Remodeling Cellular Uptake via Inducing Clustering of Urokinase Receptors in Hydrogel Microspheres for Alleviating Cartilage Senescence

Cellular senescence impairs clathrin-mediated endocytosis (CME), partly due to reduced membrane fluidity and disrupted lipid raft composition, which hinder receptor clustering and limit drug uptake. To overcome this, a urokinase receptor (µPAR) antibody-modified cationic liposome was developed loaded with plasmid of DNA-repairing enzyme Sirt6 (anti-µPAR@Lipo-Sirt6), targeting the overexpressed µPAR on senescent chondrocytes to promote receptor clustering and enhance CME. Compared to Lipo-Sirt6, anti-µPAR@Lipo-Sirt6 induced µPAR clustering and enhanced cellular uptake via the CME pathway. The results showed 1.7-2-fold higher uptake efficiency in vitro and 1.5-fold higher in vivo, leading to improved Sirt6 gene delivery. In vitro, treatment with anti-µPAR@Lipo-Sirt6 promoted DNA damage repair, significantly reduced the expression of senescence markers cyclin dependent kinase inhibitor 1a (Cdkn1a) and cyclin dependent kinase inhibitor 2a (Cdkn2a), and restored the expression of cartilage functional molecules type II collagen (Col2) and aggrecan (Acan). Encapsulation within hyaluronic acid-methacrylate (HAMA) hydrogel microspheres (anti-µPAR@Lipo-Sirt6@HMs) further prolonged retention and efficacy in vivo, significantly reducing p53 expression, preserving cartilage matrix molecules such as Col2 and Acan, delaying cartilage degradation, and restoring gait function in osteoarthritis (OA) mice. This study demonstrates that receptor clustering can be harnessed to enhance drug uptake in senescent cells, offering a promising strategy for anti-senescence therapy.