A Novel Genetically Engineered Nanovesicles (GENVs) For Targeted Cholangiocarcinoma Phototherapy

Cholangiocarcinoma(CCA)is a type of malignancy arising from the epithelial cells of the intrahepatic,perihilar and distal biliary tree.Epidemiological studies revealed that only 10-15%of the patients with CCA are surgically curative,whilethe majority was identified at an advanced stage due to the late appearance of the clinical symptoms and difficulty in the early diagnosis.Moreover,the recurrence rate is as high as 50-60%,causing the five-year overall survival(OS)beonly 30%.Human epidermal growth factor receptor2(HER2)has been considered as a potential therapeutic target in CCA.Studies showed that the overexpression of HER2 may contribute to the initiation and the development of CCAas well as the tumor metastasis.Therefore,HER2 was applied in our study as a targeting biomarker.Besides,Affibody molecules have been reported to possess a strong targeting capability for HER2,and nanoparticle-affibody conjugates have demonstrated high targeting specificity in HER2-expressing cells and tumors in vitro and in vivo.Recently,liposomes have been successfully applied in clinical diagnoses and treatments for various diseases thanks to its characteristics such as high encapsulation efficiency,high stability in biological environments,low toxicity and easeof functional modification.In addition,Photothermal therapy(PTT)has becomea hotspot in cancer therapy owing to it merits such as localized treatment,high effectiveness,little adverse effect and so on.In this study,based on the understanding of sorting and trafficking mechanisms of the signal peptide-mediated eukaryotic membraneproteinand the characteristics of biosurfactants,we reconstructedthe Affibody molecule through genetic engineering.Briefly,amembrane-anchor signal peptide sequence was added to the N-terminal of Affibody,meanwhile,a transmembrane peptide sequence as well as the green fluorescent protein(GFP)were fused with itsC-terminal.Then,the recombinant gene sequence was ligated into eukaryotic expression plasmid vectors and transfected into the host cells.Consequently,the Sig-Affibody-GFP fusion protein wasexpressed on the host cell membrane.Further through the induction of biosurfactants,we eventually extracted novel genetically engineered nanovesicles(GENVs),which were self-assembled from abiomembrane-derived phospholipid bilayer,a recombinant protein Sig-Affibody-GFP anchored to the membrane,and a biosurfactant that can control the particle size and strength.After purification,GENVs were loaded with indocyanine green(ICG)andadministrated intravenously into SK-chA-1 tumor-bearingmice for photothermal therapy.As a result,the novel GENVs-Affibody-ICG demonstrated a strongtumor-targeting capability in the CCA region,and a complete ablation of the tumor was realized afterthe photothermal therapy.