Questo lavoro è stato sostenuto da sovvenzioni Deutsche Forschungsgemeinschaft HI-698 / 10-1 e RU-1652 / 1-1. Ringraziamo Doreen maggio per l&#39;eccellente assistenza tecnica e la società DYOMICS GmbH, Jena per il loro supporto gentile.

NOTA: Tutte le procedure sono approvate dal Comitato regionale animali e in conformità con le linee guida internazionali sull&#39;uso etico degli animali. 1. Preparazione dei Materiali e strumenti <ol><li> Preparazione della dispersione delle vescicole spontaneamente formate (SFV) <ol><li> Sciogliere e preparare le soluzioni madre delle seguenti fosfolipidi: 214 mg / ml fosfatidilcolina d&#39;uovo (EPC), 134 mg / ml di colesterolo, 122 mg / ml 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N - [metossi (polietilene glicole) -2000] (sale di ammonio) (mPEG 2000 -DSPE) e 2 mg / ml di 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine- N - (7-nitro-2-1,3-benzoxadiazol-4 -yl) (sale di ammonio) (NBD-DOPE) in cloroformio e conservare in fiale di vetro. </li><li> Furnish circa 3 ml di cloroformio in un pallone a fondo rotondo e trasferire il volume appropriato di fosfolipidi magazzino soluzione nel pallone a fondo tondo di preparare liposomi composti da EPC: Chol: mPEG 2000 -DSPE ad un rapporto molare di 6,5: 3: 0.5. Per l&#39;etichettatura doppia fluorescenza di liposomi aggiungere 0,3 mol% NBD-DOPE alla soluzione lipidica. </li><li> Si evapora il cloroformio dalla soluzione fosfolipide organica a pressione ridotta (300 mbar) a 55 ° C utilizzando un evaporatore rotante. </li><li> Dopo si forma una pellicola fosfolipide omogenea, ridurre la pressione a 10 mbar per 1-2 ore per rimuovere depositi cloroformio residuo. </li><li> Mentre il cloroformio evapora, sciogliere DY-676-COOH (6,181 mM) in Tris 10 mM pH 7,4 e riempire un contenitore Dewar con azoto liquido. Accendere un bagno ad ultrasuoni e impostare a 50 ° C. </li><li> Trasferire un volume adeguato (0,5-1 ml) di DY-676-COOH (6,181 micron) soluzione del pallone a fondo tondo per idratare la pellicola fosfolipide asciutto e vortice vigorosamente fino a vescicole spontaneamente formate (SFV) forme di dispersione. Assicurarsi che tutti i fosfolipidi sono dispersi per evitare la perdita dei lipidi. </li><li> Tra attenzionensfer il pallone a fondo rotondo contenente la dispersione SFV in azoto liquido e congelare la dispersione per 3-5 min. Posizionare il pallone a fondo tondo in un bagno ad ultrasuoni a 50 ° C per scongelare la dispersione quindi agitare la dispersione vigorosamente per 1-2 min. Ripetere questa procedura sei volte, per un totale di sette cicli di gelo e disgelo. </li></ol></li><li> Estrusione di SFV per formare vescicole di liposoma omogenei <ol><li> Trasferire la dispersione SFV in una siringa da 1 ml (siringa a) e estrudere la dispersione attraverso una membrana di policarbonato 100 nm con un estrusore LiposoFast-Basic nella siringa-b. </li><li> Estrudere la dispersione dalla siringa-b, di nuovo in una siringa, quindi ripetere il ciclo di dieci volte. A causa di estrusione, la soluzione nei cambiamenti siringa da un aspetto opalescente a una dispersione chiara con il tempo. Dopo dieci cicli (venti singoli gradini estrusione) rimuovere la siringa-b dal dispositivo e estrudere la dispersione per l&#39;ultima volta da una siringa direttamente in un sterile1,5 ml tubo di reazione. </li></ol></li><li> Purificazione di liposomiale incapsulato DY-676-COOH dal colorante libero <ol><li> Preparare una colonna cromatografica di gel utilizzando G25 perline imbevuti di 10 Tris mM tampone pH 7.4 (colonna lunghezza 28 cm, diametro 0,8 centimetri). </li><li> Trasferire 0,5 ml di estrusi di dispersione delle vescicole sul letto di gel e lasciare che la fuga del campione nella matrice gel. </li><li> Eluire liposomi con Tris 10 mM pH 7,4 (Figura 1A) e lavare la colonna fino scarichi colorante libero completamente dalla colonna. Se necessario, raccogliere e riciclare il colorante libero da dissalazione e disidratazione secondo le istruzioni del produttore. </li><li> Concentrare liposomi eluiti mediante ultracentrifugazione (200.000 xg, 2 ore a 8 ° C) poi disperdono in adeguato volume di 10 mM Tris sterile tampone