This work was financed through the Bristol Centre for Functional Nanomaterials, sponsored by the Engineering and Physical Sciences Research Council (EPSRC grant code EP/G036780/1).

人类骨髓间充质干细胞（hMSC）作为从接受全髋关节置换手术，完全按照布里斯托尔Southmead旅馆医院研究伦理委员会的指导方针（参考＃078/01），得到病人的同意后，骨关节炎患者股骨近端骨髓收获。 1. Magnetoferritin合成和纯化<ol><li> 总论 <ol><li>在65℃氮气氛下在一个密封的，双夹套反应容器中执行magnetoferritin合成以限制金属前体的氧化。通过接入端口在盖成使用注射泵将反应容器注入金属盐溶液和过氧化氢。 </li><li> magnetoferritin合成后，通过净化阴离子交换色谱蛋白质（参见1.4节）删除没有用蛋白质纳米腔，其次是尺寸排阻色谱法（见1.5）分离蛋白单体。德使用Bradford测定termine蛋白浓度（参见1.6节）。 </li></ol></li><li> 准备工作之前，合成 <ol><li>通过将连接到在水中氮气气缸的管，密封与保鲜膜容器，并通过氮气进行大约60分钟鼓泡脱氧500毫升去离子水。 </li><li>热连接到双夹套反应容器到65℃的水浴中。加75毫升50毫4-（2-羟乙基）-1-哌嗪乙磺酸（HEPES）缓冲液（pH 8.6）到反应容器中，通过经由缓冲溶液约20分钟，使氮气鼓泡密封容器中，和去氧。在同一时间，搅拌使用磁力搅拌器的缓冲溶液。 </li><li>脱氧的HEPES缓冲溶液后，取出管从缓冲器供给氮气，并保持它悬浮在保持氮气气氛的容器中。添加脱辅基铁蛋白来实现的3毫克/米的最终浓度湖继续磁力搅拌，但如果发生发泡降低搅拌速度。 </li><li>通过在50毫升脱氧去离子水0.19克溶解制备硫酸钴七水合物的25mM储液。 </li><li>通过在100毫升脱氧去离子水0.98克溶解制备的铁硫酸铵六水合溶液的25mM溶液。 </li><li>中除去2.5毫升铁硫酸铵六水合溶液和2.5毫升硫酸钴七水合物的替换。 </li><li>通过首先将965微升过氧化氢溶液（30％W / W）到9毫升脱氧去离子水，然后加入1ml这个分库存来99毫升制备的脱氧去离子水中的过氧化氢的8.33毫溶液脱氧去离子水。 </li></ol></li><li> Magnetoferritin合成 <ol><li>注入10.1毫升两个铁 - 钴前体与过氧化氢的同时进入脱辅基铁蛋白溶液（磁小号tirred）在以0.15毫升使用两个注射泵/分钟的流速（总体积注射20.2毫升）中。 </li><li>在注射期间，脱氧另外500毫升去离子水。 注意:反应容器的内容物逐渐采用暗褐色随着反应的进行。 </li><li>不久第一次注射完成之前，准备另一个百毫升铁 - 钴前体，并使用新鲜的脱氧去离子水的过氧化氢溶液。 </li><li>重复使用铁 - 钴和过氧化氢的新制备的溶液在1.3.1中描述的注射步骤。 </li><li>在注射期间，脱氧另外500毫升去离子水，并在1.3.3和1.3.4所述进行操作。 </li><li>在第三次注射后，离开溶液成熟搅拌下15分钟。 </li><li>加为1.5ml的1M柠檬酸钠溶液到反应容器中以螯合游离在溶液中的金属离子。 </li><li>除去从反应后的溶液容器到50ml离心管，离心在4,350 xg离心30分钟，并通过0.22μm注射过滤器传递上清液。在这一阶段，经过滤的上清液可以储存于4℃直至纯化。 </li></ol></li><li> 离子交换色谱法 <ol><li>加载样品在含有用蠕动泵以10毫升/分钟的流速的阳离子基质的柱（直径为2.5厘米，长20厘米）。 </li><li>与有约100ml用梯度泵以10毫升/分钟的流速运行缓冲液（50mM三（羟甲基）氨基甲烷（TRIS）缓冲液，pH 8.0）洗涤柱。 </li><li>为了洗脱蛋白质，以10ml /分钟洗涤柱子用氯化钠（NaCl）的Tris缓冲液浓度的增加:150，500和1000毫最终NaCl浓度，加入150ml各浓度。 </li><li>作为蛋白质洗脱在500毫的NaCl浓度，使用自动级分收集器收集它在50毫升馏分。 注:对于离子交换层析的详细的协议协商先前公布的协议。 26 </li></ol></li><li> 尺寸排阻色谱法 <ol><li>浓缩150毫升magnetoferritin来的使用一个15毫升的离心过滤器单元随后4ml的体积单元约2毫升体积。参考离心过滤单元的制造商的说明（参见材料清单）此过程的详细协议。 </li><li>加载浓缩样品到使用注射循环凝胶过滤柱。 </li><li>用运行缓冲液（50mM Tris缓冲液，pH值8.0，含有150mM NaCl）中以1.3毫升/分钟的流速洗柱。 </li><li>收集用自动级分集流管6毫升级分。蛋白质单体洗脱过去的。在这一点上，纯化magnetoferritin可以储存在4℃直至阳离子。 注:对于使用凝胶过滤柱的尺寸排阻色谱法的详细协议协商previously出版协议。 27 </li></ol></li><li> 确定蛋白浓度 <ol><li>制备使用市售马脾铁蛋白的0.06，0.125，0.25,0.5和1mg / ml，在50mM Tris缓冲液铁蛋白的标准。 注:可商购的铁蛋白的蛋白质浓度在瓶子中指出。如果没有，与供应商联系以获得该信息。 </li><li>稀释未知浓度的magnetoferritin样品在50mM Tris缓冲，直到溶液的颜色为0.5mg / ml标准的颜色大致相匹配。记下稀释倍数。 </li><li>添加10微升一式三份每个标准和magnetoferritin试样到96孔平板的孔中。 </li><li>加入200μl现成Bradford试剂以每个孔（参考材料清单用于Bradford测定试剂详细信息）。 </li><li>在室温下孵育8分钟。 </li><li>使用测量在λ= 595nm处的吸光度酶标仪。 </li><li>积的铁蛋白的标准平均吸光度作为蛋白质浓度的函数，并使用线性拟合的斜率和截距来计算magnetoferritin样品的浓度。 注意:考虑到这是用来调整magnetoferritin样品，通过标准曲线稀释因子。 </li></ol></li></ol> 2. Magnetoferritin阳离子化<ol><li> 总论 注:对于magnetoferritin阳离子，N，N-二甲基-1,3-丙二胺（DMPA）偶联到使用N-对MF表面上天冬氨酸和谷氨酸残基- （3-二甲氨基丙基） - N&#39;-ethylcarbodiimide盐酸盐（EDC）。 <ol><li>进行在室温下在搅拌下进行反应。下面给出的协议为10毫克magnetoferritin的在5毫升 （蛋白质浓度2毫克/毫升） 的总体积的阳离子化。然而，放大或缩小通过保持蛋白质:DMPA:EDC比率相一致，以及缓冲和magnetoferritin溶液的体积。 </li><li>之前的阳离子化反应中，透析的magnetoferritin样品到含有50mM NaCl的50mM磷酸盐缓冲液（pH 7）。这是很重要的，除去的Tris洗脱缓冲液，避免的Tris胺和EDC·活化的羧酸残基之间的不希望的反应。确定蛋白质浓度透析后，把它调整到阳离子/ ml的前4毫克。 </li></ol></li><li> 阳离子协议 <ol><li>称出374毫克的DMPA和2.5毫升的200mM 2-（N-吗啉代）乙磺酸（MES）缓冲液溶解。 </li><li>调整用浓（6M）盐酸（HCl）溶液的pH值至约7。 注意:执行在通风橱中此步骤中，作为添加酸的DMPA释放有毒气体！ </li><li>添加2.5的magnetoferritin溶液混合物在4毫克/毫升（最终concenMES的tration为100mM，magnetoferritin的最终浓度为2毫克/毫升，蛋白质的总量为10毫克）。 </li><li>加磁力搅拌器和搅拌2小时达到平衡。 </li><li>仔细调整使用1M的HCl溶液至pH 5.0。 </li><li> 141毫克EDC粉添加到DMPA / magnetoferritin解决方案。 </li><li>继续搅拌3.5小时。 </li><li>过滤通过0.22微米的注射器过滤溶液以除去任何沉淀物。 </li><li>添加该溶液至透析管与12-14 kDa的分子量截止和透析针对4升含有50mM NaCl的2天，4℃50mM磷酸盐缓冲液（pH 7），取代的透析缓冲液的至少三个在此期间倍。 