A light-activated theranostic nanoagent for targeted macrophage ablation in inflammatory atherosclerosis.

General All chemicals and solvents were purchased from Fisher or Sigma Aldrich and used as received without further purification. Silica gel (Sorbent Technologies, 60 Å, 40–63 μm, 230 × 400 mesh) was used for column chromatography. UV-vis spectra were recorded on a Varian Cary 50 UV-vis spectrophotometer. Fluorescence data were collected from a Varian Cary Eclipse fluorescence spectrophotometer. Absorption and fluorescence spectra were collected in DMF at room temperature unless noted otherwise. The absorption plate reader utilized was a Tecan Safire. LCMS data were collected form a Water 2695 HPLC equipped with a 2996 diode array detector, a Micromass ZQ4000 ESI-MS module, and a Grace-Vydac RPC18 column (model 218TP5210) at a flow rate of 0.3 mL/min. Gradients were run with buffer A (H 2 O/0.1% trifluoroacetic acid (TFA)) and buffer B (90% acetonitrile/10% H 2 O/0.1% TFA). For analytical HPLC a C-18 reverse phase column (Varian) was used with dimensions of 250 mm × 4.6 mm. For semi-preparative HPLC a C-18 reverse phase column (Varian) was used with dimensions of 250 mm × 21.2 mm. High-resolution electrospray ionization (ESI) mass spectra were obtained from a Bruker Daltonics APEXIV 4.7 T Fourier transform ion cyclotron resonance spectrometer (FT-ICR-MS) in the Department of Chemistry Instrumentation Facility (DCIF) at the Massachusetts Institute of Technology. All 1 H NMR spectra (500 MHz) and 13 C NMR spectra (125 MHz) were collected in the solvents noted. Particle size measurements were performed on a Malvern Zetasizer Nano. Crosslinked dextran-coated iron oxide nanoparticles (35 nm hydrodynamic radius) were obtained from the chemistry core at the Center for Molecular Imaging Research.[ 11 ] meso -tetra( m -methoxyphenyl)porphyrin[ 24 ] and fluorenylmethyl-protected glutaric acid, Fm-Glu [ 15 ] were synthesized as described previously. The procedure for singlet oxygen quantum yields determinations has been reported.[ 25 ] 5-(4-amino-3-methoxyphenyl)-10,15,20-tri(3-methoxyphenyl)porphyrin, 3 To a stirring solution of 1 (1.50 g, 2.04 × 10 −3 mol) in 150 mL trifluoroacetic acid was added sodium nitrite (257 mg, 1.8 eq). The reaction was allowed to proceed for 4 min, at which time it was poured into 1500 mL distilled water. The resulting precipitate was filtered through Celite to remove the majority of the water, redissolved in CH 2 Cl 2 /MeOH (9:1), dried over anhydr MgSO 4 , and evaporated to dryness under reduced pressure to give the crude nitrated product 2 . The resulting solid was then dissolved in conc HCl (150 mL) and heated to 60 °C under inert atmosphere while stirring. To this solution was added SnCl 2 ⊕2H 2 O (3.67 g, 8 equiv). The reaction proceeded for 3 h at which time it was quenched by pouring the solution into 1500 mL distilled H 2 O, and neutralization with conc NH 4 OH. The resulting precipitate was filtered through Celite to remove the majority of the water, redissolved in CH 2 Cl 2 /MeOH (9/1), dried