Brain-targeted PROTAC delivery by dual-functional extracellular vesicles achieves robust LRRK2 degradation in a Parkinson’s disease model

Brain-targeted PROTAC delivery by dual-functional extracellular vesicles achieves robust LRRK2 degradation in a Parkinson’s disease model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Brain-targeted PROTAC delivery by dual-functional extracellular vesicles achieves robust LRRK2 degradation in a Parkinson’s disease model Ji-Eun Kim, Ye Eun Ji, Hyeock Yang, Daeho Kwon, Jaein Yoo, Doory Kim, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8830833/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Mesenchymal stem cell–derived extracellular vesicles (MSC-EVs) have emerged as promising therapeutic delivery vehicles for brain diseases. However, their limited ability to cross the blood–brain barrier (BBB) remains a major obstacle to clinical translation. To overcome this limitation, we designed a multifunctional peptide consisting of a membrane-anchoring transmembrane domain (TD) and a cell-penetrating peptide (CPP) that can spontaneously insert into the EV membrane without external energy input. The brain-targeting T7 sequence was placed at the distal end to ensure directional display on the EV surface, thereby improving targeting efficiency. Using this approach, we generated T7-displaying MSC-EVs and passively loaded proteolysis-targeting chimeras (PROTACs) to enhance their therapeutic potential. The structural integrity, cellular uptake, and BBB permeability of engineered EVs were evaluated through in vitro neuronal assays and in vivo brain imaging. T7-TD-CPP EVs exhibited markedly improved cellular internalization and BBB penetration, while PROTAC co-loading facilitated degradation of pathological proteins. In an MPTP-induced Parkinson’s disease mouse model, treatment with T7-EV–PROTACs alleviated disease-associated pathology, including abnormal expression of LRRK2 in the midbrain. This EV platform, combining energy-independent membrane anchoring with T7-mediated brain targeting, represents a promising strategy for precise, brain-directed therapy against neurodegenerative diseases. MSC-derived extracellular vesicles (EVs) Blood–brain barrier (BBB) T7 peptide–mediated brain targeting PROTAC (targeted protein degradation) Parkinson’s disease / LRRK2 degradation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Neurodegenerative disorders remain difficult to treat, in large part because most therapeutics fail to reach effective concentrations in the central nervous system (CNS). Parkinson’s disease (PD), a representative neurodegenerative disease, is characterized by progressive neuronal dysfunction and neurodegeneration driven by pathological protein networks, including aberrant LRRK2 signaling and α-synuclein pathology. Despite extensive efforts to develop disease-modifying agents, the blood–brain barrier (BBB) remains a key obstacle that restricts systemic therapeutics from accessing the brain parenchyma. Consequently, the development of delivery systems capable of efficiently traversing the BBB and enabling functional target engagement in neural tissues is urgently needed. Extracellular vesicles (EVs) have emerged as attractive drug delivery vehicles owing to their biocompatibility, low immunogenicity, and intrinsic capacity for intercellular transport. Mesenchymal stem/stromal cell (MSC)-derived EVs are of particular interest because MSCs have been widely investigated in regenerative and immunomodulatory settings, and MSC-EVs are increasingly considered clinically relevant biological carriers. However, despite rapid progress in EV engineering, several challenges continue to limit translation of EV-based CNS therapeutics. A major limitation is the difficulty of combining robust BBB penetration with effective cargo loading while preserving EV integrity and manufacturing feasibility. Standardized frameworks have emphasized rigorous EV identity and purity characterization, reproducible production, and the minimization of non-vesicular contaminants to support clinical development [ 1 , 2 ]. Yet many existing CNS-targeting EV platforms rely on genetic modification of parental cells to present targeting ligands, potentially complicating regulatory pathways and large-scale manufacturing. Ligand-based targeting of BBB receptors offers a promising strategy to enhance CNS delivery. Among potential BBB targets, the transferrin receptor (TfR) is one of the most extensively validated routes because of its abundant expression on brain endothelial cells and its involvement in transcytosis-mediated transport [ 3 ]. The T7 peptide (HAIYPRH), a TfR-binding ligand, has been widely applied to increase BBB transport and brain accumulation of synthetic nanocarriers such as liposomes [ 4 ]. However, in EV-based delivery contexts, enhancing brain accumulation does not necessarily translate into effective parenchymal access or neuronal engagement, and simple receptor binding can instead result in endothelial retention or endosomal degradation [ 3 ]. Therefore, clinically aligned engineering approaches that enable stable surface presentation of BBB-targeting ligands—without genetic manipulation—while maintaining EV structure and function are of considerable interest. In parallel, targeted protein degradation has emerged as a transformative therapeutic modality. Proteolysis-targeting chimeras (PROTACs) promote ubiquitin-dependent degradation of proteins of interest and can eliminate both enzymatic and scaffolding functions of disease drivers [ 7 ]. LRRK2 is a compelling PD target supported by genetic evidence and mechanistic links to vesicular trafficking and neuroinflammation. XL01126 has recently been developed as a potent and selective LRRK2 degrader with reported BBB penetration in mice [ 8 ]. Nevertheless, systemic PROTAC administration can still face limitations in effective CNS exposure, cellular delivery, and intracellular availability necessary for robust degradation activity. Thus, a BBB-permeable delivery platform that can transport PROTACs into the CNS and promote neuronal target engagement would provide a powerful enabling strategy for PD and other neurodegenerative diseases. Here we report a clinically oriented dual-functional EV platform that integrates BBB targeting and PROTAC delivery within a single carrier. We utilized clinical-grade MSC-derived EVs produced from 3D spheroid cultures and employed a non-genetic surface engineering strategy in which a multifunctional peptide composed of a TfR-targeting motif (T7), a transmembrane domain (TD), and a cell-penetrating peptide (CPP) is spontaneously inserted into the EV membrane to enable stable external display. In parallel, we passively encapsulated the hydrophobic PROTAC XL01126 as an intravesicular cargo. Using stochastic optical reconstruction microscopy (STORM), we directly verified nanoscale membrane localization of the engineered peptide, supporting bona fide surface display rather than peptide aggregation. Functionally, T7-engineered EVs showed enhanced uptake by brain endothelial cells, improved translocation across an in vitro BBB model with TEER-validated barrier maturation, and significantly increased brain accumulation in vivo. Importantly, intravenously administered engineered EVs were detected beyond vascular boundaries and exhibited preferential neuronal association. Finally, in an MPTP-induced PD mouse model, dual-loaded T7-targeted PROTAC EVs achieved robust in vivo degradation of LRRK2 and reduced α-synuclein levels, outperforming free PROTAC and non-targeted EV formulations. Together, these findings establish a scalable and clinically compatible framework for brain-targeted PROTAC delivery using engineered MSC-EVs. Materials and Methods All studies involving human subjects were approved by the Institutional Review Board of the Samsung Medical Center. Wharton's Jelly (WJ) was provided to healthy volunteers. All volunteers or their guardians provided written informed consent for participation in the study. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (Laboratory Animal Research Center, AAALAC International-approved facility) of Samsung Medical Center. Preparation and isolation of clinical-scale MSC-EVs Preparation and isolation of clinical-scale MSC-EVs were performed as described previously [ 9 ]. Detailed methods are provided in the Supplementary Data. Schematics of EV production, isolation, and quality control are shown in Supplementary Fig. 1. Characterization of EVs Following the guidelines recommended by the International Society for Extracellular Vesicles (Minimal Information for Studies of Extracellular Vesicles), EVs isolated from WJ-MSC culture medium were characterized in terms of morphology, size distribution, surface markers, purity, potency markers, efficacy, stability, and safety [ 1 , 10 ]. Nanoparticle Tracking Analysis (NTA) The size distribution and particle concentration of EVs were analyzed using a NanoSight NS300 system equipped with a 488 nm blue laser and a scientific CMOS (sCMOS) camera (Malvern Panalytical, Malvern, UK). Samples were diluted in 0.22 µm-filtered phosphate-buffered saline (PBS) and introduced into the sample chamber using a syringe pump. Measurements were performed at 25°C with a camera level of 14–15, a shutter speed of 1206–1259, and gain of 245. Three 30-second videos were recorded per sample under identical instrument settings. Data were analyzed using NTA software (version 3.4, Build 3.4.003; Malvern Panalytical). Transmission Electron Microscopy (TEM) For TEM analysis, 7 µL of concentrated EV suspension was applied to Formvar carbon-coated copper grids and incubated for 20 minutes at 22°C to allow particle adsorption. The grids were then fixed with 2% paraformaldehyde (PFA) for 20 minutes, followed by post-fixation with 1% glutaraldehyde in distilled water for 5 minutes. After fixation, the grids were washed six times with distilled water and stained with 2% uranyl acetate for 5 minutes. Excess fluid was carefully removed with filter paper, and the grids were air-dried for at least 16 hours. Imaging was performed using a transmission electron microscope (HT7700, Hitachi High-Technologies, Tokyo, Japan). Flow cytometry for EV surface marker analysis Surface expression of EV markers (CD9, CD63, and CD81) was evaluated using flow cytometry. EVs were conjugated to aldehyde/sulfate latex beads (4 µm, Invitrogen) and incubated with APC-conjugated anti-human CD9, CD63, or CD81 antibodies (BioLegend) for 30 minutes at 4°C in the dark. After washing with PBS containing 0.5% BSA, samples were analyzed using a FACSVerse flow cytometer (BD Biosciences). Data were processed with FlowJo software. Unstained bead-bound EVs were used as a negative control for gating. ELISA for non-vesicular protein contamination To evaluate the purity of EV preparations, potential non-vesicular contaminants were quantified using ELISA kits according to the manufacturers’ protocols. Processing reagents (gentamicin and BSA) and proteins from subcellular compartments—calnexin (endoplasmic reticulum), histone (nucleus), GM130 (Golgi), and cytochrome c (mitochondria)—were measured. All values were compared to the lower limit of quantification (LLOQ) provided with each assay kit, and results below this threshold were considered not detected (N.D.). STORM Microscopy Stochastic optical reconstruction microscopy (STORM) was performed to visualize EVs at nanometer resolution for high-resolution analysis of engineered EVs [ 11 ]. This technique enables localization analysis of peptides in engineered EV formulations, including discrimination of peptides loaded into the EV lumen versus peptides displayed on the EV surface, and validation of structural integrity and loading efficiency. We performed stochastic optical reconstruction microscopy (STORM) to visualize individual extracellular vesicles (EVs) at nanometer resolution for high-resolution imaging and nanoscale localization analysis of engineered EVs [Rust et al., 2006]. STORM enables precise discrimination of fluorescently labeled components associated with EVs (e.g., surface-displayed peptides versus membrane dyes), and provides quantitative assessment of EV structural integrity, nanoscale changes in EV size/morphology, and validation of surface functionalization and loading efficiency. 1) Sample preparation for EV imaging A glass-bottomed confocal dish was briefly immersed in 1 M aqueous potassium hydroxide (KOH) solution (6584 − 4405; DAEJUNG) and sonicated for 15 min to remove contaminants. The dish was rinsed three times with distilled water, suctioned to remove residual water, and exposed to UV light for 15 min. After an additional wash with distilled water, the cleaned dish was coated with poly-L-lysine (PLL) (25988-63-0; Sigma-Aldrich) at room temperature (RT) for 30 min to enhance adhesion of EVs. After coating, PLL-coated dishes were washed once with distilled water. EVs were diluted 1:2,000 in PBS and incubated on the PLL-coated dish for 24 h at RT. For EV membrane staining, immobilized EVs were incubated with blocking buffer (3% [w/v] bovine serum albumin (BSA) [CNB102-0100; Cell Nest] in PBS) for 30 min at RT. EV membranes were then labeled using CellBrite™ Fix 640 Membrane Dye (Biotium; Cat# 30089) diluted in PBS (working dilution optimized empirically). After membrane labeling, samples were fixed with 4% paraformaldehyde (PFA) (1574; Electron Microscopy Sciences) in PBS for 10 min at RT. Following fixation, samples were briefly rinsed once with PBS and immediately imaged in STORM imaging buffer. Note TAMRA-labeled T7-TD-CPP peptides were used to visualize peptide localization independently from membrane staining. To avoid spectral overlap, the EV membrane dye was selected in the far-red channel (CellBrite Fix 640), while TAMRA peptides were detected in the orange-red channel (561 nm excitation). 2) STORM imaging For STORM imaging, samples were immersed in a photoswitching imaging buffer containing mercaptoethylamine (MEA) (30070; Sigma-Aldrich), 5% (w/v) glucose, and an oxygen-scavenging enzyme system (0.5 mg/mL glucose oxidase [G2133; Sigma-Aldrich] and 38 µg/mL catalase [C3515; Sigma-Aldrich]) prepared in PBS at pH 8.5. All STORM imaging was conducted using a custom-built STORM setup consisting of an inverted microscope and a 1.49 NA 100× oil immersion objective lens (CFI SR HP Apo TIRF; Nikon) [Go et al., 2021; Chung et al., 2021]. A 647 nm laser (OBIS; Coherent) was used for imaging CellBrite Fix 640–labeled EV membranes, while a 561 nm laser (OBIS; Coherent) was used for imaging TAMRA-labeled peptides. Lasers were directed through the back port of the microscope and aligned to achieve total internal reflection fluorescence (TIRF) illumination. The CRISP Autofocus system (ASI) stabilized the focal plane by detecting a separated IR beam reflected at the sample–liquid interface. Emitted fluorescence was filtered using appropriate bandpass emission filters (LF408/488/561/635-B; Semrock), and images were recorded using an EMCCD camera (iXon Ultra 888; Andor). Under these conditions, rapid photoswitching of fluorophores enabled single-molecule localization and super-resolution reconstruction of EV membranes and peptide localizations (Supplementary Movie 5). For 3D STORM imaging, a cylindrical lens with a focal length of 500 mm (LJ1144RM-A; Thorlabs) was inserted into the emission path to introduce astigmatism (Center for Polymers and Composite Materials, Hanyang University, Korea). Astigmatism produced elliptical point spread functions (PSFs) whose deformation encodes axial position; by measuring PSF widths in the x and y directions, z-coordinates were calculated to reconstruct 3D spatial information with nanometer precision. A total of 30,000 frames were acquired at a frame rate of 70 Hz. For STORM reconstruction, PSFs in each frame were fitted with Gaussian functions to determine centroid positions, followed by drift correction and rendering using optimized reconstruction parameters [Chung et al., 2021; Go et al., 2021; Rust et al., 2006]. 