Small-Animal PET of Tumor Damage Induced by Photothermal Ablation with ⁶⁴Cu-Bis-DOTA-Hypericin

Synthesis, Characterization, and Radiolabeling of Bis-DOTA-Hypericin

Supplemental Figure 1 (supplemental materials are available online only at http://jnm.snmjournals.org) shows the synthesis scheme for bis-DOTA-hypericin. Detailed synthetic procedures are also provided in the supplemental materials. For radiolabeling, ⁶⁴CuCl₂ (18.5–148 MBq) in 0.1 M sodium acetate (pH 5.2) was added to 12.5 μg of bis-DOTA-hypericin in water. The reaction mixture was incubated at 70°C for 45 min. The progress of the reaction was monitored by reversed-phase high-performance liquid chromatography with a radio detector.

Synthesis of CuS Nanoparticles

A detailed procedure for the synthesis of citrate-coated CuS nanoparticles in water has been described previously (9). Briefly, 1 mL of sodium sulfide solution (Na₂S, 1 M) under stirring at room temperature was added into 1,000 mL of an aqueous solution of CuCl₂ (0.1345 g, 1 mmol) and sodium citrate (0.2 g, 0.68 mmol). The pale blue CuCl₂ solution turned dark brown immediately after the sodium sulfide was added. Five minutes later, the reaction mixture was heated to 90°C and stirred for 15 min until a dark green solution was obtained. The mixture was transferred to ice-cold water. The citrate-coated nanoparticles were obtained and stored at 4°C. To introduce polyethylene glycol coating, about 1 mg of sulfhydryl-polyethylene glycol (SH–PEG) was added into the citrate–CuS nanoparticle solution (1.42 × 10¹⁵ nanoparticles in 1.0 mL of water). The reaction was allowed to proceed overnight at room temperature. Supplemental Figure 2 shows a transmission electron microscopy image and the absorption spectrum of citrate–CuS nanoparticles.

Cell Line and Animal Models

To create the BT474 breast tumor xenografts, we used 6- to 8-wk-old female nude mice (Charles River Laboratories). All animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee. The human mammary cancer BT474 cell line was obtained from the American Type Culture Collection. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) and F-12 medium (Invitrogen Corp.) supplemented with 10% fetal bovine serum (FBS) (Hyclone) and 1% penicillin–streptomycin and were incubated at 37°C with 5% CO₂ and 100% humidity. One 17β-estradiol pellet (5 mg/pellet, catalog no. SE-121; Innovative Research of America) was implanted subcutaneously in the back of each mouse 1 d before subcutaneous injection of tumor cells (1 × 10⁷ cells) in the right front flank. When the tumors reached approximately 8–10 mm in diameter, the mice were randomly divided into a treated group (TG) and nontreated group (N-TG; controls). All mice in the TG received an intratumoral injection of 10 μL of CuS nanoparticle solution (4 × 10¹³ nanoparticles/mL), and their tumors were irradiated with an NIR laser (808 nm, 12 W/cm² for 3 min) 24 h later. The mice in both groups were intravenously injected with ⁶⁴Cu-Bis-DOTA-hypericin (24 h after laser treatment in the TG).

Laser Treatment

A continuous-wave GCSLX-05-1600m-1 fiber-coupled diode laser with a center wavelength of 808 ± 10 nm was used. It was powered by a DH 1715A-5 dual-regulated power supply (15PLUS Laser; Diomed). A 5-m, 600-μm core BioTex LCM-001 optical fiber (BioTex Inc.) was used to transfer laser power from the laser unit to the target. The output power was independently calibrated using a handheld model 840-C optical power meter (Newport Corp.) and was found to be 1.5 W for a spot diameter of 4 mm (∼12 W/cm²) and a 2-amp supply current. The end of the optical fiber was attached to a retort stand using a movable clamp and positioned directly above the tumor.

