Copper Nanoparticles With Intrinsic Protease-like Activity Inhibits Human Cervical Cancer Cells

Protease is an important industrial enzyme that can catalyze the hydrolysis of proteins and peptides,and is also involved in metabolism and biological regulation in vivo.As a biocatalyst,protease has the advantages of high efficiency,fast rate and good specificity.However,due to the complex extraction process,high price,easy deactivation of the reaction,limited reaction conditions and non-recyclability,its application is limited.Based on this,we synthesized cheap and readily available elemental metal catalyst copper nanoparticles（Cu NPs）as a protease substitute to overcome the above shortcomings,and further used in vivo catalytic reaction for tumor therapy.Previous studies in our laboratory have found that metal complexes（Cu-MOF,Cu₂O/CQDs,etc.）with Cu as active centers not only have similar protease activity,but also selectively inhibit human ovarian cancer cells.However,no individual mono-metallic catalysts of copper-based materials for the study of literature reported.Therefore,in this paper,copper nanoparticles（Cu NPs）were used for the first time to simulate protease-catalyzed hydrolysis of bovine albumin（BSA）and Casein,and on this basis,the modified folic acid-copper-polyvinylpyrrolidone（FA-Cu-PVP）were used for anti-tumor.The main contents include:（1）The copper nanoparticles（Cu NPs）prepared by ultrasonic hydrothermal method were used for the hydrolysis of bovine serum albumin（BSA）and Casein.The effects of reaction time,temperature,p H value and material concentration on hydrolysis were studied.It was found that Cu NPs could hydrolyze protein under physiological conditions,and its affinity to BSA was 165,000 times higher than trypsin,which had a wide temperature range and good stability.A series of experiments have proved that Cu NPs not only have high catalytic activity,but also can be used for many times,which is a good substitute for protease.（2）Anticancer activity of folic acid（FA）and polyvinylpyrrolidone（PVP）modified copper nanoparticles（FA-Cu-PVP）.The inhibitory effects of materials on different cells（human cervical cancer cells（He La）,human ovarian cancer cells（SKOV3）,lung cancer human alveolar basal epithelial cells（A549）,and human colon cancer cells（HCT-116）and mouse embryonic fibroblasts（BABL-3T3））were tested by MTT method,which have shown that FA-Cu-PVP not only has a high inhibitory effect on He La cells,but also has a low toxicity to BABL-3T3.（3）Study on the mechanism of FA-Cu-PVP inhibiting the proliferation of He La cells.AO/EB staining,Hoechst 33342 staining,flow cytometry,in vitro scratch test and Western Blot showed that FA-Cu-PVP could block He La cells in S phase and inhibit the migration activity of cancer cells.Moreover,FA-Cu-PVP could induce apoptosis and autophagy of He La cells and inhibit their growth and proliferation by down-regulating the expression of MMP-2/MMP-9 and anti-apoptotic factor（Bcl-2）,up-regulating the expression of pro-apoptotic factor（Bax）and autophagy-producing marker protein LC3BII.