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、形状和组成可以通过改变肽、疏水尾或胶束浓度来调节, 从而为各种应用提供最佳的化学和物理特性22,33。
应提及本议定书的一些局限性。首先, 由于 PAMs 是由疏水相互作用驱动的, 所以有必要构建具有亲水性的靶向肽, 从而允许在胶束表面呈现靶向基团。如果肽序列是疏水的, 在 C 末端增加一个或两个亲水氨基酸可以容纳, 但氨基酸的加法可能影响多肽二级结构在胶束之内, 因此改变物产肽在它的本机状态在蛋白质44,45中。其次, 在低浓度时, PAMs 在水环境中的稳定性较差, 因为它们高度依赖 CMC, PAs 形成胶束结构所需的最低浓度为46。在 CMC 下面, 胶束分解为单个 PA 单体, 并限制胶束作为载体, 在特定的站点47上卸载药物。为了缓解这个缺点, CMC 可以通过增加疏水尾部的链长48,49来减少。最后, 不推荐 PAMs 在溶液中的长期贮存, 因为 PA 二次结构可以随时间变化, 从而影响胶束结构和稳定性, 以及配体结合的效率26。
总之, 该协议证明了一个健壮的, 自组装肽 micelle-based 纳米的成像动脉粥样硬化的策略。MCP-1 PAMs 是生物相容性的, 可降解的, 无毒的, 因为胶束的成分来自于体内的元素。更重要的是, MCP-1 PAMs 对单核细胞有优先的约束力, 这增加了与斑块进展的比例, 促进了非侵入性的方法来区分动脉粥样硬化斑块的分期。由于该平台的模块化, PAMs 可以纳入治疗, 额外的靶向肽, 成像基, 和核酸。因此, 鉴于这些多功能胶束的动态能力, 我们相信这些粒子对动脉粥样硬化和其他疾病的临床翻译有很大的希望。