di pH, 7,4. </li></ol></li><li> Quantificazione dei incapsulato concentrazione DY-676-COOH <ol><li> Preparare una curva di calibrazione sciogliendo DY-676-COOH (0, 82, 124,247, 494, 988 nM) in tampone Tris 10 mM, pH 7,4 contenente 0,1% Triton X100. </li><li> Sciogliere 2 microlitri (100 nmol finale lipidi del 50 mmol / L stock) dei liposomi per 5 minuti a temperatura ambiente in 100 microlitri tampone Tris contenente 1% Triton X100 per distruggere le vescicole e rilasciare il colorante incapsulato. Poi diluire campioni con tampone Tris 10 mM, pH 7,4 ad una concentrazione finale Triton-X100 del 0,1% (v / v) fare un volume totale di 1 ml. Preparare tutti i campioni in duplicato. </li><li> Misurare l&#39;assorbimento e l&#39;emissione di tutti i campioni (gratuito DY-676-COOH e Triton-X100 trattati liposomi) ad una eccitazione λ = 645 nm e un&#39;emissione λ = 700 nm. Stabilire e utilizzare una curva di taratura del colorante libero per determinare la concentrazione di colorante incapsulato. </li></ol></li><li> Caratterizzazione Liposome <ol><li> Determinare la dimensione e zeta potenziale di liposomi di dispersione della luce dinamica. Diluire i campioni liposomiali con filtro sterilizzato (0,2 micron) Tris 10 mM a pH 7,4 per aconcentration di 100-300 micron (lipidi). Trasferire i campioni diluiti in un volume basso cuvetta monouso e misurare il campione in base alle istruzioni del produttore. </li><li> Caratterizzare i liposomi mediante microscopia elettronica a sostegno delle dimensioni, l&#39;integrità e l&#39;omogeneità delle vescicole liposomiali secondo protocolli standard. </li></ol></li></ol> 2. Convalida della fluorescenza tempra e attivazione di liposomi preparati <ol><li> Analisi fisico-chimiche di fluorescenza tempra e l&#39;attivazione <ol><li> Preparare due da 1,5 ml tubi per il Lip-Q e 2 tubi per la libera DY-676-COOH. Trasferire 100 nmol lipidi totali (2.38 microlitri di una / L Lip-Q soluzione stock 42 mmol, contenenti 138 mg / ml di incapsulato DY-676-COOH) nelle provette corrispondenti. Trasferimento libero DY-676-COOH equivalente al contenuto di colorante di Lip-Q (0.38 mg che risulta ad esempio da 138 mg / ml 1,000 x 2,38 microlitri Lip-Q utilizzato). Incubare una vascae di ogni sonda a 4 ° C e congelare il secondo tubo a -80 ° C per una notte (16 ore). </li><li> Riscaldare un blocco riscaldante a 30 ° C. Riempire una scatola di raffreddamento con ghiaccio tritato e equilibrare una aliquota di 10 mM Tris pH 7,4 tampone a temperatura ambiente. </li><li> Rimuovere le sonde da 4 ° C ed equilibrare a temperatura ambiente (avvolti in carta stagnola per proteggere dalla luce) e rapidamente scongelare sonde da -80 ° C a 30 ° C per 5 min. Raffreddare le sonde scongelati in ghiaccio per 1 minuto prima di trasferirli a temperatura ambiente (anche avvolti in carta stagnola per proteggere dalla luce). </li><li> Aggiungere 10 mM tampone Tris (pH 7,4) per ciascuna delle sonde al volume finale di 100 microlitri ed equilibrare tutte le sonde a RT per 10 min. </li><li> Pipettare 80 ml di ciascuna sonda in un bicchiere provetta basso volume e misurare l&#39;assorbimento di ogni sonda 400-900 nm su uno spettrometro. Ritorna la sonda di loro tubi corrispondenti. </li><li> Trasferire 80 ml di ciascuna sonda nella cuvetta bicchiere adattoe misurare l&#39;emissione di fluorescenza su un spettrofluorimetro eccitando le sonde a 674 nm e misurando la fluorescenza 694-800 nm. </li></ol></li><li> Assorbimento cellulare e l&#39;attivazione di fluorescenza <ol><li> Prendi e cultura le seguenti linee cellulari in loro terreni di coltura corrispondente in base alle condizioni standard (37 ° C, 5% di CO 2 e il 95% atmosfera umidificata). Qui, usare la linea cellulare murina J774A.1 macrofagi (Dulbecco modificati su terreno di Eagle integrato con 10% (v / v) di siero fetale di vitello), la linea cellulare glioblastoma umano, U-118mg (MEM contenente vitamine essenziali e 10% (v / v) di siero fetale di vitello) e la linea cellulare fibrosarcoma umano, HT-1080 (RPMI con FCS 5%). </li><li> Cappotto 8-ben diapositive camera con poli-L-lisina (aggiungere 100 ml 0,001% poli-L-lisina in ciascun pozzetto e incubare