Esta pesquisa foi financiada pela Fundação Nacional de Ciências Naturais da China (31700840); o Principal Projeto de Pesquisa Científica da Província de Henan (18B430013, 18A150049). Esta pesquisa foi apoiada pelo Programa de Estudiosos de Nanhu para Jovens Estudiosos da XYNU. Os autores gostariam de agradecer ao estudante de bacharel Zebo Qu, da Faculdade de Ciências da Vida em XYNU, por seus trabalhos úteis. Os autores gostariam de reconhecer o Centro de Análise & Testes da XYNU para o uso de seus equipamentos.

ATENÇÃO: Certifique-se de consultar todas as folhas de dados de segurança do material relevante (MSDS) antes de usar todos os produtos químicos. Vários dos produtos químicos usados para preparar copolímeros e nanopartículas são agudamente tóxicos. As nanopartículas também têm perigos potenciais. Certifique-se de usar todas as práticas de segurança apropriadas e equipamentos de proteção individual, incluindo luvas, jaleco, capuzes, calças de comprimento integral e sapatos de dedos próximos.
1. Síntese de polietileno glicol de carboxil duplo (CT-PEG)33
<ol><li>Adicione 1,46 g (14,6 mmol) de anidrido succinico (SA) e 209 mgs (1,71 mmol) de 4 dimetilaaminopirridina (DMAP) a um frasco fundo redondo de 100 mL.</li>
	<li>Adicione 15 mL de tetrahidrofurano anidrofurano (THF) ao frasco usado na etapa 1.1 e coloque uma rolha de vidro. Mantenha o frasco a 0 °C por 30 min.</li>
	<li>Adicione 4,28 g (4,28 mmol) de polietileno glicol (PEG) e 1,8 mL (12,8 mmol) de trietilamina (TEA) a um novo frasco.</li>
	<li>Adicione 15 mL de THF anidro ao frasco usado na etapa 1.3 e coloque uma rolha de vidro. Transfira a solução lentamente para o frasco usado na etapa 1.2, usando uma seringa sob atmosfera de nitrogênio.</li>
	<li>Mexa a solução a 0 °C por 2h e continue a reação à temperatura ambiente (RT) durante a noite.</li>
	<li>Utilizando um evaporador rotativo (40 °C, 0,1 MPa), concentre a solução de reação e remova o solvente THF.</li>
	<li>Na RT, dissolva a solução de reação da etapa 1.6 em 15 mL de 1,325 g/mL de diclorometano (DCM), em seguida, adicione 15 mL de éter de dietil frio (Et2O) para obter o produto de precipitação (diacíclico polietilenoglicol). Remova o solvente através de papel filtro. NOTA: O passo de precipitação pode ser repetido 3x.</li>
	<li>Seque os precipitados sob vácuo em RT por 48 h.</li>
</ol>2. Síntese do copolímero PEI-g-PEG
<ol><li>Adicione 305,47 mg de CT-PEG da etapa 1.8 e 5 mL de sulfóxido de dimetila (DMSO) a um frasco e mexa no RT para garantir que o CT-PEG seja totalmente dissolvido no DMSO.</li>
	<li>Dissolver 49,46 mg de 1-(3-dimetilaminapropyl)-3-etilcarbodimídeo hydrochloride (EDC) em 5 mL de DMSO, em seguida, adicione a solução ao frasco usado na etapa 2.1 e mexa por 30 min na RT.</li>
	<li>Dissolver 29,69 mg de N-hidroxisuccinimida (NHS) em 5 mL de DMSO e adicionar a solução ao frasco utilizado na etapa 2.1. Continue mexendo no RT por 3 h.</li>
