作者要感谢南加州大学, 国家心脏, 肺和血液研究所 (aha), R00HL124279, Eli 和伊蒂丝广泛创新奖的财政支持, 以及力惠蒂尔基金会非癌症翻译研究奖授予 EJC。作者感谢中心的电子显微镜和微量分析, 卓越中心的 NanoBiophysics, 卓越中心的分子特性, 和翻译成像中心在南加州大学的援助仪器设置。

注: 阅读试剂的 MSDS, 并按照当地机构的要求, 遵守所有化学安全措施。
1. MCP-1 PAMs 的制备
<ol><li>
MCP-1 肽的制备
	<ol>
<li>在反应容器 (RV) 中, 称 n-芴甲氧羰基-l-赖氨酸 (Boc)-摩尔0.25。用化学油烟机冲洗5毫升 dimethylforamide (DMF) 的 RV 侧面。</li>
		<li>将 RV 加载到自动台式多肽合成器上。载入预包装的氨基酸瓶, n "-CYNFTINRKISVQRLASYRRITSS-c", 从 c 到 N 末端。在最后的脱步骤中包括一个空瓶子。 注意: 一个序列, N "-CNNSIIRSKQVLRSTRYFRSATYK-C" 的炒多肽, 也可以使用相同的协议合成。</li>
		<li>一旦合成完成, 转移到树脂的多肽从 RV 到闪烁瓶与2-3 毫升甲醇, 直到所有的树脂转移。在树脂沉到底部后, 除去甲醇上清液, 蒸发剩余的直至干燥。真空干燥额外的2小时或隔夜在室温下。</li>
		<li>制作12毫升解理液含 trifluoracetic 酸 (TFA): 乙: 水: triisopropylsilane 在 94:2. 5:2. 5:1% 在化学油烟罩中。旋流的裂解液, 以确保它是均匀混合。 警告: 由于 triisopropylsilane 和乙是敏, 氩清除库存瓶的 triisopropylsilane 和乙1分钟之前, 把他们回来。</li>
		<li>将合成容器 (sv) 放在靠近 SV 的下半部分的位置上。转移肽树脂到 SV 与2毫升的裂解液, 直到所有的肽树脂转移。</li>
		<li>用3毫升解理液清洗 SV 的两侧。关闭 SV 的盖子, 在300振荡/分钟晃动4小时。</li>
		<li>称量出一个空的50毫升离心管。记录质量。</li>
		<li>4小时后, 将从 SV 的裂解肽溶液排出到50毫升离心管中。用剩余的溶液冲洗几次 SV 。</li>
		<li>蒸发裂解肽溶液与氮气, 直到有少于4毫升剩余在离心管。</li>
		<li>将36毫升的 ice-cold 醚加入到多肽裂解溶液中。</li>
		<li>漩涡的解决方案, 直到它是完全白色或黄色。离心机在 3000 x g 为5分钟在4° c, 和醒酒上清液。</li>
		<li>加入40毫升 ice-cold 醚的管。漩涡和几种再次。重复离心过程。醒酒上清。</li>
		<li>用氮气气体吹干粗 PA 颗粒, 直到醚明显蒸发。</li>
		<li>加入20毫升双水, 漩涡和几种, 直到肽完全溶解在溶液中。</li>
		<li>冷冻溶液, lyophilize 直至干燥。</li>
		<li>一旦该肽被晒干, 称量出含有该粗肽的离心管的质量。将粗肽溶于5毫克/毫升水中。</li>
		<li>在5毫升双水中溶解25毫克的粗 MCP-1 肽, 使总浓度为5毫克/毫升。用高效液相色谱 (HPLC) 纯化 MCP-1 肽, 用 0.1% TFA 水 (溶剂 a) 和 0.1% TFA 在乙腈 (溶剂 B) 中作为流动相在 C8 反相柱上。将5毫升的肽注入 HPLC。 注: 初始流动阶段由100% 溶剂 a 组成, 流速为9.0 毫升/分钟. 减少溶剂 a 慢慢地到30% 在 27 min 并且举行在这个构成为 3 min. 返回梯度到100% 溶剂 a 在 3 min 并且举行在这个构成为 1 min 给总运行时34分钟保持柱温在55° c。使用正离子源模式进行分析。如果 TFA 对 HPLC 柱的酸性太强, 可用于代替 TFA。建议有机溶剂组成的坡道速度不应超过 2%/分钟, 以便更好地分离。</li>
