Study On Anti-lung Cancer Activity Of Sodium New Houttuyfonate Combined With Cisplatin And Preparation Of Co-loaded Liposomes

Objective:To study the inhibitory effects of sodium new houttuyfonate（SNH）combined with cisplatin（CDDP）on non-small cell lung cancer A549 cells,and explore the possible anti-lung cancer mechanism of the combination of the two drugs.The thin-film dispersion method was used to prepare liposomes loading sodium new houttuyfonate and cisplatin（SNH-CDDP-Lp）and the pharmacokinetics were investigated.It can provide new ideas to combination Chemotherapy of clinical non-small cell lung cancer.Methods:The inhibition effects of SNH and CDDP alone or in combination on A549 cells proliferation was detected by MTT method,which to screen out the optimal combined ratio.The apoptosis and cell cycle effects of each administration group on A549 cells were investigated by flow cytometry.Western Blot technology and QPCR technology were used to study the mechanism of A549 cells apoptosis in each administration group at the protein and gene levels,respectively.Acute toxicity experiments were conducted to investigate the toxicity of SNH and CDDP.To investigate anti-tumor effect of each administration group,a nude mouse tumor-bearing model was established.After preparing SNH-CDDP-Lp by thin-film dispersion method,the prescription was optimized by response surface method.The particle size and Zeta potential were measured by particle size analyzer.The morphology of SNH-CDDP-Lp was observed by transmission electron microscope.A high performance liquid chromatograph（HPLC）method was established for determining the encapsulation efficiency and total drug loading of SNH and CDDP in SNH-CDDP-Lp.The dynamic dialysis method was employed to investigate the cumulative release rate of SNH-CDDP-Lp in Physiological saline solution containing1.0%Tween 80,and to investigate the stability of SNH-CDDP-Lp.Took SD rats as a model to investigate the pharmacokinetics of SNH-CDDP-Lp,the drug concentrations in blood of sodium new houttuyfonate and cisplatin were measured with HPLC method and the concentrationtime curve was drawn.The pharmacokinetic parameters were calculated and analyzed by DAS 3.2.8 software.Results:In A549 cells,the IC₅₀values of SNH at 24,48,and 72 hours were higher than that of CDDP.The IC₅₀values of SNH at 24,48,and 72 hours were（42.39±2.86）,（29.08±0.22）,（16.21±0.78）μg/m L,respectively.The IC₅₀values of CDDP at 24,48,and 72 hours were（8.95±0.67）,（3.03±0.11）,（1.08±0.09）μg/m L,respectively.When the combined ratio of SNH to CDDP was 4:1,the combined index（CI）of each administration group was less than 1,and the CI value of the 10μg/m L SNH+2.5μg/m L CDDP group was 0.84,with the inhibition rate of（54.68±0.88）μg/m L.Compared with the Control group,the apoptotic rates of the SNH group,CDDP group,and SNH+CDDP group all increased,with apoptotic rates of（17.10±1.05）,（45.67±1.33）,（59.07±4.60）μg/m L,respectively.The difference was statistically significant（P<0.01）.Compared with the Control group,the proportion of cells in the G0/G1 phase of the SNH group increased by 21.23%,and the S phase decreased by19.03%.The CDDP group and the SNH+CDDP group decreased by 6.74%and20.07%in the G0/G1 phase（P<0.01）,respectively;increased by 9.77%and 24.17%in the S phase（P<0.01）,respectively.Compared with the Control group,the other three groups all increased the expression of Bax,P53,Cleaved-Caspase3 protein and genes（P<0.05）,and all reduced the expression of Bcl-2,Caspase3 protein and genes（P<0.05）).In addition,no significant down-regulation of NF-κB protein and gene expression was observed in the CDDP group,but significant down-regulation was seen in the SNH group and SNH+CDDP group.The LD₅₀values of the Kunming group in SNH group,CDDP group and SNH+CDDP group were 166.7mg/kg,13.1mg/kg and 213.2 mg/kg+16.8 mg/kg,respectively.The antitumor experiment showed that the antitumor effects in SNH+CDDP_Lgroup and SNH+CDDP_Hgroup were better than those in single drug groups.Compared with other groups,SNH+CDDP_Hgroup had the highest tumor inhibition rate,which was 79.72%.The tumor inhibition rate in SNH+CDDP_Lgroup was similar to that in CDDP_Hgroup,which was 66.67%and 69.32%,respectively.The body weight of the nude mice in the CDDP_Lgroup and CDDP_Hgroup continuously decreased,and the body weight in the other administration groups slowly increased.When the mass fraction of PEG was 4.92%,the ratio of drug and carrier was24.04:1,and the ratio of phospholipid and cholesterol was 3.70:1,there was an optimal prescription of the prepared liposomes co-loaded with SNH and CDDP.The mean diameter of liposome was（112.40±1.30）nm with Pd I of 0.188±0.004 and zeta potential of（-34.0±0.10）m V.Under the transmission electron microscope,the liposomes were Spheroid with evenly distributed.The encapsulation rates of SNH and CDDP in SNH-CDDP-LP were（89.43±0.91）%and（92.30±1.08）%,respectively,and the total drug loading was（12.25±0.09）%.The cumulative release rates of SNH and CDDP in SNH-CDDP-LP were（58.58±1.97）%and（62.66±3.56）%,respectively.The encapsulation efficiency and drug loading of SNH and CDDP in liposomes decreased significantly under high temperature and strong light.When placed at 4℃for 90 days,the encapsulation efficiency and drug loading of SNH and CDDP in the liposomes remained basically unchanged.When placed at room temperature for 90 days,the encapsulation efficiency and drug loading of SNH and CDDP gradually decreased.The pharmacokinetic results showed that compared with the sodium new houttuyfonate-cisplatin-solution（SNH-CDDP-SOL）group,the AUC and T_1/2of the SNH-CDDP-LP group were significantly increased.The AUC _0-tof SNH-CDDP-LP:LP-SNH was 7.53 times that of SOL-SNH,and LP-CDDP was 5.70 times that of SOL-CDDP;the T_1/2of SNH-CDDP-LP:LP-SNH was 1.62 times that of SOL-SNH,and LP-CDDP was 1.93 times that of SOL-CDDP.Conclusion:The inhibition ability of CDDP on A549 cell proliferation was stronger than that of SNH,but the cytotoxicity of CDDP was greater than that of SNH.The combination of the two drugs enhanced the anti-lung cancer activity,and when the combination ratio was 4:1,there had best synergistic effect.A549 cells apoptosis in SNH+CDDP group was higher than that in single drug group.SNH and CDDP block at different stages of the cell cycle,preventing the synthesis of m RNA and protein and the replication of DNA,leading to abnormal cell division and inducing cell apoptosis.The mechanism of SNH and CDDP inducing cell apoptosis may be related to the regulation of protein and gene expression in the two signal pathways of NF-κB and apoptosis.Compared with the SNH group and the CDDP group,the toxicity of the SNH+CDDP group was reduced.The combination of SNH and CDDP enhanced the anti-tumor effect in vivo,and maintained the curative effect while reducing the CDDP dose and adverse reactions.The encapsulation rate of SNH and CDDP co-loaded liposomes meeted the requirements and had a certain sustained release effect,which could improve the pharmacokinetic process of the two drugs,release the drugs in proportion,and significantly improve the bioavailability of the two drugs in rats.