这项研究得到了 NRF 大学研究补助金 (2016) 和纳米材料技术开发项目的支持, 通过由科学和信息和通信技术部 (2017M3A7B8061942) 资助的韩国国家研究基金会 ()。

1. MWCNT 氧化
<ol><li>测量 30-50 毫克的 MWCNT 成一个50毫升的瓶子。缓慢地添加8毫升的 H2, 所以4: HNO3解决方案 (90% 初始浓度, 3:1 卷/卷) 的吸管与1毫升吸管提示。 注意: 此制剂必须在化学油烟机中进行。<ol>
<li>允许30分钟的放热反应完成。</li>
	</ol>
</li>
	<li>油脂实验溶液在 60-80 °c 和 160 W 为1小时, 直到 MWCNT 安定在瓶的底部。 注意: sonicator 的水位应该低于瓶子的顶端, 这样水就不会被引入瓶子里。</li>
	<li>把溶液转移到离心管上。<ol>
<li>用微, 加入30毫升蒸馏水滴到 MWCNT-COOH 溶液中。 注意: 在加蒸馏水的过程中会发生强烈的放热反应。慢慢加入蒸馏水, 让时间冷却。</li>
	</ol>
</li>
	<li>离心机的 MWCNT-COOH 溶液在 1.2万 x g 和室温15分钟。 注意: 在操作离心机时, 要确保离心机保持平衡, 在取样管对面放置一个与重量相同的管。</li>
	<li>用吸管和1毫升吸管端取出上清液。添加5毫升乙醇与吸管和1毫升吸管提示。用额外的 pipetted 乙醇冲洗瓶子的壁。离心溶液在 1.2万 x g 和室温15分钟。<ol>
<li>用 ph 纸测定上清液的 ph 值。重复洗涤和离心, 直到上清液 pH 值为中性。</li>
	</ol>
</li>
	<li>移除所有上清。加入1毫升蒸馏水, 在溶液中均匀沉淀和分散 MWCNT-COOH。冷冻干燥溶液在-60 摄氏度真空下直到干燥。</li>
</ol>2. 纳米银在 MWCNTs 上的沉积
<ol><li>将10毫克的 MWCNT-COOH 在50毫升圆锥管中, 稀释30毫升蒸馏水。油脂实验溶液在60°c 和 160 W 为 1 h。</li>
	<li>在50毫升圆锥管中放置6毫升的 0.1 N AgNO3 , 然后用18毫升蒸馏水稀释。油脂实验溶液在60°c 和 160 W 为 1 h。 警告: 添加 AgNO3与 MWCNTs 总质量的比例, 使 Ag 的总质量不超过20%。</li>
	<li>在添加 AgNO1后, 将 AgNO3解决方案添加到带吸管和1毫升吸管尖端的 MWCNTs 溶液中, sonicating 60 摄氏度和 160 w 的混合物继续 sonicating 3 h。</li>
	<li>离心溶液在 1.2万 x g 和室温15分钟, 并清除上清。离心溶液, 并去除上清液两次。</li>
	<li>将1毫升蒸馏水添加到银 MWCNTs 混合物中, 在溶液中均匀地分散沉淀。添加5毫升乙醇, 并允许反应在室温下进行1小时。</li>
	<li>离心溶液在 1.2万 x g 和室温15分钟, 并清除上清。</li>
	<li>用 ph 纸测定上清液的 ph 值。重复洗涤和离心, 直到清液 pH 值为中性。</li>
	<li>将1毫升蒸馏水加入银 MWCNTs, 均匀分散在溶液中。冷冻干燥溶液在-70 摄氏度真空下24小时。</li>
</ol>3. 抑制试验区
<ol><li>将18.12 克的 R2A 琼脂和1升的蒸馏水混合在烧瓶中。在121摄氏度的蒸压釜中消毒混合物15分钟。</li>
	<li>允许凝胶在室温下冷却。在硬化前, 在培养皿中放置10毫升凝胶以制备固体培养基。</li>
	<li>在固体培养基上放置100µL 细菌。使用吊具将细菌传播到固体介质中。 注意: 此程序必须在用酒精灯点燃的化学油烟罩中进行。</li>
	<li>在30摄氏度的盘子里孵化48小时。</li>
	<li>将3.12 克的 R2A 汤与1升的蒸馏水混合在一个烧瓶中。在121摄氏度的蒸压釜中消毒混合物15分钟。</li>
	<li>使用循环和针将生长的菌落转移到液体培养基中, 在 180 rpm 和30摄氏度的孵化器中孵化。</li>
	<li>以每小时 600 nm 的紫外分光光度法记录 OD (光学密度) 值以测量细菌生长。 注: R2A 琼脂是用于本试验, 而不是标准的米勒-韩丁琼脂, 因为 R2A 是首选琼脂生长的Methylobacterium.
</li>