a 37 ° C per 10 min. Soluzione Aspirare e lasciare che i vetrini camera ad asciugare per almeno 4 ore a RT su un banco di lavoro sterile. Sciacquare i vetrini camera 3volte con soluzione salina tamponata 200 microlitri di Hank poi sigillare con paraffina e un foglio di alluminio e conservare a 4 ° C fino al necessario. </li><li> Mentre le diapositive da camera si stanno prosciugando, filtro sterilizzare i liposomi e soluzione colorante e conservare a 4 ° C fino al necessario. </li><li> Per l&#39;analisi della sonda assorbimento con tutto il corpo in vivo sistema di imaging di fluorescenza NIR, preparare 5 piccole fiasche di coltura per ciascuna delle linee cellulari 3 di prova (15 boccette in totale). Seed 2 x 10 6 J774A.1, U-118mg e cellule HT-1080 per pallone di coltura con 5 ml del rispettivo mezzo di coltura (in quintuplica) e crescere per 16-24 ore. Parallelamente alla cultura palloni, sementi 30.000 cellule di ciascuna linea cellulare (J774A.1 e HT-1080) o 20.000 celle (U-118mg) per 2 pozzi della slitta da camera rispettivamente e crescere in 500 microlitri terreno di coltura per 16-24 ore . </li><li> Il giorno successivo, aggiungere 100 nmol (importo lipidi finale) di Lip-Q per 2 palloni per linea cellulare e ad un pozzo di ciascuna linea cellulare sul vetrino della camera.Immediatamente trasferire 1 pallone per linea cellulare a 4 ° C e il secondo pallone indietro al incubatore. NOTA: Il volume delle sonde aggiunto nei flaconi e le diapositive camera sono gli stessi, rendendo la concentrazione su vetrini camera 10 volte superiore (5 ml è di 500 microlitri terreno di coltura). Questo è necessario perché la rilevazione microscopica è meno sensibile rispetto al sistema di imaging di fluorescenza NIR dove vengono esposte pellet cellulari dai fiaschi. </li><li> Aggiungere libera DY-676-COOH in concentrazione pari al contenuto di colorante di Lip-Q alle cellule in 2 palloni per linea cellulare e ad un pozzetto di ciascuna linea cellulare sul vetrino camera, poi trasferire immediatamente 1 pallone per linea cellulare a 4 ° C (svuotamento di energia) e l&#39;altra beuta con la slitta camera posteriore al incubatore. Incubare tutte le cellule per 24 ore nelle condizioni corrispondenti. Le cellule nel pallone senza sonde servono come controllo non trattato. </li></ol></li><li> NIRF imaging e di analisi semi-quantitativa <ol><li> Dopo 24 ore la durata dell&#39;incubazione, raccogliere le cellule in fiaschi lavando cellule 2 volte con Hanks tamponata soluzione salina (HBSS) poi raschiare le cellule in 500 microlitri HBSS e pellet per centrifugazione (5 min a 200 xg) in tubi microlitri 500. </li><li> Posizionare i tubi con il pellet di cellule (e HBSS) in un imager NIRF e immagini utilizzando filtri di eccitazione (615-665 nm) e l&#39;emissione (taglio&gt; 700 nm). </li><li> Sottrarre autofluorescenza e valutare l&#39;intensità del target rispetto autofluorescenza secondo le istruzioni del produttore. Questo darà i livelli semi-quantitativi della intensità di fluorescenza come segnale medio (conteggi scalati / sec), che rappresenta i livelli di conteggio dopo la scalatura per tempo di esposizione, guadagno della telecamera, binning e profondità di bit, in modo che le misurazioni sono confrontabili tra loro. </li></ol></li><li> Analisi microscopica confocale <ol><li> Dopo 24 ore di incubazione, raccogliere le cellule su vetrini da camera lavando 2 volte con 500 microlitri HBSS. </li><li> Fissare il cells con 200 microlitri HBSS contenente 3,7% (v / v) di formaldeide per 30 minuti a RT. </li><li> Mentre è in corso di fissazione, diluire la macchia DNA, Hoechst-33258 1:50 con soluzione di montaggio. </li><li> Dopo la fissazione, lavare le cellule due volte con HBSS quindi separare le camere dai vetrini. Aggiungere soluzione di montaggio 50 ml contenente il DNA-macchia su ogni posto corrispondente ai pozzi delle diapositive da camera. Coprire le cellule con vetrino, sigillare i bordi con smalto trasparente e asciugare per 10 minuti a RT (scuro). </li><li> Celle di immagine su un microscopio a fluorescenza adatto o microscopio confocale. Utilizzare le seguenti impostazioni di eccitazione e di emissione per