这项工作部分由PHRMA基金会资助，马里兰大学帕克学院颁发的研究和学者奖。我们感谢达雷尔·欧文教授支持完成"通过在体内注入辅助释放聚合物颗粒来原位 工程淋巴结微环境"的初步工作。5

本议定书中的所有动物研究都是按照联邦、州和地方准则完成的，并使用马里兰大学机构动物护理和使用委员会 （IACUC） 审查和批准的协议。
1. 脂质稳定微粒子和纳米粒子的合成
<ol><li>在 7 毫升玻璃瓶中，以 60：20：20 摩尔比率将 DOPC、DSPE-PEG 和 DOTAP 脂质组合在一起，以准备主脂质混合物。<ol>
<li>要合成单个样本：使用 2 毫升玻璃血清移液器分别将 DOPC、DSPE-PEG 和 DOTAP 的 242.9 μl、287.4μl 和 71.9μl 传输到小瓶中。</li>
		<li>要合成多个样本：将上面的每个脂质体积乘以样品数量，并组合成一小瓶，然后将这种脂质混合物的等量液化为与每个样品相对应的小瓶中。</li>
		<li>在温和的氮气流下干燥脂质10分钟，或在真空烤箱中放置过夜。</li>
	</ol>
</li>
	<li>在一个空的20毫升玻璃瓶中，溶解80毫克PLGA在5毫升二氯甲烷为每个粒子样品产生一个16毫克/毫升的聚合物库存溶液。</li>
	<li>将 5 毫升聚合物溶液添加到含有干脂、盖子和漩涡的瓶中，30 秒。</li>
	<li>合成微粒：<ol>
<li>使用声波器在 12W 时开始在冰上对含有聚合物、脂质和其他水溶性货物的有机相进行声波化。</li>
		<li>使用移液器添加 500 μl 蒸馏 H2O 或 H2O，其中含有 1 毫克肽、蛋白质或其他水溶性货物，从而创建油中水 （w/o） 乳液。</li>
		<li>在冰上12 W处继续声波30秒，轻轻上下摇动小瓶，并一边绕声波器尖端，以确保完全乳化。</li>
		<li>通过将乳液倒入 40 毫升 H2O 中的 150 毫升烧杯中，创建水中油中水（w/o/w）乳液。</li>
		<li>使用数字同质化器在 16，000 rpm 下均质 3 分钟。</li>
		<li>加入磁搅拌棒，将烧焦器转移到搅拌板上，让乳液在一夜之间搅拌，以去除多余的溶剂。</li>
	</ol>
</li>
	<li>合成纳米粒子：<ol>
<li>在14W开始在冰上对含有聚合物、脂质和其他水不溶性货物的有机相进行声波化。</li>
		<li>使用移液器添加 500 μl 蒸馏 H2O 或 H2O，其中含有 1 毫克肽、蛋白质或其他水溶性货物，从而创建无乳液。</li>
		<li>在冰上14 W继续声波30秒。</li>
		<li>通过将湿乳液倒入 150 毫升烧杯中的 40 毫升 H2O，并在冰上 16 W 时发出 5 分钟的声波，创建 w/o/w 乳液。轻轻地上下摇动小瓶，并围绕声波器尖端左右，以确保完全乳化。</li>
		<li>加入磁搅拌棒，将烧瓶转移到搅拌板上，让乳液在一夜之间搅拌，以去除多余的溶剂。</li>
	</ol>
</li>
	<li>第二天早上，清洗和收集颗粒：<ol>
<li>将乳液通过 40μm 尼龙网状细胞过滤器倒入 50 毫升圆锥管中。</li>
		<li>离心机颗粒5分钟，微粒5，000 x 克，纳米粒子5分钟，纳米粒子24，000×克。</li>
		<li>在 1 毫升 H2O 中重新悬念，降低超自然性并清洗颗粒。</li>
		<li>将悬浮颗粒转移到 1.5 毫升微中微管。</li>
		<li>离心机5分钟，微粒5，000 x 克，纳米粒子5分钟，纳米粒子24，500×克。</li>
		<li>通过去除超自然物、在 1ml H2O 中重新悬念和像步骤 1.6.5 那样离心来洗涤颗粒两次。清洗后，将颗粒悬浮在 1 毫升 H2O 中，立即使用，或用于扩展存储的嗜血。</li>
	</ol>
</li>
</ol>2. 综合产量测量
<ol><li>预制一个空的20毫升玻璃瓶。用微皮管上下吹管后，将 100 μl 的颗粒悬浮加入预加的小瓶中。</li>
	<li>将颗粒合二为一或在温和的氮流下干燥。</li>
	<li>称量含有干燥聚合物的瓶。通过从含有干燥颗粒的小瓶质量中减去小瓶的原始重量，确定小瓶中的颗粒产量。</li>
	<li>通过稀释因子将小瓶中的粒子质量乘以，确定整体粒子产量。要确定百分比产量，将粒子质量除以最大理论输入质量，乘以100%。</li>
</ol>3. 粒子大小的确定
