Questo lavoro è stato sostenuto dalla Fondazione Ospedale Principessa Margaret, la National Science and Engineering Research Council of Canada, Canadian Institute for Health Research, la Fondazione canadese per l&#39;innovazione e il Joey e Toby Tanenbaum / brasiliano Ball Chair in Prostate Cancer Research, concedere dalla Research Foundation SUNY e supporto all&#39;avvio da University at Buffalo.

1. Sintesi di Pyropheophorbide lipidi <ol><li> Unire 200 mmol pyropheophorbide (preparato da Spirulina Pacifica alghe come descritto in precedenza,... Zheng et al, Bioconj Chem, 2002, 13 -392) 4, 98,7 mg 16:00 lysophophatidylcholine (1-palmitoil-2-idrossi-sn-glycero -3-fosfocolina, Avanti Polar Lipidi # 855675), 76,3 mg di EDC, 48,7 mg DMAP (4 - (dimetilammino) piridina) in 5 ml amylene stabilizzato cloroformio per un rapporto di 1:1:2:2 Pyro: Lipid: EDC : DMPA. </li><li> Mescolare la miscela di reazione a temperatura ambiente sotto argon per 24 h. Si evapora il cloroformio sotto pressione ridotta utilizzando un evaporatore rotante con riscaldamento a 40 ° C. La miscela di isomeri piro-lipidico (pyro attaccati alla posizione sn-1 e sn-2) viene prodotto. </li><li> Risospendere piro-lipidico miscela di isomeri in 1 mM CaCl 2, Tris 50 mM (pH 8), 0,5% Triton X-100 e 10% di metanolo alla concentrazione di 5 mg / ml. Aggiungi fosfolipasi A2 (PLA2), dal miele al veleno d&#39;api0,1 mg concentrazione ml / e incubare la soluzione a 37 ° C per 24 ore per digerire sn-1 piro-lipidico e per produrre il puro sn-2 piro-lipidico isomero. </li><li> Aggiungere ulteriori 2 volumi di cloroformio e di 1,25 volumi di metanolo, estratto, e quindi rimuovere il solvente con evaporatore rotante a pressione ridotta a 40 ° C. Risospendere il prodotto di scissione in 1% MeOH in DCM e purificare su una piccola colonna di silice diolo per la isomericamente puro piro-lipidico. </li></ol> 2. Preparazione di Porphysomes <ol><li> Sciogliere pyropheophorbide-lipide, distearoil-sn-glicero-3-fosfoetanolamina-N-metossi (polietilene glicole) (PEG-2000-PE) e colesterolo in cloroformio separatamente, e registrare le concentrazioni rispettivamente. </li><li> Preparare porphysome film lipidico in una provetta 12x75 mm prova borosilicato con un peso lipidico totale di 5 mg / tubo combinando il 65% molare porfirina-lipide, 5% molare PEG-2000-PE e il 30% molare colesterolo. </li><li> Asciugare i film lipidici sotto un getto di nitrogas gen e farli ulteriormente essiccati sotto vuoto per 1 ora. Memorizzare i film lipidici a -20 ° C sotto argon fino futuro idratazione ed estrusione. </li><li> Aggiungere 1 ml di tampone fosfato salino (PBS, 150 mM NaCl, 10 mM fosfato, pH 7.4) a ciascuna provetta film lipidico. Congelare la provetta in azoto liquido fino a completo congelato, e poi scongelare il tubo 65 ° C bagnomaria finché il campione è tutto sciolto. Vortex il tubo per un totale di 30 sec (3x a 10 sec ciascuno, con 5 sec ri-riscaldamento a 65 ° C in bagno d&#39;acqua tra ogni vortex). </li><li> Ripetere il congelamento-scongelamento per 5 cicli o più al momento necessaria fino a quando non grandi aggregati possono essere osservati nella soluzione idratata. Lasciare la sospensione porphysome sul ghiaccio quando fatto. </li><li> Montare l&#39;estrusore ad alta pressione (10 ml) con due filtri in policarbonato 100 nm accatastati