The authors would like to thank Drs. Helene Salmon and Miriam Merad from Icahn School of Medicine at Mount Sinai for providing the B16-F10-YFP cells and for their expert advice on melanoma mouse models. The authors further thank the Animal Imaging Core Facility, the Radiochemistry and Molecular Imaging Probes Core Facility, and the Molecular Cytology Core Facility at Memorial Sloan Kettering Cancer Center (MSK) for their support. This work was supported by National Institutes of Health grants NIH 1 R01 HL125703 (W.J.M.M.), R01CA155432 (W.J.M.M.), K25 EB016673 (T.R.) and P30 CA008748 (MSK Center Grant). The authors also thank the Center for Molecular Imaging and Nanotechnology (CMINT) at MSK for their financial support (T.R.).

La procedura consiste nella etichettatura duplice radioattiva e fluorescenti di nanoparticelle, imaging in vivo PET-CT, ex vivo misure biodistribuzione, e ex vivo immunostaining e citometria a flusso analisi. Tutti gli esperimenti sugli animali sono stati approvati dal Comitato di Cura e uso istituzionale degli animali del Memorial Sloan Kettering Cancer Center. 1. Preparazione di liposomi Dual-etichettati NOTA: singenico B16 melanoma può essere indotta da iniezione di 300.000 cellule B16-F10 nei fianchi posteriori di topi C57BL / 6 sotto anestesia da inalazione di 2% di ossigeno -isoflurane contenenti. Utilizzare reagenti e strumenti sterili in un&#39;area di lavoro sterile durante l&#39;intervento chirurgico. Dopo l&#39;intervento chirurgico, osservare attentamente gli animali fino al loro recupero dall&#39;anestesia. Mettere gli animali nel loro gabbie solo quando riacquistano piena coscienza e la mobilità. li ospitare in camere sterili per animali con tumori xenotrapiantati. Tumori con una dimensionedi 300 mm 3 sono ideali per la caratterizzazione di nanoparticelle a causa delle loro pronunciate permeabilità e ritenzione effetti migliorati (EPR), che possono aumentare l&#39;accumulo di nanoparticelle. I protocolli dettagliati sono forniti di seguito. <ol><li> In un pallone a fondo tondo, dissolvere una miscela di 20 mg di lipidi in 2 mL di cloroformio contenente 1,2-dipalmitoyl- sn -glycero-3-phophocholine (DPPC), colesterolo, 1,2-disearoyl- sn -glycero- 3-fosfoetanolamina-poli (glicole etilenico) 2000 (DSPE-PEG2000), DSPE-deferoxamina (DSPE-DFO) 8, e l&#39;acido 1,1-diododecyl-3,3,3,3-tetramethylindodicarbocyanine-5,5-disolfonico ( DiIC12 [5] -ds) in rapporti molari di 61,3%, 33,4%, 5, 0,2% e 0,1% rispettivamente. NOTA: Questa composizione lipidica è molto simile alla formulazione liposomiale di doxorubicina attualmente utilizzati in clinica 2. I liposomi risultanti dalla nostra procedura sarà strettamente imitare la performance in vivo del nanomedic clinicaine. </li><li> Utilizzare un evaporatore rotante per rimuovere il solvente organico e formare un film lipidico sottile a temperatura ambiente. Aggiungere 20 ml di PBS e ultrasuoni per 30 minuti per generare nanoparticelle liposomiali utilizzando una punta sonicazione 3,8 mm e un&#39;ampiezza di 30 W, con sufficiente raffreddamento sul ghiaccio. </li><li> Centrifugare la soluzione di liposomi a 4.000 g per 10 minuti per rimuovere gli aggregati e lavare le nanoparticelle con PBS usando filtrazione centrifuga (peso molecolare di cut-off (MWCO:. 