Berberine-loaded liquid crystalline nanoparticles inhibit non-small cell lung cancer proliferation and migration in vitro

Abstract

Non-small cell lung cancer (NSCLC) is reported to have a high incidence rate and is one of the most prevalent types of cancer contributing towards 85% of all incidences of lung cancer. Berberine is an isoquinoline alkaloid which offers a broad range of therapeutical and pharmacological actions against cancer. However, extremely low water solubility and poor oral bioavailability have largely restricted its therapeutic applications. To overcome these limitations, we formulated berberine-loaded liquid crystalline nanoparticles (LCNs) and investigated their in vitro antiproliferative and antimigratory activity in human lung epithelial cancer cell line (A549). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), trypan blue staining, and colony forming assays were used to evaluate the anti-proliferative activity, while scratch wound healing assay and a modified Boyden chamber assay were carried out to determine the anti-migratory activity. We also investigated major proteins associated with lung cancer progression. The developed nanoparticles were found to have an average particle size of 181.3 nm with spherical shape, high entrapment efficiency (75.35%) and have shown sustained release behaviour. The most remarkable findings reported with berberine-loaded LCNs were significant suppression of proliferation, inhibition of colony formation, inhibition of invasion or migration via epithelial mesenchymal transition, and proliferation related proteins associated with cancer progression. Our findings suggest that anti-cancer compounds with the problem of poor solubility and bioavailability can be overcome by formulating them into nanotechnology-based delivery systems for better efficacy. Further in-depth investigations into anti-cancer mechanistic research will expand and strengthen the current findings of berberine-LCNs as a potential NSCLC treatment option.

Keywords: Berberine; Liquid crystalline nanoparticles; Lung cancer; Migration; Proliferation, Epithelial mesenchymal transition.

Fig. 1

The chemical structure of berberine

Fig. 2

Pictorial description for the preparation of berberine-LCNs

Fig. 3

Molecular mapping and geometrical positioning of berberine with a monoolein, and b monoolein and PF127, after molecular mechanics simulations in vacuum

Fig. 4

a Contour plots of PS for berberine-loaded MO-LCNs. b Contour plots of EE for berberine-loaded MO-LCNs

Fig. 5

a TEM image of berberine-loaded MO-LCNs with a scale bar of 200 nm; b TEM image of berberine-loaded MO-LCNs with a scale bar of 100 nm

Fig. 6

In vitro release profile of berberine LCNs

Fig. 7

Anti-proliferative activity of berberine-LCNs in A549 cells. a MTT cell viability assay. b Cell count after trypan blue staining. ***p < 0.001, ****p < 0.0001 vs control (without berberine-LCNs treatment). Values are expressed as mean ± SEM, n = 3 independent experiments. Analysis was done by one-way ANOVA followed by Dunnett’s multiple comparison test

Fig. 8

Anti-migratory activity of berberine-LCNs in A549 cells. a Wound was created by scratching with a sterile tip on a confluent A549 in six-well plate and treated with or without various doses of berberine-LCNs for 24 h. Photographs were taken on 10 × magnification. b The distance between two edges of wound was measured for 0 and 24 h to calculate the percentage wound closure. c A549 cells were seeded in a transwell chamber and treated with or without various doses of berberine-LCNs. Cells were allow to migrate through the membrane for 24 h. Migrated cells were stained with haematoxylin and eosin and photographed under a microscope. d The cells on the outer layer of the membrane after migration were counted in 5 random positions under a high-power field. Values are expressed as mean ± SEM (n = 3 independent experiments); *p < 0.05, **p < 0.01 vs control (without berberine-LCNs treatment); magnification 20 × . Analysis was performed by one-way ANOVA followed by Dunnett’s multiple comparison test

Fig. 9

Colony formation activity of berberine-LCNs in A549 cells. A549 cells were seeded in a six-well plate and treated with or without various doses of berberine-LCNs for 24 h. Cells were stained with crystal violet staining solution, after which the six-well plate was inverted to capture the image of an individual well

Fig. 10

Inhibition of expression of EMT related proteins a SNAIL, b P27, and c Vimentin upon treatment with berberine-LCNs on A549 cells. Values are expressed as mean ± SEM (n = 2); **P < 0.01 vs control (without berberine-LCNs treatment). Analysis was performed by a 2-tailed Student’s t-test

Fig. 11

Inhibition of expression of protein a PDGF-AA, b Axl, c BCLx, d Cathepsin S, e Galectin-3, f Survivin, g CEACAM5, h Progranulin, and i ERBB3 upon treatment with berberine-LCNs on A549 cells. Values are expressed as mean ± SEM (n = 2); *p < 0.05, **p < 0.01 vs control (without berberine-LCNs treatment). Analysis was performed by a 2-tailed Student’s t-test

Fig. 12

Anti-cancer mechanism of action of berberine LCNs. Lung cancer progression is a result of uncontrolled cell proliferation and migration. Proliferation of A549 cell is mediated overexpression of growth factor such as PDGF-AA and EGFR as well as Axl and progranulin protein. Similarly, migration of A549 is induced by upregulation of Galectin-3, CTSS, and CEACAM5 protein. Furthermore, metastasis of A549 cell is promoted by protein involved in EMT such as SNAIL, p27, and Vimentin. Protein such as survivin and BCLx increase cancer cell survival by resisting cell to undergo apoptosis. Berberine-LCNs showed anti-proliferative and anti-migratory activity by inhibiting aforementioned proliferation and migration-related protein respectively. Additionally, berberine-LCNs also inhibited protein involved EMT and protein make cancer cell resistance to apoptosis. Overall, the potent anti-cancer activity results in inhibition of cancer progression