Tat Peptide Directs Enhanced Clearance and Hepatic Permeability of

Patrick Wunderbaldinger,† Lee Josephson,*,† and Ralph Weissleder. From the Center of Molecular Imaging Research, Massachusetts General Hospital, ...
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Bioconjugate Chem. 2002, 13, 264−268

Tat Peptide Directs Enhanced Clearance and Hepatic Permeability of Magnetic Nanoparticles Patrick Wunderbaldinger,† Lee Josephson,*,† and Ralph Weissleder From the Center of Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachussets. Received October 5, 2001

Superparamagnetic nanoparticles have a number of important biomedical applications, serving as MR contrast agents for imaging specific molecular targets, as reagents for cell labeling and cell tracking, and for the isolation of specific classes of cells. We have determined the physical and biological properties of MION-47 and amino-CLIO, nanoparticles which serve as precursors for the synthesis of targeted MR contrast agents, and Tat-CLIO, a nanoparticle used as a cell labeling reagent. Blood half-lives for MION-47 and amino-CLIO were 682 ( 34 and 655 ( 37 min, respectively. The attachment of 9.7 tat peptides per crystal to amino-CLIO resulted in a reduction in blood half-life to 47 ( 6 min. MION-47, amino-CLIO, and Tat-CLIO were present in highest concentrations in liver and spleen and lymph nodes, where concentrations for all three nanoparticles ranged from 8.80 to 6.11% of injected dose per gram. Twenty-four hours after the intravenous injection of amino-CLIO, the nanoparticle was concentrated in cells surrounding hepatic blood vessels (endothelial and Kupffer cells), in a fashion similar to that obtained with other nanoparticle preparations. In contrast, Tat-CLIO was present as numerous discrete foci of intense fluorescence throughout the parenchyma. Using the peptide as a component of future nanoparticles, it might be possible to design sensors for the detection of macromolecules present in intracellular compartments.

INTRODUCTION

The ability to induce the internalization of therapeutic agents by attachment to positively charged peptides has suggested a variety of new approaches to drug design including new forms of cyclosporin A (1), improved radiotherapy based on the nuclear localization of toxic radioisotopes (2), and the enhanced delivery of therapeutic proteins or DNA (3-5). Peptides with membrane translocating properties include the tat peptide from the HIV tat protein, polyarginyl peptides, and penetrin (the third helix homeodoman of antenapedia) (1, 6, 7). We have shown that the attachment of tat peptides to a superparamagnetic iron oxide known as Tat-CLIO (Tatcross-linked iron oxide) induces the intracellular accumulation of iron oxide and makes cells highly detectable by magnetic resonance imaging (8). Uses of TatCLIO include labeling stem cells homing to bone marrow (9), labeling T cells homing to the spleen (10), labeling neuroprogenitor cells migrating in the developing mouse brain (11), and labeling T cells invading diabetic insulitic lesions (12) (Table 1). Tat-CLIO uptake is not associated with toxicity (8-10). A key question concerning the usefulness of the tat peptide for the delivery of diagnostic and sensing agents involves the fate of tat peptide conjugates after clearance from the vascular compartment. If tat-conjugates move beyond cells bordering the vascular compartment, a new capability to deliver cell impermeable drugs to a wide range of different cell types throughout an organ can be imagined. We therefore chose to examine the in vivo * Corresponding author: Lee Josephson, Ph.D. MGH-CMIR; Building 149, 13th Street, Room 5406, Charlestown, MA 02129. Office: 617-726-5788. Fax: 617-726-5708. E-mail: josephso@ helix.mgh.harvard.edu. † P.W. and L.J. contributed equally.

behavior of Tat-CLIO, a nanoparticle developed for cell labeling applications. We examined the physical and biological properties of Tat-CLIO by comparison with two nanoparticles, MION-47 and amino-CLIO, which have served as precursors for the synthesis of Tat-CLIO and targeted nanoparticles generally. Here we report that the attachment of the tat peptide enhances nanoparticle clearance from the vascular compartment and that nanoparticles were found throughout the liver, rather than in cells adjacent to the vascular compartment (endothelial cells/Kupffer cells). This observation opens exciting opportunities for the delivery of intracellular sensing molecules that will allow in vivo molecular imaging. EXPERIMENTAL PROCEDURES

