Name: selective cell elimination from mixed 3d culture using a near infrared photoimmunotherapy technique
Uploaded: 2016-03-14T14:00:00.000+00:00
Duration: 5 min 18 s
Description: Watch this Scientific Journal Video about Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique at JoVE.com

이 연구는 암 연구를위한 국립 보건원 (National Institutes of Health)의 교내 연구 프로그램, 국립 암 연구소, 센터에 의해 지원되었다.

참고 : 다음 프로토콜은 NIR-PIT를 사용하여 특정 세포를 제거하는 데 필요한 단계를 설명합니다. 컨트롤 및 NIR-PIT 및 세포 생존에 대한 기타 세부 사항은 다른 곳에서 18 찾을 수 있습니다. 단일 클론 항체에 IR700 1. 활용 (단클론 항체) <ol><li> 2-5 밀리그램 / 0.1 M 나 2 HPO 4 (PH 8.6) 용액 ㎖의이자의 단클론 항체를 준비합니다. </li><li> microcentrifuge 관에 0.1 M 나 2 HPO 4 용액 (PH 8.6)에 10 mM의 IR700의 30.8 nmol의와 단클론 항체의 6.8 nmol의 혼합과 알루미늄 호일로 덮여 1 시간, 실온에서 품어. </li><li> 두 번, 15 ml의 PBS와 PD-10 컬럼 (자재 / 장비의 표 참조) 씻으십시오. 단계 1.2에서 샘플을로드합니다. </li><li> 제조업체의 지시에 따라 PBS 용출하여 PD-10 컬럼을 통해 정제하여 혼합물. 참고 : 여기에, 용출 IR700의 밴드 색깔을 따라했다. 300 ㎕의 샘플의 경우, PBS 용출 분획을 4.4 ml를 전형적으로 2.5 ㎖의 사이이다. </li><li> 결정분광 광도계 (9) 595 nm에서 흡광도를 측정하여 쿠마시 염색으로 단백질 농도. 각각의 단클론 항체 분자에 접합 구 형광 분자의 수를 확인하는 689 nm에서의 흡수율 IR700의 농도를 결정한다. 주 : 분광 한 단클론 항체에 IR700 분자 최적 접합 수를 결정하는 것이 중요하다. 일반적으로, 1 단클론 항체 분자의 3 주위 IR700 분자는 모두 생체 외 및 생체 작업에 최적입니다. HPLC 및 SDS-PAGE 방법은 단클론 항체 및 IR700가 결합 여부를 여부를 확인하는 데 사용할 수 있습니다. </li><li> 단백질 농도를 측정 한 후 4 ℃에서 보관하십시오. </li></ol> 혼합 된 3 차원 세포 배양 2. 준비 (회전 타원체 혼합) <ol><li> 드롭 플레이트 매달려의 판 저수지 섹션에 멸균 수 (약 1 ml)에 적용합니다. </li><li> A431-루크-GFP 세포와 3T3-RFP 세포 - 다양한 관심의 세포 유형의 비율을 준비 -배지를 50 μL에 현탁 총 5000 세포를 포함하는 각 샘플. 참고 : 문화 미디어에 대한 관심의 세포 유형의 비율이 세포 유형에 따라 등 100, 100, 10 : 100, 25 : 100, 50 (1)을 준비합니다. </li><li> 37 ° C, 5 % 이산화탄소에서 가습 된 배양기에서 96 웰 플레이트에 매달려 드롭 5-7 일간 인큐베이션 믹스. 