Framework\nNucleic Acid-Nanobody Fusion Probe-Based\nPharmacokinetics Modulation and Analysis for Efficient Positron Emission\nTomography Imaging

Nanobodies\nare promising for immunoPET imaging due to their excellent\nantigen recognition and tumor targeting, yet rapid clearance limits\ntheir tumor accumulation. Although multimerization and albumin binding\ncan extend their circulation time and improve tumor targeting, a simple\nand universal method for creating protein multimers is still needed.\nHere, we leveraged the facile synthesis, controllable size, and precise\nassembly of DNA nanotechnology to construct CD47-targeted framework\nnucleic acid-nanobody fusion probes with multiple valences and sizes.\nFollowing comprehensive structural characterization, in vitro specificity\nassessment and <i>in vivo</i> PET/CT imaging analysis were\nconducted on a colorectal cancer LS174T mouse model. Furthermore,\na pharmacokinetic model was developed and fitted with considerable <i>in vivo</i> data to prove its rationality, followed by testing\nthe effects on tumor uptake prediction by changing different pharmacokinetic\nparameters. Indeed, by manipulating the size of the nucleic acid scaffolding\nand the number of attached nanobodies, we could precisely modulate\nthe accumulation of probes at the tumor site. Overall, this study\nnot only developed an efficient strategy for constructing nanobody\nmultimers but also provided a pharmacokinetic model, allowing profound\ninsight into the multidimensional data obtained experimentally and\ninforming the design of future imaging probes with predictable delivery\nefficacies.