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

본 연구는 보조금없이 의해 지원되었다. SNUBH 연구 기금 14-2014-017. 저자는이 원고의 자신의 프로 보노 편집 J. 패트릭 배런, 명예 교수, 도쿄 의과 대학 겸임 교수, 분당 서울 대학교 병원 신세를하고 있습니다.

참고 : 모든 동물 연구 프로토콜 분당 서울 대학교 병원의 기관 동물 관리 및 사용위원회 (BA1308-134 / 072-01)에 의해 승인되었다. 히알루 론산 - 세라마이드 1. 합성 (HACE) <ol><li> 이중 증류수 (DDW)를 60 ml의 테트라 n 개의 -butylammonium 수산화물 (TBA)의 12.21 히알루 론산 (HA) 올리고머 밀리몰 및 9.77 밀리몰 가용화. 30 분 동안 교반한다. </li><li> 의 DS-Y30 링커를 합성 테트라 히드로 푸란 25 ㎖ (THF)에 트리 에틸 아민 8.59 DS-Y30의 세라마이드 밀리몰 및 9.45 밀리몰 용해합니다. THF에 4 chloromethylbenzoyl 클로라이드 8.59 밀리몰와 함께 섞는다. 60 ° C에서 6 시간 동안 교반한다. </li><li> THF의 혼합물 및 아세토 니트릴 DS-Y30 링커의 합성 HA-TBA의 8.10 밀리몰와 0.41 밀리몰 용해 (4 : 1, v / v)로. 40 ° C에서 5 시간 동안 교반한다. </li><li> 필터 제로 여과하여 불순물을 제거하고, 진공 증착법에 의해 유기 용매를 제거한다. (투석막을 이용하여 생성물을 정제하여분자량 컷 - 오프 : 3.5 kDa의)과 동결 건조. </li></ol> 나노 입자의 제조 2 <ol><li> HB 1 mg을 디메틸 설폭 사이드 (DMSO)에 0.5 ml의 파클리탁셀 1 mg을 용해시키고 볼텍스 혼합하여 5 분 동안 DDW 0.5 ㎖의 혼합. 이어서, 추가의 5 분 동안 소용돌이 혼합하여 그 혼합물을 용해 HACE. </li><li> 질소의 부드러운 흐름하에 4 시간 동안 70 ° C에서 용매, 열을 제거한다. </li><li> DDW 1 mL로 HACE, HB 이루어지는 막 및 파클리탁셀을 재현 탁. 봉입 약물 제거 주사기 필터 (0.45 ㎛의 공극 크기)에 필터. </li></ol> 3. 체외 광독성 <ol><li> 폐암 세포주에서의 나노 입자의 흡수 <ol><li> RPMI-1640 배지를 함유 10 % (v / v)의 소 태아 혈청, 1 % (w / v)를 페니실린 - 스트렙토 마이신을 준비한다. </li><li> 각 / 웰의 1 × 105 세포의 밀도로 24- 웰 세포 배양 플레이트에 시드 A549 세포 (삼중그룹). 가습 된 5 % CO 2 및 95 % 공기 분위기하에 37 ℃에서 24 시간 동안 인큐베이션. </li><li> 세포 부착 후, 배지를 제거하고 인산 완충 식염수 (PBS) 1 ㎖를 첨가하여 세포를 세척 하였다. </li><li> 2 μM / ㎖의 HB의 최종 농도로 PBS에 나노 입자를 녹인다. 그리고, 어두운 곳에서 4 시간 동안 각 웰에 PBS 빈 NP에, HB-NP에 또는 HB-P-NP에 1 ml의 세포를 배양한다. </li><li> 용액을 모두 제거하고 차가운 PBS 1 ㎖를 첨가하여 세포를 세척 하였다. 한 번 더 세척 단계를 반복합니다. 신선한 문화 매체를 추가합니다. </li></ol></li><li> 세포 생존 분석 <ol><li> PDT 섬유에서 세포 배양 접시를 놓습니다 (우물에 PDT 섬유에서 거리 1cm 포함). 레이저 안전 안경을 착용 다양한 시간에 대한 암의 PDT 레이저 (630 내지 400 mW의 / cm 2)로 세포를 밝히는 0, 5, 10, 20, 30 및 40 초 (0, 2, 4 8, 12 및 16 J / cm 2). 그리고, 어두운 곳에서 24 시간 동안 세포를 배양한다. </li><li>배지를 흡인하고 차가운 PBS 1 ㎖를 첨가하여 세포를 세척 하였다. 