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

Abstract

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.

Graphical abstract

Keywords

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

1. Introduction

Neonatal hypoxic–ischemic encephalopathy (HIE), triggered by perinatal asphyxia, poses a critical threat to neonatal health and represents a major cause of neonatal mortality and childhood disability [1, 2, 3, 4]. Its incidence ranges from 1–8% in developed countries and is reportedly as high as 26% in underdeveloped regions, with global mortality rates of 10–60%. Among survivors, at least 25% of affected infants suffer long-term neurodevelopmental sequelae, with 15–28% of pediatric cerebral palsy cases attributed to HIE [5, 6]. The pathological mechanisms involve energy metabolism dysfunction, excitotoxicity, oxidative stress, neuroinflammation, and apoptosis.

Current therapeutic options for HIE remain limited. While therapeutic hypothermia—the clinical gold standard—has demonstrated neuroprotective effects via the attenuation of astrocyte overactivation, 44–53% of treated infants still experience mortality or severe neurological deficits [7]. Stem cell transplantation, constrained by a narrow therapeutic window, dosage uncertainties, and individualized strategy challenges, faces limited clinical applicability [8]. These limitations underscore the urgent need for novel therapeutic approaches for HIE.

Astrocytes exhibit dual roles in central nervous system (CNS) inflammation [9, 10, 11, 12, 13]. While they are initially protective during lesion isolation and blood–brain barrier (BBB) restoration [14, 15], they become harmful under ischemic stress in HIE. In the latter context, overactivated astrocytes release pro-inflammatory cytokines such as tumor necrosis factor-\alpha (TNF-\alpha) and interleukin-1\beta (IL-1\beta), disrupt BBB integrity, and amplify neuroinflammation, perpetuating a pathological cascade [16, 17, 18, 19, 20].

Circular RNAs (circRNAs), which are covalently closed structures lacking 5^′ caps and 3^′ poly(A) tails, have emerged as key regulators in brain disorders due to their exceptional stability [21, 22, 23]. For example, circHIPK2 has been identified as a key regulator of astrocyte activation through a molecular mechanism involving miR-124-2HG sequestration and subsequent Sigma non-opioid intracellular receptor 1 (SIGMAR1)-mediated endoplasmic reticulum stress/autophagy pathway activation [24]. In lipopolysaccharide-induced neuroinflammation models, activation of the cortical circHIPK2/SIGMAR1 axis correlates with upregulated TNF-\alpha and IL-1\beta expression, whereas circHIPK2 knockdown effectively suppresses astrocyte activation and inflammatory responses [25]. These findings suggest that circHIPK2 silencing may represent a promising therapeutic strategy for HIE.

The BBB, although a critical protective mechanism, severely hinders drug delivery to the CNS [26, 27, 28, 29]. RNA interference (RNAi) technology offers therapeutic potential, yet the application of small interfering RNA (siRNA) as a drug faces clinical translation challenges due to rapid in vivo degradation and poor cellular uptake [30, 31]. Delivery of siRNA within nanocarriers can address these limitations through enhanced biocompatibility and modifiability, enabling efficient siRNA encapsulation and delivery [32, 33]. Mesoporous silica nanoparticles (MSNs) offer tunable pore structures and high drug-loading capacity, making them promising gene vectors [34, 35, 36, 37, 38]. Lipid nanoparticles (LNPs) have been validated for safety and efficacy and shown to improve targeted delivery while minimizing off-target effects [32, 39, 40, 41]. For example, glioma-targeting LNPs developed by Kuang et al. [42] demonstrated low systemic toxicity in clinical applications. However, delivery systems specifically for modulation of circHIPK2 expression remain unexplored, hindering the translational progress for circHIPK2-based therapies.

Recent advances have highlighted RVG29, a 29-amino-acid peptide derived from rabies virus glycoprotein, as a BBB-penetrating ligand. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly (ethylene glycol)-neurotropic virus-derived peptide (DSPE-PEG2000-RVG29) binds to nicotinic acetylcholine receptors on endothelial cells of the BBB and facilitates receptor-mediated transcytosis [43, 44, 45, 46, 47]. Concurrently, overexpression of mannose receptor on astrocytes enables targeted delivery by mannose-decorated nanoparticles [48, 49, 50, 51].

In the present study, we engineered a dual-targeted siRNA nanoplatform consisting of RVG29 peptide/mannose-functionalized LNPs loaded with circHIPK2 siRNA (M-R@siC-NPs) as a potential therapy for HIE. This approach attempted to combine the advantages of LNPs and MSNs to achieve precise astrocyte targeting, suppress their pathological activation, and ameliorate neonatal hypoxic–ischemic brain injury. We tested the efficacy of M-R@siC-NPs both in vitro and in vivo, and the results indicate that this innovative strategy may provide novel therapeutic avenues for HIE and advance RNAi-based targeted gene therapy.

2. Materials and Methods

2.1 Materials

Aminated dendritic MSNs (NH2-MSNs, cat no. R-SG-89NH2), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, cat no. LP-R4-057), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000, cat no. R-H54510), DSPE-PEG2000-RVG29 (cat no. R9985), Cy5.5 (cat no. D10061), and M-PEG-lipid (cat. no. LP-R4-18) were purchased from Ruixi Biotechnology Co., Ltd. (Xi’an, Shaanxi, China). Three siRNA (cat no. 15258) sequences targeting circHIPK2 (circHIPK2 siRNA) and a non-homologous negative control (siNC) without sequence homology to the target gene, along with fluorescein amidite (FAM)-labeled siRNA, were obtained from Qingke Biotechnology Co., Ltd. (Beijing, China). The siRNA-mate Plus transfection kit was acquired from GenePharma Co., Ltd. (cat no. G04026, Shanghai, China). Chloroform (CHCl₃ , cat no. CX1060) and other analytical-grade reagents were sourced from Chongqing Chuandong Chemical Group Co., Ltd. (Chongqing, China). Fetal bovine serum (FBS) was acquired from Lanzhou Minhai Bio-Engineering Co.Ltd. (cat no. SA211.02, Lanzhou, Gansu, China). Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12, cat no. 11320033), and 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA, cat no. 25200056) were purchased from Gibco BRL (Grand Island, NY, USA). The Cell Counting Kit-8 (CCK-8) was obtained from Boster Biological Technology Co., Ltd. (cat no. AR1199, Wuhan, Hubei, China). Trizol (cat no. RR036A), PrimeScript RT reagent Kit (cat no. RR092S), and SYBR Green PCR Master Mix (cat no.RR820A) were supplied by Takara Bio, Inc. (Shiga City, Shiga Prefecture, Japan). Quantitative real-time polymerase chain reaction (qPCR) primers (cat no. TSE20240906-029) were synthesized by ZerKcorp Biotech (Xi’an, Shaanxi, China). Agarose powder (cat no. GC205013), SerRed Nucleic Acid Stain (10,000\times, water-soluble, cat no. G3606), phosphate-buffered saline (PBS, cat no. G4202), Tris-buffered saline with Tween 20 (TBST, cat no. G2157), Tris-glycine sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) high-resolution rapid electrophoresis (cat no. G2081)/transferring buffer(cat no. G2164), paraformaldehyde fixative (cat no. G1101), hematoxylin and eosin (H&E) staining solutions (cat no. G1076), and cell culture consumables were purchased from Servicebio (Wuhan, Hubei, China). C57BL/6 (C57) neonatal mice were obtained from the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Lanzhou, Gansu, China). Glial fibrillary acidic protein (GFAP) antibody (Abcam, cat no. ab7260, Shanghai, China; 1:10,000 dilution), IL-1\beta antibody (Proteintech, Wuhan, Hubei, China; 1:5000 dilution), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Proteintech, cat no. 60004-1-Ig, Wuhan, Hubei, China; 1:5000 dilution) were used for immunoblotting.

