Blood-brain barrier-penetrative lipid nanoparticles enable systemic delivery of TRIM11 mRNA to disaggregate Tau in Alzheimer’s disease models

TL;DR

A new nanoparticle delivery system enables effective brain delivery of mRNA to clear Tau aggregates in Alzheimer's disease models.

Contribution

A ligand-free lipid nanoparticle is developed for efficient blood-brain barrier crossing and targeted mRNA delivery to neurons.

Findings

PLNPs increase hippocampal mRNA accumulation and neuronal transfection compared to unformulated mRNA.

TRIM11 mRNA reduces Tau aggregates and neuroinflammation in Alzheimer’s disease mice.

Prophylactic dosing prevents Tau pathology and preserves cognitive function in young mice.

Abstract

Hyperphosphorylated Tau aggregates are a central pathological hallmark of Alzheimer’s disease (AD), yet no approved therapy directly targets this process. mRNA therapeutics provide a transient and non-viral option but are limited by the blood-brain barrier (BBB). TRIM11 is an ATP-independent disaggregase that dissolves pathological Tau fibrils and promotes proteasomal clearance. Here, a ligand-free lipid nanoparticle (PLNP) is developed with zwitterionic, acetylcholine-mimetic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) as a core component and leverages interactions with nAChRs and chTs to enable BBB transcytosis. Systemic PLNP delivery of TRIM11 mRNA yields an 8.1-fold increase in hippocampal accumulation and >30-fold higher neuronal transfection than unformulated mRNA. In 3×Tg-AD mice, PLNP-mTRIM11 reduces P-Ser396- and AT8-positive Tau aggregates, attenuates neuroinflammation, restores synaptic/neuronal integrity, and improves cognition and nest building for ≥3 months. Early prophylactic dosing prevents Tau pathology and preserves cognitive function, supporting PLNP-mTRIM11 for tauopathy therapy.

•PLNPs achieves efficient BBB transcytosis and neuron-targeted mRNA delivery•TRIM11 mRNA enables controllable restoration of Tau disaggregase for Tau clearance•Systemic administration provides therapeutic and prophylactic benefit in AD mice

PLNPs achieves efficient BBB transcytosis and neuron-targeted mRNA delivery

TRIM11 mRNA enables controllable restoration of Tau disaggregase for Tau clearance

Systemic administration provides therapeutic and prophylactic benefit in AD mice

Shi et al. develop a ligand-free, zwitterionic lipid nanoparticle (PLNP) that enables systemic, BBB-penetrant delivery of mRNA with enhanced hippocampal deposition. TRIM11 efficiently reduces pathological Tau aggregates and neuroinflammation and restores cognition in 3×Tg-AD mice, with prophylactic benefit in younger cohorts, supporting PLNP-mTRIM11 as safe and effective for tauopathies.

Figures

PLNP-mRNA exhibits enhanced BBB penetration and robust gene transfection *in vitro* and *in vivo*(A) Schematic representation of features of PLNP-mRNA.(B) Flow cytometry analysis of cellular uptake of Cy5-labeled PLNP-mRNA in SH-SY5Y cells after 4-h incubation.(C) Corresponding confocal fluorescence images of intracellular Cy5 signal. Scale bars, 10 μm (*n* = 3).(D) GFP mRNA transfection efficiency of PLNPs in SH-SY5Y cells after 24-h incubation as determined by flow cytometry and confocal microscopy (inset). Scale bars, 50 μm (*n* = 3).(E) *In vivo* whole-brain fluorescence imaging (left) and

PLNP-mTRIM11 enables efficient TRIM11 expression and facilitates the disaggregation of phosphorylated Tau and Tau tangles *in vitro*(A) Schematic structure of the pcDNA3.1-cFLAG-TRIM11-GFP plasmid used for *in vitro* transcription.(B and C) Western blot analysis of FLAG and TRIM11 protein expression in SH-SY5Y cells 24 h post-transfection with Lipo_2000_+pcDNA3.1-cFlag-TRIM11-GFP plasmid or PBS (*n* = 3).(D) Quantitative RT-PCR analysis of TRIM11 mRNA levels in SH-SY5Y cells following transfection with Lipo_2000_ + T7-cFLAG-TRIM11 mRNA or PBS (*n* = 4).(E) Confocal images showing co-localizati

PLNP-mTRIM11 treatment leads to significant and sustained improvements in cognitive and behavioral performance in 3×Tg-AD mice(A) Schematic of the treatment and behavioral assessment timeline. Seven-and-a-half-month-old 3×Tg-AD mice received intravenous injections of PLNP-mTRIM11 (2 mg mRNA equiv/kg) on days 0, 7, and 14. Control groups included 3×Tg-AD mice treated with PLNP-mGFP or PBS and WT mice. Behavioral evaluations—OFT, MWM, NOR, and nesting test—were performed on day 16 (first behavioral test, 1#).(B) Total distance traveled in the OFT (*n* = 10).(C) Number of platform crossings in th

PLNP-mTRIM11 treatment ameliorates Tau pathology, suppresses neuroinflammation, and protects neuronal integrity in 3×Tg-AD mice(A) Schematic overview of the experimental design for assessing insoluble Tau pathology in the hippocampus following treatment; mRNA dosage: 2 mg equiv/kg per injection.(B) IHC staining of phosphorylated Tau (P-ser396), Tau tangles (AT8), and TRIM11 in hippocampal sections collected 28 days after the final injection of PLNP-mTRIM11 or controls. Scale bars, 50 μm.(C–E) Quantification of TRIM11, P-ser396, and AT8 staining intensities in the hippocampus (*n* = 3).(F) West

PLNP-mTRIM11 treatment prevents early Tau pathology and preserves cognitive and behavioral function in young 3×Tg-AD mice(A) Experimental timeline: 5.5-month-old 3×Tg-AD mice received three intravenous injections of PLNP-mTRIM11 (2 mg mRNA equiv/kg) on days 0, 7, and 14. Behavioral and cognitive assessments were conducted starting on day 47.(B) Total distance traveled in the OFT (*n* = 8).(C and D) (C) Discrimination index and (D) PI in the NOR test (*n* = 8).(E and F) (E) Total platform crossings (*n* = 8) and (F) latency (*n* = 6) to platform in the MWM test.(G) Nesting behavior scores (*n*

Full text

Introduction

Alzheimer’s disease (AD) affects more than 50 million individuals worldwide and still lacks an effective cure.1^,^2^,^3^,^4^,^5 Current pharmacological options, including acetylcholinesterase inhibitors (e.g., donepezil and galantamine) and NMDA receptor antagonists (e.g., memantine), provide only short-term symptomatic improvement and do not modify the underlying neuropathology.6^,^7^,^8 More recently, monoclonal antibodies targeting amyloid-β (Aβ) pathology, such as aducanumab and lecanemab, have been approved by the US Food and Drug Administration (FDA). However, their modest clinical benefit, significant cost, and adverse events—such as amyloid-related imaging abnormalities—have sparked debate regarding their therapeutic value.9^,^10^,^11 Accumulating evidence suggests that Tau pathology—most notably the formation of neurofibrillary tangles driven by hyperphosphorylated Tau aggregation—tends to track cognitive decline more closely than Aβ deposition.12^,^13 While Tau-targeted therapeutics are advancing, most strategies focus on inhibiting phosphorylation or blocking Tau propagation, with limited success in reversing established aggregates.14^,^15^,^16^,^17 Crucially, no currently approved therapy actively disaggregates pathogenic Tau.

TRIM11, a member of the tripartite motif family of E3 ligases, was recently identified as an ATP-independent protein disaggregase capable of dissolving misfolded Tau fibrils and directing their proteasomal degradation.18 TRIM11 is downregulated in AD brains, and its overexpression in neuronal models reduces Tau aggregation, restores synaptic integrity, and improves cell survival.19 These findings establish TRIM11 as a potent neuroprotective factor and underscore its potential as a therapeutic target for tauopathies.20^,^21 Delivering TRIM11 mRNA enables rapid restoration of its expression and clearance of pathological Tau, a strategy particularly valuable in diseases marked by protein loss or dysfunction.22^,^23 As a transient, non-viral, and non-invasive modality, mRNA therapeutics provide a safer alternative, yet effective targeting of the brain across the blood-brain barrier (BBB) remains a major challenge.

