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Bioconjugate Chem. 2002, 13, 840−844
Differential Conjugation of Tat Peptide to Superparamagnetic Nanoparticles and Its Effect on Cellular Uptake Ming Zhao,*,† Moritz F. Kircher,† Lee Josephson, and Ralph Weissleder Center for Molecular Imaging Research, Harvard Medical School, Room 5404, Building 149, 13th Street, Charlestown, Massachusetts 02169. Received February 27, 2002; Revised Manuscript Received April 19, 2002
Surface modification of superparamagnetic contrast agents with HIV-1 tat peptide has emerged as a promising means for intracellular magnetic labeling and noninvasive tracking of a large number of cell types with MRI. To achieve efficient intracellular delivery of the nanoparticles, we investigated the effect on cellular uptake of superparamagnetic iron oxide particles by varying the number of attached tat peptides. First, we report here a modified P2T method in measuring the numbers of surface attachments per particle through disulfide linkage. The method was shown to have desirable simplicity and reproducibility. With the P2T method as a tool, conjugates with progressively higher ratios of peptide-to-particle were synthesized. We were able to demonstrate that higher numbers of tat peptide facilitate the cellular uptake of iron oxide nanoparticles in a nonlinear fashion. Cells labeled with these optimized preparations were readily detectable by MR imaging. The increase in sensitivity could allow in vivo tracking of 100-fold lower cell concentration than currently described.
INTRODUCTION
Magnetic resonance imaging (MRI) has emerged as a potentially powerful tool in the investigation of cell migration in vivo (1). With a growing array of cell labeling techniques, cells tagged with various monocrystalline MR probes have been evaluated in the past in their detection both in vitro and in vivo (1-4). Among these, HIV-1 tat peptide, which carries a transmembrane and a nuclear localization signal within its sequence (5), is capable of translocating exogenous molecules into cells (6-10). Due to its membrane translocation function, tat peptide has been used to derivatize the surface of magnetic pharmaceuticals and substantially facilitated their uptake into target cells. For instance, using tat conjugated 1,4,7,10-tetraazacyclododecane-N,N′N′′N′′′-tetraacetic acid (DOTA), paramagnetic chelates were delivered to intracellular compartments (2). In addition, tat-derivatized CLIO (cross-linked superparamagnetic iron oxide), which consists of dextrancoated superparamagnetic iron oxide nanoparticles and is a potent T2 shortening agent, has been shown to achieve high loading and allowed ex vivo tracking of progenitor cells to bone marrow (1). In a continuous effort to further improve cell labeling conditions and to achieve maximum detectability of labeled cells, it has become necessary to understand how the number of tat peptide per CLIO particle may influence cellular uptake of the complex. To do so, it is imperative to have a method which can accurately and conveniently determine the peptide-to-CLIO ratio. Here we report such a protocol adapted to measure the degree of conjugation on CLIO through disulfide linkage with simplicity, reliability, and reproducibility. Subsequently, a series of CLIO-tat conjugates with progressively higher numbers of tat peptide per CLIO particle were synthesized based on the assumption that higher num* Corresponding author. E-mail: mingzhao@ helix.mgh.harvard.edu. † These authors made an equal contribution to the study.