2.2 Screening of siRNAs Targeting circHIPK2

Specific siRNAs were designed to silence the expression of circHIPK2. The efficacy of circHIPK2 silencing was evaluated using quantitative reverse transcription PCR (qRT-PCR) and western blotting. A total of four groups were established: three experimental groups transfected with each siRNA targeting circHIPK2 (circHIPK2-1, circHIPK2-2, and circHIPK2-3) and one control group without siRNA transfection. The siRNA-mate Plus transfection reagent was used to deliver the siRNAs into astrocytes. The siRNA demonstrating the most efficient silencing effect was selected for subsequent experiments (Table 1).

2.3 Electrophoretic Mobility Shift Assay (EMSA)

Agarose gel electrophoresis was performed to evaluate the siRNA loading capacity of NH₂-MSNs. At room temperature, siRNA (218 µg/mL) and NH₂-MSNs (5 mg/mL) were mixed at mass ratios (1:0, 1:10, 1:20, 1:25, 1:30, w/w), followed by incubation for 30 min. After incubation, 6\times DNA loading buffer was added to the mixture. Samples were separated on a 3% agarose gel at 100 V for 20 min. The gel was visualized using a gel imaging system (version S2101237, ChampGel6000, Beijing, China) to observe band shifts of NH₂-MSN-siRNA complexes. The optimal binding ratio was determined to guide the preparation of M-R@siC-NPs.

2.4 Preparation of M-R@siC-NPs

Liposomes capable of crossing the BBB were prepared by mixing DPPC, cholesterol, M-PEG-lipid, and DSPE-PEG2000-RVG29 at a mass ratio of 3:1:1:1, dissolving the mixture in chloroform, and evaporating the solvent under vacuum to form a lipid film. The film was hydrated with 4 mL PBS and sonicated for 20 min to obtain a nanoemulsion. The emulsion was then emulsified under ice-cold conditions with 125 W ultrasonication for 8 min, followed by centrifugation at 6100 \timesg for 10 min at 4 °C (repeated three times). The resulting product was purified via dialysis using a 100-nm cellulose membrane to obtain the liposomes.

For nanoparticle assembly, circHIPK2 siRNA and NH₂-MSNs were mixed at a mass ratio of 1:30, incubated at room temperature for 10 min, and electrostatically bound to form NH₂-MSN-siRNA complexes. The liposomes were then combined with NH₂-MSN-siRNA at a 1:1 mass ratio, incubated at room temperature for 10 min, and subjected to ultrasonication (3 min pulses with 5-s intervals). After centrifugation at 6100 \timesg for 10 min, the precipitate was resuspended and stored at 4 °C.

As controls, target-free LNPs (siC-NPs) were prepared using the same method but omitting DSPE-PEG2000-RVG29 and M-PEG-lipid. The full name of siC-NPs is non-targeted modified circHIPK2 siRNA-lipid nanoparticles, which serve as non-targeted control nanoparticles in this study. Their core carrier components are consistent with those of M-R@siC-NPs (mannose/RVG29 peptide dual-modified circHIPK2 siRNA-lipid nanoparticles), with the only difference being the lack of two targeted ligands, namely M-PEG-lipid and DSPE-PEG2000-RVG29. Both siC-NPs and M-R@siC-NPs carry circHIPK2 siRNA3. Fluorescently labeled Cy5.5-conjugated targeting (Cy5.5-M-R@siC-NPs) and non-targeting nanoparticles (Cy5.5-siC-NPs) were also synthesized for fluorescence imaging.

2.5 Nanoparticle Characterization

The Malvern Nano analyzer (cat no. ZEN-3600, Zeta-Sizer, Malvern Instruments, Malvern, UK) was used to measure particle size and surface zeta potential. For sample preparation, 10 µL of nanoparticle solution was spotted onto a 400-mesh carbon-coated copper grid, air-dried at room temperature, and imaged using transmission electron microscopy (TEM, cat no. HT7800, Hitachi, Tokyo Metropolis, Japan) to observe the morphology, structure, and size of NH₂-MSNs and M-R@siC-NPs.

2.6 Stability Assessment

The colloidal stability of M-R@siC-NPs was evaluated by monitoring hydrodynamic particle size changes via The Malvern Nano analyzer over 14 days (0, 2, 4, 6, 8, 10, 12, and 14 days). Additionally, M-R@siC-NPs suspensions were separately incubated in DMEM/F12 cell culture medium, PBS (pH 7.4), pure FBS, and artificial cerebrospinal fluid (aCSF) at ambient temperature (25 °C). Morphological changes, including macroscopic aggregation or precipitation, were observed and recorded daily for 14 days. This temperature represents the typical ambient temperature used for simulating the storage of nanoformulations and in vitro experiments, and it is also consistent with the specifications on room temperature conditions for long-term stability testing as outlined in the “Guidelines for the Stability Testing of Pharmaceutical Preparations” of the Chinese Pharmacopoeia (25 °C \pm 2 °C).

2.7 Encapsulation Efficiency and Drug Loading Capacity Determination

The siRNA concentration was quantified using a microvolume UV-Vis spectrophotometer (cat no. DENOVIX DS-11, DENOVIX, Wilmington, DE, USA) at 260 nm. Encapsulation efficiency (EE, %) was calculated via centrifugation (14,000 \timesg, 30 min) to separate unencapsulated siRNA. The formula used to calculate EE was:

\text{EE(\%)}=\frac{\text{Total siRNA in NPs}-\text{Free siRNA}}{\text{Total siRNA in NPs}}\times 100

Drug-loading capacity (DLC, %) was calculated using the following formula:

\mathrm{DLC}(\%)=\frac{\mathrm{Total}\mathrm{siRNA}\text{in NPs}\times \text{EE(\%)}}{\text{Total lipid nanoparticle mass}}\times 100

2.8 Experimental Animals

C57BL/6 neonatal mice (specific pathogen-free grade) were purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. All histopathological evaluations were guided by faculty members specializing in pathology from the School of Basic Medical Sciences, Lanzhou University. The animal experimental protocols were approved by the Animal Ethics Committee of the Second Hospital of Lanzhou University (approval number: D2024-439).

2.9 Cell Viability Assay

Primary astrocytes were seeded into 96-well plates at a density of 1 \times 10⁴ cells/mL (100 µL per well). Edge wells were filled with sterile PBS to minimize edge effects. Cells were allowed to adhere for 24 h at 37 °C in an incubator with 5% CO₂. Solutions containing PBS (control), siC-NPs, and M-R@siC-NPs were prepared at gradient concentrations (0, 20, 40, 60, 80, 100, and 120 µg/mL). Primary astrocytes were treated with the respective solutions, with three replicates per group, and incubated for an additional 24 h. Prior to measurement, the supernatant was aspirated, and 10 µL of CCK-8 reagent was added to each well followed by 90 µL of serum-free medium. The plates were incubated in the dark for 2 h. Cell viability was assessed using a microplate reader (cat no. ELX808, Bio Tek Instruments, Inc., Windsor, VT, USA) across four groups (normal control (NC), oxygen-glucose deprivation (OGD), siC-NPs, M-R@siC-NPs) to evaluate nanoparticle cytotoxicity. During the culture and identification of astrocytes, detailed descriptions have been provided regarding their cellular morphology and the expression of the specific marker GFAP, and the result of mycoplasma testing was confirmed to be negative.

