The Evaluation the Efficiency of Photodynamic Therapy with Meso-Tetraphenylporphirin As a Photosensitizer and Modified Graphene Oxide As a Drug Carrier Using Microfluidic Device

Abstract

Introduction Despite certain advances in medicine cancer diseases are still an urging problem. There is a great need to develop new or improve already existing anticancer therapies. Due to its high surface area, abundance of oxygen groups and affinity towards cancer cells, graphene oxide (GO) has a great potential to found an application as a novel drug carrier in photodynamic therapy (PDT).In this study we examined the efficiency of photodynamic therapy using meso-tetraphenylporphirin (TPP) as a photosensitizer and modified graphene oxide as a drug carrier. Graphene oxide was functionalized with ethylenediamine via carbon – nitrogen bond (GO1) and with tetraethylenepentamine (GO2). The cytotoxicity tests and cellular uptake examination were conducted using coculture of normal (HMF) and cancer (MCF-7) breast cell lines. Cells were cultured in a microfluidic Lab-on-a-chip device as monolayer (2D) and spheroids (3D). The use of the microfluidic device provided conditions which better mimicked in vivo environment than standard macroscale cell cultures. The design of the microfluidic device A main microchannel allowed to introduce solutions and cell suspensions into microfluidic device. In order to avoid presence of air bubbles it was equipped with a venting system. The main microchannel split into two pairs of parallel microchannels with three microchambers in each channel. In six microchambers cells were cultured as spheroids and in the other six as monolayer. Microchambers for spheroid culture contained 19 microwells with semicircular bottom. In addition their surface was modified with poli(vinylalcohol) (PVA). Microchambers designed for monolayer culture had flat bottom and their surface was modified with fibronectin. The layout of microchambers corresponded to layout of wells in 384-well plate. Therefore, spectrofluorimetric measurements could be performed in standard multi-well plate reader using dedicated chip holder. The design of the microfluidic device is illustrated on the Figure 1. Method On the first day of the experiment the microfluidic device was fabricated and modified with PVA and fibronectin. Then, cell suspension was introduced into the microsystem and incubated for 24h. After that GO1-TPP or GO2-TPP and negative control solution were introduced into the. In order to evaluate efficiency of photodynamic therapy using GO1-TPP and GO2-TPP cell viability, cellular uptake and reactive oxygen species (ROS) generation were examined. Cell cultures were irradiated (640 nm, 40 mWcm-2, 10 min.). Then cell cultures were incubated for another 24h. After that photocytotoxicity was evaluated using AlamarBlue® test. TPP accumulation in cells were measured 24h after introduction of GO1-TPP or GO2-TPP solutions into microsystem. Cell culture medium was changed to fresh and fluorescence intensity of TPP was measured (EEX=514 nm, EEM=650 nm). In addition, intracellular localization and uptake ware examined using confocal microscopy. Reactive oxygen species generation was measured 24h after introducing examined compounds into the microsystem. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay was used to examine ROS generation before and after cell irradiation (640 nm, 40 mWcm-2, 10 min.). All results were presented as a percentage of negative control. Results and Conclusions Non-irradiated cells incubated with GO1-TPP and GO2-TPP solutions did not exhibit significant decrease in cell viability. However, examined compounds in the same samples after irradiation showed enhanced cytotoxicity towards cell cultures. In addition, efficiency of photodynamic using GO1-TPP and GO2-TPP was greater comparing to free PTT. Cellular uptake studies are consistent with these results. Only TPP bound to the drug carrier was able to penetrate cell membrane. What is more, ROS generation was also greater in case of GO1-TPP and GO2-TPP comparing to free TPP. What is interesting 2D cell culture (monolayer) was more resistant to examined compounds than 3D cell culture (spheroids). This shows the importance of applying various cellular models in preclinical studies. The results of our experiment shows that examined graphene oxide derivatives can be efficient drug carriers in PDT. This work was financially supported within a frame of OPUS 11 program No. UMO-2016/21/B/ST5/01774. Figure 1