Vascular Accessibility of Endothelial Targeted Ferritin Nanoparticles

Abstract

Targeting nanocarriers to the endothelium, using affinity ligands to cell adhesion molecules such as ICAM-1 and PECAM-1, holds promise to improve the pharmacotherapy of many disease conditions. This approach capitalizes on the observation that antibody-targeted carriers of 100 nm and above accumulate in the pulmonary vasculature more effectively than free antibodies. Targeting of prospective nanocarriers in the 10-50 nm range, however, has not been studied. To address this intriguing issue, we conjugated monoclonal antibodies (Ab) to ICAM-1 and PECAM-1 or their single chain antigen-binding fragments (scFv) to ferritin nanoparticles (FNPs, size 12 nm), thereby producing Ab/FNPs and scFv/FNPs. Targeted FNPs retained their typical symmetric core-shell structure with sizes of 20-25 nm and ∼4-5 Ab (or ∼7-9 scFv) per particle. Ab/FNPs and scFv/FNPs, but not control IgG/FNPs, bound specifically to cells expressing target molecules and accumulated in the lungs after intravenous injection, with pulmonary targeting an order of magnitude higher than free Ab. Most intriguing, the targeting of Ab/FNPs to ICAM-1, but not PECAM-1, surpassed that of larger Ab/carriers targeted by the same ligand. These results indicate that (i) FNPs may provide a platform for targeting endothelial adhesion molecules with carriers in the 20 nm size range, which has not been previously reported; and (ii) ICAM-1 and PECAM-1 (known to localize in different domains of endothelial plasmalemma) differ in their accessibility to circulating objects of this size, common for blood components and nanocarriers.

Figure 1

Quantitative analysis of number of radiolabeled whole antibody and scFv moieties conjugated using HPLC. HPLC radiotrace of a) radiolabeled-IgG conjugated to ferritin. The IgG^I125/FNP conjugate peak is indicated with number (1) and an overlay of (2) HPLC absorbance trace of IgG (red color). Green colored dashed line indicates the cut-off line used for calculation of areas under the curve (AUC) for conjugate vs. free antibody peaks. Area under the curve (AUC) for b) conjugated vs. free antibody. HPLC radiotrace of c) radiolabeled-scFv conjugated to ferritin. The scFv^I125/FNP conjugate peak indicated is with number (1) and an overlay of (2) HPLC absorbance trace of scFv (red color). Area under the curve (AUC) for c) conjugated vs. free scFv.

Figure 2

TEM images and size analysis of FNPs. TEM images of a) FNP, b) anti-ICAM Ab/FNP, c) anti-PECAMscFv/FNP, Scale bar: 100 nm (a, b), and 50 nm (c). Particle size analysis (size distribution by number) obtained by DLS for d) FNP, anti-ICAM Ab/FNP, and anti-PECAMscFv/FNP. Illustration of e) FNP, f) whole antibody/FNP and g) scFv antibody fragment/FNP conjugates. Size and polydispersity index (PDI) for h) FNP and IgG/FNP. DLS frequency curve for i) FNP and IgG/FNP.

Figure 3

Binding of targeted FNPs to ICAM positive, PECAM positive, and negative REN cells, as well as to HUVECs. FNP was ¹²⁵I-labelled prior to antibody conjugation. Cells were grown to confluence and incubated with ferritin nanoparticles for 1 h at 37 °C. Bound radiolabeled nanoparticles were measured by gamma counter. Binding of a) anti-ICAM Ab/FNP and c) anti-ICAMscFv/FNP to REN and REN-ICAM cells. Binding of b) anti-PECAMscFv/FNP to REN and REN-PECAM cells. Binding of d) free ferritin was examined on HUVECs against IgG/FNP. Ferritin binding to HUVECs was evaluated using e) human anti-ICAM Ab/FNP and f) human anti-PECAM Ab/FNP.

Figure 4

Targeting of FNPs to ICAM-1. a) Biodistribution of ¹²⁵I-labeled anti-ICAM mAb and IgG FNPs in mice at 30 min. Tissue uptake is indicated as mean ± SEM (n=3). b) Localization ratio of selected organs. Significant differences determined by t-test with Bonferroni correction to account for multiple comparisons.

Figure 5

Targeting of FNPs to PECAM-1. a) Biodistribution of ¹²⁵I-labeled anti-PECAM mAb and IgG FNPs in mice at 30 min. Tissue uptake is indicated as mean ± SEM (n=3). b) Localization ratio of selected organs. Significant differences determined by t-test with Bonferroni correction to account for multiple comparisons.