over anhydr MgSO 4 , and evaporated to dryness under reduced pressure. The product was purified by flash chromatography (CH 2 Cl 2 , silica gel). All fractions containing the product were combined, evaporated to dryness, redissolved in CH 2 Cl 2 , and crystallized by slow solvent exchange into MeOH to give 3 as a purple microcrystalline powder in 51 % yield (0.67 g). A fraction containing the starting material 1 was also recovered (0.39 g, 26 %). UV-vis (DMF) λ max (log ε): 426 (5.4), 518 (4.0), 560 (4.1), 599 (3.8), 653 (3.3) nm; 1 H NMR (300 MHz, CDCl 3 , δ) −2.73 (s, 2H), 4.01 (m, 12H), 4.18 (s, 2H), 7.11 (d, J = 7.8 Hz, 1H), 7.36 (m, 3H), 7.68 (m, 5H), 7.84 (m, 6H), 8.91 (br s, 6H), 9.00 (d, J = 4.8 Hz, 2H) ppm; 13 C NMR (100 MHz, CDCl 3 , δ) 55.5. 55.8, 113.2, 113.6, 117.7, 119.4, 120.5, 127.7, 132.4, 135.8, 143.6, 145.6, 158.0 ppm; +ESI-MS (30 V, CH 3 CN/0.1% TFA) m/z = 750.5 (MH + ). Fluorenylmethyl-protected 4-[2-methoxy-4-(10,15,20-tri(3-methoxyphenyl)porphyrin-5-yl)-phenylcarbamoyl]-butyric acid, 4 To an 80 mL microwave reaction vessel was added 3 (0.38 g, 5.07 × 10 −4 mol), dicyclohexylcarbodiimide (DCC, 0.63 g, 6 equiv), Fm-Glu (0.94 g, 6 equiv), and a CHCl 3 /Pyridine solution (25 mL, 95:5). The resulting solution was subject to microwave irradiation while stirring, with the following settings: T = 100 °C, t = 30 min, P max = off. Once the reaction had cooled, it was filtered to remove the resulting insoluble dicyclohexylurea (DCU), diluted with DMF (25 mL) and evaporated to dryness. In order to remove the excess Fm-Glu, which is of similar polarity to the product, the crude product was subject to column chromatography (CH 2 Cl 2 /MeOH/TFA, 98/1.5/0.5, silica gel). The fractions containing the crude product were combined and the total volume was reduced to 25 mL, at which point the TFA was neutralized by addition of 5 mL DMF/Et 3 N (95/5). The resulting solution was evaporated to dryness, dissolved in EtOAc and washed thrice with water, dried over anhydr MgSO 4 , and evaporated to dryness. The pure product was then dissolved in CH 2 Cl 2 and precipitated with hexanes to yield 4 as a purple powder (0.48 g, 91%). UV-vis (DMF) λmax (log ε): 421 (5.6), 515 (4.2), 552 (3.9), 591 (3.7), 647 (3.5) nm; 1 H NMR (500 MHz, CDCl 3 , δ) −2.56 (s, 2H), 2.22 (m, 2H), 2.57 (m, 2H), 2.68 (m, 2H), 4.07 (s, 12H), 4.29 (m, 1H), 4.57 (d, J = 6.5 Hz, 2H), 7.47 (m, 8H), 7.72 (m, 6H), 7.82 (d, J = 7.0 Hz, 2H), 7.94 (m, 8H), 8.04 (d, J = 7.5 Hz, 1H), 8.84 (d, J = 7.5 Hz, 1H), 9.11 (m, 9H) ppm; 13 C NMR (125 MHz, CDCl 3 , δ) 20.7, 33.4, 36.6, 47.0, 55.6, 56.1, 66.3, 113.5, 113.8, 117.1, 118.0, 119.9, 120.0, 120.1, 120.2, 120.6, 120.8, 125.1, 127.2, 127.5, 127.7, 127.8, 127.9, 128.0, 137.8, 141.4, 143.6, 143.8, 146.3, 158.1, 170.7, 173.2 ppm. Fluorenylmethyl-protected 4-[2-methoxy-4-(2,3-dihydroxy-10,15,20-tri(3-methoxyphenyl)chlorin-5-yl)-phenylcarbamoyl]-butyric acid, 5I, and fluorenylmethyl-protected 4-[2-methoxy-4-(12,13-dihydroxy-10,15,20-tri(3-methoxyphenyl)chlorin-5-yl)-phenylcarbamoyl]-butyric acid, 5II To a solution of 