注意:在这一点上，阳离子magnetoferritin可以储存在4℃直至进一步使用。 </li></ol></li></ol> 3.人力间充质干细胞标记与阳离子化Magnetoferritin <ol><li> 总论 <ol><lI&gt;在37℃和5％二氧化碳的气氛下进行使用II类层流柜和加湿培养箱所有细胞培养物。 </li><li>培养细胞作为使用单层175厘米2烧瓶和20毫升培养基，将其每3补充的- 4天。培养基由Dulbecco改进的Eagle培养基（DMEM），含有1000毫克/升葡萄糖，10％（体积/体积）胎牛血清，1％（体积/体积）青霉素/链霉素溶液，1％（V / V）L-丙氨酰-L-谷氨酰胺的溶液和5纳克/毫升新鲜补充人类成纤维细胞生长因子。 </li></ol></li><li> 的hMSC磁标记与阳离子化magnetoferritin <ol><li>种子150000的hMSCs成为25cm 2烧瓶中并离开粘附过夜，在37℃。 </li><li>过滤通过0.22微米的注射器过滤器消毒的阳离子化magnetoferritin并确定蛋白质浓度。 </li><li>保持无菌的条件下，准备阳离子化magnetoferri的0.5微米的解决方案在磷酸锡缓冲盐水（PBS）。保持封在无菌培养罩直到使用。 </li><li>洗铺板的细胞用2ml室温PBS中。 </li><li> 1毫升灭菌阳离子magnetoferritin溶液添加到接种的细胞孵育所需的时间段（1分钟至1小时）。 </li><li>通过加入0.5ml的胰蛋白酶/乙二胺四乙酸（EDTA），并在37℃孵育5分钟，用PBS洗细胞，然后收获细胞。 </li><li>加入1ml培养基的灭活胰蛋白酶/ EDTA，在524×g下将溶液转移至15毫升离心管中并离心5分钟。 </li><li>弃去上清液并重新悬浮于0.5ml的细胞沉淀磁选缓冲液，由0.5％（重量/体积）胎牛血清和2mM EDTA的PBS中。 </li></ol></li><li> 磁性细胞分离 <ol><li>磁体连接到所述多台，和一个磁选列添加到磁体。放在日的预剥离过滤器Ë列。 </li><li>放置0.5毫升磁选缓冲液中预分离滤波器，让它通过滤波器和清洗和湿它们列都运行。 </li><li>放置一个15毫升的离心管列之下。 </li><li>加入0.5毫升细胞悬浮液中的磁性分离柱的过滤器储存器。 </li><li>当水库是空的，将0.5ml磁分离缓冲。 </li><li>当水库是空的，进一步增加0.5毫升磁选缓冲。 </li><li>重复一次（用于洗涤应1.5毫升磁选缓冲器的总体积）。此洗涤步骤洗脱所有非磁化细胞从塔（未磁化细胞级分）。 </li><li>从磁体取出柱，并将其放置在一个新的15ml离心管中。从塔储除去的过滤器。 </li><li> 1毫升磁选缓冲器添加到储存器和通过该柱使用由制造所提供的柱塞立即推河此洗脱从柱到离心管（磁化细胞级分）的磁化的细胞。 </li><li>离心两管在524×g下5分钟。 </li><li>弃去上清液并重新悬浮在0.3ml毫升的PBS细胞沉淀。 </li><li>计数细胞数在每个级分使用血球数。 </li><li>确定磁化细胞级分中，M（％），使用以下等式: <img alt="方程" src="/files/ftp_upload/54785/54785eq1.jpg" /> 其中，n（M）是细胞中的磁化馏分和n的数量（M + NM）是细胞中的磁化和未磁化的级分的数目的总和。 </li></ol></li><li> 制备使用电感耦合等离子体发射光谱标记为铁量化阳离子化magnetoferritin的hMSCs（ICP-OES） 注:该协议可能需要适于其他ICP-OES仪器。这是明智的重新咨询sponsible技术员。 <ol><li>离心的已知数量的细胞在524×g下5分钟。 </li><li>除去PBS上清，加入为0.25毫升50％（体积/体积）硝酸。 </li><li>涡混合酸化细胞悬液。 </li><li>在室温下孵育过夜。 </li><li>加4.75毫升蒸馏水向每个样品并涡旋混合。 </li><li>用ICP-OES分析。 28 </li><li>归一化测定铁含量对细胞的数目，以确定蜂窝铁含量的值。 </li></ol></li></ol>

