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 estudo foi apoiado por nenhuma subvenção. 14-2014-017 do Fundo de Investigação SNUBH. Os autores agradecem a J. Patrick Barron, professor emérito da Universidade de Tóquio Médico e Professor Adjunto, Seoul National Bundang Hospital Universitário para a sua edição pro bono deste manuscrito.

NOTA: Todos os protocolos de estudo com animais foram aprovados pelo Comitê Cuidado e Uso Institucional animal de Seul Bundang National University Hospital (BA1308-134 / 072-01). 1. Síntese de ácido hialurônico-ceramida (HACE) <ol><li> Solubiliza-se 12,21 mmol de ácido hialurónico (HA) de oligómero e 9,77 mmol de tetra-n-butilamónio hidróxido (TBA) em 60 ml de água duplamente destilada (ADD). Agita-se durante 30 min. </li><li> Para sintetizar o ligante DS-Y30, dissolve-se 8,59 mmol de ceramida DS-Y30 e 9,45 mmol de trietilamina em 25 ml de tetra-hidrofurano (THF). Misturar com 8,59 mmol de cloreto de 4-clorometilbenzoilo em THF. Agita-se durante 6 h a 60 ° C. </li><li> Dissolve-se a 8,10 mmol sintetizado de HA-TBA e 0,41 mmol de ligante DS-Y30 em uma mistura de THF e acetonitrilo (4: 1, v / v). Agita-se durante 5 h a 40 ° C. </li><li> Remover as impurezas por filtração com um filtro de agente, e eliminar o solvente orgânico por evaporação sob vácuo. Purifica-se o produto usando uma membrana de diálise (peso molecular de corte: 3,5 kDa) e liofilizar. </li></ol> 2. Preparação das nanopartículas <ol><li> Dissolve-se 1 mg de HB e 1 mg de paclitaxel em 0,5 ml de sulfóxido de dimetilo (DMSO) e misturam-se com 0,5 ml de DDW por vortex para mistura durante 5 min. Em seguida, solubilizar HACE nessa mistura por vórtice de mistura durante mais 5 min. </li><li> Para eliminar o solvente, aquece-se a 70 ° C durante 4 horas sob uma corrente suave de azoto gasoso. </li><li> Ressuspender o filme composto por HACE, HB, e paclitaxel com 1 ml de DDW. Filtrar com um filtro de seringa (0,45 um de tamanho de poro) para remover droga não encapsulados. </li></ol> 3. A fototoxicidade in vitro <ol><li> A absorção de nanopartículas em linhas de células de câncer de pulmão <ol><li> Prepare (v / v) de soro de meio RPMI-1640 a 10% contendo meio de bovino fetal e 1% (w / v) de penicilina-estreptomicina. </li><li> Semente células A549 em placas de cultura celular de 24 poços a uma densidade de 1 x 10 5 células / poço (triplicados para cadagrupo). Incubar durante 24 horas a 37 ° C numa atmosfera humidificada com 5% de CO 2 e 95% de ar. </li><li> Após a fixação das células, remover o meio e lava-se as células por adição de 1 ml de solução salina tamponada com fosfato (PBS). </li><li> Dissolve-se as nanopartículas em PBS para uma concentração final de 2 jiM / ml HB. Em seguida, incubar as células com 1 ml de PBS, o NPS vazios, HB-NPs, ou HB-P-NPs em cada poço durante 4 horas no escuro. </li><li> Remove toda a solução e lava-se as células por adição de 1 ml de PBS frio. Repetir o passo de lavagem mais uma vez. Adicionar meio de cultura fresco. </li></ol></li><li> ensaio de viabilidade celular <ol><li> Colocar a placa de cultura celular sob a fibra PDT (com 1 cm de distância a partir da fibra TFD ao poço). Usar óculos de segurança do laser e iluminar as células com um laser PDT (630 nm, 400 mW / cm2), no escuro durante vários períodos de tempo: 0, 5, 10, 20, 30, e 40 seg (0, 2, 4 , 8, 12, e 16 J / cm 2). Em seguida, as células incubar durante 24 horas no escuro. </li><li>Aspirar o meio e lava-se as células por adição de 1 ml de PBS frio. Repetir o passo de lavagem mais uma vez. </li><li> Adicionar 10 ul de solução de medição da citotoxicidade de cada poço. Incubar no escuro durante 2 horas. </li><li> Medir a absorvância a 450 nm utilizando um leitor de microplacas. </li></ol></li><li> A análise microscópica <ol><li> Colocar a placa de cultura celular sob a fibra PDT (com 1 cm de distância a partir da fibra TFD ao poço). Use óculos de segurança a laser e iluminar as células com um laser PDT (630 nm, 400 mW / cm 2) no escuro, durante 0, 20 ou 40 segundos (0, 8, ou 16 J / cm 2). Em seguida, as células incubar durante 24 horas no escuro. </li><li> Aspirar o meio e lava-se as células por adição de 1 ml de PBS frio. Repetir o passo de lavagem mais uma vez. </li><li> Adicionar 50? L de anexina V-FITC e 50 ul de 4 &#39;, 6-diamidino-2-fenilindole (DAPI, 1,5 ug / ml). Agitar suavemente a placa e incubar-lo por 15 minutos à temperatura ambiente no escuro. NOTA: Use anexina V-FITC a partir do kit de microscópio de fluorescência. </li><li> Lavam-se as células por adição de 1 ml de PBS frio. Repetir o passo de lavagem mais uma vez. Manter as células em PBS fresco. Identificar as células em apoptose utilizando um microscópio óptico com ampliação de 100X. </li></ol></li><li> separação de células activadas por fluorescência (FACS) <ol><li> Colocar a placa de cultura celular sob a fibra PDT (com 1 cm de distância a partir da fibra TFD ao poço). Use óculos de segurança a laser e iluminar as células com um laser PDT (630 nm, 400 mW / cm 2) para 0, 20 ou 40 segundos (0, 8, ou 16 J / cm 2). Em seguida, as células incubar durante 24 horas no escuro. </li><li> Aspirar o meio e lava-se as células por adição de 1 ml de PBS frio. Repetir o passo de lavagem mais uma vez. </li><li> Ressuspender as células em 1 ml de 1 × tampão de ligação (diluído 1 parte