High-Efficiency Intracellular Magnetic Labeling with Novel

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Bioconjugate Chem. 1999, 10, 186−191

High-Efficiency Intracellular Magnetic Labeling with Novel Superparamagnetic-Tat Peptide Conjugates Lee Josephson, Ching-Hsuan Tung, Anna Moore, and Ralph Weissleder* Center for Molecular Imaging Research, Massachusetts General Hospital, Boston, Massachusetts 02129.

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Received October 19, 1998; Revised Manuscript Received January 4, 1999

A biocompatible, dextran coated superparamagnetic iron oxide particle was derivatized with a peptide sequence from the HIV-tat protein to improve intracellular magnetic labeling of different target cells. The conjugate had a mean particle size of 41 nm and contained an average of 6.7 tat peptides. Derivatized particles were internalized into lymphocytes over 100-fold more efficiently than nonmodified particles, resulting in up to 12.7 × 106 particles/cell. Internalized particles localized in cytoplasm and nuclear compartments as demonstrated by fluorescence microscopy and immunohistochemistry. Labeled cells were highly magnetic, were detectable by NMR imaging, and could be retained on magnetic separation columns. The described method has potential applications for in vivo tracking of magnetically labeled cells by MR imaging and for recovering intracellularly labeled cells from organs.

INTRODUCTION

Cell labeling with ferro/supermagnetic substances is an increasingly common method for in vitro cell separation (1). Magnetically labeled cells can also be detected by magnetic resonance imaging (2-5). Most current labeling techniques utilize one of two approaches: (i) attaching magnetic particles to the cell surface (6) or (ii) internalizing biocompatible magnetic particles by fluid phase endocytosis (3, 5), receptor mediated endocytosis (7) or phagocytosis (2). The first approach is limited to ex vivo use (8). The second approach is limited by the fact that many cells, such as lymphocytes, do not possess high-efficiency internalizing receptors and that the fluid phase pathway is relatively inefficient (5). It would therefore be highly desirable to develop a technique of high efficiency intracellular magnetic labeling for in vivo magnetic separation and/or imaging of a wide variety of cells. Such techniques are expected to be of particular importance as cell-based genetic therapies are being developed. We hypothesized that a superparamagnetic iron oxide with a membrane translocating signal (MTS) peptide attached might be capable of producing high levels of cell internalization. Several MTSs have been described including the third helix of the homeodomain of Antennapedia (9), peptide derived from anti-DNA monoclonal antibody (10), VP22 herpes virus protein (11, 12), and HIV-1 tat peptide (13). HIV-1 tat protein is an 86 amino acid polypeptide and is essential for viral replication. It has been shown to freely travel through cellular and nucleic membranes (14, 15). Its membrane translocational property is dominated by a short signal, GRKKRRQRRR (amino acid residues 48-57). More recently, it has been demonstrated that the core peptide itself rather than the entire protein is capable of translocating a variety of molecules, including fluorescent probes (16), peptides (17), pathogenic epitopes (18), and proteins (13, 19-21). Here we report that tat peptide (residues 48* To whom correspondence should be addressed. Tel: (617) 726-8226. Fax: (617) 726-5708. E-mail: [email protected]. harvard.edu.

57) derivatized superparamagnetic iron oxide nanoparticles (41 nm) are internalized into cells over 100-fold more efficiently than has been reported to date. The novel compound may thus serve as a reagent for magnetic resonance imaging of cell trafficking and/or magnetic separation of in vivo homed cells. EXPERIMENTAL PROCEDURES

