The authors gratefully acknowledge PRIN 2010-2011 NANOMED prot. 2010 FPTBSH and PRIN 2010-2011 PROxy prot. 2010PFLRJR_005 for funding.

1. CDNSEDTA聚合物的合成<ol><li>干β环糊精（β-CD）在烘箱中在80℃使用前4小时。 500ml干燥的二甲基亚砜（DMSO）和100毫升三乙胺（乙基3 N）在分子筛（4埃）24小时在该协议使用之前。 </li><li>引入25毫升DMSO中入50毫升单颈圆底烧瓶中。在磁力搅拌，加5.675克β-CD（5毫摩尔）的。为了减少团块的形成，在小部分以DMSO添加β-CD粉。 </li><li>大约30分钟后，用10毫升刻度吸管添加的Et 3 N的将6mL的均相溶液。保持在RT下搅拌15分钟下的混合物。投身烧瓶到室温水浴。 注:β-CD和EDTA之间的反应是放热的。因此，浸入烧瓶放入水浴利于热交换避免反应混合物的过热。 </li><li>添加5.124克（20毫摩尔，准备CDNSEDT的A 1:4）或10.248克（40毫摩尔，准备CDNSEDTA 1:8）的下强烈搅拌EDTA二酐。 </li><li> 3小时后，除去固体物质（CDNSEDTA 1:4或CDNSEDTA 1:8）从使用刮刀并用研钵和研杵严重粉碎它烧瓶中。 </li><li>在RT（100毫升×5次），用丙酮洗涤固体材料到滤纸，用HCl的0.1M（200毫升×5次）和去离子水（200ml×3次）。 </li><li>最后，干燥全部空气中的固体材料在室温48小时，细粉碎成一个研钵和杵，然后保持真空（&lt;15毫巴）下2小时，在45℃。 </li></ol><img alt="图1" src="/files/ftp_upload/53769/53769fig1.jpg" /> 图1:该CDNSEDTA聚合物的示意图示意图合成路线。左:单体β环糊精（β-CD）和交联剂EDTA的二酐的分子结构。箭头上的整体的反应条件。对:交联聚合物的草图<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53769/53769fig1large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> 2. HR-MAS NMR测量<ol><li> HR-MAS NMR样品制备 <ol><li>准备在重水（99.8％）布洛芬钠盐（IP）中的溶液0.27微米。 </li><li> 20毫克CDNSEDTA 1的添加:4和2mg无水碳酸钠（ 的 Na 2 CO 3），以150微升在点2.1.1制备的溶液）到2ml玻璃小瓶中。混合用小铲将小瓶中的内容，以使之均匀。使用此过程形成的凝胶前等待2小时。重复此点，为CDNSEDTA 1:8的聚合物。 </li><li>插入在适合于使用小刮勺的HR-MAS NMR实验采用5mm NMR转子的凝胶。凝胶使用的总量取决于转子的内部容积（推荐12微升）。 </li></ol></li><li> HR-MAS 1 H-NMR实验 <ol><li>在MAS气动控制单元，在可变温度单位样品温度在305 K 4千赫的转子旋转速度:设置以下仪器参数。 </li><li>获得在CDNSEDTA布洛芬的1 H HR-MAS NMR光谱（1:4）和CDNSEDTA（1:8），使用的质子共振的常规脉冲序列的聚合物体系。 <ol><li>创建一个新的数据集。点击"AcquPars"标签。选择PULPROG:ZG。 </li><li>选择的扫描次数（NS = 4），并将其（D 1 = 5秒）。设置谱宽（SW = 8ppm的）之间的时间延迟，时域（TD = 16K）和所述接收器增益（RG = 32 ）。 </li><li>键入控制台上的"ZG"并且会有一个自由感应衰减（FID）在屏幕上。要处理"ProcPars"选项卡上的数据点击。设置的光谱的大小（SI = 32K），一个指数乘法窗口函数（WDW = EM）和线展宽（LB = 1）。键入"FT"来执行傅立叶变换。相使用屏幕上的相位片的光谱。获取高分辨率很好地解决频谱。 </li></ol></li></ol></li></ol><img alt="图2" src="/files/ftp_upload/53769/53769fig2.jpg" /> 图2:双极脉冲对纵向涡流延迟（BPPLED）脉冲序列用于进行PFGSE实验脉冲序列的示意图。对于90°脉冲的相位周期是:P1:（0）16，P2:（0022）4，P 3:（0）4（2）4（1）4（3）4，P4:（0202 2020 1313 3131 ），P5:（0）4（2）4（1）4（3）4。 