Development Of Endogenous Protein Sulfcnic Acid-reactive Gold Nanoparticles For Enhanced Tumor CT Imaging And Radiotherapy

Background:Malignant tumors are currently one of the major diseases that seriously endanger human life and health.It has the characteristics of unlimited cell replication,continuous proliferation signals,induction of angiogenesis,resistance to apoptosis,easy invasion and metastasis and unlimited growth.Radiation therapy,as one of the three main methods of tumor treatment commonly used in clinical practice,plays an important role in treating tumors,alleviating patient suffering,and extending survival.The use of radiation sensitizers can cause the same dose of radiation to cause greater damage to tumor cells,and it has a significant effect on improving-the efficacy of tumor radiotherapy.Inorganic nanoparticles composed of high atomic number elements are good radiosensitizers.Gold nanoparticles modified with long-chain PEG have excellent biocompatibility,stability,inertness and extremely low cytotoxicity.And they have been widely used in biomedical research fields in recent years.Compared with large-sized gold nanoparticles,small-sized gold nanoparticles are more easily taken up by tumors under the effect of EPR.Because of their small size,they also have the disadvantage of being easily removed by the body.Therefore,prolonging the blood circulation cycle of small-sized gold nanoparticles in the body and increasing its enrichment in tumor lesions is an effective way to improve the effect of tumor diagnosis and treatment.At present,the commonly used method is to modify specific responsive molecules on the particle surface,so that under the action of stimulators such as pH,enzymes,light,etc.,they can selectively aggregate into particle aggregates in the tumor to improve the accumulation and retention of nanoparticles in the tumor.In this paper,the folic acid receptor is modified to target folic acid molecules on the surface of small-sized gold nanoparticles(25 nm),and the tumor's active targeting of nanoparticles is improved.3,5-dioxocyclohexanecarboxylic acid(DHCA)achieves tumor anchoring of gold nanoparticles,increases the accumulation of probes in tumor lesions and prolongs their retention time,and improves the CT imaging and radiotherapy effects of tumors.Research purposes:This paper constructs a protein sulfenic acid reactive tumor-targeting gold nanoprobe--AuNPs-FA,which modifies tumor-targeted folate molecules(FA)and 3,5-dioxocyclohexanecarboxylic acid which can react with sulfenic acid on the surface of gold nanoparticles.It uses folic acid to improve the active targeting of the probe to the tumor.Through the cross-linking with the high concentration of protein sulfenic acid in the tumor microenvironment,it can increase the accumulation and retention time of gold nanoparticles in the tumor,thereby achieving enhanced CT imaging and improving curative effect of radiotherapyMethods:(1)Uniformly sized gold nanoparticles were prepared by the classic trisodium citrate reduction method and the surface was modified with SH-PEG-NH2 The ROS sensitive molecule 3,5-dioxocycloalkylcarboxylic acid was modified by dehydration condensation reaction and then was modified by the targeted folate molecule.It was characterized by DLS,UV spectrometer and FTIR spectrometer.(2)DLS and TEM were used to determine the cross-linking of gold nanoparticles and bovine serum albumin after hydrogen oxide.(3)The stability of dAuNPs-FA in different solvents was measured by DLS and UV spectrometer.(4)The cytotoxicity of dAuNPs and dAuNPs-FA to mouse embryonic fibroblasts 3T3,mouse gastric cancer cell MFC and human gastric cancer cell MKN-45 was verified by MTT assay.(5)Dark field microscope,ICP-MS and ?-H2AX recognition antibody immunofluorescence method were used to study that dAuNPs co-incubated with 4T1 cells treated with hydrogen peroxide at different concentrations and the retention effect produced by cross-linking in the cells.(6)The uptake of dAuNPs-FA and dAuNPs by cells was observed by dark field microscope.(7)Cell cycle experiments were used to verify the blocking effect of dAuNPs and dAuNPs-FA on the cell cycle(8)Radiosensitization of dAuNPs-FA after cross-linking in 4T1 cells was investigated by Live/Dead experiment,apoptosis experiment,y-H2AX recognition antibody immunofluorescence method,single cell gel electrophoresis experiment and clone formation experiment.(9)CT imaging and TEM were used to determine the retention of dAuNPs-FA after intratumoral cross-linking.(10)The radiotherapy effects of dAuNPs-FA and AuNPs-FA in 4T1 tumor-bearing mice were compared and investigated.The tumor radiation therapy effect was evaluated by tumor volume change,metastasis and tissue section analysisResults:In vitro studies showed that gold nanoparticles dAuNPs-FA and dAuNPs were able to cross-link with bovine serum albumin afier hydrogen peroxide pretreatment.Cell experiments found that dAuNPs and dAuNPs-FA have little cytotoxicity and good biocompatibility,and dAuNPs showed significant uptake and long retention in 4T1 cells treated with hydrogen peroxide.With the further introduction of folic acid,dAuNPs-FA was captured more by tumor cells.Under the same X-ray irradiation,dAuNPs-FA,which has undergone protein sulfinic acid cross-linking,has a greater damage effect on cells.In vivo studies in mice found that the gold nanoprobe dAuNPs-FA has significant high uptake and long retention in living tumors,which significantly improves the CT imaging and radiosensitization effects of tumors,and has a certain inhibitory effect on distant tumor metastasis.Conclusion:The protein sulfenic-responsive tumor-targeting gold nanoprobe dAuNPs-FA has significantly higher tumor accumulation and long retention under the mediation of H2O2 in the tumor microenvironment.It can effectively improve the CT imaging and radiation treatment effects of tumors in vivo and provides a new plan for the efficient radiation treatment of tumors.