この研究は、中国の国立自然科学財団(31700840)によって資金提供されました。河南省の主要な科学研究プロジェクト(18B430013、18A150049)。この研究は、XYNUの若い学者のための南湖学者プログラムによって支えられた。著者たちは、XYNUのライフサイエンス大学の学士学生ゼボ・クに、彼の役に立つ作品に感謝したいと思います。著者らは、XYNUの分析・試験センターが機器の使用を認めることを望んでいる。

注意: すべての化学物質を使用する前に、関連するすべての材料安全データシート(MSDS)を必ずご確認ください。コポリマーおよびナノ粒子の調製に使用される化学物質のいくつかは、急性毒性である。ナノ粒子には潜在的な危険もあります。手袋、ラボコート、フード、フルレングスパンツ、近い靴など、適切な安全対策と個人用保護具をすべて使用してください。
1. 二重カルボキシルポリエチレングリコール(CT-PEG)33の合成
<ol><li>コハク酸無水(SA)1.46g(14.6 mmol)と4-ジメチルアミノピリジン(DMAP)の209mg(1.71ミリモル)を100 mLラウンドボトムフラスコに加えます。</li>
	<li>ステップ1.1で使用したフラスコに無水テトラヒドロフラン(THF)の15mLを加え、ガラスストッパーを装着します。フラスコを0°Cで30分間おいて下します。</li>
	<li>ポリエチレングリコール(PEG)4.28g(4.28 mmol)とトリエチルアミン(TEA)の1.8 mL(12.8 mmol)を新しいフラスコに加えます。</li>
	<li>ステップ1.3で使用したフラスコに無水THFの15 mLを加え、ガラスストッパーを合わせます。ステップ1.2で使用したフラスコに溶液をゆっくりと移し、窒素雰囲気下の注射器を用いた。</li>
	<li>溶液を0°Cで2時間撹拌し、室温(RT)で一晩反応を続ける。</li>
	<li>ロータリーエバポレーター(40°C、0.1 MPa)を使用して、反応液を濃縮し、THF溶媒を除去します。</li>
	<li>RTでは、ステップ1.6から15mLの1.325g/mLジクロロメタン(DCM)を溶解し、15mLのコールドジエチルエーテル(Et2O)を加えて沈殿物(ポリエチレングリコール二酸)を得る。フィルターペーパーで溶剤を取り除きます。 注:降水ステップは3倍に繰り返すことができます。</li>
	<li>RTで真空下で沈殿物を48時間乾燥させます。</li>
</ol>2. PEI-g-PEG共重合体の合成
<ol><li>ステップ1.8および5 mLのジメチルスルホキシド(DMSO)から305.47mgのCT-PEGをフラスコに加え、RTで攪拌してCT-PEGがDMSOに完全に溶解していることを確認します。</li>
	<li>DMSOの5mLで1-(3-ジメチルアミノプロピル)-3-エチルカルボジイミド(EDC)の49.46mgを5mLに溶解し、ステップ2.1で使用されるフラスコに溶液を加え、RTで30分間かき混ぜます。</li>
	<li>29.69 mgのN-ヒドロキシスクッチイミド(NHS)をDMSOの5 mLに溶解し、ステップ2.1で使用するフラスコに溶液を加えます。RTで3時間かき混ぜ続けます。</li>
	<li>28.6 μLのポリエチレンイミン(PEI)をDMSOの10 mLに溶解し、ステップ2.1で使用するフラスコに溶液を滴下して添加します。少なくとも3日間かき混ぜます。</li>
