用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素
Summary
工程纳米材料中内毒素的检测是纳米医学领域面临的重大挑战之一。在这里, 我们提出了一个案例研究, 描述了由三种不同的 lal 格式组成的框架, 以估计纳米颗粒中潜在的内毒素污染。
Abstract
当存在于医药产品中时, 革兰氏阴性细菌细胞壁成分内毒素 (通常也称为脂多糖) 可引起炎症、发热、低血压或高血压, 在极端情况下, 可导致组织和器官损伤, 从而可能导致组织和器官损伤。变得致命。因此, 对药品中的内毒素含量进行了严格的监管。在可用于内毒素检测和定量的方法中, 利穆鲁斯·阿米巴细胞裂解液 (lal) 法在全球范围内得到了广泛的应用。虽然任何药品都会干扰 lal 检测, 但由于其复杂性, 纳米配方是一个特殊的挑战。本文的目的是为在估算工程纳米材料和纳米颗粒配方药物中的内毒素方面经验不足的研究人员提供实用的指导。在此, 讨论了执行三种 lal 格式的实用建议, 包括浊度、显色性和凝胶凝块检测。这些检测可用于测定纳米技术药物产品、疫苗和佐剂中的内毒素污染。
Introduction
内毒素是革兰氏阴性细菌细胞壁 1,2的一个组成部分。它可以在非常低的 (皮图) 浓度 1,2激活免疫细胞。细胞对内毒素产生的促炎介质 (细胞因子、白三烯、eicooid等) 导致发烧、低血压、高血压和更严重的健康问题, 包括多器官衰竭1,2,3. 内毒素引发的免疫介导的副作用的严重程度取决于其效力, 这些副作用由内毒素组成和结构决定, 并以国际内毒素单位 (iu 或 eu)3进行测量。每公斤体重单位的数量被用来设定一个阈值的热原剂量内毒素。对于通过除鞘内路线以外的所有途径给药的药物产品, 这一剂量为5欧元/千克。每平方米的药物体表面、眼内液体、放射性药物和通过鞘内途径施用的产品具有不同的阈值热原剂量, 即 100 EU/mL、0.2 euml、175 欧元 (其中 v 是用于管理的产品的体积), 分别为 0.2 eu/kg.关于各种药物产品和设备的阈值热原剂量的更多细节, 在其他地方提供和讨论。
动物对内毒素介导的反应的敏感性差别很大。人类、非人类灵长类动物和兔子是对内毒素3最敏感的物种之一.为了避免患者内毒素介导的副作用, 并防止临床前毒性和疗效研究的不准确结论, 必须准确地检测和量化临床和临床前等级配方中的内毒素。目前有几种可用的方法可以完成这项任务。其中之一是利穆鲁斯阿米巴细胞裂解液 (lal) 检测, 它在全球范围内常用用于筛选生物医学产品的潜在内毒素污染, 以及检测细菌感染7,8,9。裂解液是由存在于北美大陆东岸的马蹄蟹血液中的肌细胞组成的, 这种细胞存在于马蹄形细胞中。有趣的是, 在亚洲有几个不同种类的马蹄蟹 (马蹄形蟹和三头肌) 。在几个亚洲国家, 细胞裂解液 (tal) 被用来检测内毒素, 类似于其他国家使用 lal 的方式.裂解物 (lal 和 tal) 含有一组蛋白质, 在激活时产生蛋白酶活性。其中一种蛋白质, 所谓的因子 c 在与内毒素接触时被激活。活化因子 c 裂解因子 b, 进而成为蛋白酶, 并裂解促进凝血酶产生凝血酶。这种反应链的结果是形成了凝胶, 增加了样品的浊度, 并且在有显色底物的情况下, 出现了有色产品, 作为凝胶凝块、浊度和显色检测的基础,分别。虽然没有强制性的 lal 格式, 美国食品药品监督管理局 (fda) 在行业文件的指导意见中解释说, 如果不同的 lal 格式之间的测试结果不一致, 决定是根据凝胶凝块检测5.
