We demonstrate that quantum dots injected into two model tumors rapidly migrate to sentinel lymph nodes. PEG-coated quantum dots having terminal carboxyl, amino, or methoxyl groups all migrated from the tumor to surrounding lymph nodes similarly. Passage from the tumor through lymphatics to adjacent nodes could be visualized dynamically through the skin; at least two nodes could usually be defined. Imaging during necropsy confirmed confinement of the quantum dots to the lymphatic system and demonstrated easy tagging of sentinel lymph nodes for pathology. Examination of the sentinel nodes identified by quantum dot localization showed that at least some contained metastatic tumor foci.

We report that a symmetric small molecule ligand mediates the assembly of antibody light chain variable domains (VLs) into a correspondent symmetric ternary complex with novel interfaces. The L5* Fluorogen Activating Protein (FAP) is a VL domain that binds malachite green dye (MG) to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two VL domains. Unliganded L5* VL domains crystallize as a similar antiparallel VL/VL homodimer. The complementarity determining regions (CDRs) are spatially oriented to form novel VL/VL and VL/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable VL/MG/VL complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two VL domains into a single protein tightens MG binding over 1,000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high affinity protein tags and capture reagents.

Quantum dots are a powerful fluorophore family with desirable attributes for fluorescence imaging. They have been used in several animal models with direct clinical relevance, including sentinel lymph node mapping, tracing vasculature and lymphatics, and targeting specific lesions for diagnosis and removal [1][2][3][4][5][6][7][8][9][10][11][12] . Despite significant interest for use in translational applications, little is known about the persistence and long-term fate of quantum dots in vivo. We have observed fluorescence of quantum dots injected into Balb/c and nude mice for up to two-years post injection using both wholebody and microscopic fluorescence techniques. Two-photon spectral microscopy was used to verify the existence of quantum dots within two-year tissues, but also revealed a range of significantly blue shifted emission peaks with increased bandwidths. Systemically administered quantum dots persist and retain fluorescence for up to two-years in vivo, but with significantly blue-shifted emission. KeywordsQuantum dots; in vivo; imaging; whole animal fluorescence; microscopy; two-photon; spectral analyses Quantum dots were supplied by Quantum Dot Corporation, now a division of Invitrogen Corporation (Eugene, OR). For the experiments reported here, 655nm emitting ZnS capped CdSe quantum dots were used exclusively. The coating was the standard primary amphiphilic polymer coat 13 , with mPEG-5000 conjugated to the outer coating, analogous to the Q-Tracker non-targeted quantum dots available from Invitrogen (Eugene, OR). These quantum dots are rod-shaped with an aspect ratio of ~2.2:1 which has previously been confirmed by Transmission Electron Microscopy (TEM) 14 .All animal experiments were approved by the IACUC at Carnegie Mellon University. Mice (BALB/c and nude) were obtained from Harlan Sprague Dawley (Indianapolis, IN). Nude mice were used to minimize any background due to intrinsic fluorescence from fur. Quantum dots (typically 480 picomoles, ~170 μg cadmium ion; equivalent to 8.5mg/kg as Cd ++ , about 2 lethal doses of Cd ++ as CdCl 2 i.v. 15 ) were injected by tail vein. Whole-animal imaging was as described; 3 briefly, images were acquired using a either a Photometrics C258 deep-cooled †fitzp@andrew.cmu.edu. Brattleboro, VT). Imaging times varied with time post-injection, and were adjusted on the fly to give maximum dynamic range, but were typically 0.5s; acquisition times of 0.2, 1.0, and 5s were sometimes used to bracket the large range in intensities between different sites of deposition or to reveal faint fluorescence.All spectral measurements were undertaken using an inverted Axio Observer Z1 motorized microscope with an LSM 510 Meta NLO confocal scan head (Carl Zeiss, Jena, Germany) coupled to a Chameleon Ultra-II femtosecond titanium sapphire laser (Coherent Laser Inc., Santa Clara, CA) as the two-photon excitation source. Once a particular area within a given tissue sample had been identified by both brightfield (targeting of specific cellular structures, e.g., germinal cente...