This work was supported by grants from the National Research Foundation of Korea, funded by the government of the Republic of Korea, (Grant nos. 2012M2B2B1055245 and 2012M2A2A6011335) and by the RI-Biomics Center of Korea Atomic Energy Research Institute.

Cuidado: A forma oxidada de iodo radioativo é bastante volátil e devem ser manuseados com escudos de chumbo adequados e frascos de chumbo. Todos os passos radioquímica deve ser levada a cabo em uma capa filtrou-carvão bem ventilado, e os procedimentos experimentais devem ser monitorizadas por meio de dispositivos de detecção de radioactividade. 1. Preparação dos produtos químicos e o cartucho de fase reversa para a síntese da azida 125 I-rotulados <ol><li> Preparação de reagentes em solução <ol><li> Dissolve-se 1 mg de azida do precursor (2) em 150 ul de etanol absoluto (Figura 1). NOTA: Um procedimento sintético detalhado para o precursor azida (2) foi relatada em estudo anterior 22. </li><li> Dissolve-se 1 mg de cloramina T em 20 ul de 1x tampão salino de fosfato (pH = 7,4). </li><li> Dissolve-se 2 mg de metabissulfito de sódio em 20 mL de H 2 O. </li></ol></li><li> Preparação do cartucho <ol><li> Lavar o cartucho de TC18 com 10 ml de etanol absoluto seguida por 10 ml de H 2 O. Não se secar a matriz de o cartucho com ar. </li></ol></li></ol> 2. radiossï¿½tese do protético grupo azida marcada com 125I <ol><li> Radioiodação reacção do precursor <ol><li> Adicionar a solução precursora de azida (1 mg em 150 ul de etanol absoluto) e ácido acético (10 ul) para um tubo de microcentrífuga de 1,5 ml. </li><li> Adicionar 150 MBq de [125I] NaI em NaOH 0,1 M (50 mL) à mistura de reacção. </li><li> Adicionar uma solução de cloramina T (1 mg em 20 ul de 1x tampão de fosfato salino) e fechar-se a mistura de reacção tubo de microcentrífuga contendo. </li><li> Incubar a mistura de reacção à temperatura ambiente durante 15 min, até que a reacção é completada a radioiodinação. </li><li> Adicionar uma solução de metabissulfito de sódio (2 mg em 20 ul de H2O) à mistura reaccionalpara extinguir a reação radioiodação. </li><li> Retirar 0,2 mL do produto em bruto e depois dilui-se com 100 ul de solução (H 2 O / CH 3 CN, 1: 1) para análise por HPLC. NOTA: Para todas as experiências de HPLC, utilizar 0,1% de ácido fórmico contendo H2O (solvente A) e 0,1% de ácido fórmico contendo acetonitrilo (solvente B) como eluentes. </li><li> Analisar o produto em bruto diluído usando um de fase inversa analítica rádio-HPLC (C18 de fase reversa; taxa de fluxo: 1 ml / min; gradiente de eluente: 20% de solvente B durante 0-2 min, 20-80% de solvente B durante 2-22 minutos, 80-100% de solvente B durante 22-23 min, e 100% de solvente B durante 23-28 min; tempo de retenção: 16,4 min) (Figura 2). </li></ol></li><li> A purificação do produto em bruto com uma HPLC preparativa NOTA: Fornecer suficiente blindagem de chumbo em torno de partes de HPLC tal como o injector, coluna, detector, frasquinhos de colheita, e o recipiente no qual o efluente é recolhido. <ol><li> retirar thum e todo mistura reaccional para dentro de um frasco de HPLC. Lavar o tubo de reacção com acetonitrilo (0,5 mL) e adicionar a água de lavagem para o mesmo frasco de injecção. Dilui-se a solução recolhida com H 2 O (1 mL). </li><li> Injectar o produto em bruto para um rádio-preparativa de HPLC (coluna C18 de fase inversa; caudal: 10 ml / min; gradiente de eluente: 20% de solvente B durante 0-2 min, 20-80% de solvente B durante 2-22 min, 80-100% de solvente B durante 22-23 min, e 100% de solvente B durante 23-28 min). </li><li> Recolhe-se o pico que representa a azida radioactivos 125I-marcado (1) (tR sob estas condições de HPLC é 17,8-18,8 min) num tubo de ensaio de vidro (Figura 2). </li><li> Medir o rendimento radioquímico da fracção utilizando um calibrador de doses de radioactividade de acordo com o protocolo do fabricante. </li><li> Injectar o produto purificado sobre um rádio-HPLC analítica usando as mesmas condições