Name: preparation and characterization of novel hdl-mimicking nanoparticles for nerve growth factor encapsulation
Uploaded: 2017-05-22T15:00:00.000+00:00
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这项工作由NIH R03 NS087322-01支持给Dong，X

注意:所有程序中包含的动物研究已获得北德州健康科学中心大学机构动物护理和使用委员会的批准。 NGF HDL模拟纳米颗粒的制备<ol><li>在乙醇中溶解赋形剂，磷脂酰胆碱（PC），鞘磷脂（SM），磷脂酰丝氨酸（PS），胆固醇油酸酯（CO）和D-α-生育酚聚乙二醇琥珀酸酯（TPGS）），以制备1mg / mL的储备溶液。 注意:将储备溶液等分并储存在-20°C。将胆甾醇油酸酯储存在黑暗的瓶子中。 PC，SM，PS和CO储备溶液在-20℃下分别稳定高达6,3,12和12个月。 TPGS溶液在-20℃下稳定至少12个月。 </li><li>将10μLNGF（1mg / mL水溶液）与10μL鱼精蛋白USP（1mg / mL水溶液）在1.5mL微量离心管中混合，并在室温下放置10分钟形成复合体。 注意:将NGF和鱼精蛋白储备溶液等分并储存在-20℃。对NGF原料不推荐重复冻融。 </li><li>向玻璃瓶中加入59μLPC，11μLSM，4μLPS，15μLCO和45μLTPGS。在温和的氮气流下混合并蒸发乙醇约5分钟;所有赋形剂应在玻璃小瓶的底部形成油性薄膜。 </li><li>向小瓶中加入1mL超纯水（1型）水，并在室温下以9,500rpm（8,600×g）均化5分钟，形成原型NP。 </li><li> 将步骤1.2中制备的复合物加入到原型NP中，并在玻璃小瓶中使用小的搅拌棒在37℃下搅拌孵育30分钟。 <ol><li>通过在室温下搅拌另外30分钟将NP降低。冷却后，加入106μLApo AI（1.49 mg / mL），室温搅拌过夜，形成最终NGF HDL模拟NP。 </li></ol></li></ol> NGF HDL模拟纳米颗粒的表征<ol><li> 根据制造商的说明书使用颗粒分析仪测量颗粒尺寸和ζ电位（参见材料表）。 </li><li>使用交联的琼脂糖凝胶过滤层析柱从NGF HDL模拟的NPs分离未负载的NGF并确定NGF的包埋效率。 <ol><li>对于柱制备，将15 mL Sepharose 4B-CL悬浮液转移到50 mL烧杯中。使用玻璃棒搅拌珠悬浮液，并将其倒入柱子（直径30厘米，直径1厘米，底部有玻璃料）。轻轻点击色谱柱以除去气泡。使溶剂排出，珠子沉降数分钟。 </li><li>继续添加剩余的暂停。冲洗柱内壁清洁珠子。排出溶剂直到溶剂水平稍微a摆在固定相的顶部。用20mL 1x磷酸缓冲盐水（PBS，含有137mM NaCl，2.7mM KCl，8mM Na 2 HPO 4和2mM KH 2 PO 4 ）洗涤并调节柱子。 </li><li>为了确定含有未加载NGF的级分，将200μLNGF溶液（10μg/ mL）加到凝胶过滤柱（25cm长×1cm直径）上，并用1x PBS洗脱。 </li><li>收集总共12个部分（每个部分1 mL），并使用三明治ELISA试剂盒测量NGF在每个级分中的浓度。 </li><li>使用三明治ELISA试剂盒从6到10检测NGF。获取柱上未加载NGF的色谱图。用20 mL PBS洗柱。 </li><li>为了确定含有NGF HDL-模拟NP的级分，将200μL的NP载入柱上，并用1x PBS洗脱。收集总共12个级分（每个级分1 mL），并测量每个级分u中的颗粒强度唱出粒度分析仪。从2级到4级检测NGF NP。 注意:强度是由粒子分析仪测量的参数，它说明了测试溶液中可能存在多少纳米颗粒。溶液中存在的NP越多，强度越高。通过这种方法，我们可以确认该柱可以将NGF NP与未载入的NGF分离。 </li><li>为了测量NGF的包埋效率，将200μLNGF HDL模拟的NP载入柱上，并用1x PBS洗脱。共收集12个级分（每个级分1 mL）。 </li><li>使用三明治ELISA试剂盒测量未分级6至10的未加载NGF浓度。 </li><li>将从部分6至10测量的NGF量作为未加载的NGF一起加入，并使用以下等式计算NGF的包载效率。 ％EE =（1-未加载的NGF /加入NP中的总NGF）×100％等式（1） </li></ol></li></ol> 3. 体外释放NGF HDL- 纳米微粒<ol><li>平行研究游离NGF（水中10μg/ mL; n = 4）和NGF HDL-模拟NP（10μg/ mL; n = 4）。 </li><li>按照制造商的说明预处理8个透析管（分子量截止值:300 kDa）。 </li><li>在PBS中制备5％牛血清白蛋白（BSA）作为释放培养基。将30 mL释放培养基加入到50 mL离心管中，并以135 rpm的振荡速度在振荡器中将其升温至37°C。将另外50 mL的释放介质置于振荡器中进行更换。 </li><li>将400μL释放培养基加入透析管，并快速加入200μL经过测试的样品，以制备足够的透析体积。 </li><li>关闭管子并迅速将透析管放入释放介质（离心管）中。从外部透析管中快速取出100μL的释放介质作为样品，时间为0.立即将样品置于-20°C进行后续分析。加入100μL新鲜的将介质放入离心管中以取代抽出的样品。启动计时器</li><li>在1,2,4,6,8,24,48和72小时，取出100μL释放培养基，并用100μL新鲜培养基代替。立即将取出的样品放入-20°C进行后续分析。 </li><li> 72小时后，将所有样品从-20°C取出，并在室温下解冻。使用三明治ELISA试剂盒测量释放样品的NGF浓度。 </li></ol> NGF HDL模拟纳米颗粒的生物活性（Neurite Outgrowth Study） <ol><li>在补充有10％热灭活马血清，5％胎牛血清，100μg/ mL链霉素和100单位/ mL青霉素的RPMI-1640培养基中培养PC12细胞。将细胞保持在37℃和5％CO 2的潮湿培养箱中。 </li><li>当细胞达到〜70-80％汇合时，取出培养基，用1×PBS洗涤细胞（约10mL /sup&gt; 2培养表面积）。取出洗液。通过向烧瓶中加入0.25％胰蛋白酶-EDTA溶液（0.25％胰蛋白酶和1mM EDTA; 0.5mL / 10cm 2培养表面积）将细胞胰蛋白酶化，并在37℃下孵育直至大部分细胞脱离。 </li><li>加入两卷培养基，并以180×g离心5分钟。去除上清液并将细胞沉淀重悬于5mL培养基中。通过22 G，½英寸针头过滤细胞以破坏细胞簇。 </li><li>将细胞以1:3的比例分裂成新的细胞培养瓶。孵育过夜后，取出未附着的PC12细胞。使附着的细胞保持进一步生长。 </li><li>重复此过程3次，以选择具有强粘附性的PC12的传代培养。用800μL/孔的大鼠尾部I型胶原（100μg/ mL）预涂6孔板。 </li><li>如步骤4.2所述对所选PC12细胞进行胰蛋白酶化，并通过a过滤22 G，½寸针打破细胞簇。用血细胞计数器计数细胞。在步骤4.5中在预涂布的6孔板中以10,000个细胞/孔的密度种细胞过夜，以使细胞附着于平板。 </li><li>用培养基（步骤4.1中所述）稀释游离NGF（10μg/ mL）和NGF HDL-模拟NP（10μg/ mL），以制备0.5,1,5,10,50和100ng / mL浓度。从每个孔中取出2 mL培养基，并用2 mL稀释的NGF或NGF HDL模拟NP替代。文化4天。 </li><li>在第4天，将培养基更换为含有相应治疗的新鲜培养基（如步骤4.1所述），并继续治疗3天。 </li><li>在第7天，用倒置的光学显微镜观察细胞，并以10X的放大倍数随机斑点图像。 </li></ol> NGF HDL模拟纳米颗粒的生物分布<ol><li>使用成年BALB / c小鼠（雄性，25-30g）至t估计NGF NPs的组织分布。随机将小鼠分成3组，每组3只。使用锥形塑料约束（ 例如，十倍体）来约束小鼠并用乙醇擦拭尾巴以促进血管舒张和静脉的可见性。 </li><li>通过尾静脉，以40μg/ kg的NGF剂量向每组小鼠（3组）注射100μL盐水，游离NGF或NGF HDL模拟NP。使用连接到1 mL注射器的30½G针。 </li><li> 注射后30分钟，用3％吸入的异氟烷麻醉小鼠（氧气，流速为2L / min）。执行尾部和脚趾捏，以确定麻醉深度。通过心脏穿刺收集血液。 <ol><li>从心脏取出大约1毫升的血液。通过颈椎脱位安乐死每只老鼠。 </li></ol></li><li>将小鼠胴体置于背侧卧位。使用手术剪刀打开腹部，将脂肪和肠子移开使用棉签将肝脏，脾脏和肾脏暴露出来。收获这些组织，并在1x PBS中冲洗干净血液。 </li><li>立即以3,400 xg和4℃离心血液样品5分钟，以获得血浆。将血浆和组织储存在-80°C直到分析。 </li><li>为了分析组织样品，将样品从-80℃移动到4℃，并将100毫克组织样品悬浮在10倍体积的提取缓冲液（0.05M乙酸钠，1.0M氯化钠，1％triton X-100， 1％BSA，0.2mM苯基甲磺酰氟和0.2mM苄索氯铵）。在10,000rpm和4℃均质5分钟。 </li><li>牺牲两只未处理的小鼠以收集空白血浆和组织，如步骤5.3和5.4所述。使用空白血浆或空白组织匀浆制备NGF标准溶液。 </li><li>如上所述，使用夹心ELISA试剂盒确定血浆和组织匀浆中NGF的浓度。 </li></ol>

