Matrix Metalloproteinases-14 Activated Ag₂S Probe For Early Detection Of Living Neuroblastoma

Objective:The aim of this study was to demonstrate a novel matrix metalloproteinases-14 activated Ag2S QD-based probe(A1094@Ag2S-AF7P)for early detection of neuroblastoma.After successfully transmitted to localization of tumor tissues by neuroblastoma-targeted peptide(AF7P),it could be cleaved by high-expressed matrix metalloproteinases 14 anchored on tumor cell membrane.Then,the fluorescence would recover and the probe could be taken into cell to accurately localized neuroblastoma tissues and residual tumors in vivo and in vitro.Methods:1.Construction and characterization of the matrix metalloproteinases-14 activated Ag2S fluorescent probe:the oil phase DT-coated Ag2S quantum dots were ostensibly functionalized by DSPE-PEG2K-MAL,then the enzyme-activated nanoprobe A1094@Ag2S-AF7P was successfully synthesized through hydrophilic and hydrophobic interaction.Besides,we tested the morphology features,optical properties,stability and the feature of fluorescent quench-recover of A1094@Ag2S-AF7P nanoparticle.2.Fluorescence imaging of matrix metalloproteinases-14 activated Ag2S fluorescent probe for certification of targeting capability towards neuroblastoma in vivo and in vitro:The MMP 14-positive KP-N-NS cells and MMP 14-negative MCF-7 cells were incubated with A1094@Ag2S-AF7P to observe the fluorescent signals.After successfully constructing of xenograft subcutaneous tumor model,the as-prepared fluorescence nanoparticle probe A1094@Ag2S-AF7P was injected intravenous into tumor-bearing animals to certify the targeting capability of A1094@Ag2S-AF7P towards neuroblastoma in vivo.3.In vivo Fluorescence imaging of matrix metalloproteinases-14 activated Ag2S fluorescent probe for neuroblastoma and residual tumors:peritoneal tumor model was successfully established by injection of KP-N-NS cells,then the fluorescence signals was observed by IP injection of A1094@Ag2S-AF7P to confirm the capability of distinguishing tumor nodules from normal organs and achieving precise localization of tumor boundaries in the surgery.The excised tumor tissues obtained from patient were incubated with A1094@Ag2S-AF7P with or without MMPs inhibitor GM6001 to monitor the near-infrared florescence signals and further verify capability of precisely recognizing NB from tissues obtained from clinical patient.Results:1.The matrix metalloproteinases-14 activated nanoparticles(A1094@Ag2S-AF7P)constructed successfully showed a shape of cluster,an optical characteristic of fluorescence energy resonance transfer and maintenance of stability under different PH conditions and within a specific time range.2.The near-infrared imaging result of tumor cell in vitro showed the fluorescence intensity of NB KP-N-NS cells was about seven-fold of MCF-7 cells after incubated with A1094@Ag2S-AF7P.The near-infrared imaging result of subcutaneous tumor model in vivo showed fluorescence signals of neuroblastoma tissues is stronger than breast cancer tissues in vivo after injection of A1094@Ag2S-AF7P.The results of MTT assay confirmed that when compared with control group,state of cellular activities was more than 90%with different concentrations of A1094@Ag2S-AF7P.And Annexin V-FITC/PI apoptosis detection kit confirmed that the percentage of normal cells in both groups were nearly more than 90%with different concentrations of A1094@Ag2S-AF7P.3.The near-infrared imaging result of in vivo diagnosis in a model of peritoneal metastasis illustrated the excised tumor nodules with fluorescence and then were confirmed by HE staining.The tumor border showed by HE staining was consistent with the result of fluorescence imaging.The near-infrared imaging result of tumor tissues obtained from patient were also verified,the fluorescence intensity of tumor tissues incubated with MMPs inhibitor(GM6001)was significantly weaker than those without being incubated with GM6001.The results of blood and biochemical test demonstrated the slight rise in number of neutrophils and white blood cells compared with PBS group,which was within the normal fluctuation range.And there was no difference between the other blood and biochemical indicators of the two groups.Conclusion:1.The novel metalloproteinases-14 activated nanoparticle(A1094@Ag2S-AF7P)we constructed exhibits decent biocompatibility,excellent NIR-? fluorescence properties.2.The probe A1094@Ag2S-AF7P showed targeting capability towards subtype of NB cells in vivo and in vitro.3.The probe A1094@Ag2S-AF7P demonstrated ability of precisely localization of residual tumors and tumor borders in vivo.