de HPLC para determinar a pureza radioquímica do produto. </li></ol></li><li> Extracção em fase sólida do produto <ol><li> Dilui-se a fracção que contém o produto desejado (1) com 40 ml de H2O puro </li><li> Adicionar a solução diluída em um cartucho de TC18 pré-condicionados. </li><li> Lavar o cartucho com um adicional de 15 ml de H 2 O. </li><li> Elui-se o produto (1) preso no cartucho com 2 ml de acetona para um frasco de vidro de 10 ml, que é protegido por um escudo de chumbo. Medir a radioactividade do produto eluído utilizando um calibrador de doses de radioactividade de acordo com o protocolo do fabricante. NOTA: Dimetil sulfóxido (DMSO) ou etanol absoluto, também pode ser usado para eluição do produto a partir do cartucho. Cerca de 5-10% da radioactividade normalmente adere ao cartucho, e o produto marcado radioactivamente restante não pode ser totalmente fluido utilizando quantidades em excesso de solvente orgânico. </li><li> Evapora-se a acetona, com uma corrente de azoto ou árgon gasoso. </li><li> Dissolve-se o residue com DMSO (100-200 uL) para o próximo passo de radiomarcação. </li></ol></li></ol> 3. Síntese de DBCO-grupo conjugado Nanopartículas de Ouro <ol><li> Modificação da superfície de ouro de 13 nm de tamanho nanopartículas com DBCO contendo grupo polietilenoglicol <ol><li> Prepare-de sódio estabilizado citrato de nanopartículas de ouro (3) (média size = 13 nm) de acordo com um relatório anterior 24. </li><li> Adicionar uma solução aquosa de Tween 20 (1 mM, 1,5 mL) às nanoparticulas de ouro estabilizadas-citrato (10 nM, 15 ml). Agitar a solução durante 20 min num agitador orbital. </li><li> Adicionar uma solução aquosa de DBCO contendo-grupo tiol polietileno glicol (peso molecular médio = 5000, 100 ^ M, 1,5 ml). Agitar a solução durante 2 h num agitador orbital. </li></ol></li><li> A purificação das nanopartículas de ouro DBCO-modificado com grupo <ol><li> Purificar o nanopartículas de ouro DBCO-modified-grupo (4) &lt;/ Strong&gt; por centrifugação sucessiva (11.400 xg, 15 min x 3). </li><li> Decantar o sobrenadante e adicionar água pura para a ressuspensão das peletes de nanopartulas de ouro. </li></ol></li></ol> 4. A marcação radioactiva de DBCO-modified-grupo do ouro nanopartículas através do livre-Cobre Clique Reaction <ol><li> Síntese de nanopartículas de ouro 125 marcado com I utilizando a azida de 125I-marcado (1) <ol><li> Prepara-se uma solução concentrada de nanopartículas de ouro DBCO-modificado com grupo usando centrifugação (11400 xg, 15 min), e ajustar a concentração das nanopartículas de ouro de 2 uM. </li><li> Adicionar 4,1 MBq de azida marcada com 125 I (1) em DMSO (5 ul) a uma suspensão de nanopartículas de ouro (4) (2 ^ M, 50 ul). </li><li> Incubar a mistura de reacção resultante a 40 ° C durante 60 min. </li><li> Retirar uma aliquota (0,2 pi) do produto em bruto e aplicá-lo sobre um gel co-cromatografia em camada fina ated (TLC) placa. </li><li> Desenvolver a placa de TLC, utilizando acetato de etilo como a fase móvel. </li><li> Colocar a placa de TLC em um scanner de rádio-TLC e executar o scanner para monitorizar a reacção de marcação radioactiva (Figura 3) de acordo com o protocolo do fabricante. </li></ol></li><li> A purificação do produto em bruto <ol><li> Purifica-se a mistura de reacção contendo os 125 marcado com I nanopartículas de ouro (4) por centrifugação (11400 xg, 15 min). </li><li> Decantar o sobrenadante e adicionar água pura para a ressuspensão das peletes de nanopartulas de ouro. </li><li> Retirar uma aliquota (0,2 pi) do produto purificado e aplicá-lo sobre uma placa de CCF de sílica-revestido. </li><li> Desenvolver a placa de TLC, utilizando acetato de etilo como a fase móvel. </li><li> Colocar a placa de TLC em um scanner de rádio-TLC e executar a análise para determinar o rendimento radioquímico e pureza radioquímica do gol 125 marcado com ID (4) nanopartículas (Figura 3) de acordo com o protocolo do fabricante. </li></ol></li></ol>

