Abstract
BACKGROUND: Endoplasmin (ENPL), a heat shock protein 90 family member, promotes chondrogenic differentiation of stem cells by inhibiting ERK1/2 phosphorylation and inducing endoplasmic reticulum stress. However, its large size limits cellular uptake and therapeutic potential. To overcome this challenge, a cationic lipid nanoparticle (C_LNP) system was designed to deliver ENPL intracellularly, enhancing its effects on human tonsil-derived mesenchymal stem cells (hTMSCs). METHODS: ENPL-loaded cationic lipid nanoparticles (ENPL_C_LNP) were synthesized to facilitate intracellular ENPL delivery. The delivery efficiency and cytotoxicity were assessed in vitro using hTMSCs. Additionally, ENPL_C_LNPs were incorporated into a hyaluronic acid and chondroitin sulfate-based injectable hydrogel and tested for chondrogenic differentiation potential in a mouse subcutaneous model. RESULTS: ENPL_C_LNP achieved over 80% intracellular protein delivery efficiency with no cytotoxic effects. Co-cultured hTMSCs exhibited increased glycosaminoglycans (GAGs) and collagen expression over 21 days. In vivo, the hydrogel-embedded ENPL_C_LNP system enabled stable cartilage differentiation, evidenced by abundant cartilage-specific lacuna structures in regenerated tissue. CONCLUSION: Combining ENPL_C_LNP with an injectable hydrogel scaffold supports chondrogenic differentiation and cartilage regeneration, offering a promising strategy for cartilage tissue engineering.