本研究由中国国家自然科学基金委员会（31700840）资助:河南省重点科研项目（18B430013，18A150049）。这项研究得到了南湖青年学者项目的支持。作者要感谢来自XYNU生命科学学院的本科生曲泽波的乐于助人的作品。作者希望确认XYNU的分析和测试中心使用他们的设备。

注意:在使用所有化学品之前，请务必查阅所有相关材料安全数据表 （MSDS）。用于制备共聚物和纳米粒子的几种化学物质具有剧毒性。纳米粒子也有潜在的危害。确保使用所有适当的安全做法和个人防护设备，包括手套、实验室外套、头罩、全长裤子和近身鞋。
1. 双碳水化合物聚乙二醇（CT-PEG）合成33
<ol><li>将 1 . 46 克（ 14 . 6 mmol ）的氢化物（ SA ）和 209 毫克（ 1 . 71 mmol ）的 4 二甲基甲基二胺（ DMAP ）添加到 100 mL 圆形底部烧瓶中。</li>
	<li>将 15 毫升的无水四氢氢素 （THF） 添加到步骤 1.1 中使用的烧瓶中，并安装玻璃塞。将烧瓶保持在 0 °C 30 分钟。</li>
	<li>将 4.28 克（4.28 mmol） 聚乙二醇 （PEG） 和 1.8 mL （12.8 mmol） 添加到新的烧瓶中。</li>
	<li>将 15 毫升的无水 THF 添加到步骤 1.3 中使用的烧瓶中，并安装玻璃塞。使用氮气下的注射器，将溶液缓慢地转移到步骤 1.2 中使用的烧瓶中。</li>
	<li>在 0 °C 搅拌溶液 2 小时，然后在室温 （RT） 下持续反应过夜。</li>
	<li>使用旋转蒸发器（40 °C，0.1 MPa），浓缩反应溶液并去除THF溶剂。</li>
	<li>在RT，溶解步骤1.6在1.325克/毫升二氯甲烷（DCM）的15毫升反应溶液，然后加入15毫升的冷二乙醚（Et2O）以获得降水产品（聚乙二醇二恶英）。通过滤纸去除溶剂。 注:降水步骤可重复 3 倍。</li>
	<li>在RT的真空下干燥沉淀物48小时。</li>
</ol>2. PEI-g-PEG共聚体的合成
<ol><li>将 305.47 毫克 CT-PEG 从步骤 1.8 和 5 mL 的二甲基硫化物 （DMSO） 添加到烧瓶中，并在 RT 搅拌，以确保 CT-PEG 在 DMSO 中完全溶解。</li>
	<li>在 5 mL DMSO 中溶解 49.46 毫克 1-（3-二甲基丙烯酸丙基）-3-乙基二氧化硫 （EDC），然后将溶液添加到步骤 2.1 中使用的烧瓶中，并在 RT 搅拌 30 分钟。</li>
	<li>在 5 mL DMSO 中溶解 29.69 毫克的 N-羟基硫酰胺 （NHS），并将溶液添加到步骤 2.1 中使用的烧瓶中。继续在RT搅拌3小时。</li>
	<li>在 DMSO 的 10 mL 中溶解 28.6 μL 聚乙烯 （PEI），并将溶液顺向添加到步骤 2.1 中使用的烧瓶中。至少搅拌3天。</li>
	<li>将反应溶液从步骤 2.4 转移到透析袋（1，000 分子重量截止 [MWCO]）。将透析袋放入 1 升烧嘴中，并放入 500 毫升超纯水作为透析剂。每12小时更换一次超纯水3天。</li>
	<li>将第 2.5 步的溶液转移到另一个透析袋 （10，000 MWCO）。将透析袋放入 1 升烧嘴中，并放入 500 毫升超纯水作为透析剂。每12小时更换一次超纯水3天。</li>
	<li>使用旋转蒸发器（40 °C，0.1 MPa）浓缩步骤2.6的溶液，并冷冻干燥样品以获得PEI-g-PEG粉末。</li>
</ol>3. PEI-g-PEG@AuNPs综合
<ol><li>将5毫克准备好的PEI-g-PEG（步骤2.7）溶解在5毫升的超纯水中，放入新的烧瓶中，并配有玻璃塞。</li>
	<li>将 100 mL 的 0.3 mM HAuCl4 溶液添加到烧瓶中，并在 RT 搅拌解决方案 3 小时。 注意:溶液的颜色应立即从黄色更改为橙色。</li>
	<li>在烧瓶中加入 1 毫克/mL NaBH4 溶液，在 RT 搅拌溶液 3 小时。 注意:反应解决方案应立即变成酒红色。</li>
	<li>使用透析袋 （1，000 MWCO） 对反应产品进行透析，为期 3 天，如步骤 2.5 所述，以获得 PEI-g-PEG@AuNPs 解决方案。</li>
</ol>4. 多克斯-格-爱德华-格-PEG@AuNPs综合
