Name: synthesis and characterization of placental chondroitin sulfate a (plcsa)-targeting lipid-polymer nanoparticles
Uploaded: 2018-09-18T14:00:00
Description: Watch this Scientific Journal Video about Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles at JoVE.com

这项工作得到了中国国家重点研究开发项目 (2016YFC1000402)、国家自然科学基金 (81571445 和 81771617) 和广东省自然科学基金 (2016A030313178) 的资助, 以范与深圳基础研究基金 (JCYJ20170413165233512) 范

1. 库存解决方案的编制
<ol><li>用100毫升的超纯水稀释4毫升的绝对乙醇, 制备4% 乙醇的水溶液。将解决方案存储在摄氏4摄氏度。 注: 超纯水被定义为无污染物的水, 如细菌、微粒、离子或核酸。从水净化系统中获得了超纯水, 其目标电阻率高达 18.2 mΩ·cm, 这意味着低阴离子污染。</li>
	<li>通过将20毫克的大豆卵磷脂溶解在4% 乙醇的20毫升水溶液中, 制备1毫克/毫升的大豆卵磷脂溶液。将大豆卵磷脂?...

<ol>
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该协议为合成 plCSA-BP 共轭脂质高分子纳米粒子提供了一种高效、重现的方法。制备脂质高分子纳米粒子的单步法超声波方法快速、重现性好, 不同于典型的 nanoprecipitation 方法, 包括加热、涡流或蒸发。因此, 所开发的方法大大降低了合成时间。此外, 该协议中使用的 bioconjugate/NHS 是一种常用的和方便的技术, 用于共轭肽和抗体的纳米粒子。因此, 将单步法超声波法与 bioconjugate 技术相结合, 合成 plCSA ...

有效的癌症治疗方法可以减少和消除全身毒性最小的肿瘤。因此, 选择性肿瘤靶向是探索成功治疗方法的关键。纳米微粒为癌症治疗提供了一个有希望的机会, 不同功能组的分子组合将提高药物功效并减少相关的副作用1,2。此外, 纳米微粒系统主要利用被动和主动靶向靶向肿瘤3。
被动靶向利用纳米粒子的固有特性和增强的渗透性和保留 (EPR) 效应, 以达到肿瘤细胞。阳离子脂质体已成功地用于向肿瘤提供各种抗癌药物的临床应用4,5,6。尽管潜在的有效的癌症治疗效果, 低药物浓度的肿瘤地区和无法区分肿瘤细胞从正常组织是两个主要的限制, 被动靶向纳米粒子7。
主动靶向策略利用抗原抗体, 配体受体和其他分子识别相互作用, 专门向肿瘤提供药物8。多糖胎盘硫酸软骨素 A (plCSA) 广泛表达于大多数癌细胞和胎盘滋养。此外, 疟疾蛋白 VAR2CSA 可以专门绑定到 plCSA9,10。因此, VAR2CSA 可以作为靶向人类癌细胞的工具。然而, 当 VAR2CSA 与纳米粒子结合时, 全长蛋白可能会限制纳米粒子对肿瘤细胞的穿透。最近, 我们发现了一种 plCSA 结合肽 (plCSA BP), 来源于疟疾蛋白 VAR2CSA。plCSA-BP-共轭脂质高分子纳米粒子迅速结合到绒细胞, 显著增加阿霉素 (DOX)在体内的抗癌活性11;这些微粒也专门与胎盘滋养结合, 可作为靶向胎盘12提供药物的工具。
脂质高分子纳米颗粒由脂质单层壳和疏水性高分子核组成, 是一种新的药物传递载体。这些纳米粒子结合了脂质体和高分子 nanocarriers 的优点, 如纳米颗粒的大小、生物相容性高、缓释药、药载效率高 (LE)、稳定性好13。在这项工作中, 我们采用单步超声波法合成脂质高分子纳米粒子。该方法快速、方便、适用于规模化, 已广泛用于制备脂质高分子纳米粒子, 由我们的11组,14和其他15,16,17,18.
1-乙基 3-(3-dimethylaminopropyl) carbodiimide 盐酸盐 (EDC) 是一种常用的 carbodiimide 作为交联剂的共轭生物大分子含有胺和羧酸19。除 EDC 外, n-hydroxysulfosuccinimide (NHS) 是最常见的共轭试剂在表面和纳米粒子共轭反应20,21。NHS 可以减少侧向反应次数, 提高酯中间体22、23的稳定性和产量。
在这里, 我们描述了合成 plCSA 靶向脂聚合物纳米粒子的协议。首先, 介绍了 DOX 负载脂质高分子纳米粒子 (DNPs) 的单步法超声波合成方法。然后介绍了一种用于生成 plCSA-BP-共轭脂质高分子纳米粒子的 bioconjugate/NHS 技术。这种 bioconjugate 技术也可用于将其他抗体和多肽与纳米粒子结合。最后, 我们描述了用于表征 plCSA 靶向脂质高分子纳米粒子的理化性质和体外检测方法。我们认为, 这些 plCSA 靶向的脂质高分子纳米粒子可以构成一个有效的系统, 以靶向大多数人类癌症提供药物, 并有针对性地向胎盘提供有效载荷, 以治疗胎盘紊乱。