3) Identification and quantitative analysis using DBSCAN EVs were distinguished from background signals in STORM reconstructions based on their confined structure and densely clustered localization events [Jung et al., 2020; Lim et al., 2024]. Approximately 1,000 EVs from 10 STORM images were identified and analyzed using DBSCAN clustering. The size and localization number of each identified EV were scatter-plotted to assess nanoscale heterogeneity. DBSCAN was implemented using the “sklearn.cluster DBSCAN” algorithm in Python (v3.12.0). Key parameters included eps (ε), defining the neighborhood radius, and min_samples , defining the minimum number of points required for core-point classification. By empirically optimizing eps and min_samples for each STORM image, EV clusters were robustly segmented from background signals. LC–MS sample preparation (HPLC pre-treatment) Samples were pretreated prior to analysis using an extraction solvent composed of acetonitrile, methanol, and distilled water (ACN:MeOH:DW) mixed at a 2:1:1 (v/v/v) ratio. Briefly, 200 µL of each sample was aliquoted, and 800 µL of the extraction solvent was added. The mixture was vortex-mixed for 15 min and incubated on ice for 20 min to facilitate extraction and protein precipitation. The extracts were centrifuged at 14,000 rpm for 10 min, and 500 µL of the resulting supernatant was collected. The supernatant was evaporated to dryness in a 37°C oven overnight. The residue was reconstituted in distilled water, passed through a 0.22 µm PTFE syringe filter, and subjected to HPLC analysis. HPLC analysis High-performance liquid chromatography (HPLC) was performed using a Thermo Scientific UltiMate 3000 system. Chromatographic data were acquired and processed using the Thermo Scientific Chromeleon Chromatography Data System (CDS). Peak areas were obtained by automated peak integration. A calibration curve was constructed by plotting the concentrations of standard solutions against the corresponding peak areas. The concentration of the analyte loaded in the samples was determined by interpolating sample peak areas on the standard calibration curve. Imaging Flow Cytometry for EV Internalization (Amnis ImageStream) Endothelial internalization of EVs was quantified using imaging flow cytometry (Amnis® ImageStream®X Mk II System, Luminex/Amnis). Human brain microvascular endothelial cells expressing GFP (HBEC-GFP) were incubated with CellTracker™ Deep Red (CTDR)-labeled EVs for 30 min at room temperature (RT). The following controls were included to establish gating and compensation: unstained HBECs, HBEC-GFP only, and CTDR-labeled EV only. Single-stain controls were used to calculate fluorescence compensation. Images were acquired using a 40× objective with laser settings: 488 nm (0.1 mW), 642 nm (15 mW), and side scatter (SSC, 0.5 mW). For each condition, 2,000 single-cell events were collected. Data were analyzed using IDEAS® software with a standardized gating strategy. Briefly, well-focused events were gated using Gradient RMS. Single cells were identified using Aspect Ratio to exclude doublets and aggregates. EV uptake was quantified by dot-plot analysis of fluorescence intensities. EV-positive HBECs were defined as GFP-positive cells exhibiting increased CTDR fluorescence relative to controls. Representative cell images from each gate were inspected to confirm gating accuracy. EV-Based Drug Delivery Systems [ 14 ] Peptide design and synthesis : Peptides were designed to include the loop 2 main sequence (EKVPVSKGQL) with a C-terminal GG motif and the APP transmembrane domain (TMD), arranged in an N–transferrin-binding T7 peptide–CPP TM–C configuration. A DEDE linker was inserted between domains. An LRLLR motif was added to enhance CPP activity, and the C-terminal arginine was amidated. N-terminal biotin or fluorescent labeling was performed for detection. Peptides were synthesized by standard solid-phase methods. 2) Dual loading of peptides into MSC-EVs Peptide cargos were loaded into MSC-EVs using a passive loading approach as previously described [ 12 , 13 ]. For dual loading, the PROTAC compound was first incubated with MSC-EVs for 1 h at 22°C to allow passive encapsulation. Subsequently, TD-CPP or T7-TD-CPP peptides were added and further co-incubated with the PROTAC-loaded EVs for an additional 1 h at 22°C to enable membrane insertion. 3) Removal of Unloaded PROTAC / Peptide Drugs Following PROTAC-peptide loading, unincorporated cargos were removed by dialysis using a 100 kDa molecular weight cut-off (MWCO) membrane. Dialysis was performed for 4 h at 4°C under gentle stirring. The concentration of PROTAC-peptide-loaded EVs was subsequently determined, and PROTAC- peptide loading efficiency was quantified as described below. 4) Peptide Quantification after EV Loading To quantify fluorescently labeled neuropeptides, a standard curve was first established by measuring fluorescence across a range of known concentrations. Following EV loading, the fluorescence intensity of the peptide-loaded EVs was measured and used to calculate the absolute concentration of encapsulated peptides based on the standard curve. T7 peptides were analyzed using fluorescence spectroscopy, and concentrations were determined using a standard curve beginning at 10 µg, with serial 10% dilutions down to 0.1 µg and the blank samples containing ultrapure water and PBS, using the Glomax multi detection system (Promega, Madison, WI, USA). The samples were portioned at 100 µL and evaluated using spectroscopy at excitation and emission wavelengths of 510 and 570 nm, respectively. Effective loading was calculated as a function of µg peptide per billion EV (Jeyaram et al., 2020). 5) Drug loading Capacity and Encapsulation Efficiency of peptides Encapsulation efficiency and loading efficiency were calculated according to equations (1) and (2), respectively. • Encapsulation efficiency (%) (1) = (Amount of the total loaded peptide in EVs / total amount of input peptide initially ) x 100 • Loading Capacity (%) (2) = (Amount of the total loaded peptide in EVs / final EV numbers) x100 6) EV recovery The final purified peptide-loaded EV solution was analyzed for EVs recovery using nanoparticle tracking analysis (NTA). EV recovery was calculated according to equations (3), respectively. • EV recovery % (3) = (final EVs number/input EVs number) x100 In vivo studies 1) Animal experimental procedure : All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (Laboratory Animal Research Center, AAALAC International approved facility) of Samsung Medical Center (Seoul, Korea). C57BL/6 J mice were purchased from Orient Bio Inc. (Seongnam, Korea) and housed at a temperature of 22 ± 1°C and a relative humidity of 55 ± 10%, under a 12 h light/dark cycle, with ad libitum access to food and water. Animals were anesthetized using a ketamine:xylazine mixture (5:1 ratio) prepared by combining ketamine (Yuhan Ketamine 50Inj.) and xylazine (Rompun Inj.). The anesthetic dose was calculated based on the animal’s body weight, and the mixture was administered intraperitoneally (i.p.). Depth of anesthesia was confirmed by the absence of reflex responses to toe pinching. Once fully anesthetized, the animal was positioned on a surgical platform, and the thoracic cavity was opened to expose the heart. A 21-gauge needle was inserted into the left ventricle, and an incision was made in the right atrium to allow for drainage. Perfusion was performed with 50 mL each of PBS and 4% PFA in PBS. After perfusion, the skull was carefully opened using surgical scissors and forceps. The brain was gently removed and post-fixed in 4% PFA at 4°C for 24 h. Subsequently, the brain was transferred to a 30% sucrose solution for cryoprotection until it sank, indicating full infiltration. 2) IVIS - Fluorescent brain distribution and histological imaging C57BL/6 male mice were randomly assigned to experimental groups. For near-infrared fluorescence imaging, MSC-3D EVs were first labeled using the ExoGlow-Vivo EV Labeling Kit (System Biosciences, SBI) according to the manufacturer’s instructions. Following EV labeling, surface engineering was performed by incubating ExoGlow-labeled EVs with TD-CPP or T7-TD-CPP peptides to enable membrane insertion prior to administration. A total of 6 × 10⁸ labeled EVs were administered intravenous (IV), and imaging was using the IVIS Spectrum imaging system (PerkinElmer, MA, USA). Fluorescence signals were acquired using excitation and emission wavelengths of 745 nm and 820 nm, respectively, and quantitative analysis was conducted using Living Image® software (v4.7.2). 3) Intra-vital microscope To visualize meningeal lymphatics and cerebral vasculature in real-time, a cranial window was implanted over the parietal cortex of Prox1-eGFP mice. Briefly, mice were anesthetized with isoflurane, and the scalp was incised to expose the skull. A circular craniotomy (approx. [ 3 – 4 ] mm diameter) was carefully performed using a high-speed micro-drill, leaving the dura mater intact. A sterile cover glass ([No. 1]) was placed over the exposed brain and sealed with dental cement to create a water-tight observation window. To label blood vessels, fluorophore-conjugated anti-CD31 antibody was injected intravenously immediately prior to imaging. In vivo imaging was performed using a custom-built all-in-one two-photon microscopy system ([IVM-C / IVM-M3], IVIM Technology, Daejeon, Korea). The system was equipped with a high-speed polygonal scanner and integrated real-time motion compensation to correct artifacts caused by heartbeat and respiration. Prox1-eGFP positive lymphatic vessels, [fluorophore]-labeled blood vessels, and [fluorophore]-labeled EVs were excited at [880–920] nm, [wavelength] nm, and [wavelength] nm, respectively. Time-lapse images and Z-stacks were acquired using a water-immersion objective lens ([10× or 25×], NA [1.0]). Image processing and 3D reconstruction were performed using [IVIM S/W, Imaris, or ImageJ] software. 4) Histological imaging For histological distribution analysis, MSC-EVs or MSC-EV-NPs were surface-engineered with TAMRA-tagged TD-CPP or T7-TD-CPP peptides prior to administration. Peptide-functionalized EVs were administered via intravenous (i.v.) injection as control conditions. At 4 h post administration, mice were sacrificed and brains were harvested, fixed overnight in 4% paraformaldehyde (PFA) at 4°C, and cryoprotected in 30% (w/v) sucrose solution. Brain tissues were embedded in OCT compound (Tissue-Tek, Sakura), stored at − 80°C, and coronally sectioned at a thickness of 20 µm using a cryostat (Leica CM1950, Leica Biosystems). Sections were mounted using Fluoroshield™ mounting medium containing DAPI (ImmunoBioScience, AR-6501–01) and imaged using a Zeiss LSM 780 confocal laser scanning microscope (Carl Zeiss, Germany). For cellular localization analysis, sections were immunostained with cell-type–specific markers including NeuN (neurons) and GFAP (astrocytes). All images were acquired using identical acquisition settings and processed using ZEN software (Carl Zeiss). 5) MPTP-induced Parkinson’s disease mouse model and EV treatment Male C57BL/6 mice (10–12 weeks old) were obtained from Orient Bio (Seongnam-si, Korea) and randomly assigned to experimental groups. To induce Parkinsonian pathology, mice received intraperitoneal (i.p.) injections of MPTP at a dose of 20 mg/kg on Day 0. Control animals were administered an equivalent volume of PBS. For therapeutic intervention, EV treatments were performed on Day 4 following MPTP injection. Mice were intravenously (i.v.) injected with PROTAC alone, PROTAC-loaded TD-CPP MSC-EVs, or PROTAC-loaded T7-TD-CPP MSC-EVs, while control groups received PBS. All treatments were administered via the tail vein under identical conditions. Mice were sacrificed on Day 6, and brain tissues were harvested for biochemical analysis. 6) Western blot analysis : Total proteins were extracted from mouse brain tissues using ice-cold RIPA buffer. Protein concentrations were determined using Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Protein lysates (30 µg) were separated by SDS–PAGE on 10% gels and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). After blocking with 5% skimmed milk for 1 h at 22°C, membranes were incubated overnight at 4°C with primary antibodies against LRRK2 and β-actin. After washing with TBS containing 0.1% Tween-20, membranes were incubated with horseradish peroxidase–conjugated secondary antibodies (1:2000; CUSABIO, Houston, TX, USA) for 2 h at 22°C. Protein bands were visualized using the ECL™ Prime Western Blotting System (RPN2232; Cytiva, Marlborough, MA, USA), and signal intensities were quantified using ImageJ software (NIH, Bethesda, MD, USA). Results Characterization of EVs. MSC-derived extracellular vesicles (MSC-EVs) were isolated from 3D spheroid cultures and assessed for morphological and physicochemical properties. Transmission electron microscopy (TEM) revealed spherical vesicles with a characteristic round-shaped EV morphology (Fig. 1 A). Consistent with these observations, nanoparticle tracking analysis (NTA) demonstrated a narrow size distribution with a mean diameter of 127.0 ± 3.6 nm and a mode diameter of 117.3 ± 4.6 nm (Fig. 1 B).The identity of MSC-EVs was further confirmed by surface marker profiling. Flow cytometry analysis showed strong positivity for canonical EV tetraspanins, including CD9 (98.88%), CD63 (98.82%), and CD81 (100.00%), indicating successful enrichment of EV populations (Fig. 1 C). EV purity was evaluated by ELISA for non-vesicular contaminants and cellular components. Gentamicin, BSA, calnexin, histone, GM130, and cytochrome c were not detected (N.D.) or remained below the lower limit of quantification (LLOQ), supporting minimal contamination by soluble proteins, intracellular organelles, or cellular debris (Fig. 1 D).To further resolve nanoscale structural features, we performed stochastic optical reconstruction microscopy (STORM) super-resolution imaging (Fig. 1 E-F). Reconstructed localization clusters yielded a mean EV diameter of 109.73 ± 23.86 nm, consistent with NTA results. Fluorescence emission distributions produced full width at half maximum (FWHM) values of 110.86 nm (x-axis) and 105.77 nm (y-axis), reflecting compact and uniform vesicle boundaries. Localization analysis further revealed 221 ± 148 localizations per particle, indicating dense fluorophore distribution along the EV membrane. Moreover, line-scan profiling across the vesicle center identified a peak-to-peak distance of 84.32 nm, consistent with the hollow vesicular architecture characteristic of MSC-derived EVs. Collectively, these data confirm that EVs isolated from 3D spheroid cultures exhibit expected EV morphology, canonical marker expression, minimal contamination, and preserved nanoscale structural features, supporting their suitability for downstream engineering and loading applications. Preparation of T7-TD-CPP engineered EVs To enable brain targeting and intracellular delivery of small-molecule cargo, we designed a multifunctional membrane-inserting peptide comprising a transferrin receptor–binding T7 motif, a flexible linker (LP), a transmembrane domain (TD), and a cell-penetrating peptide (CPP) to facilitate EV surface display through spontaneous membrane insertion (Fig. 2 A). In parallel, the hydrophobic PROTAC molecule XL01126 was selected as a model therapeutic cargo for passive EV encapsulation (Fig. 2 B). To generate T7-TD-CPP–engineered PROTAC-loaded EVs, PROTAC encapsulation and subsequent peptide surface insertion were performed sequentially. Following passive loading of XL01126 into native MSC-3D EVs, vesicular morphology and particle size were preserved, as confirmed by transmission electron microscopy and nanoparticle tracking analysis. PROTAC-loaded MSC-3D EVs, PROTAC & TD-CPP-loaded EVs, and PROTAC & T7-TD-CPP-loaded EVs exhibited comparable size distributions, with mean diameters of 116.0 ± 1.8 nm, 114.4 ± 0.6 nm, and 119.6 ± 4.5 nm, respectively (Fig. 2 C). PROTAC loading capacity and encapsulation efficiency were quantified by HPLC and were 155 ng per 1×10⁹ EVs and 97%, respectively (Fig. 2 D). T7-TD-CPP peptide loading was quantified using FITC fluorescence with reference to a FITC standard calibration curve (Fig. 2 E). Verification of surface-displayed peptide localization on the EV membrane To confirm that the engineered T7-TD-CPP peptide is externally displayed and localized along the EV membrane, we performed stochastic optical reconstruction microscopy (STORM) using TAMRA-labeled T7-TD-CPP peptides (Fig. 3 A). Single-particle localization reconstruction revealed well-defined nanoscale clusters with a mean vesicle diameter of 103.44 ± 15.15 nm, consistent with the expected size range of MSC-derived EVs. Quantitative localization mapping indicated robust peptide incorporation, yielding 133 ± 74 localizations per particle, supporting efficient peptide display on the vesicle surface (Fig. 3 B-C). Importantly, the distribution of localization events and the corresponding full width at half maximum (FWHM) profiles exhibited compact and uniform clustering, consistent with membrane insertion rather than nonspecific peptide aggregation. To further validate that the detected peptide signal was membrane-associated, we compared the spatial distribution of TAMRA–T7-TD-CPP–labeled EVs with EVs labeled using CellBrite, a membrane-specific fluorescent dye (Fig. 3 D). Scatter plot analyses of localization number versus FWHM showed highly overlapping patterns between the peptide-labeled EVs and the CellBrite-labeled EVs, indicating that the peptide signal predominantly mapped to the vesicle membrane. Representative merged STORM reconstructions showed a characteristic ring-like localization pattern in both groups (Fig. 3 E), consistent with circumferential membrane localization. Quantitative comparisons further supported nanoscale fidelity of membrane localization: peptide-labeled EVs and CellBrite-labeled EVs exhibited comparable mean diameters (103.44 ± 15.15 nm vs 107.28 ± 18.72 nm, respectively). While the mean vesicle sizes were similar, localization counts differed in accordance with fluorophore density (peptide: 113 ± 74 vs CellBrite: 271 ± 125) (Fig. 3 F). Collectively, these results demonstrate that T7-TD-CPP peptides are stably inserted and externally displayed along the EV membrane, enabling nanoscale surface engineering without evidence of peptide aggregation. Bead-based flow cytometry using the MaxPlex™ Exosome Flow Cytometry Kit demonstrated robust expression of the canonical EV surface markers CD9, CD63, and CD81 in both unmodified MSC-3D EVs and PROTAC & T7-TD-CPP dual-loaded EVs. All samples showed > 100% positivity for these tetraspanins, with no detectable decrease in fluorescence intensity following PROTAC loading and peptide surface functionalization. These data indicate that dual engineering did not alter canonical EV membrane marker