Name: synthesis of functionalized 10-nm polymer-coated gold particles for endothelium targeting and drug delivery
Uploaded: 2018-01-15T15:00:00.000+00:00
Duration: 10 min 38 s
Description: Watch this Scientific Journal Video about Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery at JoVE.com

这项工作得到了东北大学化学工程部、启动基金和东北大学教务处、NIH K01 HL125499 和 NSF-IGERT 赠款 NSF/DGE-096843 的1级试点研究补助金的支持。作者还要感谢托马斯 j ·韦伯斯特和他的实验室的帮助, 以及东北大学的纳米科学和技术中心和制药科学系。

1. 库存解决方案的准备或采购 (储存在室温或冻结使用之前)
<ol><li>在室温下, 在10毫升超纯水中溶解400毫克氢氧化钠, 制备出1米氢氧化钠 (氢氧化钠) 溶液, 并将物料混合均匀。 注: 超纯水是指无杂质的水, 如微粒、细菌、核酸、离子或有机物的痕迹。纯化系统用于获得超纯水, 其电阻率可达 18.2 mΩ·cm, 表明低阴离子污染。不推荐使用商用瓶装超纯水。从今以后, 这种超纯水将被称为水, 除非另有规定。</li>
	<li>在10毫升的水中溶解2.4 克氢氧化钠, 并在室温下储存溶液, 以制备一个6M 的氢氧化钠溶液。</li>
	<li>在10.16 毫升的水中溶解1克干 HAuCl4 , 制备金酸 (HAuCl4) 的250毫米库存溶液。稀释1毫升的250毫米 HAuCl4与9毫升水, 以获得工作浓度 25 mm HAuCl4。在室温下存储 HAuCl4解决方案。</li>
	<li>在 ph 值8.8 处制备0.1 米碳酸盐缓冲液, 在10毫升水中溶解84毫克碳酸氢钠, 并在步骤8.8 中加入6米氢氧化钠, 调整 ph 值至1.2。在室温下储存碳酸盐缓冲液。</li>
	<li>混合20毫升的唑化合物, 3-(45-dimethylthiazol-2-基)-5-(3-carboxymethoxyphenyl)-2-(4-磺)-2 h-唑盐 (MTS), 具有稳定剂, 嗪基 (PES) (请参阅材料表)。将 MTS/PES 混合物储存在等分 (最多10冻融循环) 在-20 ° c 的黑暗中。</li>
</ol>2. 金纳米粒子芯的合成
<ol><li>准备一个稀释的 THPC 溶液, 在室温下, 在离心管中加入12µL 80% THPC 水到1毫升的水中。</li>
	<li>在磁搅拌板的中心上固定一个100毫升的圆形底烧瓶。将45毫升的水倒入烧瓶中, 用一个小的蛋形磁力搅拌棒搅动 300 rpm 的水。加入0.5 毫升1米氢氧化钠和1毫升稀释 THPC 溶液。继续积极搅拌反应混合物5分钟。</li>
	<li>继续搅拌混合物, 并添加2毫升的 25 mM HAuCl4解决方案。混合物的颜色会在几秒钟内从黄色变成深褐色, 这表明 THPC 盖 AuNPs 的形成是负电荷。搅拌的混合物为一个额外的15分钟, 以允许完全形成的黄金核心。</li>
</ol>3. 在金纳米粒子周围添加聚合物电晕
<ol><li>在步骤2.3 中描述的15分钟内, 通过在4毫升的玻璃瓶中溶解下面的1毫升水中的每一个, 准备 peg 解决方案:30 毫克 NH2-peg-SH;7.5 毫克 m-PEG-SH;7.5 毫克羧基-SH。 注: 所产生的浓度应为8.8 米 NH2-peg-sh, 3.75 m m-sh, 和3.75 米羧基-sh。</li>
	<li>在将圆底烧瓶中的溶液搅拌速度降低到 100 rpm 后, 在 THPC 帽 AuNPs 滴的溶液中加入 PEG 溶液。继续在室温下轻轻搅拌, 以确保高效去除 THPC 和用 PEG 代替。</li>
	<li>在接下来的72小时, 使用 12-14 kDa 分子量截止纤维素膜透析搅拌混合物对4升桶水搅拌 60 rpm。通过透析夹将膜的两端系在一起, 然后通过吸管转移溶液。 注: 这一透析过程与 12-14 kDa 截止膜只去除未 PEG, THPC, 和其他化学反应物, 通过扩散沿浓度梯度。</li>
	<li>为了保持高浓度梯度和促进连续扩散, 改变水每 2 h 为前 6 h 和每 6-12 h 为剩余的 66 h。 注: 本水变化计划的目的是为了适应早期发生的快速扩散, 因为样品中最初的高浓度。上述的透析过程使纳米粒子溶液 semi-purified, 因为纳米颗粒、沉淀物、聚合体和其他大尺寸的杂质不能通过 12-14 kDa 截止膜的孔隙。</li>