<ol><li>通过加满除离子水和用棉签擦拭子来清洁供应的 cuvette 风格的玻璃碎块。将 10 毫升蒸馏 H2O 转移到清洁的派系细胞中，加入磁性微搅拌棒，并将分数细胞加载到粒子大小分析仪的细胞安装中。</li>
	<li>调整粒子分析仪中的磁搅拌速度，在分数单元中实现完全混合并关闭隔间门。</li>
	<li>使用仪器软件接口将激光器与分数单元对齐。</li>
	<li>使用仪器软件界面记录基线读数，分数单元仅包含蒸馏 H2O。</li>
	<li>派佩特原来的粒子悬架上下与微皮条混合。</li>
	<li>将10μl颗粒悬架（通常约为0.5毫克）放入分数细胞中。确保添加到细胞中的粒子样本体积足以在仪器软件界面上指示的适当范围内产生信号强度。所需粒子的实际质量取决于百分比产量和粒子样本的光学特性。</li>
	<li>关闭粒子大小分析器舱门，使用 PLGA 的折射指数 1.60 测量粒子大小。</li>
	<li>使用软件界面使用数字基础计算粒子直径。</li>
</ol>4. 粒子可视化
<ol><li>派佩特粒子悬架上下与微皮素混合。在阳离子化水中将颗粒悬浮稀释至 1 毫克/毫升。</li>
	<li>通过添加 3μl 的稀释颗粒悬浮，并在 45° 角度安装盖片以避免气泡形成，从而准备显微镜滑梯。使用适用于每个荧光货物的适当滤光片将幻灯片放在显微镜舞台和图像上。</li>
</ol>5. 为 i.LN 准备小鼠。注射
<ol><li>准备示踪剂染料解决方案：<ol>
<li>通过用 10 毫升蒸馏 H2O 溶解 10 毫克染料粉，准备 0.1% （w/v） 的示踪剂染料解决方案。</li>
		<li>使用 0.2μm 注射器过滤器将染料溶液消毒到玻璃瓶中。</li>
	</ol>
</li>
	<li>注射前一天，根据IACUC批准的动物协议，用异氟素麻醉小鼠。要评估麻醉深度，请执行脚趾捏反射测试并监测呼吸速率，以确保呼吸速度约为每分钟 100-140 次。</li>
	<li>在老鼠麻醉时，用剪子在尾巴和后四肢底部剃头发。从动物的腹侧去除头发，横向到后腿（臀部）关节上方的背侧。</li>
	<li>注射示踪剂染料。<ol>
<li>每次注射染料时，使用微皮将 10 μl 的染料溶液转移到微中福格管中，并将整个 10 μl 吸气到连接到 1 毫升注射器的 31G 针中。</li>
		<li>在剪头发的尾部基座的每一侧皮下注射 10 μl 染料溶液，在注射之间重新加载。</li>
	</ol>
</li>
	<li>通过棉签涂抹轻度脱毛霜，去除剩余的头发。一定要涂抹在大腿后部和腹部之间的区域。</li>
	<li>允许去皮膏在皮肤上孵育3分钟。孵化后，湿手套手与温暖的H2O，轻轻地擦去皮膏到皮肤。</li>
	<li>立即去除脱毛霜，用温暖的H2O润湿戴手套的手，揉尾巴底座和后四肢。重复，直到多余的脱毛被移除，确保保持手湿，以避免刺激。</li>
	<li>通过用温暖的 H2O 润湿软布或纸巾，并以单个动作擦拭鼠标的下半部分，从鼠标上去除残留的除湿剂。避免摩擦运动，以防止擦伤或皮肤损伤的鼠标。</li>
	<li>允许鼠标在热灯下恢复并恢复保持。</li>
</ol>6. 即粒子注射
<ol><li>第二天，根据IACUC批准的动物协议，用异氟素麻醉小鼠。</li>
	<li>检查鼠标，确认示踪剂染料排入每个淋巴结。淋巴结应作为大腿后部和腹部附近的黑点可见。</li>
	<li>准备粒子注射解决方案：<ol>
<li>以所需的注射浓度在蒸馏H2O中补充颗粒。每次注射时，使用微皮将 10 μl 的颗粒溶液转移到微中微管中。</li>
		<li>将整个 10 μl 吸进附着在 1 毫升注射器上的 31G 胰岛素针中。</li>
	</ol>
</li>
	<li>注射颗粒剂量：<ol>
<li>可视化 LN 后，使用拇指、食指和中指收紧 LN 周围的皮肤，以拉动皮肤嘲讽，并允许可控制注射量的位置。</li>
		<li>用针头以 90° 角接近 LN 皮肤，并将皮肤穿透染色 LN 的深度为 1 mm。</li>
		<li>慢慢注入整个体积。注射期间，通过皮肤观察LN体积，通过可见的LN放大确认注射。</li>
	</ol>
</li>
	<li>允许鼠标在热灯下恢复，并返回保持或进行其他测试。</li>
</ol>有关分析技术（如组织学、流动细胞测量）请参阅JoVE第 265 条、1743 和 3054 条以及免疫学的当前协议，第 5 章和第 21 章15-22。

