The authors thank Prof. Ralph Weissleder, Dr. Pat Zanzonico, and Dr. NagaVaraKishore Pillarsetty for helpful conversations and the NIH for funding (BMZ: 1K99CA178205-01A1)

ETICA DICHIARAZIONE: Tutti gli esperimenti sugli animali in vivo descritti sono stati eseguiti secondo un protocollo approvato e alle linee guida etiche del Memorial Sloan Kettering Cancer Center Institutional Animal Care and Use Committee (IACUC). 1. Sintesi di Tz-Bn-NOTA <ol><li> In un piccolo recipiente di reazione, sciogliere 7 mg NH 2--BN NOTA (1,25 x 10 -2 mmoli) in 600 microlitri tampone di NaHCO3 (0,1 M, pH 8,1). Controllare il pH della soluzione. Se necessario, regolare il pH della soluzione a 8,1 utilizzando piccole aliquote di 0,1 M Na 2 CO 3. </li><li> Aggiungere la soluzione di NH 2 -BN-NOTA a 0,5 mg Tz-NHS (1,25 x 10 -3 mmol) in una provetta 1,7 ml microcentrifuga. NOTA: Il Tz-NHS può essere sia pesato secco o aggiunta di una soluzione madre di DMF secco o DMSO (&lt;50 ml). </li><li> Lasciare la soluzione di reazione risultante a reagire per 30 minuti a RTcon agitazione lieve. </li><li> Dopo 30 minuti, purificare il prodotto con fase inversa C 18 cromatografia HPLC per rimuovere reagito NH 2 -BN-NOTA. L&#39;NH 2 -BN-NOTA può essere monitorato alla lunghezza d&#39;onda di 254 nm, mentre il TZ-NHS e Tz-Bn-NOTA sono meglio controllati alla lunghezza d&#39;onda di 525 nm. NOTA: I tempi di ritenzione sono ovviamente fortemente dipendente dalla configurazione apparecchiature HPLC di ogni laboratorio (pompe, colonne, tubi, ecc). Tuttavia, per presentare un esempio, se un gradiente di 0: / H 2 O (entrambi con 0,1% TFA) a 100 100 MeCN: 0 MeCN / H 2 O oltre 25 min ed una analitica 4,6 x 250 millimetri C 18 colonna viene utilizzata , i tempi di ritenzione di Tz-Bn-NOTA, Tz-NHS, e NH 2 -BN-NOTA si aggirano intorno a 15 min, 16.5 min, e 10 minuti rispettivamente. Il prodotto può essere purificato dagli altri componenti della reazione in un singolo run o più corse con un C 18 colonna HPLC semi-preparativa o preparativo. 1 H-NMR, analeytical HPLC, e ESI-MS sono tutti i metodi che possono essere utilizzati per verificare la purezza del completamento precursore Tz-Bn-NOTA. 11 </li><li> Congelare l&#39;eluente HPLC raccolti con azoto liquido. </li><li> Avvolgere il tubo di raccolta congelato in un foglio di alluminio opaco. </li><li> Posizionare il tubo di raccolta congelati in un recipiente liofilizzazione O / N per rimuovere la fase mobile HPLC. </li><li> Conservare il prodotto purificato (un rosa brillante solido) al buio a -80 ° C. NOTA: Questo è un punto di sosta accettabile nella procedura. Il precursore Tz-Bn-NOTA completato è stabile per almeno 1 anno in queste condizioni. </li></ol> 2. Preparazione di huA33-TCO immunoconiugato <ol><li> In una provetta 1,7 ml, preparare un 1 mg / ml (2,7 mM) soluzione TCO-NHS in secca DMF. </li><li> In una provetta 1,7 ml microcentrifuga, preparare un 2 mg / ml (13,3 micron) di huA33 in 1 ml di tampone fosfato, pH 7.4 (0,01 M PO 4 3-, </sub&gt; 0,154 M NaCl). </li><li> Utilizzando piccole aliquote (&lt;5 microlitri) di 0,1 M Na 2 CO 3, regolare il pH della soluzione a 8,8-9,0 anticorpi. Utilizzare la carta pH o pH-metro con un microelettrodo per monitorare il pH, e fare attenzione a non permettere il pH di elevarsi al di sopra di pH 9,0. </li><li> Una volta che la soluzione di anticorpo è alla giusta pH, aggiungere un volume della soluzione TCO-NHS corrispondenti a 8 equivalenti molari di dell&#39;estere attivato. Ad esempio, aggiungere 7,9 ml di 1 mg / ml