Name: a tripeptide-stabilized nanoemulsion of oleic acid
Uploaded: 2019-02-27T13:00:00
Description: Watch this Scientific Journal Video about A Tripeptide-Stabilized Nanoemulsion of Oleic Acid at JoVE.com

我们感谢国家癌症研究所提供的财政支持, 提供 sc2ca206194。没有宣布相互竞争的金融利益。

1. 油酸的合成–铂 (ii) 共轭
<ol><li>顺铂的激活<ol>
<li>在60°c 水中 (例如纳米颗粒) 悬浮50毫克 (0.167 mmol) 顺铂。</li>
		<li>在0.5 毫升的水中加入液位55.2 毫克 (0.325 mmol)agno 3, 在顺铂溶液中搅拌反应至少2小时。agcl 的白色沉淀将形成, 表明反应的进展。</li>
		<li>要确定活化反应是否完成, 请用10% 的 hcl 进行测试, 以确定溶液中是否存在游离银+离子。测试应该是阴性的 (不应该形成额外的 a...

纳米乳液合成的关键步骤包括调整基材的摩尔比, 在油酸-pt (ii) 加法期间保持温度和流速控制, 为自组装提供足够的时间, 并使用离心式集中机柱。这些参数影响 kyf-pt-ne 的大小和形态;因此, 保持适当的摩尔比, 正确调整合成条件尤为重要。
纳米乳液合成过程中基板的比例 (步骤 3) 对自组装过程至关重要, 并决定了产品的最终尺寸。kyf 与油酸-pt (ii) 摩尔比为 1:3, 水中三聚酰亚胺?...

近年来, 人们对用于药物输送和生物成像等生物医学应用的纳米粒子的工程越来越感兴趣, 这些应用包括 1、2、3、4。基于纳米粒子的系统的多功能往往需要将多个组件纳入一个配方。以脂质或聚合物为基础的构建块往往因其物理化学特性及其生物相容性和生物降解性而不同, 这最终可能影响纳米结构1的功能, 5,6。生物衍生材料, 如蛋白质和肽, 由于其序列的灵活性7,8,早已被公认为多功能纳米结构中很有前途的成分。多肽自组装成高度有序的超分子结构, 形成螺旋丝带9,10, 纤维支架11,12和更多, 从而为建设铺平道路采用自下而上的方法13的生物分子基混合纳米结构.
多肽已被探索在医学和生物技术中的应用, 特别是用于抗癌治疗14和心血管疾病15 , 以及抗生素开发16,17, 代谢疾病18, 感染19。有一百多个小肽疗法正在进行临床试验20。多肽易于修改和快速合成在较低的成本。此外, 它们还可生物降解, 极大地促进了它们的生物和制药应用 21,22.多肽作为结构成分的使用包括反应灵敏的、基于肽的纳米粒子和水凝胶库的工程, 用于控制释放 23、24、25、26,27、基于肽的生物传感器28、29、30、31或生物电子设备32、33、34.重要的是, 即使是含有两到三个氨基酸残留物 (包括苯基丙氨酸) 的短肽, 也能指导自组装过程35、36、37, 并产生稳定的乳液38.
铂基药物由于其高效, 在许多癌症治疗方案中单独使用, 并与其他药物结合使用 39,40。铂化合物通过形成单加合物和链内或链间交联来诱导 dna 损伤。pt-dna 病变被细胞机械识别, 如果不修复, 会导致细胞凋亡。最重要的机制, pt (ii) 贡献导致癌细胞死亡, 是 dna 转录41,42的抑制。然而, 铂治疗的好处是减少的系统毒性的 pt (ii), 引发严重的副作用。这导致较低的临床剂量的 pt (ii)43, 这往往导致亚治疗浓度的铂到达 dosing。因此, 随后的 dna 修复有助于癌细胞的存活和获得 pt (ii) 的抵抗力。铂耐药是抗癌治疗中的一个主要问题, 也是治疗失败的主要原因 44,45。
我们开发了一种稳定的纳米系统, 该系统封装了 pt (ii) 剂, 以便在系统循环中提供屏蔽效果, 并减少 pt (ii) 诱导的副作用。该系统是基于油酸-pt (ii) 核心稳定与 kyf 三肽, 形成纳米乳液 (kyf-pt-ne)46。kyf-pt-ne 的构建块, 三肽的氨基酸, 以及油酸, 具有公认的安全 (gras) 地位, 在食品药品监督管理局 (fda)。采用纳米沉淀法47制备了 kyf-pt-ne。简而言之, 油酸-pt (ii) 共轭溶解在有机溶剂中, 然后在37°c 下滴入水 kyf 溶液 (图 1)。该溶液搅拌数小时, 以便 pyf-pt-ne 自行组装。纳米乳液集中在 10 kda 离心式集中器中, 用清水清洗3次。通过对 kyf 的化学改性和荧光粉, 可以合成适用于生物医学成像的荧光 fitc-kyf-pt-ne。

