这项工作得到了补助1P01DA028555 - 01A1，2R01 NS034239，2R37 NS36126，P01 NS31492，P20RR 15635，P01MH64570，并从美国国立卫生研究院P01 NS43985（HEG）。作者感谢罗宾泰勒女士的手稿和杰出的图形和文学支持的关键读。我们要感谢他的湿铣削的专业知识，史蒂夫Grzelak。我们还要感谢汉臣的内布拉斯加 - 林肯电子显微镜的核心设施大学博士提供的扫描和透射电子显微镜图像。最后，我们想感谢她的专业知识采用现场共聚焦显微镜梅根Marquart。

NanoART制造 1。 NETZSCH MicroCer方法湿磨NanoART <ol><li>称取药物的结晶和各种表面活性剂，将用于nanoformulations使用分析天平和10 mM的HEPES缓冲液，pH值7.8使用T - 18 Ultraturrax调音台，直至完全分散混合。在制定该药物的％应在1 - 2％之间。 </li><li>对于连续铣削，确保冷水机组和压缩机。出口压力应开始磨你的样品之前，大约100 PSI。 </li><li>打开控制单元，使研磨介质，可装载在坦克和旋转的研磨罐。 </li><li>添加50毫升的珠子粉碎室，利用一个漏斗。确保有研磨腔以外的线程或任何没有珠子，以避免在机械密封泄漏。 </li><li>确保O形圈在其研磨腔。选择一个合适的筛网的大小和使用适当的盖子大会和安全的盖子，拧紧螺母。筛网的大小应至少有一半的珠子的大小。使用大屏幕的网目尺寸，以避免堵塞过滤器而连续铣削的产品。 </li><li>降低研磨室，使横向使用室顶部的旋钮，并确保产品出口管进入收集容器清空。 </li><li>添加不同量的表面活性剂产品入口药物停用。暂停的最低量应该是100毫升。这将防止过热研磨腔，也避免因零件磨损少珠污染</li><li>启动泵使用的控制单元。流速为您的配方（〜50 - 150毫升/分钟）的要求，可以是多种多样的。 </li><li>打开搅拌器和速度控制单元上的调整。从620转的速度可提高到4320 MicroCer的转速。监测温度，使用温度计附着在上面的研磨腔，并确保有没有过热。 </li><li>穆勒以不同的速度和流速的样品，并采取了制定使用衍射光散射布鲁克海文国家实验室的激光粒度仪（见表1）的大小和电荷测量等分的小（〜1毫升）。 </li><li>获得所需的大小后，停止使用控制单元的铣床。泵，一个烧瓶中收集到的样本。允许的药物，以完全排入样品采集瓶。 </li><li>关闭泵。要消除的珠子，地方单位收集盘。松开前面板上的盖子大会上的螺母，并收集在托盘的珠子。起飞前板，并使用去离子水瓶，洗珠入盘内。 </li><li>转移到50 mL离心管球磨悬挂和设置离心机10,000 RPM为30分钟（10174 RCF）。离心机周期完成后，测量上清液的数量和相同数量的新鲜表面活性剂溶液取代。 </li><li>一旦再悬浮完成后，将暂停10分钟的研磨腔和磨。等分（1ML），并再次测量的大小和样品费，使用激光粒度仪（见表1）。 </li><li>执行MicroCer使用去离子水和70％的乙醇后使用清洁。 </li><li>测量浓度采用HPLC nanoART配方。在我们的例子中，我们评估样品反相高效液相色谱法使用一式三份到一个YMC辛基C8柱与C8保护柱（WATERS公司，米尔福德，马萨诸塞州）20μL，注射。流动相组成为48％乙腈/ 52％25mm的KH 2 PO 4，pH值4.15，0.4毫升/分钟，在272纳米的紫外/可见检测的泵浦。所有药物的测量，定量确定的每一个免费的药物（0.025-100微克/毫升）的甲醇配制的标准曲线比较。 </li></ol> 2。 NanoART同质化使用Avestin EmulsiFlex C5 <ol><li>衡量药物的晶体和各种表面活性剂将用于nanoformulations使用分析天平和10 mM的HEPES缓冲液，pH值7.8使用T - 18 Ultraturrax龙头，以获得完整的分散混合。 </li><li>传输的悬浮液的均质容器，并开始回流。确保冷水机组开始前均质样品</li><li>增加的压力，才渐渐米读取20000 ± 2000 PSI，继续同质化，直到实现目标粒径。这通常需要45 - 60分钟之间。定期采取等份（约1毫升）和测量使用激光粒度仪（见表1）样本的规模和负责。 </li><li>匀浆器匀浆悬浮转移至50 mL离心管中，并设置离心机10,000 RPM为30分钟（10174 RCF）。完成后离心机的周期，测量上清量，更换新鲜的表面活性剂溶液等体积。 </li><li>再悬浮后，将悬挂在C5匀浆。重新启动的流通和20000 ± 2000 PSI返回均质压力为30分钟，均质。测量使用的激光粒度仪（见表1）制定的大小和电荷。 </li><li>执行的单位后，与去离子水和乙醇为溶剂清洗。 </li><li>测量使用高效液相色谱法样品的浓度。 </li></ol> 3。超声使用科尔帕默超声处理器<ol><li>测量一个玻璃烧杯50 mL二氯甲烷。测量6克聚（乳酸 - 乙醇酸）（PLGA）使用分析天平，添加二氯甲烷，搅拌至完全溶解观察。 </li><li>测量1.25克毒品晶体，并添加二氯甲烷/ PLGA的解决方案。