Protamine as an Efficient Membrane-Translocating Peptide

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Bioconjugate Chem. 2005, 16, 1240−1245

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Protamine as an Efficient Membrane-Translocating Peptide Fred Reynolds, Ralph Weissleder, and Lee Josephson* Center for Molecular Imaging Research, Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts 02129. Received May 19, 2005; Revised Manuscript Received August 11, 2005

Protamine, a mixture of positively charged proteins from salmon roe used in diverse pharmaceutical applications, was reacted with the N-hydroxysuccinimide ester of tetramethylrhodamine to yield tetramethylrhodamine-labeled protamines (Pro(Rh)) containing one mole of fluorochrome per mole of protein. The internalization of tetramethylrhodamine-labeled protamine (Pro(Rh)) and the fluoresceinlabeled tat peptide (Tat(Fl)) showed a similar dependence on time and concentration. Pro(Rh) and Tat(Fl) showed strong nuclear localizations, evident with both live cells and fixed cells costained with DAPI, a nuclear stain. The loss of fluorescence when cells were loaded with Pro(Rh) or Tat(Fl) was similar, further supporting a strong similarity between these two materials. Finally, when Pro(Ph) was covalently attached to the amino-CLIO nanoparticle, the cellular uptake of the nanoparticle was greatly enhanced. All experiments were performed with HeLa and CaCo-2 cells with similar results. These observations imply that protamine, a protein in regular clinical use, might be used for the design of novel membrane translocating/nuclear localizing pharmaceuticals whose development was initiated with other membrane-translocating peptides. In addition, the fluorescent protamines developed here might be used to further our understanding of this important pharmaceutical.

INTRODUCTION

Protamine, a mixture of proteins extracted from salmon roe, is in regular clinical use as an excipient in insulin formulations (1, 2) and to reverse the anticoagulant properties of heparin (3). The biological role of protamine is to bind DNA and to provide a highly compact configuration of chromatin in the nucleus of the sperm (4, 5). Protamine is arginine rich, a feature it shares with some viral proteins which utilize arginine rich sequences of amino acids to provide membrane-translocating and/or nuclear-localizing activities (6), see Table 1. We noted that protamine was not only arginine rich, but in common with viral translocation sequences had short sequences consisting of seven or more arginine residues (6). In addition, protamine has a single reactive amine at the N-terminus and a single carboxyl group at the Cterminus, a situation that might facilitate site-specific modification and the design of protamine-based probes. We therefore investigated the membrane-translocating activity of protamine to add to the understanding of this important pharmaceutical and because protamine might be superior to other membrane-translocating peptides for drug delivery applications due to its ease of modification, low cost, availability, sterility, and GMP manufacture. Synthetic polyarginyl peptides have been exploited to provide improved forms of cyclosporine (7), improved radiotherapeutic agents (8), and improved methods of intracellular delivery of DNA and enzymes (9-11). Using fluorescent protamines, we show that protamine has a membrane-translocating activity comparable to that of the HIV1 tat peptide and therefore might be an ideal * To whom correspondence should be addressed. Tel. 617-7266478; Fax. 617-726-5708; e-mail: [email protected]. 1 Abbreviations: CLIO, cross-linked iron oxide; Pro, protamine; Rh, 5-carboxytetramethylrhodamine; Fl, 5-carboxyfluorescein; HIV, human immunodeficiency virus; HTLV-II, human T-cell lymphotrophic virus type-II; BMV, brome mosaic virus; FHV, flock house virus.

carrier for drug delivery applications. Studies following the intracellular disposition of fluorescent protamines in vivo may prove helpful in understanding the fate of this widely used pharmaceutical after injection. MATERIALS AND METHODS