매 2 일 문화 매체를 변경합니다. 참고 : 세포를 포함하는 상품으로, 3D 정밀하게 회전 타원체 수 있도록 부드럽게 판을 처리하기 위해 3 차원 형상을 형성하기 전에 쉽게 떨어져. </li><li> 40X 배율 - 10 배에서 반전 시야 현미경을 사용하여 회전 타원체의 형태와 크기를 관찰한다. 참고 : 세포 유형과 매달려 드롭의 크기에 따라 다르지만, 구형의 직경도 감소 플레이트 매달려 96 7 일간 배양 한 후, 약 400 내지 600 ㎛의 것을 보장한다. </li></ol> 혼합 된 3 차원 세포 배양을위한 체외 NIR-PIT 3. <ol><li> 헥타르의 미디어 변경10 μg의 / ㎖ 항체 photoabsorber 복합체 (APC)를 포함하는 미디어 드롭 플레이트 플 레이어, 37 ° C, 5 % 이산화탄소에서 가습 된 배양기에서 6 시간 동안 배양한다. </li><li> 6 시간 배양 한 후, 신선한 문화 매체 (페놀 레드 무료)로 두 번 회전 타원체를 씻는다. 끝이 절단 부드럽게 멸균 200 μL 피펫 팁을 사용하여 100 ㎕의 신선한 페놀 레드 무료 문화 매체와 유리 바닥 50mm 접시에 회전 타원체를 전송합니다. 각각의 접시에 한 회전 타원체를 놓습니다. </li><li> 형태의 변화를 검출하는 반전 시야 현미경 타원체를 관찰한다. 다음 필터 설정을 형광 현미경을 사용하여 광 기자 (예 : GFP 및 RFP)를 관찰 : GFP - 469 nm의 여기 필터, 525 nm의 방출 필터; RFP - 559 nm의 여기 필터, 630 nm의 방출 필터. </li><li> 사출 용 유리 - 바닥 접시 위에 발광 다이오드 (LED)를 배치했다. 주 : 타원체는 NIR에 노출 될 수있다현미경 또는 층류 후드에 하나 켜집니다. <ol><li> 광 파워 미터 (9) 근적외선 광의 파워 밀도를 측정한다. 본 측정에 따르면, 2 J / cm 2에서의 670 내지 710의 파장에서 광을 방출하는 발광 다이오드 (LED)를 통해 근적외선 광을 조사한다. 참고 : LED 조명은 약 5 인치 최대 깊이 침투 할 수 있습니다. NIR-PIT의 세포 독성 효과에 관계없이 전력 밀도와 노출 23 시간 만 주어진 에너지에 의존한다. </li></ol></li><li> 조사 후, 신선한 배지를 50 μL로 새로운 매달아 놓기 플레이트에 회전 타원체를 전송하고 37 ° C, 5 % 이산화탄소에서 가습 된 배양기에서 하루 동안 배양한다. </li><li> 조심스럽게 잘라 끝 멸균 200 μL 피펫 팁을 사용하여 신선한 문화 매체 (페놀 레드 무료) 100 ㎕와 유리 바닥 접시에 회전 타원체를 전송합니다. NIR-PIT 사용 Fi를 한 후 형광 현미경 일일으로 회전 타원체를 관찰LTER 설정 단계 3.3에 기재된. <ol><li> 또는 형광 현미경 (도 2B)를 이용하여 세포질 14,18,22 GFP 형광의 감소에 의해 2 μg / ML의 최종 농도에서 미디어 프로피 듐 요오다 이드를 첨가함으로써 사균을 검출한다. </li></ol></li><li> 표적 세포가 완전히 제거되지 않은 경우 반복 3.1-3.6 단계를 반복합니다. </li></ol>