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The authors have nothing to disclose.

与金硫葡萄糖染色magnetoferritin样品的TEM揭示了蛋白质内笼的纳米粒子成功矿化。电子衍射和纳米颗粒核的拉曼分析表明钴铁氧体的情况下，指示与钴的纳米颗粒核的成功掺杂。这表明混合氧化物纳米颗粒能成功是APO-铁腔内矿化。此外，我们已经表明先前钴掺杂可以通过改变加入到反应混合物中，使磁特性的调谐的钴前体的量而变化。 18 Magnetoferritin合成可在各种容器，只要它们是紧密地密封，并具有通过它可以引入的反应物（ 例如 ，一个三颈圆底烧瓶中）接入端口来执行。反应温度应在65℃下或者通过放置在容器中保持水/油浴中或使用双夹套容器中。这里，我们使用的双夹套电化学电池设置以执行合成。为了保证成功地合成，保持正确的pH值，并避免水溶液的氧污染是至关重要的。金属盐溶液应随时准备使用，而不是提前新鲜之前。此外，商业脱铁铁蛋白溶液可以在质量发生变化，影响合成的结果（ 例如 ，纳米颗粒核矿化的大小）。它可以帮助在脱铁铁蛋白溶液透析到合成之前的50mM HEPES缓冲液（pH 8.6），以除去由生产商所用的任何残留的还原剂。它是使一便条用于合成的脱辅基铁蛋白溶液的批号是有用的，因此它可以从制造商应该附加材料必须被购买具体要求。此外，市售的脱辅基铁蛋白的蛋白质浓度应在瓶子中说明，它可以用于计算所需的合成脱辅基铁蛋白溶液的体积。如果不是这种情况，与供应商联系以获得该信息。 如在这里和在先前的报告中提出- -逐渐加入金属盐和过氧化氢的优点是，纳米颗粒核的矿化可以被控制，使得不同的负载系数（ 即，纳米颗粒大小）就可以实现。 33此外，有可能以纯化magnetoferritin进一步使用磁性分离柱， 例如，填充有电磁铁内固定不锈钢粉末的柱。 34因此，高度的单分散纳米粒子核可以被从散装magnetoferritin样品中分离。然而，对于磁性细胞标记如这里提出这不是必需的。 magnetoferritin合成的限制是10％左右的相对低的合成产率，以及商业脱辅基铁的成本相对较高的rritin解决方案。然而，脱辅基铁蛋白也可从由以下建立脱矿化协议铁蛋白便宜地提供马脾制备。 16 magnetoferritin的阳离子通过加入DMPA 250分子和每个负电荷的残基的EDC 50分子的摩尔比来实现（计算基于马脾铁蛋白的氨基酸序列）。这样，多余的试剂在蛋白质导致了高效率的阳离子化，也可比以往报告的铁蛋白的阳离子化结果。 35对于MALDI-TOF分析，脱铁铁蛋白和脱铁铁蛋白的阳离子化进行，因为magnetoferritin芯的过度分子量的使用。为了产生高效率的阳离子化，最适pH也是至关重要的。 EDC介导的交联是温和的酸性条件下最有效，而我们发现，pH为5，得到为magnetoferritin最佳阳离子化的结果。然而，对于其他蛋白质cationization pH值可能需要进行优化。阳离子化在或接近蛋白质的等电点应该避免，因为这可能会导致严重的沉淀。 与阳离子化magnetoferritin干细胞磁化是高效，并可能使用的温育时间远远低于30分钟达到。甚至一分钟孵育导致3.6皮克，这是影响用于MRI T2和T2 *对比所需的报告的范围内的蜂窝铁含量。 36,37这也是显着的，这有效的标记与外低铁浓度达到。例如，使用阴离子纳米粒子以往的研究报告有30分钟的潜伏期为5毫米的铁后，每单元10微克的铁含量。 38相比较而言，孵育用含有0.5μM的蛋白质的阳离子magnetoferritin溶液对应于孵育约0.2mM的铁和也产生约10皮克每CEL铁升在30分钟后。我们无法清楚地确定用TEM的内吞囊泡。然而，使用阳离子化铁以往的研究发现，前十分钟的暴露内发生内化。 39,40阳离子化铁蛋白能够在小泡，网格蛋白说明或小窝依赖的内吞进行本地化。同一研究还报告说，孵育30分钟后，阳离子铁蛋白仍然存在于细胞表面上，以及在多泡体，类似溶酶体。 进一步的应用可包括装载有其他纳米颗粒和/或功能分子，如抗癌药物41或量子点42脱辅基铁蛋白笼的阳离子。这些铁蛋白结构的阳离子，可能会导致他们的货物细胞的更快，更高效地交付。

