Name: evaluation of polymeric gene delivery nanoparticles by nanoparticle tracking analysis and high-throughput flow cytometry
Uploaded: 2013-03-01T12:00:00
Description: Watch this Scientific Journal Video about Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry at JoVE.com

作者感谢的TEDCO的MSCRF（2009-MSCRFE-0098-00）和美国国立卫生研究院R21CA152473的支持。

1。细胞接种<ol><li>不要让细胞增长到overconfluency。早期传代细胞转染原代细胞时使用。 </li><li>二十四小时，在转染前，trypsinize细胞，用血细胞计数器计数的细胞，并稀释以达到所需的细胞密度（细胞/体积）的细胞悬浮液与媒体。种子细胞转化为明确的组织培养处理的水库和多道移液器使用的平底96孔板。选择的密度应得到70-80％汇合的转染当天，。例如，如在表1中显示，细胞稀释?...

<ol>
	<li>Putnam, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymers+for+gene+delivery+across+length+scales.">Polymers for gene delivery across length scales.</a> Nature Materials. 5, 439-451 (2006).</li><li>Sheyn, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+modified+cells+in+regenerative+medicine+and+tissue+engineering.">Genetically modified cells in regenerative medicine and tissue engineering.</a> Advanced Drug Delivery Reviews. 62, 683-698 (2010).</li><li>Boussif, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+vector+for+gene+and+oligonucleotide+transfer+into+cells+in+culture+and+in+vivo:+polyethylenimine.">A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.</a> Proc. Natl. Acad. Sci. U.S.A. 92, 7297-7301 (1995).</li><li>Green, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rita+Schaffer+Lecture:+Nanoparticles+for+Intracellular+Nucleic+Acid+Delivery.">Rita Schaffer Lecture: Nanoparticles for Intracellular Nucleic Acid Delivery.</a> Ann. Biomed. Eng. 40, 1408-1418 (2011).</li><li>Lynn, D. M., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradable+poly(beta-amino+esters):+Synthesis,+characterization,+and+self-assembly+with+plasmid+DNA.">Degradable poly(beta-amino esters): Synthesis, characterization, and self-assembly with plasmid DNA.</a> J. Am. Chem. Soc. 122, 10761-10768 (2000).</li><li>Sunshine, J. C., Peng, D. Y., Green, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+and+transfection+with+polymeric+nanoparticles+are+dependent+on+polymer+end-group+structure,+but+largely+independent+of+nanoparticle+physical+and+chemical+properties.">Uptake and transfection with polymeric nanoparticles are dependent on polymer end-group structure, but largely independent of nanoparticle physical and chemical properties.</a> Mol. Pharm. , (2012).</li><li>Shmueli, R. B., Sunshine, J. C., Xu, Z., Duh, E. J., Green, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+nanoparticles+specific+for+human+microvasculature+and+macrovasculature.">Gene delivery nanoparticles specific for human microvasculature and macrovasculature.</a> Nanomedicine: Nanotechnology, Biology, and Medicine. , (2012).</li><li>Bhise, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+terminal+functionalization+and+molecular+weight+of+a+gene+delivery+polymer+and+transfection+efficacy+in+mammary+epithelial+2-D+cultures+and+3-D+organotypic+cultures.">The relationship between terminal functionalization and molecular weight of a gene delivery polymer and transfection efficacy in mammary epithelial 2-D cultures and 3-D organotypic cultures.</a> Biomaterials. 31, 8088-8096 (2010).</li><li>Tzeng, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-viral+gene+delivery+nanoparticles+based+on+poly(beta-amino+esters)+for+treatment+of+glioblastoma.">Non-viral gene delivery nanoparticles based on poly(beta-amino esters) for treatment of glioblastoma.</a> Biomaterials. 32, 5402-5410 (2011).</li><li>Sunshine, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-Molecule+End-Groups+of+Linear+Polymer+Determine+Cell-Type+Gene-Delivery+Efficacy.">Small-Molecule End-Groups of Linear Polymer Determine Cell-Type Gene-Delivery Efficacy.</a> Adv. Mater. 21, 4947 (2009).</li><li>Bhise, N. S., Shmueli, R. B., Gonzalez, J., Green, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+quantifying+the+number+of+plasmids+encapsulated+by+polymer+nanoparticles.">A novel assay for quantifying the number of plasmids encapsulated by polymer nanoparticles.</a> Small. 8, 367-373 (2012).</li><li>Yu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+induced+pluripotent+stem+cells+free+of+vector+and+transgene+sequences.">Human induced pluripotent stem cells free of vector and transgene sequences.</a> Science. 324, 797-801 (2009).</li><li>Ho, Y. P., Chen, H. H., Leong, K. W., Wang, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+intracellular+stability+and+unpacking+of+DNA+nanocomplexes+by+quantum+dots-FRET.">Evaluating the intracellular stability and unpacking of DNA nanocomplexes by quantum dots-FRET.</a> Journal of Controlled Release: Official journal of the Controlled Release Society. 116, 83-89 (2006).</li><li>Kreiss, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmid+DNA+size+does+not+affect+the+physicochemical+properties+of+lipoplexes+but+modulates+gene+transfer+efficiency.">Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency.</a> Nucleic Acids Research. 27, 3792-3798 (1999).</li><li>Pitard, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-sized+self-assembling+lamellar+complexes+between+plasmid+DNA+and+cationic+micelles+promote+gene+transfer.">Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 14412-14417 (1997).</li><li>Collins, L., Kaszuba, M., Fabre, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+in+solution+of+(Lys)(16)-containing+bifunctional+synthetic+peptide/DNA+nanoparticles+for+gene+delivery.">Imaging in solution of (Lys)(16)-containing bifunctional synthetic peptide/DNA nanoparticles for gene delivery.</a> Biochimica et Biophysica Acta. 1672, 12-20 (2004).</li><li>Green, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+polymeric+vectors+for+gene+delivery+to+human+endothelial+cells.">Biodegradable polymeric vectors for gene delivery to human endothelial cells.</a> Bioconjug. Chem. 17, 1162-1169 (2006).</li></ol>