组合，以获得完全溶解。 </li><li> 1％的聚乙烯醇（PVA）表面活性剂的解决方案，并用冰浴冷却。确保除了包含溶解药物的有机溶液，表面活性剂溶液冷却后再。 </li><li> 1％PVA表面活性剂溶液倒入药物溶液，并开始在50％幅度的ultrasonicator。确保表面活性剂溶液，放置在冰浴。 sonicator幅度可以减少〜30％的幅度为小批量的样品。超声为10分钟的样品。 </li><li>取出一个小粒度分析（见表1）等分（〜1毫升）。如果粒子的大小是大于1.5微米，超声样品在2分钟的时间间隔最多共16分钟。在20X和文件的意见（图1A），在显微镜下观察的样本。 </li><li>使用搅拌板创建其余悬挂提供足够的旋涡，并继续在室温下混合过夜。 </li><li>称取8克甘露醇烧瓶，添加80毫升的RO水。混合直到完全溶解，并在室温下保存。这将用于离心后，如果样品需要冻干再悬浮。 </li><li>收集24小时后暂停，并倒入50 mL离心管。样品离心20分钟8000转的速度在5 ° C。 75毫升过滤反渗透（RO）水和75毫升0.5％的十六烷基三甲基铵（CTAB）表面活性剂的另一半，倒出上清，悬浮其中一半的样本。 </li><li>再次在20分钟8000转的速度离心样品在5 ° C，共20毫升甘露醇溶液重悬颗粒。 </li><li>第二次再悬浮后，测量使用激光粒度仪（见表1）粒子的大小。 </li><li>使用合适的小瓶，把剩余的悬浮液，并放入冻干机。 </li><li>测量使用高效液相色谱法样品的浓度。 </li></ol> 4。 NanoART形态<ol><li>采取nanoART悬挂和简要超声5秒用一个超声探头在20％的幅度。转移到1.5 mL的RO水10μL的悬浮液，在1.7 ml离心管和10秒的旋涡。 </li><li>以水的nanoART解决方案，并转移到一个Swinnex 13 0.2μm的聚碳酸酯滤膜（Nuclepore轨道蚀刻）聚丙烯过滤器组装持有人组成的一个过滤装置的50μL样品。过滤装置是与50μL的0.2微米过滤蒸馏水稀释药物悬挂前催芽。真空，然后将其应用于仪器，直到整个溶液的体积已经完全拉过去的过滤膜。 </li><li>一旦膜是干的，它是铝针存根使用双节棍导电碳胶带贴和溅射涂层与钯。标本存根，然后成像的JEOL JSM6300F低电压，场发射扫描电子显微镜（图2），在我们的情况下，使用。 </li></ol> 5。 NanoART可视化<ol><li>以准备nanoART悬浮液和超声为10秒，他们用一个超声探头在20％的幅度。 </li><li>载玻片盖玻片，放置在一个倒置显微镜。 15μL与100μL磷酸盐nanoART悬挂在盖玻片混合缓冲液（PBS）和混合移液器向上和向下几次。使用20倍的目标（图3）可视化的纳米粒子。 </li></ol> 对nanoART的生物学 6。血源性单核细胞和单核细胞衍生的巨噬细胞（MDM）的分离和制备<ol><li>人力leukapheresis从HIV - 1和肝炎血清阴性的捐助者获得的单核细胞和净化的细胞逆流离心淘洗。评估抗CD68（克隆KP免疫标记细胞纯度-1），赖特 - 染色cytospins 1。 </li><li>辅以10％热灭活汇集人类血清，1％谷氨酰胺，50微克/毫升庆大霉素，10微克/毫升环丙沙星和1000 U / mL的重组人巨噬细胞集落刺激因子（MCSF）DMEM培养液培养纯化单核细胞在浓度为1 × 10 6细胞/ mL，在37 ° C在与5％的CO 2饱和湿度。板每孔2 × 10 6细胞在6孔板和培养细胞中，以7天为使单核细胞分化成巨噬细胞1。 （图3A） </li></ol> 7。细胞摄取及征收<ol><li>与培养基混合nanoART（无MCSF）至终浓度为100μm和治疗液添加1毫升，每口井。 </li><li>在所需的时间点，或细胞完全符合nanoART（图2）加载时，删除所有治疗介质，并用1 mL PBS洗涤细胞3次，以去除颗粒细胞在尚未采取。在1 mL的PBS液，删除通过使用一个细胞升降机井底的贴壁细胞和它们放入1.7 ml离心管2-4。 </li><li>离心机在1000 RCF的细胞在4 ° C下10分钟。去除上清，加入200μL100％甲醇。裂解细胞，溶解简要nanoART（2-5秒），用一个超声探头超声沉淀在20％的幅度。商店样品-80 ° C直到准备来分析药物含量。 </li></ol> 8。细胞内保留的NanoART <ol><li>治疗如7.1中所述的MDM。 </li><li>在所需的时间点，洗如7.2中所述的细胞，但立即刮单元格，而不是固定在0.1 M磷酸缓冲液（pH值7.4）的3％戊二醛溶液在室温下15分钟的细胞，并进一步修复10 10分钟，在0.1 M磷酸缓冲液（pH 7.4）中，用1％四氧化锇。 </li><li>删除固定液在1 mL PBS和刮细胞如7.2中所述。此外，收集和离心机在7.2中描述的细胞，但加入甲醇沉淀，而不是添加戊二醛固定液200μL。 </li><li>一个切片，切成薄（80 nm）的部分细胞沉淀。用醋酸铀和柠檬酸铅染色的部分。观察染色切片使用透射电子显微镜（图4）。 </li></ol> 9。