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.

1. DOX-PA的合成<ol><li>称量390毫克的阿霉素和243毫克棕榈酸酰肼，并转移到一个圆底烧瓶中。 </li><li>添加150毫升无水甲醇中，用玻璃注射器烧瓶中。添加39微升三氟乙酸（TFA）的用移液管。用磁力搅拌器，搅拌在RT在黑暗中18小时，将反应混合物。 注意:反应材料的量可被放大或缩小，以获得不同量的DOX的功率放大器。反应物的比例应在相同的比例被维持。使用DOX量在78毫克至1,170毫克的范围内的反应可在常规的化学实验室中进行。 </li><li>用硅胶柱DOX-PA的纯化。17 <ol><li>用旋转蒸发器除去在反应混合物中的溶剂。加入3克硅胶的混合物的体积减少到大约20之后ml。继续旋转蒸发，得到干的粉末，并允许将T他吸附产品走上硅胶。保持在真空下将样品另外的30分钟，形成了干燥粉末后。 </li><li>包50克硅胶成使用二氯甲烷作为溶剂的柱。小心加入含吸附水的产品到柱硅胶样本。 </li><li>洗脱用二氯甲烷和甲醇的混合物的塔，同时逐渐增加甲醇的比例，从而增加溶剂极性（ 表1）。 </li><li>收集在试管洗脱液级分（25毫升/管）并监测通过薄层色谱（TLC）的进展。 </li><li>结合含有纯DOX-PA的所有级分，并用旋转蒸发器，直至形成干粉除去溶剂。进一步干燥真空O / N下的产物。 </li></ol></li><li>分析DOX-PA通过TLC的。 <ol><li>切TLC板的4cm的×8cm的部分。从在用TLC点样毛细管板的底部斑点样品溶液0.5厘米使用满足hanol作为溶剂。 </li><li>放置TLC板到含有二氯甲烷和甲醇的混合物中的显影腔室（3/1，体积/体积）。的溶剂中的深度应只是比0.5厘米以下。 </li><li>从显影腔移除所述板当溶剂前沿到达板的顶部。标记溶剂前沿用铅笔的位置，并允许所述板干燥。放置TLC板到含有以显现样品饱和碘蒸气染色室中。 </li></ol></li><li> 1 H-核磁共振光谱（1H-NMR）DOX-PA分析。18 <ol><li>溶解15mg的DOX的PA在1ml甲基亚砜-D6（DMSO）中的和样品转移到一个NMR管中。 </li><li>将NMR管进入核磁共振仪器的磁铁。测量质子光谱，选择DMSO作为溶剂。从磁体取出NMR管。分析核磁共振结果18。 </li></ol></li></ol> 2。 DOX-PA胶束由薄膜分散法制备<ol><li>溶解DSPE-PEG（40毫克）和DOX-PA（4毫克）在10毫升玻璃小瓶2ml甲醇。 </li><li>使用旋转蒸发，直到在小瓶的薄膜形成真空下除去有机溶剂。 注意:可替换地，蒸发的有机溶剂在惰性气体下（ 例如 ，氩气或氮气），以形成薄膜，并保持小瓶在真空干燥器以进一步除去残留的溶剂。 </li><li>转印2毫升Dulbecco氏磷酸盐缓冲盐水（pH值7.4，DPBS）的玻璃小瓶中。 </li><li>放置在小瓶在超声浴中3分钟在室温下，以产生胶束。 注:超声波浴的不同型号之间的超声波功率变化。选择能产生足够的超声功率驱散薄的聚合物/药物薄膜的单位。在这个协议中使用超声波浴的输出功率为110 W. </li><li>保持胶束在4℃下短期储存和-20℃下长期-term存储。 注意:可替换地，微胶粒还可以是冷冻干燥并在使用前用水重构。通常情况下，需要对这一提法没有冷冻保护剂或冻干保护剂。 </li></ol> 3. DOX-PA胶束的表征<ol><li> DOX-PA浓度的胶束和药物包封率的测定 <ol><li> 1微克/毫升，5微克/毫升，20微克/毫升，50微克/毫升和100微克/毫升:溶解DOX-PA在DMSO中前面的步骤，制备了五种不同浓度的DOX-PA的溶液合成。测量DOX的PA解决方案的吸收与在490nm处的UV-VIS光谱仪。基于所述DOX-PA的药物浓度和在490nm处的相应吸收率的标准曲线。 </li><li>稀释25微升载药胶束的500微升DMSO的。测量在490nm处用UV-VIS分光计的吸收。计算药物浓度与3.1.1中产生的标准曲线。 </li><li>使用以下等式计算包封效率: 药物包封率（％）=（在胶束药物量）/（加入的药物量）×100％ </li></ol></li><li> 粒径用动态光散射表征（DLS） <ol><li>稀释用DPBS（pH 7.4）中的胶束为1毫克/毫升的最终的DSPE-PEG浓度。分析2ml样品用粒度分析仪得到的Z均尺寸和多分散指数（PDI）。 </li></ol></li><li> 体外抗癌活性的评价 注意:使用适当的无菌技术和生物安全柜的内部运作。 <ol><li>从细胞培养烧瓶中取出细胞培养基（ 例如，T25）含有DU-145人前列腺癌细胞并用2ml的DPBS（pH7.4）中冲洗细胞。 </li><li>抽吸DPBS，加入1毫升胰蛋白酶溶液（0.25％），并在37℃孵育2分钟以分离细胞。 </li><li>添加10米细胞培养基的L（RPMI 1640 + 10％胎牛血清+ 1％抗菌 - 抗霉菌），当大多数细胞从烧瓶脱落。转移细胞到15毫升离心管中并离心将细胞以1000 xg离心5分钟。 </li><li>重悬用5ml细胞培养基的细胞沉淀并除去的样​​品用于与血球计数细胞数。稀释用细胞培养基将细胞以50,000细胞/ ml的密度。添加稀释的细胞悬液到96孔细胞培养板（100μl/孔）。孵育在细胞培养孵化器中的细胞（37℃，5％CO 2）18 ​​小时，以允许细胞附着。 </li><li>稀DOX二甲基亚砜（DMSO）溶液和DOX-PA DMSO溶液用细胞培养基分别以获得0.1微米的最终药物浓度，0.5μM，2微米，5微米和10微米。保持最终DMSO浓度以上所有的样品中至0.5％。稀DOX-PA胶束用细胞培养基以得到最终的药物共0.1μMncentrations，分别为0.5微米，2微米，5微米和10微米。使用空白细胞培养液控制。 </li><li>从培养箱中取出96孔细胞培养板，并用100微升培养基含有在步骤3.3.5制备（n = 4时为每个组）不同处理剂更换细胞培养基。孵育在细胞培养孵化器中的细胞（37℃，5％CO 2）的额外72小时。 </li><li>吸出培养基并加入100μl的培养基中含有0.5mg / ml的3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑鎓溴化物（MTT）。 </li><li>孵育额外的2小时的细胞培养孵化细胞。小心取出介质，并添加100微升DMSO来溶解甲晶体。 </li><li>在570纳米的波长和670nm的参考波长测定用微孔板分光光度计吸光度。 </li><li>使用下列公式计算细胞存活率: <strong&gt;的细胞活力（％）=（A 测试 / 控制 ）×100％ 注意:使用方差分析（ANOVA）统计检验的单向分析比较不同组之间的细胞生存力。基于细胞生存力相对于药物浓度的数据计算IC 50。 </li></ol></li></ol>

