Name: construction of cyclic cell-penetrating peptides for enhanced penetration of biological barriers
Uploaded: 2022-09-19T13:00:00
Description: Watch this Scientific Journal Video about Construction of Cyclic Cell-Penetrating Peptides for Enhanced Penetration of Biological Barriers at JoVE.com

这项工作得到了国家自然科学基金（21708031）、中国博士后科学基金（BX20180264，2018M643519）和中央高校基本科研业务费基金（2682021ZTPY075）的支持。

1. 设备准备
注意：使用合适的个人防护设备在操作通风橱中执行所有程序。
<ol><li>将手动肽合成装置组装在通风橱中（图1）。将三通旋塞阀（见材料表）放在真空歧管上（见材料表）并连接到氮气（N2）。确保盖上未使用的入口。</li>
	<li>将 10 mL 聚丙烯色谱柱（参见 材料表）连接到三通旋塞?...

<ol>
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通过结合构象约束来稳定肽已被证明是提高肽26的稳定性和细胞通透性的有效策略。在该协议中，描述了用于合成具有芳香交联的环状CPPs并评估其跨生物屏障的渗透性的分步程序。与亲水性内酰胺或三唑交联22，27相比，芳族交联的掺入（用于本研究）提高了CPP的整体疏水性，从而显着提高了其细胞通透性。另一方面，肽环化可以通过?...