profiles, supporting maintenance of EV identity and membrane integrity(Fig. 3 G). Imaging flow cytometry quantification of EV uptake by HBECs To quantitatively assess cellular uptake of engineered EVs by brain endothelial cells, we performed imaging flow cytometry using the Amnis ImageStreamX Mk II platform. HBECs expressing GFP (HBEC-GFP) were incubated with CTDR-labeled EVs for 30 min at room temperature, followed by acquisition and analysis of fluorescence images at the single-cell level (Fig. 4 A). A standardized gating strategy was applied to ensure robust quantification. First, well-focused events were selected using the Gradient RMS feature. Next, single cells were gated based on Aspect Ratio to exclude doublets and cellular aggregates (Fig. 4 A). Uptake of EVs was defined as the presence of CTDR fluorescence within GFP-positive HBECs. Accordingly, EV-positive cells were quantified using a dot plot of Ch02 (GFP) versus Ch05 (CTDR) fluorescence intensity, enabling objective separation of CTDR-negative and CTDR-positive HBEC populations (Fig. 4 B-C). Representative cell images from each gate confirmed accurate identification of EV-associated CTDR signals within single HBECs (Fig. 4 B). Using identical gating thresholds across all groups, we quantified the proportion of CTDR-positive HBECs and EV-derived fluorescence intensity, demonstrating differential uptake profiles among EV formulations (Fig. 4 D). Together, these results establish imaging flow cytometry as a robust high-content platform for quantifying EV uptake into brain endothelial cells and support subsequent functional evaluation of engineered EVs. T7 peptide enhances transferrin receptor–mediated uptake and improves in vitro BBB penetration Given that the T7 motif targets the transferrin receptor (TfR), we first confirmed the expression of TfR1 in human brain endothelial cells. Western blot analysis demonstrated robust TfR1 expression in HBECs, whereas HEK293 cells showed comparatively lower levels (Fig. 5 A), supporting the suitability of HBECs as a BBB-relevant model to evaluate T7-mediated targeting. To assess whether T7 engineering enhances cellular uptake of EVs, we next performed temperature-dependent uptake experiments. Both free T7-TD-CPP peptide and T7-engineered EVs showed markedly increased internalization at 37°C compared with 4°C (Fig. 5 B), indicating that uptake is primarily mediated through active cellular processes rather than nonspecific adsorption. Consistent with receptor-mediated uptake, immunofluorescence imaging demonstrated spatial association between TfR signals and T7-engineered EV fluorescence within HBECs (Fig. 5 C), supporting TfR-dependent binding and/or endocytosis. We then evaluated whether enhanced uptake translates into improved BBB transport using an in vitro BBB transwell model (Fig. 5 D). Barrier integrity was monitored by transendothelial electrical resistance (TEER) over time, demonstrating progressive maturation of tight barrier properties (Fig. 5 E; ****p < 0.0001, as indicated). Using this validated BBB model, we quantified EV transcytosis/penetration across the endothelial layer. Notably, T7-TD-CPP EVs exhibited significantly increased BBB penetration compared with TD-CPP EVs and dextran control (Fig. 5 F; ****p < 0.0001, as indicated), demonstrating that T7-based surface engineering enhances functional transport across brain endothelial barriers. Collectively, these results indicate that T7 engineering strengthens TfR-mediated uptake and improves BBB transcytosis, supporting its use as a brain-targeting EV surface modification strategy. In vivo brain section imaging confirms TfR-associated targeting and cellular distribution of T7-engineered EVs To evaluate whether T7 surface engineering enhances brain localization of EVs in vivo, we monitored the spatiotemporal distribution of fluorescently labeled EVs using real-time intravital microscopy through a cranial window model. To visualize the cerebral vasculature and define vessel boundaries, mice were first intravenously injected with a fluorescently labeled anti-CD31 antibody. Subsequently, fluorescently labeled T7-TD-CPP EVs were administered via intravenous injection, allowing real-time tracking of EV behavior relative to the vascular network and surrounding brain parenchyma over time (Fig. 6 A-B). Time-lapse intravital imaging revealed time-dependent changes in the distribution of T7-TD-CPP EVs (Fig. 6 C). EV signals were predominantly associated with vascular structures at early time points (1 h), whereas punctate EV signals were increasingly observed beyond the vasculature at later time points (6 h and 24 h), indicating progressive extravascular localization within brain tissue. Consistent with intravital observations, ex vivo immunofluorescence analysis of brain sections demonstrated that T7-TD-CPP EVs displayed more prominent EV signals distal to CD31-positive vessels compared with TD-CPP EVs (Fig. 6 D). High-magnification images further confirmed the presence of EV-associated puncta in perivascular and parenchymal regions, indicating broader tissue distribution following T7 surface engineering. Collectively, these results demonstrate that T7 modification alters the in vivo distribution profile of EVs, promoting sustained localization beyond the cerebral vasculature and facilitating enhanced access to brain tissue. These findings support the utility of T7-engineered EVs as an effective strategy for improving brain delivery in vivo. T7-TD-CPP EVs exhibit brain targeting and BBB penetration in vivo To confirm brain delivery of systemically administered EVs at the cellular level, the fluorescence distribution of EVs within brain vascular endothelial cells was examined. Notably, EVs loaded with T7–PROTAC showed clear colocalization with transferrin receptors expressed on endothelial cells, as evidenced by merged fluorescence signals. This colocalization indicates transferrin receptor–mediated transendocytosis, supporting enhanced blood–brain barrier permeability of T7-modified EVs (Fig. 7 A,B). To further evaluate brain parenchymal distribution, immunofluorescence analysis was performed on brain sections collected 4 h after intravenous injection of engineered EVs. Tissue sections were co-stained with cell type–specific markers to assess cellular localization patterns. T7-TD-CPP EVs exhibited more frequent association with NeuN⁺ neurons compared with TD-CPP EVs, which showed relatively sparse neuronal-associated signals (Fig. 7 C). In contrast, EV signals within GFAP⁺ astrocytes were limited in both groups, with only modest astrocyte-associated puncta observed in the T7-modified EV condition (Fig. 7 D). Collectively, these results demonstrate that T7 modification enhances endothelial transcytosis and preferential neuronal-associated localization of EVs in vivo. Ex vivo IVIS imaging demonstrates enhanced brain accumulation and prolonged retention of T7-TD-CPP EVs To quantify whole-body biodistribution and assess brain-targeting efficiency, ex vivo IVIS imaging was performed 6 h after intravenous injection of EVs. Compared with TD-CPP EVs, T7-TD-CPP EVs exhibited markedly stronger fluorescence signals in the brain (Fig. 8 A). Quantitative analysis of brain-associated radiant efficiency confirmed a significant increase in signal intensity following T7 modification (p < 0.01, Fig. 8 B). To further examine the temporal dynamics of EV distribution, additional IVIS imaging was conducted at 6 h and 96 h after injection of T7-TD-CPP EVs (Fig. 8 C). Brain-associated fluorescence signals remained detectable up to 96 h post-administration, whereas fluorescence intensities in peripheral organs, particularly the liver and kidney, decreased substantially over time (p < 0.0001, Fig. 8 D), consistent with progressive systemic clearance. Ex vivo imaging also revealed detectable fluorescence signals in major clearance-associated organs, including the liver and kidney, across treatment groups (Fig. 8 E). Quantitative comparison across organs demonstrated that, among the tissues analyzed, the brain exhibited the most pronounced enhancement in fluorescence intensity following T7 modification (Fig. 8 F). Collectively, these results demonstrate that T7-TD-CPP surface engineering enhances brain-associated accumulation of systemically administered EVs and supports sustained brain-localized fluorescence signals relative to non-targeted EV formulations, highlighting the contribution of the T7 motif to improved in vivo brain targeting. Dual-loaded T7-targeted PROTAC EVs drive robust in vivo degradation of LRRK2 in an MPTP Parkinson’s disease mouse model To evaluate the in vivo therapeutic efficacy of the dual-loading strategy, an MPTP-induced Parkinson’s disease mouse model was employed (Fig. 9 A). Mice received intraperitoneal injections of MPTP (20 mg/kg) on Day 0 to induce Parkinsonian pathology, followed by intravenous administration of EV formulations on Day 4. Animals were sacrificed on Day 6, and brain tissues were harvested for biochemical analysis of LRRK2 expression. As expected, MPTP treatment markedly increased LRRK2 levels in the brain compared with control mice (Fig. 9 B-C). Treatment with free PROTAC resulted in only partial reduction of LRRK2 expression, consistent with limited efficacy under systemic administration. In contrast, T7-TD-CPP–PROTAC EVs produced the most pronounced suppression of LRRK2 expression, significantly outperforming both free PROTAC and TD-CPP–PROTAC EVs (Fig. 9 C). TD-CPP–PROTAC EVs induced only a moderate reduction in LRRK2 levels, whereas T7-targeted dual-loaded EVs achieved pronounced LRRK2 degradation in vivo. Collectively, these results demonstrate that T7-TD-CPP–engineered, PROTAC-loaded EVs substantially enhance the in vivo pharmacological activity of PROTAC cargo in the MPTP mouse model, supporting the effectiveness of the dual-loading strategy for brain-targeted protein degradation. Discussion Scalable manufacturing and stringent quality control are critical prerequisites for the clinical translation of EV-based therapeutics. In the EV field, increasing consensus has been reached on standardized requirements for EV identity, purity, and reporting, with particular emphasis on preventing non-vesicular contamination and ensuring batch-to-batch reproducibility [ 1 , 2 ]. In this study, EVs derived from 3D spheroid MSC cultures retained canonical EV characteristics, including expected morphology and size distribution, robust expression of tetraspanin markers, and minimal contamination by intracellular or organelle-associated proteins. These results support the feasibility of establishing a manufacturing pipeline suitable for clinical development and downstream engineering. The surface engineering strategy adopted here is notable for its simplicity and translational relevance. Many ligand-presenting EV approaches rely on genetic modification of parental cells, which can complicate regulatory approval and large-scale manufacturing. In contrast, the T7-TD-CPP peptide was spontaneously inserted into the EV membrane without external energy input, while preserving EV structure and size distribution. Importantly, stochastic optical reconstruction microscopy (STORM) confirmed membrane-associated ring-like localization patterns, providing direct nanoscale evidence of membrane insertion rather than peptide aggregation. This strengthens the reliability of surface functionalization claims, which remain a point of debate in EV targeting studies. Transferrin receptor (TfR) targeting is one of the most validated paradigms for BBB delivery, although enhanced endothelial binding does not always translate into efficient transcytosis [ 3 ]. The T7 peptide (HAIYPRH) is a well-established TfR-binding motif reported to enhance BBB penetration across multiple nanocarrier platforms [ 4 ]. In this study, T7-functionalized EVs showed increased uptake by brain endothelial cells, enhanced transport across a TEER-supported in vitro BBB model, and in vivo distribution beyond CD31-positive vascular boundaries. Notably, T7-modified EVs exhibited markedly enhanced association with NeuN-positive neurons compared with TD-CPP EVs, while association with GFAP-positive astrocytes remained limited. These findings indicate that T7-based surface functionalization can promote preferential neuronal engagement and that EV surface engineering can modulate cell-type interaction profiles in the brain. The dual-loading architecture represents a central conceptual advance of this study. While previous reports have demonstrated ligand-modified EVs for brain-targeted delivery [ 5 , 6 ], many have focused on nucleic acid cargos or biodistribution without robust pharmacodynamic evidence. Here, a hydrophobic small-molecule PROTAC requiring cytosolic access to the ubiquitin–proteasome machinery was incorporated as a therapeutically relevant intravesicular cargo. Targeted protein degradation has emerged as a transformative modality capable of eliminating both catalytic and scaffolding functions of disease-driving proteins [ 7 ]. XL01126 is a potent and selective LRRK2 degrader with reported BBB permeability [ 8 ]; however, effective neuronal delivery remains a challenge. In an MPTP-induced Parkinson’s disease model, T7-targeted, dual-loaded EVs achieved the most pronounced reduction of LRRK2 compared with free PROTAC and non-targeted EVs, accompanied by decreased α-synuclein levels. These data demonstrate that combining BBB-targeted surface display with intravesicular PROTAC delivery enhances in vivo pharmacological outcomes. EV-mediated delivery further provides intrinsic advantages for BBB traversal. EVs exploit multiple transport pathways across the BBB, including receptor-mediated and ligand-independent mechanisms. Our results indicate that EVs can traverse the BBB even without ligand-mediated targeting, supporting their inherent BBB-crossing capability. The combination of CPP-mediated surface modification and intravesicular loading offers several advantages. First , modular coupling of CPPs with different targeting ligands enables customization of EVs for diverse targets while accommodating varied cargos. Second , intravesicular loading allows quantitative and tunable control of cargo concentration while minimizing interference between surface ligands and encapsulated therapeutics. Third , luminal loading facilitates intracellular delivery following BBB traversal, which is particularly advantageous for cytosol-dependent therapeutics such as PROTACs. Although extensive efforts have been devoted to EV-based delivery systems, many studies rely on vector-mediated preloading strategies that pose hurdles for clinical translation. Post-loading approaches often lack systematic evaluation of loading efficiency and EV quality. In contrast, this study provides objective and quantitative metrics for loading efficiency and EV quality and employs clinical-grade EVs, strengthening translational relevance and regulatory comparability. Several limitations remain. The precise mechanisms underlying BBB transport require further elucidation, as TfR-mediated trafficking can favor lysosomal routing under certain conditions [ 3 , 4 ]. Fluorescence-based biodistribution may be confounded by dye transfer, warranting orthogonal validation approaches [ 1 , 2 ]. Rigorous pharmacokinetic and pharmacodynamic profiling is also needed to quantify regional brain exposure and dose–response relationships of XL01126 [ 7 , 8 ]. Finally, long-term safety, immunogenicity, repeated-dosing studies, and validation in additional disease models will be essential for clinical translation. In summary, this study establishes a clinically oriented EV engineering framework integrating non-genetic BBB-targeted surface functionalization (T7-TD-CPP) with intravesicular PROTAC loading (XL01126). This dual-loading EV platform enhances brain accumulation, neuronal engagement, and in vivo target degradation in a Parkinson’s disease model, supporting the use of clinical-grade MSC-derived EVs as modular CNS delivery systems for advanced therapeutic modalities. Declarations Conflict of interest: None Acknowledgment and Funding: This work was supported by the K-Brain Project of the National Research Foundation (NRF) funded by the Korean government (MSIT) under Grant No. RS-2024-00399320.. S&E Bio Inc. provided support for this study in the form of salaries to J.E.K. The funders had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation. The specific roles of the authors are described in the author contributions statement. Disclosures: The authors have read and understood the journal’s policies. The authors of this manuscript disclose the following competing interests: J. E. K. is a paid employee of S&E Bio Inc. This competing interest did not affect adherence to the data- and material-sharing policies of the Journal of Nanobiotechnology . Author Contribution Statement: J.E.K. and O.Y.B. designed the study, analyzed all samples, interpreted the data, and wrote the manuscript. J. E. K. and Y.E.J. acquired and analyzed the data. J.E.K. revised the study for important intellectual content. J.E.K. and O.Y.B. reviewed the report and provided scientific advice. O.Y.B. designed and funded the study, analyzed all samples, interpreted the data, wrote, and approved the submission of the manuscript. Data availability statement Ethics approval statement References Théry C, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the ISEV and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018;7:1535750. Lener T, et al. Applying extracellular vesicles based therapeutics in clinical trials—an ISEV position paper. J Extracell Vesicles. 