Synthesis of Magnetic Nanoparticles. The synthesis of nanoparticles used here, MION-47, amino-CLIO, and Tat-CLIO, follows the scheme shown in Figure 1. Amino-CLIO was synthesized by cross-linking MION-47 in strong base with epichlorohydrin followed by reaction with ammonia to provide primary amino groups (8, 13, 14). The original synthesis of Tat-CLIO (8) was modified to ensure that FITC, used to tract the nanoparticle in fluorescent microscopy studies, could not be cleaved from the nanoparticle in vivo. We used succinimidyl iodoacetate (SIA) as a bifunctional conjugating reagent because it forms a stable thioether linkage between peptide and amino-CLIO. Our earlier synthesis used N-succinimidyl 3-(-pyridyldithio)propionate (SPDP), which results in a reducing-environment-sensitive, disulfide linkage between peptide and nanoparticle. Second, our earlier synthesis used GGCGRKKRRQRRRK(FITC)-NH2 as the tat peptide, which features a cysteine residue near the N terminus and a C-terminal lysine modified by FITC. The current study used the peptide GRKKRRQRRRGYK-

10.1021/bc015563u CCC: $22.00 © 2002 American Chemical Society Published on Web 02/12/2002

Properties of Tat Peptide−Nanoparticles

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Table 1. Summary of Magnetic Nanoparticles Used in Current and Previous Studies compound MION-47 Amino-CLIO Tat-CLIO

primary use monocrystalline iron oxide nanoparticles (MION) used as a blood pool agent and as a lymphotrophic conrast agent an aminated, cross-linked iron oxide (CLIO) used as a precursor in the synthesis of other nanoparticles a cell labeling and cell tracking reagent synthesized from amino-CLIO

(FITC)C-NH2. The C-terminal cysteine side chain is modified by the attachment to the CLIO nanoparticle, while the penultimate lysine is modified by attachment of FITC (Figure 1). Unlike our original Tat-Clio nanoparticle, cleavage of the naturally occurring amino acids providing the membrane translocation sequence will result not result in a separation of FITC from the nanoparticle. To verify the stability of the current TatCLIO, the nanoparticle was incubated with 10 µg/mL trypsin, 10 µg/mL chymotrypsin, or 10 mM DTT at 37 °C for 4 h. All treatments failed to separate FITC from the nanoparticle, based on its inability to pass through an ultrafiltration membrane. GRKKRRQRRRGYK(FITC)C-NH2 was synthesized with Fmoc chemistry and labeled with FITC, MALDIMS (M+1)+: 2237.54 (calc.), 2237.2 (found) as described (8). Italicized residues are the membrane translocation sequence of the tat peptide. One and a half milliliters of amino-CLIO (39 mg Fe, 0.70 mmol) in 5 mM sodium citrate, pH 8 was reacted with succinimidyl-iodoacetate or SIA (42 mg, 0.12 mmol) (Molecular Biosciences, Boulder CO) dissolved in DMSO (3 mL). Reaction was for 2 h at room temperature. The mixture was separated on a Sephadex G-25 column (30 × 1.5 cm) and the void volume with iron oxide was collected. To the iron oxide was added 13 mg of peptide dissolved in 3 mL of 0.02 M

Figure 1. Scheme for the synthesis of Tat-CLIO.

ref 15, 23 8, 13, 14 9

citrate, pH 6.5 and the mixture was allowed to stand for 3 h at room temperature. The mixture was then applied to the column above to remove unreacted peptide. Isotopic Labeling of Magnetic Nanoparticles. To prepare indium-labeled MION-47 or indium-labeled aminoCLIO, nanoparticles were dialyzed against 0.01M sodium carbonate, pH 11-11.5. To 0.5 mL of nanoparticle (10 mg Fe, 0.178 mmol) was added diethylenetriaminpentaacetic acid dianhydride (DPTA anhydride, Aldrich, Milwaukee, WI), in 100 µL of DMSO (0.5 mg, 1.4 µmol of DTPA anhydride). The mixture was allowed to stand for 90 min, and the addition of DTPA anhydride was repeated. To remove unreacted DTPA, the mixture was applied to Sephadex G-25 column PD-10 columns equilibrated with 0.02 M citrate, pH 6.5, and the dark void volume was collected. One millicurie of 111InCl3 (NEN, Boston MA) was then added and the mixture allowed to stand at room temperature for 4 h. The mixture was added to a Sephadex G-25 PD-10 column equilibrated with the above buffer and which had been spun (4 min × 2000 g) to remove entrapped fluid. The sample was added and the column spun again and the flow through taken. For indium labeled Tat-CLIO, to 0.6 mL Tat-CLIO (2.1 mg Fe, 0.038 mmol) was added to 0.1 mL of 0.1 M Borate, pH 8.5, and 0.07 mL of 2.8 mM DTPA anhydride (0.20

