Lc 20at system

The ¹H NMR spectroscopy were applied on a Bruker DRX-400 spectrometer using D₂O with concentration of 10 mg/ml. Scanning electron microscopy (SEM) images were obtained on a JSM-6700F microscope (JEOL, Japan), and the morphologies of the hydrogel surfaces were observed at an accelerating voltage of 10 kV. The freeze-dried samples were sputter-coated with a thin layer of Pt for 90 s to make the conductive sample before testing. The compressive profiles of GelMA@OSA and GelMA@OSA-ALN hydrogels were measured with a beam velocity of 1 mm/min using a testing machine of Instron 3,365. The diameter and thickness of cylindrical samples were 10 mm and 5 mm, respectively. Five samples were measured for each group. Confocal laser scanning microscopy (CLSM) image was obtained on a Zeiss LSM 510 microscope. The ALN release was measured by high performance liquid chromatography (HPLC) on a Shimadzu LC-20AT system with UV detection at 266 nm.

HPLC analyses were performed on a Shimadzu, LC-20AT system (model DGU 20A3, SHIMADZU corporation, Kyoto, Japan) equipped with LC-20AT quaternary pump, a DGU20A3/DGU-20A5 on-line degasser, an SPD-20A photodiode array detector, and a CBM-20A/20A interface. The data were acquired and processed using Lab Solution software. The active fraction was analyzed using a reverse-phase HPLC column SHIMADZU corporation, Kyoto, Japan), SunfireTM prep C18 (10 mm × 250 mm, 5 µm) column (Waters, Ireland). The mobile phase was composed of 30% H₂O with 0.1% TFA (ACROS ORGANICS) and 70% acetonitrile (LC-MS CHROMASOLVR, Fluka) with 0.1% TFA. Prior to HPLC injection, samples were filtered through a CHROMAFIL R Xtra H-PTFI filter (pore size 0.45 µm, filter 13 mm, (MACHEREY-NAGEL, Düren, Germany). Two mL of sample was injected and run for 41 min at 20 °C with a flow rate of 4 mL/min [68 (link)].

The permeation studies through the hydrogels were performed with a series of beta-blocking agents with a homologous structure and different degree of hydrophilicity—atenolol, betaxolol hydrochloride, penbutolol sulphate, and timolol maleate—using horizontal permeation cells. The cells were composed of two parts, a donor and a receiving compartment. The hydrogels were mounted between the compartments and one side was filled with the drug solution (the donor); meanwhile, the other side contained the Ringer–Krebs buffer (RKB, the receiving phase). The process was carried out at a constant temperature of 32 °C, using a temperature-controlled water bath.
At appropriate time intervals, 1 mL of the receiving solution was withdrawn and replaced with the same volume of fresh RKB. Each experiment was conducted for 4 h and was performed in triplicate.
The withdrawn volume was analyzed by HPLC using a LC-20AT system equipped with an SPD-10A UV detector, a CBM-20A interface (Shimadzu, Kyoto, Japan), and a 20 μL Rheodyne injection valve. The analyses were conducted with a C18 Bondclone (10 μm, 300 × 3.9 mm; Phenomenex, Torrance, CA, USA) column and the conditions are listed in Table 2.
From the obtained data, the apparent permeability coefficient (P_app) values were calculated according to Fick’s first law.

All chemicals (reagent grade) were purchased from Sigma-Aldrich (Burlington, MA, USA), Alfa Aesar (Ward Hill, MA, USA), and Merck (Burlington, MA, USA) without being further purified. Reaction progress was monitored by thin layer chromatography (TLC) with precoated silica gel 60 F254 plates of a thickness of 0.25 mm (Merck), and spots were detected with UV light (254 nm and/or 360 nm). Column chromatography was performed on silica gel (70–230 mesh and 230–400 mesh). ¹H- and ¹³C-NMR spectra were recorded on a Bruker AMX-400 spectrometer, using a deuterated solvent as the internal standard. Standard pulse sequences and parameters were used for the NMR experiments, and all chemical shifts are reported in parts per million (ppm, δ). Splitting patterns were designed as s, singlet; d, doublet; dd, doublet of doublet; ddd, doublet of doublet of doublet; t, triplet; m, multiplet; and br, broadband. The purity of all compounds was confirmed to be higher than 95% by means of analytical HPLC performed with a Shimadzu LC-20AT system and an SPD-20A UV detector. High-resolution mass spectra were measured in the instrument center of National Sun Yat-sen University (Bruker FT-MS SolariX). Capillarisin was provided by courtesy of Prof. Tian-Shung Wu’s lab and was isolated from A. capillaris [24 (link)].

Validation HPLC method was performed on a Shimadzu (Kyoto, Japan) LC-20AT system, which was equipped with a degasser, a binary pump, an autosampler and a diode array detector. The herbal extract was separated on Agilent ZORBAX SB-C18 (250 mm × 4.6 mm, 5 μm) column. The mobile phase was composed of acetonitrile (A) and 10 mmol/L ammonium acetate (B) using the following gradient program: 0–1.2 min, 5% A; 1.2–2 min, 5–20% A; 2–4 min, 20–40% A; 4–8 min, 40% A; 8–10 min, 40–95% A; 10–17 min, 95% A; 17–20 min, 5% A; the flow rate was 0.8 ml/min; the injection volume was 5 μL. A Shimadzu mass spectrum (LC-2020) equipped with an ESI ion source was operated in positive and negative modes, and the selected ion monitoring was used. The drying gas temperature was 350 °C; drying gas flow: 1.5 L/min; nebulizer pressure: 35 psi; capillary voltage: 3500 V. Shimadzu Mass workstation software was used for data acquisition and processing.

An appropriate amount of AMP-N-a-1 was dissolved in 0.2 M NaCl solution to prepare a sample solution at a concentration of 5 mg mL⁻¹. The solution was filtered through a 0.22 μM filtration membrane (Millex-LG hydrophilic PTFE membrane, Millipore, United States), and then, the residue was analyzed using high-performance gel permeation chromatography (HPGPC), in which an LC-20AT system of Shimadzu company, a RID-20A differential refractive detector, and a TSK-gel G-4000 PW_XL chromatographic column (7.8 mm × 300 mm) were used. The column temperature was 40°C, 0.2 M NaCl was used as a mobile phase, the flow rate was 0.6 mL min⁻¹, and the injection volume was 20 μL. The dextran with molecular weights of 1, 5, 12, 25, and 50 kDa was used for calibration to draw the standard curve.