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

Cette étude a été soutenue par aucune subvention. 14-2014-017 du Fonds pour la recherche SNUBH. Les auteurs remercient J. Patrick Barron, professeur émérite, Université de médecine de Tokyo et professeur adjoint, Hôpital Bundang Université nationale de Séoul pour sa pro bono édition de ce manuscrit.

NOTE: Tous les protocoles d&#39;études animales ont été approuvés par le soin et l&#39;utilisation des animaux Comité institutionnel de l&#39;hôpital Bundang Université nationale de Séoul (BA1308-134 / 072-01). 1. Synthèse de l&#39;Acide Hyaluronique-céramide (HACE) <ol><li> Solubiliser 12,21 mmol d&#39;acide hyaluronique (HA) oligomère et 9,77 mmol de tétra n - butylammonium hydroxyde (TBA) dans 60 ml d&#39;eau distillée deux fois (DDW). On agite pendant 30 min. </li><li> Pour synthétiser l&#39;agent de liaison DS-Y30, on dissout 8,59 mmol de DS-Y30 céramide et 9,45 mmol de triéthylamine dans 25 ml de tétrahydrofuranne (THF). Mélanger avec 8,59 mmol de chlorure de 4-chlorométhylbenzoyle dans le THF. Agiter pendant 6 heures à 60 ° C. </li><li> On dissout le 8,10 mmol synthétisé de HA-TBA et 0,41 mmol de liaison DS-Y30 dans un mélange de THF et d&#39;acétonitrile (4: 1, v / v). Remuez pendant 5 heures à 40 ° C. </li><li> Éliminer les impuretés par filtration avec un agent de filtration et éliminer le solvant organique par évaporation sous vide. On purifie le produit en utilisant une membrane de dialyse (poids moléculaire de coupure: 3,5 kDa) et lyophiliser. </li></ol> 2. Préparation des nanoparticules <ol><li> Dissoudre 1 mg de HB et 1 mg de paclitaxel dans 0,5 ml de diméthylsulfoxyde (DMSO) et mélanger avec 0,5 ml de DDW par vortex le mélange pendant 5 min. Ensuite, solubiliser HACE en ce que le mélange par tourbillons engendrés par le mélange pendant encore 5 min. </li><li> Pour éliminer le solvant, la chaleur à 70 ° C pendant 4 heures sous un léger courant d&#39;azote gazeux. </li><li> Reprendre le film composé de HACE, HB, et le paclitaxel avec 1 ml de DDW. Filtrer avec un filtre à seringue (0,45 um de taille des pores) pour éliminer les médicaments non encapsulés. </li></ol> 3. In Vitro phototoxicité <ol><li> L&#39;absorption des nanoparticules dans des lignées cellulaires de cancer du poumon <ol><li> Préparer (v / v) de sérum de fœtus bovin RPMI-1640 contenant 10% de milieu et 1% (p / v) de pénicilline-streptomycine. </li><li> Cellules de pépins A549 dans des plaques à 24 puits de culture de cellules à une densité de 1 x 10 5 cellules / puits ( en triple pour chaquegroupe). Incuber pendant 24 heures à 37 ° C dans un CO 2 et 95% d&#39;une atmosphère d&#39;air humidifiée à 5%. </li><li> Après la fixation des cellules, éliminer le milieu et laver les cellules en ajoutant 1 ml de solution saline tamponnée au phosphate (PBS). </li><li> Dissoudre les nanoparticules dans du PBS à une concentration finale de 2 uM / ml de HB. Ensuite, incuber les cellules avec 1 ml de PBS, NPs vides, HB-NPs, ou HB-P-NPs dans chaque puits pendant 4 heures dans l&#39;obscurité. </li><li> Retirez tout de la solution et laver les cellules en ajoutant 1 ml de PBS froid. Répéter l&#39;étape de lavage une fois de plus. Ajouter milieu de culture frais. </li></ol></li><li> test de viabilité cellulaire <ol><li> Placer la plaque de culture cellulaire sous la fibre PDT (avec 1 cm de distance de la fibre PDT pour le bien). Porter des lunettes de sécurité laser et illuminer les cellules avec un laser PDT (630 nm, 400 mW / cm 2) dans l&#39;obscurité pendant diverses périodes de temps: 0, 5, 10, 20, 30, et 40 sec (0, 2, 4 , 8, 12 et 16 J / cm 2). Ensuite, incuber les cellules pendant 24 heures dans l&#39;obscurité. </li><li>Aspirer le milieu et laver les cellules en ajoutant 1 ml de PBS froid. Répéter l&#39;étape de lavage une fois de plus. </li><li> Ajouter 10 pi de solution cytotoxicité de mesure dans