	<li>Dissolva 28,6 μL de polietileneimina (PEI) em 10 mL de DMSO e adicione a solução dropwise ao frasco usado na etapa 2.1. Mexa por 3 dias, pelo menos.</li>
	<li>Transfira a solução reagida da etapa 2.4 para um saco de diálise (corte de peso molecular de 1.000 [MWCO]). Coloque o saco de diálise em um béquer de 1 L com 500 mL de água ultrapura como o dialise. Troque a água ultrauso a cada 12 h por 3 dias.</li>
	<li>Transfira a solução na etapa 2.5 para outra bolsa de diálise (10.000 MWCO). Coloque o saco de diálise em um béquer de 1 L com 500 mL de água ultrapura como o dialise. Troque a água ultrauso a cada 12 h por 3 dias.</li>
	<li>Concentre a solução a partir da etapa 2.6 usando um evaporador rotativo (40 °C, 0,1 MPa) e congele a amostra para obter o pó PEI-g-PEG.</li>
</ol>3. Síntese de PEI-g-PEG@AuNPs
<ol><li>Dissolva 5 mg de PEI-g-PEG preparado (passo 2.7) em 5 mL de água ultrapura em um novo frasco e ajuste com uma rolha de vidro.</li>
	<li>Adicione 100 mL de solução HAuCl4 de 0,3 mM ao frasco e mexa a solução por 3 h na RT. NOTA: A cor da solução deve mudar imediatamente de amarelo para laranja.</li>
	<li>Adicione 1 mL de solução NaBH4 de 1 mg/mL ao frasco e mexa a solução por 3 h na RT. NOTA: A solução de reação deve instantaneamente virar borgonha.</li>
	<li>Dialise o produto de reação usando um saco de diálise (1.000 MWCO) durante 3 dias, conforme descrito na etapa 2.5 para obter a solução PEI-g-PEG@AuNPs.</li>
</ol>4. Síntese de DOX-g-PEI-g-PEG@AuNPs
<ol><li>Adicione 1 mL de solução DOX de 2,2 mg/mL e 20 mL de solução PEI-g-PEG@AuNPs a um novo frasco e ajuste com uma rolha de vidro.</li>
	<li>Dissolva 0,727 mg de EDC em 1 mL de água ultrauso e adicione a solução EDC ao frasco utilizado na etapa 4.1.</li>
	<li>Dissolva 0,437 mg de NHS em 1 mL de água ultrapura. Adicione a solução NHS ao frasco e mexa no RT por 1h.</li>
	<li>Dilífique o produto de reação usando um saco de diálise (1.000 MWCO) durante 3 dias, conforme descrito na etapa 2.5 para obter a solução DOX-g-PEI-g-PEG@AuNPs.</li>
</ol>5. Síntese de AS1411-g-DOX-g-PEI-g-PEG@AuNPs
<ol><li>Adicione 20 mL de solução DOX-g-PEI-g-PEG@AuNPs e 4OD de AS1411 (OD = densidade óptica; 1OD ≈ 33 μg) a um novo frasco.</li>
	<li>Dissolva 28,76 mg de EDC em 1 mL de água ultrauso e adicione a solução EDC ao frasco usado na etapa 5.1.</li>
	<li>Dissolva 17,27 mg de NHS em 1 mL de água ultrapura. Adicione a solução NHS ao frasco usado na etapa 5.1 e mexa a reação por 1 h no RT.</li>
	<li>Dilífique o produto de reação usando um saco de diálise (1.000 MWCO) durante 3 dias, conforme descrito na etapa 2.5 para obter AS1411-g-DOX-g-PEI-g-PEG@AuNPs.</li>
</ol>6. Caracterização da amostra
<ol><li>Dissolva o polímero CT-PEG (etapa 1.8) e o copolímero PEI-g-PEG (etapa 2.7) em tubos de clorofórmio-d em tubos de ressonância magnética nuclear (RMR), respectivamente. Analise as amostras usando um espectrômetro NMR de 600 MHz equipado com um ímã supercondutor de 14,09 T e uma sonda de alta resolução de banda larga Z de 5,0 mm 600 MHz para confirmar a estrutura química34.</li>
