Mesenchymal Stem Cell-derived exosomes loaded Alitretinoin for TBI repair treatment

Mesenchymal Stem Cell-derived exosomes loaded Alitretinoin for TBI repair treatment | 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 Method Article Mesenchymal Stem Cell-derived exosomes loaded Alitretinoin for TBI repair treatment Songyu Chen, Junyu Lin, Xiaxuan Zhang, Peng Yu, Yida Wang, Xianyu Deng, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4573349/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Traumatic brain injury (TBI) remains a leading cause of mortality and morbidity worldwide. Current clinical treatments often target singular pathological processes, resulting in suboptimal outcomes due to a lack of comprehensive neuroprotective effects. To address this critical gap, we have developed an innovative therapeutic approach utilizing mesenchymal stromal cell (MSC)-derived exosome (Mexo)-coated albumin nanospheres loaded with Alitretinoin (Ali-NPs@Mexo). This novel design aims to enhance neural repair mechanisms, offering a multifaceted approach to neuroprotection and recovery in TBI patients. Our in vitro and in vivo experiments demonstrated that Ali-NPs@Mexo effectively modulates the TBI immuno-microenvironment by attenuating oxidative stress and neuroinflammatory responses. Treatment with Ali-NPs@Mexo was also found to reduce the abnormal activation of astrocytes, which contribute to glial scar formation that hinders neuronal repair. Furthermore, this nanomedicine promoted the proliferation and repair of oligodendrocytes, neural stem cells, and neurons. These findings underscore the potential of Ali-NPs@Mexo as a robust therapeutic strategy for TBI, combining targeted delivery with comprehensive anti-inflammatory, neuroprotective, and reparative effects. The innovative use of MSC-derived exosome-coated nanoparticles ensures enhanced brain targeting and prolonged therapeutic action, making this nanomedicine a promising candidate for clinical translation in the treatment of TBI. Traumatic brain injury Mesenchymal stem cell Exosomes Alitretinoin Nerve repair Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Traumatic brain injury (TBI) is one of the most serious injuries to the central nervous system, and millions of people suffer TBI every year from accidents, sports, and military conflicts [ 1 ]. TBI may result in permanent motor, sensory, and autonomic dysfunction [ 2 , 3 ]. Due to the limited self-repair capacity of the nerves and the complex pathological changes, there is no effective treatment strategy. Clinically available treatments, including high-dose glucocorticoid shock therapy, are mostly palliative and have little effect on recovery of sensory and motor function [ 4 ]. Reducing glial scar hyperplasia and maximizing neuronal axonal growth within ischemic regions remain major challenges in the treatment of TBI. Cell therapies, particularly those utilizing mesenchymal stromal cells (MSCs), have shown significant promise in regenerative medicine for treating various diseases, including TBI [ 5 ]. Administration of MSCs through various routes including intraarterial, intravenous, and intracereb ral has demonstrated significant therapeutic potential in experimental models of TBI [ 6 – 8 ]. However, each route presents specific limitations. Intracranial injection is constrained by the small amount of MSCs that can be administered, posing a challenge for achieving effective concentrations at the injury site [ 9 ]. Intraarterial injection, while potentially delivering MSCs more directly to the brain, carries the risk of inducing brain ischemia [ 10 ]. Intravenous administration, though less invasive, leads to a widespread distribution of MSCs, resulting in suboptimal localization and retention in the targeted brain regions [ 7 ]. MSC-derived exosomes (Mexo), which serve as nanocarriers for proteins, RNA, and lipids, have been demonstrated to modulate immune responses, and reduce inflammation [ 9 – 11 ]. Neovascularization is critical for TBI repair, as it provides the oxygen and nutrients necessary for neurogenesis, especially given the cerebral vasculature damage following the injury [ 12 ]. Mexo have been proven to promote angiogenesis, thereby accelerating nerve repair and neurological functional recovery after brain injury by fostering the growth of neuronal dendrites and axons [ 13 ]. These delivery challenges of MSCs highlight the promise of Mexo as a therapeutic alternative for TBI. Exosomes, being smaller and more stable than whole cells, can potentially overcome these hurdles by facilitating targeted delivery and retention at lesion sites [ 16 ]. Nonetheless, the development of efficient delivery systems and strategies to enhance the retention and efficacy of Mexo in the injured brain tissue remains a critical area for further research. Addressing these issues could significantly advance the therapeutic application of Mexo in TBI and improve outcomes for patients suffering from this condition. The blood-brain barrier (BBB) serves as a critical defense mechanism, restricting most therapeutic agents from entering the brain and thus maintaining brain homeostasis. However, this selective permeability poses a significant challenge for the treatment of brain diseases [ 14 ]. Notably, during TBI, the integrity of the BBB is compromised, creating a window of opportunity for therapeutic agents to reach the lesion sites [ 15 , 16 ]. Bovine serum albumin-based nanocarriers (BSA NPs), with particle sizes less than 100 nm, have demonstrated the capability to penetrate the BBB effectively [ 17 ]. This property positions them as promising vehicles for enhancing drug delivery to the brain. Research has indicated that albumin-based drug delivery nanosystems can substantially improve the efficiency of drug entry and retention within brain tissues [ 18 , 19 ]. Therefore, albumin-based nanoparticles are one of the best options for brain drug delivery. Alitretinoin, a derivative of all-trans retinoic acid (9-cis-retinoic acid), has demonstrated potential in neuroprotection and nerve regeneration in several studies [ 23 ]. Retinoic acid is known to protect against central nervous system damage by promoting neuronal survival and growth and reducing inflammatory responses. Similarly, alitretinoin may influence the repair process following nerve injury through its anti-inflammatory and immunomodulatory properties. In this study, we encapsulated the small-molecule nerve repair drug Alitretinoin (Ali) within bovine serum albumin (BSA) nanoparticles to form Ali-NPs, which can penetrate the blood-brain barrier. Subsequently, these Ali-NPs were further encapsulated with mesenchymal stem cell-derived exosomes (Mexo) through a co-extrusion process, resulting in the formation of Ali-NPs@Mexo (Scheme 1 A). This novel nanosystem is designed to reduce glial scar hyperplasia in the TBI area and promote the growth of neuronal axons, thereby facilitating nerve repair (Scheme 1 B). Our results demonstrate that this dual-encapsulation strategy not only effectively delivers Alitretinoin to the targeted brain regions but also leverages the unique properties of exosomes to modulate the inflammatory response and reshape the microenvironment, achieved a synergistic neuroprotective effect, highlighting its potential in the treatment of TBI. Materials and methods Materials BSA was purchased from sigma (a1933). Alitretinoin was purchased from Sinopharm Chemical Reagent (Shanghai, China). Annexin V-FITC/PI apoptosis detection kit was purchased from Dalian Meilun Biotechnology Co., Ltd (Dalian, China). 4′,6‐diamidino‐2‐phenylin dole (DAPI) was purchased from Sigma‐Aldrich (St. Louis, USA). RIPA lysis solution, nuclear and cytoplasmic protein extraction kit, and BCA protein quantification kit were purchased from Beyotime (Shanghai, China). Mouse TNF‐α, IL‐10 and IL‐1β ELISA kits were purchased from R&D Systems (Minnesota, USA). Animals C57 BL/6J (male, 8weeks) mouse were purchased from Shanghai Tenth People’s Hospital Laboratory Animal Technology Co., Ltd. (Shanghai, China). Production for MSC Exosomes Mesenchymal stem cells (MSCs) were isolated from mouse bone marrow and cultured according to established protocols. To harvest exosomes, we replaced the conventional culture medium with an exosome-depleted fetal bovine serum medium once the cells reached 60%-80% confluence. After an additional 24 hours of incubation, the culture medium was collected and subjected to a series of centrifugation steps to isolate the exosomes. Specifically, the medium was centrifuged at 3000g for 30 minutes to remove cell debris, followed by centrifugation at 10,000g for 1 hour to eliminate larger vesicles and particles. Finally, the supernatant was ultracentrifuged at 100,000g for 2 hours to pellet the exosomes. The exosome pellet was then resuspended in PBS at 4°C, and bacterial contamination was prevented by passing the suspension through a 0.22 µm filter. The filtered exosome solution was subsequently stored at -80°C for future use. Preparation of BSA, Ali-NPs, and Ali-NPs@MSCs For BSA activation, 120 mg of BSA, 60 mg of sodium dodecyl sulfate (SDS), and 4.4 mg of dithiothreitol (DTT) were added to a 5 mL sample vial. This mixture was fully dissolved in 3 mL of ultrapure water and then reacted for 2 hours in an oil bath. To prepare the MES buffer solution (0.1 M), 0.97 g of MES was dissolved in 50 mL of ultrapure water and the pH was adjusted to 4.6 with 0.1 M NaOH. To synthesize Ali-NPs, the drug was thoroughly mixed with 25 µL of Ca solution, 25 µL of Te solution, and 950 µL of MES buffer. Then, 25 µL of the activated BSA solution was added, maintaining a BSA-to-drug concentration ratio of 10:1, and the mixture was shaken for 4 hours. Finally, Ali-NPs@MSCs were obtained by mixing the synthesized Ali-NPs with MSCs in a specific proportion and extruding the mixture 30 times using a liposome extruder. Cytotoxicity The cell viability was investigated with Cell Counting Kit-8 (CCK-8) assay. The treated cells were resuspended and seeded into 96-well plates with a density of 3×10 3 cells per well. After incubation for 0, 12, 24, 48 h, the previous medium was discarded, and 100 µL of fresh medium containing 10 µL of CCK-8 was added to each well. After incubation for another 3 h, the cells were washed with PBS and added with 100 µL DMSO to dissolve the formed formazan. The absorbance at 450 nm was recorded after shaking at 100 rpm for 10 min, and the viability of cells without any treatments was used as a control. ROS detection was determined by DCFH-DA The treated cells were inoculated in 48-well plates. According to the instructions, the dilution ratio of the DCFH-DA reagent is 1:2000. 