Luteolin-Loaded TGN/RAP12 Dual-Peptide Functionalized Nanoparticles: Synergistic Enhancement of BBB Penetration and Microglia Targeting in Alzheimer’s Disease

TL;DR

Researchers developed a nanoparticle delivery system to improve luteolin's ability to cross the blood-brain barrier and target microglia in Alzheimer’s disease.

Contribution

A novel nanoparticle system combining RBC membrane coating and dual peptides enhances BBB penetration and microglia targeting for Alzheimer’s treatment.

Findings

TGN/RAP12-RBC-NPs@Ltn significantly improved BBB permeability and microglia targeting.

Encapsulated luteolin enhanced cognitive function and reduced mitochondrial dysfunction in AD mice.

RBC membrane coating reduced macrophage phagocytosis and improved nanoparticle immune evasion.

Abstract

Luteolin (Ltn), a natural flavonoid, effectively inhibits microglial activation in Alzheimer’s disease (AD) with promising therapeutic potential, but its efficacy is severely limited by the blood–brain barrier (BBB). To overcome this obstacle, this study prepared poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs)—designated as TGN/RAP12-RBC-NPs@Ltn—which were coated with red blood cell membranes (RBCm) functionalized with two peptides, TGN (TGNYKALHPHN) and RAP12 (EAKIEKHNHYQK). The results demonstrated that TGN significantly enhanced BBB permeability, while RAP12 enabled effective targeting and delivery of TGN/RAP12-RBC-NPs@Ltn to microglial mitochondria in the brain. In addition, the presence of RBCm significantly inhibited the phagocytosis of NPs by macrophages, exerting a notable role in immune evasion. Meanwhile, the study confirmed that encapsulating Ltn within NPs significantly enhanced cognitive function in APP/PS1 mice, modulated the expression of key mitochondrial metabolic enzymes—pyruvate dehydrogenase (PDH) and its phosphorylated forms (pS232PDH, pS293PDH, pS300PDH)—in microglia, thereby ameliorating mitochondrial dysfunction and effectively regulating the neuroinflammatory environment in the mouse brain, and ultimately contributed to therapeutic efficacy. From this, it could be seen that TGN/RAP12-RBC-NPs@Ltn could significantly enhance the therapeutic effect of Ltn on AD, providing an effective treatment strategy for delaying the progression of AD.

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1. Introduction

Alzheimer’s disease (AD) is an aging-related neurodegenerative disease [1,2]. The high incidence rate of AD and irreversible cognitive dysfunction seriously affect the quality of life of patients. Currently, treatment options for AD remain limited, primarily due to two key challenges. Firstly, the existence of the blood–brain barrier (BBB) limits the delivery efficiency of drugs to the central nervous system (CNS) [3]. Second, even when certain drugs manage to cross the BBB, their non-specific distribution within brain tissues often leads to subtherapeutic local drug concentrations, which fail to achieve the threshold required for effective treatment, thereby diminishing therapeutic efficacy.

To address this challenge, it has been discovered that nano-drugs using nanomaterials have become an effective approach for treating AD. The key lies in penetrating the BBB through receptor-mediated endocytosis (e.g., via lat1, GLUT-1, LDL, or transferrin receptor TfR) to enhance drug delivery efficiency [4,5]. A functional TGN peptide (TGNYKALHPHN) can be recognized by the TfR receptor on the BBB and has great potential in brain transport [6]. Another functional peptide, RAP12 (EAKIEKHNHYQK), has strong targeted delivery capabilities and neuroimmune regulatory effects [7,8].

Red blood cells (RBCs) are present in large numbers in the blood, possessing excellent biocompatibility, biodegradability, and a long circulation half-life [9,10]. The red blood cell membrane (RBCm) can not only confer similar “immune evasion” capabilities to nanocarriers but can also facilitate the transmembrane transport of small molecules [11]. Moreover, a large amount of RBCm is conducive to achieving high loading capacity, enhancing the stability of nanoparticles (NPs), prolonging their storage time in vitro, and preventing aggregation. Although there are some drugs for treating AD in clinical practice, they have not fundamentally solved this problem.

Microglia, as one of the most important components of the CNS, exhibits morphological changes that are a hallmark of neurodegenerative diseases [12,13]. This type of cell’s own function maintenance and activation are highly dependent on energy metabolism [14,15]. In the CNS, there are two opposite types of microglia, the M1 phenotype and the M2 phenotype. Activated (M1-polarized) microglia release pro-inflammatory factors, while M2-polarized microglia achieve neuroprotection by promoting tissue repair and regeneration [16,17]. Previous studies have shown that many neurodegenerative diseases are accompanied by abnormal brain glucose metabolism in the early stage [18,19,20]. The Pyruvate Dehydrogenase (PDH) within microglia not only serves as a “metabolic bridge” connecting glycolysis and the TCA cycle, but it also acts as a “core molecular switch” that regulates the metabolic phenotype, supports core functions, and maintains survival homeostasis. In AD patients, the activity of the metabolic enzyme complex in the TCA cycle is reduced, especially the pyruvate dehydrogenase complex (PDHC) [21]. Under aerobic conditions, PDH, as a key metabolic enzyme in the mitochondria, is responsible for converting pyruvate into acetyl coenzyme A, releasing CO_2_ and generating Nicotinamide Adenine Dinucleotide (NADH), which then enters the TCA cycle [22,23,24]. When microglia are overly activated, the activity of PDH within them is inhibited and metabolic disorders are promoted, further advancing the progression of AD. Additionally, impaired PDH function may lead to mitochondrial dysfunction [25,26]. This can trigger inflammatory responses and further damage neurons.

To more effectively regulate the energy metabolism within microglia cells, luteolin (3′,4′,5,7-tetrahydroxyflavone, Ltn) [27], as a kind of microglial cell inhibitor and a type of flavonoid widely present in plants, has a significant therapeutic effect on AD [28]. According to the literature, Ltn in AD exerts its effects by directly protecting the structure and function of mitochondria, regulating metabolic signaling pathways, and combating mitochondrial damage [29,30]. It also inhibits pro-inflammatory mediators, including TNF-α, IL-β, and IL-6. In several in vitro AD models [31,32,33,34], it has anti-inflammatory effects and inhibits the activation of microglia.

