Innovative Construction Of Novel AIE-based Organosilver Fluorescent Molecular Probes For The Diagnosis And Their Application In The Diagnosis And Treatment Of Bacterial Biofilms

Objectives:Infectious diseases caused by pathogenic bacteria,especially multi-drug resistant（MDR）bacteria,have become a serious threat to the global public health.As one of the major factors causing the bacterial resistance,biofilm formation has been regarded as an underlying contributor of the development of chronic clinical infectious diseases and recurrent refractory infectious diseases.Unfortunately,conventional antibiotic treatment is generally found ineffective against multi-drug resistant bacteria and bacterial biofilms.To alleviate the issue,early diagnosis emerges as an effective pathway to prevent the worsening and further spread of infection,while targeted and rational use of drugs can reduce the risk of bacterial resistance as well.Thus,there is an urgent need to develop new strategies and treatments to combat drug-resistant bacteria and biofilm infections,ultimately reducing the development of clinical resistance.To address the challenge in combating drug-resistant bacteria and their biofilm infections and promoting wound healing in the treatment of chronic,refractory and recurrent wounds and subcutaneous infections,a series of novel organosilver AIE fluorescent molecular probes were designed and constructed for a)visualizing Gram-negative and positive bacteria and their biofilms,b)investigating the antibacterial activities and involving antimicrobial mechanism,the inhibition on MRSA biofilms growth of drug-resistant bacteria and the removal activity of mature biofilms,and further c)exploring the antibacterial,anti-biofilm and pro-healing functions and in vivo biosafety in chronic,refractory and hard-to-heal wounds infected with drug-resistant bacteria and their biofilms.This study would lay the foundation for the research and development of novel diagnostic and therapeutic tools for bacterial and biofilm infections,and provide new ideas for the development of novel antibiotics.Methods:（1）On basis of the scaffold tetraarylimidazole with AIE effect,the organosilver AIE fluorescent probe APDIS was constructed by introducing a methylimidazole through a dodecyl chain,which was further bonded to silver as an azacyclic complex.（2）On basis of the scaffold trianiline with AIE effect,a photosensitive fluorophore TPIM with near-infrared fluorescence emission was constructed under light of a D-π-A strategy.Further,different alkyl chain substituted imidazoles were introduced as azacyclic complexes bonded to silver to afford a series of organosilver near-infrared AIE photosensitizers TPIMS-n（n=2,8,14）.（3）The morphological structure and physical photochemical properties,photochemical conversion,and photoactivated silver ion release efficiency of APDIS and TPIMS-induced self-aggregating organic nanomaterials were characterized and tested by high-resolution transmission electron microscopy,UV-vis spectrophotometer,fluorescence spectrophotometer,dynamic light scattering instrument,high-performance liquid chromatography,and nuclear magnetic resonance hydrogen spectroscopy.（4）In vitro bacterial imaging experiments,the capabilities of APDIS for ultra-fast wash-free bacterial imaging and bacterial classification and detection were evaluated and examined by quantitative and qualitative analysis using fluorescence spectrophotometer and laser confocal imaging.Meanwhile,the difference of TPIMS-n（n=2,8,14）with different alkyl chains were systematically examined in bacterial near-infrared fluorescence no-clean imaging.（5）In vitro bacterial,biofilm and cellular models,the in vitro antibacterial and anti-biofilm efficacy and mechanism of APDIS and TPIMS were evaluated and examined by statistical quantitative analysis using turbidimetric method,smear plate method,scanning electron microscopy,dead-live staining method and laser confocal imaging.（6）In BALB/c mice subcutaneous infection with drug-resistant bacteria MRSA,MRSA biofilm catheter graft infection and MRSA mature biofilm subcutaneous chronic infection animal models,the antibacterial,anti-biofilm and pro-healing efficacy of these organosilver AIE probes in vivo were evaluated by monitoring the size of infected wound area,degree of inflammation and recovery of septic area,wound closure rate,tissue homogenization at the infection site and pathological histological analysis of model mice.