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Table 2. Physical and Biological Properties of Magnetic Nanoparticles agent

size (nm)

R1 (mM-1 s-1)

R2 (mM-1 s-1)

peptides per crystal

blood T1/2 (min)

MION-47 Amino-CLIO Tat-CLIO

27.5 ( 6.8 29.8 ( 5.0 65.2 ( 7.3

21.9 ( 3.6 23.2 ( 1.9 22.9 ( 1.3

44.6 ( 7.1 54.9 ( 15 73.4 ( 17.8

NA NA 11.6 ( 1.7

682 ( 34 655 ( 37 47 ( 6

µmol) in DMSO. The mixture was allowed to stand 4 h at room temperature and separated on a PD-10 column equilibrated as above. One millicurie of 111InCl3 was added and the mixture allowed to stand overnight. Removal of unreacted indium was by centrifugation as above. To prepare fluorescein isothiocyanate (FITC)-labeled CLIO, 1 mL of amino-CLIO (26 mg Fe, 0.464 mmol) was dialyzed against sodium carbonate as above and 1 mL of 0.01 M FITC (10 µmol) in sodium carbonate was added. The mixture was allowed to stand for 1 h at room temperature and FITC removed with a PD-10 G-25 column in citrate as above. Characterization of Nanoparticles. Size was determined by laser light scattering using a Coulter N4 particle size analyzer. Relaxivity (R1 and R2) were measured at 0.47 T as described. Iron was measured spectrophotometrically. Details are provided elsewhere (8, 13). The number of tat peptides attached was determined from the OD493 of a solution of Tat-CLIO of known iron concentration. The OD493 of a similar iron concentration of amino-CLIO was subtracted and the peptide concentration calculated from the difference using an extinction coefficient for fluorescein of 73000 M-1 cm-1. Results are expressed as the number of peptides per crystal assuming 2064 iron atoms per crystal (15). Animal Studies. For biodistribution studies, female Balb C mice (six animals per agent) (Charles River Breeding Laboratories, NC) (weight 20-30 g, retired breeders) were used. Mice were anesthetized by an intraperitoneal (IP) injection of ketamine (80 mg(kg) and xylazine (12 mg/kg; Parker-Davis, Morris Plains, NJ/ Miles, Shawnee Mission, KS). They were then injected intravenously (iv) through the tail vein with 111 In-labeled contrast agents at a dose of 10 mg Fe/kg. Twenty-four hours later animals were sacrificed by an ip injection of pentobarbital (Anpro Pharmaceutical, Arcadia, CA). Brain, heart, intestine, liver, spleen, kidney, muscle, bone, lung, paraaortic and inguinal lymph nodes, and body fluids (blood and urine) were excised, weighed, sealed in test tubes, and counted in a well counter (Wallace, Turku, Finland). Tissue concentration was expressed as percentage of injected dose per gram of tissue (%ID/g). Blood half-life was calculated from blood radioactivity at various time points after animals were injected. Blood (120 µL) was withdrawn at various known time points between 1 and 24 h postinjection for five animals per agent (MION-47 and amino-CLIO: 1, 5, 8, 12, 18, and 24 h; Tat-CLIO: 1, 2, 4, 8, 12, 18, and 24 h). Half-life was obtained by fitting blood radioactivity to a singleexponential equation use for a one-compartment open pharmacokinetic model:

C(t) ) C(0)e-kt C(t) is blood radioactivity at time t after injection, C(0) is the concentration of the colloid in the blood at the moment of injection time, k is the decay constant, and t is time after injection. Animal protocols were approved by the Institutional Review Committee on Animal Care and were conducted in accordance with National Animal Welfare guidelines.

