The financial support of the Max-Planck Society, the Turkish Ministry of National Education (PhD fellowship to S. G.), and the German Exchange Academic Service (DAAD, PhD fellowship to T. S.) are gratefully acknowledged.

1. DCA的制备<ol><li> 单体单元4 6的合成。 <ol><li> 4-（4-硝基苯基）-2-（4,7,10-三- 叔丁氧基羰基环十二烷-1,4,7,10-四氮杂-1-基）丁酸叔丁基酯（2）。 <ol><li>溶解（4,7-双- 叔丁氧基羰基- 1,4,7,10-四氮杂cyclododec -1-基）在N -乙酸叔丁基酯1（1.00克，1.94毫摩尔），N-二甲基甲（ DMF，5ml）中，加入碳酸钾（0.67克，4.86毫摩尔，2.5当量）和在室温下搅拌45分钟该混合物。 注:大环1从CYCLEN制备和叔根据先前公布的程序7 -丁基溴。 </li><li>添加叔丁基-2-溴-4-（4-硝基苯基）丁酸甲酯（0.87克，2.53毫摩尔，1.3当量）分批1小时。继续下ŧ搅拌混合物他相同的反应条件下18小时。 注: 叔丁基-2-溴-4-（4-硝基苯基）丁酸根据先前公布的程序8由4-（4-硝基苯基） -丁酸，亚硫酰氯和溴制备。 </li><li>通过球对球真空蒸馏的手段在40-60℃9除去DMF。 </li><li>将该残余物通过柱色谱法（硅胶，7％甲醇/二氯甲烷），得到产物2，为棕色无定形固体（1.09克，72％）10。 </li></ol></li><li> 4-（4-氨基苯基）-2-（4,7,10-三- 叔丁氧基羰基环十二烷-1,4,7,10-四氮杂-1-基）丁酸叔丁基酯（3）。 <ol><li>溶解硝基苯衍生物2（1.00克，1.28毫摩尔）在乙醇（10毫升）和甲醇（150微升）的7N氨溶液。活性炭上添加钯作为催化剂（钯/炭，150毫克，15重量％）至soluti上。 </li><li>摇在帕尔氢化装置中在氢气氛（2.5巴）下16小时的非均相混合物。 </li><li>通过它在乙醇中悬浮，并通过烧结的玻璃漏斗过滤该悬浮液制备硅藻土饼。倾从1.1.2.2悬浮在所制备的滤饼通过过滤除去Pd / C催化剂。 </li><li>在旋转蒸发器（水浴温度〜40℃）除去通过温和蒸馏溶剂，得到化合物3，为棕色无定形固体（0.91克，95％）。 </li></ol></li><li>的4-（4-异硫氰基）合成-2-（4,7,10-三- 叔丁氧基羰基- 1,4,7,10-四氮杂-1-基）丁酸叔丁基酯（4）。 <ol><li> （0.124毫升，1.58毫摩尔，1.3当量）加入二氯硫化碳为3的三乙胺的混合物（0.91克，1.22毫摩尔）和（0.685毫升，4.87毫摩尔，4当量）的二氯甲烷（15毫升）中。 </li><li>大力搅拌反应英里在室温下用磁力搅拌器16小时夹具。 </li><li>在旋转蒸发器（水浴温度〜40℃）除去通过温和蒸馏出溶剂，然后纯化通过柱色谱法（硅胶，5％甲醇/二氯甲烷）粗产物，得到产物4，为浅棕色无定形固体（0.51克，53％）。 </li></ol></li></ol></li><li> 树枝状DCA的合成。 <ol><li>树枝状聚合物5的合成。 <ol><li>取G4-PAMAM树枝状聚合物（667毫克，在甲醇中10％的树枝状聚合物的溶液，4.67微摩尔），蒸发通过温和蒸馏甲醇用旋转蒸发器（水浴温度〜40℃），并溶解于DMF，残余物（4毫升）中。 </li><li>添加三乙胺（0.105毫升，0.75毫摩尔，160当量），在60℃下搅拌45分钟，并添加异硫氰酸酯4（354毫克，0.45毫摩尔，1.5当量相对于树枝状聚合物的氨基表面基团）分批OVE，R 1小时。 </li><li>搅拌用磁力搅拌器将反应混合物在45℃下48小时。 </li><li>在40-60℃除去的球对球真空蒸馏装置中的溶剂。 </li><li>净化用亲脂性凝胶过滤介质和甲醇作为洗脱剂通过大小排阻层析残余物。收拾列中，不施加压力溶胀该过滤介质在甲醇中在室温下（&gt;4毫升每1g粉末的甲醇）至少3小时。通过收集1毫升分数进行重力分离。 </li><li>分析用薄层色谱（TLC）所收集的级分。开发在15％甲醇/二氯甲烷的TLC板（仅位于基线时的最极性点是从树枝状产物衍生）。蒸发在旋转蒸发器（水浴温度〜40℃）通过温和蒸馏收集到的馏分，得到产物5（270毫克，91％）。 </li></ol></li><li>树枝状聚合物的合成<strong&gt; 6。 <ol><li>溶解在甲酸（5毫升）中的受保护的树枝状螯合剂5（270毫克，4.23微摩尔），搅拌在60℃下该混合物24小时。 </li><li>蒸发在旋转蒸发（约15毫巴的压力，水浴温度〜40℃）通过蒸馏甲酸并冷冻干燥该产物，得到6-（压力〜0.2毫巴）9。 </li></ol></li><li>所述树枝状造影剂的合成（DCA） <ol><li>溶解在水中的树枝状螯合剂6（4.35微摩尔）和将pH调节至7.0与0.1M氢氧化钠。 </li><li>溶解GdCl 3·6H 2 O（113毫克，304微摩尔）的水（1毫升）中，并添加其滴加至螯合剂6的历时4小时的溶液;保持与氢氧化钠水溶液（0.05M），通过测量pH值用pH计的pH值在7.0。 </li><li>搅拌在室温吨磁力搅拌器将混合物emperature 24小时。 </li><li>乙二胺四乙酸（EDTA，158毫克，426微摩尔）的溶液中分批添加经4小时以除去过量的Gd（III），同时保持与氢氧化钠水溶液（0.05M）pH在7.0。在室温下搅拌该混合物24小时。 </li><li>执行大小排阻层析以除去大部分GdEDTA的和过量的EDTA。在使用水溶胀亲水凝胶过滤介质收拾列。混合物降低至合适的体积，并装载柱。洗脱用离子交换水列不施加压力。 </li><li>使用3 kDa的离心过滤单元30分钟，在离心力1800 XG删除GdEDTA和EDTA的残留离心机样品。重复此步骤（左右五次）直到滤液显示不存在EDTA和GdEDTA的。将样品转移到一个烧瓶中，蒸发，然后冷冻干燥该溶剂，得到灰白色产物作为最终的DCA（186毫克，71％）。 注:ESI-MS的方法检查没有EDTA和GdEDTA的。 </li><li>确认没有钆（III）的作为使用二甲酚橙测试一个自由离子。溶解在醋酸盐缓冲液（pH 5.8），滤液（0.5毫升）中。添加二甲酚橙溶液几滴和跟踪变色（黄色或紫色表明不存在或游离钆（Ⅲ）分别离子在溶液中，存在）11。 </li></ol></li></ol></li></ol> 2.树枝状产品在体外表征<ol><li> 联接到PAMAM树枝状聚合物（具有DOTA状的大环化合物的树枝状聚合物的载量）大环DOTA-单元的数目的估计 <ol><li>估计用1 H-NMR（核磁共振-核磁共振光谱）。 注意:此过程有可能在树枝状5和6，但不是在DCA。 <ol><li>记录1 H NMR谱12。 </li><li>整合芳香族区和两个单独的脂族区（1脂族树枝状聚合物和大环的质子的信号; 2。第t -Bu基的信号）为树枝状聚合物5和6或只是一个脂族区域，分别。 注意:没有单独的信号中的脂族区来自第t -Bu组树枝状6，因为它们已被水解。 </li><li>运用EQ。 1或公式。 2来估计大环单元（n），其中R =积分的比值的数量（脂族/在等式1或脂族-树枝状芳香族/脂肪- 叔卜在公式2），H DEND =质子数在树枝状聚合物， 高度 的Ar =芳族质子的数量，H t中 BU =质子在叔 -Bu组的数量，和H MAC =在一个大环的质子数。 注意:无论是公式。 1或公式。 2 FO可以使用ř树枝状5，而只有等式1可用于树枝状聚合物6。由于可交换质子（在胺，酰胺，硫脲，或羧酸盐）通常被用氘取代，他们没有在计算假定。这里，H DEND = 1128（5）或1000（6），H 氩 = 4，和H MAC = 27被使用。 <img alt="式（1）" src="/files/ftp_upload/54776/54776eq1.jpg" /> （1） <img alt="公式（2）" src="/files/ftp_upload/54776/54776eq2.jpg" /> （2） </li></ol></li><li>通过使用氮气的比率为硫从元素分析估计。 <ol><li>执行对固体样品枝（DCA在此工作）的元素分析。 </li><li>运用EQ。 3估计大环单元（n），其中R =测定％N和％S，N DEND或S DEND的比例数=数氮或树枝状聚合物中的硫原子，和N MAC或S MAC =氮或硫原子的一个大环单元的数目。 注意:因子2.29是从硫和氮的原子质量的比获得。在这项工作中，N- DEND = 250，S DEND = 2，N MAC = 5和S MAC = 1被使用。 <img alt="公式3" src="/files/ftp_upload/54776/54776eq3.jpg" /> （3） </li></ol></li><li>估计用的飞行基质辅助激光解吸/电离飞行时间（MALDI-TOF）。 <ol><li>执行MALDI-TOF MS分析13。 </li><li>计算大环单元（n）的根据式的数目。 4，其中M Z =所观察到的质量（M / Z）中，z =电荷的物种中，M DEND =树枝状部分的质量，以及M MAC =一个大环单元的质量。 没有TE:M = DEND 14,306和M MAC = 719在这项工作中使用。 <img alt="公式4" src="/files/ftp_upload/54776/54776eq4.jpg" /> （4） </li></ol></li></ol></li><li> DCA浓度（[DCA]）的测定:散装磁化率测量（BMS） <ol><li>在一个塑料瓶中管（[DCA]〜5-10毫摩尔）溶解在水（360微升）的DCA（5-10毫克）。 注:[DCA]应在5-10毫的范围，以避免叔丁醇共振的可能的重叠在样品浓度&gt; 15毫米，与水的谐振在δ= 4.7 ppm的。 </li><li>添加60微升D 2 O中的: 叔丁醇混合物（2:1体积/体积）至DCA的水溶液，并使用涡旋混合器将所得的溶液（420微升）混合。 </li><li>转移400微升样品放入一个外NMR管，并放置一同轴NMR插入管与叔丁醇:H 2 O的混合物（10:90体积/体积）到样品管中。 </li><li>记录1 H NMR谱，并测量在内部和外部的NMR管中（参考）12从叔丁醇衍生谐振信号之间的频移。 </li><li>运用EQ。 5，以确定[DCA]，其中T =绝对温度，Δχ=记录的移位，μ 有效 =有效磁的镧系元素离子为钆（III）14，和s =一个常数依赖力矩（μ 有效 = 7.94在样品的形状和其在磁场（0，1/3，和在一个球体，平行于气缸，和气缸垂直于磁场的情况下的1/6，分别地）的位置。 注:对于[DCA]得到的计算值应该被校正的原始浓度由于加入的D 2 O中的: 叔丁醇溶液（60微升）。 <img alt="公式5" src="/files/ftp_upload/54776/54776eq5.jpg" /> （5） </li></ol></li><li> 动态光散射（DLS）测量。 <ol><li>制备过滤DCA溶液（0.2微米聚四氟乙烯/ PTFE过滤，每钆0.75毫（III））在4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES）缓冲液（25mM，pH7.4）中，并将其转移到一个比色皿为DLS测量。 </li><li>放置反应杯插入DLS装置和设置以下参数:30秒之前记录5次重复的15扫描（1次扫描= 12秒，折射率= 1.345，吸收= 1％），而不会在扫描之间，并与温度平衡延时。 </li><li>导出所获取的数据和通过绘制人口（％），为大小（流体动力学直径）的函数得到的粒度分布的直方图。 </li></ol></li><li> 纵向和横向弛豫的测量。 注意:使用弛豫时间分析仪15用类似的方法已经描述;使用具有的Topspin 300MHz的NMR波谱仪进行此过程软件。 <ol><li>准备一套H中DCA解决方案2 O的:D 2 O（500微升，10％D 2 O H中2 O，[DCA] = 2.0，2.5，3.0，3.5，4.0，4.5和5.0毫米，[HEPES ] = 25毫米）从DCA股票样本（见第2.2节）。 </li><li>转移450微升溶液进入核磁共振管，并将其放入仪器中。 </li><li>优化采集参数（90°激励脉冲持续时间（P1），以及辐射频率偏移（O 1）），然后执行T使用反转恢复（IR）和台车式赛尔Meiboom -吉尔和 T2实验（CPMG ）的脉冲序列。 </li><li>的T1和T2 2弛豫时间测定。 <ol><li>选择所记录的测量，过程在F2尺寸的二维频谱，并执行交互式相位校正。 </li><li>在分析/ T选择适当的切片（最大强度峰值） <sub&gt; 1 / T 2松弛窗口，整合它，以及该地区出口到放松模块。 </li><li>选择合适的拟合函数（invrec或uxnmrt2红外和CPMG实验，分别），得到的T 1或T 2弛豫时间。 </li></ol></li><li>重复步骤2.4.4.2-2.4.4.4的所有剩余[DCA]解决方案。 </li><li>计算从所获得的Ť1值的弛豫率（R 1和R 2）（R 1,2 = 1 / T 1,2）。 </li><li>情节的 R 1和R 2（秒-1），为钆（III）浓度的在毫函数。 </li><li>确定纵向和横向弛豫，R 1和R 2（毫-1秒-1），从拟合直线的斜率，由式所定义。 6，其中R I，观测值 =纵向（ⅰ= 1）或横向（ⅰ= 2）抗磁松弛率水在实验中所用的情况下顺磁物质的和[钆] =钆（III）的浓度。 <img alt="公式6" src="/files/ftp_upload/54776/54776eq6.jpg" /> （6） </li></ol></li></ol> 3. 在体外 MRI; DCA和GdDOTA的比较<ol><li> 管幻影的制备 <ol><li>为两组实验其中计算造影剂的浓度制备的DCA（4×350微升）和GdDOTA（4×350微升）以及水样（4×350微升）的水溶液:（3.1.1.1）每钆（III）或每分子（3.1.1.2）。 <ol><li>准备两个样品DCA和两个GdDOTA 样品的分别每钆0.5和1.0毫米（III）的浓度。此外，准备两个水样（如控制管）。 </li><li>准备两个DCA样品（2.5和每钆（III）或0.05 5.0毫和每个树枝状分子0.1毫摩尔），二GdDOTA样品（0.25，0.5毫摩尔）和两个水样（CONTRO升管）。 注意:适当的DCA和GdDOTA浓度应通过稀释各自的股票样本通过BMS法测定浓度制备（参见2.2节）与HEPES缓冲液（pH 7.4）。为了简化计算，假设为平均每树枝状聚合物分子的大环单元的N = 50。因此，DCA的比率:GdDOTA为1:5，计算上的每个分子基础。 </li></ol></li><li>放置在300μl的塑料瓶管中的样品，从而避免气泡的在溶液中的存在。 注:在塑料小瓶管的尺寸取决于所使用的射频线圈的类型和大小（在此，与卷线圈给出一个例子）。 </li><li>插入样品的注射器（60 ml，此）的内部，用1mM GdDOTA填充 溶液，并将其放置在扫描仪。 注:样品置于GdDOTA的水溶液，以避免易感性的影响（在磁场的Field变化对发生的不同磁化率的物质之间的接口附近）trength。 </li></ol></li><li> 参数优化和成像。 <ol><li>使用解剖扫描（航向/ Tripilot）与样品的注射器放置在磁体的等角点。 </li><li>按红绿灯（调整扫描）来执行调整为垫补整个卷，中心频率（O 1），接收器增益（RG），并且所述发射增益（TX0和TX1）的（磁场均匀的调整）。 </li><li>用于T 1 -加权（T 1W）成像，选择快速低角度拍摄（FLASH）法。 </li><li>选择在扫描仪使用扫描定位器垂直放置（注射器水平）样本冠状切片。 </li><li>运用EQ。 7对比度-噪声（CNR）采集的优化参数16，其中α=翻转角，TE =回波时间TR =重复时间，T 1，A，T 1，B =第t样品A的1倍（T 1，A）和样品B（T 1，B）该CNR应该最大化（同样是有效的ŧ2次:T 2，A和T 2，B）。 注:t 1和T 2弛豫时间应设置为从纵向和横向弛豫（第2.4节）的测量获得的值，而TE，TR，α应距离CNR优化计算而获得。 <img alt="公式7" src="/files/ftp_upload/54776/54776eq7.jpg" /> （7） </li><li>取得使用在前面的步骤（3.2.5）中获得的参数的图像。 </li><li>计算信噪比（SNR）。 <ol><li>加载收购牛逼 1瓦特图像（扫描）转换成图像显示及处理窗口，单击定义感兴趣区域 （ROI） 的区域 。 </li><li>选择一个圆形的投资回报率，并在样品位置和背景绘制。接着，点击显示以获得的平均信号振幅（S 信号 ）和背景（S 噪声 ）的标准偏差。 </li><li>重复步骤3.2.7.2对于DCA，GdDOTA和水样。 </li><li>用计算公式SNR:SNR = S 信号 / S 的噪音 。 </li></ol></li><li>经过稍微修改程序，用快速采集弛豫增强（RARE）方法执行T2加权（T 2W）成像。对于CNR采集参数的优化，运用EQ。 8。 <img alt="式（8）" src="/files/ftp_upload/54776/54776eq8.jpg" /> （8） </li></ol></li></ol>

