| In the understanding of Traditional Chinese Medicine theory,the nose is a clear orifice which its qi is connected to the brain.The nasal cavity is directly connected with the brain through the Du meridian and the full bladder meridian.,so nasal drug delivery can be used to treat brain diseases.The treatment of CNS disease by nasal administration of traditional Chinese medicine has become a hot topic in Chinese medicine pharmacy in recent years.The traditional Chinese medicine compound nasal administration to the central nervous system,which is represented by ischemic cerebral apoplexy,has the advantages of high efficiency,high safety and good compliance.Tongqiaosanyu fang which consists of pueraria,mint and paeonia is an effective experience in the treatment of acute phase of ischemic stroke.The present study based on the theory of traditional Chinese medicine and modern technology,preparation research study the effective constituent of Tongqiaosanyu fang microemulsion transport from the nose to the brain characteristics and to elucidate the influence mechanism of puerarin transport.The drug "nose-brain" delivery is a complex process.The drug can be absorbed by intracellular transport and cell bypass in the nasal endothelium or ingested by the olfactory nerves in the form of phagocytosis or pinocytosis and passed into the brain by the axon of the nerve cell and the above process is the "nose-brain" direct pathway.The olfactory nerve pathway can bypass the blood-brain barrier and realize rapid and efficient delivery,which is the key to the brain targeting of the nasal administration.The mucosal epithelial cells of the drug cross-respiratory tract are absorbed into the blood circulation and then enter the brain tissue through the blood-brain barrier,which is the main "nose-brain" indirect pathway of the nasal drug targeting central nervous system.After the drug is administered through the nasal cavity,the process of the drug transport to the brain depends on the speed and extent of the two approaches.The present research model of "nose-brain" delivery is mainly in animal model and in vitro cell model.Because of the physiological differences between the nasal structures and human beings,the results of the study cannot fully characterize the human nasal absorption,and it is not conducive to the study of mechanism.In vitro cell model has a homogeneous genetic background and repeatability,which is suitable for rapid evaluation and screening of drug transport and it is a powerful tool for the study of the nasal infiltration of drugs.The existing model of primary nasal mucosal epithelial cell model,human lung adenocarcinoma cell model and so on can be used for the study of the indirect pathway of nasal-brain.There is no external model of the "nasal-brain" direct pathway.The appropriate "naso-brain" direct pathway in vitro model should be able to reflect the characteristics of the drug through the olfactory nerve transport.Therefore,the present study proposes to establish a cell model combination with"nose-brain" multichannel(direct and indirect pathway)transport characteristics,which can be used to simulate nasal administration and evaluation in vitro.It is of great significance for the development of new nasal preparation,especially Chinese medicine compound.The blood-brain barrier is one of the main barriers to drug transport across the "nasal-brain"indirect pathway.The pathologic process of ischemic cerebral apoplexy in the central nervous system can lead to significant changes in the structure of the blood-brain barrier which directly impact on drug absorption and transport.Normal blood-brain barrier cell model can be used to study drug transport characteristics and drug screening.The establishment of the sugar-oxygen deprivation model can predict the absorption of drugs under pathological conditions and guide the clinical medication.Based on the characteristics of "nose-brain" delivery,this paper established a multi-pathway cell combination that can be reflected in the nasal absorption of the nasal administration.The MDCK-MDR1 cells were selected as the extracorporeal cell model of the blood-brain barrier and then establish the normal-pathological BBB model.We examined the mechanism of the effective constituent of Tongqiaosanyu fang microemulsion transport to brain by nasal administration as well as investigate the effect of cerebral ischemia on drug transport by BBB.Finally,a small animal imaging technique was used to investigate the distribution of the"nose to brain" delivery in different rat models,and the reliability of the cell model was evaluated.1.The establishment of the combination of "nose-brain" multi-channel extracorporeal cell model and the study on the absorption characteristics and mechanism of the main components of Tongqiaosanyu fang.The establishment of the "nose-brain" multi-pathway in vitro cell model combination is based on the calu-3 model which can reflect the indirect pathway of the "nose-brain" and the OECs model that can reflect the direct pathway of the nasal-brain.Using MTT assay to determine the appropriate drug concentration.In this study,the characteristics of puerarin in the microemulsion were studied,and the mechanism of the multichannel transport of puerarin through the "nose-brain" was studied.This study was based on the nasal septum of the newborn rat,and it was obtained by using trypsin digestion method to get