并且他们在那里是安全的。</li>
		<li>以低功率百分比打开磁铁。</li>
		<li>确保光纤温度探头记录温度变化。一旦AMF被激活，温度就会随着磁场的增加而升高。确保动物的核心温度保持在38°C。 使用调节空气夹套调节核心温度。</li>
		<li>使用内置控制拨盘通过改变发电机上的功率来调整磁场强度，进而控制肿瘤中的温度水平。</li>
		<li>一旦在肿瘤内达到先前由用户确定的所需剂量（例如CEM43 40），关闭AMF。</li>
		<li>AMF 关闭后，从线圈中取出管子。</li>
		<li>从管中取出鼠标，提取各种探针和导管。如有必要，用新的金属耳标标记动物。</li>
		<li>处理完成后，关闭冷却器。</li>
		<li>从麻醉中恢复动物，确保没有并发症。监控他们的行为以确保恢复正常。</li>
	</ol></li>
</ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+physical+lines+of+force.">On physical lines of force.</a> The London, Edinburgh, and Dublin Philosophical Magazine and Journal of Science. 21 (139), 161-175 (1861).</li></ol>

作者没有什么可透露的。

该系统的设计和实施提供了进行准确和可重现的体外和体内磁性纳米颗粒热疗实验的能力。至关重要的是，该系统的设计使AMF频率和场强与磁性纳米颗粒类型，浓度以及所需的组织位置和温度充分匹配。此外，实时精确监测温度对于安全和计算准确的热剂量（43 °C/CEM 下的累积当量分钟）至关重要。如图1所示，探头的放置允许实时监测热剂量和核心体温，如图<strong class="xfig...

热疗历来用于癌症治疗，无论是单独使用还是与其他治疗联合使用。尽管它有很长的使用历史，但提供这种治疗的最有利方法仍在争论中，并且取决于疾病部位和位置。热疗方法包括微波、射频、聚焦超声、激光和金属纳米颗粒（如金或氧化铁）1，2，3，4。这些输送方法可以导致从发烧水平到数百摄氏度的一系列治疗温度。热疗的生物学效应主要取决于使用的温度和治疗的持续时间5。对于这份手稿和目的，我们专注于磁性纳米颗粒热疗（mNPH）。该方法允许使用无毒、FDA批准的氧化铁纳米颗粒进行集中，局部，良好监测和控制的温度变化。
其他热疗方式的一个缺陷是缺乏精确的细胞靶向;热疗没有固有的高治疗比例，因此，仔细的测温和靶向是必要的6.mNPH允许全身或肿瘤内注射mNPs，仅在mNPs所在的位置产生热量，从而直接针对肿瘤进行治疗。当磁性纳米颗粒位于细胞内部或外部时，mNPH可能有效。对于癌症治疗，mNPH的一般概述是注射磁性纳米颗粒（肿瘤内或静脉内），然后施加交变磁场，使纳米颗粒磁极不断重新排列，导致与纳米颗粒相关的细胞和组织的局部加热7，8.通过调整纳米颗粒的体积和交变磁场（AMF）的频率/强度，可以仔细控制组织内产生的温度。
这种治疗在靠近体表的肿瘤中效果很好，因为更深的肿瘤需要更强的AMF，因此涡流加热的风险增加9。有证据表明热疗在临床上被用作单一疗法，然而，通常热疗与放射治疗或化疗相结合，导致更有针对性的抗癌作用10，11，12。热疗与放疗联合使用的临床证据在先前的出版物13中进行了回顾。我们的实验室使用mNPH方法12，14，15成功治疗了各种动物，从小鼠到猪和自发性犬癌。该方案专为那些有兴趣研究局部热疗效果的人而设计，无论是单独使用还是与其他疗法联合使用。
热疗最重要的因素之一是能够实时测量和理解传递到靶标/肿瘤组织的热剂量。计算和比较剂量的标准方法是通过演示在43°C下加热的累积等效分钟数;该算法允许比较独立于输送系统、最高和最低温度（在特定范围内）和加热/冷却参数5，16 的剂量。CEM计算最适合39-57°C之间的温度5。例如，在我们进行的一些研究中，我们选择了CEM43 30的热剂量（即在43°C下30分钟）。选择这种剂量使我们能够在体外观察安全，有效的免疫遗传效应，无论是单独使用还是与单剂量辐射结合使用17。
对于磁性纳米颗粒热疗，在构建适当的输送系统时需要考虑几个因素。仪器设计包括重要的安全因素，例如使用冷却器来确保磁场输送设备即使在高功率下运行时也能保持冷却，以及故障安全程序，如果所有温度、功率评估和控制系统尚未激活，则防止系统打开。此外，对于体内和体外情况，需要考虑一些重要的生物学因素。使用培养细胞时，有必要在生长培养基中处理并保持一致的活温度，以避免可能影响结果的生理变化。对于单个纳米颗粒类型，在计算基于AMF的加热参数时，了解比吸收率（SAR）非常重要。同样，了解细胞和组织中的mNP / Fe浓度对于实现所需的加热是必需的也很重要。体内方法需要更加注重细节，因为在治疗期间必须将动物保持在麻醉下，并且在整个治疗过程中动物的核心体温保持在正常水平。允许动物的体温下降，就像在麻醉下发生的那样，会影响整体结果，相对于被治疗组织的热剂量。
在这篇手稿中，我们讨论了用于设计和构建多功能磁性纳米颗粒热疗系统的方法，以及需要考虑的重要使用因素。所描述的系统允许稳健、一致、生物学上适当、安全和控制良好的磁性纳米颗粒热疗输送。最后，应该注意的是，我们进行的mNPH研究通常涉及其他疗法，例如放疗，化学疗法和免疫疗法。为了使这些结果有意义，重要的是要确定传递的热量如何影响其他方式的功效和/或安全毒性（反之亦然）以及动物的健康。由于这个原因以及前面提到的剂量学和治疗情况，必须严格注意磁性纳米颗粒热疗的剂量精度以及连续的核心和目标温度测量。该协议的目标是为输送安全有效的磁性纳米颗粒热疗提供一种简单，一致的方法和描述。

