制备和表征亲脂阿霉素前药胶束
Summary
一个亲脂性的阿霉素前体药物的制备和表征协议加载1,2- distearoyl- SN -glycero -3- phosphoethanolamine- N – [氨基（聚乙二醇）-2000]（DSPE-PEG）胶束描述。
Abstract
Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous environment through self-assembly. The hydrophobic core of micelles functions as a drug reservoir and encapsulates hydrophobic drugs. The hydrophilic shell prevents the aggregation of micelles and also prolongs their systemic circulation in vivo. In this protocol, we describe a method to synthesize a doxorubicin lipophilic pro-drug, doxorubicin-palmitic acid (DOX-PA), which will enhance drug loading into micelles. A pH-sensitive hydrazone linker was used to conjugate doxorubicin with the lipid, which facilitates the release of free doxorubicin inside cancer cells. Synthesized DOX-PA was purified with a silica gel column using dichloromethane/methanol as the eluent. Purified DOX-PA was analyzed with thin layer chromatography (TLC) and 1H-Nuclear Magnetic Resonance Spectroscopy (1H-NMR). A film dispersion method was used to prepare DOX-PA loaded DSPE-PEG micelles. In addition, several methods for characterizing micelle formulations are described, including determination of DOX-PA concentration and encapsulation efficiency, measurement of particle size and distribution, and assessment of in vitro anticancer activities. This protocol provides useful information regarding the preparation and characterization of drug-loaded micelles and thus will facilitate the research and development of novel micelle-based cancer nanomedicines.
Introduction
化疗通常用于治疗各种形式的癌症。大多数，如果不是全部，化疗药物具有毒性副作用可以从管理次要条件，如恶心和腹泻而变化，更危及生命的状况。因为大多数抗癌药物的毒性，这些药物非选择性暴露于正常组织不可避免地会导致毒性。因此，为治疗性的方法，可以选择性地递送药物进入肿瘤细胞非常需要。与抗肿瘤药物的管理的另一个挑战是他们的水溶性差。一般，需要增溶剂来配制这些难溶性药物。然而，大多数的增溶剂，如二甲基亚砜（DMSO），聚氧乙烯蓖麻油，和聚山梨醇酯80（吐温80）可能引起肝，肾毒性，溶血，急性过敏反应和周围神经病变。1因此，需要对安全性和生物相容性制剂临床使用的差LY水溶性抗癌药物。纳米载体是有希望的药物递送系统用于解决上述挑战。这些纳米载体包括脂质体， 纳米粒子，3胶束，4-7聚合物药物结合物，8和无机材料。9一些纳米产品（ 如 DOXIL，Abraxane的，和Genexol）已通过监管机构来治疗癌症病人。 10
聚合物胶束是有希望的纳米级药物递送载体，其中已被成功地用于抗癌药物的输送。通过自组装过程从两亲聚合物制备4-7,11,12典型聚合物胶束。芯 – 壳结构的聚合物胶束包括亲水壳和疏水核心。亲水壳可以在空间上稳定胶束并延长其在血液中循环。疏水核可以有效地封装疏水ð地毯。因为胶束（通常小于200nm）和长循环特性的小尺寸的，聚合物胶束被认为达到肿瘤通过增强的渗透性和保留（EPR）效果（被动靶向肿瘤）定位。
载药量稳定对肿瘤靶向胶束的能力至关重要。以达到最佳的肿瘤定位，微胶粒应具有最小的药物泄漏在到达肿瘤部位之前，尚未进入癌细胞后迅速释放药物。此外，制剂稳定性也是产品开发的一项基本要求，因为制剂稳定性决定产品开发的可行性，以及开发的产品的保质期。最近，许多努力已经取得了改善的药物装载到递送载体。所述亲脂性前药的方法是已被探索，以改善药物加载到脂质纳米颗粒和乳剂的策略。13,14的连词用药物脂质ugation可以显著改善他们的亲脂性，提高装载和固定在纳米载体的亲脂成分。
在这里，我们描述了准备亲脂阿霉素前药胶束的协议。首先，对于阿霉素亲脂性前药的合成方法进行说明。然后，被引入用于产生与一个薄膜分散法胶束的协议。这种方法已在我们以前的研究已成功地用于5 DSPE-PEG，被选定为，因为它已被成功地用于胶束药物递送制备胶束载体材料15,16最后，我们描述了用于表征胶束几个体外测定制剂，并评价抗癌活性。
Protocol
Representative Results
Discussion
在这项工作中，我们描述了微胶粒的制备的简单，快速的膜分散法。这种方法利用一个两亲性聚合物的自组装特性（ 如 DSPE-PEG）以形成在含水环境中的核-壳结构的胶束。此胶束制备方法具有几个优点。 1.它涉及一个简单配制方法，它避免了使用脂质体，纳米颗粒和纳米乳剂。19 2的制备常用复杂小型化步骤（如挤出或均化）。它具有良好的重复性。一旦该制剂进行了优化，并建立?…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported by the following grants: NIH-SC3 grant, NSF-PREM grant, Hampton University Faculty Research Grant. We would like to thank Mrs. Michele A. Cochran at Virginia Institute of Marine Science (VIMS) for the use of the particle size analyzer. We would also like to thank Mrs. Corinne R. Ramaley for reviewing the manuscript.
Materials
| DSPE-PEG2K | Cordenpharm | LP-R4-039 | >95% |
| Doxorubicin | LC Laboratories | D-4000 | >99% |
| Palmitic Acid Hydrazide | TCI AMERICA | P000425G | >98.0% |
| Methanol | ACROS Organics | 610981000 | Anhydrous |
| Methylene chloride | FISHER | D151-4 | 99.90% |
| Methyl sulfoxide-d6 | ACROS Organics | AC320760075 | NMR solvent |
| Trifluoroacetic Acid | ACROS Organics | AC293811000 | 99.50% |
| Silica Gel | FISHER | L-7446 | 230-400 mesh |
| BAKER FLEX TLC PLATES | FISHER | NC9990129 | |
| DPBS | Sigma-Aldrich | D8537 | |
| DU 145 Prostate Cancer Cells | ATCC | HTB-81 | |
| MTT | ACROS Organics | 158990050 | 98% |
| RPMI 1640 Medium | MEDIATECH INC | 10041CV | |
| Antibiotic-Antimycotic | LIFE TECHNOLOGIES | 15240062 | 100x stock solution |
| Fetal Bovine Serum | LIFE TECHNOLOGIES | 10437077 | |
| Nuclear Magnetic Resonance Spectroscopy | Varian, Inc | 300 NMR | |
| Büchi R-3 Rotavapor | Buchi | 1103022V1 | Rotary evaporator |
| Ultrasonic Bath | BRANSON ULTRASONICS CORPORATION | CPX952318R | |
| UV-VIS spectrometer Biomate 3 | Thermo Spectronic | ||
| Zetasizer Nano ZS90 | Malvern Instruments | Particle Size Analyer | |
| Microplate Spectrophotometer | Rio-Rad | Benchmark Plus | |
| Cell Culture Incubator | Napco | CO2 6000 | |
| Biological Safety Cabinet | Nuaire | ||
| SigmaPlot | Systat Software, Inc. | Analytical Software | |
| 96-Well Cell Culture Plate | Becton Dickinson | 353072 | |
| Trypsin 0.25% | Corning Cellgro | 25-053-CI |
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