	<li>Disperse AuNPs, DOX e AS1411, e prepare pei-g-PEG@AuNPs (etapa 3.4), DOX-g-PEI-g-PEG@AuNPs (etapa 4.4) e AS1411-g-DOX-g-PEI-g-PEG@AuNPs (etapa 5.4), respectivamente em água ultrapura. Em seguida, transfira para cuvetas e registos ultravioleta-visível (UV-vis) usando um espectrômetro UV-vis.</li>
	<li>Conecte um adesivo de dupla face (~2 mm x 2 mm) à folha de alumínio e mergulhe a solução de amostra (PEI-g-PEG@AuNPs, DOX-g-PEI-g-PEG@AuNPs e AS1411-g-DOX-g-PEI-g-PEG@AuNPs) na fita inteira uniformemente. Analise as amostras usando um analisador de espectroscopia de fotoeletrâncro de raios-X.</li>
	<li>Disperse as soluções PEI-g-PEG@AuNPs, DOX-g-PEI-g-PEG@AuNPs e AS1411-g-DOX-g-PEI-g-PEG@AuNPs, respectivamente em água ultrauso. Em seguida, transfira para cuvetas e avalie a distribuição de tamanho usando dispersão dinâmica de luz.</li>
	<li>Disperse as soluções PEI-g-PEG@AuNPs, DOX-g-PEI-g-PEG@AuNPs e AS1411-g-DOX-g-PEI-g-PEG@AuNPs em água ultrauso (uma gota de amostra por 5 mL de água ultrauso para cada amostra). Sonicate por 2 h. Mergulhe a rede de cobre em soluções de amostra e seque sob uma lâmpada infravermelha. Caracterize a morfologia usando um microscópio eletrônico de transmissão.</li>
	<li>Injete 1 mg de AS1411-g-DOX-g-PEI-g-PEG@AuNPs em um de diálise MWCO de 20 kDa, em seguida, coloque em 80 mL de salina tamponada de fosfato (PBS) com 5% de albumina de soro bovino (BSA). Mexa a 37 °C.</li>
	<li>Nos pontos de tempo predeterminados, colete alíquotas de 100 μL e substitua por PBS fresco. Use um espectrofotômetro UV-vis para medir a intensidade de fluorescência do DOX das alíquotas.</li>
</ol>7. Ensaio CCK-8 de nanopartículas AS1411-g-DOX-g-PEI-g-PEG@AuNPs
<ol><li>Cultivar células A549 no meio modificado da Águia (DMEM) modificado de Dulbecco ( DMEM) suplementado com 10% de soro bovino fetal, penicilina U/mL de 100 U/mL e 100 μg/mL estreptomicina sob uma atmosfera umidificada de 95% de ar e 5% de CO2 a 37 °C. Substitua o meio de cultura a cada 2 dias. Use células na passagem 5 para a proliferação celular e ensaios de citotoxicidade para avaliar quantitativamente a citotoxicidade das nanopartículas AS1411-g-DOX-g-PEI-g-PEG@AuNPs.</li>
	<li>Adicione 100 μL de solução de nanopartículas em cada poço que contabilizando 1 mL de meio celular. Depois de cultivar por 24h e 48 h, remova a mídia de cultura das placas de cultura celular, em seguida, adicione 300 μL de mídia de cultura fresca e 30 μL de soluções de kit-8 (CCK-8) de contagem celular imediatamente para cada poço. Incubar por 4h em uma incubadora de CO2 a 37 °C.</li>
	<li>Transfira 200 μL de soluções de reação da etapa 7.2 para uma placa de poço 96. Leia a densidade óptica (OD) de cada poço a 570 nm com um leitor de microplacas.</li>
	<li>Observe a morfologia das células a 24 h e 48 h sob um microscópio.</li>
</ol>

