Co-delivery Of Chemotherapeutic Drugs And Genes Based On Folate Modified Amphiphilic Chitosan Nanocarrier For Enhanced Antitumor Efficacy

Due to the complexity of signaling network, tumor cells may develop several pathways to escape from death induced by chemotherapeutics. The curative effect of conventional monotherapy hitting single target in tumor cells is therefore severely limited. The combination of chemotherapy and gene therapy provides a promising modality to improve the therapeutic index through simultaneous modulation of multiple signaling pathways in cancer cells. However, chemotherapy has suffered from the poor specificity and severe side effects. Gene therapy also has obstacles in its application because of its poor stability and cellular uptake efficiency. The drug delivery systems which are characterized by improvement of in vivo antitumor efficacy are therefore needed. Chitosan derivatives have been widely applied as the carrier of chemotherapeutic drug and gene due to their satisfactory biocompatibility and biodegradability, the absence of immunogenicity, as well as the multifunctional modification. In our previous study, amphiphilic linoleic acid (LA) and poly (β-malic acid) (PMLA) double grafted chitosan (LMC) could self-assemble into nanoparticles (NPs) in aqueous phase. LMC has been applied in the effective delivery of paclitaxel (PTX) and enhanced green fluorescence protein expressing plasmid (EGFP). LMC could serve as a promising platform for the co-delivery of antitumor drug and gene due to the properties of hydrophilic, hydrophobic modifications and the facility of ligand modification.Herein, LMC nanoparticles (NPs) with various grafting degrees of LA and PMLA were prepared. Folate (FA) was further conjugated to endow LMC NPs with the active-targeting capacity. PTX and surviving shRNA-expressing plasmid (iSur-pDNA), served as antitumor drug and gene, were loaded into LMC NPs to obtain LMC/PTX/pDNA NPs, and investigate the influence of the antitumor efficiency exerted by hydrophilic, hydrophobic and FA modifications both in vitro and in vivo.LMC/PTX/pDNA NPs exhibited particle sizes of 151-220 nm and zeta potentials of 41-46 mV. Particle sizes of LMC/PTX/pDNA NPs decreased with the increasing grafting degree of LA and the decreasing grafting degree of PMLA. PTX was effectively loaded into LMC NPs through sonication and the encapsulation efficiencies were above 80%. The loading capacity of PTX was enhanced with the increasing grafting degree of LA, the decreasing grafting degree of PMLA and FA modification. The strong binding affinity of LMC NPs to iSur-pDNA was confirmed by the encapsulation efficiency, which exceeded 80% in all formulations. LMC/PTX/pDNA NPs could effectively protect iSur-pDNA from enzymatic degradation, in which LMC3/PTX/pDNA NPs showed the strongest protection of iSur-pDNA. The ability to resist non-specific protein adsorption was improved with the higher grafting degree of LA and PMLA, as well as FA modification. The cellular uptake of PTX and iSur-pDNA in both QGY-7703 and L02 cells was raised with the increasing grafting degree of LA, while was declined with the increasing grafting degree of PMLA. FA modification dramatically enhanced the cellular uptake in QGY-7703 cells. LMC/PTX/pDNA NPs mainly internalized through energy-dependent, clathrin-mediated and cholesterol-dependent endocytosis while were irrelevant of caveolin-mediated endocytosis, cytoskeleton reorganization and macropinocytosis. The increment of LA and PMLA grafting degrees and FA modification were responsible for the tardy PTX release and rapid iSur-pDNA release. After release from LMC/PTX/pDNA NPs, more than 90% of PTX was distributed into cytoplasm which was beneficial for the combination between PTX and microtubule. Nuclear accumulation of iSur-pDNA positively correlated with the grafting degree of LA, but negatively correlated with PMLA. After FA modification, the nuclear accumulation of iSur-pDNA was further elevated. The transfection efficiency of LMC/PTX/pDNA NPs was remarkably enhanced with increasing grafting degree of LA, deceasing grafting degree of PMLA and FA modification. The cell apoptosis and cytotoxicity assays demonstrated an enhanced antitumor efficiency with increasing grafting degree of LA, decreasing grafting degree of PMLA and FA modification. In vivo antitumor efficiency of LMC/PTX/pDNA NPs was evaluated in tumor-bearing mice. The enhanced antitumor efficacy induced by the synergistic effect of PTX and iSur-pDNA was proven by the superior tumor inhibition and prolonged survival period exhibited in H22 tumor-bearing mice as compared with LMC3/PTX/pDNA NPs. As compared with FA-LMC3/PTX/pGL NPs or FA-LMC3/pDNA NPs, FA-LMC3/PTX/pDNA NPs showed the highest antitumor efficiency and longest survival time of tumor-bearing mice, in accordance with the results of in vitro cell apoptosis and cytotoxicity assays. In addition, it was worth noting that the TIR and survival ability of mice treated with LMC3/PTX/pDNA NPs was much higher than those treated with FA-LMC3/PTX/pGL NPs, suggesting that the synergistic effect of PTX and iSur-pDNA induced by targeting multiple mechanisms was more effective than improvement in the monotherapy through ligand modification.In summary, folate modified LA and PMLA double grafted chitosan NPs, amphiphilic polymeric NPs, could be served as nanocarriers for enhance antitumor efficacy induced by the synergistic effect of antitumor drugs and genes.