Therefore, in this study, targeting Ltn was employed, combined with the bionic RBCm coating, TGN peptide was used as the blood–brain barrier penetration peptide, and RAP12 peptide was used as the targeting peptide for microglia cells in the brain. A nanodelivery system for Ltn, TGN/RAP12-RBC-NPs@Ltn, was constructed (Scheme 1). The released Ltn can directly and effectively act on the mitochondria of microglia cells in the mouse brain. By effectively reversing the mitochondrial damage of microglia cells in the hippocampus of AD mice and improving the inflammatory microenvironment in the brain, it can thereby alleviate the occurrence of AD.

2. Results

2.1. Preparation and Characterization of TGN/RAP12-RBC-NPs@Ltn

TGN/RAP12-RBC-NPs@Ltn was prepared by an optimized emulsification solvent evaporation method, as shown in Figure 1a. To enhance the targeting efficiency of the drug delivery NPs, functional conjugates DSPE-PEG_2000_-Mal/TGN and DSPE-PEG_2000_-Mal/RAP12 were synthesized (Figure S1). The transmission electron microscopy (TEM) results showed that NPs, RBC-NPs@Ltn, and TGN/RAP12-RBC-NPs@Ltn all presented spherical morphology and relatively uniform size (Figure 1b). The DLS results indicated that the average hydrodynamic diameter of NPs@Ltn was 196.19 ± 2.92 nm. After being coated with red blood cell membranes, the average size of RBC-NPs@Ltn increased to 198.39 ± 1.38 nm and further increased to 203.63 ± 3.79 nm after the connection of TGN and RAP12 peptides. Moreover, the polydispersity index (PDI) of each nanoparticle was less than 0.3, indicating a uniform distribution. The zeta potential of TGN/RAP12-RBC-NPs@Ltn was (−11.88 ± 0.79 mV), which was lower than that of NPs (11.25 ± 1.46 mV) and RBC-NPs@Ltn (−10.75 ± 0.70 mV). This was because TGN and RAP12 were covalently coupled to the surface of the NPs via Mal, reducing the overall charge. Furthermore, through screening, the optimal ratio of the dosage was determined to be 3:9. The encapsulation efficiency (EE) of NP was on average 77.82%, and the drug loading (DL) was on average 20.55% (Table S1).

To verify the integrity of the red blood cell membrane, we analyzed the proteins on the membrane. We conducted a parallel analysis of the proteins in different samples using SDS-PAGE. The results showed that the protein bands on the cell membrane were all located at the same position (Figure 1c); this indicates that the RBCm encapsulation was successful, and TGN and RAP12 did not have any impact during the modification process.

In order to study the coupling conditions of TGN peptide and RAP12 peptide with different NPs, FT-IR spectroscopy was conducted. The infrared spectrum showed a characteristic amide bond absorption at 1456 cm^−1^ (Figure S2). Compared with RBC-NPs, TGN/RAP12-RBC-NPs@Ltn has two characteristic peaks simultaneously, indicating that TGN and RAP12 have successfully bound to the surface of the NPs. We used a simple BCA protein assay method to calculate the binding efficiency. By using this method, we could determine that the binding efficiencies of the targeting peptides of TGN and RAP12 on RBC-NPs were 84.12 ± 1.45% and 77.23 ± 1.23%, respectively, and the peptide density was 12.02 ± 0.21% (by weight ratio).

Based on the ^1^H-NMR verification that the peptide and DSPE-PEG_2000_-MAL were successfully coupled, the nuclear magnetic resonance results showed that when DSPE-PEG_2000_ was not connected, the hydrogen at the sulfur atom in the peptide showed a peak at 1.5. However, after the connection of DSPE-PEG_2000_, the hydrogen at the sulfur atom was replaced and no peak was displayed. Through comparative analysis, it indicated that the coupling of the peptide and the NPs was successful (Figure S3).

To investigate the stability of the NPs, the changes in their particle size, PDI and encapsulation rate over time were measured. The results showed that all of them tended to stabilize, indicating that the stability of the NPs was good (Figure 1d).

To study the controlled drug release, the release curves of Ltn from different formulations were analyzed using the PBS release medium (pH 7.4). The Free Ltn was almost completely released within 1 h, indicating that there were no diffusion barriers in the dialysis bag. Compared with the Free Ltn, TGN-RBC-NPs@Ltn and RAP12-RBC-NPs@Ltn exhibited controlled drug release, reaching approximately 60% within 24 h without an initial burst release. A similar release pattern was observed in TGN/RAP12-RBC-NPs@Ltn, indicating that the modifications of TGN and RAP12 have little effect on the release of Ltn (Figure 1e).

2.2. The Biological Functions of RBCm on NPs

To investigate whether the RBCm coupled to NPs can inhibit the phagocytic action of phagocytes and inherit the long circulation characteristics of the RBCm, we conducted an in vitro anti-phagocytosis experiment. FITC-NPs, RBC-FITC-NPs, RAP12-RBC-FITC-NPs, TGN-RBC-FITC-NPs, and TGN/RAP12-RBC-FITC-NPs loaded with fluorescein isothiocyanate (FITC) were added to RAW264.7 cells. Compared with FITC-NPs, RBC-FITC-NPs, RAP12-RBC-FITC-NPs, TGN-RBC-FITC-NPs, and TGN/RAP12-RBC-FITC-NPs exhibited weakened phagocytic activity of macrophages (Figure 2g), demonstrating that the RBCm coating can effectively prevent macrophages from non-specifically taking up NPs.

2.3. Evaluation of BBB Permeation Efficiency

Firstly, the BBB model was successfully established (Figure 2a) and the cytotoxicity was measured by the methyl thiazolyl tetrazolium (MTT) assay. As shown in Figure S4, the survival rates of hCMEC/D3 cells, U-118 MG cells, and BV2 cells after incubation with different NPs ranged from 87% to 97%. The toxicity of these NPs on the cells can be considered negligible.

The success of the BBB model was evaluated by the changes in TEER. As shown in Figure 2b, the TEER values were measured continuously for seven days. The results indicated that it increased over time and reached its maximum value (183.55 ± 0.378 Ω·cm^2^) on the 4th day. As shown in Figure 2c, the liquid levels inside and outside the Transwell blood–brain barrier model were both maintained at a position of 0.4–0.5 cm, and this liquid level difference was consistently maintained. This indicates that a tight cellular barrier has been formed. Additionally, fluorescein sodium showed a good linear relationship (Figure 2d), with a correlation coefficient greater than 0.999. As shown in Figure 2e, although the permeability increased with time, it significantly decreased after 30 min. This result indicates that the BBB successfully blocked the free transport of small molecules. The above results collectively show that the BBB model was successfully established.

