SPHK-1 SiRNA Delivery Against Cerebral Ischemia-reperfusion Injury Mediated By Biomimetic Nanocomplexes

Cerebral ischemia-reperfusion(CIR)injury is a pathological phenomenon in which the degree of tissue damage is further exacerbated by the resumption of blood perfusion in brain tissue that has been ischemic for a prolonged period.During CIR injury,an overactive inflammatory cascade causes damage to the neurovascular unit,resulting in the blood-brain barrier(BBB)disruption and neuronal loss,ultimately leading to functional impairment.In the acute phase of pathogenesis,microglia,as intrinsic immune cells in the brain,are first activated and polarized into a proinflammatory phenotype,initiating a cascade of inflammatory responses.Therefore,inhibiting the inflammatory response and improving the inflammatory microenvironment by modulating microglia is significant for the treatment of CIR injury.Currently,CIR injury is mainly treated clinically by small-molecular inhibitors,whose clinical application is severely limited due to the limited therapeutic targets and side effects.Small interfering RNA(siRNA)is widely applied in the treatment of various diseases owing to its abundant targets,high specificity,and long-lasting efficacy.Nevertheless,siRNA generally has the issues of poor stability,poor targeting and poor membrane permeability.It is crucial to develop safe and efficient delivery vectors for potentiating siRNA therapy.Cationic polymers are important gene delivery vehicles that facilitate efficient gene silencing for the treatment of disease.Unfortunately,the existence of multiple physiological barriers in vivo severely hampers the application of cationic delivery vehicles.Based on the physiological barriers and the microenvironmental characteristics of the focal site,cationic delivery carriers can be modified by functional group or membrane coating technology to modulate the interaction between cation delivery carriers and nucleic acids as well as the microenvironment of the body,thus crossing the physiological barriers and achieving targeted delivery of siRNA.The thesis focused on cross-barriers delivery and inflammation regulation to design a ROS-responsive delivery system targeting microglia to enhance the intracytoplasmic gene silencing effect of sphingosine kinase 1 siRNA(siSPHK-1),reversing the pro-inflammatory state of microglia,improving the inflammatory microenvironment,and mitigating neurovascular unit damage.The main research of this thesis is summarized as follows:In Chapter 1,CIR injury,RNA interference technology,and brain delivery barriers for siRNA are outlined and described.Then,the current representative gene delivery vectors for the treatment of brain diseases and cell membrane coating technology are listed and illustrated.Subsequently,cationic polymer modification strategies for crossing the in vivo delivery barriers are described.In Chapter 2,inflammation-targeting,ROS-responsive dissociated biomimetic nanocomplexes are designed and synthesized for achieving efficient siRNA encapsulation and ROS-responsive release.Firstly,ROS-responsive hyperbranched poly(β-amino ester)s(BS)were synthesized by Michael addition reaction,followed by condensation of siSPHK-1 to form cationic BS/siSPHK-1 nanocomplexes(BSsS NCs)with a particle size of 127.2 nm and a zeta potential of 29.6 mV.BSsS NCs could release siRNA rapidly upon H2O2 treatment(100μM,4h).Subsequently,the hybrid membrane(HM)of platelet membrane(PM)and BV-2 cell membrane(BM)prepared by membrane fusion via ultrasonication was coated onto the surface of BSsS NCs to obtain biomimetic nanocomplexes(HBSsS NCs)with a particle size of 161.1 nm and a zeta potential of-9.3 mV.HBSsS NCs had good serum stability and retained characteristic HM membrane surface proteins,including the PM-specific protein(CD61)and the BMspecific protein(Ibal).In Chapter 3,the biomimetic nanocomplexes(HBSsS NCs)constructed in Chapter 2 are utilized to overcome multiple delivery barriers and deliver siSPHK-1 into microglia for the anti-inflammatory treatment of CIR injury.Upon systemic administration post CIR injury,HBSsS NCs featured long blood circulation due to HMassisted avoidance of opsonization.Subsequently,HBSsS NCs could rely on the stepwise targeting of the outer HM coating to sequentially penetrate the multiple physiological barriers and eventually deliver siSPHK-1 into microglia.Particularly,by inheriting the microthrombus targeting ability of platelets,HBSsS NCs enabled accumulation around the microthrombus.Subsequently,HBSsS NCs recognized CD 106 on the surface of vascular endothelial cells via CD29 on HM and efficiently penetrated the BBB via receptor-mediated transcytosis.Upon entry into brain tissue,HBSsS NCs were able to trigger receptor-mediated endocytosis with the aid of microglia membrane-mediated homotypic targeting and CD29-mediated CD 106 recognition,resulting in efficient uptake by microglia.In the cytosol of inflamed microglia,the high intracellular ROS concentration triggered degradation of BS to liberate siSPHK-1,ultimately provoking efficient SPHK-1 silencing to ameliorate the inflammatory microenvironment,relieve the oxidative stress,attenuate neurovascular unit damage,and promote brain function recovery.In the in vitro BBB model,compared to those of uncoated BSsS NCs,the penetration efficiency and cellular uptake level of HBSsS NCs were increased by 2.9 and 2.7 folds,respectively.In OGD/R-challenged BV-2 cells,the gene silencing efficiency of HBSsS NCs reached 72%.In CIR-injured mouse model,HBSsS NCs inhibited SPHK-1 mRNA level by 68%,and reduced the levels of pro-inflammatory cytokines(TNF-α,IL-6,IL-1β and IL-17A),proteases(MMP-9 and iNOS),and H2O2 in the infarcted tissues by inhibiting the NF-κB signaling pathway.Ultimately,HBSsS NCs improved the inflammatory microenvironment,thereby reducing neuronal and endothelial cell apoptosis and reducing the size of the cerebral infarction to 23%.In addition,the results of the morris water maze experiment demonstrated that the cognitive function of CIR-injured mice was significantly improved after treatment with HBSsS NCs.In Chapter 4,the thesis was summarized and the future work that needed to be improved was discussed.In summary,based on the targeting and modulation of the CIR injury microenvironment,the HM-coated biomimetic siRNA delivery system was developed for crossing the in vivo delivery barriers of siSPHK-1,achieving gene silencing in microglia,improving the inflammatory microenvironment at the site of CIR injury,and achieving an effective treatment of CIR injury in an animal model.This thesis provided new ideas for overcoming biological barriers and inflammatory microenvironment modulation in vivo,as well as new insights into the design of biomimetic intelligent gene delivery vehicles.