A Hierarchical Microglial-Targeting Nanoplatform for the Therapy of Parkinson’s Disease by Modulating Mitochondrial Dysfunction

Abstract

Background: Mitochondrial dysfunction in microglia is an important pathogenic factor inducing the onset of Parkinson’s Disease (PD). To address this challenge, a novel hierarchical nano-delivery system was developed to deliver a PD therapeutic agent, wedelolactone (WED) to modulate mitochondrial dysfunction. Methods: The nano-delivery system (WED@RBCm-B6&RAP12-NPs) was coated with red blood membrane (RBCm) to avoid immune clearance and conjugated with the BBB-penetrating peptide CGHKAKGPRK (B6) and the microglia targeting peptide EAKIEKHNHYQK (RAP12). Results: The experimental results demonstrated that this novel nano-delivery system could increase its half-life in blood circulation effectively via evading immune recognition and clearance and enhanced its brain distribution by synergistic effect of B6 and RAP12. By specifically targeting microglia in PD mouse brain, the system increased pyruvate dehydrogenase (PDH) activity, leading to mitochondrial structural repair, reduced secretion of pro-inflammatory cytokines, and improved the inflammatory microenvironment. Conclusions: The result first designed and synthesis a dual targeting drug delivery system WED@RBCm-B6&RAP12-NPs which significantly alleviated mitochondrial dysfunction and warranted further study to develop therapeutic agent for PD treatment.

1. Introduction

Parkinson’s disease (PD) ranks as the second most prevalent neurodegenerative disorder worldwide [1,2], and is primarily identified by the progressive loss of dopaminergic neuronal cells within the substantia nigra [3,4]. To this day, there are no reliable curative therapies available, most notably due to the blood–brain barrier (BBB) that enforces stringent regulatory constraints on how molecules move from systemic circulation into the central nervous system [5]. Furthermore, the majority of pharmacological agents demonstrate limited target specificity and insufficient BBB penetration, posing a substantial hurdle to the advancement of workable clinical therapies for PD [6,7].

In recent years, nanoparticles (Nps) have demonstrated remarkable advantages, including the ability to cross the BBB, active targeting capabilities, high drug-loading capacity, favorable stability, and low toxicity [8]. The surface of red blood cell membrane (RBCms) is enriched with CD47 protein, which specifically interacts with the SIRPα receptor expressed on macrophages. By coating nanoparticles with RBCm, a strategy referred to as “natural camouflage” can be employed to enable immune evasion, enhance biocompatibility, and substantially improve both the in vivo efficacy and safety profile of the nanoparticles [9,10,11]. Poly (lactic-co-glycolic acid) (PLGA) is an ideal polymeric carrier for nanoscale drug delivery systems, owing to its outstanding biocompatibility, controlled biodegradability, and tunable drug release kinetics. Peptide-mediated transport represents a critical mechanism of active targeting. Liu et al. demonstrated that the BBB-penetrating peptide CGHKAKGPRK (B6) exhibits a high affinity for the transferrin receptor (TfR). B6-modified nanoparticles display significantly enhanced accumulation in brain capillary endothelial cells via lipid raft-mediated and clathrin-mediated endocytosis, and also exhibit increased brain accumulation in vivo [12]. These findings suggest that B6 is a promising peptide capable of traversing the BBB, leveraging TfR as a selective target domain to drive nanoparticle translocation into the central nervous system [13,14,15]. Sun et al. demonstrated that the peptide EAKIEKHNHYQK (RAP12) demonstrates strong binding affinity toward lipoprotein receptor-related protein-1 (LRP1), a receptor that is abundantly expressed on microglial cells. The uptake of nano-biological signal proteins modified with RAP12 by microglial cells was significantly enhanced, indicating the specific targeting capability of RAP12 toward microglia [16].

Microglia are now understood to carry out an essential mediating role in the pathological progression of neurodegenerative illnesses by undergoing morphological transformation [17,18,19]. Upon activation, microglia exhibit distinct phenotypes: one is associated with the release of pro-inflammatory mediators, including TNF-α, iNOS, and IL-1β, whereas the other exerts neuroprotective effects through endogenous cellular secretion of anti-inflammatory effector molecules, such as Arg-1, IL-10, and TGF-β [20,21,22]. Research by Xin Zhong et al. has shown that mitochondrial impairment within microglial cells can amplify cellular secretion of pro-inflammatory effector cytokines, notably TNF-α, iNOS and IL-1β [23]. Pyruvate dehydrogenase (PDH) complex, a protein assembly susceptible to phosphorylation and inactivation via pyruvate dehydrogenase kinase (PDK), serves as a key regulatory enzyme in mitochondrial energy metabolism [24]. Therefore, targeting PDH to restore mitochondrial function and regulating microglial phenotypic polarization acts as a viable prospective therapeutic approach for neurodegenerative diseases.

Wedelolactone (WED) is primarily extracted from plants belonging to the Asteraceae family, such as Achyranthes bidentata and plants of the Eclipta genus. Research by Shruti et al. demonstrated that WED enhanced mitochondrial function by attenuating oxidative stress, thereby ameliorating PD in a Caenorhabditis elegans model. Furthermore, WED supplementation has been shown to significantly decrease levels of neutral lipids and triglycerides in PD patients, as well as reduce protein carbonyl content [25]. Previous studies from our laboratory had revealed that Erzhi Pill can enhance the activity of tyrosine hydroxylase (TH), serving as a key biomarker for PD progression. More importantly, WED had been identified as one of the major bioactive constituents responsible for the therapeutic effects of the Erzhi pill [26]. Despite its therapeutic potential, WED exhibited extremely low oral bioavailability (with a peak plasma concentration of only 15.22 mg/L following oral administration of 50 mg/kg in rats) [27], and its brain concentration was less than one-tenth of that observed in the liver [28]. The limited capacity of WED to traverse the BBB is considered the primary factor restricting its therapeutic efficacy.