the olfactory receptor neurons(ORNs).The cell was isolated by flow separation,and the high purity ORNs cells were obtained.However,ORNs is highly differentiated neurons,which cannot be propagated and proliferated,with low content and high cost.Therefore,olfactory nerve sheath cells(OECs)with glial nerve cell characteristics were selected as neuronal pathway cell models.The OECs were obtained by using trypsin digestion which based on the olfactory bulb of neonatal rats.The cells were initially purified with DMEM/F12 medium(including 15%fetal bovine serum),and the cell was labeled with P75 antibody,and the cell purity was 99.7%.The OECs were selected for the polycarbonate cultured bottle and cultivated with DMEM/F12 medium(including 10%FBS,2 mu M forskolin/L,20ng/mL BPE).The OECs culture adherent growth after 36 h,cells in good condition,present a bipolar or three pole shape,high transparency,strong refraction,the method of primitive culture OECs activity is higher,good repeatability.The MTT colorimetry was to determine the concentration of microemulsion transport to the combination of "nose-brain" cell models.This paper investigation the cytotoxicity of Calu-3 and OECs with the blank microemulsion,puerarin microemulsion,puerarin compatibility menthol microemulsion,puerarin compatibility paeoniflorin microemulsion and puerarin compatibility menthol and paeoniflorin micro emulsion,in order to determine the dosage of drug administration for the subsequent transport and uptake study and we also made a preliminary evaluation of the nasal irritant of the tongqiaosanyu microemulsion.The effect of microemulsion dilution on calu-3 cells was 24h.The blank microemulsion,puerarin microemulsion,puerarin compatibility menthol microemulsion,puerarin compatibility paeoniflorin microemulsion and puerarin compatibility menthol and paeoniflorin microemulsion all were not significantly toxic to cells by HBSS diluted 500 times.The microemulsion incubate with OECs 4h to investigate the toxic effect,and the blank microemulsion,puerarin microemulsion and puerarin compatibility paeoniflorin microemulsion behave not significantly toxic to cells by HBSS diluted 300 times,and the dilution times was 400 on the effect of puerarin compatibility menthol microemulsion and puerarin compatibility menthol and paeoniflorin microemulsion.The toxicity of microemulsion in each group was mainly derived from the component of microemulsion,and the toxicity of microemulsion decreased with the dilution multiple.In the following experiments,the attenuated microemulsion was selected without cytotoxicity.Calu-3 cell lines are derived from human lung adenocarcinoma,and in this study group,Calu-3 cells were cultured in MEM culture medium with NEAA.When the cells were 90%confluence,the cells with an appropriate density were digested and adjusted to the bottom of the small chamber of the 12-transwell.After 4-6h cells adhered to the wall,the transwells were flipped for liquid-liquid culture,when the TEER values come to 700-800 Ω cm2 the culture model replaced with gas-liquid culture for 24h and the "nose-brain" multi-channel extracorporeal cell models were built futher more.The Papp value of puerarin solution was 0.91 × 10-6cm·s-1 on the combination of "naso-brain" multi-pathway cell model,suggesting that puerarin was difficult to absorb.Puerarin uptake by OECs can be balanced of 90min,and its intake is 0.1785mg/g(drug/protein).The Papp value of puerarin was between 1.497×10-6 cm·s-1 and 1.54×10-6 cm·s-1 for absorption direction of microemulsion in each group.This may be related to the effect of microemulsion matrix(propanediol,etc.)on the single layer of cells.The drug uptake of puerarin microemulsion was significantly higher in OECs than in the puerarin solution group.OECs uptake puerarin in each group of microemulsion reached a balance at 60min and the uptake speed and degree were higher than the puerarin solution group which suggested that microemulsion plays a role in promoting the uptake of drug cells.The mechanism may be increasing the fluidity of cell membrane in the microemulsion matrix and promoting microemulsion endocytosis.Endocytosis is the main way for nerve cells to ingest exogenous substances.The microemulsion of coumarin was found to accumulate in OECs for only 5min.The mechanism of endocytosis is studied through inhibitors and OECs is mainly dependent on the caveolin-mediated endocytosis and macropinocytosis,and the process is accompanied by energy dependence.The inhibition rate of the microemulsion uptake of OECs was shown as follows:D deoxyglucose group was 39.39%;Non-law group 81.18%;Cell relaxin D group was 47.91%.The advantages of nasal delivery to brain targeting are reflected in the intracerebral delivery of macromolecular preparations.Therefore,the combination of "nose-brain" multi-pathway cell model should be able to reflect the characteristics of nasal absorption of large molecules and the transport characteristics of the agents related to particle size.This paper regard fluorescein isothiocyanate right-lateral glycosidase(FD4 FD10,FD20,FD40)and different particle size(20 nm,40 nm,60 nm,80 nm to 100 nm)fluorescent silver nanoparticles as model drug and study the transport in "nose-brain" combined cell models:The Papp value of FD4-fFD0 across the cells monolayer of calu-3 cells absorption direction was 0.0056 × 10-6 cm·s-1-0.0392 × 10-6 cm×s-1,showing the characteristics