<ol>
	<li>Merbach, A. E., Helm, L., T&#243;th, &. #. 2. 0. 1. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The chemistry of contrast agents in medical magnetic resonance imaging. 2nd ed. , (2013).</li><li>Geraldes, C. F. G. C., Laurent, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+and+basic+properties+of+contrast+agents+for+magnetic+resonance+imaging.">Classification and basic properties of contrast agents for magnetic resonance imaging.</a> Contrast Media Mol. Imaging. 4 (1), 1-23 (2009).</li><li>Caravan, P., Ellison, J. J., McMurry, T. J., Lauffer, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gadolinium(III)+chelates+as+MRI+contrast+agents:+Structure,+dynamics,+and+applications.">Gadolinium(III) chelates as MRI contrast agents: Structure, dynamics, and applications.</a> Chem. Rev. 99 (9), 2293-2352 (1999).</li><li>Villaraza, A. J. L., Bumb, A., Brechbiel, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecules,+Dendrimers,+and+Nanomaterials+in+Magnetic+Resonance+Imaging:+The+Interplay+between+Size,+Function,+and+Pharmacokinetics.">Macromolecules, Dendrimers, and Nanomaterials in Magnetic Resonance Imaging: The Interplay between Size, Function, and Pharmacokinetics.</a> Chem. Rev. 110 (5), 2921-2959 (2010).</li><li>Langereis, S., Dirksen, A., Hackeng, T. M., van Genderen, M. H. P., Meijer, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrimers+and+magnetic+resonance+imaging.">Dendrimers and magnetic resonance imaging.</a> New J. Chem. 31 (7), 1152-1160 (2007).</li><li>G&#252;nd&#252;z, S., Power, A., Maier, M. E., Logothetis, N. K., Angelovski, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+Characterization+of+a+Biotinylated+Multivalent+Targeted+Contrast+Agent.">Synthesis and Characterization of a Biotinylated Multivalent Targeted Contrast Agent.</a> ChemPlusChem. 80 (3), 612-622 (2015).</li><li>Pope, S. J. A., Kenwright, A. M., Heath, S. L., Faulkner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+luminescence+properties+of+a+kinetically+stable+dinuclear+ytterbium+complex+with+differentiated+binding+sites.">Synthesis and luminescence properties of a kinetically stable dinuclear ytterbium complex with differentiated binding sites.</a> Chem. Commun. (13), 1550-1551 (2003).</li><li>Vibhute, S. M., 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=Synthesis+and+characterization+of+pH-sensitive,+biotinylated+MRI+contrast+agents+and+their+conjugates+with+avidin.">Synthesis and characterization of pH-sensitive, biotinylated MRI contrast agents and their conjugates with avidin.</a> Org. Biomol. Chem. 11 (8), 1294-1305 (2013).</li><li>Vogel, A. I., Furniss, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Vogel's textbook of practical organic chemistry. 5th ed. , (1989).</li><li>Lundanes, E., Reubsaet, L., Greibrokk, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chromatography : basic principles, sample preparations and related methods. , (2013).</li><li>Barge, A., Cravotto, G., Gianolio, E., Fedeli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+determine+free+Gd+and+free+ligand+in+solution+of+Gd+chelates.+A+technical+note.">How to determine free Gd and free ligand in solution of Gd chelates. A technical note.</a> Contrast Media Mol. Imaging. 1 (5), 184-188 (2006).</li><li>Keeler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Understanding NMR spectroscopy. 2nd ed. , (2010).</li><li>Hillenkamp, F., Peter-Katalini&#263;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> MALDI MS : a practical guide to instrumentation, methods and applications. , (2007).</li><li>Peters, J. A., Huskens, J., Raber, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lanthanide+induced+shifts+and+relaxation+rate+enhancements.">Lanthanide induced shifts and relaxation rate enhancements.</a> Prog. Nucl. Magn. Reson. Spectrosc. 28, 283-350 (1996).</li><li>Averill, D. J., Garcia, J., Siriwardena-Mahanama, B. N., Vithanarachchi, S. M., Allen, 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=Preparation,+Purification,+and+Characterization+of+Lanthanide+Complexes+for+Use+as+Contrast+Agents+for+Magnetic+Resonance+Imaging.">Preparation, Purification, and Characterization of Lanthanide Complexes for Use as Contrast Agents for Magnetic Resonance Imaging.</a> J. Vis. Exp. (53), e2844 (2011).</li><li>Hagberg, G. E., Scheffler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+r1+and+r2+relaxivity+of+gadolinium-based+contrast+agents+on+the+T1-weighted+MR+signal+at+increasing+magnetic+field+strengths.">Effect of r1 and r2 relaxivity of gadolinium-based contrast agents on the T1-weighted MR signal at increasing magnetic field strengths.</a> Contrast Media Mol. Imaging. 8 (6), 456-465 (2013).</li><li>Boswell, C. A., 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=Synthesis,+characterization,+and+biological+evaluation+of+integrin+alpha(v)beta(3)-targeted+PAMAM+dendrimers.">Synthesis, characterization, and biological evaluation of integrin alpha(v)beta(3)-targeted PAMAM dendrimers.</a> Mol. Pharmaceut. 5 (4), 527-539 (2008).</li><li>Sherry, A. D., Caravan, P., Lenkinski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer+on+Gadolinium+Chemistry.">Primer on Gadolinium Chemistry.</a> J. Magn. Reson. Imaging. 30 (6), 1240-1248 (2009).</li><li>Caki&#263;, N., G&#252;nd&#252;z, S., Rengarasu, R., Angelovski, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+strategies+for+preparation+of+cyclen-based+MRI+contrast+agents.">Synthetic strategies for preparation of cyclen-based MRI contrast agents.</a> Tetrahedron Lett. 56 (6), 759-765 (2015).</li><li>Polasek, M., Hermann, P., Peters, J. A., Geraldes, C. F. G. C., Lukes, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAMAM+Dendrimers+Conjugated+with+an+Uncharged+Gadolinium(III)+Chelate+with+a+Fast+Water+Exchange:+The+Influence+of+Chelate+Charge+on+Rotational+Dynamics.">PAMAM Dendrimers Conjugated with an Uncharged Gadolinium(III) Chelate with a Fast Water Exchange: The Influence of Chelate Charge on Rotational Dynamics.</a> Bioconjugate Chem. 20 (11), 2142-2153 (2009).</li><li>Ali, M. M., 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=Synthesis+and+relaxometric+studies+of+a+dendrimer-based+pH-responsive+MRI+contrast+agent.">Synthesis and relaxometric studies of a dendrimer-based pH-responsive MRI contrast agent.</a> Chem. Eur. J. 14 (24), 7250-7258 (2008).</li><li>Jackson, C. L., 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=Visualization+of+dendrimer+molecules+by+transmission+electron+microscopy+(TEM):+Staining+methods+and+Cryo-TEM+of+vitrified+solutions.">Visualization of dendrimer molecules by transmission electron microscopy (TEM): Staining methods and Cryo-TEM of vitrified solutions.</a> Macromolecules. 31 (18), 6259-6265 (1998).</li><li>Jain, K., Kesharwani, P., Gupta, U., Jain, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrimer+toxicity:+Let's+meet+the+challenge.">Dendrimer toxicity: Let's meet the challenge.</a> Int. J. Pharm. 394 (1-2), 122-142 (2010).</li><li>Rudovsky, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAMAM+dendrimeric+conjugates+with+a+Gd-DOTA+phosphinate+derivative+and+their+adducts+with+polyaminoacids:+The+interplay+of+global+motion,+internal+rotation,+and+fast+water+exchange.">PAMAM dendrimeric conjugates with a Gd-DOTA phosphinate derivative and their adducts with polyaminoacids: The interplay of global motion, internal rotation, and fast water exchange.</a> Bioconjugate Chem. 17 (4), 975-987 (2006).</li><li>Xu, H., 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=Toward+improved+syntheses+of+dendrimer-based+magnetic+resonance+imaging+contrast+agents:+New+bifunctional+diethylenetriaminepentaacetic+acid+ligands+and+nonaqueous+conjugation+chemistry.">Toward improved syntheses of dendrimer-based magnetic resonance imaging contrast agents: New bifunctional diethylenetriaminepentaacetic acid ligands and nonaqueous conjugation chemistry.</a> J. Med. Chem. 50 (14), 3185-3193 (2007).</li><li>Nwe, K., Bryant, L. H., Brechbiel, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(amidoamine)+Dendrimer+Based+MRI+Contrast+Agents+Exhibiting+Enhanced+Relaxivities+Derived+via+Metal+Preligation+Techniques.">Poly(amidoamine) Dendrimer Based MRI Contrast Agents Exhibiting Enhanced Relaxivities Derived via Metal Preligation Techniques.</a> Bioconjugate Chem. 21 (6), 1014-1017 (2010).</li><li>Livramento, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+in+vivo+MRI+assessment+of+a+self-assembled+metallostar+compound+endowed+with+a+remarkable+high+field+relaxivity.">First in vivo MRI assessment of a self-assembled metallostar compound endowed with a remarkable high field relaxivity.</a> Contrast Media Mol. Imaging. 1 (1), 30-39 (2006).</li><li>Norek, M., Kampert, E., Zeitler, U., Peters, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+of+the+Size+of+Dy2O3+Nanoparticles+for+Optimal+Performance+as+an+MRI+Contrast+Agent.">Tuning of the Size of Dy2O3 Nanoparticles for Optimal Performance as an MRI Contrast Agent.</a> J. Am. Chem. Soc. 130 (15), 5335-5340 (2008).</li></ol>

