J.W.S. 是霍华德休斯医学研究所的一名调查员。这项工作部分是由西蒙斯基金会的赠款 (290363) 支持 J.W.S。A.E.E. 和 K.P.A. 都承认明尼苏达大学启动基金的支持。

1. 囊泡制剂
<ol><li>
薄膜的制备
	<ol>
<li>使用气密注射器将表 1.1中描述的某些脂质转移到玻璃瓶中的氯仿。</li>
		<li>在通风罩中的氮气或氩流下蒸发产生的溶液。</li>
		<li>将所产生的薄膜与室内真空至少2小时, 以去除任何氯仿残渣。在这一点上, 脂质可以留在真空下过夜。</li>
	</ol>
</li>
	<li>
囊泡补液
	<ol>
<li>10毫升5毫米 calcein 250 毫米三 hcl ph 8.0 水合缓冲液溶解31毫克的 calcein 粉末2.5 毫升1米三盐酸 ph 8.0 缓冲和添加另7.5 毫升的去离子水 (DI)。</li>
		<li>吸管250µL 以上水化缓冲成空管, 并添加1.875 µL 10 米氢氧化钠为最终75毫米基地 (½相当于总脂肪酸)。将溶液转化为干脂薄膜, 形成囊泡, 总脂质浓度为0.15 米。</li>
		<li>使用高速 (> 3000 rpm) vortexer 涡的结果混合物为 4-5 s。</li>
		<li>将脂质缓冲液保持在低速 (约 30 rpm) 旋转振动筛水化, 至少一夜之间。</li>
	</ol>
</li>
	<li>
水泡的大小 (可选)
	<ol>
<li>使用镊子, 将一个过滤器支持应用到挤出机的注射器端口的每个内表面。</li>
		<li>每一个过滤器支持250毫米三 HCl, pH 值8。</li>
		<li>使用镊子, 应用一个轨道蚀刻100纳米聚碳酸酯膜的一个挤出 O 形环和过滤器支持。注意不要撕裂或刺穿膜, 轻轻地将薄膜推入 O 形环, 以便在两个表面之间进行良好的接触。</li>
		<li>组装挤出机, 注意不要取代膜和过滤器的支持。</li>
		<li>用约0.5 毫升的250毫米三盐酸, pH 8 来填充挤出机注射器。将注射器插入挤出机的一侧。</li>
		<li>将空注射器插入挤出机的另一侧。</li>
		<li>用手把装有缓冲液的注射器的柱塞推慢 (≤50µL/秒), 并验证是否有电阻, 表明轨道蚀刻膜就位并完好无损。在没有膜的情况下练习这一步是有帮助的, 以评估预期的阻力水平。</li>
		<li>取出并清空两个注射器。在这个阶段没有必要清洗两个注射器, 因为它们含有与脂质体制剂相同成分的缓冲剂。</li>
		<li>将脂质体制剂装入两个注射器中的一个。</li>
		<li>将挤出机与含有囊泡样品的注射器重新组装在左侧, 并将空注射器放在右侧。</li>
		<li>用手, 把含有囊泡样品的注射器的柱塞压得非常慢 (10-25 µL/秒)。</li>
		<li>仔细观察挤出机右侧的注射器;清除缓冲区最初将进入挤出机的右侧 (约50µL, 这是由于挤出机的内部死容量), 其次是挤压脂质体的小羽状。立即停止推, 在这一点上, 取出注射器的右侧的挤出机, 并放弃这个解决方案。</li>
		<li>更换挤出机右侧的注射器并挤出, 直到左注射器为空。</li>
		<li>反转挤出机的方向, 然后重复步骤13。继续所需的周期数 (通常为7、9或 11);奇数总是用来确保未被挤出的脂质体不收集从最初包含前瘦身脂质体准备的注射器。</li>
		<li>将正确的注射器的内容轻轻地放进一个离心的管子里, 把管子放在低速 (约 30 rpm) 旋转的振动筛上, 大约0.5 小时。</li>
		<li>进行囊泡纯化, 如第2节所述。挤出泡囊应在24小时内使用, 或在下次使用前重新挤出。</li>
	</ol>
</li>
</ol>2. 囊泡纯化
<ol><li>
囊泡净化移动相的制备
	<ol>
<li>5毫升250毫米的三盐酸 ph 8 水合缓冲, 加入1.25 毫升的1米三盐酸 ph 值8缓冲, 3.75 毫升的 DI 水到15毫升猎鹰管。然后添加37.5 µL 10 米氢氧化钠 (½当量相对于 unesterified 脂肪酸) 到水化缓冲。吸管235µL 纯油酸直接进入猎鹰管产生的囊泡溶液与0.15 米总脂。</li>
		<li>使用高速 (> 3000 rpm) vortexer 涡混合 4-5 s, 然后在低速旋转振动筛上翻滚至少2小时。在这一点上, 可以在旋转的振动筛上过夜的脂制剂。在使用前过滤0.22 微米注射器过滤器的移动相, 以去除任何潜在的骨料。</li>
	</ol>
</li>
	<li>
琼脂糖囊泡的纯化
	<ol>
<li>使用吸管从瓶子中取出约5毫升的乙醇琼脂糖4B 浆。将其应用于一次性的10毫升色谱柱。</li>
		<li>允许泥浆沉淀和乙醇流动, 直到乙醇接近树脂床的顶端。</li>
		<li>将5毫升的去离子水应用到树脂的顶部, 让水流冲走乙醇残渣。</li>
		<li>应用250毫米三 HCl, pH 8 在1-2 毫升部分到树脂的顶部, 每次液位接近树脂床的顶部时应用一个新的部分。重复3-5 次。</li>
		<li>将该列夹在反击架上, 然后将柱头与旋塞阀连接器和油管连接到分数收集器。添加另一个缓冲部分冲洗油管, 当柱液位接近树脂顶部时, 关闭旋塞阀。</li>
		<li>使用200µL pipettor, 将挤出泡从1部份涂到树脂顶部, 注意在不接触树脂床或柱壁的情况下尽可能均匀地将囊泡制剂应用于树脂。</li>
		<li>打开旋塞阀开始流动, 开始收集分数成一个96井板。在 0.5-1 毫升部分, 将囊泡净化移动相应用于树脂床的顶部, 当缓冲器耗尽时, 注意不要让树脂床干燥。收集五滴分数, 收集至少36口井 (三行96井板)。</li>
	</ol>
</li>
	<li>
纯化馏分的荧光表征
	<ol>
<li>从上一节中取96井板, 并在板读取器 (λex= 485 毫微米, λem= 515 nm) 上读取它。</li>
		<li>将所生成的荧光数据绘制为荧光与分数号。泡洗脱首先, 后跟 unencapsulated 分数 (图1)。</li>
	</ol>
</li>
	<li>