that could hardly be absorbed;OECs can uptake FD4-FD10,and its intake decreased with the increase of molecular weight,indicating that the water-soluble macromolecules are scarcely absorbed in the nasal administration which the main pathway of absorption is the uptake of olfactory pathway.The absorption results of different particle size fluorescence nanoparticles in the combined model showed with the Papp value of the particle size of the nanoparticles below 40nm was greater than 1 × 10-6 cm·s-1 through the Calu-3 monolayer.The uptake of nanoparticles by OECs decreases with the increase of particle size,suggesting that nanoparticles with smaller size are beneficial to nasal absorption,and the nasal absorption of large particle size nanoparticles is mainly through the uptake of olfactory nerves.The mechanism of Tongqiaosanyu fang main ingredient microemulsion transport and uptake on the combination "nasal-brain" multi-pathway cell model is as follows:Puerarin solution incubated on the Calu-3 monolayer for 3h,it had no significant effect on the tight junction(TJ).However,there was significant damage to claudin-1,Occludin,ZO-1,and F-actin after the blank microemulsion and each group of medicine containing microemulsion incubated on the Calu-3 monolayer.It is expressed as a partial fracture of the tight junction protein,the fluorescent stripe is blurred,the cells are loosely connected and the intercellular space increased,which suggested that the effect of microemulsion on the absorption of puerarin in Calu-3 cells is related to the open function of its TJ.Compared with the control group of HBSS,the cell membrane potential increased after the incubated with puerarin solution and increased drug transport barriers.The effect of puerarin solution was reversed by the effect of both blank microemulsion and each group of medicine containing microemulsion which could lead to depolarization of the cell,decrease of membrane potential,decrease the viscosity of cell membrane,and increase its permeability.In addition,the results of laser photobleaching recovery method for the determination of cell membrane fluidity showed that microemulsion could also improve cell membrane fluidity and promote the transcellar absorption of puerarin.Puerarin transport is a passive diffusion process without the use of ATP energy.The activity of Na+,K+-ATPase and Ca2+-ATPase can be enhanced after microemulsion cultured cells,suggesting that the drug with active carrier transport in the microemulsion preparation can promote absorption.2.Study on the transport of the effective component of Tongqiaosanyu microemulsion on the in vitro blood brain barrier cell model.Drug transport on blood-brain barrier is the key to the "nose to brain" indirect pathway targeting the brain.The appropriate model of the blood brain barrier in vitro is a powerful tool for the study of the drug "nose to brain" delivery.MDCK cellsand MDCK-MDR1 cells which transfected with multidrug resistance gene MDR1,are commonly used in vitro blood brain barrier cell models of epithelial sources.HBMECs are derived from the human brain microvascular endothelium,which is a common BBB endothelial cell model in vitro.In this study,we compared the TEER values of the three normal model cell models,study the leakage test of fluorescein isothiocyanate dextral glucoside,test the fluorescent nanosilver particle transport experiments with different particle sizes(20nm-100nm),investigated the distribution of TJ proteins,determined of p-gp activity and based on the above experiments,the characteristics of blood brain barrier were evaluated.The TEER values of MDCK cell confluence monolayer was stable in 200-300 Ω·cm2;MDCK-MDR1 cells form a complete monolayer with a TEER value about 250-350 Ω·cm2;while the TEER value of HBMECs monolayer was about 250-350 Ω·cm2.The Papp values of FD4,FD10,FD20,FD40 on MDCK,MDCK-MDR1,and HBMECs cells were less than 1×10-6 cm/s that almost impossible to pass through the cell monolayer.It show that the above three cells are closely connected and all of them can be used to simulate in vitro blood-brain barrier.The results of different particle size nano-silver transport showed that when the nanoparticles were less than 40nm,the Papp value was greater than 1 × 10-6 cm/s.Compared the Papp values of different particle size fluorescence nanoparticles with HBMECs,MDCK and MDCK-MDR1 cells,MDCK-MDR1 cell with closely TJ was more suitable for simulating blood brain barrier in vitro model.Immunohistochemical results indicated that claudin-1 Occludin,zo-1,and f-actin were highly expressed in HBMECs,MDCK-MDR1 and MDCK cells.P-gp activity evaluation results showed that,compared with MDCK and HBMECs,MDCK-MDR1 cells were more highly expressed P-gp.In the pathological state of ischemic stroke,the blood-brain barrier changes.We used physical anoxia and chemical hypoxia to establish in vitro blood-brain barrier MDCK,MDCK-MDR1 and HBMECs OGD models.After OGD,the permeability of the cell monolayer of the three cells increased,showing the leakage of FD4 in the cell monolayer,and Papp value was 3.77×10-6cm/s、2.75×1 0-6 cm/s、6.02×10-6cm/s,which suggest the paracellar was free by increasing the particle size of nanoparticles,and the tight junction protein was broken.In summary,we choose MDCK-MDR1 as the simulation of the blood brain barrier in vitro cell model of follow-up experiments for stability batches,compact connection,forming