Name: evaluation of the in vivo antitumor activity of polyanhydride il-1&#945; nanoparticles
Uploaded: 2021-06-28T14:30:00
Description: Watch this Scientific Journal Video about Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1ÃŽÂ± Nanoparticles at JoVE.com

这项工作得到了美国优异审查奖#I01BX004829的部分支持。退伍军人事务部，生物医学实验室研究与发展服务，并通过爱荷华大学霍顿综合癌症中心获得Mezhir奖计划的支持。

本研究中使用的所有 体内 程序均已获得爱荷华大学机构动物护理和使用委员会（IACUC）的批准。
1. HNSCC细胞系的制备与维护
注意：在本研究中，将使用HPV E6和E7以及hRas和荧光素酶（mEERL）稳定转化的小鼠口咽上皮细胞系。该细胞系由C57BL / 6J小鼠株开发而成，是Paola D. Vermeer博士（美国南达科他州南达科他州南达科他州桑福德医学院外科系）的礼物。<...

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该协议将允许任何研究人员在 体内 肿瘤小鼠模型系统中研究抗肿瘤活性和免疫调节药物的一些潜在机制。在这里，使用了同源皮下肿瘤模型，该模型与原位模型相比具有几个优点，包括其技术上简单的方案，易于监测肿瘤生长，更少的动物发病率和更高的可生产性。皮下肿瘤模型也可以通过在左翼和右翼注射肿瘤细胞来修改为双侧肿瘤模型。在这种双侧肿瘤模型中，可以对肿瘤内的一个?...

癌症免疫疗法的新兴领域之一是使用炎性细胞因子来激活患者的免疫系统对抗其肿瘤细胞。几种促炎细胞因子（即干扰素-α（IFNα）、白细胞介素-2（IL-2）和白细胞介素-1（IL-1））可以产生显着的抗肿瘤免疫力，这引起了人们对探索抗肿瘤特性以及基于细胞因子的药物的安全性的兴趣。特别是白细胞介素-1α（IL-1α）是一种促炎细胞因子，称为炎症1的主细胞因子。自 1970 年代后期发现这种细胞因子以来，它已被研究为抗癌剂以及治疗化疗负面影响的造血药物2.在1980年代后期，进行了几项临床前和临床研究，以确定IL-1α3，4，5，6的抗癌作用。这些研究发现重组IL-1α（rIL-1α）对黑色素瘤、肾细胞癌和卵巢癌具有有希望的抗肿瘤活性。然而，通常观察到毒性，包括发热、恶心、呕吐、流感样症状和最严重的剂量限制性低血压。不幸的是，这些剂量相关的毒性抑制了进一步临床使用rIL-1α的热情。
为了解决IL-1α介导的毒性的关键问题，将研究允许通过表面侵蚀动力学控制IL-1α释放的聚酐纳米颗粒（NP）配方。这些NP制剂旨在获得IL-1α抗肿瘤特性的益处，同时减少剂量限制性副作用7。聚酐是FDA批准的聚合物，通过表面侵蚀降解，导致封装剂8，9，10，11，12的释放几乎为零级。据报道，含有1，8-双-（对羧基苯氧基）-3，6-二氧杂辛烷（CPTEG）和1，6-双（对羧基苯氧基）己烷（CPH）的两亲性聚酐共聚物是肿瘤学和免疫学研究中各种有效载荷的出色递送系统8，12。在以下方案中，20：80 CPTEG：CPH NPs将加载1.5wt.%rIL-1α（IL-1α-NPs）用于研究该细胞因子在HNSCC小鼠模型中的抗肿瘤活性和毒性。
以下程序的总体目标是评估IL-1α-NPs对HNSCC的抗肿瘤活性。所描述的程序，包括评估肿瘤生长和存活，可以应用于任何感兴趣的免疫调节剂。这些程序应在具有完整免疫系统的同系小鼠模型中进行13 ，以最大限度地提高临床相关性。IL-1α-NP毒性也将通过测量促炎细胞因子循环水平和动物体重的变化来评估。测定 体内 药物毒性的方法有很多种;然而，最广泛使用的方法涉及测量血清酶的器官毒性和这些器官的组织学变化。但是，要进行组织学分析，需要牺牲动物，这将影响实验的存活曲线。因此，该协议将包括用于从活小鼠收集血液以测量血清样品中的细胞因子的方案。收集的血清可用于测量任何所需的血清分析物的器官毒性。多色流式细胞术将用于了解肿瘤微环境中免疫细胞群的变化以及免疫细胞向淋巴结的迁移。其他方法可用于鉴定免疫细胞，包括保存部分的免疫组织化学和/或免疫荧光14。然而，这些技术在大量动物身上执行可能既耗时又乏味。总体而言，以下方法将使研究人员能够研究抗肿瘤免疫反应和免疫刺激剂用于癌症治疗的潜在机制。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bio-Plex 200 Systems</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>The system was provided from the Flow Cytometry Facility University of IOWA Health Care</td>
		</tr>
		<tr>
			<td>Bio-Plex Pro Mouse Cytokine 23-plex Assay</td>
			<td>Bio-Rad</td>
			<td>M60009RDPD</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J Mice</td>
			<td>Jakson Labs</td>
			<td>664</td>
			<td>4 to 6 weeks old</td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#39;s Modified Eagle Medium)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/Hams F12 (Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Millipore Sigma</td>
			<td>SRP3196-500UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Millipore Sigma</td>
			<td>12103C-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin sulfate solution</td>
			<td>IBI Scientific</td>
			<td>IB02030</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS Dissociator</td>
			<td>Miltenyi biotec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hand-Held Magnetic Plate Washer</td>
			<td>Thermo Fisher Scientific</td>
			<td>EPX-55555-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Millipore Sigma</td>
			<td>H6909-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Millipore Sigma</td>
			<td>I0516-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine/xylazine</td>
			<td></td>
			<td></td>
			<td>Injectable anesthesia</td>
		</tr>
		<tr>
			<td>MEERL cell line</td>
			<td></td>
			<td></td>
			<td>Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells</td>
		</tr>
		<tr>
			<td>Portable Balances</td>
			<td>Ohaus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scienceware&#160;Digi-Max&#160;slide caliper</td>
			<td>Millipore Sigma</td>
			<td>Z503576-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile alcohol prep pad (70% isopropyl alcohol)</td>
			<td>Cardinal</td>
			<td>COV5110.PMP</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin Human</td>
			<td>Millipore Sigma</td>
			<td>T8158-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-iodothyronin</td>
			<td>Millipore Sigma</td>
			<td>T5516-1MG</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of the in vivo antitumor activity of polyanhydride il-1&#945; nanoparticles

