Name: targeted plasma membrane delivery of a hydrophobic cargo encapsulated in a liquid crystal nanoparticle carrier
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这项工作是由夺标基地的经费计划（工作单位MA041-06-41-4943）的支持。 ON由国家研究理事会博士后研究会士的支持。

1. DIO-LCNP和DIO-LCNP-PEG-澈的制备<ol><li>溶解液晶二丙烯酸酯交联剂（DACTP11，45毫克），3,3&#39;- dioctadecyloxacarbocyanine高氯酸（DIO，2毫克），和自由基引发剂（偶氮二异丁腈，1毫克）聚合于2ml氯仿中。此添加到丙烯酸酯官能化的表面活性剂（AC10COONa，13毫克7毫升）的水溶液。 </li><li>搅拌1小时，并在80％振幅的混合物超声处理5分钟以产生由通过聚合表面活性剂在水包围的有机材料的小滴的细乳液。 </li><li>加热该混合物至64℃的油浴中 以引发两者交联剂和表面活性剂的聚合作为氯仿慢慢蒸发，留下一个由表面活性剂稳定的含DIO-NP悬浮液。 </li><li>通过0.2μm注射过滤器过滤的NP悬浮液（3次），以去除任何聚集。申通再在4℃下将过滤的NP溶液直至进一步使用。 </li><li> PEG-澈的偶联通过EDC耦合DIO-LCNP。 <ol><li>溶解澈-PEG-NH 2·HCl的（PEG-澈，0.9毫摩尔）在25mM HEPES缓冲液（pH7.0）中。 </li><li>制备含有N-羟基磺基琥珀酰亚胺钠盐（NHSS，40毫摩尔），并在从浓缩储备溶液HEPES缓冲液1-乙基-3-（3-（二甲基氨基） -丙基）碳化二亚胺盐酸盐（EDC，400毫摩尔）的工作溶液。 </li><li>立即加入20微升NHSS / EDC的新鲜制备的工作溶液到1.0 DIO-LCNP毫升在HEPES缓冲液中，并搅拌5分钟。 </li><li>加入20μl的澈-PEG-NH 2·HCl的原液至该混合物并搅拌2小时。 </li><li>用台式小型离心机，并通过与平衡的PD-10尺寸排阻层析柱13传递上清液短暂离心以最大速度（〜2000 xg离心）将反应混合物进行30秒Dulbecco氏磷酸盐缓冲盐水（DPBS，0.1倍）。 </li></ol></li></ol> 2. DIO-LCNP和DIO-LCNP-PEG-澈的表征<ol><li> 确认通过凝胶电泳PEG-澈成功的共价结合到DIO-LCNP表面。 <ol><li>溶解0.5克琼脂糖在三 - 硼酸盐电泳50毫升（1×）（TBE; 89毫摩尔Tris（pH值7.6），89毫硼酸，和2mM EDTA）缓冲液中。加热微波炉中的溶液以溶解琼脂糖。让溶液稍微冷却并倒入内容到在电泳箱的凝胶板。将凝胶梳子创建样品孔。 </li><li>一旦凝胶固化，添加所需的量（足以淹没在腔室中的凝胶）电泳缓冲液的室TBE的。 </li><li>添加35微升DIO-LCNP样品（修订与甘油，5％体积/体积）至凝胶的孔中，并在110伏的电压20分钟运行</li><li>图片使用凝胶成像系统无线凝胶个激发和发射滤光片分别在488纳米和500-550纳米。 </li></ol></li><li>通过稀释在PBS中DIO-LCNP溶液（〜200倍稀释）（pH约8，0.1×）和一DLS仪器14测量评估通过动态光散射（DLS）的颗粒尺寸和分布。