Injectable protein hydrogel microspheres with reactive oxygen species-responsive nitric oxide release for cardiac protection against ischemia/reperfusion injury

Nitric oxide (NO) can alleviate cardiac ischemia/reperfusion injury with its anti-inflammatory, antioxidant, and angiogenic effects. However, local NO availability is limited due to its short half-life, reduced production, and consumption by excess reactive oxygen species (ROS) generated in injured myocardium. Here, we designed an injectable hydrogel microsphere system (WPI-H-N) based on acrylated whey protein (WPI), onto which phenylborate ester group was attached and served as a ROS-cleavable linker for 5-isosorbide mononitrate (ISMN), a NO donor. This injectable hydrogel microsphere system was designed to scavenge excess ROS, and release NO in response to oxidative stress in the niche in order to achieve on-demand NO release, reduce NO depletion by ROS, and prolong NO retention in the infarcted myocardium. In a rat I/R model, WPI-H-N protected cardiomyocytes from apoptosis, attenuated cardiac oxidative injury and improved angiogenesis in the infarcted myocardium. These results demonstrate that the combination of ROS scavenging and responsive NO release can simultaneously overcome the two major limitations of NO therapy, supporting the development of more efficient NO delivery strategies. STATEMENT OF SIGNIFICANCE: This study presents an injectable hydrogel microsphere system that synergistically scavenges reactive oxygen species (ROS) and enables on-demand nitric oxide (NO) release for cardiac protection against ischemia/reperfusion injury. Unlike existing NO delivery platforms, the ROS-responsive phenylborate ester linkage ensures spatiotemporally controlled NO release, minimizing premature consumption by ROS and secondary nitrosative stress. The microspheres' dual functionality-simultaneously neutralizing oxidative stress and promoting angiogenesis-addresses critical limitations of conventional NO therapies. In vivo results demonstrate significant reductions in cardiomyocyte apoptosis, oxidative damage, and infarct size, alongside improved cardiac function and vascularization. This strategy offers a potentially translatable approach for local and controlled NO release to achieve cardiac repair. The work holds broad implications for ROS-related pathologies and precision therapeutic delivery in regenerative medicine.