Enhanced Cellular Uptake with a CobaltacarboranePorphyrinHIV-1

containing a HIV-1 Tat 48-60 peptide sequence linked via a low molecular weight poly(ethylene glycol) (PEG) spacer. The cellular uptake, cytotoxicity,...
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Bioconjugate Chem. 2006, 17, 928−934

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Enhanced Cellular Uptake with a Cobaltacarborane-Porphyrin-HIV-1 Tat 48-60 Conjugate Martha Sibrian-Vazquez, Erhong Hao, Timothy J. Jensen, and M. Grac¸ a H. Vicente* Department of Chemistry, Louisiana State University, Baton Rouge LA, 70803. Received February 24, 2006; Revised Manuscript Received April 24, 2006

A series of four porphyrin-cobaltacarborane conjugates have been synthesized, containing three or four cobaltabisdicarbollide anions linked by O(CH2CH2O)2 groups to the porphyrin macrocycle and one of them containing a HIV-1 Tat 48-60 peptide sequence linked via a low molecular weight poly(ethylene glycol) (PEG) spacer. The cellular uptake, cytotoxicity, and preferential sites of intracellular localization of the conjugates were evaluated in human HEp2 cells. All conjugates are nontoxic in the dark at the concentrations studied. Upon exposure to low light dose (1 J cm-2) only the porphyrin-cobaltacarborane-HIV-1 Tat 48-60 conjugate showed 30% inhibition of cell proliferation at a concentration of 10 µM. The cellular uptake was dependent on the number of carborane cages and was significantly enhanced by the presence of the cell penetrating peptide sequence HIV-1 Tat 48-60. All conjugates preferentially localized in the cell lysosomes.

INTRODUCTION The efficient cellular delivery of porphyrin sensitizers for the boron neutron capture therapy (BNCT) and the photodynamic therapy (PDT) of tumors has been the subject of increasing interest in recent years (1-3). BNCT (4-6) and PDT (7, 8) are binary therapies for cancer treatment that involve the activation of a tumor-localized sensitizer with low-energy neutrons (in BNCT) or with red light (in PDT), which generates highly cytotoxic species that cause tumor destruction. The main advantage of these localized therapeutic modalities is the selectivity of the treatment; upon activation of the sensitizing drug only those cells that incorporate it are killed. Both the amount and biodistribution of the porphyrin sensitizer within tumor tissue determine its biological effectiveness and, consequently, the outcome of the BNCT and PDT treatments (9, 10). To date, two porphyrin-based drugs, Photofrin and Visudyne, have been approved by the Federal Drug Administration (FDA) for the PDT treatment of various cancers and the wet form of macular degeneration, respectively, but no porphyrin-based sensitizer has yet been approved for use in BNCT. One of the main challenges in BNCT is the discovery of boron-containing drugs capable of selectively delivering therapeutic amounts of boron-10, between 15 and 30 µg of boron/(g of tumor), depending on the exact location of the boron atoms, to target cells with minimum systemic toxicity (11, 12). Porphyrin derivatives containing multiple boron clusters are particularly promising delivery vehicles for BNCT because of the usually low dark toxicity of this type of macrocycle, their ready detection by fluorescence, and their inherent ability to accumulate within tumors and to persist there during the entire radiation treatment (1, 13, 14). In addition, boronated porphyrin derivatives are being investigated as dual sensitizers for the BNCT and PDT of tumors (15-17). Because of the high amount of boron needed for effective BNCT, the cellular uptake of boron-containing porphyrins is a crucial parameter in determining their biological effectiveness. Lipid vehicles such as Cremophor EL and liposomes have been used to enhance the solubility and tumor delivery of boronated * Corresponding author. Phone: (225) 578 7405. Fax: (225) 578 3458. E-mail: [email protected].

