Tat-Conjugated Synthetic Macromolecules Facilitate Cytoplasmic

Our data indicate the transport of these conjugates by a single Tat molecule to both the cytoplasm and nucleus via a nonendocytotic and concentration ...
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Bioconjugate Chem. 2003, 14, 44−50

Tat-Conjugated Synthetic Macromolecules Facilitate Cytoplasmic Drug Delivery To Human Ovarian Carcinoma Cells Aparna Nori,† Keith D. Jensen,† Monica Tijerina,† Pavla Kopecˇkova´,†,‡ and Jindrˇich Kopecˇek*,‡ Department of Pharmaceutics and Pharmaceutical Chemistry/CCCD, and Department of Bioengineering, University of Utah, Salt Lake City, Utah 84112. Received August 1, 2002; Revised Manuscript Received October 3, 2002

We have synthesized N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-cell penetrating peptide Tat conjugates and evaluated their subcellular distribution in A2780 human ovarian carcinoma cells by confocal fluorescence microscopy and subcellular fractionation. Our data indicate the transport of these conjugates by a single Tat molecule to both the cytoplasm and nucleus via a nonendocytotic and concentration independent process. The uptake was observed to occur within 3 min, as confirmed by live cell microscopy. In contrast, HPMA copolymers lacking the Tat peptide were internalized solely by endocytosis. For the first time, Tat-mediated cytoplasmic delivery of a polymer bound anticancer drug, doxorubicin, was also demonstrated. These findings establish the feasibility of overcoming major cellular and subcellular obstacles to intracellular macromolecular delivery and hold great promise for the development of polymer-based systems for the cytoplasmic delivery of therapeutic molecules.

INTRODUCTION

Water-soluble polymers such as N-(2-hydroxypropyl)methacrylamide (HPMA)1 copolymers are frequently employed as drug carriers because of their ability to improve the solubility of hydrophobic compounds, reduce nonspecific toxicity, and increase the therapeutic index of low molecular weight anticancer drugs (1, 2). Anticancer drugs bound to water-soluble polymeric carriers such as HPMA copolymers have exhibited decreased systemic toxicity, a result of the altered biodistribution of polymer-bound drugs as compared to free drugs. Further, cationic polymers poly(L-lysine) and polyethyleneimine are being increasingly investigated as nonviral vectors for nucleic acids and oligonucleotides due to the immunogenicity and toxicity problems associated with conventional viral vectors. Binding a drug or nucleic acid to a polymer directs the conjugate to the lysosomes by endocytosis. Due to the impermeable nature of the lysosomal membrane, such vesicular entrapment poses a hindrance to molecules expressing biological activity in the cytoplasm or nucleus, thus necessitating the endosomal escape of the polymer conjugate. Drug release from the lysosomes can be facilitated by introducing drug-polymer linkages such as the tetrapeptide spacer glycylphenylalanylleucylglycine (GFLG), which are stable in the bloodstream but susceptible to enzymatic cleavage in the lysosomes (3). However, this approach cannot be implemented for the delivery of therapeutic molecules such as oligonucleotides * Corresponding author. Phone: (801) 581-4532. Fax: (801)581-7848. E-mail: [email protected]. † Department of Pharmaceutics and Pharmaceutical Chemistry/ CCCD. ‡ Department of Bioengineering. 1 Abbreviations: AIBN, 2,2′-azo-bis-isobutyronitrile; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; Dox, Doxorubicin, HPMA, N-(2-hydroxypropyl)methacrylamide; MWCO, molecular weight cut off; SAMSA, 5-((2-S-acetylmercapto)succinoyl)amino fluorescein; SMCC, succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate; TR, Texas Red.

