Regression of Melanoma Following Intravenous Injection of Plumbagin Entrapped in Transferrin-Conjugated, Lipid-Polymer Hybrid Nanoparticles

Abstract

Background: Plumbagin, a naphthoquinone extracted from the officinal leadwort presenting promising anti-cancer properties, has its therapeutic potential limited by its inability to reach tumors in a specific way at a therapeutic concentration following systemic injection. The purpose of this study is to assess whether a novel tumor-targeted, lipid-polymer hybrid nanoparticle formulation of plumbagin would suppress the growth of B16-F10 melanoma in vitro and in vivo.

Methods: Novel lipid-polymer hybrid nanoparticles entrapping plumbagin and conjugated with transferrin, whose receptors are present in abundance on many cancer cells, have been developed. Their cellular uptake, anti-proliferative and apoptosis efficacy were assessed on various cancer cell lines in vitro. Their therapeutic efficacy was evaluated in vivo after tail vein injection to mice bearing B16-F10 melanoma tumors.

Results: The transferrin-bearing lipid-polymer hybrid nanoparticles loaded with plumbagin resulted in the disappearance of 40% of B16-F10 tumors and regression of 10% of the tumors following intravenous administration. They were well tolerated by the mice.

Conclusion: These therapeutic effects, therefore, make transferrin-bearing lipid-polymer hybrid nanoparticles entrapping plumbagin a highly promising anti-cancer nanomedicine.

Keywords: cancer therapy; lipid–polymer hybrid nanoparticles; plumbagin; transferrin; tumor targeting.

Figure 1

Optimization of lipid–polymer hybrid nanoparticles entrapping plumbagin: effects of lipid: PLGA-COOH weight ratio (A) and HSPC: DSPE-PEG2000 molar ratio (B), on particle size, zeta potential and drug entrapment efficiency (n=3).

Figure 2

TEM pictures of Tf-bearing (left) and control (right) lipid–polymer hybrid nanoparticles entrapping plumbagin (Bar: 200 nm).

Figure 3

Drug release profile of plumbagin formulated as Tf-bearing lipid–polymer hybrid nanoparticles (▲, blue), control lipid–polymer hybrid nanoparticles (●, red) or as free drug in solution (■, black) in phosphate buffer at pH 7.4 (A) and pH 5.5 (B) over 24 h (n=3).

Figure 4

Cellular uptake of plumbagin (10 µg/well) formulated as Tf-bearing lipid–polymer hybrid nanoparticles (dark grey), control lipid–polymer hybrid nanoparticles (grey) or as solution (white), in B16-F10, A431 and T98G cancer cell lines (n=5) (*p<0.05 vs Tf-bearing lipid–polymer hybrid nanoparticles).

Figure 5

Uptake of coumarin-6 loaded in Tf-bearing or control lipid–polymer hybrid nanoparticles, or as solution, by B16-F10 cells: (A) quantitative analysis of the mean fluorescence intensity of coumarin-6 in the cells, by flow cytometry (n=9) (*p<0.05 vs Tf-bearing lipid–polymer hybrid nanoparticles), (B) qualitative analysis by confocal microscopy (magnification: 40x).

Figure 6

Relative cellular uptake of coumarin-6 loaded in Tf-bearing lipid–polymer hybrid nanoparticles (dark grey) or control lipid–polymer hybrid nanoparticles (light grey), in the presence of endocytosis inhibitors, in B16-F10 cells (n = 3) (*p<0.05 vs No inhibitor).

Figure 7

Apoptosis induction of B16-F10, A431 and T98G cells following treatment with plumbagin (1 µg/mL, corresponding to 5 µM) loaded in Tf-bearing or control lipid–polymer hybrid nanoparticles, or in solution. (A) Flow cytometric plots showing the percentage of specific cell populations in live, early apoptosis, late apoptosis and necrosis. (B) Percentage of total apoptotic cells (n = 3) (*P< 0.05 vs Tf-bearing lipid–polymer hybrid nanoparticles).

Figure 8

(A) Tumor growth studies in a B16-F10 murine model following systemic injection of transferrin-bearing lipid–polymer hybrid nanoparticles loading plumbagin (2 mg/kg of body weight/injection) (■, dark green), control lipid–polymer hybrid nanoparticles entrapping plumbagin (●, red), blank lipid–polymer hybrid nanoparticles (▲, blue), plumbagin solution (▼, orange), untreated tumors (■, black) (relative tumor volume rel. Volt_x = Volt_x/Volt₀) (n=10). (B) Variations of the body weight of the mice during the treatment (Color coding as in (A)). (C) Overall tumor response to treatments at the end of the study (green: complete response, yellow: partial response, orange: stable response and red: progressive response). (D) Time to disease progression. The Y-axis indicates the proportion of surviving mice over time. Animals were removed from the study once their tumor size reached 10 mm diameter in any direction. (Color coding as in (A)).