Intranasal delivery of LB244-loaded M2 microglia membrane nanoparticles for targeted treatment of neuroinflammation after I/R brain injury

Abstract

Ischemic stroke following revascularization therapy inevitably leads to varying degrees of ischemia/reperfusion (I/R) injury. The complexity of post-I/R inflammatory processes have hindered the development of effective strategy to improve functional recovery in stroke patients. Extensive evidence has confirmed that activation of the STING pathway and microglial pyroptosis exacerbates neuroinflammatory responses and neuronal damage in cerebral I/R injury. LB244, a potent STING inhibitor, shows therapeutic promise but suffers from poor solubility and bioavailability. Here, we developed LB244@M2 nanoparticles by encapsulating LB244 in M2 microglia membrane vesicles. Intranasal administration of LB244@M2 enabled direct nose-to-brain delivery, achieving 1.7-fold higher brain accumulation than intravenous injection within 2 h. By inhibiting the STING pathway, LB244@M2 attenuated pyroptosis-induced microglial inflammation and promoted the phenotypic shift from pro-inflammatory (M1) to anti-inflammatory (M2) states. This conferred significant neuroprotection, reducing neuroinflammatory cell death by 67.9 % and infraction volume by 49.8 % compared to saline-treated controls. Moreover, LB244@M2-treated I/R mice exhibited 53.3 % improvement in Bederson neurological score at 7 days post-treatment, with no observed adverse effects. These findings highlighted LB244@M2 as a promising nanotherapeutic strategy to mitigate neuroinflammation and enhance post-stroke recovery.

Graphical abstract

Intranasally delivered M2 microglia-membrane-camouflaged LB244 nanoparticles target I/R brain lesions, suppressing microglial pyroptosis and neuroinflammation in ischemic stroke.

1. Introduction

Ischemic stroke (IS) remains one of the leading causes of global mortality and disability, imposing significant burdens on global healthcare systems [1]. Current revascularization therapies, such as thrombolysis with recombinant tissue plasminogen activator (rtPA) and endovascular thrombectomy, are considered essential for restoring blood flow following cerebral ischemia, the reperfusion process itself has been demonstrated to trigger cerebral ischemia/reperfusion (I/R) injury [2]. This secondary injury, driven by oxidative stress, inflammatory cascades and programmed cell death, significantly diminishes the benefits of revascularization and constitutes a major limitation of current stroke treatments.

Cerebral I/R injury involves a complex cascade of pathophysiological process that mainly includes excitotoxicity, oxidative stress, and inflammatory response [3]. Inflammation plays a pivotal role in exacerbating secondary brain injury, impairing tissue repair, and prolonging neurological deficits [4]. Microglia, the resident immune cells of the central nervous system (CNS), are key contributors to neuroinflammation following IS [5]. Upon I/R injury, microglia undergo rapid activation, shifting predominantly into the pro-inflammatory M1 phenotype, which secretes high levels of cytokines such as TNF-α, IL-6, and IL-1β [4]. This inflammatory environment further exacerbates neuronal death and disrupts the blood-brain barrier (BBB), amplifying CNS injury.

Recent studies have identified the Stimulator of Interferon Genes (STING) pathway as a central regulator of innate immunity and neuroinflammation, making it a promising target for therapeutic intervention [6]. The STING pathway is a critical component of innate immunity, activated by cytosolic DNA through the cyclic GMP-AMP synthase (cGAS). Mitochondrial dysfunction caused by I/R injury leads to the release of mitochondrial DNA (mtDNA) into the cytosol, which activates the cGAS-STING axis in microglia [7]. The cGAS-STING axis activation triggers downstream signaling cascades, resulting in the robust production of type I interferons and pro-inflammatory cytokines such as TNF-α and IL-6 [8]. These factors collectively promote the formation of a pro-inflammatory microenvironment. Importantly, excessive STING activation has been implicated in pyroptosis, a lytic and highly inflammatory form of programmed cell death driven by the NLRP3 inflammasome-caspase-1-gasdermin D (GSDMD) axis [9]. The cleaved GSDMD oligomerizes in the cell membrane to form pores to initiate microglial pyroptosis and release pro-inflammatory cytokines such as IL-1β and IL-18 [10]. This process propagates a self-sustaining cycle of inflammation and neuronal injury [11]. Thus, targeting STING-mediated pyroptosis represents a novel and promising approach for mitigating I/R-induced neuroinflammation.

Although STING inhibitors have shown therapeutic potential in inflammatory and autoimmune diseases, their application in IS remains underexplored. Small-molecule STING inhibitors, such as C-176 [12], H-151 [13], and SN-011 [14], have demonstrated anti-inflammatory effects in mouse models of STING-dependent inflammatory diseases. Recent experimental studies have revealed that STING antagonist post-treatment ameliorated neuroinflammation and neuronal apoptosis after traumatic brain injury [12]. Furthermore, Ding et al. reported that siRNA mediated inhibition of STING could suppress microglial pyroptosis and reduce neuroinflammation in preclinical models of IS [11]. More recently, LB244, a first-in-class irreversible STING antagonist, exhibits nanomolar potency and enhanced proteome-wide selectivity through covalent modification of C292 — a key regulatory site controlling STING oligomerization. By impairing protein multimerization, this unique mechanism disrupts STING-dependent signaling cascades, distinguishing LB244 from conventional STING inhibitors. LB244 demonstrates robust efficacy in suppressing pathological inflammatory responses, highlighting its therapeutic potential for CNS disorders driven by aberrant STING activation [15]. LB244 was well tolerated in cell viability assays. Despite these advantages, LB244 faces significant pharmacokinetic limitations, including poor oral bioavailability, rapid clearance, and inadequate BBB penetration [16], which hinder its therapeutic efficacy in IS.

Nanotechnology-based delivery systems present a potential solution. While exosomes have shown promise as natural nanocarriers for IS treatment by preserving drug stability and enhancing brain-targeted delivery [17], their clinical application is limited by inherent challenges such as compositional complexity and low production yields [18]. Recent advances have introduced cell-membrane-cloaked nanoparticles as a superior alternative. These bioengineered nanoparticles combine the advantages of biocompatibility, high stability, and versatile drug-loading capacity [19,20]. Their lipid bilayer of the cell membrane endows them with the ability to overcome the BBB [21]. Crucially, they can also be engineered to precisely deliver drugs to cerebral infarction sites and selectively target specific cell populations. Supporting this approach, our previous work demonstrated that macrophage membrane-derived nanovesicles effectively delivered Dasatinib to ischemic regions, where they were internalized by neurons [22]. Building on this foundation, we proposed M2 microglial membrane-coated nanovesicles as an advanced delivery strategy. Given that M2-polarized microglia inherently possess anti-inflammatory properties, including secretion of anti-inflammatory cytokines IL-10 and TGF-β [23], and expression of high levels of Arg-1 and CD206, this platform offers a promising strategy to both deliver STING inhibitor and modulate neuroinflammation.

