| Objective:The periprosthetic joint infection(PJI)after the surgery is the most catastrophic deep joint infection,leading to a heavy health and financial burden for patient Some studies have found that the PJI cases are mainly induced by Staphylococcus aureus and nearly 60%of them are methicillin-resistant Staphylococcus aureus(MRSA),which leads to a high failure rate of the PJI treatment.Besides the joint revision surgery,the current antibiotic treatment mainly utilizes vancomycin,but there have been reports of vancomycin-resistant enterococci..At the same time,vancomycin may also bring some serious side effects,such as ototoxicity and nephrotoxicity,to the patients during the antibacterial treatments.To overcome these disadvantages,photodynamic therapy(PDT)is a good candidate.In the photodynamic therapy,the photosensitizer was exposed to the oxygen and proper wavelength light to produce the singlet oxygen that can bring about damage effects on the bacteria.Curcumin is a kind of yellow pigment isolated from plant Curcuma Longa and has excellent antibacterial and anti-inflammatory properties.In recent years,some studies have found that the curcumin is also a novel photosensitive dye with high efficiency and low toxicity.However,there is few report on using curcumin for PDT in the deep tissue infection treatment such as PJI.The main reason is that the excitation light wavelength of curcumin is located at 200-230nm?410-450nm while the penetration depth of the visible light is low and easy to be disturbed by tissues.Although the penetration depth of near-infrared light(NIR)like 980nm NIR is much larger than that of visible light,its energy is not high enough to activate the curcumin-induced PDT.Upconversion nanoparticles(UCNPs)can absorb NIR light and convert it to more energetic ultraviolet or visible light.based on this characteristic,UCNPs used for PDT has attracted much attention in recent years.Therefore in this study,(1)we study the antibacterial effect of curcumin and the PDT effect it induced and test its antibacterial effect on MRSA and its biofilm structure in vitro,(2)we synthesized the curcumin-UCNP nanocomposites by connecting the curcumin onto the upconversion nanoparticles,testify its safety and effectivness.(3)we peform the photodynamic ablation experiment from the curcumin itself and the PDT it induced in vitro(4)Furthermore,we constructed the MRSA-induced PJI models with SD rat and peform the PDT experiments against MRSA-induced PJI in vivo.Methods:1.We perform the test that curcumin can produce reactive oxygen species by the 450nm laser excitation.First,the reactive oxygen detection fluorescent probe(SOSG)was used to detect the active oxygen produced by the curcumin under 450 nm laser excitation MRSA suspension models were prepared as the subject that divided into 4 groups respectively,which was curcumin group,curcumin-laser group,laser-solo group and negtive group.The survival rate of MRSA planktonic bacteria was detected by dilution plate method.2.We synthesized the UCNPs by following previous protocols with slight Then modifications and connected the curcumin onto the upconversion nanoparticles.The morphology of the UCNPs and curcumin-UCNPs nanocomposites was characterized by SEM(JSM-6700F)and TEM(Tecnai F20).Zeta Potential was characterized by Malvern laser particle size analyzer(LPSA2000).The photoluminescence spectra were tested by a Shimadzu UV 3600Plus spectrophotometer with a 980 nm laser as light source.And fluorescence decay was measured by UltraFast lifetime Spectrofluorometer(Delta flex).After that we test ROS production of curcumin-UCNPs with the SOSG fluorescent probe at the 525nm under the 980nm infared light.3.MRSA were collected with inoculating loops from agar medium,inoculated in TSB medium in a biochemical incubator.The suspension was diluted with sterile water to 1×10~8CFU/mL with the McFarland Standard method.The growth rate of the strains was determined by measuring the OD of solution at 450 nm wavelength with a microplate reader(Rayto RT-6100)and figure out the minimum inhibitory concentration(MIC)of the nanocomposites and the appropriate irradiation time.The photodynamic ablation experiment in vitro was carried out in the 96-well plate.The wells were evenly covered with a mixture of 100?L MRSA suspension and the same amount of phosphate buffer saline(PBS),200?g/mL UCNPs,200?g/mL curcumin,100?g/mL curcumin-UCNPs and200?g/mL curcumin-UCNPs respectively and all processes were carried out at room temperature.The treated bacterial suspensions were subjected to gradient dilution in the dark or under the NIR respctively 10?L of the diluted solution was dropped into the culture plate and the heated sterile Trypticase Soytone agar(TSA)medium was poured into it whilst stirring until the agar solidified before performing colony counting.4.We constructed PJI animal model with rats and studied the antibactericidal effect of curcumin-UCNPs on PJI animal models in vivo.Firstly,the rat model of MRSA prosthesis infection was constructed and divided into differernt groups.Different concentrations of curcumin-UCNPs were injected into the rats knee cavity and then irradiated with 980 nm near-infrared light.3 days later,all the animals were sacrificed.The synovial tissue was taken out,the homogenate was weighed,the bacterial content was cultured to calculate the bacterial survival rate,and the other part of the synovial tissue was histologically subjected to HE staining and Gram staining to evaluate the bacterial condition and the inflammatory reaction in the tissue.In addition,blood was taken from the abdominal aorta,and the samples were collected in an EDTA tube,and the inflammatory factors were detected using an XFA 6030 blood analyzer.Results:1.Curcumin can produce ROS under NIR with concentration dependence in the same time.The concentrations were statistically significant compared with DMSO(P<0.001).When the concentration of curcumin was constant,the amount of ROS produced increased with time and stabilized after 5 min.In the experimental concentration range,curcumin-mediated PDT killing MRSA showed curcumin concentration-dependent,and the intervention of 25?M curcumin PDT significantly inhibited the MRSA floating bacteria and biofilm MRSA.The survival rate of planktonic bacteria decreased to(0.38±0.32)%,and the