Name: fabrication of a functionalized magnetic bacterial nanocellulose with iron oxide nanoparticles
Uploaded: 2016-05-26T13:00:00.000+00:00
Duration: 8 min 59 s
Description: Watch this Scientific Journal Video about Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles at JoVE.com

This work was funded by Department of Defense under contract No. W81XWH-11-2-0067

1.细菌纳米纤维素的制备（BNC） 注意:所有的步骤都在无菌条件下进行的，除非另有说明。 <ol><li>准备培养基。 <ol><li>通过组合25克酵母提取物，15g的蛋白胨，125.0克甘露醇，和500毫升的高纯度的水制成500毫升之液体培养基。高压釜在120℃下该混合物在4℃下20分钟，并存储。 </li><li>通过加入琼脂15克的5.0克酵母提取物，3.0克胨25.0克甘露醇，和100毫升高纯水中制备100ml半固体介质。高压釜在120℃下该混合物20分钟。一旦高压灭菌，存款将5ml混合物中的90mm×16毫米的塑料陪替氏培养皿。允许在该温度下在4℃下该溶液至凝胶和存储直至进一步使用。 </li></ol></li><li>补充水分G. xylinus应变加入1 ml液体培养基和吹吸在冷冻干燥的小瓶保存和下来，按照制造商的说明来表示。 </li><li>接种含有半固体培养基用接种环菌悬液的小液滴的培养皿。确保接种涵盖从边缘在Z字形方向移动所述环的菜中心的整个培养皿。 </li><li>孵化培养皿在26℃下72小时在恒温箱不用的 CO 2。一旦潜伏期结束后，小白色菌落是可见的。如果菌落不立即使用，通过密封用封口膜盖和放置碗碟倒置储存于4℃的培养皿中。菌落可以存储在这种方式长达6个月。 </li><li>传送2毫升步骤（1.1.1）中制备的液体培养基在24孔组织培养板的每个孔中的。举两个菌落从接种的培养皿步骤的接种针（1.3），并将它们放到组织培养板的第一口井。 - [REPEAT对于剩余23孔相同的步骤。 </li><li>孵育在30℃下的组织培养板7天。这将产生共24的BNC药膜为16毫米直径，厚度直径约为2-3毫米的如在图1中描绘。 注:在期间潜伏期任何一点摇动板请勿打扰的细菌培养，例如。在潜伏期，G. xylinus挤出吡喃葡萄糖糖分子，以在气-液界面，即采用静态培养条件下的形状和在烧瓶大小的聚合物结晶的网格。这种聚合物基质，称为细菌纳米纤维素（BNC），是在培养期结束显眼。 </li><li>从生长培养基收集在BNC药膜和在50℃下消毒他们在200ml的1％的NaOH溶液1小时，以除去G的所有痕迹xylinus。任选地，使用磁力棒搅拌以300rpm此溶液和搅拌板。丢弃该NaOH溶液和加入200毫升新制备的1％的NaOH溶液。重复同样的过程再一次，或直至溶液中的BNC药膜获得一个半透明的外观。 </li><li>冲洗BNC药膜用水三次，并在室温在高纯度的水将其存储。确保在BNC药膜是在水中完全浸没并且不允许在任何时间进行干燥。 </li><li>高压釜在121℃下在BNC药膜20分钟。 注:由Märtson和他的同事进行的老鼠的体内皮下研究表明后60周植入BNC非降解的迹象。的确，BNC是由微生物和真菌酶，它们是在哺乳动物中不存在于自然界降解的。另一方面，在BNC的生物降解性可以是机械，化学，和削弱体内 4的微纤维网的生物过程的结果。 </li></ol> 2.合成聚合物涂层氧化铁纳米粒子及其在细菌纳米纤维素膜沉积<ol><li>气泡1,000ml中，用氮气高纯度水，以便除去任何溶解的氧气在水中，并用氮气替换。 </li><li>使用的三颈圆底烧瓶中，以在一个2制备溶液:铁（III），氯化六水合物（的FeCl 3·6H 2 O）和铁（II）四水合氯化的1摩尔比（的FeCl 2·4H 2 O）稀释与脱氧高纯度的水。例如，使用5.4克三氯化铁3·6H 2 O和10毫升脱氧高纯度水1.98克的FeCl 2·4H 2 O的。如果这个准备转向过于粘稠，不易搅拌均匀，用0.54克三氯化铁3·6H 2 O和0.198克的FeCl 2·4H 2 O在20毫升脱氧高纯度的水。 注意:通过称量此通道缩小的FeCl 2·4H的曝光时间2 O至空气emical化合物尽可能快。一旦在该三颈圆底烧瓶中引入，关闭与隔膜止动件的三颈圆底烧瓶中，直至其被连接到氮气供给和冷凝管。 </li><li>使用该容器的两个颈部通过连接氮气供给到在一个隔塞冲孔并固定在容器的颈部的针提供恒定的入口和氮气的输出。 </li><li>放置其在与反应物的容器中而制备先前在步骤1.5（直径为15.6毫米，厚度为2-3毫米）1个BNC防护膜。确保将样品中的液体完全浸没。 </li><li>容器的剩余颈部连接到一个冷凝管。此外，使用填充有在冷凝器管顶部的无水硫酸钙干燥管。通过冷凝器管运行的水。 </li><li>全密封玻璃接缝的真空硅脂。 </li><li>使用搅拌在硅油浴将溶液加热至80℃烤盘和保持此温度直到步骤2.10。用一个小的磁力搅拌棒以350转5分钟的反应物混合。