	<li>在培养皿中放置10毫升准备好的 R2A 琼脂, 准备下琼脂。<ol>
<li>将3.12 克 R2A 汤和8克琼脂和1升蒸馏水混合在一起。在121摄氏度的高压釜中消毒混合物15分钟. 在底部琼脂上放置10毫升的凝胶。</li>
	</ol>
</li>
	<li>用微在无菌纸盘上加载50µL 银 MWCNTs 溶液。<ol>
<li>在紫外光照射下, 在真空室中干燥和消毒银 MWCNTs 样品30分钟。</li>
	</ol>
</li>
	<li>在琼脂上放置银 MWCNTs 样品。</li>
	<li>在30摄氏度孵育琼脂盘48小时。</li>
	<li>抑制12的度量区域。 注意: 引用中包含用于软件的说明。</li>
</ol>4. 抗菌试验
<ol><li>重复步骤3.1 至 3.7, 准备细菌培养。</li>
	<li>在最大 OD, 接种100µL 细菌成3毫升新鲜液体培养基与吸管和100µL 微吸管提示。</li>
	<li>在15毫升圆锥管中准备五50µL 的控制溶液样品。将以下各管加入: 1) 甲醇;2) 1000 µg/毫升 MWCNT COOH;3) 50 µg/毫升银 MWCNTs;4) 40 µg/毫升银 MWMWCNTs;5) 30 µg/毫升银 MWCNTs。</li>
	<li>孵育在180转每分钟和30°c, 直到控制是最大地活跃的由紫外分光光度的决定如上文所述。</li>
	<li>测量每个样品的 OD 值并计算麦克风浓度。OD 值与样品汤中的细菌细胞数量直接相关。</li>
	<li>将培养出来的细菌传播到固体培养基上。</li>
	<li>在30摄氏度孵化出48小时的菜肴。</li>
	<li>计数细菌菌落数和测量 CFU 值以确定抗菌活性12。 注意: 引用中包含用于软件的说明。</li>
</ol>5. 生存能力测定
<ol><li>从孵化器中取出培养细胞后, 吸入培养基, 用5毫升 PBS 的滴状洗涤清洗三次。</li>
	<li>在 PBS 中加入1毫升的胰蛋白酶-EDTA, 对洗涤后的细胞进行1分钟的吸入。<ol>
<li>点击盘子移除卡住的细胞。</li>
	</ol>
</li>
	<li>添加5毫升的 DMEM (Dulbecco 的改良鹰的培养基), 然后取出细胞。将样品离心 200 x 克3分钟, 取出上清液。<ol>
<li>加入1毫升的 DPBS 和混合。</li>
		<li>用自动计数机对解决方案的10µL 中的单元格进行计数。 注意: 使用的单元计数机的说明包含在引用13中。</li>
	</ol>
</li>
	<li>在含有 DMEM 溶液的六井板中, 每井放置 1 x 105的 AML12 细胞。培养基中含有 10% (v/v) 胎牛血清和1% 无菌抗生素。</li>
	<li>在 5% CO2/95% 空气环境中, 在37摄氏度孵育 8-12 小时的孵化板。</li>
	<li>用吸尘口从水井中吸取上清。<ol>
<li>添加每个样本包括控制, NMS-二甲基亚砜) 和30-40 µg/毫升银 MWCNT 到1毫升的 DMEM。</li>
		<li>37°c 在 5% CO2/95% 空气环境中孵化 8 h。</li>
	</ol>
</li>
	<li>清除上清液, 用5毫升的 PBS 冲洗样品。</li>
	<li>添加1毫升准备活/死套件 (20 µL 2 毫米 EthD-1 与5µL 4 毫米 calcein 是在亚砜在10毫升 PBS) 滴向细胞。</li>
	<li>在室温下孵育 30-45 分钟或37摄氏度, 用标准荧光显微镜在100X 或300X 测量荧光。</li>
</ol>6. 台盼蓝检测
<ol><li>根据步骤5.1 通过以上5.6.2 准备样品。</li>
	<li>移除上清。</li>
	<li>1毫升 1x DPBS 和醒酒的洗涤板。</li>
	<li>添加1毫升的胰蛋白酶到板块和孵化3分钟, 以分离细胞从文化。用细胞培养基冲洗板材, 收集细胞。将收集到的细胞放置在15毫升的试管中。</li>
	<li>离心管在 200 x g 和4°c 为3分钟。</li>
	<li>放弃上清。将1毫升新鲜培养基添加到细胞颗粒中, 重新分散细胞。</li>
	<li>添加10µL 的台盼蓝与10µL 分散细胞和计数的细胞与自动计数机。 注: 所使用的细胞计数机的说明包括在参考资料中。</li>
</ol>