Peptide Synthesis. A peptide containing the translocation sequence of the tat peptide was synthesized on an automatic synthesizer (PS3, Rainin, Woburn, MA) by Fmoc chemistry using 2-(1H-benzotriazole-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate (HBTU)/Nhydroxybenzotriazole (HOBt) as activating agent. The peptide is referred to as tat(FITC). The sequence is GlyGly-Cys-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg- Lys(FITC)-NH2 (the italicized amino acids correspond to residues 48-57 of the tat protein). Fmoc-Lys(Dde) was anchored to 0.1 mmol of Rink amide MBHA resin (NovaBiochem, San Diego, CA) and followed with other amino acids, e.g., Fmoc-Arg(Pbf), Fmoc-Gln(Trt), FmocLys(Boc), Fmoc-Gly, and Fmoc-Cys(Trt). The N-terminal was finally capped with t-Boc-Gly. Thereafter, the Dde group on the C-terminal lysine residue was selectively removed with 10 mL of 2% hydrazine in DMF (2 × 3 min) and the deprotected amino group was reacted with 0.4 mmol of fluorescein isothiocyanate (FITC) (Aldrich, Milwaukee, WI) in 5 mL of DMSO/diisopropylethylamine (20% v/v) overnight. The peptide was cleaved by 5 mL of TFA/thioanisole/ethandithiol/anisole (90/5/3/2) and purified by C18 reversed-phase HPLC. MALDI-MS (M + H)+: 2130.4 (calc.), 2130.3 (found). Synthesis of Amine Terminated Cross-Linked Superparamagnetic Iron Oxide (CLIO-NH2) (II). A monodispersed superparamagnetic iron oxide colloid (MION) I was synthesized and cross-linked with epichlohydrin (Aldrich) to prepare II as described (22). Briefly, amination was achieved by the addition of 50 mL of concentrated ammonia/200 mL of the MION colloid, followed by heating at 37 °C overnight. The mixture was incompatible with ultrafiltration equipment, so low molecular weight materials were removed by dialysis against

10.1021/bc980125h CCC: $18.00 © 1999 American Chemical Society Published on Web 02/26/1999

Superparamagnetic-Tat Peptide Conjugate Labeling

water using dialysis tubing (12-14K cutoff) (Spectra/Por, Laguna Hills, CA). Air was then bubbled through the colloid for 24 h at 37 °C. The colloid was subjected to pressure dialysis with the addition of 10 vol of 5 mM sodium citrate, pH 8. These steps fully oxidize any ferrous iron and remove traces of low molecular weight materials. Final concentration was 26 mg Fe/mL. Synthesis of 2-Pyridyl Disulfide Derivatized Superparamagnetic Iron Oxide (III). To 1.2 mL of II (31.2 mg Fe, 557 µmol) was added 1.2 mL of 0.1 M phosphate buffer, pH 7.4, and 2 mL of N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (Molecular Biosciences, Boulder, CO) in DMSO (25 mM, 50 µmol). The mixture was allowed to stand for 60 min at room temperature. Low molecular impurities were removed by PD-10 columns (Sigma Chemical, St. Louis, MO) equilibrated with 0.01 M Tris and 0.02 M citrate, pH 7.4 buffer. The pooled void volume of 4.4 mL was recovered, containing III at a concentration of 7 mg Fe/mL. To measure the number of 2-pyridyl disulfide groups attached, 0.2 mL of the solution above was added to 0.2 mL of 0.05 M DTT in 0.1 M phosphate, pH 7.4. The mixture was allowed to stand for 30 min at room temperature. A microconcentrator, 30 kDa cutoff (Amicon, Beverly, MA) was used to separate the product of the reduction, pyridine-2-thione (P2T), from iron. The concentration of P2T was determined using an extinction coefficient at 343 nm of 8100 M-1 cm-1. Results are expressed as the number of SPDP groups per particle, i.e., per crystal, assuming 2064 Fe atoms/crystal (23). Synthesis of Superparamagnetic Iron Oxide-Tat(FITC) (CLIO-Tat) (IV). To 2.3 mL of III (16.1 mg/Fe, 288 µmol) was added 1.9 mL of tat(FITC), (803 µM, 1.52 µmol, as determined by FITC absorbance, extinction coefficient of 73 000 M-1 cm-1 at 494 nm in 0.1 M phosphate buffer). The mixture was allowed to react overnight at room temperature. The solution was applied to PD-10 columns equilibrated as above and the excluded volume containing IV saved. To measure the number of tat(FITC) groups attached, the colloid was reacted with DTT and applied to a microconcentrator as above. Released peptide in the filtrate was quantitated using FITC fluorescence read against a standards of FITC. 111In Labeling of IV. To permit IV to chelate indium, it was reacted with DTPA dianhydride (Aldrich). IV was exhaustively dialyzed against 2.5 mM citrate, pH 6.5 (Spectra/Por membrane, 12-14 kDa cutoff). To 0.6 mL of iron oxide (17.1 µmol of Fe, 8.28 nmol of crystal with 56 nmol of tat-peptide attached) were added 0.1 mL of 0.1 M tetraborate buffer, pH 8.5, and 0.2 mL of 0.14 mM DTPA dianhydride in DMSO (0.028 µmol). The reaction was allowed to proceed at room temperature for 60 min. Unreacted DTPA was removed by using a PD-10 column, equilibrated with 0.02 M sodium citrate, pH 6.5. The samples were applied to the columns which was spin for 5 min at 800g. To the filtrate was added 0.72 mCi of 111 In (NEN Life Sciences, Boston, MA), which was allowed to react for 3 h at room temperature. The separation procedure was repeated with 45-50% of the radioactivity passing through the column. Cellular Uptake Assays. Lymphocytes were prepared from mouse spleen as previously described (5). Briefly, mice (n ) 2) were sacrificed under general anesthesia by lethal intravenous injection of sodium pentobarbital (220 mg/kg, Anpro, Arcadia, CA) and their spleens removed using aseptic procedures. Lymphocytes were obtained by disrupting the spleen between sterile frosted histology slides and dispersed in RPMI 1640 medium (Cellgro, Mediatech, Washington, DC) supplemented with

