Design, Synthesis and Anti-Cancer Studies of Fluorescent 1,2,4-Triazole Peptide Conjugates

Abstract

Considering the non-selectivity and lack of targeting ability of currently available anti-cancer drugs, and the blood-brain barrier (BBB) obstacles for the treatment of pediatric brain tumors, it is necessary to explore new conjugates and targeted delivery models for anti-cancer agents. This research work was focused on design, synthesis and anti-cancer studies of double conjugates of fluorescent carbon dots (CDs) with L-carnosine dipeptide (LC) as cancer targeting ligand and new 1,2,4-triazole derivatives as anti-cancer agents for the development of target specific anti cancer drugs. A panel of 25 new 1,2,4-triazole derivatives were synthesized after screening through molecular docking procedures with αvβ6 integrin. 1,2,4-Triazoles showed better docking score as compared to anti-cancer drug temozolomide (TM). Similarly, the title conjugates displayed significantly higher molecular affinity towards αvβ6 integrin than corresponding 1,2,4-triazole derivatives and TM alone due to their perfect size and potential synergistic effects of LC. 1,2,4-Triazole derivatives and their conjugates were synthesized by employing reported methods and further characterized by spectroscopy, mass spectrometry (MS) and electron microscopy. The physical data, NMR spectroscopy, FTIR spectroscopy and mass spectrometry results confirmed their successful formation. The as synthesized CDs and their conjugates emitted bright photoluminescence (PL) when excited under UV lamp (λmax = 365 nm). The CDs were spherical, highly hydrophilic and quantum-sized (4-5 nm). The corresponding conjugates were also nanodrug delivery systems having particles size between 7-9 nm. Drug loading and in vitro drug release studies revelead that 1,2,4-triazole derivatives were effectively loaded onto CDs with high loading capacity (~300 µg of the drug for 1 mg of the corresponding conjugate). Furthermore, there was a continuous release of drug with time at pH 7.4 and 5.0 as shown by the drug release profiles. However, at physiological pH (7.4), the drug release was very slow (~30% after 80 hours). On the other hand, at low pH value (5.0), the drug release significantly increased (~60%) within the same time span. In vitro cells viability assay (MTS assay) and fluorescence microscopy of two pediatric glioblastoma multiforme (GBM) cell lines (CHLA200 & SJGBM2) and non-cancerous human embryonic kidney cell line-HEK293 have shown significantly higher cytotoxicity and selectivety of 1,2,4-triazole derivatives and corresponding conjugates against both cancerous cell lines as x compared to control HEK293 cells. All the conjugates induced significantly higher cytotoxicity (greater than 50%) in both cancerous cell lines as compared to non-treated controls at 50 µg/ml concentration. Furthermore, The title conjugates were 20-30% more cytotoxic than corresponding 1,2,4-triazole derivatives and anti-cancer drug TM against both cancerous cell lines. The CDs were non-toxic and LC only increased the therapeutic efficacy of 1,2,4-triazole derivatives, displaying possible synergistic effects. The title conjugates also exhibited excellent fluorescence properties after selective internalization via endocytosis into the CHLA200 cells as compared to HEK293 cell line. This fluorescence is highly beneficial for the bioimaging of cells. The results displayed that the conjugates have potent cytotoxicity as the cells viability decreased progressively than non-treated controls with increasing the drug concentration. Furthermore, cytotoxic effects of conjugates on CHLA200 cells were more pronounced than corresponding 1,2,4-triazole derivatives within concentration range of 10-50 µg/ml. The results of fluorescene microscopy were consistent with MTS assay data and further augment the findings of cells viability assay. The 9- and 10-series conjugates displayed high molecular affinity (< -17 kcal/mol binding energy) towards αvꞵ6 integrin as well as good bioactivity (~50% cytotoxicity) for both cancer cell lines as compared to corresponding 5- and 7-series 1,2,4-triazole derivatives, respectively. These CDs-based materials alongwith anti-cancer activity of 1,2,4-triazoles and tailored targeting ability of LC will help to develop excellent alternatives for currently avaiable drug delivery models and anti-cancer agents. In conclusion, the title conjugates can be useful carriers of anti cancer drugs for in vivo models, making advances towards a new class of chemotherapeutics. The synthesis of new constructs possessing only triazole-CD and peptide-CD conjugates, in vivo biological studies of already synthesized conjugates, evaluation of their potential to cross the blood-brain barrier (BBB) and clinical studies are future prospective of this research project.