Regulation Of Cell Fate By Multifunctional Calcium Nervonate Nanoparticles And Its Biomedical Applications

Small organic molecules and inorganic metal ions of biological origin can regulate the fate of cells by regulating the chemical microenvironment of cells.Because of their abundant sources and low cost,they have important application prospects in cell therapy and tissue repair.However,due to some lipid soluble organic molecules and metal ions have their own property problems such as poor solubility and difficulty in entering cells,when they are applied in vitro to regulate cell fate,their availability is still very low even if by increas ing the concentration of material.When small organic molecules and metal ions are injected into the regulatory cell microenvironment in vivo in the form of dispersing liquid,the concentration of material at the target site usually decreases rapidly due to the diffusion of the injection.Besides,the material cannot be locally retained for a long time,there will be side effects on non-target cells and tissues.Therefore,functional small molecules and metal ions can be solid-state by bonding,and small molecules-metal nanoparticles can be prepared by nanomatization.Moreover,the regulation of functional small molecules and metal ions on intracellular microenvironment and cell fate can be realized through the endocytosis of nanoparticles by cells and the intelligent release of nanoparticles under the acidic pH microenvironment of lysosomes.This way based on the endocytosis of cells and the response of materials to the intracellular microenvironment can enable small molecules and metal ions to regulate cells locally,quickly and quantitatively.It has universal applicability for the fate regulation of cells that can endocytose nanomaterials,including stem cells,immune cells,tumor cells,etc.,and has important application prospects in the fields of tissue repair and immune regulation related to cell fate regulation.Therefore,more targeted,more efficient and more controllable nanomaterials can be designed according to the functions of small organic molecules and inorganic metal ions of biological origin,so as to achieve precise regulation of cell fate.Among many diseases,bone defection-related diseases and neurodegenerative diseases are respectively the most common and the most difficult to cure in daily life.With the development of regenerative medicine,the treatment of related diseases using cell-level tissue repair has made rapid progress and attracted more and more attention.As the core of tissue repair,seed cells are widely used in various fields of regenerative medicine.The fate regulation of seed cells is a key step to achieve repair and regeneration.Among them,stem cells are often used as seed cells in bone tissue and nerve tissue repair due to their excellent proliferative ability and multidirectional differentiation potential.Therefore,based on the above content,it is of great significance to explore functional small molecular-metal nanoparticles that can regulate the directed differentiation of stem cells in various tissue repair and the treatment of related diseases.In addition,macrophages,as one of the immune cells in body,can cause the host’s inflammatory response after tissue injury.At the same time,the host’s inflammatory response will affect stem cell-based tissue repair,which plays a key role in the initiation,maintenance and dissipation of tissue repair.Therefore,in order to achieve more efficient stem cell-based tissue repair and treatment of related diseases,functional small molecular-metal nanoparticles should regulate the phenotype and function of macrophages while regulating the directed differentiation of stem cells.To solve the above problems,in this dissertation,the author selected nervonic acid and calcium ions to construct calcium nervonate nanoparticles,and explored the role of calcium nervonate nanoparticles integrating the functions of nervonic acid and calcium ions in the fate regulation of stem cells and macrophages.The main research contents of this dissertation are as follows:(1)The osteogenic and immune multifunctional regulation of calcium nervonate nanoparticles promoted bone tissue regeneration.Calcium nervonate nanoparticles were prepared by nano-precipitation method using nervonic acid and calcium acetate.The size and structure of the nanoparticles were characterized by scanning electron microscopy(SEM),fourier transform infrared spectrometer(FTIR),electron probe X-ray microanalyzer(EPMA),X-ray diffractometer(XRD)and other instruments.Experiments have shown that calcium nervonate nanoparticles,after endocytosis,will decompose in the acidic environment of lysosomes with pH≈5.5,thus realizing the intracellular release of nervonic acid molecules and calcium ions.In vitro,mesenchymal stem cells(MSCs)were cultured with calcium nervonate nanoparticles,and the osteogenic differentiation levels of MSCs were characterized by quantitative real-time PCR(qPCR),Western blot and immunofluorescence staining(IF).In vitro experiments showed that the expression of osteoblast-related genes and proteins in stem cells,such as OPN and OCN,were increased under the mediation of calcium nervonate nanoparticles,and the intracellular calcium deposition increased,indicating