Preparation Of CEL-loaded Reduction-sensitive Polymer Micelles And Evaluation Of Its Anti-ovarian Cancer In Vitro

The National Cancer Center states that ovarian cancer has the highest mortality rate in gynecological malignancies.At present,the treatment of ovarian cancer is mainly surgery and chemotherapy.The surgery is only a small range of simple resection,and the recurrence rate is high after the chemotherapy drugs enter the body.Tissue causes toxic side effects.Celastrol?CEL?has a strong anti-ovarian cancer effect,but it has poor water solubility,low bioavailability,and high toxicity.Overexpression of integrin ?_??₅ receptor on the surface of ovarian cancer cells,and ubiquitin ligase TRAF6 in cells.The reducing substance GSH concentration in tumor cells is 2-10 m M,which is about 1000 times the GSH concentration?2-20 ?M?outside the tumor cells.In order to overcome the shortcomings of CEL,a reduction-sensitive nanocarrier with active targeting function was designed and synthesized in this thesis for targeted delivery of CEL tumor cells and intracellular responsive drug release.This topic is divided into four chapters:In the first chapter,m PEG-C₁₈,m PEG-SS-C₁₈,cRGD-PEG-C₁₈?TIP-PEG-C₁₈ were prepared.The structure of the synthesized product was verified by 1H-NMR spectrum,and the grafting ratio of the peptide was calculated.The results show that each polymer molecule has been successfully synthesized,the grafting rate of cRGD in cRGD-PEG-C₁₈ is 37.5%,and the grafting rate of TIP in TIP-PEG-C₁₈ is 20.2%.In the second chapter,the dual-targeted reduction-sensitive polymermicelles cRGD/TIP/m PEG-SS-C₁₈ were prepared.And the non-sensitive and target-free polymer micelles m PEG-C₁₈ and the sensitive and target-free polymer micelles m PEG-SS-C₁₈ were prepared,which served as comparison.First,the optimal graft density of cRGD peptide was screened,and then the polymer micelles were characterized.The results showed that the optimal graft densities of cRGD peptide was 12%.The average particle diameters of m PEG-C₁₈,m PEG-SS-C₁₈,cRGD/TIP/m PEG-SS-C₁₈ polymer micelles were 290.60 nm,269.35 nm,224.33 nm;PDI is 0.259,0.202,0.248 respectively;Zeta potentials are-9.74 m V,-5.84 m V,-7.66 m V,respectively;micelles present a uniform and regular spherical structure;The CMC values of m PEG-C₁₈,m PEG-SS-C₁₈,cRGD/TIP/m PEG-SS-C₁₈ polymer micelles were measured by Pyrene fluorescence probe method,which were 85 mg/L,68 mg/L,and 63 mg/L,respectively,which Shows good dilution stability;After polymer micelles are co-cultured with monocyte macrophage RAW264.7 cells and ovarian cancer cell A2780 cells,the cell survival rate was above 82%.The results of hemolysis experiments showed that the HR% of polymer micelles were less than 5%.Cytotoxicity experiments and hemolysis experiments have verified that polymer micelles have good biocompatibility.In the third chapter,m PEG-C₁₈/CEL ? m PEG-SS-C₁₈/CEL ?cRGD/TIP/m PEG-SS-C₁₈/CEL micelles were prepared and characterized.The results showed that the average particle sizes of m PEG-C₁₈/CEL,m PEG-SS-C₁₈/CEL and cRGD/TIP/m PEG-SS-C₁₈/CEL micelles are 282.0 nm,304.1 nm,and 215.2 nm,respectively,and PDI is 0.39,0.38,0.28,Zeta potential is-9.53 m V,-13.6 m V,-6.51 m V,respectively;micelles are regular spherical,round appearance,uniform dispersion;The drug loadings of m PEG-C₁₈/CEL,m PEG-SS-C₁₈/CEL and cRGD/TIP/m PEG-SS-C₁₈/CEL micelles were 3.08%,2.63%,3.02%,respectively,the encapsulation efficiency of m PEG-C₁₈/CEL,m PEG-SS-C₁₈/CEL and cRGD/TIP/m PEG-SS-C₁₈/CEL micelles were 99.63%,85.23%,and 87.34%;Within 60 hours of the in vitro release experiment,the cumulative release rate of cRGD/TIP/m PEG-SS-C₁₈ is 81%,which is 1.56 times that of m PEG-C₁₈,showing obvious reduction sensitivity characteristics;The HR% of the CEL-loaded micelles are all less than 5%,which proves that the CEL-loaded micelles also have good biocompatibility;The CEL-loaded micelles examined by DLS have long-term stability.In the fouth chapter,the toxicity and cell uptake of m PEG-C₁₈/CEL,m PEG-SS-C₁₈/CEL and cRGD/TIP/m PEG-SS-C₁₈/CEL micelles on A2780 cells were investigated.Cytotoxicity experiments showed that compared with m PEG-C₁₈/CEL and m PEG-SS-C₁₈/CEL micelles,cRGD/TIP/m PEG-SS-C₁₈/CEL micelles have a significant inhibitory effect on the growth of A2780 cells.CLSM shows that the binding of targeted polypeptides is conducive to enhancing the tumor cell's uptake of drugs,and cRGD/TIP/m PEG-SS-C₁₈/CEL micelles have active targeting.The dual-targeted reduction-sensitive polymer CEL-loaded micelles cRGD/TIP/m PEG-SS-C₁₈/CEL constructed in this paper can improve the solubility of hydrophobic drugs,improve the uptake efficiency of tumor cells to nanoparticles,and have good antitumor properties effect.It provides a certain experimental basis for the clinical treatment of ovarian cancer and has broad application prospects.