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><tbody><tr><td>1,2-ethanedithiol</td><td>VWR</td><td>E0032</td><td>for peptide synthesis</td></tr><tr><td>10 mL disposable serological pipets</td><td>VWR</td><td>89130-898</td><td>for cell culture</td></tr><tr><td>15 mL centrifuge tubes, polypropylene</td><td>VWR</td><td>89401-566</td><td>for various applications</td></tr><tr><td>2,5-dihydroxybenzoic acid, 99%</td><td>Fisher Scientific</td><td>AC165200050</td><td>for MALDI</td></tr><tr><td>25 mL disposable serological pipets</td><td>VWR</td><td>89130-900</td><td>for cell culture</td></tr><tr><td>2-Mercaptoethanol, 50 mM</td><td>ThermoFisher Scientific</td><td>31350010</td><td>for cell culture</td></tr><tr><td>5 mL disposable serological pipets</td><td>VWR</td><td>89130-896</td><td>for cell culture</td></tr><tr><td>50 mL centrifuge tubes</td><td>VWR</td><td>89039-658</td><td>for various applications</td></tr><tr><td>75 cm2 culture flask</td><td>Fisher Scientific</td><td>13-680-65</td><td>for cell culture</td></tr><tr><td>75 mL reaction vessel</td><td>Protein Technologies</td><td>3000005</td><td>for peptide synthesis</td></tr><tr><td>96-wells cell culture plate</td><td>VWR</td><td>40101-346</td><td>for MTS assay</td></tr><tr><td>Acetonitrile, HPLC grade</td><td>Fisher Scientific</td><td>A998SK-4</td><td>for HPLC purification</td></tr><tr><td>Borosilicate glass, 1 dram</td><td>VWR</td><td>66011-041</td><td>for PAM synthesis</td></tr><tr><td>Borosillicate glass pipet, Long tips</td><td>VWR</td><td>14673-043</td><td>for various applications</td></tr><tr><td>Coverslip, 0.16-0.19 mm, 22 x 22 mm</td><td>Fisher Scientific</td><td>12-542B</td><td>for confocal microscopy</td></tr><tr><td>Cy5 amine</td><td>Abcam</td><td>ab146463</td><td>for peptide conjugation</td></tr><tr><td>Diethyl ether, ACS grade</td><td>Fisher Scientific</td><td>E138-1</td><td>for peptide precipitation</td></tr><tr><td>Disposable syringes, 20 mL</td><td>Fisher Scientific</td><td>14-817-54</td><td>for HPLC purification</td></tr><tr><td>Double neubauer ruled hemocytometer</td><td>VWR</td><td>63510-13</td><td>for cell counting</td></tr><tr><td>DSPE-PEG(2000) amine</td><td>Avanti</td><td>880128P</td><td>for peptide conjugation</td></tr><tr><td>DSPE-PEG(2000) maleimide</td><td>Avanti</td><td>880126P</td><td>for peptide conjugation</td></tr><tr><td>DSPE-PEG(2000)-NHS ester&amp;nbsp;</td><td>Nanocs</td><td>PG2-DSNS-10K</td><td>for conjugation to Cy5</td></tr><tr><td>Dulbecco&#039;s modified eagle medium-high glucose</td><td>Sigma Aldrich</td><td>D5796-500ML</td><td>for cell culture</td></tr><tr><td>Fetal bovine serum, qualified, heat inactivated</td><td>ThermoFisher Scientific</td><td>10438026</td><td>for cell culture</td></tr><tr><td>Fmoc-L-Ala-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-A</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Arg(Pbf)-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-RBF</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Asn(Trt)-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-NT</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Cys(Trt)-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-CT</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Gln(Trt)-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-QT</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Ile-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-I</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Leu-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