| Objective:Clinically,open wounds can be caused by tooth extraction,accidental trauma,surgical nidus resection or plastic repair surgery,and the open wounds are vulnerable to oral and external bacterial infection,which hinders wound healing.Antibacterial photodynamic therapy(aPDT)has become a promising strategy for the treatment of wound infection due to its spatio-temporal selectivity,minimal invasion,low systemic toxicity and no induction of bacterial resistance.In this study,a novel photodynamic antibacterial agent(QMC nanoparticles)was synthesized using curcumin(Cur),quaternized chitosan(QCS)and mesoporous poly-dopamine(MPDA)nanoparticles.We explored the biological safety of QMC nanoparticles and evaluated their photodynamic antibacterial activity against Staphylococcus aureus(S.aureus)and Escherichia coli(E.coli)under the irradiation of 450 nm blue light(1 W/cm2)in vitro.Method:1.MPDA nanoparticles were synthesized by one-pot method.Cur was loaded into MPDA nanoparticles through π-π stacking and hydrogen bonding,i.e.,MPDA-Cur nanoparticles.Then the QCS was attached to the surface of the drug-loaded nanoparticles by electrostatic adsorption and Schiff base and/or Michael addition reaction to synthesize QMC nanoparticles.2.The dispersibility,particle size and surface morphology of the nanoparticles were observed by transmission electron microscopy(TEM).The hydrated particle size,dispersibility and Zeta potential were analyzed by the nano-particle size and Zeta potential analyzer.The loading rate and the encapsulation rate of the drug-loaded nanoparticles were counted by the ultraviolet-visible(UV-Vis)spectrophotometer,and the optimal reaction condition for drug loading was determined through screening.The chemical composition of the nanoparticles was analyzed by ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy(FT-IR).3.To screen the optimal antibacterial and biosafety concentration of QMC nanoparticles,the cytotoxicity of QMC nanoparticles with different concentrations was studied for 1 day and 3 days by cell counting kit-8(CCK-8)experiment,and the survival rates of S.aureus and E.coli at different concentrations of QMC nanoparticles were calculated by colony-forming unit count before and after light irradiation.4.The biological safety of different nanoparticles was evaluated by co-culturing MPDA,MPDA-Cur,QCS-MPDA and QMC nanoparticles(150 μg/mL)with L929 cells for 1 and 3 days using CCK-8 experiments and cell live/dead staining.5.MPDA,MPDA-Cur,QCS-MPDA,and QMC nanoparticles(150 μg/mL)were cocultured with S.aureus and E.coli in vitro.The colony-forming unit count is adopted to calculate the survival rate of bacteria before and after illumination(450 nm,1 W/cm2).The capability of resisting bacterial biofilm before and after illumination of the nanoparticles is detected through bacterial live/dead staining.The effect of nanoparticles on the metabolic activity of bacterial biofilm before and after light irradiation was evaluated by MTT experiment.And then the influence of nano-particles on bacterial morphology before and after illumination was observed by SEM.Results:1.Material characterization showed that the curcumin-loaded mesoporous polydopamine nanoparticles modified by quaternized chitosan,i.e.,QMC nanoparticles,have been successfully synthesized.TEM images showed that the QMC nanoparticles were uniformly dispersed,and the particle size reached 161.3 ± 5.22 nm.The particle size test results showed that the hydrated particle size of QMC nanoparticles was 207.0±1.03 nm,and the dispersibility was 0.04±0.007.The Zeta potential results showed that the Zeta potential of QMC nanoparticles was 31.8 ± 1.35 mV,which was higher positive potential than MPDA and MPDA-Cur nanoparticles,indicating that the QCS modification was successful.The UV-Vis spectrophotometer results showed that when the molar ratio of Cur to MPDA was 1.5:1,the encapsulation efficiency and drug loading rate reached 35.7±2.23%and 34.8±1.43%,respectively.The UV-Vis spectrum showed that both MPDA-Cur and QMC nanoparticles exhibited the same characteristic absorption peaks as the Cur spectrum at 425 