Construction Of Polylysine-SN38 Targeting Polymer Micelle Modified By Hyaluronic Acid And Its Anti-lung Cancer Effect

Objective:Polylysine was modified by 7-ethyl-10-hydroxy camptothecin（SN38）and hyaluronic acid（HA）to construct tumor-targeting polymer micelles polylysine-SN38 for delivering programmmed death-ligand 1 small interfering RNA（si PD-L1）and fibrinogen like protein 1 small interfering RNA（si FGL1）.The gene delivery ability and tumor targeting of the polymer micelles,and the antitumor effect of chemotherapy combining immunotherapy in mouse lung cancer LLC cells were investigated.Methods:（1）Polylysine-SN38 polymer（L-S）was prepared by connecting polylysine with SN38.The structure of the polymer was identified by MS,¹H-NMR and IR.（2）Polymer micelle L-S was constructed by self-assembly of polymer L-S,and then modified with different proportions of HA to construct tumor-targeting polymer micelles L-S-HA.Particle size,Zeta potential,stability in PBS buffer and serum of the polymer micelles were measured by laser nanoscale analyzer.The morphology of polymer micelles was observed by transmission electron microscopy（TEM）,and the drug loading of polymer micelles was detected using ultraviolet spectrophotometry.（3）The effects of SN38,polymer micelle L-S and targeted polymer micelle L-S-HA on the proliferation of LLC cells were detected by CCK-8 assay.The fluorescence inverted microscope was used to observe the uptake efficiency of L-S and L-S-HA loading si RNA^FAM in LLC cells.（4）Gene silencing efficiency of si PD-L1 and si FGL1 in LLC cells was determined by RT-QPCR.（5）Western blot was used to detect the effects of polymer micelles on two immune checkpoint pathways,including programmmed death-1/programmmed death-ligand 1（PD-1/PD-L1）and fibrinogen-like protein1/lymphocyte-activation gene 3（FGL1/LAG-3）.The effect of polymer micelles on apoptotic proteins Bax and Bcl2 was also detected by Western blot.（6）The hemolysis of polymer micelles in mouse red blood cells was detected.LLC cells were inoculated in the armpit of C57BL/6 mice to establish a xenograft transplanted tumor model.SN38,L-S,L-S-HA,L-S-HA/si PD-L1,L-S-HA/si FGL1,L-S-HA/si PD-L1/si FGL1 were administered by tail vein every other day and for seven times,the dosage of SN38 and si RNA was 5 mg/kg and 0.33 mg/kg respectively in each group.The mice were sacrificed two days after the last injection.The antitumor effect and safety of targeted polymer micelle were assessed according to the tumor volume,body weight,organ weight,pathological section and blood biochemical indexes of the mice.Results:（1）The polymer L-S was synthesized and its structure was verified by MS,¹H-NMR and IR.（2）Polymer micelle L-S and targeted polymer micelle L-S-HA loading HA with different ratio were obtained by self-assembly,in which the drug loading of SN38 was 19.4%.The optimal ratio of HA to L-S in targeting polymer micelles was 2:1.Polymer micelles L-S and L-S-HA were nearly spherical and distributed uniformly,and had good stability in PBS buffer and serum.（3）CCK-8 assay showed that the polymer micelles had better inhibitory effect than SN38 on the proliferation of LLC cells.The cell uptake experiment indicated that the uptake efficiency of targeting polymer micelle L-S-HA was the strongest in tumor cells.（4）RT-QPCR experiment revealed that the gene expression rates of PD-L1 and FGL1 were48%and 41%after transfection of si PD-L1 and si FGL1,respectively.（5）Western blot results showed that SN38 could up-regulate the expression of Bax and down-regulate the expression of Bcl2,si PD-L1 and si FGL1 could down-regulate the expression of PD-L1 and FGL1,respectively.（6）Hemolysis experiment illustrated that the hemolysis rate of both polymer micelles were lower than 5%,and L-S-HA could significantly reduce the cytolytic rate.Tumor suppression experiment demonstrated that L-S-HA/si PD-L1/si FGL1 had the strongest tumor inhibition effect.The main organs had no significant damage,the blood and serum biochemical indexes had no significant changes,indicating that the polymer micelles had good safety in vivo at the administrated dosage.Conclusion:HA-modified polylysine-SN38 targeted polymer micelle was constructed successfully.The targeted polymer micelle could deliver drugs and genes simultaneously to realize the synergistic effect of chemotherapy and gene immunotherapy.It showed stronger inhibiton on LLC cells in vitro and in vivo,good tumor-targeting and safety.