Multifunctional Nanoparticle:Combination Of Photothermal Effect And Reactive Oxygen Species Generation For Dual-modality Cancel Therapy And Heterogenous Expression Of LOX Family Proteins

This thesis consists of two parts:1)the study of a multifunctional nanoparticle:combination of photothermal effect and reactive oxygen species generation for dual-modality cancer therapy and 2)heterogenous expression of lysyl oxidase(LOX)family proteins.Cancer is the second leading cause of death globally,and was responsible for 8.8 million deaths in 2015 according to WHO data.Nanomaterials have been considered as one of the main propellants for strategy development in disease diagnosis and therapy,especially in the prolonged battle against cancer.Cancer cells possess some inherent properties,such as glucose-dependence,exogenous reactive oxygen species(ROS)and heat intolerance.In the first part,we developed a strategy to target these vulnerable properties of cancer cells using glucose oxidase(GOx)and polydopamine functionalized iron oxide nanoparticles(Fe3O4@PDA/GOx NPs).This system is characterized by the covalently linked GOx which can competitively consume glucose and spontaneously generate ROS hydrogen peroxide(H2O2)through biochemical reactions.The enzyme was decorated on iron oxide NPs through the cross-linking between the protein and polydopamine(PDA)which deposits in advance on the surface of iron oxide NPs during the self-polymerization of monomer dopamine.In this system,the PDA along with iron oxide NPs served as the photothermal transfer materials in converting NIR radiation into heat.In addition,the synergistic effect of this system was achieved through the conversion of H2O2 to more toxic hydroxyl free radicals(·OH)catalyzed by ferrous ions releasing from iron oxide NPsIn our research,two sizes of Fe3O4@PDA/GOx NPs(200 nm and 20 nm in diameter)were successfully synthesized.The nanoparticals possessed strong NIR absorption,high photothermal conversion and ROS generation ability.Fe3O4@PDA/GOx NPs could significantly inhibit the growth of breast cancer cells MDA-MB-231 through dual-modality ROS-photothermal effect in vitro,and displayed very low toxicity to normal cells MCF-10A.The half-maximal inhibitory concentrations(IC50)of 200 nm Fe3O4@PDA/GOx NPs with respect to MDA-MB-231 were calculated to be 25.82 ?g/mL and 20.98 ?g/mL without and with laser irradiation,respectively.While IC50S of 20 nm Fe3O4@PDA/GOx NPs were 70.56 ?g/mL and 64.77 ?g/mL without and with laser irradiation,respectively,which were 2.7 and 3 fold higher than those of 200 nm.After incubation with Fe3O4@PDA/GOx NPs,the iron levels of cancer cells and normal cells were evaluated using the Iron Analysis Kit and Prussian blue staining.The iron concentration of the normal cells treated with Fe3O4@PDA/GOx NPs remained almost unchanged.In contrast,the iron concentration of cancer cells sequentially increased with the increased dose of Fe3O4@PDA/GOx NPs.We then investigated the generation of ROS by Fe3O4@PDA/GOx NPs in vitro,the results indicated the Fe3O4@PDA/GOx,especially the 200 nm NPs,could preferentially increase the H2O2 and·OH levels in the media and within cancer cells.In summary,compare with 20 nm NPs,200 nm Fe3O4@PDA/GOx NPs displayed higher selective inhibition protential on cancer cells.This may be due to its higher affinity ability and more ROS induction capacity with cancer cells.TUNEL staining showed that the 200 nm Fe3O4@PDA/GOx NPs could induce chromosome breakage in cancer cells.AnnexinV-FITC/PI staining showed that the 200 nm Fe3O4@PDA/GOx NPs induced cencer cell apoptosis in a dose and time-dependent manner.We further assessed their dual-modality therapy effects in vivo,Fe3O4@PDA/GOx NPs could significantly inhibit the growth of tumor or complete ablation of tumor under the NIR,and five from the eight mice of treated group showed the complete ablation of tumor.In addition,the Fe3O4@PDA/GOx NPs didn't induce obvious histological damage to the major organs of mice including under tail intravenous injection.The complete blood tests showed no obvious interference when compared with the control group and the body weights of the treated group gradually increased in a manner similar to those of the control group.The versatility of the functionalization strategy reported in this study will contribute to developing efficient therapies in selective cancer treatment.In the second part of this thesis,we focused on the heterogenous expression of lysyl oxidase(LOX)family proteins.Lysyl oxidase(LOX)is a series of copper-dependent extracellular matrix enzymes,and can oxidize primary amine to reactive aldehydes.LOX proteins play a critical role in promoting the maturation of extracellular matrix(EMC)and maintaining its stability by catalyzing the covalent crosslinking of collagen and elastin.In the human lysyl oxidase protein family,there are five members,named hLOX and hLOXL1-4.hLOX and hLOXL2 are two important proteins which have been reported involved in the proliferation,adhesion,malignant metastasis,invasion and angiogenesis of various tumors.They both contain a highly conserved C-terminal which includes a copper binding site,a lysine tyrosylquinone(LTQ)cofactor and a cytokine receptor-like domain(CRL).In addition,both of them have three predicted N-glycosylation sites.The main difference between them is that in N-terminal region,hLOXL2 possesses four SRCR domains,but hLOX contains a propeptide.Since till now,there is no effective expression system,it remains extremely difficult to achieve the soluble and active recombinant proteins,which have been precluding the studies of structure-function relationship of LOX family proteins.In order to obtain a large amount of soluble and active recombinant LOX proteins,we selected prokaryotic and eukaryotic expression systems for LOX family proteins expression.In the prokaryotic system,we employed the molecular chaperones and fusion tags to assist the expression of soluble recombinant LOX proteins in E.coli.In the eukaryotic system,we used the Pichia expression system to realize the posttranslational modification of LOX proteinsWith the assisted of fusion tag,such as the Trx-tag and GST-tag,and the two molecular chaperone systems DnaK,DnaJ and GrpE as well as GroEL and GroES,we have successfully expressed the soluble truncated hLOXL2 protein(D1-2 hLOXL2),and detected the LOX activity from the purified recombinant protein.We also attempted to express recombinant LOX proteins in three types of Pichia host strains GS115,SMD1168,and KM71.Using the expression vector pPIC9K and pPICZaA,we detected the recombinant protein D1-2 hLOXL2 as the glycosylated and soluble form in the cytosol of P.pastoris KM71.Since the low expression level of the target protein,we couldn't get the purified recombinant D1-2 hLOXL2 protein.We successfully expressed and purified the soluble and active recombinant hLOXL2 protein using the prokaryotic expression systems,which could promote further studies on the biochemical properties and the physiological functions of LOX family proteins.