艺术发行<ol><li>治疗细胞在7.1-7.2描述，但是，洗涤后收集细胞，而不是取代PBS 2 mL细胞培养基无MCSF没有nanoART。 </li><li>在表示天，或如细胞培养转向黄色，一半的介质交换的介质。 </li><li>在所需的一天，收集1 mL的细胞液中中，以及在7.2-7.3所述的复制细胞。 </li><li>在7.3中描述的过程单元。中等样品150μL，加入1毫升100％甲醇和10秒的全速涡的过程介质。然后离心10分钟，在21000 RCF的样本。去除上清转移到新管。甲醇蒸发真空离心机，这可能需要从1到4小时，根据样品。商店样品在-80 ° C，直到准备好药物分析。 </li></ol> 10。共聚焦显微镜实时摄取的NanoART <ol><li>从成熟的MDM 6.2步骤和标签使用电池的标签解决方案，如Vybrant细胞标记解决方案制造的指令后的细胞。冲洗细胞3次，用1毫升培养液中，以消除任何多余的染料。 </li><li>与培养基混合，在步骤7.1中使用荧光标记的nanoART nanoART。 </li><li>添加标记nanoART前夕共聚焦显微镜成像处理介质。使用60X的目标5（视频1＆2）4个小时，每30秒拍摄图像。 </li></ol> HIV - 1感染和感染性的实验 11。 HIV - 1 ADA的感染<ol><li>治疗在9.1-9.3所述的细胞。 </li><li>所需的天数，删除所有中等和中等更换，没有MCSF，包含在0.01病毒颗粒/细胞孵育24小时1的多重感染HIV - 1 ADA的。删除病毒的媒介和更换新鲜，无病毒的传播媒介。 10天培养细胞交换的一半中期隔日或在必要时保持细胞存活（图5）6。 </li><li>感染后的十天收集10μL介质从每口井，地点在96孔板，并储存在-80 ° C，直到准备好执行逆转录病毒（反向）转录分析。 </li></ol> 12。逆转录酶（RT）的含量<ol><li>每个步骤11.3 10μL样品，10μL一个解决方案，包含100毫米的Tris - HCl（pH值7.9），氯化钾300毫米，10毫米数码地面电视，0.1％，壬基酚phenoxylpolyethoxylethanol - 40（NP - 40），和水混合。孵育15分钟，这种混合物在37℃7。 </li><l<html>我&gt;然后添加25μL的解决方案，其中包含50毫米的Tris - HCl（pH值7.9），氯化钾150毫米，5毫米数码地面电视，MgCl 2的 15毫米，0.05％NP - 40，10微克/毫升聚（一），0.250寡核苷酸的U / mL的D（T）12-18，和10μCi/ 毫升 3的H - TTP的每口井和孵化，在37 ° C时18小时7 。 </li><li>孵化后，每孔加50μL冰冷的10％三氯乙酸（TCA）的。收获到玻璃纤维过滤器的井，并评估β-闪烁光谱7 3H - TTP的法团的过滤器。 </li></ol> 13。 HIV - 1p24侦测<ol><li>洗净从逆转录病毒酶液样品1 mL的PBS 3次液冲洗步骤11.3删除复制细胞。 </li><li>一夜之间修复细胞，用4％多聚甲醛4 ° C。第二天早晨，用PBS冲洗细胞3次。 </li><li>使用鼠标的HIV - 1 P24（1:10，Dako公司，Carpinteria的CA）的单克隆抗体，感染艾滋病毒的可视化。明场下的图像细胞，使用40倍的目标（图6）。 </li></ol> 14。代表性的成果： <table border="1"><tbody><tr><td> 药物 </td><td> 尺寸 （NM） </td><td> 交车 </td><td> 充电 （MV） </td><td> 表面活性剂 </td></tr><tr><td> IDV </td><td> 1052 </td><td> 0.286 </td><td> -24.58 </td><td> P188，5.0％SDS，9.25％，蔗糖0.5％ </td></tr><tr><td>室温</td><td> 233 </td><td> 0.273 </td><td> -7.47 </td><td> P188，0.5％，9.25％蔗糖</td></tr><tr><td> ATZ </td><td> 840 </td><td> 0.192 </td><td> 25.47 </td><td> P188 0.5％，0.1％MPEG 2000 - DSPE，1.0％DOTAP，9.25％蔗糖</td></tr><tr><td> EFV </td><td> 347 </td><td> 0.235 </td><td> -13.52 </td><td> 1.0％PVA </td></tr></tbody></table> nanoART物理特性见表1。 表显示使用指定的表面活性剂生产nanoART配方的物理特性，潜在的代表值。值包括根据散射光的强度，多分散指数（PDI，粒度分布的估计），以及各种nanoformulation样品获得的zeta电位值的平均直径。