Repurification 囊泡对囊泡渗漏的监测
	<ol>
<li>重复2.2 和2.3 节中的纯化和表征步骤。保持其含量的囊泡不会出现 unencapsulated 溶质的峰值 (或者是小泡峰强度的 5-10%)。</li>
	</ol>
</li>
</ol>3. 在镁存在的情况下使用囊泡
<ol><li>
unliganded 镁的使用
	<ol>
<li>如1、2.1 和2.2 节所述, 制备和纯化囊泡。</li>
		<li>在250毫米三 hcl ph 值8.0 缓冲器中加入250µL, 1 米氯化镁8.0至50µL 的 DI 水, 准备1毫升的50毫米氯化镁2溶液。涡流混合良好。</li>
		<li>为了使所需的镁浓度为5毫米, 混合0.9 毫升纯化的囊泡和100µL 的镁溶液, 迅速搅拌, 以减少囊泡的瞬态暴露, 以高局部浓度的镁。 注: 快速混合镁溶液对囊泡稳定性至关重要。如果镁和囊泡不是快速混合的直接涡流, 一些囊泡将暴露在较高浓度的镁, 导致不一致的结果从样品到样品。</li>
		<li>在 repurification 前将囊泡样品留在滚筒上至少30分钟, 如2.4 所述, 检查是否有渗漏。添加相同浓度的镁, 如在囊泡样品到 repurification 移动相。</li>
	</ol>
</li>
	<li>
liganded 镁的使用
	<ol>
<li>如1、2.1 和2.2 节所述, 制备和纯化囊泡。 注: 使用½当量 KOH 代替氢氧化钠 deprotonate 脂肪酸;发现, 这在螯合氯化镁2系统中产生了更稳定的脂质体。</li>
		<li>准备2M 柠檬酸钾溶液, 用 KOH 调整 pH 值为8.0。</li>
		<li>预混料氯化镁2和柠檬酸钾的指定比例 (约1:4 用于稳定油酸泡) 在250毫米三盐酸 pH 值8.0 缓冲。</li>
		<li>添加预混柠檬酸镁溶液的囊泡样品, 简要涡。 注: 总预混料镁和配体溶液。不要单独暴露泡 unchelated 镁溶液。</li>
		<li>在 repurification 前, 将囊泡样品留在滚筒上至少30分钟, 如2.4 所述。加入相同浓度的镁和柠檬酸在囊泡样品到 repurification 移动相。</li>
	</ol>
</li>
</ol>4. 囊泡中的非酶 RNA 复制
<ol><li>
单分散 RNA 封装囊泡的制备
	<ol>
<li>准备一份干油酸膜, 如第1.1 节所述。</li>
		<li>用50µM 荧光标记的 rna 底漆, 150 µM rna 模板和250毫米三盐酸 pH 8.0 含½当量的油酸, 制备囊泡补液缓冲液。</li>
		<li>将250µL 的补液缓冲液添加到脂质膜中, 并跟随步骤1.2.2 和 1.2.3, 使囊泡具有总0.15 米的脂质浓度。</li>
		<li>为了使小 unilamellar 单分散囊泡, 按照1.3 节为囊泡挤压。</li>
	</ol>
</li>
	<li>
枸橼酸镁加和囊泡纯化
	<ol>
<li>预混料镁和柠檬酸盐库存, 然后混合这个与囊泡样品到最后的脂质浓度的 0.1 m。</li>
		<li>纯化琼脂糖4B 大小排除柱上的泡, 具有250毫米三盐酸 pH 8.0, 0.1 米脂, 并给予镁和柠檬酸盐作为流动相。</li>
		<li>通过以下2.3 节收集囊泡分数。</li>
	</ol>
</li>
	<li>
底漆延伸反应
	<ol>
<li>准备200毫米 2-MeImpG (鸟苷 5 '-单磷酸 2-methylimidazolide) 的股票溶液与250毫米三 HCl pH 值8.0。(注意: 按照以前发布的协议10进行 2 MeImpG 合成。</li>
		<li>要启动底漆的延伸反应, 转移150µL 2 MeImpG 的股票溶液到450µL 囊样, 达到最终浓度75毫米脂和50毫米活化单体。开始计时器, 并保持反应样品翻滚所有的时间。</li>
		<li>选)对于连续的新鲜单体喂养, 将300µL 的反应溶液转移到实验室的一个室中, 构建了小体积脂质体透析11 , 并将300µL 喂养溶液放入另一室中。喂养溶液应包含与反应溶液相同浓度的所有成分, 但替换含有空洞囊泡的 RNA。每轮透析需要1-24 小时, 具体取决于所用的分析物和膜。</li>
		<li>对于动力学研究, 采取100µL 整除在每个时间点, 并 repurify 整除通过琼脂糖4B 大小排除柱与250毫米三 HCl pH 8.0 作为移动相。</li>
		<li>收集1.5 毫升离心管内的囊泡成分。</li>
		<li>吸管到囊泡馏分到最后大约 0.1% v/v。</li>
		<li>在试管中加入0.5 毫升的冷乙醇, 在-20 摄氏度孵育至少2小时。</li>
		<li>离心机所有样品在16.1×1000区域合作框架 (16.1 x g) 为10分钟和轻轻地吸出液体。再用70% 冷乙醇和离心机清洗 rna 颗粒5分钟. 丢弃液体, 将管与 rna 丸放在80°c 的热量块上, 以蒸发残留乙醇。在50µL 8 米尿素中溶解颗粒, 1xTBE 加载缓冲。</li>
	</ol>
</li>
	<li>
页面分析
	<ol>
<li>20% 页凝胶通过混合200毫升的 UreaGel 系统浓缩, 25 毫升 UreaGel 系统稀释剂, 25 毫升10xTBE 缓冲器, 80 µL TEMED 和250毫克的过硫酸铵。快速倒入夹紧的凝胶板 (35 厘米 x 45 厘米) 之间的凝胶混合物, 并插入梳子。等待至少30分钟, 直到完全聚合。</li>
		<li>将凝胶板组装到凝胶支架上, 用1xTBE 凝胶器运行缓冲器填充凝胶盒。摘下梳子, 用注射器冲洗水井。预运行的凝胶与 60 W 为30分钟。</li>
		<li>在80摄氏度热块上加热样品2分钟, 每个样品每井装5µL。</li>
		<li>打开凝胶电源盒, 并设置凝胶运行以恒定的瓦特在 60 W 为2.5 小时。</li>
		<li>将凝胶板从凝胶支架上拆卸, 擦拭玻璃, 将整片板放入凝胶扫描仪中, 开始凝胶扫描。</li>