soluzione TCO-NHS (1,07 x 10 -7 mol TCO-NHS) per il 1 ml di soluzione di 2 mg / ml di anticorpi huA33 (1.33 x 10 -8 mol huA33). Non superare il 5% in volume DMF nella soluzione. </li><li> Mescolare delicatamente la soluzione invertendo la provetta diverse volte. </li><li> Avvolgere la provetta in un foglio di alluminio opaco. </li><li> Lasciare che la soluzione per incubare per 1 ora a temperatura ambiente con agitazione lieve. </li><li> Dopo 1 ora a RT, purificare l&#39;immunoconiugato risultante con un siz getta preconfezionatoe l&#39;esclusione colonna dissalazione. Innanzitutto, lavare la colonna esclusione dimensionale come descritto dal fornitore per rimuovere eventuali conservanti presenti nella colonna durante la conservazione. Quindi, aggiungere la miscela di reazione alla colonna esclusione dimensionale, lavare la colonna con 1,5 ml 0,9% soluzione salina sterile, e successivamente raccogliere il prodotto con 2 ml di 0,9% soluzione salina sterile come eluente. NOTA: Questo passaggio produrrà il completamento huA33-TCO come soluzione 2 ml. </li><li> Misurare la concentrazione della risultante huA33-TCO su un UV-Vis spettrofotometro. </li><li> Se una concentrazione maggiore è desiderato, concentrare la soluzione huA33-TCO utilizzando un&#39;unità filtro centrifugo con un peso molecolare di 50.000 cut-off. NOTA: E &#39;importante notare che, mentre huA33 e una varietà di altri anticorpi noti (ad esempio, bevacizumab, trastuzumab, cetuximab, e J591) sono molto tolleranti di essere concentrato, l&#39;aggregazione e la precipitazione può verificarsi dalla concentrazione in altri casi. I ricercatori di tentare questo procedUre con un nuovo anticorpo dovrebbe fidarsi letteratura o la propria conoscenza di anticorpo in questione per quanto riguarda se non concentrare l&#39;anticorpo. </li><li> Conservare il completamento immunoconiugato huA33-TCO a 4 ° C al buio. NOTA: Questo è un punto di sosta accettabile nella procedura. Il completamento coniugato mAb-TCO deve essere stabile per almeno 3 mesi in queste condizioni di conservazione. </li></ol> 3. 64 Cu radiomarcatura di Tz-Bn-NOTA NOTA: Questo passaggio del protocollo comporta il trattamento e la manipolazione di radioattività. Prima di eseguire questi passi - o l&#39;esecuzione di qualsiasi altro lavoro con radioattività - i ricercatori dovrebbero consultarsi con il Dipartimento di sicurezza di radiazione del proprio istituto. Adottare tutte le misure possibili per ridurre al minimo l&#39;esposizione alle radiazioni ionizzanti. <ol><li> In una provetta 1,7 ml, preparare un 0,5 mg / ml (723 um) soluzione di Tz-Bn-NOTA. </li><li> In un MicroC 1,7 mltubo entrifuge, aggiungere 10 ml di soluzione di Tz-Bn-NOTA (5 mg) per 400 ml di 0,2 M NH 4 OAc pH 5,5 tampone. </li><li> Nell&#39;interesse di una corretta radiochimica nota-keeping, misurare e registrare la quantità di radioattività nel campione con un calibratore di dosi prima e dopo i passi successivi nel protocollo di seguito (3,4-3,8). Ciò contribuirà con la determinazione precisa dei rendimenti radiochimica. </li><li> Aggiungi 2.000 uCi (74 MBq) di 64 Cu alla soluzione Tz-Bn-NOTA. NOTA: in genere, [64 Cu] CuCl 2 viene fornito in un piccolo volume (&lt;30 ml) di 0,1 N HCl, e quindi solo piccoli volumi (&lt;10 ml) di questa soluzione madre sono necessarie per la reazione radiomarcatura. Se sono necessari grandi volumi della [64 Cu] CuCl 2 stock, la reazione radiomarcatura è tollerante di aumentare il volume di reazione complessiva. Tuttavia, il pH della soluzione di reazione