<table>
	<tbody>
		<tr>
			<td>2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium 
			tetrafluoroborate (TBTU)</td>
			<td>ANASPEC INC.:</td>
			<td>AS-20376</td>
			<td>SPPS</td>
		</tr>
		<tr>
			<td>4-well chamber confocal dish</td>
			<td>Lab-Tek II, Thermo Fisher Scientific</td>
			<td>154526</td>
			<td>For imaging</td>
		</tr>
		<tr>
			<td>6-bromohexanoic acid</td>
			<td>Chem-Impex INT&#8217;L INC.</td>
			<td>24477</td>
			<td>Click modification for peptide</td>
		</tr>
		<tr>
			<td>A2780</td>
			<td>Generously doanted by professor John Martignetti from The Mount Sinai Hospital</td>
			<td></td>
			<td>Ovarian cancer cell line</td>
		</tr>
		<tr>
			<td>Barnstead Nanopure</td>
			<td>Thermo Fisher</td>
			<td>D11901</td>
			<td>water filtration system</td>
		</tr>
		<tr>
			<td>BUCHI rotavapor R-3</td>
			<td>Buchi</td>
			<td>Z568090</td>
			<td>For solvent removal and sample drying</td>
		</tr>
		<tr>
			<td>Centrifuge 5810 R</td>
			<td>eppendorf</td>
			<td>5811F</td>
			<td>For platinum complex separation</td>
		</tr>
		<tr>
			<td>Cis-dichlorodiamineplatinum (II) 99%</td>
			<td>Acros Organics</td>
			<td>19376-0050</td>
			<td>in vitro tests</td>
		</tr>
		<tr>
			<td>CP70</td>
			<td>Generously doanted by professor John Martignetti from The Mount Sinai Hospital</td>
			<td></td>
			<td>Ovarian cancer cell line</td>
		</tr>
		<tr>
			<td>Digital water bath</td>
			<td>VWR</td>
			<td>97025-134</td>
			<td>For warming up media for cell culture</td>
		</tr>
		<tr>
			<td>Dynamic Light Scattering (DLS)</td>
			<td>Brookhaven Instrument Corporation</td>
			<td></td>
			<td>For nanoparticle size measurments</td>
		</tr>
		<tr>
			<td>ES-2</td>
			<td>ATCC</td>
			<td>CRL-1978</td>
			<td>ovarian cancer cell line</td>
		</tr>
		<tr>
			<td>Fmoc-L-Lys(Boc)-OH 99.79%</td>
			<td>Chem-Impex INT&#8217;L INC.</td>
			<td>00493</td>
			<td>SPPS</td>
		</tr>
		<tr>
			<td>Fmoc-L-Phe 4-alkoxybenzyl alcohol resin (0.382 meq/g),</td>
			<td>Chem-Impex INT&#8217;L INC.</td>
			<td>01914</td>
			<td>SPPS</td>
		</tr>
		<tr>
			<td>Fmoc-LTyr(tBu)-OH 98%</td>
			<td>Alfa Aesar</td>
			<td>H59730</td>
			<td>SPPS</td>
		</tr>
		<tr>
			<td>HERACELL 150i CO2 incubator</td>
			<td>Thermo Scientific Fisher</td>
			<td></td>
			<td>incubator</td>
		</tr>
		<tr>
			<td>High pressure syringe pump</td>
			<td>New Era</td>
			<td>1010-US</td>
			<td>For platinum complex addition in nanoparticle synthesis</td>
		</tr>
		<tr>
			<td>Hotplate/stirrer</td>
			<td>VWR</td>
			<td>12365-382</td>
			<td>For sample stirring and heating</td>
		</tr>
		<tr>
			<td>LAMP-1 Antibody(cojugated with Alexa Fluor 647)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-18821 AF647</td>
			<td>For imaging</td>
		</tr>
		<tr>
			<td>N,N-diisopropylethylamine (DIPEA)</td>
			<td>Oakwood Chemical</td>
			<td>005027</td>
			<td>SPPS</td>