2.10 Hemolysis Assay

Nanoparticle solutions (20, 40, 60, 80, and 100 µg/mL), PBS (negative control), or triple-distilled water (positive control) were added to 1.5-mL eppendorf tubes (1 mL per group). Blood was collected from the orbital venous plexus of healthy C57BL/6 mice using EDTA anticoagulant tubes. After mixing, the blood was centrifuged at 3000 \timesg for 15 min at room temperature. The plasma was discarded, and 20 µL of red blood cell pellet was resuspended in the respective solutions. The mixtures were incubated at 37 °C for 4 h before repeated centrifugation at 3000 \timesg for 15 min. The supernatant (100 µL) was transferred to a 96-well plate, and absorbance (optical density, OD) was measured at 540 nm using a microplate reader (Bio Tek Instruments, Inc.). Hemolysis rates were calculated using the standard formula:

\text{Hemolysis rate}(\%)=\frac{\mathrm{OD}(\text{sample})-\mathrm{OD}(\mathrm{PBS})}{\mathrm{OD}\left({\mathrm{ddH}}_2\mathrm{O}\right)-\mathrm{OD}(\mathrm{PBS})}\times 100\%

2.11 Toxicity Assessment

In strict accordance with the guidelines on non-clinical safety studies of pharmaceuticals issued by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), these guidelines explicitly require that the potential effects of pharmaceuticals on major physiological functions (cardiovascular, respiratory, central nervous systems) be evaluated. Additionally, the study design and implementation refer to the Good Laboratory Practice for Non-Clinical Studies (GLP) promulgated by the National Medical Products Administration (NMPA) of China. Although this study does not belong to the anti-tumor field, the requirement in this guideline that nanopharmaceuticals should focus on carrier accumulation, organ-targeted distribution, and long-term toxicity is universally applicable. To evaluate the systemic toxicity of the nanoparticles, 10-day-old C57BL/6 neonatal mice were randomly divided into four groups (n = 3 per group, statistical analysis is conducted based on the complete set of sample data): a NC group, PBS group, siC-NPs group (80 µg/mL), and M-R@siC-NPs group (80 µg/mL). Nanoparticles were administered intravenously for 20 consecutive days. Blood samples were collected for complete blood count (CBC) analysis using a Mindray Veterinary Automated Hematology Analyzer (cat no. BC-2800vet, Mindray Biomedical Electronics Co., Ltd. Shenzhen, Guangdong, China). Serum was obtained via refrigerated centrifugation, and biochemical parameters were analyzed using an Automated Biochemical Analyzer (cat no. Chemray 240, Rayto Life Technology Co., Ltd. Shenzhen, Guangdong, China). Mice were anesthetized via intraperitoneal injection of 1% sodium pentobarbital at a dose of 50 mg/kg body weight. Anesthesia was confirmed when the mice exhibited no response to toe pinch and loss of corneal reflex. Subsequently, the mice were euthanized by cervical dislocation, in compliance with the Animal Ethics Guidelines of the Second Hospital of Lanzhou University. Death was verified by the absence of breathing and heartbeat for 5 consecutive minutes. After euthanasia, heart, liver, spleen, lung, kidney, and brain tissues were collected, fixed, and stained with H&E. Histopathological changes were quantitatively analyzed using the TissueFAXS Plus panoramic tissue cytometry system (cat no. TissueFAXS Plus, TissueGnostics GmbH, Vienna, Vienna State, Austria).

2.12 In Vitro Fluorescence Imaging

Primary astrocytes were seeded on confocal dishes at a density of 1 \times 10⁵ cells/mL and allowed to adhere overnight in an incubator. Experimental groups included: a PBS group, Cy5.5-siC-NPs group (80 µg/mL), and Cy5.5-M-R@siC-NPs group (80 µg/mL). After 24 h in culture, Hoechst 33258 (cat no. BL804A, Biosharp Biotechnology Co., Ltd. Nanjing, Jiangsu, China) was used to stain cell nuclei. Fluorescence imaging of the cells was performed 10 min after staining using a laser scanning confocal microscope (version ZEN Blue 2.3, Zeiss LSM880, Jena, Germany).

2.13 In Vivo Fluorescence Imaging

Ten-day-old C57BL/6 neonatal mice were randomly divided into three groups (n = 9 per group): a PBS group, Cy5.5-siC-NPs group, and Cy5.5-M-R@siC-NPs group. The corresponding agents were intraperitoneally injected into each group. At time points of 0, 1, 2, 4, 6, 8, 10, 12, 24, and 48 h post-injection, in vivo fluorescence imaging was performed using a small-animal imaging system (Vie-works Smart-LF, Eschborn, Hessen, Germany) to monitor nanoparticle accumulation and assess targeting specificity. Mice were euthanized when the peak intracranial fluorescence intensity was achieved, and brain tissues were harvested for ex vivo imaging. Heart, liver, spleen, kidney, and lung tissues were also collected and imaged using the Vie-works Smart-LF small-animal live imaging system to observe the Cy5 fluorescence distribution in major organs [52]. Brain tissue sections were further processed with 4’,6-Diamidino-2-Phenylindole (DAPI, cat no. D1306, Thermo Fisher Scientific Inc. Waltham, MA, USA) staining for pathological examination.

2.14 Primary Astrocyte Isolation, Culture, and Characterization

Primary astrocytes were isolated from postnatal day 2–3 C57BL/6 neonatal mice anesthetized with isoflurane and disinfected with 75% ethanol. Whole brains were dissected under a biosafety cabinet, minced in pre-cooled Hank’s Buffered Salt Solution (HBSS) buffer, and digested with 0.25% trypsin-EDTA at 37 °C for 5 min. The digestion was halted by adding DMEM/F12 medium containing 10% FBS, followed by centrifugation at 1200 \timesg for 5 min for the collection of cell pellets. Cells were resuspended and cultured at 37 °C in an incubator with 5% CO₂. After 3–4 days in culture, contaminating oligodendrocytes and microglia were removed by shaking at of the culture plates 260 rpm for 12 h using a THZ-98A thermomixer (cat no. THZ-98A, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd., Shanghai, China). High-purity astrocytes were characterized via GFAP immunofluorescence staining, according to the following procedure. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, incubated overnight with rabbit anti-GFAP primary antibody (1:10,000) at 4 °C, and labeled with Alexa Fluor 488-conjugated secondary antibody (1:1000, Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature in the dark. Nuclei were counterstained with DAPI for 5 min. Stained samples were viewed using a Zeiss LSM880 laser scanning confocal microscope, and GFAP-positive cell proportions were quantified using ImageJ software (Version number: 1.54 g, NIH, Bethesda, MD, USA).

2.15 In Vitro and In Vivo Model Establishment

For the in vitro hypoxia–ischemia (HI) model, the cell culture medium was switched to serum-free medium 24 h before the cells were subjected to OGD. After two washes with PBS, the cells were incubated in serum-free and glucose-free medium and placed in a hypoxic chamber (1% O₂, 5% CO₂, 94% N2, cat no. Coy-HC-05, Coy Laboratory Products, Inc. Grass Lake, MI, USA) at 37 °C for 6 h. The medium was then exchanged with complete medium for 24–48 h of recovery culture before collection for further analysis.