As such, within this current research, a newly engineered nanodelivery system has been constructed (WED@RBCm-B6&RAP12-NPs). The system was constructed using poly PLGA nanoparticles as the core structure, which were coated with RBCm to evade immune phagocytosis and extend the duration of drug circulation in the bloodstream. Furthermore, the surface of RBCm was functionalized with B6 and RAP12 peptides to enhance BBB penetration and target specificity, thereby enabling efficient delivery of WED to microglial cells in the brain. The therapeutic effects of the nano-delivery system on microglial modulation and pathological outcomes were subsequently evaluated in PD mouse models.

2. Materials and Methods

2.1. Chemical Reagents

Detailed information is presented in

Table A1. Chemical reagents.

Chemical Reagents	Company	Catalog No
DSPE-PEG2000-Mal	Shanghai Yuanye Bio-Technology Co., Ltd.	474922-22-0
B6	China Peptide Co., Ltd., Hangzhou, Zhejiang, China	CGHKAKGPRK
RAP12	China Peptide Co., Ltd., Hangzhou, Zhejiang, China	EAKIEKHNHYQK
BCA Protein Concentration Assay Kit (Enhanced)	Beyotime Biotechnology (Shanghai, China)	P0009
RIPA Lysis Solution (strong)	Beyotime Biotechnology (Shanghai, China)	P0013B
Tanon™ Femto-sig ECL Chemiluminescent Substrate	Shanghai Tanon Science & Technology Co., Ltd., Shanghai, China	180-506
Frozen Section Embedding Agent (OCT, Clear)	Beyotime Biotechnology (Shanghai, China)	C0171A
HRP-conjugated goat anti-rabbit IgG (H+L) (affinity purified)	Beyotime Biotechnology (Shanghai, China)	A0216
Anti-β-actin antibody	Beyotime Biotechnology (Shanghai, China)	AF2815
Anti-IL-10 antibody	Biosynthesis Biotechnology Co., Ltd. (Beijing, China)	bs-0698R
Anti-TNF-α antibody	Biosynthesis Biotechnology Co., Ltd. (Beijing, China)	bs-10802R
Anti-IL-1β antibody	Biosynthesis Biotechnology Co., Ltd. (Beijing, China)	bs-0812R
Anti-iNOS antibody	Biosynthesis Biotechnology Co., Ltd. (Beijing, China)	bs-0162R
Anti-TGF-β antibody	Biosynthesis Biotechnology Co., Ltd. (Beijing, China)	bs-20413R
Anti-Arg-1 antibody	Biosynthesis Biotechnology Co., Ltd. (Beijing, China)	bs-100491P
Anti-TH antibody	Cell Signaling Technology (CST, Shanghai, China)	58844
Anti-IBA1 antibody	Cell Signaling Technology (CST, Shanghai, China)	17198
Anti-PDH antibody	Cell Signaling Technology (CST, Shanghai, China)	3820
Anti-PDH (Ser293) antibody	Cell Signaling Technology (CST, Shanghai, China)	37115
Anti-PDH (Ser232) antibody	Cell Signaling Technology (CST, Shanghai, China)	15289
Anti-PDH (Ser300) antibody	Proteintech Group, Inc. (Wuhan, China)	29583-1-AP

.

2.2. Cell Culture

BV2, hCMEC/D3, U-118MG, and RAW 264.7 cell lines were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and Guangzhou Saiweier Biotechnology Co., Ltd. (Guangzhou, China), respectively. All the cell lines were placed in a 37 °C, 5% carbon dioxide incubator for cultivation.

2.3. Preparation of Nanoparticle WED-NPs

WED (1 mg) was solubilized in 0.2 mL of DMSO, while PLGA (50:50, 2 mg) was dissolved in 0.8 mL of dichloromethane. The two solutions were then sonicated for 5 min to form the oil phase, which was slowly added dropwise into 4 mL of 0.5% porloxacinum aqueous solution (aqueous phase) under gentle shaking in an ice bath with ultrasonic treatment for 4 min. The mixture was further rotary evaporated for 20 min to remove the organic solvents and then solidified at −20 °C. After 10 min, nanoparticles were collected by centrifugation (4 °C, 12,000 rpm for 30 min).

FITC-NPs and DiR-NPs were prepared by labeling nanoparticles with fluorescein isothiocyanate or the near-infrared dye, respectively. In these formulations, WED was replaced with equimolar amounts of the corresponding fluorescent dye.

2.4. RBCm Derivation

Blood was collected from mice and then subjected to centrifugation (4 °C 2000 rpm for 15 min) in sodium heparin-coated tubes. The erythrocyte pellet was carefully removed and washed three times with pre-cooled PBS by centrifugation (4 °C 5000 rpm for 10 min). The erythrocytes were subsequently resuspended in a 40-fold volume of Tris-HCl buffer (0.01 mol/L) and incubated at 4 °C for 2 h to induce membrane rupture. Then, the RBCms suspension was washed by centrifugation (10,000 rpm for 10 min) until the pellet appeared milky white. The purified RBCms were resuspended in PBS and kept at −80 °C for subsequent.