测量使用合适的zeta电位的测量仪器的DIO-LCNPs的ζ电位。 </li></ol> 3.细胞培养皿中准备交货的实验和成像注:DIO-LCNP标签上的HEK 293T / 17的人胚胎肾细胞（通道5和15之间），为先前4描述的是培养的进行。执行输送实验和如下所述的随后的细胞成像。 <ol><li>通过用牛纤连蛋白（涂覆它们在20微克的浓度制备直径35 mm组织培养皿（装有14性mm 1号盖玻片插入）〜100微升/ ml）的DPBS为在37℃下2小时。 </li><li>除去从培养皿纤连蛋白涂布溶液。收获HEK 293的T1 / 7细胞从T-25烧瓶中由第一洗涤用3ml的DPBS中，然后通过加入2ml胰蛋白酶-EDTA（0.5％胰蛋白酶0.25％的EDTA）的细胞单层。 </li><li>在37℃下〜3分钟的烧瓶中。除去胰蛋白酶-EDTA将烧瓶返回到培养箱中。一旦细胞从烧瓶中脱离，通过加入4 ml完全培养基中和胰蛋白酶（; DMEM; Dulbecco改良的Eagle培养基修正，以含有10％胎牛血清，5％丙酮酸钠和5％抗生素/抗真菌剂）向烧瓶中。通过在细胞计数器计数他们确定在悬浮液中的细胞浓度。 </li><li>调节细胞浓度至〜8×10 4个细胞/用生长培养基毫升。加入3毫升细胞悬浮液，以在孵化过夜盘和文化;第二天，细胞应在约70％汇合，并应准备使用。充足细胞密度为细胞的高百分比的健壮和有效的标记是至关重要的。 </li></ol> 4. DIO和DIO-LCNPs的细胞交付和固定细胞成像<ol><li>在送介质准备DIO，DIO-LCNP和DIO-LCNP-PEG-澈1毫升的解决方案（HEPES-DMEM，含25毫米的HEPES DMEM）稀释DIO 自由和DIO-LCNPs股票方案;适当的DIO浓度对培养细胞会〜1-10微米（表示为在DIO DIO-LCNP的形式免费或DIO任浓度）。 </li><li>使用血清吸管除去从培养皿生长培养基洗细胞单层（参见步骤3）的HEPES的DMEM（每次2毫升清洗）两次。轻轻添加和使用移液管向/从在餐具的边缘除去的HEPES的DMEM进行洗涤。 </li><li>增加±毫升准备好的DIO 免费或DIO-LCNP交付解决方案的培养皿中心和菜回到我ncubator为一段适当的时间（通常为15或30分钟，这取决于实验的需要）。潜伏期较长时间增加的非膜领域更非特异性细胞标记（ 如细胞质）。 </li><li>潜伏期后，取出使用血清吸管的交付解决方案。用DPBS（每次2毫升洗涤）洗涤细胞单层两次。轻轻从盘的边缘添加和删除DPBS到/执行洗涤。 </li><li>固定用4％多聚甲醛（在DPBS中制备）在室温下15分钟的细胞单层。 注意:多聚甲醛是易燃品，对呼吸道有刺激性，并且可能致癌。 </li><li>除去用吸管的多聚甲醛溶液，并通过加入并使用移液管向/从在餐具的边缘除去DPBS轻轻洗涤细胞1次用DPBS（2毫升）中。 </li><li>固定细胞准备使用共聚焦激光扫描被成像为膜状荧光信号的存在显微镜（CLSM）。立即执行的摄像，或者，替换用含有0.05％NaN 3的DPBS介质和菜肴存储在4℃。 注意:为NaN 3是有毒的;使用NaN的3时谨慎操作。 注:以这种方式存储固定样本应48小时为最佳结果中进行成像。 </li></ol>活细胞DIO和DIO-LCNPs和FRET成像5.