Name: anticancer efficacy of photodynamic therapy with lung cancer-targeted nanoparticles
Uploaded: 2016-12-01T13:00:00.000+00:00
Duration: 8 min 3 s
Description: Watch this Scientific Journal Video about Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles at JoVE.com

Questo studio è stato sostenuto dalla concessione n. 14-2014-017 dal Fondo di ricerca SNUBH. Gli autori sono in debito con J. Patrick Barron, professore emerito, Tokyo Medical University e Professore, Seoul National University Hospital Bundang per il suo pro bono la modifica di questo manoscritto.

NOTA: Tutti i protocolli di studio sugli animali sono stati approvati dal Comitato di Cura e uso istituzionale degli animali di Seoul National University Hospital Bundang (BA1308-134 / 072-01). 1. Sintesi di acido ialuronico-Ceramide (HACE) <ol><li> Solubilizzare 12.21 mmoli di acido ialuronico (HA) oligomero e 9.77 mmoli di tetra- idrossido n -butylammonium (TBA) in 60 ml di acqua bidistillata (DDW). Agitare per 30 min. </li><li> Per sintetizzare il linker DS-Y30 sciogliere 8.59 mmoli di DS-Y30 ceramide e 9.45 mmoli di trietilammina in 25 ml di tetraidrofurano (THF). Mescolare con 8.59 mmol di cloruro di 4-chloromethylbenzoyl in THF. Mescolare per 6 ore a 60 ° C. </li><li> Sciogliere il sintetizzato 8.10 mmoli di HA-TBA e 0,41 mmoli di DS-Y30 linker in una miscela di THF e acetonitrile (4: 1, v / v). Mescolare per 5 ore a 40 ° C. </li><li> Rimuovere impurità filtrando con un agente di filtro, ed eliminare il solvente organico mediante evaporazione sotto vuoto. Purificare il prodotto utilizzando una membrana di dialisi (peso molecolare cut-off: 3,5 kDa) e liofilizzato. </li></ol> 2. Preparazione delle nanoparticelle <ol><li> Sciogliere 1 mg di HB e 1 mg di paclitaxel in 0,5 ml di dimetilsolfossido (DMSO) e miscelare con 0,5 ml di DDW da vortex-miscelazione per 5 min. Poi, solubilizzazione HACE nella mischia dal vortice di miscelazione per un ulteriore 5 min. </li><li> Per eliminare il solvente, il calore a 70 ° C per 4 ore sotto leggera corrente di azoto. </li><li> Risospendere il film composto da HACE, HB, e paclitaxel con 1 ml di DDW. Filtrare con un filtro a siringa (0,45 micron dimensione dei pori) per rimuovere farmaci incapsulati. </li></ol> 3. In Vitro Fototossicità <ol><li> L&#39;assorbimento di nanoparticelle in linee cellulari di cancro al polmone <ol><li> Preparare RPMI-1640 (v / v) di siero fetale bovino medio contenente 10% e 1% (w / v) di penicillina-streptomicina. </li><li> Cellule Seed A549 in piastre di coltura cellulare di 24 pozzetti ad una densità di 1 × 10 5 cellule / pozzetto (triplicato per ognigruppo). Incubare per 24 ore a 37 ° C in un umidificata 5% aria atmosfera di CO 2 e il 95%. </li><li> Dopo l&#39;adesione cellulare, rimuovere il terreno e lavare le cellule aggiungendo 1 ml di soluzione salina tamponata con fosfato (PBS). </li><li> Sciogliere le nanoparticelle in PBS ad una concentrazione finale di 2 micron / ml HB. Poi, incubare le cellule con 1 ml di PBS, NP vuoti, HB-NP, o HB-P-NP in ciascun pozzetto per 4 ore al buio. </li><li> Rimuovere tutta la soluzione e lavare le cellule aggiungendo 1 ml di PBS freddo. Ripetere la fase di lavaggio, ancora una volta. Aggiungere mezzo di coltura fresco. </li></ol></li><li> saggio vitalità cellulare <ol><li> Posizionare la piastra di coltura cellulare sotto la fibra PDT (con 1 cm di distanza dalla fibra PDT al pozzo). Indossare occhiali di protezione laser e illuminare le cellule con un laser PDT (630 nm, 400 mW / cm 2) al buio per diversi periodi di tempo: 0, 5, 10, 20, 30, e 40 sec (0, 2, 4 , 8, 12, e 16 J / cm 2). Poi, incubare le cellule per 24 ore al buio. </li><li>Aspirare il medio e lavare le cellule con l&#39;aggiunta di 