porphyrin derivatives (18-20). The syntheses of carboranylporphyrins containing either a methoxy poly(ethylene glycol) (PEG) chain (21) or a positively charged peptide sequence (PKKKRKV) (22) have also been reported but their biological properties have not yet been studied. A particularly attractive strategy for increasing the cellular uptake of porphyrin macrocycles consists of their conjugation to a fusogenic or cell-penetrating peptide sequence, such as that found in the human immunodeficiency virus I transcriptional activator (HIV-1 Tat) (23). This short peptide sequence has been successfully used to enhance the cellular uptake of several drugs and macromolecules (24, 25), and we have recently shown that it efficiently delivers a nonboronated porphyrin intracellularly (26). Although the porphyrin-HIV-1 Tat conjugate is expected to be taken up by both tumor and normal cells, a leaky tumor vasculature and poor lymphatic clearance might result in favorable differential boron concentrations between tumor and normal tissues and therefore selective tumor accumulation. Furthermore, the conjugation of a boronated porphyrin to the HIV-1 Tat peptide is expected to significantly enhance the delivery of boron to target tumors via convection enhanced delivery (6, 27). Herein we report the synthesis of a new boronated porphyrin-HIV-1 Tat 48-60 conjugate containing PEG linkers between the macrocycle, the peptide sequence (GRKKRRQRRRPPQ), and the cobaltabisdicarbollide anions. We have previously shown that porphyrincobaltacarborane conjugates are efficiently prepared in high yields from reaction of a nucleophilic porphyrin with zwitterionic cobaltacarborane [3,3′-Co(8-C4H8O2-1,2-C2B9H10)(1′,2′C2B9H11)] (28). Furthermore we showed that the cellular uptake of a series of conjugates containing one to four cobaltabisdicarbollide anions increases with the number of carborane cages (29). In our continuing investigation of structure/activity relationships in carborane-containing porphyrins, we now report the synthesis and characterization of four new porphyrincobaltacarborane conjugates containing three or four cobaltabisdicarbollide anions and compare their cellular uptake, cytotoxicity, and intracellular localization in human HEp2 cells. We show that the presence of the HIV-1 Tat 48-60 peptide sequence significantly enhances the cellular uptake of porphyrin-cobaltacarborane conjugates while it does not alter the preferential sites of intracellular localization.

10.1021/bc060047v CCC: $33.50 © 2006 American Chemical Society Published on Web 06/29/2006

Uptake with Cobaltacarborane−Porphyrin Conjugates

EXPERIMENTAL PROCEDURES Syntheses. All commercially available starting materials were used directly without further purification, unless otherwise indicated. Reactions under anhydrous conditions were performed in dried and distilled solvents under an argon atmosphere. All reactions were monitored by thin-layer chromatography (TLC) using Sorbent Technologies 0.25 mm silica gel plates with UV indicator (60F-254). Silica gel Sorbent Technologies 32-63 µm was used for flash column chromatography. 