installati dentro Collegare l&#39;estrusore thermobarrel ad un circolatore termostatato a 65 ° C in circolazione bagnomaria, e collegare il sistema estrusore ad un serbatoio di azoto per alta pressionesicuro approvvigionamento. </li><li> Trasferire la sospensione porphysome alla camera estrusore con una pipetta di trasferimento vetro 9 pollici. Impostare la pressione ruotando la manopola sul regolatore serbatoio di azoto fino a una lettura tra 200 e 300 psig è osservato come pressione di estrusione iniziale. Aprire la valvola del regolatore di pressione gradualmente fino portata porphysome sufficiente è raggiunto o viene raggiunto un massimo di 800 psig (5.440 kPa) di pressione. </li><li> Dopo il primo passaggio di estrusione, chiudere la valvola di controllo della pressione e rilasciare la pressione dell&#39;estrusore aprendo la valvola limitatrice della pressione sulla parte superiore estrusore. Riempire il estrusore con la soluzione porphysome appena estruso, e ripetere l&#39;estrusione per almeno 10 volte per assicurare la soluzione estruso finale è omogeneo. </li><li> Determinare la concentrazione della soluzione finale porphysome misurando l&#39;assorbimento di un campione diluito in metanolo per UV-Vis spettrometro. Calcolare la concentrazione utilizzando la seguente equazione with il coefficiente di estinzione di 97.000 M -1 cm -1 a 410 nm per pyropheophorbide-lipidico. </li></ol><img alt="Equazione 1" src="/files/ftp_upload/50536/50536eq1.jpg" /><ol start="10"><li> Misurare la distribuzione finale dimensioni di porphysomes utilizzando un Malvern Zetasizer ZS90 dalla dispersione della luce dinamica. Diluire la soluzione porphysome in PBS e di eseguire tre misurazioni con 15 corse ciascuno per i risultati medi. </li><li> Determinare il quenching di fluorescenza porphysome confrontando l&#39;emissione di fluorescenza di porphysome temprato (1 mM in PBS) e porphysome unquenched (1 mM in 1% Triton X-100) utilizzando Spectrofluorometer. Prendere gli spettri prima e dopo il detersivo (Tirton X-100) si aggiunge, e normalizzare il segnale di lettura massima fluorescenza. Impostare l&#39;eccitazione di essere 410 nm, e l&#39;emissione gamma di lunghezze d&#39;onda per essere 600-800 nm. </li><li> Tenere soluzione porphysomes sotto argon a 4 ° C fino all&#39;utilizzo futuro, e proteggerlo dalla luce in alluminio folio. </li></ol> 3. Preparazione di Animal Xenograft Modello <ol><li> Cultura le cellule KB in RPMI-1640 (con 10% FBS), e sostenere le cellule in 5% di CO 2 e l&#39;atmosfera idratata a 37 ° C. </li><li> Cellule Harvest KB utilizzano tecniche di coltura cellulare standard. Mantenere la sospensione cellulare (20.000 cellule / ml) in tampone fosfato (PBS) in ghiaccio per la conservazione a breve termine fino al momento dell&#39;iniezione. </li><li> Preparare un KB xenotrapianti in topi nudi. Topi nudi sono acquistati da Charles River Laboratories quando sono di 20 g (Categoria # Crl: NU (NCR)-Foxn1 nu, 6-7 settimane di età), data dieta standard e biancheria da letto (Harlan Laboratories, Dieta: irradiato LM-485, # 7912; Letti: biancheria da letto Pannocchia, ¼ &quot;, # 7097). Sterilizzare tutti gli strumenti di gestione degli animali (siringhe, pinze e pastiglie animale) prima di esperimenti. Anestetizzare topi con 2% isoflurano v / v in 100% di ossigeno gassoso. Tutti i