<table>
	<tbody>
		<tr>
			<td>plCSA peptide</td>
			<td>Shanghai GL Biochem</td>
			<td>573518</td>
			<td>for peptide synthesis</td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Sinopharm Chemical</td>
			<td>10009218</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Soybean lecithin</td>
			<td>Avanti Polar Lipids</td>
			<td>441601</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>DSPE-PEG-COOH</td>
			<td>Avanti Polar Lipids</td>
			<td>880125</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Doxorubicin</td>
			<td>JKChemical</td>
			<td>113424</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Shanghai Lingfeng</td>
			<td>1008621</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>PLGA</td>
			<td>Sigma-Aldrich</td>
			<td>719897</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Ultrasonic processor</td>
			<td>Sonics</td>
			<td>VCX130</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Centrifuge filter (MWCO 10 kDa)</td>
			<td>Millipore</td>
			<td>UFC801024</td>
			<td>for nanoparticles purification</td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Sigma</td>
			<td>3-18KS</td>
			<td>for nanoparticles purification</td>
		</tr>
		<tr>
			<td>2-[morpholino]ethanesulfonic acid(MES)</td>
			<td>Sigma-Aldrich</td>
			<td>M3671</td>
			<td>for peptide conjugation</td>
		</tr>
		<tr>
			<td>1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)</td>
			<td>Sigma-Aldrich</td>
			<td>3450</td>
			<td>for peptide conjugation</td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide (NHS)</td>
			<td>Sigma-Aldrich</td>
			<td>56480</td>
			<td>for peptide conjugation</td>
		</tr>
		<tr>
			<td>Dialysis bags</td>
			<td>Spectrum</td>
			<td>132592T</td>
			<td>for nanoparticles purification</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Hyclone</td>
			<td>SH30028.01</td>
			<td>for cell culture</td>
		</tr>
		<tr>
			<td>10 mL centrifuge tubes, polypropylene</td>
			<td>Aladdin</td>
			<td>S-025</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes, polypropylene</td>
			<td>Corning</td>
			<td>430791</td>
			<td>for various applications</td>
		</tr>
		<tr>
			<td>0.22 &#956;m sterile syringe filter</td>
			<td>Millipore</td>
			<td>SLGV033RB</td>
			<td>for nanoparticles purification</td>
		</tr>
		<tr>
			<td>1 ml syringe, polypropylene</td>
			<td>BD</td>
			<td>328421</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Malvern Zetasizer</td>
			<td>Malvern</td>
			<td>Nano ZS</td>
			<td>for particle size analyer</td>
		</tr>
		<tr>
			<td>Phosphotungstic acid</td>
			<td></td>
			<td></td>
			<td>for TEM</td>
		</tr>
		<tr>
			<td>TEM grid</td>
			<td>EMCN</td>
			<td>BZ10024a</td>
			<td>for TEM</td>
		</tr>
		<tr>
			<td>UV-VIS spectrometer</td>
			<td>Leagene</td>
			<td>DZ0035</td>
			<td>for TEM</td>
		</tr>
		<tr>
			<td>Transmission 
			electron microscope</td>
			<td>JEOL</td>
			<td>JEM-100CXII</td>
			<td>for particle size analyer</td>
		</tr>
		<tr>
			<td>BCA reagent A</td>
			<td>Thermo Fisher Scientific</td>
			<td>23228</td>
			<td>for BCA assay</td>
		</tr>
		<tr>
			<td>BCA reagent B</td>
			<td>Thermo Fisher Scientific</td>
			<td>23224</td>
			<td>for BCA assay</td>
		</tr>
		<tr>
			<td>96-Well Plates</td>
			<td>Corning</td>
			<td>3599</td>
			<td>for BCA assay</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Thermo Fisher Scientific</td>
			<td>Multiskan&#8482; GO</td>
			<td>for BCA assay</td>
		</tr>
		<tr>
			<td>12-well plates</td>
			<td>Corning</td>
			<td>3513</td>
			<td>for cell culture</td>
		</tr>
		<tr>
			<td>JEG3 cell</td>
			<td>Cell Bank of the Chinese Academy of Sciences</td>
			<td>TCHu195</td>
			<td>Human placenta</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Hyclone</td>
			<td>SH30272.01</td>
			<td>phenol red-free</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>GIBCO</td>
			<td>10100</td>
			<td>for cell culture</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>GIBCO</td>
			<td>15070063</td>
			<td>for cell culture</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>OLYMPUS</td>
			<td>CKK53</td>
			<td>for celluar uptake</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Shanghai Lingfeng</td>
			<td>1372021</td>
			<td>for celluar uptake</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sangon Biotech</td>
			<td>A606584</td>
			<td>for celluar uptake</td>
		</tr>
		<tr>
			<td>Mounting medium</td>
			<td>Life</td>
			<td>P36961</td>
			<td>for celluar uptake</td>
		</tr>
	</tbody>
</table>

