Dual-drug-loaded nanohydrogel for intraoperative local application: sequential release-mediated spatiotemporal targeting of diverse secondary injury mechanisms to improve long-term prognosis in traumatic brain injury

Bin Zhang; Mengshi Yang; Shunchang Ma; Yumei Wang; Xueling Zhang; Can Zhu; Shuai Zheng; Xiyu Chen; Yaxuan Zhang; Miao Bai; Guangzhi Shi

doi:10.1186/s12916-026-05034-0

. 2026 Jun 30;24:500. doi: 10.1186/s12916-026-05034-0

Dual-drug-loaded nanohydrogel for intraoperative local application: sequential release-mediated spatiotemporal targeting of diverse secondary injury mechanisms to improve long-term prognosis in traumatic brain injury

Bin Zhang ^1,^2,^#, Mengshi Yang ^1,^#, Shunchang Ma ^3,^#, Yumei Wang ^1,^#, Xueling Zhang ¹, Can Zhu ⁴, Shuai Zheng ⁵, Xiyu Chen ¹, Yaxuan Zhang ¹, Miao Bai ⁶, Guangzhi Shi ^1,^2,^✉

PMCID: PMC13587472 PMID: 42381038

Abstract

Background

Traumatic brain injury (TBI) can induce both primary and secondary brain injuries. Hampered by a multitude of constraints, including the complexity of secondary injury pathophysiological mechanisms, the selective permeability of the blood-brain barrier (BBB), and the side effects of therapeutic agents, systemic monotherapy has demonstrated limited efficacy in improving the long-term prognosis of TBI patients. Therefore, there is an urgent need to develop novel therapeutic strategies that can bypass the BBB, avoid systemic complications, and target multiple secondary injury mechanisms simultaneously.

Methods

An injectable dual-drug-loaded nanohydrogel system was synthesized and its characteristics were evaluated. A mouse controlled cortical impact (CCI) model was established, and the nanohydrogel was locally administered intraoperatively. The neurological prognosis of mice was observed in both acute and chronic phases. Multiple methods, including evans blue assay, magnetic resonance imaging, transmission electron microscopy, western blot, enzyme-linked immunosorbent assay, and immunofluorescence staining, were used to evaluate the cerebral edema, BBB integrity, neuroinflammation, neuronal death/survival, angiogenesis, and neurogenesis after TBI.

Results

The nanohydrogel hybridizes hemoglobin (Hb) nanoparticles (NPs) with brain-derived neurotrophic factor (BDNF), uses these as the core to synthesize polydopamine (PDA) NPs, and loads dexamethasone (DEX) on their surface. In vitro drug release experiments confirmed that BDNF@Hb-PDA@DEX@gel had a high drug-loading rate and sequential sustained-release characteristics. The hydrogel matrix exhibited hemostatic and antibacterial effects. Both in vitro and in vivo experiments showed that the nanohydrogel could effectively reduce the acute-phase inflammatory response, protect BBB integrity, and alleviate cerebral edema by releasing DEX. In the late stage, it could promote brain tissue repair by releasing BDNF, including angiogenesis, neurogenesis, and neuron survival. The therapeutic efficacy of this dual-drug sequential delivery system was significantly superior to that of DEX monotherapy, and it could improve both acute and chronic neurological functions.

Conclusions

By virtue of local sequential and sustained release of multiple drugs, the injectable nanohydrogel-based dual-drug delivery system can target multiple secondary injury mechanisms of TBI and exert spatiotemporal therapeutic effects, which provides a new strategy for the effective management of complex secondary brain injury and the improvement of long-term prognosis in TBI.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12916-026-05034-0.

Keywords: Traumatic brain injury, Glucocorticoids, Neuroinflammation, Brain-Derived Neurotrophic Factor, Nanohydrogel Drug Delivery System, Neurorepair

Background

Traumatic brain injury (TBI) is a leading cause of injury-related mortality and disability globally [1]. TBI induces primary injury occurring instantaneously post-insult and secondary injury, an intricate cascade of pathophysiological processes elicited by primary injury. Secondary injury encompasses an interconnected sequence of complex pathophysiological events, including neuroinflammation, cerebral edema, ischemia, hemorrhage, calcium overload, and oxidative stress [2]. For patients with moderate-to-severe TBI, craniotomy for evacuation of necrotic brain tissue and hematomas, serves as primary treatment modalities in the acute phase. However, these surgical interventions cannot fully halt the progression of secondary injury, which can persist for days, weeks, or even decades, resulting in sustained neuronal loss and the development of various neurodegenerative diseases. These consequences not only profoundly impair patients’ quality of life but also impose considerable socioeconomic burdens [3, 4].

However, despite extensive experimental and clinical studies on the treatments of secondary brain injuries, there are still no drugs that can effectively target the complex mechanisms of secondary injuries. Many neuroprotective agents that were proven effective in basic experimental studies have failed to be beneficial in clinical trials [5–16]. For instance, high-dose glucocorticoids, due to their potent anti-inflammatory, edema reduction, and blood-brain barrier (BBB) protection properties, have been widely used in TBI patients. However, due to their severe systemic side effects and low BBB permeability, they are not recommended for TBI treatment by the current guidelines unless new compounds or new mechanisms are discovered [17–19]. Moreover, a majority of basic studies have shown that neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), have strong neuroprotective effects and can promote the regeneration of the neurovascular unit and the remodeling of neurites. However, BDNF is a large basic protein dimer with high hydrophilicity, incapable of traversing the BBB through passive diffusion. Following intravenous injection, only a negligible fraction reaches the brain parenchyma, far below the therapeutically effective concentration. Moreover, BDNF displays an extremely short circulating half-life of minutes to hours. It is susceptible to proteolytic degradation, with further reduced stability in the post-injury cerebral microenvironment. Even direct high-dose intracerebroventricular or intraparenchymal injection leads to rapid clearance and fails to sustain local drug levels over time, necessitating continuous delivery to preserve effective concentrations. Thus, despite the robust neuroprotective and restorative properties of BDNF, these limitations collectively impede its clinical translation and use [20–23].

In addition to BBB impermeability and the intrinsic side effects of therapeutic agents, the complexity and spatiotemporal specificity of secondary injury mechanisms represent another pivotal factor underlying the inefficacy of single-drug therapeutic strategies. The pathophysiological processes vary across different phases of TBI, which in turn demand distinct pharmacological intervention. For instance, neuroinflammation persists throughout the entire process of secondary injury. It is one of the core pathological mechanisms that interacts and cooperates with other pathological processes [24]. However, in the context of secondary injury, neuroinflammation acts as a double-edged sword [25, 26]. In the early stage of inflammation, overactive inflammatory cells and elevated levels of pro-inflammatory cytokines impair the integrity of the blood-brain barrier, aggravate brain edema, and lead to neuronal cell death [27, 28].

On the contrary, in the late inflammatory phase, these immune cells polarize toward an anti-inflammatory phenotype and secrete a repertoire of neurotrophic factors, including BDNF, vascular endothelial growth factor (VEGF), and transforming growth factor-β (TGF-β), which promote tissue repair and improve neurological function [29–31]. Previous investigations revealed that the anti-inflammatory effects mediated by DEX preserved BBB integrity and alleviated cerebral edema in the acute phase, but significantly suppressed brain tissue repair in the subacute phase [32, 33]. These findings indicate that monotherapy cannot meet the stage-specific requirements of TBI treatment. In contrast, sequential combination therapy emerges as a more feasible and promising therapeutic strategy. To date, no precise sequential therapeutic strategy targeting different pathological stages of TBI has been approved for clinical application. Therefore, to overcome the limitations of the blood-brain barrier, avoid systemic adverse reactions of the drug, and target the spatiotemporal specific secondary injury mechanism, local multiple sequential administration of drugs may be a key strategy to improve the therapeutic efficiency of pharmacological intervention after TBI.

Furthermore, repeated intracranial local injections are associated with high surgical risks and poor clinical translatability. For moderate to severe TBI patients requiring surgical intervention, single-dose local sustained-release delivery of therapeutics simultaneously with surgical debridement represents the most rational and clinically feasible strategy in both basic research and translational medicine. Over the past decade, organic, inorganic and hybrid nanoparticle-based carriers have propelled transformative advances in active-targeting drug delivery systems (DDS), with particularly prominent breakthroughs in cancer chemotherapy. These next-generation DDS are rationally engineered to possess a spectrum of optimized properties: reduced particle size, enhanced tissue permeability and drug solubility, improved physicochemical stability, precise site-specific targeting, prolonged sustained release, and diminished systemic toxicity, all of which collectively elevate therapeutic efficacy. As a result, these modern formulations substantially outperform traditional dosage forms in clinical and preclinical studies [34, 35]. Currently, representative DDS platforms include red blood cell membrane-camouflaged NPs [36], hyaluronic acid-based nanocarriers [37], polymer-lipid hybrid NPs [38], self-microemulsifying systems [39] and in situ forming gels, each offering unique advantages for specific clinical applications. For more than 60 years, the development of reproducible, controlled-release drug delivery systems has remained a central focus of pharmaceutical research [40]. In this context, in situ gel drug delivery has stood out as a particularly transformative strategy. Distinguished by its ability to undergo rapid sol-to-gel transition in response to physiological cues, this system not only achieves prolonged and tunable drug release at the target site but also significantly improves patient adherence and comfort by reducing dosing frequency [41]. Injectable self-healing hydrogels, with excellent biocompatibility and biodegradability, have become ideal carriers for local drug delivery and also serve as a soft supporting matrix for the brain cavity after TBI [42, 43]. However, despite the promising prospects, the local application scenarios within the brain require extremely high standards for hydrogel materials and properties, including appropriate elasticity and compliance, low expansion rate, shape adaptability, injectability, and biodegradability, and such materials must overcome the high infection rate and bleeding risks associated with material implantation.

Herein, we design and synthesize a novel nanohydrogel-based DDS, which possesses both therapeutic and functional properties. The hydrogel matrix is composed of carboxymethyl chitosan (CMC) and oxidized dextran (OD), endowed with injectability, in-situ gelation, and self-healing capacities. This hydrogel has a low swelling ratio and mechanical characteristics similar to those of the brain and can completely adhere and fill the irregular lesion cavities in the injured brain. Notably, by grafting antibacterial and hemostatic peptides onto the matrix, this gel system acquires strong antibacterial and hemostatic activities, capable of dealing with surgery-associated infections and hemorrhage risks. The nano-structural component of the DDS features a core-shell structure: with a core of hemoglobin (Hb)-loaded BDNF NPs and a shell of polydopamine (PDA)-encapsulated DEX (BDNF@Hb-PDA@DEX). In organic nanomaterials, PDA stands out due to its high drug-loading capacity, excellent biocompatibility, good biodegradability and adhesion performance, making it a highly promising candidate material. This rational core-shell structure enables sequential release of the encapsulated drugs, ensuring that neuroprotective and neurorestorative agents exert their respective therapeutic effects in a phase-specific manner aligned with the dynamic pathological cascade of TBI. We hypothesized that this local combination drug delivery system effectively inhibited the acute-phase inflammatory response, protected the BBB, reduced cerebral edema, and promoted neuron survival and brain tissue repair in the later stages, these pleiotropic effects might collectively contribute to the improvement of long-term neurological prognosis after TBI.

Methods

Preparation and Characterization of BDNF@Hb-PDA@DEX NPs

To activate Hb (H7379, Sigma-Aldrich, USA), Hb (120 mg), sodium dodecyl sulfate (SDS, 60 mg, N159397, Aladdin, Shanghai, China), and dithiothreitol (DTT, 4.4 mg), were transferred into a 5 mL sample vial with ultrapure water (3 mL), and then the vial was mixed thoroughly until all components were completely dissolved, resulting in a homogeneous solution. Subsequently, the vial was placed in an oil bath (at 90 °C) and the mixture was incubated at a stirring speed of 180 revolutions per minute (rpm) for 2 h. Finally, once the incubation was finished, sterile ultrapure water was used to dilute the reacted mixture, and the final concentration of the recombinant human BDNF (rhBDNF, ab151895, Abcam, UK) solution was adjusted to 0.15 mg/mL. To prepare BDNF@HB NPs, taking a 1 mL system as an example, 25 μL of activated HB solution (40 mg/mL) was added to a 24-well plate. Then 875 μL of MES and 100 μL (150 μg/mL, prepared in vehicle of 0.1% BSA) of BDNF solution were added under shaking conditions. Finally, the mixture was shaken at 37℃, 800 rpm for 4 h. The synthesized NPs were washed three times with a 100 kD ultrafiltration tube at 4000 rpm for 10 min, and then refrigeration at 4 °C was selected as the storage condition. For long-term storage, a white solid was obtained after being lyophilized and stored in a − 20 °C refrigerator.

To Synthesize the BDNF@Hb-PDA@DEX NPs, 1 mL of BDNF@Hb solution (Hb concentration: 1 mg/mL; BDNF concentration: 15μg/mL) was added to a 5 mL reaction vial, followed by the addition of 2 mL of Tris-HCl buffer (1 M, pH 8.5) and 12 mg of dopamine (H8502, Sigma-Aldrich, USA). The reaction mixture was stirred for 8 h. Subsequently, 1 mg of DEX (D4902, Sigma-Aldrich, USA) solution was added to the reaction, and the reaction was allowed to continue for 12 h. Then, the reaction mixture was centrifuged at a speed of 135,000 rpm for 15 min. The precipitate obtained was washed three times to obtain BDNF@Hb-PDA@DEX NPs. These particles were resuspended in ultrapure water for storage.

The particle size and zeta potential of BDNF@HB and BDNF@Hb-PDA@DEX NPs were tested by dynamic light scattering (DLS, Zetasizer Nano ZS90, Malvern Company, UK) (n = 3). The morphology of the NPs was characterized by transmission electron microscopy (TEM, FEI Tecnai G2 F20 S-Twin, FEI, USA). In addition, circular dichroism (CD) spectrometer (J-1500, JASCO, Japan) was used to analyze optically active substances of NPs, aiming to obtain information on their molecular configuration and conformation [44].

The loading and encapsulation rate of DEX and BDNF in NPs were quantitatively analyzed by high performance liquid chromatography (HPLC, LD- 20AD, Shimadzu, Japan) (n = 3). The calculation formulas are as follows: Drug loading efficiency = (weight of loaded drug) / (weight of NPs) × 100%. Encapsulation rate = (weight of loaded drug) / (weight of added drug) × 100%.

Preparation and Characterization of BDNF@Hb-PDA@DEX@gel

RADARGDK-OD/EPDMDAC-CMC (RAD-OD/EPD-CMC) hydrogel was prepared according to previous reported procedures [27]. Briefly, EPDMDAC (EPD, GL Biochem Ltd, Shanghai, China) (30 mg) was incubated with 5 mL CMC (3%) solution for 4 h, followed by dialysis for 24 h with a 1000 kDa dialysis bag to obtain EPD-CMC. OD was synthesized by oxidizing dextran with sodium periodate. For the preparation of RAD-OD, 10 mg of the hemostatic peptide RADARGDK (RAD, GL Biochem Ltd, Shanghai, China) was incorporated into 5 mL of 5% oxidized dextran solution, followed by a 2-hour Schiff base reaction. Subsequently, 2% EPD-CMC dispersion and 5% RAD-OD solution were blended at a 1:3 volume ratio to yield the blank control hydrogel. To obtain BDNF@Hb-PDA@DEX@gel, an equal volume of BDNF@Hb-PDA@DEX solution was first mixed with 2% EPD-CMC dispersion; the resultant mixture was then combined with RAD-OD solution at a 2:3 volume ratio. A colloidal transition was observed within 30 s of homogenization. The gelation time of hydrogel was determined by the tube inversion method. The formation of hydrogel was determined if no flow was observed after inverting the vial. The gelation time was recorded [45].

Transmission Electron Microscopy (TEM) (Tecnai G2 F20 S-Twin FEI, USA) was used to observe the morphological characteristics of hydrogel. For the identification of chemical functional groups in the hydrogels, a Tensor II Fourier transform infrared (FTIR) spectrometer (Nicolet IS 10, Thermo-Fisher Co., USA) was utilized [44, 45].

The elemental contents of carbon, nitrogen, oxygen and chlorine on the surfaces of different hydrogels were detected by X-ray photoelectron spectroscopy (XPS, ESCALAB 250XI, Thermal Metro USA) [44, 46].

The swelling ratio of the samples (n = 3) was assessed through the following steps [47]: the pre-frozen hydrogels were freeze-dried, and their initial mass was recorded as W₀. The samples after freeze-drying were placed in nylon bags and incubated in PBS at 37 °C for 24 h until complete swelling. At the preset time points, the hydrogels were taken out and weighed until a stable mass was reached. Before weighing, the excess water on the surface was gently wiped with filter paper, and the mass was recorded as Wₜ. Swelling ratio = (Wₜ – W₀) / W₀ × 100%.

The degradation behavior of the hydrogels (n = 3) was evaluated through the following scheme [45]: Hydrogels with a predefined initial mass (W₀) were incubated in PBS (pH 7.4) at 37 °C for different time points. After each incubation time, the hydrogels were taken out; the surface moisture was gently wiped with absorbent paper before weighing (recorded as Wₜ). The degradation rate was calculated by the formula: Degradation rate = (W₀ – Wₜ) / W₀ × 100%. Thermogravimetric analysis (TGA) was performed using a TGA2 SF/1100 instrument (Mettler-Toledo, Switzerland) under both air and nitrogen atmospheres. The analysis was conducted over a temperature range of 30–700 °C with a heating rate of 10 °C/min. Rheological properties of the hydrogels (diameter: 20 mm, height: 2 mm) were characterized using a MARS 60 rheometer (Haake, Germany).

