| Objective To prepare ANG-PEG-PAMAM doxorubicin nano-complex,we take polyamido-amine of polyethylene glycol(PEG-PAMAM)as carrier material,coupling targeted functional group Angiopep-2(ANG)and fluorescent markers fluorescein isothiocyanate(FITC)or loading anti-cancer drug doxorubicin(doxorubicin,DOX)as model drug.Borneol as a"messenger drugs" of glioma targeted drug delivery system,combining nano-complex with borneol to evaluate its effect in vivo and in vitro,and provide a new ideas of combining glioma targeted drug deliver system with traditional Chinese medicine for the treatment of glioma.Methods Synthesized ANG-PEG-PAMAM with methoxy sealing side mPEG3000,double functional group MAL-PEG3500-NHS and G5 PAMAM as materials.1H-NMR was used to identificate the structure,as well as ensuring grafting rate,nano particle-potential analyzer was used to measure the size and potential;Transmission electron microscopy(TEM)observed its shape.Nano-carrier materials were flagged by FITC and identified their structure with 1H-NMR.We measured its DOX drug loading capacity with fluorescence spectrophotometry and investigated in vitro drug releasing characteristics with dialysis bag method.The brain capillary endothelial cells(HBMEC)and glioma cells(C6)was used to measure the cytotoxicity of carrier materials;Flow cytometry instrument tested the ability of cells uptaking nano-carriers and the effect of borneol on them;we constructed in vitro BBB cells model,and verified effect of borneol on the FITC-nano-carriers transport across BBB.Besides,we set up the co-culture model of HBMEC and C6 cells and examined the effect of borneol on nano-complex anti-C6 cells after transport across BBB.We examined the pharmacokinetic behavior in vivo of the rat which after vein injection of DOX nano-complex combined with lavage of borneol.Results ANG-PEG-PAMAM was synthesized successfully and each PAMAM approximately coupled 31 PEG and 4 ANG.DLS result showed ANG-PEG-PAMAM average particle size was(74.15±1.16)nm,and the average potential was(19.90±2.87)mV.TEM showed that particle was spherical appearance.The drug loading of ANG-PEG-PAMAM/DOX was 6.95%.The results of release in vitro showed that the ANG-PEG-PAMAM/DOX had pH-responsive and sustained characteristics.Besides,PEG modification significantly reduced the toxicity of PAMAM(P<0.05).Cells uptake experiments showed that the uptake of drug carrier had time dependence,with the extension of incubation time,uptake rate increased.The rate of ANG modified carrier was obviously higher than that of the unmodified group(P<0.05).However,compared with add borneol treatment group,borneol did not promote cell uptake.The specific mechanism remains further research.Across BBB transport:according to the results of across BBB after 4 h,ANG-PEG-PAMAM-FITC and without ANG modified group’s transport rate had significant difference(P<0.05),and borneol incubation can improve ANG-PEG-PAMAM-FITC transport rate.HBMEC cells short-term toxicity,according to the results of treatment group compared with blank control group had no significant difference(P>0.05).Across BBB,according to the results of C6 cells survival rate after ANG-PEG-PAMAM/DOX and without ANG qualities and technical group compared with the results of difference to be markedly,and the incubation group compared with borneol,cell survival results have significant difference(P<0.05).Rat caudal vein Injection respectively DOX,PEG-PAMAM/DOX,ANG-PEG-PAMAM/DOX or combined lavage of borneol,compared with DOX solution,the t1/2,AUC and MRT of nano-complex groups increased.Conclusion ANG-PEG-PAMAM was successfully synthesized,and DOX nano-complexs’encapsulation efficiency and drug loadings were higher which had pH-responsive and sustained characteristics.PEG significantly reduced the toxicity of PAMAM;Borneol and Angiopep-2 improved nano-carriers transport across BBB,and enhanced sequential targeting to C6 cells in vitro.The pharmacokinetic behavior of nano-complexs showed long circulation,and borneol changed the distribution and metabolism in vivo. |
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