		</tr>
		<tr>
			<td>Ninhydrin 99%</td>
			<td>Alfa Aesar</td>
			<td>A10409</td>
			<td>Kaiser test</td>
		</tr>
		<tr>
			<td>Oleic acid</td>
			<td>Chem-Impex INT&#8217;L INC.</td>
			<td>01421</td>
			<td>For platinum complex synthesis</td>
		</tr>
		<tr>
			<td>OV90</td>
			<td>ATCC</td>
			<td>CRL-11732</td>
			<td>Ovarian cancer cell line</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td>For cell wash</td>
		</tr>
		<tr>
			<td>Permount mounting medium</td>
			<td>Fisher Chemical</td>
			<td>SP15-100</td>
			<td>For imaging</td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Fisher Chemical</td>
			<td>A92500</td>
			<td>Kaiser test</td>
		</tr>
		<tr>
			<td>Phosphotungstic acid</td>
			<td>Fisher Chemical</td>
			<td>A248-25</td>
			<td>negative stain for TEM</td>
		</tr>
		<tr>
			<td>Piperidine 99%</td>
			<td>BTC</td>
			<td>219260-2.5L</td>
			<td>SPPS</td>
		</tr>
		<tr>
			<td>Platinum AAS standard soultion</td>
			<td>Alfa Aesar</td>
			<td>88086</td>
			<td>1000ug/ml for calibration curve</td>
		</tr>
		<tr>
			<td>Propargyl bromide 97%</td>
			<td>Alfa Aesar</td>
			<td>L10595</td>
			<td>For alkyne modification of fluoresceine</td>
		</tr>
		<tr>
			<td>Scientific biological cabinet</td>
			<td>Thermo Scientific Fisher</td>
			<td>1385</td>
			<td>Bio-hood for cell culture</td>
		</tr>
		<tr>
			<td>Self-Cleaning Vacuum System</td>
			<td>Welch</td>
			<td>2028</td>
			<td>Vacuum pump for rotavapor</td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Acros Organics</td>
			<td>19768-0250</td>
			<td>Cisplatin activation</td>
		</tr>
		<tr>
			<td>SKOV3</td>
			<td>ATCC</td>
			<td>HTB-77</td>
			<td>Ovarian cancer cell line</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S313-1</td>
			<td>For platinum complex synthesis</td>
		</tr>
		<tr>
			<td>Tin (II) chloride</td>
			<td>Sigma Aldrich</td>
			<td>208256</td>
			<td>Test for Platinum presence</td>
		</tr>
		<tr>
			<td>TOV21G</td>
			<td>ATCC</td>
			<td>CRL-11730</td>
			<td>Ovarian cancer cell line</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid 99% (TFA)</td>
			<td>Alfa Aesar</td>
			<td>L06374</td>
			<td>SPPS</td>
		</tr>
		<tr>
			<td>Triisopropylsilane (TIPS)</td>
			<td>Chem-Impex INT&#8217;L INC.</td>
			<td>01966</td>
			<td>SPPS</td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma Aldrich</td>
			<td>T8787-100ML</td>
			<td>For imaging</td>
		</tr>
		<tr>
			<td>Uranine powder 40%</td>
			<td>Fisher Scientific</td>
			<td>S25328A</td>
			<td>For alkyne modification of fluoresceine</td>
		</tr>
		<tr>
			<td>Vivaspin 20 (10000 MWCO)</td>
			<td>Sartorious</td>
			<td>VS2001</td>
			<td>For Nanoparticle wash and condensation</td>
		</tr>
		<tr>
			<td>VWR Inverted Microscope</td>
			<td>VWR</td>
			<td>89404-462</td>
			<td>For cell culture monitoring</td>
		</tr>
	</tbody>
</table>

