Alliance 2795

Molecular size distributions were assessed using a chromatographic system consisting of a Waters 2795 Alliance followed by a Waters 2996 Photodiode Array Detector set at 280 nm. Size exclusion separation occurred through a XBridge BEH 200 Å SEC 3.5 µm 7.8x150 mm column (Waters).

Analysis of DSPC and DOTAP was conducted using a Waters 2795 Alliance module including a quaternary pump, mobile phase degasser, autosampler, and column thermostat. Separation was carried out on a Luna C18 analytical column (150 × 4.6 mm i.d., 3 µm particle size, 100 Å pore size) supplied by Phenomenex. The analytical method, derived from Zhong et al. [36 (link)], consisted of a tertiary gradient, where A was 0.1% (v/v) FA in hexane, B was 0.2% (v/v) FA in methanol, and C was 0.1% (v/v) FA in water. Gradient was 88% B and 12% C for 3 min, changed to 13% A, 82% B, and 5% C over 1 min, and changed back to initial solvent mixture after 6 min. The total run time was 15 min. The column temperature was 60 °C and sample temperature was 30 °C. The flow rate was 1.4 mL/min, and a sample injection volume of 20 µL was used. The electrospray ionization mass spectrometer used to detect lipids was a Waters Micromass ZQ analyzer. Nitrogen gas was used at 450 L/h, and capillary and cone voltages were set to 3 kV and 50 V. The desolvation temperature and source temperature were 220 °C and 100 °C, respectively. Quantification was carried out using SIR mode (single ion recording) in positive mode at m/z = 790.1 for DSPC and 662.0 for DOTAP. Data were processed using Waters Empower 2 software version number 6.20.00.00 (Water Corporation, Milford, CT, USA).

All NMR spectra were recorded at ambient temperature on a 300 MHz Bruker DRX Spectrometer equipped with a 5 mm BBFO probe and a SampleExpress for automated sample handling. Proton (δH) chemical shifts are quoted in ppm and are internally referenced to the residual protonated solvent signal. Resonances are described as s (singlet), d (doublet), t (triplet) and so on. Coupling constants (J) are given in Hz and are rounded to the nearest 0.1 Hz.
Compound purity and identity were determined by LC–MS (Alliance 2795, Waters, Milford, MA). Purity was measured by UV absorbance at 210 nm. Mobile phase A consisted of 0.01% formic acid in water, while mobile phase B consisted of 0.01% formic acid in acetonitrile. The gradient ran from 5 to 95% mobile phase B over 1.75 min at 1.75 ml/min. An Agilent Poroshell 120 EC-C18, 2.7 µm, 3.0 × 30 mm column was used with column temperature maintained at 40 °C. 2.1 µL of sample solution were injected. RT refers to the retention time for the compound under the above conditions. Identity was determined on a SQ mass spectrometer by positive and negative electrospray ionization. m/z values are reported in Daltons, with the relevant fragment ions quoted in parentheses.

NP size and polydispersity index (PDI) were determined by 3 ways: (1) dynamic light scattering (DLS) (Brookhaven 90 plus Particle Analyzer, Brookhaven Instruments, Holtsville, NY) at an angle of 90°; (2) nanoparticle tracking analysis (NTA) (LM-10, Malvern Instruments, Westborough, MA); and (3) transmission electron microscopy (TEM). Surface morphology was analyzed by TEM. TEM samples were prepared by staining with 0.1% w/v phosphotungstic acid. Zeta potential was determined by Zeta PALS (Brookhaven Instruments). ARV loading and in vitro release were characterized by high performance liquid chromatography (HPLC) (Alliance 2795, Waters Corporation, Milford, MA).

To ensure the consistency of herbal preparation, the quantitative analysis of the rosmarinic acid content in the 70% EtOH extract of C. myxa leaves was performed using Waters Alliance 2795 and Waters 996 PDA. MassLynx version 4.1 software operated the system control and was used for data analysis. The analytical chromatogram was developed using an Eclipse XD8-C18 column (Agilent Technologies, 150 × 4.6 mm I.D., 5 μm, Santa Clara, CA, USA). The mobile phase consisted of water containing 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B) and isocratic elution was performed at a flow rate of 0.5 ml/min using the following conditions: 21% B (0–25 min) and 100% B (25–30 min). The column was equilibrated at the initial conditions for 15 min before the next injections. The injection volume was 10 μL, the detection wavelength was set to 330 nm, and the column temperature was 35 °C. Rosmarinic acid and the extract were diluted with methanol and filtered with a 0.20 μm syringe filter (Whatman Inc., Maidstone, UK) before the HPLC analysis. The analysis was repeated three times to check its reproducibility.