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

Diese Studie wurde von Zuschuss nicht unterstützt. 14-2014-017 von der SNUBH Research Fund. Die Autoren danken J. Patrick Barron, Professor Emeritus, Tokyo Medical University und Adjunct Professor, Seoul National University Bundang Krankenhaus für seine Pro - bono - Bearbeitung dieses Manuskript.

HINWEIS: Alle Tierstudie Protokolle, die von der Institutional Animal Care und Use Committee von der Seoul National University Bundang Krankenhaus genehmigt wurden (BA1308-134 / 072-01). 1. Synthese von Hyaluronsäure-Ceramid (HACE) <ol><li> Solubilisieren 12,21 mmol von Hyaluronsäure (HA) Oligomeren und 9,77 mmol Tetra- n -butylammonium Hydroxid (TBA) in 60 ml doppelt destilliertem Wasser (DDW). Rühre 30 min. </li><li> Um die DS-Y30-Linker synthetisiert, aufzulösen 8,59 mmol DS-Y30 Ceramid und 9,45 mmol Triethylamin in 25 ml Tetrahydrofuran (THF). Mix mit 8,59 mmol 4-Chlormethylbenzoylchlorid in THF. Rühre 6 Stunden bei 60 ° C. </li><li> Löse das synthetisierte 8.10 mmol HA-TBA und 0,41 mmol des DS-Y30 Linkers in einem Gemisch aus THF und Acetonitril (4: 1, v / v). Rühre 5 Stunden bei 40 ° C. </li><li> Entfernen von Verunreinigungen mit einem Filtermittel Filterung und Beseitigung des organischen Lösungsmittels durch Vakuumverdampfung. Reinige das Produkt eine Dialysemembran (Molekulargewichts-Cut-off: 3,5 kDa) und lyophilisieren. </li></ol> 2. Herstellung der Nanoteilchen <ol><li> Man löst 1 mg HB und 1 mg Paclitaxel in 0.5 ml Dimethylsulfoxid (DMSO) und Mischung mit 0,5 ml DDW durch Vortex-Mischen für 5 min. Dann solubilisieren HACE in diesem Gemisch durch Wirbelmischen für weitere 5 min. </li><li> Um das Lösungsmittel, Wärme bei 70 ° C für 4 Stunden unter einem schwachen Stickstoffgasstrom zu eliminieren. </li><li> Resuspendieren Film, der aus HACE, HB, und Paclitaxel mit 1 ml DDW. Filter mit einem Spritzenfilter (0,45 &amp; mgr; m Porengröße) zu offenen Drogen zu entfernen. </li></ol> 3. In - vitro - Phototoxizität <ol><li> Aufnahme von Nanopartikeln in Lungenkrebszelllinien <ol><li> Bereiten RPMI-1640-Medium enthaltenden 10% (v / v) fötalem Rinderserum und 1% (w / v) Penicillin-Streptomycin. </li><li> Seed A549 - Zellen in 24-well Zellkulturplatten in einer Dichte von 1 × 10 5 Zellen / Vertiefung (Triplikaten für jedeGruppe). In einem befeuchteten 5% CO 2 und 95% Luftatmosphäre bei 37 ° C für 24 Stunden inkubieren. </li><li> Nach Zellanheftung, um das Medium zu entfernen und die Zellen zu waschen, indem 1 ml phosphatgepufferter Kochsalzlösung (PBS). </li><li> Man löst die Nanopartikel in PBS auf eine Endkonzentration von 2 &amp; mgr; M / ml HB. Dann brüten die Zellen mit 1 ml PBS, leer NPs, HB-NPs oder HB-P-NPs in jeder Vertiefung für 4 Stunden im Dunkeln. </li><li> Entfernen all der Lösung und Waschen der Zellen durch Zugabe von 1 ml kaltem PBS. Wiederholen Sie den Waschschritt noch einmal. In frischem Kulturmedium. </li></ol></li><li> Die Lebensfähigkeit der Zellen-Assay <ol><li> Legen Sie die Zellkulturplatte unter der PDT-Faser (mit 1 cm Abstand von der PDT Faser zum Brunnen). Tragen Laserschutzbrille und beleuchten die Zellen mit einem PDT - Laser (630 nm, 400 mW / cm 2) im Dunkeln für verschiedene Zeiträume: 0, 5, 10, 20, 30 und 40 Sekunden (0, 2, 4 , 8, 12 und 16 J / cm 2). Dann brüten die Zellen 24 Stunden lang im Dunkeln. </li><li>Saugen Sie das Medium und waschen, indem die Zellen mit 1 ml kaltem PBS hinzufügen. Wiederholen Sie den Waschschritt noch einmal. </li><li> Fügen Sie 10 &amp; mgr; l der Zytotoxizität Messlösung in jede Vertiefung. Inkubieren