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The authors have nothing to disclose.

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，可以实现优异的批与批之间的一致性。该协议是耐受在制备过程变量的变化，如超声处理的强度和时间。尽管其它方法（如透析法或乳液聚合的溶剂蒸发的方法）可用于制备胶束，膜分散方法是更加方便快捷。因此，它具有适于在制药工业中的大型制造胶束使用的巨大潜力。该方法的限制在于它依赖于载体材料的自组装特性，因此被限制为具有这样的特性的材料。高分子材料的不当选择可以导致失败在产生令人满意的胶束制剂。此外，该制备方法依赖于超声分散聚合物/药物膜，并促进胶束的形成。大多数市售因为只需要一个弱超声波功率形成胶束可用超声浴是合适的。但是，如果使用具有非常弱的功率的超声波浴中它可能是一个问题。 普药量和过早的药物释放是胶束给药重大关切。在这个协议中，我们开发了一种亲脂性药物前体策略，提高负荷ING阿霉素进入DSPE-PEG胶束。与多柔比星脂质的缀合显著改善与DSPE-PEG胶束的脂质核心药物兼容性和互动。的前药，DOX-PA，被通过的酸性pH响应腙连接体缀合脂质阿霉素合成。此接头是在酸性pH值在中性pH下是稳定和可切割的。20,21因此，DOX被稳定地在DSPE-PEG胶束加载在中性pH前药，具有在储存期间和在血液循环中最小的药物泄漏。一旦DOX-PA的胶束通过内吞作用进入肿瘤细胞，DOX-PA前药将被切割，并且响应于酸性核内体环境（pH为5-6）转化成游离DOX。由于DOX比DOX-PA更具亲水性，这种转换会扰乱DOX和胶束的疏水核心之间的相互作用，从而有利于DOX从胶束的快速释放。这种创新的设计特点将避免不完全的药物释放，这与使用不可切割或缓慢裂解连接子-药物偶联物相关的主要问题。22这种方法也可应用到其他药物的输送。合适的连接体的选择是对这种方法的成功是至关重要的。的接头应该是稳定的在生理环境中，以最大限度地提高制剂的稳定性。所述接头也应响应被有效地裂解在肿瘤微环境（ 如，pH值， 酶等 ）以释放母体药物触发。 在这个协议中，DOX-PA被有效装入具有高包封率（〜100％）胶束。胶束制剂也表现出优异的稳定性和无药物沉淀或减少在药物浓度为至少三周保持完整。这是由于脂质部分之间的前药和DSPE-PEG胶束的脂质核心增强的相互作用。工程的兼容耗材用药物，以提高它们的相互作用的载体分子的lity是用于提高微胶粒和其他纳米载体的性能有前途的策略。这种方法可以提高药物负载和最小化这些纳米载体预成熟释放药物23的适当的药物/聚合物对的选择是在设计胶束制剂的关键步骤。药物分子的结构可以修改（前药的方法）或载体材料的设计可以提高相容性，并提高药物/载体相互作用得到优化。23 24此外，计算建模可以协助一个有用的工具在纳米载体的和最优化使得能够制备特定的特定药剂的量身设计纳米载体。 总之，我们在这里描述，以合成多柔比星的一个亲脂性前药的方法。药物胶束的制备和表征协议也是DESCRIBED。薄膜分散方法是一种制备多种纳米尺寸自组装递送系统的一个简单的和有希望的方法。这里所描述的表征方法可在体外试验被用来作为标准来确定纳米医学的属性，从 ​​而可以促进制定和产品开发流程的优化。

