ARTICLE pubs.acs.org/bc
Intracellular Protein Target Detection by Quantum Dots Optimized for Live Cell Imaging Youngseon Choi,*,† Keumhyun Kim,† Sukmin Hong,† Hichul Kim,‡ Yong-Jun Kwon,‡ and Rita Song*,† †
Chemical Biology Laboratory Discovery Biology Group Institut Pasteur Korea (IP-K), 696 Sampyeong-dong, Bundang-gu, Seongnam-Si, Gyeonggi-Do, 463-400, South Korea
‡
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bS Supporting Information ABSTRACT: 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 (ETAR), 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 welldefined 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-ETAR 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.
’ INTRODUCTION 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 in cell biology and drug discovery.1 Highly photolumminescent and water-soluble semiconductor nanocrystal quantum dots (QDs) have been extensively exploited for the various bioimaging applications because of their narrow spectra, tunable fluorescence, and long-term photostability.25 Despite the great success in biological applications, QDs without proper surface modification suffer from nonspecific binding as well as instability in the cells, resulting in incapability of subcellular targeting in complex intracellular environments.6,7 Endosomal trapping and subsequent transport to the perinuclear region has been known to hamper the specific targeting of the functionalized nanoparticles.8,9 This inherent r 2011 American Chemical Society
endocytosis process of nanosized QDs and the active machinery of the endocytosis trafficking system remain problems for the application of QDs toward efficient intracellular labeling of specific targets in live cells.5,1014 Recent advancements in conjugation chemistry and live cell imaging techniques, however, have allowed us to significantly address these issues. Nonspecific binding of QDs can be significantly reduced by appropriate PEGylation (7506000 Da) because of the well-known stealth effect of the PEG molecules.15,16 Bentzen et al. also reported the efficacy of PEGylated QDs in HEK and 3T3 cell lines, where they found that the nonspecific uptake of Received: March 15, 2011 Revised: June 14, 2011 Published: June 30, 2011 1576
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Bioconjugate Chemistry QDs depends on cell types.17 Most recently, Tan et al. showed that the cellular interaction of oleyl-modified QDs (2050 nm) depends on the balance between surface charge and hydrophobicity.18 Numerous studies have corroborated the fact that the cellular interaction of nanoparticles depends on the size, charge, shape, and surface properties,14,19,20 reflecting the importance of surface chemistry as well as complexity of nanoparticle cell interactions. With the reduced nonspecific binding and cytotoxicity of the PEGylated QD surface, various efforts have been made to image and track cell-surface membrane proteins such as integrins,21 folate receptors,22 neurotransmitter receptors,23 epidermal growth factor receptor (EGFR),24,25 and G-protein coupled receptors (GPCRs)26 using the PEG-QDs conjugated with receptor-specific small molecules or antibodies. The QDs used in these studies are designed to bind the receptors on the cellular surface membrane, where the QDs conjugated with small ligands or antibodies are delivered into cells via receptormediated endocytosis. This approach, however, may not be employed for directly detecting targets of interest if the targets exist in the cytosol. Therefore, another approach has been implemented to deliver various nanoparticles into cells using a cell-penetrating peptide (CPP).2731 In particular, a CPP derived from human immunodeficiency virus-1 transactivator (TAT) protein, or TAT peptide, has been widely used to facilitate delivery of various nanoparticles into cells, where TAT-conjugated QDs has been known to be uptaken via macropinocytosis, followed by accumulation in perinuclear region.3234 Though intracellular distribution, cytotoxicity as well as mechanistic studies of the QDs have been extensively performed, few studies have yet explored the specific detection of targets located inside living cells. This may be potentially due to the difficulty of avoiding nonspecific uptake which interferes the correct interpretation of target localization by live cellular imaging. Herein, we report the study on the detection of the specific intracellular target protein in live cells based on TAT-conjugated QDs. First, to find optimum surface chemistry for polymercoated QD (pcQD) which allows minimization of nonspecific uptake and cytotoxicity, we investigated several aspects of cellular interaction of pcQD in live cells. The surface functionality, concentration, incubation time, and cytotoxicity were considered important factors for affecting the intracellular behavior of QDs. We performed controlled PEGylation of pcQDs (PEG-pcQDs), followed by stepwise modification with TAT and an antibody against a specific target protein. To demonstrate the specific binding capability of the QD conjugate, we employed the HEK293 cell line overexpressing green fluorescent protein (GFP) as our intracellular target of interest, which is genetically engineered to be tethered to the cytosplasmic c-terminal site of endothelin A receptor (ETAR). ETAR is one of two mammalian receptor subtypes (ETAR and ETBR) of 7-transmembrane GPCR family, widely present in most cells and tissues related with cardiovascular and renal diseases and cancer.35,36 Given the biological complexity of living cells, we designed a simplified cellular model system, where the target is a fluorescent protein and the intracellular behavior of the target can be easily detected by fluorescence imaging. Thus, we constructed the fluorescent target protein which is fused with the cytoplasmic region of the GPCR. This cellular model may help us to identify the specific binding of QD probe to the target via colocalization of the GFP signal with the different-colored
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QD signals (red emission in this study). Also, the well-defined mechanisms of cell-surface expression, internalization, and agonist-induced translocation of the GPCR allows us to confirm the specific binding of the probe in a reliable manner.37,38 Further, the emerging interest of GPCRs as a major therapeutic target in drug discovery may provide additional significance for this study using a cellular model overexpressing GPCR transmembrane protein. Using confocal fluorescence microscopy, we could observe the PEG-pcQDs modified with TAT and anti-GFP antibody specifically bind the intracellular target GFP in live cells. The antiGFPTAT-PEG-pcQD which initially colocalized with the GFP at the cellular membrane also translocated with the GFP after the ET-1 agonist treatment in agreement with the characteristic of the GPCR. These results suggest that the controlled functionalization of PEGylated pcQD with both TAT and an antibody against a specific target protein can afford efficient cellular entry of QDs as well as specific detection of an intracellular target without significant nonspecific uptake or binding.