The N-hydroxysuccinimide esters of 5-carboxytetramethylrhodamine and 5-carboxyfluorescein were from Molecular Probes (Eugene, OR). Protamine sulfate was from American Pharmaceutical Partners (Schaumburg, IL). Carbodiimide (1-ethyl-3(3′dimethylaminopropyl)carbodiimide) was from Novabicohem (LaJolla, CA). The Tat(Fl) peptide, see Table 1, was synthesized on an Advanced Chemtech Apex 396 peptide synthesizer (Advanced Chemtech, Louisville, KY) as described (12), except that an N-hydroxysuccinimide ester of fluorescein rather than fluorescein isothiocyanate was used. Tat(Fl)CLIO was prepared as described (13). To synthesize fluorescent protamines, 0.5 mg of the N-hydroxysuccinimide ester of either tetramethylrhodamine or fluorescein in 100 µL of DMSO was added to 5 mg of protamine (in 500 µL of 0.85 M NaCl as supplied) and 100 µL of PBS, and allowed to react overnight at 4°C. Peptides were purified using a reverse phase C18 column with acetonitrile/water gradients, and products were characterized by MALDI-MS. The molecular weights (M + 1) of the fluorescent protamines we synthesized were within 1 Da of the expected values (Table 1). Fluorescein-labeled protamine is denoted Pro(Fl), and tetramethylrhodamine-labeled protamine is denoted Pro(Rh). To synthesize protamine-derivatized nanoparticles, amino-CLIO (14) was first reacted with Cy5.5 to yield CLIO(Cy5.5) as described (15). Unreacted Cy5.5 was removed by Sephadex G-25 gel filtration and Cy5.5 concentration determined spectrophotometrically (absorption at 675 nm, extinction coefficient of 250 000 L/mol-cm). Since there are 200-240 amines per 8000 Fe with the CLIO nanoparticle (16, 17), and 8.0 were

10.1021/bc0501451 CCC: $30.25 © 2005 American Chemical Society Published on Web 09/02/2005

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Membrane Translocation of Protamine Table 1. Protamine and Fluorescent Protaminesa name protamine 1 protamine 2 protamine 3 protamine 4 protamine 1-tetramethylrhodamine protamine 1-fluorescein HIV-1 Tat (48-60) HIV Rev (34-50) FHV coat (35-49) BMV Gag (7-25) HTLV-II Rex (4-16)

sequence PRRRR SSSRP VRRRR RPRVS RRRRR RGGRR RR (22R’s of 32) PRRRR SSSRP IRRRR PRRAS RRRRR RGGRR RR (22R’s of 32) P(Rh)PRRRR SSSRP VRRRR RPRVS RRRRRRRGGR RRR (22R’s of 32) P(Fl)RRRR SSSRP VRRRR RPRVS RRRRR RRGGR RRR (22R’s of 32) GRKKR RQRRR RGP (7R’s of 13) TRQAR RNRRR RWRER QR (10R’s of 17) RRRRN RTRRN RRRVR (11R’s of 15) KMTRA QRRAA ARRNR WTAR (7R’s of 19) TRRQR TRRAR RNR (8R’s of 13)

mass (M + 1) exp 4252.1 4237.5 4321.5 4065.5 4664.6 4610.8 NA NA NA NA NA

a Protamine is a mixture of four peptides, protamines 1, 2, 3, and 4. Also shown are the arginyl rich sequences of peptides derived from viral genes that show a strong nuclear localizing activity (6). Numbers in parentheses are the numbers of arginines and total amino acids.