细胞交付注:在该方法中，将细胞colabeled 6μM每个DIO-LCNP-PEG-澈（其中DIO是FRET供体）和1,1&#39;-双十八烷基-3,3,3&#39;，3&#39;- tetramethylindocarbocyanine高氯酸（DII 免费 ，其中dii是FRET受体）。 DIO从DIO-LCNP-PEG-澈和其掺入释放到质膜是通过在从DIO施主的能量转移到所述的DiI受体的观察增加确认。 <ol><li>与DIO-LCNP-PEG-澈和FRE的DiI顺序标签细胞e。使用在步骤4中描述的方法。 </li><li>染色后，清洗细胞用血清吸管1次用DPBS（2ml）中，并用2毫升活细胞成像溶液（LCIS）更换洗涤缓冲液。 </li><li>在装有加热的孵育室，图像通过在488纳米的4小时的时间内通过激励DIO施主使用共聚焦显微镜（60X物镜）具有FRET成像配置以30分钟的间隔，并收集全发射的活细胞样本的显微镜阶段无论是DIO捐助者和从490-700纳米的DiI受体配备了32通道光谱探测器的光谱。 </li><li>注:有关FRET成像的更多信息，请参考15。 </li><li>确定两个DIO给体和从与DIO-LCNP-PEG-澈和的DiI染色的细胞的DiI受体的时间分辨发射强度。计算时间分辨受主/施主FRET比率（DII EMI / DIO EMI）的图像，这将稳步增加，并最终板盟一旦DIO给体划分成的膜的数量已经达到最大16。 </li></ol> 6. DIO的细胞毒性检测和DIO-LCNPs到HEK 293T / 17细胞注:DIO-LCNP材料的细胞毒性使用基于染料四唑鎓增殖测定17评估。细胞，在不同浓度的物质的存在下模拟交付/标记条件下多孔板中培养。然后将细胞72小时培养以允许增殖。的染料（MTS，（3-（4,5-二甲基-2-基）-5-（3-羧基甲氧基）-2-（4-磺苯基）-2H-四唑）然后被加入到孔中，以及代谢活性细胞将染料成蓝色甲产物。颜色形成的量成正比的活细胞的数量。 <ol><li>收获HEK 293的T1 / 7细胞从T-25烧瓶中通过以下步骤3.2.Seed中描述的方法HEK 293T / 17细胞（5,000个细胞/100μl/孔），以24小时96孔组织培养处理板与文化的井。 </li><li>完全从使用微量孔除去培养基和添加的HEPES的DMEM中加入50μl含有游离 DIO，DIO-LCNPs，或在递增浓度复制井DIO-LCNP-PEG-澈。孵育的细胞在37℃下进行30分钟。 注意:通常情况下，以一式三份或一式四份重复做是足以产生统计学上可靠的数据。 <ol><li>培养后，除去含有使用微量的物料的输送介质并用100微升生长培养基的更换。培养细胞72小时。 </li></ol></li><li>加入20μl四唑基片的至每个孔，将板返回孵化器，并允许颜色形成，以在37℃下继续进行4小时。读出的甲臜产物的吸光度（ABS）在570nm（吸收极小对该STU使用的DIO-LCNPs使用微量滴定板读数器DY）和650纳米（对于非特异性背景吸收的减法）。 </li><li>积微分吸光度值（绝对570 -绝对650）相对于材料浓度，其结果作为控制细胞增殖（在仅细胞培养基的细胞增殖的程度）百分之报告。 </li></ol> 7.数据分析<ol><li>统计学分析数据以方差的单变量分析（ANOVA）。多重比较，应用邦费罗尼的事后检验。提供所有的平均值作为除另有说明外（SEM）的平均值±标准误差。 注:意义可接受的概率P &lt;0.05。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

