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

Este estudio fue apoyado por ninguna concesión. 14-2014-017 del Fondo de Investigación SNUBH. Los autores están en deuda con J. Patrick Barron, Profesor Emérito de la Universidad Médica de Tokio y Profesor Adjunto, Hospital Bundang Universidad Nacional de Seúl, por su pro bono edición de este manuscrito.

NOTA: Todos los protocolos de los estudios con animales fueron aprobados por el Comité de Cuidado y Uso de Animales institucional del Hospital Bundang Universidad Nacional de Seúl (BA1308-134 / 072-01). 1. Síntesis de ácido hialurónico-ceramida (ECA) <ol><li> Solubilizar 12,21 mmol de ácido hialurónico oligómero (HA) y 9,77 mmol de hidróxido de tetra- n -butilamonio (TBA) en 60 ml de agua doblemente destilada (DDW). Se agita durante 30 min. </li><li> Para sintetizar el enlazador DS-Y30, se disuelven 8,59 mmoles de ceramida DS-Y30 y 9.45 mmol de trietilamina en 25 ml de tetrahidrofurano (THF). Mezclar con 8,59 mmol de cloruro de 4-clorometilbenzoilo en THF. Se agita durante 6 horas a 60 ° C. </li><li> Se disuelve el 8,10 mmol sintetizado de HA-TBA y 0,41 mmol de enlazador DS-Y30 en una mezcla de THF y acetonitrilo (4: 1, v / v). Se agita durante 5 horas a 40 ° C. </li><li> Eliminar las impurezas mediante el filtrado con un agente de filtro, y eliminar el disolvente orgánico por evaporación a vacío. Se purifica el producto usando una membrana de diálisis (peso molecular de corte: 3,5 kDa) y se liofiliza. </li></ol> 2. Preparación de las nanopartículas <ol><li> Disolver 1 mg de HB y 1 mg de paclitaxel en 0,5 ml de sulfóxido de dimetilo (DMSO) y se mezclan con 0,5 ml de DDW por vórtice de mezcla durante 5 min. Entonces, solubilizar HACE en la mezcla por vórtice mezcla durante otros 5 min. </li><li> Para eliminar el disolvente, el calor a 70 ° C durante 4 h bajo una corriente suave de gas nitrógeno. </li><li> Resuspender la película compuesta de ECA, HB, y paclitaxel con 1 ml de DDW. Filtrar con un filtro de jeringa (0,45 micras de tamaño de poro) para eliminar los medicamentos no encapsulados. </li></ol> 3. En Vitro fototoxicidad <ol><li> La adopción de las nanopartículas en líneas celulares de cáncer de pulmón <ol><li> Preparar (v / v) de suero RPMI-1640 10% que contenía medio-bovino fetal y 1% (w / v) de penicilina-estreptomicina. </li><li> Células A549 semillas en placas de cultivo celular de 24 pocillos a una densidad de 1 x 10 5 células / pocillo (por triplicado para cadagrupo). Incubar durante 24 horas a 37 ° C en una atmósfera de aire de CO2 y 95% al 5% humidificado. </li><li> Después de la unión celular, se elimina el medio y se lavan las células mediante la adición de 1 ml de solución salina tamponada con fosfato (PBS). </li><li> Disolver las nanopartículas en PBS a una concentración final de 2 M / ml HB. A continuación, se incuban las células con 1 ml de PBS, NP vacías, HB-PN, o HB-P-NP en cada pocillo durante 4 horas en la oscuridad. </li><li> Quitar toda la solución y se lavan las células mediante la adición de 1 ml de PBS frío. Repetir la etapa de lavado una vez más. Añadir medio de cultivo fresco. </li></ol></li><li> ensayo de viabilidad celular <ol><li> Coloque la placa de cultivo celular debajo de la fibra TFD (con 1 cm de distancia de la fibra PDT para el bienestar). Use gafas de seguridad láser e iluminar las células con un láser PDT (630 nm, 400 mW / cm2) en la oscuridad durante varios períodos de tiempo: 0, 5, 10, 20, 30 y 40 segundos (0, 2, 4 , 8, 12, y 16 J / cm 2). A continuación, se incuban las células durante 24 horas en la oscuridad. </li><li>Aspirar el medio y lavar las células mediante la adición de 1 ml de PBS frío. Repetir la etapa de lavado una vez más. </li><li> Añadir 10 l de solución de medición citotoxicidad a cada pocillo. Incubar en la oscuridad durante 2 hr. </li><li> Medir la absorbancia a 450 nm usando un lector de microplacas. </li></ol></li><li> El análisis microscópico <ol><li> Coloque la placa de cultivo celular debajo de la fibra TFD (con 1 cm de distancia de la fibra PDT para el bienestar). Use gafas de seguridad láser e iluminar las células con un láser PDT (630 nm, 400 mW / cm2) en la oscuridad durante 0, 20, o 40 segundos (0, 8, o 16 J / cm2). A continuación, se incuban las células durante 24 horas en la oscuridad. </li><li> Aspirar el medio y lavar las células mediante la adición de 1 ml de PBS frío. Repetir la etapa de lavado una vez más. </li><li> Añadir 50 l de anexina V-FITC y 50 l de 4 &#39;, 6-diamidino-2-fenilindol (DAPI, 1,5 mg / ml). Agitar suavemente la placa y se incuba durante 15 minutos a temperatura ambiente en la oscuridad. NOTA: Utilice la anexina V-FITC del kit microscopio de fluorescencia. </li><li> Lavar las células mediante la adición de 1 ml de PBS frío. Repetir la etapa de lavado una vez más. Mantener las células en PBS fresco. Identificar las células apoptóticas utilizando un microscopio óptico a un aumento de 100X. </li></ol></li><li> clasificación de células activadas por fluorescencia análisis (FACS) <ol><li> Coloque la placa de cultivo celular debajo de la fibra TFD (con 1 cm de distancia de la fibra PDT para el bienestar). Use gafas de seguridad láser e iluminar las células con un láser PDT (630 nm, 400 mW / cm2) durante 0, 20, o 40 segundos (0, 8, o 16 J / cm2). A continuación, se incuban las células durante 24 horas en la oscuridad. </li><li> Aspirar el medio y lavar las células mediante la adición de 1 ml de PBS frío. Repetir la etapa de lavado una vez más. </li><li> Resuspender las células en 1 ml de tampón de unión 1 x (diluir 1 parte