consumed by reaction with Cy5.5, the remaining amines were available to react with the C-terminal carboxyl group of Pro(Rh). The CLIO(Cy5.5) nanoparticle (1 mg Fe, 5.73 mg Fe/mL) was added to 200 µL of Pro(Ph) (2.65 mM). Eight milligrams of 1-ethyl-3(3′dimethyl-aminopropyl)carbodiimide in 1 mL of MES buffer (0.1 M, pH 4.9) were added, the mixture allowed to sit overnight, and unreacted Pro(Rh) was removed by gel filtration on Sephadex G50. There were 28 Pro(Rh) attached per 8000 Fe atoms determined from tetramethylrhodamine absorption (565 nm, extinction coefficient of 91 000 L/molcm). The nanoparticle is denoted Pro(Rh)-CLIO(Cy5.5). CaCo-2 and HeLa cells were from ATCC (Manassas, VA) and grown according to ATCC specifications, except for the addition of penicillin/streptomycin. Cell culture reagents were from CellGrow (Herndon, VA). Cell uptake of peptides or nanoparticles was determined using 24-well plates (250 µL media/well). Cells were incubated with peptide or nanoparticle for the indicated times and concentrations. Cells were washed three times with PBS and lysed (0.1% Triton X-100 µL in PBS), and fluorescence was determined on a plate reader (Molecular Devices, Sunnyvale, CA) (Figure 3). Cells were trypsinized for FACS measurements (FACS Caliber, Becton Dickinson, San Jose, CA) (Figure 1C). Cell counts were made with a Beckman Coulter Z1 cell counter (San Jose, CA). Amounts of cell associated peptide were determined by use of standards of fluorescent peptides. To obtain the time course of fluorescence loss (Figure 4), cells were allowed to internalize peptide, washed, and incubated with media for varying times. Cell-associated fluorescence was determined after trypsinization and FACS. To demonstrate the uptake of nanoparticles (Figure 5), cells were trypsinized and cell-associated fluorescence determined by FACS. A portion of cells was spun down and lysed, and the iron uptake was obtained by calibration with fluorescent standards of each nanoparticle. For microscopy, cells were grown on glass slides (Fisher Scientific, Pittsburgh, PA), exposed to 5 µM peptide for 4 h, and washed three times with PBS. Unfixed cells (Figure 1) were examined using a Nikon Eclipse TE2000-S epifluorescence microscope with long focal length optics (Torrance, CA). Cells were also fixed with 70% ethanol at 4 C for 90 min, washed with PBS, mounted using Vector Shield with DAPI (Vector Laboratories, Burlingame, CA), and examined by epifluorescence (Figure 2). Fluorescence microscopy used a Nikon Eclipse 80i epifluorescence microscope (Torrance, CA).

RESULTS

Protamine is a mixture of four proteins listed in the order of their peak height determined by mass spectrometry in Table 1. The mass of protamine 1 is that of the protamine denoted AI, gene accession number P02327 (NCBI-protein database). The mass of protamine 2 is that of the protamine denoted AII, gene accession number B02669 (NCBI-protein database). The composition of protamine 3 and protamine 4 is not known. Also shown are the arginyl rich sequences derived from viral genes that show a strong nuclear localizing activity (6) and membrane-translocating sequence of the tat peptide (18). Numbers in parentheses are the number of arginines and total number of amino acids for the peptide. Both protamine and viral-translocating sequences share an overall arginine rich character that consists of seven or more arginines. Fluorescent protamines were synthesized by reacting the N-hydroxysuccinimidyl esters of 5-carboxytetramethylrhodamine and 5-carboxyfluorescein with protamine, to produce proteins containing a single fluoro-

Figure 1. Internalization of rhodamine-labeled protamine and fluorescein-labeled tat peptide. HeLa cells were incubated (2 µM, 4 h) with either Pro(Rh) (A) or Tat(Fl) (B). Phase contrast images on unfixed cells were taken, and images from rhodamine channel (A) or fluorescein channel (B) were superimposed. Scale marker is 10 µm. (C) HeLa cells labeled with Pro(Ph) as above were trypsinized and analyzed by FACS.

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Figure 2. Nuclear localization of protamine or tat peptide. Cells were incubated with Pro(Rh) (A, B, C) or Tat(Fl) (D, E, F) at 2 µM fluorochrome for 4 h. Cells were washed, fixed, and stained with DAPI, a nuclear stain. (A and D) Cell nuclei shown by the DAPI (ultraviolet) channel. (B) Pro(Rh) visualized in rhodamine channel. (C) Overlay of parts A and B. (E) Tat(Fl) visualized in the fluorescein channel. (F) Overlay of parts D and E. Scale marker is 10 µm.

