An Experimental Study On Bonetargeted Engineered Exosome Platform Delivering SiRNA To Treat Osteoporosis

Background With the aged tendency of population deteriorating,osteoporosis has become a serious public health problem endangering human health.However,the pathogenesis of osteoporosis is complex,involving multiple aspects such as bone remodeling,bone resorption and bone marrow microenvironment angiogenesis.At present,the commonly used drugs cannot cover each mechanism and accumulated in bone tissue specifically,which lead to a poor efficacy and side effects on non-bone tissue or organs.Therefore,it is of great significance to develop bone-targeting drugs that can cover multiple mechanisms of osteoporosis and specifically act on bone tissues,so as to achieve bone-targeting "precise" treatment of osteoporosis.Objective This study was to develop a bone-targeted engineered exosome platform delivering siRNA to osteoblasts specifically for osteoporosis treatment based on exosomes of MSCs derived from i PSCs,which covered the major therapeutic mechanisms of osteoporosis including enhancing the osteogenic differentiation,promoting type H vessels formation and inhibiting osteoclast formation.Methods(1)Commercial kits were used to differentiate i PSCs into i MSCs and a i MSCs culture.Flow cytometry and three-line differentiation experiments were used to authenticate the i MSCs.Exosomes were extracted with ultracentrifuge,identified using transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA)and Western blot assay.The osteoblast targeting peptide SDSSD was inserted into the surface of exosome membrane by the efficient insertion of DSPE-PEG into the phospholipid bilayer,and bone targeting exosome BT-Exo was obtained.The successful insertion of targeted peptide was detected by immunomagnetic bead capture of exosomes.siRNA was loaded into BT-Exo using electroporation.The particle size and surface potential were measured by dynamic light scattering(DLS),and the loading efficiency and release kinetics were measured using PCR.Serum stability of RNA loaded into exosomes was assessed using agarose gel electrophoresis.The mi RNA expression profiles were determined using the next-generation sequencing(NGS)technology with Illumina Hi Seq sequencer.(2)Flow cytometry and laser confocal microscopy were used to show the osteoblastic uptake of BT-Exo.In vivo imaging system and tissue sections immunofluorescence staining were used to investigate the bone targeting ability and specific delivery to osteoblasts.(3)The osteogenic effect of BT-Exo-si Shn3 was evaluated using alizarin red staining,ALP activity analysis and immunofluorescence staining.The pro-angiogenic effect was evaluated using tube forming experiment.The effect of BT-Exo-si Shn3 on osteoclast formation was evaluated with TRAP staining and F-actin staining.(4)Ovariectomy was used to construct the mouse model of osteoporosis.Micro-CT and dynamic bone fluorescence labeling experiments were used to evaluate the therapeutic effect of BT-Exo-si Shn3 on osteoporosis.Immunofluorescence staining and TRAP staining were used to label type H vessels and osteoclasts,respectively.Results(1)The culture system of i PSCs cell lines differentiation to i MSCs was successfully established and large number of i MSCs were obtained.Exosomes of i MSCs(Exo)was successfully extracted using ultracentrifuge.TEM showed that Exo present a typical cup-shape range of ～100 nm.DLS and NTA manifested the mean diameter of Exo was 99.8 ± 34 nm.Western blotting results confirmed that the specific protein markers Alix,CD81,CD63 were highly expressed in Exo.The BT-Exo-si Shn3 bone-targeting engineered exosome was successfully constructed.The green FITC fluorescence and orange-red Cy3 fluorescence were observed after the immunomagnetic beads were captured.DLS showed that the average diameter of BTExo and BT-Exo-si Shn3 was 108 ± 37 and 118 ± 42 nm,respectively.The hydrodynamic diameter of BT-Exo-si Shn3 was slightly changed from 114.6 ± 2.2 nm to 121.4 ± 4.0 nm,the surface zeta potential fluctuated from-16.8 ± 1.8 m V to-21.3 ± 2.1 m V fetal bovine serum(FBS)for 7 consecutive days by DLS,which indicated that BT-Exo-si Shn3 had a physical stability.The loading efficiency of BT-Exo determined by Q-PCR was 18%.After incubation for 2 h,the RNA efflux was approximately 11% in PBS and 9% in 50% FBS,respectively.After incubation for 24 h,the release saturated at about 95% in PBS and 98% in FBS,respectively.Agarose gel electrophoresis showed that exosomes had good serum stability and could protect the internal siRNA from being completely degraded within 16 h.The mi RNA expression profile of the vector system was determined by the second-generation high-throughput sequencing technology,which showed no statistical difference from the original IMSCExo,and the endogenous mi RNA carried by the vector system could promote bone formation,promote angiogenesis and inhibit osteoclasts.NGS data showed the exosome possessed endogenous mi RNA profiles of anti-osteoporosis effects,and the engineering procedures would not change the endogenous mi RNA.(2)Flow cytometry and laser confocal microscopy showed specific uptake of BT-Exo by osteoblasts,and the uptake was periostin-dependent.In vivo imaging system showed that bone-targeted modification could increase the accumulation in bone tissues.Tissue sections immunofluorescence staining showed that BT-Exo could deliver si Shn3 to bone formation surface and osteoblasts specifically osteoblasts.(3)Alizarin red staining,ALP activity analysis and immunofluorescence staining showed that BT-Exo-si Shn3 could promote osteogenic differentiation of pre-osteoblasts.Image J quantitative analysis of tube formation showed that BT-Exo-si Shn3 promoted angiogenesis.TRAP staining and F-actin staining showed that BT-Exo-si Shn3 could inhibit osteoclast formation.(4)The mouse model of osteoporosis was constructed with ovariectomy successfully.Micro-CT and dynamic bone fluorescence labeling experiments indicated BT-Exosi Shn3 showed great therapeutic effects on osteoporosis.Immunofluorescence staining indicated BT-Exo-si Shn3 promoted the type H vessels formation.TRAP staining indicated BT-Exo-si Shn3 inhibited osteoclast formation.Conclusion In this study,we develop a bone-targeting engineered exosome which could deliver siRNA to osteoblasts specifically and mediate the osteoblastic Shn3 gene silencing,exerting multiple effects of enhancing the osteogenic differentiation,inhibiting osteoclast formation and promoting type H vessels formation,providing a new strategy for the treatment of osteoporosis.