Preparation And Antitumor Pharmacodynamics Evaluation Of Two Kinds Of Doxorubicin-dihydroartemisinin Conjugated Prodrug Self-assembled Nanoparticles

Objective:The purpose of our study is to reduce the drug resistance rate of doxorubicin（DOX）,reduce the toxic and side effects of DOX,and better play the anti-tumor effect of DOX.Therefore,we introduced dihydroartemisinin（DHA）using a two-drug combination idea in our study.DOX-DHA conjugated prodrug with disulfide bond and succinic anhydride as the linking arm were synthesized.The self-assembled nanoparticles were prepared by nanoprecipitation method.The formulation properties of the two nanoparticles and the toxicity and cellular uptake characteristics in 4T1cells,MCF-7 cells and H9C2 cells were also investigated.In addition,the distribution and pharmacodynamics of both nanoparticles in 4T1-bearing BABL/c mice were studied and evaluated.We hope that by chemically combining DOX and DHA and preparing them into prodrug self-assembled nanoparticles.It provides an experimental basis for the combination of the two drugs to improve antitumor activity and reduce toxic side effects.Methods:1.Preparation and characterization of two kinds of Doxorubicin-Dihydroartemisinin conjugated prodrugs.Doxorubicin and dihydroartemisinin were used as raw materials for the synthesis of conjugated prodrugs with disulfide bonds as linking arms（DOX-SS-DHA）.Artesunate and doxorubicin were used as parent drugs for the synthesis of conjugated prodrugs with succinic anhydride as the linker arm（DOX-DHA）.HR-MS,¹H-NMR,¹³C-NMR and FT-IR were used to identify and characterize the structures.2.Preparation and prescription optimization of self-assembled nanoparticles of two kinds of doxorubicin-dihydroartemisinin conjugated prodrugs.In vitro analysis of DOX-SS-DHA and DOX-DHA was established by UV-vis spectrophotometry.In vitro analysis of DOX was established by HPLC.Prodrug self-assembled nanoparticles were prepared by nanoprecipitation method.Single-factor optimization was used to screen out the superior prescriptions.The encapsulation rate（EE）and drug loading capacity（DL）of the nanoparticles were determined by low-speed centrifugation.The morphology of nanoparticle appearance was observed by electron scanning transmission electron microscopy（TEM）.The long-term stability of the nanoparticles was investigated using particle size and PDI as evaluation indexes.The in vitro release characteristics were investigated by using the dialysis bag method.3.Evaluation of in vitro antitumor activity of self-assembled nanoparticles of two kinds of doxorubicin-dihydroartemisinin conjugated prodrugs.Mouse breast cancer cells（4T1）,human breast cancer cells（MCF-7）and rat cardiac cells（H9C2）were used as model cells to investigate the proliferation inhibitory effects of DOX-SS-DHA NPs and DOX-DHA NPs on the three cells.The intracellular uptake and uptake mechanism of DOX-SS-DHA NPs and DOX-DHA NPs were analyzed by using High Content Imaging Analysis System（Molecular Devices）.4.In vivo distribution test and antitumor efficacy evaluation of self-assembled nanoparticles of two kinds of doxorubicin-dihydroartemisinin conjugated prodrugs.4T1 tumor-bearing BALB/c mice model was established.The in vivo anti-tumor effect study was used to evaluate the anti-tumor activities of DOX-SS-DHA NPs and DOX-DHA NPs.Saline,doxorubicin solution（DOX-sol）,doxorubicin and dihydroartemisinin physical mixture solution（MIX-sol）,DOX-SS-DHA NPs and DOX-DHA NPs were administered via tail vein every three days for five times.The tumor volume changes,body weight changes,survival rate,tumor weight of the mice were observed and tumor load was calculated.In addition,H&E staining histopathological results of mice to evaluate the in vivo antitumor activity of DOX-SS-DHA NPs and DOX-DHA NPs.Results:1.Preparation and characterization of two kinds of doxorubicin-dihydroartemisinin conjugated prodrugs.Both conjugated prodrugs were successfully synthesized by HR-MS,¹H-NMR,¹³C-NMR and FT-IR.DOX-SS-DHA(C₄₆H₅₉NO₁₈S₂)was synthesized by a two-step method.The m/z of DOX-SS-DHA by HR-MS was 1024.30658,which is[M+Na]⁺peak.The yield was 60.5%.DOX-DHA(C₄₆H₅₅NO₁₈)was synthesized by a one-step method.The m/z 932.33114 was the[M+Na]⁺peak.The yield was 58.0%.2.Preparation and prescription optimization of self-assembled nanoparticles of two kinds of doxorubicin-dihydroartemisinin conjugated prodrugs.In vitro analytical methods for DOX-SS-DHA,DOX-DHA and DOX were developed.The optimal prescriptions of DOX-SS-DHA NPs and DOX-DHA NPs were screened by single-factor optimization method.The prepared prodrugs self-assembled nanoparticle dispersions were all red in appearance and the particle size was less than 200 nm.Specifically,the particle size,PDI,zeta potential,EE