Glass microfiber chromatography paper

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).

Human cancer cell lines BxPC-3 and DU145 were purchased from American type tissue culture collection (ATTC via LGC Promochem, Borås, Sweden).
Metal contaminants were removed from buffers with Chelex100 Resin (Sigma Aldrich, St.Louis, MO, USA).
ITLC was used to measure the distribution of activity to determine labeling yield and stability of the radiolabeled compounds. For analysis, 1 µl of the sample was added to strips made of silica gel-impregnated glass microfiber chromatography paper (Agilent Technologies, Santa Clara, CA, USA). Citric acid (0.2 M) was used for elution. With this method, free gallium-68 will move to the front of the strips and the radiolabeled affibody will stay at the application point. Distribution of activity was measured in the Cyclone Storage Phosphor System and analyzed with OptiQuant image analysis software (Perkin Elmer, Waltham, MA, USA).
Activity in cells and organs samples was measured with an automated gamma counter with a 3-inch NaI(Tl) detector (1480 Wizard; Wallac Oy, Turku, Finland). Raw data were corrected for decay.
Statistical significance for in vitro and in vivo specificity was tested with two-tailed, unpaired t-test. Comparison of the different groups in the biodistribution was done with 1-way ANOVA and post-hoc t-test corrected for multiple comparisons with the Bonferroni method.

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.