or proteins, as these molecules not only lack inherent membrane penetrating capabilities but are susceptible to both the acidic environment of the lysosomes and enzymatic degradation (4). Recently, the ability of several peptides, called cell penetrating peptides or protein transduction domains (PTDs), to translocate across the cell membrane into the cytoplasm in an energy-independent (5), receptor-less manner has been discovered (6). These peptides which have been successfully employed as vectors for cytoplasmic and nuclear macromolecular transport include synthetic/chimeric peptides and protein derived peptides such as the Drosophila homeotic transcription factor ANTP, the herpes simplex virus type-1 transcription factor VP22 and the Tat peptide from the HIV-1 transactivating factor Tat. While the Tat protein itself has been proven to be taken up rapidly by cells in culture (7, 8), shorter sequences such as the Tat peptide (Y47GRKKRRQRRR57) have also been shown to transport full-length proteins such as a β-galactosidase-Tat fusion protein across not only the plasma membrane in different tissues but also the blood brain barrier, in mice (9). Additionally, the Tat PTD has successfully ferried derivatized nanoparticles (45 nm) into progenitor cells, thus allowing their facile detection by magnetic resonance imaging (10). Tat peptide containing peptide-based chelates of technetium-99m and rhenium have also been efficiently delivered to the cytoplasm and nucleus of living human cells (11). Both the Tat and ANTP peptides have shown prowess in delivering 2′-O-methyl phosphorothioate antisense oligonucleotides to the cytoplasm and nucleus, resulting in an increase in pharmacological activity without an accompanying loss in specificity of the antisense oligonucleotides (12). λ bacteriophages expressing the Tat peptide on their surface have also been shown to deliver DNA intracellulary (13). Recently, 200 nm liposomes bearing a Tat peptide-modified surface (500 Tat peptide molecules per liposome) were transported intracellularly in an energy independent fashion (14). Additionally, the Tat peptide has facilitated the

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uptake of cysteine-tagged antibody fragments in a compartmentalized manner; however, this suggests that the transport capabilities of the Tat peptide may be limited by size or the biological activity of the cargo (15). We hypothesized that attachment of the Tat peptide to a water-soluble synthetic macromolecule will result in cytoplasmic delivery of the conjugate. To test this hypothesis, we synthesized several HPMA copolymerTat peptide conjugates and evaluated their internalization and subcellular trafficking in A2780 human ovarian carcinoma cells by confocal fluorescence microscopy and subcellular fractionation. We showed that these conjugates were localized to the cytoplasm and nucleus by a Tat-facilitated pathway as opposed to conjugates without the Tat peptide, which accumulated only in endocytotic vesicles. EXPERIMENTAL PROCEDURE

Synthesis of Polymer Precursors. The HPMA copolymer containing amino groups in the side chains (P-NH2, where P represents the polymeric backbone), was prepared by copolymerization of HPMA and 3-(aminopropyl)methacrylamide hydrochloride (MA-AP), using 2,2′-azo-bis-isobutyronitrile (AIBN, Fluka) as the initiator and 3-mercaptopropionic acid (MPA) as the chain transfer agent (molar ratio 93:7:5:0.5, respectively) in methanol (10 wt % monomers in the mixture) at 50 °C for 24 h. The polymer was precipitated into ether, extensively dialyzed (MWCO 6-8 kDa), and freeze-dried. The maleimide containing HPMA copolymer (P-MAL)was obtained by reaction of 160 mg P-NH2 (0.070 mmol NH2 groups) with 46.8 mg (0.140 mmol) of succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, Molecular Probes) and 22 mg (0.18 mmol) of diisopropylethylamine in DMF at room temperature for 12 h with stirring. The solvent was removed in vacuo, and the polymer was precipitated into acetone. Synthesis of Polymer Conjugates. P-Tat-FITC. Tatcontaining HPMA copolymer (P-Tat-FITC) was obtained by reaction of 40 mg (0.015 mmol maleimide groups) of P-MAL in 0.8 mL PBS buffer (pH 7.2), with 11.4 mg (0.0052 mmol) of Tat-FITC (Emory University Microchemical Facility, Atlanta) dissolved in 0.2 mL PBS (under nitrogen). The reaction mixture was stirred overnight in dark at room temperature. Five microliters of mercaptoethanol was added to inactivate the residual maleimide groups, and the polymer was then separated on a PD-10 column in PBS. P-Tat-FITC was isolated by dialysis (MWCO 6-8 kDa) against DI water and freezedried. P(TR)-Tat(FITC). Double-label HPMA copolymer-Tat conjugate P(TR)-Tat(FITC) containing TR-labeled polymer backbone and Tat-FITC was prepared in several steps. HPMA copolymer containing TR and NH2 (P-TR-NH2) was obtained by reaction of 100 mg P-NH2 (0.044 mmol NH2) with 5 mg Texas Red succinimidyl ester (Molecular Probes) (0.0061 mmol) and 6 mg diisopropylethylamine (8 µl) in 0.8 mL DMSO with stirring for 3 h. The mixture was then diluted with ethanol/H2O (1:1, 5 mL) and dialyzed against water acidified with dilute HCl (pH 3) followed by extensive dialysis against water. The content of TR in the freeze-dried polymer determined spectrophotometrically ( 1.16 × 105 M-1 cm-1, λmax 586 nm) in MeOH was 0.055 mmol/g. The residual amino groups present in P-TR-NH2 were reacted with SMCC to yield a polymer containing both maleimide groups as well as TR. This polymer was treated with Tat-FITC to form a thioether linkage between the Tat and the polymer using similar procedures as described above.