To evaluate the permeability of the in vitro BBB model of the NPs, Figure 2f shows that the fluorescence intensities of FITC-NPs and RBC-FITC-NPs remained at a low level throughout the monitoring period. The NPs functionalized with TGN and RAP12 demonstrated markedly improved BBB transmigration capability. These results indicated that after TGN and RAP12 modification, the biomimetic nanosystem had a stronger ability to penetrate the BBB.

In order to study the penetration ability of NPs through the BBB, FITC was used to mark different NPs, and the uptake ability of hCMEC/D3 cells and U-118 MG cells was studied. The fluorescence intensity was observed by fluorescence microscopy (Figure 2h,i). The fluorescence of TGN/RAP12-RBC-FITC-NPs, RAP12-RBC-FITC-NPs, and TGN-RBC-FITC-NPs was significantly stronger than that of FITC-NPs and RBC-FITC-NPs. The results showed that NPs modified by TGN and RAP12 could significantly increase the intracellular drug accumulation concentration by accelerating the uptake process and enhancing the ability to penetrate the BBB.

To further demonstrate its targeting ability, we conducted in vivo validation (Figure 3a). As shown in Figure 3b, the fluorescence signal of TGN/RAP12-RBC-NPs@Ltn in the mouse brain was stronger. This result indicates that the NPs modified by TGN and RAP12 have a stronger advantage in penetrating the BBB (Figure 3c,d). Moreover, the brain tissues treated by each nanoparticle group also supported this view (Figure 3e). At the same time, to comprehensively evaluate the distribution of the NPs in the mouse body, it was found that there were also significant differences in various organs. These results collectively indicate that, compared with DiR-NPs, TGN/RAP12-RBC-DiR-NPs have higher targeting efficiency and better BBB penetration ability.

2.4. Biodistribution of Ltn in Brain Tissue

To evaluate the content of Ltn in different NPs in the mouse brain, we determined the biodistribution of Ltn in the mouse brain after tail vein injection by UPLC-MS/MS (Figure 3f). The results showed that the concentration of Ltn in the brain of NPs connected with TGN/RAP12 was significantly higher than that of Free Ltn, while the Ltn content in the brain of NPs@Ltn and RBC-NPs@Ltn was slightly higher than that of Free-Ltn. Thus, it could be seen that TGN/RAP12-RBC-NPs@Ltn could increase the accumulation concentration of Ltn in the brain, and TGN/RAP12 could effectively enhance the transport capacity of the BBB.

2.5. The Targeted Effect of TGN/RAP12-RBC-NPs@Ltn on Microglia Cells

To confirm microglial targeting, FITC-labeled NPs were fabricated for assessing cellular uptake in BV2 cells (Figure 3g). As illustrated in Figure 3h, the fluorescence intensity of TGN/RAP12-RBC-FITC-NPs, RAP12-RBC-FITC-NPs, and TGN-RBC-FITC-NPs was markedly higher compared to that of unmodified FITC-NPs and RBC-FITC-NPs. These results indicate that dual functionalization with TGN and RAP12 promotes efficient recognition by microglia.

To further explore the targeting ability of different formulations of NPs to microglia, we injected the drugs labeled with the red fluorescent probe DiR into mice and observed the co-localization of microglial marker (Iba1) and NPs through confocal microscopy. The results are shown in Figure 3i; DiR-NPs and RBC-DiR-NPs showed almost no red fluorescence expression, while TGN-RBC-DiR-NPs and RAP12-RBC-DiR-NPs showed a relatively small amount of red fluorescence expression. In sharp contrast, the fluorescence intensity of TGN/RAP12-RBC-DiR-NPs was the strongest. The results indicated that TGN/RAP12-RBC-DiR-NPs had a strong targeting ability for microglia cells in the mouse brain tissue.

2.6. Improving Learning and Memory Deficits in APP/PS1 Mice

To assess the impact of TGN/RAP12-RBC-NPs@Ltn on cognitive function in AD mice, behavioral evaluations—including the Morris water maze and novel object recognition assays—were performed following a 30-day therapeutic intervention. The in vivo experimental process is shown in Figure 4a. For the Morris water maze training test, the movement trajectories of mice in each group are shown in Figure 4b. Control and RBC-NPs@Ltn- and TGN/RAP12-RBC-NPs@Ltn-treated mice showed significantly reduced latency during the 5-day training period, indicating that RBC-NPs@Ltn- and TGN/RAP12-RBC-NPs@Ltn-treated mice exhibited better spatial learning ability than NPs@Ltn-treated mice (Figure 4c). On the 6th day, the platform was removed, and a memory recall assessment probe test was conducted. Compared with the AD mice treated with Free Ltn and NPs@Ltn, the mice treated with RBC-NPs@Ltn and TGN/RAP12-RBC-NPs@Ltn spent more time crossing the platform (Figure 4d), had a longer stay time in the target quadrant (Figure 4e), and swam a greater total distance (Figure 4f). These data indicate that RBC-NPs@Ltn and TGN/RAP12-RBC-NPs@Ltn significantly rescued the memory deficits in AD mice, with TGN/RAP12-RBC-NPs@Ltn showing the most significant effect on the cognition and memory of AD mice. Novel object recognition test results revealed a significantly reduced recognition index for novel objects in the model group mice compared to the control group, and the recognition indices of the RBC-NPs@Ltn and TGN/RAP12-RBC-NPs@Ltn treatment groups increased successively, indicating that TGN/RAP12-RBC-NPs@Ltn could significantly improve the novel object recognition ability of AD model mice (Figure 4g). These results collectively demonstrated that RBC-NPs@Ltn and TGN/RAP12-RBC-NPs@Ltn could significantly rescue the memory deficits in AD mice, among which TGN/RAP12-RBC-NPs@Ltn showed the most significant impact on the cognition and memory of AD mice.