de tampão de ligação 10x; 0,1 M de Hepes / NaOH (pH 7,4), NaCl 1,4 M, e 25 mM de CaCl 2 a 9 partes de água destilada) e transferência de 100 uL da solução de amostra para a 5 mltubo de cultura. </li><li> Adicionar 5 uL de anexina V-FITC e 5 ul de iodeto de propídio (PI). Suavemente vortex do tubo e incubar durante 15 min à temperatura ambiente (TA) no escuro. Adicionar 400 mL de 1 × tampão de ligação. NOTA: Use anexina V-FITC e PI a partir do kit microscópio de fluorescência. </li><li> Identificar as células apoptóticas por SCAF usando 14. As linhas de laser de excitação PI e anexina V-FITC são 488 nm e 635 nm, respectivamente. Medir a emissão de fluorescência de PI e anexina V-FITC a 610 ± 20 nm e 660 ± 20 nm, respectivamente. Recolher as células adquiridas no citômetro de fluxo por 10.000 eventos. </li></ol></li></ol> 4. eficácia anticancerígena in vivo In em ratos portadores de tumores <ol><li> modelo do rato induzida por câncer de pulmão <ol><li> Prepare 1 × 10 6 células A549 em 0,1 ml de meio RPMI-1640; mantê-lo em gelo. </li><li> Anestesiar os ratos com uma injecção IP de xilazina e uma uma mistura de tiletamina e zolazepam (1: 2, 1 ml /kg). Confirmar anesthetization adequada por beliscar suavemente uma pequena dobra de pele mouse. Use vet pomada sobre os olhos para evitar a secura e sob anestesia. NOTA: Se não se observa qualquer movimento, o animal é anestesiado suficientemente para iniciar as experiências. </li><li> Injectar as células subcutaneamente nos flancos de ratinhos BALB restantes ratinhos nu / C macho (6 - 7 semanas de idade, 20-22 g). </li><li> Mantenha observando os ratos até que eles começam a se mover ao redor da gaiola. NOTA: Não deixe um animal sem supervisão até que tenha recuperado a consciência suficiente para manter decúbito esternal. Não devolva um animal que foi submetido a uma cirurgia para a companhia de outros animais até que ele se recuperou totalmente. Mantenha os ratos sob condições específicas isentos de agentes patogénicos (SPF). </li><li> Medir o tamanho do tumor com compassos de calibre de cada dia. Calcular o volume do tumor (mm 3) como (comprimento x largura 2) / 2. Quando o tamanho do tumor atingir cerca de 200 mm 3 em volume, a iniciar a experiência. </li&gt; </ol></li><li> estudo de eficácia Anticancer <ol><li> Aleatoriamente dividir os ratos em 4 grupos (n = 10 para cada grupo). </li><li> Anestesiar os ratos com uma injecção IP de xilazina e uma uma mistura de tiletamina e zolazepam (1: 2, 1 ml / kg). Confirmar anesthetization adequada por beliscar suavemente uma pequena dobra de pele mouse. Use vet pomada sobre os olhos para evitar a secura e sob anestesia. NOTA: Se não se observa qualquer movimento, o animal é anestesiado suficientemente para iniciar as experiências. </li><li> Dissolve-se as nanopartículas em PBS para uma concentração final de 2 mg / ml HB. Injectar PBS, livre HB, HB-NPs, ou HB-P-PN através da veia caudal (2 mg / kg como HB) duas vezes nos dias 0 e 7. </li><li> Mantenha observando os ratos até que eles começam a se mover ao redor da gaiola. NOTA: Não deixe um animal sem supervisão até que tenha recuperado a consciência suficiente para manter decúbito esternal. Não devolva um animal que foi submetido a uma cirurgia para a companhia de outros animais até que esteja totalmenterecuperado. Mantenha as gaiolas escuro e em condições SPF específicos. </li><li> 24 h após cada injecção, anestesiar os ratos com uma injecção IP de xilazina e uma uma mistura de tiletamina e zolazepam (1: 2, 1 ml / kg). Confirmar anesthetization adequada por beliscar suavemente uma pequena dobra de pele mouse. Use vet pomada sobre os olhos para evitar a secura e sob anestesia. NOTA: Se não se observa qualquer movimento, o animal é anestesiado suficientemente para iniciar as experiências. </li><li> Colocar o sítio do tumor sob a fibra PDT (com 1 cm de distância a partir da fibra de PDT para o tumor). Use óculos de segurança laser, desligar o interruptor, e iluminar o tumor com um laser PDT (630 nm, 400 mW / cm 2) para 500 segundos (200 J / cm 2) duas vezes nos dias 1 e 8. </li><li> Mantenha observando os ratos até que eles começam a se mover ao redor da gaiola. Não deixe um animal sem supervisão até que tenha recuperado a consciência suficiente para manter decúbito esternal. Não devolva um animal que tem undergone cirurgia para a companhia de outros animais até que esteja totalmente recuperado. </li><li> Mantenha as gaiolas em condições SPF. Manter as gaiolas no escuro durante 24 horas após o tratamento a laser. </li><li> Monitorizar visualmente o volume do tumor e as alterações no local do tumor a cada dia. Medir o tamanho do tumor com compassos de calibre, e calcular o volume como (comprimento x largura 2) / 2 (mm 3). Tirar fotos dos locais de tumor todos os dias para verificar se há alterações de superfície do tumor após PDT. </li><li> No dia 16, sacrifica cinco ratinhos por grupo por anestesia terminal usando isoflurano. </li><li> Com uma pinça e tesouras, corte a pele exterior e expor os tumores 15. Cuidadosamente colhê-las. Corrigi-los em formol 10%, incorporá-los em parafina e manchá-las com hematoxilina e eosina (H &amp; E) para a análise histológica 16. </li><li> Após 45 dias de monitoramento, sacrificar os ratinhos restantes pela anestesia terminal usando isoflurano. </li></ol></li></ol>