180°脉冲+ X。 （从ref.18修改） <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53769/53769fig2large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <ol start="3"><li> HR-MAS 1 </s达&gt; H-NMR实验PGSE 注意:使用在图 2中所报告的BPPLED脉冲序列18执行的PGSE实验。这是一个伪二维实验用梯度的斜坡的2％，在间接尺寸线性增加至100％。取决于扩散时间Δ和梯度脉冲δ的信号强度被衰减。这些参数的优化运行正确一个PGSE实验前是必需的。该优化是通过运行在其中Δ保持恒定几个1D测量完成，而δ是多种多样的。 <ol><li> 参数优化 <ol><li>创建一个新的数据集 - 实验编号1.单击"AcquPars"标签。选择PULPROG:ledbpgp2s1d扩散优化1D脉冲序列。 </li><li>选择（NS = 16）扫描的数目和它们之间的时间延迟（D1 = 10秒）。设置的光谱宽度（SW = 8ppm的），叔他的时域（TD = 16K）和所述接收器增益（RG = 32）。 </li><li>设置Δ等于恒定值和δ（P30）到试验值（该序列中D20）。启动值Δ= 50毫秒，δ（高分辨率仪器最大允许值）= 3毫秒。 </li><li>读谱频率（SFO1）的，由1 H实验值和现在使用此值。设置GPZ6梯度强度至2％。重复步骤2.2.2.3。使用这个频谱作为用于优化参考。 </li><li>在同一数据集建立实验号2.遵守所有的实验参数。增加GPZ6梯度强度至95％。重复步骤2.2.2.3。使用双显示图标比较该光谱与参考光谱和观察的信号强度的变化。 注:与参考频谱相比，减毒以及频谱应该有大约5％的残余信号强度。如果信号强度丢失，降低的δ和水库的值直到δ正确的价值被发现从点2.3.1.3挞的第2.3.1节的过程。 </li><li>重复第2.3.1节中的参数优化程序为所有五个Δ值。 注:选择Δ= 50，80，110，140 5值和170毫秒和优化相应δ3，2.7，2.4，2.1，1.8毫秒（知识产权在CDNSEDTA 1:8）和成δ至2.7，2.4 2，1.7，1.4（知识产权在CDNSEDTA 1:4）。 </li></ol></li><li> 二维扩散数据集采集 <ol><li>在相同的数据集创建实验编号3中，所有的1D实验参数将被加载。键入"EDA"。选择PULPROG:ledbpgp2s二维脉冲序列，改变parmode为2D。 </li><li>设置FnMODE = QF。设置的时间域的TD在F2中尺寸等于32的梯度步骤的数目。所有其他的参数设置是否正确。输入"DOSY"和梯度的斜坡将被生成并存储在一个文件中。圣艺术和斜坡（2 - 95）的最终值给出作为输入参数。此次收购已启动。 </li></ol></li></ol></li><li> 数据处理 <ol><li>键入"XF2"中的F2维度来执行傅立叶变换。键入"ABS2"在F2尺寸进行基线校正。类型"setdiffparm"召回实验参数（Δ，δ，和梯度清单）为下一个处理步骤。 </li><li>在分析标签点击"的T1 / T2弛豫模块"，并定义使用2D实验的第一频谱嵌合的峰值。定义峰值范围和执行拟合。获得在每个施加梯度步骤的信号强度。 注意:信号强度I（Q，T d）中 ，对于每个Δ值，取决于实验变量:根据施加脉冲日提交的梯度（克），时间变量（δ），magnetogyric比（γ）Q =（γgδ）在FOllowing公式: <img alt="公式1" src="/files/ftp_upload/53769/53769equation1.jpg" /> 与分子的MSD = Z 2。 </li><li>导出的信号强度在电子表格中，并执行该数据的线性拟合，以获得Z 2的值对每一个观察到的扩散时间td。 注:MSD值与根据观测时间t D: <img alt="方程2" src="/files/ftp_upload/53769/53769equation2.jpg" /></li><li>执行Z 2对T d设定的每个实验T d设定值数图。指数α值是线性回归的斜率。上述报道方程的物理方面更为详尽的讨论可以在文献中找到。 21，并在其中的参考文献。 注意:根据指数α的值，扩散机制的定义是:i）在α= 1，ⅱ）异常subdiffusiv各向同性无限制扩散0 &lt;α&lt;1，III）异常superdiffusive为α&gt; 1政权Ë制度。 </li></ol></li></ol>