许多常用的实验室化学品 (如edta) 和已知的药物产品 (如青霉素) 会干扰 lal 检测11。这种干扰通常是通过评估在已知浓度下刺入含有测试材料的溶液中的内毒素标准的回收情况来识别的。如果穗恢复小于50% 或大于 200%, 则给定测试材料的 lal 检测结果是无效的, 原因是抑制或增强, 分别为 4。基于纳米技术的配方通常是复杂的, 并通过各种机制12,13,14干扰 lal。已经描述了许多克服干扰的方法: 样品在特定的缓冲液和表面活性剂中的重组, 蛋白质通过加热失活, 通过加热和补充样品过量来破坏以脂质为基础的空心材料二价阳离子5,12,13,14,15。还介绍了 lal 干扰无法克服的情况下的替代方法: elisa、hek-tlr4 报告细胞系检测和16、17、18、 19岁
本文介绍了进行凝胶凝块、浊度和显色性 lal 检测的实验过程。这些检测也可在纳米技术表征实验室 (ncl) 网站20上获得, 协议为 ste1.2 (浊度 lal)、ste3 (凝胶-凝体 lal) 和 ste1.4 (显色性 lal)。建议至少采用两种不同的格式来表征相同的纳米配方。当浊度和色原性 lal 的结果不一致时, 凝胶凝块的结果被认为是5。当两种 lal 格式的结果不一致时, 使用单核细胞活化试验 (mat) 或兔热原测试 (rpt) 来验证 lal 的结果进行了21项额外的研究。需要注意的是, 用于内毒素检测和热原性评估的每一种方法都有优点和局限性 21、22、23、24.认识到用于表征特定纳米技术配方的程序的局限性对于获得科学的理由来证明该程序的使用是最佳的, 该纳米制剂。
本研究采用聚乙二醇化脂质体多沙比星作为纳米颗粒的模型制剂。这种制剂于1995年获得美国 fda 的批准, 用于治疗全世界25 例癌症患者。
Protocol
1. 纳米粒子样品的制备 在 lal 级水中准备研究样本。 如果样品 ph 值在6-8 范围之外, 请使用无热原氧化钠或盐酸调整 ph 值。 使用 lal 级的水制备了几个稀释的研究样本。确保最高稀释量不超过最大有效稀释 (mvd)。有关 mvd 估计的详细信息, 请参阅讨论部分。 2. lal 格式之间常用试剂的制备 使用无热剂 lal 试剂水稀释浓缩氢氧化钠库存, 制备浓度…
Representative Results
表 1显示了在 lal 检测中测试此配方后生成的数据示例。聚乙二醇脂质体多索比星在稀释5时干扰显色性 lal。然而, 这种干扰是通过更大的稀释克服的。当这种配方在浊度和显色性 lal 的稀释50和500中进行稀释测试时, 以及在浊度 lal 中的稀释5时, 尖刺回收率在50% 至200% 之间。当通过稀释因子进行调整时, 两个检测中的稀释结果是一致的。此外, 三种检测方法的结果…
Discussion
本议定书中提供的信息在15、26之前就已经描述过, 并依赖于美国食品药品监督管理局 (fda 或 fda) 和美国药典 (usp)公布的若干监管文件4,5,6,27, 也可在 ncl 网站20协议ste2 (浊度 lal), ste3 (凝胶-凝胶 lal) 和 ste1.4 (显色性 lal)。
…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了国家癌症研究所联邦基金的支持, 该研究的合同是 HHSN261200800001E。本出版物的内容不一定反映卫生与公众服务部的观点或政策, 提及商品名称、商业产品或组织也不一定意味着美国政府的认可。
Materials
| Turbidity LAL Assay | |||
| Sodium Hydroxide | Sigma | S2770 | When needed, it is used to adjust sample pH to be between 6-8 |
| Hydrochloric acid | Sigma | H9892 | When needed, it is used to adjust sample pH to be between 6-8 |
| LAL Reagent | Associates of Cape Cod | T0051 | This reagent can be used with turbidity assay only |
| Control Endotoxin Standard | Associates of Cape Cod | E0005 | This reagent can be used with turbidity and gel-clot assays |
| LAL grade water | Associates of Cape Cod | WP0501 | This reagent can be used with any LAL format |
| Glucashield Buffer | Associates of Cape Cod | GB051-25 | Used to prevent false-positive response from beta-glucans |
| Disposable endotoxin-free glass dilution tubes 12 x 75 mm | Associates of Cape Cod | TB240 | These tubes can be used with all three assays |
| Disposable endotoxin-free glass reaction tubes 8 x 75 mm | Associates of Cape Cod | TK100 | These tubes can be used with turbidity and chromogenic assays |
| Pyrogen-free tips with volumes 0.25 and 1.0 mL | RAININ | PPT25, PPT10 | Tips and pipettes may adsorb endotoxin and release leachables which interfere with LAL assay. These RAININ tips are used because their optimal performance in the LAL assay was verified and confirmed |
| Pyrogen-free microcentrifuge tubes, 2.0 mL | Eppendorf | 22600044 | Other equivalent supplies can be used |
| Pyrogen-fee combitips, 5mL | Eppendorf | 30089669 | Other equivalent supplies can be used |
| Repeat pipettor | Eppendorf | 4982000020 | Other equivalent supplies can be used |
| Microcetrifuge | any brand | Any brand can be used | |
| Refrigerator, 2-8 C | any brand | Any brand can be used | |
| Vortex | any brand | Any brand can be used | |
| Freezer, -20 C | any brand | Any brand can be used | |
| Pyros Kinetix or Pyros Kinetix Flex reader | Associates of Cape Cod | PKF96 | Other instruments can be used. However, LAL reagents and endotoxin standards used in this assay may require optimization. When other instrumentation is used, please refer to the instrument and LAL kit manufacturers for instructions |
| Chromogenic LAL Assay | |||
| Pyrochrome LAL Reagent | Associates of Cape Cod | CG1500-5 | This reagent is specific to the Chromogenic Assay |
| Control Endotoxin Standard | Associates of Cape Cod | EC010 | This standard is different than that used for turbidity and gel-clot LALs; it is optimized for optimal performance in the chromogenic assay |
| Sodium Hydroxide | Sigma | S2770 | When needed, it is used to adjust sample pH to be between 6-8 |
| Hydrochloric acid | Sigma | H9892 | When needed, it is used to adjust sample pH to be between 6-8 |
