High pressure pumps

The mole percentages of CHOL, DPPC, and SPC in the lipids were, respectively, 50%, 25%, and 25% for LIP loaded with CRM (CRM-LIP). The mole percentages of CL, CHOL, DPPC, and SPC in the lipids were, respectively, 20%, 40%, 20%, and 20% for CRM-CL/LIP. The lipids were mixed with an additional 3% (mol) DSPE-PEG(2000) and 2% (mol) CRM. To prepare fluorescent LIP, 0.1% (w/v) FITC-D(70000) was added to the mixture. The mixture was dissolved in chloroform and condensed using a rotary evaporator (Panchum, Kaohsiung, Taiwan). The deposited solid thin film was hydrated with DPBS and compressed through a polycarbonate membrane containing pores 100 nm in size using an extruder set (Avanti Polar Lipids). The effluents of CRM-CL/LIP were added to methanol at 37°C for 10 min, analyzed using a reverse-phase BDS Hypersil C-18 column warmed by a column heater (Alltech, Deerfield, IL, USA) at 37°C in a high-performance liquid chromatograph (HPLC; Jasco, Tokyo, Japan) and detected using an ultraviolet (UV) detector (Jasco) at 430 nm. The mobile phase utilized ultra-pure water containing citric acid at pH 3 associated with a tetrahydrofuran gradient from 5% to 40% (v/v) for 20 min and was driven using 2 high pressure pumps (Jasco) in a series at a flow rate of 1 mL/min. The encapsulation efficiency of CRM was defined as ([weight of encapsulated CRM]/[weight of total CRM]) ×100%.

To a 3.3 30 cm² membrane tube in a flask with 20 mL of DPBS at pH 7.4, 1 mg/mL of LIP carriers in 2 mL of DPBS containing 0.05% sodium azide at pH 7.4 was added and shaken in a bath-reciprocal shaker at 50 rpm and 37°C for 2 days. Then, 100 µL of the sample containing CRM or NGF was treated with 1% (v/v) Triton-X-100 at 4°C for 1 h. The volume of the medium was compensated with 100 µL of fresh DPBS when sampling. To evaluate the quantity of released CRM, the sample was treated with methanol at 37°C for 30 min, analyzed using a HPLC (Jasco) with a reverse-phase BDS Hypersil C-18 column (Thermo Hypersil-Keystone, Bellefonte, PA, USA) at 37°C, and detected using an UV detector (Jasco) at 430 nm. The mobile phase used ultra-pure water (Barnstead) containing citric acid at pH 3 with a tetrahydrofuran gradient from 5% to 40% (v/v), driven using 2 high pressure pumps (Jasco) at a flow rate of 1 mL/min for 20 min. To assess the release of NGF, the sample was analyzed with a human NGF kit using an ELISA spectrofluorometer at 450 nm.