确保在BNC适当与亚铁溶液浸渍和反应物完全溶解。保持搅拌该混合物，直至实验结束。 注:利用温度计来验证硅油的温度。它应该是稳定至80℃。 </li><li>使用移液针，这一直增加搅拌速度至700转，并添加（滴加），在（NH 4 OH，14％）的5分钟，加入5ml氢氧化铵的时间间隔，以10毫升亚铁溶液还砸出一个隔塞。加入氢氧化铵后，将溶液的颜色从黄色/橙色变为黑色。 </li><li>继续在80℃搅拌该溶液另外5分钟。避免高速蛛丝马迹以保持样品的完整性。高速行驶时， 即高于1000转，可以摧毁例子。 </li><li>降低溶液的温度升高到30℃，用搅拌加热板的温度控制底部，并保持搅拌另外5分钟。然后，关闭热板。在这一点上，IONP已纳入在BNC网格。 </li><li>冷却该混合物至RT和磁性纳米颗粒（MNP）和BNC使用强永久磁铁（ 例如 ，1特斯拉）分开。要做到这一点，将混合物转移到一个容器烧瓶中，然后，在保持磁铁靠近容器，握住的MNP和BNC到位而倾析上清液。 注:在处理强磁铁时，因为不正确使用时，他们可能是有害的注意。对于步骤（2.12） - （2.14）和（2.16）使用（2.1）先前准备的脱氧高纯度的水，以防止氧化颗粒。 </li><li>重悬的MNP和BNC在100毫升水中。轻轻摇动解决方案删除所有未强烈纳入BNC的的MNP。倒出标通过使用磁铁持有的MNP和BNC的地方再次upernatant。 </li><li>用水洗涤的MNP和BNC几次直至上清液达到中性pH（pH约7），如用比色条进行测定。 </li><li>用镊子独立于的MNP磁功能化的BNC或磁细菌纳米纤维素（MBNC），并用清水冲洗MBNC几次，直到水变清。 </li><li>由MBNC O / N暴露于紫外线（110-280纳米）消毒MBNC​​。 </li><li>在120℃下进行20分钟的高压釜500毫升脱氧高纯度水，并存储MBNC在20ml这种水。 </li><li>无菌，浸入所述样品中聚乙二醇的1％，并搅拌在RT（37℃）2小时。此过程改善沉积在BNC的氧化铁纳米颗粒，特别是那些在表面5-7露出的生物相容性和稳定性。所述PEG涂层将在MBNC三维网络进行分发。 注:裸IONP很容易在空气中氧化因为他们的高化学活性8。即使PEG被认为是一种非生物降解的材料，其化学稳定性取决于所施加的生物条件，例如水含量，pH值，温度，酶的存在下，活性氧，活性氮种类和其他9。 </li></ol> 3. BNC和MBNC药膜的表征<ol><li> 机械性能 <ol><li>执行正常装载和使用Berkovich压头卸载纳米压痕试验。 Berkovich压金刚石压头的半径为20纳米。 </li><li>用熔融硅和钨的接触面积标定，因为在室温压痕深度的函数。在测试过程中，用胶安装在缩进的样品。压头接触在其厚度方向上的样品。 </li><li>样本表面上随机选择缩进位置。保持2缩进之间的间距200-300毫米之间。 </li><li>适用的负载的样品中的步骤，并记录该压头的相应位移。分析负载VS深度的情节找到了杨氏模量。 </li><li>通过施加0.0001 MN /秒和0.005 MN /秒之间的负荷率，用0.01 mN与0.60 MN之间高峰负荷进行在去离子水（DI水），以及测试存在下样品的纳米压痕测试。 </li><li>使用液体池，并保持样品液体环境下。对于浸在液体环境中的纳米机械表征这种独特的设置是理想的有效模拟BNC和MBNC膜的覆盖面生物力学功能。 </li></ol></li><li> 结构表征SEM <ol><li>通过扫描电子显微镜（SEM）表征纳米纤维素纤维结构。 </li><li>冻干的样品在-80℃下24小时。然后安装在SEM螺栓，用金钯薄膜溅射10秒，并用扫描电镜分析。 </li><li>在22,000X的放大倍数取图像和60,000，用5千伏的加速电压。 </li></ol></li><li> 磁畴 <ol><li>允许MBNC药膜完全干燥在RT，并随后暴露5分钟以一永久磁铁（1特斯拉）。 </li><li>立即，进行使用根据制造商的协议的生物AFM磁力测量。 </li><li>对于每次测量，第一捕捉地形特征和第二遍期间获取磁畴。获得在非接触模式与生物AFM两种测量。 </li><li>纳米颗粒的磁特性在物理性能的测量系统的量子设计 （PPMS），使用振动样品磁强计（VSM）在-10,000至10,000奥斯特的范围内进行的，在室温（300K），用磁场。 </li></ol></li><li> 细胞相容性 <ol><li>种子的人主动脉平滑肌细胞（HASMC）在6孔组织培养板在1.0×10的密度<sup&gt; 3细胞/ cm 2，并在试验样品的存在下培养24小时:BNC和MBNC药膜（每个15.6毫米直径的的）。 </li><li>分别使用未经处理的和过氧化氢处理的细胞群体作为阴性和阳性对照。 </li><li>根据制造商的协议和由A. Azqueta＆AR柯林斯10建议的准则进行彗星测定。 </li><li>使用核酸染料SYBR金在该试验中，以相互插入和荧光标记载根据制造商的协议的电泳样品中的DNA。 注意:不进行在BNC和MBNC样品存在任何DNA损伤的细胞，会呈现荧光绿色圆形类核，而DNA受损细胞将长彗星 - 阳性样本将有类核（彗星头部），其次是含有DNA碎片材料（尾部DNA的百分比）的尾巴。 </li></ol></li></ol>

