提交人承认, 资助 RHC 由 NIH 赠款 # 的 AI095382, EB021230, CA198880, 国家粮食和农业研究所, 以及芬兰杰出的教授计划。

1. 昆虫细胞的 HEVNP 生产
注意: 所有以下步骤都应在单元格培养罩中执行。有关更详细的 HEVNP 生产过程11, 请参阅我们以前的出版物。
<ol><li>在昆虫细胞介质中培养 Sf9 细胞 (参见材料表), 以 50-75% 汇流在6井板上。</li>
	<li>利用昆虫细胞转染试剂根据制造商的协议, 染 Bacmids 含有 HEVNP-573C ORF2 到 Sf99 细胞生产重组杆状病毒。根据细胞的生存能力, 在27摄氏度孵育转染细胞, 3-6 天。</li>
	<li>在感染后 3-6 天收集上清液 (转染后的细胞因杆状病毒感染裂解) 为 P0 杆状病毒。</li>
	<li>在25厘米2单层烧瓶中应用200µL P0 库存到 50-75% 汇合 Sf9 细胞之前, 移除培养基。每15分钟摇动烧瓶, 确保接种的全部覆盖。重复4次, 总共60分钟。</li>
	<li>添加2毫升的昆虫细胞培养培养基到烧瓶, 并保持在27摄氏度 3-6 天, 取决于细胞的生存能力, 放大杆状病毒, 以更高的效价。</li>
	<li>进行斑块化验以获得杆状病毒滴度阅读12。</li>
	<li>培养悬浮的 Tn5 昆虫细胞 (材料表) 与100毫升昆虫细胞媒介在250毫升瓶并且在27°c 在150转到一个滴度 0.5 x 105 -1 x 106为接种。</li>
	<li>添加杆状病毒在 5-10 到100毫升 Tn5 细胞在250毫升瓶中的多重感染 (语言)。接种后, 在27°c 以150转每分钟晃动 5-7 天。</li>
	<li>一旦大多数细胞出现囊泡, 70-90% 的细胞在光学显微镜观察下死亡, 收集 Tn5 细胞并转移到33毫升 ultracentrifuge 管。将 ultracentrifuge 管放在摆动斗转子中, 平衡, 并将细胞碎片和重组杆状在 1万 x g 处旋转90分钟, 达到25摄氏度。</li>
	<li>将含有释放 HEVNPs 的上清液保持在4摄氏度, 以进一步纯化。为进一步贮存杆状病毒上清, 添加蛋白酶抑制剂。</li>
</ol>2. HEVNP 纯化
<ol><li>用氯化铯 (中海) 梯度分离 HEVNPs 颗粒和分离:<ol>
<li>转移收集到的上清 (从步骤 1.10), 并添加 20% NP-40 到每个管, 使最终浓度 0.5% NP-40, 以溶解任何剩余的细胞膜。轻轻地混合吹打和孵化至少30分钟在25摄氏度。</li>
		<li>Ultracentrifuge HEVNPs 在 112400 x g 在摆动的桶式转子为2小时在4°c 从上清的 HEVNPs 颗粒下来。离心后丢弃上清, 在每管200µL 10 毫米的 MES 缓冲 pH 值 6.2 (O/N) 在4摄氏度的情况下, 轻轻地重新悬浮原油颗粒。运行 SDS 页 (步骤 3.1) 以确认 ORF2 在粗颗粒中存在 HEVNP, 作为 52 kDa 带。</li>
		<li>在6.2 毫升 ultracentrifuge 管中混合1.96 克中海, 再悬浮的粗球团, 4 毫升0.01 米的 MES pH 5, 准备 38.5% (w/v) 中海梯度。平衡管和地方在摆动斗转子。Ultracentrifuge 在 14.7万 x g 16 小时在4摄氏度。</li>
	</ol>
</li>
	<li>收集了中海梯度后的分数:<ol>
<li>丢弃前500µL 分数, 这主要是轻量细胞膜碎片。收集500µL 分数, 从管的顶端开始, 并改变每个分数之间的提示。将每个分数放入编号/标记为1.5 毫升的管。 注: 由于中海中高浓度, HEVNP ORF2 的存在, 不能通过运行 SDS 页凝胶来检测。中海中的每一个分数可以被删除或稀释通过遵循中海清洁程序。另外, 中海梯度可由 10-40% 蔗糖梯度13取代, 以避免中海中残留。</li>
		<li>把每一个分数转移到一个5毫升 ultracentrifuge 管和稀释中海中与4.5 毫升10毫米 MES, pH 值6.2。平衡管和地方在摆动斗转子。Ultracentrifuge 在 14.7万 x g 2 小时, 在4°c, 以球团下来的 HEVNPs。</li>
		<li>放弃上清, 并轻轻地重新悬浮在100µL 10 毫米 MES pH 6.2 在每管 HEVNPs。覆盖管, 以避免蒸发和孵化4摄氏度的 O/N。</li>