	<li>将 semi-purified 纳米微粒溶液过滤成50毫升的锥形管, 使用0.2 µm 注射器过滤器除去剩余的沉淀物、骨料和其他大尺寸杂质。</li>
	<li>将锥形管置于-80 ° c 的冷冻库中, 并允许纯化的乙二醇 AuNP (PEG-AuNP) 溶液冻结约5小时。</li>
	<li>使用冷冻干燥机 lyophilize 纯化的 PEG-AuNP。</li>
	<li>将干燥、纯化的 PEG AuNP 在4° c 贮存, 直至使用。</li>
</ol>4. 共轭荧光使用 n-羟基琥珀酰亚胺 (NHS) 酯到 NH2组上的 PEG-AuNP
<ol><li>在磁搅拌板的中心上固定一个50毫升的圆形底烧瓶。用18毫升的水灌满烧瓶, 用一个蛋形的磁力搅拌棒将水搅拌100分钟。</li>
	<li>重2毫克的冻干 PEG AuNP 在4毫升锥管, 使用高精度台式规模。使用防静电枪, 以消除静电电荷和附着的 PEG AuNP 粉的管面。把2毫升的水加到管子里。将2毫升 PEG-AuNP 溶液倒入圆底烧瓶中, 以便在总共20毫升的水中进行进一步的重组。继续搅拌的混合物在 100 rpm 使用一个鸡蛋形的磁力搅拌棒。</li>
	<li>添加1毫升0.1 米, pH 8.8 碳酸盐缓冲到20毫升重组 AuNP 包含在圆底烧瓶。继续搅拌。</li>
	<li>在二甲基亚砜中添加5µL 10 毫克/毫升荧光琥珀酰亚胺酯。 注: 任何荧光染料都可以在这个过程中使用。在一夜之间搅动荧光钉。保持室温, 用铝箔盖住。</li>
	<li>在接下来的72小时中, 使用步骤3.3 中描述的协议删除多余的荧光。简要地, 透析搅拌混合使用4升水桶和 12-14 kDa 分子量截止纤维素膜。改变的水每2小时的前6小时和每6-12 小时的剩余66小时。</li>
	<li>获得1毫升的纯化荧光 PEG-AuNP 溶液, 将用于在下文5节描述的特性。如步骤 3.5-3.7 所述, 冻结并 lyophilize 余下的溶液以粉状形式获得荧光纳米粒子以贮存在4° c。</li>
	<li>使用0.2 µm 注射器过滤器, 以消毒和消除任何潜在的沉淀物一旦重组为细胞培养应用。</li>
</ol>5. 荧光乙二醇金纳米粒子的表征
<ol><li>利用透射电子显微镜 (TEM) 对金纳米粒子的核心进行可视化, 并对其大小进行量化<ol>
<li>将纯化的荧光 PEG-AuNP 溶液滴10µL 在碳涂层网格上。</li>
		<li>5分钟后, 用一张滤纸仔细地将多余的液体从 TEM 网格的边缘吸走, 直到一部含有荧光钉 AuNPs 的胶片被留下。</li>
		<li>查看荧光 PEG-AuNPs 在 80 kV 和放大5000万。拍摄数码照片。 注: 只有金芯才可见, 因为在 TEM 上, PEG 不是电子致密的。</li>
		<li>使用测量软件, 设置与刻度条相关的测量刻度。计算直径约20金芯, 然后确定平均金芯直径。 注: TEM 成像系统自动在数字图像上放置刻度线。</li>
	</ol></li>
	<li>动态光散射 (dl) 法测量水动力纳米粒尺寸<ol>
<li>使用步骤4.2 中描述的方法将1毫克冻干 THPC 涂层 AuNPs 到离心管中。将1毫克冻干的 PEG AuNP 在单独的试管中。在每根管子上加1毫升的水, 并将每个 AuNP 溶液转换为1毫升的透明塑料。</li>
		<li>将试管放入 dl 仪器中, 并使用内置在 dl 系统中的粒子分析器软件测量球形水动力直径。</li>
		<li>使用直方图格式绘制测量的水动力直径。 注: 直方图将按大小对纳米粒子进行分组。最大的一组纳米粒子将澄清的直径, 最好的描述 AuNPs 的大小的合成在这个协议。</li>
	</ol></li>
	<li>荧光萤光确认 注: 同一样品和试管从步骤5.2 可以用来测量荧光信号。<ol>
<li>从 dl 中删除试管并插入到光中。将激发波长设置为 633 nm, 并沿着 650-800 nm 波长范围读取发射的荧光信号。</li>
		<li>确认峰值大约为 665 nm, 这与 NHS 的排放峰值有关。 注意: 根据过程中使用的荧光调整激发和发射波长。</li>
	</ol></li>
	<li>MTS 检测11评估生物相容性<ol>