la visualizzazione dei componenti corrispondenti: nuclei (Hoechst-33258: eccitazione 405 nm, emissione 420-480 nm). NBD-DOPE (liposomiale lipidico: eccitazione 488 nm, emissione 530 nm). DY-676-COOH (colorante fluorescente NIR: eccitazione 633-645 nm, emissione 650-700 nm). NOTA: Assicurarsi che la fluorescenza del microscopio available è dotato di filtri idonei che consentono di eccitazione ed emissione di lunghezze d&#39;onda superiore a 630 nm. </li></ol></li></ol> 3. liposomi a base di In Vivo imaging di fluorescenza di infiammazione <ol><li> Preparazione degli animali e materiali <ol><li> Casa 8-12 week-old topi NMRI maschi di peso di circa 36 g in condizioni standard con cibo e acqua ad libitum. </li><li> Sette giorni prima dell&#39;inizio della sperimentazione, invia tutti i topi una dieta povera pheophorbide per ridurre autofluorescenza tessuti. </li><li> Ventiquattro ore prima dell&#39;inizio di ogni esperimento, radere topi nella zona desiderata (ad esempio, l&#39;intera area del lotto se si desidera l&#39;imaging di edema hind gamba). </li><li> Pesare gli animali e calcolare la quantità di sonde da iniettare per mouse (Lip-Q e Lip-DQ a 10 micromol (concentrazione di lipidi) per kg di peso e senza DY-676-COOH (equivalente a tingere contenuto di Lip-Q utilizzata) . </li><li> Immagine gli animali inun corpo intero NIR Fluorescenza imager con le stesse impostazioni utilizzate per i pellet cellulari. Questa misurazione fornisce autofluorescenza degli animali. </li><li> Disciogliere 10 mg zymosan-A in 1 ml di soluzione salina isotonica e memorizzare una notte a 4 ° C. </li></ol></li><li> Induzione di infiammazione e in vivo NIRF immagini <ol><li> Preparare 3 siringhe per topo contenenti le seguenti soluzioni. Riempire una siringa con 50 microlitri zymosan-Una soluzione (10 mg / ml) e la seconda siringa con 50 microlitri di soluzione salina isotonica. Riempire la terza siringa con le sonde, per cui Lip-Q e Lip-DQ (10 mmol / kg di peso (lipidi)) sono designati per gli animali di prova e la connessione DY-676-COOH (concentrazione in Lip-Q) per gli animali di controllo. Assicurarsi che le sonde sono diluiti con sterili HBSS a 150 microlitri volume finale. </li><li> Applicare la crema occhio gli occhi degli animali per evitare secchezza e anestetizzare gli animali con il 2% isoflurano fino a quando non sono profondamente addormentati e non reagiscono al tocco sulle zampe (questo richiede circa 2 minuti). </li><li> Posizionare il mouse su una stuoia calda (ancora sotto anestesia) e iniettare il zymosan-A sottocutanea soluzione sul zampa posteriore destra e la soluzione salina sulla zampa posteriore sinistra. Iniettare immediatamente la sonda per via endovenosa e l&#39;immagine dell&#39;animale, successivamente poi tempo record di iniezione / misura (come t = 0 ore). Salvare le immagini risultanti come cubetti di immagine e ripetere i passi precedenti per tutti gli altri animali e le rispettive sonde. </li><li> Fotografia gli animali ogni 2 hr per 10 ore dopo l&#39;iniezione e poi a 24 ore dopo l&#39;iniezione, facendo in modo che la fase della camera di misura è caldo (per esempio, posizionando una stuoia calda sotto), al fine di evitare l&#39;ipotermia. Dopo ogni misura, mettere gli animali in una gabbia con cibo e acqua ad libitum e posizionare la gabbia in una camera di animali temperato. Eutanasia gli animali di primo anestetizzante con il 2% isoflurano fino gli animali reagiscono più a toccare, poi eutanasia con anidride carbonica per 5-10 minuti, rendendoAssicurarsi che gli animali smettono di respirare completamente e si verifica rigor mortis. </li><li> Staccare i topi secondo protocolli standard che possono essere valutati online (http://www.freebookez.com/mouse-dissection-lab-report/) e l&#39;immagine gli organi. </li><li> Valutare i risultati delle misure secondo le istruzioni del produttore per la prima sottraendo la fluorescenza complessiva degli animali (unmixing) poi regioni assegnazione di interesse per autofluorescenza (sinistra zampa posteriore con soluzione salina) e l&#39;obiettivo di fluorescenza di zona infiammata (destra zampa posteriore con zymosan-A). </li></ol></li></ol>