a tripeptide-stabilized nanoemulsion of oleic acid

我们描述了一种方法, 以产生纳米乳液组成的油酸-pt (ii) 核和赖氨酸-酪氨酸-苯基丙氨酸 (kyf-pt-ne) 涂层。kyf-pt-ne 封装 pt (ii) 为 10 wt%, 直径为 107±27 nm, 表面电荷为负。kyf-pt-ne 在水和血清中都是稳定的, 具有生物活性。荧光体与 kyf 的结合允许合成适用于生物成像的荧光纳米乳液。纳米乳液的合成是在水环境中进行的, kyf-pt-ne 是通过短 kyf 肽和油酸-铂 (ii) 共轭的自组装形成的。自组装过程取决于溶液的温度、基板的摩尔比和基板添加的流速。关键的步骤包括在合成过程中保持最佳的搅拌速率, 允许足够的时间进行自组装, 以及在离心式集中器中逐步预浓缩纳米乳液。

图 2a 显示了使用该协议编写的 kyf-pt-ne 的代表性 tem 图像。kyf-pt-ne 在形态上呈球形, 分散良好, 尺寸均匀。kyf-pt-ne 的核心直径为 107±27 nm, 直接从三个 tem 图像中测量, 至少完成了200次测量。利用动态光谱 (dls) 对 kyf-pt-ne 的流体动力直径进行了分析, 发现其动态直径为240纳米, 多分散性指数为0.156。确定了 kyf-pt-ne 在水中的 zeta 电位, 确定了三个独立合成?...

Watch this Scientific Journal Video about A Tripeptide-Stabilized Nanoemulsion of Oleic Acid at JoVE.com

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

一种三肽稳定的油酸纳米乳液

该方案描述了一种合成与赖氨酸-酪氨酸-苯基丙氨酸 (kyf) 三肽稳定的油酸-铂 (ii) 共轭纳米乳液的有效方法。纳米乳液通过 kyf 和共轭的自组装在温和的合成条件下形成。

