Antibacterial,Antioxidant And Anti-inflammatory Effects Of Minocycline-loaded Seif-assembled Nanoparticles

Background and ObjectiveSevere periodontitis is not only capable of causing tooth loss and impairing oral function,but is also an important risk factor for many systemic diseases such as cardiovascular diseases and diabetes.Currently,for severe periodontitis,it is difficult to obtain the desired effect by mechanical treatment alone,and topical medication is often required.Topical administration is often used to control periodontal infections by delivering antimicrobial agents to the base of the periodontal pockets at antibacterial or bactericidal concentrations through special dosage forms,thus reducing systemic side effects and obtaining better patient compliance.Minocycline(MC),a broad-spectrum antibiotic,is commonly used in clinical practice for the treatment of periodontitis under the trade name of Periocline.Periocline can release MC locally in the periodontal pocket to exert antibacterial effects.However,the current delivery system has limitations,including difficulties in maintaining effective concentrations in the periodontal pockets due to rapid diffusion and bacterial resistance due to overdose.In addition,periodontal tissues remain inflamed after basic treatment,which can continue to cause tissue damage.Although MC has certain antioxidant and anti-inflammatory ability,the regulatory efficiency is low.Therefore,it is important to construct a new system of loading MC with low dosage,high antibacterial efficiency and synergistic antioxidant and anti-inflammatory functions to optimize the application of MC in periodontal treatment.Based on metal coordination effect,a double-liganded self-assembled nanosystem of MC,tannic acid(TA)and Zn2+,namely Zn2+-tannic acid-minocycline nanoparticles(Zn-TA-MC NPs,ZTM NPs),was constructed in this project by a one-step method.During the construction of the self-assembled system,on the one hand,MC as a ligand molecule directly participates in the formation of the nanosystem,which improves the yield and stability of the material itself;on the other hand,the ligand effect greatly increases the drug loading.Meanwhile,due to the restriction of coordination bond and supramolecular network,this nanoparticle is able to release the active ingredient stably and realize the long-term antibacterial effect of the drug.Based on this,this project explores the antibacterial antioxidant and anti-inflammatory properties of this self-assembled nanosystem,and lays the foundation for the subsequent treatment of periodontitis.Material and MethodPartⅠSynthesis optimization and physicochemical characterization of ZTM NPs.The ZTM NPs with different drug loadings were constructed by adjusting the MC content in the reaction system,and the optimal synthesis ratio was screened.The physical properties of the ZTM NPs were measured by scanning electron microscope(SEM)to observe the morphology of the prepared ZTM NPs;the size,dispersion and zeta potential of the ZTM NPs were characterized by particle size potentiometry;fourier transform infrared spectroscopy(FTIR)was used to observe the characteristic peaks of functional groups;X-ray diffractometer(XRD)(40 kV,200 mA)was used to obtain the crystalline phases of the nanopowders;X-ray photoelectron spectroscopy(XPS)and energy dispersive X-ray spectroscopy(EDS)were used to analyze the characteristic elemental species,the distribution of each element and the distribution of each element in the NPs.Part Ⅱ1.Cytocompatibility of ZTM NPs.Human periodontal ligament cells(PDLCs)were cultured in α-MEM medium containing ZTM NPs at different concentrations(0,5,10,25,50,100,150,200 μg/mL).After 24 and 48 hours,cell status was characterized by live-dead cell staining and cell activity was quantified by cell counting kit 8(CCK-8).2.In vitro antibacterial effect of ZTM NPs was investigated.ZTM NPs at different concentrations(0,2.5,5,10,25,50 μg/mL)were co-cultured with Gram-positive Staphylococcus aureus(S.aureus)and Gram-negative Porphyromonas gingivalis(P.gingivalis),and the effect of different concentrations on the growth of colonies was examined by the plate coating method,and the effective antibacterial concentration was selected for the subsequent experiments;at this concentration,the control(Ctrl),MC and ZTM NPs groups were set up,Ctrl group contained only bacterial solution,MC group added bacterial solution and equivalent concentration of MC,and ZTM NPs group added bacterial solution and ZTM NPs.The effect of ZTM NPs on the growth of S.aureus and P.gingivalis colonies was further examined by using the plate coating method;the effect of ZTM NPs on the morphology of bacteria was observed under SEM.3.The antioxidant effect of ZTM NPs.PDLCs were infected with P.gingivalis with a multiplicity of infection(MOI)of 100 to establish an in vitro inflammation model.First,PDLCs were treated with different concentrations(0,5,10,25 and 50 μg/mL)of ZTM NPs for 24 hours.The optimal antioxidant concentrations were screened by quantitative real-time polymerase chain reaction(qRT-PCR)for subsequent experiments.Next,different material groups were treated with PDLCs for 12 and 24 hours.Groupings were set:Ctrl group(containing PDLCs only),P.gingivalis group(PDLCs+P.gingivalis),MC group(PDLCs+P.gingivalis+equivalent concentration of MC)and ZTM NPs group(PDLCs+P.gingivalis+5μg/mL ZTM NPs).The expression levels of messenger ribonucleic acid(mRNA),factors related to oxidative stress,was measured by qRT-PCR;the expression levels of related antioxidant factors were measured by western blot(WB);and the production of intracellular superoxide radicals and peroxides were measured by reactive oxygen species(ROS)assay kit.4.The anti-inflammatory effect of ZTM NPs on PDLCs.PDLCs were infected with P.gingivalis with MOI of 100 to establish an in vitro inflammation model.First,PDLCs were treated with different concentrations(0,5,10,25 and 50 μg/mL)of ZTM NPs for 24 hours.The optimal anti-inflammatory