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

这项研究是由赠款没有支持。 14-2014-017从SNUBH研究基金。 该作者感谢J.帕特里克·巴伦，名誉教授，日本东京医科大学兼职教授，首尔国立大学盆唐医院这个手稿他的无偿编辑。

注:所有动物研究方案是由首尔国立大学盆唐医院的机构动物护理和使用委员会（BA1308-134 / 072-01）的批准。 1.玻尿酸，神经酰胺的合成（HACE） <ol><li>在60的双蒸水（DDW）毫升溶解12.21毫摩尔透明质酸（HA）寡聚物和9.77毫摩尔四- 正丁基氢氧化铵（TBA）的。搅拌30分钟。 </li><li>以合成的DS-Y30连接体，溶解8.59毫摩尔的DS-Y30神经酰胺和9.45毫摩尔的三乙胺在25毫升四氢呋喃（THF）。用8.59毫摩尔的THF 4-氯甲基苯甲酰氯混合。搅拌在60℃下6小时。 </li><li>溶解所合成的8.10毫摩尔的HA-TBA和0.41毫摩尔的DS-Y30接头中和THF的混合物中的乙腈（4:1，体积/体积）。搅拌在40℃5小时。 </li><li>通过与过滤剂过滤除去杂质，并消除由真空蒸发有机溶剂。净化用渗析膜产物（分子量截止:3.5 kDa的），并冷冻干燥。 </li></ol> 2.纳米颗粒的制备<ol><li>溶解1毫克的HB和在0.5ml二甲基亚砜（DMSO）的1毫克的紫杉醇和通过涡旋混合5分钟，用0.5毫升DDW的混合。然后，溶解HACE在通过涡旋混合该混合物另外5分钟。 </li><li>为了消除在氮气缓流在70℃下4小时的溶剂，热。 </li><li>悬浮脑水肿，HB组成的薄膜，紫杉醇用1毫升DDW的。用注射器过滤器（0.45微米孔径大小）过滤以除去未包封的药物。 </li></ol> 3. 在体外光毒性<ol><li>在肺癌细胞系中的纳米颗粒的摄取<ol><li>制备的RPMI-1640含10％中等（体积/体积）胎牛血清和1％（重量/体积）青霉素 - 链霉素。 </li><li>种子A549细胞在24孔细胞培养板中以1×10 5个细胞的密度/孔（一式三份对各组）。在湿润的5％CO 2和95％空气气氛中孵育在37℃下24小时。 </li><li>细胞附着后，除去培养基并加入1 ml磷酸盐缓冲盐水（PBS）洗细胞。 </li><li>溶解在PBS中，纳米颗粒为2μM/ ml的HB的终浓度。然后，孵育将细胞用1ml的PBS，空纳米颗粒，HB-纳米粒，或HB-P-纳米粒的各孔在黑暗中4小时。 </li><li>除去所有溶液，并通过加入1ml冷PBS洗细胞。重复洗涤步骤一次。添加新鲜的培养基。 </li></ol></li><li>细胞生存力测定法<ol><li>放置PDT纤维下的细胞培养板（以1厘米的距离从PDT光纤到孔）。戴激光安全眼镜，并与在黑暗中的不同时间一个PDT激光器（630纳米，400毫瓦/厘米2）照射细胞:0，5，10，20，30，和40秒（0，2，4 ，8，12，和16焦耳/厘米2）。然后，孵育所述细胞在黑暗中24小时。 </li><li>吸出培养基并加入1 ml冷PBS洗细胞。重复洗涤步骤一次。 </li><li>添加10微升的细胞毒性测量溶液每个孔中。孵育在黑暗中搅拌2小时。 </li><li>用酶标仪测量450nm处的吸光度。 </li></ol></li><li>显微分析<ol><li>放置PDT纤维下的细胞培养板（以1厘米的距离从PDT光纤到孔）。戴激光安全眼镜和照亮与在黑暗中的PDT激光器（630纳米，400毫瓦/厘米2）的细胞为0，20，或40秒（0，1,8或16焦耳/厘米2）。然后，孵育所述细胞在黑暗中24小时。 </li><li>吸出培养基并加入1 ml冷PBS洗细胞。重复洗涤步骤一次。 </li><li>添加膜联蛋白V-FITC的50微升和50微升4&#39;，6-二脒基-2-苯基吲哚（DAPI，1.5微克/毫升）的。轻轻摇动板和孵化它在黑暗中室温下15分钟。 注:使用膜联蛋白V-FITC从荧光显微镜套件。 </li><li>通过加入1ml冷PBS洗细胞。重复洗涤步骤一次。保持所述细胞在新鲜的PBS中。确定用光学显微镜放大100倍的凋亡细胞。 </li></ol></li><li>荧光激活细胞分选（FACS）分析<ol><li>放置PDT纤维下的细胞培养板（以1厘米的距离从PDT光纤到孔）。