<ol><li>将 1 毫升 2.2 毫克/毫升 DOX 解决方案和 20 mL 的 PEI-g-PEG@AuNPs 溶液添加到新的烧瓶中，并配有玻璃塞子。</li>
	<li>在 1 mL 的超纯水中溶解 0.727 毫克 EDC，并将 EDC 解决方案添加到步骤 4.1 中使用的烧瓶中。</li>
	<li>在 1 mL 的超纯水中溶解 0.437 毫克 NHS。将 NHS 解决方案添加到烧瓶中，在 RT 搅拌 1 小时。</li>
	<li>通过使用透析袋（1，000 MWCO）对反应产品进行透析，为期3天，如第2.5步所述，以获得DOX-g-PEI-g-PEG@AuNPs解决方案。</li>
</ol>5. AS1411-g-DOX-g-PEI-g-PEG@AuNPs综合
<ol><li>将 20 mL 的 DOX-g-PEI-g-PEG@AuNPs 溶液和 4OD AS1411 （OD = 光学密度;1OD ≈ 33 μg） 添加到新的烧瓶中。</li>
	<li>在 1 mL 的超纯水中溶解 28.76 毫克 EDC，并将 EDC 解决方案添加到步骤 5.1 中使用的烧瓶中。</li>
	<li>在 1 mL 的超纯水中溶解 17.27 毫克 NHS。将 NHS 解决方案添加到步骤 5.1 中使用的烧瓶中，并在 RT 中搅拌反应 1 小时。</li>
	<li>通过使用透析袋（1，000 MWCO）对反应产品进行透析3天，如步骤2.5所述，获得AS1411-g-DOX-g-PEI-g-PEG@AuNPs。</li>
</ol>6. 样本特征
<ol><li>分别在核磁共振（NMR）管中溶解氯仿中的CT-PEG聚合物（步骤1.8）和PEI-g-PEG共聚物（步骤2.7）。使用配备 14.09 T 超导磁体和 5.0 mm 600 MHz 宽带 Z 梯度高分辨率探头的 600 MHz NMR 光谱仪分析样品，以确认化学结构34。</li>
	<li>分散 AuNP、DOX 和 AS1411，并在超纯水中分别准备 PEI-g-PEG@AuNPs（步骤 3.4）、DOX-g-PEI-g-PEG@AuNPs（第 4.4 步）和 AS1411-g-DOX-g-PEI-g-PEG@AuNPs（步骤 5.4）。然后，转移到紫外线，并使用紫外线-维斯光谱仪记录紫外线可见光谱（UV-vis）。</li>
	<li>在铝箔上附加双面胶粘剂（~2 mm x 2 mm），并将样品溶液（PEI-g-PEG@AuNPs、DOX-g-PEI-g-PEG@AuNPs和AS1411-g-DOX-g-PEI-g-PEG@AuNPs）均匀地浸入整个磁带上。使用 X 射线光电子光谱分析仪分析样品。</li>
	<li>分别在超纯水中分散 PEI-g-PEG@AuNPs、DOX-g-PEI-g-PEG@AuNPs和 AS1411-g-DOX-g-PEI-PEG@AuNPs解决方案。然后，转移到小面包，并使用动态光散射来评估大小分布。</li>
	<li>将 PEI-g-PEG@AuNPs、DOX-g-PEI-g-PEG@AuNPs和 AS1411-g-DOX-g-PEI-g-PEG@AuNPs溶液分散在超纯水中（每个样品每 5 mL 超纯水一滴样品）。声波2小时。将铜网格浸入样品溶液中，并在红外灯下干燥。使用传输电子显微镜描述形态。</li>
	<li>将 1 毫克 AS1411-g-DOX-g-PEI-g-PEG@AuNPs注入 20 kDa MWCO 透析盒中，然后放入 80 mL 磷酸盐缓冲盐水 （PBS） 中，加入 5% 牛血清白蛋白 （BSA）。在37°C搅拌。</li>
	<li>在预先确定的时间点，收集 100 μL 别名，代之以新鲜的 PBS。使用紫外线光谱仪测量音素的 DOX 荧光强度。</li>
</ol>7. CCK-8 对AS1411-g-DOX-g-PEI-g-PEG@AuNPs纳米粒子的检测
<ol><li>在杜尔贝科改良的鹰介质 （DMEM） 中生长 A549 细胞，在 95% 空气和 5% CO2 在 37 °C 的潮湿大气中补充 10% 胎儿牛血清、100 U/mL 青霉素和 100μg/mL 链霉素。 每 2 天更换一次文化介质。使用通道 5 中的细胞进行细胞增殖和细胞毒性检测，定量评估制备 AS1411-g-DOX-g-PEI-g-PEG@AuNPs纳米粒子的细胞毒性。</li>