protocolli animali sono approvati dall&#39;istituzione (University Health NeTwork in questo caso) la cura degli animali e l&#39;uso commissione. </li><li> Iniettare cellule tumorali sospese in PBS per via sottocutanea al fianco destro del mouse. I topi sono pronti per essere utilizzati dopo 10 giorni quando tumore raggiunge diametro di 4-5 mm e spessore di 2-3 mm. </li></ol> 4. In vivo Photothermal Therapy <ol><li> Iniettare porphysomes (contenenti 750 nmol pyro al volume di 200 ml) per via endovenosa via vena della coda. Condurre laser irradiazione 24 ore dopo l&#39;iniezione di porphysomes. </li><li> In questa sezione, eseguire tutte le procedure in una camera di sicurezza laser chiusa. Indossare occhiali di protezione laser durante l&#39;irraggiamento laser. </li><li> Impostare l&#39;apparecchiatura irraggiamento laser. Tenere la fibra laser e un diffusore da un morsetto su un supporto. Impostare la potenza del laser a basso livello e accendere il laser. Regolare l&#39;altezza della fibra laser per ottenere una superficie di irradiazione del 9 mm di diametro (DPSS Laser, LaserGlow Technologies, Toronto, Canada), calibrare la potenza del laser di 750 mW (1.18 W / cm 2) con apower metro e poi spegnere il laser per preparare gli animali. </li><li> Anestetizzare gli animali con 2% isoflurano v / v in 100% di gas di trasporto di ossigeno attraverso un&#39;ogiva non scavenging evidente di isoflurano un pericolo rischio per la salute umana). Verificare che l&#39;animale viene anestetizzato abbastanza profondo eseguendo un pizzico punta con una pinza o dita zampe posteriori dell&#39;animale. Gli animali sono pronti per irraggiamento laser quando sono insensibili a questa procedura. </li><li> Monitorare la temperatura del tumore da una telecamera a infrarossi termica (Mikroshot, LumaSense Technologies). Posizionare la fotocamera vicino al tumore, concentrarsi bene e prendere una immagine pre PTT così come un normale immagine a luce bianca prima di PTT inizio di irradiazione. </li><li> Accendere il laser di nuovo, e aumentare il potere della luce a 750 mW. Posizionare il tumore nel centro del raggio laser per ottenere il tumore completamente coperto, e iniziare a programmare il calendario di irradiazione. Acquisire l&#39;immagine temperatura tumore ogni 5 secondi per un trattamento laser un minuto. Spegnere il laser a 1 min. Stop la fornitura di gas del 2% isoflurano, attendere fino a quando le recupera animali provenienti anestesia e la rimise alla gabbia. </li><li> Mantenere i topi a normothermia stanza degli animali sotto 00:12 h ciclo buio e luce secondo protocolli animali approvati dalla University Health Network. Iniettare buprenorfina (0,05 mg / kg, per via sottocutanea due volte al giorno per una settimana dopo l&#39;irradiazione laser per ridurre il dolore causato dalla cicatrice. Ogni due giorni, misurare il diametro del tumore usando un calibro e scattare una foto. Calcolare il volume del tumore usando l&#39; seguente equazione: V = π / 6 · a · b 2, dove a è il diametro lungo, e b è quello corto. </li><li> Sacrificare gli animali mettendoli in CO 2 camera quando la lunga diametro del tumore raggiunge 10 mm, in quanto è il punto finale definito nel nostro laboratorio. Eseguire dislocazione cervicale dopo il trattamento CO2. Tracciare la curva di sopravvivenza. </li></ol>