The authors have nothing to disclose.

所述树枝状MRI造影剂的制备需要的单体单元的适当选择（ 即，螯合剂为钆（III））。它们减少这种顺磁性离子的毒性，迄今为止，各种各样的无环的和大环螯合剂用于此目的1-3。在这些中，大环DOTA型螯合剂具有最高热力学稳定性和动力学惰性的，因此，是惰性MRI造影剂1,18的制剂最优选的选择。此外，它们易发生各种合成转化，这导致双官能螯合剂，能够链接到各种功能分子（ 例如，靶向载体或纳米载体），同时仍形成稳定的Gd（III）络合物19。为此，在此过程中所描述的DOTA型单体单元是从DO3A- 叔丁基酯，公共的和容易得到的前体，和的溴衍生物制备的4-（4-硝基苯基）丁酸。这种分子是从DOTA衍生的并具有相似的结构，以协调的Gd（Ⅲ）。合成修改的目的是使该螯合剂容易耦合到各种功能分子和运营商的反应。即，在螯合剂DOTA的改性分子结果的制备仍然使用可协调到钆4羧基（Ⅲ），以形成一种惰性复杂和正交硝基苯基，其在转换重视这个螯合剂到树枝状聚合物的表面。此过程还允许在正交反应性基团的选择的灵活性（ 例如，NH 2或COOH），它可以用来耦合的Gd（III）螯合剂到所需载体在一个优选的方式。 所得到的双功能螯合剂可以以两种不同的方式（ 即，合成方法）耦合到其它分子。当硝基还原为氨基，将得到的苯胺可以根据去与其他分子8的羧酸基的缩合反应。此外，在硫光气的存在下的芳族伯胺官能团可容易地转化成异硫氰酸酯，它容易与在极性有机溶剂以及水的胺反应，为单体单元的至树枝状聚合物17偶联，提供更多的反应的可能性的基团，20,21。 用于耦合的双功能螯合剂的树枝状载体，一个合适的树枝状骨架应选择。关系到最终树枝状聚合物缀合物的结构和期望的应用的几个因素应该在此步骤中被考虑。由于树枝状载体的广泛的商业可用性，产品具有不同的核心结构，表面反应性基团，或世代可以选择。因此，偶联反应将取决于表面基团的树枝状聚合物和螯合剂的正交基，而最终结合物可以是中性的，带电荷的，或具有不同的大小（可达15-20纳米，这取决于树枝状代）22。所有这些方面，应考虑制备树枝状CA之前，因为它们可能影响溶解性，弛豫（MRI信号增强），扩散，和造影剂，这可能危及其在MRI中的应用的其他药物代谢动力学性质。例如，阳离子树枝状聚合物可以在生物系统中表现出毒性。然而，可以通过树枝状聚合物表面上带负电荷的基团的缀合可以减少这种影响，从而降低了其总体正电荷23。 在这个协议中，我们已经使用其中单体大环化合物4的异硫氰酸酯基偶联到装有64伯胺表面基团的商业胱胺核G4-PAMAM的方法制备的树枝状造影剂的DCA。该hydroph的初步纯化OBIC树枝状产物5是为了除去大部分未反应的单体单元的使用具有亲脂性凝胶过滤介质和甲醇作为洗脱剂的柱通过柱色谱法进行的。 叔丁基酯的用甲酸水解是直接的，从而导致可与大小排阻层析使用亲水性凝胶过滤介质中纯化的水溶性树枝状产物。被同时保持在为了便于形成复合物在中性pH的溶液进行用钆（III）的多聚体和树枝状螯合剂的络合。否则，钆的络合（Ⅲ）（加入作为氯化物盐）降低pH值，减缓该反应。最后，值得注意的是，在树枝状聚合物芯胺基也往往具有钆（III），以协调，但只与过量的，可能无法与DOTA单位螯合。避免钆（III）的DOTA螯合剂以外的存在是必要的，因为文件从CA钆（III）的akage可以具有不希望的影响;即，它可以在体内 18诱导的毒性。过量的钆（III）可通过用EDTA接着GdEDTA的超滤和使用3- kDa的分子量截止（MWCO）过滤器无EDTA的络合被有效地除去。当树枝状缀合物具有较低的分子量下MWCO过滤器可能被使用。 有相关DCA的筹备工作两大问题进行故障排除。由于对核磁共振信号的Gd（Ⅲ）的大扩大效果，DCA的通过NMR光谱的装置的分析并不丰富。相反，该分析应在前面的步骤（化合物5和6）来执行。接着，monomacrocyclic单元与树枝状聚合物表面缀合不会与100％转化来实现，但它很可能是50-90％之间（见下文）。通常情况下，反应产率可提高通过添加monomeri的第二部分C反应单元树枝状聚合物和单体单元的第一缀合完成之后24。然而，每一个制备批次的结果在缀合树枝状聚合物表面上螯合剂的稍微不同的平均数目，即使当相同的树枝状聚合物和DOTA的单元被用作用于耦合材料。虽然钆（III）的存在于DCA最终量可以经由BMS方法独立地确定（见2.2节），为树枝状偶联物的更好表征，有必要在每次新一批的执行结合单体单元的估计DCA准备（见2.1和讨论如下）。 分离树枝状产品的分析表征可通过1 H NMR谱（仅在制品5和6），元素分析，和MALDI-TOF MS的手段来进行。氨基表面基团的转化率典型收率位于50-90％之间，这取决于树枝状聚合物generati上，螯合剂的类型，和所使用的反应条件（溶剂，温度）6,20,24,25。在这个特殊的情况下，从联合分析得到的计算出的质量对应于平均的49单体螯合物被连接到树枝状聚合物（ 即，〜树枝状聚合物表面的胺的75％占有率）。虽然在反应氨基的最终数量稍有失配可能这些方法25之间所预期的，其直接比较提供了用于形成所需的DCA的具有特定平均数目附螯合单元的合理的证据。 在体外表征旨在评估DCA的潜在增强的MRI实验的对比度由DLS，relaxometric和MRI实验。 DCA的流体动力学直径被确定为通过DLS测量7.2±0.2纳米，这是在协议与这类以前报道偶联物与G4第4代树形分子26。 DCA的纵向弛豫的测定接着先前描述的步骤15和揭示的6.2±0.1毫米-1秒-1每钆（III）中的值。增强在顺磁钆（III）的DCA相对第r 1到相似类型（ 例如，GdDOTA）的小尺寸分子的约50％可与树枝状造影剂的中间大小进行说明。即，附着于树枝状聚合物的表面的钆螯合物的减少运动增加了旋转相关时间，因此，R 1;这种效果仍然可以在更小的纳米尺寸剂高磁场被观察到。否则，在旋转相关时间的增加显性有助于在低磁场27，R 1的增强。另一方面，所述树枝状造影剂的大小对横向RELA有显着影响xivity 28，产生30.5±0.6毫米-1秒-1的值每钆（III）。总之， 在体外 DCA的评估方法是直接的，只需要仔细的样品制备，所以获得的数据和分析结果时没有任何困难的预期。 为了证明树枝状造影剂和它的力量影响图像的对比度的效果，我们使用新制备的造影剂的DCA执行上管幻影的MRI实验。我们还使用了可商购的和临床批准MRI对比剂，GdDOTA的溶液中，作为比较和管以水为对照。在加权的MRI实验中第一T 1中 ，当被用于相等的Gd（III）的浓度（0.5或1钆毫（Ⅲ）在DCA或GdDOTA），信噪比与DCA管已经高达12％是由于较高的在DCA相比GdDOTA的纵向弛豫（增加约50％Figure 5A）。第二T 1 -加权的MRI实验的目的是要证明的DCA时每分子计算的浓度的影响。虽然相比GdDOTA施加5倍以下的DCA（50与250微米或100与500微米的DCA与GdDOTA，分别），DCA与钆一个高负荷（Ⅲ）导致了图象对比度的显著增量，这反过来又导致所观察到的SNR值是在填充有DCA幻像管高至少三倍。果然，无论是T2加权MRI实验表现出充满DCA和GdDOTA幻象管之间的SNR大（3-20倍）的差异。 总之，这个协议描述了使用普通的合成方法，以提供DCA相比于小尺寸的CA改善的性质用于MRI树枝状的CA的一个方便的制剂。时相比，DCA展品首选热力学稳定性和动力学惰性其单体CA类似物。尽管如此，DCA和，的多价，因此，在目标区域中的顺磁性物质的高局部浓度诱导的MR图像的高对比度。考虑到往往是最好的药代动力学特性 （ 如更长的组织中的保留时间）相比，其单体CA类似物，或执行进一步的功能的能力（ 例如，有针对性的载体），这些树枝状，大环偶联代表一个有前途的，有价值的类造影剂为未来的各种MRI和分子成像应用。