The authors have nothing to disclose.

Dal liposomi possono anche servire come sistemi di consegna per tinture fluorescenti, che permettono l&#39;imaging di malattie in questione. L&#39;incapsulamento di alte concentrazioni di coloranti fluorescenti come il colorante NIRF, DY676-COOH usato qui, porta ad un elevato livello di estinzione della fluorescenza del colorante intrappolato. Fluorescenza tempra, un fenomeno visto con molti fluorofori ad alta concentrazione può essere sfruttata in diversi in applicazioni di imaging in vivo, dove è r...

I liposomi sono stati intensamente studiato e servire come uno dei sistemi di rilascio di farmaci biomedici più biocompatibili per applicazioni cliniche 1,2. Essi sono composti principalmente di fosfolipidi e colesterolo, che sono entrambi composti biocompatibili mimano parti di membrane cellulari naturali. Considerando sostanze idrofile possono essere intrappolati all&#39;interno acquosa, agenti lipofili possono essere incorporati all&#39;interno liposomiale doppio strato fosfolipidico 3. Incapsulamento di sostanze entro l&#39;interno acquosa di liposomi garantisce la protezione contro la degradazione in vivo e impedisce anche il sistema host da effetti tossici di farmaci citotossici utilizzati per la terapia di malattie, per esempio chemioterapici volti a distruggere le cellule tumorali. La modifica della superficie liposomiale con polimeri come polietilenglicole (PEGylation) si estende ulteriormente il tempo di circolazione sanguigna liposomiale in vivo a causa di stabilizzazione sterica 4. Moreover, liposomi possono sequestrare elevate concentrazioni di varie sostanze quali proteine, sostanze idrofile 5,6 7,8 e 9 enzimi. Servono pertanto strumenti terapeutici e diagnostici clinici affidabili che meritano la loro approvazione per la consegna di farmaci citotossici, come doxorubicina per la terapia del cancro 4. Grazie alla loro flessibilità, liposomi possono essere caricati con fluorocromi per uso chirurgico di diagnostica e di immagine-guidata. Imaging di fluorescenza prevede un costo-efficace e non invasivo in strumento diagnostico vivo che però, richiede alcuni requisiti di base. Si potrebbe dimostrare che fluorocromi che si adattano meglio per l&#39;imaging in vivo hanno assorbimento caratteristico e massimi delle emissioni nel range in cui dispersione della luce e dispersione, nonché autofluorescenza dei tessuti d&#39;origine da acqua ed emoglobina è bassa. Pertanto, tali sonde hanno il loro massimi abs / em tra 650 e 900 nm 10.<html> Oltre a questo, la stabilità di fluorocromi sia in vitro che in vivo è critica, come opsonizzazione e rapida clearance possono limitare notevolmente la loro applicazione per imaging in vivo 11. Altri effetti quali la scarsa stabilità e bassa sensibilità o effetti citotossici sugli organi bersaglio come si è visto con verde di indocianina (ICG) 12-16, sono indesiderati e devono essere presi in considerazione quando si progetta le sonde per l&#39;imaging in vivo. Queste osservazioni hanno portato allo sviluppo attivo di diversi fluorocromi NIR preclinici, nanoparticelle nonché nuove tecniche di imaging in vivo di processi infiammatori, cancro e per immagine guidata chirurgia 17-20. Nonostante la stabilità di più (fluorescenza vicino infrarosso) NIRF preclinico coloranti in vitro, la perfusione rapida e passaggio attraverso il fegato ei reni impediscono il loro uso in imaging ottico in vivo di malattie e processi infiammatori. S copi &quot;&gt; Abbiamo quindi presentiamo un protocollo per l&#39;incapsulamento di fluorocromi come il ben caratterizzato vicino infrarosso colorante fluorescente DY-676-COOH, noto per la sua tendenza ad auto-estinzione alle concentrazioni relativamente elevate di 21 in liposomi. Ad alte concentrazioni H- formazione di dimero e / o-pi stacking interazioni tra molecole fluoroforo situate all&#39;interno di ciascun altro risultato raggio Förster in Förster trasferimento di energia di risonanza (FRET) tra le molecole fluorocromi. In bassa concentrazione lo spazio tra le molecole fluoroforo aumenta, impedendo in tal modo l&#39;interazione-pi impilamento e H-dimero formazione e la conseguente elevata emissione di fluorescenza. La commutazione tra alta e bassa concentrazione e l&#39;accompagnamento della fluorescenza e l&#39;attivazione è una strategia promettente che può essere sfruttata per l&#39;imaging ottico 22. A questo proposito, incapsulamento di alte concentrazioni di colorante NIRF DY-676-COOH all&#39;interno acquosa di liposomi è più favorable per l&#39;imaging in vivo che il colorante libera. La sfida del metodo consiste innanzitutto nel incapsulamento corretta e in secondo luogo, la validazione dei benefici derivanti da incapsulare alte concentrazioni di colorante. Confrontando le proprietà di imaging di liposomi bonifica con quello del colorante libero ed anche con una formulazione non quenched liposomi con basse concentrazioni di colorante è indispensabile. Mostriamo da un protocollo pellicola idratazione ed estrusione semplice, ma molto efficace in combinazione con gelo e disgelo alternative che l&#39;incapsulamento delle concentrazioni tempra di DY-676-COOH in liposomi è fattibile. Altri metodi utilizzati per preparare liposomi come il metodo di evaporazione a fase inversa 23 nonché il metodo di iniezione di etanolo 24 consentono la preparazione di liposomi con alte efficienze di incapsulamento per molte sostanze idrofile. Tuttavia, la natura della sostanza da incapsulare può influenzare l&#39;efficienza di incapsulamento. In effetti,il protocollo pellicola idratazione ed estrusione presentato qui ha rivelato la più alta efficienza per l&#39;incapsulamento di DY-676-COOH. Per illustrare i vantaggi di incapsulamento liposomiale di DY-676-COOH, un modello edema zymosan indotta, che consente lo studio dei processi infiammatori in poche ore, è stato utilizzato. Qui, è dimostrato che liposomi con alte concentrazioni della incapsulato DY-676-COOH sono più adatti per tutto il corpo in imaging ottico vivo di processi infiammatori rispetto colorante libero o la formulazione liposomiale non quenched con concentrazioni basse colorante. Così il protocollo sottostante fornisce un metodo semplice e veloce per produrre liposomi fluorescenti bonifica e la validazione della loro attivazione e di imaging potenziale sia in vitro che in vivo.