chrome as shown in Table 1. The site of attachment is presumed to be N-terminal proline residue, since none of the other amino acids have a reactive primary amine. The amines of the guanidinium group of arginine are not chemically reactive (19). After reaction with 5-carboxytetramethylrhodamine, the four protamines exhibited an increase in molecular weight consistent with the addition of a residue weight of tetramethylrhodamine (C25H20O4, 412.45 Da). Similarly, after reaction with 5-carboxyfluorescein, four proteins were identified with an increase in molecular weight consistent with the addition of a residue of 5-carboxyfluorescein (C21H10O6, 358.3 Da). The molecular weights obtained by reaction of protamine 1 with tetramethylrhodamine or fluorescein are given in Table 1. Protamine labeled with tetramethylrhodamine is denoted Pro(Rh), fluorescein-labeled protamine is denoted Pro(Fl), and fluorescein-labeled tat peptide is denoted Tat(Fl). The uptake of Pro(Rh) and Tat(Fl) by HeLa cells is shown in Figure 1A and B, respectively after 4 h of incubation with each peptide and after cells were washed with PBS (three times) to remove any loosely adsorbed peptide. Phase contrast images and fluorescence micrographs in the rhodamine channel were obtained for cells exposed to Pro(Rh) and the images superimposed as shown in part A. Under these conditions, an intense fluorescence was seen in the center of the cell, which appeared to represent a nuclear accumulation as is discussed further below. Fluorescence was not associated with the cell membrane or in a punctate pattern in the cytoplasm typical of endosomal accumulation. A similar pattern was seen when HeLa cells were incubated with the Tat(Fl) peptide and the fluorescence from the fluorescein channel superimposed on phase contrast images (B). Pro(Rh)-treated HeLa cells were then trypsinized and the distribution of fluorescence obtained by FACS, as shown in Figure 1C. Pro(Rh) uniformly labeled cells in a manner to that seen with Tat(Fl) labeling; see for example Figure 2 of ref 15. All experiments in this report were performed on HeLa cells and CaCo-2 cells, with similar results, an example of which is shown in Figure 3. To confirm that fluorescence was nuclear, the nuclei of cells incubated with Pro(Rh) or Tat(Fl) were stained

with the nuclear stain DAPI. Figures 2A and 2D show the well-defined nuclei visualized as the fluorescence from DAPI nuclear stain. Figure 2B shows the tetramethylrhodamine fluorescence from cells incubated with Pro(Ph), which, based on the overlay, appeared to be nuclear (Figure 2C). Figure 2D shows the result obtained when cells were incubated with Tat(Fl) (fluorescein channel), which, based on the overlay with the DAPI stain, again appeared to be nuclear (Figure 2E). Thus, Rh(Pro) was internalized by cells and showed a nuclear accumulation in a manner similar to Tat(Fl). We next examined the time course of internalization of the fluorescent protamines (Pro(Rh) and Pro(Fl)) and compared it with tat peptide (Tat(Fl)) as a function of time (Figure 3A) and concentration (Figure 3B). The internalization of Pro(Rh) was complete within 1 h and did not show saturation behavior with respect to concentration. Previous studies with arginine rich peptides have shown internalization within 1 h and either a linear dependence of uptake on peptide concentration or some evidence of saturation at high peptide concentrations (6, 20, 21). The internalization of protamine was strongly dependent on the attached fluorochrome, with the uptake of Pro(Rh) being far greater than Pro(Fl), which may reflect an interaction between the negative charge on fluorescein and the positive charges on protamine. Data comparing the uptake of Pro(Ph) with HeLa cells and CaCo-2 cells is shown as example of the data we obtained showing the similar behavior of all proteins and peptides with these two cell types in all experiments. Data shown in Figure 3 is the cell-associated fluorescence measured after lysis and determination of fluorescence with a microtiter plate reader and standards, a procedure which permits expression of results in moles per cell. Trypsinization and FACS yielded similar relationships about the time course and amounts of uptake for the three peptides examined (data not shown). Thus, the kinetics of uptake and relative amounts of cell-associated Tat(Fl) and Pro(Ph) were similar when fluorescence was determined by lysis of adherent cells or with trypsinization and FACS (data not shown). We next examined the loss of cell-associated fluorescence after HeLa cells were loaded with fluorescent

