Research On The Application Of Mesoporous Materials In The Diagnosis And Therapy Of Cancer Based On Targeting GSH Strategy

Mesoporous materials are a kind of porous materials with pore size between 2?50 nm.They show many excellent physiochemical properties involving high surface area and large pore volume,narrow pore size distribution and continuous adjustment,high chemical and thermal stability,easy surface modification and good biocompatibility.In recent years,the development of mesoporous nanomaterials and its deep integration with biomedical diagnosis and therapy technologies have provide a new approach and strategy for the diagnosis and therapy of cancer,and have application prospects in the field of biomedicine.Mesoporous materials provide two solutions for the diagnosis and therapy of cancer.On one hand,mesoporous materials-based biosensors are designed to detect tumor markers,which is used in the early diagnosis of cancer.On the other hand,mesoporous materials can be used as a carrier in nanoparticulate drug delivery system?NDDS?design.NDDS can achieve targeting tumor tissue,increasing the concentration of antitumor drugs in cancer cells,reducing side effects,and reversing multidrug resistance by virtue of its specific tumor tissue enhanced permeability and retention?EPR?effect.In this paper,mesoporous Mn O₂ with sensitive oxidase activity was used to construct a simple and rapid biosensor for glutathione?GSH?detection.The constructed sensor was used to evaluate the difference of GSH level between normal cells and cancer cells.Based on this,mesoporous silicon?MSN?-based NDDS with targeting cancer cells and GSH responsive release drug was designed to reverse drug resistance.Part one:A colorimetric sensor based on mesoporous Mn O₂ for the detection of intracellular GSHObjective:A biosensor based on oxidase activity of mesoporous Mn O₂was designed to detect intracellular GSH levels in cancer cells and normal cells,developing a new path for identifying cancer cells in biomedical diagnosis and providing the basis for designing NDDS based on targeting GSH strategy.Methods:Using Mn Cl₂.4H₂O and?NH₄?₆Mo₇O₂₄.4H₂O as raw materials,mesoporous Mn O₂ was synthesized by inorganic homogeneous method.The as-prepared mesoporous Mn O₂ was characterized by scanning electron microscopy?SEM?,transmission electron microscopy?TEM?,zeta potential,infrared spectroscopy?FT-IR?,x-ray powder diffraction?XRD?,brunner emmet teller?BET?,and x-ray Photoelectron spectroscopy?XPS?.Then,the catalytic substrate 3,3',5,5'-tetramethylbenzidine?TMB?was reacted with mesoporous Mn O₂ to explore the catalytic activity of simulated enzymes.For conditional optimization,the effects of different p H,TMB concentration,temperature and reaction time on the catalytic activity of mesoporous Mn O₂were investigated.Subsequently,the enzyme activity of mesoporous Mn O₂inhibited by GSH was experimentally verified.The linear range and detection limit of GSH was obtained using linear regression of the GSH concentration?C?versus the absorbance?A?at 652 nm.The interference of other substances in cells was excluded by specificity experiment.In addition,the cell lysates of hepatocellular carcinoma cells Bel-7402 and human umbilical vein endothelial cells HUVEC were obtained by repeated freeze-thaw method,and the GSH concentration of the lysates was determined to compare the GSH levels in cancer cells and normal cells.Finally,using the Bel-7402 cell lysate as a model,the standard addition method was used to perform the recovery test,which evaluated the feasibility of this method in the analysis of complex biological samples.Results:Mesoporous Mn O₂ particles were spherical with a diameter of about 50 nm and the pore size distribution of about 3?4 nm.The crystal state of as-prepared Mn O₂ was?-Mn O₂.FT-IR,Zeta potential and XPS further confirmed the successful synthesis of mesoporous Mn O₂.The optimum reaction conditions for mesoporous Mn O₂ in catalyzing the oxidation of TMB were as follows:p H value was 4?Na Ac buffer?,the concentration of TMB was 200?M,reaction time was 5 min and the temperature was 25?.GSH effectively inhibited the oxidation of the substrate TMB to produce blue products ox TMB.Under the optimum conditions,the sensor showed a linear range of 0.1?10 n M with a detection limit of 55 n M for GSH detection.Selective experiment showed that the interference of other substances in the cell was negligible,and the method was specific.By evaluating the intracellular GSH content using the designed sensor,it showed that the GSH levels in cancer cells is higher than that of normal cells,which provides a simple way to identify cancer cells.Conclusion:GSH sensor based on oxidase activity of mesoporous Mn O₂was simple,convenient,sensitive,flexible and stable.It could be used to evaluate GSH levels in cells.The detection limit and range had advantages over those of other nanomaterial-based colorimetric biosensors.Using biosensor based on mesoporous Mn O₂,simple UV colorimetric method was used to verify that GSH concentration in tumor cells was higher than that of normal cells,providing novel path for cancer diagnosis,drug delivery,and other biomedical applications.Part two:The construction of mesoporous silica nanocarrier for to overcome 5-fluorouracil drug resistanceObjective:To achieve the targeting tumor cell,responsive releasing of drugs in tumor cell microenvironment,sequential releasing of CDDP and5-FU,reversing multidrug resistance,a smart response NDDS anti-tumor platform was designed based on MSNs with GSH and p H response,folic acid acceptor targeting,and simultaneous loading with cisplatin?CDDP?and5-fluorouracil?5-FU?.Methods:MSNs were synthesized by solvent gel method and modified by amino group to achieve amino-functionalized MSNs?MSN-NH₂?.CDDP prodrug,monocarboxylic acid chain oxidized cisplatin?MDDP?,and folate modified polyethylene glycol?PEG-FA?were synthesized by oxidation and condensation reactions.MDDP and PEG-FA were modified on MSN-NH₂ by amide condensation,and 5-FU was encapsulated inside the pores of MSN to obtain MDDP-MSN?5-FU?@PEG-FA nano-dual-loading NDDS.TEM,small angle x-ray scattering?SAXS?,BET,zeta potential,thermogravimetric analysis?TGA?,FT-IR,ultraviolet-visible spectrum?UV-Vis?,¹H-NMR,electrospray ionization mass spectrometry?ESI-MS?,and high performance liquid chromatography-mass spectrometry?HPLC-MS?were used to characterize and verify as-prepared nanomaterials and compounds.The concentration of 5-FU drug was measured by UV-Vis,and MDDP content was determined by inductively coupled plasma mass spectrometry?ICP-MS?.GSH and p H-dependent drug release in vitro from NDDS was evaluated by dialysis method.The MTT method was used to evaluate the cytotoxicity of NDDS for human hepatoma cell sensitive strain bel-7402 and 5-FU-resistant strain bel-7402/5-FU.Uptake by human hepatoma cell sensitive strain bel-7402 was observed by fluorescence microscopy.Results:MSNs were spherical with a diameter of 140 nm,specific surface area of 813 m²/g and pore size of about 2.28 nm.ESI-MS and¹H-NMR verified the successful synthesis of the prodrug MDDP.HPLC-MS verification showed that MDDP was effectively reduced to active CDDP in the presence of GSH.Both ¹H-NMR and UV-Vis analysis suggested that PEG-FA was successfully synthesized.The loading contents of 5-FU and CDDP in NDDS were 78.26 and 5.83?g/mg,respectively.In the presence of 5.0 m M GSH and at p H of 5,the accumulated release of CDDP at 24 h was 3.00?g/mg.Rhodamine B?Rhb?was loaded in the MSN@PEG and MSN@PEG-FA nanoparticles to achieve Rhb-loaded NDDSs?MSN?Rhb?@PEG and MSN?Rhb?@PEG-FA?.It showed that the Rhb-loaded NDDSs were quickly taken by cancer cells.MSN?Rhb?@PEG-FA sample showed stronger endocytosis due to the effect of folic acid targeting the folate receptor on the surface of cancer cells.The cytotoxicity results of MSN@PEG nanoparticles showed that the NDDS had high biocompatibility.Cytotoxicity results of six groups of samples 5-FU,CDDP,5-FU+CDDP,MSN?5-FU?,MDDP-MSN?5-FU?,and MDDP-MSN?5-FU?@PEG-FA showed that the combination of 5-FU and CDDP reversed drug resistance and enhanced cytotoxicity.The 5-FU and CDDP co-loaded MDDP-MSN?5-FU?@PEG-FA system displayed a lower resistance index than that of physical mixture of5-FU and CDDP.Conclusion:The 5-FU and CDDP dual-loaded NDDS based on MSN was designed.After the NDDS endocytosed by the tumor cells,the outer layer of cisplatin prodrug MDDP was reduced to active CDDP in the presence of the high GSH concentration in the tumor cells.With the release of the outer layer of CDDP,5-FU in the inner hole was slowly released.The sequential administration pattern of 5-FU combined with low-dose cisplatin showed a higher inhibition rate on Bel-7402/5-FU resistant strains and a significant effect of reversing drug resistance.