The radiochemical purity of [99mTc]Tc-NLCS-IMT formulations was assessed by RTLC (Bioscan AR 2000, Bioscan, Inc., Washington, DC) up to 6 h. Instant thin layer chromatography-silica gel coated fiber sheets (ITLC-SG) was used as stationary phase. The free [99mTc]Tc and reduced/hydrolyzed (R/H) [99mTc]Tc were determined by using saline and pyridine:acetic acid:water (3:5:1.4)) as mobile phases. The radiochemical purity percentage (RP%) was calculated from the following equation (İlem-Özdemir et al., 2016 (link)):
Ar 2000
Manufactured by Bioscan
Sourced in United States, France, Canada
The AR-2000 is a versatile laboratory equipment designed for accurate and reliable analysis. It utilizes advanced spectroscopy techniques to measure and quantify various analytes in a wide range of samples. The core function of the AR-2000 is to provide precise and reproducible analytical data to support research, quality control, and other scientific applications.
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Lab products found in correlation
Itlc sg, by Agilent Technologies (3 mentions)
Gc1020 7500sl, by Mirion Technologies (2 mentions)
Pierce pre coated iodination tubes, by Thermo Fisher Scientific (1 mentions)
6 well plate, by Sarstedt (1 mentions)
Lc 10at, by Shimadzu (1 mentions)
Spectrasystem p4000, by Thermo Fisher Scientific (1 mentions)
Zetasizer nano zs90, by Malvern Panalytical (1 mentions)
Tin 2 chloride, by Merck Group (1 mentions)
P scn bn chx a dtpa, by Macrocyclics (1 mentions)
Sephadex g 25 pd 10 desalting column, by GE Healthcare (1 mentions)
Dotatoc, by Horiba (1 mentions)
Flexanalysis 3, by Bruker (1 mentions)
Silica gel impregnated glass fiber sheets, by Pall Corporation (1 mentions)
Accumet basic ab15 ph meter, by Thermo Fisher Scientific (1 mentions)
Anhydrous ch3cn, by Thermo Fisher Scientific (1 mentions)
Tlc silica gel 60 f254, by Merck Group (1 mentions)
Isomed 1010, by Mirion Technologies (1 mentions)
Fluka traceselect, by Merck Group (1 mentions)
Dotatoc, by Bachem (1 mentions)
Sodium citrate, by Merck Group (1 mentions)
36 protocols using ar 2000
1
Radiochemical Purity Assessment of [99mTc]Tc-NLCS-IMT Formulations
2
Validating JAM-A mAb/IR700 Binding Assay
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As a quality control of the JAM-A mAb/IR700, the levels of immune-binding of intact JAM-A mAb and JAM-A mAb/IR700 conjugates to alive PC3pip cells in vitro were assessed. First, JAM-A mAb and JAM-A mAb/IR700 conjugates were labeled with 125I Na using a direct protein iodination. Briefly, aliquots of 250-μCi 125I Na (PerkinElmer, Akron, OH) in Tris-Iodination Buffer were activated in the Pierce Pre-Coated Iodination Tubes (Thermo Scientific, Rockford, IL) followed by mixing with 40-μg of JAM-A mAbs or IR700 conjugates per tube. After incubation with Scavenging Buffer, mixes were purified with Zeba spin desalting columns at 1000 g for 2-min and elution fractions were collected. Next, for immune-binding assay, the purified radiolabeled JAM-A mAbs or IR700 conjugates were mixed with alive culture of PC3pip cells for 1-h at 4 °C followed by washing and radioactive scan of the collected pellets (Bioscan AR 2000, Bioscan Inc., Washington, DC). PC3pip cells that were blocked by an excess of JAM-A mAb prior these procedures were used as internal controls.
3
Inhibition of COX-1 and COX-2 in Intact Cells
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We evaluated
the ability of test compounds1–7 to inhibit COX-1
and COX-2 in intact cells using a published procedure.24 (link) Briefly, both OVACR3 cells and 1483 HNSCC cell
lines were plated in 6-well plates (Sarstedt) and grown to 60% confluence.
Serum-free medium (2 mL) was added per well with 0, 0.04, 0.2, 0.5,
1.0, and 5.0 μM final concentration of FDF for 30 min at 37
°C. The final concentration of DMSO was 0.5% in all wells. The
medium (1 mL) was removed, and [1-14C]-arachidonic acid
(57 mCi/mmol, 2.11 GBq/mmol, NEC661, PerkinElmer)
was added to provide a 4 μM final concentration. At the end
of the 30 min, 37 °C incubation, an aliquot of the medium (0.4
mL) was removed from each well and added to a 0.6 mL ice-cold termination
solution (Et2O/CH3OH, 1 mol/L citrate 30:4:1,
pH 4). The organic layer was spotted on 20 × 20 TLC plates, developed,
and quantitated on the Bioscan AR-2000 for the remaining arachidonic
acid substrate compared to the vehicle-only cells.
the ability of test compounds
and COX-2 in intact cells using a published procedure.24 (link) Briefly, both OVACR3 cells and 1483 HNSCC cell
lines were plated in 6-well plates (Sarstedt) and grown to 60% confluence.
Serum-free medium (2 mL) was added per well with 0, 0.04, 0.2, 0.5,
1.0, and 5.0 μM final concentration of FDF for 30 min at 37
°C. The final concentration of DMSO was 0.5% in all wells. The
medium (1 mL) was removed, and [1-14C]-arachidonic acid
(57 mCi/mmol, 2.11 GBq/mmol, NEC661, PerkinElmer)
was added to provide a 4 μM final concentration. At the end
of the 30 min, 37 °C incubation, an aliquot of the medium (0.4
mL) was removed from each well and added to a 0.6 mL ice-cold termination
solution (Et2O/CH3OH, 1 mol/L citrate 30:4:1,
pH 4). The organic layer was spotted on 20 × 20 TLC plates, developed,
and quantitated on the Bioscan AR-2000 for the remaining arachidonic
acid substrate compared to the vehicle-only cells.
