Development of functional Prussian blue nanocomposites for biomedical applications

Abstract

Prussian blue (PB) nanozyme, a biocompatible iron hexacyanoferrate coordination complex, possesses multienzyme-like activities including those mimicking peroxidase, catalase, and superoxide dismutase. These inherent properties enable it to act as a highly efficient reactive oxygen species (ROS) scavenger. Its ability to degrade H2O2 and scavenge superoxide radicals allows it to mitigate excessive intracellular ROS under oxidative stress, exhibiting a significant cytoprotective effect. Furthermore, the PB nanozyme demonstrates remarkable anti-inflammatory efficacy, as evidenced by its capacity to suppress inflammatory mediators in immune cells. Based on the synergistic effect of its ROS-scavenging and anti-inflammatory activities, PB nanozyme has been extensively utilized in biomedical applications, including wound healing and tissue regeneration, as well as the treatment of ROS-related inflammatory diseases such as osteoarthritis and acute kidney injury. The precise control over the size and colloidal stability of PB nanocomposites using various templating materials is a key aspect that broadens their application in biomedical fields. Functional PB nanocomposites have been studied for biomedical applications in this thesis. In Chapter 2, we developed novel electrospun nanofibers (NFs) by integrating ROS-scavenging chitosan-templated PB nanoparticles (PBChi NPs) into a poly(vinyl alcohol) matrix to impart antioxidant and proliferative properties. The PBChi NPs were synthesized using chitosan of varying molecular weights, and their optimal loading within the PVA matrix was optimized to maintain a well-defined NF structure. Both in situ and in vitro assays confirmed the effective ROS elimination capacity of PBChi/PVA NFs. Notably, NFs prepared with lower molecular weight chitosan (PBChi10k/PVA) exhibited superior antioxidant activity with excellent biocompatibility. Treatment with the PBChi/PVA NFs significantly enhanced cell proliferation in an in vitro scratch assay. These findings establish PBChi/PVA NFs as a promising, ROS-scavenging wound dressings with significant regenerative potential. In Chapter 3, we prepared a novel PB nanocomposites through the controlled nucleation and stabilization of PB within Pluronic micelles (PPBzymes). The resulting nanocomposites maintained its uniform sizes under both aqueous and biological conditions, confirming their excellent colloidal stability for biomedical uses. In vitro assessments revealed that PPBzymes possess the capacity to both promote cartilage generation and mitigate degradation. Importantly, intra-articular administration in mouse joints showed that the PPBzymes maintained long-term stability and were effectively internalized by the cartilage matrix. Furthermore, these localized injections attenuated cartilage degradation without inducing systemic cytotoxicity in the synovial membrane, lungs, or liver. Further investigation using proteome microarray identified that PPBzymes specifically block the phosphorylation of JNK, thereby modulating the inflammatory pathogenesis characteristic of osteoarthritis (OA). These results suggest that PPBzymes are a biocompatible and effective nanotherapeutic agent for OA treatement. In Chapter 4, we used levan polysaccharide to stabilize PB nanozymes (L-PB), resulting in a nanocomposites with improved colloidal stability, biocompatibility, and the capacity for inflammation site-targeting. L-PB maintained a stable hydrodynamic size in physiological media for more than two weeks. In vitro assays confirmed their safety, high ROS-scavenging efficiency, and markedly enhanced cellular uptake when compared to the non-targeting control, B-PB (PB stabilized with bovine serum albumin). In a murin model of glycerol-induced acute kidney injury (AKI), L-PB demonstrated selective accumulation within CD44 receptor-overexpressing injured kidneys. This targeting resulted in superior therapeutic benefits, significantly reducing both oxidative stress and inflammation, with minimal systemic toxicity relative to B-PB. These findings demonstrate L-PB as a novel, CD44-actively targeted therapeutic candidate for AKI, highlighting its potential for treating various inflammatory diseases. In conclusion, this thesis highlights the potential of PB nanocomposites as a therapeutic nanozyme platform in various biomedical applications. The PB nanocomposites were successfully used as a therapeutic nanozyme platform for wound healing, OA, and AKI.