100 µL DCFH-DA working solution was added to each well and stained at 37°C for 30 min. PBS was used to remove the unstained DCFH-DA reagent and immediately photographed. Western Blot Total protein from Mexo and MSCs cells was isolated. After being quantified with Thermo Fisher Scientific's BCA Protein Assay Reagent, identical aliquots of total protein (30–60 g) were electrophoresed on SDS-PAGE before being transferred to PVDF membranes. After indoor incubation in blocking buffer containing NaCl (150 mmol L − 1 ), skim milk (5%), Tris-HCl (20 mmol L − 1 ) Tween-20 (0.05%, pH 7.6) for a period of 2 hours, the membranes were subjected to overnight cultivation (4℃) with first antibodies directed against ACTIN, CD9, TSG101, and Calnexin (dilution 1:1000; all from Cell Signaling Technology Inc) in 5% BSA. After three washes of 10 minutes each in saline plus Tween-20 (1x TBST), they were cultivated with a secondary antibody (1:1000; Cell Signaling Technology) for a period of 2 hours, followed by chemiluminescence visualization. ImageJ software was used for the densitometric analysis of immunoreactive bands. Immunofluorescence analysis The expressions of GFAP, MAP2 and Iba1 were determined by immunofluorescence. After fixation and permeability, brain tissue slices of each group were taken and cultured overnight at 4℃ with anti-GFAP, MAP2 and Iba1 antibodies (dilution 1:500). GFAP, MAP2 and Iba1 were labeled with goat anti-mouse antibodies. After that, slice with DAPI reverse dye (blue). Finally, fluorescence images were captured using confocal laser scanning microscopy (Leica TCS SP5, Leica Co. Ltd). RNA extraction and qRT-PCR Total RNA from tissues and cells was isolated using Trizol reagent (Gibco, Grand Island, USA), and RNA from sev was extracted using MiRNeasy micro kit (Qiagen, Germany). MiRNA was converted to cDNA using MicroRNA reverse transcription kit (Qiagen, Germany). Quantitative PCR was performed using the SYBR Green PCR Core reagent kit (Applied Biosystems,Foster City,CA,USA). cDNA amplification was performed on MicroAmp optical 96-well reaction plates (Applied Biosystems) and ABI PRISM 7700 sequence detection systems (Applied Biosystems). The reaction mixture (20 µL) consisted of 10 µL 2 x SYBR Green mix, 0.4 µL (10 µmol) of each primer, 2 µL cDNA and nuclease-free H 2 O. The initial reaction conditions were 95 ℃ for 10 min, then 20 s at 95 ℃, 1 min at 55 ℃, 30 s at 55 ℃, and 40 cycles. Elevated plus maze The Elevated plus maze consists of 2 closed arms (arm length 50 × arm width 10 × arm height 29 cm) and 2 open arms (arm length 50 × arm width 10 cm) and a central area (10 × 10 cm). Place the animal facing the open arm in the center. The camera system was used to record the behavior changes of the animals for 5 minutes, including the time to enter the open arm and the closed arm. Every remove feces after the end of the animal experiment, spray the bottom of the tank with 75% ethanol and wipe dry with clean gauze to avoid the previous one. The residual odor of an animal caused the influence of this experiment. During the experiment, keep quiet and avoid the sound to the experiment effect of result. Histological Analysis. The tissue samples isolated from mouse were taken and fixed in formaldehyde (4%) before paraffin embedding. (5 µm thick cross-sections were prepared. It was followed by hematoxylin and eosin (H&E) staining and the subsequent photographing with the Nikon brand optical microscope at the original magnification of 200X. Targeting Capability of Ali-NPs@MSCs Nanoparticles In Vivo mouse was injected with fluorescent probe (Cy5) labled BSA and Ali-NPs@MSCs nanoparticles with BSA labeled by the probe for imaging in vivo imaging system (IVIS) (Caliper) in 6, 8 and 12h, and then the heart, liver, spleen, lung, kidney, and brain were taken for imaging with IVIS (Caliper) in vitro. Statistical Analysis All statistical analysis were performed with GraphPad Prism software 9.0, and all results were reported as means ± standard deviation (SD). The statistical significance between groups were studied by unpaired t-test, which was presented as * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Results and discussion Preparation and characterization of Ali-NPs@Mexo BSA and Ali-NPs were prepared by the classical method, and the optimal particle size was obtained by adjusting the reaction pH. After that, the surface of Ali-NPs was coated with an exosome membrane by co-extrusion, named Ali-NPs@Mexo. As shown in Fig. 1 A, Transmission electron microscopy (TEM) images showed that BSA and Ali-NPS were spherical with good dispersity and rough surface, with particle size between 30 and 40nm. When coated with exosomes, Ali-NPs@Mexo particle size did not change much, and the surface was still rough. The hydrodynamic size (Fig. 1 B) of the nanoparticles was about 30 nm as measured by dynamic light scattering (DLS). The small particle size and narrow particle size distribution facilitate BBB penetration. The potential (Fig. 1 C) of BSA, Ali-NPS and Ali-NPs@Mexo were − 11.33 Mv, -20.4 Mv and − 25.5 mV, respectively. The content and release of Alitretinoin were measured by UV. The encapsulation efficiency and loading efficiency were 8.81 and 96.69% (Fig. 1 D-E), respectively. In addition, the hydrodynamic diameters of Ali-NPs@Mexo at 24 h were measured by DLS and did not change significantly (Fig. 1 F), indicating their good stability. Western blotting and quantification confirmed the enrichment of CD9, TSG101 in Mexo and Ali-NPs@Mexo. The biomarker of Calnexin was only in CM (Fig. 1 G-H). Ali-NPs@Mexo promoted anti-inflammatory and nerve repair The biocompatibility of the materials was assessed using a CCK-8 assay to evaluate cell viability. The results demonstrated that BSA, Alitretinoin (Ali), Ali-NPs, and Ali-NPs@Mexo did not exhibit significant cytotoxicity on BV2 cells at various time points, confirming their biocompatibility (Fig. 2 A, Figure S1 A ). Flow cytometry analysis revealed that the uptake of Ali-NPs@Mexo by BV2 cells was time-dependent, indicating efficient cellular internalization over time (Fig. 2 B). To investigate the anti-inflammatory and neurorepair functions of Ali-NPs@Mexo, a BV2 cell injury model was established via repeated LPS administration. Immunofluorescence confocal microscopy was employed to detect the anti-inflammatory marker YM1/2 (red fluorescence) and the pro-inflammatory marker COX2 (green fluorescence) after treatment with different nanomedicine groups (Fig. 2 C). Post-LPS treatment, RNA was extracted from the cells and qRT-PCR was conducted to quantify the expression of pro-inflammatory cytokines, including INOS, TNF-α, COX2, CD16, and CD32. The results showed that the Ali-NPs@Mexo group had a significant reduction in the expression of these cytokines, indicating a substantial decrease in the inflammatory response in BV2 cells (Fig. 2 D). Furthermore, treatment with Ali-NPs significantly reduced ROS levels in BV2 cells ( Figure S1 B-C ). This reduction in ROS is anticipated to facilitate the phenotypic shift of microglia from the pro-inflammatory M1 state to the anti-inflammatory M2 state, thereby enhancing the overall neuroprotective and repair mechanisms. This comprehensive approach highlights the potential of Ali-NPs@Mexo as a promising therapeutic strategy for mitigating inflammation and promoting nerve repair in TBI. To establish a neuronal cell injury model, LPS was administered repeatedly ( Figure S2, S3A ). Following this, primary neuronal cells were successfully isolated and used for subsequent experiments ( Figure S3C ). The biocompatibility of the materials was first evaluated using a CCK-8 assay, which demonstrated that the Ali-NPs@Mexo nanoparticles had minimal impact on neuronal viability (Fig. 3 A). To further explore the cellular uptake of Ali-NPs@Mexo nanomedicine, a phagocytosis experiment was conducted where FITC was conjugated to the nanomedicine. The mean fluorescence intensity (MFI) peaked when cells were incubated with Ali-NPs@Mexo for 8 hours, indicating efficient internalization by neurons (Fig. 3 B). Light microscopy images of neuronal cultures showed that the Ali-NPs@Mexo group alleviated cell damage and promoted neuronal repair, confirming the successful induction of the injury model ( Figure S3B ). To assess the changes in reactive oxygen species (ROS) levels within the cells, the ROS indicator probe DCFH-DA was used. The results demonstrated a significant reduction in ROS levels induced by LPS following treatment with Ali-NPs@Mexo, suggesting a decrease in the inflammatory response (Fig. 3 C). To further verify the