Abstract
The objectives of this research are the preparation, characterization and in vitro cell tests of two drug delivery systems. Since the conventional drugs belong to the dump system, people have to take more than the predicted amounts of drugs to reach the therapeutic effect. Hence, this study has developed two types of carbon materials as drug carriers to overcome the drawbacks of conventional drugs. The first one is multi-walled carbon nanotubes (MWCNTs) based drug delivery system and the second one is graphene oxides (GOs) based drug delivery system. In the aspect of carbon nanotubes, multi-walled carbon nanotubes (MWCNTs) was combined with the iron oxide magnetic nanoparticles (Fe3O4), anti-cancer drug doxorubicin (DOX), targeting molecule folic acid (FA), and fluorescent dye (FITC) to form a multifunctional MWCNT drug carrier system. This study includes the following subjects: 1. Acid oxidation and purification of the MWCNTs to reduce toxicity and to increase the biocompatibility and aqueous solibility. 2. Supramolecular modification of the MWCNT surface with poly acrylic acid (PAA) was conducted to obtain the buffer dispersible PAA-CNT. The possibility of the following magnetic particle decoration was evaluated. 3. Conjugation with folic acid, FITC and Fe3O4 (magnetic particle) were grafted on the PAA-CNT surface to acquire the ability of magnetic guidance and primary targeting. Accompanying the high drug loading ability of the MWCNT, the chemotherapeutic ability during co-culture with the U87MG glioma cells was evaluated and the cell survival was investigated. PAA-CNTs were analyzed by X-ray photoelectron (XPS) and thermogravimetric analysis (TGA). The morphology of PAA-CNTs-Fe was observed by Transmission electron microscope (TEM). Through TGA analysis, the content of PAA on MWCNT was estimated about 23.1wt%. From XPS analysis, it was found that the additional XPS peaks of PAA-MWCNTs appeared at 285.2, 286.1eV, originated from the PAA main chain; these peaks confirmed that MWCNTs were successfully functionalized. Then from the TEM image of PAA-CNTs-Fe, one can observe the uniform dispersion of Fe3O4 nanoparticles on the PAA-MWCNT surface. PAA-MWCNTs exhibit much higher DOX loading efficiency than that of pristine-MWCNTs. The DOX loading efficiencies of pristine-MWCNTs and PAA-MWCNTs were 95% and 187.6%, respectively. This suggests that PAA polymer chain on MWCNTs can provide excellent electrostatic interaction with positively charged DOX due to the negatively charged functional groups such as carboxyl group. Thus, PAA-MWCNTs can combine electrostatic and stacking interactions to increase the efficiency of DOX loading. Furthermore, the release of DOX from PAA-MWCNTs was pH-dependent. The release of DOX was fast in acid environment but slow in neutral environment. This phenomenon suggested that PAA-MWCNTs were promising delivery materials for anticancer drug DOX. It is intend to investigate the function of the targeting molecules FA which plays the key role of receptor-mediated endocytosis. U87MG cells were incubated with PAA-CNTs-Fe-FITC and PAA-CNTs-FA-Fe-FITC, respectively. By confocal fluorescence microscopy imaging, one can observe that both PAA-CNTs-Fe and PAA-CNTs-FA-Fe can be transported into cells via endocytosis. However, PAA-CNTs-FA-Fe-FITC revealed intense green fluorescence than PAA-CNTs-Fe-FITC in U87MG cells. It means that the effect of FA can assist the PAA-CNTs to enter the cell efficiently. This important result will help to develop MWCNT functionalization schemes with specific materials for recognizing and targeting different types of tumor cell. In the tumor cell toxic tests, PAA-CNTs-FA-Fe was designed to assist the antitumor drugs DOX to eliminate the brain tumor cells (U87MG) with less side reaction, and this drug delivery system exhibited the highest cytotoxicity against U87MG cells. In comparison with free drug, the IC50 (half-maximum inhibitory drug concentration) value for PAA-CNTs-FA-Fe-DOX (~28μg/ml) is much lower than that of free DOX (~89μg/ml), suggesting the PAA-CNTs-FA-Fe loaded with DOX can enter into cell effectively and release DOX into cell nuclei rapidly. Finally, the magnetic targeting test was conducted to confirm the magnetism of PAA-CNTs based drug delivery system and its effect on cell toxic tests. For U87MG cells which was treated by PAA-CNTs-Fe-DOX or PAA-CNTs-FA-Fe-DOX with an external magnetic field, it can be observed that almost no U87MG cells were located on the outer of culture mediums