Methods
L-α-phosphatidylcholine (from chicken eggs), 1,2-dioleoyl-sn-dlycero-3-[phospho-L-serine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Cholesterol, azelaic acid and all other chemicals were obtained from Sigma-Aldrich Corporation (St Louis, MO, USA); Gd-diethylenetriaminepentaacetic acid distearylamide (Gd-DTPA-SA) was synthesized according to methods described in,[16] RPMI 1640, fetal bovine serum and phosphate-buffered saline (PBS) were purchased from Cellgro Mediatech Inc. (Herndon, VA, USA); penicillin-streptomycin, glutamine and sodium pyruvate were from Gibco/InVitrogen (Carlsbad, CA, USA).
1H and 13C NMR spectra were recorded on a Bruker AVANCE-400 spectrometer. Transmission electron microscopy (TEM) was performed using a FEI Technai G2 Spirit TEM operating at 80 kV. Samples negatively stained with ammonium molybdate were applied on Formvar-coated copper grids and analyzed.
Mass spectra were acquired in positive ion mode using an Applied Biosystems 3200 QTRAP coupled with electrospray ionization (ESI) source. Capillary temperature was set at 300°C, and spray voltage was set at 5.5 kV. Nebulizer gas and auxiliary gas flow was set to 60 and 20 arbitrary units, respectively. Curtain gas was set to 15.00 and collision gas settings were on medium.
Synthesis of 9-CCN
To a solution of cholesterol (1 g, 2.6 mmol) and azelaic acid (1.4 g, 7.5 mmol) in the 20 ml of mixture of acetone-chloroform (1:1 by volume), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (1.9 g, 10 mmol) and 4-(dimethylamino)pyridine (610 mg, 5 mmol) were added and flushed with nitrogen. The mixture was vigorously stirred overnight, concentrated and purified by column chromatography on silica gel using dichloromethane-methanol mixture as the eluent (9:1 by volume) to give 9-CCN (1.05 g, 74%) as a colorless solid. The product was found to be more than 95% pure by 1H-NMR and high-performance liquid chromatography (HPLC).
1H-NMR (400 MHz, chloroform-d), δ ppm 0.66 (s, 3 H), 0.85 (dd, J = 6.6, 1.8 Hz, 4 H), 0.89 (d, J = 6.6 Hz, 4 H), 1.00 (s, 3 H), 1.12 (m, 4 H), 1.33 (m, 12 H), 1.49 (s, 6 H), 1.59 (m, 4 H), 1.82 (m, 2 H), 1.98 (m, 2 H), 2.13 (s, 3 H), 2.29 (m, 8 H), 2.99 (s, 2 H), 3.65 (s, 1 H), 4.59 (m, 1 H), 5.35 (d, J = 3.8 Hz, 1 H); 13C-NMR (100 MHz, chloroform-d), δ ppm 11.83, 18.69, 19.30, 21.01, 22.53, 22.79, 23.81, 24.26, 24.58, 24.83, 24.86, 26.52, 27.79, 27.98, 28.20, 28.80, 31.75, 31.89, 33.90, 34.61, 35.36, 36.16, 36.98, 38.13, 39.50, 39.72, 42.30, 50.01, 52.36, 56.12, 56.68, 73.73, 79.96, 122.57, 139.69 and 173.29. High resolution mass spectrometry (ESI mass spectrometry [ESI-MS]): calculated for C36H59O4: 555.4413 [M-H]+; found: 555.4465.
Synthesis of (3b)-cholest-5-en-3-yl Methyl Azelaate (9-CCN-OMe)
Synthesis was performed in the Aldrich MNNG diazomethane generation apparatus according to manufacturer instructions. Briefly, to the outside tube of the apparatus, 2 ml of anhydrous ether and 0.5 ml of chloroform solution of 9-CCN (6 mg/ml, 30.5 mg, 0.06 mmol) was added. The lower part of the assembled apparatus was immersed in the ice and diazomethane was generated by addition of 1 ml of concentrated potassium hydroxide into the inside tube containing a solution of N-methyl-N-nitroso-p-toluenesulfonamide (0.122 g, 0.57 mmol) in 0.5 ml ethyldiglycol. Apparatus was kept in ice for 2 h and then solvents were evaporated under a gentle stream of nitrogen to give 9-CCN-OMe (33.8 mg, 99%) as a yellowish solid. The crude product was purified by column chromatography on silica gel using dichloromethane as the eluent yielded 32.9 mg (96%) of greater than 99% pure 9-CCN-OMe.