2.5. Preparation of WED@RBC-B6&RAP12-NPs

DSPE-PEG-MAL-B6 and DSPE-PEG-MAL-RAP12 were synthesized via conjugation of DSPE-PEG-MAL with B6 and RAP12, respectively. Both reactions were carried out by dissolving B6 or RAP12 with DSPE-PEG-MAL in PBS (1:1.5). The reaction mixtures were stirred magnetically for 24 h at ambient room temperature under a nitrogen gas atmosphere, with light exclusion implemented to avert sample degradation. Subsequently, the unreacted components were removed through dialysis to ensure high purity of the final product. The purified products were then freeze-dried and analyzed by ¹H NMR spectroscopy (JEOL Ltd., Tokyo, Japan).

RBCms were subjected to sonication at a power of 200 W for 6 min. Erythrocyte vesicles (RVs) were generated by extruding the sonicated membranes through a microextruder (Avanti Polar Lipids, Alabaster, AL, USA) using polycarbonate filters.

A mixture of WED-NPs and RVs was sonicated at 200 W for 4 min and extruded for 10 cycles to form WED-RBC-NPs. DSPE-PEG-MAL-B6 and DSPE-PEG-MAL-RAP12 were then introduced to the WED-RBC-NPs and incubated in PBS for 30 min to yield WED@RBC-B6&RAP12-NPs. All formulations, including DSPE-PEG-MAL-B6, DSPE-PEG-MAL-RAP12, WED@RBC-NPs, WED@RBC-RAP12-NPs, WED@RBC-B6-NPs, and WED@RBC-B6&RAP12-NPs, were successfully prepared, lyophilized into powder form, and subsequently used for FT-IR analysis.

2.6. Characterization of the Nanosystems

The particle size (nm) was determined by dynamic light scattering (DLS) through analysis of the diffusion coefficient after appropriate dilution of the nanoparticle suspension. ζ-potential (mV) was measured using electrophoretic light scattering to assess the surface charge and colloidal stability of the particles. The morphological characteristics of WED@RBC-B6&RAP12-NPs were analyzed by TEM, Brookhaven Instruments Corporation, Holtsville, NY, USA.

RBCm protein concentration in the bionanoparticles and native erythrocyte membranes was assessed for protein concentration via a validated BCA protein assay kit, Beyotime Biotechnology, Shanghai, China. The protein profile of RBCm-coated nanoparticles was characterized through SDS-PAGE separation with subsequent visualization by Coomassie blue staining.

The encapsulation efficiency (EE) and drug loading (DL) of WED nanoparticles were quantified using HPLC. Separation was performed over a C18 analytical column, employing an acetonitrile-water mobile phase with 0.1% formic acid added (4:6, v/v) for fine-tuning the pH of the aqueous phase. Detection was carried out at a wavelength of 350 nm.

The protein conjugation efficiency was assessed using the BCA Protein Assay Kit. B6 and RAP12 peptides were separated from the nanoparticles via ultracentrifugation at 100,000× g for 30 min, following the instructions.

The WED formulations were loaded into pretreated dialysis bags and subsequently immersed in PBS maintained at 37 °C under continuous stirring. The PBS samples were analyzed by HPLC at predetermined time points to quantify the released WED.

The stability of WED@RBC-B6&RAP12-NPs was evaluated by monitoring particle size changes over 7 days. Particle size was measured at regular intervals using dynamic light scattering.

EE% = (Wtotal WED − free WED)/Wtotal WED × 100%

DL% = (Wtotal WED − Wfree WED)/Wtotal WED and carriers × 100%

2.7. Cell Viability Assay

BV2, hCMEC/D3 and U-118MG cells were pre-incubated for an initial 24 h period, with diverse WED formulations added to separate culture wells, followed by continued incubation under consistent culture conditions for a further 24 h. MTT solution was subsequently added to all wells, and the absorbance optical density was assessed at a wavelength of 490 nm.

2.8. Phagocytosis Assay

RAW 264.7 cells were seeded and incubated for 24 h to allow adherence. Subsequently, the cells were co-incubated with FITC-labeled nanoparticles for 2 h. For qualitative observation via fluorescence microscopy: after incubation, the culture medium was discarded, and cells were gently washed with pre-chilled PBS (3 times) to remove unbound nanoparticles. The cells were then fixed with 4% paraformaldehyde for 15 min at room temperature, followed by counterstaining with DAPI staining reagent (1 μg/mL) for 5 min in the dark. After washing with PBS to remove excess DAPI, the cells were observed and imaged under a fluorescence microscope (Olympus, Tokyo, Japan). Meanwhile, the cells were inoculated into 6-well plates and incubated with the aforementioned different nanoparticles for 90 min. Finally, the living cells were collected and resuspended in PBS, and quantitative analysis was performed using a flow cytometer (FCM).

2.9. Cellular Uptake

BV2, hCMEC/D3, and U-118MG cells were incubated and stained following the same procedure outlined in Method 2.8.

2.10. Cell Co-Culture BBB Model

hCMEC/D3 cells were seeded into the upper chamber of the Transwell insert with 2 × 10⁴ cells per well, while U-118MG cells were seeded into the lower chamber with 1 × 10⁴ cells per well. The Transwell system was incubated for 7 days at 37 °C in a humidified atmosphere containing 5% CO₂. TEER across the membrane was measured using a cell resistance meter to confirm the successful formation of the BBB model, and a time-dependent TEER curve was plotted accordingly.