Bioconjugate Chem., Vol. 10, No. 2, 1999 187

10% fetal bovine serum. After an initial centrifugation, erythrocytes were lysed by resuspending the cell pellet in 0.83% ammonium chloride in distilled water. To remove the majority of the monocyte/macrophage population, the nonadherent cells were transferred into a new flask after 1 h and counted. This method yielded a cell population containing about 95% lymphocytes, as determined by morphology. Cells were counted and their viability was determined with the Trypan Blue exclusion assay. The uptake of 111In-labeled IV in mouse lymphocytes was measured as previously described (5). To determine the amount of superparamagnetic particle associated with cells, we used a simple cell binding assay. Cell suspensions (106 cells) were placed into plastic tubes containing 1 mL of the media with different concentrations of 111In-IV. Cells were incubated at 37° C for 1 h and then washed three times by centrifugation through a step gradient of 40% Histopaque-1077 (Sigma) in Hanks’ balanced salt solution (Mediatech). After the last centrifugation and aspiration of the supernatant, cell pellets were counted in a gamma counter (1289 Compugamma LS; Wallac, Turku, Finland). Fluorescence Microscopy. Lymphocytes, natural killer (NK) cells, and HeLa cells were further used for fluorescent microscopy. HeLa cells were grown to confluence on glass cover slips in 12 well plates (Becton Dickinson, Bedford, MA). The culture MEM (Minimum essential medium) was replaced with 500 µL of IV in MEM media, 0.2 mg Fe/mL. Same amount of IV was added to cell suspensions of NK cells and mouse lymphocytes. After 30 min at 37 °C the cells were washed 3 times with HBSS and analyzed by fluorescence microscopy. All images were obtained using a cooled charge coupled device (Photometrics, Tucson, AZ), operated via a linked Power Macintosh 7600/120 computer. Immunohistochemical Stain for Dextran. To evaluate cellular localization of IV, HeLa cells were plated on glass cover slips in a 12 well plate at 60% confluency 24 h before the experiment. Cells were incubated with IV for 1 h at 20 µg of Fe, washed extensively with HBSS and fixed with MeOH:acetone (1:1) mixture for 10 min at -80° C. After fixation cells were incubated with antidextran IgA antibody W1329 (generous gift from Dr. Glaudemans) (24), followed by incubation with secondary biotinylated goat anti-mouse IgA antibody (Sigma). Biotin was probed for with avidin peroxidase (Bio-Rad, Hercules, CA). The color reaction was revealed with DAB Fast substrate tablets (Sigma). Physical Properties of the Superparamagnetic Iron Oxide Particles. Relaxivity, iron concentration, and size by light scattering were measured as described (23). Special phantoms were constructed for NMR imaging that consisted of 107 cells embedded in agar to prevent drying and susceptibility artifacts. Freshly isolated lymphocytes were labeled with IV or I, and sedimented cell pellets were resuspended in 30 µL of warm agarose and placed into holes previously stenciled into the agarose. Each well was then sealed with an additional 0.5 mL of agarose. NMR imaging was performed with a 1.5 T superconducting magnet (Signa 5.0; GE Medical Systems, Milwaukee, WI) using a 5 in. surface coil. The imaging protocol consisted of coronal T1 weighted spin-echo (SE 300/11 where the first number represents the repetition time, TR, and the second number the echo time, TE), T2 weighted SE (SE 2000/102) or T2* gradient echo sequence (Steady-State Free Precession pulse sequence with TR/TE 50/60 and a flip angle of 20°) representative of commonly used pulse sequences in