（7）The biosafety and biocompatibility of these organosilver AIE probes were evaluated by means of body weight monitoring,hemolysis assay,and histological examination of major organs.Results:In this study,two types of organosilver AIE fluorescent molecular probes were successfully constructed.The water-soluble Bola-type fluorescent probes APDIS exhibits excellent bacterial imaging capabilities and antibacterial activity.At low concentrations（1μg/m L,<MIC）,APDIS was able to identify Gram-positive bacteria S.aureus,S.pneumoniae and Gram-negative bacteria E.coli（within 60 s）),with advantages of wash-free imaging,ultra-sensitive detection（with detection limits as low as 10³ CFU/m L）and excellent bacterial biofilm imaging（25μm depth）.For antimicrobial therapy,APDIS exhibits broad-spectrum antibacterial activity,with good antibacterial activity against E.coli（MIC=10μg/m L）,S.aureus（MIC=5μg/m L）and S.pneumoniae（MIC=5μg/m L）,and more importantly,excellent anti-MRSA activity（MIC=5μg/m L）.In a mouse subcutaneous MRSA infection model,APDIS was shown to be effective in eliminating MRSA infections,while promoting wound healing in infected wounds.In terms of anti-biofilm,APDIS was effective in inhibiting the growth of MRSA biofilm in vitro,penetrating the mature MRSA biofilm barrier,disrupting the biofilm matrix,killing the MRSA-retentive bacteria inside the biofilm and completely removing the MRSA biofilm.The near-infrared organic silver AIE photosensitizer TPIMS-n（n=2,8,14）provides photo-activated the release of Ag⁺and synergistic singlet oxygen production for the combined chemical/photodynamic treatment of bacterial infections.TPIMS-n not only enables near-infrared fluorescence imaging of S.aureus,S.pneumoniae,E.coli and MRSA,but also enables effective inhibition of their growth.Further upon light illumination,TPIMS-n（n=2,8,14）released Ag⁺and produced singlet oxygen,synergistically enhancing the bactericidal effect.Due to the structural differences,the antibacterial activity of the series compounds varied slightly,and the overall antibacterial activity was ranked in order of magnitude:TPIMS-8>TPIMS-2>TPIMS-14>TPIM-8>TPIM-2>TPIM-14.Among them,compound TPIMS-8 exhibited much higher anti-MRSA activity(MIC_Dark=1μM,MIC_Light=0.1μM),a value lower than the positive drug control vancomycin（MIC=2μM）.In the in vitro anti-MRSA biofilm assay,TPIMS-8 was effective in inhibiting the growth and formation of MRSA biofilm;in mature MRSA biofilm,TPIMS-8 was able to effectively penetrate the MRSA biofilm barrier and completely remove the MRSA biofilm by synergistic chemical/photodynamic disruption of the biofilm matrix and killing the MRSA-retentive bacteria within the biofilm.TPIMS-8 can effectively kill MRSA and remove MRSA biofilm after external light activation,and the light-induced release of Ag⁺can promote the regeneration of skin cells,tissues and vascular endothelial cells,thus effectively accelerating the chronic wound healing.Animal studies have also shown that the potency of TPIMS-8 is higher than vancomycin at the same concentration in terms of anti-biofilm activity and biosafety.None of the above probes exhibited significant biological toxicity to body weight and major organs of mice at therapeutic doses.Conclusion:In this study,to address the limitation in the rapid diagnostic and treatment of recurrent refractory chronic infections caused by drug-resistant bacteria and their biofilms,two types of organosilver AIE small molecule complex were constructed.One of candidates APDIS exhibited robust features including ultra-fast wash-free imaging,bacterial detection and identification,broad-spectrum antibacterial activity,high potency to MRSA and its biofilm,and promotion of wound healing.Another one TPIMS-n（n=2,8,14）showed the advantages of simultaneous NIR bacterial fluorescence imaging,detection,synergistic light-activated silver ion release,singlet oxygen generation,dark antimicrobial activity against MRSA biofilms and promotion of wound healing.The development of these probes demonstrates an integration of diagnosis and treatment of bacterial and biofilm infections without significant toxic effects on surrounding normal tissues and major organs.More importantly,This study provides a promising candidate for clinical translation in the treatment of bacterial infections,especially intractable,refractory,and chronic refractory infections caused by drug-resistant bacteria and their biofilms,and also provide new ideas and theoretical guidance for the development of novel antibiotics.This dissertation contains 94 Figures,7 Tables and 234 references.