AgCa-PLGA Submicron Particles Inhibit The Growth Of Enterococcus Faecalis And Porphyromonas Gingivalis

Object:The purpose of this study was to synthesize and characterize the PLGA submicron particles carrying both Ag⁺and Ca²⁺and investigated its ability to infiltrate into dentinal tubules using ultrasound devices,antibacterial effects against E.faecalis and P.gingivalis in both planktonic and biofilm forms and its mineralization properties in vitro.Materials and Methods:AgCa-PLGA submicron particles were synthesized using a water/oil/water（w/o/w）emulsification solvent evaporation method.The particle size distribution,Z-average particle-diameter,polydispersity-index（PDI）and Zeta-potential of particles were measured using dynamic light scattering（DLS）.Characterization and the element of particles was then assessed using field–emission scanning electron microscopy（FE-SEM）and energy dispersive spectrometry（EDS）,respectively.The encapsulation efficiency（EE）and the ion release profiles of AgCa-PLGA and Ca-PLGA particles were measured using inductively coupled plasma optical emission spectrometry（ICP-OES）.The pH changes of particles were measured every 3 days using a pH meter.The antibacterial effects of particles against planktonic E.faecalis and P.gingivalis for the 24-h and 7-day were investigated using a CFU counting method.The cell cytotoxicity of particles on MC3T3-E1 in vitro was assessed using the CCK-8method.Infiltration and antibacterial effects of particles against E.faecalis and P.gingivalis on dentin slices were observed using FE-SEM.Meanwhile,the OD value of E.faecalis within 8 h and of P.gingivalis within 48h after the direct soaking of dentin slices from different groups in fresh liquid basal media were assessed using a spectrometer.To assess the mineralization ability of the AgCa-PLGA submicron particles,30 mg of particles was compacted to a tablet and soaked in simulated body fluid（SBF）for 3 weeks at 37°C.ICP was used to quantify the Ca content.Tablets in each group were analyzed for the formation of mineral crystals on the surface using FE-SEM and EDS.Results:FE-SEM images showed that the AgCa-PLGA submicron particles were in a spherical shape with a homogenous particle size.The Z-average particle-diameter and PDI of AgCa-PLGA was 239.30±13.79 nm and 0.347±0.03,respectively.The Zeta potentials of particles were all negative.EDS analysis revealed the existence of C,O,Ag and Ca elements in AgCa-PLGA.The AgCa-PLGA could release both Ag⁺and Ca²⁺over 30 days in a sustained manner.The pH of AgCa-PLGA decreased quickly in the first 18 days and maintained at approximately 4.7.In the test on antibacterial effects against planktonic E.faecalis and P.gingivalis,PLGA and Ca-PLGA showed no antibacterial activity（P>0.05）.AgCa-PLGA showed higher antibacterial effects with the concentration of 5 mg/mL and 10 mg/mL（p<0.05）.The highest antibacterial efficiencies against E.faecalis were 43.73%and 57.51%for the 24-h and 7-day antibacterial tests,respectively.The highest antibacterial efficiencies against P.gingivalis were 99.89%and 99.58%for the 24-h and 7-day antibacterial tests,respectively.After being treated with AgCa-PLGA by ultrasonic activation,FE-SEM images showed that numerous particles were observed in tubule openings and on the axial sections of tubules.For the E.faecalis,the OD value measurement revealed that the dentin slices treated by AgCa-PLGA showed significantly less bacteria than other groups at the stage of 4 to 7 h（P<0.05）,especially at 6 h（P<0.001）.For P.gingivalis,the OD values of the AgCa-PLGA did not increase during the 48 h（P<0.01）.The FE-SEM images all confirmed the better antibacterial effects of AgCa-PLGA than other groups,and AgCa-PLGA could induce HA-like crystal formation.CCK-8 test showed no cytotoxicity on MC3T3-E1.Conclusions:From the perspective of antibacterial agent development,we successfully synthesized the AgCa-PLGA submicron particles.The AgCa-PLGA submicron particles could release both Ag⁺and Ca²⁺over 30 days in a sustained manner and exhibited strong antibacterial effects against both E.faecalis and P.gingivalis.SEM images showed that AgCa-PLGA particles could infiltrate into dentinal tubules by ultrasonic activation and inhibit the adherence and colonization of both E.faecalis and P.gingivalis on dentin.AgCa-PLGA particles showed no cytotoxicity on osteoblastic cells and induced HA-like crystal formation on its surface.AgCa-PLGA submicron particles are a potentially efficient biomaterial for both infection control and hard tissue regeneration in dentistry and other related medical areas.