<li>在一个96井清底无菌组织培养处理板, 吸管100µL Dulbecco 氏改良鹰的培养基 (DMEM; 补充10% 胎牛血清和1% 青霉素/链霉素) 和 3.2 x 103细胞到每个井。 注: 本研究采用大鼠脂肪垫内皮细胞。总共21口井用于允许7种不同的纳米颗粒浓度的3个复制 (参见步骤5.4.3 的特定浓度的兴趣)。</li>
		<li>允许细胞生长到70-100 在湿度和 5% CO2控制孵化器在37° c11。</li>
		<li>准备单独的离心管, 每一个都含有300µL 的 DMEM 和不同浓度的纳米颗粒。这项研究需要7不同的 DMEM 与以下 PEG-AuNP 浓度: 0 µg/毫升, 10 µg/毫升, 50 µg/毫升, 100 µg/毫升, 250 µg/毫升, 500 µg/毫升, 和1000µg/毫升。</li>
		<li>从试管中抽出 DMEM。用100µL 的 DMEM 介质填入井中, 其中含有0、10、50、100、250、500或1000µg/毫升 AuNP。</li>
		<li>允许细胞和纳米粒子在预定的时间长度 co-incubate, 这里为16小时。</li>
		<li>在潜伏期接近尾声, 解冻的 MTS/PES 混合物从步骤1.5。</li>
		<li>在孵化后, 吸入 DMEM 和悬浮的, 松散附着的纳米颗粒从井。添加100µL 的新鲜 DMEM 连同20µL 的 mts/pes 解决方案获得 1:5 mts/pes 的 DMEM 比根据制造商的协议。用 MTS/PES 在37° c 下孵育2小时的细胞。 注: 这 2 h 潜伏期由内皮细胞的新陈代谢活动决定, 并且可以增加到 4 h 为其他细胞类型。在2小时内, 该 MTS 被内皮细胞代谢成有色甲, 与 DMEM 混合。AuNP 的生物相容性和细胞活力的可能性将被证明有更多的有色甲。PEG-AuNP 的毒性和细胞死亡的几率会显示出不那么强烈的颜色。</li>
		<li>使用兼容的平板阅读器读取 492 nm 的吸光度, 对每个井进行比色分析。</li>
		<li>对于每个纳米粒子的浓度, 计算三个吸光度值的平均吸收量。将平均吸光度除以在含0µg/毫升纳米颗粒的油井中的吸光度平均值。 注: 这些不含 AuNPs 的水井是用于计算在其他油井中施用纳米颗粒处理后的细胞存活率的控制措施。</li>
	</ol></li>
	<li>荧光共聚焦显微镜对黏附或功能失调的内皮细胞摄取纳米颗粒的糖11<ol>
<li>种子 1.0 x 104细胞/cm2大鼠脂肪垫内皮细胞到无菌12毫米玻璃片和饲料细胞与 DMEM 补充10% 胎牛血清和1% 青霉素/链霉素。允许细胞生长到完全70-100 在湿度和 CO2控制孵化器在37° c, 大约3天。</li>
		<li>添加治疗, 以修改糖, 如果适用。例如, 2 h 治疗的培养内皮细胞与 2.5 x 10-6酶 III 酶降解糖通过专门裂解其素硫酸盐成分。 注意: 健康的糖可以通过生长5.5.1 中所描述的内皮细胞来模拟, 而不需要添加降解酶。</li>
		<li>内皮糖保持完整或功能失调, 孵育的内皮细胞与荧光 AuNPs 在550µg/毫升16小时或其他需要的时间。</li>
		<li>用2毫升37° c 1% 牛血清白蛋白在磷酸盐缓冲盐水 (PBS; 含100毫克/升钙和100毫克/升镁) 轻轻冲洗细胞。在 PBS 中浸没2毫升2% 甲醛和0.1% 戊二醛的细胞, 并允许细胞在室温下固定30分钟。</li>
		<li>取出醛固定液, 用室温 PBS 清洗细胞三5分钟。</li>
		<li>在初级抗体应用前, 在 PBS 中, 在室温下30分钟将内皮细胞暴露在2% 山羊血清中, 以阻断非特异性结合位点。单元格必须完全由阻塞解决方案覆盖。</li>
		<li>在4° c 的 PBS 中, 用 1% anti-heparan 硫酸根原抗体在2% 山羊血清中孵育内皮细胞, 以标记糖的素硫酸盐成分。确保固定细胞与抗体溶液完全接触。</li>
		<li>第二天, 用室温 PBS 清洗抗体三10分钟。</li>
		<li>孵育内皮细胞与 1:1, 000 稀释适当的荧光二次抗体在室温下, 在黑暗中或用铝箔覆盖的30分钟。</li>
		<li>用室温 PBS 清洗二次抗体三10分钟的周期。</li>
		<li>将片安装在显微镜上, 使用含有4的防、6-diamidino-2-吲、二盐酸盐 (DAPI) 进行细胞核染色的安装介质。用指甲油封住片的边缘。</li>
		<li>图像荧光 immunostained 内皮细胞样品与共聚焦显微镜, 使用蓝色, 绿色和远红色通道, 以可视化的细胞核, 素硫酸糖, 和纳米粒子, 分别。</li>
	</ol></li>
</ol>