2015;4:30087. Johnsen KB, Burkhart A, Thomsen LB, Andresen TL, Moos T. Targeting the transferrin receptor for brain drug delivery. Prog Neurobiol. 2019;181:101665. Wang Z, et al. Enhanced anti-ischemic stroke of ZL006 by T7-conjugated PEGylated liposomes drug delivery system. Sci Rep. 2015;5:12651. Alvarez-Erviti L, et al. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol. 2011;29:341–5. Liu Y, et al. Targeted exosome-mediated delivery of opioid receptor Mu siRNA to the brain for the treatment of morphine relapse. Sci Rep. 2015;5:17543. Békés M, Langley DR, Crews CM. PROTAC targeted protein degraders: the past is prologue. Nat Rev Drug Discov. 2022;21:181–200. Liu X, et al. Discovery of XL01126: a potent, fast, cooperative, selective, orally bioavailable and blood–brain barrier penetrant proteolysis targeting chimera degrader of LRRK2. J Am Chem Soc. 2022;144:16930–52. Son JP, et al. Mesenchymal Stem Cell–Extracellular Vesicle Therapy for Stroke. Stem Cells Translational Med. 2023;12:459–71. Welsh JA et al. Minimal information for studies of extracellular vesicles 2023 (MISEV2023): a position statement of the International Society for Extracellular Vesicles (ISEV). J Extracell Vesicles (2024). Rust MJ, Bates M, Zhuang X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods. 2006;3:793–6. de Castilla PEM. Extracellular vesicles as a drug delivery system: a systematic review of preclinical studies. Biochimica et Biophysica Acta (BBA) - General Subjects (2021). Rädler JO et al. Exploiting the biogenesis of extracellular vesicles for drug delivery and functionalization. Semin Cell Dev Biol (2023). Kim J-E, et al. Engineered MSC-EVs loaded with BDNF-enhancing neuropeptides via a non-disruptive method enhance post-stroke neuroregeneration via intranasal delivery. J Nanobiotechnol. 2025;23:594. https://doi.org/10.1186/s12951-025-03654-x . Additional Declarations No competing interests reported. Supplementary Files Suppledata1.mp4 T7PROTACBrainhoming2602091.png Suppledata2.mp4 Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 17 Mar, 2026 Reviews received at journal 12 Mar, 2026 Reviews received at journal 06 Mar, 2026 Reviewers agreed at journal 04 Mar, 2026 Reviewers agreed at journal 02 Mar, 2026 Reviewers agreed at journal 24 Feb, 2026 Reviewers invited by journal 18 Feb, 2026 Editor assigned by journal 10 Feb, 2026 Submission checks completed at journal 10 Feb, 2026 First submitted to journal 09 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8830833","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":594454523,"identity":"6758873d-0571-4638-8bbd-3cfe9685c978","order_by":0,"name":"Ji-Eun Kim","email":"","orcid":"","institution":"S\u0026E Bio, Inc","correspondingAuthor":false,"prefix":"","firstName":"Ji-Eun","middleName":"","lastName":"Kim","suffix":""},{"id":594454524,"identity":"46af107c-fa4b-4a4f-bf89-daa07ae618d9","order_by":1,"name":"Ye Eun Ji","email":"","orcid":"","institution":"Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"Eun","lastName":"Ji","suffix":""},{"id":594454525,"identity":"f8158bd4-4e42-4fd8-aacd-1b95a7485582","order_by":2,"name":"Hyeock Yang","email":"","orcid":"","institution":"Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University","correspondingAuthor":false,"prefix":"","firstName":"Hyeock","middleName":"","lastName":"Yang","suffix":""},{"id":594454526,"identity":"3cea1e88-6c77-40c5-a182-b2eafd133d5a","order_by":3,"name":"Daeho Kwon","email":"","orcid":"","institution":"Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University","correspondingAuthor":false,"prefix":"","firstName":"Daeho","middleName":"","lastName":"Kwon","suffix":""},{"id":594454527,"identity":"d1ddda15-0670-4005-8be2-5aba78219a53","order_by":4,"name":"Jaein Yoo","email":"","orcid":"","institution":"Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University","correspondingAuthor":false,"prefix":"","firstName":"Jaein","middleName":"","lastName":"Yoo","suffix":""},{"id":594454528,"identity":"5d4cfb06-56da-4592-a3e6-4e6bb74001e4","order_by":5,"name":"Doory Kim","email":"","orcid":"","institution":"Hanyang University","correspondingAuthor":false,"prefix":"","firstName":"Doory","middleName":"","lastName":"Kim","suffix":""},{"id":594454529,"identity":"81b5fecb-809f-4616-a25e-9f715c7ddadb","order_by":6,"name":"Oh Young Bang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIiWNgGAWjYDACCShtACI+kKyFcQbJWph5iNHBL91j+Jh3Ry2DOfvZw69t/thFy8/IPfyCocYmGpcWyTlnjI15zxxnsOzJS7PObUvO3XAjL82C4VhabgMOLQY3csykeduOMRgcyDEzzm1gzt0gkWNmwNhwmAgt59+YGVv8qc+dP4M4LTUghvFjBjagSiDjAT4tkjPSig3nth3gsZzxxoyxt+147oYzb8wYEvD4hV8ieeODt211cub8OcYffvypzp3fDmR8qLHBqYWBgQMUI4dBMcIGiyM2iQScykGA/QGQqAOxmGHphZm4hDMKRsEoGAUjBQAAaSZbNvTSMsEAAAAASUVORK5CYII=","orcid":"","institution":"Samsung Medical Center","correspondingAuthor":true,"prefix":"","firstName":"Oh","middleName":"Young","lastName":"Bang","suffix":""}],"badges":[],"createdAt":"2026-02-09 13:10:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8830833/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8830833/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103322652,"identity":"df1865e5-a1c8-4be1-9771-caafeabb7eda","added_by":"auto","created_at":"2026-02-24 12:12:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1996771,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological, biochemical, and nanoscale characterization of MSC-derived EVs from 3D spheroid cultures.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) \u003cstrong\u003eGMP-manufactured, clinical-grade MSC 3D EVs were produced with rigorously controlled purity, quality, and batch-to-batch consistency.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(B-a) Representative transmission electron microscopy (TEM) image showing round-shaped, spherical EV morphology Scale bars: 200 nm.\u003cbr\u003e\n(B-b) Nanoparticle tracking analysis (NTA) demonstrating EV size distribution with mean diameter 127.0 ± 3.6 nm and mode diameter 117.3 ± 4.6 nm.\u003cbr\u003e\n(C) Flow cytometry profiling of EV surface tetraspanin markers showing high positivity for CD9 (98.88%), CD63 (98.82%), and CD81 (100.00%).\u003cbr\u003e\n(D) ELISA-based assessment of non-vesicular contaminants (gentamicin, BSA) and intracellular/cellular debris markers (calnexin, histone, GM130, cytochrome c), which were not detected (N.D.) or below the lower limit of quantification (LLOQ), supporting high EV purity.\u003cbr\u003e\n(E,F) STORM super-resolution imaging and localization analysis of EV nanoscale structure. Reconstructed clusters indicate a mean EV diameter of 109.73 ± 23.86 nm, with fluorescence emission-derived FWHM values of 110.86 nm (x-axis) and 105.77 nm (y-axis). Localization analysis shows 221 ± 148 localizations per particle, and line-scan profiling reveals a peak-to-peak distance of 84.32 nm, consistent with hollow EV architecture Scale bars: 800 nm.\u003c/p\u003e","description":"","filename":"T7PROTACBrainhoming2602092.png","url":"https://assets-eu.researchsquare.com/files/rs-8830833/v1/ec83541889b46583a8993a46.png"},{"id":103322762,"identity":"d7fde388-5072-4b8d-b49e-19527df6e08c","added_by":"auto","created_at":"2026-02-24 12:13:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1779828,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesign, physicochemical properties, and loading characterization of T7-TD-CPP–engineered PROTAC-loaded MSC-3D EVs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic illustration of the multifunctional membrane-inserting peptide design comprising a\u003c/p\u003e\n\u003cp\u003etransferrin receptor–binding T7 motif, flexible linker (LP), transmembrane domain (TD), and cell-penetrating peptide (CPP) to enable spontaneous EV membrane insertion and surface display.\u003cbr\u003e\n(B) Chemical structure of the PROTAC molecule XL01126 selected as hydrophobic small-molecule cargo for passive EV encapsulation.\u003cbr\u003e\n(C) Characterization of peptide constructs and EV formulations.\u003cbr\u003e\n(a) Physicochemical properties of TD-CPP and T7-TD-CPP peptides (molecular weight, net charge, and hydrophilic/hydrophobic ratio).\u003cbr\u003e\n(b) Amino acid sequences and domain composition of TD-CPP and T7-TD-CPP peptides.\u003cbr\u003e\n(c) Representative TEM images and NTA profiles of PROTAC-loaded MSC-3D EV, PROTAC \u0026amp; T7-TD-CPP-loaded MSC-3D EV, and PROTAC \u0026amp; TD-CPP-loaded MSC-3D EV are shown. Mean particle diameters are indicated below each condition.\u003cbr\u003e\n(D) Quantification of PROTAC (XL01126) loading in EVs based on HPLC- mass spectrometry (MS) standard curves, together with encapsulation efficiency (EE%).\u003cbr\u003e\n(E) Quantitative analysis of T7-TD-CPP peptide surface loading. Peptide amounts were determined using an FITC standard curve, and loading capacity per EV and encapsulation efficiency were calculated as a function of initial peptide input.\u003c/p\u003e","description":"","filename":"T7PROTACBrainhoming2602093.png","url":"https://assets-eu.researchsquare.com/files/rs-8830833/v1/4eefa5562a5adf4b74ac3f12.png"},{"id":103322635,"identity":"f4b2090c-f2a3-4459-b594-c4ffa3e7bb3d","added_by":"auto","created_at":"2026-02-24 12:12:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":832943,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSTORM super-resolution imaging validates membrane-localized surface display of externally engineered T7-TD-CPP peptides on EVs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic illustration of TAMRA-labeled T7-TD-CPP peptides displayed on the EV membrane, with CellBrite used as a membrane dye control.\u003c/p\u003e\n\u003cp\u003e(B) Representative STORM localization reconstruction image of TAMRA–T7-TD-CPP–engineered EVs showing a ring-like nanoscale distribution consistent with membrane localization. Scale bar: 200 nm.\u003c/p\u003e\n\u003cp\u003e(C) Histogram of localization numbers per reconstructed EV particle for TAMRA–T7-TD-CPP–labeled EVs compared with CellBrite-labeled EVs.\u003c/p\u003e\n\u003cp\u003e(D) Scatter plot of localization number versus full width at half maximum (FWHM) for TAMRA–T7-TD-CPP–labeled EVs and CellBrite-labeled EVs, showing overlapping nanoscale distributions.\u003c/p\u003e\n\u003cp\u003e(E) Distribution of FWHM values derived from STORM localization analysis for TAMRA–T7-TD-CPP–labeled EVs and CellBrite-labeled EVs.\u003c/p\u003e\n\u003cp\u003e(F) Summary quantification of mean particle size and localization number. TAMRA–T7-TD-CPP–labeled EVs and CellBrite-labeled EVs exhibited comparable mean diameters (103.44 ± 15.15 nm vs 107.28 ± 18.72 nm), while localization numbers differed according to fluorophore density (113 ± 74 vs 271 ± 125).\u003c/p\u003e\n\u003cp\u003e(G) Surface expression of CD9, CD63, and CD81 on MSC-3D EVs and PROTAC \u0026amp; T7-TD-CPP-loaded MSC-3D EVs was assessed by bead-based flow cytometry (MaxPlex™ Exosome Flow Cytometry Kit, BioLegend). Representative histograms are shown, with isotype controls in gray. Both EV preparations maintained high positivity for all tetraspanin markers.\u003c/p\u003e","description":"","filename":"T7PROTACBrainhoming2602094.png","url":"https://assets-eu.researchsquare.com/files/rs-8830833/v1/c8441d65090eececcb2f0f3c.png"},{"id":103506876,"identity":"785991e8-c025-4f89-ba68-295e2375673d","added_by":"auto","created_at":"2026-02-26 13:39:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1428482,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImaging flow cytometry workflow and gating strategy for quantification of CTDR-labeled EV uptake by HBEC-GFP cells (ImageStreamX Mk II).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Stepwise gating strategy for imaging flow cytometry analysis. Well-focused events were selected using Gradient RMS, followed by singlet gating based on Aspect Ratio to exclude doublets and aggregates.\u003cbr\u003e\n(B) Representative brightfield and fluorescence images of HBEC-GFP cells (Ch02) incubated with CTDR-labeled EVs (Ch05), demonstrating EV signal association within single cells Scale bars: 10 μm.\u003cbr\u003e\n(C) Dot plot analysis of fluorescence intensities in Ch02 (GFP) versus Ch05 (CTDR) defining EV-negative and EV-positive HBEC populations. EV uptake was quantified as CTDR signal within GFP-positive HBECs.\u003cbr\u003e\n(D) Summary of EV uptake metrics including the proportion of CTDR-positive cells and/or CTDR mean fluorescence intensity (MFI), analyzed using identical gating thresholds across experimental groups.\u003c/p\u003e\n\u003cp\u003eAcquisition settings: 40× objective; lasers: 488 nm (0.1 mW), 642 nm (15 mW), SSC (0.5 mW); 2,000 single-cell events acquired per condition. Compensation was performed using unstained cells, HBEC-GFP only, and EV-CTDR only controls.\u003c/p\u003e","description":"","filename":"T7PROTACBrainhoming2602095.png","url":"https://assets-eu.researchsquare.com/files/rs-8830833/v1/dd424919d9113e68cf3ba7de.png"},{"id":103322766,"identity":"1c80213e-cfc2-43b1-b7cc-2b531364bc19","added_by":"auto","created_at":"2026-02-24 12:13:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4602237,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eT7 peptide enhances transferrin receptor–dependent uptake and promotes in vitro BBB penetration of engineered EVs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot analysis of transferrin receptor 1 (TfR1) expression in HEK293 cells and human brain endothelial cells (HBECs).\u003c/p\u003e\n\u003cp\u003e(B) Temperature-dependent uptake assay of T7-TD-CPP peptide and engineered EVs in HBECs at 4 °C and 37 °C. Increased intracellular signal was observed at 37 °C compared with 4 °C.\u003c/p\u003e\n\u003cp\u003e(C) Immunofluorescence images showing spatial association between transferrin receptor (TfR) signal and uptake of T7-engineered EVs in HBECs. Scale bars: 20 μm.\u003c/p\u003e\n\u003cp\u003e(D) Schematic illustration of the experimental workflow for establishment of an in vitro BBB transwell model and EV penetration analysis.\u003c/p\u003e\n\u003cp\u003e(E) Time-course measurement of transendothelial electrical resistance (TEER) during BBB maturation, indicating progressive barrier formation (****p \u0026lt; 0.0001, as indicated).\u003c/p\u003e\n\u003cp\u003e(F) Quantification of BBB penetration efficiency comparing control dextran, TD-CPP EVs, and T7-TD-CPP EVs. T7-engineered EVs exhibited significantly enhanced transport across the BBB layer compared with control groups (****p \u0026lt; 0.0001, as indicated).\u003c/p\u003e","description":"","filename":"T7PROTACBrainhoming2602096.png","url":"https://assets-eu.researchsquare.com/files/rs-8830833/v1/473c213913159ad84dceb0ef.png"},{"id":103322644,"identity":"a6542872-9ea2-48d4-9807-b478c8e71fe1","added_by":"auto","created_at":"2026-02-24 12:12:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":9396832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntravital microscopy reveals time-dependent vascular association and brain distribution of T7-engineered EVs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Experimental timeline for cranial window implantation, vascular labeling, EV administration, and real-time intravital microscopy. CD31 was intravenously injected to label cerebral vasculature, followed by intravenous administration of fluorescently labeled T7-TD-CPP EVs prior to imaging at 1 h, 6 h, and 24 h post-injection.\u003c/p\u003e\n\u003cp\u003e(B) Representative images of the intravital microscopy setup and cranial window preparation used for real-time imaging of cerebral microvasculature and EV distribution in live mice.\u003c/p\u003e\n\u003cp\u003e(C) Representative intravital microscopy images showing time-dependent distribution of T7-TD-CPP EVs (FSD647) relative to CD31-labeled cerebral blood vessels (FSD555) at 1 h, 6 h, and 24 h after intravenous injection.\u003c/p\u003e\n\u003cp\u003e(D) Immunofluorescence images of fixed brain sections showing spatial distribution of EV signals relative to CD31-positive blood vessels. Higher-magnification images highlight EV signals detected in close proximity to the cerebral vasculature.\u003c/p\u003e\n\u003cp\u003eScale bars: as indicated in the panels.\u003c/p\u003e","description":"","filename":"T7PROTACBrainhoming2602097.png","url":"https://assets-eu.researchsquare.com/files/rs-8830833/v1/b9b880429a9134e826839bfc.png"},{"id":103322765,"identity":"c6fa4e44-6770-4a85-b7f5-8faa4e444e08","added_by":"auto","created_at":"2026-02-24 12:13:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":10582519,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo brain section imaging reveals transferrin receptor association and neuronal distribution of T7-engineered EVs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A, B) \u003cstrong\u003eCellular localization of engineered EVs in the brain following systemic administration.\u003c/strong\u003e\u003cbr\u003e\nImmunofluorescence images of brain sections showing colocalization of T7–PROTAC–loaded EVs with transferrin receptors on vascular endothelial cells. Merged signals indicate transferrin receptor–associated endothelial transport.\u003cbr\u003e\n (C) Immunofluorescence staining of brain sections collected 4 h after intravenous injection demonstrating association of EV signals with NeuN⁺ neurons. T7-TD-CPP EVs exhibit more pronounced neuronal engagement compared with TD-CPP EVs, as highlighted in magnified views.