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The authors have nothing to disclose.

Em geral, o rendimento radioquímico observada da azida 125 purificada marcada com I (1) foi de 75 ± 10% (n = 8). A marcação radioactiva foi feita com 50-150 MBq de radioactividade, e os resultados são bastante consistente radioquímica. Se [125I] NaI (T 1/2 = 59,4 d) que foram submetidos a decaimento radioactivo durante mais de um mês foi utilizado na reacção de radioiodação, observou-se o rendimento radioquímico de 1 a ser ligeiramente reduzida (53-65%). Portanto, recomenda-se que o [125I] NaI ser utilizado logo que é produzida, ou é enviada para o laboratório para se obter um rendimento optimizado radioquímica. Em adição, uma solução de cloramina T preparada de fresco também deve ser usado na reacção para obter o rendimento radioquímico desejado. Uma vez que o precursor (2) era muito hidrofóbico, 150 ul de etanol absoluto deve ser adicionada para dissolver 1 mg de <stRong&gt; 2 na reacção de radioiodação. Caso contrário, toda a mistura de reacção se tornar turva poderia após a adição de uma solução aquosa do oxidante e [125I] NaI. A diminuição da solubilidade do precursor resulta frequentemente em baixo rendimento radioquímico de 1. DMSO também pode ser utilizado para dissolver a dois no passo de radiomarcação. Além disso, o ácido acético deve ser adicionado à solução precursora para obter um rendimento elevado radioquímica no passo de radioiodação. Antes utilizando HPLC preparativa para a purificação do produto em bruto contendo 1, a coluna de HPLC de fase inversa tem de ser lavada com solventes A e B (taxa de fluxo: 10 ml / min; gradiente de eluente: de 100% de solvente B durante 0-10 min, 100 -0% de solvente B durante 10-25 min, e 0% de solvente B durante 25-30 min) para remover quantidades vestigiais de impurezas a partir do sistema. Em seguida, a coluna de HPLC de fase reversa foi equilibrada com 20% de solvente B em 80% de solvente A durante pelo menos 20 minutos para se obtero tempo de retenção de 1 consistente. A fracção contendo 1 purificada deve ser diluída com mais de 4 vezes o volume de H 2 O no procedimento de extracção em fase sólida. Caso contrário, uma parte do produto purificado não possa ficar preso no cartucho TC18. Quando acetona é usada para eluir purificada a partir do cartucho 1, o volume final pode ser reduzido por evaporação da acetona com uma corrente de azoto ou de árgon à temperatura ambiente. Entre os vários iodos radioactivos, 125 I foi selecionado e utilizado na pesquisa atual. Diferentes tipos de radioisótopos de iodo precisam ser testados utilizando o presente método em outros estudos biológicos e médicos (por exemplo, 124I para imagiologia por PET, 131 I para fins terapêuticos). Tanto quanto sabemos, o presente protocolo de marcação radioactiva é o primeiro relatório que descreve syntheti detalhadapassos C por um grupo azida marcada com radioiodo. Recentemente, publicou um outro grupo prostético azida, que tem uma estrutura diferente 23. No entanto, a azida radiomarcado (1) no método corrente fornecida ligeiramente melhores resultados radioquímica do que o outro, em termos de eficiência de marcação radioactiva com moléculas contendo grupos DBCO. Existente grupos prostéticos (ou seja, N-hidroxissuccinimida e maleimida) para a rotulagem de iodo radioativo não poderia fornecer especificidade do local. No entanto, o presente método demonstra a eficiência de radiomarcação directa juntamente com excelente bioorthogonality. Desde que o grupo funcional azida é conhecido por ser altamente estável em condições fisiológicas e em ambientes in vivo, o produto marcado radioactivamente (1) podem ser utilizados em pré-alvo de estudos de imagiologia in vivo. Prevemos que este método serão eficientemente aplicada a ambos in vitro e in vivo radioisótopo de iodo labeling de biomoléculas e dos nanomateriais que contêm uma estrutura cyclooctyne tensa. Com base na radioactividade específica de 1, a razão molar calculada de 125 I e nanopartículas de ouro é ~ 1:. 1 125 nanopartículas de ouro marcado com I (4) pode ser utilizado em estudos de imagem e de biodistribuição moleculares dos nanomateriais. O actual método também pode ser aplicado a rotulagem iodo radioactivo dos diferentes tamanhos e formas dos nanomateriais ouro.