		<li>用 ImageQuant TL 1D 分析对凝胶进行量化。绘制在给定时间点上剩余的底漆量比率的自然对数, 指向初始量的底漆与时间, 适合直线, 并计算伪一阶反应速率 (图 2)。</li>
	</ol>
</li>
</ol>5. 锤囊中的锤头 RNA 自裂
<ol><li>囊泡中核酶的制备与纯化<ol>
<li>准备一个脂质薄膜 (OA: GMO=9:1), 如1.1 节中的组件, 如表 1.2.注: 温暖的转基因至少60摄氏度, 以完全熔化前使用。</li>
		<li>用每个锤头核酶链的5µM 和250毫米的三盐酸 pH 值8.0 制备囊泡补液缓冲液。</li>
		<li>将250µL 的补液缓冲液添加到脂质膜中, 并按照步骤1.2.2 和 1.2.3, 使囊泡与0.15 米总脂浓度。</li>
		<li>为了使小 unilamellar 单分散囊泡, 按照1.3 节为囊泡挤压。</li>
		<li>在琼脂糖4B 尺寸排除柱上纯化泡, 以250毫米三盐酸 pH 8.0, 0.15 M 混合脂作为流动相。</li>
	</ol>
</li>
	<li>
锤头核酶自解理反应
	<ol>
<li>准备镁溶液, 并与3.1 节中描述的纯化泡混合, 开始自解裂反应。</li>
		<li>对于动力学研究, 采取100µL 的混合物在每个时间点, 并直接 repurify 这整除通过琼脂糖4B 大小排除柱与250毫米三 HCl pH 8.0 作为移动相。收集囊泡分数。</li>
	</ol>
</li>
	<li>
页面分析
	<ol>
<li>按照步骤 4.3.6 4.3.8 准备 RNA 加载样品。</li>
		<li>将商业铸造的15% 尿素凝胶放入凝胶盒中, 用1xTBE 凝胶运行缓冲器填充凝胶盒。</li>
		<li>在80摄氏度热块上加热样品1分钟, 每个样品每井装5µL。</li>
		<li>运行的凝胶与恒定的 200 V 约1小时。</li>
		<li>扫描凝胶。</li>
		<li>用分析软件 (如 ImageQuant TL 1D) 量化凝胶, 通过比较剩余基底 rna 带和分裂 rna 片段带 (图 3) 的强度来测量裂解的程度。</li>
	</ol>
</li>
</ol>6. 巨大的脂肪酸囊泡显微术
<ol><li>
巨脂肪酸囊泡的制备
	<ol>
<li>按照1.1 和表 1.3的方法, 用 0.2 (摩尔) 的荧光标记脂制备油酸薄膜, 以便更好地进行膜观察。</li>
		<li>水化脂膜与500µL 250 毫米的三盐酸 pH 8.0, 使囊泡样本10毫米的脂肪总数。如果需要, 缓冲液可能含有荧光染料或 RNA 分子。让水泡样品一夜之间翻滚。</li>
		<li>用470µL 油酸和150毫升的250毫米三盐酸盐 pH 8.0, ½当量氢氧化钠, 制备150毫升纯脂肪酸透析缓冲器, 总脂质为10毫米。</li>
		<li>通过10µm 聚碳酸酯膜挤出囊泡样品, 去除大骨料。</li>
		<li>如前所述, 将囊泡样品转移到1µm 大孔透析盒中。12
</li>
		<li>将透析盒放入含有30毫升透析缓冲器的100毫升烧杯中。将烧杯轻轻地搅动在桌上的振动筛上, 转速在80-100 转。</li>
		<li>每30分钟更换透析缓冲液5次, 去除游离染料或 RNA 和小泡。</li>
		<li>用注射器小心地从透析盒中取出囊泡样本, 然后转移到离心管。</li>
	</ol>
</li>
	<li>
显微观察
	<ol>
<li>吸管10µL 的泡在一个干净的标准显微镜幻灯片上, 并放置一个玻璃盖子滑过样品。带透明指甲油的密封盖玻璃。</li>
		<li>使用适当的激光源荧光标记的膜、RNA 或封装染料 (图 4) 观察与60X 油分散或类似目标的囊泡。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Hanczyc, M. M., Fujikawa, S. M., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Models+of+Primitive+Cellular+Compartments:+Encapsulation,+Growth,+and+Division.">Experimental Models of Primitive Cellular Compartments: Encapsulation, Growth, and Division.</a> Science. 302, 618-622 (2003).</li><li>Mansy, S. S., Schrum, J. P., Krishnamurthy, M., Tob&#233;, S., Da Treco, ., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Template-directed+synthesis+of+a+genetic+polymer+in+a+model+protocell.">Template-directed synthesis of a genetic polymer in a model protocell.</a> Nature. 454 (7200), 122-125 (2008).</li><li>Apel, C. L., Deamer, D. W., Mautner, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembled+vesicles+of+monocarboxylic+acids+and+alcohols:+conditions+for+stability+and+for+the+encapsulation+of+biopolymers.">Self-assembled vesicles of monocarboxylic acids and alcohols: conditions for stability and for the encapsulation of biopolymers.