| Part ?:Construction of photothermal/pharmaceutical synergistic antibacterial system based on gold nanocages and its application in the treatment of periodontitis.Purposes:The purpose of this study is to construct a photothermal/pharmaceutical synergistic antibacterial system based on gold nanocage and to explore its application in the treatment of periodontitis.Materials and Methods:The phase change material PCM was used as the medium to help load the antibiotic TC into the gold nanocage.Then the thermal sensitive polymer PND was modified on the surface to obtain the photothermal/pharmaceutical synergistic antibacterial system TC-PCM@GNC-PND.The morphology of GNC and TC-PCM@GNC-PND were observed by SEM and TEM.Their absorption spectra,hydrodynamic particle size,and Zeta-potential were measured respectively.The long-term stability of the nanoparticles was evaluated by measuring the change in particle size,solution color,and Tyndall effect after storing for four weeks in PBS.The photothermal properties were evaluated by measuring the heating curve under the near-infrared laser irradiation,and the influence of concentrations and irradiation intensities were measured.The photothermal stability of TC-PCM@GNC-PND was evaluated through seven consecutive photothermal cycle experiments.The thermal sensitivity of PND were tested using UV-visible spectrophotometer,and the sol-gel phase transition ability of PND was studied by bottle reversal experiment.Methylene blue was used as the simulated pharmaceutical to study the NIR-PTT controlled release profile.Different concentrations of TC-PCM@GNC-PND were co-cultured with rat MSCs and GFs respectively,and CCK-8 kit was used to evaluate the cytotoxicity of TC-PCM@GNC-PND.The in vitro antibacterial effect of TC-PCM@GNC-PND was evaluated by bacterial growth curve,plate counting method and live-dead fluorescence staining.Different materials were used to treat the experimental periodontitis in rats.Methylene blue staining,micro-CT scan and reconstruction,as well as H&E staing were used to measure the antibacterial effect in vivo.The weight of the rats were recorded during the whole experiment,and the whole blood was taken for blood routine and blood biochemical tests at the end of the experiment to evaluate the biocompatibility in vivo.Results:SEM and TEM observation showed that GNC and TC-PCM@GNC-PND were both cubic nanoparticles with regular morphology and good uniformity.GNC had a porous and hollow structure,while TC-PCM@GNC-PND particles were filled.The UV-visible absorption spectra of GNC and TC-PCM@GNC-PND showed that the LSPR peaks of both were around 800nm.The hydrodynamic diameter of GNC was about 93nm,while TC-PCM@GNC-PND was about 123nm.The size distribution of both particles presented a single-peak normal distribution with good dispersion.Zeta-potential of GNC was-33m V,while TC-PCM@GNC-PND was+8m V.Both GNC and TC-PCM@GNC-PND were stable in PBS solution,with no significant change in the particle size after 4 weeks,and both of them had good Tyndall effect.Both of them shown good photothermal properties,causing rapid temperature rise under 808nm laser irradiation,and the rate and amplitude of temperature rise were correlated with material concentration and laser irradiation intensity.TC-PCM@GNC-PND has good photothermal stability.The temperature sensitivity of PND was verified by the curve of transmittance change with temperature."Stepped"pharmaceutical release were observed under pulsed laser irradiation,proving that the accurate controlled release of pharmaceutical molecules can be achieved by regulating the near-infrared laser.TC-PCM@GNC-PND cooperating with 808nm laser irradiation has the best bactericidal effect in vitro,and can significantly reduce the degree of bone loss caused by experimental periodontitis in rats.Conclusion:In this experiment,we found that the obtained TC-PCM@GNC-PND has stable structure and properties,good biocompatibility,high photothermal conversion efficiency,and can realize the NIR-PTT controlled on-demand release of encapsulated pharmaceuticals,precisely synchronizing with the photothermal effect.Synergistic antibacterial effect were observed both in vitro and in experimental periodontitis.Part ?:Construction of photothermal/pharmaceutical synergistic antibacterial system based on black phosphorus quantum dots and its application in the treatment of MRSA related subcutaneous abscess.Purposes:The purpose of this study was to construct a photothermal/pharmaceutical synergistic antibacterial system based on black phosphorus quantum dots,and to explore the application in the treatment of MRSA related subcutaneous abscess.Materials and Methods:BPQDs was obtained by liquid phase exfoliation