Lipid nanoparticles (LNPs) have revolutionized mRNA therapeutics, as exemplified by their success in SARS-CoV-2 vaccines. However, conventional LNPs exhibit poor BBB penetration, limiting their application for central nervous system (CNS) disorders.24^,^25^,^26^,^27^,^28 Here, we report a rationally engineered, zwitterionic LNP designated PLNP by employing PMPC (poly(2-methacryloyloxyethyl phosphorylcholine)) as a core component. PMPC, as an acetylcholine analog, facilitates effective BBB penetration and neuron-targeted delivery by interacting with nAChRs and chTs expressed on BBB endothelial cells and neurons without any ligand modification (Figure 1).29^,^30^,^31 We demonstrate that intravenous administration of PLNP-encapsulated TRIM11 mRNA (PLNP-mTRIM11) achieves efficient brain transfection in vivo, with >7.5-fold enrichment in hippocampal tissue and >30-fold higher expression compared to naked mRNA. In aged 3×Tg-AD mice, three systemic doses of PLNP-mTRIM11 significantly reduced AT8- and P-Ser396-positive Tau aggregates, suppressed neuroinflammatory cytokines (tumor necrosis factor TNF-α and interleukin IL-6), and reversed behavioral deficits in multiple cognitive tests. Importantly, a prophylactic treatment in 5.5-month-old pre-symptomatic mice attenuated the development of Tau pathology and preserved memory and learning capacity. The PLNP system uses FDA-approved excipients, exhibits high serum stability, and is biodegradable, enhancing its translational relevance.Figure 1PLNP-mRNA exhibits enhanced BBB penetration and robust gene transfection in vitro and in vivo(A) Schematic representation of features of PLNP-mRNA.(B) Flow cytometry analysis of cellular uptake of Cy5-labeled PLNP-mRNA in SH-SY5Y cells after 4-h incubation.(C) Corresponding confocal fluorescence images of intracellular Cy5 signal. Scale bars, 10 μm (n = 3).(D) GFP mRNA transfection efficiency of PLNPs in SH-SY5Y cells after 24-h incubation as determined by flow cytometry and confocal microscopy (inset). Scale bars, 50 μm (n = 3).(E) In vivo whole-brain fluorescence imaging (left) and (right) quantification of Cy5-labeled PLNP-mRNA distribution in C57BL/6 mice 0.5 h post-intravenous injection (1 mg Cy5-mRNA equiv./kg), showing enhanced brain accumulation relative to controls (n = 3).(F and G) (F) Ex vivo fluorescence imaging of brains isolated from treated mice and (G) quantification of Cy5 fluorescence intensity specifically in the hippocampus. Scale bars: 1 mm (left) and 300 μm (right) (n = 3).(H) Representative confocal images showing co-localization of Cy5 signal with neurons (β3-Tubulin), astrocytes (GFAP), and microglia (Iba-1). Scale bars, 50 μm (n = 3).(I and J) (I) Quantification of Cy5 fluorescence associated with each cell type and (J) fold change in Cy5-positive cell populations (n = 3 independent experiments).(K) Schematic showing PMPC-mediated targeting of PLNP-mRNA to the BBB via binding to chTs and nAChRs on endothelial cells.(L) Brain-wide GFP expression in C57BL/6 mice 24 h after systemic administration of PLNP-GFP mRNA, visualized by ex vivo fluorescence imaging. Scale bars: 1 mm (left) and 300 μm (right).(M and N) (M) Quantitative analysis of GFP fluorescence and (N) fold-change in GFP-positive cells in the hippocampal region, compared with LNP-mRNA controls (n = 3).(O) Representative images showing GFP expression and phosphorylated Tau (AT8) in 3×Tg-AD mice following PLNP-mGFP administration. Scale bars, 150 μm.(P) Quantification of GFP fluorescence intensity in treated mice (n = 3).Data are presented as mean ± SD. For in vitro studies (Figures 2B–2D), mRNA concentration was 2 μg/mL. For in vivo experiments (Figures 2E–2P), dosage was 1 mg mRNA equiv./kg.

Results

PLNP-mRNA achieves blood-brain barrier permeability to mediate efficient mRNA transfection

To enable brain-targeted mRNA delivery, we engineered an LNP formulation with DSPE-poly(2-methacryloyloxyethyl phosphorylcholine (DSPE-PMPC) (Figures 1A and S1), referred to as PMPC-LNPs (PLNPs). This design leveraged the interactions with nAChRs and chTs, which are highly expressed on neurons, glial cells, and brain endothelial cells (Figure S2A). The optimized PLNP formulation consisted of SM-102, DSPC, cholesterol, DMG-PEG, and DSPE-PMPC in a molar ratio of 50:5:38.5:1.5:5, respectively, and was complexed with mRNA at a mass ratio of 40.7:1. Non-targeted control LNPs were prepared identically but excluded DSPE-PMPC.

PLNP-mRNA particles displayed a homogeneous size (∼81 nm), low polydispersity index (PDI), and a slightly negative surface charge, as assessed by dynamic light scattering with morphological confirmation by transmission electron microscopy (Figures S2B–S2E). Gel electrophoresis and NanoDrop quantification confirmed high mRNA encapsulation efficiency, which was 1.5-fold higher than that of non-targeted commercial LNPs (Figures S2F–S2G). Furthermore, PLNP-mRNA retained stability under physiological conditions, with minimal mRNA degradation.

To evaluate cellular uptake and transfection efficiency, we tested PLNP-mRNA in mouse (Neuro2A) and human neuroblastoma (SH-SY5Y) cell lines, as well as in BV2 microglial cells. Flow cytometry confirmed that all three cell types express high levels of nAChRs. PLNP-mRNA demonstrated significantly enhanced cellular uptake, with 2.5- and 6.1-fold greater internalization in Neuro2A and SH-SY5Y cells, respectively, compared to non-targeted LNPs (Figures 1B and S2H). Similar effects were observed in BV2 microglial cells, further supporting the superior cellular uptake of PLNP-mRNA (Figure S2J). Confocal imaging corroborated these results, revealing brighter intracellular Cy5 fluorescence in cells treated with PLNPs compared to LNPs (Figures 1C and S2I). Free mRNA exhibited negligible uptake, consistent with its instability and charge-based repulsion. In addition, PLNP-mRNA rapidly escaped the endo/lysosomal pathway, facilitating efficient cytosolic delivery (Figure S2K).

We next assessed transfection efficiency using GFP-mRNA. PLNPs achieved 97.4% GFP-positive cells in SH-SY5Y cells and comparable results in Neuro2A cells (Figure 1D, S2L, and S2M). Varying the PMPC ratio identified 5% as optimal for maximal encapsulation and uptake. These data suggest that the combination of PMPC targeting, rapid endosomal escape, and robust mRNA release contributes to the high transfection efficacy of PLNPs.

To further evaluate if the interaction between PLNP-mRNA and nAChRs/chTs facilitates effective BBB penetration, we conducted competitive inhibition, transporter activity blockade, as well as in vitro BBB penetration assays in both SH-SY5Y neuroblastoma and bEnd.3 endothelial cell lines. Notably, Cy5-labeled mRNA was used for cell endocytosis and BBB penetration studies, whereas GFP mRNA was used for transfection assays. Specifically, we pre-incubated the cells with a 10-fold excess of DSPE-PMPC32 and hemicholinium chloride, a well-established choline transporter inhibitor,33 to assess the impact of receptor and transporter inhibition on PLNP’s uptake and transfection. Flow cytometry results revealed a significant reduction in PLNP-mRNA uptake in the pretreated groups, emphasizing the critical role of nAChRs and choline transporters in mediating endocytosis (Figures S3A–S3D). Furthermore, the transfection efficiency in the pretreated groups was reduced by 17.1% and 14.4%, respectively, compared with the PLNP-mRNA group without pretreatment (Figures S3E–S3H). Moreover, Transwell assays corroborated these results and showed a 7.6% decrease in BBB permeability in the pretreated groups upon receptor and transporter inhibition (Figure S3I and S3J). Collectively, all these results provide compelling evidence that PLNP-mRNA traverses the BBB through its interaction with nAChRs and chTs.

To evaluate BBB permeability in vivo, we intravenously administered Cy5-labeled PLNP-mRNA to C57BL/6 mice. Strong Cy5 fluorescence was detected in the brain within 30 min post-injection, exceeding that of LNPs or free mRNA (Figure 1, Figure 2, Figure 3, Figure 4, Figure 5K and S3L). Ex vivo imaging confirmed preferential accumulation in the brain, although signals were also present in the liver and spleen, suggesting clearance via the reticuloendothelial system (Figures S3M and S3N). Quantitative biodistribution analysis showed that PLNP-mRNA reached a brain accumulation of 6.9% injected dose per gram (%ID/g), markedly higher than LNPs (1.9% ID/g) and free mRNA (1.2% ID/g) (Figure 1E).

Imaging of brain sections revealed widespread distribution of PLNP-mRNA throughout the brain, with pronounced accumulation in the hippocampus (Figure 1F). Quantification showed an 8.1-fold and 41.2-fold increase in hippocampal fluorescence for PLNP-mRNA over LNPs and naked mRNA, respectively (Figure 1G). Co-staining with cellular markers confirmed that PLNPs not only preferentially targeted neurons but also localized to astrocytes and microglia (Figures 1H–1J), likely mediated by interactions with nAChRs and chTs highly expressed on brain endothelium (Figure 1K).

To evaluate in vivo transfection, PLNPs encapsulating GFP mRNA were administered intravenously. Twenty-four hours after administration, robust and widespread GFP fluorescence was observed across the brain, including the hippocampus (Figure 1L), confirming efficient in vivo mRNA transfection. Quantitative analysis revealed that PLNP-mRNA achieved 7.9- and 30.5-fold higher transfection than LNP-mRNA and free mRNA, respectively (Figures 1M and 1N). Flow cytometric analysis of single-cell suspensions obtained from brain tissues further corroborated that the PLNP-mRNA treatment achieved 16.7- and 148.1-fold higher transfection enhancement than that in the LNP-mRNA and free mRNA controls, respectively (Figure S3O and S3P). Co-localization of GFP expression with P-Tau (AT8) in hippocampal neurons (Figures 1O and 1P) further confirmed target engagement and translation. Together, these results demonstrate that PMPC-functionalized PLNPs enable efficient systemic delivery of mRNA across the BBB, with selective neuronal transfection and widespread distribution throughout the brain.

