Synthesis of Stable Multifunctional Perfluorocarbon Nanoemulsions for Cancer Therapy and Imaging

Nanotechnology provides a promising platform for drug-delivery in medicine. Nanostructured materials can be designed with desired superparamagnetic or fluorescent properties in conjunction with biochemically functionalized moieties (i.e., antibodies, peptides, and small molecules) to actively bind to target sites. These multifunctional properties make them suitable agents for multimodal imaging, diagnosis, and therapy. Perfluorohexane nanoemulsions (PFH-NEs) are novel drug-delivery vehicles and contrast agents for ultrasound and photoacoustic imaging of cancer <i>in vivo</i>, offering higher spatial resolution and deeper penetration of tissue when compared to conventional optical techniques. Compared to other theranostic agents, our PFH-NEs are one of the smallest of their kind (<100 nm), exhibit minimal aggregation, long-term stability at physiological conditions, and provide a noninvasive cancer imaging and therapy alternative for patients. Here, we show, using high-resolution imaging and correlative techniques, that our PFH-NEs, when in tandem with silica-coated gold nanoparticles (scAuNPs), can be used as a drug-loaded therapeutic via endocytosis and as a multimodal imaging agent for photoacoustic, ultrasound, and fluorescence imaging of tumor growth

Synthesis of Stable Multifunctional Perfluorocarbon Nanoemulsions for Cancer Therapy and Imaging

Nanotechnology provides a promising platform for drug-delivery in medicine. Nanostructured materials can be designed with desired superparamagnetic or fluorescent properties in conjunction with biochemically functionalized moieties (i.e., antibodies, peptides, and small molecules) to actively bind to target sites. These multifunctional properties make them suitable agents for multimodal imaging, diagnosis, and therapy. Perfluorohexane nanoemulsions (PFH-NEs) are novel drug-delivery vehicles and contrast agents for ultrasound and photoacoustic imaging of cancer <i>in vivo</i>, offering higher spatial resolution and deeper penetration of tissue when compared to conventional optical techniques. Compared to other theranostic agents, our PFH-NEs are one of the smallest of their kind (<100 nm), exhibit minimal aggregation, long-term stability at physiological conditions, and provide a noninvasive cancer imaging and therapy alternative for patients. Here, we show, using high-resolution imaging and correlative techniques, that our PFH-NEs, when in tandem with silica-coated gold nanoparticles (scAuNPs), can be used as a drug-loaded therapeutic via endocytosis and as a multimodal imaging agent for photoacoustic, ultrasound, and fluorescence imaging of tumor growth

Single-Molecule Analysis of the Supramolecular Organization of the M₂ Muscarinic Receptor and the Gα_i1 Protein

G protein-coupled receptors constitute the largest family of transmembrane signaling proteins and the largest pool of drug targets, yet their mechanism of action remains obscure. That uncertainty relates to unresolved questions regarding the supramolecular nature of the signaling complex formed by receptor and G protein. We therefore have characterized the oligomeric status of eGFP-tagged M₂ muscarinic receptor (M₂R) and G_i1 by single-particle photobleaching of immobilized complexes. The method was calibrated with multiplexed controls comprising 1–4 copies of fused eGFP. The photobleaching patterns of eGFP-M₂R were indicative of a tetramer and unaffected by muscarinic ligands; those of eGFP-G_i1 were indicative of a hexamer and unaffected by GTPγS. A complex of M₂R and G_i1 was tetrameric in both, and activation by a full agonist plus GTPγS reduced the oligomeric size of G_i1 without affecting that of the receptor. A similar reduction was observed upon activation of eGFP-Gα_i1 by the receptor-mimic mastoparan plus GTPγS, and constitutively active eGFP-Gα_i1 was predominantly dimeric. The oligomeric nature of G_i1 in live CHO cells was demonstrated by means of Förster resonance energy transfer and dual-color fluorescence correlation spectroscopy in studies with eGFP- and mCherry-labeled Gα_i1; stochastic FRET was ruled out by means of non-interacting pairs. These results suggest that the complex between M₂R and holo-G_i1 is an octamer comprising four copies of each, and that activation is accompanied by a decrease in the oligomeric size of G_i1. The structural feasibility of such a complex was demonstrated in molecular dynamics simulations