All the experimental mice in this study were healthy mice of the same age and weight; before the formal experiments, the living conditions and autonomous activity abilities of all mice were comprehensively evaluated through food intake, water intake, and visual function; moreover, all behavioral experiments were conducted in a unified environment, with consistent lighting and temperature, in order to minimize the influence of confounding factors on the experimental results of the mice.

2.7. Improvement of Pathological Conditions in APP/PS1 Mice by TGN/RAP12-RBC-NPs@Ltn

Through H&E staining, it could be clearly observed that the CA1, CA3, and dentate gyrus regions of the hippocampus in the control group mice were uniformly stained, with dense and orderly cell nuclei, round shapes, and clear boundaries between the nucleus and the cell membrane. In the APP/PS1 mouse model, the mice showed smaller cell volumes, irregular shapes, and severe nuclear atrophy. Compared to the model group mice, the other treatment groups improved the hippocampal neurons of the mice to varying degrees, and the TGN/RAP12-RBC-NPs@Ltn treatment group showed the most significant improvement (Figure 4h). This indicated that TGN/RAP12-RBC-NPs@Ltn could improve the pathological changes in the hippocampal region of APP/PS1 mice.

2.8. The Regulatory Effect of TGN/RAP12-RBC-NPs@Ltn on PDH and Mitochondria in the Brains of APP/PS1 Mice

PDH is a key enzyme in the cellular energy metabolism process, participating in the conversion of pyruvate to acetyl-CoA, which then enters the tricarboxylic acid cycle to provide energy for the cell. In AD, the activity of PDH in microglial mitochondria is reduced, which leads to energy metabolism imbalance and mitochondrial damage, and these changes are related to pathological changes such as neuronal damage in AD; notably, pyruvate dehydrogenase PDK2 can inhibit PDH activity by phosphorylating PDHA1, the E1 subunit of PDH. Therefore, in order to verify whether the prepared NPs can effectively regulate PDH and phosphorylation expression and restore mitochondrial damage (Figure 5a), the immunofluorescence experiment showed that compared with the control group, the TGN/RAP12-RBC-NPs@Ltn treatment group could significantly enhance the fluorescence signal of PDH (Figure 5b). Next, WB was used to determine PDH, PS-232PDH, PS-293PDH, and PS-300PDH (Figure 5c). The quantitative results indicated that, compared to the control group, TGN/RAP12-RBC-NPs@Ltn could more significantly enhance the expression of PDH and down-regulate the expression of PDH phosphorylation (Figure 5d). To further demonstrate that TGN/RAP12-RBC-NPs@Ltn can enhance PDH activity, we also measured the PDH activity and ultimately obtained consistent results with the above, which more strongly indicates that TGN/RAP12-RBC-NPs@Ltn can significantly up-regulate PDH expression (Figure 5e).

In order to investigate whether TGN/RAP12-RBC-NPs@Ltn can further alleviate the mitochondrial damage within microglia cells, transmission electron microscopy was used to observe that the mitochondrial morphology was intact and the matrix was uniform in the control group mice (Figure 6a). In the MX group, the mitochondria were swollen and ruptured, with a more severe degree of damage. However, the NPs treatment group could reverse this damage, and TGN/RAP12-RBC-NPs@Ltn was the most significant (Figure 6b). These results collectively indicate that TGN/RAP12-RBC-NPs@Ltn can effectively inhibit PDH phosphorylation and thereby improve the mitochondrial damage within microglia cells.

2.9. Effect of TGN/RAP12-RBC-NPs@Ltn on Reversing Brain Inflammation in APP/PS1 Mice

The NPs linking TGN and RAP12 exerted a regulatory influence on the neuroinflammatory environment in the brains of APP/PS1 mice; the schematic diagram is shown in Figure 7a. ELISA results indicated that, compared with the control group, the hippocampus of APP/PS1 mice exhibited elevated expression levels of pro-inflammatory markers, including TNF-α, iNOS, and IL-1β. Treatment with TGN/RAP12-RBC-NPs@Ltn significantly attenuated this upregulation. Conversely, the expression levels of anti-inflammatory factors—TGF-β, Arg-1, and IL-10—were reduced in the hippocampus of APP/PS1 mice relative to the control group. Similarly, treatment with TGN/RAP12-RBC-NPs@Ltn resulted in a significant upregulation of anti-inflammatory factor expression (Figure 7b). These findings were further corroborated by WB analysis. Compared with control mice, APP/PS1 mice exhibited increased hippocampal expression levels of pro-inflammatory markers iNOS, IL-1β, and TNF-α, while the expression of anti-inflammatory markers Arg-1, IL-10, and TGF-β was decreased (Figure 7c,d). Collectively, these results indicated that NPs conjugated with both TGN and RAP12 peptides could effectively modulate the overexpression of inflammatory mediators. This suggested that TGN/RAP12-RBC-NPs@Ltn might ameliorate the neuroinflammatory environment in the hippocampus of AD mice.

2.10. Safety Assessment

In order to assess the safety of the treated groups on the organs of the mice, H&E staining was performed on the organs of the mice in each treatment group. The results showed that the NPs prepared had almost no damage to the organs of the mice, proving that the safety of the NPs was good (Figure S5).

3. Materials and Methods

3.1. Materials

Ltn (purity 95%) was provided by Shanghai Yuanfan Biotechnology Co., Ltd., Shanghai, China. DSPE-PEG_2000_-Mal (molecular weight: 2805.5 Da) was purchased from Chongqing Yusi Pharmaceutical Technology Co., Ltd., Chongqing, China. Poly (lactic acid)-glycolic acid (PLGA; 5000 Da; 50:50 PLA: PGA, (w/w)) was purchased from Jinan Daigang Biomedical Materials Co., Ltd., Jinan, China. The RAP12 peptide (EAKIEKHNHYQK, MW: 1627.82 Da) and TGN peptide (TGNYKALHPHNG, MW: 1308.4 Da) were obtained from China Petides Co., Ltd. (Shanghai, China). The sodium fluorescein was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). The FITC was acquired from Beijing Sun Biotechnology Co., Ltd. (Beijing, China). DiR was provided by Beijing Innovation Chemical Co., Ltd., Beijing, China. DAPI, penicillin-streptomycin, HRP-labeled Goat Anti-Rabbit IgG(H+L) and Mouse Anti-βaction mAb were supplied by Beyotime Biotechnology Co., Ltd., Shanghai, China. Pyruvate Dehydrogenase (C54G1), Phospho-Pyruvate Dehydrogenase α1 (Ser293) Rabbit mAb and iNOS (D6B6S) Rabbit mAb were purchased from Cell Signaling Technology Co., Ltd., Danvers, MA, USA. Phospho-PDH E1 α (Ser300) pAb, TGF β 1 rAb, IBA1 pAb, and Arginase-1 pAb were purchased from Proteintech Group Co., Ltd. PDH1 (Ser232) pAb was purchased from Abmart Co., Ltd., Shanghai, China. IL-1 β Rabbit pAb, TNF α Rabbit pAb, and IL-10 Rabbit pAb were obtained from Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China.