-L</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Lys(Boc)-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-KBC</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Phe-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-F</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Ser(tBu)-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-SB</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Thr(tBu)-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-TB</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Tyr(tBu)-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-YB</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-L-Val-OH /HBTU</td><td>Protein Technologies</td><td>PS3-H5-V</td><td>for peptide synthesis</td></tr><tr><td>Fmoc-Lys(Boc)-wang resin, 100-200 mesh</td><td>Novabiochem</td><td>856013</td><td>for peptide synthesis</td></tr><tr><td>Formic acid, optima LC/MS grade</td><td>Fisher Scientific</td><td>A117-50</td><td>for HPLC purification</td></tr><tr><td>Glycerol</td><td>VWR</td><td>M152-1L</td><td>for confocal microscopy</td></tr><tr><td>Hand tally counter</td><td>Fisher Scientific</td><td>S90189</td><td>for cell counting</td></tr><tr><td>Magnetic stir bars, egg-shaped</td><td>VWR</td><td>58949-006</td><td>for peptide conjugation</td></tr><tr><td>Methanol, ACS certified</td><td>Fisher Scientific</td><td>A412-4</td><td>for PAM synthesis</td></tr><tr><td>MTS cell proliferation colorimetric assay kit</td><td>VWR</td><td>10191-104</td><td>for MTS assay</td></tr><tr><td>N,N-Dimethylformamide, sequencing grade</td><td>Fisher Scientific</td><td>BP1160-4</td><td>for peptide synthesis</td></tr><tr><td>N-Methylmorpholine</td><td>Protein Technologies</td><td>S-1L-NMM</td><td>for peptide synthesis</td></tr><tr><td>Paraformaldehyde</td><td>Fisher Scientific</td><td>AC416780250</td><td>for fixing cells</td></tr><tr><td>PBS, pH 7.4</td><td>ThermoFisher Scientific</td><td>10010049</td><td>for various applications</td></tr><tr><td>Penicillin/streptomycin, 10,000 U/mL</td><td>ThermoFisher Scientific</td><td>15140122</td><td>for cell culture</td></tr><tr><td>Peptide synthesis vessel, 25 mL&amp;nbsp;</td><td>Fisher Scientific</td><td>CG186011</td><td>for peptide synthesis</td></tr><tr><td>Phosphotungstic acid&amp;nbsp;</td><td>Fisher Scientific</td><td>A248-25</td><td>for TEM</td></tr><tr><td>Piperidine</td><td>Spectrum</td><td>P1146-2.5LTGL</td><td>for peptide synthesis</td></tr><tr><td>Plain glass microscope slide 75 x 25 mm</td><td>Fisher Scientific</td><td>12-550-A3</td><td>for confocal microscopy</td></tr><tr><td>Reagent reservoirs, sterile&amp;nbsp;</td><td>VWR</td><td>95128093</td><td>for cell culture</td></tr><tr><td>Self-closing tweezer</td><td>TedPella</td><td>515</td><td>for TEM</td></tr><tr><td>TEM support film</td><td>TedPella</td><td>01814F</td><td>for TEM</td></tr><tr><td>Trifluoroacetic acid&amp;nbsp;</td><td>Fisher Scientific</td><td>BP618-500</td><td>for peptide cleavage and HPLC purification</td></tr><tr><td>Triisopropylsilane</td><td>VWR</td><td>TCT1533-5ml</td><td>for peptide cleavage</td></tr><tr><td>Trypan blue solution, 0.4%</td><td>ThermoFisher Scientific</td><td>15250061</td><td>for cell counting</td></tr><tr><td>Tweezer, general purpose-serrated</td><td>VWR</td><td>231-SA-SE</td><td>for confocal microscopy</td></tr><tr><td>WEHI-274.1</td><td>ATCC</td><td>ATCC&amp;nbsp;CRL-1679</td><td>murine monocyte</td></tr><tr><td>automated benchtop peptide synthesizer</td><td>Protein Technologies</td><td></td><td>PS3 Benchtop Peptide Synthesizer</td></tr><tr><td>&amp;alpha;- cyano- 4- hydroxycinnamic acid, 99%</td><td>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。含半胱氨酸的肽被共轭到 DSPE-PEG2000-maleimide 上, 通过硫链的连接产生 DSPE-PEG (2000)-肽共轭物。在室温24小时后, 用 HPLC (图 2) 对原油进行提纯。DSPE-PEG (2000)-Cy5 是用琥珀酰亚胺酯反应合成的。单核细胞靶向 PAMs co-assembled 与 DSPE peg (2000)-MCP-1 和 DSPE-peg (2000)-Cy5 是由溶解在甲醇, 然后蒸发了 N2, 并产生的薄膜在真空干燥过夜和自组装在水或PBS 通过疏水相互作用的 "尾群" 形成 MCP-1 PAMs。