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Figure 2. Uptake of IV and I by mouse lymphocytes at 37 °C for 1 h. I was prepared as described in ref 5. Note the logarithmic Y axis. Cellular uptake of control peptide-particles were similar to I (data not shown).

Figure 1. Synthetic scheme of superparamagnetic iron oxideTat peptide conjugate

clinical imaging. Slice thickness was 3 mm. The field of view (FOV) was 10 or 12 cm2, using an 256 × 192 imaging matrix and 2-4 acquisition averages. RESULTS AND DISCUSSION

A variety of MTS peptides have been used as vectors to carry various payloads into cells (10, 25-27). These reports inspired us to test whether MTS conjugated to nanometer sized superparamagnetic iron oxide particles could be used for intracellular magnetic cell labeling. Among the reported MTS peptides, HIV-1 tat is unique in that it is one of the shortest MTS signals. Tat peptides have been used to internalize different compounds into cells, such as peptides (17) and proteins (13, 19-21). Although it has been reported that tat peptide with extensions on both ends has a higher efficiency (13), we chose to use the shortest tat peptide (residues 48-57) (17) for our conjugate. The tat peptide was modified to carry a fluorescein isothiocyanate (FITC) tag, to follow the peptide during conjugation and as a marker for fluorescence microscopy. A Gly-Gly-Cys peptide was incorporated to the N-terminus to introduce a mono reactive sulfhydryl group. The fluorescent peptide was attached to the amino group of a cross-linked dextran iron oxide (1) using SPDP (Figure 1). Reacting II with SPDP, the amino groups on II were converted to sulfhydryl groups, which were initially protected with 2-pyridyl disulfide. The degree

