The long-term objective of this R21 proposal is to develop a new class of nanoparticle (NP)-based positron emission tomography (PET) probes that enable high-performance tumor imaging. We propose to adopt a pretargeted imaging strategy to decouple the NP components from their corresponding radiolabeled reporters. First, a pair of tumor-targeting NP component and radiolabeled reporter with desired PKs will be synthesized separately via rational molecular designs. We will then modulate the interplay between other experimental variables such as injection times and dosage in order to achieve optimal PET imaging outcomes. A prerequisite to successful pretargeted imaging is to accomplish selective and irreversible coupling of the tumor- targeting NP and sequentially injected radiolabeled reporter in vivo. We thus exploit the use of a bioorthogonal conjugation chemistry based on a pair of reactive motifs, i.e., trans-cyclooctene (TCO) and tetrazine (Tz), which have fast reaction kinetics and biological stability. Future progress in PET imaging will involve designing molecular imaging probes that preferentially accumulate in tumors. Aside from small molecule and affinity ligand-based PET imaging probes, NPs exhibiting unique enhanced permeability and retention (EPR) effects represent a new category of PET probes capable of passively targeting leaky vasculature - a universal characteristic observed for most solid tumors. While a variety of NP PET probes have been examined in pre-clinical setting, challenges remain to further improve tumor uptake and reduce nonspecific distribution in other organs. In our molecular design, the TCO motif is covalently attached onto a polymer building block of supra-molecular nanoparticle (SNP). Self-assembly of the molecular building blocks leads to encapsulation of TCO to yield TCO-encapsulated SNP (TCO?SNP) as the tumor-targeting NP component. Further, the radiolabeled reporter is composed of the complementary Tz motif and 18F-tag. In the proposed PET imaging study, TCO?SNP is first administered to an animal. When the TCO?SNPs approach their optimal accumulation in tumor, the radiolabeled reporter is then injected. In vivo bio-orthogonal reaction occurs instantaneously, resulting in high-contras PET imaging. Our joint team has some preliminary data supporting the feasibility of this new class of NP PET imaging probes. We will implement the following two Specific Aims to accomplish our research endeavors, 1) Prepare and select TCO?SNPs and radiolabeled reporters with optimal PKs, and 2) In vivo demonstration of pretargeted PET imaging using pairs of TCO?SNPs and radiolabeled reporters. This proposal brings together the expertise of four research groups (PI and 3 co-investigators) covering the fields of supramolecular chemistry, nanoparticle, radiochemistry, molecular imaging and cancer biology. We envision that the successful demonstration of our proposed research could change current paradigm in oncologic PET imaging, and open up new opportunities for pretargeted drug delivery.