磁共振成像（MRI）是广泛用于生物医学研究和临床诊断的有力和非电离成像技术，由于其非侵入性和优良特性的软组织的对比。最常用的MRI方法利用从水的质子获得的信号的基础上，在水信号的密度差异的组织中提供高清晰度的图像和详细的信息。的信号强度和在MRI实验的特异性可以使用造影剂（CA）的进一步提高。这些都是影响的纵向（T 1）和横向分别1,2-顺磁性或超顺磁性物质（T 2）弛豫时间。 用聚氨基聚羧酸配体的镧系元素离子钆配合物是最常用的Ť1的CA。钆（III）缩短的T 1松弛因此水的质子的时间，增加的MRI实验3中的信号对比度。然而，钆离子是有毒的;它的大小近似于钙（Ⅱ），它严重影响钙辅助细胞信号传导。因此，无环和大环螯合物被用来抵消这种毒性。各种多齿配体迄今已开发的，导致具有高的热力学稳定性和动力学惰性1钆（Ⅲ）配合物。那些基于所述12元azamacrocycle CYCLEN，特别是其四衍生物DOTA（1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸）是此CA类的最研究和应用的复合物。 尽管如此，GdDOTA型CA是低分子量的系统，其中显示某些缺点，例如低对比度效率和快速的肾排泄。大分子和多价的CA可能是一个很好的解决了这些问题，4。由于CA biodistribu化主要是由它们的大小决定的，大分子的CA显示组织中更长的保留时间。同样重要的是，在单体的MR 探测器 （ 如 GdDOTA复合），基本上改善所获取的MR信号和测量质量的增加的局部浓度这些试剂的结果的多价。 树枝状聚合物之间的编写多价的CA用于MRI 4,5最好支架。这些具有良好定义的尺寸的高度支化的大分子易于在其表面上的不同的偶合反应。在这项工作中，我们报道的制备，纯化和树枝状的CA用于MRI由第4代（G4）聚（酰氨基胺），耦合到GdDOTA状螯合物（DCA）（PAMAM）树枝状聚合物的表征。我们描述了反应性的DOTA衍生物的合成及其与PAMAM树枝状耦合。在与钆（III），标准的理化性质络合procedu再DCA的进行。最后，进行核磁共振实验证明DCA的以产生具有比从低分子量的CA获得的那些较强的对比度的MR图像的能力。