100 kDa) per rimuovere i lipidi liberi e solvente organico residuo concentrare il liposomi, che sarà rimangono nella camera superiore, e trasferirli in una nuova provetta. i liposomi possono essere memorizzati in un frigorifero in PBS fino a 1 settimana prima delle fasi successive. </li><li> Per il controllo di qualità, caratterizzare i liposomi dalla dispersione della luce dinamica (DLS). In particolare, mescolare 50 ml di liposomi con 950 ml di PBS, e poi trasferire il composto nella cuvetta dimensionamento. Posizionare la cuvetta nell&#39;analizzatore e misurare la particelladimensioni e la loro distribuzione dimensionale. Le dimensioni delle particelle e la loro distribuzione delle dimensioni (indice di polidispersità (PDI)) dovrebbero essere 90-120 nm e 0.1 - 0.2, rispettivamente. </li><li> Per radiomarcatura, miscelare i liposomi purificati, pari a 2 mg di lipidi, in PBS a pH compreso tra 6,9 e 7,1 con 1 mCi 89 Zr-ossalato a 37 ° C per 2 h in una provetta da 1,5 mL, (volume totale: ~ 100 ml). Rimuovere la libera, che non ha reagito 89 Zr mediante filtrazione centrifuga (MWCO = 100 kDa), simile al punto 1.3. Lavare il retentato con PBS sterile e diluire al volume desiderato. La resa radiochimica dovrebbe essere 80-95%. ATTENZIONE! Lavorare con materiali radioattivi è altamente regolamentato. Le istituzioni devono essere certificati e di fornire i servizi necessari, formazione del personale, e rigoroso monitoraggio dell&#39;uso della radioattività. I ricercatori hanno bisogno di ricevere una formazione adeguata e indossare dispositivi di protezione individuale e anelli dosimetria / badge durante la manipolazione di radioattività. </li><li&gt; Determinare la purezza radiochimica dei liposomi radiomarcati per dimensione esclusione radio-HPLC, che in genere dovrebbe essere superiore al 98% 10. purezze radiochimici inferiori al 95% sono indicativi di un lavaggio insufficiente nel passaggio 1.5. </li><li> Dopo radiomarcatura, determinare le dimensioni delle nanoparticelle di dispersione della luce dinamica. La dimensione e la PDI dovrebbero essere all&#39;interno dello stesso intervallo come le misure in fase 1.4 10. NOTA: Se i campioni radioattivi non sono ammessi nel citofluorimetro o microscopio a fluorescenza, NAT non radioattivo Zr-ossalato può essere usato per etichettare liposomi nella fase 1.5. Questi liposomi non radioattive hanno caratteristiche identiche a loro analoghi radioattivi. Una rappresentazione di questa procedura è fornito in Figura 2. </li></ol> 2. In Vivo PET-CT Imaging e Biodistribuzione di liposomi Dual-etichetta <ol><li> Iniettare circa 100 ml di liposomi dual-marcato Wesimo circa 300 pCi di radioattività in topi di melanoma trattenuto (C57BL / 6, di sesso femminile, 10 - 16 settimane) nelle loro vene coda, a circa 10 mCi 89 Zr / kg di peso corporeo. </li><li> Per determinare il tempo di dimezzamento, prelevare il sangue dalla vena della coda da animali anestetizzati di sotto del 2% isoflurano contenenti ossigeno utilizzando 28-G siringhe da insulina. Raccogliere il sangue (10-20 ml) in tubi pre-pesato a 2 min, 15 min, 1 h, 4 h, 8 h, 24 h, e 48 ore dopo l&#39;iniezione. Pesare il sangue per differenza, determinare il contenuto di radioattività in un contatore gamma, e calcolare la concentrazione di radioattività come percentuale della dose iniettata per grammo (% ID / g). NOTA: Eseguire la procedura in una stanza dotata di un sistema di anestesia, gabbie di recupero, riscaldamento, e un cappuccio a rischio biologico. Assicurarsi che gli animali recuperano completamente e contare su una mobilità normale dopo la procedura prima del trasferimento dalle gabbie di recupero a tenere gabbie. Casa gli animali portatori di tumore in camere apposite per gli animali somministrato conmateriali radioattivi. </li><li> 24 o 48 ore dopo l&#39;iniezione del liposomi, immagine i topi in un piccolo animale microPET-CT scanner 10. Posto l&#39;animale anestetizzato orizzontalmente sulla sua pancia e tenerlo anestetizzato. Somministrare 2% isoflurano-ossigeno attraverso un cono di naso. Applicare una pomata oftalmica per gli occhi prima della scansione per impedire loro di essiccazione. </li><li> Eseguire un corpo intero PET registrazione scansione statica di almeno 50 milioni di eventi coincidenti, con una durata approssimativa di 15 