| OBJECTIVE To establish a stable,repeatable model of blood-brain barrier?BBB?,which would be used to evaluate the effect of size and surface modification on nano-drug BBB permeability.Synthesis of high/low water-soluble nano-drugs as well as the assessment of their BBB permeability would be run on this model so as to provide guidance for the research of central targeting nano-drugs.METHODS 1.Highly purified rat brain microvascular endothelial cells?RBMECs?were obtained from 2-week-old SD rats via double digestion with type-II collagenase and collagenase-dispase,following by high-speed centrifugation and gradient separation.And the RBMECs were identified by immunofluorescence via VIII factor-associated antigen.Furthermore,the tight junctions between cells was observed by transmission electron microscopy?TEM?.2.RBMECs within 5 generations were then inoculated into the upper chamber of Trans well to establish the BBB model.Classic 4h-leakage test,trans-epithelial electrical resistance experiment?TEER?as well as fluorescein disodium salt?FLU?permeability assay were adopted to verify the establishment of the model.The standard positive drug colchicine that can penetrate the BBB and doxorubicin that cannot penetrate the BBB were used to evaluate the established model.3.Penetration screening and assessing of nanoparticles,central targeting nano-drugs and high lipid-soluble nano-drugs?nano-etoposide?with various sizes and surface-modifications were then carried out based on this BBB model.RESULTS 1.The obtained RBMECs exhibited typical monolayer"paving stone"morphology,with a purity over 90%identified by factor VIII-related antigen test.Tight junctions among RBMECs were also observed under TEM,suggesting the superiority of RBMECs cultivation.2.RBMECs were then inoculated into the upper chamber of Transwell 6-well plate with a suitable density.The model then underwent assessments after the cell fusion was observed under microscope.3.A liquid level difference above 0.5cm between the upper and lower grid was well maintained in4h-leakage test,showing obvious barricading effect of the model.The effective electrical resistance value could be up to 200350?·cm2 in TEER,suggesting a compact growth of the cells.Besides,non-central targeting drugs?FLU,DOX?showed penetrating coefficients all lower than 1.0?10-3cm·min-1 within 1h,while BBB penetrating colchicine achieved approximately 3.0?10-3 cm·min-1.Results above showed low permeability and intact barricading function of the model,meeting the BBB model requirement in vitro.3.?1?The TEM and LSCM methods were used to qualitatively evaluate the nanoparticles that penetrated the in vitro BBB model.Observed results showed that 5nm Si NP as well as 100nm Tf-SiNP nanoparticles could penetrate the BBB,while 100nm SiNP,360nm Tf-Si NP and 360nm Si NP nanoparticles cannot.?2?Assessment with this BBB model also suggested that the central targeting nano-drugs Tf-MSN-HI-6 had a penetration coefficient of?6.3±0.7?×10-3cm·min-1,while this number for reactivator HI-6 was?1.2±0.2?×10-3cm·min-1,which meant that the former is 5 times of the later?p<0.01?.?3?The anti-tumor drug Etoposide showed fine penetrating ability against BBB model in vitro,as well as an increase in the release rate and also a significant enhancement in cell killing effect once being prepared as nanoparticale suspension,comparing to that as ordinary powder or solution.CONCLUSION 1.In the process of isolating brain tissue from RBMECs,a high vitality,high purity RBMECs were successfully isolated and cultured by a method of cell purification by selecting appropriate week-old animals,two enzymatic digestions,gradient centrifugation,and subsequent puromycin addition to remove pericytes.2.After comparison,it was found that the P0 generation cells had high viability,good modeling effect,and the cells were inoculated on Transwell following the principle of?flat,gentle,and stable?.Finally,the in vitro BBB model was successfully established,and the classic 4h leakage experiment and TEER experiment were used to complete a series of evaluation and verification of its function.3.Innovative methods of TEM and LSCM were used to qualitatively evaluate the in vitro BBB model.In particular,the use of LSCM enables a simpler,clearer,and more intuitive way of judging how large the size and surface modification nano-drugs can penetrate the in vitro BBB model,solving the difficult problem of difficulty in characterization of nanoparticle central targeting.4.Synthesize silicon nanoparticles with different size and surface modification,and successfully explore the rules and conditions that must be satisfied to penetrate the BBB nanoparticles by in vitro BBB model.5.According to this information,the use of nanotechnology to nanometerize high water-soluble drugs and low water-soluble anti-cancer drugs that can not penetrate the BBB has a good central targeting effect after evaluation in the in vitro BBB model. |
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