chaque puits. Incuber dans l&#39;obscurité pendant 2 heures. </li><li> Mesurer l&#39;absorbance à 450 nm en utilisant un lecteur de microplaques. </li></ol></li><li> L&#39;analyse microscopique <ol><li> Placer la plaque de culture cellulaire sous la fibre PDT (avec 1 cm de distance de la fibre PDT pour le bien). Porter des lunettes de sécurité laser et illuminer les cellules avec un laser PDT (630 nm, 400 mW / cm 2) dans l&#39;obscurité pendant 0, 20, ou 40 sec (0, 8, ou 16 J / cm 2). Ensuite, incuber les cellules pendant 24 heures dans l&#39;obscurité. </li><li> Aspirer le milieu et laver les cellules en ajoutant 1 ml de PBS froid. Répéter l&#39;étape de lavage une fois de plus. </li><li> Ajouter 50 pi d&#39;annexine V-FITC et 50 ul de 4 &#39;, 6-diamidino-2-phénylindole (DAPI, 1,5 pg / ml). Secouer doucement la plaque et incuber pendant 15 min à température ambiante dans l&#39;obscurité. REMARQUE: Utilisez annexine V-FITC du kit fluorescence microscope. </li><li> Laver les cellules en ajoutant 1 ml de PBS froid. Répéter l&#39;étape de lavage une fois de plus. Gardez les cellules dans du PBS frais. Identifier les cellules apoptotiques en utilisant la microscopie optique à grossissement 100X. </li></ol></li><li> tri Fluorescence cellulaire activé (FACS) <ol><li> Placer la plaque de culture cellulaire sous la fibre PDT (avec 1 cm de distance de la fibre PDT pour le bien). Porter des lunettes de sécurité laser et illuminer les cellules avec un laser PDT (630 nm, 400 mW / cm 2) pour 0, 20, ou 40 sec (0, 8, ou 16 J / cm 2). Ensuite, incuber les cellules pendant 24 heures dans l&#39;obscurité. </li><li> Aspirer le milieu et laver les cellules en ajoutant 1 ml de PBS froid. Répéter l&#39;étape de lavage une fois de plus. </li><li> Remettre en suspension les cellules dans 1 ml de 1 x tampon de liaison (dilution 1 partie du tampon de liaison 10x, 0,1 M HEPES / NaOH (pH 7,4), 1,4 M de NaCl et 25 mM de CaCl 2 à 9 parties d&#39; eau distillée) et transférer 100 pl de la solution d&#39;échantillon à une 5 mltube de culture. </li><li> Ajouter 5 pi d&#39;annexine V-FITC et 5 pi d&#39;iodure de propidium (PI). vortexer doucement le tube et incuber pendant 15 min à température ambiante (RT) dans l&#39;obscurité. Ajouter 400 pi de 1 x tampon de liaison. REMARQUE: Utilisez annexine V-FITC et PI à partir du kit de fluorescence au microscope. </li><li> Identifier les cellules apoptotiques en utilisant FACS 14. Les rayons laser d&#39;excitation et de l&#39;IP à l&#39;annexine V-FITC sont 488 nm et 635 nm, respectivement. Mesurer l&#39;émission de fluorescence de PI et de l&#39;annexine V-FITC à 610 ± 20 nm et 660 ± 20 nm, respectivement. Recueillir les cellules acquises sur le cytomètre de flux pour 10.000 événements. </li></ol></li></ol> 4. anticancéreuse in vivo l&#39; efficacité chez les souris porteuses de tumeurs <ol><li> modèle de souris induite par le cancer du poumon <ol><li> Préparer 1 × 10 6 cellules A549 dans 0,1 ml de milieu RPMI-1640; le garder dans la glace. </li><li> Anesthésier les souris avec une injection intrapéritonéale de xylazine et un mélange de tilétamine et zolazépam (1: 2, 1 ml /kg). Confirmez anesthetization appropriée en pinçant doucement un petit pli de la peau de la souris. Utilisez vétérinaire pommade sur les yeux pour prévenir la sécheresse sous anesthésie. NOTE: Si aucun mouvement est observé, l&#39;animal est suffisamment anesthésié pour commencer les expériences. </li><li> Injecter les cellules sous-cutanée dans les flancs gauche de BALB / C mâle souris nude (6 - 7 semaines, 20 - 22 g). </li><li> Gardez l&#39;observation de la souris jusqu&#39;à ce qu&#39;ils commencent à se déplacer autour de la cage. REMARQUE: Ne pas laisser un animal sans surveillance jusqu&#39;à ce qu&#39;il ait repris connaissance suffisante pour maintenir décubitus sternale. Ne retournez pas un animal qui a subi une intervention