The strain-promoted copper-free click reaction between azides and cyclooctynes has been extensively applied to the efficient bioorthogonal labeling of a wide range of biomolecules, nanomaterials, and living subjects1-7. Due to the excellent site-specificity and rapid reaction rate of this conjugation reaction, it has also been used to synthesize radiolabeled tracers. A few 18F-labeled azide or DBCO prosthetic groups have been prepared for in vitro labeling of various cancers targeting peptides and antibodies, as well as for in vivo pre-targeted imaging of tumors8-13. In addition to these examples, the same conjugation reaction was applied to the metal-radioisotope-labeling of nanomaterials for positron emission tomography (PET) imaging studies14-16.
For several decades, radioactive iodines have been used for biomedical research and clinical trials through PET imaging (124I), single-photon emission computed tomography (SPECT) imaging (123I, 125I), and thyroid cancer treatment (131I)17-21. Therefore, an efficient method for radioactive iodine labeling is fundamentally important for various investigations, including molecular imaging studies, analysis of organ distribution of biomolecules, biomarker identification, and drug development. A copper-free click reaction strategy could be used in radioactive iodine labeling. However, this application has not been investigated as extensively as 18F-labeled biomolecules22-23. Here, we will provide a step-by-step protocol for the synthesis of an 125I-labeled azide for radiolabeling of DBCO-group-derived molecules. The procedures in the present report will include radioiodination of the stannylated precursor, purification steps with HPLC, and solid phase extraction. We also demonstrate efficient radiolabeling of DBCO-group-modified 13-nm-sized gold nanoparticles using the 125I-labeled azide. The detailed protocol in this report will help synthetic chemists understand a new radiolabeling methodology for the synthesis of radiolabeled products.

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			<td height="21" style="height:21px;width:255px;">Chloramine T trihydrate</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">402869</td>
			<td style="width:167px;"></td>
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		<tr height="24">
			<td height="24" style="height:24px;width:255px;">[125I]NaI in aq. NaOH</td>
			<td style="width:101px;">Perkin-Elmer</td>
			<td style="width:160px;">NEZ033A010MC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Sodium metabisulfite&#160;</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">S9000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Formic acid</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">251364</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Sep-Pak tC18 plus cartridge</td>
			<td style="width:101px;">Waters</td>
			<td>WAT036800</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Dimethyl sulfoxide&#160;</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Acetone</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">650501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Ethanol</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">459844</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Gold(III) chloride trihydrate</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">520918</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tween 20&#160;</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:160px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DBCO PEG SH (MW 5000)</td>
			<td style="width:101px;">NANOCS</td>
			<td style="width:160px;">PG2-DBTH-5k</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:255px;">TLC silica gel 60 F254</td>
			<td style="width:101px;">Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Analytical HPLC</td>
			<td style="width:101px;">Agilent</td>
			<td style="width:160px;">1290 Infinity</td>
			<td>Model number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Preparative HPLC</td>
			<td style="width:101px;">Agilent</td>
			<td style="width:160px;">1260 Infinity</td>
			<td>Model number</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Analytical C18 reverse-phase column</td>
			<td style="width:101px;">Agilent</td>
			<td style="width:160px;">Zorbax Eclipse XDB-C18</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Preparative C18 reverse-phase column</td>
			<td style="width:101px;">Agilent</td>
			<td style="width:160px;">PrepHT XDB-C18</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Radio TLC scanner</td>
			<td style="width:101px;">Bioscan</td>
			<td style="width:160px;">AR-2000</td>
			<td>Model number</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Radioisotope dose calibrator</td>
			<td style="width:101px;">Capintec, Inc</td>
			<td style="width:160px;">CRC -25R dose calibrator</td>
			<td>Model number</td>
		</tr>
	</tbody>
</table>

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.