1H-NMR (400 MHz, chloroform-d), δ ppm 1H-NMR 0.69 (s, 3 H), 0.89 (dd, J = 6.6, 1.8 Hz, 4 H), 0.94 (d, J = 6.6 Hz, 4 H), 1.04 (s, 3 H), 1.17 (m, 6 H), 1.35 (m, 12 H), 1.59 (m, 13 H), 1.87 (m, 2 H), 2.02 (m, 2 H), 2.31 (m, 8 H), 3.69 (s, 3 H), 4.63 (m, 1 H), 5.39 (d, J = 4.5 Hz, 1 H); 13C-NMR (100 MHz, chloroform-d), δ ppm 11.84, 18.71, 19.31, 21.02, 22.55, 22.80, 23.82, 24.27, 24.85, 24.83, 24.94, 27.81, 27.99, 28.21, 28.87, 28.93, 31.86, 31.89, 34.03, 34.63, 35.78, 36.18, 36.59, 36.99, 38.15, 39.51, 39.73, 42.30, 50.03, 51.42, 56.13, 56.68, 73.70, 122.58, 139.71 and 173.20. High resolution mass spectrometry (ESI-MS): calculated for C37H64O4: 572.4805 [M+H]+; found: 572.4824.
Synthesis of 9-(cholest-5-en-3-yloxy)-9-oxononanoic Acid-d7 (9-CCN-d7)
9-CCN-d7 was synthesized as described previously for the nondeuterated analogous using 10 mg of cholesterol-d7 as a precursor. The product was purified by thin-layer chromatography followed by preparative HPLC to give 5 mg (36%) of greater than 99% pure 9-CCN-d7.
1H-NMR (400 MHz, chloroform-d), δ ppm 0.69 (s, 3 H), 0.90 (d, J = 6.6 Hz, 4 H), 1.00 (d, J = 4.3 Hz, 4 H), 1.11 (m, 10 H), 1.32 (m, 10 H), 1.54 (m, 15 H), 1.83 (m, 4 H), 1.98 (m, 3 H), 2.29 (m, 7 H), 3.52 (m, 1 H), 3.66 (s, 1 H), 4.60 (m, 1 H), 5.35 (m, 1 H); 13C-NMR (100 MHz, chloroform-d), δ ppm 11.85, 18.71, 19.39, 21.08, 23.78, 24.29, 24.60, 24.94, 27.81, 28.22, 28.84, 28.89, 31.65, 31.91, 33.67, 34.31, 35.79, 36.20, 36.51, 36.60, 37.25, 39.27, 39.79, 42.32, 50.14, 56.17, 71.84, 73.74, 117.17, 121.73 and 173.23. High resolution mass spectrometry (ESI-MS): calculated for C36H53D7O4: 581.5275 [M+NH4]+; found: 581.5275.
Synthesis of Liposomes
Liposomes were prepared by mixing lipid solutions in chloroform in molar ratios as indicated in Table 1 . Solvent was evaporated in vacuo and the obtained lipid film was hydrated with 20 mM HEPES buffered saline (pH = 7.35; 140 mM NaCl). Next, liposomal suspension was sonicated with probe-type sonicator (Sonicator 3000, Misonix, Inc. Farmingdale, NY, USA) maintaining mixture in the flow of nitrogen gas and cooling with the ice bath. After vesicles are formed, the pH of obtained liposomes was adjusted to 7.20–7.35 by addition of 0.1 M sodium bicarbonate.
Characterization of Liposomes
Physical properties of paramagnetic liposomes (i.e., oxPL, PL and mePL) were characterized with respect to size, lamellarity and longitudinal relaxation properties. Size measurements were performed using dynamic light scattering technique on Nano S Zetasizer system (Malvern Instruments, Worcestershire, UK). In addition, size and lamellarity were confirmed by electron microscopy. Longitudinal relaxivity (r1) measurements were conducted using a 1.5 T scanner at 25°C (Magnetom, Avanto MRI scanner, Siemens Medical Solutions, Erlangen, Germany). Samples were prepared based on Gd concentrations (as determined by inductively coupled plasma mass spectrometry [ICP-MS], see below) as series of dilutions ranging from 1000 to 100 ppm of Gd. Eppendorf tubes were filled with liposomal samples and placed in a styrene box filled with water. Relaxation curves for T1 were obtained by 2D imaging with an inversion-recovery turbo spin-echo pulse sequence. The following inversion times were applied: 30, 60, 90, 120, 150, 250, 400, 600, 800, 1200, 1600 and 2000 ms. Relaxation times (r1) were calculated from each slope of the linear relation between 1/T1 and Gd concentration.
