Aluminum Fluoride-18 Labeled Mannosylated Dextran: Radiosynthesis and Initial Preclinical Positron Emission Tomography Studies

Materials

The mannosylated and cysteinylated dextran derivative D10CM (21.3 kDa) was synthesized with a method similar to that previously described [3, 5, 6]. The compound 2,2′-(7-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl) diacetic acid (NOTA-NHS, N-hydroxysuccinimide-activated ester of NOTA) was purchased from CheMatech (Dijon, France). All other chemicals were purchased from commercial sources.

Preparation of NOTA-D10CM

NOTA-NHS ester (117 mg) dissolved in 2 mL of dimethyl sulfoxide was added to a solution of D10CM (100 mg) in 4 mL of borate buffer (0.1 M, pH 9.0). The reaction mixture was stirred at room temperature for 18 hours and then transferred to an ultrafiltration cell (model 8050, Millipore Corp, Bedford, MA) fitted with an ultrafiltration membrane (YM03). The volume was adjusted to 50 mL with deionized water, and the solution was then concentrated to 5 mL by applying nitrogen gas under pressure directly to the ultrafiltration cell. The procedure was repeated four times, and the residue was finally lyophilized to give the product as a white solid (100 mg).

Preparation of AlCl3 Stock Solution in Acetate Buffer

The stock solution of AlCl3 (2 mM) was prepared in acetate buffer (pH 4.0, 1 M). The acetate buffer was prepared by dissolving sodium acetate (8.2 g, 100 mmol) in TraceSELECT water. The pH was carefully adjusted to 4.0 with acetic acid and magnetic stirring, and the final volume was adjusted to 100 mL. Aluminum chloride hydrate (AlCl3 6.5H2O, 50.1 mg, 200 μmol) was added to the acetate buffer. The stock solution was divided into aliquots and stored at 4°C.

Radiosynthesis of Al[18F]F-NOTA-D10CM

Radiosynthesis was performed with a custom-made remote-controlled device (DM Automation, Nykvarn, Sweden), as previously reported [7]. [18F]Fluoride was produced in an 18O(p,n)18F nuclear reaction with a cyclotron using H2[18O]O as the target and extracted into an anion-exchange cartridge (Chromafix 30 PS-HCO3−, Macherey-Nagel, Düren, Germany; preconditioned with 1 mL of ethanol followed by 1 mL of water). Subsequently, physiological saline (9 mg/mL, 220 μL) was used to elute [18F]fluoride into the reaction vial prefilled with NOTA-D10CM (1.5 mg) in 50 μL TraceSELECT water (Honeywell, Morristown, NJ), ascorbic acid (150 mM, 40 μL), AlCl3 (2 mM) in sodium acetate buffer (pH 4.0, 1 M, 25 μL), and acetonitrile (50 mM, 60 μL). The mixture was heated at 100°C for 13 minutes and cooled down to 40°C before addition of water (810 μL) containing 0.1% trifluoroacetic acid (TFA) and ascorbic acid in water (150 mM, 90 μL). The product was purified using high-performance liquid chromatography (HPLC) equipped with a semipreparative C18 Jupiter Proteo column (250 × 10 mm, 4 μm, 90 Å; Phenomenex, Torrance, CA) with mobile phases of water containing 0.1% TFA (solvent A) and acetonitrile containing 0.1% TFA (solvent B). From 0 to 1 minute, the flow rate was increased from 1 to 4 mL/minute with 5% solvent B and 95% solvent A. The solvent B content was increased to 30% over 1–20 minutes, and the fraction of Al[18F]F-NOTA-D10CM was collected to a vial prefilled with water (30 mL) and ascorbic acid (150 mM, 400 μL). The product was extracted into a preconditioned (5 mL of ethanol followed by 10 mL of water) tC18 cartridge (Waters, Milford, CT), which was subsequently rinsed with 5 mL of water. Then, the final product was eluted out from the tC18 cartridge with 1 mL of 30% ethanol (300 μL ethanol, 100 μL 150 mM ascorbic acid in water, and 600 μL water) into the end product bottle, which was prefilled with 15 mM ascorbic acid in saline (1.5 mL), and 0.1 M phosphate-buffered saline (PBS) was then used to adjust the pH to make it suitable for animal studies.

Quality Analysis of Al[18F]F-NOTA-D10CM

The radiochemical purity of the end product sample (0.3-0.8 MBq) was determined using an HPLC system (Hitachi; Merck, Darmstadt, Germany) equipped with a Jupiter Proteo column (reversed-phase C18, 150 × 4.6 mm, 300 Å; Phenomenex) and a radioactivity detector (Radiomatic 150TR flow-through, Packard, Meriden, CT). Solvent A was water containing 0.1% TFA, and solvent B was acetonitrile containing 0.1% TFA. In the elution protocol, the content of solvent B was increased from 8% to 40% over 0–9 minutes and then maintained at 40% over 9–10 minutes at a flow rate of 1 mL/minute. The stability of Al[18F]F-NOTA-D10CM in the final product formulation buffer was assessed by HPLC analysis of aliquots taken at 1, 2, 3, and 4 hours after the end of synthesis (EOS).

LogD Measurement

PBS (600 μL) and 1-octanol (600 μL) were added to a 1.5 mL Eppendorf tube followed by the addition of 5 kBq of Al[18F]F-NOTA-D10CM. The mixture was vortexed for 5 minutes, and then the water and organic phases were separated by centrifugation at 14,100 ×g for 3 minutes at room temperature. A 400 μL aliquot was taken from both phases for gamma counting (Wizard 3″, PerkinElmer/Wallac, Turku, Finland). The measurements were performed in triplicate. The LogD value was calculated using the formula: \(logD=_\left(\frac\right)\).

