这项工作得到了加利福尼亚大学、河畔分校和美国农业部食品与农业研究所的支持, 孵化项目 1009710 J.P.G.。该材料的基础是国家科学基金会根据1817363号赠款资助的 J.P.G. 的工作。

1. 种植芥的植物
<ol><li>在5厘米 x 5 厘米的一次性花盆中播种一个.把32这些花盆放入一个装满水的塑料托盘 (0.5 厘米深), 把塑料托盘和植物转移到植物生长室。<ol>
<li>设置生长室设置如下: 200 µmol/ms 光合活性辐射 (PAR), 24 1 °c 日和 21 @ 1 °c 夜, 60% 湿度, 14/10 h 天/夜光政权分别。</li>
	</ol>
</li>
	<li>在一周的发芽之后, 每壶都要细细地留下一株。注意保持每个花盆中大小相同的幼苗。</li>
	<li>每两天将自来水直接浇在塑料托盘上, 浇壶水。种植植物四周。南芥植物已准备好进一步使用。</li>
</ol>2. 民警的综合和特性
<ol><li>在50毫升圆锥管中, 将硝酸铈 (III.) 重1.08 克, 并将其溶解在2.5 毫升的分子生物学级水中。</li>
	<li>在50毫升圆锥管中, 将4.5 克的聚 (丙烯酸) 酸和溶解在5毫升分子生物级水中。</li>
	<li>使用数字涡旋混合器, 将这两个解决方案彻底地混合在 2000 rpm 15 分钟。</li>
	<li>将15毫升的氢氧化铵溶液 (7.2 米) 转移到50毫升玻璃烧杯中。</li>
	<li>搅拌在 500 rpm, 加入混合物从步骤2.3 滴到氢氧化铵溶液和搅拌在 500 rpm 在室温下24小时的通风罩。</li>
	<li>用一张纸盖上烧杯, 以避免夜间反应中的大量溶液损失。</li>
	<li>24小时后, 将产生的溶液转移到50毫升圆锥管, 并将其离心在 3900 x g 处, 以去除任何可能的碎片和大团聚体。</li>
	<li>将这22.5 毫升的上清液转化为三15毫升 10 kDa 过滤器, 用分子级水填充过滤器的其余部分, 使总稀释45毫升。</li>
	<li>用台式离心机在15毫升 10 kDa 过滤器中加入上清液, 用 3900 x g 的离心机在15分钟内, 从游离聚合物和其他试剂中纯化上清溶液. 重复此步骤至少六次。</li>
	<li>用 220-700 nm 的紫外-可见光分光光度计测量每个周期中淋洗的吸光度, 以确保最终的民警溶液中没有游离聚合物和其他试剂。</li>
	<li>将收集的民警解决方案放入5毫升注射器中, 并用20纳米孔径注射器过滤器进行过滤。在50毫升锥形管中收集经过过滤的民警解决方案。</li>
	<li>在塑料试管中采用稀释后的最终民警解决方案, 用 220-700 nm 的紫外可见光分光光度计测量其吸光度。刚果国民吸光度峰值为 271 nm。</li>
	<li>用啤酒-兰伯特定律计算其浓度: A = εCL。a 是给定样本峰值的吸光度, ɛ是国民 (cm-1米-1) 的摩尔吸收系数, L 是光路长度 (试管宽度, 1 厘米), C 是被测纳米粒子的摩尔浓度。</li>
	<li>使用粒子大小和泽塔电位分析仪测量合成的民警的水动力直径和泽塔电位 (图 1)。</li>
	<li>将最终的民警解决方案存储在冰箱 (4 摄氏度), 直到进一步使用。 注: 请参阅吴等。8有关民警特性的更多协议详细信息。</li>
</ol>3. 用 DiI 荧光染料标记民警
<ol><li>混合0.4 毫升5毫米 (58 毫克/升) 民警与3.6 毫升分子生物学级水在20毫升玻璃瓶和搅拌在 500 rpm。</li>