1H and 13C NMR were obtained on either a DPX-250 or ARX-300 Bruker spectrometer. Chemical shifts (δ) are given in parts per million relative to acetone-d6 (2.05 ppm, 1H; 29.92 ppm, 13C), unless otherwise indicated. Electronic absorption spectra were measured on a Perkin-Elmer Lambda 35 UV-vis spectrophotometer. Mass spectra were obtained on an Applied Biosystems QSTAR XL, a hybrid QqTOF mass spectrometer with a MALDI ionization source using either CCA or dithranol as the matrix. HPLC separation and analysis were carried out on a Dionex system equipped with P680 pump and a UVD340U detector. Semipreparative HPLC was carried out using a Luna C18 100 Å, 5 µm, 10 × 250 mm (Phenomenex, USA) column and a stepwise gradient; analytical HPLC was carried out using a Delta Pak C18 300 Å, 5 µm, 3.9 × 150 mm (Waters, USA) column and a stepwise gradient. 5,10,15,20-Tetra(4-hydroxyphenyl)porphyrin (1) was purchased from Aldrich. 5,10,15-Tri(4-hydroxyphenyl)-20-(4-methoxyphenyl)porphyrin (2). To a solution of porphyrin 1 (170 mg, 0.250 mmol) in anhydrous dimethyl sulfoxide (DMSO; 40 mL) was added K2CO3 (280 mg, 2.03 mmol). After heating this mixture to 60 °C for 15 min under argon, iodomethane (0.016 mL, 0.26 mmol) was added in one portion and the final mixture was stirred at 60 °C overnight. The reaction mixture was cooled to room temperature, poured into 200 mL of brine, extracted with ethyl acetate until colorless, and dried over anhydrous sodium sulfate. The solvent was removed under vacuum, and the resulting purple residue was purified by flash column chromatography on silica gel using a gradient of chloroform/ethyl acetate (1:1 to 100% ethyl acetate) for elution. The title porphyrin was collected and further purified by recrystallization from ethyl acetate/hexanes to give 42.6 mg, 25% yield, of dark purple crystals. The 1H NMR and UV-vis for the title porphyrin are in agreement with those reported in the literature (30). Mp > 300 °C; HRMS (MALDI-TOF) m/z 693.2468 (M + H)+, calcd for C45H32N4O4 693.2501. 5,10,15-Tri(4-hydroxyphenyl)-20-[4-(tert-butoxycarbonylmethyl)phenyl]porphyrin (3). To a solution of porphyrin 1 (500 mg, 0.737 mmol) in anhydrous DMSO (15 mL) was added Cs2CO3 (480 mg, 1.47 mmol). The mixture was heated at 50 °C for 1 h under argon before tert-butyl bromoacetate (287 mg, 1.473 mmol) was added in one portion and the final mixture heated at 60 °C for 2 h. After cooling to room temperature, ethyl acetate (100 mL) was added to the reaction mixture and the organic phase was washed with water (3 × 100 mL) and dried over anhydrous Na2SO4. The solvent was removed under vacuum, and the title porphyrin was purified by flash column chromatography on silica gel using chloroform/methanol 95/5 for elution to give 175 mg, 30% yield, of pure title porphyrin residue, mp > 300 °C. HPLC tR ) 5.90 min. UV-vis (CHCl3) λmax (/(M-1 cm-1)) 421 (401 690), 522 (28 400), 553 (21 950), 596 (10 500), 654 (11 000). 1H NMR (CDCl3, 300 MHz): δ 8.90-8.93 (m, 8H, β-H), 8.00-8.05 (m, 8H, PhH), 7.14-7.27 (m, 8H, PhH), 4.87 (s, 2H, CH2), 1.56 (s, 9H, tBu), -2.68 (s, 2H, NH). 13C NMR (CDCl3, 75 MHz): δ 168.80, 159.18, 158.45, 136.52, 136.24, 135.84, 134.08, 121.13, 120.34, 114.75, 113.90, 82.39, 66.54. 28.38. HRMS (MALDI-TOF) m/z 793.3048 (M + H+), calcd for C50H41N4O6, 793.3026.

