In vitro stability of 111In-DOTA-EB-cRGDfK was evaluated by incubation with normal saline (in 1:1 vol ratio) or rat plasma (in 1:19 vol ratio) at room temperature. The radiochemical purity was determined by iTLC analysis as previously described [43 (link)]. At desired times (0, 2, 4, 24, 48, 72, and 96 h), 1 μL was using for iTLC on the glass microfiber chromatography paper impregnated with a silica gel (Agilent Technologies, Santa Clara, CA, USA), whereas the mobile phase was used as 0.1 M citric acid/0.1 M sodium citrate (v/v = 2.1/7.9) buffer. Then, the sheets were measured using a radioactive scanner (AR-2000 radio-TLC Imaging Scanner, Bioscan, France).
Silica gel
Manufactured by Agilent Technologies
Sourced in United States
Silica gel is a desiccant material composed of porous, amorphous silicon dioxide. It is a commonly used adsorbent material in various laboratory and industrial applications due to its ability to adsorb moisture and other polar compounds. Silica gel has a high surface area and a porous structure, allowing it to effectively remove water and other polar contaminants from gas or liquid streams.
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Lab products found in correlation
Itlc sg glass microfiber chromatography paper, by Agilent Technologies (3 mentions)
Glass microfiber chromatography paper, by Agilent Technologies (3 mentions)
Ar 2000 radio tlc imaging scanner, by Bioscan (1 mentions)
Zeba desalting column, by Thermo Fisher Scientific (1 mentions)
Df bz ncs, by Macrocyclics (1 mentions)
6540 q tof, by Agilent Technologies (1 mentions)
1260 hplc system, by Agilent Technologies (1 mentions)
Cholesteryl palmitate, by Merck Group (1 mentions)
α cholestanol, by Merck Group (1 mentions)
Crc 25r, by Mirion Technologies (1 mentions)
Iodogen, by Merck Group (1 mentions)
Amicon ultra, by Merck Group (1 mentions)
Sodium acetate, by Thermo Fisher Scientific (1 mentions)
Edta ph 7, by Merck Group (1 mentions)
Psma 617, by Horiba (1 mentions)
12 protocols using silica gel
1
Evaluating In Vitro Stability of 111In-DOTA-EB-cRGDfK
2
Radiochemical Purity and Stability of 89Zr-CD133 IgG
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89Zr-CD133 IgG was tested for radiochemical purity and stability with radio-instant thin layer chromatography (radio-iTLC). The radiotracer was incubated in phosphate-buffered saline (PBS) at 37°C for indicated durations. Radio-iTLC was then performed using 50 mM ethylene diamine tetraacetic acid (EDTA, pH 5.5) as eluent on an iTLC-SG glass microfiber chromatography paper infused with silica gel (Agilent Technologies, CA). Under these conditions, an intact radiolabeled antibody remained at baseline while free 89Zr4+ ions and [89Zr]-EDTA migrated to the solvent front.
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89Zr-CD133 IgG was tested for radiochemical purity and stability with radio-instant thin layer chromatography (radio-iTLC). The radiotracer was incubated in phosphate-buffered saline (PBS) at 37°C for indicated durations. Radio-iTLC was then performed using 50 mM ethylene diamine tetraacetic acid (EDTA, pH 5.5) as eluent on an iTLC-SG glass microfiber chromatography paper infused with silica gel (Agilent Technologies, CA). Under these conditions, an intact radiolabeled antibody remained at baseline while free 89Zr4+ ions and [89Zr]-EDTA migrated to the solvent front.
3
Radiolabeling of Ontuxizumab with Zirconium-89
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89Zr labeling was performed according to previously published methods [25 , 26 (link)]. Briefly, Ontuxizumab (5 mg/ml) was conjugated to isothiocyanatobenzyl-desferrioxamine-NCS (Df-Bz-NCS) (Macrocyclics, Dallas, TX) at 1:10 (Antibody) Ab to chelate ratio for 1 hour at 37°C in the presence of 0.1 M sodium carbonate buffer (pH 9). The unreacted chelate was removed utilizing Zeba desalting columns (40,000 MW cut off, 0.5 mL volume, ThermoFisher Scientific, Rockford, IL). The Ab-Df-Bz-NCS conjugate was combined with 89Zr oxalate at 1:1 or 1:5 mg/mCi and pH 7.1. The mixture was incubated for one hour at 37°C. The labeling efficiency was confirmed by radio-iTLC on glass microfiber chromatography paper impregnated with silica gel (Agilent Technologies, Lake Forest, CA) with 50 mM diethylene triamine pentaacetic acid (DTPA) eluent solvent and a Bioscan AR- 2000 radio-TLC scanner equipped with a 10% methane/argon gas supply. The specific activity obtained was 1-5 μCi/μg with labeling efficiency of 98-100%; therefore, the compounds were used with no further purification.