A reacção de radioiodação do precursor estanilado (2) foi realizado utilizando 150 MBq de [125I] NaI, ácido acético, e cloramina T à temperatura ambiente durante 15 min para proporcionar o produto marcado radioactivamente (1). Depois de purificação por HPLC preparativa da mistura em bruto, o produto desejado foi obtido com 75 ± 10% (n = 8) de rendimento radioquímico. HPLC analica revelou que a pureza radioquímica do produto marcado com 125I foi mais do que 99% (Figura 2), e a radioactividade específica observada de um produto é 40,7 MBq / nmol. Extracção em fase sólida de a fracção que contém o produto purificado usando o cartucho fornecida uma solução de acetona de 1. Usando um fluxo de azoto ou árgon gasoso, o solvente orgânico pode ser evaporada, e depois o resíduo pode ser novamente dissolvido em DMSO ou etanol absoluto para o próximo step. Para 125I-marcação de nanopartículas de ouro polietileno-modificado com glicol, as nanopartículas de ouro DBCO-grupo-modificados foram preparados pelo processo mostrado na Figura 1. Uma quantidade em excesso de tiol de polietileno glicol (MW 5000) com grupos DBCO foi feito reagir com o citrato, estabilizado nanopartículas de ouro de 13 nm. Após a etapa de modificação, o produto foi purificado por centrifugação sucessiva para dar as nanopartículas de ouro-funcionalizado DBCO (3). No passo de marcação radioactiva, 3,7 MBq de 1 foi adicionada a 2 uM de 3 (~ 400 uM dos grupos DBCO), e a reacção de marcação foi realizada a 40 ° C durante 1 h. Radio-TLC análise mostrou que foi feito reagir mais do que 95% de uma com as nanopartículas DBCO-funcionalizado por grupo de ouro (3) dentro de 60 min. A reacção foi realizada durante 60 min, e, em seguida, o produto em bruto foi purficada por centrifugação. 125 nanopartículas de ouro marcadas com I (4) foram obtidos com&gt; 99% (n = 4) rendimento radioquímico, determinado por rádio TLC (Figura 3). <img alt="figura 1" src="/files/ftp_upload/54759/54759fig1.jpg" /> Figura 1. radiossï¿½tese da azida 125 marcado com I (1) e marcada com 125I nanopartículas de ouro (4). Reagentes e condições: (a) [125I] NaI, ácido acético, cloramina T, TA, 15 min, 75 ± 10% (n = 8) rendimento radioquímico; (B) DBCO-PEG-SH (MW 5000), H2O, RT, 2 h; ~ 40 ° C, 60 min,&gt; 99% de rendimento radioquímico. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54759/54759fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img al<html> t = &quot;Figura 2&quot; src = &quot;/ files / ftp_upload / 54759 / 54759fig2.jpg&quot; /&gt; Figura 2. Cromatograma de HPLC analítica da azida 125 marcado com Eu (1). (A) radiocromatograma do produto em bruto. (B) radiocromatograma do produto purificado. (C) cromatograma UV (254 nm) do produto purificado. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54759/54759fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/54759/54759fig3.jpg" /> Figura 3. Resultados de rádio-125 por CCF da nanopartículas de ouro marcado com I (4) (R f = 0,05 de 4, Rf de 1 = 0,45, eluente: acetato de etilo) (a) após uma reacção de 60 min e (b) após purificação./ecsource.jove.com/files/ftp_upload/54759/54759fig3large.jpg &quot;target =&quot; _ blank &quot;&gt; Clique aqui para ver uma versão maior desta figura.

Watch this Scientific Journal Video about An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group at JoVE.com

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

Um procedimento pormenorizado para a síntese de uma azida marcado com 125I e a radiomarcação de dibenzocyclooctyne (DBCO) -Grupo-conjugados, nanopartículas de ouro de 13 nm de tamanho, utilizando uma reacção de clique sem cobre é descrito.