	<li>反応した溶液をステップ2.4から透析袋(1,000分子量カットオフ[MWCO])に移します。透析袋を1 Lビーカーに入れ、500mLの超純水を透析液として入れます。超純水を12時間ごとに3日間交換します。</li>
	<li>ステップ 2.5 のソリューションを別の透析バッグ (10,000 MWCO) に移します。透析袋を1 Lビーカーに入れ、500mLの超純水を透析液として入れます。超純水を12時間ごとに3日間交換します。</li>
	<li>回転エバポレーター(40°C、0.1 MPa)を使用してステップ2.6から溶液を濃縮し、サンプルを凍結乾燥してPEI-g-PEG粉末を得る。</li>
</ol>3. PEI-g-PEG@AuNPsの合成
<ol><li>5 mgの調製した PEI-g-PEG (ステップ 2.7) を 5 mL の超純水に新しいフラスコに溶かし、ガラス栓でフィットさせます。</li>
	<li>フラスコに0.3 mM HAuCl4溶液の100 mLを加え、RTで3時間攪拌します。 注: ソリューションの色は、すぐに黄色からオレンジ色に変更されます。</li>
	<li>フラスコに1mg/mL NaBH4 溶液1 mLを加え、RTで3時間攪拌します。 注:反応溶液は即座にバーガンディを回す必要があります。</li>
	<li>ステップ2.5で説明したように3日間透析バッグ(1,000MWCO)を用いた反応生成物を透析し、PEI-g-PEG@AuNPs溶液を得た。</li>
</ol>4. DOX-g-PEI-g-PEG@AuNPsの合成
<ol><li>2.2 mg/mL DOX溶液1 mLと20 mLのPEI-g-PEG@AuNPs溶液を新しいフラスコに加え、ガラス栓に合わせます。</li>
	<li>0.727 mgのEDCを1mLの超純水に溶解し、ステップ4.1で使用するフラスコにEDC溶液を加えます。</li>
	<li>1 mLの超純水に0.437mgのNHSを溶かします。フラスコにNHS溶液を加え、RTで1時間かき混ぜます。</li>
	<li>手順2.5に記載した3日間の透析袋(1,000MWCO)を用いて反応生成物を透析し、DOX-g-PEI-g-PEG@AuNPs溶液を得た。</li>
</ol>5. AS1411-g-DOX-g-PEI-g-g-PEG@AuNPsの合成
<ol><li>20 mL の DOX-g-PEI-g-PEG@AuNPs溶液と 4OD AS1411 (OD = 光学密度; 1OD ≈ 33 μg) を新しいフラスコに加えます。</li>
	<li>28.76 mgのEDCを1mLの超純水に溶解し、ステップ5.1で使用するフラスコにEDC溶液を加えます。</li>
	<li>1mLの超純水に17.27mgのNHSを溶かします。ステップ5.1で使用したフラスコにNHS溶液を加え、RTで1時間反応をかき混ぜる。</li>
	<li>透析用バッグ(1,000 MWCO)をステップ2.5に記載した3日間用いて反応生成物を透析し、AS1411-g-g-g-PEI-g-PEG@AuNPsを得た。</li>
</ol>6. サンプルの特性評価
<ol><li>CT-PEGポリマー(ステップ1.8)とPEI-g-PEG共重合体(ステップ2.7)をそれぞれ核磁気共鳴(NMR)チューブでクロロホルムdに溶解する。14.09 T超伝導磁石と5.0mm 600 MHzの広帯域Z勾配高分解能プローブを搭載した600MHz NMR分光計を用いてサンプルを分析し、化学構造34を確認する。</li>