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The authors have nothing to disclose.

我们提出了一个实验方法来确定封装内CDNSEDTA水凝胶的两个有代表性的剂型有小毒分子的扩散制度。 HR-MAS PGSE核磁共振允许在给定的扩散时间的小分子的均方位移的确定（在几毫秒的上升到第二范围内），在微米尺度然后监测距离。中观察到的范围内（50 - 170毫秒）只有一种类型的运动的观察对每个研究的系统。然而，应当强调的是，更长的观察时间不同的扩散制度之间的过渡，可以观察到22。因此，这里报导的结果被称为时间尺度由可用于实验，用在我们的处置仪器硬件决定。 对于这项工作的每一个步骤 - 高分子合成，水凝胶装载，HR-MAS NMR数据收集和处理，我们在这里列举三个主要的问题D中的相应的解决方案。 纯化 根据在点1.6中概述的过程的材料的纯化允许DMSO中的消除，的Et 3 N和未反应的EDTA最终载到反应产物。特别是，用0.1M盐酸水溶液洗涤导致完全除去的Et 3 N的仅丙酮洗涤该材料是没有效率用于此目的。实际上，的Et 3 N是部分地存在于与抗衡离子的反应结束的Et 3 NH + COO-由交联的EDTA提供。 NMR样品制备 转子馅的样品制备（参见2.1.3）的关键一步。必须避免气泡的存在。如果存在气泡，一个不正确的纺丝一般检测（ 例如 ，最大纺丝速度没有达到，纺丝不能启动所有，不UNIFORM纺纱速度）。在非均匀的纺丝或启动失败纺纱转子的情况下，提取样品并重复转子更仔细地填充。 NMR数据分析 在点2.4.2中描述了如何对来自强度衰减曲线获得MSD的值。很多时候实验点似乎并不遵循完全的线性趋势VS Q 2。在这种情况下，仅使用数据集的线性部分和运行线性回归。 所得到的结果可总结如下: D 2 O解决方案 在这个简单的例子，对MSD示出了在扩散时间Δ和IP分子线性关系经历无限制扩散在溶液中。这种情况下，通常被称为"正常"的扩散。自扩散系数可以从一个单一的测量在任何观测Ť直接估计IME T d设定。在一般的方法中，在各向同性的分子溶剂扩散的测量可以作为参考，调查该支架上的封装位的传输性质的影响。 CDNSEDTA 1:4聚合物 知识产权包封到聚合物水凝胶的影响相比于水溶液中的药物输运性质。当检测到与α= 0.64 subdiffusive运动。的药物分子的平移运动是由不同尺寸的纳米孔的存在妨碍了源自在交联过程。 CDNSEDTA（1:8）聚合物 在这种情况下，意外的值α= 1.06被确定，因此显示出略微superdiffusive运动制度。因此，在颗粒上的MSD的加速度效果观察（ 表1）。这种效应可以归因于该聚合物b的负电位ackbone通过在CDNS聚合物的某些部分带负电荷的羧酸基团产生的。带负电荷的IP分子的静电相互作用提供了运动的superdiffusive组件的驱动力。 描述关于通过将药物在对应于两个水凝胶的不同分子的环境经历的扩散制度这里提供的信息的方法，通过以上讨论的α指数的值制剂。这种方法是普遍适用的，可以理直气壮地提出作为调查包封药物输运性质的工具，有趣的属于淘汰的给药控释系统的设计。然而，应该记住的是，这里给出的结果从所用的仪器的硬件限制受到影响。 此外，对于该方法的普遍适用性一些限制因素可能是低thesized:用亲脂性和/或不带电荷的药物，该药物对聚合物骨架的附着力强的可能性，从而导致未解决的NMR信号，并在pH控制困难载药凝胶的制备。最后，应该提及的是，扩散NMR数据跨越毫秒的时间尺度，而经典的体外药物释放实验依赖更长 ​​的时间窗口。这是研究和讨论的开放点对象。作为一个可能的贡献，以解决这个问题，我们最近检查模型药物的扩散数据在基于碳水化合物的水凝胶的库和衍生的数学模型在与动力学数据23分子水平连接的测量。扩散数据和支架的更广泛的图书馆的馆藏较大正在调查，以改进和验证模型。

