作者要感谢美国心脏协会国家科学家开发格兰特（10SDG2600001），斯坦福大学Bio-X中心的跨学科倡议计划，以及斯坦福大学医学院的学者研究计划提供资金。

1。高分子合成<ol><li>在通风橱中，称出3,523毫克丁二醇酯（C），并转移到装有搅拌棒玻璃闪烁瓶中。 </li><li>预热5 - 氨基-1 - 戊醇（32）到90℃以溶解盐，然后在通风橱中，称出1533毫克32，并添加到含有C.这种方法的闪烁瓶中，将导致以摩尔比的C：32 = 1:1.2。 </li><li>马上含有这两种解决方案的小瓶放置到一个搅拌盘。以600rpm设定搅拌速度。 </li><li>在闪烁瓶中转移到烘箱设定在90℃。设定搅拌速度为1000 rpm离心4小时。如果搅拌棒卡住，降低速度。 4小时后，降低速度至300rpm，并在90℃下保持12-16小时。 </li><li>添加5克C32的在装有搅拌棒玻璃闪烁小瓶10毫升无水四氢呋喃（THF）。包装在铝箔和涡高至完全溶解。 </li><li>在一个单独的玻璃闪烁瓶中CONTA伊宁搅拌棒，加10毫Tetraethyleneglycoldiamine的（122）。加入40毫升THF。 </li><li>广场上的轰动板两个小瓶（C32和122）5分钟，然后结合在了一起。覆盖箔片并在室温下以400rpm的转速进行24小时搅拌离开。最终产物被称作C32-122。 </li><li>称取5×50毫升猎鹰管，每次5克配料C32-122。注意，在笔记本每个管的质量。 </li><li>转移30ml无水乙醚中，以每次50ml Falcon管中。 </li><li>转让10毫升C32-122至每次50毫升猎鹰管。 </li><li>涡猎鹰管大力然后离心2500转2分钟。注意：该提取的聚合物将收集在试管的底部。 </li><li>弃去上层溶液，并重复步骤1.9和1.11的2次以上。 </li><li>将含有该提取C32-122在干燥器和真空通宵开放的管子。确保所有管避光。 </li><li>称取含C32-122所有的管子，以确定最终质量提取的聚合物。 </li><li>溶解在无水DMSO提取的聚合物在100毫克/毫升的浓度。 </li><li>溶解的聚合物储存在-20℃下避光，防潮保护。 </li></ol> 2。细胞接种<ol><li>预涂一个T-150细胞培养瓶中用0.1％（重量/体积）明胶的基极。明胶应该留在烧瓶中30-45分钟。明胶涂层有助于脂肪干细胞的黏附。 </li><li>抽吸明胶从预涂层的T-150烧瓶中。 </li><li>添加4×10 6个脂肪干细胞（≤通道3），以含10％FBS，1％青霉素/链霉素和10毫微克/毫升的bFGF烧瓶中在20ml的DMEM。 </li><li>将T-150烧瓶中，在培养箱中在37℃，5％CO 2中过夜。 </li><li>开始转染过程中的第二天。 </li></ol> 3。纳米粒子的制备和转染<ol><li>下面的程序是含有纳米粒子每16.1微克质粒DNA的制备1.0×10 6细胞在聚合物质粒30:1重量比。 </li><li>稀释质粒DNA（1毫克/毫升，pVEGF165，Aldevron，ND，USA），以在15毫升Falcon管中120微克/毫升的乙酸钠（25毫摩尔）的终浓度。 </li><li>稀C32-122（100毫克/毫升），以在第二15毫升Falcon管中3.6毫克/毫升的乙酸钠（25毫摩尔）的终浓度。 </li><li>结合各Falcon管中的内容为紧接高，持续10秒单只15毫升猎鹰管和旋涡。 </li><li>让所述管在室温下放置10分钟，对纳米颗粒的形成发生。 </li><li>同时孵化，在组织培养瓶中，7.8毫升完全补充的DMEM每1.0×10 6细胞转染更换培养基。 </li><li>转移的纳米颗粒溶液到组织培养烧瓶中，并向前倾斜或向后移动，以均匀地分散。 </li><li>放置在组织培养瓶中在培养箱中在37℃，5％CO 2的4小时。 </li><li> 2小时后，更换纳米颗粒ticle包含与DMEM培养基。 </li><li>以下进一步的2小时温育，在37℃，5％CO 2中，细胞准备用于体内注射。 </li></ol> 4。后肢缺血过程<ol><li>所有实验均按照斯坦福大学动物护理和使用委员会的指导方针和APLAC认可的协议进行的。产生后肢缺血的小鼠模型中进行，如前所述。8请参考详细说明参考。一个简短的描述如下。 </li><li>通过吸入和1升/分钟氧气流量将鼠标在麻醉下用1-3％剂量的异氟醚。按捏脚趾，降低呼吸频率，昏睡确保麻醉深度。 </li><li>从鼠标剃须膏的腹部和后肢两个方面消除头发。 </li><li>使切口在大腿内侧向中线结构居中，然后通过覆脂肪垫EX切工造成股动脉。 </li><li>结扎动脉在两个站点：远端部位（靠近膝盖）和近端部位（只是远至腹股沟韧带）。 </li><li>要创建结扎，从静脉分离动脉，穿入动脉周围5-0丝线缝合。摘下领带动脉有两个结，使结扎。 </li><li>卸下两个结扎点之间的动脉。 </li><li>用PBS冲洗手术区，然后缝合切口闭合。 </li><li>麻醉现在可以移除。按住鼠标热情，直到重新觉醒。对于手术后的护理，2-4毫克/公斤利多卡因可在皮下切口部位注射前重新觉醒。更多的痛苦管理人员可包括皮下交付0.05-0.1毫克/公斤丁丙诺啡在12和24小时。通过观察在改变呼吸方式，明显的疼痛或痛苦，和肤色监测小鼠每天一次的健康状况七天。 </li><li>通过激光多普勒血流灌注成像确认后肢缺血。</li></ol> 5。细胞注射<ol><li>当转染的细胞和小鼠都准备好了，洗转染的细胞用PBS，胰蛋白酶消化，离心，再算上。 </li><li>重悬的细胞以每毫升10×10 6细胞在PBS中的浓度。 </li><li>细胞悬浮液应在110微升体积被分离成单独的Eppendorf管中。这将确保各小鼠接收单元的量相等。 </li><li>将每个鼠在麻醉下（2.5％异氟烷，1升/分钟氧气流量）。 </li><li>清洁注射部位用酒精擦拭。 </li><li>利用一个27政结核菌素注射器，向上和向下绘制细胞进入注射器，以确保均匀的悬浮液来实现。制定100μl的细胞悬浮液体积的入注射器。 </li><li>注入一半的细胞悬液放入收肌区域，然后将另一半注入小腿肌肉区域。注射后，留在原地的注射器，至少15-30秒，以防止细胞悬浮液的泄漏。 </li><li>适当的控制，对于动物研究包括：1）转染的非编码的质粒（ 例如质粒，无生物学活性）的细胞，2）单独的细胞，以及3）的PBS中。 </li><li>当注射完成后，从麻醉中取出鼠标和御寒，直到鼠标重新醒来。 </li></ol> 6。生物发光成像<ol><li>开始生物发光成像（BLI），麻醉上述老鼠。 </li><li>清洁注射部位用酒精擦拭。 </li><li>使用胰岛素注射器，负载100微升15毫克/毫升的D-荧光素（底物为萤火虫荧光素酶）。保持基板避光保存。 </li><li>要执行腹腔注射，通过它们的背部皮肤保持老鼠拉其前皮肤拉紧。针插入腹腔入腹部，并注入100微升底物。 </li><li>将鼠标放置在荧光素酶的IVIS成像腔麻醉下。 </li><lI&gt;图像的小鼠的1.0分钟的曝光时间。当参数设置，开始采集图像。 </li><li>当获取的图像，标记感兴趣区域来测量荧光素酶的信号。在这种情况下，标记的细胞注射部位的缺血肢体。 </li><li>继续下去，直到信号达到峰值并开始下降来测量荧光素酶信号每隔3-5分钟。这是用于跨越小鼠和在几个时间点的比较值。 </li><li>当完成时，从所述腔室和麻醉取出小鼠。养老鼠温暖，直到重新觉醒。 </li></ol> 7。激光多普勒灌注成像<ol><li>激光多普勒灌注成像是如先前所述进行。8的步骤，这里将简要说明。 </li><li>在此之前多普勒成像鼠标应该是刮得干净的区域覆盖两个后肢和腹部，防止工件因头发。 </li><li>当准备多普勒图像，谅解备忘录如上所述，本质上应该被麻醉。 </li><li>保暖鼠标至36°C上，而由直肠温度计监测体温热板。 </li><li>将鼠标上的非反射的黑色表面，并通过鼻锥保持麻醉。鼠标应放在其背水面，其四肢暴露均匀。 </li><li>定位激光多普勒传感器约15厘米以上的小鼠和从对小鼠的腹股沟区域中的传感器将激光指针。 </li><li>当鼠标和多普勒传感器的位置，图像可以采取利用LDPIwin软件按开始按钮。 </li><li>相对灌注测量可以采取通过指示两个后肢（缺血性和非缺血性）作为利益区（ROI）。局部缺血的非缺血后肢的平均灌注的比率将指示相对灌注。 </li></ol> 8。组织收获过程<ol><li>当准备收获鼠标那朵控告的组织学和/或生化处理，鼠标应该安乐死。这里，鼠标是通过暴露于5％异氟醚为3-5分钟，然后通过颈脱位安乐死的小鼠处以安乐死。 </li><li>切割沿圆周方向包围后肢在远端腹部区域和近端后肢上覆皮。皮肤是从腹侧切向背侧表面，使得皮肤能够被拉回在后肢的足。 </li><li>当提拉肌肤背部，四肢可以利用钝性分离剪刀剪开在骨盆与股骨关节截肢。 </li><li>两个主要的组织被用于分析：内侧内收肌和腓肠肌肌肉。 </li><li>对于组织学，在最佳切割温度（OCT）为cryosectioning嵌入一个或两个组织。 </li><li>用于生化分析，收集一个或两个组织到一个1.5ml离心并浸没入液氮浴中至少2分钟。样品可以是储存于-80℃以备将来处理。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

