Cancer Targeting And Synergistic Chemotherapy Of Self-assembly Of Hyaluronic Hyaluronic Acid-Cu(Ⅱ)-Quercetin Coordination Polymer Nanoparticles

Part I Preparation and Characterization of Hyaluronic Acid-Cu(II)-Quercetin Coordination Polymer NanoparticlesObjective:To prepare and characterize Hyaluronic Acid-Cu(II)-Quercetin coordination polymer nanoparticles.Methods:Hyaluronic Acid-Cu(II)-Quercetin coordination polymer Nanoparticles(HA-Cu-Qu NPs)were prepared by self-assembly method,and the morphology was observed by TEM.Then its particle size,Zeta potential,copper ion valence and content,general properties of nanoparticle composition,drug loading,and stability at room temperature were detected.Results:The prepared Hyaluronic Acid-Cu(II)-Quercetin coordination polymer nanoparticles of similar morphology had a uniform particle size distribution with an average value of(78.62 ± 1.60)nm,and the Zeta potential with(-7.29 ± 0.73)MV.XPS results showed that the copper ion valence is +2 and +1 valence.133 μg of copper ions permilliliter of nanoparticles was detected by ICP-MS.FT-IR results showed that quercetin and hyaluronic acid were contained in the structure of the nanoparticles.The total drug loading of quercetin and copper ions was 9%.DLS results showed that HA-Cu-Qu NPs can be stable at room temperature for more than 7 days.Conclusion:Hyaluronic Acid-Cu(II)-Quercetin coordination polymer nanoparticles were successfully prepared in this experiment,and the results of various characterization experiments showed that the nanoparticles have uniform particle size,high drug loading,and good room temperature stability.All of the abrove characters suggested that it can be used as basis for drug research in vivo and in vitro.Part II Preliminary experimental Study on the Antitumor Effect of Hyaluronic Acid-Cu(II)-Quercetin Copolymer Nanoparticles on Breast CancerObjective: To investigate the ability of HA-Cu-Qu NPs to producing ROS inside and outside cells,the process of HA-Cu-Qu NPs cellular uptaking,the synergistic inhibitory effect and biocompatibility on breast cancer MDA-MB-231 cells.Methods: Outside the cell,the ability of HA-Cu-Qu NPs to react with different concentrations of hydrogen peroxide(H2O2)to generate hydroxyl radicals(· OH)was measured by MB ultraviolet absorption method;and inside the cell,the ability of HA-Cu-Qu NPs to react with 50μM H2O2(the concentration of H2O2 in the tumor microenvironment)to generate ROS was qualitatively detected by fluorescence intensity of DCFH-DA.Flow cytometry was used to detect the uptake of HA-Cu-Qu NPs at different time points;the concentration of H2O2 in the tumor microenvironment(50μM)was simulated,then the CCK-8 methods was used to evaluate the inhibitory effects of different concentrations of HA-Cu-Qu NPs and HA-Cu-Qu NPs + H2O2 on breast cancer MDA-MB-231 cells,moreover to analyze their synergistic antitumor effect and dose-effect relationship;the CCK-8 method was used to evaluate the toxic effect of different concentrations of HA-Cu-Qu NPs on normal liver cells LO2,depending this tests to analyze their biocompatibility.Results: Outside the cell,HA-Cu-Qu NPs could react with H2O2 to form Strongly oxidative ·OH through Fenton-like reaction,and the amount of ·OH produced more with the increasing of H2O2 concentration,which indicating that HA-Cu-Qu NPs had a good ability to generate ROS.Under the simulated tumor microenvironment,HA-Cu-Qu NPs also had the ability to convert H2O2 into strongly oxidative ·OH.With the time going on,HA-Cu-Qu NPs accumulated in MDA-MB-231 cells and reached the maximum accumulation concentration in about 0.5 hours.In the tumor microenvironment(+H2O2),HA-Cu-Qu NPs had a significant synergistic inhibitory effect,and with concentration increasing,the inhibitory effect was stronger.The results also showed that it has a significant dose-effect relationship;HA-Cu-Qu NPs at different concentrations had no obvious toxicity to LO2 cells,indicating that it had good biocompatibility.Conclusion: Hyaluronic Acid-Cu(II)-Quercetin coordination polymer nanoparticles could efficiently generate ROS through Fenton-like reaction intracellular or extracellular,which provided evidences for CDT.Moreover it had significant synergistic inhibitory effect on breast cancer MDA-MB-231 cells.In addition,the nanoparticles also had good biocompatibility.