Ultrasonic Endocavitation Mediated BMP2 Gene Transfection Into BMSCs With Nucleophilic Gas Vesicles To Promote Bone Regeneration

BackgroundBone defects are prone to poor healing due to the destruction of bone tissue.Bone mesenchymal stem cells(BMSCs)transfected with bone morphogenetic proteins 2(BMP2)gene bring hope for the treatment of bone defects.Ultrasoundtargeted microbubble destruction(UTMD)is a safe new gene transfection method proposed in recent years.However,because the cavitation nucleus loaded with gene is located outside the cell,it is difficult to deliver the gene to the cell nucleus in a targeted and localized way,so the transfection efficiency is low.ObjectiveTo construct a nucleophilic nano-gene delivery system BMP2CNGVs@BMSCs(BMP2-Cationic nucleophilic gas vesicles@BMSCs),and to stimulate the intracellular cavitation effect in cooperation with UTMD technology,effectively improve the permeability of nuclear membrane of BMSCs,mediate efficient nuclear transfection of BMP2 gene,and provide a promising targeted gene delivery strategy for promoting bone defect repair.Methods1、To construct a nucleophilic nano-gene delivery system BMP2-CNGVs@BMSCs:firstly,gas vesicles(GVs),Polyethylenimine(PEI),Nuclear localization signal peptide,NLS)Cationic nucleophilic nano-gene carrier CNGVs was prepared by electrostatic adsorption method,and the ability of loading BMP2 plasmid on CNGVs was detected by agarose gel electrophoresis experiment to form transfection complex BMP2-CNGVs,and the incubation time and ultrasonic parameters of transfection complex BMP2-CNGVs and BMSCs were optimized to improve transfection efficiency.2、Study on NLS-targeted nuclear membrane and its opening effect:The effect of NLS-targeted nuclear membrane was detected by confocal fluorescence microscope;Transmission electron microscope,real-time confocal imaging and three-dimensional confocal imaging were used to detect the nuclear membrane opening effect,plasmid nucleation effect and plasmid effective nuclear localization.3、In vitro experiment of Intra-ultrasound cavitation combined with NLSmediated BMP2 plasmid transfection into BMSCs:①The gene transfection efficiency was qualitatively and quantitatively analyzed by inverted fluorescence microscope and flow cytometry.②qRT-PCR and Elisa were used to detect the transcription of BMP2 mRNA and the expression of target protein.③The osteogenic efficiency and matrix mineralization ability of engineered BMSCs were determined by alkaline phosphoric acid and alizarin red staining.4、In vivo experiment of Intra-ultrasound cavitation combined with NLSmediated BMP2 plasmid transfection into BMSCs:3.2 mm rat femoral defect model was constructed,BMP2-CNGVs@BMSCs was injected in situ,bone regeneration was detected by Micro-CT and histology,the secretion of BMP2,CD 34 vascular endothelial growth factor and alkaline phosphatase protein was evaluated by immunohistochemistry,and the biological safety was evaluated by HE staining.Results1、The cationic nucleophilic nano-gene vector CNGVs was successfully constructed,with a surface Zeta potential of(26.1±2.2)mv,an average particle size of(330±1.4)nm,and high plasmid carrying capacity.The CNGVs containing 20 μg PEI can carry 10μg BMP2 plasmid to form BMP2-CNGVs.Further,it was screened out that the incubation time for BMSCs to maximize the phagocytosis of BMP2-CNGVs was 9 h,and BMP2-CNGVs@BMSCs was constructed,and the best ultrasonic irradiation parameters were screened out as follows:central frequency 1.5 MHZ,duty ratio 20%,sound pressure 120 mvpp and irradiation time 2.5 min.2、Under confocal microscope,it was observed that the plasmids in BMP2CNG Vs group could be enriched on the cytoplasm side of BMSCs nuclear membrane faster than those in BMP2-CGVs group,and even a small amount of plasmids entered the nucleus,which confirmed that NLS had the characteristics of targeting nuclear membrane.After ultrasonic-mediated intracellular cavitation,290 nm nuclear membrane open pores were observed under transmission electron microscope,and the dynamic nucleation of plasmid was observed by confocal microscope real-time imaging,and the permeability of nuclear membrane was obviously improved.3、The nuclear introduction rate of plasmid in Intra-ultrasonic cavitation with NLS group was 82.7%,which was 3.9 times higher than that in traditional Extraultrasonic cavitation group,and the transfection efficiency was 61%.After at least 14 days of continuous expression,the expression of BMP2 gene mRNA was 37.3 times that in BMP-2 plasmid group only,and the expression of BMP-2 protein in supernatant remained at a high level,reaching the threshold dose of stimulating osteogenic effect,and improving the osteogenic efficiency of engineered BMSCs.4、Micro-CT and histological analysis show that compared with the traditional Extra-ultrasonic cavitation group and Intra-ultrasonic cavitation group,Intra-ultrasonic cavitation with NLS group can significantly improve the angiogenesis and bone regeneration of femoral defect which provides a safe and effective targeted gene delivery strategy for promoting bone defect repair.ConclusionBased on the characteristic that BMSCs can devour nanobubbles,a nucleophilic targeted nanogene delivery system(BMP2-CNGVs@BMSCs)was successfully constructed,which was enriched around the nuclear membrane,and then combined with UTMD technology to stimulate intracellular cavitation effect to induce gene transfection at a fixed point,so as to realize safe,efficient and controllable gene transfection and stable and continuous expression of BMP2,thus improving the osteogenic environment of bone defects and promoting the repair of bone defects.It is expected that gene therapy,microbiology and materials science will be combined to provide an all-round,multi-level and wide-ranging new treatment strategy for clinic.