		<li>用基体辅助激光解吸/电离 (MALDI) 质谱分析每一个分数, 以达到预期产品的2890。 注: 在乙腈/水 (50:50%) 中, 以1毫克/毫升溶解α-氰基-4-肉桂酸为基体溶液。点0.75 µL 的矩阵解到 MALDI 板上, 其次是0.75 µL 的每个分数。在500-5、000 Da 的质量范围内采用正反射离子模式进行分析。</li>
		<li>结合产品的分数, 吹灭乙腈, 冷冻, 和 lyophilize 纯化多肽直到干燥。</li>
	</ol>
</li>
	<li>
MCP-1 PA 的研制
	<ol>
<li>准备1.1 摩尔超过 12-硬脂酸-sn-磷脂-3-乙醇-N-[氨基 (聚乙二醇)-2000] DSPE-PEG (2000)-亚在单独的闪烁小瓶中的多肽。溶解在双水中的两个化合物, 使〜15毫克/毫升。几种15分钟或直到两个解决方案都完全清楚。 注: DSPE peg (2000)-Cy5 是用同一个协议合成的 DSPE peg (2000)-胺和 n-羟基琥珀酰亚氨酸酯官能化 Cy5, 除了步骤 1.2.3, 其中 pH 值的解决方案被调整到8.5。</li>
		<li>添加 DSPE-PEG (2000)-亚溶液的肽溶液。漩涡和几种。</li>
		<li>添加1米氢氧化钠滴 (1 µL) 的时间, 直到溶液的 pH 值是7。</li>
		<li>氮气净化液5分钟搅拌溶液过夜。</li>
		<li>冷冻溶液, lyophilize 直至干燥。</li>
		<li>一旦原油干燥, 溶解固体在5毫克/毫升水。</li>
		<li>用高效液相色谱法纯化。 注: 游离肽和 DSPE (2000) 应洗在15-30% 有机浓度之间 (%), 而 DSPE-peg (2000)-MCP-1 共轭应在40-50% 有机浓度之间洗。</li>
		<li>用 MALDI 质谱法分析对应的 m/z 的每个分数. DSPE-PEG (2000)-MCP-1 应该有一个宽的 m/z 峰值在5760。 注:25-苯甲酸酸最适用于 DSPE-PEG (2000)-MCP-1 与 MALDI 的矩阵。</li>
	</ol>
</li>
	<li>
MCP-1 PAMs 的组装
	<ol>
<li>用3毫升甲醇在 1 dram 瓶中溶解1.75 毫克 MCP-1 PAs。几种直到 MCP-1 PA 完全溶解。 注: Cy5 分子可并入 10:90 MCP-1 PA 的摩尔比率, 用于成像应用。</li>
		<li>将甲醇在氮气下以圆周运动的形式蒸发, 同时冲洗瓶子一侧的剩余甲醇, 直到瓶底形成均匀的薄膜。真空干燥这部电影过夜。</li>
		<li>用3毫升的水或磷酸盐缓冲盐水 (PBS) 将薄膜水合成100的 MCP-1 PAMs 的µM 溶液。轻轻的漩涡和几种的解决方案, 直到清除。孵育在80° c 为30分钟。 注: 升高的温度已显示加速自组装过程, 并允许肽组分形成最稳定的二级结构, 同时降低临界胶束浓度 (CMC)42,43。</li>
		<li>冷却结果分散到室温。</li>
	</ol>
</li>
</ol>2. MCP-1 PAMs 的表征
<ol><li>
动态光散射 (dl) 和泽塔电位
	<ol>
<li>根据 dl 或泽塔电位仪的指示, 在试管中放置100µM MCP-1 pam 或炒 pam。 注: dl 和泽塔电位试管可以根据仪器的指示不同而有所不同。</li>
		<li>将仪器平衡到室温。测量 PAM 的尺寸和表面电荷。</li>
	</ol>
</li>
	<li>
透射电子显微镜 (TEM)
	<ol>
<li>添加7µL 50 µM MCP-1 pam 或炒 pam 到一个 TEM 网格悬浮在一个自动关闭的镊子为5分钟灯芯远离的水滴与一个微妙的任务擦拭。</li>
		<li>加7µL 水清洗网格。灯芯远离滴。</li>
		<li>添加7µL 1% 磷钨酸2分钟灯芯远离滴。重复步骤2.2.2。</li>
		<li>在 tem 分析之前, 先将 tem 网格烘干。</li>