<ol>
	<li>Lovell, J. 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=Porphysome+nanovesicles+generated+by+porphyrin+bilayers+for+use+as+multimodal+biophotonic+contrast+agents.">Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents.</a> Nat. Mater. 10, 324-332 (2011).</li><li>Lovell, J. 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=Enzymatic+regioselection+for+the+synthesis+and+biodegradation+of+porphysome+nanovesicles.">Enzymatic regioselection for the synthesis and biodegradation of porphysome nanovesicles.</a> Angew Chem Int Ed Engl. 51, 2429-2433 (2012).</li><li>Liu, T. W., MacDonald, T. D., Shi, J., Wilson, B. C., Zheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsically+Copper-64-Labeled+Organic+Nanoparticles+as+Radiotracers.">Intrinsically Copper-64-Labeled Organic Nanoparticles as Radiotracers.</a> Angewandte Chemie. , (2012).</li><li>Zheng, G., 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=Low-density+lipoprotein+reconstituted+by+pyropheophorbide+cholesteryl+oleate+as+target-specific+photosensitizer.">Low-density lipoprotein reconstituted by pyropheophorbide cholesteryl oleate as target-specific photosensitizer.</a> Bioconjug Chem. 13, 392-396 (2002).</li><li>Chen, B., Pogue, B. W., Hasan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+delivery+of+photosensitising+agents.">Liposomal delivery of photosensitising agents.</a> Expert Opin Drug Deliv. 2, 477-487 (2005).</li><li>Jin, C. S., Zheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+nanostructures+for+photosensitizer+delivery.">Liposomal nanostructures for photosensitizer delivery.</a> Lasers Surg Med. 43, 734-748 (2011).</li><li>Olson, F., Hunt, C. A., Szoka, F. C., Vail, W. J., Papahadjopoulos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+liposomes+of+defined+size+distribution+by+extrusion+through+polycarbonate+membranes.">Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes.</a> Biochim Biophys Acta. 557, 9-23 (1979).</li><li>Berger, N., Sachse, A., Bender, J., Schubert, R., Brandl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filter+extrusion+of+liposomes+using+different+devices:+comparison+of+liposome+size,+encapsulation+efficiency,+and+process+characteristics.">Filter extrusion of liposomes using different devices: comparison of liposome size, encapsulation efficiency, and process characteristics.</a> Int J Pharm. 223, 55-68 (2001).</li><li>Jesorka, A., Orwar, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes:+technologies+and+analytical+applications.">Liposomes: technologies and analytical applications.</a> Annu Rev Anal Chem (Palo Alto Calif). 1, 801-832 (2008).</li><li>Lapinski, M. M., Castro-Forero, A., Greiner, A. J., Ofoli, R. Y., Blanchard, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+liposomes+formed+by+sonication+and+extrusion:+rotational+and+translational+diffusion+of+an+embedded+chromophore.">Comparison of liposomes formed by sonication and extrusion: rotational and translational diffusion of an embedded chromophore.</a> Langmuir. 23, 11677-11683 (2007).</li><li>Liu, D., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+Homogeneity+of+a+Liposome+Preparation+is+Crucial+for+Liposome+Biodistribution+In+Vivo.">Size Homogeneity of a Liposome Preparation is Crucial for Liposome Biodistribution In Vivo.</a> Journal of Liposome Research. 2, 57-66 (1992).</li><li>Zhang, Y., Lovell, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porphyrins+as+Theranostic+Agents+from+Prehistoric+to+Modern+Times.">Porphyrins as Theranostic Agents from Prehistoric to Modern Times.</a> Theranostics. 2, 905-915 (2012).</li></ol>

