Assessment of nanoparticles permeation in cartilage involved incubating free rhodamine B (RHB) solution, RHB-labeled PAMAM (RHB@PEG-PA) and RHB-labeled targeting liposome nanoparticles (RHB@P-Lipo) (1 μg/mL of RHB) with porcine cartilage tissue disks. Following a 12 h incubation period, tissue sections were imaged utilizing a laser scanning confocal microscope (Leica, TCS SP8, Germany). Images were captured at sequential 20 μm intervals from the surface of the porcine cartilage tissue. The intensity of RHB fluorescence was quantified using the corresponding software (LAS X). Additionally, x-z axis sections were orthogonally reconstructed at the same depth using LAS X. In addition, we further performed cartilage tissue cross-sectional imaging based on a reported method [32 ]. Briefly, porcine cartilage explants were incubated with Free RHB, RHB@PEG-PA and RHB@P-Lipo (1 μg/mL of RHB) for 24 h. After washing with PBS three times and embedded in Tissue-Tek OCT compound (Surgipath®Leica FSC 22, USA) for frozen sectioning. The frozen slices were sealed by antifade mounting medium with DAPI and imaged with fluorescence microscope (Nikon, Japan).
For the evaluation of nanoparticles retention time in rat joints, male Sprague-Dawley (SD) rats (210–230 g) were utilized to create an OA model via anterior cruciate ligament transection (ACLT) in the right knee joints. After a 2-month duration, the rats were randomly assigned to five groups and intra-articular injected with free DiR, DiR@PEG-PA, DiR@Lipo, DiR@P-Lipo, and DiR@PEG-PA@P-Lipo, respectively. The DiR dosage administered was 12.5 μg/kg. Subsequently, the rats were imaged on days 0.5, 1, 2, 4, and 7 using an in vivo imaging system (Flex, Maestro, USA).