concentrations were screened by qRT-PCR and nitric oxide(NO)secretion assay for subsequent experiments.Next,different material groups were treated with PDLCs for 12 and 24 hours.The settings were grouped as above.The mRNA levels of relevant pro-inflammatory and anti-inflammatory factors were detected by qRT-PCR;the protein levels were detected by WB and enzyme linked immunosorbent assay(ELISA);the expression of intracellular inflammatory factor-inducible nitric oxide synthase(iNOS)was detected using cellular immunofluorescence staining,and the level of NO produced by the cells was also measured.ResultsPart ⅠThe optimal group of ZTM NPs was successfully prepared,screened and characterized.During the preparation process,it was found that the parameters of material stability and drug loading were optimized when the mass ratio of Zn,TA and MC were 1:2:2(ZTM-2),and the physical properties of different groups of ZTM NPs were similar.The FTIR of the NPs showed the C=O and C=C characteristic peaks of tannic acid,which confirmed the successful coordination of tannic acid;no crystalline peaks were observed in the XRD spectra,indicating that the ZTM NPs were amorphous;XPS elemental analysis and EDS spectroscopy confirmed the presence of characteristic elements of nitrogen(from MC)and zinc in the NPs and the uniform distribution of various elements in the material.Part Ⅱ1.ZTM NPs have good cytocompatibility.PDLCs were in good activity status in αMEM medium containing different concentrations of ZTM NPs.CCK-8 and live-dead cell staining results showed that low concentrations(5-50 μg/mL)of ZTM NPs promoted the proliferation of PDLCs,while the proliferation of PDLCs was slightly inhibited at concentrations as high as 200 μg/mL.2.ZTM NPs were significantly antibacterial in vitro.According to the colony growth,the ZTM NPs concentration up to 5 μg/mL had a significant antibacterial effect on Grampositive S.aureus and Gram-negative P.gingivalis;meanwhile,the smear results showed that the ZTM NPs group inhibited the growth of S.aureus and P.gingivalis colonies better than the MC group,and SEM observation showed that the ZTM NPs group had a greater effect on the bacterial morphology destruction of S.aureus and P.gingivalis.3.ZTM NPs were significantly antioxidant in vitro.After 24 hours of P.gingivalis induction in vitro inflammatory environment and treatment with different concentrations of ZTM NPs,the results showed that the mRNA levels of the antioxidant factors superoxide dismutase-1(SOD-1)and catalase(CAT)increased to different degrees.The differences between ZTM NPs groups at different concentrations(5,10,25 and 50 μg/mL)were not significant,and the optimal antioxidant effect was achieved at the concentration of 5 μg/mL.The ZTM NPs concentration of 5 μg/mL was selected for the next experiment,after 12 and 24 hours,qRT-PCR results showed that ZTM NPs promoted the expression of antioxidant factors SOD-1 and CAT mRNA levels which was down-regulated by P.gingivalis at different time points;WB results showed that ZTM NPs also significantly increased the expression of antioxidant factors SOD-1 protein levels inhibited by P.gingivalis;ROS detection results showed that the intracellular fluorescence intensity was the weakest in the ZTM NPs group,which proved that the intracellular production of superoxide radicals and peroxides were the least.The above results were consistent with each other,and although the MC group had a similar trend of action,the effect was not as good as that of the ZTM NPs group,which proved that the antioxidant effect of the ZTM NPs group was better than that of the MC group.4.ZTM NPs were significantly anti-inflammatory in vitro.After 24 hours of P.gingivalis induction in vitro inflammatory environment,the expression of pro-inflammatory factor iNOS and interleukin-8(IL-8)mRNA levels were reduced to different degrees after treatment with different concentrations of ZTM NPs;the expression of anti-inflammatory factor arginase-1(Arg-1)and transforming growth factor-β(TGF-β)mRNA levels increased to different degrees.The differences between the different concentrations of ZTM NPs(5,10,25,50 μg/mL)were not significant,and the optimal anti-inflammatory effect was achieved at a concentration of 5 μg/mL.Meanwhile,the degree of inhibition of NO production in PDLCs by different concentrations of ZTM NPs tended to be consistent,and no significant differences were observed between groups,which was consistent with the qRT-PCR results.We subsequently selected a concentration of 5 μg/mL of ZTM NPs for the next experiment.After 12 and 24 hours,qRT-PCR results showed that ZTM NPs inhibited the expression of P.gingivalis-stimulated pro-inflammatory factors IL-6 and IL-8 mRNA levels,while significantly increasing the expression of anti-inflammatory factors Arg-1 and TGF-β mRNA;WB and ELISA results showed that ZTM NPs decreased the expression of P.gingivalisinduced pro-inflammatory factors IL-6 and IL-8 protein levels and increased the expression of P.gingivalis-inhibited anti-inflammatory factor TGF-β protein levels;cellular immunofluorescence staining showed that the intracellular fluorescence intensity was significantly reduced in the ZTM NPs group,suggesting that the expression of iNOS was reduced,and the ZTM NPs group inhibited NO production in PDLCs to a greater extent.The MC group was less effective than the ZTM NPs group,proving that the ZTM NPs group had a better anti-inflammatory effect.ConclusionThe multifunctional ZTM NPs are successfully constructed with good biocompatibility and antibacterial activity,which can exert multiple effects of antibacterial,antioxidant and anti-inflammation;scavenge ROS,reduce cell death;inhibit the production of inflammatory factors by PDLCs and reduce inflammatory response;and inhibit NO production induced by P.gingivalis.The novel MC drug system constructed has good potential for the treatment of periodontitis and provides a useful exploration of new dosage forms for the topical administration of periodontitis.