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저자는 공개 아무것도 없어.

우리는 NIR-PIT를 사용하여 비 - 표적 세포의 손상없이 혼합 3D 세포 배양으로부터 특정 세포를 제거하는 방법을 보여준다. 지금까지 실제적인 세포 제거 방법 조직이 설립되면 또는 이식 후가 없습니다. 따라서, NIR-PIT이 작업을 수행 할 수있는 유망한 방법이다. 장갑차는 단클론 항체 자체와 유사한 약물 동태를 보여 때문에이 기술은도 18, 22 생체 내에서 이용 될 수있다. 표적 세포 형은 다양한 APC로 구성 될 수있다. 21 - 항 EGFR, 안티 PSMA, 안티 mesothelin, 안티 CEA 등 다양한 항체 또는 항체 단편은 이미 장갑차 (15)을 사용 하였다. 또한, NIR-조사의 영역을 변경하면 치료 영역을 최소화 할 수 있습니다. 여기서, 표적 세포에서 루시퍼 라제 활성의 정량 형광 특정 세포의 완전한 제거가 확인되었다. NIR-PIT 따라서 중요한 점은 t이며, 광 기반 치료입니다빛 에너지와 역 제곱 법칙 (단계 3.4)에 따라, 따라서 세포 괴사 감소 이후 NIR 빛 소스와 대상 사이 그는 거리. 3D 정밀 플레이트가 처리되어야하며, 가능한 한 부드럽게 배양 회전 타원체 확인하십시오. 방울 포함하는 세포는 쉽게 파괴 또는 3D 형상을 형성하기 전에 약간의 충격에 의해 떨어져있다. NIR-PIT는 몇 가지 독특한 장점이 있습니다. 첫째, NIR-PIT 장갑차 인해 세포와 최소한의 비 표적 축적을 대상으로 바인딩 매우 구체적인 이어질 IR700의 친수성 ​​특성에 부모의 비공 항체와 유사한 정맥 주사 약물 동태를 보여줍니다. 따라서, 심지어 조직 이식 후, NIR-PIT는 NIR 관심 영역의 노광 하였다 APC의 정맥 내 주사를 통해 표적 세포를 치료할 수있다. 둘째, NIR - 빛이 자외선이나 가시 광선보다 조직에 더 깊이 침투 할 수 있으므로, NIR-PIT 표면뿐만 아니라 enti뿐만 아니라 치료를 할 수표면 NIR을 조사함으로써 이식 조직의 두께를 재. 마지막으로, NIR-PIT 반복 제한없이 표적 세포를 제거하는데 사용될 수있다. 표적 세포가 세포 표면 (18)상의 표적 분자의 충분한 복사 표현할 때이 방법은 유용하다. 그러나,이 기술에 대한 몇 가지 제한 사항이 있습니다. 이 큰 분자이기 때문에 먼저, APC의 투과성이 제한된다. 이 문제를 극복하기 위해, NIR-PIT 처리 또는 항체 단편은 18,19,22 악용 될 수 반복했다. 또 다른 한계는 빛이 침투합니다. NIR 광이 제대로 체외 배양 등 타깃 영역에 가까운 광원을 이동하는 광통신로 소화 내시경, 기관지 또는 직접 흉강경 같은 적응 방식을 필요로 생체 내 치료에 역 침투 수 있지만. NIR-PIT의 또 다른 적응 지역 및 사양 모두이 방법의 사용을 허용합니다26 - 이러한 면역 또는 종양 미세 환경 (24) 등 많은 분야에서 IFIC 세포 제거.

다른 세포에 손상을주지 않고 특정 세포를 제거하는 것은 특히 설립 조직에서는 매우 곤란하며, 조직 공학 분야에서 세포 제거 방법이 절실히 요구되고있다. 3 - 현재 재생 의학 분야에서, 배아 줄기 세포 (ES), 다 능성 줄기 세포 (PSC를) 또는 유도 된 다 능성 줄기 세포 (IPS)를 이용하여 조직 배양 유망한 재료 1이다. 이러한 조직 재생 유망하지만, 원치 않는 세포 오염의 주요 관심사이다. 또한, 이식 4,5 후 종양 유발의 안전 우려가있다. 많은 연구는 이러한 문제에 집중하고 있지만, 특히 재생 의료 6, 특정 세포를 제거 - (8) 실제적인 방법이 개발되지 않았다. 근적외선 photoimmunotherapy (NIR-PIT)는 항체 - photoabsorber의 conjugat에 따라 치료E (APC). APC는 세포 특이 단일 클론 항체 (단클론 항체)와 photoabsorber, IR700로 구성되어 있습니다. IR700은 친수성 실리카 - 프탈로시아닌 유도체이며, 자체 9 광독성을 유발하지 않습니다. IR700는 공유 라이신 분자의 측쇄상의 아미드 잔기를 통해 항체에 접합된다. APC는 세포막을 표적 분자에 결합하고 690 nm에서 NIR 광에 노출 후 거의 즉각적으로 세포 괴사를 유도한다. 14 - NIR 광, 세포 사멸 (9)에 이르는 세포 멤브레인 파열로 노광시. 21 - NIR-PIT는 항 EGFR, 항 HER2, PSMA 방지, 항 CD25, 항 mesothelin, 항 GPC3 및 안티 CEA (15)를 포함한 다수의 항체 또는 항체 단편으로 효과적인 것으로 입증되었다. 따라서, NIR-PIT는 표적 분자의 다양한 대해 사용될 수있다. 또한, NIR-PIT은 NIR-조명 관리를 제한하여 특정 지역의 선택적 치료를 할 수있는 잘 조절 치료입니다t 조사 18, 22. 여기서는 3D 혼합 배양에서 NIR-PIT를 사용하여 특정 세포를 제거하는 방법을 제시한다.

