Anti-Tumor Activity of Orally Administered Gefitinib-Loaded Nanosized Cubosomes against Colon Cancer

Abstract

Gefitinib (GFT) is a tyrosine kinase inhibitor drug used as a first-line treatment for patients with advanced or metastatic non-small cell lung, colon, and breast cancer. GFT exhibits low solubility and hence low oral bioavailability, which restricts its clinical application. One of the most important trends in overcoming such problems is the use of a vesicular system. Cubosomes are considered one of the most important vesicular systems used to improve solubility and oral bioavailability. In this study, GFT cubosomal nanoparticles (GFT-CNPs) were prepared by the emulsification method. The selected formulation variables were analyzed and optimized by full factorial design and response surface methodology. Drug entrapment efficiency (EE%), transmission electron microscopy, particle size, polydispersity index, in vitro release and its kinetics, and the effect of storage studies were estimated. The chosen GFT-CNPs were subjected to further investigations as gene expression levels of tissue inhibitors of metalloproteinases-1 (TIMP-1) and matrix metalloproteinases-7 (MMP-7), colon biomarkers, and histopathological examination of colon tissues. The prepared GFT-CNPs were semi-cubic in shape, with high EE%, smaller vesicle size, and higher zeta potential values. The in vivo data showed a significant decrease in the serum level of embryonic antigen (CEA), carbohydrate antigen 19-9 (CA 19-9), and gene expression level of TIMP-1 and MMP-7. Histopathological examination showed enhancement in cancer tissue and highly decreased focal infiltration in the lamina propria after treatment with GFT-CNPs.

Keywords: colon cancer; gefitinib; gene expression; histopathological examination; nanoparticles; nanosized cubosomes.

Figure 1

Chemical structure of (a) Gefitinib, (b) Glyceryl monooleate, and (c) Poloxamer 407.

Figure 2

DSC thermograms of (a) pure GFT, (b) GMO, (c) P407, and (d) optimized GFT-CNP formulation (GC9).

Figure 3

FTIR spectra of pure GFT, GMO, P407, and optimized GFT-CNP formula (GC9).

Figure 4

TEM micrograph of (A) GFT-CNP plain optimized formulation (GC9), (B) GFT-CNP, GC8, and (C) GFT-CNP optimized formulation (GC9).

Figure 5

EE% of GFT-CNP formulae (GC1–GC9).

Figure 6

Main effects (A) and response surface plots (B) of GMO/P407 and P407% on EE%.

Figure 7

Main effects (A) and response surface plots (B) of GMO/P407 and P407% on particle size.

Figure 8

Particle size distribution of GFT-CNP formulation (GC9).

Figure 9

Zeta potential of GFT-CNP formulation (GC9).

Figure 10

In vitro release profiles of pure GFT-suspension and GFT-CNP formulation (GC1–GC9) in phosphate buffer, pH7.4, at 37 ± 0.5 °C (n = 3; the data are expressed as mean ±SD).

Figure 11

Main effects (A) and response surface plots (B) of GMO/P407 and P407% on cumulative percentage GFT released.

Figure 12

Stability profiles of optimum GFT-CNPs (GC9) under storage at 4 °C and 25 °C for 6 months (mean ± SD, n = 3).

Figure 13

Effects of GFT and/or GFT-CNPs on gene expression levels of (a) TIMP-1 and (b) MMP-7, and serum levels of (c) CEA and (d) CA19-9. Data are presented as mean ± SEM (n = 12). ☆, ✺ and $ indicate significant change from Control group, DMH group and DMH + GFT suspension respectively. ☆ and $, indicate significant change at p < 0.05; ☆☆☆ and ✺✺✺ indicate significant change at p < 0.001.

Figure 14

Histopathological changes of colon tissues: (a,b) colon tissues of control animals showing normal colon tissue with no histopathological alteration (H&E staining; scale bar, 200 µm), (c,d) colon tissues of diseased animals showing focal ulceration in the lining mucosal epithelium associated with inflammatory cell infiltration in the underlying lamina propria (H&E staining; scale bar, 200 µm), (e) colon tissues of diseased animals treated with GFT suspension showing focal inflammatory cell infiltration in the lamina propria (H&E staining; scale bar, 200 µm), (f) colon tissues of infected animals treated with GFT-CNPs showing enhancement and decrease in the focal inflammatory cell infiltration in the lamina propria (H&E staining; scale bar, 200 µm).