Based On The Au-Se Bond, Nano-fluorescent Probes Are Constructed For The Detection Of Caspase Family Proteases And Their Application In Tumor Photothermal Therapy

Cancer is a major disease that endangers human health.Research shows that,there are more than 10 million new cancer patients and more than 6 million deaths worldwide each year.The number of cases and deaths each year increased by about 22 percent compared with 1990.It is predicted that by 2020,new cancer cases will reach 15.4 million per year.Apoptosis is involved in the occurrence and development of many diseases.It is of great significance to study apoptosis for the diagnosis and treatment of cancer.Nanotechnology is a highly interdisciplinary and comprehensive subject,which involves many fields such as chemistry,engineering,biology and medicine.Nanomaterials have great potential in early diagnosis and personalized treatment of cancer.Nanoparticles,typically smaller than a few hundred nanometers,are comparable to larger biomolecules such as enzymes,receptors and antibodies in size.They are biocompatible and can interact with cell surface and internal molecules to enhance permeability and retention effects,allowing nanocarriers to passively target tumor cells.The targeting groups can also be modified on the surface of the nanocarrier to achieve active targeting,so as to further improve the enrichment of nanocarrier in tumor tissue,develop effective integrated methods of diagnosis and treatment and provide a new approach for cancer treatment.Based on the excellent chemical stability,controllable morphology,unique optical and electrical properties and good biocompatibility of gold nanomaterials,nanosensors and nanodrug carriers constructed by Au-S bond have been widely used in the diagnosis and treatment of cancer.Nanosensor is usually to modify the sulfhydryl group of DNA,RNA,polypeptide or other molecules on the surface of gold nanoparticles based on the Au-S bond to assemble sensors or probes for the detection of target molecules.However,a large number of experiments have shown that,as gold nanosensors are applied in complex biological systems,biothiols of high concentration can partially replace the biothiols modified functional recognition unit,resulting in distortion of sensing signals.In order to solve this problem,we propose to replace Au-S bond with Au-Se bond to reconstruct gold nanosensor,resulting the fidelity of detection signal.In this paper,we designed a gold nanoscale fluorescence probe(GNP-Se-Casp)based onthe Au-Se bond instead of Au-S bond for the detection of caspase cascade activation and realized the real-time observation of caspase-3/8/9 level changes during apoptosis,verifying the upstream and downstream relationship of caspase-3/8/9 activation during apoptosis.Also,based on the Au-Se bond,we modified the surface of gold nanorods with RGD(one specific recognition molecular of tumor cells)and peptide chain specific to caspase-9,built the treatment and tracer dual function probe and realized the accurate targeting the tumor area,photothermal therapy and tracing the degree of tumor cell apoptosis,free from the interference of biothiols during the long-term in vivo circulation.The main research contents of this paper include the following two parts:1.A multicolor fluorescent nano-probe GNP-Se-Casp was designed and synthesized based on the gold-selenium bond for real-time in-situ monitoring of caspase in the process of apoptosis.Real-time monitoring of the upstream and downstream relationship of caspase-3/8/9in apoptosis was achieved by using this probe.The probe was assembled with three different specific identification of caspase-3/8/9 peptide chain on the gold nanopartilces via Au-Se bond.In the absence of targeted caspases,GNP-Se-Casp showed no fluorescence due to the efficient quenching effect of GNPs.The blue and green fluorescence,corresponding to casp-8 and casp-9sequentially turned on,followed by appearance of the red fluorescence of casp-3.The experiment showed that in the process of STS(staurosporine)inducing Hela cell apoptosis,the fluorescence signal of the dye could be obtained in real time through the fluorescence confocal microscope,which realized the real-time in-situ monitoring of the cell apoptosis process.In addition,the fluorescence signal was not interfered by glutathione under the simulated physiological condition.Thus,GNP-Se-Casp is an effective tool for real time in situ monitoring caspase cascade activation in the apoptosis process of tumor cells.This design strategy is easily adaptable to in situ detection of other signal molecules,especially those with upstream and downstream activation relationships.2.Based on the Au-Se bond,we modified the surface of gold nanorods with RGD(one specific recognition molecular of tumor cells)and peptide chain specific to caspase-9,built the treatment and tracer dual function probe.gold-sulfur nano-fluorescence probe(RGD@AuNRsS-peptide-FITC)and gold-selenium nano-fluorescence probe(RGD@AuNRs-Se-peptide-FITC)were constructed to avoid interference of high level mercaptol complex in the long-termcirculation of mice,and the probe targeted tumor areas accurately and obtained high-fidelity fluorescence signals.Compared with gold-sulfur nano-probe,gold-selenium nano-probe has significant anti-biothiol interference advantages,RGD peptides consist of arginine,glycine and aspartic acid(Arg-Gly-Asp)sequence of the peptide chain and is used to target the tumor cell.The tumor area was irradiated with near-infrared light at a specific wavelength,and the tumor cell was killed by the heat energy which was converted from the light energy because of the gold nanorods in near-infrared light super absorption,and can convert light energy into heat energy,kill tumor cells,During apoptosis,the release of caspase protein breaks the peptide chain and promotes the fluorescence recovery.The probe can accurately target the tumor region with photothermal therapy and track the apoptosis degree of tumor cells,and the circulation in vivo is not disturbed by biothiols for a long time.