	<li>AuNPs、DOX、およびAS1411を分散し、PEI-g-PEG@AuNPs(ステップ3.4)、DOX-g-PEI-g-PEG@AuNPs(ステップ4.4)、AS1411-g-DOX-g-PEI-g-g-PEG@AuNPsをそれぞれ超純水中に調製した。次に、UV-vis分光光度計を用いて、キュベットに移し、紫外線可視(UV-vis)スペクトルを記録します。</li>
	<li>両面接着剤(2mm×2mm)をアルミ箔に取り付け、サンプル溶液(PEI-g-PEG@AuNPs、DOX-g-PEI-g-PEG@AuNPs、AS1411-g-DOX-g-PEI-g-PEG@AuNPs)をテープ全体に均一に浸します。X線光電子分光分析装置を用いてサンプルを分析します。</li>
	<li>PEI-g-PEG@AuNPs、DOX-g-PEI-g-PEG@AuNPs、AS1411-g-doX-g-PEI-g-PEG@AuNPs溶液をそれぞれ超純水に分散させます。次いで、キュベットに移し、動的光散乱を用いてサイズ分布を評価する。</li>
	<li>PEI-g-g-PEG@AuNPs g-PEG@AuNPs、AS1411-g-DOX-g-PEI-g-PEG@AuNPs溶液を超純水(各サンプルに対して5mLあたり1滴サンプル)の超純水に分散させます。2時間の超音波処理。銅グリッドをサンプル溶液に浸し、赤外線ランプで乾燥させます。透過型電子顕微鏡を用いて形態を特徴付け</li>
	<li>AS1411-g-DOX-g-PEI-g-PEG@AuNPs g-20 kda MWCO透析カセットに1mgを注入し、5%ウシ血清アルブミン(BSA)でリン酸緩衝生理食塩液(PBS)80 mLに入れます。37°Cでかき混ぜる。</li>
	<li>所定のタイムポイントで、100 μLのアリコートを収集し、新鮮なPBSに交換します。UV-vis分光光度計を使用して、アリコートのDOX蛍光強度を測定します。</li>
</ol>7. As1411-g-DOX-g-PEI-g-PEG@AuNPsナノ粒子のCCK-8アッセイ
<ol><li>10%の胎児ウシ血清、100 U/mLペニシリン、100 μg/mLストレプトマイシンを37°Cで95%空気、5%CO2の加湿雰囲気下で補ったダルベックコの修飾イーグル培地(DMEM)でA549細胞を増殖させます。 2日ごとに培地を交換してください。細胞増殖および細胞毒性アッセイの継代時の細胞を使用して、調製したAS1411-g-g-g-g-PEI-g-PEG@AuNPsナノ粒子の細胞毒性を定量的に評価する。</li>
	<li>ナノ粒子溶液を100μLずつ、細胞培地1mLを各ウェルに加えます。24時間と48時間培養した後、培養液を細胞培養プレートから取り出し、300μLの新鮮な培養培地と30μLの細胞計数キット-8(CCK-8)キット溶液を各ウェルに直ちに添加します。CO2インキュベーターで4時間インキュベートを37°Cでインキュベートする。</li>
	<li>ステップ7.2から96ウェルプレートに200 μLの反応溶液を移します。マイクロプレートリーダーで570 nmの各井戸の光学密度(OD)を読み取ります。</li>
	<li>顕微鏡下で24時間と48時間で細胞の形態を観察します。</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>

