Am. led to the development of a diverse array of nanocarriers, which include liposomes; YC-1 (Lificiguat) dendrimers; polymeric micelles; silica; metallic nanoparticles (e.g. silver, gold, iron oxide); and carbon nanotubes.4C6 Almost all currently existing nanocarriers require the use of carrier materials, such as amphiphiles, to solubilize their cargo. Recently, we introduced a new class of nanoparticles, whereby amphiphilic functional dyes such as the near-infrared fluorescent dye Indocyanine Green (ICG) and the photosensitizer Protoporphyrin IX (PpIX) (i.e., YC-1 (Lificiguat) clinically-used functional materials) are used to drive the formation of stable nanoemulsions, without the use of any additional amphiphilic polymers, lipids, or surfactants.7,8 Hydrophobic materials such as superparamagnetic iron oxide nanoparticles (SPIONs) can be encapsulated in the nanoemulsions to confer additional functionality. It has also been shown that small-molecule hydrophobic drugs can be packaged into nanoemulsions using a similar approach.9 These novel dyestabilized nanoemulsions allow for extremely high drug payloads and have been shown to exhibit improved efficacy compared with free drug 9 and even analogous micelle carriers, due to their exceptional stability and reduced drug leakage.8 The attachment of targeting ligands to nanoparticles is desirable because it has the potential to increase both tumor accumulation and specificity, and ultimately the therapeutic index.10C12 While many nanoparticles rely primarily on enhanced permeability and retention (EPR) for preferential accumulation at tumor sties,13 active targeting is generally preferred to more specifically deliver drugs to the desired cell type based on its molecular profile through ligand-receptor or antibody-antigen interactions.14,15 Targeting has also been shown to trigger cellular uptake for more effective delivery of drug to intracellular targets.16C18 Despite the benefits of targeting, low bioconjugation efficiencies, high batch-to-batch variability, and the inability to control the orientation and density of the targeting ligands on the nanoparticle surface slows clinical translation.9,19,20 Dye-stabilized nanoemulsions have yet to be functionalized with any disease-associated targeting ligands. The surface chemistry and chemical handles available for bioconjugation are dependent on the dye used and for some dyes no chemical handle is available for subsequent bioconjugations. Preferably, a bioorthogonal chemical Mouse monoclonal to CRKL handle would be available for the attachment of targeting ligands via click-chemistry.21 Click chemistry is a highly efficient and specific reaction chemistry that has become the preferred approach for bioconjugations. One of the most popular click chemistry reactions occurs between an azide and a constrained alkyne, with efficiencies nearing 100%, without copper catalysts.22 Herein, we describe a strategy for the site-specific and efficient attachment of targeting ligands onto carrier-free, ICG- and PpIX-stabilized nanoemulsions. Azide-handles for click-chemistry were introduced onto the surface of dye-stabilized nanoemulsions, by first preparing azidemodified variants of ICG and PpIX (Figure 1). The azide was introduced near the hydrophilic sulfate and carboxyl groups of ICG and PpIX, YC-1 (Lificiguat) respectively, to increase the likelihood that it would be exposed to the surrounding aqueous medium and available for subsequent conjugations. The structure of the azide variants was confirmed by ESI-MS and 1H NMR (Supporting information, Section S1 and Figure S1). The absorbance and fluorescence spectra of the azide variants and the free dyes YC-1 (Lificiguat) (in DMSO) were identical (Figure S2). Azide-functionalized nanoemulsions were formed by first dissolving the azide-dyes at a 1:20 molar ratio with unmodified dye in Dimethyl sulfoxide (DMSO). This dye mixture was combined with SPION (SPIONs, diameter = 7.6 1.0 nm; Figure S3) in toluene at a ratio of 1 1:1 w/w. No additional amphiphiles or carrier.