Framework Nucleic Acid-Nanobody Fusion Probe-Based Pharmacokinetics Modulation and Analysis for Efficient Positron Emission Tomography Imaging

Nanobodies are promising for immunoPET imaging due to their excellent antigen recognition and tumor targeting, yet rapid clearance limits their tumor accumulation. Although multimerization and albumin binding can extend their circulation time and improve tumor targeting, a simple and universal method for creating protein multimers is still needed. Here, we leveraged the facile synthesis, controllable size, and precise assembly of DNA nanotechnology to construct CD47-targeted framework nucleic acid-nanobody fusion probes with multiple valences and sizes. Following comprehensive structural characterization, in vitro specificity assessment and <i>in vivo</i> PET/CT imaging analysis were conducted on a colorectal cancer LS174T mouse model. Furthermore, a pharmacokinetic model was developed and fitted with considerable <i>in vivo</i> data to prove its rationality, followed by testing the effects on tumor uptake prediction by changing different pharmacokinetic parameters. Indeed, by manipulating the size of the nucleic acid scaffolding and the number of attached nanobodies, we could precisely modulate the accumulation of probes at the tumor site. Overall, this study not only developed an efficient strategy for constructing nanobody multimers but also provided a pharmacokinetic model, allowing profound insight into the multidimensional data obtained experimentally and informing the design of future imaging probes with predictable delivery efficacies.