Name: preparation and characterization of novel hdl-mimicking nanoparticles for nerve growth factor encapsulation
Uploaded: 2017-05-22T15:00:00
Description: Watch this Scientific Journal Video about Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation at JoVE.com

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，...

<ol>
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Modulation of release rate.</a> J Control Release. 67 (2-3), 249-260 (2000).</li><li>Patel, A., Patel, M., Yang, X., Mitra, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+protein+and+peptide+drug+delivery:+a+special+emphasis+on+polymeric+nanoparticles.">Recent advances in protein and peptide drug delivery: a special emphasis on polymeric nanoparticles.</a> Protein Pept lett. 21 (11), 1102-1120 (2014).</li><li>Kuai, R., Li, D., Chen, Y. E., Moon, J. J., Schwendeman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-density+lipoproteins:+nature's+multifunctional+nanoparticles.">High-density lipoproteins: nature's multifunctional nanoparticles.</a> ACS Nano. 10 (3), 3015-3041 (2016).</li><li>Gursky, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+stability+and+functional+remodeling+of+high-density+lipoproteins.">Structural stability and functional remodeling of high-density lipoproteins.</a> FEBS Lett. 589 (19 Pt A), 2627-2639 (2015).</li><li>McMahon, K. M., Thaxton, C. 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G., F&#246;rtsch, V., Hodler, G., Bolli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+characterization+of+a+reconstituted+high+density+lipoprotein+for+therapeutic+applications.">Production and characterization of a reconstituted high density lipoprotein for therapeutic applications.</a> Vox Sang. 71 (3), 155-164 (1996).</li><li>Zhang, Z., Chen, J., Ding, L., Jin, H., Lovell, J. F., Corbin, I. R., Cao, W., Lo, P. C., Yang, M., Tsao, M. S., Luo, Q., Zheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDL-mimicking+peptide-lipid+nanoparticles+with+improved+tumor+targeting.">HDL-mimicking peptide-lipid nanoparticles with improved tumor targeting.</a> Small. 6 (3), 430-437 (2010).</li><li>Kim, Y., Fay, F., Cormode, D. P., Sanchez-Gaytan, B. L., Tang, J., Hennessy, E. J., Ma, M., Moore, K., Farokhzad, O. C., Fisher, E. A., Mulder, W. J., Langer, R., Fayad, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+step+reconstitution+of+multifunctional+high-density+lipoprotein-derived+nanomaterials+using+microfluidics.">Single step reconstitution of multifunctional high-density lipoprotein-derived nanomaterials using microfluidics.</a> ACS Nano. 7 (11), 9975-9983 (2013).</li><li>Prathipati, P., Zhu, J., Dong, X. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+novel+HDL-mimicking+&#945;-tocopherol-coated+nanoparticles+to+encapsulate+nerve+growth+factor+and+evaluation+of+biodistribution.">Development of novel HDL-mimicking &#945;-tocopherol-coated nanoparticles to encapsulate nerve growth factor and evaluation of biodistribution.</a> Eur J Pharm and Biopharm. 108, 126-135 (2016).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Recombinant Human Beta-NGF</td>
			<td>Creative Biomart</td>
			<td>NGF-05H</td>
			<td></td>
		</tr>
		<tr>
			<td>L-a-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-&#945;-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 &#38; Technology</td>
			<td>16-16-120101</td>
			<td>1mg in 671 &#181;l 10 mM NH4HCO3, pH 7.4</td>
		</tr>
		<tr>
			<td>Sepharose 4B-CL</td>
			<td>Sigma</td>
			<td>CL4B200</td>
			<td>Cross-linked agarose,&#160; gel filtration chromatography column filling material</td>
		</tr>
		<tr>
			<td>Sandwich ELISA Kit for NGF</td>
			<td>R&#38;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</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>&#160;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 ...

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