	</ol>
</li>
	<li>
圆形二 (CD) 光谱学
	<ol>
<li>在使用仪器前, 打开氮气5-10 分钟。</li>
		<li>将300µL 的空白 (水或 PBS) 放在试管。</li>
		<li>测量光谱在190-265 毫微米, 0.2 毫米路径长度以 1 s 集成时间和1毫微米带宽在室温下。收集3复制。</li>
		<li>测量100µM MCP-1 肽, MCP-1 pam, 炒多肽, 并在步骤2.3.3 描述的相同条件下的炒 pam。从空白处减去。</li>
		<li>使用赖氨酸基谱的线性插值来拟合数据。</li>
		<li>关掉仪器在关闭氮气之前等待10分钟。</li>
	</ol>
</li>
</ol>3.体外MCP-1 PAMs 的分析
<ol><li>
体外生物相容性
	<ol>
<li>培养和扩展 WEHI 274.1 小鼠单核细胞在 Dulbecco 的改良鹰培养基中补充10% 胎牛血清 (FBS), 1% 青霉素-链霉素, 和0.05 毫米 2-基在37° c 以下 5% CO2到段落5。 注: WEHI 274.1 是悬浮细胞。当培养时, 媒体应每2-3 天补充一次。</li>
		<li>吸管单核细胞在培养基中变成无菌50毫升离心管。离心机在 300 x g 为5分钟在4° c。</li>
		<li>吸取旧媒体和重在10毫升新鲜媒体进入离心管。慢慢混合, 直到细胞均匀分布在介质中。使用台盼蓝染色和例计数细胞。</li>
		<li>稀释的细胞, 使每90µL 的介质有4000单核。在96孔板的井中加入90µL 的细胞。孵育24小时。 注意: 避免在板的外边缘使用水井, 以避免在蒸发和板热变化的差异。用100µL PBS 填充外部井。</li>
		<li>将0.15 µmol MCP-1 肽, MCP-1 pam, 炒多肽, 或炒 pam 在150µL PBS 在单独的, 灭菌的离心管中溶解。执行连续稀释, 使每个样品的 1, 10 和100µM 的65µL。</li>
		<li>在含 WEHI 274.1 的油井中加入10µL PBS (总体积: 100 µL, 6 条线, 96 个总井)。孵育72小时。</li>
		<li>每井添加10µL (3-(45-dimethylthiazol-2-基)-5-(3-carboxymethoxyphenyl)-2-(4-磺)-2 h-唑) (MTS)。孵育1小时。</li>
		<li>使用 490 nm 的平板阅读器或根据制造商的说明分析吸光度。</li>
	</ol>
</li>
	<li>
体外胶束绑定
	<ol>
<li>种子50万单核细胞在每6个井板2毫升的培养基中含有100µM Cy5-labeled MCP-1 PAM (10:90 摩尔比 DSPE-PEG (2000)-Cy5 和 MCP-1 PA)。孵育1小时。</li>
		<li>收集 WEHI 274.1 由离心 300 x g 为5分钟在4° c。吸媒体。</li>
		<li>在2毫升 PBS 中重和冲洗细胞。离心机在 300 x g 为5分钟在4° c。吸入 PBS重复洗涤过程与2毫升 PBS。吸入 PBS</li>
		<li>加入2毫升的冷4% 甲醛修复细胞。室温孵育10分钟。</li>
		<li>将固定细胞吸管在一个6孔板的井内的无菌玻璃片上。离心机在 400 x g 为5分钟。</li>
		<li>删除甲醛。用2毫升 PBS 清洗和冲洗一次。重1毫升 PB 注意: 冲洗时, 避免干扰片上的细胞。</li>
		<li>将10µL 90% 甘油放在 PBS 的一张幻灯片上。用针和镊子拿起片。擦干片的边缘。轻轻地把片放到一张朝下的滑梯上。</li>
		<li>用清晰的指甲油轻轻封住片的边缘。放置在冰箱中, 直到准备好成像。</li>
		<li>使用共焦激光扫描显微镜 (CLSM) 安装与激光器为 Cy5 在λ励磁= 650 nm。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