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作者没有什么可以披露的。

在本研究中，我们展示了制备用于NGF包封的HDL模拟NP的简单方法。已经研究了各种NP递送系统来递送蛋白质。目前，许多NP制剂涉及透析，溶剂沉淀和膜水合。这些过程在扩大规模时通常是复杂和具有挑战性的。在该NP开发中，确定了脂质对容器的玻璃壁具有很强的粘附性，这导致难以水合薄膜并有效地混合赋形剂。鉴于天然HDL NPs中的多种组分，混合变得非常具有挑战性，并且对于制备HDL模拟NPs至?...

大分子如蛋白质，肽和核酸已经成为有希望的药物，并在过去几十年中获得了相当大的关注。由于其高效和特异的作用方式，它们对治疗癌症，免疫疾病，艾滋病毒及相关病症3，4表现出很大的治疗潜力。然而，物理化学性质如其分子大小，三维结构，表面电荷和亲水性质使得这些大分子的体内递送非常具有挑战性。这大大阻碍了他们的临床应用。药物递送系统如微粒，聚合物纳米颗粒（NP），脂质体和脂质NPs的最新进展克服了这些挑战，并显着地改善了大分子的体内递送。何wever，已经揭示了这些运送货物的一些缺点，包括低的药物负载能力，低的包埋效率，短的半衰期，生物活性的丧失和不良的副作用5,6,7,8。有效的运营商系统仍然是研究兴趣领域。此外，开发用于表征药物负载NP的分析方法对于大分子而言比对小分子更具挑战性。 高密度脂蛋白（HDL）是由脂质核心组成的天然NP，其由载脂蛋白和磷脂单层包被。内源性HDL通过与目标受体（如SR-BI，ABCAI和ABCG1）的相互作用在脂质，蛋白质和核酸的转运中起关键作用。已经被探索作为递送不同治疗剂的载体9， 10,11,12 。已经开发了各种制备HDL模拟NP的方法。透析是一种受欢迎的方法。在该方法中，通过使用胆酸钠溶液水合脂质膜来形成NP。然后通过用三个缓冲液13进行2天透析除去盐。超声处理方法通过在加热条件下超声处理脂质混合物60分钟制备NP;通过凝胶色谱法14进一步纯化NP。微流体通过微流体装置产生NP，其通过在聚焦模式15中产生微量涡来混合磷脂和载脂蛋白AI（Apo AI）溶液。显然，这些方法对于工业规模来说可能是耗时，苛刻和困难的。 在本文中，我们介绍了新型HDL模拟NPs神经的制备和表征生长因子（NGF）封装。 NGF是含有两个13.6-kDa多肽单体的二硫键连接的多肽同源二聚体。开发了通过均质化制备NPs的新方法，随后将NGF包被到NP中。 NGF HDL模拟NPs的特征在于粒度，大小分布，ζ电位和体外释放。对PC12细胞中的神经突生长评估了其生物活性。将NGF HDL模拟NPs的生物分布与静脉注射后的游离NGF的生物分布进行比较。