Capitalizing on the properties of engineered nanocarrier properties, intranasal administration has emerged as an attractive route for CNS-targeted drug delivery. The non-invasive nature of this approach allows for potential self-administration without the need for sterile equipment, thereby substantially improving treatment accessibility. More importantly, the nose-to-brain pathway provides direct CNS access through the peripheral olfactory and trigeminal neural pathways, effectively bypassing the BBB [24,25]. This unique feature enables rapid drug penetration into brain tissues, a crucial advantage for time-sensitive conditions such as IS. Compared with systemic delivery, the intranasal route exhibits in substantially lower systemic exposure, thereby minimizing potential adverse effects on peripheral organs, including the hematological and cardiovascular systems. When integrated with nanotechnology-based delivery systems, this route synergistically enhances drug stability, bioavailability, and brain targeting, showing great promise for significantly improving outcomes in I/R brain injury [26].

In this study, we developed a novel M2 microglia membrane vesicles-based STING inhibitor (LB244@M2) nanoplatform for targeting neuroprotection after I/R injury. By leveraging the anti-inflammatory properties of M2 microglial membranes and the precise brain-targeting capability of intranasal administration, LB244@M2 could effectively suppress STING-mediated neuroinflammation, attenuate pyroptosis, and improve neurological outcomes in IS (Fig. 1). Our study aims to provide a comprehensive nanotherapeutic strategy that integrates a cutting-edge STING inhibitor with an advanced drug delivery approach, paving the way for novel interventions in ischemic stroke treatment.

Fig. 1.

Intranasal administration of LB244@M2 as a therapeutic strategy for IS. A schematic illustration depicting the preparation, administration, and neuroprotective mechanism of LB244@M2 for treating IS. M2 microglial cell membranes were extracted and used to encapsulate the STING inhibitor LB244, forming LB244@M2 nanoparticles. The LB244@M2 solution was intranasally administered to I/R mice, enabling targeted delivery to the ischemic brain region. LB244@M2 exerted neuroprotective effects by suppressing microglial pyroptosis and neuroinflammation, thereby promoting functional recovery post-ischemic injury.

2. Materials and methods

2.1. Animals and middle cerebral artery occlusion/reperfusion (MACO/R) model

All experiments conducted in this study were approved by the Animal Ethics Committee of the Fifth Affiliated Hospital of Sun Yat-sen University. The implementation process complied with the requirements of experimental animal welfare ethics. Male C57BL/6 mice (aged 8–12 weeks, weight 20–25g) were obtained from Guangdong Animal Experimental Center. The animals were maintained under controlled temperature conditions with a standardized 12-h light/dark cycle.

To establish a mouse model of cerebral ischemia-reperfusion (I/R) injury, transient middle cerebral artery occlusion (MCAO) was performed. The experimental procedure involved anesthetizing mice with isoflurane inhalation, followed by surgical exposure of the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). After ligating the distal CCA and ECA with 4-0 silk sutures, a silicone-coated filament was advanced into the ICA to occlude the middle cerebral artery for 45 min. Subsequent filament withdrawal permitted cerebral reperfusion. Sham-operated control animals underwent identical surgical procedures without filament insertion and ischemic induction.

2.2. Cell culture and oxygen-glucose deprivation/reoxygenation (OGD/R) model

The BV2 cell line, obtained from Wuhan Procell Biological Co., Ltd., was employed as an alternative model to primary microglia for in vitro studies. Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10 % fetal bovine serum (FBS) under standard culture conditions (37 °C, 5 % CO₂ humidified atmosphere).

The OGD/R model was established following previously described protocols with modifications. Briefly, cells were maintained in deoxygenated glucose-depleted DMEM medium (serum-free) under hypoxic conditions using a tri-gas incubator (Binder, Germany) programmed with 37 °C, 5 % CO₂, 94 % N₂, and 1 % O₂ for 2 h. Subsequently, cells underwent reoxygenation by replacing the medium with complete culture medium (containing or excluding LB244) and returning to normoxic conditions (5 % CO₂/95 % air) for 24 h.

2.3. Cell counting kit-8 (CCK-8) assay

The CCK-8 Assay (MIKX, China) was used to measure the BV2 cells viability after different treatment. BV2 cells were seeded in 96-well plates at the density of 5 × 10³ cells/well, then exposed to OGD/R and LB244 treatment for 24 h. Next, 10 μL of CCK8 reagent was added into each well. After the cells were incubated for 2 h in the 37 °C incubator, the absorbance of each well was detected at 450 nm using EnVision multimode plate reader (PerkinElmer, USA).

2.4. Preparation and characterization of LB244@M2 nanoparticles

The primary microglia were isolated from cerebral cortices from newborn mice (1–2-day old) according to the published protocol [27]. Briefly, the cerebral cortex was dissected under sterile conditions, mechanically dissociated on ice, and cultured in DMEM/F12 medium supplemented with 20 % fetal bovine serum and 1 % penicillin-streptomycin for 7 days to enrich glial cells. Microglia were separated from astrocytes by shaking the flasks and identified by immunostaining with the marker Iba1 (≥95.0 % purity). For M2 polarization, the primary microglia were treated with 20 ng/mL recombinant IL-4 (NovoProtein, China) for 48 h, a well-established protocol that robustly induces the M2 phenotype. This approach aligns with previous study demonstrating ∼89.1 % M2 conversion under identical conditions [28].

M2 microglia were disrupted by sonication on ice and centrifuged at 12,000 rpm for 20 min. Then, the supernatant was centrifuged at 4 °C at 110,000×g for 1 h to obtain cell membrane fragments. The protein concentration of the membrane fraction was quantified using BCA assay (Beyotime, China) to standardize membrane mass. LB244 was mixed with the M2 cell membrane at a precise 1:2 mass ratio (drug: membrane), and the mixture was extruded through a 200 nm polycarbonate membrane using a liposome extrusion system to form LB244@M2 nanoparticles. The final nanoparticles were snap-frozen in liquid nitrogen and lyophilized using a benchtop lyophilizer (Ningbo Scientz, China), with dry weight measurements recorded for subsequent experiments. Transmission electron microscopy (JEM-1200EX, Japan) was used to characterize the morphology of the nanoparticles. The particle size distribution and concentration of the nanoparticles were characterized using nanoparticle tracking analysis (NanoSight NS300, UK).

The drug encapsulation efficiency (EE) and loading efficiency (LE) of LB244 in M2 vesicles were quantitatively determined using UV-vis spectroscopy. These critical pharmaceutical parameters were calculated according to the following equations (Eqs. (1), (2)):

$$\text{LE}(\%)=\frac{\text{Weight}\text{of}\text{LB}244\text{enveloped}\text{in}\mathrm{M}2\text{vesicles}}{\text{Weight}\text{of}\mathrm{M}2\text{vesicles}-\text{Weight}\text{of}\text{LB}244}\times 100\%$$ (1)

$$\text{EE}(\%)=\frac{\text{Weight}\text{of}\text{LB}244\text{enveloped}\text{in}\mathrm{M}2\text{vesicles}}{\text{Weight}\text{of}\text{initially}\text{added}\text{LB}244}\times 100\%$$ (2)

2.5. In vivo distribution of LB244@M2 nanoparticles

Excessive amounts of Cy5-NHS ester (Meilunbio, China) were added to the LB244@M2 solution (mass ratio of 10:1) on a rotating mixer for 30 min at room temperature (RT). Then, the unconnected Cy5-NHS ester was removed using a high-speed centrifugation system. The precipitate was re-suspended in sterile PBS and named Cy5-labeled LB244@M2.