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作者声明他们没有竞争的金融利益。

该技术是一种合成可定制的超 PEG 涂层 AuNPs 的有效方法。这个过程的一个重要部分是最初形成的 THPC 上限金纳米粒子, 可以确认的颜色变化, 从黄色到褐色, 将发生后, HAuCl4 已添加到内容的圆底烧瓶 (协议步骤 2.3)。没有颜色变化表明没有形成纳米粒子, 并且在继续之前应检查和重复初始步骤。在这种情况下, 颜色变化的东西, 而不是棕色, 如葡萄酒红色或灰色, 产生的粒子可能不会在目标 2.5 nm 和一个新的批次, 也应该作出。
在金芯形成后, THPC 的交换和纯化程序包含了成功完成该议定书的几个关键步骤。隔夜混合允许替换反应去完成。如果透析水不随规定的频率改变, 就会发生失败的净化。如果在透析中留下的微粒超过72小时, 粒子的聚集和沉淀也会发生。在冷冻干燥过程中可以观察到其他潜在的问题。如果超 PEG 涂层的 AuNP 溶液未完全冻结, 或者如果未正确设置冻, 样品可能会丢失。参考冻手册, 因为有些设备可能需要不同的样品准备。
合成的方便性和由此产生的微粒的生物相容性代表了使用这些 PEG-AuNPs 的优势。此外, 这些纳米粒子具有能够与纳米细胞结构相互作用的优势, 如通过吸收这些纳米粒子来识别降解糖的能力所证明的那样。这一优势可以用于开发新的动脉粥样硬化治疗和预防措施。除了我们这里, 这个协议的另一个优点是, 它允许广泛定制的粒子, 以及增加的稳定性和存储能力, 通过附加硫醇含有 PEG 到金纳米颗粒4。另一端的 PEG 链可以包含任何功能基团, 和无数的分子可以共轭这些群体。在本协议中, 所有三常见的功能组都附有 (甲基、羧基和氨基)。选择 PEG 的比例是先确定荧光检测的优先级, 然后再利用羧酸基团加入次级靶向基团的能力。这些基团的比例可以根据应用进行调整, 而且聚合物的长度和形状也可以调节。
为了测量粒子的摄取量, 我们将一个荧光探针与一个功能基团结合在一起。应该指出的是, 任何超越我们所描述的结合将导致纳米粒子的表面性质的变化。每一次迭代的纳米粒子与额外的成分和共轭反应应测试的期望属性。
该方法产生超金纳米粒子, 旨在克服内皮细胞外糖的防御性, 这阻碍了常规大小纳米颗粒的摄取。然而, 小尺寸借给困难的成像和药物加载方面。这些微粒明显小于典型的纳米粒子的大小范围, 因此, 可用于治疗和靶基的附着面面积大大减少。这可能会导致很难在成像应用中提取单个信号, 尽管在共焦图像中显示的粒子簇仍然可以很容易识别。靶向配体和疗法的附着物的减少表面可能需要更多的微粒来达到靶向剂量的要求。但是, 当考虑到糖时, 较小的粒子在交付时会更有效。
这些新的超粒子能够交付到难以达到的纳米区域内的体内微环境的破坏。加入 PEG 可以增加生物相容性, 并为不同应用的颗粒重定制提供功能基团。较小的尺寸与典型的纳米粒子相比有一些缺点, 但如果发展战略, 超粒子是一个有前途的方法, 以适应难以穿透, 复杂, 脆弱的糖血管靶向和药物传递。

纳米粒子已被应用于药物传递和成像, 它能够通过身体导航到达目标区域1,2。这些微粒可能在肿瘤中通过漏脉积聚, 或在目标配体表达和暴露的地方进行定位。黄金, 特别是, 已成为一种常用的纳米颗粒材料, 因为其独特的化学和物理性能影响的运输和释放疗法3。金是一种有效的纳米颗粒材料, 因为它的表面可以被修改以结合到硫, 并且具有很高的生物相容性, 因为它的低毒性4。AuNPs 有能力成为大型生物分子药物的载体, 并成功地提供了多肽、核酸和蛋白质, 允许 AuNPs 为目标2,4。
不幸的是, 纳米颗粒药物的传递效果受到了负电荷糖的阻碍, 这是大多数哺乳动物细胞膜上的胞外涂层, 其孔径大小高达 7 nm5,6。这种孔径比大多数纳米微粒药物载体都小, 它们的直径从50-200 纳米不等。在疾病的情况下, 这些糖毛孔变得更大由于退化, 通过增加渗透到内皮细胞。然而, 大多数纳米粒子仍然是太大, 以利用这种结构变化的糖。这种大小不匹配的一个含义是, 常规大小的粒子不会与血管内皮细胞良性互动。这就影响了静脉输液的微粒进入内皮, 也可以说是通过血脑屏障传递的粒子7,8,9,10。
解决这个问题的一种方法是利用较小的粒子通过糖中的小孔隙。在这里, 我们合成了10.5 纳米超金纳米颗粒, 这通常应该被完整的, 健康的糖的威慑。一旦糖开始被破坏, 纳米颗粒就可以通过增加的孔隙大小很容易地穿透细胞。本协议详细介绍了用 PEG 包覆的超金芯的合成, 提高了生物相容性, 降低了系统间隙4。PEG 还可以包含多种类型的功能基团, 为靶向配体、荧光和疗法的结合开辟了途径。先前公布的结果表明, 这些超纳米粒子往往更有利于在破坏的区域内皮糖功能, 即使没有任何积极的目标4,11。这表明了使用正确尺寸的颗粒用于交付应用的可行性和重要性。下面的协议介绍了 peg 涂层 AuNPs (peg-AuNP) 的合成、纯化和表征, 并讨论了如何剪裁功能基团和动词其他应用。