4
TLC Analysis of Radiolabeled Metabolites
5
In-111 Production and Characterization
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In-111 was produced at the Agricultural, Medical and Industrial Research School (AMIRS), 30 MeV cyclotron (Cyclone-30, IBA) using natCd(p,x)111In reaction. Natural cadmium sulfate with a purity of more than 99% was obtained from Merck Co. Germany. All chemicals were purchased from Sigma-Aldrich Chemical Co. U.K. Radio-chromatography was performed by Whatman paper using a thin layer chromatography scanner, Bioscan AR2000, Paris, France. Analytical HPLC to determine the specific activity was performed by a Shimadzu LC-10AT, armed with two detector systems, flow scintillation analyzer (Packard-150 TR) and UV-visible (Shimadzu) using Whatman Partisphere C-18 column 250 × 4.6 mm (Whatman Co. NJ, USA). Calculations were based on the 172 keV peak for In-111. All values were expressed as mean ± standard deviation (Mean ± SD) and the data were compared using student T-test. Animal studies were performed in accordance with the United Kingdom Biological Council's Guidelines on the Use of Living Animals in Scientific Investigations, second edition.
6
Radiolabeling for Molecular Imaging
7
Preparation and Characterization of 188Re-Liposomes
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The procedure of 188Re-liposomal preparation has been reported before [23 (link)]. In brief, 188Re was milked from the 188W/188Re generator system (Institute National des Radioelements, Fleurus, Belgium) and conjugated with sodium perrhenate. Moreover, 188Re was conjugated with N,N-bis(2-mercapatoethly)-N′,N′-diethylenediamine (BMEDA, ABX GmbH, Radeberg, Germany), and the quality of 188Re-BMEDA was validated by using the instant thin-layer chromatography (iTLC) followed by a radioactive scanner (Bioscan AR2000; Bioscan, TriFoil Imaging Inc., Chatsworth, CA, USA). Furthermore, PEGylated liposome (NanoX; Taiwan Liposome Co. Ltd., Taipei, Taiwan) was used to encapsulate 188Re-BMEDA and eluted using the PD-10 column (GE Health BioSciences, Pittsburgh, PA, USA) (Supplementary Figure S1 ). The average molecular weight of polyethylene glycol (PEG) was 2000. The particle size (84.6 ± 4.12 nm) and surface charge (1.1 ± 1.9 mV) were measured by the dynamic light scattering apparatus (Zetasizer Nano ZS90, Malvern Panalytical Ltd., Malvern, UK). The in vitro stabilities of 188Re-liposome in normal saline and rat plasma were, respectively, over 92% and 82% in 72 h as reported before [20 (link)].
8
Radioactive Labeling of Extracellular Vesicles
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Immediately before use, 5 mg of sulfosuccinimidyl-3-(4-hydroxypheynyl) propionate (sulfo-SHPP, ThermoFisher, Rockford, IL, USA) was dissolved in 1 mL of PBS. 100 µL of sulfo-SHPP were added to 100 µL of EVs (1 mg/mL) and incubated in 4° C for 3 h with gentle mixing. The sample was centrifuged at 100,000 × g in 4° C for 60 min and the pellet was resuspended in 100 µL of PBS (SHPP-EVs). Pierce Pre-Coated Iodination Tubes, formerly called “IODO-GEN”, were used for radioiodination. Pre-coated iodination tube was wet with PBS and 10 µL (37 MBq) of I-131 or I-125 was added to the tube. The tube was gently mixed for 5 min at room temperature to activate radioiodine. Then, prepared SHPP labeled EVs were added to the tube and incubated for 15 min at room temperature with gentle mixing. The sample was centrifuged at 100,000 × g in 4 °C for 60 min and the pellet was resuspended in 100 µL of PBS. Radiochemical purity was measured by instant thin-layer chromatography (ITLC) using 0.9% NaCl solution as an eluent for each column. Radioactivity of the column was counted using a TLC imaging scanner (AR-2000, Bioscan, Poway, CA, USA). Radiochemical yield was calculated as radioactivity of radioiodine labeled EVs after purification divided by radioactivity of pre-labeled radioiodine (n = 3).
9
Technetium-99m Labeling Stability
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One hundred-microliter samples of [99mTc]Tc-gluconate and [99mTc]Tc-DTPA were mixed with one hundred microliter of 0.2 mM NaHS in 0.2 M sodium phosphate buffer (pH 7.4). The mixtures were vortexed and incubated at 37 °C for 10 min, spotted onto ITLC plates, and eluted with saline. The ITLC plates were scanned using the Bio-Scan AR-2000. The insoluble fraction was analyzed by calculating the percentage of remaining radioactivity at the origin.
10
Technetium-99m Labeling of RBC-EMs
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RBC-EMs were incubated with 0.01% Tin (II) chloride (Sigma, United States) in a shaker for 5 min, technetium-99m, 99mTc (RBC-EMs, 100 μg: 99mTc, 111 MBq) was added to the RBC-EMs, and they were further incubated in the shaker for 20 min. Labeling efficiency of RBC-EMs by 99mTc was measured by instant thin-layer chromatography (TLC) using 0.9% NaCl solution as an eluent for each column, and the radioactivity of the column was detected with a radio-TLC imaging scanner (AR-2000, Bioscan, Poway, CA, United States) (Hwang et al., 2015 (link)). Stability was determined by the percent change in the radiochemical purity of 99mTc-RBC-EMs over time.
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