Summary
Blood–mind barrier (BBB) disruption and extreme neuroinflammation are pivotal drivers of cerebral ischemia-reperfusion harm. Though simvastatin (SV) possesses potent pleiotropic results in selling BBB restore and attenuating irritation, its scientific translation for ischemic stroke is severely hampered by poor BBB penetration, low lesion accumulation, and the want for excessive systemic doses that enhance the threat of off-target toxicity (e.g., myopathy). To deal with these boundaries, herein, we developed a platelet membrane–biomimetic nanoparticle system (pmPLGA@SV) designed to leverage the innate affinity of platelets for injured vasculature for lesion-targeted SV delivery at a decrease efficient dose. Physicochemical characterization confirmed the profitable cloaking of platelet membranes onto SV-loaded PLGA cores. In vitro, pmPLGA@SV demonstrated superior therapeutic versatility: it successfully scavenged reactive oxygen species in oxygen glucose deprivation/reoxygenation-treated PC12 cells and orchestrated microglial repolarization from a pro-inflammatory M1 phenotype towards an anti-inflammatory M2 state in BV2 cells, considerably modulating the secretion of IL-1β and IL-10. In vivo, pmPLGA@SV treatment dramatically diminished the cerebral infarct quantity, exhibiting vital superiority over free SV. Moreover, longitudinal behavioral assessments over 21 days demonstrated that pmPLGA@SV markedly accelerated motor and sensory-motor practical restoration, accompanied by constant physique weight regain and improved neurological scores. Mechanistically, pmPLGA@SV facilitates a synergistic therapeutic method by mitigating neuronal oxidative stress and reworking the inflammatory microenvironment. This examine demonstrates that pmPLGA@SV serves as a strong biomimetic platform for the built-in treatment of neurovascular unit injury, providing a promising technique for long-term neuroprotection and practical rehabilitation following ischemic stroke.
Quotation: Li Q, Li R, Lin L, Gong M, Liang Y, Sui D (2026) Platelet membrane biomimetic nanoparticle-based targeted delivery system of simvastatin for the treatment of ischemic stroke. PLoS One 21(7):
e0354184.
https://doi.org/10.1371/journal.pone.0354184
Editor: Anisha DSouza, Mass Eye Infirmary, Harvard Medical College / Northeastern College, UNITED STATES OF AMERICA
Acquired: March 18, 2026; Accepted: July 3, 2026; Revealed: July 23, 2026
Copyright: © 2026 Li et al. That is an open entry article distributed beneath the phrases of the Creative Commons Attribution License, which allows unrestricted use, distribution, and copy in any medium, supplied the unique creator and supply are credited.
Information Availability: All related knowledge are inside the manuscript and its Supporting Information recordsdata.
Funding: This examine was supported by A hospital-level analysis undertaking at the Normal Hospital of the Western Theater Command (No. 2024-YGLC-B18 to DS). The funders had no function in examine design, knowledge assortment and evaluation, determination to publish, or preparation of the manuscript.
Competing pursuits: The authors have declared that no competing pursuits exist.
Introduction
IIschemic stroke is a serious trigger of world mortality and long-term incapacity worldwide. It’s characterised by the sudden interruption of blood circulate to the mind, leading to neural tissue dying [1,2]. Though well timed reperfusion by pharmacological thrombolysis or mechanical thrombectomy is the scientific gold commonplace, its software is severely restricted by a slender therapeutic window and the threat of ischemia–reperfusion harm [3,4]. Accumulating proof means that the main ischemic insult is quickly adopted by a posh cascade of secondary accidents, together with blood–mind barrier (BBB) disruption and uncontrolled neuroinflammation, which considerably exacerbate neurological deficits and impede practical restoration [5].
The neurovascular unit (NVU), comprising neurons, endothelial cells, and glial cells, performs a vital function in sustaining mind homeostasis [6,7]. Submit-stroke, the overproduction of reactive oxygen species (ROS) instigates vital oxidative injury to neurons and exacerbates the subsequent neuroinflammatory cascade [8,9]. Concurrently, microglia, the resident immune cells of the mind, are activated and polarized into distinct phenotypes: the pro-inflammatory M1 phenotype, which secretes deleterious cytokines like IL-1β, and the anti-inflammatory M2 phenotype, which promotes tissue restore and neuroprotection [10,11]. Consequently, modulating microglial polarization from M1 to M2 and mitigating neuronal oxidative stress have emerged as promising synergistic methods for stroke remedy [12,13].