</a> Biochim. Biophys. Acta. 1559 (1), 1-9 (2002).</li><li>Walde, P., Wick, R., Fresta, M., Mangone, A., Luisi, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autopoietic+Self-Reproduction+of+Fatty+Acid+Vesicles.">Autopoietic Self-Reproduction of Fatty Acid Vesicles.</a> J. Am. Chem. Soc. 116 (26), 11649-11654 (1994).</li><li>Sulston, J., Lohrmann, R., Orgel, L. E., Todd Miles, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonenzymatic+Synthesis+of+Oligoadenylates+on+a+Polyuridylic+Acid+Template.">Nonenzymatic Synthesis of Oligoadenylates on a Polyuridylic Acid Template.</a> Proc. Natl. Acad. Sci. U. S. A. 59 (3), 726-733 (1967).</li><li>Adamala, K., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonenzymatic+template-directed+RNA+synthesis+inside+model+protocells.">Nonenzymatic template-directed RNA synthesis inside model protocells.</a> Science. 342 (6162), 1098-1100 (2013).</li><li>Uhlenbeck, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+catalytic+oligoribonucleotide.">A small catalytic oligoribonucleotide.</a> Nature. 328 (6131), 596-600 (1987).</li><li>Chen, I. A., Salehi-Ashtiani, K., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+catalysis+in+model+protocell+vesicles.">RNA catalysis in model protocell vesicles.</a> J. Am. Chem. Soc. 127 (38), 13213-13219 (2005).</li><li>Adamala, K. P., Engelhart, A. E., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collaboration+between+primitive+cell+membranes+and+soluble+catalysts.">Collaboration between primitive cell membranes and soluble catalysts.</a> Nat. Commun. 7, 1-7 (2016).</li><li>Joyce, G. F., Inoue, T., Orgel, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Template-directed+Synthesis+on+Random+Copolymers.">RNA Template-directed Synthesis on Random Copolymers.</a> J. Mol. Biol. 176, 279-306 (1984).</li><li>Adamala, K., Engelhart, A. E., Kamat, N. P., Jin, L., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+liposome+dialyzer+for+the+preparation+of+high-value,+small-volume+liposome+formulations.">Construction of a liposome dialyzer for the preparation of high-value, small-volume liposome formulations.</a> Nat. Protoc. 10 (6), 927-938 (2015).</li><li>Zhu, T. F., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+large+monodisperse+vesicles.">Preparation of large monodisperse vesicles.</a> PloS one. 4 (4), e5009 (2009).</li><li>Zhu, T. F., Budin, I., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle+extrusion+through+polycarbonate+track-etched+membranes+using+a+hand-held+mini-extruder.">Vesicle extrusion through polycarbonate track-etched membranes using a hand-held mini-extruder.</a> Methods Enzym. 533, (2013).</li></ol>

