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

この研究は、助成金なしによってサポートされていました。 SNUBH研究基金から14-2014-017。 著者は、この原稿の彼のプロボノ編集のためにJ.パトリック・バロン、名誉教授、東京大学医科大学と非常勤講師、盆唐ソウル大病院にお世話になっています。

注：すべての動物実験プロトコルは、盆唐ソウル大病院（BA1308-134 / 072から01）の施設内動物管理使用委員会によって承認されました。 ヒアルロン酸セラミドの1の合成（HACE） <ol><li>二重蒸留水（DDW）の60ミリリットルでヒアルロン酸の12.21ミリモル（HA）オリゴマー及びテトラn個の -butylammonium水酸化物（TBA）の9.77ミリモルを可溶化します。 30分間撹拌しました。 </li><li> DS-Y30リンカーを合成するために、テトラヒドロフラン25ミリリットル（THF）中のDS-Y30セラミドの8.59ミリモルおよびトリエチルアミン9.45ミリモルを溶解します。 THF中の4-クロロメチルベンゾイルクロリドの8.59ミリモルと混合。 60℃で6時間撹拌します。 </li><li> THFの混合物及びアセトニトリルでDS-Y30リンカーの合成されたHA-TBAの8.10ミリモル及び0.41ミリモル溶解（4：1、v / v）でした。 40℃で5時間撹拌します。 </li><li>フィルター剤で濾過することにより不純物を除去し、真空蒸着法により、有機溶媒を除去します。 （透析膜を使用して生成物を精製分子量カットオフ：3.5キロダルトン）および凍結乾燥。 </li></ol>ナノ粒子の作製<ol><li> HB 1mgのジメチルスルホキシド（DMSO）0.5mlのパクリタキセル1mgを溶解し、ボルテックス混合し、5分間によってDDW 0.5mlでブレンド。その後、さらに5分間ボルテックス混合することにより、その混合物中のHACEを可溶化します。 </li><li>窒素ガスの穏やかな流れの下で4時間、70℃で溶剤、熱を除去するために。 </li><li> DDW 1mlでHACE、HBからなる膜、及びパクリタキセルを再懸濁します。封入されていない薬物を除去するために、シリンジフィルター（孔径0.45μm）でフィルタします。 </li></ol> 3. インビトロ光毒性<ol><li>肺癌細胞株中のナノ粒子の取り込み<ol><li> RPMI-1640培地を含む10％の（v / v）のウシ胎児血清および1％（w / v）のペニシリン - ストレプトマイシンを準備します。 </li><li> 1×10 5細胞/ウェルの各々について（三連の密度で24ウェル細胞培養プレート中の種子A549細胞グループ）。加湿5％CO 2及び95％空気雰囲気中37℃で24時間インキュベートします。 </li><li>細胞付着後に、培地を除去し、リン酸緩衝生理食塩水（PBS）を1ml添加して細胞を洗浄します。 </li><li> 2μM/ mlのHBの最終濃度になるようにPBS中のナノ粒子を溶解させます。その後、暗所で4時間、各ウェルにPBS、空のNP、HB-のNP、またはHB-P-NPの1ミリリットルで細胞を培養します。 </li><li>ソリューションのすべてを削除し、冷PBSの1ミリリットルを追加することによって、細胞を洗浄。もう一度洗浄工程を繰り返します。新鮮な培地を追加します。 </li></ol></li><li>細胞生存率アッセイ<ol><li> （ウェルにPDTファイバからの距離から1cmで）PDT繊維の下で細胞培養プレートを置きます。レーザー安全メガネを着用し、種々の時間暗所でPDTレーザー（630 nmで、400ミリワット/ cm 2）を用いて細胞を照らす：0、5、10、20、30、および40秒（0、2、4 、8、12、16 J / cm 2）でした。その後、暗所で24時間細胞をインキュベートします。 </li><li>培地を吸引し、冷PBSを1mlを加えることによって細胞を洗浄します。もう一度洗浄工程を繰り返します。 </li><li>各ウェルに細胞毒性測定溶液10μlを加えます。 2時間暗所でインキュベートします。 </li><li>マイクロプレートリーダーを用いて450nmで吸光度を測定します。 </li></ol></li><li>顕微分析<ol><li> （ウェルにPDTファイバからの距離から1cmで）PDT繊維の下で細胞培養プレートを置きます。レーザー安全メガネを着用し、0、20、または40秒（0、8、または16 J / cm 2）を、暗所でのPDTレーザー（630 nmで、400ミリワット/ cm 2）を用いて細胞を照らします。その後、暗所で24時間細胞をインキュベートします。 </li><li>培地を吸引し、冷PBSを1mlを加えることによって細胞を洗浄します。もう一度洗浄工程を繰り返します。 </li><li>アネキシンV-FITCを50μlと4 &#39;、6-ジアミジノ-2-フェニルインドール（DAPI、/ mlの1.5μgの）の50μLを加えます。静かにプレートを振る、暗所で室温で15分間静置します。 注：アネキシンV-FIを使用してください蛍光顕微鏡キットからTC。 </li><li>冷PBSの1ミリリットルを追加することによって、細胞を洗浄。もう一度洗浄工程を繰り返します。新鮮なPBSで細胞を保管してください。 100Xの倍率で光学顕微鏡を用いて、アポトーシス細胞を識別します。 </li></ol></li><li> （FACS）分析、蛍光活性化細胞選別<ol><li> （ウェルにPDTファイバからの距離から1cmで）PDT繊維の下で細胞培養プレートを置きます。レーザー安全メガネを着用し、0、20、または40秒（0、8、または16 J / cm 2）をするためのPDTレーザー（630 nmで、400ミリワット/ cm 2）を用いて細胞を照らします。その後、暗所で24時間細胞をインキュベートします。 </li><li>培地を吸引し、冷PBSを1mlを加えることによって細胞を洗浄します。もう一度洗浄工程を繰り返します。 </li><li>転送100μlを、1×結合緩衝液1mlに細胞を再懸濁（0.1MのHepes / NaOHで（pHは7.4）、1.4 MのNaCl、および25mMのCaCl 2 9重量部を蒸留水10×結合緩衝液の1部を希釈） 5ミリリットルの試料溶液の培養管。 </li><li>アネキシンV-FITCおよびヨウ化プロピジウム（PI）を5μlの5μlを添加します。静かにチューブをボルテックスし、暗所で室温（RT）で15分間インキュベートします。 1×結合緩衝液の400μlのを追加します。 注：蛍光顕微鏡キットから、アネキシンV-FITCおよびPIを使用してください。 </li><li> FACS 14を使用して、アポトーシス細胞を識別します。 PIおよびアネキシンV-FITCの励起レーザー線は、それぞれ、488 nmおよび635 nmです。それぞれ、610±20 nmおよび660±20 nmでPIとアネキシンV-FITCの蛍光発光を測定します。 10,000事象ごとのフローサイトメーター上で獲得した細胞を収集します。 </li></ol></li></ol>担癌マウスのin vivo抗腫瘍効果4. <ol><li>肺癌誘発性マウスモデル<ol><li> 0.1ミリリットルRPMI-1640培地に1×10 6 A549細胞を準備し;氷の中に保管してください。 </li><li>キシラジンの腹腔内注射およびチレタミンとゾラゼパム（1の混合物をマウスに麻酔：2、1ミリリットル/kg）。優しくマウスの皮膚の小さな倍をつまんで適切な麻酔を確認してください。麻酔下ながら乾燥を防ぐために、目に獣医軟膏を使用してください。 注：移動が観察されない場合、動物は、実験を開始するのに十分な麻酔です。 </li><li> （ - 7週齢、20〜22グラム6）BALB / C雄ヌードマウスの左脇腹に皮下細胞を注入します。 </li><li>彼らはケージの周りに移動を開始するまで、マウスを観察してください。 注：それは胸骨横臥位を維持するのに十分な意識を取り戻したまで無人の動物を放置しないでください。それは完全に回復するまで他の動物の会社に手術を受けた動物を返さないでください。特定病原体フリー（SPF）の条件下でマウスを保管してください。 </li><li>毎日ノギスで腫瘍の大きさを測定します。腫瘍サイズが体積で約200ミリメートル3に達したとき（長さ×幅2）/ 2として腫瘍体積（mm 3で）を計算し 、実験を開始します。 </李&gt; </ol></li><li>抗がん効果の研究<ol><li>無作為に4群（各群n = 10）にマウスを分けます。 </li><li> （：2、1ミリリットル/キログラム1）キシラジンの腹腔内注射およびチレタミンとゾラゼパムの混合物をマウスに麻酔。優しくマウスの皮膚の小さな倍をつまんで適切な麻酔を確認してください。麻酔下ながら乾燥を防ぐために、目に獣医軟膏を使用してください。 注：移動が観察されない場合、動物は、実験を開始するのに十分な麻酔です。 </li><li> 2 mg / mlでのHBの最終濃度になるようにPBS中にナノ粒子を溶解します。 0日と7日に二回、尾静脈（HBとしての2mg / kg）を介して、PBS、無料のHB、HB-のNP、またはHB-P-のNPを注入します。 </li><li>彼らはケージの周りに移動を開始するまで、マウスを観察してください。 注：それは胸骨横臥位を維持するのに十分な意識を取り戻したまで無人の動物を放置しないでください。完全になるまで他の動物の会社に手術を受けた動物を返さないでください。回収されました。暗く、特定のSPF条件下でケージを保管してください。 </li><li> （：2、1ミリリットル/キログラム1）各注射後24時間は、キシラジンおよびチレタミンとゾラゼパムの混合物の腹腔内注射でマウスを麻酔します。優しくマウスの皮膚の小さな倍をつまんで適切な麻酔を確認してください。麻酔下ながら乾燥を防ぐために、目に獣医軟膏を使用してください。 注：移動が観察されない場合、動物は、実験を開始するのに十分な麻酔です。 </li><li> （腫瘍に対するPDTファイバからの距離から1cmで）PDT繊維の下に腫瘍部位を配置します。 、レーザー安全メガネを着用してスイッチをオフにして、1日目と8日に二回500秒（200 J / cm 2）をするためのPDTレーザー（630 nmで、400ミリワット/ cm 2）を用いて腫瘍を照らします。 </li><li>彼らはケージの周りに移動を開始するまで、マウスを観察してください。それは胸骨横臥位を維持するのに十分な意識を取り戻したまで無人の動物を放置しないでください。 undergonを持つ動物を返さないでください。他の動物の会社に電子手術は完全に回復するまで。 </li><li> SPF条件下でケージを保管してください。レーザー治療後24時間暗所でケージを維持します。 </li><li>視覚的に毎日の腫瘍体積と腫瘍部位での変化を監視します。ノギスで腫瘍の大きさを測定し、（長さ×幅2）/ 2（ミリメートル3）などの体積を計算します。 PDT後の腫瘍表面変化を確認するために、毎日の腫瘍部位の写真を撮ります。 </li><li> 16日目には、イソフルランを用いて、端末麻酔によって群当たり5匹のマウスを生け贄に捧げます。 </li><li>鉗子やハサミで、外側の皮膚をカットし、腫瘍15を露出させます。慎重にそれらを収穫。 、10％ホルマリンでそれらを修正し、パラフィン中にそれらを埋め込む、組織学的分析16のためにヘマトキシリンおよびエオシン（H＆E）でそれらを染色します。 </li><li>モニタリングの45日後に、イソフルランを用いて、端末麻酔によって残りのマウスを生け贄に捧げます。 </li></ol></li></ol>

