A material designed to change acidity inside cells was associated with less inflammatory microglial activity and better recovery measures after ischemic stroke in experiments with mouse cells and mice. The nanoparticle treatment, called sulfonated Nano Proton Scavengers, or sNPS, was linked to smaller areas of brain injury, better tissue preservation, higher survival and improved neurological scores in the study’s mouse model.
The work focuses on endosomal acidity, or the pH inside endosomes and lysosomes, the cell compartments examined in the experiments. The researchers asked whether changing that environment could alter microglial behavior through TLR3 and TLR4 pathways, two signaling routes examined in the study. The results support a possible materials-based way to temper damaging inflammation, but they do not show that sNPS causes the same benefits in people.
Changing the cell’s internal environment
sNPS formed nanoscale particles with a reported hydrodynamic size of 101.6 ± 25.65 nm. The study also reported an isoelectric point of about pH 8.78 and a critical micelle concentration of 72.65 µg mL −1, while its absolute zeta potential exceeded 30 mV below pH 7.66.
The experiments combined mouse-derived BV2 microglia, primary microglia, neuronal cells, neural stem cells and blood-brain barrier cocultures with mice subjected to transient middle cerebral artery occlusion, a procedure used here to model ischemic stroke. The researchers compared sNPS with PBS and unassembled sulfonated chitosan, or SCS, and used pharmacological and inflammatory controls in cell experiments.
In the primary-microglia tests, the researchers used a ratio between a signal tracking overall dextran uptake and a signal that indicates acidic endosomal and lysosomal compartments. LPS-associated inflammation corresponded to a lower ratio, while sNPS was associated with a marked increase. The strongest reported response came from combining sNPS with BFA1, a pharmacological inhibitor used to block endosomal acidification.
The strongest clues came from cell signaling
Under the reported conditions, sNPS was associated with stronger attenuation of the LPS-related TLR4/NF-κB pathway and the poly(I:C)-related TLR3/IRF3 pathway than SCS. The response was comparable to BFA1. In these assays, the researchers assessed activated signaling proteins relative to their total amounts, including , the phosphorylated p65 signal divided by total p65, and , the corresponding IRF3 ratio.
Other measurements pointed to changes in the way endosomes matured and interacted with the proton-pumping machinery that helps acidify them. Relative to the LPS-associated pattern, sNPS was associated with higher Rab5 signal, lower Rab7 and LAMP1 signals, and lower recruitment of the V1A part of V-ATPase to membranes. Measured total V-ATPase abundance did not differ significantly among groups, suggesting that the reported pattern involved where the machinery was located rather than how much of it was present overall.
After systemic administration to stroke-model mice, fluorescent sNPS became progressively more concentrated in the hemisphere on the same side as the injury at 12 and 24 hours. Free SCS showed no comparable hemispheric preference. The labeled brain populations were mainly microglia, neutrophils and macrophages, although the measurements could not separate immune-cell carriage from blood-brain barrier disruption, leakage, retention or lingering signal in blood vessels.
Better recovery measures, with important gaps
The biological changes coincided with a broad set of tissue and behavior findings. sNPS-associated microglial changes coincided with more favorable neural stem-cell marker profiles, better preservation of MAP-2-associated neuronal structure and preservation or remodeling of dendritic spines. In the tMCAO experiments, it was also associated with lower infarct injury, better histological preservation, higher survival and Garcia neurological scores during the 14-day observation, plus improvement in selected sensorimotor and open-field outcomes. The size and consistency of the effect varied by endpoint.
The study also reported changes in gene activity across whole brain hemispheres. Compared with PBS, sNPS was associated with 1,288 lost expressed genes and 1,066 additional expressed genes, including 111 significantly upregulated and 144 downregulated genes. A lower fraction of CD86-positive microglia was most evident on days 1 and 3, while a CD86-negative/CD206-negative population was higher. These markers offer a broad view of microglial states, not a complete map of their behavior.
These results should be read as preclinical findings, because the experiments used cells and mice and the mouse endpoints came from separate, assay-specific cohorts. The reported cohorts included four to five mice for TTC, five for histology, 50 for survival, five to seven for sensorimotor tests and five for open-field testing. Figures marked thresholds ranging from to , where p is the reported p-value, but the specific statistical tests and corrections were not reported.
Whole-hemisphere RNA sequencing cannot show that the gene changes came from microglia themselves, and the fluorescence results could not determine whether the signal represented intact particles or other material. The study therefore supports a preclinical research direction, not clinical efficacy or safety. Cell-type-specific sequencing, better tracking of how long buffering persists inside cells and longer-term studies would be needed before the approach could be judged for translation to human stroke care.
Paper data and sources
Original title: Nano Proton Scavengers Modulate Endosomal pH to Inhibit Microglial Activation and Enhance Stroke Recovery.
Authors: Xuanlin Wang, Yujing Shi, Zehua Gao et al.
Journal/Repository: Advanced science (Weinheim, Baden-Wurttemberg, Germany)
Status: Peer-reviewed
First online: 2026-08-21
DOI: 10.1002/advs.77311
Original paper