	<li>将 100 μL 的纳米粒子溶液添加到每口井中，其中含有 1 mL 的细胞介质。培养 24 小时和 48 小时后，从细胞培养板中去除培养介质，然后立即向每口井添加 300 μL 的新鲜培养介质和 30 μL 的细胞计数套件-8 （CCK-8） 套件解决方案。在 37 °C 的 CO2 孵化器中孵化 4 小时。</li>
	<li>将 200 μL 的反应解决方案从步骤 7.2 转移到 96 井板中。用微板读取器阅读每口井的光学密度 （OD）在 570 nm。</li>
	<li>在显微镜下以24小时和48小时观察细胞的形态。</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanoparticles+in+delivery+applications.">Gold nanoparticles in delivery applications.</a> Advanced Drug Delivery Reviews. 60 (11), 1307-1315 (2008).</li><li>Vandghanooni, S., Eskandani, M., Barar, J., Omidi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antisense+LNA-loaded+nanoparticles+of+star-shaped+glucose-core+PCL-PEG+copolymer+for+enhanced+inhibition+of+oncomiR-214+and+nucleolin-mediated+therapy+of+cisplatin-resistant+ovarian+cancer+cells.">Antisense LNA-loaded nanoparticles of star-shaped glucose-core PCL-PEG copolymer for enhanced inhibition of oncomiR-214 and nucleolin-mediated therapy of cisplatin-resistant ovarian cancer cells.</a> International Journal of Pharmaceutics. 573, 118729 (2020).</li><li>Andghanooni, S., Eskandani, M., Barar, J., Omidi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AS1411+aptamer-decorated+cisplatin-loaded+poly(lactic-co-glycolic+acid)+nanoparticles+for+targeted+therapy+of+miR-21-inhibited+ovarian+cancer+cells.">AS1411 aptamer-decorated cisplatin-loaded poly(lactic-co-glycolic acid) nanoparticles for targeted therapy of miR-21-inhibited ovarian cancer cells.</a> Nanomedicine. 13 (21), 2729-2758 (2018).</li><li>Palmieri, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+anti-nucleolin+recombinant+immunoagent+for+cancer+therapy.">Human anti-nucleolin recombinant immunoagent for cancer therapy.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (30), 9418-9423 (2015).</li><li>Pichiorri, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+NCL+targeting+affects+breast+cancer+aggressiveness+through+miRNA+regulation.">In vivo NCL targeting affects breast cancer aggressiveness through miRNA regulation.