PLNP-mTRIM11 promotes intracellular clearance of insoluble Tau aggregates in vitro

Given the robust transfection efficiency demonstrated by PLNPs using GFP mRNA, we next evaluated their therapeutic potential by delivering mRNA encoding tripartite motif-containing protein 11 (TRIM11), a disaggregase known to modulate pathological protein accumulation. TRIM11 mRNA (mTRIM11) was synthesized via in vitro transcription based on a plasmid construct encoding TRIM11 fused with GFP and tagged with 3×Flag (Figure 2A). The construct was validated across multiple cell lines (SH-SY5Y, RAW264.7, Neuro2A, HA1800, 293T, U87-luc, and HeLa) (Figures S4A and S4B). Reverse-transcription PCR (RT-PCR) detected abundant TRIM11 transcripts in transfected cells, and western blotting showed strong TRIM11/Flag signals, together indicating successful transcription of the delivered mRNA and subsequent protein production (Figures 2B–2D). SH-SY5Y cells treated with PLNP-mTRIM11 showed strong upregulation of Flag and TRIM11 proteins, exceeding the levels achieved by non-targeted LNP-mTRIM11 (Figure S4C and S4D). Control nanoparticles carrying mGFP did not produce detectable expression of TRIM11, supporting the sequence specificity of transfection. Comparable results were observed in Neuro2A cells treated with PLNP-mTRIM11 (Figure S4E and S4F).Figure 2PLNP-mTRIM11 enables efficient TRIM11 expression and facilitates the disaggregation of phosphorylated Tau and Tau tangles in vitro(A) Schematic structure of the pcDNA3.1-cFLAG-TRIM11-GFP plasmid used for in vitro transcription.(B and C) Western blot analysis of FLAG and TRIM11 protein expression in SH-SY5Y cells 24 h post-transfection with Lipo_2000_+pcDNA3.1-cFlag-TRIM11-GFP plasmid or PBS (n = 3).(D) Quantitative RT-PCR analysis of TRIM11 mRNA levels in SH-SY5Y cells following transfection with Lipo_2000_ + T7-cFLAG-TRIM11 mRNA or PBS (n = 4).(E) Confocal images showing co-localization of translated TRIM11 protein (green) with phosphorylated Tau (P-Tau, red) in SH-SY5Y cells 24 h after PLNP-mTRIM11 treatment. Scale bars, 20 μm (n = 3).(F) Schematic of Tau pathology induction using OA (100 nM) and PFFs (400 nM) to promote intracellular phosphorylated Tau accumulation and aggregation.(G and H) Western blot quantification of insoluble Tau species (P-ser396 and AT8) in SH-SY5Y cells treated with PLNP-mTRIM11 or indicated controls after OA (100 nM) and PFFs (400 nM) for 18-h induction (n = 3).(I and J) RT-PCR analysis of Tau mRNA levels after treatment with PLNP-mTRIM11, PLNP-mGFP, LNP-mTRIM11, or PBS, following OA/PFF induction (n = 3).(K and M) Representative confocal images of phosphorylated Tau (P-ser396-Tau, red) and AT8-positive Tau aggregates in SH-SY5Y cells treated with PBS, LNP-mTRIM11, or PLNP-mTRIM11. Cells were pretreated with OA (100 nM) and PFFs (400 nM) for 18 h. Scale bars, 50 μm (n = 3).(L and N) Quantification of fluorescence intensity corresponding to P-Tau and AT8 aggregates, respectively (n = 3).All data are presented as mean ± SD. The concentration of mRNA used in all experiments was 2 μg/mL.

To investigate whether TRIM11 overexpression could mediate disaggregation of pathological Tau, we fused GFP to TRIM11 to facilitate intracellular tracking. Phosphorylated Tau accumulation was induced using the phosphatase inhibitor okadaic acid (OA), a well-established method to model Tau pathology in vitro. Confocal microscopy revealed extensive co-localization between GFP-TRIM11 and phosphorylated Tau, with a Pearson’s correlation coefficient of 0.65—significantly higher than that observed with non-targeted LNP delivery (Figures 2E, S4G, and S4H). These results confirm that PLNP-delivered TRIM11 localizes to and interacts with intracellular Tau aggregates in SH-SY5Y and Neuro2A cells (Figure S4I).

We next established a robust model of Tau aggregation by combining OA with preformed Tau fibrils (PFFs), which induced intracellular accumulation of insoluble phosphorylated Tau (P-Tau, detected by anti-P-ser396) and Tau tangles (detected by AT8 staining) in SH-SY5Y cells (Figure 2F). Remarkably, treatment with PLNP-mTRIM11 led to a 65.1% reduction in P-ser396 and a 62.5% reduction in AT8-positive Tau aggregates, demonstrating potent disaggregase activity (Figures 2G, 2H, S4J, and S4K). In contrast, LNP-mTRIM11 induced only modest reductions in P-ser396 (30.1%) and AT8 immunoreactivity (41.1%). In vitro, TRIM11-mediated clearance of insoluble Tau was accompanied by a measurable decline in total Tau levels (Figures 2I and 2J), with similar Tau-degrading activity observed in Neuro2A cells (Figure S4L–S4O). Taken together, these data indicate that efficient, brain-targeted delivery is critical to fully realize the therapeutic potential of mTRIM11.

As expected, in the absence of OA and PFFs induction, only weak background staining for P-Tau and AT8 was detected (Figures 2K and 2L). In OA- and PFFs-treated cells, free mTRIM11 mRNA failed to suppress Tau aggregation and instead showed intense P-Tau and AT8 immunofluorescence (Figure 2M and 2N). In sharp contrast, PLNP-mTRIM11 treatment resulted in substantially diminished P-Tau and AT8 staining, supporting efficient intracellular translation and functional action of TRIM11 in degrading insoluble Tau aggregates.

Together, these findings demonstrate that PLNPs enable functional delivery of TRIM11 mRNA to neuronal cells, resulting in effective clearance of phosphorylated and aggregated Tau. These results establish a molecular foundation for the therapeutic use of mRNA-encoded disaggregases in neurodegenerative tauopathies.

PLNP-mTRIM11 treatment results in sustained cognitive improvement in 3×Tg-AD mice

To evaluate the in vivo therapeutic potential of TRIM11 mRNA, we utilized the triple-transgenic AD mouse model (3×Tg-AD), which harbors APPSwe, PS1M146V, and TauP301L mutations and recapitulates hallmark AD pathology, including Tau tangles.34^,^35 Mice at 7.5 months of age—when substantial Tau aggregation is evident in the hippocampus—were intravenously administered PLNP-mTRIM11 (2 mg mRNA equiv/kg) three times over a defined treatment window (Figure 3A). Following treatment, a battery of behavioral tests was conducted to assess locomotion, anxiety, learning, memory, and executive function.Figure 3PLNP-mTRIM11 treatment leads to significant and sustained improvements in cognitive and behavioral performance in 3×Tg-AD mice(A) Schematic of the treatment and behavioral assessment timeline. Seven-and-a-half-month-old 3×Tg-AD mice received intravenous injections of PLNP-mTRIM11 (2 mg mRNA equiv/kg) on days 0, 7, and 14. Control groups included 3×Tg-AD mice treated with PLNP-mGFP or PBS and WT mice. Behavioral evaluations—OFT, MWM, NOR, and nesting test—were performed on day 16 (first behavioral test, 1#).(B) Total distance traveled in the OFT (n = 10).(C) Number of platform crossings in the MWM probe trial (n = 10).(D) Representative trajectory maps during the MWM probe trial.(E) Preference index (PI) for novel object interaction in the NOR test (n = 7).(F) Representative movement paths in the NOR test.(G and H) (G) Nest-building scores and (H) representative images of nests constructed 48 h after paper towel placement in home cages (n = 10). To assess long-term effects, behavioral assessments were repeated on day 96 (second behavioral test, 2#).(I) Total distance moved in the OFT (n = 6).(J) Platform crossings in the MWM probe trial (n = 6).(K) PI in the NOR test (n = 5).(L) Nest-building scores (n = 6).Data are presented as mean ± SD. mRNA dosage: 2 mg equiv/kg per injection.

In the open field test (OFT), PLNP-mTRIM11-treated 3×Tg-AD mice spent longer in the center zone and traveled significantly farther than PBS- or PLNP-mGFP-treated controls, with locomotor tracks resembling those of wild-type (WT) mice. (Figures 3B and S5A). This normalization of center exploration and ambulatory activity is consistent with attenuated anxiety-like behavior and restored exploratory drive.20^,^36

Spatial learning and memory were evaluated with the Morris water maze (MWM). PBS-treated AD mice displayed longer escape latencies and less-efficient navigation than WT mice, consistent with impaired cognitive performance. In contrast, PLNP-mTRIM11 treatment significantly shortened escape latency, indicating improved spatial learning (Figures 3C and S5B). In the probe trial (platform removed), PLNP-mTRIM11-treated mice made more frequent platform crossings and spent longer time in the target quadrant than control groups, further confirming enhanced memory retention (Figure 3D).

In the novel object recognition (NOR) test, which evaluates recognition memory and novelty discrimination, PLNP-mTRIM11-treated mice demonstrated a higher preference for new objects, with elevated discrimination indices and exploratory time, suggesting restored recognition memory (Figures 3E, 3F, and S5C). Control mice failed to distinguish novel from familiar objects, a hallmark of AD-associated memory decline.

Cognitive function was further evaluated using the nesting test, which assesses executive function, motivation, and planning. PBS-treated mice scored lowest on the nesting scale, often leaving nesting material untouched. In contrast, mice receiving PLNP-mTRIM11 constructed partially formed nests using shredded paper, achieving significantly higher behavioral scores (Figures 3G and 3H). These behaviors suggest improvement in higher-order functional deficits typically observed in AD models.

To determine the durability of these therapeutic effects, a second round of behavioral assessments was conducted 96 days after the final treatment. Notably, PLNP-mTRIM11-treated mice retained enhanced performance across all metrics: increased locomotion and center exploration in the OFT (Figures 3I, and S5D), more frequent platform crossings in the MWM (Figure 3J, S5E, and S5F), sustained novel object preference in the NOR test in the expanded cohort (Figures 3K, S5G, and S5H), and consistently higher nesting scores (Figures 3L and S5I). Representative trajectory maps reaffirmed the enduring improvement in cognitive and behavioral performance.