The hydrogel rheology (20 mm in diameter, 2 mm in height) was analyzed to assess the sol–gel transition, injectable, and self-healing properties of the hydrogels using MARS 60 rheometer (Haake, Germany) [48]. The storage modulus G′ and loss modulus G′′ were measured via sweep frequency tests at 25 °C. Strain amplitude sweep (γ = 0.1 − 1000%) tests were conducted on the hydrogel samples at 25 °C to obtain the linear viscoelastic region. Then, alternate step strain sweep test was performed with the amplitude strains switched from low strain (1%) to high strain (300%) and with a duration of 100 s for each strain.

A cut/heal test was also conducted to investigate the macroscopic self-healing behavior of the hydrogel. Two hydrogels were labeled with and without rhodamine B, a red fluorescent dye. After complete gelation, the two hydrogels were cut with a scalpel into two pieces separately and then the four hydrogels with different colors were put together in the mold for 6 h at 25 °C, followed by static incubation. Then, external stress was applied to the healed hydrogel sample to check the stability [49].

The compression tests were performed on cylindrical hydrogel samples (n = 3) [44, 45]. According to previous procedures, hydrogels (10 mm diameter, 5 mm high) were subjected to compression tests using 10 N load cell at 1 mm/min until the sample ruptured or the compression strain reached 60%. Each hydrogel sample was tested 3 times to obtain the average values. The compressive rate was 1 mm/min. The compressive stress was obtained by dividing the load by the initial cross-sectional area. Each test was repeated at least three times. For cyclic compressive tests, cylindrical hydrogels (diameter of 10 mm, height of 5 mm) were cyclically compressed to a strain of 50% and returned to the initial height.

The tissue adhesion capability of the as-prepared hydrogel was further verified. Briefly, the fresh BDNF@Hb-PDA@DEX@gel was prepared. Fresh mouse organs were gently attached to the surface of the freshly prepared nanohydrogel. All tested tissues adhered firmly to the hydrogel, and no detachment of the organs was observed even after inversion, indicating that the hydrogel can tightly adhere to various fresh organs [45, 50].

In vitro drug release study

In vitro drug release behaviors of DEX and BDNF from BDNF@Hb-PDA@DEX@gel, were investigated according to previous procedures [51], with 100 µL of each formulation used for testing (n = 3). The drug-loaded nanohydrogel was transferred to a sealed centrifuge tube, added with 3mL volume of 37℃ pre-warmed PBS (pH 7.0), and tightly sealed. Samples were incubated at 37℃ in a constant-temperature shaking incubator at 50–100 rpm (to avoid shear-induced structural damage to the hydrogel). At each scheduled sampling time point, the whole release medium was collected, immediately replaced with an equal volume of isothermal fresh medium, and the tube was re-sealed and returned to the incubator. The drug concentration in all collected samples was quantified, and the cumulative drug release rate was calculated.

Each hydrogel sample was incubated in 3 mL of PBS (pH 7.0, 37℃), and the soaking solutions (100 µL) at different time points were collected for concentration determination.Specifically, sampling time points were set as 1, 2, 3, 4, 5, 6, 7, 14 and 21 days. The concentrations of DEX and BDNF in the collected solutions were quantified using a HPLC system (Waters e2695, USA).

In vitro antibacterial and hemostatic experiments

The agar diffusion assay was employed to evaluate the in vitro antibacterial activity of the hydrogels according to previous reported procedures [43]. Staphylococcus aureus (S. aureus, ATCC6538) and Escherichia coli (E. coli, ATCC25922) suspensions were inoculated onto Luria Bertani (LB, Shanghai Yuanye Bio-Technology Co., Ltd.) solid medium. Disc-shaped hydrogels (1 cm in diameter, 0.2 g in weight) (n = 3) were placed on the medium, and then incubated in the incubator at 37 °C. After 24 h of incubation, inhibition zones were observed to confirm the antibacterial efficacy.

The liver injury model and tail amputation model are the most commonly used methods for evaluating the in vivo hemostatic performance of biomaterials, which can be used to verify the hemostatic effects of materials on active and exudative bleeding. In the present study, to evaluate the hemostatic ability of hydrogels, liver injury (n = 3) and tail amputation models were conducted using SD rats (male, weighing 200–220 g, Beijing Vital River Technology, Ltd., Beijing, China) according to the established protocol [49]. The rats were anesthetized with 2% pentobarbital and then fixed on a corkboard. The rat tail was cut in the middle with surgical scissors and the liver surface was incised. The bleeding site was immediately covered with 500 µL of hydrogel. The hemostatic time and the amount of blood loss were recorded.

Additionally, whole blood clotting index (BCI) tests were carried out following standardized procedures [52]. Briefly, CaCl2 (0.2 M, 30 ul) was added to a mixture of whole blood (0.4 mL) and citrated (0.04 mL, 38 mg/mL) and then injected into each hydrogel sample in a centrifuge tube. After incubation for 2 min at 37 °C, deionized water (10 mL) was added to the tube and incubated for another 5 min, after which the supernatant was collected and centrifuged. The absorbance values were recorded (B). The absorbance of whole blood in deionized water (10 mL) was used as a negative control (A). The BCI was calculated as BCI (%) = B/A × 100%.

All experimental protocols were approved by the Institutional Animal Care and Use Committee of Capital Medical University.

In vivo and in vitro biocompatibility

The Cell Counting Kit-8 (CCK-8) assay was employed to assess the in vitro biocompatibility of the hydrogels [49]. Briefly, 293T (CRL-3216, ATCC, USA) cells were first seeded and incubated at 37 °C for 24 h, followed by replacement of the culture medium with fresh MEM containing 10 µL of the hydrogel. After further incubation for 2, 4, 6, 8, 12, and 24 h, 10% CCK-8 reagent (C0048, Beyotime, Nantong, China) was subsequently added to each well, and the mixture was incubated for an additional 4 h. The optical density (OD) value at 450 nm was determined using an ultraviolet-visible (UV-Vis) spectrophotometer (Uv-2600i, SHIMADZU, Japan). Cell viability was calculated by the formula: Cell viability = (A₁/A₀) × 100%, where A₁ and A₀ represent the absorbance values of the hydrogel-treated group and the untreated control group, respectively.

To assess the in vivo biocompatibility, hematoxylin-eosin (H&E) and enzyme-linked immunosorbent assay (ELISA) were used to evaluate the impact of the materials on the functions of major organs (heart, liver, lung, and kidney) (n = 6). Organs and blood samples were collected, then the histological changes and the levels of serum biochemical parameters (alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and urea nitrogen (BUN) were quantified on post-injury day 3.

ROS assay

The intracellular reactive oxygen species (ROS) levels were quantitatively determined using an ROS detection kit (S0033M, Beyotime, Shanghai, China) [53]. According to the manufacturer’s protocol, BV2 cells were cultured overnight, and then the culture medium was replaced with fresh medium with hydrogel (10 µl) for 4 h. Subsequently, DCFH-DA and DAPI were added to the culture medium, and the cells were incubated for 30 min to observe the intracellular ROS levels. Fluorescence images were obtained using a confocal laser scanning microscope (A1, Nikon, Japan).

After being stimulated with Lipopolysaccharide (LPS) (1 µg/mL; Sigma, L4391), BV2 cells were divided into five groups: (1) control group; (2) LPS group; (3) Hb-PDA@gel group; (4) BDNF@Hb-PDA@gel group; and (5) BDNF@Hb-PDA@DEX@gel group. Except for the sham group, all groups are treated with LPS in addition to the different formulations.

Controlled cortical impact (CCI) animal models

Adult male C57BL/6 mice (aged 6 − 8 weeks, 20–22 g) were purchased from Beijing Vital River Technology, Ltd. (Beijing, China) for this study.

PCI3000 PinPoint Precision Cortical Impactor (Hatteras Instruments, Cary, NC, USA) was used to establish a severe TBI model as we previously described [27]. Briefly, mice were anesthetized with 2% isoflurane (continuous delivery) and fixed in a stereotactic frame (RWD Life Science Co., Shenzhen, Guangdong, China). A midline incision was made to fully expose the skull, and then a right craniectomy was performed in the middle of the parietal bone (diameter 4 mm), while maintaining the integrity of the dura mater. A 3 mm circular impact head was used for injury induction, with parameters identical to those previously reported: speed 3.5 m/s, compression time 400 ms, and depth 2 mm. For the sham group, mice underwent craniotomy without CCI. Body temperature was maintained using a thermal pad throughout the surgical process.

Animal experimental groups and treatments

The mice were randomly divided into the following groups: (1) the sham group; (2) the CCI group; (3) the Hb-PDA@gel treatment group, (4) the Hb-PDA@DEX@Gel treatment group, and (5) the BDNF@Hb-PDA@DEX@gel treatment group. After debridement of the contused brain tissue and hematoma, an irregular cavity was formed at the cortical injury site. Two liquid hydrogels components were injected into the lesion cavity using a dual-lumen syringe (50 µl, DEX: 0.5 mg/mL gel; BDNF: 10ug/mL gel). After approximately 30 s, the liquid nanohydrogel underwent in situ gelation to form a gel state. The doses of DEX and BDNF were determined based on preliminary experiments and previous studies [43, 54].

To quantify the newly generated mature neurons, 5-bromo-2′-deoxyuridine (BrdU; B5002, Sigma-Aldrich, USA) was administered intraperitoneally twice daily at 8-h intervals after TBI for 7 consecutive days (50 mg/kg in saline). This 7-day BrdU labeling period (days 1–7 post-injury) ensured capture of the major proliferative phase induced by TBI.

Cavity volume

To quantify the lesion volume, serial coronal brain sections were cut at 30 µM on a cryostat (Leica, Germany) and collected every 300 µM. The slices were stained with HE. The lesion volume was traced and quantified by ImageJ [55].

ELISA assays

Inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were detected in the ipsilateral hemisphere. Tissue samples from the ipsilateral hemisphere were collected at 3 days post-injury. After homogenization, supernatants were obtained and used for further analysis. Cytokine levels were measured using specific ELISA kits (IL-1β: KGC1401-96, TNF-α: KGC8204-48, IL-6: KGC1404-96, Nanjing KeyGen Biotech. Co., Ltd.) in accordance with the manufacturer’s instructions.

Immunofluorescence staining in vivo and vitro

Paraffin-embedded brain slices were prepared for immunofluorescence staining. Briefly, brain slices were stained singly (CD31, NeuN, SOX2, DCX, ) or doubly (CD16/32 and Iba-1, CD34/PDGFRβ, BrdU/NeuN) on post-injury day 3 and 21 for immunohistochemical evaluation. Briefly, brain slices were incubated with primary antibodies overnight at 4 °C, followed by a 1-hour room-temperature incubation with Alexa Fluor-conjugated secondary antibodies (488: 1:500, ab150113 and ab150077; 594: 1:500, ab150080, both from Abcam, UK). Nuclei were counterstained with DAPI (Sigma-Aldrich) for 10 min to visualize cell populations.

Antibodies are listed as follows: rabbit polyclonal anti-NeuN (1:200, Abcam, ab177487, UK), anti-CD34 (1:200, Abcam, ab98395, UK), anti-CD31 (1:200, Abcam, ab98395, UK), anti-PDGFRβ (1:500, Abcam, ab69506, UK), anti-CD16/32 (1:500, Abcam, ab223200, UK), and anti-Iba-1(1:200, Abcam, ab178846, UK), anti-SOX2 (1:200, Abcam, ab92494, UK), anti-DCX (1:500, Abcam, ab18723, UK), anti-BrdU (1:200, Abcam, ab152095, UK).

TUNEL assay

TUNEL assay was employed for apoptotic cell detection using the TUNEL Detection Kit (11684817910, Roche, USA) on post-injury day 3 in accordance with the manufacturer’s instructions. Briefly, tissue sections were dewaxed, rehydrated, and subjected to proteinase K digestion for antigen retrieval. Subsequently, TUNEL reaction cocktail (comprising terminal deoxynucleotidyl transferase and dUTP) was added to the sections, followed by incubation at 37 °C for 2 h. After two washes with PBS, sections were incubated with peroxidase-conjugated anti-fluorescein antibody (Converter-POD) at 37 °C for 1 h to visualize labeled DNA breaks. Nuclei were counterstained with DAPI.

Image acquisition and cell counting

Full-field digital pictures of brain slices were captured using a MIDI FL system (3D Histech, Hungary). Quantification of cells was conducted in the ipsilateral cortex and hippocampus. Three equally sized, non-overlapping regions in the perilesional cortex or ipsilateral hippocampus were counted in three tissue sections per mouse (at 50 µM intervals). Data were expressed as the number of positive cells per mm² in both regions; additionally, the density of neurons and TUNEL-positive cells in the hippocampus was quantified as the number of positive cells per mm. Separately, the number of SOX2⁺, DCX⁺, and BrdU⁺ cells was counted in the dentate gyrus subregion of the hippocampus.

Determination of brain water content

Brain water content was evaluated by measuring the wet and dry weights of brain tissues at 3 days post-TBI. Mouse brain tissues were removed without transcardial perfusion, after which the injured cerebral hemisphere was dissected and weighed to determine the wet weight. The dissected tissue was then dried at 80 °C for 72 h until a constant weight was achieved, defined as the dry weight. Brain water content percentage was calculated using the formula: brain water content (%) = [(wet weight − dry weight)/wet weight] × 100%.

Evans blue extravasation

Extravasation of Evans Blue (EB, Sigma-Aldrich, USA) was used to assess the permeability of BBB in the injured side of the brain on post-injury day 3. A 4% EB solution (3 mL/kg) was administered via the tail vein of the mice. Four hours after the injection, the animals were anesthetized and underwent cardiac perfusion with PBS. Then, the injured side of the brain was isolated, weighed, and homogenized in 72 °C with formamide for 3 days. After centrifugation, the supernatant was collected and its absorbance at 620 nanometers was measured using a spectrophotometer (Bio-Rad, Hercules, CA, USA).

Western blot analysis

Three days post-TBI, the ipsilateral cortex and hippocampus were isolated and Total protein was extracted (n = 6). After determining protein concentration by BCA assay, Western blot analysis was conducted according to the procedures described previously [27]. Briefly, equal protein amounts (30 µg) were separated via SDS-PAGE, then electro-transferred onto methanol-activated PVDF membranes at 400 mA for 90 min. Membranes were blocked overnight at 4 °C in TBST with 5% non-fat dry milk (rocking platform) to block non-specific binding, followed by overnight incubation with primary antibodies at 4 °C. After three washes, membranes were incubated with corresponding secondary antibodies (1:5000, anti-mouse ab6728/anti-rabbit ab205718, Abcam, UK) in blocking buffer for 1 h at room temperature. Protein bands were visualized with an ECL system (1:1 reagent mix) and imaged using the Bio Spectrum 500 Imaging System (UVP Co., Upland, CA, USA). GAPDH was used as an internal loading control.

The primary antibodies are as follows: rabbit monoclonal anti-occludin (1:1,000, ab224526, Abcam, UK), rabbit monoclonal anti-claudin-5 (1:1,000, ab131259, Abcam, UK), anti-ZO-1 (1:1,000, ab276131, Abcam, UK), and anti-GAPDH (1:5,000, ab8245, Abcam, UK). Protein blots were visualized via chemiluminescence using a Bio Spectrum 500 Imaging System (UVP Co., Upland, CA, USA). Relative band densities were quantified with ImageJ software (version 1.49).

Magnetic Resonance Imaging (MRI)

In the present study, a 7.0 T animal MRI scanner (Bruker, Germany) was employed to evaluate brain edema volume on post-injury day 3. Briefly, all animals were anesthetized with pentobarbital. Axial T1/T2/ADC imaging was performed for each group with parameters as follows: field of view = 35 mm × 35 mm; layer thickness = 1 mm. MRI images were analyzed using RadiAnt DICOM viewer (version 4.6.9, Medixant, Poznan, Poland) and annotated by radiologists.

Transmission Electron Microscopy

On post-injury day 3, mice were transcardially perfused sequentially with 0.1 M PBS and a 4% paraformaldehyde/2.5% glutaraldehyde fixative in 0.1 M PBS. Ipsilateral cortical and hippocampal tissues adjacent to the injury core were dissected and post-fixed in the fixative at 4 °C for 24 h. The specimens were then trimmed into 1 mm³ tissue blocks and rinsed three times with 0.1 M PBS (10 min per rinse). Tissue blocks were dehydrated via a graded ethanol gradient (50%, 70%, 90%, 100%, v/v) and embedded in Epon 812 resin (Electron Microscopy Sciences). Ultrathin Sects. (70–90 nm) were sectioned with a diatome diamond knife using a Leica UC7 ultramicrotome and mounted onto copper grids. Sections were stained sequentially with 2% uranyl acetate (15 min) and lead citrate (5 min) for contrast enhancement. BBB ultrastructure, encompassing endothelial tight junctions, basement membrane integrity, and perivascular astrocyte endfeet, was examined via a Hitachi HT7800 transmission electron microscope (operating voltage: 80 kV). Images were acquired at 10,000×–30,000× magnification, with a focus on regions displaying tight junctions and astrocyte endfoot swelling.

Neurological function assessments

The Morris water maze (MWM) test was performed to evaluate spatial learning and memory capacity, following previously established protocols [43]. Each mouse was subjected to 4 daily trials over 5 successive days (preinjury days 7–3) to locate the submerged platform. Twenty-four hours post training, the submerged platform was withdrawn, and probe trials were performed at 1 day preinjury as well as 3 and 21 days post-injury. A video tracking system was used to quantify the time spent in the target quadrant. Sucrose preference test (SPT) and forced swim test (FST) were performed to evaluate depression-like behaviors (anhedonia and behavioral despair, respectively) post-TBI. For SPT, single-housed mice were pre-adapted with two bottles of 1% sucrose solution and tap water for 12 h first; after 24 h of food and water deprivation, the formal test was conducted for 2 days (12 h per day, with bottle positions swapped in the second 12 h), and sucrose preference (%) was calculated as (sucrose consumption/total consumption) × 100% by recording bottle weights pre- and post-test. For FST, each mouse was placed in a Perspex cylinder (30 cm height × 18 cm diameter) filled with 16 cm-deep water (22 ± 0.5 °C) for 6 min, and the durations of immobility and swimming were recorded via the SMART 3.0 imaging system (Panlab, Germany).