作者声明他们没有竞争的财政利益。

由脂肪酸组成的脂质体由于其高的渗透性和动态特性, 被许多人认为是原始细胞的潜在模型。单链脂肪酸的羧酸头组只允许在有限的 pH 范围内将自组装成膜, 由此产生的膜对盐的存在相当敏感。因此, 与磷脂囊泡相比, 脂肪酸泡需要不同的制备和处理方法。
在本议定书中, 虽然我们用油酸作为脂质体形成的例子, 其他长链不饱和脂肪酸 (C14) 及其衍生物 (肉豆蔻油酸, 棕榈油酸, 相应的醇和甘油酯) 也形成囊泡采用薄膜补液法, 只要总脂浓度高于 cmc, 水化缓冲 pH 值接近膜内脂肪酸的 pKa。除了在本协议中使用的三 HCl 缓冲区之外, 还报告了其他缓冲系统 (ca bicine、磷酸盐、硼酸盐) 支持脂肪酸囊形成, 尽管磷酸盐或硼酸盐缓冲液中形成的囊泡通常是相当漏水的13。在补液后产生的脂肪酸囊多分散和 multilamellar, 但容易转化为小单分散 unilamellar 囊挤压, 如所述。与超声波作为小泡产生的替代方法相比, 挤压为不同孔径膜的囊泡大小的控制提供了更多的选择。挤压后的囊泡通常比膜孔尺寸稍大, 但通过增加挤出循环的次数, 可以获得较窄的粒度分布的囊泡和平均尺寸接近膜孔径的大小。
为了合成功能性原始细胞, 脂肪酸囊需要宿主特定的生化反应产生的 RNA 或其他积木的封装。薄膜补液方法为形成含有所需封装材料的囊泡提供了一种简便的方法。然而, 封装效率相对较低, 而在纯化过程中, RNA 等珍贵材料中的一大小部分通常会丢失。在某些情况下, 在挤出前重复冻融循环可以适度地提高封装效率。微流控法用于磷脂脂质体的高产制备, 允许近100% 的封装效率, 但是还没有开发出用于脂肪酸泡的类似方法。
当处理原始细胞与螯合或自由镁2+, 净化后, 镁溶液添加和 repurification 之前, 每个时间点确保清除泄漏的封装材料可能影响的准确性的反应速率在囊泡内测量。由于每种纯化都需要至少10分钟才能取得良好的分离, 并收集囊泡馏分, 快速反应的分析是困难的, 必须在柱 repurification 之前停止反应。
我们在这里所提供的协议非常适合于构建脂肪酸脂质体, 宿主反应模仿那些可能发生在原始细胞中。我们的协议还可以在开发生物医学交付系统和其他生化反应反应器方面发挥潜在的应用价值。

脂质体, 或囊泡-由两亲脂组成的双层膜所包围的隔间, 在许多生物医学应用中被用作药物、细胞膜模型和合成细胞.我们和其他人也在早期使用脂质体作为原始细胞膜的模型。1,2,3,4通常, 在这种系统中, 我们使用单链 amphiphiles, 仅包含一个脂质烃尾 (例如, 油酸), 因为这些分子在没有现代细胞所使用的编码蛋白酶的益处的情况下更容易合成。
由单链脂组成的脂质体类似于从 diacylphospholipids (例如、1-棕榈酰-2-油酰-sn glycero-3-phosphocholine 或 POPC) 中形成的, 因为边界由双层膜组成。由两种脂质组成的脂质体可以保留溶解的有效载荷, 并可通过不同的技术进行裁减和纯化。由于单链脂质的独特化学特性, 有几个重要的差异。磷脂形成的囊泡在广泛的 ph 范围内是稳定的, 而脂肪酸泡只在中性至温和的碱性 ph 值 (约 7-9) 稳定, 这需要一定的 ph 缓冲剂进行囊泡制备。在大多数情况下, 这个缓冲也可能包含特定的可溶性分子囊泡封装, 它可以是功能性材料 (例如, RNA) 为分开的生物化学反应或简单的荧光染料 (例如, calcein) 用于囊泡特征。
只有单一的碳氢化合物链的存在, 产生的膜, 这是一个更渗透到溶质, 以及更多的动态。此外, 富含脂肪酸的羧酸头组产生的囊泡对盐和价阳离子的存在更敏感 (例如, Mg2 +)。镁是催化原始细胞生化反应的最重要的价阳离子之一, 用于生命起源的研究。在早期的生活中, 在成熟的蛋白质酶进化之前, RNA 可能是主要的聚合物, 由于它的双重能力自我复制和执行催化作用。一个有代表性的镁需要 rna 相关反应的例子是非酶 rna 复制, 第一次证明在二十世纪六十年代。5当化学活化的 RNA 核苷酸 (即2-methylimidazolide 核苷酸) 与原有的底漆模板复合物绑定时, 底漆的 3 '-羟基基团攻击相邻活性单体的 5 '-磷酸盐, 以取代离开组 (即2 甲基咪唑), 并形成新的磷酸二酯键。这种 RNA 复制化学需要高浓度的 Mg2 +, 这需要螯合, 以配合脂肪酸原始细胞。6另一个 Mg2 +依赖的反应是由锤头核酶催化的, 这也许是最具特色的催化 RNA。这种核酶可以从两个短寡核苷酸中重组, 执行一种自解切反应, 便于通过凝胶移位来监测。因此, 它经常被用作生物起源研究中的模型核酶。7由于此核酶对 unliganded 镁的要求, 脂质体通常是由脂肪酸和脂肪酸甘油酯的混合物构建的, 对镁更稳定。8,9在本协议中, 我们介绍了我们为制备、操作、表征这些囊泡而开发的技术, 并演示了这些囊泡作为原始细胞, 用于宿主非酶 RNA 复制和锤头核酵素的应用。催化.