在这里，我们报告一个编程成体干细胞利用非病毒，可生物降解纳米粒子过度治疗因素的方法。此平台可用于治疗疾病，其中的干细胞可以自然的家，如局部缺血和癌症中特别有用。9-10此外，非病毒基因递送平台允许对治疗因素，这是适用于大多数组织再生和伤口瞬时表达愈合过程。转染过程取决于有效的DNA进入细胞时，一般在积极分裂的细胞类型，在非分裂细胞的效果很好，但不为好?...

这项技术的总体目标是使用非病毒编程干细胞过表达治疗因素在缺血的部位，以促进治疗性血管新生。用在实验室中合成的可生物降解的纳米颗粒的干细胞进行了修改， 先体外后体内 ，然后再移植到后肢缺血的小鼠模型来验证他们的潜在增强血管生成和组织打捞。 控制血管生长是成功的组织再生的重要组成部分，以及用于治疗各种缺血性疾病，如中风，肢体局部缺血和心肌梗死。多种策略已经被开发，以促进血管生长，包括生长因子递送和基于细胞的疗法。1尽管在动物疾病模型中观察到的功效，这些方法仍然面临如需要超生理剂量的生长因子递送的限制，或没有足够的旁分泌单靠细胞释放。为克服上述限制的一个潜在策略是结合干细胞治疗和基因治疗，其中的干细胞是基因体内之前的前向移植编程以过表达所需的治疗因子。这种方法已被证明在多种疾病模型包括后肢缺血2，心脏疾病3，骨愈合4和神经损伤5， 等等 。然而，大多数的基因治疗技术依赖于病毒载体，这是与安全性问题，例如潜在的免疫原性和插入突变相关联。生物材料介导的非病毒基因递送可以克服这些限制，但通常从低转染效率受到影响。要加快新型生物材料的发现为有效的非病毒基因传递，最近的研究采用组合化学和高通量筛选方法。可生物降解聚合物的库，如聚（β-氨基ES字符）（PBAE）已开发和筛选，从而导致了与显着增强的转染效率比传统的聚合物载体同行领先的聚合物的发现。6-7 本文中，我们描述PBAE的合成和验证其转染脂肪来源的干细胞（ADSC中） 在体外能力，其次是基因修饰脂肪干细胞在后肢缺血的小鼠模型中过量表达血管内皮生长因子（VEGF）的后续的移植。该结果由追踪细胞命运利用生物发光成像，采用激光多普勒血流灌注成像（LDPI）评估组织灌注，并确定血管生成和组织打捞组织学评价。

