Hybrid nanoparticulate system of Fluvastatin loaded phospholipid, alpha lipoic acid and melittin for the management of colon cancer

Abstract

As a hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, Fluvastatin (FLV) is used for reducing low-density lipoprotein (LDL) cholesterol as well as to prevent cardiovascular problems. FLV showed cell line cytotoxicity and antitumor effect. Melittin (MEL) exhibits antineoplastic activity and is known to be promising as a therapeutic option for cancer patients. The aim of this work was to investigate the combination of FLV with MEL loaded hybrid formula of phospholipid (PL) with alpha lipoic acid (ALA) nanoparticles to maximize anticancer tendencies. This study examines the optimization of the prepared formulation in order to minimize nanoparticles size and maximize zeta potential to potentiate cytotoxic potentialities in colon cancer cells (Caco2), cell viability, cell cycle analysis and annexin V were tested. In addition to biological markers as P53, Bax, bcl2 and Caspase 3 evaluation The combination involving FLV PL ALA MEL showed enhanced cytotoxic potentiality (IC50 = 9.242 ± 0.35 µg/mL), about twofold lower, compared to the raw FLV (IC50 = 21.74 ± 0.82 µg/mL). According to studies analyzing cell cycle, optimized FLV PL ALA MEL was found to inhibit Caco2 colon cancer cells more significantly than other therapeutic treatments, wherein a higher number of cells were found to accumulate over G2/M and pre-G1 phases, whereas G0/G1/S phases witnessed the accumulation of a lower number of cells. The optimized formulation may pave the way for a novel and more efficacious treatment for colon cancer.

Figure 1

Diagnostic plots for FLV-PL-ALA-MEL nanoparticles size. (A) Normal probability plot (B) studentized residuals vs. predicted values plot (C) externally studentized residuals vs. run number plot and (D) predicted vs. actual values plot.

Figure 2

Diagnostic plots for zeta potential of FLV-PL-ALA-MEL nanoparticles. (A) Normal probability plot (B) studentized residuals vs. predicted values plot (C) externally studentized residuals vs. run number plot and (D) predicted vs. actual values plot.

Figure 3

Perturbation plot (A) and Contour 2D-plots (B–D) showing the effect and interaction between investigated variables on the size of FLV-PL-ALA-MEL nanoparticles.

Figure 4

Perturbation plot (A) and Contour 2D-plots (B–D) showing the effect and interaction between investigated variables on the zeta potential of FLV-PL-ALA-MEL nanoparticles.

Figure 5

FLV-PL-ALA-MEL (A,D), FLV (B,E), and Plain formula (C,F) IC50 levels in Caco2 and EA.hy926 cells, respectively.

Figure 6

The effect of the FLV-PL-ALA-MEL over the phases of Caco2 cell cycle. With a view to making more than one comparison, we used the Tukey post hoc test. *Significantly different versus control (p < 0.05). ^#Significantly different versus Plain formula (p < 0.05). ^$Significantly different versus FLV-R (p < 0.05). (a) control, (b) Plain formula, (c) FLV and (d) FLV-PL-ALA-MEL.

Figure 7

The effect of plain formula, FLV raw and FLV-PL-ALA MEL treatments, on apoptotic/necrotic Caco2 cells. Early refers to the early phase (apoptotic) while late denotes the late phase. On the other hand, total denotes the combination of necrosis and apoptosis. With a view to making more than one comparison, we used the Tukey post hoc test. *Significantly different versus control (p < 0.05). ^#Significantly different versus Plain formula (p < 0.05). ^$Significantly different versus FLV raw.

Figure 8

Expression of apoptotic markers (A) Bax, (B) Bcl-2 (C) caspase-3, and (D). p53 within Caco2 cells. Data denote the mean of four independent experiments ± SD. With a view to making more than one comparison, we used the Tukey post hoc test. ^#Significantly different versus plain formula (p < 0.05). *Significantly different versus control (p < 0.05). ^$Significantly different versus FLV-R (p < 0.05).

Figure 9

Modulation of treatments using the plain formula, FLV-raw and FLV-PL-ALA-MEL over Expression level of inflammatory markers (A) TNF-α and (B) NF-kB. Data denote the mean of four independent experiments ± SD. To make several comparisons, we used the Tukey post hoc test. *Significantly different versus control (p < 0.05). ^#Significantly different versus plain formula (p < 0.05). ^$Significantly different versus FLV raw (P < 0.05).

Figure 10

Bcl-2, Bax, Caspase 3, and p 53 proteins expression, Western blots (A) and histogram (B) for the groups (I) control (II) plain formula, (III) FLV and (IV) FLV-PL-ALA-MEL. *Significantly different versus control (p < 0.05). ^#Significantly different versus plain formula (p < 0.05). ^$Significantly different versus FLV raw (P < 0.05).

Figure 11

The effect of optimized FLV-PL-ALA MEL on Caco2 cells’ MMP variation. Normationalization of values took place with respect to untreated (control) Caco2 cells and expressed as variation percentage. Data denote the mean of four independent experiments ± SD. To make several comparisons, we used the Tukey post hoc test. *Significantly different versus control (p < 0.05). ^#Significantly different versus Plain formula (p < 0.05).