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General Procedure for Conjugation of Cobaltacarborane to a 4-Hydroxyphenylporphyrin. To a solution of 4-hydroxyphenylporphyrin (0.1 mmol) in acetone (20 mL) was added K2CO3 (0.6 mmol), and the mixture was heated to reflux for 30 min under argon. After cooling to room temperature [3,3′-Co(8-C4H8O2-1,2-C2B9H10(1′,2′-C2B9H10)] (0.3 mmol) was added and the final mixture was refluxed under argon overnight. A second portion of K2CO3 (0.3 mmol) and [3,3′-Co(8-C4H8O21,2-C2B9H10(1′,2′-C2B9H10)] (0.15 mmol) were added to the reaction mixture, and reflux continued for 1 day. A third portion of [3,3′-Co(8-C4H8O2-1,2-C2B9H10(1′,2′-C2B9H10)] (0.1 mmol) was added, and reflux continued for another day, while monitoring the reaction by HPLC. After 3 days the mixture was cooled to room temperature, the acetone removed under vacuum, and the residue dissolved in ethyl acetate. The organic phase was washed with water and dried over anhydrous Na2SO4 and the solvent removed under vacuum. The resulting solid residue was washed with ethyl ether (5 × 5 mL) to remove impurities, and the porphyrin-cobaltacarborane conjugate was dried under vacuum at 50 °C for 2 days. Porphyrin Conjugate 4. This conjugate was prepared from porphyrin 2 and [3,3′-Co(8-C4H8O2-1,2-C2B9H10(1′,2′-C2B9H10)] as described above and obtained in 93% (64.2 mg) yield, mp ) 230 °C (dec). HPLC tR ) 11.04 min. UV-vis (acetone) λmax (/(M-1 cm-1)) 419 (593 000), 516 (23 900), 553 (18 000), 593 (9 000), 651 (8 900). 1H NMR (acetone-d6): δ 8.91-8.94 (m, 8H, β-H), 8.14 (d, 8H, J ) 7.9 Hz, o-PhH), 7.34-7.37 (m, 8H, m-PhH), 4.45-4.50 (m, 6H, OCH2), 4.37 (br, 6H, OCH2), 4.30 (br, 6H, OCH2), 4.11 (s, 3H, OCH3), 4.02-4.06 (m, 6H, OCH2), 3.74 (br, 12H, carborane-H), 1.30-1.60 (br, 51H, BH), -2.68 (s, 2H, NH). 13C NMR (acetone-d6) 160.3, 136.6, 135.5, 121.1, 114.2, 113.5, 73.4, 70.8, 69.8, 69.0, 60.9, 55.5, 47.7. HRMS (MALDI-TOF) m/z 1992.1954 (M - 3K+ + 3Na+ + H+)+, calcd for C69H117B54N4O10Co3Na3 1992.1899. Porphyrin Conjugate 5. This conjugate was prepared from porphyrin 3 and [3,3′-Co(8-C4H8O2-1,2-C2B9H10(1′,2′-C2B9H10)] as described above, and the corresponding tert-butyl protected conjugate 5 was obtained in 91% yield, mp ) 262-265 °C (dec). HPLC tr ) 12.52 min. UV-vis (acetone) λmax (/(M-1 cm-1)) 419 (396 700), 516 (26 000), 552 (21 200), 593 (8 600), 650 (9 000). 1H NMR (acetone-d6, 250 MHz): δ 8.90-8.93 (m, 8H, β-H), 8.13 (d, 8H, J ) 7.9 Hz, o-PhH), 7.36-7.43 (d, 8H, J ) 8.5 Hz, m-PhH), 4.92 (s, 2H, CH2), 4.50 (s, 6H, OCH2), 4.33 (s, 6H, OCH2), 4.28 (s, 6H, OCH2), 4.04 (s, 6H, OCH2), 3.75 (s, 12H, carborane-H), 1.6-3.0 (br, 51H, BH), 1.59 (s, 9H, CH3), -2.70 (s, 2H, NH). 13C NMR (acetone-d6, 63 MHz): δ 168.5, 159.7, 158.9, 136.1, 135.5, 134.9, 131.8, 120.6, 120.2, 113.7, 82.1, 72.8, 70.2, 69.2, 68.5, 66.3, 65.8, 61.6, 54.8, 47.1, 28.1. LRMS (MALDI-TOF) m/z 2139.003 (M+), calcd for C74H124N4O12B54Co3K3 2139.602. To a solution of this conjugate (100 mg, 0.0467 mmol) in chloroform (5 mL) was added TFA (5 mL) and the final mixture stirred at room temperature for 4 h. The solvent was removed under vacuum and the residue triturated with 5 mL of Et2O. The resulting green precipitate was washed with Et2O (6 × 10 mL) to remove residual TFA and dried under vacuum to give 93 mg, 95% yield, of pure conjugate 5, mp ) 283-285 °C (dec). HPLC tr ) 16.59 min. UV-vis (acetone) λmax (/(M-1 cm-1)) 419 (408 000), 516 (17 800), 553 (14 250), 593 (8 300), 650 (8 800). 1H NMR (acetone-d6, TFA): δ 8.78 (br, 8H, β-H), 8.48 (br, 8H, o-PhH), 7.64 (br, 8H, m-PhH), 5.10 (s, 2H, CH2), 4.53 (s, 6H, OCH2), 4.36 (s, 6H, OCH2), 4.29 (s, 6H, OCH2), 4.05-4.06 (m, 6H, OCH2), 3.73 (s, 12H, carborane-H), 0.77-2.43 (br, 51H, BH). LRMS (MALDI-TOF) m/z 2083.518 (M + H)+, calcd for C70H116N4O12B54Co3K3 2083.498. Porphyrin Conjugate 6. Peptidyl resin (0.02 mmol) was introduced into a glass synthesizer, swelled in dimethylforma-