著者らは開示するものは何もない。

1HNMRスペクトル(図2)は、CT-PEG共重合体とPEI-g-PEG共重合体の合成に成功したことを確認した。PEGとPEIの分子量はそれぞれ1,000と1,200であった。さらに、EDC/NHS触媒系を用い、アミド反応を介してPEI-g-PEG共重合体を合成した。なお、PEGとPEIの分子量がPEI-g-PEG共重合体を合成するために変化した場合、反応時間と触媒系を再評価する必要がある。また、AUNPs上のPEI-g-PEG共?...

世界的に主要な公衆衛生上の問題である癌は、治癒率が低く、再発率が高く、死亡率が高いとして広く特徴づけられている。現在の従来の抗癌法には、手術、化学療法、および放射線療法3が含まれており、その中で化学療法は、クリニック4における癌患者に対する主要な治療法である。臨床用抗癌剤は主に、パクリタキセル(PTX)5およびドキソルビシン(DOX)6,7を含む。DOXは、抗新生剤である、癌細胞毒性および癌細胞増殖の阻害の利点のために、臨床化学療法に広く適用されている8,9。しかし、DOXは心毒性10、11を引き起こし、DOXの短い半減期は、診療所12での適用を制限する。したがって、DOXをロードし、標的領域に制御された方法で潜伏放出するために分解性薬物キャリアが必要である。
ナノ粒子は標的薬物送達システムで広く使用されており、がん治療にはいくつかの利点があります(すなわち、大きな表面対体積比、小型、様々な薬物をカプセル化する能力、および調整可能な表面化学など)13、14、15。特に、金ナノ粒子(AuNPs)は、光熱癌治療16,17などの生物及び生物医学的用途において広く用いられている。AuNPsのユニークな特性は、ファシリティ合成や一般表面官能化など、がん治療18の臨床分野において優れた見通しを持っている。また、AuNPは、多くの研究で薬物送達戦略を特定し、腫瘍を診断し、抵抗を克服するために使用されてきました19,20.
それにもかかわらず、AuNPは、標的化およびアクセシビリティ特性などの強化された浸透および保持(EPR)を介して腫瘍病変で高い局所放出を介して薬剤耐性を克服するためにさらに調整される必要がある。ポリマー機能性のAUNPsは、疎水性抗癌剤の水溶性の改善や長期の循環時間21,22などの独自の利点を発揮している。ポリエチレングリコール(PEG)、ポリエチレンイミン(PEI)、ヒアルロン酸、ヘパリン、キサンタンガムなどの様々な生体適合性ポリマーがAuNPコーティングに使用されています。その後、AuNPsの安定性とペイロードが23を改善します。具体的には、PEIは、一次、二次、および第三級アミンの多くの繰り返し単位で構成される高度に分岐したポリマーである24。PEIは、優れた溶解性、低粘度、高い機能性を有しており、AUNP上でのコーティングに適しています。
一方、抗癌剤は、癌細胞に直接送達する必要があり、負荷効率を向上させ、原発性および進行転移性腫瘍25を治療するための毒性を低くする必要がある。標的リガンドは、抗癌剤標的送達システム26に大きな可能性を秘めている。標的分子結合に対するその選択性は、特異性を標的とする抗癌薬を付与し、疾患組織における薬物濃縮を増加させる27。よりリガンドには、抗体、ポリペプチド、および小分子が含まれる。他のリガンドと比較して、核酸アプタマーは、インビトロで合成することができ、変更が容易である。AS1411は、癌細胞28、29、30上の過剰発現標的核タンパク受容体に特異的に結合する安定な二量体G-四量体構造を形成する非修飾26bpホスホジエステルオリゴヌクレオチドチドである。AS1411は多くの癌細胞の増殖を阻害するが、健康な細胞31、32の増殖には影響しない。その結果、AS1411は理想的な標的薬物送達システムを作製するために使用されてきた。
本研究では、PEI-g-PEG共重合体をアミド反応を介して合成し、次いでPEI-g-PEG共重合体被覆金ナノ粒子(PEI-g-PEG@AuNPs)を作製した。さらに 、DOXと AS1411 は、図 1 に示すように、準備された PEI-g-PEG@AuNPsに順次リンクされています。この詳細なプロトコルは、研究者がDOXとAS1411を搭載した新しいPEI-g-PEG@AuNPsの製造に関連する多くの一般的な落とし穴を避けるのを助けることを目的としています。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;-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&#39;s modified Eagle&#39;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&#39;-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 H2O</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&#8194;Fisher&#8194;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

