构建环状细胞穿透肽以增强生物屏障的穿透力
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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。 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。 将 10 mL 聚丙烯色谱柱（参见 材料表）连接到三通旋塞?…
Representative Results
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…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 1,2-ethanedithiol | Aladdin | K1722093 | stench |
| 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) | HEOWNS | A-0443697 | |
| 4,4'-bis(bromomethyl)biphenyl | TCI | B1921 | |
| 4T1 cells | ATCC | 4T1 cells were cultured in DMEM medium supplemented with 10% FBS (Hyclone) in a 37 °C humidified incubator containing 5% CO2. | |
| Acetonitrile | Adamas | 1484971 | toxicity |
| Dichloromethane | Energy | W330229 | skin harmful |
| Diethyl ether | Aldrich | 673811 | flammable |
| Dimethyl sulfoxide | Beyotime | ST038 | skin harmful |
| Dulbecco’s Modified Eagle Medium (DMEM) | Gibco | ||
| Electrospray Ionization Mass Spectrometer | Waters | G2-S Tof | |
| Ethylene Diamine Tetraacetic Acid (EDTA) | BioFroxx | 1340 | |
| Fetal bovine serum (FBS) | HyClone | ||
| Flow cytometer | Beckman Coulter | CytoFLEX | |
| Fluorescein isothiocyanate isomer (FITC) | Energy | E0801812500 | |
| Fluorescent microscope | Carl Zeiss | Axio Observer 7 | |
| Fmoc-Arg(Pbf)-OH | HEOWNS | F-81070 | |
| Fmoc-Cys(Trt)-OH | GL Biochem | GLS201115-35202 | |
| Fmoc-βAla-OH | Adamas | 51341C | |
| HeLa cells | ATCC | HeLa cells were cultured in DMEM supplemented with 10% FBS (Hyclone) in a 37 °C humidified incubator containing 5% CO2. | |
| High-Performance Liquid Chromatography | Agilent | Agilent 1260 | |
| High-Performance Liquid Chromatography column | Agilent | Poroshell EC-C18 120, 4.6 × 150 mm (pore size 120 Å, particle size 4 μm) | |
| Lyophilizer | SP Scientific | Vir Tis | |
| Methanol | Aldrich | 9758 | toxicity |
| Microtiter plate | Thermo μdrop plate | N12391 | |
| Morpholine | HEOWNS | M99040 | irritant |
| Multi-technology microplate reader | Thermo | VARIOSKAN LUX | |
| N,N-Diisopropylethylamine | HEOWNS | E-81416 | irritant |
| N,N-Dimethyl formamide | Energy | B020051 | harmful to skin |
| Poly-Prep column | Bio-Rad | 7321010 | polypropylene chromatography columns |
| Rink Amide MBHA resin (0.572 mmol/g) | GL Biochem | GLS180301-49101 | |
| Three-way stopcocks | Bio-Rad | 7328107 | |
| Tissue culture plate insert | LABSELECT | 14211 | |
| Trifluoroacetic acid | HEOWNS | T63278 | corrosive |
| Triisopropylsilane | HEOWNS | T-0284475 | |
| Trypsin | BioFroxx | 1004 | |
| Vacuum manifold | Promega | A7231 |
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Cite This Article
Fang, D., Wang, R., Yu, X., Tian, Y. Construction of Cyclic Cell-Penetrating Peptides for Enhanced Penetration of Biological Barriers. J. Vis. Exp. (187), e64293, doi:10.3791/64293 (2022).