synthesis and characterization of placental chondroitin sulfate a (plcsa)-targeting lipid-polymer nanoparticles

有效的癌症治疗方法可以减少和消除全身毒性最小的肿瘤。积极靶向纳米粒子为癌症治疗提供了一种有前景的方法。多糖胎盘硫酸软骨素 A (plCSA) 表达在广泛的癌细胞和胎盘滋养, 和疟疾蛋白 VAR2CSA 可以专门绑定到 plCSA。报告的胎盘硫酸软骨素 a 结合肽 (plCSA BP), 来源于疟疾蛋白 VAR2CSA, 也可以具体绑定到 plCSA 癌症细胞和胎盘滋养。因此, plCSA-BP-共轭纳米颗粒可作为靶向药物向人类癌症和胎盘滋养的工具。在本协议中, 我们描述了一种合成 plCSA-BP-共轭脂质聚合物纳米粒子的方法, 载有阿霉素 (plCSA-DNPs);该方法由单超声波步骤和 bioconjugate 技术组成。此外, 还介绍了几种表征 plCSA DNPs 的方法, 包括测定其理化性质和胎盘绒 (JEG3) 细胞的细胞吸收。

在该协议中, PLGA、DSPE-COOH 和大豆卵磷脂分别是具有代表性的聚合物, 脂聚乙二醇-COOH 共轭和脂质。plCSA 靶向脂质高分子纳米粒子的合成,通过单步超声波法和一项 EDC/NHS 技术, 如图 1所示。首先, 在超声波条件下, 大豆卵磷脂、PLGA 和 DSPE-COOH 自组装形成核壳结构 DNPs。该芯由 PLGA 和封装 DOX 组成, 壳体由 DSPE-COOH 组成, 大豆卵磷脂单层位于壳体和芯?...

Watch this Scientific Journal Video about Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles at JoVE.com

Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles

在这里, 我们提出了一个协议, 以合成胎盘软骨素 a 结合肽 (plCSA BP) 共轭脂质高分子纳米粒子通过单步超声波和 bioconjugate 技术。这些微粒构成了一种新的工具, 用于靶向大多数人类肿瘤和胎盘滋养治疗癌症和胎盘疾病的治疗。

胎盘硫酸软骨素 A (plCSA) 靶向脂质高分子纳米粒的合成与表征

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising ...