在表中使用的缩写：IDV：茚; RTV：利托;亚视：阿扎那韦; EFV：effavirenz;聚乙烯醇：聚乙烯醇; SDS十二烷基硫酸钠; P188：泊洛沙姆188（也称为聚醚F68），MPEG 2000 - DSPE：聚甲基（1,2 - 乙烯 - 乙二醇）distearoyl -磷脂乙醇胺; DOTAP：（1 -油酰- 2 - [6 - [（7 - 硝基- 2 - 1 ,3 - benzoxadiazol -4 - 基）氨基] hexanoyl] - 3 - 三甲基丙烷。 <img src="/files/ftp_upload/2460/2460fig1.jpg" alt="图1" /> 图1总结用于制造nanoART各种方法的流程图。 图1总结了各种方法用来制造nanoART。流程图包括各自方法的每个关键阶段的预期时间分配。 <img src="/files/ftp_upload/2460/2460fig2.jpg" alt="图2" /> 图2。受欢迎和不受欢迎的NanoART形态的代表图像。 ，扫描电子显微镜分析的RTV nanoART（放大倍率，15000 X）生产的同质化，湿磨，超声​​和0.2μm的聚碳酸酯滤膜上。测量栏等于2.0微米的所有帧。可取nanoART组成，平均而言，小（≤2微米）自足的颗粒，边缘光滑往往有相同或相似的的形状。不良nanoART的大小和形状差别很大，保险丝和/或粘到另一个。 <img src="/files/ftp_upload/2460/2460fig3.jpg" alt="图3" /> 图3。可取nanoART解决方案和MDM的高达nanoART的图像。明场显微镜图像（所有使用20倍的目标收购）的匀浆的RTV - NP和MDM高达nanoART。一个玻璃盖滑的顶部与100μL的PBS 10μLRTV - NP的解决方案相结合后，粒子是可见的，只有几个较大的颗粒被单独识别（一）类似的沙子。图像完全分化和主轴nanoART治疗前（二）形的MDM。后，细胞已nanoART，他们变深，其原子核变得更加明显，由于nanoART核周分布，但是，他们仍然保持他们的主轴形细胞体（C）。一旦细胞超载与nanoART，变得模糊不清的核;细胞变得圆润，失去了自己的主轴形结构，并可能从底部以及（四）分离。 4“/&gt; 图4。nanoART蜂窝纳入到MDM的确认。透射电子显微镜（放大倍率，15000 X）演示湿磨（二），超声（三）RTV - NP的，未经处理的细胞和（D从同质化（A）的RTV - NP暴露的RTV - NP的MDM nanoART摄取）。 nanoART细胞内，应易于识别其几何形状。请注意没有任何明显的几何结构，在控制细胞（四）。已经概述了每个粒子的一个例子：为同质化（一）红色，蓝色为湿磨（二），（三）超声绿色。颗粒结构应类似，这是使用SEM。测量栏等于5.0微米的所有帧。 <img src="/files/ftp_upload/2460/2460fig5.jpg" alt="图5" /> 图5。nanoART抗逆转录病毒药物的疗效测试方法示意图。为了测试nanoART能力，抑制病毒复制的MDM必须首先与nanoART治疗，然后暴露于HIV - 1 ADA的。类似的艺术释放研究，MDM与nanoART加载，然后洗净，以消除任何没有内在的颗粒。 NanoART载货MDM再培养一个半换液隔日至15天。在这段时间内（一般为1天，5，10和15）的MDM与HIV - 1 ADA的挑战。每个病毒接触细胞培养后，再过10天，以便感染的进展。第10天感染后的媒体样本收集RT分析和p24抗原的固定和染色的细胞。非nanoART处理MDM中，感染和未感染，也培养并行存在p24抗原和RT活性测试。 <img src="/files/ftp_upload/2460/2460fig6.jpg" alt="图6" /> 图6。HIV - 1感染的MDM P24染色nanoART效果的测试。 RTV - NP（20X目标）明场图像处理的MDM后10日内与HIV - 1 ADA的挑战。无感染时存在细胞virus1一天后nanoART治疗（一）挑战;注意的NP的大多数细胞（插图用箭头表示）细胞质内的存在。当细胞暴露nanoART治疗（二）后10天病毒感染目前，虽然少得多比不nanoART治疗的细胞（D），注意保持的NP在某些细胞（乙插图用箭头表示）的存在，但少于每日1次后nanoART治疗（插图）。既不nanoART也不感染（三）处理的细胞图像;注意缺乏细胞的细胞质内的NP（插图）。没有与nanoART治疗，但暴露于HIV - 1 ADA（四）的细胞的图像。 视频1。活细胞MDM共聚焦显微镜差RTV - NP的摄取。 MDM的荧光显微镜Vybrant迪欧细胞标记的解决方案，并标有绿色，红色标记的RTV - NP治疗。一个图像60X放大倍率为4小时，每隔30 <a href="http://www.jove.com/files/ftp_upload/2460/Video%201.mov">秒点击这里观看</a>视频 视频（2）活细胞共聚焦显微镜成功的RTV - NP MDM的摄取。 MDM的荧光显微镜Vybrant迪欧细胞标记的解决方案，并标有绿色，红色标记的RTV - NP治疗。一个图像60X放大倍率为4小时，每隔30 <a href="http://www.jove.com/files/ftp_upload/2460/Video%202.mov">秒点击这里观看</a>视频