Annexin V Binding Assays
Annexin-V-Biotin (BioVision Inc, USA) was added to streptavidin coated #1 coverslips (Xenopore corp., NJ, USA) and incubated for 10 min at room temperature followed by extensive washing with PBS. Next, 25 µl of vesicle formulations with or without PS was added and incubated for 5 min at room temperature. After five washing steps in PBS, coverslips were mounted on microscope slides and examined under Olympus FV1000 spectral confocal microscope using 100× objective. Quantification of rhodamine fluorescence was performed with ImageJ software (NIH, Bethesda, MD, USA).
Ex vivo Gd Release
Cumulative release of Gd from liposomal particles (release of free Gd and Gd-DTPA-SA) was determined by dynamic dialysis. In short, aliquots (50 µl) of liposomal formulations were dialyzed (membrane MW cut-off: 12 kDa) against 50 ml of human plasma (obtained from American Red Cross) at 37°C. Plasma sampling was performed at different time intervals and Gd concentrations were obtained using ICP-MS.
Determination of Gd Content in Liposomes & Plasma Samples
Plasma samples were digested in a mixture of 70% perchloric acid and 65% nitric acid at 80°C for 8 h. Digested samples were diluted with deionized water and concentration of Gd was determined using Perkin-Elmer Elan 6100 ICP-MS.
Animals
Watanabe heritable hyperlipidemic rabbits (WHHL; Brown Family Enterprises, LLC, Odenville, AL, USA) were fed regular rabbit diet for 10 months. Animals ranging from 3.2 to 3.5 kg were used in in vivo experiments. The Institutional Animal Care and Use Committee (IACUC) at The Ohio State University, UH, USA, approved the experimental animal protocols.
Magnetic Resonance Imaging
The animals were anesthetized with a subcutaneous injection of xylazine (10 mg/kg) and ketamine (50 mg/kg), and placed into the phased array cardiac coil (Siemens, Erlangen, Germany). MRI was performed on 1.5 T Siemens clinical scanner (Magnetom, Avanto) using a coronal gradient echo, T1-weighted localizing sequence (repetition time/echo time = 800/1.0; field of view = 94 × 125; matrix = 288 × 384). A total of 30 4-mm-thick axial slices spanning approximately from the iliac bifurcation to the superior pole of the topmost kidney were obtained using a T1-weighted gradient echo turbo FLASH protocol (repetition time/echo time = 230/5; three averages, slice thickness of 4.0 mm with a 5.2-mm gap between slices and a time of acquisition of ~11 m). After precontrast acquisition, 10 ml of oxPL or PL (0.02 mmol/kg of Gd, 10 mmol/kg of total lipids) were injected through marginal ear vein and flushed with 5 ml of normal saline. Images were acquired immediately after the contrast administration for approximately 1 h using repetitive acquisitions of 6-min duration. MRI scans were repeated 12 and 24 h postcontrast administration time points using a single acquisition.
MR images were analyzed using OsiriX image analysis software (The Osirix Foundation, Geneva, Switzerland). The aortic wall was identified and signal intensities of enhanced regions of interest (ROI) were obtained for three slices at each time point. Blind to histopathological data observer performed all ROI measurements. Signal to noise ratio (SNR) of each ROI was calculated as the average signal intensity divided by the standard deviation of the noise level. Relative enhancement ratios with respect to averaged SNR (RERSNR) were calculated and plotted as a percentage decrease in SNR over the time by the formula: % RERSNR = 100 × (SNRpost/SNRpre). SNRpre was obtained from images acquired before contrast agent administration, while SNRpost represents SNRs calculated from images acquired 1, 12 and 24 h postinjection. Series of identified ROIs, which matched to histopathological sections, were used for immunostaining followed by confocal microscopy.
Pharmacokinetics Studies
All animals subjected to MRI with contrast agent (oxPL or PL) were examined with respect to pharmacokinetics of injected liposomes. The blood was collected from a catheter placed in the marginal ear vein of rabbits by gentle withdrawal with syringe. Sampling was performed before administration of paramagnetic liposomes as well as 1, 12 and 24 h after contrast agent injection. The plasma was isolated by centrifugation (1000 g, 10 min) and stored at -80oC until analyzed. Gd concentrations in plasma were determined by ICP-MS. Concentrations of 9-CCN in all plasma samples were analyzed by ESI-MS after separation by reverse-phase HPLC. Deuterated internal standard (9-CCN-d7) was included at a concentration of 2.5 ppm in all plasma samples. Next, proteins were precipitated by adding three volumes of methanol, and supernatant was collected after centrifugation. Pellet was extracted three times with chloroform/methanol mixture (1:1) and combined extracts were evaporated to dryness under a stream of nitrogen. The residue was reconstituted with 90% methanol by sonication followed by isocratic separation on Ascentis 50 × 2.1 mm, 3 µm C18 HPLC column using 90% methanol with 0.1% ammonium acetate as eluent at 0.3 ml/min flow rate. Liquid chromatography was performed using Shimadzu LC-20AD pump interfaced to a Shimadzu CBM-20A system controller (Shimadzu, Columbia, MD, USA). Effluent was analyzed by Applied Biosystems 3200 QTRAP system (Applied Biosystems, Foster, CA, USA) operated in multiple reactions monitoring-positive ionization mode. Specific monitor Q1/Q3 ion pairs were m/z 574.6→369.6 for 9-CCN and m/z 581.6→376.6 for 9-CCN-d7. Concentrations of 9-CCN were calculated using Analyst software (version 1.4.2, Applied Biosystems). The percentage of injected liposomes at different time points was calculated as described before and based on the average amount of blood plasma in animals of appropriate weight. Plasma volume was assumed to be 3.3% of rabbit body weight.