For the in vivo HIBD model, 6–8-day-old C57BL/6 neonatal mice were anesthetized with 2% isoflurane. The left common carotid artery was permanently ligated with an 8-0 surgical suture. Post-surgery, mice were exposed to a hypoxic environment (8% O₂, 92% N₂, flow rate 1.5 L/min) in a chamber for 2 h. Successful establishment of the HIBD model was verified using laser speckle imaging. Mice were randomized to four groups: the sham group, HIBD group, siC-NPs group, and M-R@siC-NPs group. Brain morphology was observed at 3, 5, and 7 days post-surgery. Brain tissues were collected, sectioned, and subjected to H&E staining and immunofluorescence analysis for assessment of pathological changes.

2.16 Western Blot Analysis

After treatment, brain tissues and cells were washed 2–3 times with pre-cooled PBS. Protein lysates were prepared using radioimmunoprecipitation assay (RIPA) lysis buffer (Ailv Life, AIWB-012, Wuhan, Hubei, China) containing protease inhibitors, incubated on ice for 30 min, and centrifuged at 12,000 \timesg for 20 min at 4 °C for the collection of supernatant. Total protein concentration was determined using the BCA assay ( PC0020, Solarbio,, Beijing, China) and normalized. Equal amounts of proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, ISEQ00010, Darmstadt, Hessen, Germany). Membranes were blocked with QuickBlock blocking buffer (Servicebio, G2052-500ML) for 10 min at room temperature, followed by overnight incubation at 4 °C with primary antibodies: GFAP (1:10,000), IL-1\beta (Proteintech, 26048-1-AP; 1:5000), and GAPDH (Proteintech, CL594-60004; 1:5000). After washing with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000) at room temperature for 1 h. Protein bands were detected using a chemiluminescence imaging system (cat no. Tanon 5200 Multi, Shanghai Tanon Science & Technology Co., Ltd., Shanghai, China), and gray value quantification analysis was performed using ImageJ software.

2.17 Behavioral Assessments

Mice in the siC-NPs and M-R@siC-NPs groups (n = 12/group) received intraperitoneal injections of LNPs (80 µg/mL) for 5 consecutive days, followed by a 2-day pause, totaling four cycles over 28 days (100 µL per dose). Post-treatment, three mice per group were euthanized for histopathological analysis, including H&E staining and GFAP immunofluorescence staining to assess glial activation. Samples from parallel groups (n = 3/group) were used for western blot analysis to quantify GFAP and IL-1\beta expression in brain tissues.

2.17.1 Righting Reflex Test

This test was administered at the end of the first treatment cycle (day 5). Mice were placed ventral-side down on a soft pad, and the time to resume prone position was recorded (60 s threshold). The results from three trials per mouse were averaged for analysis.

2.17.2 Negative Geotaxis Test

This test was conducted post-modeling at the end of the first treatment cycle (day 5). Mice were positioned head-down at a 15° incline on a 30-cm slope. Latency to complete a 180° turn toward the top was measured (cutoff: 60 s). The results from three trials per mouse were averaged.

2.17.3 Morris Water Maze

This test was performed on post-treatment day 28 using a 120-cm diameter pool (water depth: 50 cm, temperature: 21 \pm 1 °C) containing a hidden platform (10-cm diameter, submerged 0.5 cm) in Quadrant 2. Six mice per group underwent four daily training sessions (120-s limit, 1-h intervals) over 4 days. On day 5, probe trials were initiated with platform removal. Target quadrant dwell time and swimming trajectories were tracked using the VisuTrack System (Shanghai Xinsoft, V3.0-2112, Shanghai, China).

2.18 Statistical Analysis

Statistical analysis was performed using GraphPad Prism 10.1.2 (GraphPad Software, Inc. La Jolla, CA, USA). All data are presented as mean \pm standard deviation (SD). Differences among multiple groups were compared using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. Pairwise comparisons between two groups were performed using Student’s t-test. Values of p 0.05 indicated statistical significance.

3. Results

3.1 Screening of Silencing Sequences for circHIPK2

In the development of functional nanoparticles for this study, the first challenge was to identify highly efficient circHIPK2 silencing sequences. For this purpose, primary astrocytes were transfected with three distinct circHIPK2 siRNAs (circHIPK2-1, circHIPK2-2, and circHIPK2-3). FAM-labeled siRNAs were utilized to track transfection efficiency (Fig. 1A). qRT-PCR results showed that compared with the Control group (without siRNA transfection), all three circHIPK2 siRNAs significantly reduced the expression level of circHIPK2 mRNA in primary astrocytes. Among them, circHIPK2 siRNA-3 exhibited the optimal silencing efficiency, mediating an 80.13% decrease in circHIPK2 mRNA expression. This efficiency was significantly higher than that of circHIPK2 siRNA-1 and circHIPK2 siRNA-2, with a statistically significant difference (p 0.0001), confirming that siRNA3 has the strongest ability to specifically recognize and degrade the target gene mRNA (Fig. 1D). We further evaluated the effect of the three siRNAs on GFAP expression via Western blot. The results demonstrated that the GFAP protein expression in the circHIPK2 siRNA-3 group was 40% lower than that in the Control group. This inhibitory effect was significantly more pronounced than those in the siRNA-1 and siRNA-2 groups (p 0.001), and was positively correlated with the high degradation efficiency at the mRNA level (Fig. 1B,C). These findings confirm that the silencing effect of siRNA-3 can be effectively transmitted to the protein expression level, forming a complete functional chain of gene silencing functional inhibition. circHIPK2 siRNA-3 is identified as the optimal sequence for silencing circHIPK2, with its gene sequences being 5^′-GCUUAGUCUUUGAGAUGUU-3^′ (sense strand) and 5^′-AACAUCUCAAAGACUAAGC-3^′ (antisense strand). Based on these findings, circHIPK2-3 was selected for use in the subsequent experiments.

3.2 Characterization of Nanoparticles

The physicochemical properties of the different synthesized nanoparticles are summarized in Fig. 2. Dynamic light scattering (DLS) analysis revealed that the hydrodynamic diameters of the NH₂-MSNs, NH₂-MSN-siRNA, and M-R@siC-NPs were 115.80 \pm 9.65 nm, 118.40 \pm 7.21 nm, and 134.78 \pm 10.25 nm, respectively, with polydispersity index (PDI) values of 0.206 \pm 0.041, 0.211 \pm 0.019, and 0.205 \pm 0.089, respectively (Fig. 2A). The narrow PDI values indicated homogeneous particle size distribution and high-quality synthesis. Zeta potential measurements further demonstrated surface charges of 20.00 \pm 0.24 mV for NH₂-MSNs, –3.2 \pm 0.86 mV for NH₂-MSN-siRNA, and 4.3 \pm 0.48 mV for M-R@siC-NPs (Fig. 2B). The transition from positive to negative zeta potentials confirmed electrostatic binding between the positively charged NH₂-MSNs and negatively charged siRNA. TEM images revealed spherical morphologies for both NH₂-MSNs (Fig. 2C) and M-R@siC-NPs (Fig. 2D), with uniform diameters less than 200 nm and no apparent aggregation or fragmentation. The consistent particle size, comparable zeta potentials, and excellent colloidal stability of M-R@siC-NPs align with the requirements for biomedical applications. These physicochemical properties are critical for the performance of these nanoparticles in subsequent gene delivery and biosensing experiments, influencing parameters such as blood circulation time, cellular uptake, and biodistribution.