<table>
	<tbody>
		<tr>
			<td>.25% Trypsin</td>
			<td>Corning</td>
			<td>45000-664</td>
			<td>available from many companies</td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>Eppendorf</td>
			<td>Eppendorf 22363204</td>
			<td>available from many companies</td>
		</tr>
		<tr>
			<td>B16F10 murine melanoma cells</td>
			<td>American Type Culture Collection</td>
			<td>CRL-6475</td>
			<td></td>
		</tr>
		<tr>
			<td>C57/Bl6 mice</td>
			<td>Charles river</td>
			<td>027C57BL/6</td>
			<td>6-week-old female mice</td>
		</tr>
		<tr>
			<td>Chiller</td>
			<td>Thermal Care</td>
			<td>NQ 5 series</td>
			<td>chiller that cools the coil</td>
		</tr>
		<tr>
			<td>Coolant fluid</td>
			<td>Dow Chemical Company</td>
			<td>Dowtherm SR-1</td>
			<td>antenna cooling fluid</td>
		</tr>
		<tr>
			<td>Fetal Bovine serum</td>
			<td>Hyclone</td>
			<td>SH30071</td>
			<td>available from many companies</td>
		</tr>
		<tr>
			<td>fiber optic probes, software and chassis</td>
			<td>FISO</td>
			<td></td>
			<td>FISO evolution software used to read the temperatures</td>
		</tr>
		<tr>
			<td>IR camera</td>
			<td>Flir</td>
			<td></td>
			<td>infrared camera to monitor unintentional heating</td>
		</tr>
		<tr>
			<td>iron oxide nanoparticles</td>
			<td>micromod Partikeltechnologie GmbH</td>
			<td>Bionized NanoFerrite</td>
			<td>dextran coated iron oxide nanoparticles</td>
		</tr>
		<tr>
			<td>mouse coil, solenoid</td>
			<td>Fluxtrol</td>
			<td>custom built</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>Corning</td>
			<td>45000-652</td>
			<td>available from many companies</td>
		</tr>
		<tr>
			<td>RF generator</td>
			<td>Huttinger</td>
			<td>TIG 10/300</td>
			<td>power source</td>
		</tr>
		<tr>
			<td>RPMI media</td>
			<td>Corning</td>
			<td>45000-396</td>
			<td>available from many companies</td>
		</tr>
	</tbody>
</table>

in vitro and in vivo delivery of magnetic nanoparticle hyperthermia using a custom-built delivery system

