Biocompatible Inorganics Target Mineralization To Cancer Cell Membrane And The Influencing Mechanism

At present,great progress has been made in the study of biomineralization,and various materials with excellent properties have been synthesized by biomimetic mineralization.However,there are a few studies on abnormal biomineralization which may cause various diseases.Farthermore,the mechanism of biomineralization needs to be further studied.Inspired by the abnormal biomineralization,it was found that in situ mineralization of biocompatible calcium phosphate on cancer cell membrane could inhibit the proliferation of cancer cells,that is leading to cancer cells"getting sick",and yet no obvious inhibition and toxic and side effects were found in normal cells and tissues.However,after this finding,few studies have been published in this field so far.Under such background,this thesis studied the targeted in situ mineralization on the membrane surface of various cancer cells by different biocompatible minerals,such as calcium phosphate,calcium carbonate,silicon dioxide,etc.and the effect of mineralization on the growth of cancer cells,and further studied the affected protein and related mechanisms.Some significant results were obtained.Our results not only have important theoretical significance in revealing the mechanism of biomineralization,but also provide a meaningful reference for understanding the factors and methods that affect the proliferation of cancer cells.The main contents of the thesis are as follows:Firstly,we studied the methods targeted in situ mineralization of calcium phosphate（CaP）on the surface of cancer cells and the mechanism of inducing apoptosis of cancer cells at the molecular level.Using biocompatible calcium salts and phosphates as raw materials,we achieved targeted in situ biomineralization of CaP on the cell membrane of solid tumor under the guidance of folate（FA）.The results of FE-SEM,EDS,CLSM,XRD and FT-IR showed that the mineralized layer of amorphous CaP was formed on the surface of cancer cells.The MTT results showed that CaP significantly inhibited the proliferation of cancer cells,while there was no significant inhibition in normal cells.Mechanism studies showed that targeted in situ biomineralization of CaP mainly downregulated Annexin A2 and other transfer proteins on the membrane of cancer cells,and inhibited their functions,damaged cell membrane integrity,led to lactate dehydrogenase（LDH）leakage,inhibited glycolysis of cancer cells,blocked cell cycle in G0/G1 phase,and induced apoptosis.Secondly,we studied the method of calcium phosphate（CaP）targeted in situ mineralization of leukemia cell membrane with folic acid（FA）as the targeting reagent and the mechanism of inducing leukemia cell apoptosis.Based on the previous study on adherent cancer cells,we used biocompatible calcium salts and phosphates as raw materials to achieve targeted in situ mineralization of CaP on leukemia cell membranes by induction of FA.The results of FE-SEM,EDS,CLSM,XRD and FT-IR showed that the mineralized layer of amorphous CaP was formed on the surface of leukemia cells.The result of MTT showed that the proliferation of leukemia cells in vitro was significantly inhibited by CaP,while there was no significant inhibition in normal cells.Mechanism studies showed that CaP damaged the integrity of leukemia cell membrane,led to lactate dehydrogenase（LDH）leakage,inhibited glycolysis of leukemia cells,blocked cell cycle in S phase,and induced apoptosis.Thirdly,we investigated the methods targeted in situ mineralization of calcium carbonate（CaCO₃）on the surface of cancer cells and the mechanism of inducing apoptosis of cancer cells at the molecular level.Taking biocompatible calcium salts and carbonates as raw materials and using chondroitin sulfate（CS）as targeting reagent and linker,we achieved targeted in situ mineralization of CaCO₃ on cancer cell membrane,and studied the influence of raw material concentration,targeting reagent and other factors on its mineralization.The results of FE-SEM,EDS,CLSM,XRD and FT-IR showed that the mineralized layer of amorphous CaCO₃ was formed on the surface of cancer cells.The binding membrane proteins are mainly proteins containing N-H,P-O-C,NH2 and other functional groups,and the mineralization mechanism is discussed.The results of MTT and in vivo experiments showed that CaCO₃ significantly inhibited the proliferation of cancer cells and the growth of S-180 tumor in mice,while no obvious inhibition and toxic and side effects were found in normal cells and tissues.Mechanism studies showed that the CaCO₃ mainly blocked Na,K-ATPase and other transfer proteins on the membrane of cancer cells,collapsed the mitochondrial membrane potential（MMP）,damaged cell membrane integrity,led to lactate dehydrogenase（LDH）leakage,disruptd cell cycle,and induced apoptosis.Fourthly,we investigated the methods targeted in situ mineralization of silicon（SiO₂）on the surface of cancer cells induced by the targeting reagent hyaluronic acid（HA）and mechanism of inducing apoptosis of cancer cells.Taking biocompatible tetramethyl silicate（TMOS）and（3-mercapto propyl）trimethoxysilane（MPTMS）as raw materials and using HA as targeting reagent and linker,we achieved targeted in situ mineralization of SiO₂ on cancer cell（Hep G2,A-549,S-180）membrane.The results of FE-SEM,EDS,CLSM,XRD and FT-IR showed that the mineralized layer of amorphous SiO₂ which bound to membrane proteins was formed on the surface of cancer cells.The results of MTT and in vivo experiments showed that SiO₂ significantly inhibited the proliferation of cancer cells and the growth of S-180 tumor in mice,while no obvious inhibition and toxic and side effects were found in normal cells and tissues.Mechanism studies showed that the SiO₂ mainly bound to membrane proteins of functional groups such as N-H and C-N,damaged cell membrane integrity,led to lactate dehydrogenase（LDH）leakage,inhibited glycolysis of cancer cells,blocked cell cycle in S phase,and induced apoptosis.Finally,the results of this thesis were summarized.Our research showed that:1)Inorganic substances with biocompatibility（calcium phosphate,calcium carbonate,silica etc.）can be targeted in situ mineralization on cancer cell membranes under the induction of targeting reagent,and then inhibited the proliferation of cancer cells.2)In terms of cell types,both adherent cells and suspended cells could be targeted in situ mineralization,and their proliferation was significantly inhibited.3)Mineralization significantly inhibited the proliferation of cancer cells in vitro and tumor growth in vivo,while no obvious inhibition and toxic and side effects were found in normal cells and tissues.4)Mechanism studies showed that the inorganic substances affected the transfer proteins on the cancer cell membrane like Annexin A2and Na⁺,K⁺-ATPase,destroyed the integrity of cell membrane,led to lactate dehydrogenase（LDH）leakage,damaged the membrane function,inhibited the glycolysis of cancer cells,and disrupted the cell cycle and induced apoptosis.Our results on targeted in situ mineralization on membrane of cancer cell that inducing cancer cell apoptosis and inhibition mechanism could provide experimental basis and theoretical reference for understanding the biomineralization and the mechanism of inhibiting cancer cell proliferation.