All assessments were performed by observers blinded to the experimental procedure.

Statistical analysis

All data are expressed as mean ± standard deviation (SD). Statistical analyses were performed via SPSS 26.0 software (IBM Corporation, USA). Latency data from the MWM test were analyzed using repeated-measures analysis of variance (rmANOVA) coupled with Bonferroni post hoc correction. For all remaining datasets, one-way ANOVA was conducted, followed by Tukey’s post hoc test for pairwise comparisons (Welch ANOVA with Dunnett’s T3 test was used for heterogeneous variance). Statistical significance was defined as a two-tailed P-value < 0.05.

Results

Formulation and characteristics of BDNF@Hb-PDA@DEX NPs

The synthesis process and neuroprotective effects of BDNF@Hb-PDA@DEX@gel were illustrated in Fig. 1. To prepare BDNF@Hb-PDA@DEX NPs, Hb and BDNF were first assembled into hybrid NPs. Subsequently, dopamine hydrochloride was polymerized onto these hybrids under magnetic stirring, forming a PDA shell for core encapsulation. Finally, DEX was loaded onto their surface via hydrophobic interactions and ionic adsorption. The particle size, zeta potential and morphology of the as-prepared NPs were characterized using DLS and TEM at 24 h post-reaction. TEM images revealed that the Hb, BDNF@Hb, and BDNF@Hb-PDA@DEX composite NPs all displayed uniform spherical structures (Fig. 2A-C). DLS revealed that BDNF@Hb NPs had a mean diameter of 47.44 nm, while the BDNF@Hb-PDA@DEX composite NPs showed a significantly increased size of 119.45 nm (Fig. 2D, E, P < 0.05).

The dispersion state of these NPs remained stable for up to 48 h, with a polydispersity index (PDI) below 0.3. (Fig. 2D, F). Zeta potential measurements were used to verify the successful synthesis of BDNF@Hb-PDA@DEX. Significant shift in zeta potential was observed among different NPs, confirming the successful assembly of the composite nanostructure (Fig. 2G).

FTIR spectroscopy was conducted to evaluate the chemical interactions between Hb, BDNF@Hb, and BDNF@Hb-PDA@DEX. As shown in Fig. 2H, Hb showed obvious signals at 1656 cm⁻¹ (amide I, -COO⁻ stretching) and 1541 cm⁻¹ (amide II, N-H bending). In the BDNF@Hb-PDA@DEX group, the Hb-derived amide II peak was weakened and blue-shifted to 1523 cm⁻¹, indicating the reactivity of the amino group of hemoglobin. Additionally, the phenolic C-O stretching band of PDA underwent a red shift from 1290 cm⁻¹ to 1299 cm⁻¹. The presence of DEX in the composite was confirmed by the residual C = O delocalization peak at 1663 cm⁻¹ (decreased intensity), further verifying the success of the assembly of the composite.

TGA was performed to assess the thermal stability of the samples. As shown in Fig. 2I, The BDNF@Hb complex showed mass loss between 150 and 480 °C, which was attributed to the degradation of its protein backbone, including the breakage of hydrogen bonds, disulfide bonds, and hydrophobic interactions. For BDNF@Hb-PDA@DEX, the decomposition of the PDA that was bound to the surface of BDNF@Hb began at 150 °C. At 790 °C, the mass loss of BDNF@Hb-PDA@DEX was lower than that of BDNF@Hb, suggesting that surface modification with PDA and DEX significantly improved the thermal stability of the composite material. The residual mass percentages of BDNF@Hb and BDNF@Hb-PDA@DEX were 21.80% and 33.93% at 790 °C, respectively.

The CD spectroscopy was shown in Additional file 1: Fig. S1, Hb showed a mixed structure of β-sheet and β-turn, and it presented a positive peak corresponding to the n − π* transition at 199 nm and a negative peak at 227 nm. Compared with Hb, Hb NPs exhibited a typical α-helix structure, and there was a positive peak at 194 nm, and two negative peaks at 209 nm and 222 nm, respectively, corresponding to the π − π* transition and the n − π* transitions. While the spectral lines of PDA and Hb@PDA showed relatively weak overall CD signals, PDA only had a weak positive peak at around 199 nm, indicating that the optical activity of PDA had little characteristic absorption to light. After Hb bound to PDA, the CD signal of Hb@PDA at 199 nm slightly increases, indicating that Hb@PDA has been successfully constructed, and the binding of Hb to PDA had enhanced the CD signal of PDA.

The drug loading efficiency and encapsulation efficiency of DEX were 21.28% and 84.97%, respectively, whereas for BDNF, the corresponding drug loading efficiency and encapsulation efficiency were 7.53% and 79.36%, respectively.

Synthesis and characteristics of the BDNF@Hb-PDA@DEX@gel

Postoperative bleeding and intracranial infection are the main complications of neurosurgery, and the implant materials will further increase these risks. In order to enhance the hemostatic and antibacterial properties of the nanohydrogel, we developed a hydrogel system with hemostatic and antibacterial activities. OD was grafted with a hemostatic peptide to form RAD-OD, while CMC was combined with an antibacterial quaternary ammonium salt to generate EPD-CMC. Finally, EPD-CMC and RAD-OD were cross-linked by aldehyde-amide Schiff base, resulting in a modified hydrogel system with combined hemostatic and antibacterial functions, suitable for neurosurgical applications.

As shown in Fig. 3A, B, the nanohydrogel used in this study is composed of two components, CMC and OD, both of which are liquid precursors before mixing. After mixing, the solution can undergo rapid dynamic cross-linking via schiff base bonds in approximately 30 s, and gradually change from a fluid to a colloid. The hydrogel exhibited a brownish-yellow color after the incorporation of composite NPs.

Two liquid hydrogels components were injected into the lesion cavity using a dual-lumen syringe. Benefiting from this feature of in situ injection and immediate gelation, the fluid precursor can adhere to the injured brain tissue and fill the irregular traumatic cavities upon injection, followed by rapid solidification. This is also one of the core advantages of injectable in situ hydrogels. The microscopic morphology of the gels was observed using SEM. The results are shown in Fig. 3C. The hydrogel presented a homogenous three-dimensional network structure with mutual cross-linking. Compared to the blank gel, the NPs were uniformly dispersed throughout the hydrogel in the BDNF@Hb-PDA@DEX@gel group, indicating that the hybrid NPs have successfully been loaded in the gel pores.

The physical photos and mass loss curves of Gel, BDNF@HB@gel, DEX@PDA@gel and BDNF@HB-DEX@PDA@gel hydrogels after degradation in PBS (pH = 7.4) are shown in Fig. 3B, D. The residual gel mass of the hydrogels gradually decreases with the increase of incubation time, and the degradation rates of blank Gel, and BDNF@Hb-PDA@DEX@gel hydrogels at 21 days are around 80 and 50% respectively. Compared to blank Gels, the mass loss rate of BDNF@Hb-PDA@DEX@gel under the same conditions is significantly reduced, which might be attributed to the addition of PDA. PDA NPs promoted the formation of more cross-linking points within the gel matrix, thereby making the internal structure of the gel more stable and thus resulting in a lower loss rate of the gel.

Expansion without dissolution is a key characteristic of hydrogel. Figure 3E showed the swelling test results of dry gels and wet gels. In the PBS solution, all gels absorbed water rapidly within 10 s and swelled, and the swelling ratio of the gels, BDNF@Hb@gel, DEX@PDA@gel, and BDNF@Hb-DEX@PDA@gel were 1700%, 1650%, 1400%, and 1430% respectively. Due to the swelling property of gels, which may cause a mass effect and increase the intracranial pressure, thereby affecting their local application in the brain after TBI, we further evaluated the volume changes of the wet gels over time in the PBS solution. The results showed that the cylindrical wet gel (diameter: 1 cm; height: 1 cm) had no significant volume change within 30 min. After 24 h, the gel blocks began to soften but did not dissolve, and this characteristic effectively avoided the intracranial volume occupation effect.

In addition, the rheological properties of the hydrogel were evaluated. As shown in Fig. 3F, within the strain range of 0.1% − 100%, the G’ (30–40 Pa) of the hydrogel was always higher than G’’ (0–10 Pa), indicating that it mainly exhibited elastic deformation and structural integrity. When the strain exceeded 100%, G’ and G’’ began to cross (gel-sol transition point); thereafter, G’ was smaller than G’’, indicating that it mainly exhibited viscous deformation and structural collapse. Notably, the incorporation of PDA NPs increased the G’ of the hydrogel, which may be due to the hydroxyl groups of PDA enhancing hydrogen bonding and providing additional Schiff base reaction sites, thereby overall improving the internal structural stability. With the change of shear rate (fixed shear strain), the viscosity was observed to change (Fig. 3G). This hydrogel exhibited shear-thinning behavior, the viscosity decreased with the increase of shear stress, confirming the injectability of the hydrogel.

The self-healing ability of hydrogels is one of their core functional characteristics, which can resist the flushing of cerebrospinal fluid and the degradation of extracellular matrix and prevents drug leakage due to gel rupture and ensures the stability of local drug concentration. As shown in Fig. 3H, the Stepwise dynamic strain amplitude assays (with an interval of 100 s between each step) showed that once the applied shear strain increased from 1% to 300%, the storage modulus (G′) dropped sharply and even be lower than the loss modulus (G′′). This obvious transformation behavior revealed that the hydrogel underwent a structural transition from a stable elastic gel network to a viscous sol state. Notably, after restoring the shear strain to 1%, G′ and G′′ rapidly returned to their original baseline values, which validated the efficient reconstruction of the damaged hydrogel network. Moreover, this reversible gel-sol transition could be stably and continuously maintained in consecutive cycles, thereby highlighting the inherent superior self-repairing ability of hydrogels.

Moreover, a cut/heal test was also conducted to investigate the macroscopic self-healing behavior of the hydrogel. The hydrogel was labeled with rhodamine B, a red fluorescent dye. After complete gelation, the hydrogel was cut with a scalpel and then tightly spliced with an unlabeled hydrogel block, followed by static incubation. As shown in Additional file 1: Fig. S2A, obvious fluorescence fusion was observed at the splicing interface, indicating the dynamic diffusion and rearrangement of polymer chains across the interface and effective healing of the spliced gap. which confirmed the excellent self-healing ability of this hydrogel.

Moreover, to examine the mechanical properties of these hydrogels, the compression properties were tested (Additional file 1: Fig. S2B). Compression stress–strain curves are shown in Fig. 2C. The results showed that addition of NPs increased the compressive modulus of blank hydrogel. The compressive modulus of BDNF@Hb-DEX@PDA@gel (38.33士9.48 kPa) and DEX@PDA@gel (37.28士7.93 kPa) were significantly higher than the blank gel (9.86士3.13 kPa) (P < 0.05, Additional file 1: Fig. S2D).

Finally, the tissue adhesion ability of the nanohydrogel was further verified, with the results presented in Additional file 1: Fig. S2E. Fresh mouse organs were attached to freshly prepared BDNF@Hb-PDA@DEX@Gel. All organs adhered firmly to the gel, and no detachment was observed even when the gel-organ were inverted, demonstrating the gel’s strong adhesive capacity for various fresh tissues. The incorporation of PDA endowed the hydrogel with enhanced tissue adhesion. Specifically, the phenolic hydroxyl/quinone groups on PDA can react with the amino groups of carboxymethyl chitosan and those in fresh tissues (e.g., via Schiff base formation), in addition to mediating reversible non-covalent interactions.

In vitro drug release test

HPLC was employed to quantify the in vitro drug release profiles of BDNF@Hb-DEX@PDA@gel. As shown in Fig. 3I, BDNF@Hb-DEX@PDA@gel mediated continuous and controlled DEX release, with approximately 57.75% cumulative release achieved within 3 days and nearly complete release attained by day 21. In contrast, BDNF displayed a substantially delayed release profile compared to DEX, with only 14.89% cumulative release over the same period. In contrast, BDNF release from the composite gel (BDNF@Hb-DEX@PDA@gel) was further regulated by the hydrogel network. A notable surge in BDNF release was not observed until day 3, followed by a sustained slow-release phase that reached 80% cumulative release by day 21. This sequential release was attributed to the structure of the NPs. DEX was attached to the PDA NPs and released rapidly in the acute phase. The BDNF@HB NPs are encapsulated by PDA. Therefore, the release of BDNF was limited by PDA and was released slowly as PDA gradually disintegrates. This release pattern was consistent with the regulatory role of the nanohydrogel network in regulating the spatiotemporal release of multiple drugs.

Antibacterial and hemostatic properties of the BDNF@Hb-DEX@PDA@gel

In this study, we utilized the EPD-CMC/RAD-OD hydrogel system, which was designed as the carrier for the nanomedicine. The experimental results showed that the hydrogel exhibited antibacterial activity against both Staphylococcus aureus and Escherichia coli (Additional file 1: Fig. S3). Meanwhile, BDNF@Hb-PDA@DEX@gel demonstrated reliable hemostatic performance, effectively reducing bleeding volume, shortening bleeding time, and lowering BCI, which was consistent with the previously verified functions (Additional file 1: Fig. S4).

Biocompatibility and antioxidation of BDNF@Hb-PDA@DEX@gel in vitro and in vivo

To evaluate the antioxidant capacity of Hb@PDA@gel and BDNF@Hb-DEX@PDA@gel in vitro, the DCFH-DA fluorescent probe was used to detect the reactive oxygen species (ROS) levels within BV2 microglial cells activated by LPS. As shown in Additional file 1: Fig. S5A, B, compared to the untreated control group, LPS stimulation induced a significant increase in the intracellular ROS level (p < 0.05), indicating severe oxidative stress in the activated microglial cells. Hb@PDA@gel treatment significantly reduced the accumulation of ROS (p < 0.05) compared to LPS group, confirming the inherent antioxidant potential of blank nanohydrogel. Moreover, BDNF@Hb-DEX@PDA@gel exhibited a more significant antioxidant effect, further reducing the intracellular ROS levels compared to the Hb@PDA@gel group (p < 0.05), verifying the synergistic antioxidant activity of the composite drug gel.

The CCK-8 assay was utilized to evaluate the in vitro cytotoxicity of different hydrogels by detecting 293T cell proliferation rates. As presented in Additional file 1: Fig. S5C, at different time points, the cell proliferation rates in the blank hydrogel, Hb@PDA@gel group and BDNF@Hb-DEX@PDA@gel group showed no significant differences compared with the control group, and all maintained high viability. These results demonstrated that hydrogels possessed favorable cytocompatibility, laying a solid foundation for subsequent in vivo applications.

Moreover, the in vivo biosafety of the hydrogels was comprehensively evaluated to lay a foundation for their potential clinical translation. Histopathological examination results showed that no obvious abnormal pathological changes were observed in the tissues of the hydrogel-treated groups, and the tissue morphology was consistent with that of the control group. Meanwhile, we detected key blood biochemical indicators to assess the functional status of major organs: AST and ALT (reflecting liver function), as well as Cr and BUN (indicating renal function). Quantitative analysis revealed no statistical differences in the levels of these indicators among all groups (P > 0.05). Collectively, these in vivo findings fully confirm the excellent biosafety of the BDNF@Hb-DEX@PDA@gel (Additional file 1: Fig. S6).

BDNF@Hb-PDA@DEX@gel promoted neuronal survival and improved neurological functions after TBI

Even after surgical removal of hematomas and necrotic brain tissues, secondary brain injury processes persist. In mice TBI model, we observed a continuous expansion of the lesion volume over time, accompanied by progressive impairment of neurological function, as evidenced by behavioral tests and histological analyses (Fig. 4). MWM, SPT, and FST were performed to evaluated spatial learning and memory abilities and depressive-like behaviors after CCI.

Fig. 4 — BDNF@Hb-PDA@DEX@gel treatment promotes brain tissue repair and ameliorates long-term neurological function after TBI. (A, B) Representative images of NeuN + and TUNEL+ cells (green fluorescence; Scale bar: 50 µm); (C, D) Quantification of NeuN+ cells in ipsilateral cortex and hippocampus on post-injury day 3 and 21 [one-way ANOVA, F (4, 25) = 77.38, P < 0.0001, η²=0.92 for cortex on post-injury day 3; F (4, 25) = 193.7, P < 0.0001, η²=0.97 for cortex on post-injury day 21; F (4, 25) = 66.42, P < 0.0001, η²=0.91 for hippocampus on post-injury day 3; F (4, 25) = 55.07, P < 0.0001, η²= 0.90 for hippocampus on post-injury day 21].; (E, F) Quantification of TUNEL+ cells in ipsilateral cortex and hippocampus on post-injury day 3 [one-way ANOVA, F (4, 25) = 581.4, P < 0.0001, η²=0.99 for cortex; F (4, 25) = 208.9, P < 0.0001, η²=0.97 for hippocampus]; (G) Representative HE images of injury lesion on post-injury day 21 (Scale bar: 1000 µM); (H) Quantification of the lesion volume on post-injury day 21[one-way ANOVA, F (4, 25) = 62.47, P < 0.0001, η²=0.91]; (I) The escape latency before CCI [Repeated-measures ANOVA, F (16, 100) = 0.093, P > 0.9999, η²=0.014]; (J) Percentage of time in goal quadrant before and after CCI [one-way ANOVA, F (4, 25) = 2.043, P = 0.119, η²= 0.25 for pre-injury day 1; F (4, 25) = 32.33, P < 0.0001, η²=0.84 for post-injury day 3; F (4, 25) = 70, P < 0.0001, η²=0.92 for post-injury day 21]; (K) Sucrose preference on post-injury day 3 and 21[one-way ANOVA, F (4, 25) = 26.47, P < 0.0001, η²=0.81 for post-injury day 3; F (4, 25) = 26.76, P < 0.0001, η²=0.81 for post-injury day 21]; and (L) The FST immobility time on post-injury day 3 and 21 [one-way ANOVA, F (4, 25) = 25.96, P < 0.0001, η²=0.81 for post-injury day 3; F (4, 25) = 44.85, P < 0.0001, η²=0.88 for post-injury day 21]. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. The data are presented as the means ± SD (n = 6)

Our results showed that Hb-PDA@gel treatment exhibited a certain degree of improvement in acute-phase neurological functions in TBI mice. Compared with the CCI control group, the blank gel group showed increased time percentages spent in the target quadrant, preference of sucrose, and reduced FST immobility time on post-injury day 3. In addition, Hb-PDA@gel reduced cell apoptosis and increased the number of survival neurons. However, these behavioral and histological improvements did not reach statistical significance (p > 0.05). These neuroprotective effects could potentially be attributed to the inherent biocompatibility of the nano-hydrogel matrix, which might have provided physical support for the surviving neural tissues and constructed a favorable local microenvironment, thereby mitigating the secondary impact of the initial traumatic injury to some extent.