		<li>使用分光光度计记录 A280 读数和 A260/A280 nm 比值。确定 ORF2 的近似浓度为: <img alt="Equation" src="/files/ftp_upload/57020/57020eq1.jpg"> 每个 ORF2 将包含1胱氨酸站点和1赖氨酸站点为化学共轭。 注: HEVNP ORF2 的摩尔消光系数为 60280, 相当于1.019 毫克/毫升 x A280。这是如此接近 1:1, HEVNPs 的浓度 (在镁/毫升) 可以接近的 A280, 因此, ORF2 的浓度由上述方程式。例如, A280 读数为1的 HEVNP 将有1毫克/毫升的浓度, 相当于18.8 µM ORF2。</li>
		<li>准备一个 SDS 页面。使用每个分数的6µL 样本来确定含有 53.3 kDa HEVNP ORF2 蛋白的分数 (步骤 3.1)。HEVNPs 应该被发现在分数 3-5, 与密度为 ~ 1.25 克/毫升。</li>
		<li>通过 TEM 观察, 确定 HEVNPs 的存在和纯度。准备或稀释 HEVNP 样品到 0.5-2.0 毫克/毫升的 TEM。HEVNPs 以透射电镜显示为空 icosahedral 蛋白, 直径约 27 nm (图 2)。一些蛋白质污染物可能留在分数, 并将在 TEM 下观察 (步骤 3.2)。</li>
	</ol>
</li>
	<li>在透射电镜下存在杂质的情况下, 重复步骤 2.1.3 2.2.6, 以提高 HEVNPs 的纯度。 注: 通过中海梯度的额外净化可能导致 HEVNPs 的产量损失。另外, 中海梯度纯化可以用 10-40% 蔗糖梯度代替, 以避免中海中的残留13。</li>
</ol>3. HEVNP 特征
<ol><li>准备 SDS 页4-12% 双三蛋白凝胶, 1.0 毫米, 17 井 (见材料表) 根据用户手册14:<ol>
<li>添加2µL 4x 加载缓冲到6µL 蛋白质样品。将样品混合物在热块中孵育10分钟, 在100摄氏度变性蛋白质。将蛋白质样品装载到凝胶上。</li>
		<li>通过将 DC 电源设置为10分钟, 然后 150 v 为45分钟, 运行 SDS 页面, 直到样品在凝胶底部的约1厘米处运行为止。</li>
		<li>染色 SDS 页凝胶与考马斯蓝色 (0.25% (瓦特/v) 考马斯明亮的蓝色 R250, 30% (v/v) 甲醇, 10% (v/v) 乙酸), 1 h。</li>
		<li>染色程序后, 去除考马斯蓝染色, 并应用去除染色缓冲器 (30% (v/v) 甲醇, 10% (v/v) 醋酸) 到蛋白质凝胶 > 12 小时室温。</li>
		<li>在白光下记录下凝胶以确认在 52 kDa 带 HEVNP ORF2 的存在。</li>
	</ol>
</li>
	<li>用 TEM 观察 HEVNPs。<ol>
<li>将 HEVNP 样品准备或稀释为 0.5-2 毫克/毫升, 10 毫米 MES pH 6.2 用于 TEM 成像。</li>
		<li>电离 40 mA 辉光放电的碳涂层网格在三十年代生产一个亲水性的碳表面。辉光放电设备在材料表中描述。 注: 在辉光放电处理后, 网格的亲水性碳表面只能持续30分钟。</li>
		<li>按住镊子, 在网格中添加2µL 的 HEVNP 样品, 等待15三十年代, 并用滤纸涂抹。</li>
		<li>立即用 ddH20 清洗网格, 然后用过滤纸涂抹。</li>
		<li>立即在网格中加入2µL 2% 的醋酸铀, 等待十五年代, 然后用过滤纸涂抹。将样品栅格放在电子除湿干燥柜中, 使其干燥。</li>
		<li>将网格转换成透射电镜和10-80k 放大图像。HEVNPs 出现在透射电镜作为空的 icosahedral 蛋白, 是27毫微米直径, 由于缺乏病毒 RNA。</li>
	</ol>
</li>
</ol>4. HEVNPs 与生物素、肿瘤靶向配体、显影的化学共轭
<ol><li>执行一步共轭 HEVNPs 和 maleimide 链接生物素。<ol>
<li>缓冲变化: 根据制造商的协议 (材料表), 将 HEVNPs 在迷你透析单元和透析中应用于室温下的0.01 米 PBS pH 值为1小时。将 HEVNPs 转移到1.5 毫升管, 用分光光度计测量 280 nm 的蛋白质浓度。</li>
		<li>将 HEVNP 混合到1毫克/毫升, 相当于18.8 µM 的胱氨酸反应站点 (参见步骤2.2.4 中的详细信息), 在0.01 米 PBS, pH 为 7.4, 以相等的 maleimide-生物素 (100 µM), 使1:5 摩尔比;反应4摄氏度。根据制造商的协议 (材料表), 删除未绑定 maleimide-生物素与 40 K 截留分子量自旋脱盐塔工艺。</li>