MCP-1 PAM 的表征 单核靶 PAMs 的 dl 数据和 TEM 图像显示分散, 直径为15.3 ± 2.0 nm 的球形纳米颗粒 (图 3和表 1)。两个 MCP-1 PAMs 和炒 PAMs 显示了轻微的正面泽塔电位12.1 ± 3.1 mv 和13.7 ± 1.9 mv, 分别 (表 1)。CD 显示, MCP-1 肽在胶束中的加入增强了二级结构 (表 2, MCP-1 PAMs: 46.2 ±0.9% β页, 53.1 ±1.4% 随机线圈, 自由 MCP1: 34.2 ±3.5% β页, 65.8 ±3.5% 随机线圈)。
体外MCP-1 PAM 分析 体外MCP-1 PAMs 的生物相容性和单核细胞结合的共聚焦显微镜观察。小鼠单核细胞 MCP-1, MCP-1 pam, 炒多肽, 和炒 pam 的72小时的浓度增加, 并发现其可存活和生物相容性高达100µM (图 4)。此外, 共聚焦显微镜显示单核细胞与 MCP-1 PAMs 的特异结合, 与炒 PAMs (图 5)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56625/56625fig1.jpg"> 图 1:HPLC 在 220 nm (肽键的波长) 和 254 nm (酪氨酸波长) 的 MCP-1 肽 (A) 和炒多肽 (B) (盒装峰值在 7.4 min)。MALDI MCP-1 肽 (C) 和炒多肽 (D) 在500-5、000 Da 的范围内的质量频谱, 显示在 m/z 2888 (预期 2890) 的 [m + H]+的峰值。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56625/56625fig1large.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/56625/56625fig2.jpg"> 图 2:HPLC 色谱在 220 nm (肽键的波长) 和 254 nm (酪氨酸波长) 的 DSPE peg (2000)-MCP-1 (A) 和 DSPE-peg (2000)-炒 (B)。(装箱峰值在16.9 分钟)。(C) MALDI-DSPE-PEG (2000)-MCP-1 的质量谱在 100万 Da 的范围内显示了 [M + H] 的峰值+在 m/z 2888 (预期 2890) 为 MCP-1 和 m/z 5758 (预期 5760) 为 DSPE-PEG (2000)-MCP-1。(D) MALDI-DSPE-PEG (2000) 的质量谱--在 100万 Da 的范围内进行炒, 显示 [M + H] 的峰值]+在 m/z 2887 (预计为 2890) 的炒多肽和 m/z 5761 (预期 5760) 为 DSPE-PEG-炒。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56625/56625fig2large.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/56625/56625fig3.jpg"> 图 3:MCP-1 pam (A) 和已炒 pam (B) 的 TEM 图像。缩放条形图 = 100 nm。数字-MCP-1 pam (C) 和通过 dl (D) 的已加密 pam 的平均大小分布。数据是平均±法令 (n = 3)。(E) CD 分析 MCP-1 和炒多肽和 PAMs (n = 3)。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56625/56625fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56625/56625fig4.jpg"> 图 4:体外WEHI 274.1 小鼠单核细胞存活72小时后接触 MCP-1 肽、MCP-1 pam、炒多肽和炒 pam.数据是平均±法令 (n = 6)。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56625/56625fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56625/56625fig5.jpg"> 图 5:CLSM MCP-1 pam (A) 和被炒的 pam (B) 的图像与10摩尔 Cy5 PA (红色) 一起孵化与 WEHI 274.1 为 1 h. 刻度栏 = 20 µm.<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56625/56625fig5large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>pams</td>
			<td>数字大道直径 (nm)</td>
			<td>pdi</td>
			<td>泽塔势 (mV)</td>
		</tr>
<tr>
<td>MCP-1</td>
			<td>15.3 ±2。0</td>
			<td>0.119 ±0.006</td>
			<td>12.1 ±3。1</td>
		</tr>
<tr>
<td>炒</td>
			<td>16.9 ±1。4</td>
			<td>0.232 ±0.019</td>
			<td>13.7 ±1。9</td>
		</tr>
<tr>
<td colspan="4">在 PBS 缓冲区中测量。数据以平均± SD 表示。</td>
		</tr>
</tbody></table>表 1:PAMs 的大小、多和泽塔电位.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td>a-螺旋 (%)</td>
			<td>b 页 (%)</td>
			<td>随机线圈 (%)</td>
		</tr>
<tr>
<td>MCP-1 肽</td>
			<td>0.0 ±0。0</td>
			<td>36.2 ±2。2</td>
			<td>63.8 ±1。8</td>
		</tr>
<tr>
<td>MCP-1 PAM</td>
			<td>0.7 ±0。6</td>
			<td>46.2 ±0。9</td>
			<td>53.1 ±1。4</td>
		</tr>
<tr>
<td>炒多肽</td>
			<td>0.0 ±0。0</td>
			<td>43.7 ±2。1</td>
			<td>56.3 ±3。5</td>
		</tr>
<tr>
<td>炒 PAM</td>
			<td>0.0 ±0。0</td>
			<td>60.7 ±0。2</td>
			<td>39.3 ±0。2</td>
		</tr>
</tbody></table>表 2:多肽和 PAMs 的二级结构组成.