Hb-PDA@DEX@gel (DEX-loaded hydrogel) treatment showed neuroprotective efficacy in the acute phase after TBI. On post-injury day 3, histological examination revealed a significant (P < 0.05) reduction in neuronal apoptosis and increase in neuronal survival in the acute phase after TBI. Additionally, behavioral tests showed significantly enhanced spatial learning and memory abilities and reduced depressive-like behaviors (P < 0.05). However, Hb-PDA@DEX@gel treatment did not significantly increase the number of neurons, reduce lesion volume, and improve neurological functions compared to the CCI group on post-injury day 21(P < 0.05). These results indicated that the treatment using DEX alone failed to promote long-term neural repair and improve the overall prognosis in the chronic stage after traumatic brain injury, which suggest that using dexamethasone alone may not be sufficient to induce significant brain tissue repair.

In contrast, the composite BDNF@Hb-PDA@DEX@gel significantly improved neurological functions during both the acute and chronic phases after TBI. Histological analysis further confirmed these findings, showing that the neuronal survival rate significantly increased, and the volume of traumatic injury was significantly reduced on post-injury day 21 after TBI (P < 0.05).

The composite hydrogel likely promoted neural tissue repair through the combination of DEX’s anti-inflammatory effect in the acute phase and BDNF’s neurotrophic support in the subacute and chronic phase.

BDNF@Hb-PDA@DEX@gel Inhibited neuroinflammation after TBI

In the acute phase after TBI, a robust inflammatory cascade is initiated, characterized by the activation and proliferation of resident brain immune cells and substantial infiltration of peripheral immune cells within the perilesional tissue. As the brain’s key innate immune guardians, microglia trigger robust inflammatory cascades in response to the recognition of damage-associated molecular patterns following TBI. Such microglial-initiated immune activation not only induces BBB breakdown but also facilitates the infiltration of peripheral leukocytes, thereby aggravating the progression of secondary neuronal injury. Immunohistochemical results revealed a significant increase in the number of Iba-1⁺ microglia and activated Iba-1⁺/CD16/32⁺ microglia in the ipsilateral cortex and hippocampus of TBI mice compared to sham-operated controls, consistent with the well-documented sterile inflammatory response triggered by mechanical brain injury (Fig. 5).

Fig. 5 — BDNF@Hb-PDA@DEX@gel treatment inhibits neuroinflammation after TBI. (A, B) Representative images of Iba+ (red fluorescence) and CD16/32 + cells (green fluorescence) (Scale bar: 50 µm); (C-F) Quantification of Iba+, and Iba+/CD16/32 + cells in ipsilateral cortex and hippocampus on post-injury day 3 [one-way ANOVA, F (4, 25) = 302.4, P < 0.0001, η²=0.98 for cortex; F (4, 25) = 331.1, P < 0.0001, η²=0.98 for hippocampus]; (G-I) Quantification of IL-1β, IL-6, and TNF-α levels in the ipsilateral hemisphere [one-way ANOVA, F (4, 25) = 72.2, P < 0.0001, η²=0.92 for IL-1β; F (4, 25) = 93.32, P < 0.0001, η²=0.94 for IL-6; F (4, 25) = 52.53, P < 0.0001, η²=0.89 for TNF-α ]. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. The data are presented as the means ± SD (n = 6)

TBI triggers a robust inflammatory response in the acute phase. The inherent immune cells, such as microglia, are first activated and proliferate, simultaneously releasing high levels of inflammatory factors, increasing the permeability of the blood-brain barrier, and promoting the infiltration of peripheral immune cells into the surrounding tissues of the injury site. This inflammatory cascade reaction not only increases cerebral edema and intracranial pressure, but also aggravates neuron damage. In this study, ELISA and immunohistochemical results showed that the expression levels of proinflammatory factors (IL-1β, IL-6, and TNF-α), the number of Iba-1⁺ microglia and activated Iba-1⁺/CD16/32⁺ microglia were significantly increased in the ipsilateral cerebral cortex and hippocampus on post-injury day 3. Hb-PDA@gel exerted anti-inflammatory effect and significantly reduced the activation of microglia and the expression levels of proinflammatory factors (P < 0.05) in the density of activated microglia compared to CCI group, suggesting that the nanohydrogel matrix itself exerts intrinsic anti-inflammatory properties. Notably, both Hb-PDA@DEX@gel and BDNF@Hb-PDA@DEX@gel treatment exerted stronger anti-inflammatory effects, which induced significant decrease in the number of activated microglia and downregulation of pro-inflammatory cytokine expression compared to Hb-PDA@DEX@gel group (P < 0.05), suggesting the enhanced anti-inflammatory effect by addition of DEX to nanohydrogel.

BDNF@Hb-PDA@DEX@gel restored BBB integrity, decreased BBB permeability, and reduced brain edema after TBI

In the present study, dry/wet ratio analysis and MRI was used to evaluate the effects of nanohydrogels on brain edema at 3 days post-injury (Fig. 6A). The results showed that TBI induced significant increase in brain water content (p < 0.05) and MRI edema volume (p < 0.05) in the injured hemisphere compared to sham group. Treatment with Hb-PDA@gel treatment reduced these parameters, but there was no statistical difference compared to CCI group (p > 0.05). In contrast, both Hb-PDA@DEX@gel and BDNF@Hb-PDA@DEX@gel treatment attenuated brain edema, significantly reduced brain water content and edema volume (p < 0.05) compared with CCI control group (Fig. 6C, D).

The disruption of BBB structural integrity and increased permeability promote the inflammatory response and aggravate brain edema after TBI. In this study, EB extravasation assays, TEM, and WB were used to detect the permeability and structural integrity of the BBB on post-injury day 3. The results showed that EB leakage significantly increased after TBI compared with the sham operation group animals, (p < 0.05, Fig. 6B, E,F). In contrast, both Hb-PDA@DEX@gel and BDNF@Hb-PDA@DEX@gel treatments reduced BBB permeability and significantly decreased EB penetration (p < 0.01). Moreover, TEM further confirmed the BBB damage after TBI at the ultrastructural level, including a significant decrease in tight junction density and extensive swelling of the end-foot of astrocytes (p < 0.05). Both Hb-PDA@DEX@gel and BDNF@Hb-PDA@DEX@gel treatment significantly increased the number of tight junctions and reduced the swelling of the end-foot (p < 0.05) (Additional file 1: Fig. S7A-C). Additionally, WB results showed that TBI induced a significant downregulation of ZO-1, occludin and claudin-5 compared to sham controls. Hb-PDA@DEX@gel and BDNF@Hb-PDA@DEX@gel treatments significantly upregulated these proteins (p < 0.05, Additional file 1: Fig. S7D, E), suggesting improved BBB structural integrity at the molecular level.

Collectively, the above results suggested that local administration of nanohydrogels containing DEX effectively reduced BBB permeability and preserved BBB integrity after TBI.

Local administration of BDNF@Hb-PDA@DEX promoted angiogenesis and the proliferation and differentiation of neural stem cell after TBI

TBI triggers both damage and repair responses, among which the repair of the neurovascular unit is the most important component. Angiogenesis and neurogenesis exhibit crosstalk and mutually reinforce each other, thereby synergistically facilitating neural tissue repair during the late phase post-TBI. The results showed that TBI caused severe damage to the blood vessel, the number of CD34⁺ mature endothelial cells and PDGFRβ⁺ pericytes in the injured cortex and hippocampus on post-injury day 3 decreased significantly (P < 0.05) (Additional file 1: Fig. S8). However, the number of CD31⁺ newborn cells increased significantly (P < 0.05), indicating that angiogenesis and vascular repair response were active after TBI (Fig. 7). Accordingly, TBI significantly induced the proliferation of SOX2⁺ neural stem cells (NSC) in the dentate gyrus (DG) area of the ipsilateral hippocampus, while the number of DCX⁺ immature neurons did not increase significantly, indicating that TBI mainly induced the proliferation of NSC rather than differentiation in the acute phase (Fig. 8). The above results suggested that endogenous reparative mechanisms were already initiated concurrently with injury in the acute phase after TBI, acting to counteract the associated damage.

Fig. 7 — Effects of BDNF@Hb-PDA@DEX@gel treatment on angiogenesis after TBI. (A) Representative images of CD31 + cells in the ipsilateral cortex and hippocampus (red fluorescence) (Scale bar: 50 µm); (B-E) Quantification of CD31 + cells in the ipsilateral cortex and hippocampus on post-injury day 3 and 21 [one-way ANOVA, F (4, 25) = 74.19, P < 0.0001, η²=0.92 for cortex on post-injury day 3; F (4, 25) = 48.06, P < 0.0001, η²=0.88 for hippocampus on post-injury day 3; F (4, 25) = 47.87, P < 0.0001, η²=0.88 for cortex on post-injury day 21; F (4, 25) = 45.58, P < 0.0001, η²=0.87 for hippocampus on post-injury day 21]. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. The data are presented as the means ± SD (n = 6)

Fig. 8 — Effects of BDNF@Hb-PDA@DEX@gel treatment on neurogenesis after TBI. (A) Representative images of NeuN+ (green fluorescence), BrdU+ (red fluorescence), SOX2+ (red fluorescence) and DCX+ (red fluorescence) cells in hippocampal DG area (Scale bar: 100 µm) ; (B) Quantification of BrdU+/NeuN+ cells on post-injury day 21 [one-way ANOVA, F (4, 25) = 59, P < 0.0001, η²=0.9]; (C) Quantification of SOX2 + cells on post-injury day 3 and 21 [one-way ANOVA, F (4, 25) = 327.4, P < 0.0001, η²=0.98 for post-injury day 3; F (4, 25) = 307.8, P < 0.0001, η²=0.98 for post-injury day 21]; (D) Quantification of DCX+ cells on post-injury day 3 and 21 [one-way ANOVA, F (4, 25) = 59.35, P < 0.0001, η²=0.9 for post-injury day 3; F (4, 25) = 423.7, P < 0.0001, η²=0.99 for post-injury day 21]. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. The data are presented as the means ± SD (n = 6)

However, although Hb-PDA@DEX@gel treatment showed significant protective effects on the BBB by promoting the survival of CD34⁺ endothelial cells and PDGFRβ⁺ pericytes on post-injury day 3, but it also inhibited angiogenesis by suppressing the proliferation of CD31⁺ cells both in acute (3 day) and chronic (21 day) phase after TBI which may be attributed to its anti-inflammatory properties dampening pro-angiogenic signals during tissue repair. Accordingly, the number of CD34⁺ cells and PDGFR⁺ cells were significantly reduced after Hb-PDA@DEX@gel treatment on post-injury day 21. In terms of neurogenesis and neuronal survival, this Hb-PDA@DEX@gel significantly reduces the number of SOX2 + and DCX⁺ cell in the DG area on post-injury day 3 and 21 compared to the TBI control group (p < 0.05), suggesting inhibition of both NSC proliferation and differentiation, and fails to improve long-term neuronal survival and significantly reduced the number of NeuN+/BrdU newborn cells in DG area on post-injury day 21, consistent with its inhibitory effect on neurogenesis.

In contrast, the BDNF@Hb-PDA@DEX@gel exerted a dual therapeutic effect: it not only preserved BBB structural integrity during the acute phase but also significantly promotes angiogenesis and vascular maturation. The results showed that BDNF@Hb-PDA@DEX@gel significantly increased the number of CD34+, PDGFRβ⁺, and CD31 + cells compared to the Hb-PDA@DEX@gel group. For neurogenesis and neuronal survival, the composite hydrogel significantly increased the number of SOX2⁺ and DCX⁺ cells on post-injury day 3 and 21 (p < 0.05), enhancing NSC proliferation and differentiation. Accordingly, BDNF@Hb-PDA@DEX@gel treatment significantly increase the number of NeuN+/BrdU newborn cells in DG area on post-injury day 21 (p < 0.05). These effects are likely due to the synergistic action of dexamethasone suppressing excessive inflammation and BDNF promoting vascular maturation and neuronal lineage commitment, highlighting its potential as a promising therapeutic approach for TBI. The above results indicated that the addition of BDNF to the DEX nanohydrogel effectively avoided the side effect of excessive anti-inflammatory action and exhibited multiple synergistic effects, providing an effective strategy for the treatment of TBI.

Discussion

Due to the intricate mechanisms of secondary injury, BBB impermeability, and the systemic complications of drugs, effective neuroprotective agents for TBI remain scarce until now. In order to overcome the above limitations, we developed a nanohydrogel dual-drug delivery system for intraoperative local administration after TBI. The core design feature of this DDS was a core-shell nanoparticle with the internal core composed of BDNF-conjugated Hb NPs and the external layer encapsulated by DEX-loaded PDA NPs. This integrated formulation possessed comprehensive combined antioxidative, anti-inflammatory, and neurotrophic capacities. The CMC/OD hydrogel matrix served as the carrier for the nano-drugs and has the properties of injectability, in situ gelation, compliance similar to brain tissue, and the ability to fully fill the irregular injury cavities without mass effect. By grafting with antibacterial and hemostatic peptides, it exhibited potent hemostatic and antibacterial capabilities. In vitro and in vivo evaluations showed that BDNF@Hb-PDA@DEX@gel had high drug loading efficiency and spatiotemporally controlled sequential release characteristics. Functionally, it effectively alleviated acute neuroinflammation, maintained the integrity of the blood-brain barrier, reduced brain edema by releasing DEX, and promoted angiogenesis and neurogenesis by releasing BDNF, which might collectively contribute to the improved neurological functions. Additionally, this local sequential drug delivery strategy could effectively alleviate systemic complications, bypass the obstruction of the blood-brain barrier, and increase local drug concentration. By exerting spatiotemporal therapeutic effects on the intricate multiphase secondary injury cascade, it represented a promising therapeutic approach for the treatment of TBI.

For moderate to severe TBI, surgical treatments can reduce the mortality rate during the acute stage, but they fail to halt the occurrence and development of secondary injuries after surgery. Therefore, pharmacotherapy becomes extremely important. Targeting different secondary injury mechanisms after TBI, numerous basic and clinical studies have previously focused on drug treatments, but most of them have failed to yield positive results [5–16]. The low permeability of BBB and the systemic complications of the drugs are regarded as the key factors underlying the failure of drug therapy. However, the complexity and spatiotemporal specificity of the secondary injury mechanisms have been largely ignored in most basic and clinical experiments. Monotherapy, inappropriate administration methods, and suboptimal dosing timing and sequencing all contribute to the negative outcomes of current clinical trials. Therefore, in light of these challenges, future drug treatment research should prioritize the development of more targeted and efficacious therapeutic strategies, such as modifying drug delivery modalities to reduce systemic complications and increase local drug concentration in brain tissue, as well as designing combinatorial medication regimens tailored to the complexity and spatiotemporal specificity of secondary injury mechanisms.

Currently, the main clinical administration routes for pharmacological interventions in traumatic brain injury include intravenous infusion, intrathecal injection, and direct intraoperative administration. However, the neuroprotective effects of both glucocorticoids and BDNF require long-term and sustained drug exposure. Repeated intracranial local injections are associated with high risks of surgical trauma and infection and exhibit extremely poor clinical operability and translational potential. Meanwhile, BDNF suffers from an inherent short half-life, failing to exert long-lasting and stable neuroprotection in the injured region. Even direct high-dose intracerebroventricular or intraparenchymal injection leads to rapid clearance and fails to sustain local drug levels over time, necessitating continuous delivery to preserve effective concentrations. For moderate to severe TBI patients requiring surgical intervention, single-dose local sustained-release delivery of therapeutics simultaneously with surgical debridement represents the most rational and clinically feasible strategy in both basic research and translational medicine.

In recent years, the development of medical materials has created new avenues for breaking through the limitations of current drug therapies for TBI [56–58]. Nanohydrogels combine the advantages of natural hydrogels and nanostructures, offering a multifunctional and highly promising platform in biomedical applications. These hybrid systems combine the biocompatibility and biodegradability of hydrogels with the high drug-loading capacity and tunable release characteristics of NPs, achieving synergistic enhancement in therapeutic effects and safety. They have great application prospects and can enable local multi-drug delivery, bypass the blood-brain barrier, enable the synergistic use of different drugs, and promote sequential drug release [59–61]. In addition, the three-dimensional porous structure of nanohydrogels provides an ideal microenvironment for tissue repair and drug delivery, making them applicable in a wide range of fields from cancer treatment to wound healing and neurological diseases [62]. However, local dual-loaded drug nanohydrogel systems have been predominantly applied in local chemoradiotherapy in the tumor surgical area. In contrast, research on local nanohydrogel-based therapies after TBI have mostly focused on mono-therapeutic regimens or the therapeutic effects of the material itself, and there is no nanohydrogel system that has been clinically proven effective. Therefore, the role of nanohydrogel dual-loaded drug systems in TBI treatment needs further study.