<table border="0" cellpadding="0" cellspacing="0" width="921">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Cyclen</td>
			<td style="width:275px;">CheMatech</td>
			<td style="width:136px;">C002</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">tert-Butyl bromoacetate&#160;</td>
			<td style="width:275px;">Alfa Aesar</td>
			<td style="width:136px;">A14917</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">N,N-Dimethylformamide</td>
			<td style="width:275px;">Fluka</td>
			<td style="width:136px;">40248</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Potassium carbonate</td>
			<td style="width:275px;">Sigma-Aldrich</td>
			<td style="width:136px;">209619</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">4-(4-Nitrophenyl)butryic acid</td>
			<td style="width:275px;">Aldrich</td>
			<td style="width:136px;">335339</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Thionyl chloride&#160;</td>
			<td style="width:275px;">Acros Organics</td>
			<td style="width:136px;">382662500</td>
			<td style="width:259px;">Note: Corrosive substance; toxic if inhaled</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Bromine</td>
			<td style="width:275px;">Acros Organics</td>
			<td style="width:136px;">402841000</td>
			<td style="width:259px;">Note: causes severe skin burns, fatal if inhaled&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Diethyl ether</td>
			<td style="width:275px;">any source</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Sodium sulphate</td>
			<td style="width:275px;">Acros Organics</td>
			<td style="width:136px;">196640010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Chloroform&#160;</td>
			<td style="width:275px;">VWR Chemicals</td>
			<td style="width:136px;">22711.29</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">tert-Butyl 2,2,2-trichloroacetimidate</td>
			<td style="width:275px;">Aldrich</td>
			<td style="width:136px;">364789</td>
			<td style="width:259px;">Note: flammable substance; irritrant to skin and eyes</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:251px;">Boron trifluoride etherate</td>
			<td style="width:275px;">Acros Organics</td>
			<td style="width:136px;">174560250</td>
			<td style="width:259px;">48 % BF3. Note: Flammable substance; causes skin burns, fatal if inhaled&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Sodium bicarbonate</td>
			<td style="width:275px;">Acros Organics</td>
			<td style="width:136px;">424270010</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Ethyl-acetate</td>
			<td style="width:275px;">any source</td>
			<td style="width:136px;"></td>
			<td>For column chromatography</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">n-Hexane</td>
			<td style="width:275px;">any source</td>
			<td style="width:136px;"></td>
			<td>For column chromatography</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Bulb-to-bulb (Kugelrohr) distillation apparatus</td>
			<td style="width:275px;">B&#252;chi</td>
			<td style="width:136px;"></td>
			<td>Model type: Glass oven B-585</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Silicagel</td>
			<td style="width:275px;">Carl Roth GmbH</td>
			<td style="width:136px;">P090.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Methanol</td>
			<td style="width:275px;">any source</td>
			<td style="width:136px;"></td>
			<td>For column chromatography</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Dichloromethane&#160;</td>
			<td style="width:275px;">any source</td>
			<td style="width:136px;"></td>
			<td>For column chromatography</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Ethanol</td>
			<td style="width:275px;">VWR Chemicals</td>
			<td style="width:136px;">20821.296</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Ammonia</td>
			<td style="width:275px;">Acros Organics</td>
			<td style="width:136px;">428381000</td>
			<td style="width:259px;">7N Solution in Methanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Palladium&#160;</td>
			<td style="width:275px;">Aldrich</td>
			<td style="width:136px;">643181</td>
			<td style="width:259px;">15 % wet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Hydrogenation apparatus PARR</td>
			<td style="width:275px;">PARR Instrument Company</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Celite 503</td>
			<td style="width:275px;">Aldrich</td>
			<td style="width:136px;">22151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Sintered glass funnel</td>
			<td style="width:275px;">any source</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Thiophosgen</td>
			<td style="width:275px;">Aldrich</td>
			<td style="width:136px;">115150</td>
			<td style="width:259px;">Note: irritrant to skin; toxic if inhaled</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Triethylamine</td>
			<td style="width:275px;">Alfa Aesar</td>
			<td style="width:136px;">A12646</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Dichloromethane&#160;</td>
			<td style="width:275px;">Acros Organics</td>
			<td style="width:136px;">348460010</td>
			<td style="width:259px;">Extra dry&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Magnetic stirrer</td>
			<td style="width:275px;">any source</td>
			<td style="width:136px;"></td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">PAMAM G4 Dendrimer</td>
			<td style="width:275px;">Andrews ChemService</td>
			<td style="width:136px;">AuCS - 297</td>
			<td style="width:259px;">&#160;10 % wt. solution in MeOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Lipophylic Sephadex LH-20</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:136px;">LH20100</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Thin-layer chromatography plates</td>
			<td style="width:275px;">Merck Millipore</td>
			<td style="width:136px;">1.05554.0001</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Formic acid</td>
			<td style="width:275px;">VWR Chemicals</td>
			<td style="width:136px;">20318.297</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Lophylizer&#160;</td>
			<td style="width:275px;">any source</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Gadollinium(III) chloride hexahydrate</td>
			<td style="width:275px;">Aldrich</td>
			<td style="width:136px;">G7532</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Sodium hydroxide</td>
			<td style="width:275px;">Acros Organics</td>
			<td style="width:136px;">134070010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">pH meter</td>
			<td style="width:275px;">any source</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Ethylenediaminetetraacetic acid disodium salt dihydrate</td>
			<td style="width:275px;">Aldrich</td>
			<td style="width:136px;">E5134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Mass spectrometer (ESI)</td>
			<td style="width:275px;">Agilent</td>
			<td style="width:136px;"></td>
			<td>Ion trap SL 1100&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Acetate buffer</td>
			<td style="width:275px;">any source</td>
			<td style="width:136px;"></td>
			<td>pH 5.8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Xylenol orange</td>
			<td style="width:275px;">Aldrich</td>
			<td style="width:136px;">52097</td>
			<td>20 &#956;M in acetate buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Hydrophylic Sephadex G-15</td>
			<td style="width:275px;">GE Healthcare</td>
			<td style="width:136px;">17-0020-01</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Amicon Ultra-15 Centrifugal Filter Unit</td>
			<td style="width:275px;">Merck Millipore</td>
			<td style="width:136px;">UFC900324</td>
			<td style="width:259px;">Ultracel-3 membrane (MWCO 3000)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Centrifuge</td>
			<td style="width:275px;">any source</td>
			<td style="width:136px;"></td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">NMR spectrometer&#160;</td>
			<td style="width:275px;">Bruker</td>
			<td style="width:136px;"></td>
			<td style="width:259px;">Avance III 300 MHz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Topspin</td>
			<td style="width:275px;">Bruker</td>
			<td style="width:136px;"></td>
			<td style="width:259px;">version 2.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Combustion analysis instrument</td>
			<td style="width:275px;">EuroVector SpA</td>
			<td style="width:136px;"></td>
			<td style="width:259px;">EuroEA 3000 Elemental Analyser&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">MALDI-ToF MS instrument</td>
			<td style="width:275px;">Applied Biosystems</td>
			<td style="width:136px;"></td>
			<td style="width:259px;">Voyager-STR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Deuteriumoxid</td>
			<td style="width:275px;">Carl Roth GmbH</td>
			<td style="width:136px;">6672.3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">tert-Butyl alcohol</td>
			<td style="width:275px;">Carl Roth GmbH</td>
			<td style="width:136px;">AE16.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Vortex mixer</td>
			<td style="width:275px;">any source</td>
			<td style="width:136px;"></td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Norell NMR tubes</td>
			<td style="width:275px;">Deutero GmbH</td>
			<td style="width:136px;">507-HP-7</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">NMR coaxial tube</td>
			<td style="width:275px;">Deutero GmbH</td>
			<td style="width:136px;">coaxialb-5-7</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">DLS instrument</td>
			<td style="width:275px;">Malvern</td>
			<td style="width:136px;"></td>
			<td style="width:259px;">Zetasizer Nano ZS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">0.20 &#956;m PTFE filter&#160;</td>
			<td style="width:275px;">Carl Roth GmbH</td>
			<td style="width:136px;">KC94.1</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">HEPES</td>
			<td style="width:275px;">Fisher BioReagents</td>
			<td style="width:136px;">BP310</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Plastic tube vials</td>
			<td style="width:275px;">any source</td>
			<td style="width:136px;"></td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Dotarem</td>
			<td style="width:275px;">Guerbet</td>
			<td style="width:136px;"></td>
			<td style="width:259px;">NDC 67684-2000-1</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:251px;">MRI scanner</td>
			<td style="width:275px;">Bruker</td>
			<td style="width:136px;"></td>
			<td style="width:259px;">BioSpec 70/30 USR magnet (7 T). Note: potential hazards related to high magnetic fields</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">RF coil</td>
			<td style="width:275px;">Bruker</td>
			<td style="width:136px;"></td>
			<td style="width:259px;">dual frequency volume coil (RF RES 300 1H/19F 075/040 LIN/LIN TR)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Paravision (software)</td>
			<td style="width:275px;">Bruker</td>
			<td style="width:136px;"></td>
			<td style="width:259px;">Version 5.1</td>
		</tr>
	</tbody>
</table>

preparation and in vitro characterization of dendrimer-based contrast agents for magnetic resonance imaging

