This work was supported by the Sheikh Zayed Institute for Pediatric Surgical Innovation (RAC Awards #30000174 and 30001489).

1.合成PB纳米粒子，GdPB和MnPB的的纳米颗粒（纳米颗粒的PB，GdPB或MnPB）的合成是使用一釜合成方案通过执行下面详述的步骤来实现的： <ol><li>制备溶液“A”含有在去离子（DI）水加入5ml的5mM亚铁氰化钾（II）中。取决于纳米颗粒的类型被合成 - PB纳米粒，GdPB或MnPB，制备溶液“B”，如下所示： <ol><li>为 PB纳米粒：制备10ml含有2.5mM的铁（III），氯化在去离子水中的溶液。 </li><li>对于GdPB纳米粒：制备10ml含有2.5mM的每种钆（Ⅲ）硝酸盐，铁（III），氯化在DI水中的溶液</li><li>对于MnPB纳米粒：制备10ml含有2.5mM的每种锰（II），氯化铁（III），氯化在DI水中的溶液。 </li></ol></li><li>溶液“B”添加到一个圆底烧瓶中，并在室温下搅拌该烧瓶内容物（RT）和1000转。添加解决方案&#39;A&#39;逐滴成圆底含液瓶&#39;B&#39;。控制流速通过蠕动泵设置为分配约10毫升/ hr的加入溶液“A”至“B”。 </li><li>继续以1,000rpm在室温加入溶液“A”到“B”之后搅拌另外的30分钟，就完成了。停止搅拌，收集混合物。 </li><li>转移混合物的等分试样到微量离心管中以冲洗游离的未反应成分的纳米颗粒。添加5M的氯化钠（0.2毫升氯化钠/毫升反应混合物）至每个等分试样离心，以协助粒子集合。 </li><li>离心机纳米颗粒的每等份为至少10分钟，在20000×g下。离心后，小心地取出上清。 </li><li>通过使用微尖（超声脉冲超声处理悬浮在1ml去离子水各纳米粒子沉淀的开/关= 1/1秒，振幅= 50％，持续时间=5秒，超声仪额定功率= 125 W），打破了球。 </li><li>重复步骤1.4-1.6至少3×，以确保纳米颗粒是自由的初始反应和反应副产物的组分。最后一次离心后，悬浮在1ml去离子水中的颗粒。 </li></ol> 2. Biofunctionalization PB纳米粒子，GdPB和MnPB的纳米颗粒的Biofunctionalization涉及纳米粒子“芯”与抗生物素蛋白和生物素化的加入配位体的涂层，如下所述： <ol><li>纳米粒子与荧光抗生物素蛋白涂层 用荧光抗生物素蛋白的纳米颗粒的涂层是使用静电自组装来实现，其中带正电荷的抗生物素蛋白（PI〜10.5）上涂覆到带负电荷的纳米颗粒如下18： <ol><li>制备该PB的NP，GdPB或MnPB在1ml去离子水中的悬浮液。过滤的Alexa Fluor 488标记的抗生物素蛋白（A488;重新溶解在DI水中）通过在14000×g的10分钟，用0.​​2μm尼龙微量过滤。添加≤0.2毫克亲/毫克纳米粒子。单独添加A488的每一个过滤分装到PB纳米粒子，GdPB，或MnPB的等分。 </li><li>联系与A488的纳米粒子2-4小时用在4℃轻轻摇动或转动。防止光使用铝箔的样品。 注：此步骤产量A488涂PB纳米粒子（PB-A488），GdPB（GdPB-A488），并MnPB（MnPB-A488）。 </li></ol></li><li>生物素标记的抗体附着 生物素化的靶向抗体上的抗生物素蛋白包覆的纳米微粒的附着用实现抗生物素蛋白 - 生物素的相互作用，如下所示： <ol><li>制备的抗生物素蛋白包覆的纳米微粒的悬浮液 - PB-A488，GdPB-A488，和MnPB-A488在1ml去离子水中。