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Surface Coating Directed Cellular Delivery of TAT-Functionalized Quantum Dots Yifeng Wei, Nikhil R. Jana,*,† Shawn J. Tan, and Jackie Y. Ying* Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669. Received September 6, 2008; Revised Manuscript Received June 18, 2009
TAT peptide functionalized shell-core ZnS-CdSe quantum dots (QDs) have been prepared by three different methods, direct ligand exchange with cysteine-terminated TAT (TAT-QDlig exch), and covalent conjugation to QDs coated with silanes (TAT-QDsilica) and polyacrylate derivatives (TAT-QDpolyacrylate). The silica and polyacrylate coatings incorporated multiple primary and secondary amines, introducing positive surface charges onto the QDs, providing high water solubility and sites for peptide conjugation, while inducing the “proton sponge effect”. The different coating methods produced particles of different sizes, surface charges, and colloidal stability; these factors jointly influenced the cellular uptake and subcellular localization of these particles. As the particle size increased, (TAT-QDlig exch (6 nm) < TAT-QDsilica (10 nm) < QDpolyacrylate (25 nm)), both the particle surface charge and cellular uptake increased. The smaller TAT-QDlig exch and TAT-QDsilica particles were localized mainly in the perinuclear regions, while the larger TAT-QDpolyacrylate particles were localized in both the perinuclear regions and the lysosomes. Compared to the other TAT-QDs, TAT-QDlig-exch has a lower colloidal stability and was more cytotoxic due to the weak binding of the ligands.
INTRODUCTION QDs are of interest to cell imaging due to their bright, sharp, stable, and tunable fluorescence, which can be excited by UV and visible light (1-4). Current research efforts are focused on greener QD syntheses (5), cadmium-free QDs (6), biocompatible coatings that allow for QD-bioconjugates (7-13), reduction of nonspecific binding (14), and bioaffinity molecules attachment to improve the labeling specificity and cellular trafficking of QDs (15-19). Although there have been many examples of QDbased labeling of fixed cells and tissues and of cell surface proteins, there has been limited success with live cell imaging using QDs (3, 4). One major difficulty is the delivery of QDbioconjugates into live cells intact, as QDs usually either aggregate within the cytoplasm due to the instability of their coating (9) or are trafficked to vesicles or endosomes (3, 4). Thus, there is a need to develop coatings that can protect QDs from aggregation within cells and facilitate endosomal escape (12). Cationic cell-penetrating peptides are widely used as cell transfection reagents for delivering macromolecules, nanoparticles, and QDs (20-36). Commonly used peptides include TAT peptide (a nuclear localization signal (NLS) peptide derived from the HIV-1 transcriptional activator protein TAT), SV40 NLS peptide, and oligoarginine or other variants (20-36). There are various ways to prepare peptide-functionalized QDs (31-36), e.g., biotin-streptavidin linkage (33, 36), covalent coupling with a terminal cysteine residue (34), self-assembly of terminal oligohistidine (35), or ligand exchange with cysteine-terminated peptide (32). The self-assembly and ligand-exchange methods are simple and produce smaller particles (80% labeling efficiency. Effect of Coating on QD-based Cell Labeling. TAT peptide has been used in the intracellular delivery of macromolecules, nanoparticles, and QDs (22-36). It generally enhances the cargo uptake and, in certain cases, causes it to localize in the perinuclear region. The delivery process starts with the binding of TAT to the plasma membrane due to its highly cationic nature associated with the arginine and lysine residues (20, 28). Arginine differs from other cationic amino acids, as it can make strong bidentate hydrogen bonds with sulfate, phosphate, and carboxylate anions via its guanidium group (20, 28). This feature leads to many possible interactions of TAT with the cell membrane, such as with the acidic regions of proteins, sulfated glycans, membrane phospholipid head groups, or a combination of these. Such interactions are responsible for the enhanced cellular interaction of TAT-QDs, as well as their membrane translocation. We noted that the uptake of TAT-QDs was significantly reduced at 4 °C, as observed for other TAT-conjugated cargos,
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Figure 4. Fluorescence micrographs of TAT-QDsilica labeled HepG2 cells, showing the time-dependent migration of QDs toward the nucleus. Cell nucleus was colocalized with Hoechst 33342 dye (blue) in all cases. A QD concentration of 20 nM was employed. Figure 3. Fluorescence micrographs of TAT-QD labeled HepG2 cells. Green QDs were used for the TAT-QDlig exch and TAT-QDpolyacrylate systems, and orange QDs were used for the TAT-QDsilica system. Cell nucleus was colocalized with Hoechst 33342 dye (blue) in all cases. For TAT-QDpolyacrylate, the lysotracker red was used to label lysosome, in addition to the Hoechst dye. A QD concentration of 20 nM and an incubation time of 12 h were employed for all cases.
suggesting that the uptake was an active process (28). A variety of endocytic routes has been suggested earlier for TAT-mediated cargo delivery, such as caveolae-mediated endocytosis of TATprotein and localization in the perinuclear region via actin cytoskeleton-mediated mechanism (25), clathrin-mediated endocytosis of TAT-peptide (27), lipid raft dependent macropinocytosis of TAT-protein (26), and macropinocytosis-directed uptake of TAT-QD via vesicle generation from plasma membrane, followed by active transport of QD-containing vesicle from cell periphery to the perinuclear region (36). The cargo size and nature, surface charge of TAT-cargo conjugate, and nature of cell are important factors determining the endocytic pathway taken. Earlier studies showed that the endocytosis mechanism is particle size dependent (49, 50). Clathrin- and caveolin-dependent endocytosis usually occurs with
