The recent breakthrough advances on nucleic acid-based therapies serve as a glimpse into the tremendous potential of this therapeutic approach. Indeed, this class of therapeutic agents have been exploited to address a wide variety of oncological and gene defect-related diseases. Furthermore, nucleic acid-based therapies have gained prominence in the specific area of prophylactic vaccination of infectious diseases [1]. However, the intrinsic lability of these nucleic acids in the presence of physiological nucleases often leads to their premature degradation, severely hampering their potential delivery in an unprotected form [2]. In this context, nanomedicines, and more precisely polymeric nanoparticles (NPs), have become a go-to vector for both DNA and RNA in vivo delivery [3]. Among the possible polymeric carriers, linear poly(β-aminoesters) (L-PBAEs, see Scheme A), a well-established family of biocompatible and biodegradable polymers [4], are particularly promising as vectors in this regard. Due to the presence of basic amine moieties, the cationic (protonated) form of such polymer is capable of complexing the negatively charged genetic material in the form of nanometric polyplexes known to exhibit a high matrix-assisted encapsulation effectiveness.4a In addition, their overall high transfection efficiency can be further fine-tuned by a wide range of synthetic modifications to achieve more cell-type selective formulations [5], either at the poly(β-amino ester) backbone or at the polymer termini [6]. We have been actively working in the field, including the development of a wide library of bioinspired linear oligopeptide-modified PBAEs (OM-PBAEs). This new family of poly(β-aminoesters) differs from the PBAEs reported by other research groups [7] on the use of these end-terminal oligopeptides as cationic moieties [2,9a,9b] The introduction of a peptide-based moiety resulted in a clear advantage thanks to the low toxicity and biocompatibility associated to the peptides in comparison with other polyamine synthetic fragments. Besides, the high tendency of peptides like Lys, Arg and His to be protonated at ensodomal pH makes these peptides as candidates of choice for this application. The protonation is specifically important after the endocytic uptake of polyplexes to ensure the endosomal escape by virtue of the proton sponge effect [8]. Next, expression of the encoded nucleic acids is properly achieved [2]. Consequently, these OM-PBAE polymers demonstrated quite useful for applications as varied as the protection of mRNAs for vaccination purposes [2], the encapsulation of interference RNAs for cardiovascular diseases treatment,[9c] the incorporation of the various gene editing systems (CRISPR/Cas) for the treatment of rare monogenic diseases,[9d] the formulation of hydrogels for breast cancer local siRNA treatment or their use for microneedles loading of immunomodulatory components,[9b among others.[9e,9f] To keep advancing these L-PBAE systems in their translational preclinical phases, it would be highly valuable to gain accurate insights of their in vivo pharmacokinetic profile. We have recently reported a subcellular-level super resolution microscopy (dSTORM) tracking study of the hydrolytic kinetics of these systems once they reach the cytoplasm [10]. However, tracking the behavior of the polymeric vector on an in vivo level would be particularly informative. In this context, Positron Emission Tomography (PET), a non-invasive nuclear imaging technique widely used in clinical setting, could provide valuable information for this purpose. PET is an ultra-sensitive and fully quantitative technique that enables the quantification of sub-pharmacological amounts of radiolabeled species at the whole-body level. We considered that the most appropriate radiotracer tool to this end could be a silent 18F-labelling linear poly(β-aminoester). Interestingly, despite the enormous number of studies showing the indisputable therapeutic efficacy of the PBAE family, the capacity of this compound class as tracking agents has not yet been explored. Among the possible 18F-labelling strategies, our attention was drawn away from the common 18F‑carbon bond-forming processes, focusing instead on the [19F]-to-[18F]-fluor isotopic exchange using ammonium trifluoroborate functionalities (AMBF3). These types of prosthetic groups, introduced by the Perrin laboratory [11], allow for efficient, single-step, aqueous 18F-labelling using directly the cyclotron-generated aqueous [18F]fluoride, thus avoiding time-consuming evaporation/solvent exchange steps. We note that this approach is highly complementary to the more classical click-based radiolabelling used in the context of PET imaging, a topic that has been reviewed by Elsinga and co-workers [12]. Herein, we describe the synthesis and 18F efficient radiolabeling of PBAE derivative (called C32) and its use, for the first time for this compound class, as PET-tracking vector probe. As a proof of concept, the incorporation of the newly designed and synthesized 18F-C32-PBAE into a model nanoformulation demonstrates to be fully compatible with the formation of the polyplexes, their biophysical characterization, and all their in vitro and in vivo functional features, an essential requirement for an ideal PET-oriented radiotracer.