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本协议中描述的技术允许将疫苗控制地输送到LN和LN驻地抗原呈现细胞。生物材料封装货物可在 LN 内本地化，从而能够操纵交付给 LN 微环境的一种或多种类型的货物的剂量。聚合物颗粒的本地化和受控释放已被证明能以明显低于传统方法的剂量产生强大的细胞和幽默免疫反应。此外，通过生物材料载体尺寸的操纵，可以在直接吸收纳米粒子或从较大的微粒5释放的细胞外货物之间调节细胞处理的主要模式。这些结果确定了 作为 治疗性疫苗交付平台的生物材料交付的可行性。
乳液/溶剂蒸发合成PLGA颗粒已广泛应用于药物输送应用23，24。因此，与这项技术相关的潜在挑战主要与LN靶点疫苗的成功识别和沉积有关。虽然使用示踪剂染料有助于靶向导入 LN 的可视化，但皮肤下方的目标大小和深度较小。因此，作者建议分配时间和小鼠练习小鼠的制备和注射。在动物准备（即剃须和应用去皮化）时，应注意不要割伤动物腹侧的老鼠，因为腿部与腹部的角度使皮肤更容易受到剪子的伤害。此外，所有脱毛应用温水去除，以防止动物在正常梳妆行为期间摄入奶油。要练习 LN 注射，可以管理更高的示踪剂染料浓度，练习动物可以安乐死，然后多次注射。注射后的小鼠可以被坏死，注射动物的LNs大小可以与未弹出的控制LN进行比较。此技术的一个限制是可加载到 LN 结构中的注射体积的物理限制。我们的协议建议小鼠的注射量为10微克，尽管其他研究报告注射量较大，至少高达20微克。13 然而，通过 i.LN. 注射直接交付疫苗可以大幅减少剂量，因此这些疫苗的功能一般不应受到量限制。
如前所述，改变粒子（即大小）的物理特性是改变生物材料和LN组织中封装货物诱发的通路或结果的有效机制。乳液/溶剂蒸发协议可以很容易地修改，以改变物理或化学性质，如表面电荷或功能，和生物降解/货物释放率23，24。例如，释放动力学可以通过替代聚合物成分进行调整，并且使用改性脂质成分或聚（乙烯基酒精）改变表面功能。装载在颗粒中的货物可以很容易地纵，以包含目标病原的不同抗原或辅助剂。这种方法的优势是通过将 i.LN. 交付与生物材料中货物的局部、可控释放相结合来实现。这种协同作用建立了一个平台，可以利用该平台，利用微量剂量和减少非特异性/系统副作用，高效生成自适应免疫反应。