MCP-1 PAMs 是一个充满希望的分子成像平台, 包括一个亲水性靶向肽和疏水尾巴, 驱动自组装性质的纳米颗粒。这种单核细胞靶向胶束可以通过简单的合成和纯化步骤 MCP-1 肽和 DSPE-PEG (2000)-MCP-1。PAMs 有许多有益的特征为在体内分子成像, 如他们的自组装在温和的条件下, 内在生物降解性, 和结构和化学多样性允许纳入其他成像基或靶向多肽选择性地传递到特定的感兴趣的地点。它们的粒度、形状...

心血管疾病仍然是全球死亡的主要原因, 全世界约有1730万人死亡,1。心血管疾病是由动脉粥样硬化造成的, 一种情况是斑块在动脉中形成, 从而抑制血液和氧气流向身体的细胞2,3。动脉粥样硬化的进展是由炎症反应、不规则脂代谢和斑块堆积引起的动脉增厚和硬化, 导致斑块破裂和心肌梗死4,5。内皮细胞表达细胞因子和黏附分子, 其中包括 MCP-1, 结合到 c-c 趋化因子受体 (CCR2) 的表面发现单核的6,7,8。氧化胆固醇在斑块形成的早期将单核细胞转化为巨噬生物, 从而放大了该地区的炎症反应, 导致组织损伤, 形成不稳定或易受伤害的斑块9,10。
传统上, 动脉粥样硬化是评估腔狭窄的解剖成像使用血管造影或超声11,12。然而, 这些方法只能确定严重的动脉壁狭窄, 而不是动脉粥样硬化的早期阶段, 因为最初的斑块生长导致动脉重塑, 以维持动脉大小和血流率12,13, 14。因此, 造影 underrepresent 动脉粥样硬化的患病率。此外, 非侵入性成像技术, 如单光子发射计算机断层扫描, 正电子发射断层扫描和磁共振成像最近被用来表征斑块形态, 因为它们可以提供初步的细节和斑块的特征。然而, 这些模式往往受限于缺乏灵敏度, 空间分辨率, 或要求使用电离辐射, 使成像斑块进展在不同阶段更具挑战性的15,16, 17。一个成像传递系统, 将专门确定斑块在不同阶段的动脉粥样硬化仍有待开发。
纳米粒子已显示为体内斑块靶和诊断的一个新兴平台18,19,20,21。特别是, PAMs 是有利的, 因为他们的化学多样性和能力, 以适应各种基, 组成, 大小, 形状和表面功能化22。多肽分子 (PAs) 由亲水性, 多肽 "headgroup" 附着在疏水的尾巴上, 通常是脂质;这两亲结构赋予自功能, 并允许在粒子的表面上的多肽的多显示22,23,24。肽 headgroups 可通过折叠和多肽25之间的氢键作用影响颗粒形状。通过β-片相互作用折叠的多肽已经被证明形成拉长的胶束, 而α-螺旋确认可以形成球形和拉长的胶束22,23,24, 25,26,27。聚乙二醇 (PEG) 连接, 屏蔽了肽的表面电荷可以放在亲水性肽和 PAMs 的疏水尾之间, 增强了纳米颗粒在系统循环中的可用性28,29,30,31. PAMs 也很有利, 因为它们具有生物相容性, 并被证明具有广泛的应用范围3233。胶束的水溶性比其他纳米系统的优势, 如某些高分子纳米颗粒, 不溶于水, 必须在增悬浮在注射剂34。此外, 创建 PAMs 的能力, 以响应特定的刺激, 使 PAMs 一个有吸引力的候选人, 控制细胞内药物传递35。
通过结合 CCR2 受体和积累在主动脉弓, PAMs 以前开发的单核细胞靶向监测不同阶段的动脉粥样硬化病变的主动脉9。在载脂蛋白-/-小鼠中, 单核细胞积累成正比地增加到斑块级数36。此外, 发现有易破裂的晚期斑块的患者有较高的单核细胞数量37。因此, 对 PAMs 的修改, 以纳入 MCP-1 是有用的, 因为它允许更大的目标特异性和分化的早期和晚期动脉粥样硬化病变。这些概念验证研究还证实了 PAMs 是足够安全的, 可以使用 pre-clinically 并清除 renally38。由于单核细胞和炎症的特点, 其他疾病, MCP-1 PAMs 有潜力用于治疗和诊断的应用以外的其他疾病的动脉粥样硬化8,39,40,41。
在此, 我们报告了高可伸缩性和自组装 MCP-1 PAMs 的制作, 证明了粒子的最佳大小, 表面电荷, 选择性靶向单核细胞的增强成像应用在动脉粥样硬化。

