The Efficacy Of Nanoparticle Delivery System As Carriers For Anticancer Drugs On Laryngeal Cancer Cells

Objective The nanoparticle delivery system,one of the outcomes and focuses of nanotechnology,can increase the regional drug concentration and reduce the systematic toxicity,which was usually caused by traditional drug delivery systems. Considering the important role of the tumor blood vessels in the tumor growth and metastasis,we set the purpose of the thesis to explore the effect of nanoparticle delivery system loaded with cytotoxic drug and vascular disrupting agent on the laryngeal cancer Hep-2 cells.We used paclitaxel(PTX) as a model of cytotoxic drug,and incorporated it into biodegradable polymer-methoxy poly(ethylene glycol)-poly(lactide-co-glycolide) (MPEG-PLGA) to synthesize the controlled released paclitaxel-loaded MPEG-PLGA nanoparticles(PTX NPs).The NPs were prepared by emulsification-solvent evaporation method,and the preparation condition was optimized.The characterization,in vitro release profile of NPs was evaluated.The in vitro cytotoxicity and uptaken was investigated with the human laryngeal cancer Hep-2 cells.We also prepared and characterized MPEG-DSPE liposome system for combretastatin A4(CA4),which serves a model of vascular disrupting agents.To identify the in vivo efficacy of PTX and CA4 against laryngeal cancer,PTX NPs and CA4 liposme were intravenously injected into Hep-2 cell xenograft nude mice model, with PTX and CA4 parenteral solutions as comparison.Methods 1.The NPs were prepared by emulsification-solvent evaporation method, the preparation conditions were optimized,such as the molecular weight and concentration of PVA,the concentration of MPEG-PLGA,the ratio of oil and water et. al..The nanoparticle was characterized by dynamic light scattering technique for size and size distribution,TEM for morphological properties,DSC and XRD for the formation properties.The drug loading efficiency,drug encapsulation efficiency and in vitro drug release kinetics were measured by high performance liquid chromatography(HPLC).To evaluate the in vitro cytotoxicity,PTX NPs,paclitaxel injection and blank MPEG-PLGA NPs at various concentrations were added to treat Hep-2 cells.And MPEG-PLGA NPs loaded with coumarin-6 were synthesized to investigate the intracellular uptake by Hep-2 cells.Visual evidence of NPs'uptake was obtained by laser scanning confocal microscope.The acute toxicity of PTX NPs was also test in mice compared with paclitaxel injection.2.The PEGylated CA4 long-circulating liposomes were prepared by thin-film hydration method.The orthogonal test design was used to optimize the technology of preparation.In vitro characteristics of the MPEG-DSPE liposome were investigated.3.BALB/c nude mice bearing Hep-2 laryngeal squamous cell cancer were randomly assigned into seven treatment groups:(1) PTX NPs(10mg/kg)+CA4 liposome (40mg/kg);(2) PTX NPs,10mg/kg;(3) CA4 liposome,40mg/kg;(4) paclitaxel injection,10mg/kg;(5) CA4 injetion,40mg/kg;(6) paclitaxel injection(10mg/kg)+ CA4 injection(40mg/kg);(7) Saline.All the animals were intravenously injected every six days for 3 times.The systemic toxic reactions,body weight,tumor size,and tumor weight and were observed.The tumor inhibition rate(IR) was calculated.Results 1.The PTX NPs particle size and Zeta potential were around 153.3±41.7 nm and-5.36 mV.Transmission electron microscopy(TEM) showed the NPs were homogeneous and spherical in shape.Differential scanning calorimeters(DSC) and X-ray powder diffractogram(XRD) did not detect any crystalline drug in the NPs samples.The drug loading efficiency was(5.35±0.75)%and encapsulation efficiency was(75.56±2.61)%.In the in vitro drug release study,paclitaxel was released from the NPs in a slow but time-dependent manner.Both of the PTX NPs and paclitaxel injection inhibited Hep-2 cell growth in a dose-and time-dependent manner.The PTX NPs showed higher or comparable cytotoxicity and IC₅₀ value against human laryngeal cancer cell Hep-2 versus the commercial Cremopher EL-based paclitaxel formulation.At low concentration and shorter time(1-10nmol/l,24h),the PTX NPs had higher cytotoxicity against paclitaxel injection.No significant cytotoxicity was observed in blank MPEG-PLGA NPs.For the intracellular uptake study,NPs entered the cells and gathered in the cytoplasm.In the safety test,little side effect was observed in the PTX NPs group.It was considered PTX NPs was better tolerant in mice than paclitaxel injection.2.The optimized formulation ratio was SPC:Chol:MPEG-DSPE=71:24:5,drug to lipid ratio 1:10,and the optimized final total drug concentration at 60 mmol/l.The size distribution of the liposome was 113.4±40.6 nm and the entrapment efficiency was 71.20±0.31%.The prepared liposomes showed stable physicochemical properties when stored at 4℃for 7 days.3.On the 18th day,there was no significant difference in body weight among all the groups.The tumor volume in PTX NPs+CA4 Lipo group,PTX NPs group and CA4 liposome group was significantly smaller than the saline group(P＜0.05).There was significant difference in the tumor volume between the PTX NPs+CA4 liposome group and the PTX NPs group(P＜0.05).Significance also can be seen between the PTX NPs+CA4 liposome group and the CA4 liposome group(P＜0.05).The tumor volume in the PTX NPs+CA4 liposome group was significantly smaller.The tumor volume and weight of the PTX NPs+CA4 liposome group was significantly smaller than the paclitaxel injection+CA4 injection group(P＜0.05).There was significant difference in tumor weight between the PTX NPs+CA4 liposome group and saline group.The tumor weight in PTX NPs group and PTX injection group was similar (P＞0.05).The tumor inhibition rate for the PTX NPs+CA4 liposome was 65.03%, which was the highest among the groups.Conclusion Nanoparticle delivery system as carders for the small molecular VDAs-CA4 and the cytoxicity agent-paclitaxel,can significantly inhibit the Hep-2 tumor growth in nude mice and reduce the drug toxicity.Our study suggests that the nanoparticle targeting delivery system is prospective for laryngeal cancer chemotherapy.