</a> Journal of Experimental Medicine. 210 (5), 951-968 (2013).</li><li>Hou, S., McCauley, L. K., Ma, P. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+erosion+properties+of+PEG-containing+polyanhydrides.">Synthesis and erosion properties of PEG-containing polyanhydrides.</a> Macromolecular Bioscience. 7 (5), 620-628 (2007).</li><li>Nie, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+Vaginal+Hydrogels+as+a+Multi-Drug+Carrier+for+Contraception.">Injectable Vaginal Hydrogels as a Multi-Drug Carrier for Contraception.</a> Applied Sciences. 9 (8), 1638 (2019).</li><li>Zou, P., Suo, J., Nie, L., Feng, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-responsive+biodegradable+star-shaped+block+copolymers+for+vaginal+gels.">Temperature-responsive biodegradable star-shaped block copolymers for vaginal gels.</a> Journal of Materials Chemistry. 22 (13), 6316-6326 (2012).</li><li>Etrych, T., &#352;ubr, V., Laga, R., &#344;&#237;hov&#225;, B., Ulbrich, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+conjugates+of+doxorubicin+bound+through+an+amide+and+hydrazone+bond:+Impact+of+the+carrier+structure+onto+synergistic+action+in+the+treatment+of+solid+tumours.">Polymer conjugates of doxorubicin bound through an amide and hydrazone bond: Impact of the carrier structure onto synergistic action in the treatment of solid tumours.</a> European Journal of Pharmaceutical Sciences. 58, 1-12 (2014).</li><li>Safari, F., Tamaddon, A. M., Zarghami, N., Abolmali, S., Akbarzadeh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyelectrolyte+complexes+of+hTERT+siRNA+and+polyethyleneimine:+Effect+of+degree+of+PEG+grafting+on+biological+and+cellular+activity.">Polyelectrolyte complexes of hTERT siRNA and polyethyleneimine: Effect of degree of PEG grafting on biological and cellular activity.</a> Artificial Cells, Nanomedicine, and Biotechnology. 44 (6), 1561-1568 (2016).</li></ol>