Um protocolo otimizado para a radiomarcação eficiente de nanopartículas de ouro usando um<sup&gt; 125</sup&gt; Marcado com Eu Azida grupo prostético

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

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Nanyang Technological University

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Chemistry

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

8.1K visualizações.  Korea Atomic Energy Research Institute.  Um procedimento pormenorizado para a síntese de uma azida marcado com 125I e a radiomarcação de dibenzocyclooctyne (DBCO) -Grupo-conjugados, nanopartículas de ouro de 13 nm de tamanho, utilizando uma reacção de clique sem cobre é descrito. 

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

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing and maintaining primary and pluripotent stem cell derived hepatocytes. While biological matrices are useful, they permit short term culture of hepatocytes, limiting their widespread application. We have attempted to overcome the limitations using a synthetic polymer coating. Polymers represent one of the broadest classes of biomaterials and possess a wide range of mechanical, physical and chemical properties, which can be fine-tuned for purpose. Importantly, such materials can be scaled to quality assured standards and display batch-to-batch consistency. This is essential if cells are to be expanded for high through-put screening in the pharmaceutical testing industry or for cellular based therapy. Polyurethanes (PUs) are one group of materials that have shown promise in cell culture. Our recent progress in optimizing a polyurethane coated surface, for long-term culture of human hepatocytes displaying stable phenotype, is presented and discussed.

This article will focus on developing polymer coated surfaces for long-term, stable culture of stem cell derived human hepatocytes.

Estabilização hepatocelular Fenótipo Usando superfícies sintéticas otimizados

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing ...

Química

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 ...

A Protocol for the Production of Gliadin-cyanoacrylate Nanoparticles for Hydrophilic Coating

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic by a simple spray coating. These nanoparticles are produced by the polymerization reaction of alkyl cyanoacrylate on the surface of cereal protein (gliadin) molecules. Alkyl cyanoacrylate is a monomer that instantly polymerizes at RT when it is applied to the surface of materials. Its polymerization reaction is initiated by the trace amounts of weakly basic or nucleophilic species on the surface, including moisture. Once polymerized, the polymerized alkyl cyanoacrylates show a strong affinity with the object materials because nitrile groups are in the backbone of poly (alkyl cyanoacrylate). Proteins also work as initiator for this polymerization because they contain amine groups that can initiate the polymerization of cyanoacrylate. If aggregated protein is used as an initiator, protein aggregate is surrounded by the hydrophobic poly(alkyl cyanoacrylate) chains after the polymerization reaction of alkyl cyanoacrylate. By controlling the experimental condition, particles in the nanometer range are produced. The produced nanoparticles readily adsorb to the surface of most materials including glass, metals, plastics, wood, leather, and fabrics. When the surface of a material is sprayed with the produced nanoparticle suspension and rinsed with water, the micellar structure of nanoparticle changes its conformation, and the hydrophilic proteins are exposed to the air. As a result, the nanoparticle-coated surface changes to hydrophilic.

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic. The produced nanoparticle is an assembly of gliadin-cyanoacrylate diblock copolymers. Spray coating with the produced nanoparticle changes the surface of target material to a hydrophilic surface.

Um protocolo para a produção de nanopartículas Gliadina-cianoacrilato para hidrofílico Coating

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic by a simple spray ...

A Simple and Efficient Protocol for the Catalytic Insertion Polymerization of Functional Norbornenes

Norbornene can be polymerized by a variety of mechanisms, including insertion polymerization whereby the double bond is polymerized and the bicyclic nature of the monomer is conserved. The resulting polymer, polynorbornene, has a very high glass transition temperature, Tg, and interesting optical and electrical properties. However, the polymerization of functional norbornenes by this mechanism is complicated by the fact that the endo substituted norbornene monomer has, in general, a very low reactivity. Furthermore, the separation of the endo substituted monomer from the exo monomer is a tedious task. Here, we present a simple protocol for the polymerization of substituted norbornenes (endo:exo ca. 80:20) bearing either a carboxylic acid or a pendant double bond. The process does not require that both isomers be separated, and proceeds with low catalyst loadings (0.01 to 0.02 mol%). The polymer bearing pendant double bonds can be further transformed in high yield, to afford a polymer bearing pendant epoxy groups. These simple procedures can be applied to prepare polynorbornenes with a variety of functional groups, such as esters, alcohols, imides, double bonds, carboxylic acids, bromo-alkyls, aldehydes and anhydrides.

