| Objective:PTX is an important drug for the clinical treatment of lung cancer.At present,the clinical PTX preparation has selected the polyoxyethylene castor oil and ethanol as a solvent,however the solvent is hypersensitivity and neurotoxic.A large number of studies have shown that combination of CUR and PTX has the effect of synergistic and detoxification.CUR is a safe antitumor candidate with multiple targets for anti-tumor effect.However,because of the poor solubility and low bioavailability of CUR,its clinical application is limited.And it is very important to find a new dosage form of CUR-PTX compound that can improve its solubility and safety and efficiency.Methods:1.CUR lipid nanoparticles(CUR-LN)were prepared by thin film dispersion method.The optimum prescription was selected by process screening,single factor investigation and orthogonal test.PTX lipid nanoparticles(PTX-LN)were prepared by thin film dispersion method.The optimum prescription was selected by process screening,single factor investigation and orthogonal test.2.MTT method for detected the inhibitory effect of CUR and PTX at different concentrations and different ratios(5:1,10:1,40:1,80:1,w/w)on lung cancer A549 cell and A549-T cell at 72 h;MTT method for detected the inhinitory effect of CUR-LN and PTX-LN at different concentrations and different ratios(5:1,10:1,40:1,80:1,w/w)on lung cancer A549 cell and A549-T cell at 72 h.3.The target sites of CUR,PTX and CUR+PTX in A549 and A549-T cells were detected by laser confocal microscopy.4.The effects of CUR,PTX and CUR+PTX on the apoptosis of A549 and A549-T were detected by flow cytometry.5.Using rats as animal model,CUR were administered by tail vein(30 mg/kg),PTX(30 mg/kg),CUR-PTX(30:30 w/w),CUR-LN(about CUR mg/kg)PTX-LN(about PTX 30 mg/kg).And at 5 min,10 min,15 min,30 min,45 min,60,min 120 min,240 min,480 min,1440 min draw blood samples.The drug concentrations were determined by HPLC.The pharmacokinetic parameters were calculated by software DAS 2.Results:1.CUR-LN was prepared by thin film dispersion method.After a single factor investigation,a better preparation process was obtained,namely the rotational evaporation temperature was 45?,the ultrasonic time of the probe was 7 min,and the ultrasonic intensity was 50%.The better prescriptions were PVPK15 30 mg,HSPC 60 mg,and CUR 5 mg.Through orthogonal test,the best prescription was selected as CUR=5 mg,PVPKi5=25 mg,HSPC=55 mg.PTX-LN was prepared by thin film dispersion method.After a single factor investigation,a better preparation process was obtained,namely the rotational evaporation temperature was 35?,the ultrasonic time of the probe was 5 min,and the ultrasonic intensity was 50%.The better prescriptions were HSPC 25 mg,F68 60 mg,and PTX 5 mg.Through orthogonal test,the best prescription was selected as PTX=5 mg,HSPC=17.14 mg,F68=42.86 mg the ultrasonic intensity was 55%and the ultrasonic time was 6 min.2.For A549 cells,with the increase of CUR:PTX ratio,the activity of combined administration increased 4.58-1.40 times compared with CUR.With the increase of CUR:PTX ratio,the activity of combined administration increased 52.38-250.89 times combined with PTX.Compared with CUR,CUR-LN,PTX and PTX-LN,the activity were increased 11.07-2.11 times,11.07-2.11 times,132.42-397.25 times and 4.47-13.42 times respectively after the combination of CUR-LN and PTX-LN in different proportions.For A549-T cells,with the increase of CUR:PTX ratio,the activity of combined administration increased 2.95-1.50 times combined with CUR.With the increase of CUR:PTX ratio,the activity of combined administration increased 38.00-285.00 times combined with PTX.After combining CUR-LN and PTX-LN in different proportions,the activity increased 7.35-2.30 times,5.81-1.82 times,95.00-482.31 times and 21.29-108.08 times compared with