Construction And Anti-tumor Activity Of Dual-targeting Ph-sensitive Paclitaxel-loaded Lipoprotein-mimicking Nanocarrier

In the present study,we constructed a tumor-targeting and pH-sensitive lipoprotein-mimic nanocarrier containing paclitaxel(FA-BSA-LC/DOPE-PTX).The bovine serum albumin(BSA)was used as the protein model for constructing the lipoprotein-mimic nanocarrier.Albumin had been shown to be non-toxic,biocompatible In addition,as endogenous albumin,BSA had long residence time in blood increased the stability in vivo.BSA specifically targeted to tumor by increased transendothelial gp60-mediated transport and increased intratumoral accumulation as a result of the SPARC-albumin interaction.The further conjugating folic acid to bovine serum albumin achieved the dual active targeting.The lipid core(LC/DOPE)of lipoprotein-mimic nanocarrier was consisted of the pH-sensitive lipid dioleoyl-phosphatidylethalonamine(DOPE)and a stabilizer amphiphile oleic acid,to facilitate endo/lysosomal escape Therefore,FA-BSA-LC/DOPE as biocompatible,tumor-targeting and pH-sensitive lipoprotein-mimic nanocarrier is a promising system for effective intracellular delivery of PTX to tumor with optimal anti-tumor efficacy.The chemical conjugate of FA-BSA was synthesized by grafting the carboxyl group of FA with an amine group of BSA in the presence of EDC and NHS.The molecular structure of FA-BSA was evaluated by UV-vis absorption spectrophotometer,FTIR measurement and differential scanning calorimetry,indicating the successful conjugation of folic acid to BSA(FA-BSA).The results supported the successful synthesis of the FA-BSA.To quantify the substitution degree of folic acid to BSA,we used the TNBS method and the recovery method calculated the un-reacted FA,in this study,FA was attached to BSA by amide bond at a binding rate of 12-13 per BSA,so the nanocarrier could exhibit good targeting effect.The lipid core(LC/DOPE-PTX)of lipoprotein-mimick nanocarrier was composed of PTX,DOPE,OA,cholesterol and OL,with the optimized ratio and method.Same modicum of OL was incorporated to increase the positive charge of the lipid core.As the PI(isoelectric point)of BSA is about 4.9-5.3,the protein would be negatively charged in PBS(pH 7.4).The lipid core exhibited positive charge to facilitate FA-BSA attachment through electrostatic attraction.The amount of protein attached on the lipid core was determined by Coomassie(Bradford)Protein Assay.The coupling efficiency of protein into FA-BSA-LC/DOPE-PTX,BSA-LC/DOPE-PTX and BSA-LC/SPC-PTX were 83.8%,85.9%and 85.2%,respectively,which indicated these nanocarriers possessed high coupling efficiency of protein.Transmission electron microscopy of FA-BSA-LC/DOPE-PTX proved that the particles were generally spheroids.The mean diameter of LC/DOPE-PTX was about 104 nm,while the mean diameter of FA-BSA-LC/DOPE-PTX was about 116 nm,which conspicuously increased compared with LC/DOPE-PTX.This indicated that the particle size significantly increased after being coated by the modified protein.The results showed that FA-BSA-LC/DOPE-PTX had high EE and DL,which was suitable for PTX delivery.In vitro drug release study demonstrated that paclitaxel(PTX)was released from FA-BSA-LC/DOPE in a pH-dependent manner.The particle size distribution of the FA-BSA-LC/DOPE-PTX in this study showed that there was no significant change when incubated plasma,suggesting that the high coupling amounts of folic acid modified BSA,and very weak positive charge of FA-BSA-LC/DOPE-PTX could increase the stability in plama.In vitro cytotoxicity assays showed that all blank nanocarriers were nontoxic and biocompatible and could be used as a delivery system for anticancer agents.BSA-LC/SPC-PTX,BSA-LC/DOPE-PTX and FA-BSA-LC/DOPE-PTX exhibited higher cytotoxicity than Taxol(?)even in a low PTX concentration below 0.1 ?g/mL.BSA-LC/DOPE-PTX and FA-BSA-LC/DOPE-PTX inhibited the proliferation more effective than BSA-LC/SPC-PTX.BSA-LC/DOPE-PTX and FA-BSA-LC/DOPE-PTX could escape quickly from lysosomes and rapidly release PTX into the cytoplasm because of the pH-sensitive nature of DOPE.The cellular inhibition of FA-BSA-LC/DOPE-PTX with BSA and FA targeting agents was greater than that of BSA-LC/DOPE-PTX and LC/DOPE-PTX.The cellular uptake study showed that FA-BSA-LC/DOPE-PTX modified with dual-targeting agents BSA and FA,had a faster and greater cellular uptake when compared to BSA-LC/DOPE-PTX and LC/DOPE-PTX,resulting in enhanced cytotoxicity against MCF-7 tumor cells.The Confocal microscopy studies showed that the cellular uptakes of coumarin-6-loaded BSA-LC/SPC,BSA-LC/DOPE and FA-BSA-LC/DOPE were time-dependent;BSA-LC/DOPE and FA-BSA-LC/DOPE were able to effectively deliver coumarin-6 to the cytoplasm.The subcellular localization and intracellular drug release behavior of FA-BSA-LC/DOPE were evaluated by LSCM,These results suggested that FA-BSA-LC/DOPE could facilitate the capacity of endosomal escape,rapidly released of the loaded agents into the cytoplasm under acid conditions in lysosomes.The in vivo targeting activity showed DiR-loaded BSA-LC/SPC,BSA-LC/DOPE and FA-BSA-LC/DOPE all had relative long residence at the tumor tissue,the fluorescence could last for more than 48 h at the tumor tissue.In this study,FA-BSA-LC/DOPE exhibited high tumor targeting ability in vivo,for the BSA layer prolonged the residence time in blood and accumulated in tumor.Besides,folate receptor-mediated endocytosis and pH-sensitive release of loaded agent into cytoplasm aslo contributed to the increased tumor targeting ability of FA-BSA-LC/DOPE.In vivo anti-tumor activity study,the increase of body weight in the mice groups treated with BSA-LC/SPC-PTX,BSA-LC/DOPE-PTX and FA-BSA-LC/DOPE-PTX indicated that the coated BSA could increase the safety of nanocarriers and reduce effectively the side effects and toxicity induced by free PTX.FA-BSA-LC/DOPE-PTX exhibited outstanding tumor inhibition effect with tumor growth inhibition rates 79.3%,The stained tumor tissue section by hematoxylin and eosin(H&E)is showed that the necrosis area in the FA-BSA-LC/DOPE-PTX group was the largest among the tested groups,which presented a substantial evidence of the marked antitumor activity of FA-BSA-LC/DOPE-PTX in vivo.