4
Instant Thin Layer Chromatography Analysis
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The RCP was determined by instant thin layer chromatography (ITLC) using a Scan-RAM radio-TLC scanner (LabLogic) and Laura software (LabLogic, Version 6.0.3). ITLC analyses were performed on dried ITLC-SG Glass microfiber chromatography paper coated with silica gel (Agilent Technologies, Folsom, CA 95,630). The radiotracers were analyzed by a retention factor (Rf), reflecting the migration distance of the compound relative to the spotting line.
5
NMR and Mass Spectrometry Analysis of Deuterated Tocopherols
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1H NMR and 13C NMR spectra were obtained in chloroform-d on a Bruker DRX 600 spectrophotometer using TMS as the
internal standard. Mass spectra were acquired on a 6540 Q-TOF (Agilent).
Column chromatography was performed using silica gel (Qingdao Marine
Chemical Co., Ltd., China). The purity of target compounds was determined
using an Agilent 1260 HPLC system which was equipped with an autoinjector,
a binary pump, and a diode array detector. The chromatographic separation
was achieved on a Diamonsil-C18 column (250 × 4.6 mm, 5 μm).
The mobile phase system consisted of A (water) and B (acetonitrile).
The linear gradient program was set as follows: time (min)/% of mobile
phase B: 0/5; 60/90 forD3-T ∼D6-T (0/30; 60/95 for D7-T ∼D12-T ). The
detection wavelength and flow rate were 210 nm and 1 mL/min, respectively.
All commercially available reagents were used without further purification
unless otherwise stated. The progress of the reactions was monitored
by analytical thin-layer chromatography (TLC) performed on homemade
HSGF254 precoated silica gel plates. Visualization was performed by
UV or development using vanillin solution in sulfuric acid and ethanol
(4/1 v/v). Yields were reported after chromatographic purification.
internal standard. Mass spectra were acquired on a 6540 Q-TOF (Agilent).
Column chromatography was performed using silica gel (Qingdao Marine
Chemical Co., Ltd., China). The purity of target compounds was determined
using an Agilent 1260 HPLC system which was equipped with an autoinjector,
a binary pump, and a diode array detector. The chromatographic separation
was achieved on a Diamonsil-C18 column (250 × 4.6 mm, 5 μm).
The mobile phase system consisted of A (water) and B (acetonitrile).
The linear gradient program was set as follows: time (min)/% of mobile
phase B: 0/5; 60/90 for
detection wavelength and flow rate were 210 nm and 1 mL/min, respectively.
All commercially available reagents were used without further purification
unless otherwise stated. The progress of the reactions was monitored
by analytical thin-layer chromatography (TLC) performed on homemade
HSGF254 precoated silica gel plates. Visualization was performed by
UV or development using vanillin solution in sulfuric acid and ethanol
(4/1 v/v). Yields were reported after chromatographic purification.
6
Lipid Extraction Protocol
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All chemicals and reagents were obtained from Sigma-Aldrich Chemical Co., Ltd. (Milan, Italy). The isooctane (2,2,5 tri-methyl-pentane), n-heptane, n-hexane, and diethyl ether were of analytical grade. Internal standards α-cholestanol and cholesteryl palmitate were purchased from Sigma-Aldrich (Milan, Italy). Internal standards were prepared weekly using n-heptane as the diluting solvent. All solutions were stored at 4 °C. Silica gel (pore size 60 Å, 0.04–0.015 µm particle size) was purchased from Agilent (Milan, Italy).
