L. Kоbylinska, R. Panchuk, N. Skorohyd, Y. Senkiv, P. Heffeter, W. Berger, N. Boiko, N. Mitina, A. Zaichenko, R. Lesyk, B. Zіmenkovsky, R. Stoika, G.S. Vari
Danylo Halytsky Lviv National Medical University,
Keywords: polyethylene glycol (PEG), anticancer drugs, drug delivery, biomarkers, apoptosis
Summary:In the last decade, the development of targeted drug delivery with conjugated nanoparticles brings more drug molecules to the diseased sites, at the same time reducing a systemic drug exposure. Polyethylene glycol (PEG) conjugation is one of the options for targeted drug delivery already used to overcome the limitation of traditional drug delivery methods. A novel nanocarrier of 5-tert-butyl-peroxy-5-methyl-1-hexene-3-yne (VEP) and glycidyl methacrylate (GMA) co-polymer containing PEG brushes was synthesized, and Doxorubicin (Dox) and synthetic 4-thiazolidinone derivatives - ID3882, ID3288 and ID3833 - were immobilized on this nanocarrier. The synthetic 4-thiazolidinone derivatives demonstrated anti-neoplastic effect in vitro when tested by the NCI, NIH (Bethesda, USA). The activity of biochemical indicators reflecting toxic actions in vitro and in vivo of traditional and experimental drugs was tested. In the first step, rat glioma C6 and human U251 glioblastoma cells were treated with ID3882, ID3288, ID3833 compounds and doxorubicin, and their cytotoxicity was studied using MTT assay and Trypan blue exclusion test, light and fluorescent microscopy, Western-blot analysis, and flow cytometry study of cell cycling and apoptosis. Following, tested compounds were injected in laboratory rat’s vein, and levels of malonic dialdehyde, activity of superoxide dismutase, catalase, and glutathione peroxydase were measured in animal blood serum. Status of small signaling molecules such as OH*, O2-, H2O2, NO2-, NO3-, H2S has been also monitored, and several biochemical indicators of general toxicity were determined. After that, the antitumor effect of these derivatives was evaluated in BALB/C mice that were grafted with murine NK/Ly lymphoma. In these studies, the universal binding characteristics of the developed polymeric nanocarrier were demonstrated. The nanocarrier-immobilized anticancer drugs enhanced the uptake and cytotoxicity compared to free drugs. The nanocarrier complexes with roughly 10 times lower doses had similar effects as free form of drugs cell cycle arrest, DNA damage, as well as the apoptotic cell death (caspase 7 and PARP cleavage) were measured. In vitro, the nanocarrier complexes with Dox and 4-thiazolidinone derivatives decreased the activity of metabolic enzymes compared to free forms of drugs. In vivo, the drug-loaded nanocarrier decreased uptake by reticuloendothelial system and the activity of metabolic enzymes, therefore, significantly abolishing the cardio-, hepato- and nephrotoxic effects of these agents. The new polymeric drug delivery system distinctly enhanced the antineoplastic activity of Dox. In the in vivo experimental model, the polymeric nanocarrier with Dox 100% cured animals at low doses (0.1 mg/kg), while free Dox solely extended a survival time of murine NK/Ly lymphoma and L1210 leukemia. The polymeric nanocarrier loaded with ID3288 drug demonstrated an advantage in the apoptosis mechanisms in eliminating NK/Ly lymphoma and L1210 leukemia cells, measured by Western-blot analysis of cleaved pro-caspase 3, principle biomarker of apoptosis. The polymeric nanocarrier with anticancer ID3882, ID3288 and ID3833 compounds and Doxorubicin are biocompatible, demonstrate a reduced general toxicity, and enhanced efficacy of anticancer action at treatment of drug-sensitive and drug-resistant cancers, therefore could improve the outcomes of the onco-therapeutics.