CUR,CUR-LN,PTX and PTX-LN,respectively.3.The main coloration of CUR is the nucleus of A549,the main coloration of CUR is the nucleus and cytoplasm A549-T the coloration of PTX is mainly in the cytoplasm of A549 and A549-T.The nucleus and cytoplasm of the coloration of A549 and A549-T,and the color intensity of the target is increased.4.The apoptosis rate of PTX induced by 24h at 10,20 and 40 g/mL was 20.61%,44.50%and 46.79%,respectively,in A549.The apoptosis rates of A549 cells induced by different concentrations of PTX(10,20,40 g/mL)were 20.61%,44.50%and 46.79%at 24h,respectively.The apoptosis rates of A549 cells induced by different concentrations of CUR(5,10,20 g/mL)were 23.56%,32.60%and 40.26%at 24h,respectively.The apoptosis rates of A549 cells induced by different concentrations of 5 ?g/mL(CUR)+l ?g/mL(PTX)?10?g/mL(CUR)+2 ?g/mL(PTX)?20?g/mL(CUR)+4 ?g/mL(PTX)were 24.58%,50.95%and 72.35%at 24h,respectively.The apoptosis rates of A549-T cells induced by different concentrations of PTX(10,20,40 g/mL)were 16.18%,20.44%and 27.12%at 24h,respectively.The apoptosis rates of A549-T cells induced by different concentrations of CUR(5,10,20 g/mL)were 7.58%,24.19%and 25.06%at 24h,respectively.The apoptosis rates of A549 cells induced by different concentrations of 5 ?g/mL(CUR)+1?g/mL(PTX)?10?g/mL(CUR)+2 ?g/mL(PTX)?20 ?g/mL(CUR)+4 ?g/mL(PTX)were 21.02%,28.43 and 65.34%at 24h,respectively.5.The AUC(0-t),MRT,tl/2z,and Cmax of CUR-LN are 9.55,11.82,5.67,and 2.84 times of CUR,respectively,and the clearance rate of CUR(CLz)is 7.33 times that of CUR-LN.The AUC(0-t),MRT,t1/2z,and Cmax of PTX-LN are 7.76,12.88,15.67,and 2.24 times of PTX,and PTX clearance(CLz)is 9.36 times that of PTX-LN.Conclusions:1.The HPLC analysis method of CUR-LN and PTX-LN were successfully established,CUR-LN and PTX-LN was successfully prepared by thin film dispersion.2.After combined use of CUR and PTX at different ratio,the inhibitory effects on human lung cancer A549 cell were significantly increased.The inhibitory effect of combine CUR-LN with PTX-LN on A549 cells was better than combined CUR with PTX.It is suggested that CUR-LN and PTX-LN have a good inhibitory effect on A549 cells,and significantly increases the efficacy of CUR and PTX.After combined use of CUR and PTX at different ratio,the inhibitory effects on human lung cancer A549-T cell were significantly increased,and reversal of drug resistance.After the preparation of CUR into CUR-LN and PTX into PTX-LN,the inhibition of A549-T was significantly increased.The inhibitory effect of combine CUR-LN with PTX-LN on A549-T cells was better than combined CUR with PTX.It is suggested that CUR-LN and PTX-LN have a good inhibitory effect on A549-T cells and reverse the reverse effect of A549-T,meanwhile,significantly increases the efficacy of CUR and PTX.3.The target site of CUR is in the nucleus of A549,the target site of CUR is in the nucleus and cytoplasm of A549-T,and the target site of PTX is in the cytoplasm of A549 and A549-T.When CUR combined with PTX,the target site is in the nuclei and cytoplasm of A549 and A549-T,and the intensity is increased.4.The combination of CUR and PTX,compared with the same concentration of CUR,the ability of inducing apoptosis of A549 and A549-Ttumor cells increased significantly.5.When CUR and PTX(1:1)were administered at the same time,the pharmacokinetics of plasma did not interfere with each other;while CUR-LN and PTX-LN(1:1)were administered simultaneously,the pharmacokinetics of plasma of both did not interfere with each other.After the preparation of CUR and PTX into CUR-LN and PTX-LN,the blood drug concentration in the animals increased,the half-life prolonged,the retention time increased,and the clearance rate decreased.The effect of CUR and PTX was significantly improved. |
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