Fluorescent nanodiamonds engage innate immune effector cells: A potential vehicle for targeted anti-tumor immunotherapy.

Fluorescent nanodiamonds (FNDs) are nontoxic, infinitely photostable, and emit fluorescence in the near infrared region. Natural killer (NK) cells and monocytes are part of the innate immune system and are crucial to the control of carcinogenesis. FND-mediated stimulation of these cells may serve as a strategy to enhance anti-tumor activity. FNDs were fabricated with a diameter of 70±28 nm. Innate immune cell FND uptake, viability, surface marker expression, and cytokine production were evaluated in vitro. Evaluation of fluorescence emission from the FNDs was conducted in an animal model. In vitro results demonstrated that treatment of immune cells with FNDs resulted in significant dose-dependent FND uptake, no compromise in cell viability, and immune cell activation. FNDs were visualized in an animal model. Hence, FNDs may serve as novel agents with "track and trace" capabilities to stimulate innate immune cell anti-tumor responses, especially as FNDs are amenable to surface-conjugation with immunomodulatory molecules.

Wide-field in vivo background free imaging by selective magnetic modulation of nanodiamond fluorescence.

The sensitivity and resolution of fluorescence-based imaging in vivo is often limited by autofluorescence and other background noise. To overcome these limitations, we have developed a wide-field background-free imaging technique based on magnetic modulation of fluorescent nanodiamond emission. Fluorescent nanodiamonds are bright, photo-stable, biocompatible nanoparticles that are promising probes for a wide range of in vitro and in vivo imaging applications. Our readily applied background-free imaging technique improves the signal-to-background ratio for in vivo imaging up to 100-fold. This technique has the potential to significantly improve and extend fluorescent nanodiamond imaging capabilities on diverse fluorescence imaging platforms.

Silica encapsulation of fluorescent nanodiamonds for colloidal stability and facile surface functionalization.

Fluorescent nanodiamonds (FNDs) emit in the near-IR and do not photobleach or photoblink. These properties make FNDs better suited for numerous imaging applications compared with commonly used fluorescence agents such as organic dyes and quantum dots. However, nanodiamonds do not form stable suspensions in aqueous buffer, are prone to aggregation, and are difficult to functionalize. Here we present a method for encapsulating nanodiamonds with silica using an innovative liposome-based encapsulation process that renders the particle surface biocompatible, stable, and readily functionalized through routine linking chemistries. Furthermore, the method selects for a desired particle size and produces a monodisperse agent. We attached biotin to the silica-coated FNDs and tracked the three-dimensional motion of a biotinylated FND tethered by a single DNA molecule with high spatial and temporal resolution.

Quantitative characterization of fluorophores in multi-component nanoprobes by single-molecule fluorescence.

Multi-modal nanoparticles incorporating fluorophores are increasingly being used for medical applications. The number of fluorophores incorporated into the nanoparticles during synthesis is stochastic, leaving some nanoparticles devoid of fluorophores. Determining the number, the brightness and the photostability of the fluorophores incorporated, and the percentage of labeled nanoparticles (labeling efficiency) remains challenging. We have determined the aforementioned quantities for two synthesized multi-modal nanoparticles by exploiting the photobleaching of fluorophores at the single-molecule level using a total internal reflection fluorescence microscope. Labeling efficiency was determined by fitting the distribution of incorporated fluorophores with a statistical model and verified by independent experiments.

Trafficking of a dual-modality magnetic resonance and fluorescence imaging superparamagnetic iron oxide-based nanoprobe to lymph nodes.

PURPOSE: This study aims to develop and characterize the trafficking of a dual-modal agent that identifies primary draining or sentinel lymph node (LN). PROCEDURE: Herein, a dual-reporting silica-coated iron oxide nanoparticle (SCION) is developed. Nude mice were imaged by magnetic resonance (MR) and optical imaging and axillary LNs were harvested for histological analysis. Trafficking through lymphatics was observed with intravital and ex vivo confocal microscopy of popliteal LNs in B6-albino, CD11c-EYFP, and lys-EGFP transgenic mice. RESULTS: In vivo, SCION allows visualization of LNs. The particle's size and surface functionality play a role in its passive migration from the intradermal injection site and its minimal uptake by CD11c+ dendritic cells and CD169+ and lys+ macrophages. CONCLUSIONS: After injection, SCION passively migrates to LNs without macrophage uptake and then can be used to image LN(s) by MRI and fluorescence. Thus, SCION can potentially be developed for use in sentinel node resections or for intralymphatic drug delivery.

Macromolecular and dendrimer-based magnetic resonance contrast agents.

Magnetic resonance imaging (MRI) is a powerful imaging modality that can provide an assessment of function or molecular expression in tandem with anatomic detail. Over the last 20-25 years, a number of gadolinium-based MR contrast agents have been developed to enhance signal by altering proton relaxation properties. This review explores a range of these agents from small molecule chelates, such as Gd-DTPA and Gd-DOTA, to macromolecular structures composed of albumin, polylysine, polysaccharides (dextran, inulin, starch), poly(ethylene glycol), copolymers of cystamine and cystine with GD-DTPA, and various dendritic structures based on polyamidoamine and polylysine (Gadomers). The synthesis, structure, biodistribution, and targeting of dendrimer-based MR contrast agents are also discussed.

Synthesis of a cross-bridged cyclam derivative for peptide conjugation and 64Cu radiolabeling.

The increased use of copper radioisotopes in radiopharmaceutical applications has created a need for bifunctional chelators (BFCs) that form stable radiocopper complexes and allow covalent attachment to biological molecules. Previous studies have established that 4,11-bis-(carbo- tert-butoxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (H 2CB-TE2A), a member of the ethylene "cross-bridged" cyclam (CB-cyclam) class of bicyclic tetraaza macrocycles, forms highly kinetically stable complexes with Cu(II) and is less susceptible to in vivo transchelation than its nonbridged analogue, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA). Herein, we report a convenient synthesis of a novel cross-bridged BFC that is structurally analogous to CB-TE2A in that it possesses two coordinating acetate arms, but in addition possesses a third orthogonally protected arm for conjugation to peptides and other targeting agents. Application of this strategy to cross-bridged chelators may also enable the development of even further improved agents for (64)Cu-mediated diagnostic positron emission tomography (PET) imaging as well as for targeted radiotherapeutic applications.