ARTICLES

Applications of Mesenchymal Stem Cells Labeled with Tat PeptideConjugated Quantum Dots to Cell Tracking in Mouse Body

Yun Lei,†,‡ Haiyang Tang,§ Lide Yao,† Richeng Yu,‡ Meifu Feng,*,§ and Bingsuo Zou*,†

Micro-Nano Technologies Research Center and State Key Lab of CBSC, Hunan University, Changsha 410082, China, Instituteof Physics and Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China. Received February 28, 2007;Revised Manuscript Received September 15, 2007

Fluorescent quantum dots have great potential in cellular labeling and tracking. Here, PEG encapsulated CdSe/ZnS quantum dots have been conjugated with Tat peptide, and introduced into living mesenchymal stem cells.The Tat peptide conjugated quantum dots in mesenchymal stem cells were assessed by fluorescent microscopy,laser confocal microscope and. flow cytometry. The result shows that Tat peptide conjugated quantum dots couldenter mesenchymal stem cells efficiently. The Tat-quantum dots labeled stem cells were further injected into thetail veins of NOD/SCID beta2 M null mice, and the tissue distribution of these labeled cells in nude mice wereexamined with fluorescence microscope. The result shows that characteristic fluorescence of quantum dots wasobserved primarily in the liver, the lung and the spleen, with little or no quantum dots accumulation in the brain,the heart, or the kidney.

INTRODUCTION

Stem cells have generated a great deal of excitement as apotential source of cell-based therapies (1, 2). The developmentof stem cell therapies requires a quantitative and qualitativeassessment of stem cell fate and their distribution in the hostorganism. The uses of traditional organic fluorophores and greenfluorescent protein are subject to certain limitations in livingcells. Quantum dots (QDs) as a new generation of fluorescentmarkers have shown tremendous advantage in biologicalresearch, especially cellular labeling and imaging (3, 4). Manyefforts have been directed toward introducing QDs into cells.Strategies have been used such as microinjection (5), electropo-ration (6), phagocytosis (7, 8), cationic lipid transportation (9),and peptide-mediated transfection (10–15). Therefore, peptide-mediated transfection has attracted more and more attention forthe efficient cellular transmembrane delivery and labeling (16, 17).Though the mechanism of peptide-mediated membrane trans-location remains unclear, increasing evidence indicates theinvolvement of several types of endocytosis (18–20).

Here, we used the cell-penetrating peptide Tat modified witha linker and the C-terminus followed by a reactive cysteine,which was covalently attached to QDs via the cross-linkingreagent. Moreover, considering the cytotoxicity of QDs in thebiological system, CdSe/ZnS QDs have been investigated fordifferent surface modification (21) such as coating with mer-captoacid, silane, and polymer. Recently, PEG-coated QDs wereinvestigated (22, 23), and the results show that PEG-coated QDshave minimal impact on cells. Thus, in our work, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly(ethylene gly-col)) 2000] (DSPE-PEG 2000 amine) was selected to coat QDs

with amine groups as functional groups for further covalentbonding to the cysteine-modified Tat peptide. PEG-coated QD-Tat peptide complexes were transported into living mesenchymalstem cells (MSCs) and assessed by fluorescent microscopy, laserconfocal microscopy, and flow cytometry. The QD–Tat peptidelabeled MSCs were further assessed with stem cell engraftmentin nude mice, and the result shows that Tat peptide covalentlyconjugated QDs may not only act as an effective probe for stemcell observation in vitro, but also serve as a long-term tracerfor stem cell based transplantation in the mouse body. Thus,the purpose of our study was to evaluate the internalization ofTat peptide conjugated QDs MSCs and the tissue distributionof these labeled stem cells in a nude mouse.

MATERIALS AND METHODS

Materials. n-Trioctylphosphine oxide (TOPO) (90%), bis(tri-methylsilyl) sulfide (95%), Cd(CH3)2 (97% hexane solution),Zn(CH3)2 (1.0 M heptane solution), and n-trioctylphosphine(TOP) were purchased from Acros Chemicals Corporation. Se

* E-mail: zoubs@aphy.iphy.ac.cn (B.Z.); fengmf@ioz.ac.cn (M.F.).† Hunan University.‡ Institute of Physics, Chinese Academy of Sciences.§ Institute of Zoology, Chinese Academy of Sciences.