Collectively, these findings demonstrate that systemic administration of PLNP-mTRIM11 not only restores cognitive and executive functions in 3×Tg-AD mice but also confers durable behavioral benefits lasting at least 3 months post-treatment. These results support the potential of PLNP-mediated mRNA delivery as a non-invasive and sustained therapeutic strategy for neurodegenerative tauopathies.

PLNP-mTRIM11 mitigates Tau pathology and neuroinflammation in 3×Tg-AD mice

To elucidate the mechanisms underlying the cognitive benefits observed following PLNP-mTRIM11 treatment, we conducted comprehensive histological and molecular analyses (Figure 4A). Immunohistochemistry (IHC) revealed that PBS- and PLNP-mGFP-treated 3×Tg-AD mice retained extensive accumulation of phosphorylated Tau (P-ser396) and aggregated Tau tangles (AT8) in the hippocampus, consistent with progressive AD pathology. In contrast, PLNP-mTRIM11 administration led to a marked reduction in both phosphorylated and aggregated Tau species (Figure 4B). Robust TRIM11 expression was detected within hippocampal lesions, confirming successful delivery and translation of exogenous mRNA (Figure 4C). Quantitative image analysis demonstrated 5.9- and 2.2-fold reductions in P-ser396 and AT8 immunoreactivity, respectively, relative to PBS-treated controls (Figures 4D and 4E). Western blot analysis further corroborated the histological findings. Mice treated with PLNP-mTRIM11 showed strong TRIM11 protein expression in the hippocampus, alongside significant downregulation of both P-ser396 and AT8, whereas TRIM11 was virtually undetectable in PLNP-mGFP and PBS groups. Total Tau levels showed a modest decrease in the PLNP-mTRIM11 group, further supporting a reduction in Tau pathology (Figures 4F and S6A–S6D).37 The persistence of Tau aggregates in these control groups reinforces the link between unresolved Tau pathology and the behavioral impairments observed earlier.Figure 4PLNP-mTRIM11 treatment ameliorates Tau pathology, suppresses neuroinflammation, and protects neuronal integrity in 3×Tg-AD mice(A) Schematic overview of the experimental design for assessing insoluble Tau pathology in the hippocampus following treatment; mRNA dosage: 2 mg equiv/kg per injection.(B) IHC staining of phosphorylated Tau (P-ser396), Tau tangles (AT8), and TRIM11 in hippocampal sections collected 28 days after the final injection of PLNP-mTRIM11 or controls. Scale bars, 50 μm.(C–E) Quantification of TRIM11, P-ser396, and AT8 staining intensities in the hippocampus (n = 3).(F) Western blot analysis of Tau, TRIM11, phosphorylated Tau (P-ser396), and Tau tangle (AT8) expression in hippocampal lysates (n = 3).(G) Schematic illustration of TRIM11-mediated reduction in neuroinflammation via cytokine modulation.(H–J) ELISA quantification of (H) anti-inflammatory IL-10, (I) pro-inflammatory TNF-α, and (J) IL-6 levels in hippocampal tissue following treatment (n = 3).(K–M) (K) Representative IHC images and quantification of (L) astrocytic (GFAP) and (M) microglial (Iba-1) immunoreactivity in the hippocampus. Red arrows indicated activated microglia (Iba-1) and astrocytes (GFAP). Scale bars, 500 μm (n = 3).(N) Schematic representation of TRIM11’s neuroprotective function through suppression of NFL secretion.(O) Confocal imaging of NFL in hippocampal neurons reveals preserved axonal structure following PLNP-mTRIM11 treatment. Scale bars, 50 μm (n = 3).Data are presented as mean ± SD. mRNA dosage: 2 mg equiv./kg per injection.

In addition to its Tau-disaggregating effects, PLNP-mTRIM11 significantly attenuated neuroinflammation. IHC staining showed that GFAP expression—an indicator of astrocyte activation—was markedly elevated in PBS-treated mice but significantly reduced in those receiving PLNP-mTRIM11 (Figure 4G). To further evaluate the neuroimmune response, cytokine levels were measured via enzyme-linked immunosorbent assay (ELISA). PLNP-mTRIM11 treatment led to an upregulation of the anti-inflammatory cytokine IL-10 (Figure 4H) and concurrent suppression of the key pro-inflammatory cytokines TNF-α, IL-6, and IL-1β (Figures 4I, 4J, and S6F). These findings suggested that PLNP-mediated TRIM11 expression not only targets pathological Tau but also modulates the neuroinflammatory milieu, likely by alleviating Tau-induced immune activation. Consistent with the above, PLNP-mTRIM11 was associated with lower indices of glial activation, reflected by reduced Iba-1-positive microglia and fewer GFAP-positive astrocytes compared with controls (Figure 4K). Correspondingly, both microglia and astrocytes exhibited a shift toward less-reactive morphologies after treatment with TRIM11 mRNA liquid nanoparticles (Figure S7). Quantification of GFAP and Iba1 immunoreactivity confirmed significant suppression of both astrocyte and microglial activation compared to control groups (Figures 4L and 4M). These changes are indicative of a broad neuroprotective effect that extends beyond Tau clearance.

Neurofilament light chain (NFL), a cytoskeletal component of axons, is commonly elevated in neurodegenerative states due to axonal injury. In the present study, NFL expression was markedly decreased in PLNP-mTRIM11-treated brains compared to PBS or PLNP-mGFP groups, with immunofluorescence levels comparable to WT mice (Figures 4N, 4O, and S6E), suggesting preserved neuronal integrity and reduced axonal degeneration. Importantly, PLNP-mTRIM11-mediated reductions in phosphorylated Tau and glial activation were not limited to the hippocampus. Similar effects were observed in the cerebral cortex, indicating a broad therapeutic impact across multiple brain regions affected in AD (Figure S8A). H&E-stained sections of major organs did not reveal apparent abnormalities in PLNP-mTRIM11-treated mice, indicating no obvious systemic toxicity under the tested conditions (Figure S8B).

Together, these results demonstrate that systemically administered PLNP-mTRIM11 effectively reduces insoluble Tau aggregates and suppresses neuroinflammatory responses in the AD brain. The targeted delivery of TRIM11 enables dual therapeutic action: direct clearance of pathogenic Tau via proteasomal degradation pathways and attenuation of secondary inflammation through inhibition of glial activation. Together, these multi-pronged effects suggest that PLNP-mTRIM11 may serve as a non-invasive therapeutic platform capable of simultaneously mitigating core pathology and associated secondary disease processes in AD and other tauopathies.

PLNP-mTRIM11 delays cognitive decline and Tau pathology progression in early-stage AD

Beyond its capacity to disaggregate existing Tau tangles, TRIM11 has been reported to interfere with the seeding and propagation of pathological Tau species. To explore whether PLNP-mediated delivery of TRIM11 mRNA could prevent or delay the onset of neurodegenerative features, we administered PLNP-mTRIM11 to 5.5-month-old 3×Tg-AD mice, a stage at which initial Tau pathology is emerging.38^,^39 Mice received three intravenous doses of PLNP-mTRIM11, and behavioral performance was assessed using the same battery of tests employed in older animals (Figure 5A).Figure 5PLNP-mTRIM11 treatment prevents early Tau pathology and preserves cognitive and behavioral function in young 3×Tg-AD mice(A) Experimental timeline: 5.5-month-old 3×Tg-AD mice received three intravenous injections of PLNP-mTRIM11 (2 mg mRNA equiv/kg) on days 0, 7, and 14. Behavioral and cognitive assessments were conducted starting on day 47.(B) Total distance traveled in the OFT (n = 8).(C and D) (C) Discrimination index and (D) PI in the NOR test (n = 8).(E and F) (E) Total platform crossings (n = 8) and (F) latency (n = 6) to platform in the MWM test.(G) Nesting behavior scores (n = 8), indicating motivation and executive function.(H) Quantitative expression of Tau, TRIM11, phosphorylated Tau (P-ser396), and Tau tangles (AT8) in hippocampal tissue following treatment (n = 3).(I) Representative immunohistochemical images of hippocampal sections stained for P-Ser396, AT8, GFAP (astrocytes), Iba-1 (microglia; scale bars, 100 μm), and NFL (axons; scale bars, 200 μm). Red arrows indicate positive immunostaining for AT8 (P-Ser202/Thr205), P-Ser396, Iba-1, GFAP, and NFL.(J) Quantification of Iba-1-positive microglia in the hippocampus. ELISA quantification of pro-inflammatory cytokines.(K–M) (K) TNF-α, (L) IL-1β, and (M) IL-6 in brain homogenates (n = 3).All data are expressed as mean ± SD.

In the OFT, PLNP-mTRIM11-treated 3×Tg-AD mice exhibited locomotor activity comparable to WT mice, traveling an average distance of 1,452 cm, which was significantly greater than that of the non-targeted PLNP-mGFP group (780 cm; Figure 5B). This normalization of ambulatory distance and exploratory behavior suggests both improved motor function and attenuation of anxiety-like phenotypes. By contrast, PBS-treated mice displayed pronounced hypoactivity and reduced exploration of the central zone, consistent with heightened anxiety-like behavior (Figure S9A). In the NOR test, PLNP-mTRIM11-treated mice showed a distinct preference for the novel object, as reflected by both increased exploration time and a higher discrimination index, while PBS and PLNP-mGFP groups exhibited suppressed novelty-seeking behavior (Figures 5C, 5D, and S9B).

During MWM training, escape latencies were similar across all groups, indicating comparable learning acquisition (Figure 5E). Notably, in the probe trial after platform removal, PLNP-mTRIM11 increased target-quadrant occupancy compared with controls, consistent with improved spatial memory recall (Figures 5F and S9C). Nesting performance was also markedly enhanced in the PLNP-mTRIM11 group, supporting improvements in executive function and motivation (Figures 5G and S9D).