健康な細胞における薬剤耐性および毒性のために、ドキソルビシン(DOX)の使用は臨床癌治療において限られている。このプロトコルは、ポリエチレングリコール(PEI-g-PEG)共重合体機能化金ナノ粒子(AuNPs)をアミド反応を介してアプタマー(AS1411)とDOXを移植したポリ(エチレニミン)の設計を記述する。AS1411は、がん細胞上の標的ヌクレオリン受容体と特異的に結合し、DOXが健康な細胞の代わりに癌細胞を標的とするようにする。まず、PEGはカルボキシ化され、次いで分枝状PEIに移植し、PEI-g-PEG共重合体を得、これは 1HNMR分析によって確認される。次に、PEI-g-PEG共重合体被覆金ナノ粒子(PEI-g-PEG@AuNPs)が合成され、DOXとAS1411はアミド反応を介して徐々にAuNPsに共有結合される。調製されたAS1411-g-DOX-g-PEI-g-g-PEG@AuNPsの直径は、-29.3mVのゼータ電位を有する、ナノ粒子が水および細胞媒体において安定であることを示す。.細胞細胞毒性アッセイは、新たに設計されたDOX搭載AuNPsが癌細胞を殺すことができることを示している(A549)。この合成は、連続アミド反応によって達成されるAuNPs上のPEI-g-PEGコポリマー、アプタマー、およびDOXの繊細な配置を示しています。このようなアプタマー-PEI-g-PEG機能化されたAuNPsは、がん治療における標的薬物送達のための有望なプラットフォームを提供する。

1H NMR分光法は、CT-PEGポリマーとPEI-g-PEG共重合体の合成に成功したことを確認するために用いた(図2)。図2aは、δ=3.61ppmでのメチレンプロトン信号とδ=2.57ppmのカルボキシルプロトン信号がCT-PEGポリマーの合成に成功したことを確認することを示す。 図2bは、δ=2.6ppmでのPEGのメチレンプロトンシグナル?...

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

このプロトコルでは、ドキソルビシンを装填したAS1411-g-PEI-g-PEG変性金ナノ粒子は、3段階のアミド反応を介して合成される。次いで、ドキソルビシンをロードし、癌治療のために標的癌細胞に送達する。

標的薬物送達のためのドキソルビシンを装填したアプタマー-PEI-G-PEG改変金ナノ粒子の合成

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

Research

JoVE Journal

Chemistry

5.8K ビュー.  Xinyang Normal University. このプロトコルでは、ドキソルビシンを装填したAS1411-g-PEI-g-PEG変性金ナノ粒子は、3段階のアミド反応を介して合成される。次いで、ドキソルビシンをロードし、癌治療のために標的癌細胞に送達する。

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

Template Directed Synthesis of Plasmonic Gold Nanotubes with Tunable IR Absorbance

A nearly parallel array of pores can be produced by anodizing aluminum foils in acidic environments1, 2. Applications of anodic aluminum oxide (AAO) membranes have been under development since the 1990's and have become a common method to template the synthesis of high aspect ratio nanostructures, mostly by electrochemical growth or pore-wetting. Recently, these membranes have become commercially available in a wide range of pore sizes and densities, leading to an extensive library of functional nanostructures being synthesized from AAO membranes. These include composite nanorods, nanowires and nanotubes made of metals, inorganic materials or polymers 3-10. Nanoporous membranes have been used to synthesize nanoparticle and nanotube arrays that perform well as refractive index sensors, plasmonic biosensors, or surface enhanced Raman spectroscopy (SERS) substrates 11-16, as well as a wide range of other fields such as photo-thermal heating 17, permselective transport 18, 19, catalysis 20, microfluidics 21, and electrochemical sensing 22, 23. Here, we report a novel procedure to prepare gold nanotubes in AAO membranes. Hollow nanostructures have potential application in plasmonic and SERS sensing, and we anticipate these gold nanotubes will allow for high sensitivity and strong plasmon signals, arising from decreased material dampening 15.

Solution-suspendable gold nanotubes with controlled dimensions can be synthesized by electrochemical deposition in porous anodic aluminum oxide (AAO) membranes using a hydrophobic polymer core. Gold nanotubes and nanotube arrays hold promise for applications in plasmonic biosensing, surface-enhanced Raman spectroscopy, photo-thermal heating, ionic and molecular transport, microfluidics, catalysis and electrochemical sensing.

