Alliance e2695 hplc

The dried extracts were dissolved in 2 mL of DMSO, filtrated through, and analyzed with high-performance liquid chromatography (Alliance HPLC e2695, PDA 2998, Waters, Milford, MA, USA) and electrospray ionization mass spectrometry (ACQUITY QDa Mass Detector, Waters, Milford, MA, USA). The chromatographic separation was achieved with a column (Symmetry C18, 150 × 4.6 mm, 5 μm) at a flow of 0.8 mL/min with a linear gradient system for 30 min (A: 0.1% formic acid in Water, B: methanol). The column temperature was adjusted to 40 °C, and the injection volume was 5 μL. The exact analytical conditions are described in Supplementary Figures S3–S6. For mass detection, the positive-ion ESI mode was used. The rest of the capillary voltage settings were: Pos: 0.8 kV, gain: 1, and probe: 600 °C, while the cone voltage was set to 15 V.

Dynamic light scattering with a Zetasizer Nano ZS90 (Malvern, UK) was used to determine the particle size, polydispersity index, and zeta potential for all liposomes, while transmission electron microscopy (TEM) was used to observe their morphologies (HT7800, Hitachi, Tokyo, Japan). Furthermore, high-performance liquid chromatography (HPLC) was used for measuring the TP content (Alliance HPLC E2695, Waters Corporation, Milford, MA, USA). The unencapsulated drug was separated from the liposome solution by centrifugal ultrafiltration (4500 rpm, 5 min) (TG16-WS, Changsha, China), and the encapsulation rate was determined by HPLC. Specifically, the method involved the addition of 20 volumes of methanol to the liposome solution, followed by demulsification using ultrasound for 20 min and measurement of the total TP amount (Wtotal). In addition, 0.2 mL liposome solution was measured precisely into an ultrafiltration centrifuge tube (MWCO 30,000 Da), diluted to 1 mL with PBS, and centrifuged (4500 rpm, 5 min) to obtain the unwrapped TP. The unwrapped drug content (W_{Unwrapped drug}) was determined, and the encapsulation efficiency (EE) was calculated as:
where W_Unwrapped and W_total represent the unencapsulated amount and the total amount of the drug, respectively. The drug loading (DL%) for TP-loaded liposomes was then calculated using the following equation:

Fourier transform infrared spectroscopy measurements (FT-IR, NICOLET IS50, Thermo Fisher Scientific) were conducted to identify the functional group of Lf-GL. The molecular weight of Lf-GL was determined by SDS-PAGE (12%-gel) and matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF). MALDI-TOF was conducted at Seoul National University (Korea) using a MALDI-TOF Voyager DE-STR (Applied Biosystems, MA, USA). A sinapinic acid (Sigma-Aldrich) aqueous solution containing about 30% acetonitrile in 0.15% trifluoroacetic acid (Millipore) was used as a matrix. High performance liquid chromatography (Alliance HPLC e2695, Waters, MA, UK) using a gel permeation chromatography (GPC) column was performed to verify GL content in the Lf-GL conjugate. The mobile phase was composed of methanol, acetonitrile, water, and acetic acid in a ratio of 55:23.69:19.63:0.68. GL was dissolved in the mobile phase in different concentrations (2.5 to 200 × 10^–6 M), and Lf-GL (25 × 10^–6 M) was also dissolved in the mobile phase. Ultrahydrogel 120 Column (Waters) was used as a column, and the flow rate was 1 mL min⁻¹. Then, absorbance was measured at 254 nm, and the results were calibrated with Empower software (Waters).