有在设计和能够截留的聚合系统的配制剂的兴趣日益增​​长，通过非共价相互作用，小分子具有潜在生化活性。这种材料预期找到的活性成分运输至选择性目标应用程序和在外部刺激，例如pH的变化，温度， 等。在这样的背景下的动作释放，水凝胶被证明是对纳米灵活和强大的材料鉴于药物1控释的。聚合物水凝胶的形成可通过用i）物理的，非共价相互作用，如氢键，ⅱ）共价导致能够在存在膨胀的三维网络的链的交联的高分子链相互连接来实现的水溶液或iii）的两个上述方法2-4的组合。 特别多才多艺类三角钱8引起环糊精纳米海绵（CDNS） - nsional，用于有机和无机物质的封装溶胀性聚合物可以从天然β环糊精（β-CD）通过缩合具有四羧酸5的合适的，活化的衍生物开始来获得的。 CDNS的合成，表征和应用是我们小组的综合研究的主题。在过去的几年的结果表明，CDNS显示肿胀，吸收/增列化学品，以及小分子药物释放的有趣特性，在药物活性成分9控释应用- 11和环境化学12 - 14。 鉴于这些前提，两个主要的问题，以在聚合物凝胶和溶质流动在凝胶矩阵15的改进的了解来解决关注的活性化合物的有效载荷</s了&gt;。文献同时提供了实验研究和大分子网络16,17与小分子扩散机制的理论。脉冲场梯度自旋回波（PGSE）NMR谱是广泛用于研究小分子在溶剂18中的平移扩散或纯液体的自扩散行之有效的结构的方法。最近的高分辨率魔角旋转（HR-MAS）NMR技术的发展使人们有可能收集移动分子的高分辨率核磁共振数据在异构的悬浮液19，凝胶和溶胀性聚合物20,21。事实上，实验装置结合HR-MAS核磁共振光谱和PGSE脉冲序列提供了观察主机的分子环境中的溶质分子的独特机会。因此，可以得到在凝胶基质中包裹的药物分子的输运性质的重要数据。因此高品质的实验数据可以是obtai斯内德让纳米主客体系统的设计更加合理。 在目前的工作中，我们描述了详细方案为以下步骤:i）合成和CDNS的两个不同的制剂纯化用EDTA聚合物（ 图1）的交联，称为CDNSEDTA，和其特征在于由不同的CD /交联剂摩尔比率:1:4（CDNSEDTA 1:4）和1:8（CDNSEDTA 1:8）;二）载药凝胶的制备CDNSEDTA 1两种:4和CDNSEDTA 1:8。在这个步骤中，我们使用作为模型药物分子，流行的非甾体抗炎布洛芬钠盐（IP）;三）知识产权的CDNSEDTA内部的传输性能严查通过PGSE-HRMAS核磁共振光谱水凝胶。我们在这里提出的方法是基于在水凝胶接着将MSD的时间依赖性的分析内包封的药物的均方位移（MSD）的测量。 我们都很开心。十岁上下强调，上面概述的方法 - 这是集中在药物的的MDS矩阵中的时间依赖性 - 提供相比仅基于药物的扩散系数的测定的综合方法的信息更广泛。我们最近展示了21，允许通过IP在CDNS水凝胶密闭经历正常和反常扩散制度的歧视，这种做法。 因此，我们认为，高分子合成/净化，形成载药水凝胶，HR-MAS NMR表征和MDS数据进行数据处理，的一步一步的描述是有意刻画的纳米系统科学家强大的工具包约束和小分子的释放。