这种方法可以帮助回答药物输送领域的关键问题，例如有针对性地向人类癌症和胎盘细胞细胞提供治疗。该技术的主要优点是，合成其他颗粒中结合的肽是一种简单易重复的方法。首先，在无菌的10毫升离心管中加入3毫米的4%乙醇。然后，加入90微克大豆卵磷脂库存溶液、210微克的DSPE-PEG-碳氧酸库存溶液和750微克的DEX库存溶液。使用一毫升注射器移液两毫克的PLGA库存溶液。将离心管放在超声波处理器上的冰浴中，并发出两分钟的声波。然后，将 PLGA 库存溶液滴入离心管。要净化 DNP，请使用离心过滤器在 0.1 摩尔 MES 缓冲液中清洗溶液。然后，在 1000 G 下将溶液离心，在 4 摄氏度下将溶液离心 3 分钟。要开始酯激活，请向一毫升的 DNP 中加入 0.4 毫克 EDC。然后，在反应中加入0.24毫克的NHS。然后，让反应在黑暗中室温下发生长达一小时。在此之后，彻底混合反应部件，将反应放在摇床上。要开始胺反应，请使用PBS将缓冲pH值从7.2提高至7.5。然后，在反应溶液中加入0.5毫克的pICSA靶向肽。将溶液混合好，放在摇床上。然后，让反应在黑暗中一夜之间在四摄氏度下继续。第二天，用结合溶液填充透析袋。在黑暗中用 PBS 缓冲液在室温下用 PICSA DNP 净化 24 小时。根据文本协议培养细胞后，取出介质，添加一毫升新鲜、冷介质，并加入 5% FBS 和 pICSA DNP 或 DNP。然后，在4摄氏度下孵育细胞和纳米粒子混合物一小时。在此之后，取出介质，用 PBS 清洗细胞三次。然后，加入一毫升新鲜介质，在37摄氏度下孵育细胞30分钟。在此之后，取出介质，用 PBS 清洗细胞三次。然后，加入两毫升的冷，百分之四的PFA，并在室温下孵育混合物10分钟。取出PFA，用两毫升PBS清洗细胞。然后，添加一毫升的PBS与DAPI核染色。让混合物在室温下孵育10分钟。最后，添加安装介质，用荧光显微镜对荧光进行成像。使用此协议，DNP 已成功编写并结合到 pICSA BP 中。结合后，初级 DNP 的直径增加到大约 109 纳米。DNP 和 pICSA DNP 的 TEM 图像表明，这些粒子分布良好，一般表现出球形形态。纳米粒子的泽塔电位为负20.1和29.9毫伏，表明纳米粒子高度稳定。JEG3细胞吸收测定表明，在30分钟内，与JEG3细胞绑定的pICSA DNP。因此，pICSA BP与表面有效结合。在尝试此过程时，在添加 PLGA 之前，必须记住对混合物进行一到两分钟的声波化。按照此过程，可以执行 HPLC 测定等方法，以便更准确地确定结合效率。该技术开发后，为生殖生物学领域的研究人员探索妊娠并发症胎盘靶向治疗的可能性铺平了道路。

Research

JoVE Journal

Cancer Research

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NF&#954;B Activity and Breast Cancer Stem Cells

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NFÃƒÆ’Ã…Â½Ãƒâ€šÃ‚ÂºB Activity and Breast Cancer Stem Cells

合成与阿司匹林富马酸酯前药的表征抑制NFκB活动和乳腺癌干细胞

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an ...

科研

癌症研究

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

光动力疗法的抗癌功效肺癌靶向纳米粒子

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

单细胞下游特征的靶细胞预富集与全基因组扩增

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

细胞膜延伸特性及其在癌细胞黏附动力学中的作用研究

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

用医用金属丝捕获循环肿瘤细胞的肿瘤细胞: 基于免疫荧光和 DNA 的3D 方法

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

来自沙柯玛患者衍生异种移植物的肿瘤启动细胞的分离和表征

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

肿瘤免疫和非免疫微环境在小鼠皮下肿瘤中的富集与鉴定

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

小肠神经内分泌肿瘤类球体的建立与特征

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited ...

Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models

患者衍生胰腺腺腺腺腺癌有机体模型的分离和特征

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...