minuti. Impostare la finestra di temporizzazione energia e coincidenza a 350-700 keV e 6 ns rispettivamente. Successivamente, eseguire una scansione a 5 min tomografia computerizzata (CT). Impostare il tubo a raggi X ad una tensione di 80 kV e una corrente di 500 μA; la TAC utilizza 120 gradini di rotazione per un totale di 220 gradi, con circa 145 ms per frame 10. </li><li> Dopo la scansione PET-CT, sacrificare subito i topi di anidride carbonica per asfissia e confermare le morti per Pinching le zampe degli animali. Una volta confermata, eseguire lussazioni cervicali. </li><li> Rimuovere il sangue nei tessuti di topo dal primo Tagliando l&#39;atrio destro e poi iniettando 20 ml di PBS nel ventricolo sinistro, e successivamente raccogliere i tessuti rilevanti come tumore, fegato, milza, polmone, rene, muscolo, altri 16. </li><li> Pesare i tessuti, misurare il loro contenuto radioattività gamma contando 8, 9, e calcolare l&#39;accumulo di tessuto radioattività come percentuale della dose iniettata per g (% ID / g). </li></ol> 3. Ex Vivo Citometria a Flusso e Immunocolorazione NOTA: Iniettare circa 100 ml di liposomi fluorescenti in topi di melanoma attraverso la vena della coda alla dose di 0,5 mg di colorante per kg di peso corporeo. Se i campioni radioattivi non sono ammessi nel citofluorimetro e microscopio a fluorescenza, devono essere utilizzati liposomi non radioattivi. <ol><li> Sacrifica l&#39;iniezione dopo 24 ore i topi, come al punto 2.5, e raccogliere i tumori, che devono essere memorizzati in PBS freddo. </li><li> Per citometria a flusso, attenersi alla seguente procedura 17: <ol><li> Preparare un cocktail enzimatico costituito da una miscela di purificato collagenasi I e II (4 U / mL), ialuronidasi (60 U / mL), e DNasi I (60 U / ml) in citometria a flusso tampone (PBS con 0,5% BSA e 1 mM EDTA). </li><li> Miscelare 100 mg di tessuto tumorale con 200 ml di tampone cocktail di enzimi in una provetta da 1,5 ml e dadi tessuto a piccoli pezzi con forbici sottili. Aggiungere un altro 1,3 ml di cocktail di enzimi nel tubo e trasferire il contenuto di un 6-pozzetti. </li><li> Agitare la piastra da 6 pozzetti su un agitatore orizzontale posta all&#39;interno di un forno 37 C ° per 60 min a 60 rpm. </li><li> Lavare il tessuto omogenato con citometria a flusso buffer di 3 volte per ottenere una sospensione singola cella. </li><li> Colorare le cellule con un cocktail di anticorpi specifici per biomarcatori tra cui CD45, CD11b, Ly6C, CD11c,CD64, CD3, MHCII, e CD31 (diluizione 1: 200 per tutti gli anticorpi; informazioni clone è fornita nel foglio di calcolo dei materiali). Identificare i tipi di cellule rilevanti e determinare la loro intensità di fluorescenza media (MFI) di DiIC12 (5) -ds in ogni popolazione di cellule con un flusso adeguato di citometria software di analisi. </li></ol></li><li> Per immunostaining, attenersi alla seguente procedura: <ol><li> Mettere un tumore in una cassetta piena di medio taglio ottimale (OCT) e congelarlo in ghiaccio secco. </li><li> Fare 6-micron sezioni congelate di tessuto tumorale e metterli su vetrini istologici; sezioni dovrebbero coprire più grandi sezioni di tumore, che forniscono le informazioni più rappresentative sul tessuto. </li><li> Macchiare le biomarcatori (ad esempio, CD31 per le cellule endoteliali) di interesse con i corrispondenti anticorpi (ad esempio, anti-CD31, clone MEC13.3). Fissare le sezioni con paraformaldeide; bloccarli con siero corrispondenza dell&#39;origine delle specie degli anticorpi secondari; incubmangiato con anticorpi primari per 12 ore a 4 ° C e quindi con anticorpi secondari per 2 h; e, infine, lavare con PBS e coprire con coprioggetto. Immagine i vetrini colorati con una Leica confocale in posizione verticale SP5 microscopio 13, 16. </li></ol></li></ol>

