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.

Attenzione: la forma ossidata di iodio radioattivo è molto volatile e deve essere maneggiato con adeguati schermi di piombo e fiale di piombo. Tutti i passaggi radiochimiche dovrebbero essere eseguite in una cappa carbone filtrata ben ventilata, e le procedure sperimentali devono essere controllati da dispositivi di rilevazione di radioattività. 1. Preparazione della Chimica e della cartuccia Reverse Phase per la sintesi del 125 Azide I-marcato <ol><li> Preparazione dei reagenti in soluzione <ol><li> Sciogliere 1 mg del precursore azide (2) in 150 ml di etanolo assoluto (Figura 1). NOTA: Una procedura di sintesi dettagliata per il precursore azide (2) è stato riportato nel precedente articolo 22. </li><li> Sciogliere 1 mg cloramina T in 20 ml di 1x tampone fosfato isotonico (pH = 7,4). </li><li> Sciogliere 2 mg di sodio metabisolfito in 20 ml di H 2 O. </li></ol></li><li> preparazione della cartuccia <ol><li> Lavare la cartuccia TC18 con 10 ml di etanolo assoluto seguito da 10 ml di H 2 O. Non asciugare la matrice della cartuccia con aria. </li></ol></li></ol> 2. radiosintesi del Azide Protesi Gruppo marcato 125 I <ol><li> Reazione Radioiodination del precursore <ol><li> Aggiungere la soluzione azide precursore (1 mg in 150 ml di etanolo assoluto) e acido acetico (10 ml) in una provetta da 1,5 ml microcentrifuga. </li><li> Aggiungere 150 MBq di [125 I] NaI in 0,1 M NaOH (50 ml) alla miscela di reazione. </li><li> Aggiungere una soluzione cloramina T (1 mg in 20 ml di 1x tampone fosfato salino) e chiudere la miscela di reazione provetta contenente. </li><li> Incubare la miscela di reazione a temperatura ambiente per 15 minuti fino a quando la reazione è completa radioiodination. </li><li> Aggiungere una soluzione di sodio metabisolfito (2 mg in 20 ml H 2 O) alla miscela di reazioneper placare la reazione radioiodination. </li><li> Prelevare 0,2 ml di prodotto grezzo e diluire con 100 ml di soluzione di (H 2 O / CH 3 CN, 1: 1) per l&#39;analisi HPLC. NOTA: Per tutti gli esperimenti HPLC, usa acido formico 0,1% contenente H 2 O (solvente A) e 0,1% di acido formico contenente acetonitrile (solvente B) come eluenti. </li><li> Analizzare il prodotto grezzo diluito utilizzando una fase inversa analitica radio-HPLC (C18 a fase inversa colonna; portata: 1 ml / min; eluente pendenza: 20% B solvente per 0-2 min, 20-80% di solvente B per 2-22 min, 80-100% B solvente per 22-23 min, e il 100% di solvente B per 23-28 min; tempo di ritenzione: 16,4 min) (Figura 2). </li></ol></li><li> La purificazione del prodotto grezzo con una HPLC preparativa NOTA: fornire abbastanza schermatura di piombo intorno parti HPLC quali l&#39;iniettore, colonne, rivelatore, fiale di raccolta, il contenitore in cui l&#39;effluente viene raccolto. <ol><li> ritirare °una e intera miscela di reazione in una fiala HPLC. Lavare il tubo di reazione con acetonitrile (0,5 ml) e aggiungere il risciacquo nello stesso flaconcino. Diluire la soluzione raccolta con H 2 O (1 ml). </li><li> Iniettare il prodotto grezzo su una preparativa radio-HPLC (C18 a fase inversa colonna; la portata: 10 ml / min; gradiente eluente: 20% di solvente B per 0-2 min, 20-80% di solvente B per 2-22 min, 80-100% B solvente per 22-23 min, e il 100% B solvente per 23-28 min). </li><li> Raccogliere il picco radioattivi rappresenta il azide marcato 125 I (1) (t R in queste condizioni HPLC è 17,8-18,8 min) in una provetta di vetro (Figura 2). </li><li> Misurare la resa radiochimica della frazione con un calibratore di dosi di radioattività secondo il protocollo del produttore. </li><li> Iniettare il prodotto purificato su una analisi radio-HPLC utilizzando le stesse condizioni HPLC per determinare la purezza radiochimica del prodotto. </li></ol></li><li> Estrazione in fase solida del prodotto <ol><li> Diluire la frazione