Experimental Study On Targeting Therapy Of Colorectal Cancer With Lyp-1 Targeted Paclitaxel Nanomicelles

Objective In this study,the hydrophobic hydrophobicity of paclitaxel was solved by using a film-forming hydration method to prepare paclitaxel-polyethylene glycol monomethyl ether poly lactic acid nanomicelles(Lyp-1-PTX-mpeg-pla)carrying a Lyp-1 target and the in vitro and in vivo experiments were performed to investigate the efficacy of the nanomicelles.The two effects act simultaneously on colorectal cancer C26 cells,in order to explore the therapeutic effect of Lyp-1 target paclitaxel polyethylene glycol monomethyl ether polylactic acid nanomicelles on colorectal cancer.Methods1.Preparation of anti-tumor drugs:paclitaxel polyethylene glycol monomethyl ether polylactic acid micelles(PTX-mpeg-pla),containing Lyp-1 target paclitaxel polyethylene glycol monomethyl ether polylactic acid micelles(Lyp-1-PTX-mpeg-pla).The optimum preparation conditions of the two nanomicelles were investigated by measuring the particle size and potential of the prepared micelles.The entrapment efficiency and drug loading of the two micelles were determined.The morphology of the micellar nanoparticles was evaluated by electron microscopy.2.In vitro experiments(1)The cell proliferation inhibition experiment was conducted to evaluate the drug efFect by inhibiting the cells under the same concentration gradient of the PTX,the PTX-mpeg-pla and the target Lyp-1-PTX-mpeg-pla.(2)Cell migration experiments were divided into saline group,PTX group,PTX-mpeg-pla group and target group.The drug concentration was set at the same concentration of paclitaxel.The inhibitory effect of each group of drugs on the scratch of C26 cells in 6-well plates was observed(3)Laser confocal experiments,coumarin-6 instead of paclitaxel,prepared coumarin-6 polyethylene glycol monomethyl ether polylactic acid micelles(c6-mpeg-pla)and contained Lyp-1 target incense.The coumarin-6 polyethylene glycol monomethyl ether polylactic acid micelles(c6-mpeg-pla)and Lyp-1 target coumarin-6 polyethylene glycol monomethyl ether polylactide(Lyp-1-c6-mpeg-pla)by the same method.We can observe the ability of coumarin-6 to enter C26 cells by laser microscope.(4)The apoptosis was measured by flow cytometry.The drug was divided into normal saline group,PTX group,PTX-mpeg-pla group and target group.The same concentration of drug(paclitaxel content).Finally,the cells were digested in 12-well plates and detected by the light sources of a 488nm Argon ion laser.The images of C26 cells apoptosis in different drug groups were analyzed.3.In vivo experiments(1)Establishment of colorectal cancer mouse model in situ.Male BALB/c mice weighing 16-17 g were selected(SPF grade).After one week of adaptation in the animal room,each mouse was injected subcutaneously with an average of 1.5×106colorectal cancer C26 cells.After the tumor became long enough to be visible to the naked eye,one mouse was randomly selected to be killed and dissected to evaluate whether the model was successfully modeled.(2)The successful modeling of mice grouping,measuring tumor,weight measurement,administration,sacrifice,observe the pathological sections.Results1.Under the conditions of paclitaxel and polyethylene glycol monomethyl ether polylactic acid ratio 1:10,water bath temperature 50℃,rotation speed 70r/min,and hydration volume 2ml,Paclitaxel polyethylene glycol monomethyl ether polylactic acid micelles group(PTX-mpeg-pla)drug loading was 6.17 ± 0.37%,encapsulation efficiency was 96.22 ± 1.22%,particle size was 15.91 ± 1.5nm,PDI was 0.090±0.001,zeta potential-1.70 ± 0.4mv by the film dispersion method Under the same conditions,the Lyp-1 target paclitaxel polyethyleneglycol monomethyl ether polylactic acid micelles group average particle size of 16.02 ± 0.09nm,PDI of 0.092 ± 0.003,zeta potential of-2.95 ±0.25mv,encapsulation efficiency of 95.82 ± 0.38%,Drug loading 5.92 ± 0.07%.2.In vitro experiments,containing Lyp-1 target paclitaxel polyethylene glycol monomethyl ether polylactic acid micelles and paclitaxel polyethylene glycol monomethyl ether polylactic acid micelles than the drug-drug group has a better inhibition of colon cancer C26 cell,inhibition of cell migration more significant role in faster access to colorectal cancer cells,and enhance the role of apoptosis in the late.containing Lyp-1 target paclitaxel polyethylene glycol monomethyl ether polylactic acid micelles group than the simple use of polyethylene glycol monomethyl ether polylactic acid paclitaxel micellar group paclitaxel micelles in the inhibition of cell proliferation inhibition effect is more pronounced,to the late apoptosis stronger.3.In vivo experiments:Four groups of successfully established BALB/c mouse colorectal cancer model containing Lyp-1 target paclitaxel polyethylene glycol monomethyl ether polylactic acid micelles than paclitaxel polyethylene glycol monomethyl ether polylactic acid micelles group,raw drug group,saline control group tumor size smaller(P<0.05),no liver metastasis at high magnification.Paclitaxel polyethylene glycol monomethyl ether polylactic acid micelles group and paclitaxel polyethylene glycol monomethyl ether polylactic acid micelles group containing Lyp-1 targeting target paclitaxel drug group had less toxicity(P<0.05).Conclusion1.Lyp-1-PTX-mpeg-pla has good stability,encapsulation efficiency of more than 96%,drug loading more than 6%,particle size of 16nm,and uniform distribution of nanoparticles.2.Lyp-1-PTX-mpeg-pla nanomicelles can enter tumor cells and induce growth inhibition of tumor cells by inducing apoptosis pathway.3.Lyp-1-PTX-mpeg-pla nanomicelles can inhibit tumor growth and inhibit metastasis better than other control drugs,with less toxicity.