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

鉴于短肽序列的范围很广，这种方法可以扩展到许多其他纳米粒子设计。肽修饰可以使系统适合诊断目的。将抗癌铂金封装到具有可生物降解、低毒性稳定剂的药物中，与药物应用具有高度相关性。卵巢癌的初步体内研究是有希望的，将进一步研究。首先，在60摄氏度的超纯水中悬浮50毫克的顺铂。将硝酸银溶液滴入顺铂溶液中，以300至400 RPM的转速在黑暗中60摄氏度搅拌混合物两小时。之后，使用10%的重量盐酸，以确认溶液中没有自由银离子。如果添加盐酸后未产生额外的氯化银，则将混合物在重力为 3，220 倍的离心10分钟。去掉上清液，然后通过0.2微米注射器过滤器过滤。然后，在铂金滤液中，将两到三滴到10个氯化物晶体中。如果悬架变成深棕色或橙色，铂金是存在的。接下来，在一毫升水中加入13.3毫克氢氧化钠，在60摄氏度的三毫升水中悬浮油酸94.2毫克。在60摄氏度下搅拌混合物两小时。然后，关闭热量，在室温下继续搅拌16至24小时，以获得原油作为油性黄褐色沉淀物。将反应混合物在重力的3，220倍下离心10分钟，然后取出上一代。在25摄氏度的旋转蒸发器上干燥产品。通过涡旋将原油悬浮在五毫升乙酰丙尼三叶草中，通过离心回收产品。重复此洗涤过程至少两次三次，以获得纯，油酸铂II结合作为淡黄色固体。首先，将5.7克Fmoc保护的L-苯丙氨酸功能化王树脂浸泡在25毫升二甲基甲酰胺中一小时。然后，将树脂浸泡在 DMF 中 15 毫升 20% 管中，持续 5 分钟，同时以 80 RPM 的速度摇动，以解热胺。排空树脂并重复除保护 20 分钟。之后用D0F和异丙醇清洗树脂。一旦分离保护被确认，加入Fmoc保护L-酪氨酸TBTU和DIPEA，搅拌混合物16至24小时，以执行耦合。用 DMF 和异丙醇清洗树脂。执行凯撒测试，继续使用前文所述的去保护和清洗。夫妇和去保护Fmoc保护Lysine的方式相同。之后用D0F、IPA、甲醇、二氯甲烷和二乙醚清洗树脂。留出一克 KYF 轴承树脂进行 FITC 改性。将剩余的树脂浸泡在95%的三氟乙酸中。2.5%三异丙基硅烷和2.5%水三小时，从树脂中切出三肽。用二乙醚沉淀粗肽冷却至20摄氏度。在30至40毫升的冷二乙醚中离心，在真空中干燥三次沉淀物。接下来，将剩余的KYF树脂与120.1毫克6-乙酸甲酸混合在30毫升D分甲酸与TBTU和DIPEA中。在室温下搅拌混合物16至24小时，以获得阿齐德改性三肽。然后，将253毫克的这种树脂与3.78毫克的固体铜（I）碘化物71.9毫克的丙丙基花环和2.24毫克的IPEA。让混合物在摇晃24小时时做出反应。然后，彻底清洗 FITC 改性 KYF 轴承树脂。从树脂中切出 FITC 的 KYF，并像未经过修饰的三肽一样进行纯化。要开始制备纳米，将10毫克的油酸铂二结合在1.5毫升的 IPA 中。将溶液绘制到 5 毫升注射器中，连接 20 量针，然后将注射器安装在反应区域的注射器泵上。将注射器泵设置为每分钟 0.1 毫升。对于使用 FITC 改性 KYF 的纳米乳液，在 20 毫升水中溶解 1 毫克 FITC 改性 KYF 和 1 毫克未改性 KYF。对于未修改的 KYF 的纳米乳液，在 20 毫升水中溶解两毫克 KYF。将 FITC 修改或未修改的 KYF 溶液加热至 37 摄氏度。确保 KYF 溶液以约 400 RPM 的转速搅拌，并确保注射器与溶液对齐。然后，启动注射器泵开始添加油酸铂二结合滴明智。添加偶联体后，将搅拌速度降低至 150 RPM，使溶液冷却至室温。继续搅拌 16 至 24 小时。然后，将纳米乳化液集中在一个10，000道尔顿离心过滤器单元中。用四毫升的超纯水在离心过滤器单元中洗三次纳米乳液。然后把它储存在摄氏四度的水溶液中。最终产品为KYF纳米粒子的蛋白石，以及用于FTIC改性纳米粒子的荧光。用未经修改的KYF准备的纳米乳液中的液滴是球形的，分散的，大小相对均匀。根据透射电子显微镜图像，核心直径约107纳米。FITC改性纳米乳液，以绿色出现在这里，可以进入各种卵巢癌细胞系的细胞。虽然两种制剂的水动力直径在四个月的蓄水期间增加，但总体尺寸得到保留。此外，在20%的胎儿牛血清中孵育一天后，未观察到乳液的蛋白化。乳液中铂金的释放速度最慢，为pH7.4，4小时后仅释放20.8%的铂金。随着pH下降，铂释放加速。含铂金的KYF涂层纳米乳液降低了这些卵巢癌细胞系的生存能力，其程度远远大于与临床相关的模拟类碳铂。纳米化也显示出可比或更大的生存能力下降比顺铂在六个细胞系中的五个。KYF 涂层纳米乳液持续导致与油酸铂 II 结合剂相比，其可行性具有可比性或更大性。这是一种简单、有效的方法，用于在三肽基纳米粒子支架中封装疏水疗法。短肽可生物降解，毒性低，对医学和生物技术具有重要意义。在烟罩下执行此程序，避免接触有机溶剂。铂油酸和KYF铂纳米乳液具有抗癌特性，因此应特别注意处理。该方法在低纤维活性和设备方面很简单，但依赖于合成策略和基本化学概念的正确应用。我们的方法可以扩展，以提供其他小药物分子。纳米粒子芯由油酸组成，因此适用于疏水剂的封装。