contenente il prodotto desiderato (1) con 40 ml puro H 2 O. </li><li> Aggiungere la soluzione diluita in una cartuccia TC18 condizionato. </li><li> Lavare la cartuccia con un ulteriore 15 ml H 2 O. </li><li> Eluire il prodotto (1) intrappolato nella cartuccia con 2 ml di acetone in una fiala di vetro da 10 ml che è protetto da uno scudo piombo. Misurare la radioattività del prodotto eluito utilizzando un calibratore di dosi di radioattività secondo il protocollo del produttore. NOTA: dimetilsolfossido (DMSO) o etanolo assoluto può essere utilizzato anche per eluizione del prodotto dalla cartuccia. Circa il 5-10% della radioattività attacca normalmente alla cartuccia, e il prodotto radiomarcato rimanente non può essere completamente eluito utilizzando quantità in eccesso di solvente organico. </li><li> Evaporare l&#39;acetone con un flusso di azoto o gas argon. </li><li> Sciogliere il residue con DMSO (100-200 ml) per il prossimo passo radiomarcatura. </li></ol></li></ol> 3. Sintesi di DBCO-gruppo-coniugato oro Nanoparticelle <ol><li> Modifica della superficie di oro 13-nm di dimensioni nanoparticelle con DBCO-gruppo contenente glicole polietilenico <ol><li> Nanoparticelle d&#39;oro Preparare sodio-citrato-stabilizzato (3) (dimensione media = 13 nm), secondo un rapporto precedente 24. </li><li> Aggiungere una soluzione acquosa di Tween 20 (1 mM, 1,5 ml) per le nanoparticelle di oro citrato-stabilizzato (10 nM, 15 ml). Agitare la soluzione per 20 minuti in un agitatore orbitale. </li><li> Aggiungere una soluzione acquosa di DBCO-contenente gruppi tiolo polietilenglicole (peso molecolare medio = 5000, 100 mM, 1,5 ml). Agitare la soluzione per 2 ore su un agitatore orbitale. </li></ol></li><li> Purificazione delle nanoparticelle di oro DBCO-gruppo modificati <ol><li> Purificare il nanoparticelle d&#39;oro DBCO-gruppo-modificato (4) &lt;/ Strong&gt; per centrifugazione successivi (11.400 xg, 15 min x 3). </li><li> Decantare il surnatante e aggiungere acqua pura per la risospensione del pellet delle nanoparticelle d&#39;oro. </li></ol></li></ol> 4. radiomarcatura di DBCO-gruppo-modificato oro nanoparticelle tramite la libera-Rame Clicca Reazione <ol><li> Sintesi di nanoparticelle d&#39;oro 125 I-etichettati utilizzando l&#39;azide marcato 125 I (1) <ol><li> Preparare una soluzione concentrata di nanoparticelle di oro DBCO-gruppo-modificato usando centrifugazione (11.400 xg, 15 min) e regolare la concentrazione delle nanoparticelle di oro a 2 mM. </li><li> Aggiungere 4,1 MBq del azide I-marcato 125 (1) in DMSO (5 ml) ad una sospensione di nanoparticelle di oro (4) (2 mM, 50 microlitri). </li><li> Incubare la miscela di reazione risultante a 40 ° C per 60 min. </li><li> Prelevare un&#39;aliquota (0,2 ml) dal prodotto grezzo e applicarlo su una silice-coated cromatografia su strato sottile (TLC) piatto. </li><li> Sviluppare la piastra TLC usando acetato di etile come fase mobile. </li><li> Posizionare la piastra TLC su uno scanner radio TLC ed eseguire lo scanner per monitorare la reazione radiomarcatura (figura 3) secondo il protocollo del produttore. </li></ol></li><li> La purificazione del prodotto grezzo <ol><li> Purificare la miscela di reazione contenente 125 nanoparticelle I marcato con oro (4) mediante centrifugazione (11.400 xg, 15 min). </li><li> Decantare il surnatante e aggiungere acqua pura per la risospensione del pellet delle nanoparticelle d&#39;oro. </li><li> Prelevare un&#39;aliquota (0,2 ml) dal prodotto purificato e applicarlo su una piastra TLC rivestiti di silice. </li><li> Sviluppare la piastra TLC usando acetato di etile come fase mobile. </li><li> Posizionare la piastra TLC su uno scanner radio TLC ed eseguire lo scanner per determinare la resa radiochimica e purezza radiochimica del gol marcato 125 Id nanoparticelle (4) (Figura 3) secondo il protocollo del produttore. </li></ol></li></ol>