<table border="0" cellpadding="0" cellspacing="0" width="834">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">1,2-ethanedithiol</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">E0032</td>
			<td style="width:259px;">for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 mL disposable serological pipets</td>
			<td>VWR</td>
			<td>89130-898</td>
			<td>for cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL centrifuge tubes, polypropylene</td>
			<td>VWR</td>
			<td>89401-566</td>
			<td>for various applications</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2,5-dihydroxybenzoic acid, 99%</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>AC165200050</td>
			<td>for MALDI</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">25 mL disposable serological pipets</td>
			<td>VWR</td>
			<td>89130-900</td>
			<td>for cell culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">2-Mercaptoethanol, 50 mM</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">31350010</td>
			<td>for cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5 mL disposable serological pipets</td>
			<td>VWR</td>
			<td>89130-896</td>
			<td>for cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 mL centrifuge tubes</td>
			<td>VWR</td>
			<td>89039-658</td>
			<td>for various applications</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">75 cm2 culture flask</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:119px;">13-680-65</td>
			<td>for cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">75 mL reaction vessel</td>
			<td>Protein Technologies</td>
			<td>3000005</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-wells cell culture plate</td>
			<td style="width:147px;">VWR</td>
			<td>40101-346</td>
			<td>for MTS assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile, HPLC grade</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>A998SK-4</td>
			<td>for HPLC purification</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Borosilicate glass, 1 dram</td>
			<td style="width:147px;">VWR</td>
			<td>66011-041</td>
			<td>for PAM synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Borosillicate glass pipet, Long tips</td>
			<td style="width:147px;">VWR</td>
			<td>14673-043</td>
			<td>for various applications</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coverslip, 0.16-0.19 mm, 22 x 22 mm</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>12-542B</td>
			<td>for confocal microscopy</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Cy5 amine</td>
			<td style="width:147px;">Abcam</td>
			<td style="width:119px;">ab146463</td>
			<td>for peptide conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Diethyl ether, ACS grade</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:119px;">E138-1</td>
			<td>for peptide precipitation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Disposable syringes, 20 mL</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>14-817-54</td>
			<td>for HPLC purification</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Double neubauer ruled hemocytometer</td>
			<td style="width:147px;">VWR</td>
			<td>63510-13</td>
			<td>for cell counting</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">DSPE-PEG(2000) amine</td>
			<td>Avanti</td>
			<td style="width:119px;">880128P</td>
			<td>for peptide conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">DSPE-PEG(2000) maleimide</td>
			<td>Avanti</td>
			<td style="width:119px;">880126P</td>
			<td>for peptide conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DSPE-PEG(2000)-NHS ester&#160;</td>
			<td>Nanocs</td>
			<td>PG2-DSNS-10K</td>
			<td>for conjugation to Cy5</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Dulbecco&#39;s modified eagle medium-high glucose</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">D5796-500ML</td>
			<td>for cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum, qualified, heat inactivated</td>
			<td>ThermoFisher Scientific</td>
			<td>10438026</td>
			<td>for cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Ala-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td>PS3-H5-A</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Arg(Pbf)-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td style="width:119px;">PS3-H5-RBF</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Asn(Trt)-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td style="width:119px;">PS3-H5-NT</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Cys(Trt)-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td style="width:119px;">PS3-H5-CT</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Gln(Trt)-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td>PS3-H5-QT</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Ile-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td style="width:119px;">PS3-H5-I</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Leu-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td>PS3-H5-L</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Lys(Boc)-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td>PS3-H5-KBC</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Phe-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td style="width:119px;">PS3-H5-F</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Ser(tBu)-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td style="width:119px;">PS3-H5-SB</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Thr(tBu)-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td>PS3-H5-TB</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Tyr(tBu)-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td>PS3-H5-YB</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fmoc-L-Val-OH /HBTU</td>