method,and thermal sensitive liposomes were prepared by thin film hydration method.BPQDs and vancomycin were loaded into the thermal sensitive liposome to obtain the photothermal/pharmaceuticalsynergisticantimicrobialsystem BPQDs-vanco@liposome.The morphology and diameter of BPQDs were observed and measured by TEM.The surface morphology and thickness of BPQDs were observed by AFM.The morphology of BPQDs-vanco@liposome was observed by CLSM.The major elements of BPQDs-vanco@liposome were analyzed under SEM.The Raman spectra of BPQDs and BPQDs-vanco@liposome were determined respectively.Absorption spectra of BPQDs-vanco@liposome at different concentrations were determined by UV-visible spectrophotometer.The photothermal properties of BPQDs-vanco@liposome were evaluated by monitoring temperature changes under NIR irradiation using an infrared thermal imager,and the heating curves under different concentrations and irradiation intensities were measured.The long-term stability of BPQDs and BPQDs-vanco@liposome were evaluated.FITC was used as a simulated pharmaceutical to detect the pharmaceutical release profile under NIR irradiation.Different concentrations of BPQDs-vanco@liposome were co-cultured with MSCs and GFs,and cell proliferation was detected by CCK-8 kit to evaluate the cytotoxicity.MRSA was used as the model microorganism to evaluate the synergistic antibacterial effect of BPQDs-vanco@liposome in vitro by the detection of bacterial growth curve after treatment with different materials,plate counting method and live-dead fluorescence staining.Photothermal effects of BPQDs-vanco@liposome were tested by the infrared thermal imager.By subcutaneous injection of MRSA,a mouse model of MRSA related subcutaneous abscess was established.Different materials were injected into the abscess to treat the subcutaneous abscess with or without NIR irradiation.The local conditions of the abscess were observed,and the abscess tissue was homogenated and spread on a dish to judge the effect of different treatments.Finally,the possible mechanisms of synergistic antibacterial effect of BPQDs-vanco@liposome under NIR irradiation was explored by means of SEM observation of morphologic changes,detection of the amount of nucleic acid in supernatant after treatment,examination of the production of reactive oxygen species,and the integrity of bacterial DNA.Results:TEM image showed that the obtained BPQDs had uniform morphology.AFM cross-sectional analysis showed that the thickness of BPQDs was about1.9-3.4nm.Photos of CLSM showed BPQDs-vanco@liposome had uniform morphology and good dispersion.SEM element mapping showed that phosphorus was concentrated in the center of the circular area,while carbon and oxygen,the main components of the liposome,were distributed around the circle,showing a typical shell-core structure.Both BPQDs and BPQDs-vanco@liposome had three distinct characteristic peaks,which were 359.7 cm-1,436.2 cm-1,463.5 cm-1,respectively.It can be seen from the absorption spectrum that BPQDs-vanco@liposome had a wide absorption band from the ultraviolet region to the near infrared region,and the absorption intensity was concentration dependant.The extinction coefficient at the near infrared region(808nm)was large,about 9.6l g-1 cm-1.The heating curve and photothermal cycle experiment results indicate that BPQDs-vanco@liposome caused robust temperature rise under NIR irradiation,demonstrating the high photothermal conversion efficiency and good photothermal stability.BPQDs-vanco@liposome was more stable than BPQDs by comparing the color and photothermal curves of BPQDs and BPQDs-vanco@liposome solution stored for a period of time.As can be seen from the pharmaceutical release experiment,the release of the pharmaceutical molecules was controlled by NIR.Low cytotoxicity of BPQDs-vanco@liposome was verified.BPQDs-vanco@liposome exhibited excellent antibacterial effect towards MRSA under NIR irradiation in vitro as well as in subcutaneous abscess,through the synergistic effect of pharmaceutical therapy and photothermal therapy.The destroy of bacteria membrane,overflow of nucleic acid,elevated level of ROS and destruction of DNA structure were proved to be the antibacterial mechanism of BPQDs-vanco@liposome.Conclusion:In the present study,we found that BPQDs-vanco@liposome has good biocompatibility,high photothermal conversion efficiency,strong photothermal stability,and can achieve the on demand release of pharmaceuticals under near-infrared laser.Synergistic antibacterial effect towards MRSA were observed in vitro and in vivo on account of the precise synchronization of antibiotics release and photothermal effects. |
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