Simvastatin (SV), a broadly used HMG-CoA reductase inhibitor, has gained vital consideration for its potent pleiotropic results past ldl cholesterol decreasing [14–17]. Current research have highlighted its capability to attenuate oxidative stress, stabilize the BBB, and orchestrate immunomodulatory responses in numerous neurological problems [18,19]. Nonetheless, the scientific translation of SV for the treatment of acute ischemic stroke is severely hampered by its suboptimal pharmacokinetic profile, which incorporates low aqueous solubility, fast systemic clearance, and significantly poor penetration throughout the BBB [20,21]. As a way to obtain the requisite therapeutic concentrations inside the ischemic penumbra, excessive systemic doses are continuously required. Nonetheless, this inevitably will increase the threat of unwanted effects, together with myopathy and hepatic dysfunction [22,23]. These points spotlight the pressing want for a targeted delivery technique that may focus SV at the ischemic lesion whereas lowering systemic publicity [20,21].
To beat these delivery boundaries—particularly the poor BBB penetration and inadequate lesion accumulation that necessitate excessive systemic doses of SV—nanotechnology-based platforms have been developed to boost drug stability and focusing on [24,25]. Amongst these, biomimetic camouflaging—particularly the use of platelet membranes (pm)—represents a paradigm shift in drug delivery [26,27]. Platelets possess an inherent organic tendency to stick to injured vascular endothelium and inflammatory websites, a property that’s ruled by particular floor receptors [28,29]. By cloaking SV-loaded Poly lactic-co-glycolic acid (PLGA) nanoparticles with platelet membranes (pmPLGA@SV), it’s potential to bypass immune surveillance, delay systemic circulation, and obtain lesion-specific drug accumulation by lively homing mechanisms, thereby maximizing therapeutic efficacy whereas minimizing off-target toxicity [30,31]. Consequently, this biomimetic platform permits SV to exert its pleiotropic results at a considerably decrease efficient dose, circumventing the high-dose toxicity that has restricted its scientific software in acute stroke.
In the current examine, we developed a platelet membrane–biomimetic nanoparticle system (pmPLGA@SV) for the targeted treatment of ischemic stroke. We hypothesized that this biomimetic platform may successfully ship SV to the ischemic mind, concurrently mitigating oxidative stress and reworking the inflammatory microenvironment. In vitro, we investigated the ROS-scavenging functionality of pmPLGA@SV in oxygen glucose deprivation/reperfusion (OGD/R)-treated PC12 cells, in addition to its impression on M1/M2 polarization in BV2 microglia. In vivo, utilizing a mouse mannequin of transient center cerebral artery occlusion (tMCAO), we comprehensively evaluated its results on infarct quantity discount and, critically, on long-term neurological practical restoration. This examine supplies a strong technique for integrating biomimetic nanotechnology with pleiotropic drug remedy to advertise neurovascular restoration and practical rehabilitation.
Supplies and strategies
Supplies
PLGA (75:25 MW = 15000) was bought from Jinan Daigang Biomaterial Co., Ltd. (Jinan, China). Acetone was bought from Adamas Reagent, Ltd. (Shanghai, China). Reagents for western blot evaluation, together with RIPA lysis buffer, protein loading buffer, and TBST (Tris-buffered saline with Tween® 20) had been bought from GenScript Biotech Company (China).
Cell strains and tradition circumstances
Mouse microglia BV2 cells, mouse mind capillary endothelial bEnd. 3 cells, and PC12 paraganglioma cell strains had been obtained from iCell Bioscience Inc (Shanghai, China). BV2 and bEnd.3 had been cultured in DMEM (Gibco) containing 10% fetal bovine serum (FBS) (ExCell Bio) at 37℃ with 5% CO2. PC12 cells had been cultured in 1640 medium (Gibco) with 10% FBS.
Animals
C57BL/6J mice (25 ~ 30 g, male) had been bought from Gempharmatech Co., Ltd. (Nanjin, China; license: SCXK (Chuan) 2020−034) had been utilized in the experiments. All animals had been maintained beneath 12 h light-dark cycle at 25 ± 1℃ and 50% ~ 60% humidity with entry to meals and water advert libitum. All experiments strictly adopted the 3R ideas for the use of experimental animals and provision of humane care. The strategy of euthanasia was carried out in accordance with the AVMA Tips for the Euthanasia of Animals (2020 version) and is detailed in the Tissue assortment and sacrifice subsection beneath.
Characterization of pmPLGA@SV
PLGA nanoparticles had been ready by solvent evaporation methodology. 50 mg PLGA had been dissolved in 5 mL acetone, added dropwise into 50 mL water, and stirred for 4 h at room temperature. The spherical nanoparticles could possibly be obtained with the evaporation of acetone. For simvastatin-loaded nanoparticles, the drug and PLGA had been dissolved in acetone at a weight ratio of 1:10 (SV: PLGA). The answer was then added dropwise into water beneath stirring. The ensuing nanoparticle suspension was dialyzed towards 1 L of distilled water utilizing a dialysis membrane with a molecular weight cutoff (MWCO) of 3.5 kDa at 4℃ for 24 h, with water modified each 6 h, to take away unloaded simvastatin and residual acetone.
