Name: protein kinase c-delta inhibitor peptide formulation using gold nanoparticles
Uploaded: 2019-03-09T14:00:00
Description: Watch this Scientific Journal Video about Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles at JoVE.com

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。

1. 多肽溶液的制备
<ol><li>从-20°c 的冷冻机中提取多肽 (p2:caaaae, p4:caaaaw, pkkcci:csfnsyelgsl), 并在室温下解冻 (rt)。 注: 保持瓶子关闭, 以防止水分凝结在肽上。</li>
	<li>在微观上称量每个肽 0.01 g。将每个肽放入一个单独的50毫升锥形管。</li>
	<li>在 p2 管中加入18.74 毫升的去离子水。</li>
	<li>在 p4 管中加入16.93 毫升的 di 水。</li>
	<li>在 di 水中稀释50% 乙腈8.21 毫升至 pkcc i 管中。</li>
	<li>短暂的旋涡肽?...

<ol>
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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...

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。

<table border="0" cellpadding="0" cellspacing="0" style="width:603px;" width="603">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">negatively charged glutamic acid peptide (P2)</td>
			<td style="width:189px;">CanPeptide</td>
			<td></td>
			<td style="width:197px;">Sequence: CAAAAE-NH2 
			Length: 6aa 
			Modification: C-terminal amidation 
			Quantity: 50mg 
			Purity: &#62;95%</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">hydrophobic tryptophan peptide (P4)</td>
			<td>CanPeptide</td>
			<td></td>
			<td style="width:197px;">Sequence: CAAAAW-NH2 
			Length: 6aa 
			Modification: C-terminal amidation 
			Quantity: 50mg 
			Purity: &#62;95%</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">&#948;PKCi peptide</td>
			<td>CanPeptide</td>
			<td></td>
			<td style="width:197px;">Seqeuence: CSFNSYELGSL-NH2 
			Length: 11aa 
			Modification: C-terminal amidation 
			Quantity: 50mg 
			Purity: &#62;95%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conical tube(50ml)</td>
			<td>Corning Life Sciences</td>
			<td align="right" style="width:119px;">3582070</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conical tube(15ml)</td>
			<td>Corning Life Sciences</td>
			<td align="right" style="width:119px;">3582096</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Acetonitrile</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">271004-100ML</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sonicator</td>
			<td style="width:71px;">Branson Ultrasonics Corp.</td>
			<td style="width:119px;">Branson 2510MTH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microtube</td>
			<td>Diamed.ca</td>
			<td style="width:119px;">AD 150-N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gold nanoparticle solution</td>
			<td>Ted Pella</td>
			<td style="width:119px;">15705-5</td>
			<td>A particle size is 20nm</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Rocking Platform shaker</td>
			<td style="width:71px;">VWR international</td>
			<td style="width:119px;">40000-304&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microcentrifuge</td>
			<td style="width:71px;">Eppendorf</td>
			<td style="width:119px;">5417R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Acryl cuvette</td>
			<td style="width:71px;">SARSREDT</td>
			<td align="right" style="width:119px;">67.758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV-Vis spectrophotometer</td>
			<td style="width:71px;">Agilent</td>
			<td style="width:119px;">Caty 60 UV-Vis</td>
			<td></td>
		</tr>
	</tbody>
</table>

protein kinase c-delta inhibitor peptide formulation using gold nanoparticles

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。

应注意评估 gnpmci 杂交种的生物物理特性, 因为 gnp 倾向于在溶剂中聚合。当 gnp 聚合时, 溶液的颜色将从粉红色变为紫色 (图 1a)。uv-vis 分光光度计能够更灵敏地检测到变化。如果 gnp/pkci 不聚合, 吸收的峰值应为 525 nm (图 1b)。如果国民生产总值是汇总的, 吸收的高峰将转移到右边。作为另一种分析方法, 当聚合物形成了光学密度 (o...

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Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

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。

金纳米粒子的蛋白质激酶 c-三角洲抑制剂肽配方

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a ...

