| In recent years, nanoparticles have led to new and exciting developments with great potential application in biology and medicine. However, due to the high surface energy, nanoparticles would easily aggregate without protection or passivation of their surfaces, which severely restricts its application. Thus, it is of great importance to comprehensively consider the size, shape, synthesis and dispersion of the nanoparticles in the further biomedical application to avoid the cytotoxicity. Meanwhile, some noxious dispersants, such as mercaptan and cationic surface active agents, should also be avoided. Various approaches have been developed to prevent the nanoparticles from aggregation. One ideal way is the addition of biomacromolecule, owing to its excellent biocompatibility. Lentinan, an occurring natural polymer, adopts triple helical conformation,which shows excellent bioactivities such as anti-tumor, anti-inflammation, and so forth. Especilly, Lentinan with most hydroxy groups provides lots of binding sites for nanoparticles, and the nanoparticles can be well enwrapped into the hydrophobic cavity of triplex during the renaturation process. Therefore, in this thesis, we aimed to develop an stratage to well disperse the nanoparticles by uitilizing the nature of denaturation and renaturation for triple helical Lentinan in water, and the structure and functions of the nanocomposites were also extensively investigated.The innovative points of this thesis are as follows.(1) A new approach to the reduction and dispersion of gold nanoparticles (AuNPs) with different size and shape by taking the advantage of lentinan was developed for the first time, and Au/lentinan nanocomposite nanowire was also obtained by entrapping the AuNPs into the hydrophobic cavity of renatured triple helical Lentinan via the hydrophobic interaction;(2) It was confirmed that Au/lentinan nanocomplex showed great ability in killing tumor cells without cytotoxicity against normal cells such as macrophages RAW264.7, and the anti-tumor mechanism was also investigated;(3) Se/lentinan nanocomplex was successfully prepared based on the nature of denaturation-renaturation for triple helical Lentinan, and the dispersion mechanism and stability of Se/lentinan were illustrated;(4) The effects of particle size on the anti-tumor activity for Se/lentinan and the corresponding mechanism were discussed;(5) Carboxyl and amino single-walled carbon nanotube (SWNT) were successfully wrapped with single chains of lentinan (s-LNT), which can improve the water solubility of SWNT, and reduce cytotoxicity of SWNT.The main contents and conclusions in this work are divided into the following parts. A green approach was described for the synthesis and dispersion of AuNPs in aqueous medium. AuNPs was successfully synthesized from Au3+using s-LNT. It was determined that LNT has certain reducibility. The reduction of Au3+was shown to be dependent on s-LNT concentration, reaction time, and temperature. A higher ?-glucan concentration and reaction temperature led to quicker formation of reduced AuNPs with uniform size and spherical shape. Prolongation of the reaction time was also found favorable for the formation of spherical AuNPs. AuNPs formed nanobelts in aqueous solution at low s-LNT concentrations and spherical AuNPs at relatively high s-LNT concentrations. AuNPs were also seen to arrange sequentially in the hydrophobic cavity of the renatured triple helical LNT (r-LNT) to form stable Au nanowire. This effect was more prominent at high s-LNT concentrations. Furthermore, both the hydrophobic interaction and the interaction between AuNPs and the hydroxyl groups of LNT were established as contributing factors in the stable dispersion of AuNPs in water. More specifically, Au3+could be reduced and stabilized by s-LNT, and the reduced Au could be manipulated into nanobelt, spherical shape, or nanowire morphology through the s-LNT concentration, reaction time, and temperature. s-LNT could thus be used as either a reducing or stabilizing agent to prepare stable AuNPs in a water system. Neither organic nor toxic agents were used in this process, and the reduction reaction was performed in an aqueous medium. This method was thus considered environmentally friendly and safe, which is very important for application in biotechnology and biomedicine.It was proved that Au/lentinan complex showed great inhibition of tumor cells (Hela) proliferation by the in-vitro biological test, and little cytotoxicity against normal cells of macrophages RAW264.7. Based on the variation of intracellular ROS, GSH/GSSG, mitochondrial membrane potential and caspase-9activity, the anti-cancer mechanism was proposed as follows. The Au/lentinan complex could induce oxidative stress in both Hela and RAW264.7cells. While the RAW264.7cells with higher antioxidant ability than Hela cells could clear the overexpressed ROS immediately. The higher content in Hela cells with long duration could cause the damage of mitochondria, following the increase in permeability of mitochondrial membrane. The further release of cytochrome C could stimulate the caspase-9, leading to the apoptosis of tumor cells and antitumor activity. Se nanoparticles (SeNPs) was successfully synthesized from Se4+under the reduction of vitamin C and stabilized by s-LNT base on the denaturation and renaturation of triple helical Lentinan. The formed Se/s-LNT complex was thoroughly investigated through the TEM, EDX, FT-IR, DLS and Viscometry, including the nanocomposite structure, dynamic formation and stabilization. All the results revealled that SeNPs with the diameter of28nm could be well stabilized by s-LNT; The Se---O-H interaction between hydroxyl groups of LNT and SeNPs contributed to the stabilization; The hydroxyl groups of LNT can adsorb the SeO32-, which offered the reaction sites, then s-LNT could wrap SeNPs during the renaturation process to avoid aggregation, resulting in the homogenous Se/s-LNT nano-complex formation. Due to the immunomodulatory activity for Lentinan, the as-prepared Se/s-LNT complex can be further applicated in the biomedical field with goog biocompatibility.SeNPs with different particle size were prepared by simply controlling the reduction rate of SeO32-. The average diameter of SeNPs was aslo evaluated by DLS and TEM. In-vitro biological tests indicated that the SeNPs with smaller size could be distributed uniformly in complex and showed higher anti-tumor activity. Treated by the same quality of SeNPs, the smaller size particle could stimulate Hela cells to produce more ROS, resulting in the damage of mitochondrial and lower transmembrane potential. It was suggested that Se/s-LNT with smaller particle size showed great anticancer activity compared with the larger one.Single-walled carbon nanotubes (SWNT) was treated with the concentrated nitric acid to obtain carboxyl carbon nanotubes (SWNT-COOH), and the amination carbon nanotube (SWNT-NH2) was obtained following the modification with octadecyl amine group. Compared with the unmodified SWNT, SWNT-COOH and SWNT-NH2showed better dispersibility, which could be further stabilized by s-LNT. Among them, SWNT-NH2with the best dispersibility, could be well dispersed by s-LNT avoiding the aggregation. The s-LNT in the renaturation process could wrap around the SWNT to form carbon thread structure. In-vitro experimental tests showed that covalently derivatized SWNT can improve the dispersibility, but it would increase the cytotoxicity of SWNT. s-LNT could be well used to stabilize the SWNT for better compatibility and decreasing cytotoxicity.Summarily, the results of this foundation research mentioned above presented an effective, simple, green, safe and good biocompatibility way to dispersed nanoparticles, resulting in different nanoparticles/LNT complex. Meanwhile, the antitumor activity, the mechanism of anti-tumor and cell toxicity were well investigated, which provides an important scientific evidence for the nanoparticles in the biomedical application. Therefore, it has great important academic values and potential applications. |
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