Binding Affinity and Transport Studies of Engineered Photocrosslinkable Affibody-enzyme-nanoparticle Constructs

Nanoparticle accumulation at tumor sites has been well reported , where targeting typically shows increased retention, but challenges remain for clinical translation. This work examines the effect of targeting ligand binding affinities and nanoparticle size on retention and transport through a solid tumor. We first show using cell affinity assays that modifying a wildtype (WT) anti-epidermal growth factor receptor (EGFR) affibody-enzyme fusion protein into a UV-photocrosslinkable (N23BP) version led to a significant decrease in affinity, whether as a free protein or as a conjugate to silica nanoparticles. Despite the reduced EGFR affinity, all protein conjugated nanoparticles showed binding and uptake to EGFR-overexpressing HTB9 bladder cancer cells as detected by confocal microscopy and flow cytometry. Next, transport studies of the protein conjugated nanoparticles using monoculture spheroids revealed that spheroid binding was higher for 17 nm particles bound with the WT proteins than N23BP, which was expected based on their respective values. However, the 17 nm particles conjugated with the photocrosslinkable N23BP affibody-enzymes showed an altered distribution profile that peaked further into the spheroid than the WT nanoparticle conjugates or in the absence of UV treatment. We correlate this finding with increased transport and retention of the photocrosslinked N23BP-nanoparticle conjugates in 3D spheroids to both the lower binding affinity of the affibodies for EGFR and the ability to introduce covalent linkages between the affibody and cell receptor. The larger 40 nm protein-conjugated nanoparticles showed limited penetration regardless of affinity or photocrosslinking on a 12 h timescale but did show overall increased transport after 24 h.