한 번 더 세척 단계를 반복합니다. </li><li> 각 웰에 세포 독성 측정 용액 10 μl를 추가합니다. 2 시간 동안 어두운 데에서 인큐베이션. </li><li> 마이크로 플레이트 리더를 사용하여 450 nm에서 흡광도를 측정한다. </li></ol></li><li> 현미경 분석 <ol><li> PDT 섬유에서 세포 배양 접시를 놓습니다 (우물에 PDT 섬유에서 거리 1cm 포함). 레이저 안전 안경을 착용하고 0, 20, 또는 40 초 (0, 8, 16 J / cm 2)의 어둠 속에서 PDT 레이저 (630 nm의, 400 mW의 / cm 2)와 세포를 조명. 그리고, 어두운 곳에서 24 시간 동안 세포를 배양한다. </li><li> 배지를 흡인하고 차가운 PBS 1 ㎖를 첨가하여 세포를 세척 하였다. 한 번 더 세척 단계를 반복합니다. </li><li> 아 넥신 V-FITC의 50 μL, 4 &#39;, 6-diamidino-2-페닐 인돌 (DAPI, / ㎖ 1.5 μg의) 50 μl를 추가합니다. 가볍게 흔들어 플레이트 어둠 속에서 실온에서 15 분 정도 배양한다. 참고 : 넥신 V-FI를 사용하여형광 현미경 키트에서 TC. </li><li> 차가운 PBS 1 ㎖를 첨가하여 세포를 세척 하였다. 한 번 더 세척 단계를 반복합니다. 신선한 PBS에 세포를 보관하십시오. 100X 배율 광학 현미경을 사용하여 세포 사멸 세포를 식별합니다. </li></ol></li><li> 형광 활성화 된 셀 정렬 (FACS) 분석 <ol><li> PDT 섬유에서 세포 배양 접시를 놓습니다 (우물에 PDT 섬유에서 거리 1cm 포함). 레이저 안전 안경을 착용하고 0, 20, 또는 40 초 (0, 8, 16 J / cm 2)에 대한 PDT 레이저 (630 nm의, 400 mW의 / cm 2)와 세포를 조명. 그리고, 어두운 곳에서 24 시간 동안 세포를 배양한다. </li><li> 배지를 흡인하고 차가운 PBS 1 ㎖를 첨가하여 세포를 세척 하였다. 한 번 더 세척 단계를 반복합니다. </li><li> 및 전송 100 μL, 1 × 결합 완충액 1 ㎖로 세포를 재현 탁 (0.1 M 헤 페스 / 수산화 나트륨 (PH 7.4), 1.4 M의 NaCl, 25 mM의 CaCl2를 9 중량 부, 증류수 10 배 결합 완충액 1 부 희석) 5 ml의에 검액문화 튜브. </li><li> 아 넥신 V-FITC의 5 μL 및 요오드화 프로피 듐 (PI)의 5 μl를 추가합니다. 조심스럽게 튜브를 와동과 어두운 데서 실온 (RT)에서 15 분 동안 배양한다. 1 × 바인딩 버퍼 400 μl를 추가합니다. 참고 : 형광 현미경 키트에서 넥신 V-FITC와 PI를 사용합니다. </li><li> FACS (14)를 사용하여 사멸 세포를 식별합니다. PI 및 아 넥신 V-FITC의 여기 레이저 라인은 각각 488 nm 내지 635 nm 인이다. 각각 610 ± 20 nm의 660 ± 20 nm에서 PI와 아 넥신 V-FITC의 형광 방출을 측정합니다. 10000 이벤트에 따라 유동 세포 계수기에 획득 한 세포를 수집합니다. </li></ol></li></ol> 종양 함유 마우스 (4)에 생체 내 항암 효과 <ol><li> 폐암 유도 마우스 모델 <ol><li> 0.1 ㎖의 RPMI-1640 배지에 1 × 106 세포 A549를 준비; 얼음에 보관합니다. </li><li> 자일 라진의 IP 주입 및 tiletamine 및 zolazepam ((1)의 혼합물로 생쥐를 마취 : 2, 1 ㎖ /킬로그램). 부드럽게 마우스 피부의 작은 배를 곤란하게하여 적절한 마취를 확인합니다. 마취 동안 건조를 방지하기 위해 눈에 수의사 연고를 사용합니다. 