im Dunkeln für 2 Stunden. </li><li> Messen Sie die Absorption bei 450 nm im Mikrotiterplatten-Reader. </li></ol></li><li> Die mikroskopische Analyse <ol><li> Legen Sie die Zellkulturplatte unter der PDT-Faser (mit 1 cm Abstand von der PDT Faser zum Brunnen). Tragen Laserschutzbrille und beleuchten die Zellen mit einem PDT - Laser (630 nm, 400 mW / cm 2) im Dunkeln für 0, 20 oder 40 Sekunden (0, 8, oder 16 J / cm 2). Dann brüten die Zellen 24 Stunden lang im Dunkeln. </li><li> Saugen Sie das Medium und waschen, indem die Zellen mit 1 ml kaltem PBS hinzufügen. Wiederholen Sie den Waschschritt noch einmal. </li><li> Je 50 ul von Annexin V-FITC und 50 ul 4 &#39;, 6-Diamidino-2-phenylindol (DAPI, 1,5 ug / ml). Die Platte vorsichtig schütteln und für 15 Minuten bei Raumtemperatur im Dunkeln inkubiert. HINWEIS: Verwenden Sie Annexin V-FITC aus dem Fluoreszenzmikroskop-Kit. </li><li> Waschen Sie die Zellen durch Zugabe von 1 ml kaltem PBS. Wiederholen Sie den Waschschritt noch einmal. Halten Sie die Zellen in frischem PBS. Identifizieren Sie die apoptotischen Zellen unter Verwendung von Lichtmikroskopie bei 100-facher Vergrößerung. </li></ol></li><li> Fluoreszenz-aktivierte Zellsortierung (FACS) -Analyse <ol><li> Legen Sie die Zellkulturplatte unter der PDT-Faser (mit 1 cm Abstand von der PDT Faser zum Brunnen). Tragen Laserschutzbrille und beleuchten die Zellen mit einem PDT - Laser (630 nm, 400 mW / cm 2) für 0, 20 oder 40 Sekunden (0, 8, oder 16 J / cm 2). Dann brüten die Zellen 24 Stunden lang im Dunkeln. </li><li> Saugen Sie das Medium und waschen, indem die Zellen mit 1 ml kaltem PBS hinzufügen. Wiederholen Sie den Waschschritt noch einmal. </li><li> Resuspendieren der Zellen in 1 ml 1 × Bindungspuffer (verdünnt 1 Teil des 10x Bindungspuffer, 0,1 M Hepes / NaOH (pH 7,4), 1,4 M NaCl, und 25 mM CaCl 2 bis 9 Teile destilliertes Wasser) und den Transfer 100 ul der Probenlösung auf eine 5-mlKulturröhrchen. </li><li> In 5 ul von Annexin V-FITC und 5 ul Propidiumiodid (PI). Ziehen Sie das Rohr verwirbeln und für 15 Minuten bei Raumtemperatur (RT) im Dunkeln inkubiert. In 400 ul 1 × Bindungspuffer. HINWEIS: Verwenden Sie Annexin mit V-FITC und PI aus dem Fluoreszenzmikroskop-Kit. </li><li> Identifizieren Sie die apoptotischen Zellen durch FACS unter Verwendung von 14. Die Anregungslaserlinien von PI und Annexin V-FITC sind 488 nm und 635 nm. Messen der Fluoreszenzemission von PI und Annexin V-FITC bei 610 ± 20 nm und 660 ± 20 nm. Sammeln Sie die erworbenen Zellen auf dem Durchflusszytometer pro 10.000 Veranstaltungen. </li></ol></li></ol> 4. In vivo Wirksamkeit gegen Krebs in Tumor-tragenden Mäusen <ol><li> Lungenkrebs-induzierte Mausmodell <ol><li> Vorbereitung 1 × 10 6 A549 - Zellen in 0,1 ml RPMI-1640 - Medium; halten Sie es in Eis. </li><li> Anästhesieren die Mäuse mit einer ip Injektion eines Xylazin und einer Mischung von Tiletamin und Zolazepam (1: 2, 1 ml /kg). Bestätigen Sie die richtige Betäubung durch sanft eine kleine Falte der Mäusehaut Kneifen. Verwenden Sie Tierarzt Salbe auf die Augen Trockenheit während der Narkose zu verhindern. HINWEIS: Wenn keine Bewegung festgestellt wird, ist das Tier ausreichend narkotisierten die Experimente zu starten. </li><li> Injizieren Sie die Zellen subkutan in die linken Flanken von Balb / C männliche Nacktmäuse (6 - 7 Wochen alt, 20 bis 22 g). </li><li> Halten Sie die Mäuse beobachtet, bis sie um den Käfig zu bewegen beginnen. HINWEIS: Verwenden Sie ein Tier nicht unbeaufsichtigt lassen, bis es genügend Bewusstsein wiedererlangt hat Brustlage zu halten. Nicht