NMDD的一个持续目标是控制目标和药物制剂的细胞和组织的递送，并同时提高药物疗效相结合。对于此已经构成了显著挑战的一种特定类的药物分子是有节制地在水介质中不溶于疏水性药物/显像剂。这个问题一直困扰的强效药物的转变，从体外细胞培养系统，临床上并导致了一些有前途的药物分子被"搁置"，或遗弃，而不是在临床上进一步推行。渥曼青霉素（Wtmn），例如是磷脂酰肌醇3&#39;?...

由于与活细胞的接口的纳米材料（材料≤100纳米的至少一个维度）的出现，持续目标是采取纳米颗粒（纳米颗粒），用于各种应用的独特大小依赖特性的优势。这些应用包括细胞和组织的标记/成像（ 在体外和在体内 ），实时检测和的药物和其它货物1的受控递送。这种相关的NP性质的实例包括半导体纳米晶体的尺寸依赖性发射（量子点，量子点）;金纳米颗粒的光热特性;脂质体的水性核心的大负荷能力;和碳同素异形体，例如单壁碳纳米管和石墨烯的弹道导电性。 最近，显著的兴趣已经出现在药品和其他货物，如对比度/成像的控制调制使用核动力源男厕所。在这里，基本原理是显著提高/通过提供它作为一个NP制剂优化整体溶解度，递送剂量，循环时间，并最终清除药物的货物。这已经到了被称为NP介导的药物递送（NMDD），并有目前有7种FDA批准的药物NP制剂在临床上用于治疗各种癌症和数百个临床试验的各个阶段。在本质上，目标是"实现事半功倍;"也就是说，要使用NP作为支架通过取较大的表面积的优点用较少的给药施用以提供更多的药物:体积（ 如 ，硬颗粒，如量子点和金属氧化物）纳米粒子的或它们的大的内部容积为装载大货的有效载荷（ 例如 ，脂质体或胶束）。这里的目的是为了减少多全身递送给药方案的必要性，而在同一时间促进水溶液稳定性和增强的循环，特别是用于有挑战性疏水药物货物，虽然高效，是在水性介质中微溶。 因此，本文所描述的工作的目的是确定使用一种新的NP支架，用于向亲脂质膜双层的具体和受控递送疏水货物的生存能力。对工作的动机是固有的有限的溶解度和难于从含水介质递送疏水性分子的细胞。典型地，这样的疏水性分子的递送需要使用的有机溶剂（ 例如 ，DMSO）或两亲性表面活性剂（ 例如 ，泊洛沙姆），这可能是有毒的和妥协的细胞和组织的生存力2，或胶束载体，其可以具有有限的内部负载能力。这里所选择的NP载体是一种新型液晶的NP（LCNP）先前开发3制剂和先前已示出，实现了〜40倍<html>改进，在抗癌药物阿霉素的培养细胞4的功效。 在本文所述的工作中，所选择的代表性的货物是电位膜染料，3,3&#39;- dioctadecyloxacarbocyanine高氯酸（DIO）。 DIO是已经用于生活和固定神经细胞，膜电位测量顺行和逆行追踪不溶于水的染料，和一般的膜标记5，6，7，8，9。由于其疏水特性，DIO通常直接加入到细胞单层或组织中的结晶形式10，或它在非常高的浓度下培养 （〜1-20μM）从浓度原液11,12稀释后。 内容"&gt;这里，方法是利用以LCNP平台，一个多功能的NP，其内芯是完全疏水性的，其表面是同时的亲水性和适合于生物耦合，作为递送载体用于DIO。DIO掺入合成期间LCNP芯和对NP表面，然后用聚乙二醇化的胆固醇部分官能化，以促进膜的DIO-LCNP合奏的质膜的结合。这种方法产生了一个输送系统，该系统划分的DIO到质膜以更大的保真度和膜滞留时间比DIO的游离形式从本体溶液递送（DIO 免费 ）。另外，该方法表明，DIO的LCNP介导的递送基本调制并驱动染料的特定分区的速率进入亲脂质膜双层。这是实现同时伴随减少游离药物的细胞毒作用〜40％，通过提供它作为一个LCNP配方。