钆（III）与无环或大环螯合物的顺磁性配合物是用于磁共振成像（MRI）中最常用的造影剂（CA）的。他们的目的是提高在组织的水质子的弛豫率，从而增加了MR图像对比度和磁共振测量的特异性。当前临床批准的造影剂是正在迅速从体内清除的低分子量分子。作为顺磁性螯合剂的载体的使用树枝状聚合物可以在更高效的MRI造影剂的未来发展中发挥重要作用。具体地，增加在一个较高的信号对比度顺磁物质的结果的局部浓度。此外，此CA提供由于其高分子量和尺寸较长的组织的保留时间。这里，我们证明了基于聚（酰氨基胺）大分子MRI造影剂（PAMAM）与monomacro树枝状聚合物的制备便利过程环状DOTA型螯合剂（DOTA - 1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸）。螯合单元通过利用朝向PAMAM树枝状聚合物的胺基表面基团的异硫氰酸酯（NCS）基团的反应性，以形成硫脲桥追加。树枝状产物纯化并通过核磁共振谱，质谱和元素分析法分析。最后，高清晰度的MR图像记录和从制备树枝状和市售单体剂获得的信号反差进行比较。

DCA的制备包括两个阶段:1）的单体的DOTA型螯合剂的合成（ 图1）和2）与G4 PAMAM树枝状聚合物和树枝状钆（III）络合物的后续制备螯合剂的耦合（ 图2） 。在第一阶段，制备含有四个羧酸的基于CYCLEN-DOTA型螯合剂和适合于进一步合成修饰的正交基。从1（DO3A- 叔丁基酯）7开始制备，这是烷基化叔丁基2-溴-4-（4-硝基苯基）丁8提供DOTA衍生物2。钯催化氢化还原芳族硝基在2得到苯胺3。的3与二氯硫化碳的转换导致的异硫氰酸酯4，which的先前用作树枝状的CA 17的制备的胺反应剂。 在下面的阶段中，大环化合物4作为基本单体单元中的偶联反应，以市售的G4 PAMAM树枝状聚合物。树枝状聚合物的胺基表面基团在碱的存在下单体4的异硫氰酸酯基团反应。用甲醇作为洗脱剂的亲脂性凝胶过滤介质通过大小排阻色谱法除去过量的4。所获得的树枝状大环共轭5的叔丁基酯用甲酸进行水解，得到6，然后将其冷冻干燥，不经纯化用于下一步骤。钆（III）DOTA型大环化合物的复合物的形成是通过将GdCl 3·6H 2 O的水溶液ö进行F 6，同时保持pH值在约7。过量的Gd（Ⅲ），用共同的螯合剂乙二胺四乙酸（EDTA）络合。的GdEDTA复杂和过量的EDTA采用以水为洗脱剂的亲水性凝胶过滤介质从系统中通过大小排阻层析去除。剩下的小尺寸的杂质，从由使用3- kDa的离心过滤单元离心溶液中除去。 继树枝状大分子大环化合物结合物的合成，组合的分析方法已被用来表征产物。以确定的5和6的表面胺占用，1 H NMR谱进行了分析。的结果进行了比较，并与最终产物（DCA），其中具有大环化合物的树枝状聚合物的装载已使用元素分析和MALDI-TOF质谱法估计（ 图证实3）。这三种方法的组合导致了平均的49大环单元被缀合到G4树枝状聚合物，其对应于〜75％的胺表面基团而定。 所述树枝状复杂的进一步表征包含在弛豫值的确定，从而产生6.2±0.1毫米-1秒-1每钆（III）（或大致约300毫-1秒-1每个树枝状聚合物）为纵弛豫和30.5± 0.6毫-1秒-1每钆（III）（几乎1500毫-1秒-1每个树枝状聚合物）为横向弛豫。 DLS测定法显示的7.2±0.2纳米的流体动力学直径为DCA（ 图4）。 最后，以证明树枝状MRI造影剂的作用，于两套幻影与DCA和诊所进行MR成像同盟可用GdDOTA用于比较（ 图5）。第一组幻影被用于在相同的Gd（III）的浓度比较这两个对比剂的目的制备，而第二组的目的是要表明的效果在树枝状和单体造影剂的可比较的分子的浓度，分别。 <img alt="图1" src="/files/ftp_upload/54776/54776fig1.jpg" /> 图 1: 大环DOTA型螯合剂4试剂，条件和分离收率的合成 :（ⅰ） 叔丁基2-溴-4-（4-硝基苯基）丁酸乙酯，K 2 CO 3，DMF，45℃ ，16小时，72％; （二） 的 H 2，钯/碳，乙醇，室温，16小时，95％; （三）中海集运2等3 N，RT，2小时，53％。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54776/54776fig1large.jpg" target="_blank">请点击此处查看该网络的放大版本</a><html>古尔。 <img alt="图2" src="/files/ftp_upload/54776/54776fig2.jpg" /> 图 2: 树枝状MRI对比剂的DCA试剂和条件的合成 :（ⅰ）4，的Et 3 N，DMF，45℃，48小时，91％; （ⅱ）甲酸，60℃，24小时，定量; （三）GdCl 3∙6H 2 O，pH值7.0，室温，24小时，71％。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54776/54776fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54776/54776fig3.jpg" /> 图 3: 通过MALDI-TOF质谱法的装置的树枝状产物的表征 DCA获得的典型的MALDI-TOF质谱。rget ="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/54776/54776fig4.jpg" /> 图4: 动态光散射（DLS）DCA（HEPES，pH值7.4）的DLS测量手段的树枝状产品的表征。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54776/54776fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54776/54776fig5.jpg" /> 图 5: 管幻影 体外 实验MRI在7牛逼磁场 DCA和GdDOTA的（A，B） 笔 1 -加权和（C，D）T2加权MRI。每个MRI实验以进行<html>两种不同浓度造影剂组成:（a，c）与可比的Gd（III）的浓度（HEPES，pH7.4）中; （B，D）用DCA:1的GdDOTA浓度比:5（HEPES，pH7.4）中。的浓度每分子表达和SNR值显示在括号中。在这些实验中所用的参数为:场的视场（FOV）= 40×40 平方毫米，层厚= 0.5毫米，激励（NEX）= 30的数; （ 一 ）矩阵大小（MTX）= 256×256，重复时间（TR）= 100毫秒，回波时间（TE）= 2.95毫秒，翻转角（FA）= 90°，采集时间（TA）= 12分48秒; （ 二 ）MTX = 256×256，TR / TE = 20 / 2.95毫秒，FA = 90°，TA = 2分34秒; （ 三 ）MTX = 512×512，TR / TE = 10,000 / 130毫秒，稀有因子（RF）= 16，TA = 26分40秒; （ 四 ）MTX = 512×512，TR / TE = 10,000 / 100毫秒，RF = 16，TA = 26分40秒。776fig5large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging at JoVE.com

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

本协议描述携带CYCLEN基于大环螯合物协调顺钆离子树枝状磁共振成像（MRI）造影剂的制备和表征。在一系列的体外 MRI实验中，相对于可商购的单体类似物时此代理产生的放大的磁共振信号。

制备及<em&gt;体外</em&gt;磁共振成像基于树状大分子造影剂的表征

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance ...

preparation-vitro-characterization-dendrimer-based-contrast-agents

Nanyang Technological University

Research

JoVE Journal

Chemistry

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

9.8K 次观看.  Max Planck Institute for Biological Cybernetics. 本协议描述携带CYCLEN基于大环螯合物协调顺钆离子树枝状磁共振成像（MRI）造影剂的制备和表征。在一系列的体外 MRI实验中，相对于可商购的单体类似物时此代理产生的放大的磁共振信号。 

视频: Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

The development of fluorescent indicators represented a revolution for life sciences. Genetically encoded and synthetic fluorophores with sensing abilities allowed the visualization of biologically relevant species with high spatial and temporal resolution. Synthetic dyes are of particular interest thanks to their high tunability and the wide range of measureable analytes. However, these molecules suffer several limitations related to small molecule behavior (poor solubility, difficulties in targeting, often no ratiometric imaging allowed). In this work we introduce the development of dendrimer-based sensors and present a procedure for pH measurement in vitro, in living cells and in vivo. We choose dendrimers as ideal platform for our sensors for their many desirable properties (monodispersity, tunable properties, multivalency) that made them a widely used scaffold for several biomedical devices. The conjugation of fluorescent pH indicators to the dendrimer scaffold led to an enhancement of their sensing performances. In particular dendrimers exhibit reduced cell leakage, improved intracellular targeting and allow ratiometric measurements. These novel sensors were successfully employed to measure pH in living HeLa cells and in vivo in mouse brain.

