| Stroke is a cerebrovascular disease with extremely high incidence,fatality,and disability in the world today.Among them,ischemic stroke accounts for more than 80% of stroke cases.Clinically,patients with ischemic cerebrovascular disease have spontaneous reperfusion,which in turn causes a series of secondary injuries,including oxidative stress,inflammation,and excitatory amino acid toxicity.Therefore,the study of ischemic stroke is of great significance.Traditional Chinese medicine(TCM)compound formula has rich experience in the prevention and treatment of central nervous system diseases through multi-level and multi-target therapeutic methods.Huanglian Jiedu Decoction(HLJDD)was derived from The Handbook of Prescriptions for Emergencies,which was a representative prescription for clearing away heat and detoxification.Compatible composition of four TCMs of Rhizoma Coptidis,Radix Scutellariae,Cortex Phellodendri amurensis and Fructus Gardeniae.Studies have shown that HLJDD has anti-inflammatory,anti-oxidant,anti-bacterial,anti-tumor effects,and could significantly protect brain nerves and improve acute cerebral ischemia reperfusion injury.The blood-brain barrier(BBB)is a special structure between brain tissue and blood,mainly composed of brain capillary endothelial cells and their tight junctions.BBB can restrict toxic substances from entering the central nervous system and prevent almost all molecules from entering the brain.Due to the existence of BBB,drugs used to treat brain diseases can not exert better effects.As an inorganic nanocarrier,mesoporous silica nanoparticles(MSN)has been widely used in the field of biomedicine due to its large specific surface area,adjustable particle size and pore volume,easy surface modification and high biological safety.In this paper,we constructed a MSN co-modified by transferrin receptor monoclonal antibody(OX26)and apolipoprotein E(Apo E)(OX26/Apo E-HLJDD-MSN)to explore the protective effect in the treatment of acute cerebral ischemia reperfusion injury.In this paper,carboxyl functionalized MSN were prepared by an improved hydrothermal synthesis method,OX26/Apo E were coupled to the surface by the carbodiimide method,and the effective part of HLJDD was encapsulated in the pores by the dipping method to prepare into OX26/Apo E-HLJDD-MSN.The results showed that the prepared carrier has a small particle size and uniform dispersion,and the surface electronegativity was easily excreted without causing accumulated toxicity.Phosphate buffered saline(PBS,pH 7.4)and artificial cerebrospinal fluid(ACSF)were used as release media to investigate the in vitro release behavior of the formulation.The results showed that compared with the solution group and the physical mixture group,HLJDD-MSN showed a certain slow-release effect in the two release media.Using a in vivo imaging system(IVIS),and considering Cy5-hydrazide as a fluorescent probe to investigate distribution of Cy5 solution,Cy5-MSN,OX26/Apo E-Cy5-MSN in BALB/c-nu mice at different times after administration,aim at evaluating the in vivo brain targeting of the OX26/Apo E co-modified carrier.The results showed that Cy5 solution had been distributed throughout the body of mice at 1 h,and the fluorescence intensity gradually decreased with the increase of time,and the metabolism was basically complete at 24 h,while Cy5-MSN and OX26/Apo E-Cy5-MSN had a longer circulation in the body,and OX26/Apo E-Cy5-MSN could observe more obvious fluorescence in the brain at 8 and 24 h,indicating that OX26 and Apo E could be recognized by receptors to increase the drugs aggregation concentration in the brain,and has certain brain targeting.Coumarin 6(Cou-6)was used as a fluorescent probe instead of drug encapsulated in MSN,and the mouse brain microvascular endothelial cell line(bEnd.3)was used as a preparation in vitro targeting evaluation model.The effects of MSN and OX26/Apo E-MSN on the activity of bEnd.3cells were evaluated by the CCK-8 method.The high-content imaging system was used to observe and record the the uptake effects of bEnd.3 cells on Cou-6 solution and Cou-6@MSN and Cou-6@OX26/Apo E-MSN,and evaluated the in vitro brain targeting of OX26/Apo E co-modified MSN.The results showed that the content of Cou-6 in MSN and OX26/Apo E-MSN were non-toxic to bEnd.3 cells at a concentration of 0.05 μg/m L,the cell survival rate was greater than 80%.And the fluorescence intensity of the preparations in each group increased with 2,4,and 6 h after administration.The time has increased,and the fluorescence intensity of the antibody-modified group was significantly stronger than that of the solution group and the unmodified group,and the difference was significant(p<0.05).At the same time,the uptake of the antibody-modified preparation by bEnd.3 cells was significantly higher than that of the unmodified group.The remaining two groups showed that the MSN co-modified by OX26 and Apo E can increase the drug uptake ability of bEnd.3 cells through receptor-mediated endocytosis.Divide rats into Model group and Sham group,the cerebral ischemia-reperfusion(MCAO)model was prepared by occluding the middle cerebral artery in rats with a suture method.Three groups of HLJDD solution,HLJDD-MSN,and OX26/Apo E-HLJDD-MSN were administered by tail vein injection,and reperfused 24 hours after administration,and then the behavioral functional deficits were scored by Zea Longa method.The rats were sacrificed and the brains were taken for frozen sections,stained with 2% TTC,and the infarct area was calculated.The results showed that compared with the Model group,the three groups of preparations could reduce the neurobehavioral deficits of the MCAO model mice to a certain extent,reduce the area of cerebral infarction,and have a certain improvement effect on the neuronal cell status of the hippocampus of the brain.At the same time,the area of cerebral infarction in the OX26/Apo E-HLJDD-MSN group was about 10% less than that in the Model group,indicating that the prepared preparation has obvious protective effect on acute cerebral ischemia-reperfusion injury.In summary,the OX26/Apo E-HLJDD-MSN constructed in this experiment could improve the stability of the drugs,increase the content of the drug into the brain through receptor-specific recognition,make the drug accumulate in the brain and play a role,and has obvious effects in improving the acute cerebral ischemia perfusion injury. |
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