Preparation And Brain Targeting Evaluation Of Cyclovirobuxine D Nanoparticles

Objective:Nasal drug delivery system is one of the most effective administration way which can improving brain targeting function. It is reported that drug after nasal drug delivery in addition to directly through the blood brain barrier, still can bypass the blood brain barrier directly into the brain tissue, improve the brain targeting drug. At the same time, the application of nano preparation which as a new type of targeted formulations in the nasal drug administration way has caused more and more attention. Its preparation has small molecular, high bioavailability and can easily through the blood brain barrier into the brain tissue. Enhancing the efficacy and reducing the toxic effect, provide in vivo pharmacodynamics and metabolic dynamics with new characteristics. Therefore, its application in targeted brain has a great deal of research prospects. From the above, we studied the topic on "Preparation and brain targeting evaluation of cyclovirobuxine D nanoparticles. " Combined the advantage of intranasal drug delivery and nano preparation, establishing the quality standards of cyclovirobuxine D nanoparticles. Before the formulation prepared, we establish the local pharmacokinetic study using brain microdialysis technology to explore brain targeting advantage of CVB-D after intranasal drug delivery. After the formulation prepared, we did an comprehensive and system evaluation of CVB-D nanoparticles on its brain targeting.Method:Is Established HPLC analysis method for CVB-DCVB-D does not contain a double bond in the molecular structure, so directly measured with UV detector belongs to end absorption with poor specificity and low sensitivity. This study used the characteristics of CVB-D containing p-amin in its molecular structure. HPLC-UV determination was carried out after pre-column derivatization method, using phenyl isocyanate as a derivatization reagent. And the chromatographic conditions, standard curve, precision and accuracy, stability, etc were examined.2、Established HPLC-MS/MS analysis method for CVB-DCVB-D belongs to the fat-soluble alkaloid. Microdialysis perfusion fluid is water solubility which close to the internal environment. Therefore, recovery rate of the probe is low, so the study also established HPLC-MS/MS analysis method for CVB-D. The method has high specificity, high sensitivity, and can also eliminate operations error. Therefore, It can accurately measure CVB-D concentration in the microdialysis samples and improve data reliability. The study also examined the specificity of the chromatographic conditions, standard curve, precision and accuracy, stability, extraction recovery and matrix effects etc.3、Application modifier perfusion fluid for microdialysis sampling and established reverse microdialysis sampling method of CVB-DMicrodialysis perfusion fluid including artificial cerebrospinal fluid and ringer’s solution is close to the body environment perfusion fluid. The solubility of CVB-D in the water is 0.105 mg/mL, so low solubility lead to low probe recovery. Based on the previous research(Perfusion liquid modifier contain ethanol, B-cyclodextrin, glycerin, dimethyl sulfoxide were investigated), this study determined using 30% ethanol as perfusion fluid modifier. (Details can refer to doctoral dissertation of the team members xin-guo Liu).4、The holistic and local pharmacokinetic of CVB-D in rats were compared via intragastric, intranasal, and intravenous drug delivery routesCombined with microdialysis technology and LC-MS/MS analysis method, microdialysis sampling were established in both plasma and brain of the rat after intragastric, intranasal, and intravenous administration for CVB-D. Then we compared the pharmacokinetic parameters of brain and blood, AUCbrain/AUCblood, DTE%, DTP%, oral absolute bioavailability, etc.Explore whether there is a certain brain targeting after intranasal drug delivery compared with other two delivery ways.5、The preparation of nanoparticles of CVB-D based on chitosanThis study prepared chitosan nanoparticles using modified ionic crosslinking method. The preparation process of chitosan nanoparticles were optimized, and the trapping efficiency and drug-loading rate is target. Through examine a variety of factors, such as:CS and TPP(poly sodium phosphate) ratio, the quality of the CS concentration, stirring speed and time, concentration of solution pH value and added drug concentration ect.6, Brain targeting property evaluation of CVB-D nanoparticles.The CVB-D loaded CS-NPs and the CVB-D solution was administered in a single nostril. The CVB-D loaded CS-NPs was injected through the tail vein. After administrationed, rats blood was collected by abdominal aortic method at different time points. At the same time, silk, hippocampus and the rest of the brain were further isolated, respectively. Blood and brain tissue were analysised after slurry and