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The authors have no disclosure to make.

I passaggi critici all&#39;interno del protocollo: La qualità dei liposomi dual-marcato è la chiave per produrre risultati costanti per un lungo periodo di tempo. Coloranti fluorescenti liberi o 89 ioni Zr possono generare totalmente diversi modelli di targeting e devono essere completamente rimosso durante la fase di purificazione. Inoltre, se il sistema immunitario influisce significativamente sperimentale prestazioni cancro nanomedicina, l&#39;utilizzo di modelli di topo immunoc...

Nanomedicina si sta spostando il paradigma dello sviluppo trattamento del cancro 1. Ispirato al tremendo impatto clinico di precedenti nanomedicina cancro, come liposome- e nanotherapies albumina a base di 2, 3, molte nuove formulazioni sono state prodotte negli ultimi dieci anni. Tuttavia, recenti analisi del successo di traduzione clinica di questi nanomedicina cancro indicano che solo pochi di essi sono stati approvati per l&#39;uso clinico 4, 5. Uno dei principali ostacoli alla traduzione clinica di nuovi nanomedicina cancro è la loro limitata miglioramento dell&#39;indice terapeutico rispetto alla gestione diretta dei composti terapeutici liberi 6. Come tale, la valutazione accurata delle prestazioni in vivo di nanomedicine a sistemica, tessuti, e livello cellulare in modelli animali preclinici è essenziale per identify quelli con indici terapeutici ottimali per future Traduzione clinica. I nanomateriali possono essere radioattivo per la caratterizzazione quantitativa in animali che vivono con la tomografia ad emissione di positroni (PET), che ha superba sensibilità e riproducibilità tra tutte le modalità di imaging clinico 7. Ad esempio, 89 Zr-etichettato lungo circolanti nanomedicine sono stati caratterizzati in modelli murini di cancro 8, 9, 10, così come in altri modelli di malattia 11. Inoltre, il sangue emivita e biodistribuzione delle nanomedicine possono essere valutate utilizzando ex vivo misure della radioattività in tessuti 8. Pertanto, radiomarcatura consente la valutazione quantitativa delle nanomedicine a concentrazioni sistemiche e tissutali. È importante sottolineare che, radiolabeled nanomedicine generalmente non possono essere analizzate a singola cella o livelli subcellulari causa della limitata risoluzione spaziale del segnale radioattivo. Pertanto, marcatura fluorescente rivela essere una modalità complementare per la valutazione delle nanoparticelle con tecniche di imaging ottico come citometria di flusso e microscopia a fluorescenza 12. A tal fine, le nanoparticelle marcate con radioisotopi e tag fluorescenti possono essere quantitativamente valutati in vivo mediante imaging nucleare e ex vivo mediante conteggio della radioattività, e possono anche essere ampiamente caratterizzati a livello cellulare imaging ottico. In precedenza, abbiamo sviluppato procedure modulari per incorporare etichette fluorescenti radioattive e varie nanoparticelle, tra lipoproteine ad alta densità (HDL) 11, liposomi 9, 10, nanoparticelle polimeriche, frammenti di anticorpi, e nanoemulsions 10, 13. Queste nanoparticelle etichettati hanno consentito per la caratterizzazione quantitativa in modelli animali pertinenti a diversi livelli, che hanno guidato l&#39;ottimizzazione di questi nanomateriali per le loro applicazioni specifiche. Nel corso di studio, l&#39;obiettivo è quello di utilizzare le nanoparticelle-the liposomiali piattaforma di nanomedicina più affermati 14 -come un esempio per dimostrare le procedure complete per generare una nanoparticella dual-etichettati e per caratterizzare a fondo in un classico singenici melanoma B16-F10 modello di topo 15 . Dai risultati, siamo fiduciosi questo approccio caratterizzazione di nanoparticelle può essere adattato per valutare altre nanomedicine cancro in modelli murini rilevanti.