Biochemical analyses demonstrated strong hippocampal induction of TRIM11 in PLNP-mTRIM11-treated mice, accompanied by pronounced reductions in P-Ser396- and AT8-reactive Tau aggregates. Consistently, total Tau levels were also modestly reduced in the PLNP-mTRIM11 group (Figure 5H and S9E–S9H),suggesting that TRIM11 overexpression can help lower the overall Tau burden.37 IHC confirmed a reduction in insoluble Tau accumulation in hippocampal sections. In PBS- and PLNP-mGFP-treated 3×Tg-AD mice, hippocampal NFL immunostaining was markedly increased and appeared coarse and discontinuous (red arrowheads), consistent with heightened axonal stress. By contrast, PLNP-mTRIM11-treated 3×Tg-AD mice and WT controls exhibited weaker, more continuous NFL labeling, indicative of preserved neuroaxonal integrity and the absence of treatment-associated axonal injury (Figure 5I).40 Furthermore, Iba-1 and GFAP immunoreactivity were markedly diminished, consistent with reduced activation of microglia and astrocytes, respectively (Figure 5J and S9I–S9L).

Consistent with reduced glial activation and inflammation, ELISA assays showed decrease in pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6 in PLNP-mTRIM11-treated mice compared to PLNP-mGFP controls (Figures 5K–5M). Importantly, comprehensive safety evaluations including H&E staining of major organs, blood biochemistry profiles (day 1, 7, and 30), complete blood counts, and systemic cytokine assessments revealed no adverse effects or immune-related toxicity associated with PLNP-mTRIM11 (Figure S10).

Together, these results demonstrate that early systemic administration of PLNP-mTRIM11 effectively prevents the progression of Tau pathology and delays the emergence of cognitive and behavioral impairments in 3×Tg-AD mice. By targeting Tau aggregation at early stages and modulating neuroinflammation, TRIM11 mRNA delivery via PLNP offers a disease-modifying strategy for preclinical intervention in AD.

Discussion

Although TRIM11 has emerged as a compelling therapeutic candidate for AD due to its ability to disaggregate pathological Tau, modulate neuroinflammation, and preserve neuronal integrity, its clinical deployment remains hindered by the absence of safe, effective, and brain-penetrant delivery systems. Adeno-associated virus (AAV) vectors, while widely used in preclinical models, present several limitations: their limited packaging capacity constrains the delivery of complex or regulatory elements, their prolonged and often irreversible expression may lead to loss of temporal control over dosage, and their potential immunogenicity raises safety concerns in humans, particularly when repeated or systemic dosing is required.41^,^42 These characteristics render AAVs suboptimal for delivering plasmids like TRIM11 that require finely tuned transient expression in sensitive neural environments. In contrast, mRNA-based therapeutics offer distinct advantages, including flexible and transient protein expression, rapid design and production cycles, and a reduced risk of insertional mutagenesis. However, translating these benefits to the CNS has remained a formidable challenge due to poor BBB permeability, susceptibility to nuclease degradation, and inefficient neuronal uptake.43 Moreover, currently available LNP platforms lack the requisite targeting specificity and often accumulate in off-target organs such as the liver.

To address these unmet needs, we developed a zwitterionic LNP (PLNP) with poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) for the targeted delivery of TRIM11 mRNA to the brain. The PMPC-modified ligand-free LNP interact with nAChRs and chTs, which are abundantly expressed on neurons and brain endothelial cells. This targeting strategy significantly enhances BBB penetration and neuronal internalization. The PLNP formulation exhibits favorable colloidal stability, narrow size distribution, and sustained structural integrity, retaining >80% mRNA encapsulation after 21 days of storage at 4°C, outperforming several commercial LNP platforms. The internal volume and surface properties of PLNPs contribute to superior mRNA protection in circulation and rapid endosomal escape post-cellular uptake.

Using the 3×Tg-AD mouse model, we demonstrated that intravenous administration of PLNP-encapsulated TRIM11 mRNA leads to robust brain-wide transgene expression, preferential accumulation in neurons, and effective degradation of phosphorylated Tau aggregates. The clearance of Tau pathology is accompanied by the attenuation of glial activation, reduction of pro-inflammatory cytokines, and marked improvements in behavioral outcomes across multiple cognitive domains. Importantly, our results extend beyond therapeutic benefit in established AD pathology: when administered at earlier stages of disease, PLNP-mTRIM11 significantly delayed cognitive decline, prevented Tau accumulation, and suppressed the onset of neuroinflammatory changes. These findings suggest that TRIM11 mRNA delivery may serve both therapeutic and prophylactic roles in tauopathy progression.

In summary, we report the development of a biodegradable, brain-targeted mRNA delivery system based on PLNPs that enables non-invasive, systemic administration of TRIM11 mRNA. This strategy achieves effective BBB penetration, neuron-specific transfection, and robust degradation of pathological Tau, with strong safety and efficacy profiles demonstrated in both therapeutic and preventive AD models. The PLNP-mRNA platform represents a versatile and clinically promising vehicle for CNS gene therapy, with broad applicability to a range of neurodegenerative disorders beyond Tauopathies.

Limitations of the study

This study has several limitations that should be considered when extrapolating toward clinical translation. First, successful deployment of this platform will likely require (1) the development of conformation-selective Tau probes that preferentially direct TRIM11 toward pathological Tau assemblies while sparing physiological Tau isoforms, thereby reducing the risk of off-target or indiscriminate protein degradation, and (2) systematic characterization and optimization of PLNP-mRNA stability under ambient and stress conditions to support long-term storage and large-scale distribution in real-world settings. Second, our conclusions are intentionally focused on BBB transcytosis and Tau target engagement/modulation in a mid-stage 3×Tg-AD model, rather than on global disease modification or efficacy in late-stage neurodegeneration. Because the 3×Tg-AD model relies on overexpression of APPswe, PS1M146V, and TauP301L and may not fully capture the heterogeneity of sporadic late-onset AD, the present results are most appropriately interpreted as mechanistic proof of feasibility generated under pre-specified, blinded workflows and viewed in the context of known constraints of this model, including transgene overexpression, mixed genetic background, and sex/age heterogeneity. Going forward, we will extend these studies to older 3×Tg-AD cohorts and additional models to assess the durability and generalizability of the observed effects. We further plan to validate the platform in knock-in and Tau-dominant models, include female and aged cohorts, and align outcome measures with clinically relevant biomarkers (e.g., plasma/CSF p-Tau217/181, NFL, Tau-PET) to strengthen its translational robustness.44^,^45

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. B. Shi (oceanorbing.shi@uts.edu.au).

Materials availability

All materials and reagents used in this study are available from the lead contact upon request.

Data and code availability

• •This study did not generate code.
• •Study data are available within the article and its supplementary materials. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

This work was supported by 10.13039/501100001809National Natural Science Foundation of China (NSFC 32271463), the 10.13039/501100006407Natural Science Foundation of Henan Province (242300421089 and 232301420064), and the 10.13039/501100004773Henan University Double First-Class Foundation. Cartoons in figures were created with BioRender.com.

