Kromasil 100 5c18

The folate standards: 10-formyl-folic acid (10–CHO-PteGlu; 10–CHO–FA), 5,10-methenyl-5,6,7,8-tetrahydrofolate (5,10–CH=H₄PteGlu; 5,10–CH=THF), 5-formyl-tetrahydrofolate (5–CHO-H₄PteGlu; 5–CHO–THF), 5-methyl-tetrahydrofolate (5–CH₃-H₄PteGlu; 5–CH₃-THF), dihydrofolate (H₂PteGlu; DHF), folic acid (PteGlu; FA), tetrahydrofolate (H₄PteGlu; THF), and methotrexate (MTX) were purchased from Schircks Laboratories (Jona, Switzerland) and MeFox, an oxidation product of 5–CH₃-THF, was obtained from Toronto Research Chemicals (Toronto, Canada). Sodium phosphate dibasic (Na₂HPO₄), sodium phosphate monobasic (NaH₂PO₄), sodium ascorbate, and β-mercaptoethanol were obtained from the Sigma-Aldrich Chemical. The endogenous folates in rat serum were removed by incubation with one-tenth (w/w) of activated charcoal for 1 h on ice, followed by centrifuging at 13,000 rpm at 4°C for 30 min (Sigma 3K15, Osterode am Harz, Germany), and the supernatant was used for the following incubation experiment. Acetonitrile and formic acid (LC-MS grade) were obtained from Fisher Scientific (Geel, Belgium). The HPLC analytical column (Kromasil 100-5 C18, 2.1 × 50 mm, 2.5 μM particle size) was purchased from Akzo Nobel (Stockholm, Sweden), and an Agilent SB-C18 pre-column (2.1 × 5 mm, 2.7 μM particle size) was acquired from Agilent Technologies (California, USA).

[¹⁸F]CPFPX was synthesized in house as described previously [10 (link)]. [¹⁸F]CBX and [¹⁸F]MCBX were synthesized analogous to [¹⁸F]CPFPX with minor adjustments of the chromatographic separation procedure (HPLC column: Kromasil 100-5 C18, 250 × 10 mm (AkzoNobel, Bohus, Sweden); eluent: MeOH/H₂O/HAc 60:40:0.2 (v/v/v)). Radiochemical purity of all batches used for pharmacokinetic studies was >95%.

FDPC-NPs (0.1mM, calculated as DOX) were dissolved in PBS (pH7.4). After 50-fold dilution with PBS (pH7.4), particle sizes were measured at 0.25, 1, 3, 6, 12 h by DLS, and 10 μL of the samples were removed at each time point. TEM and DLS were used to evaluate their particle sizes and morphologies. To mimic intravascular microenvironment, FDPC-NPs (1 mM, calculated as DOX) or DOX (1 mM) was co-incubation with 2 mL of rat plasma in dialysis bag (Mw: 2000 Da) in 8mL of PBS (pH7.4) at 37 °C. At designated time points i.e. 0.5, 1, 2, 4, 6, 8 and 12 h, samples were removed from the incubator, centrifuged for 10 min at 3000 rpm and filtered. The amount of DOX was quantified using high performance chromatography (HPLC) (Shimadzu, Japan). Chromatographic study was performed at 30 °C on a reversed-phase column (Kromasil 100-5-C 18, AKZO NOBEL, Sweden). The mobile phase consisted of acetonitrile and 0.1% trifluoroacetic acid (TFA), and a gradient method was employed for the analysis (20-70% acetonitrile over 0-25 min). Detection was performed at a wavelength of 480 nm (Ultra Violet detector, Shimadzu, Japan). Specificity, linearity, recovery, precision, accuracy and solution stability were performed for validation.

All the reagents were obtained commercially and used without further purification. Thiazolidine-2,4-dione 1 and (4-oxo-2-thioxo-3-thiazolidinyl)acetic acid 3 were purchased from Sigma-Aldrich. Purity of the compounds was checked by thin layer chromatography using silica-gel 60 F254-coated Al plates (Merck) and spots were observed under UV light. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker Avance spectrometer (600 and 150 MHz, respectively) and Varian VXR-400 spectrometer at 400 MHz and 101 MHz, respectively, at 298 K in CDCl₃ or DMSO-d₆ at a concentration of samples of 5–15 mmol, with TMS as internal reference for ¹H and ¹³C NMR spectra. The signal assignments of compound 11, 12, 15, and 17 were performed using 2D spectra (DQF-COSY, ¹³C–¹H HSQC, and ¹³C–¹H HMBC); the chemical shifts are expressed in ppm (δ scale) using DMSO and CDCl₃ as an internal standard, and the coupling constants are expressed in Hz. The mass-spectral measurements were carried out by ESI method on micrOTOF-QII (Brucker Daltonics GmbH). Analytical HPLC was performed on a Shimadzu LC-20AD system using Kromasil-100-5-C18 (Akzo-Nobel) column, 4.6 × 250 mm, temperature 20 °C, UV detection, mobile phase A—0.2% HCOONH₄), mobile phase B-MeCN, (pH 7.4), and fl-1ml/min.

pHLIP (Var7) and the control sequence kVar7 (7 (link)) were synthesized by Shanghai Science Peptide Biological Technology Co., Ltd. (Shanghai, China) using solid-phase peptide synthesis. To facilitate ^99mTc labeling, four amino acids, Gly-(D)-Ala-Gly-Gly, were attached to the N-terminus of pHLIP (Var7) and kVar7 to form a strong chelating group containing an N4 structure. To prevent any spatial obstruction, gamma-aminobutyric acid (GABA) was introduced as a spacer between the N4 structure and pHLIP (Var7)/kVar7. The modified pHLIP (Var7) and kVar7 sequences were as follows:pHLIP (Var7), GaGG-GABA-ACEEQNPWARYLEWLFPTETLLLEL-NH₂; kVar7, GaGG-GABA-ACEEQNPWARYLKWLFPTKTLLLKL-NH₂.
pHLIP (Var7) and kVar7 were purified via high-performance liquid chromatography (HPLC) using a Shimadzu-LC2010 instrument (Shimadzu Corporation, Japan) equipped with a Kromasil 100-5C18 chromatographic column (AkzoNobel, Sweden) (5 µm, 4.6 × 150 mm). The peptides were eluted with a gradient from 25 to 85% solvent A (0.1% trifluoroacetic in 100% acetonitrile) and 75 to 15% solvent B (0.1% trifluoroacetic in 100% water) at 1.0 ml/minute, with monitoring at 220 nm. The identities of pHLIP (Var7) and kVar7 were confirmed by mass spectrometric analysis using an Agilent 6125B mass spectrometer (Agilent Technologies, Inc., USA).

The monosaccharide compositions of DOP-40, DOP-50, DOP-60, and DOP-70 were analyzed by HPLC equipped with a Kromasil 100-5C18 chromatographic column (4.6 × 250 nm, 5 μm, AKZO NOBEL, Sweden) [22 (link)]. The compositions of mobile phase were acetonitrile (solvent A) and 0.1 M aqueous KH₂PO₄ acetonitrile (solvent B), the flow rate was 0.8 ml/min, and the wavelength of detection was 250 nm. Five monosaccharide standards (d-mannose, l-rhamnose, d-galactose, d-glucose, and d-arabinose) were used to establish standard curves, and the monosaccharide compositions and molar ratio of DOP-40, DOP-50, DOP-60, and DOP-70 were determined.