del tampón de unión 10x; 0,1 M Hepes / NaOH (pH 7,4), 1,4 M NaCl, y 25 mM CaCl 2 a 9 partes de agua destilada) y la transferencia de 100 l de la solución de muestra a una 5-mltubo de cultivo. </li><li> Añadir 5 l de anexina V-FITC y 5 l de yoduro de propidio (PI). vórtice suavemente el tubo y se incuba durante 15 min a temperatura ambiente (RT) en la oscuridad. Añadir 400 l de tampón de unión 1 ×. NOTA: Utilice la anexina V-FITC y PI del kit microscopio de fluorescencia. </li><li> Identificar las células apoptóticas mediante FACS 14. Las líneas de láser de excitación de PI y la anexina V-FITC son 488 nm y 635 nm, respectivamente. Medir la emisión de fluorescencia del PI y la anexina V-FITC a 610 ± 20 nm y 660 ± 20 nm, respectivamente. Recoger las células adquiridas en el citómetro de flujo por cada 10.000 eventos. </li></ol></li></ol> 4. Eficacia in vivo contra el cáncer en ratones portadores de tumores <ol><li> modelo de ratón inducida por el cáncer de pulmón <ol><li> Preparar 1 × 10 6 células A549 en 0,1 ml de medio RPMI-1640; mantenerlo en hielo. </li><li> Anestesiar a los ratones con una inyección ip de un xilazina y una mezcla de tiletamina y zolazepam (1: 2, 1 ml /kg). Confirmar la anestesia adecuada pellizcando suavemente un pequeño pliegue de piel de ratón. Utilice ungüento veterinario en los ojos para evitar la sequedad, mientras que bajo anestesia. NOTA: Si no se observa movimiento, el animal es suficientemente anestesiado para comenzar los experimentos. </li><li> Inyectar las células por vía subcutánea en los flancos izquierdo de / C macho ratones desnudos BALB (6 - 7 semanas de edad, 20 - 22 g). </li><li> Mantener la observación de los ratones hasta que empiezan a moverse alrededor de la jaula. NOTA: No deje un animal sin vigilancia hasta que se haya recuperado el conocimiento suficiente para mantener decúbito esternal. No devuelva un animal que ha sido sometido a una cirugía para la compañía de otros animales hasta que se haya recuperado por completo. Mantener a los ratones bajo condiciones libres de patógenos específicos (SPF). </li><li> Medir el tamaño del tumor con calibres cada día. Calcular el volumen del tumor (mm 3) como (longitud x anchura 2) / 2. Cuando el tamaño del tumor alcanza aproximadamente 200 mm 3 en volumen, iniciar el experimento. </li&gt; </ol></li><li> estudio de eficacia contra el cáncer <ol><li> Aleatoriamente dividir los ratones en 4 grupos (n = 10 para cada grupo). </li><li> Anestesiar a los ratones con una inyección ip de un xilazina y una mezcla de tiletamina y zolazepam (1: 2, 1 ml / kg). Confirmar la anestesia adecuada pellizcando suavemente un pequeño pliegue de piel de ratón. Utilice ungüento veterinario en los ojos para evitar la sequedad, mientras que bajo anestesia. NOTA: Si no se observa movimiento, el animal es suficientemente anestesiado para comenzar los experimentos. </li><li> Disolver las nanopartículas en PBS a una concentración final de 2 mg / ml HB. Se inyecta PBS, libre de HB, HB-PN, o HB-P-NP a través de la vena de la cola (2 mg / kg como HB) dos veces en los días 0 y 7. </li><li> Mantener la observación de los ratones hasta que empiezan a moverse alrededor de la jaula. NOTA: No deje un animal sin vigilancia hasta que se haya recuperado el conocimiento suficiente para mantener decúbito esternal. No devuelva un animal que ha sido sometido a una cirugía para la compañía de otros animales hasta su completarecuperado. Mantener las jaulas oscuridad y bajo condiciones SPF específicos. </li><li> 24 horas después de cada inyección, anestesiar a los ratones con una inyección ip de un xilazina y una mezcla de tiletamina y zolazepam (1: 2, 1 ml / kg). Confirmar la anestesia adecuada pellizcando suavemente un pequeño pliegue de piel de ratón. Utilice ungüento veterinario en los ojos para evitar la sequedad, mientras que bajo anestesia. NOTA: Si no se observa movimiento, el animal es suficientemente anestesiado para comenzar los experimentos. </li><li> Coloque el sitio del tumor bajo la fibra PDT (de 1 cm de distancia de la fibra PDT al tumor). Use gafas de seguridad láser, apague el interruptor, e iluminar el tumor con un láser PDT (630 nm, 400 mW / cm2) durante 500 seg (200 J / cm2) dos veces en los días 1 y 8. </li><li> Mantener la observación de los ratones hasta que empiezan a moverse alrededor de la jaula. No deje un animal sin vigilancia hasta que se haya recuperado el conocimiento suficiente para mantener decúbito esternal. No devuelva un animal que tiene undergone cirugía para la compañía de otros animales hasta que esté completamente recuperado. </li><li> Mantener las jaulas en condiciones SPF. Mantener las jaulas en la oscuridad durante 24 horas después de tratamiento con láser. </li><li> supervisar visualmente el volumen del tumor y los cambios en el sitio del tumor cada día. Medir el tamaño del tumor con calibradores, y calcular el volumen como (longitud x anchura 2) / 2 (mm 3). Tome fotografías de los sitios tumorales todos los días para verificar si hay alteraciones de la superficie del tumor después de la TFD. </li><li> En el día 16, sacrificar cinco ratones por grupo con anestesia terminales utilizando isoflurano. </li><li> Con pinzas y tijeras, corte la piel exterior y exponer los tumores 15. Cuidadosamente la cosecha. Fijarlos en formalina al 10%, incrustarlos en parafina, y teñirlos con hematoxilina y eosina (H &amp; E) para el análisis histológico 16. </li><li> Después de 45 días de seguimiento, sacrificar los ratones restantes por la anestesia terminales utilizando isoflurano. </li></ol></li></ol>

