Multimodal nanoparticles have been extensively studied for target-specific imaging and therapy of various diseases, including cancer. In this study, radiolabeled arginine-glycine-aspartic acid (RGD)-functionalized Er 31 /Yb 31 co-doped NaGdF 4 upconversion nanophosphors (UCNPs) were synthesized and evaluated as a multimodal PET/MR/optical probe with tumor angiogenesisspecific targeting properties. Methods: A dimeric cyclic RGDyk ((cRGDyk) 2 ) peptide was conjugated to polyacrylic acid-coated NaGdF 4 :Yb 31 /Er 31 UCNPs along with polyethylene glycol molecules and was consecutively radiolabeled with 124 I. In vitro cytotoxicity testing was performed for 3 d. Upconversion luminescence imaging of (cRGDyk) 2 -UCNP was performed on U87MG cells with a laboratory-made confocal microscope. In vivo small-animal PET and clinical 3-T T1-weighted MR imaging of 124 I-labeled RGD-functionalized UCNPs was acquired with or without blocking of cyclic RGD peptide in a U87MG tumor model. Inductively coupled plasma mass spectrometry and biologic transmission electron microscopy were done to evaluate gadolinium concentration and UCNP localization, respectively. Results: Polymer-coated UCNPs and dimeric RGD-conjugated UCNPs were monodispersely synthesized, and those of hydrodynamic size were 30 6 8 nm and 32 6 9 nm, respectively. (cRGDyk) 2 -UCNPs have a low cytotoxic effect on cells. Upconversion luminescence signals of (cRGDyk) 2 -UCNP were specifically localized on the surface of U87MG cells. 124 I-c(RGDyk) 2 -UCNPs specifically accumulated in U87MG tumors (2.8 6 0.8 vs. 1.3 6 0.4 percentage injected dose per gram in the blocking experiment), and T1-weighted MR images showed significant positive contrast enhancement in U87MG tumors. Tumor localization of 124 I-c(RGDyk) 2 -UCNPs was confirmed by inductively coupled plasma mass spectrometry and biologic transmission electron microscopy analysis. Conclusion: These results suggest that 124 Ilabeled RGD-functionalized UCNPs have high specificity for a v b 3 integrin-expressing U87MG tumor cells and xenografted tumor models. Multimodal UCNPs can be used as a platform nanoparticle with multimodal imaging for cancer-specific diagnoses.

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Synthesis of biologically active antibody conjugated quantum dots (QDs) has been of great importance in cellular imaging and diagnostics. Cetuximab (or Erbitux) is the first monoclonal antibody drug which targets the epidermal growth factor receptor (EGFR) overexpressed in most cancer cells. In the present work, we investigated three different conjugation strategies to obtain the biologically functional QD-cetuximab conjugates for the tumor-specific imaging. Successful conjugation of cetuximab to QDs was achieved using PEG conjugated polymer-coated QDs and two long-chain heterobifunctional linkers, sulfo-LC-SPDP and sulfo-SMCC. The dissociation constant of the QD-cetuximab conjugates to EGFR was determined to be 0.61 +/- 0.28 nM. The cancer cell-specific binding ability of the QD-cetuximab conjugates was evaluated in vitro, and the cellular internalization of the QD-cetuximab conjugates was clearly demonstrated in live cells by confocal microscopy. The cellular imaging experiments using the QD-cetuximab conjugates showed a clear endocytosis pathway, which was evidenced by the colocalization of the QD-cetuximab conjugates with dye-labeled transferrin. These results suggest that the QD-cetuximab conjugates as an imaging modality for tumor EGFR overexpression can be expected to provide important information on the expression levels of EGFR on the cancer cells.

Imaging of specific intracellular target proteins in living cells has been of great challenge and importance for understanding intracellular events and elucidating various biological phenomena. Highly photoluminescent and water-soluble semiconductor nanocrystal quantum dots (QDs) have been extensively applied to various cellular imaging applications due to the long-term photostability and the tunable narrow emission spectra with broad excitation. Despite the great success of various bioimaging and diagnostic applications, visualization of intracellular targets in live cells still has been of great challenge. Nonspecific binding, difficulty of intracellular delivery, or endosomal trapping of nanosized QDs are the main reasons to hamper specific target binding in live cells. In this context, we prepared the polymer-coated QDs (pcQD) of which the surface was optimized for specific intracellular targeting in live cells. Efficient intracellular delivery was achieved through PEGylation and subsequent cell penetrating peptide (i.e., TAT) conjugation to the pcQD in order to avoid significant endosomal sequestration and to facilitate internalization of the QDs, respectively. In this study, we employed HEK293 cell line overexpressing endothelin A receptor (ET(A)R), a family of G-protein coupled receptor (GPCR), of which the cytosolic c-terminal site is genetically engineered to possess green fluorescent protein (GFP) as our intracellular protein target. The fluorescence signal of the target protein and the well-defined intracellular behavior of the GPCR help to evaluate the targeting specificity of QDs in living cells. To test the hypothesis that the TAT-QDs conjugated with antibody against intracellular target of interest can find the target, we conjugated anti-GFP antibody to TAT-PEG-pcQD using heterobifunctional linkers. Compared to the TAT-PEG-pcQD, which was distributed throughout the cytoplasm, the antiGFP-functionalized TAT-PEG-pcQD could penetrate the cell membrane and colocalize with the GFP. An agonist (endothelin-1, ET-1) treatment induced GFP-ET(A)R translocation into pericentriolar region, where the GFP also significantly colocalized with antiGFP-TAT-PEG-pcQD. These results demonstrate that stepwise optimization of PEG-pcQD conjugation with both a cell penetrating peptide and an antibody against a target of interest allows specific binding to the intracellular target protein with minimized nonspecific binding.