The Preliminary Study On Antitumor Effect Of Mannose-conjugated Chitosan Zoledronic Nanoparticles

Objective: it is to know the uptake condition of different types of macrophage on zoledronic acidnanoparticle by inducing U937 cells into M0 macrophage,M1 macrophage and M2 macrophage;to know the influence of zoledronic acidnanoparticle on tumor cells' metastasis and invasion in co-culture condition;to know the inhibitioncondition of the zoledronic acidnanoparticle on tumor cells' proliferation.Methods : 1.Macrophages inducing: U937 cells were paved in 6-well plate and phorbol ester(PMA)was added in to induce for 6 hours;2.M1 macrophages induction: U937 cells were paved in 6-well plate and phorbol ester(PMA)(200ng/ml)was added in to induce for 6 hours;then lipopolysaccharide(LPS)(100ng/ml)and IFN-?(20ng/ml)were added in to induce for 66 hours(totally 72 hours).3.M2 macrophages induction:U937 cells were paved in 6-well plate and phorbol ester(PMA)(200ng/ml)was added in to induce for 6 hours;then IL-4(20ng/ml)?IL-13(20ng/ml)were added in to induce for 66 hours(totally 72 hours).Expression of Arg-1 enzyme and i NOS were detected to check whether the induction was successful.4.Uptake experiment: human body fibroblasts,M1 macrophages,M2macrophages,SW620 colon cancer cells,were selected and added with zoledronic acidnanoparticle labelled by fluorescence.After 3 hours of reaction,Dil was adopted to stain cell membrane.The uptake condition of four cells on zoledronic acidnanoparticle was observed under fluorescence microscope.5.CCK-8proliferation and inhibitionexperiment: by interaction of zoledronic acidnanoparticle with different cells,to know its influence on cells' proliferation ability;6.Cell scratch test and Transwell chamber metastasis experiment: SW620 colon cancer group,SW620+M1macrophages group,SW620+M2macrophages group were set and each group was divided into the group with zoledronic acidnanoparticle and the group without zoledronic acidnanoparticle(20ng/ul).There were six experiment groups;to observe the influence of different factors on tumor cells' metastasis ability.7.Invasion experiment: SW620 colon cancer cells were placed in the upper room of Transwell chamber and the lower rooms were separately the blank group(single culture medium),the zoledronic acidnanoparticle group,M1 macrophages group,M1macrophages+zoledronic acidnanoparticle group,M2 macrophages group,M2 macrophages+zoledronic acidnanoparticle group;giemsa staining method was adopted to count and to know the influence of different factors on invasion ability of tumor cells.Results:1.Arg-1 and i NOS testing results showed that immunohistochemistryof M2 macrophages presented positive reaction,while the other two groups presented negativereaction.iNOS expression of M1 macrophages(74.4%)was much higher than the other two groups(14.0% and 14.7%),confirming that the inducing of M1 and M2 macrophages is successful.2.Fluorescence intensity of M2 macrophages absorbing nanoparticleswas much higher than the other cells;3.M1 macrophages inhibited the metastasis of tumor cells;M2 macrophagespromoted the metastasis of tumor cells and its interaction with zoledronic acidnanoparticle can inhibit this kind of promoting effect of M2 macrophages(P<0.01);4.M1 macrophagesinhibited the invasion ability of tumor cells;M2macrophages promoted the invasion of tumor cells and its interaction with zoledronic acidnanoparticle can inhibit this kind of promoting effect of M2 macrophages(P<0.01);5.Nanoparticles can influence tumor cells' proliferation by inhibiting M2 macrophages(P<0.01).Conclusions:zoledronic acidnanoparticle can be specifically absorbed by M2 macrophages and inhibit the proliferation of M2 macrophages.M2macrophages can promote tumor's metastasis,invasion and proliferation.Zoledronic acidnanoparticle can inhibit tumor cells' metastasis,invasion and proliferationindirectly by inhibiting M2 macrophages.