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Nello sviluppo di tecnologie di drug delivery, nanoparticelle multifunzionali sono attualmente in fase di ampie ricerche per il monitoraggio accurato del veicolo di consegna della droga, pur mantenendo un elevato carico utile di droga. Porfirinici liposomi caricati sono stati sviluppati per migliori proprietà farmacocinetiche e consegna più efficiente rispetto alla somministrazione diretta di porfirina, ma esistono ostacoli, compresa la quantità di carico limitato di porfirina e rapida ridistribuzione di porfirina da...

Porphysomes sono nanovesicles nuovi multifunzionali che abbiamo recentemente sviluppato, che sono in grado di imaging e terapia multimodale 1. Esse sono formate da doppi strati porfirinici auto-assemblati e contengono una altissima densità di porfirina (oltre 83, 000/porphysome particella), che genera grande coefficiente di estinzione e risultati in unica struttura-dipendente fluorescenza auto-spegnimento. Porphysomes hanno buone in vivo farmacocinetica e biodistribuzione proprietà: essi mostrano un sangue emivita di 12 ore dopo la somministrazione sistematica, e passivamente accumulano nei tumori trapiantati con 7,5% ID / g in 24 ore dopo l&#39;iniezione 2. La loro struttura unica e le proprietà fisico-chimiche fanno porphysome un buon candidato per l&#39;imaging multimodale e la terapia immagine-guida. Prima di tutto, contenente porfirina, porphysomes sono intrinsecamente adatti per imaging di fluorescenza dei tumori sulla accumulo tumore 1.Inoltre, ogni porfirina ha un sito stabile per radioisotopi chelanti, dunque, porphysomes possono essere facilmente etichettati con radioisotopi come 64Cu per l&#39;imaging PET 3. Inoltre, l&#39;energia luminosa assorbita viene dissipata termicamente sotto irraggiamento laser di esposizione quando porphysome struttura è intatta, così porphysomes mostrano anche immagini fotoacustica unico e funzionalità PTT. E &#39;stato dimostrato che 24 ore dopo l&#39;iniezione endovenosa di porphysomes, irradiazione laser del tumore porphysome accumulata ha indotto un aumento di temperatura rapido e forte fototermico ablazione del tumore. Ciò ha dimostrato che porphysomes sono stimolatori fototermiche efficienti con coefficiente di estinzione alto come le nanoparticelle d&#39;oro (AuNPs) 1. D&#39;altra parte, in confronto con altri agenti fototermiche inorganici, incluse AuNPs, porphysomes mostrano un vantaggio eccezionale nei biosicurezza a causa della loro natura organica. Porphysomes sono enzimaticamente biodegradabili e inducono minimo toxicit acutay in topi con dosi endovenose alto come 1,000 mg / kg 1. Inoltre, simile a liposomi, il grande nucleo acquosa di porphysomes potrebbe essere passivamente o attivamente caricato con agenti terapeutici o di imaging. Le proprietà ottiche e biocompatibilità di porphysomes dimostrano il potenziale multimodale di nanoparticelle organiche per l&#39;imaging e la terapia BioPhotonic. In questo lavoro, si introduce il metodo di sintesi di coniugati pyropheophorbide-lipidico, la fabbricazione e il metodo di caratterizzazione di porphysomes utilizzando estrusione ad alta pressione. PTT in vivo su topi è condotta anche per dimostrare l&#39;efficienza di porphysome abilitati PTT nel tumore trattamento con un modello di tumore xenotrapianto sottocutanea.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>Thermo-Savant</td>
			<td>SPD131DDA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Votex</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td>Model number: G560</td>
		</tr>
		<tr>
			<td>High pressure extruder</td>
			<td>LIPEX, Northern Lipids Inc.</td>
			<td>T.001</td>
			<td>10 ml Thermobarrel Extruder</td>
		</tr>
		<tr>
			<td>Heated bath circulators (Thermostatted circulator)</td>
			<td>Thermo SCIENTIFIC</td>
			<td>SC100-S5P</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polycarbonate filters</td>
			<td>Avanti Polar Lipids</td>
			<td>610005</td>
			<td>Pore size 100 nm, and membrane diameter 19 mm</td>
		</tr>
		<tr>
			<td>Zetasizer</td>
			<td>Malvern Instruments</td>
			<td>ZS90</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>UV/Visible Spectrophototmeter</td>
			<td>Varian Australia</td>
			<td>Cary 50 Bio UV/ Visible Spectrophotometer</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spectrofluorometer</td>
			<td>HORIBA Scientific</td>
			<td>FluoroMax-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Transmission Electron Microscopy (TEM)</td>
			<td>Hitachi</td>
			<td>H-7000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>SANYO</td>
			<td>MCO-18AIC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Powermeter</td>
			<td>Thorlabs</td>
			<td>PM100D</td>
			<td>with sensor S142C</td>
		</tr>
		<tr>
			<td>671nm Laser</td>
			<td>LaserGlow Technologies</td>
			<td>LRS-0671_PFN-02000-05</td>
			<td>S/N:10097270</td>
		</tr>
		<tr>
			<td>Infrared Thermometer</td>
			<td>Mikroshot, LUMASENSE Technologies</td>
			<td>9102409</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laser protective eye goggles</td>
			<td>LaserGlow Technologies</td>
			<td>AGF6602XX</td>
			<td>Optical Density: 1.5+ at 630-700 nm</td>
		</tr>
	</tbody>
</table>

