In vivo fluorescence energy transfer imaging of nanoparticle behavior in tumor-bearing mice

Introduction: In the last decade self-assembled lipidic nanoparticles (SALNPs) have shown broad applicability as intravenously injectable agents for biomedical purposes. SALNPs can serve as molecular imaging probes as well as delivery vehicles for agents, ranging from cytostatic agents to proteins and small interfering RNAs (siRNA). However, little is known about the dissociation (kinetics) of SALNPs upon entering the circulation and subsequent trafficking of the individual SALNP components. Despite the fact that for successful delivery of the imaging agents/therapeutic molecules it is crucially important to understand decomposition dynamics, studies addressing this issue are scarce. Here, we present an innovative near infrared (NIR) in vivo fluorescence imaging method based on Förster resonance energy transfer (FRET) [1] to visualize the dissociation and delivery dynamics of SALNPs in tumor bearing mice in vivo. Methods and Results: PEGylated NIR SALNPs with Cy7-labeled lipids that contain a NIR emitting quantum dot core (QD710-Cy7-PEG) were synthesized (Fig. a). The large spectral separation between QD and Cy7-labeled lipids allows tracking the nanoparticle components simultaneously, while FRET between the QD-core donor and the Cy7-lipid acceptor enables sensitive and semi-quantitative monitoring of the dissociation of the lipid corona from the QD core in the NIR region.(Fig. b) [2] QD710-Cy7-PEG was injected in tumor bearing mice to image their trafficking, delivery and intactness by whole body NIRF imaging (Fig. c) and intravital microscopy (S. Fig. 1). We observed QD710-Cy7-PEG to be dynamic structures that progressively disintegrate due to a lipid exchange process after intravenous administration. Upon vascular extravasation and accumulation in the tumor interstitium this process continued. In tumor, QD signal kept increasing over 24 h due to dequenching, while the FRET signal decreased gradually, indicating the dissociation of the nanoparticles and the migration of coating lipids (Fig. d). By injecting this probe in the periphery of solid tumors, we followed the lymphatic drainage and observed the dissociating dynamics of the nanoparticles in the sentinel lymph node(S. Fig. 2). In case of the QD core SALNPs used in the current study, cellular internalization caused the QD cores to sequester in the tumor, lymph nodes and MPS organs, while their coating lipids partially followed different clearance kinetics in the circulatory system and were also be cleared renally.[3] Conclusion: Our multi-faceted strategy to assess the in vivo stability by FRET imaging is flexible and applicable to a wide variety of SALNPs, including other type of lipid-based nanoparticles, lipid-polymer hybrid nanoparticles and lipoprotein-derived nanoparticles. It represents a modular in vivo optical imaging tool to visualize the behavior of self-assembled nanoparticles in real-time and may contribute to enhancing the therapeutic outcome or improving the molecular imaging signature of this widely used class of nanoparticles. (Figure Presented).