<ol>
	<li>Saxena, I. M., Brown, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+bacterial+cellulose.">Biosynthesis of bacterial cellulose.</a> Bacterial Nanocellulose: A Sophisticated Multifunctional Material. , 1-18 (2012).</li><li>Chan-Park, M. B., Shen, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+control+of+vascular+smooth+muscle+cell+morphology+and+phenotype+for+functional+tissue-engineered+small-diameter+blood+vessels.">Biomimetic control of vascular smooth muscle cell morphology and phenotype for functional tissue-engineered small-diameter blood vessels.</a> J.Biomed.Mater.Res.A. 88, 1104-1121 (2009).</li><li>Barud, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocellulose-based+flexible+magnetic+paper.">Biocellulose-based flexible magnetic paper.</a> J. Appl. Phys. 117, (2015).</li><li>M&#228;rtson, M., Viljanto, J., Hurme, T., Laippala, P., Saukko, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+cellulose+sponge+degradable+or+stable+as+implantation+material?+An+in+vivo+subcutaneous+study+in+the+rat.">Is cellulose sponge degradable or stable as implantation material? An in vivo subcutaneous study in the rat.</a> Biomaterials. 20, 1989 (1999).</li><li>Ill&#233;sa, E., Tomb&#225;cza, E., Szekeresa, M., T&#243;tha, I., Szab&#243;b, &. #. 1. 9. 3. ;., Iv&#225;n, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+carboxylated+PEG-coating+on+magnetite+nanoparticles+designed+for+biomedical+applications.">Novel carboxylated PEG-coating on magnetite nanoparticles designed for biomedical applications.</a> J. Magn. Magn. Mater. 380, 132 (2015).</li><li>Torrisi, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preventing+corona+effects:+multiphosphonic+acid+poly(ethylene+glycol)+copolymers+for+stable+stealth+iron+oxide+nanoparticles.">Preventing corona effects: multiphosphonic acid poly(ethylene glycol) copolymers for stable stealth iron oxide nanoparticles.</a> Biomacromolecules. 15, 3171 (2014).</li><li>Cai, Z., Kim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+cellulose/poly(ethylene+glycol)+composite:+characterization+and+first+evaluation+of+biocompatibility.">Bacterial cellulose/poly(ethylene glycol) composite: characterization and first evaluation of biocompatibility.</a> Cellulose. 17, 83 (2010).</li><li>Wu, W., He, Q., Jiang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+iron+oxide+nanoparticles:+synthesis+and+surface+functionalization+strategies.">Magnetic iron oxide nanoparticles: synthesis and surface functionalization strategies.</a> Nanoscale Res. Lett. 3, 397-415 (2009).</li><li>Ulbricht, J., Jordan, R., Luxenhofer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+biodegradability+of+polyethylene+glycol,+polypeptoids+and+poly+(2-oxazoline)s.">On the biodegradability of polyethylene glycol, polypeptoids and poly (2-oxazoline)s.</a> Biomaterials. 35, 4848 (2014).</li><li>Azqueta, A., Collins, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essential+comet+assay:+a+comprehensive+guide+to+measuring+DNA+damage+and+repair.">The essential comet assay: a comprehensive guide to measuring DNA damage and repair.</a> Arch. Toxicol. 87 (6), 949-968 (2013).</li><li>Scherner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+application+of+tissue-engineered+blood+vessels+of+bacterial+cellulose+as+small+arterial+substitutes:+proof+of+concept.">In vivo application of tissue-engineered blood vessels of bacterial cellulose as small arterial substitutes: proof of concept.</a> J. Surg. Res. 189, 340 (2014).</li><li>Bodin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+cultivation+conditions+on+mechanical+and+morphological+properties+of+bacterial+cellulose+tubes.">Influence of cultivation conditions on mechanical and morphological properties of bacterial cellulose tubes.</a> Biotechnol Bioeng. 97, 425 (2007).</li><li>Zaborowska, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microporous+bacterial+cellulose+as+a+potential+scaffold+for+bone+regeneration.">Microporous bacterial cellulose as a potential scaffold for bone regeneration.</a> Acta Biomaterialia. 6, 2540 (2010).</li><li>Karimi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+on+the+mechanical+properties+of+the+umbilical+vein+and+umbilical+artery+under+uniaxial+loading.">A comparative study on the mechanical properties of the umbilical vein and umbilical artery under uniaxial loading.</a> Artery Res. 8, 51 (2014).</li><li>Lina, F., Ping, Z., Shengmin, Z., Guang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+bacterial+nanocellulose-based+uniform+wound+dressing+for+large+area+skin+transplantation.">Evaluation of bacterial nanocellulose-based uniform wound dressing for large area skin transplantation.</a> Mater. Sci. Eng. C. 33, 2995 (2013).</li><li>Olsson, R. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+flexible+magnetic+aerogels+and+stiff+magnetic+nanopaper+using+cellulose+nanofibrils+as+templates.">Making flexible magnetic aerogels and stiff magnetic nanopaper using cellulose nanofibrils as templates.</a> Nature Nanotech. 5 (8), 584-588 (2010).</li><li>Torre, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+force+microscopy+and+energy+loss+imaging+of+superparamagnetic+iron+oxide+nanoparticles.">Magnetic force microscopy and energy loss imaging of superparamagnetic iron oxide nanoparticles.</a> Sci. Rep. 1 (202), 1-8 (2011).</li></ol>