<table border="0" cellpadding="0" cellspacing="0" width="872">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Name of Material/ Equipment</td>
			<td style="width:179px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:267px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:309px;">Materials and equipments for preparation of liposomes</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">egg phospahtidylcholine</td>
			<td>Avanti Polar Lipids</td>
			<td>840051P</td>
			<td>Dissolve in Chloroform and store in glass vials (214 mg/ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cholesterol</td>
			<td>Sigma</td>
			<td>C8667</td>
			<td>Dissolve in Chloroform and store in glass vials (134 mg/ml)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt)</td>
			<td>Avanti Polar Lipids</td>
			<td>880120P</td>
			<td>Dissolve in Chloroform and store in glass vials (122 mg/ml)</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:309px;">1,2-dioleoyl-snglycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt)</td>
			<td>Avanti Polar Lipids</td>
			<td>810145P</td>
			<td>Dissolve in Chloroform and store in glass vials (2mg/ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Sartorius MC1 (d = 0.01 mg)</td>
			<td>Sartorius AG</td>
			<td>Research RC 210 P</td>
			<td>used for weighing the phospholipids</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Rotavapor</td>
			<td>B&#252;chi Labortechnik AG</td>
			<td>R-114</td>
			<td>used for hydration of phospholipid film</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Waterbath</td>
			<td>B&#252;chi Labortechnik AG</td>
			<td>R-481</td>
			<td>used for hydration of phospholipid film</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Vacuum Controller</td>
			<td>B&#252;chi Labortechnik AG</td>
			<td>B-720</td>
			<td>used for hydration of phospholipid film</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Vacobox</td>
			<td>B&#252;chi Labortechnik AG</td>
			<td>B-177</td>
			<td>used for hydration of phospholipid film</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Circulation Chiller</td>
			<td style="width:179px;">LAUDA DR. R. WOBSER 
			GMBH &#38; CO. KG</td>
			<td>WKL 230</td>
			<td>used for hydration of phospholipid film</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DY-676-COOH</td>
			<td>Dyomics GmbH</td>
			<td>676-00</td>
			<td>Dissolve in 10 mM Tris and store stock at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-(Hydroxymethyl)-aminomethan</td>
			<td>Applichem</td>
			<td>A1086</td>
			<td>buffer 10 mM, pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichlormethan</td>
			<td>Carl Roth GmbH + Co. KG</td>
			<td>Y015.2</td>
			<td>used for liposome preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sonicator</td>
			<td>Merck Eurolab GmbH</td>
			<td>USR 170 H</td>
			<td>used for liposome preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex Genie 2 (Pop-off Cup, No. 146-3011-00)</td>
			<td>Scientific Industries Inc.</td>
			<td>SI-0256</td>
			<td>used for liposome preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sephadex G25 medium&#160;</td>
			<td>GE Healthcare Europe GmbH</td>
			<td>17-0033-01</td>
			<td>used for liposome purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X100</td>
			<td>Ferak Berlin GmbH</td>
			<td>505002</td>
			<td>used to destruct liposomes&#160; for dye quantification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LiposoFast-Basic</td>
			<td>Avestin Inc.</td>
			<td style="width:119px;"></td>
			<td>used for the extrusion of liposomes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polycarbonate filter membrane, 100 nm (Whatman Nucleopore Trans Etch Membrane, NUCLEPR PC 19 MM, 0.1 U)</td>
			<td>VWR</td>
			<td style="width:119px;"></td>
			<td>used for the extrusion of liposomes via LiposoFast-Basic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluostar Optima</td>
			<td>BMG Labtech</td>
			<td style="width:119px;"></td>
			<td>used for dye quantification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zetasizer Nano ZS</td>
			<td>Malvern</td>
			<td style="width:119px;"></td>
			<td>used for the determination of liposome size and zetapotential</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Ultracentrifuge&#160;</td>
			<td style="width:179px;">Beckmann Coulter GmbH</td>
			<td style="width:119px;">XL 80</td>
			<td>used for concentration of the samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Rotor</td>
			<td style="width:179px;">Beckmann Coulter GmbH</td>
			<td style="width:119px;">SW 55 TI</td>
