Deciphering the physical binding mechanism of enzyme–photosensitizer facilitates catalysis-augmented photodynamic therapy

Research Background

Photodynamic therapy (PDT), characterized by its non-invasive nature and precise spatiotemporal selectivity, has garnered considerable attention for its applications in tumor and anti-infection therapies. However, the clinical translation of PDT continues to encounter several challenges. Firstly, traditional photosensitizers (PS) frequently display poor solubility and insufficient stability. Additionally, the complex pathological microenvironment can hinder PS delivery and release, while tissue hypoxia markedly inhibits the generation of reactive oxygen species (ROS), ultimately leading to therapeutic failure. Enzyme–PS conjugates offer a promising strategy to address these limitations. Enzyme carriers can improve the physicochemical properties of PS, while their biocatalytic activity facilitates the remodeling of the pathological microenvironment, thereby enabling catalysis-augmented PDT. However, conventional covalent conjugation methods are prone to compromising enzyme activity and may lead to undesirable side effects. In contrast, physical binding approaches not only better preserve enzyme activity but also simplify the operational procedures. However, the physical binding mechanisms between enzymes and PS, which encompass various weak forces, are complex and remain insufficiently explored. Therefore, a comprehensive understanding of the physical binding mechanisms between enzymes and PS will offer valuable insights for the rational design of enzyme–PS conjugates, thereby opening new avenues for the development of catalysis-augmented PDT.

Research Progress

Through the integration of molecular dynamics (MD) simulations and experimental studies, this study systematically investigated the interaction mechanisms between three representative enzymes—lysozyme (Lys), glucose oxidase (GOx), and catalase (CAT)—and the chlorin e6 (Ce6) (Figure 1). The results demonstrated that the initial binding of Ce6 to enzymes was primarily driven by electrostatic interactions, leading to the formation of a loose complex that was subsequently stabilized by hydrophobic interactions and hydrogen bonds (Figure 2).

Through free energy calculations and residue analysis of enzyme–Ce6 binding sites, the results demonstrated that positively charged and hydrophobic residues on the enzyme surface was critical determinants of enzyme–Ce6 binding (Figure 3). Furthermore, the inclusion of three enzymes—horseradish peroxidase (HRP), alkaline phosphatase (ALP), and lactate dehydrogenase A (LDHA)—which exhibited substantial differences in molecular weight and possessed significant biomedical relevance, further corroborated these findings.

By examining the relationship between the binding affinity of Ce6 for enzymes and the surface areas of positively charged and hydrophobic residues, the authors demonstrated that the surface area of positively charged regions on the enzyme was strongly correlated with its binding affinity. Considering intravenous administration, this study further incorporated human serum albumin (HSA) and established a systematic criterion for assessing the binding strength between enzymes and Ce6 (Figure 4).

Based on this criterion, the authors further developed catalase (CAT)-Ce6 nanoconjugates (CAT-Ce6 NCs) that exhibited excellent stability and high biocompatibility (Figure 5). In vitro experiments demonstrated that CAT-Ce6 NCs efficiently catalyze the conversion of excess H₂O₂ to O₂ in the bacterial microenvironment, thereby alleviating hypoxia and enhancing the production of ROS (Figure 6). In a murine subcutaneous abscess model infected with methicillin-resistant Staphylococcus aureus (MRSA), CAT-Ce6 NCs effectively modulated the hypoxic pathological microenvironment and eradicated the bacteria, thereby enabling catalysis-augmented PDT (Figure 7).

Future Prospects

Through a systematic investigation of the physical interactions between enzymes and Ce6 photosensitizer, this study revealed for the first time that positively charged and hydrophobic residues on the enzyme surface were key structural determinants of their binding. Furthermore, this study proposed that the area of positively charged regions on the enzyme surface can be used as a criterion to evaluate and predict the binding affinity of enzymes and Ce6. Based on this criterion, the authors successfully developed CAT-Ce6 NCs with exceptional stability, which efficiently remodeled the hypoxic pathological microenvironment and completely eradicated pathogens, thereby achieving catalysis-augmented PDT of bacterial infections. This study establishes a theoretical framework to guide the design of physically bonded enzyme–PS conjugates and facilitate catalysis-augmented PDT.

Brief Introduction of the Corresponding Author

Dr. Yong-Qiang Li, a professor at Shandong University, mainly focuses on research in the fields of nanomaterials and biophysics. He has received the Second Prize of Jiangsu Province's Scientific Research Achievements (2018), the Third Prize of Jiangsu Province's Science and Technology (2023), the Third Prize of Natural Science of Hubei Province (2021), and the Imagination Award from Ocean Optics (2021). He serves as a reviewer for the National Natural Science Foundation of China, a communication and meeting reviewer for the Ministry of Science and Technology's Key Research and Development Program, a young editorial board member for Research, Nano-Micro Letters, and Interdisciplinary Medicine, and a standing committee member of the Tissue Regeneration Branch of the China Association for Medical Equipment. He has published over 80 academic papers in international authoritative SCI journals such as PNAS, Adv. Mater., Angew. Chem. Int. Ed., ACS Nano, and Adv. Sci., with a total citation of over 5,000 times. He has been granted 8 national invention patents (2 of which have been transferred), and has led multiple projects including the National Natural Science Foundation of China, the Key Research and Development Program of the Ministry of Science and Technology, the Youth Taishan Program, the Jiangsu Province's Outstanding Youth Program, and the Tang Zhongying Foundation Research Project.

Dr. Yanmei Yang, a professor at Shandong Normal University, mainly focuses on the application of multifunctional nanomaterials in the biomedical field, including mass spectrometry detection, disease diagnosis and treatment, and proteomics. She has led and participated in numerous national and provincial-level scientific research projects, published nearly 50 academic papers (including 3 ESI highly cited papers), with over 3,600 citations (H-index = 29), and has been granted 6 national invention patents. She was selected for Jiangsu Province's "Double Innovation Plan - Overseas World-Class University Doctoral Program", Suzhou's High-level Talent Shortage in Higher Education Institutions and Research Institutes, and Shandong Province's Introduction of High-level Talents, and was also selected as a Young Expert of Shandong Province's Taishan Scholars Program.

Sources: https://doi.org/10.34133/research.0732