| Objectives: To synthesize tumor microenvironment-responsive single-walled carbon nanotubes(SWCNTs) loading doxorubicin(DOX) and survivin siRNA, and to characterize the physicochemical properties and evaluate the drug release, cytotoxicity, cellular uptake, intracellular localization, expression of protein and apoptosis effect of functionalized SWCNTs in vitro. In addition, the anti-tumor effects of functionalized SWCNTs were also studied. This study can provide experimental evidence for the co-delivery of anticancer drug and siRNA.Methods: Raw SWCNTs were purified by an acid oxidation to form the oxidized SWCNTs(O-SWCNTs). Polyetherimide(PEI) was covalently modified with Betaines and the resulting conjugate PEI-Betaines(PB) was synthesized with O-SWCNTs to form SWCNT-PEI-Betaines(SPB), which exhibits a pH-responsive lysosomal escape. DOX was conjugated with the SPB via π-π stacking interaction to form DOX-SWCNTs-PEI-Betaines(DOX-SPB), and further noncovalently functionalized with DSPE-PEG2000-Mal modified with the targeting penetrating peptide BR2 to get DOX-SWCNT-PEI-Betaine-BR2(DOX-SPBB). Subsequently, survivin siRNA was adsorbed onto DOX-SPBB to form DOX-SWCNTs-PEI-Betaines-BR2-siRNA(DOX-SPBB-siRNA). SPB were characterized by fourier transform infrared resonance spectroscopy(FT-IR), ultravio let-visible spectrophotometry(UV-Vis), XRD spectroscopy, transmission electron microscopy(TEM), thermal gravity analysis(TGA). The cytotoxicity and cellular efficacy of SPB and DOX-SPBB were evaluated in A549 cells using a WST-1 assay. The cellular uptake, intracellular localization, endosomal escape, gene silencing and cell apoptosis of the functionalized SWCNTs were performed using flow cytometry, confocal laser scanning microscope(CLSM), and Western blot analysis. The anti-tumor effects of DOX-SPBB-siRNA were evaluated in A549 tumor-bearing nude mice. Finally, H&E staining method was employed to investigate the influence of functionalized SWCNTs on the tissues and organs of mice.Results: According to a series of modification and characterization, DOX-SPBB- siRNA was successfully synthesized. BR2 displayed more effective targeting ability than RGD in A549 cells. The SPB showed lower toxicity to A549 cells and 293 T cells compared to SP. The DOX-SPBB displayed relatively higher IC50 value of 63.5 μg/mL. SPBB showed significantly higher uptake of siRNA than SP or SPB. The cellular uptake siRNA by DOX-SPBB-siRNA were 20% lower than that by SPBB-siRNA, while the uptake of DOX showed no significant difference. The endosomal escape of SPB was faster than that of SP, the intracellular localization of DOX-SPBB-siRNA showed that siRNA and DOX could effectively arrived in the cytoplasm and nuclear of A549 cells at 4h, respectively. On the other hand, SPBB-siRNA showed significantly lower survivin expression and apoptotic index than Lipofectamine 2000. Compared to SPBB-siRNA or DOX-SPBB, DOX-SPBB-siRNA significantly reduced tumor volume in A549 lung cancer cells-bearing nude mice, showing synergestic effects of DOX and survivin siRNA. H&E staining also indicated that the most potential therapeutic effects of DOX-SPBB-siRNA on the tumor without distinct damages to normal tissues.Conclusion: Novel functionalized SWCNTs loading DOX and survivin siRNA(DOX-SPBB-siRNA) were successfully synthesized. The functionalized SWCNTs showed effective anti-tumor effects both in vitro and in vivo, and may also provide an effective stragy for the co-delivery of anti-cancer drug and gene. |
