G 25 medium

Urine collection was performed before treatment after a written informed consent of participants in accordance with the protocol approved by the Ethical Committee (Record No12 from 17 November 2016) of V.I. Kulakov’s National Medical Research Center of Obstetrics, Gynecology and Perinatology.
Urine samples were centrifuged (2000 g, 10 min, 4 °C) within 20 min after collection, and the supernatant was stored at −80 °C. The peptide fraction was obtained as described earlier [45 (link),49 (link)]. Particularly, 1.5 mL of urine was diluted with 3 volumes of denaturing buffer (4M urea, 20 mM ammonium hydroxide, 0.2% sodium dodecyl sulfate), transferred to Vivaspin-4 10 kDa MWCO (Sartorious) filters and centrifuged at 4000 g for 20 min at room temperature. The filtrate (2.5 mL) was further subjected to gel-filtration on a PD-10 Column (GE Healthcare; Sephadex™ G-25 Medium, equilibration and elution with 0.01% ammonium hydroxide). An amount of 2 mL of the eluate was lyophilized and dissolved in 100 µl of deionized water prior to analysis.

The non-encapsulated drugs were removed from the liposomes after passage through a gel filtration chromatography column (GE Healthcare, UK), with 5 kDa cut-off (PD-10 Desalting columns containing 8.3 mL of Sephadex™ G-25 Medium) and eluted with PBS buffer for all liposome. The concentration of MTX and TAM encapsulated was measured by proton nuclear magnetic resonance (¹H NMR) using a Bruker Avance III Instrument, operating at 400 MHz (Guimarães et al., 2019 (link)). Powder liposomes containing drug were dissolved in deuterium oxide (for MTX) or deuterated chloroform (for TAM) to determine the amount of drug in the liposomal formulation. Pyridine was used as internal standard. Quantification of DOX was evaluated by UV–vis spectrophotometry measuring the absorbance at 490 nm. UV–vis spectra of liposomes encapsulated DOX were recorded on spectrophotometer BioTek Synergy™ HT using a plastic microplate. The final DOX concentration was determined based on the respective calibration curve.
The drug leakage was determined as follows:

Liposomes composed of DOPE/Cholesterol/DSPE-mPEG (54:36:10, molar ratio) [21 (link)] were produced by a pre-concentration ethanol injection method [24 (link)]. Briefly, the lipid components were dissolved in ethanol (20% of the final volume) to obtain a 1:1 initial ratio of organic:aqueous phase (v/v). The organic phase was added to an aqueous phase containing the drug MTX and folate-peptide (0.75% w/v) dissolved in phosphate-buffered saline (PBS) buffer, under vigorous magnetic stirring, at 70 °C. After ethanol reduction, the liposomal suspension was diluted five times with PBS buffer. The non-encapsulated MTX and residual ethanol were removed from the liposomes after passage through a gel filtration chromatography column (GE Healthcare, Hatfield, UK), with 5 kDa cut-off (PD-10 Desalting Columns containing 8.3 mL of Sephadex™ G-25 Medium). After separation, the concentration of the encapsulated MTX was determined by measuring the absorbance at 303 nm, the maximum wavelength of MTX in PBS. The data were recorded on spectrophotometer BioTek Synergy™ HT (Winooski, VT, USA) using a quartz microplate.