合成阿普塔默-PEI-g-PEG改性金纳米粒子装载多索鲁比辛用于靶向药物输送
Summary
在此协议中，多索鲁比辛加载 AS1411-g-PEI-g-PEG 改性金纳米粒子通过三步阿米内反应合成。然后，多索鲁比辛被加载并交付给靶向癌细胞进行癌症治疗。
Abstract
由于健康细胞的耐药性和毒性，多索鲁比辛（DOX）在临床癌症治疗中的使用受到限制。该协议描述了用聚乙二醇（PEI-g-PEG）二甘醇（PEI-g-PEG）共聚聚（乙二烯氨酸）嫁接的聚（乙二烯氨酸）与加载的阿普塔默（AS1411）和DOX通过阿米特反应进行功能化金纳米粒子（AuNPs）的设计。AS1411与癌细胞上的靶向核素受体特别结合，使DOX针对的是癌细胞而不是健康细胞。首先，PEG 是盒装的，然后嫁接到分支 PEI 以获得 PEI-g-PEG 聚合物，这一点由 1H NMR 分析确认。其次，PEI-g-PEG copolymer 涂层金纳米粒子 （PEI-g-PEG@AuNPs） 进行合成，DOX 和 AS1411 通过阿米德反应逐渐与 AuNP 粘合。制备的 AS1411-g-DOX-g-PEI-g-PEG@AuNPs直径为 39.9 nm，Zeta 电位为 -29.3 mV，表明纳米粒子在水和细胞介质中是稳定的。细胞毒性检测表明，新设计的DOX加载的AuNPs能够杀死癌细胞（A549）。这种合成展示了PEI-g-PEG共聚物、贴花和DOX在AuNP上的微妙排列，这些排列是通过连续的阿米德反应实现的。这种阿普塔默-PEI-g-PEG功能化的 AuNP 为癌症治疗中的靶向药物输送提供了一个很有前途的平台。
Introduction
癌症是世界性的主要公共卫生问题，其广泛特征是治愈率低、复发率高、死亡率高。目前传统的抗癌方法包括手术、化疗和放疗3种，其中化疗是4号诊所癌症患者的主要治疗方法。临床使用的抗癌药物主要包括帕利塔塞尔（PTX）5和多索鲁比辛（DOX）6，7。DOX，一种抗肿瘤药物，由于癌症细胞毒性和抑制癌细胞增殖8，9的优点，已广泛应用于临床化疗。然而，DOX导致心毒性10，11，和DOX的短半生限制其应用在诊所12。因此，需要降解的药物携带者以可控的方式将 DOX 加载并以子等方式释放到目标区域。
纳米粒子已广泛应用于靶向药物输送系统，在癌症治疗方面具有若干优势（即表面与体积比大、体积小、封装各种药物的能力、可调谐表面化学等）。13，14，15.特别是金纳米粒子（AuNPs）已广泛应用于生物和生物医学应用，如光热癌治疗16，17。AuNPs的独特特性，如简体合成和一般表面功能化，在癌症治疗18的临床领域具有良好的前景。此外，AuNP已经被用来识别药物输送策略，诊断肿瘤，并克服阻力在许多研究19，20。
尽管如此，AuNP 需要进一步定制，通过增强渗透和保留 （EPR）（如靶向和辅助功能）在肿瘤病变时高局部释放来克服耐药性。聚合物功能化 AuNP 具有独特的优势，如疏水抗癌药物水溶解性提高，循环时间延长21、22。各种生物相容聚合物已用于 AuNP 涂料，如聚乙二醇 （PEG）、聚乙烯胺 （PEI）、透明质酸、肝素和黄豆胶。然后，AuNP的稳定性和有效载荷得到很好的改善。具体来说，PEI是一种高度分支的聚合物，由许多重复单位的初级，二级和三级胺24组成。PEI 具有极好的溶解度、低粘度和高功能性，适合涂在 AuNPs 上。
另一方面，抗癌药物需要直接输送到癌细胞，提高负荷效率，降低毒性，治疗原发性肿瘤和晚期转移性肿瘤25。靶向配体具有巨大的潜力，抗癌药物靶向输送系统26。其靶分子结合的选择性赋予抗癌药物针对特异性，并增加药物在病组织中的丰富性27。更多的配体包括抗体、多肽和小分子。与其他配体相比，核酸贴合剂可以在体外合成，易于修改。AS1411是一种未经改性26b磷化物寡核苷酸，形成稳定的二元G-四聚氰胺结构，专门与癌细胞28、29、30上过度表达的目标核蛋白受体结合。AS1411抑制许多癌细胞的增殖，但不影响健康细胞31，32的生长。因此，AS1411 被用来制造理想的有针对性的药物输送系统。
在这项研究中，PEI-g-PEG共聚物通过阿米德反应合成，然后制造PEI-g-PEG共聚物涂层金纳米粒子（PEI-g-PEG@AuNPs）。此外，DOX 和 AS1411 与准备好的 PEI-g-PEG@AuNPs有顺序链接，如 图 1所示。此详细的协议旨在帮助研究人员避免与制造装有 DOX 和 AS1411 的新 PEI-g-PEG@AuNPs相关的许多常见陷阱。
Protocol
注意：在使用所有化学品之前，请务必查阅所有相关材料安全数据表 （MSDS）。用于制备共聚物和纳米粒子的几种化学物质具有剧毒性。纳米粒子也有潜在的危害。确保使用所有适当的安全做法和个人防护设备，包括手套、实验室外套、头罩、全长裤子和近身鞋。 1. 双碳水化合物聚乙二醇（CT-PEG）合成33 将 1 . 46 克（ 14 . 6 mmol ）的氢化物（ SA ）和 209…
Representative Results
1H NMR光谱学用于确认CT-PEG聚合物和PEI-g-PEG共聚合物（图2）的成功合成。图2a显示，δ的甲基质子信号=3.61ppm，δ=2.57ppm的纸箱质子信号确认CT-PEG聚合物的成功合成。图2b显示PEG的乙烯质子信号在δ=2.6ppm，PEI的质子信号在δ=1.66ppm确认PEI-g-PEG共聚物的合成。 紫外线光谱学的进行，…
Discussion