of substitution was determined by reduction with DTT followed by ultrafiltration to free solutions of iron. Measuring the specific absorption of the released pyridine-2-thione at 343 nm, an average of 14 sulfhydryl groups was determined per particle. Tat(FITC) was attached to activated III at pH 7.4 through a disulfide exchange reaction. After separating the free peptide from the conjugate IV by a size-exclusion column, a small portion of IV was used to determine the number of tat(FITC) per particle using the DTT reduction reaction and ultrafiltration. Released peptide was quantitated by determining FITC fluorescence against a FITC standard at 494 nm. An average of 6.7 peptides were thus attached per particle. The physical properties of the superparamagnetic iron oxide colloids involved in this study are summarized in Table 1. No significant difference in the particle size was observed after peptide attachment. In addition, the similar relaxivity (values) of II, III, and IV indicate that the magnetic property of the colloid is unaffected by the reactions performed to attach tat(FITC). A high cellular uptake of IV could be demonstrated in all three cell types tested in this study: murine lymphocytes, human natural killer cells and HeLa cells. With murine lymphocytes, the uptake of radioactive IV was quantified and compared to I (5) (Figure 2). Over a wide concentration range, the uptake of IV was about 100fold higher than that of a conventional nonmodified magnetic particle. At the highest concentration of IV employed, 100 µg of Fe/million cells, there was an uptake of 2535 ng of iron/million cells corresponding to 12.7 × 106 particles/cells. Under similar conditions but using I, mouse peritoneal macrophages internalized 970 ng (2), while splenic lymphocytes, human and rat lymphocytes took up only 18.7, 27.9, and 11.5 ng, respectively (5). The concentration dependence of the uptake of IV was such that even at 20 µg of Fe/million cells, mouse lymphocytes internalized 981 ng/million cells (Figure 2), an amount equivalent to that obtained with macrophages at 100 µg of Fe/million cells.

Table 1: Physical Properties of Superparamagnetic Iron Oxide Colloids compd

surface

size (nm)

2-pyridyl per particle

tat(FITC) per particle

R1a

R2a

II III IV

cross-linked aminated dextran cross-linked dextran-S-S-2Py cross-linked dextran-tat(FITC)

37.1 ND 41.3

ND 14.3 ND

ND ND 6.7

22.4 22.4 22.3

76.7 74.2 71.9

a

mM-1 s-1.

Superparamagnetic-Tat Peptide Conjugate Labeling

Bioconjugate Chem., Vol. 10, No. 2, 1999 189

Figure 3. Fluorescence microscopy of murine lymphocytes (left) or activated human NK cells (right) 30 min after incubation with IV. The label is clearly contained within lymphocytes, particularly the nucleus and nucleoli. The shape of activated NK is slightly irregular representing terminal tufts. No significant intracellular fluorescence was observed when cells were labeled with FITC-II under identical conditions (not shown).

Figure 4. Fluorescence microscopy of HeLa cells incubated with IV.

Figure 3 shows the intracellular distribution of IV by fluorescence microscopy in murine lymphocytes and activated human NK cells. Fluorescence signal was distributed in the cytoplasm with particularly high concentrations in the nucleus and nucleolus. There was a notable lack of punctate staining as is observed with lysosomal accumulation where receptor-mediated or fluid

phase endocytosis pathways are used for magnetic cell labeling (5). Intracellular location of IV was also examined in HeLa cells, which are commonly used in studies of MTS biology. Figure 4 shows HeLa cells after an incubation of 30 min with a concentration of 0.2 mM of Fe IV. A pronounced nuclear accumulation was observed in virtually all cells examined. Cells were not fixed to

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Figure 5. Anti-dextran staining of HeLa cells incubated with IV. (Inset) Magnification at 600×. The brown reaction product represents dextran within cell and the nucleus.

avoid possible alterations due to the fixing procedure. No significant cellular internalization was observed for the control compounds consisting of GRGDSPGGCK(FITC)NH2-II conjugate or FITC-II. The linkage between the superparamagnetic iron and tat (FITC) peptide of conjugate IV is a reversible disulfide bond. Under physiological conditions, it is possible that the peptide is released from the particle. Although the fluorescence signal was clearly intracellular, we performed additional experiments to exclude the possibility of peptide dissociation. Briefly, cells were stained for dextran using a monoclonal antibody (24). As shown in Figure 5, dextran was concentrated in and distributed through the nucleus thus confirming internalization of the entire construct IV. This suggests that the uptake and nuclear localization of IV seen by fluorescence microscopy corresponds to the intact particle and not the dissociated peptide-tat (FITC). Finally, we compared the magnetic effects of cellular internalization of IV and I. These studies were performed with lymphocytes incubated with either compound and then subjected to MR imaging using different pulse sequences. Figure 6 summarizes these results. Signal intensity differences were most marked with the T2 and T2* weighted sequences as predicted by theory. For example, IV-labeled cells produced a much lower signal intensity than did I-labeled cells (Figure 6). These results are entirely consistent with quantitative cell uptake studies (Figure 2) and attest to the feasibility of MR imaging of labeled cells. Furthermore, 95% of lymphocytes labeled with IV were retained on magnetic separation columns whereas cells labeled with I were not (data not shown). We describe the synthesis and properties of a functionalized, biocompatible iron oxide particle containing multiple copies of a membrane translocating tat peptide.