作者没有什么可透露的。

1H NMR光谱（图2）确认CT-PEG共聚合体和PEI-g-PEG共聚合体的成功合成。PEG和PEI的分子量分别为1，000和1，200。此外，EDC/NHS催化系统还用于通过阿米德反应合成PEI-g-PEG共聚物。需要注意的是，如果PEG和PEI的分子量因合成PEI-g-PEG共聚物而发生变化，则需要重新评估反应时间和催化系统。此外，需要进一步调整AUNPs上PEI-g-PEG共聚合体涂层的反应条件，主要是因为PEG-g-PEI聚合体?...

癌症是世界性的主要公共卫生问题，其广泛特征是治愈率低、复发率高、死亡率高。目前传统的抗癌方法包括手术、化疗和放疗3种，其中化疗是4号诊所癌症患者的主要治疗方法。临床使用的抗癌药物主要包括帕利塔塞尔（PTX）5和多索鲁比辛（DOX）6，7。DOX，一种抗肿瘤药物，由于癌症细胞毒性和抑制癌细胞增殖8，9的优点，已广泛应用于临床化疗。然而，DOX导致心毒性10，11，和DOX的短半生限制其应用在诊所12。因此，需要降解的药物携带者以可控的方式将 DOX 加载并以子等方式释放到目标区域。
纳米粒子已广泛应用于靶向药物输送系统，在癌症治疗方面具有若干优势（即表面与体积比大、体积小、封装各种药物的能力、可调谐表面化学等）。13，14，15.特别是金纳米粒子（AuNPs）已广泛应用于生物和生物医学应用，如光热癌治疗16，17。AuNPs的独特特性，如简体合成和一般表面功能化，在癌症治疗18的临床领域具有良好的前景。此外，AuNP已经被用来识别药物输送策略，诊断肿瘤，并克服阻力在许多研究19，20。
尽管如此，AuNP 需要进一步定制，通过增强渗透和保留 （EPR）（如靶向和辅助功能）在肿瘤病变时高局部释放来克服耐药性。聚合物功能化 AuNP 具有独特的优势，如疏水抗癌药物水溶解性提高，循环时间延长21、22。各种生物相容聚合物已用于 AuNP 涂料，如聚乙二醇 （PEG）、聚乙烯胺 （PEI）、透明质酸、肝素和黄豆胶。然后，AuNP的稳定性和有效载荷得到很好的改善。具体来说，PEI是一种高度分支的聚合物，由许多重复单位的初级，二级和三级胺24组成。PEI 具有极好的溶解度、低粘度和高功能性，适合涂在 AuNPs 上。
另一方面，抗癌药物需要直接输送到癌细胞，提高负荷效率，降低毒性，治疗原发性肿瘤和晚期转移性肿瘤25。靶向配体具有巨大的潜力，抗癌药物靶向输送系统26。其靶分子结合的选择性赋予抗癌药物针对特异性，并增加药物在病组织中的丰富性27。更多的配体包括抗体、多肽和小分子。与其他配体相比，核酸贴合剂可以在体外合成，易于修改。AS1411是一种未经改性26b磷化物寡核苷酸，形成稳定的二元G-四聚氰胺结构，专门与癌细胞28、29、30上过度表达的目标核蛋白受体结合。AS1411抑制许多癌细胞的增殖，但不影响健康细胞31，32的生长。因此，AS1411 被用来制造理想的有针对性的药物输送系统。
在这项研究中，PEI-g-PEG共聚物通过阿米德反应合成，然后制造PEI-g-PEG共聚物涂层金纳米粒子（PEI-g-PEG@AuNPs）。此外，DOX 和 AS1411 与准备好的 PEI-g-PEG@AuNPs有顺序链接，如 图 1所示。此详细的协议旨在帮助研究人员避免与制造装有 DOX 和 AS1411 的新 PEI-g-PEG@AuNPs相关的许多常见陷阱。