1 ml di PBS freddo. Ripetere la fase di lavaggio, ancora una volta. </li><li> Aggiungere 10 ml di soluzione di misura citotossicità in ciascun pozzetto. Incubare al buio per 2 ore. </li><li> Misurare l&#39;assorbanza a 450 nm usando un lettore di micropiastre. </li></ol></li><li> analisi microscopica <ol><li> Posizionare la piastra di coltura cellulare sotto la fibra PDT (con 1 cm di distanza dalla fibra PDT al pozzo). Indossare occhiali di protezione laser e illuminare le cellule con un laser PDT (630 nm, 400 mW / cm 2) al buio per 0, 20, o 40 sec (0, 8, o 16 J / cm 2). Poi, incubare le cellule per 24 ore al buio. </li><li> Aspirare il medio e lavare le cellule con l&#39;aggiunta di 1 ml di PBS freddo. Ripetere la fase di lavaggio, ancora una volta. </li><li> Aggiungere 50 ml di annessina V-FITC e 50 ml di 4 &#39;, 6-diamidino-2-fenilindolo (DAPI, 1,5 mg / ml). Agitare delicatamente la piastra e incubare per 15 min a temperatura ambiente al buio. NOTA: Utilizzare annessina V-FITC dal kit microscopio a fluorescenza. </li><li> Lavare le cellule con l&#39;aggiunta di 1 ml di PBS freddo. Ripetere la fase di lavaggio, ancora una volta. Mantenere le cellule in PBS fresco. Identificare le cellule apoptotiche mediante microscopia ottica a ingrandimento 100X. </li></ol></li><li> ordinamento fluorescenza delle cellule attivate (FACS) Analisi <ol><li> Posizionare la piastra di coltura cellulare sotto la fibra PDT (con 1 cm di distanza dalla fibra PDT al pozzo). Indossare occhiali di protezione laser e illuminare le cellule con un laser PDT (630 nm, 400 mW / cm 2) per 0, 20, o 40 sec (0, 8, o 16 J / cm 2). Poi, incubare le cellule per 24 ore al buio. </li><li> Aspirare il medio e lavare le cellule con l&#39;aggiunta di 1 ml di PBS freddo. Ripetere la fase di lavaggio, ancora una volta. </li><li> Risospendere le cellule in 1 ml di 1 × tampone di legame (diluire 1 parte di tampone legante 10x; 0,1 M Hepes / NaOH (pH 7,4), 1,4 M NaCl, e 25 mM CaCl 2 a 9 parti di acqua distillata) e trasferimento 100 microlitri della soluzione campione 5 mltubo di cultura. </li><li> Aggiungere 5 ml di annessina V-FITC e 5 ml di ioduro di propidio (PI). Agitare delicatamente la provetta e incubare per 15 minuti a temperatura ambiente (RT) al buio. Aggiungere 400 ml di 1 × tampone vincolante. NOTA: Utilizzare annessina V-FITC e PI dal kit microscopio a fluorescenza. </li><li> Identificare le cellule apoptotiche utilizzando FACS 14. Le linee laser di eccitazione di PI e annessina V-FITC sono 488 nm e 635 nm, rispettivamente. Misurare l&#39;emissione di fluorescenza di PI e annessina V-FITC a 610 ± 20 nm e 660 ± 20 nm, rispettivamente. Raccogliere le cellule acquisite sul citometro di flusso per 10.000 eventi. </li></ol></li></ol> 4. In vivo l&#39;efficacia antitumorale nei topi portatori di tumore <ol><li> Lung modello murino di cancro indotto <ol><li> Preparare 1 × 10 6 cellule A549 in 0,1 ml di mezzo RPMI-1640; tenerlo in ghiaccio. </li><li> Anestetizzare i topi con una iniezione ip di xilazina e una miscela di tiletamina e zolazepam (1: 2, 1 ml /kg). Verificare il corretto anestesia pizzicando delicatamente una piccola piega della pelle del mouse. Utilizzare veterinario pomata per gli occhi per prevenire la secchezza mentre sotto anestesia. NOTA: Se si osserva alcun movimento, l&#39;animale è sufficientemente anestetizzati per avviare gli esperimenti. </li><li> Iniettare le cellule per via sottocutanea nei fianchi sinistra del BALB / C maschile topi nudi (6 - 7 settimane di vita, 20 - 22 g). </li><li> Mantenere osservando il mouse fino a quando iniziano a muoversi intorno alla gabbia. NOTA: Non lasciare un animale incustodito fino a quando non ha ripreso conoscenza sufficiente a mantenere