戴激光安全眼镜和照亮用PDT激光（630纳米，400毫瓦/厘米2）的细胞为0，20，或40秒（0，1,8或16焦耳/厘米2）。然后，孵育所述细胞在黑暗中24小时。 </li><li>吸出培养基并加入1 ml冷PBS洗细胞。重复洗涤步骤一次。 </li><li>悬浮细胞在1ml 1×结合缓冲液（稀释10倍的结合缓冲液的1份; 0.1M HEPES / NaOH水溶液（pH为7.4），1.4M的NaCl和25mM的氯化钙2至9份蒸馏水）和转让100微升样品溶液至5毫升培养管。 </li><li>添加膜联蛋白V-FITC的5和5μl碘化丙啶（PI）的。轻轻涡旋管孵育在黑暗中室温（RT）15分钟。添加400μl的1×结合缓冲液。 注:从荧光显微镜套件使用膜联蛋白V-FITC和PI。 </li><li>通过使用FACS 14识别凋亡细胞。 PI和膜联蛋白V-FITC的激发激光线分别是488纳米和635纳米。测量PI和膜联蛋白V-FITC的荧光发射在610±20纳米和660±20纳米，分别。收集每10,000事件流式细胞仪获得的细胞。 </li></ol></li></ol>在荷瘤小鼠4. 在体内的抗癌功效<ol><li>肺癌引起的小鼠模型<ol><li>制备1×10 6个 A549细胞在0.1ml RPMI-1640培养基;保持它在冰上。 </li><li>用甲苯噻嗪的腹腔注射和替来他明和唑拉西泮（1的混合物麻醉小鼠:2,1毫升/公斤）。轻轻捏小鼠皮肤小褶皱确认正确的麻醉。使用对眼睛兽医药膏，以防止干燥时的麻醉下。 注意:如果没有观察到运动，动物是充分麻醉开始实验。 </li><li>注入细胞皮下到BALB / c雄性裸鼠的左侧面（6 - 7周龄，20 - 22克）。 </li><li>保持观察老鼠，直到他们开始在笼子里移动。 注:直到它重新获得足够的意识，以保持胸骨斜卧不要离开无人看管的动物。不要返回已动过手术，以公司的其他动物，直到它已经完全康复的动物。保持无特定病原体（SPF）的条件下的小鼠。 </li><li>每天测量肿瘤大小与卡钳。计算肿瘤体积（毫米3）为（长×宽2）/ 2，当肿瘤大小在体积达到约200mm 3，开始实验。 </LI&gt; </ol></li><li>抗癌功效研究<ol><li>随机划分小鼠分成4组（n = 10每组）。 </li><li>麻醉小鼠用甲苯噻嗪的腹腔注射和替来他明和唑拉西泮的混合物（1:2,1毫升/公斤）。轻轻捏小鼠皮肤小褶皱确认正确的麻醉。使用对眼睛兽医药膏，以防止干燥时的麻醉下。 注意:如果没有观察到运动，动物是充分麻醉开始实验。 </li><li>溶解在PBS中，纳米颗粒为2毫克/毫升的HB的终浓度。通过尾静脉（2毫克/公斤为HB）第0天及7两次注射的PBS，无HB，HB-纳米粒，或HB-P-纳米粒。 </li><li>保持观察老鼠，直到他们开始在笼子里移动。 注:直到它重新获得足够的意识，以保持胸骨斜卧不要离开无人看管的动物。不要返回已动过手术公司其他动物直至完全的动物恢复。保持笼黑暗和具体的SPF条件下。 </li><li>每次注射后24小时，麻醉小鼠用甲苯噻嗪的腹腔注射和替来他明和唑拉西泮的混合物（1:2,1毫升/公斤）。轻轻捏小鼠皮肤小褶皱确认正确的麻醉。使用对眼睛兽医药膏，以防止干燥时的麻醉下。 注意:如果没有观察到运动，动物是充分麻醉开始实验。 </li><li>放置PDT纤维下的肿瘤部位（以1厘米的距离从PDT光纤到肿瘤）。戴激光安全眼镜，关掉开关，并用PDT激光（630纳米，400毫瓦/厘米2）500秒（200焦耳/厘米2）1和8天两次照射肿瘤。 </li><li>保持观察老鼠，直到他们开始在笼子里移动。无人看管，直到它已经恢复了足够的意识，以保持胸骨斜卧不要让动物。不要返回具有undergon动物Ë手术给公司其他动物，直到完全康复。 </li><li>保持SPF条件下的笼子里。保持在黑暗中的笼激光治疗后24小时。 </li><li>直观地监视肿瘤体积，并在每天肿瘤部位的变化。测量肿瘤大小用卡尺，并计算出体积为（长×宽2）/ 2（毫米3）。就拿肿瘤部位的照片，每天检查后PDT肿瘤表面的改变。 </li><li>在第16天，通过使用异氟烷终端麻醉牺牲每组五只小鼠。 </li><li>随着镊子和剪刀，剪开外皮和揭露肿瘤15。仔细收获他们。解决这些问题，在10％福尔马林，在石蜡中嵌入其中，和与用于组织学分析16苏木精和曙红（H＆E）染色它们。 </li><li>监测后45天，用异氟烷麻醉终端牺牲剩余的老鼠。 </li></ol></li></ol>