<table><tbody><tr><td>Sodium hydroxide (NaOH)</td><td>Sigma Aldrich</td><td>795429</td><td></td></tr><tr><td>Gold (III) Chloride trihydrate (HAuCl&lt;sub&gt;4&lt;/sub&gt;.3H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma Aldrich</td><td>520918</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma Aldrich</td><td>S5761</td><td></td></tr><tr><td>Tetrakis (hydroxymethyl) phosphnium chloride</td><td>Sigma Aldrich</td><td>404861</td><td></td></tr><tr><td>Mono-functional mPEG-thiol</td><td>Layson Bio Inc.</td><td>MPEG-SH-2000-1g</td><td>Mw: 2,000 Da</td></tr><tr><td>hetero bi-functional anime-PEG-thiol</td><td>Layson Bio Inc.</td><td>NH2-PEG-SH-3400-1g</td><td>Mw: 3,400 Da</td></tr><tr><td>Carboxymethyl-PEG-thiol</td><td>Layson Bio Inc.</td><td>CM-PEG-SH-2000-1g</td><td>Mw: 2,000 Da</td></tr><tr><td>Cellulose dialysis membrane (12-14 kDa)</td><td>Sigma Aldrich</td><td>D9777</td><td></td></tr><tr><td>Zerostat anti-static instrument</td><td>Sigma Aldrich</td><td>Z108812</td><td></td></tr><tr><td>Alexa Fluor 647 (AF647) carboxylic acid succinimidyl ester</td><td>Fisher</td><td>A20006</td><td>Fluorophore</td></tr><tr><td>Fisherbrand Qualitative Grade Plain Filter Paper Circles - P5 grade</td><td>Thermo Fisher Scientific</td><td>09-801-B</td><td></td></tr><tr><td>Transmission electron microscopy</td><td>JEOL USA</td><td>JEOL JEM-1000</td><td>TEM</td></tr><tr><td>Dynamic Light Scattering</td><td>Brookhaven Instruments Corporation</td><td>Brookhaven 90 Plus Particle Size Analyzer</td><td>DLS</td></tr><tr><td>Fluorometer</td><td>Horiba Scientific</td><td>Jobin Yvon Fluromax 4</td><td>Fluorometer</td></tr><tr><td>CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS)</td><td>Promega</td><td>G3582</td><td>MTS</td></tr><tr><td>Plate reader</td><td>Molecular Devices</td><td>SpectraMax M4</td><td>Plate reader</td></tr><tr><td>10E4 epitope HS mouse monoclonal IgM antibody (primary antibody)</td><td>Amsbio</td><td>370255</td><td>Primary antibody</td></tr><tr><td>Alexa Fluor 488 goat anti-mouse IgG (secondary antibody)</td><td>Thermo Fisher Scientific</td><td>R37120</td><td>Secondary antibody</td></tr><tr><td>VECTASHIELD mounting medium with DAPI</td><td>Vector Laboratories</td><td>H-1000</td><td>With DAPI</td></tr><tr><td>Confocal Microscope</td><td>Carl Zeiss Meditex AG</td><td>Zeiss LSM 700</td><td>Confocol microscopy</td></tr></tbody></table>

synthesis of functionalized 10-nm polymer-coated gold particles for endothelium targeting and drug delivery

金纳米粒子 (AuNPs) 由于其大小、生物相容性和可修改的表面, 在医学研究中得到了广泛的应用。特异靶向和药物传递是这些 AuNPs 的一些应用, 但内皮细胞胞外基质的防御特性阻碍了粒子的摄取。为了解决这个问题, 我们描述了一种超金纳米颗粒的合成方法, 以改善血管的传递, 可定制的功能基团和聚合物长度作进一步的调整。该协议产生 2.5 nm AuNPs, 其上限为四 (羟甲基) 磷氯 (THPC)。在 AuNP 表面上用异功能聚乙二醇 (PEG) 取代 THPC, 可将水动力半径提高到 10.5 nm, 同时在表面上提供各种功能基团。该协议的最后一部分包括一个可选的荧光, 允许 AuNPs 在荧光下被可视化来跟踪纳米粒子的摄取。用透析和冻干法纯化和分离 AuNPs。这些荧光纳米粒子可以在体外和体内实验中可视化, 这是由于生物相容性的 PEG 涂层和荧光探针。此外, 这些纳米微粒的大小范围使他们成为探索糖的理想候选者而不干扰正常的血管功能, 这可能导致改进的分娩和治疗。

