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，细胞稀释至25至50个细胞/μL的转染在这里示出的数据。 </li></ol> 2。细胞转染<ol><li>稀释的聚合物和DNA股。解冻后的聚合物与DNA原液在室温（RT）。在明确的96 - 孔板，使用一个12通道移液器稀释聚合物原液和DNA原液，分开，与适当的溶剂中的所需浓度的DNA的重量比（重量/重量），以获得所需的聚合物重量。在这种情况下，所选择的溶剂是25 mM乙酸钠缓冲液（pH = 5.2时）。 <ol><li> DNA稀释。通常情况下，存储在1毫克/毫升的DNA是在乙酸钠缓冲液稀释，在清晰的非组织培养处理的96孔板中（一个孔为一个单一的制剂）中的浓度为0.03〜0.06毫克/毫升。 表2所示为一个单一的制剂，用于转染4复制井从步骤1与细胞在96孔板中接种的典型的DNA稀释协议。 </li><li>聚合物稀释。 100毫克/毫升聚合物/ DMSO溶液在乙酸钠缓冲液稀释，根据所要求的浓度，以获得所需的聚合物，DNA的重量/重量比。通常用于与聚（β-氨基酯）秒（PBAEs）的基因传递的范围内的重量/重量比为20〜100。 PBAE聚合物首先稀释至10毫克/毫升，然后由稀释P议定如表3中所示。的聚合物稀释，可以执行在一个明确的非组织培养处理的96孔板中的DNA稀释板的取向一致的样本。 </li></ol></li><li>纳米粒子的形成。加入等体积的质粒DNA溶液使用12通道移液器PBAE溶液并剧烈混合。让混合物在室温孵育10分钟，以允许自组装。 </li><li>纳米粒子转染。 20微升的纳米粒子的自组装，每孔加入到培养基中滴加使用12通道移液器。复制井不及时治疗或用市售的转染试剂作为对照组。转染的细胞保温在37℃，两到四个小时，然后在各孔所取代用新鲜培养基（100μl/孔）。孵育时间的选择将依赖于细胞系，培养条件和转染系统。转染中的差异将被量化的效率和细胞毒性的结果不同潜伏期，通过分析在步骤3中描述的协议。 </li></ol> 3。转染效率和细胞毒性分析转染效率用荧光显微镜视觉分析，并用流式细胞仪转染后48小时，定量。细胞毒性分析用荧光显微镜可视化和量化的使用CellTiter 96水溶液测定24小时转染后的。 <ol><li>荧光显微镜。可视化地分析井使用的适当的荧光通道的转染报道基因（例如，绿色荧光蛋白或红色荧光蛋白）的表达。采集图像的每个孔中，选择的视场，适当地表示为特定的配方（ 图1）的转染效率。请记下任何肉眼观察的细胞毒性。 </li><li>流式细胞仪。 <ol><li>使用准备96孔板流式细胞仪的多道移液器。用PBS洗涤，用30μl/孔的胰蛋白酶/ EDTA，与170微升的FACS缓冲液（PBS +2％胎牛血清）中，移液管和各孔中，包括在边缘周围trypsinize，和转移的整个200微升体积的在一个圆底或V形底96孔板的各孔成相应的井。 </li><li>离心板，在130×g离心5分钟，在4℃下</li><li>从每口井和吸液重悬细胞，同时避免气泡中取出170微升媒体。使用可行性，如碘化丙啶（PI）染色，FACS缓冲液+ PI（50:1 FACS缓冲液:稀释PI PI股票浓度为1毫克/毫升），每孔加入10μl。立即放在冰，覆盖了从光。 </li><li>启动C6 Accuri的流式细胞仪，附件96的HyperCyt，超级软件。使用超级设计"和"议定书"选项卡，选择适当的P晚型，板布局，和其他设置，如抖动/ SIP /清洗/摇晃时间。对于上述的协议，使用了以下的参数，预板总理一分钟，并在2400转30秒预板晃动。品尝时间设置为15秒，15转，探头清洗每2秒，并，井间摇晃，持续12秒，每12口井在2400转。重要的是要保持细胞分散在媒体上，以及改进的能力来处理数据通过包括定时井组（在这种情况下，12秒）的时间间隔，井间的晃动。 </li><li>使用超级井标识"选项卡来处理数据到适当的孔中（ 图2）和单独的。为了确定单井，它可以帮助使用该软件的噪声滤波器，以及包括基地连过滤器的高级设置。 </li><li>量化比例正转染的活细胞，通过适当的门来分隔不同的弹出ulations（ 图3）。首先查看流量数据上的FSC与SSC分散的情节，以不同的细胞碎片（ 图3A）。如果使用碘化丙啶（PI），栅极个别的细胞群体，并检视，为了分离从死细胞和门的活细胞的活细胞上的FSC对比FL3情节数据。为了量化转染细胞的数量，查看现场的细胞群，在适当的渠道，例如，GFP可以看出，在FL1。使用未处理的人口，栅极的未处理的细胞，以便确定的背景信号（ 图3B）。正转染细胞，然后可以使用该栅极分离和通过查看使用散和等高线图（ 图3C和3D）转染的种群。有可能是一个连续的正转染的细胞，不同的细胞将被转染到不同程度的荧光。 </li></ol></li><html><li> CellTiter 96水分析<ol><li>转染在完全相同的方式进行细胞毒性测量另一个板。 </li><li>预混和物1毫升的CellTiter 96水溶液的一个测定溶液用10ml的新鲜培养基。从细胞中移除介质，并使用多道移液器加110微升的预混液+每口井的媒体。 </li><li>每一个小时的时间间隔为1至4小时，直到在490nm处的吸光度与未处理的条件从阱是在测定的线性范围内，测量吸光度。包括4个控制井，唯一的解决办法+媒体背景吸光度读数。 </li><li>平均吸光度值从四个重复的井每条件和减去平均背景吸收，从每个平均。规范化校正为每个处理的条件下的平均校正的未处理的条件来确定的％的细胞存活率的平均。 </li></ol></li></ol>_title"&gt; 4。纳米粒子的大小调整有形资产净值及质粒每个粒子计算<ol><li>总理的NANOSIGHT NS500射流系统运行稀释剂泵，约五分之一的最大速度和采样泵落后，约十分之一的最大速度。继续，直到射流系统与稀释液冲洗。 </li><li>准备以进行分析的纳米颗粒。以箱子PBAEs，分别稀释存货质粒DNA（1毫克/毫升）和股票PBAE（100毫克/毫升）在乙酸钠缓冲液（25mM的，pH值为5）在Eppendorf管中。 </li><li>稀释的质粒DNA的浓度为0.06毫克/毫升。可以变化，这取决于所需的聚合物的重量的重量比为60 wt / wt的颗粒的DNA（例如，聚合物浓度，聚合物稀释至3.6毫克/毫升）。 </li><li>的聚合物溶液加入到等体积的质粒DNA溶液，并剧烈混合。