dense cell monolayer,and high expression of P-gp,sensitive to sugar oxygen deprivation intervention.The drug concentration was determined by MTT assay.The effect of microemulsion on MDCK-MDR1 cells was test for 24h.Blank microemulsion,puerarin microemulsion,puerarin compatibility menthol(1:0.5)microemulsion,puerarin compatibility paeoniflorin(1:0.4)microemulsion and puerarin compatibility menthol and paeoniflorin(1:0.5:0.4)microemulsion with HBSS 500 times dilution had no significant toxicity to cells.The transport results of microemulsion effect on puerarin in vitro blood-brain barrier model MDCK-MDR1 are as follows:The Papp value of puerarin solution in normal MDCK-MDR1 monolayer was 1.04×10-6 cm/s(AP-BL)and 1.05×10-6 cm/s(BL-AP),and the external row rate is less than 2.Puerarin absorption is passive diffusion,without active process participation.The microemulsion of each group significantly increased the bilateral transport of puerarin in the normal cell model of MDCK-MDR1,suggesting that microemulsion had the effect of promoting the absorption of puerarin paracellar.Its mechanism may be associated with nonspecific permeability mediated,high concentration of surfactant in the micro emulsion EL35 and 1,2 propanediol synergy,cause nonspecific penetration,and increased the drug transport across the blood-brain barrier.Compared with puerarin microemulsion,the Papp of puerarin transport on the normal cell model was significantly increased in menthol microemulsion.The reason may be the release of menthol in the process of microemulsion transport has the synergistic effect on the cell monolayer,and further promote the absorption of puerarin,indicating the synergistic effect of Chinese medicine compatibility.The Papp values of the puerarin solution transport on the OGD model were 1.34×10-6 cm/s(AP-BL)and 1.39×1 0-6 cm/s(BL-AP)respectively.The Papp values both side were significantly higher than the normal model.Compared with the normal model,there was a significant increase in the bilateral Papp values in the OGD MDCK-MDR1 transport by the microemulsion groups.It was suggested that under pathological conditions,both the solution and the microemulsion were easier to cross the blood-brain barrier into the central nervous system.Compared with puerarin solution and each group microemulsion preparation,it found that microemulsion can further promote puerarin transport in pathological conditions.The mechanism of microemulsification promote puerarin absorption is closely related to the destruction of claudin-1,Occludin,zo-1 and f-actin.After the culture of microemulsion,the intercellular connections were loose and the gap between the cells increased,suggesting that the microemulsion increased the transport of puerarin in MDCK-MDR1 by paracellar.The membrane potential of cells was also significantly affected by microemulsion.Compared with puerarin solution,the diluted microemulsion can depolarize the cell,reduce the membrane potential,reduce the viscosity of the cell membrane,and increase its permeability.In addition,the results of laser photobleaching recovery method for the determination of cell membrane fluidity showed that microemulsion could improve cell membrane fluidity and promote the transcellar transport of puerarin.Microemulsion can improve the activity of Na+,K+-ATPase and Ca2+-ATPase in MDCK-MDR1 cells,suggesting that the drug with active carrier transport in microemulsion preparation can promote absorption.3.Using vivo imaging technology to investigate the characteristics and distribution of the intracerebral after microemulsion nasal deliveryThe vivo imaging technology was used to test the fluorescence distribution intracerebral after microemulsion nasal delivery with normal model rats,stroke model(MCAO)rats and olfactory nerve block model rats as experimental animals and compared with the results of in vitro transport on cell model to verify the reliability of the cell model.After microemulsion nasal administration the fluorescence was mainly distributed in the brain in the normal rats and no distribution was found in other tissues,showed significant brain targeting.The microemulsion was first distributed in the olfactory bulb,and then distributed from the olfactory bulb to the brain which suggested that microemulsion can be transported into the brain with the characteristics of rapid targeting by the "nose-brain" olfactory neural pathway.This was consistent with the results of the previous study on the OECs uptake of coumarin microemulsion.Olfactory nerve block model rats still have fluorescence distribution in the brain,suggesting that the indirect pathway of "nose-brain" is involved in microemulsion delivery into the brain.However,the diffusion rate and degree of microemulsion was lower than that of normal model rats,which could be speculated that the "nose-brain" olfactory neural pathway played an important role in the delivery of nasal and brain.Compared with normal model,MCAO rats had faster brain speed and greater degree than normal model.It is suggested that the lesion of blood-brain barrier in rats with pathological model was revealed that increasing the infiltration of microemulsion paracellar transport and increase the level of brain delivery.The results were consistent with the results of the study on the increase of the puerarin transport on the blood brain barrier cell model. |
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