Experimental Study On Photodynamic Removal Of Enterococcus Faecalis And Its Biofilm By Novel Upconversion Nanoparticles

Objective:A quaternized chitosan（QCh）-modified upconversion nanoparticles（UCNP）was prepared and coated with a silica（SiO₂）layer and a photosensitizer methylene blue（MB）.The small particle size,excellent water solubility and stable optical properties could be used as a new root canal irrigant for the photodynamic treatment of refractory periapical inflammation.The physicochemical properties of the nanoparticles were characterised to verify their effectiveness in the removal of Enterococcus faecalis（E.faecalis）and its biofilm,and the biocompatibility was tested.Methods:In our study,UCNP was synthesized by seed growth method,loaded with a thin layer of silica（SiO₂）and a photosensitizer methylene blue（MB）,and finally modified by electrostatic adsorption on quaternized chitosan（UCNP@SiO₂/MB@QCh）.UCNP@SiO₂/MB@QCh was detected using transmission electron microscopy（Transmission electron microscope,TEM）,dynamic light scattering（DLS）,zeta potential detection,X-ray diffraction detection（XRD）,fourier transform infrared spectrometer（FTIR）,test fluorescence instrument and other instruments.Material morphology,particle size,surface positive and negative charges,lattice structure,chemical composition,upconversion luminescene（UCL）.The biocompatibility of the nanoparticle was tested by CCK-8 method.The antibacterial effect of UCNP@SiO₂/MB@QCh on free E.faecalis was studied by plate count method（CFU）,and finally its antibacterial ability was tested again by in vitro anti-E.faecalis biofilm test.Results:（1）Through TEM test,it was found that pure UCNP was in the form of a hexagonal phase.It presented a double-layer core-shell structure,the outer layer was light gray,and the surface was smooth.After combining with QCh,a three-layer core-shell structure was formed,and the outermost layer of QCh was not smooth.XRD confirmed that UCNP@SiO₂/MB@QCh still showed a hexagonal phase structure even after wrapping the silicon layer and QCh,ensuring that its optical properties could not change.FTIR confirmed that UCNP was successfully encapsulated in the silicon layer and QCh,and MB was encapsulated in the silicon layer,which could not be detected by this method.The constituent elements of UCNP@SiO₂/MB@QCh were detected by EDS,which confirmed the successful modification of SiO₂ and QCh as well.It was confirmed by UCL detection that UCNP had a strong emission peak at 650 nm.After modification of SiO₂ and MB,because the absorption peak of MB was around 650 nm,fluorescence resonance energy transfer（FRET）occurs between the two above,and the fluorescence intensity decreases,which could excite MB to generate ROS.After modification of QCh,the fluorescence intensity of UCNP@SiO₂/MB@QCh further decreased at 650 nm,confirming that QCh could promote FRET and enhanced the ability of MB to produce ROS.In vitro ROS detection confirmed that UCNP@SiO₂/MB could generate ROS,and the efficiency was effectively improved after loading QCh.（2）The biocompatibility of UCNP@SiO2/MB@QCh was tested by CCK-8 method,which confirmed that the material has excellent biocompatibility.（3）By intermittently irradiating free E.faecalis with pure near-infrared light（NIR）,it was confirmed that when the light source was irradiated for 10 and 20 minutes,NIR could not have an antibacterial effect on E.faecalis,excluding the photothermal effect of the light source.（4）E.faecalis and UCNP@SiO2/MB@QCh were co-cultured and then irradiated by NIR,which confirmed that 10 minutes was an appropriate light time.（5）The antibacterial experiments were carried out on the free bacteria and biofilms of E.faecalis,and it was proved that UCNP@SiO2/MB@QCh may produce excellent antibacterial properties at lower concentrations through the dual antibacterial effects of QCh and ROS.Conclusions:UCNP@SiO2/MB@QCh nanoparticles could be prepared by seed growth method and electrostatic adsorption method.The addition of QCh made UCNP@SiO2/MB@QCh exhibit a three-layer core-shell structure,which could make UCNP@SiO2/MB@QCh exhibit a positive charge.In addition,it may effectively remove E.faecalis free bacteria and biofilm,and have excellent biocompatibility.Therefore,the UCNP@SiO2/MB@QCh prepared in this study has great potential for the treatment of refractory apical periodontitis.