	<li>添加24µL 11 '-dioctadecyl 33, 3 ', 3 '-tetramethylindocarbocyanine 高氯酸染料溶液 (DiI, 2.5 毫克/毫升, 稀释在亚砜) 到176µL 的亚砜 (二甲基亚砜), 使 DiI 染料溶液。</li>
	<li>将 DiI 染料滴入到民警溶液中, 在室温下搅动 1000 rpm 1 分钟。</li>
	<li>将由此产生的混合物转化为15毫升 10 kDa 过滤器, 并用分子生物学级水填充管顶, 使总稀释15毫升。</li>
	<li>从亚砜和任何可能的游离 DiI 染料中纯化 DiI 标记的民警 (DiI) 溶液, 其台式离心与15毫升 10 kDa 过滤器在 3900 x g 处5分钟。<ol>
<li>重复步骤3.5 至少五次。</li>
	</ol>
</li>
	<li>通过20纳米孔径注射器过滤器过滤最终的 DiI-民警解决方案。</li>
	<li>用紫外-可见光分光光度法测量最终 DiI 的吸光度, 并根据啤酒-兰伯特定律计算其浓度 (图 2)。有关详细信息, 请参阅步骤2.13。</li>
	<li>将其存储在4摄氏度的冰箱中以供进一步使用。</li>
</ol>4. 植物叶片与民警的渗透
<ol><li>添加0.1 毫升的渗透缓冲 (100 毫米的附加费, 100 毫米氯化镁2, pH 值 7.5, 由 HCl 调整) 成0.9 毫升的0.5 毫米民警或 DiI-民警解决方案和涡旋。使用10毫米的污水附加入渗缓冲液作为负控制。</li>
	<li>将0.2 毫升的民警或 DiI 渗透液转移到1毫升无菌无针注射器。点击删除任何可能的气泡。</li>
	<li>在渗透纳米微粒之前, 从生长室中提取植株, 以避免在室温条件下可能的气孔闭合。</li>
	<li>在入渗前, 用叶绿素计测定拟南芥叶片的叶绿素含量。测量每片叶子的三个复制 (每个复制包括至少三测量)14。选择具有相似叶绿素含量的拟南芥叶, 进行浸润试验。</li>
	<li>用最近准备好的民警或 DiI--民警解决方案缓慢地将无针注射器的尖端向下渗透到叶子叶片的底部 (背面) 并压低柱塞 (图 3A)。</li>
	<li>用一个微妙的任务雨刷 (图 3C) 轻轻地擦掉残留在叶片表面上的多余溶液 (图 3B), 并给植物贴上标签。为每组叶子使用新的微妙的任务湿巾。</li>
	<li>在3小时内, 将 DiI 的植物放在长凳上进行叶适应和孵化. 注: 渗透到南芥植物, 然后准备进一步使用 (图 3D)。</li>
</ol>5. 共焦显微镜用叶样的制备
<ol><li>将豌豆大小的观察凝胶卷到大约1厘米半径 (图 4a), 然后将其摊开, 直到它在玻璃滑块上1毫米薄 (图 4B)。</li>
	<li>使用软木塞 (直径0.3 厘米) 在玻璃滑动的观察凝胶的中心切出圆形截面 (图 4C)。</li>
	<li>完全用 perfluorodecalin (PFD) 填充切口, 以便在叶组织中更深入和更好地共聚焦成像分辨率。</li>
	<li>使用软木塞 (直径0.2 厘米) 从适应的 DiI 收集叶盘--民警渗入了南芥植物 (图 4D)。</li>
	<li>在 PFD 填充井中安装叶盘;面对渗透 (背面) 的叶子的一侧。</li>
	<li>将方形盖玻片放在叶盘的顶部, 轻轻按下滑动盖玻片, 将其与观察凝胶的井封住, 确保没有气泡滞留 (图 4E)。</li>
</ol>6. 用共焦显微镜成像 DiI 叶组织中的民警
<ol><li>在反向激光扫描共聚焦显微镜中使用40X 物镜。</li>
	<li>在40X 物镜的顶部放置两到三滴 ddH2O。</li>
	<li>将准备好的 DiI 在倒置的40X 物镜上, 将已制备的叶片样品滑动放在上面。<ol>