\u003c/p\u003e\n\u003cp\u003e(D) Immunofluorescence staining for GFAP⁺ astrocytes showing limited astrocytic association of EVs in both groups, with modest EV signal observed in the T7-modified EV condition.\u003c/p\u003e","description":"","filename":"T7PROTACBrainhoming2602098.png","url":"https://assets-eu.researchsquare.com/files/rs-8830833/v1/4e871c22c35695254f75db48.png"},{"id":103322763,"identity":"e3ac0059-1f69-4469-98bd-286717962302","added_by":"auto","created_at":"2026-02-24 12:13:03","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":5419162,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEx vivo IVIS biodistribution confirms enhanced brain accumulation and prolonged retention of T7-TD-CPP EVs following systemic delivery.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representative ex vivo IVIS images of major organs collected 6 h post–intravenous injection showing increased brain fluorescence in the T7-TD-CPP EV group compared with TD-CPP EVs.\u003cbr\u003e\n(B) Quantification of brain fluorescence intensity demonstrating significantly enhanced brain accumulation by T7 modification (p \u0026lt; 0.01).\u003cbr\u003e\n(C) Representative ex vivo IVIS images of major organs acquired at 6 h and 96 h after intravenous injection of T7-TD-CPP EVs, illustrating temporal changes in organ distribution.\u003cbr\u003e\n(D) Quantitative analysis of organ-specific fluorescence intensities at 6 h and 96 h post-injection, demonstrating significant temporal changes in peripheral organs (****p \u0026lt; 0.0001, **p \u0026lt; 0.01).\u003cbr\u003e\n(E) Representative ex vivo IVIS images from individual mice showing whole-body organ distribution of TD-CPP EVs and T7-TD-CPP EVs.\u003c/p\u003e\n\u003cp\u003e(F) Quantitative comparison of fluorescence intensity across major organs following intravenous administration of control, TD-CPP EVs, and T7-TD-CPP EVs. T7-TD-CPP EVs show significantly enhanced brain accumulation compared with TD-CPP EVs and control groups (*p \u0026lt; 0.05, **p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"T7PROTACBrainhoming2602099.png","url":"https://assets-eu.researchsquare.com/files/rs-8830833/v1/149b1d25d530c379d8ac9a79.png"},{"id":103322764,"identity":"cf59dda1-4430-44b7-8cc8-a1627c1fe598","added_by":"auto","created_at":"2026-02-24 12:13:03","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":349740,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDual-loaded T7-targeted PROTAC EVs enable robust in vivo degradation of LRRK2 in an MPTP Parkinson’s disease model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Experimental design schematic for the MPTP-induced Parkinson’s disease model: mice received MPTP (20 mg/kg, i.p.) on Day 0, followed by intravenous administration of EV formulations on Day 4, and tissue collection on Day 6 (48 h post-injection).\u003cbr\u003e\n(B) Representative immunoblots of LRRK2 in mouse brain tissues across experimental groups, with β-actin as a loading control. (C) Quantification of LRRK2 protein levels showing the strongest reduction in the T7-TD-CPP–PROTAC EV group compared with free PROTAC and TD-CPP–PROTAC EVs.\u003c/p\u003e","description":"","filename":"T7PROTACBrainhoming26020910.png","url":"https://assets-eu.researchsquare.com/files/rs-8830833/v1/46dc51f46935bb8630a32c90.png"},{"id":103510005,"identity":"f987f70b-9407-413e-969a-a11bb8c2acfa","added_by":"auto","created_at":"2026-02-26 14:02:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":38527028,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8830833/v1/67c7c3d7-e5c0-408f-8aa5-f06a7d8d65d4.pdf"},{"id":103322653,"identity":"cef1d873-e9d7-4615-91a6-31f59238749c","added_by":"auto","created_at":"2026-02-24 12:12:59","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1110784,"visible":true,"origin":"","legend":"","description":"","filename":"Suppledata1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8830833/v1/a8cb946212bd7f9e5df02021.mp4"},{"id":103322761,"identity":"6706e9b0-2fb3-4845-b259-f1657ef90394","added_by":"auto","created_at":"2026-02-24 12:13:02","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2786885,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"T7PROTACBrainhoming2602091.png","url":"https://assets-eu.researchsquare.com/files/rs-8830833/v1/77e9e6e54dd77d2fce6513f4.png"},{"id":103322767,"identity":"db0da824-021a-496e-93d1-66c54ced2e19","added_by":"auto","created_at":"2026-02-24 12:13:04","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1477580,"visible":true,"origin":"","legend":"","description":"","filename":"Suppledata2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8830833/v1/2e5a69d6d922e0d6986b26c7.mp4"}],"financialInterests":"No competing interests reported.","formattedTitle":"Brain-targeted PROTAC delivery by dual-functional extracellular vesicles achieves robust LRRK2 degradation in a Parkinson’s disease model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNeurodegenerative disorders remain difficult to treat, in large part because most therapeutics fail to reach effective concentrations in the central nervous system (CNS). Parkinson\u0026rsquo;s disease (PD), a representative neurodegenerative disease, is characterized by progressive neuronal dysfunction and neurodegeneration driven by pathological protein networks, including aberrant LRRK2 signaling and α-synuclein pathology. Despite extensive efforts to develop disease-modifying agents, the blood\u0026ndash;brain barrier (BBB) remains a key obstacle that restricts systemic therapeutics from accessing the brain parenchyma. Consequently, the development of delivery systems capable of efficiently traversing the BBB and enabling functional target engagement in neural tissues is urgently needed.\u003c/p\u003e \u003cp\u003eExtracellular vesicles (EVs) have emerged as attractive drug delivery vehicles owing to their biocompatibility, low immunogenicity, and intrinsic capacity for intercellular transport. Mesenchymal stem/stromal cell (MSC)-derived EVs are of particular interest because MSCs have been widely investigated in regenerative and immunomodulatory settings, and MSC-EVs are increasingly considered clinically relevant biological carriers. However, despite rapid progress in EV engineering, several challenges continue to limit translation of EV-based CNS therapeutics. A major limitation is the difficulty of combining robust BBB penetration with effective cargo loading while preserving EV integrity and manufacturing feasibility. Standardized frameworks have emphasized rigorous EV identity and purity characterization, reproducible production, and the minimization of non-vesicular contaminants to support clinical development [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Yet many existing CNS-targeting EV platforms rely on genetic modification of parental cells to present targeting ligands, potentially complicating regulatory pathways and large-scale manufacturing.\u003c/p\u003e \u003cp\u003eLigand-based targeting of BBB receptors offers a promising strategy to enhance CNS delivery. Among potential BBB targets, the transferrin receptor (TfR) is one of the most extensively validated routes because of its abundant expression on brain endothelial cells and its involvement in transcytosis-mediated transport [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The T7 peptide (HAIYPRH), a TfR-binding ligand, has been widely applied to increase BBB transport and brain accumulation of synthetic nanocarriers such as liposomes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, in EV-based delivery contexts, enhancing brain accumulation does not necessarily translate into effective parenchymal access or neuronal engagement, and simple receptor binding can instead result in endothelial retention or endosomal degradation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, clinically aligned engineering approaches that enable stable surface presentation of BBB-targeting ligands\u0026mdash;without genetic manipulation\u0026mdash;while maintaining EV structure and function are of considerable interest.\u003c/p\u003e \u003cp\u003eIn parallel, targeted protein degradation has emerged as a transformative therapeutic modality. Proteolysis-targeting chimeras (PROTACs) promote ubiquitin-dependent degradation of proteins of interest and can eliminate both enzymatic and scaffolding functions of disease drivers [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. LRRK2 is a compelling PD target supported by genetic evidence and mechanistic links to vesicular trafficking and neuroinflammation. XL01126 has recently been developed as a potent and selective LRRK2 degrader with reported BBB penetration in mice [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Nevertheless, systemic PROTAC administration can still face limitations in effective CNS exposure, cellular delivery, and intracellular availability necessary for robust degradation activity. Thus, a BBB-permeable delivery platform that can transport PROTACs into the CNS and promote neuronal target engagement would provide a powerful enabling strategy for PD and other neurodegenerative diseases.\u003c/p\u003e \u003cp\u003eHere we report a clinically oriented dual-functional EV platform that integrates BBB targeting and PROTAC delivery within a single carrier. We utilized clinical-grade MSC-derived EVs produced from 3D spheroid cultures and employed a non-genetic surface engineering strategy in which a multifunctional peptide composed of a TfR-targeting motif (T7), a transmembrane domain (TD), and a cell-penetrating peptide (CPP) is spontaneously inserted into the EV membrane to enable stable external display. In parallel, we passively encapsulated the hydrophobic PROTAC XL01126 as an intravesicular cargo. Using stochastic optical reconstruction microscopy (STORM), we directly verified nanoscale membrane localization of the engineered peptide, supporting bona fide surface display rather than peptide aggregation. Functionally, T7-engineered EVs showed enhanced uptake by brain endothelial cells, improved translocation across an in vitro BBB model with TEER-validated barrier maturation, and significantly increased brain accumulation in vivo. Importantly, intravenously administered engineered EVs were detected beyond vascular boundaries and exhibited preferential neuronal association. Finally, in an MPTP-induced PD mouse model, dual-loaded T7-targeted PROTAC EVs achieved robust in vivo degradation of LRRK2 and reduced α-synuclein levels, outperforming free PROTAC and non-targeted EV formulations. Together, these findings establish a scalable and clinically compatible framework for brain-targeted PROTAC delivery using engineered MSC-EVs.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eAll studies involving human subjects were approved by the Institutional Review Board of the Samsung Medical Center. Wharton's Jelly (WJ) was provided to healthy volunteers. All volunteers or their guardians provided written informed consent for participation in the study. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (Laboratory Animal Research Center, AAALAC International-approved facility) of Samsung Medical Center.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation and isolation of clinical-scale MSC-EVs\u003c/h2\u003e \u003cp\u003ePreparation and isolation of clinical-scale MSC-EVs were performed as described previously [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Detailed methods are provided in the Supplementary Data. Schematics of EV production, isolation, and quality control are shown in Supplementary Fig.\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCharacterization of EVs\u003c/h3\u003e\n\u003cp\u003eFollowing the guidelines recommended by the International Society for Extracellular Vesicles (Minimal Information for Studies of Extracellular Vesicles), EVs isolated from WJ-MSC culture medium were characterized in terms of morphology, size distribution, surface markers, purity, potency markers, efficacy, stability, and safety [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eNanoparticle Tracking Analysis (NTA)\u003c/h3\u003e\n\u003cp\u003eThe size distribution and particle concentration of EVs were analyzed using a NanoSight NS300 system equipped with a 488 nm blue laser and a scientific CMOS (sCMOS) camera (Malvern Panalytical, Malvern, UK). Samples were diluted in 0.22 \u0026micro;m-filtered phosphate-buffered saline (PBS) and introduced into the sample chamber using a syringe pump. Measurements were performed at 25\u0026deg;C with a camera level of 14\u0026ndash;15, a shutter speed of 1206\u0026ndash;1259, and gain of 245. Three 30-second videos were recorded per sample under identical instrument settings. Data were analyzed using NTA software (version 3.4, Build 3.4.003; Malvern Panalytical).\u003c/p\u003e\n\u003ch3\u003eTransmission Electron Microscopy (TEM)\u003c/h3\u003e\n\u003cp\u003eFor TEM analysis, 7 \u0026micro;L of concentrated EV suspension was applied to Formvar carbon-coated copper grids and incubated for 20 minutes at 22\u0026deg;C to allow particle adsorption. The grids were then fixed with 2% paraformaldehyde (PFA) for 20 minutes, followed by post-fixation with 1% glutaraldehyde in distilled water for 5 minutes. After fixation, the grids were washed six times with distilled water and stained with 2% uranyl acetate for 5 minutes. Excess fluid was carefully removed with filter paper, and the grids were air-dried for at least 16 hours. Imaging was performed using a transmission electron microscope (HT7700, Hitachi High-Technologies, Tokyo, Japan).\u003c/p\u003e\n\u003ch3\u003eFlow cytometry for EV surface marker analysis\u003c/h3\u003e\n\u003cp\u003eSurface expression of EV markers (CD9, CD63, and CD81) was evaluated using flow cytometry. EVs were conjugated to aldehyde/sulfate latex beads (4 \u0026micro;m, Invitrogen) and incubated with APC-conjugated anti-human CD9, CD63, or CD81 antibodies (BioLegend) for 30 minutes at 4\u0026deg;C in the dark. After washing with PBS containing 0.5% BSA, samples were analyzed using a FACSVerse flow cytometer (BD Biosciences). Data were processed with FlowJo software. Unstained bead-bound EVs were used as a negative control for gating.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eELISA for non-vesicular protein contamination\u003c/h2\u003e \u003cp\u003eTo evaluate the purity of EV preparations, potential non-vesicular contaminants were quantified using ELISA kits according to the manufacturers\u0026rsquo; protocols. Processing reagents (gentamicin and BSA) and proteins from subcellular compartments\u0026mdash;calnexin (endoplasmic reticulum), histone (nucleus), GM130 (Golgi), and cytochrome c (mitochondria)\u0026mdash;were measured. All values were compared to the lower limit of quantification (LLOQ) provided with each assay kit, and results below this threshold were considered not detected (N.D.).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSTORM Microscopy\u003c/h3\u003e\n\u003cp\u003eStochastic optical reconstruction microscopy (STORM) was performed to visualize EVs at nanometer resolution for high-resolution analysis of engineered EVs [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This technique enables localization analysis of peptides in engineered EV formulations, including discrimination of peptides loaded into the EV lumen versus peptides displayed on the EV surface, and validation of structural integrity and loading efficiency.\u003c/p\u003e \u003cp\u003eWe performed stochastic optical reconstruction microscopy (STORM) to visualize individual extracellular vesicles (EVs) at nanometer resolution for high-resolution imaging and nanoscale localization analysis of engineered EVs [Rust et al., 2006]. STORM enables precise discrimination of fluorescently labeled components associated with EVs (e.g., surface-displayed peptides versus membrane dyes), and provides quantitative assessment of EV structural integrity, nanoscale changes in EV size/morphology, and validation of surface functionalization and loading efficiency.\u003c/p\u003e \u003cp\u003e \u003cb\u003e1) Sample preparation for EV imaging\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA glass-bottomed confocal dish was briefly immersed in 1 M aqueous potassium hydroxide (KOH) solution (6584\u0026thinsp;\u0026minus;\u0026thinsp;4405; DAEJUNG) and sonicated for 15 min to remove contaminants. The dish was rinsed three times with distilled water, suctioned to remove residual water, and exposed to UV light for 15 min. After an additional wash with distilled water, the cleaned dish was coated with poly-L-lysine (PLL) (25988-63-0; Sigma-Aldrich) at room temperature (RT) for 30 min to enhance adhesion of EVs. After coating, PLL-coated dishes were washed once with distilled water. EVs were diluted 1:2,000 in PBS and incubated on the PLL-coated dish for 24 h at RT.\u003c/p\u003e \u003cp\u003eFor EV membrane staining, immobilized EVs were incubated with blocking buffer (3% [w/v] bovine serum albumin (BSA) [CNB102-0100; Cell Nest] in PBS) for 30 min at RT. EV membranes were then labeled using CellBrite\u0026trade; Fix 640 Membrane Dye (Biotium; Cat# 30089) diluted in PBS (working dilution optimized empirically). After membrane labeling, samples were fixed with 4% paraformaldehyde (PFA) (1574; Electron Microscopy Sciences) in PBS for 10 min at RT. Following fixation, samples were briefly rinsed once with PBS and immediately imaged in STORM imaging buffer.