주 : 사용 이동이 관찰되지 않은 경우, 동물 실험을 시작하기에 충분히 마취. </li><li> (- 7 주, 20~22g 6) 피하 BALB / C 남성 누드 마우스의 왼쪽 측면에 세포를 주입한다. </li><li> 그들은 새장 주위를 이동하기 시작 때까지 쥐를 관찰하십시오. 참고 :이 흉골 드러 누움을 유지하기 위해 충분한 의식을 회복 할 때까지 무인 동물을 두지 마십시오. 완전히 회복 될 때까지 다른 동물의 회사에 수술을 한 동물을 반환하지 않습니다. 특정 병원균이없는 (SPF) 상태에서 마우스를 유지합니다. </li><li> 매일 캘리퍼스와 종양의 크기를 측정한다. 종양의 크기는 부피 약 200 mm 3에 도달 할 때, 실험을 시작 (폭 × 길이 2) / 2로 종양 체적 (mm 3)을 계산한다. </리&gt; </ol></li><li> 항암 효능 연구 <ol><li> 무작위로 4 개의 그룹 (각 그룹 N = 10)로 쥐를 나눕니다. </li><li> (2, 1 ㎖ / kg 1) 자일 라진의 IP 주입 및 tiletamine zolazepam과의 혼합물로 생쥐를 마취. 부드럽게 마우스 피부의 작은 배를 곤란하게하여 적절한 마취를 확인합니다. 마취 동안 건조를 방지하기 위해 눈에 수의사 연고를 사용합니다. 주 : 사용 이동이 관찰되지 않은 경우, 동물 실험을 시작하기에 충분히 마취. </li><li> 2 ㎎ / ㎖의 HB의 최종 농도로 PBS에 나노 입자를 녹인다. 두 번 일 0에서 7에 꼬리 정맥 (2 ㎎ / ㎏ HB)를 통해 PBS, 무료 HB, HB-NP에, 또는 HB-P-NPS를 주입한다. </li><li> 그들은 새장 주위를 이동하기 시작 때까지 쥐를 관찰하십시오. 참고 :이 흉골 드러 누움을 유지하기 위해 충분한 의식을 회복 할 때까지 무인 동물을 두지 마십시오. 완전히 때까지 다른 동물의 회사에 수술을 한 동물을 반환하지 않습니다회복되었다. 어두운 특정 SPF 조건 하에서 새장을 유지합니다. </li><li> (2, 1 ㎖ / kg 1) 24 시간 각각 주입 한 후, 자일 라진의 IP 주입 및 tiletamine zolazepam과의 혼합물로 생쥐를 마취. 부드럽게 마우스 피부의 작은 배를 곤란하게하여 적절한 마취를 확인합니다. 마취 동안 건조를 방지하기 위해 눈에 수의사 연고를 사용합니다. 주 : 사용 이동이 관찰되지 않은 경우, 동물 실험을 시작하기에 충분히 마취. </li><li> (종양의 PDT 섬유에서 거리 1cm 포함) PDT 섬유에서 종양 부위를 놓습니다. 레이저 안전 안경을 착용 스위치를 끄고, 두 번 일 1 8 500 초 (200 J / cm 2)에 대한 PDT 레이저 (630 nm의, 400 mW의 / cm 2)으로 종양을 조명. </li><li> 그들은 새장 주위를 이동하기 시작 때까지 쥐를 관찰하십시오. 이 흉골 드러 누움을 유지하기 위해 충분한 의식을 회복 할 때까지 무인 동물을 두지 마십시오. undergon이있는 동물을 반환하지 않습니다다른 동물의 회사에 전자 수술은 완전히 회복 될 때까지. </li><li> SPF 조건에서 케이지를 유지합니다. 레이저 치료 후 24 시간 동안 어둠 속에서 새장을 유지한다. </li><li> 시각 종양 부피 및 종양 부위에서 매일의 변화를 감시한다. 캘리퍼스와 종양의 크기를 측정하고 (폭 2 × 길이) / 2 (mm 3)로 볼륨을 계산합니다. PDT 후 종양 표면의 변화를 확인하기 위해 매일 종양 사이트의 사진을 촬영합니다. </li><li> 날 16 일, 이소 플루 란을 사용하여 단말기 마취에 의해 그룹에 5 쥐를 희생. </li><li> 집게와 가위, 외부 피부를 잘라 종양 (15)를 노출합니다. 조심스럽게 그들을 수확. 10 % 포르말린을 고정 파라핀을 포함하고, 조직 학적 분석을 16 헤 마톡 실린 및 에오신 (H &amp; E)로 얼룩. </li><li> 모니터링 45 일 후, 이소 플루 란을 사용하여 단말기 마취하여 나머지 쥐를 희생. </li></ol></li></ol>