To organize platelet membrane-coated nanoparticles, 1 mL of mouse complete blood was first centrifuged (1,500 × g, 15 min) to isolate platelet-rich plasma. Platelets had been then collected and lysed through probe sonication (4℃, 10 × 30 s pulses) to yield membrane vesicles. The vesicles had been resuspended in 1 mL of PBS and mixed with 1 mL of PLGA suspension (1 mg/mL). The combination was subsequently subjected to co-extrusion by 200 nm polycarbonate membranes for 10 cycles to acquire pmPLGA nanoparticles. pmPLGA@SV was ready following an identical protocol.
The particle measurement, PDI and zeta potential of the nanoparticles had been decided by dynamic mild scattering (DLS) (Zeta-Sizer, Malvern Nano-ZS90, Malvern, Ltd., UK). The morphology of nanoparticles was examined by transmission electron microscopy (TEM, TECNAI G2F20FEI, USA). The drug loading content material (LC%) and entrapment effectivity (EE%) of the nanoparticles had been detected by UV-vis (UV-2550 Shimadzu，Japan). DL and EE had been calculated by the following components:
The place m is the mass (mg) of simvastatin encapsulated in the nanoparticles, mA is the complete mass of the drug-loaded nanoparticles, and mR is the preliminary mass (mg) of simvastatin utilized in the preparation.
Zeta potential measurement: Zeta potential of PLGA@SV and pmPLGA@SV was measured at 25℃ utilizing a Zetasizer (Malvern Nano-ZS90) after dilution in PBS (pH 7.4). Three impartial batches had been measured, every in triplicate.
Stability evaluation: Freshly ready pmPLGA@SV was saved as a sterile suspension in PBS (pH 7.4) at 4℃. Aliquots had been taken at 0, 1, 3, 7, 14 d to measure encapsulation effectivity (EE%) and hydrodynamic diameter.
In vitro launch examine: One milliliter of pmPLGA@SV suspension (1 mg/mL) was dialyzed towards 10 mL of PBS (pH 7.4, 0.1% Tween 80) at 37℃ with mild shaking (100 rpm). At predetermined time factors (0, 6, 12, 24, 48, 72 h), 1 mL of launch medium was withdrawn and changed with recent PBS. SV focus in the withdrawn samples was quantified by UV-vis. The cumulative launch share was calculated. All measurements had been carried out in triplicate.
Characterization of platelet membrane coating
To substantiate the profitable coating of platelet membrane proteins on nanoparticles, gel electrophoresis and western blot had been carried out. The nanoparticle answer was centrifuged (12,000 × g, 15 min) and lysed with RIPA buffer, and the supernatant containing membrane proteins was collected after a second centrifugation. Protein concentrations had been quantified by BCA assay and adjusted to 1 μg/μL. After denaturation, samples had been separated on a 12% polyacrylamide gel (80 V for 30 min, then 120 V for 45 min). For Coomassie Blue staining, gels had been destained till clear. For western blot, proteins had been transferred to a PVDF membrane at 400 mA for 20 min, blocked with 5% skim milk, and sequentially incubated with main antibody (in a single day, 4℃) and secondary antibody (2 h, room temperature). Alerts had been detected by ECL chemiluminescence.
Cell OGD/R mannequin
The bEnd.3 cells or BV2 cells had been cultured in incubator for 24 h of attachment. Then the tradition medium was changed with glucose-free and serum-free DMEM medium. After that, the cells had been cultured in 95% N2 and 5% CO2 atmosphere to simulate low oxygen situation. 3 h later, the medium was changed with regular medium and the cells had been transferred to regular incubator to simulate reperfusion harm.
Cell phagocytosis experiment
The bEnd. 3 cells had been seeded in 6-well plates (1 × 105 cells/properly) for 24 h of attachment. After OGD/R, cells had been handled with rhodamine-loaded PLGA or pmPLGA for 2 h. Then the cells had been washed and noticed utilizing fluorescence microscopy and circulate cytometry.
In vitro BBB mannequin
The BBB mannequin was established by seeding bEnd.3 cells onto Transwell inserts and monitoring integrity through TEER measurements. Upon profitable mannequin development (TEER > 200 Ω/cm2), cells had been subjected to OGD/R treatment. Subsequently, rhodamine-loaded PLGA or pmPLGA had been added to the apical chamber. After half-hour, the fluorescence depth in the basolateral chamber was quantified utilizing a microplate reader to judge nanoparticle transport throughout the injured barrier.