Figure 1. TEM images of the as-prepared TOPO-capped CdSe/ZnSQDs (left, about 3 nm) and DSPE-PEG-coated QDs (right, about 4nm).

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10.1021/bc0700685 CCC: $40.75 2008 American Chemical SocietyPublished on Web 12/15/2007

powder (99%), CHCl3 (AR), and methanol (AR) were purchasedfrom Beijing Chemical Reagents Corporation. Sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC)were purchased from Pierce Biotechnology. Cell-penetratingpeptide Tat custom-modified with a linker at the C-terminusfollowed by a reactive cysteine was purchased from BeijingScilight Biotechnology Limited Corporation. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly(ethylene gly-col)) 2000] (DSPE-PEG 2000 amine) was purchased fromAvanti Polar Lipids Inc.

Preparation of DSPE-PEG-Coated CdSe/ZnS QDs. Anencapsulation strategy was used for hydrophobic CdSe/ZnSQDs. This approach exploits the hydrophobic nature of thenanoparticle surface by utilizing an amphiphilic poly(ethyleneglycol)-phospholipid (PEG-phospholipid), whose hydrophobicportion interacts with the nanoparticle surface to create micelles,resulting in a self-assembled monolayer coating. The PEGportion of the coating confers them aqueous solubility andbiocompatibility, at the same time the use of modified PEGallows for bioconjugation (24).

CdSe/ZnS QDs were prepared using a synthetic route reportedearlier (25, 26). Since the quality of TOPO affected thenucleation and growth process seriously, TOPO was purifiedat the temperature of 400 °C in argon before use. All otherreagents were used without any further purification. Theprepared TOPO-capped QDs were added with equal volume ofmethanol and cooled to -10 °C for 2 h. After vigorouscentrifugation, the precipitation from the cold solution wascollected for the further use.

50 mg DSPE-PEG 2000 amine was dissolved in 2 mLchloroform, and 2.5 mg TOPO-capped CdSe/ZnS QDs were

added. QDs coated with DSPE-PEG 2000 amine was driedunder argon gas and left in a vacuum desiccator for 48 h toremove all trace of organic solvent.

Bioconjugation of DSPE-PEG-Coated CdSe/ZnS QDs. Tofunctionalize CdSe/ZnS QDs for bioconjugation and subsequentdelivery and imaging studies, we used the cysteine-modifiedTat peptide and heterobifunctional cross-linking reagent Sulfo-SMCC. First, Sulfo-SMCC was dissolved in 500 µL phosphate-buffered saline (PBS, pH 7.4, NaCl 50 mM) at a concentrationof 20 µM, and then DSPE-PEG-coated CdSe/ZnS QDs weredispersed in the PBS solution of Sulfo-SMCC cross-linker at aconcentration of 1 µM. After incubating for 60 min at roomtemperature, excess cross-linker molecules were removed byusing a Sephadex G-25 column. The 10-fold concentration ofTat peptide (10 µM) was added to the above PBS solution andreacted overnight with the DSPE-PEG-coated CdSe/ZnS QDsvia the Sulfo-SMCC cross-linker.

Cell Cultures. MSCs were isolated from fetal bone marrow.The cells were grown in complete Dulbecco’s Modified Eagle’SMediumshigh glucose (10% fetal bovine serum, 100 U/mLpenicillin, and 100U/mL streptomycin sulfate) on a 6-well platepredeposited with a slide.

Cell Labeling with Bioconjugated CdSe/ZnS QDs. 500 µLTat peptide-conjugated QDs were added to the culture of MSCs(5 × 106). The Tat peptide-conjugated QDs and MSCs wereincubated at 37 ° for 60 min.

MSC Transplantation and Bioluminescent Imaging. Forstudies on systemic distribution and engraftment of QD–Tatpeptide labeled stem cells, approximately 106 labeled stem cellssuspended in 0.2 mL PBS were injected intravenously into thetail veins of NOD/SCID beta2 M null mice (male, 8 weeks of

Figure 2. Schematic illustration of peptide-conjugated QDs for stem cell targeting and imaging. The first step involves the coating of TOPO-capped QDs with DSPE-PEG 2000 NH2. After coating, DSPE-PEG-coated QDs are water-soluble with amine on the surface. Then, the DSPE-PEG-coated CdSe/ZnS QDs were reacted overnight with the cysteine-modified Tat peptides via the Sulfo-SMCC cross-linker. Sulfo-SMCC has anN-hydroxysuccinimide (NHS) ester at one end, which reacts with a primary amine on the QDs to form an amide bond and a maleimide group atthe other, which reacts with a thiol on the cysteine-modified Tat peptide to form a thioether.