我们独特的药物输送系统，在两种特定的短肽中使用20纳米金颗粒，可以稳定生理溶液中的金纳米颗粒，增强细胞吸收，从而有效地将肽或其他药物转化为细胞。我们的GNP肽杂交是易于合成和稳定。利用金纳米粒子肽杂交作为药物载体，可以有效、丰富地将肽药物转移到细胞中。我们用 GNP/PKCi 杂交治疗肺部缺血-再灌注损伤。GNP 将 PKC 抑制剂肽引入细胞质。这种分娩方法能够减少更严重的肺损伤使用较低的PKCi剂量比其它分娩方法。要开始此过程，请从零下 20 摄氏度的冰柜中取出肽，并在室温下解冻。使用微量表称每个解冻肽 0.01 克，将每种肽转移到单独的 50 毫升锥形管中。在P2管中加入18.74毫升的去水，在P4管中加入16.93毫升的去水。将8.21毫升的50%乙酰酸二醇稀释在去维化水中添加到PKC三角洲抑制剂管中。短暂地涡旋肽溶液。然后将管子放入40兆赫的声波机中5分钟。在此之后，将解决方案转移到生物安全柜。将 50 mL 圆锥管放入声子中 5 分钟至关重要。如果跳过此步骤，GNP/PKCi 将形成聚合。将每一多肽溶液的一毫升转移到其新的50毫升锥形管中。在含有P2和P4的管子中加入19毫升的去化水。并在含有PKC三角洲抑制剂的管子中加入19毫升50%乙酰三叶酸。将 475 毫升的 P2 溶液、25 微升的 P4 溶液、500 微升的 PKC 增量抑制剂溶液加入到 15 毫升管中。然后在同一管中加入9毫升20纳米GNP溶液。从生物安全柜中去除组合溶液，用铝箔包裹管子。将管子放在摇床上过夜。第二天，将样品退回生物安全柜。将 GNP/PKCi 溶液的一毫升等分液转移到 1.5 毫升微管中。在微离心机时以15294倍g和4摄氏度离心，30分钟。将微管重新转移到生物安全柜中，从每个管中去除超高动管，同时确保 GNP 颗粒不受干扰。根据所需的浓度，将每个颗粒重新排放到所需的溶剂中。要评估 GNP/PKCi 杂交溶解度，请将 0.5 毫升的 GNP/PKCi 溶液倒入应计式库中。将库放在 UV-Vis 分光光度计上，并测试峰值吸收。在这项研究中，蛋白激酶C-delta抑制剂与金纳米粒子结合，形成GNP/PKCi杂交。在评估混合体的生物物理特性时，必须注意，因为金纳米粒子往往在溶剂中聚合。当纳米粒子聚集时，溶液的颜色将从粉红色变成紫色。用UV-Vis分光光度计对样品进行分析，该光谱仪能够以高度的灵敏度进行检测。如果未聚合混合动力车，吸收的峰值应为 525 纳米。如果 GNP 聚合，吸收峰值将向右移动。可以看到，当聚合形成时，增量光密度会降低。在制定 GNP 肽杂交体时，重要的是要记住肽与 GNP 溶液的比例应为 1 比 9。此 GNP/PKCi 配方可用于肺以外的器官系统中的缺血-再灌注损伤。如果这些肽在N-1INus有半胱氨酸残留物，任何肽都可以附着在GNP上。这意味着GNP肽杂交可用于将其他肽药物运送到细胞中。合理设计的肽是新技术，可以带来特定的蛋白质-蛋白质相互作用在这些细胞内。这项技术，在这里开发，可用于交付新的肽类药物或现有药物的改性版本。

Research

JoVE Journal

Bioengineering

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

制定通过Nanoprecipitation技术嵌段聚合物纳米粒子

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

科研

生物工程

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

单层的合成，装配和表征蛋白质单分子膜的电化学保护的金纳米粒子薄膜

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

分析有针对性的病毒蛋白的纳米粒子递送HER2 +肿瘤

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

细胞物质相互作用研究微细的纳米多孔金模式

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

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, ...

Harmonic Nanoparticles for Regenerative Research

纳米谐波再生研究

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

化学共轭法在戊型肝炎病毒纳米粒子表面功能化中的应用

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

在肽膜前体的维西图洛合成中,用于自主囊泡生长

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

大肠杆菌-表达自组装蛋白纳米颗粒的生产，用于需要三聚体异体的疫苗

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...