In vitro microglia polarization
PBS, PLGA@SV, and pmPLGA@SV had been incubated with OGD/R handled BV2 cells for 24 h. Then, the cells had been washed, digested to cell suspension, and labeled with CD206-FITC (1:100, eBioscience) and CD86-PE (1:100, Abcam) for circulate cytometry (CytoFLEX, Beckman Coulter, USA) evaluation. The gating technique was as follows: dwell cells had been gated on FSC-A/SSC-A, doublets had been excluded utilizing FSC-A/FSC-H and FSC-H/FSC-W, after which CD206 and CD86 expression had been analyzed on the singlet inhabitants. The polarization ratio of BV2 cells had been calculated in line with the share of completely different phenotypes.
ELISA
The degrees of cytokines, together with IL-1β, IL-10, had been measured utilizing business ELISA kits (Shanghai Enzyme-linked Biotechnology Co.). The supernatant was collected and centrifuged at 3000 rpm for 10 min. All supernatants had been measured in line with the producer’s directions.
In vitro ROS scavenging
PC12 (1 × 105 cells per properly) cells had been seeded in 6-well plates. After cell attachment, cells had been incubated beneath OGD situation for 2 h and in regular tradition situation for 24 h. Then saline, PBS, PLGA@SV, and pmPLGA@SV had been incubated with the cells. 6 h later, cells had been washed, incubated with DCFH-DA (10 µM), and imaged by fluorescence microscopy.
The tMCAO mannequin
All animal procedures had been accredited by the Moral Committee on Animal Testing at the Normal Hospital of Western Theater Command (Ethics Approval No. 2023ky119−1) and had been carried out in accordance with the ARRIVE tips.
Male C57BL/6J mice (25–30 g) had been used. Anesthesia and analgesia: Anesthesia was induced with 5% isoflurane in 100% oxygen and maintained with 1.5–2% isoflurane in 100% oxygen through a nostril cone all through the surgical procedure. The depth of anesthesia was confirmed by the absence of a pedal withdrawal reflex. For postoperative analgesia, buprenorphine (0.05 mg/kg, subcutaneously) was administered instantly after surgical procedure and once more at 12 hours post-surgery. Efforts to alleviate struggling: Physique temperature was maintained at 37.0 ± 0.5℃ utilizing a rectal probe and a feedback-controlled heating pad throughout surgical procedure and till full restoration. After surgical procedure, animals had been positioned in a restoration cage with a warming pad and monitored each 2 hours for the first 24 hours for indicators of ache or misery (e.g., hunched posture, diminished locomotion, piloerection, decreased meals/water consumption). Any animal exhibiting extreme misery would have acquired extra buprenorphine (0.05 mg/kg, s.c.) or been humanely euthanized earlier; nonetheless, no animal met the early elimination standards on this examine.
For tMCAO induction, the exterior carotid artery (ECA) and the inner carotid artery (ICA) on the proper facet had been separated. Then, a specialised filament (wire clot) was inserted from the ECA into the ICA till the origin of the center cerebral artery (MCA) was blocked. After 1 hour of ischemia, the filament was eliminated to permit reperfusion. Sham-operated mice underwent the identical surgical process (anesthesia, neck incision, and carotid artery isolation) besides that the filament was not inserted to induce occlusion. The Sham group served as a non-ischemic detrimental management.
Tissue assortment and sacrifice: At the predetermined endpoint, mice had been deeply anesthetized with 5% isoflurane in 100% oxygen. The chemical agent used for euthanasia was 5% isoflurane (inhalation overdose). After confirming the loss of pedal reflex, animals had been decapitated, and the brains had been quickly eliminated. Decapitation was carried out beneath deep isoflurane anesthesia to make sure that the animals didn’t expertise ache or misery. Dying was confirmed by cessation of heartbeat and respiration. Thus, the strategies of sacrifice included an overdose of isoflurane as the main chemical agent, adopted by decapitation as a secondary bodily methodology.
TTC staining
Following humane euthanasia, all mind tissue assortment was carried out strictly in accordance with the 3R ideas and the AVMA Tips for the Euthanasia of Animals to make sure moral compliance. 2,3,5-Triphenyl-2H-Tetrazolium Chloride (TTC) staining was employed to judge cerebral infarction. Mouse brains had been collected and sectioned, adopted by incubation in 2% TTC answer at 37℃ for 30 min. Viable tissue stained crimson crimson resulting from the enzymatic discount of TTC to formazan, whereas infarcted areas, devoid of dehydrogenase exercise, remained pale white.
Behavioral evaluation
A sequence of behavioral exams had been carried out to judge neurological restoration in mice. The modified neurological severity rating (mNSS), has a complete of 18 factors, together with 6 factors for motor take a look at, 2 factors for sensory take a look at, 6 factors for the steadiness beam take a look at, and 4 factors for reflex loss and irregular motion. For the rotarod take a look at assessing motor coordination and endurance, mice had been positioned on a rotating rod that accelerated step by step from 5 to twenty rotations/min. Previous to formal testing, all mice underwent a 7-day coaching interval to make sure they may stay on the rod for a most of 240 seconds. The imply period spent on the rod was recorded as the latency to fall. For the cylinder take a look at assessing forepaw use asymmetry, mice had been positioned in a clear cylinder for a 10-min session, which was recorded utilizing a video digital camera. Forepaw contacts towards the cylinder wall had been quantified as follows: L (quantity of left forepaw contacts), R (quantity of proper forepaw contacts), and B (quantity of simultaneous contacts with each forepaws). The asymmetry price was calculated utilizing Equation (5).
