前药染料纳米组件的简单制备和光活化
Summary
该协议描述了光响应前药染料纳米组装的制造和表征。明确描述了通过光触发拆卸从纳米颗粒释放药物的方法，包括光照射设置。光照射后从纳米颗粒释放的药物对人结直肠肿瘤细胞表现出优异的抗增殖作用。
Abstract
自组装是构建纳米级药物递送系统的一种简单而可靠的方法。光活化前药能够在光照射调节的靶位点从纳米载体中可控地释放药物。在该协议中，提出了一种通过分子自组装 制造 可光活化前药染料纳米颗粒的简便方法。详细介绍了前药合成、纳米颗粒制造、纳米组件的物理表征、光切割演示和 体外 细胞毒性验证的程序。首先合成了可光裂解的硼-二吡咯甲烷-苯丁酸氮芥（BC）前药。BC和近红外染料IR-783以优化的比例可以自组装成纳米颗粒（IR783 / BC NPs）。合成的纳米颗粒的平均尺寸为87.22 nm，表面电荷为-29.8 mV。纳米颗粒在光照射下分解，可以通过透射电子显微镜观察到。BC的光裂解在10 min内完成，苯丁酸氮芥的回收率为22%。与未辐照的纳米粒子和无辐照的BC前药相比，纳米粒子在530 nm的光照射下表现出更强的细胞毒性。该协议为光响应性药物递送系统的构建和评估提供了参考。
Introduction
化疗是一种常见的癌症治疗方法，它使用细胞毒性药物杀死癌细胞，从而抑制肿瘤生长1。然而，由于化疗药物的脱靶吸收，患者可能会遭受诸如心脏毒性和肝毒性等副作用2，3，4。因此，通过肿瘤中药物释放/活化的时空控制进行局部药物递送对于最大限度地减少正常组织中的药物暴露至关重要。
前药是化学修饰的药物，在正常组织中表现出降低的毒性，同时在激活后保留其在病变中的作用5，6。前药可以对多种刺激有反应，如pH7，8，酶9，10，超声11，12，热13和光14，15，16，并在病变中特异性释放其母体药物。然而，许多前药表现出固有的缺点，如溶解度差、吸收率不正确和早期代谢破坏，这可能会限制它们的发育17。在这种情况下，前药纳米组件的形成具有减少副作用、原位药物释放、更好的保留以及治疗和成像相结合等优点，表明这些纳米组件具有巨大的应用潜力。已经开发出许多用于疾病治疗的前药纳米组件，包括阿霉素前药纳米球、姜黄素前药胶束和喜树碱前药纳米纤维18。
在该协议中，我们提出了一种制备前药染料纳米组件的简单方法，该方法具有高前药含量，良好的水分散性，长期稳定性和灵敏的反应能力。IR783是一种水溶性近红外染料，可作为纳米组件的稳定剂19。纳米组件的另一个成分是硼 – 二吡咯甲烷 – 苯丁酸氮芥（BODIPY-Cb，BC），这是一种前药，设计有两个主要原因。由于苯丁酸氮芥（Cb） 在体内表现出全身毒性，前药形式可以降低其毒性20。BC前药可以使用针对疾病病变的530nm光照射进行光切割，从而实现Cb的局部释放。另一方面，Cb在水性环境中容易水解，可以通过将其转化为前药形式来保护21。因此，BC前药和IR-783染料的共组装有望形成稳定有效的药物递送纳米系统（图1A）。这种前药染料纳米组装提高了前药分子的分散性和稳定性，表明其在光控药物递送中的应用潜力。BC前药的光切割能够分解纳米颗粒并在病变中光控制Cb的释放（补充图1）。
Protocol
1. 硼-二吡咯甲烷-苯丁酸氮芥（BC）前药的合成（图2）22 BODIPY-OAc的合成称取1.903g2，4-二甲基吡咯，并在氮气气氛下将其溶解在圆底烧瓶中的20mL无水二氯甲烷（DCM）中。称取1.638g乙酰氧基乙酰氯，并将其滴加到溶液中。在室温下继续搅拌10分钟，然后在40°C下回流溶液1小时。 将混合物冷却至室温。称取5.170克N <e…
Representative Results
本研究采用闪速降水法成功制备了IR783/BC NPs。合成的IR783 / BC NPs呈紫色溶液，而IR783的水溶液为蓝色（图4A）。 如图4B所示，IR783/BC NPs的平均尺寸约为87.22 nm，多分散指数（PDI）为0.089，尺寸分布较窄。IR783/NPs的表面电荷约为-29.8 mV（图4C），这可能归因于IR783带负电荷的磺酸盐基团。 图4D 显示了IR783/BC NP的稳定…
Discussion
该协议概述了用于制造前药物染料纳米颗粒的简便闪蒸沉淀方法，该方法为纳米颗粒形成提供了一种简单方便的方法。此方法有几个关键步骤。首先，对于合成、制造和表征的所有步骤，微管等容器应用箔覆盖，以避免环境光对BC前药进行不必要的光切割。此外，在闪速沉淀步骤中，应将含有IR-783溶液的微管稳定地放置在涡旋混合器上，同时缓慢加入BC前药溶液。这样，BC前药溶液（在DMSO中）可以…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢香港大学李嘉诚医学院核心设施的协助。我们感谢香港大学的车志明教授提供人类HCT116细胞系。这项工作得到了刘明伟修复医学中心准会员计划和香港研究资助局（早期职业计划，第27115220号）的支持。
Materials
| 1260 Infinity II HPLC | Agilent Technologies | ||
| 2,4-Dimethyl pyrrole | J&K Scientific | 315305 | |
| 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) | Gibco | M6494 | |
| 4-Dimethylaminopyridine (4-DMAP) | J&K Scientific | 212279 | |
| 90 mm Petri Dish Clear Treated Sterile | SPL | 11090 | |
