Biocompatibility and properties of carbon nanotubes in the biological systems

Abstract

The potential widespread use of nanomaterials has led to a strong public concern about their impact on human health and the environment. Carbon nanotubes (CNTs) are nano-sized hollow graphite cylinders and their unique physical and chemical properties have raised great expectations for numerous applications in medicine, chemistry, electronic, materials and the environment. As the production and applications of CNTs increase, the introduction of CNTs into the environment will also occur more frequently. However, information on their potential environmental and health impact is still insufficient, and the biocompatibility and safety concern have limited their applications. In this thesis, the in vitro and in vivo biocompatibility and properties of CNTs in the biological systems were investigated using various mammalian cells and the zebrafish. Raw CNTs are strongly hydrophobic and not soluble in water. After certain purification and functionalization procedures, raw CNTs can be modified into functionalized CNTs (f-CNTs) differing from raw CNTs in their surface properties and purities. While f-CNTs exhibit a larger spectrum of biomedical and pharmaceutical applications, the biocompatibility and the intracellular fate and behaviour of f-CNTs are not fully understood yet. Here time-lapse fluorescence microscopy was used to investigate the intracellular dynamic distribution of f-CNTs in living cells. The results demonstrated the differences of distribution and accumulation profiles of f-CNTs between primary cells and transformed cells. Functionalized CNTs entered the cancer cell nucleus and accumulated in the primary cytoplasm in an energy-dependent process. The presence of f-CNTs in the transformed cell nucleus did not cause discernible changes in the nuclear organization and had no effect on the growth kinetics and cell cycle distribution for up to 4 days. Upon removal of the f-CNTs from the culture medium, f-CNTs rapidly moved out of the nucleus from the transformed cells and slowly moved out of the cytoplasm from the primary cells. Thus, the intracellular f-CNTs were highly dynamic and divergent in different cell types. The cellular removal of f-CNTs from the cells suggested that the cell penetration of f-CNTs were bi-directional, indicating a possible controllable clearance of f-CNTs after the delivery. Furthermore, the cellular accumulation study in two multi-drug resistant systems showed f-CNTs will not be pumped out by the efflux pumps, and achieved similar accumulation in both the sensitive and drug resistant cell lines. These observations suggested the intrinsic transporting capabilities of f-CNTs combined with their effective transporting in the resistant cancer cells can potentially lead to novel drug delivery and cancer therapy. The in vivo biocompatibility and properties of f-CNTs were investigated in developing zebrafish from early embryonic stage through three different exposure schemes. All tested f-CNTs displayed good in vivo biocompatibility and did not show any obvious toxic effects in zebrafish. When injected into the embryos at 1-cell stage, f-CNTs accumulated in the blastoderm cells but not in the yolk cell in zebrafish embryos, and distributed evenly among the blastoderm cells by the cytoplasmic stream of the yolk during embryogenesis. These injected f-CNTs translocated into the nuclear membrane and entered the nucleus of the blastoderm cells. The embryonic cells produced vesicles to wrap the f-CNTs upon loading, and the embryos exerted immune response to the loading of f-CNTs by relocating the circulating white blood cells along the trunk region. The reproductive systems of the treated zebrafish, including the production of primordial germ cells, were not affected. The whole life cycle analysis demonstrated zebrafish loaded with f-CNTs had similar survival rates as the untreated control, and they formed proper reproduction systems and produced second generations. Functionalized CNTs displayed good in vivo biocompatibility in the whole life cycle in the loaded zebrafish themselves, but the implanting of f-CNTs in zebrafish at 1-cell stage affected their second generation survival rates. When f- CNTs were delivered into the circulation systems at 72 hpf by intravascular loading, they moved easily in the compartments and gradually accumulated in the swimbladder structured region within 24h, and maintained a circulation time more than 48 h in the cardiovascular systems in the larvae. The larvae finally managed to clean up the loaded f-CNTs at around 96h after the loading, indicating a cleaning up mechanism. When f-CNTs were put into aquatic environment, they formed small bundles rather than behaving as single nanotubes. When zebrafish embryos were exposed to these small bundles of f-CNTs in aquatic environment, the embryonic chorion prevented them to get into direct contact with the inside embryos. Thus, f- CNTs did not affect the normal embryonic development of the exposed embryos in aquatic environment. The present study indicated the good biocompatibility of f- CNTs for in vivo applications. The impact of raw CNTs on the aquatic environment was investigated by examining the properties of raw CNTs under several environmental conditions and using developing zebrafish (Danio rerio) embryos. Raw CNTs formed aggregates in aquatic environment, and the agglomerate size for single walled CNTs (SWCNTs) was significantly larger at pH ≥11 and was stable at temperatures from 4 °C to 40 °C and salinities from 0 ppt to 30 ppt. Exposure to SWCNTs induced a significant hatching delay in zebrafish embryos between 52 to 72 hours post-fertilization (hpf) at concentrations greater than 120 mg/L, but 99% of the exposed embryos hatched by 75 hpf. Double-walled carbon nanotubes (DWCNTs) also induced a hatching delay at concentrations greater than 240 mg/L, but carbon black did not affect hatching at the concentrations tested. Molecular and cellular analysis showed that the embryonic development of the exposed embryos up to 96 hpf was not affected at SWCNT concentrations up to 360 mg/L. Scanning electron microscope inspection showed that the size of the pores on the embryo chorion was nano-scaled and the size of SWCNT agglomerates was micro-scaled or larger, indicating that the chorion of zebrafish embryos was an effective protective barrier to SWCNT agglomerates. The hatching delay observed was likely induced by the Co and Ni catalysts used in the production of SWCNTs that remained at trace concentrations after purification. This study suggests that materials associated with raw SWCNTs, perhaps metal contaminants, have the potential to affect aquatic life when released into the aquatic environment. The present study demonstrated that well purified CNTs were biocompatible in the biological systems but the associated contaminations can induce adverse toxic effects. For example, CNTs are frequently contaminated with metals, amorphous carbon and other materials in the preparations processes. The findings can help understand the in vivo and in vitro biocompatibility and properties of CNTs in the biological systems. The present study can also help understand the interactions between manufactured nanomaterials and the biological systems. This thesis can be a reference for the green chemistry of nanotechnology and nanomaterials, especially for the further biomedical applications using f-CNTs.