bers of peptides would facilitate cellular targeting with greater density of affinity ligands and/or multivalency effects. We observed that high (above 10 per CLIO) numbers of tat peptide increased the intracellular accumulation of CLIO-tat (up to 100-fold) compared to prior preparations, and that this greatly enhanced the detection threshold of labeled cells. MATERIALS AND METHODS
General. A modified tat peptide was synthesized and purified as previously described (3), with a sequence GRKKRRQRRRGYK(FITC)C-NH2. The inclusion of FITC in the peptide sequence is essential for measuring peptide-to-CLIO ratios using the fluorescent absorption method, which is to be compared with the new P2T method (see below). The iodination site (tyrosine), FITC modified lysine, and site of attachment to the nanoparticle (cysteine) are at the C terminal end and separated from the residues involved in membrane translocation by a spacer glycine. The synthesis of amino-CLIO and reaction with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) have been described elsewhere (3). Measuring the Degree of Conjugation Using the P2T Method. Tat peptide of appropriate amount was dissolved in DMSO (Fisher) to make a concentrated stock. 37.5 µL of tat solution was mixed with 500 µL of CLIOP2T (1.5 mg/mL Fe). The mixture was allowed to stand for 2 h at room temperature. At the end of the incubation, 70 µL of the reaction mixture was withdrawn for the measurement of pyridine-2-thione (P2T). A microconcentrator (50 kDa molecular weight cut off, Amicon, Beverly, MA) was used to remove CLIO, and the fraction containing P2T was collected as flow-through. A 50 µL amount of the P2T fraction was diluted to 400 µL using 1 × PBS, pH 7.4. The absorbance of P2T was determined at 343 nm on a spectrophotometer, and its concentration was calculated using an extinction coefficient of 8100 M-1cm-1. Tat-to-CLIO ratio was subsequently deduced with known iron concentration in the reaction mixture. To validate the P2T method, the degree of conjugation was also quantified using fluorescent absorption from
10.1021/bc0255236 CCC: $22.00 © 2002 American Chemical Society Published on Web 06/08/2002
Cell Labeling with Differentially Conjugated CLIO−tat
FITC. Briefly, for a suspension of CLIO-tat, the absorbance was determined at 494 nm and the concentration of FITC was calculated using an extinction coefficient of 73000 M-1 cm-1. Synthesis of CLIO-tat Series with Progressively Higher Ratios. A calibration curve was constructed by setting up a series of reactions in smaller scale with a range of peptide concentrations, while keeping all other conditions constant. In detail, 7.5 µL of each peptide stock solution was mixed with 100 µL aliquots of CLIO-P2T (1.5 mg/mL Fe). The reactions were allowed to proceed as above. In due course, tat-to-CLIO ratios were determined to establish a relationship between the amount of peptide used and the resulting ratios. According to the calibration curve, CLIO-tat conjugates of expected ratios were synthesized and characterized. The products were cleaned of unreacted free peptide and low molecular weight impurities using 1.5 × 5 cm gel filtration column (Sephadex G-50) equilibrated with 1 × PBS, pH 7.4. Control conjugates were synthesized using a peptide (Gly-Arg-Arg-Lys(FITC)-Cys-NH2) that lacks the transmembrane and nuclear localization sequence of tat. Two control conjugates were synthesized with 9.7 and 12.9 peptides per CLIO particle, respectively. The products were treated with Bolton-Hunter reagent, SHPP (Pierce) to introduce a phenolic group for iodination (see below). In detail, 50 µL of 8 mg/mL of SHPP was mixed with 500 µL of conjugates at pH 8.0. The reaction was allowed to proceed for 2 h at room temperature. The modified product was purified with 1.5 × 5 cm gel filtration column (Sephadex G-50) equilibrated with 1 × PBS, pH 7.4. Isolation of Lymphocytes. Lymphocytes were obtained from the spleen of C57 Bl/6 mice (National Cancer Institute, Bethesda, MA). Donor animals were sacrificed by inhalation of an overdose of Halothane, and their spleens were removed using aseptic procedures. The spleens were disrupted between frosted histology slides and dispersed in RPMI 1640 medium (Cellgro; Mediatech, Washington, DC) supplemented with 10% fetal bovine serum (FBS; Cellgro; Mediatech). Cells were filtered through a cell-strainer with 30 µm mesh size (Miltenyi Biotech, Auburn, CA) and washed. Erythrocytes were lysed by resuspending the cell pellet in 0.83% ammonium chloride in distilled water. After being washed further, the cells were incubated in RPMI 1640 supplemented with 10% FBS in 175 cm2 tissue culture flasks (Falcon; Becton Dickinson Labware, Bedford, MA). To remove the majority of the monocyte/macrophage population, the nonadherent cell population was