decubito sternale. Non restituire un animale che ha subito un intervento chirurgico per la compagnia di altri animali fino a quando non ha pienamente recuperato. Mantenere i topi in condizioni specifiche esenti da organismi patogeni (SPF). </li><li> Misurare la dimensione del tumore con pinze ogni giorno. Calcolare il volume del tumore (mm 3) come (lunghezza x larghezza 2) / 2. Quando la dimensione del tumore raggiunge circa 200 mm 3 di volume, iniziare l&#39;esperimento. </li&gt; </ol></li><li> studio di efficacia antitumorale <ol><li> Casualmente dividere i topi in 4 gruppi (n = 10 per ciascun gruppo). </li><li> Anestetizzare i topi con una iniezione ip di xilazina e una miscela di tiletamina e zolazepam (1: 2, 1 ml / kg). Verificare il corretto anestesia pizzicando delicatamente una piccola piega della pelle del mouse. Utilizzare veterinario pomata per gli occhi per prevenire la secchezza mentre sotto anestesia. NOTA: Se si osserva alcun movimento, l&#39;animale è sufficientemente anestetizzati per avviare gli esperimenti. </li><li> Sciogliere le nanoparticelle in PBS ad una concentrazione finale di 2 mg / ml HB. Iniettare PBS, libero HB, HB-NP, o HB-P-NP attraverso la vena della coda (2 mg / kg come HB) due volte nei giorni 0 e 7. </li><li> Mantenere osservando il mouse fino a quando iniziano a muoversi intorno alla gabbia. NOTA: Non lasciare un animale incustodito fino a quando non ha ripreso conoscenza sufficiente a mantenere decubito sternale. Non restituire un animale che ha subito un intervento chirurgico per la compagnia di altri animali fino al completorecuperato. Mantenere le gabbie buio e in condizioni SPF specifiche. </li><li> 24 ore dopo ciascuna iniezione, anestetizzare i topi con una iniezione ip di xilazina e una miscela di tiletamina e zolazepam (1: 2, 1 ml / kg). Verificare il corretto anestesia pizzicando delicatamente una piccola piega della pelle del mouse. Utilizzare veterinario pomata per gli occhi per prevenire la secchezza mentre sotto anestesia. NOTA: Se si osserva alcun movimento, l&#39;animale è sufficientemente anestetizzati per avviare gli esperimenti. </li><li> Posizionare il sito del tumore sotto della fibra PDT (di 1 cm di distanza dalla fibra PDT al tumore). Indossare occhiali di sicurezza laser, spegnere l&#39;interruttore, e illuminare il tumore con un laser PDT (630 nm, 400 mW / cm 2) per 500 sec (200 J / cm 2) due volte nei giorni 1 e 8. </li><li> Mantenere osservando il mouse fino a quando iniziano a muoversi intorno alla gabbia. Non lasciare un animale incustodito fino a quando non ha ripreso conoscenza sufficiente a mantenere decubito sternale. Non restituire un animale che ha oggetto d&#39;importanti lavorie un intervento chirurgico per la compagnia di altri animali fino alla completa guarigione. </li><li> Mantenere le gabbie in condizioni SPF. Mantenere le gabbie al buio per 24 ore dopo il trattamento laser. </li><li> Visivamente controllare il volume del tumore ed i cambiamenti al sito del tumore ogni giorno. Misurare la dimensione del tumore con pinze, e calcolare il volume (lunghezza x larghezza 2) / 2 (mm 3). Scattare foto dei siti tumorali ogni giorno per verificare la presenza di alterazioni della superficie del tumore dopo PDT. </li><li> Il giorno 16, sacrificare cinque topi per gruppo da anestesia terminali che utilizzano isoflurano. </li><li> Con pinze e forbici, tagliare la pelle esterna ed esporre i tumori 15. li raccolto con cura. Fissarli in formalina al 10%, incorporarli in paraffina, e li macchiare con ematossilina e eosina (H &amp; E) per l&#39;analisi istologica 16. </li><li> Dopo 45 giorni di monitoraggio, sacrificare i topi rimanenti anestesia terminali che utilizzano isoflurano. </li></ol></li></ol>