and the number of tumor cell decreasing significantly in magnetic zones, which implies that these zones attracted more drug carriers and thus possessed higher drug concentration to inhibit the survival of tumor cells. These phenomena indicate that magnetic MWCNTs drug delivery system is a very promising nano-platform for future cancer therapeutics. In the aspect of graphene oxides, GOs were combined with anti-cancer drug Carmustine (BCNU) to form a drug carrier system. This study includes the following subjects: 1. Preparation of the acid-oxided graphene (GOs), the length of GOs was shortened by ultrasonication, then the toxicity was also reduced and the biocompatibility will be increased via purification. 2. Supramolecular modification of the GO surface with poly acrylic acid (PAA) to obtain the biocompatible GO, rendering stability in physiological solutions including serum. 3. In order to reach the target of fluorescent detection during cell tests, Cyanine-5(Cy5) was decorated on the surface of PAA-GOs and carried on the incubation with GL261 glioma cells to observe cell endocytosis of PAA-GOs. 4. To form a drug delivery system, BCNU was attached on the PAA-GOs surface. The chemotherapeutic ability of PAA-GOs-BCNU during co-culture with the GL261 glioma cells was evaluated in comparison with free BCNU. PAA-GOs were analyzed by X-ray photoelectron (XPS) and thermogravimetric analysis (TGA). The morphology and dimension of PAA-GOs were observed by Transmission electron microscope (TEM) and Atomic force microscope (AFM). Through TGA analysis, the content of PAA on GO was estimated about 15.7 wt%. From XPS analysis, it was found that the additional XPS peaks of PAA-GOs appeared at 285.1, 285.9 eV, which were originated from the PAA main chain, these peaks confirmed GOs were successfully functionalized. Then from the TEM image of GO and PAA-GO, one can observe that GO was a thin material with wrinkles while PAA-GO showed obvious PAA-covered area. Finally, from the AFM image GO and PAA-GO one can find the dimensions of GO and PAA-GO were less than 60nm, suggesting that continued ultrasonication helped reduce the dimensions of both materials. The thickness of PAA-GO was about twice of GO, this also confirmed that PAA was grafted on GO successfully. In order to confirm that PAA-GOs can be transported into glioma cells, GL261 cells were incubated with PAA-GOs-Cy5 to proceed the fluorescent detection. Cy5 (Cyanine-5) is a kind of synthetic red fluorescent dye which is often used in biomedical imaging. By confocal fluorescence microscopy imaging, one can observe that PAA-GOs were transported into GL261 cells via endocytosis and spread uniformly inside cells. In order to confirm whether pure PAA-GOs were toxic to tumor cells or not, PAA-GOs were incubated with GL261 cells at first. Results showed that even when the concentration of PAA-GO reached up to 200μg/ml, cell survival was still very high (>70%). This suggested pure PAA-GOs did not induce cell death. Hence, PAA-GOs was combined with the antitumor drugs BCNU to proceed the following tumor cell toxic tests. In comparison with free BCNU, when the concentration of BCNU reached up to 50μg/ml, the number of cells treated with PAA-GOs-BCNU only remained 20%. At the same concentration of BCNU, the number of cells treated with free BCNU still remained 75%. These results suggest that PAA-GO drug delivery system with less BCNU can reach the better cancer therapeutics. This can reduce the usage of drugs and prevent the waste of drugs. In this study, two types of carbon material (MWCNT and GO) based drug delivery systems have been developed to increase their effect in killing tumor cells and to decrease the usage of drugs at the same time. Regarding MWCNT-based drug delivery system, the hydrophilic, pH sensitive, and dual targeting drug vehicle, PAA-CNTs-FA-Fe-DOX was designed to eliminate U87MG cells in vitro. From the results it was found that doxorubincin could focus on the tumor region and decreased unnecessary peripheral toxicity by the magnetic guidance (primary targeting) and the affinity of FA (secondary targeting). Regarding GO-based drug delivery system, PAA-GO was combined with BCNU to form PAA-GO and it was used to eliminate GL261 cells in vitro. From the results it was found that PAA-GO with less BCNU could kill GL261 cells effectively in comparison with free BCNU. It is anticipated that both of these materials can become very promising drug delivery platforms for cancer therapeutics effectively.