1H NMR光谱（图2）确认CT-PEG共聚合体和PEI-g-PEG共聚合体的成功合成。PEG和PEI的分子量分别为1，000和1，200。此外，EDC/NHS催化系统还用于通过阿米德反应合成PEI-g-PEG共聚物。需要注意的是，如果PEG和PEI的分子量因合成PEI-g-PEG共聚物而发生变化，则需要重新评估反应时间和催化系统。此外，需要进一步调整AUNPs上PEI-g-PEG共聚合体涂层的反应条件，主要是因为PEG-g-PEI聚合体?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究由中国国家自然科学基金委员会（31700840）资助：河南省重点科研项目（18B430013，18A150049）。这项研究得到了南湖青年学者项目的支持。作者要感谢来自XYNU生命科学学院的本科生曲泽波的乐于助人的作品。作者希望确认XYNU的分析和测试中心使用他们的设备。
Materials
| 4-Dimethylaminopyridine | Macklin | D807273 | |
| A549 cell | ATCC CCL-185TM | ||
| AS1411 | BBI Life Sciences Corporation | 5'-d (TTTGGTGGTGGTGGTTGTGGTGGTGGTGG) FL-AS1411 (fluorophore-labeled AS1411) | |
| Anhydrous Tetrahydrofuran (THF) | SinoPharm Chemical Reagent Co., Ltd | ||
| Cell counting kit-8 (CCK-8) | Sigma Aldrich | 96992-500TESTS-F | |
| Dichloromethane | Traditional Chinese medicine | 80047318 | |
| Diethyl ether (Et2O) | SinoPharm Chemical Reagent Co., Ltd | ||
| Dimethyl sulfoxide | Macklin | D806645 | |
| Dulbecco's modified Eagle's medium (DMEM) | Sigma Aldrich | ||
| Doxorubicin hydrochloride | Rhawn | R017518 | |
| Ether absolute | Traditional Chinese medicine | 80059618 | |
| Field Emission Transmission Electron Microscope | FEI Company | Tecnai G2 F 20 | |
| Gold(III) chloride trihydrate | Rhawn | R016035 | |
| Laser Particle-size Instrument | Malvern Instruments Ltd | ZetasizerNanoZS/Masterszer3000E | |
| Microplate Reader | Molecular Devices | SpectraMax 190 | |
| N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride | Macklin | N808856 | |
| N-Hydroxysuccinimide | Macklin | H6231 | |
| NMR software | Delta 5.2.1 | ||
| Nuclear Magnetic Resonance Spectrometer | JEOL | JNM-ECZ600R/S3 | |
| Origin 8.5 | OriginLab | ||
| Penicillin | Sigma Aldrich | V900929-100ML | |
| Phosphate-buffered saline | Sigma Aldrich | P4417-100TAB | |
| Poly(ethylene glycol) | Sigma Aldrich | 81188 | BioUltra, average Mn ~ 1000 |
| Poly (ethyleneimine) solution | Sigma Aldrich | 482595 | average Mn ~ 1200, 50 wt.% in H2O |
| Sodium borohydride, powder | Acros | C18930 | |
| Streptomycin | Sigma Aldrich | 85886-10ML | |
| Succinic anhydride | Traditional Chinese medicine | 30171826 | |
| Tetrahydrofuran | Traditional Chinese medicine | 40058161 | |
| Triethylamine | Traditional Chinese medicine | 80134318 | |
| UV/VIS/NIR Spectrometer | Lambda950 | Lambda950 | |
| X-ray Photoelectron Spectrometer | Thermo Fisher Scientific | K-ALPHA 0.5EV |
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Cite This Article
Nie, L., Sun, S., Sun, M., Zhou, Q., Zhang, Z., Zheng, L., Wang, L. Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery. J. Vis. Exp. (160), e61139, doi:10.3791/61139 (2020).