<ol>
	<li>Gendelman, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+and+propagation+of+human+immunodeficiency+virus+on+recombinant+colony-stimulating+factor+1-treated+monocytes.">Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor 1-treated monocytes.</a> J Exp Med. 167 (4), 1428-1441 (1988).</li><li>Dou, H. <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+a+macrophage-based+nanoparticle+platform+for+antiretroviral+drug+delivery.">Development of a macrophage-based nanoparticle platform for antiretroviral drug delivery.</a> Blood. 108 (8), 2827-2835 (2006).</li><li>Dou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+investigations+for+the+morphologic,+pharmacokinetic,+and+anti-retroviral+properties+of+indinavir+nanoparticles+in+human+monocyte-derived+macrophages.">Laboratory investigations for the morphologic, pharmacokinetic, and anti-retroviral properties of indinavir nanoparticles in human monocyte-derived macrophages.</a> Virology. 358, 148-158 (2007).</li><li>Dou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+delivery+of+nanoformulated+antiretroviral+drug+to+the+brain+in+a+murine+model+of+neuroAIDS.">Macrophage delivery of nanoformulated antiretroviral drug to the brain in a murine model of neuroAIDS.</a> J Immunol. 183 (1), 661-669 (2009).</li><li>Nowacek, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoformulated+Antiretroviral+Drug+Combinations+Extend+Drug+Release+and+Antiretroviral+Responses+in+HIV-1-Infected+Macrophages:+Implications+for+NeuroAIDS+Therapeutics.">Nanoformulated Antiretroviral Drug Combinations Extend Drug Release and Antiretroviral Responses in HIV-1-Infected Macrophages: Implications for NeuroAIDS Therapeutics.</a> J Neuroimmune Pharmacol. , (2010).</li><li>Nowacek, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoART+synthesis,+characterization,+uptake,+release+and+toxicology+for+human+monocyte-macrophage+drug+delivery.">NanoART synthesis, characterization, uptake, release and toxicology for human monocyte-macrophage drug delivery.</a> Nanomed. 4 (8), 903-917 (2009).</li><li>Kalter, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+HIV+replication+in+macrophage+colony-stimulating+factor-treated+monocytes.">Enhanced HIV replication in macrophage colony-stimulating factor-treated monocytes.</a> J Immunol. 146 (1), 298-306 (1991).</li></ol>