<table><tbody><tr><td>DPPC</td><td>Avantilipids</td><td>850355</td><td></td></tr><tr><td>Cholesterol</td><td>Sigma-Aldrich</td><td>C8667</td><td></td></tr><tr><td>DSPE-PEG2000</td><td>Avantilipids</td><td>880120P</td><td></td></tr><tr><td>DSPE-DFO</td><td>Home made</td><td>110634</td><td>Perez-Medina &lt;em&gt;et al.&lt;/em&gt;, JNM, 2014</td></tr><tr><td>DiIC12[5]-DS</td><td>AAT Bioquest</td><td>22051</td><td></td></tr><tr><td>Centrifugal filter</td><td>Vivaproducts</td><td>VS2061</td><td></td></tr><tr><td>Rotary evaporator</td><td>Buchi</td><td>R-100</td><td></td></tr><tr><td>Radio-HPLC</td><td>Shimadzu HPLC with 2 LC-10AT pumps</td><td>N/A</td><td></td></tr><tr><td>&lt;sup&gt;89&lt;/sup&gt;Zr-oxalate</td><td>MSKCC</td><td>Synthesized in house</td><td>TR19/9 variable beam cyclotron (Ebco Industries Inc.)</td></tr><tr><td>Micro PET-CT</td><td>Siemens</td><td>Inveon Micro-PET/CT</td><td></td></tr><tr><td>Gamma counter</td><td>PerkinElmer</td><td>2470-0150</td><td></td></tr><tr><td>Flow cytometry</td><td>BD Biosciences</td><td>Fortessa</td><td>Any multi-parametric flow cytometry analyzers would suffice</td></tr><tr><td>C57BL/6 mice</td><td>Jackson Laboratories</td><td></td><td></td></tr><tr><td>B16-YFP melanoma cells</td><td>Home made</td><td>N/A</td><td>Salmon &lt;em&gt;et al.&lt;/em&gt;, Immunity, 2016</td></tr><tr><td>Ly6C (clone HK1.4)--APC-Cy7</td><td>128025</td><td>Biolegend</td><td></td></tr><tr><td>MHCII (M5/114/152)--APC</td><td>107613</td><td>Biolegend</td><td></td></tr><tr><td>CD45 (30-F11)--BV510</td><td>103137</td><td>Biolegend</td><td></td></tr><tr><td>CD64 (X54-5/7.1)--PE-Cy7</td><td>139313</td><td>Biolegend</td><td></td></tr><tr><td>CD11b (M1/70)--BV605</td><td>101237</td><td>Biolegend</td><td></td></tr><tr><td>CD3 (17A2)--BV711</td><td>100241</td><td>Biolegend</td><td></td></tr><tr><td>CD31 (13.3)--PE</td><td>561073</td><td>Biolegend</td><td></td></tr><tr><td>CD11c (M418)--PerCP-Cy5.5</td><td>117327</td><td>BD Biosciences</td><td></td></tr><tr><td>CD31 (13.3) no fluorophore</td><td>550274</td><td>BD Biosciences</td><td></td></tr></tbody></table>

a comprehensive procedure to evaluate the in vivo performance of cancer nanomedicines