在过去的几十年里，用于药物递送的细胞穿透肽（CPP）的开发取得了快速进展。CPP已被广泛用作分子转运蛋白，用于治疗一系列危及生命的疾病，包括神经系统疾病1，2，心脏病3，糖尿病4，皮肤病5和癌症6，7。尽管开展了广泛的研究工作，但癌症仍然是一个全球健康负担，发病率和死亡率很高8。治疗癌症的一个严重障碍是，由于生理屏障，如紧凑的细胞外基质（ECM）、异常的肿瘤脉管系统、多重膜屏障和高组织液压力（IFP）9，治疗药物对更深的肿瘤细胞的获取有限。因此，开发具有跨越生物屏障运送货物的卓越能力的新型CPP被认为是癌症治疗的基本策略10，11。
CPP 根据其理化性质可分为阳离子性、两亲性和疏水性 CPP12。其中，带正电荷的HIV-TAT肽和合成的聚精氨酸在生物医学研究中具有相当的重要性，并且已被广泛研究以促进细胞内药物递送13。Tunnemann等人报告说，基于使用R3至R12肽14进行的细胞通透性研究，八个精氨酸的最小长度对于合成聚精氨酸肽的有效细胞渗透至关重要。然而，这些CPP由于其 在体内快速水解，通常具有较短的血浆半衰期。此外，关于优化CPP的化学结构以增加其跨屏障能力知之甚少，因为穿透多个细胞膜具有挑战性15。因此，强烈希望开发能够穿透生物屏障的新型分子转运蛋白以提高药物递送效率。2020 年，Komin 等人 16 发现了一种名为 CL 肽的 CPP，其中包含用于穿过上皮单层的螺旋基序 （RLLRLLR） 和聚精氨酸尾部 （R7）。通过改变螺旋模式，还合成了一组CL肽变体。这一探索可以成为开发新的CPPs的重要指南，用于跨越生物屏障运送货物。此外，Dietrich等人优化了StAX肽的细胞通透性，通过增加肽的整体疏水性来抑制Wnt/β-连环蛋白信号通路17。
通过环化对非结构化线性肽进行构象限制是增强其蛋白水解稳定性和渗透性的有效途径18，19，20。结构增强增加了环肽的蛋白酶抗性，使其在体内比线性对应物更稳定。此外，肽的环化可以通过促进分子内氢键来潜在地掩盖极性肽主链，从而增加肽的膜通透性21。在过去的二十年中，化学选择性环化方法已成为构建具有不同结构的环肽的有效策略，例如全烃，内酰胺，三唑，间二甲苯，全氟芳基和其他交联22，23。复杂的肿瘤微环境所施加的生物屏障可以减少药物在实体瘤中的渗透24。我们之前发现，环状CPPs对酶消化的抵抗力优于线性对应物20。此外，肽的整体疏水性对于增强细胞通透性至关重要22。基于上面讨论的研究，可以假设带正电模式、升高的整体疏水性和增强的蛋白水解稳定性的组合可以增加 CPP 跨越生物屏障的渗透性。在最近的一项研究中，我们确定了两种在位置i和i + 7具有芳香交联的环状CPP，与线性对应物相比，它们在肿瘤细胞和组织中表现出更好的通透性15。本文介绍了用于合成荧光标记环状CPP的简明合成方案以及研究其渗透性的关键步骤。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-ethanedithiol</td>
			<td>Aladdin</td>
			<td>K1722093</td>
			<td>stench</td>
		</tr>
		<tr>
			<td>2-(7-Azobenzotriazole)-N,N,N&#39;,N&#39;-tetramethyluronium hexafluorophosphate (HATU)</td>
			<td>HEOWNS</td>
			<td>A-0443697</td>
			<td></td>
		</tr>
		<tr>
			<td>4,4&#39;-bis(bromomethyl)biphenyl</td>
			<td>TCI</td>
			<td>B1921</td>
			<td></td>
		</tr>
		<tr>
			<td>4T1 cells</td>
			<td>ATCC</td>
			<td></td>
			<td>4T1 cells were cultured in DMEM medium supplemented with 10% FBS (Hyclone) in a 37 &#176;C humidified incubator containing 5% CO2.</td>
		</tr>
		<tr>
			<td>Acetonitrile&#160;</td>
			<td>Adamas</td>
			<td>1484971</td>
			<td>toxicity</td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Energy</td>
			<td>W330229</td>
			<td>skin harmful</td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Aldrich</td>
			<td>673811</td>
			<td>flammable</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Beyotime</td>
			<td>ST038</td>
			<td>skin harmful</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electrospray Ionization Mass Spectrometer</td>
			<td>Waters</td>
			<td>G2-S Tof</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene Diamine Tetraacetic Acid (EDTA)</td>
			<td>BioFroxx</td>
			<td>1340</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>HyClone</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>Beckman Coulter</td>
			<td>CytoFLEX</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate isomer (FITC)</td>
			<td>Energy</td>
			<td>E0801812500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Carl Zeiss</td>
			<td>Axio Observer 7</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Arg(Pbf)-OH</td>
			<td>HEOWNS</td>
			<td>F-81070</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Cys(Trt)-OH</td>
			<td>GL Biochem</td>
			<td>GLS201115-35202</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-&#946;Ala-OH</td>
			<td>Adamas</td>
			<td>51341C</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa cells</td>
			<td>ATCC</td>
			<td></td>
			<td>HeLa cells were cultured in DMEM supplemented with 10% FBS (Hyclone) in a 37 &#176;C humidified incubator containing 5% CO2.</td>
		</tr>
		<tr>
			<td>High-Performance Liquid Chromatography</td>
			<td>Agilent</td>
			<td>Agilent 1260</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Performance Liquid Chromatography column</td>
			<td>Agilent</td>
			<td>Poroshell EC-C18 120, 4.6 &#215; 150 mm (pore size 120 &#197;, particle size 4 &#956;m)</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td>SP Scientific</td>
			<td>Vir Tis</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Aldrich</td>
			<td>9758</td>
			<td>toxicity</td>
		</tr>
		<tr>
			<td>Microtiter plate</td>
			<td>Thermo &#956;drop plate</td>
			<td>N12391</td>
			<td></td>
		</tr>
		<tr>
			<td>Morpholine</td>
			<td>HEOWNS</td>
			<td>M99040</td>
			<td>irritant</td>
		</tr>
		<tr>
			<td>Multi-technology microplate reader</td>
			<td>Thermo</td>
			<td>VARIOSKAN LUX</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Diisopropylethylamine</td>
			<td>HEOWNS</td>
			<td>E-81416</td>
			<td>irritant</td>
		</tr>
		<tr>
			<td>N,N-Dimethyl formamide</td>
			<td>Energy</td>
			<td>B020051</td>
			<td>harmful to skin</td>
		</tr>
		<tr>
			<td>Poly-Prep column</td>
			<td>Bio-Rad</td>
			<td>7321010</td>
			<td>polypropylene chromatography columns</td>
		</tr>
		<tr>
			<td>Rink Amide MBHA resin (0.572 mmol/g)</td>
			<td>GL Biochem</td>
			<td>GLS180301-49101</td>
			<td></td>
		</tr>
		<tr>
			<td>Three-way stopcocks</td>
			<td>Bio-Rad</td>
			<td>7328107</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plate insert</td>
			<td>LABSELECT</td>
			<td>14211</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>HEOWNS</td>
			<td>T63278</td>
			<td>corrosive</td>
		</tr>
		<tr>
			<td>Triisopropylsilane</td>
			<td>HEOWNS</td>
			<td>T-0284475</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>BioFroxx</td>
			<td>1004</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum manifold</td>
			<td>Promega</td>
			<td>A7231</td>
			<td></td>
		</tr>
	</tbody>
</table>

construction of cyclic cell-penetrating peptides for enhanced penetration of biological barriers