3.3 Encapsulation Efficiency and Drug Loading Capacity of Nanoparticles

To determine the optimal mass ratio for binding circHIPK2 siRNA (218 µg/mL) with NH₂-MSNs (5 mg/mL), agarose gel electrophoresis was performed. The results demonstrated that when the mass ratio of siRNA to NH₂-MSNs reached 1:30 or higher, the fluorescence intensity of the siRNA band decreased (Fig. 3A), indicating successful electrostatic binding between the positively charged NH₂-MSN and negatively charged siRNA. Thus, a 1:30 mass ratio was employed in the preparation of nanoparticles for subsequent experiments. To evaluate the encapsulation efficiency and drug loading capacity of the nanoparticles, ultramicroscopic UV-Vis spectrophotometry was used to quantify siRNA content (R² = 0.9983). The calculated encapsulation efficiency and drug loading capacity were 75.28% \pm 0.06% and 15.63% \pm 0.27%, respectively (Fig. 3B).

3.4 Stability of Nanoparticles

To evaluate the stability and biocompatibility of nanoparticles in physiological environments, the morphology and dispersion of nanoparticle suspensions in DMEM/F12, PBS, FBS, and aCSF were observed over 14 days. The results demonstrated that M-R@siC-NPs maintained a uniform dispersion without significant aggregation or precipitation in all tested media. Notably, no evident interaction between nanoparticles and serum proteins or culture medium components was observed in FBS and DMEM/F12, confirming effective surface modification (Fig. 4A–C). These findings indicate that M-R@siC-NPs exhibit excellent long-term stability, making them suitable for in vitro cell culture and in vivo CNS delivery applications. Additionally, DLS analysis revealed no significant fluctuations in the hydrodynamic diameter of M-R@siC-NPs throughout the 14-day study period (p \ge 0.05, Fig. 4D). Particle size measurements at specific time points (day 0, 2, 4, 6, 8, 10, 12, and 14) were as follows: 134.78 \pm 10.25 nm, 135.12 \pm 9.87 nm, 136.54 \pm 10.13 nm, 137.21 \pm 9.92 nm, 136.89 \pm 10.31 nm, 137.56 \pm 10.05 nm, 138.12 \pm 9.78 nm, and 138.45 \pm 10.22 nm. These consistent measurements further confirm the structural integrity of M-R@siC-NPs under physiological conditions.

3.5 Nanoparticle Biocompatibility

3.5.1 In Vitro Biocompatibility Assessment

The biocompatibility of LNPs was evaluated by adding them to cultures of primary astrocytes for 24 h, followed by cell viability analysis using the CCK-8 assay. The results demonstrated that astrocytes retained over 90% cell viability upon exposure to siC-NPs and M-R@siC-NPs at concentrations of 20–80 µg/mL. However, with nanoparticle concentrations of 100 µg/mL, astrocyte viability decreased to 89.01% (siC-NPs) and 87.59% (M-R@siC-NPs), and these reductions were significant compared with the viability of the control group (Fig. 5A). Based on these findings, 80 µg/mL was selected as the optimal concentration for subsequent experiments to ensure the biocompatibility of nanoparticle treatments (Fig. 5B).

3.5.2 In Vivo Biocompatibility Assessment

To evaluate the in vivo safety of the nanoparticles, a peripheral blood hemolysis assay was conducted to assess whether free nanoparticles in the bloodstream would induce acute erythrocyte destruction. At nanoparticle concentrations of 20, 40, 60, 80, and 100 µg/mL, the hemolysis rates remained less than 1.5% (Fig. 6A,B). From the appearance of centrifuged samples in Fig. 6A, the supernatants of all nanoparticle concentration groups remained colorless and transparent, which was completely consistent with the PBS negative control group. In contrast, the dark red supernatant observed in the positive control group (triple-distilled water) was absent in all nanoparticle groups. This macroscopic result directly confirms that even at the highest tested concentration (100 µg/mL), the nanoparticles did not induce visible red blood cell (RBC) lysis, indicating no significant hemolysis and initially verifying their hemocompatibility (Fig. 6A). The absorbance of the supernatants was measured at a wavelength of 540 nm using a microplate reader, and the hemolysis rate was calculated according to the formula. The results showed that the hemolysis rate of each concentration group was below 1.5%, which was far lower than the 5% safety threshold for hemolysis rate of biomaterials (Fig. 6B). The slight upward trend of hemolysis rate with increasing concentration was attributed to the slightly increased probability of non-specific contact between nanoparticles and RBC membranes as the nanoparticle concentration increased (from 20 to 100 µg/mL). This contact led to the release of a minimal amount of hemoglobin; however, the release volume was extremely low and did not reach the threshold for macroscopically visible color changes. These results confirmed that the nanoparticles did not induce erythrocyte destruction, indicating they offer an excellent in vivo safety profile.

For in vivo analysis of nanoparticle biocompatibility, this study evaluated the functional status of major physiological organs in experimental animals after daily intravenous injection of nanoparticles for 20 days. Blood routine parameters (white blood cell [WBC] count, red blood cell [RBC] count, hemoglobin [HGB], and platelet [PLT] count) and serum biochemical markers (alanine aminotransferase [ALT], aspartate aminotransferase [AST], total bilirubin [TBIL], albumin [ALB], alkaline phosphatase [ALP], creatinine [CREA], blood urea nitrogen [BUN], and uric acid [UA]) were analyzed in both control and LNP-treated groups. All values fell within the reference ranges for healthy mice, with no adverse effects observed (Fig. 7). Statistical analysis revealed no significant differences between groups (p > 0.05), collectively indicating that the LNP complexes exhibited no detectable hepatotoxicity or nephrotoxicity.

To assess the in vivo biosafety of the LNPs, histopathological analysis was performed on major organs (heart, liver, spleen, lung, kidney, and brain) harvested after 20 days of intravenous injection of the different particles. The H&E staining results demonstrated that, compared with the NC group, the nanoparticle-treated groups exhibited no significant histopathological abnormalities, showing no inflammatory cell infiltration, cellular edema, or necrosis in any organ (Fig. 8). These morphological observations confirmed that the LNPs caused no detectable cytotoxicity or structural damage to vital organs, underscoring their excellent in vivo biocompatibility and safety profile.

3.6 Nanoparticle Targeting Efficiency

3.6.1 In Vitro Targeting Assessment

Evaluations of cellular nanoparticle uptake efficiency and targeting revealed distinct fluorescence patterns. No fluorescent signals were detected in the PBS control group under laser scanning confocal microscopy (LSCM). In contrast, significant intracellular fluorescence accumulation was observed in the groups treated with Cy5.5-siC-NPs (80 µg/mL) and Cy5.5-M-R@siC-NPs (80 µg/mL). Quantitative analysis demonstrated that the mean fluorescence intensity in these experimental groups was statistically higher than that in the control group (Fig. 9A,B), with cellular uptake efficiency reaching 95.2% \pm 2.7%. These results confirm that the LNP carriers effectively penetrate cell membranes and thus, offer sufficient transmembrane transport capacity.

