Magnetic Porous Iron Oxide Nanoprticles As Drug Carriers

Nanomedicine is defined as the application of nanotechnology in medicine field, including medical diagnostics and therapy. Nanosized drug carriers, widely applied in the delivery of chemotherapeutics, can preferably accumulate at tumor tissue through a phenomenon known as the enhanced permeability and retention (EPR) effect, and realize the controlled drug release, resulting in increasing the circulation time of anticancer drugs and reducing side effects to normal cells. In particular, magnetic iron oxide porous nanoparticle have several advantages, including non-toxic (body’s iron stores and eventually incorporated as hemoglobin), large surface area and pore volume for maximal drug loading, easy synthesis and modification of the active surface for maximum signal, environmentally friendly. In this dissertation, we prepared a series of iron oxide nanoparticle, and chose a magnetic iron oxide porous nanorod to construct different nanoparticles systemic drug delivery for cancer therapy by various methods. These delivery systems could achieve to control drug release and exhibit remarkable cancer cells proliferation inhibition capacity. The main work includes:In chapter1, the development of nano-drug delivery carrier in the recent years was summarized, including the passive and active targeting of nanoparticles, the classification, function modification and application of nanoparticles in nano-delivery system.In chapter2, we have used different mole ratio of FeCl2·4H2O and FeCl3·6H2O to prepare spherical and rod-shaped iron oxide nanoparticles with different magnetism and size by coprecipitation approach and calcine. It was observed that the amount of FeCl2improved the crystallinity and shape, and the resulted samples were had high productivity magnetism and crystallinity. As the increase of the ferrous ions, the shape of nanoparticle changed from spherical to rod, finally to smaller spherical, when the mole ratio of Fe2+/Fe3+was15%or more, the nanoparticles shown superparamagnetic, and the saturation magnetization of nanoparticles was increased with the increase of the ferrous ions. When the mole ratio was30%or less, the nanoparticles exhibited porous structure. We chose15%mole ratio nanorods (15%MNRs), with lengths ranging from40nm to60nm, width ranging from10to20nm and pore diameters ranging from5nm to10nm, to achieve load small anti-cancer drug DOX successfully (DOX@15%MNRs). The release rate of DOX was slightly faster in acidity (pH=5.3) condition than that in neutral (pH=7.4) condition. The15%MNRs and DOX@15% MNRs could effectively internalize in the case of HeLa cells, and the amount of cellular uptake with an external magnetic field was much higher than that without an external magnetic field. This indicated that an external magnetic field not only facilitated the nanorods to target cells, but also increased the yield of internalization. As a result, in the presence of external magnetic field, DOX@15%MNRs exhibited much higher cytotoxicity than that without an external magnetic field. We discussed the three uptake pathways of the nanorods, which were clathrin-mediated, caveolae-mediated and macropinocytosis,15%MNRs and DOX@15%MNRs entered cells via the three pathways and there was no significant differences whether an external magnetic field or not. Moreover, we selected a model macromolecular drug FITC-dextran to be loaded into15%MNRs, which confirmed15%MNRs could help macromolecular drug to overcome biological barriers and enter the cell successfully, achieving a macromolecular drug delivery. An external magnetic field would increase the efficiency of macromolecular drug delivery. These results confirmed15%MNRs could be effectively uptake in the case of HeLa cells in order to achieve the small and macromolecular drug delivery, and in response to an external magnetic field.Although nanoparticles can accumulate at tumor site by EPR effect, there are some limitations. In chapter3, we used15%MNRs to construct an active targeting drug delivery system. The15%MNRs was further modified with NH2-PEG-FA (FA-coated15%MNRs) for ligand targeting and modified with NH2-PEG-OCH3(PEG-coated15%MNRs) as a control. The two kinds of nanoparticles was non-toxic in HeLa, MCF-7and COS7cells. Instead of chemical bonding, doxorubicin (DOX), a low water solubility anticancer drug, was loaded in the pores of the modified IOPNR because of their porous structure and high porosity. The release of DOX in acidic PBS solution (pH=5.3) was faster than that in neutral (pH=7.4) solution. Compared with PEG-coated15%MNRs, the presence of FA on the surface of the nanorods increased the cellular uptake of nanorods in the case of HeLa cells, a folate receptor (FR)-positive cell line. When free FA was added in the media, this specific binding was inhibited, so the amount of uptake was sharply reduce as same as the amount of PEG-coated15%MNRs uptake. In contrast, for COS7cells, a FR-negative cell line, FA ligand on the surface of the nanorods showed no effect on the cellular uptake. MTT assay indicated that the cytotoxicity of DOX loaded in FA-coated15%MNRs to HeLa cells was