’ EXPERIMENTAL SECTION Materials. Cadmium nitrate (99.99%), sodium myristate (99%), hexamethyldisilathiane ((TMS)2S), diethylzinc (ZnEt2), trioctylphosphine (TOP, 90%), trioctylphosphine oxide (TOPO, 99%), 1-octadecene (ODE), selenium powder (99.9%), poly(acrylic acid) (PAA, M.W. = 1800 g/mol), and octylamine (99%) were purchased from Aldrich. All organic solvents were purchased from Junsei (Japan). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and sodium tetraborate were purchased from Sigma. Monoamino methoxy PEGs (PEG molecular weights 750, 2000, 5000 Da) and diamino-PEG5000 were purchased from SunBio (Korea). Alexa Fluor 634-labeled human transferrin conjugate (AF634-transferrin), 20 -(4-ethoxyphenyl)5-(4-methyl-1-piperazinyl)-2,50 -bi-1H-benzimidazole trihydrochloride trihydrate (Hoechst33342), and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) were obtained from Invitrogen (USA). Sulfo-succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) and sulfosuccinimidyl 6-(30 -[2-pyridyldithio]-propionamido)hexanoate (sulfo-LC-SPDP) were purchased from Pierce (Thermo Scientific, USA). Monoclonoal anti-GFP antibody was obtained from Calbiochem (USA). The cystein-modified cell penetrating peptide TAT possessing the sequence Ac-RKKRRQRRRPPQCCONH2 (Nterminal acetyl, C-terminal amide capping; c-TAT) was obtained from Peptron Inc. (Daejon, Korea). All the chemicals were used without further purification. All the solutions were prepared using deionized water (18 MΩ) or autoclaved water. General Characterization Methods. All absorption spectra were obtained in a quartz cuvette using Cary 5000 UVvis spectrometer (Varian Inc., CA, USA) with a spectral range of 200800 nm, a scan rate of 600 nm/min, and a slit width of 2 nm. Agarose gel electrophoresis was performed with 1% agarose in 0.5 Tris-borate EDTA buffer (pH 8.0) at 25 V cm1 for 50 min. The resulting gel image was obtained using LAS 3000 gel imager (Fuji, Japan). Dynamic light scattering (DLS) measurements were performed using Nano-ZS (Malvern, UK) at room temperature. NH2-PEG Modification of Carboxylated Polymer-Coated QDs. Polymer-coated QDs possessing carboxyl terminal groups (pcQD) were prepared according to the procedure described in the literature.36 The pcQDs (100 pmol) were reacted with 1577
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Bioconjugate Chemistry Scheme 1. Synthetic Scheme for Anti-GFP AntibodyConjugated TAT-PEG-pcQDa
a
Diamino-PEG5k modified pcQD (NH2-PEG-pcQD, 1) was reacted with sulfo-SMCC to give SMCC-PEG-pcQD 2, followed by coupling reaction with cystein-modified TAT peptide. The partially modified TAT-PEG-pcQD 3 was further reacted with SPDP-modified antiGFP 4 to yield antiGFP-TAT-PEG-pcQD 5. As a control, antiGFP-pcQD without TAT was synthesized by reacting 2 with 4 to yield antiGFPPEG-pcQD.
20 000 equiv of diamino-PEG5k in 10 mM borate buffer (pH 8) in the presence of 5000 equiv of EDC for 16 h at room temperature. The reaction mixture was purified by membrane filtration using Microcon (MWCO, 100 kDa, Milipore) with 5 centrifugation in deionized water. The final conjugate (NH2-PEG-pcQD, 1) in Scheme 1 was characterized using agarose gel electrophoresis for the electrophoretic mobility and DLS for the hydrodynamic size. The QD concentration was determined by the absorption at the first exciton peak using the extinction coefficent according to the literature.39,40 TAT Peptide Conjugation of NH2-PEG-pcQD. To conjugate TAT peptide to the amine-functionalized PEG-pcQD, 100 pmol of QD 1 was first reacted with 5000 equiv of sulfo-SMCC in 1 mL of 1PBS (10 mM phosphate buffer, 150 mM NaCl, pH 7.4) for 1 h at room temperature before purification with membrane filter (MWCO 100 kDa, Milipore). Twenty picomoles of SMCCmodified PEG-pcQDs (SMCC-PEG-pcQD, 2) was then added to the cystein-modified TAT peptide (c-TAT; QPPRRRQRRKKR;
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1 nmol) in 200 μL of 1PBS buffer and reacted for 1 h at room temperature. The partially modified TAT-PEG-pcQD 3 conjugate was purified five times with 1PBS by using 100 kDa MWCO membrane filter. The filtrate was collected and concentrated for determination of the number of TAT molecules per QD using BCA assay. Anti-GFP antibody Conjugation of TAT-PEG-pcQD. First, the anti-GFP antibody (molecular weight 150 kDa, 67 pmol) was activated with 1000 equiv of sulfo-LC-SPDP in 1PBS for 30 min at room temperature. To avoid oxidation of the resulting thiol group of the SPDP-modified anti-GFP antibody (SPDPantiGFP, 4), 0.1 M DTT (10 μL) was added and reacted for an additional 10 min and the conjugate was purified three times with 1 PBS using 30 kDa MWCO membrane filter. Finally, 4 equiv of 4 was added to the partially modified TAT-PEG-pcQD 3 and incubated for 3 h at room temperature with gentle shaking. The reaction mixture was then purified using a size exclusion column filled with S-200 resin. The fluorescent fractions monitored by hand-held UV lamp for QD fluorescence were collected and concentrated using 100 kDa MWCO membrane filter in 1PBS before agarose gel electrophoresis analysis and DLS measurement. Cell Culture. The human epidermal carcinoma cell line HeLa was cultured at 37 °C in a humidified atmosphere and 5% CO2 in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 IU/mL streptomycin (all from Invitrogen). HEK293 cells were cultured in DMEM/F12 medium supplemented with 10% fetal calf serum, 1% penicillin streptomycin, and 1% Geneticin under humidified air containing 5% CO2 at 37 °C. Construction of a Stable HEK293 Cell Line Expressing GFP-Tagged ETAR. A human endothelin A receptor (ETAR) cDNA clone (www.cDNA.org) was fused to the eGFP coding sequence of the pEGFP-N1 vector (Clontech, USA) and ligated in the pCDNA3.1(-) vector. The resulting endothelin A-EGFP DNA was cloned, checked by sequencing, and used for transfection of HEK293 cells using standard protocols. A recombinant clone was obtained through several selections with 1% of v/v Geneticin (Invitrogen). Cytotoxicity Test. MTT assay was performed to evaluate the cytotoxicity of the pcQDs at various concentrations (10 nM to 1 μM). HeLa cells were incubated with the QDs in a dosedependent manner for 1 and 5 days and the percentage of viable cell was measured by MTT assay. At each time point, MTT (0.5 mg/mL) was added to each well, and the plates were incubated at 37 °C for 4 h. Spectrophotometric data were obtained by Victor3 microplate reader (Perkin-Elmer, USA) at 531 nm. The MTT assay was performed in duplicates for each condition. Live Cell Imaging. Cellular uptake of the QDs in live cells was investigated by confocal laser scanning fluorescence microscope (LSM5 Live, Carl Zeiss) in HeLa cell line. HeLa cells (5000 cells/ well) were seeded in glass-bottomed 8-well chamber slide (LabTek) with 0.4 mL DMEM medium. For the study on nonspecific endocytosis of the QDs, the cells were treated with various concentrations of the QDs (10500 nM) and incubated up to 24 h at 37 °C in the CO2 incubator. For the targeting of the intracellular GFP tethered to the c-terminal region of the ETAR, antiGFP-TAT-PEG-pcQD conjugate solution (2.5 μM) was added to the HEK293 cells expressing GFP-tagged ETAR with the final concentration 10 nM for 30 min. For endosomal labeling, 1 μL of AF634-transferrin (1 mg/mL in 1PBS) was 1578
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Figure 1. Gel electrphoresis analysis for various QD conjugation reactions: (A) conjugation of pcQD with diamno-PEG5k, (B) sulfo-SMCC conjugation to NH2-PEG-pcQD 1, (C) c-TAT conjugation to SMCC-PEG-pcQD 2. 1% agarose gel was run for 50 min at 50 V in 0.5TBE buffer (pH 8).