4 (191 mg, 1.8 × 10 −4 mol) in CH 2 Cl 2 /pyridine (10 mL, 95/5) was added OsO 4 in CH 2 Cl 2 /pyridine (0.78 mL, 0.83 equiv, 0.05 g/mL). The reaction was allowed to proceed for 24 h, at which time it was diluted with DMF (10 mL), and evaporated to dryness. The crude product was then dissolved in CH 2 Cl 2 and H 2 S was bubbled through the reaction for 5 min, at which time the reaction was allowed to proceed for 45 min. The solution was evaporated to dryness, redissolved in CH 2 Cl 2 , filtered through celite, and evaporated to dryness. The products were purified by column chromatography (silica gel, CH 2 Cl 2 /MeOH (98/2)), and the fractions containing the respective isomer were combined and evaporated to dryness. The products were redissolved in CH 2 Cl 2 and precipitated with hexanes to give isomer I (31 mg, 19%), and isomer II (36 mg, 22%). 5I UV-vis (DMF) λ max (log ε): 422 (5.3), 519 (4.2), 548 (4.1), 596 (3.8), 648 (4.4) nm; 1 H NMR (500 MHz, CDCl 3 , δ) −1.76 (s, 2H), 2.18 (m, 2H), 2.56 (t, J = 6.5 Hz, 2H), 2.64 (t, J = 6.5 Hz, 2H), 3.28 (br s, 2H), 3.99 (m, 13H), 4.29 (t, J = 6.5 Hz, 1H), 4.53 (d, J = 6.5 Hz, 2H), 6.43 (d, J = 23.0 Hz, 2H), 7.31 (m, 3H), 7.38 (t, J = 7.0 Hz, 2 H), 7.46 (t, J = 7.0 Hz, 2H), 7.54 (d, J = 18.5 Hz, 2H), 7.73 (m, 14 H), 8.08 (s, 1H), 8.42 (d, J = 11.0 Hz, 2H), 8.57 (s, sH), 8.74 (br s, 3H) ppm; 13 C NMR (125 MHz, CDCl 3 , δ) 20.7, 33.3, 36.6, 46.9, 55.5, 56.0, 66.3, 74.1, 74.3, 113.0, 113.4, 113.6, 113.8, 116.3, 116.5, 117.9, 118.5, 119.8, 120.1, 122.8, 122.9, 124.4, 125.1, 126.5, 127.2, 127.4, 127.6, 127.9, 128.2, 128.6, 128.8, 132.7, 135.6, 135.7, 137.2, 140.7, 141.4, 142.4, 143.2, 143.8, 146.2, 153.0, 153.2, 158.0, 158.7, 159.1, 161.4, 170.5, 173.2 ppm; +ESI-MS (30 V, CH 3 CN/0.1% TFA) m/z = 1076.7 (MH + ). 5II UV-vis (DMF) λ max (log ε): 422 (5.4), 519 (4.1), 548 (4.1), 596 (3.9), 648 (4.4) nm; 1 H NMR (500 MHz, CDCl 3 , δ) −1.78 (s, 2H), 2.16 (m, 2H), 2.53 (s, 2H), 2.62 (s, 2H), 3.99 (s, 13H), 4.28 (s, 1H), 4.52 (s, 2H), 6.42 (d, J = 21.5 Hz, 2H), 7.53 (m, 26H), 8.06 (s, 1H), 8.44 (s, 2H), 8.56 (s, 2H). 8.73 (m, 3H) ppm; 13 C NMR (125 MHz, CDCl 3 , δ) 20.7, 33.3, 36.6, 46.9, 55.5, 56.1, 66.3, 74.1, 74.4, 113.0, 113.7, 113.8, 114.2, 116.3, 118.8, 120.1, 122.9, 124.2, 125.1, 126.7, 126.9, 127.2, 127.6, 127.9, 128.2, 132.7, 135.6, 136.6, 140.6, 140.9, 141.4, 142.3, 143.2, 143.8, 146.7, 153.1, 158.0, 170.5, 173.1 ppm; +ESI-MS (30 V, CH 3 CN/0.1% TFA) m/z = 1076.7 (MH + ). 4-[2-hydroxy-4-(2,3-dihydroxy-10,15,20-tri(3-hydroxyphenyl)chlorin-5-yl)-phenylcarbamoyl]-butyric acid, 6I, and 4-[2-hydroxy-4-(12,13-dihydroxy-10,15,20-tri(3-hydroxyphenyl)chlorin-5-yl)-phenylcarbamoyl]-butyric acid, 6II To a solution of 5I or 5II (31.1 mg, 2.9 × 10 −5 mol) in anhydr CH 2 Cl 2 (10 mL) under an argon atmosphere was added BBr 3 (300 μL, 1 M in CH 2 Cl 2 , 12 equiv). The reaction is allowed to proceed for 1 h, at which time it is quenched by addition of MeOH (5 mL), and neutralized with DMF/Et 3 N (10 mL, 95/5). The solution was evaporated to dryness, dissolved in EtOAc, washed thrice with water, dried