For the adhesive take a look at assessing the tactile response and sensorimotor perform, a 5 × 5 mm2 patch was connected to the paralyzed forepaw of every mouse. The latency for the mouse to fully take away the patch was recorded as the elimination time.
Statistical evaluation
Statistical analyses had been carried out utilizing GraphPad Prism 10. For datasets satisfying assumptions of normality and homogeneity of variance, inter-group imply comparisons had been carried out utilizing unpaired Pupil’s t-test. Between-group variations had been evaluated through one-way ANOVA. Quantitative outcomes are expressed as imply ± SD, with all determine captions specifying the corresponding p values.
Outcomes
Preparation and characterization of pmPELA@SV
The physicochemical properties of the synthesized nanoparticles had been systematically characterised. TEM photographs revealed that PLGA, PLGA@SV, and pmPLGA@SV all exhibited a uniform spherical morphology (Fig 1a). Notably, pmPLGA@SV displayed a definite core-shell construction, with a skinny membrane layer efficiently encapsulated round the PLGA core (Fig 1a, magnified view). DLS evaluation confirmed that the hydrodynamic diameters of PLGA and PLGA@SV had been roughly 150 nm, whereas the measurement of pmPLGA@SV roughly elevated considerably to 165 nm (p = 0.0110), according to the addition of the membrane coating (Figs 1b and 1c, S1 Table). Zeta potential shifted from −32.2 mV (PLGA@SV) to −22.9 mV after platelet membrane coating (Fig 1d, S1 Table). The short-term stability of pmPLGA@SV was assessed at 4℃ in PBS (pH 7.4). The polydispersity index (PDI) values for PLGA, PLGA@SV, and pmPLGA@SV had been all beneath 0.3, indicating slender measurement distribution for all formulations (Fig 1e, S1 Table).
Fig 1. Physicochemical characterization of pmPLGA@SV.
(a) Consultant TEM photographs of PLGA, PLGA@SV, and pmPLGA@SV nanoparticles. (b) Hydrodynamic measurement distribution measured by DLS (consultant of three impartial batches). (c) Imply particle measurement of completely different nanoparticle formulations. (d) Zeta potential of PLGA, PLGA@SV and pmPLGA@SV. (e) Polydispersity index (PDI) of PLGA, PLGA@SV, and pmPLGA@SV. (f) Drug loading (DL%) and (g) entrapment effectivity (EE%) of SV in PLGA@SV and pmPLGA@SV. (h) In vitro cumulative launch profile of SV from pmPLGA@SV in PBS (pH 7.4, 0.1% Tween 80, 37℃). (i) SDS-PAGE evaluation of protein compositions from platelet membrane (lane 1) and pmPLGA@SV (lane 2). Numbers above bars point out p-values. All quantitative knowledge are introduced as imply ± SD from three impartial nanoparticle batches (n = 3).
Moreover, the EE and DL of SV in each PLGA@SV and pmPLGA@SV had been assessed. Each formulations exhibited excessive EE (~65%) and DL (~6.2%), with no vital variations noticed between the two teams, indicating that the membrane coating course of didn’t induce untimely drug leakage (Figs 1f and 1g). The soundness of pmPLGA@SV was evaluated at 4℃ in PBS (pH 7.4). The encapsulation effectivity remained secure over 24 h with lower than 10% drug leakage, and the hydrodynamic diameter confirmed no vital change (Supplementary S1 and S2 Figs). The in vitro launch profile of SV from pmPLGA@SV exhibited an preliminary burst launch (~18% inside 24 h) adopted by a sustained launch section (Fig 1h), indicating a secure and managed launch habits of the nanoparticles.
To confirm the profitable translocation of platelet membrane proteins onto the nanoparticles, SDS-PAGE was carried out (Supplementary S3 Fig). The protein profile of pmPLGA@SV carefully matched that of the platelet membrane, confirming that the important membrane proteins had been efficiently retained on the floor of the nanoparticles (Fig 1i).