表面结合或超顺磁性氧化铁纳米颗粒（SPIONs）的内部，使多种类型的细胞的磁化的应用，如成像和远程操作。 1然而，实现充分的蜂窝磁化可能是一个挑战，尤其是当SPION和细胞表面之间的相互作用弱。 2在过去，长时间暴露或高SPION浓度已用作策略来克服这一挑战。 3,4-然而，这些策略是有问题的，因为它们增加毒性5,6-并在细胞类型具有低的内化速率，如淋巴细胞非常有限的成功。 7为了提高SPIONs的细胞摄取，几个表面官能化的方法已被探讨。例如，抗体已经被用于促进受体介导的内吞作用，8而能使用的转染一个来实现非特异性摄取绅士9,10或细胞穿透物种，例如HIV TAT肽。 11,12然而，抗体和肽官能的方法是通过昂贵的试剂和复杂的合成制备的限制，而转染试剂可诱导的纳米颗粒的沉淀和细胞功能产生不利影响。 13,14 我们最近报道化学阳离子magnetoferritin的合成中，使用的孵育时间短至一分钟新颖SPION这是在磁化的人类间充质干细胞中高度有效（hMSC）的培养。 15 Magnetoferritin被重构的储铁蛋白铁蛋白的去离子腔内SPION合成。 16该蛋白质基SPION结合了许多特性使其非常适合用于细胞磁化，如在由蛋白质外壳赋予的磁芯，17-19和生物相容性和水溶性的磁特性的控制。进一步以上，表面官能化是容易由于寻址氨基酸可以是化学20-22或遗传修饰实现的。 23-25我们已经表明，该蛋白质壳的酸性氨基酸残基的化学阳离子产生容易与细胞表面导致快速和持续的细胞的磁化上的阴离子域相互作用稳定纳米颗粒。这个过程消除了费力的官能化和冗长孵育协议的需要，并且由于非特异性标记机制此快速磁化战略应找到在其他细胞类型广泛的应用。在这里，我们提出了超快速的细胞标记方法的深度报告，包括阳离子magnetoferritin的合成，提纯和表面功能的详细协议。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>Fisher Scientific</td>
			<td>BPE310-1</td>
			<td>powder; prepare a 1 M stock solution at pH 8.6 and dilute to 50 mM prior to use. Check the pH carefully prior to synthesis!</td>
		</tr>
		<tr>
			<td>apoferritin from equine spleen</td>
			<td>Sigma Aldrich</td>
			<td>A3641</td>
			<td>we used LOT# 081M7011V</td>
		</tr>
		<tr>
			<td>cobalt sulfate heptahydrate&#160;</td>
			<td>Sigma Aldrich</td>
			<td>C6768</td>
			<td>prepare fresh solutions from the salt prior to synthesis</td>
		</tr>
		<tr>
			<td>ammonium iron sulphate hexahydrate&#160;</td>
			<td>Sigma Aldrich</td>
			<td>F1543</td>
			<td>prepare fresh solutions from the salt prior to synthesis</td>
		</tr>
		<tr>
			<td>hydrogen peroxide solution (30%)</td>
			<td>Sigma Aldrich</td>
			<td>216763</td>
			<td>prepare fresh solutions from the salt prior to synthesis</td>
		</tr>
		<tr>
			<td>sodium citrate</td>
			<td>Sigma Aldrich</td>
			<td>S1804</td>
			<td>powder; a 1 M solution can be prepared and kept at room temperature for several months</td>
		</tr>
		<tr>
			<td>Millex GP filter unit, 0.22 micron</td>
			<td>Merck Millipore</td>
			<td>SLGP033RS</td>
			<td>syringe filter</td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma Aldrich</td>
			<td>T1503</td>
			<td>powder; prepare a 1 M stock solution at pH 8.0 and dilute to 50 mM prior to use</td>
		</tr>
		<tr>
			<td>sodium chloride</td>
			<td>Sigma Aldrich</td>
			<td>31434</td>
			<td>poweder; add to buffers as required</td>
		</tr>
		<tr>
			<td>Centriprep centrifugal filter units</td>
			<td>Merck Millipore</td>
			<td>4310</td>
			<td>Ultracel YM-50 membrane, 12 mL volume; use for initial concentration until the magnetoferritin solution has been concentrated from about 150 mL to 20 mL</td>
		</tr>
		<tr>
			<td>Amicon Ultra-4 centrifugal filter untis</td>
			<td>Merck Millipore</td>
			<td>UFC801024</td>
			<td>Ultracel-10 membrane, 4 mL volume; use to concentrate magnetoferritin solution from about 20 mL to 2 mL</td>
		</tr>
		<tr>
			<td>ANX Sepharose 4 Fast Flow</td>
			<td>GE Healthcare</td>
			<td>17-1287-04</td>
			<td>we packed this column ourselves</td>
		</tr>
		<tr>
			<td>HiPrep 26/60 Sephacryl S-300 HR column&#160;</td>
			<td>GE Healthcare</td>
			<td>17-1196-01</td>
			<td>this column was bought ready packed</td>
		</tr>
		<tr>
			<td>&#196;KTA purifier system</td>
			<td>GE Healthcare</td>
			<td>28406264</td>
			<td></td>
		</tr>
		<tr>
			<td>sample pump P-960</td>
			<td>GE Healthcare</td>
			<td>18-6727-00</td>
			<td>load sample at a flow rate of 10 mL/min</td>
		</tr>
		<tr>
			<td>automated fraction collector Frac-950</td>
			<td>GE Healthcare</td>
			<td>18-6083-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford assay reagent</td>
			<td>Sigma Aldrich</td>
			<td>B6916</td>
			<td>solution ready to use</td>
		</tr>
		<tr>
			<td>Ferritin, Type I: from horse spleen</td>
			<td>Sigma Aldrich</td>
			<td>F4503</td>
			<td>prepare ferritin standards from this solution to determine magnetoferritin concentration</td>
		</tr>
		<tr>
			<td>N,N-dimethyl-1,3-propanediamine (DMPA)</td>
			<td>Sigma Aldrich</td>
			<td>308110</td>
			<td>CAUTION: when adjusting the pH of a DMPA solution, perform this step in a fume hood</td>
		</tr>
		<tr>
			<td>N-(3-dimethylaminopropyl)-N&#8217;-ethylcarbodiimide hydrochloride (EDC)</td>
			<td>Sigma Aldrich</td>
			<td>E6383</td>
			<td>keep in freezer but bring to room temperature before opening the bottle</td>
		</tr>
		<tr>
			<td>&#160;2-(N-morpholino)ethanesulfonic acid (MES)</td>
			<td>AppliChem</td>
			<td>A0689,0500</td>