<ol>
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作者没有什么可透露的。

在这里, 我们报告了一个简单的方法来制备 MWCNTs 与沉积银纳米粒子。这种含银的纳米材料显示了大量的抗菌活性和极少的潜力, 不受控制地吸收银纳米粒子在体内。我们证明, 30 µg/毫升的合成银-MWCNTs 是一个有效的水平的抗菌活性的Methylobacterium. , 对哺乳动物肝细胞的毒性微乎其微。虽然在商业部门扩大之前需要对银 MWCNTs 进行额外的改进和生物安全评估, 但使用乙醇作为减少剂可以帮助生产环保和廉价的银 MWCNTs。
下面是演示的要点。用所开发的纳米材料可以破坏各种类型的细菌。有可能在纳米管表面上设计多个位置, 在这些地方可以沉积银纳米粒子。抗菌性能也可以通过控制在表面沉积的银纳米粒子的大小和数量来量身定做。所开发的纳米材料对细菌细胞是有毒的, 但在适当浓度下对人体和动物细胞无毒。
为抗菌活性提出了以下机制。银 MWCNT 纳米结构直接接触细菌细胞表面, 破坏细胞壁, 导致活性氧的二次氧化;这些过程导致氧化应激。银-MWCNT 纳米结构已被证实释放银离子, 抑制的法定人数传感的Methylobacterium , 从而抑制基因的表达, 控制生物膜的形成。
目前的协议的局限性将包括在人体试验中允许的不确定的最大数量的银纳米粒子和银 MWCNTs。在本议定书中, 银-MWCNTs 30 µg/毫升样品中所含的银色纳米粒子的数量平均估计为0.4 µg/毫升。这种浓度被视为与哺乳动物细胞的生物相容, 正如以前研究报告的6,7,8,9。MWCNTs 40 µg/毫升浓度的细胞毒性机制尚不确定, 但建议在培养皿的底部不附着血细胞可增加银 MWCNT 接触的可能性。今后可以对不同培养条件下的多种细胞类型的银纳米粒子进行详细的毒性研究。这些研究可以提供更多的信息, 关于银 MWCNT 与细胞相互作用的机制。

这项研究的最终目的是制造环境友好的抗菌纳米材料, 从而抑制形成生物膜的细菌的生长。这些抗菌纳米材料有可能克服常用化学药品或抗生素化合物的毒性和耐药性问题。生物膜是由多糖、蛋白质、核酸和血脂组成的水合胞外高分子物质 (EPS),1,2。生物膜防止外来物质的侵入, 帮助细菌大力生长3,4。生物膜引起气味和慢性传染性疾病5,6。例如, Methylobacterium通过附着在水始终存在的地方或难以确保连续进行细菌根除 (如空调热交换器、淋浴室和医疗设备) 而生长。这些类型的生物膜引起气味和慢性传染性疾病5,6。
通常, 化学物质或抗生素化合物用于抑制形成生物膜的细菌的生长。抗生素耐药性细菌的出现和对化学品的体内安全性的担忧促使人们需要开发新材料, 以防止生物膜的形成和抑制细菌的生长。
在本研究中, 合成了抗菌纳米材料, 无抗生素耐药性和毒性。银是一种众所周知的抗菌物质, 最近的纳米和纳米技术的发展导致了对金属纳米粒子的抗菌作用的积极研究7,8。最近的研究报告说, 纳米粒子的体积小, 体积比大, 导致抗菌活性增加9,10,11。
本文介绍的纳米材料将银纳米粒子与增强的抗菌性能和碳纳米管结合在一起, 从而增加了单位体积的表面积。制备的银纳米粒子-碳纳米管复合材料具有很大的抗菌性能和对人体和动物细胞的最小毒性。以前研究中的合成过程使用了诸如 NaBH4、甲酰胺、甲基酰胺和肼等有害还原剂。这个过程很复杂, 很危险, 而且耗时。这里报告的合成过程使用乙醇作为一种显著减少危险的还原剂。
测定了银 MWCNTs 的抑菌区和最低杀菌浓度;用活/死和台盼蓝法测定毒性和抗菌性能。最低抑制浓度 (MIC) 和线粒体毒性 (MTT) 检测没有执行, 因为不寻常的颜色的碳纳米管, 这将干扰的化验。最后, 确定了在不影响哺乳动物细胞的情况下, 防止Methylobacterium生长的最小浓度。