NanoART旨在改善HIV - 1病毒疗法。作为第一步，以供临床使用这种技术的发展和作为一个用于nanoformulated药物开发的实验室测试系统，我们提出一个基于单核细胞 - 巨噬细胞的药物输送系统。我们过去的作品表明，该系统是这样一个应用程序 5,6可行的。 在目前的报告，我们已经说明了合成常用的HIV - 1药物（茚地那韦，IDV; ritonovir，室温;依法韦仑，依非韦伦和阿扎那韦，亚视）的NP方法。这是通过湿磨，同质化和超声。重要的是，我们的做法，允许修改nanoART物理特性，如大小，形状和电荷5,6。通过精心挑选的表面活性剂，可以控制高度负电荷的粒子从的中性非常积极的。通过改变处理的时间和强度，控制规模，并呈现小≤200纳米或为≥3微米大的粒子。也可以控制粒子的形状，但以一个较小的程度上比其他的物理特性。例如，球形颗粒，可通过与聚合物超声可以通过湿磨或同质化生产，而多面的几何形状。湿磨和同质化生产不同形状的颗粒，但这个过程是依赖于分馏法，并不能直接控制的。这些属性的制造方法可以使研究容易产生nanoART精确的设计规格。自nanoART的理化性质，其稳定后，双方有一个强大的效果，以及如何与细胞相互作用，这是非常重要的的。例如，我们已经表明nanoART约1微米大小的，具有很强的正电荷，并更迅速地采取多方面的几何形状是由巨噬细胞，一旦细胞内更稳定，并在较长时期内公布时间6。 开发一种测试nanoART方法是，允许粒子的能力应采取的简单筛选，保留，以及由巨噬细胞，原则靶细胞的HIV - 1之一，除了自己的能力，抑制病毒复制，发布。这可以轻松区分nanoART基于性能，并确定那些已经成功地在体内模型，因此，增加发展nanoART人类使用的效率和速度的最佳机会。 它已被证明小鼠骨髓骨髓巨噬细胞，当满载着nanoART 体外静脉注射过继转移到人性化的艾滋病毒1感染小鼠旅游活动性感染的网站，包括中枢神经系统，并释放药物长达2周抑制病毒复制2-4。此外，这些nanoART加载细胞也能有效地降低血浆中的病毒感染的细胞数量，淋巴结，脾，肝和肺以及保护CD4 +细胞2。 ，虽然是初步的，这些研究表明，一个细胞介导的​​nanoART交付系统可以分发药物临床大量血液和受感染的组织。由于从HIV - 1感染小鼠的研究令人鼓舞的初步结果，我们目前正在开发一个猴免疫缺陷病毒感染的猕猴模型进一步检验的nanoART供临床使用的潜力。