chirurgicale à la compagnie d&#39;autres animaux jusqu&#39;à ce qu&#39;il ait complètement récupéré. Gardez les souris sous (SPF) conditions exempts d&#39;agents pathogènes spécifiques. </li><li> Mesurer la taille de la tumeur avec étriers tous les jours. Calculer le volume de la tumeur (mm 3) (longueur x largeur 2) / 2. Lorsque la taille de la tumeur atteint environ 200 mm3 en volume, de commencer l&#39;expérience. </li&gt; </ol></li><li> Anticancer étude d&#39;efficacité <ol><li> Diviser aléatoirement les souris en 4 groupes (n = 10 pour chaque groupe). </li><li> Anesthésier les souris avec une injection ip d&#39;un xylazine et un mélange de tilétamine et zolazépam (1: 2, 1 ml / kg). Confirmez anesthetization appropriée en pinçant doucement un petit pli de la peau de la souris. Utilisez vétérinaire pommade sur les yeux pour prévenir la sécheresse sous anesthésie. NOTE: Si aucun mouvement est observé, l&#39;animal est suffisamment anesthésié pour commencer les expériences. </li><li> Dissoudre les nanoparticules dans du PBS à une concentration finale de 2 mg / ml de HB. Injecter PBS, libre HB, HB-NPs, ou HB-P-NPs via la veine de la queue (2 mg / kg comme HB) deux fois aux jours 0 et 7. </li><li> Gardez l&#39;observation de la souris jusqu&#39;à ce qu&#39;ils commencent à se déplacer autour de la cage. REMARQUE: Ne pas laisser un animal sans surveillance jusqu&#39;à ce qu&#39;il ait repris connaissance suffisante pour maintenir décubitus sternale. Ne retournez pas un animal qui a subi une intervention chirurgicale à la compagnie d&#39;autres animaux jusqu&#39;à leur entièrerétabli. Gardez les cages sombres et dans des conditions SPF spécifiques. </li><li> 24 heures après chaque injection, anesthésier les souris avec une injection intrapéritonéale de xylazine et un mélange de tilétamine et zolazépam (1: 2, 1 ml / kg). Confirmez anesthetization appropriée en pinçant doucement un petit pli de la peau de la souris. Utilisez vétérinaire pommade sur les yeux pour prévenir la sécheresse sous anesthésie. NOTE: Si aucun mouvement est observé, l&#39;animal est suffisamment anesthésié pour commencer les expériences. </li><li> Placer le site de la tumeur sous la fibre de TPD (1 cm de distance de la fibre TPD à la tumeur). Porter des lunettes de sécurité laser, éteindre l&#39;interrupteur, et illuminer la tumeur avec un laser PDT (630 nm, 400 mW / cm 2) pour 500 sec (200 J / cm 2) deux fois aux jours 1 et 8. </li><li> Gardez l&#39;observation de la souris jusqu&#39;à ce qu&#39;ils commencent à se déplacer autour de la cage. Ne pas laisser un animal sans surveillance jusqu&#39;à ce qu&#39;il ait repris connaissance suffisante pour maintenir décubitus sternale. Ne retournez pas un animal qui a undergonchirurgie de e à la compagnie d&#39;autres animaux jusqu&#39;à ce que complètement rétabli. </li><li> Gardez les cages dans des conditions SPF. Maintenir les cages dans l&#39;obscurité pendant 24 heures après le traitement au laser. </li><li> contrôler visuellement le volume de la tumeur et les changements au niveau du site de la tumeur chaque jour. Mesurer la taille de la tumeur avec des étriers, et calculer le volume que (longueur x largeur 2) / 2 (mm 3). Prenez des photos de sites tumoraux tous les jours pour vérifier les modifications de la surface de la tumeur après PDT. </li><li> Le jour 16, sacrifier cinq souris par groupe par l&#39;anesthésie terminaux utilisant l&#39;isoflurane. </li><li> Avec des pinces et des ciseaux, couper la peau extérieure et exposer les tumeurs 15. les récolter avec précaution. Les fixer dans 10% de formaline, les incorporer dans de la paraffine, et les tacher avec de l&#39; hématoxyline et de l&#39; éosine (H &amp; E) pour l&#39;analyse histologique 16. </li><li> Après 45 jours de surveillance, sacrifier les souris restantes par l&#39;anesthésie terminaux utilisant l&#39;isoflurane. </li></ol></li></ol>