On the evaluation of the targeted delivery performance of the nanoparticles, the surface-modified Cy5.5-M-R@siC-NPs exhibited distinct advantages. LSCM imaging revealed significantly stronger fluorescence signals in the group treated with Cy5.5-M-R@siC-NPs compared with that treated with Cy5.5-siC-NPs. Fluorescence quantification confirmed that the intensity of Cy5.5-M-R@siC-NPs was three-fold higher than that of Cy5.5-siC-NPs (Fig. 9A,B), with a statistically significant difference observed. This outcome substantiates that the active targeting capability of M-R@siC-NPs can achieve drug delivery specificity.

3.6.2 In Vivo Targeting Efficiency Evaluation

In vivo live imaging revealed that intraperitoneally administered targeted nanoparticles (Cy5.5-M-R@siC-NPs) exhibited peak fluorescence intensity in the brain at 4 hours post-injection, with complete clearance observed by 48 hours (Fig. 10A-III). In contrast, non-targeted Cy5.5-siC-NPs displayed a delayed peak fluorescence at 6 hours, reduced intensity, and rapid clearance within 24 hours (Fig. 10A-II,C). Statistical analysis demonstrated that the brain accumulation of targeted nanoparticles was significantly higher than that of the non-targeted counterparts from 4–10 hours post-administration. These results confirm that the M-R@siC-NPs with brain-targeting modifications effectively penetrated the BBB, potentially offering enhanced cerebral bioavailability and an extended therapeutic window.

Ex vivo fluorescence quantification further validated the brain-specific targeting efficacy of the modified nanoparticles (Fig. 10). Quantitative analysis of brain tissues revealed markedly stronger Cy5.5-M-R@siC-NPs fluorescence compared with Cy5.5-siC-NPs fluorescence (Fig. 10B,D), underscoring their superior BBB-penetrating capacity. Systemic biodistribution analysis (Fig. 10B,E) showed uniform distribution of the non-targeted nanoparticles across major organs, whereas Cy5.5-M-R@siC-NPs demonstrated selective enrichment in the brain, with statistically significant differences in fluorescence intensity observed. The results of this spatial distribution analysis support the notion that the brain-targeting ligands on M-R@siC-NPs minimize peripheral organ exposure while enhancing brain-specific delivery.

Microscopic localization in brain tissues provided insight into the BBB-penetration mechanism of M-R@siC-NPs (Fig. 11). Frozen sections stained with DAPI (blue fluorescence, nuclear counterstain) revealed distinct red fluorescence signals from Cy5.5-M-R@siC-NPs, with pronounced accumulation in the brain parenchyma and perivascular regions. Compared with controls and non-targeted nanoparticles, targeted nanoparticles exhibited concentrated fluorescence in the brain parenchyma, particularly near the BBB. The BBB restricts passive diffusion of most molecules; however, the enhanced retention of Cy5.5-M-R@siC-NPs in brain tissues suggests active transport mechanisms. The unique lipid composition and surface modifications of these nanoparticles likely enable receptor-mediated interactions with endothelial cells lining the BBB, facilitating transcytosis and sustained cerebral retention (Fig. 11).

3.7 Cellular Experiments to Test the Therapeutic Efficacy of M-R@siC-NPs

3.7.1 Characterization of Primary Astrocytes

Primary astrocytes were successfully isolated and purified from the cerebral tissues of C57BL/6 neonatal mice. Immunofluorescence staining confirmed astrocyte identity: GFAP-labeled astrocytic processes (green, Fig. 12A-I), phalloidin-stained F-actin cytoskeleton (red, Fig. 12A-II), and DAPI-stained nuclei (blue, Fig. 12A-III). Merged images (Fig. 12A-IV) revealed that primary astrocytes exhibited characteristic star-shaped or polygonal morphology with large cell bodies, prominent nuclei, and multiple filopodia-like processes in culture. Quantitative analysis demonstrated that >90% of cells displayed strong GFAP positivity in both the cell body and processes. Statistical results confirmed a purity of 94.65% GFAP-positive cells (Fig. 12B,C), confirming high-quality astrocyte cultures.

3.7.2 In Vitro Therapeutic Efficacy Evaluation

To assess the therapeutic potential of M-R@siC-NPs, primary astrocytes were subjected to OGD treatment. The experimental groups included NC, OGD (model), siC-NPs-treated, and M-R@siC-NPs-treated groups. The outcomes were evaluated based on GFAP expression, IL-1\beta level, and cell viability via CCK-8 assay (Fig. 13A–D).

In the OGD model of primary astrocytes, Western blot results demonstrated that the OGD model group exhibited significantly increased protein levels of GFAP and IL-1\beta. This elevation was attributed to astrocyte activation and subsequent inflammation triggered by hypoxia-ischemia, confirming the successful establishment of the OGD model. After treatment with siC-NPs and M-R@siC-NPs, siC-NPs slightly reduced the expression of GFAP and IL-1\beta, while M-R@siC-NPs showed a significantly superior inhibitory effect (Fig. 13A–C, the original western blot images can be found in the Supplementary Materials). This difference stemmed from the dual modification of M-R@siC-NPs with M-PEG-lipid and DSPE-PEG2000-RVG29. Specifically, this dual modification enhanced the targeting ability of M-R@siC-NPs to astrocytes and improved their cellular uptake efficiency, thus confirming that targeted modification is the core factor for enhancing in vitro therapeutic efficacy. The CCK-8 assay results further showed that OGD exposure drastically reduced cell viability, whereas treatment with siC-NPs partially restored viability. Notably, M-R@siC-NPs treatment resulted in the most significant recovery of cell viability (Fig. 13D).

These findings demonstrate that M-R@siC-NPs can alleviate astrocyte activation and neuroinflammation via targeted gene silencing, while promoting the functional recovery of damaged cells.

3.8 Animal Experiments to Test the Therapeutic Efficacy of M-R@siC-NPs

3.8.1 Establishment of the HIBD Model

The murine HIBD model was established using the Rice-Vannucci method [53]. Cortical blood flow was quantified using laser speckle flowmetry at 24 hours post-surgery. Compared with those in the sham-operated group, HIBD mice exhibited a statistically significant reduction in left cortical blood flow (Fig. 14A,B), confirming successful model induction.

Post-mortem examination of brain tissues revealed progressive pathological changes. Sham-operated mice displayed normal brain morphology, characterized by a uniform texture, smooth surfaces, and an intact structure. In contrast, the brains of HIBD mice exhibited time-dependent deterioration, including mild cerebral edema at 3 days post-HIBD (indicative of ischemia–hypoxia injury), ipsilateral infarction foci at 5 days (reflecting vascular occlusion and neuronal necrosis), and exacerbated tissue damage at 7 days, including softening of the brain parenchyma, regional necrosis, and structural disintegration (Fig. 14C). These findings collectively validate the reliability of the HIBD model for studying hypoxic–ischemic brain injury progression.

3.8.2 In Vivo Therapeutic Efficacy Evaluation

To build upon our in vitro findings, the therapeutic efficacy of M-R@siC-NPs was evaluated in a C57BL/6 HIBD model. The mice were divided into four groups: sham-operated, HIBD, siC-NPs-treated, and M-R@siC-NPs-treated. At 5 days post-treatment, brain tissues were subjected to H&E staining. The brain tissues of sham-operated mice exhibited normal cellular morphology with tightly arranged neurons and no injury. The brain tissues of HIBD mice showed pyknotic nuclei, deep staining, and inflammatory cell infiltration. While the brain tissues of siC-NPs-treated mice displayed reduced injury severity, including decreased inflammatory infiltration and improved edema, the brain tissues of M-R@siC-NPs-treated mice demonstrated significant improvement, including an intact brain architecture, minimal neuronal damage, orderly cellular arrangement, and markedly reduced inflammatory cell infiltration (Fig. 15A).