In the present study, the nanohydrogel system we developed was composed of several key components, each of which has its unique advantages. Hb NPs have excellent biocompatibility and oxygen-carrying capacity, which not only can mimic the function of red blood cells to increase oxygen delivery to the injured brain tissue, but also can serve as a drug carrier. Nanomaterial-related Hb-based oxygen carriers (Nano-HBOCs) are novel oxygen carriers fabricated by the organic combination of nanomaterials and Hb through strategies such as encapsulation, self-assembly, bioconjugation, embedding, or surface attachment [63]. They can effectively overcome the side effects of conventional HBOCs, including vasoconstriction, oxidative stress toxicity, and rapid oxidation of Hb, while prolonging in vivo half-life. Among them, PDA coated Hb (PDA-Hb) represents a classic Nano-HBOCs [64, 65]. PDA NPs are another critical component, which have excellent adhesion properties, antioxidant and anti-inflammatory capabilities. Therefore, PDA NPs have high drug loading capacity and can provide a sustained release platform for drugs [66]. The hydrogel matrix was composed of CMC and OD. CMC has good biocompatibility, biodegradability, and antibacterial properties. OD can form dynamic Schiff base bonds with CMC, endowing the hydrogel with properties of in situ gelation and self-healing. This hydrogel matrix has mechanical properties similar to brain tissue, alleviating mechanical damage to surrounding tissue during injection and providing a soft and compliant microenvironment that is conducive to brain tissue repair.

Our results showed that this core-shell nanostructure had high loading rates and enabled the sequential sustained release of BDNF and DEX at different time points. Hb@PDA@gel exhibited strong adhesion to fresh brain tissue and resistance to cerebrospinal fluid scouring, ensuring stable localization at the irregular injury cavity and preventing drug leakage. The integration of PDA enhanced the hydrogel’s mechanical and adhesive properties by increasing the number of Schiff-base reaction sites between PDA and the CMC/OD matrix. Functionally, In vitro and in vivo evaluations suggested that Hb@PDA@gel itself exhibited inherent antioxidative and anti-inflammatory effects, which attenuated the activation of inflammatory cells and the release of pro-inflammatory cytokines after TBI, thereby exerting a synergistic effect with DEX. Hemorrhage and implant-related intracranial infection are the most common complications of craniocerebral surgery. Chitosan/dextran hydrogel exerts antibacterial effects through multiple mechanisms, including electrostatic interaction, membrane disruption, DNA inhibition, and synergistic effects, and possesses broad-spectrum antibacterial activity and favorable biocompatibility. On this basis, we further introduced the antimicrobial peptide EPD to significantly enhance the overall antibacterial performance of the system. The in vitro results revealed that the addition of hemostatic peptide RAD and antibacterial peptide EPD enhanced the hemostatic and antibacterial properties of the composite nanohydrogel, which could reduce the incidence of infection and hemorrhage risks associated with implantation of biomedical materials. However, in vivo results showed that monotherapy with Hb@PDA@gel alone was insufficient to promote robust brain tissue repair and improve long-term prognostic outcomes.

TBI induces sterile inflammation by damage-associated molecular patterns released by injured cells, which activate resident microglia and astrocytes. The activated innate immune cells in the brain secrete pro-inflammatory cytokines (IL-1α, IL-1β, TNF-α), recruit peripheral neutrophils/monocytes and eventually form a feedforward loop of inflammatory response exacerbating tissue damage [67]. Neuroinflammation permeates nearly the entire course of secondary injury after TBI. As one of the pivotal pathological mechanisms, this inflammatory cascade interacts synergistically with other key pathologies, inducing BBB disruption through cytokine-mediated tight junction breakdown and upregulated endothelial adhesion molecules, triggering vasogenic edema and cytotoxic edema, and reinforcing oxidative stress to collectively promote neuronal and white matter loss, leading to progressive neurological deficits [68]. Thus, targeting inflammation is therapeutically critical, as anti-inflammatory strategies preserve BBB integrity, reduce both vasogenic and cytotoxic edema, mitigate oxidative stress, and limit intracranial pressure elevation and ischemia to improve outcomes.

Glucocorticoids were once widely considered for TBI due to purported anti-inflammatory effects (targeting post-traumatic cytokine release/immune activation) and potential BBB protection/anti-edema properties [69, 70]. However, the CRASH trial showed higher 2-week mortality with 48-hour methylprednisolone (MP) infusion for patients with TBI [9]. Therefore, current guidelines now advise against routine use. Possible reasons for glucocorticoid failure include: (1) Low BBB permeability of synthetic glucocorticoids, failing to reach therapeutic parenchymal concentrations even with partial BBB disruption [71, 72]; (2) Peripheral complications that exacerbate TBI patients’ vulnerability; (3) High-dose DEX and MP inhibited the activation of HPA axis and increased the incidence of corticosteroid insufficiency and mortality [73].

Building on previous work, a mesoporous PDA (mPDA)-integrated nanocomposite hydrogel has been developed for localized long-term sustained delivery of DEX in TBI [43]. This system effectively elevated local DEX concentrations while circumventing systemic side effects by confining drug release to the injury site, protected BBB, reduced brain edema, and improve neurofunctions in acute phase after TBI. In the present study, we made the following modifications to the NPs: (1) Replacing mesoporous polydopamine (mPDA) used in previous experiments with PDA. While mPDA exhibits excellent uniform and sustained drug release performance, this study requires most of the DEX to be released within the first 3 days of the acute phase to fully exert its effects in protecting the blood-brain barrier, anti-inflammation, and reducing brain edema, with a small dose released in the later stage to minimize its side effect of inhibiting brain tissue repair while maintaining anti-inflammatory effects. (2) The inner layer of the NPs consists of Hb NPs combined with BDNF, where Hb functions as a drug carrier and to increase local oxygen content. However, Hb is prone to oxidation to methemoglobin, thereby losing its oxygen-carrying capacity. In this study, Hb@PDA NPs were synthesized by encapsulating Hb in PDA, which effectively prevents the autoxidation of Hb and enhances its local oxygen-carrying capacity. In addition to intravenous systemic administration [74], Previous research findings have shown that the local application of Hb NPs can promote the entry of oxygen from the blood into tissues and effectively bind oxygen, thereby increasing the oxygen content of local tissues [75, 76]. As hypothesized, in vitro and in vivo release profiles confirmed that DEX from the Hb-PDA@DEX@gel was predominantly released within 3 days post-administration, which aligns precisely with the peak of TBI-induced neuroinflammation and vasogenic edema. This acute release profile translated to robust therapeutic effects: Hb-PDA@DEX@gel inhibited the activation of pro-inflammatory immune cells in the peri-injury cortex and hippocampus, with a concurrent decrease in the levels of pro-inflammatory cytokines. Furthermore, this anti-inflammatory activity is coupled with preservation of BBB integrity, reduction in cerebral edema, and improvement in neurological function. Overall, Hb-PDA@DEX@gel combines the multifaceted properties of DEX and the nanohydrogel, which effectively target the key pathological hallmarks in acute phase after TBI (neuroinflammation, BBB impairment, and cerebral edema).

However, our results also revealed the limitation of this monotherapeutic strategy. Hb-PDA@DEX@gel failed to improve long-term prognosis, and continuous chronic-phase neuronal loss and neurological function deficits were still unresolved. This discrepancy highlights the complexity of secondary injury, such as the duality of the inflammatory response. This process is detrimental during the acute phase but is indispensable in the later stage of tissue repair. Mechanistically, DEX monotherapy failed to promote long-term recovery due to its excessive inflammation inhibition, thereby disrupting the reparative functions of immune cells and glial cells in the late phase of inflammation.

As resident CNS immune cells, microglia undergo phenotypic switching: while pro-inflammatory microglia dominate the acute phase and exacerbate tissue damage, anti-inflammatory microglia emerge in the later phase and secrete neurotrophic factors, such as BDNF and VEGF that support angiogenesis, neurogenesis, and synaptic remodeling.

This anti-reparative effect of DEX aligns with broader evidence linking GCs to impaired tissue healing. Preclinical studies have shown that GCs inhibit skin wound repair and graft survival by downregulating pro-angiogenic cytokines (e.g., VEGF, fibroblast growth factor-2) and suppressing endothelial cell proliferation. Previous study found that DEX not only suppressed pathogenic M1 activation but also abrogates the transition to reparative M2 microglia and inhibited post-TBI angiogenesis by reducing the release of neurotrophic factors [32]. The current study extends these findings: immunofluorescence staining revealed a decrease in the number of CD31 + newborn endothelial cells, SOX2 + NSC, and DCX newborn neurons after Hb-PDA@DEX@gel treatment at 21 days post-TBI, suggesting that DEX’s anti-inflammatory efficacy was accompanied by unintended suppression of the neurovascular repair cascade, a key barrier to long-term functional recovery.

BDNF serves as a pivotal mediator in brain tissue repair by binding to its high-affinity receptor TrkB, triggering downstream Ras-MAPK, PI3K-Akt, and PLCγ pathways to regulate neurogenesis, neuronal survival, axonal sprouting, remyelination, and synaptic plasticity [77]. Its therapeutic potential has been widely validated in preclinical models of diverse neurological disorders, including promoting endogenous neural stem cell proliferation, axon regeneration in spinal cord injury, Alzheimer’s disease, TBI and depression [21, 78–81]. In addition, studies have shown that BDNF can not only act directly as an angiogenin to promote angiogenesis, but also stimulate the release of VEGF, which act synergistically on the neurovascular unit, effectively promoting angiogenesis and an increase in neuron numbers [82], thereby repairing damaged brain tissue. However, the clinical administration of BDNF is hindered by its inherent limitations as a macromolecular protein with poor BBB permeability, short in vivo half-life, and rapid degradation, which fail to maintain effective concentrations at lesion sites. Recently, BDNF-loaded nanohydrogel systems have emerged as a promising carrier via local application strategies.

Over recent years, biomaterial-based drug delivery systems have revolutionized the delivery of neurotrophic and angiogenic factors (e.g., BDNF, VEGF) for brain injury repair by overcoming critical barriers such as poor BBB permeability, rapid systemic clearance, and off-target toxicity. For instance, poly lactide-co-glycolide NPs coated with poloxamer 188 have been shown to markedly enhance BBB penetration of BDNF, significantly increasing cerebral BDNF levels and improving neurological and cognitive outcomes in mice with TBI [83]. Similarly, nanocarrier-mediated delivery of VEGF and other pro-angiogenic factors has promoted neuroprotection and vascular remodeling in ischemic stroke models. Parallel advances in hydrogel technology have demonstrated remarkable efficacy in spinal cord injury, TBI, and stroke: alginate hydrogels loaded with VEGF provided nearly complete neurological and anatomical protection in a rat model of cerebral ischemia; hyaluronic acid–poly lactide-co-glycolide composite hydrogels co-delivering VEGF and angiopoietin-1 enhanced angiogenesis and functional recovery in stroke mice [84]; hyaluronan-methylcellulose hydrogels co-loading BDNF and anti-inflammatory peptides reduced inflammation, cystic cavitation, and glial scarring while promoting axonal regeneration in spinal cord injury rats [85]; and epicortically implanted silk fibroin films stabilized CXCL12 to enhance stem cell homing and functional recovery after stroke [86]. Furthermore, heparin nanoparticle-integrated HA hydrogels enabled clustered VEGF presentation to revascularize stroke cavities and support axonal ingrowth [87].

Despite the above progress, single-delivery systems still present critical defects that restrict their clinical translation. Intravenous nanoparticle-based BDNF delivery suffers from rapid proteolytic breakdown in the systemic circulation; attempting to compensate with higher doses only increases systemic adverse effects. While hydrogels permit localized administration, they commonly exhibit uncontrolled burst release of encapsulated protein therapeutics [88]. A more fundamental limitation is that conventional hydrogels poorly encapsulate hydrophobic drugs and cannot regulate spatiotemporal release at injury sites, further exacerbating burst release and compromising both therapeutic efficacy and safety. This early, uncontrolled drug loss wastes bioactive factors and prevents stable, therapeutic concentrations from being maintained in the injured microenvironment over the extended timeline of neural repair.

In the present study, to overcome the above limitations, we developed the BDNF@Hb-PDA@DEX@gel nanoplatform. This core-shell design utilizes the PDA shell for acute DEX release and subsequent gradual BDNF release from the core after PDA degradation, which could enhance BDNF stability and targeted accumulation. By separating acute anti-inflammatory therapy from late-stage repair, this sequential delivery system preserves DEX’s benefits and overcomes its anti-reparative drawbacks, targeting the spatiotemporal specificity of the secondary injury processes. In vitro drug release assays confirmed the platform’s programmed kinetics: DEX (on the PDA shell) was mainly released within the first 3 days, coinciding with peak TBI-induced neuroinflammation and vasogenic edema. In contrast, Hb-encapsulated BDNF showed delayed release-rates increased sharply from day 3 post-injury, with cumulative release exceeding 70% by day 21. Consistently, in vivo results indicated that BDNF@Hb-PDA@DEX@gel treatment not only exerted potent anti-inflammatory effects, preserved BBB integrity, and alleviated cerebral edema in the acute phase but also significantly promoted angiogenesis and neurogenesis, reduced sustained neuronal loss, and improved tissue repair around the injured area, which collectively might account for the enhanced long-term neurological outcomes. These findings suggest that the structural design and drug release mode of this nanohydrogel achieved a transition from early-stage anti-inflammatory effect to late-stage repair promotion, effectively counteracting the key limitation of DEX monotherapy. Additionally, this structural design enhanced BDNF stability, enabling its sustained and stable release around the injury foci, significantly increasing the local concentration of BDNF and duration of action.

Conclusions

In conclusion, we developed an injectable BDNF@Hb-PDA@DEX@gel nanoplatform for the intraoperative local administration of DEX and BDNF after TBI. The hierarchical architecture of this nanohydrogel realized the co-encapsulation and sequential controlled release of multiple therapeutics. DEX, loaded on the surface of PDA NPs, was released in the acute phase to suppress inflammation and protect the BBB. BDNF was encapsulated with HB NPs and was gradually released in the sub-acute and chronic phases of TBI, promoting brain tissue repair and long-term prognosis. This spatiotemporal control over drug delivery is crucial for addressing the complex and dynamic pathophysiological processes of TBI, which involve different stages of injury and repair. Our study provides a clinically relevant strategy to improve both short- and long-term TBI outcomes, addresses the “one-size-fits-all” limitation of single-agent or non-temporal delivery systems, and offers a new direction for multi-modal TBI drug delivery system design.

Limitations

Several limitations should be acknowledged in this study. First, the in vivo observation was limited to 21 days post-injury. Longer-term neurological function recovery and complete degradation of the hydrogel material are lacking, which restricts our comprehensive understanding of the system’s long-term safety and efficacy. Second, the doses of DEX and BDNF were determined based on preliminary experiments and previous literature, and the efficacy of different dose combinations was not systematically explored to identify the optimal therapeutic regimen. Third, this is a preliminary exploratory study with a small sample size (n = 3 or 6 per group), which may compromise the statistical robustness and generalizability of the conclusions, and larger-scale validation studies are required in subsequent work. Fourth, some claims in the manuscript were not directly supported by the experimental evidence. Specifically, the increased expression of DCX and CD31 suggests enhanced neurogenesis-associated and angiogenic responses, but these findings do not provide direct evidence of structural or functional regeneration of integrated neurovascular networks. The observed functional recovery could be attributed to multiple mechanisms including tissue remodeling and plasticity-related processes, which were not specifically disentangled in this study. Therefore, future studies will address these gaps by extending the observation period to include long-term follow-up, conducting dose-gradient experiments to optimize the therapeutic regimen, increasing the sample size to enhance statistical robustness, performing large animal experiments to further validate the long-term safety and efficacy of this system for subsequent clinical trials, and generating more rigorous experimental evidence to support the proposed mechanisms.

Supplementary Information

Below is the link to the electronic supplementary material.

12916_2026_5034_MOESM1_ESM.docx^{(5.4MB, docx)}

Supplementary Material 1: Additional File 1: Figure S9

12916_2026_5034_MOESM2_ESM.docx^{(3.7MB, docx)}

Supplementary Material 2: Additional File 1: Figures S1-S8

Acknowledgements

We thank the Beijing neurosurgical institute consultation and instrument availability that supported this work. The authors would like to acknowledge the efforts of all the staff who contributed to this study.

Abbreviations

ALT

Alanine aminotransferase

AST

Aspartate aminotransferase

BBB

Blood-brain barrier

BCI

Blood clotting index

BDNF

Brain derived neurotrophic factor

BrdU

5-bromo-2′-deoxyuridine

BUN

Urea nitrogen

CCI

Controlled cortical impact

CCK-8

Cell Counting Kit-8

CD

Circular dichroism

CMC

Carboxymethyl chitosan

CREA

Creatinine

DDS

Drug delivery systems

DEX

Dexamethasone

DLS

Dynamic light scattering

EB

Evans Blue

ELISA

Enzyme-linked immunosorbent assay

FST

Forced swim test

FTIR

Fourier transform infrared

Hb

Hemoglobin

HBOCs

Hb based oxygen carriers

HPLC

High performance liquid chromatography

IL-1β

Interleukin-1β

IL-6

Interleukin-6

LPS

Lipopolysaccharide

MRI

Magnetic Resonance Imaging

MWM

Morris water maze

NPs

Nanoparticles

NSC

Neural stem cells

OD

Oxidized dextran

PDA

Polydopamine

ROS

Reactive oxygen species

SPT

Sucrose preference test

TBI

Traumatic brain injury

TEM

Transmission electron microscopy

TGA

Thermogravimetric analysis

TGF-β

Transforming growth factor-β

TNF-α

Tumor necrosis factor-alpha

VEGF

Vascular endothelial growth factor

XPS

X-ray photoelectron spectroscopy

Author contributions

BZ and GZS designed the study. BZ, SCM, MSY, YMW, and GZS planned, and organized all experiments and results, including the writing of the manuscript. XLZ, CZ, SZ, and YXZ performed all experiments and provided technical guidance. MSY, MB, XYC, and SCM performed statistical analysis. BZ, SCM, MSY, and YMW wrote the original draft. BZ and GZS supervised the study and reviewed the article for submission. All authors read and approved the final manuscript.

