Enhanced Proapoptotic Effects of Water Dispersed Complexes of 4-Thiazolidinone-Based Chemotherapeutics with a PEG-Containing Polymeric Nanocarrier

Abstract

Aim: To study whether water formulation of the complex of 4-thiazolidinone derivatives with a PEG-containing polymeric nanocarrier enhances their pro-apoptotic action towards rat glioma C6 cells.

Methods: Mechanisms of antineoplastic effects of 4-thiazolidinone derivatives were investigated in vitro with rat glioma C6 cells. Cell nativity, cell cycling pattern, and Annexin V expression were evaluated and DNA damage was estimated by DNA comet analysis. A novel water-based formulation of 4-thiazolidinone derivatives complexed with a polymeric nanocarrier was utilized for enhancing pro-apoptotic action towards C6 cells.

Results: The studied 4-thiazolidinone derivatives use apoptosis mechanisms for killing rat glioma C6 cells, as confirmed by FACS analysis of these cells in pre-G1 stage, the appearance of Annexin V positive C6 cells, and an increased number of DNA comets of higher classes. Complexation of the studied compounds with a PEG-containing polymeric nanocarrier significantly increased pro-apoptotic effects in rat glioma C6 cells measured by all methods mentioned above.

Conclusion: Complexation of 4-thiazolidinone derivatives with a PEG-containing polymeric nanocarrier provided them with water solubility and enhanced pro-apoptotic effects in rat glioma C6 cells.

Keywords: 4-thiazolidinones; Apoptosis; Polyethylene glycol; Polymeric nanocarrier; Rat glioma C6 cells.

Fig. 1

Structure of the investigated compounds—Les-3288 and Les-3833

Fig. 2

Schematic structure of the PNC — poly(VEP-co-GMA)-graft-PEG with the main chain on the base of a co-polymer of unsaturated peroxide of 2-tert-butylperoxy-2-methyl-5-hexen-3-in (VEP, denoted by “k”), with side chains of glycidyl methacrylate (GMA, denoted by “l”) and polyethylene glycole (PEG, denoted by “m”)

Fig. 3

Transmission electron microscopy (TEM) images of the PNC (left) and complex of PNC + Les-3833 (right)

Fig. 4

DLS study of hydrodynamic diameters of PNC and complexes (a) and dependence of average sizes of PNC-drug complexes on dispersion dilution (b): water dispersions of PNC (1), complexes of Les-3833+PNC (2), and Les-3288+PNC (3), as well as hydrodynamic sizes of Les-3833+PNC (1–3) and Les-3288+PNC (4–6) dispersions after storage for 2 days (1.4), 4 months (2.5), and 6 months (3.6) (c)

Fig. 5

Number of living rat glioma C6 cells measured by Trypan blue exclusion test after treatment for 24 h and 48 h with different concentrations (0.1, 0.5, and 1.0 μM) of Les-3288 alone compared with the action of its complex with the polymeric nanocarrier (PNC) *P < 0.05; **P < 0.01; ***P < 0.001 (difference in comparison to the control, 100%)

Fig. 6

Number of living rat glioma C6 cells measured by Trypan blue exclusion test after treatment for 24 h and 48 h with different concentrations (0.1, 0.5, and 1.0 μM) of Les-3833 alone compared with the action of its complex with the polymeric nanocarrier (PNC) *P < 0.05; **P < 0.01; ***P < 0.001 (difference in comparison to the control, 100%)

Fig. 7

Impact on cell cycle progression of 4-thiazolidinone derivatives Les-3288 and Les-3833 in free form and in complex with the polymeric nanocarrier (PNC) in C6 rat glioma cells. Propidium iodide (PI) staining, flow cytometry. R2, pre-G1; R3, G1; R4, S; R5, G2 phase. FL2-H, peak emission values of the second channel (585/40 filter) of flow cytometer

Fig. 8

Comparison of the pro-apoptotic activity of Les-3288 (a) and Les-3833 (b) compounds and their complexes with the polymeric nanocarrier (PNC) towards rat glioma C6 cells. Annexin V/PI double staining, flow cytometry. PI, propidium iodide

Fig. 9

DNA damage was evaluated using the Olive tail moment in rat glioma C6 cells treated for 3 h with experimental antineoplastic compounds used at a 0.5 μg/mL concentration. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. PNC, polymeric nanocarrier; Dox, doxorubicin (positive control)

Fig. 10.

Presence of DNA in comet tail of rat glioma С6 cells after treatment for 3 h with (a) control (untreated cells), Les-3288 (b), Les-3288+PNC (c), Les-3833 (d) Les-3833 + PNC (e), PNC (f) and doxorubicin (g), used at a 0.5 μg/mL concentration

Fig. 11

DNA damage was evaluated using the Olive tail moment in rat glioma C6 cells treated for 6 h with experimental antineoplastic compounds used at a 0.5 μg/mL concentration. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. PNC, polymeric nanocarrier; Dox, doxorubicin (positive control)

Fig. 12

Presence of DNA in comet tail of rat glioma С6 cells after treatment for 6 h with (a) control (untreated cells), Les-3288 (b), Les-3288 + PNC (c), Les-3833 (d) Les-3833 + PNC (e), PNC (f), and doxorubicin (g), used at a 0.5 μg/mL concentration

Fig. 13

DNA damage evaluated using the % of DNA in comet tail of C6 rat glioma cells treated with experimental antineoplastic compounds during 3 h at a concentration of 0.5 μg/mL. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. PNC, polymeric nanocarrier; Dox, doxorubicin (positive control)

Fig. 14

DNA damage evaluated using the % of DNA in comet tail of C6 rat glioma cells treated with experimental antineoplastic compounds during 6 h at a concentration of 0.5 μg/mL. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. PNC, polymeric nanocarrier; Dox, doxorubicin (positive control)

Fig. 15

Replacement of methyl green dye intercalated into the DNA of salmon sperm by Les-3288 and Les-3833 compounds (1.0 μg/mL) and ethidium bromide (EtBr) used as a positive control