		<li>通过标准的减少 SDS 页面 (步骤 3.1) 分析样品。</li>
		<li>使用标准程序, 用酶联的链亲和素制备一种化学发光印迹。通过 x 射线胶片捕获化学发光信号 (图 2)。</li>
	</ol>
</li>
	<li>在混合动力核动力源上执行两步 LXY30 共轭到表面暴露的半胱氨酸 (图 5)。<ol>
<li>缓冲交换: 应用 HEVNPs 在微型透析单位和透析对 0.01 M PBS pH 值7.4 在室温下为 1 h. 将 HEVNPs 转移到1.5 毫升管, 用分光光度计测定 280 nm 的蛋白质浓度。</li>
		<li>添加650µM maleimide-叠氮化物和650µM alkyne-LXY3010在 0.01 M PBS pH 7.4 与200µM 丘索4和1毫米抗坏血酸形成 maleimide 链接的 LXY30 (Mal-LXY30) 在650µM. 孵育混合物在4°c 为 O/N。</li>
		<li>混合 HEVNP 到1毫克/毫升, 相当于18.8 µM 的胱氨酸反应站点 (见细节在步骤 2.2.4), 约10% 卷 Mal-LXY30 (650 µM) 在0.01 米 PBS pH 7.4, 使一个1:3 摩尔比率;反应4摄氏度。 注: 由于 Mal-LXY30 浓度相对较高, 反应物的最终浓度 (如丘索4) 在混合后减少约10倍, 以避免其对 HEVNPs 的损害。另一个选项是无铜共轭方法15。</li>
		<li>根据制造商的协议删除未绑定 maleimide-click-LXY30 与 40 K 截留分子量自旋脱盐塔 (请参阅材料表)。保持 LXY30-linked HEVNPs (LXY30-HEVNPs) 在4摄氏度。</li>
	</ol>
</li>
	<li>执行一步共轭 LXY30-HEVNPs 和 Cy5.5 nhs 酯 (nhs-Cy5.5)<ol>
<li>混合 LXY30-linked HEVNPs (LXY30-HEVNPs) 到1毫克/毫升, 这相当于胱氨酸反应站点的18.8 µM (参见步骤2.2.4 中的详细信息), 在0.01 米 PBS (100 Cy5.5) 的同等数量的 NHS-µM 的 pH 7.4, 使1:5 摩尔比率;反应4摄氏度。</li>
		<li>根据制造商的协议, 删除未绑定 Cy5.5-NHS 与40K 截留分子量自旋脱盐塔程序 (请参阅材料表)。保持 LXY30, Cy5.5 链接 HEVNPs (LXY30 HEVNP Cy5.5) 在4摄氏度。</li>
	</ol>
</li>
</ol>5. HEVNP 对 MDA-MB231 乳腺癌细胞的束缚和内化
<ol><li>种子 MDA-MB231 乳腺癌细胞成一个35毫米的玻璃底部菜肴 (5 x 104每碟) O/N 在哺乳动物细胞培养柜。</li>
	<li>对于细胞结合实验, 请按照步骤 4.2-4.3 准备 LXY30-HEVNP Cy5.5。稀释 LXY30-HEVNP-Cy5.5 0.01 毫克/毫升, 相当于 HEVNP ORF2 的0.188 µM (指步骤 2.2.4), 250 µL 0-1% 的血清/DMEM。<ol>
<li>在0.01 毫克/毫升 HEVNP Cy5.5 的情况下, 按步骤 4.3, 但与 NHS-Cy5.5 染料共轭, (HEV-Cy5.5), 准备阴性对照样品。稀释 HEVNP-Cy5.5 到0.01 毫克/毫升, 相当于 HEVNP ORF2 的0.188 µM (指步骤 2.2.4), 250 µL 10% 的血清补充 DMEM。</li>
	</ol>
</li>
	<li>通过应用250µL 1 米 PBS 缓冲液, pH 值7.4 来清洗细胞。清洗后取出 PBS 缓冲器, 同时在细胞培养皿中保留一些缓冲液。</li>
	<li>应用250µL 的 LXY30-HEVNP Cy5.5 在10% 的血清补充 DMEM 或 HEVNP Cy5.5 在10% 的血清补充 DMEM 的培养 MDA-MB231 乳腺癌细胞。用铝箔保护细胞培养皿。</li>
	<li>将细胞培养皿保持在37摄氏度细胞培养柜内, 用于内部化1小时。</li>
	<li>在冰上冲洗培养的细胞3次, 每洗5分钟, 250 µL 1 米 PBS, pH 7.4。</li>
	<li>在1米 pbs 中固定4% 只粉煤灰中的细胞, ph 7.4 为20分钟, 然后用250µL 的1米 pbs, ph 7.4 清洗一次。 注意: 细胞现在已经准备好被共聚焦显微镜成像。代表性数据显示在图 5中。</li>
</ol>

