Prenatal Delivery of HIF-1α siRNA Using Transferrin-Modified Lipid Nanoparticles Alleviates Hypoxia-Induced Neurodevelopmental Abnormalities via PTEN/PI3K/AKT Signaling

Purpose: Prenatal hypoxia is a major environmental risk factor for neurodevelopmental disorders, yet effective prenatal therapeutic strategies remain lacking. This study aimed to develop a transferrin-modified lipid nanoparticle platform for targeted delivery of HIF-1α siRNA to the fetal brain and to evaluate its therapeutic efficacy and molecular mechanisms. Methods: Transferrin-modified lipid nanoparticles encapsulating HIF-1α siRNA were intravenously administered to pregnant rats prior to hypoxic exposure. Biodistribution, gene silencing efficiency, molecular signaling alterations, neuronal structural changes, and behavioral outcomes in offspring were systematically assessed. Results: In this study, we developed a transferrin-modified lipid nanoparticle system for non-invasive, transplacental delivery of HIF-1α small interfering RNA to the fetal brain, achieving a siRNA encapsulation efficiency of 84.18% and a loading capacity of 2.5%. Systemic administration to pregnant rats prior to hypoxic exposure resulted in preferential accumulation of nanoparticles in fetal brain tissue and effective suppression of HIF-1α expression in the fetal hippocampus without overt effects on offspring survival, as evidenced by comparable offspring survival rates across all groups (p > 0.05). Prenatal hypoxia induced sustained elevation of HIF-1α protein (p < 0.001), impaired phosphatase and tensin homolog (PTEN) activity through increased phosphorylation (p < 0.01), aberrant activation of the PI3K/AKT signaling pathway (p < 0.05), and deficits in hippocampal neuronal structural plasticity, including reduced dendritic spine density (p < 0.0001) and dendritic complexity (p < 0.0001), accompanied by autism-like behaviors in offspring, including impaired social preference (p < 0.01), prolonged self-grooming (p < 0.0001), and increased marble-burying (p < 0.001). Prenatal HIF-1α silencing restored PTEN functional status, normalized PI3K/AKT signaling, improved dendritic architecture to levels comparable to controls (p > 0.05 vs control), and significantly ameliorated behavioral abnormalities (social preference and stereotyped behaviors, p < 0.0001). Mechanistic analyses revealed that although HIF-1α binds to the PTEN promoter (ChIP-qPCR, p < 0.01), prenatal hypoxia did not alter PTEN transcript or total protein levels (p > 0.05), indicating that HIF-1α primarily regulates PTEN function at the post-transcriptional level in vivo. Conclusion: These findings identify a HIF-1α/PTEN/PI3K/AKT signaling axis as a key molecular pathway underlying hypoxia-associated neurodevelopmental impairment and demonstrate the feasibility of targeted prenatal gene modulation using transferrin-modified lipid nanoparticles. This work provides a nanomedicine-based framework linking environmental risk factors to early-life preventive strategies for neurodevelopmental disorders. The diagram illustrates the effects of prenatal hypoxia on fetal development and behavior in rats. On the left, a rat is shown experiencing low oxygen levels, labeled as ‘Prenatal Hypoxia’. This condition leads to increased HIF-1 alpha in the fetal hippocampus. An intervention using tLNP/siHIF is depicted to inhibit PTEN functionally. This process affects the PI3K, AKT and p-AKT pathways. On the right, impaired dendritic plasticity and ASD-like behaviors are shown as outcomes, with two rats depicted to represent these behaviors.Diagram of prenatal hypoxia effects on fetal hippocampus and ASD-like behaviors in rats.