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。

<table><tbody><tr><td>DSPE-PEG2K</td><td>Cordenpharm</td><td>LP-R4-039</td><td>&gt;95%</td></tr><tr><td>Doxorubicin</td><td>LC Laboratories</td><td>D-4000</td><td>&gt;99%</td></tr><tr><td>Palmitic Acid Hydrazide</td><td>TCI AMERICA&amp;nbsp;&amp;nbsp;</td><td>P000425G</td><td>&gt;98.0%</td></tr><tr><td>Methanol</td><td>ACROS Organics</td><td>610981000</td><td>Anhydrous</td></tr><tr><td>Methylene chloride&amp;nbsp;</td><td>FISHER&amp;nbsp;</td><td>D151-4</td><td>99.90%</td></tr><tr><td>Methyl sulfoxide-d&lt;sub&gt;6&lt;/sub&gt;</td><td>ACROS Organics</td><td>AC320760075</td><td>NMR solvent</td></tr><tr><td>Trifluoroacetic Acid&amp;nbsp;</td><td>ACROS Organics</td><td>AC293811000</td><td>99.50%</td></tr><tr><td>Silica Gel</td><td>FISHER&amp;nbsp;</td><td>L-7446</td><td>230-400 mesh</td></tr><tr><td>Baker Flex TLC Plates</td><td>FISHER&amp;nbsp;</td><td>NC9990129</td><td></td></tr><tr><td>DPBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>DU 145&amp;nbsp; Prostate Cancer Cells</td><td>ATCC</td><td>HTB-81</td><td></td></tr><tr><td>MTT</td><td>ACROS Organics</td><td>158990050</td><td>98%</td></tr><tr><td>RPMI 1640 Medium</td><td>MEDIATECH INC&amp;nbsp;</td><td>10041CV</td><td></td></tr><tr><td>Antibiotic-Antimycotic&amp;nbsp;</td><td>LIFE TECHNOLOGIES&amp;nbsp;</td><td>15240062</td><td>100x stock solution</td></tr><tr><td>Fetal Bovine Serum</td><td>LIFE TECHNOLOGIES&amp;nbsp;</td><td>10437077</td><td></td></tr><tr><td>Nuclear Magnetic Resonance Spectroscopy</td><td>Varian, Inc</td><td></td><td>300 NMR&amp;nbsp;</td></tr><tr><td>B&amp;uuml;chi R-3 Rotavapor</td><td>Buchi</td><td>1103022V1&amp;nbsp;</td><td>Rotary evaporator</td></tr><tr><td>Ultrasonic Bath</td><td>BRANSON ULTRASONICS CORPORATION&amp;nbsp;</td><td>CPX952318R</td><td></td></tr><tr><td>UV-VIS spectrometer Biomate 3</td><td>Thermo Spectronic</td><td></td><td></td></tr><tr><td>Zetasizer Nano ZS90&amp;nbsp;</td><td>Malvern Instruments</td><td></td><td>Particle Size Analyer</td></tr><tr><td>Microplate Spectrophotometer&amp;nbsp;</td><td>Rio-Rad</td><td>Benchmark Plus&amp;nbsp;</td><td></td></tr><tr><td>Cell Culture Incubator</td><td>Napco</td><td>CO2 6000</td><td></td></tr><tr><td>Biological Safety Cabinet</td><td>Nuaire</td><td></td><td></td></tr><tr><td>SigmaPlot&amp;nbsp;</td><td>Systat Software, Inc.</td><td></td><td>Analytical Software</td></tr><tr><td>96-Well Cell Culture Plate</td><td>Becton Dickinson</td><td>353072</td><td></td></tr><tr><td>Trypsin&amp;nbsp; 0.25%</td><td>Corning Cellgro</td><td>25-053-CI</td><td></td></tr></tbody></table>

preparation and characterization of lipophilic doxorubicin pro-drug micelles

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.