over anhydr MgSO 4 , and evaporated to dryness. The crude product was then dissolved in DMF/piperidine (4:1) to effect deprotection of the fluorenylmethyl group, reacted for 20 min, and evaporated to dryness. The products were purified by HPLC (80% Buffer A to 20% Buffer A), with the fractions containing the pure product being combined and evaporated to dryness. The product was precipitated from i PrOH with hexanes to give the product in 31 % yield (9 mg). 6I UV-vis (DMF) λ max (log ε): 420 (5.1), 550 (3.9), 547 (3.9), 595 (3.6), 648 (4.2) nm; 1 H NMR (500 MHz, Methanol-d 4 , δ) 2.02 (m, 2H), 2.36 (m, 2H), 2.55 (t, J = 7.5, 2H), 6.25 (m, 2H), 7.03 (d, J = 7.5, 1H), 7.10 (d, J = 7.5, 2H), 7.26 (m, 2H), 7.33 (m, 1H), 7.49 (m, 8H), 8.34 (d, J = 5.0, 1H), 8.40 (m, 3H), 8.63 (d, J = 5.0, 2H) ppm; 13 C NMR (125 MHz, Methanol-d 4 , δ) 23.3, 36.3, 37.2, 75.4, 75.6, 115.0, 115.4, 115.5, 115.7, 116.0, 120.9, 121.8, 122.6, 123.1, 125.3, 126.7, 128.9, 129.0, 129.2, 129.5, 133.5, 136.6, 142.0, 142.1, 144.2, 144.5, 154.3, 157.2, 157.4, 157.8, 164.0, 164.3, 174.9, 175.2 ppm; +ESI-MS (30 V, CH 3 CN/0.1% TFA) m/z = 842.5 (MH + ). 6II UV-vis (DMF) λ max (log ε): 421 (5.1), 520 (4.0), 548 (4.0), 596 (3.7), 648 (4.2) nm; 1 H NMR (500 MHz, Methanol-d 4 , δ) 2.11 (t, J = 8.5 Hz, 2H). 2.47 (m, 2H), 2.65 (t, J = 8.5 Hz, 2H), 6.33 (d, J = 12.5 Hz, 2H), 7.11 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 9.0 Hz, 1H), 7.33 (s, 2H), 7.57 (m, 9H), 8.01 (s, 1H), 8.42 (d, J = 6.0 Hz, 2H), 8.50 (d, J = 5.5 Hz, 1H), 8.53 (d, J = 6.0 Hz, 1H), 8.71 (d, J = 6.0 Hz, 1H), 8.75 (d, J = 6.0 Hz, 1H) ppm; 13 C NMR (125 MHz, Methanol-d 4 , δ) 21.6, 34.3, 35.7, 74.1, 113.9, 114.0, 114.2, 114.5, 119.4, 120.7, 121.9, 123.7, 125.5, 127.5, 127.7, 128.0, 132.0, 135.1, 135.2, 139.0, 140.5, 142.7, 143.0, 146.8, 152.9, 155.7, 156.0, 156.3, 162.5, 173.4 ppm; +ESI-MS (30 V, CH 3 CN/0.1% TFA) m/z = 842.5 (MH + ). General synthesis of succinimidyl esters of 6I or 6II To 6I or 6II (~10 mg) in DMF (5 mL) was added DCC (4 equiv), 4-dimethylaminopyridine (0.4 equiv), and N-hydroxysuccinimide (4 equiv). The reaction was allowed to proceed for 2 h, at which time the solution was filtered through a plug of cotton and evaporated to dryness. The solid was then dissolved in anhydr DMSO (2 mL) and filtered through a plug of cotton to remove any residual solid. The resulting solution was used for conjugation to the nanoparticle without purification. Dye labelling of CLIO To 20 mg CLIO (12.12 mg Fe mL −1 ) in PBS was added AlexaFluor 750 (1 mg, Molecular Probes, Eugene, OR). The reaction was allowed to proceed for 16 h, at which time the suspension was filtered through Sephadex G-25 to give CLIO-AF750 . The resultant solution was divided, with one half kept as the control agent, and the remainder reacted with the chlorin. To CLIO-AF750 (10 mg) was added 7I (2 mg) dissolved in anhydr DMSO (300 μL). The reaction was allowed to proceed for 16 h, at which time it was filtered through Sephadex G-25 to give the final product, CLIO-THPC . The number of dyes per particle was determined as described previously.