The authors have nothing to disclose.

在BNC防护膜的厚度和尺寸可以通过改变培养时间和其所静态培养中生长的烧瓶的尺寸来容易地操纵。在BNC的microproperties，如孔隙率，可以通过在静态培养改变氧气比修改。高浓度的氧气产生更严厉的BNC 11。 A.布丹和同事制作了一个爆破压力BNC的直径达880毫米汞柱通过在G的发酵过程中改变从大气中的氧气到100％氧气的氧气比率xylinus 12。同样地，在BNC的孔隙率，也可通?...

所述bacterian纳米纤维素（BNC）由木醋杆菌菌株，也称为Gluconacetobacter xylinus合成，并且静置培养期间沉积在空气-液体界面上的薄膜或药膜的形式。这些BNC药膜采用在那里它们被生长的容器的形式，并且它们的厚度取决于天在培养数。A. xylinus通过聚合和随后的结晶的方法使用在用于纤维素微纤丝的合成培养基中的葡萄糖。葡萄糖残基的聚合是在细菌细胞外膜，其中葡聚糖链从分布在细胞被膜单个细孔挤出进行。纤维素微纤维的结晶发生在与通过范德华键合形成葡聚糖链片材随后通过氢键1堆叠片材的胞外空间。 磁铁集成到一个BNC矩阵集成电路纳米颗粒可容易地通过外部磁场，以增加包含磁性纳米粒子需要引导和限制平滑肌细胞的力（平滑肌细胞），在动脉壁的损伤部位进行操作。这种策略保持平滑肌远离其它组织，并保持细胞在抵抗由血液流动所施加的力的地方。它已经显示，平滑肌细胞在血管，在那里形成主要位于中膜2丰富层的vasoelasticity发挥重要的作用。 用于MBNC的合成方法涉及的BNC薄膜浸入并在80℃下铁（III）六水合氯化铁（Ⅱ）四水合氯化的溶液中搅拌。加入氢氧化铵以形成在BNC网格内的氧化铁纳米颗粒。加入氢氧化铵的变化从橙色到黑色的溶液的颜色。该IONPs紧凑沿着BNC纤维一起s的一个非均匀分布。 此协议的重点是细菌纳米纤维素磁性纳米颗粒薄膜，这是我们命名磁性细菌纳米纤维素（MBNC），它的目的是为丢失，损坏或损伤的小直径的血管的替代物使用的设计。 HS Barud和同事最近发表了类似的工作通过在PEG和超顺磁性氧化铁纳米微粒3的稳定的水性分散体混合的BNC药膜，以产生一个基于BNC柔性磁性纸。这里，我们描述了生产细菌纤维素和其就地与磁性纳米颗粒浸渍。基于检测单个DNA链断裂的细胞毒性测定用于测试的BNC和MBNC药膜的生物相容性。

<table><tbody><tr><td>&lt;em&gt;Glucoacetobacter xylinus&lt;/em&gt;</td><td>ATCC</td><td>700178</td><td></td></tr><tr><td>Agar</td><td>Sigma Aldrich</td><td>A1296-500G&amp;nbsp;</td><td></td></tr><tr><td>D-Mannitol Bioxtra</td><td>Sigma Aldrich</td><td>M9546-250G&amp;nbsp;</td><td></td></tr><tr><td>Yeast Extract</td><td>BD Biosciences</td><td>212750</td><td></td></tr><tr><td>Bacteriological Peptone</td><td>Sigma Aldrich</td><td>P0556</td><td></td></tr><tr><td>Sodium Hydroxide, 50% Solution In Water</td><td>Sigma Aldrich</td><td>158127-100G</td><td></td></tr><tr><td>Iron(III) Chloride Hexahydrate</td><td>Sigma Aldrich</td><td>236489-100G&amp;nbsp;</td><td></td></tr><tr><td>Ammonium Hydroxide&amp;nbsp;</td><td>Macron Fine Chemicals</td><td>6665-46</td><td></td></tr><tr><td>Poly(Ethylene Glycol), Average Mn 400</td><td>Sigma Aldrich</td><td>202398-250G&amp;nbsp;</td><td></td></tr><tr><td>Iron (II) chloride