图1示出DOX的PA的合成方案。 DOX-PA用棕榈酸的偶联通过pH敏感腙键阿霉素合成。稍微过量的棕榈酸酰肼的使用，以促进该反应的完成。此反应方法具有非常高的效率，仅阿霉素的少量残留的18小时的反应（ 图2）之后。产率是大约88％。在反应结束时，DOX-PA用硅胶柱纯化，干燥，得到纯的红色固体产物。 DOX-PA用薄层色谱法，这表明对纯化的DOX的PA（ 图2）的单个点进行分析。 图3示出了DOX-PA的1 H-NMR谱。观察来自阿霉素和棕榈酸的特征NMR峰，这进一步证实了偶联反应的成功。 图4。空白DSPE-PEG胶束未经药物显示为透明的液体。 DOX-PA DSPE-PEG胶束显示为红色液体;红色是由于胶束加载的DOX的PA中。粒度分析的代表性结果示于图5。的Z均平均粒度为空白的DSPE-PEG胶束为17.0±0.5纳米（PDI = 0.034±0.019）。 DOX-PA的负荷略有增加胶束粒度; Z均平均粒度为DOX-PA的DSPE-PEG胶束为25.7±1.6纳米（PDI = 0.407±0.035）。 基于在490nm处的吸收测定在胶束制剂中的DOX的PA浓度。 DSPE-PEG具有在该波长可忽略的吸收，从而在DOX-PA浓度的分析不干扰。在胶束制剂中的DOX的PA浓度为1.99±0.11毫克/毫升和药物装载效率为99.3±5.7％。高包封效率是由于DOX-PA，增强其在胶束的疏水核心保留的亲脂性特征。该制剂表现出优良的稳定性。 4℃3周 - 当储存在没有可见的沉淀。在储存期间没有观察到药物浓度没有显著变化。 DU-145人前列腺癌的细胞用不同浓度的游离的阿霉素，游离DOX-PA和DOX-PA DSPE-PEG胶束处理。细胞存活率在使用MTT测定（ 图6）72小时处理结束确定。在细胞活力浓度依赖性减少通过用游离阿霉素（IC 50 = 0.10微米），游离DOX-PA（IC 50 = 0.33微米），或DOX-PA胶束（IC 50 = 0.25μM）处理DU145细胞来实现的。虽然免费阿霉素是<html>比游离DOX的PA或在较低浓度DOX-PA胶束（0.1μM和0.5μM）更有效，DOX-PA基团表现出细胞存活率比在较高浓度（2-10微米）自由阿霉素更大的减少。还有，似乎是在更高和更低浓度的DOX的PA和DOX-PA胶束之间没有显著差异。空白DSPE-PEG胶束显示没有毒性（数据未显示），这表明良好的生物相容性和DSPE-PEG的安全作为药物递送载体的材料。 <img alt="图1" src="/files/ftp_upload/54338/54338fig1.jpg" /> 阿霉素的亲脂性前药（DOX-PA）图1.合成试剂和条件:（PA）三氟乙酸（TFA）溶于甲醇阿霉素（DOX），棕榈酸酰肼，并在搅拌18小时在RT黑暗。<html> T ="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54338/54338fig2.jpg" /> 图2薄层色谱（TLC）。（1）多柔比星的薄层，（2）原料的反应混合物，（3）棕榈酸酰肼，以及（4）DOX-PA的缀合物。样品用二氯甲烷和甲醇的混合物开发（3/1，V / V），并用碘染色。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54338/54338fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54338/54338fig3.jpg" /> 图3. 1 DOX-PA的在氘代DMSO中H-NMR特征峰的来自DOX和PA的存在证明了偶联反应的成功。://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54338/54338fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54338/54338fig4.jpg" /> 图4. 外观胶束。（A）DSPE-PEG胶束和（B）DOX-PA DSPE-PEG胶束。代表性的人物都表现出空白DSPE-PEG胶束和DOX-PA DSPE-PEG胶束的外观。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54338/54338fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54338/54338fig5.jpg" /> 图5. 粒度和胶束粒度分布:（A）DSPE-PEG胶束和（B）DOX<html> -PA DSPE-PEG胶束。胶束尺寸通过动态光散射来确定。代表性的人物呈现展示颗粒尺寸和尺寸分布。提出的数据是平均值±SD（N = 3）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54338/54338fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54338/54338fig6.jpg" /> 图6. 胶束制剂的抗癌活性。DU145细胞72小时和细胞活力处理使用MTT测定法测定的。在图中给出的数据是平均值±标准差（n = 4）。 *，P &lt;0.01，相比于DOX治疗组。有治疗组DOX-PA和DOX-PA胶束差异无统计学意义。集成电路50是基于细胞生存力相对于药物浓度的数据计算出来的。p_upload / 54338 / 54338fig6large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="表格1" src="/files/ftp_upload/54338/54338table1.jpg" /> 表1 的 CH 2 Cl 2 / CH 3 OH洗脱，DOX的PA的纯化中使用的溶剂。

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Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

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。

制备和表征亲脂阿霉素前药胶束

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous ...