テンプレートには、調整可能な赤外線吸光度モニック金ナノチューブの合成を監督

A nearly parallel array of pores can be produced by anodizing aluminum foils in acidic environments1, 2. Applications of anodic aluminum oxide (AAO) ...

化学

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

Preparation and Characterization of SDF-1&amp;#945;-Chitosan-Dextran Sulfate Nanoparticles

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

SDF-1α-キトサン硫酸デキストランナノ粒子の調製とキャラクタリゼーション

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like oligomeric clusters of these yellow nanoparticles of narrow size distribution are synthesized under ambient conditions via two methods. The delay-time method controls the number of subunits in the oligoclusters by varying the time between the addition of HAuCl4 to alkaline solution and the subsequent addition of reducing agent, NaSCN. The yellow oligoclusters produced range in size from ~3 to ~25 nm. This size range can be further extended by an add-on method utilizing hydroxylated gold chloride (Na+[Au(OH4-x)Clx]-) to auto-catalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3 nm to 70 nm. The crude oligocluster preparations display narrow size distributions and do not require further fractionation for most purposes. The oligoclusters formed can be concentrated &#62;300 fold without aggregation and the crude reaction mixtures remain stable for weeks without further processing. Because these oligomeric clusters can be concentrated before derivatization they allow expensive derivatizing agents to be used economically. In addition, we present two models by which predictions of particle size can be made with great accuracy.

We describe a simple method for producing highly stable oligomeric clusters of gold nanoparticles via the reduction of chloroauric acid (HAuCl4) with sodium thiocyanate (NaSCN). The oligoclusters have a narrow size distribution and can be produced with a wide range of sizes and surface coats.

周囲条件下での金ナノ粒子の安定オリゴマークラスターのサイズ制御された合成のための簡単​​な方法

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like ...

Hydroquinone Based Synthesis of Gold Nanorods

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the synthetic methods for the preparation of gold nanorods are still not fully optimized. In this paper we describe a new, highly efficient, two-step protocol based on the use of hydroquinone as a mild reducing agent. Our approach allows the preparation of nanorods with a good control of size and aspect ratio (AR) simply by varying the amount of hexadecyl trimethylammonium bromide (CTAB) and silver ions (Ag+) present in the "growth solution". By using this method, it is possible to markedly reduce the amount of CTAB, an expensive and cytotoxic reagent, necessary to obtain the elongated shape. Gold nanorods with an aspect ratio of about 3 can be obtained in the presence of just 50 mM of CTAB (versus 100 mM used in the standard protocol based on the use of ascorbic acid), while shorter gold nanorods are obtained using a concentration as low as 10 mM.

This paper describes a protocol for the synthesis of gold nanorods, based on the use of hydroquinone as reducing agent, plus the different mechanisms for controlling their size and aspect ratio.

金ナノロッドのヒドロキノンベースの合成

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the ...

Chemical Precipitation Method for the Synthesis of Nb2O5 Modified Bulk Nickel Catalysts with High Specific Surface Area

We demonstrate a method for the synthesis of NixNb1-xO catalysts with sponge-like and fold-like nanostructures. By varying the Nb:Ni ratio, a series of NixNb1-xO nanoparticles with different atomic compositions (x = 0.03, 0.08, 0.15, and 0.20) have been prepared by chemical precipitation. These NixNb1-xO catalysts are characterized by X-ray diffraction, X-ray photoelectron spectroscopy, and scanning electron microscopy. The study revealed the sponge-like and fold-like appearance of Ni0.97Nb0.03O and Ni0.92Nb0.08O on the NiO surface, and the larger surface area of these NixNb1-xO catalysts, compared with the bulk NiO. Maximum surface area of 173 m2/g can be obtained for Ni0.92Nb0.08O catalysts. In addition, the catalytic hydroconversion of lignin-derived compounds using the synthesized Ni0.92Nb0.08O catalysts have been investigated.