All statistical calculations were made using SAS software (SAS Institute, Cary, NC). Results are expressed as mean ((SD). Fluorescence Microscopy. To visualize the intrahepatic distribution of iron oxides, fluorescence microscopy in the green channel was used (excitation at 485 nm, emission at 530 nm). Mice were injected with FITC labeled amino-CLIO or Tat-CLIO, which has an FITC residue on the tat peptide, at 10 mg/kg and sacrificed 24 h later. Immediately after sacrifice animals were perfused with heparinized 5% glucose to reduce background fluorescence from blood. The liver was then removed, placed in freezing mounting liquid, and frozen in liquid nitrogen. The frozen tissue was cut into 5 µm thick sections for microscopic examination. Fluorescence microscopy on liver tissue was performed on air-dried, unembedded, and unfixed sections. This was done to prevent leaching of Tat-CLIO from the tissue during fixation, bleaching artifacts, and other distortions. HeLa cell images are from ref 8 and were obtained under similar conditions (unfixed, unembedded). Images were collected using a cooled, charge-coupled device (Photometrics, Tucson, AZ) with appropriate filters for excitation and emission (Omega Optics, Brattleboro, VT). RESULTS AND DISCUSSION

The sizes, magnetic properties and numbers of tat peptides for MION-47 and amino-CLIO and Tat-CLIO are shown in Table 2. Values are the means plus/minus one standard deviation for four lots of MION-47 or aminoCLIO and for three lots of Tat-CLIO. Values are consistent with earlier studies of the physical properties of these materials (8, 16-18). Biological data were obtained with one of the three lots of Tat-CLIO that had 9.7 tat peptides attached per crystal. The blood half-lives of MION-47, amino-CLIO, and TatCLIO were determined by sampling blood radioactivity at various time points after injection with results summarized Table 2. Data fit the one compartment model well, with correlation coefficients (r2 values) of 0.959 for MION-47, 0.997 for amino-CLIO, and 0.98 for Tat-CLIO. Half-lives for MION-47 and amino-CLIO were similar, 682 ( 34 min and 655 ( 37 min, respectively. Thus the cross-linking and amination of the dextran surface of MION-47, to yield amino-CLIO (Figure 1), did not alter the physical properties (size and relaxivity) or the blood half-life. MION has a long blood half-life in rats and humans, i.e., 252 min in rats (18) and greater than 24 h in humans (19). The attachment of tat peptides resulted in a reduction of blood half-life from 655 ( 37 for aminoCLIO to 47 ( 6 min for Tat-CLIO. MION-47, amino-CLIO, and Tat-CLIO had similar biodistributions; see Table 3. Each had the highest concentrations in the liver, spleen and lymph nodes, ranging from 8.80 to 6.11% ID/g, which suggests that all three nanoparticles were removed by phagocytes of the reticuloendothelial system. The enhanced clearance of Tat-CLIO is likely due to the enhanced interaction of TatCLIO with phagocytes mediated by the tat peptide itself or by enhanced opsonin-mediated phagocytosis. To compare the distributions of Tat-CLIO and aminoCLIO after clearance, fluorescence microscopy of the liver

Properties of Tat Peptide−Nanoparticles

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Figure 2. Fluorescence microscopy of livers of mice injected with amino-CLIO or Tat CLIO. Line is a scale marker indicating 50 microns.

Figure 3. Fluorescence microscopy of livers of mice injected with amino-CLIO and HeLa cells in culture incubated with tat-CLIO. White line indicates 10 microns. Table 3. Biodistribution of Nanoparticlesa organ

MION-47 (%ID/g)

Amino-CLIO (%ID/g)

Tat-CLIO (%ID/g)

liver lymph node spleen kidney lung bone fat muscle brain blood

8.8 ( 0.27 8.79 ( 0.21 6.13 ( 0.21 2.94 ( 0.78 1.82 ( 0.21 1.69 ( 0.49 1.84 ( 0.17 1.97 ( 0.31 0.38 ( 0.09 2.23 ( 0.45

8.28 ( 0.45 8.75 ( 0.26 6.11 ( 0.35 2.9 ( 0.51 1.92 ( 0.27 1.79 ( 0.25 1.81 ( 0.41 1.91 ( 0.25 0.17 ( 0.03 2.2 ( 0.3

8.53 ( 0.3 6.4 ( 0.1 6.27 ( 0.18 1.87 ( 0.38 1.92 ( 0.39 1.0 ( 0.21 0.72 ( 0.13 0.92 ( 0.19 0.09 ( 0.02 0.49 ( 0.19

a

%ID/g ) percent injected dose per gram.