			<td>used for concentration of the samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;"></td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:309px;">Materials and equipments for the evaluation of liposome and optical imaging&#160;</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;"></td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Zymosan-A from Saccharomyces cereviciae</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:119px;">Z4250-250MG</td>
			<td>used for induction of inflammation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Isotonic Saline (0.9)</td>
			<td style="width:179px;">Fresenius GmbH</td>
			<td style="width:119px;">PZN-2159621</td>
			<td>used for the dilution of Zymosan-A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Isoflurane vaporizer</td>
			<td>Ohmeda Isotec 4</td>
			<td style="width:119px;"></td>
			<td>used for anesthesizing animals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Isoflurane</td>
			<td>Actavis GmbH&#160;</td>
			<td style="width:119px;">PZN-7253744</td>
			<td>anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Mat Pro 20 W</td>
			<td style="width:179px;">Lucky Reptile</td>
			<td style="width:119px;">61202-HTP-20</td>
			<td>used to keep animals warm during anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Omnican-F (1 ml injection)&#160;</td>
			<td style="width:179px;">Braun</td>
			<td style="width:119px;">PZN-3115465</td>
			<td>used for subcutaneous and intravenous application of probes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Panthenol eye cream</td>
			<td style="width:179px;">Jenapharm</td>
			<td style="width:119px;">PZN-3524531</td>
			<td>used to prevent dryness of the eyes of animals during anesthesia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:309px;">Hanks buffered saline solution</td>
			<td style="width:179px;">PAA Laboratories /Biochrom AG</td>
			<td style="width:119px;">L2045</td>
			<td>w/o Mg2+, Ca2+ and phenol red. For dilution of probes and for washing of cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">8-Well chamber slides</td>
			<td style="width:179px;">BD Biosciences</td>
			<td style="width:119px;">354108</td>
			<td>used for cell culture followed by microscopy&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Cell culture flasks</td>
			<td style="width:179px;">Greiner BioOne</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture media</td>
			<td colspan="2">Gibco (life technologies GmbH)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal calf serum&#160;</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-Lysine solution (0,01% - 50&#160;ml)</td>
			<td>Sigma</td>
			<td style="width:119px;">P4832</td>
			<td>used to coat cell culture chamber slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mountant Permafluor</td>
			<td>ThermoScientific&#160;</td>
			<td>S21022-3</td>
			<td>Mounting solution for microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Hoechst-33258</td>
			<td style="width:179px;">AppliChem</td>
			<td style="width:119px;"></td>
			<td>DNA stain for microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;"></td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Hera-Safe</td>
			<td>Heraeus Instruments</td>
			<td style="width:119px;"></td>
			<td>sterile work bench used for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HERA cell</td>
			<td>Heraeus Instruments</td>
			<td style="width:119px;"></td>
			<td>Incubator used for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">LSM510-Meta</td>
			<td style="width:179px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>used for confocal microscopy</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:309px;">Maestro-TM in vivo fluorescence imaging system</td>
			<td style="width:179px;">CRi, Woburn</td>
			<td style="width:119px;"></td>
			<td>used for whole body fluorescence imaging of small animals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Spectrophotometer (Ultrospec 4300 pro UV)</td>
			<td style="width:179px;">GE Healthcare</td>
			<td style="width:119px;"></td>
			<td>used for measurement of absorption</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Spectrofluorometer (Jasco FP-6200)</td>
			<td style="width:179px;">Jasco</td>
			<td style="width:119px;"></td>
			<td>used for measurement of fluorescence emission</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;"></td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Animals</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NMRI mice (8-12 weeks old, male)</td>
			<td style="width:179px;">Elevage Janvier, France</td>
			<td style="width:119px;"></td>
			<td>used for inflammation trials</td>
		</tr>
	</tbody>
</table>

