Studies On The Preparation,Characterization, Cytotoxicity, And PEGFP Transfection Ability Of MWCNT-CS-FA Hybrids

In this paper, we combined the advantages of multi-walled carbon nanotubes (MWCNTs), chitosan (CS), and folic acid (FA) to synthesize the MWCNT-CS-FA NP hybrids. Firstly, the MWCNTs were treated with mixed acids (concentrate HNO3+H2SO4, v/v,1:3) for different time to obtain carboxylated MWCNTs with different length. Based on these carboxylated MWCNTs, the chitosan-folic acid nanoparticles (CS-FA NPs) and the MWCNT-CS-FA NP hybrids were synthesized by ionotropic gelation process. The morphologies/microstructures, surface groups, and surface potential of the CS-FA NPs and MWCNT-CS-FA NP hybrids were characterized by field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy (FTIR), and Zeta potential instruments. Additionally, their cytotoxicity and transfection ability for enhanced green fluorescence protein plasmid (pEGFP) in mammalian cells were investigated in detail. The main works are summarized as follows:1. The MWCNTs were treated with mixed acids (concentrate HNO3:H2SO4, v/v,1:3) for24、36、40, and48h, respectively to prepare four kinds of carboxylated MWCNTs with different length. The micro-morphologies, surface functional groups, and the surface potential of these MWCNTs were characterized by FESEM, FTIR, and Zeta potential instruments. The cytotoxicity to HeLa cells and the pEGFP gene transfection property of the MWCNTs treated with mixed acids for different time were investigated by fluorescence microscopy and MTT assays. The results demonstrate that the mixed acid treatment can functionalize carboxylate or hydroxyl groups onto the surface of MWCNTs. With the prolonging of the treating time, the length of the MWCNTs decreases; and the tubular morphology of the MWCNTs has no obvious change. In the studied concentration range, the carboxylated MWCNTs show some cytotoxicity to HeLa cells. The carboxylated MWCNTs with shorter length show larger cytotoxicity and higher transfection efficiency than those of longer MWCNTs.2. The MWCNT-CS-FA NP hybrids and CS-FA NPs were prepared by ionotropic gelation process. The morphologies, surface functional groups, and Zeta potential of these hybrids were characterized by FESEM, FTIR, and Zeta potential instruments. The results suggest that the CS-FA NPs with small diameter were efficiently tethered onto the surface of MWCNTs in relatively high density and with uniform coverage. The MWCNT-CS-FA NP hybrids are positively charged in aqueous solutions.3. The prepared MWCNT-CS-FA NP hybrids and CS-FA NPs were used as delivery vectors to transport the pEGFP-N1gene into cultured HeLa cells and MCF-7cells. Their transfection properties were investigated in detail. The cytotoxicity of the above two nanomaterials to HeLa cells and MCF-7cells were also investigated by MTT assays. Additionally, the effects of the mixed acid treating time and TPP concentration on the pEGFP-N1transfection ability and cytotoxicity were evaluated. The transfection property and cytotoxicity of the MWCNT-CS-FA NP hybrids were compared with those of the CS-FA NPs. The results suggest that the transfection efficiency of CS-FA NPs to pEGFP-N1is lower than that of MWCNT-CS-FA NP hybrids and in the same concentration range, the CS-FA NPs show larger cytotoxicity to HeLa cells than that of MWCNT-CS-FA NP hybrids. When the TPP concentration is1.0mg mL and the mixed acid treating time is48h, the prepared MWCNT-CS-FA NPs possess largest transfection efficiency to pEGFP-N1.