| Stroke is one of the major diseases threatening human health,with high morbidity,high disability rate,high recurrence rate and high mortality.Puerarin(Pue)is the main component of Chinese herbal medicine puerarin,which can treat ischemic stroke,improve myocardial contraction,reduce myocardial oxygen consumption and promote microcirculation.However,due to the nature of drugs and the barrier function of blood-brain barrier(BBB),by the ways of oral and intravenous,Pue is difficult to enter brain lesions quickly and effectively,which greatly restricts the therapeutic effect.Pharmacokinetic studies showed that the brain target of purarin was less than 2%for intravenous administration.It is valuable to seek a way of administration with high safety,good compliance,rapid exertion and good brain targeting for puerarin.Nasal administration has the advantages of quick effect and high bioavailability in the treatment of brain diseases.Traditional Chinese medicine has the theory of "nose orifices through brain",and modern medicine has also proved that there is a pathway between the nose and brain,which provides a theoretical basis for the treatment of encephalopathy by nasal administration.Puerarin is isoflavone glycoside with low solubility(4.6 mg/mL),low absolute bioavailability of oral administration,poor cell absorption and mucosal permeability.Although organic solvent(eg.1,2-propanedio)can improve the above defects,it can cause the change of cell physiological structure,which can not be ignored.How to increase the solubility of puerarin and improve its cell absorption ability is of great significance for the exertion of puerarin’s pharmacodynamics.Cubic liquid crystal nanocrystalline(LCN)is a kind of double continuous cubic liquid crystal dispersion system formed spontaneously by amphiphilic lipids and surfactants in water.LCN encapsulate water-soluble drugs in waterways and liposoluble drugs in lipid bilayer to increase solubilization of different polar drugs.LCN has affinity with biofilm and can promote drug entry into cells.At the same time,LCN also has good bioadhesive,biodegradability,safety and other characteristics.It is a new drug nano-carrier.In order to improve the absorption of Pue in nasal cavity and brain,and to improve the low solubility,poor cell permeability of Pue,toxicity of organic solvent compatibilization,etc,in this paper,puerarin cubic liquid crystal nanocrystalline Pue-LCNA was prepared.The formulation and preparation process of Pue-LCN were determined,and its pharmacological properties,nasal absorption,nasal irritation,absorption and mechanism across blood brain barrier were studied.The specific contents as follows:1.Preparation and pharmacological properties of Pue-LCNFirstly,a method for the determination of puerarin in Pue-LCN was established.The free and encapsulated puerarin was separated by ultrafiltration and centrifugation.LCNs were prepared by GMO/F127/glycerol/H2O system,thin-film dispersion method and the ultrasonic time in the preparation process was investigated.Then,taking the encapsulation rate as the index,the prescription ranges of GMO,F127 and 5%glycerol-H2O were investigated by single factor.The results are 1%～4.5%GMO,1%～3%F127,3mg/g～5mg/g Pue.Then according to the single factor experiment results,by the optimal mixing method of Design expert software,in which the drug loading,the encapsulation efficiency and the particle size were taken as the indexes,the prescription was optimized.The best prescription is 2%GMO,2.13%F127,5mg/g Pue and the rest is 5%glycerol-H2O.In order to improve the stability of Pue-LCN,1%vitamin E acetate was selected as the stabilizer of cubic liquid crystal nanoparticles from several stabilizers,which improved the stability of the drug loading,the encapsulation efficiency and the particle size in a week at least.The final prescription is 0.5%Pue,2%GMO,2.13%F127,1%vitamin E acetate,94.37%5%glycerol-H2O.Finally,Pue-LCNs were preliminarily evaluated from the micromorphology,particle size,potential and crystal structure.The appearance of Pue-LCN is milky white,and shape can be observed by projective microscope.The polarizing microscope proves the existence of the crystal,and the X ray diffraction proves LCN is cubic liquid crystal.The particle radius and potential of Pue-LCN is 97.87±0.3386nm and-33.78±2.942mV.Encapsulation efficiency and drug loading are 95.69±0.1377%and 0.5205±0.0187%.2 Circulation absorption in rats of Pue-LCNIn order to evaluate the ability of Pue-LCN to pass through nasal mucosal cells,theabsorption rate of Pue