<table>
	<tbody>
		<tr>
			<td>0.1 N silver nitrate</td>
			<td>SIGMA-ALDRICH</td>
			<td>1090811000</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon nanotube, multi-walled</td>
			<td>Tokyo Chemical Industry Co., LTD</td>
			<td>308068-56-6</td>
			<td></td>
		</tr>
		<tr>
			<td>R2A agar</td>
			<td>MBcell</td>
			<td>MB-R1129</td>
			<td></td>
		</tr>
		<tr>
			<td>R2A broth</td>
			<td>MBcell</td>
			<td>MB-R2230</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylobacterium spp.</td>
			<td>KCTC</td>
			<td>12618</td>
			<td>from Korea Collection for Type Cultures Daejeon Korea 12618, Daejon, Korea</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Cell imaging Kit</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>R37601</td>
			<td></td>
		</tr>
		<tr>
			<td>AML12</td>
			<td></td>
			<td></td>
			<td>from Chungnam University, Dajeon, Korea</td>
		</tr>
		<tr>
			<td>human PBMC</td>
			<td>ATCC</td>
			<td>PCS-800-011</td>
			<td></td>
		</tr>
		<tr>
			<td>TEM</td>
			<td>JEOL</td>
			<td>JEM-2100F</td>
			<td></td>
		</tr>
		<tr>
			<td>XRD</td>
			<td>Rigaku</td>
			<td>D/MAX 2500</td>
			<td>Cu K photon source (40kV, 100mA)</td>
		</tr>
		<tr>
			<td>JuLI Br</td>
			<td>NanoEnTek</td>
			<td>JULI-BRSC&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis of multi-walled carbon nanotubes modified with silver nanoparticles and evaluation of their antibacterial activities and cytotoxic properties

本研究采用水硫酸溶液处理多壁碳纳米管 (MWCNTs), 形成氧基功能基团。银 MWCNTs 是由还原沉积银从 AgNO3的水溶液中氧化 MWCNTs。鉴于碳纳米管的独特颜色, 不可能将其应用于最小抑制浓度或线粒体毒性检测, 以评估毒性和抗菌性能, 因为它们会干扰化验。测定了银 MWCNTs 的抑菌区和最低杀菌浓度, 并利用台盼蓝法测定了其毒性和抗菌性能, 不影响碳纳米管的颜色。

透射电镜 (TEM) 图像确认银 MWCNTs 的形成 (图 1A 和 1B)。表面电荷的变化证实了它们的成功合成。计算了 MWCNTs 上沉积的 Ag 粒子的大小 (图 1C)。平均微粒大小是大约3.83 毫微米。该合成银 MWCNTs 的 XRD 模式显示在图 1D中。峰值在 20-30°对应于 MWCNT;其余的峰值对应于 Ag。抗菌活动数据显示在图 2中。细菌菌落数量由Methylobacterium控件确认 (图 2A);增加甲醇减少了人口 103次 (图 2B), 并且加法 MWCNT-COOH 减少了人口 108时间 (图 2C)。在50µg/ml ag MWCNT (图 2D) 和40µg/ml ag MWCNT (图 2E) 样品中无法识别菌落。在20µg/mL Ag MWCNT 样品中, 人口减少了 105次 (图 2F)。在针对Methylobacterium (图 3) 的抑制测试区域中, 在添加了10µg/mL 时确定了抑制区域。在1µg/毫升浓度下未观察到抑制区。在细胞毒性试验中, 活体/死法 (图 4) 确认阴性控制 (图 4A) 中没有细胞毒性, 在使用甲醇时阳性控制 (图 4B) 中存在细胞毒性。增加了40µg/毫升 ag MWCNTs (图 4C) 揭示了一些细胞毒性, 然而增加30µg/毫升 ag MWCNTs (Figure 4D) 没有显示大量的细胞毒性。对该控件执行了一个台盼蓝检测 (图5) (图 5A), 甲醇 (图 5B), 40 µg/毫升 ag MWCNTs (图 5C) 和30µg/毫升 ag MWCNTs (图 5D) 样品。结果证实, 在30µg/毫升银 MWCNTs 中, 细胞毒性很少。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57384/57384fig1.jpg"> 图 1: 银 MWCNTs 的物理化学特性.(A) 和 (B)银 MWCNTs 的透射电镜图像;(C)通过 TEM 评估的银 MWCNTs 的大小分布 (n = 100);MWCNTs 的(D) XRD 模式<a href="/files/ftp_upload/57384/57384fig1large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57384/57384fig2.jpg"> 图 2: 银 MWCNTs 相对于Methylobacterium的抗菌性评价。(A)控件, (B)甲醇, (C) MWCNT-COOH, (D) 50 微克/毫升的银 MWCNTs, (E) 40 微克/毫升的银 MWCNTs, 和(F) 30 微克/毫升的银 MWCNTs.<a href="/files/ftp_upload/57384/57384fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57384/57384fig3.jpg"> 图 3: 银 MWCNTs 的抑制试验区。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57384/57384fig4.jpg"> 图 4: MWCNTs 显微照片的细胞生存性测定 AML12 荧光标记为死 (红) 和活 (绿) 细胞。(A)控件, (B)甲醇, (C) 40 微克/毫升 MWCNTs, 和(D) 30 微克/毫升的银 MWCNTs.<a href="/files/ftp_upload/57384/57384fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57384/57384fig5.jpg"> 图 5:30 和 40μg/毫升银 MWCNTs 的台盼蓝化验.(A)控件, (B)甲醇, (C) 40 微克/毫升的银 MWCNTs, 和(D) 30 微克/毫升的银 MWCNTs. (红色: 死细胞; 蓝色: 活细胞)<a href="/files/ftp_upload/57384/57384fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties at JoV

