TAT-chitosan Functionalized Multiwalled Carbon Nanotubes As Delivery Systems

Recent progress in nanotechnology enables creation of targeted drug delivery system which can effectively delivery anti-cancer drugs to the specific tissues, organs and cells with minimal systemic toxicity. Functionalized carbon nanotubes (f-CNTs) displayed great potential in biomedical applications due to their unique features and functionality, especially used as an effective carrier for gene delivery or drug delivery. However, poor dispersion of carbon nanotubes in aqueous solution limits their application in the biomedical field to a certain extent.High cellular uptake rate and good bio-security are very important in the development and application of carbon nanotubes as delivery system. In this study, a novel carbon nanotubes carrier modified with water-soluble chitosan (CS) and TAT polypeptides were investigated. Water-soluble CS has attractive biocompatibility, biodegradability, nontoxicity and excellent hydrophilicity. Carbon nanotubes modified with water-soluble CS have showed highly improved dispersibility without any damage to their unique physical features such as light and heat behavior. Functionalized carbon nanotubes can be further crosslinked with specific targeting molecules, such as monoclonal antibodies, peptides and saccharides.The capability of efficiently penetrating into cells is one of the prerequisites for the drug/gene delivery vehicles. TAT (transactivator of transcription) peptide, a widely accepted cell penetrating peptide which can penetrate across the cell membrane in less than one minute, has been successfully utilized to modify several polymers to enhance their cell penetrating capability. In the present study, low molecular weight chitosan (LMWC) conjugated with TAT peptide was used for non-covalent functionalization of multiwalled carbon nanotubes (MWCNTs), aiming at providing a more efficient drug delivery vehicle for cancer therapy. The TAT-chitosan conjugated MWCNTs (MWCNTs-TC) were further investigated for their water solubility, cytotoxicity, cell penetrating capability and in vivo accumulation in the target tumor tissues. The work mainly includes the following three parts:1. Preparation and characterization of MWCNTs-TC and MWCNTs-CSTC was synthesized by covalently coupling the TAT peptide with LMWC using N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) as the linking agent,'H-NMR spectroscopic analysis was conducted to characterize TC and CS, the substitution degree of TC was about 4.1%. MWCNTs-TC with a TC/MWCNTs (w/w) ratio of 10:1 was most water soluble and remained stable for at least 2 months, which was similar to MWCNTs-CS, while the p-MWCNTs precipitated very soon after the preparation. Transmission electron microscopic observation showed that MWCNTs-CS and MWCNTs-TC were better dispersed than the p-MWCNTs, but no obvious morphological difference was found among the three types of MWCNTs. TGA results showed that the amount of CS and TC coated on MWCNTs calculated from the difference in the weight loss were 58.54 wt.% and 59.79 wt.% respectively. The average zeta potential was 47.7 ±1.96 for MWCNTs-TC and 46.1±1.83 for MWCNTs-CS, which were much higher than that for p-MWCNTs (13±1.32), indicating that coating of the positively charged CS or TC elevated the zeta potential of the MWCNTs.2.The study in vitro for MWCNTs-TC as a carrierInternalization of MWCNTs-TC or MWCNTs-CS into MD-MBA-231 cells was observed with laser confocal microscope and it showed that both MWCNTs-TC and MWCNTs-CS could efficiently enter the tumor cells and mainly accumulated in the cytoplasm. Flow cytometric analysis demonstrated that the cellular uptake rate of MWCNTs-TC were moderately higher (84.69±5.03%) than that of MWCNTs-CS (72.85 ±4.95%). Particularly important was the finding that the fluorescent intensity in the MD-MBA-231 cells incubated with MWCNTs-TC was about 25 times higher than that in the cells incubated with MWCNTs-CS, revealing that more MWCNTs-TC were taken up by each cell as compared with MWCNTs-CS. The toxicity of p-MWCNTs, MWCNTs-TC or MWCNTs-CS against two typical normal cell lines (HUVEC, L929) and one breast cancer cell line MD-MBA-231 was assessed by the CCK-8 assay. The results showed that both MWCNTs-TC and MWCNTs-CS were less toxic against the three types of cell as compared with p-MWCNTs, and MWCNTs-CS was the least cytotoxic. In addition, we compared the impact of pristine MWCNTs(p-MWCNTs) and carboxylic acid functionalized MWCNTs(MWCNTs-COOH) on RAW264.7 cells by looking at the cell viability, phagocytic activity, production of cytokines (IL-1?, IL-10, IL-12 and TNF-a) and intracellular reactive oxygen species (ROS). It was revealed that exposure to either p-MWCNTs or MWCNTs-COOH induced decreased viability of murine macrophage RAW 264.7 cells, as well as moderately elevated phagocytic activity of murine peritoneal macrophages, but no statistical significance was found between the two groups. Increased production of ROS in macrophages was induced after exposure to either p-MWCNTs or MWCNTs-COOH. However, no significantly elevated production of cytokines (IL-1?, IL-10, IL-12, and TNF-?) was observed from RAW 264.7 cells after exposure to the CNTs.3.1n vivo distribution study for MWCNTs-TCMWCNTs-TC or MWCNTs-CS labeled with Alexa Fluor(?) 700 NHS Esterwere intravenously injected into tumor-bearing mice with NS treated mice as negative control. Living animal imaging was conducted at 24,48 and 72 h after CNTs administration. Living animal imaging on the breast cancer-bearing mice was conducted to investigate the in vivo distribution of intravenously administered fluorescent MWCNTs-TC or MWCNTs-CS. In the MWCNTs-TC group, strong fluorescence in tumor tissues and kidney was observed at 24 h after injection, and visible fluorescence was still found in the tumor tissues and kidney at 48 h and 72 h after the injection, although the fluorescent intensity gradually attenuated with time. In the MWCNTs-CS group, visible fluorescence was only observed in kidney at 24 and 48 h after the injection, which diminished at 72 h after the injection. Direct imaging on the harvested tissues was conducted, it was demonstrated that higher amount of MWCNTs-TC was distributed in tumor, liver and lung as compared with that of MWCNTs-CS at 24 and 48 h after administration. Obvious kidney distribution was observed in both MWCNTs-TC group and MWCNTs-CS group, and no dramatic difference was found between the two groups. No heart and spleen distribution was found in either MWCNTs-TC or MWCNTs-TC group.In conclusion, the preliminary data collected from the present study demonstrated that MWCNTs-TC were essentially non-toxic with perfect water solubility, and were more efficient in term of cancer targeted intracellular transport both in vitro and in vivo as compared with MWCNTs-CS, suggesting the great potential of MWCNTs-TC in cancer therapy. However, further investigation on drug loading capacity, targeting specificity and tumor suppressive effect should be conducted before their application in the clinic.