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

Published: January 13, 2023
doi:

Özet

在这里,我们描述了一种新颖,简单且低成本的设备,可以使用二维HeLa细胞培养物和维特泊芬作为光敏剂成功进行 体外 光动力疗法(PDT)测定。

Abstract

本文描述了一种新颖、简单且低成本的体 光动力疗法 (PDT) 测定设备,名为 PhotoACT。该器件使用一组传统的可编程发光二极管(LED)、液晶显示器(LCD)模块和连接到商用微控制器板的光传感器构建。原型的盒式结构由中密度纤维板(MDF)制成。内部隔室可同时分配四个细胞培养多孔微孔板。

作为概念验证,我们研究了光敏剂(PS)维特泊芬在二维(2D)培养中对HeLa细胞系的细胞毒性作用。用增加浓度的维替泊芬处理HeLa细胞24小时。弃去含药物的上清液培养基,用磷酸盐缓冲盐水(PBS)洗涤贴壁细胞,加入无药培养基。在这项研究中,使用红绿蓝(RGB)值255、255和255(平均通量为49.1±0.6J / cm2)检查维替泊芬对细胞的影响。24小时后,通过3-(4,5-二甲基-2-噻唑基)-2,5-二苯基四唑溴化物(MTT)测定评估细胞活力。

实验结果表明,用维替泊芬处理的细胞暴露于来自装置的光下,通过由活性氧(ROS)介导的机制 增强了 药物的细胞毒性作用。此外,通过将结果与商用PDT设备进行比较,验证了这项工作中描述的原型的使用。因此,这种基于LED的光动力疗法原型代表了PDT体 研究的良好替代方案。

Introduction

在最致命的非传染性疾病中,癌症是导致过早死亡的全球主要原因。2020年,它造成近1000万人死亡,约占全球六分之一的死亡1。此外,多重耐药性(MDR)现象构成了巨大的公共卫生威胁,因为批准的化疗方案未能达到该临床状况的缓解阶段2。癌细胞可以通过多种机制对化疗产生耐药性;然而,一些ATP结合盒(ABC)转运蛋白(ATP依赖性外排泵)的过表达被认为是肿瘤微环境中MDR发展的主要原因3。除耐多药外,其他癌症并发症,如复发和转移,也加强了对开发和改进治疗方法以克服这一肿瘤学挑战的迫切需求。

光的治疗利用已经实践了几个世纪4,光动力疗法(PDT)代表了临床认可的实体瘤治疗方法。PDT结合光敏剂(PS)的施用,然后进行光照射以产生活性氧(ROS),以在肿瘤细胞中发挥选择性细胞毒性。这种治疗方法优于传统方法,包括手术、放疗和化疗5;它是一种微创技术,在结缔组织中显示出较低的细胞毒性6。直接在肿瘤或其微环境中的光应用和PS积累确保了精确的靶向,因此,轻微的,不良的全身副作用7以及在同一部位重复治疗的可能性。此外,成本低于其他方法。由于其有希望的特性,PDT可以被认为是单一的适当选择,特别是在无法手术的肿瘤的情况下,或辅助癌症治疗7,并且代表了与化疗相关的MDR的替代方法89

第一份报告显示使用PDT的高客观反应率于1975年在小鼠和大鼠模型10中描述。从那时起,使用PDT进行了研究,在2D细胞培养物中与人肿瘤细胞系在体内和体外均取得了积极的结果711,12考虑到临床认可的PS的广泛适用性,无论其特定的积累途径和吸收峰的波长范围如何,一般过程如下:(i)PS摄取,(ii)肿瘤或其微环境中PS浓度的峰值,(iii)光应用,(iv)PS光相互作用,(v)PS激发态能量转移到组织底物或周围的氧分子, (vi) ROS产生,涉及单线态氧或超氧阴离子,(vii)肿瘤细胞死亡,本质上坏死或凋亡(直接死亡),自噬(细胞保护机制),组织缺血(血管损伤),免疫调节或这些机制的重叠7。在这个最后阶段,特定细胞死亡途径的激活取决于许多因素,例如细胞特征,实验设计以及最重要的是PS细胞内定位和PDT相关的靶向损伤13

维替泊芬是第二代PS,在挪威和中国被监管机构批准用于临床使用7。据报道,给药后,该前药在线粒体14 中部分积累并诱导细胞蛋白酪氨酸磷酸化和DNA片段化,导致肿瘤细胞凋亡1516。孵育24小时后用于维特泊芬内化,建议使用690nm波长设置的PDT方案,以实现向相邻分子717的有效电磁辐射转移水平。

关于PDT的光源,经典的二极管激光系统通常价格昂贵,技术复杂,尺寸过大,因此不便携1819。由于其单波长分布(也可以在基于LED的PDT设备中观察到),每个光敏剂应用对独立单元的需求使得二极管激光系统的使用更加复杂,经济上不可行2021。因此,利用LED机械被认为是最有前途的替代方案,不仅可以解决成本22和维护问题,而且还要提供高功率输出和危害较小的23以及更宽的照明能力24,25,2627

尽管基于LED的设备可以为PDT实验28提供潜在的贡献,但大多数商业选择仍然存在缺点,例如缺乏便携性,高成本以及复杂的建筑项目和操作29。这项工作的主要目的是为 体外 PDT测定提供一种简单可靠的工具。本文介绍了 PhotoACT,这是一种内部构建的基于 LED 的 PDT 设备,价格低廉、用户友好且便于携带。作为概念验证,该装置被证明可以增强维替泊芬在2D细胞培养模型中的细胞毒性,因此可以用作PDT实验中的研究工具。