淋巴结 （LNs） 是免疫系统的指挥中心。在这个免疫学站点，抗原呈现细胞对特定的外来抗原原始天真淋巴细胞，以激活细胞和幽默免疫反应。因此，LN已成为疫苗和免疫疗法的有吸引力的目标。不幸的是，大多数疫苗策略导致低效的，暂时交付抗原和辅助剂淋巴组织1。因此，改进LN疫苗成分的靶向和保留的方法可能对新疫苗的效力和效率产生重大影响。
一种绕过LN靶向挑战的策略，已经显示出对新的临床试验的极大兴趣是直接，LN（lN.）注射2-4。这些试验采用超声波指导，将疫苗输送到LN，作为简单的门诊程序。与传统的外周注射途径相比，这种方法在治疗环境中，包括过敏和癌症2-4，产生了显著的剂量节省和提高疗效。这些研究采用 了lN. 注射可溶性疫苗（即无生物物质疫苗），这些疫苗通过淋巴排泄迅速清除。因此，多次注射 -或多次注射周期 - 被管理，以实现这些令人印象深刻的治疗效果。LN的保留性提高可以增强抗原和/或佐剂与免疫细胞之间的相互作用，进一步提高免疫细胞的启动能力。最近的研究表明，抗原和佐剂输送的动力学在确定产生5-7的特定免疫反应方面起着至关重要的作用，这支持了这一潜力。此外，本地化和尽量减少药物和疫苗剂量可以减少或消除系统性影响，如慢性炎症。
生物材料已广泛研究，以提高疫苗的效力和效率1，8，9。生物材料载体的封装或吸附可以物理保护货物免受降解，并克服溶解性限制。生物材料载体的另一个显著特点，如聚合物微粒或纳米粒子，是能够共装几类货物，然后通过受控间隔释放这些货物。然而，继续阻碍体内生物材料疫苗和免疫疗法的重大 限制是免疫细胞的低效靶向和对淋巴结的贩运有限。例如，通过常规途径（如皮肤内、肌肉内）进行生物材料疫苗的外围注射通常表现出不良的LN靶向，注射地剩余的注射材料高达99%为4，10。最近，生物材料疫苗携带者的规模已调整，以改善这些疫苗的优先贩运或排水到LN通过间歇流动8，10。这些进展提高了细胞和幽默免疫反应，强调了针对LN环境为新疫苗设计的重要性。
本文提出了一个疫苗接种协议，结合脂质稳定的聚合物颗粒和i.LN.交付产生控制释放疫苗库5，11。以最近的研究为基础，采用外科技术为i.LN。在小鼠6，7，12，13，我们开发了一个快速，非手术策略，注射生物材料疫苗的小动物5。结合i.LN.交付与生物材料疫苗载体有力地增强了CD8 T细胞反应后7天内，一次注射控制释放疫苗库5。也产生了强烈的幽默反应（即抗体滴定器）：这两种增强都与淋巴结中疫苗成分的保留增加有关，淋巴结是由生物材料载体的控制释放所调解的。有趣的是，疫苗颗粒的大小改变了这些材料的命运一次在LNs：纳米级粒子显示细胞直接吸收增加，而较大的微粒留在细胞外LN环境中，并释放货物（如辅助剂），由LN驻地抗原呈现细胞5。这些数据表明，通过控制注射的i.LN生物材料的大小，可以利用两种途径来获得新疫苗。
本文采用改性液策略5 ， 11合成可生物降解脂质稳定聚合物颗粒（微尺度和纳米尺度）。粒子特性的特点是激光衍射和显微镜。然后，这些颗粒被直接注射到非手术识别的内在LN中，使用一种常见的无毒示踪剂染料14。通过组织学或流动细胞学对 LN 进行注射后分析可用于验证 LN 环境中粒子的分布，以及监测颗粒的细胞吸收和保留情况。对于详细的组织学处理和流动细胞测量的协议，读者被引用到最近的JoVE文章和期刊报告15-22。典型结果表明，这些库的局部 LN 目标可被利用来实现有效、高效的免疫反应或为目标病原体量身定做免疫力。

