Construction And Antitumor Activity Evaluation Of Multiple Stimulus-responsive Hydroxyethyl Starch-doxorubicin Conjugate

Chemotherapy is one of the main methods for cancer treatment at present.Traditional chemotherapeutic drugs have disadvantages such as poor water solubility,low tumor enrichment,small molecules,and easy to be cleared by the kidney and so on.Therefore,the clinical use of chemotherapeutic drugs is often accompanied by serious side effects,and the clinical dose is limited.In recent years,many nano anti-tumor chemotherapeutic drugs based on macromolecular carriers have been developed by researchers to improve the shortcomings of traditional chemotherapeutic drugs.Among them,some studies,taking the advantage of biochemical characteristics of cancer lesions,linked chemotherapeutic drugs to the carrier through stimulus-responsive chemical bonds to design intelligent responsive nanodrugs.Intelligent responsive nanodrugs can achieve specific stimulus-responsive release of active drugs in cancer lesions,so as to improve drug distribution,enhance drug efficacy and reduce side effects.However,relying on a single response condition will result in insufficient drug release and untimely release.In addition,the release of non-original drug structure after bond breakage will lead it unable to reach specific subcellular organelles,resulting in poor therapeutic effect.Considering polysaccharide polymer hydroxyethyl starch（HES）as drug carrier,four kinds of tumor specific hydroxyethyl starch-doxorubicin conjugates（HES-R-DOX）were designed and synthesized by delivering clinical first-line anticancer drug doxorubicin（DOX）.Their properties,anti-tumor effects and safety were studied,and their anti-tumor activities were compared.Hydroxyethyl starch-doxorubicin conjugate HES-R-DOX was synthesized and characterized.Four hydroxyethyl starch doxorubicin conjugates HES-R-DOX were designed as hydroxyethyl starch-carbamate-disulfide-urea-doxorubicin（HES-CM-SS-UR DOX）,hydroxyethyl starch-disulfide-doxorubicin（HES-SS-DOX）,Hydroxyethyl starch-carbonate-dicarbon-doxorubicin（HES-CN-CC-CM-DOX）and hydroxyethyl starch-carbonate-disulfide-carbamate-doxorubicin（HES-CN-SS-CM-DOX）.A synthetic route was developed for the synthesis of HES and DOX derivatives by HES activation,DOX amino derivatization and nucleophilic reaction.Nuclear magnetic resonance（¹H NMR）and fluorescence spectra showed the connection of DOX.The drug loading of HES-R-DOX determined by the absorbance at 500nm via UV-Vis spectrophotometer is as follows:HES-CM-SS-UR-DOX is 3.01%,HES-SS-DOX is 3.92%,HES-CN-CC-CM-DOX is 2.44%,HES-CN-SS-CM-DOX is 2.64%.The hydrated particle size of HES-R-DOX measured by laser particle size analyzer is as follows:HES-CM-SS-UR-DOX is 33 nm,HES-SS-DOX is 28 nm,HES-CN-CC-CM-DOX is 33 nm,HES-CN-SS-CM-DOX is 43 nm.The in vitro drug release experiments showed that HES-CM-SS--DOX had triple stimulation responsiveness of p H,oxidation and reduction.Within 48hours,44%of the DOX was released in the release solution of p H 5.0,32%in the release solution of 5 m M H₂O₂ and 48%in the release solution of 10 m M DTT.The HES-SS-DOX drug had dual responsiveness of oxidation and reduction responsiveness.within 48 hours,86%of the DOX was released in the release solution of 10 m M DTT and46%was released in the release solution of 5 m M H₂O₂.The HES-CN-CC-CM-DOX drug had acidic p H responsiveness,and 65%of the DOX was released from the release solution of p H 5.0 within 48 hours.The HES-CN-SS-CM-DOX drug had triple stimulation responsiveness of acid p H,oxidation and reduction.Within 48 hours,70%of the DOX was released from the release solution of p H 5.0,46%in the release solution of 10 m M DTT,and 42%of the DOX was released in the release solution of 5 m M H₂O₂.Anti-tumor activity and safety of hydroxyethyl starch-doxorubicin conjugate HES-R-DOX in vivo and in vitro were studied.Flow cytometry showed that the cellular uptake of HES-R-DOX conjugate by 4T1 cells was positively correlated with time.Cytotoxicity experiment showed that HES-CM-SS-UR-DOX,HES-SS-DOX,HES-CN-CC-CM-DOX and HES-CN-SS-CM-DOX all could kill 4T1 cells.Among them,the killing effect of HES-CN-SS-CM-DOX was the strongest.In the 4T1 orthotopic tumor model,HES-CM-SS-UR-DOX,HES-SS-DOX,HES-CN-CC-CM-DOX and HES-CN-SS-CM-DOX could inhibit the growth of 4T1 tumor,and HES-CN-SS-CM-DOX had the highest tumor inhibition rate.After 3 times of administration within 12 days,the tumor inhibition rate reached 55%,which was higher than that of DOX alone（36%）.The results of blood biochemical index and blood routine index analysis showed that HES-CM-SS-UR-DOX,HES-SS-DOX,HES-CN-CC-CM-DOX and HES-CN-SS-CM-DOX had no significant effect on liver,kidney and heart of mice,and had no significant effect on blood routine index.The body weight of mice in HES-R-DOX groups was not significantly affected,and the tissue sections of heart,liver,spleen,lung and kidney showed that the viscera of mice were basically normal,which indicated that HES-R-DOX was safe.In view of the disadvantages of the traditional nano-drug delivery system,a kind of hydroxyethyl starch-doxorubicin conjugate with multiple stimulus response is provided in this paper.The anti-tumor activities of drugs of triple response connectors,double response connectors and single response connectors,drugs releasing raw drugs and drugs released by non-original drugs were discussed in detail.The results show that the HES-CN-SS-CM-DOX drugs with low p H,oxidation and reduction triple stimulus responsiveness and raw drug release behavior have the best anti-tumor activity.In addition,the results showed that after DOX is conjugated to HES,the toxicity and side effects are impaired and the safety is increased.