radiomarcatura devono essere monitorati attentamente per garantireche non scenda al di sotto di pH 4,0. </li><li> Lasciare che la soluzione per incubare per 10 min a RT con agitazione lieve. </li><li> Dopo 10 min di incubazione, purificare il prodotto con fase inversa C 18 cromatografia HPLC. I tempi di ritenzione sono ovviamente altamente dipendenti dalla configurazione apparecchiature HPLC di ogni laboratorio (pompe, colonne, tubi, ecc). Tuttavia, per presentare un esempio, se un gradiente di 5:95 MeCN / H 2 O (entrambi con 0,1% TFA) a 95: 5 MeCN / H 2 O oltre 15 min viene utilizzato, il tempo di ritenzione di 64 Cu-Tz- Bn-NOTA dovrebbe essere di circa 9,8 minuti, mentre la connessione, non complessato 64 Cu si eluire con il fronte del solvente a circa 2-4 min. </li><li> Utilizzando un evaporatore rotante, rimuovere l&#39;eluente HPLC. </li><li> Riprendere il prodotto 64 Cu-Tz-Bn-NOTA nello 0,9% soluzione salina sterile. NOTA: Dato il 12,7 hr fisica emivita di 64 Cu, questo non è un punto di arresto accettabile nel procedimento. Eseguire la sintesi di 64 Cu-Tz-Bn-NOTA immediatamente prima dell&#39;iniezione del radiolegante, e seguire passo 3.7 immediatamente by Step 4.5. </li></ol> 4. In Vivo Pretargeted PET Imaging NOTA: Come nella sezione protocollo 3, questa fase del protocollo comporta la gestione e la manipolazione della radioattività. Prima di effettuare questa procedura i ricercatori dovrebbero consultarsi con il Dipartimento di sicurezza di radiazione del proprio istituto. Adottare tutte le misure possibili per ridurre al minimo l&#39;esposizione alle radiazioni ionizzanti. <ol><li> In una femmina atimici topo nudo, impianto sottocute 1 x 10 6 SW1222 cellule tumorali del colon-retto e permettere a questi di crescere in un xenotrapianto 100-150 mm 3 (9-12 giorni dopo l&#39;inoculazione). 11 </li><li> Diluire un&#39;aliquota della soluzione huA33-TCO dalla sezione protocollo 2 ad una concentrazione di 0,5 mg / ml in 0,9% soluzione salina sterile. </li><li> Iniettare 200 microlitri della soluzione di huA33-TCO (100 ug) nella vena della coda del topo xenotrapianto-cuscinetto. </li><li> Consentire 24 ore per l&#39;accumulo del huA33-TCO nel tumore del mouse. </li><li> Diluire il 64 Cu-Tz-Bn-NOTA radioligando ad una concentrazione di 1,5 mCi / ml in 0,9% soluzione salina sterile. </li><li> Iniettare 200 ml di soluzione di radiolegante 64 Cu-Tz-Bn-NOTA (300 uCi; 11,1 MBq; 1.6 nmol di 64 Cu-Tz-Bn-NOTA, assumendo una specifica attività di 6,7 MBq / nmol) nella vena della coda del xenotrapianto-cuscinetto topi. </li><li> Al punto di tempo di imaging desiderato (ad esempio, 2, 6, 12 o 24 ore post-iniezione), anestetizzare il mouse con isoflurano 2%: miscela di gas ossigeno. </li><li> Posizionare il mouse sul letto del piccolo scanner PET animali. Mantenere l&#39;anestesia durante la scansione utilizzando una isoflurane 1%: miscela di ossigeno. Prima di mettere l&#39;animale sul piatto dello scanner, verificare l&#39;anestesia con il metodo punta pinch e applicare pomata veterinaria agli occhi del mouse per evitare l&#39;essiccazione durante l&#39;anestesia. </li><li> Acquisire i dati di PET per il mouse con una staticascansione con un minimo di 20 milioni di eventi coincidenti utilizzando una finestra di energia di 350-700 keV ed una finestra di temporizzazione coincidenza di 6 nsec. Dopo aver completato l&#39;acquisizione delle immagini, non lasciare incustodito il mouse e non metterlo in una gabbia con altri topi fino a quando non ha ripreso conoscenza. </li></ol>