transferred into a new flask after 1 h. This method yielded a cell population containing about 95% lymphocytes, as described (11). Preparation of 125I-Labeled CLIO-tat. Radioactive labeling of CLIO-tat was performed according to the Chizzonite indirect method using IODO-GEN precoated tubes (Pierce, Rockford, IL) following closely the protocol of the manufacturer (Pierce). Briefly, for each sample of CLIO-tat with different tat-to-CLIO ratios 1 mCi 125I was activated for 7 min at RT in IODO-GEN tubes in 100 µL of Tris iodination buffer. The activated 125I was added to 100 µg of CLIO-tat and reacted for 10 min, followed by a reaction with scavenging buffer (50 µL/ sample) for 5 min. The samples were added to 1.5 × 5 cm gel filtration columns (Sephadex G-50) and centrifuged at 400g for 5 min and the eluates collected. Immediately before performing cell uptake assays, the labeled samples were again purified and equilibrated to the culture conditions with 1.5 × 5 cm gel filtration
Bioconjugate Chem., Vol. 13, No. 4, 2002 841
columns (Sephadex G-50) equilibrated with RPMI-1640 supplemented with 10% FBS. Cell Uptake Assay. To quantify the cellular uptake of the different CLIO-tat conjugates, 20 µg 125I-CLIOtat was added to lymphocytes in RPMI-1640 supplemented with 10% FBS at a final concentration of 106 cells/ ml. Cells were incubated for 2 h in coated (1% BSA in Hanks’ balanced salt solution (HBSS; Cellgro; Mediatech)) 5 mL polystyrene culture tubes (Fisher Scientific, Pittsburgh, PA) and incubated at 37 °C, 5% CO2 in a humidified atmosphere. After an initial washing step with 4 mL HBSS cells were washed 3 times by centrifugation through a step gradient of 40% Histopaque-1077 (Sigma; St. Louis, MO) in HBSS. The cell-pellets were then transferred to a new set of culture tubes and washed again in HBSS. Cell numbers per tube and numbers of viable lymphocytes were determined by trypan blue stain exclusion in a hemocytometer (Fisher Scientific). After aspiration of the supernatant, cell pellets were counted in a gamma counter (Model 1282 Compugamma CS; LKB Wallac, Turku, Finland). Background counts were subtracted, and cell uptake was calculated by scaling cpm/ tube to the number of cells/tube. All experiments were performed in triplicate. Conjugates with thioether (SC) linkage were synthesized using a heterobifunctional cross linker N-succinimidyl iodoacetate (SIA). Briefly, CLIO-NH2 was treated with SIA to introduce iodoacetyl groups, which then reacted with free SH on tat peptides to form SC bonds. The conjugates were purified, radiolabeled, and tested for cellular uptake as described above. MR Imaging. Phantoms were prepared by immobilizing 10 million cells in 0.8% low-melting agarose (Bio-Rad, Hercules, CA) in small plastic tubes in a final volume of 100 µL per sample. The tubes were embedded in 1% agarose (Bio-Rad) and imaged with a 1.5 T superconducting magnet (Signa 5.0; GE Medical Systems, Milwaukee, WI) using a 3-in. surface coil. A T2-weighted spin-echo sequence with the timing parameters 3000/ 60 (TR/TE) was used. Slice thickness was 1 mm, field of view (FOV) 7 × 7 cm, imaging matrix 256 × 256, and number of excitations (NEX) 2. RESULTS AND DISCUSSION
Noninvasive cell tracking using MRI is being established as a powerful tool in monitoring cell migration in real time in vivo and native environments. Surface modification with HIV-1 tat peptide on a number of MR probes has resulted in substantially enhanced uptake and hence improved MR detection of labeled cells in tissue. In the current study, we set out to optimize CLIO-tat preparations by systematically altering the degree of tat attachment on the surface of CLIO, a potent nanometer T2/T2* contrast agent. To meet this goal, a P2T method was adapted and evaluated in measuring the degree of conjugation on CLIO through disulfide linkage. Instead of directly assaying for the amount of attached peptide, the method determines the average number of pyridine-2-thione (P2T) groups released during conjugation reaction (Figure 1) (12). The number of displaced P2T is strictly equal to that of disulfide bonds formed, which in turn reflects the amount of peptide covalently attached on the surface of CLIO. The usefulness of the P2T method can be summarized as follows. First, P2T is relatively stable, and the absorbance of P2T at 343 nm varied less than 5% within a 24-hour period in aqueous solution, at room temperature
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Figure 3. Cellular uptake of radio-labeled CLIO-tat as a function of tat-to-CLIO ratio. The uptake levels of different conjugates are expressed in relative to the conjugate with the lowest tat-to-CLIO ratio. Error bars indicate the SD (n ) 3). Figure 1. Synthesis of CLIO-tat with disulfide linkage, with P2T as a measurable byproduct.