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14.8K 次观看.  Hampton University. 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。 

视频: Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

可逆二硫键交联胶束生产的简便和有效途径

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

科研

生物化学

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its therapeutic benefit. Nanoparticle-based drug combination has been shown to mitigate the problems encountered by free drug combination therapy. Our previous studies have shown that the combination of two anticancer drugs, doxorubicin (DOX) and mitomycin C (MMC), produced a synergistic effect against both murine and human breast cancer cells in vitro. DOX and MMC co-loaded polymer-lipid hybrid nanoparticles (DMPLN) bypassed various efflux transporter pumps that confer multidrug resistance and demonstrated enhanced efficacy in breast tumor models. Compared to conventional solution forms, such superior efficacy of DMPLN was attributed to the synchronized pharmacokinetics of DOX and MMC and increased intracellular drug bioavailability within tumor cells enabled by the nanocarrier PLN. 
To evaluate the pharmacokinetics and bio-distribution of co-administered DOX and MMC in both free solution and nanoparticle forms, a simple and efficient multi-drug analysis method using reverse-phase high performance liquid chromatography (HPLC) was developed. In contrast to previously reported methods that analyzed DOX or MMC individually in the plasma, this new HPLC method is able to simultaneously quantitate DOX, MMC and a major cardio-toxic DOX metabolite, doxorubicinol (DOXol), in various biological matrices (e.g., whole blood, breast tumor, and heart). A dual fluorescent and ultraviolet absorbent probe 4-methylumbelliferone (4-MU) was used as an internal standard (I.S.) for one-step detection of multiple drug analysis with different detection wavelengths. This method was successfully applied to determine the concentrations of DOX and MMC delivered by both nanoparticle and solution approaches in whole blood and various tissues in an orthotopic breast tumor murine model. The analytical method presented is a useful tool for pre-clinical analysis of nanoparticle-based delivery of drug combinations.

This protocol describes an efficient and convenient analytical process of sample extraction and simultaneous determination of multiple drugs, doxorubicin (DOX), mitomycin C (MMC) and a cardio-toxic DOX metabolite, doxorubicinol (DOXol), in the biological samples from a preclinical breast tumor model treated with nanoparticle formulations of synergistic drug combination.

纳米颗粒对荷瘤小鼠药相结合的样品提取及同时色谱定量分析

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its ...

癌症研究

Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar concentration forming hydrophobic cores surrounded by hydrophilic shells in aqueous environments. The core of these micelles can be utilized to load hydrophobic, poorly water soluble drugs like docetaxel (DTX) and everolimus (EVR). Systematic characterization of the micelle structure and drug loading capabilities are important before in vitro and in vivo studies can be conducted. The goal of the protocol described herein is to provide the necessary characterization steps to achieve standardized micellar products. DTX and EVR have intrinsic solubilities of 1.9 and 9.6 &#181;g/ml respectively Preparation of these micelles can be achieved through solvent casting which increases the aqueous solubility of DTX and EVR to 1.86 and 1.85 mg/ml, respectively. Drug stability in micelles evaluated at room temperature over 48 hr indicates that 97% or more of the drugs are retained in solution. Micelle size was assessed using dynamic light scattering and indicated that the size of these micelles was below 50 nm and depended on the molecular weight of the polymer. Drug release from the micelles was assessed using dialysis under sink conditions at pH 7.4 at 37 oC over 48 hr. Curve fitting results indicate that drug release is driven by a first order process indicating that it is diffusion driven.

The goal of this protocol is to describe the preparation and characterization of physically entrapped, poorly water soluble drugs in micellar drug delivery systems composed of amphiphilic block copolymers.

制备及个体表征和多载药物理截留聚合物胶束

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar ...