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E., 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=A33+antigen+displays+persistent+surface+expression.">A33 antigen displays persistent surface expression.</a> Cancer Immunology and Immunotherapy. 57, 1017-1027 (2008).</li><li>Carrasquillo, J. A., 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=124I-huA33+antibody+PET+of+colorectal+cancer.">124I-huA33 antibody PET of colorectal cancer.</a> Journal of Nuclear Medicine. 52, 1173-1180 (2011).</li><li>Sakamoto, J., 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=A+phase+I+radioimmunolocalization+trial+of+humanized+monoclonal+antibody+huA33+in+patients+with+gastric+carcinoma.">A phase I radioimmunolocalization trial of humanized monoclonal antibody huA33 in patients with gastric carcinoma.</a> Cancer Science. 97, 1248-1254 (2006).</li><li>Rossin, R., Lappchen, R., vanden Bosch, S. M., LaForest, R., Robillard, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diels-Alder+reaction+for+tumor+pretargeting:+In+vivo+chemistry+can+boost+tumor+radiation+dose+compared+with+directly+labeled+antibody.">Diels-Alder reaction for tumor pretargeting: In vivo chemistry can boost tumor radiation dose compared with directly labeled antibody.</a> Journal of Nuclear Medicine. 54, 1989-1995 (2013).</li><li>Rossin, R., 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=Highly+reactive+trans-cyclooctene+tags+with+improved+stability+for+Diels-Alder+chemistry+in+living+systems.">Highly reactive trans-cyclooctene tags with improved stability for Diels-Alder chemistry in living systems.</a> Bioconjugate Chemistry. 34, 1210-1217 (2014).</li><li>Emmetiere, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=18F-labeled-bioorthogonal+liposomes+for+in+vivo+targeting.">18F-labeled-bioorthogonal liposomes for in vivo targeting.</a> Bioconjugate Chemistry. 24, 1784-1789 (2013).</li></ol>

