Study On Caspase-3 Gene Silencing For The Treatment Of Ischemic Stroke Based On DNA “Nanoflowers”

Objective:To design and construct a DNA nanoflowers delivery system that combines blood-brain barrier（BBB）penetration and caspase-3 silencing,and investigate the neuroprotective effects of DNA nanoflowers on ischemic stroke（IS）through in vivo and in vitro pharmacodynamic experiments.Methods:1.Design and construction of DNA nanoflowers.（1）A DNA template strand encoding a complementary sequence of transferrin receptor（TfR）aptamer and caspase-3 antisense DNA sequence was designed to assemble DNA nanoflowers（TD）by rolling circle amplification（RCA）reaction.（2）The size and morphology of TD were characterized by agarose gel electrophoresis,nanoparticle size analyzer（DLS）,scanning electron microscopy（SEM）and transmission electron microscopy（TEM）;The elemental composition of the TD and the amount of Mg²⁺released at different pH conditions were analyzed by X-ray spectroscopy（EDS）,mapping and inductively coupled plasma mass spectrometry（ICP-MS）.（3）The stability of TD was examined by DLS,agarose gel electrophoresis and SEM.（4）The adsorption capacity of caspase-3 mRNA was detected by enzyme plate analyzer.In addition,two template strands with only caspase-3 antisense DNA complementary sequence and only TfR aptamer complementary sequence were designed,using the same method as TD,and D and T nanoflowers were synthesized as controls for subsequent experiments.2.Neuroprotective effects of DNA nanoflowers in vitro.An oxidative stress injury model was established by H₂O₂（200μM）induction in rat adrenal pheochromocytoma cells（PC12）.（1）PC12 cell uptake and lysosomal escape were determined by flow cytometry and confocal microscopy.（2）RT-RCR and Western Blot experiments to determine the change of caspase-3 content in model cells;The cytotoxicity of TD and its protective effect on model cells were detected by CCK-8 assay;The experiment of Calcein-AM/PI staining was conducted to investigate the effect of TD on the apoptosis of model cells.（3）In vitro BBB crossing experiment to examine the brain targeting of nanoflowers.3.Neuroprotective effects of DNA nanoflowers in vivo.（1）Twelve male SD rats were randomly divided into transient middle cerebral artery occlusion（MCAO）+D and MCAO+TD groups by body weight.Six in each group.The two groups of animals were injected with 1 ml of the corresponding Cy5 short-chain D and TD nanoflowers（2μM）respectively in the tail vein,and the small animal in vivo imaging instrument was used for the injection at 1,2,4,8 and 12 h to investigate its brain-targeting effect.（2）Seventy-two male rats were randomly divided into sham operation group,MCAO group,MCAO+D group and MCAO+TD group.Eighteen in each group.Except the rats in the sham operation group and MCAO group were injected with the same amount of normal saline through the tail vein,the rats in the other groups were injected with the corresponding D or TD solution（both 2μM）.After 24 hours of administration,the neuroprotective effects of TD nanoflowers on MCAO rats was evaluated by neurological function score,brain tissue TTC,TUNEL,Nissl staining,histopathological examination,RT-PCR and Western Blot,and the biological safety of DNA nanoflowers was preliminarily evaluated.Results:1.Design and construction of DNA nanoflowers.（1）The results of agarose gel electrophoresis,SEM,TEM and DLS showed that the surface of TD was petal-like,with a particle size of about 250 nm and a potential of about-19.6 mV.（2）EDS and Mapping element analysis showd that TD contains C,N,O,Mg,P elements,indicating that it is composed of DNA and magnesium pyrophosphate;ICP-MS results showed that TD disintegrated at pH 5.0 and released Mg²⁺of about 58.5μg/ml,suggesting that it may have good pH sensitivity in lysosomes.（3）The results of DLS,agarose gel electrophoresis and SEM showd that TD has good stability.（4）The results of Cy5 fluorescence intensity suggested that TD could adsorb caspase-3 mRNA in large quantities.2.Neuroprotective effects of DNA nanoflowers in vitro.（1）PC12 cell uptake and lysosomal escape experiments showd that TD can enter cells in large quantities and achieve lysosomal escape under acidic lysosomal conditions（pH 5.0）,laying the foundation for its function.（2）The results of RT-RCR and Western Blot experiments showed that compared with the H₂O₂ group,TD could significantly reduce the content of caspase-3 in PC12 cells,laying a foundation for the treatment of IS by reducing nerve cell apoptosis;The results of cytotoxicity and H₂O₂-induced PC12 nerve cell protection experiments showed that TD had no obvious toxicity to PC12 cells after 24hours,and there was a significant dose-effect relationship between the survival rate of model cells and TD,and the highest survival rate could reach 88.8%;Cell live/death staining experiment showed that compared with H₂O₂ group,TD could reduce cell apoptosis and protect nerve cells.（3）In vitro cell BBB crossing experiments showed that at 12 h,the fluorescence intensity of the TD group was approximately 6.4 times that of the D group.3.Neuroprotective effects of DNA nanoflowers in vivo.（1）In vivo imaging experiments showed that TD nanoflowers were more concentrated in the brain in the presence of TfR aptamer.（2）The neurological function score and brain tissue TTC staining results showed that compared with the MCAO group,the neurological function score of rats in the TD group was significantly reduced,and the cerebral infarction area was significantly reduced;The results of TUNEL and Nissl staining of the rat brain and brain histopathological examination showed that compared with the MCAO group,the apoptosis of nerve cells in the TD group was significantly reduced;RT-PCR and Western Blot results showed that the content of caspase-3 in the TD group was significantly lower than that in the MCAO group.The histopathological examination results of the heart,liver,spleen,lung,and kidney showed that there were no obvious pathological changes in the important organs of rats in each group,indicating that DNA nanoflowers had good biological safety.Conclusion:In this project,a pure DNA nanosystem with both BBB passing through and silencing caspase-3 in nerve cells was constructed.The system used TfR aptamers to improve the efficiency of nanoflowers BBB crossing,and efficiently loads ASO to silence caspase-3 gene.Both in vivo and in vitro pharmacodynamic experiments showd that DNA nanoflowers had a significant protective effect on nerve cells,and provide new ideas for the treatment of IS.