合成的 AuNPs, 涂有 THPC 或 PEG (图 1A和 图 1B分别), 是用 tem 成像的, 粒子大小是用 tem 和 dl 来测量的, 以确保纳米粒度的分布。图 2显示了 THPC-AuNP 样品在 80 kV 和150,000x 放大率下的 TEM 图像。THPC-AuNP 粒子的直径从2-3 纳米, 基于 TEM 图像中的校准条。在图 2中显示的 dl 大小测量直方图中, 这种 THPC-AuNP 大小也很明显, 其中 THPC 涂层 AuNP 显示出峰值在 2.5 nm。聚在 TEM 下不可见, 因为聚合物不是电子致密的。TEM 成像的 peg-AuNP 样品简单地证实存在个别分散粒子, 这是预期的, 因为 peg 聚合物电晕周围的纳米粒子的援助, 防止聚集。对于这些 PEG-AuNP 样本, dl 大小测量直方图显示在峰值的变化, 到约 10.5 nm 的 dl。将进一步的配体或药物附着在功能基团上, 也会影响纳米颗粒的大小, 在测量直径时应考虑到。
荧光加法 (图 1C) 通过使用荧光测量纳米粒子的荧光信号来确认, 如图 3所示。当激发与波长为 633 nm, 发射被测量有最大在660和672毫微米之间, 与制造商的产品信息匹配最大放射在665毫微米。应以类似的方式检查其他荧光探针的附着, 以确保荧光反应。
用 mts 法测定了 16 h 孵化后不同浓度的纳米粒子的相对细胞代谢情况, 对颗粒的生物相容性和相关细胞活性进行了评估。荧光共轭乙二醇 AuNPs 没有明显的毒性, 如在所有浓度高达1毫克/毫升 (图 4A) 的细胞活力的类似水平表明。这种生物相容性可能会改变, 取决于治疗或附加到其余功能基团的配体进一步定制的粒子。药物附着物往往会增加毒性, 但根据工作浓度的不同, 它可能不会对生存能力产生显著影响。
通过粘附细胞摄取荧光、乙二醇 AuNPs (图 1C), 使用具有完整或功能失调的糖 (图 4B) 的培养的大鼠脂肪垫内皮细胞进行评估。功能失调的糖条件是通过添加酶 III 酶的文化, 导致素硫酸盐糖成分的退化和损害矩阵12。图 4B显示了 AuNPs 的不同摄取水平, 即代表内皮细胞横断面上的红点。一个健康的糖阻止摄取这些金纳米颗粒, 但当酶被使用11时观察到大量的增加。这一结果突出了这些超纳米粒子的潜能, 以一种以糖健康为基础的不同相互作用控制的内皮细胞提供治疗方法。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56760/56760fig1.jpg"> 图 1: 金纳米粒子示意图(A) THPC 在 PEG 置换前涂上 AuNP 纳米。(B) 乙二醇 AuNP 具有3种类型的 PEG 终端, 包括羧基、NH2和 CH3。蓝色波浪代表聚合物。(C) 用于荧光成像的共轭纳米粒子。红星显示荧光共轭到 NH2功能组。<a href="//cloudflare.jove.com/files/ftp_upload/56760/56760fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56760/56760fig2.jpg"> 图 2: 金纳米颗粒的尺寸测量左: 在 80 kV 和150,000X 放大前加入 PEG 的金纳米粒子的 TEM, 显示了刻度条。右: 在 (AuNP) 和后 (peg-AuNP) THPC 置换与 peg 测量的纳米粒大小的直方图。THPC 涂层 AuNP 的 dl 结果量化了 TEM 的可视化。聚乙二醇涂层 AuNP 的结果克服了无法通过 TEM 对 peg 进行可视化的挑战, 因为聚合物不是电子致密的。AuNP 的 TEM 将只显示核心的金纳米粒子, 看起来和 THPC 的 AuNP 一样。<a href="//cloudflare.jove.com/files/ftp_upload/56760/56760fig2large.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/56760/56760fig3.jpg"> 图 3: 荧光钉 AuNP 的萤光数据.荧光峰在 667 nm 匹配的发射荧光共轭 PEG AuNP。a.u: 任意单位。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56760/56760fig4.jpg"> 图 4: 与荧光 PEG AuNP 的细胞相互作用.(A) 细胞活力 (MTS 代谢) 阴谋的大鼠脂肪垫内皮细胞后 16 h co-incubation 与荧光 PEG-AuNP。(B) 固定大鼠脂肪垫内皮细胞的共焦横断面图像, 染色 DAPI 为核 (蓝色) 和抗体抗素硫酸, 糖成分 (绿色)。顶部图像是一个健康的糖和底部是一个退化的糖层;样品中的纳米粒子具有明显的红色荧光, 其降解糖。缩放条是10µm.<a href="//cloudflare.jove.com/files/ftp_upload/56760/56760fig4large.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 Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery at JoVE.com

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

我们描述了一种合成具有生物相容性的10纳米金纳米粒子的方法, 其功能是在表面上涂上聚乙二醇。这些微粒可以使用在体外和在体内提供治疗的纳米细胞和细胞外空间, 难以获得常规纳米颗粒的大小。

用于内皮靶向和药物传递的功能化10纳米高分子涂层金粒子的合成

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting ...