<table><tbody><tr><td>HR-MAS probe</td><td>BRUKER</td><td>N/A</td><td>Probe for NMR measurements on semi-solid samples</td></tr><tr><td>NMR Spectrometer</td><td>BRUKER</td><td>DRX 500</td><td>FT NMR spectrometer for liquid ans semi-solis state</td></tr><tr><td>&amp;beta;-cyclodextrin (&amp;beta;-CD)</td><td>Alfa-Aesar</td><td>J63161</td><td>Reagent</td></tr><tr><td>Ethylenediaminetetracetic (EDTA) dianhydride</td><td>Sigma-Aldrich</td><td>332046</td><td>Reagent</td></tr><tr><td>Dimethylsulfoxide (DMSO)</td><td>Alfa-Aesar</td><td>D0798</td><td>Solvent</td></tr><tr><td>Triethylamine</td><td>Sigma-Aldrich</td><td>471283</td><td>Base (reagent)</td></tr><tr><td>Ibuprofen (IP) sadium salt</td><td>Sigma-Aldrich</td><td>I1892</td><td>Antinflammatory drug</td></tr><tr><td>Excel 2010</td><td>Microsoft</td><td>N/A</td><td>speadsheet for data analysis</td></tr><tr><td>Origin 8 SR0</td><td>OriginLab Co.</td><td></td><td>speadsheet for data analysis</td></tr></tbody></table>

transport properties of ibuprofen encapsulated in cyclodextrin nanosponge hydrogels: a proton hr-mas nmr spectroscopy study

β环糊精（β-CD）与乙二胺四羧酸二酐（EDTA）的化学交联导致称为环糊精纳米海绵（CDNSEDTA）支化聚合物。 4和1:8的CD-EDTA的摩尔比两个不同的制剂以1所述。相应的交联的聚合物用布洛芬钠盐（IP）的主导的同质，无色，载药凝胶0.27 1M水溶液接触。 高分辨魔角旋转（HR-MAS）核磁共振光谱系统进行了表征。脉冲梯度场自旋回波（PGSE）核磁共振光谱来测定IP的聚合物凝胶内的均方位移（MSD）在不同的观察时间T d设定 。 MSD = F（T d）段 :数据以研究MSD的时间依赖关系被进一步处理。该建议的方法是表征不同的扩散制度有用，在原则上，溶质可以在水凝胶，即正常或异常的扩散内体验。全协议，包括在聚合物制备和纯化，载药凝胶的获得，该NMR样品制备，MSD的通过HR-MAS NMR谱的测定和最终数据处理实现的MSD的时间依赖性在这里报告并讨论。所呈现的实验表示一个典范壳体和数据的创新的方法来封装旅客的输运性质的表征方面用于药物递送的潜在应用的聚合物主体内讨论。

