Construction Of In Situ Self-assembling Peptide Probe With Aggregation-induced Emission And Its Application In Diagnosis And Treatment Of Bacterial Infections

Objective:This subject is to construct an in situ self-assembling peptide-based multifunctional probe（AIEgen-peptide-Van）for detecting and fighting Gram-positive bacterial infections.The probe was designed with a short self-assembling peptide as backbone,an aggregation-induced luminescence molecule（AIEgen,TPEPy-Butyne）as responsive fluorescent signal,and vancomycin as targeting group.The targeted binding of probe with D-Ala-D-Ala in the Gram-positive bacterial wall induced its self-assembly which turned on its fluorescence by restricting AIEgen’s intra-molecular rotation.At the same time,the self-assembly of probe enhanced its ability to produce reactive oxygen species.Eventually,the specific detection and efficient photodynamic killing of Gram-positive bacteria were achieved both in vitro and in vivo.Methods:1.Probe synthesis and characterization:Firstly,self-assembling peptide N₃-^DF^DF^DY^DEG^DK was synthesized by solid-phase peptide synthesis method（SPPS）.Vancomycin was covalently conjugated to the side chain of lysine（K）by maleic anhydride activated ester to generate N₃-^DF^DF^DY^DEG^DK-Van.AIEgen（TPEPy-Butyne）with electron donor-acceptor structure was synthesized and then connected with N₃-^DF^DF^DY^DEG^DK-Van by clicking chemical reaction to obtain AIEgen-peptide-Van.Nuclear magnetic resonance and high-resolution mass spectrometry were used to characterize the intermediates and final product.2.In vitro properties study of the probe:The structure and fluorescence propertyof the probe were further studied.The self-assembly ability of the probe in vitro was investigated by dynamic light scattering.The ability of self-assembly induced luminescence of probe in vitro was verified by co-assembly with Nap-G^DF^DFG.The ROS production of the probe（including singlet oxygen ¹O₂ and superoxide O₂^-）was quantitatively studied.3.In vitro bacterial detection and killing of the probe:The specificity of the probe binding to different types of bacteria was studied by confocal laser scanning microscopy and the peak changes of fluorescence emission spectrum.The microscopic morphology of the assemblies were investigated by transmission electron microscope.The chem-iluminescent S.aureus Xen 36 strain was used as the model bacteria to study the sensitivity of the probe.4.In vivo bacterial infection detection and bactericidal properties of the probe:The biocompatibility of the probe were evaluated through cell viability assay,hemolysis experiment,and H&E staining.A variety of Gram-positive bacterial and Gram-negative bacterial infection mice models were prepared to study the in vivo detection and bactericidal property of the probe by small animal live fluorescence imaging,live two-photon confocal microscopy imaging,and small animal chemiluminescence imaging.Results:1.Synthesis and characterization of probes:¹H NMR,¹³C NMR and LC-MS characterization data indicated the successful synthesis of AIEgen,N₃-^DF^DF^DY^DEG^DK,N₃-^DF^DF^DY^DEG^DK-Van and the final product.2.In vitro performance study of the probe:Dynamic light scattering data showed that the key self-assembly concentration of the probe was 14.52μM.The 600 nm characteristic peak of the fluorescence emission spectrum increased sharply after in vitro assembly,indicating the assembly induced turn-on propterty of the probe.The efficient production of ¹O₂ and O₂^-indicated that self-assembly enhances AIEgen’s ROS production capacity.3.In vitro bacterial detection and killing of the probe:CLSM imaging showed that the probe can targeted imaging Gram-positive bacteria.Preliminary results indicated the probe intensities were directly related to the resistance of the strain to vancomycin.The detection limit of the probe to S.aureus Xen 36 is 2.4×10³ CFU/m L,indicating that the probe has high sensitivity.TEM results showed that the probe assembled on the surface of Gram-positive bacteria to form spherical,amorphous nano-assemblies.4.In vivo bacterial infection detection and bactericidal properties of the probe:In vivo fluorescence imaging and in vivo two-photon confocal microscopy imaging results showed that the probe can accumulate at the sites of Gram-positive bacterial infection after the tail vein injection.The fluorescence intensity at the infection sites reached the highest level at 2 h,indicating that the probe can detect Gram-positive bacterial infections quickly and with high sensitivity.Chem-iluminescence imaging showed that bacterial infections can basically be cured after two photodynamic treatments.Conclusion:In this study,based on vancomycin-modified self-assembled peptides and AIEgen with fluorescence and therapeutic functions,in situ self-assembled probes were developed for highly sensitive diagnosis and efficient treatment of Gram-positive bacterial infections.The probe has high detection sensitivity and strong targeting ability in vitro and in vivo.Since the structure of probe based on self-assembling peptide is highly modifiable,various strain-specific probes can be obtained by modifying different targeting groups and connecting different AIEgens.Therefore,our design can be developed into a general strategy for constructing multifunctional bacterial diagnostic probes to guide the rational use of clinical antibiotics.In addition,the good biocompatibility in vitro and in vivo also indicated that the probe shows great potentials in the diagnosis and treatment or visual treatment of bacterial infections.