In Vitro Study of PTA with CuS Nanoparticles in BT474 Cells

BT474 cells were seeded onto a 96-well plate with a density of 10,000 cells per well 1 d before the experiment. Cells were washed 3 times with Hanks balanced salt solution (Sigma-Aldrich) and then were treated with CuS nanoparticles alone (1 mM), NIR laser alone, or CuS nanoparticles followed by NIR laser 2 h after CuS nanoparticle treatment. Nontreated cells were used as a control. For laser treatment, cells were irradiated with a diode NIR laser (15PLUS Laser; Diomed) centered at 808 nm at an output power of 12 W/cm² for 3 min. The laser was coupled to a 1-m-long, 2-mm-core-diameter fiber, which delivered a circular laser beam (diameter, 4 mm), covering the central area of the microplate well. After treatment, cells were resupplied with RPMI 1640 (Invitrogen) without phenol red containing 10% FBS. Twenty-four hours later, the cells were washed with Hanks balanced salt solution and stained with calcein AM for visualization of viable cells and with EthD-1 for visualization of dead cells according to the manufacturer's suggested protocol (Invitrogen). Cells were examined under an Axio Observer.Z1 fluorescence microscope (Carl Zeiss MicroImaging GmbH). Experiments were performed in triplicate.

Cell Uptake

BT474 cells in a 12-well plate were incubated with approximately 3 × 10¹¹ CuS nanoparticles/mL in 0.2 mL of DMEM at 37°C for 2 h. Cells were washed once with phosphate-buffered saline, and then DMEM supplemented with 10% FBS without phenol red was added. Cells either were not treated with the NIR laser or were irradiated at a dose of 12 W/cm² for 3 min. After irradiation, the cells were cultured with fresh complete DMEM supplemented with 10% FBS for 24 h. Each experiment was performed in triplicate. The cells were scraped and transferred into 2-mL tubes and coincubated with 0.37 MBq/μL (10 μCi/μL) of ⁶⁴Cu-bis-DOTA-hypericin at 37°C for 1 h. Next, 500 μL of a 75:25 mixture of silicon oil (density, 1.05; Sigma-Aldrich) and mineral oil (density, 0.872; Acros) were added into the cell suspension. The mixture was centrifuged at 14,000 rpm for 10 min. The tubes were frozen in liquid nitrogen, the bottom tips containing the cell pellet were cut off, and the cell pellets and supernatants were collected and counted for radioactivity. The data were expressed as activity ratios of the cell pellet to the medium ([cpm/ug of protein in pellet/cpm/ug of medium] × 100%).

Preparation of Small Unilamellar Vesicles

Small unilamellar vesicles were prepared as described previously (24). Briefly, rotary evaporation under reduced pressure of the chloroform phospholipid stock solution (Avanti Polar Lipids Inc.) resulted in a thin lipid film on the sides of a round-bottom flask. The lipid films were thoroughly dried to remove residual organic solvent by rotation under a vacuum for 2 h. Hydration of each lipid film was achieved by the addition of 10 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) buffer (pH 7.4) containing 2.5 mM CaCl₂ and 100 mM NaCl to give a final 5 mM concentration of phospholipid vesicles. This suspension was then thoroughly mixed for 2 h on a rotary evaporation system without a vacuum. The resultant hydrated vesicles were then stored on dry ice overnight. Before loading to the L1 chip surface, the vesicles were extruded 20 times through a 50-nm polycarbonate filter using an extrusion apparatus to give small unilamellar vesicles composed of phosphatidylserine only, phosphatidylethanolamine only, or phosphatidylcholine only or composed of a mixture of phosphatidylcholine and phosphatidylethanolamine (phosphatidylethanolamine/phosphatidylcholine, 1:1).