Enhanced mobile uptake and trans-BBB transport of pmPLGA nanoparticles
To additional examine the delivery effectivity beneath pathological circumstances, an in vitro ischemic BBB mannequin was established utilizing OGD/R-treated bEnd.3 monolayers (Fig 2a). Rhodamine B (RhoB) was encapsulated into the nanoparticles as a fluorescent tracer. Mobile uptake evaluation through circulate cytometry first confirmed that pmPLGA@RhoB considerably elevated internalization by bEnd.3 cells in comparison with PLGA@RhoB (Fig 2b). The imply fluorescence depth (MFI) of the pmPLGA@RhoB group was roughly 2.48-fold increased than that of the PLGA@RhoB group (p < 0.0001, Fig 2c). Crucially, the trans-barrier transport functionality was quantified by measuring the fluorescence depth in the basolateral chamber. In the OGD/R-injured mannequin, pmPLGA@RhoB exhibited a considerably increased translocation effectivity than that of the PLGA@RhoB group (p < 0.0017, Fig 2d). This outcome signifies that the platelet membrane coating not solely enhances mobile affinity but in addition facilitates the environment friendly penetration of nanoparticles throughout the compromised BBB. Complementary confocal laser scanning microscopy (CLSM) imaging additional visualized the strong accumulation of pmPLGA@RhoB inside the endothelial layer (Fig 2e), collectively demonstrating the superior brain-targeting potential of the biomimetic formulation beneath stroke-like circumstances.
Fig 2. Mobile uptake and trans-barrier transport of biomimetic nanoparticles.
(a) Schematic of the BBB mannequin and the transport assay protocol. (b, c) Movement cytometry histograms and quantitative evaluation of RhoB uptake in bEnd.3 cells (n = 3). (d) Fluorescence depth of RhoB in the basolateral chamber, representing the trans-barrier effectivity throughout the OGD/R-treated bEnd.3 monolayer (n = 3). (e) Consultant CLSM photographs of bEnd.3 cells exhibiting the intracellular distribution of RhoB-labeled nanoparticles (crimson) and DAPI-stained nuclei (blue) (Consultant photographs from three impartial experiments (n = 3)). Numbers above bars point out p-values. All measurements had been carried out utilizing three impartial batches (n = 3). Information are introduced as imply ± SD.
Regulation of microglial polarization and neuroprotection towards oxidative stress by pmPLGA@SV
To guage the multifaceted therapeutic results of the biomimetic nanoparticles, we investigated their impression on microglial polarization and neuronal oxidative stress beneath OGD/R circumstances (Supplementary S4 Fig). First, the immunomodulatory impact of pmPLGA@SV on BV2 cells had been assessed through circulate cytometry (Fig 3a). OGD/R insult considerably polarized BV2 cells towards a pro-inflammatory M1 phenotype, with the share of CD86 ⁺ cells reaching 56.07% (Fig 3b). Nonetheless, treatment with pmPLGA@SV successfully reversed this pattern, considerably lowering M1 markers whereas selling the transition to an anti-inflammatory M2 phenotype (CD206⁺). This phenotypic change was additional confirmed by the cytokine profiles. pmPLGA@SV treatment markedly downregulated the expression of the pro-inflammatory cytokine IL-1β and upregulated the anti-inflammatory cytokine IL-10 in BV2 cells (Fig 3c), demonstrating a superior capability to transform the inflammatory microenvironment in comparison with the naked PLGA@SV group.
Fig 3. pmPLGA@SV modulates microglial polarization and alleviates neuronal oxidative stress post-OGD/R.
(a) Consultant circulate cytometry scatter plots and (b) statistical evaluation of M1 (CD86+) and M2 (CD206⁺) marker expression in BV2 microglia (n = 3). (c) Ranges of pro-inflammatory (IL-1β) and anti inflammatory (IL-10) cytokines in BV2 cells throughout completely different treatment teams (n = 3). (d) Consultant fluorescence photographs exhibiting intracellular ROS ranges (inexperienced) in PC12 cells after numerous remedies (Consultant photographs from three impartial experiments (n = 3)). Numbers above bars point out p-values. All measurements had been carried out utilizing three impartial batches (n = 3). Information are introduced as imply ± SD.
Past its anti-inflammatory function, we additional explored the direct neuroprotective potential of pmPLGA@SV by monitoring ROS ranges in PC12 cells. As proven in Fig 3d, OGD/R treatment induced a large accumulation of intracellular ROS. Notably, the administration of pmPLGA@SV considerably attenuated the ROS-associated fluorescence, indicating its potent antioxidant functionality in defending neuronal-like cells from oxidative injury. Collectively, these findings counsel that pmPLGA@SV exerts a dual-functional therapeutic impact by concurrently suppressing microglial-mediated neuroinflammation and mitigating neuronal oxidative stress.