and DL results of DOX-SS-DHA NPs were（139.00±2.68）nm,0.10±0.01,（-13.30±0.87）m V,（75.03±0.14）%and（52.25±0.18）%,respectively.The results of particle size,PDI,zeta potential,EE and DL of DOX-DHA NPs were（121.50±3.57）nm,0.14±0.02 and（-7.05±0.45）m V,（79.14±0.21）%and（67.42±0.20）%,respectively.Both DOX-SS-DHA NPs and DOX-DHA NPs at 4℃storage for 20 d was stability.DOX-SS-DHA NPs released DHA and DOX-SH in release media containing 1 m M and 10 m M GSH.The cumulative release of DHA at 48 h reached（72.32±1.01）%and（93.73±1.49）%,respectively.In addition,the cumulative release of DOX-SH at 48 h reached（75.79±0.12）%and（89.73±2.29）%,respectively.Compared to DOX-SS-DHA NPs,DOX-DHA NPs showed no reduction response in the presence of GSH.Their release of DOX and DHA was less in the release medium of 30%ethanol aqueous solution.The released cumulative percentages of DOX and DHA at 48 h reached（34.16±0.95）%and（36.03±2.28）%,respectively.3.Evaluation of in vitro antitumor activity of self-assembled nanoparticles of two kinds of doxorubicin-dihydroartemisinin conjugated prodrugs.The MTT results showed a dose-dependent inhibition of cell proliferation by DOX-sol,MIX-sol,DOX-SS-DHA NPs and DOX-DHA NPs.The IC₅₀values of DOX-SS-DHA NPs at 24 h were 4.58±1.58,10.08±0.88 and 11.61±0.93μmol/L in 4T1 cells,MCF-7 cells and H9C2 cells,respectively.The IC₅₀values of DOX-DHA NPs were 8.37±2.86,6.65±1.54 and 9.25±1.00μmol/L in 4T1 cells,MCF-7 cells and H9C2 cells,respectively.The proliferation inhibitory effects of DOX-SS-DHA NPs and DOX-DHA NPs were smaller than those of DOX-sol and MIX-sol.The cellular uptake assay indicated that DOX-SS-DHA NPs and DOX-DHA NPs were mainly distributed in the cytoplasm of cells.Cellular uptake mechanism assays demonstrated that both DOX-SS-DHA NPs and DOX-DHA NPs entered MCF-7 cells via the caveolae-mediated endocytic pathway.And they entered 4T1cells via caveolae-and clathrin-mediated pathway.In addition,the results suggested that the entry of DOX-SS-DHA NPs into H9C2 cells was mainly mediated by caveolae-and clathrin-mediated pathway,while DOX-DHA NPs were mainly mediated by clathrin-mediated.4.In vivo antitumor efficacy evaluation of self-assembled nanoparticles of two kinds of doxorubicin-dihydroartemisinin conjugated prodrugs.The results of in vivo antitumor test showed that,on day 21,the relative tumor volume growth multiple（measured volume/initial tumor volume,V/V₀）was observed in the following order:Saline（11.58±3.13 times）>DOX-sol（5.47±0.83 times）>MIX-sol（5.24±0.58 times）>DOX-DHA NPs（4.99±0.43 times）>DOX-SS-DHA NPs（4.7±0.14 times）.The body weight of mice in DOX-sol and MIX-sol groups decreased significantly,while the body weight of mice in Saline,DOX-SS-DHA NPs and DOX-DHA NPs groups basically did not change.However,the tumor load value of Saline group was the highest.Therefore,we believe that DOX-SS-DHA NPs and DOX-DHA NPs have better anti-tumor effects.Conclusion:1.Preparation and characterization of two kinds of doxorubicin-dihydroartemisinin conjugated prodrugs.The successful synthesis of doxorubicin-dihydroartemisinin conjugated prodrugs（DOX-SS-DHA and DOX-DHA）was confirmed by HR-MS,¹H-NMR,¹³C-NMR and FT-IR spectroscopy.2.Preparation and prescription optimization of self-assembled nanoparticles of two kinds of doxorubicin-dihydroartemisinin conjugated prodrugs.Prodrug self-assembled nanoparticles were prepared by nanoprecipitation method to evaluate their physicochemical properties and to investigate in vitro release behavior and preliminary stability.DOX-SS-DHA NPs and DOX-DHA NPs were spherical in appearance,with uniform particle size distribution,high encapsulation efficiency and drug loading capacity.The particle size remained stable for 20 d at 4℃.The in vitro release results showed that DOX-SS-DHA NPs were reduction-responsive and released DOX-SH and DHA faster and more at high concentrations of GSH,while DOX-DHA NPs did not have GSH-responsive performance but had certain slow-release properties.3.Evaluation of in vitro antitumor activity of self-assembled nanoparticles of two kinds of doxorubicin-dihydroartemisinin conjugated prodrugs.DOX-SS-DHA NPs and DOX-DHA NPs enter 4T1 cells,MCF-7 cells and H9C2cells by endocytosis to release the drug,and for a certain period of time,DOX-SS-DHA NPs and DOX-DHA NPs are less toxic to cells than DOX-sol and MIX-sol.4.In vivo antitumor efficacy evaluation of self-assembled nanoparticles of two kinds of doxorubicin-dihydroartemisinin conjugated prodrugs.In vivo pharmacodynamic tests,the anti-tumor ability of DOX-SS-DHA NPs and DOX-DHA NPs was slightly higher than that of DOX-sol group and MIX-sol group.And DOX-SS-DHA NPs and DOX-DHA NPs had no significant effect on the body weight of mice and greatly improved the survival quality of mice.