Ispirato dal successo delle precedenti nanomedicina cancro nella clinica, i ricercatori hanno generato un gran numero di nuove formulazioni negli ultimi dieci anni. Tuttavia, solo un piccolo numero di nanomedicine sono stati approvati per l&#39;uso clinico, mentre la maggior parte dei nanomedicine sotto sviluppo clinico hanno prodotto risultati deludenti. Uno dei principali ostacoli alla traduzione clinica di successo di nuovi nanomedicina cancro è la mancanza di una comprensione accurata delle loro prestazioni in vivo. Questo articolo presenta una procedura rigorosa per caratterizzare il comportamento in vivo della nanomedicina in topi portatori di tumore al sistemico, tessuti, cella singola, e livelli subcellulare tramite l&#39;integrazione di emissione di positroni tomografia tomografia-computerizzata (PET-CT), metodi di radioattività di quantificazione , citometria a flusso, e microscopia a fluorescenza. Usando questo approccio, i ricercatori possono valutare con precisione le formulazioni su scala nanometrica romanzo in modelli murini pertinenti cator. Questi protocolli possono avere la capacità di identificare i nanomedicine cancro più promettenti ad alto potenziale o traslazionale per aiutare nella ottimizzazione della nanomedicine cancro per la traduzione futuro.

La Figura 1 mostra una panoramica della procedura. Figura 2 presenta la procedura di sintesi schematica dei liposomi dual-marcato descritte nel passaggio 1 10. La figura 3 mostra un rappresentante PET-CT immagine (Figura 3a), la radioattività quantificazione da immagini PET (figura 3b), il sangue emivita (Figura 3c), e biodistribuzione (figura 3d)</strong...

Watch this Scientific Journal Video about A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines at JoVE.com

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

Una procedura completa per valutare il<em&gt; In Vivo</em&gt; Prestazioni del Cancro nanomedicina

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

a-comprehensive-procedure-to-evaluate-vivo-performance-cancer

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

8.8K Views.  Memorial Sloan Kettering Cancer Center. The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

Video: A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light.
To increase the therapeutic effect of PDT, we developed both photosensitizer- and anticancer agent-loaded lung cancer-targeted nanoparticles. Both enhanced permeability and retention (EPR) effect-based passive targeting and hyaluronic-acid-CD44 interaction-based active targeting were applied. CD44 is a well-known hyaluronic acid receptor that is often introduced as a biomarker of non-small cell lung cancer. 
In addition, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may increase anticancer therapeutic effects and reduce adverse reactions. 
We chose hypocrellin B (HB) as a novel photosensitizer in this study. It has been reported that HB causes higher anticancer efficacy of PDT compared to hematoporphyrin derivatives1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for lung cancer2. 
The antitumor efficacies of photosensitizer (HB) solution, photosensitizer encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT were compared both in vitro and in vivo. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
The HB-P-NP treatment group showed the most effective anticancer effect after PDT. In conclusion, the HB-P-NPs prepared in the present study represent a potential and novel photosensitizer delivery system in treating lung cancer with PDT.

Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

Anticancro Efficacia della terapia fotodinamica con cancro del polmone con targeting nanoparticelle

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

Ricerca

Ricerca sul cancro

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its therapeutic benefit. Nanoparticle-based drug combination has been shown to mitigate the problems encountered by free drug combination therapy. Our previous studies have shown that the combination of two anticancer drugs, doxorubicin (DOX) and mitomycin C (MMC), produced a synergistic effect against both murine and human breast cancer cells in vitro. DOX and MMC co-loaded polymer-lipid hybrid nanoparticles (DMPLN) bypassed various efflux transporter pumps that confer multidrug resistance and demonstrated enhanced efficacy in breast tumor models. Compared to conventional solution forms, such superior efficacy of DMPLN was attributed to the synchronized pharmacokinetics of DOX and MMC and increased intracellular drug bioavailability within tumor cells enabled by the nanocarrier PLN. 
To evaluate the pharmacokinetics and bio-distribution of co-administered DOX and MMC in both free solution and nanoparticle forms, a simple and efficient multi-drug analysis method using reverse-phase high performance liquid chromatography (HPLC) was developed. In contrast to previously reported methods that analyzed DOX or MMC individually in the plasma, this new HPLC method is able to simultaneously quantitate DOX, MMC and a major cardio-toxic DOX metabolite, doxorubicinol (DOXol), in various biological matrices (e.g., whole blood, breast tumor, and heart). A dual fluorescent and ultraviolet absorbent probe 4-methylumbelliferone (4-MU) was used as an internal standard (I.S.) for one-step detection of multiple drug analysis with different detection wavelengths. This method was successfully applied to determine the concentrations of DOX and MMC delivered by both nanoparticle and solution approaches in whole blood and various tissues in an orthotopic breast tumor murine model. The analytical method presented is a useful tool for pre-clinical analysis of nanoparticle-based delivery of drug combinations.

This protocol describes an efficient and convenient analytical process of sample extraction and simultaneous determination of multiple drugs, doxorubicin (DOX), mitomycin C (MMC) and a cardio-toxic DOX metabolite, doxorubicinol (DOXol), in the biological samples from a preclinical breast tumor model treated with nanoparticle formulations of synergistic drug combination.

Esempio di estrazione e quantificazione simultanea cromatografica di doxorubicina e mitomicina C successivi alla consegna combinazione farmaco in nanoparticelle topi del tumore-cuscinetto

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its ...

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Valutazione della captazione nanoparticelle nei tumori in tempo reale utilizzando intravitale Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Medicina

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 ...

Bioingegneria

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

Valutazione dell'attività antitumorale in vivo di nanoparticelle di polianidride IL-1α

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Valutare l&#39;efficacia delle Cancer Drug Sensibilizzazione<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

Validazione di Nanobody e anticorpi Based<em&gt; In Vivo</em&gt; Esperimenti tumore xenotrapianto NIRF imaging nei topi mediante<em&gt; Ex Vivo</em&gt; Citometria di flusso e microscopia

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

<meta charset="utf8"></head><body>Nanofarmaco mirato al miRNA-10b oncogenico per il trattamento del carcinoma mammario metastatico

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or &#34;nanodrug&#34;) administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

<meta charset="utf8"></head><body>Monitoraggio dell'accumulo di nanofarmaci nelle metastasi del carcinoma mammario murino

Here, we describe the protocol for in vivo delivery of magnetic iron oxide nanoparticles carrying RNA oligomers to metastatic breast cancer in animal models, providing a clinically viable approach for the therapeutic silencing of oncogenic nucleic acids.

Monitoraggio dell'accumulo di nanofarmaci nelle metastasi del carcinoma mammario murino

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have ...

Una procedura completa per valutare il

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Anticancro Efficacia della terapia fotodinamica con cancro del polmone con targeting nanoparticelle

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Nanoparticelle virali per

Valutazione dell'attività antitumorale in vivo di nanoparticelle di polianidride IL-1α

Valutare l'efficacia delle Cancer Drug Sensibilizzazione

Validazione di Nanobody e anticorpi Based

Monitoraggio dell'accumulo di nanofarmaci nelle metastasi del carcinoma mammario murino

Monitoraggio dell'accumulo di nanofarmaci nelle metastasi del carcinoma mammario murino

Monitoraggio dell'accumulo di nanofarmaci nelle metastasi del carcinoma mammario murino