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作者声明他们没有竞争的兴趣。

与耗时的基因工程程序, 这通常需要几个星期, 在这里我们演示简单的两步和一步化学共轭程序, 可以在3天内完成, 增加癌症靶向配体和/或荧光检测染料到胱氨酸/赖氨酸 HEVNPs 的部位。该技术可用于筛选候选池中的最佳配体目标, 从而在合理的成本和交付时间内利用可用的肽/小分子合成服务。
不同于传统的基因工程, 它只能将多肽插入到感兴趣的蛋白质中, 化学共轭可以将各种...

在治疗和诊断分娩中纳米载体的发展, 被称为 nanotheranostics, 已经把大部分生物医学领域从广义治疗转移到目标传递1。靶向 nanotheranostic 交付将纳米载体 (纳米粒子) 与 theranostic 分子结合在一起, 稳定地直接 theranostic 分子到特定患病组织或生物化学途径2,3,4.纳米医学已经走到了目标交付的前列, 因为最佳尺寸的纳米粒子有能力稳定 theranostic 分子的循环, 有选择地靶向细胞表面分子呈现在患病的组织上。许多 nanotheranostic 平台仍然遭受被动细胞吸收, 前成熟退化, 毒性和与 theranostic 分子不充分的联系。VLPs 克服了目标交付中的许多障碍。它们被用作 nanocarriers 来显示外来的表位和/或提供小分子: 一种可以用来对抗许多疾病的方案1。该应用主要依赖于自组装的特性以及遗传修饰的方便性, 以满足给定 vip 的设计应用。与基因工程相比, 国外多肽对 vip 的化学共轭显示了一个重要的优势, 因为它允许多种实体, 如多肽或寡糖, 被共轭到 VLPs 的表面, 在调制和灵活的方式, 不改变贵宾大会。
HEVNPs, 来自重组的混合动力衣壳蛋白, 2nd开放阅读框架 (ORF2), 是非传染性的, 自组装衣壳能够细胞结合和进入。由于混合动力汽车进化为黏膜传输, 组装的衣壳蛋白是同样稳定的水解和酸性粘膜条件5。HEVNPs 形成一个空心, T = 1 icosahedral 衣壳, 由60个相同的单位组成,6,7 ORF2, 使其在储存和严酷的生理条件下高度稳定。由于缺乏任何病毒遗传元素, 通过昆虫细胞的杆状病毒表达系统实现了高效、高产的生产。由于其蛋白水解稳定性, 自组装 HEVNPs 提取和纯化的细胞上清, 大大减少必要的净化步骤。此外, HEVNPs 拥有一个表面暴露的凸出域 (P 域) 通过一个灵活的铰链连接到一个稳定的 icosahedral 基础。p 域在 icosahedral 基础上形成表面暴露的尖峰, 而柔性铰链使得在不损害基 icosahedral 结构的情况下可以显著地修改 P 域。与60个重复的单位, 单一站点特定的修改结果在60个对称站点的化学调制。最近, 我们提出了一个纳米平台使用 HEVNP, 可以化学共轭配体或小分子的 theranostic 应用。这是通过取代单一氨基酸与半胱氨酸的突出领域的混合动力-vip 作为一个反应地点与 maleimide 链接的肽或分子。基于以前对混合动力-vip 和研究良好的免疫表位8,9的结构分析, 以下五个 hev-vip 氨基酸以半胱氨酸取代为潜在候选者: Y485C、T489C、S533C、N573C 和 T586C (图 1)。通过透射电镜 (TEM) 观察 (图 2) 对昆虫细胞的表达和纯化后的 vip 形成进行了验证, 并在 maleimide 链接生物素后对暴露的半胱氨酸部位进行了分析。共轭 (图 2)。在五变种人中, HEVNP-573C 显示了 maleimide-生物素共轭 (图 2) 的最强信号, 并被用来作为乳腺癌细胞靶向4 (图 3) nanocarrier 的后续演示。
该协议描述了化学共轭方法, 通过表面半胱氨酸共轭将肿瘤靶向分子附着在 HEVNPs 上。我们详细的肿瘤靶向和检测分子结合的肿瘤传递与重组 HEVNPs 含有半胱氨酸在 N573 (HEVNP-573C)。我们专注于两步点击化学共轭过程, 以绑定乳腺癌肿瘤靶向肽, LXY3010到 HEVNPs 形成 LXY30-HEVNP (图 4)。随后, n-hydroxysuccimide (NHS)-Cy5.5 被共轭到单独的赖氨酸站点在 HEVNPs 建立 LXY30 HEVNP-Cy5.5 为荧光检测的在体外(图 5) 和在体内4.