Author contributions

Y.Z. and B.S. conceived and designed the project. Y.S. was responsible for maintaining the 3×Tg-AD mice. Y.S. and Y.Z. performed the experiments and analyzed the data. Y.S. and Y.Z. prepared the first draft of the manuscript. B.S. and M.Z. reviewed the manuscript and contributed to the final version.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCESOURCEIDENTIFIERAntibodiesPhospho-Tau (P-Ser396)HUABIOCat#ET1611-68;RRID:AB_3070047TauHUABIOCat#ET1612-44;RRID:AB_3070118DYKDDDDK Tag FlagCell Signaling TechnologyCat#8146;RRID:AB_10950495Phospho-Tau (Ser202, Thr205) Monoclonal Antibody (AT8)ThermofisherCat#MN1020;RRID:AB_223647NF-L Monoclonal AntibodyHUABIOCat#HA721538;RRID:AB_3072654GFAP Polyclonal antibodyProteintechCat#16825-1-AP;RRID:AB_2109646Iba-1 Polyclonal antibodyProteintechCat#10904-1-AP;RRID:AB_2224377β3-Tubulin Monoclonal AntibodyHUABIOCat#ET1602-4;RRID:AB_2938592GAPDH Monoclonal AntibodyHUABIOCat#HA721136;RRID:AB_3072260TRIM11ProteintechCat#10851-1-AP;RRID:AB_2209210Bacterial and virus strainspCDNA3.0AddgeneAddgene plasmid#136622Chemicals, peptides and recombinant proteinsOkadaic acidYuanyeCat#S30686;CAS:78111-17-8Tau-441/2N4R Pre-formed Fibr-ils Protein (PFFs)AcrobiosystemsCat#TAU-H5115Distearoyl Phosphatidylcholine (DSPC)MacklinCat#D742457;CAS:816-94-4N,N′-dicyclohexylcarbodiimide (DCC)MacklinCat#N806920;CAS:538-75-0CholesterolMacklinCat#C769358;CAS:57-88-51,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG_2000_)MacklinCat#D890330;CAS:160743-62-4Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102)MacklinCat#H960267;CAS:2089251-47-62,2′-Azobis(2-methylpropionitrile) (AIBN)MacklinCat#A800353;CAS:78-67-1Phosphoric Acid 2-(Methacryloyloxy)ethyl 2-(Trimethylammonio)ethyl Ester (MPC)AladdinCat#M158414;CAS:67881-98-54-Cyano-4-(phenylcarbonothioylthio)pentanoic Acid (CPADN)MacklinCat#C833222;CAS:201611-92-9N-hydroxysuccinimide (NHS)MacklinCat#130672;CAS:6066-82-61,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)MacklinCat#D130471;CAS:1069-79-0Paraformaldehyde (PFA)ServicebioCat#G1101RIPA bufferSolarbioCat#R0020Lipofectamine 2000InvitrogeneCat#11668-019Trypsin-EDTA (0.25%)GibcoCat#A11105-014′,6-diamidino-2-phenylindole(DAPI, Thermo-FisherScientific)SolarbioCat#C0065Cy5-conjugated goat anti-rabbit IgGServicebioCat#GB27303Cy5-conjugated goat anti-mouse IgGServicebioCat#GB27301FITC-conjugated goat anti-mouse IgGServicebioCat#GB22301FITC-conjugated goat anti-rabbit IgGServicebioCat#GB22303PMSF (100 mM)ServicebioCat#G2008Triton X-100BioBasicCat#BT0198Skim milkServicebioCat#GC310001FBSGibcoCat#A5256701High Glucose DMEMPricellaCat#PM150210RIPA bufferSolarbioCat#R0020Critical commercial assaysTransStart FastPfu DNA PolymeraseTRANCat#AP221-12Mouse tumor necrosis factor-α (TNF-α) ELISA kitGelatinsCat#JLC-R13963Mouse interleukin-6 (IL-6) ELISA kitGelatinsCat#JLC-R13878Mouse interleukin-1β (IL-1β) ELISA kitGelatinsCat#JLC-R13796Mouse interleukin-10 (IL-10) ELISA kitGelatinsCat#JLC-R13907HisScribe T7 mRNA KitNEWENGLAND BiolabsCat#E2080Total RNA Extraction KitSolarbioCat#R1200Experimental models: Cell linesMouse: Neuro2AATCCCat#CCL-131™Human: U87-MGThis paperN/AMouse: BV2This paperN/AHuman: SH-SY5YATCCCat#CRL-2266™Mouse: bEnd.3ATCCCat#CRL-2299™Mouse: RAW264.7ATCCCat#TIB-7™Human: HEK293TProcellsystemCat#CL-0005Human: Human AstrocytesProcellsystemCat#CP-H122Human: HelaATCCCat#CRM-CCL-2™Experimental models: Organisms/strains3×Tg-AD miceShulaibao (Wuhan) Biotechnology Co., Ltd.N/AC57BL/8M miceSPF (Beijing)Biotechnology Co.Ltd.N/AOligonucleotidesqRT-PCR primersSangonsee Table S1PCR primersSangonsee Table S1mRNA sequencesNCBIsee Table S2Software and algorithmsImageJNIHhttps://imagej.nih.gov/ij/GraphPad Prism 8.0Graphpadhttps://www.graphpad.com/FlowJo software v10.6.2FlowJohttps://www.flowjo.comLiving Image softwareCaliper Life Scienceswww.caliperls.comZEN Microscopy SoftwareZeisshttps://www.zeiss.com.cn/SnapGeneSnapGenehttps://www:snapgene.comCytExpert software v1.0Beckmanhttps://www.beckman.com/flow cytometry/research-flow cytometers/cytoflex/softwareOtherEcoR1NEWENGLAND BiolabsCat#R0101SXho1NEWENGLAND BiolabsCat#R0146SXba1NEWENGLAND BiolabsCat#R0145SPVDF membranesMilliporeCat#IPVH0001096-well platesFalcon®Cat#330096-well black platesLABSELECTCat#31112

Experimental model and study participant details

Cell lines and cell culture

U87-MG and BV2 cells were obtained as described in our previous report.46 HEK293T cells and human astrocytes were purchased from Procellsystem. Human SH-SY5Y, bEnd.3, Neuro2A, RAW264.7, and HeLa cell lines were purchased from the American Type Culture Collection (ATCC). All cells were maintained under standard culture conditions in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin, and were incubated at 37°C in a humidified atmosphere containing 5% CO_2_. Cultures were routinely monitored to ensure logarithmic growth and were regularly tested to confirm the absence of mycoplasma contamination.

In vivo animal model

All animal procedures were approved by the Medical and Scientific Research Ethics Committee of Henan University School of Medicine, P.R. China (HUSOM-2018-354). 3×Tg-AD mice (Shulaibao (Wuhan) Biotechnology Co., Ltd.) were maintained as a single-source, specific-pathogen-free colony, and all experimental animals (3×Tg-AD mice and C57BL/8M) used in this study were male. Animals were group-housed in individually ventilated cages under standardized conditions (22 ± 2°C; 45–65% relative humidity; 12:12 h light–dark cycle; matched chow and bedding lot numbers), and all testing was performed at a fixed zeitgeber time. Animals were age-matched (±0.2 months) and weight-matched, and were assigned to experimental groups by block randomization stratified by litter and cage with allocation concealment. In linear mixed-effects analyses, cohort/batch (and cage, when applicable) was specified a priori as a random effect to account for inter-batch and housing-unit variability.

Method details

Synthesis of DSPE-PMPC

Firstly, we synthesized the PMPC-CPADN. 2,2′-Azobis(2-methylpropionitrile) (AIBN, 1.69 mg, 0.01 mmol) was used as an initiator, then 4-Cyano-4-(phenylcarbonothioylthio)pentanoic Acid (CPADN, 10 mg, 0.036 mmol) and Phosphoric Acid 2-(Methacryloyloxy)ethyl 2-(Trimethylammonio)ethyl Ester (MPC, 225 mg, 0.762 mmol) were added to anhydrous methanol. Vacuum freeze-thaw degassing was required during reaction with a temperature of 70°C and 600 rpm for 24 h. The RAFT polymerization method was used to synthesize PMPC-CPADN with a target molecular weight of 5 kDa. The reaction mixture was then transferred to a dialysis bag with a molecular weight of 3.5 kDa and dialyzed with anhydrous ethanol for 24 h, followed by dialysis with ultrapure water for 24 h.

DSPE-PMPC was synthesized subsequently. N-hydroxysuccinimide (NHS, 0.92 mg, 0.008 mmol) and N,N′-dicyclohexylcarbodiimide (DCC, 1.65 mg, 0.008 mmol) were used to activate the carboxyl functional groups of PMPC-CPADN (20 mg, 0.004 mmol) in anhydrous methanol, then reacted at room temperature for 24 h. After 24 h, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-NH_2_) (3.6 mg, 0.0048 mmol) was dissolved in 1 mL of anhydrous DMF and added to the previous reaction mixture, simultaneously 200 μL of triethylamine was added and reacted at 40°C for 24 h. After the reaction was completed, the reaction mixture was filtered through a 0.22 μm to remove the precipitate, then dialyzed with 75% ethanol for 24 h and ultrapure water for 24 h. Finally, the sample was lyophilized.

Preparation of PLNP-mRNA

Firstly, 8 mM solution of lipid in ethanol was prepared with a molar ratio of (SM-102) 50%: (DSPC) 5%: (Cholesterol) 38.5%: (DSPE-PMPC) 5%: (DMG-PEG_2000_) 1.5%. Subsequently, mRNA was dissolved in a 50 mM citrate buffer (pH = 4) with a concentration of 0.054 μg/μL. mRNA-citrate buffer and the lipid-ethanol solution were mixed at a volumetric ratio of 1:3. Finally, PLNP-mRNA was ultrafiltrated with a 100 kDa cutoff using the citrate buffer, PBS and sucrose protection solution.

Characterization of PLNP-mRNA

The size and zeta potential of nanoparticles were determined at 25°C using dynamic light scattering (DLS, Nano-Zen 3600, Malvern Instruments, UK). The double-layered vesicular structure of liquid nanoparticles was observed by transmission electron microscopy (TEM). Confocal laser scanning micros-copy images of cells and tissues were taken using a Zeiss Confocal Microscope system (Zeiss 880). The fluorescence was quantitatively measured by flow cytometry (BD FACS Calibur, San Jose).

Encapsulation efficiency

mRNA (0.054 μg/μL) citrate solution was mixed with liquid solution at mass ratio of 1:40.7, then ultrafiltered sequentially with citrate buffer (pH = 4), PBS buffer (pH = 7.4), and 2% sucrose protective solution to ultimately obtain PLNP-mRNA. Subsequently, the RNA concentration of the lipid nanoparticles was compared using Nanodrop. The PLNP-mRNA and LNP-mRNA were electrophoresed through a 2% agarose gel containing Gel Red at 110 V in TAE solution (40 mM tris-HCl, 1% acetic acid (v/v), and 1 mM EDTA). The gel electrophoresis images were taken by Molecular Imager FX (Bio-Rad, Hercules, CA).

Cellular uptake

N2A, SH-SY5Y and BV2 cells were seeded in a 12-well plate (1 × 10^5^ cells per well) and incubated with PBS, free mRNA, 5%PLNP-mRNA or LNP-mRNA in 1 mL media (Cy5 mRNA: 2 μg/mL) at 37°C for 4 h. The cells were digested by 0.25% (w/v) trypsin and 0.03% (w/v) EDTA. The suspensions were centrifuged at 1000 rpm for 3 min, washed twice with PBS, and then resuspended in 200 μL of PBS. Fluorescence histograms were immediately recorded with a BD FACS Calibur flow cytometer (Becton Dickinson, USA) and analyzed using CellQuest software based on 10,000 gated events. The gate was set for the detection of Cy5 fluorescence. The competitive inhibition and Hemicholinium chloride (HC-3) experiments follow a protocol similar to the one described above. Cells were pre-incubated with DSPE-PMPC (12 μM) or HC-3 (100 μM) for 24 h.

The cellular uptake was also detected by confocal imaging. Similarly, the culture media was removed after 4 h incubation with different samples, microscope plates were then washed with PBS for three times, fixed with 4% paraformaldehyde solution for 15 min. The cell nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI) for 10 min. Fluorescence images were obtained using a confocal microscope (Zeiss 880).