我们首先应用这个方法，以溶解在水中的溶液，以验证这种方法的可行性的IP药物分子。代表结果的完整描述可以在文献中找到。 21.相反，我们将在这里集中在方法学方面，螺母和螺栓的方式进行数据收集和数据分析。 如图3所示，在一个半对数刻度，标准化的实验信号衰减I（Q，T D） / I（0，T D）为q 2的功能（根据第2.4节）。在表1中报道了每个扩散时间Δ观测MSD的值。 Z 2与T d设定 （ 图3）的数图给出了R 2 = 0.999（根据第2.4节）一行。对于IP获得一个标度指数α= 1溶解在D 2 O解决方案，指示在液体溶液高斯运动0.27 1M浓度。只有在这种情况下的自扩散系数可以相应地计算为D = 4.1 * 10 -10 m 2的仲- 1，是独立于观测时间Δ的。 <img alt="表格1" src="/files/ftp_upload/53769/53769table1.jpg" /> 表1: 实验MSD和 Δ 值 MSD在几个Δ（毫秒）测量值（M 2）的IP在D 2 O中的溶液，IP中CDNS（1:4）和IP中CDNS（1:8）的水凝胶。 （从参考文献21修改）。 <img alt="图3" src="/files/ftp_upload/53769/53769fig3.jpg" /> 图3:核磁共振信号衰减和MSD时间相关的情节 ）归核磁共振信号衰减I（Q，T D）在D 2 O解决方案Q 2的功能IP 二 ）对数VS扩散MSD的阴谋对于时间tdIP在D 2 O解决方案。 （从参考文献21修改） <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53769/53769fig3large.jpg" target="_blank">点击此处查看该图的放大版本。</a> 的实验步骤已被用于研究包封在CDNSEDTA 1的IP药物分子的扩散运动:4和CDNSEDTA 1:8的水凝胶图4示出了归一化的实验信号衰减I（Q，T D）/ I（0，T d设定）作为q 2的功能。使用衰减的线性部分进行的线性拟合，然后计算将MSD的值CDNS 1:从线性拟合的斜率8个样本（ 表1）:4和CDNS 1。 MSD的与T d设定的log-log图（ 图5）给出，其中R 2 = 0.981在CDNSEDTA线性相关于IP（1:4）指示博士的子扩散运动和一个标度指数α= 0.64<html>微克的聚合物凝胶。使用类似的过程，知识产权在CDNSEDTA运动制度（1:8）测定的聚合物。该数据的线性拟合，得到定标指数α= 1.06，其中R 2 = 0.972。聚合物网络施加的小分子药物略有superdiffusive运动。因而所提出的方法给出了数据的一个两步骤的处理获得在点2.4.5报道方程式的α指数。 α值是针对给定的基质和客体分子中观察到的扩散机制的一个描述符。 <img alt="图4" src="/files/ftp_upload/53769/53769fig4.jpg" /> 图4:在CDNSEDTA 1 的核磁共振信号衰减归核磁共振信号衰减I（Q，T D）作为Q 2的功能的 IP 简介 :4（a）和CDNSEDTA 1:8（b）中 。 （从REF修改。21）平直"&gt;点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/53769/53769fig5.jpg" /> 图5:MDS的时间相关性对数VS封装在CDNSEDTA的IP扩散时间td MSD的情节1:4（a）和CDNSEDTA 1:8（B）。 （从参考文献21修改） <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53769/53769fig5large.jpg" target="_blank">点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Transport Properties of Ibuprofen Encapsulated in Cyclodextrin Nanosponge Hydrogels: A Proton HR-MAS NMR Spectroscopy Study at JoVE.com

Transport Properties of Ibuprofen Encapsulated in Cyclodextrin Nanosponge Hydrogels: A Proton HR-MAS NMR Spectroscopy Study

布洛芬包封在β环糊精纳米海绵聚合物网络的运动制度使用脉冲场梯度自旋回波（PGSE）NMR技术分析。合成，纯化，载药量，实施核磁共振脉冲序列和数据分析在几个观察时间制定出药物的均方位移的详细描述。

布洛芬封装的输运性质环糊精Nanosponge凝胶:质子HR-MAS NMR光谱研究

The chemical cross-linking of &#946;-cyclodextrin (&#946;-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as ...

transport-properties-ibuprofen-encapsulated-cyclodextrin-nanosponge

Nanyang Technological University

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JoVE Journal

Bioengineering

10.2K 次观看.  Politecnico di Milano. 布洛芬包封在β环糊精纳米海绵聚合物网络的运动制度使用脉冲场梯度自旋回波（PGSE）NMR技术分析。合成，纯化，载药量，实施核磁共振脉冲序列和数据分析在几个观察时间制定出药物的均方位移的详细描述。 

视频: Transport Properties of Ibuprofen Encapsulated in Cyclodextrin Nanosponge Hydrogels: A Proton HR-MAS NMR Spectroscopy Study

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 ...