ein Tier zurück, die Operation an die Firma von anderen Tieren unterzogen wurde, bis sie vollständig erholt hat. Halten Sie die Mäuse unter spezifischen pathogenfreien (SPF) Bedingungen. </li><li> Messen Sie die Größe des Tumors mit täglich Sätteln. Berechnen Sie den Tumorvolumen (mm 3) als (Länge × Breite 2) / 2. Wenn die Tumorgröße etwa 200 mm 3 im Volumen erreicht, das Experiment zu starten. </li&gt; </ol></li><li> Anti-Krebs-Wirksamkeitsstudie <ol><li> Teilen Randomly die Mäuse in 4 Gruppen (n = 10 pro Gruppe). </li><li> Anästhesieren die Mäuse mit einer ip Injektion eines Xylazin und einer Mischung von Tiletamin und Zolazepam (1: 2, 1 ml / kg). Bestätigen Sie die richtige Betäubung durch sanft eine kleine Falte der Mäusehaut Kneifen. Verwenden Sie Tierarzt Salbe auf die Augen Trockenheit während der Narkose zu verhindern. HINWEIS: Wenn keine Bewegung festgestellt wird, ist das Tier ausreichend narkotisierten die Experimente zu starten. </li><li> Man löst die Nanopartikel in PBS auf eine Endkonzentration von 2 mg / ml HB. Injizieren PBS, frei HB, HB-NPs oder HB-P-NPs über die Schwanzvene (2 mg / kg als HB) zweimal an den Tagen 0 und 7. </li><li> Halten Sie die Mäuse beobachtet, bis sie um den Käfig zu bewegen beginnen. HINWEIS: Verwenden Sie ein Tier nicht unbeaufsichtigt lassen, bis es genügend Bewusstsein wiedererlangt hat Brustlage zu halten. Nicht ein Tier zurück, die Operation an die Firma von anderen Tieren, bis sie vollständig durchlaufen hatzurückgewonnen. Halten Sie die Käfige dunkel und unter bestimmten Bedingungen SPF. </li><li> 24 Stunden nach jeder Injektion anästhesieren die Mäuse mit einer ip Injektion eines Xylazin und einer Mischung von Tiletamin und Zolazepam (1: 2, 1 ml / kg). Bestätigen Sie die richtige Betäubung durch sanft eine kleine Falte der Mäusehaut Kneifen. Verwenden Sie Tierarzt Salbe auf die Augen Trockenheit während der Narkose zu verhindern. HINWEIS: Wenn keine Bewegung festgestellt wird, ist das Tier ausreichend narkotisierten die Experimente zu starten. </li><li> Platzieren Sie den Tumor unter der PDT-Faser (mit 1 cm Abstand von der PDT Faser auf den Tumor). Tragen Laserschutzbrille, drehen Sie den Schalter ab und beleuchten den Tumor mit einer PDT - Laser (630 nm, 400 mW / cm 2) für 500 sec (200 J / cm 2) zweimal an den Tagen 1 und 8. </li><li> Halten Sie die Mäuse beobachtet, bis sie um den Käfig zu bewegen beginnen. Verwenden Sie kein Tier unbeaufsichtigt lassen, bis es genügend Bewusstsein zu halten Brustlage wiedergewonnen hat. Nicht ein Tier zurück, die Renovierungsarbeite hate Chirurgie an der Gesellschaft von anderen Tieren, bis sie vollständig erholt. </li><li> Halten Sie die Käfige unter SPF Bedingungen. Pflegen Sie die Käfige im Dunkeln für 24 Stunden nach der Laserbehandlung. </li><li> Optisch überwachen jeden Tag das Tumorvolumen und die Änderungen an der Tumorstelle. Messen Sie die Größe des Tumors mit einer Schieblehre, und berechnen Sie die Lautstärke (Länge × Breite 2) / 2 (mm 3). Machen Sie Fotos von den Tumorstellen für Tumoroberflächenveränderungen nach der PDT jeden Tag überprüfen. </li><li> Am Tag 16, opfern fünf Mäuse pro Gruppe von Terminal Anästhesie mit Isofluran. </li><li> Mit einer Pinzette und Schere, schneiden Sie die Außenhaut und setzen die Tumore 15. Sorgfältig ernten. Fix sie in 10% Formalin, betten sie in Paraffin und färben sie mit Hämatoxylin und Eosin (H &amp; E) für die histologische Analyse 16. </li><li> Nach 45 Tagen der Überwachung, opfern, um die restlichen Mäuse durch Klemme Narkose mit Isofluran. </li></ol></li></ol>

