Blood-nanoparticle interactions and in vivo biodistribution: impact of surface PEG and ligand properties

Abstract

Theranostic nanoparticles (NPs) cannot reach their target tissue without first passing through blood; however, the influence of blood protein and blood cell interactions on NP biodistribution are not well understood. The current work shows that 30 nm PEGylated gold NPs (GNPs) interact not only with blood proteins as thought before but also with blood cells (especially platelets and monocytes) in vivo and that longer blood circulation correlates strongly with tumor uptake. Further, GNP surface properties such as negative charge or lyophilization had either a minimal (i.e., charge) or 15-fold increase (i.e., fresh vs lyophilized) in blood retention times and tumor uptake. Tumor accumulation was increased over 10-fold by use of a bioactive ligand (i.e., TNF) on the lyophilized GNP surface. Resident macrophages were primarily responsible for the bulk of GNP uptake in liver while spleen uptake was highly surface property dependent and appears to involve macrophages and cellular interaction between the red and white pulp. This study shows that the PEG layer and ligand on the surface of the NP are critical to blood interactions and eventual tumor and RES organ biodistribution in vivo.

Figure 1. Quantitative in vivo organ distribution of intravenously injected GNPs in

A) Blood; B) Tumor; C) Liver; D) Spleen. Blood, tumor, liver, and spleen accumulation of GNPs varied based on their surface properties. ICP-MS (blood) or ICP-AES (other organs) were used for quantitative analysis. L-GNP* is 27nm in core diameter as supplied by CytImmune Sciences, Inc. E) Biodistribution of plasma sensitized GNPs. GNPs were incubated with mouse plasma before injection (p-F-GNP, p-F-GNP⁻). Gold concentration is measured 1 hour post-injection. *indicated significant difference of p-GNPs from F-GNPs. F) Complement activation. No significant differences were observed between control and treated animals for C3a activation. Control animals were not treated with any GNPs. Accumulation is indicated as a percentage of injected dose (% ID/g tissue). Data are given in mean ± SEM, n=3–5. Statistical significance, p<0.05.

Figure 2. TEM imaging of GNP-blood cell interaction in vitro and in vivo

TEM micrographs of blood cells in vitro (A), or in vivo (B, C, D) post-GNP exposure. (A) GNPs bind to the surface of washed platelets after 15 minute of incubation. L-GNP shows highest binding and F-GNP⁻ shows no binding. (B) Circulating platelets show GNP uptake in OCS 1 hour post-injection in tumor bearing mice. (C) Except for F-GNP⁻ circulating RBCs show no interactions with GNPs. F-GNP⁻ bind to the RBC surface membrane. (D) All GNPs show uptake in circulating blood monocytes. GNPs are located in small vacuoles, a characteristic of monocytes. Black arrows indicate GNP localization.

Figure 3. Histological analysis of GNP tumor distribution over time

Light micrographs of silver enhanced tumor sections show GNP accumulation/distribution changes overtime with the type of GNP used. H&E counter-stain allows identification of various features of the tissue, including blood vessels. GNPs mainly accumulate around blood vessels (BV) at 1 and 8 hours, and distribute away from BV at 24 hours. Silver enhanced GNPs show up as black dots (indicated with arrows where not obvious). Enlarged images at 24hr show the spreading of GNPs around BV. Arrowheads indicate GNP binding to tumor cells. [Scale bar = 5 micron unless indicated otherwise]

Figure 4. Histological analysis of GNP liver distribution over time

Light micrographs of silver enhanced liver sections show GNP accumulation/distribution changes overtime with the type of GNP used. Sections were counter-stained with H&E for identification of various features of the tissue. Silver enhanced GNPs show up as black dots. GNPs mainly accumulate in Kupffer cells with rare binding to hepatocytes at 24 hours. L-GNP shows highest and F-GNP shows the least accumulation at all time points. Kupffer cells stain completely black in L-GNP condition due to high uptake of GNPs. L-GNP^TNF and F-GNP⁻ show similar distributions, which increase overtime. [Magnification = 40x, Scale bar = 20 micron]

Figure 5. Histological analysis of GNP spleen distribution over time

Light micrographs of silver enhanced spleen sections show GNP accumulation/distribution changes overtime with the type of GNP used. Sections were counter-stained with H&E for identification of various features of the tissue. Silver enhanced GNPs show up as black dots. The distribution of GNPs shifts from red-pulp (R) to follicular regions (F). “Anatomical zones” shows where majority of GNPs are located at each time point and the corresponding row shows high magnification image for each particle of that zone. Images were acquired at 100x. R, red pulp; M, marginal zone; F, follicle. [Scale bar = 10 micron unless indicated otherwise]