<table><tbody><tr><td>Recombinant Human Beta-NGF</td><td>Creative Biomart</td><td>NGF-05H</td><td></td></tr><tr><td>L-&amp;alpha;-Phosphatidylcholine (PC)</td><td>Avanti</td><td>131601P</td><td>95%, Egg, Chicken</td></tr><tr><td>Sphingomyelin (SM)</td><td>Avanti</td><td>860062P</td><td>Brain, Porcine</td></tr><tr><td>Phosphatidylserine (PS)</td><td>Avanti</td><td>840032P</td><td>Brain, Porcine</td></tr><tr><td>Cholesteryl oleate (CO)</td><td>Sigma</td><td>C9253</td><td></td></tr><tr><td>D-&amp;alpha;-Tocopheryl polyethylene glycol succinate (TPGS)</td><td>BASF</td><td>9002-96-4</td><td>Vitamin E Polyethylene Glycol Succinate</td></tr><tr><td>Protamine sulfate</td><td>Sigma</td><td>P3369</td><td>meets USP testing specifications</td></tr><tr><td>Apolipoprotein A1, Human plasma</td><td>Athens Research &amp;amp; Technology</td><td>16-16-120101</td><td>1 mg in 671 &amp;micro;L 10 mM NH&lt;sub&gt;4&lt;/sub&gt;HCO&lt;sub&gt;3&lt;/sub&gt;, pH 7.4</td></tr><tr><td>Sepharose 4B-CL</td><td>Sigma</td><td>CL4B200</td><td>Cross-linked agarose,&amp;nbsp; gel filtration chromatography column filling material</td></tr><tr><td>Sandwich ELISA Kit for NGF</td><td>R&amp;amp;D system</td><td>DY008</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>RPMI-1640 medium</td><td>GE Healthcare Life Science</td><td>SH30096.02</td><td></td></tr><tr><td>Horse serum</td><td>GE Healthcare Life Science</td><td>SH30074.03</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>10082147</td><td></td></tr><tr><td>PC12 cells</td><td>ATCC</td><td>CRL-1721</td><td></td></tr><tr><td>Rat tail collagen type I</td><td>Sigma</td><td>C3867</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma</td><td>S2889</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>31414</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Phenylmethanesulfonyl fluoride (PMSF)</td><td>Sigma</td><td>P7626</td><td></td></tr><tr><td>Benzethonium chloride</td><td>Sigma</td><td>B8879</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Homogenizer</td><td>Tekmar</td><td>T 25-S1</td><td></td></tr><tr><td>Delsa Nano HC particle analyzer</td><td>Beckman-Coulter</td><td>Delsa Nano HC</td><td></td></tr><tr><td>Float-A-Lyzer G2 Dialysis Device</td><td>Spectrum Laboratories</td><td>G235036</td><td>Molecule Cutoff 300 kDa</td></tr><tr><td>Centrifuge</td><td>Eppendoff</td><td>5424R</td><td></td></tr><tr><td>Polytron homogenizer</td><td>Kinematica</td><td>PT 1200C</td><td></td></tr><tr><td>DecapiCone</td><td>&amp;nbsp;Braintree Scientific Inc.</td><td>DC-M200</td><td></td></tr></tbody></table>

preparation and characterization of novel hdl-mimicking nanoparticles for nerve growth factor encapsulation