癌症一直是全球卫生领域的一大挑战。然而，复杂的肿瘤微环境通常会限制治疗药物进入更深的肿瘤细胞，导致肿瘤复发。为了克服生物屏障的有限渗透，已经发现了具有出色膜易位能力的细胞穿透肽（CPP），并已成为将各种货物输送到细胞中的有用分子转运蛋白。然而，传统的线性CPP通常表现出蛋白水解稳定性受损，这限制了它们跨越生物屏障的渗透性。因此，开发能够穿透生物屏障并表现出增强的蛋白水解稳定性的新型分子转运蛋白是非常需要的，以促进生物医学应用中的药物递送效率。我们之前已经合成了一组具有芳香交联的短环CPP，与线性对应物相比，其在癌细胞和组织中表现出优异的渗透性。在这里，描述了用于合成荧光标记的环状精氨酸R8肽及其线性对应物的简明方案，以及研究其细胞通透性的关键步骤。

在该协议中，提出了一种将线性聚精氨酸R8限制为其环状形式的合成程序。SPPS是使用简单的设备手动进行的（图1）。SPPS的详细合成过程如图2所示。简而言之，树脂充分膨胀，然后对N α-Fmoc保护基团进行脱保护。然后，将受N α-Fmoc保护的氨基酸锚定在树脂上，直到肽组装完成（图2中的步骤1-4）。然后...

Watch this Scientific Journal Video about Construction of Cyclic Cell-Penetrating Peptides for Enhanced Penetration of Biological Barriers at JoVE.com

Construction of Cyclic Cell-Penetrating Peptides for Enhanced Penetration of Biological Barriers

该协议描述了具有芳香交联的环细胞穿透肽的合成以及它们跨生物屏障的渗透性的评估。

构建环状细胞穿透肽以增强生物屏障的穿透力

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper ...

癌症治疗的一个严重障碍是由于生理障碍，治疗药物对更深的肿瘤细胞的访问有限。因此，强烈希望开发能够治疗生物屏障的新型分子转运蛋白以提高药物递送效率。该程序的目的是合成环状细胞穿透肽以增强细胞间的渗透。该方法的优点是肽环化可以通过与半胱氨酸和树脂的取代反应轻松实现，而无需任何金属催化剂。与亲水性内酰胺或三唑交联相比，芳香交联的掺入改善了肽的整体疏水性，从而增强了其渗透性。首先，在通风橱中组装手动肽合成装置。将三通旋塞阀放在真空歧管上并将它们连接到氮气。确保盖上未使用的入口。将 10 毫升聚丙烯柱连接到三通旋塞阀上，并使用橡胶移液器灯泡或通过废物收集器真空从聚丙烯柱中排出反应混合物或溶剂。为了制备用于肽合成的树脂，将4至5毫升DMF加入所需量的树脂中，并将其转移到聚丙烯柱中，轻轻的氮气鼓泡30分钟以使树脂充分膨胀。沥干DMF，向树脂中加入4至5毫升50%吗啉/ DMF。轻轻地将氮气吹泡30分钟以除去N端Fmoc基团，然后沥干混合物。通过向色谱柱中加入4至5毫升DMF并每次用氮气冒泡至少1分钟，彻底洗涤树脂三次。以同样的方式，用二氯甲烷洗涤树脂三次，用DMF洗涤三次。接下来，将 648.8 毫克 Fmoc 和 PBF 保护的精氨酸和 372.6 毫克 HATU 溶解在离心管中的 5 毫升 DMF 中。加入348.4微升DIPEA以活化偶联反应，并将反应混合物转移到装有树脂的聚丙烯柱中。用氮气鼓泡轻轻搅拌混合物1至2小时，然后重复偶联反应一次。排干反应混合物，依次用DMF，二氯甲烷和DMF洗涤树脂三次，每次至少1分钟。去除Fmoc保护基团并按照前面显示的程序清洗树脂，然后再偶联下一个氨基酸。接下来，使用与氨基酸偶联相同的过程将β-丙氨酸偶联作为FITC标记的间隔物，然后在黑暗中将FITC，DIPEA和DMF的混合物添加到聚丙烯柱中，以对树脂上的肽进行FITC标记。反应需要将近8个小时。为了进行线性肽的环化，将三氟乙酸，三异丙基硅烷和二氯甲烷的混合物加入聚丙烯柱中2分钟，以选择性地除去半胱氨酸的三苯甲基保护基团。沥干混合物并重复该过程，直到淡黄色溶液变为无色，以完全去除三苯甲基保护基团。用DMF和二氯甲烷连续洗涤树脂至少三次，然后用DIPEA将4，4'双溴甲基联苯溶解在DMF中。将溶液加入色谱柱中并反应4小时。用4至5毫升甲醇洗涤树脂两次，每次五分钟，然后用连续的氮气流干燥。对于含有半胱氨酸的肽，用三氟乙酸、三异丙基硅烷和水或三氟乙酸、三异丙基硅烷、1，2-乙二硫醇和水的有效裂解混合物处理树脂。将肽结合树脂处理2至3小时以裂解肽，然后用氮气流小心地除去三氟乙酸。为了获得粗肽，向裂解的肽制剂中加入4至5毫升乙醚以沉淀粗肽，并以10， 000×g离心4分钟。小心地丢弃上清液，并在有效的通风橱中风干肽3分钟。将小规模粗肽溶解在800微升乙腈中，然后用反相高效液相色谱和液相色谱-质谱分析。在带有组织培养板插入物的 12 孔室中的 2 毫升 DMEM 中接种 100， 000 个 HeLa 细胞，并在含有 37% 二氧化碳的 57 摄氏度加湿培养箱中孵育 24 小时。取出培养基，并在无FBS的DMEM中用1毫升10微摩尔FITC-R8或FITC-sR8-4在室中孵育细胞1小时。之后，除去含有肽的培养基，并用1毫升PBS洗涤细胞三次。向腔室中加入 1 毫升新鲜的不含 FBS 的 DMEM，然后将 HeLa 细胞与组织培养板插入物在腔室中共同孵育，将 HeLa 细胞放在底部的圆形盖玻片上 2 小时。用2.5%戊二醛将HeLa细胞固定在圆形盖玻片上15分钟，然后用DAPI染色细胞15分钟。最后，在荧光显微镜下观察盖玻片上的HeLa细胞。这里给出了FITC标记的线性R8肽和FITC标记的吻合R8肽的合成示意图。线性R8肽和吻合R8肽的HPLC和MS谱图如图所示。吻合肽的保留时间明显长于线性类似物的保留时间，表明与疏水交联环合后肽的整体疏水性增强。该图形表示线性肽和吻合肽在25%FBS存在下的稳定性。与FBS孵育4小时后，环状R8保持77.3%的完整，而其线性对应物大部分降解，表明环状R8肽的蛋白水解稳定性增强。此处显示了与3微摩尔FITC标记的线性R8肽和FITC标记的吻合R8肽孵育1小时后HeLa细胞和4T1细胞的活细胞荧光显微镜图像。可以看出，用芳香交联环状R8处理的细胞比用线性对应物处理的细胞表现出更高的细胞内荧光。流式细胞术分析也获得了类似的结果。为了进一步研究环状R8是否赋予增强的细胞间渗透性，使用Transwell模型来模拟肽从一个细胞层到另一个细胞层的屏障通透性。环状R8明显表现出比线性R8肽更高的跨屏障穿透力，如细胞内荧光的显着增加所示。半胱氨酸和树脂的三苯甲基的完全脱保护对于随后的环化步骤很重要。此外，由于空间位阻效应，环化效率还取决于特定的序列和肽的长度。在这种情况下，使用具有较低负载能力的树脂或在溶液相中稀释浓度下环化肽将是有帮助的。与线性肽相比，这些环肽显示出增强的细胞间通透性。我们相信它们在克服生物屏障方面具有巨大的希望，并将用于分子在药物递送领域的进一步应用。