Immunofluorescence analysis revealed distinct astrocyte activation states across groups (Fig. 15B). Weak GFAP fluorescence in brain tissues from sham-operated mice indicated quiescent astrocytes. In contrast, intense GFAP signals in brain tissues from HIBD mice reflected hypoxia–ischemia-induced astrocyte activation and secondary neuroinflammation. While treatment with siC-NPs reduced the GFAP intensity compared with that in HIBD mice, treatment with M-R@siC-NPs achieved the most pronounced suppression, with significantly weaker GFAP fluorescence than in both the HIBD and siC-NPs-treated groups, underscoring the superior efficacy of targeted nanoparticles for inhibiting astrocyte activation.

Western blot analysis confirmed these trends in an astrocyte activation marker (GFAP) and an inflammatory cytokine (IL-1\beta). Compared with the brain tissues of sham-operated mice, those of HIBD mice exhibited upregulated GFAP and IL-1\beta expression, confirming hypoxia–ischemia-driven astrocyte activation and neuroinflammation. Both siC-NPs and M-R@siC-NPs treatment reduced the levels of these proteins, but treatment with M-R@siC-NPs achieved significantly greater suppression (Fig. 15C–E, the original western blot images can be found in the Supplementary Materials). Statistical analysis revealed that the GFAP and IL-1\beta levels in the M-R@siC-NPs group were markedly lower than those in the HIBD and siC-NPs groups. These findings collectively demonstrate that M-R@siC-NPs effectively alleviate neuroinflammation and suppress astrocyte activation, thereby mitigating brain injury.

3.9 Behavioral Assessments to Investigate the Therapeutic Efficacy of M-R@siC-NPs

3.9.1 Effects of Nanoparticle Treatment on Short-Term Behaviors

The righting reflex test evaluates sensorimotor development by measuring the latency to rightward rotation after placement in a supine position. Sham-operated mice exhibited rapid righting reflexes with significantly shorter latency times compared with HIBD mice (Fig. 16A), which showed prolonged latencies, indicating impaired neuromuscular control and balance due to neurological injury. Treatment with siC-NPs and M-R@siC-NPs reduced latency times, with the M-R@siC-NPs-treated group demonstrating the most marked improvement.

Using the negative geotaxis test, the mice in each group were assessed for their ability to climb upward on an inclined plane. The HIBD mice displayed significantly longer escape latencies than the sham-operated control mice (Fig. 16B), reflecting compromised neuromuscular strength and coordination. While both treatment groups showed improved latencies compared with the HIBD mice, treatment with M-R@siC-NPs led to statistically significant recovery, suggesting enhanced motor function restoration.

3.9.2 Effects of Nanoparticle Treatment on Long-Term Behaviors

To evaluate spatial learning and memory recovery, the Morris water maze test was performed over 5 days (Fig. 16C). During training (days 1–4), sham-operated mice exhibited progressively shorter escape latencies, indicating efficient spatial memory formation. In contrast, the HIBD mice displayed prolonged latencies (Fig. 16D) and disorganized swimming paths, reflecting hippocampal dysfunction. Treatment with siC-NPs or M-R@siC-NPs reduced latency times and increased platform-crossing frequency. Notably, M-R@siC-NPs-treated mice showed the greatest improvements in learning and memory retention during the probe trial (day 5, Fig. 16E).

In the mouse HIBD model, the siC-NPs group exhibited significantly less improvement in brain pathological lesions and neurological function recovery compared to the M-R@siC-NPs group. Combined with in vivo fluorescence imaging results—where the cerebral accumulation of M-R@siC-NPs was 3 times that of siC-NPs—it was further confirmed that the BBB penetration capability and astrocyte-targeting ability, mediated by M-PEG-lipid and DSPE-PEG2000-RVG29, are the key factors underlying the superior in vivo therapeutic efficacy of the targeted nanoparticles. Moreover, the presence of siC-NPs clearly verifies that this efficacy advantage originates from the targeted modification, rather than the carrier or siRNA itself.

4. Discussion

Current HIE therapies are associated with multiple limitations. For example, the gold standard approach, hypothermia, can only be employed in neonates within 6 h post-birth, and its use is still associated with 44–53% mortality/severe disability rates and infection risks [7, 54, 55, 56, 57, 58]. While experimental drugs (allopurinol, melatonin) have shown promise, long-term safety data for these therapeutics are lacking [59, 60, 61]. Stem cell therapies encounter ethical/immunological barriers [62, 63]. This study successfully constructed brain-targeting LNPs (M-R@siC-NPs) for astrocyte-specific delivery of circHIPK2 siRNA, which exhibited significant therapeutic efficacy in a murine HIBD model. The dual-targeting nanoplatform efficiently traversed the BBB, silenced circHIPK2 expression, inhibited astrocyte activation, and attenuated neuroinflammation, providing a novel strategy for neonatal HIE therapy (Fig. 17). In contrast with existing therapies, M-R@siC-NPs offer superior targeting, efficacy, and safety. Compared with glioma-targeted liposomes specifically, M-R@siC-NPs exhibit greater siRNA encapsulation and brain accumulation (RVG29-mediated hippocampal targeting), minimizing systemic toxicity.

Neuroinflammation is central to ischemia–reperfusion injury in HIE. During the pathogenesis of HIE, astrocytes undergo aberrant activation, characterized by upregulated GFAP expression and the release of pro-inflammatory mediators (e.g., TNF-\alpha, IL-1\beta), which exacerbates neuronal damage and disease progression [64]. By precisely suppressing astrocyte activation, M-R@siC-NPs effectively disrupted this pathological cascade, offering innovative therapeutic potential. While previous studies have explored astrocyte modulation, our dual-targeted delivery system introduces unique advancements in carrier design and specificity. Composed of DPPC, cholesterol, DSPE-PEG2000, DSPE-PEG2000-RVG29, and mannose-PEG-lipid, this system involves loading circHIPK2 siRNA into NH₂-MSNs, forming core-shell LNPs (Fig. 17).

The BBB poses a critical barrier to CNS drug delivery, limiting cerebral bioavailability while increasing systemic toxicity [65, 66]. Although RNA interference holds promise, the application of siRNA as therapeutic agents has faced limitations including poor membrane permeability, rapid degradation, mononuclear phagocyte system clearance, renal excretion, and off-target effects [67, 68]. To address these limitations, we developed M-R@siC-NPs incorporating MSNs. MSNs exhibit exceptional drug-loading capacity due to their ordered mesopores, high surface area, and modifiable silanol groups [34, 69, 70, 71, 72, 73, 74]. In this study, positively charged NH₂-MSNs achieved high siRNA loading via electrostatic interactions with negatively charged siRNA.

Liposomes, with their amphiphilic lipid bilayer structure, enable co-delivery of hydrophilic/hydrophobic agents while enhancing BBB penetration [75, 76]. Targeted modifications using ligands (e.g., polymers, peptides) further improve brain delivery [38, 77]. M-R@siC-NPs were functionalized with a Glucose Transporter 1 (GLUT1)-targeting M-PEG-lipid and the BBB-penetrating DSPE-PEG2000-RVG29. RVG29 mediates BBB transcytosis via receptor recognition, while M-PEG-lipid directs astrocyte targeting, conferring superior specificity compared with non-targeted siC-NPs.