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P-GFLG-Dox-Tat. Dox containing HPMA copolymerTat conjugate (P-GFLG-Dox-Tat) was synthesized in several steps. First, the Dox containing monomer Nmethacryloylglycylphenylalanylleucylglycyldoxorubicin (MA-GFLG-DOX) was synthesized (16) and copolymerized with HPMA and MA-AP (molar ratio 90.5:2.5:7) using AIBN as the initiator in methanol (12.5 wt % monomers in the mixture) at 50 °C for 24 h. The volume of the polymerization mixture was reduced by half, and the product precipitated in acetone/ether (3:1). The polymer was first dialyzed against 50% ethanol/H2O (containing 0.1% HCl) and then against DI water. The polymer isolated by freeze-drying contained 0.38 mmol/g (6.1 mol %) of the amino group containing side chains as determined by the Ninhydrin assay. The content of Dox determined spectrophotometrically ( ) 11 000 M-1 cm-1) in water was 0.11 mmol Dox/g (1.8 mol %; 6.4 wt %). The HPMA copolymer containing both Dox and maleimide groups (P-Mal-Dox) was then obtained by modification of amine groups with SMCC as described above. To purify the conjugate from free Dox, the polymer was purified twice on a Sephadex LH-20 column using MeOH/DMSO/ CH3COOH (89:10:1) solvent mixture. The polymer was isolated after MeOH evaporation and precipitation in acetone/ether mixture. In the last step, the Tat peptide was conjugated to P-Mal-Dox via a thioether linkage as described above. The product was purified by dialysis and isolated by freeze-drying. Control Polymers. The control polymers P-FITC and P-GFLG-Dox lacking the Tat peptide were synthesized as described (16, 17). Characterization of Polymer Precursors and Polymer Conjugates. The polymer precursor P-NH2 contained 6.6 mol % (0.44 mmol/g) of the amino group containing side chains as determined by the Ninhydrin assay. The molecular weight determined by size exclusion chromatography (SEC) on a Superose 6 (HR 10/30) column, AKTA system, PBS buffer was 26 000 Da; Mw: Mn)1.5 and dn/dc ) 0.156, using MiniDawn laser light scattering detector (Wyatt Technology, Santa Barbara). The maleimide content of the polymer precursor P-MAL as determined by the SAMSA assay (Molecular Probes) was 0.37 mmol/g. The polymer did not contain any residual amine groups as determined by the Ninhydrin assay. P-Tat-FITC. The Tat-FITC content in the polymer was determined by spectrophotometric determination of FITC ( ) 80 000 M-1 cm-1, λmax 500 nm) in 0.1 M borate buffer (pH 9.2). P(TR)-Tat(FITC). The TR and Tat-FITC contents in the final polymer were determined spectrophotometrically at λmax ) 588 nm,  ) 1.16 × 105 M-1 cm-1 and λmax ) 504 nm,  ) 80 000 M-1 cm-1, respectively. P-GFLG-Dox-Tat. The content of Dox and Tat-FITC in this polymer was measured by two methods: (a) UV spectra in alkaline conditions (0.01 M NaOH), where the λmax of Dox shifts to 590 nm and can be separated from FITC (λmax ) 500 nm); (b) amino acid analysis after acidic hydrolysis (Glu, Tyr). Cell Lines. The A2780 human ovarian carcinoma cell line obtained from Dr. T. C. Hamilton (Fox Chase Cancer Center) was cultured in RPMI 1640 media (Sigma) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 10 µg/mL insulin and kept at 37 °C in a humidified atmosphere of 5% CO2. Confocal Fluorescence Microscopy. For all studies, 500 000 A2780 cells were seeded on previously sterilized glass coverslips in 35 mm dishes (Becton Dickinson) 24 h before incubation to reach 90% confluence.