also added to the cell medium. After incubation for 20 min at 37 °C, the cells were washed three times with fresh DMEM medium. Fluorescence Microscopy and Image Analysis. Fluorescence images of the cells were obtained with the confocal microscope at 37 °C in a live cell chamber using a 63/1.4 NA oil immersion objective and scan speed of 15 frame/s. For the excitation of QD, 488 Ar-ion laser line was used with emission bandpass filter (550615 nm), while the AF634transferrin conjugate was excited with a 633 HeNe laser and observed with a long pass filter (650 nm). The confocal fluorescence images were processed using Zen 2008 software (Carl Zeiss).
’ RESULTS AND DISCUSSION Synthesis and Characterization of AntiGFP-TAT-pcQD Conjugate 5. Conjugation of anti-GFP antibody to TAT-
PEG-pcQD 3 was performed using two different heterobifunctional linkers, sulfo-SMCC and sulfo-LC-SPDP, as shown in Scheme 1. Diamino-PEG Conjugation to pcQD. The carboxyl groups of the pcQD (50 pmol) were reacted with various amounts of diamino-PEG5k (50100 k equiv) in the presence of EDC (25 nmol). The mobility of pcQD was highly reduced after conjugation of PEG molecules due to the decrease of negative charges and the increase of the size of the pcQD. The pcQD activated with EDC alone were destabilized and aggregated, while PEGylated pcQDs 1 showed high stability in various buffer conditions showing the decrease in mobility with the increase in molar ratios of the PEG to pcQD (Figure 1A). DLS analysis also showed the increase of hydrodynamic diameter from 12 ( 1 nm (pcQD) to 14 ( 1.5 nm. The electrophoretic mobility was maximized at the molar ratios of PEG over 10k, with the apparent mobility of 2.0 109 cm V1 s1. Considering the potential cytotoxic effects, the decreased negative charges inducing colloidal instability, and the higher content of PEGs, we used
NH2-PEG-pcQD 2 prepared with 1:500 molar ratio of QD to PEG for further reaction. See Supporting Information Figure S1 for the agarose gel electrophoresis characterization of PEGylated pcQDs with different molecular weights of PEGs. Sulfo-SMCC Conjugation to QD 1. The N-hydroxyl succinimide group of the sulfo-SMCC reacts with the amino groups of QD 1. The agarose gel electrophoresis exhibited the increasing mobility of sulfo-SMCC activated QD 2 with the increase of the molar ratio of sulfo-SMCC to QD 1 (1004000) (Figure 1B). It may be due to the fact that the overall negative charges relatively increase as the SMCC reacts with amino groups of QD 1. On the basis of this result, the molar ratio of 1000 (SMCC to QD 1) was used for further conjugation. TAT Peptide Conjugation to Sulfo-SMCC-PEG-QD 2. QD 2 bearing an optimized number of SMCC moieties was used for both TAT and anti-GFP antibody conjugation. We first performed a titration experiment using c-TAT with the increasing molar ratios of the c-TAT to QD 2 (202000 equiv). The mobility of QD 2 gradually decreased as more c-TAT molecules reacted with QD 2 (Figure 1C) due to the positively charged arginine-rich TAT peptide. This result allowed us to determine the amount of c-TAT per QD 2 for partial TAT modification, while the remaining SMCC groups can be further conjugated to the anti-GFP antibody. In the molar ratio of 200, we found that approximately 50 TAT peptides were conjugated to QD 2, which was determined by BCA protein assay. Anti-GFP Antibody Conjugation to TAT-PEG-pcQD 3. To minimize the steric hindrance of antibody, we introduced a long chain heterobifunctional linker, sulfo-LC-SPDP, to the anti-GFP antibody to afford antibody 4. The 4-fold molar excess of SPDPanti-GFP antibody 4 was then allowed to react with QD 3 for 3 h, followed by purification with S-200 size exclusion column equilibrated with 1PBS (pH 7.4) to remove unreacted antiGFP antibodies. The successful conjugation of antibody to QD 3 was confirmed through both agarose gel electrophoresis and 1579
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Figure 2. Characterization of anti-GFP antibody-conjugated TAT-PEG-pcQD using agrose gel electrophoresis (A,B) and dynamic light scattering analysis (C). (A) Fluorescence image of the gel with lane 1: NH2-PEG-pcQD 1 (Em = 600 nm), lane 2: SMCC-pcQD 2, lane 3: partially modified TATPEG-pcQD 3, lane 4: antiGFP-PEG-pcQD, lane 5: antiGFP-TAT-pcQD 5, lane 6: anti-GFP antibody (10 μg). 1% agarose gel was run at 50 V for 60 min in 0.5TBE buffer (pH 8). The loading amount of QD was 13 pmol per well, and the same gel was consecutively stained with colloidal blue staining kit. (C) The conjugates showed the size range of 1819 nm, compared with the starting NH2-PEG-pcQD 1 (∼14 nm). The values were the average of five measurements in 1PBS at room temperature.
