Preparation Of The Fluorescence Resonance Energy Transfer Probe Targeting MMP-2 And Its Application In The Detection Of Cervical Carcinoma

BackgroundAbout 300,000 women die of cervical carcinoma every year worldwide,which is one of the most common gynecological malignancies.The disease develops from cervical intraepithelial neoplasia to cervical carcinoma that takes about 10²0 years,this period is an important stage for the early prevention of cervical carcinoma.At present,there are many methods used for the screening of cervical carcinoma,but they has many defects.Therefore,the exploration of fluorescence probes with high sensitivity and specificity for the early detection and diagnosis methods of cervical carcinoma has become a hot spot.The expression of matrix metalloproteinase-2?MMP-2?in normal cervical epithelial cells is low or absent.However,in cervical intraepithelial neoplasia and cervical carcinoma,MMP-2 is over-expressed,which can be used for the differential diagnosis of benign cervical lesions and cervical carcinoma.Meanwhile,it provides a foundation for the cervical carcinoma detection methods based on targeting MMP-2.However,none of these methods used to detect MMP-2 can be used for the analysis of living cells or animals.The application of the probe of CdTe quantum dots probe targeting MMP-2 was limited due to its high level of toxicity.ObjectivesIn this research,the fluorescence resonance energy transfer probe targeting MMP-2was prepared by coupling a fluorophore?7-[diethylamino]-2-oxo-2H-chromene-3-succinimidyl ester?with a quencher [4-?4-[dimethylamino] phenylazo?benzoic acid N-succinimidyl ester] via a polypeptide chain?GPLGVRGKGG?that can be cleaved byMMP-2.The aims are to detect the feasibility of cervical carcinoma cells?CaSki cells,SiHa cells,C33-A cells and HeLa cells?and cervical carcinoma tumor-bearing mice in vivo imaging,which provides scientific basis for cervical carcinoma detection.Methods1.Synthesis of 7-[diethylamino]-2-oxo-2H-chromene-3-succinimidyl ester.2.Coupling a fluorophore?7-[diethylamino]-2-oxo-2H-chromene-3-succinimidyl ester?with a quencher [4-?4-[dimethylamino]phenylazo?benzoic acid N-succinimidyl ester]via a polypeptide chain?GPLGVRGKGG?that can be cleaved by MMP-2 to prepare of the fluorescence resonance energy transfer probe targeting Matrix Metalloproteinase-2.3.The fluorescence responses of the probe targeting Matrix Metalloproteinase-2 to MMP-2,MMP-1,and biologically relevant chemical species were done.4.Reverse transcription-polymerase chain reaction?RT-PCR?,real-time quantitative PCR?qPCR?and western blotting were used to test the MMP-2 expression level in cervical carcinoma cell lines.5.The cervical carcinoma cell lines were incubated with MMP-2 fluorescence probe and then the cells imaging was observed using confocal laser scanning microscopy.6.The establishment of cervical carcinoma SiHa and HeLa tumor-bearing mice.Taking SiHa and HeLa tumor-bearing mice as research object,the fluorescence resonance energy transfer probe targeting Matrix Metalloproteinase-2 was injected into mice via the tail vein and intratumoral,then the in vivo imaging was observed by vivo imager.Results1.The fluorophore?7-[diethylamino]-2-oxo-2H-chromene-3-succinimidyl ester?was prepared conveniently and characterized by1 H nuclear magnetic resonance hydrogen spectrum?¹H NMR?,¹³ C nuclear magnetic resonance spectrum(¹³C NMR)and electrospray ionization mass spectrometry?ESI-MS?.The UV-vis absorption spectrum of?7-[diethylamino]-2-oxo-2H-chromene-3-succinimidyl ester??10 ?mol/L?in DMSO: H₂O?1: 1000,v/v?solution exhibited a broad coumarin-based ?-?* transition band around 445 nm.The fluorescence spectrum of the same solution showed a broad emission peak around480 nm with strong green fluorescence.The experimental results provide the basis for synthesis of probe.2.The fluorescence resonance energy transfer probe targeting Matrix Metalloproteinase-2 was constructed and characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry?MALDI-TOF?and high-pressure liquid chromatography?HPLC?.3.?1?The results from the fluorescence responses suggested that: After the probe incubated with MMP-2,the fluorescence intensity of the probe itself was increased obviously?about 10-fold?with emission at 480 nm as time went on and then leveled off about 2 h.?2?The results from the selective experiments displayed that: When other biologically relevant species such as MMP-1?10 nM?,K⁺,Mg²⁺,Na⁺,Fe³⁺,Cu²⁺,Zn²⁺,Ca²⁺,L-tryptophan?L-Trp?,L-serine?L-Ser?,L-aspartic acid?L-Asp?,HCO₃^-,NO₃^-,ClO₄^-,F-,and Br-?all 10 mM?were incubated with the probe?20 nM?at 37? for 2 h,no obvious changes in the fluorescence spectra were observed.4.The qPCR,RT-PCR,Western blotting results showed that MMP-2 expression in cervical carcinoma cell lines were ordered as CaSki > Si Ha > C33-A > HeLa?P<0.05?.5.The results from cells imaging indicated that: When the cervical carcinoma cell lines were incubated individually with the fluorescence probe for 120 min at 37? and observed by confocal laser scanning microscopy,the fluorescence intensities were in the following order: CaSki > SiHa > C33-A > HeLa,which were consistent with the experimental results of qPCR,RT-PCR,and western blotting.6.The cervical carcinoma tumor-bearing mice of SiHa and HeLa were successfully established.The results from in vivo imaging experiments indicated that the strong fluorescence signals were observed in Si Ha and HeLa tumor-bearing mice,while the the fluorescence intensities of SiHa tumor-bearing mice were higher than HeLa tumor-bearingmice.Conclusions1.A coumarin-based fluorescence resonance energy transfer probe targeting Matrix Metalloproteinase-2 was prepared.2.The MMP-2 probe could be used for cerical carcinoma live cell imaging and tumor-bearing mice in vivo imaging.