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age). Mice were killed after 24 h, and major organs (heart, lung,spleen, liver, kidney, and brain) were removed and washed threetimes with PBS to remove retained erythrocytes. Collectedtissues were frozen in liquid nitrogen and embedded intoparaffin. To assess histological QD uptake and distribution oftransplanted stem cells, tissue collections were cryosectionedinto sections 5–10 µm thick, fixed with acetone at 0 °C, andexamined with a fluorescence microscope.

Characterizations. The morphologies of CdSe/ZnS QDswere observed on a JEM2010 transmission electron micro-scope. The photoluminescence (PL) spectra were recordedon a PTI-601 fluorescence spectrophotometer. The fluores-cence images were taken on a fluorescence microscope(Olympus IX71) excited by an ultraviolet source, and theconfocal images were recorded on a Zeiss LSM510 laserconfocal microscope excited by an argon laser (488 nm).

To investigate the location of QD–Tat peptides into MSCs,the MSCs were treated for transmission electron microscope(TEM) observation. MSCs were fixed with 2.5% glutaralde-hyde for 4 h at room temperature and rinsed with phosphate-buffered saline (PBS), and then the cells were fixed with 1%osmium tetraoxide for 1 h and washed with distilled water.The samples were dehydrated through a graded ethanol andacetone series, and finally embedded with epoxy resin. Theepoxy resin samples were polymerized at 60 °C for 24 h.

Thin sections were made using an ultramicrotome andcollected on copper grids. The samples on copper grids wereobserved on a Philips CM200 transmission electron micro-scope (TEM) at 80 KV.

To assess the labeling efficiency of QD–Tat peptides, theMSCs incubated with QD–Tat peptides were trypinized andwashed with PBS three times, and then analyzed using flowcytometry (FACSCaliblur, BD Biosciences, San Diego, CA,USA) with a 488 nm Ar laser.

RESULTS AND DISCUSSION

Monodispersed CdSe/ZnS QDs capped with TOPO werecharacterized by transmission electron microscopy (Figure 1left), and the corresponding size distribution (σ) is 2.72%. Aftercoating with DSPE-PEG 2000 amine (Figure 1 right), the sizedistribution of DSPE-PEG-coated QDs is 11.3% due to the effectof the solvent polarity.

The scheme of DSPE-PEG coating and Tat peptide conjuga-tion for cellular targeting and imaging is shown in Figure 2.The first step involves the coating of TOPO-capped QDs withDSPE-PEG 2000 amine. After coating, DSPE-PEG-coated QDsare water-soluble with amine on the surface. Then, the DSPE-PEG-coated CdSe/ZnS QDs reacted overnight with the cysteine-modified Tat peptides via the Sulfo-SMCC cross-linker.

Tat peptide conjugated QDs were prepared with two distinctQDs (QDs-520 and QDs-645). The PL spectra were measuredafter the first reaction with DSPE-PEG 2000 amine, and thePL intensity of QDs changed during the peptide couplingprocess. As shown in Figure 3a, the PL intensity of QDs-520was enhanced remarkably after the secondary coupling with theTat peptides, increasing by 41%. Clearly, this increase was alsoobserved in QDs-645 (Figure 3b) after conjugating with Tatpeptides, enhanced by 22%. The enhancement might be at-tributed to the covalent bonding between the thiol groups ofcysteine-modified Tat peptides and the amide groups of DSPE-PEG 2000 amine capped QDs. Coating QD with a highmolecular weight polymer such as polypeptides might prohibitelectron leakage through the surface NH2 groups and mightcontribute to the continuously electron-rich condition of wholeQDs (10).