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			<td height="84" style="height:84px;width:216px;">1,2-dioleoyl-sn-glycero-3-phosphocholine1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)</td>
			<td style="width:71px;">Avanti Polar Lipids</td>
			<td style="width:119px;">850375</td>
			<td style="width:167px;">10 mg/ml stock in chloroform</td>
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		<tr height="105">
			<td height="105" style="height:105px;width:216px;">1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] ammonium salt (DSPE-PEG)</td>
			<td style="width:71px;">Avanti Polar Lipids</td>
			<td style="width:119px;">880128</td>
			<td style="width:167px;">10 mg/ml stock in chloroform</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">1,2-Dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP)</td>
			<td style="width:71px;">Avanti Polar Lipids</td>
			<td style="width:119px;">890890</td>
			<td style="width:167px;">10 mg/ml stock in chloroform</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Polylactic-co-glycolic acid (PLGA)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2191</td>
			<td style="width:167px;">Lactide:Glycolide (50:50). MW 30,000-60,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dichloromethane (DCM)</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">BDH1113</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoflurane</td>
			<td style="width:71px;">Vetone</td>
			<td style="width:119px;">502017</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nair</td>
			<td style="width:71px;">Nair</td>
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			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:216px;">Evans blue tracer dye</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">AAA16774-09</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">U-100 BD Ultra-Fine Short Insulin Syringes, 31 G 5/16 in needle</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">BD328418</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Single-Use Needles, BD Medical, 21 G, 1.5 in needle</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">BD305167</td>
			<td style="width:167px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syringes with BD Luer-Lok Tip, BD Medical, 1 ml</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">BD309628</td>
			<td style="width:167px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Falcon Cell Strainers, Sterile, Corning, 40 &#181;m</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">21008-949</td>
			<td style="width:167px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vybrant DiD Cell-Labeling Solution</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">V-22887</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fluoresbrite YG Microspheres 6.00 &#181;m</td>
			<td style="width:71px;">Polysciences</td>
			<td style="width:119px;">17149</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fluoresbrite YG Microspheres 0.05 &#181;m</td>
			<td style="width:71px;">Polysciences</td>
			<td style="width:119px;">17156</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Ovalbumin, Purified</td>
			<td style="width:71px;">Worthington Biochemical</td>
			<td style="width:119px;">LS003056</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Qsonica Sonicator Ultrasonic Processor Q125</td>
			<td style="width:71px;">Qsonica</td>
			<td style="width:119px;">Q125</td>
			<td style="width:167px;">1/8 in diameter microtip probe</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultra-Turrax T 25 digital homogenizer</td>
			<td style="width:71px;">IKA</td>
			<td style="width:119px;">YO-04739-22</td>
			<td style="width:167px;">10 G dispersing element</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescent Microscope</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">IX-83</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Laser Diffraction Particle Size Distribution Analyzer</td>
			<td style="width:71px;">Horiba</td>
			<td style="width:119px;">LA-950</td>
			<td style="width:167px;">Including provided cuvette-style glass fraction cell</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Professional 8685 Peanut Classic Clippers</td>
			<td style="width:71px;">Wahl</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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intra-lymph node injection of biodegradable polymer particles

自适应免疫反应的产生依赖于高效排水或将抗原贩运到淋巴结，以便处理和将这些外来分子呈现给T和B淋巴细胞。因此，淋巴结已成为新疫苗和免疫疗法的关键目标。最近针对这些组织的一项战略是直接注射可溶性疫苗成分的淋巴结，涉及该技术的临床试验很有希望。还研究了几种生物材料战略，以改善淋巴结靶向，例如调整颗粒大小，以最佳排水生物材料疫苗颗粒。本文介绍了一种将直接淋巴结注射与可富含抗原、佐剂或其他疫苗成分的可生物降解聚合物颗粒相结合的新方法。在这种方法中，聚合物微粒或纳米粒子通过结合脂质稳定剂的改性液协议合成。粒子特性（如大小、货物装载）分别通过激光衍射和荧光显微镜得到确认。然后，通过非毒示踪染料的外周注射识别小鼠淋巴结，从而可可视化目标注射部位，并随后在淋巴结中沉积聚合物颗粒。这项技术允许直接控制生物材料和疫苗成分输送到淋巴结的剂量和组合，并可用于开发新的生物材料疫苗。