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JoVE Journal

Bioengineering

12.5K 次观看.  Northeastern University. 我们描述了一种合成具有生物相容性的10纳米金纳米粒子的方法, 其功能是在表面上涂上聚乙二醇。这些微粒可以使用在体外和在体内提供治疗的纳米细胞和细胞外空间, 难以获得常规纳米颗粒的大小。

视频: Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Template Directed Synthesis of Plasmonic Gold Nanotubes with Tunable IR Absorbance

A nearly parallel array of pores can be produced by anodizing aluminum foils in acidic environments1, 2. Applications of anodic aluminum oxide (AAO) membranes have been under development since the 1990's and have become a common method to template the synthesis of high aspect ratio nanostructures, mostly by electrochemical growth or pore-wetting. Recently, these membranes have become commercially available in a wide range of pore sizes and densities, leading to an extensive library of functional nanostructures being synthesized from AAO membranes. These include composite nanorods, nanowires and nanotubes made of metals, inorganic materials or polymers 3-10. Nanoporous membranes have been used to synthesize nanoparticle and nanotube arrays that perform well as refractive index sensors, plasmonic biosensors, or surface enhanced Raman spectroscopy (SERS) substrates 11-16, as well as a wide range of other fields such as photo-thermal heating 17, permselective transport 18, 19, catalysis 20, microfluidics 21, and electrochemical sensing 22, 23. Here, we report a novel procedure to prepare gold nanotubes in AAO membranes. Hollow nanostructures have potential application in plasmonic and SERS sensing, and we anticipate these gold nanotubes will allow for high sensitivity and strong plasmon signals, arising from decreased material dampening 15.

Solution-suspendable gold nanotubes with controlled dimensions can be synthesized by electrochemical deposition in porous anodic aluminum oxide (AAO) membranes using a hydrophobic polymer core. Gold nanotubes and nanotube arrays hold promise for applications in plasmonic biosensing, surface-enhanced Raman spectroscopy, photo-thermal heating, ionic and molecular transport, microfluidics, catalysis and electrochemical sensing.

的模板导演电浆黄金纳米管的合成与可调谐红外吸收

A nearly parallel array of pores can be produced by anodizing aluminum foils in acidic environments1, 2. Applications of anodic aluminum oxide (AAO) ...

科研

化学

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 ...

生物工程

Synthesis, Characterization, and Functionalization of Hybrid Au/CdS and Au/ZnS Core/Shell Nanoparticles

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large extinction coefficients which can be tuned across the visible spectrum. Research into the plasmonic enhancement of optical transitions has become popular, due to the possibility of altering and in some cases improving photo-absorption or emission properties of nearby chromophores such as molecular dyes or quantum dots. The electric field of the plasmon can couple with the excitation dipole of a chromophore, perturbing the electronic states involved in the transition and leading to increased absorption and emission rates. These enhancements can also be negated at close distances by energy transfer mechanism, making the spatial arrangement of the two species critical. Ultimately, enhancement of light harvesting efficiency in plasmonic solar cells could lead to thinner and, therefore, lower cost devices. The development of hybrid core/shell particles could offer a solution to this issue. The addition of a dielectric spacer between a gold nanoparticles and a chromophore is the proposed method to control the exciton plasmon coupling strength and thereby balance losses with the plasmonic gains. A detailed procedure for the coating of gold nanoparticles with CdS and ZnS semiconductor shells is presented. The nanoparticles show high uniformity with size control in both the core gold particles and shell species allowing for a more accurate investigation into the plasmonic enhancement of external chromophores.

The synthesis of uniform gold nanoparticles coated with semiconductor shells of CdS or ZnS is performed. The semiconductor coating is conducted by first depositing a silver sulfide shell and exchanging the silver cations for zinc or cadmium cations.

合成，表征和混合金/硫化镉和Au / ZnS核/壳纳米颗粒功能化

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large ...

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like oligomeric clusters of these yellow nanoparticles of narrow size distribution are synthesized under ambient conditions via two methods. The delay-time method controls the number of subunits in the oligoclusters by varying the time between the addition of HAuCl4 to alkaline solution and the subsequent addition of reducing agent, NaSCN. The yellow oligoclusters produced range in size from ~3 to ~25 nm. This size range can be further extended by an add-on method utilizing hydroxylated gold chloride (Na+[Au(OH4-x)Clx]-) to auto-catalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3 nm to 70 nm. The crude oligocluster preparations display narrow size distributions and do not require further fractionation for most purposes. The oligoclusters formed can be concentrated &#62;300 fold without aggregation and the crude reaction mixtures remain stable for weeks without further processing. Because these oligomeric clusters can be concentrated before derivatization they allow expensive derivatizing agents to be used economically. In addition, we present two models by which predictions of particle size can be made with great accuracy.