3.2. Synthesis of NPs

To enhance the penetration ability of the drug delivery system through the BBB, the maleimide group at the end of DSPE-PEG_2000_-Mal reacted with the thiol group at the peptide end to form DSPE-PEG_2000_/RAP12 and DSPE-PEG_2000_/TGN. TGN, RAP12 and DSPE-PEG_2000_-Mal (in a ratio of 1.5:1 M) were dissolved in PBS. Under nitrogen and dark conditions, the mixture was stirred with a magnetic stirrer for 4 h. The reaction mixture was subsequently subjected to dialysis against deionized water at 4 °C for 48 h using a membrane with a molecular weight cutoff of 3.0 kDa to eliminate unreacted components.

Next, the preparation of TGN/RAP12-RBC-NPs@Ltn was carried out. First, the whole blood of the rats was taken and transferred into a centrifuge tube containing heparin sodium. It was mixed with 2 mL of 1×PBS solution and centrifuged at 2000× g rpm for 15 min at 4 °C. The supernatant and the white substance at the interface were discarded. The RBC were washed three times with pre-cooled PBS solution (5000 r/min, 10 min), and the supernatant was discarded to obtain free RBC. RBC was mixed with a Tris-HCl buffer solution at a ratio of 1:40. It was left to stand for 1 h and then centrifuged (at 12,000 r/min for 20 min). The supernatant was discarded, and the above steps were repeated until the supernatant was almost colorless. This yielded RBCm. A total of 3 mg of Ltn was taken and dissolved in 1.5 mL of acetone. A total of 9 mg of poly (lactic-co-glycolic acid) (PLGA) was dissolved in 0.9 mL of acetone and ultrasonicated for 2 min at 700 rpm. It was dropped into 5 mL of 0.5% PVA under liquid condition, ultrasonicated for 5 min, and then rotary evaporated for 20 min to remove the organic solvent. It was left to solidify at −20 °C for 10 min and centrifuged at 12,000 rpm at 4 °C for 30 min. The lower layer precipitate PLGA-NPs@Ltn was obtained, and the optimal synthesis scheme was screened through orthogonal experiments. The collected RBCm was subjected to ultrasonic treatment at 200 W for 5 min. Then, the obtained RBC-derived vesicles were repeatedly extruded from 400 nm and 200 nm polycarbonate membranes using a small extruder (Avanti Polar Lipids, Alabaster, AL, USA). The RBCm vesicles were mixed with the NPs@Ltn suspension and ultrasonic treatment was performed (for 5 min). A liposome extruder was used to repeatedly extrude the mixed suspension through a 400 nm polycarbonate membrane to obtain the biomimetic NPs (NPs@Ltn). Then, DSPE-PEG_2000_-RAP12 and DSPE-PEG_2000_-TGN were added to RBC-NPs@Ltn (in a 4:1 ratio, with the mass ratio of Ltn to peptide), and the mixture was incubated in a water bath at 37 °C for 30 min. Finally, TGN-RBC-NPs@Ltn and RAP12-RBC-NPs@Ltn were successfully prepared.

3.3. Characterization of the NPs

3.3.1. Determination of TEM

The prepared NPs were passed through a copper mesh and then stained with phosphotungstic acid solution (1%, w/w). After drying, the morphology of the NPs was characterized using a transmission electron microscope (TEM) (Hitachi, Japan).

3.3.2. Determination of Size, PDI and Zeta Potential

The obtained NPs were suspended in PBS, and their particle size, polydispersity index (PDI), and zeta potential were measured using a particle size and potential analyzer, Brookhaven Instruments, Holtsville, NY, USA.

3.3.3. Determination of Encapsulation Rate (EE%) and Drug Loading Capacity (DL%)

The EE% and DL% of NPs were determined by high-performance liquid chromatography (HPLC). The HPLC chromatographic conditions were as follows: a C18 column (Agela, 200 × 4.6 mm, 5 μm), mobile phase of acetonitrile-water (50:50, v/v), detection wavelength of 254 nm, and flow rate of 1 mL/min. The formula is as follows:

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3.3.4. Determination of FTIR

The freeze-dried powders of each NP were mixed with dry potassium bromide powder (1:100 v/v), then pressed into tablets. The coupling of NPs and peptides was verified using the IRTracer-100 FTIR spectrometer (Shimadzu, Kyoto, Japan).

3.3.5. 1H-NMR Characterization of NPs

TGN and RAP12 were respectively dissolved in PBS with DSPE-PEG2000-MAL (in a 1:1.5 M ratio). The solutions were stirred under nitrogen protection and in the dark for 24 h. The reactants were dialyzed in deionized water using a dialysis membrane (with a molecular weight cut-off of 3.0 KDa) at 4 °C for 48 h to remove uncoupled reactants. The resulting solution was freeze-dried and subjected to HNMR analysis.

3.3.6. Determination of Peptide Binding Efficiency

The unbound free TGN, RAP12, and the bound NPs (with MWCO of 10 kDa) were separated by ultrafiltration centrifugation in a centrifuge tube. After centrifugation (at 10,000 rpm for 10 min), the lower layer was the uncoupling peptide. The protein concentration was determined using the BCA protein concentration kit (Birgen Biotechnology Co., Ltd., Shanghai, China). The peptide binding efficiency was calculated as follows:

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3.3.7. Determination of Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

The protein content of the NPs was determined using a BCA protein assay kit. SDS-PAGE combined with Coomassie Brilliant Blue staining was employed to verify the integrity of the erythrocyte membrane proteins, followed by imaging analysis.

3.3.8. Determination of Stability

The prepared NPs were suspended in PBS buffer solution and placed at 4 °C. Samples were taken at 0, 1, 3, 6, 12, 24, 48 and 72 days. The particle size and PDI index as well as EE% at different times were analyzed using a particle size and potential analyzer.