We describe the catalytic insertion polymerization of 5-norbornene-2-carboxylic acid and 5-vinyl-2-norbornene to form functional polymers with a very high glass transition temperature.

Um protocolo simples e eficiente para a polimerização por inserção catalítica de norbornenos funcionais

Norbornene can be polymerized by a variety of mechanisms, including insertion polymerization whereby the double bond is polymerized and the bicyclic ...

A Protocol for Safe Lithiation Reactions Using Organolithium Reagents

Organolithium reagents are powerful tools in the synthetic chemist&#39;s toolbox. However, the extreme pyrophoric nature of the most reactive reagents warrants proper technique, thorough training, and proper personal protective equipment. To aid in the training of researchers using organolithium reagents, a thorough, step-by-step protocol for the safe and effective use of tert-butyllithium on an inert gas line or within a glovebox is described. As a model reaction, preparation of lithium tert-butyl amide by the reaction of tert-butyl amine with one equivalent of tert-butyl lithium is presented.

The safe and proper use of organolithium reagents is described.

Um protocolo para reações seguras litiação Usando organolï¿½io Reagentes

Organolithium reagents are powerful tools in the synthetic chemist&#39;s toolbox. However, the extreme pyrophoric nature of the most reactive reagents ...

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 ...

The MPLEx Protocol for Multi-omic Analyses of Soil Samples

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and characterize microbial communities in environmental and biological systems. These measurements are even enabling enhanced analyses of complex soil microbial communities, which are the most complex microbial systems known to date. Multi-omic analyses, however, do have sample preparation challenges, since separate extractions are typically needed for each omic study, thereby greatly amplifying the preparation time and amount of sample required. To address this limitation, a 3-in-1 method for the simultaneous extraction of metabolites, proteins, and lipids (MPLEx) from the same soil sample was created by adapting a solvent-based approach. This MPLEx protocol has proven to be both simple and robust for many sample types, even when utilized for limited quantities of complex soil samples. The MPLEx method also greatly enabled the rapid multi-omic measurements needed to gain a better understanding of the members of each microbial community, while evaluating the changes taking place upon biological and environmental perturbations.

A protocol is presented for simultaneously extracting metabolites, proteins, and lipids from a single soil sample, allowing reduced sample preparation times and enabling multi-omic mass spectrometry analyses of samples with limited quantities.

O protocolo de mplexas para Multi-omic análises de amostras de solo

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and ...

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of Gold(III)

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em = 640 nm) of BSA-Au(III) complexes. The method adjusts the pH to show that the emergence of the red fluorescence is correlated with the pH-induced equilibrium transitions of the BSA conformations. Red fluorescent BSA-Au(III) complexes can only be formed with an adjustment of pH at or above 9.7, which corresponds to the &#34;A-form&#34; conformation of BSA. The protocol to adjust the BSA to Au molar ratio and to monitor the time-course of the process of Au(III) binding is described. The minimum number of Au(III) per BSA, to produce the red fluorescence, is less than seven. We describe the protocol in steps to illustrate the presence of multiple Au(III) binding sites in BSA. First, by adding copper (Cu(II)) or nickel (Ni(II)) cations followed by Au(III), this method reveals a binding site for Au(III) that is not the red fluorophore. Second, by modifying BSA by thiol capping agents, another nonfluorophore-forming Au(III) binding site is revealed. Third, changing the BSA conformation by cleaving and capping of the disulfide bonds, the possible Au(III) binding site(s) are illustrated. The protocol described, to control the BSA conformations and Au(III) binding, can be generally applied to study the interactions of other proteins and metal cations.

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII

The protocols for studying the binding of gold cations (Au(III)) to various conformations of bovine serum albumin (BSA) as well as for characterizing the conformational dependent unique BSA-Au fluorescence are presented.

Lumíforo formação em várias conformações de albumina de soro bovino por ligação de Gold(III)

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em ...

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 ...

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

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

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 ...