Hyaluronic Acid Modified Erythrocyte Membrane Coated Paclitaxel-loaded Lipid Liquid Crystal Nanoparticles For Targeted Treatment Of Lung Tumors

Objective:Nanocarrier-based drug delivery systems（DDS）have been widely used in anti-tumor researches to improve the therapeuticeffect of the drug.However,nanoparticles are easily recognized and cleared by the immune system and have a short circulation time in the blood.In this paper,hyaluronic acid-modified erythrocyte membrane-coated lipid liquid crystal nanoparticles（HA&RBCm-LCNPs）were designed as a carrier for injection,which could bypass macrophage phagocytosis and achieve long-circulation.At the same time,modification of hyaluronic acid on the surface of the membrane could enhance the targeting of A549 tumor cells with high expression of CD44 protein.Methods:HA was covalently grafted onto dioleoyl phosphatidylethanolamine（DOPE）molecules,and the synthetic phospholipid material（HA-DOPE）was inserted into the red blood cell membranes（RBCm）by co-incubation to form HA modified red blood cell membranes（HA&RBCm）.Then the paclitaxel-loaded lipid liquid crystal nanoparticles（LCNPs）were co-extruded with HA&RBCm to obtain HA&RBCm-LCNPs.The drug loading was used as an indicator to optimize the formulation of paclitaxel-loaded lipid liquid crystal nanoparticles by single factor investigation and response surface design.The particle size,zeta potential,appearance morphologyand surface proteinsof the nanocarriers were characterized,and plasma stability and in vitro hemolysis experiments were performed to evaluate the stability and biosafety of HA and RBCm-LCNPs.A model of A549 cells cultured in vitro was established.The active targeting behavior of HA&RBCm-LCNPs on tumor cells was assessed by examining cellular uptake and intracellular colocalization.We evaluated the cytotoxicity of HA&RBCm-LCNPsand explored its role in promoting tumor cell apoptosis.Cell scratch test was used to investigate the effect of drug-loaded preparations on tumor cell migration.The in vivo pharmacokinetic behavior of HA&RBCm-LCNPs and reference preparations was compared to investigate the long-circulating effects of erythrocyte membrane coating.Immunogenicity experiments were carried out by in vitro culture of RAW264.7 cells to analyze the long-circulating mechanism of the preparations.The distribution of PTX injection,LCNPs,RBCm-LCNPs and HA&RBCm-LCNPs in tumor-bearing nude micewas investigated by in vivo imaging experiments.The nude mouse xenograft model was used to investigate the tissue distribution in the nude mice and the inhibition of tumor growth after tail vein injection.Results:The synthesis of HA-DOPE was verified by FT-IR.The optimized formulation consisted of phosphatidylcholine（S100）:86.18 mg;glycerol dioleate（GDO）:165.82 mg;PTX:30 mg;ethanol（Et OH）:50 mg;Tween 80（P80）:75 mg;water phase:10 m L.Preferred LCNPs had an average particle size of 192.83 nm,a Z-potential valueof-15.33 m V,a drug loading of 8.57%for paclitaxelrespectively,and the encapsulation efficiency is 99.9%.The nuclear-membrane structure of RBCm-LCNPs was observed by transmission electron microscopy,with uniformdistributionandno adhesion with each other.Western Blot results showed that most of the erythrocyte membrane surface proteins including CD47 were retained on RBCm-LCNPs and HA&RBCm-LCNPs.No significant aggregation and sedimentation occurred after 72 hours of the formulations settled in the plasma,indicating that the formulation had good stability.The hemolysis test showed that the concentration of paclitaxel in the preparations of RBCm-LCNPs and HA&RBCm-LCNPs did not show hemolysis from 0.5 to 50μg/m L,demonstrating that the preparations had good blood compatibility.The results of CCK-8 experiments showed that the unloaded lipid liquid crystal nanocarriers had no obvious cytotoxicity,indicating that the carrier had good biocompatibility.The IC50 valueof the HA&RBCm-LCNPs group was smaller than those of the other groups,indicating that they had greater lethality against A549 cells.Flow cytometry and laser confocal results showed that HA modification could significantly increase the uptake of RBCm-LCNPs by A549 cells.Apoptosis experiments showed that HA&RBCm-LCNPs had a stronger effect on inducing apoptosis than the reference groups.In addition,HA&RBCm-LCNPs significantly inhibited the migration of A549 cells.The results of in vitro cellular immunogenicity experiments indicated that HA&RBCm-LCNPs could effectively reduce the uptake of macrophages,evade the clearance of the immune system,and reduce the immunogenicity.The pharmacokinetic results in rats showed that the values of T_1/2,MRT_0-t,AUC_0-tand C_max of HA&RBCm-LCNPs and RBCm-LCNPs groups were significantly higher than those of the commercially available（PTX Injection）group and the uncoated nanocarriers（LCNPs）group,the CL values were significantly lower than that in the PTX Injection and LCNPs groups,indicating that the coating of the erythrocyte membrane could effectively prolong the half-life of the drug,improve the bioavailability of the drug,and achieve better therapeutic effects.The modification of HA had no significant effect on the pharmacokinetic behavior of RBCm-LCNPs.The results of in vivo targeting and pharmacodynamic evaluation showed that the HA&RBCm-LCNPs group had stronger drug accumulation and fluorescence intensity at the tumor site than other groups,which could exert its active targeting effect and concentrate on tumor tissues.The tumor volume and weight of the HA&RBCm-LCNPs group were the smallest in all groups,showing the strongest tumor suppressing effect.There were no obvious pathological changes in the heart,liver,spleen,lung and kidney tissues of HA&RBCm-LCNPs group.HA&RBCm-LCNPs effectively prevented the pathological changes of tumor-bearing nude mice and had good in vivo safety.Conclusions:The erythrocyte membrane-coated lipid liquid crystal nanoparticles could effectively escape the phagocytosis of the immune system and achieve long-term circulation.At the same time,the modification of HA could effectively target tumor tissues to achieve better anti-tumor effects.