a-tripeptide-stabilized-nanoemulsion-of-oleic-acid

Research

JoVE Journal

Bioengineering

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Micropatterned表面透明质酸与肿瘤细胞相互作用的研究

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

科研

生物工程

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

细胞内胆汁酸动态实时监控使用遗传编码的FRET为基础的胆汁酸传感器

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of this new field of biomaterials research is due to the necessity to develop implants capable of mimicking the complex functionality of the various tissues, including a continuous change from one structure or composition to another. In this latter context, one topic of main interest concerns the design of appropriate scaffolds for bone-cartilage interface tissue. In this study, three-layered scaffolds with graded pore size were achieved by melt mixing poly(lactic acid) (PLA), sodium chloride (NaCl) and polyethylene glycol (PEG). Pore size distributions were controlled by NaCl granulometry and PEG solvation. Scaffolds were characterized from a morphological and mechanical point of view. A correlation between the preparation method, the pore architecture and compressive mechanical behavior was found. The interface adhesion strength was quantitatively evaluated by using a custom-designed interfacial strength test. Furthermore, in order to imitate the human physiology, mechanical tests were also performed in phosphate buffered saline (PBS) solution at 37 &#176;C. The method herein presented provides a high control of porosity, pore size distribution and mechanical performance, thus offering the possibility to fabricate three-layered scaffolds with tailored properties by following a simple and eco-friendly route.

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

In this work, poly(lactic acid)/polyethylene glycol (PLA/PEG) scaffolds were prepared by using a combination of melt mixing and selective leaching. The method herein discussed permitted to develop three-layer scaffolds by highly controlling both porosity and pore size. The mechanical properties were also evaluated in a physiological environment.

的简便和环保路线制造聚乳酸支架与分级孔径

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of ...

Sustained Administration of &#946;-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic &#946;-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to &#946;-cells in culture and in transplanted tissue models. These technologies allow the study of candidate &#946;-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating &#946;-cell proliferation in a culture system is critical before moving forward to in vivo models. Herein, we describe a method to co-culture intact mouse islets with biodegradable compound of interest (COI)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for the purpose of assessing the effects of sustained in situ release of mitogenic factors on &#946;-cell proliferation. This technique describes in detail how to generate PLGA microspheres containing a desired cargo using commercially available reagents. While the described technique uses recombinant human Connective tissue growth factor (rhCTGF) as an example, a wide variety of COI could readily be used. Additionally, this method utilizes 96-well plates to minimize the amount of reagents necessary to assess &#946;-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.

Sustained Administration of &amp;#946;-cell Mitogens to Intact Mouse Islets &lt;em&gt;Ex Vivo&lt;/em&gt; Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of &#946;-cell proliferation. This method is suitable for determining the efficacy of candidate &#946;-cell mitogens.

β细胞的有丝分裂原持续管理局完整小鼠胰岛<em&gt;离体</em&gt;使用可生物降解的聚（乳酸 - 共 - 乙醇酸）微球

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the ...

