Name: preparation, purification, and use of fatty acid-containing liposomes
Uploaded: 2018-02-09T13:00:00.000+00:00
Duration: 10 min 43 s
Description: Watch this Scientific Journal Video about Preparation, Purification, and Use of Fatty Acid-containing Liposomes at JoVE.com

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>
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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&#039;Cy3-GCG UAG ACU GAC UGG</td><td></td></tr><tr><td>RNA template</td><td>IDT</td><td>5&#039;-AAC CCC CCA GUC AGU CUA CGC</td><td></td></tr><tr><td>hammerhead substrate strand</td><td>IDT</td><td>5&#039;Cy3-GCG CCG AAA CAC CGU GUC UCG AGC</td><td></td></tr><tr><td>hammerhead ribozyme strand</td><td>IDT</td><td>5&#039;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/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/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/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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

41.8K 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

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

恒压控制的挤压方法制备纳米脂质体

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

科研

生物工程

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

无洗涤剂超快重组膜蛋白成脂双层使用融合补充充电 Proteoliposomes。

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

生物化学

Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and lipids during endocytosis, sorting within the endosomal system, and autophagy. Central to SNX-BAR protein function is the ability to form homodimers or heterodimers that bind membranes using highly conserved phox-homology (PX) and BAR (Bin/Amphiphysin/Rvs) domains. In addition, oligomerization of SNX-BAR dimers on membranes can elicit the formation of membrane tubules and vesicles and this activity is thought to reflect their functions as coat proteins for endosome-derived transport carriers. Researchers have long utilized in vitro binding studies using recombinant SNX-BAR proteins on synthetic liposomes or giant unilamellar vesicles (GUVs) to reveal the precise makeup of lipids needed to drive membrane remodeling, thus revealing their mechanism of action. However, due to technical challenges with dual expression systems, toxicity of SNX-BAR protein expression in bacteria, and poor solubility of individual SNX-BAR proteins, most studies to date have examined SNX-BAR homodimers, including non-physiological dimers that form during expression in bacteria. Recently, we have optimized a protocol to overcome the major shortcomings of a typical bacterial expression system. Using this workflow, we demonstrate how to successfully express and purify large amounts of SNX-BAR heterodimers and how to reconstitute them on synthetic liposomes for binding and tubulation assays.

Here, we present a workflow for the expression, purification and liposome binding of SNX-BAR heterodimers in yeast.

萌芽酵母 SNX-BAR 异体体的表达、纯化和脂质结合

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and ...

遗传学

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

单脂蛋白的SNARE介导融合在一个微流控流细胞拴支持双分子层通过偏光显微镜TIRF监控

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

神经科学

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the endoplasmic reticulum.

人工脂质纳米管网作为内质神经的底部模型的自发形成与重排

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

生物活性亲水球状蛋白的纳米脂质体制备与表征

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

含脂脂的微流体生产-温度敏感脂质体

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...

Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome formulation, liposomes may be preferentially taken up by different cell types in the body. This may influence the efficacy of the therapeutic particle as progression of different diseases is tissue- and cell-type-specific. In this protocol, we present one method for synthesizing and fluorescently labeling liposomes using DSPC, cholesterol, and PEG-2000 DSPE and the lipid dye DiD as a fluorescent label. This protocol also presents an approach for delivering liposomes in vivo and assessing cell-specific uptake of liposomes using flow cytometry. This approach can be used to determine the types of cells that take up liposomes and quantify the distribution and proportion of liposome-uptake across cell types and tissues. While not mentioned in this protocol, additional assays such as immunofluorescence and single-cell fluorescence imaging on a cytometer will strengthen any findings or conclusions made as they permit assessment of intracellular staining. Protocols may also need to be adapted depending on the tissue(s) of interest.

The goal of this protocol is to synthesize fluorescently-labeled liposomes and use flow cytometry to identify in vivo localization of liposomes at a cellular level.

荧光染料标记脂质体体内组织特异性细胞摄取的制备、给药和评估

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome ...

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

细胞模拟支持和悬浮脂质双分子层模型的组装用于分子相互作用研究

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...

Examining BCL-2 Family Function with Large Unilamellar Vesicles

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis1. Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)1. After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues2.
In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)3-6. Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation7,8. In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members7,9. As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane.
 Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization10. LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)10. This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for kinetic analyses of protein interactions and real-time measurements of membrane permeabilization, which is based on LUVs containing a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (DPX: p-xylene-bis-pyridinium bromide)11. As the LUVs permeabilize, ANTS and DPX diffuse apart, and a gain in fluorescence is detected. Here, commonly used recombinant BCL-2 family protein combinations and controls using the LUVs containing ANTS/DPX are described.

Biochemically-defined large unilamellar vesicles (LUVs) are a convenient model system to analyze BCL-2 family interactions with immediate implications in better understanding the mitochondrial pathway of apoptosis. A method to produce LUVs, along with standard BCL-2 family protein combinations and controls to examine LUV permeabilization, are presented.