Determination of Molar Activity

The molar activity was determined by means of HPLC analysis using a wavelength of 220 nm under UV detection. Five NOTA-D10CM samples at concentrations of 1, 2, 4, 6, and 8 μM were analyzed in triplicate and used to generate a calibration curve. The HPLC method was the same as that described above for quality control. Then, 50 μL samples of radioactivity-decayed final product were analyzed by HPLC, and the concentration (c, nmol/mL) of the product was calculated using the calibration curve. Finally, the molar activity at EOS was calculated as follows:

$$_m=\frac\right)\times volume\ (mL)\ of\ final\ product\ at\ EOS}$$

PET Studies

Healthy male Sprague-Dawley rats (n = 6, body weight 268.3 ± 31.8 g, age 7–8 weeks) and smaller sized rats of the same strain for the in vivo blocking study (n = 4, body weight 94.9 ± 3.7 g, age 4 weeks) were obtained from the Central Animal Laboratory, University of Turku, Turku, Finland. The PET/computed tomography (CT) imaging was acquired using an Inveon Multimodality scanner (Siemens Medical Solutions, Knoxville, TN). Rats were anesthetized with isoflurane (4-5% for induction and 1.5-2% for maintenance), and the tail vein was cannulated. A CT scan was first performed for attenuation correction and anatomical reference, and then a 60 minute PET acquisition was started at the time of intravenous injection of Al[18F]F-NOTA-D10CM (51.6 ± 1.8 MBq, 431.1 ± 233.4 μL). To perform the blocking experiment, 100-fold molar excess of mannan from Saccharomyces cerevisiae (Sigma-Aldrich M7504)) in saline (400 μL) was injected 15 minutes before the Al[18F]F-NOTA-D10CM injection. PET data obtained in a list-mode were reconstructed with an ordered subsets expectation maximization 3-dimensional (OSEM-3D) algorithm into 30 × 3 s, 9 × 10 s, 4 × 30 s, 5 × 60 s, and 10 × 300 s time frames.

After the PET/CT imaging, animals were euthanized, and various tissues were collected, weighed, and measured with a gamma counter (Triathler 3″, Hidex, Turku, Finland). The radioactivity of the excised tissues was expressed as a percentage of the injected radioactivity dose per gram of tissue (%ID/g), corrected for the radioactivity remaining in the cannula and tail.

PET/CT image analysis was performed using Carimas 2.10 software (Turku PET Centre, Turku, Finland, www.turkupetcentre.fi/carimas/). Regions of interest (ROIs) were manually defined in main organs using CT for anatomical reference. Time-activity curves were extracted from the 60 minute PET data and expressed as standardized uptake value (SUV) versus time after injection.

All animal experiments were approved by the national Project Authorization Board in Finland (license number ESAVI/43134/2019) and carried out in compliance with the EU Directive 2010/EU/63 on the protection of animals used for scientific purposes.

In Vivo Stability of Al[18F]F-NOTA-D10CM

Healthy male Sprague-Dawley rats (n = 6, body weight 457.5 ± 15.5 g, age 15 weeks) were intravenously injected with Al[18F]F-NOTA-D10CM (dose 49.1 ± 2.1 MBq), and blood samples were collected into heparinized tubes at 5, 15, 30, 45 and 60 minutes (n = 3-4 for each time point) postinjection, using the tail-cut method. Plasma and cells were separated by centrifugation (5 minutes at 14,000 ×g at 4°C) and gamma counted (3″ NaI system, Triathler, Hidex Oy, Turku, Finland). Plasma proteins were precipitated with 10% sulfosalicylic acid and pelleted by centrifugation (2 minutes at 14,000 ×g at room temperature). The radioactivity concentration of the supernatant and the pellet were determined by gamma counting. The plasma supernatant was filtered through a 0.45 μm Minispike filter (Waters Corporation, Milford, MA), diluted with 0.1% TFA in water to 1 mL, and analyzed by radio-HPLC with a C18 Jupiter Proteo semipreparative column (Phenomenex, 250 × 10 mm, 5 μm, 90 Å) and conditions of 0.1% TFA in water (solvent A) and 0.1% TFA in acetonitrile (solvent B), a gradient of 5% B from 0-3 minutes gradually increasing from 5% to 60% B until 15 minutes, and a 5 mL/minute flow rate.

Immunohistochemical Staining

Liver, spleen, and bone marrow samples were formalin-fixed, paraffin-embedded, and cut into 4-μm sections. The slides were deparaffinized and rehydrated, followed by antigen retrieval (citrate buffer, pH 6.0, 20 minutes in pressure cooker). After washing (Tris-HCl 0.05 M, pH 7.6 with 0.05% Tween 20) and blocking endogenous peroxidase (3% hydrogen peroxidase), the slides were incubated with the anti-mannose receptor (CD206/MRC1) antibody (ab64693, working dilution 1:10000; Abcam, Cambridge, UK) for 60 minutes at room temperature. After washes, the slides were incubated with BrightVision horseradish peroxidase conjugated goat anti-mouse secondary antibody (DPVR110HRP; WellMed, Duiven, The Netherlands) for 30 minutes at room temperature. After 3,3-diaminobenzidine reaction (BrightDAB, BS04-110; WellMed), the sections were counterstained with Mayer’s hematoxylin, and mounted with Pertex. The slides dried overnight were scanned with a digital slide scanner (Pannoramic P1000, 3DHistech Ltd., Budapest, Hungary).

Statistical Analysis

Results are expressed as mean ± standard deviation (SD). The significance of differences between the groups was determined with unpaired Student’s t tests. P-values <0.05 were considered statistically significant.

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