Figure 3. Comparison of nonspecific uptake of various concentrations of unmodified pcQD (A) with NH2-PEG-pcQD (B). The green QDs (emission peak at 525 nm) were incubated with live HeLa cells for 3 h before confocal fluorescence imaging. The images are the overlay of differential interference contrast (DIC) image (for cell morphology), green channel (for QDs) and red channel (for endosomes labeled with AF634-transferrin). Scale bar = 20 μm.
dynamic light scattering analysis (Figure 2). The electrophoretic mobility of the antiGFP-TAT-PEG-pcQD (lane 5) slightly increased when compared to the TAT-PEG-pcQD (lane 3), and also showing increasing hydrodynamic size (19 ( 1.5 nm). Importantly, high purity of antiGFP-TAT-PEG-pcQD 4 was clearly shown after the column purification, which can be compared with the free antibody control (lane 6) stained with colloidal blue. The number of TAT peptides and anti-GFP
antibodies per QD was determined to be 50 ( 5 and 2 ( 0.8, respectively, based on the calibration curve of antibody and TAT peptide according to BCA assay. Nonspecific Uptake of NH2-PEG-pcQD versus Unmodified pcQD. The nonspecific endocytosis of unmodified polymercoated QDs with carboxyl groups are problematic in the case where the specific binding to a target of interest should be observed in cellular imaging. To avoid these undesirable 1580
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Figure 4. Twenty-four hour incubation of unmodified pcQD (left, 50 nM) and PEG-pcQD (middle and right, 100 nM). The green pcQDs (emission peak at 525 nm) were accumulated predominantly in the perinuclear region and colocalized with late endosomal vesicles. The endosomes were labeled with the red AF634-transferrin conjugate (1 μg/mL). N indicates nucleus region marked with dotted line. The 3D projection of the boxed area in the middle image is represented in the right with only green and red channels for clarity. The scale bar indicates 10 μm.
Figure 5. MTT assay of unmodified pcQD (black bar) and NH2-PEGpcQD (gray bar) for cytoxicity evaluation.
interactions of nanoparticles with cells, various PEG molecules have been used in biology, pharmaceutics, and polymer chemistry.41 Figure 3 shows the comparison of the nonspecific uptake of PEGylated pcQDs with the unmodified pcQD after 3 h incubation in live HeLa cells. The uptake of the pcQD with carboxyl surface (pcQD) was first observed at 10 nM, significantly accumulating in the cells at 50 nM. However, the PEGylated pcQD (NH2-PEGpcQD 1) exhibited no significant cellular uptake in the concentration range 150 nM. However, at a higher concentration range (100200 nM), the PEGylated pcQD showed a slight cellular uptake, though the number of cells showing the QD signal was less than 1% of total cell population (∼10 000 cells per well). The uptake of PEG-pcQD 1 was hardly observed in the cell even after 24 h incubation at a concentration of 50 nM, which is in contrast with the abundant distribution of carboxylated pcQDs in the cytoplasm, mostly in the perinuclear region (Figure 4). A few PEG-pcQDs 1 found in the cytoplasm were not colocalized with endosomes labeled with AF634-transferrin. Three-dimensional cellular image analysis clearly showed the separate location of PEG-pcQDs from endosomes, indicating that the PEGylation helps to avoid not only nonspecific cellular uptake, but also endosomal sequestration. In addition, the effect of different molecular weights of PEG molecules (750, 2000, 5000) on the cellular uptake of pcQDs was negligible (see Supporting Information Figure S2). Cytotoxicity of PEGylated pcQD. To quantitatively assess the cytotoxicity of NH2-PEG-pcQD 1 compared with pcQD, MTT assay was performed according to the standard protocol. The unmodified pcQDs showed significant cytotoxicity with an effective concentration at 50% cell survival (EC50) of ∼120 nM (Figure 5). The NH2-PEG-pcQDs revealed less cytotoxic
effect than pcQD overall, while exhibiting similar cytotoxicity with the unmodified pcQD in the range 1050 nM. This result indicates that the amino-PEG can reduce the cytotoxicity of pcQD. It is worth noting that the survival ratio of the cell in the presence of PEG-QD was kept in a constant range (∼60%) at the entire concentration range tested (10500 nM). However, the cytotoxicity of pcQD remarkably increased depending on the concentration. In a separate experiment, we also prepared methoxy PEG-modified pcQD which showed significantly reduced cytotoxicity (see the Supporting Information for the preparation of methoxy PEG-pcQD). Efficient Intracellular Delivery of PEG-pcQDs via TAT Peptide. The significantly reduced nonspecific binding and the low cytotoxicity of the PEG-pcQDs enabled us to pursue an efficient intracellular delivery using a well-known cell penetrating peptide, human immunodeficiency virus-1 transactivator protein (TAT) peptide. The arginine-rich TAT peptide (RKKRRQRRRPPQ) has been successfully used to promote the internalization of a range of polymeric or inorganic nanoparticles into living cells.5,28,42,43 Figure 6 showed the cellular uptake of TAT-PEG-pcQD 3 which was remarkably facilitated, exhibiting characteristic punctate distribution of the QDs throughout the cytoplasm even at 1 nM after 30 min incubation. This facilitated delivery of the PEGylated pcQDs is attributed to the presence of the cationic TAT, which has been known to enhance the interactions with negatively charged cellular membranes.31 In the live cell experiment using HeLa cells, the QDs delivered via TAT peptide were not significantly colocalized with endosomes labeled with AF634-transferrin (red fluorescence). This observation potentially indicates that the TAT-PEG-pcQDs enter into cells via the pathway independent of clathrin-mediated endocytosis because transferrin has been well-documented for the clathrin-mediated internalization pathway.43 Though still being elucidated for the mechanisms of nanoparticle uptake via cell penetrating peptides, recent reports have shown that TAT conjugation to streptavidin-QDs allows for the QDs to follow lipid-dependent macropinocytosis and active transport into cytosol.29,32,33 Specific Targeting of Cytoplasmic GFP. Despite the proven efficiency of TAT-mediated facilitated delivery of pcQDs, TATPEG-pcQDs alone cannot recognize any specific intracellular targets since they are easily taken up into cells and nonspecifically distributed in the cytoplasm in live cells. However, we hypothesized that the TAT-PEG-pcQD conjugated with an antibody against a specific target protein can find the intracellular target in 1581
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Figure 6. Facilitated intracellular delivery of PEGylated pcQDs mediated by TAT peptide. (Left) Live HeLa cells were incubated with varying concentration of the TAT-PEG-pcQD (150 nM) for 30 min at 37 °C before confocal imaging. The representative images at each concentration (1, 5, 20, 50 nM) were the overlays of DIC, green channel for QD, and red channel for endosomes labeled with transferrin-AF634 conjugates. (Right) To quantify the green channel fluorescence per cell, the average pixel areas per cell (n = 1015) were obtained from the z-section images (10 μm depth with 1 μm interval) stacked in Zen 2008 software using ImageJ plug-in (v 1.43).
