αvβ3 nanobody-conjugated PLGA nanoparticles for molecular imaging of angiogenesis

[Introduction] For effective diagnosis of pathologic tissues using functionalized nanoparticles, diverse ligands such as antibody, peptide, and aptamer, are being explored as promising recognition moieties that can bind to... [ view full abstract ]

[Introduction] For effective diagnosis of pathologic tissues using functionalized nanoparticles, diverse ligands such as antibody, peptide, and aptamer, are being explored as promising recognition moieties that can bind to selective targets with high affinity and specificity. Among them, antibody-based targeting strategies were widely used for imaging and delivering therapeutics. However, there are limitations of conventional antibodies including relatively large size and high Fc-mediated aspecific binding. Nanobodies (Nbs) are single-domain antigen-binding fragments (15 KDa) derived from camelid heavy-chain-only antibodies, showing unique characteristics such as small size, high stability and specificity. We have recently developed α_vβ₃ integrin-specific nanobodies (α_vβ₃-Nbs) as targeting ligand for α_vβ₃ integrin receptor, which is overexpressed at sites of angiogenesis in many solid tumors. In this work, we investigate their targeting capabilities functionalized with biocompatible nanoparticles for near-infrared (NIR) imaging of neovascularization related with a variety with tumor malignancy.

[Methods] PLGA nanoparticles loaded with ICG (PLGA NPs) were prepared by single-step nanoprecipitation methods and α_vβ₃-Nbs were subsequently conjugated on the surface of PLGA NPs by EDC/NHS reactions. The obtained α_vβ₃-Nbs functionalized PLGA nanoparticles (α_vβ₃-Nbs-PLGA NPs) were characterized by dynamic light scattering (DLS), zeta potential, transmission electron microscopy (TEM), UV-Vis spectroscopy, and XPS analysis. The targeting capabilities and cytotoxicity of α_vβ₃-Nbs-PLGA NPs were assessed both in vitro/in vivo in U87-MG cancer cells.

[Results and Discussion] 

The results showed that PLGA NPs and α_vβ₃-Nbs-PLGA NPs were dispersed as individual nanoparticles with a well-defined spherical shape and size distribution ranged from 180 to 200 nm, showing the effective ICG encapsulation and α_vβ₃-Nbs conjugation. The obtained α_vβ₃-Nbs-PLGA NPs exhibited the significant targeting capabilities compared with PLGA NPs in vitro and in vivo. The results suggest that α_vβ₃-Nbs serve as a great potential to target α_vβ₃ integrin receptor as an alternative ligand, and α_vβ₃-Nbs functionalized PLGA NPs can be used as non-invasive in vivo imaging of tumors and lesions related with α_vβ₃ integrin expression.