anti-inflammatory and nerve repair functions of the nanomedicine, we examined the expression of Nestin and MAP2 through immunofluorescence confocal microscopy (Fig. 3 D). Nestin is an intermediate filament protein mainly expressed in proliferating or migrating neural stem cells/neural precursor cells during central nervous system development, while MAP2 is a microtubule-associated protein used as a marker of mature neurons. The results indicated that both Ali and Mexo alone exhibited certain therapeutic effects, with stronger red and green fluorescence signals compared to the LPS-induced group ( Figure S4 ). Notably, the Ali-NPs@Mexo nanomedicine group significantly promoted axonal growth and nerve repair. The MFI data provided additional confirmation, reflecting increased fluorescence intensity of Nestin and MAP2, thus illustrating the enhanced neuroprotective and regenerative effects of the Ali-NPs@Mexo nanomedicine. These findings underscore the potential of Ali-NPs@Mexo as a promising therapeutic approach for reducing inflammation and promoting neuronal repair in models of neuronal injury. Behavioral tests after treatment in brain injury model mice Barnes Maze was used to measure spatial learning and memory. After treatment (Fig. 4 A-B ) , the learning and memory impairment of TBI model mice were alleviated to a certain extent. Under the training condition, the number of reaching the dark box increased, and the residence time in the quarter area near the dark box (that is, the Barnes maze was divided into four quadrants) was also relatively prolonged. After treatment in the Ali-NPs@Mexo group, TBI mice showed a significant increase in occupancy of the quadrant close to the dark box, a residence time about five times longer than that of the model group, and a significant increase in the number of visits to the target dark box, indicating that TBI mice were greatly moderated after treatment. The elevated plus maze (EPM) is a widely utilized experimental method to assess anxiety-like responses in rodents. In our study, the TBI model group exhibited a lower exposure rate to the open arms compared to the treatment group (Fig. 4 C-D), indicating heightened anxiety and abnormal behavioral patterns post-injury. This reduced exploration of open areas is indicative of increased anxiety-like behavior. In the EPM experiment, mice in the TBI + Ali-NPs@Mexo group demonstrated a significant increase in both the time spent and the frequency of entries into the open arms compared to the TBI group. These findings suggest that the treatment with Ali-NPs@Mexo ameliorates anxiety-like behaviors induced by TBI. TBI is known to be associated with oxidative stress damage in brain tissue, which can lead to anxiety-like behavior and impaired spatial memory in mice. The administration of Ali-NPs@Mexo appears to mitigate oxidative stress damage in brain tissue, thereby exerting a neuroprotective effect. This neuroprotection is evidenced by the improved behavioral outcomes in the EPM, highlighting the potential of Ali-NPs@Mexo as a therapeutic strategy to address both the psychological and physiological sequelae of TBI. Brain targeting and neural repair ability of Ali-NPs@Mexo Coating mesenchymal stem cell-derived exosomes (Mexo) onto the surface of nanocarriers has been reported to confer them with natural properties from the original cell membrane. This not only reduces immunogenicity and prolongs their half-life, but also enhances their targeting capability [ 20 , 21 ]. We further assessed the brain enrichment capability of Ali-NPs@Mexo nanoparticles in a mouse model of TBI. Fluorescence imaging of the probe Cy5 in the brain post-TBI was performed at various time points after the nanoparticle injection. Ali-NPs@Mexo exhibited prominent accumulation in the brain compared to free Cy5, free BSA (Cy5 labled) at 8 hours post-injection, with significant retention observed up to 12 hours (Fig. 5 A). To confirm the targeting ability of Ali-NPs@Mexo to the brain, a tissue distribution experiment was conducted. As shown in Fig. 5 B, the accumulation of Ali-NPs@Mexo nanoparticles in the brain was significantly higher compared to the BSA group. Furthermore, cortical tissues from different groups of mice were obtained and examined via brain electron microscopy (Fig. 5 C). Quantitative analysis of synaptic density, dense material, length of synaptic gap, and number of synapses demonstrated significant recovery of neural synaptic structures following Ali-NPs@Mexo treatment. Additionally, Golgi staining images (Fig. 5 D) of cerebral cortical tissues, along with the quantification of neuronal branch lengths, indicated that Ali-NPs@Mexo significantly promoted neural synaptic plasticity. This evidence supports the potential of Ali-NPs@Mexo to enhance neural repair and recovery in TBI. Ali-NPs@Mexo Relieved the TBI and Reversed the Proinflammatory Microenvironment We subsequently evaluated the nerve repair efficacy of Ali-NPs@Mexo in mouse models of traumatic brain injury (TBI). As illustrated in Fig. 6 A, treatment with Ali-NPs@Mexo significantly ameliorated the pathological state induced by TBI. Confocal imaging further revealed that the expressions of myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and Nestin in the Ali-NPs@Mexo-treated group were comparable to those in the normal group (Figs. 6 B and 6 C). Ali-NPs@Mexo effectively inhibited the abnormal activation of astrocytes and reduced the expression of GFAP, a key marker of astrocyte activation. Moreover, Ali-NPs@Mexo promoted the expression of MBP, a marker of oligodendrocytes, thereby enhancing cell regeneration and the myelination of nerve cells. This treatment also stimulated the generation of neural stem cells. In addition to these regenerative effects, Ali-NPs@Mexo reduced the expression of pro-inflammatory cytokines, including IFN-γ ( Figure S5A ), IL-1β ( Figure S5B ), IL-6 ( Figure S5C ), and TNF‐α ( Figure S5D ), fostering an anti-inflammatory microenvironment. These results validate that Ali-NPs@Mexo treatment can achieve a synergistic neuroprotective effect in TBI therapy by simultaneously reducing oxidative stress and modulating the inflammatory microenvironment. The combination of these mechanisms underpins the potential of Ali-NPs@Mexo as a promising therapeutic strategy for enhancing neural repair and functional recovery following TBI. The protein contents of MBP, Tuj1, GFAP, Nestin, and IBA1 in brain tissue were assessed using immunofluorescence confocal microscopy. The results demonstrated that Ali-NPs@Mexo nanomedicine upregulated the expression of MBP and Tuj1 proteins (Fig. 7 A) while downregulating the expression of IBA1 proteins (Fig. 7 B). Further analysis through PCR ( Figure S6 ) and Western blot ( Figure S7 ) confirmed the changes in MBP, GFAP, and β3-Tubulin protein levels in brain tissue. The Ali-NPs@Mexo nanomedicine group showed increased levels of MBP and β3-Tubulin and decreased levels of GFAP. These findings indicate that Ali-NPs@Mexo can mitigate the inflammatory state of glial cells, promote the production of tubulin and related proteins, and facilitate myelin sheath formation, thereby promoting neuronal axon growth and nerve repair. Additionally, hematoxylin–eosin (H&E) staining of tissue sections revealed no significant abnormalities, suggesting that intravenous administration of the nanomedicine did not induce organ toxicity ( Figures S8 ). Collectively, these results highlight the therapeutic potential and favorable safety profile of the Ali-NPs@Mexo nanomedicine, underscoring its effectiveness in reducing inflammation, enhancing neuronal repair, and ensuring biocompatibility without adverse organ effects. Conclusion Traumatic brain injury (TBI) represents a widespread challenge globally, yet effective treatment options remain elusive [ 22 ]. TBI often results in a cascade of primary and secondary injuries, triggering the activation of astrocytes, microglia, and immune cells, leading to the production of pro-inflammatory cytokines [ 23 ]. Mexo, a promising form of bioactive nanomaterial, exhibit remarkable regulatory functions through the release of active cargo, rendering them versatile bioactive vehicles. In light of these insights, we have pioneered the development of Mexo-disguised albumin nanospheres loaded with Alitretinoin (Ali-NPs@Mexo) as a groundbreaking strategy to promote anti-inflammatory responses and nerve repair in TBI treatment. Our meticulously crafted nanomedicine, Ali-NPs@Mexo, demonstrates exceptional biocompatibility and precise brain targeting capabilities, particularly in penetrating the blood-brain barrier. Moreover, Ali-NPs@Mexo exhibits an extraordinary ability to actively accumulate within the brain, directly shielding neurons by mitigating reactive oxygen species (ROS) accumulation, thereby mitigating TBI-induced damage and alleviating microcirculation dysfunction [ 24 ]. Consequently, it fosters the growth of neuronal axons, facilitating nerve repair. This innovative nanomedicine heralds a paradigm shift in the landscape of TBI treatment, offering a multifaceted approach to address the complex pathophysiology of nerve injury. By leveraging the unique properties of MSC-derived exosomes, Ali-NPs@Mexo presents a novel strategy for multitargeted combined treatment, ushering in a new era of therapeutic possibilities for brain diseases. Declarations Acknowledgments This work was sponsored by the Shanghai Pujiang Programme (grant number 23PJD078). Author contributions Conceptualization, Lei Li and Songyu Chen; methodology, Junyu Lin and Xiaxuan Zhang; software, Peng Yu; validation, Yida Wang and Xianyu Deng; formal analysis, Xiaxuan Zhang; investigation, Lin Zhou; resources, Dongming Gao; data curation, Liang Gao; writing—original draft preparation, Songyu Chen; writing—review and editing, Junyu Lin and Xiaxuan Zhang; visualization, Songyu Chen; supervision, Lei Li.; project administration, Liang Gao; funding acquisition, Lei Li. All authors have read and agreed to the published version of the manuscript. Funding This work was sponsored by the Shanghai Pujiang Programme (grant number 23PJD078). Availability of data and materials Data, further information, and requests for resources and reagents supporting the findings of this study are available from the corresponding authors, Lei Li ( [email protected] ) and Liang Gao( [email protected] ). Ethics approval and consent to participate This study was approved by the Ethics Committee of Shanghai Tenth People’s Hospital, Tongji University School of Medicine (ethical approval number for animal research: SHDSYY-2022-6426). Consent for publication Not applicable. Competing interests The authors declare no competing interests regarding the publication of this paper. Author details a Department of Neurosurgery, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, China b Department of Neurosurgery, Shanghai Clinical Medical College, Anhui Medical University, Anhui, China. References Roozenbeek B, Maas AIR, Menon DK: Changing patterns in the epidemiology of traumatic brain injury. 