通过0.2μm尼龙微量过滤器过滤生物素化的抗体（由生产商提供），在14000×g的10分钟。 注：在此，本研究采用比奥inylated抗 - 神经元 - 神经胶质抗原2（ANG2）靶向细胞和中枢神经系统的组织和内NG2过表达生物素化的抗 - 人趋化因子-3（Eot3）靶向受体过表达对嗜酸性粒细胞或嗜酸性细胞系。 </li><li>单独添加生物素化的抗体的每个滤波分量，以抗生物素​​蛋白包覆的纳米微粒的等分试样。添加≤0.05毫克生物素化抗体/毫克抗生物素蛋白包覆的纳米微粒。接触抗生物素蛋白包覆的纳米微粒与生物素化的抗体（ANG2或Eot3）2-4小时与在4℃下轻轻摇动或旋转。防止光使用铝箔的样品。 注：此步骤的产量抗体涂覆的纳米颗粒（ 如 GdPB-A488-Eot3和MnPB-A488-ANG2）。 </li></ol></li></ol>纳米粒子的3上浆，Zeta电位和时间稳定性的粒度分布的纳米粒子，电荷和稳定性是使用动态测光散射（DLS）的方法如下所述： <ol><li>纳米粒子的大小 上浆纳米颗粒是使用动态光散射如下来实现的： <ol><li>加10微升的纳米颗粒样品（1毫克/毫升），以990微升去离子水在一个一次性塑料杯中。 注：这是一个很好的信号DLS典型值。 </li><li>帽试管，涡旋拌匀。放置反应杯在用于分析的粒径，以便测量所述纳米颗粒的尺寸的系统。进行粒度分析，在173°的测量角度。 </li></ol></li><li>纳米粒子的Zeta电位 纳米粒子的ζ电位是使用相位分析光散射测量如下： <ol><li>添加100微升的纳米颗粒样品（1毫克/毫升），以900微升去离子水在一个一次性塑料毛细管单元。此值是代表一个好的zeta电位的测量。 </li><li>将毛细ý细胞中用于分析，以测量使用默认参数的纳米粒子的ζ电位在25℃下的ζ电位的系统。 </li></ol></li><li>纳米粒子的时空稳定性 纳米颗粒的经时稳定性，使用动态光散射如下测定： <ol><li>通过测量所述纳米颗粒的尺寸在DI水中以及Dulbecco氏改良的Eagle氏培养基（DMEM），为3.1节中所述评估纳米颗粒的稳定性。 </li><li>重复在DI水和DMEM一次/天的规模测量历时5天。 </li></ol></li></ol> 4.细胞毒性的纳米粒子的纳米颗粒的细胞毒性是使用XTT细胞增殖试验测定如下： <ol><li>种子10,000-15,000细胞/孔的每种细胞类型，在96孔板的研究（的Neuro2A，BSG D10，EOL-1，和OE21）。确保种子细胞的总数量不éxceed0.2毫升/孔。孵育接种的细胞过夜，在37℃和5％的CO 2。 </li><li>接触的纳米粒子与细胞： <ol><li>孵育细胞与不同浓度的纳米颗粒（0.01 - 0.5毫克/毫升）。参照表1作为代表性表描述了纳米颗粒的量（50微升）加入细胞中去除50微升培养基/孔后。 <ol><li>考虑到在测定中的纳米粒子的任何干扰吸光度，添加空白组成的纳米颗粒的适当浓度的（0-0.5毫克/毫升;在等价于添加到细胞中的量），而不细胞培养基。 </li></ol></li><li>孵育细胞过夜纳米颗粒，在37℃和5％的CO 2。抽吸培养基从每个孔和冲洗用在磷酸盐缓冲溶液（PBS）中含有5％胎牛血清（FBS）的染色缓冲液中。加入100微升培养基不含酚红和孵育在37℃和5％CO 2的16-18小时。 </li></ol></li><li>通过混合该试剂盒组分（XTT试剂和XTT活化剂）按照制造商的说明书制备XTT细胞增殖试验工作溶液。从孔中吸出培养基，并添加100微升的RPMI（RPMI W / O型酚红+ 10％FBS + 1×青霉素 - 链霉素），或用于研究的细胞类型合适的培养基。添加50微升XTT工作液到每个孔中，并孵育在37℃和5％CO 2的2-2.5小时。 </li><li>测量490nm处的吸光度，用630nm的参照波长以校正指纹或污迹。计算每口井的校正吸光度（A 490 -A 630）和重复平均读数。 </li><li>减去来自每个井值空白读数来计算最终校正吸光度值。由纳米颗粒未经标准化的最后校正吸光度到未处理的细胞计算存活百分比对每个样品。苏积rvival百分比作为纳米粒子的浓度的函数。 </li></ol> 5. MRI弛豫的PB纳米颗粒的，GdPB和MnPB 核磁共振弛豫是对采用T 1测量-和T 2 -加权序列由如下所述制备的MRI“幻影”使用96孔板含有纳米颗粒： <ol><li>幻影准备<ol><li>制备一个96孔板每孔含有100微升的纳米颗粒（纳米颗粒的PB，GdPB或MnPB）在适当的浓度。用0.4mM的每种类型的纳米颗粒的浓度开始，连续地稀释使用去离子水的纳米粒子2×直至2.4×10 -5 M的达到的浓度（这需要14稀释和15孔）。 </li><li>加入100微升的去离子水熔融1％琼脂糖溶液到每口井拌匀。使凝胶凝固在4℃下12小时。 注意：这产生含有连续稀释的假想纳米粒子在凝固的琼脂糖。 </li><li>放置幻象在水平3个T临床磁铁2％琼脂（150 毫升 ）的一个固体块下。安全的8声道HD大脑线圈中心内琼脂的幻象和块。测量，以0.5毫米厚的冠状切片，在96孔板11的中间高度的弛豫时间。 </li></ol></li><li>牛逼1 -和T2加权序列 使用下面的代表设置用于获取所述序列在临床磁体。 注意：这些值对于在本研究中使用的纳米颗粒的优化： <ol><li>获得Ť1 -加权使用下列液体衰减反转恢复（FLAIR）序列（T1W）MR图像：回声列车（ET）= 8，重复时间（TR）= 2300毫秒，回声时间（TE）= 24.4毫秒，矩阵大小= 512×224，视场（FOV）= 16×16 平方厘米。 </li><li>使用下面的快速relaxa获得T2加权（T2W）MR图像化快速自旋回波（FRFSE）序列：ET = 21; TR = 3500毫秒; TE = 104毫秒;矩阵大小= 512×224; FOV = 16×16 平方厘米。 </li><li>获得在127兆赫（3 T）T1W和T2W磁共振成像在以下反转时间：50，177，432，942，1961，4000毫秒。 </li></ol></li><li>测量MRI弛豫<ol><li>获取使用5.2节中描述的序列T1W和T2W图像后，测量每个使用以及ImageJ的信号强度，从此指定为感兴趣区域（ROI）通过位于主工具栏上的椭圆形按钮作物选择每个ROI区域，然后选择分析&gt;测量。 注：弹出应标有“平均值”栏下显示的每个投资回报率的平均信号强度。 </li><li>对于每一个投资回报率，暗算其特定的反转时间测量信号强度。如果需要的话，反转点（读）产生一个初始的“泡沫”或离群，在剧情的开始（下反转倍）。 注：在低反转时间产生这些读数，因为MRI测量图像，而不是他们的实际（负）值，其中需要进行核算/ 19修正的幅度或绝对值。 </li><li>准备一个指数适合的投资回报（SI）与反转时间（T I）的信号强度每个小区在5.2节所述。