			<td>powder; prepare a 200 M stock solution at pH 5</td>
		</tr>
		<tr>
			<td>dialysis tubing cellulose membrane</td>
			<td>Sigma Aldrich</td>
			<td>D9652</td>
			<td>soak 10 min in deionized water before use</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM), 1000 mg/L glucose</td>
			<td>Sigma Aldrich</td>
			<td>D5546</td>
			<td>warm in 37 &#176;C&#160; water bath before use</td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Sigma Aldrich</td>
			<td>F7524</td>
			<td>add to stock DMEM bottle, 10 % (v/v) final concentration</td>
		</tr>
		<tr>
			<td>penicillin/streptomycin solution</td>
			<td>Sigma Aldrich</td>
			<td>P0781</td>
			<td>add to stock DMEM bottle, 1 % (v/v) final concentration</td>
		</tr>
		<tr>
			<td>glutamax solution</td>
			<td>Gibco</td>
			<td>35050-087</td>
			<td>add to stock DMEM bottle, 1 % (v/v) final concentration</td>
		</tr>
		<tr>
			<td>human fibroblast growth factor</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td>add to DMEM freshly into cell culture flask with each media change; final concentration 5 ng/mL</td>
		</tr>
		<tr>
			<td>phosphate buffered saline</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td>sterile solution, for cell cultrue</td>
		</tr>
		<tr>
			<td>trypsin/EDTA solution</td>
			<td>Sigma Aldrich</td>
			<td>E5134</td>
			<td>keep in freezer and defrost in 37 &#176;C&#160; water bath before use</td>
		</tr>
		<tr>
			<td>ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma Aldrich</td>
			<td>E5134</td>
			<td>powder; make a 2 mM solution in PBS&#160;</td>
		</tr>
		<tr>
			<td>bovine serum albumin</td>
			<td>Sigma Aldrich</td>
			<td>A7030</td>
			<td>add 0.5 % (w/v) into 2 mM EDTA solution in PBS; carefully stir with magnetic stirrer, avoid foaming; filter sterilize through a 0.22 micron syringe filter</td>
		</tr>
		<tr>
			<td>MACS multi stand&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td>for attachment of MACS magnet</td>
		</tr>
		<tr>
			<td>MACS MS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td>disposable; intended for single use, but if sterility is not required, they can re-used: wash with deionized water and 100 % ethanol, and placed in a drying oven; discard if you observe rusty patches</td>
		</tr>
		<tr>
			<td>MiniMACS separator magnet</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td>can be bought as a starter kit, together with columns and stands</td>
		</tr>
		<tr>
			<td>MACS column pre-separation filter</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-407</td>
			<td>30 mm filter</td>
		</tr>
		<tr>
			<td>Nitric acid solution, 64-66%</td>
			<td>Sigma Aldrich</td>
			<td>7006</td>
			<td></td>
		</tr>
		<tr>
			<td>Titrando 907, syringe pump</td>
			<td>Metrohm</td>
			<td>2.907.0020</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Equipment used to characterize magnetoferritin and cationized magnetoferritin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax M5</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Used to measure absorbance in the Bradford assay</td>
		</tr>
		<tr>
			<td>JEM 1200 EX</td>
			<td>JEOL</td>
			<td></td>
			<td>Used for TEM imaging of magnetoferritin</td>
		</tr>
		<tr>
			<td>InVia Raman spectrometer</td>
			<td>Renishaw</td>
			<td></td>
			<td>Used for Raman spectroscopy</td>
		</tr>
		<tr>
			<td>Torus DPSS laser</td>
			<td>Laser Quantum</td>
			<td></td>
			<td>Used for Raman spectroscopy</td>
		</tr>
		<tr>
			<td>Bruker UltrafleXtreme</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td>Used for MALDI-TOF analysis of apoferritin and cationized apoferritin</td>
		</tr>
		<tr>
			<td>ZetaSizer Nano-ZS</td>
			<td>Malvern Instruments</td>
			<td></td>
			<td>Used to measure hydrodynamic diameter and zeta potential of magnetoferritin and cationized magnetoferritin</td>
		</tr>
		<tr>
			<td>Magnetic Property Measurement System</td>
			<td>Quantum Design</td>
			<td></td>
			<td>Used to measure magnetic saturation moment and magnetic susceptibility</td>
		</tr>
		<tr>
			<td>Magnetom Skyra</td>
			<td>Siemens</td>
			<td></td>
			<td>Used to determine longitudinal and transverse relaxivity&#160;</td>
		</tr>
		<tr>
			<td>Tecnai 12 BioTwin Spirit</td>
			<td>FEI</td>
			<td></td>
			<td>Used for TEM imaging of hMSC labeled with cationized magnetoferritin</td>
		</tr>
		<tr>
			<td>710 ICP-OES</td>
			<td>Agilent</td>
			<td></td>
			<td>Used to determine iron content in cells</td>
		</tr>
	</tbody>
</table>