Chemical Precipitation Method for the Synthesis of Nb2O5 Modified Bulk Nickel Catalysts with High Specific Surface Area

A protocol for the synthesis of sponge-like and fold-like Ni1-xNbxO nanoparticles by chemical precipitation is presented.

Nb2O5の合成化学沈殿法変更高表面積一括ニッケル触媒

We demonstrate a method for the synthesis of NixNb1-xO catalysts with sponge-like and fold-like nanostructures. By varying the Nb:Ni ratio, a series of ...

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good&#39;s buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.

原金ナノ粒子合成による急速なナノプローブ信号強調

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...

Methionine Functionalized Biocompatible Block Copolymers for Targeted Plasmid DNA Delivery

Reversible addition-fragmentation chain transfer (RAFT) polymerization integrates the advantages of radical polymerization and living polymerization. This work presents the preparation of methionine functionalized biocompatible block copolymers via RAFT polymerization. Firstly, N,N-bis(2-hydroxyethyl)methacrylamide-b-N-(3-aminopropyl)methacrylamide (BNHEMA-b-APMA, BA) was synthesized via RAFT polymerization using 4,4&#8217;-azobis(4-cyanovaleric acid) (ACVA) as an initiating agent and 4-cyanopentanoic acid dithiobenzoate (CTP) as the chain transfer agent. Subsequently, N,N-bis(2-hydroxyethyl)methacrylamide-b-N-(3-guanidinopropyl)methacrylamide (methionine grafted BNHEMA-b-GPMA, mBG) was prepared by modifying amine groups in APMA with methionine and guanidine groups. Three kinds of block polymers, mBG1, mBG2, and mBG3, were synthesized for comparison. A ninhydrin reaction was used to quantify the APMA content; mBG1, mBG2, and mBG3 had 21%, 37%, and 52% of APMA, respectively. Gel permeation chromatography (GPC) results showed that BA copolymers possess molecular weights of 16,200 (BA1), 20,900(BA2), and 27,200(BA3) g/mol. The plasmid DNA (pDNA) complexing ability of the obtained block copolymer gene carriers was also investigated. The charge ratios (N/P) were 8, 16, and 4 when pDNA was complexed completely with mBG1, mBG2, mBG3, respectively. When the N/P ratio of mBG/pDNA polyplexes was higher than 1, the Zeta potential of mBG was positive. At an N/P ratio between 16 and 32, the average particle size of mBG/pDNA polyplexes was between 100-200 nm. Overall, this work illustrates a simple and convenient protocol for the block copolymer carrier synthesis.

This work presents the preparation of methionine functionalized biocompatible block copolymers (mBG) via the reversible addition-fragmentation chain transfer (RAFT) method. The plasmid DNA complexing ability of the obtained mBG and their transfection efficiency were also investigated. The RAFT method is very beneficial for polymerizing monomers containing special functional groups.

標的プラスミドDNA送達に対するメチオニン機能性生体適合性共重合体

Reversible addition-fragmentation chain transfer (RAFT) polymerization integrates the advantages of radical polymerization and living polymerization. This ...

Synthesis and Characterization of Amphiphilic Gold Nanoparticles

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of their interactions with cell membranes, lipid bilayers, and viruses. The hydrophilic ligands make these particles colloidally stable in aqueous solutions and the combination with hydrophobic ligands creates an amphiphilic particle that can be loaded with hydrophobic drugs, fuse with the lipid membranes, and resist nonspecific protein adsorption. Many of these properties depend on nanoparticle size and the composition of the ligand shell. It is, therefore, crucial to have a reproducible synthetic method and reliable characterization techniques that allow the determination of nanoparticle properties and the ligand shell composition. Here, a one-phase chemical reduction, followed by a thorough purification to synthesize these nanoparticles with diameters below 5 nm, is presented. The ratio between the two ligands on the surface of the nanoparticle can be tuned through their stoichiometric ratio used during synthesis. We demonstrate how various routine techniques, such as transmission electron microscopy (TEM), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and ultraviolet-visible (UV-Vis) spectrometry, are combined to comprehensively characterize the physicochemical parameters of the nanoparticles.

