Design And Detection Of Intelligent Fluorescent Nanomaterical Based On MMP-2 Tumor-biomark For Tumor Mouse In Vitro And Vivo

It is necessary to find a sensitive diagnosis and treat way for the tumor,as the main factors that cause the death of people.Nano-drug delivery system has become a popular strategy for tumor detection due to its high drug-loading property,biocompatibility and low biological damage.Presently,Matrix metalloproteinase-2(MMP-2,tumor marker)has been served as a popular target for tumor deteciton.The purpose of this study was to structure a efficient MMP-2-based Nano-drug delivery system for the detection of tumor cells and in-vivo tumor.1.Construction and study of release-controled and MSN-based MMP-2-sensitive Nano-delivery system for monitoring of tumor cells.A porous silica nano-material(MSN)as carrier and a pair of fluorescence resonance energy transfer(quenching agent BHQ as acceptor and fluorescent substance Cy5 as the donor)was constructed to produce a signal firstly.And MMP-2-sensitive antibody was introducted to serve as a "bio-lid" and construct a release-controlled MMP-2-based nanosensor for monitoring of MMP-2 in tumor cells.In normal cells,MMP-2 antibody is encapsulated on the surface of nano-particles by electrostatic adsorption in "OFF" state,while fluorescence resonance energy transfer occurs in the nano-pore size to produce the fluorescence quenching and no fluorescence.In the presence of the MMP-2 protein in tumor cells,specific bond between MMP-2 antibody and MMP-2 protein occurs and leads to the shedding of the "bio-gate" MMP-2 antibody from the probe at "ON" state,which damages fluorescence resonance energy transfer between BHQ and Cy5 to cause the return of fluorescent signal of Cy5.Protein addition test in vitro demonstrated the release ability of the MMP-2-responsive probe at the presence of MMP-2 protein,specificity and feasibility of the probe for tumor cells detection.Then the model of MMP-2 changes in tumor cells was constructed to further verify the sensitivity of the MMP-2-responsive probe to the fluctuation of MMP-2 in tumor cells.Experimental results show that a large amount of fluorescent material loaded in MSN can improve the ability of the probe to monitor the level of MMP-2 in tumor.Preparation of the MMP-2-responsive is helpful to early diagnosis of bladder cancer and other kinds of tumor,and lay a solid foundation for discovery of new anti-tumor drug.2.Construction and study of targeted MMP-2-based drug delivery system for monitoring of tumor in-vivo.In the design of MMP-2-sensitive,the fluorescent nano-particle was served as the carrier,and hydrophilic group PEG(polyethylene glycol)was introduced to improve the hydrophilicity and biocompatibility of the nano-material,and then(FAM/Dabcyl)FRET-modified MMP-2-sensitive substract peptide introduced by click-on reaction and tumor-targeted peptide iRGD was modified on the surface of nanomaterical to construct a novel MMP-2-sensitive(TPFNs)biosensor for tumor detection in vivo.Michaelis constant was obtained by the kinetic reaction of substrate enzyme,demonstrating that the signal of FAM from the MMP-2-sensitive probe in urine could serve as the index for effectiveness evaluation of tumor detection.Results of cell experiment showed that the MMP-2-sensitive probe has a the targeting(tumor targeting peptide)and specificity(the presence of MMP-2 substrate)to tumor cells;In animal experiment,breast cancer model is constructed,then diagnosis effect of MMP-2-sensitive TPFNs to tumor is evaluated at four stages with the growth of the tumor in mice.In each stage,MMP-2-sensitive TPFNs biosensor was injected by tail vein in mouse and collected the urine after 2 h of administration for urine measurement.Compared with the traditional breast cancer detection method(CA15-3 kit),MMP-2-sensitive sensor has good diagnostic capability for tumors with a volume of 15 mm3.The success of the preparation of MMP-2-sensitive biosensor will be beneficial to the identification of the tumor,early diagnosis in clinic and efficacy evaluation of tumor treatment,and lay the foundation for the discovery of new anti-tumor drags.New methods for detection of tumor cells and the tumor in vivo will be served as a solid foundation for early detection of tumors in clinic and provide a new idea for treatment of the tumor and for the detection of tumor cells and living tumors.