上述的协议描述评估转染效率的纳米颗粒的制剂，以及一种方法来表征的纳米粒子的粒径和DNA装载的方法。每个粒子的数目的质粒是一个重要的参数，该参数可以帮助预测粒子的有效性，也可以被用于剂量测定。纳米粒子跟踪分析，可以执行在一个范围内的不同的水溶液，如那些不同的盐浓度。这通常表征在PBS中进行，到模仿生理盐水。虽然在PBS中的尺寸可以得到一个良好的估计媒体或生理盐水?...

每纳米粒子的复合物的质粒的数量的测定是很重要的设计有效的纳米粒子为基础的基因传递策略，特别是对于合作提供多个质粒给同一小区的目标，由于经常需要在干细胞重编程的研究12。已经描述了几种方法，来计算与一个单一纳米粒子的数目的质粒，每种方法都有缺点，用于估计13-16中所使用的技术。量子点（QD）标签结合TEM被用来估计每个粒子的质粒在基于壳聚糖的纳米粒子。估计这QD技术是复杂的，由于需要标记的DNA，这可能会改变其自组装特性;封装的未标记的DNA还没有直接检测的可能性;可能重叠的质粒和量子点在2D的颗粒的TEM图像;其他简化的假设13。另一种方法，是一个pplicable当排列的微颗粒中存在时，已被用来研究Lipopolyamine-DNA复合物，通过冷冻透射电子显微镜（冷冻TEM），X-射线散射，和动态光散射（DLS）14,15。不幸的是，这里考察的聚合物纳米颗粒材料，如用这种方法并不适用。 Collins 等人在另一项研究中，使用流动式粒子图像分析技术来研究（Lys）的16的含肽/ DNA复合物，但是，他们的方法只能评估较大，微米大小的颗粒16。因此，我们最近开发出一种新颖，灵活的检测，以量化的数量每11纳米粒子的质粒。