Transfection assay

N2A and SH-SY5Y cells were incubated in 12-well plates at a density of 1×10^5^ cells and allowed to grow until they were approximately 80% confluent. GFP mRNA (mGFP) loaded liquid nanoparticles 5% PLNP-mRNA, LNP-mRNA or free mRNA (2 μg/mL) were added to cells and incubated for 24 h, then cells were collected and suspended in 200 μL PBS. Fluorescence histograms were recorded immediately as above. The competitive inhibition and Hemicholinium chloride (HC-3) inhibition assays were performed following a protocol similar to the one described above. Cells were pre-incubated with DSPE-PMPC (12 μM) or HC-3 (100 μM) for 24 h. Transfection was also studied by confocal microscopy. Similarly, after 24 h incubation with different samples, the cells on microscope plates were washed, fixed with 4% paraformaldehyde, stained with DAPI, respectively. Fluorescence images were obtained using a confocal microscope.

In vitro BBB penetration

The in vitro BBB model was constructed with endothelial bEnd.3 cells using a transwell cell culture system. bEnd.3 cells were seeded at a density of 1× 10^5^ cells per insert in 300 μL of DMEM supplemented with 10% FBS, and and 800 μL of medium was added to the basolateral chamber. bEnd.3 cells were cultured for 5–6 days until a confluent monolayer formed. The transendothelial electrical resistance (TEER) instrument (World Precision Instruments Inc. Sarasota, FL, USA) was used to monitor the intactness of the cell monolayer. The following experiments were carried out only when the TEER value of the endothelial bEnd.3 cell monolayer was above 200 Ω·cm^2^. Then used HC-3 (100 μM) for 24 h of pre-incubation to inhibit acetylcholine transporter activity. Cy5 mRNA were used for BBB penetration evaluation. free mRNA, LNP-mRNA, PLNP-mRNA and PLNP-mRNA+HC-3 were added to the upper chamber. Then, FBS-free media was added to the lower chamber. After 1, 2, 4, 6, 8, 10, 12 and 24 h of incubation, 200 μL of solution is sampled from the lower chamber and replaced with 200 μL of fresh solution, the supernatant in the upper chamber and the media in the lower chamber were extracted with dimethyl sulfoxide. The amounts of Cy5 mRNA in the supernatant and filtered compartments were determined using a standard microplate assay. Using a microplate reader (Bio Tek, USA) with excitation at 640 nm and emission at 670 nm. The transport ratio was determined by collecting the fluorescence intensity of sample in different intervals, acquiring the cumulative concentration at different time points and finally obtaining the percentage of transport ratio relative to initial concentration.

In vivo and ex vivo imaging of PLNP-mRNA

To evaluate the in vivo brain targeting ability, C57BL/6 mice were intravenously injected with PLNP-mRNA, LNP-mRNA or free mRNA (Cy5-mRNA: 1 mg/kg) and monitored at different time points using a Lumina IVIS III Imaging System (excitation = 620 nm; emission = 670 nm).

For the biodistribution study, normal C57BL/6 mice were randomly divided into three groups (n = 3 per group) and intravenously injected with Cy5-labeled mRNA formulations—free mRNA, LNP-mRNA, or PLNP-mRNA—at a dose of 1 mg Cy5-mRNA equivalent per kg. At 0.5 h post-injection, the mice were euthanized, and major organs (heart, liver, spleen, lung, kidney, and brain) were harvested, thoroughly washed, and weighed (Step 1). To extract Cy5-mRNA, 300 μL of 1% Triton X-100 and dimethyl sulfoxide were added to each sample, followed thorough homogenization using a tissue grinder at 70 Hz for 15 min. The samples were then centrifuged at 13,000 rpm for 30 min, and the supernatants were transferred to a black 96-well plate (LABSELECT). In parallel, a Cy5-mRNA standard curve was prepared using serial dilutions to ensure that all measured fluorescence intensities fell within the linear range of the standard curve. Fluorescence was measured using a microplate reader (BioTek, USA) with an excitation wavelength of 640 nm and an emission wavelength of 670 nm (Step 2). Additionally, during sampling, PBS-treated control organs and brain tissues were collected as baseline samples. The final fluorescence values for each organ were corrected by subtracting the corresponding baseline values to minimize potential interference from residual blood (Step 3). The corrected fluorescence intensities were then converted to Cy5-mRNA concentrations using the standard curve and multiplied by the detection volume to calculate the total amount of Cy5-mRNA in each organ (Step 4). This amount was subsequently divided by the initial injected mRNA dose to obtain the fraction of injected dose (ID) in each organ (Step 5). Finally, the fraction of ID was normalized to the respective organ weight and multiplied by 100 to yield the percentage of injected dose per gram of tissue (%ID/g) (Step 6). The calculation formula:

$$[eqn]$$

ODdetected: Fluorescence value of the experimental group.

ODPBS: Fluorescence value of PBS.

Vdetected: Detection volume of fluorescence value

k: Slope of the standard curve

b: Intercept of the standard curve

m1: Total mass of Cy5 mRNA injected into each mouse

m2: Mass of each organ.

In vivo transfection and targeting of the hippocampal region of PLNP-mRNA

C57BL/6 mice received a single intravenous injection of PLNP–mRNA, LNP–mRNA, or free Cy5-labeled mRNA (1 mg/kg). Thirty minutes after dosing, the mice were euthanized, brains were rapidly removed, and post-fixed overnight in 4% paraformaldehyde at 4°C. Tissues were then cryoprotected by sequential immersion in 20% and 30% sucrose, embedded, and cut into fresh frozen coronal sections (∼4 μm thick) for immunofluorescence analysis. Nuclei were counterstained with DAPI (5 μg/mL, 10 min), and images were acquired using a Zeiss LSM 880 confocal laser scanning microscope.

Under the same experimental conditions, 9-month-old 3×Tg-AD mice were treated by tail-vein injection as described above. Following brain fixation, cryoprotection, and preparation of coronal frozen sections, immunofluorescence staining was performed by incubation with AT8 primary antibody, followed by an appropriate FITC-conjugated secondary antibody, with DAPI used for nuclear counterstaining. Confocal images were collected using identical acquisition settings.

Targeted delivery of PLNP-mRNA in neurons and microglial cells

Similar as above, the obtained frozen sections were incubated with specific antibodies for neurons (β3-Tubulin), microglia (Iba-1), and astrocytes (GFAP) at 4°C overnight. After three washes with PBS, the slides were incubated with fluorescent secondary antibodies (FITC) at room temperature for 1 h. Then nucleus was stained with DAPI (5 μg/mL) for 10 min, and slides observed using a CLSM imaging system (Zeiss 880).

Synthesis of TRIM11 mRNA

To obtain the mouse TRIM11 sequence, we screened different cells including SH-SY5Y, RAW, HA1800, 293FT, U87MG and HeLa cells to identify and acquire the target sequence from SH-SY5Y cells. Then T4 ligase was used to insert the target sequence into the PCDNA3.1vector, finally yielding T7-mTRIM11-mRNA-cFlag (mTRIM11) by in vitro transcription (IVT). To further verify the expression of mTRIM11, SH-SY5Y cells (1×10^5^/well) were incubated in 12-well plates, then Lipo(mTRIM11) (2 μg/mL) was added and incubated for 12 h. Afterward, the cells were collected by centrifugation at 1000 rpm for 3 min, 1% PMSF (Phenylmethanesulfonyl fluoride; protease inhibitor), high-efficiency RIPA (Radioimmunoprecipitation assay) buffer were used to lyse the mixture on ice for 30 min. The supernatant was centrifuged for 20 min (4°C, 13000 rpm) to obtain the protein lysate pellet and the concentration was determined by a BSA protein standard curve. Standard Western blot electrophoresis was then performed, with proteins transferred onto polyvinylidene difluoride membranes (Millipore 0.22 μm) and immunoblotted. Primary antibody TRIM11, Flag-labled, GAPDH, and mouse or rabbit secondary antibody were used. Data quantification was performed by ImageJ software.

The RNA sequence of mTRIM11 was further verified, similarly as above, total RNA was extracted by the RNAprep Pure Cell/Tissue Kit (CWBIO, China) and reverse transcription to CDNA by the PrimeScript RT kit (Takara, Kyoto, Japan). The amounts of TRIM11 mRNA were quantified by real-time PCR and normalized to the amount of GAPDH mRNA.

TRIM11 expression assay by quantitative real-time PCR

N2A and SH-SY5Y cells were seeded in a 12-well plate (1 × 10^5^ cells per well) in media for 24 h. The media was removed and replenished with fresh media (1 mL) containing PLNP-mRNA, LNP-mRNA and free mRNA (2 μg/mL). After 24 h, the cells were washed with PBS and total RNA was extracted using RNAprep Pure Cell/Tissue Kit (CWBIO China). Reverse transcription and qPCR were carried out by following reverse transcription protocol (vazyme) and SYBR Green Gene Expression Assays Protocol (Takara) using the Roche Light Cycler 480 RT-PCR System. mGAPDH was used as an endogenous housekeeping gene to normalize the Bace1 mRNA. The mRNA expression level was calculated based on comparative Ct method.

TRIM11 and Tau co-localization assay

To assess co-localization of endogenous TRIM11-GFP and Tau, untreated SH-SY5Y and N2A cells were fixed for 10 min in 4% paraformaldehyde at room temperature, permeabilized with 0.5% Triton X-100 for 5 min, and blocked for 30 min with 5% BSA. Cells were incubated with rabbit anti-Tau antibody (for all cells), and at 4°C overnight. After being washed three times with PBS, cells were incubated with Cy5 fluorescence anti-rabbit secondary antibodies at room temperature for 1 h. After being washed three times with PBS, nuclei were stained with DAPI (5 μg/mL) for 10 min, coverslips were then mounted on the glass slides and fluorescence images were captured using a confocal microscope.