Cellular uptake of nanoparticles is influenced by the physicochemical properties of the nanoparticles, such as surface charge and size [78, 79, 80, 81]. While sub-100 nm particles are better able to penetrate the BBB, 100–150-nm particles exhibit prolonged circulation. However, even larger particles (>250 nm) undergo rapid clearance [82, 83, 84]. Positively charged nanoparticles offer good cellular adhesion via electrostatic interactions [85]. Moreover, PEGylation can reduce aggregation, prolonged circulation, and minimize mononuclear phagocyte system–mediated clearance [86, 87, 88, 89]. Based on these effects of nanoparticle size and properties, the M-R@siC-NPs developed in this study were optimized to 130–140 nm in diameter (Malvern Nano analysis) with a narrow polydispersity and ability to remain stable for more than 14 days.

The assessment of “nanorisk” requires a comprehensive evaluation of nanoparticle trafficking dynamics, organ-specific accumulation patterns, and toxicity mechanisms in biological systems [90]. Safety assessments in the present study revealed that after treatment with M-R@siC-NPs at concentrations of 20–80 µg/mL, >90% of astrocytes remained viable (CCK-8 assay), negligible hemolysis occurred, and hematological/biochemical indices were normal. Histopathological analysis confirmed the absence of organ toxicity after M-R@siC-NPs treatment.

Activated astrocytes critically regulate sterile inflammation and IL-1\beta release in HIE [17, 91, 92, 93]. Previous studies have demonstrated that circHIPK2 inhibits astrocyte activation and alleviates neuroinflammation through multiple mechanistic pathways [24, 25, 94]. The experiments in the present study provided evidence that the developed M-R@siC-NPs effectively silenced circHIPK2 in vitro and in vivo, suppressing GFAP/IL-1\beta expression and improving cell viability. In HIBD model mice, treatment with M-R@siC-NPs reduced neuroinflammation and restored spatial learning/memory (shortened Morris water maze latency; prolonged target quadrant duration). The dual RVG29/mannose-based targeting showed greater efficacy than previous mono-targeted approaches. Mechanistically, circHIPK2 inhibition may preserve hippocampal synaptic plasticity by attenuating IL-1\beta–driven inflammation. In the present study, the HIBD model exhibited a dynamic pattern of macroscopic pathological changes, specifically edema at 3 days, infarction at 5 days, tissue lysis at 7 days, which is highly consistent with the classic findings of neonatal HIBD model studies in the field. The Rice-Vannucci method for establishing HIBD models, first developed by Rice et al. [95], has clearly demonstrated a consistent pathological sequence in the ischemic cerebral hemisphere of neonatal rats: edema at 3 days post-surgery, infarction formation at 5 days, and tissue necrosis at 7 days. This model and its associated pathological characteristics have become a consensus in the field, laying a critical foundation for subsequent research [95]. Additionally, studies on neonatal mouse HIBD models have further confirmed that the ischemic cerebral hemisphere exhibits localized tissue loss at 7 days post-surgery due to infarct expansion, and this change shows a significant negative correlation with the reduction in cerebral blood flow (r = –0.87, p 0.001) [96]. Motor coordination improvements (righting reflex/negative geotaxis tests) suggest cerebellar–vestibular pathway repair, although the long-term cognitive effects require further evaluation.

Currently, the treatment of HIBD primarily relies on two major strategies: neuronal protection and broad-spectrum anti-inflammation, both of which have significant limitations. On one hand, neurons exhibit extremely poor tolerance to hypoxia-ischemia—intracellular ATP depletion exceeds 80% within 2 hours of ischemia. Traditional neurotrophic factors such as brain-derived neurotrophic factor (BDNF) can only delay neuronal apoptosis by approximately 15%–20% due to restricted BBB permeability and downregulation of target receptors. Although antioxidants like N-acetylcysteine can temporarily reduce oxidative stress, they fail to block cascading necrosis via the mitochondrial pathway; clinical translation trials have shown that their 90-day neurological function improvement rate is only 5.7% [97, 98, 99]. On the other hand, while broad-spectrum anti-inflammatory strategies can inhibit early pro-inflammatory factors IL-1\beta and TNF-\alpha, they simultaneously disrupt late-stage reparative inflammation, such as M2 microglial polarization, leading to a 50% reduction in neural stem cell proliferation (p 0.01) and significant delays in the recovery of motor and cognitive functions [100].

As core regulators of CNS homeostasis, astrocytes play a crucial role in HIBD. They clear over 80% of glutamate in the synaptic cleft by highly expressing glutamine synthetase (GS), thereby inhibiting excitotoxicity; secrete IL-6 and C-X-C motif chemokine ligand 12 (CXCL12) to regulate neuroinflammation; and maintain BBB integrity through the modulation of vascular endothelial growth factor (VEGF) and TGF-\beta [101]. However, traditional intervention methods mostly involve non-specific inhibition—for example, the pan-glial inhibitor minocycline. While suppressing the excessive activation of astrocytes, it non-selectively impairs the functions of normal glial cells, resulting in a 60% reduction in hippocampal neurogenesis in the late stage (p 0.001) and a 40% prolongation of escape latency in the Morris water maze test [102].

In this study, a novel mannose/RVG29 peptide dual-modified lipid nanoparticle M-R@siC-NPs was innovatively constructed to overcome the bottlenecks of traditional interventions. Leveraging the high expression of mannose receptors (MR) on the surface of astrocytes, the mannose modification enables specific endocytosis of the nanoparticles; combined with the RVG29 peptide, which enhances brain penetration capability, this system can efficiently deliver circHIPK2 siRNA into astrocytes. M-R@siC-NPs demonstrate great potential in inhibiting astrocyte activation, exerting anti-inflammatory effects, and alleviating HIBD-induced brain damage, holding promise as an innovative strategy for the treatment of HIE.

The present study has some limitations that must be considered and addressed in future research. The sample size in this study was small, and the long-term biodistribution and toxicity of M-R@siC-NPs were not evaluated. Moreover, the mechanisms by which M-R@siC-NPs interact with the BBB remain unclear. Future work should include expanded cohorts, assessment of scale-up feasibility, proteomics analyses for pathway elucidation, and in vivo imaging to visualize BBB traversal.

5. Conclusions

In conclusion, this study successfully developed M-R@siC-NPs with dual functions based on the inclusion of RVG29 peptide and mannose-PEG-lipid, offering good BBB penetration and astrocyte-targeting efficiency. These nanoparticles achieved efficient circHIPK2 silencing while exhibiting excellent stability and biosafety. Mechanistically, the results of this study indicate that M-R@siC-NPs reduce astrocyte activation and mitigate neuroinflammation, effectively ameliorating brain injury in HIBD model mice. This work not only establishes a novel RNA interference-based targeted therapy for neuroinflammation but also provides a foundation for the clinical translation of circHIPK2 as a therapeutic target in HIE. Overall, M-R@siC-NPs represent a breakthrough in HIE therapy, combining BBB penetration, astrocyte specificity, and robust siRNA delivery. This dual-targeted system overcomes multiple limitations of conventional treatments, demonstrating transformative potential for precision gene therapy in neonatal brain injury.

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