2.11. Animals

Eight-month-old C57BL/6J mice were purchased from Jiangsu Jicere Pharmacogenetics Co., Ltd., Nanjing, China. Ethical approval was granted for all animal experimental procedures undertaken by the Animal Ethics Committee of Heilongjiang University of Traditional Chinese Medicine (Approval No.: 2024042623).

2.12. In Vivo Targeted Imaging Effects

Four hours after intravenous injection of DiR-labeled WED nanocapsules into the mice, in vivo fluorescence imaging was conducted with a small animal imager, followed by the collection of brains and organs for ex vivo imaging to evaluate biodistribution.

2.13. UPLC-MS/MS Analysis of WED in the Brain

C57BL/6J mice were randomly allocated into four separate experimental groups (6 animals per group). and administered WED, WED-NPs, WED-RBC-NPs, or WED-RBC-B6&RAP12-NPs via tail vein injection. One hour post-injection, the brain tissues of mice were homogenized in a fixed volume of distilled water (1:5, w/v) and the supernatants were collected. To each sample, 20 μL of luteolin (2 μg/mL) and 1 mL of methanol were added, followed by thorough vortex mixing. After a second centrifugation at 14,000× g for 15 min, the supernatants were transferred and evaporated to dryness under vacuum using a centrifugal concentrator. The residues were reconstituted in 150 μL of methanol and centrifuged again under the same conditions. The final supernatants were collected for UPLC-MS/MS analysis.

2.14. Behavioral Testing

C57BL/6J mice in the model group received daily intraperitoneal injections of MPTP (30 mg/kg, dissolved in 0.2 mL of physiological saline) for 10 consecutive days, whereas control animals were administered 0.2 mL of physiological saline via the same route on the same schedule. Starting on day 11, the treatment group received tail vein administration of API (167 μg/mL, 12 mL/kg), and the non-administered group received daily tail vein injections of saline as a control for 4 consecutive weeks [29,30,31]. Behavioral assessments were carried out using three standardized tests: (1) Mice were suspended by their front paws on a wire positioned 30 cm above the ground, and the latency to fall was recorded. (2) Rotarod test: Mice were trained at a fixed speed of 5 rpm for the first two days. On day three, they were placed on a rotor starting at 4 rpm, which gradually accelerated to 40 rpm. Each mouse’s residence time on the rotor was recorded. Three trials were conducted per mouse with 15 min intervals, and the average was calculated. (3) Balance beam test: Each mouse’s latency to cross a 1-meter beam section was documented across three separate test trials, with a 10 min rest interval between individual trials, and mean values were subsequently derived from these recordings. The procedure was conducted over three consecutive days, with the first two days designated for training.

2.15. ELISA

Equal masses of mouse brain tissue were homogenized in equal volumes of normal saline, followed by centrifugation to collect the supernatant. The experiments were performed in strict accordance with the manufacturer’s instructions, and absorbance measurements were collected via a microplate spectrophotometer.

2.16. Hematoxylin-Eosin Staining

Following behavioral testing, mice were humanely euthanized and dissected. Histological sections were prepared from the substantia nigra region of mouse brain tissue and from major organs (including heart, liver, spleen, lung, and kidney). Sections were deparaffinized, dehydrated, and stained with H&E. Then the slides were coverslipped and examined under a microscope.

2.17. Immunohistochemical Staining

After fixing the slices of the mouse substantia nigra with sodium citrate, hydrogen peroxide and goat serum blocking solution were applied, followed by incubation with TH primary antibody diluted at 1:500 for an overnight period. The next day, the sections were incubated with secondary antibody for 2 h. Afterward, the sections were rinsed again with PBS, chromogenic development was performed with DAB substrate for 2 min, and nuclear counterstaining was completed using hematoxylin stain over a 3 min incubation period. Then the slides were coverslipped and examined under a microscope.

2.18. Immunohistofluorescence Staining

Mouse brain tissues were embedded using a cryostat and sectioned to include the substantia nigra. Sections were incubated with 3% hydrogen peroxide for 10 min, blocked with goat serum containing 0.1% Triton X-100 for 2 h, and incubated overnight with PDH antibody (1:100 dilution). The following day, sections were incubated with fluorescent secondary antibody for 2 h, mounted with DAPI-containing solution, and imaged under a fluorescence microscope (Olympus, Tokyo, Japan).

2.19. Immunofluorescence Co-Localization

Inject DIR-labeled nanomedicine into mice, and the antibody used was Iba1 (1:200 dilution). The procedure followed the same protocol as described in Section 2.17. Immunofluorescence colocalization images were quantified using ImageJ 1.54f software.

2.20. Western Blotting

The substantia nigra homogenates from mouse brains were collected, and supernatants were obtained. The samples were followed by gel electrophoresis and transferred to PVDF membranes. Membranes were probed with primary antibodies targeting PDH, pS232-PDH, pS293-PDH and pS300-PDH (each diluted 1:500), followed by incubation with HRP-linked secondary antibody at a 1:1000 dilution ratio. Protein band immunoreactivity was detected through ECL chemiluminescent detection reagent, and blot imaging was conducted on the Tanon Chemi Doc ULTRA RGB imaging system. Target protein expression levels were quantified by normalizing band intensities to β-actin using ImageJ software (v1.54f).