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	<li>Shang, L., Zhou, N., Gu, Y., Liu, F., Zeng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+on+killing+effect+of+esophageal+cancer+cell+line+between+hypocrellin+B-photodynamic+therapy+and+hematoporphyrin+derivative-photodynamic+therapy.">Comparative study on killing effect of esophageal cancer cell line between hypocrellin B-photodynamic therapy and hematoporphyrin derivative-photodynamic therapy.</a> Chin J Cancer Prev Treat. 12, 1139-1142 (2005).</li><li>Huisman, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paclitaxel+triggers+cell+death+primarily+via+caspase-independent+routes+in+the+non-small+cell+lung+cancer+cell+line+NCI-H460.">Paclitaxel triggers cell death primarily via caspase-independent routes in the non-small cell lung cancer cell line NCI-H460.</a> Clin Cancer Res. 8 (2), 596-606 (2002).</li><li>Dolmans, D. E., Fukumura, D., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+for+cancer.">Photodynamic therapy for cancer.</a> Nat Rev Cancer. 3 (5), 380-387 (2003).</li><li>Pass, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+in+oncology:+mechanisms+and+clinical+use.">Photodynamic therapy in oncology: mechanisms and clinical use.</a> J Natl Cancer Inst. 85 (6), 443-456 (1993).</li><li>Sutedja, T. G., Postmus, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+in+lung+cancer.+A+review+.">Photodynamic therapy in lung cancer. A review .</a> J. Photochem. Photobiol. 36 (2), 199-204 (1996).</li><li>Peng, C. L., Shieh, M. J., Tsai, M. H., Chang, C. C., Lai, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembled+star-shaped+chlorin-core+poly(epsilon-caprolactone)-poly(ethylene+glycol)+diblock+copolymer+micelles+for+dual+chemo-photodynamic+therapies.">Self-assembled star-shaped chlorin-core poly(epsilon-caprolactone)-poly(ethylene glycol) diblock copolymer micelles for dual chemo-photodynamic therapies.</a> Biomaterials. 29 (26), 3599-3608 (2008).</li><li>Chen, B., Pogue, B. W., Hasan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+delivery+of+photosensitising+agents.">Liposomal delivery of photosensitising agents.</a> Expert Opin Drug Deliv. 2 (3), 477-487 (2005).</li><li>Lee, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+photosensitizer+loaded+and+conjugated+glycol+chitosan+nanoparticles+for+cancer+therapy.">Comparative study of photosensitizer loaded and conjugated glycol chitosan nanoparticles for cancer therapy.</a> J Control Release. 152 (1), 21-29 (2011).</li><li>Chang, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticancer+efficacy+of+photodynamic+therapy+with+hematoporphyrin-modified,+doxorubicin-loaded+nanoparticles+in+liver+cancer.">Anticancer efficacy of photodynamic therapy with hematoporphyrin-modified, doxorubicin-loaded nanoparticles in liver cancer.</a> J. Photochem. Photobiol. 140, 49-56 (2014).</li><li>Chang, J. E., Cho, H. J., Yi, E., Kim, D. D., Jheon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypocrellin+B+and+paclitaxel-encapsulated+hyaluronic+acid-ceramide+nanoparticles+for+targeted+photodynamic+therapy+in+lung+cancer.">Hypocrellin B and paclitaxel-encapsulated hyaluronic acid-ceramide nanoparticles for targeted photodynamic therapy in lung cancer.</a> J. Photochem. Photobiol. 158, 113-121 (2016).</li><li>Wang, A. Z., Langer, R., Farokhzad, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+delivery+of+cancer+drugs.">Nanoparticle delivery of cancer drugs.</a> Annu. Rev. Med. 63, 185-198 (2012).</li><li>Matsumura, Y., Maeda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+concept+for+macromolecular+therapeutics+in+cancer+chemotherapy:+mechanism+of+tumoritropic+accumulation+of+proteins+and+the+antitumor+agent+smancs.">A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs.</a> Cancer Res. 46 (12 Pt 1), 6387-6392 (1986).</li><li>Penno, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+CD44+in+human+lung+tumors.">Expression of CD44 in human lung tumors.</a> Cancer Res. 54 (5), 1381-1387 (1994).</li><li>Wilkins, R., Kutzner, B., Truong, M., Sanchez-Dardon, J., McLean, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+radiation-induced+apoptosis+in+human+lymphocytes:+Flow+cytometry+using+Annexin+V+and+propidium+iodide+versus+the+neutral+comet+assay.">Analysis of radiation-induced apoptosis in human lymphocytes: Flow cytometry using Annexin V and propidium iodide versus the neutral comet assay.</a> Cytometry. 48 (1), 14-19 (2002).</li><li>Bannas, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+nanobody+and+antibody+based+in+vivo+tumor+xenograft+NIRF-imaging+experiments+in+mice+using+ex+vivo+flow+cytometry+and+microscopy.">Validation of nanobody and antibody based in vivo tumor xenograft NIRF-imaging experiments in mice using ex vivo flow cytometry and microscopy.</a> J Vis Exp. (98), e52462 (2015).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematoxylin+and+eosin+staining+of+tissue+and+cell+sections.">Hematoxylin and eosin staining of tissue and cell sections.</a> Cold Spring Harbor Protocols. (5), (2008).</li><li>Lam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+of+lung+cancer.">Photodynamic therapy of lung cancer.</a> Semin Oncol. 21 (6 Suppl 15), 15-19 (1994).</li><li>Simone, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+for+the+treatment+of+non-small+cell+lung+cancer.">Photodynamic therapy for the treatment of non-small cell lung cancer.</a> J Thorac Dis. 4 (1), 63-75 (2012).</li></ol>