<li>确保盖玻片一侧, 但不是玻璃滑动接触直接与 ddH2O 的镜头。</li>
	</ol>
</li>
	<li>在显微镜下, 用激光光或明亮的磁场找到一个感兴趣的区域。</li>
	<li>启动显微镜软件, 打开氩激光 (设定在 20%)。</li>
	<li>设置针孔, 以收集小于2µm 的光学切片, 线平均为4。</li>
	<li>图像的共聚焦显微镜设置的样品: 514 nm 激光激发 (30%);Z 堆叠截面厚度: 2 µm;PMT1: 550-615 nm (用于 DiI-民警成像);PMT2: 700-800 nm (用于叶绿体成像)。</li>
	<li>采取代表性的共焦图像的叶子样本从不同的人, 至少三生物复制。</li>
</ol>7. 用共焦显微镜对活体活性氧清除成像的实验观察
<ol><li>准备25µM 2 ', 7 '-dichlorodihydrofluorescein 双乙酸 (H2DCFDA, 一染料为表示一般 ROS) 和10µM dihydroethidium (DHE, 一种指示超氧化物阴离子) 染料的染料在工商业污水附加剂渗透缓冲 (pH 7.5) 在1.5 毫升离心管,分别。</li>
	<li>使用软木钻 (直径0.2 厘米) 从适应的民警中收集叶盘渗透到南芥植物中.<ol>
<li>使用钳尖尖, 使叶片上三到四孔, 加速染料加载过程。</li>
	</ol>
</li>
	<li>将叶盘转移到离心管, 分别用 H2DCFDA 和 DHE, 在黑暗下孵化30分钟。</li>
	<li>孵化后, 用 ddH2O 三次冲洗叶盘, 用观察凝胶将其装入玻璃滑梯 (见协议部分 5)。</li>
	<li>将幻灯片放在共焦显微镜上, 并手动聚焦到叶肉细胞的区域。有关详细信息, 请参阅协议6节。</li>
	<li>将叶盘暴露在紫外-A (405 nm) 激光器上, 3 分钟产生 ros, 并记录每叶圆盘的时间序列 ("xyt") 的 ros 信号强度变化。</li>
	<li>用共聚焦显微镜对叶盘进行图像设置: 40X 水目标;496 nm 激光激发;PMT1: 500-600 nm (用于 DHE 和 DCFDA 染料检测);PMT2: 700-800 nm (用于叶绿体检测)。使用仅渗透缓冲液渗透的植物作为阴性控制。</li>
</ol>8.从体外清除 H2O2
<ol><li>按照以前出版物3、8、15中的方法, 在体外进行合成民警的猫 (过氧化氢酶) 模拟活动
</li>
	<li>添加45.4 µL 的1x 附加费渗透缓冲 (10 毫米的附加费, 10 毫米氯化镁2, pH 7.5, 由 HCl 调整), 民警 (60 毫微米, 3 µL) 和 H2O2 (2 µM, 1 µL) 入井 (白色圆的底部96井板), 并且轻轻地混合它由吹打。</li>
	<li>加入 10-乙酰基 37-dihydroxyphenoxazine (工作浓度100µM, 0.5 µL) 和辣根过氧化物酶 (HRP; 工作浓度 0.2 U/毫升, 0.1 µL) 入井, 轻轻地混合它由吹打, 孵化它为30分钟 10-乙酰基 37-dihydroxyphenoxazine反应与 H2O2和被转换成试卤灵在存在的 HRP。<ol>
<li>在孵化过程中, 用铝箔包装盘子以避免光线。</li>
		<li>用反应缓冲器或水代替 H2O2, 准备一个负控制。</li>
		<li>除了库存解决方案, 在室温下准备所有其他解决方案。</li>
	</ol>
</li>
	<li>孵化后, 与一个板块阅读器, 监测吸光度在 560 nm 使用试卤灵, 以表明水平的 H2O2。设置时间制度在0、2、5、10、20和30分钟。</li>
</ol>