<table><tbody><tr><td>4-Dimethylaminopyridine</td><td>Macklin</td><td>D807273</td><td></td></tr><tr><td>A549 cell</td><td></td><td>ATCC CCL-185TM</td><td></td></tr><tr><td>AS1411</td><td>BBI Life Sciences Corporation</td><td></td><td>5&#039;-d (TTTGGTGGTGGTGGTTGTGGTGGTGGTGG) FL-AS1411 (fluorophore-labeled AS1411)</td></tr><tr><td>Anhydrous Tetrahydrofuran (THF)</td><td>SinoPharm Chemical Reagent Co., Ltd</td><td></td><td></td></tr><tr><td>Cell counting kit-8 (CCK-8)</td><td>Sigma Aldrich</td><td>96992-500TESTS-F</td><td></td></tr><tr><td>Dichloromethane</td><td>Traditional Chinese medicine</td><td>80047318</td><td></td></tr><tr><td>Diethyl ether (Et2O)</td><td>SinoPharm Chemical Reagent Co., Ltd</td><td></td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Macklin</td><td>D806645</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle&#039;s medium (DMEM)</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Doxorubicin hydrochloride</td><td>Rhawn</td><td>R017518</td><td></td></tr><tr><td>Ether absolute</td><td>Traditional Chinese medicine</td><td>80059618</td><td></td></tr><tr><td>Field Emission Transmission Electron Microscope</td><td>FEI Company</td><td>Tecnai G2 F 20</td><td></td></tr><tr><td>Gold(III) chloride trihydrate</td><td>Rhawn</td><td>R016035</td><td></td></tr><tr><td>Laser Particle-size Instrument</td><td>Malvern Instruments Ltd</td><td>ZetasizerNanoZS/Masterszer3000E</td><td></td></tr><tr><td>Microplate Reader</td><td>Molecular Devices</td><td>SpectraMax 190</td><td></td></tr><tr><td>N-(3-Dimethylaminopropyl)-N&#039;-ethylcarbodiimide hydrochloride</td><td>Macklin</td><td>N808856</td><td></td></tr><tr><td>N-Hydroxysuccinimide</td><td>Macklin</td><td>H6231</td><td></td></tr><tr><td>NMR software</td><td>Delta 5.2.1</td><td></td><td></td></tr><tr><td>Nuclear Magnetic Resonance Spectrometer</td><td>JEOL</td><td>JNM-ECZ600R/S3</td><td></td></tr><tr><td>Origin 8.5</td><td>OriginLab</td><td></td><td></td></tr><tr><td>Penicillin</td><td>Sigma Aldrich</td><td>V900929-100ML</td><td></td></tr><tr><td>Phosphate-buffered saline</td><td>Sigma Aldrich</td><td>P4417-100TAB</td><td></td></tr><tr><td>Poly(ethylene glycol)</td><td>Sigma Aldrich</td><td>81188</td><td>BioUltra, average Mn ~ 1000</td></tr><tr><td>Poly (ethyleneimine) solution</td><td>Sigma Aldrich</td><td>482595</td><td>average Mn ~ 1200, 50 wt.% in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Sodium borohydride, powder</td><td>Acros</td><td>C18930</td><td></td></tr><tr><td>Streptomycin</td><td>Sigma Aldrich</td><td>85886-10ML</td><td></td></tr><tr><td>Succinic anhydride</td><td>Traditional Chinese medicine</td><td>30171826</td><td></td></tr><tr><td>Tetrahydrofuran</td><td>Traditional Chinese medicine</td><td>40058161</td><td></td></tr><tr><td>Triethylamine</td><td>Traditional Chinese medicine</td><td>80134318</td><td></td></tr><tr><td>UV/VIS/NIR Spectrometer</td><td>Lambda950</td><td>Lambda950</td><td></td></tr><tr><td>X-ray Photoelectron Spectrometer</td><td>Thermo&amp;ensp;Fisher&amp;ensp;Scientific</td><td>K-ALPHA 0.5EV</td><td></td></tr></tbody></table>

synthesis of aptamer-pei-g-peg modified gold nanoparticles loaded with doxorubicin for targeted drug delivery

由于健康细胞的耐药性和毒性，多索鲁比辛（DOX）在临床癌症治疗中的使用受到限制。该协议描述了用聚乙二醇（PEI-g-PEG）二甘醇（PEI-g-PEG）共聚聚（乙二烯氨酸）嫁接的聚（乙二烯氨酸）与加载的阿普塔默（AS1411）和DOX通过阿米特反应进行功能化金纳米粒子（AuNPs）的设计。AS1411与癌细胞上的靶向核素受体特别结合，使DOX针对的是癌细胞而不是健康细胞。首先，PEG 是盒装的，然后嫁接到分支 PEI 以获得 PEI-g-PEG 聚合物，这一点由 1H NMR 分析确认。其次，PEI-g-PEG copolymer 涂层金纳米粒子 （PEI-g-PEG@AuNPs） 进行合成，DOX 和 AS1411 通过阿米德反应逐渐与 AuNP 粘合。制备的 AS1411-g-DOX-g-PEI-g-PEG@AuNPs直径为 39.9 nm，Zeta 电位为 -29.3 mV，表明纳米粒子在水和细胞介质中是稳定的。细胞毒性检测表明，新设计的DOX加载的AuNPs能够杀死癌细胞（A549）。这种合成展示了PEI-g-PEG共聚物、贴花和DOX在AuNP上的微妙排列，这些排列是通过连续的阿米德反应实现的。这种阿普塔默-PEI-g-PEG功能化的 AuNP 为癌症治疗中的靶向药物输送提供了一个很有前途的平台。

1H NMR光谱学用于确认CT-PEG聚合物和PEI-g-PEG共聚合物（图2）的成功合成。图2a显示，δ的甲基质子信号=3.61ppm，δ=2.57ppm的纸箱质子信号确认CT-PEG聚合物的成功合成。图2b显示PEG的乙烯质子信号在δ=2.6ppm，PEI的质子信号在δ=1.66ppm确认PEI-g-PEG共聚物的合成。 
紫外线光谱学的进行，...