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Dai Y, Dong J, Wu Y, Zhu M, Xiong W, Li H, Zhao Y, Hammock BD, Zhu X: Enhancement of the liver’s neuroprotective role ameliorates traumatic brain injury pathology. Proceedings of the National Academy of Sciences 2023, 120: e2301360120. Kong L-Z, Zhang R-L, Hu S-H, Lai J-B: Military traumatic brain injury: a challenge straddling neurology and psychiatry. Military Medical Research 2022, 9: 2. Han L, Jiang C: Evolution of blood–brain barrier in brain diseases and related systemic nanoscale brain-targeting drug delivery strategies. Acta Pharmaceutica Sinica B 2021, 11: 2306-2325. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx scheme.jpg Scheme 1. (A) Schematic representation of the preparation process for Ali-NPs@Mexo nanoparticles. (B) Illustration of the therapeutic application of Ali-NPs@Mexo for TBI. 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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-4573349","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":329602275,"identity":"a1f98bba-8a52-4797-9a69-afc480f56936","order_by":0,"name":"Songyu Chen","email":"","orcid":"","institution":"Department of Neurosurgery, Shanghai Tenth People’s Hospital, Tongji University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Songyu","middleName":"","lastName":"Chen","suffix":""},{"id":329602277,"identity":"d5dcfe40-2942-4f71-84e3-fa04a4b24714","order_by":1,"name":"Junyu Lin","email":"","orcid":"","institution":"Department of Neurosurgery, Shanghai Tenth People’s Hospital, Tongji University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Junyu","middleName":"","lastName":"Lin","suffix":""},{"id":329602278,"identity":"1cacb1d9-c003-45d9-9b39-f89a28efef7f","order_by":2,"name":"Xiaxuan Zhang","email":"","orcid":"","institution":"Department of Neurosurgery, Shanghai Clinical Medical College, Anhui Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaxuan","middleName":"","lastName":"Zhang","suffix":""},{"id":329602283,"identity":"54a7d33b-a33c-40d2-ba45-a36d4f83818d","order_by":3,"name":"Peng Yu","email":"","orcid":"","institution":"Department of Neurosurgery, Shanghai Tenth People’s Hospital, Tongji University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Yu","suffix":""},{"id":329602288,"identity":"8b814799-ae8f-424e-83f9-c95c3dc1ab2d","order_by":4,"name":"Yida Wang","email":"","orcid":"","institution":"Department of Neurosurgery, Shanghai Tenth People’s Hospital, Tongji University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yida","middleName":"","lastName":"Wang","suffix":""},{"id":329602292,"identity":"a6709ffb-7aaa-445d-9c05-838e0b6bf002","order_by":5,"name":"Xianyu Deng","email":"","orcid":"","institution":"Department of Neurosurgery, Shanghai Tenth People’s Hospital, Tongji University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xianyu","middleName":"","lastName":"Deng","suffix":""},{"id":329602295,"identity":"fe9abcb3-3755-497d-9c4c-9cd0b959824f","order_by":6,"name":"Lin Zhou","email":"","orcid":"","institution":"Department of Neurosurgery, Shanghai Tenth People’s Hospital, Tongji University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lin","middleName":"","lastName":"Zhou","suffix":""},{"id":329602298,"identity":"321b8dc7-ecff-4417-aa45-956bf749645e","order_by":7,"name":"Dongming Gao","email":"","orcid":"","institution":"Department of Neurosurgery, Shanghai Clinical Medical College, Anhui Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dongming","middleName":"","lastName":"Gao","suffix":""},{"id":329602299,"identity":"5fba6536-b0ca-43bd-b9be-ee44e2e990eb","order_by":8,"name":"Liang Gao","email":"","orcid":"","institution":"Department of Neurosurgery, Shanghai Tenth People’s Hospital, Tongji University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Gao","suffix":""},{"id":329602301,"identity":"909e1e15-726f-4acd-892e-efea0b76bfb8","order_by":9,"name":"Lei Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCUlEQVRIiWNgGAWjYBACAwbGBmYGAxsGPhAvASKYcICBgZmQljQGNogWA2K0gGUPQ7RA+GCARwt7M9CWgvPybBLZaQ8eMPyRM+df8PAAQ4V1YgP72QNYtfAcBDnstmGbRO52A6DDjC1nPAA67Ex6YgNPXgJWLRKJ7b+BWhiBWrZJALUkbrhxIOEAY9vhxAYJHgOsWuQfgmw5Z4+m5R8eLRLgEDuQiNByvgGopQGPFp5EkJbk5Daet0AtBsbGBjeAgZxwLN24jScHqxb79uMPmH/8sbPtZ8/dJvmjQk7O4PyZ5A8faqxl+9nPYNWCbikQS+QkgJMBGxHqoYD/+AHiFY+CUTAKRsFIAAA49Vz30E2OZQAAAABJRU5ErkJggg==","orcid":"","institution":"Department of Neurosurgery, Shanghai Tenth People’s Hospital, Tongji University School of Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-06-13 03:53:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4573349/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4573349/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60853057,"identity":"8921d6f2-829f-4ca5-aea7-d002407aecff","added_by":"auto","created_at":"2024-07-22 21:14:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2237070,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Representative TEM images of BSA, Ali-NPs, Mexo, Ali-NPs@Mexo (scale bar=50 nm). (B) Size distribution and (C) Zeta‐potential of as‐prepared BSA, Ali-NPs, Mexo, Ali-NPs@Mexo. (D) UV absorption of BSA, Ali-NPs, Mexo, Ali-NPs@Mexo. (E) Load rate and packet rate of Ali from Ali-NPs@Mexo. (F) The size stability of Ali-NPs@Mexo in 10% FBS over 48h. (G-H) Western blotting and quantification of CD9, TSG101, Calnexin expression in MSCs (CM), Mexo, Ali-NPs@Mexo. Results are presented as means ± SD, n = 3.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4573349/v1/2b8b435ef20060cc8a8b1ff1.png"},{"id":60853061,"identity":"359f1d93-401f-4ad7-bbfb-43ea6d8a9a4a","added_by":"auto","created_at":"2024-07-22 21:14:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4744198,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The cell viability of BV2 cells incubated with Ali and Ali-NPs@Mexo of different times. Data are presented as means ± SD, n = 5. (B) The flow cytometry results of cellular uptake, treated with Ali-NPs@Mexo for different time. (C) Representative fluorescence images and quantification of YM1/2 and Cox2 in BV2 co-cultured with Ali, Mexo and Ali-NPs@Mexo. (D) The expression level of INOS, TNF-α, COX2, CD16 and CD32 detected by qRT-PCR. * P \u0026lt; 0.05, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001, **** P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4573349/v1/eeed41f4646be7f5665a9ca6.png"},{"id":60853430,"identity":"4f054bc6-961c-4881-ae35-aed222729725","added_by":"auto","created_at":"2024-07-22 21:22:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6953286,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The cell viability of neuronal cells incubated with Ali and Ali-NPs@Mexo of different times. Data are presented as means ± SD, n = 5. (B) The flow cytometry results of cellular uptake, treated with Ali-NPs@Mexo for different time. (C) Representative fluorescence images and quantification of ROS which monitored using DCFH-DA (green). (D) Representative fluorescence images and quantification of Nestin and MAP2. * P \u0026lt; 0.05, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001, **** P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4573349/v1/1bc77495617bfbe75e53170a.png"},{"id":60853062,"identity":"d14e64e7-7fad-46cb-b6f9-f0e845cb53f3","added_by":"auto","created_at":"2024-07-22 21:14:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3376568,"visible":true,"origin":"","legend":"\u003cp\u003e(A-B). Statistical analysis of the escape latency and the total number of errors in the Barnes maze test. (C-D) Statistical analysis of the residence time in open area and the ratio of residence time in the closed to open area in the Elevated plus maze. * P \u0026lt; 0.05, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001, **** P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4573349/v1/96df55b7d3cc0b8d34020c4b.png"},{"id":60853065,"identity":"e439d861-db85-40f5-9813-c36682abdf17","added_by":"auto","created_at":"2024-07-22 21:14:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":16620101,"visible":true,"origin":"","legend":"\u003cp\u003e(A-B) The pharmacokinetic profiles of Ali-NPs@Mexo. (C) Electron micrographs of the brain and statistics of the number, density, width, and length of synapses. Data are