Figure 7. Targeting of the GFP tagged at the c-terminal cytoplamic site of the endothelin A receptor (ETAR) of HEK293 cells using antiGFP-TATPEG-pcQD 5 conjugate (20 nM, emission peak at 605 nm). (AC) After 30 min incubation, the red QD 5 were delivered to the plasma membrane (white arrows), preferentially localized with GFP signal as indicated by overlaid yellow color; QDs are often observed in the cytosol (red arrow); see Supporting Information Figures S3 and S4 for the separate channel images to identify QD colocalizing with GFP. QDs seemingly entrapped in the plasma membrane were moving rapidly along the membrane or traversing the filipodia of the cells (A; dotted line with asterisk) or along the cellular contacts (C; dotted line with asterisk); see the Supporting Information for the movies (Movie S1 for A and Movie S2 for C). (D,E) Control cell images at the same conditions with the antiGFP-PEG-pcQD (without TAT) showing nonsignificant endocytosis (D) and with the TAT-PEG-pcQD displaying punctuate and evenly distributed cellular uptake mostly in the cytoplasm (E). The images are overlays of blue channel (Hoechst33342 for nucleus), green channel (GFP), and red channel (pcQD). The scale bars correspond to 10 μm.
live cells. We tested this hypothesis by employing the GFP-ETAR cell model, where the GFP as our target protein is designed to be tethered at the cytoplasmic c-terminal site of the transmembrane receptor ETAR. To allow for the TAT-PEG-pcQD to target the GFP located in the intracellular region after membrane
penetration, we conjugated anti-GFP antibody to the TATPEG-pcQD using heterobifunctional linker strategy as schematically shown in Scheme 1. As shown in Figure 7AC, antiGFP-TAT-PEG-pcQDs were initially bound to the plasma membrane (white arrows) and 1582
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Figure 8. Confocal microscope images of live GFP-ETAR expressing HEK293 cells incubated with 30 nM antiGFP-TAT-PEG-pcQD (red) for 1 h, followed by an agonist treatment (40 nM ET-1) for additional 1 h (A). The colocalization of the red QD mostly with the GFP cluster shown in yellow color was observed in the pericentriolar region. Notice the concomitant decrease of membrane GFP signal, as the GFP-ETAR translocated. The characteristic perinuclear clustering of the translocated GFP-ETAR was shown (B) along with the untreated HEK293 expressing GFP-ETAR (C). The images are overlays of blue channel (Hoechst33342 nuclear stain), green channel (GFP), and red channel (QD). The scale bars correspond to 10 μm.
some QDs were also observed in the cytoplasm (red arrow) after 30 min incubation at 37 °C. To ensure that the QDs are specifically bound with the target GFP of our interest, the control cellular images were also obtained. The antiGFP-PEG-pcQD (without TAT) showed negligible cellular uptake due to the presence of PEG molecules (Figure 7D). As expected, the nontargeted TAT-PEG-pcQD (without antiGFP) showed punctate and even distribution mostly in the cytoplasm with no significant colocalization with GFP or membrane (Figure 7E). These results indicate that the localization of the antiGFP-TATPEG-pcQD in the membrane region can be the result of the facilitated internalization of the QDs owing to TAT conjugation and target binding due to the presence of anti-GFP antibody. Since our target GFP is tethered to the membrane receptor ETAR and the anti-GFP antibody is bound to QD, it may be difficult to bind efficiently to each other because of steric hindrance. Accordingly, we noticed that some of GFP signal seemed not to colocalize with the QD, which may be due to the decreased affinity of the conjugated GFP antibodies. In addition, the faster photobleaching of GFP compared to the stable QD signal during the confocal imaging, which is an inherent problem of most fluorescent proteins, could not make complete colocalization of the QD with the GFP signal. Despite this, as noticed from the separate fluorescence channel images of Figure 7AC in the Supporting Information (Figure S3 and S4), it seemed more evident that the majority of the QD signal was colocalized with the GFP. In addition, the separate channel image of the TAT-PEG-pcQD control (Figure 7E) clearly exhibited cytoplasmic distribution mainly surrounding the nucleus with no significant QD signal at the membrane region (see the Supporting Information Figure S5). It is noteworthy that a portion of the antiGFP-TAT-PEGpcQD also appeared colocalized with the GFP in the cytoplasmic endosomal structures as indicated by the red arrows (Figure 7B, C), while some remained on the cellular membrane. This may be related with heterogeneous expression of GFP-ETAR, which is also in agreement with data in the literature, where CFP- or YFPtagged ETAR distribution were found mainly on plasma membrane and slightly within endosomal structure.44 Time-Course Imaging. We also performed the time-course imaging to monitor the intracellular behavior of the antiGFP-TATPEG-pcQD which is associated with GFP. Tracking of QDs in live cells in real time may provide information on the mechanism of binding of the QD probe to the target given the possibilities of
receptor-mediated endocytosis or nonspecific endocytosis (nonTAT mediated); for example, the receptor-mediated endocytosis leads to perinuclear accumulation of QD-receptor after 30 min to 1 h (data not shown), while the nonspecific endocytosis requires at least 3 h to show the similar phenomena as demonstrated in Figure 4. During the 30 min to 1 h period of imaging time, we found that QD controls without anti-GFP antibody showed negligible binding (PEG-pcQD) or facilitated uptake (TAT-PEG-pcQD) into the cytoplasm. Unlike these control QDs, the colocalized antiGFP-TAT-QD and GFP were moving rapidly along the membrane with an average speed of 0.15 μm/s (n = 10) at the membrane region. Some are traversing between the two cells through the filipodia of the cells (Figure 7A; dotted line with asterisk) or along the cell periphery (Figure 7C; dotted line with asterisk); see the Supporting Information movie for lateral movement of QD along the membrane: Movie S1 for Figure 7A and Movie S2 for Figure 7C. These types of lateral movement on the membrane were not observed in the case of the TAT-PEG-pcQD or other cases of nonspecific endocytosis using, i.e., carboxylated