Preparation Of A Novel Hydrogen Sulfide Donor HA-ADT And Its Anti-breast Cancer Mechanism

The occurrence of cancer is extremely harmful to individuals,families and society.Currently,chemotherapy and radiation therapy are the two main ways to treat cancer,and the primary method is chemotherapy.Although chemotherapy can inhibit the growth of cancerous cells to a certain extent,in the process of treating cancer,because traditional anti-tumor drugs have no ability to distinguish between cancerous cells and normal cells,therefore,when a drug kill cancer cells,it also affects the growth of normal cells,which in turn causes side effects.And because of the complex internal structure of tumor cells and tissues,a single drug medical treatment has no significant effect.Therefore,more researchers are concerned about the targeted treatment of drugs.Targeted drug delivery is based on the cell level,which binds the drug to the tumor tissue or cells in a targeted manner,so that a large number of anticancer drugs are concentrated in the cancerous site,thereby avoiding damage to normal cells.In addition,chemotherapeutic drugs generally have disadvantages such as low solubility in water,inability to circulate in the body for a long period of time,and poor bioavailability.In recent years,natural polymer hyaluronic acid and its derivatives have high hydrophilicity,high viscoelasticity,biodegradability,hypoallergenicity,good biocompatibility and ability to bind to cell surface specific receptors is considered a good pharmaceutical carrier.OBJECTIVE:In this paper,HA and the poorly soluble small hydrogen sulfide donor ADT-OH are esterified to synthesize water-soluble HA-ADT,which increases the water solubility and targeting of ADT-OH.Increase the bioavailability of hydrogen sulfide donors.METHODS:Acetone was reacted with sulfur to synthesize ADT;ADT was demethylated to form ADT-OH;finally,ADT-OH was grafted onto HA to form HA-ADT.The structure and molecular weight of the compounds were characterized by ¹H-NMR,LCMS and DSC;The particle size,zeta potential and PDI of the prepared HA-ADT were measured by a laser particle size analyzer;The H₂S release behavior of HA-ADT was investigated by ELISA;The in vitro safety of HA-ADT was evaluated by hemolytic assay.In vitro experiments:The effects of HA-ADT on proliferation of breast cancer cells were detected by MTT and EdU methods;Scratch,migration,invasion and soft agar assay were used to detect the effects of migration and invasiveness on human breast cancer cells treated with HA-ADT.The effect of HA-ADT on the clonal ability of breast cancer cells was detected by plate cloning;The Tunel kit detected the effect of HA-ADT on apoptosis of human breast cancer cells;finally,the effect of HA-ADT on the expression of related proteins such as apoptotic protein,PI3K/AKT/mTOR and Ras/Raf/Mek/Erk signaling pathway were determined by Western blot.In vivo experiment:The effect of HA-ADT on the tumorigenic ability of human breast cancer cells was detected by injecting human breast cancer cells into the armpit of nude mice;Histological morphology was observed by HE staining,immunohistochemical staining of Ki67 and CD31 of tumor tissues;finally,the toxicity of HA-ADT was evaluated by analyzing the body weight of the tumor-bearing nude mice and the organ index.Results:1.From the nuclear magnetic spectrum,we successfully synthesized HA-ADT with a graft ratio of 20%.2.HA-ADT had an average particle diameter of 81.98 nm,a PDI of 0.293,and a potential of-28.8 mV.3.HA-ADT release was relatively stable and persists within 48 hours.4.The hemolysis rates of HA-ADT at 100,200,and 300?mol/L were 0.74%,1.47%,and 1.54%,respectively.5.The results of MTT and EdU showed that the HA-ADT group had a significant inhibitory effect on the proliferation of human breast cancer cells compared with the control group.6.By cell scratch and cell migration,the cell migration ability of the HA-ADT group was significantly weaker than that of the control group.7.From the cell invasion and soft agar experiments,it showed that the HA-ADT had a weaker effect on cell invasion compared with the control group.8.The results of plate cloning experiments showed that the cloning ability of the HA-ADT group was significantly lower than that of the control group.9.The results of Tunel experiment and expression of apoptotic protein levels showed that HA-ADT significantly promoted apoptosis of breast cancer cells compared with the control group.10.Western blot results showed that HA-ADT reduced the expression of p-PI3K/p-AKT/p-mTOR and Ras/p-Raf/p-Mek/p-Erk protein compared to the control.11.The tumor formation experiment showed that the volume of the underarm tumor of the nude mice administered with HA-ADT group was significantly smaller than that of the control group.12.The results of hematoxylin-eosin staining showed that compared with the control group,the HA-ADT group had smaller nuclei,more interstitial,less nuclear division,and a larger range of necrosis.13.From the results of immunohistochemistry of Ki67 and CD31,it can be seen that the number of nuclei and microvessel density in the tumor tissue of the HA-ADT group were significantly reduced compared with the control group.14.Nude mouse body weight and organ index statistics showed no statistical difference between the groups.Conclusion:HA-ADT was successfully prepared,which had uniform particle size distribution,good stability and good sustained release.Cell experiments and animal experiments showed that HA-ADT had better anticancer effects than NaHS and GYY4137 at the same concentration,and significantly reduced the side effects of drug treatment.Therefore,HA-ADT can be considered as a new type of anti-tumor preparation with potential value.