| Nanocarrier-based tumor targeted drug delivery systems have been extensively investigated to overcome the poor selectivity and severe side effect of chemotherapeutic agents in cancer treatment. Specific ligands and hydrophilic polymers are commonly used in the surface modification of nanocarriers to improve the active and passive tumor targeting efficacy, respectively. It is known that surface modification may alter the particle size, surface charge, protein adsorption, drug release rate, cellular uptake, and biodistribution of nanocarriers. Therefore, it is necessary to evaluate the effect of surface modification on the in vitro and in vivo tumor targeting efficacy of nanocarriers.Linoleic acid and poly (β-malic acid) double grafted chitosan (LMC) can self-assemble into nanoparticles (NPs) in aqueous condition, which were proved to be efficient in the tumor targeted delivery of paclitaxel (PTX). In the present study, biotin (BT), folic acid (FA), and methoxyl(poly(ethylene glycol)) (PEG) were conjugated to LMC NPs, to evaluate the effect of dual-ligand and PEG modification on tumor targeting efficacy. LMC NPs with single or dual-ligand (BT-LMC NPs, FA-LMC NPs, and FA,BT-LMC NPs) and FA-LMC NPs with various densities of PEG (3.7%,8.9%, and 15.1%) were synthesized. Fourier transform infrared spectrum (FTIR) and nuclear magnetic resonance (’H NMR) studies showed that BT, FA, and PEG were successfully conjugated to LMC NPs. The DS of BT in BT-LMC NPs and FA,BT-LMC NPs was 7.5%. The DS of FA in FA-LMC NPs, FA,BT-LMC NPs, and PEG modified FA-LMC NPs was 7.5%,4.9%, and 7.5%, respectively. The DS of PEG in PEG1,FA-LMC NPs, PEG2,FA-LMC NPs, and PEG3,FA-LMC NPs were 3.7%,8.9%, and 15.1%, respectively. Significantly increased particle sizes of LMC NPs were detected after BT modification, while the contribution of FA modification to the particle size was negligible. Particle sizes of FA-LMC NPs were increased with the increased density of PEG. Surface modified LMC NPs exhibited decreased negative surface charge and enhanced hydrophilicity.BSA served as model protein to evaluate the effect of surface modificatioin on protein adsorption of NPs. The BSA adsorption of BT-LMC NPs, FA-LMC NPs and FA,BT-LMC NPs were 60.2%,59.1%, and 85.3% to that of LMC NPs, respectively, indicating that protein adsorption of NPs was suppressed after ligands modification. For PEG modified FA-LMC NPs, BSA adsorption was restrained with the increased density of PEG. PTX was used to investigate the effect of surface modification on the in vitro drug release. After 12 h, the PTX accumulative release rate for LMC NPs, BT-LMC NPs, FA-LMC NPs and FA,BT-LMC NPs were 79.8%,58.7%,69.7%, and 40.2%, respectively. Decreased release rate was also detected in PEG modified FA-LMC NPs, particularly for PEG3,FA-LMC NPs.Cellular uptake was conducted on SMMC-7721 cells (human hepatoma cell line), L02 cells (human liver cell line), and Raw 264.7 cells (mouse leukaemic monocyte macrophage cell line). The results showed that dual-ligand modification was a more effective method to improve the tumor cellular uptake selectivity of LMC NPs than single-ligand modification. It was also observed that the macrophages uptake of LMC-NPs was notably promoted by ligands modification. Cellular uptake of FA-LMC NPs was inversely correlated with the density of PEG modification.In vivo tumor inhibition and PTX accumulation were monitored in H22 tumor bearing mice. The tumor growth inhibition ratio (TIR) of PTX-loaded LMC NPs was enhanced by single-ligand modification and further improved after dual-ligand modification, which were correlated well with the results of in vitro cell uptake and in vivo PTX accumulation. For PEG modification, the phagocytosis of FA-LMC NPs by the spleen was decreased with the increased density of PEG modification. Combined the results of TIR and PTX accumulation, it was exhibited that FA-LMC NPs with moderate density of PEG was more favorable for tumor targeted delivery of PTX than high and low density of PEG modified FA-LMC NPs.
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