2.21. Mitochondrial Electron Microscope

Mice were euthanized, and brains were immediately excised and placed on ice. The substantia nigra was microdissected and cut into tissue blocks of approximately 1 mm³. Hippocampal tissue specimens from mice were subjected to cold fixation with 2.5% glutaraldehyde at 4 °C for an overnight incubation, then processed for secondary fixation using 1% osmium tetroxide solution over a 1–2 h period. Tissues were then dehydrated through a graded ethanol series (30–100%) and transferred to acetone. Finally, they were embedded in epoxy resin and sectioned. Sections were mounted on copper grids and doubly stained with uranyl acetate and lead citrate to enhance electron contrast. Mitochondrial ultrastructure in microglia of the substantia nigra was examined by transmission electron microscopy.

2.22. Statistical Analysis

Experimental data are presented as mean ± SEM. Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test. Differences were considered statistically significant at p < 0.05.

3. Results

3.1. Characterization of Nanocarriers

The schematic diagram of WED@RBC-B6&RAP12-NPs preparation, as presented in Figure 1a and Figure A1, confirms that the successful conjugation of WED@RBC-B6&RAP12-NPs, FT-IR and ¹H NMR analyses were performed. ¹H NMR analysis demonstrated the absence of characteristic peaks corresponding to the sulfhydryl group region (1.6–2 ppm) in DSPE-PEG-Mal-B6 and DSPE-PEG-Mal-RAP12 (Figure A2), indicating successful conjugation between DSPE and the peptide chains to form DSPE-PEG-Mal-B6 and DSPE-PEG-Mal-RAP12. FT-IR analysis further demonstrated that the SH stretching vibration peak at approximately 2551 cm⁻¹ was absent in WED@RBC-NPs (Figure A3), suggesting that PEG had undergone nucleophilic substitution with the peptides. These findings confirmed that DSPE-PEG-Mal-B6 and DSPE-PEG-Mal-RAP12 were successfully conjugated to WED@RBC-NPs.

To evaluate the effects of RBCm, B6 peptide, and RAP12 peptide on WED-NPs, TEM morphology and zeta potential measurements were conducted. TEM images revealed that all nanoparticles exhibited smooth surfaces, regular spherical shapes, and suitable particle sizes (Figure 1b,c), with diameters below 220 nm (

Table A2. Characterization of different nanoparticle formulations.

Formulation	Particle Diameters (nm)	Zeta Potential (mV)	EE%	DL%
WED-NPs	185.93 ± 5.70	−6.00 ± 0.87	85.09 ± 0.39	25.97 ± 0.32
WED@RBC-NPs	192.18 ± 7.30	−7.83 ± 1.31	84.33 ± 0.60	24.25 ± 0.18
WED@RBC-B6&RAP12-NPs	215.23 ± 6.87	−9.87 ± 0.87	84.11 ± 0.26	23.12 ± 0.35

). The zeta potential analysis determined that the ζ-potential of WED@RBC-B6&RAP12-NPs was slightly lower than that of WED@RBC-NPs, suggesting that the modification with B6 and RAP12 was facilitated by electrostatic attraction. Moreover, WED@RBC-B6&RAP12-NPs exhibited good stability in PBS solution at 4 °C (Figure 1d), indicating that RBCm, B6, and RAP12 did not significantly affect the physical stability of WED-NPs.

To assess the integrity of RBCm, protein profiles of different samples were analyzed by SDS-PAGE. Distinct RBCm protein bands were observed in RVs, WED@RBC-NPs, WED@RBC-B6-NPs, WED@RBC-RAP12-NPs, and WED@RBC-B6&RAP12-NPs compared to WED-NPs (Figure 1e). Covalent conjugation efficiency was determined by the BCA protein assay. The attachment efficiencies of B6 and RAP12 peptides on RBC-NPs were 92.58% and 89.52%, respectively, with peptide densities of 4.62% and 5.25% (w/w), indicating successful encapsulation of RBCm and effective conjugation of B6 and RAP12 without interference between the peptides.

In vitro release profiles of WED and WED-loaded nanodrugs were evaluated to investigate their drug release characteristics. As shown in Figure 1f, the release rates of the three nanoparticle formulations were significantly slower compared to free WED. However, WED-NP and RBCm-WED-NP showed no significant release profile difference, as RBCm only served as an outer shell on WED-NP without altering its core structure. This physical coating did not affect the core’s drug release-related physicochemical properties and drug diffusion channels, thus ensuring consistent drug release for both formulations. Moreover, drug release in the free WED group was driven by concentration gradient-dependent free diffusion, yielding a cumulative release rate exceeding 80% at 10 h. In contrast, the presence of PLGA generated diffusion resistance in the nanoparticle formulation group, giving a cumulative WED release rate of no more than 60% at 10 h. The slow hydrolysis of PLGA also drastically slowed the diffusion of the encapsulated WED. These results demonstrated that the WED-loaded nanomedicines exhibited sustained release behavior, which was beneficial for enhancing drug accumulation at the target site.

3.2. Long Cycle Characterization of RBCm

The anti-phagocytic and long-circulation properties of nanomedicine were assessed using RAW 264.7 cells. As shown in Figure 2a, the fluorescence intensity of nanoparticles without RBCm coating was significantly higher than that of RBCm-coated nanoparticles. As shown in Figure 2e, the results of flow cytometry indicated that the fluorescence of the nanoparticle group coated with RBCms was significantly lower than that of the uncoated group. The possible reason is that the RBCm releases the “don’t eat me” signal CD47, which protects the nanoparticles from being phagocytosed by RAW246.7. These results demonstrate that RBCm coating inhibits nanoparticle uptake by RAW 264.7 macrophages, which is beneficial for immune evasion.