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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>

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O espectro de 1H NMR (Figura 2) confirma a síntese bem sucedida do copolímero CT-PEG e do copolímero PEI-g-PEG. Os pesos moleculares de PEG e PEI foram de 1.000 e 1.200, respectivamente. Além disso, o sistema catalítico EDC/NHS foi usado para sintetizar o copolímero PEI-g-PEG através de reações de amida. Deve-se notar que se os pesos moleculares de PEG e PEI mudaram para sintetizar o copolímero PEI-g-PEG, então o tempo de reação e o sistema catalítico precisam ser re...

Sendo o maior problema de saúde pública em todo o mundo, o câncer é amplamente caracterizado como tendo uma baixa taxa de cura, alta taxa de recidiva e alta taxa de mortalidade1,2. Os métodos anti-câncer convencionais atuais incluem cirurgia, quimioterapia e radioterapia3, entre os quais a quimioterapia é o tratamento primário para pacientes com câncer na clínica4. Os medicamentos anticancerígenos usados clinicamente incluem paclitaxel (PTX)5 e doxorubicina (DOX)6,7. O DOX, um medicamento antineoplásico, tem sido amplamente aplicado na quimioterapia clínica, devido às vantagens da citotoxicidade do câncer e da inibição da proliferação de células cancerosas8,9. No entanto, o DOX causa cardiotoxicidade10,11, e a curta meia-vida do DOX restringe sua aplicação na clínica12. Portanto, os transportadores de drogas degradáveis são necessários para carregar o DOX e liberar subeqüentemente de forma controlada para uma área alvo.
As nanopartículas têm sido amplamente utilizadas em sistemas de entrega de medicamentos direcionados e têm várias vantagens no tratamento do câncer (ou seja, proporção superfície-volume considerável, tamanho pequeno, capacidade de encapsular várias drogas e química de superfície incapaz, etc.) 13,14,15. Em particular, as nanopartículas de ouro (AuNPs) têm sido amplamente utilizadas em aplicações biológicas e biomédicas, como a terapia fototérmica do câncer16,17. As propriedades únicas dos AuNPs, como síntese fácil e funcionalização geral da superfície, têm excelentes perspectivas no campo clínico da terapiaoncológica 18. Além disso, os AuNPs têm sido utilizados para identificar estratégias de entrega de medicamentos, diagnosticar tumores e superar a resistência em muitos estudos19,20.
Não obstante, os AuNPs precisam ser mais adaptados para superar a resistência medicamentosa através da alta liberação local em lesões tumorais por meio de uma maior permeação e retenção (EPR), como as propriedades de segmentação e acessibilidade. Os AuNPs funcionais de polímeros apresentaram vantagens únicas, como a melhoria da solubilidade da água de medicamentos anticâncer hidrofóbicos e o tempo de circulação prolongado21,22. Vários polímeros biocompatíveis têm sido usados para revestimentos AuNP, como polietileno glicol (PEG), polietileneimina (PEI), ácido hialurônico, heparina e goma xantana. Em seguida, a estabilidade, assim como a carga útil, de AuNPs é melhorada bem23. Especificamente, o PEI é um polímero altamente ramificado que é composto por muitas unidades repetitivas de aminas primárias, secundárias e terciárias24. PEI tem excelente solubilidade, baixa viscosidade e alto grau de funcionalidade, que é adequado para revestimento em AuNPs.
Por outro lado, os medicamentos anticâncicos precisam ser entregues diretamente às células cancerosas com maior eficiência de carregamento e com menor toxicidade para o tratamento de tumores metastáticos primários e avançados25. Ligantes-alvo têm grande potencial para sistemas de entrega direcionados a medicamentos anticâneos26. Sua seletividade para a ligação de moléculas-alvo confere especificidade de especificidade de medicamentos anticâncer e aumenta o enriquecimento medicamentoso em tecidos doentes27. Mais ligantes incluem anticorpos, polipeptídeos e pequenas moléculas. Comparados com outros ligantes, os aptamers de ácido nucleico podem ser sintetizados in vitro e são fáceis de modificar. AS1411 é um oligonucleotídeo de 26 bp fosfodiester não modificado que forma uma estrutura G-tetramer dimeric estável para se ligar especificamente a um receptor de proteína nuclear de alvo superexpresso em células cancerosas28,29,30. AS1411 inibe a proliferação de muitas células cancerígenas, mas não afeta o crescimento de células saudáveis31,32. Como resultado, o AS1411 tem sido usado para fabricar um sistema ideal de entrega de medicamentos direcionado.
Neste estudo, um copolímero PEI-g-PEG é sintetizado através de uma reação de amida, então as nanopartículas de ouro revestidas de copolímero PEI-g-PEG (PEI-g-PEG@AuNPs) são fabricadas. Além disso, o DOX e o AS1411 estão sequencialmente ligados ao PEI-g-PEG@AuNPs preparado, como mostrado na Figura 1. Este protocolo detalhado destina-se a ajudar os pesquisadores a evitar muitas das armadilhas comuns associadas à fabricação de novas PEI-g-PEG@AuNPs carregadas com DOX e AS1411.