<ol>
	<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.

Der wichtigste Schritt in dieser Studie ist die Auswahl der richtigen Laserbedingungen: Wellenlänge, Leistung und Bestrahlungszeit. Die richtige Wellenlänge des Lichts, geeignet für die spezifische Photosensibilisator für die PDT notwendig. Wir haben eine 630-nm - Laser, der für Hypokrellinverbindung B. Die Ausgangsleistung angemessen war , war ein weiterer wichtiger Faktor, der bei 400 mW / cm 2 basiert auf vielen Pilotstudien festgelegt wurde. Ausgangsleistungen von mehr als 400 mW / cm 2 , ...

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.

Wir bereiteten sowohl HB-NPs und HB-P-NPs mit den oben genannten Techniken. Die mittleren Durchmesser von HB-NPs und HB-P-NPs waren 220,9 ± 3,2 nm und 211,9 ± 1,6 nm. Die Lebensfähigkeit der Zellen von A549 - Zellen nach 4 h Inkubation mit PBS, leere NPs, HB-NPs und HB-P-NPs durch Bestrahlung mit Licht gefolgt (0 bis 16 J / cm 2) ist in Abbildung 1. Ohne Licht gezeigt, ist die HB-NP zeigte Behand...

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.

Anti-Krebs-Wirksamkeit der Photodynamischen Therapie mit Lungenkrebs-Targeted-Nanopartikel

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

anticancer-efficacy-photodynamic-therapy-with-lung-cancer-targeted

Research

JoVE Journal

Cancer Research

9.0K Aufrufe.  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.

Serie: 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.

Ein umfassendes Verfahren zur Bewertung der<em&gt; In Vivo</em&gt; Performance von Krebs Nanomedizin

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

Forschung

Krebsforschung

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).

Photodynamische Therapie mit Blended leitendes Polymer / Fullerene Nanopartikel Photosensibilisatoren

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

Chemie

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;

Isolierung primärer krebsseziensässiger Fibroblasten aus einem syngenen murinen Modell von Brustkrebs zur Untersuchung gezielter Nanopartikel

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>5-Aminolävulinsäure-vermittelte photodynamische Therapie an primären Knochentumor- und Knochenmetastasenzelllinien

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.

5-Aminolävulinsäure-vermittelte photodynamische Therapie an primären Knochentumor- und Knochenmetastasenzelllinien

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.

Zytotoxische Wirksamkeit von Photodynamische Therapie in Osteosarkomzellen<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 ...

Medizin

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.

Ein selbst gebautes photodynamisches Therapiegerät auf Basis von Leuchtdioden zur Verbesserung der Verteporfin-Zytotoxizität in einem 2D-Zellkulturmodell

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 ...

Biochemie

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.

Plasmonische photothermische Krebstherapie: Nanopartikel-eingebettete Tumorgewebe-imitierende Phantome zur Visualisierung der photothermischen Temperaturverteilung

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.

Nahinfrarot-Photoimmuntherapie für Mausmodelle der Pleuraverbreitung

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.

Anti-Krebs-Wirksamkeit der Photodynamischen Therapie mit Lungenkrebs-Targeted-Nanopartikel

Zusammenfassung

Weitere Videos entdecken

PLAYLIST