QDs. This spatiotemporal information from the time-course imaging further indicated that the localization of the antiGFPTAT-PEG-pcQD is associated with the intracellular target GFP. Despite the limit of optical resolution of confocal fluorescence microscope for the plasma membrane with a thickness of approximately 10 nm, it is technically challenging to differentiate the location of QDs at the inner membrane region from the outer membrane region. However, the unique photochemical properties of QD such as great brightness and resistance to photobleaching allowed for imaging the lateral movement of the antiGFP-TAT-PEG-pcQD in the membrane region. Interestingly, these features of QD movements seem to reflect the dynamic nature of GPCR on the plasma membrane as described in the literature.38,4548 It has been reported that various GPCRs including β-adrenergic and GABA receptors, and TGF β-receptors have shown surface mobility with the lateral diffusion coefficients ((112) 109 cm2/s).45,4951 Though the measurement of the exact diffusion coefficient of the GFP-ETAR is not within the scope of this study, our relevant data (0.15 μm/s of QD movement along the membrane) apparently yields 5.6 1010 cm2/s, when applied with a free diffusion equation; diffusion length = 4Dt, where D is diffusion coefficient (cm2/s) and t is the time (s). It is noteworthy that the present GPCR tracking method can be applied to GPCR-related drug screening if the condition for quantifying the endocytosis of GPCR is established. 1583
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Bioconjugate Chemistry Ligand-Induced Translocation of Target Protein. To further confirm that QD 5 is specifically bound with the GFP of the ETAR, we treated the cells with a well-known agonist, endothelin-1 (ET-1), which stimulates the ETAR to be internalized and translocated into pericentriolar recycling endosomes.5254 It was reported that endocytosis of ETAR occurs in an agonist-dependent manner, and the ETAR bound to ET-1 remains a stable complex for at least 2 h in the cell.55 As shown in Figure 8, ET-1 treatment on the cells preincubated with QD 5 (30 nM) for 1 h resulted in the decrease of membrane GFP signal and the increase of perinuclear GFP signal. The majority of the red QDs colocalized with the green ETAR cluster as appeared in yellowish color in the pericentriolar region (Figure 8A). This is attributed to the ET-1 induced ETAR translocation from cellular membrane to perinuclear region, which allows for the GFP tethered to ETAR to translocate together. See the Supporting Information Figure S5 for separate channel images for identification of colocalization. Also, we found that a small portion of QDs are still present in the membrane region at the different focal plane. This may be due to a heterologous translocation pattern of the ETAR, or potential oligomerization of the ETAR, leading to the aggregation on the surface.38 See the Supporting Information Figure S6 for the zsection images of Figure 8A. For comparison purposes, the characteristic perinuclear clustering of the translocated ETAR after ET-1 treatment was shown in Figure 8B. Also, no activated translocation of GFP-ETAR without agonist treatment during the 2 h was observed in the naïve cells (Figure 8C). These results suggest that the QD signal is associated with ET-1-dependent ETAR receptor pathway, confirming the specific binding of the antiGFP-TAT-PEG-pcQD to the target GFP.
’ CONCLUSIONS Specific intracellular target detection in live cells using QD was demonstrated by the antibody-functionalized TAT-PEG-pcQD prepared by stepwise optimization of the conjugation of pcQDs with PEG, TAT, and anti-GFP antibody. To demonstrate the intracellular targeting specificity of the QD conjugate, a model cell line overexpressing cytoplasmic GFP as an intracellular target at the c-terminal site of endothelin A receptor (ETAR) was constructed. The GFP-ETAR cellular model allowed for identification of specific binding of the red-emitting antiGFP-TATPEG-pcQD with the cytoplasmic GFP of the ETAR via colocalization. To further confirm the specificity of the probe to the target GFP, the ETAR agonist (ET-1)-induced translocation phenomenon of GPCR was utilized. When compared with the controls (PEG-pcQD, TAT-PEG-pcQD, and antiGFP-PEGpcQD), the antiGFP-TAT-PEG-pcQD was significantly colocalized with the translocated GFP-ETAR cluster in the pericentriolar region after the ET-1 treatment. High luminescence and photostability of the QDs also allowed us to obtain spatiotemporal information of the target protein in live cells, offering the potential of the QD for target visualization in living cells. These results suggested successful specific binding of the antiGFP-TAT-PEG-pcQD with the intracellular target GFP in living cells. This study demonstrated that controlled functionalization of PEGylated pcQDs with both TAT and an antibody against a target of interest can afford efficient cellular entry of QDs as well as specific detection of an intracellular target without significant nonspecific uptake or binding. Further studies are underway to explore the potential of the TAT-PEG-pcQD-based
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probes for visualizing multiple targets in live cells. The detection of target proteins without any fluorescent tagging should be the ultimate goal of this study. We are currently applying this strategy to pursue drug-target identification in live cells.
’ ASSOCIATED CONTENT
bS
Supporting Information. Details of synthesis and characterization of polymer-coated quantum dot and methoxy PEGylated pcQDs; cellular uptake of PEGylated pcQDs with different molecular weights of PEG molecules; separate confocal fluorescence channel images of Figure 7A,B,C, and Figure 8A; z-section image of Figure 8A; cellular entry of TAT-PEG-pcQD. This material is available free of charge via the Internet at http:// pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Dr. Youngseon Choi, Chemical Biology Laboratory, Institut Pasteur Korea (IP-K), 696 Sampyeong-dong, Bundang-gu, Seongnam-Si, Gyeonggi-Do, 463-400, South Korea; (Tel) +82-318018-8234, (Fax) +82-31-8018-8014, E-mail:
[email protected]. Dr. Rita Song, Chemical Biology Laboratory, IP-K; (Tel) +82-318018-8230, (Fax) +82-31-8018-8014, E-mail:
[email protected].