<table><tbody><tr><td>1-ethyl-3-(3-(dimethylamino)-propyl)carbodiimide hydrochloride (EDCA)</td><td>ThermoFisher</td><td>E2247</td><td></td></tr><tr><td>3,3&amp;prime;-dioctadecyloxacarbocyanine perchlorate (DiO)</td><td>Sigma Aldrich</td><td>D4292-20MG</td><td>Hazardous; make stock solution in DMSO</td></tr><tr><td>Cholesterol poly(ethylene glycol) amine hydrochloride</td><td>Nanocs, Inc.</td><td>PG2-AMCS-2k</td><td></td></tr><tr><td>Countess automated cell counter</td><td>ThermoFisher</td><td>C10227</td><td></td></tr><tr><td>Dioctadecyl-3,3,3&amp;prime;&amp;#8288;,3&amp;prime;-tetramethylindocarbocyanine perchlorate (DiI)</td><td>Sigma Aldrich</td><td>468495-100MG</td><td>Hazardous; make stock solution in DMSO</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>ThermoFisher</td><td>21063045</td><td>Warm in 37 &amp;deg;C before use</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (DPBS)</td><td>ThermoFisher</td><td>14040182</td><td>Warm in 37 &amp;deg;C before use</td></tr><tr><td>Dynamic light scattering instrument</td><td>ZetaSizer NanoSeries (Malvern Instruments Ltd., Worcestershire, UK)</td><td></td><td></td></tr><tr><td>Fibronectin Bovine Protein, Plasma</td><td>ThermoFisher</td><td>33010018</td><td>Make stock solution 1 mg/ml using DPBS. Use 20-30 &amp;micro;g/ml for coating MetTek dish, 2 hr at 37 &amp;deg;C</td></tr><tr><td>Formaldehyde (16%, W/V)</td><td>ThermoFisher</td><td>28906</td><td>Hazardous; dilute to 4% using DPBS</td></tr><tr><td>Human embryonic kidney cells (HEK 293T/17)</td><td>American Type Culture Collection</td><td>ATCC&amp;reg; CRL-11268&amp;trade;</td><td></td></tr><tr><td>Live cell imaging solution (LCIS)</td><td>ThermoFisher</td><td>A14291DJ</td><td>Warm in 37 &amp;deg;C before use</td></tr><tr><td>MatTek 14 mm # 1.0 coverglass insert cell culture dish</td><td>MatTek corporation</td><td>P35G-1.0-14-C</td><td></td></tr><tr><td>Modified Eagle Medium (DMEM) containing 25 mM HEPES</td><td>ThermoFisher</td><td>21063045</td><td>Warm in 37 &amp;deg;C before use</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-hydroxysulfosuccinimide sodium salt (NHSS)</td><td>ThermoFisher</td><td>24510</td><td></td></tr><tr><td>Nikon A1si spectral confocal microscope</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>Trypan Blue Stain (0.4%)&amp;nbsp;</td><td>ThermoFisher</td><td>T10282</td><td>mix as a 50% to the cell suspension before counting the cells</td></tr><tr><td>Zeta potential instrument</td><td>ZetaSizer NanoSeries (Malvern Instruments Ltd., Worcestershire, UK)</td><td></td><td></td></tr><tr><td>Ultrasonic Processor</td><td>Sonics and Materials Inc</td><td>GEX 600-5</td><td></td></tr><tr><td>Mini Cetntrifuge</td><td>Benchmark</td><td>Mini-fuge-04477</td><td></td></tr><tr><td>PD-10 Sephadex&amp;trade; G-25 Medium</td><td>GE Healthcare</td><td>17-0851-01</td><td></td></tr><tr><td>Bio-Rad ChemiDoc XRS Imaging System</td><td>Bio-RAD</td><td>76S/07434</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>ThermoFisher</td><td>25200056</td><td></td></tr></tbody></table>

targeted plasma membrane delivery of a hydrophobic cargo encapsulated in a liquid crystal nanoparticle carrier

药物/成像剂对细胞的受控递送是治疗学的发展和对细胞信号过程的研究是至关重要的。最近，纳米粒子（NPS）都表现出这样的运载系统的发展显著的承诺。这里，液晶的NP（LCNP）系递送系统已被用于不溶于水的染料，3,3&#39;- dioctadecyloxacarbocyanine高氯酸（DIO）的受控递送，从NP芯内的等离子体的疏水区双层膜。在纳米颗粒的合成中，将染料有效地掺入疏水LCNP芯，如确认由多个光谱分析。 PEG化的胆固醇衍生物的NP表面（DIO-LCNP-PEG-澈）的缀合使染料加载纳米粒的结合在HEK 293T / 17细胞质膜。时间分辨激光扫描共聚焦显微镜和荧光共振能量转移（FRET）成像证实通从LCNP芯和其插入质膜双层香港专业教育学院DIO的流出。最后，迪欧作为LCNP-PEG-澈交付衰减DIO的细胞毒作用;相比DIO 无批量交付解决方案DIO的NP形式展出少〜30％-40％的毒性。这种做法表明了LCNP平台的效用的有效模式疏水分子货物的具体膜交付和调制。

制备LCNPs其中对NP的疏水核心中装入一个代表膜标记探针来演示LCNP的效用作为有效递送载体为疏水货物。为此目的，选择的货物是高度不溶于水的电位膜标记染料，DIO。 DIO-装载LCNPs（DIO-LCNPs）用与化学成分DACTP11，AC10COONa，和DIO两相微乳液技术合成， 如图 1 18。在这个NP系统中，结晶交联剂DACTP11的共价连接的聚合物网络提供其中DIO驻留的交联网络中的间?...