情节T I的x轴，SI在y轴; ɑ和Δ是通过回归确定的常数，和T 1或T 2是要解决的变量： <img alt="式（1）" fo:content-width="1.75in" src="/files/ftp_upload/52621/52621eq1.jpg" /> 其中t = T 1，T 2。 </li><li>解决对于T 1或T 2使用上述方程和绘制的计算值（1 / T 1和1 / T 2）针对每个ROI中使用的纳米颗粒的浓度的倒数。 </li><li>计算出的线性图的斜率产生的弛豫R 1和R 2。 注：该协议的以下部分描述了纳米粒子的荧光标记和靶细胞产生MRI造影的应用。 </li></ol></li></ol>有针对性的细胞6.荧光标记使用纳米粒子 - 共聚焦显微镜注：纳米颗粒（纳米颗粒的PB，GdPB和MnPB）可用于荧光标记的靶细胞（通过共聚焦显微镜监测）如下的群体： <ol><li>荧光纳米颗粒的合成<ol><li>通过以下在部分1和2详述的步骤合成GdPB-A488，GdPB-A488-Eot3，MnPB-A488，MnPB-A488-ANG2用于靶向细胞群的荧光标记。 </li></ol></li><li>细胞准备荧光瞄准<ol><li>大衣没有。 1.5微盖玻片浸渍它们在0.002％的聚（L-赖氨酸）氢溴酸盐的90分钟的溶液中。从溶液中取出盖玻片，并使其干燥24小时。 </li><li>种子的细胞（ 如报废-1，BSG D10，SUDIPG1）上涂布的盖玻片放置在6孔板，孵育在37℃和5％CO 2中的至少16小时。冲洗细胞与1×PBS液和（可选），在15分钟的中性缓冲液10％甲醛固定。染色的细胞用5μM的红橙色细胞穿透物染料的PBS，在37℃下进行30分钟。随后，冲洗细胞，用PBS。 </li></ol></li><li> &gt;目标细胞的荧光标记<ol><li>添加1％牛血清白蛋白（BSA;在去离子水）至细胞中以减少非特异性的细胞结合，并在37℃进行1小时。 </li><li>孵育细胞在1％BSA的纳米颗粒1小时。作为EOL-1：孵育0.2毫克/毫升GdPB-A488或GdPB-A488-Eot3;为BSG D10和SUDIPG1：孵育0.2毫克/毫升MnPB-A488或MnPB-A488-ANG2。冲洗细胞，PBS 3×删除unbouND纳米粒子。 </li><li>小心倒置护罩玻璃（含有细胞和纳米颗粒）在显微镜载玻片，以确保没有气泡。通过仔细运用透明指甲油密封盖玻片和载玻片之间的边缘。图像使用共聚焦激光扫描显微镜的细胞。 </li></ol></li></ol>靶细胞7.荧光标记使用纳米 - 流式细胞计数的纳米颗粒（纳米颗粒的PB，GdPB和MnPB）可用于荧光标记的目标细胞群（通过流式细胞术监测）如下： <ol><li>细胞的制备的悬浮液被靶向于PBS中。对于纯文化的研究中，悬浮液由单个细胞类型（ 如 BSG D10）的;悬浮BSG D10细胞在PBS中的细胞沉淀以100,000细胞/ ml（10毫升总）。为混合培养的研究中，悬浮液由至少两种细胞类型（ 如报废-1和OE的21）;类似于BSG D10，EOL-1和OE21在PBS中重悬细胞沉淀以100,000个细胞/ ml的每种（10毫升总）。 <ol><li>制备变报废-1的比率：OE21 1:0（2毫升报废-1），3：1（1.5毫升报废-1，0.5毫升OE21），1：1（1 ml的每种报废-1和OE21的）， 1：3（0.5毫升报废-1，将1.5ml OE21），0：1（2毫升OE21）。 </li></ol></li><li>阻断细胞（纯的和混合的悬浮液）被靶向的纳米颗粒与2ml的5％的BSA，以减少非特异性结合。 </li><li>加入1毫升（1毫克/毫升）的纳米颗粒向细胞孵育1小时。对于BSG D10，培养细胞MnPB-A488，MnPB-A488-ABC（对照抗体），或MnPB-A488-ANG2）。作为EOL-1和OE21的混合物，孵育GdPB-A488-Eot3固定量的混合物。 </li><li>漂洗未结合的纳米颗粒的细胞通过降低盘片样品在1000×g下5分钟，在至少3×以除去未结合的颗粒。重悬细胞于1ml PBS中并用在PBS中的10％的甲醛固定。染色的细胞用10微克/毫升孵育7- Aminoactinomytcin D的PBS在冰上30分钟。冲洗用PBS，然后使用流式细胞仪分析万门控的细胞从每个样品。 </li></ol>在目标单元格中​​的纳米粒子生成8 MRI对比的纳米颗粒（纳米颗粒的PB，GdPB和MnPB）可以被用于产生MRI对比（在两者是叔1 -和T 2 -加权序列）在靶向细胞群如下： <ol><li>幻影准备<ol><li>生长细胞（ 如 BSG D10）在T75瓶中，直到〜80％ ​​汇合。使用适当的生长培养基和条件。为BSG D10，生长的细胞在DMEM + 10％FBS + 1×青霉素-链霉素，在37℃和5％的CO 2。 </li><li>冲洗培养基的细胞用5ml PBS中并阻断细胞与在PBS5毫升的1％BSA进行1小时，以减少非特异性结合。加入5 ml（0.5毫克/毫升）的纳米颗粒的细胞。对于BSG D10，孵化细胞MnPB-A488，MnPB-A488-ABC（对照抗体）和锰过氧化物酶B-A488-ANG2 1小时。 </li><li>冲洗细胞3×5毫升PBS以除去未结合的纳米颗粒。通过孵育细胞用2ml胰蛋白酶的EDTA的0.25％溶液1×5分钟，在37℃和5％的CO 2，以从烧瓶幻像制备分离它们Trypsinize细胞。添加8毫升DMEM中以淬灭细胞的胰蛋白酶消化。 </li><li>通过在1000×g下5分钟离心它们收集细胞;吸出上清液。重悬细胞于1ml PBS中并加入1毫升10％甲醛的PBS固定细胞。添加100微升各样品至96孔板的单独的井。 </li><li>加入100微升的在去离子水中的熔融的1％琼脂糖溶液导入各孔中，并通过上下吹打混匀。使凝胶凝固在4℃下12小时。 注意：这会产生含有细胞在凝固的琼脂糖连接纳米粒子的幻象。 </li><li>放置幻象在水平3个T临床磁铁旁边的2％琼脂的固体块（150厘米<sup&gt; 3）。安全的8声道HD大脑线圈中心内琼脂的幻象和块。测量使用0.5毫米厚的冠状切片，在96孔板11的中间高度的弛豫时间。 </li></ol></li><li>牛逼1 -和T2加权序列 使用以下设置获取临床MRI磁体的序列： <ol><li>使用下面的自旋回波序列T1W获得磁共振图像：回波序列（ET）= 1，重复时间（TR）= 650毫秒，回声时间（TE）= 11毫秒，矩阵大小= 320×256，视场（FOV） = 10×10 平方厘米。 </li><li>使用以下FRFSE序列获得T2W磁共振图像：ET = 28; TR = 3000毫秒; TE = 101毫秒;矩阵大小= 384×288; FOV = 10×10 平方厘米。 </li></ol></li><li>收购后的处理<ol><li>为了方便阅读，转换原有的灰度图像转换成使用ImageJ彩色图像规模：选择图片&gt;类型&gt; 8位，将图像转换为灰度。 </li><li>减去从琼脂糖溶液中的信号的贡献后计算出归一化强度对每个样品。 </li></ol></li></ol>