In vivo neuroprotective efficacy of pmPLGA@SV in tMCAO Mice
To guage the in vivo therapeutic potential of the biomimetic nanoparticles, we established a tMCAO mouse mannequin. Mind infarct volumes had been visualized utilizing TTC staining 24 hours post-reperfusion (Fig 4a). As anticipated, no infarct was noticed in the Sham group. In distinction, the MCAO group exhibited a considerable and well-defined infarct space (white area), with a mean infarct share of roughly 27.8% (Fig 4b). Treatment with free SV considerably diminished the infarct space in comparison with the MCAO group (p = 0.0072). Notably, pmPLGA@SV treatment demonstrated the most pronounced neuroprotective impact, dramatically shrinking the infarct space to roughly 13.2% (p < 0.0001, vs. MCAO group). The therapeutic efficacy of pmPLGA@SV was additionally considerably superior to that of free SV (p = 0.0001), indicating that the platelet membrane coating remarkably enhances the brain-targeted delivery and bio-availability of SV. These outcomes present strong proof that the pmPLGA@SV nano-system can successfully mitigate ischemic mind harm and protect neural tissue integrity.
Fig 4. In vivo neuroprotective results of pmPLGA@SV in tMCAO mouse mannequin.
(a) Consultant photographs of TTC-stained coronal mind sections from the Sham, MCAO, SV, and pmPLGA@SV teams. The white areas characterize the infarcted mind tissue (Consultant photographs from three impartial experiments (n = 3)). (b) Quantitative evaluation of the infarct space as a share of the complete mind quantity (n = 3). Numbers above bars point out p-values. All measurements had been carried out utilizing three impartial batches (n = 3). Information are introduced as imply ± SD.
Lengthy-term practical restoration and neuro-rehabilitation facilitated by pmPLGA@SV
To additional consider the impression of pmPLGA@SV on long-term neurological restoration, we carried out a sequence of behavioral assessments over a 21-day interval following MCAO harm. Initially, the rotarod take a look at was employed to evaluate motor coordination and steadiness (Fig 5a). The MCAO group exhibited a pointy decline in the latency to fall in comparison with the Sham group. Whereas free SV treatment led to average enhancements, mice handled with pmPLGA@SV confirmed a considerably quicker and extra strong restoration of motor perform all through the statement interval, with the latency to fall approaching Sham ranges by day 21. Sensory-motor integration was evaluated utilizing the adhesive elimination take a look at (Fig 5b). Submit-tMCAO, mice required considerably extra time to take away the adhesive tape. Nonetheless, treatment with pmPLGA@SV markedly diminished this elimination time, outperforming the free SV group and suggesting accelerated sensory-motor rehabilitation. Equally, the cylinder take a look at revealed a excessive uneven price in the MCAO group, indicating impaired spontaneous forelimb use (Fig 5c). Administration of pmPLGA@SV successfully mitigated this lateralization, as evidenced by a constant lower in the uneven price in comparison with each MCAO and SV teams.
Fig 5. Lengthy-term neurological practical restoration in tMCAO mice.
(a) Motor coordination evaluation through the rotarod take a look at (n = 3). (b) Sensory-motor integration analysis through the adhesive elimination take a look at (n = 3). (c) Spontaneous forelimb use asymmetry assessed by the cylinder take a look at (n = 3). (d) Longitudinal neurological deficit scores following tMCAO (n = 3). (e) Physique weight modifications recorded over the 21-day statement interval (n = 3). Numbers above bars point out p-values. Information are introduced as imply ± SD. Statistical significance is indicated solely for the comparability between pmPLGA@SV and MCAO teams at day 21.
The mNSS additional corroborated these findings (Fig 5d). The pmPLGA@SV group demonstrated a gradual and vital decline in deficit scores, indicating a steady enchancment in general neurological standing. Moreover, the restoration of physique weight (Fig 5e), which serves as an indicator of basic well being and vitality, was most outstanding in the pmPLGA@SV group, with their weights practically returning to baseline by the finish of the examine. Collectively, these outcomes display that the biomimetic pmPLGA@SV nano-system considerably enhances long-term practical restoration and promotes neuro-rehabilitation following ischemic stroke.
Dialogue
Ischemic stroke triggers a devastating cascade of occasions, the place the neurovascular unit (NVU) is compromised by oxidative stress, BBB breakdown, and chronic neuroinflammation [12,32]. On this examine, we efficiently engineered a platelet membrane–biomimetic nanoparticle system, pmPLGA@SV, for the targeted delivery of SV to the ischemic mind. Our findings point out that pmPLGA@SV not solely successfully reduces acute infarct quantity but in addition considerably promotes long-term neurological restoration over a 21-day interval. The noticed therapeutic efficacy is related to a synergistic mechanism that probably entails the lively homing of platelet membranes to injured vasculature, the ROS-scavenging functionality of SV, and the shift of microglial polarization towards a pro-healing M2 phenotype [12,14,15].
