Background: With the intensification of population aging, osteoporosis has become one of the significant public health issues affecting human health. Currently available medications for treating osteoporosis are associated with various adverse effects and resistance issues. Oligonucleotide drugs show great potential. Effective delivery systems are essential to enhance the stability, bioavailability, and targeting of sRNA drugs. Lipid nanoparticles (LNPs) show promise as alternative osteoporosis therapeutics. This study explores the potential of LNPs as an effective delivery system to treat osteoporosis. Methods: LNPs were prepared using microfluidic techniques with varying lipid compositions, and characterized in terms of size, zeta potential, and entrapment efficiency (EE%). Dynamic light scattering (DLS) was employed to determine the size of the LNPs. The zeta potential was measured using electrophoretic light scattering. The pharmacodynamic effects and safety were then evaluated in a mouse model through intravenous administration. Results: Several lipid nanoparticle (LNP) formulations with different nitrogen/phosphorus ratios and different DMG-PEG2000 ratios were examined, and a lead candidate that supports delivery of sRNA in animal models of osteoporosis was identified. In OVX mice, LNP-sRNA significantly improved bone mineral density (BMD), trabecular microstructure, and biomechanical strength. Safety assessments revealed no systemic toxicity. It is shown that the optimized LNPs can serve as a promising delivery system to mediate sRNA delivery to bone tissue. Conclusions: After comparison of in vitro and in vivo properties, the optimized LNPs demonstrated good comprehensive performance as a delivery system for osteoporosis treatment. These results highlight the potential of the optimized LNPs as an ideal delivery system for osteoporosis, offering improved therapeutic efficacy and reduced systemic side effects.

Osteoarthritis (OA) is one of the most prevalent degenerative joint diseases, and there is no approved pharmacological therapy that can reliably reverse cartilage degeneration or restore cartilage structure. Allogeneic mesenchymal stromal cells (MSCs) have shown therapeutic potential for OA, but inflammatory conditions can enhance HLA expression and increase allogeneic immune recognition risk. Here, we generated B2M and CIITA double-knockout induced pluripotent stem cells (dKO-iPSCs) using CRISPR/Cas9 and subsequently differentiated them into dKO-iMSCs. B2M/CIITA double-knockout did not affect iPSC pluripotency, karyotype, or teratoma formation. dKO-iMSCs retained characteristic MSC phenotypes, exhibited greater proliferative capacity than tissue-derived MSCs, and could be expanded to passage 18. Notably, HLA-ABC and HLA-DR expression remained undetectable in dKO-iMSCs even after IFN-γ stimulation. In co-culture assays with PHA-L-stimulated allogeneic PBMCs, dKO-iMSCs suppressed PBMC and T-cell proliferation as effectively as wild-type iMSCs, indicating preservation of their immunomodulatory activity. Intra-articular administration of dKO-iMSCs significantly reduced cartilage degeneration in the destabilization of the medial meniscus (DMM) mouse model of OA. The treatment showed greater therapeutic efficacy than wild-type iMSCs and tissue-derived MSCs and did not cause evident systemic organ toxicity. Single-cell RNA sequencing of BM-MSCs and dKO-iMSCs after in vitro IFN-γ stimulation revealed enhanced extracellular matrix-associated cell–cell communication and markedly reduced IL1B expression in dKO-iMSCs, indicating a less pro-inflammatory and more matrix-supportive cellular state under inflammatory conditions. Collectively, B2M/CIITA double-knockout enables sustained reduction in HLA expression while preserving the MSC phenotype and immunomodulatory activity, and enhances the therapeutic efficacy of iMSCs in the mouse OA model, providing a potential strategy for developing iPSC-derived MSCs with reduced HLA expression for allogeneic cell therapy.