热疗长期以来一直用于治疗癌症。技术从热铁棒的肿瘤内插入到全身递送的肿瘤抗体靶向磁性纳米颗粒，温度从39°C（发烧水平）到1，000°C（电灼），治疗时间从几秒钟到几小时不等。温度-时间关系（热剂量）决定了高热剂量导致组织消融和低热剂量导致亚致死效应的效果，例如血流量增加、药物积累和免疫刺激。目前最有前途的医学疗法之一是磁性纳米颗粒热疗（mNPH）。该技术涉及激活磁性纳米颗粒，该纳米颗粒可以通过非侵入性，无毒的交变磁场进行全身或肿瘤内递送。磁性纳米颗粒的大小、构造和关联以及磁场的频率和场强是主要的加热决定因素。我们开发了先进的仪器和技术，用于在大型和小型动物模型和培养细胞中提供可重复的磁性纳米颗粒热疗。这种方法在多个位置使用连续的实时温度监测，允许将明确定义的热剂量输送到目标组织（肿瘤）或细胞，同时限制非目标组织加热。精确控制和监测多个站点的温度，并使用行业标准算法（43 °C /CEM43 下的累积等效分钟数），可以准确测定和量化热剂量。我们的系统允许各种温度、热剂量和生物效应，是通过商业收购和内部工程和生物学开发相结合而开发的。该系统已经过优化，允许在离体、体外和体内技术之间快速转换。该协议的目标是演示如何设计，开发和实施有效的技术和系统，以提供可重复和准确的磁性纳米颗粒治疗（mNP）热疗。

体外研究 只有当磁性纳米颗粒/铁和AMF的量和浓度适当匹配时，细胞才能达到并保持所需的温度和热剂量。当使用磁性纳米颗粒在体外（和体内）加热细胞时，应该注意的是，为了在具有内化磁性纳米颗粒的细胞中实现热疗，需要特定水平的细胞内mNP / Fe，并且mNP负载细胞的数量和接近度将是必要的。如果靶细胞/组织中的mNP/Fe水平足以达到加热效果，则可以调节磁场频率和强度以达...

Watch this Scientific Journal Video about In Vitro and In Vivo Delivery of Magnetic Nanoparticle Hyperthermia Using a Custom-Built Delivery System at JoVE.com

In Vitro and In Vivo Delivery of Magnetic Nanoparticle Hyperthermia Using a Custom-Built Delivery System

该协议提供了使用复杂的输送和监测系统准确输送磁性纳米颗粒热疗所需的技术和方法。

使用定制的输送系统进行磁性纳米颗粒热疗的体外和体内输送

Hyperthermia has long been used in the treatment of cancer. Techniques have varied from the intra-tumoral insertion of hot iron rods, to systemically ...

in-vitro-vivo-delivery-magnetic-nanoparticle-hyperthermia-using

Research

JoVE Journal

Medicine

4.2K 次观看.  Dartmouth College. 该协议提供了使用复杂的输送和监测系统准确输送磁性纳米颗粒热疗所需的技术和方法。

视频: In Vitro and In Vivo Delivery of Magnetic Nanoparticle Hyperthermia Using a Custom-Built Delivery System

Ex Vivo Culture of Patient Tissue &amp; Examination of Gene Delivery

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial.

Ex Vivo Culture of Patient Tissue &amp; Examination of Gene Delivery

This article describes the culture of patient tissue slices for gene delivery studies and subsequent analysis of gene expression using IVIS bioluminescence imaging.

患者组织及基因传递考试前的体外培养

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery.  Fresh patient tissue obtained at the time of surgery ...

科研

医学

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.
Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.
Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.

Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

心肌内细胞交货：观察在小鼠心脏

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

通过40W的Elvax缓释给药的大鼠视网膜：对慢性疾病的治疗

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a &#62;98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

插管介导气管内（IMIT）灌输：一种无创，龙专用输送系统

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

生物血管内送货到大鼠肾脏

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice.

使用多氟化胆汁酸和<em&gt;在体内</em&gt;磁共振成像来衡量胆汁酸运输

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

经优化的 LC/ms 方法对囊性纤维化患者 Ivacaftor 及其主要代谢物和 Lumacaftor 的高通量分析

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

一种基于猪脑内皮细胞的体外血脑屏障模型的改进方法

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

聚乙胺包覆氧化铁纳米粒子作为载体, 在体外和体内向巨噬细胞输送小干扰 rna

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...