Funding

This work was partially supported by the Capital’s Funds for Health Improvement and Research (2026-M-2-107-1), the Natural Science Foundation of Capital Medical University (PYZ25133), and National Natural Science Foundation of China (No. 82502357).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

All animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of Beijing Neurosurgical Institute (Committee approval No. 202204007).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Bin Zhang, Mengshi Yang, Shunchang Ma and Yumei Wang contributed equally to this work.

References

1.Maas AIR, Menon DK, Manley GT, Abrams M, Akerlund C, Andelic N, et al. Traumatic brain injury: progress and challenges in prevention, clinical care, and research. Lancet Neurol. 2022;21:1004–60. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Wilson L, Stewart W, Dams-O’Connor K, Diaz-Arrastia R, Horton L, Menon DK, et al. The chronic and evolving neurological consequences of traumatic brain injury. Lancet Neurol. 2017;16:813–25. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Zima L, Moore AN, Smolen P, Kobori N, Noble B, Robinson D, et al. The evolving pathophysiology of TBI and the advantages of temporally-guided combination therapies. Neurochem Int. 2024;180:105874. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Halabi C, Izzy S, DiGiorgio AM, Mills H, Radmanesh F, Yue JK, et al. Traumatic Brain Injury and Risk of Incident Comorbidities. JAMA Netw Open. 2024;7(12):e2450499. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Morris GF, Bullock R, Marshall SB, Marmarou A, Maas A, Marshall LF. Failure of the competitive N-methyl-D-aspartate antagonist Selfotel (CGS 19755) in the treatment of severe head injury: results of two phase III clinical trials. Selfotel Investigators J Neurosurg. 1999;91(5):737–43. [DOI] [PubMed] [Google Scholar]
6.Marmarou A, Nichols J, Burgess J, Newell D, Troha J, Burnham D, et al. Effects of the bradykinin antagonist Bradycor (deltibant, CP-1027) in severe traumatic brain injury: results of a multi-center, randomized, placebo-controlled trial. American Brain Injury Consortium Study Group. J Neurotrauma. 1999;16(6):431–44. [DOI] [PubMed] [Google Scholar]
7.Maas AI, Murray G, Henney H, Kassem N, Legrand V, Mangelus M, et al. TBI investigators. Efficacy and safety of dexanabinol in severe traumatic brain injury: results of a phase III randomised, placebo-controlled, clinical trial. Lancet Neurol. 2006;5(1):38–45. [DOI] [PubMed] [Google Scholar]
8.Marshall LF, Maas AI, Marshall SB, Bricolo A, Fearnside M, Iannotti F, et al. A multicenter trial on the efficacy of using tirilazad mesylate in cases of head injury. J Neurosurg. 1998;89(4):519–25. [DOI] [PubMed] [Google Scholar]
9.CRASH-3 trial collaborators. Effects of tranexamic acid on death, disability, vascular occlusive events and other morbidities in patients with acute traumatic brain injury (CRASH-3): a randomised, placebo-controlled trial. Lancet. 2019;394(10210):1713–23. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Nichol A, French C, Little L, Haddad S, Presneill J, Arabi Y, et al. Erythropoietin in traumatic brain injury (EPO-TBI): a double-blind randomised controlled trial. Lancet. 2015;386(10012):2499–506. [DOI] [PubMed] [Google Scholar]
11.Roquilly A, Moyer JD, Huet O, Lasocki S, Cohen B, Dahyot-Fizelier C, et al. effect of continuous infusion of hypertonic saline vs standard care on 6-month neurological outcomes in patients with traumatic brain injury: the cobi randomized clinical trial. JAMA. 2021;325(20):2056–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Hatton J, Kryscio R, Ryan M, Ott L, Young B. Systemic metabolic effects of combined insulin-like growth factor-I and growth hormone therapy in patients who have sustained acute traumatic brain injury. J Neurosurg. 2006;105(6):843–52. [DOI] [PubMed] [Google Scholar]
13.Zafonte RD, Bagiella E, Ansel BM, Novack TA, Friedewald WT, Hesdorffer DC, et al. Effect of citicoline on functional and cognitive status among patients with traumatic brain injury: Citicoline Brain Injury Treatment Trial (COBRIT). JAMA. 2012;308(19):1993–2000. [DOI] [PubMed] [Google Scholar]
14.Vergouwen MD, Vermeulen M, Roos YB. Effect of nimodipine on outcome in patients with traumatic subarachnoid haemorrhage: a systematic review. Lancet Neurol. 2006;5(12):1029–32. [DOI] [PubMed] [Google Scholar]
15.Skolnick BE, Maas AI, Narayan RK, van der Hoop RG, MacAllister T, Ward JD, et al. SYNAPSE Trial Investigators. A clinical trial of progesterone for severe traumatic brain injury. N Engl J Med. 2014;371(26):2467–76. [DOI] [PubMed] [Google Scholar]
16.Giacino JT, Whyte J, Bagiella E, Kalmar K, Childs N, Khademi A, et al. Placebo-controlled trial of amantadine for severe traumatic brain injury. N Engl J Med. 2012;366(9):819–26. [DOI] [PubMed] [Google Scholar]
17.Rhodes JK. Actions of glucocorticoids and related molecules after traumatic brain injury. Curr Opin Crit Care. 2003;9(2):86–91. [DOI] [PubMed] [Google Scholar]
18.Edwards P, Arango M, Balica L, Cottingham R, El-Sayed H, Farrell B, et al. Final results of MRC CRASH, a randomised placebo-controlled trial of intravenous corticosteroid in adults with head injury-outcomes at 6 months. Lancet. 2005;365:1957–9. [DOI] [PubMed] [Google Scholar]
19.Alderson P, Roberts I. Corticosteroids for acute traumatic brain injury. Cochrane Database Syst Rev. 2005;20051:CD000196. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Gustafsson D, Klang A, Thams S, Rostami E. The Role of BDNF in Experimental and Clinical Traumatic Brain Injury. Int J Mol Sci. 2021;22(7):3582. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Giesler LP, Mychasiuk R, Shultz SR, McDonald SJ. BDNF: New Views of an Old Player in Traumatic Brain Injury. Neuroscientist. 2024;30(5):560–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Nagahara AH, Merrill DA, Coppola G, Tsukada S, Schroeder BE, Shaked GM, et al. Neuroprotective effects of brain-derived neurotrophic factor in rodent and primate models of Alzheimer’s disease. Nat Med. 2009;15(3):331–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Matsuoka Y, Nishi D, Tanima Y, Itakura M, Kojima M, Hamazaki K, et al. Serum pro-BDNF/BDNF as a treatment biomarker for response to docosahexaenoic acid in traumatized people vulnerable to developing psychological distress: a randomized controlled trial. Transl Psychiatry. 2015;5(7):e596. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Shi FD, Yong VW. Neuroinflammation across neurological diseases. Science. 2025;388(6753):eadx0043. [DOI] [PubMed] [Google Scholar]
25.Kalra S, Malik R, Singh G, Bhatia S, Al-Harrasi A, Mohan S, et al. Pathogenesis and management of traumatic brain injury (TBI): role of neuroinflammation and anti-inflammatory drugs. Inflammopharmacology. 2022;30(4):1153–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Jassam YN, Izzy S, Whalen M, McGavern DB, El Khoury J. Neuroimmunology of Traumatic Brain Injury: Time for a Paradigm Shift. Neuron. 2017;95(6):1246–65. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Hsueh SC, Parekh P, Batsaikhan B, Vargesson N, Tweedie D, Luo W, et al. Targeting neuroinflammation: 3-monothiopomalidomide a new drug candidate to mitigate traumatic brain injury and neurodegeneration. J Biomed Sci. 2025;32(1):57. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Meng S, Cao H, Huang Y, Shi Z, Li J, Wang Y, et al. ASK1-K716R reduces neuroinflammation and white matter injury via preserving blood-brain barrier integrity after traumatic brain injury. J Neuroinflammation. 2023;20(1):244. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Wu H, Zheng J, Xu S, Fang Y, Wu Y, Zeng J, et al. Mer regulates microglial/macrophage M1/M2 polarization and alleviates neuroinflammation following traumatic brain injury. J Neuroinflammation. 2021;18(1):2. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Willis EF, MacDonald KPA, Nguyen QH, Garrido AL, Gillespie ER, Harley SBR, et al. Repopulating Microglia Promote Brain Repair in an IL-6-Dependent Manner. Cell. 2020;180(5):833–e84616. [DOI] [PubMed] [Google Scholar]
31.He X, Huang Y, Liu Y, Zhang X, Wang Q, Liu Y, et al. Astrocyte-derived exosomal lncRNA 4933431K23Rik modulates microglial phenotype and improves post-traumatic recovery via SMAD7 regulation. Mol Ther. 2023;31(5):1313–31. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Yang M, Bai M, Zhuang Y, Lu S, Ge Q, Li H, et al. High-dose dexamethasone regulates microglial polarization via the GR/JAK1/STAT3 signaling pathway after traumatic brain injury. Neural Regen Res. 2025;20(9):2611–23. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Zhang B, Zhu X, Wang L, Hao S, Xu X, Niu F, et al. Dexamethasone impairs neurofunctional recovery in rats following traumatic brain injury by reducing circulating endothelial progenitor cells and angiogenesis. Brain Res. 2019;1725:146469. [DOI] [PubMed] [Google Scholar]
34.Uzakova AB, Yergaliyeva EM, Yerlanuly A, Mukatayeva ZS. A Systematic Review of Advanced Drug Delivery Systems: Engineering Strategies, Barrier Penetration, and Clinical Progress (2016-April 2025). Pharmaceutics. 2025;18(1):11. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Ezike TC, Okpala US, Onoja UL, Nwike CP, Ezeako EC, Okpara OJ, et al. Advances in drug delivery systems, challenges and future directions. Heliyon. 2023;9(6):e17488. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Vincy A, Mazumder S, Amrita, Banerjee I, Hwang KC, Vankayala R. Recent Progress in Red Blood Cells-Derived Particles as Novel Bioinspired Drug Delivery Systems: Challenges and Strategies for Clinical Translation. Front Chem. 2022;10:905256. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Yu T, Li Y, Gu X, Li Q. Development of a Hyaluronic Acid-Based Nanocarrier Incorporating Doxorubicin and Cisplatin as a pH-Sensitive and CD44-Targeted Anti-Breast Cancer Drug Delivery System. Front Pharmacol. 2020;11:532457. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Weng Q, Wang X, Wang X, Bando Y, Golberg D. Functionalized hexagonal boron nitride nanomaterials: emerging properties and applications. Chem Soc Rev. 2016;45(14):3989–4012. [DOI] [PubMed] [Google Scholar]
39.Mohanty A, Uthaman S, Park IK. Utilization of Polymer-Lipid Hybrid Nanoparticles for Targeted Anti-Cancer Therapy. Molecules. 2020;25(19):4377. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Vigani B, Rossi S, Sandri G, Bonferoni MC, Caramella CM, Ferrari F. Recent Advances in the Development of In Situ Gelling Drug Delivery Systems for Non-Parenteral Administration Routes. Pharmaceutics. 2020;12(9):859. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Nazemidashtarjandi S, Larsen B, Cheng K, Faulkner S, Peppas NA, Parekh SH, et al. Near-infrared light-responsive hydrogels for on-demand dual delivery of proangiogenic growth factors. Acta Biomater. 2024;183:61–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Han Y, Gu J, Xu M, Ma Y, Weng W, Feng Q, et al. Intraoperative application of an antioxidant nanoparticle-hydrogel targeting microglia regulates neuroinflammation in traumatic brain injury. J Nanobiotechnol. 2025;23(1):599. [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Zhang B, Bai M, Yang M, Wang Y, Chen X, Liu B, et al. Injectable nanocomposite hydrogel for localized precision delivery of dexamethasone after traumatic brain injury: dual modulation of neuroinflammation and blood-brain barrier restoration. J Transl Med. 2025;23(1):579. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Deng QS, Gao Y, Rui BY, Li XR, Liu PL, Han ZY, et al. Double-network hydrogel enhanced by SS31-loaded mesoporous polydopamine nanoparticles: Symphonic collaboration of near-infrared photothermal antibacterial effect and mitochondrial maintenance for full-thickness wound healing in diabetes mellitus. Bioact Mater. 2023;27:409–28. [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Ren H, Zhang Z, Cheng X, Zou Z, Chen X, He C. Injectable, self-healing hydrogel adhesives with firm tissue adhesion and on-demand biodegradation for sutureless wound closure. Sci Adv. 2023;9(33):eadh4327. [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Li M, Liang YP, Liang YQ, Pan GY, Guo BL. Injectable Stretchable Self-Healing Dual Dynamic Network Hydrogel as Adhesive Anti-Oxidant Wound Dressing for Photothermal Clearance of Bacteria and Promoting Wound Healing of MRSA Infected Motion Wounds. Chem Eng J. 2022;427:32039. [Google Scholar]
47.Hao Y, Zhao W, Zhang H, Zheng W, Zhou Q. Carboxymethyl chitosan-based hydrogels containing fibroblast growth factors for triggering diabetic wound healing. Carbohydr Polym. 2022;287:119336. [DOI] [PubMed] [Google Scholar]
48.Zhao X, Wu H, Guo B, Dong R, Qiu Y, Ma PX. Antibacterial anti-oxidant electroactive injectable hydrogel as self-healing wound dressing with hemostasis and adhesiveness for cutaneous wound healing. Biomaterials. 2017;122:34–47. [DOI] [PubMed] [Google Scholar]
49.Zhu Z, Zhang K, Xian Y, He G, Pan Z, Wang H, et al. A Choline Phosphoryl-Conjugated Chitosan/Oxidized Dextran Injectable Self-Healing Hydrogel for Improved Hemostatic Efficacy. Biomacromolecules. 2023;24(2):690–703. [DOI] [PubMed] [Google Scholar]
50.Qi X, Xiang Y, Cai E, You S, Gao T, Lan Y, et al. All-in-one: Harnessing multifunctional injectable natural hydrogels for ordered therapy of bacteria-infected diabetic wounds. Chem Eng J. 2022;439:135691. [Google Scholar]
51.Meng J, Yang X, Huang J, Tuo Z, Hu Y, Liao Z, et al. Ferroptosis-Enhanced Immunotherapy with an Injectable Dextran-Chitosan Hydrogel for the Treatment of Malignant Ascites in Hepatocellular Carcinoma. Adv Sci (Weinh). 2023;10(20):e2300517. [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Zhao X, Guo B, Wu H, Liang Y, Ma PX. Injectable antibacterial conductive nanocomposite cryogels with rapid shape recovery for noncompressible hemorrhage and wound healing. Nat Commun. 2018;9(1):2784. [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Han Y, Weng W, Zhang Y, Feng Q, Ma Y, Quan A, et al. Intraoperative application of intelligent, responsive, self-assembling hydrogel rectifies oxygen and energy metabolism in traumatically injured brain. Biomaterials. 2024;306:122495. [DOI] [PubMed] [Google Scholar]
54.Yu Q, Liu S, Guo R, Chen K, Li Y, Jiang D, et al. Complete Restoration of Hearing Loss and Cochlear Synaptopathy via Minimally Invasive, Single-Dose, and Controllable Middle Ear Delivery of Brain-Derived Neurotrophic Factor-Poly (dl-lactic acid-co-glycolic acid)-Loaded Hydrogel. ACS Nano. 2024;18(8):6298–313. [DOI] [PubMed] [Google Scholar]
55.Sun YX, Su YA, Wang Q, Zheng JY, Zhang CC, Wang T, et al. The causal involvement of the BDNF-TrkB pathway in dentate gyrus in early-life stress-induced cognitive deficits in male mice. Transl Psychiatry. 2023;13(1):173. [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Chi W, He Y, Chen S, Guo L, Yuan Y, Li R, et al. Advances in research on biomaterials and stem cell/exosome-based strategies in the treatment of traumatic brain injury. Acta Pharm Sin B. 2025;15(7):3511-3544. [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Li Y, Zhang Y, Fu Y, Lu Y, Jiang K, Li L, et al. Innovative breakthroughs in novel biomaterials for traumatic brain injury and cranial repair. Rare Met. 2025;44:4315–45. [Google Scholar]
58.Gao Y, Tang Z, Wu Z, Zhang J, Ma J, Liu S, et al. Biomaterials and stem cells targeting the microenvironment for traumatic brain injury repair: progress and prospects. Biomater Sci. 2025;13(19):5278–96. [DOI] [PubMed] [Google Scholar]
59.Zhou X, Ma P, Liu Y, Wang Z, Chen S, Cheng Z, et al. Sprayable hydrogel sponge for neurovascular microenvironment reconstruction and inflammation modulation in diabetic wound healing. Bioact Mater. 2025;54:352–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
60.Xu X, Wu Y, Gu R, Zhang Z, Liu X, Hu Y, et al. Nanoparticle-hydrogel composite as dual-drug delivery system for the potential application of corneal graft rejection. Eur J Pharm Biopharm. 2024;201:114351. [DOI] [PubMed] [Google Scholar]
61.Zhu H, Zheng J, Oh XY, Chan CY, Low BQL, Tor JQ, et al. Nanoarchitecture-Integrated Hydrogel Systems toward Therapeutic Applications. ACS Nano. 2023;17(9):7953–78. [DOI] [PubMed] [Google Scholar]
62.Wu D, Zhou J, Zheng Y, Zheng Y, Zhang Q, Zhou Z, et al. Pathogenesis-adaptive polydopamine nanosystem for sequential therapy of ischemic stroke. Nat Commun. 2023;14(1):7147. [DOI] [PMC free article] [PubMed] [Google Scholar]
63.Zhu K, Wang L, Xiao Y, Zhang X, You G, Chen Y, et al. Nanomaterial-related hemoglobin-based oxygen carriers, with emphasis on liposome and nano-capsules, for biomedical applications: current status and future perspectives. J Nanobiotechnol. 2024;22(1):336. [DOI] [PMC free article] [PubMed] [Google Scholar]
64.Hu J, Wang Q, Wang Y, You G, Li P, Zhao L, et al. Polydopamine-based surface modification of hemoglobin particles for stability enhancement of oxygen carriers. J Colloid Interface Sci. 2020;571:326–36. [DOI] [PubMed] [Google Scholar]
65.Wang Q, Zhang R, Lu M, You G, Wang Y, Chen G, et al. Bioinspired Polydopamine-Coated Hemoglobin as Potential Oxygen Carrier with Antioxidant Properties. Biomacromolecules. 2017;18(4):1333–41. [DOI] [PubMed] [Google Scholar]
66.Battaglini M, Emanet M, Carmignani A, Ciofani G. Polydopamine-based nanostructures: A new generation of versatile, multi-tasking, and smart theranostic tools. Nano Today. 2024;55:102151. [Google Scholar]
67.Morganti-Kossmann MC, Semple BD, Hellewell SC, Bye N, Ziebell JM. The complexity of neuroinflammation consequent to traumatic brain injury: from research evidence to potential treatments. Acta Neuropathol. 2019;137:731–55. [DOI] [PubMed] [Google Scholar]
68.Alam A, Thelin EP, Tajsic T, Khan DZ, Khellaf A, Patani R, et al. Cellular infiltration in traumatic brain injury. J Neuroinflammation. 2020;17(1):328. [DOI] [PMC free article] [PubMed] [Google Scholar]
69.Renaudin J, Fewer D, Wilson CB, Boldrey EB, Calogero J, Enot KJ. Dose dependency of decadron in patients with partially excised brain tumors. J Neurosurg. 1973;39:302–5. [DOI] [PubMed] [Google Scholar]
70.Reichardt HM, Gold R, Luhder F. Glucocorticoids in multiple sclerosis and experimental autoimmune encephalomyelitis. Expert Rev Neurother. 2006;6:1657–70. [DOI] [PubMed] [Google Scholar]
71.Mason BL, Pariante CM, Thomas SA. A Revised Role for P-Glycoprotein in the Brain Distribution of Dexamethasone, Cortisol, and Corticosterone in Wild-Type and ABCB1A/B-Deficient Mice. Endocrinology. 2008;149:5244–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
72.De Kloet ER. Why Dexamethasone Poorly Penetrates in Brain. Stress. 1997;2:13–20. [DOI] [PubMed] [Google Scholar]
73.Zhang B, Bai M, Xu X, Yang M, Niu F, Gao F, et al. Corticosteroid receptor rebalancing alleviates critical illness-related corticosteroid insufficiency after traumatic brain injury by promoting paraventricular nuclear cell survival via Akt/CREB/BDNF signaling. J Neuroinflammation. 2020;17:318. [DOI] [PMC free article] [PubMed] [Google Scholar]
74.Yin N, Zhao Y, Liu C, Yang Y, Wang ZH, Yu W, et al. Engineered Nanoerythrocytes Alleviate Central Nervous System Inflammation by Regulating the Polarization of Inflammatory Microglia. Adv Mater. 2022;34(27):e2201322. [DOI] [PubMed] [Google Scholar]
75.Chen Y, Ma X, Hu Y, Wang H, Zhou P, Tian X et al. A pH/ROS dual-responsive hydrogel promotes diabetic wound healing by controlled release of O2 and EGCG to induce angiogenesis and immunomodulation, Chemical Engineering Journal. 2025; 526: (2025).
76.Cai W, Sun Y, Xu W, Kuang X, Zhang Z, Cao X, et al. Ascorbic Acid-Derived Supramolecular Gels Induce Immunogenic Ferroptosis in Cancer Cells to Potentiate Tumor Immunotherapy. ACS Nano. 2025;19(36):32405–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
77.Hohn A, Leibrock J, Bailey K, Barde YA. Identification and characterization of a novel member of the nerve growth factor/brain-derived neurotrophic factor family. Nature. 1990;344(6264):339–41. [DOI] [PubMed] [Google Scholar]
78.Panda HS, Dhokne MD, Thakur S, Singh N, Datusalia AK, Panda JJ. BDNF Loaded Amino Acid-Catecholamine Hybrid Nanoparticles as Curative Agents Against Cognitive Decline in Alzheimer’s Disease. Small. 2025;21(42):e2411701. [DOI] [PubMed] [Google Scholar]
79.Hakami A, Narasimhan K, Comini G, Thiele J, Werner C, Dowd E, et al. Cryogel microcarriers for sustained local delivery of growth factors to the brain. J Control Release. 2024;369:404–19. [DOI] [PubMed] [Google Scholar]
80.Huang Z, Li J, Wo J, Li CL, Wu ZC, Deng XH, et al. Intranasal Delivery of Brain-Derived Neurotrophic Factor (BDNF)-Loaded Small Extracellular Vesicles for Treating Acute Spinal Cord Injury in Rats and Monkeys. J Extracell Vesicles. 2025;14(4):e70066. [DOI] [PMC free article] [PubMed] [Google Scholar]
81.Nagahara AH, Merrill DA, Coppola G, Tsukada S, Schroeder BE, Shaked GM, et al. Neuroprotective effects of brain-derived neurotrophic factor in rodent and primate models of Alzheimer’s disease. Nat Med. 2009;15(3):331–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
82.Mori A, Nishioka Y, Yamada M, Nishibata Y, Masuda S, Tomaru U, et al. Brain-derived neurotrophic factor induces angiogenin secretion and nuclear translocation in human umbilical vein endothelial cells. Pathol Res Pract. 2018;214(4):521–6. [DOI] [PubMed] [Google Scholar]
83.Khalin I, Alyautdin R, Wong TW, Gnanou J, Kocherga G, Kreuter J. Brain-derived neurotrophic factor delivered to the brain using poly (lactide-co-glycolide) nanoparticles improves neurological and cognitive outcome in mice with traumatic brain injury. Drug Deliv. 2016;23(9):3520–8. [DOI] [PubMed] [Google Scholar]
84.Emerich DF, Silva E, Ali O, Mooney D, Bell W, Yu SJ, et al. Injectable VEGF hydrogels produce near complete neurological and anatomical protection following cerebral ischemia in rats. Cell Transpl. 2010;19(9):1063–71. [DOI] [PubMed] [Google Scholar]
85.He Z, Zang H, Zhu L, Huang K, Yi T, Zhang S, et al. An anti-inflammatory peptide and brain-derived neurotrophic factor-modified hyaluronan-methylcellulose hydrogel promotes nerve regeneration in rats with spinal cord injury. Int J Nanomed. 2019;14:721–32. [DOI] [PMC free article] [PubMed] [Google Scholar]
86.Lekouaghet A, Sánchez-Díez M, Pérez-Rigueiro J, Rojo FJ, Ramírez-Castillejo C, Ruiz-León Y, et al. Stabilization of CXCL12 (SDF-1α) via silk fibroin films enhances stem cell migration/retention and functional recovery after stroke. Regen Biomater. 2025;13:rbaf129. [DOI] [PMC free article] [PubMed] [Google Scholar]
87.Nih LR, Gojgini S, Carmichael ST, Segura T. Dual-function injectable angiogenic biomaterial for the repair of brain tissue following stroke. Nat Mater. 2018;17(7):642–51. [DOI] [PMC free article] [PubMed] [Google Scholar]
88.Ding C, Liu X, Zhang S, Sun S, Yang J, Chai G, et al. Multifunctional hydrogel bioscaffolds based on polysaccharide to promote wound healing: A review. Int J Biol Macromol. 2024;259:129356. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

12916_2026_5034_MOESM1_ESM.docx^{(5.4MB, docx)}

Supplementary Material 1: Additional File 1: Figure S9

12916_2026_5034_MOESM2_ESM.docx^{(3.7MB, docx)}

Supplementary Material 2: Additional File 1: Figures S1-S8

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

[CR1] 1.Maas AIR, Menon DK, Manley GT, Abrams M, Akerlund C, Andelic N, et al. Traumatic brain injury: progress and challenges in prevention, clinical care, and research. Lancet Neurol. 2022;21:1004–60. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR2] 2.Wilson L, Stewart W, Dams-O’Connor K, Diaz-Arrastia R, Horton L, Menon DK, et al. The chronic and evolving neurological consequences of traumatic brain injury. Lancet Neurol. 2017;16:813–25. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR3] 3.Zima L, Moore AN, Smolen P, Kobori N, Noble B, Robinson D, et al. The evolving pathophysiology of TBI and the advantages of temporally-guided combination therapies. Neurochem Int. 2024;180:105874. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR4] 4.Halabi C, Izzy S, DiGiorgio AM, Mills H, Radmanesh F, Yue JK, et al. Traumatic Brain Injury and Risk of Incident Comorbidities. JAMA Netw Open. 2024;7(12):e2450499. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR5] 5.Morris GF, Bullock R, Marshall SB, Marmarou A, Maas A, Marshall LF. Failure of the competitive N-methyl-D-aspartate antagonist Selfotel (CGS 19755) in the treatment of severe head injury: results of two phase III clinical trials. Selfotel Investigators J Neurosurg. 1999;91(5):737–43. [DOI] [PubMed] [Google Scholar]

[CR6] 6.Marmarou A, Nichols J, Burgess J, Newell D, Troha J, Burnham D, et al. Effects of the bradykinin antagonist Bradycor (deltibant, CP-1027) in severe traumatic brain injury: results of a multi-center, randomized, placebo-controlled trial. American Brain Injury Consortium Study Group. J Neurotrauma. 1999;16(6):431–44. [DOI] [PubMed] [Google Scholar]

[CR7] 7.Maas AI, Murray G, Henney H, Kassem N, Legrand V, Mangelus M, et al. TBI investigators. Efficacy and safety of dexanabinol in severe traumatic brain injury: results of a phase III randomised, placebo-controlled, clinical trial. Lancet Neurol. 2006;5(1):38–45. [DOI] [PubMed] [Google Scholar]

[CR8] 8.Marshall LF, Maas AI, Marshall SB, Bricolo A, Fearnside M, Iannotti F, et al. A multicenter trial on the efficacy of using tirilazad mesylate in cases of head injury. J Neurosurg. 1998;89(4):519–25. [DOI] [PubMed] [Google Scholar]

[CR9] 9.CRASH-3 trial collaborators. Effects of tranexamic acid on death, disability, vascular occlusive events and other morbidities in patients with acute traumatic brain injury (CRASH-3): a randomised, placebo-controlled trial. Lancet. 2019;394(10210):1713–23. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR10] 10.Nichol A, French C, Little L, Haddad S, Presneill J, Arabi Y, et al. Erythropoietin in traumatic brain injury (EPO-TBI): a double-blind randomised controlled trial. Lancet. 2015;386(10012):2499–506. [DOI] [PubMed] [Google Scholar]

[CR11] 11.Roquilly A, Moyer JD, Huet O, Lasocki S, Cohen B, Dahyot-Fizelier C, et al. effect of continuous infusion of hypertonic saline vs standard care on 6-month neurological outcomes in patients with traumatic brain injury: the cobi randomized clinical trial. JAMA. 2021;325(20):2056–66. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR12] 12.Hatton J, Kryscio R, Ryan M, Ott L, Young B. Systemic metabolic effects of combined insulin-like growth factor-I and growth hormone therapy in patients who have sustained acute traumatic brain injury. J Neurosurg. 2006;105(6):843–52. [DOI] [PubMed] [Google Scholar]

[CR13] 13.Zafonte RD, Bagiella E, Ansel BM, Novack TA, Friedewald WT, Hesdorffer DC, et al. Effect of citicoline on functional and cognitive status among patients with traumatic brain injury: Citicoline Brain Injury Treatment Trial (COBRIT). JAMA. 2012;308(19):1993–2000. [DOI] [PubMed] [Google Scholar]

[CR14] 14.Vergouwen MD, Vermeulen M, Roos YB. Effect of nimodipine on outcome in patients with traumatic subarachnoid haemorrhage: a systematic review. Lancet Neurol. 2006;5(12):1029–32. [DOI] [PubMed] [Google Scholar]

[CR15] 15.Skolnick BE, Maas AI, Narayan RK, van der Hoop RG, MacAllister T, Ward JD, et al. SYNAPSE Trial Investigators. A clinical trial of progesterone for severe traumatic brain injury. N Engl J Med. 2014;371(26):2467–76. [DOI] [PubMed] [Google Scholar]

[CR16] 16.Giacino JT, Whyte J, Bagiella E, Kalmar K, Childs N, Khademi A, et al. Placebo-controlled trial of amantadine for severe traumatic brain injury. N Engl J Med. 2012;366(9):819–26. [DOI] [PubMed] [Google Scholar]

[CR17] 17.Rhodes JK. Actions of glucocorticoids and related molecules after traumatic brain injury. Curr Opin Crit Care. 2003;9(2):86–91. [DOI] [PubMed] [Google Scholar]

[CR18] 18.Edwards P, Arango M, Balica L, Cottingham R, El-Sayed H, Farrell B, et al. Final results of MRC CRASH, a randomised placebo-controlled trial of intravenous corticosteroid in adults with head injury-outcomes at 6 months. Lancet. 2005;365:1957–9. [DOI] [PubMed] [Google Scholar]

[CR19] 19.Alderson P, Roberts I. Corticosteroids for acute traumatic brain injury. Cochrane Database Syst Rev. 2005;20051:CD000196. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR20] 20.Gustafsson D, Klang A, Thams S, Rostami E. The Role of BDNF in Experimental and Clinical Traumatic Brain Injury. Int J Mol Sci. 2021;22(7):3582. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR21] 21.Giesler LP, Mychasiuk R, Shultz SR, McDonald SJ. BDNF: New Views of an Old Player in Traumatic Brain Injury. Neuroscientist. 2024;30(5):560–73. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR22] 22.Nagahara AH, Merrill DA, Coppola G, Tsukada S, Schroeder BE, Shaked GM, et al. Neuroprotective effects of brain-derived neurotrophic factor in rodent and primate models of Alzheimer’s disease. Nat Med. 2009;15(3):331–7. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR23] 23.Matsuoka Y, Nishi D, Tanima Y, Itakura M, Kojima M, Hamazaki K, et al. Serum pro-BDNF/BDNF as a treatment biomarker for response to docosahexaenoic acid in traumatized people vulnerable to developing psychological distress: a randomized controlled trial. Transl Psychiatry. 2015;5(7):e596. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR24] 24.Shi FD, Yong VW. Neuroinflammation across neurological diseases. Science. 2025;388(6753):eadx0043. [DOI] [PubMed] [Google Scholar]

[CR25] 25.Kalra S, Malik R, Singh G, Bhatia S, Al-Harrasi A, Mohan S, et al. Pathogenesis and management of traumatic brain injury (TBI): role of neuroinflammation and anti-inflammatory drugs. Inflammopharmacology. 2022;30(4):1153–66. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR26] 26.Jassam YN, Izzy S, Whalen M, McGavern DB, El Khoury J. Neuroimmunology of Traumatic Brain Injury: Time for a Paradigm Shift. Neuron. 2017;95(6):1246–65. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR27] 27.Hsueh SC, Parekh P, Batsaikhan B, Vargesson N, Tweedie D, Luo W, et al. Targeting neuroinflammation: 3-monothiopomalidomide a new drug candidate to mitigate traumatic brain injury and neurodegeneration. J Biomed Sci. 2025;32(1):57. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR28] 28.Meng S, Cao H, Huang Y, Shi Z, Li J, Wang Y, et al. ASK1-K716R reduces neuroinflammation and white matter injury via preserving blood-brain barrier integrity after traumatic brain injury. J Neuroinflammation. 2023;20(1):244. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR29] 29.Wu H, Zheng J, Xu S, Fang Y, Wu Y, Zeng J, et al. Mer regulates microglial/macrophage M1/M2 polarization and alleviates neuroinflammation following traumatic brain injury. J Neuroinflammation. 2021;18(1):2. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR30] 30.Willis EF, MacDonald KPA, Nguyen QH, Garrido AL, Gillespie ER, Harley SBR, et al. Repopulating Microglia Promote Brain Repair in an IL-6-Dependent Manner. Cell. 2020;180(5):833–e84616. [DOI] [PubMed] [Google Scholar]

[CR31] 31.He X, Huang Y, Liu Y, Zhang X, Wang Q, Liu Y, et al. Astrocyte-derived exosomal lncRNA 4933431K23Rik modulates microglial phenotype and improves post-traumatic recovery via SMAD7 regulation. Mol Ther. 2023;31(5):1313–31. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR32] 32.Yang M, Bai M, Zhuang Y, Lu S, Ge Q, Li H, et al. High-dose dexamethasone regulates microglial polarization via the GR/JAK1/STAT3 signaling pathway after traumatic brain injury. Neural Regen Res. 2025;20(9):2611–23. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR33] 33.Zhang B, Zhu X, Wang L, Hao S, Xu X, Niu F, et al. Dexamethasone impairs neurofunctional recovery in rats following traumatic brain injury by reducing circulating endothelial progenitor cells and angiogenesis. Brain Res. 2019;1725:146469. [DOI] [PubMed] [Google Scholar]

[CR34] 34.Uzakova AB, Yergaliyeva EM, Yerlanuly A, Mukatayeva ZS. A Systematic Review of Advanced Drug Delivery Systems: Engineering Strategies, Barrier Penetration, and Clinical Progress (2016-April 2025). Pharmaceutics. 2025;18(1):11. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR35] 35.Ezike TC, Okpala US, Onoja UL, Nwike CP, Ezeako EC, Okpara OJ, et al. Advances in drug delivery systems, challenges and future directions. Heliyon. 2023;9(6):e17488. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR36] 36.Vincy A, Mazumder S, Amrita, Banerjee I, Hwang KC, Vankayala R. Recent Progress in Red Blood Cells-Derived Particles as Novel Bioinspired Drug Delivery Systems: Challenges and Strategies for Clinical Translation. Front Chem. 2022;10:905256. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR37] 37.Yu T, Li Y, Gu X, Li Q. Development of a Hyaluronic Acid-Based Nanocarrier Incorporating Doxorubicin and Cisplatin as a pH-Sensitive and CD44-Targeted Anti-Breast Cancer Drug Delivery System. Front Pharmacol. 2020;11:532457. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR38] 38.Weng Q, Wang X, Wang X, Bando Y, Golberg D. Functionalized hexagonal boron nitride nanomaterials: emerging properties and applications. Chem Soc Rev. 2016;45(14):3989–4012. [DOI] [PubMed] [Google Scholar]