3.3. Safety Evaluation

3.3.1. Cytotoxicity

The cytotoxicity of WED and WED nanodrugs was determined by MTT assay. Co-incubation of hCMEC/D3, U-118MG and BV2 cells with different concentrations of free WED and WED-containing nanomedicines, respectively. As shown in Figure A4, the cell viability of all three cell lines remained within the range of 88% to 97% after incubation. The cell survival of nanodrugs was slightly lower than that of free drugs, but the difference was relatively small, especially at low concentrations. In summary, the toxicity of nanocarriers is negligible.

3.3.2. Pathological Safety Assessment

The pathological changes in major organs—including the heart, liver, spleen, lung, and kidney—of mice treated with nanomedicine were assessed by H&E staining to evaluate the biocompatibility and safety of the nano-materials. The results, as shown in Figure A5, showed that none of the HE staining results of the organs of the drug-treated mice showed obvious tissue damage or lesions, which was consistent with the blank group. This result indicated that the drug treatment has good biological safety.

3.4. Targeted Capability Verification

3.4.1. Cellular Uptake

To evaluate the drug targeting capability, hCMEC/D3, U-118MG, and BV12 cells were incubated with FITC-labeled nanomedicines, and the results are presented in Figure 2b–d. The RBC-B6&RAP12-NPs group exhibited significantly higher cellular uptake across all cell lines than the other nanomedicine groups. As shown in Figure 2e, Cell uptake was quantified by flow cytometry. The results showed that the fluorescence intensity of the nanoparticle groups linked with targeting peptides was significantly different from that of the Nps-FITC group in the three types of cells. Moreover, the RBC-B6&RAP12-NPs-FITC group had the strongest fluorescence intensity in the uptake of the three types of cells. In the uptake of hCMEC/D3 and U-118MG cells, the fluorescence of the RBC-B6-NPs-FITC group was higher than that of the RBC-RAP12-NPs-FITC group. The main reason is that the B6 peptide can specifically bind to the LRP1 receptor, which is highly expressed in hCMEC/D3 and U-118MG cells. However, in BV2 cells, the uptake fluorescence intensity of the RBC-RAP12-NPs-FITC group was close to that of the dipeptide group. The possible reason might be that the RAP12 peptide played a significant role in the uptake of BV2 cells. Collectively, these results demonstrated cell-type-specific uptake: B6/RAP12 co-modification enhances nanoparticle internalization in endothelial and glioma cells, while RAP12 drives targeted uptake in BV2 microglia and B6 mediates microglial immune evasion—supporting the brain-targeting potential of the co-engineered nanosystem.

3.4.2. BBB Transport

The structural integrity of the BBB (Figure 2g) was assessed via transendothelial electrical resistance (TEER) measurements, while nanoparticle delivery efficiency was determined by collecting the culture medium from the BBB model and quantifying fluorescence intensity at predetermined time points. As shown in Figure A6, the TEER value of the BBB model on the fourth day was 183.55 ± 0.378 Ω·cm; this indicated gradual maturation of the BBB barrier and a concomitant enhancement in its structural integrity over the culture period. All nanomedicine transport rate assays were performed on day 4, when the BBB exhibited maximal structural integrity. As presented in Figure 2f, fluorescence intensity quantification revealed a time-dependent increase in the transport rate of all four nanomedicines across the BBB: The transport rate of Nps-FITC was 5.52% at 12 h. RBC-Nps-FITC achieved a transport rate of 4.72% at 12 h, and notably, RBC-B6&RAP12-NPs-FITC exhibited a transport rate of 33.46% at 12 h—this value was significantly higher than those of the other three groups. The slightly lower transport rate of RBC-Nps-FITC compared with Nps-FITC was likely due to the modest increase in nanoparticle size caused by RBC membrane coating.

Collectively, these results confirmed that the dual-peptide-modified biomimetic nanosystem has markedly enhanced BBB penetration capacity and improved drug delivery efficiency relative to the other formulations.

As shown in Figure 3a, nanomedicine effectively crosses the BBB to specifically target microglia, demonstrating its potential for precise central nervous system delivery. To assess brain targeting, mice were injected with DIR-labeled nanoparticles and monitored for in vivo fluorescence distribution. As illustrated in Figure 3b, the brain fluorescence intensity in the RBC-B6&RAP12-NPs-DiR group was significantly greater than that in the other groups. To facilitate a more precise comparison of DiR accumulation in the brain, brain tissues were excised and subjected to ex vivo imaging analysis. The results demonstrated a significantly enhanced DiR signal in the brain following administration of the B6&RAP12-modified nanosystems (Figure 3c,e). Additionally, organs and brains were harvested from mice administered with Dir-labeled nanoparticles for subsequent imaging analysis. As shown in Figure 3d, the nanocarriers were predominantly distributed in the liver and spleen, indicating that these organs were the primary sites of metabolic clearance. Minimal fluorescence signals were detected in the heart and kidneys. As shown in Figure 3f, following intravenous administration via the tail vein, the biodistribution analysis of WED in the murine brain demonstrated that the concentration of WED delivered by WED@RBC-B6&RAP12-NPs was significantly higher than that of free WED. Compared with free WED, the brain levels of WED derived from WED@RBC-NPs and WED-NPs were moderately elevated. These findings collectively suggested that the synergistic action of B6 and RAP12 peptides enabled the nanocarriers to effectively cross the BBB and accumulate in the brain, thereby achieving superior brain-targeting performance.

3.4.3. In Vivo Validation of Targeted Microglia

To assess the microglia-targeting efficacy of the nanocarriers, DIR-labeled nanopreparations were intravenously administered via tail vein injection in mice, and the targeting performance was evaluated using immunofluorescence co-localization analysis. As shown in Figure 3g, the co-localization of drug fluorescence (red) with microglial markers (green) was significantly enhanced in the WED@RBC-B6&RAP12-NPs-DiR group compared to other treatment groups. As shown in Figure 3h, quantitative results further confirmed that the WED@RBC-B6&RAP12-NPs-DiR group exhibited the highest fluorescence colocalization efficiency between microglia and nanomedicines. Among the single peptide-modified groups, the colocalization efficiency of the WED@RBC-RAP12-NPs-DiR group was significantly higher than that of the WED@RBC-B6-NPs-DiR group, which may be attributed to the stronger targeting affinity of the RAP12 peptide for microglia. In addition, the WED@RBC-NPs-DiR group showed relatively low fluorescence colocalization efficiency, while the WED-NPs-DiR group had the lowest efficiency. These results demonstrated that the WED@RBC-B6&RAP12-NPs-DiR nanocarrier system could effectively traverse the BBB and specifically target microglial cells.

3.5. Behavioral Assessment

The timeline for mouse model establishment and drug administration is presented in Figure 4a. As shown in Figure 4b, the behavioral schematic illustrates the experimental design. Muscle strength and motor function were assessed using the suspension test (Figure 4c). Mouse motor coordination and balance were assessed using the rotarod and balance beam tests (Figure 4d,e). The results indicated that the suspension time and fall latency in the model group were markedly shorter relative to both the control and treatment groups, while the time required to traverse the balance beam was markedly increased. These impairments were alleviated following drug administration, with the WED@RBC-B6&RAP12-NPs group showing the most pronounced improvement. These findings demonstrated that WED effectively restored motor function and balance in the mouse model.

3.6. Reversing Neuroinflammation

To explore the anti-neuroinflammatory effects and underlying mechanisms of WED, PDH protein expression and inflammatory responses were analyzed using Western blot and ELISA (Figure A7). As shown in Figure 5a, PDH expression was markedly reduced in the model group compared to the control group, while phosphorylated PDH isoforms (PDHSer300, PDHSer293, and PDHSer232) were markedly increased. Immunofluorescence analysis further confirmed that PDH fluorescence intensity was markedly decreased in the model group and was restored following nanomedicine treatment (Figure 5b). Furthermore, transmission electron microscopy analysis (Figure 5b) revealed that mitochondria in the substantia nigra tissue of mice in the blank group exhibited a typical double-ellipsoidal shape, with intact outer membranes and a regular, dense arrangement of inner membrane cristae. No ultrastructural abnormalities—such as swelling, vacuolization, or membrane rupture—were observed. In contrast, mitochondria in the model group generally showed significant swelling, local rupture of the outer membrane, dissolution or disappearance of inner membrane cristae, and vacuolar changes, indicating severe impairment of mitochondrial structural integrity. This pathological alteration was markedly ameliorated following nano-drug treatment, with the WED@RBC-B6&RAP12-NPs group exhibiting the most pronounced structural restoration. Consistent with these findings, Western blot (Figure 5a) and ELISA (Figure A6) data revealed that pro-inflammatory cytokines (iNOS, TNF-α, IL-1β) were markedly upregulated in the model group, while anti-inflammatory markers (Arg-1, TGF-β, IL-10) were downregulated. The condition showed improvement following drug administration. These findings demonstrate that WED exerts neuroprotective effects by inhibiting PDH phosphorylation, thereby restoring mitochondrial structure and attenuating neuroinflammatory responses (Figure 5b).

3.7. PD Treatment Effectiveness Assessment

To evaluate the therapeutic potential of PD, the morphological characteristics of mouse substantia nigra tissue and TH levels were analyzed. Immunohistochemical analysis revealed that, compared with the control group, the TH optical density in the model group was significantly decreased, and this reduction was markedly reversed following nanomedicine treatment (Figure 5b). HE staining showed that neurons in the substantia nigra were well-organized, densely packed, and exhibited regular morphology and uniform distribution without evident neuronal damage in the control group. In contrast, the model group displayed disorganized neuronal architecture, shrunken nuclei, intensified nuclear staining, and pronounced neuronal damage. Following nanomedicine administration, the neuronal arrangement was notably improved compared to the model group, with a reduction in nuclear shrinkage and a mitigation of neuronal damage (Figure 5b). These findings confirmed that WED effectively alleviated neuronal injury and contributed to the therapeutic management of PD (Figure 5c).

4. Discussion

To address the key challenges in the clinical treatment of PD—poor BBB penetration, insufficient lesion targeting, and limited therapeutic efficacy—this study used WED as the model drug to design and construct a biomimetic nano-delivery system: WED@RBC-B6&RAP12-NPs. This system combines RBCm coating with dual-peptide modification (B6 and RAP12). Among them, RBCm-modified nanoparticles can prolong the circulation time in the blood, achieve immune escape, and PLGA features favorable biocompatibility and biodegradability with tunable drug release behavior, serving as an ideal carrier for nanoparticles. We conducted systematic investigations into the formulation’s preparation and characterization, targeting verification, in vivo efficacy evaluation, and mechanism of action, forming a complete research chain from formulation development to mechanism elucidation. This work offers new experimental evidence and strategic insights for the targeted treatment of PD and the central nervous system (CNS) delivery of active components from traditional Chinese medicine.

For the preparation and characterization of the biomimetic nano-delivery system, we successfully synthesized the targeting molecules DSPE-PEG2000-B6 and DSPE-PEG2000-RAP12, extracted and purified mouse RBC membranes, and optimized the preparation process through single-factor experiments. This yielded WED-NPs and WED@RBC-B6&RAP12-NPs with uniform particle size and regular morphology.

Characterization results showed the target formulation possessed ideal encapsulation efficiency and drug loading capacity, along with good in vitro stability and satisfactory dual-peptide conjugation efficiency. FT-IR and NMR analyses confirmed the successful conjugation of targeting molecules and effective coating of RBC membranes. In vitro release experiments demonstrated that the nano-formulation enabled sustained and slow release of WED, laying the foundation for its long-acting in vivo performance.

Targeting studies revealed that WED@RBC-B6&RAP12-NPs exhibited no significant cytotoxicity against BV2 microglia, hCMEC/D3 BBB endothelial cells, or U-118MG glioma cells, while their cellular uptake efficiency was significantly higher than that of unmodified or single-peptide modified nano-formulations. In vitro BBB model assays confirmed markedly enhanced BBB penetration efficiency of the nanosystem, with FITC-labeled nanoparticle transport rates rising slowly at 2, 4 and 6 h and peaking at 12 h. This time-dependent trend originates from the cumulative nature of transcellular transcytosis—the core pathway for nanoparticles to cross the intact BBB—since the recognition, uptake and transendothelial transport of nanoparticles by BBB endothelial cells form a gradual biological process whose cumulative effect is fully manifested at 12 h. Additionally, the system exhibits prominent long-circulating properties, which mitigate non-specific phagocytosis by the reticuloendothelial system. In vivo bioimaging and biodistribution experiments further confirmed that the formulation significantly increased brain-drug accumulation and reduced non-specific distribution in organs such as the liver and spleen. Immunofluorescence colocalization assays clearly demonstrated its precise targeting of microglia in the substantia nigra of PD mice, providing core support for precise drug action at lesion sites.

In vivo efficacy evaluation was performed using PD mouse models. Results showed that model mice exhibited typical PD-related behavioral abnormalities: significantly shortened rotarod fall time, prolonged beam walking time, and reduced tail suspension immobility time. Pathologically, neurons in the substantia nigra showed shrinkage and degeneration, with a significant decrease in dopaminergic neuron count, confirming stable and reliable model establishment. After administration, all treatment groups alleviated the aforementioned pathological and behavioral abnormalities to varying degrees. The WED@RBC-B6&RAP12-NPs group showed the most prominent improvements, with mouse phenotypes approaching normal levels. All three behavioral indicators were significantly superior to those of the WED group, WED-NPs group, and WED@RBC-NPs group. Neuronal morphology in the substantia nigra was largely restored, and dopaminergic neuron counts rebounded substantially. Safety evaluations confirmed that at the experimental dose, all formulations caused no significant toxic damage to the major organs of mice, indicating good in vivo biocompatibility.

Mechanistic investigations focused on targeted regulation, energy metabolism, and the inflammatory microenvironment. Results demonstrated that WED@RBC-B6&RAP12-NPs achieved precise targeting of microglia in the substantia nigra through dual-peptide (B6 and RAP12) modification, enhancing local drug accumulation efficiency. Additionally, the formulation significantly restored the normal expression of PDH in the substantia nigra of PD mice, inhibited its excessive phosphorylation, improved mitochondrial morphological abnormalities, and repaired mitochondrial dysfunction to restore energy metabolic balance. Furthermore, the formulation significantly downregulated the expression of pro-inflammatory cytokines (TNF-α, iNOS, IL-1β) and upregulated anti-inflammatory cytokines (TGF-β, Arg-1, IL-10) in the substantia nigra, effectively balancing the local inflammatory microenvironment and reducing inflammatory damage to dopaminergic neurons. Ultimately, through a synergistic mechanism—“precise microglial targeting → regulating the PDH pathway to improve mitochondrial function → balancing the inflammatory microenvironment”—the formulation protects dopaminergic neurons and alleviates motor deficits in PD mice.

The synergistic delivery system constructed in this study—integrating RBC membrane biomimetic coating and dual-peptide targeted modification—simultaneously achieves long-circulating drug properties, efficient BBB penetration, and precise targeting of microglia in the substantia nigra. It significantly enhances the brain delivery efficiency and anti-PD efficacy of WED. Moreover, this study is the first to clarify the synergistic anti-PD mechanism of WED by regulating the PDH pathway, mitochondrial function, and inflammatory microenvironment. This work not only provides a new formulation with clinical translation potential for the targeted treatment of PD but also offers novel ideas and experimental support for the modern development of active components from traditional Chinese medicine and targeted delivery research for CNS diseases. A key limitation of this study is that the PD mouse model cannot fully replicate the complexity of human PD, and the translational potential of WED@RBC-B6&RAP12-NPs thus requires further validation in more clinically relevant models.