The biodistribution of Cy5-labeled LB244@M2 in MCAO/R mice were imaged by an IVIS Spectrum imaging system (PerkinElmer, USA). Cy5-labeled LB244@M2 (containing 100 μg of LB244) were administered intravenously (i.v.) via tail veins (100 μL) or intranasally (i.n.) by spray (10 μL) after reperfusion in each MCAO/R mouse. The mice were anesthetized and sacrificed at 0, 0.5, 1, 2, and 4 h after administration. The brain, heart, lungs, liver, spleen, and kidneys were dissected and imaged.

2.6. Intranasal administration

Intranasal administration was performed on MCAO/R mice immediately after reperfusion using an optimized protocol adapted from established methods [29]. M2 vesicles or LB244@M2 nanoparticles (30 μg in 2 μL PBS) were administered dropwise into alternating nostrils with spray every 2 min for 10 min (total 150 μg/day). This dosing regimen was designed to: (1) maintain therapeutic equivalence with systemic administration (5 mg/kg free LB244) accounting for 63.5 % drug loading; (2) optimize absorption using fractionated delivery within the demonstrated safe volume range; and (3) follow successful treatment durations from comparable studies. The regimen was repeated daily for 3 consecutive days.

2.7. Immunofluorescence staining

Mice were sacrificed by an overdose of anesthesia at the experimental endpoint. Transcardial perfusion with prechilled 0.9 % saline followed by 4 % paraformaldehyde (PFA) was performed to fix the mice brains. Brains were removed and post-fixed in 4 % PFA overnight at 4 °C, followed by a graded series of sucrose gradient dehydration. OTC-embedded brain tissues were cut into frozen 50-μm-thick coronal sections. The sections were washed with PBS and incubated with blocking solution (bovine serum albumin 1 % and goat serum 5 % in PBS) at RT for more than 1 h. Then, these sections were incubated with the primary antibodies (anti-Iba1,1:100; anti-GSDMD, 1:200; anti-p-STING, 1:200; anti-iNOS, 1:200; anti-Arg1, 1:100) overnight at 4 °C. After washing with PBS, the fluorescent-labeled secondary antibody (anti-rabbit or anti-mouse, 1:200) was added to incubated with the sections at RT for 1 h in the dark. Finally, the sections were taped to the glass slides and sealed with a fluorescence quenching sealing tablet containing 4′, 6-diamidino-2-phenylindole (DAPI). Laser-scanning confocal microscope (Zeiss LSM 880, Germany) was used to image the stained sections.

2.8. Triphenyl tetrazolium chloride (TTC) staining and neurological evaluation

On the 7th day of treatment, the mice were euthanized and their brains were collected.

The brain tissues were rapidly sectioned into 2-mm-thick coronal slices using a rodent brain matrix and subsequently stained with 2 % TTC solution. The sections were incubated in TTC-containing PBS at 37 °C for 15 min under light-protected conditions. This staining protocol resulted in differential tissue coloration, with infarcted regions appearing pale white in contrast to the brick-red coloration of viable brain tissue.

Also, the modified Bederson scoring system was employed to evaluate neurological outcomes in MCAO/R model mice post-treatment, with the following criteria: 0 score: Complete absence of spontaneous motor activity; 1 score: Spontaneous ipsilateral circling during free ambulation; 2 score: Impaired forelimb function manifesting as contralateral limb dragging during locomotion; 3 score: Diminished resistance to lateral pressure applied to the paretic forelimb during the paper grip test; 4 score: Persistent forelimb flexion with concomitant shoulder adduction and internal rotation; 5 score: Normal forelimb extension upon tail suspension.

2.9. Behavioral tests

• (1)Open field test (OFT):

OFT was used to detect the movement and anxiety of ischemic stroke mice. The mice were placed in the center of an opaque plexiglass box (40 × 40 × 40 cm) and allowed to explore freely for 5 min. The empty space was divided into a 20 × 20 cm central zone (center) and a surrounding boundary zone (peripheral). The motion trajectory of mice was recorded by a computer-operated camera system (Nanjing Calvin Biotechnology Co., Ltd, China). And the software system is used to collect information such as total distance (mm), time of movement (sec), time spent in the center area (sec), etc.

• (2)Y-maze spontaneous alternation test (YMT):

YMT has been reported for memory and cognitive assessment in mice after ischemic stroke. The Y maze consists of three identical arms, each with an angle o 120°. In this test, mice were placed on one arm end of the Y maze, and allowed to freely explore all three arms for 8 min. The number and order in which the mice entered each arm was recorded to calculate the rate of spontaneous alternations. The spontaneous alternation refers to the process in which mice select an unvisited arm each time while exploring three arms in succession. Total entry, alternate and preferred arms were recorded to evaluate spatial memory and exploratory behavior.

2.10. Statistical analysis

All data are expressed as mean ± standard deviation (SD). Statistical analyses were conducted using Prism 8.01 software (GraphPad Software, CA) with each experiment independently repeated at least three times. Intergroup comparisons were performed using two-tailed Student's t-test for two-group analyses. For multi-group comparisons, one-way analysis of variance (ANOVA) was employed. Statistical significance was defined as P < 0.05.

3. Results

3.1. LB244 exhibited protective effects against hypoxia-induced pyroptosis

Previous study have demonstrated that the number of pyroptotic cells was increases over a time course from day 1 to day 7 following brain I/R injury [30]. The protein expressions of STING was reported to strikingly increase, peaking at 3 days post-injury [11]. Therefore, we first evaluated the microglial pyroptosis and STING activation in MCAO/R mice at day 3 post-injury. As shown in Fig. 2A, I/R injury upregulated the expression of GSDMD (a pyroptosis marker) in Iba1⁺ microglia, indicating that I/R injury promoted microglial pyroptosis in the ischemic brain region. Similarly, elevated STING expression was observed in microglia form the MCAO/R group (Fig. 2B). Quantitative analysis revealed that the density of GSDMD/Iba1 double-positive cell density (100.5 ± 17.2 positive cells/mm²) was approximately 3.2-fold higher in the peri-infarct region of MCAO/R mice compared to Sham controls (Fig. 2C). Consistently, the mean fluorescence intensity (MFI) of STING in Iba1⁺ microglia was elevated by ∼2.6-fold after MCAO/R (Fig. 2D), confirming STING pathway upregulation during microglial activation at this critical phase. These findings were consistent with recent reports demonstrating that STING activation drives microglial pyroptosis and inflammation during the acute phase of I/R brain injury [11,[31], [32], [33]].

Fig. 2.