MSCs were incubated with Tat peptide-conjugated QDs andobserved by fluorescent microscopy. The fluorescent microscopyimages (Figure 4a–c) show that MSCs incubated with QD–Tatpeptides emit fluorescence. A punctate and vesicular QDfluorescence pattern was observed within perinuclear spaces.For comparison, DSPE-PEG-coated QDs were directly incubatedwith MSCs without the assistance of Tat peptides. Thefluorescent microscopy images are shown in Figure 4d–f. Nointracellular QD fluorescence was observed in MSCs. Similarly,control experiments performed with a random peptide resultedin no noticeable cellular uptake (Figure 4g–i). The resultssuggest that Tat peptide is an effective vehicle for labeling QDsentering stem cells. The reactive cysteine sequence allows Tatpeptides to conjugate with DSPE-PEG-coated QDs via cross-linking reagent while the oligoarginine sequence allows QDdelivery across the cellular membrane and intracellular labeling.

The QD–Tat peptide labeled stem cells were further studiedon a laser confocal microscope (Figure 5). In contrast to ourprevious observations using fluorescence microscopy, the moresensitive confocal observation technique used here indicated ahigher intensity of intracellular QD fluorescence than what waspreviously measured above (a punctate and vesicular QDfluorescence pattern in Figure 4). The fluorescent QD–Tatpeptides dispersed nearly homogeneously throughout the cyto-plasm in the periphery of the nucleus. To demonstrate the useof QD–Tat peptides in a multiplexing experiment, two colorquantum dots (QDs-520 and QDs-645) are designed to bind to

Figure 3. PL spectra of DSPE-PEG-coated QDs and Tat peptide-conjugated QDs. The PL intensity of Tat peptide-conjugated QDs-520(solid line) enhances by 41% compared to simple DSPE-PEG-coatedQDs-520 (dotted line). Similarly, the PL intensity of Tat peptide-conjugated QDs-645 (solid line) increases by 22% as compared tosimple DSPE-PEG-coated QDs-645 (dotted line). The PL spectra wererecorded with an excitation at 380 nm.

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Tat peptides and introduced into the same stem cells simulta-neously. After excitation with a 488 nm laser line, bothpopulations of bioconjugated QDs were identified in stem cells

using confocal laser microscopy. The confocal images of MSCslabeled with two color QD–Tat peptides are shown in theSupporting Information. The green (Figure S1a) and red (FigureS1b) fluorescence of QD–Tat peptides are superimposed asyellow dots, which are shown in the merged fluorescent imageof Figure S1d.

The internalization of QD–Tat peptides was further observedby transmission electron microscopy, which provided an evenhigher resolution than confocal microscopy. Figure 6a,b providesTEM images of intracellular QD–Tat peptides. The whitearrowheads in Figure 6a indicate endosomes, which are magni-

Figure 4. Fluorescent microscopy images of MSCs incubated with DSPE-PEG-coated QDs. With the use of Tat peptides, MSCs labeled by thegreen QD–Tat peptides are shown in parts a–c. As compared, DSPE-PEG-coated QDs were directly incubated with MSC without the assistance ofTat peptides, and the images are shown in parts d–f. A control experiment was performed to use QDs with a random peptide, and the images areshown in parts g–i. Therefore, a, d, and g are the images in the bright field; b, e, and h are the corresponding fluorescent images; and c, f, and Iare the merged images.

Figure 5. Confocal images of MSCs labeled by QD–Tat peptides. (a)MSCs labeled by green QD–Tat peptides in bright field. (b) Greenfluorescent image. (c) Merged image of parts a and b.

Figure 6. TEM analysis of the internalized QD–Tat peptides in MSCs.(a) TEM images of MSCs labeled by QD–Tat peptides, and the whitearrowheads indicate endosomes. (b) A magnified view of endosomesin (a). QD–Tat peptides are seen as black dots surrounding the interiorof the vesicles.

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fied in Figure 6b. QD–Tat peptides are seen as black dots,surrounding the interior of the endosomes. The electronmicrographs provide direct evidence that at least a portion ofthe internalized QD–Tat peptides were colocalized withinendosomes. This result is consistent with the work reported byDelehanty et al. (12). They not only confirmed that QD signaldispersed nearly homogeneously throughout the cytoplasm onconfocal microscopy, but also observed partial colocalizationof QDs within endosomes. The partial colocalization of QDs

within endosomes suggests a role for endocytosis in the uptakeof the QD–Tat peptides. Other reports using live-cell imagingand FACS analysis have also established the role of endocytosisin the uptake of arginine-rich cell-penetrating peptides and theircargos (27–29).