<ol>
	<li>Shang, L., Zhou, N., Gu, Y., Liu, F., Zeng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+on+killing+effect+of+esophageal+cancer+cell+line+between+hypocrellin+B-photodynamic+therapy+and+hematoporphyrin+derivative-photodynamic+therapy.">Comparative study on killing effect of esophageal cancer cell line between hypocrellin B-photodynamic therapy and hematoporphyrin derivative-photodynamic therapy.</a> Chin J Cancer Prev Treat. 12, 1139-1142 (2005).</li><li>Huisman, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paclitaxel+triggers+cell+death+primarily+via+caspase-independent+routes+in+the+non-small+cell+lung+cancer+cell+line+NCI-H460.">Paclitaxel triggers cell death primarily via caspase-independent routes in the non-small cell lung cancer cell line NCI-H460.</a> Clin Cancer Res. 8 (2), 596-606 (2002).</li><li>Dolmans, D. E., Fukumura, D., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+for+cancer.">Photodynamic therapy for cancer.</a> Nat Rev Cancer. 3 (5), 380-387 (2003).</li><li>Pass, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+in+oncology:+mechanisms+and+clinical+use.">Photodynamic therapy in oncology: mechanisms and clinical use.</a> J Natl Cancer Inst. 85 (6), 443-456 (1993).</li><li>Sutedja, T. G., Postmus, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+in+lung+cancer.+A+review+.">Photodynamic therapy in lung cancer. A review .</a> J. Photochem. Photobiol. 36 (2), 199-204 (1996).</li><li>Peng, C. L., Shieh, M. J., Tsai, M. H., Chang, C. C., Lai, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembled+star-shaped+chlorin-core+poly(epsilon-caprolactone)-poly(ethylene+glycol)+diblock+copolymer+micelles+for+dual+chemo-photodynamic+therapies.">Self-assembled star-shaped chlorin-core poly(epsilon-caprolactone)-poly(ethylene glycol) diblock copolymer micelles for dual chemo-photodynamic therapies.</a> Biomaterials. 29 (26), 3599-3608 (2008).</li><li>Chen, B., Pogue, B. W., Hasan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+delivery+of+photosensitising+agents.">Liposomal delivery of photosensitising agents.</a> Expert Opin Drug Deliv. 2 (3), 477-487 (2005).</li><li>Lee, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+photosensitizer+loaded+and+conjugated+glycol+chitosan+nanoparticles+for+cancer+therapy.">Comparative study of photosensitizer loaded and conjugated glycol chitosan nanoparticles for cancer therapy.</a> J Control Release. 152 (1), 21-29 (2011).</li><li>Chang, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticancer+efficacy+of+photodynamic+therapy+with+hematoporphyrin-modified,+doxorubicin-loaded+nanoparticles+in+liver+cancer.">Anticancer efficacy of photodynamic therapy with hematoporphyrin-modified, doxorubicin-loaded nanoparticles in liver cancer.</a> J. Photochem. Photobiol. 140, 49-56 (2014).</li><li>Chang, J. E., Cho, H. J., Yi, E., Kim, D. D., Jheon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypocrellin+B+and+paclitaxel-encapsulated+hyaluronic+acid-ceramide+nanoparticles+for+targeted+photodynamic+therapy+in+lung+cancer.">Hypocrellin B and paclitaxel-encapsulated hyaluronic acid-ceramide nanoparticles for targeted photodynamic therapy in lung cancer.</a> J. Photochem. Photobiol. 158, 113-121 (2016).</li><li>Wang, A. Z., Langer, R., Farokhzad, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+delivery+of+cancer+drugs.">Nanoparticle delivery of cancer drugs.</a> Annu. Rev. Med. 63, 185-198 (2012).</li><li>Matsumura, Y., Maeda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+concept+for+macromolecular+therapeutics+in+cancer+chemotherapy:+mechanism+of+tumoritropic+accumulation+of+proteins+and+the+antitumor+agent+smancs.">A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs.</a> Cancer Res. 46 (12 Pt 1), 6387-6392 (1986).</li><li>Penno, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+CD44+in+human+lung+tumors.">Expression of CD44 in human lung tumors.</a> Cancer Res. 54 (5), 1381-1387 (1994).</li><li>Wilkins, R., Kutzner, B., Truong, M., Sanchez-Dardon, J., McLean, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+radiation-induced+apoptosis+in+human+lymphocytes:+Flow+cytometry+using+Annexin+V+and+propidium+iodide+versus+the+neutral+comet+assay.">Analysis of radiation-induced apoptosis in human lymphocytes: Flow cytometry using Annexin V and propidium iodide versus the neutral comet assay.</a> Cytometry. 48 (1), 14-19 (2002).</li><li>Bannas, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+nanobody+and+antibody+based+in+vivo+tumor+xenograft+NIRF-imaging+experiments+in+mice+using+ex+vivo+flow+cytometry+and+microscopy.">Validation of nanobody and antibody based in vivo tumor xenograft NIRF-imaging experiments in mice using ex vivo flow cytometry and microscopy.</a> J Vis Exp. (98), e52462 (2015).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematoxylin+and+eosin+staining+of+tissue+and+cell+sections.">Hematoxylin and eosin staining of tissue and cell sections.</a> Cold Spring Harbor Protocols. (5), (2008).</li><li>Lam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+of+lung+cancer.">Photodynamic therapy of lung cancer.</a> Semin Oncol. 21 (6 Suppl 15), 15-19 (1994).</li><li>Simone, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+for+the+treatment+of+non-small+cell+lung+cancer.">Photodynamic therapy for the treatment of non-small cell lung cancer.</a> J Thorac Dis. 4 (1), 63-75 (2012).</li></ol>

