Na Ren, Jing Jianmei, Yang Hua, Li Ye, Yuan Xiaofeng, Sun Xue, Han Jiangfan, Wang Jiajun, Tong Zhenhua, He Guangbin, Ye Weiliang
Department of Pharmaceutics, School of Pharmacy, Fourth Military Medical University, Xi'an, 710032, People's Republic of China.
Department of Ultrasound Diagnosis, Xijing Hospital, Fourth Military Medical University, Xi'an, 710038, People's Republic of China.
Int J Nanomedicine. 2025 Sep 8;20:10977-10998. doi: 10.2147/IJN.S532163. eCollection 2025.
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.
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.
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 (<0.05) and more effectively inhibited the growth (<0.05), migration (<0.05), and secretion of inflammatory cytokines (significantly reduced levels of IL-1β and IL-6, <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 (<0.05), demonstrating good targeting. Moreover, it enhanced the anti-inflammatory effects of methotrexate in AIA rats, significantly reducing paw swelling (<0.05) and joint clinical scores (<0.05). Importantly, it had no significant effect on the blood routine indicators of rats (>0.05), indicating no obvious toxicity.
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.
甲氨蝶呤（MTX）广泛用于治疗类风湿关节炎（RA），但靶向性差且副作用显著。本研究制备了用PVCAM-1肽修饰的载甲氨蝶呤脂质纳米粒（MTX@LNP-PVCAM-1），以增强靶向性并降低毒性。
采用薄膜分散法制备MTX@LNP-PVCAM-1。通过动态光散射（DLS）和透射电子显微镜（TEM）评估粒径和形态。使用人脐静脉内皮细胞（HUVEC）和溶血试验检测生物相容性。通过荧光显微镜检查细胞摄取情况，同时使用CCK-8和划痕试验评估细胞毒性和细胞迁移抑制。通过酶联免疫吸附测定（ELISA）测量炎性细胞因子（IL-1β、IL-6）。使用体内成像观察其在佐剂诱导的关节炎（AIA）大鼠体内的分布，并通过血液检测、爪体积、关节评分和组织学评估安全性和抗炎效果。
MTX@LNP-PVCAM-1的平均粒径为168.5 nm，多分散指数（PDI）为0.142，zeta电位为-12.1 mV，呈球形形态。它表现出pH响应性和良好的生物相容性。与未修饰的MTX@LNP相比，PVCAM-1表面修饰显著提高了细胞摄取效率（<0.05），并更有效地抑制了滑膜成纤维细胞的生长（<0.05）、迁移（<0.05）以及炎性细胞因子的分泌（IL-1β和IL-6水平显著降低，<0.05）。在动物实验中，MTX@LNP-PVCAM-1在炎症部位的蓄积明显高于MTX@LNP（<0.05），显示出良好的靶向性。此外，它增强了甲氨蝶呤对AIA大鼠的抗炎作用，显著减轻了爪肿胀（<0.05）和关节临床评分（<0.05）。重要的是，它对大鼠血常规指标无显著影响（>0.05），表明无明显毒性。
MTX@LNP-PVCAM-1结合了被动靶向和主动靶向，能将MTX高效递送至炎症部位并降低毒性。该方法增强了对AIA大鼠的抗炎作用，为低毒性RA治疗提供了一种潜在策略。