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing termini of oligo- and polysaccharides, as well as its unique chemical characteristics, sialic acid is involved in a multitude of different receptor-ligand interactions. By modifying the expression of sialic acid on the cell surface, sialic acid-dependent interactions will consequently be influenced. This can be helpful to investigate sialic acid-dependent interactions and has the potential to influence certain diseases in a beneficial way. Via metabolic glycoengineering (MGE), the expression of sialic acid on the cell surface can be modulated. Herein, cells, tissues, or even entire animals are treated with C2-modified derivatives of N-acetylmannosamine (ManNAc). These amino sugars act as sialic acid precursor molecules and therefore are metabolized to the corresponding sialic acid species and expressed on glycoconjugates. Applying this method produces intriguing effects on various biological processes. For example, it can drastically reduce&#160;the expression of polysialic acid (polySia) in treated neuronal cells and thus affects neuronal growth and differentiation. Here, we show the chemical synthesis of two of the most common C2-modified N-acylmannosamine derivatives, N-propionylmannosamine (ManNProp) as well as N-butanoylmannosamine (ManNBut), and further show how these non-natural amino sugars can be applied in cell culture experiments. The expression of modified sialic acid species is quantified by high performance liquid chromatography (HPLC) and further analyzed via mass spectrometry. The effects on polysialic acid expression are elucidated via Western blot using a commercially available polysialic acid antibody.

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid is a typical monosaccharide-unit found in glycoconjugates. It is involved in a plethora of molecular and cellular interactions. Here we present a method to modify cell surface sialic acid expression using metabolic glycoengineering with N-acetylmannosamine derivatives.

利用N酰基修饰 Mannosamines 对唾液酸的代谢 Glycoengineering

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

异条件下具有不同刚度的聚乙烯醇-钴-衣康酸水凝胶的人多能干细胞培养

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

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

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these 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 ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

透明质酸基水凝胶对3维患者源性胶质瘤细胞培养的研究

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Measurement of the Acid-Resistant Fluorescent Reporter Protein mCherry2

Pick any colony from the MRS agar plate containing electroporated L.reuteri cells and inoculate into a 96 well microplate containing filter sterilized MRS broth per well and an appropriate selection antibiotic. Incubate aerobically for 24 hours at 37 degrees Celsius without shaking. The next day, L.reuteri will precipitate out of the media in the stationary phase.Resuspend the culture by pipetting. Add 200 microliters of culture into a flat, clear, bottom 96 well plate. Transfer the plate to a plate reader and measure the optical density and fluorescence of M cherry two and other relevant reporters.Colony morphology noticeably changes in the presence of oxygen. Under low oxygen conditions colonies appear white, opaque, smooth and round. Under partial or leaky conditions, colonies appear undulate, and umbanet.Whereas under atmospheric oxygen levels colonies always appear low bait, round and dry. In wild Type L.rueteri, growth and reporter protein expression vary between colonies making accurate deductions difficult. Plasmid curing, and re-transformation can result in more stable expression but it is an uncommon and unique occurrence reliant on more stable mutant phenotypes spontaneously occurring.

耐酸荧光报告蛋白mCherry2的测量

<meta charset="utf8"></head><body>优化的基于 CRISPR 的分子诊断方案，适用于床旁检测

Point-of-care CRISPR-based Diagnostics with Premixed and Freeze-dried Reagents

Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes that are suitable for use at point-of-care settings such as fast turnaround times for results, convenient simple readouts, and no requirement of complicated instruments. However, the reactions can be cumbersome to perform at the point of care due to their many components and manual handling steps. Herein, we provide a step-by-step, optimized protocol for the robust detection of disease pathogens and genetic markers with recombinase-based isothermal amplification and CRISPR-based reagents, which are premixed and then freeze-dried in easily stored and ready-to-use formats. Premixed, freeze-dried reagents can be rehydrated for immediate use and retain high amplification and detection efficiencies. We also provide a troubleshooting guide for commonly found problems upon preparing and using premixed, freeze-dried reagents for CRISPR-based diagnostics, to make the detection platform more accessible to the wider diagnostic/genetic testing communities.

<meta charset="utf8"></head><body>作者聚焦：开发基于 CRISPR 的简化检测方法，用于传染病的快速检测

Here, we present the step-by-step preparation of premixed, lyophilized recombinase-based isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based reactions, which can be used for the detection of nucleic acid biomarkers of infectious disease pathogens or other genetic markers of interest.

使用预混和冻干试剂进行基于 CRISPR 的即时诊断

Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes ...