Effect of PEG surface conformation on anticancer activity and blood circulation of nanoemulsions loaded with tocotrienol-rich fraction of palm oil

Abstract

Tocotrienol-rich fraction of palm oil, which contains the isomers of vitamin E, was shown to possess potent anticancer activity against mammary adenocarcinoma cell lines. Its clinical use, however, is limited by poor oral bioavailability and short half-life. Previously, we developed tocotrienol-rich lipid nanoemulsions for intravenous administration. The objective of this study was to investigate the effect of surface grafted polyethylene glycol (PEG) on the properties of the nanoemulsions. PEGylation was achieved by the addition of equimolar PEG groups using poloxamer or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)2000] (PEG2000-DSPE). The effect of PEG surface topography on the antiproliferative activity of nanoemulsions against mammary adenocarcinoma cells, their susceptibility to protein adsorption, and its effect on blood hemolysis and circulation time was investigated. Nanoemulsions PEGylated with poloxamer or PEG2000-DSPE were stable under physical stress. Poloxamer nanoemulsion, however, displayed higher uptake and potency against MCF-7 tumor cells in 2D and 3D culture and increased hemolytic effect and susceptibility to IgG adsorption, which was reflected in its rapid clearance and short circulation half-life (1.7 h). Conversely, PEGylation with PEG2000-DSPE led to a 7-fold increase in mean residence time (12.3 h) after IV injection in rats. Reduced activity in vitro and improved circulation time suggested strong shielding of plasma proteins from the droplets. Differences between the nanoemulsions were attributed to polymer imbibitions and the differences in PEG conformation and density on the surface of the droplets.

Fig. 1

Droplet size of poloxamer 188 and PEG₂₀₀₀-DSPE stabilized TRF nanoemulsions immediately after preparation and after a exposure to physical stress by mechanical shaking for 24 h or incubation at 37°C for 24 h and b dilution in isotonic 5% dextrose solution and cell culture media. Vertical bars are expressed as the mean ± SD (n = 3)

Fig. 2

a % hemolysis of poloxamer 188 and PEG₂₀₀₀-DSPE stabilized TRF nanoemulsions as a function of time and b droplet size of poloxamer 188 and PEG₂₀₀₀-DSPE stabilized TRF nanoemulsions as a function of IgG concentration. Vertical bars are expressed as the mean ± SD (n = 3). b P < 0.001; c P < 0.05 versus PEG₂₀₀₀-DSPE of the same concentration or time point

Fig. 3

Schematic presentation of PEG conformations and their affinity for protein (IgG) adsorption in 1 poloxamer 188 stabilized nanoemulsion droplet and 2 PEG₂₀₀₀-DSPE stabilized nanoemulsion droplet. Showing in the figure are the PEG conformation parameters A, D, and L where A is the area occupied by one PEG chain (in square nanometers), D is the distance between PEG grafts, and L is the length/thickness of the grafted PEG layer

Fig. 4

Dose–response relationship showing the antiproliferative activity of a TRF and α-tocopherol-loaded poloxamer 188 and PEG₂₀₀₀-DSPE nanoemulsions in 2D cell culture using 96-well plates and b TRF-loaded poloxamer 188 and PEG₂₀₀₀-DSPE nanoemulsions in 3D cell culture using the Alvetex® scaffolds against the estrogen-receptor positive MCF-7 cells. Vertical bars indicate the mean cell count ± SD (n = 6). a P < 0.0001; b P < 0.001; c P < 0.05 versus PEG₂₀₀₀-DSPE of the same concentration

Fig. 5

Cellular uptake of poloxamer 188 and PEG₂₀₀₀-DSPE stabilized nanoemulsions by MCF-7 cells after 2 and 4 h incubation as a function of TRF concentration. Vertical bars indicate the mean fluorescence intensity ± SD (n = 6). a P < 0.0001; b P < 0.001; c P < 0.05 versus PEG₂₀₀₀-DSPE of the same concentration

Fig. 6

Image of MCF-7 cells stained with KaryoMAX® Giemsa and grown in 2D (left) and 3D (right) culture plates. The geometry and shape of cultured cells in the two environments were significantly different. Cells in 2D environment adopt flattened morphology in a mono-layer, while in 3D cells maintain their natural 3D ellipsoidal shape and structure. Images were taken with Nikon Eclipse TS-1000 inverted microscope at ×40 magnification

Fig. 7

Plasma concentration (in nanograms per milliliter) versus time (in hours) profiles of γ-T3 after IV administration of 10 mg/kg of TRF loaded in non-PEGylated micelles or PEGylated poloxamer 188 or PEG₂₀₀₀-DSPE nanoemulsions to rats (n = 3). The plots represent profiles of the IV data. Each value represents the mean ± SD