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作者没有什么可透露的。

在本协议中, 我们描述了在植物叶肉细胞内的纳米粒子的合成、表征、荧光染料标记和共焦成像, 以展示其体内活性氧清除活性。从硝酸铈的混合物和氢氧化铵中的临机溶液中合成了民警。民警的特点是吸收 spectrophotomery 和浓度确定使用啤酒 Lamberts 法。泽塔电位测量证实了民警的负电荷表面, 以增强对叶绿体8的传递。用荧光 DiI 染料对民警进行标记,在叶片叶肉细胞?...

氧化铈纳米粒子 (nanoceria) 广泛应用于生物, 从基础研究到生物工程, 由于其独特的催化活性氧 (ROS) 清除能力1,2,3。Nanoceria 有 ROS 清除能力由于大量表面氧气空缺交替在二氧化状态 (ce3 +和 Ce4 +) 4,5,6。Ce3 +悬垂键有效地清除 ROS, 而纳米晶格菌株通过氧化还原循环反应7促进这些缺陷部位的再生。Nanoceria 最近也被用于研究和工程植物功能8,9。非生物胁迫下的植物积累了 ROS, 对脂质、蛋白质和 DNA 造成氧化损伤10。在 nanoceria 植物中, 在体内活性氧的催化清除能在高光、高温和冷应力下改善植物光合作用8。nanoceria 对土壤的施用也增加了小麦的生物质量和籽粒产量11;nanoceria 处理的油菜 (甘蓝) 植株在盐胁迫下具有较高的植物生物量12。
Nanoceria 提供生物工程师和植物生物学家一个基于纳米技术的工具, 以了解非生物胁迫反应和提高植物非生物胁迫耐受性。Nanoceria 的体内活性氧清除能力是独立于植物物种, 并在植物组织的简便交付有潜力, 使广泛应用外的模型有机体。不像其他基于基因的方法, nanoceria 不需要产生植物线与抗氧化酶的过度表达, 以提高 ROS 清除能力13。nanoceria 对植物的叶片浸润是实验室研究的一种实用方法。
本议定书的总目标是描述 1) 带负电荷的聚 (丙烯酸) 酸 nanoceria (民警) 的合成和表征, 2) 在整个叶细胞内提供和跟踪全国民警, 3) 监测在体内。本协议合成了带负电荷的聚丙烯酸 (nanoceria) 酸 (民警), 并以其吸收谱、流体力学直径和泽塔电位为特征。我们描述了一种简单的叶片入渗方法, 以提供民警到植物叶组织。在体外显像的纳米颗粒在叶肉细胞中的分布, 用荧光染料 (DiI) 标记民警 (DiI), 并通过共焦荧光显微镜观察纳米粒子。最后, 我们解释如何通过共聚焦显微镜监测体内的ROS 清除。

<table border="0" cellpadding="0" cellspacing="0" style="width:1225px;" width="1225">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Cerium (III) nitrate hexahydrate</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">238538-100G</td>
			<td style="width:265px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Molecular Biology Grade Water, Corning</td>
			<td style="width:228px;">VWR</td>
			<td style="width:165px;">45001-044&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Falcon 50 mL Conical Centrifuge Tubes</td>
			<td style="width:228px;">VWR</td>
			<td style="width:165px;">14-959-49A</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Poly (acrylic acid) 1,800 Mw</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">323667-100G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Fisherbrand Digital Vortex Mixer</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">02-215-370</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Fisherbrand Digital Vortex Mixer Accessory, Insert Retainer</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">02-215-391</td>
			<td></td>
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			<td height="21" style="height:21px;width:567px;">Fisherbrand Digital Vortex Mixer Accessories: Foam Insert Set</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">02-215-395</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Ammonium hydroxide solution</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">05002-1L</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">PYREX Griffin Beakers, Graduated, Corning</td>
			<td style="width:228px;">VWR</td>
			<td style="width:165px;">13912-149&#160;</td>
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			<td height="21" style="height:21px;width:567px;">RCT basic</td>
			<td style="width:228px;">IKA</td>
			<td style="width:165px;">3810001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Eppendorf Microcentrifuge 5424</td>
			<td style="width:228px;">VWR</td>
			<td style="width:165px;">80094-126</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Amicon Ultra-15 Centrifugal Filter Units</td>
			<td style="width:228px;">Millipore-Sigma</td>
			<td style="width:165px;">UFC901024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Allegra X-30 Series Benchtop Centrifuge</td>
			<td style="width:228px;">Beckman Coulter</td>
			<td style="width:165px;">B06314</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">UV-2600 Sptecrophotometer</td>
			<td style="width:228px;">Shimadzu</td>
			<td style="width:165px;">UV-2600 120V</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Whatman Anotop 10 syringe filter</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">WHA68091102</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">BD Disposable Syringes with Luer-Lok Tips</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">14-829-45</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:567px;">Zetasizer Nano S</td>
			<td style="width:228px;">Malvern Panalytical</td>
			<td style="width:165px;">Zen 1600</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:567px;">1,1&#8242;-Dioctadecyl-3,3,3&#8242;,3&#8242;-tetramethylindocarbocyanine perchlorate</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">42364-100MG</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Dimethyl Sulfoxide, ACS</td>
			<td style="width:228px;">VWR</td>
			<td style="width:165px;">BDH1115-1LP</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Sunshine Mix #1 LC1</td>
			<td style="width:228px;">Green Island Distributors, Inc</td>
			<td style="width:165px;">5212601.CFL080P</td>
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			<td height="21" style="height:21px;width:567px;">Adaptis 1000</td>
			<td style="width:228px;">Conviron</td>
			<td style="width:165px;">A1000</td>
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			<td height="21" style="height:21px;width:567px;">TES, &#62;99% (titration</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">T1375-100G</td>
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			<td height="21" style="height:21px;width:567px;">Magnesium chloride</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">M8266-1KG</td>
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			<td height="21" style="height:21px;width:567px;">Air-Tite All-Plastic Norm-Ject Syringe</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">14-817-25</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:567px;">Kimberly-Clark Professional Kimtech Science Kimwipes Delicate Task Wipers</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">06-666A</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:567px;">Carolina Observation Gel</td>
			<td style="width:228px;">Carolina</td>
			<td style="width:165px;">132700</td>
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			<td height="21" style="height:21px;width:567px;">Corning microscope slides, frosted one side, one end</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">CLS294875X25-72EA</td>
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			<td height="21" style="height:21px;width:567px;">Cork Borer Sets with Handles</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">S50166A</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:567px;">Perfluorodecalin</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">P9900-25G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Micro Cover Glasses, Square, No. 1</td>
			<td style="width:228px;">VWR</td>
			<td style="width:165px;">48366-045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Leica Laser Scanning Confocal Microscope TCS SP5</td>
			<td style="width:228px;">Leica Microsystems</td>
			<td style="width:165px;">TCS SP5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">2&#8242;,7&#8242;-Dichlorofluorescin diacetate</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">D6883-250MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Dihydroethidium</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">D7008-10MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Fisherbrand Premium Microcentrifuge Tubes: 1.5 mL</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Eppendorf Uvette cuvettes</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">Z605050-80EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Chlorophyll meter&#160;</td>
			<td style="width:228px;">Konica Minolta</td>
			<td style="width:165px;">SPAD-502</td>
			<td></td>
		</tr>
	</tbody>
</table>