synthesis of cationized magnetoferritin for ultra-fast magnetization of cells

许多重要的生物医学应用，如细胞成像和远程操作，可以通过用超顺磁性氧化铁纳米颗粒（SPIONs）标记的细胞来实现。实现SPIONs的足够细胞摄取是历来满足通过使细胞暴露于升高的浓度SPIONs的或通过延长曝光时间（最多72小时）是一个挑战。然而，这些策略可能介导的毒性。在这里，我们提出了基于蛋白质的SPION magnetoferritin的合成以及一个浅显的表面功能化协议，使细胞快速磁化使用低接触浓度。 magnetoferritin的SPION核心由掺钴 - 铁氧化物与马脾脱辅基铁蛋白的腔内部8.2纳米矿化的平均颗粒直径。 magnetoferritin的化学阳离子产生使用温育时间作为一种新型的，高的膜 - 活性SPION该磁化人类间质干细胞（hMSCs）短短一分钟的铁浓度低点0.2毫米。

TEM被用来确认脱铁铁蛋白腔内纳米颗粒矿化和确定平均芯尺寸（ 图1A和1B）。未染色magnetoferritin样品的图像分析，得到的8.2±0.7纳米的平均芯径，和金硫葡萄糖染色证实纳米颗粒的蛋白质笼子内的存在。注意，图像显示用磁选分离均一纳米粒子核被进一步纯化magnetoferritin样品。没有磁净化的Magnetoferritin样品具有稍宽核心尺寸分布。采用选区电子衍射的magnetoferritin芯的结构29分析表明基于磁铁矿的反尖晶石结构的铁 （Fe 3 O 4）和/可能存在或磁赤铁矿（γ- 的 Fe 2 O 3），以及尖晶石结构由于共3 </子&gt; O 4。此外，拉曼光谱揭示归因于四氧化三铁，少量的γ- 的 Fe 2 O 3的，和钴铁氧体（ 图1 C）的峰。 magnetoferritin的ICP-OES分析表明，铁的102微克和0.9微克每magnetoferritin毫克钴的平均值。 示意图包括，示出了随后的阳离子化步骤（ 图2的A）。 magnetoferritin和阳离子magnetoferritin的流体动力学直径分别为11.8±1.1纳米和12.5±1.4纳米，如通过动态光散射来确定。共价DMPA耦合的阳离子化效率magnetoferritin使用ζ电电位和基质辅助激光解吸电离时间飞行（MALDI-TOF）质谱法进行评估。 Zeta电位从-10.5 mV的改变MF到+ 8.3毫伏阳离子化magnetoferritin，确认一在从负的表面电势改变为阳性（ 表1）。质谱实验中发现20.1 kDa的亚基分子量为本地APO-铁蛋白和21.1 kDa的对阳离子APO-铁蛋白（ 图2 B）。该质量增加对应于每蛋白质亚基大约12耦合DMPA分子和288残基上的整个24亚基蛋白的阳离子化。 磁饱和度和磁化率是用SQUID磁力测定，并且使用磁共振成像测定的横向和纵向弛豫。磁特性分别为magnetoferritin和阳离子magnetoferritin相似，这表明阳离子对封闭SPION（ 表1）的磁特性的影响可以忽略。此外，这些性能类似于其他氧化铁基纳米颗粒，19,30证明，认为阳离子马格纳toferritin将如适合作为增强成像对比常规的基于SPION-MRI造影剂。 30分钟的曝光后，细胞表面密布着阳离子magnetoferritin（ 图3 A）。然而，在一周后，没有纳米颗粒在细胞表面（ 图3B）上找到。阳离子magnetoferritin是在磁标记的hMSCs非常有效。值得注意的是，将细胞暴露于阳离子magnetoferritin一分钟导致的细胞群体的92％的磁化强度和3.6皮克每细胞的铁的递送。增加孵育时间来导致整个细胞群体（ 图3 C）的磁化15分钟。 <img alt="图1" src="/files/ftp_upload/54785/54785fig1.jpg" /> 图1:magnetof 表征<html> erritin芯掺杂有5％的钴。与金硫葡萄糖（A）和未染色（B）染色magnetoferritin的TEM照片。插图显示了磁铁矿指数对应的电子衍射。比例尺:20纳米。 （C）为拉曼光谱magnetoferritin。箭头指示钴铁氧体（T 2G），磁铁矿和赤铁矿（既是1G）的主要拉曼振动模式。 31,32所使用的激光波长为532纳米。 （图片改编自Okuda等18）。请注意，此magnetoferritin样品已经使用磁分离，其分离均匀加载magnetoferritin颗粒被进一步纯化。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54785/54785fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 785fig2.jpg"/&gt; 图2:magnetoferritin 的阳离子。 A）的溶剂可及表面积的表示示出酸性（红色的蛋白质表面上的分布）和基本的（黄色）的氨基酸残基。 Magnetoferritin（1）通过对蛋白质表面（3）上的酸性氨基酸残基的DMPA碳二亚胺介导的交联修饰以阳离子magnetoferritin（2）。 B）的脱辅基铁蛋白和阳离子脱辅基铁蛋白亚基的质谱分析。通过MALDI-TOF产生质量 - 电荷（M / Z）脱铁铁蛋白的谱（APOF）和阳离子脱铁铁蛋白（猫APOF）。从20.1 kDa的的质量增加21.1 kDa的是阳离子后观察。 （图片改编自科雷亚的Carreira 等 15） <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54785/54785fig2large.jpg" target="_blank">点击此处查看该图的放大版本。</a> 图3: 磁性标签和阳离子化magnetoferritin培养的hMSCs细胞分离。 A）与阳离子magnetoferritin 30分钟孵育后的hMSCs的TEM图像。箭头指示密密麻麻在细胞表面上magnetoferritin芯的存在。比例尺:200纳米。标记后的hMSC一周B）的 TEM图像。细胞表面是明确的阳离子magnetoferritin的。 C）的调查磁性标记的快速性:细胞群体的92％是一分钟的曝光与阳离子magnetoferritin 0.5微米后磁化，并且整个细胞群体的15分钟内被磁化。每个细胞的铁含量使用ICP-OES确定的。从三个生物学重复平均值和标准偏差被示出。 （图片改编自科雷亚的Carreira 等人15） <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54785/54785fig3large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td> MF </td><td> CAT-MF </td></tr><tr><td> 流体动力学直径[nm]的 </td><td> 11.8±1.1 </td><td> 12.5±1.4 </td></tr><tr><td> Zeta电位[MV] </td><td> （ - ）10.4±0.2 </td><td> 8.3±0.7 </td></tr><tr><td> 磁饱和磁矩[AM2 公斤 -1] </td><td> 54.9±1.6 </td><td> 55.3±1.4 </td></tr><tr><td> 质量磁化率[×10 4 m 3的kg的-1] </td><td> 1.75±0.08 </td><td> 1.75±0.07 </td></tr><tr><td>纵向弛豫[MM -1秒-1] </td><td> 2.6±0.1 </td><td> 2.3±0.1 </td></tr><tr><td> 横向弛豫[MM -1秒-1] </td><td> 44.6±1.0 </td><td> 52.8±0.8 </td></tr></tbody></table> 表1:magnetoferritin 的物化表征（MF）和阳离子magnetoferritin（CAT-MF）。 （改编自科雷亚的Carreira 等表15）

