Breaking Immunosuppression Against Intracellular Bacteria Using Thermo-Responsive Nanoparticles

Intracellular bacteria are the main reason for the recurrence and persistence of bacterial infections in clinical situations.They exist in the host cells and are difficult to be cleared effectively due to the barrier effect of the host cell membrane that prevents antibiotics from penetrating into the cells,and also interfere with the innate immune response of the host cells,and the emergence of antibiotic-resistant strains exacerbates this problem and even causes life-threatening infections.Objective:Our experiment aims to exploit a controlled drug-loaded nanoparticle that can delivery antibiotics into cells,increase the effective intracellular antibiotic concentration,and break immunosuppression to enhance the innate immunity of the host cell itself,providing a novel approach to the treatment of intracellular bacteria.Methods:（1）Using a hydrolysis-precipitation method to synthesize kernel CaO₂-Vancomycin NPs（（CaO₂-Vanco NPs,CV NPs））,a suitable ratio of eutectic mixture of lauric acid and stearic acid was selected as a shell for a thermally responsive phase transition to synthesize CaO₂-Vanco@PCM NPs（CVP NPs）with controlled release at the desired melting point.The morphological structures of the CVP NPs were characterized using transmission electron microscopy（TEM）,scanning electron microscopy（SEM）,X-ray diffraction mapping（XRD）and Fourier infrared transform spectroscopy（FI-TR）,and fluorescence co-localization analysis was performed using confocal fluorescence microscopy（CLSM）and flow cytometry.（2）Measure the rate of vancomycin release from CVP NPs at different pH and temperature;assay the time-resolved and spatially resolved vancomycin release from CVP NPs using cold-thermal cycling drug release and 96-well plate release assays;co-culture with RAW 264.7 cells at different temperatures to assess the thermo-responsive drug release from CVP NPs at the cellular level;and assess the oxygen release efficiency using an oxygen electrode.（3）The effects of different concentration gradients of nanocomplexes on the growth activity of RAW 264.7 cells and L-929 cells at 37℃to 40℃were examined using CCK-8,cell morphology fluorescence imaging and hemolysis assays.（4）Evaluation of the killing of planktonic bacteria by the CVP+HTD（CVP+Heated Treatment）treatment group by colony plate counting,FDA/PI live-dead staining,flow cytometry and SEM imaging.Biofilm damage by CVP+HTD was assessed using FDA/PI live-dead staining CLSM fluorescence imaging and SEM imaging.（5）Establish MRSA-infected RAW264.7 model and assess the efficiency of CVP NPs into cells by CLSM imaging and flow cytometry technique;assess the effect of CVP+HTD clearance of intracellular bacteria using plate count after lysing cells bacteria and CLSM fluorescence imaging.（6）The intracellular lactate and H⁺were detected in different treatment groups to assess the effect of CVP+HTD de-acidification;the expression of i NOS,Arg-1 and HIF-1αin each group of cells was detected using protein blotting（Western Blot,WB）to determine the polarization status of macrophages;the major antibacterial substances Reactive Oxygen Species（ROS）and Reactive nitrogen species（RNS）production in macrophages were detected.（7）To verify the effectiveness of CVP+HTD in clearing intracellular bacteria in vivo,a septic arthritis model was established using BABL/c mice,and the effectiveness of in vivo treatment was judged by observing localized joint erythema,joint destruction,bone adhesions in joint gap radiographs,clearance of intracellular bacteria in frozen section fluorescence imaging,and tissue paraffin staining results.Results:（1）The synthesized CV NPs have homogeneous size and well-dispersed spherical structure;the synthesized CVP NPs have a highly stable spherical core-shell structure with CV NPs as the core and fatty acids as the shell.（2）The loading of vancomycin in CVP NPs is about 56.39%.With a good oxygen release effect,vancomycin release is accelerated at high temperature（40℃）and low pH environment,CVP NPs release with high temporal and spatial resolution,and this property is also maintained at the cellular level.（3）The cell viability of RAW 264.7 and L-929 cells after co-culture with different components of nanocomposites at 40℃was above 90%,and the percentage of hemolysis in all groups ranged from 1.46-4.96%,which were less than 5%,indicating that CVP NP has good cytocompatibility and hemocompatibility.（4）The minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）of CVP+HTD against planktonic MRSA were 3.90μg/m L and7.80μg/m L,respectively,which were significantly smaller than those of the non-heated group.Even if the initial bacterial density reached 10⁸CFU/m L,which was equivalent to 100 times of the MIC measurement,the bactericidal rate of CVP+HTD was more than 99%and had a high biofilm disruption effect.（5）Under the treatment of CVP+HTD,the transmembrane transport of vancomycin could be efficiently achieved and released on demand by heat-responsive phase change to increase the effective intracellular bactericidal concentration,and almost all intracellular MRSA（>99%）were killed,and no antibiotic tolerance was developed.（6）CVP+HTD can produce Ca（OH）₂through redox reactions with surrounding water and H⁺,and acid-base neutralization reactions with lactic acid to consume accumulated lactic acid.It can also reduce the production of lactic acid associated with bacterial metabolites by killing intracellular MRSA,overcome lactic acid-related immunosuppression,and promote M1 polarization of macrophages.（7）In vivo experiments showed that the CVP+HTD treatment group could effectively eliminate planktonic MRSA and intracellular MRSA in the synovial fluid and synovium and reverse the local immunosuppression of the lesion.Mice treated with CVP+HTD had a better prognosis and excellent biocompatibility,providing a promising strategy for the treatment of intracellular bacteria.Conclusions:CVP NPs have excellent thermo-responsive drug release properties and effectively neutralize lactate,attenuate lactate-associated immunosuppression increase ROS and RNS output,and provide a safe and effective protocol for intracellular bacterial therapy.