科研

化学

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the average content of fluorinated drugs in their formulated drug product forms: tablets or capsules. This method is specific to fluorinated drugs because it detects only the content of fluorine, avoiding interference from the excipients that lack fluorine. The advantages of measuring the active content of fluorinated drugs using low-field 19F TD-NMR versus high-field 19F solid-state (SS) NMR are the simplicity of the method; the low cost; and the non-destructive nature of the technique, with all samples recoverable in intact forms (e.g., powders, tablets, and capsules), making this technique affordable for any laboratory.
We have tested the method with three fluorinated drug products available on the market - cinacalcet, lansoprazole, and ciprofloxacin - with doses ranging from 15 to 500 mg. The results of the analyses, measured by low-field 19F TD-NMR, supported the reported label claims for the average drug content.
Based on the simplicity and reproducibility of the analysis, we envision this methodology being implemented in any laboratory, including manufacturing plants, as a process analytical technology (PAT) tool in the pharmaceutical industry.

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

A simple and non-destructive technique that measures the average content of drug substances in formulated drug products containing fluorine using low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) is presented here. The technique can be applied to the development and manufacturing of drugs in the pharmaceutical industry.

应用程序和非破坏性19F 时间域核磁共振技术的方法来衡量中含氟药物产品的内容

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

生物工程

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

一种快速定量荧光方法蛋白靶向小分子药物筛选

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Magnetic and Thermal-sensitive Poly(N-isopropylacrylamide)-based Microgels for Magnetically Triggered Controlled Release

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were designed and fabricated for magnetically triggered release. PNIPAAm-based magnetic microgels with a spherical structure were produced via a temperature-induced emulsion followed with physical-crosslinking by mixing PNIPAAm, polyethylenimine (PEI), and Fe3O4-NH2 magnetic nanoparticles. Because of their dispersity, the Fe3O4-NH2 nanoparticles were embedded inside the polymer matrix. The amine groups exposed on the Fe3O4-NH2 and PEI surface supported the spherical structure by physically crosslinking with the amide groups of the PNIPAAm. The hydrophobic anti-cancer drug curcumin can be dispersed in water after encapsulation into the microgels. The microgels were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), and UV-Vis spectral analysis. Furthermore, magnetically triggered release was studied under an external high frequency magnetic field (HFMF). A significant &#34;burst release&#34; of curcumin was observed after applying the HFMF to the microgels due to the magnetic inductive heating (hyperthermia) effect. This manuscript describes the magnetically triggered controlled release of Cur-PNIPAAm/Fe3O4-NH2 encapsulated curcumin, which can be potentially applied for tumor therapy.

Magnetic and Thermal-sensitive PolyN-isopropylacrylamide-based Microgels for Magnetically Triggered Controlled Release

This manuscript describes the preparation of magnetic and thermal-sensitive microgels via a temperature-induced emulsion without chemical reaction. These sensitive microgels were synthesized by mixing poly(N-isopropylacrylamide) (PNIPAAm), polyethylenimine (PEI) and Fe3O4-NH2 nanoparticles for the potential use in magnetically and thermally triggered drug-release.

磁性和热敏性聚（<em&gt;ñ</em基于异丙基丙烯酰胺）的微凝胶用于磁触发控制释放

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were ...