The authors have nothing to disclose.

Il principale vantaggio di questa strategia PET pretargeted è che è in grado di delineare tumori con contrasto immagine target-to-background soltanto ad una frazione della dose di radiazione di fondo prodotto da anticorpi marcati direttamente. Ad esempio, nel sistema di imaging del cancro del colon-retto qui descritto, i dati provenienti da esperimenti biodistribuzione acuti sono stati impiegati per eseguire i calcoli di dosimetria per la strategia basata pretargeting-Cu 64 con direttamente marcato 64<...

Negli ultimi trent&#39;anni, tomografia ad emissione di positroni (PET) è diventato uno strumento indispensabile clinica nella diagnosi e gestione del cancro. Anticorpi sono stati a lungo considerati vettori promettenti per la fornitura di radioisotopi ad emissione di positroni per tumori a causa della loro affinità squisita e specificità per biomarcatori tumorali. 1,2, tuttavia, relativamente lenti farmacocinetica in vivo di anticorpi mandati l&#39;uso di radioisotopi con più giorni emivita fisica. Questa combinazione può produrre alte dosi di radiazioni agli organi non bersaglio di pazienti, una complicazione importante che è di particolare rilevanza clinica poiché radioimmunoconjugates vengono iniettati per via endovenosa e quindi - a differenza parziali scansioni corpo CT - risultato in dosi assorbite in ogni parte del corpo, indipendentemente tessuti interrogati. Per evitare questo problema, notevole sforzo è stato dedicato alla svipment di strategie di imaging PET che disaccoppiare radioisotopo e la frazione di targeting, sfruttando in tal modo le proprietà vantaggiose di anticorpi e contemporaneamente costeggiando i loro limiti farmacocinetiche intrinseche. Queste strategie - spesso definito pretargeting o il targeting più fasi - tipicamente impiegano quattro fasi: (1) la somministrazione di un anticorpo in grado di legare sia un antigene ed un radioligando; (2) l&#39;accumulo dell&#39;anticorpo nel tessuto bersaglio e la sua clearance dal sangue; (3) la somministrazione di una piccola molecola radioligando; e (4) la legatura in vivo del radioligando all&#39;anticorpo seguita dalla rapida clearance dell&#39;eccesso radioligando. 3-8 In alcuni casi, un agente di compensazione addizionale viene iniettato tra i passaggi 2 e 3, al fine di accelerare l&#39;escrezione di qualsiasi anticorpo che deve ancora impegnare il tumore e rimane nel sangue. 5 In generale, two tipi di strategie pretargeting sono più diffusi in letteratura. Mentre entrambi hanno dimostrato con successo in modelli preclinici, possiedono anche limitazioni chiave che hanno impedito la loro applicabilità clinica. La prima strategia si basa sul alta affinità tra streptavidina anticorpi coniugati e radiolabels biotina-modificato; Tuttavia, l&#39;immunogenicità degli anticorpi streptavidina-modificato ha dimostrato di essere un problema preoccupante per quanto riguarda la traduzione. 5,6,9,10 La seconda strategia, al contrario, impiega anticorpi bispecifici geneticamente ingegnerizzati per legare sia un cancro biomarker antigene e una piccola aptene radiomarcato molecola. 3,11-14 Mentre quest&#39;ultimo rotta è certamente creativo, la sua ampia applicabilità è limitata dalla complessità, spesa, e la mancanza di modularità del sistema. Recentemente, abbiamo sviluppato e pubblicato una metodologia di imaging PET pretargeted basato sulla domanda inversa elettrone Diels-Alder (IEDDA) reazione di cicloaddizione tra -cyclooctene trans (TCO) e tetrazina (Tz;. Figura 1) 11 Mentre la reazione si è stato conosciuto per decenni, IEDDA chimica ha conosciuto una rinascita negli ultimi anni come una tecnica bioconjugation click chemistry, come illustrato da l&#39;affascinante lavoro dei gruppi di Ralph Weissleder, Giuseppe Volpe, e Peter Conti, tra gli altri. 12-15 L&#39;IEDDA cicloaddizione è stato applicato in una vasta gamma di impostazioni, tra cui imaging di fluorescenza con peptidi, anticorpi, e nanoparticelle e di imaging nucleare . con entrambi radiohalogens e radiometalli 16-26 La legatura è ad alto rendimento, pulito, veloce (k 1&gt; 30.000 M -1 sec -1), selettivo, e - critica -. bioorthogonal 27 E mentre un certo numero di tipi di click chemistry - compresi azide-alkyne cicloaddizioni Cu-catalizzata, cicloaddizioni azide-alkyne ceppo-promossa, e Staudinger ligzioni -. bioorthogonal sono pure, è la combinazione unica di cinetica di reazione veloci e bioorthogonality che rende IEDDA chimica così adatto per applicazioni pretargeting in organismi interi 28,29 Lungo queste linee, è importante notare che la recente relazione nostro laboratori non è stato il primo ad applicare IEDDA chimica per pretargeting: il primo rapporto delle immagini pretargeted con IEDDA sorto dal lavoro di Rossin, et al e caratterizzato da una metodologia SPECT impiegando un 111 In marcato tetrazina 30.. Come abbiamo discusso in precedenza, la metodologia pretargeting ha quattro abbastanza semplici passaggi (Figura 2). Nel protocollo a portata di mano, sarà descritta una strategia pretargeted per imaging PET di tumore del colon-retto, che si avvale di un Cu-NOTA-marcato radiolegante 64 tetrazina e un coniugato TCO modificato dell&#39;anticorpo huA33. Tuttavia, in ultima analisi, la modularità di questa metodologia è uno dei suoi grbeni ne mangerai, come la frazione -cyclooctene trans possono essere aggiunti a qualsiasi anticorpo non interiorizzare, e la tetrazina può essere collegato a una grande varietà di giornalisti radioattive.