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td>磷酸盐缓冲盐水（PBS，1个） </td><td> Invitrogen公司</td><td> 10010 </td><td></td></tr><tr><td> EGM-2MV BulletKit的</td><td>龙沙</td><td> CC-3202 </td><td></td></tr><tr><td>胰蛋白酶</td><td> Invitrogen公司</td><td> 25300 </td><td></td></tr><tr><td>醋酸钠缓冲液</td><td> Sigma-Aldrich公司</td><td> S7899 </td><td>在去离子水中稀释至的25mM </td></tr><tr><td>二甲基亚砜</td><td> Sigma-Aldrich公司</td><td> 276855 </td><td></td></tr><tr><td> PEGFP DNA </td><td>以琳生物制药</td><td> NA </td><td></td></tr><tr><td>红色荧光蛋白DNA </td><td> Addgene </td><td> 21718 </td><td></td></tr><tr><td> PEI，支链</td><td> Sigma-Aldrich公司</td><td> 408727 </td> &lt;TD&gt; </td></tr><tr><td> CellTiter 96水一</td><td> Promega公司</td><td> G3580 </td><td></td></tr><tr><td></td><td></td><td></td><td> 物料 </td></tr><tr><td>清除平底96孔板中，无菌</td><td>萨尔斯塔特</td><td> 82.1581.001 </td><td></td></tr><tr><td>晴圆底96孔板中，无菌</td><td>萨尔斯塔特</td><td> 82.1582.001 </td><td></td></tr><tr><td> 12通道Finnpipette </td><td> Thermo Scientific的</td><td> NA </td><td> 5-50和50-300微升</td></tr><tr><td>荧光显微镜</td><td>蔡司</td><td> NA </td><td>产品型号：AX10 </td></tr><tr><td> C6的Accuri流仪</td><td> BD Biosciences公司</td><td> NA </td><td></td></tr><tr><td> HyperCyt附件</td><td> Intellicyt </td><td> NA </td><td></td></tr><tr><td> NS500</td><td> NANOSIGHT </td><td> NA </td><td></td></tr></tbody></table>

evaluation of polymeric gene delivery nanoparticles by nanoparticle tracking analysis and high-throughput flow cytometry

使用高分子纳米粒子的非病毒基因传递已经成为一个有吸引力的方法对基因疗法治疗遗传性疾病，再生医学的技术。与病毒不同，它具有显着的安全问题，聚合物纳米颗粒可以被设计为是无毒的，非免疫原性的，非诱变性，更容易合成，化学通用的，能够承载较大的核酸货物和可生物降解的和/或环境响应。的阳离子聚合物与带负电荷的DNA通过静电相互作用形成复合物的顺序为100nm的，通常被称为聚合物纳米颗粒的自组装。生物材料，用于以形成纳米级的聚阳离子基因传递纳米粒子的实例包括多聚赖氨酸，polyphosphoesters，聚（酰氨基胺）和聚乙烯亚胺（PEI），这是一种不可降解的关闭的，现成的阳离子性聚合物的核酸转1,3常用。聚（β-氨基酯）（PBAEs）是一个较新的类的阳离子聚合物4的水解降解的5,6，并已被证明是有效的基因传递到硬-转染的细胞类型，如人视网膜内皮的细胞（HRECs）7，小鼠乳腺上皮细胞，人脑肿瘤细胞和大血管内皮细胞（人类脐静脉内皮细胞）10。 一种新的协议来表征高分子纳米粒子，利用纳米粒子跟踪分析（NTA）。在这种方法中，得到的粒度分布和分布的每个粒子的数目的质粒11。此外，高吞吐量的96 - 孔板的聚合物纳米颗粒的转染效率进行快速筛选转染。在这个协议中，作为模型聚合物和人视网膜内皮的细胞（HRECs）的聚（β-氨基酯）（PBAEs）用作莫德尔人类细胞。这个协议可以很容易地适于评估任何聚合物纳米颗粒和感兴趣的任何类型的细胞中的多穴板的格式。

图1示出了荧光显微镜图像的一个例子的一个成功的转染与EGFP质粒HRECs。亮场图像是有帮助的，以确保细胞保持自己平时的形态。此外，细胞活力检测，如MTS或类似的检测，可以用来评估的纳米粒子毒性7。流式细胞术，如所述，可以用来量化的转染效率。当使用HyperCyt的多井板附件，数据将需要进行适当的处​​理，为了正确地识别各孔。中可以看出， 图2中 ，?...

Watch this Scientific Journal Video about Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry at JoVE.com

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

纳米粒子跟踪分析（NTA）和高通量流式细胞仪来评价聚合物纳米基因传递的协议。 NTA用于表征的纳米粒子的粒径分布和每个粒子分布的质粒。高通量的基因传递的生物材料库，流式细胞仪，定量的转染效率评价。

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Research

JoVE Journal

Bioengineering

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

制定通过Nanoprecipitation技术嵌段聚合物纳米粒子

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

科研

生物工程

Polymer Microarrays for High Throughput Discovery of Biomaterials

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

组合合成和高通量的蛋白质释放的聚合物薄膜和纳米粒子库

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

碳水化合物和聚酸酐纳米粒子功能化的高通量合成

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Polymeric Microneedle Array Fabrication by Photolithography

聚合物微针阵列制造用光刻

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

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 ...

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

高通量自动 Microbioreactor 系统在 CHO 细胞模型 IgG1 生产中的应用

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

闪速纳米沉淀法包聚物纳米粒子中疏水亲水化合物的作用

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...