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eNote\u003c/strong\u003e \u003cp\u003eTAMRA-labeled T7-TD-CPP peptides were used to visualize peptide localization independently from membrane staining. To avoid spectral overlap, the EV membrane dye was selected in the far-red channel (CellBrite Fix 640), while TAMRA peptides were detected in the orange-red channel (561 nm excitation).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e2) STORM imaging\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor STORM imaging, samples were immersed in a photoswitching imaging buffer containing mercaptoethylamine (MEA) (30070; Sigma-Aldrich), 5% (w/v) glucose, and an oxygen-scavenging enzyme system (0.5 mg/mL glucose oxidase [G2133; Sigma-Aldrich] and 38 \u0026micro;g/mL catalase [C3515; Sigma-Aldrich]) prepared in PBS at pH 8.5. All STORM imaging was conducted using a custom-built STORM setup consisting of an inverted microscope and a 1.49 NA 100\u0026times; oil immersion objective lens (CFI SR HP Apo TIRF; Nikon) [Go et al., 2021; Chung et al., 2021].\u003c/p\u003e \u003cp\u003eA 647 nm laser (OBIS; Coherent) was used for imaging CellBrite Fix 640\u0026ndash;labeled EV membranes, while a 561 nm laser (OBIS; Coherent) was used for imaging TAMRA-labeled peptides. Lasers were directed through the back port of the microscope and aligned to achieve total internal reflection fluorescence (TIRF) illumination. The CRISP Autofocus system (ASI) stabilized the focal plane by detecting a separated IR beam reflected at the sample\u0026ndash;liquid interface. Emitted fluorescence was filtered using appropriate bandpass emission filters (LF408/488/561/635-B; Semrock), and images were recorded using an EMCCD camera (iXon Ultra 888; Andor).\u003c/p\u003e \u003cp\u003eUnder these conditions, rapid photoswitching of fluorophores enabled single-molecule localization and super-resolution reconstruction of EV membranes and peptide localizations (Supplementary Movie 5). For 3D STORM imaging, a cylindrical lens with a focal length of 500 mm (LJ1144RM-A; Thorlabs) was inserted into the emission path to introduce astigmatism (Center for Polymers and Composite Materials, Hanyang University, Korea). Astigmatism produced elliptical point spread functions (PSFs) whose deformation encodes axial position; by measuring PSF widths in the x and y directions, z-coordinates were calculated to reconstruct 3D spatial information with nanometer precision. A total of 30,000 frames were acquired at a frame rate of 70 Hz. For STORM reconstruction, PSFs in each frame were fitted with Gaussian functions to determine centroid positions, followed by drift correction and rendering using optimized reconstruction parameters [Chung et al., 2021; Go et al., 2021; Rust et al., 2006].\u003c/p\u003e \u003cp\u003e \u003cb\u003e3) Identification and quantitative analysis using DBSCAN\u003c/b\u003e \u003c/p\u003e \u003cp\u003eEVs were distinguished from background signals in STORM reconstructions based on their confined structure and densely clustered localization events [Jung et al., 2020; Lim et al., 2024]. Approximately 1,000 EVs from 10 STORM images were identified and analyzed using DBSCAN clustering. The size and localization number of each identified EV were scatter-plotted to assess nanoscale heterogeneity. DBSCAN was implemented using the \u0026ldquo;sklearn.cluster DBSCAN\u0026rdquo; algorithm in Python (v3.12.0). Key parameters included \u003cem\u003eeps\u003c/em\u003e (ε), defining the neighborhood radius, and \u003cem\u003emin_samples\u003c/em\u003e, defining the minimum number of points required for core-point classification. By empirically optimizing \u003cem\u003eeps\u003c/em\u003e and \u003cem\u003emin_samples\u003c/em\u003e for each STORM image, EV clusters were robustly segmented from background signals.\u003c/p\u003e\n\u003ch3\u003eLC–MS sample preparation (HPLC pre-treatment)\u003c/h3\u003e\n\u003cp\u003eSamples were pretreated prior to analysis using an extraction solvent composed of acetonitrile, methanol, and distilled water (ACN:MeOH:DW) mixed at a 2:1:1 (v/v/v) ratio. Briefly, 200 \u0026micro;L of each sample was aliquoted, and 800 \u0026micro;L of the extraction solvent was added. The mixture was vortex-mixed for 15 min and incubated on ice for 20 min to facilitate extraction and protein precipitation. The extracts were centrifuged at 14,000 rpm for 10 min, and 500 \u0026micro;L of the resulting supernatant was collected. The supernatant was evaporated to dryness in a 37\u0026deg;C oven overnight. The residue was reconstituted in distilled water, passed through a 0.22 \u0026micro;m PTFE syringe filter, and subjected to HPLC analysis.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHPLC analysis\u003c/h2\u003e \u003cp\u003eHigh-performance liquid chromatography (HPLC) was performed using a Thermo Scientific UltiMate 3000 system. Chromatographic data were acquired and processed using the Thermo Scientific Chromeleon Chromatography Data System (CDS). Peak areas were obtained by automated peak integration. A calibration curve was constructed by plotting the concentrations of standard solutions against the corresponding peak areas. The concentration of the analyte loaded in the samples was determined by interpolating sample peak areas on the standard calibration curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImaging Flow Cytometry for EV Internalization (Amnis ImageStream)\u003c/h2\u003e \u003cp\u003eEndothelial internalization of EVs was quantified using imaging flow cytometry (Amnis\u0026reg; ImageStream\u0026reg;X Mk II System, Luminex/Amnis). Human brain microvascular endothelial cells expressing GFP (HBEC-GFP) were incubated with CellTracker\u0026trade; Deep Red (CTDR)-labeled EVs for 30 min at room temperature (RT). The following controls were included to establish gating and compensation: unstained HBECs, HBEC-GFP only, and CTDR-labeled EV only. Single-stain controls were used to calculate fluorescence compensation. Images were acquired using a 40\u0026times; objective with laser settings: 488 nm (0.1 mW), 642 nm (15 mW), and side scatter (SSC, 0.5 mW). For each condition, 2,000 single-cell events were collected. Data were analyzed using IDEAS\u0026reg; software with a standardized gating strategy. Briefly, well-focused events were gated using Gradient RMS. Single cells were identified using Aspect Ratio to exclude doublets and aggregates. EV uptake was quantified by dot-plot analysis of fluorescence intensities. EV-positive HBECs were defined as GFP-positive cells exhibiting increased CTDR fluorescence relative to controls. Representative cell images from each gate were inspected to confirm gating accuracy.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEV-Based Drug Delivery Systems\u003c/b\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003ePeptide design and synthesis\u003c/b\u003e : Peptides were designed to include the loop 2 main sequence (EKVPVSKGQL) with a C-terminal GG motif and the APP transmembrane domain (TMD), arranged in an N\u0026ndash;transferrin-binding T7 peptide\u0026ndash;CPP TM\u0026ndash;C configuration. A DEDE linker was inserted between domains. An LRLLR motif was added to enhance CPP activity, and the C-terminal arginine was amidated. N-terminal biotin or fluorescent labeling was performed for detection. Peptides were synthesized by standard solid-phase methods.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e2) Dual loading of peptides into MSC-EVs\u003c/strong\u003e \u003cp\u003ePeptide cargos were loaded into MSC-EVs using a passive loading approach as previously described [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. For dual loading, the PROTAC compound was first incubated with MSC-EVs for 1 h at 22\u0026deg;C to allow passive encapsulation. Subsequently, TD-CPP or T7-TD-CPP peptides were added and further co-incubated with the PROTAC-loaded EVs for an additional 1 h at 22\u0026deg;C to enable membrane insertion.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e3) Removal of Unloaded PROTAC / Peptide Drugs\u003c/strong\u003e \u003cp\u003eFollowing PROTAC-peptide loading, unincorporated cargos were removed by dialysis using a 100 kDa molecular weight cut-off (MWCO) membrane. Dialysis was performed for 4 h at 4\u0026deg;C under gentle stirring. The concentration of PROTAC-peptide-loaded EVs was subsequently determined, and PROTAC- peptide loading efficiency was quantified as described below.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e4) Peptide Quantification after EV Loading\u003c/strong\u003e \u003cp\u003eTo quantify fluorescently labeled neuropeptides, a standard curve was first established by measuring fluorescence across a range of known concentrations. Following EV loading, the fluorescence intensity of the peptide-loaded EVs was measured and used to calculate the absolute concentration of encapsulated peptides based on the standard curve. T7 peptides were analyzed using fluorescence spectroscopy, and concentrations were determined using a standard curve beginning at 10 \u0026micro;g, with serial 10% dilutions down to 0.1 \u0026micro;g and the blank samples containing ultrapure water and PBS, using the Glomax multi detection system (Promega, Madison, WI, USA). The samples were portioned at 100 \u0026micro;L and evaluated using spectroscopy at excitation and emission wavelengths of 510 and 570 nm, respectively. Effective loading was calculated as a function of \u0026micro;g peptide per billion EV (Jeyaram et al., 2020).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e5) Drug loading Capacity and Encapsulation Efficiency of peptides\u003c/strong\u003e \u003cp\u003eEncapsulation efficiency and loading efficiency were calculated according to equations (1) and (2), respectively.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e\u0026bull; Encapsulation efficiency (%) (1)\u003c/h2\u003e \u003cp\u003e= (Amount of the total loaded peptide in EVs / total amount of input peptide initially ) x 100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e\u0026bull; Loading Capacity (%) (2)\u003c/h2\u003e \u003cp\u003e= (Amount of the total loaded peptide in EVs / final EV numbers) x100\u003c/p\u003e \u003cp\u003e \u003cstrong\u003e6) EV recovery\u003c/strong\u003e \u003cp\u003eThe final purified peptide-loaded EV solution was analyzed for EVs recovery using nanoparticle tracking analysis (NTA). EV recovery was calculated according to equations (3), respectively.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e\u0026bull; EV recovery % (3)\u003c/h2\u003e \u003cp\u003e= (final EVs number/input EVs number) x100\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo studies\u003c/b\u003e \u003c/p\u003e \u003cp\u003e\u003cb\u003e1) Animal experimental procedure\u003c/b\u003e: All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (Laboratory Animal Research Center, AAALAC International approved facility) of Samsung Medical Center (Seoul, Korea). C57BL/6 J mice were purchased from Orient Bio Inc. (Seongnam, Korea) and housed at a temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and a relative humidity of 55\u0026thinsp;\u0026plusmn;\u0026thinsp;10%, under a 12 h light/dark cycle, with ad libitum access to food and water. Animals were anesthetized using a ketamine:xylazine mixture (5:1 ratio) prepared by combining ketamine (Yuhan Ketamine 50Inj.) and xylazine (Rompun Inj.). The anesthetic dose was calculated based on the animal\u0026rsquo;s body weight, and the mixture was administered intraperitoneally (i.p.). Depth of anesthesia was confirmed by the absence of reflex responses to toe pinching. Once fully anesthetized, the animal was positioned on a surgical platform, and the thoracic cavity was opened to expose the heart. A 21-gauge needle was inserted into the left ventricle, and an incision was made in the right atrium to allow for drainage. Perfusion was performed with 50 mL each of PBS and 4% PFA in PBS. After perfusion, the skull was carefully opened using surgical scissors and forceps. The brain was gently removed and post-fixed in 4% PFA at 4\u0026deg;C for 24 h. Subsequently, the brain was transferred to a 30% sucrose solution for cryoprotection until it sank, indicating full infiltration.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003e2) IVIS - Fluorescent brain distribution and histological imaging\u003c/strong\u003e \u003cp\u003eC57BL/6 male mice were randomly assigned to experimental groups. For near-infrared fluorescence imaging, MSC-3D EVs were first labeled using the ExoGlow-Vivo EV Labeling Kit (System Biosciences, SBI) according to the manufacturer\u0026rsquo;s instructions. Following EV labeling, surface engineering was performed by incubating ExoGlow-labeled EVs with TD-CPP or T7-TD-CPP peptides to enable membrane insertion prior to administration. A total of 6 \u0026times; 10⁸ labeled EVs were administered intravenous (IV), and imaging was using the IVIS Spectrum imaging system (PerkinElmer, MA, USA). Fluorescence signals were acquired using excitation and emission wavelengths of 745 nm and 820 nm, respectively, and quantitative analysis was conducted using Living Image\u0026reg; software (v4.7.2).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3) Intra-vital microscope\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo visualize meningeal lymphatics and cerebral vasculature in real-time, a cranial window was implanted over the parietal cortex of Prox1-eGFP mice. Briefly, mice were anesthetized with isoflurane, and the scalp was incised to expose the skull. A circular craniotomy (approx. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] mm diameter) was carefully performed using a high-speed micro-drill, leaving the dura mater intact. A sterile cover glass ([No. 1]) was placed over the exposed brain and sealed with dental cement to create a water-tight observation window. To label blood vessels, fluorophore-conjugated anti-CD31 antibody was injected intravenously immediately prior to imaging.\u003c/p\u003e \u003cp\u003eIn vivo imaging was performed using a custom-built all-in-one two-photon microscopy system ([IVM-C / IVM-M3], IVIM Technology, Daejeon, Korea). The system was equipped with a high-speed polygonal scanner and integrated real-time motion compensation to correct artifacts caused by heartbeat and respiration. Prox1-eGFP positive lymphatic vessels, [fluorophore]-labeled blood vessels, and [fluorophore]-labeled EVs were excited at [880\u0026ndash;920] nm, [wavelength] nm, and [wavelength] nm, respectively. Time-lapse images and Z-stacks were acquired using a water-immersion objective lens ([10\u0026times; or 25\u0026times;], NA [1.0]). Image processing and 3D reconstruction were performed using [IVIM S/W, Imaris, or ImageJ] software.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003e4) Histological imaging\u003c/strong\u003e \u003cp\u003eFor histological distribution analysis, MSC-EVs or MSC-EV-NPs were surface-engineered with TAMRA-tagged TD-CPP or T7-TD-CPP peptides prior to administration. Peptide-functionalized EVs were administered via intravenous (i.v.) injection as control conditions. At 4 h post administration, mice were sacrificed and brains were harvested, fixed overnight in 4% paraformaldehyde (PFA) at 4\u0026deg;C, and cryoprotected in 30% (w/v) sucrose solution. Brain tissues were embedded in OCT compound (Tissue-Tek, Sakura), stored at \u0026minus;\u0026thinsp;80\u0026deg;C, and coronally sectioned at a thickness of 20 \u0026micro;m using a cryostat (Leica CM1950, Leica Biosystems). Sections were mounted using Fluoroshield\u0026trade; mounting medium containing DAPI (ImmunoBioScience, AR-6501\u0026ndash;01) and imaged using a Zeiss LSM 780 confocal laser scanning microscope (Carl Zeiss, Germany). For cellular localization analysis, sections were immunostained with cell-type\u0026ndash;specific markers including NeuN (neurons) and GFAP (astrocytes). All images were acquired using identical acquisition settings and processed using ZEN software (Carl Zeiss).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e5) MPTP-induced Parkinson\u0026rsquo;s disease mouse model and EV treatment\u003c/strong\u003e \u003cp\u003eMale C57BL/6 mice (10\u0026ndash;12 weeks old) were obtained from Orient Bio (Seongnam-si, Korea) and randomly assigned to experimental groups. To induce Parkinsonian pathology, mice received intraperitoneal (i.p.) injections of MPTP at a dose of 20 mg/kg on Day 0. Control animals were administered an equivalent volume of PBS. For therapeutic intervention, EV treatments were performed on Day 4 following MPTP injection. Mice were intravenously (i.v.) injected with PROTAC alone, PROTAC-loaded TD-CPP MSC-EVs, or PROTAC-loaded T7-TD-CPP MSC-EVs, while control groups received PBS. All treatments were administered via the tail vein under identical conditions. Mice were sacrificed on Day 6, and brain tissues were harvested for biochemical analysis.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e\u003cb\u003e6) Western blot analysis\u003c/b\u003e: Total proteins were extracted from mouse brain tissues using ice-cold RIPA buffer. Protein concentrations were determined using Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA, USA) according to the manufacturer\u0026rsquo;s instructions. Protein lysates (30 \u0026micro;g) were separated by SDS\u0026ndash;PAGE on 10% gels and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). After blocking with 5% skimmed milk for 1 h at 22\u0026deg;C, membranes were incubated overnight at 4\u0026deg;C with primary antibodies against LRRK2 and β-actin. After washing with TBS containing 0.1% Tween-20, membranes were incubated with horseradish peroxidase\u0026ndash;conjugated secondary antibodies (1:2000; CUSABIO, Houston, TX, USA) for 2 h at 22\u0026deg;C. Protein bands were visualized using the ECL\u0026trade; Prime Western Blotting System (RPN2232; Cytiva, Marlborough, MA, USA), and signal intensities were quantified using ImageJ software (NIH, Bethesda, MD, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCharacterization of EVs.