Immunohistochemistry & Confocal Microscopy
Segments of abdominal aorta were embedded in optimal cutting temperature compound (Tissue-Tek, Sakura Finetek USA Inc, Torrance, CA, USA) and then frozen in dry ice. The embedded tissues were sectioned at a thickness of 8 µm and placed into cold ethanol. Air-dried slides were hydrated with PBS and treated with 0.2% TritonX-100 in PBS for 2 min. After subsequent washing with PBS and blocking in 1.5% horse serum for 20 min, the slides were stained with RAM-11 primary antibody specific to rabbit macrophages (mouse antirabbit, DAKO, Carpinteria, CA, USA; dilution 1:75) overnight at 4°C. Next, slides were incubated with fluorescently labeled secondary antibody (Cy-5 conjugated goat antimouse, Jackson Immuno-Research, West Grove, PA, USA; dilution 1:200) for 90 min at 25°C followed by nuclei staining with Hoescht 33342 (Invitrogen, Carlsbad, CA, USA; dilution 1:1500) for 10 min. Slides were mounted and examined with Zeiss LSM 510 confocal microscope equipped with Argon (458, 477, 488 and 514 nm), green HeNe (543 nm) and red HeNe (633 nm) lasers. For Hoechst 33342 fluorescence Titanium Sapphire two-photon laser was used.
Purification, Culture & Uptake Studies in Macrophages Derived from Peripheral Blood Monocytes
Monocytes were isolated from buffy coats obtained from the American Red Cross using Ficoll-Hypaque. The cells were further purified using magnetic cell isolation system and CD14 microbeads (Miltenyi Biotec, Auburn, CA, USA). After isolation, monocytes were resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum (heat inactivated) and seeded in six-well plates. After 2 h, the nonadherent cells were removed and cells were washed with warm medium. The cells were cultured in RPMI supplemented with 10% fetal bovine serum, 1% PSA (penicillin G sodium, streptomycin sulfate and amphotericin B), 10 µg/ml of polymyxin B and 20 ng/ml of M-CSF for 5 days in 37°C with 5% CO2. The differentiated macrophages were serum-starved for 1 day at 37°C prior to use.
Cells were treated with oxPL or PL formulations and incubated for 30–120 min. Labeled cells were washed, detached and analyzed by flow cytometry using FL2 rhodamine channel (Accuri Cytometers, Ann Arbor, MI, USA). The capacity of macrophages to ingest oxPL in the presence of a number of antibodies was determined after 1 h of incubation and all data were normalized to control PL uptake values that were obtained in the absence of antibodies. The macrophages were pretreated with anti-αvβ3, -αvβ5, -CD36 or -scavenger receptor type-1 antibody (all from Millipore, Billerica, MA, USA) for 1 h. The control group was treated with matching isotype (IgG1) antibody.
Lipoproteins
Human low-density lipoprotein (LDL) was isolated from plasma by density ultracentrifugation.[17] Its binding to vesicles was analyzed after separation of vesicle–lipoprotein mixture by ultracentrifugation followed by Lowry protein assay of the vesicle fraction.
Tissue/cell Processing & Microscopy
Abdominal aorta was sectioned, fixed and stained using previously described methods.[18–20] Cells were grown in glass bottomed dishes (MatTek, Ashland, MA, USA) and treated with BODIPY-LDL or DiI-acLDL (Invitrogen, Carlsbad, CA, USA) with and without vesicles. Confocal fluorescence was performed on a Zeiss LSM 510 microscope.
Nanomedicine. 2010;5(9):1341-1356. © 2010
Future Medicine Ltd.
Cite this: Detection of Macrophages via Paramagnetic Vesicles Incorporating Oxidatively Tailored Cholesterol Ester: An Approach for Atherosclerosis Imaging - Medscape - Nov 01, 2010.
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