Dual-Modified Mannose/RVG29 Peptide-Functionalized Lipid Nanoparticles Loaded With circHIPK2 siRNA Ameliorate Hypoxic-Ischemic Brain Damage in Neonatal Mice by Suppressing Astrocyte Activation

Background: To address the unmet need for targeted therapeutic strategies for neonatal hypoxic-ischemic encephalopathy (HIE), we developed a brain-targeting lipid nanoparticle delivery system capable of silencing circular RNA homeodomain-interacting protein kinase 2 (circHIPK2) in astrocytes and investigated its ability to mediate neuroinflammation and improve neurological outcomes.

Methods: Dual-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly (ethylene glycol)-neurotropic virus-derived peptide (DSPE-PEG2000-RVG29) peptide/mannose-functionalized lipid nanoparticles loaded with circHIPK2 small interfering RNA (M-R@siC-NPs) were constructed, and their physicochemical properties, stability, and biocompatibility were characterized. Using an in vitro oxygen-glucose deprivation (OGD) model and a neonatal murine hypoxic-ischemic brain damage (HIBD) model, we evaluated the effects of circHIPK2 silencing by the M-R@siC-NPs on the expression of two astrocyte activation markers, glial fibrillary acidic protein (GFAP) and interleukin-1β (IL-1β), via western blotting, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and immunofluorescence staining. Neurobehavioral recovery was assessed through righting reflex, negative geotaxis, and Morris water maze tests.

Results: M-R@siC-NPs exhibited a uniform size distribution (134 nm), good blood-brain barrier penetrability, and astrocyte-targeting specificity. The nanoparticles effectively silenced circHIPK2 while demonstrating excellent colloidal stability and biosafety. In vitro, circHIPK2 knockdown by M-R@siC-NPs markedly suppressed OGD-induced astrocyte activation, reducing GFAP and IL-1β expression (p < 0.01). In HIBD mice, M-R@siC-NPs attenuated hippocampal astrocyte activation and improved motor coordination (shortened righting reflex latency, p < 0.0001) and spatial memory (increased platform crossings in Morris water maze, p < 0.0001).

Conclusions: The RVG29/mannose dual-modified M-R@siC-NPs precisely regulated astrocyte activation and attenuated neuroinflammation, effectively ameliorating brain injury in HIBD mice. This study establishes a novel RNA interference-based therapeutic strategy for targeted neuroinflammatory modulation, providing a promising translational platform for HIE treatment.

circHIPK2; astrocyte activation; hypoxic–ischemic encephalopathy; lipid nanoparticles; targeted gene therapy.