3.3.9. In Vitro Release Profile

The study on the release of NPs was conducted using the in vitro dialysis method. 1 mL of freshly prepared NPs suspension was added to a sealed dialysis bag immersed in PBS and placed in a 37 °C constant temperature water bath. Samples were taken in parallel from the solution outside the dialysis bag at 0, 10, 20, 30, 40, 50, 60, 70, and 80 h. Equal amounts of fresh PBS were added, the content of Ltn in the release medium was determined by HPLC (with the same conditions as above), and the cumulative release rate was calculated.

3.4. Cell Culture

BV2, hCMEC/D3, U-118MG and RAW 264.7 cells were obtained from Hunan Fenghui Biotechnology Co., Ltd. BV2, RAW 264.7 and U-118MG were cultured in DMEM with 10% FBS and 1% penicillin-streptomycin, while hCMEC/D3 cells were grown in EGM-2 medium containing VEGF, IGF-1, EGF, bFGF, hydrocortisone, penicillin-streptomycin and 2.5% FBS. All cells were maintained at 37 °C in a humidified 5% CO_2_ incubator.

3.5. Cytotoxicity Assay

To detect the cell viability of different cells, 96-well plates were seeded with BV2 cells, hCMEC /D3 cells and U-118MG cells (density of 5 × 10^3^ cells per well) in a logarithmic growth phase. The cells were cultured under 5% CO_2_ at 37 °C until they adhered to the plate. Then, different concentration gradients of nanocarriers were added and incubated for 24 h. An amount of 20 μL of MTT solution (5 mg/mL) was added to each well and incubated for another 4 h. The supernatant was discarded, 150 μL of DMSO solution was added to each well, and the plates were shaken at low speed for 10 min. Finally, the absorbance was measured at 490 nm using the MK3 microplate reader Shanghai Thermal Instrument Co., Ltd., Shanghai, China), and the blank medium wells were used for background zeroing. Then, the cell survival rate was calculated using the following formula:

$$[eqn]$$

3.6. Cellular Uptake

In order to observe the uptake of NPs by cells, BV2, hCMEC /D3 and U-118MG cells were seeded into 6-well plates (1 × 108 cells per well) and incubated (at 37 °C, 5% CO_2_). After adherence, the cells were incubated with FITC-labeled NPs at a concentration of 50 μL/mL for 90 min. Subsequently, the cells were fixed with 4% paraformaldehyde for 15 min, followed by nuclear staining with DAPI for 5 min, and then the intracellular fluorescence intensity of the FITC-labeled nanoparticles was observed under the fluorescence microscope (Olympus Corporation, Tokyo, Japan). Subsequently, the living cells were collected and resuspended in PBS, and then quantitative analysis was performed using the Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA).

3.7. Phagocytosis Assay

In order to verify the phagocytic effect of macrophages on various NPs, RAW 264.7 cells were inoculated into 24-well plates and cultured in a 37 °C, 5% CO_2_ incubator. Amounts of 500 μL of 50 μg/mL FITC-NPs, RBC- FITC-NPs, RAP12-RBC-FITC-NPs, TGN-RBC- FITC-NPs, and TGN/RAP12-RBC-FITC-NPs were added to each well and incubated for 2 h. Subsequently, cells were fixed with 4% paraformaldehyde for 15 min and counterstained with DAPI for 5 min. Nuclear fluorescence was visualized using an Olympus fluorescence microscope (Tokyo, Japan).

3.8. Construction of the BBB Model and Determination of Transportation Capacity

To construct an in vitro BBB model, U-118MG cells (1 × 10^4^ cells/mL) were seeded in the lower layer of the Transwell chamber, and hCMEC/D3 cells (1 × 10^4^ cells/mL) were seeded in the upper layer; they were then placed in a 12-well plate. After 7 days of cultivation, the trans-epithelial resistance (TEER) value was measured using the Millicell ERS-2 resistance measurement instrument (Millipore, Molsheim, France) to evaluate the sealing performance of the BBB model. The formula for calculating trans-epithelial resistance is as follows: TEER (Ω·cm^2^) = (measured resistance value (Ω)—background resistance value (Ω)) × membrane area (1.12 cm^2^).

To further verify the integrity of the BBB model, a leakage experiment was conducted when the TEER value was at its maximum. The BBB model was placed in a cell culture box at 37 °C and containing 4% CO_2_. After 4 h, the height difference of the liquid levels between the co-culture group and the blank group was measured. Subsequently, a permeability experiment was conducted. A certain concentration of sodium fluorescein was added to the BBB chamber. Under the condition of 37 °C and 5% CO_2_, samples were taken at 15, 30, and 60 min, and their fluorescence intensity was measured using a spectrophotometer (PerkinElmer Company, Shelton, CT, USA). This was performed to further confirm the successful establishment of the BBB model. When the resistance reached its peak, the BBB transport experiment was conducted. FITC-labeled NPs were used to replace the upper layer of the culture medium in the chamber. Four hundred microliters of the solution was collected at different time points, and fresh culture medium was replenished simultaneously. Finally, the fluorescence intensity of the sample solution was measured using a fluorescence spectrophotometer.

3.9. Assay of Ltn in the Brain with UPLC-MS/MS

Male C57BL/6 mice (20–25 g) were randomly allocated into four groups (n = 6) and intravenously injected via the tail vein with Ltn at an equivalent dose of 20 mg/kg, formulated as follows: (i) Free Ltn, (ii) NPs@Ltn, (iii) RBC-NPs@Ltn, and (iv) TGN/RAP12-RBC-NPs@Ltn, all suspended in sterile PBS. At one hour post-administration, mice were humanely euthanized; brains were rapidly dissected, weighed, and homogenized in ice-cold saline (1:4 w/v). Homogenates were centrifuged at 14,000 rpm for 10 min at 4 °C. A 250 μL aliquot of the supernatant was spiked with 50 μL internal standard solution (apigenin, 2 μg/mL), followed by an addition of 1 mL pre-chilled methanol. The mixture was vortexed for 2 min then re-centrifuged under identical conditions. The supernatant was collected, dried under a gentle nitrogen stream, and the residue reconstituted in 150 μL of methanol. After vortexing for 1 min and final centrifugation (14,000 rpm, 10 min, 4 °C), the supernatant was filtered through a 0.22 μm membrane and analyzed quantitatively by UPLC-MS/MS.

Ltn quantification was carried out via UPLC-MS/MS using a Waters ACQUITY H-Class PLUS system interfaced with an AB SCIEX QTRAP^®^ 5500 mass spectrometer operating in negative-ion multiple reaction monitoring (MRM) mode. Apigenin was employed as the internal standard. Chromatographic resolution was achieved on an ACQUITY UPLC BEH C18 column (1.7 μm, 50 mm × 2.1 mm) held at 40 °C, with a 5 μL injection volume. The mobile phase—comprising water and acetonitrile (20:80, v/v)—was delivered at 0.6 mL/min. Mass spectrometric settings included the following transitions: apigenin, m/z 367.1 → 217.0 [M − H]^−^; Ltn, m/z 285.1 → 133.1 [M − H]^−^; capillary voltage, −4.50 kV; cone voltage, 20 V; collision energy, 25 eV; dwell time, 100 ms; source temperature, 120 °C; desolvation gas temperature, 400 °C; desolvation and cone gases, nitrogen; collision gas, argon (≈2.61 × 10^−3^ mbar).

3.10. In Vivo Imaging

To deeply explore the biological distribution of NPs during the brain-targeting process, the various NPs encapsulated with the fluorescent probe DiR were injected into the tail veins of mice and the organs were taken for fluorescence measurement. Twelve hours later, the mice were imaged in vivo using the IVIS Lumina III (PerkinElmer, Shelton, CT, USA).

3.11. Experimental Animals and Dosing Regimens

Male APP/PS1 mice aged 8 weeks (GemPharmatech Co., Ltd., Nanjing, China), and male c57 mice aged 8 weeks (Second Affiliated Hospital of Harbin Medical University, Heilongjiang, China). Thirty 7-month-old APP/PS1 mice were randomly divided into the following 5 groups: APP/PS1 group (n = 6), free Ltn group (n = 6), NPs@Ltn group (n = 6), RBC-NPs@Ltn group (n = 6), and TGN/RAP12-RBC-NPs@Ltn group (n = 6), and the C57 mice with matching age were assigned to the control group (n = 6). The mice were subjected to continuous intravenous tail injections (2 mg/kg/day) for 30 days. Behavioral experiments were conducted. Finally, the mice were euthanized and their tissues were taken for pathological analysis.

The animals were housed under controlled 12:12 h light–dark cycles and provided ad libitum access to standard chow and fresh water. All experimental procedures strictly adhered to the “Guidelines for the Care and Use of Laboratory Animals” promulgated by the Ministry of Science and Technology of the People’s Republic of China. All the animal experiments were approved by the Animal Experiment Ethics Committee of Heilongjiang University of Traditional Chinese Medicine (Approval Number: 2024042623).

3.12. Behavioral Experiment

In this study, the Morris water maze experiments and the new object recognition experiments were conducted using a double-blind method to completely eliminate the subjective biases of the experimenters and data analysts. Independent researchers who were not involved in the behavioral testing conducted the coding of all the experimental mice according to the random number table. The personnel responsible for the experimental operation and data recording only carried out the experiments based on the coding and were unaware of the group information of the mice; at the same time, the data analysts remained blind to the group information during the statistical processing stage. All the experiments and data analyses were completed before the group decoding was carried out.

3.12.1. Water Maze Experiment

A pool with a diameter of 100 cm was filled with water (at 22–24 °C). A platform was fixed in the second quadrant. During the positioning navigation experiment, the mice were trained for five consecutive days, and the time it took for them to find the platform was recorded. If they failed to find the platform within 90 s, they were guided to stay on the platform for 15 s. On the sixth day, the platform was removed and a spatial exploration experiment was conducted. The swimming trajectory within 90 s, the number of times crossing the platform, the total swimming distance, and the time spent in the target quadrant were recorded.

3.12.2. New Object Recognition

The mice were placed in an empty identification box for two days (10 min per day). On the third day, two identical objects were placed in the test box, and the experimental animals were put in to explore for 5 min. The exploration time or frequency of the mice for the two objects was recorded. One hour later, one of the objects was replaced with a new one, and the above experiment was repeated. In the experiment, high-definition cameras installed on the experimental device were used to capture the videos, and the video trajectories of the mice were automatically tracked and data were collected using EthoVision XT behavioral analysis software 11.4 (from Noldus, Gelderland, The Netherlands). The cognitive index (Recognition Index, RI) was calculated using the following formula: RI = New object exploration time/(New object exploration time + Old object exploration time) × 100%.

3.13. H&E Staining

In order to observe the various organizational forms, the mouse tissues were fixed with 4% paraformaldehyde, dehydrated, then embedded and fixed, and cut into 5 μm wax sections. These sections were placed at 37 °C overnight. Subsequently, the wax sections were successively treated with xylene I, graded ethanol, water, safranin, water, mounting solution, water, eosin, water, graded ethanol, xylene I, and xylene II solution. They were then sealed with neutral gum and observed under a microscope.

3.14. Transmission Electron Microscope

The damage of mitochondria in microglia cells of mouse hippocampal tissue was detected by transmission electron microscopy. The mouse hippocampus was fixed with a 4% paraformaldehyde and 2.5% glutaraldehyde (in a 1:1 ratio) solution. After dehydration, embedding, and curing, the ultra-thin sections were made and placed under the ultrathin sectioning machine for slicing, staining, washing, and drying at room temperature overnight. The mitochondria were observed under the transmission electron microscope and photographed.

3.15. Immunofluorescence Analysis

The expression of PDH in the hippocampus region of the brain was observed through immunofluorescence. After the mouse brain tissue was fixed, it was placed in a 30% sucrose solution for sugar fixation. The dehydrated brain tissue was then embedded in OCT and cut into 6 μm sections. The samples were washed with PBS for 15 min and incubated with goat serum at room temperature for 1 h. The primary antibody was incubated at 4 °C overnight. The next day, the sections were washed with PBS for 15 min and then incubated with a fluorescent-labeled secondary antibody in the dark for 2 h, and finally, a mounting solution containing DAPI was added for mounting and used for microscopic observation.