<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

Devido à resistência e toxicidade de medicamentos em células saudáveis, o uso de doxorubicina (DOX) tem sido limitado na terapia clínica do câncer. Este protocolo descreve a concepção de poli (etilenimina) enxertada com polietileno glicol (PEI-g-PEG) nanopartículas de ouro funcionalizadas (AuNPs) com aptámero carregado (AS1411) e DOX através de reações de amida. O AS1411 é especificamente ligado a receptores de nucleoslinas direcionados em células cancerosas para que o DOX alveja células cancerígenas em vez de células saudáveis. Primeiro, o PEG é carboxilado, depois enxertado em PEI ramificado para obter um copolímero PEI-g-PEG, que é confirmado pela análise de 1H NMR. Em seguida, as nanopartículas de ouro revestidas de copolímero PEI-g-PEG (PEI-g-PEG@AuNPs) são sintetizadas, e DOX e AS1411 são covalentemente ligados aos AuNPs gradualmente através de reações de amida. O diâmetro do AS1411-g-DOX-g-PEI-g-PEG@AuNPs é de ~39,9 nm, com potencial zeta de -29,3 mV, indicando que as nanopartículas estão estáveis em água e meio celular. Ensaios de citoxicidade celular mostram que os AuNPs carregados de DOX recém-projetados são capazes de matar células cancerígenas (A549). Esta síntese demonstra o delicado arranjo de copolímeros PEI-g-PEG, aptamers e DOX em AuNPs que são obtidos por reações sequenciais de amida. Tais AuNPs funcionalizados aptamer-PEI-g-PEG fornecem uma plataforma promissora para a entrega de medicamentos direcionados na terapia contra o câncer.

1 A espectroscopia H NMR foi utilizada para confirmar a síntese bem sucedida de polímeros CT-PEG e copolímeros PEI-g-PEG(Figura 2). A Figura 2a mostra que o sinal de próton de metileno em δ = 3,61 ppm e sinal de próton carboxil em δ = 2,57 ppm confirmam a síntese bem sucedida dos polímeros CT-PEG. A Figura 2b mostra que o sinal de próton de metileno de PEG em δ = 2,6 ppm e sinal de pr...

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

Neste protocolo, as nanopartículas de ouro modificadas AS1411-g-PEI-g-PEG são sintetizadas através de reações de meio-médio de três etapas. Em seguida, a doxorubicina é carregada e entregue às células cancerígenas alvo para a terapia contra o câncer.

Síntese de Nanopartículas de ouro modificada Aptamer-PEI-g-PEG carregadas com doxorubicina para entrega de drogas direcionadas

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 visualizações.  Xinyang Normal University. Neste protocolo, as nanopartículas de ouro modificadas AS1411-g-PEI-g-PEG são sintetizadas através de reações de meio-médio de três etapas. Em seguida, a doxorubicina é carregada e entregue às células cancerígenas alvo para a terapia contra o câncer.

Vídeo: 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.

Dirigido modelo de síntese de nanotubos de Ouro plasmônicos com Absorbância IR Tunable

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

Química

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.

Preparação e Caracterização de SDF-1α-Quitosana As nanopartículas de sulfato de dextrano

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.

Um método simples para o tamanho Síntese controlada de estáveis ​​Oligoméricas Clusters de nanopartículas em condições ambientais

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.

Hidroquinona baseada Síntese de ouro Nanorods

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.

Método de precipitação química para a síntese do Nb2O5 modificado catalisadores de níquel em massa com alta área de superfície específica

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.

Sinal de nanossondas rápida realce pela síntese de nanopartículas de ouro em Situ

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.

Methionine Funcionalizou o bloco biocompatible copolímeros para a entrega alvejada do ADN do plasmid

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.

Síntese e caracterização de nanopartículas de ouro Anfífilos

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.

Nanopartícula de ouro modificado microeletrodos de fibra de carbono para detecção neuroquímica reforçada

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.

Methods Article

Síntese de Nanopartículas de ouro modificada Aptamer-PEI-g-PEG carregadas com doxorubicina para entrega de drogas direcionadas