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Figure 1. Structures of HPMA copolymer Tat conjugates (A) P-Tat-FITC, (B) P(TR)-Tat(FITC), and (C) P-GFLG-Dox-Tat.

For live microscopy (17), the cells were cultured in MEM Eagle media (Sigma) without phenol red, buffered with 0.25 mM HEPES (Sigma), containing 10% FBS and 0.292 mg/mL L-glutamine (Hyclone) in open air. A BioRad MRC 1024 confocal system with a krypton-argon laser, and a Nikon Diaphot microscope (100 X plan-apo objective, NA ) 1.3, oil; for FITC, excitation ) 488 nm, emission ) 515 nm long pass filter) was used. For all fixed cell microscopy studies, cells were washed with DPBS after incubation with the polymer conjugates, fixed with 3% paraformaldehyde for 20 min at room temperature, mounted with SlowFade Light antifade medium, and sealed. In case of the nuclear localization study, cells were first incubated with 7.3 µM P-Tat-FITC and then permeated with 0.1% Triton-X (Sigma). They were then treated with RNAse in 2X SSC buffer (Sigma) at 37 °C for 20 min, followed by incubation with 1 µM nuclear marker propidium iodide (Molecular Probes). Time de-

pendence studies were conducted by incubating cells with 7.3 µM P-Tat-FITC for different time periods from 5 min to 1 h. For the concentration dependence studies, cells were incubated with concentrations ranging from 1.8 to 18 µM P-Tat-FITC for 1 h at 37°C. For all studies, the cells were imaged on a Zeiss (Thornwood, NY) LSM 510 confocal imaging system with an Axioplan 2 microscope (100× plan-apo objective, NA ) 1.4, oil) and an argon laser. (FITC, excitation ) 488 nm, emission ) 505 nm long-pass filter; propidium iodide, excitation ) 543 nm, emission ) 650 nm long-pass filter; Texas Red and Rhodamine, excitation ) 543 nm, emission ) 560 nm long-pass filter). The settings for all the confocal systems were adjusted so that control cells always yielded dark images. Subcellular Fractionation. Percoll was used as the separation medium for subcellular fractionation. Ten million A2780 cells were cultured in 75 cm2 flasks for 24

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h prior to exposure to polymer-Tat conjugates. Cells were exposed to the conjugate P(TR)-Tat(FITC) for 4 h at 37 °C and then treated to the procedure as previously described(21) to obtain the lysosomal, plasma membrane, cytoplasmic and nuclear fractions. Fluorescence of FITC in each of these subcellular fractions was analyzed with an LS 55 luminescence spectrometer (Perkin-Elmer) (excitation-495 nm, emission-518 nm) and normalized to the protein content of each fraction (Coomassie Plus Protein Assay, Pierce). The fluorescence per microgram of protein associated with each fraction was expressed as a percentage of the total fluorescence per microgram of all fractions. RESULTS AND DISCUSSION