Figure 6. Comparative NMR imaging of I or IV labeled lymphocytes. (Top) T1 weighted spin-echo sequence (SE 300/ 11); middle T2 weighted sequences (FSE 2000/102); (bottom) T2* weighted sequence (SSFP 50/60/20°). Each well measures 2 cm in diameter and each agar plug contains a 20 µL insert with 107 labeled cells. Cell concentration and incubation conditions among the two samples are identical.

Superparamagnetic-Tat Peptide Conjugate Labeling

To the best of our knowledge, our study is the first to show that relatively short MTS signals can internalize entire nanometer sized magnetic particles into cells. A second type of tat (FITC) conjugate was synthesized devoid of the C-terminal GGC sequence. Both conjugates were internalized in a similar manner (data not shown). Utilizing the above-described approach, a variety of studies are currently being conducted to further explore the exact mechanism of cellular internalization of IV conjugates. Preliminary trypan blue exclusion toxicity studies showed no cellular toxicity at incubation concentrations used for MR imaging (up to 30 µg of Fe). Apart from more elaborate toxicity studies, additional MR imaging studies are underway to determine the threshold of cell detection by NMR imaging. Initial feasibility studies have also shown that magnetically labeled cells are indeed recoverable by magnetic cell separation technique after intravenous administration. LITERATURE CITED (1) Olsvik, O., Popovic, T., Skjerve, E., Cudjoe, K. S., Hornes, E., Ugelstad, J., and Uhlen, M. (1994) Magnetic separation techniques in diagnostic microbiology. Clin. Microbiol. Rev. 7, 43-54. (2) Weissleder, R., Cheng, H. C., Bogdanova, A., and Bogdanov, A. (1997) Magnetically labeled cells can be detected by MR imaging. J. Magn. Reson. Imaging 7, 258-63. (3) Yeh, T. C., Zhang, W., Ildstad, S. T., and Ho, C. (1993) Intracellular Labeling of T-Cells with Superparamagnetic Contrast Agents. Magn. Reson. Med. 30, 617-25. (4) Yeh, T. C., Zhang, W., Ildstad, S. T., and Ho, C. (1995) In Vivo Dynamic MRI Tracking of Rat T-Cells Labeled with Superparamagnetic Iron-Oxide Particles. Magn. Reson. Med. 33, 200-208. (5) Schoepf, U., Marecos, E., Melder, R., Jain, R., and Weissleder, R. (1998) Intracellular magnetic labeling of lymphocytes for in vivo trafficking studies. BioTechniques 24, 642-51. (6) Handgretinger, R., Lang, P., Schumm, M., Taylor, G., Neu, S., Koscielnak, E., Niethammer, D., and Klingebiel, T. (1998) Isolation and transplantation of autologous peripheral CD34+ progenitor cells highly purified by magnetic-activated cell sorting. Bone Marrow Transpl. 21, 987-93. (7) Moore, A., Basilion, J. P., Chiocca, E. A., and Weissleder, R. (1998) Measuring transferrin receptor gene expression by NMR imaging. Biochim. Biophys. Acta 1402, 239-49. (8) Tsai, E., Bogdanov, A., Papisov, M., Brady, T. J., and Weissleder, R. (1992) Lymphocytes as drug carriers for MR contrast agents. Society of Magnetic Resonance in Medicine, 11th Annual Meeting, August 8-14, Berlin. (9) Derossi, D., Calvet, S., Trembleau, A., Brunissen, A., Chassaing, G., and Prochiantz, A. (1996) Cell internalization of the third helix of the Antennapedia homeodomain is receptorindependent. J. Biol. Chem. 271, 18188-93. (10) Avrameas, A., Ternynck, T., Nato, F., Buttin, G., and Avrameas, S. (1998) Polyreactive anti-DNA monoclonal antibodies and a derived peptide as vectors for the intracytoplasmic and intranuclear translocation of macromolecules. Proc. Natl. Acad. Sci. U.S.A. 95, 5601-6. (11) Phelan, A., Elliott, G., and O’Hare, P. (1998) Intercellular delivery of functional p53 by the herpesvirus protein VP22. Nat. Biotechnol. 16, 440-3. (12) Elliott, G., and O’Hare, P. (1997) Intercellular trafficking and protein delivery by a herpesvirus structural protein. Cell 88, 223-33.