LB244 exhibited protective effects against hypoxia-induced pyroptosis. (A) Representative immunofluorescence images of microglia (Iba1, green) and GSDMD (red) in the ischemic penumbra at day 3 post-injury. Scale bar: 20 μm. (B) Representative immunofluorescence images of microglia (Iba1, green) and STING (red) in the ischemic penumbra at day 3 post-injury. Scale bar: 20 μm. (C) Quantification of GSDMD⁺/Iba1⁺ cell density (cells/mm²). N = 5 per group. (D) STING relative fluorescence intensity in Iba1⁺ microglia. N = 5 per group. GSDMD and STING expression increased, and colocalized with microglia in the ischemic brain region following I/R injury. (E) Two-dimensional chemical structure of LB244. (F) Optical microscopy images showed the morphological changes of BV2 cells in the Control group (untreated cells), the OGD/R group (cells subjected to 2 h OGD followed by 24 h reoxygenation), and the LB244 group (cells treated with 0.5 μM LB244 during the reoxygenation period after OGD). Yellow arrows indicate membrane bubbles characteristic of pyroptotic BV2 cells. Scale bar: 20 μm. (G) Cell viability analysis of BV2 cells across treatment groups, assessed by CCK-8 assay. N = 6 per group. (H) Representative immunofluorescence images of BV2 cells (Iba1, green) co-stained with GSDMD (red) following various treatment. (I) Representative immunofluorescence images of BV2 cells (Iba1, green) co-stained with STING (red) following various treatment. LB244 inhibited STING and GSDMD expression in BV2 cells compared to OGD/R treatment. Statistical significance: ∗∗P < 0.01, ∗∗∗P < 0.001 vs. Sham or Control group. ^###P < 0.001 vs. OGD/R group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

LB244, a BB-Cl-amidine analogue, is a highly potent STING inhibitor that block the covalent modification of C292, as recently identified by Paul et al. (Fig. 2E). To explore the therapeutic potential of LB244 in acute IS, an OGD/R cell model was established to evaluate its effects. Cytotoxicity assay indicated that 0.5 μM LB244 did not significantly impair the viability of BV2 cells, prompting its selection for subsequent experiments (Fig. S1). Typical morphological features of cell pyroptosis, including cell swelling and the presence of substantial, bulging bubbles on the plasma membrane, were observed in most of BV2 cells following OGD/R treatment (Fig. 2F). Notably, the number of BV2 cells undergoing pyroptosis after OGD/R was significantly reduced after LB244 treatment, and cytotoxicity assay confirmed that the viability of BV2 cells was significantly rescued after OGD/R (Fig. 2F and G). We further validated that hypoxia-reoxygenation injury activates STING and induces pyroptosis in microglia. Immunofluorescence analysis of BV2 cells revealed elevated STING and GSDMD signals co-localize with Iba1⁺ signals in the OGD/R group. Of note, the expression of STING and GSDMD in OGD/R cells was considerably suppressed by LB244 (Fig. 2H and I). Statistical analysis showed that LB244 treatment significantly reduced OGD/R-induced STING MFI by 60.3 % (Fig. S2A), accompanied by a 33.1 % decrease in GSDMD⁺/Iba1⁺ cell proportion (Fig. S2B), indicating concurrent suppression of both STING activation and pyroptosis. Moreover, GSDMD gene expression was also downregulated in the LB244-treated group (Fig. S3A). Specifically, the mRNA expression levels of inflammasome-related markers, including NLRP3, ASC, Caspase-1, IL-1β, and IL-18, were notably decreased after treating with LB244 (Fig. S3B–F), suggesting that LB244 attenuates NLRP3 inflammasome-mediated inflammation and cytokine production. These in vitro experiments demonstrated that LB244 significantly inhibited OGD/R-induced microglial pyroptosis and inflammation. Therefore, it is reasonable to hypothesize that the STING inhibitor LB244 potentially exerts anti-inflammatory effect by inhibiting pyroptosis in microglia in vivo.

3.2. Preparation and characterization of LB244@M2 nanoparticles

Previous study has shown that LB244 exhibits low oral bioavailability (t_½ = 2.8 h) with high clearance (2854.3 mL/min/kg), primarily due to extensive first-pass metabolism [15]. These unfavorable pharmacokinetic properties significantly limited its therapeutic efficacy. To address the dilemma of LB244, we developed membrane-camouflaged nanoparticles (M2 microglia membrane vesicles) to encapsulate the LB244 and employed intranasal administration for efficient and targeted drug delivery to brain tissue.

The membrane-camouflaged nanoparticles were engineered to selectively target microglia in I/R-injured regions. Primary microglia were extracted from neonatal mice (Fig. S4A and B). M2 microglia, characterized by high expression of CD206 and Arg1, were generated through IL-4 stimulation (Fig. S4C). Subsequently, cell membranes with anti-inflammatory and homologous microglia-targeting properties were isolated from M2 microglia. LB244 was then encapsulated within the vesicles using repeated coextrusion and ultrasonication. The drug-loaded vesicles (LB244@M2) retained a similar morphology and maintained a good vesicular structure in PBS compared to empty M2 vesicles (Fig. 3A). The nanoparticle tracking analysis (NTA) revealed that both M2 vesicles and LB244@M2 displayed unimodal particle size distribution. The modal size distribution of M2 vesicles was 136.1 ± 7.0 nm, while that of LB244@M2 was 146.7 ± 8.9 nm (Fig. 3B). These findings suggested that the process of drug loading did not result in any deleterious alterations to the morphology or size of the vesicles. Moreover, the cell membrane surficial proteins bands in the LB244@M2 were nearly identical to that of M2 microglia membrane (Fig. 3C). These results indicated that the LB244@M2 was successfully enveloped by M2 microglia membrane, providing a structural basis for homologous targeting.

Fig. 3.

Preparation and characterization of LB244@M2. (A) Representative TEM images of M2 vesicles and LB244@M2 nanoparticles. Scale bar: 200 nm. (B) Size distribution of M2 vesicles and LB244@M2 nanoparticles detected by NTA. (C) SDS-PAGE electrophoresis analysis of protein composition in M2 microglia, M2 membranes, and LB244@M2 nanoparticles. (D) Images and illustrations of LB244, M2 vesicles and LB244@M2 nanoparticles dispersed in 0.9 % NaCl solution. (E) In vitro release profile of LB244@M2 under different pH conditions over 48 h. (F) Cell viability of BV2 cells treated with PBS, M2 vesicles (0.42 μg/mL), or LB244@M2 nanoparticles (equivalent to 0.5 μM free LB244). Viability was assessed by CCK-8 assay. N = 6 per group. (G) Schematic representation of MCAO/R mouse experimental design. (H) Hematoxylin and eosin (H&E) staining of the major organs after 7 days administration of various drugs. Scale bar: 500 μm. Statistical significance: n.s: no significance vs. Control group.

Vesicle encapsulation significantly improved the aqueous solubility of LB244, which is otherwise insoluble in 0.9 % NaCl and forms a white precipitate. Encapsulation of LB244 in M2 vesicles remarkably prevented precipitation, as illustrated in Fig. 3D. In addition, a detailed characterization of the drug loading rate and the drug release ability of the vesicles was conducted. UV absorption spectroscopy assay revealed a drug loading efficiency of ∼63.5 % and an encapsulation efficiency of ∼32.7 % (Fig. S5A and B). The release rate of the vesicle drug at pH 5.0 was estimated to be approximately 77.3 % within 10 h. Conversely, under normal physiological conditions (pH 7.4), the drug release was approximately 29.7 % over the same period (Fig. 3E). These results suggested that LB244@M2 exhibits pH-sensitive drug release, with enhanced release under acidic conditions. Finally, an investigation was conducted into the biosafety of LB244@M2 in both in vitro and in vivo contexts. Our findings demonstrated that the encapsulation of LB244 in M2 vesicles did not compromise cell viability (Fig. 3F) and did not induce any alterations in tissue structure (Fig. 3G and H).

