Functional Doxorubicin-Loaded Omega-3 Unsaturated Fatty Acids Nanoparticles in Reversing Hepatocellular Carcinoma Multidrug Resistance

Abstract

BACKGROUND This study investigated a nanoparticle drug delivery system to reverse multidrug resistance (MDR) and assessed its anticancer efficacy in hepatocellular carcinoma (HCC). MATERIAL AND METHODS Docosahexaenoic acid (DHA) was used as the functional excipient and doxorubicin (DOX) as the chemotherapeutic drug to synthesize DOX nanoparticles (DOX-nano). The human HCC cell line HepG2 was used for experiments. HepG2/DOX, HepG2+DOX, HepG2+DOX-nano, HepG2/DOX+DOX, and HepG2/DOX+DOX-nano groups cells were treated with DOX or DOX-nano (5 μg/mL). Nude mice bearing a HepG2/DOX xenograft were divided into model, DOX, vector-nano, and DOX-nano groups and injected with saline, DOX reagent, vector-nano, and DOX-nano (2 mg/kg), respectively. Next, cytotoxicity, cellular uptake, cell apoptosis and migration, fluorescence imaging, TUNEL assay, and tumor inhibition effects were assessed in vitro and in vivo. Furthermore, expression of MDR-related proteins was also detected using western blotting. RESULTS Fluorescence imaging showed that the DOX uptake in the DOX-nano-treated group was the strongest in the HCC cells or tumors. Cell apoptosis was significantly increased in DOX-nano-treated HepG2/DOX cells and tumors, and cell migration was significantly inhibited in the DOX-nano-treated HepG2/DOX cells compared with the other groups. The tumor inhibitory rate in DOX-nano-injected tumors was also significantly higher than in other groups. The expression of breast cancer resistance protein, B-cell lymphoma 2, lung resistance protein, multidrug resistance protein, and protein kinase C alpha was significantly decreased in DOX-nano-treated HepG2/DOX cells and xenograft tumors. Significantly better antitumor and MDR-reversing effects were also observed in the HepG2+DOX group compared with the HepG2/DOX group. CONCLUSIONS This study revealed the potential efficacy of a DOX-nano drug delivery system for the treatment of HCC, using HepG2/DOX cells and nude mice bearing HepG2/DOX xenografts.

Figure 1

Cytotoxicities of the DOX-nano in HepG2/DOX cells. HepG2/DOX cells were treated with DOX-nano reagent at different dosages (1, 2, 4, 8, 16 μg/mL) for 4 h, and the MTT assay was used to detect the cytotoxicities and IC50 values. DOX – doxorubicin; DOX-nano – doxorubicin-loaded docosahexaenoic acid nanoparticles.

Figure 2

Cellular uptake of DOX-nano. Cells in the HepG2+DOX, HepG2+DOX-nano, HepG2/DOX+DOX, and HepG2/ADM+DOX-nano groups were treated with DOX reagent (5 μg/mL) or DOX-nano (5 μg/mL) for 2, 8, and 24 h after incubation, and fluorescence intensity of DOX was detected. DOX – doxorubicin; DOX-nano – doxorubicin-loaded docosahexaenoic acid nanoparticles.

Figure 3

Analysis of apoptotic HepG2 or HepG2/DOX cells using annexin V-fluorescein isothiocyanate/propidium iodide flow cytometry assay. Compared with the HepG2/DOX group, ^@ P<0.05, ^@@ P<0.01; compared with the HepG2+DOX-nano group, ^# P<0.05, ^## P<0.01; compared with the HepG2/DOX+DOX group, * P<0.05, ** P<0.01. DOX – doxorubicin; DOX-nano – doxorubicin-loaded docosahexaenoic acid nanoparticles.

Figure 4

(A, B) Analysis of HepG2 or HepG2/DOX cells migration. Pictures of the scratches were photographed at 0 and 24 h. Compared with the HepG2/DOX group, ^@ P<0.05, ^@@ P<0.01; compared with the HepG2+DOX-nano group, ^# P<0.05, ^## P<0.01; compared with the HepG2/DOX+DOX group, * P<0.05, ** P<0.01. DOX – doxorubicin; DOX-nano – doxorubicin-loaded docosahexaenoic acid nanoparticles.

Figure 5

Protein expressions of drug efflux transporters, MRP, LRP, BCRP, Bcl-2, and PKC-α in HepG2 or HepG2/DOX cells. The protein expression was detected using western blotting, and β-actin was used as the internal control. Compared with the HepG2/DOX group, ^@ P<0.05, ^@@ P<0.01; compared with the HepG2+DOX-nano group, ^# P<0.05, ^## P<0.01; compared with the HepG2/DOX+DOX group, * P<0.05, ** P<0.01. BCRP – breast cancer resistance protein; Bcl-2 – B-cell lymphoma 2; DOX – doxorubicin; DOX-nano – doxorubicin-loaded docosahexaenoic acid nanoparticles; LRP – lung resistance protein; MRP – multidrug resistance protein; PKC-α – protein kinase C alpha.

Figure 6

In vivo fluorescence imaging (A) and TUNEL assay (B). In vivo fluorescence imaging showing tumor accumulation of DOX-nano after intravenous injection. TUNEL assay observed cell apoptosis in tumor tissues with DOX-nano injection. DOX-nano – doxorubicin-loaded docosahexaenoic acid nanoparticles.

Figure 7

In vivo anticancer efficacy of DOX-nano in nude mice bearing HepG2/DOX xenograft. Compared with the model group, ^& P<0.05, ^&& P<0.01; compared with the DOX group, ^# P<0.05, ^## P < 0.01; compared with the vector-nano group, * P<0.05, ** P<0.01. DOX – doxorubicin; DOX-nano – doxorubicin-loaded docosahexaenoic acid nanoparticles.

Figure 8

Protein expressions of drug efflux transporters, MRP, LRP, BCRP, Bcl-2, and PKC-α in tumor tissues. The protein expressions were detected using western blotting, and β-actin was used as the internal control. Compared with the model group, ^& P<0.05, ^&& P<0.01; compared with the DOX group, ^# P<0.05, ^## P<0.01; compared with the vector-nano group, * P<0.05, ** P<0.01. BCRP – breast cancer resistance protein; Bcl-2 – B-cell lymphoma 2; LRP – lung resistance protein; MRP – multidrug resistance protein; PKC-α – protein kinase C alpha.