<table><tbody><tr><td>IRDye 700DX Ester Infrared Dye</td><td>LI-COR Bioscience (Lincoln, NE, USA)</td><td>929-70011</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>SIGMA-ALDRICH (St. Louis, MO, USA)</td><td>S9763</td><td></td></tr><tr><td>Sephadex G25 column (PD-10)&amp;nbsp;</td><td>GE Healthcare (Piscataway, NJ, USA)</td><td>17-0851-01</td><td></td></tr><tr><td>Coomassie (bradford) Plus protein assay</td><td>Thermo Fisher Scientific Inc (Waltham, MA, USA)</td><td>PI-23200</td><td></td></tr><tr><td>Perfecta3D 96-Well hanging Drop Plates</td><td>3D Biomatrix Inc (Ann Arbor, MI, USA)</td><td>HDP1096-8</td><td></td></tr><tr><td>Optical power meter</td><td>Thorlabs (Newton, NJ, USA)</td><td>PM100</td><td></td></tr><tr><td>LED: L690-66-60</td><td>Marubeni America Co. (Santa Clara, CA, USA)</td><td>L690-66-60</td><td></td></tr><tr><td>Vectibix (panitumumab)</td><td>Amgen (Thousand Oaks, CA, USA)</td><td></td><td></td></tr><tr><td>35&amp;nbsp;mm glass bottom dish, dish size 35&amp;nbsp;mm, well size 10&amp;nbsp;mm</td><td>Cellvis (Mountain View, CA, USA)</td><td>D35-10-0-N</td><td></td></tr></tbody></table>

selective cell elimination from mixed 3d culture using a near infrared photoimmunotherapy technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

광학 EGFR 과발현 NIR-PIT, A431 세포주의 효과를 모니터링하기 위해 유전자 또한 GFP와 루시 페라 제 (A431 뤽-GFP)을 표현하기 위해 수정되었습니다. NIR-PIT의 비 표적으로의 Balb / 3T3 세포주 광학적 RFP (3T3-RFP)를 표현하기 위해 변경되었다. APC, panitumumab-IR700 (팬-IR700)를 합성 하였다. 세포 (A431 뤽-GFP 및 3T3-RFP)의 다양한 비율로 구성 된 혼합 타원체는이 프로토콜 (그림 1)에 따라 제조 하였다. 반복 NIR-PIT는 팬 IR700 (도 2a에 처방 참조)와 인큐베이션 하였다. 이 혼합 3D 배양에서 표적 세포의 제거는 달성 형광 루시퍼 활동 (도 2B, C)로 모니터링 하였다. 반면에, 비 표적 세포는 성장을 계속했다. <img alt="그림 1" src="/files/ftp_upload/53633/53633fig1.jpg" /> <str옹&gt; 혼합 타원체의 다양한 비율 그림 1. 대표 이미지. 혼합 된 타원체의 대표 이미지는 A431-루크-GFP 세포 및 7 일에 3T3-RFP 세포의 다양한 비율이 표시됩니다. 스케일 바 = 200 μm의. 이 수치가 16에서 수정되었습니다. <a href="https://www.jove.com/files/ftp_upload/53633/53633fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/53633/53633fig2.jpg" /> 3D 세포 타원체 그림 2. 대상 셀 제거 (A431 뤽-GFP 및 3T3-RFP 세포의 혼합 회전 타원체). (A) NIR-PIT (2 J / cm 2)식이 요법이 표시됩니다. NIR-PIT 반복 (B)는 완전히 혼합 3D 회전 타원체에, 비 표적 세포 (3T3-RFP)없이 해와 표적 세포 (A431 뤽-GFP)을 제거. 바 = 200 μm의. 루시퍼 활동 (C) 정량 (RLU 비)표적 세포 (N = 10, 각 그룹의 회전 타원체)의 완전한 제거를 보여 주었다. 이 그림은 18에서 수정되었습니다. 데이터는 ± SEM는 의미로 표현 <a href="https://www.jove.com/files/ftp_upload/53633/53633fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique at JoVE.com