3.3. Targeting ability of LB244@M2 to microglia in the ischemic brain region

To assess cellular uptake, LB244@M2 was labeled with Cy5 dye and incubated with OGD/R-treated BV2 cells for 2 h. Confocal imaging confirmed the presence of LB244@M2 within BV2 cells, suggesting that it can be taken up by BV2 cells with ease (Fig. 4A). To further evaluate the targeting capability of LB244@M2 in vivo, Cy5-labeled LB244@M2 was administered to MCAO/R mice via intravenous (i.v.) or intranasal (i.n.) routes. Fluorescence imaging was performed using the IVIS detection system. Administration of Cy5-labeled LB244@M2 (150 μg vesicles) was conducted immediately upon reperfusion, and fluorescence signals were assessed after 10 min. Mice treated with Cy5-labeled LB244@M2 exhibited stronger fluorescence signals than those in the Sham and Cy5-only groups (Fig. S6A). The fluorescence intensity in the i.v. group was significantly higher than that in the i.n. group (P < 0.05; Fig. S6B). At 2 h post-administration, mice were sacrificed, and the brain, heart, liver, kidneys, and spleen were harvested for fluorescence imaging. Notably, Cy5 fluorescence remained highly concentrated in the ischemic brain region of mice in the i.n. group, whereas significantly lower fluorescence was observed in the brains of i.v.-treated mice (Fig. 4B). Quantitative analysis revealed that the fluorescence intensity in the brain of i.n.-treated mice was 1.7-fold higher than that in the i.v. group (Fig. 4C). To further investigate the brain distribution of LB244@M2, frozen brain sections were analyzed via confocal microscopy. As shown in Fig. 4D, E, a higher concentration of Cy5-labeled LB244@M2 was detected in the ipsilateral ischemic region of the i.n. administration group (184.7 ± 11.5 particles) compared to i.v. administration group (93.3 ± 9.5 particles). Moreover, in the i.n. group, the fluorescence intensity of Cy5-labeled LB244@M2 in the ischemic region (Ipsilateral) was significantly higher than that in non-ischemic region (Contralateral, 147.0 ± 3.0 particles) (Fig. 4E). Importantly, the red fluorescence of Cy5-labeled LB244@M2 co-localized with Iba1-positive microglia, indicating microglial uptake of LB244@M2. The number of Cy5-labeled LB244@M2 particles engulfed by microglia was significantly greater in the ipsilateral brain region (4.2 ± 0.7 particles) than in the contralateral brain region (1.4 ± 1.0 particles) (Fig. 4F), confirming effective microglial targeting by LB244@M2.

Fig. 4.

Targeting ability of LB244@M2 to microglia in the ischemic brain region. (A) Confocal microscopy images showing the uptake of Cy5-labeled LB244@M2 (red) by BV2 cells (Iba1, green) and their localization around the nucleus (blue). White arrows indicate the LB244@M2 within the cytoplasm of BV2 cells. Scale bar: 20 μm. (B) Representative IVIS images of dissected mice brains 2 h post-administration of PBS, Cy5 and Cy5-labeled LB244@M2. (C) Fluorescence intensity in all groups of brain was quantified. N = 6 per group. (D) Fluorescence microscopy images of bilateral brain sections from MCAO/R mice collected at 2 h after intravenous (i.v.) or intranasal (i.n.) administration of Cy5-labeled LB244@M2. (E–F) Quantitative analysis of Cy5-labeled LB244@M2 accumulation in the brain (N = 3 per group) and microglia uptake (N = 9 per group) is shown. (G–H) Ex vivo fluorescence imaging showing Cy5 fluorescence intensity in major organs (liver, lungs, kidneys, spleen, and heart) of each group at 2 h post-administration. Fluorescence intensity of organs was quantified. N = 3 per group. (I) Time-dependent accumulation of Cy5-labeled LB244@M2 in the brain following i.n. administration, as assessed by IVIS imaging. (J) The line chart shows the average radiance after subtraction of background fluorescence from untreated mice brains at different time points. N = 6 per group. Statistical significance: n.s: no significance; ∗P < 0.05, ∗∗∗P < 0.001 vs. Sham group or Ipsilateral (i.v.) group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. Cy5-labeled LB244@M2 (i.v.) group or Ipsilateral (i.n.) group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

To assess systemic distribution, fluorescence signals in peripheral organs were analyzed 2 h post-administration. The i.v. group exhibited markedly enhanced fluorescence in the liver, lungs, and kidneys (Fig. 4G and H), indicating rapid hepatic and renal clearance. In contrast, i.n. administration resulted in prolonged fluorescence retention in the brain, with reduced accumulation in peripheral organs, suggesting enhanced brain-targeted delivery and reduced systemic exposure. We next monitored the temporal profile of Cy5-LB244@M2 accumulation in the brain following i.n. administration. Quantitative fluorescence analysis revealed a peak signal at 0.5 h post-administration, followed by a gradual decline over 24 h (Fig. 4I and J). Despite the decreasing intensity, sustained fluorescence was detectable throughout the observation period, indicating prolonged nanoparticle retention in the brain.

Collectively, these findings demonstrated that intranasal administration of LB244@M2 provides a non-invasive and efficient delivery route for targeted brain delivery, achieving sustained nanoparticle presence in the ischemic brain for up to 24 h while minimizing peripheral clearance—an advantageous profile for time-sensitive neuroprotective interventions.

3.4. LB244@M2 enhances neurological function recovery following I/R brain injury

To investigate the neuroprotective effects of LB244@M2 in ischemic stroke, we assessed neurological function recovery by analyzing infarct volume and neurological scores in MCAO/R mice. Firstly, MCAO mice were randomly assigned into four groups. The initial treatment was administered immediately after reperfusion. A 5 mg/kg dose of LB244 was administrated intraperitoneally (i.p.) to the MCAO/R mice in the LB244 group. Vesicles with or without LB244 (150 μg total, 10 μL) were administered intranasally to MCAO/R mice in the M2 vesicles and LB244@M2 groups. Additionally, mice in Sham and MCAO/R groups received saline (10 μL) by i.n. administration. Each group received treatment once daily for three consecutive days.

On day 7 post-treatment, infarct volumes were quantified by TTC staining. LB244@M2-treated MCAO/R mice exhibited a 49.8 % reduction in infarct volume compared with saline-treated controls (18.9 % vs. 37.7 %, P < 0.001). M2 vesicles alone (37.1 %) did not differ significantly from the MCAO/R group (P > 0.05), whereas free LB244 achieved a moderate reduction (31.0 %, P < 0.01). Notably, LB244@M2 provided significantly greater neuroprotection than both M2 vesicles and free LB244, underscoring the enhanced efficacy of the nanoparticle-based delivery strategy (Fig. 5A and B). This structural neuroprotection was accompanied by functional improvement. Bederson's neurological scores improved by 53.3 % in the LB244@M2 group (1.4 ± 0.5) relative to MCAO/R controls (3.0 ± 0.7, P < 0.001), outperforming free LB244 (23.3 % improvement, P < 0.05) and M2 vesicles (no improvement, P > 0.05) (Fig. 5C). Consistent results were observed in the modified Neurological Severity Score (mNSS) test, where LB244@M2 yielded the lowest scores, indicating the most substantial neurological recovery among all treatment groups (Fig. S7). Collectively, these results demonstrated that LB244@M2 not only achieved superior infarct volume reduction but also delivered the most pronounced functional recovery, validating the therapeutic advantage of M2-membrane-coated nanoparticle delivery in IS.