nm.Finally,the FT-IR spectrum showed that the QMC nanoparticles exhibited the same characteristic absorption peaks as those of Cur and QCS,indicating that Cur was successfully loaded and QCS was successfully modified.2.With the increasing concentration of QMC nanoparticles,the cell compatibility is gradually reduced and the antibacterial activity is gradually enhanced.L929 cell activity was more than 90%of that in the control group after 1 and 3 days of co-culture when the concentration of QMC was 150 μg/mL.After treatment with the same concentration of QMC nanoparticles,compared with the dark treatment group,the bacterial survival rates were significantly decreased after light treatment.When the concentration was 150 μg/mL,the survival rate of S.aureus and E.coli after blue light irradiation was only 2.9%and 3.3%,respectively.QMC at 150 μg/mL exhibited both good biosafety and antibacterial activity.3.CCK-8 and cell live/dead staining experiments showed that after 1 day and 3 days of co-culture with different materials,compared with the control group and the MPDA group,the L929 cell viability of the MPDA-Cur and QMC groups was more than 90%,which showed a better biocompatibility than that of the QCS-MPDA group.4.Preliminary analysis of antibacterial activity in vitro and antibacterial mechanism of Control,MPDA,MPDA-Cur,QCS-MPDA and QMC nanoparticles against S.aureus and E.coli before and after light irradiation(450 nm,1 W/cm2):(1)Count of colony-forming units:Under dark treatment,the survival rates of bacteria of QCS-MPDA and QMC groups were decreased significantly.bacteria survival rates were significantly lower in the MPDA-Cur and QMC groups following blue light treatment than in the dark treatment.In addition,after blue light treatment,the antibacterial activity of MPDA-Cur against E.coli was lower than that of S.aureus,but the antibacterial activity of QMC group against E.coli and S.aureus was significant,and there was no significant difference in survival rate.(2)Bacterial live/dead staining:Before and after the blue light treatment,the bacterial biofilm in the Control group and the MPDA group showed large areas of green fluorescence(live bacteria).The QCS-MPDA group before and after blue light treatment,the QMC group after dark treatment,and the MPDA-Cur group after blue light treatment showed a certain anti-biofilm effect,and the bacterial biofilm presented a certain amount of red fluorescence(dead bacteria).After blue light treatment,the bacterial biofilm of the QMC group exhibited a large area of red fluorescence,and the anti-biofilm effect was the most significant.(3)Biofilm metabolic activity:The QCS-MPDA group before and after blue light treatment,the MPDA-Cur group after blue light treatment,and the QMC group after dark treatment showed a certain inhibitory effect on biofilm metabolic activity.After blue light treatment,the anti-biofilm metabolic activity of the bacteria in the QMC group was significantly higher than that in the MPDA-Cur group and the the QMC group after dark treatment.(4)Bacterial morphology:Both S.aureus and E.coli in the MPDA-Cur nanoparticle group after laser treatment experienced deformation and collapse,or even bacterial membrane rupture.In addition,in the QCS-MPDA group and the QMC group,the close distribution of the QCS-MPDA nanoparticles and the QMC nanoparticles around the bacteria was observed regardless of the light treatment.In which that bacterial morphology showed shrinkage and pitting,and even the bacterial membrane rupture caused the exudation of the contents.This result indicated that after photodynamic treatment,Cur cooperated with QCS to produce serious damage to bacterial cell wall.Conclusion:In this study,a novel photodynamic antibacterial QMC nanoparticle was successfully prepared,which could significantly improve the water dispersibility of curcumin.QMC nanoparticles with the concentration of 150 μg/mL exhibited both good biocompatibility and antibacterial activity.And QMC nanoparticles can effectively capture and enrich bacteria in a short time,and obtain enhanced photodynamic antibacterial activity in vitro. |