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The authors have nothing to disclose.

In generale, la resa radiochimica osservato del purificato 125 azide I-marcato (1) è stato 75 ± 10% (n = 8). La radiomarcatura è stata compiuta con 50-150 MBq di radioattività, ed i risultati radiochimiche sono abbastanza coerenti. Se [125 I] NaI (t 1/2 = 59,4 d) sottoposto decadimento radioattivo per più di un mese è stato utilizzato nella reazione radioiodination, la resa radiochimica 1 è stata osservata essere leggermente diminuita (53-65%). Pertanto, si raccomanda di [125 I] NaI essere utilizzato non appena viene prodotto o viene consegnato al laboratorio per ottenere ottimizzata resa radiochimica. Inoltre, una soluzione cloramina T appena preparata deve essere utilizzato nella reazione per ottenere la resa radiochimica desiderato. Poiché il precursore (2) era molto idrofoba, 150 ml di etanolo assoluto dovrebbe essere aggiunto per sciogliere 1 mg di <strong&gt; 2 nella reazione radioiodination. Altrimenti, la miscela di reazione insieme potrebbe diventare torbida dopo l&#39;aggiunta di una soluzione acquosa di ossidante e [125 I] NaI. Solubilità Diminuzione del precursore si traduce spesso in bassa resa radiochimica di 1. DMSO può anche essere usato per sciogliere 2 nella fase radiomarcatura. Inoltre, l&#39;acido acetico deve essere aggiunto alla soluzione precursore di ottenere un&#39;elevata resa radiochimica nel passaggio radioiodination. Prima di utilizzare HPLC preparativa per la purificazione del prodotto grezzo contenente 1, la colonna HPLC in fase inversa deve essere lavato con solventi A e B (portata: 10 ml / min; eluente gradiente: 100% B solvente per 0-10 min, 100 -0% B solvente per 10-25 min, e 0% B solvente per 25-30 min) per rimuovere quantità in tracce di impurità dal sistema. Successivamente, la colonna HPLC in fase inversa viene equilibrato con 20% di solvente B in 80% Un solvente per almeno 20 min per ottenereil tempo di ritenzione coerente 1. La frazione contenente purificata 1 deve essere diluito con più di 4 volte il volume di H 2 O nella procedura di estrazione in fase solida. Altrimenti, alcuni dei prodotti purificato non può essere intrappolati nella cartuccia TC18. Quando acetone viene usato per eluire purificata 1 dalla cartuccia, il volume finale può essere ridotto mediante evaporazione di acetone con un flusso di azoto o gas argon a temperatura ambiente. Tra le varie iodio radioattivo, 125 sono stata selezionata e utilizzato nella ricerca attuale. Hanno bisogno di essere testati con il metodo presente in altri studi biologici e medici diversi tipi di radioisotopi di iodio (ad esempio, 124 sono per l&#39;imaging PET, 131 sono a scopo terapeutico). Per quanto si capisce, il presente protocollo radiomarcatura è il primo rapporto che descrive syntheti dettagliatapassaggi c per un gruppo azide radioiodio marcato. Recentemente, abbiamo pubblicato un altro gruppo prostetico azide, che ha una struttura diversa 23. Tuttavia, l&#39;azide radiomarcato (1) nel metodo corrente disponibile leggermente migliori risultati radiochimica rispetto all&#39;altra in termini di radiomarcatura efficienza con DBCO-contenenti gruppi molecole. Esistenti gruppi prostetici (vale a dire, N -hydroxysuccinimide e maleimmide) per l&#39;etichettatura di iodio radioattivo non ha potuto fornire sito-specificità. Tuttavia, il presente metodo dimostra semplice efficienza radiomarcatura insieme ad un eccellente bioorthogonality. Poiché il gruppo funzionale azide è noto per essere altamente stabile in condizioni fisiologiche e in ambienti vivo, il prodotto radiomarcato (1) può essere utilizzato in pre-targeting in studi di imaging in vivo. Prevediamo che questo metodo verrà applicato efficacemente sia in vitro che in vivo radioisotopi di iodio labeling di biomolecole e dei nanomateriali che contengono una struttura cyclooctyne tesa. Sulla base della radioattività specifica di 1, il rapporto molare calcolato di 125 nanoparticelle I e oro è ~ 1:. 1 125 nanoparticelle di oro I-etichettati (4) possono essere utilizzati in molecolari di imaging e biodistribuzione studi di nanomateriali. L&#39;attuale metodo può essere applicato anche all&#39;etichettatura iodio radioattivo di diverse dimensioni e forme di nanomateriali oro.