Bioconjugate Chem., Vol. 10, No. 2, 1999 191 (13) 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, 664-8. (14) Frankel, A. D., and Pabo, C. O. (1988) Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55, 1189-93. (15) Mann, D. A., and Frankel, A. D. (1991) Endocytosis and targeting of exogenous HIV-1 Tat protein. EMBO J. 10, 1733-9. (16) 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. 272, 16010-7. (17) Vives, E., Granier, C., Prevot, P., and Lebleu, B. (1997) Structure-activity relationship study of the plasma membrane translocating protential of a short peptide from HIV-1 Tat protein. Lett. Pept. Sci. 4, 1-8. (18) Schluesener, H. J. (1996) Protection against experimental nervous system autoimmune diseases by a human immunodeficiency virus-1 Tat peptide-based polyvalent vaccine. J. Neurosci. Res. 46, 258-62. (19) Efthymiadis, A., Briggs, L. J., and Jans, D. A. (1998) The HIV-1 Tat nuclear localization sequence confers novel nuclear import properties. J. Biol. Chem. 273, 1623-8. (20) Pepinsky, R. B., Androphy, E. J., Corina, K., Brown, R., and Barsoum, J. (1994) Specific inhibition of a human papillomavirus E2 trans-activator by intracellular delivery of its repressor. DNA Cell Biol. 13, 1011-9. (21) Anderson, D. C., Nichols, E., Manger, R., Woodle, D., Barry, M., and Fritzberg, A. R. (1993) Tumor cell retention of antibody Fab fragments is enhanced by an attached HIV TAT protein-derived peptide. Biochem. Biophys. Res. Commun. 194, 876-84. (22) Palmacci, S., and Josephson, L. (1993) Synthesis of polysaccharide covered superparamagnetic oxide colloids, U.S. Patent 5,262,176. (23) Shen, T., Weissleder, R., Papisov, M., Bogdanov, A., and Brady, T. J. (1993) Monocrystalline iron oxide nanocompounds (MION): physiocochemical properties. Magn. Reson. Med. 29, 599-604. (24) Nashed, E. M., and Glaudemans, C. P. (1996) Observations on the binding of four anti-carbohydrate monoclonal antibodies to their homologous ligands. J. Biol. Chem. 271, 820914. (25) Pooga, M., Soomets, U., Hallbrink, M., Valkna, A., Saar, K., Rezaei, K., Kahl, U., Hao, J. X., Xu, X. J., WiesenfeldHallin, Z., Hokfelt, T., Bartfai, T., and Langel, U. (1998) Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo. Nat. Biotechnol. 16, 857-61. (26) Rojas, M., Donahue, J. P., Tan, Z., and Lin, Y. Z. (1998) Genetic engineering of proteins with cell membrane permeability. Nat. Biotechnol. 16, 370-5. (27) Chaloin, L., Vidal, P., Lory, P., Mery, J., Lautredou, N., Divita, G., and Heitz, F. (1998) Design of carrier peptideoligonucleotide conjugates with rapid membrane translocation and nuclear localization properties. Biochem. Biophys. Res. Commun. 243, 601-8.

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