higher than that of DOX loaded in PEG-coated15%MNRs. In the case of COS7cells, no significant difference between the cytotoxicity of DOX loaded in FA-coated15%MNRs and PEG-coated15%MNRs was found. EPR effect and active targeting of FA-coated15%MNRs was confirmed in biodistribution of solid tumors. These results suggested that FA-coated15%MNRs had the potential for target delivery of chemotherapeutic into cancer.In chapter4, two kinds of cationic polyamideamine with or without disulfide linkage in the main chains were synthesized. We used the two kinds of polycations and anionic bovine serum albumin to modify the surface of15%MNRs or DOX@15%MNRs by layer by layer self-assembly. In DTT solution, the size of15%MNRs-ss and DOX@15%MNRs-ss changed, while the size of15%MNRs-cc DOX@15%MNRs-cc was not affected. In vitro release studies revealed that in the presence of DTT, DOX@15%MNRs-ss released DOX more rapidly compared with DOX@15%MNRs-cc, whereas they own the similar release rate in the absence of DTT. The two kinds of nanorods had almost the same amount uptake in the case of HeLa cells. Once internalized in the cells, the S-S bond in DOX@15%MNRs-ss was degraded by the high concentration of GSH, resulting in rapid release of DOX and accumulating into cytoplasm and nuclei, whileDOX@15%MNRs-cc was not affected. So DOX@15%MNRs-ss displayed higher cytotoxicity than DOX@15%MNRs-cc. These results proved that the iron oxide nanoparticles with modification by disulfide bonds showed fast drug release and killing cancer cells effectively.Drug synergy can reduce the dose of drugs for cancer treatment, which lower side effects on normal tissues and cells, however, drug carriers were rarely used for the treatment of diseases using combined drugs therapy, which lead that drug carrier dose not embody the advantages in treatment of cancer. In chapter5, we used previously synthesized15%MNRs as a carrier of arsenic trioxide (ATO) and/or cisplatin (CDDP). CDDP was incorporated into15%MNRs by nanoprecipitation method, whereas ATO was loaded into15%MNRs by the strong interaction between As and iron nanocrystal. Both of free drugs and drugs-loaded15%MNRs showed a synergistic effect in the case of HeLa and HepG2cells, and in the case of A549cells there was a synergistic effect in free drugs, but the drugs-loaded IOPNR emerged drug antagonism. That was to say, drug synergism was closely related to cell types. Cellular uptake experiments proved drugs-loaded nanorods could be effective internalized into HeLa cells. It had been found that drugs-loaded15%MNRs could inhibit the proliferation of cancer cells by inducing apoptosis. Morphological changes of apoptosis were included nuclear chromatin condensation, nuclear cleavage fragments, cristae of mitochondria decrease and disorder reduction, cytoplasmic vacuoles and endoplasmic reticulum dilated at different times. Additionally, when drugs were combination used, they induced apoptosis pathway not only by increasing caspase-3expression but also reducing the ratio of Bcl-2and Bax expression, so there was a synergistic effect of drugs in HeLa cells. These results demonstrated that ATO and CDDP synergy effect existed in some cells, and the drugs combination could reduce the doses of drugs for cancer treatment.In the last chapter, based on previous work in our lab, we explored mitochondria and endoplasmic reticulum in the HeLa apoptosis which was induced by ATO-loaded rod-like or spherical nanoparticles (ATO-loaded HSA-n-PHNRs and ATO-loaded HSA-n-PHNSs). When HeLa cell was treated with free ATO and ATO-loaded nanoparticles, the cell experiment confirmed that the morphology of mitochondria and endoplasmic reticulum changed. The potential of mitochondrial membrane and the activity of caspase-3significantly decreased, the decrease in ATO group was most obvious. These phenomena indicated ATO induced apoptosis through classic mitochondrial pathway. In addition, intracellular Ca2+concentration and ROS levels also increased, the increase in ATO-loaded HSA-n-PHNRs group was more obvious than ATO-loaded HSA-n-PHNSs group. The extra Ca2+was released by endoplasmic reticulum primarily, the uptake of Ca2+by mitochondria would increase, resulting in more sensitive to apoptosis. Although the process of apoptosis was involved the endoplasmic reticulum stress, the important GRP78which was the endoplasmic reticulum chaperone protein had not changed, so there was no direct evidence of the occurrence of endoplasmic reticulum pathway. In animal experiments, after iv injection, it was found that ATO accumulations in tumor tissue in the groups of ATO-loaded HSA-n-PHNRs and ATO-loaded HSA-n-PHNSs were higher than that of free ATO group, because of EPR effect of nanoparticles. ATO-loaded HSA-n-PHNRs exhibited much higher of ATO accumulation than ATO-loaded HSA-n-PHNSs. After33days of inhibition experiment, the ability of ATO-loaded HSA-n-PHNRs groups in inhibition tumor growth was twice higher than that of ATO-loaded HSA-n-PHNSs groups, and was four times higher than that of free ATO group. Theas phenomena indicated the rod-like nanoparticle was more suitable than spherical nanoparticle as an ATO carrier.