Construction Of Active And Passive Dual-targeted Nanomedicine And The Anti-hepatocellular Carcinoma Effect

Nanomedicine design is an important means to improve the efficiency of chemotherapy drugs and reduce the toxic side effects.However,compared with traditional small-molecule drugs,tumor tissue penetration,cellular uptake and drug release are the main factors that limit the clinical therapeutic effect of nanomedicine.Therefore,the development of active and passive dual-targeted nanomedicine is of great significance to overcome multiple delivery barriers and improve the treatment efficiency.Herein,when chitosan oligosaccharides（COS）were used as the carrier material,poly（gamma-glutamic acid）（γ-PGA）,phenylboronic acid（PBA）and disulfide bonds were introduced into COS,endowing the nano-system with active-passive dual-targeting characteristics.The responsive release of doxorubicin（DOX）was investigated in the presence of different p H,active oxygen species（ROS）,gamma-glutamyl transpeptidase（GGT）and glutathione（GSH）.Furthermore,the hepatocellular carcinoma-targeted delivery mechanism was also investigated through analyzing the cytotoxicity,intracellular distribution and endocytosis pathways of nanomedicine in breast cancer（MCF-7）,cervical cancer（He La）and hepatocellular carcinoma（HepG2）cells.This research was discussed from the following three aspects:（1）PBA-COS was successfully synthesized by atom transfer radical polymerization when the molar ratio of COS,ceric ammonium nitrate（CAN）,PBA and N,N-diethyl aminoethyl acrylate（DEAEA）was 1:1:1:1.Then,the p H/ROS/GGT active-passive dual-targeted nanoparticles（PBA-COS/γ-PGA NPs）were prepared using sodium tripolyphosphate（TPP）as the ionic crosslinking agent.The effects of raw material concentration,rotational speed and reaction time on the particle size and potential of PBA-COS/γ-PGA were investigated respectively,and the in vitro drug release properties of nanoparticles were preliminarily analyzed.The results showed that the concentrations of PBA-COS,TPP andγ-PGA had large effects on particle size and potential.The particle size and zeta potential of PBA-COS/γ-PGA NPs was about 141.60 nm and-19.60 m V after magnetic stirring for 3 h at 600 rpm when the C_COS,C_TPP and Cγ_-PGA were 0.75,2.00 and 1.50mg/m L,respectively.The encapsulation and drug loading rate were increased to 93.74%and28.84%,respectively,at the C_DOX of 1.00 mg/m L.Meanwhile,particle size and zeta potential of DOX@PBA-COS/γ-PGA NPs were 164.20 nm and-11.40 m V,respectively.The drug release rate（90.36%）of DOX@PBA-COS/γ-PGA in the simulated tumor microenvironment（p H 6.5,100μM H₂O₂ and 10 U/m L GGT）was approximately 2.22 times higher than that in the normal tissue environment（p H 7.4）.（2）Furthermore,the tumor-targeting specificity of the p H/ROS/GGT active-passive dual-targeting nanomedicine delivery system to MCF-7,He La and HepG2 cells was further investigated by analyzing the cytotoxicity and intracellular uptake in the tumor microenvironment（p H,H₂O₂,GGT）.The results showed that DOX@PBA-COS/γ-PGA NPs exhibited relatively strong effects on HepG2 cells compared to MCF-7 and He La cells,with IC₅₀ as low as 0.26μg/m L,and its GGT sensitivity was significantly better than PBA sensitivity.The uptake of DOX@PBA-COS/γ-PGA NPs in HepG2 cells was significantly enhanced with a fluorescence intensity of 7.62×10⁵,which was 1.58 and 1.48 times higher than that in MCF-7 and He La cells after 24 h incubation at p H 6.5,10μM H₂O₂ and 10U/m L GGT.DOX@PBA-COS/γ-PGA NPs showed lysosomal escape after 3 h of incubation in HepG2 cells and were able to successfully escape from the lysosomes into the nucleus after 8 h.The intracellular uptake of DOX@PBA-COS/γ-PGA NPs was temperature-dependent.Clathrin-mediated endocytosis was dominant in HepG2 cells,while microtubule polymerization/vesicular transport-mediated endocytosis was enhanced upon the GGT addition,indicating that GGT-responsive drug delivery was associated with microtubule polymerization/vesicular transport-mediated endocytosis.（3）GSH-responsive prodrugs DOX-ss and active-passive dual-targeted-enhanced PBA-COS-ss-DOX/γ-PGA NPs were prepared by connecting DOX with disulfide bonds to improve the drug delivery efficiency,prevent premature leakage of DOX in the normal physiological environment and achieve efficient enrichment at tumors sites.PBA-COS-ss-DOX/γ-PGA NPs showed better biocompatible and significantly higher drug release rates in a simulated tumor microenvironment（p H 6.5,10 m M GSH,100μM H₂O₂and 10 U/m L GGT）,approximately 6.87 times higher than in normal tissue（p H 7.4）.Moreover,the cytotoxicity of PBA-COS-ss-DOX/γ-PGA NPs was the strongest at p H 6.5,10m M GSH,10μM H₂O₂ and 10 U/m L GGT,and the fluorescence intensity was about twice higher than that at p H 7.4,which exhibiting a significant GSH responsiveness.Moreover,the ROS intensity in this condition was much higher than that in normal conditions,indicating that the introduction of disulfide bonds could induce significant ROS production in hepatocellular carcinoma cells through redox,which would make an effective foundation for subsequent studies.