and Pue-LCN was compared with that of rat nasal circulation perfusion model in vivo.Firstly,a method for the determination of Pue in nasal perfusion fluid was established.The irritation of Pue-LCN and its diluent was investigated in detail and intuitively by BCA kit and nasal septum tissue section.The results show that Pue-LCN diluted with normal saline for 10 times and Pue normal saline solution at same concentration cause less irritation,so these two solutions can be used in the experiment of nasal cavity absorption comparison.Finally,the absorption rates of Pue-LCN saline solution in the nasal cavity of rats is 2.480±0.3033 × 10-3·min-1,significantly higher than that in the group of Pue 1.380±0.3033×10-3·min-1.It suggests that the cubic liquid crystal nanoparticles system can increase the permeability of puerarin in rat nasal mucosa and promote the absorption of drugs with poor mucosal permeability in nasal cavity.3 Effects of Pue-LCN on the ciliates of the upper palate toad and irritation of rat mucous membranePue-LCN can increase the absorption of puerarin in nasal cavity of rats,we need to reserch on if it can cause irritarion to on mucous membrane.Two animal models,toad and rat,were used to study the effects of Pue-LCN and Pue-5%1,2-propanedio.In toad cilia toxicity test,the stopping time of cilia in Pue-LCN and Pue-5%1,2-propanedio solution group was 92.90±2.650%and 66.01±8.146%,respectively.The liquid crystal nanoparticles showed less cilia toxicity.In nasal irritation test of rats,both Pue-LCN and Pue-5%propanedio were irritated one day after administration,but Pue-LCN group was less than 5%propanedio group.On the 8th day after administration,Pue-LCN group recovered well,but there was no significant difference in irritation score between Pue-LCN group and normal saline group.Pue-5%propanedio group recovered hardly.It is suggested that the Pue-LCNs compared with Pue-5%propanedio in the same concentration of puerarin are less irritating and easier to recover.4 A reserch on Pue-LCN ransport and cubic liquid crystal nanoparticle uptake on MDCK-MDR1To study the ability of Pue-LCN to penetrate blood-brain barrier and whether cubic liquid crystal nanoparticles can increase the intracellular endocytosis of water-soluble drugs,we use MDCK-MDR1 as in vitro cell model,Pue-LCNs transport on MDCK-MDR1 cells and cubic liquid crystal nanoparticles uptake and pathway were studied.Firstly,a stable cell culture method was established,which can be used as BBB in vitro model.In transport experiments,the cell MTT toxicity test of Pue-LCN shows that when Pue-LCN dilute more than 25 times(Pue<200 μg/ml),it is not toxic to cells.The methods for the determination of Pue by HPLC were established.The results of monolayer transport experiments show that the transport transmittance of Pue-LCN is higher than that of Pue.Papp on absorption direction is 1.799± 0.13 55×10-6 cm·s-1 and on efflux directoion is 1.982±0.1181× 10-6 cm·s-1,which increase 34.92%and 18.64%compared with the same concentration of Pue solution respectively.The increase of Papp prove that LCNs can promote puerarin through blood-brain barrier cells and promote the absorption in brain.The ER of Pue-LCN is less than 2,the result suggestes that the transmembrane transport of Pue was passive diffusion.In uptake experiments,a suitable water-soluble drug calcium was selected to prepare liquid crystal nanoparticles(Cal-LCN).Firstly,Cal-LCN were prepared according to the preparation method of Pue-LCN.Encapsulation efficiency and drug loading are 91.20±0.5829%and 0.2473±0.0106%.Concentration of calcium in Cal-LCN is 250μg/mL.Particle size is 115.9±2.325 nm and PDI is 0.2683±0.0025.In the MTT results,Cal-LCN diluted more than 25 times and 10 ug/mL cal solution are safe on MDCK-MDR1,which can be used on MDCK-MDR1 cell uptake experiment.In the results of uptake experiment,the cubic liquid crystal nanoparticles not only increase the amount of calcein into the cell,but also change the rate of entry into the cell.After that pathway inhibitors(Filipin,cytochalasin D,chlorpromazine,2-D-deoxyglucose)were co-cultured with cells to find the pathway LCN go through MDCK-MDR1.Intracellular fluorescence intensity was determined by flow cytometry.In the results,uptake pathway is related on chlorpromazine and 2-D-deoxyglucose.These results suggest that cubic liquid crystal nanoparticles can increase the ability of water-soluble drugs to enter cells through energy-dependent grid protein mediated endocytosis pathway. |
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