化学

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

内部自组装脂质颗粒的简易制备碳纳米管稳定

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

Anionic Polymerization of an Amphiphilic Copolymer for Preparation of Block Copolymer Micelles Stabilized by &#960;-&#960; Stacking Interactions

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE). Characterization of the copolymer revealed a narrow molecular distribution (PDI &#60; 1.03) and confirmed the degree of polymerization of mPEG122-b-(PheGE)15. The critical micelle concentration of the copolymer was evaluated using an established fluorescence method with the aggregation behavior evaluated by dynamic light scattering and transmission electronic microscopy. The potential of the copolymer for use in drug delivery applications was evaluated in a preliminary manner including in vitro biocompatibility, loading and release of the hydrophobic anti-cancer drug doxorubicin (DOX). A stable micelle formulation of DOX was prepared with drug loading levels up to 14% (wt%), drug loading efficiencies &#62; 60% (w/w) and sustained release of drug over 4 days under physiologically relevant conditions (acidic and neutral pH, presence of albumin). The high drug loading level and sustained release is attributed to stabilizing &#960;-&#960; interactions between DOX and the core-forming block of the micelles.

The key steps of living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE) are described. The resulting block copolymer micelles (BCMs) were loaded with doxorubicin 14% (wt%) and sustained release of drug over 4 days under physiologically relevant conditions was obtained.

一个两亲性共聚物的阴离子聚合通过π-π堆积作用稳定嵌段共聚物胶束的制备

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl ...

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

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

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...

Phase Change Dimethyldioctadecylammonium-Shelled Microdroplets as a Promising Drug Delivery System: Results on 3D Spheroids of Mammalian Tumor Cells

Significant improvement of phase-change perfluorocarbon microdroplets (MDs) in the vast theranostic scenario passes through the optimization of the MDs composition with respect to synthesis efficiency, stability, and drug delivery capability. To this aim, decafluoropentane (DFP) MDs stabilized by a shell of dimethyldioctadecylammonium bromide (DDAB) cationic surfactant were designed. A high concentration of DDAB-MDs was readily obtained within a few seconds by pulsed high-power insonation, resulting in low polydisperse 1 &#181;m size droplets. Highly positive &#950;-potential, together with a long, saturated hydrocarbon chains of the DDAB shell, are key factors to stabilize the droplet and the drug cargo therein. The high affinity of the DDAB shell with cell plasma membrane allows for localized chemotherapeutics delivery by increasing the drug concentration at the tumor cell interface and boosting the uptake. This would turn DDAB-MDs into a relevant drug delivery tool exhibiting high antitumor activity at very low drug doses.
In this work, the efficacy of such an approach is shown to dramatically improve the effect of doxorubicin against 3D spheroids of mammalian tumor cells, MDA-MB-231. The use of&#160;three-dimensional (3D) cell cultures developed in the form of multicellular tumor spheroids (i.e., densely packed cells in a spherical shape) has numerous advantages compared to 2D cell cultures: in addition to have the potential to bridge the gap between conventional in vitro studies and animal testing, it will improve the ability to perform more predictive in vitro screening assays for preclinical drug development or evaluate the potential of off-label drugs and new co-targeting strategies.

Decafluoropentane microdroplets developed with a shell of dimethyldioctadecylammonium bromide exhibited an exceptional colloidal stability and an actractive biointerface. DDAB-MDs proved to be efficient drug reservoirs characterized by a high affinity to plasma membranes together with enhanced uptake and antitumor activity of Doxorubicin against human triple-negative breast cancer (MDA-MB-231) 3D model.

Significant improvement of phase-change perfluorocarbon microdroplets (MDs) in the vast theranostic scenario passes through the optimization of the MDs ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

疏水货物封装有针对性的质膜交付在液晶纳米颗粒载体

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

生物工程

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

生物活性亲水球状蛋白的纳米脂质体制备与表征

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

含脂脂的微流体生产-温度敏感脂质体

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...