The authors have nothing to disclose.

La fase più critica in questo studio sta selezionando le condizioni appropriate laser: lunghezza d&#39;onda, il potere, e il tempo di irradiazione. La corretta lunghezza d&#39;onda della luce adatta per il fotosensibilizzatore specifico è necessario per la PDT. Abbiamo utilizzato un laser a 630 nm, che era appropriato per hypocrellin B. La potenza di uscita è stato un altro fattore importante, che è stato fissato a 400 mW / cm 2 sulla base di molti studi pilota. Potenze di uscita superiore a 400 mW / cm <...

Photodynamic therapy (PDT) is composed of three major factors: photosensitizers, light, and oxygen. PDT is reported as a promising treatment for various cancers3. When the photosensitizers are administered into the cancer patient, they selectively accumulate in the tumor tissues. When the proper wavelength of light is applied, the highly reactive singlet oxygen and other free radicals lead to tumor cell damage4. 
Lung cancer was introduced as one of the first applications for PDT in the early 1980s5. PDT provides several advantages in treating lung cancer. Since PDT is a non-invasive and non-surgical treatment, it is an attractive alternative choice for the patients in whom surgical resection is inappropriate.
There have been many challenges to enhance the cancer-targeting efficacy of the photosensitizers. Increasing photosensitizer accumulation in cancer sites and decreasing accumulation in normal tissues are the identical goals for the cancer-targeting studies. A variety of targeted drug delivery systems, such as polymers, liposomes, and nanoparticles are adopted as photosensitizer carriers6-8. In our previous studies, nanoparticles effectively increased the cancer-targeting abilities of the photosensitizers9,10. Nanoparticles are ideal cancer-targeting carriers since they possess both passive and active targeting abilities. The leaky tumor vessels provide opportunity for nano-sized carriers to accumulate easily in tumors, which is well-known as the enhanced permeability and retention (EPR) effect11,12. The interaction between the nanoparticles and the specific receptors on cancer cells enables active cancer targeting. In this study, we prepared hyaluronic acid-based nanoparticles to interact with CD44, the major hyaluronic acid receptor that is overexpressed on lung cancer cells13. 
To maximize the anticancer efficacy, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may permit an increased therapeutic effect. Furthermore, decreased doses of both the photosensitizer and the anticancer drug can diminish adverse effects. We selected hypocrellin B (HB) as a novel photosensitizer in the present study. HB is isolated from Chinese medicinal fungus Hypocrella bambuase. Shang et al. reported that HB-based PDT possesses a higher anticancer efficacy when compared to hematoporphyrin derivative-based PDT1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for various cancers, including lung cancer2. 
Herein, we compared the anticancer efficacies of photosensitizer (hypocrellin B, 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. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.

<table><tbody><tr><td>oligo hyaluronic acid</td><td>Bioland Co., Ltd.</td><td>_</td><td></td></tr><tr><td>DS-Y30 (ceramide 3B; mainly &lt;em&gt;N&lt;/em&gt;-oleoyl-phytosphingosine)</td><td>Doosan Biotech Co., Ltd.</td><td>_</td><td></td></tr><tr><td>adipic acid dihydrazide</td><td>Sigma Aldrich</td><td>A0638</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-(3-dimethylaminopropyl)-&lt;em&gt;N&amp;prime;&lt;/em&gt;-ethylcarbodiimide</td><td>Sigma Aldrich</td><td>39391</td><td></td></tr><tr><td>4-(chloromethyl)benzoyl chloride</td><td>Sigma Aldrich</td><td>270784</td><td></td></tr><tr><td>Tween 80</td><td>Tokyo Chemical Industry Co., Ltd.</td><td>T0546</td><td></td></tr><tr><td>syringe filter</td><td>Sartorius Stedim Biotech GmbH</td><td>17762</td><td>15 mm, RC, PP, 0.45 &amp;micro;m</td></tr><tr><td>triethylamine</td><td>Sigma Aldrich</td><td>T0886</td><td></td></tr><tr><td>Mini-GeBAflex tubes</td><td>Gene Bio-Application Ltd.</td><td>D070-12-100</td><td></td></tr><tr><td>Paclitaxel</td><td>Taihua Corporations</td><td>_</td><td></td></tr><tr><td>RPMI-1640</td><td>Gibco Life Technologies, Inc.</td><td>11875</td><td></td></tr><tr><td>Penicillin&amp;ndash;streptomycin</td><td>Gibco Life Technologies, Inc.</td><td>15070</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco Life Technologies, Inc.</td><td>16140071</td><td></td></tr><tr><td>Celite (Filter agent)</td><td>Sigma Aldrich</td><td>6858</td><td>See step 1.4</td></tr></tbody></table>