| LAL grade water | Associates of Cape Cod | WP0501 | This reagent can be used with any LAL format |
| Glucashield Buffer | Associates of Cape Cod | GB051-25 | Used to prevent false-positive response from beta-glucans |
| Disposable endotoxin-free glass dilution tubes 12 x 75 mm | Associates of Cape Cod | TB240 | These tubes can be used with all three assays |
| Disposable endotoxin-free glass reaction tubes 8 x 75 mm | Associates of Cape Cod | TK100 | These tubes can be used with turbidity and chromogenic assays |
| Pyrogen-free tips with volumes 0.25 and 1.0 ml | RAININ | PPT25, PPT10 | Tips and pipettes may adsorb endotoxin and release leachables which interfere with LAL assay. These RAININ tips are used because their optimal performance in the LAL assay was verified and confirmed |
| Pyrogen-free microcentrifuge tubes, 2.0 mL | Eppendorf | 22600044 | Other equivalent supplies can be used |
| Pyrogen-fee combitips, 5mL | Eppendorf | 30089669 | Other equivalent supplies can be used |
| Repeat pipettor | Eppendorf | 4982000020 | Other equivalent supplies can be used |
| Microcetrifuge | any brand | Any brand can be used | |
| Refrigerator, 2-8 C | any brand | Any brand can be used | |
| Vortex | any brand | Any brand can be used | |
| Freezer, -20 C | any brand | Any brand can be used | |
| Pyros Kinetix or Pyros Kinetix Flex reader | Associates of Cape Cod | PKF96 | Other instruments can be used. However, LAL reagents and endotoxin standards used in this assay may require optimization. When other instrumentation is used, please refer to the instrument and LAL kit manufacturers for instructions |
| Gel-Clot LAL Assay | |||
| LAL Reagent | Associates of Cape Cod | G5003 | This reagent is specific to the gel-clot assay |
| Control Endotoxin Standard | Associates of Cape Cod | E0005 | This reagent can be used with turbidity and gel-clot assays |
| Sodium Hydroxide | Sigma | S2770 | When needed, it is used to adjust sample pH to be between 6-8 |
| Hydrochloric acid | Sigma | H9892 | When needed, it is used to adjust sample pH to be between 6-8 |
| LAL grade water | Associates of Cape Cod | WP0501 | This reagent can be used with any LAL format |
| Glucashield Buffer | Associates of Cape Cod | GB051-25 | Used to prevent false-positive response from beta-glucans |
| Disposable endotoxin-free glass dilution tubes 12 x 75 mm | Associates of Cape Cod | TB240 | These tubes can be used with all three assays |
| Disposable endotoxin-free glass reaction tubes 10 x 75 mm | Associates of Cape Cod | TS050 | These tubes are for use with the gel-clot assay |
| Pyrogen-free tips with volumes 0.25 and 1 mL | RAININ | PPT25, PPT10 | Tips and pipettes may adsorb endotoxin and release leachables which interfere with LAL assay. These RAININ tips are used because their optimal performance in the LAL assay was verified and confirmed |
| Pyrogen-free microcentrifuge tubes, 2.0 mL | Eppendorf | 22600044 | Other equivalent supplies can be used |
| Pyrogen-fee combitips, 5mL | Eppendorf | 30089669 | Other equivalent supplies can be used |
| Repeat pipettor | Eppendorf | 4982000020 | Other equivalent supplies can be used |
| Microcetrifuge | any brand | Any brand can be used | |
| Refrigerator, 2-8 C | any brand | Any brand can be used | |
| Vortex | any brand | Any brand can be used | |
| Freezer, -20 C | any brand | Any brand can be used | |
| Water bath, 37 C | any brand | Any brand can be used, however, it is important either to switch off water circulation or use non-circualting water bath because water flow will affect clot formation and lead to false-negative results |
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Tags
Keywords:
EndotoxinNano-formulationsLimulus Amoebocyte Lysate (LAL) AssaysCalibration StandardControl Standard EndotoxinLAL Grade WaterQuality ControlInstrument SettingsTest IDData GroupHardwareLAL MethodSerial NumberSystem IDSerial PortSample IDNegative ControlStandard CurveTest SamplesInhibition Enhancement ControlDilution
Cite This Article
Neun, B. W., Dobrovolskaia, M. A. Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays. J. Vis. Exp. (143), e58830, doi:10.3791/58830 (2019).