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Membrane Translocation of Protamine

Figure 3. Uptake of protamine and tat peptide by HeLa cells. (A) Uptake of rhodamine protamine (Pro(Rh)) or fluorescein protamine (Pro(Fl)) and tat peptide (Tat(Fl)) as a function of time at 2 µM fluorochrome. (B) Uptake of Pro(Rh), Pro(Fl)), and Tat(Fl) as a function of peptide concentration at 4 h. Experiments were with HeLa cells except where indicated. Total cell-associated fluorescence was obtained after lysis.

Figure 4. Loss of fluorescence after loading with protamine and tat peptide. HeLa cells were loaded with fluorescent protamines or tat peptide (2 µM, 4 h), washed, and incubated in peptide and protein free media for the indicated time. Cells were trypsinized, and fluorescence was determined by FACS.

protamines or Tat(Fl) as shown in Figure 4. The loss of fluorescence for Pro(Rh) was fit to single-exponential decay model and yielded half-lives of 12.2 h (Pro(Rh), 10.8 h (Tat(Fl)), and 13.0 h (Pro(Fl). All fits had values of r2 greater than 0.99. Thus, the loss of cell-associated fluorescence was similar regardless of whether cells were loaded with either fluorescent protamine or the tat peptide. A feature of the tat peptide is that when it is conjugated to enzymes, nanoparticles, or liposomes, it induces internalization and/or nuclear localization of these macromolecular or supramolecular materials (22-24). To examine whether protamine could act similarly, we attached Pro(Rh) to an amino-CLIO nanoparticle labeled with Cy5.5, by using a carbodiimide to react the Cterminal carboxyl group (Table 1) with the primary amine of the nanoparticle. The resulting nanoparticles are denoted Pro(Rh)-CLIO(Cy5.5) and CLIO(Cy5.5). As shown in Figure 5, polymer-coated nanoparticles such as CLIO(Cy5.5) are not significantly internalized by nonphagocytic cells such as HeLa cells. Conjugation of the Tat(Fl) peptide greatly increases nanoparticle internalization, an effect noted earlier (13, 22). The attachment of protamine yielded a nanoparticle, Pro(Rh)-CLIO(Cy5.5), that was internalized at 4-5 times higher levels than that of the nanoparticle with the tat peptide attached, Tat(Fl)-CLIO. Although the cellular uptake of CLIO(Cy5.5) was too low to quantitate in these fluorescence-based experiments, experiments with radiolabeled nanoparticles have demonstrated that the attachment of the tat peptide induces a 100-fold increase in nanoparticle internalization (22). Protamine, like tat peptide, can be

Figure 5. Uptake of protamine nanoparticles by HeLa Cells. Pro(Rh) was conjugated to the amino-CLIO nanoparticle to obtain Pro(Rh)CLIO(Cy5.5). Incubation was for 4 h. Cells were trypsinized, and fluorescence was determined by FACS.

conjugated to nanoparticles, and the resulting peptidenanoparticle conjugate is readily internalized by cells. DISCUSSION

Our goal was to demonstrate that protamine is an efficient membrane-translocating peptide, a previously unrecognized activity of this protein, by comparing its behavior with that of the tat peptide, a peptide with a well-recognized membrane-translocating activity. When HeLa cells where incubated with either peptide for 4 h, a time chosen to allow intracellular processing of peptides to proceed, both Pro(Rh) and Tat(Fl) showed a clear nuclear localization in living, unfixed cells (Figure 1). Nuclear localization with both peptides was also seen with fixed cells (Figure 2). Both peptides reached a nearly constant level of internalization by the initial time point of 1 h (Figure 3); the rapid kinetics of tat peptide internalization at shorter time points been described in detail elsewhere (20, 26). The similar disposition of Pro(Ph) and Tat(Fl) by microscopy and their similar rates of efflux (Figure 4) indicate that both peptides are processed in a similar fashion by HeLa cells. Finally, when Pro(Rh) was attached to the magnetic nanoparticles, the cellular uptake of the nanoparticle was enhanced more than that obtained by conjugation of the tat peptide (Figure 5). The ability of the tat peptide to enhance the internalization of enzymes, liposomes, and other materials when conjugated to them is a characteristic of this peptide (23-25). Though in early studies the nuclear localization of tat peptide was not dependent on fixation (18), more recent studies have reported the tat peptide in cytoplasmic endocytic vesicles after membrane translocation and attributed the earlier nuclear localiza-