| 96-well Tissue Culture Plate Clear Treated Sterile | SPL | 30096 | |
| Acetoxyacetyl chloride | J&K Scientific | 192001 | |
| Boron trifluoride diethyl etherate | J&K Scientific | 921076 | |
| Büchner funnel | AS ONE | 3-6466-01 | |
| Chlorambucil | J&K Scientific | 321407-1G | |
| CM100 Transmission Electron Microscope | Philips | ||
| CombiFlash RF chromatography system | Teledyne ISCO | ||
| Dichloromethane | DUKSAN Pure Chemicals | JT9315-88 | |
| Dimethyl sulfoxide | DUKSAN Pure Chemicals | 2762 | |
| Disposable cuvette | Malvern Panalytical | DTS1070 | Zeta potential measurement |
| Disposable cuvette | Malvern Panalytical | ZEN0040 | |
| Empty Disposable Sample Load Cartridges | Teledyne ISCO | 693873225 | can hold up to 65 g |
| Fetal bovine serum | Gibco | 10270106 | |
| Filtering flask | AS ONE | 3-7089-03 | |
| Hexane | DUKSAN Pure Chemicals | 4198 | |
| Holey carbon film on copper grid | Beijing Zhongjingkeyi Technology Co.,Ltd | BZ10023a | |
| HPLC column (InfinityLab Poroshell 120) | Agilent Technologies | 695975-902T | |
| Integrating sphere photodiode power sensor | Thorlabs | S142C | |
| IR783 | Tokyo Chemical Industry (TCI) Co., Ltd | I1031 | |
| LED | Mightex | LCS-0530-15-11 | |
| LED Driver Control Panel V3.2.0 (Software) | Mightex | ||
| Lithium Hydroxide Anhydrous | TCI | L0225 | |
| Methylmagnesium iodide, 3M solution in diethyl ether | Aladdin | M140783 | |
| N,N-Diisopropyl ethyl amine (DIPEA) | J&K Scientific | 203402 | |
| N,N'-Dicyclohexylcarbodiimide (DCC) | J&K Scientific | 275928 | |
| penicillin–streptomycin | Gibco | 15140122 | |
| Phosphate-buffered saline (10×) | Sigma-Aldrich | P5493 | |
| Power and energy meter | Thorlabs | PM100 USB | |
| Rotavapor | BUCHI Rotavapor R300 | ||
| RMPI 1640 | Gibco | 21870076 | |
| Separatory funnel (125 mL) | Synthware | F474125L | |
| Silver Silica Gel Disposable Flash Columns, 40 g | Teledyne ISCO | 692203340 | |
| Sodium sulfate, anhydrous | Alfa Aesar | A19890 | |
| SpectraMax M4 | Molecular Devices LLC | ||
| Tetrahydrofuran (THF), anhydrous | J&K Scientific | 943616 | |
| Trypsin-EDTA (0.25%), phenol red | Gibco | 25200056 | |
| Vortex | DLAB Scientific Co., Ltd | MX-S | |
| Zetasizer Nano ZS90 | Malvern Instrument |
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Citer Cet Article
Zhang, Y., Long, K., Wang, W. Facile Preparation and Photoactivation of Prodrug-Dye Nanoassemblies. J. Vis. Exp. (192), e64677, doi:10.3791/64677 (2023).