在表中使用的缩写：IDV：茚; RTV：利托;亚视：阿扎那韦; EFV：effavirenz;聚乙烯醇：聚乙烯醇; SDS十二烷基硫酸钠; P188：泊洛沙姆188（也称为聚醚F68），MPEG 2000 - DSPE：聚甲基（1,2 - 乙烯 - 乙二醇）distearoyl -磷脂乙醇胺; DOTAP：（1 -油酰- 2 - [6 - [（7 - 硝基- 21 ,3 - benzoxadiazol -4 - 基）氨基] hexanoyl] - 3 - 三甲基丙烷）; PLGA：聚（乳酸 - 羟基酸）; CTAB法：十六烷基三甲基铵溴化物; DMEM培养液：贝科的改性鹰中等MCSF：巨噬细胞集落的刺激因子;高效液相色谱法：高压液体色谱仪; HIV - 1病毒：人类免疫缺陷病毒1

methods development for blood borne macrophage carriage of nanoformulated antiretroviral drugs

Nanoformulated药物可以提高药效和生物利用度，同时也降低抗逆转录病毒药物的药物毒性。为此，我们的实验室已应用纳米的原则，简化抗逆转录病毒疗法，如降低毒性，同时提高了遵守和药物的药代动力学。简单和可靠的方法制造nanoformulated艺术（nanoART）所示。纯药物粒子是由表面活性剂的脂质涂料薄薄的一层封装，要么湿磨或高压均质成小的分馏较大的药物晶体产生。一种替代方法是悬浮在一种聚合物液滴中游离药物。在这里，药物溶解然后由超声波激动，直到个别纳米液滴形成的聚合物内。动态光散射和镜检表征粒子的物理特性（颗粒大小，电荷和形状）。其生物学特性（细胞吸收和潴留，细胞毒性和抗逆转录病毒药物的疗效）与人类单核细胞衍生的巨噬细胞（MDM）的决定。 MDM的是来自人类从使用净化离心淘洗leukopacks分离的外周血单核细胞。这种血源性的巨噬细胞可作为nanoART分配到人类免疫缺陷病毒（HIV）感染的器官的细胞转运。我们断定临床提供抗逆转录病毒药物的重新包装成纳米粒子为HIV - 1治疗可改善合规性和积极的影响疾病结果。

Watch this Scientific Journal Video about Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs at JoVE.com

Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs

茚地那韦，利托那韦，依非韦伦和阿扎那韦的纳米颗粒制造，采用湿磨，同质化和超声。这些nanoformulations，统称nanoformulated抗逆转录病毒疗法（nanoART），评估巨噬细胞为基础的药物交付。单核细胞衍生的巨噬细胞nanoART摄取，保留和缓释进行了测定。这些初步的研究表明，nanoART临床使用的潜力。

方法Nanoformulated的抗逆转录病毒药物的血源性巨噬细胞运输的发展

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To ...

methods-development-for-blood-borne-macrophage-carriage

Research

JoVE Journal

Bioengineering

13.1K 次观看.  University of Nebraska Medical Center. 茚地那韦，利托那韦，依非韦伦和阿扎那韦的纳米颗粒制造，采用湿磨，同质化和超声。这些nanoformulations，统称nanoformulated抗逆转录病毒疗法（nanoART），评估巨噬细胞为基础的药物交付。单核细胞衍生的巨噬细胞nanoART摄取，保留和缓释进行了测定。这些初步的研究表明，nanoART临床使用的潜力。

视频: Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

荧光检测方法，为微流体液滴平台

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

科研

生物工程

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

方法表征的共同发展生物膜和生境异质性

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

在生理体外模型和病理血流应用的血管细胞重塑调查

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

全自动离心微流体装置从全血超灵敏蛋白检测

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

生长在微重力人体肠道黏膜的多细胞三维器官模型的建立

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs through the blood-brain barrier (BBB), causing difficulties for effective pharmacological therapies. In recent years, different strategies for promoting brain targeting have aimed to improve drug delivery and activity at this site, including innovative nanosystems for drug delivery across the BBB. In this context, an in vitro approach based on a simplified cellular model of the BBB provides a useful tool to investigate the effect of nanoformulations on the trans-BBB permeation of molecules. This study describes the development of a double-layer BBB, consisting of co-cultured commercially available primary rat brain microvascular endothelial cells and astrocytes. A multiparametric approach for the validation of the model, based on the measurement of the transendothelial electrical resistance and the apparent permeability of a high molecular weight dextran, is also described. As proof of concept for the employment of this BBB model to study the effect of different nanoformulations on the translocation of fluorescent molecules across the barrier, we describe the use of fluorescein isothiocyanate (FITC), loaded into ferritin nanoparticles. The ability of ferritins to improve the trans-BBB permeation of FITC was demonstrated by flux measurements and confocal microscopy analyses. The results suggest this is a useful system for validating nanosystems for delivery of drugs across the BBB.

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules.

跨越大鼠血脑屏障FITC加载铁蛋白体外渗透：一个模型来研究Nanoformulated分子的交付

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

组织工程应用中植物组织去细胞的两种方法

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial bypass. Characterization of the growth dynamics and remodeling process of degradable polymer scaffold-based tissue-engineered blood vessels (TEBVs) with pulsatile stimulation is crucial for vascular tissue engineering. Optical imaging techniques stand out as powerful tools for monitoring vascularization of engineered tissue enabling high-resolution imaging in real-time culture. This paper demonstrates a nondestructive and fast real-time imaging strategy to monitor the growth and remodeling of TEBVs in long-term culture by using optical coherence tomography (OCT). Geometric morphology is evaluated, including vascular remodeling process, wall thickness, and comparison of TEBV thickness in different culture time points and presence of pulsatile stimulation. Finally, OCT provides practical possibilities for real-time observation of the degradation of polymer in the reconstructing tissues under pulsatile stimulation or not and in each vessel segment, by compared with the assessment of polymer degradation using scanning electron microscopic(SEM) and polarized microscope.

A step by step protocol for nondestructive and long-period monitoring the process of vascular remodeling and scaffold degradation in real-time culture of biodegradable polymeric scaffold-based tissue-engineered blood vessels with pulsatile stimulation using optical coherence tomography is described here.

基于光学相干断层扫描的可降解支架组织工程血管发育的无损监测

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

心脏内侧燃光导管,用于监测细胞代谢,使用渗透哺乳动物心脏的透射吸收光谱

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...