Watch this Scientific Journal Video about Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells at JoVE.com

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

对细胞的超快磁化阳离子化Magnetoferritin的合成

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

synthesis-cationized-magnetoferritin-for-ultra-fast-magnetization

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.9K 次观看.  University of Bristol. A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

视频: Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

单层的合成，装配和表征蛋白质单分子膜的电化学保护的金纳米粒子薄膜

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

科研

生物工程

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

基于角蛋白纳米纤维生物医学工程合成

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

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

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

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

动脉粥样硬化的快速检测的氧化铁纳米颗粒微波驱动综合

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

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 ...

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 ...

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. Atherosclerosis is also the leading cause of sudden death and myocardial infarction, instigated by unstable plaques that rupture and occlude the blood vessel without warning. Current imaging modalities cannot differentiate between stable and unstable plaques that rupture. Peptide amphiphiles micelles (PAMs) can overcome this drawback as they can be modified with a variety of targeting moieties that bind specifically to diseased tissue. Monocytes have been shown to be early markers of atherosclerosis, while large accumulation of monocytes is associated with plaques prone to rupture. Hence, nanoparticles that can target monocytes can be used to discriminate different stages of atherosclerosis. To that end, here, we describe a protocol for the preparation of monocyte-targeting PAMs (monocyte chemoattractant protein-1 (MCP-1) PAMs). MCP-1 PAMs are self-assembled through synthesis under mild conditions to form nanoparticles of 15 nm in diameter with near neutral surface charge. In vitro, PAMs were found to be biocompatible and had a high binding affinity for monocytes. The methods described herein show promise for a wide range of applications in atherosclerosis as well as other inflammatory diseases.

This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes.

单核靶向肽 Amphiphile 胶束合成动脉粥样硬化的研究

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

Design and Synthesis of a Reconfigurable DNA Accordion Rack

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m resolution, show great potential in various fields of nanotechnology such as the molecular reactors, drug delivery, and nanoplasmonic systems. The reconfigurable DNA accordion rack, which can collectively manipulate a 2D or 3D nanoscale network of elements, in multiple stages in response to the DNA inputs, is described. The platform has potential to increase the number of elements that DNA nanomachines can control from a few elements to a network scale with multiple stages of reconfiguration.
In this protocol, we describe the entire experimental process of the reconfigurable DNA accordion rack of 6 by 6 meshes. The protocol includes a design rule and simulation procedure of the structures and a wet-lab experiment for synthesis and reconfiguration. In addition, analysis of the structure using TEM (transmission electron microscopy) and FRET (fluorescence resonance energy transfer) is included in the protocol. The novel design and simulation methods covered in this protocol will assist researchers to use the DNA accordion rack for further applications.

We describe the detailed protocol for design, simulation, wet-lab experiments, and analysis for a reconfigurable DNA accordion rack of 6 by 6 meshes.

可重构 DNA 手风琴架的设计与合成

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

在肽膜前体的维西图洛合成中,用于自主囊泡生长

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

生物应用近红外发光金纳米簇的合成

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...