本手稿中提出的协议的预期结果可分为三类：粒子合成、动物制备和粒子注射。
图1 描绘了可生物降解聚合物颗粒的合成和特征，由两栖脂质稳定。乳液/溶剂蒸发合成协议（图1A）的结果可以通过对产生的最终乳液进行目视检查进行定性评估：粒子批次应是同质的，稳定的乳液与不透明的外观。并发症包括乳液，奶油或絮凝，往往是由于脂质稳定器存储不当。为了避免这种不稳定，脂质应储存在 -80 °C 的脱水状态或用氮清除的密封小瓶中。粒子合成的定量评估可以使用激光衍射或动态光散射来分析大小分布（图1B）。预期结果包括分布紧密的单模态粒子大小，表明粒子数量均匀。本手稿中描述的合成参数生成以纳米粒子和微粒为中心的平均分布数，分别为约 100 nm 或 3μm。通过修改上述协议，将多种荧光货物纳入其中，可以进一步对粒子合成进行定性评估。在 图 1C中，装有荧光肽（FITC、绿色）、脂质染料（DiD、红色）和叠加图像（黄色）的微粒的显微镜图像确认在所需的大小范围内产生颗粒，并在颗粒体积内封装肽。
图2的前两个小组总结了本文所述的i.LN.注射策略动物准备的预期结果。该方法包括通过非毒示踪剂的外围注射标记内源LN，以确定随后i.LN.注入粒子的位置（图2A）5。如前所述，尾部部皮下注射后，追踪染料的排水，可可视化因子LNs（图2B）5。摄入经批准的脱毛霜可能会对小鼠造成危害。因此，应小心彻底去除所有涂有奶油的膏，特别注意爪子和小鼠的腹腔侧。脱毛应使用湿软布或湿纸巾在单一的，流畅的运动删除。避免摩擦以去除奶油，因为这可能导致小鼠暴露的皮肤擦伤。
通过观察或组织学对当地交付给因吉纳尔LN的确认进行评估。LN 体积可在注射期间进行可视监控，作为成功注射的指标。预期结果包括在整个 LN 结构中高效分配货物，不会对相邻的组织或细胞造成重大泄漏。此外，当注射的液体取代/稀释 LN 中的示踪剂时，注射后染料浓度/着色应变得不那么强烈。对组织的观察应显示一个完好无损的，但扩大LN由于液体注射。潜在的挑战包括注射太快或缺少 LN，这两种情况都可能导致体积向周围的皮下组织蒸发。这些不良结果可以通过坏死或组织学来证实，在那里粒子悬浮将观察到扩散到远离注射目标节点的细胞和组织。相比之下，由于LN结构内粒子的密封，预期的结果将是识别扩大的内在LN。切除的LNs的组织学处理可以明确确认货物运送到淋巴组织，如 图2C 和 2D所示。请注意，图 2 中的颗粒包含荧光货物，以便在注射过程中以及在组学处理和荧光显微镜期间实现货物的可视化。
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/50984/50984fig1.jpg" width="600px"> 图1。脂质稳定颗粒的合成和特征。A） 描述乳液/溶剂蒸发所制备的脂质稳定颗粒合成的原理图。B） 微粒（实心线、直径 =2.8 μm）和纳米粒子（虚线、直径 = 113 nm）的大小分布。C） 荧光显微镜图像的粒子加载荧光标签肽和荧光颗粒染料。标签：肽（绿色）和颗粒（红色）。<a href="https://www.jove.com/files/ftp_upload/50984/50984fig1highres.jpg" target="_blank">单击此处查看更大的图像</a>。
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/50984/50984fig2.jpg" width="600px"> 图2。伊琳LN内可生物降解颗粒的注入和分布。A）注射的方法。B） 通过皮肤（上图）和以下坏死（下图）5对鼠标中的 LN 进行可视化。C） LN的组织染色，确认荧光标记聚合物微粒（颗粒、绿色） 的沉积和分布：T细胞，红色：B细胞，蓝色）。D） 注射后 24 小时内在 LNs 中贴有荧光标签的纳米粒子（50 nm，左图）和微粒（6μm，右图）。<a href="https://www.jove.com/files/ftp_upload/50984/50984fig2highres.jpg" target="_blank">单击此处查看更大的图像</a>。

Watch this Scientific Journal Video about Intra-lymph Node Injection of Biodegradable Polymer Particles at JoVE.com