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The authors have nothing to disclose.

El paso más crítico en este estudio es la selección de las condiciones apropiadas láser: longitud de onda, potencia y tiempo de irradiación. La longitud de onda apropiada de luz adecuada para el fotosensibilizador específica es necesaria para la PDT. Se utilizó un láser de 630 nm que era apropiado para hipocrelina B. La potencia de salida era otro factor importante, que se fijó en 400 mW / cm2 sobre la base de muchos estudios piloto. Potencias de salida superior a 400 mW / cm2 dañadas las...

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.

Preparamos tanto HB-PN y HB-P-NP con las técnicas mencionadas anteriormente. Los diámetros medios de HB-NPs y HB-P-NP fueron 220,9 ± 3,2 nm y 211,9 ± 1,6 nm, respectivamente. La viabilidad celular de las células A549 después de 4 horas de incubación con PBS, NP vacías, HB-PN, y HB-P-NP seguido de irradiación de luz (de 0 a 16 J / cm2) se muestra en la Figura 1. Sin luz, la grupo de tratamie...

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.

Contra el cáncer eficacia de la terapia fotodinámica con nanopartículas orientada por cáncer de pulmón

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 vistas.  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.

Un procedimiento adecuado para evaluar la<em&gt; En Vivo</em&gt; Rendimiento de cáncer Nanomedicinas

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

Investigación

Investigación sobre el cáncer

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 fotodinámica con Blended polímero conductor / Fullerene nanopartículas fotosensibilizadores

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

Química

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;

Aislamiento de fibroblastos asociados al cáncer primario a partir de un modelo murino singénico de cáncer de mama para el estudio de nanopartículas dirigidas

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 fotodinámica mediada por ácido 5-aminolevulínico en líneas celulares de tumores óseos primarios y metástasis óseas

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 fotodinámica mediada por ácido 5-aminolevulínico en líneas celulares de tumores óseos primarios y metástasis óseas

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.

Citotóxica eficacia de la terapia fotodinámica en células de 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 de terapia fotodinámica construido internamente y basado en diodos emisores de luz para mejorar la citotoxicidad de la verteporina en un modelo de cultivo celular 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 ...

Bioquímica

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 fototérmica plasmónica contra el cáncer: fantasmas que imitan el tejido tumoral incrustados en nanopartículas para visualizar la distribución de la temperatura fototérmica

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.

Fotoinmunoterapia de infrarrojo cercano para modelos de ratón de diseminación pleural

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.

Contra el cáncer eficacia de la terapia fotodinámica con nanopartículas orientada por cáncer de pulmón

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