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tion to the use of fixation procedures (26, 27). In our hands, protamine and tat peptide gave similar images of nuclear localization with fixed and unfixed cells. Regardless of the final resolution of the intracellular fate of internalized tat peptide, the tat peptide is widely accepted as having a membrane-translocating activity (28-31). Since our data indicate that protamine and tat peptide behaved similarly, we therefore conclude that protamine, like other high arginine-containing peptides, is a membrane-translocating peptide. Our results indicate that protamine has a membranetranslocating activity and nuclear localizing activity in vitro and suggest that protamine could have these activities in vivo, including after its intramuscular administration in insulin formulations. The development of fluorescent protamines may lead to an improved understanding of a material long used in pharmaceutical applications. Our results also suggest the possible use of protamine for the design of new membrane-translocating peptidebased diagnostic or therapeutic pharmaceuticals, including some whose development has been initiated with other membrane-translocating peptides. The use of protamine in this fashion would have several advantages. First, the cost of the natural product protamine is currently substantially less than synthetic peptides. Protamine is available as a sterile solution (10 mg/mL, 5 mL) at less than $50/ampule compared to a price of at least $500 for 50 mg of 10 amino acid peptide made by solid-phase synthesis. This decreased cost could stimulate research or the clinical development of protamine-based conjugates. Second, protamine has a long history of use establishing its biological effects and general safety in humans. Finally protamine has only one reactive amine, the N-terminal proline, and, one carboxyl group, the C-terminus, facilitating the single point attachment of fluorochromes or drugs to protein. Our observations suggest that a protamine, a protein in regular parenteral use, might be a convenient raw material for use in the design of novel membrane-translocating/nuclear-localizing pharmaceuticals. ACKNOWLEDGMENT

This work was supported by NIH grants RO1-EB00662 and R01 EB004626. LITERATURE CITED (1) Brange, J., and Langkjaer, L. (1992) Chemical stability of insulin. 3. Influence of excipients, formulation, and pH. Acta Pharm. Nord. 4, 149-58. (2) Brange, J., and Langkjaer, L. (1997) Insulin formulation and delivery. Pharm. Biotechnol. 10, 343-409. (3) Carr, J. A., and Silverman, N. (1999) The heparin-protamine interaction. A review. J. Cardiovasc. Surg. (Torino) 40, 65966. (4) Fuentes-Mascorro, G., Serrano, H., and Rosado, A. (2000) Sperm chromatin. Arch. Androl. 45, 215-25. (5) Meistrich, M. L., Mohapatra, B., Shirley, C. R., and Zhao, M. (2003) Roles of transition nuclear proteins in spermiogenesis. Chromosoma 111, 483-8. (6) Futaki, S., Suzuki, T., Ohashi, W., Yagami, T., Tanaka, S., Ueda, K., and Sugiura, Y. (2001) Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J. Biol. Chem. 276, 5836-40. (7) Rothbard, J. B., Garlington, S., Lin, Q., Kirschberg, T., Kreider, E., McGrane, P. L., Wender, P. A., and Khavari, P. A. (2000) Conjugation of arginine oligomers to cyclosporin A