Watch this Scientific Journal Video about Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery at JoVE.com

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

在此协议中，多索鲁比辛加载 AS1411-g-PEI-g-PEG 改性金纳米粒子通过三步阿米内反应合成。然后，多索鲁比辛被加载并交付给靶向癌细胞进行癌症治疗。

合成阿普塔默-PEI-g-PEG改性金纳米粒子装载多索鲁比辛用于靶向药物输送

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...

synthesis-aptamer-pei-g-peg-modified-gold-nanoparticles-loaded-with

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

5.9K 次观看.  Xinyang Normal University. 在此协议中，多索鲁比辛加载 AS1411-g-PEI-g-PEG 改性金纳米粒子通过三步阿米内反应合成。然后，多索鲁比辛被加载并交付给靶向癌细胞进行癌症治疗。

视频: Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.

An example of a nano drug based on polymalic acid is presented towards the rational design of personalized medicine that is applicable to cancer. It describes the synthesis of a nano drug to treat Her2-positive human breast cancer in a nude mouse.

聚苹果酸为基础的生物聚合物纳米靶向的多种肿瘤标志物：用于个性化医疗的机会？

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this ...

科研

化学

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its therapeutic benefit. Nanoparticle-based drug combination has been shown to mitigate the problems encountered by free drug combination therapy. Our previous studies have shown that the combination of two anticancer drugs, doxorubicin (DOX) and mitomycin C (MMC), produced a synergistic effect against both murine and human breast cancer cells in vitro. DOX and MMC co-loaded polymer-lipid hybrid nanoparticles (DMPLN) bypassed various efflux transporter pumps that confer multidrug resistance and demonstrated enhanced efficacy in breast tumor models. Compared to conventional solution forms, such superior efficacy of DMPLN was attributed to the synchronized pharmacokinetics of DOX and MMC and increased intracellular drug bioavailability within tumor cells enabled by the nanocarrier PLN. 
To evaluate the pharmacokinetics and bio-distribution of co-administered DOX and MMC in both free solution and nanoparticle forms, a simple and efficient multi-drug analysis method using reverse-phase high performance liquid chromatography (HPLC) was developed. In contrast to previously reported methods that analyzed DOX or MMC individually in the plasma, this new HPLC method is able to simultaneously quantitate DOX, MMC and a major cardio-toxic DOX metabolite, doxorubicinol (DOXol), in various biological matrices (e.g., whole blood, breast tumor, and heart). A dual fluorescent and ultraviolet absorbent probe 4-methylumbelliferone (4-MU) was used as an internal standard (I.S.) for one-step detection of multiple drug analysis with different detection wavelengths. This method was successfully applied to determine the concentrations of DOX and MMC delivered by both nanoparticle and solution approaches in whole blood and various tissues in an orthotopic breast tumor murine model. The analytical method presented is a useful tool for pre-clinical analysis of nanoparticle-based delivery of drug combinations.

This protocol describes an efficient and convenient analytical process of sample extraction and simultaneous determination of multiple drugs, doxorubicin (DOX), mitomycin C (MMC) and a cardio-toxic DOX metabolite, doxorubicinol (DOXol), in the biological samples from a preclinical breast tumor model treated with nanoparticle formulations of synergistic drug combination.

纳米颗粒对荷瘤小鼠药相结合的样品提取及同时色谱定量分析

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its ...

癌症研究

Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications including contrast enhancement in novel magnetomotive imaging modalities, simultaneous capture&#160;and detection of circulating tumor cells (CTCs),&#160;and multimodal molecular imaging combined with photothermal therapy of cancer cells. These applications have stimulated significant interest in development of protocols for synthesis of magneto-plasmonic nanoparticles with optical absorbance in the near-infrared (NIR) region and a strong magnetic moment. Here, we present a novel protocol for synthesis of such hybrid nanoparticles that is based on an oil-in-water microemulsion method. The unique feature of the protocol described herein is synthesis of magneto-plasmonic nanoparticles of various sizes from primary blocks which also have magneto-plasmonic characteristics. This approach yields nanoparticles with a high density of magnetic and plasmonic functionalities which are uniformly distributed throughout the nanoparticle volume. The hybrid nanoparticles can be easily functionalized by attaching antibodies through the Fc moiety leaving the Fab portion that is responsible for antigen binding available for targeting.

