Design, Synthesis, and Targeted Delivery of Fluorescent 1,2,4-Triazole-Peptide Conjugates to Pediatric Brain Tumor Cells

Abstract

Most of the chemotherapeutics and drug-delivery models pose serious health problems and several undesirable side effects due to nonspecificity, lack of proper targeting system, and their large sizes. The rational design and synthesis of target-specific chemotherapeutics are highly important. This research work is focused on the rational design, synthesis, and anticancer studies of fluorescent 1,2,4-triazole-peptide conjugates for the development of target-specific anticancer drugs. Three novel 1,2,4-triazole derivatives: 4-(4-fluorobenzylidenamino)-3-hydrazino-5-mercapto-1,2,4-triazole (4FBAHMT, 2a), 4-(3,4,5-trimethoxybenzylidenamino)-3-hydrazino-5-mercapto-1,2,4-triazole (TMOBAHMT, 2b), and 4-(4-benzyloxy-2-methyloxbenzylidenamino)-3-hydrazino-5-mercapto-1,2,4-triazole (4BO2MOBAHMT, 2c) were synthesized after screening through molecular docking procedures. The docking studies were performed between ligand molecules and αvβ6 integrin protein. Fluorescent carbon nanoparticles (CNPs, 3) were conjugated with 1,2,4-triazole derivatives (2a-c) and l-carnosine (LC) dipeptide to get their corresponding conjugates (4a-c). The title double conjugates were characterized by spectroscopic (UV/vis spectroscopy, fluorescence spectroscopy, and FTIR spectroscopy) and microscopic (scanning electron microscopy, transmission electron microscopy, and atomic force microscopy) techniques. In vitro efficacy of fluorescent 1,2,4-triazole-peptide conjugates was investigated against two pediatric brain tumor cell lines (CHLA-200 & SJGBM2) and human embryonic kidney cell line (HEK293 as a control) by employing cell proliferation assay/MTS assay and fluorescence microscopy. 1,2,4-Triazole derivatives and their conjugates showed potent and selective anticancer activity against CHLA-200 and SJGBM2 cell lines. Cell proliferation assay and fluorescence microscopy results revealed that conjugates were more highly selective and cytotoxic than control drug temozolomide (TM) against both cell lines. CNPs are highly biocompatible and the quantum-sized conjugates were nontoxic for normal embryonic kidney cell line (HEK 293). The experimental results of MTS bioactivity assay and fluorescence microscopy were in close agreement with the theoretical results of molecular docking studies.

Figure 1

Commonly used anticancer drugs based on 1,2,4-triazole moiety.

Scheme 1. Synthesis of Conjugates of CNPs with 1,2,4-Triazole Derivatives and LC

Figure 2

Docking presentation of (a, b) 4BO2MOBAHMT derivative and (c, d) LC-CNP-4BO2MOBAHMT conjugate in the active binding site of αvβ6 integrin.

Figure 3

Spectroscopic and microscopic characterization of CNPs: (a) UV/vis spectra, (b) PL spectra (the inset shows the normalized spectra), (c) TEM image, (d) AFM image (the inset shows the height profile of AFM image).

Figure 4

Spectroscopic and microscopic characterization of LC-CNP-4FBAHMT conjugate: (a) UV–vis spectra, (b) PL spectra (the inset shows the normalized spectra), (c) FT-IR spectra, (d) TEM image of conjugate, and (e) AFM image of conjugate (the inset shows the extracted height profile of AFM image).

Figure 5

Kinetics of 4FBAHMT derivative release from LC-CNP-4BAHMT conjugate at different pH values.

Figure 6

Cell viability/proliferation/MTS assay of (a, d) 4FBAHMT conjugate, (b, e) TMOBAHMT conjugate, (c, f) 4BO2MOBAHMT conjugate against CHLA-200 and SJGBM2 cell lines. The cells were exposed to different concentrations (5, 10, 25, and 50 μg/mL) of CNPs, TM, LC, 1,2,4-triazole derivatives, and LC-CNP-1,2,4-triazole conjugates.

Figure 7

Microscopic images of CHLA-200 cells treated with various concentrations (10, 25, 50 μg/mL) of (a) LC-CNP-4FBAHMT conjugate and (b) 4FBAHMT derivative. Cells treated with 1,2,4-triazole derivative were stained with rhodamine 6G dye.

Figure 8

Microscopic images of CHLA-200 cells treated with various concentrations (10, 25, 50 μg/mL) of (a) LC-CNP-TMOBAHMT conjugate and (b) TMOBAHMT derivative. Cells treated with 1,2,4-triazole derivative were stained with rhodamine 6G dye.

Figure 9

Microscopic images of CHLA-200 cells treated with various concentrations (10, 25, 50 μg/mL) of (a) LC-CNP-4BO2MOBAHMT conjugate and (b) 4BO2MOBAHMT derivative. Cells treated with 1,2,4-triazole derivative were stained with rhodamine 6G dye.