supporting data for "aptamer-mediated" targeting of MMP14 as a potential therapeutics for osteoporosis

Osteoporosis is a prevalent skeletal disorder characterized by reduced bone mineral density and microarchitectural deterioration. Current systemic treatments often suffer from off-target effects, underscoring the urgent need for localized therapeutic strategies. Membrane-bound Matrix Metalloproteinase 14 (MMP14), in addition to its role in the degradation of multiple collagenous extracellular matrix (ECM) components, has also been demonstrated to be a key negative regulator of bone formation. MMP14 cleaves the parathyroid hormone 1 receptor (PTH1R), thereby attenuating parathyroid hormone (PTH) signal transduction and negatively regulating bone formation. This finding suggests that specific inhibition of MMP14 in the Hypertrophic chondrocyte (HC)-derived osteoblast lineage may be a potential therapeutic approach for bone loss disorders. However, achieving efficient and cell-specific delivery of gene-silencing therapeutics to bone remains a major obstacle.My PhD project aims to establish a novel delivery platform based on aptamer-mediated DNA nanotechnology to achieve specific MMP14 knockdown in HC-derived osteoblast lineages for the treatment of osteoporosis. Specifically, there are three main objectives of this study.The first objective is to identify and characterize a specific aptamer against mouse osteoblast (MC3T3-E1) cells. To achieve this, we employed Systematic Evolution of Ligands by Exponential Enrichment (SELEX) strategy. After 11 rounds of selection, three aptamer candidates, Apt6, Apt15, and Apt40 were identified, in which, Apt15 exhibited the highest binding affinity (33.19 nM) and membrane-binding capability to osteoblasts. Truncation of Apt15 greatly reduced binding, indicating the full-length sequence is essential for proper folding and function. Furthermore, all three aptamers appeared to recognize a common target. To investigate this, proximity labeling combined with Mass Spectrometry (MS) analysis was performed, with five potential protein targets identified for further validation. Collectively, these results confirm the success of the aptamer selection, validate the robustness of the SELEX strategy, and highlight Apt15 as a promising targeting tool for osteoblasts.The second objective is to optimize the structural geometry of DNA nanocarriers to enhance their internalization efficiency. Using flow cytometry, we evaluated the delivery efficiency of three DNA nanostructures: tetrahedron, icosahedron, and tesseract in MC3T3-E1 cells. Among these, the DNA icosahedron demonstrated the highest delivery efficiency, primarily via clathrin-mediated endocytosis and macropinocytosis. Conjugating Apt15 further enhanced uptake without altering the entry pathway. These results establish the DNA icosahedron as an efficient scaffold for targeted delivery and provide a foundation for subsequent functional evaluation of this DNA nanotechnology-based strategy. The third objective is to investigate the efficiency of MMP14 suppression by antisense oligonucleotides (ASOs) delivered via different strategies. Specific antisense oligonucleotides were designed computationally, and the inhibitory efficiency following different delivery methods was assessed using RT-qPCR and western blot analysis. The results demonstrated that Apt15-conjugated icosahedral DNA–ASO complexes (IA-15) achieved robust MMP14 silencing, comparable to that achieved with Lipofectamine 3000, but with much less cytotoxicity.In conclusion, this study demonstrates that the aptamer-functionalized DNA platform enables targeted and efficient MMP14 inhibition in osteoblasts, offering a promising anabolic strategy for osteoporosis treatment. Furthermore, this work also provides new insights into targeted drug delivery and advances the field of precision medicine.