Fig. 5.

LB244@M2 enhanced neurological function recovery following I/R brain injury. (A) Representative TTC-stained brain sections and (B) corresponding quantification of infarct volumes across different groups on day 7 post-MCAO/R. N = 6 per group. (C) All groups were evaluated for neurological deficits using the modified Bederson's scoring system (range: 0–5, where 0 = normal and 5 = severe deficit). N = 10 per group. (D) Representative H&E-stained brain sections illustrating histopathological changes in each group. The white dashed line marks the boundary of the ischemic penumbra. Scale bar: 50 μm. (E) Representative mouse trajectory in the OFT. (F–I) Quantitative analysis of locomotor activity and anxiety-like behavior in MCAO/R mice across different groups, as evaluated by: Total movement distance (F); Movement speed (G); Number of entries into the central zone (H); Time spent in the central zone (I). N = 6 per group. (J) Schematic representation of the Y-maze spontaneous alternation test (YMT). (K) Percentage of spontaneous alternation in the YMT, indicating spatial memory performance in each group. N = 6 per group. Statistical significance: n.s: no significance; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. MCAO/R group. ^##P < 0.01, ^###P < 0.001 vs. LB244 group.

Histopathological examination further supported the superior neuroprotective effect of LB244@M2. H&E staining revealed marked neuronal necrosis and cytoarchitectural disruption in MCAO/R mice. Both free LB244 and LB244@M2 treatments ameliorated these pathological changes; however, LB244@M2 demonstrated the greatest preservation of tissue integrity, characterized by minimal vacuolization and necrotic areas (Fig. 5D). Correspondingly, Nissl staining showed pronounced neuronal damage in the ischemic region of MCAO/R mice, characterized by sparse neuronal distribution and significantly diminished Nissl body counts. At 7 days post-treatment, both LB244 and LB244@M2 administration conferred significant neuroprotection, rescuing Nissl body numbers (Fig. S8A). Quantitative analysis confirmed that intranasal administration of LB244@M2 significantly outperformed free LB244 in restoring Nissl body density (P < 0.01), whereas M2 vesicles alone showed no therapeutic benefit compared to MCAO/R controls (P > 0.05) (Fig. S8B). The preservation of Nissl bodies, key markers of neuronal metabolic activity, further corroborates the superior neuroprotective effects of LB244@M2 observed in H&E staining. The absence of therapeutic effect in the M2 vesicles group indicated that LB244 is the primary active agent, while the enhanced performance of LB244@M2 over free LB244 confirmed the therapeutic advantage of nanoparticle-based intranasal delivery.

To evaluate behavioral and cognitive changes in MCAO/R mice under different treatments, the open field test (OFT) and Y‐maze test were conducted on day 7. The OFT revealed a significant decline in locomotor and exploratory activity in MCAO/R mice (Fig. 5E). LB244 and LB244@M2 treatments improved motor function in MCAO/R mice. Mice in the LB244@M2 group showed greater movement distances and speeds compared to the LB244 group (Fig. 5F and G). Additionally, LB244@M2-treated mice entered the central zone more frequently and spent more time there compared to LB244-treated mice (Fig. 5H and I). These results further suggested that LB244@M2 (i.n.) treatment could reduce the anxiety-like behavior in MCAO/R mice. Spatial memory and exploratory activity were assessed using the Y-maze continuous alternation test (YMT) (Fig. 5J). MCAO/R mice showed reduced spontaneous alternation behavior in the YMT. No significant differences were observed between the MCAO/R group and the M2 vesicles group. However, LB244@M2 treatment significantly increased alternation rates compared to LB244 alone, with a 20.9 % improvement in spatial memory performance (Fig. 5K). Collectively, these findings demonstrate that intranasal LB244@M2 administration effectively enhances neuroprotection, reduces infarct volume, improves locomotor function, and alleviates cognitive deficits in MCAO/R mice. This highlighted the superior in vivo therapeutic potential of LB244@M2 over free LB244 for IS treatment.

3.5. STING inhibition mitigated I/R-induced microglial pyroptosis and suppressed neuroinflammation

Considering the observation that LB244@M2 improved the neurobehavioral function of MCAO/R mice, we next explored its neuroprotective mechanism. We first verified the in vivo inhibition of the STING pathway by LB244@M2. Western blotting analysis showed that both free LB244 and LB244@M2 significantly inhibited the STING pathway, as seen in the reduced phosphorylation of its key components [STING, interferon regulatory factor 3 (IRF3), and nuclear factor-kappa B (NF-κB)], which are critical mediators of pro-inflammatory cytokine production (Fig. S9A–F). Of note, LB244@M2 produced the strongest suppression (P < 0.05 vs. free LB244). Consistent with this upstream inhibition, LB244@M2 also most potently reduced the mRNA expression of downstream interferon-stimulated genes (ISGs), including IFN-β, Isg15, and Isg20 (Fig. S10A–C). Collectively, these findings demonstrated that intranasal delivery of LB244@M2 significantly enhanced the inhibitory effect of LB244 on STING-driven neuroinflammation following cerebral I/R injury.

Consistent with STING pathway suppression, LB244@M2 also most effectively attenuated pyroptosis, marked by the greatest reduction (decreased by approximately 41.3 % for Cleaved GSDMD and 31.5 % for Cleaved Caspase-1) in these key proteins versus free LB244 (Fig. S11). To obtain visual evidence of this inhibition in microglia, we next performed double immunostaining for Iba1 and GSDMD across treatment groups. Immunofluorescence analysis revealed a marked reduction in GSDMD⁺/Iba1⁺ cells in both the LB244 and LB244@M2 groups compared to the MCAO/R group, indicating that LB244 effectively inhibited microglial pyroptosis in MCAO/R mice (Fig. 6A and B). Importantly, LB244@M2-treated mice exhibited a significantly lower density of GSDMD-expressing microglia in ischemic brain regions (53.1 ± 10.8 positive cells/mm²) than the LB244 group (93.0 ± 5.4 positive cells/mm²). These results collectively suggested that intranasal delivery of LB244@M2 improved the efficacy of LB244 by more effectively inhibiting STING pathway activation and consequently attenuating microglial pyroptosis after I/R injury.

Fig. 6.