Amphiphilic gold nanoparticles can be used in many biological applications. A protocol to synthesize gold nanoparticles coated by a binary mixture of ligands and a detailed characterization of these particles is presented.

親和性金ナノ粒子の合成と特性化

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of ...

Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into glass capillaries, pulled to a fine taper, and then sealed using an epoxy to create electrode materials that are used for fast scan cyclic voltammetry testing. The use of bare CFMEs has several limitations, though. First and foremost, the carbon fiber contains mostly basal plane carbon, which has a relatively low surface area and yields lower sensitivities than other nanomaterials. Furthermore, the graphitic carbon is limited by its temporal resolution, and its relatively low conductivity. Lastly, neurochemicals and macromolecules have been known to foul at the surface of carbon electrodes where they form non-conductive polymers that block further neurotransmitter adsorption. For this study, we modify CFMEs with gold nanoparticles to enhance neurochemical testing with fast scan cyclic voltammetry. Au3+ was electrodeposited or dipcoated from a colloidal solution onto the surface of CFMEs. Since gold is a stable and relatively inert metal, it is an ideal electrode material for analytical measurements of neurochemicals. Gold nanoparticle modified (AuNP-CFMEs) had a stability to dopamine response for over 4 h. Moreover, AuNP-CFMEs exhibit an increased sensitivity (higher peak oxidative current of the cyclic voltammograms) and faster electron transfer kinetics (lower &#916;EP or peak separation) than bare unmodified CFMEs. The development of AuNP-CFMEs provides the creation of novel electrochemical sensors for detecting fast changes in dopamine concentration and other neurochemicals at lower limits of detection. This work has vast applications for the enhancement of neurochemical measurements. The generation of gold nanoparticle modified CFMEs will be vitally important for the development of novel electrode sensors to detect neurotransmitters in vivo in rodent and other models to study neurochemical effects of drug abuse, depression, stroke, ischemia, and other behavioral and disease states.

In this study, we modify carbon-fiber microelectrodes with gold nanoparticles to enhance the sensitivity of neurotransmitter detection.

強化された神経化学的検出のための金ナノ粒子修飾炭素繊維マイクロ電極

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into ...

Gold Nanoparticle Synthesis

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of tetrachloroauric acid in 3.0 g (3.7 mmol, 3.6 mL) of oleylamine (technical grade) and 3.0 mL of toluene into a boiling solution of 5.1 g (6.4 mmol, 8.7 mL) of oleylamine in 147 mL of toluene. While boiling and mixing the reaction solution for 2 hours, the color of the reaction mixture changed from clear, to light yellow, to light pink, and then slowly to dark red. The heat was then turned off, and the solution was allowed to gradually cool down to room temperature for 1 hour. The gold nanoparticles were then collected and separated from the solution using a centrifuge and washed three times; by vortexing and dispersing the gold nanoparticles in 10 mL portions of toluene, and then precipitating the gold nanoparticles by adding 40 mL portions of methanol and spinning them in a centrifuge. The solution was then decanted to remove any remaining byproducts and unreacted starting materials. Drying the gold nanoparticles in a vacuum environment produced a solid black pellet; which could be stored for long periods of time (up to one year) for later use, and then redissolved in organic solvents such as toluene.

A protocol for synthesizing ~12 nm diameter gold nanoparticles (Au nanoparticles) in an organic solvent is presented. The gold nanoparticles are capped with oleylamine ligands to prevent agglomeration. The gold nanoparticles are soluble in organic solvents such as toluene.