PFFs and OA induced aggregation of endogenous Tau

Briefly, SH-SY5Y cells were transfected with PLNP-mTRIM11 or LNP-mTRIM11 (2 μg/mL), 24 h later Tau PFFs was added (which had previously been incubated with Lipo_2000_ in Opti-MEM for 20 min and then added to cells at the final concentration of 400 nM) and incubated for 18 h. Cells were then washed three times with PBS, fixed for 10 min in 4% paraformaldehyde at room temperature, permeabilized with 0.5% Triton X-100 for 5 min and blocked for 30min with 5% BSA. Cells were incubated with mouse-AT8 antibody at 4°C overnight. After three PBS wash cycles, cells were incubated with Cy5 fluorescence anti-mouse secondary antibodies at room temperature for 1 h. After three PBS wash cycles, nuclei were stained with DAPI (5 μg/mL) for 10 min, washed three times with PBS, then fluorescence images were captured using a confocal microscopy.

Similarly, SH-SY5Y cells were transfected with PLNP-mTRIM11 or LNP-mTRIM11 (2 μg/mL) for 24 h. Okadaic acid (OA) was then added to cells at the final concentration of 100 nM and incubated for 18 h. Cells were then washed three times with PBS, fixed for 10 min in 4% paraformaldehyde at room temperature, permeabilized with 0.5% Triton X-100 for 5 min and blocked for 30 min with 5% BSA. Cells were incubated with rabbit-P-ser396 antibody at 4°C overnight. After being washed three times with PBS, cells were incubated with Cy5 fluorescence anti-rabbit secondary antibodies at room temperature for 1 h. After being washed three times with PBS, nuclei were stained with DAPI (5 μg/mL) for 10 min, coverslips were mounted on the glass slides and fluorescence images were captured using a confocal microscope.

Cognitive and behavioral assessment of AD mice

All 3×Tg-AD mice were randomly divided into different treatment groups with one mouse per cage to avoid fighting. The AD mice then were i.v. injected with PBS, PLNP-mGFP or PLNP-mTRIM11 (2 mg/kg) once every 7 days for a total of three treatment cycles. The WT mice also were i.v. injected with PBS.

Open Field Test (OFT): an open box (40 × 40 cm) was used as the test apparatus. The tracking route was recorded by software to obtain a movement score. A central area (20 × 20 cm) was delineated to record the trajectory of the mouse exploring the central area. The mouse was placed in a brightly lit, enclosed arena, and its movements are recorded for a set period (5 min) using Xeye Aba behavioral analysis software (Beijing Macro Ambition S&T Development Co., Ltd). Key metrics include total distance traveled, time spent in the center (indicating lower anxiety), and rearing frequency. The arena was cleaned between trials to remove residual odors, and the test was conducted under consistent conditions to ensure reliable results. Data was analyzed to assess behavioral differences, often comparing control and experimental groups.

Novel Object Recognition (NOR): the experimental apparatus was a polyethylene white rectangular open field box. Habituation took place by exposing the animal to the experimental apparatus for 10 min in the absence of objects on the day before training. During the training phase, mice were placed in the experimental apparatus in the presence of two identical objects (odorless wood cuboid or pyramid was used to prevent mice from climbing onto the object to avoid development of object preference) and were allowed to explore the object for 10 min. After 24 h, mice were placed again in the apparatus, but one of the objects was replaced by a novel one. Mice were allowed to explore for 10 min. Discrimination Index (DI) and Preference Index (PI) were used to assess NOR; these indices accounts for differences in exploration time. DI and PI are calculated as the time spent exploring (total exploration of at least 30 s, sniffing, trying to move object, object contact with front paw were defined as exploring, but not the time spent near the objects without investigation or passing by the objects). Data were collected using tracking software and manual scoring was used to assess behaviors from videos. DI was calculated as the time spent exploring the novel object minus the time spent exploring the familiar object, divided by the total exploration time. DI and PI were calculated based on the exploration time of the novel (T_novel_) and familiar (T_familiar_) objects: DI = T_novel_ – T_familiar_/(T_novel_ + T_familiar_); PI = T_novel_ or T_familiar_/(T_novel_+T_familiar_). DI values range from −1 to +1, where positive values indicate a preference for the novel object, and zero indicates no preference. PI ranges from 0 to 1, with a value of 0.5 corresponding to chance level (no preference), and values >0.5 indicating a preference for the novel object.

Nesting: Test mice were caged and housed one mouse per cage. On the first day of the test, two pieces of paper were introduced inside the home cage to allow assessment of nest-building behavior. After 48 h, the nest was photographed and scored as follows: 0 points, The Nestlet shows little to no manipulation.; 1 point, paper towels scattered throughout the cage, but no obvious bite marks (indicating active nest construction); 2 points, paper towels are concentrated in an area of the cage, but no obvious bite marks; 3 points, the paper towel was concentrated on one side or one corner with some bite marks; 4 points, most of the paper towels were bitten and gathered together; 5 points, nestlet fully shredded and consolidated into a well-defined crater/cup-shaped nest. Paper that had been shredded into small pieces or full of holes, was defined as bitten. All results were scored blindly.

Morris water maze (MWM): a pool was used as the test equipment and divided into four quadrants affixed with various symbols. The pool was filled with water containing titanium dioxide powder and kept at 22 ± 1°C and exclude noise and strong light. The mice were trained daily for four consecutive days to seek the hidden platform. The time spent moving from the start site to the target platform (escape latency) was recorded in the first four days. After removing the platform, the moving route, number of apparent crossings of the removed platform and residence time in the target quadrant were recorded on the fifth day.

Preparation and immunofluorescence staining of brain slices

After treatment was concluded, mice were anesthetized and perfused with saline. Brains of mice were then harvested and fixed via immersion in paraformaldehyde for three days. After the dehydration treatment by gradient concentrations of alcohol, the brains were embedded into paraffin. Brain slices (4 μm thick) were prepared using a microtome (Leica RM2016) and mounted onto glass slides. The slices were deparaffinized after the successive treatment of xylene, alcohol and washing with deionized water. Then slices were immersed in the EDTA antigen retrieval buffer (pH = 8.0) and microwaved for 8 min. After being washed with PBS buffer (pH = 7.4), slides were further incubated with bovine serum albumin (BSA, 5%) for 1 h and then incubated with the diluted primary antibody in PBS overnight at 4°C. Lastly, the slices were washed and incubated with the secondary antibody for 1 h. Prior to the imaging observation using CaseViewer (Pannoramic MIDI), the slices were washed with PBS and then stained with DAPI (Servicebio G1012).

Immunohistochemical staining of brain slices

Initially, paraffin-embedded sections of 4 μm thickness were deparaffinized down to water. The sections were then incubated in 3% hydrogen peroxide (H_2_O_2_) in deionized water at room temperature for 5-10 min to quench endogenous peroxidase activity. This was followed by three rinses with phosphate-buffered saline for 5 min each. For antigen retrieval, the sections were immersed in EDTA retrieval solution and heated in a microwave oven until boiling, then the power was turned off and the sections were allowed to sit for 5–10 min intervals before repeating the retrieval process once or twice more, after which they were left to cool. 5% bovine serum albumin (BSA) blocking solution was applied and the sections were incubated at 37°C for 30 min to prevent non-specific binding. Subsequently, the appropriately diluted primary antibody was added and the sections were incubated at 37°C for 1–2 h or overnight at 4°C. Biotin-labeled goat anti-rabbit IgG was then applied and incubated at 37°C for an additional 30 min. This was followed by the addition of streptavidin-horseradish peroxidase complex (SABC) and a further incubation at 37°C for 30 min. The sections were then rinsed three times with PBS for 5 min each.

Finally, the sections were examined microscopically. After counterstaining with hematoxylin (AR0005) for 0.5–2 min to enhance contrast, the sections were mounted using neutral gum, aqueous mounting media or other appropriate mounting media to preserve the samples for further analysis.

Safety assessment

The in vivo toxicity of PLNPs was evaluated by histopathological examination and routine hematological and biochemical analyses. Healthy ICR mice were randomly divided into two groups (n = 6) and received either PLNP-mTRIM11 (2 mg/kg, mRNA equivalent) or PBS via tail vein injection. After completion of behavioral assessments, major organs were harvested for H&E staining and microscopic examination.

Blood and serum samples were collected on days 1, 7, and 30 post-injection. Serum levels of alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (ALB), urea (UREA), and creatinine (CREA) were measured using commercial kits (Wuhan Servicebio Technology Co., Ltd.) on an automated chemistry analyzer (Chemray 240, Rayto Inc.). Whole blood was also collected to determine routine hematological parameters, including white blood cells (WBC), platelets (PLT), and red blood cells (RBC), using an automated hematology analyzer (BC-2800Vet, Mindray Inc.).

On days 7 and 30, three mice from each group were euthanized, and liver and kidney tissues were collected. The mRNA expression levels of inflammatory cytokines, including IL-6, TNF-α, and IL-1β, were quantified by real-time quantitative PCR (qRT-PCR).

Quantification and statistical analysis

All data are presented as mean ± SD. Statistical analyses were performed using Microsoft Excel (2016), and GraphPad Prism 8.0. Differences between two groups were assessed using two-tailed unpaired Student’s t tests. For multiple-group comparisons with all pairwise testing, one-way ANOVA followed by Tukey’s multiple-comparisons test was used. A p value <0.05 was considered statistically significant; exact n and individual p values are provided in the figure legends.