tetrahydrate</td><td>Sigma Aldrich</td><td>44939-250G</td><td></td></tr><tr><td>Disposable Petri dish</td><td>Sigma Aldrich</td><td>BR452000</td><td></td></tr><tr><td>Disposable Inoculating Loop</td><td>Fisher Scientific</td><td>22-363-604&amp;nbsp;</td><td></td></tr><tr><td>Anhydrous Calcium Sulfate</td><td>W.A. Hammond Drierite&amp;nbsp;</td><td>13001</td><td></td></tr><tr><td>High vacuum grease</td><td>Sigma Aldrich</td><td>Z273554-1EA</td><td></td></tr><tr><td>Laboratory pipetting needle with 90&amp;deg; blunt ends</td><td>Sigma Aldrich</td><td>CAD7937-12EA</td><td></td></tr><tr><td>pH test strips&amp;nbsp;&amp;nbsp;</td><td>Sigma Aldrich</td><td>P4786-100EA</td><td></td></tr><tr><td>Round-bottom three neck angle type distilling flask</td><td>Sigma-Aldrich</td><td>CLS4965250</td><td></td></tr><tr><td>Silicone oil for oil baths</td><td>Sigma-Aldrich</td><td>85409-250ML&amp;nbsp;</td><td></td></tr><tr><td>Drying Tube</td><td>Chemglass</td><td>CG-1295-01</td><td></td></tr><tr><td>Septum Stopper, Sleeve Type</td><td>Chemglass</td><td>CG-3022-98</td><td></td></tr><tr><td>Magnetic stir bar</td><td>Chemglass</td><td>CG-2001-05</td><td></td></tr><tr><td>Condenser</td><td>Chemglass</td><td>CG-1218-01</td><td></td></tr><tr><td>Temperature Controller</td><td>BriskHeat</td><td>SDC120JC-A</td><td></td></tr><tr><td>Stirring Hotplate</td><td>Fisher Scientific</td><td>11-100-49SH&amp;nbsp;</td><td></td></tr><tr><td>Comet Assay Kit</td><td>Trevigen</td><td>4250-050-K</td><td></td></tr><tr><td>SYBR Gold Nucleic Acid Gel Stain</td><td>Life Technologies</td><td>S-11494</td><td></td></tr><tr><td>bio-AFM</td><td>JPK Instruments</td><td>NanoWizard 4a BioScience AFM</td><td></td></tr><tr><td>Nanoindenter</td><td>Micro Materials Ltd</td><td>Multi-module mechanical tester&amp;nbsp;</td><td></td></tr><tr><td>Scanning electron microscopy (SEM)</td><td>Hitachi High Technologies America</td><td>Hitachi S-4800</td><td></td></tr></tbody></table>

