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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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>oligo hyaluronic acid</td>
			<td>Bioland Co., Ltd.</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>DS-Y30 (ceramide 3B; mainly N-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>N-(3-dimethylaminopropyl)-N&#8242;-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 &#181;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&#8211;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

8.9K 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

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

폐 종양 세포 모집 분석 결과

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

연구

암 연구학

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

췌장암의 형광 동소 이식 마우스 모델

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

진공 안정 이미징 창으로 폐의 장기 고해상도 생체 내에 현미경

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

2 차원 젤 전기 이동 법 질량 분석 방법으로 인간의 뇌 Adenoma 조직 프로테옴의 분석에 대 한 결합

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

순환 종양 세포 시각화 폐 조직에 대 한 폐 식민 분석 결과의 설립

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines respond to therapeutic compounds in 2D (dimensional) monolayer cell cultures, 3D culture systems were developed to understand the efficacy of drugs in more physiologically relevant models. In recent years, a paradigm shift was observed in pre-clinical research to validate the potency of new molecules in 3D culture systems, more precisely mimicking the tumor microenvironment. These systems characterize the disease state in a more physiologically relevant manner and help to gain better mechanistic insight and understanding of the pharmacological potency of a given molecule. Moreover, with the current trend in improving in vivo cancer models, zebrafish has emerged as an important vertebrate model to assess in vivo tumor formation and study the effect of therapeutic agents. Here, we investigated the therapeutic efficacy of hydroxycoumarin OT48 alone or in combination with BH3 mimetics in lung cancer cell line A549 by using three different 3D culture systems including colony formation assays (CFA), spheroid formation assay (SFA) and in vivo zebrafish xenografts.

Here, we present the preclinical screening of anticancer coumarins using 3D culture and zebrafish.

Spheroids, 식민지 대형 분석 실험, 그리고 Zebrafish Xenografts 제 Coumarin OT48의 Bioactivity의 전 임상 평가

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines ...

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of &lt;1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

Potentiation Antineoplastic 약물으로 항 암 항 체 효능: 조합 인덱스 방정식을 사용 하 여 항 체-마약 성분의 검출

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

펄프 조직 문화와 공동 암 세포와 그들의 상호 작용을 공부 하에서 중간 엽 줄기 세포 분리

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% with current standard of care. Tumors often recur within 9 months following initial surgery, radiation and chemotherapy, at which point treatment options become limited. This highlights the pressing need for the development of better therapeutics to prolong survival and increase the quality of life for these patients.
Tumor Treating Fields (TTFields) therapy was developed to take advantage of the effect of low frequency alternating electrical fields on cells for cancer therapy. TTFields have been demonstrated to disrupt cells during mitosis and slow tumor growth. There is also growing evidence that they act through stimulating immune responses within exposed tumors. The advantages of TTFields therapy include its noninvasive approach and increased quality of life compared to other treatment modalities such as cytotoxic chemotherapies. The Food and Drug Administration approved TTFields therapy for the treatment of recurrent glioblastoma in 2011 and for newly diagnosed glioblastoma in 2015. We report on the effects of TTFields during mitosis, the results of electric fields modeling, and proper transducer array placement. Our protocol outlines the clinical application of TTFields on a patient post-surgery, using the second-generation device.

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

종양 세포종 필드 치료 치료의 임상 적용

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in most countries. The discovery of "oncogenic driver mutations," such as epidermal growth factor receptor (EGFR)-activating mutations, and subsequent development of molecular targeted agents of EGFR tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib) have dramatically altered lung cancer treatment in recent decades. However, these drugs are still not effective in patients with non-small cell lung cancer (NSCLC) carrying EGFR-activating mutations. Following acquired resistance, the systemic progression of NSCLC remains a significant obstacle in treating patients with EGFR mutation-positive NSCLC. Here, we present a stepwise dose escalation method for establishing three independent acquired afatinib-resistant cell lines from NSCLC PC-9 cells harboring EGFR-activating mutations of 15-base pair deletions in EGFR exon 19. Methods for characterizing the three independent afatinib-resistance cell lines are briefly presented. The acquired resistance mechanisms to EGFR TKIs are heterogeneous. Therefore, multiple cell lines with acquired resistance to EGFR-TKIs must be examined. Ten to twelve months are required to obtain cell lines with acquired resistance using this stepwise dose escalation approach. The discovery of novel acquired resistance mechanisms will contribute to the development of more effective and safe therapeutic strategies.

A method for establishing afatinib-resistance cell lines from lung adenocarcinoma PC-9 cells was developed, and resistant cells were characterized. The resistant cells can be used to investigate epidermal growth factor receptor tyrosine kinase inhibitor-resistance mechanisms, applicable for patients with non-small cell lung cancer.

아파티닙 내성 폐 선암 종 PC-9 세포주 의 설립 및 특성은 아파티닙의 증가 복용량으로 개발

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in ...