Nucleosil c18

Glycoconjugate 5 (10 μmol) was dissolved in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (1 mL, 50 mM, pH 7.0) and mixed with UDP-Gal (20 μmol, 11.25 mg) and β4Gal-T1 (14.75 μL of a 0.54 mg mL⁻¹ solution). After degassing the solution by sonication and purging it with argon, MnCl₂ (3 μL of a 1.0 M solution in water) was added to the solution flask. The mixture was then vortex mixed followed by incubation overnight at 37 °C. The reaction mixture was directly purified by HPLC (Macherey-Nagel Nucleosil C18, 250 × 4.6 mm, 5 μm) using portion-wise (20–80 μL) addition to an analytical column and a 40 min gradient ranging from 5% to 100% acetonitrile in water (0.5 mL min⁻¹ flow rate). The eluent was monitored by UV spectroscopy (260 nm) and the product containing fractions were collected and freeze-dried to afford a 7 : 3 mixture of compounds 5 and 9.

Peptide syntheses were performed using optimized Fmoc chemistry protocols with a multichannel peptide synthesizer [52 (link)]. Side-chain deprotection and cleavage of the peptide from the solid support were performed by treatment with reagent K (88 % TFA v/v, 2 % triisopropylsilane v/v, 5 % dithiothreitol w/v, 5 % water v/v) for 150 min at 20 °C. The VEGF receptor binding peptides (VRbp) were purified by reversed-phase HPLC (RP-HPLC) using a preparative HPLC system (Waters) on a Nucleosil C18 (1 × 30 cm) column (Macherey Nagel). The elution was achieved with a linear gradient of aqueous 0.1 % TFA (A) and 0.08 % TFA in acetonitrile (B) at a flow rate of 6 mL min⁻¹ with UV detection at 230 nm. The purity of the VEGFR-binding peptide was controlled by analytical RP-HPLC (Fig. S7) and their molecular weight was assessed by LC/MS (Fig. S8). MS (ESI, m/z): 1967.7 [M+H]⁺ for CGGGGGGHRHTKQRHTALH and 1967.0 [M+H]⁺ for HRHTKQRHTALHGGGGGGC.

HPLC analysis was performed on a Nucleosil^® C18 reversed phase column (250 × 4 mm, 5 µm; Macherey Nagel, Germany), using a Dionex system consisting of a pump P580, a UV/VIS Detector UVD 170S/340S, an ASI-100 Automated Sample Injector, a termobox COLUMN OVEN LCO 101 and an In-Line Mobile Phase Degasser Degasys DG-1210 Dionex controlled with ChromeleonTM 6.11 build 490 software. HPLC conditions were as follows: 50 % acetonitrile in HPLC water (v/v), with a linear gradient to 85 % acetonitrile in 35 min, then an isocratic elution with 85 % acetonitrile for 5 min, a linear gradient from 85 % acetonitrile to 50 % acetonitrile in 5 min, followed by an isocratic elution of 50 % acetonitrile for 5 min. Detection was by UV at 254 nm. BaP metabolite peaks were collected and analysed by NMR and/or mass spectrometry as described (Stiborova et al. 2014 (link)). The structures of BaP metabolites analysed are given in Supplementary Figure 8. The metabolite peak areas were calculated relative to the peak area of the internal standard.

The free DOX in the HA-DOX was quantified by high-performance liquid chromatography (HPLC). Conjugates were dissolved in PBS (2.0 mg/mL) and purified by vivaspin (15,000 rpm, 15 min, cut-off 10 kDa). The filtrated solution was analyzed using a Shimadzu Prominence system (Shimadzu, Kyoto, Japan). A nucleosilC18 (Macherey-Nagel, Duren, Germany) (150 × 4.6 mm; particle size 5 μm) column was used to separate the desired product. Then, 0.1% of formic acid in acetonitrile and 0.1% of formic acid in water (10%, 0 min; 90%, 13–15 min; 10%, 15–20 min) were used as mobile phases and applied in gradient mode. The quantification method was set with a detection wavelength of 480 nm, a sample injection volume of 10 μL, and a flow rate of 0.6 mL/min. Free DOX content was assessed by means of a previously performed DOX calibration curve (limit of detection (LOD) 2.4 µg/mL).

Solid phase extraction method (SPE) was used to extract HETE, HODE, and HEPE from rat serum, according to the procedure, described previously by Frohberg with slight modifications [35 (link),36 (link)]. Methanol (0.5 mL) was added to samples of serum (0.4 mL) which were then diluted with water to achieve 10% methanol solution. The samples were loaded onto the SPE C18 cartridges (Backerbond C18, 500 mg/3 mL, J.T. Baker, Holland), preconditioned with methanol, and followed by water (10 mL each). Cartridges containing applied samples were washed with 2 mL of water and 2 mL of 10% methanol, then compounds to be analyzed were eluted with 100% methanol (3 × 0.5 mL). The samples were evaporated to dryness under the nitrogen stream and redissolved in 100 μL of ethanol.
Eicosanoids were determined with high performance liquid chromatography (HPLC), using Shimadzu system comprising LC-20AD pump, DGU-20A5 degasser, UV–VIS SPD-10AV detector and CTO-10 AS VP oven. The compounds studied were separated on Nucleosil C18 column (Nucleosil C18, 100-5, 250 × 4.6 mm, 5 µm, Macherey-Nagel) held at 30 °C. Samples injected to the HPLC system were eluted with a mobile phase composed of methanol, water and acetic acid (73:27:0.01, by volume), with a flow rate of 0.8 mL/min. Detection wavelength was 235 nm. The whole analysis lasted 45 min.

The analysis was done using liquid chromatography (LC, DIONEX UltiMate 3000, Thermo Scientific UK Ltd., Hemel Hempstead) on-line coupled to the ESI-QqLIT-MS/MS (QTRAP 5500, AB Sciex UK Ltd., Warrington). Samples (20 µl in 50% aqueous methanol with 1% formic acid) were separated on the reverse phase C18 column: NUCLEOSIL® C18, 100 mm, 5 µm pore size (Macherey-Nagel, Germany) using mobile phase (A) methanol: water:formic acid (70:30:0.1, v/v) and (B) isopropanol: methanol: formic acid (90:10:0.1, v/v) and a column temperature at 45 °C. Flow rate was maintained at 200 µl/min with the gradient as follows: 84% B from 0 to 7 min, 84–76% B from 7 to 11 min, 76–100% B from 11 to 25 min, 100% B 25–30 min, 100–84% B from 30 to 32 min, 84% B 32–48 min. Acquired data were processed using Analyst Software (version 1.7, AB Sciex).