fabrication of a functionalized magnetic bacterial nanocellulose with iron oxide nanoparticles

在这项研究中，由细菌Gluconacetobacter xylinus产生细菌纳米纤维素（BNC）合成并在使用氧化铁纳米颗粒（IONP） 铁 （Fe 3 O 4） 原位浸渍，得到磁性细菌纳米纤维素（MBNC）。 MBNC的合成是一个精确而专门设计的多步骤过程。简言之，将细菌纳米纤维素（BNC）药膜是从保留G形成根据我们的尺寸和形态的实验要求xylinus菌株。铁（III），氯化六水合物（的FeCl 3·6H 2 O）和铁（II），氯化四水合物（的FeCl 2·4H 2 O）与2的溶液:1的摩尔比制备，并在脱氧的高纯度的水稀释。通过BNC防护膜，然后在与反应物的容器中引入的。此混合物搅拌并在硅油浴中和氢氧化铵（14％），在80℃下加热，然后加入通过丢弃以沉淀亚铁离子进入BNC网格。最后一步允许在原地的磁性纳米颗粒铁 （Fe 3 O 4）细菌的纳米纤维素网内部形成赋予磁特性BNC薄膜。毒理学测定用来评估BNC-IONP防护膜的生物相容性。聚乙二醇（PEG）被用来覆盖IONPs以改善其生物相容性。扫描电子显微镜（SEM）图像显示，IONP被优先位于原纤维交织在BNC矩阵的空间，但它们中的一些也沿BNC色带找到。在MBNC进行磁力显微镜测量检测存在磁畴拥有高强度弱磁场，确认MBNC薄膜的磁性质。在这项工作中获得的杨氏模量也都在这些报道的几个血管在以往的研究一个合理的协议。

G.潜伏期xylinus共9天是，但菌膜开始较早形成和后约2天是显而易见的。在BNC的宏观外观显示在图1中 ，它模仿了菜生长的培养物的形状。 图 2描述了用于生产BNC-IONP药膜，其总结涉及协议之上以及主要步骤的主要部件的配置。 SEM图像被用来解决微观结构，形态，和BNC（ ...

Watch this Scientific Journal Video about Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles at JoVE.com

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

一机能磁性纳米纤维素的细菌与氧化铁纳米粒子的制备

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

fabrication-functionalized-magnetic-bacterial-nanocellulose-with-iron

Research

JoVE Journal

Bioengineering

13.1K 次观看.  University of Illinois at Urbana-Champaign. Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh. 

视频: Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

在生物系统中的离子浓度测量荧光纳米粒子

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

科研

生物工程

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

预设计尺寸和形状的聚合物纤维和生物贿赂纤维的微流体制造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

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

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

细胞标记和超顺磁性氧化铁纳米粒子靶向

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

Iron Nanowire Fabrication by Nano-Porous Anodized Aluminum and its Characterization

Magnetic nanowires possess unique properties that have attracted the interest of different fields of research, including basic physics, biomedicine, and data storage. We demonstrate a fabrication method for iron (Fe) nanowires&#160;via electrochemical deposition into anodic&#160;alumina oxide (AAO) templates. The templates are fabricated by anodization of aluminum (Al) discs, and the pore length and diameter are controlled by changing the anodizing conditions. Pores with an average diameter of around 120 nm are created using oxalic acid as the electrolyte. Using this method, cylindrical nanowires are synthesized, which are released by dissolving the alumina using a selective chemical etchant.