7
Radiolabeling of Polymersomes with I-124
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Two milligrams of Iodogen (Sigma-Aldrich, Spain) were dissolved in 10 mL of CH2Cl2 and 20 μL of this solution was transferred to a tube and the solvent was evaporated. LinTT1-Tyr-polymersomes or Tyr-polymersomes (1mg) were mixed with Na124I (18.5MBq) and 10 μL of buffer phosphate 0.5 M in a tube containing Iodogen. After 30 min 250 μL of phosphate buffer, 1M NaCl, pH 7.4 was added to the reaction and the solution was transferred to a tube containing 50 μL of Na2S2O3 0.1 M. The radiolabeling yield was measured by TLC using glass microfiber chromatography paper impregnated with silica gel (Agilent Technologies, USA) and ethanol:water 85:15 as mobile phase. The radioactivity of the peaks was measured with a TLC reader (γ-MiniGITA, Raytest, Germany). The polymersomes were purified using centrifugal filters of 100kDa MWCO (Amicon Ultra, Merck Millipore. Ltd. Ireland) and resuspended in 0.1mL of PBS. The removal of the free 124I was confirmed by radio-TCL and the final radioactivity was measured with a dose calibrator (Capintec CRC-25R, USA).
8
In vitro stability of [89Zr]Zr-DFO-PS
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The in vitro stability of [89Zr]Zr-DFO-PS was studied in PBS pH 7.4, simulated gastric fluid pH 3.0 (SGF; Ricca Chemical Company, Arlington, TX, USA), and simulated intestinal fluid pH 6.0 (SIF; Ricca Chemical Company, Arlington, TX, USA) in triplicate. The radiolabeled PS particles (0.05 mL) were mixed with 1 mL of PBS, SGF, or SIF. At predetermined timepoints, iTLC was performed to determine the amount of free [89Zr]Zr4+ using a glass microfiber chromatography paper impregnated with silica gel (Agilent, Inc.) and an eluent of 50 mM EDTA pH 5.5.
9
Radioactive Labeling and TLC Analysis
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iTLC analysis were performed using dried iTLC-SG Glass microfiber chromatography paper impregnated with silica gel (Agilent Technologies, Folsom, CA 95630).
Detection of the radioactivity were obtained on a miniGITA scanning device (Raytest, Straubenhard, Germany) using the Gina star software after manual integration of the peaks. In this system, the [177Lu]Lu-1C1m-Fc remain at Rf = 0 while the unbound 177Lu migrate to the solvent front.
Detection of the radioactivity were obtained on a miniGITA scanning device (Raytest, Straubenhard, Germany) using the Gina star software after manual integration of the peaks. In this system, the [177Lu]Lu-1C1m-Fc remain at Rf = 0 while the unbound 177Lu migrate to the solvent front.
10
Radiolabeling of Fc-Conjugated Antibody
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64Cu dichloride (64CuCl2) in 0.1 N HCl solution was produced by the ARRONAX cyclotron (Saint Herblain, France). A calculated volume of sodium acetate 2.5 M metal-free (Alfa Aesar, Haverhill, MA, USA) was first added to the 64CuCl2 solution, followed by a calculated volume of 5 mg/mL DOTA-conjugated 1C1m-Fc in acetate buffer 0.1 M. After 30 min incubation at 42 °C, 1 mM EDTA pH 7.0 (Sigma–Aldrich, St. Quentin Fallavier, France) was added to obtain a final concentration of 0.01 mM to complex free 64Cu(II).
The radiochemical purity of [64Cu]Cu-1C1m-Fc was determined by instant thin layer chromatography (iTLC). The release criterium was ≥95%. iTLC analyses were performed using dried iTLC-SG glass microfiber chromatography paper impregnated with silica gel (Agilent Technologies, Folsom, CA 95630). Citrate buffer (0.1 M, pH 4.5) was used as eluent. In this system, [64Cu]Cu-1C1m-Fc remains at Rf = 0, while unbound [64Cu]Cu-EDTA migrates to the solvent front (Rf = 1).
The radiochemical purity after antibody radiolabeling was assessed by iTLC-SG just after radiolabeling and 24 h after.
The radiochemical purity of [64Cu]Cu-1C1m-Fc was determined by instant thin layer chromatography (iTLC). The release criterium was ≥95%. iTLC analyses were performed using dried iTLC-SG glass microfiber chromatography paper impregnated with silica gel (Agilent Technologies, Folsom, CA 95630). Citrate buffer (0.1 M, pH 4.5) was used as eluent. In this system, [64Cu]Cu-1C1m-Fc remains at Rf = 0, while unbound [64Cu]Cu-EDTA migrates to the solvent front (Rf = 1).
The radiochemical purity after antibody radiolabeling was assessed by iTLC-SG just after radiolabeling and 24 h after.
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