A serious hurdle in stroke remedy is the environment friendly delivery of neuroprotective brokers throughout the BBB to the ischemic penumbra. Standard nanocarriers typically endure from fast immune clearance and low focusing on precision [33]. Our outcomes counsel that pmPLGA@SV displays superior lesion-specific accumulation in comparison with non-coated nanoparticles. This enhanced focusing on might be mediated by the wealthy repertoire of floor proteins on the platelet membrane, which possess a pure affinity for activated endothelial cells and inflammatory websites inside the cerebral vasculature [34]. By cloaking the PLGA core with this bio-interface, pmPLGA@SV could bypass systemic immune surveillance and focus SV at the lesion web site, thereby maximizing its pleiotropic results whereas minimizing systemic toxicity. Nonetheless, it must be famous that we didn’t carry out aggressive inhibition experiments (e.g., utilizing blocking antibodies towards GPVI or P-selectin) to immediately display receptor-mediated uptake or BBB transcytosis. Such mechanistic research are warranted in future work to dissect the exact molecular pathways concerned in the lively homing of this biomimetic platform.
The interaction between oxidative stress and neuroinflammation is a trademark of ischemia–reperfusion harm [35]. In our in vitro research utilizing PC12 and BV2 cells, pmPLGA@SV demonstrated a dual-functional capability to transform the pathological microenvironment. The numerous discount in ROS ranges means that SV, when delivered through this targeted platform, exerts neuroprotective results by attenuating neuronal oxidative stress. Extra importantly, we noticed a repolarization of microglia from the pro-inflammatory M1 phenotype towards the anti-inflammatory M2 phenotype. This shift is taken into account helpful, as M2 microglia secrete neurotrophic elements and anti inflammatory cytokines (e.g., IL-10), which might mitigate secondary inflammatory injury and supply a supportive area of interest for surviving neurons [36]. Nonetheless, the exact molecular mechanisms linking SV delivery to microglial phenotypic switching stay to be elucidated; pathway-specific inhibitors or genetic fashions are required to determine causality (e.g., involvement of NF-κB, STAT3, or Nrf2 pathways).
A particular spotlight of this analysis is the complete analysis of long-term practical restoration. Whereas many research focus solely on acute-phase survival (1–7 days), our 21-day statement interval supplies extra clinically related proof of neuro-rehabilitation [37]. The constant enhancements in motor coordination (rotarod), sensory-motor integration (adhesive elimination), and spontaneous forelimb symmetry (cylinder take a look at) collectively counsel that pmPLGA@SV facilitates sustained practical restoration, not merely transient safety. The noticed correlation between diminished infarct quantity and long-term behavioral restoration helps the premise that attenuating neuronal oxidative stress and modulating the inflammatory response throughout the acute section could translate into sustained neurological enhancements [38].
A number of limitations of this examine must be explicitly acknowledged. First, we didn’t carry out formal pharmacokinetic (PK) research or quantitative biodistribution evaluation (e.g., tissue accumulation of pmPLGA@SV in mind, liver, spleen). Though our in vitro trans-BBB mannequin and in vivo therapeutic efficacy strongly counsel lesion-targeted delivery, direct proof of the nanoparticle’s distribution profile and circulation half-life is missing. Future work will embody fluorescence imaging and PK evaluation. Second, the behavioral research had been carried out with a modest pattern measurement (n = 3 per group). Whereas the impact sizes had been massive and constant throughout animals, leading to low variability, a bigger cohort (e.g., n ≥ 8 per group) is required for confirmatory research to make sure generalizability. Third, solely male mice had been used; future research ought to consider efficacy in feminine mice to evaluate potential sex-dependent variations. Fourth, aggressive inhibition experiments (e.g., utilizing blocking antibodies towards GPVI or P-selectin) weren’t carried out to immediately verify receptor‑mediated uptake or BBB transcytosis, which stays a key route for future mechanistic research. Lastly, the exact downstream signaling cascades mediating the pleiotropic results of pmPLGA@SV stay to be immediately examined.
Conclusions
In conclusion, the pmPLGA@SV biomimetic system represents a promising platform with additional scope of validation for ischemic stroke treatment. By integrating the focusing on capabilities of platelet membranes with the multi-targeted therapeutic potential of simvastatin, we achieved a synergistic impact that addresses the complexity of stroke pathology. Future research will concentrate on scientific translation, together with the scalability of the membrane-coating course of and the analysis of this platform in massive animal fashions and each sexes. Nonetheless, our findings present a compelling proof-of-concept for utilizing biomimetic nanotechnology to bridge the hole between drug discovery and efficient scientific neuroprotection.
Supporting info
S4 Fig. Consultant circulate cytometry gating technique.
Dwell cells had been gated on FSC-A/SSC-A, doublets excluded by FSC-A/FSC-H and FSC-H/FSC-W, after which CD206 vs. CD86 analyzed on singlet inhabitants. Quadrant gates primarily based on management pattern.
https://doi.org/10.1371/journal.pone.0354184.s004
(TIF)
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