Protocol

注意:有关此协议中使用的所有材料、试剂和软件的详细信息,请参阅 材料表 。 1. 设备结构 锯切3毫米厚的中密度纤维板(MDF),以获得尺寸如图 1A所示的碎片。注意: 使用矢量文件(补充文件 1)进行计算机数控 (CNC) 切割。 构建两个具有以下尺寸(长 x 宽 x 高)的盒子:较大的盒子为 330 mm…

Representative Results

最终的PDT设备名为PhotoACT,包括一个暗室,用于分配多达四个多孔微孔板,其上内表面配备了一组30个散射LED,编程为发射不同的可见光光谱(图3 和 补充文件6)。该设备使用两个相关的盒子构建:一个设计为PDT测定暗室的内部盒子,以及一个覆盖内部室并固定控制单元的外部盒子(图1B)。内部盒子被漆成黑色(图1D</strong…

Discussion

最终的PhotoACT设备使用市售的低成本组件建造起来很方便,总成本不到50美元。其他优点包括低维护需求,能够照射多种类型的培养板,每次测定同时使用多达四个单位,重量轻(2 kg)/尺寸(44 cm3),便于携带,准确且可重复的辐照(数据未显示),以及用户友好且简单的设置界面,无需连接到计算机或其他机器。

施工和运营协议的某些关键步骤在项目构思期间产生…

Açıklamalar

The authors have nothing to disclose.

Acknowledgements

我们感谢亚瑟·恩里克·戈麦斯·德·奥利维拉和卢卡斯·朱利安·克鲁兹·戈麦斯对拍摄过程的帮助。该项目得到了巴西研究委员会(CNPq,资助号400953/2016-1-404286/2021-6)和南洋杉基金会-PPSUS 2020/2021(SUS2020131000003)的支持。这项研究也部分由巴西苏必利尔尼维尔的财政代码001资助。

Materials

0.5% Trypsin-EDTA (10x) Gibco 15400054 Mammalian cell culture dissociation reagent
3D printer Flashforge Finder model
96-well plates Non-sterile, polystyrene, and high-binding surface plates with flat bottom wells used for 2D cell culture
Arduino
Brightness sensor TSL2561 model with 0.1-40.000+ lux detection levels and I2C interface
Buttons
Buzzer
Cell culture Flasks Sterile, polystyrene, rectangular bottom flask with Tissue Culture (TC)-treated surface, canted neck and vent cap (sizes)
Centrifuge Tubes Sterile, polypropylene tubes with 15/50 mL capacity used for cell culture dilution at seeding step of the assay
CO2 Incubator
Controller board ESP32
Design Software Trimble SketchUp
DMEM High Glucose Gibco 11965092 DMEM (Dulbecco's Modified Eagle Medium) is a widely used basal medium for supporting the growth of many different mammalian cells.
DMSO Sigma-Aldrich D4540-500ML Dimethyl sulfoxide, ≥99.5% (GC), suitable for plant cell culture
Fetal Bovine Serum  Gibco 12657029 FBS provides the best value by delivering consistency of cell growth over time and passages.
Gentamicin (50 mg/mL) Gibco 15750060 Water-soluble antibiotic drug originally purified from the fungus Micromonospora purpurea. Gentamicin acts by preventing cell culture contamination
Hemocytometer Neubauer patterned chamber used for cell counting at seeding step of the assay
Inverted Laboratory Microscope Leica DM IL LED
Laminar Flow Hood Cabin designed to protect the working environment from contaminants by maintaining a constant, unidirectional flow of HEPA-filtered air over the work area. Used at several steps of cell cultivation and treatment procedures
LCD display
LED RGB WS2812 5050 RGB SMD model with a built-in processor. Tape with 30 LEDs, 1 meter length and 9 watts
MDF fiberboards 3mm thickness medium-density fiberboards
Microcentrifuge Tubes Sterile, polypropylene tubes with safety lid and 1.5/2.0 mL capacity. Convenient tools for manipulating small volumes at treatment step of the assay
Microplate reader ThermoFischer Multiskan FC Microplate Photometer designed to detect a broad wavelength range of absorbance (340-850 nm). The equipment was used to evaluate cell viability after MTT incubation.
MTT Reagent Invitrogen M6494 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Used for cell viability assays
Operational System Real Time Engineers ltd. FreeRTOS
P10 micripipette Non-electronic, single-channel, 1-10 μL capacity
P1000 micropipette Non-electronic, single-channel, 10-1000 μL capacity
P200 micropipette Non-electronic, single-channel, 20-200 μL capacity
PDT Equipment LumaCare Model LC-122
Phosphate-Buffered Saline pH 7.4 Gibco 10010031 Balanced salt formulation used for washing cells during cultivation and assay procedures
Potentiometers
Tips Non-sterile, universal fit, 10/200/1000 μL maximum volumes
Verteporfin Sigma-Aldrich SML0534-5MG Verteporfin, ≥94% (HPLC)

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Bu Makaleden Alıntı Yapın
Zanzarini, I. d. S., Barbosa, G., Prado, L. d. O., Zattoni, I. F., Da Paz, G., Prado, A. L. d., Volanski, W., Lavarda, M. D., Rego, F. G. d. M., Picheth, G., Moure, V. R., Valdameri, G. An In-House-Built and Light-Emitting-Diode-Based Photodynamic Therapy Device for Enhancing Verteporfin Cytotoxicity in a 2D Cell Culture Model. J. Vis. Exp. (191), e64391, doi:10.3791/64391 (2023).

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