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.

<table border="0" cellpadding="0" cellspacing="0" width="683">
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	<tbody>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<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.

La reazione radioiodination del precursore stannylated (2) è stata effettuata utilizzando 150 MBq di [125 I] NaI, acido acetico, e cloramina T a temperatura ambiente per 15 minuti per fornire il prodotto radiomarcato (1). Dopo la purificazione preparativa HPLC della miscela grezza, il prodotto desiderato è stato ottenuto con 75 ± 10% (n = 8) della resa radiochimica. Analytical HPLC ha rivelato che la purezza radiochimica del prodotto 125 I-marcato era superiore al 99% (Figura 2), e la radioattività specifica osservata del prodotto 1 è 40,7 MBq / mol. Estrazione in fase solida della frazione contenente il prodotto purificato utilizzando la cartuccia fornito una soluzione in acetone di 1. Utilizzando un flusso di azoto o gas argon, il solvente organico può essere evaporato, e poi il residuo può essere ridisciolto in DMSO o etanolo assoluto per la prossima step. Per 125 I-etichettatura di nanoparticelle di oro polietilene-glicole-modificato, le nanoparticelle di oro DBCO-gruppo modificati sono stati preparati mediante la procedura illustrata in Figura 1. Una quantità in eccesso di polietilenglicol tiolo (MW 5000) con gruppi DBCO è stato fatto reagire con il citrato, stabilizzato nanoparticelle d&#39;oro 13-nm. Dopo la fase di modifica, il prodotto è stato purificato mediante centrifugazione successive invia le nanoparticelle di oro DBCO-funzionalizzati (3). Nella fase radiomarcatura, 3,7 MBq di 1 inserito 2 mM di 3 (~ 400 uM dei gruppi DBCO), e la reazione di marcatura viene effettuata a 40 ° C per 1 ora. Radio-TLC analisi ha mostrato che oltre il 95% di 1 è stato fatto reagire con le nanoparticelle di oro DBCO-gruppo funzionalizzato (3) entro 60 min. La reazione è stata condotta per 60 minuti, e quindi il prodotto grezzo era purified per centrifugazione. 125 nanoparticelle di oro I-marcato (4) sono stati ottenuti con&gt; 99% (n = 4) resa radiochimica come determinato via radio TLC (Figura 3). <img alt="Figura 1" src="/files/ftp_upload/54759/54759fig1.jpg" /> Figura 1. radiosintesi del azide marcato 125 I (1) e 125 nanoparticelle di oro I-etichettati (4). Reagenti e condizioni: (a) [125 I] NaI, acido acetico, cloramina T, RT, 15 min, 75 ± 10% (n = 8) resa radiochimica; (B) DBCO-PEG-SH (MW 5000), H 2 O, RT, 2 ore; ~ 40 ° C, 60 min,&gt; 99% di resa radiochimica. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54759/54759fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img al<html> t = &quot;Figura 2&quot; src = &quot;/ files / ftp_upload / 54759 / 54759fig2.jpg&quot; /&gt; Figura 2. Analytical HPLC cromatogramma della azide marcato 125 I (1). (A) cromotogramma radioattivo del prodotto grezzo. (B) cromotogramma radioattivo del prodotto purificato. (C) cromatogramma UV (254 nm) del prodotto purificato. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54759/54759fig2large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/54759/54759fig3.jpg" /> Figura 3. Radio-TLC risultati delle nanoparticelle di oro 125 I-etichettati (4) (R f 4 = 0,05, R f 1 = 0,45, eluente: acetato di etile) (a) dopo una reazione 60 min e (b) dopo la purificazione./ecsource.jove.com/files/ftp_upload/54759/54759fig3large.jpg &quot;target =&quot; _ blank &quot;&gt; Clicca qui per vedere una versione più grande di questa 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