The authors have nothing to disclose.

波長、パワー、照射時間：この研究の中で最も重要なステップは、適切なレーザー条件を選択されています。特定の光増感剤に適した光の適切な波長は、PDTのために必要です。私たちは、出力電力が多くのパイロット研究に基づいて400ミリワット/ cm 2に設定したもう一つの重要な要因であったヒポクレリンBについて適切であった630 nmのレーザーを使用していました。 400 mWの/ cm 2<...

Photodynamic therapy (PDT) is composed of three major factors: photosensitizers, light, and oxygen. PDT is reported as a promising treatment for various cancers3. When the photosensitizers are administered into the cancer patient, they selectively accumulate in the tumor tissues. When the proper wavelength of light is applied, the highly reactive singlet oxygen and other free radicals lead to tumor cell damage4. 
Lung cancer was introduced as one of the first applications for PDT in the early 1980s5. PDT provides several advantages in treating lung cancer. Since PDT is a non-invasive and non-surgical treatment, it is an attractive alternative choice for the patients in whom surgical resection is inappropriate.
There have been many challenges to enhance the cancer-targeting efficacy of the photosensitizers. Increasing photosensitizer accumulation in cancer sites and decreasing accumulation in normal tissues are the identical goals for the cancer-targeting studies. A variety of targeted drug delivery systems, such as polymers, liposomes, and nanoparticles are adopted as photosensitizer carriers6-8. In our previous studies, nanoparticles effectively increased the cancer-targeting abilities of the photosensitizers9,10. Nanoparticles are ideal cancer-targeting carriers since they possess both passive and active targeting abilities. The leaky tumor vessels provide opportunity for nano-sized carriers to accumulate easily in tumors, which is well-known as the enhanced permeability and retention (EPR) effect11,12. The interaction between the nanoparticles and the specific receptors on cancer cells enables active cancer targeting. In this study, we prepared hyaluronic acid-based nanoparticles to interact with CD44, the major hyaluronic acid receptor that is overexpressed on lung cancer cells13. 
To maximize the anticancer efficacy, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may permit an increased therapeutic effect. Furthermore, decreased doses of both the photosensitizer and the anticancer drug can diminish adverse effects. We selected hypocrellin B (HB) as a novel photosensitizer in the present study. HB is isolated from Chinese medicinal fungus Hypocrella bambuase. Shang et al. reported that HB-based PDT possesses a higher anticancer efficacy when compared to hematoporphyrin derivative-based PDT1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for various cancers, including lung cancer2. 
Herein, we compared the anticancer efficacies of photosensitizer (hypocrellin B, HB) solution, photosensitizer-encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.

<table><tbody><tr><td>oligo hyaluronic acid</td><td>Bioland Co., Ltd.</td><td>_</td><td></td></tr><tr><td>DS-Y30 (ceramide 3B; mainly &lt;em&gt;N&lt;/em&gt;-oleoyl-phytosphingosine)</td><td>Doosan Biotech Co., Ltd.</td><td>_</td><td></td></tr><tr><td>adipic acid dihydrazide</td><td>Sigma Aldrich</td><td>A0638</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-(3-dimethylaminopropyl)-&lt;em&gt;N&amp;prime;&lt;/em&gt;-ethylcarbodiimide</td><td>Sigma Aldrich</td><td>39391</td><td></td></tr><tr><td>4-(chloromethyl)benzoyl chloride</td><td>Sigma Aldrich</td><td>270784</td><td></td></tr><tr><td>Tween 80</td><td>Tokyo Chemical Industry Co., Ltd.</td><td>T0546</td><td></td></tr><tr><td>syringe filter</td><td>Sartorius Stedim Biotech GmbH</td><td>17762</td><td>15 mm, RC, PP, 0.45 &amp;micro;m</td></tr><tr><td>triethylamine</td><td>Sigma Aldrich</td><td>T0886</td><td></td></tr><tr><td>Mini-GeBAflex tubes</td><td>Gene Bio-Application Ltd.</td><td>D070-12-100</td><td></td></tr><tr><td>Paclitaxel</td><td>Taihua Corporations</td><td>_</td><td></td></tr><tr><td>RPMI-1640</td><td>Gibco Life Technologies, Inc.</td><td>11875</td><td></td></tr><tr><td>Penicillin&amp;ndash;streptomycin</td><td>Gibco Life Technologies, Inc.</td><td>15070</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco Life Technologies, Inc.</td><td>16140071</td><td></td></tr><tr><td>Celite (Filter agent)</td><td>Sigma Aldrich</td><td>6858</td><td>See step 1.4</td></tr></tbody></table>

