2.8. Transport Assay of Intact LNCs across Membranes

First, 1.5 mL of HBSS and 0.5 mL of diluted FRET-LNCs (1% v/v in HBSS) were filled into the basolateral and the apical side of the Transwell^® plates, respectively. The plates were incubated for 2 h at 37 °C with humidified air and 5% CO₂. Then, samples from the basolateral and the apical sides were collected, and fluorescence was analyzed by spectrophotometer. The TEER of all membranes were measured before and after the experiment to ensure their integrity. Membranes with TEER <300 Ω·cm² were excluded from the test.

Fluorescence emission spectra of collected samples were recorded on a FluoroMax^® 4 spectrophotometer (Horiba Jobin Yvon Inc., Piscataway, NJ, USA) at room temperature with the 548 nm excitation and 0.5 s integration time. The emission spectra were collected from 555 to 750 nm, with an increment of 1 nm. They were corrected for the lamp source fluctuations and the wavelength-dependent response of the detector. The integrity of nanoparticles was determined by FRET efficiency (proximity ratio) calculated by the following equation:

where A and D are the maximum fluorescence intensity of the acceptor (678 nm) and donor (569 nm), respectively. The particle concentration of the nanocarriers was calculated from the standard curve of the FRET acceptor signal. An acceptor signal lower than the limit of detection (LOD = 1382 cps/mA) and/or a PR lower than 0.70 was considered as zero particle concentration.

The transport efficiency (TE) of FRET-LNCs is the percentage of numbers of nanoparticles presenting at the basolateral medium compared to the initial particle concentration at the apical medium. It was calculated by the equation:

where C_f is the particle concentration at the basolateral side after 2 h, C₀ is the initial particle concentration at the apical side (particles/mL), V_A is the volume of sample at the apical side (1 mL), and V_B is the volume of sample at the basolateral side (1.5 mL).