Construction Of Ursolic Acid And Doxorubicin Loaded Ferritin Drug Delivery System And Its Synergistic Antitumor Study

Ursolic acid(UA)is a natural triterpene carboxylic acid compound found in a variety of fruits,vegetables and herbs with excellent preventive and anticancer activities.Doxorubicin(DOX)is a kind of broad-spectrum antitumor drugs in clinical use,which can inhibit RNA and DNA synthesis.It is a cyclically nonspecific drug.UA can not only induce apoptosis of cancer cells,but also improve the sensitivity of various tumor cells to DOX,effectively inhibit the growth of tumor tissues,reduce the ability of tumor cells to metastasize,and it has almost no side effects on normal cells.Although UA has great prospects and advantages in the treatment of cancer,its poor water solubility leads to low bioavailability.DOX has a great side effect on normal cells,causing myocardial damage and heart failure.The shortcomings of both drugs such as lack of targeting limit the clinical application.Ferritin is composed of 24 subunits to form a spherical cage structure,which is widely found in animals,plants and microorganisms.Human heavy chain ferritin(HFtn)has the advantages of good water solubility,good biocompatibility,good stability and uniform particle size.In addition,the surface of most tumor cells can overexpress transferrin receptor 1(Tf R1),and ferritin can specifically bind to it and effectively enter cells under the mediation of this receptor,so as to achieve active targeting.In this study,hydrophobic UA and hydrophilic DOX drugs were co-encapsulated into the intracavity of ferritin to construct ferritin-doxorubicin/ursolic acid nanodrugs(HFtn-DOX/UA),in order to improve the water solubility of UA and reduce the side effects of DOX,and to evaluate the targeting and anti-tumor effects of the nanoparticles.The results are as follows:(1)Human heavy chain ferritin(HFtn)was successfully expressed,and the target protein was obtained through the purification process of His nickel column and FPLC.The target protein was successfully expressed and purified by volume size exclusion chromatography(SEC),SDS-polyacrylamide gel electrophoresis(SDS-PAGE)and non-denaturednative polyacrylamide gel electrophoresis(Native PAGE).(2)Hydrophobic UA and hydrophilic DOX were successfully encapsulated into ferritin interior by thermal treatment at 60℃ for 4 h,and HFtn-DOX/UA interior were successfully prepared.The optimal nanoparticles encapsulated 90 UA and 18 DOX drugs per ferritin cavity.DOX and UA loading did not affect the polymeric state(24-mer),secondary structure(α-helix),and morphology(spherical cage structure)of the protein carrier.In vitro drug release results showed that HFtn-DOX/UA nanodrugs were relatively stable at p H 7.4,and the drug release was less than 20% at 48 h.Under the lysosome p H 5.0,UA and DOX release increased significantly,and the drug release reached 80% after 48 h,indicating that the release of HFtn-DOX/UA nanodrugs was p H-dependent.(3)The antitumor activity was studied for the prepared HFtn-DOX/UA nanodrugs in vitro.The cell cytotoxicity results showed that free drugs and HFtn-DOX,HFtn-UA and HFtn-DOX/UA nanodrugs had inhibitory effects on MCF-7 and A549 cells,and the inhibition rate increased with the increase of concentration.At the same concentration,the inhibition rate of HFtn-DOX/UA nanodrugs on MCF-7 and A549 cells was the highest,and the inhibition rate of co-loaded drugs was significantly higher than that of single-loaded drugs.According to the drug combination index(CI),it was proved that DOX and UA had a synergistic effect,which was strongest when the drug load ratio was 1:5.Calcein AM/PI staining of live and dead cells further proved that the prepared HFtn-DOX/UA nanodrugs had the strongest cytotoxicity effect on MCF-7 and A549 cells.Wound healing assay experiments demonstrated that HFtn-DOX/UA nanodrugs could effectively inhibit the migration ability of MCF-7 and A549 cells.Cell apoptotic results showed that HFtn-DOX/UA nanodrugs had the strongest apoptotic induction ability.3D tumor spheroids of MCF-7 cells were constructed to evaluate the inhibitory effect of the constructed nanodrugs on tumor growth.The results displayed that HFtn-DOX/UA nanodrugs had the strongest inhibitory effect on tumor spheroids,and the inhibitory effect of co-loaded drugs was higher than that of single-loaded drugs,and the inhibitory effect of drugs delivered by ferritin was better.These results indicate that HFtn-DOX/UA nanodrugs are a promising drug delivery system,providing a reference for loading hydrophilic and hydrophobic drugs simultaneously.