<table border="0" cellpadding="0" cellspacing="0" width="565">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrazine NHS Ester</td>
			<td>Sigma-Aldrich</td>
			<td>764701</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trans-cyclooctene NHS Ester</td>
			<td>Sigma-Aldrich</td>
			<td>764523</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">p-NH2-Bn-NOTA</td>
			<td>Macrocyclics</td>
			<td>B-601</td>
			<td>Store at -80 &#176;C</td>
		</tr>
	</tbody>
</table>

harnessing the bioorthogonal inverse electron demand diels-alder cycloaddition for pretargeted pet imaging

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for PET imaging. However, the necessity of labeling antibodies with radionuclides with long physical half-lives often results in high background radiation dose rates to non-target tissues. In order to circumvent this issue, we have employed a pretargeted PET imaging strategy based on the inverse electron demand Diels-Alder cycloaddition reaction. The methodology decouples the antibody from the radioactivity and thus exploits the positive characteristics of antibodies, while eschewing their pharmacokinetic drawbacks. The system is composed of four steps: (1) the injection of a mAb-trans-cyclooctene (TCO) conjugate; (2) a localization time period during which the antibody accumulates in the tumor and clears from the blood; (3) the injection of the radiolabeled tetrazine; and (4) the in vivo click ligation of the components followed by the clearance of excess radioligand. In the example presented in the work at hand, a 64Cu-NOTA-labeled tetrazine radioligand and a trans-cyclooctene-conjugated humanized antibody (huA33) were successfully used to delineate SW1222 colorectal cancer tumors with high tumor-to-background contrast. Further, the pretargeting methodology produces high quality images at only a fraction of the radiation dose to non-target tissue created by radioimmunoconjugates directly labeled with 64Cu or 89Zr. Ultimately, the modularity of this protocol is one of its greatest assets, as the trans-cyclooctene moiety can be appended to any non-internalizing antibody, and the tetrazine can be attached to a wide variety of radioisotopes.

Le prime tre fasi dell&#39;esperimento - la sintesi di Tz-Bn-NOTA, la coniugazione di TCO per huA33, e la radiomarcatura della Tz-Bn-NOTA costruiscono (figure 3 e 4) - sono altamente affidabili. Nel caso della procedura sopra, il costrutto Tz-Bn-NOTA stato sintetizzato con alta resa e purezza. L&#39;anticorpo huA33 stato modificato con 4,2 ± 0,6 TCO / mAb e Tz-Bn-NOTA era radiomarcato con 64 Cu a cedere il radiolegante purificato in&gt; 99% di purezza radiochimica,&gt; 85% d...

Watch this Scientific Journal Video about Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging at JoVE.com

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand.

Sfruttando la domanda Bioorthogonal Inverse Electron Diels-Alder cicloaddizione per Pretargeted PET Imaging

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for ...

harnessing-bioorthogonal-inverse-electron-demand-diels-alder

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JoVE Journal

Bioengineering

8.7K Views.  Memorial Sloan Kettering Cancer Center. The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand. 