STING inhibition mitigated I/R injury-induced microglial pyroptosis and suppressed neuroinflammation. (A) Representative immunofluorescence images of microglia (Iba1, green) and GSDMD (red) in the ischemic penumbra on days 7 after treatment. Scale bar: 20 μm. (B) Quantification of GSDMD⁺Iba1⁺ cells in (A). N = 6 per group. (C) Immunofluorescence staining for Iba1 (red) and iNOS (green) illustrating the presence of M1-polarized microglia (Iba1⁺/iNOS⁺) in each group. (D) Quantification of M1 microglia is shown. N = 6 per group. (E) Immunofluorescence staining for Iba1 (red) and Arg1 (green) demonstrating the presence of M2-polarized microglia (Iba1⁺/Arg1 ⁺) in each group. (F) Quantification of M2 microglia is presented. N = 6 per group. (G) Double immunofluorescence staining (TUNEL and NeuN) showing apoptotic neurons in each group. Scale bar: 20 μm. (H) Quantification of TUNEL⁺/NeuN⁺ apoptotic neurons is provided. N = 6 per group. Statistical significance: n.s: no significance; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. MCAO/R group. ^###P < 0.001 vs. LB244 group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Since pyroptotic cell death releases potent inflammatory mediators that exacerbate inflammation [34], we next hypothesized that LB244@M2's suppression of microglial pyroptosis would correspondingly ameliorate inflammatory responses following I/R injury. To test this, we assessed microglial polarization states, given their pivotal role in regulating neuroinflammatory cascades. Immunofluorescence double-staining revealed that LB244@M2 treatment significantly reduced iNOS⁺/Iba1⁺ M1 microglia, which are associated with neurotoxic inflammation (Fig. 6C and D). Conversely, a significant increase in Arg1⁺/Iba1⁺ M2 microglia was observed in the ischemic regions of LB244@M2-treated mice, indicating a shift towards an anti-inflammatory phenotype (Fig. 6E and F). To further characterize the inflammatory response, we performed real-time RT-PCR to quantify mRNA expression levels of key inflammatory cytokines in the brain tissue. LB244@M2 treatment significantly downregulated M1-associated pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6 (Fig. S12A–C). In contrast, M2-associated anti-inflammatory cytokines, such as IL-10 and IL-4, were significantly upregulated (Fig. S12D and E). These findings suggested that LB244@M2 mitigated neuroinflammation not only by promoting M2 microglial polarization but also by concurrently inhibiting M1 microglia-associated pro-inflammatory responses. This dual effect could also be achieved through its potent suppression of the STING pathway (Fig. S9), which has been shown to be a master regulator of microglial polarization [7,35].

Having established the potent inhibition of STING signal and microglial pyroptosis by LB244@M2, the subsequent examination focused on its impact on oxidative stress and neuronal survival. Given that I/R-induced reactive oxygen species (ROS) are well-known mediators of neuronal damage, we quantified ROS production in ischemic brain tissue using dihydroethidium (DHE) fluorescence staining at 7 days post-treatment (Fig. S13A). DHE analysis revealed that significant ROS elevation in the MCAO/R and M2 vesicles groups compared to the Sham group, with no statistically significant difference observed between these two experimental groups (P > 0.05). However, fluorescent intensity of ROS production was markedly reduced in MCAO/R mice treated with either LB244 or LB244@M2. Most notably, LB244@M2 demonstrated the most significant ROS suppression among all treatment groups (Fig. S13B). Immunofluorescence analysis of TUNEL⁺/NeuN⁺ apoptotic neurons yielded consistent results with our results (Fig. 6G and H). While the M2 vesicles group showed no significant reduction in apoptotic neurons compared to the MCAO/R group (P > 0.05), the LB244@M2 group achieved a striking 67.9 % decrease in apoptotic neurons (P < 0.001). The protective effect of LB244@M2 was significantly greater than that of LB244 alone, which only achieved a 35.4 % reduction (P < 0.05 vs. MCAO/R group; P < 0.001 vs. LB244@M2 group). Together, these findings further confirmed that the neuroprotective effects were attributable to LB244 rather than M2 vesicles, and that the intranasal nanoparticle delivery system markedly enhanced the therapeutic efficacy of LB244 by facilitating targeted accumulation in ischemic brain regions.

To assess the long-term therapeutic potential of LB244@M2, neurological and inflammatory outcomes were evaluated up to 28 days post-treatment. LB244@M2-treated MCAO/R mice exhibited progressive neurological improvement, with mNSS scores significantly reduced from day 7 to day 14 (P < 0.05) and day 28 (P < 0.01), followed by a plateau between day 14 and 28 (P > 0.05), indicating sustained functional recovery (Fig. S14). This improvement was accompanied by persistent anti-inflammatory effects, as evidenced by a marked reduction in iNOS⁺/Iba1⁺ M1 microglia at both 14 and 28 days compared with 7 days, with no difference between the later timepoints (Fig. S15A and B). Consistently, qPCR analysis revealed sustained downregulation of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) at 14 and 28 days relative to 7 days (Fig. S16). Collectively, these findings demonstrated that LB244@M2 conferred durable neuroprotection and anti-inflammatory effects for at least 28 days following I/R injury.

4. Discussion

CNS is a highly regulated and intricate environment that requires precise immune control to maintain neuronal function and facilitate post-injury repair. Ischemic brain injury, one of the most common CNS injuries, progress in two distinct phases: primary and secondary injury [36]. The primary phase occurs immediately after ischemia, causing cellular stress, membrane disruption, ionic imbalance, excitatory neurotransmitter release, and reactive oxygen species generation in the affected region [37]. These initial events initiate secondary injury, characterized by neuroinflammation, neuronal death, and progressive functional impairment [38], which exacerbate tissue damage in the surrounding healthy brain regions.

Microglia, the resident immune cells of the CNS, are pivotal in immune defense and neuroinflammation regulation [39]. Post-stroke microglial activation triggers a robust inflammatory response, which is a hallmark of cerebral I/R injury [40]. Emerging evidence identified the cytoplasmic DNA sensor cGAS and its downstream effector STING as key mediators of I/R-induced neuroinflammation, with both predominantly upregulated in microglia within the ischemic cortex [31,41]. STING activation drives pyroptotic cell death and inflammasome activation, amplifying pro-inflammatory cascades [42,43]. In the present study, we confirmed marked elevation of STING and GSDMD in peri-infarct microglia following I/R injury. Pyroptotic microglia are potent sources of IL-1β and IL-18, cytokines known to exacerbate secondary neuronal injury [44], and MCAO/R mice exhibited significantly elevated IL-1β and IL-18 levels relative to sham controls. This inflammatory profile corresponded with the most severe ischemic pathology, characterized by widespread neuronal apoptosis, the largest infarct volumes, and the most impaired neurological function among all experimental groups. These findings underscored STING as a central regulator of microglial pyroptosis in I/R brain injury and support its therapeutic targeting to mitigate neuroinflammation and improve post-stroke outcomes.