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

本研究通过多壁纳米碳纳米管的酸性氧化和银纳米粒子随后的还原沉积合成了抗菌纳米材料。用制备的纳米材料进行抗菌活性和细胞毒性试验。

银纳米粒子改性多壁碳纳米管的合成及其抗菌活性和细胞毒性的评价

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

synthesis-multi-walled-carbon-nanotubes-modified-with-silver

Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.2K 次观看.  Korea Institute of Science and Technology. 本研究通过多壁纳米碳纳米管的酸性氧化和银纳米粒子随后的还原沉积合成了抗菌纳米材料。用制备的纳米材料进行抗菌活性和细胞毒性试验。

视频: Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

Simultaneous Synthesis of Single-walled Carbon Nanotubes and Graphene in a Magnetically-enhanced Arc Plasma

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very promising application for molecular sensors, field effect transistor and super thin and flexible electronic devices1-4. Anodic arc discharge supported by the erosion of the anode material is one of the most practical and efficient methods, which can provide specific non-equilibrium processes and a high influx of carbon material to the developing structures at relatively higher temperature, and consequently the as-synthesized products have few structural defects and better crystallinity.
 To further improve the controllability and flexibility of the synthesis of carbon nanostructures in arc discharge, magnetic fields can be applied during the synthesis process according to the strong magnetic responses of arc plasmas. It was demonstrated that the magnetically-enhanced arc discharge can increase the average length of SWCNT 5, narrow the diameter distribution of metallic catalyst particles and carbon nanotubes 6, and change the ratio of metallic and semiconducting carbon nanotubes 7, as well as lead to graphene synthesis 8. 
 Furthermore, it is worthwhile to remark that when we introduce a non-uniform magnetic field with the component normal to the current in arc, the Lorentz force along the J&times;B direction can generate the plasmas jet and make effective delivery of carbon ion particles and heat flux to samples. As a result, large-scale graphene flakes and high-purity single-walled carbon nanotubes were simultaneously generated by such new magnetically-enhanced anodic arc method. Arc imaging, scanning electron microscope (SEM), transmission electron microscope (TEM) and Raman spectroscopy were employed to analyze the characterization of carbon nanostructures. These findings indicate a wide spectrum of opportunities to manipulate with the properties of nanostructures produced in plasmas by means of controlling the arc conditions.

Anodic arc discharge is one of the most practical and efficient methods to synthesize various carbon nanostructures. To increase the arc controllability and flexibility, a non-uniform magnetic field was introduced to process the one-step synthesis of large-scale graphene flakes and high-purity single-walled carbon nanotubes.

同时合成单壁碳纳米管和石墨在电弧等离子体磁增强

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very ...

科研

生物工程

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

黄金Nanostar合成银种子介导的生长方法

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

碳水化合物和聚酸酐纳米粒子功能化的高通量合成

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

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 ...

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 ...

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 ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

工程分子识别与单壁碳纳米管仿生聚合物

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

液体中脉冲激光烧蚀制备金属纳米粒子:研究纳米粒子抗菌性能的工具

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...