survival rate of MRSA in biofilm decreased to(34.50±1.44)%.Simple curcumin and simple irradiation treatment had no effect on bacterial activity.Scanning electron microscopy showed that curcumin-mediated PDT destroyed the biofilm,the structure was messy,and the microbial density decreased.2.1 The up-conversion luminescent nanoparticles synthesized by hydrothermal method were observed by scanning electron microscopy(SEM)and transmission electron microscopy(TEM).The particle size of the nanoparticles was about 25 nm,and the size distribution was relatively uniform.Curcumin is connected to the conjugated complex formed on the upconverting luminescent nanoparticles by PEI,and has a size of about 200nm and a uniform distribution.In addition,we tested the zeta potential of the conjugated complex to be+19 mV,and the zeta potential of MRSA bacteria was negative-32 mV.2.2 We measured the fluorescence emission peaks of the curcumin-upconverting luminescent nanoparticle conjugate complex at 980 nm excitation at 450 nm and 475 nm,respectively.In contrast to the fluorescence spectra of pure nanoparticles,we can see that the contours of the fluorescence spectra did not change significantly before and after the connection of curcumin,but the fluorescence emission intensity was significantly reduced.In further studies,we found that the fluorescence lifetime decay of the conjugated complex after coupling with curcumin became shorter,from 276.13?s of pure particles to 186.83?s of the conjugated complex,while further showing that the fluorescence decay of pure particles was Single exponential decay,and its attenuation appears as multi-exponential decay after attachment of curcumin.These experimental results confirmed that curcumin was already attached to the upper surface,and the energy of the excited state level was received by curcumin in the form of energy transfer,and the energy transfer efficiency was calculated to be 32.3%.2.3 We found that under the same 980 nm near-infrared light irradiation time,the amount of singlet oxygen production increased with the concentration of the curcumin-upconverting nanoparticle conjugate complex.As the irradiation time prolonged,the concentration of singlet oxygen initially increased rapidly,and after 20 minutes,the rate of increase gradually slowed down and tended to be gentle.2.4 We found that as the concentration of the curcumin-upconverting nanoparticle conjugate complex increased,the killing ratio of MRSA bacteria also increased.At the same concentration of the curcumin-upconverting nanoparticle conjugated complex,as the irradiation time increases,the bacterial killing ratio also increases.When the concentration of the conjugated complex is greater than 200?g/mL,the bacterial killing effect at 20 min and 30 min is close to 100%.The minimum inhibitory concentration(MIC)of the complex was 200?g/mL and the subsequent irradiation time was 20 minutes.3.We found that several experimental groups containing curcumin have a killing effect on MRSA bacteria,which is distinct from the negative control group,and has a high concentration of curcumin-upconverting luminescent nanoparticle conjugate complex plus980 nm near-infrared light illumination.The best germicidal effect.4.We found that in the hematology and synovial count tests,all three cytokines(IL-1?,IL-6 and TNF-?)and the number of bacteria were significantly increased in the negative group compared with the sham group.The bacterial count and inflammatory factors of the curcumin-upconversion luminescent nanoparticle conjugate complex with 980 nm near-infrared light irradiation group were significantly reduced,especially in the high dose group.At the same time,the simple curcumin group also has a certain killing effect on MRSA,and it can also reduce the inflammatory response of tissues.Histological examination showed that a large number of inflammatory cells occurred in the negative group,and bacteria were found in large numbers in the same part of the joint tissue.Compared with the negative control group,the bacterial content and inflammatory cell aggregation in the curcumin group were also reduced,and in the two curcumin-UCNPs groups,the amount of MRSA was significantly reduced,and inflammatory cell aggregation was significantly reduced,especially This effect is particularly pronounced in the high dose group.Conclusion:(1)Low concentration Curcumin-mediated PDT has a concentration-dependent killing effect on MRSA planktonic bacteria in vitro.(2)The up-converting luminescent nanoparticles synthesized by hydrothermal method have uniform particle size distribution and stable structure,and the Zeta potential is+19mV,indicating that our conjugated complex can be adsorbed to the surface of bacteria,which is beneficial to photodynamic generation.The singlet oxygen plays a better role in killing.At the same time,we measured the fluorescence spectra of the conjugated complex and the absorption spectrum of curcumin and the fluorescence lifetime decay measurement.It was found that after the curcumin was added,there was energy transfer between the curcumin and the conjugated complex,that is,the up-converting luminescent particles could The absorbed near-infrared light is transmitted to curcumin to exert a photo-chemical action,and the light transmission efficiency is 32.3%.(3)Our results show that curcumin itself can produce certain killing effect on MRSA bacteria,and the PDT effect of curcumin-upconverting nanoparticle conjugated compound under 980nm laser irradiation can also cause massive killing of MRSA bacteria..Therefore,in vitro experiments have shown that curcumin can kill MRSA bacteria by double antibacterial action under near-infrared light irradiation.(4)We found that PDT caused by nano-conjugated complex under NIR irradiation can kill MRSA bacteria in a large amount and reduce the inflammatory reaction of tissue.Curcumin itself has certain killing effect on MRSA,and tissue reaction also follows.The reduction proves that curcumin can effectively remove MRSA through its own antibacterial action and mediated double bactericidal action of PDT,which provides a new possible treatment for the treatment of periprosthetic infection and photodynamic therapy.The infection around the prosthesis provides some theoretical basis. |
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