one minute, sub-one-watt photothermal tumor ablation using porphysomes, intrinsic multifunctional nanovesicles

Abbiamo recentemente sviluppato porphysomes come nanovesicles intrinsecamente multifunzionali. Un fotosensibilizzante, pyropheophorbide α, è stato coniugato ad un fosfolipide e quindi auto-assemblato vescicole sferiche liposoma-like. A causa della densità estremamente elevata della porfirina nel doppio strato lipidico porfirina, porphysomes generato grandi coefficienti di estinzione, la struttura dipendente dalla fluorescenza di auto-spegnimento, e ottima efficacia fototermica. Nella nostra formulazione, porphysomes stati sintetizzati utilizzando estrusione ad alta pressione, e visualizzate una dimensione media delle particelle di circa 120 nm. Iniezione Ventiquattro ore dopo endovenosa di porphysomes, la temperatura locale del tumore è aumentata da 30 ° C a 62 ° C rapidamente su uno minuti di esposizione di 750 mW (1,18 W / cm 2), 671 nm laser irradiazione. Seguendo l&#39;ablazione termica completa del tumore, escare formata e guarito entro 2 settimane, mentre nei gruppi di controllo i tumori hanno continuato a crescere e tutti raggiunto il en definitalettera d entro 3 settimane. Questi dati mostrano come porphysomes possono essere usate come terapia photothermal potente (PTT) agenzie.

Pyropheophorbide è coniugato al fosfolipide come monomero lipidico stabile (Figura 1a) e coniugati auto-assemblano per formare porphysomes dal metodo di estrusione membrana utilizzando un estrusore ad alta pressione. Di solito è difficile estrudere sospensione porphysome lipidi durante i primi 3 cicli di estrusione con portata relativamente lento. Come più estrusi vengono ripetute, la velocità di flusso della soluzione estruso aumenta gradualmente, e la pressione può essere leggermente diminuita se...

Watch this Scientific Journal Video about One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles at JoVE.com

One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

Abbiamo sviluppato nuovi nanovesicles multifunzionali intrinseche chiamati porphysomes, che hanno struttura-dipendente fluorescenza auto-estinzione e le proprietà fototermiche uniche, fungendo così gli agenti di terapia fototermica come potenti. Abbiamo formulato porphysomes utilizzando estrusione ad alta pressione e studiato l&#39;efficacia della terapia photothermal in un modello di tumore xenotrapianto.

One Minute, Sub-One-Watt Photothermal ablazione del tumore Utilizzando Porphysomes, intrinseca multifunzionale nanovesicles

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a ...

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JoVE Journal

Bioengineering

10.7K Views.  University of Toronto.  Abbiamo sviluppato nuovi nanovesicles multifunzionali intrinseche chiamati porphysomes, che hanno struttura-dipendente fluorescenza auto-estinzione e le proprietà fototermiche uniche, fungendo così gli agenti di terapia fototermica come potenti. Abbiamo formulato porphysomes utilizzando estrusione ad alta pressione e studiato l'efficacia della terapia photothermal in un modello di tumore xenotrapianto. 

