Nanoparticle-based drug delivery systems have been developed to improve the efficacy and reduce the systemic toxicity of a wide range of drugs. Although clinically approved nanoparticles have consistently shown value in reducing drug toxicity, their use has not always translated into improved clinical outcomes. This has led to the development of “multifunctional” nanoparticles, where additional capabilities like targeting and image contrast enhancement are added to the nanoparticles. However, additional functionality means additional synthetic steps and costs, more convoluted behavior and effects in vivo, and also greater regulatory hurdles. The trade-off between additional functionality and complexity is the subject of ongoing debate and the focus of this Review.

Endothelial vascular adhesion molecule-1 (VCAM-1) is a critical component of the leukocyte–endothelial adhesion cascade, and its strict temporal and spatial regulation make it an ideal target for imaging and therapy. The goal of this study was to develop novel VCAM-1–targeted imaging agents detectable by MRI and fluorescence imaging using phage display–derived peptide sequences and multimodal nanoparticles (NPs). We hypothesized that VCAM-1–mediated cell internalization of phage display–selected peptides could be harnessed as an amplification strategy to chaperone and trap imaging agents inside VCAM-1–expressing cells, thus improving target-to-background ratios. To accomplish our goal, iterative phage display was performed on murine endothelium under physiological flow conditions to identify a family of VCAM-1–mediated cell-internalizing peptides. One specific sequence, containing the VHSPNKK motif that has homology to the α-chain of very late antigen (a known ligand for VCAM-1), was shown to bind VCAM-1 and block leukocyte–endothelial interactions. Compared with VCAM-1 monoclonal antibody, the peptide showed 12-fold higher target-to-background ratios. A VHSPNKK-modified magnetofluorescent NP (VNP) showed high affinity for endothelial cells expressing VCAM-1 but surprisingly low affinity for macrophages. In contrast, a control NP without VCAM-1–targeting sequences showed no affinity for endothelial cells. In vivo, VNP successfully identified VCAM-1–expressing endothelial cells in a murine tumor necrosis factor-α–induced inflammatory model and colocalized with VCAM-1–expressing cells in atherosclerotic lesions present in cholesterol-fed apolipoprotein E apoE^−/−mice. These results indicate that: (1) small peptide sequences can significantly alter targeting of NPs, (2) the used amplification strategy of internalization results in high target-to-background ratios, and (3) this technology is useful for in vivo imaging of endothelial markers.

Molecular beacons are increasingly being used in many applications involving nucleic acid detection and quantification. The stem-loop structure of molecular beacons provides a competing reaction for probe-target hybridization that serves to increase probe specificity, which is particularly useful when single-base discrimination is desired. To fully realize the potential of molecular beacons, it is necessary to optimize their structure. Here we report a systematic study of the thermodynamic and kinetic parameters that describe the molecular beacon structure-function relationship. Both probe and stem lengths are shown to have a significant impact on the binding specificity and hybridization kinetic rates of molecular beacons. Specifically, molecular beacons with longer stem lengths have an improved ability to discriminate between targets over a broader range of temperatures. However, this is accompanied by a decrease in the rate of molecular beacon-target hybridization. Molecular beacons with longer probe lengths tend to have lower dissociation constants, increased kinetic rate constants, and decreased specificity. Molecular beacons with very short stems have a lower signal-to-background ratio than molecular beacons with longer stems. These features have significant implications for the design of molecular beacons for various applications.