Mitochondrial Calcium Nanoregulators Reverse the Macrophage Proinflammatory Phenotype Through Restoring Mitochondrial Calcium Homeostasis for the Treatment of Osteoarthritis

Xiao Lei,1,* Guodong Tan,2,* Yiming Wang,3,* Li Chen,4,* Yuan Cao,1 Bingxin Si,2 Zhen Zhen,2 Bei Li,5 Yan Jin,5 Wei Wang,6 Fang Jin1 1Shaanxi Clinical Research Center for Oral Disease & Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China; 2Air Force Medical Center, Fourth Military Medical University, Beijing, People’s Republic of China; 3Shaanxi Clinical Research Center for Oral Diseases & Department of Oral Surgery, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China; 4Shaanxi Key Laboratory of Energy Chemical Process Intensification & Institute of Polymer Science in Chemical Engineering, School of Chemical Engineering and Technology, Xi’an Jiao Tong University, Xi’an, Shaanxi, People’s Republic of China; 5State Key Laboratory of Military Stomatology & Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China; 6State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Key Laboratory of Oral Diseases, Department of Operative Dentistry and Endodontics, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China*These authors contributed equally to this workCorrespondence: Fang Jin, Shaanxi Clinical Research Center for Oral Disease & Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China, Email fangjin191@163.com Wei Wang, State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Key Laboratory of Oral Diseases, Department of Operative Dentistry and Endodontics, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi, People’s Republic of China, Email weiwang_0510@163.comIntroduction: Osteoarthritis (OA) is a chronic degenerative joint disease accompanied by an elevated macrophage proinflammatory phenotype, which is triggered by persistent pathologically elevated calcium ion levels in mitochondria. However, existing pharmacological compounds targeting the inhibition of mitochondrial calcium ion (m[Ca2+]) influx are currently limited in terms of plasma membrane permeability and low specificity for ion channels and transporters. In the present study, we synthesized mesoporous silica nanoparticle-amidated (MSN)-ethylenebis (oxyethylenenitrilo)tetraacetic acid (EGTA)/triphenylphosphine (TPP)-polyethylene glycol (PEG) [METP] nanoparticles (NPs), which specifically target mitochondria and block excess calcium ion influx.Methods: m[Ca2+] overload in OA mouse bone marrow-derived macrophages (BMDMs) was detected by a fluorescence probe. A tissue in situ fluorescence colocalization assay was used to evaluate METP NP uptake by macrophages. BMDMs from healthy mice were pretreated with a concentration gradient of METP NPs followed by lipopolysaccharide (LPS) stimulation and detection of m[Ca2+] levels in vitro. The optimal METP NP concentration was further applied, and the endoplasmic reticulum (ER) and cytoplasm calcium levels were detected. The inflammatory phenotype was measured by surface markers, cytokine secretion and intracellular inflammatory gene/protein expression. A Seahorse cell energy metabolism assay was performed to elucidate the mechanism by which METP NPs reverse the BMDM proinflammatory phenotype.Results: The present study identified calcium overload in BMDM mitochondria of OA mice. We demonstrated that METP NPs reversed the increased m[Ca2+] levels in mitochondria and the proinflammatory phenotype of BMDMs, with both in vivo and in vitro experiments, via the inhibition of the mitochondrial aspartate-arginosuccinate shunt and ROS production.Conclusion: We demonstrated that METP NPs are effective and highly specific regulators of m[Ca2+] overload. In addition, we demonstrated that these METP NPs reverse the macrophage proinflammatory phenotype by restoring m[Ca2+] homeostasis, thereby inhibiting the tissue inflammatory response and achieving a therapeutic effect for OA.Graphical Abstract: Keywords: nanoparticle, macrophage, mitochondria, calcium homeostasis, osteoarthritis