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mide (DMF) for 1 h, and then washed with DMF (2 × 5 mL). To the resin was added 500 µL of a solution containing 0.08 mmol of FmocNH(CH2CH2O)7CH2CH2NHCOCH2OCH2CO2H, 0.120 mmol of DIEA, 0.08 mmol of HOBt, and 0.08 mmol of TBTU. The reaction mixture was allowed to react until the Kaiser test was negative, and then the resin was washed with DMF (5 × 5 mL) to remove residual reactants. The Fmoc protecting group was removed by treatment with 20% piperidine/DMF, for 40 min at room temperature. The pegylated peptidyl resin was washed with DMF (5 × 5 mL) and then 500 µL of a solution containing 0.04 mmol of conjugate 5, 0.120 mmol of DIEA, 0.04 mmol of HOBt, and 0.038 mmol of HATU was added. The reaction mixture was shaken 24 h at room temperature and then filtered to give a dark purple resin. This resin was washed to remove unreacted porphyrin, first with DMF until the filtrate was colorless, then with dichloromethane and methanol, before being dried under vacuum. Cleavage and deprotection was carried out by treatment of the dried resin with 3 mL of a mixture of TFA/phenol/TIS/H2O, 88:5:2:5 at room temperature for 4 h. The resin was filtered and washed with TFA (3 × 2 mL), and the filtrates were combined and evaporated under vacuum to give a green residue. The conjugate was precipitated by addition of cold Et2O, washed repeatedly with Et2O, and dried under vacuum. The purification of the porphyrin conjugate was accomplished by reverse phase HPLC on a Luna C18 semipreparative column (10 × 250 mm, 5 µm) (Phenomenex, USA) using water/acetonitrile both containing 0.1% TFA as the mobile phase, with a stepwise gradient from 80 to 95%. The fraction containing the conjugate was collected and lyophilized to afford pure conjugate 6 (6.9 mg) in 8% yield, mp > 300 °C. The purity of conjugate 6 was > 98% as obtained by HPLC on an analytical Delta Pak C18 (3.9 × 150 mm, 5 µm) column. HPLC tr ) 7.29 min. UV-vis (methanol) λmax (/(M-1 cm-1)) 421 (178 700), 559 (21 700), 553 (18 200), 593 (13 700), 654 (12 500). 1H NMR (CD3OH, 300 MHz): δ 7.66 (s, 8H, β-H), 7.50 (s, 8H, o-PhH), 7.33 (s, 8H, m-PhH), 3.92 (s, 6H, OCH2) 3.89 (s, 6H, OCH2), 3.63-3.77 (m, 32H, OCH2, carborane-H and peptide-H), 0.99-2.64 (m, 84H, peptide-H and BH). LRMS (MALDI-TOF) m/z 4302.084 (M - 3K+ + 7Na+)+, calcd for C160H283N42O26B54Co3Na7 4301.391. Porphyrin Conjugate 7. This conjugate was prepared from porphyrin 1 and [3,3′-Co(8-C4H8O2-1,2-C2B9H10(1′,2′-C2B9H10)] as described above and obtained in 85% (106.1 mg) yield, mp ) 290-292 °C (dec). HPLC tR ) 13.93 min. UV-vis (acetone) λmax (/(M-1 cm-1)) 419 (383 000), 516 (13 700), 552 (9 900), 595 (3 900), 651 (4 600). 1H NMR (acetone-d6, 300 MHz): δ 8.94 (s, 8H, β-H), 8.17 (d, 8H, J ) 8.5 Hz, o-PhH), 7.43 (d, 8H, J ) 8.5 Hz, m-PhH), 4.48 (s, 8H, OCH2), 4.37 (s, 8H, OCH2), 4.30 (s, 8H, OCH2), 4.05 (s, 8H, OCH2), 3.76 (s, 16H, carborane-H), 1.6-3.0 (br, 68H, BH), -2.70 (s, 2H, NH). 13C NMR (acetone-d6, 75 MHz): δ 160.5, 136.8, 135.7, 121.3, 114.4, 73.6, 71.0, 69.9, 69.3, 55.9, 47.9. HRMS (MALDI-TOF) m/z 2411.4881 (M - 4K+ + 4Na+ + H+), calcd for C76H143 N4O12B72Co4Na4 2411.4855. Cell Culture. All tissue culture media and reagents were obtained from Invitrogen. Human HEp2 cells were obtained from ATCC and maintained in a 50:50 mixture of DMEM/ Advanced MEM containing 5% FBS. The cells were subcultured biweekly to maintain subconfluent stocks. Cellular Uptake. HEp2 cells were plated at 10 000/well in a Costar 96 well plate and allowed to grow overnight. Conjugate stocks were prepared in a mixture of 9:1 DMSO/cremophore at a concentration of 1 mM and then diluted into medium to final working concentrations maintaining DMSO at 1%. The cells were exposed to 10 µM of each conjugate for 0, 1, 2, 4, 8, and 24 h. At the end of the incubation time the loading medium was removed and the cells were washed with PBS pH