Figure 2. Direct comparison between fluorescent absorption and 2 PY method. Tat-to-CLIO ratios were determined for four conjugates, a, b, c, and d, using the fluorescent absorption method (gray) and P2T method (black), respectively. Error bars indicate the SD (n ) 3).
(data not shown), whereas the conjugation reaction approaches completion in minutes. Thus uncertainties arisen from P2T degradation are negligible. Second, a distinct absorbance of P2T at 343 nm is well resolved from other chemical components in the reaction. The absorbance of peptides occurs over wavelength below 300 nm, creating little or no interference with P2T measurement. In the same sense, the P2T method could be applied equally well in the conjugation of proteins and nucleic acids, where both are commonly known to have absorbance at below 300 nm. In addition, the method measures the degree of conjugation by determining the number of disulfide bond formation, therefore eliminating the need to tag the peptide or protein solely for quantification purposes. To evaluate the effectiveness of the P2T method, the degree of conjugation determined using this method was compared with that quantified from FITC absorbance at 494 nm. Figure 2 illustrates the comparison from two preparations. The average numbers of tat peptide per CLIO particle as determined by the two methods have good agreement and are within (10% variation of each other. Quantification using FITC absorbance directly measures the number of peptide carried on CLIO and thus serves as an authentic standard in the evaluation
of the P2T method. By doing so, the latter was shown to have excellent accuracy, reliability, and reproducibility. With a reliable method to determine the degree of conjugation, a series of CLIO-tat conjugates were designed for cell uptake studies, carrying a progressively increasing number of tat per particle. A calibration curve was constructed between the amount of tat peptide used in the reaction mixture and the resultant tat-to-CLIO ratios. On the basis of the calibration curve, conjugates were synthesized with progressively higher tat/CLIO ratios. The maximum number of peptide which can be attached to CLIO is ultimately limited by the reactive sites on the surface of CLIO particles, where CLIO-NH2 carries 20-30 amine groups in average per particle. In addition, the degree of conjugation is influenced by the size and hydrophobicity of the peptide. We have observed that the solubility of CLIO can be severely lowered when attached with high number of larger, more hydrophobic peptides. With a series of CLIO-tat conjugates, the effect of increasing numbers of tat per CLIO particle on cellular uptake could be demonstrated with radiolabeled 125ICLIO-tat using mouse lymphocytes. As expected, a substantial increase in radioactivity uptake is associated with progressively higher tat-to-CLIO ratios (Figure 3). This effect was clearly not linear, and the observed exponential pattern suggested a role of multivalency. Overall, an increase of 15 tat per CLIO resulted in an elevation of radioactivity uptake of roughly 100 folds. While the current studies used a SS linkage between tat and CLIO, it could be argued that this instability would offset cellular uptake by degradation in serum, our results indicated that this was not the case during the relatively short incubation periods. In addition, comparative studies using similar CLIO-tat preparations containing SC linkage resulted in similar cellular uptake curves. These observations suggested a less important role of the linker type in cellular uptake. In control experiments, where CLIO was attached to similar degrees