| Vascular dementia(VaD)is a severe cognitive dysfunction syndrome caused by ischemic stroke,hemorrhagic stroke and cerebrovascular disease,resulting in hypoperfusion of memory,cognition,and behavior area in the brain.VaD is widely considered as the second most common cause of cognitive dysfunction following Alzheimer's disease.Currently,there is still no effective therapeutic strategy to attenuate or stop the progression for VaD clinically.Under the condition of ischemic and hypoxic stress,the expression of calpastatin is down-regulated,leading to the activation of calpain and the damage of neurons.DY-9836,a novel calmodulin antagonist,can reduce the hydrolysis of calpastatin,alleviate the damage of blood-brain barrier(BBB)induced by ischemia,reduce the infarct volume and exert neuroprotective effects.Nevertheless,low water solubility,short half-life time and poor BBB permeability restrict its further application.Nano drug delivery system is an effective strategy to improve drug bioavailability and BBB permeability.Polysialic acid(PSA)is a homogeneously polymerized biodegradable endogenous polymer.As a hydrophilic linear polymer,PSA could prevent the interaction between the drug and the proteins and cells of immune system,thus improving the stability of drug in vivo.Meanwhile.PSA is competent to ameliorate the circulatory stability and extend circulation time of drug by diminishing urinary excretion and hepatic uptake due to its large hydrated volume and negative charge.Our previous study found that PSA based micelles had the capacity of accumulation in the brain.Therefore,a nanodrug delivery system based on PSA was designed for encapsulation and delivery of DY-9836,so as to improve the BBB permeability and realize effective accumulation of drug in the brain.First,polysialic acid-octadecylamine graft(PSA-ODA)was synthesized via amide reaction between the amino group of octadecylamine(ODA)and the carboxyl group of PSA.When the feeding ratio of ODA was 10%,the degree of substitution of PSA-ODA graft was 3.9%.The critical micelle concentration of PSA-ODA was 120.0 ?g/mL.The number-average size of PSA-ODA micelle was 145.7 ± 2.1 nm.PSA-ODA micelle could effectively encapsulate DY-9836.When the drug feeding amount was 4.0%,drug loading(DL%)and encapsulation efficiency(EE%)were 3.6%and 90.0%,respectively.The particle size of DY-9836-loaded PSA-ODA micelle(PSA-ODA/DY-9836)was 107.0±4.0 um.The DY-9836 release behavior from the micelle showed a sustained release for a prolonged time up to 72 h.Then,the in vivo pharmacokinetic properties of DY-9836 and PSA-ODA/DY9836 were studied in male Sprague-Dawley(SD)rats.The concentration of DY-9836 was measured by high performance liquid chromatography-fluorescence detector(HPLC-FLD)with DY-9760e as the internal standard.The results showed that after DY-9836 was encapsulated in PSA-ODA micelle,the half-life time of DY-9836 increased from 1.27±0.27 h to 3.43 ±0.34 h;the corresponding clearance rate decreased from 2.34 ± 0.24 L/h/kg to 1.20±.0.14 L/h/kg;the mean residence time(MRT)increased from 1.67±0.17 to 3.98 ± 0.10 h.After i.p.injection,the maximum plasma concentration(Cmax)appeared at 0.33 and 0.5 h for DY-9836 and PSA-ODA/DY-9836,respectively.The Cmax of PSA-ODA/DY-9836 was 1.38-fold greater than that of free DY-9836.The area under concentration curve(AUC)calculated from time zero to infinity for free DY-9836 and PSA-ODA/DY-9836 were 437.27 ± 52.74and 836.42 ± 90.69 ?g/L/h,respectively.The cytotoxicity of PSA-ODA graft was studied by MTT method against bEnd.3 cells.The results showed that the cell survival rate was still higher than 65%even when the concentration of PSA-ODA graft reached 1000 ?g/ml,indicating that the PSA-ODA graft had low cytotoxicity.Then,the internalization ability and specific mechanism of PSA-ODA micelle were investigated by laser confocal microscopy and flow cytometry.The results demonstrated that the cellular uptake of PSA-ODA micelle was time-dependent,and the intracellular fluorescence intensity increased with time.When cells were incubated with the micelle under 4?,the fluorescence intensity was dramatically decreased,indicating that the process of cellular uptake was energy-dependent Furthermore,after pre-incubation with chlorpromazine,the amount of cellular uptake decreased by 32.1%,demonstrating that the internalization process of PSA-ODA was mainly mediated by clathrin.Afterwards,the in vitro BBB model was constructed using bEnd.3 cells to investigate the BBB penetration ability of PSA-ODA/DY-9836.The results showed that the apparent permeability coefficient(Papp)of PSA-ODA/DY-9836 was significantly higher than that of DY-9836,displaying that the BBB permeability of DY-9836 was significantly enhanced after it was encapsulated into the PSA-ODA micelleThe right unilateral common carotid arteries occlusion(rUCCAO)model was established using adult male C57BL/6J mice.The distribution of PSA-ODA micelle in the brain of rUCCAO mice was investigated by a Maestro imaging system with indocyanine green(ICG)as the fluorescent probe.The results showed that the fluorescence intensity of the brain of PSA-ODA/ICG micelles was significantly higher than that of the free ICG group after intraperitoneal administration for 2 h and 4 h.Furthermore,the fluorescence intensity of coronal brain slices showed that more micelle was distributed in the hippocampus.Using DY-9760e as the internal standard,the concentration of DY-9836 in hippocampus and other brain tissues was quantitatively detected by HPLC-FLD.The results showed that the drug concentration in hippocampus and other brain tissues firstly increased and then decreased after intraperitoneal administration of PSA-ODA/DY-9836 and DY-9836.The drug concentration in the hippocampus was higher than that of other brain tissues,and the highest drug concentration appeared at 1 h.Furthermore,the drug concentration in the hippocampus of PSA-ODA/DY-9836 group was higher than that of DY-9836 group at 0.5,1,2,4 and 6 h after administration.The results were consistent with those observed by Maestro imaging system.In conclusion,PSA-ODA graft constructed in this study could effectively encapsulate hydrophobic DY-9836 and achieve sustained drug release.After DY-9836 was encapsulated in PSA-ODA micelle,its half-life time in vivo was obviously prolonged and the area under concentration curve was significantly increased.Furthermore,BBB permeability of DY-9836 in vitro and in vivo was significantly enhanced,and drug distribution in the hippocampus was higher than that in other brain tissues.Overall,the PSA-ODA/DY-9836 nanodrug delivery system constructed in this study has a good application prospect in the treatment of VaD... |
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