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.

Abbiamo preparato sia HB-NP e HB-P-NP con le tecniche di cui sopra. I diametri medi di HB-NP e HB-P-NP erano 220,9 ± 3,2 nm e 211.9 ± 1.6 nm, rispettivamente. La vitalità cellulare delle cellule A549 dopo 4 ore di incubazione con PBS, NP vuoti, HB-NP e HB-P-NP seguito da irradiazione di luce (0 a 16 J / cm 2) è mostrato in Figura 1. Senza luce, la gruppo di trattamento HB-NP non ha mostrato cito...

Watch this Scientific Journal Video about Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles at JoVE.com

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

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. ...

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Research

JoVE Journal

Cancer Research

9.0K Views.  Seoul National University Bundang Hospital. 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.

Video: Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

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

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

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 ...

Ricerca

Ricerca sul cancro

Photodynamic Therapy with Blended Conducting Polymer/Fullerene Nanoparticle Photosensitizers

In this article a method for the fabrication and reproducible in-vitro evaluation of conducting polymer nanoparticles blended with fullerene as the next generation photosensitizers for Photodynamic Therapy (PDT) is reported. The nanoparticles are formed by hydrophobic interaction of the semiconducting polymer MEH-PPV (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene]) with the fullerene PCBM (phenyl-C61-butyric acid methyl ester) in the presence of a non-compatible solvent. MEH-PPV has a high extinction coefficient that leads to high rates of triplet formation, and efficient charge and energy transfer to the fullerene PCBM. The latter processes enhance the efficiency of the PDT system through fullerene assisted triplet and radical formation, and ultrafast deactivation of MEH-PPV excited stated. The results reported here show that this nanoparticle PDT sensitizing system is highly effective and shows unexpected specificity to cancer cell lines.

This protocol describes a method for the fabrication of conducting polymer nanoparticles blended with fullerene. These nanoparticles were investigated for their potential use as a next generation photosensitizers for Photodynamic Therapy (PDT).

Terapia fotodinamica con Blended Condurre Polimero / Fullerene nanoparticelle fotosensibilizzanti

In this article a method for the fabrication and reproducible in-vitro evaluation of conducting polymer nanoparticles blended with fullerene as the next ...

Chimica

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor cells, CAFs regulate tumor progression and provide protection from antitumor immunity. Emerging anticancer strategies aim to remodel the tumor microenvironment through the ablation of pro-tumorigenic CAFs or reprogramming of CAFs functions and their activation status. A promising approach is the development of nanosized delivery agents able to target CAFs, thus allowing the specific delivery of drugs and active molecules. In this context, a cellular model of CAFs may provide a useful tool for in vitro screening and preliminary investigation of such nanoformulations.
This study describes the isolation and culture of primary CAFs from the syngeneic 4T1 murine model of triple-negative breast cancer. Magnetic beads were used in a 2-step separation process to extract CAFs from dissociated tumors. Immunophenotyping control was performed using flow cytometry after each passage to verify the process yield. Isolated CAFs can be employed to study the targeting capability of different nanoformulations designed to tackle the tumor microenvironment. Fluorescently labeled H-ferritin nanocages were used as candidate nanoparticles to set up the method. Nanoparticles, either bare or conjugated with a targeting ligand, were analyzed for their binding to CAFs. The results suggest that ex vivo extraction of breast CAFs may be a useful system to test and validate nanoparticles for the specific targeting of tumorigenic CAFs.

This paper aims to provide a protocol for the isolation and culture of primary cancer-associated fibroblasts from a syngeneic murine model of triple-negative breast cancer and their application for the preclinical study of novel nanoparticles designed to target the tumor microenvironment.&#160;

Isolamento di fibroblasti primari associati al cancro da un modello murino singeneico di cancro al seno per lo studio di nanoparticelle mirate

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...