In this work, we describe a&#160;protocol to fabricate iron nanowires, including the formation of the porous alumina membrane that is used as the template, electrodeposition into templates using electrolyte solution, and the release of the nanowires into the solution.

纳米孔阳极氧化铝的铁纳米线制造及其特性

Magnetic nanowires possess unique properties that have attracted the interest of different fields of research, including basic physics, biomedicine, and ...

化学

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine, a siderophore recognized by Yersinia enterocolitica, using a succinyl moiety as a linker are described.
Magnetic nanoparticles (MNP) of magnetite (Fe3O4) were prepared by solvothermal method and coated with SiO2 (MNP@SiO2) using the St&#246;ber process followed by functionalization with APTES (MNP@SiO2@NH2). Then, feroxamine was conjugated with the MNP@SiO2@NH2 by carbodiimide coupling to give MNP@SiO2@NH2@Fa. The morphology and properties of the conjugate and intermediates were examined by eight different methods including powder X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-Ray (EDX) mapping. This exhaustive characterization confirmed the formation of the conjugate. Finally, in order to evaluate the capacity and specificity of the nanoparticles, they were tested in a capture bacteria assay using Yersinia enterocolitica.

This work describes protocols for the preparation of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine using a succinyl moiety as a linker. A deep structural characterization description and a capture bacteria assay using Y. enterocolitica for all the intermediate nanoparticles and the final conjugate are also described in detail.

功能化磁性纳米粒子的合成及其与侧双磷铁氧胺的结合及其细菌检测评价

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

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 ...

Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized in cells and then left within. One challenge is to assess their fate in the intracellular environment with reliable and precise methodologies. Herein, we introduce the use of the vibrating sample magnetometer (VSM) to precisely quantify the integrity of magnetic nanoparticles within cells by measuring their magnetic moment. Stem cells are first labeled with two types of magnetic nanoparticles; the nanoparticles have the same core produced via a fast and efficient microwave-based nonaqueous sol gel synthesis and differ in their coating: the commonly used citric acid molecule is compared to polyacrylic acid. The formation of 3D cell-spheroids is then achieved via centrifugation and the magnetic moment of these spheroids is measured at different times with the VSM. The obtained moment is a direct fingerprint of the nanoparticles&#8217; integrity, with decreasing values indicative of a nanoparticle degradation. For both nanoparticles, the magnetic moment decreases over culture time revealing their biodegradation. A protective effect of the polyacrylic acid coating is also shown, when compared to citric acid.

Iron oxide nanoparticles are synthesized via a nonaqueous sol gel procedure and coated with anionic short molecules or polymer. The use of magnetometry for monitoring the incorporation and biotransformations of magnetic nanoparticles inside human stem cells is demonstrated using a vibrating sample magnetometer (VSM).

使用磁力学监测化学合成氧化铁纳米粒子的细胞合并和后续生物降解

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized ...

Biofunctionalization of Magnetic Nanomaterials

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting agents is a crucial aspect to enhance their efficacy in diagnostics and treatments while minimizing the side effects. The benefit of magnetic nanomaterials compared to non-magnetic ones is their ability to respond to magnetic fields in a contact-free manner and over large distances. This allows to guide or accumulate them, while they can also be monitored. Recently, magnetic nanowires (NWs) with unique features were developed for biomedical applications. The large magnetic moment of these NWs enables a more efficient remote control of their movement by a magnetic field. This has been utilized with great success in cancer treatment, drug delivery, cell tracing, stem cell differentiation or magnetic resonance imaging. In addition, the NW fabrication by template-assisted electrochemical deposition provides a versatile method with tight control over the NW properties. Especially iron NWs and iron-iron oxide (core-shell) NWs are suitable for biomedical applications, due to their high magnetization and low toxicity.
In this work, we provide a method to biofunctionalize iron/iron oxide NWs with specific antibodies directed against a specific cell surface marker that is overexpressed in a large number of cancer cells. Since the method utilizes the properties of the iron oxide surface, it is also applicable to superparamagnetic iron oxide nanoparticles. The NWs are first coated with 3-aminopropyl-tri-ethoxy-silane (APTES) acting as a linker, which the antibodies are covalently attached to. The APTES coating and the antibody biofunctionalization are proven by electron energy loss spectroscopy (EELS) and zeta potential measurements. In addition, the antigenicity of the antibodies on the NWs is tested by using immunoprecipitation and western blot. The specific targeting of the biofunctionalized NWs and their biocompatibility are studied by confocal microscopy and a cell viability assay.