本文的目的是介绍神经生长因子（NGF），高密度脂蛋白（HDL） - 纳米颗粒（NPs）的制备和表征方法。高密度脂蛋白是内源性NPs，已被探索作为递送治疗剂的载体。已经开发了各种制备HDL模拟NP的方法。然而，它们通常是复杂的，耗时的，而且对于工业规模来说是困难的。在本研究中，使用一步均质化来混合赋形剂并形成原型NP。 NGF是26kDa的水溶性蛋白质。为了便于将NGF包封到HDL模拟NP的脂质环境中，使用鱼精蛋白USP与NGF形成离子对复合物以中和NGF表面上的电荷。然后将NGF /鱼精蛋白复合物引入原型NP。载脂蛋白AI最终涂在NP的表面上。 NGF HDL模拟NP在术语上显示出优选的性质的粒径，粒度分布，包埋效率， 体外释放，生物活性和生物分布。通过仔细设计和探索高密度脂蛋白模拟NPs的均质化，程序大大简化，NPs可扩展。此外，克服了诸如从NP中分离未负载的NGF，进行可靠的体外释放研究和测量NP的生物活性的各种挑战。

通过离子对策略制备的HDL模拟，α-生育酚包被的NGF NPs的工程方案如图1所示 。为了中和NGF的表面电荷，使用鱼精蛋白USP作为离子对试剂与NGF形成络合物。为了保护生物活性，原型HDL模拟NPs被设计，首先使用均质化;然后，NGF /鱼精蛋白复合物被封装到原型NPs中。均质化提供了足够的能量，并成功地促进了赋形剂的混合。均质3分钟后，样品NPs获得了一致的粒度（?...

Watch this Scientific Journal Video about Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation at JoVE.com

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

使用简单的均质化来制备新颖的高密度脂蛋白模拟纳米颗粒以包封神经生长因子。本文描述了纳米颗粒制备， 体外表征和体内研究的挑战，详细方案。

用于神经生长因子包封的新型HDL模拟纳米颗粒的制备和表征

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

preparation-characterization-novel-hdl-mimicking-nanoparticles-for

Research

JoVE Journal

Bioengineering

8.3K 次观看.  University of North Texas Health Science Center. 使用简单的均质化来制备新颖的高密度脂蛋白模拟纳米颗粒以包封神经生长因子。本文描述了纳米颗粒制备， 体外表征和体内研究的挑战，详细方案。 

视频: Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

SDF-1α - 壳聚糖硫酸葡聚糖纳米粒子的制备与表征

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

科研

化学

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

静电生长因子释放到微纤维支架

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

生物工程

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. ...

Formulation and Characterization of Bioactive Agent Containing Nanodisks

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle&#39;s lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk&#160;particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin&#160;B as the hydrophobic bioactive agent.

Here, we describe the production and characterization of bioactive agents containing nanodisks. Amphotericin B nanodisks are taken as an example to describe the protocol in a stepwise manner.

含纳米片生物活性剂的配制与表征

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. ...

生物化学

<meta charset="utf8"></head><body>氧化铁纳米颗粒用于小鼠胶质母细胞瘤的图像引导治疗递送

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

<meta charset="utf8"></head><body>作者聚焦:使用氧化铁纳米颗粒治疗胶质母细胞瘤的创新癌症疗法

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

癌症研究

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 ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

多孔硅薄膜作为神经生长因子载体的设计

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

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 ...

用于神经生长因子包封的新型HDL模拟纳米颗粒的制备和表征

摘要

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含纳米片生物活性剂的配制与表征

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多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

阳离子纳米脂质体的生成, 用于高效输送体外转录信使 rna

多孔硅薄膜作为神经生长因子载体的设计

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