extraction treatment. The different tissue parts local pharmacokinetic parameters were compared, such as MRT, AUCbrain/AUCblood, Cmax, Tmax, DTE% and DTP% ect. Investigating whether the prepared CS-NPs can enhance brain targeting. Discussing brain targeting mechanism of nanoparticles after analyzing the distribution of drugs in the brain tissue.Results:1、Establish the HPLC analysis method of CVB-DChromatographic conditions as follows, instrument:Agilent 1100; Chromatographic column:Elite-AAA C18 (200 × 4.6,5um); Phenomenex C18 protect column (4 × 2.0 mm); Mobile phase:methanol:water= 85:15; Flow rate:1 ml/min; Column temperature:room temperature; Sample quantity:10ul; Detection wavelength:240nm; Theoretical plate number according to the CVB-D not less than 3000.2、Establish the HPLC-MS/MS analysis method of CVB-DThe chromatographic conditionsHPLC/MS/MS:Agilent 1260 and 6460 triple quadrupole system; Chromatographic column:Agilent C18 (150 × 4.6,5 um); Phenomenex C18 protect column(4×2.0 mm); Mobile phase:methanol:water(0.1% formic acid and 5mm ammonium acetate)= 50:50; Velocity:1 ml/min, the 3-way shunt; Column temperature:40℃; Sample quantity:5ul;Mass spectrometry conditionsElectrospray ionization source(ESI) and MassHunter B 05 data collector; Spray voltage:3500v; Atomizing air pressure:45psi; Heated capillary temperature:300℃; Positive ion mode detection, scanning mode for multiple reaction monitoring (MRM); Quantitative ions are m/z 403.4-372.3 for CVB-D and m/z 380.2-243.1 for donepezil(internal standard) respectively; Scan time is 0.5s;3、On the previous research (The investigated modifier perfusion fluid contain ethanol, B-cyclodextrin, glycerin, dimethyl sulfoxide), we determined using 30% ethanol as modifying perfusion fluid by flow rate of 1.0 uL/min.4、Rat blood and brain samples were collected at the same time using micro dialysis technology. Dialysis samples were measured after disposed, local and pharmacokinetic parameters of blood such as the oral absolute bioavailability, AUCbrain/AUCblood, DTE%, DTP%, etc were compared, the results prove that nasal drug delivery has certain advantages of brain targeting when compared with other two delivery ways.5、The preparation process of chitosan nanoparticles is as follows:CS and TPP quality ratio is 2.5:1; CS concentration is 1.8 mg/ml; Solution pH is 5.0; The mixing speed is 800 r/min for 0.5 h. CVB-D concentration is 2.0 mg/ml; CVB-D loaded CS-NPs is obtained with envelopment rate 62.82±2.59, zeta potential 33.9±1.7 mV and size scope of 235.37±12.71nm.6、According to the brain tissue distribution study, the research found that Cmax significantly increased after nasal drug delivery and Tmax shortened. Moreover drug-time curve olfactory bulb, hippocampus appear double peak. We speculated that the highest peak(0.5h) could be drugs through olfactory bulb pathways enter into the brain. And through consulting relevant literature, we infer the second peak (about 2 or 4h) may be drugs reach the brain tissue by the trigeminal nerve pathways. Results show that the prepared CVB-D loaded CS-NPs has a certain brain targeting.Conclusion1、In the established chromatographic conditions, the CVB-D concentration appear good linear relationship with the corresponding peak area as well as its ratio. Furthermore, specificity meet with the requirements in biological samples and peak appear 7.2 min. Separating degree is good. It can be used for quantitative analysis of CVB-D.2、under the condition of established chromatography mass spectrometry, the corresponding peak area ratio of CVB-D and internal standard and CVB-D concentration appeared good linear relationship. Specificity, inter-and intra-day precision, stability, the matrix effect, dilution test, plasma sample processing recovery rate all can meet the requirements of biological sample testing. The HPLC-MS/MS analysis method can accurately used in CVB-D quantitative analysis.3、30% ethanol can significantly improve the recovery of the probe and can be used as perfusion liquid modifier of Lipid-soluble drugs.4、The study successfully established a micro dialysis sampling technology in rat blood and brain at the same time. Simultaneously, the micro dialysis sample appeared good stability, high specificity and recovery after disposed. The method can be used in CVB-D micro dialysis sample processing. By comparing brain local and blood pharmacokinetic parameters after dosing, results showed the higher drug concentration in the brain after intranasal drug delivery, which indicate there is a certain brain targeting.5、Through the optimization of the ionic crosslinking method, we determined the complete preparation technology of chitosan nanoparticles.6、The results prove that the prepared CVB-D loaded CS-NPs has a stronger brain targeting and drugs enterment into the brain tissue after intranasal drug delivery approach may be a comprehensive results of olfactory bulb, trigeminal nerve pathways and systemic pathways.