<table border="0" cellpadding="0" cellspacing="0" width="984">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:391px;">MINI Dialysis Units, 10K MWCO</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:265px;">69572</td>
			<td style="width:167px;">mini dialysis unit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Five Cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>B85502</td>
			<td>Tn5 cells</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SF9 Cells&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>11496015</td>
			<td>Sf9 cells</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Bac-to-Bac Baculovirus Expression System</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11101, A11100</td>
			<td>Baculovirus expression system</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Bac-to-Bac Baculovirus Expression System</td>
			<td>Life Technologies</td>
			<td>10359-016, 10360-014, 10584-027, 10712-024</td>
			<td>Bacmid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ESF921 Insect Cell Media</td>
			<td>Expression Systems LLC</td>
			<td>96-001-01</td>
			<td>insect cell media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cy5.5 NHS ester, 5mg</td>
			<td>Lumiprobe Corp</td>
			<td>27020</td>
			<td>Cy5.5 NHS ester</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zeba Spin Desalting Columns, 40K MWCO, 0.5 mL</td>
			<td>Thermo Scientific</td>
			<td>87766</td>
			<td>spin desalting column</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MES Hydrate</td>
			<td>Sigma-Aldrich Chemical Co</td>
			<td>M8250-250G</td>
			<td>MES</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultra-Clear Centrifuge Thinwall Ultra-Centrifuge Tubes</td>
			<td>Beckman Coulter, Inc</td>
			<td>Depends on Rotor</td>
			<td>ultracentrifuge tube</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NuPage 4-12% Bis-Tris Protein Gels</td>
			<td>Thermo Fisher Scientific</td>
			<td>NPO321BOX</td>
			<td>SDS protein gel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cellfectin II Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>10362100</td>
			<td>transfection reagent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:391px;">EMS Glow Discharger</td>
			<td style="width:161px;">Electron Microscopy Science</td>
			<td style="width:265px;"></td>
			<td>glow discharger</td>
		</tr>
	</tbody>
</table>

surface functionalization of hepatitis e virus nanoparticles using chemical conjugation methods