Sibrian-Vazquez et al.

) 7.4. The cells were solubilized upon addition of 100 µL of 0.25% Triton X-100 (Calbiochem) in PBS. To determine the conjugate concentration, fluorescence emission was read at 410: 650 nm (excitation/emission) using a BMG FLUOstar plate reader. Then the cell numbers were quantified using the CyQuant reagent (Molecular Probes). Cytotoxicity. The HEp2 cells were plated as described above and allowed 36 h to attach. The cells were exposed to increasing concentrations of conjugate up to 10 µM and incubated for 24 h in the dark. The loading medium was then removed, and the cells washed twice with 100 µL of growing medium containing 50 mM HEPES pH 7.4 and then fed with medium containing Cell Titer Blue (Promega) as per manufacturer’s instructions. The cell toxicity was then measured by reading the fluorescence at 520:584 nm using a BMG FLUOstar plate reader. The signal was normalized to 100% viable (untreated) cells and 0% viable (treated with 0.2% saponin from Sigma) cells. For the phototoxicity experiments the HEp2 cells were prepared as described above. After conjugate loading, the medium was removed and replaced with medium containing 50 mM HEPES pH 7.4. The cells were then placed on ice and exposed to light from a 100 W halogen lamp filtered through a 610 nm long pass filter (Chroma) for 20 min; the total light dose was approximately 1 J cm-2. An inverted plate lid filled with water to a depth of 5 mm acted as an IR filter. The cells were returned to the incubator for 24 h, and the cytotoxicity was assayed as described above. Microscopy. HEp2 cells were plated on Lab Tek II chamber coverslips and incubated for 48 h prior to exposure to conjugate. The conjugates were diluted from 10 mM stocks to a working concentration of 10 µM in medium. The cells were then loaded with conjugate by incubating overnight. Organelle tracers were obtained from Molecular Probes and incubated concurrently with conjugate for 30 min prior to microscopy. Mitochondria were visualized using MitoTracker Green FM at 250 nM, lysosomes with LysoSensor Green DND-189 at 50 nM, Endoplasmic Reticulum (ER) with ER Tracker Blue-White DPX at 100 nM, and Golgi Complex with BODIPY FL C5-ceramide at 50 nM. The cells were then washed three times with medium containing 50 mM HEPES pH 7.4. Microscopy was performed using a Zeiss Axiovert 200M inverted fluorescent microscope fitted with an AxioCam MRm CCD camera (Zeiss).

RESULTS Synthesis and Characterization. Porphyrins 2 and 3 were prepared from commercially available porphyrin 1 in 25-30% yields, by Williamson etherification using CH3I and tert-butyl bromoacetate, respectively. The zwitterionic cobaltacarborane [3,3′-Co(8-C4H8O2-1,2-C2B9H10(1′,2′-C2B9H10)] was prepared from the reaction of cobaltabisdicarbollide anion [Co(C2B9H11)2]with 1,4-dioxane in the presence of BF3•Et2O, according to the literature (31). The nucleophilic ring opening reaction of the dioxane ring of cobaltacarborane [3,3′-Co(8-C4H8O2-1,2-C2B9H10(1′,2′-C2B9H10)] using hydroxyporphyrins 1, 2, and 3 afforded the corresponding porphyrin-cobaltacarborane conjugates in 85-93% yield (28). The free carboxylic acid group of compound 5 was converted into the corresponding hydroxybenzotriazole ester and coupled to a pegylated-peptide-PAL-PEGPS resin containing the HIV-1 Tat 48-60 sequence. After cleavage and deprotection from the solid support using 88:2: 5:5 TFA/TIS/phenol/H2O, the porphyrin-peptide-cobaltacarborane conjugate 6 was isolated by reverse-phase HPLC in 8% yield. This low yield may be due to the following: (1) the weak electrophilic character of the free carboxylate, (2) steric effects imposed by the close proximity of the macrocycle to the carboxylic group during the coupling reaction, (3) the formation of aggregates in the solvent used, and/or (4) to the “PEG effect” which seems to cause several conventional reactions to go less

Uptake with Cobaltacarborane−Porphyrin Conjugates

Figure 1. Dark toxicity of porphyrin conjugates 4 (-‚‚-), 5 (- -), 6 (s), and 7 (--) toward HEp2 cells using the Cell Titer Blue assay.

Bioconjugate Chem., Vol. 17, No. 4, 2006 931

Figure 3. Time-dependent uptake of porphyrin conjugates 4 (-‚‚-), 5 (- -), 6 (s), and 7 (--) at 10 µM by HEp2 cells.

Figure 2. Phototoxicity of porphyrin conjugates 4 (-‚‚-), 5 (- -), 6 (s), and 7 (--) toward HEp2 cells using 1 J cm-2 dose light.

