金纳米粒子的蛋白质激酶 c-三角洲抑制剂肽配方
Summary
我们以前使用过一种金纳米多肽混合体静脉注射一种合成肽, 即蛋白激酶 c-三角洲抑制剂, 减少缺血再灌注引起的急性肺损伤。在这里, 我们展示了药物配方的详细协议。其他细胞内肽也可以类似地配制。
Abstract
蛋白激酶 c-三角洲抑制剂 (pkcc i) 是一种很有前途的预防缺血再灌注所致器官损伤的药物。它通常与细胞穿透性肽 tat 结合, 用于细胞内分娩。然而, tat 显示了非特异性的生物活动。金纳米颗粒 (gnps) 可用作药物输送载体, 而不具有公认的毒性。因此, 我们使用了 gnp肽杂化剂来传递 pkc i。以95:5 的比例使用了两个短肽 (p2:caaae 和 p4:caaaaw) 来修正 gnp 的表面特性。与 pkcc i (gnp/pkgi) 结合的 gnp 在蒸馏水、0.9% 氯化钠和含牛血清白蛋白或胎儿牛血清的磷酸盐缓冲盐水中均稳定。静脉注射 gnp-pki 对肺缺血再灌注损伤有明显的预防作用。本文概述了制定 gnp/pkci 和评价 gnp/pkci 理化性质的方案。我们采用了类似的方法来制定其他具有国民生产总值的含肽药物。希望本文能引起人们对这种新型细胞内药物输送技术及其在体内应用的更多关注。
Introduction
肺移植挽救终末期肺病 1.然而, 肺移植后的严重并发症仍然是一个障碍。在肺移植术后早期, 原发性移植物功能障碍是最有害的并发症1, 其主要原因是缺血再灌注 (ir) 致急性肺损伤2。
在冷保存条件下, 供体肺的代谢被限制在很低的水平。然而, 活性氧和一氧化氮合成被激活, 由于停止血液流动 3。移植后, 血液循环恢复, 冷缺血过程中产生的活性氧和一氧化氮会增强炎症和细胞死亡, 导致组织损伤。
为预防红外损伤, 在心脏、大脑和肺4、5、6、7、8中使用了蛋白激酶 c 抑制剂 (pkc i)。这些研究表明, pkcc i 减少了再灌注过程中的炎症和凋亡。它还可以防止大鼠和第6型肺移植模型的肺 ir 损伤。pkc i 通常与细胞内穿透性肽 tat 结合, 用于细胞内分娩。然而, 事实表明, 仅 tat 肽具有非特异性的生物学效应, 包括促进血管生成, 凋亡, 并抑制多种细胞因子9,10,11。纳米粒子, 直径为 12的小颗粒, 已被探索为促进药物输送的候选物质 13.特别是, 金纳米粒子 (gnps) 被认为是无创和无毒的。因此, 我们开发了 gnp 作为肽基药物的药物传递载体 14,15。
gnps 的表面可用于分子识别16、17、化学传感18、成像19和药物输送等特定应用。开发了一种 gnp 肽混合体系, 该系统以95:5 的比例, 含有20纳米 gnp 和两个短肽 (p2:caaaae 和 p4:caaaaw), 以改变 gnp 的表面特性。p2 肽, 与带负电荷谷氨酸 (e) 的末端, 稳定 gnp 在水溶液中, 而 p4 肽, 与疏水色氨酸 (w) 在末端, 帮助 gnp 进入细胞14。这些肽 n 端的半胱氨酸 (c) 残留物含有硫醇基团, 可与金表面结合 14.该杂交种进一步用于 pkcgi (csfsyelgsl) 的制备。p2:p4 与 pkcci 的优化摩尔比为4.5:2.5:50。与 pkcc i (gnp/pkci) 结合的 gnp 在蒸馏水、0.9% 氯化钠和含有牛白蛋白或胎儿牛血清的pbs 中稳定。静脉注射 gnpmci 可预防肺缺血再灌注损伤 15.本文概述了一种制备 gnp/pkci 的方法, 并对 gnp/pkci 的理化性质进行了评价。我们使用了类似的方法来制定其他与 gnp20、21、22结合的基于肽的药物。我们希望本文能引起人们对这种新型细胞内药物输送制剂的更多关注。
Protocol
Representative Results
Discussion
为了确保正确的配方, 至关重要的是, pkgi 解决方案要经历1.6 中概述的声纳步骤。pkgi 肽序列含有疏水的分子, 因此超声器有助于在50% 乙腈溶液中溶解 pkgi。此外, 如步骤2.7 中所述, 精心混合溶剂也是非常重要的。 gnp / pkci 将不是很好地制定 , 如果这些步骤不正确 , 由于聚集的 pkci 肽23。
基于 gnp 的药物配方提供了几个优点。首先, gnps 可以很容易地合成在良好的控制大小, 从…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了加拿大卫生研究所 (pjt-148847)、安大略省研究和创新部 (re-08-029) 和加拿大首次卓越研究方案—-多伦多大学设计医学方案—-的研究赠款的支持。刘明耀博士是《肺损伤、修复和再生》的詹姆斯和玛丽·戴维斯主席。
Materials
| negatively charged glutamic acid peptide (P2) | CanPeptide | Sequence: CAAAAE-NH2 Length: 6aa Modification: C-terminal amidation Quantity: 50mg Purity: >95% |
|
| hydrophobic tryptophan peptide (P4) | CanPeptide | Sequence: CAAAAW-NH2 Length: 6aa Modification: C-terminal amidation Quantity: 50mg Purity: >95% |
|
| δPKCi peptide | CanPeptide | Seqeuence: CSFNSYELGSL-NH2 Length: 11aa Modification: C-terminal amidation Quantity: 50mg Purity: >95% |
|
| Conical tube(50ml) | Corning Life Sciences | 3582070 | |
| Conical tube(15ml) | Corning Life Sciences | 3582096 | |
| Acetonitrile | Sigma-Aldrich | 271004-100ML | |
| Sonicator | Branson Ultrasonics Corp. | Branson 2510MTH | |
| Microtube | Diamed.ca | AD 150-N | |
| Gold nanoparticle solution | Ted Pella | 15705-5 | A particle size is 20nm |
| Rocking Platform shaker | VWR international | 40000-304 | |
| Microcentrifuge | Eppendorf | 5417R | |
| Acryl cuvette | SARSREDT | 67.758 | |
| UV-Vis spectrophotometer | Agilent | Caty 60 UV-Vis |
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