Targeted Fluorescence Imaging Of Novel Metal Nanomaterials In Tumor Cells And Tumor Tissues

In recent years,biology imaging technology has made breakthrough progress,with wide in-depth application in the field of biomedical,clinical diagnosis and other fields,which also provided a strong support for theoretical research and practical application.Fluorescent bio-imaging technology,as the most widely used optical bioimaging technique,can monitor the process of tumors in a real-time,dynamic,accurate and noninvasive mode,and provide the information of the kinetic changes of biological samples on the cellular and in vivo levels.Therefore fluorescent bioimaging technology is favored with researchers and medical professionals.At present,many fluorescent probes are designed for fluorescent bioimaging,such as quantum dots,precious metal nanoparticles,organic dyes,upconversion materials,and carbon-based materials.However,many probes have the defects of cytotoxicity,stability,and photobleaching.For example,although quantum dots show less toxicity through surface modification or the formation of shell structure,they can still lead to the cellular immune response and other side effects because they contained cadmium,lead and others heavy metals.Moreover,organic dye has weak anti-bleaching ability,etc.These disadvantages may limit the applications in the biomedical field.Metal nanomaterials as special materials will provide new options and possibilities to solve these problems.So far,a great many of the preparation methods of metal nanomaterials have been proposed,as a whole,which mainly included chemical and physical method.These mature techniques can be even available for large-scale syntheses,such as hydrothermal synthesis,sol-gel method,microwave-assisted synthesis,ultrasonic synthesis and so on.However,the disadvantages of these methods,for instance,environmental pollution,high cost,poor controllability,agglomeration,complexity of reactions,still greatly limit its biomedical applications.Biosynthesis nanotechnology,as a green,novel alternative,which mainly use biological metabolites,has attracted great attention.This method provides a new way for synthesis of nano-materials by using the bioactive molecule in vivo or cell-free extract(CFE)through the metabolism paths in living organism.On the other hand,the molecular mechanisms of biosynthetic fluorescent probes and the interaction between fluorescence probe and cells have been rarely reported.Therefore,in this study we have established a new and facile method for rapidly and accurately achieving tumor targeting fluorescent images by using a specifically biosynthesized fluorescent nanomaterials in cancer cells or tumor tisues,and further explored the molecular mechanisms of biosynthesis though using proteomics and genomics:Firstly,we have explored a new strategy for biosynthesis of fluorescent Eu nanocomplex in virtue of special biological microenvironments inside cancer cells and tumor tissues.The intracellular self-biosynthesized Eu nanocomplex was characterized through fluorescence spectroscopy,energy dispersive X-ray spectroscopy(EDS),and X-ray photoelectron spectroscopy(XPS),etc.Laser confocal fluorescence microscopy was used to measure and assess the bio-imaging efficiency for cancer cells.We established subcutaneously xenografted tumor mice model,s detected the fluorescence signals of tumors through local injection or tail intravenous of Eu(NO3)3 solution,and studied the biological toxicity and tumor-target ability of the biosynthesized Eu nanocomplex and the bio-distribution of Eu species.The results indicated that fluorescent Eu nanocomplex efficiently realized fluorescence imaging of cancer cells and accurate labelling of tumor area.The results of histological and hematology examinations showed that Eu nanocomplex has good biocompatibility,without causing histopathological changes and hematological abnormality.It can improve both fluorescence and biocompatibility of rare earth nanomaterials.Generally,this study shows the possibility to specifically in vivo biosynthesize fluorescent Eu nanocomplexes in cancerous cells or tumor tissues.Combined with cell imaging techniques,it provides a new idea and broad application prospect for the synthesis and application of rare earth nanomaterials in rapid and accurate/effective cancer theranostics.Secondly,although we have realized self-biosynthesized fluorescent Eu nanocomplex in HeLa cells for specifical and accurate fluorescence imaging for cancer cells,the molecular mechanism of the in situ biosynthesized process is still unclear.In order to reveal this mechanism,we have investigated whole-genome expression profiles before and after incubation of HeLa cells with Eu(NO3)3 solution for 24 h with cDNA microarrays.It is observed that 563 up-regulated genes and 274 down-regulated genes were differentially expressed.Meanwhile,quantitative RT-PCR technique was utilized to measure the expression of several important genes,which validated the results of microarray data analysis.GO analysis showed that a wide range of differential expression functional genes are involved in three groups,including cellular component,molecular function and cellular biological process.It was evident that some important biological pathways were apparently affected through KEGG pathway analysis,including focal adhesion pathway and PI3K(phosphatidylinositol 3’-kinase)-Akt signaling pathway,which can influence glycolytic metabolism and NAD(P)H-oxidases metabolic pathway.Thirdly,we have also studied the molecular mechanism of in situ biosynthesis fluorescent Eu nanocomplex by proteomics.Isobaric tags for relative and absolute quantitation(iTRAQ)technology was used to study the whole proteins of HeLa cells and analysize proteins with different expression,upon incubation Eu(NO3)3 solution for 24 h.These differentially expressed proteins were involved in a variety of biological processes,including long chain fatty-acyl-CoA biosynthetic processes,single-organism metabolic processes,cofactor metabolic and cellular metabolic processes,cell surface receptor signal transduction processes and energy generation processes The differentially expressed proteins were enriched in KEGG pathways of cell adhesion,glycolysis/gluconeogenesis,oxidative phosphorylation,calcium signaling pathway and glutathione metabolism.The results of the proteomics analysis indicated the molecular processed of biosynthesized fluorescenct Eu nanocomplex.This study provides a theoretical basis for further revealing the molecular mechanism of in situ biosynthetic systems.Finally,based on the understanding of molecular mechanisms of biosynthetic fluorescent probes in tumor cells,we have established a facile and efficient way of in situ biosynthesis copper nanoclusters(CuNCs)by the special biological environments of cancer cells for measuring intracellular temperature.The CuNCs were characterized by fluorescence spectroscopy,UV absorption spectroscopy,X-ray photoelectron spectroscopy,transmission electron microscopy(TEM)and elemental analysis.The CuNCs,with an average diameter of 2.4 ±0.4 nm,were exhibited red fluorescence emission(Em= 610 nm)and could further efficiently accumulate for bioimaging in target cancer cells.MTT assays showed that the CuNCs have good in vitro stability and biocompatibility.More importantly,the fluorescence signal of the biosynthesized CuNCs is sensitively thermo-responsive over the physiological temperature range in MDA-MB-231 cells(relative sensitivity:-3.18%per Celsius),The method was simple,green,and controllable,with high reproducibility and good biosecurity.This provides an efficient nanothermometer based on the in situ biosynthesized CuNCs for cellular fluorescence imaging and other biomedical applications.