Synthesis of Stimuli-responsive Nanogels using Aqueous One-step Crosslinking and Co-nanopolymerization

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to their versatile bottom-up synthesis and biocompatibility. While various methods have been employed for nanogel synthesis to date, very few have achieved it without the use of harsh organic solvents or high temperatures that can damage the integrity of the biological payload. In contrast, the methodology presented here accomplishes the synthesis of sub-100 nm sized, protein-loaded nanogels using mild reaction conditions. Here, we present a method for the non-covalent encapsulation of protein-based payloads within nano-gels that were synthesized using an aqueous-based, single-step, crosslinking copolymerization technique. In this technique, we initially electrostatically bind a protein-based payload to a cationic quaternary ammonium monomer and simultaneously cross-link and co-polymerize it using ammonium persulfate and N,N,N&#39;,N&#39;-tetramethylethylenediamine to form nanogels that entrap the protein payload. The size and polydispersity index of the nanogels is determined using dynamic light scattering (DLS), while the surface morphology is assessed by transmission electron microscopy (TEM). The mass of protein entrapped within nanogels is determined by calculating the encapsulation efficiency. Furthermore, the controlled-release ability of the nanogels via the gradual degradation of redox-responsive structural elements is also assessed in bioreduction assays. We provide examples of nanoparticle optimization data to demonstrate all caveats of nanogel synthesis and characterization using this technique. In general, uniformly sized nanogels were obtained with an average size of 57 nm and a polydispersity index value of 0.093. A high encapsulation efficiency of 76% was achieved. Furthermore, the nanogels exhibited controlled release of up to 86% of the encapsulated protein by gradual degradation of novel redox-responsive components in the presence of glutathione over 48 h.

Nanogels are an excellent and versatile nanoparticle platform for the delivery of biologics. Stimuli-responsive poly(ethylene) glycol-based polymeric nano-gels, capable of encapsulating protein-based payloads, were synthesized using a one-step cross-linking co-nanopolymerization strategy in aqueous conditions. The optimal fabrication and characterization of these novel nanoparticles are presented here.

使用水性一步交联和共纳米聚合合成刺激响应纳米凝胶

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

纳米传感器检测体内蛋白酶活性的无创诊断

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

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

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

闪速纳米沉淀法包聚物纳米粒子中疏水亲水化合物的作用

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

A Freeze-Thawing Method to Prepare Chitosan-Poly(vinyl alcohol) Hydrogels Without Crosslinking Agents and Diflunisal Release Studies

Chitosan-poly(vinyl alcohol) hydrogels can be produced by the freeze-thawing method without using toxic crosslinking agents. The applications of these systems are limited by their characteristics (e.g., porosity, flexibility, swelling capacity, drug loading and drug release capacity), which depend on the freezing conditions and the kind and ratio of polymers. This protocol describes how to prepare hydrogels from chitosan and poly(vinyl alcohol) at 50/50 w/w % of polymer composition and varying the freezing temperature (-4 &#176;C, -20 &#176;C, -80 &#176;C) and freeze-thawing cycles (4, 5, 6 freezing cycles). FT-IR spectra, SEM micrograph and porosimetry data of hydrogels were obtained. Also, the swelling capacity and drug loading and release of diflunisal were assessed. Results from SEM micrographs and porosimetry show that the pore size decreases, while the porosity increases at lower temperatures. The swelling percentage was higher at the minor freezing temperature. The release of diflunisal from the hydrogels has been studied. All the networks maintain the drug release for 30 h and it has been observed that a simple diffusion mechanism regulates the diflunisal release according to Korsmeyer-Peppas and Higuchi models.

A Freeze-Thawing Method to Prepare Chitosan-Polyvinyl alcohol Hydrogels Without Crosslinking Agents and Diflunisal Release Studies

The freezing-thawing method is used to produce chitosan-poly(vinyl alcohol) hydrogels without crosslinking agents. For this method, it is important to consider the freezing conditions (temperature, number of cycles) and polymer ratio, which can affect the properties and applications of the obtained hydrogels.

一种冷冻-解冻方法，用于制备奇托桑-波里（乙烯基酒精）水凝胶，无交联剂和二分释放研究

Chitosan-poly(vinyl alcohol) hydrogels can be produced by the freeze-thawing method without using toxic crosslinking agents. The applications of these ...

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

生物活性亲水球状蛋白的纳米脂质体制备与表征

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...

布洛芬封装的输运性质环糊精Nanosponge凝胶:质子HR-MAS NMR光谱研究

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