was performed because it achieved high concentrations of Tat-CLIO and because of its well-defined vasculature and large vessel-free regions. Amino-CLIO was concentrated along the hepatic vessels in endothelial cells and/ or Kupffer cells as shown in Figure 2, a pattern similar to that seen when MION accumulates in rat liver. MION shows reticuloendothelial uptake, with an absence of iron in hepatocytes (20). However, the pattern of hepatic fluorescence obtained after Tat-CLIO administration was quite different, with numerous discrete foci of intense fluorescence throughout the parenchyma and a notable absence of fluorescence surrounding blood vessels. This indicates that Tat-CLIO passes from the vascular compartment, and through cells surrounding blood vessels, to obtain a parenchymal distribution. The distribution of Tat-CLIO fluorescence was analyzed at higher mag-

nification as shown in Figure 3. The distribution of fluorescence appears to be similar to that seen when HeLa cells are incubated with Tat-CLIO, where TatCLIO shows a pronounced nuclear accumulation. It appears that attachment of the tat peptide to magnetic nanoparticles reduces blood half-life and leads to a broad parenchymal intrahepatic distribution in the liver, while the organ biodistribution is determined by the nanoparticlate rather than the presence of the tat peptide. Our results can be compared with studies using protein transduction domains such as the tat peptide to induce enzymes to penetrate tissues after injection. Fusion proteins of the tat protein with β-galactosidase or of the tat peptide with β-glucuronidase have been synthesized and exhibit enhanced tissue penetration after injection (21, 22). On the other hand a tat-β-galactosidase, prepared by the conjugation of between one and two peptides per mole of enzyme, concentrated perivascularly after injection (6). Schwarze et al. have proposed that protein denaturation plays a role in the cellular uptake of their conjugates. The label to which the tat peptide is attached here, amino-CLIO, is extremely robust and cannot undergo temperature dependent changes in physical state similar to denaturation. Cross-linked dextran-coated iron oxide (amino-CLIO) can withstand abusive treatments, such as incubation at 121 °C for 30 min, without a change in size, blood half-life, or loss of its dextran coat (Josephson, unpublished observations). Second, the use of Tat-CLIO for cell tracking by MR requires the highly magnetic form

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the iron oxide (superparamagnetic iron oxide), which requires preservation of the iron oxide crystals in their original form (greater than 3 nm in diameter with an inverse spinel crystal structure). The magnetic integrity of the iron oxide must be maintained after internalization because of the profound effect that Tat-CLIO loaded cells have on MR images (9, 10). Finally, as explained above, the FITC label of the Tat-CLIO nanoparticle used here cannot be separated from the nanoparticle with the chemistry employed. Our finding that the tat peptide can affect the delivery of magnetic nanoparticles might provide the basis for the design of new types of magnetic nanoparticle based diagnostic agents. We have recently demonstrated that magnetic nanoparticles can serve as sensitive biosensors for the sequence specific detection of nucleic acids by NMR (14). The addition of the tat peptide to such biosensors may provide a signal for their membrane translocation or nuclear localization in vivo. Alternatively, it may be possible to add the tat peptide sequence to direct the intracellular disposition of nanoparticles targeted to cells by antibodies or receptor binding ligands. ACKNOWLEDGMENT

P. Wunderbaldinger was supported by the Austrian Science Fund (FWF), Vienna, Austria. LITERATURE CITED (1) Rothbard, J. B., Garlington, S., Lin, Q., Kirschberg, T., Kreider, E., McGrane, P. L., et al. (2000) Conjugation of arginine oligomers to cyclosporin A facilitates topical delivery and inhibition of inflammation. Nat. Med. 6, 1253-7. (2) 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. (3) Ford, K. G., Souberbielle, B. E., Darling, D., and Farzaneh, F. (2001) Protein transduction: an alternative to genetic intervention? Gene Ther. 8, 1-4. (4) Eguchi, A., Akuta, T., Okuyama, H., Senda, T., Yokoi, H., Inokuchi, H., et al. (2001) Protein transduction domain of HIV-1 Tat protein promotes efficient delivery of DNA into mammalian cells. J. Biol. Chem. 9, 9. (5) 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. (6) Fawell, S., Seery, J., Daikh, Y., Moore, C., Chen, L. L., Pepinsky, B., et al. (1994) Tat-mediated delivery of heterologous proteins into cells. Proc. Natl. Acad. Sci. U.S.A. 91, 6648. (7) Derossi, D., Chassaing, G., and Prochiantz, A. (1998) Trojan peptides: the penetratin system for intracellular delivery. Trends. Cell Biol. 8, 84-7. (8) 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.

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