孵育混合物在室温下搅拌10分钟，以允许自组装。 </li><li>迪琵琶纳米颗粒溶液中的纳米粒子的浓度依次为100倍到PBS在适当的范围内，NTA，以获得最终的体积至少为500微升。 </li><li>装入到NANOSIGHT样品通过将加载管进入样品的Eppendorf管中（ 图4A）。确保不引入气泡。 </li><li>在拍摄模式下，增加相机的水平，，直到颗粒可以看出。调整的重点，使粒子看起来光滑（ 图4A和4B）。 </li><li>而在标准模式下，调整相机的水平，所有的粒子在屏幕上可以看到过去的点。然后减少相机的水平以使得颗粒都可以仍然可以观察到的最低水平。如果中间相机的水平是必要的，进入高级模式，修改相机快门，并获得到适当的水平。 </li><li>目视检查，以确保有20 - 100粒子在Screen。纳米粒子跟踪的一个理想的数字是约50颗。如果有过多或过少，冲洗NS500，调整稀释到PBS，并重新加载样本。 </li><li>调整捕捉持续时间，根据标准模式表。通常为30 - 60秒是一个适当的捕获长度（ 图4B）。 </li><li>加载下一个样品，冲洗系统，然后重新加载新的样本。 </li><li>一旦被捕获的视频，打开一个视频文件进行处理阶段。 </li><li>有许多参数，这些参数可以被调谐，以最好地处理视频（ 图4C）。我们的目标是最好的选择参数，捕捉每一个粒子在屏幕上显示一个红色的十字标记在每个粒子在屏幕上（ 图5），。 </li><li>增加屏幕增益，以更好地看到粒子。在标准模式或高级模式下，选择自动调整的参数clickin克在相应的框中。以自动设置的情况下，不充分选择颗粒，取消勾选框，并手动设置参数（ 图4C）。 </li><li>一旦所有的粒子都挑在屏幕上，单击该进程按钮的软件处理视频文件（ 图4C）。这提供的颗粒尺寸分布，尺寸的平均值，以及颗粒浓度。 </li><li>为了使粒子的浓度是准确的，改变PBS稀释，如增加了2倍的稀释，重复上述步骤。测量多个相同的样品稀释液，建议，以确保样品的浓度测量是精确的。一个很好的测量范围是10 7 -10 9个/毫升。 </li><li>验证质粒纳入凝胶电泳的纳米粒子，纳米粒子，并评估是否有任何免费的DNA是存在的。如果不是所有的质粒被封装，使用标准技术来测量为了量化总封装的质粒DNA的吸光度。 </li><li>要计算每个粒子的平均数量质粒，将总的封装质粒的浓度由国家旅游局测得的颗粒物浓度。为了估计质粒的每个粒子的样本分布，第一使用NTA粒子的大小直方图计算的体积分数各1纳米颗粒斌。总质粒量乘以体积分数分布，以获得在每个容器的质粒。除以这些数字的各bin中的颗粒数，得到的质粒，每个粒子的数目的每个粒子的大小（ 图6）。 </li></ol>

<ol>
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这组作者报告没有利益冲突。

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 ...

evaluation-polymeric-gene-delivery-nanoparticles-nanoparticle

Research

JoVE Journal

Bioengineering

15.8K 次观看.  Johns Hopkins University School of Medicine. 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。

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

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

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

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

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

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these 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

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

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

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

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 ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

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 ...

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

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases.

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with 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

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

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 ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

用于细胞质 siRNA 传递的带电反应性聚合物纳米粒子的制备

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...