Studies On Preparation, Characterization And Properties Of The Carbon Nanotubes/Polyamidoamine Dendrimer Hybrid As Gene-Delivery Vectors

In this paper, combined carbon nanotubes (CNTs) with polyamindoamine (PAMAM), the CNT-PAMAM hybrids were prepared by covalent functionalization of the CNTs with PAMAM dendrimer molecules, and the prepared CNT-PAMAM hybrids were exploited as gene supporting materials for intracellular transporting; the CNT-PAMAM hybrids were characterized by scanning electron microscopy (SEM), transimition electron microscopy (TEM), FITR, UV-vis spectroscopy, fluorescent microscopy and so on. The micromorphologies of the CNT-PAMAM hybrid, dispersive property, protein/gene immobilization ability, cellular cytotoxicity, interaction between the hybrids and live cells and the transfection performance of the CNT-PAMAM hybrids acting as gene surporting materials for intracellular transporting have been investigated in detail. Additionally, the factors that affect the transfection efficiency of the CNT-PAMAM hybrids have also been discussed. The main work is summarized as follows:1. The progress and the problems in the research of gene/protein therapy have been briefly reviewed. On the other hand, the structure, properties, surface functionalization and the application of CNTs in biomedicine field have been introduced in details.2. Polyamindoamine (PAMAM) dendrimers were covalently tethered onto the surface of multi-walled carbon nanotubes (MWCNTs). The morphology, dispersive property and peripheral functional groups of the MWCNT-PAMAM hybrids were characterized and identified. The cytotoxicity to human osteosarcoma MG-63 cells, protein and DNA immobilization ability of the hybrids were evaluated in detail. The results have showed that the dendrimers grow on the surface of MWCNTs with a high density; the MWCNT-PAMAM hybrids show good dispersibility and stability in aqueous solutions. We have found that the hybrids are biocompatible, with no obvious cytotoxicity within a given concentration (< 25μg mL-1) and long-time (24 h) exposure in cells and possess excellent biomolecule immobilization ability (bovine serum albumin and 5'-Fam-CAAggTCgTgTAAAggTCAg-3'were used here as a model). The functionalization of MWCNTs with PAMAM dendrimers improved the biomolecule immobilization ability for about 70 times and simultaneously decreased the cellular toxicity to about 30%.3. The MWCNTs functionalized with generation 4 (G4) PAMAM dendrimers was used as delivery system to transport the green fluorescence protein (GFP) gene into cultured HeLa cells. The interactions of MWCNT-PAMAM with plasmid DNA of enhanced green fluorescent protein (pEGFP-N1) and Hela cells were characterized and identified by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and ultraviolet-visible spectra (UV-vis). The intracellular trafficking of the complex of MWCNT-PAMAM with plasmid DNA was monitored with TEM. The transfection efficiency of MWCNT-PAMAM hybrid to GFP gene was improved about 2.4 and 0.9 times than those of acid-treated MWCNTs (MWCNT-COOH) and pure PAMAM dendrimers. And simultaneously the cytotoxicity of MWCNT-PAMAM was decreased about 38% compared with pure PAMAM. Our results demonstrated a simple way to increase the transfection efficiency of MWCNTs and simultaneously decrease cytotoxicity through modification of PAMAM dendrimers.4. Polyamindoamine (PAMAM) dendrimers were covalently tethered onto the surface of three different kinds of carbon nanotubes (CNTs), which were respectively denoted by L-MWNTs-1020, S-MWNTs-1020 and L-SWNTs-1. The three CNTs-PAMAM hybrids were used as delivery system to transport the green fluorescence protein (GFP) gene into cultured HeLa cells. The relationships between transfection efficiency and the properties of three CNTs-PAMAM hybrids, which involve the numbers of surface functional groups, cytotoxicity, length/diameter of CNTs, were preliminarily evaluated. The results suggested that the prepared three kinds of CNTs-PAMAM hybrids can all transport the pEGFP-N1 into Hela cells and the GFP can be expressed normally in the Hela cells. However, the three kinds of hybrids have different transfection efficiency; the order of transfection efficiency is as follows: MWCNT-PAMAM-2> SWCNT-PAMAM-3> MWCNT-PAMAM-1. The more of the quantity of the functionalized amine groups on the surface of the CNTs-PAMAM hybrids, the more of the transfection efficiency; on the other hand, the parameter (especial the length and impurity) of the used CNTs is one of the important factors that may affect the transfection efficiency; the CNTs that have relative short length, good dispersive property and no impurity will have good transfection efficiency.