Watch this Scientific Journal Video about Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier at JoVE.com

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

一种液晶纳米颗粒（LCNP）纳米载体被利用作为用于受控递送疏水货物的活细胞的质膜的车辆。

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. ...

targeted-plasma-membrane-delivery-hydrophobic-cargo-encapsulated

Research

JoVE Journal

Bioengineering

7.5K 次观看.  Naval Research Laboratory. 一种液晶纳米颗粒（LCNP）纳米载体被利用作为用于受控递送疏水货物的活细胞的质膜的车辆。 

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

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 ...

科研

化学

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising approach to cancer therapy. The glycosaminoglycan placental chondroitin sulfate A (plCSA) is expressed on a wide range of cancer cells and placental trophoblasts, and malarial protein VAR2CSA can specifically bind to plCSA. A reported placental chondroitin sulfate A binding peptide (plCSA-BP), derived from malarial protein VAR2CSA, can also specifically bind to plCSA on cancer cells and placental trophoblasts. Hence, plCSA-BP-conjugated nanoparticles could be used as a tool for targeted drug delivery to human cancers and placental trophoblasts. In this protocol, we describe a method to synthesize plCSA-BP-conjugated lipid-polymer nanoparticles loaded with doxorubicin (plCSA-DNPs); the method consists of a single sonication step and bioconjugate techniques. In addition, several methods for characterizing plCSA-DNPs, including determining their physicochemical properties and cellular uptake by placental choriocarcinoma (JEG3) cells, are described.

Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles

Here, we present a protocol for the synthesis of placental chondroitin sulfate A binding peptide (plCSA-BP)-conjugated lipid-polymer nanoparticles via single-step sonication and bioconjugate techniques. These particles constitute a novel tool for the targeted delivery of therapeutics to most human tumors and placental trophoblasts to treat cancers and placental disorders.

胎盘硫酸软骨素 A (plCSA) 靶向脂质高分子纳米粒的合成与表征

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising ...

癌症研究

Encapsulation of Cancer Therapeutic Agent Dacarbazine Using Nanostructured Lipid Carrier

The only formula of dacarbazine (Dac) in clinical use is intravenous infusion, presenting a poor therapeutic profile due to the low dispersity of the drug in aqueous solution. To overcome this, a nanostructured lipid carrier (NLC) consisting of glyceryl palmitostearate and isopropyl myristate was developed to encapsulate Dac. NLCs with controlled size were achieved using high shear dispersion (HSD) following solidification of oil-in-water emulsion. The synthesis parameters, including surfactant concentration, the speed and time of HSD were optimized to achieve the smallest NLC with size, polydispersion index and zeta potential of 155 &#177; 10 nm, 0.2 &#177; 0.01, and -43.4 &#177; 2 mV, respectively. The optimal parameters were also employed for Dac-loaded NLC preparation. The resultant NLC loaded with Dac possessed size, polydispersion index and zeta potential of 190 &#177; 10 nm, 0.2 &#177; 0.01, and -43.5 &#177; 1.2 mV, respectively. The drug encapsulation efficiency and drug loading reached 98% and 14%, respectively. This is the first report on encapsulation of Dac using NLC, implying that NLC could be a new potential candidate as drug carrier to improve the therapeutic profile of Dac.

The most commonly used method for nanostructured lipid carrier (NLC) synthesis involves oil-in-water emulsion, homogenization and solidification. This was modified here by applying high shear dispersion after solidification to achieve a NLC with desirable size, improved drug encapsulation and drug loading efficiency as a potential carrier for dacarbazine delivery.

使用纳米脂质载体癌症治疗剂，达卡巴嗪封装

The only formula of dacarbazine (Dac) in clinical use is intravenous infusion, presenting a poor therapeutic profile due to the low dispersity of the drug ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

生物工程

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

治疗的siRNA递送至CNS使用阳离子和阴离子脂质体

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

纳米丝绸制造和药物输送应用

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

快速, 可伸缩的组装和负载的生物活性蛋白和免疫增强剂到不同的合成 Nanocarriers 通过闪光 Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA.
For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

阳离子纳米脂质体的生成, 用于高效输送体外转录信使 rna

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

人骨髓间充质干细胞吸收含有法卡林多醇的新型脂质包覆纳米颗粒

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...