Design and Optimization of Orally Administered Luteolin Nanoethosomes to Enhance Its Anti-Tumor Activity against Hepatocellular Carcinoma

Abstract

Luteolin (LUT) is a natural flavonoid with low oral bioavailability with restricted clinical applications due to its low solubility. LUT shows significant anti-tumor activity in many cancer cells, including hepatocellular carcinoma (HCC). The most recent trend in pharmaceutical innovations is the application of phospholipid vesicles to improve the solubility of such hydrophobic drugs. Ethosomes are one of the most powerful phospholipid vesicles used to achieve that that target. In this study, LUT-loaded ethosomal nanoparticles (LUT-ENPs) were prepared by the cold method. Full factorial design and response surface methodology were used to analyze and optimize the selected formulation variables. Drug entrapment efficiency, vesicle size, zeta potential, Fourier transform infra-red spectroscopy, scanning electron microscopy, and cumulative percent drug released was estimated. The selected LUT-ENPs were subjected to further investigations as estimation of hepatic gene expression levels of GPC3, liver biomarkers, and oxidative stress biomarkers. The prepared LUT-ENPs were semi-spherical in shape with high entrapment efficiency. The prepared LUT-ENPs have a small particle size with high zeta potential values. The in vitro liver biomarkers assay revealed a significant decrease in the hepatic tissue nitric oxide (NO), malondialdehyde (MDA) content, and the expression of the GPC3 gene. Results showed a high increase in the hepatic tissue levels of glutathione (GSH) and superoxide dismutase (SOD). Histopathological examination showed a small number of hepatic adenomas and a significant decrease of neoplastic hepatic lesions after treatment with LUT-ENPs. Our results firmly suggest the distinctive anti-proliferative activity of LUT-ENPs as an oral drug delivery system for the treatment of HCC.

Keywords: ethosomes; hepatocellular carcinoma; luteolin; nano-sized vesicles; nanoparticle; oxidative stress biomarkers.

Figure 1

Schematic diagram showing the detailed cold method steps for preparation of luteolin-loaded ethosomal nanoparticles (LUT-ENPs).

Figure 2

FTIR spectra of: (a) luteolin (LUT), (b) SL, (c) ethanol (EtOH), (d) propylene glycol (PG), (e) cholesterol, (f) physical mixture, and (g) LUT-ENPs (F8).

Figure 3

SEM micrograph of selected LUT ethosomes (F8).

Figure 4

Response surface plots effects of different formulation factors on (a) entrapment efficiency %, (b) vesicle size (nm), (c) release 12 h, and (d) zeta potential (mV).

Figure 5

Cumulative percentage release of LUT from ethosomal formulations (mean ± SD, n = 3). Control was an aqueous suspension of the drug.

Figure 6

Stability profiles of the selected LUT-ENPs (F8) under storage at 4 °C and 25 °C for 12 weeks (mean ± S.D, n = 3).

Figure 7

Effects of LUT and/or LUT-ENPs on gene expression of GPC3. Data are presented as mean ± SEM (n = 12). *, #, and † indicate significant changes from control, DENA, and DENA+ luteolin suspension groups, respectively. † indicates significant change at p < 0.05; *** and ### indicate significant change at p < 0.001.

Figure 8

Effects of LUT and/or LUT-ENPs on (a) alanine transaminase (ALT), (b) aspartate transaminase (AST), (c) alkaline phosphatase (ALP), (d) total bilirubin, and (e) alpha fetoprotein-L3 (AFP-L3) Data are presented as mean ± SEM (n = 12). *, #, and † indicate significant changes from control, DENA, and DENA+ Luteolin suspension groups respectively. † indicates significant change at p < 0.05; †† indicates significant change at p < 0.01; *** and ### indicate significant change at p < 0.001.

Figure 9

Effects of LUT and/or LUT-ENPs on, (a) glutathione (GSH), (b) superoxide dismutase (SOD), (c) nitric oxide (NO), and (d) malondialdehyde (MDA). Data are presented as mean ± SEM (n = 12). *, # and † indicate significant changes from control, DENA, and DENA+ Luteolin suspension groups respectively. †† indicates significant change at p < 0.01; *** and ### indicate significant change at p < 0.001.

Figure 10

Histopathological changes in liver tissues; (a,b) liver of control animal appearing typical hepatocytes organized in lines around the central vein (arrow; H&E staining; scale bar, 100 µm, (c,d) liver section of the diseased animal showing HCC nodule revealing marked hepatic necrosis (arrow), H&E, bar = 100 µm., (e) liver segment of infected animal treated with LUT-suspension showing hepatic adenoma with hepatic vacuolar degenerative changes (arrow), H&E, bar = 100 µm, (f) liver segment of infected animal treated with LUT-ENPs appearing hepatic foci with marked diminish the hepatic neoplastic injuries with a little number of hepatic adenomas (arrow shows central degenerative zone), H&E, bar = 100 µm.