Prominence lc 20

Protein-NP yield was determined by UV spectrophotometry (Shimadzu UV-1800, Kyoto, Japan). The typical curve of a protein dissolved in a phosphate-salt buffer (PBS) was used as a standard reference. The protein absorbance level was recorded at 280 nm. The concentrations of standards in the range from 1 to 5 mg/mL of albumin were used to create the calibration curve (standard linear curve equation y = 0.0198x − 0.0003 (R value = 0.9973)). This formula was applied to the yield calculating.

Protein-HU-NPs were collected by centrifugation using Amicon filters (Cedarlane, Burlington, ON, Canada) with molecular weight cut-off (MWCO) 10,000 Da to find out the efficiency of hydroxyurea encapsulation in NP proteins. This made possible the nonencapsulated HU to be eluated in a tube for collection. The nonencapsulated HU concentration was found by the high-performance liquid chromatography (HPLC) method (Shimadzu LC-20 Prominence, Kyoto, Japan).

After centrifugation while the nanoparticles were being washed, the supernatant was collected, and the drug amount in the solution was determined using the high-performance liquid chromatography (HPLC) method (Shimadzu LC-20 Prominence). The amount of drug encapsulated in the nanoparticles was then determined by subtracting the amount in the supernatant (free drug) that was not entrapped in the nanoparticles. Encapsulation efficiency and production yield were calculated as follows: $$\mathrm{Encapsulation} \mathrm{efficiency} \left(\mathrm{EE}\%\right)=\frac{\mathrm{Mass} \mathrm{of} \mathrm{the} \mathrm{total} \mathrm{Drug} -\mathrm{Mass} \mathrm{of} \mathrm{free} \mathrm{Drug}}{\mathrm{Mass} \mathrm{of} \mathrm{total} \mathrm{Drug}}\times 100\%$$
$$\mathrm{Production} \mathrm{Yield} (\%)=\frac{ \mathrm{Mass} \mathrm{of} \mathrm{total} \mathrm{nanoparticles}}{\mathrm{Mass} \mathrm{of} \mathrm{the} \mathrm{total} \mathrm{Drug}+\mathrm{Mass} \mathrm{of} \mathrm{total} \mathrm{BSA}}\times 100\%$$

The composition of synthesized polymers was determined by ¹H and ³¹P NMR. Spectra were recorded on a Bruker AC-400 NMR spectrometer (Karlsruhe, Germany) at 25 °C using THF and DMSO-d₆ as solvents.
Molecular weight (M_n) and dispersity (Đ) were established using size exclusion chromatography (SEC) (Shimadzu LC-20 Prominence, Kyoto, Japan) equipped with refractometric detector (RID 10-A).
The hydrodynamic diameter and zeta potential of prepared nanoobjects were measured on a ZetasizerNano-ZS (Malvern, UK) at a scattering angle of 173° at 25 °C. The content of rhodamine 6G was determined by UV–Vis spectroscopy using a UV-1800 (Shimadzu, Kyoto, Japan) spectrometer at 527 nm.

The obtained extracts were obtained with the use of a dry oven GP-20 (Ukraine) and vacuum circulating apparatus (ZOIBKD, Zhengzhou, Henan, China).
Studies of phenolic compounds of the extracts were performed by HPLC on a Shimadzu LC20 Prominence liquid chromatograph in a modular system equipped with a four-channel pump LC20AD, column thermostat STO20A, and automatic sampler SIL20A.
The quantitative content of the basic groups of BAS (total polyphenols, flavonoids, and iridoids) in the extracts were determined via absorption spectrophotometry using the Evolution TM 60S UV–visible light spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

The molecular weight analysis of melanoidin EAM was determined using high-performance gel permeation chromatography with photodiode array detection (HPGPC-PDA) at liquid chromatograph LC-20 Prominence coupled with photodiode array detector SPD-M30A (wavelength 270 nm; all Shimadzu) and TSK-Gel G4000 SWXL column (7.8 × 300 mm; Shimadzu, Tosoh Bioscience LLC, Tokyo, Japan) at the column temperature of 35 °C. Isocratic elution with 20 mM KH₂PO₄ was used for the separation. The injection volume was 5 μL, and the flow rate was 500 μL/min. To calibrate the column, the dextrans with 10, 400, and 400 kDa were used (all Sigma-Aldrich).

The yield of the HSA-NPs was measured by UV-spectrophotometry. A standard curve of HSA dissolved in a solution of phosphate buffered saline (PBS) was used as a reference. The absorbance values for HSA were measured at 280 nm. The following equation was used for the calculation of the yield.
The HSA–HU-NPs were spin-concentrated using Amicon centrifugal filters (Cedarlane, Burlington, ON, Canada) with a molecular weight cut off (MWCO) of 10,000 Da to calculate the encapsulation efficiency of hydroxyurea in HSA-NPs. This allowed the non-encapsulated hydroxyurea drug to be eluted into the collection tube. The concentration of non-encapsulated hydroxyurea was determined by the high performance liquid chromatography (HPLC) method (Shimadzu LC-20 Prominence).

The Mg and Ti contents of the samples were determined using inductively coupled plasma -- atomic emission spectroscopy (ICP-AES) on an Optima 4300 DV (PerkinElmer) spectrometer, while the contents of DBP were determined by high-performance liquid chromatography in isocratic mode by using standard solutions of the compounds in acetonitrile. The measurements were made on an LC-20 Prominence (Shimadzu) liquid chromatograph.