anticancer efficacy of photodynamic therapy with lung cancer-targeted nanoparticles

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light.
To increase the therapeutic effect of PDT, we developed both photosensitizer- and anticancer agent-loaded lung cancer-targeted nanoparticles. Both enhanced permeability and retention (EPR) effect-based passive targeting and hyaluronic-acid-CD44 interaction-based active targeting were applied. CD44 is a well-known hyaluronic acid receptor that is often introduced as a biomarker of non-small cell lung cancer. 
In addition, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may increase anticancer therapeutic effects and reduce adverse reactions. 
We chose hypocrellin B (HB) as a novel photosensitizer in this study. It has been reported that HB causes higher anticancer efficacy of PDT compared to hematoporphyrin derivatives1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for lung cancer2. 
The antitumor efficacies of photosensitizer (HB) solution, photosensitizer encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT were compared both in vitro and in vivo. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
The HB-P-NP treatment group showed the most effective anticancer effect after PDT. In conclusion, the HB-P-NPs prepared in the present study represent a potential and novel photosensitizer delivery system in treating lung cancer with PDT.

私たちは、上記の技術とHB-のNPとHB-P-NPの両方を用意しました。 HB-のNPとHB-P-NPの平均直径はそれぞれ220.9±3.2 nmおよび211.9±1.6nmでした。 光照射（0〜16 J / cm 2）を、続いてPBS、空のNP、HB-のNP、及びHB-P-のNPとのインキュベーションの4時間後のA549細胞の細胞生存率は、光がない。 図1に示されています、 HB-P...

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.

肺癌標的化ナノ粒子と光線力学療法の抗がん効能

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 ビュー.  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.

ビデオ: 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.

評価するための総合的な手順<em&gt;インビボ</em&gt;がんNanomedicinesのパフォーマンス

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

がん研究

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).

ポリマー/フラーレンナノ粒子の光増感を実施ブレンドと光線力学療法

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;

標的ナノ粒子の研究のための乳癌の同質性マウスモデルからの原発癌関連線維芽細胞の分離

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.

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.

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.

骨肉腫細胞における光線力学療法の細胞毒性有効性<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

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

標的薬物送達のためのドキソルビシンを装填したアプタマー-PEI-G-PEG改変金ナノ粒子の合成

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...

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.

2D細胞培養モデルにおけるベルテポルフィン細胞毒性を高めるための自作発光ダイオードベースの光線力学治療装置

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 ...

生化学

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.

胸膜普及のマウスモデルに対する近赤外光免疫療法

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.

In vitro光毒性アッセイ:肺がん細胞における光増感剤の光毒性の可能性を評価するためのPDTベースの方法

Source: Chang, J. E., et al. Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles.&#160;J. Vis. Exp.&#160;(2016).
...

JoVE Encyclopedia of Experiments

肺がん

試験管研究

In Vivo Antitumor Efficacy Analysis of PDT: A Technique to Determine the Phototoxic Potential of a Photosensitizer in Tumor Bearing Mice

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> J. Vis. Exp. (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. This video describes the in vivo anti-tumor efficacy analysis using photosensitizer containing nanoparticles in a tumor-bearing mouse, to determine the phototoxic potential of the photosensitizer.

PDTのin vivo抗腫瘍効果分析:担がんマウスにおける光増感剤の光毒性能を決定する技術

Source: Chang, J. E., et al. Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles. J. Vis. Exp. (2016).
Photodynamic ...

肺癌標的化ナノ粒子と光線力学療法の抗がん効能

要約

さらに動画を探す

この動画の章

評価するための総合的な手順