The MSCs incubated with Tat peptide-conjugated QDs weretrypinized and washed with PBS three times, and then analyzedusing flow cytometry. Flow cytometry analysis (Figure 7) inthe left curve represents the MSCs treated as control, and thatin the right curve represents the responding sample treated withgreen QD–Tat peptides emitted at 520 nm. The flow cytometrydata indicate that Tat peptide-conjugated QDs could enter MSCswith high efficiency (>95%). Although flow cytometry cannotdistinguish between surface-bound and intracellular QD–Tatpeptides, confocal and TEM data do confirm the uptake of theQD–Tat peptides by the MSCs. Furthermore, steps were takento wash away the surface-bound QD–Tat peptides and ensurethat data from flow cytometry were from the internalizedQD–Tat peptides.

The internalized QD–Tat peptides in MSCs were furtherassessed with stem cell engraftment in nude mouse. Becausered luminescent QDs were easier to distinguish from the

Figure 7. Flow cytometry analysis for the Tat peptide-conjugated QDsin MSCs. The left curve represents MSCs treated as control, and theright curve represents the responding sample treated with Tat peptide-conjugated QDs.

Figure 8. Intracellular labeling of stem cells by QD–Tat peptides permits in vivo imaging despite tissue autofluorescence. MSCs labeled with Tatpeptide-conjugated QDs were intravenously injected into mice, and tissue sections of six normal host organs were observed via fluorescent microscopy.The characteristic red fluorescence of QD–Tat peptides were detected primarily in the lung (a), the liver (b), and the spleen (c), with little or no QDaccumulation in the brain (d), the heart (e), or the kidney (f). All other signals were due to background autofluorescence.

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autofluorescent tissue background than the green luminescentQDs, we injected the tail veins of NOD/SCID beta2 M nullmice with QD-645-Tat peptide-labeled MSCs. After 24 h, themice were killed and major organs (heart, lung, spleen, liver,kidney, and brain) were extracted and fixed in formalin.Representative images (Figure 8a) were obtained from 5 to 10µm optical tissue sections on a fluorescence microscope excitedby an ultraviolet source. The characteristic red fluorescenceshows the presence of labeled MSCs in the lung. Tissues fromother organs including liver (Figure 8b) and spleen (Figure 8c)were examined for the presence of QD-645-Tat peptide labeledstem cells. The presence of QD–Tat peptide labeled stem cellsin other tissues indicated that not all the stem cells are trappedin the lung. A population of stem cells is able to circumnavigatethe lung capillary network and seed other organs. However, thenumber of stem cells per field labeled with red QD–Tat peptidetracer was lower in the spleen and liver than in the lung tissues.The sections of the brain (Figure 8d), the heart (Figure 8e), orthe kidney (Figure 8f) were also observed in fluorescencemicroscopy; no red luminescent spots appeared on these threekinds of organs. The results are in line with the report by Simonet al. after injecting QDs-labeled tumor cells into the tail veinof a rat (9).

CONCLUSIONS

In summary, PEG-QDs with covalently coupled Tat peptidesproduce nanometer-sized materials with biological function,which can penetrate the cellular membrane for cellular labelingand imaging. To assess the technique qualitatively and quan-titatively, we combined the use of laser confocal microscopyand transmission electron microscopy to evaluate intracellularquantum dot localization in single cells and flow cytometry toquantify the delivery efficiency over a population of livemesenchymal stem cells. The result shows that Tat peptide-conjugated quantum dots could enter MSCs efficiently. TheQD–Tat peptide labeled stem cells were injected intravenouslyinto the tail veins of NOD/SCID beta2 M null mice, and thetissue distribution of these labeled stem cells in nude mice wasexamined by fluorescence microscopy. The result shows thatcharacteristic fluorescence of QD–Tat peptides was observedprimarily in the liver, the lung, and the spleen, with little or noQD accumulation in the brain, the heart, or the kidney. Thelabeled stem cells can be visualized by fluorescent imaging atthe single-cell level, which offers a promising approach in stemcell transplantation and will hold a significant impact on theadvancement of stem cell based therapy.

ACKNOWLEDGMENT

The authors would like to thank the 973 projects of MOST(Grant No. 2002CB713802 and No. 2005CB623602) and NSFCfund (No. 30370383, 90606001, and 90406024) of China forfinancial supports.

Supporting Information Available: Additional informationas described in the text. This material is available free of chargevia the Internet at http://pubs.acs.org.

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