In this work, we provide a protocol to biofunctionalize magnetic nanomaterials with antibodies for specific cell targeting. As examples, we utilize iron nanowires to target cancer cells.

磁性纳米材料的生物功能化

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting ...

Fabrication of Magnetic Platforms for Micron-Scale Organization of Interconnected Neurons

The ability to direct neurons into organized neural networks has great implications for regenerative medicine, tissue engineering, and bio-interfacing. Many studies have aimed at directing neurons using chemical and topographical cues. However, reports of organizational control on a micron-scale over large areas are scarce. Here, an effective method has been described for placing neurons in preset sites and guiding neuronal outgrowth with micron-scale resolution, using magnetic platforms embedded with micro-patterned, magnetic elements. It has been demonstrated that loading neurons with magnetic nanoparticles (MNPs) converts them into sensitive magnetic units that can be influenced by magnetic gradients. Following this approach, a unique magnetic platform has been fabricated on which PC12 cells, a common neuron-like model, were plated and loaded with superparamagnetic nanoparticles. Thin films of ferromagnetic (FM) multilayers with stable perpendicular magnetization were deposited to provide effective attraction forces toward the magnetic patterns. These MNP-loaded PC12 cells, plated and differentiated atop the magnetic platforms, were preferentially attached to the magnetic patterns, and the neurite outgrowth was well aligned with the pattern shape, forming oriented networks. Quantitative characterization methods of the magnetic properties, cellular MNP uptake, cell viability, and statistical analysis of the results are presented. This approach enables the control of neural network formation and improves neuron-to-electrode interface through the manipulation of magnetic forces, which can be an effective tool for in vitro studies of networks and may offer novel therapeutic biointerfacing directions.

This work presents a bottom-up approach to the engineering of local magnetic forces for control of neuronal organization. Neuron-like cells loaded with magnetic nanoparticles (MNPs) are plated atop and controlled by a micro-patterned platform with perpendicular magnetization. Also described are magnetic characterization, MNP cellular uptake, cell viability, and statistical analysis.

为互连神经元微缩组织制造磁平台

The ability to direct neurons into organized neural networks has great implications for regenerative medicine, tissue engineering, and bio-interfacing. ...

Fabrication of a Crystalline Nanocellulose Embedded Agarose Biomaterial Ink for Bone Marrow-Derived Mast Cell Culture

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto which cells are deposited. Because many biomaterial inks can be potentially cytotoxic to primary cells, it is necessary to determine the biocompatibility of these hydrogel composites prior to their utilization in costly 3D tissue engineering processes. Some 3D culture methods, including bioprinting, require that cells be embedded into a 3D matrix, making it difficult to extract and analyze the cells for changes in viability and biomarker expression without eliciting mechanical damage. This protocol describes as proof of concept, a method to assess the biocompatibility of a crystalline nanocellulose (CNC) embedded agarose composite, fabricated into a 24-well culture system, with mouse bone marrow-derived mast cells (BMMCs) using flow cytometric assays for cell viability and biomarker expression.
After 18 h of exposure to the CNC/agarose/D-mannitol matrix, BMMC viability was unaltered as measured by propidium iodide (PI) permeability. However, BMMCs cultured on the CNC/agarose/D-mannitol substrate appeared to slightly increase their expression of the high-affinity IgE receptor (Fc&#949;RI) and the stem cell factor receptor (Kit; CD117), although this does not appear to be dependent on the amount of CNC in the bioink composite. The viability of BMMCs was also assessed following a time course exposure to hydrogel scaffolds that were fabricated from a commercial biomaterial ink composed of fibrillar nanocellulose (FNC) and sodium alginate using a 3D extrusion bioprinter. Over a period of 6-48 h, the FNC/alginate substrates did not adversely affect the viability of the BMMCs as determined by flow cytometry and microtiter assays (XTT and lactate dehydrogenase). This protocol describes an efficient method to rapidly screen the biochemical compatibility of candidate biomaterial inks for their utility as 3D scaffolds for post-print seeding with mast cells.

This protocol highlights a method to rapidly assess the biocompatibility of a crystalline nanocellulose (CNC)/agarose composite hydrogel biomaterial ink with mouse bone marrow-derived mast cells in terms of cell viability and phenotypic expression of the cell surface receptors, Kit (CD117) and high-affinity IgE receptor (Fc&#949;RI).

用于骨髓源性肥大细胞培养的结晶纳米纤维素包埋琼脂糖生物材料墨水的制备

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto ...