Studies On The Multifunctional Brucea Javanica Oil Core-matched Nanoemulsions Drug Delivery

ObjectiveTo optimize the formulation and preparation of multifunctional Brucea javanica oil and Paclitaxel core-matched nanoemulsions drug delivery(PTX-OA/BJO CMNEs).The antitumor effect and mechanism of PTX-OA/BJO CMNEs was determined in HepG-2 cell lines and Hela lines in vitro and murine HepG-2-bearing BALB/c mice in vivo.Methods1.PTX-OA was determined by high performance liquid chromatography(HPLC).Oleic acid was determined by gas chromatography(GC).2.PTX-OA/BJO was synthesized by the esterification of PTX and OA.Ultrasionic emulsification was used to prepare the CMNEs,The concentration of oil phase(A),quality of polysorbate 80(B)and ultrasonic power(C)were selected as the significant factors that were applied in L,6(43)orthogonal array design with the average particle size as the criterion.In addition the physicochemical properties were tested.3.The effect of PTX-OA/BJO CMNEs,PTX-OA CMNEs,BJO CMNEs and Blank CMNEs on the growth of HepG-2 cells and Hela cells was assessed by MTT assay.Flow cytometry was applied to detect the coadministration-induced and single-induced apoptosis and analyze the cell cycle.4.HepG-2 cells were xenografted in nude mice to establish the animal models,then the inhibition rate of tumor growth,hematoxylin-yihong staining,TUNEL and immunohistochemistry were compared in the groups.Results1.Linear range of PTX-OA was 5-25 ug/mL,Y=12.709X+6.2520(r=0.999 5).Linear range of oleic acid and phenyl benzoate peak area ratio was 3-27 mg/mL,Y = 0.0312X-0.0172(r = 0.999 8).2.The optimized conditions of PTX-OA/BJO CMNEs were as followings,the concentration of oleic acid was 6.5 mg/ml,the mass ratio of polysorbate 80 to oleic acid was 3.5:6.5 and the ultrasonic power was 120 W.The CMNEs prepared by the optimal conditions showed an entrapment efficiency of(100.6 ± 1.9)%and were of nanoscale particle size(108.7±2.3)nm,PDI 0.232±0.038.The morphology of the CMNEs examined by TEM exhibited a uniform and spherical shape.In vitro drug release was up to be 67%after 48h.After PTX-OA/BJO CMNEs sealed in a bottle were stored at 4 ? for 60 d,the average particle size and entrapment efficiency had no significant change.3.MTT results showed that PTX-OA/BJO CMNEs strongly inhibited the growth of HepG-2 and Hela cells in the low concentration(HepG-2 in the range of 50-100 nmol/L,Hela in the range of 5-25 nmol/L),which implied combination therapy has a better effect.When drugs concentration was 50 nmol/L for 24 h,inhibition rate of PTX-OA/BJO CMNEs of HepG-2 was(11.6±2.1)%which was higher than PTX-OA CMNEs(2.4 ± 1.0)%(P<0.01).When drugs concentration was 50-100 nmol/L for 48 h,inhibition rate of HepG-2 had significant different in two groups(P<0.01).When drugs concentration was 5 nmol/L for 24 h,inhibition rate of PTX-OA/BJO CMNEs of Hela was(12.0±2.9)%which was higher than PTX-OA CMNEs(6.0±1.5)%(P<0.01).When drugs concentration was 5-25 nmol/L for 48 h,inhibition rate of Hela had significant different in two groups(p<0.01).HepG-2 cycle results showed that PTX-OA/BJO CMNEs had a significant increase in the G2/M population,PTX-OA/BJO CMNEs was(63.34±0.96)%,PTX-OA CMNEs was(37.82±1.32)%(P<0.05).Hela cycle results showed that PTX-OA/BJO CMNEs had significant increase S subpopulation,PTX-OA/BJO CMNEs was(40.48±0.58)%,PTX-OA CMNEs was(35.23±0.34)%(P<0.05).After HepG-2 being treatment for 48 h,the apoptotic cells of PTX-OA/BJO CMNEs were(34.45±1.34)%,PTX-OA CMNEs was(28.40±0.85)%.The apoptotic cells of groups had significant different in two groups(P<0.05).After Hela being treatment for 48 h,the apoptotic cells of PTX-OA/BJO CMNEs were(30.50±2.55)%,PTX-OA CMNEs was(19.80±1.27)%.The apoptotic cells of groups had significant different in two groups(P<0.01).4.After the final administration,the inhibition rates of tumor(%)were 24.7%,39.6%and 45.4%for Brucea javanica oil injection,taxol and PTX-OA/BJO CMNEs,which shown PTX-OA CMNEs is better than taxol(P<0.05)and Brucea javanica oil injection(P<0.01).Taxols may lead more severe side effects by losing weight.The histologicalimages(100 X)with H&E staining for heart,liver,spleen,lung,kidney showed that all of groups were normal.The density of tumor cells were more less in mice treated with PTX-OA/BJO CMNEs than that in mice with taxol at the same drug doses.Apoptotic cells for TUNEL as follow:PTX-OA/BJO CMNEs>taxol>Brucea javanica oil injection>normal saline.PCNA and Topo II order were opposite to TUNEL.Summarily,PTX-OA/BJO CMNEs is better than PTX-OA CMNEs in inducing apoptosis,inhibiting DNA duplication and cell proliferation.ConclusionThe particle size of PTX-OA/BJO CMNEs was about 100 nm which had less PDI and high entrapment efficiency.PTX-OA/BJO CMNEs has better performance than PTX-OA CMNEs in ivtro and in vivo.The anti-tumor mechenism of PTX-OA/BJO CMNEs was performed by inhibiting DNA duplication and proliferation,cell cycle arrest and inducing apoptosis.