We describe a simple method for producing highly stable oligomeric clusters of gold nanoparticles via the reduction of chloroauric acid (HAuCl4) with sodium thiocyanate (NaSCN). The oligoclusters have a narrow size distribution and can be produced with a wide range of sizes and surface coats.

根据环境条件的金纳米粒子的稳定低聚簇的大小控制合成的一种简便方法

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like ...

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising approach to cancer therapy. The glycosaminoglycan placental chondroitin sulfate A (plCSA) is expressed on a wide range of cancer cells and placental trophoblasts, and malarial protein VAR2CSA can specifically bind to plCSA. A reported placental chondroitin sulfate A binding peptide (plCSA-BP), derived from malarial protein VAR2CSA, can also specifically bind to plCSA on cancer cells and placental trophoblasts. Hence, plCSA-BP-conjugated nanoparticles could be used as a tool for targeted drug delivery to human cancers and placental trophoblasts. In this protocol, we describe a method to synthesize plCSA-BP-conjugated lipid-polymer nanoparticles loaded with doxorubicin (plCSA-DNPs); the method consists of a single sonication step and bioconjugate techniques. In addition, several methods for characterizing plCSA-DNPs, including determining their physicochemical properties and cellular uptake by placental choriocarcinoma (JEG3) cells, are described.

Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles

Here, we present a protocol for the synthesis of placental chondroitin sulfate A binding peptide (plCSA-BP)-conjugated lipid-polymer nanoparticles via single-step sonication and bioconjugate techniques. These particles constitute a novel tool for the targeted delivery of therapeutics to most human tumors and placental trophoblasts to treat cancers and placental disorders.

胎盘硫酸软骨素 A (plCSA) 靶向脂质高分子纳米粒的合成与表征

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising ...

癌症研究

Synthesis and Characterization of Amphiphilic Gold Nanoparticles

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of their interactions with cell membranes, lipid bilayers, and viruses. The hydrophilic ligands make these particles colloidally stable in aqueous solutions and the combination with hydrophobic ligands creates an amphiphilic particle that can be loaded with hydrophobic drugs, fuse with the lipid membranes, and resist nonspecific protein adsorption. Many of these properties depend on nanoparticle size and the composition of the ligand shell. It is, therefore, crucial to have a reproducible synthetic method and reliable characterization techniques that allow the determination of nanoparticle properties and the ligand shell composition. Here, a one-phase chemical reduction, followed by a thorough purification to synthesize these nanoparticles with diameters below 5 nm, is presented. The ratio between the two ligands on the surface of the nanoparticle can be tuned through their stoichiometric ratio used during synthesis. We demonstrate how various routine techniques, such as transmission electron microscopy (TEM), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and ultraviolet-visible (UV-Vis) spectrometry, are combined to comprehensively characterize the physicochemical parameters of the nanoparticles.

Amphiphilic gold nanoparticles can be used in many biological applications. A protocol to synthesize gold nanoparticles coated by a binary mixture of ligands and a detailed characterization of these particles is presented.

两栖金纳米粒子的合成与表征

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

含有金纳米棒蜂窝车辆的制备及光声分析

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

合成阿普塔默-PEI-g-PEG改性金纳米粒子装载多索鲁比辛用于靶向药物输送

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...

Gold Nanoparticle Synthesis

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of tetrachloroauric acid in 3.0 g (3.7 mmol, 3.6 mL) of oleylamine (technical grade) and 3.0 mL of toluene into a boiling solution of 5.1 g (6.4 mmol, 8.7 mL) of oleylamine in 147 mL of toluene. While boiling and mixing the reaction solution for 2 hours, the color of the reaction mixture changed from clear, to light yellow, to light pink, and then slowly to dark red. The heat was then turned off, and the solution was allowed to gradually cool down to room temperature for 1 hour. The gold nanoparticles were then collected and separated from the solution using a centrifuge and washed three times; by vortexing and dispersing the gold nanoparticles in 10 mL portions of toluene, and then precipitating the gold nanoparticles by adding 40 mL portions of methanol and spinning them in a centrifuge. The solution was then decanted to remove any remaining byproducts and unreacted starting materials. Drying the gold nanoparticles in a vacuum environment produced a solid black pellet; which could be stored for long periods of time (up to one year) for later use, and then redissolved in organic solvents such as toluene.

A protocol for synthesizing ~12 nm diameter gold nanoparticles (Au nanoparticles) in an organic solvent is presented. The gold nanoparticles are capped with oleylamine ligands to prevent agglomeration. The gold nanoparticles are soluble in organic solvents such as toluene.

金纳米粒子合成

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of ...

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKC&#948;i. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

金纳米粒子的蛋白质激酶 c-三角洲抑制剂肽配方

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a ...