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

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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

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.

An example of a nano drug based on polymalic acid is presented towards the rational design of personalized medicine that is applicable to cancer. It describes the synthesis of a nano drug to treat Her2-positive human breast cancer in a nude mouse.

聚苹果酸为基础的生物聚合物纳米靶向的多种肿瘤标志物：用于个性化医疗的机会？

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this ...

科研

化学

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

可逆二硫键交联胶束生产的简便和有效途径

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

生物化学

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

前列腺癌与小分子肽CLT1靶向造影剂的磁共振分子成像

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

医学

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the A&#946; peptide associated with Alzheimer&#39;s disease, the peptide is shown to self-assemble into cross-&#946; amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.

12-Dithiolane 改性自组装肽的合成与表征

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting ...

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 ...

生物工程

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 ...

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

病毒衍生肽修饰抗体结合物对增强细胞积累和改善肿瘤靶向性的初步评价

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

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

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices&#39; defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes.

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 ...

Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the transmigration of innate immune monocytes to inflammatory sites of deposited lipid called fatty streaks, which are present in arterial walls of medium to large arteries. The key pathogenic feature of lesions at this early stage of atherosclerosis is the maturation of monocytes which migrate into arteries to form foam cells or lipid-laden macrophages. Considerable evidence supports the hypothesis that risk of atherosclerosis is increased by chronic inflammatory conditions accompanying diseases such as rheumatoid arthritis and HIV, as well as general ageing, and that this risk is predicted by monocyte activation. While mouse models provide a good platform to investigate the role of monocytes in atherogenesis in vivo, they require genetic alteration of natural cholesterol metabolism and drastic alteration of normal mouse diets, and have limited suitability for the study of atherogenic influences of human comorbid diseases. This motivated us to develop a human in vitro model to measure the atherogenic potential of monocytes isolated from individuals with defined disease states. Currently, human in vitro models are limiting in that they evaluate monocyte transmigration and foam cell formation in isolation. Here we describe a protocol in which monocytes isolated from patient blood transmigrate across human endothelial cells into a type 1 collagen matrix, and their propensity to mature into foam cells in the presence or absence of exogenous lipid is measured. The protocol has been validated for the use of human monocytes purified from individuals with HIV infection and elderly HIV uninfected individuals. This model is versatile and allows monocyte transmigration and foam cell formation to be evaluated using either microscopy or flow cytometry as well as allowing the assessment of atherogenic factors present in serum or plasma.

We describe a protocol to measure transmigration by monocytes across human endothelial monolayers and their subsequent maturation into foam cells. This provides a versatile method to assess the atherogenic properties of monocytes isolated from people with different disease conditions and to evaluate factors in blood which may enhance this propensity.

慢性炎症状态个体的单核细胞轮回和泡沫形成的定量研究

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the ...

免疫与感染

Preparation and Characterization of Targeted Microbubbles

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of microbubble preparation and targeting ligand attachment are cumbersome, complicated, and lengthy. Therefore, there is a need to simplify the targeted microbubble preparation procedure to bring it closer to clinical translation. The purpose of this publication is to provide a detailed description and explanation of the steps necessary for targeted microbubble preparation, functional characterization and testing. A sequence of the optimized and simplified procedures is presented for two systems: a biotin-streptavidin targeting pair model, and a cyclic RGD peptide targeting the recombinant &#945;v&#946;3 protein, which is overexpressed on the endothelial lining of the tumor neovasculature.
Here, we show the following: covalent coupling of the targeting ligand to a lipid anchor, assessment of the reagent quality, and tests that confirm the successful completion of the reaction; preparation of the aqueous precursor medium containing microbubble shell components, followed by microbubble preparation via amalgamation; assessment of the efficacy of lipid transfer onto the microbubble stabilizer shell; adjustment of microbubble size distribution by flotation at normal gravity to remove larger microbubbles that might be detrimental for in vivo use; assessment of microbubble size distribution by electrozone sensing; evaluation of targeted binding of the microbubbles to receptor-coated surface in a static binding assay test (in an inverted dish); and evaluation of targeted binding of the microbubbles to receptor-coated surface in a parallel plate flow chamber test.

The goal of this protocol is to prepare, purify and characterize gas-filled microbubbles (targeted contrast agents for ultrasound molecular imaging). Two targeting systems are described: biotinylated bubbles adherent to streptavidin, and cyclic RGD peptide microbubbles that bind to &#945;v&#946;3, a known tumor neovasculature biomarker.