			<td>Protein Technologies</td>
			<td>PS3-H5-V</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Fmoc-Lys(Boc)-wang resin, 100-200 mesh</td>
			<td>Novabiochem</td>
			<td style="width:119px;">856013</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid, optima LC/MS grade</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>A117-50</td>
			<td>for HPLC purification</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Glycerol</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">M152-1L</td>
			<td>for confocal microscopy</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hand tally counter</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>S90189</td>
			<td>for cell counting</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnetic stir bars, egg-shaped</td>
			<td style="width:147px;">VWR</td>
			<td>58949-006</td>
			<td>for peptide conjugation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol, ACS certified</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>A412-4</td>
			<td>for PAM synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MTS cell proliferation colorimetric assay kit</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">10191-104</td>
			<td>for MTS assay</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N,N-Dimethylformamide, sequencing grade</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>BP1160-4</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-Methylmorpholine</td>
			<td>Protein Technologies</td>
			<td>S-1L-NMM</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Paraformaldehyde</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:119px;">AC416780250</td>
			<td>for fixing cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PBS, pH 7.4</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td>10010049</td>
			<td>for various applications</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/streptomycin, 10,000 U/mL</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td>for cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Peptide synthesis vessel, 25 mL&#160;</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:119px;">CG186011</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Phosphotungstic acid&#160;</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:119px;">A248-25</td>
			<td>for TEM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Piperidine</td>
			<td>Spectrum</td>
			<td>P1146-2.5LTGL</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plain glass microscope slide 75 x 25 mm</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>12-550-A3</td>
			<td>for confocal microscopy</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reagent reservoirs, sterile&#160;</td>
			<td>VWR</td>
			<td>95128093</td>
			<td>for cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Self-closing tweezer</td>
			<td style="width:147px;">TedPella</td>
			<td style="width:119px;">515</td>
			<td>for TEM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TEM support film</td>
			<td style="width:147px;">TedPella</td>
			<td style="width:119px;">01814F</td>
			<td>for TEM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trifluoroacetic acid&#160;</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>BP618-500</td>
			<td>for peptide cleavage and HPLC purification</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triisopropylsilane</td>
			<td style="width:147px;">VWR</td>
			<td>TCT1533-5ml</td>
			<td>for peptide cleavage</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trypan blue solution, 0.4%</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">15250061</td>
			<td>for cell counting</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tweezer, general purpose-serrated</td>
			<td style="width:147px;">VWR</td>
			<td>231-SA-SE</td>
			<td>for confocal microscopy</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">WEHI-274.1</td>
			<td>ATCC</td>
			<td style="width:119px;">ATCC&#160;CRL-1679</td>
			<td>murine monocyte</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">automated benchtop peptide synthesizer</td>
			<td>Protein Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:259px;">PS3 Benchtop Peptide Synthesizer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#945;- cyano- 4- hydroxycinnamic acid, 99%</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td>476870-2G</td>
			<td>for MALDI</td>
		</tr>
	</tbody>
</table>