effectively when there are (CH2CH2O)n motifs in the reactants. Conjugates 4 and 5 are soluble in polar organic solvents, such as acetonitrile, acetone, methanol, tetrahydrofuran (THF), and DMSO, but insoluble in water, while 6 and 7 are partially watersoluble and also show good solubility in polar organic solvents. Cellular Studies. The dark and phototoxicity of conjugates 4, 5, 6, and 7 were evaluated in human HEp2 cells, and the results obtained are shown in Figures 1 and 2, respectively. Due to their limited water solubility, both DMSO and Cremophor EL were used as delivery vehicles at low, nontoxic concentrations (0.9:0.1 DMSO/Cremophor EL) (29). At concentrations up to 10 µM (higher concentrations were not studied because of the low solubility of the conjugates in aqueous solution), all compounds are nontoxic to the cells in the dark, as determined using the Cell Titer Blue assay. Upon exposure to a low light dose (1 J cm-2), only conjugate 6 shows mild cytotoxicity, with approximately 30% inhibition of cell proliferation at the highest concentration investigated in this study (10 µM). The cellular uptake of conjugates 4-7 depends significantly on the number of cobaltacarborane moieties linked to the porphyrin ring and on the presence of the HIV-1 Tat 48-60 peptide sequence. As shown in Figure 3, conjugate 7 containing four cobaltacarborane moieties is taken up to a larger extent at all time points studied than conjugates 4 and 5, which contain only three cobaltacarborane units. The presence of an acetic acid group in place of a methyl does not have an effect on the cellular uptake nor on the dark toxicity and phototoxicity under the conditions studied. Conjugate 6 containing three cobaltacarborane moieties and the HIV-1 Tat 48-60 peptide sequence accumulated the most within cells of all conjugates at long time points (>2 h) (Figure 3). For example after 24 h, 11 times more conjugate 6 was found within cells than either 4 or 5 with the same number of cobaltacarboranes and three times more than 7, which contains one more cobaltacarborane moiety. At short time points however, the uptake of 6 was similar to that of 7, and always higher than that of conjugates 4 and 5. Conjugates 6 and 7 were taken up rapidly in the first 2 h, after which the uptake of 7 reached a plateau, whereas that of 6 continued to steadily

Figure 4. Subcellular localization of conjugate 6 in HEp2 cells at 10 µM for 18 h: (a) phase contrast, (b) overlay of 6 fluorescence and phase contrast, (c) ER Tracker Blue/White fluorescence, (e) LysoSensor Green fluorescence, (g) MitoTracker Green fluorescence, (i) BODIPY Ceramide, and (d, f, h, j) overlays of organelle tracers with 6 fluorescence. Scale bar: 10 µm.

increase. The kinetics of uptake of conjugates 4 and 5 was similar to that of 7, but the amount of conjugate taken up by cells was significantly lower than that of 7.

932 Bioconjugate Chem., Vol. 17, No. 4, 2006 Scheme 1

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a

a Conditions: (a) CH I, K CO , DMSO, 60 °C, overnight (25%); (b) BrCH COOtBu, Cs CO , DMSO, 60 °C, 2 h (30%); (c) [3,3′-Co(8-C H O 3 2 3 2 2 3 4 8 2 1,2-C2B9H10)(1′,2′-C2B9H11)], K2CO3, acetone, reflux, 72 h (85-93%); (d) TFA, CH2Cl2, rt, 4 h (95%); (e) HOBt, HATU, DIPEA, pegylatedpeptidyl PAL-PEG-PS resin, DMF, rt, 24 h then TFA/TIS/H2O/Phenol, 88:2:5:5 (8%).

The subcellular localization of conjugates 4-7 in HEp2 cells was evaluated by fluorescence microscopy, 24 h after exposure to the cells. Figure 4 shows the fluorescent patterns observed for the porphyrin-cobaltacarborane-HIV-1 Tat 48-60 conjugate 6, and its overlay with the organelle specific fluorescent probes LysoSensor Green (lysosomes), Mitotracker Green (mitochondria), ER Tracker Blue-White (ER), and BODIPY Ceramide (Golgi). Similar figures were obtained for conjugates 4, 5, and 7 (see Supporting Information). The microscopy shows punctate, predominantly perinuclear staining that localized in vesicles for all compounds. These vesicles were quite large for conjugate 6 and are clearly visible in the phase contrast micrograph. All compounds showed clear co-localization with lysosomes as evidenced by a yellow to orange color in the fluorescence signal when the green organelle tracer signal is overlaid with the red conjugate signal.