<ol>
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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=Advance+of+Molecular+Imaging+Technology+and+Targeted+Imaging+Agent+in+Imaging+and+Therapy.">Advance of Molecular Imaging Technology and Targeted Imaging Agent in Imaging and Therapy.</a> Biomed Res Int. 2014, 1-12 (2014).</li><li>Weissleder, R., Pittet, M. J. <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+the+era+of+molecular+oncology.">Imaging in the era of molecular oncology.</a> Nature. 452 (7187), 580-589 (2008).</li><li>Jaiswal, J. K., Mattoussi, H., Mauro, J. M., Simon, S. 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The authors have nothing to disclose.

本文提出的方法为一类新的基于biofunctionalized普鲁士蓝纳米颗粒的多峰分子成像剂的合成。掺入纳米颗粒中的分子成像模式是荧光成像和分子MRI检查，由于它们的互补特征。该biofunctionalized普鲁士蓝纳米粒子具有核 - 壳设计。在这些纳米颗粒的合成中的关键步骤是：1）一釜合成这产生了被包括普鲁士蓝纳米颗粒的核心（PB NPS），含钆普鲁士蓝纳米颗粒（GdPB），或含锰普鲁士蓝纳米颗粒（MnPB），2?...

分子成像是生物过程在细胞，亚细胞和分子水平1的非侵入性的和有针对性的可视化。分子成像允许样品保持其天然的微环境，而其内源性途径和机制实时进行评估。通常情况下，分子成像涉及小分子，大分子，或纳米粒子可视化，目标，和跟踪有关的生理过程的形式的外源成像剂被研究2的管理。已探索了在分子成像的各种成像方式包括MRI，CT，PET，SPECT，超声，光声学，拉曼光谱，生物发光，荧光和活体显微镜3。多峰成像是两种或多种成像模态，其中所述组合增强可视化和表征的各种生物过程和事件4的能力相结合。 Multimoda升成像利用的各个成像技术的优势，同时补偿其个人的局限性3。 本文介绍的协议biofunctionalized普鲁士蓝纳米颗粒的合成（PB NPS） - 一类新的多峰分子成像剂。该PB纳米颗粒被用于荧光成像和分子MRI。 PB是颜料由交替铁（II）和铁（III）中的原子面心立方网络（ 图1）。该PB晶格是由在铁二线性氰化物配体- CN -铁三连杆并入阳离子来平衡其三维网络5内的收费。 PB的掺入阳离子成其晶格的能力是由分别装入钆和锰离子进入该PB纳米粒为MRI造影利用。 对于追求纳米设计的MRI造影的基本原理是因为优点这种设计提供了相对于目前的MRI造影剂。美国FDA批准的MRI造影剂的绝大多数是钆螯合物是顺磁性的性质，并提供正的对比度的自旋-晶格松弛机构6,7,8。相比于单钆螯合物，具有低信号强度在其自己的，纳米颗粒的该PB晶格内的多个钆离子的掺入提供了增强的信号强度（阳性对照）3,9。此外，多个钆离子的PB晶格内的存在增加了整体的自旋密度和纳米颗粒的顺磁性的幅度，这扰乱在其附近的局部磁场，从而产生负反差的自旋 - 自旋松弛机理。因此，含钆纳米粒子两者的作用为T 1（正）和T 2（负）的造影剂10,11。 患者肾功能受损的一个子集，钆造影剂的管理已与肾系统纤维化8,12，13的发展。这一发现促使调查使用替代顺磁离子造影剂的MRI。因此，纳米颗粒的多功能设计适于该PB晶格内掺入锰离子。类似于钆螯合物，锰螯合物也顺磁性，并且通常用来提供正信号强度中的MRI 7,14。作为与含钆PB纳米粒，含锰PB纳米粒也起到为T 1（正）和T 2（负）的造影剂。 掺入 ​​荧光成像能力，所述纳米颗粒“核”涂有“生物功能”外壳组成的荧光标记的糖蛋白抗生物素蛋白（ 图1）。抗生物素蛋白不仅使荧光成像，而且也作为一个对接平台，针对特定的细胞和组织的生物素化的配体。该抗生物素蛋白-生物素键是其特征在于抗生物素蛋白和生物素15之间非常强的结合亲和力最强已知的，非共价键中的一个。生物素化配体与抗生物素蛋白包被的纳米颗粒的PB附着赋予分子的定位功能，在PB纳米颗粒。 动机使用PB纳米粒追求荧光和磁共振成像是因为这些成像模式具有互补的特点。荧光成像是一种最广泛使用的光学分子成像技术之一，并且允许对多个对象中的高灵敏度1,16,17同时可视化。荧光成像是一种安全，无创方式，但与普及率低的深度和空间分辨率1,3,16有关。另一方面，MRI产生高时间的二维空间分辨率非侵入和而不需要电离辐射1,3,16。然而磁共振患有低灵敏度。因此荧光成像和MRI被选定作为分子成像技术由于穿透深度，灵敏度和空间分辨率它们的互补特征。 本文介绍了协议的PB纳米颗粒的合成和biofunctionalization，PB纳米颗粒（GdPB）和含钆PB纳米颗粒（MnPB）10,11含锰。以下方法描述：1）测量的大小，电荷，和纳米颗粒的经时稳定性，纳米颗粒，MRI弛豫3）测定的细胞毒性的2）的评价，和4）利用所述纳米粒子为荧光和分子MR成像的的靶细胞在体外的群体。这些结果表明了纳米颗粒的电势用作体内多峰的，分子成像剂。