Video: One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

Mezzo di contrasto ecografico mirato trattamento dei gliomi nei topi tramite Drug-Bearing consegna delle nanoparticelle e ablazione microvascolari

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

Ricerca

Bioingegneria

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting airways, whereas adenocarcinomas are typically more peripheral in location. Lung malignancy detection early in the disease process may be difficult due to several limitations: radiological resolution, bronchoscopic limitations in evaluating tissue underlying the airway mucosa and identifying early pathologic changes, and small sample size and/or incomplete sampling in histology biopsies. High resolution imaging modalities, such as optical frequency domain imaging (OFDI), provide non-destructive, large area 3-dimensional views of tissue microstructure to depths approaching 2 mm in real time (Figure 1)2-6. OFDI has been utilized in a variety of applications, including evaluation of coronary artery atherosclerosis6,7 and esophageal intestinal metaplasia and dysplasia6,8-10.
Bronchoscopic OCT/OFDI has been demonstrated as a safe in vivo imaging tool for evaluating the pulmonary airways11-23 (Animation). OCT has been assessed in pulmonary airways16,23 and parenchyma17,22 of animal models and in vivo human airway14,15. OCT imaging of normal airway has demonstrated visualization of airway layering and alveolar attachments, and evaluation of dysplastic lesions has been found useful in distinguishing grades of dysplasia in the bronchial mucosa11,12,20,21. OFDI imaging of bronchial mucosa has been demonstrated in a short bronchial segment (0.8 cm)18. Additionally, volumetric OFDI spanning multiple airway generations in swine and human pulmonary airways in vivo has been described19. Endobronchial OCT/OFDI is typically performed using thin, flexible catheters, which are compatible with standard bronchoscopic access ports. Additionally, OCT and OFDI needle-based probes have recently been developed, which may be used to image regions of the lung beyond the airway wall or pleural surface17.
While OCT/OFDI has been utilized and demonstrated as feasible for in vivo pulmonary imaging, no studies with precisely matched one-to-one OFDI:histology have been performed. Therefore, specific imaging criteria for various pulmonary pathologies have yet to be developed. Histopathological counterparts obtained in vivo consist of only small biopsy fragments, which are difficult to correlate with large OFDI datasets. Additionally, they do not provide the comprehensive histology needed for registration with large volume OFDI. As a result, specific imaging features of pulmonary pathology cannot be developed in the in vivo setting. Precisely matched, one-to-one OFDI and histology correlation is vital to accurately evaluate features seen in OFDI against histology as a gold standard in order to derive specific image interpretation criteria for pulmonary neoplasms and other pulmonary pathologies. Once specific imaging criteria have been developed and validated ex vivo with matched one-to-one histology, the criteria may then be applied to in vivo imaging studies. Here, we present a method for precise, one to one correlation between high resolution optical imaging and histology in ex vivo lung resection specimens. Throughout this manuscript, we describe the techniques used to match OFDI images to histology. However, this method is not specific to OFDI and can be used to obtain histology-registered images for any optical imaging technique. We performed airway centered OFDI with a specialized custom built bronchoscopic 2.4 French (0.8 mm diameter) catheter. Tissue samples were marked with tissue dye, visible in both OFDI and histology. Careful orientation procedures were used to precisely correlate imaging and histological sampling locations. The techniques outlined in this manuscript were used to conduct the first demonstration of volumetric OFDI with precise correlation to tissue-based diagnosis for evaluating pulmonary pathology24. This straightforward, effective technique may be extended to other tissue types to provide precise imaging to histology correlation needed to determine fine imaging features of both normal and diseased tissues.

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

A method to image ex vivo pulmonary resection specimens with optical frequency domain imaging (OFDI) and obtain precise correlation to histology is described, which is essential to developing specific OFDI interpretation criteria for pulmonary pathology. This method is applicable to other tissue types and imaging techniques to obtain precise imaging to histology correlation for accurate image interpretation and assessment. Imaging criteria established with this technique would then be applicable to image assessment in future in vivo studies.

