Intracellular protein target detection by quantum dots optimized for live cell imaging.

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.