Several fluorescently labeled HPMA copolymer-Tat conjugates were synthesized by reacting the Tat peptide [H-Gly-Arg-(Lys)2-(Arg)2-Gln-(Arg)3-Gly-Tyr-Lys(FITC)Cys-OH] with maleimide containing HPMA copolymer to form a thioether linkage (Figure 1). The Tat-FITC content in the P-Tat-FITC polymer as determined by UV spectroscopy was 0.038 mmol/g (one Tat peptide molecule per chain), indicating 40% binding efficiency of the Tat peptide to the polymer backbone. The doublelabel conjugate P(TR)-Tat(FITC) contained 0.034 mmol/g of TR and 0.056 mmol/g Tat-FITC. The content of Dox and Tat-FITC in the Dox containing HPMA copolymerTat conjugate P-GFLG-Dox-Tat determined by UV spectroscopy and amino acid analysis was 0.099 mmol/g (1.7 mol %) Dox and 0.064 mmol/g (1.2 mol %) TatFITC, respectively. The control polymer P-FITC and P-GFLG-Dox contained 0.038 mmol/g of FITC and 0.121 mmol/g of Dox, respectively, as determined by UV spectroscopy. Confocal fluorescence microscopy of polymer-Tat conjugate P-Tat-FITC after 1 h incubation with the cells at 37 °C, exhibited powerful fluorescence associated with the plasma membrane. Diffuse fluorescence observed throughout the cell implied that the conjugate localized to the cytoplasm. Faint fluorescence in the nuclear region accompanied by areas of intense staining indicative of putative nucleolar accumulation were also seen (Figure 2A). Earlier reports demonstrate Tat-mediated transport of liposomes only into the cytoplasm (14); however, our data indicate both cytoplasmic as well as nuclear delivery of the polymer. The presence of punctate staining suggested that the conjugate was also internalized via endocytosis, which is typical of macromolecular uptake (18). In contrast, a control FITC-labeled polymer lacking the Tat peptide, P-FITC, was internalized solely by endocytosis even when high concentrations of the polymer were tested (Figure 2B). To determine whether the Tat peptide mediated transport was by endocytosis, cells were incubated with either P-Tat-FITC or P-FITC at 4 °C for 1 h. Endocytosis, an energy dependent process is blocked at 4 °C (19). The internalization of P-Tat-FITC by the Tat mediated pathway was not inhibited, suggesting an energy independent, nonendocytotic uptake process (Figure 2C). However, cells incubated with P-FITC did not exhibit any fluorescence, as the endocytotic uptake of P-FITC was suppressed (Figure 2D). Nuclear localization was corroborated by co-incubating the cells with P-Tat-FITC and the nuclear marker propidium iodide for 1 h at 37 °C. Confocal microscopy performed by separately tracking the FITC label on the conjugate (green) and propidium iodide (red) demonstrated areas of orange-yellow staining representing co-

Figure 2. Subcellular trafficking of various polymer conjugates incubated with A2780 cells by confocal fluorescence microscopy. (A) HPMA copolymer-Tat conjugate (P-Tat-FITC) for 1 h at 37 °C showed intense plasma membrane, cytoplasmic, and nuclear uptake. (B) FITC-labeled polymer lacking Tat peptide (P-FITC) for 1 h at 37 °C exhibited only endocytotic vesicles. (C) P-TatFITC for 1 h at 4 °C demonstrated Tat-mediated nonendocytotic transport of the conjugate. (D) P-FITC for 1 h at 4 °C showed no uptake. (E) P-Tat-FITC (green), (F) DIC image, and (G) nuclear marker propidium iodide (red) for 1 h at 37 °C. (H) Colocalization of the two dyes (orange-yellow staining) implied nuclear localization. Scale bar represents 10 µm.

localization of the two dyes in the nucleus (Figure 2E-H). One possible explanation for nuclear entry of the conjugate could be the presence of a nuclear localization signal RKKRR embedded in the Tat peptide sequence (6). Similarly, cytoplasmic import was verified by treating the cells with the membrane permeant cytoplasmic marker chloromethylbenzoylaminotetramethyl rhodamine (CMTMR) and P-Tat-FITC. The FITC label on the polymer-Tat conjugate (green) and CMTMR (red) were separately tracked and were found to co-localize,

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Figure 3. Fluorescence images of cells incubated with P-Tat-FITC after 20 min, 2 h, and 4 h. Panels A-C represent images taken at high microscope settings in order to visualize the cytoplasm and nucleus. Panels D-F are images of the same cells taken at lower settings in order to observe the plasma membrane. Scale bar represents 10 µm.

