CHARACTERIZATION OF OLAPARIB-CYSTEINE-CARBON NANOPARTICLES BASED COMBINATIONS BY BIOCHEMICAL AND IMAGING ANALYSES

Abstract

Carbon nanodots are widely used in drug delivery and imaging due to their useful properties. However, their usage as anti-cancer drugs have not been fully elucidated yet. Recently, beet carbon dots have been emerged to treat cancer cells in which they are able to induce cell death through DNA damage. PARP is an important family of proteins which play a key role in DNA repair. One of the PARP inhibitors which is approved by FDA is Olaparib which has been already applied to treat cancers like ovarian or prostate. Thus, carbon dots which exert cell toxicity by damaging DNA can be combined with PARP inhibitors to induce cell death and can be considered as a potential combinatorial compound to be used as an anti-cancer agent. In addition to this, there is a possibility of adding new compound like L-cysteine which might serve as an active phosphatase site. This means that L-cysteine can be used to dephosphorylate proteins as cell signaling cascade requires phosphorylation of proteins which are recruited to repair DNA. Therefore, based on the previous findings revealed by other studies regarding the effect of carbon dots on DNA, also hypothesizing the L-cysteine as an active phosphatase site, there is a possibility to create a new combinatorial compound which might enhance the efficiency of PARP inhibitor. This research work focuses on the combination of Olaparib together with beet carbon dots and L-cysteine and simulate the mechanism of DNA repair inhibition in vitro by complex binding analysis. Therefore, microscopic techniques including SEM, TEM, and AFM were used to characterize the morphology and size of combination of compounds, while FT-IR and Fluorescence spectroscopy were used to identify the chemical functional groups and their interaction with each other. SEM, TEM, and AFM have shown the aggregation of small bubble-like dots when 3 compounds were mixed. Moreover, FT-IR results have demonstrated the shift in peaks as well as change in FT-IR absorbance due to mix of compounds, whereas fluorescence spectroscopy has shown that fluorescence intensity of beet carbon dots can be enhanced or decreased upon addition of Olaparib or cysteine. BCIP-NBT phosphatase assay has shown that the addition of cysteine significantly increases the phosphatase activity of 3 types of nanoparticle mixture (L-cysteine, Beet CD and Olaparib) presenting strong dark-blue color, while no peroxidase activity was observed in all compounds. In addition, PARP Universal Colorimetric assay has confirmed the PARP activity of 3 types of nanoparticle mixture which was better than Olaparib alone with 2.86nM IC50 value. Along with that, results from Nanodrop have displayed the increase in absorbance at 260 nm upon addition of combination of compounds, while agarose gel electrophoresis has not presented any changes in DNA mobility regarding its size and charge. Based on findings obtained so far, the 5 proposed 3 types of nanoparticle mixture might form a complex with associations and maintain the strong phosphatase activity with enhanced PARP inhibition. However, additional experiments are needed to prove the phosphatase activity of L-cysteine. Thus, the novel combined nanoparticles complex may be applied to target PARP for further validation in vivo with promising potential for clinical application.