presented as means ± SD, n = 3. (D) the Golgi staining images of cerebral cortical tissues and the quantification of neuronal branch lengths. * P \u0026lt; 0.05, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001, **** P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4573349/v1/e45b70e5c6a4d24841111279.png"},{"id":60853066,"identity":"5be171a8-89b6-4d10-9d33-b4150d003249","added_by":"auto","created_at":"2024-07-22 21:14:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":26856882,"visible":true,"origin":"","legend":"\u003cp\u003e(A) H\u0026amp;E and Nissl staining analysis after therapy. (B). IF staining and fluorescence intensity quantification of the MBP (green) and GFAP (red) in the brain tissues after treatment. The scale bar represents 20 μm. (C) IF staining and fluorescence intensity quantification of the MBP (green) and Nestin (red) in brain tissues after treatment. The scale bar represents 20 μm. ** P \u0026lt; 0.01, *** P \u0026lt; 0.001, **** P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4573349/v1/3bb5ad824ee605f54db86c2b.png"},{"id":60853063,"identity":"ebb0ff67-207f-445d-9689-5f16b26ab38f","added_by":"auto","created_at":"2024-07-22 21:14:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":13730482,"visible":true,"origin":"","legend":"\u003cp\u003e(A) IF staining and fluorescence intensity quantification of the MBP (green) and Tuji (red) in the brain tissues after treatment. The scale bar represents 20 μm. (B) IF staining and fluorescence intensity quantification of the MBP (green) and Iba1 (red) in the brain tissues after treatment. The scale bar represents 20 μm. ** P \u0026lt; 0.01, *** P \u0026lt; 0.001, **** P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-4573349/v1/8523187b1fc39b38f84f75a7.png"},{"id":61445293,"identity":"8d95afa8-29bf-4860-98a2-34f0a3e155b3","added_by":"auto","created_at":"2024-07-30 22:02:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":78765180,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4573349/v1/d87ac7e4-1afd-4ae1-9229-e1b6eb1ffb3f.pdf"},{"id":60853058,"identity":"fc2cc9d5-69c1-4772-89e8-c60edda6faec","added_by":"auto","created_at":"2024-07-22 21:14:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2367890,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4573349/v1/5e5c900d45f4c66d24cc4319.docx"},{"id":60853431,"identity":"8bfe1166-9de9-42f2-9f2a-2d5e29fb7d2f","added_by":"auto","created_at":"2024-07-22 21:22:17","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11319866,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1.\u003c/strong\u003e (A) Schematic representation of the preparation process for Ali-NPs@Mexo nanoparticles. (B) Illustration of the therapeutic application of Ali-NPs@Mexo for TBI.\u003c/p\u003e","description":"","filename":"scheme.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4573349/v1/512dc326937eb95effeeb6ba.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mesenchymal Stem Cell-derived exosomes loaded Alitretinoin for TBI repair treatment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTraumatic brain injury (TBI) is one of the most serious injuries to the central nervous system, and millions of people suffer TBI every year from accidents, sports, and military conflicts [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. TBI may result in permanent motor, sensory, and autonomic dysfunction [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Due to the limited self-repair capacity of the nerves and the complex pathological changes, there is no effective treatment strategy. Clinically available treatments, including high-dose glucocorticoid shock therapy, are mostly palliative and have little effect on recovery of sensory and motor function [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Reducing glial scar hyperplasia and maximizing neuronal axonal growth within ischemic regions remain major challenges in the treatment of TBI.\u003c/p\u003e \u003cp\u003eCell therapies, particularly those utilizing mesenchymal stromal cells (MSCs), have shown significant promise in regenerative medicine for treating various diseases, including TBI [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Administration of MSCs through various routes including intraarterial, intravenous, and intracereb ral has demonstrated significant therapeutic potential in experimental models of TBI [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, each route presents specific limitations. Intracranial injection is constrained by the small amount of MSCs that can be administered, posing a challenge for achieving effective concentrations at the injury site [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Intraarterial injection, while potentially delivering MSCs more directly to the brain, carries the risk of inducing brain ischemia [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Intravenous administration, though less invasive, leads to a widespread distribution of MSCs, resulting in suboptimal localization and retention in the targeted brain regions [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. MSC-derived exosomes (Mexo), which serve as nanocarriers for proteins, RNA, and lipids, have been demonstrated to modulate immune responses, and reduce inflammation [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Neovascularization is critical for TBI repair, as it provides the oxygen and nutrients necessary for neurogenesis, especially given the cerebral vasculature damage following the injury [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Mexo have been proven to promote angiogenesis, thereby accelerating nerve repair and neurological functional recovery after brain injury by fostering the growth of neuronal dendrites and axons [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese delivery challenges of MSCs highlight the promise of Mexo as a therapeutic alternative for TBI. Exosomes, being smaller and more stable than whole cells, can potentially overcome these hurdles by facilitating targeted delivery and retention at lesion sites [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Nonetheless, the development of efficient delivery systems and strategies to enhance the retention and efficacy of Mexo in the injured brain tissue remains a critical area for further research. Addressing these issues could significantly advance the therapeutic application of Mexo in TBI and improve outcomes for patients suffering from this condition.\u003c/p\u003e \u003cp\u003eThe blood-brain barrier (BBB) serves as a critical defense mechanism, restricting most therapeutic agents from entering the brain and thus maintaining brain homeostasis. However, this selective permeability poses a significant challenge for the treatment of brain diseases [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Notably, during TBI, the integrity of the BBB is compromised, creating a window of opportunity for therapeutic agents to reach the lesion sites [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Bovine serum albumin-based nanocarriers (BSA NPs), with particle sizes less than 100 nm, have demonstrated the capability to penetrate the BBB effectively [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This property positions them as promising vehicles for enhancing drug delivery to the brain. Research has indicated that albumin-based drug delivery nanosystems can substantially improve the efficiency of drug entry and retention within brain tissues [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Therefore, albumin-based nanoparticles are one of the best options for brain drug delivery. Alitretinoin, a derivative of all-trans retinoic acid (9-cis-retinoic acid), has demonstrated potential in neuroprotection and nerve regeneration in several studies [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Retinoic acid is known to protect against central nervous system damage by promoting neuronal survival and growth and reducing inflammatory responses. Similarly, alitretinoin may influence the repair process following nerve injury through its anti-inflammatory and immunomodulatory properties.\u003c/p\u003e \u003cp\u003eIn this study, we encapsulated the small-molecule nerve repair drug Alitretinoin (Ali) within bovine serum albumin (BSA) nanoparticles to form Ali-NPs, which can penetrate the blood-brain barrier. Subsequently, these Ali-NPs were further encapsulated with mesenchymal stem cell-derived exosomes (Mexo) through a co-extrusion process, resulting in the formation of Ali-NPs@Mexo (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This novel nanosystem is designed to reduce glial scar hyperplasia in the TBI area and promote the growth of neuronal axons, thereby facilitating nerve repair (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Our results demonstrate that this dual-encapsulation strategy not only effectively delivers Alitretinoin to the targeted brain regions but also leverages the unique properties of exosomes to modulate the inflammatory response and reshape the microenvironment, achieved a synergistic neuroprotective effect, highlighting its potential in the treatment of TBI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eBSA was purchased from sigma (a1933). Alitretinoin was purchased from Sinopharm Chemical Reagent (Shanghai, China). Annexin V-FITC/PI apoptosis detection kit was purchased from Dalian Meilun Biotechnology Co., Ltd (Dalian, China). 