[ 14 ] Cellular uptake and phototoxicity Murine macrophage RAW 264.7 cells growing in DMEM media were resuspended in PBS and incubated with CLIO-THPC , CLIO-AF750 , or chlorin e6 at varying concentrations (50nM-5μM) for 3 hours. Cells were then washed with fresh media. At this point, selected cells were examined by fluorescence microscopy (Nikon TE2000, Nikon Instruments Inc.) by light microscopy and utilizing rhodamine (ex/em 546/590) and Cy7 (ex/em 710/810) filter cubes. Selected cells for photodynamic therapy were illuminated with a 650 nm laser, 150 mW, 3 minutes for total fluence of 6 J/cm2. Cells were returned to the incubator overnight and then evaluated by MTS assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega Corp.) for cell viability determination. MTS results were analyzed on a well plate reader (Tecan Saphire). In vivo uptake and phototoxicity Male ApoE −/− mice aged 23–29 weeks on high cholesterol diet since 13 weeks of age were injected with CLIO-THPC or CLIO-AF750 (as a control) at a dose of 10 mg Fe/kg in PBS via tail vein injection. One day post-injection mice were anesthetized with ketamine/xylazine by peritoneal injection and the carotid artery was surgically exposed. Mice were imaged with an Olympus IV100 intravital laser scanning fluorescence microscope developed for imaging small experimental animals (Olympus Inc.). Images were obtained in the AF750 channel as well as the FITC channel to detect autofluorescence. Imaging was performed before and after injection of FITC-dextran (0.5mg, MW 2,000,000, 2 mg/mL, Invitrogen Corp.) to image the vessel lumen and aid with registration of images. Following imaging, carotid ateries were illuminated externally with a 650 nm laser, 150 mW, 1 cm target, 3 minutes for total fluence of 11 J/cm2. Mice were returned to their housing for 1 day. Following IVFM or apoptosis endpoints, mice were perfused with 20 mL of cold PBS and dissected. Specimens were embedded in OCT compound and stored at −80°C. Specimens were stained with hematoxylin and eosin, Mac-3 for macrophage content, and TUNEL peroxidase staining for apoptosis (Apoptag Peroxidase, Millipore Corp.). Percent TUNEL positive cells determined in ImageJ (NIH) after Color-based Thresholding. Cohorts: CLIO-THPC - 5 mice, CLIO-AF750 - 5 mice Mouse hind paw edema model Female c57/bl6 mice were injected with CLIO-THPC at a dose of 10mg Fe/kg in PBS or chlorin e6 5mg/kg via tail vein injection. Baseline footpad thickness was measured bilaterally with a digital caliper. After a drug-light interval of 1 h or 24 h, the mice were anesthetized with inhaled isoflurane (2%) and the right footpad was illuminated with a 650 nm laser, 150 mW, 3 minutes for total fluence of 11 J/cm2. Repeat footpad measurements were performed 24 hours after light exposure. Footpad swelling was analyzed using the change in footpad thickness as a percentage of baseline, using the left (non-illuminated) footpad as a control. Cohorts: CLIO-THPC - 1 h - 3 mice; 24 h - 3 mice, Chlorin e 6 - 1 h - 5 mice, 24 h - 3 mice.