Study On The Targeted Therapy Of Alzheimer’s Disease By Ultrasound Combined With Biosynthetic Nanobubbles To Edit GSK-3β Gene In Vivo

BackgroundAlzheimer’s disease（Alzheimer’s Disease,AD）is the most common type of dementia in old age.Its main pathological features are the formation of senile plaques formed byβ-amyloid（β-amyloid,Aβ）deposition and Tau protein hyperphosphorylation.Tangles of neuron fibers.Studies have shown that the increase of glycogen synthase kinase-3β（GSK-3β）protein activity in the brain of AD patients is closely related to the two major pathological features and reducing the activity of GSK-3βcan alleviate the pathological state of AD.CRISPR/Cas9 gene editing technology provides a powerful means to repair gene dysfunction.The key to gene targeted therapy is to find a suitable gene vector and target it to the target area.However,brain diseases are due to the blood-brain barrier（BBB）.The existence of BBB makes the treatment more difficult.Previous studies have shown that ultrasound combined with microbubbles（MBs）can effectively open the BBB,but the intravascular MBs cavitation is difficult to produce sound hole effects due to the inability to contact nerve cells.Therefore,in this study,we propose a new method for AD treatment by the second ultrasound cavitation targeted gene editing GSK-3βin vivo.It is planned to use ultrasound combined with MBs to cavitation the open target area BBB,and then inject the adeno-associated virus CRISPR/Cas9(Adeno-associated virus CRISPR/Cas9（Adeno-Associated Virus CRISPR/Cas9,AC）biosynthetic nanobubbles（BNBs）,which use the open BBB to enter the brain tissue and directly contact the nerve cells,and then use the second ultrasound to induce cavitation in the BNBs,so that the cell membrane is opened and the editing system is efficiently delivered Targeted gene editing in vivo.This study will further explore the effect and mechanism of using secondary ultrasound cavitation in vivo targeted gene editing to treat AD,and provide new ideas for ultrasound targeted gene editing to treat AD.ObjectiveAfter opening the BBB with ultrasound combined with MBs,this study proposes a secondary ultrasound combined with BNBs in vivo gene editing GSK-3βmethod to treat AD,which enables cell membrane to be opened efficiently deliver the CRISPR/Cas9 gene editing system,and achieve in vivo targeted gene editing So as to knock down the therapeutic effect of GSK-3βon AD-like mice induced by Aβ_1-42 oligomers,and provide a new idea for ultrasound targeted gene editing to treat AD.Method1.Extract biosynthetic nanobubble BNBs by culturing Halobacterium NRC-1（Halo）in ATCC medium,and measure the particle size,potential,morphology and morphology of BNBs using a Malvern particle size potentiometer,electron microscope,and ultrasonic instrument.Ultrasound contrast imaging characterization;the cationic polymer PEI is coupled to the shell of BNBs to obtain positively charged BNBs（Cationic BNBs,CBNBs）,and the Malvern particle size potentiometer is used to detect the particle size,potential characterization,and agar of CBNBs Glycogel electrophoresis was used to detect the ability of CBNBs to carry plasmids.2.Incubate the aged Aβ_1-42 oligomers in N2a cells for 24h,add the nanobubble transfection complex（CBNBs-AAV-CRISPR/Cas9,CBNBs-A-C）to the cells,and perform transfection under ultrasound.Western Blot detects the protein changes related to Tau phosphorylation,GSK-3β,P-GSK-3β（Ser9）and related Tau phosphorylation sites.3.Use bEnd.3 cells to construct an in vitro BBB model,and observe the opening effect of different microbubbles on the in vitro BBB model under the action of ultrasound.4.By injecting Aβ_1-42 oligomers into bilateral hippocampal CA1 regions of C57 mice,an AD mouse model of Aβaggregation in the brain was constructed.MBs were injected through the tail vein,and after the BBB was opened under the action of a single ultrasound,the transfection complex CBNBs-A-C was continued to be delivered from the tail vein to the body.The second ultrasound combined with CBNBs-A-C increased the transfection efficiency of nerve cells.Use water maze and Y maze to detect the learning and memory ability of mice;use Western Blot to detect the proteins GSK-3β,P-GSK-3β（Ser9）and related Tau phosphorylation sites in hippocampal brain tissues Changes;immunohistochemistry was used to evaluate the expression of synaptophysin,and brain slice clamp was used to detect long-term poentiation（LTP）to evaluate the learning and memory of mice and the plasticity of synapses.Result1.BNBs is a nano-scale spindle-shaped bubble with a negative charge on the surface,and can be imaged in vivo;after using PEI to modify BNBs into positively charged BNBs（CBNBs）,the particle size of the BNBs is not changed but it is obviously The surface potential is reversed,and CBNBs can bind negatively charged plasmids well as a new type of gene carrier.2.Aβ_1-42 oligomer can activate the protein expression of GSK-3βand enhance the phosphorylation of Tau protein.Under the action of ultrasound,the transfection complex（CBNBs-A-C）can be expressed in the cell,reducing GSK-3β.The protein expression level of Tau reduces the protein expression level of Tau phosphorylation site.3.bEnd.3 cells can form an in vitro single-cell BBB model on the Transwell membrane.Under the action of ultrasound,combined with MBs has the best opening effect on BBB in vitro,but when combined with BNBs,the opening effect on BBB in vitro is not obvious.4.Injection of Aβ_1-42oligomers into the CA1 region of the hippocampus on both sides of the hippocampus activates GSK-3βin the hippocampus,leading to an increase in the phosphorylation of Tau protein and a decrease in the level of learning and memory in mice.After ultrasound combined with MBs to open the BBB,the second ultrasound combined with nanobubble complex（CBNBs-A-C）edits GSK-3βgene expression in vivo,thereby reducing GSK-3βand related Tau phosphorylation protein sites in the brains of AD-like mice;improve the learning and memory ability of mice;increase the expression of synaptophysin;enhance the LTP electrophysiological signal in the hippocampal CA1 area.ConclusionAfter ultrasound combined with MBs to open the BBB,the second ultrasound combined with biological nanobubble complex as a new gene transfection method can more efficiently transfect neuronal cells in the hippocampus,and inhibit the activation of GSK-3βthrough the CRISPR/Cas9 system,Weaken the hyperphosphorylation and aggregation of Tau protein,thereby improving the learning and memory function of AD model mice induced by Aβ_1-42 oligomers.