’ ACKNOWLEDGMENT This research was supported by the National Research Foundation of Korea funded by Ministry of Education and Science and Technology through National R&D Program (no. 2010-0019107) and Basic Science Research Program (no. 2010-0024880). ’ REFERENCES (1) Lang, P., Yeow, K., Nichols, A., and Scheer, A. (2006) Cellular imaging in drug discovery. Nat. Rev. Drug Discovery 5, 343–356. (2) Alivisatos, P. (2004) The use of nanocrystals in biological detection. Nat. Biotechnol. 22, 47–52. (3) Michalet, X., Pinaud, F. F., Bentolila, L. A., Tsay, J. M., Doose, S., Li, J. J., Sundaresan, G., Wu, A. M., Gambhir, S. S., and Weiss, S. (2005) Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307, 538–544. (4) Medintz, I. L., Uyeda, H. T., Goldman, E. R., and Mattoussi, H. (2005) Quantum dot bioconjugates for imaging, labelling and sensing. Nat. Mater. 4, 435–446. (5) Delehanty, J. B., Boeneman, K., Bradburne, C. E., Robertson, K., and Medintz, I. L. (2009) Quantum dots: a powerful tool for understanding the intricacies of nanoparticle-mediated drug delivery. Expert Opin. Drug Delivery 6, 1091–1112. (6) Kirchner, C., Liedl, T., Kudera, S., Pellegrino, T., Javier, A. M., Gaub, H. E., Stolzle, S., Fertig, N., and Parak, W. J. (2005) Cytotoxicity of colloidal CdSe and CdSe/ZnS nanoparticles. Nano Lett. 5, 331–338. (7) Gopalakrishnan, G., Danelon, C., Izewska, P., Prummer, M., Bolinger, P. Y., Geissbuhler, I., Demurtas, D., Dubochet, J., and Vogel, H. (2006) Multifunctional lipid/quantum dot hybrid nanocontainers for controlled targeting of live cells. Angew. Chem., Int. Ed. 45, 5478–5483. (8) Howarth, M., Liu, W. H., Puthenveetil, S., Zheng, Y., Marshall, L. F., Schmidt, M. M., Wittrup, K. D., Bawendi, M. G., and Ting, A. Y. (2008) Monovalent, reduced-size quantum dots for imaging receptors on living cells. Nat. Methods 5, 397–399. (9) Mok, H., Bae, K. H., Ahn, C. H., and Park, T. G. (2009) PEGylated and MMP-2 specifically dePEGylated quantum Dots: comparative evaluation of cellular uptake. Langmuir 25, 1645–1650. 1584
dx.doi.org/10.1021/bc200126k |Bioconjugate Chem. 2011, 22, 1576–1586
Bioconjugate Chemistry (10) Parak, W. J., Boudreau, R., Le Gros, M., Gerion, D., Zanchet, D., Micheel, C. M., Williams, S. C., Alivisatos, A. P., and Larabell, C. (2002) Cell motility and metastatic potential studies based on quantum dot imaging of phagokinetic tracks. Adv. Mater. 14, 882–885. (11) Jaiswal, J. K., Mattoussi, H., Mauro, J. M., and Simon, S. M. (2003) Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nat. Biotechnol. 21, 47–51. (12) Hoshino, A., Fujioka, K., Oku, T., Nakamura, S., Suga, M., Yamaguchi, Y., Suzuki, K., Yasuhara, M., and Yamamoto, K. (2004) Quantum dots targeted to the assigned organelle in living cells. Microbiol. Immunol. 48, 985–994. (13) Lovric, J., Bazzi, H. S., Cuie, Y., Fortin, G. R. A., Winnik, F. M., and Maysinger, D. (2005) Differences in subcellular distribution and toxicity of green and red emitting CdTe quantum dots. J. Mol. Med. 83, 377–385. (14) Maysinger, D., Lovric, J., Eisenberg, A., and Savic, R. (2007) Fate of micelles and quantum dots in cells. Eur. J. Pharm. Biopharm. 65, 270–281. (15) Hild, W. A., Breunig, M., and Goepferich, A. (2008) Quantum dots - Nano-sized probes for the exploration of cellular and intracellular targeting. Eur. J. Pharm. Biopharm. 68, 153–168. (16) Chang, E., Yu, W. W., Colvin, V. L., and Drezek, R. (2005) Quantifying the influence of surface coatings on quantum dot uptake in cells. J. Biomed. Nanotechnol. 1, 397–401. (17) Bentzen, E. L., Tomlinson, I. D., Mason, J., Gresch, P., Warnement, M. R., Wright, D., Sanders-Bush, E., Blakely, R., and Rosenthal, S. J. (2005) Surface modification to reduce nonspecific binding of quantum dots in live cell assays. Bioconjugate Chem. 16, 1488–1494. (18) Tan, S. J., Jana, N. R., Gao, S. J., Patra, P. K., and Ying, J. Y. (2010) Surface-ligand-dependent cellular Interaction, subcellular Localization, and cytotoxicity of polymer-coated quantum dots. Chem. Mater. 22, 2239–2247. (19) Zhang, L. W., and Monteiro-Riviere, N. A. (2009) Mechanisms of quantum Dot nanoparticle cellular uptake. Toxicol. Sci. 110, 138–155. (20) Verma, A., and Stellacci, F. (2010) Effect of surface properties on nanoparticle-cell interactions. Small 6, 12–21. (21) Winter, J. O., Liu, T. Y., Korgel, B. A., and Schmidt, C. E. (2001) Recognition molecule directed interfacing between semiconductor quantum dots and nerve cells. Adv. Mater. 13, 1673–1677. (22) Bharali, D. J., Lucey, D. W., Jayakumar, H., Pudavar, H. E., and Prasad, P. N. (2005) Folate-receptor-mediated delivery of InP quantum dots for bioimaging using confocal and two-photon microscopy. J. Am. Chem. Soc. 127, 11364–11371. (23) Gussin, H. A., Tomlinson, I. D., Little, D. M., Warnement, M. R., Qian, H. H., Rosenthal, S. J., and Pepperberg, D. R. (2006) Binding of muscimol-conjugated quantum dots to GABA(c) receptors. J. Am. Chem. Soc. 128, 15701–15713. (24) Lidke, D. S., Nagy, P., Heintzmann, R., Arndt-Jovin, D. J., Post, J. N., Grecco, H. E., Jares-Erijman, E. A., and Jovin, T. M. (2004) Quantum dot ligands provide new insights into erbB/HER receptormediated signal transduction. Nat. Biotechnol. 22, 198–203. (25) Lee, J., Choi, Y., Kim, K., Hong, S., Park, H. Y., Lee, T., Cheon, G. J., and Song, R. (2010) Characterization and cancer cell specific binding properties of Anti-EGFR antibody conjugated quantum dots. Bioconjugate Chem. 21, 940–946. (26) Zhou, M., Nakatani, E., Gronenberg, L. S., Tokimoto, T., Wirth, M. J., Hruby, V. J., Roberts, A., Lynch, R. M., and Ghosh, I. (2007) Peptide-labeled quantum dots for imaging GPCRs in whole cells and as single molecules. Bioconjugate Chem. 18, 323–332. (27) Fischer, R., Fotin-Mleczek, M., Hufnagel, H., and Brock, R. (2005) Break on through to the other side - Biophysics and cell biology shed light on cell-penetrating peptides. ChemBioChem 6, 2126– 2142. (28) Patel, L. N., Zaro, J. L., and Shen, W. C. (2007) Cell penetrating peptides: Intracellular pathways and pharmaceutical perspectives. Pharm. Res. 24, 1977–1992. (29) Richard, J. P., Melikov, K., Brooks, H., Prevot, P., Lebleu, B., and Chernomordik, L. V. (2005) Cellular uptake of unconjugated TAT