Video: Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Ad alta risoluzione in fibra ottica per Microendoscopy In situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

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Bioingegneria

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

Electrotaxis microfluidica-based per On-demand analisi quantitativa della<em&gt; Caenorhabditis elegans</em&gt; &#39;Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Creazione di sfumature adesivi e solubile per la migrazione di imaging cellulare con microscopia a fluorescenza

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Fluorescenza tempra di un vicino infrarosso Fluoroforo incapsulata all&#39;interno di liposomi come strumento per<em&gt; In Vivo</em&gt; Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Biofunctionalized Prussian Blue Nanoparticelle per applicazioni di imaging multimodale molecolari

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

Rapid Scan paramagnetica elettronica Risonanza apre nuove strade per l&#39;imaging Fisiologicamente parametri importanti<em&gt; In Vivo</em

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Microscopia a luce correlativa (CLEM) per il monitoraggio e la proteina virale di Imaging strutture in cellule di Cryo-immobilizzata associate

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various factors affect the relaxation rates, such as buffer composition, solution pH, temperature, and magnetic field. In this last regard, the spin-lattice relaxation time can be measured at clinical field strengths, but at lower fields, where these compounds are dispensed from the polarizer and transported to the MRI, the relaxation is even faster and difficult to measure. To have a better understanding of the amount of magnetization lost during transport, we used fast field-cycling relaxometry, with magnetic resonance detection of 13C nuclei at ~0.75 T, to measure the nuclear magnetic resonance dispersion of the spin-lattice relaxation time of hyperpolarized [1-13C]pyruvate. Dissolution dynamic nuclear polarization was used to produce hyperpolarized samples of pyruvate at a concentration of 80 mmol/L and physiological pH (~7.8). These solutions were rapidly transferred to a fast field-cycling relaxometer so that relaxation of the sample magnetization could be measured as a function of time using a calibrated small flip angle (3&#176;-5&#176;). To map the T1 dispersion of the C-1 of pyruvate, we recorded data for different relaxation fields ranging between 0.237 mT and 0.705 T. With this information, we determined an empirical equation to estimate the spin-lattice relaxation of the hyperpolarized substrate within the mentioned range of magnetic fields. These results can be used to predict the amount of magnetization lost during transport and to improve experimental designs to minimize signal loss.

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

We present a protocol to measure the magnetic field dependence of the spin-lattice relaxation time of 13C-enriched compounds, hyperpolarized by means of dynamic nuclear polarization, using fast field-cycled relaxometry. Specifically, we have demonstrated this with [1-13C]pyruvate, but the protocol could be extended to other hyperpolarized substrates.

Misurazione della dipendenza da campo magnetico di rilassamento Spin-Lattice di iperpolarizzato [1-13C]pyruvate

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various ...

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built high viscous injector, Lipidico, as part of the macromolecular crystallography (MX2) beamline to measure large numbers of small crystals at room temperature. The goal of this technique is to enable crystals to be grown/transferred to&#160;glass syringes to be used directly in the injector for serial crystallography data collection. The advantages of this injector include the ability to respond rapidly&#160;to changes in the flow rate without interruption of the stream. Several limitations for this high viscosity injector (HVI) exist which include a restriction on the allowed sample viscosities to &#62;10 Pa.s. Stream stability can also potentially be an issue depending on the specific properties of the sample. A detailed protocol for how to set up samples and operate the injector for serial crystallography measurements at the Australian synchrotron is presented here. The method demonstrates preparation of the sample, including the transfer of lysozyme crystals into a high viscous media (silicone grease), and the operation of the injector for data collection at MX2.

The goal of this protocol is to demonstrate how to prepare serial crystallography samples for data collection on a high viscous injector, Lipidico, recently commissioned at the Australian synchrotron.

Protocollo di iniezione lipidico per le misurazioni della cristallografia seriale presso il sincrotrone australiano

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

Imaging del flusso sanguigno con Doppler ultraveloce

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...