A versatile and tunable coating strategy allows control of nanocrystal delivery to cell types in the liver

Abstract

There are many liver diseases that could be treated with delivery of therapeutics such as DNA, proteins, or small molecules. Nanoparticles are often proposed as delivery vectors for such therapeutics; however, achieving nanoparticle accumulations in the therapeutically relevant hepatocytes is challenging. In order to address this issue, we have synthesized polymer coated, fluorescent iron oxide nanoparticles that bind and deliver DNA, as well as produce contrast for magnetic resonance imaging (MRI), fluorescence imaging, and transmission electron microscopy (TEM). The composition of the coating can be varied in a facile manner to increase the quantity of poly(ethylene glycol) (PEG) from 0% to 5%, 10%, or 25%, with the aim of reducing opsonization but maintaining DNA binding. We investigated the effect of the nanoparticle coating on DNA binding, cell uptake, cell transfection, and opsonization in vitro. Furthermore, we exploited MRI, fluorescence imaging, and TEM to investigate the distribution of the different formulations in the liver of mice. While MRI and fluorescence imaging showed that each formulation was heavily taken up in the liver at 24 h, the 10% PEG formulation was taken up by the therapeutically relevant hepatocytes more extensively than either the 0% PEG or the 5% PEG, indicating its potential for delivery of therapeutics to the liver.

Figure 1

A) Left, cellular structure around a liver sinusoid and right, nanoparticle interactions at the sinusoid. B) Schematic depiction of the polymer-coated iron core, fluorescent nanoparticles used as gene delivery agents in this study. C) Depiction of nanoparticle synthesis and subsequent complexation with plasmid DNA.

Figure 2

A–D) Negative stain TEM images of the different nanoparticles. The same scale is used in each image. Insets are a 2× magnification of a region of the main image. E) Phosphorous analysis of the different formulations indicating successful DSPC-PEG inclusion. F) Gel electrophoresis of DNA-NP complexes. G) Effect of incubation with FBS on nanoparticle size.

Figure 3

A) Cell viability of the different nanoparticle formulations as expressed as a percentage of cells incubated with media only. B) MR images of pellets of 293T cells incubated under different conditions. The three pellets in each column represents triplicate experiments. C) TEM images depicting nanoparticle uptake in 293T cells. D) Confocal microscopy of GFP transfection of 293T cells where green is GFP and blue is DAPI staining (nuclei).

Figure 4

A) MR images of the livers of mice pre- and 24hr post-injection with plasmid carrying iron oxide nanoparticles. B) Cy5.5 channel fluorescence images of liver of mice. C) TEM images of sections of liver tissue indicating the nanoparticle cellular distribution. For each row of images, the scale is indicated on the rightmost image.

Figure 5

Analysis of TEM images of >150 cells/formulation for hepatocyte uptake.