Una procedura dettagliata per la sintesi di un azide 125 I-etichettati e la radiomarcatura di dibenzocyclooctyne (DBCO) -gruppo-coniugati, nanoparticelle d&#39;oro 13-nm dimensioni utilizzando una reazione click senza il rame è descritto.

Un protocollo ottimizzato per la radiomarcatura efficiente di oro nanoparticelle utilizzando un<sup&gt; 125</sup&gt; Azide gruppo I-etichettati protesico

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

an-optimized-protocol-for-efficient-radiolabeling-gold-nanoparticles

Nanyang Technological University

Research

JoVE Journal

Chemistry

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

8.1K Views.  Korea Atomic Energy Research Institute.  Una procedura dettagliata per la sintesi di un azide 125 I-etichettati e la radiomarcatura di dibenzocyclooctyne (DBCO) -gruppo-coniugati, nanoparticelle d'oro 13-nm dimensioni utilizzando una reazione click senza il rame è descritto. 

Video: 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.

Stabilizzazione epatocellulare Fenotipo Utilizzando superfici sintetiche ottimizzati

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

Ricerca

Chimica

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.

Un metodo semplice per la dimensione sintesi controllata di stabili oligomeriche Grappoli di oro nanoparticelle in condizioni ambientali

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.

Un protocollo per la produzione di nanoparticelle gliadina-cianoacrilato per rivestimento idrofilo

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.

Un protocollo semplice ed efficiente per l&#39;inserimento catalitica polimerizzazione di norborneni funzionali

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.

Un protocollo per reazioni sicuro litiazione Utilizzando organolitio Reagenti

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.

Segnale Nanosonda rapido aumento di sintesi di nanoparticelle di oro In 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.

Il protocollo MPLEx Multi-omic nell'analisi di campioni di terreno

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.

Luminophore formazione in varie conformazioni di albumina di siero bovino di legame dell'oro

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.

Sintesi e caratterizzazione delle nanoparticelle d'oro dell'anfifile

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.

Sintesi di Nanoparticelle d'oro modificate Aptamer-PEI-g-PEG Caricate con doxorubicina per la consegna mirata di farmaci

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