with a short peptide lacking the transmembrane and nuclear localization sequences, the uptake was near background level. This result indicated that neither the disulfide linker nor FITC contributes to the internalization of CLIO-tat complex. A dramatically elevated cellular uptake at high tatto-CLIO ratios is further demonstrated using T2 weighted MRI (Figure 4). After a 2-h incubation in 20 ng/mL Fe with each of the conjugates, the cells were washed thoroughly and fixed in a phantom, and T2-weighted MR images were acquired. Changes in signal intensity from
Cell Labeling with Differentially Conjugated CLIO−tat
b
Bioconjugate Chem., Vol. 13, No. 4, 2002 843
where cell proliferation is accompanied by a decrease of mean signal intensity and increase in signal heterogeneity (Weissleder, unpublished observations). In addition, the material is clearly biodegradable as demonstrated in nondividing cells and by experiments in vivo (data not shown). We had previously shown that the iron from a CLIO precursor gradually enters global iron metabolism. This is evident from observed decrease in the apparent T2 effect from injected iron oxide contrast agent over a period of days in vivo and that 59Fe radio tracer became incorporated into hemoglobin of red blood cells (14). In conclusion, we describe a P2T method adapted to determine the degree of conjugation in peptide/protein and CLIO complex with disulfide linkage. The method was shown with simplicity, accuracy, and reproducibility. Using a series of conjugates with progressively increasing numbers of tat peptide per CLIO particle, we demonstrated the multivalent effect of peptide conjugation on cellular uptake, which led to a vastly improved CLIOtat preparation with 100-fold increase in cell labeling efficiency. This would translate directly into lower detection thresholds of labeled cells in vivo, where imaging of individual, magnetically labeled cells has been a long cherished goal, particularly in tracking immune cells and stem cells. ACKNOWLEDGMENT
Figure 4. T2 weighted spin-echo images of cells incubated with a series of CLIO-tat conjugates of progressively increasing tat-to-CLIO ratios. A, 10 million cells were immobilized in 0.8% agar in 100 µL volume. The top row includes images of unlabeled cells (left) and plain CLIO (right). Images in the bottom row were from cells incubated with identical iron concentration of CLIO-tat with increasing tat-to-CLIO ratios. B, relative signal intensities were averaged and plotted as a function of tat-toCLIO ratios.
cells labeled with plain CLIO were barely detectable compared with unlabeled cells. Magnetic effects from the low tat-to-CLIO conjugates produced little detectable effect. In particular, one to three tat peptides per CLIO generated little difference upon visual inspection of the images. With increasing numbers of tat, the magnetic effect becomes significantly more prominent. At the highest ratio, there were no signs of toxic effects observed with trypan blue exclusion staining. Finally, the MRI data is consistent with the result from radioactivity uptake experiments in that the loss in signal intensity is clearly associated with progressively increasing tat-to-CLIO ratios. A cluster of basic amino acids in tat peptide is thought to be crucial for membrane translocation (13). In addition, the GRKKR nuclear localization signal within its sequence may play a role in the rapid accumulation in the nuclear regions (13). Indeed, using a monoclonal antibody against dextran, which is the polysaccharide coating of CLIO, it has been demonstrated that CLIO-tat particles are internalized and localized in and through the nucleus (3). These reports confirmed that the observed elevation in radioactivity uptake and the loss of MR signal intensity are caused by the intracellular accumulation of CLIO-tat particles, and that the number of tat peptide on the surface of CLIO dramatically influences uptake of the contrast agent. Once internalized, CLIO-tat is distributed to daughter cells during cell divisions. This can be seen in flow cytometric studies of CLIO-tat labeled cells