Research

JoVE Journal

Chemistry

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

保利的微波辅助功能化（乙二醇）和On-树脂链聚合反应，并形成水凝胶的肽使用

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

科研

化学

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

生物分子有序结构由短肽的自组装形成

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Preparation of Hydrophobic Metal-Organic Frameworks via Plasma Enhanced Chemical Vapor Deposition of Perfluoroalkanes for the Removal of Ammonia

增强型氟烷烃的化学气相沉积等离子通过疏水金属 - 有机骨架的制备氨的去除

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high ...

Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides

对磷酸增强富集制备高度多孔配位聚合物涂层的孔聚合物石柱的

We describe a protocol for the preparation of hybrid materials based on highly porous coordination polymer coatings on the internal surface of macroporous ...

A Filter-based Surface Enhanced Raman Spectroscopic Assay for Rapid Detection of Chemical Contaminants

基于过滤器的表面增强拉曼光谱检测方法的化学污染物快速检测

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be ...

Construction of Models for Nondestructive Prediction of Ingredient Contents in Blueberries by Near-infrared Spectroscopy Based on HPLC Measurements

基于HPLC测量通过近红外光谱对在蓝莓成分的含量无损预测模型的构建

Nondestructive prediction of ingredient contents of farm products is useful to ship and sell the products with guaranteed qualities. Here, near-infrared ...

Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level

环状聚合物的合成及其在熔融状态扩散运动的表征在单分子水平

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

洞察氨基酸和多肽与无机材料的使用相互作用的单分子力谱

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Observation and Analysis of Blinking Surface-enhanced Raman Scattering

闪烁表面增强拉曼散射的观测与分析

From a single molecule at a silver nanoaggregate junction, blinking surface-enhanced Raman scattering (SERS) is observed. Here, a protocol is presented on ...