Surface Plasmon Resonance Measurements

A Biacore 3000 apparatus (Biacore Life Sciences) equipped with an L1 chip was used for surface plasmon resonance measurements using 10 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (pH 7.4, 100 mM NaCl), with or without 2.5 mM CaCl₂ as the running buffer. The buffer solution was filtered through a 0.22-μm filter and degassed thoroughly. The L1 chips were coated with vesicles by injecting 20 μL of 5 mM phospholipid vesicle suspension at a rate of 5 μL/min for 20 min, conditioned with 3 consecutive injections of isopropanol/50 mM NaOH (2:3, v/v), each at 30 μL/min for 1 min. Solutions of bis-DOTA-hypericin at various concentrations in the running buffer were injected over the flow cells at a flow rate of 20 μL/min for predetermined periods. The bis-DOTA-hypericin solution was then replaced with the running buffer to allow it to dissociate. Binding levels expressed as a response unit (RU) were recorded for all injections before and after the onset of dissociation. The specific binding profiles of the peptides to phosphatidylserine were obtained by subtracting the response signal of the control flow cell coated with phosphatidylcholine from the total phosphatidylserine binding signal. The analysis was performed in duplicate at concentrations ranging from 0.325 to 25 μM. A sensorgram was obtained by plotting the RU against time. An increase in RU from the injection point and a decrease in RU from the beginning of the washing step, as measures of association and dissociation, respectively, were determined and used to compare binding affinities. After each injection–dissociation cycle, the flow cell surfaces were regenerated by injection of isopropanol and 50 mM NaOH solution.

Small-Animal PET and Biodistribution

An R4 microPET unit (Siemens Medical Solutions USA, Inc.), which has an approximate resolution of 2 mm in each axial direction, was used for imaging. After a tail vein injection of ⁶⁴Cu-bis-DOTA-hypericin (5.55–7.4 MBq/0.1 mL [150–200 μCi/0.1 mL]), a 20-min prone scan was obtained at 2, 6, and 24 h after injection. Mice were maintained under anesthesia with 1%–2% isoflurane, and a heating lamp was used to maintain their body temperature during acquisition. Small-animal PET images were reconstructed with the ordered-subsets expectation maximization algorithm using 16 subsets and 4 iterations.

Irregular 3-dimensional regions of interest were manually drawn around the edge of the tumor activity by visual inspection using ASIpro VM software (Analysis Tools and System Setup/Diagnostics Tool; Siemens Medical Solutions USA). Separate regions of interest were drawn for each scan because the tumor size tended to change 24 h after treatment. The mean activities within the regions of interest were recorded. Assuming a tissue density of 1 g/mL, the regions of interest were converted to MBq/g and then divided by the administered activity to obtain a percentage injected dose per gram of tissue (%ID/g) derived from the imaging region of interest.

Immediately after the last imaging session at 24 h after injection, the mice were killed. Blood, heart, liver, spleen, kidney, lung, stomach, intestine, muscle, bone, brain, and tumor tissues were removed and weighed, and radioactivity was measured with a Cobra γ-counter (Packard Cobra). The uptake of ⁶⁴Cu-labeled hypericin in the various organs was calculated as the %ID/g.

Histopathology and Autoradiography

Of each tumor collected at 24 h after injection, one half was used to measure the radioactivity count and the other half was snap-frozen with optimum-cutting-temperature compound (Sakura Finetek) and sectioned into 6–8 consecutive 5-μm slices. One slice was stained with hematoxylin and eosin (H&E), and the other slices were stained for autoradiography. For autoradiography, the slices were dried at 40°C in open air. The sections were then photographed and exposed on BAS-SR 2025 Fuji phosphorous film, and the film was scanned with the FLA5100 Multifunctional Imaging System (Fujifilm Medical Systems USA).

Statistical Analysis

Group variation was described as the mean ± SD. Statistical analyses were performed to compare the uptake at different time points in the TG and N-TG at 2, 6, and 24 h. Means were compared using 1-way ANOVA. A P value of less than 0.05 was assigned statistical significance.