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

Fluorescence sensors are powerful tools in life science. Here we describe a methodology to synthesize and use dendrimer-based fluorescent sensors to measure pH in living cells and in vivo. The dendritic scaffold enhances the properties of conjugated fluorescent dyes leading to improved sensing properties.

合成，细胞和交货<em&gt;在体内</em&gt;的树状为基础的pH值传感器应用

The development of  fluorescent indicators represented a revolution for life sciences. Genetically  encoded and synthetic fluorophores with sensing ...

科研

化学

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

SDF-1α - 壳聚糖硫酸葡聚糖纳米粒子的制备与表征

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar concentration forming hydrophobic cores surrounded by hydrophilic shells in aqueous environments. The core of these micelles can be utilized to load hydrophobic, poorly water soluble drugs like docetaxel (DTX) and everolimus (EVR). Systematic characterization of the micelle structure and drug loading capabilities are important before in vitro and in vivo studies can be conducted. The goal of the protocol described herein is to provide the necessary characterization steps to achieve standardized micellar products. DTX and EVR have intrinsic solubilities of 1.9 and 9.6 &#181;g/ml respectively Preparation of these micelles can be achieved through solvent casting which increases the aqueous solubility of DTX and EVR to 1.86 and 1.85 mg/ml, respectively. Drug stability in micelles evaluated at room temperature over 48 hr indicates that 97% or more of the drugs are retained in solution. Micelle size was assessed using dynamic light scattering and indicated that the size of these micelles was below 50 nm and depended on the molecular weight of the polymer. Drug release from the micelles was assessed using dialysis under sink conditions at pH 7.4 at 37 oC over 48 hr. Curve fitting results indicate that drug release is driven by a first order process indicating that it is diffusion driven.

The goal of this protocol is to describe the preparation and characterization of physically entrapped, poorly water soluble drugs in micellar drug delivery systems composed of amphiphilic block copolymers.

制备及个体表征和多载药物理截留聚合物胶束

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

超分子胶体的合成与表征

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Low-energy Cathodoluminescence for (Oxy)Nitride Phosphors

Nitride and oxynitride (Sialon) phosphors are good candidates for the ultraviolet and visible emission applications. High performance, good stability and flexibility of their emission properties can be achieved by controlling their composition and dopants. However, a lot of work is still required to improve their properties and to reduce the production cost. A possible approach is to correlate the luminescence properties of the Sialon particles with their local structural and chemical environment in order to optimize their growth parameters and find novel phosphors. For such a purpose, the low-voltage cathodoluminescence (CL) microscopy is a powerful technique. The use of electron as an excitation source allows detecting most of the luminescence centers, revealing their luminescence distribution spatially and in depth, directly comparing CL results with the other electron-based techniques, and investigating the stability of their luminescence properties under stress. Such advantages for phosphors characterization will be highlighted through examples of investigation on several Sialon phosphors by low-energy CL.

Low-energy Cathodoluminescence for OxyNitride Phosphors

An excellent chemical and luminescence stabilities of (oxy)nitride phosphors present it as an promising alternative to currently used sulfide and oxide phosphors. In this paper, we present the way to investigate its local luminescence properties using low-energy cathodoluminescence (CL).

低能量的阴极射线（氧）氮化物荧光粉

Nitride and oxynitride (Sialon) phosphors are good candidates for the ultraviolet and visible emission applications. High performance, good stability and ...

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with nitroxide spin probe 3-(2-Iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IPSL) as a guest inside the periodic mesoporous organosilica (PMO) aerogel UKON1-GEL as a host and ethanol as a solvent is used as an example to describe the protocol. Data is shown from a previous publication, where the protocol has been applied to both IPSL and Tris(8-carboxy-2,2,6,6-perdeutero-tetramethyl-benzo[1,2-d:4,5-d&#8242;]bis(1,3)dithiole) methyl (Trityl) as guest molecules and UKON1-GEL and SILICA-GEL as host systems. 
A method is shown to prepare aerogel samples that cannot be synthesized directly in the sample tube for measurement due to a size change during synthesis. The aerogel is attached to sample tubes using heat shrink tubing and a pressure cooker to reach the necessary temperature without evaporating the solvent in the process. The method does not assume a clearly defined initial distribution of guest molecules at the start of the measurement. Instead, it requires a reservoir on top of the aerogel and experimentally determines the influx rate during data analysis.
The diffusion is monitored continually over a period of 20 hr by recording the 1d spin density profile within the sample. The spectrometer settings for the imaging experiment are described quantitatively. Data analysis software is provided to take the resonator sensitivity profile into account and to numerically solve the diffusion equation. The software determines the macroscopic translational diffusion coefficient by least square minimization of the difference between the experiment and the numerical solution of the diffusion equation.

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

A protocol for the in situ monitoring of the diffusion of guest molecules in porous media using electron paramagnetic resonance (EPR) imaging is presented.

在多孔介质中使用电子顺磁共振成像客体分子扩散的原位监测

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

动态和电泳光散射的 DNA-磁性颗粒结合分析

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We present methods for depositing and passively manipulating C60 molecules on rippled graphene that advance the pursuit of realizing applications involving 1D C60/graphene hybrid structures. The techniques applied in this exposition are geared towards high vacuum systems with preparation areas capable of supporting molecular deposition as well as thermal annealing of the samples. We focus on C60 deposition at low pressure using a homemade Knudsen cell connected to a scanning tunneling microscopy (STM) system. The number of molecules deposited is regulated by controlling the temperature of the Knudsen cell and the deposition time. One-dimensional (1D) C60 chain structures with widths of two to three molecules can be prepared via tuning of the experimental conditions. The surface mobility of the C60 molecules increases with annealing temperature allowing them to move within the periodic potential of the rippled graphene. Using this mechanism, it is possible to control the transition of 1D C60 chain structures to a hexagonal close packed quasi-1D stripe structure.

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Here we present a protocol for the fabrication of C60/graphene hybrid nanostructures by physical thermal evaporation. Particularly, the proper manipulation of deposition and annealing conditions allow the control over the creation of 1D and quasi 1D C60 structures on rippled graphene.

C 型60/石墨烯混合纳米结构的制备与表征

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound electrons with neutral molecules/atoms. State-of-the-art in vacuo anion generation techniques allow application to a broad range of atomic, molecular, and cluster anion systems. These are separated and selected using time-of-flight mass spectrometry. Electrons are removed by linearly polarized photons (photo detachment) using table-top laser sources which provide ready access to excitation energies from the infra-red to the near ultraviolet. Detecting the photoelectrons with a velocity mapped imaging lens and position sensitive detector means that, in principle, every photoelectron reaches the detector and the detection efficiency is uniform for all kinetic energies. Photoelectron spectra extracted from the images via mathematical reconstruction using an inverse Abel transformation reveal details of the anion internal energy state distribution and the resultant neutral energy states. At low electron kinetic energy, typical resolution is sufficient to reveal energy level differences on the order of a few millielectron-volts, i.e., different vibrational levels for molecular species or spin-orbit splitting in atoms. Photoelectron angular distributions extracted from the inverse Abel transformation represent the signatures of the bound electron orbital, allowing more detailed probing of electronic structure. The spectra and angular distributions also encode details of the interactions between the outgoing electron and the residual neutral species subsequent to excitation. The technique is illustrated by the application to an atomic anion (F&#8722;), but it can also be applied to the measurement of molecular anion spectroscopy, the study of low lying anion resonances (as an alternative to scattering experiments) and femtosecond (fs) time resolved studies of the dynamic evolution of anions.

Here, we present a protocol for photoelectron imaging of anionic species. Anions generated in vacuo and separated by mass spectrometry are probed using velocity mapped photoelectron imaging, providing details of anion and neutral energy levels, anion and neutral structure and the nature of the anion electronic state.

由 F− 310 Nm 支队所示的阴离子光电子成像

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...