Fluorescence Visualization Of Cell Behaviour Regulated By Intracellular Communication

Cancer in humans is a highly fatal disease with poor prognosis and the most life-threatening aspect of cancer is tumor metastasis which requires cellular migratory and invasive capacities.In the field of cancer treatment,it is often necessary to provide some information related to tumor development earlier so as to facilitate the individualized treatment of cancer patients,judge the prognosis of patients,and provide an effective basis for the selection of appropriate adjuvant treatment for patients at high risk of recurrence and metastasis,which finally can improve the cure rate and survival rate of patients.With the deepening of research,researchers have gradually realized that the communication mechanisms involved in genes,proteins and other biomolecules play an important role in the occurrence,development and invasion,metastasis and recurrence of tumor cells.It was reported that miR-221 promoted cellular migration and invasion via directly repressing PTEN and TIMP3 expression leading to the aberrant activation of the PI3K/AKT pathway and MMP-9 expression.Tumor progression toward invasion and metastasis is a stepwise and multistage process and the epithelial-mesenchymal transition(EMT)process is essential for invasive and metastatic properties.The current widely used approaches to analyze genes and proteinases in signaling pathways are based on traditional techniques.However,the operation of these methods is cumber-some and may cause cell structure destruction which make it impossible to study the dynamic changes and natural situation of these molecules.Therefore,it is imperative to develop a new strategy for detecting proliferative and invasive abilities of living cells for more comprehensive understanding of cancer occurrence and development,rapid and reliable antitumor drugs screening,and more accurate assessment of therapeutic effects.Fluorescence image analysis provides stimulating opportunities to visually detect components in living cells and in vivo due to its excellent advantages of rapidity,damage free,and visual identification.In recent years,with the development of nanotechnology,fluorescent nanoprobes have been widely used in the fields of analytical chemistry,biology and medicine.Among these nanomaterials,gold nanoparticles(AuNPs)have attracted much attention due to their unique biological affinity and optical properties and been applied in biomolecular detection,cell and in vivo imaging,cancer detection and diagnosis.In order to better understand the mechanism of cancer development,evaluate the therapeutic effect of cancer more accurately,and screen new anti-tumor drugs more quickly and reliably,it is necessary to develop a fluorescent nanoprobe which can simultaneously detects multiple molecules in signaling pathways to visualize the changes in cell activity.In this paper,we designed and synthesized a series of novel composite fluorescent nanoprobes based on AuNPs,molecular beacons(MB)and peptide chains.These nanoprobes can be used for simultaneous visual detection of intracellular microRNAs,mRNAs and proteases in cells.According to the changes in fluorescence signals of these molecules,the cell properties and activities were examined,and the efficacy of the drugs was further evaluated.It mainly includes the following sections:1.We construct a multicolor fluorescent nanoprobe for investigating cellular migration and invasion through detecting miRNA,mRNA and MMP involved in the PI3K/AKT pathway of living cells.The nanoprobe was composed of AuNPs anchored with a dense layer of oligonucleotide molecular beacons(MBs)and a peptide,modified step-by-step via Au-S bond formation.The MBs could respond specifically to miR-221 and PTEN mRNA targets and the peptide could be specifically cleaved by MMP-9.In the absence of the targets,the fluorescence of three fluorophores linked with the MBs and the peptide was efficiently quenched by AuNPs.In the presence of the corresponding targets,the MBs were forced to unwind and the peptide was cleaved by MMP-9,thus leading to fluorescence recover.The nanoprobe can investigate cellular migration and invasion by simultaneously monitoring multiple components involved in the PI3K/AKT pathway.The multicolor nanoprobe could provide an easy,noninvasive,instantaneous and intuitive way to evaluate cellular mobility and invasiveness and further establish effective targeted therapeutic strategy to limit cancer metastasis.2.A new multicolor fluorescent nanoprobe capable of simultaneously and visually detecting the proliferation marker Ki-67 mRNA and invasion marker uPA protein was designed and prepared.The nanoprobe was prepared by immobilizing the synthetic DNA molecular beacons(MBs)and peptides on the gold nanoparticles(AuNPs),step-by-step via Au-S bond formation.The MBs can identify Ki-67 mRNA,and the peptides can be specifically cleaved by uPA.The multicolor nanoprobe.In the presence of the corresponding targets,the MBs were forced to unwind and the peptide was cleaved by MMP-9,thus leading to fluorescence recover.After treated with the anticancer drugs tamoxifen and curcumin,the changes in cancer cell proliferation and invasion properties were visually detected and therapeutic effects of corresponding drugs were further assessed in vitro and in vivo.The design of the fluorescent nanoprobe opens up an avenue for investigating unscheduled proliferation,invasion,and metastasis in living cells and in vivo and as such will be a promising tool to screen antitumor drugs and evaluate drug efficiency in an extremely efficient manner.3.We designed a multicolor fluorescent nanoprobe for simultaneously imaging the epithelial biomarker E-cadherin mRNA,the mesenchymal marker vimentin mRNA and the apoptotic marker Caspase-3.The nanoprobe was formulated by gradually modified the AuNPs with two MBs and a peptide.Generally,the fluorescence of three dye molecules connected with a peptide and two MBs is well extinguished by AuNPs.The MBs could specifically respond to E-cadherin mRNA and vimentin mRNA tar gets,and the peptide chain was cleaved by Caspase-3,generating the three fluorophores’ fluorescent signals.The nanoprobe can simultaneously detect the processes of EMT and apoptosis in living cells,which were used to study the effect of EMT on apoptosis and further assess the influence of EMT on drug efficacy in different cancer cells.The designed nanoprobe can offer a new strategy for visualizing EMT and apoptosis in tumor cells and will be a promising tool to investigate the efficiency of drugs targeting EMT-related therapies in living cells.4.We demonstrated a novel Au-Se nanoprobe for real-time in situ detection of two proteins and their upstream and downstream regulatory relationship.The nanoprobe was prepared by capping the AuNPs with two selenol-modified peptide chains.In the presence of uPA and MMP-9,the peptides could be specifically cleaved by uPA and MMP-9,.thus leading to fluorescence recover.Compared to the conventional Au-S nanoprobe,the Au-Se nanoprobe possesses better resistance to GSH interference,and can thus be used in complicated biological environments.Moreover,the Au-Se nanoprobe can observe the successive expression of uPA and MMP-9 in situ,and identify that downstream MMP-9 can be activated by upstream uPA according to the change in protein expression.The design strategy of such a novel Au-Se nanoprobe is generally enough to be extended to the real-time investigation of multiple signal molecules in signalling pathways in situ,and offers avenues for studying the regulatory relationship between signal molecules in signalling pathways.