While a considerable number of pharmaceutical agents have been identified as cGAS–STING pathway inhibitors with demonstrated therapeutic potential in autoimmune diseases, cancers, and infectious conditions [45], the role in CNS injury remains poorly understood. LB244, a novel highly selective STING inhibitor developed for treating STING-dependent inflammatory diseases, has demonstrated low toxicity but presents pharmacokinetic limitations, including poor oral bioavailability, short half-life, and rapid systemic clearance [15]. Furthermore, its efficacy in CNS injuries remains unexplored. Our study provided the first evidence that LB244 effectively suppresses STING expression and inhibits OGD/R-induced pyroptosis in microglia. To enhance its brain-targeted delivery, we developed LB244@M2, a nanovesicle-based system derived from M2 microglial cell membranes, designed to efficiently transport LB244 to microglia in the ischemic brain. Considering the pro-inflammatory microenvironment in ischemic regions, we polarized primary microglia with IL-4 to induce an M2 phenotype, thereby enhancing the anti-inflammatory properties of LB244@M2. Structurally, LB244@M2 mimicked the morphology and biophysical characteristics of exosomes, offering aqueous solubility, stability, and biocompatibility, while retaining source cell membrane properties, facilitating ischemic tissue homing. Furthermore, LB244@M2 possessed a rapid pH-responsive drug release property under acidic condition (pH 5.0), which closely mimics the pathological microenvironment of ischemic brain regions [46]. The typical acidic microenvironment, primarily caused by lactic acid accumulation following ischemia, facilitated nanoparticle membrane dissociation, thereby accelerating targeted LB244 release in ischemic areas. Our results confirmed that this pH-dependent selective release mechanism not only significantly enhanced local drug (Cy5-labeled LB244@M2) concentration in the I/R injury region, while simultaneously reducing nonspecific drug distribution in normal brain tissue (pH 7.0–7.4).

Intranasal (i.n.) administration has drawn attention as an effective, non-invasive approach for CNS drug delivery, as it enables direct nose-to-brain transport while bypassing the blood-brain barrier (BBB) [47]. The close anatomical connection between the nasal cavity and the brain facilitates direct nose-to-brain drug delivery. The olfactory and trigeminal nerves, nasal lymphatic system, and perivascular spaces are involved in the rapid intranasal delivery and diffusion of drugs within the brain parenchyma [48]. In our study, we compared the distribution of LB244@M2 following intravenous (i.v.) and intranasal (i.n.) administration. Both routes successfully delivered LB244@M2 to microglia in I/R brain regions. However, i.v. administration resulted in significant drug clearance via hepatic and renal metabolism, leading to minimal drug accumulation in the brain within 2 h post-injection. Conversely, i.n. administration bypassed systemic metabolism, allowing greater LB244@M2 retention in the ischemic brain. These findings underscored the advantages of intranasal drug delivery in achieving efficient and sustained CNS targeting while minimizing systemic toxicity.

Additionally, we systematically evaluated the therapeutic effects of M2 vesicles, free LB244 and LB244@M2 nanoparticles in the MCAO/R model through systematic in vivo experiments. While M2 vesicles physically resemble exosomes, their therapeutic performance showed no statistically significant improvement over the untreated MCAO/R group, likely due to the absence of functional miRNA and protein cargo characteristic of natural M2-derived exosomes. In accordance with established protocols [15], LB244 were administered intraperitoneally (i.p.) to optimize systemic bioavailability. Our experiments confirmed that free LB244 exerted notable neuroprotective effects, including significant inhibition of microglial pyroptosis, promotion of microglial polarization from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype, reduction of neuronal apoptosis, and consequent improvement in neurological function.

Remarkably, intranasal delivery of LB244@M2 nanoparticles conferred superior therapeutic benefits in MCAO/R mice. Through efficient encapsulation of LB244 and leveraging the specific targeting capability of M2 microglia membranes, LB244@M2 nanoparticles were selectively taken up by microglia while achieving precise drug release triggered by the acidic microenvironment in ischemic regions, without observable toxicity. Mechanistically, LB244@M2 more efficiently inhibited STING pathway activation and suppressed microglial pyroptosis than LB244 alone. This dual action led to significantly attenuated neuroinflammation, as evidenced by promoted microglial polarization toward the M2 phenotype, decreased expression of pro-inflammatory cytokines, and reduced ROS production in the ischemic brain. These mechanistic advantages translated into a further 12.1 % reduction in infarct volume compared with LB244 treatment, alongside greater neurological and cognitive recovery. Long-term assessment demonstrated that both neuroprotective and anti-inflammatory effects were sustained over 28 days. Collectively, these findings strongly supported the translational potential of LB244@M2 as a targeted therapeutic platform for I/R-induced brain injury.

Despite these encouraging results, several limitations need to be addressed. First, the optimal therapeutic dosage of LB244@M2 remains undefined, necessitating further investigations to identify the most effective and safe concentration for neuroprotection. Second, the efficiency of intranasal delivery may be influenced by interindividual anatomical differences and variations in mucosal barrier properties, which could affect therapeutic consistency. Third, large-scale production of LB244@M2 nanoparticles poses translational challenges, particularly in ensuring batch-to-batch reproducibility, long-term stability, and cost-effectiveness. Fourth, the clinical efficacy and safety of LB244@M2 have yet to be validated, requiring rigorously designed randomized controlled trials in stroke patients. Finally, although LB244@M2 exhibits potent neuroprotective and anti-inflammatory effects, the precise molecular mechanisms underlying its modulation of neuroinflammatory responses remain to be fully elucidated.

5. Conclusion

In this study, we developed a biomimetic drug delivery system based on M2 microglia membrane. LB244-loaded M2 vesicles (LB244@M2) could be successfully targeted-delivery of LB244 to the microglia in I/R brain region by intranasal administration. LB244 exerted anti-inflammatory effect through suppressing STING expression and microglial pyroptosis. The therapeutic potential of LB244 treatment in I/R brain injury was greatly improved with this efficient delivery strategy of LB244@M2 intranasal administration. The neuroprotective effect of LB244@M2 were manifested in the reduction of microglial cell pyroptosis and neuroinflammation, and the improvement of neurological and cognitive functions. Compared to LB244, LB244@M2 exerted better neuroprotective effects by inhibiting microglial pyroptosis and inflammation in MCAO/R mice. Importantly, the safety and biocompatibility of the LB244@M2 nanoparticles were confirmed with no significant adverse effects in treated mice. The combination of targeted non-invasive delivery mechanisms has the potential to provide an innovative pharmaceutical approach for CNS disorders. The development of LB244@M2 for intranasal administration catered to the need for safely and effectively targeting the I/R brain. This therapeutic strategy may promise to be extend to the treatment of the other CNS disorders characterized by inflammation and immune dysregulation.

CRediT authorship contribution statement

Xiaoting Zhang: Writing – review & editing, Writing – original draft, Visualization, Validation, Formal analysis, Data curation, Conceptualization. Jingpei Guo: Visualization, Validation, Formal analysis, Data curation. Yun Zhang: Visualization, Validation, Formal analysis. Shengchao Zhao: Validation, Formal analysis. Jiawen Chen: Validation, Formal analysis. Jiawei Jiang: Writing – review & editing. Xiaojun Hu: Resources, Methodology. Bin Zhou: Writing – review & editing, Supervision, Resources. Ke Zhang: Writing – review & editing, Writing – original draft, Project administration, Conceptualization.

Ethics approval statement

The animal experiments in this study were approved by the Animal Ethics Committee of the Fifth Affiliated Hospital of Sun Yat-sen University (Approval number: 00629).

Funding

This study was supported by the National Natural Science Foundation of China (No. 82102164 and 82170405), and the Natural Science Foundation of Guangdong Province (2025A1515010670).

Declaration of competing interest

The authors declare that they have no competing interests.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.