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
1Laboratory for Reproductive Health, Institute of Biomedicine and Biotechnology,Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS), 2Guangdong Key Laboratory of Nanomedicine, CAS Key Lab for Health Informatics,Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (CAS), 3Department of Chemistry,Guangdong Medical University
Summary
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。
Abstract
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。
Introduction
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。
Protocol
1. 库存解决方案的编制 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。 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?…
Representative Results
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?…
Discussion
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 …
Disclosures
The authors have nothing to disclose.
Acknowledgements
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
Materials
| plCSA peptide | Shanghai GL Biochem | 573518 | for peptide synthesis |
| Ethanol absolute | Sinopharm Chemical | 10009218 | for nanoparticles synthesis |
| Soybean lecithin | Avanti Polar Lipids | 441601 | for nanoparticles synthesis |
| DSPE-PEG-COOH | Avanti Polar Lipids | 880125 | for nanoparticles synthesis |
| Doxorubicin | JKChemical | 113424 | for nanoparticles synthesis |
| Acetonitrile | Shanghai Lingfeng | 1008621 | for nanoparticles synthesis |
| PLGA | Sigma-Aldrich | 719897 | for nanoparticles synthesis |
| Ultrasonic processor | Sonics | VCX130 | for nanoparticles synthesis |
| Centrifuge filter (MWCO 10 kDa) | Millipore | UFC801024 | for nanoparticles purification |
| centrifuge | Sigma | 3-18KS | for nanoparticles purification |
| 2-[morpholino]ethanesulfonic acid(MES) | Sigma-Aldrich | M3671 | for peptide conjugation |
| 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) | Sigma-Aldrich | 3450 | for peptide conjugation |
| N-hydroxysuccinimide (NHS) | Sigma-Aldrich | 56480 | for peptide conjugation |
| Dialysis bags | Spectrum | 132592T | for nanoparticles purification |
| PBS | Hyclone | SH30028.01 | for cell culture |
| 10 mL centrifuge tubes, polypropylene | Aladdin | S-025 | for nanoparticles synthesis |
| 15 mL centrifuge tubes, polypropylene | Corning | 430791 | for various applications |
| 0.22 μm sterile syringe filter | Millipore | SLGV033RB | for nanoparticles purification |
| 1 ml syringe, polypropylene | BD | 328421 | for nanoparticles synthesis |
| Malvern Zetasizer | Malvern | Nano ZS | for particle size analyer |
| Phosphotungstic acid | for TEM | ||
| TEM grid | EMCN | BZ10024a | for TEM |
| UV-VIS spectrometer | Leagene | DZ0035 | for TEM |
| Transmission electron microscope |
JEOL | JEM-100CXII | for particle size analyer |
| BCA reagent A | Thermo Fisher Scientific | 23228 | for BCA assay |
| BCA reagent B | Thermo Fisher Scientific | 23224 | for BCA assay |
| 96-Well Plates | Corning | 3599 | for BCA assay |
| Plate reader | Thermo Fisher Scientific | Multiskan™ GO | for BCA assay |
| 12-well plates | Corning | 3513 | for cell culture |
| JEG3 cell | Cell Bank of the Chinese Academy of Sciences | TCHu195 | Human placenta |
| DMEM/F12 | Hyclone | SH30272.01 | phenol red-free |
| Fetal bovine serum (FBS) | GIBCO | 10100 | for cell culture |
| Penicillin/streptomycin | GIBCO | 15070063 | for cell culture |
| Fluorescence microscope | OLYMPUS | CKK53 | for celluar uptake |
| Paraformaldehyde | Shanghai Lingfeng | 1372021 | for celluar uptake |
| DAPI | Sangon Biotech | A606584 | for celluar uptake |
| Mounting medium | Life | P36961 | for celluar uptake |
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Cite This Article
Zhang, B., Zheng, M., Cai, L., Fan, X. Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles. J. Vis. Exp. (139), e58209, doi:10.3791/58209 (2018).