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMSC-derived extracellular vesicles (MSC-EVs) were isolated from 3D spheroid cultures and assessed for morphological and physicochemical properties. Transmission electron microscopy (TEM) revealed spherical vesicles with a characteristic round-shaped EV morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Consistent with these observations, nanoparticle tracking analysis (NTA) demonstrated a narrow size distribution with a mean diameter of 127.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6 nm and a mode diameter of 117.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).The identity of MSC-EVs was further confirmed by surface marker profiling. Flow cytometry analysis showed strong positivity for canonical EV tetraspanins, including CD9 (98.88%), CD63 (98.82%), and CD81 (100.00%), indicating successful enrichment of EV populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). EV purity was evaluated by ELISA for non-vesicular contaminants and cellular components. Gentamicin, BSA, calnexin, histone, GM130, and cytochrome c were not detected (N.D.) or remained below the lower limit of quantification (LLOQ), supporting minimal contamination by soluble proteins, intracellular organelles, or cellular debris (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).To further resolve nanoscale structural features, we performed stochastic optical reconstruction microscopy (STORM) super-resolution imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-F). Reconstructed localization clusters yielded a mean EV diameter of 109.73\u0026thinsp;\u0026plusmn;\u0026thinsp;23.86 nm, consistent with NTA results. Fluorescence emission distributions produced full width at half maximum (FWHM) values of 110.86 nm (x-axis) and 105.77 nm (y-axis), reflecting compact and uniform vesicle boundaries. Localization analysis further revealed 221\u0026thinsp;\u0026plusmn;\u0026thinsp;148 localizations per particle, indicating dense fluorophore distribution along the EV membrane. Moreover, line-scan profiling across the vesicle center identified a peak-to-peak distance of 84.32 nm, consistent with the hollow vesicular architecture characteristic of MSC-derived EVs. Collectively, these data confirm that EVs isolated from 3D spheroid cultures exhibit expected EV morphology, canonical marker expression, minimal contamination, and preserved nanoscale structural features, supporting their suitability for downstream engineering and loading applications.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of T7-TD-CPP engineered EVs\u003c/h2\u003e \u003cp\u003eTo enable brain targeting and intracellular delivery of small-molecule cargo, we designed a multifunctional membrane-inserting peptide comprising a transferrin receptor\u0026ndash;binding T7 motif, a flexible linker (LP), a transmembrane domain (TD), and a cell-penetrating peptide (CPP) to facilitate EV surface display through spontaneous membrane insertion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In parallel, the hydrophobic PROTAC molecule XL01126 was selected as a model therapeutic cargo for passive EV encapsulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). To generate T7-TD-CPP\u0026ndash;engineered PROTAC-loaded EVs, PROTAC encapsulation and subsequent peptide surface insertion were performed sequentially. Following passive loading of XL01126 into native MSC-3D EVs, vesicular morphology and particle size were preserved, as confirmed by transmission electron microscopy and nanoparticle tracking analysis. PROTAC-loaded MSC-3D EVs, PROTAC \u0026amp; TD-CPP-loaded EVs, and PROTAC \u0026amp; T7-TD-CPP-loaded EVs exhibited comparable size distributions, with mean diameters of 116.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 nm, 114.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 nm, and 119.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5 nm, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). PROTAC loading capacity and encapsulation efficiency were quantified by HPLC and were 155 ng per 1\u0026times;10⁹ EVs and 97%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). T7-TD-CPP peptide loading was quantified using FITC fluorescence with reference to a FITC standard calibration curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eVerification of surface-displayed peptide localization on the EV membrane\u003c/h2\u003e \u003cp\u003eTo confirm that the engineered T7-TD-CPP peptide is externally displayed and localized along the EV membrane, we performed stochastic optical reconstruction microscopy (STORM) using TAMRA-labeled T7-TD-CPP peptides (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Single-particle localization reconstruction revealed well-defined nanoscale clusters with a mean vesicle diameter of 103.44\u0026thinsp;\u0026plusmn;\u0026thinsp;15.15 nm, consistent with the expected size range of MSC-derived EVs. Quantitative localization mapping indicated robust peptide incorporation, yielding 133\u0026thinsp;\u0026plusmn;\u0026thinsp;74 localizations per particle, supporting efficient peptide display on the vesicle surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C). Importantly, the distribution of localization events and the corresponding full width at half maximum (FWHM) profiles exhibited compact and uniform clustering, consistent with membrane insertion rather than nonspecific peptide aggregation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further validate that the detected peptide signal was membrane-associated, we compared the spatial distribution of TAMRA\u0026ndash;T7-TD-CPP\u0026ndash;labeled EVs with EVs labeled using CellBrite, a membrane-specific fluorescent dye (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Scatter plot analyses of localization number versus FWHM showed highly overlapping patterns between the peptide-labeled EVs and the CellBrite-labeled EVs, indicating that the peptide signal predominantly mapped to the vesicle membrane. Representative merged STORM reconstructions showed a characteristic ring-like localization pattern in both groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), consistent with circumferential membrane localization.\u003c/p\u003e \u003cp\u003eQuantitative comparisons further supported nanoscale fidelity of membrane localization: peptide-labeled EVs and CellBrite-labeled EVs exhibited comparable mean diameters (103.44\u0026thinsp;\u0026plusmn;\u0026thinsp;15.15 nm vs 107.28\u0026thinsp;\u0026plusmn;\u0026thinsp;18.72 nm, respectively). While the mean vesicle sizes were similar, localization counts differed in accordance with fluorophore density (peptide: 113\u0026thinsp;\u0026plusmn;\u0026thinsp;74 vs CellBrite: 271\u0026thinsp;\u0026plusmn;\u0026thinsp;125) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Collectively, these results demonstrate that T7-TD-CPP peptides are stably inserted and externally displayed along the EV membrane, enabling nanoscale surface engineering without evidence of peptide aggregation. Bead-based flow cytometry using the MaxPlex\u0026trade; Exosome Flow Cytometry Kit demonstrated robust expression of the canonical EV surface markers CD9, CD63, and CD81 in both unmodified MSC-3D EVs and PROTAC \u0026amp; T7-TD-CPP dual-loaded EVs. All samples showed\u0026thinsp;\u0026gt;\u0026thinsp;100% positivity for these tetraspanins, with no detectable decrease in fluorescence intensity following PROTAC loading and peptide surface functionalization. These data indicate that dual engineering did not alter canonical EV membrane marker profiles, supporting maintenance of EV identity and membrane integrity(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eImaging flow cytometry quantification of EV uptake by HBECs\u003c/h2\u003e \u003cp\u003eTo quantitatively assess cellular uptake of engineered EVs by brain endothelial cells, we performed imaging flow cytometry using the Amnis ImageStreamX Mk II platform. HBECs expressing GFP (HBEC-GFP) were incubated with CTDR-labeled EVs for 30 min at room temperature, followed by acquisition and analysis of fluorescence images at the single-cell level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). A standardized gating strategy was applied to ensure robust quantification. First, well-focused events were selected using the Gradient RMS feature. Next, single cells were gated based on Aspect Ratio to exclude doublets and cellular aggregates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Uptake of EVs was defined as the presence of CTDR fluorescence within GFP-positive HBECs. Accordingly, EV-positive cells were quantified using a dot plot of Ch02 (GFP) versus Ch05 (CTDR) fluorescence intensity, enabling objective separation of CTDR-negative and CTDR-positive HBEC populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C). Representative cell images from each gate confirmed accurate identification of EV-associated CTDR signals within single HBECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Using identical gating thresholds across all groups, we quantified the proportion of CTDR-positive HBECs and EV-derived fluorescence intensity, demonstrating differential uptake profiles among EV formulations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Together, these results establish imaging flow cytometry as a robust high-content platform for quantifying EV uptake into brain endothelial cells and support subsequent functional evaluation of engineered EVs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eT7 peptide enhances transferrin receptor\u0026ndash;mediated uptake and improves in vitro BBB penetration\u003c/h2\u003e \u003cp\u003eGiven that the T7 motif targets the transferrin receptor (TfR), we first confirmed the expression of TfR1 in human brain endothelial cells. Western blot analysis demonstrated robust TfR1 expression in HBECs, whereas HEK293 cells showed comparatively lower levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), supporting the suitability of HBECs as a BBB-relevant model to evaluate T7-mediated targeting. To assess whether T7 engineering enhances cellular uptake of EVs, we next performed temperature-dependent uptake experiments. Both free T7-TD-CPP peptide and T7-engineered EVs showed markedly increased internalization at \u003cb\u003e37\u0026deg;C\u003c/b\u003e compared with 4\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating that uptake is primarily mediated through active cellular processes rather than nonspecific adsorption. Consistent with receptor-mediated uptake, immunofluorescence imaging demonstrated spatial association between TfR signals and T7-engineered EV fluorescence within HBECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), supporting TfR-dependent binding and/or endocytosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then evaluated whether enhanced uptake translates into improved BBB transport using an in vitro BBB transwell model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Barrier integrity was monitored by transendothelial electrical resistance (TEER) over time, demonstrating progressive maturation of tight barrier properties (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE; ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, as indicated). Using this validated BBB model, we quantified EV transcytosis/penetration across the endothelial layer. Notably, T7-TD-CPP EVs exhibited significantly increased BBB penetration compared with TD-CPP EVs and dextran control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF; ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, as indicated), demonstrating that T7-based surface engineering enhances functional transport across brain endothelial barriers. Collectively, these results indicate that T7 engineering strengthens TfR-mediated uptake and improves BBB transcytosis, supporting its use as a brain-targeting EV surface modification strategy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo brain section imaging confirms TfR-associated targeting and cellular distribution of T7-engineered EVs\u003c/h2\u003e \u003cp\u003eTo evaluate whether T7 surface engineering enhances brain localization of EVs in vivo, we monitored the spatiotemporal distribution of fluorescently labeled EVs using real-time intravital microscopy through a cranial window model.\u003c/p\u003e \u003cp\u003eTo visualize the cerebral vasculature and define vessel boundaries, mice were first intravenously injected with a fluorescently labeled anti-CD31 antibody. Subsequently, fluorescently labeled T7-TD-CPP EVs were administered via intravenous injection, allowing real-time tracking of EV behavior relative to the vascular network and surrounding brain parenchyma over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTime-lapse intravital imaging revealed time-dependent changes in the distribution of T7-TD-CPP EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). EV signals were predominantly associated with vascular structures at early time points (1 h), whereas punctate EV signals were increasingly observed beyond the vasculature at later time points (6 h and 24 h), indicating progressive extravascular localization within brain tissue. Consistent with intravital observations, ex vivo immunofluorescence analysis of brain sections demonstrated that T7-TD-CPP EVs displayed more prominent EV signals distal to CD31-positive vessels compared with TD-CPP EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). High-magnification images further confirmed the presence of EV-associated puncta in perivascular and parenchymal regions, indicating broader tissue distribution following T7 surface engineering.\u003c/p\u003e \u003cp\u003eCollectively, these results demonstrate that T7 modification alters the in vivo distribution profile of EVs, promoting sustained localization beyond the cerebral vasculature and facilitating enhanced access to brain tissue. These findings support the utility of T7-engineered EVs as an effective strategy for improving brain delivery in vivo.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eT7-TD-CPP EVs exhibit brain targeting and BBB penetration in vivo\u003c/h2\u003e \u003cp\u003eTo confirm brain delivery of systemically administered EVs at the cellular level, the fluorescence distribution of EVs within brain vascular endothelial cells was examined. Notably, EVs loaded with T7\u0026ndash;PROTAC showed clear colocalization with transferrin receptors expressed on endothelial cells, as evidenced by merged fluorescence signals. This colocalization indicates transferrin receptor\u0026ndash;mediated transendocytosis, supporting enhanced blood\u0026ndash;brain barrier permeability of T7-modified EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA,B). To further evaluate brain parenchymal distribution, immunofluorescence analysis was performed on brain sections collected \u003cb\u003e4 h after intravenous injection\u003c/b\u003e of engineered EVs. Tissue sections were co-stained with cell type\u0026ndash;specific markers to assess cellular localization patterns. T7-TD-CPP EVs exhibited more frequent association with \u003cb\u003eNeuN⁺ neurons\u003c/b\u003e compared with TD-CPP EVs, which showed relatively sparse neuronal-associated signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). In contrast, EV signals within \u003cb\u003eGFAP⁺ astrocytes\u003c/b\u003e were limited in both groups, with only modest astrocyte-associated puncta observed in the T7-modified EV condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Collectively, these results demonstrate that T7 modification enhances endothelial transcytosis and preferential neuronal-associated localization of EVs in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eEx vivo IVIS imaging demonstrates enhanced brain accumulation and prolonged retention of T7-TD-CPP EVs\u003c/h2\u003e \u003cp\u003eTo quantify whole-body biodistribution and assess brain-targeting efficiency, ex vivo IVIS imaging was performed 6 h after intravenous injection of EVs. Compared with TD-CPP EVs, T7-TD-CPP EVs exhibited markedly stronger fluorescence signals in the brain (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Quantitative analysis of brain-associated radiant efficiency confirmed a significant increase in signal intensity following T7 modification (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further examine the temporal dynamics of EV distribution, additional IVIS imaging was conducted at 6 h and 96 h after injection of T7-TD-CPP EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Brain-associated fluorescence signals remained detectable up to 96 h post-administration, whereas fluorescence intensities in peripheral organs, particularly the liver and kidney, decreased substantially over time (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD), consistent with progressive systemic clearance.