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

근적외선 Photoimmunotherapy 기술을 사용하여 혼합 된 3D 문화에서 선택적 세포 제거

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

selective-cell-elimination-from-mixed-3d-culture-using-near-infrared

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.8K 조회수.  National Cancer Institute. Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT). 

비디오: Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

종양 스페로이드 모델에서 항체 의존성 세포 독성 정량화: 약물 발견을 위한 응용

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of ...

연구

암 연구학

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

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

Photocontrolled, biologically active compounds are an emerging class of &#34;smart&#34; drug candidates. They provide additional safety in systemic chemotherapy due to their precise spatiotemporal activation by directing a benign, non-ionizable light to a specific location within the patient&#39;s body. This paper presents a set of methods to evaluate the in vitro potency and ex vivo efficiency of the photoactivation of photocontrolled, biologically active compounds as well as the in vivo efficacy at early stages of drug development. The methodology is applied to anticancer cytotoxic peptides, namely, the diarylethene-containing analogs of a known antibiotic, gramicidin S. The experiments are performed using 2D (adherent cells) and 3D (spheroids) cell cultures of a cancer cell line (Lewis lung carcinoma, LLC), live tissue surrogates (pork meat mince), and an allograft cancer model (subcutaneous LLC) in immunocompetent mice. The selection of the most effective compounds and estimation of realistic phototherapeutic windows are performed via automated fluorescence microscopy. The photoactivation efficiency at varying illumination regimens is determined at different depths in a model tissue, and the optimal light dosage is applied in the final therapeutic in vivo experiment.

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

This protocol presents a set of experiments adopted for the evaluation of photoswitchable anticancer peptides, that can be used in the preclinical screening of such compounds. This includes cytotoxicity assessment in 2D and 3D cell cultures, the evaluation of ex vivo (model tissue) photoisomerization efficiency, and in vivo efficacy.

광제어 생물학적 활성 화합물의 시험관 내 및 생체 내 평가 - 암 광약리학을 위한 잠재적 약물 후보

Photocontrolled, biologically active compounds are an emerging class of &#34;smart&#34; drug candidates. They provide additional safety in systemic ...

화학

LED-Based In Vitro Screening for Assessing Photoactivable Molecules in Bacterial Photodynamic Inactivation

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic inactivation of bacteria (aPDI). This process involves the administration of a photosensitizer (PS) activated by a suitable visible light source, producing exacerbated levels of reactive oxygen species (ROS) that damage crucial cellular biomolecules, ultimately causing bacterial cell death. It is crucial to create standardized, easy-to-use, and reproducible initial tests to assess and compare the effectiveness of light-induced phototoxicity of potential photosensitizers. This study introduces a simple and efficient in vitro technique for assessing photodynamic activity against planktonic bacterial cells. By employing a 96-well microplate format along with a large LED panel, the system facilitates the systematic evaluation of various compounds. Such a configuration allows for high-throughput screening of potential photosensitizers in a controlled and consistent environment, simplifying the process of identifying promising candidates for further development. This flexible platform serves as an important step in advancing the development of innovative photodynamic therapies for managing antibiotic-resistant infections.

This protocol describes a straightforward and economical method for evaluating the effectiveness of potential photosensitizers in antibacterial photodynamic inactivation (aPDI), using a 96-well plate format combined with an LED panel light source. This approach enables the simultaneous testing of multiple experimental conditions, including different concentrations, compounds, and bacterial strains.

