Preparation And Evaluation Of Cyclovirobuxined Brain Targeting Liposomes

Objective:Cerebral disease is one of the most common diseases,which brings heavy burden to society.Cyclovirobuxine D(CVB-D)is a kind of steroidal alkaloid extracted from Chinese medicinal herb Buxux microphylla,which can improve myocardial ischemia and cerebral ischemia.Huangyang tablet is the main formulation of CVB-D.However,its poor water solubility and low bioavailability limit the application.Moreover,because of the blood brain barrier(BBB),it is difficult to delivery most drugs to brain.Liposome was a research hotspot in the field of formulation in recent years.It has the advantages of good biocompatibility,lower side effect and better targeting.Therefore,choosing Liposome as the carrier for delivering CVB-D can effectively avoid the above disadvantages.Based on the previous study,we find that Compared with gastric and intravenous administration,nasal administration has more brain targeting.Therefore,based on the special physiological structure of nose and BBB,formulation modification with nasal administration was chosen to improve the brain targeting of CVB-D.In conclusion,on the basis of previous study,CVB-D brain-targeting liposomes were prepared and evaluated.Futhermore,targeting in vitro and in vivo distribution via Intranasal Administration were evaluated by microdialysis.Method:1?Established HPLC analysis method for CVB-DBecause there is no double bond in the structure of CVB-D,it can not be detected by UV-detector directly.According to the characteristics of its structure,HPLC-UV determination was carried out after pre-column derivatization method which benzene isocyanate was used as a derivatization reagent.2?Preparation and evaluation of Angiopep-2 conjugated Polysorbate80Coated LiposomesThe connection between DSPE-PEG-MAL and Angiopep-2 was confirmed by nuclear magnetic resonance.The Liposome was prepared by thin film evaporation-probe ultrasonic method.Encapsulation efficiency as index,the mass ratio of phospholipid to cholesterol,phospholipid to CVB-D,ultrasonic time,The molar ratio of phospholipid to DSPE-PEG2000,phospholipid to tween80 were optimized by the single factor method.The preparation was evaluated by liposome morphology,particle size,potential,polydispersity index(PDI),stability and hemolysis.3?In vitro brain-targeting evaluation of CVB-D liposomesThe experimental model of blood brain barrier in vitro was established by permanent mouse microvascular endothelial cells(bEnd.3).The formation of blood brain barrier was evaluated by resistance,4h liquid surface leakage test and HRP permeability test.After the safe concentration given by MTT cytotoxicity test,CVB-D solution,CVB-D liposomes and CVB-D brain-targeting liposomes were given to detect the amount of drugs passing through the blood-brain barrier.4?Established HPLC-MS/MS analysis method for CVB-DCVB-D has strong lipid solubility,and in microdialysis,the fluid is water solubility.Therefore,the drug concentration in the sample is low.In order to determine the drug content accurately,CVB-D HPLC-MS/MS analysis method is established.In vivo brain-targeting evaluation of CVB-D liposomesThe CVB-D brain-targeting liposomes,CVB-D liposomes and CVB-D solution was administered in a single nostril.CVB-D brain-targeting liposomes were injected through the femoral vein.After administrationed,rats blood and cerebrospinal fluid(CSF)were collected by probe at different time points.After determination of drug content,brain targeting can be evaluated by pharmacokinetic parameters and brain targeting index.Results:1?Establish HPLC analysis method of CVB-DChromatographic conditions as follows,instrument:Shimadzu LC-20A;Chromatographic column:Elite-AAA C18(200 x 4.6,5?m);Phenomenex C18protect column(4 x 2.0 mm);Flow rate:1 mL/min;Mobile phase:methanol:water=85:15;Column temperature:room temperature;Sample quantity:10ul;Detection wavelength:240nm;2?The optimal preparation of Angiopep-2 conjugated Polysorbate80 Coated LiposomesMass ratio of 20:1 for phospholipids to CVB-D,7:1 for phospholipids to cholesterol,20 mg/mL phospholipids,3 min for probe ultrasound,and with a molar ratio of 1:0.1 for phospholipids to DSPE-PEG 2000,and 1:0.2 for phospholipids to Polysorbate80.The size of the preparation was(72.03±0.33)nm,zeta potential was(-15.23±0.61)mV,PDI was(0.301±0.01)and the encapsulation efficiency was(92.39±1.29)%.3?Established the BBB model in vitro and evaluated the brain targetingThe cell resistance was higher than 200?.cm~2,the difference of liquid surface was higher than 0.5 cm and the HRP permeability was less than 5%,which indicated the successful establishment of blood brain barrier in vitro.The results of drug transpotation test showed that(70.15±1.23)%CVB-D brain-targeting liposomes could pass through the blood brain barrier,which was significantly higher than CVB-D solution and CVB-D liposomes.4?Evaluated brain targeting in vivo by microdialysisThrough microdialysis sampling technology,both cerebral and blood samples were collected.After pretreatment,the samples were analyzed by HPLC-MS/MS.AUCbrain/AUC blood,DTI%and DTP%indicated that CVB-D brain-targeting liposomes can delivery CVB-D to brain via nasal administration.