Here, we describe a protocol for synthesis of magneto-plasmonic nanoparticles with a strong magnetic moment and a strong near-infrared (NIR) absorbance. The protocol also includes antibody conjugation to the nanoparticles through the Fc moiety for various biomedical applications which require molecular specific targeting.

Immunotargeted的合成磁等离子团簇

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications ...

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. ...

生物工程

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. HerDox is formed by non-covalent self-assembly of a tumor targeted cell penetration protein with the chemotherapy agent, doxorubicin, via a small nucleic acid linker. A combination of electrophilic, intercalation, and oligomerization interactions facilitate self-assembly into round 10-20 nm particles. HerDox exhibits stability in blood as well as in extended storage at different temperatures. Systemic delivery of HerDox in tumor-bearing mice results in tumor-cell death with no detectable adverse effects to non-tumor tissue, including the heart and liver (which undergo marked damage by untargeted doxorubicin). HER2 elevation facilitates targeting to cells expressing the human epidermal growth factor receptor, hence tumors displaying elevated HER2 levels exhibit greater accumulation of HerDox compared to cells expressing lower levels, both in vitro and in vivo. Fluorescence intensity imaging combined with in situ confocal and spectral analysis has allowed us to verify in vivo tumor targeting and tumor cell penetration of HerDox after systemic delivery. Here we detail our methods for assessing tumor targeting via multimode imaging after systemic delivery.

This article details the procedures for optical imaging analysis of the tumor-targeted nanoparticle, HerDox. In particular, detailed use of the multimode imaging device for detecting tumor targeting and assessing tumor penetration is described here.

分析有针对性的病毒蛋白的纳米粒子递送HER2 +肿瘤

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. ...

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

一个优化的协议通过使用金纳米粒子的放射性标记效率<sup&gt; 125</sup&gt;我标记的叠氮辅基

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

含有金纳米棒蜂窝车辆的制备及光声分析

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous environment through self-assembly. The hydrophobic core of micelles functions as a drug reservoir and encapsulates hydrophobic drugs. The hydrophilic shell prevents the aggregation of micelles and also prolongs their systemic circulation in vivo. In this protocol, we describe a method to synthesize a doxorubicin lipophilic pro-drug, doxorubicin-palmitic acid (DOX-PA), which will enhance drug loading into micelles. A pH-sensitive hydrazone linker was used to conjugate doxorubicin with the lipid, which facilitates the release of free doxorubicin inside cancer cells. Synthesized DOX-PA was purified with a silica gel column using dichloromethane/methanol as the eluent. Purified DOX-PA was analyzed with thin layer chromatography (TLC) and 1H-Nuclear Magnetic Resonance Spectroscopy (1H-NMR). A film dispersion method was used to prepare DOX-PA loaded DSPE-PEG micelles. In addition, several methods for characterizing micelle formulations are described, including determination of DOX-PA concentration and encapsulation efficiency, measurement of particle size and distribution, and assessment of in vitro anticancer activities. This protocol provides useful information regarding the preparation and characterization of drug-loaded micelles and thus will facilitate the research and development of novel micelle-based cancer nanomedicines.

A protocol for the preparation and characterization of lipophilic doxorubicin pro-drug loaded 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) micelles is described.

制备和表征亲脂阿霉素前药胶束

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous ...

Primer-Free Aptamer Selection Using A Random DNA Library

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues.
Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10.
We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format.

SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.

引物的适体的选择，使用一个随机DNA文库

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified ...

生物学

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices&#39; defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes.

用于内皮靶向和药物传递的功能化10纳米高分子涂层金粒子的合成

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting ...