Development Of A Drug Delivery System Based On Magnesium-aluminum Hydrotalcite Nanosheets And Its Application In The Treatment Of Cancer With Mutant P53

The p53 protein encoded by the Tp53 gene,as an important tumor suppressor,plays a key role in preventing the occurrence of tumors.Studies have shown that Tp53 gene mutation occurs in more than 50% of cancers,and its transcribed mutant p53(Mutp53)exhibits ultra-high stability in protein structure,so it accumulates in tumor cells.Mutp53 lost its original tumor suppressor activity and instead acquired some new functions,such as promoting tumor cell growth,proliferation,infiltration,and metastasis.At present,some small molecule compounds have been developed for the treatment of Mutp53 tumors,but the lack of specificity of these small molecule compounds limits their clinical application.In addition,studies have shown that Mutp53 needs to form a molecular chaperone complex with HSP90 to maintain its protein structure stability.This mechanism of action prevents Mutp53 from being degraded by MDM2-mediated ubiquitination-proteasome pathway,thus causing it accumulates in large amounts in cells.It is well known that the expression of HSP90 is affected by ATP content.Therefore,we propose to deliver a highly catalytically active Glucose oxidase(GOx)into Mutp53 tumor cells to reduce the supply of intracellular ATP by catalytically decomposing Glucose.When the content of ATP in cells is reduced,the expression of HSP90 is inhibited,which in turn destroys the structural stability of Mutp53 protein.Unfortunately,as a biological macromolecular protein,GOx cannot directly enter cells.So we tried to use a carrier to help GOx enter the cell and induce the degradation of Mutp53.Magnesium-Aluminum Layered Double Hydroxide(MgAl-LDH)is widely used in the field of pharmaceutical delivery due to its unique physicochemical properties.Therefore,MgAl-LDH was selected as the drug carrier in this study for the delivery of GOx.In the second chapter,MgAl-LDH with high dispersity was synthesized by coprecipitation and hydrothermal two-step method.The MgAl-LDH was then characterized by TEM,DLS,and Zeta potential,showing that the synthesized MgAl-LDH was a nanosheet with a regular hexagonal morphology,a size of about 78 nm,and a surface charge of +32.9 m V.Next,X-ray diffraction showed that the synthesized material was indeed MgAl-LDH.Using energy-dispersive X-ray spectroscopy to further characterize MgAl-LDH,it was found that the material contained magnesium(Mg),aluminum(Al),and oxygen(O),and no other impurities peak appeared,which indicates that the prepared MgAl-LDH is of high purity.By optimizing the initial reaction concentration and time of GOx and MgAl-LDH,a magnesium-aluminum hydrotalcite@Glucose oxidase(MgAl-LDH@GOx)complex with a loading of 68.96 μg/mg was obtained.Infrared absorption spectroscopy and sodium salt-polyacrylamide gel electrophoresis confirmed the loading of GOx by MgAl-LDH.In cell experiments,it was found that the endocytosis of materials by cells was mainly mediated by clathrin,and the materials escaped lysosomes 6 hours after entering cells.Finally,the cytotoxicity of MgAl-LDH@GOx on Mutp53 and Wtp53 cell lines was tested by cytotoxicity proliferation assay.Interestingly,in the two types of cell lines treated under the same conditions,MgAl-LDH@GOx only produced significant killing on the Mutp53 type cell line,but had little effect on the Wtp53 type cell line.Therefore,we further explore the reasons for this selective killing in next chapter.In the third chapter,the Mutp53 and Wtp53 cell lines were first treated with MgAl-LDH@GOx,and the p53 content in these two types of cell lines was analyzed by Western Blotting,and it was found that MgAl-LDH@GOx only had obvious scavenging effect on Mutp53,and with the increase of the concentration of the added material and the incubation time,the Mutp53 showed a dependent downward trend,and the effect on Wtp53 was almost negligible.It was subsequently found that MgAl-LDH@GOx reduced the intracellular ATP content after entering cells.At the same time,it was confirmed by Co-Immunoprecipitation that a molecular chaperone complex was formed between Mutp53 and HSP90,but this was not the case for Wtp53.Therefore,based on the above conclusions,we speculate that when MgAl-LDH@GOx is used to reduce the intracellular ATP content,the expression of HSP90 will be inhibited,resulting in the inability of Mutp53 to form a stable complex with HSP90,which in turn causes Mutp53 to be degraded by the ubiquitination-proteasome pathway.The results of cell viability experiments showed that along with the degradation of Mutp53 induced by MgAl-LDH@GOx,the growth,proliferation and migration of cells were also inhibited,but the viability of Wtp53 cells was not significantly affected by the material.All in all,this study provides a precise tumor treatment strategy,which provides an important reference value for the study of Mutp53.