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.

L&#39;incapsulamento di alte concentrazioni di coloranti fluorescenti come la NIRF dye DY676-COOH qui utilizzato all&#39;interno acquosa di liposomi porta ad un elevato livello di estinzione della fluorescenza. Fluorescenza tempra, un fenomeno visto con molti fluorofori ad alta concentrazione, può essere sfruttata in diversi in applicazioni di imaging in vivo in cui una elevata sensibilità e rilevamento affidabile dell&#39;area di destinazione sono richiesti. L&#39;uso di liposomi fornisce anche una ...

Watch this Scientific Journal Video about Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging at JoVE.com

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 ...

fluorescence-quenching-liposomal-encapsulated-near-infrared

Research

JoVE Journal

Bioengineering

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

16.1K Views.  Jena University Hospital. 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. 

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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Tridimensionale con risoluzione ottica Microscopia fotoacustico

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Ricerca

Bioingegneria

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

Un Cre-Lox P approccio ricombinazione per la rilevazione di fusione cellulare In Vivo</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

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 ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

Imaging denaturate collagene fili<em&gt; In vivo</em&gt; E<em&gt; Ex vivo</em&gt; Via Photo-triggered ibridazione di Caged collagene Mimetic peptidi

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

Selective cellulare eliminazione dalla misto Cultura 3D utilizzando una tecnica Near Infrared Photoimmunotherapy

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparazione del 3D collagene Gel e microcanali per lo studio delle interazioni 3D<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

La sintesi di RGD-funzionalizzati idrogeli come strumento per applicazioni terapeutiche

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

Targeted membrana plasmatica di consegna di un idrofobo Cargo incapsulato in un cristallo nanoparticelle Carrier Liquid

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Nanotubi di DNA come un Versatile strumento per lo studio di polimeri semiflessibili

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.