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/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/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...

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 border="0" cellpadding="0" cellspacing="0" width="657">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HR-MAS probe</td>
			<td style="width:113px;">BRUKER</td>
			<td style="width:161px;">N/A</td>
			<td style="width:167px;">Probe for NMR measurements on semi-solid samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NMR Spectrometer</td>
			<td style="width:113px;">BRUKER</td>
			<td style="width:161px;">DRX 500</td>
			<td>FT NMR spectrometer for liquid ans semi-solis state</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#946;-cyclodextrin (&#946;-CD)</td>
			<td style="width:113px;">Alfa-Aesar</td>
			<td style="width:161px;">J63161</td>
			<td>Reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethylenediaminetetracetic (EDTA) dianhydride</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td align="right" style="width:161px;">332046</td>
			<td>Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethylsulfoxide (DMSO)</td>
			<td style="width:113px;">Alfa-Aesar</td>
			<td style="width:161px;">D0798</td>
			<td>Solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triethylamine</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td align="right" style="width:161px;">471283</td>
			<td>Base (reagent)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ibuprofen (IP) sadium salt</td>
			<td style="width:113px;">Sigma-Aldrich</td>
			<td style="width:161px;">I1892</td>
			<td>Antinflammatory drug</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Excel 2010</td>
			<td style="width:113px;">Microsoft</td>
			<td style="width:161px;">N/A</td>
			<td>speadsheet for data analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Origin 8 SR0</td>
			<td style="width:113px;">OriginLab Co.</td>
			<td style="width:161px;"></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）。?...

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

Research

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

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3 and agarose4, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5 which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact &beta;-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80&deg; C as many 150 &mu;L aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL. 
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1&alpha; for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.

A method for photo-encapsulation of cells in a crosslinked PEG hydrogel is described. Hypoxic signaling within encapsulated murine insulinoma (MIN6) aggregates is tracked using a fluorescent marker system. This system allows serial examination of cells within a hydrogel scaffold and correlation of hypoxic signaling with changes in cell phenotype.

在封装的细胞聚集跟踪缺氧信号

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

科研

生物工程

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 ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

在生物膜的力学性能原位制图粒子追踪Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

RGD功能化水凝胶作为治疗应用的工具合成

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

疏水货物封装有针对性的质膜交付在液晶纳米颗粒载体

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

水凝胶的合成与防污性能作为膜的净水

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

3D 染色细胞的包覆和超弹性蛋白样凝胶的制备

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

定量荧光显微镜为基础的单脂体测定，用于检测单个脂质体之间的成分不均匀性

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...