<table>
	<tbody>
		<tr>
			<td>sephorose 4B</td>
			<td>SIGMA-ALDRICH INC</td>
			<td>4B200</td>
			<td></td>
		</tr>
		<tr>
			<td>calcein</td>
			<td>SIGMA-ALDRICH INC</td>
			<td>C0875-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>tris-HCl pH8.0 1M</td>
			<td>LIFE TECHNOLOGIES CORP</td>
			<td>AM9851</td>
			<td></td>
		</tr>
		<tr>
			<td>citric acid</td>
			<td>SIGMA-ALDRICH INC</td>
			<td>251275-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium hydroxide</td>
			<td>SIGMA-ALDRICH INC</td>
			<td>71690-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium hydroxide</td>
			<td>Sigma</td>
			<td>30614</td>
			<td></td>
		</tr>
		<tr>
			<td>oleic acid</td>
			<td>Nu-Chek</td>
			<td>U-46-A</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol monooleate</td>
			<td>Nu-Chek</td>
			<td>M-239</td>
			<td></td>
		</tr>
		<tr>
			<td>Liss Rhodamine-PE</td>
			<td>LIFE TECHNOLOGIES CORP</td>
			<td>L1392</td>
			<td></td>
		</tr>
		<tr>
			<td>magnesium chloride</td>
			<td>Fisher/Thermo Fisher Scientific</td>
			<td>AM9530G</td>
			<td></td>
		</tr>
		<tr>
			<td>sequagel concentrate</td>
			<td>National Diagnostics</td>
			<td>EC-830</td>
			<td></td>
		</tr>
		<tr>
			<td>sequagel DILUENT</td>
			<td>National Diagnostics</td>
			<td>EC-840</td>
			<td></td>
		</tr>
		<tr>
			<td>15% TBE-UREA GEL</td>
			<td>Thermo Fisher Scientific</td>
			<td>EC68852BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>urea</td>
			<td>Sigma Aldrich</td>
			<td>U6504-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>titon-100x</td>
			<td>SIGMA-ALDRICH INC</td>
			<td>T9284-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA primer</td>
			<td>IDT</td>
			<td>5&#39;Cy3-GCG UAG ACU GAC UGG</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA template</td>
			<td>IDT</td>
			<td>5&#39;-AAC CCC CCA GUC AGU CUA CGC</td>
			<td></td>
		</tr>
		<tr>
			<td>hammerhead substrate strand</td>
			<td>IDT</td>
			<td>5&#39;Cy3-GCG CCG AAA CAC CGU GUC UCG AGC</td>
			<td></td>
		</tr>
		<tr>
			<td>hammerhead ribozyme strand</td>
			<td>IDT</td>
			<td>5&#39;GGC UCG ACU GAU GAG GCG CG</td>
			<td></td>
		</tr>
		<tr>
			<td>vesicle extruder set</td>
			<td>AVANTI POLAR LIPIDS</td>
			<td>610000</td>
			<td></td>
		</tr>
		<tr>
			<td>fraction collector</td>
			<td>Gilson, Inc.</td>
			<td>171041</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Fisher</td>
			<td>NC9995941/675</td>
			<td></td>
		</tr>
		<tr>
			<td>plate reader</td>
			<td>Molecular Devices</td>
			<td>SpectraMax i3</td>
			<td></td>
		</tr>
		<tr>
			<td>confocal microscope</td>
			<td>Nikon</td>
			<td>Nikon A1R MP Confocal</td>
			<td></td>
		</tr>
		<tr>
			<td>gel scanner</td>
			<td>GE Healthcare Life Sciences</td>
			<td>Typhoon 9410 scanner</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation, purification, and use of fatty acid-containing liposomes

含有单链 amphiphiles 的脂质体, 特别是脂肪酸, 与含有 diacylphospholipids 的人相比, 由于这些 amphiphiles 的独特化学性质而具有明显的特性。特别是脂肪酸脂质体由于单链 amphiphiles 的溶解度较高而增强了动态特性。同样, 含有游离脂肪酸的脂质体对盐和价阳离子更敏感, 这是由于羧酸头基团与金属离子之间的强相互作用。在这里, 我们说明了制备、纯化和使用由单链 amphiphiles (例如、油酸) 组成的脂质体的技术。