Reynolds et al. facilitates topical delivery and inhibition of inflammation. Nat. Med. 6, 1253-7. (8) Polyakov, V., Sharma, V., Dahlheimer, J. L., Pica, C. M., Luker, G. D., and Piwnica-Worms, D. (2000) Novel Tatpeptide chelates for direct transduction of technetium-99m and rhenium into human cells for imaging and radiotherapy. Bioconjugate Chem. 11, 762-71. (9) 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, 26204-10. (10) Ford, K. G., Souberbielle, B. E., Darling, D., and Farzaneh, F. (2001) Protein transduction: an alternative to genetic intervention? Gene Ther. 8, 1-4. (11) Snyder, E. L., and Dowdy, S. F. (2001) Protein/peptide transduction domains: potential to deliver large DNA molecules into cells. Curr. Opin. Mol. Ther. 3, 147-52. (12) Wunderbaldinger, P., Josephson, L., and Weissleder, R. (2002) Tat peptide directs enhanced clearance and hepatic permeability of magnetic nanoparticles. Bioconjugate Chem. 13, 264-8. (13) Zhao, M., Kircher, M. F., Josephson, L., and Weissleder, R. (2002) Differential conjugation of tat Peptide to superparamagnetic nanoparticles and its effect on cellular uptake. Bioconjugate Chem. 13, 840-4. (14) Josephson, L., Perez, J. M., and Weissleder, R. (2001) Magnetic nanosensors for the detection of oligonucleotide sequences. Agnew Chem. Int. Ed. 40, 3204-3206. (15) Koch, A. M., Reynolds, F., Kircher, M. F., Merkle, H. P., Weissleder, R., and Josephson, L. (2003) Uptake and metabolism of a dual fluorochrome Tat-nanoparticle in HeLa cells. Bioconjugate Chem. 14, 1115-21. (16) Reynolds, F., O’Loughlin, T., Weissleder, R., and Josephson, L. (2005) Method of determining nanoparticle core weight. Anal. Chem. 77, 814-7. (17) Koch, A. M., Reynolds, F., Merkle, H. P., Weissleder, R., and Josephson, L. (2005) Transport Of Surface-Modified Nanoparticles Through Cell Monolayers. Chembiochem 6, 337-345. (18) 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. (19) Hermanson, G. T. (1996) Bioconjugate Techniques, Academic Press, San Diego CA. (20) Suzuki, T., Futaki, S., Niwa, M., Tanaka, S., Ueda, K., and Sugiura, Y. (2002) Possible existence of common internalization mechanisms among arginine-rich peptides. J. Biol. Chem. 277, 2437-43. (21) Mann, D. A., and Frankel, A. D. (1991) Endocytosis and targeting of exogenous HIV-1 Tat protein. EMBO J 10, 17339. (22) 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, 186-91. (23) 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. (24) Schwarze, S. R., and Dowdy, S. F. (2000) In vivo protein transduction: intracellular delivery of biologically active proteins, compounds and DNA. Trends Pharmacol. Sci. 21, 45-8. (25) Torchilin, V. P., Rammohan, R., Weissig, V., and Levchenko, T. S. (2001) TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors. Proc Natl. Acad. Sci. U. S. A. 98, 8786-91. (26) 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, 585-90.

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Membrane Translocation of Protamine (27) Lundberg, M., Wikstrom, S., and Johansson, M. (2003) Cell surface adherence and endocytosis of protein transduction domains. Mol. Ther. 8, 143-50. (28) Lindgren, M., Hallbrink, M., Prochiantz, A., and Langel, U. (2000) Cell-penetrating peptides. Trends Pharmacol. Sci. 21, 99-103. (29) Vives, E., Richard, J. P., Rispal, C., and Lebleu, B. (2003) TAT peptide internalization: seeking the mechanism of entry. Curr. Protein Pept. Sci. 4, 125-32.

(30) Futaki, S. (2005) Membrane-permeable arginine-rich peptides and the translocation mechanisms. Adv. Drug Delivery Rev. 57, 547-58. (31) Wadia, J. S., and Dowdy, S. F. (2005) Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer. Adv. Drug Delivery Rev. 57, 579-96.

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