Optical Frequency Domain Imaging di<em&gt; Ex vivo</em&gt; I campioni polmonari Resezione: Ottenere One to One immagine per Istopatologia Correlazione

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

Nanoparticelle virali per<em&gt; In vivo</em&gt; Imaging Tumore

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Construction of a Preclinical Multimodality Phantom Using Tissue-mimicking Materials for Quality Assurance in Tumor Size Measurement

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for radiologic assessment of solid tumors in response to anti-tumor drug therapy in the 1980s and 1990s, respectively. WHO criteria measure solid tumors in two-dimensions, whereas RECIST measurements use only one-dimension which is considered to be more reproducible 1, 2, 3,4,5. These criteria have been widely used as the only imaging biomarker approved by the United States Food and Drug Administration (FDA) 6. In order to measure tumor response to anti-tumor drugs on images with accuracy, therefore, a robust quality assurance (QA) procedures and corresponding QA phantom are needed.
To address this need, the authors constructed a preclinical multimodality (for ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI)) phantom using tissue-mimicking (TM) materials based on the limited number of target lesions required by RECIST by revising a Gammex US commercial phantom 7. The Appendix in Lee et al. demonstrates the procedures of phantom fabrication 7. In this article, all protocols are introduced in a step-by-step fashion beginning with procedures for preparing the silicone molds for casting tumor-simulating test objects in the phantom, followed by preparation of TM materials for multimodality imaging, and finally construction of the preclinical multimodality QA phantom. The primary purpose of this paper is to provide the protocols to allow anyone interested in independently constructing a phantom for their own projects. QA procedures for tumor size measurement, and RECIST, WHO and volume measurement results of test objects made at multiple institutions using this QA phantom are shown in detail in Lee et al. 8.

This paper describes in-house procedures of constructing a preclinical multimodality phantom made of tissue-mimicking (TM) materials for quality assurance (QA) of tumor size measurement in animal imaging modalities such as ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI).

Costruzione di un Phantom multimodalità preclinici Uso dei materiali di tessuti che imitano per l&#39;assicurazione della qualità delle dimensioni del tumore Misura

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for ...

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

Produzione di grandi numeri di-Size controllata Tumore Spheroids Uso micropiastra

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

PLGA nanoparticelle formate da singola o doppia emulsione con Vitamina E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

Misurazione longitudinale di matrice extracellulare rigidità in 3D Tumore modelli utilizzando-particelle inseguimento Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

Monitoraggio in tempo reale di alta intensità ultrasuoni focalizzati (HIFU) Ablazione<em&gt; In Vitro</em&gt; Canine Livers Uso Harmonic Imaging Movimento per Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Ingegneria dei tessuti da parte intrinseca Vascolarizzazione in un<em&gt; In Vivo</em&gt; Camera ingegneria dei tessuti

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis of the intrinsic photothermal response of hemoglobin molecules, the sensor enables high-sensitivity, chemical-free measurement of [Hb]. [Hb] detection capability with a limit of 0.12 g/dl over the range of 0.35 - 17.9 g/dl has been demonstrated previously. The method can be readily implemented using inexpensive consumer electronic devices such as a laser pointer and a webcam. The use of a micro-capillary tube as a blood container also enables the hemoglobin assay with a nanoliter-scale blood volume and a low operating cost. Here, detailed instructions for the PT-AS optical setup and signal processing procedures are presented. Experimental protocols and representative results for blood samples in anemic conditions ([Hb] = 5.3, 7.5, and 9.9 g/dl) are also provided, and the measurements are compared with those from a hematology analyzer. Its simplicity in implementation and operation should enable its wide adoption in clinical laboratories and resource-limited settings.

A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

Una rapida e chimico-libera emoglobina Assay con Photothermal angolare Light Scattering

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis ...