Effects Of Ginsenoside Rg3-Loaded Carbon Nanozyme On The Antitumor Activity Of Gemcitabine

BackgroundCancer is currently one of the main diseases threatening human life and health in the world.Although many substantial progresses have been made in the treatment of cancer,it is still difficult to cure.One of the most important reasons is that patients have developed resistance to chemotherapy.According to the estimate by the American Cancer Society,more than 90%of cancer patients die from varying degrees of multidrug resistance.So far,domestic and foreign researchers have not found effective strategies to overcome tumor resistance.Therefore,how to reduce tumor multidrug resistance has become one of the research hotspots in tumor treatment.Recent studies have shown that there are many bacteria in human tumors,and the effect of bacteria on chemotherapeutic drugs is often one of the reasons for tumor resistance.As a new cytosine nucleoside derivative,gemcitabine is clinically active against a variety of solid tumors,But gemcitabine(2’,2’-difluorodeoxycytidine)can be metabolized by bacteria to its inactive form of 2’,2’-difluorodeoxyuridine(dFdU)through secreting cytidine deaminase(CDD).Therefore,finding an effective cytidine deaminase inhibitor will be one of the effective ways to improve gemcitabine resistance.Nanozyme is a kind of mimetic enzyme which not only has the unique properties of nanomaterials,but also has the catalytic function.Researchers have discovered that in addition to the biocatalytic function that mimics natural enzymes,nanozyme can also selectively bind to different parts of the enzyme,acting as an inhibitor to affect the activity of enzymes.Compared with small molecules,they have a large surface and variable function,which can bind and change enzyme activity very effectively.Because this inhibition occurs mainly depends on the electrostatic interaction,hydrophobic interaction and space blocking.The surface of carbon-based nanozymes is usually hydrophobic,and the hydrophobic interaction between carbon spheres and cytidine deaminase is expected to effectively inhibit the activity of cytidine deaminase.In addition,in the early research work of our research group,it was found that carbon nanozymes have multienzyme-like activities.In the acidic microenvironment of tumors,they can catalyze endogenous H2O2 or O2 with production of a large amount of ROS to kill tumors.Taken together,CNzymes played a dual role in tumor therapy.On the one hand,CNzymes acted as a catalysis with the capability of converting H2O2 into·OH;on the other hand,CNzymes as an enzyme inhibitor was able to inhibit the bacteria to metabolize Gem into dFdu.It is worth emphasizing that the large specific surface area and controllable pore size distribution of CNzymes provide the possibility of efficiently loading small molecule drugs.Compared with traditional drug carriers such as phospholipids or macromolecular polymers,carbon nanozymes as drug carriers can not only deliver drugs to target sites to play pharmacological effects,but also produce ROS through their own enzyme catalytic activity to achieve synergistic therapeutic effects.Therefore,we introduced ginsenoside Rg3 into the carbon nanozyme system to construct a drug-loaded carbon nanozyme(Rg3/CNzymes).Ginsenoside Rg3 is an effective chemical trace component extracted from ginseng.Due to its ability to inhibit tumor growth and angiogenesis and the ability to induce apoptosis in various tumors,it has been proven to have good antitumor effects.In addition,ginsenoside Rg3 also has a good immunomodulatory effect,which can increase the body’s immunity.Therefore,ginsenoside Rg3 can be efficiently loaded into CNzymes,which can not only exert the tumor catalytic therapy of CNzymes,but also realize tumor chemotherapy by releasing Rg3.In addition,as a cytidine deaminase inhibitor,CNzymes can reduce the metabolism of gemcitabine by bacteria in tumors.The effective integration of the above three therapeutic strategies is expected to improve the actual therapeutic effect of tumor.Based on the above research background,in this paper,CNzymes was prepared by direct carbonization of polyaniline-polypyrrole(PACP)hollow spheres,and its morphology,particle size,structure,multienzyme activity and antitumor ability were evaluated in detail.Subsequently,the ability of CNzymes as a cytidine deaminase inhibitor was studied.On the one hand,cytidine deaminase was directly selected to explore the effect of cytidine deaminase on the metabolism of gemcitabine in the presence of CNzymes.On the other hand,we chose E.coli as an example,which typically expresses cytidine deaminase activity,to discuss the influence of E.coli on the metabolism of gemcitabine in the presence of CNzymes.The inhibition mechanism and in vivo efficacy were evaluated.Taken together,the ginsenoside Rg3 loaded CNzymes was a good antitumor adjuvant of gemcitabine,with the synergistic antitumor effects.Purpose(1)Carbon nanomaterials have been confirmed to have multienzyme-like activities.The paper uses a one-step method to prepare carbon nanozyme and explores the application of its enzyme catalytic function in antitumor.(2)Nanomaterials play an effective role as enzyme inhibitors through the steric blockage of active sites and multivalent interactions.In this paper,the interaction between CNzymes and cytidine deaminase was explored to investigate the effect of CNzymes as enzyme inhibitors on the antitumor activity of gecitabine in vivo and in vitro.This exploration further expand the application of nanozymes in the biomedical field.(3)Utilizing the drug-loading capacity of CNzymes,the antitumor drug ginsenoside Rg3 was efficiently loaded into CNzymes.The inhibitory effect of Rg3/CNzymes in combination with gemcitabine on tumor growth in vivo and in vitro was investigated,which laid an experimental foundation for the development of multifunctional nanozymes.MethodsChapter 1:Preparation and antitumor activity of CNzymesIn this chapter,a simple template-free strategy was used to prepare CNzymes.First,the morphology and size of CNzymes were characterized by transmission electron microscopy(TEM)and scanning electron microscopy(SEM).Subsequently,the structure and composition of the material were analyzed in detail through X-ray diffraction spectroscopy(XRD)and X-ray photoelectron spectroscopy(XPS),and then the ability of CNzymes to mimic the peroxidase and consume glutathione was investigated.Meanwhlie,electron paramagnetic resonance(ESR)and ROS fluorescent probe(DCFH-DA)were used to detect the free radicals catalyzed by CNzymes.Finally,the in vitro anti-tumor effect and biocompatibility of CNzymes were evaluated.Chapter 2:CNzymes as a cytidine deaminse inhibitor against the metabolism of gemcitabine for tumor therapyIn this chapter,CNzymes as a enzyme inhibitors were evaluated.Three different reaction systems were designed,namely Gem,Gem+CDD and Gem+CDD+CNzymes.After co-incubation under the same conditions,the filtrate was taken out.On the one hand,the antitumor activity of Gem in filtrate of each group was evaluated by MTT and other methods.On the other hand,using HPLC/MS to quantitatively detect the concentrations of Gem and its metabolites dFdU in filtrate of each group.Then,the CDD metabolism of Gem and the effect of CNzymes on the recovery of the Gem were systematically evaluated.In addition,E.coli expressing CDD was used to replace CDD to repeat the above experiment to further improve the reliability of the experiment.The mice subcutaneous tumor and in situ tumor(tumor containing bacteria)models were established to evaluate the inhibition effect of tumor growth with different treatments.Finally,the biocompatibility of CNzymes was evaluated by H&E staining of main organs in mice.Chapter 3:The combination of Rg3/CNzymes and Gem for tumor chemo/catalytic therapyIn this chapter,the antitumor drug ginsenoside Rg3 was loaded into CNzymes to construct a drug-loaded carbon nanozyme by using the larger specific surface area of carbon materials.Firstly,Rg3/CNzymes were characterized by Fourier transform infrared spectroscopy.and the drug loading rate of the drug-loaded carbon nanozyme was measured.Then,MTT,cell death staining,and mitochondrial membrane potential were used to evaluate the inhibitory effect of Rg3/CNzymes on tumors.Finally,a mouse subcutaneous tumor model(with bacteria inside the tumor)was constructed to explore the antitumor effect of the Rg3/CNzymes in combination with gemcitabine.ResultChapter 1:The CNzymes prepared in this chapter exhibited a well defined nano spherical morphology,with an average particle size of 100 um.Through XPS,XRD and Raman analysis,it was found that the carbon nanozyme was composed of three elements:C,N,and O,showing a good degree of graphitization.Enzymatic analysis showed that CNzymes had a pH-dependent peroxidase-like activity.In addition,CNzymes had a good ability to scavenging glutathione,which could not only catalyze the production of ROS to kill tumor cells,but also avoid the scavenging effect of glutathione on free radicals in tumor sites,exerting the anti-tumor ability.MTT assay,intracellular ROS production and intracellular uptake of CNzymes proved that CNzymes could enter into cells to catalyze ROS production and thus played a better antitumor effect.At the same time,in the biological safety evaluation,various biochemical indicators and H&E section staining results showed that CNzymes had no obvious damage to the biochemical function and main organs of mice,and had high biological safety.Chapter 2:In this chapter,CNzymes was used as a cytidine deaminase(CDD)inhibitor to assist gemcitabine in antitumor therapy.Three different reaction systems,namely Gem,Gem+CDD and Gem+CDD+CNzymes were set up.After co-incubation under the same conditions,the filtrate was taken out.First,the antitumor activity of Gem in each group of filtrate was evaluated by qualitative methods,and the results showed that CNzymes could inhibit the metabolism of Gem by CDD to maintain high antitumor activity;secondly,HPLC/MS was used to quantitatively detect the contents of Gem and its metabolite dFdU in each group of filtrates.The experimental results showed that the presence of CNzymes increased the content of Gem,indicating that CNzymes effectively inhibited the activity of CDD to metabolize gemcitabine.In addition,the CDD-expressing E.coli was used to replace CDD to repeat the above experiments.The similar experimental results were obtained.However,CNzymes with PEG modification had no inhibitory activity towards the CDD to metabolize Gem into dFdU.The possible mechanism was that CNzymes blocked the binding site of CDD and Gem through hydrophobic interaction.Finally,through establishment of subcutaneous tumors and in situ tumor models,we found that CNzymes could first efficiently catalyze the limited H2O2 in the tumor microenvironment to produce suficient cytotoxic·OH for tumor treatment.Second,we discovered the CNzymes could act as an enzyme inhibitor to inhibit the bacteria in tumor sites to metabolize Gem into its inactive form dFdU,abolishing the Gem-metabolizing activity.Our observation that antitumor drug responses can be potentiated by coadministration of CNzymes suggests that such combinations merit additional exploration in the preclinical and clinical setting.Chapter 3:In this chapter,CNzymes was used as a drug carrier,and the advantages of its larger specific surface were fully exploited to efficiently load ginsenoside Rg3 to obtain the drug-loading carbon nanozymes(Rg3/CNzymes).FTIR results showed that ginsenoside Rg3 was successfully loaded into CNzymes,with a drug loading efficiency of 28.95%.Subsequently,we evaluated the in vitro antitumor effect of Rg3/CNzymes in combination with gemcitabine.The results of MTT,mitochondrial membrane potential,and apoptotic protein expression showed that compared with the CNzymes,Rg3/CNzymes induced more tumor apoptosis.With further combination of gemcitabine,Rg3/CNzymes exhibited enhanced antitumor activity.Finally,a mouse subcutaneous tumor model(with bacteria inside the tumor)was also established for evaluating the combined therapeutic effects of Rg3/CNzymes and gemcitabine.Compared with the results in the previous chapter,it was found that the combination of Rg3/CNzymes and gemcitabine was a safe and more effective antitumor strategy.In the biosafety evaluation,H&E staining results also showed that the combination of gemcitabine and Rg3/CNzymes had no obvious damage to mouse organs,indicating no significant toxic and side effects on mice with different treatments.