catalytic scavenging of plant reactive oxygen species in vivo by anionic cerium oxide nanoparticles

活性氧 (ROS) 的积累是植物非生物胁迫反应的标志。ROS 在植物中起着双重作用, 在低水平上充当信号分子, 在高水平上破坏分子。在被胁迫的植物中, ROS 的积聚会破坏代谢物、酶、脂类和 DNA, 从而减少植物生长和产量。氧化铈纳米粒子 (nanoceria) 在体内催化清除活性氧的能力为理解和生物工程植物非生物胁迫耐受提供了独特的工具。在这里, 我们提出了一个合成和表征聚 (丙烯酸) 酸涂层 nanoceria (民警) 的协议, 通过叶片渗透与植物界面的纳米微粒, 并监测其分布和 ROS 清除在体内使用共焦显微镜。目前用于操作植物中 ROS 积累的分子工具仅限于模型物种, 需要费力的转化方法。本协议的体内ROS 清除有可能适用于野生类型的植物, 宽叶和叶结构, 如拟南芥。

民警的合成和表征. 民警的合成、纯化和特点遵循了议定书第2节所述方法。图 1A显示了硝酸铈、临机局、硝酸铈和临机局的混合物和民警的溶液的着色。在合成了民警后, 从白色到浅黄色的颜色变化是可见的。用 10 kDa 过滤器纯化后, 民警的特征是紫外-可见光分光光度计。在 271 nm (图 1</st...

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Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

阴离子氧化铈纳米粒子催化清除体内植物活性氧的研究

在这里, 我们提出了一种合成和表征氧化铈纳米粒子 (nanoceria) 的协议, 用于在体内清除活性氧, nanoceria 在植物组织中进行共焦显微成像, 并在体内用共焦显微镜监测 nanoceria ROS 的清除。

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...

catalytic-scavenging-plant-reactive-oxygen-species-vivo-anionic

Research

JoVE Journal

Bioengineering

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

8.8K 次观看.  University of California. 在这里, 我们提出了一种合成和表征氧化铈纳米粒子 (nanoceria) 的协议, 用于在体内清除活性氧, nanoceria 在植物组织中进行共焦显微成像, 并在体内用共焦显微镜监测 nanoceria ROS 的清除。

视频: Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

科研

生物工程

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 ...