细胞因子治疗是一种很有前途的免疫治疗策略，可以在癌症患者中产生强大的抗肿瘤免疫反应。促炎细胞因子白细胞介素-1α（IL-1α）已在多项临床前和临床研究中被评估为抗癌剂。然而，剂量限制性毒性，包括流感样症状和低血压，削弱了对这种治疗策略的热情。在这种情况下，基于聚酐纳米颗粒（NP）的IL-1α递送将代表一种有效的方法，因为这可能允许IL-1α的缓慢和受控释放，同时减少毒副作用。这里描述了头颈部鳞状细胞癌（HNSCC）同系小鼠模型中负载IL-1α的聚酐NPs的抗肿瘤活性分析。将稳定表达HPV16 E6/E7的小鼠口咽上皮细胞与hRAS和荧光素酶（mEERL）细胞一起皮下注射到C57BL/6J小鼠的右胁中。一旦肿瘤在任何方向上达到3-4mm，就向小鼠腹膜内施用1.5%IL-1a - 负载20：80 1，8-双（对羧基苯氧基）-3，6-二氧杂辛烷：1，6-双（对羧基苯氧基）己烷（CPTEG：CPH）纳米颗粒（IL-1α-NP）制剂。连续测量肿瘤大小和体重，直到肿瘤大小或体重减轻达到安乐死标准。通过下颌下静脉穿刺采集血液样本以评估抗肿瘤免疫应答，并通过细胞因子多重测定法测定炎性细胞因子。切除肿瘤和腹股沟淋巴结，均质化成单细胞悬液，通过多色流式细胞术分析各种免疫细胞。这些标准方法将使研究人员能够研究免疫刺激NPs和其他免疫治疗剂用于癌症治疗的抗肿瘤免疫反应和潜在机制。

本研究研究了聚酐IL-1α在HNSCC同系小鼠模型中的抗肿瘤活性。重组IL-1α（rIL-1α）显着减缓了mEERL肿瘤的生长（图1A），尽管在治疗小鼠中观察到体重减轻，并且在治疗停止后恢复（图1B）。与盐水或空白-NPs相比，IL-1α-NPs没有诱导显着的抗肿瘤作用（图1A），并且伴随着一些体重减轻，尽管不如rIL-1α突出（图1B...

Watch this Scientific Journal Video about Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1ÃŽÂ± Nanoparticles at JoVE.com

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1ÃƒÅ½Ã‚Â± Nanoparticles

描述了一种标准方案来研究HNSCC同系小鼠模型中IL-1α的抗肿瘤活性和相关毒性。

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Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal ...

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