<table border="0" cellpadding="0" cellspacing="0" width="854">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:455px;">Name of Material/ Equipment</td>
			<td style="width:115px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium hexacyanoferrate (II) trihydrate (K4Fe(CN)6 &#183; 3H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>P9387</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Manganese (II) chloride tetrahydrate (MnCl2 &#183; 4H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>221279</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gadolinium (III) nitrate hexahydrate (Gd(NO3)3 &#183; 6H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>211591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iron (III) chloride hexahydrate (FeCl3 &#183; 6H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>236489</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-NG2 Chondroitin Sulfate Proteoglycan, Biotin Conjugate Antibody</td>
			<td>Millipore</td>
			<td>AB5320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotinylated Anti-Human Eotaxin-3</td>
			<td>Peprotech</td>
			<td>500-P156GBT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neuro-2a Cell Line</td>
			<td>ATCC</td>
			<td>CCL-131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSG D10 Cell Line</td>
			<td>Lab stock</td>
			<td>---</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OE21 Cell Line</td>
			<td>Sigma-Aldrich</td>
			<td>96062201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SUDIPG1 Neurospheres</td>
			<td>Lab stock</td>
			<td>---</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eol-1 Cell Line</td>
			<td>Sigma-Aldrich</td>
			<td>94042252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly(L-lysine) hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>P1399</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aminoactinomycin D</td>
			<td>Sigma-Aldrich</td>
			<td>A9400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTrace Calcein Red-Orange, AM</td>
			<td>Life Technologies</td>
			<td>C34851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Avidin-Alexa Fluor 488</td>
			<td>Life Technologies</td>
			<td>A21370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peristaltic Pump</td>
			<td>Instech</td>
			<td>P270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zetasizer Nano ZS</td>
			<td>Malvern</td>
			<td>ZEN3600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sonicator</td>
			<td>QSonica</td>
			<td>Q125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hot Plate/Magnetic Stirrer</td>
			<td>VWR</td>
			<td>97042-642</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra Clean Aluminum Foil</td>
			<td>VWR</td>
			<td>89107-732</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex Mixer</td>
			<td>VWR</td>
			<td>58816-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.7 mL conical microcentrifuge tubes</td>
			<td>VWR</td>
			<td>87003-295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL conical centrifuge tubes</td>
			<td>VWR</td>
			<td>21008-918</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tube holders</td>
			<td>VWR</td>
			<td>82024-342</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable plastic cuvettes</td>
			<td>VWR</td>
			<td>7000-590 (/586)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zetasizer capillary cell</td>
			<td>VWR</td>
			<td>DTS1070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifugal Filters, 0.2 micrometer spin column</td>
			<td>VWR</td>
			<td>82031-356</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well cell culture tray</td>
			<td>VWR</td>
			<td>29442-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin EDTA 0.25% solution 1X</td>
			<td>JR Scientific</td>
			<td>82702</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Culture Grade PBS (1X)</td>
			<td>Life Technologies</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XTT Cell Proliferation Assay Kit</td>
			<td>Trevigen</td>
			<td>4891-025-K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T75 Flask</td>
			<td>89092-700</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Biowhitaker</td>
			<td>12-604Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10437-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pen-Strep 1X</td>
			<td>Life Technologies</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluoview FV1200 Confocal Laser Scanning Microscope</td>
			<td>Olympus</td>
			<td>FV1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chambered Microscope Slides</td>
			<td>Thermo Scientific</td>
			<td>154534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Cover Glasses, Square, No. 1.5</td>
			<td>VWR</td>
			<td>48366-227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope Slides</td>
			<td>VWR</td>
			<td>16004-368</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI</td>
			<td>Sigma-Aldrich</td>
			<td>R8758&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACSCalibur Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 T Clinical MRI Magnet</td>
			<td>GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mL round-bottom flask</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

