| Background:Osteoporosis is a systemic metabolic bone disease characterized by decreased bone mass,destruction of bone tissue microstructure,and increased susceptibility to fracture.Its clinical manifestations mainly include limb fatigue,waist and back pain,generalized bone pain,and spinal deformation.Osteoporotic fracture is the most serious complication of osteoporosis,which generally leads to restricted activity,reduced quality of life,and shortened life expectancy,as well as imposes a heavy economic burden on families and society.Currently,there are more than 200 million people suffering from osteoporosis worldwide.This number continues to climb as the process of population aging society intensifies.Postmenopausal osteoporosis in women and degenerative osteoporosis in the elderly are both commonly encountered in clinical practice as primary osteoporosis.Postmenopausal osteoporosis is associated with estrogen deficiency and belongs to the high-conversion type of osteoporosis.The main manifestation is increased in both bone resorption and bone formation,but bone resorption is comparatively accelerated,which destroys the balance of bone conversion and eventually leads to osteoporosis.Senile osteoporosis is related to degenerative factors and is classified as low-conversion osteoporosis.The main manifestation is weakened of both bone resorption and bone formation,but bone formation is relatively weaker,and osteoblasts cannot repair bone loss caused by osteoclasts,resulting in osteoporosis.The increased bone brittleness following osteoporosis predisposes to osteoporotic fractures in the spine,hip,distal radius,and proximal humerus.Alendronate（ALN）,as the third-generation bisphosphonate bone resorption inhibitor,is a commonly prescribed drug for the treatment of osteoporosis and is available in both tablet and injection dosage forms.Currently,the ALN tablet is the most widely applied,but its oral administration requirements are relatively strict,with a bioavailability of only 0.7%,and it is prone to numerous gastrointestinal-related adverse reactions[1].The ALN injection has severely restricted its clinical popularity owing to its rapid pharmaceutical metabolism,short half-life,and nephrotoxicity[2].Purpose:To solve the practical issues in the application of ALN,we prepared 4 samples of multifunctional polymers by ALN functionalized modification of phosphatidylethanolamine PEG active lipids.The therapeutic effects of the multifunctional polymers were compared and analyzed in ovariectomized（OVX）mice osteoporosis model,and the mechanism of multifunctional polymers in the therapy of osteoporosis was investigated.Methods:（1）The dimyristoylphosphatidylethanolamine PEG active lipid（DMPE-PEG-NHS）,dioleoylphosphatidylethanolamine PEG active lipid（DOPE-PEG-NHS）,dipalmitoylphosphatidylethanolamine PEG active lipid（DPPE-PEG-NHS）and distearoylphosphatidylethanolamine PEG active lipid（DSPE-PEG-NHS）were respectively modified by ALN functionalization,4 kinds of multifunctional polymers with high cell membrane affinity,long-circulating property,bone targeting characteristic,and superior biological safety were prepared.Furthermore,the effects on osteoblasts and osteoclasts were investigated in vitro cellular assays.（2）The model of osteoporosis was established in BALB/c mice aged 6-7months.To determine whether the mice osteoporosis modeling was successful utilizing bone mineral density（BMD）detection,microfocus computed tomography（Micro-CT）analysis,and histopathological examination.The mice were subsequently grouped and treated according to predetermined time points.The therapeutic effects of multifunctional polymers on osteoporosis in OVX mice were verified by BMD detection,Micro-CT analysis,histopathological examination,western blot（WB）,enzyme-linked immunosorbent assay（ELISA）,and three-point bending test.Finally,the biological safety of multifunctional polymer was evaluated by weight monitoring,routine blood analysis,and histopathological examination of vital organs in mice.（3）Metabolomic assays were performed on mice femoral tissues to screen for significantly different metabolites involved in the pathogenesis and treatment of osteoporosis and to perform cluster analysis,correlation analysis,and KEGG metabolic pathway analysis.The mechanism of action of multifunctional polymers in the therapy of osteoporosis in mice was investigated at the level of small molecule metabolites.Results and Conclusion:Functionalized modification of phosphatidylethanolamine PEG active lipids with ALN successfully fabricated 4 types of multifunctional polymers.In vitro cellular assays,the multifunctional polymer promoted osteoblast accretion and mineralization.Moreover,it significantly inhibited osteoclast formation and reduced osteoclast activity compared with ALN.In the OVX mice osteoporosis model,multifunctional polymers partially reversed osteoporotic bone loss and had stronger effects in inhibiting bone resorption,reducing bone turnover rate,and improving bone biomechanical strength than ALN.In addition,metabolomics provided a meaningful exploration of the variations in bone metabolism during the pathogenesis and therapy of osteoporosis.The significantly different metabolites involved in the therapy of osteoporosis by multifunctional polymers include inosine,seryl-histidine dipeptide,and isoleucyl-proline.Besides,the significantly different metabolic pathways enriched include the histidine metabolic pathway and ATP binding cassette（ABC）transporter protein pathway.Metabolomics demonstrated that multifunctional polymers reversed the disorder of bone metabolism after osteoporosis by regulating the expression of significantly diverse metabolites and metabolic pathways.In summary,multifunctional polymers effectively reversed osteoporotic bone loss in OVX mice,promoted bone regeneration and remodeling after osteoporosis,and demonstrated superior therapeutic effects compared to ALN in the therapy of osteoporosis in mice. |
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