Preparation And Application Of Tumor Associated Protease-Activated Quantum Dots Prodrug-type Nanoprobe

Objective:Tumor-associated proteases(TAP)have attracted great attention active in recent research as diagnostic and prognostic biomarkers as well as potential drug targets.The TAPs such as matrix metalloproteinase 2(MMP2)and legumain play an important role in tumorigenesis,and their existence is featured by overexpression in tumor tissue but deficiency in normal organs.It makes them suitable as target for design of activatable probes.MMP2 is up-regulated in the process of tumorigenesis,invasion and metastasis among many kinds of tumors.Legumain is associated with malignant tumor invasion,proliferation and metastasis,and highly expressed in tumor cells,neovascular endothelial cells,tumor-associated macrophage and solid tumors,including breast,colon and prostate cancers.Here we present the design,optimization,fluorescent activation,in situ and in vivo application of the prodrug-type,protease-responsive hybrid nanoprobe system for detecting TAP-overexpressed tumor.Methods:1.In the study we designed and synthesized the MMP2-activatable QDs nanoprobe.Firstly,LMWH-modified QDs were synthesized in aqueous solution and modified by ligand exchange reaction with LMWH.It was characterized by infrared spectroscopy(IR),ultraviolet-visble spectrophotometer(UV-VIS),fluorospectrophotometer,sulphur content determination,Bancroft reagent inspection,agarose gel electrophoresis(AGE),thermogravimetric analysis(TGA)and transmission electron microscopy(TEM).Secondly,ALMWP-QSY was synthesized by chemical coupling method and verified by high performance liquid chromatography(HPLC)and mass spectrometry(MS).Thirdly,the QD-based.nanoprobes were self-assembled by electrostatic interaction of LMWH/LMWP.Characterization studies contained fluorospectrophotometer,protamine activation test,particle size,zeta potential,and TEM.2.In vitro activation of the MMP2 nanoprobe was qualitatively and quantitatively tested in nontumoral 293T cells and various tumor cells including HT1080 cells,MCF7 cells,B16 cells,Hela cells,and SW620 cells,and verified by western blot.3.In vivo imaging of the MMP2-activatable nanoprobes were further investigated using HT1080,SW620 and MCF7 tumor-bearing mice by local delivery(intramuscular injection)and systemic administration(tail vein injection).4.Biosafety of the nanoprobe was evaluated in both cellular and animal level.5.Moreover the adaptability of the hybrid nanoprobe strategy was tested for applying in detection of various tumor proteases,by using different protease-substrate peptide-conjugated QSY quencher.Legumain-activatable QDs prodrug-type nanoprobe was designed and synthesized for characterization and experiments both in vivo and in vitro.Results:1.The 200 run hybrid nanoprobe system was successfully built with good form,uniform dispersion and good stability.This hybrid nanoprobe system is comprised of two major components.One is the fluorescence donor moiety-low molecular weight heparin(LMWH)modified quantum dots(QDs),and another is cleavable quencher moiety-low molecular weight protamine(LMWP)-MMP2 substrate-conjugated fluorescent quencher QSY21.Via the strong affinity binding of LMWH/LMWP,the hybrid nanoprobe thereby forms a fluorescence resonance energy transfer(FRET)system.2.This nanoprobe was highly efficiently activated in the MMP2-overexpressed HT1080 cells,but slightly activated in other tumor cells with mild MMP2 expression.By contrast,the nanoprobe remained in a quenched state in 293T cells.3.The nanoprobe can specifically response to MMP2 by showing full fluorescence recovery,but be resistant to the trypsin-mediated non-specific enzymatic activation.4.The nanoprobe remained quenched by intramuscular injection,demonstrated the MMP2-activated specificity.The systemic nanoprobe can accumulate and be activated in the tumor especially from 2 h to 4 h after administration,clearly distinguishing the tumor from its adjacent tissues.5.The results showed that QD-LMWH and MMP2-activatable QDs nanoprobe were biocompatible.6.The legumain-activatable QDs nanoprobe was designed and synthesized based on the similar structure.Its remarkable imaging function and good biological safety was also demonstrated.Conclusion:TAP has been widely investigated as the diagnostic and prognostic biomarkers as well as potential drug targets.Real-time,in vivo detecting and imaging of TAP provides a useful tool for early diagnosis and screening of cancer.Our study showed that the TAP-activatable nanoprobe system could potentially serve as a tumor imaging and detection technique.