| Photodynamic therapy(PDT)is a minimally invasive,highly biosafety combination therapy that has received extensive attention in the biomedical field.However,since most of the photosensitizers(PS)on which PDT therapy relies are hydrophobic,they are prone to aggregation and quenching,leading to significantly reduced utilization efficiency of PS and the therapeutic effect of PDT.As the main component of biological metabolism,protease has good biocompatibility and extremely high catalytic activity and can be used as an ideal photosensitizer carrier to realize catalytically enhanced PDT.However,due to the relatively weak stability of proteases,conventional chemical coupling methods tend to impair their biological activities,thus affecting their practical applications.The development of new theories,new methods,and new structures for efficient PS delivery is an urgent need for biomedical applications and an important scientific issue for basic research.Therefore,we propose a method that relies on the adsorption method of the non-covalent interaction(hydrogen bonding,electrostatic interactions,π-π stacking,and van der Waals interactions)between the protein and the photosensitizer,it may facilitate the assembly between photosensitizers and enzymes while avoiding the adverse effects of chemical modification on the stability and activity of the protease.Based on this,this thesis firstly studied in detail the affinity propensity of the secondary structures on catalase(CAT),glucose oxidase(GOx),and lysozyme(LZ)to the photosensitizer chlorin e6(Ce6)and developed a general synthesis strategy of protease nano-photosensitizer,and further systematically discussed its PDT antibacterial effect.The main research contents and results of this paper are as follows:1.Through molecular dynamics simulation,we systematically studied the interaction mechanism and binding process of Ce6 molecule with three proteases:catalase(CAT),glucose oxidase(GOx)and lysozyme(LZ).Our simulation results have shown that although all proteases can interact with and bind to small molecules,the Helix structure on the protein is easier to interact and bind with small molecules than the β-sheet structure,and it may be related to the fact that the Helix structure can form more hydrogen bonds with Ce6 molecules than the β-sheet structure.To further verify that the Helix structure is indeed more likely to interact with small molecules,we selected HP35 protein containing only the Helix structure and aβ42 protein containing only β-sheet structure for more in-depth studies.Through the evolution of the binding position of Ce6 molecule to protein,we found that Ce6 molecule showed a higher binding probability with HP35 protein compared with aβ42 protein,which further verified our previous conclusion.Moreover,we also found that the electrostatic interaction between Ce6 molecule and protease also has a modest effect on their binding.The simulation results show that the more positively charged regions are distributed on the protein surface,the more likely the Ce6 molecule is to bind to the protein.2.Based on the binding propensity of Helix domain on protease to Ce6,we developed a general strategy for the preparation of protease nano-photosensitizers.Under the action of polyvinylpyrrolidone(PVP),three proteases of CAT,GOx and LZ can stably combine with Ce6 and form protease nano-photosensitizers CAT-Ce6 NPs,GOx-Ce6 NPs and LZ-Ce6 NPs.Experiments show that the formed protease nano-photosensitizer has good biocompatibility,and the loading of Ce6 and the coating of PVP can further improve the stability of protease.Moreover,catalytic experiments show that the prepared three protease nano-photosensitizers have significant catalytic activities of CAT,GOx,and LZ,respectively.It indicated that the loading of Ce6 and the coating of PVP did not affect the catalytic ability of the corresponding protease.3.To investigate the PDT effect of the prepared protease nano-photosensitizers,we took Staphylococcus aureus as the research object and evaluated the PDT antibacterial effect of CAT-Ce6 NPs.Through bacterial morphological studies,we found that CAT-Ce6 NPs can effectively destroy bacterial cell walls and cell membranes and have excellent photodynamic antibacterial effects.By simulating the bacterial infection microenvironment,we confirmed that CAT-Ce6 NPs can provide oxygen(O2)for photodynamic therapy by decomposing hydrogen peroxide(H2O2)in the environment,thereby improving the hypoxic microenvironment and enhancing the antibacterial effect of PDT.Animal experiments show that CAT-Ce6 NPs can effectively alleviate the hypoxic microenvironment of bacterial infection(subcutaneous abscess model)in mice,thereby enhancing the antibacterial effect of PDT and achieving effective,rapid treatment of abscesses.In conclusion,through systematically exploring the physical interactions between proteases and photosensitizers,we achieved the controllable assembly of protease nano-photosensitizers and the enhanced antibacterial effect of PDT.The research results not only contribute to the prediction and regulation of protein photosensitizer functions,but also have theoretical guiding significance for the design and development of smart nano-photosensitizers with high biocompatibility and rich biological functions. |
