Preparation Of Casein Nanospheres And Their Cellular Uptake Study

Drug nanocarriers have attracted increasing attention in recent years since they can increase drug solubility, improve drug absorption and bioavailability, and enhance drug targeting transportation. Thus, nano drug delivery systems as a new dosage form of drugs attract more and more wide attention. Biodegradable polymers, due to decomposition in vivo, participation in the metabolism of organisms, and eventually elimination from the body, become important materials as drug carriers. But nanocarrier materials with good biocompatibility and biodegradable property are limited. Natural polymers due to derivation from nature are abundant and easily accessible. Meanwhile because of the good biocompatibility, biodegradability and low toxicity, natural polymers as a drug carrier material have comparatively obvious advantages.Protein drugs, DNA, RNA and the other biologically active molecules often require access to the cells or the nucleus before playing a role, but they can not enter into cells by themselves alone, and thus their functions are affected. The current transfer technologies through the cell membrane to interior of cell include the electroporation, microinjection, lipofection and viral carrier, etc. However, electroporation and microinjection often cause comparatively strong damage to cells. Most drug nanocarriers which enter the cells through endocytosis route are often confined to the endosome and degraded in lysosome finally. Thus, the drug efficacy of the effector molecules is affected. Therefore it is necessary to establish and develop the efficient, safe and practical drug carriers, which can efficiently introduce the drug into cells to play its full role. In this dissertation, casein, the main milk protein, was used as the raw material to synthesize casein-polyacrylic acid nanospheres (casein-PAA nanospheres), and then the PAA was removed from the casein-PAA nanospheres to obtain full casein nanospheres. We found that casein nanospheres were internalized into the cells by a temperature-, energy- or cell type-independent manner, and the casein nanospheres were mainly distributed in the cytoplasm. This dissertation is involved in the research on following four aspects:(1) Casein-PAA nanospheres were synthesized by polymerization of acrylic acid monomer in the reaction system comprised of casein-acrylic acid biopolymer-monomer pair. It is found that the size of nanospheres is controlled by the preparation conditions, such as the concentration of casein, the molar ratio of acrylic acid to casein, temperature, initiator and the molar ratio of initiator to acrylic acid, as well as reaction time. And also, crosslinking casein with glutaraldehyde improves the pH stability of the casein-PAA nanospheres, and eventually the casein nanospheres showed good biocompatibility and could be used as a carrier for the sustained release of cisplatin (CDDP). Cytotoxicity test showed that CDDP-loaded casein nanospheres have higher cytotoxicity than free CDDP at the same concentration.(2) By added propionic acid into the reaction system, hollow casein-PAA nanospheres were synthesized. It is found that propionic acid can not only increase the water solubility of casein, but also adjust the electric interaction between casein and PAA. Changing the polymerization conditions can regulate the size of casein-PAA nanospheres and make the morphology of the casein-PAA nanospheres change from solid to hollow. Furthermore, after removal of PAA component from casein-PAA nanospheres by dialysis method, the hollow full casein nanospheres without cytotoxicity were obtained. These solid or hollow nanospheres can be loaded with CDDP and make the sustained release of CDDP. In addition, the cytotoxicity of the CDDP-loaded casein nanospheres is higher than that of free CDDP at the same concentration.(3) Cellular uptake of casein nanospheres was investigated. It was found that casein,αs1-casein,β-casein,κ-casein and their nanospheres could enter the cell at 37℃and 4℃. By laser scanning confocal microscopy and flow cytometry examination, it was found that at 4℃or in the presence of various endocytosis inhibitors the casein nanospheres could still enter the cells effectively, and the capability of casein nanospheres to enter cells was not affected by cell types, temperature and energy. Also, laser scanning confocal microscopy (LSCM) and transmission electron microscopy observation showed casein nanospheres mainly existed in the cytoplasm, and the internalization course of casein nanospheres was monitored by LSCM. Base on the results we studied, a probable mechanism of the casein nanospheres to enter the cells is:first, casein nanospheres adsorb on the cell membrane, then fuse with the membrane and eventually penetrate through the cell bilayer membrane.(4) The crosslinking reaction of casein nanospheres with different crosslinking agents (namely CaSO4, genipin and transglutaminase) were investigated. The effects of crosslinking conditions on the stability of nanospheres were studied detailedly, and the optimized crosslinking conditions for the casein-PAA hollow nanospheres were obtained. Compared to hollow casein nanospheres crosslinked by glutaraldehyde, the hollow casein nanospheres crosslinked by Ca2+ or transglutaminase have much better biocompatibility. Finally, gold-loaded casein-PAA composite nanospheres were prepared by in situ reducing HAuCl4 in casein-PAA composite nanospheres using casein as the reductant. Furthermore, it was found that the size of gold nanoparticles could be regulated by temperature, reaction time, and the mass ratio of HAuCl4 to casein in the reaction system.