我们通常在尺寸排除柱上执行脂质体纯化。典型的脂质体制剂含有某种荧光。当脂质体被生成和挤出时, 被封装的物种就存在于脂质体的内外。通过在尺寸排除树脂上纯化脂质体 (琼脂糖 4B), unencapsulated 溶质保留在树脂的毛孔内, 而较大的脂质体则不和洗脱首先(图 1A)。收集分数和绘制荧光与分数号 (图 1B) 通常产生两个峰值的跟踪, 与脂质体对应的早期洗脱分数, 然后收集并在后续应用中使用。
我们经常检查非酶促引伸反应, 这是一种可能的方法 rna 复制之前, 核酶和蛋白为基础的 rna 聚合酶的出现。这些反应通常使用荧光标记底漆 (图 2A), 这是由活化单体扩展。这些反应可以通过凝胶电泳 (图 2B) 来监测, 结果 electropherograms 集成在给定反应条件下获得速率常数 (图 2C)。
为了证明 rna 能在原始细胞内起作用, 我们使用锤头核酶自解裂 (图3A) 作为一种模型 rna 催化反应。这种反应需要免费的 Mg2 + , 以促进催化, 因此我们使用 OA/转基因泡, 因为它们是稳定的存在5毫米毫克2 +。与引物延伸反应相似, 锤头核酶的自解理反应也可以通过凝胶电泳 (图 3B) 进行监测, 随后分析以获取特定条件下的速率常数 (图 3C)。
我们的图像脂质体采用荧光和透射光。脂质体可以使用荧光脂来标记, 它给出一个膜标签 (图 4A), 或者在其流明内使用荧光溶质 (图 4B)。透射光也可以用来观察水泡 (也显示在图 4B中)。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="2">表1.1 氯仿中的纯油酸</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td colspan="2">组件</td>
			<td>股票</td>
			<td>量</td>
		</tr>
<tr>
<td colspan="2">油酸</td>
			<td>> 99%</td>
			<td>11.7 µL</td>
		</tr>
<tr>
<td colspan="2">氯仿</td>
			<td></td>
			<td>1毫升</td>
		</tr>
<tr>
<td colspan="2">表1.2 氯仿中油酸和甘油油酸酯 (9:1)</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td colspan="2">组件</td>
			<td>股票</td>
			<td>量</td>
		</tr>
<tr>
<td colspan="2">油酸</td>
			<td>> 99%</td>
			<td>10.5 µL</td>
		</tr>
<tr>
<td colspan="2">甘油油酸酯</td>
			<td>> 99%</td>
			<td>1.4 µL</td>
		</tr>
<tr>
<td colspan="2">氯仿</td>
			<td></td>
			<td>1毫升</td>
		</tr>
<tr>
<td colspan="2">表1.3 氯仿中的 0.2mol%rhodamine 油酸</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td colspan="2">组件</td>
			<td>股票</td>
			<td>量</td>
		</tr>
<tr>
<td colspan="2">油酸</td>
			<td>> 99%</td>
			<td>1.6 µL</td>
		</tr>
<tr>
<td colspan="2">罗丹明-氯仿中的 PE</td>
			<td>10毫米</td>
			<td>20µL</td>
		</tr>
<tr>
<td colspan="2">氯仿</td>
			<td></td>
			<td>1毫升</td>
		</tr>
</tbody></table>表1。脂肪酸氯仿溶液。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57324/57324fig1.jpg"> 图1。纯化分数的囊泡纯化及荧光表征。a.琼脂糖4B 柱上的游离 calcein 中含有 calcein 的囊泡的分离。B.囊泡和自由 calcein 峰值检测通过在每一个井数采集后绘制出荧光量与井号。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57324/57324fig1large.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/57324/57324fig2.jpg"> 图2。非酶 RNA 复制在 OA 囊泡。A.非酶 RNA 引物延伸方案。B.在纯油酸泡内引伸反应的页面图像, 其条件与第4节中的情况相同。C.在给定时间内剩余的底漆的自然对数的线性拟合, 指向初始量的底漆与时间超过 30 h. 反应速率, 从 ln (p/p0) 与时间的斜率计算, 为 0.058 h-1。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57324/57324fig2large.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/57324/57324fig3.jpg"> 图3。锤头核酶在 OA/转基因囊中裂解。A.锤头核酶裂解荧光标记衬底链 (顶部) 的方案。B. 5 毫米毫克2 +的 OA/转基因囊中锤头核酶裂解的页面图像。C.核酶在囊泡内活动。在给定时间内剩余的基底量比值的自然对数的线性拟合点与初始的基底量相对于前 4 h. 反应速率, 从 ln (s/s0) 与时间的斜率计算, 为 0.36 h-1。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57324/57324fig3large.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/57324/57324fig4.jpg"> 图4。巨大的脂肪酸囊, 用于显微术。a.一种罗丹明 PE 标记为油酸囊的共焦显微图像, 鳞片10微米。B.在传输探测器 (TD) 通道中显示的含有 Alexa488 标记 RNA 的油酸囊的共焦显微图像, 刻度条5微米。

Watch this Scientific Journal Video about Preparation, Purification, and Use of Fatty Acid-containing Liposomes at JoVE.com