DISCUSSION Porphyrin-cobaltacarborane conjugates 4-7 were synthesized as shown in Scheme 1 from commercially available tetra(hydroxyphenyl)porphyrin 1. These conjugates were designed to have either three or four cobaltacarborane moieties linked to the porphyrin macrocycle via O(CH2CH2O)2 chains, and, in the case of 6, a HIV-1 Tat 48-60 peptide sequence attached via a

low molecular weight PEG spacer. The PEG-based linkages were chosen because they tend to increase the water solubility of porphyrin macrocycles and to minimize their aggregation and intramolecular interactions in aqueous media (26). Furthermore, PEG-drug conjugates are known to exhibit enhanced serum life and tumor accumulation (32). Low molecular weight PEGs were preferred over high molecular weight PEGs because of the polydispersity of the latter. The starting porphyrin 1 was monofunctionalized using the Williamson etherification reaction to produce porphyrins 2 and 3 in 25-30% yield (Scheme 1). The conjugation of porphyrins 1-3 with cobaltacarborane was performed in a single step in 85-93% yield, by the nucleophilic ring-opening of the dioxane ring of zwitterionic [3,3′-Co(8-C4H8O2-1,2-C2B9H10)(1′,2′C2B9H11)], as we have previously reported (28). The porphyrincobaltacarborane-peptide conjugate 6 was prepared by coupling conjugate 5 with the pegylated HIV-1 Tat 48-60 sequence on solid support, following our recently published procedure (26). All conjugates were isolated in >98% purity as determined by reverse phase HPLC and were characterized by MS, UV-vis, and NMR spectroscopy. The achievement of therapeutic boron concentrations (1530 µg/(g of tumor)) within tumors is still a major challenge in BNCT (6). Porphyrin-cobaltacarborane conjugates of high

Uptake with Cobaltacarborane−Porphyrin Conjugates

boron content and with no dark cytotoxicity are potential promising boron delivery drugs for BNCT. We have previously reported that the cellular uptake of a series of zwitterionic porphyrin-cobaltacarborane conjugates increases with the number of cobaltacarborane moieties linked to the porphyrin macrocycle (29). To further increase the uptake of this type of compound, we designed and synthesized conjugate 6, bearing a cell-penetrating peptide sequence. Indeed, conjugate 6 was taken up by cells to a significantly higher extent than conjugates 4 and 5, containing the same number of cobaltacarborane moieties (Figure 3). At short time points (2 h), conjugate 6 was taken up to a much larger extent than 7. These results are in agreement with previous reports by us (26) and others (33-37) showing that the Tat 48-60 peptide fragment from the HIV-1 Tat protein has the ability to efficiently translocate cellular membranes. Our results are also in agreement with our previous observation that the cellular uptake increases with the number of cobaltacarborane residues attached to the porphyrin macrocycle (29); at all time points studied the uptake of conjugate 7, bearing four cobaltacarboranes, was always significantly higher than that of conjugates 4 and 5, containing three cobaltacarboranes and therefore one less negative charge. On the other hand, the replacement of a methyl group (in 4) for an acetic acid group (in 5) had no effect on cellular uptake. All conjugates were found to be nontoxic in the dark at the concentrations studied (Figure 1) and only conjugate 6 showed mild phototoxicity at low light dose (1 J cm-2) and concentrations > 5 µM (Figure 2), probably as a result of its large accumulation within cells. Porphyrin-cobaltacarborane conjugates 4-7 preferentially localize within cells in vesicles and lysosomes, as evaluated by fluorescence microscopy (Figure 4), probably as a result of an endocytotic mechanism of uptake of all conjugates into cells. These results are in agreement with previous reports showing that negatively charged carboranyl-containing porphyrins localize mainly in the cell lysosomes (38-41). The presence of the HIV-1 Tat 48-60 peptide sequence in conjugate 6 significantly increased the cellular uptake of porphyrin-cobaltacarborane conjugates but did not alter their preferential sites of intracellular localization. Since the cytotoxic particles produced in BNCT, 4He2+, and 7Li3+ have average ranges in tissue of 9 and 5 µm, respectively, 10B-loaded vesicles and lysosomes in the vicinity of tumor cell nuclei could potentially lead to effective tumor destruction upon activation with low-energy neutrons. The amount of boron needed for effective BNCT could be substantially lower than 30 µg/(g of tumor) if the boron-10 localizes within tumor cells rather than extracellularly, and further reduced if the boron-10 localizes inside or in the close proximity to the cell nucleus (11, 12, 42). Conjugates of boronated agents to the HIV-1 Tat peptide sequence could induce higher accumulation of boron within target tumors due to enhanced tumor retention compared with normal tissues, therefore resulting in a more efficient BNCT treatment.

ACKNOWLEDGMENT The authors thank Martha Juban for HIV-1 Tat 48-60 peptide synthesis. This work was supported by the National Science Foundation, Grant No. CHE-304833, and the National Institutes of Health, Grant No. CA098902. Supporting Information Available: Traces of HPLC analysis for porphyrins 3-7 and microscopy images for conjugates 4, 5, and 7. This material is available free of charge via the Internet at http://pubs.acs.org.

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