Intra-lymph Node Injection of Biodegradable Polymer Particles

淋巴结是协调免疫反应的免疫组织，是疫苗的关键目标。生物材料已被用于更好地瞄准淋巴结和控制抗原或辅助剂的传递。本文描述了一种将这些想法相结合的技术，将生物相容的聚合物颗粒注入淋巴结。

可生物降解聚合物颗粒的淋巴内结注射

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these ...

intra-lymph-node-injection-of-biodegradable-polymer-particles

Research

JoVE Journal

Bioengineering

14.3K 次观看.  University of Maryland, College Park. 淋巴结是协调免疫反应的免疫组织，是疫苗的关键目标。生物材料已被用于更好地瞄准淋巴结和控制抗原或辅助剂的传递。本文描述了一种将这些想法相结合的技术，将生物相容的聚合物颗粒注入淋巴结。

视频: Intra-lymph Node Injection of Biodegradable Polymer Particles

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

一个单粒子为基础的微流控液力陷阱

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

科研

生物工程

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, 'SurgiLux': Preparation and Demonstration

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures can act as a nidus for infection and in many procedures are unable to effect wound repair or interfere with functional tissue regeneration.1 Surgical glues and adhesives, such as those based on fibrin and cyanoacrylates, have been developed as alternatives to sutures for the repair of such wounds. However, current commercial adhesives also have significant disadvantages, ranging from viral and prion transfer and a lack of repair strength as with the fibrin glues, to tissue toxicity and a lack of biocompatibility for the cyanoacrylate based adhesives. Furthermore, currently available surgical adhesives tend to be gel-based and can have extended curing times which limit their application.2 Similarly, the use of UV lasers to facilitate cross-linking mechanisms in protein-based or albumin 'solders' can lead to DNA damage while laser tissue welding (LTW) predisposes thermal damage to tissues.3 Despite their disadvantages, adhesives and LTW have captured approximately 30% of the wound closure market reported to be in excess of US $5 billion per annum, a significant testament to the need for sutureless technology.4
In the pursuit of sutureless technology we have utilized chitosan as a biomaterial for the development of a flexible, thin film, laser-activated surgical adhesive termed 'SurgiLux'. This novel bioadhesive uses a unique combination of biomaterials and photonics that are FDA approved and successfully used in a variety of biomedical applications and products. SurgiLux overcomes all the disadvantages associated with sutures and current surgical adhesives (see Table 1).
In this presentation we report the relatively simple protocol for the fabrication of SurgiLux and demonstrate its laser activation and tissue weld strength. SurgiLux films adhere to collagenous tissue without chemical modification such as cross-linking and through irradiation using a comparatively low-powered (120 mW) infrared laser instead of UV light. Chitosan films have a natural but weak adhesive attraction to collagen (~3 KPa), laser activation of the chitosan based SurgiLux films emphasizes the strength of this adhesion through polymer chain interactions as a consequence of transient thermal expansion.5 Without this 'activation' process, SurgiLux films are readily removed.6-9 SurgiLux has been tested both in vitro and in vivo on a variety of tissues including nerve, intestine, dura mater and cornea. In all cases it demonstrated good biocompatibility and negligible thermal damage as a consequence of irradiation.6-10

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, &#039;SurgiLux&#039;: Preparation and Demonstration

The fabrication of a novel, flexible thin film surgical adhesive from FDA approved ingredients, chitosan and indocyanine green is described. Bonding of this adhesive to collagenous tissue through a simple activation process with a low-powered infra-red laser is demonstrated.

基于几丁聚糖，激光激发薄膜手术胶粘剂，“SurgiLux的制备及示范

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

协议相对液力评估的三单张聚合物阀

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

编程干细胞的治疗性血管新生使用可生物降解高分子纳米微球

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together.
Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together.
The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

制造业的鲁棒天然纤维预制体的利用细菌纤维素为粘结剂

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect &#8220;self-fitting.&#8221; In this way, following exposure to warm saline (~60 &#186;C), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 &#186;C) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.

Fabrication of a Bioactive, PCL-based &quot;Self-fitting&quot; Shape Memory Polymer Scaffold

Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

生物活性，PCL为基础的“自我装配”形状记忆聚合物支架的制备

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to ...

