Ren Na,1,* Jianmei Jing,2,* Hua Yang,3,* Ye Li,3 Xiaofeng Yuan,1 Xue Sun,4 Jiangfan Han,1 Jiajun Wang,1 Zhenhua Tong,4 Guangbin He,2 Weiliang Ye1 

1Department of Pharmaceutics, School of Pharmacy, Fourth Military Medical University, Xi’an, 710032, People’s Republic of China; 2Department of Ultrasound Diagnosis, Xijing Hospital, Fourth Military Medical University, Xi’an, 710038, People’s Republic of China; 3Department of Medical Affairs, No. 968 Hospital of Chinese People’s Liberation Army, Jinzhou, 121000, People’s Republic of China; 4Department of Pharmacy, General Hospital of Northern Theater Command, Shenyang, 110016, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Guangbin He, Email guangbinhe@fmmu.edu.cn Weiliang Ye, Email yaojixue@fmmu.edu.cn

Objective: Methotrexate (MTX) is widely used for rheumatoid arthritis (RA) but has poor targeting and significant side effects. This study developed MTX-loaded lipid nanoparticles modified with PVCAM-1 peptide (MTX@LNP-PVCAM-1) to enhance targeting and reduce toxicity.
Methods: MTX@LNP-PVCAM-1 was prepared using the thin-film dispersion method. Particle size and morphology were assessed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Biocompatibility was tested using human umbilical vein endothelial cells (HUVEC) and hemolysis assays. Cellular uptake was examined via fluorescence microscopy, while cytotoxicity and cell migration inhibition were evaluated using CCK-8 and scratch assays. Inflammatory cytokines (IL-1β, IL-6) were measured by ELISA. Distribution in adjuvant-induced arthritis (AIA) rats was observed using in vivo imaging, and safety and anti-inflammatory effects were assessed through blood tests, paw volume, joint scores, and histology.
Results: MTX@LNP-PVCAM-1 had an average particle size of 168.5 nm, PDI of 0.142, and zeta potential of − 12.1 mV, with spherical morphology. It exhibited pH responsiveness and good biocompatibility. Compared with unmodified MTX@LNP, PVCAM-1 surface modification significantly increased cellular uptake efficiency (p< 0.05) and more effectively inhibited the growth (p< 0.05), migration (p< 0.05), and secretion of inflammatory cytokines (significantly reduced levels of IL-1β and IL-6, p< 0.05) of synovial fibroblasts. In animal experiments, the accumulation of MTX@LNP-PVCAM-1 in inflamed sites was significantly higher than that of MTX@LNP (p< 0.05), demonstrating good targeting. Moreover, it enhanced the anti-inflammatory effects of methotrexate in AIA rats, significantly reducing paw swelling (p< 0.05) and joint clinical scores (p< 0.05). Importantly, it had no significant effect on the blood routine indicators of rats (p> 0.05), indicating no obvious toxicity.
Conclusion: MTX@LNP-PVCAM-1 combines passive and active targeting, delivering MTX efficiently to inflamed sites and reducing toxicity. This approach enhances anti-inflammatory effects in AIA rats, offering a potential strategy for low-toxicity RA treatment.

Keywords: synovial fibroblasts, PVCAM-1, lipid nanoparticles, methotrexate, rheumatoid arthritis