박테리아 광역학 불활성화에서 광활성 분자를 평가하기 위한 LED 기반 체외 스크리닝

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

자기 고열증 및 포토열 조합 암 치료를 위한 자기, 음향 및 광학 삼중 반응 마이크로 버블

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

생체공학

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce cell apoptosis. The Federal Drug Administration has approved PDT for the treatment of actinic keratosis, and clinical guidelines recommend PDT as a treatment for certain non-melanoma skin cancers and acne vulgaris. PDT is an advantageous therapeutic modality as it is low cost, non-invasive, and associated with minimal adverse events and scaring. In the first step of PDT, a PS is applied and allowed to accumulate intracellularly. Subsequent light irradiation induces reactive oxygen species formation, which may ultimately lead to cell apoptosis, membrane disruption, mitochondrial damage, immune modulation, keratinocyte proliferation, and collagen turnover. Herein, we present an in vitro method to study PDT in an adherent cell line. This treatment protocol is designed to simulate PDT and may be adjusted to studying the use of PDT with various cell lines, photosensitizers, incubation temperatures, or photoactivation wavelengths. Squamous cell carcinoma cells were incubated with 0, 0.5, 1.0, and 2 mM 5-aminolevulinic acid (5-ALA) for 30 min and photoactivated with 417 nm blue light for 1,000 s. The primary outcome measure was apoptosis and necrosis, as measured by annexin-V and 7-aminoactinomycin D flow cytometry. There was a dose-dependent increase in cell apoptosis following thirty-minute incubation of 5-ALA. To achieve high inter-test validity, it is important to maintain consistent incubation and light parameters when performing in vitro PDT experiments. PDT is a useful clinical procedure and in vitro research may allow for the development of novel PSs, optimization of protocols, and new indications for PDT.

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce apoptosis. We present an in vitro PDT protocol designed to simulate PDT that may be used to study differences in PS incubation and light treatment parameters.

Photodynamic 치료 체 외에서 접근

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light ...

의학

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 ...

칸디다 글라브라타(Candida glabrata)의 몸집이 작은 콜로니 형성 및 약물 내성 분석

Induction of Petite Colonies in Candida glabrate via Rose Bengal-Mediated Photodynamic Therapy

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial photodynamic therapy (aPDT) targets multiple fungal structures, unlike antibiotics/antifungals, potentially thwarting resistance. Traditional methods for inducing petite colonies rely on ethidium bromide or fluconazole, which can influence drug susceptibility and stress responses. This study investigated the application of green light (peak 520 nm) and rose bengal (RB) photosensitizer to combat a drug-resistant Candida glabrata isolate. The findings revealed that aPDT treatment significantly inhibited cell growth (&#8805;99.9% reduction) and effectively induced petite colony formation, as evidenced by reduced size and loss of mitochondrial redox indicator staining. This study provides initial evidence that aPDT can induce petite colonies in a multidrug-resistant C. glabrata strain in vitro, offering a potentially transformative approach for combating resistant fungal infections.

저자 스포트라이트: 항진균 연구를 위한 약물 저항성 칸디다 균에서 몸집이 작은 식민지를 유도하기 위한 새로운 접근 방식으로서의 광역학 요법

The significance of petite colonies in Candida spp. drug resistance has not been fully explored. Antimicrobial photodynamic therapy (aPDT) offers a promising strategy against drug-resistant fungal infections. This study demonstrates that rose bengal-mediated aPDT effectively deactivates Candida glabrata and induces petite colonies, presenting a unique procedure.

로즈 벵골 매개 광역학 요법을 통한 칸디다 글라브레이트의 쁘띠 콜로니 유도

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial ...

근적외선 Photoimmunotherapy 기술을 사용하여 혼합 된 3D 문화에서 선택적 세포 제거

요약

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박테리아 광역학 불활성화에서 광활성 분자를 평가하기 위한 LED 기반 체외 스크리닝

자기 고열증 및 포토열 조합 암 치료를 위한 자기, 음향 및 광학 삼중 반응 마이크로 버블

Photodynamic 치료 체 외에서 접근

흉막 보급의 마우스 모형을 위한 근적외선 광면역요법

로즈 벵골 매개 광역학 요법을 통한 칸디다 글라브레이트의 쁘띠 콜로니 유도

로즈 벵골 매개 광역학 요법을 통한 칸디다 글라브레이트의 쁘띠 콜로니 유도

로즈 벵골 매개 광역학 요법을 통한 칸디다 글라브레이트의 쁘띠 콜로니 유도