ARTICLE
peptide involves clathrin-dependent endocytosis and heparan sulfate receptors. J. Biol. Chem. 280, 15300–15306. (30) Richard, J. P., Melikov, K., Vives, E., Ramos, C., Verbeure, B., Gait, M. J., Chernomordik, L. V., and Lebleu, B. (2003) Cell-penetrating peptides - A reevaluation of the mechanism of cellular uptake. J. Biol. Chem. 278, 585–590. (31) Wadia, J. S., Stan, R. V., and Dowdy, S. F. (2004) Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat. Med. 10, 310–315. (32) Ruan, G., Agrawal, A., Marcus, A. I., and Nie, S. (2007) Imaging and tracking of tat peptide-conjugated quantum dots in living cells: new insights into nanoparticle uptake, intracellular transport, and vesicle shedding. J. Am. Chem. Soc. 129, 14759–14766. (33) Chen, B., Liu, Q. L., Zhang, Y. L., Xu, L., and Fang, X. H. (2008) Transmembrane delivery of the cell-penetrating peptide conjugated semiconductor quantum Dots. Langmuir 24, 11866–11871. (34) Wei, Y., Jana, N. R., Tan, S. J., and Ying, J. Y. (2009) Surface coating directed cellular delivery of TAT-functionalized quantum dots. Bioconjugate Chem. 20, 1752–1758. (35) Paasche, J. D., Attramadal, T., Kristiansen, K., Oksvold, M. P., Johansen, H. K., Huitfeldt, H. S., Dahl, S. G., and Attramadal, H. (2005) Subtype-specific sorting of the ETA endothelin receptor by a novel endocytic recycling signal for G protein-coupled receptors. Mol. Pharmacol. 67, 1581–1590. (36) Bhowmick, N., Narayan, P., and Puett, D. (1998) The endothelin subtype A receptor undergoes agonist- and antagonist-mediated internalization in the absence of signaling. Endocrinology 139, 3185–3192. (37) Drake, M. T., Shenoy, S. K., and Lefkowitz, R. J. (2006) Trafficking of G protein-coupled receptors. Circ. Res. 99, 570–582. (38) Bohme, I., and Beck-Sickinger, A. G. (2009) Illuminating the life of GPCRs. Cell Commun. Signal 7, 16. (39) Choi, Y., Kim, H. P., Hong, S. M., Ryu, J. Y., Han, S. J., and Song, R. (2009) In situ visualization of gene expression using polymer-coated quantum-dot-DNA conjugates. Small 5, 2085–2091. (40) Yu, W. W., Qu, L. H., Guo, W. Z., and Peng, X. G. (2003) Experimental determination of the extinction coefficient of CdTe, CdSe, and CdS nanocrystals. Chem. Mater. 15, 2854–2860. (41) Otsuka, H., Nagasaki, Y., and Kataoka, K. (2003) PEGylated nanoparticles for biological and pharmaceutical applications. Adv. Drug Delivery Rev. 55, 403–419. (42) Kaplan, I. M., Wadia, J. S., and Dowdy, S. F. (2005) Cationic TAT peptide transduction domain enters cells by macropinocytosis. J. Controlled Release 102, 247–253. (43) Traub, L. M. (2009) Tickets to ride: selecting cargo for clathrinregulated internalization. Nat. Rev. Mol. Cell Biol. 10, 583–596. (44) Gregan, B., Jurgensen, J., Papsdorf, G., Furkert, J., Schaefer, M., Beyermann, M., Rosenthal, W., and Oksche, A. (2004) Ligand-dependent differences in the internalization of endothelin A and endothelin B receptor heterodimers. J. Biol. Chem. 279, 27679–27687. (45) Pooler, A. M., and McIlhinney, R. A. J. (2007) Lateral diffusion of the GABA(B) receptor is regulated by the GABA(B2) C terminus. J. Biol. Chem. 282, 25349–25356. (46) Ostrom, R. S., and Insel, P. A. (2004) The evolving role of lipid rafts and caveolae in G protein-coupled receptor signaling: implications for molecular pharmacology. Br. J. Pharmacol. 143, 235–245. (47) Hur, E. M., and Kim, K. T. (2002) G protein-coupled receptor signaling and cross-talk - Achieving rapidity and specificity. Cell Signal 14, 397–405. (48) Nelson, S., Horvat, R. D., Malvey, J., Roess, D. A., Barisas, B. G., and Clay, C. M. (1999) Characterization of an intrinsically fluorescent gonadotropin-releasing hormone receptor and effects of ligand binding on receptor lateral diffusion. Endocrinology 140, 950–957. (49) Barak, L. S., Ferguson, S. S. G., Zhang, J., Martenson, C., Meyer, T., and Caron, M. G. (1997) Internal trafficking and surface mobility of a functionally intact beta(2)-adrenergic receptor-green fluorescent protein conjugate. Mol. Pharmacol. 51, 177–184. (50) Hebert, T. E., Gales, C., and Rebois, R. V. (2006) Detecting and imaging protein-protein interactions during g protein-mediated signal 1585
dx.doi.org/10.1021/bc200126k |Bioconjugate Chem. 2011, 22, 1576–1586
Bioconjugate Chemistry
ARTICLE
transduction in vivo and in situ by using fluorescence-based techniques. Cell Biochem. Biophys. 45, 85–109. (51) Ma, X. Y., Wang, Q., Jiang, Y. X., Xiao, Z. Y., Fang, X. H., and Chen, Y. G. (2007) Lateral diffusion of TGF-beta type I receptor studied by single-molecule imaging. Biochem. Biophys. Res. Commun. 356, 67–71. (52) Chun, M. Y., Lin, H. Y., Henis, Y. I., and Lodish, H. F. (1995) Endothelin-induced endocytosis of cell-surface ET(a) receptors endothelin remains intact and bound to the ET(a) receptor. J. Biol. Chem. 270, 10855–10860. (53) Abe, Y., Nakayama, K., Yamanaka, A., Sakurai, T., and Goto, K. (2000) Subtype-specific trafficking of endothelin receptors. J. Biol. Chem. 275, 8664–8671. (54) Bagnato, A., Spinella, F., and Rosano, L. (2008) The endothelin axis in cancer: the promise and the challenges of molecularly targeted therapy. Can. J. Physiol. Pharmacol. 86, 473–484. (55) Chun, M. Y., Liyanage, U. K., Lisanti, M. P., and Lodish, H. F. (1994) Signal transduction of a G-protein-coupled receptor in caveolae colocalization of endothelin and its receptor with caveolin. Proc. Natl. Acad. Sci. U. S. A. 91, 11728–11732.
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