synthesis of monocyte-targeting peptide amphiphile micelles for imaging of atherosclerosis

动脉粥样硬化是心血管疾病的主要贡献者, 是全世界死亡的首要原因, 每年有1730万人丧生。动脉粥样硬化也是导致猝死和心肌梗塞的主要原因, 由不稳定的斑块引起, 破裂并咬合血管, 无预警。目前的成像模式不能区分稳定和不稳定的斑块破裂。多肽分子胶束 (PAMs) 可以克服这一缺点, 因为它们可以修改的各种靶向基, 具体绑定到病变的组织。单核细胞已被证明是动脉粥样硬化的早期标记物, 而单核细胞的大量积累与易破裂的斑块有关。因此, 能够靶向单核细胞的纳米粒子可以被用来鉴别动脉粥样硬化的不同阶段。为此, 在这里, 我们描述了一个协议为单核细胞靶向 PAMs (核细胞趋化蛋白 protein-1 (MCP-1) PAMs) 的制备。MCP-1 PAMs 是通过合成在温和条件下形成纳米颗粒的 15 nm 直径与近中性表面电荷。在体外中, PAMs 被发现具有生物相容性, 对单核细胞具有较高的亲和力. 本文所描述的方法对动脉粥样硬化以及其他炎症性疾病的广泛应用有广阔的前景。

MCP-1 PAM 的制备 MCP-1 蛋白 [YNFTNRKISVQRLASYRRITSSK] 或炒多肽 [YNSLVFRIRNSTQRKYRASIST] 的 CCR2-binding 母题 (残留 13-35) 通过在 N 端部添加半胱氨酸残留物而改变。采用全自动肽合成器, 用甲氧羰基介导的固相法合成了 MCP-1 肽。用反相高效液相色谱法在50° c 的 C8 柱上用 0.1% TFA 在乙腈/水混合物中纯化了粗肽, 并以 MALDI 质谱 (图 1) 为特征。含半胱氨酸的...

Watch this Scientific Journal Video about Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis at JoVE.com

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

本文介绍了一种涉及单核细胞靶向肽 amphiphile 胶束的合成和表征方法, 并对胶束与单核细胞结合的生物相容性和能力进行了相应的测试。

单核靶向肽 Amphiphile 胶束合成动脉粥样硬化的研究

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

synthesis-monocyte-targeting-peptide-amphiphile-micelles-for-imaging

Research

JoVE Journal

Bioengineering

7.2K 次观看.  University of Southern California. 本文介绍了一种涉及单核细胞靶向肽 amphiphile 胶束的合成和表征方法, 并对胶束与单核细胞结合的生物相容性和能力进行了相应的测试。

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

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

在三维自组装多肽水凝胶的人类神经祖细胞的培养

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

科研

生物工程

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

宽带超声医疗成像应用，光学检测仪

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

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

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

基于角蛋白纳米纤维生物医学工程合成

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

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

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

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

动脉粥样硬化的快速检测的氧化铁纳米颗粒微波驱动综合

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

Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous environment through self-assembly. The hydrophobic core of micelles functions as a drug reservoir and encapsulates hydrophobic drugs. The hydrophilic shell prevents the aggregation of micelles and also prolongs their systemic circulation in vivo. In this protocol, we describe a method to synthesize a doxorubicin lipophilic pro-drug, doxorubicin-palmitic acid (DOX-PA), which will enhance drug loading into micelles. A pH-sensitive hydrazone linker was used to conjugate doxorubicin with the lipid, which facilitates the release of free doxorubicin inside cancer cells. Synthesized DOX-PA was purified with a silica gel column using dichloromethane/methanol as the eluent. Purified DOX-PA was analyzed with thin layer chromatography (TLC) and 1H-Nuclear Magnetic Resonance Spectroscopy (1H-NMR). A film dispersion method was used to prepare DOX-PA loaded DSPE-PEG micelles. In addition, several methods for characterizing micelle formulations are described, including determination of DOX-PA concentration and encapsulation efficiency, measurement of particle size and distribution, and assessment of in vitro anticancer activities. This protocol provides useful information regarding the preparation and characterization of drug-loaded micelles and thus will facilitate the research and development of novel micelle-based cancer nanomedicines.

A protocol for the preparation and characterization of lipophilic doxorubicin pro-drug loaded 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) micelles is described.

制备和表征亲脂阿霉素前药胶束

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

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

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

对细胞的超快磁化阳离子化Magnetoferritin的合成

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

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

水凝胶的合成与防污性能作为膜的净水

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Design and Synthesis of a Reconfigurable DNA Accordion Rack

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

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

可重构 DNA 手风琴架的设计与合成

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

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

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

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

在肽膜前体的维西图洛合成中,用于自主囊泡生长

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

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

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

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

生物应用近红外发光金纳米簇的合成

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