病毒样粒子 (VLPs) 被用作 nanocarriers, 用于显示异物抗原和/或在各种疾病的检测和治疗中提供小分子。该应用依赖于基因修饰、自组装和半胱氨酸共轭来实现重组 VLPs 的肿瘤靶向应用. 与单基因修饰相比, 国外多肽对 VLPs 的化学共轭提供了一种很大的优势, 因为它允许多种实体, 如合成肽或寡糖, 以调制和灵活的方式共轭到 VLPs 表面, 而不改变 vip 组装。
在这里, 我们演示如何使用 E 型肝炎病毒纳米粒子 (HEVNP), 一个模块化的 theranostic 胶囊, 作为一个多功能运送载体。HEVNPs 的功能包括组织靶向、影像学和治疗性分娩。在 HEVNP 的结构研究基础上, 选取了结构上独立的和表面暴露的残留物作为 maleimide 连接化学基团的共轭点, 通过硫醇选择性的联系来选择半胱氨酸置换。一种特殊的半胱氨酸修饰 HEVNP (胱氨酸取代天门冬在 573 aa (HEVNP-573C)) 被共轭到乳腺癌细胞特异配体, LXY30 和标记为近红外线 (近红外) 荧光染料 (Cy5.5), 呈现肿瘤靶向HEVNPs 作为有效的诊断胶囊 (LXY30-HEVNP Cy5.5)。类似的工程策略可以与其他大分子复合物与众所周知的原子结构, 以探索潜在的应用 theranostic 交付。

类似于 HEV-VLPs, 所有胱氨酸改性 HEVNPs 形成可溶性 icosahedral 衣壳, 并没有聚集在溶液中的生产或纯化。在单步 maleimide-生物素共轭之前和之后, 每个胱氨酸修改后的 HEVNPs 在负染色 EM (图 2) 中都与混合动力 VLPs 不区分。Maleimide-生物素共轭效率胱氨酸改性 HEVNPs 首次测试与西方印迹通过化学发光链亲和素结合。继 maleimide-生物素共轭后, 胱氨酸修饰 HEVNPs 显...

Watch this Scientific Journal Video about Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods at JoVE.com

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

化学共轭法在戊型肝炎病毒纳米粒子表面功能化中的应用

我们已将戊型肝炎病毒的衣壳蛋白作为 theranostic 纳米微粒 (HEVNP) 进行了设计。HEVNP 自组装成一个稳定的 icosahedral 笼在黏膜分娩。在这里, 我们描述了 HEVNPs 的改变的肿瘤靶向性的变异表面暴露残留到半胱氨酸, 共轭合成配体, 具体绑定肿瘤细胞。

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

surface-functionalization-hepatitis-e-virus-nanoparticles-using

Research

JoVE Journal

Bioengineering

6.8K 次观看.  University of California Davis. 我们已将戊型肝炎病毒的衣壳蛋白作为 theranostic 纳米微粒 (HEVNP) 进行了设计。HEVNP 自组装成一个稳定的 icosahedral 笼在黏膜分娩。在这里, 我们描述了 HEVNPs 的改变的肿瘤靶向性的变异表面暴露残留到半胱氨酸, 共轭合成配体, 具体绑定肿瘤细胞。

视频: Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

软平版功能化和图案无氧化硅和锗

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

科研

生物工程

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

收获小鼠肺泡巨噬细胞和评价聚酸酐纳米粒子诱导的细胞活化

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

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

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

碳水化合物和聚酸酐纳米粒子功能化的高通量合成

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

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry

The recent rise in virus-like particles (VLPs) in biomedical and materials research can be attributed to their ease of biosynthesis, discrete size, genetic programmability, and biodegradability. While they&#39;re highly amenable to bioconjugation reactions for adding synthetic ligands onto their surface, the range in bioconjugation methodologies on these aqueous born capsids is relatively limited. To facilitate the direction of functional biomaterials research, non-traditional bioconjugation reactions must be considered. The reaction described in this protocol uses dibromomaleimides to introduce new functionality in the solvent exposed disulfide bonds of a VLP based upon Bacteriophage Q&#946;. Furthermore, the final product is fluorescent, which has the added benefit of generating a trackable in vitro probe using a commercially available filter set.

Here, we present a procedure to fluorescently functionalize the disulfides on Q&#946; VLP with dibromomaleimide. We describe Q&#946; expression and purification, the synthesis of dibromomaleimide-functionalized molecules, and the conjugation reaction between dibromomaleimide and Q&#946;. The resulting yellow fluorescent conjugated particle can be used as a fluorescence probe inside cells.

Dibromomaleimide 二硫化学制备共轭诱导荧光 PEGylated 病毒样粒子

The recent rise in virus-like particles (VLPs) in biomedical and materials research can be attributed to their ease of biosynthesis, discrete size, ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

闪速纳米沉淀法包聚物纳米粒子中疏水亲水化合物的作用

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

用于细胞质 siRNA 传递的带电反应性聚合物纳米粒子的制备

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

大肠杆菌-表达自组装蛋白纳米颗粒的生产，用于需要三聚体异体的疫苗

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

使用微流体混合平台为基因传递制订和定性脂质纳米粒子

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...