4\u0026prime;,6‐diamidino‐2‐phenylin dole (DAPI) was purchased from Sigma‐Aldrich (St. Louis, USA). RIPA lysis solution, nuclear and cytoplasmic protein extraction kit, and BCA protein quantification kit were purchased from Beyotime (Shanghai, China). Mouse TNF‐α, IL‐10 and IL‐1β ELISA kits were purchased from R\u0026amp;D Systems (Minnesota, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eC57 BL/6J (male, 8weeks) mouse were purchased from Shanghai Tenth People\u0026rsquo;s Hospital Laboratory Animal Technology Co., Ltd. (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eProduction for MSC Exosomes\u003c/h2\u003e \u003cp\u003eMesenchymal stem cells (MSCs) were isolated from mouse bone marrow and cultured according to established protocols. To harvest exosomes, we replaced the conventional culture medium with an exosome-depleted fetal bovine serum medium once the cells reached 60%-80% confluence. After an additional 24 hours of incubation, the culture medium was collected and subjected to a series of centrifugation steps to isolate the exosomes. Specifically, the medium was centrifuged at 3000g for 30 minutes to remove cell debris, followed by centrifugation at 10,000g for 1 hour to eliminate larger vesicles and particles. Finally, the supernatant was ultracentrifuged at 100,000g for 2 hours to pellet the exosomes. The exosome pellet was then resuspended in PBS at 4\u0026deg;C, and bacterial contamination was prevented by passing the suspension through a 0.22 \u0026micro;m filter. The filtered exosome solution was subsequently stored at -80\u0026deg;C for future use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of BSA, Ali-NPs, and Ali-NPs@MSCs\u003c/h2\u003e \u003cp\u003eFor BSA activation, 120 mg of BSA, 60 mg of sodium dodecyl sulfate (SDS), and 4.4 mg of dithiothreitol (DTT) were added to a 5 mL sample vial. This mixture was fully dissolved in 3 mL of ultrapure water and then reacted for 2 hours in an oil bath. To prepare the MES buffer solution (0.1 M), 0.97 g of MES was dissolved in 50 mL of ultrapure water and the pH was adjusted to 4.6 with 0.1 M NaOH. To synthesize Ali-NPs, the drug was thoroughly mixed with 25 \u0026micro;L of Ca solution, 25 \u0026micro;L of Te solution, and 950 \u0026micro;L of MES buffer. Then, 25 \u0026micro;L of the activated BSA solution was added, maintaining a BSA-to-drug concentration ratio of 10:1, and the mixture was shaken for 4 hours. Finally, Ali-NPs@MSCs were obtained by mixing the synthesized Ali-NPs with MSCs in a specific proportion and extruding the mixture 30 times using a liposome extruder.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity\u003c/h2\u003e \u003cp\u003eThe cell viability was investigated with Cell Counting Kit-8 (CCK-8) assay. The treated cells were resuspended and seeded into 96-well plates with a density of 3\u0026times;10\u003csup\u003e3\u003c/sup\u003ecells per well. After incubation for 0, 12, 24, 48 h, the previous medium was discarded, and 100 \u0026micro;L of fresh medium containing 10 \u0026micro;L of CCK-8 was added to each well. After incubation for another 3 h, the cells were washed with PBS and added with 100 \u0026micro;L DMSO to dissolve the formed formazan. The absorbance at 450 nm was recorded after shaking at 100 rpm for 10 min, and the viability of cells without any treatments was used as a control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eROS detection was determined by DCFH-DA\u003c/h2\u003e \u003cp\u003eThe treated cells were inoculated in 48-well plates. According to the instructions, the dilution ratio of the DCFH-DA reagent is 1:2000. 100 \u0026micro;L DCFH-DA working solution was added to each well and stained at 37\u0026deg;C for 30 min. PBS was used to remove the unstained DCFH-DA reagent and immediately photographed.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eWestern Blot\u003c/h2\u003e \u003cp\u003eTotal protein from Mexo and MSCs cells was isolated. After being quantified with Thermo Fisher Scientific's BCA Protein Assay Reagent, identical aliquots of total protein (30\u0026ndash;60 g) were electrophoresed on SDS-PAGE before being transferred to PVDF membranes. After indoor incubation in blocking buffer containing NaCl (150 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), skim milk (5%), Tris-HCl (20 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) Tween-20 (0.05%, pH 7.6) for a period of 2 hours, the membranes were subjected to overnight cultivation (4℃) with first antibodies directed against ACTIN, CD9, TSG101, and Calnexin (dilution 1:1000; all from Cell Signaling Technology Inc) in 5% BSA. After three washes of 10 minutes each in saline plus Tween-20 (1x TBST), they were cultivated with a secondary antibody (1:1000; Cell Signaling Technology) for a period of 2 hours, followed by chemiluminescence visualization. ImageJ software was used for the densitometric analysis of immunoreactive bands.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eImmunofluorescence analysis\u003c/h2\u003e \u003cp\u003eThe expressions of GFAP, MAP2 and Iba1 were determined by immunofluorescence. After fixation and permeability, brain tissue slices of each group were taken and cultured overnight at 4℃ with anti-GFAP, MAP2 and Iba1 antibodies (dilution 1:500). GFAP, MAP2 and Iba1 were labeled with goat anti-mouse antibodies. After that, slice with DAPI reverse dye (blue). Finally, fluorescence images were captured using confocal laser scanning microscopy (Leica TCS SP5, Leica Co. Ltd).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and qRT-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA from tissues and cells was isolated using Trizol reagent (Gibco, Grand Island, USA), and RNA from sev was extracted using MiRNeasy micro kit (Qiagen, Germany). MiRNA was converted to cDNA using MicroRNA reverse transcription kit (Qiagen, Germany). Quantitative PCR was performed using the SYBR Green PCR Core reagent kit (Applied Biosystems,Foster City,CA,USA). cDNA amplification was performed on MicroAmp optical 96-well reaction plates (Applied Biosystems) and ABI PRISM 7700 sequence detection systems (Applied Biosystems). The reaction mixture (20 \u0026micro;L) consisted of 10 \u0026micro;L 2 x SYBR Green mix, 0.4 \u0026micro;L (10 \u0026micro;mol) of each primer, 2 \u0026micro;L cDNA and nuclease-free H\u003csub\u003e2\u003c/sub\u003eO. The initial reaction conditions were 95 ℃ for 10 min, then 20 s at 95 ℃, 1 min at 55 ℃, 30 s at 55 ℃, and 40 cycles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eElevated plus maze\u003c/h2\u003e \u003cp\u003eThe Elevated plus maze consists of 2 closed arms (arm length 50 \u0026times; arm width 10 \u0026times; arm height 29 cm) and 2 open arms (arm length 50 \u0026times; arm width 10 cm) and a central area (10 \u0026times; 10 cm). Place the animal facing the open arm in the center. The camera system was used to record the behavior changes of the animals for 5 minutes, including the time to enter the open arm and the closed arm. Every remove feces after the end of the animal experiment, spray the bottom of the tank with 75% ethanol and wipe dry with clean gauze to avoid the previous one. The residual odor of an animal caused the influence of this experiment. During the experiment, keep quiet and avoid the sound to the experiment effect of result.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHistological Analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe tissue samples isolated from mouse were taken and fixed in formaldehyde (4%) before paraffin embedding. (5 \u0026micro;m thick cross-sections were prepared. It was followed by hematoxylin and eosin (H\u0026amp;E) staining and the subsequent photographing with the Nikon brand optical microscope at the original magnification of 200X.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTargeting Capability of Ali-NPs@MSCs Nanoparticles In Vivo\u003c/h2\u003e \u003cp\u003emouse was injected with fluorescent probe (Cy5) labled BSA and Ali-NPs@MSCs nanoparticles with BSA labeled by the probe for imaging in vivo imaging system (IVIS) (Caliper) in 6, 8 and 12h, and then the heart, liver, spleen, lung, kidney, and brain were taken for imaging with IVIS (Caliper) in vitro.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll statistical analysis were performed with GraphPad Prism software 9.0, and all results were reported as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The statistical significance between groups were studied by unpaired t-test, which was presented as * P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, **** P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003ePreparation and characterization of Ali-NPs@Mexo\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eBSA and Ali-NPs were prepared by the classical method, and the optimal particle size was obtained by adjusting the reaction pH. After that, the surface of Ali-NPs was coated with an exosome membrane by co-extrusion, named Ali-NPs@Mexo. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Transmission electron microscopy (TEM) images showed that BSA and Ali-NPS were spherical with good dispersity and rough surface, with particle size between 30 and 40nm. When coated with exosomes, Ali-NPs@Mexo particle size did not change much, and the surface was still rough. The hydrodynamic size (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) of the nanoparticles was about 30 nm as measured by dynamic light scattering (DLS). The small particle size and narrow particle size distribution facilitate BBB penetration. The potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) of BSA, Ali-NPS and Ali-NPs@Mexo were \u0026minus;\u0026thinsp;11.33 Mv, -20.4 Mv and \u0026minus;\u0026thinsp;25.5 mV, respectively. The content and release of Alitretinoin were measured by UV. The encapsulation efficiency and loading efficiency were 8.81 and 96.69% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-E), respectively. In addition, the hydrodynamic diameters of Ali-NPs@Mexo at 24 h were measured by DLS and did not change significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), indicating their good stability. Western blotting and quantification confirmed the enrichment of CD9, TSG101 in Mexo and Ali-NPs@Mexo. The biomarker of Calnexin was only in CM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAli-NPs@Mexo promoted anti-inflammatory and nerve repair\u003c/h2\u003e \u003cp\u003eThe biocompatibility of the materials was assessed using a CCK-8 assay to evaluate cell viability. The results demonstrated that BSA, Alitretinoin (Ali), Ali-NPs, and Ali-NPs@Mexo did not exhibit significant cytotoxicity on BV2 cells at various time points, confirming their biocompatibility (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003c/b\u003e). Flow cytometry analysis revealed that the uptake of Ali-NPs@Mexo by BV2 cells was time-dependent, indicating efficient cellular internalization over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). To investigate the anti-inflammatory and neurorepair functions of Ali-NPs@Mexo, a BV2 cell injury model was established via repeated LPS administration. Immunofluorescence confocal microscopy was employed to detect the anti-inflammatory marker YM1/2 (red fluorescence) and the pro-inflammatory marker COX2 (green fluorescence) after treatment with different nanomedicine groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Post-LPS treatment, RNA was extracted from the cells and qRT-PCR was conducted to quantify the expression of pro-inflammatory cytokines, including INOS, TNF-α, COX2, CD16, and CD32. The results showed that the Ali-NPs@Mexo group had a significant reduction in the expression of these cytokines, indicating a substantial decrease in the inflammatory response in BV2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Furthermore, treatment with Ali-NPs significantly reduced ROS levels in BV2 cells (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB-C\u003c/b\u003e). This reduction in ROS is anticipated to facilitate the phenotypic shift of microglia from the pro-inflammatory M1 state to the anti-inflammatory M2 state, thereby enhancing the overall neuroprotective and repair mechanisms. This comprehensive approach highlights the potential of Ali-NPs@Mexo as a promising therapeutic strategy for mitigating inflammation and promoting nerve repair in TBI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo establish a neuronal cell injury model, LPS was administered repeatedly (\u003cb\u003eFigure S2, S3A\u003c/b\u003e). Following this, primary neuronal cells were successfully isolated and used for subsequent experiments (\u003cb\u003eFigure S3C\u003c/b\u003e). The biocompatibility of the materials was first evaluated using a CCK-8 assay, which demonstrated that the Ali-NPs@Mexo nanoparticles had minimal impact on neuronal viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To further explore the cellular uptake of Ali-NPs@Mexo nanomedicine, a phagocytosis experiment was conducted where FITC was conjugated to the nanomedicine. The mean fluorescence intensity (MFI) peaked when cells were incubated with Ali-NPs@Mexo for 8 hours, indicating efficient internalization by neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Light microscopy images of neuronal cultures showed that the Ali-NPs@Mexo group alleviated cell damage and promoted neuronal repair, confirming the successful induction of the injury model (\u003cb\u003eFigure S3B\u003c/b\u003e). To assess the changes in reactive oxygen species (ROS) levels within the cells, the ROS indicator probe DCFH-DA was used. The results demonstrated a significant reduction in ROS levels induced by LPS following treatment with Ali-NPs@Mexo, suggesting a decrease in the inflammatory response (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). To further verify the anti-inflammatory and nerve repair functions of the nanomedicine, we examined the expression of Nestin and MAP2 through immunofluorescence confocal microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Nestin is an intermediate filament protein mainly expressed in proliferating or migrating neural stem cells/neural precursor cells during central nervous system development, while MAP2 is a microtubule-associated protein used as a marker of mature neurons. The results indicated that both Ali and Mexo alone exhibited certain therapeutic effects, with stronger red and green fluorescence signals compared to the LPS-induced group (\u003cb\u003eFigure S4\u003c/b\u003e). Notably, the Ali-NPs@Mexo nanomedicine group significantly promoted axonal growth and nerve repair. The MFI data provided additional confirmation, reflecting increased fluorescence intensity of Nestin and MAP2, thus illustrating the enhanced neuroprotective and regenerative effects of the Ali-NPs@Mexo nanomedicine. These findings underscore the potential of Ali-NPs@Mexo as a promising therapeutic approach for reducing inflammation and promoting neuronal repair in models of neuronal injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral tests after treatment in brain injury model mice\u003c/h2\u003e \u003cp\u003eBarnes Maze was used to measure spatial learning and memory. After treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B\u003cb\u003e)\u003c/b\u003e, the learning and memory impairment of TBI model mice were alleviated to a certain extent. Under the training condition, the number of reaching the dark box increased, and the residence time in the quarter area near the dark box (that is, the Barnes maze was divided into four quadrants) was also relatively prolonged. After treatment in the Ali-NPs@Mexo group, TBI mice showed a significant increase in occupancy of the quadrant close to the dark box, a residence time about five times longer than that of the model group, and a significant increase in the number of visits to the target dark box, indicating that TBI mice were greatly moderated after treatment.\u003c/p\u003e \u003cp\u003eThe elevated plus maze (EPM) is a widely utilized experimental method to assess anxiety-like responses in rodents. In our study, the TBI model group exhibited a lower exposure rate to the open arms compared to the treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D), indicating heightened anxiety and abnormal behavioral patterns post-injury. This reduced exploration of open areas is indicative of increased anxiety-like behavior. In the EPM experiment, mice in the TBI\u0026thinsp;+\u0026thinsp;Ali-NPs@Mexo group demonstrated a significant increase in both the time spent and the frequency of entries into the open arms compared to the TBI group. These findings suggest that the treatment with Ali-NPs@Mexo ameliorates anxiety-like behaviors induced by TBI. TBI is known to be associated with oxidative stress damage in brain tissue, which can lead to anxiety-like behavior and impaired spatial memory in mice. The administration of Ali-NPs@Mexo appears to mitigate oxidative stress damage in brain tissue, thereby exerting a neuroprotective effect. This neuroprotection is evidenced by the improved behavioral outcomes in the EPM, highlighting the potential of Ali-NPs@Mexo as a therapeutic strategy to address both the psychological and physiological sequelae of TBI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eBrain targeting and neural repair ability of Ali-NPs@Mexo\u003c/h2\u003e \u003cp\u003eCoating mesenchymal stem cell-derived exosomes (Mexo) onto the surface of nanocarriers has been reported to confer them with natural properties from the original cell membrane. This not only reduces immunogenicity and prolongs their half-life, but also enhances their targeting capability [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. We further assessed the brain enrichment capability of Ali-NPs@Mexo nanoparticles in a mouse model of TBI. Fluorescence imaging of the probe Cy5 in the brain post-TBI was performed at various time points after the nanoparticle injection. Ali-NPs@Mexo exhibited prominent accumulation in the brain compared to free Cy5, free BSA (Cy5 labled) at 8 hours post-injection, with significant retention observed up to 12 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). To confirm the targeting ability of Ali-NPs@Mexo to the brain, a tissue distribution experiment was conducted. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, the accumulation of Ali-NPs@Mexo nanoparticles in the brain was significantly higher compared to the BSA group. Furthermore, cortical tissues from different groups of mice were obtained and examined via brain electron microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Quantitative analysis of synaptic density, dense material, length of synaptic gap, and number of synapses demonstrated significant recovery of neural synaptic structures following Ali-NPs@Mexo treatment. Additionally, Golgi staining images (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) of cerebral cortical tissues, along with the quantification of neuronal branch lengths, indicated that Ali-NPs@Mexo significantly promoted neural synaptic plasticity. This evidence supports the potential of Ali-NPs@Mexo to enhance neural repair and recovery in TBI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eAli-NPs@Mexo Relieved the TBI and Reversed the Proinflammatory Microenvironment\u003c/h2\u003e \u003cp\u003eWe subsequently evaluated the nerve repair efficacy of Ali-NPs@Mexo in mouse models of traumatic brain injury (TBI). As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, treatment with Ali-NPs@Mexo significantly ameliorated the pathological state induced by TBI. Confocal imaging further revealed that the expressions of myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and Nestin in the Ali-NPs@Mexo-treated group were comparable to those in the normal group (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Ali-NPs@Mexo effectively inhibited the abnormal activation of astrocytes and reduced the expression of GFAP, a key marker of astrocyte activation. Moreover, Ali-NPs@Mexo promoted the expression of MBP, a marker of oligodendrocytes, thereby enhancing cell regeneration and the myelination of nerve cells. This treatment also stimulated the generation of neural stem cells. In addition to these regenerative effects, Ali-NPs@Mexo reduced the expression of pro-inflammatory cytokines, including IFN-γ (\u003cb\u003eFigure S5A\u003c/b\u003e), IL-1β (\u003cb\u003eFigure S5B\u003c/b\u003e), IL-6 (\u003cb\u003eFigure S5C\u003c/b\u003e), and TNF‐α (\u003cb\u003eFigure S5D\u003c/b\u003e), fostering an anti-inflammatory microenvironment. These results validate that Ali-NPs@Mexo treatment can achieve a synergistic neuroprotective effect in TBI therapy by simultaneously reducing oxidative stress and modulating the inflammatory microenvironment. The combination of these mechanisms underpins the potential of Ali-NPs@Mexo as a promising therapeutic strategy for enhancing neural repair and functional recovery following TBI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe protein contents of MBP, Tuj1, GFAP, Nestin, and IBA1 in brain tissue were assessed using immunofluorescence confocal microscopy. The results demonstrated that Ali-NPs@Mexo nanomedicine upregulated the expression of MBP and Tuj1 proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) while downregulating the expression of IBA1 proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Further analysis through PCR (\u003cb\u003eFigure S6\u003c/b\u003e) and Western blot (\u003cb\u003eFigure S7\u003c/b\u003e) confirmed the changes in MBP, GFAP, and β3-Tubulin protein levels in brain tissue. The Ali-NPs@Mexo nanomedicine group showed increased levels of MBP and β3-Tubulin and decreased levels of GFAP. These findings indicate that Ali-NPs@Mexo can mitigate the inflammatory state of glial cells, promote the production of tubulin and related proteins, and facilitate myelin sheath formation, thereby promoting neuronal axon growth and nerve repair. Additionally, hematoxylin\u0026ndash;eosin (H\u0026amp;E) staining of tissue sections revealed no significant abnormalities, suggesting that intravenous administration of the nanomedicine did not induce organ toxicity (\u003cb\u003eFigures S8\u003c/b\u003e). Collectively, these results highlight the therapeutic potential and favorable safety profile of the Ali-NPs@Mexo nanomedicine, underscoring its effectiveness in reducing inflammation, enhancing neuronal repair, and ensuring biocompatibility without adverse organ effects.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTraumatic brain injury (TBI) represents a widespread challenge globally, yet effective treatment options remain elusive [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. TBI often results in a cascade of primary and secondary injuries, triggering the activation of astrocytes, microglia, and immune cells, leading to the production of pro-inflammatory cytokines [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Mexo, a promising form of bioactive nanomaterial, exhibit remarkable regulatory functions through the release of active cargo, rendering them versatile bioactive vehicles. In light of these insights, we have pioneered the development of Mexo-disguised albumin nanospheres loaded with Alitretinoin (Ali-NPs@Mexo) as a groundbreaking strategy to promote anti-inflammatory responses and nerve repair in TBI treatment. Our meticulously crafted nanomedicine, Ali-NPs@Mexo, demonstrates exceptional biocompatibility and precise brain targeting capabilities, particularly in penetrating the blood-brain barrier. Moreover, Ali-NPs@Mexo exhibits an extraordinary ability to actively accumulate within the brain, directly shielding neurons by mitigating reactive oxygen species (ROS) accumulation, thereby mitigating TBI-induced damage and alleviating microcirculation dysfunction [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Consequently, it fosters the growth of neuronal axons, facilitating nerve repair. This innovative nanomedicine heralds a paradigm shift in the landscape of TBI treatment, offering a multifaceted approach to address the complex pathophysiology of nerve injury. By leveraging the unique properties of MSC-derived exosomes, Ali-NPs@Mexo presents a novel strategy for multitargeted combined treatment, ushering in a new era of therapeutic possibilities for brain diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was sponsored by the Shanghai Pujiang Programme (grant number 23PJD078).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, Lei Li and Songyu Chen; methodology, Junyu Lin and Xiaxuan Zhang; software, Peng Yu; validation, Yida Wang and Xianyu Deng; formal analysis, Xiaxuan Zhang; investigation, Lin Zhou; resources, Dongming Gao; data curation, Liang Gao; writing\u0026mdash;original draft preparation, Songyu Chen; writing\u0026mdash;review and editing, Junyu Lin and Xiaxuan Zhang; visualization, Songyu Chen; supervision, Lei Li.; project administration, Liang Gao; funding acquisition, Lei Li. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was sponsored by the Shanghai Pujiang Programme (grant number 23PJD078).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData, further information, and requests for resources and reagents supporting the findings of this study are available from the corresponding authors, Lei Li ([email protected]) and Liang Gao([email protected]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of Shanghai Tenth People\u0026rsquo;s Hospital, Tongji University School of Medicine (ethical approval number for animal research: SHDSYY-2022-6426).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests regarding the publication of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea Department of Neurosurgery, Shanghai Tenth People\u0026rsquo;s Hospital, Tongji University School of Medicine, Shanghai 200072, China\u003c/p\u003e\n\u003cp\u003eb Department of Neurosurgery, Shanghai Clinical Medical College, Anhui Medical University, Anhui, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRoozenbeek B, Maas AIR, Menon DK: \u003cstrong\u003eChanging patterns in the epidemiology of traumatic brain injury.\u003c/strong\u003e \u003cem\u003eNature Reviews Neurology \u003c/em\u003e2013, 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\u003cstrong\u003e9:\u003c/strong\u003e2.\u003c/li\u003e\n\u003cli\u003eHan L, Jiang C: \u003cstrong\u003eEvolution of blood\u0026ndash;brain barrier in brain diseases and related systemic nanoscale brain-targeting drug delivery strategies.\u003c/strong\u003e \u003cem\u003eActa Pharmaceutica Sinica B \u003c/em\u003e2021, \u003cstrong\u003e11:\u003c/strong\u003e2306-2325.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Traumatic brain injury, Mesenchymal stem cell, Exosomes, Alitretinoin, Nerve repair","lastPublishedDoi":"10.21203/rs.3.rs-4573349/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4573349/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTraumatic brain injury (TBI) remains a leading cause of mortality and morbidity worldwide. Current clinical treatments often target singular pathological processes, resulting in suboptimal outcomes due to a lack of comprehensive neuroprotective effects. To address this critical gap, we have developed an innovative therapeutic approach utilizing mesenchymal stromal cell (MSC)-derived exosome (Mexo)-coated albumin nanospheres loaded with Alitretinoin (Ali-NPs@Mexo). This novel design aims to enhance neural repair mechanisms, offering a multifaceted approach to neuroprotection and recovery in TBI patients. Our in vitro and in vivo experiments demonstrated that Ali-NPs@Mexo effectively modulates the TBI immuno-microenvironment by attenuating oxidative stress and neuroinflammatory responses. Treatment with Ali-NPs@Mexo was also found to reduce the abnormal activation of astrocytes, which contribute to glial scar formation that hinders neuronal repair. Furthermore, this nanomedicine promoted the proliferation and repair of oligodendrocytes, neural stem cells, and neurons. These findings underscore the potential of Ali-NPs@Mexo as a robust therapeutic strategy for TBI, combining targeted delivery with comprehensive anti-inflammatory, neuroprotective, and reparative effects. The innovative use of MSC-derived exosome-coated nanoparticles ensures enhanced brain targeting and prolonged therapeutic action, making this nanomedicine a promising candidate for clinical translation in the treatment of TBI.\u003c/p\u003e","manuscriptTitle":"Mesenchymal Stem Cell-derived exosomes loaded Alitretinoin for TBI repair treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-22 21:14:12","doi":"10.21203/rs.3.rs-4573349/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f401dcd9-6738-457e-9514-23a159887bac","owner":[],"postedDate":"July 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-30T21:53:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-22 21:14:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4573349","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4573349","identity":"rs-4573349","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}