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The authors have nothing to disclose.

在这项研究中最关键的步骤是选择合适的激光条件:波长，功率和照射时间。适合于特定的光敏剂的光的适当波长是必要的PDT。我们使用了一个630纳米的激光，是适当的竹红菌素B的输出功率是另一个重要的因素，这是基于许多试验性研究设定在400毫瓦/厘米2。输出功率超过400毫瓦/厘米2受损细胞或动物的皮肤表面由于照射本身，而低于400毫瓦/厘米2的输出功率太弱，以显示?...

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.

我们制备的两个HB-NP和HB-P-纳米粒与上述技术。 HB-NP和HB-P-纳米粒子的平均粒径分别为220.9±3.2 nm和211.9±1.6纳米。 后用PBS，空纳米颗粒，HB-纳米粒，和HB-P-纳米粒孵育4小时，然后通过光照射（0至16焦耳/厘米2）A549细胞的细胞活力在图1中示出，如果没有光，则HB-NP治疗组无细胞毒性可言，而HB-P-NP-处理的细胞81....

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.

光动力疗法的抗癌功效肺癌靶向纳米粒子

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 次观看.  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.

视频: 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.

综合程序，以评估<em&gt;在体内</em&gt;癌症纳米医学的性能

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

科研

癌症研究

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).

光动力疗法与混合导电高分子/富勒烯纳米颗粒光敏剂

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

化学

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;

将原发性癌症相关纤维细胞与乳腺癌的合成素 Murine 模型分离，用于靶向纳米粒子的研究

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

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

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

<meta charset="utf8"></head><body>5-氨基乙酰丙酸介导的光动力疗法治疗原发性骨肿瘤和骨转移细胞系

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

5-氨基乙酰丙酸介导的光动力疗法治疗原发性骨肿瘤和骨转移细胞系

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.

光动力疗法治疗骨肉瘤细胞的细胞毒功效<em&gt;体外</em

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

医学

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.

一种内部构建的基于发光二极管的光动力治疗设备，用于增强维替泊芬在 2D 细胞培养模型中的细胞毒性

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 ...

生物化学

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.

等离子体光热癌症疗法：用于可视化光热温度分布的纳米颗粒包埋肿瘤组织模拟模型

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.

近红外光免疫疗法为小鼠模型的胸膜传播

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.

光动力疗法的抗癌功效肺癌靶向纳米粒子

摘要

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综合程序，以评估

光动力疗法与混合导电高分子/富勒烯纳米颗粒光敏剂

将原发性癌症相关纤维细胞与乳腺癌的合成素 Murine 模型分离，用于靶向纳米粒子的研究

5-氨基乙酰丙酸介导的光动力疗法治疗原发性骨肿瘤和骨转移细胞系

5-氨基乙酰丙酸介导的光动力疗法治疗原发性骨肿瘤和骨转移细胞系

光动力疗法治疗骨肉瘤细胞的细胞毒功效

一种内部构建的基于发光二极管的光动力治疗设备，用于增强维替泊芬在 2D 细胞培养模型中的细胞毒性

等离子体光热癌症疗法：用于可视化光热温度分布的纳米颗粒包埋肿瘤组织模拟模型

近红外光免疫疗法为小鼠模型的胸膜传播

体外光毒性测定:一种基于 PDT 的方法，用于评估光敏剂在肺癌细胞中的光毒性潜力