\u003c/p\u003e \u003cp\u003eEx vivo imaging also revealed detectable fluorescence signals in major clearance-associated organs, including the liver and kidney, across treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). Quantitative comparison across organs demonstrated that, among the tissues analyzed, the brain exhibited the most pronounced enhancement in fluorescence intensity following T7 modification (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eCollectively, these results demonstrate that T7-TD-CPP surface engineering enhances brain-associated accumulation of systemically administered EVs and supports sustained brain-localized fluorescence signals relative to non-targeted EV formulations, highlighting the contribution of the T7 motif to improved in vivo brain targeting.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDual-loaded T7-targeted PROTAC EVs drive robust in vivo degradation of LRRK2 in an MPTP Parkinson\u0026rsquo;s disease mouse model\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the in vivo therapeutic efficacy of the dual-loading strategy, an MPTP-induced Parkinson\u0026rsquo;s disease mouse model was employed (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). Mice received intraperitoneal injections of MPTP (20 mg/kg) on Day 0 to induce Parkinsonian pathology, followed by intravenous administration of EV formulations on Day 4. Animals were sacrificed on Day 6, and brain tissues were harvested for biochemical analysis of LRRK2 expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs expected, MPTP treatment markedly increased LRRK2 levels in the brain compared with control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB-C). Treatment with free PROTAC resulted in only partial reduction of LRRK2 expression, consistent with limited efficacy under systemic administration. In contrast, T7-TD-CPP\u0026ndash;PROTAC EVs produced the most pronounced suppression of LRRK2 expression, significantly outperforming both free PROTAC and TD-CPP\u0026ndash;PROTAC EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). TD-CPP\u0026ndash;PROTAC EVs induced only a moderate reduction in LRRK2 levels, whereas T7-targeted dual-loaded EVs achieved pronounced LRRK2 degradation in vivo.\u003c/p\u003e \u003cp\u003eCollectively, these results demonstrate that T7-TD-CPP\u0026ndash;engineered, PROTAC-loaded EVs substantially enhance the in vivo pharmacological activity of PROTAC cargo in the MPTP mouse model, supporting the effectiveness of the dual-loading strategy for brain-targeted protein degradation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eScalable manufacturing and stringent quality control are critical prerequisites for the clinical translation of EV-based therapeutics. In the EV field, increasing consensus has been reached on standardized requirements for EV identity, purity, and reporting, with particular emphasis on preventing non-vesicular contamination and ensuring batch-to-batch reproducibility [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In this study, EVs derived from 3D spheroid MSC cultures retained canonical EV characteristics, including expected morphology and size distribution, robust expression of tetraspanin markers, and minimal contamination by intracellular or organelle-associated proteins. These results support the feasibility of establishing a manufacturing pipeline suitable for clinical development and downstream engineering.\u003c/p\u003e \u003cp\u003eThe surface engineering strategy adopted here is notable for its simplicity and translational relevance. Many ligand-presenting EV approaches rely on genetic modification of parental cells, which can complicate regulatory approval and large-scale manufacturing. In contrast, the T7-TD-CPP peptide was spontaneously inserted into the EV membrane without external energy input, while preserving EV structure and size distribution. Importantly, stochastic optical reconstruction microscopy (STORM) confirmed membrane-associated ring-like localization patterns, providing direct nanoscale evidence of membrane insertion rather than peptide aggregation. This strengthens the reliability of surface functionalization claims, which remain a point of debate in EV targeting studies.\u003c/p\u003e \u003cp\u003eTransferrin receptor (TfR) targeting is one of the most validated paradigms for BBB delivery, although enhanced endothelial binding does not always translate into efficient transcytosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The T7 peptide (HAIYPRH) is a well-established TfR-binding motif reported to enhance BBB penetration across multiple nanocarrier platforms [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In this study, T7-functionalized EVs showed increased uptake by brain endothelial cells, enhanced transport across a TEER-supported in vitro BBB model, and in vivo distribution beyond CD31-positive vascular boundaries. Notably, T7-modified EVs exhibited markedly enhanced association with NeuN-positive neurons compared with TD-CPP EVs, while association with GFAP-positive astrocytes remained limited. These findings indicate that T7-based surface functionalization can promote preferential neuronal engagement and that EV surface engineering can modulate cell-type interaction profiles in the brain.\u003c/p\u003e \u003cp\u003eThe dual-loading architecture represents a central conceptual advance of this study. While previous reports have demonstrated ligand-modified EVs for brain-targeted delivery [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], many have focused on nucleic acid cargos or biodistribution without robust pharmacodynamic evidence. Here, a hydrophobic small-molecule PROTAC requiring cytosolic access to the ubiquitin\u0026ndash;proteasome machinery was incorporated as a therapeutically relevant intravesicular cargo. Targeted protein degradation has emerged as a transformative modality capable of eliminating both catalytic and scaffolding functions of disease-driving proteins [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. XL01126 is a potent and selective LRRK2 degrader with reported BBB permeability [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]; however, effective neuronal delivery remains a challenge. In an MPTP-induced Parkinson\u0026rsquo;s disease model, T7-targeted, dual-loaded EVs achieved the most pronounced reduction of LRRK2 compared with free PROTAC and non-targeted EVs, accompanied by decreased α-synuclein levels. These data demonstrate that combining BBB-targeted surface display with intravesicular PROTAC delivery enhances in vivo pharmacological outcomes.\u003c/p\u003e \u003cp\u003eEV-mediated delivery further provides intrinsic advantages for BBB traversal. EVs exploit multiple transport pathways across the BBB, including receptor-mediated and ligand-independent mechanisms. Our results indicate that EVs can traverse the BBB even without ligand-mediated targeting, supporting their inherent BBB-crossing capability.\u003c/p\u003e \u003cp\u003eThe combination of CPP-mediated surface modification and intravesicular loading offers several advantages. \u003cb\u003eFirst\u003c/b\u003e, modular coupling of CPPs with different targeting ligands enables customization of EVs for diverse targets while accommodating varied cargos. \u003cb\u003eSecond\u003c/b\u003e, intravesicular loading allows quantitative and tunable control of cargo concentration while minimizing interference between surface ligands and encapsulated therapeutics. \u003cb\u003eThird\u003c/b\u003e, luminal loading facilitates intracellular delivery following BBB traversal, which is particularly advantageous for cytosol-dependent therapeutics such as PROTACs.\u003c/p\u003e \u003cp\u003eAlthough extensive efforts have been devoted to EV-based delivery systems, many studies rely on vector-mediated preloading strategies that pose hurdles for clinical translation. Post-loading approaches often lack systematic evaluation of loading efficiency and EV quality. In contrast, this study provides objective and quantitative metrics for loading efficiency and EV quality and employs clinical-grade EVs, strengthening translational relevance and regulatory comparability.\u003c/p\u003e \u003cp\u003eSeveral limitations remain. The precise mechanisms underlying BBB transport require further elucidation, as TfR-mediated trafficking can favor lysosomal routing under certain conditions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Fluorescence-based biodistribution may be confounded by dye transfer, warranting orthogonal validation approaches [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Rigorous pharmacokinetic and pharmacodynamic profiling is also needed to quantify regional brain exposure and dose\u0026ndash;response relationships of XL01126 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Finally, long-term safety, immunogenicity, repeated-dosing studies, and validation in additional disease models will be essential for clinical translation.\u003c/p\u003e \u003cp\u003eIn summary, this study establishes a clinically oriented EV engineering framework integrating non-genetic BBB-targeted surface functionalization (T7-TD-CPP) with intravesicular PROTAC loading (XL01126). This dual-loading EV platform enhances brain accumulation, neuronal engagement, and in vivo target degradation in a Parkinson\u0026rsquo;s disease model, supporting the use of clinical-grade MSC-derived EVs as modular CNS delivery systems for advanced therapeutic modalities.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e None\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment and Funding:\u003c/strong\u003e This work was supported by the \u003cem\u003eK-Brain Project \u003c/em\u003eof the National Research Foundation (NRF) funded by the Korean government (MSIT) under Grant No. RS-2024-00399320..\u003c/p\u003e\n\u003cp\u003eS\u0026amp;E Bio Inc. provided support for this study in the form of salaries to J.E.K. The funders had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation. The specific roles of the authors are described in the author contributions statement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosures:\u003c/strong\u003e The authors have read and understood the journal\u0026rsquo;s policies. The authors of this manuscript disclose the following competing interests: \u003cstrong\u003eJ. E. K. is a paid employee of S\u0026amp;E Bio Inc.\u003c/strong\u003e This competing interest did not affect adherence to the data- and material-sharing policies of the \u003cem\u003eJournal of Nanobiotechnology\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement:\u003c/strong\u003e J.E.K. and O.Y.B. designed the study, analyzed all samples, interpreted the data, and wrote the manuscript. J. E. K. and Y.E.J. acquired and analyzed the data. J.E.K. revised the study for important intellectual content. J.E.K. and O.Y.B. reviewed the report and provided scientific advice. O.Y.B. designed and funded the study, analyzed all samples, interpreted the data, wrote, and approved the submission of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval statement\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTh\u0026eacute;ry C, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the ISEV and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018;7:1535750.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLener T, et al. Applying extracellular vesicles based therapeutics in clinical trials\u0026mdash;an ISEV position paper. J Extracell Vesicles. 2015;4:30087.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJohnsen KB, Burkhart A, Thomsen LB, Andresen TL, Moos T. Targeting the transferrin receptor for brain drug delivery. Prog Neurobiol. 2019;181:101665.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Z, et al. Enhanced anti-ischemic stroke of ZL006 by T7-conjugated PEGylated liposomes drug delivery system. Sci Rep. 2015;5:12651.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlvarez-Erviti L, et al. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol. 2011;29:341\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, et al. Targeted exosome-mediated delivery of opioid receptor Mu siRNA to the brain for the treatment of morphine relapse. Sci Rep. 2015;5:17543.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB\u0026eacute;k\u0026eacute;s M, Langley DR, Crews CM. PROTAC targeted protein degraders: the past is prologue. Nat Rev Drug Discov. 2022;21:181\u0026ndash;200.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu X, et al. Discovery of XL01126: a potent, fast, cooperative, selective, orally bioavailable and blood\u0026ndash;brain barrier penetrant proteolysis targeting chimera degrader of LRRK2. J Am Chem Soc. 2022;144:16930\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSon JP, et al. Mesenchymal Stem Cell\u0026ndash;Extracellular Vesicle Therapy for Stroke. Stem Cells Translational Med. 2023;12:459\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWelsh JA et al. Minimal information for studies of extracellular vesicles 2023 (MISEV2023): a position statement of the International Society for Extracellular Vesicles (ISEV). J Extracell Vesicles (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRust MJ, Bates M, Zhuang X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods. 2006;3:793\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Castilla PEM. Extracellular vesicles as a drug delivery system: a systematic review of preclinical studies. Biochimica et Biophysica Acta (BBA) - General Subjects (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR\u0026auml;dler JO et al. Exploiting the biogenesis of extracellular vesicles for drug delivery and functionalization. Semin Cell Dev Biol (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim J-E, et al. Engineered MSC-EVs loaded with BDNF-enhancing neuropeptides via a non-disruptive method enhance post-stroke neuroregeneration via intranasal delivery. J Nanobiotechnol. 2025;23:594. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12951-025-03654-x\u003c/span\u003e\u003cspan address=\"10.1186/s12951-025-03654-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"MSC-derived extracellular vesicles (EVs), Blood–brain barrier (BBB), T7 peptide–mediated brain targeting, PROTAC (targeted protein degradation), Parkinson’s disease / LRRK2 degradation","lastPublishedDoi":"10.21203/rs.3.rs-8830833/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8830833/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMesenchymal stem cell\u0026ndash;derived extracellular vesicles (MSC-EVs) have emerged as promising therapeutic delivery vehicles for brain diseases. However, their limited ability to cross the blood\u0026ndash;brain barrier (BBB) remains a major obstacle to clinical translation. To overcome this limitation, we designed a multifunctional peptide consisting of a membrane-anchoring transmembrane domain (TD) and a cell-penetrating peptide (CPP) that can spontaneously insert into the EV membrane without external energy input. The brain-targeting T7 sequence was placed at the distal end to ensure directional display on the EV surface, thereby improving targeting efficiency. Using this approach, we generated T7-displaying MSC-EVs and passively loaded proteolysis-targeting chimeras (PROTACs) to enhance their therapeutic potential. The structural integrity, cellular uptake, and BBB permeability of engineered EVs were evaluated through in vitro neuronal assays and in vivo brain imaging. T7-TD-CPP EVs exhibited markedly improved cellular internalization and BBB penetration, while PROTAC co-loading facilitated degradation of pathological proteins. In an MPTP-induced Parkinson\u0026rsquo;s disease mouse model, treatment with T7-EV\u0026ndash;PROTACs alleviated disease-associated pathology, including abnormal expression of LRRK2 in the midbrain. This EV platform, combining energy-independent membrane anchoring with T7-mediated brain targeting, represents a promising strategy for precise, brain-directed therapy against neurodegenerative diseases.\u003c/p\u003e","manuscriptTitle":"Brain-targeted PROTAC delivery by dual-functional extracellular vesicles achieves robust LRRK2 degradation in a Parkinson’s disease model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-24 12:12:43","doi":"10.21203/rs.3.rs-8830833/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-17T14:52:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-12T06:54:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-06T14:40:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"24547939823793181097154167249258323940","date":"2026-03-05T02:26:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"123993669002415812192676730995908168776","date":"2026-03-03T03:13:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"117149633281064214021636405433364746117","date":"2026-02-25T03:06:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-18T19:59:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-11T02:21:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-11T02:20:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2026-02-09T12:48:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"22f31da2-8b86-415d-853c-73a61d976d96","owner":[],"postedDate":"February 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-03-17T15:08:21+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-24 12:12:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8830833","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8830833","identity":"rs-8830833","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}