Transferrin-conjugated lipid-coated PLGA nanoparticles for targeted delivery of aromatase inhibitor 7alpha-APTADD to breast cancer cells

Abstract

Transferrin (Tf)-conjugated lipid-coated poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles carrying the aromatase inhibitor, 7alpha-(4'-amino)phenylthio-1,4-androstadiene-3,17-dione (7alpha-APTADD), were synthesized by a solvent injection method. Formulation parameters including PLGA-to-lipid, egg PC-to-TPGS, and drug-to-PLGA ratios and aqueous-to-organic phase ratio at the point of synthesis were optimized to obtain nanoparticles with desired sizes and drug loading efficiency. The optimal formulation had a drug loading efficiency of 36.3+/-3.4%, mean diameter of 170.3+/-7.6nm and zeta potential of -18.9+/-1.5mV. The aromatase inhibition activity of the nanoparticles was evaluated in SKBR-3 breast cancer cells. IC(50) value of the Tf-nanoparticles was ranging from 0.77 to 1.21nM, and IC(50) value of the nanoparticles was ranging from 1.90 to 3.41nM (n=3). The former is significantly lower than the latter (p<0.05). These results suggested that the aromatase inhibition activity of the Tf-nanoparticles was enhanced relative to that of the non-targeted nanoparticles, which was attributable to Tf receptor (TfR) mediated uptake. In conclusion, Tf-conjugated lipid-coated PLGA nanoparticles are potential vehicles for improving the efficiency and specificity of therapeutic delivery of aromatase inhibitors.

Fig. 1

Effects of four preparation variables on the particle size (◆) and loading efficiency () of the 7α-APTADD loaded nanoparticle. PLGA-to-lipid (wt/wt) (A), egg PC-to-TPGS ratio (wt/wt) (B), drug-to-PLGA ratio (wt/wt) (C) and aqueous-to-organic phase ratio at the point of synthesis (v/v) (D) (n = 3).

Fig. 2

The cryo-TEM images of the 7α-APTADD loaded Tf-nanoparticles (A), liposomes (B) and nanoparticles (C).

Fig. 3

Colloidal stability of the 7α-APTADD loaded nanoparticle (▲) and the 7α-APTADD Tf-loaded nanoparticle (○) during storage at 4 °C. The values in the plot were the means of three separate experiments. Error bars shown are the standard deviations.

Fig. 4

Binding of FITC-Tf-to-TfR on SKBR-3 cells. Cells were incubated with FITC-Tf and cellular fluorescence was determined by flow cytometry. Results are shown in histogram with the X-axis indicating the cellular fluorescence intensity and the Y-axis indicating the cell count.

Fig. 5

Uptake of the nanoparticles by SKBR-3 cells. Cells were treated with calcein labeled nanoparticles and Tf-nanoparticles with a Tf-DOPE molar percentage of 0.6 mol%, and cellular fluorescence was measured by flow cytometry. Results are shown in histogram with the X-axis indicating the cellular fluorescence intensity and the Y-axis indicating the cell count. (A) Cells were treated with the nanoparticles and the Tf-nanoparticles and (B) cells treated with the Tf-nanoparticles were cultured in media with or without 20 μM Tf.

Fig. 6

MFI of cells treated by the Tf-nanoparticles with different molar percentages of Tf-DOPE.

Fig. 7

Uptake of R18 labeled nanoparticle and Tf-nanoparticle in SKBR-3 cells. Cells were cultured in media with or without 20 μM Tf and visualized on a Zeiss LSM 510 META laser scanning confocal microscope.

Fig. 8

Relative viability of cells treated by 7α-APTADD/DMSO solution (■), 7α-APTADD loaded Tf-nanoparticles (○) and empty nanoparticles (▲). The controls of the three samples were DMSO, empty nanoparticles and water, respectively.

Fig. 9

Inhibition of aromatase activity in SKBR-3 cells cultures. Cells were treated with 7α-APTADD (■), the nanoparticles (▼) and the Tf-nanoparticles (○) at concentrations from 10 pM to 1 μM. The value for 100% estradiol formation in control cultures (no inhibitor) was 10.6±3.3 pmol/ng DNA/h. Each point represents the average of nine determinations and the error bar shown was the standard error.