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Enhanced darkfield microscopy and hyperspectral imaging with spectral mapping enable screening, localization, and identification of nanoscale materials in histological samples with improved speed and accuracy over traditional methods. The goal of this paper is to provide methods for darkfield imaging and hyperspectral mapping of metal oxide nanoparticles in histological samples.

金属氧化物纳米颗粒的组织学样品鉴定增强暗场显微镜和高光​​谱遥感制图

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in 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 ...

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 ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

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 ...

Manganese Oxide Nanoparticle Synthesis by Thermal Decomposition of Manganese(II) Acetylacetonate

For biomedical applications, metal oxide nanoparticles such as iron oxide and manganese oxide (MnO), have been used as biosensors and contrast agents in magnetic resonance imaging (MRI). While iron oxide nanoparticles provide constant negative contrast on MRI over typical experimental timeframes, MnO generates switchable positive contrast on MRI through dissolution of MnO to Mn2+ at low pH within cell endosomes to &#8216;turn ON&#8217; MRI contrast. This protocol describes a one-pot synthesis of MnO nanoparticles formed by thermal decomposition of manganese(II) acetylacetonate in oleylamine and dibenzyl ether. Although running the synthesis of MnO nanoparticles is simple, the initial experimental setup can be difficult to reproduce if detailed instructions are not provided. Thus, the glassware and tubing assembly is first thoroughly described to allow other investigators to easily reproduce the setup. The synthesis method incorporates a temperature controller to achieve automated and precise manipulation of the desired temperature profile, which will impact resulting nanoparticle size and chemistry. The thermal decomposition protocol can be readily adapted to generate other metal oxide nanoparticles (e.g., iron oxide) and to include alternative organic solvents and stabilizers (e.g., oleic acid). In addition, the ratio of organic solvent to stabilizer can be changed to further impact nanoparticle properties, which is shown herein. Synthesized MnO nanoparticles are characterized for morphology, size, bulk composition, and surface composition through transmission electron microscopy, X-ray diffraction, and Fourier-transform infrared spectroscopy, respectively. The MnO nanoparticles synthesized by this method will be hydrophobic and must be further manipulated through ligand exchange, polymeric encapsulation, or lipid capping to incorporate hydrophilic groups for interaction with biological fluids and tissues.

Manganese Oxide Nanoparticle Synthesis by Thermal Decomposition of ManganeseII Acetylacetonate

This protocol details a facile, one-pot synthesis of manganese oxide (MnO) nanoparticles by thermal decomposition of manganese(II) acetylacetonate in the presence of oleylamine and dibenzyl ether. MnO nanoparticles have been utilized in diverse applications including magnetic resonance imaging, biosensing, catalysis, batteries, and waste water treatment.

锰氧化物纳米粒子合成锰（II）乙酰酸盐的热分解

For biomedical applications, metal oxide nanoparticles such as iron oxide and manganese oxide (MnO), have been used as biosensors and contrast agents in ...

阴离子氧化铈纳米粒子催化清除体内植物活性氧的研究

摘要

探索更多视频

此视频中的章节

病毒粒子为

一机能磁性纳米纤维素的细菌与氧化铁纳米粒子的制备

细胞标记和超顺磁性氧化铁纳米粒子靶向

金属氧化物纳米颗粒的组织学样品鉴定增强暗场显微镜和高光谱遥感制图

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

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

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

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

锰氧化物纳米粒子合成锰（II）乙酰酸盐的热分解

阴离子氧化铈纳米粒子催化清除体内植物活性氧的研究

摘要

探索更多视频

此视频中的章节

病毒粒子为

一机能磁性纳米纤维素的细菌与氧化铁纳米粒子的制备

细胞标记和超顺磁性氧化铁纳米粒子靶向

金属氧化物纳米颗粒的组织学样品鉴定增强暗场显微镜和高光​​谱遥感制图

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

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

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

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

锰氧化物纳米粒子合成锰（II）乙酰酸盐的热分解

金属氧化物纳米颗粒的组织学样品鉴定增强暗场显微镜和高光谱遥感制图