The effect of nanoparticle polyethylene glycol surface density on ligand-directed tumor targeting studied in vivo by dual modality imaging

Abstract

The development and application of nanoparticles as in vivo delivery vehicles for therapeutic and/or diagnostic agents has seen a drastic growth over the last decades. Novel imaging techniques allow real-time in vivo study of nanoparticle accumulation kinetics at the level of the cell and targeted tissue. Successful intravenous application of such nanocarriers requires a hydrophilic particle surface coating, of which polyethylene glycol (PEG) has become the most widely studied and applied. In the current study, the effect of nanoparticle PEG surface density on the targeting efficiency of ligand-functionalized nanoemulsions was investigated. We synthesized 100 nm nanoemulsions with a PEG surface density varying from 5 to 50 mol %. Fluorescent and paramagnetic lipids were included to allow their multimodal detection, while RGD peptides were conjugated to the PEG coating to obtain specificity for the α(v)β(3)-integrin. The development of a unique experimental imaging setup allowed us to study, in real time, nanoparticle accumulation kinetics at (sub)-cellular resolution in tumors that were grown in a window chamber model with confocal microscopy imaging, and at the macroscopic tumor level in subcutaneously grown xenografts with magnetic resonance imaging. Accumulation in the tumor occurred more rapidly for the targeted nanoemulsions than for the nontargeted versions, and the PEG surface density had a strong effect on nanoparticle targeting efficiency. Counterintuitively, yet consistent with the PEG density conformation models, the highest specificity and targeting efficiency was observed at a low PEG surface density.

Figure 1

Nanoemulsion schematics and characteristics. A: Cartoons of the nanoemulsions with the different PEG2000-DSPE content and mushroom/brush configuration indicated. B: Lipid and soybean oil content of the different nanoemulsions. C: Hydrodynamic diameters of the nanoemulsions as a function of the PEG2000-DSPE content as measured with dynamic light scattering. D: Polydispersity indexes (PDI) as a function of the PEG2000-DSPE content of the measured diameters as reported in figure C. E: Zeta potentials as a function of the PEG2000-DSPE content. For C–E: white bars: CTRL nanoemulsion, black bars: RGD nanoemulsion.

Figure 2

Cellular uptake as a function of PEG2000-DSPE content. A: Logarithmic flow cytometry histograms for the nanoemulsions with the different PEG2000-DSPE content. B: Normalized cellular uptake as the median fluorescence intensity of the positive cells divided by the median of the cellular autofluorescence for CTRL (white bars) and RGD (black bars) nanoemulsion. The error bars represent the standard deviation (n=6). C: Cellular uptake ratio between RGD and CTRL nanoemulsions. The error bars represent the standard deviation (n=6).

Figure 3

HUVEC incubated with P5 nanoemulsion for 3 h. White bars represent 20 µm. A–B: RGD nanoemulsions (A) and CTRL nanoemulsions (B) which were double labelled with rhodamine-PE (green) and NIR664-PEG2000-DSPE (red). 2 h post incubation RGD emulsions remained intact (yellow in overlay) whereas the CTRL emulsions partially disintegrated. C: HUVEC simultaneously incubated with RGD nanoemulsion (red) and CTRL nanoemulsion (green) demonstrating different intracellular localization. D: Cellular uptake for CTRL (white bars) and RGD (black bars) nanoemulsions when no endocytosis blocking was performed (None), when clathrin-dependent pathways were blocked (CL) and when caveolae-dependent pathways were blocked (CA). (§: CTRL None vs CA p=0.0001 and *: RGD None vs CA p=0.004). The error bars represent the standard deviation (n=4). E–F: HUVEC with lysosomes labelled in green incubated with RGD nanoemulsions (red in E) and CTRL nanoemulsions (red in F).

Figure 4

Nanoemulsion distribution imaged by intravital microscopy. Images obtained after injection of P5 nanoemulsions (A–D) and P50 nanoemulsions (E). RGD nanoemulsion in blue, CTRL in red and the vasculature in green. Scale bars represent 100 µm. A: At 6 h post injection, the P5 RGD nanoemulsion was confined to the vessel wall, whereas the CTRL nanoemulsion extravasated. B: A region with extensive extravasation of the P5 CTRL nanoemulsion, where the P5 RGD nanoemulsion was confined to the vessel wall 6 h post injection. C–D: 3D reconstructions from z-stacks obtained 4 h post injection of P5 RGD nanoemulsion (C) and 8 h post injection of P5 CTRL nanoemulsion (D). E: At 6 h post injection of P50 nanoemulsions the distribution of the RGD and the CTRL nanoemulsions was very similar as evident from the pink color in the overlay. In the magnified insets the arrows indicate RGD nanoemulsion localizing to the vessel wall.

Figure 5

P5 nanoemulsion accumulation kinetics imaged by intravital microscopy. Nanoemulsions in red and the vasculature in green. White bars represent 100 µm. A: Already at 10 and 30 min post injection of RGD nanoemulsions, a speckled accumulation pattern was observed and 2 h post injection and onwards a clear binding to the vasculature wall occurred. B: Up to 4 h post injection, the extravasation of CTRL nanoemulsion was very heterogeneous as shown in the multiple images at those time points. Regions with significant accumulations and extravasation, visible as high fluorescence intensity foci, and also regions with hardly any fluorescence were observed. At 8 h post injection and onwards the CTRL nanoemulsion had extravasated throughout the tumor.

Figure 6

MRI results obtained with P5 nanoemulsion containing Gd-DTPA-DSA. A: T1-weighted images of pellets of HUVEC incubated with either no (blank), or RGD or CTRL nanoemulsion. B: T1 and R1 (1/T1) values in the cell pellets (n=3) and the calculated cellular Gd concentration. The uptake ratio was determined by dividing the Gd concentration in RGD incubated pellets by the Gd concentration in CTRL incubated pellets. C: DCE-MRI curves in the tumor rim in mice injected with CTRL nanoemulsion (blue, n=3) or RGD nanoemulsion (red, n=3). The error bars represent the standard deviations.¬ The inset shows a high resolution T1-weighted image of a tumor on a mouse flank obtained 25 min post RGD nanoemulsion injection. The color coded overlay shows the relative signal intensity in the DCE-MRI in the same units as the curves.