biofunctionalized prussian blue nanoparticles for multimodal molecular imaging applications

多峰的，分子成像允许在使用多个互补的成像技术的蜂窝，亚细胞和分子水平的分辨率的生物过程的可视化。这些显像剂促进途径和机制在体内 ，这对提高诊断和治疗功效的实时评估。本文介绍的协议biofunctionalized普鲁士蓝纳米颗粒的合成（PB NPS） - 一类新的药物用于在多峰分子成像应用。在纳米颗粒，荧光成像和磁共振成像（MRI）掺入所述成像模态，具有互补的特性。该PB纳米粒具有一个核-壳的设计，其中钆和锰离子在PB晶格的间隙空间内掺入产生MRI对比，二者T中1和T 2 -加权序列。该PB纳米粒涂覆有荧光抗生物素蛋白使用静电自如sembly，使荧光成像。该抗生物素蛋白涂覆的纳米颗粒修饰赋予分子的定位功能，以纳米颗粒的生物素化配体。纳米粒子的稳定性和毒性测定，以及它们的核磁共振驰豫。这些biofunctionalized PB纳米颗粒的多，分子成像能力，然后利用他们为荧光成像和体外分子MRI表现。

使用一锅合成方案中，纳米粒子的PB纳米粒（平均直径78.8纳米，多分散性指数（PDI）= 0.230;通过动态光散射仪器来计算），GdPB（平均直径164.2纳米，PDI = 0.102），或MnPB（平均直径122.4纳米，PDI = 0.124），它们是单分散（如通过DLS测得的）可以被一致地合成（ 图2A）。合成纳米颗粒的测得的ζ电位小于-30毫伏（ 图2B），这表明基于它们的表面电荷的粒子的适度稳定性。合成的?...

Watch this Scientific Journal Video about Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications at JoVE.com

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Biofunctionalized普鲁士蓝纳米粒子多式联运分子成像应用

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

biofunctionalized-prussian-blue-nanoparticles-for-multimodal

Research

JoVE Journal

Bioengineering

10.2K 次观看.  Children's National Medical Center. This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting. 

视频: Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

在活细胞中的荧光寿命成像分子转子

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

科研

生物工程

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

宽带超声医疗成像应用，光学检测仪

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

纳米谐波再生研究

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

细胞标记和超顺磁性氧化铁纳米粒子靶向

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

动脉粥样硬化的快速检测的氧化铁纳米颗粒微波驱动综合

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

多峰成像和光谱学纤维束显微内窥镜纲要非侵入性，<em&gt;在体内</em&gt;组织分析

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

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.