5-Aminolevulinic Acid-mediated Photodynamic Therapy on Primary Bone Tumor and Bone Metastases Cell Lines

Bone metastases are associated with poor prognosis and low quality of life for the affected patients. Photodynamic therapy (PDT) emerges as a noninvasive therapy that can target local metastatic bone lesions. This paper presents an in vitro method to study the PDT effect in adherent cell lines. To this end, we demonstrate a step-by-step approach to subject both primary (giant cell bone tumor) and human bone metastatic cancer cell lines (derived from a primary invasive ductal breast carcinoma and renal carcinoma) to 5-aminolevulinic acid (5-ALA)-mediated PDT. 
After 24 h post 5-ALA-PDT irradiation (blue light-wavelength 436 nm), the therapeutic effect was assessed in terms of cell migration potential, viability, apoptotic features, and cellular growth arrest (senescence). Post 5-ALA-PDT irradiation, musculoskeletal-derived cell lines respond differently to the same doses and exposure of PDT. Depending on the extent of cellular damage triggered by PDT exposure, two different cell fates-apoptosis and senescence were noted. Variable sensitivity to PDT therapy among different bone cancer cell lines provides useful information for selecting more appropriate PDT settings in clinical settings. This protocol is designed to exemplify the use of PDT in the context of musculoskeletal neoplastic cell lines. It may be adjusted to investigate the therapeutic effect of PDT on various cancer cell lines and various photosensitizers and light sources.

<meta charset="utf8"></head><body>Terapia fotodinamica mediata dall'acido 5-aminolevulinico su linee cellulari di tumore osseo primario e metastasi ossee

This protocol presents a step-by-step methodological approach of exposing bone metastatic cells lines and primary bone tumors to 5-aminolevulinic acid-mediated photodynamic therapy (PDT). The effects on cell migration potential/invasiveness, viability, apoptosis, and senescence potential are also analyzed following PDT exposure.

Terapia fotodinamica mediata dall'acido 5-aminolevulinico su linee cellulari di tumore osseo primario e metastasi ossee

Bone metastases are associated with poor prognosis and low quality of life for the affected patients. Photodynamic therapy (PDT) emerges as a noninvasive ...

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

Citotossico Efficacia della terapia fotodinamica in cellule di osteosarcoma<em&gt; In Vitro</em

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

Medicina

An In-House-Built and Light-Emitting-Diode-Based Photodynamic Therapy Device for Enhancing Verteporfin Cytotoxicity in a 2D Cell Culture Model

This paper describes a novel, simple, and low-cost device to perform in vitro photodynamic therapy (PDT) assays, named the PhotoACT. The device was built using a set of conventional programmable light-emitting diodes (LEDs), a liquid crystal display (LCD) module, and a light sensor connected to a commercial microcontroller board. The box-based structure of the prototype was made with medium-density fiberboards (MDFs). The internal compartment can simultaneously allocate four cell culture multiwell microplates.
As a proof of concept, we studied the cytotoxic effect of the photosensitizer (PS) verteporfin against the HeLa cell line in two-dimensional (2D) culture. HeLa cells were treated with increasing concentrations of verteporfin for 24 h. The drug-containing supernatant medium was discarded, the adherent cells were washed with phosphate-buffered saline (PBS), and drug-free medium was added. In this study, the effect of verteporfin on cells was examined either without light exposure or after exposure for 1 h to light using red-green-blue (RGB) values of 255, 255, and 255 (average fluence of 49.1 &#177; 0.6 J/cm2). After 24 h, the cell viability was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Experimental results showed that exposure of cells treated with verteporfin to the light from the device enhances the drug&#39;s cytotoxic effect via a mechanism mediated by reactive oxygen species (ROS). In addition, the use of the prototype described in this work was validated by comparing the results with a commercial PDT device. Thus, this LED-based photodynamic therapy prototype represents a good alternative for in vitro studies of PDT.

Here, we describe a novel, simple, and low-cost device to successfully perform in vitro photodynamic therapy (PDT) assays using two-dimensional HeLa cell culture and verteporfin as a photosensitizer.

Un dispositivo di terapia fotodinamica costruito internamente e basato su diodi emettitori di luce per migliorare la citotossicità delle verteporfine in un modello di coltura cellulare 2D

This paper describes a novel, simple, and low-cost device to perform in vitro photodynamic therapy (PDT) assays, named the PhotoACT. The device was built ...

Biochimica

Plasmonic Photothermal Cancer Therapy: Nanoparticle-embedded Tumor-tissue-mimicking Phantoms for Visualizing Photothermal Temperature Distribution

Plasmonic photothermal therapy (PPTT), an emerging cancer treatment, involves delivering nanoparticles (NPs) to a tumor, followed by near-infrared (NIR) irradiation to generate localized heat that destroys cancer cells. Before administering PPTT, the therapeutic parameters -- NP concentration, irradiation intensity, and duration -- need to be estimated. For this, numerical simulations are performed. However, to ensure robust computation, these simulations must be validated through photothermal experiments on tumor-tissue-mimicking phantoms replicating the optical properties of tumor tissue. For PPTT, therapeutic parameters are governed by the scattering and absorption of incident radiation by the tissue and NPs. Therefore, validation experiments can be conducted on phantoms mimicking the reduced scattering coefficient (&#181;s&#39;) and absorption coefficient (&#181;a) of the target tumor/tissue.
Specifically, this protocol provides instructions for preparing phantoms mimicking &#181;s&#39; and &#181;a of breast tumor injected with gold nanorods, surrounded by normal breast tissue. The protocol also details NIR irradiation, temperature monitoring, and validation of numerical results by comparing spatiotemporal temperatures with those measured using thermocouples. The protocols presented in this study facilitated the preparation of hydrogel-based cylindrical breast tumor-tissue phantoms with dimensions (&#981;40 x 12 mm) and a central tumor region (&#981;20 x 6 mm), comprising 1% agarose as the base matrix and intralipid as the scattering constituent and tumor region embedded with gold nanorods at 25 &#181;g/mL concentration. Representative results from a case study illustrate the application of fabricated phantoms for validating numerical simulations for PPTT. The study concludes that the demonstrated protocols are valuable for conducting photothermal experiments aimed at optimizing and planning therapeutic parameters prior to in vivo experiments and validating numerical simulations for PPTT.

This article presents the protocol for preparing tumor-tissue phantoms that replicate optical properties for plasmonic photothermal therapy. It details phantom preparation, photothermal evaluations, and validation of the developed numerical model based on photothermal temperature measurements for assessing therapeutic parameters, offering an ethical, cost-effective alternative to in vivo studies for preliminary testing.

Terapia plasmonica fototermica del cancro: fantocci che imitano il tessuto tumorale inclusi in nanoparticelle per la visualizzazione della distribuzione fototermica della temperatura

Plasmonic photothermal therapy (PPTT), an emerging cancer treatment, involves delivering nanoparticles (NPs) to a tumor, followed by near-infrared (NIR) ...

Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination model can be used for the evaluation of treatment methods for intrathoracic diseases such as lung cancer or malignant pleural mesothelioma.
Near-infrared photoimmunotherapy (NIR-PIT) is a recently developed cancer treatment strategy that combines the specificity of tumor-targeting antibodies with toxicity caused by a photoabsorber (IR700Dye) after exposure to NIR light. The efficacy of NIR-PIT has been reported using various antibodies; however, only a few reports have shown the therapeutic effect of this strategy in an orthotopic model. In the present study, we demonstrate an example of efficacy evaluation of the pleural disseminated lung cancer model, which was treated using NIR-PIT.

Near-infrared photoimmunotherapy (NIR-PIT) is an emerging cancer therapeutic strategy that utilizes an antibody-photoabsorber (IR700Dye) conjugate and NIR light to destroy cancer cells. Here, we present a method to evaluate the antitumor effect of NIR-PIT in a mouse model of pleural disseminated lung cancer and malignant pleural mesothelioma using bioluminescence imaging.

Fotoimmunoterapia nel vicino infrarosso per modelli murini di disseminazione pleurica

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination ...

In Vitro Phototoxicity Assay: A PDT-based Method to Evaluate the Phototoxic Potential of a Photosensitizer in Lung Cancer Cells

Source: Chang, J. E., et al. <a href="https://www.jove.com/v/54865/anticancer-efficacy-photodynamic-therapy-with-lung-cancer-targeted">Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles.</a>&#160;J. Vis. Exp.&#160;(2016).
Photodynamic therapy (PDT) is a non-invasive and non-surgical method 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. In this protocol video, PDT-based method is used to evaluate the phototoxic potential of a potential photosensitizer in lung cancer cells.

Saggio di fototossicità in vitro: un metodo basato sulla PDT per valutare il potenziale fototossico di un fotosensibilizzatore nelle cellule di cancro del polmone

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

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