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The authors have nothing to disclose.

L&#39;étape la plus critique dans cette étude est de choisir les conditions de laser appropriés: longueur d&#39;onde, la puissance et le temps d&#39;irradiation. La longueur d&#39;onde appropriée de lumière appropriée pour le photosensibilisateur spécifique est nécessaire pour la PDT. Nous avons utilisé un laser 630-nm qui était approprié pour hypocrelline B. La puissance de sortie est un autre facteur important, qui a été fixé à 400 mW / cm 2 sur la base de nombreuses études pilotes. Puissanc...

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.

Nous avons préparé deux HB-NPs et HB-P-NPs avec les techniques mentionnées ci-dessus. Les diamètres moyens de HB-NPs et HB-P-NPs étaient 220,9 ± 3,2 nm et 211,9 ± 1,6 nm, respectivement. La viabilité cellulaire des cellules A549 après 4 heures d&#39;incubation avec du PBS, NPs vides, HB-NPs et HB-P-NPs suivie d&#39; une irradiation de lumière (0 à 16 J / cm 2) est représentée sur la figure 1.</s...

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 efficacité de la thérapie photodynamique avec Lung Cancer-ciblée Nanoparticules

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 vues.  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.

Vidéo: 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.

Une procédure exhaustive pour évaluer la<em&gt; In Vivo</em&gt; Performance du Cancer nanomédicaments

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

Recherche

Recherche en cancérologie

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).

La thérapie photodynamique avec Blended polymère conducteur / Fullerene nanoparticules photosensibilisants

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

Chimie

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;

Isolement de fibroblastes primaires associés au cancer à partir d’un modèle murin syngénique du cancer du sein pour l’étude de nanoparticules ciblées

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>Thérapie photodynamique médiée par l’acide 5-aminolévulinique sur les lignées cellulaires de tumeurs osseuses primaires et de métastases osseuses

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.

Thérapie photodynamique médiée par l’acide 5-aminolévulinique sur les lignées cellulaires de tumeurs osseuses primaires et de métastases osseuses

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.

Cytotoxique efficacité de la thérapie photodynamique dans les cellules ostéosarcome<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 ...

Médecine

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.

Dispositif de thérapie photodynamique construit en interne et à base de diodes électroluminescentes pour améliorer la cytotoxicité des vertéporfines dans un modèle de culture cellulaire 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 ...

Biochimie

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.

Thérapie photothermique plasmonique contre le cancer : fantômes imitant les tissus tumoraux incorporés dans des nanoparticules pour visualiser la distribution photothermique de la température

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.

Photoimmunothérapie proche infrarouge pour des modèles murins de dissémination pleurale

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.

Essai de phototoxicité in vitro : une méthode basée sur la PDT pour évaluer le potentiel phototoxique d’un photosensibilisant dans les cellules cancéreuses du poumon

Anticancer efficacité de la thérapie photodynamique avec Lung Cancer-ciblée Nanoparticules

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