TAPE: A Biodegradable Hemostatic Glue Inspired by a Ubiquitous Compound in Plants for Surgical Application

This video describes the simplest protocol for preparing biodegradable surgical glue that has an effective hemostatic ability and greater water-resistant adhesion strength than commercial tissue adhesives. Medical adhesives have attracted great attention as potential alternative tools to sutures and staples due to their convenience in usage with minimal invasiveness. Although there are several protocols for developing tissue adhesives including those commercially available such as fibrin glues and cyanoacrylate-based materials, mostly they require a series of chemical syntheses of organic molecules, or complicated protein-purification methods, in the case of bio-driven materials (i.e., fibrin glue). Also, the development of surgical glues exhibiting high adhesive properties while maintaining biodegradability is still a challenge due to difficulties in achieving good performance in the wet environment of the body. We illustrate a new method to prepare a medical glue, known as TAPE, by the weight-based separation of a water-immiscible supramolecular aggregate formed after a physical mixing of a plant-derived, wet-resistant adhesive molecule,&#160;Tannic Acid (TA), and a well-known biopolymer, Poly(Ethylene) glycol (PEG). With our approach, TAPE shows high adhesion strength, which is 2.5-fold more than commercial fibrin glue in the presence of water. Furthermore, TAPE is biodegradable in physiological conditions and can be used as a potent hemostatic glue against tissue bleeding. We expect the widespread use of TAPE in a variety of medical settings and drug delivery applications, such as polymers for muco-adhesion, drug depots, and others.

We describe the simplest protocol to prepare biodegradable medical glue that has an effective hemostatic ability. TAPE is a water-immiscible supramolecular aggregate prepared by mixing of tannic acid, a ubiquitous compound found in plants, and poly(ethylene) glycol, yielding a 2.5 times greater water-resistant adhesion compared with commercial fibrin glue.

TAPE：一种生物可降解止血胶由无处不在的复方植物外科应用的启发

This video describes the simplest protocol for preparing biodegradable surgical glue that has an effective hemostatic ability and greater water-resistant ...

Sustained Administration of &#946;-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic &#946;-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to &#946;-cells in culture and in transplanted tissue models. These technologies allow the study of candidate &#946;-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating &#946;-cell proliferation in a culture system is critical before moving forward to in vivo models. Herein, we describe a method to co-culture intact mouse islets with biodegradable compound of interest (COI)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for the purpose of assessing the effects of sustained in situ release of mitogenic factors on &#946;-cell proliferation. This technique describes in detail how to generate PLGA microspheres containing a desired cargo using commercially available reagents. While the described technique uses recombinant human Connective tissue growth factor (rhCTGF) as an example, a wide variety of COI could readily be used. Additionally, this method utilizes 96-well plates to minimize the amount of reagents necessary to assess &#946;-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.

Sustained Administration of &amp;#946;-cell Mitogens to Intact Mouse Islets &lt;em&gt;Ex Vivo&lt;/em&gt; Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of &#946;-cell proliferation. This method is suitable for determining the efficacy of candidate &#946;-cell mitogens.

β细胞的有丝分裂原持续管理局完整小鼠胰岛<em&gt;离体</em&gt;使用可生物降解的聚（乳酸 - 共 - 乙醇酸）微球

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the ...

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a high-protein crambe meal that has limited value, as it is not fit for food or feed use. However, it contains proteins that could potentially make it a suitable raw material for higher-value products. The purpose of this study was to find methods of making this side stream into extruded films, showing that products with a higher value can be produced. The study mainly considered the development of material compositions and methods of preparing and extruding the material. Wheat gluten was added as a supportive protein matrix material, together with glycerol as a plasticizer and urea as a denaturant. The extrudate was evaluated with respect to mechanical (tensile testing) and oxygen barrier properties, and the extrudate structure was revealed visually and by scanning electron microscopy. A denser, more homogeneous material had a lower oxygen transmission rate, higher strength, and higher extensibility. The most homogeneous films were made at an extruder die temperature of 125-130 &#176;C. It is shown here that a film can be extruded with promising mechanical and oxygen barrier properties, the latter especially after a final compression molding step.

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

The side stream from plant oil production of Crambe abyssinica has limited value. The purpose of this study was to find methods for extruding materials based on this side stream, demonstrating that products with a higher value can be produced. The extrudates were found to have promising properties.