thus confirming the cytoplasmic delivery of P-Tat-FITC (data not shown). Recent studies have exhibited that fixing agents such as methanol, commonly employed for fixing cells in microscopy studies, lead to artificial cytoplasmic and nuclear accumulation as a consequence of membrane permeabilization (20). Microscopy of live cells, which precludes the use of fixing agents, was utilized to examine the subcellular distribution of the polymer-Tat conjugate. In agreement with data from fixed cell microscopy, cytoplasmic as well as nuclear uptake of the conjugate was observed, thus excluding the likelihood of artificial subcellular localization. Live microscopy studies also revealed internalization within 3 min, thus verifying the rapidity of the process (see movie in Supporting Information). The time and concentration dependence of the internalization process was also investigated. Increasing incubation periods of P-Tat-FITC with the cells resulted in persistent membrane staining as well as a proportional increase in the fluorescence intensity within the cytoplasm and the nucleus, signifying augmented distribution of the conjugates with time. Images of the cells were taken at both low as well as high microscope settings in order to better visualize the changes in the plasma membrane and cytoplasm at different time points. Accumulation of the conjugate in the cytoplasm and nucleus was observed as quickly as 5 min. Endocytotic vesicles were more prominent at time points greater than 1 h as expected (Figure 3A-F). In the concentration dependent study, cells incubated with higher concentrations of P-Tat-FITC showed stronger fluorescence as compared to cells incubated with lower concentrations. Cytoplasmic and nuclear localization was observed for the entire concentration range (1.8-18 µM) tested, indicating concentration-independent subcellular fate (Figure 4A,B). In the above experiments, the FITC label on the Tat peptide was traced, as the polymeric backbone was unlabeled. One concern was that subcellular proteolytic enzymes could potentially cleave the labeled Tat peptide from the polymeric backbone, resulting in the release of free, labeled Tat peptide within the cells. Thus, fluorescence in the cytoplasm and nucleus could represent the subcellular distribution of the cleaved fluorescent Tat peptide and not the polymer-Tat conjugate itself. To

Figure 4. Fluorescence images of cells incubated with (A) 1.8 and (B) 18 µM P-Tat-FITC show both cytoplasmic as well as nuclear uptake. Scale bar represents 10 µ.

address this problem, a double-label polymer-Tat conjugate [P(TR)-Tat(FITC)] was synthesized (Figure 1B). This conjugate bore a Texas Red (TR) label attached to the polymeric backbone in addition to the FITC label on the Tat peptide. Its subcellular distribution was evaluated by fluorescence microscopy. Similar uptake patterns in the cytoplasm and nucleus were observed with both dyes, establishing the Tat mediated cytoplasmic and nuclear delivery of the polymer-Tat conjugate (Figure 5A,B). The ability of the Tat peptide to efficiently transport a drug across the plasma membrane into the cytoplasm was also assessed. To this end, Dox containing HPMA copolymer-Tat conjugate [P-GFLG-Dox-Tat] was synthesized (Figure 1C). This system was chosen as a model for demonstrating the significant nuclear and cytoplasmic transport achieved by the Tat peptide in comparison with that of the GFLG spacer. The subcellular distribution pattern in A2780 cells was studied by tracking the inherent fluorescence of Dox by confocal microscopy. At 37 °C, cytoplasmic as well as nuclear uptake of P-GFLGDox-Tat was observed (Figure 5C). The intensity of the Dox fluorescence signal was significantly stronger than that of the control P-GFLG-Dox copolymer lacking the Tat peptide (Figure 5D). This enhancement in the Dox uptake has two independent componentssthe enzymatic hydrolysis of the lysosomally degradable spacer GFLG (3) and Tat-mediated cytoplasmic delivery of Dox; a large contribution was attributed to the Tat mediated transport, as evidenced by studies at 4 °C. The intense fluorescent signal of P-GFLG-Dox-Tat observed at 4 °C

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Figure 6. Subcellular fractionation showing the subcellular distribution of the polymer-Tat conjugate and control polymer P-FITC in the different organelle fractions.