3.16. Immunofluorescence Co-Localization

In order to verify the targeting ability of NPs to microglia, each NP carrying the red fluorescent probe DiR was injected into the tail vein of the mice. Samples were taken 24 h later. All other procedures were the same as those in 2.15, except that they were incubated with the green fluorescent Iba1 antibody. Finally, microscopic observation was conducted.

3.17. ELISA Assay

The cytokine levels (TNFα, iNOS, IL-1β, TGF-1β, Arg-1, IL-10) in the brain tissues of each group of mice were detected using the ELISA double-antibody sandwich method. The specific operation was carried out in accordance with the instructions of the reagent kit (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China).

3.18. Western Blot (WB)

The protein contents were determined by WB. The hippocampal tissue was homogenized and centrifuged using RAPI buffer. The supernatant was quantified using the BCA protein assay kit. Then, it was subjected to 10% SDS-PAGE gel electrophoresis (80 V for 30 min, 120 V for 60 min), transferred onto a PVDF membrane, and incubated for 30 min with blocking solution. After that, it was incubated with target proteins such as PDH and β-actin overnight at 4 °C. The next day, it was incubated with secondary antibodies for 2 h. After ECL detection, the bands were obtained using Tanon Chemi Dog Ultra Tra, and images were captured and quantified using Image J (Version 1.54f, National Institutes of Health, Bethesda, MD, USA). This version supports routine image analysis in the field of biomedicine. The accuracy of converting RGB images to grayscale images is high, and the stability of the software operation is also high. The target protein was standardized using β-actin as the protein loading control.

3.19. Determination of PDH Activity

The PDH activity in the hippocampal tissues of different NP mice was determined using the Pyruvate Dehydrogenase (PDH) Activity Assay Kit and following the instructions provided.

3.20. Statistical Analysis

All experiments were repeated at least three times, and the results are presented as the mean ± standard deviation. Normality and homogeneity of variance tests were performed on all experimental data to ensure the rationality of the statistical method selection. One-way ANOVA was used to test the overall significant differences among multiple groups; finally, Tukey’s multiple comparison test was used to compare the differences among groups. A p value < 0.05 was considered to indicate a statistically significant difference between treatments. All quantitative analyses were conducted using ImageJ (Version 1.54f, National Institutes of Health, Bethesda, MD, USA).

4. Conclusions

This study successfully developed a biomimetic nanocarrier system coated with RBCm and functionalized with TGN and RAP12 peptides, enabling precise and efficient delivery of Ltn into microglia in the brains of APP/PS1 mice for the targeted treatment of AD. The in vitro cellular uptake experiments in D3 and U-118-BV2 cells, combined with in vivo small-animal live imaging, collectively demonstrated that the developed NPs effectively crossed the BBB and specifically targeted microglia. Furthermore, TGN/RAP12-RBC-NPs@Ltn exhibited enhanced targeting efficacy compared to RBC-NPs@Ltn. The results of the water maze test, novel object recognition test and HE staining showed that TGN/RAP12-RBC-NPs@Ltn could reverse the cognitive function of APP/PS1 mice and exhibit significant neuroprotective effects. The WB and immunofluorescence results indicated that TGN/RAP12-RBC-NPs@Ltn could significantly reverse the downregulation of PDH and the upregulation of PDH phosphorylation in AD. More importantly, TGN/RAP12-RBC-NPs@Ltn effectively alleviated mitochondrial damage, thereby regulating the energy metabolism within the brain. Furthermore, the levels of inflammation-related factors in the hippocampus of mice were determined by WB and ELISA. The results indicated that TGN/RAP12-RBC-NPs@Ltn effectively improved the inflammatory microenvironment in the brain. The pro-inflammatory factors iNOS, IL-1β and TNF-α were significantly reduced, while the anti-inflammatory factors Arg-1, IL-10 and TGF-β were significantly increased. Meanwhile, mice treated with this NP exhibited excellent safety profiles across all major organs.

Currently, there are many nano-drugs targeting AD with single specificity. Our dual-peptide synergistic effect has increased the uptake rate of BV2 cells by over 30% compared to single-ligand modification, significantly outperforming the “single homologous targeting” strategy proposed by Junyang Chen et al., providing a new approach for the precise treatment of AD. Compared with previous single-targeting strategies, such as attaching a single TGN peptide, although it can penetrate the blood–brain barrier and enter the brain, it cannot effectively improve the inflammatory environment of microglia. The TGN/RAP12-RBC-NPs@Ltn we have prepared not only penetrates the BBB but also enables the nanoparticles to more precisely reach microglia in the brain, more effectively improving the inflammatory microenvironment within the brain. These results collectively indicate that the biomimetic nanocomposite material enhances BBB permeability and improves microglia-targeting efficacy, thereby augmenting the therapeutic effect of Ltn in AD and opening new avenues for its treatment.

This study has confirmed that TGN/RAP12-RBC-NPs@Ltn can effectively alleviate microglial inflammation and mitochondrial damage in the AD model. Recent research has shown that, in AD, microglial inflammation, mitochondrial dysfunction, lysosomal processing, and innate immune activation are closely networked. The intracellular nucleic acid sensing pathways (such as cGAS-STING, TLR9) are the key hubs connecting lysosomal dysfunction and microglial activation; abnormal activation of this pathway not only directly upregulates the expression of inflammatory factors, but it also further exacerbates mitochondrial dysfunction by inhibiting mitochondrial biosynthesis [35]. Some studies have also shown that lysosomes maintain mitochondrial quality control through mitochondrial autophagy, and their dysfunction can lead to mitochondrial dysfunction; at the same time, increased lysosomal membrane permeability (LMP) can trigger excessive activation of the innate immune response. Thus, the inflammation of microglia in AD is not caused by a single abnormal pathway, but it is the result of the interaction between lysosomal dysfunction, mitochondrial damage and excessive activation of the immune pathway [36].

The TGN/RAP12-RBC-NPs@Ltn prepared in this study has regulatory effects on microglial cell inflammation, key enzymes PDH in the TCA cycle, and mitochondrial damage. It is speculated that this is related to the following mechanisms: on the one hand, it may improve the function of lysosomes; on the other hand, it may directly or indirectly regulate the excessive activation of the innate immune system, ultimately breaking the pathological cycle. Although the specific regulatory targets still need to be further verified through experiments, this study provides new ideas and a new experimental basis for the multi-targeted collaborative treatment of AD.