<table border="1">
	<tbody>
		<tr>
			<td>
				Name of the Reagent</td>
			<td>
				Company</td>
			<td>
				Catalogue Number</td>
			<td>
				Comments (optional)</td>
		</tr>
		<tr>
			<td>
				DMEM</td>
			<td>
				Invitrogen</td>
			<td>
				11965</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Fetal Bovine Serum</td>
			<td>
				Invitrogen</td>
			<td>
				10082</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Penicillin/Streptomycin</td>
			<td>
				Invitrogen</td>
			<td>
				15070</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Basic Fibroblast Growth Factor</td>
			<td>
				Peprotech</td>
			<td>
				100-18B</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				1,4-Butanediol Diacrylate (90%)</td>
			<td>
				Sigma Aldrich</td>
			<td>
				411744</td>
			<td>
				Acronym: C</td>
		</tr>
		<tr>
			<td>
				5-amino-1-pentanol (97%)</td>
			<td>
				Alfa Aesar</td>
			<td>
				2508-29-4</td>
			<td>
				Acronym: 32</td>
		</tr>
		<tr>
			<td>
				Tetraethyleneglycoldiamine &gt;99%)</td>
			<td>
				Molecular Biosciences</td>
			<td>
				17774</td>
			<td>
				Acronym: 122</td>
		</tr>
		<tr>
			<td>
				Sodium Acetate</td>
			<td>
				G-Biosciences</td>
			<td>
				R010</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Phosphate Buffered Saline</td>
			<td>
				Invitrogen</td>
			<td>
				14190-144</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Tetrahyofuran Anhydrous (&gt;99.9%)</td>
			<td>
				Sigma Aldrich</td>
			<td>
				401757</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Diethyl Ether Anhydrous (&gt;99%)</td>
			<td>
				Fisher Scientific</td>
			<td>
				E138-4</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				DMSO Anhydrous (&gt;99.9%)</td>
			<td>
				Sigma Aldrich</td>
			<td>
				276855</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Gelatin</td>
			<td>
				Sigma Aldrich</td>
			<td>
				G9391</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Trypsin-EDTA</td>
			<td>
				Invitrogen</td>
			<td>
				25200</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				D-luciferin</td>
			<td>
				GoldBio</td>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Optimal Cutting Temperature (O.C.T)</td>
			<td>
				Tissue-Tek</td>
			<td>
				4583</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Rat anti-Mouse CD31</td>
			<td>
				BD Pharmingen</td>
			<td>
				550274</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Alexa Fluor 594 anti-rat IgG</td>
			<td>
				Invitrogen</td>
			<td>
				A11007</td>
			<td>
				&nbsp;</td>
		</tr>
	</tbody>
</table>

	&nbsp;

programming stem cells for therapeutic angiogenesis using biodegradable polymeric nanoparticles

控制血管生长是成功的组织再生和伤口愈合，以及用于治疗局部缺血性疾病，如中风，心脏发作，或外周动脉疾病的关键。直接输送的血管生成生长因子具有刺激新血管生长的潜力，但通常与诸如缺乏靶向和半衰期短的体内的局限性有关。基因治疗提供了一种替代的方法通过提供基因编码血管生成因子，但常常需要使用病毒，并通过安全的担忧限制。在这里，我们描述了最近开发的战略，刺激血管生长，通过编程的干细胞使用可生物降解的聚合物纳米粒子以过度血管生成因子原位 。特别是我们的战略，利用其迁移往体内缺血组织能力优势利用干细胞作为运载工具。采用优化的聚合载体，脂肪来源干细胞被修饰而过表达的血管生成的基因，编码血管内皮生长因子（VEGF）。我们描述了用于聚合物的合成中，纳米粒子的形成， 在体外转染的干细胞，以及用于验证的VEGF表达干细胞的功效在鼠后肢缺血模型中促进血管生成的方法的过程。

在混合在一起时，带正电荷的聚合物（C32-122）和带负电的DNA质粒自组装成纳米颗粒。纳米粒子的形成可通过电泳分析得到证实，即 C32-122和质粒DNA之间的络合作用将防止在电泳过程中动员的DNA。该聚合物可作为转染试剂，以促进DNA的增强吸收到靶细胞和编码蛋 ​​白质的后续表达（ 图2）。细胞可被转染的任何治疗性基因或报道DNA，例如绿色荧光蛋白（GFP），以方便使用荧光?...

Watch this Scientific Journal Video about Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles at JoVE.com

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

我们描述了编程干细胞的方法，使用可生物降解的聚合物纳米颗粒以过表达的治疗因子的血管生成。所述工艺包括聚合物的合成，转染脂肪来源的干细胞在体外和验证编程的干细胞的功效，以促进血管生成的小鼠后肢缺血模型。

编程干细胞的治疗性血管新生使用可生物降解高分子纳米微球

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

programming-stem-cells-for-therapeutic-angiogenesis-using

Research

JoVE Journal

Bioengineering

11.0K 次观看.  Stanford University. 我们描述了编程干细胞的方法，使用可生物降解的聚合物纳米颗粒以过表达的治疗因子的血管生成。所述工艺包括聚合物的合成，转染脂肪来源的干细胞在体外和验证编程的干细胞的功效，以促进血管生成的小鼠后肢缺血模型。 

视频: Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

制定通过Nanoprecipitation技术嵌段聚合物纳米粒子

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

科研

生物工程

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

交付使用表皮生长因子在人胰腺癌细胞转染基因受体有针对性的明胶基于工程Nanovectors

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

订货单细胞和单胚胎的三维约束：高内涵筛选新设备

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

聚合物微针阵列制造用光刻

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

制造超疏水高分子材料生物医学应用

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

使用多色荧光检测液滴微流控光纤

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

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

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

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

闪速纳米沉淀法包聚物纳米粒子中疏水亲水化合物的作用

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

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

人骨髓间充质干细胞吸收含有法卡林多醇的新型脂质包覆纳米颗粒

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...