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The authors have nothing to disclose.

O passo mais crítico neste estudo é selecionar as condições de laser adequados: comprimento de onda, poder e tempo de irradiação. O comprimento de onda de luz apropriado adequado para o fotossensibilizador específico é necessário para o PDT. Nós usamos um laser de 630 nm, que era apropriado para hipocrelina B. A potência de saída foi outro fator importante, que foi fixado em 400 mW / cm 2 com base em vários estudos-piloto. Potências de saída superior a 400 mW / cm 2 danificado as cé...

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.

Nós preparamos tanto HB-NPs e HB-P-NPs com as técnicas acima mencionadas. Os diâmetros médios de HB-NPs e HB-P-NPs foram 220,9 ± 3,2 nm e 211,9 ± 1,6 nm, respectivamente. A viabilidade celular das células A549 após 4 h de incubação com PBS, o NPS vazios, HB-PN, e HB-P-NPs seguido por irradiação de luz (0-16 J / cm 2) é mostrada na Figura 1. Sem luz, o grupo de tratamento HB-NP mostrou n...

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.

Anticancer eficácia da terapia fotodinâmica com Nanopartículas Lung Cancer segmentadas

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 visualizações.  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.

Vídeo: 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.

Um método completo para avaliar o<em&gt; In Vivo</em&gt; Performance of Cancer Nanomedicinas

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

Pesquisa do câncer

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 Fotodinâmica com Blended polímero condutor / Fullerene Nanoparticle Fotossensibilizadores

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;

Isolamento de fibroblastos associados ao câncer primário de um modelo de murina síndica do câncer de mama para o estudo de nanopartículas-alvo

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 fotodinâmica mediada por ácido 5-aminolevulínico em linhas celulares de tumor ósseo primário e metástases ósseas

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 fotodinâmica mediada por ácido 5-aminolevulínico em linhas celulares de tumor ósseo primário e metástases ósseas

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.

Citotóxicos Eficácia da Terapia Fotodinâmica no osteossarcoma Células<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.

Um dispositivo de terapia fotodinâmica interno construído internamente e baseado em diodo emissor de luz para melhorar a citotoxicidade da verteporfina em um modelo de cultura de células 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 Plasmônica Fototérmica do Câncer: Fantasmas que imitam o tecido tumoral embebido em nanopartículas para visualizar a distribuição fototérmica da 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.

Fotoimunoterapia infravermelha próxima para modelos de mouse de disseminação 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.

Ensaio de fototoxicidade in vitro: um método baseado em PDT para avaliar o potencial fototóxico de um fotossensibilizador em células de câncer de pulmão

Anticancer eficácia da terapia fotodinâmica com Nanopartículas Lung Cancer segmentadas

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