that calcium nervonate nanoparticles promoted the differentiation of MSCs into osteoblasts.After bone injury,a large number of inflammatory cells,such as pro-inflammatory M1 macrophages,will infiltrate the injured site,and severe inflammatory response will lead to the formation of fibrous tissue and the failure of biological materials,which is damaged to bone repair.Therefore,the author continued to explore the effect of calcium nervonate nanoparticles on macrophages.After cultured macrophages with calcium nervonate nanoparticles,the polarization state of macrophages was characterized by qPCR,Western blot and IF.The test results showed that the expression of genes and proteins related to the polarization of macrophages M1 were inhibited,such as iNOS and COX2.These results indicate that calcium nervonate nanoparticles can inhibit the M1 polarization of macrophages and inhibit the expression of inflammatory factors.By using nervonic acid and calcium acetate to culture MSCs and macrophages,the authors found that nervonic acid can inhibit M1 polarization of macrophages,and calcium acetate can promote osteogenic differentiation of MSCs.Therefore,the function of promoting stem cell osteogenic differentiation and regulating macrophage polarization of calcium nervonate nanoparticles are derived from intracellular release of calcium ions and nervonic acid,respectively.This is its unique mechanism of action.In vivo,compared with the control group,the nervonic acid group and the calcium acetate group,calcium nervonate nanoparticles have the most significant effect on promoting bone regeneration in the skull defect of rats,which indicates that calcium nervonate nanoparticles with the combination of anti-inflammatory function and osteogenic induction function are more conducive to bone regeneration.In this dissertation,calcium ions and nervonic acid were integrated in a high safety and low expenditure way,and solid phase multifunctional nanomaterial was prepared by nanomaterial.The material has good biocompatibility and degradability,which can be used as bone induction preparation and immunomodulatory preparation to effectively promote bone regeneration in bone defects,and has a good application prospect in the treatment of bone defects related diseases.(2)The neural induction function of calcium nervonate nanoparticles promotes neural stem cells differentiation.Stem cell therapy is considered a potential cell-mediated treatment for neurodegenerative diseases.Among all kinds of stem cells,neural stem cells(NSCs)possess high self-renewal ability and can differentiate into neurons and glial cells,which holds great potential in the treatment of neurodegenerative diseases.The regulation of NSCs differentiation into neurons has become an important research direction in neurodegenerative diseases and neural tissue engineering.Based on the neuroprotective effects of neuronic acid and calcium ions and the promotion of neuronal differentiation,we explored the regulatory effects of calcium nervonate nanoparticles on the fate of NSCs.After co-culture of calcium nervonate nanoparticles and NSCs,the neural differentiation level of NSCs was characterized by qPCR and IF methods.qPCR results showed that calcium nervonate nanoparticles promoted the expression of mRNA of neuronal markers TUJ1 and MAP2 in NSCs.IF results showed that,under the mediation of calcium nervonate nanoparticles,The expression levels of these two markers were also increased,indicating that calcium nervonate nanoparticles promoted the differentiation of NSCs into neurons.Lysosome co-localization experiment showed that calcium nervonate nanoparticles would enter lysosomes after endocytosis.Due to the intelligent response of lysosome pH(pH≈5.5)of the nanoparticles,nervonic acid and calcium ions were immediately released into the cells.Nervonic acid and calcium ions could achieve neuronal regeneration based on their neuroprotective function and promoting neuronal differentiation.This multifunctional nanomedicine provides a new idea for the design of smart nanomaterials for the treatment of neurodegenerative diseases and a new platform for the clinical application of nerve tissue repair and regeneration.In summary,this dissertation aims to explore the application of organic functional small molecules and metal ions in cell fate regulation and tissue repair.By nanosizing organic small molecules and metal ions,intracellular delivery of materials is achieved.Then,the response of nanoparticles to the endogenous microenvironment of cells is utilized to realize the intracellular release of functional small molecules and metal ions,thus regulating the cell microenvironment and cell fate.Later,in order to seek more effective treatments for bone defect-related diseases and neurodegenerative diseases,calcium nervonate nanoparticles were taken as an example to explore their role in the fate regulation and tissue repair of MSCs,NSCs and macrophages.This material has good biocompatibility and can regulate the fate of stem cells and immune cells.It provides a new idea and method for the regulation of cell fate,and provides a novel nano and localized therapeutic strategy for the treatment of tissue damage related diseases and inflammation,which has a good clinical application prospect.