Figure 5. Confocal fluorescence images of A2780 cells incubated with double label HPMA copolymer-Tat conjugate (P(TR)Tat(FITC) for 1 h at 37 °C. The two dyes (A) FITC and (B) TR show similar distribution, confirming the intracellular delivery of the polymer-Tat conjugate. Fluorescence images of cells incubated with different polymer conjugates containing anticancer drug doxorubicin with or without Tat. (C) P-GFLG-DoxTat for 4 h at 37 °C showed both cytoplasmic as well as nuclear localization. (D) P-GFLG-Dox for 4 h at 37 °C showed weaker signal. (E) P-GFLG-Dox-Tat at 4 °C continued to show cytoplasmic uptake in contrast with (F) P-GFLG-Dox at 4 °C, where uptake was completely abolished. Scale bar represents 10 µm.

proved that the Tat peptide could successfully transport polymer bound doxorubicin into the cytoplasm independent of the GFLG spacer (Figure 5E). In contrast, no signal was visible with P-GFLG-Dox, as endocytosis was inhibited, as expected (Figure 5F). The use of the PGFLG-Dox-Tat model system clearly demonstrates the superior transducing capabilities of the Tat peptide. The cytoplasmic and nuclear uptake of the polymerTat conjugate was independently verified by subcellular fractionation. The polymer-Tat conjugate mainly amassed in the lysosomal and plasma membrane fraction after 4 h incubation. Further, the conjugate localized in smaller fractions in the cytoplasm and nucleus. This subcellular distribution pattern was a significant improvement over that of the P-FITC conjugate, which localized to the lysosomes (Figure 6). Our approach utilizing subcellular fractionation in conjunction with live and fixed cell confocal microscopy shows advantages over conventional methods such as flow cytometry (which represents surface binding and not internalization) and functional assays, which may be typical only of a sub-population of cells (20). In conclusion, these results validate our hypothesis that the Tat peptide can transport a synthetic macromolecule across the plasma membrane and deliver it into the cytosol as shown by confocal fluorescence microscopy and subcellular fractionation. Our data indicated that

incubation of HPMA copolymer-Tat conjugate with human ovarian carcinoma cells resulted in internalization of the conjugate by two processes, namely, endocytosis and a Tat-mediated nonendocytotic process. The Tat-mediated process, characterized by intense membrane staining and cytoplasmic and nuclear uptake, was not inhibited at 4°C, indicating an energy independent pathway. Studies performed so far have demonstrated the delivery of proteins, nanoparticles, and liposomes; however, this is the first time the Tat peptide has been shown to efficiently transport a polymer bound drug to the cytosol by a nonendocytotic pathway. These systems hold great potential, due to the immense utility of water soluble polymers as drug carriers, mainly attributed to their ability to alter the biodistribution and reduce nonspecific toxicity of low molecular weight anticancer drugs. Though the lack of cell specificity of the Tat peptide makes its applicability universal, it is this very property of the Tat peptide that also causes it to be disadvantageous. The use of water-soluble polymers which allow the facile attachment of targeting moieties such as carbohydrates and antibodies may well render a Tat containing conjugate selectively biorecognizable to a specific cell population resulting in a change in both its uptake and distribution. Further, the binding of nonimmunogenic polymers to antibodies results in the decreased immunogenecity of the antibody due to steric hindrance of the polymer conjugate (1). Although further investigations are warranted, these results suggest that the development of polymer-Tat based cytoplasmic delivery systems, especially for gene and antisense therapy, which combine the advantages of macromolecules and exploit the unique transport capabilities of the Tat peptide, will be beneficial. ACKNOWLEDGMENT

We thank Drs. E. J. King and C. Anderson for their assistance with the confocal microscopy studies. This work was supported in part by NIH Grant CA51578 from the National Cancer Institute. Supporting Information Available: Movie demonstrating the intracellular uptake of the HPMA copolymerTat conjugate in A2780 cells by live microscopy. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Putnam, D.; Kopecˇek, J. (1995) Polymer conjugates with anticancer activity. Adv. Polym. Sci. 122, 55-123.

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