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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.

이 연구에서 가장 중요한 단계는 적절한 레이저 조건을 선택한다 : 파장, 전력 및 조사 시간. 구체적인 감광제에 적합한 조명의 적절한 파장은 PDT 필요하다. 우리는 출력 전력이 많은 파일럿 연구에 기초하여 400 mW의 / cm 2로 설정 한 다른 중요한 요인이었다 hypocrellin의 B. 적합한 선수 630 nm의 레이저를 사용 하였다. 400 mW의 / cm 2 이하의 출력 파워는 임의의 치료 효과를 표시하기에 ?...

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.

우리는 상기 언급 된 기법 및 NP는 HB-HB-P-NP에 모두를 준비했다. HB-NP에와 HB-P-NP에의 평균 직경은 각각 220.9 ± 3.2 nm의 211.9 ± 1.6 nm이었다. 광 조사에 의해 다음 PBS 빈 NP에, HB-NP에 및 HB-P-NP에 함께 배양 4 시간 (0 J 16 / cm 2) 후의 A549 세포의 세포 생존율은 램프 없음.도 1에 도시되고 상기 HB-NP-P 처리 된 세포는 81.2...

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.

폐암 표적 나노 입자와 광 역학 치료의 항암 효능

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 조회수.  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.

비디오: 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.

포괄적 인 절차를 평가하는<em&gt; 생체</em&gt; 암 나노 의학의 성능

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

연구

암 연구학

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).

혼합 폴리머 / 풀러렌 나노 입자 감광제를 실시와 광 역학 치료

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

화학

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;

표적 나노 입자의 연구를 위한 유방암의 합성 Murine 모형에서 1 차적인 암 관련섬유세포의 격리

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>원발성 골종양 및 뼈 전이 세포주에 대한 5-Aminolevulinic Acid 매개 광역학 요법

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.

원발성 골종양 및 뼈 전이 세포주에 대한 5-Aminolevulinic Acid 매개 광역학 요법

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.

골육종 세포의 광 역학 치료의 세포 독성 효능<em&gt; 체외</em

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

의학

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 세포 배양 모델에서 Verteporfin 세포 독성을 향상시키기 위한 자체 제작 및 발광 다이오드 기반 광역학 치료 장치

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 ...

생화학

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.

플라즈모닉 광열 암 치료: 광열 온도 분포 시각화를 위한 나노 입자 내장 종양 조직 모방 팬텀

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.

흉막 보급의 마우스 모형을 위한 근적외선 광면역요법

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.

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