[CR39] 39.Mohanty A, Uthaman S, Park IK. Utilization of Polymer-Lipid Hybrid Nanoparticles for Targeted Anti-Cancer Therapy. Molecules. 2020;25(19):4377. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR40] 40.Vigani B, Rossi S, Sandri G, Bonferoni MC, Caramella CM, Ferrari F. Recent Advances in the Development of In Situ Gelling Drug Delivery Systems for Non-Parenteral Administration Routes. Pharmaceutics. 2020;12(9):859. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR41] 41.Nazemidashtarjandi S, Larsen B, Cheng K, Faulkner S, Peppas NA, Parekh SH, et al. Near-infrared light-responsive hydrogels for on-demand dual delivery of proangiogenic growth factors. Acta Biomater. 2024;183:61–73. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR42] 42.Han Y, Gu J, Xu M, Ma Y, Weng W, Feng Q, et al. Intraoperative application of an antioxidant nanoparticle-hydrogel targeting microglia regulates neuroinflammation in traumatic brain injury. J Nanobiotechnol. 2025;23(1):599. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR43] 43.Zhang B, Bai M, Yang M, Wang Y, Chen X, Liu B, et al. Injectable nanocomposite hydrogel for localized precision delivery of dexamethasone after traumatic brain injury: dual modulation of neuroinflammation and blood-brain barrier restoration. J Transl Med. 2025;23(1):579. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR44] 44.Deng QS, Gao Y, Rui BY, Li XR, Liu PL, Han ZY, et al. Double-network hydrogel enhanced by SS31-loaded mesoporous polydopamine nanoparticles: Symphonic collaboration of near-infrared photothermal antibacterial effect and mitochondrial maintenance for full-thickness wound healing in diabetes mellitus. Bioact Mater. 2023;27:409–28. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR45] 45.Ren H, Zhang Z, Cheng X, Zou Z, Chen X, He C. Injectable, self-healing hydrogel adhesives with firm tissue adhesion and on-demand biodegradation for sutureless wound closure. Sci Adv. 2023;9(33):eadh4327. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR46] 46.Li M, Liang YP, Liang YQ, Pan GY, Guo BL. Injectable Stretchable Self-Healing Dual Dynamic Network Hydrogel as Adhesive Anti-Oxidant Wound Dressing for Photothermal Clearance of Bacteria and Promoting Wound Healing of MRSA Infected Motion Wounds. Chem Eng J. 2022;427:32039. [Google Scholar]

[CR47] 47.Hao Y, Zhao W, Zhang H, Zheng W, Zhou Q. Carboxymethyl chitosan-based hydrogels containing fibroblast growth factors for triggering diabetic wound healing. Carbohydr Polym. 2022;287:119336. [DOI] [PubMed] [Google Scholar]

[CR48] 48.Zhao X, Wu H, Guo B, Dong R, Qiu Y, Ma PX. Antibacterial anti-oxidant electroactive injectable hydrogel as self-healing wound dressing with hemostasis and adhesiveness for cutaneous wound healing. Biomaterials. 2017;122:34–47. [DOI] [PubMed] [Google Scholar]

[CR49] 49.Zhu Z, Zhang K, Xian Y, He G, Pan Z, Wang H, et al. A Choline Phosphoryl-Conjugated Chitosan/Oxidized Dextran Injectable Self-Healing Hydrogel for Improved Hemostatic Efficacy. Biomacromolecules. 2023;24(2):690–703. [DOI] [PubMed] [Google Scholar]

[CR50] 50.Qi X, Xiang Y, Cai E, You S, Gao T, Lan Y, et al. All-in-one: Harnessing multifunctional injectable natural hydrogels for ordered therapy of bacteria-infected diabetic wounds. Chem Eng J. 2022;439:135691. [Google Scholar]

[CR51] 51.Meng J, Yang X, Huang J, Tuo Z, Hu Y, Liao Z, et al. Ferroptosis-Enhanced Immunotherapy with an Injectable Dextran-Chitosan Hydrogel for the Treatment of Malignant Ascites in Hepatocellular Carcinoma. Adv Sci (Weinh). 2023;10(20):e2300517. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR52] 52.Zhao X, Guo B, Wu H, Liang Y, Ma PX. Injectable antibacterial conductive nanocomposite cryogels with rapid shape recovery for noncompressible hemorrhage and wound healing. Nat Commun. 2018;9(1):2784. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR53] 53.Han Y, Weng W, Zhang Y, Feng Q, Ma Y, Quan A, et al. Intraoperative application of intelligent, responsive, self-assembling hydrogel rectifies oxygen and energy metabolism in traumatically injured brain. Biomaterials. 2024;306:122495. [DOI] [PubMed] [Google Scholar]

[CR54] 54.Yu Q, Liu S, Guo R, Chen K, Li Y, Jiang D, et al. Complete Restoration of Hearing Loss and Cochlear Synaptopathy via Minimally Invasive, Single-Dose, and Controllable Middle Ear Delivery of Brain-Derived Neurotrophic Factor-Poly (dl-lactic acid-co-glycolic acid)-Loaded Hydrogel. ACS Nano. 2024;18(8):6298–313. [DOI] [PubMed] [Google Scholar]

[CR55] 55.Sun YX, Su YA, Wang Q, Zheng JY, Zhang CC, Wang T, et al. The causal involvement of the BDNF-TrkB pathway in dentate gyrus in early-life stress-induced cognitive deficits in male mice. Transl Psychiatry. 2023;13(1):173. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR56] 56.Chi W, He Y, Chen S, Guo L, Yuan Y, Li R, et al. Advances in research on biomaterials and stem cell/exosome-based strategies in the treatment of traumatic brain injury. Acta Pharm Sin B. 2025;15(7):3511-3544. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR57] 57.Li Y, Zhang Y, Fu Y, Lu Y, Jiang K, Li L, et al. Innovative breakthroughs in novel biomaterials for traumatic brain injury and cranial repair. Rare Met. 2025;44:4315–45. [Google Scholar]

[CR58] 58.Gao Y, Tang Z, Wu Z, Zhang J, Ma J, Liu S, et al. Biomaterials and stem cells targeting the microenvironment for traumatic brain injury repair: progress and prospects. Biomater Sci. 2025;13(19):5278–96. [DOI] [PubMed] [Google Scholar]

[CR59] 59.Zhou X, Ma P, Liu Y, Wang Z, Chen S, Cheng Z, et al. Sprayable hydrogel sponge for neurovascular microenvironment reconstruction and inflammation modulation in diabetic wound healing. Bioact Mater. 2025;54:352–70. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR60] 60.Xu X, Wu Y, Gu R, Zhang Z, Liu X, Hu Y, et al. Nanoparticle-hydrogel composite as dual-drug delivery system for the potential application of corneal graft rejection. Eur J Pharm Biopharm. 2024;201:114351. [DOI] [PubMed] [Google Scholar]

[CR61] 61.Zhu H, Zheng J, Oh XY, Chan CY, Low BQL, Tor JQ, et al. Nanoarchitecture-Integrated Hydrogel Systems toward Therapeutic Applications. ACS Nano. 2023;17(9):7953–78. [DOI] [PubMed] [Google Scholar]

[CR62] 62.Wu D, Zhou J, Zheng Y, Zheng Y, Zhang Q, Zhou Z, et al. Pathogenesis-adaptive polydopamine nanosystem for sequential therapy of ischemic stroke. Nat Commun. 2023;14(1):7147. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR63] 63.Zhu K, Wang L, Xiao Y, Zhang X, You G, Chen Y, et al. Nanomaterial-related hemoglobin-based oxygen carriers, with emphasis on liposome and nano-capsules, for biomedical applications: current status and future perspectives. J Nanobiotechnol. 2024;22(1):336. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR64] 64.Hu J, Wang Q, Wang Y, You G, Li P, Zhao L, et al. Polydopamine-based surface modification of hemoglobin particles for stability enhancement of oxygen carriers. J Colloid Interface Sci. 2020;571:326–36. [DOI] [PubMed] [Google Scholar]

[CR65] 65.Wang Q, Zhang R, Lu M, You G, Wang Y, Chen G, et al. Bioinspired Polydopamine-Coated Hemoglobin as Potential Oxygen Carrier with Antioxidant Properties. Biomacromolecules. 2017;18(4):1333–41. [DOI] [PubMed] [Google Scholar]

[CR66] 66.Battaglini M, Emanet M, Carmignani A, Ciofani G. Polydopamine-based nanostructures: A new generation of versatile, multi-tasking, and smart theranostic tools. Nano Today. 2024;55:102151. [Google Scholar]

[CR67] 67.Morganti-Kossmann MC, Semple BD, Hellewell SC, Bye N, Ziebell JM. The complexity of neuroinflammation consequent to traumatic brain injury: from research evidence to potential treatments. Acta Neuropathol. 2019;137:731–55. [DOI] [PubMed] [Google Scholar]

[CR68] 68.Alam A, Thelin EP, Tajsic T, Khan DZ, Khellaf A, Patani R, et al. Cellular infiltration in traumatic brain injury. J Neuroinflammation. 2020;17(1):328. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR69] 69.Renaudin J, Fewer D, Wilson CB, Boldrey EB, Calogero J, Enot KJ. Dose dependency of decadron in patients with partially excised brain tumors. J Neurosurg. 1973;39:302–5. [DOI] [PubMed] [Google Scholar]

[CR70] 70.Reichardt HM, Gold R, Luhder F. Glucocorticoids in multiple sclerosis and experimental autoimmune encephalomyelitis. Expert Rev Neurother. 2006;6:1657–70. [DOI] [PubMed] [Google Scholar]

[CR71] 71.Mason BL, Pariante CM, Thomas SA. A Revised Role for P-Glycoprotein in the Brain Distribution of Dexamethasone, Cortisol, and Corticosterone in Wild-Type and ABCB1A/B-Deficient Mice. Endocrinology. 2008;149:5244–53. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR72] 72.De Kloet ER. Why Dexamethasone Poorly Penetrates in Brain. Stress. 1997;2:13–20. [DOI] [PubMed] [Google Scholar]

[CR73] 73.Zhang B, Bai M, Xu X, Yang M, Niu F, Gao F, et al. Corticosteroid receptor rebalancing alleviates critical illness-related corticosteroid insufficiency after traumatic brain injury by promoting paraventricular nuclear cell survival via Akt/CREB/BDNF signaling. J Neuroinflammation. 2020;17:318. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR74] 74.Yin N, Zhao Y, Liu C, Yang Y, Wang ZH, Yu W, et al. Engineered Nanoerythrocytes Alleviate Central Nervous System Inflammation by Regulating the Polarization of Inflammatory Microglia. Adv Mater. 2022;34(27):e2201322. [DOI] [PubMed] [Google Scholar]

[CR75] 75.Chen Y, Ma X, Hu Y, Wang H, Zhou P, Tian X et al. A pH/ROS dual-responsive hydrogel promotes diabetic wound healing by controlled release of O2 and EGCG to induce angiogenesis and immunomodulation, Chemical Engineering Journal. 2025; 526: (2025).

[CR76] 76.Cai W, Sun Y, Xu W, Kuang X, Zhang Z, Cao X, et al. Ascorbic Acid-Derived Supramolecular Gels Induce Immunogenic Ferroptosis in Cancer Cells to Potentiate Tumor Immunotherapy. ACS Nano. 2025;19(36):32405–21. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR77] 77.Hohn A, Leibrock J, Bailey K, Barde YA. Identification and characterization of a novel member of the nerve growth factor/brain-derived neurotrophic factor family. Nature. 1990;344(6264):339–41. [DOI] [PubMed] [Google Scholar]

[CR78] 78.Panda HS, Dhokne MD, Thakur S, Singh N, Datusalia AK, Panda JJ. BDNF Loaded Amino Acid-Catecholamine Hybrid Nanoparticles as Curative Agents Against Cognitive Decline in Alzheimer’s Disease. Small. 2025;21(42):e2411701. [DOI] [PubMed] [Google Scholar]

[CR79] 79.Hakami A, Narasimhan K, Comini G, Thiele J, Werner C, Dowd E, et al. Cryogel microcarriers for sustained local delivery of growth factors to the brain. J Control Release. 2024;369:404–19. [DOI] [PubMed] [Google Scholar]

[CR80] 80.Huang Z, Li J, Wo J, Li CL, Wu ZC, Deng XH, et al. Intranasal Delivery of Brain-Derived Neurotrophic Factor (BDNF)-Loaded Small Extracellular Vesicles for Treating Acute Spinal Cord Injury in Rats and Monkeys. J Extracell Vesicles. 2025;14(4):e70066. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR81] 81.Nagahara AH, Merrill DA, Coppola G, Tsukada S, Schroeder BE, Shaked GM, et al. Neuroprotective effects of brain-derived neurotrophic factor in rodent and primate models of Alzheimer’s disease. Nat Med. 2009;15(3):331–7. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR82] 82.Mori A, Nishioka Y, Yamada M, Nishibata Y, Masuda S, Tomaru U, et al. Brain-derived neurotrophic factor induces angiogenin secretion and nuclear translocation in human umbilical vein endothelial cells. Pathol Res Pract. 2018;214(4):521–6. [DOI] [PubMed] [Google Scholar]

[CR83] 83.Khalin I, Alyautdin R, Wong TW, Gnanou J, Kocherga G, Kreuter J. Brain-derived neurotrophic factor delivered to the brain using poly (lactide-co-glycolide) nanoparticles improves neurological and cognitive outcome in mice with traumatic brain injury. Drug Deliv. 2016;23(9):3520–8. [DOI] [PubMed] [Google Scholar]

[CR84] 84.Emerich DF, Silva E, Ali O, Mooney D, Bell W, Yu SJ, et al. Injectable VEGF hydrogels produce near complete neurological and anatomical protection following cerebral ischemia in rats. Cell Transpl. 2010;19(9):1063–71. [DOI] [PubMed] [Google Scholar]

[CR85] 85.He Z, Zang H, Zhu L, Huang K, Yi T, Zhang S, et al. An anti-inflammatory peptide and brain-derived neurotrophic factor-modified hyaluronan-methylcellulose hydrogel promotes nerve regeneration in rats with spinal cord injury. Int J Nanomed. 2019;14:721–32. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR86] 86.Lekouaghet A, Sánchez-Díez M, Pérez-Rigueiro J, Rojo FJ, Ramírez-Castillejo C, Ruiz-León Y, et al. Stabilization of CXCL12 (SDF-1α) via silk fibroin films enhances stem cell migration/retention and functional recovery after stroke. Regen Biomater. 2025;13:rbaf129. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR87] 87.Nih LR, Gojgini S, Carmichael ST, Segura T. Dual-function injectable angiogenic biomaterial for the repair of brain tissue following stroke. Nat Mater. 2018;17(7):642–51. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR88] 88.Ding C, Liu X, Zhang S, Sun S, Yang J, Chai G, et al. Multifunctional hydrogel bioscaffolds based on polysaccharide to promote wound healing: A review. Int J Biol Macromol. 2024;259:129356. [DOI] [PubMed] [Google Scholar]

PERMALINK

Dual-drug-loaded nanohydrogel for intraoperative local application: sequential release-mediated spatiotemporal targeting of diverse secondary injury mechanisms to improve long-term prognosis in traumatic brain injury

Bin Zhang

Mengshi Yang

Shunchang Ma

Yumei Wang

Xueling Zhang

Can Zhu

Shuai Zheng

Xiyu Chen

Yaxuan Zhang

Miao Bai

Guangzhi Shi

Abstract

Background

Methods

Results

Conclusions

Supplementary Information

Background

Methods

Preparation and Characterization of BDNF@Hb-PDA@DEX NPs

Preparation and Characterization of BDNF@Hb-PDA@DEX@gel

In vitro drug release study

In vitro antibacterial and hemostatic experiments

In vivo and in vitro biocompatibility

ROS assay

Controlled cortical impact (CCI) animal models

Animal experimental groups and treatments

Cavity volume

ELISA assays

Immunofluorescence staining in vivo and vitro

TUNEL assay

Image acquisition and cell counting

Determination of brain water content

Evans blue extravasation

Western blot analysis

Magnetic Resonance Imaging (MRI)

Transmission Electron Microscopy

Neurological function assessments

Statistical analysis

Results

Formulation and characteristics of BDNF@Hb-PDA@DEX NPs

Fig. 1.

Fig. 2.

Synthesis and characteristics of the BDNF@Hb-PDA@DEX@gel

Fig. 3.

In vitro drug release test

Antibacterial and hemostatic properties of the BDNF@Hb-DEX@PDA@gel

Biocompatibility and antioxidation of BDNF@Hb-PDA@DEX@gel in vitro and in vivo

BDNF@Hb-PDA@DEX@gel promoted neuronal survival and improved neurological functions after TBI

Fig. 4.

BDNF@Hb-PDA@DEX@gel Inhibited neuroinflammation after TBI

Fig. 5.

BDNF@Hb-PDA@DEX@gel restored BBB integrity, decreased BBB permeability, and reduced brain edema after TBI

Fig. 6.

Local administration of BDNF@Hb-PDA@DEX promoted angiogenesis and the proliferation and differentiation of neural stem cell after TBI

Fig. 7.

Fig. 8.

Discussion

Conclusions

Limitations

Supplementary Information

Acknowledgements

Abbreviations

Author contributions

Funding

Data availability

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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