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

含有单链 amphiphiles 的脂质体, 特别是脂肪酸, 与含有 diacylphospholipids 的单链 amphiphiles 的独特化学性质相比, 具有明显的特性。在这里, 我们描述的技术, 以制备, 纯化和使用脂质体组成的部分或整个这些 amphiphiles。

含脂肪酸脂质体的制备、纯化及应用

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

preparation-purification-and-use-of-fatty-acid-containing-liposomes

Research

JoVE Journal

Bioengineering

39.2K 次观看.  Massachusetts General Hospital. 含有单链 amphiphiles 的脂质体, 特别是脂肪酸, 与含有 diacylphospholipids 的单链 amphiphiles 的独特化学性质相比, 具有明显的特性。在这里, 我们描述的技术, 以制备, 纯化和使用脂质体组成的部分或整个这些 amphiphiles。

视频: Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

人类血管周围干细胞用于骨再生

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

科研

生物工程

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration1,2 and efficient separation and extraction based on ionic mobility.3 For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation4-8 and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood.9-12
ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.13). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species.
We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation.
We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.

Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.

对芯片等速电泳离子的分离和纯化核酸

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

表达，分离，纯化可溶性和不溶性蛋白质生物素对神经组织再生

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect resulting in significant reductions in systemic toxicity. Nonetheless, insufficient release of encapsulated drug from liposomes has limited their clinical efficacy. Temperature-sensitive liposomes have been engineered to provide site-specific release of drug in order to overcome the problem of limited tumor drug bioavailability. Our lab has designed and developed a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, to provide triggered release of CDDP at solid tumors. Heat-activated delivery in vivo was achieved in murine models using a custom-built laser-based heating apparatus that provides a conformal heating pattern at the tumor site as confirmed by MR thermometry (MRT). A fiber optic temperature monitoring device was used to measure the temperature in real-time during the entire heating period with online adjustment of heat delivery by alternating the laser power. Drug delivery was optimized under magnetic resonance (MR) image guidance by co-encapsulation of an MR contrast agent (i.e.,&#160;gadoteridol) along with CDDP into the thermosensitive liposomes as a means to validate the heating protocol and to assess tumor accumulation. The heating protocol consisted of a preheating period of 5 min prior to administration of HTLC and 20 min heating post-injection. This heating protocol resulted in effective release of the encapsulated agents with the highest MR signal change observed in the heated tumor in comparison to the unheated tumor and muscle. This study demonstrated the successful application of the laser-based heating apparatus for preclinical thermosensitive liposome development and the importance of MR-guided validation of the heating protocol for optimization of drug delivery.

AN MRI-compatible&#160;custom-designed laser-based heating apparatus has been developed to provide local heating of subcutaneous tumors in order to activate release of agents from thermosensitive liposomes specifically at the tumor region.

定制设计的基于激光的加热装置顺铂触发释放的热敏脂质体与磁共振成像制导

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect ...

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

在脉冲激光激发金纳米粒子集成光电响应脂质体及其微泡空化的测量的合成

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

治疗的siRNA递送至CNS使用阳离子和阴离子脂质体

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  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 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 ...

Use of Micro X-ray Computed Tomography with Phosphotungstic Acid Preparation to Visualize Human Fibromuscular Tissue

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate structures&#160;while processing and histological observation provide limited information through cross-sectional imaging. Micro X-ray computed tomography (microCT) is an effective tool for obtaining three-dimensional information without damaging specimens. However, it shows limited efficiency in differentiating soft tissue parts. Use of contrast-enhancing agents, like phosphotungstic acid (PTA), can solve this problem by improving soft tissue contrast. We implemented microCT with PTA to investigate the human orbicularis retaining ligament (ORL), which is a delicate structure in the orbit area. In this method, harvested specimens are fixed in formalin, dehydrated in serial ethanol solutions, and stained with a PTA solution. After staining, microCT scanning, 3D reconstruction, and analysis are performed. Skin, ligaments, and muscles can be clearly visualized using this method. The specimen size and duration of staining are essential features of the method. The suitable specimen thickness was about 5&#8211;7 mm, above which the process was slowed, and the optimum duration was 5&#8211;7 days, below which an empty hole in the central area occasionally occurred. To maintain the location and direction of small pieces during cutting, sewing on the same region of each part is recommended. Furthermore, preliminary analyses of the anatomical structure are needed to correctly identify each piece. Parafilm can be used to prevent drying, but care should be taken to prevent specimen distortion. Our multidirectional observation showed that the ORL is composed of a multilayered meshwork of continuous plates, rather than thread-like fibers, as reported previously. These results suggest that microCT scanning with PTA is useful for examining specific compartments within complex structures of human tissue. It may be helpful in the analyses of cancer tissues, nerve tissues, and various organs, like the heart and liver.

Micro X-ray computed tomography is effective in obtaining three-dimensional information from undamaged human specimens but has limited success in observing soft tissues. The use of phosphotungstic acid contrast agent can resolve this issue. We implemented this contrast agent to examine human delicate fibromuscular tissues (the orbicularis retaining ligament).

使用微X射线计算机断层扫描与磷酸制备可视化人类纤维肌组织

Manual dissection and histological observation are common methods used to investigate human tissues. However, manual dissection can damage delicate ...

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

定量荧光显微镜为基础的单脂体测定，用于检测单个脂质体之间的成分不均匀性

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...