We thank Alexander Petrovski for assistance with MR imaging. This work was funded in part by NIH P50 CA86355. M.Z. was supported by T32 CA 79443. M.F.K. was supported by Deutsche Forschungsgemeinschaft (DFG). LITERATURE CITED (1) Lewin, M., Carlesso, N., Tung, C. H., Tang, X. W., Cory, D., Scadden, D. T., and Weissleder, R. (2000) Tat peptidederivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat. Biotech. 18, 410-414. (2) Bhorade, R., Weissleder, R., Nakakoshi, T., Moore, A., and Tung, C. H. (2000) Macrocyclic chelators with paramagnetic cations are internalized into mammalian cells via a HIV-tat derived membrane translocation peptide. Bioconjugate Chem. 11, 301-305. (3) Josephson, L., Tung, C. H., Moore, A., and Weissleder, R. (1999) High-efficiency intracellular magnetic labeling with novel superparamagnetic-Tat peptide conjugates. Bioconjugate Chem. 10, 186-191. (4) Weissleder, R., Cheng, H. C., Bogdanova, A., and Bogdanov, A., Jr. (1999) Magnetically labeled cells can be detected by MR imaging. J. Magn. Reson. Imag. 7, 258-63. (5) Vives, E., Brodin, P., and Lebleu, B. (1997) A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem. 27, 16010-16017. (6) Caron, N. J., Torrente, Y., Camirand, G., Bujold, M., Chapdelaine, P., Leriche, K., Bresolin, N., and Tremblay, J. P. (2001) Intracellular delivery of a Tat-eGFP fusion protein into muscle cells. Mol. Ther. 11, 301-308. (7) Eguchi, A., Akuta, T., Okuyama, H., Senda, T., Yokoi, H., Inokuchi, H., Fujita, S., Hayakawa, T., Takeda, K., Hasegawa, M., and Nakanishi, M. (2001) Protein transduction domain of HIV-1 Tat protein promotes efficient delivery of DNA into mammalian cells. J. Biol. Chem. 276, 26204-26210. (8) Kim, D. T., Mitchell, D. J., Brockstedt, D. G., Fong, L., Nolan, G. P., Fathman, C. G., Engleman, E. G., and Rothbard, J. B. (1997) Introduction of soluble proteins into the MHC class I pathway by conjugation to an HIV tat peptide. J. Immunol. 159, 1666-1668. (9) Fawell, S., Seery, J., Daikh, Y., Moore, C., Chen, L. L., Pepinsky, B., and Barsoum, J. (1994) Tat-mediated delivery of heterologous proteins into cells. Proc. Natl. Acad. Sci. U.S.A. 91, 664-668.
844 Bioconjugate Chem., Vol. 13, No. 4, 2002 (10) Wender, P. A., Mitchell, D. J., Pattabiraman, K., Pelkey, E. T., Steinman, L., and Rothbard, J. B. (2000) The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc. Natl. Acad. Sci. U.S.A. 97, 13003-13007. (11) Schoepf, U., Marecos, E. M., Melder, R. J., Jain, R. K., and Weissleder, R. (1998) Intracellular magnetic labeling of lymphocytes for in vivo trafficking studies. Biotechniques 24, 642-6, 648-51. (12) Ngo, T. T. (1986) A simple spectrophotometric determination of solid supported amino groups. J. Biochem. Biophys.
Zhao et al. Methods 12, 349-354. (13) Efthymiadis, A., Briggs, L. J., Jans, D. A. (1998) The HIV-1 Tat nuclear localization sequence confers novel nuclear import properties. J. Biol. Chem. 273, 1623-1628. (14) Weissleder, R., Stark, D. D., Engelstad, B. L., Bacon, B. R., Compton, C. C., White, D. L., Jacobs, P., and Lewis, J. (1989) Superparamagnetic iron oxide: pharmacokinetics and toxicity. AJR Am. J. Roentgenol. 152, 167-73.
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