A reactive oxygen species- and acidic pH-responsive hydrogel loaded with Tempol and poly-lysine enhances spinal cord injury repair in rat models

A reactive oxygen species- and acidic pH-responsive hydrogel loaded with Tempol and poly-lysine enhances spinal cord injury repair in rat models | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A reactive oxygen species- and acidic pH-responsive hydrogel loaded with Tempol and poly-lysine enhances spinal cord injury repair in rat models Gushang Xia, Renjie Shuai, Ze Li, Changlin Tang, Yaowen Zhang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6993042/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Spinal cord injury (SCI) is characterized by excessive reactive oxygen species (ROS) and an acidic pH, which hinder repair. This study aimed to develop and evaluate a local therapeutic strategy using a temperature-sensitive hydrogel loaded with antioxidant and acid-neutralizing nanoparticles to form a DT NP–Hydrogel designed to clear ROS, neutralize acidity, and promote neurological recovery after SCI. Both in vitro and in vivo , DT NP and the local administration of DT NP–Hydrogel significantly reduced ROS levels (4-hydroxynonenal), cell apoptosis (caspase-3), and connexin 43 expressions in primary spinal cord neuron models induced by hydrogen peroxide, and in SCI rat models caused by fall injury, while increasing class III β-tubulin expression. Moreover, our findings also revealed that the locally administered DT NP–Hydrogel exhibited no side effects, making it a promising therapeutic alternative for SCI. Spinal cord injury Oxidative stress Hydrogel Tempol Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Spinal cord injury (SCI) is a potentially fatal condition commonly caused by various traumatic factors, such as falls and traffic accidents, which hinder nerve signal transmission, result in severe spinal cord dysfunction, and place a heavy burden on patients’ families and society 1 , 2 . Statistically, the annual global incidence of traumatic SCI ranges from 3.3 to 195.4 per million, causing permanent disability in 2–3 million individuals worldwide 3 – 5 . Due to its high mortality and disability rates, the clinical therapy of SCI remains a major global challenge 6 . Current clinical therapies—including surgical decompression, methylprednisolone administration, and physical rehabilitation—aim to reduce secondary injury, repair spinal cord damage, and improve neurological function after SCI 7 . However, the effectiveness of these interventions remains limited due to sustained secondary injury, which results in a series of complex cascade reactions, such as oxidative stress, inflammatory response, and apoptosis in the injured area. Given that existing treatment cannot eradicate the complex microenvironment of the injured site—which further aggravates ongoing damage—strategies aimed at improving the local microenvironment present an effective approach to promote SCI recovery 8 , 9 . During sustained secondary injury, activated inflammatory factors and cytokines are released into the injured site, leading to intracellular mitochondrial dysfunction. This, in turn, generates a large amount of reactive oxygen species (ROS), which induce overdue oxidative stress, inflammatory response, and various regulatory cell death pathways—ultimately damaging spinal tissues and hindering regeneration 2 , 10 , 11 . Given the complexity of SCI pathophysiology, current clinical therapies cannot effectively restore neurological function 12 . In addition, oxygen consumption at the injury site increases sharply, resulting in lactic acid accumulation and the formation of a locally acidic microenvironment, which further aggravates spinal cord damage 13 . Therefore, eradicating ROS and neutralizing acidity at the injury site may be an effective therapy to relieve secondary injury and promote spinal cord recovery. Recently, 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol [TPL]), one of the strongest antioxidants, has been commonly utilized to scavenge ROS by capturing free radicals through its ring structure. Moreover, Tempol can help regulate the local pH by consuming ROS, thereby reducing both acidity and alkalinity 14 – 16 . However, the systemic administration of Tempol in SCI therapy is limited by its short half-life and low targeting. Therefore, developing a biocompatible hydrogel incorporating Tempol-coupled lipid nanoparticles for ROS depletion provides a potential therapeutic strategy for SCI. Recently, nanoparticle drug delivery technology has been widely used in various diseases, such as cancer, cardiovascular disease, and chronic inflammation 17 – 19 . The nanoparticle-based drug-loading platform can effectively encapsulate or link therapeutic molecules, including chemical drugs, proteins or peptides, and nucleic acids 20 . This closed spherical structure greatly enhances the solubility of lipophilic drugs, the stability of easily degraded drugs, and the targeting efficiency of otherwise non-specific drugs 21 , 22 . Consequently, antioxidant drugs such as curcumin 23 , metallic nanozymes 24 , glutathione (GSH) 25 , and microRNA 26 have been used for ROS clearance in nervous system injury. These active ingredients are administered through nanomedicine at the local injury site, enabling effective treatment through slow release 21 , ROS elimination 27 , and antioxidant enzyme enhancement 28 . In this therapeutic study of SCI, we attached Tempol to the poly-lysine (PLL)-docosahexaenoic acid (DHA) nanoparticle framework using strong succinic acid (SA) linkages to synthesize antioxidant nanoparticles (DT NP), leveraging Tempol’s strong ROS-scavenging ability and the presence of hydroxyl groups 16 , 29 . Additionally, the ammonium-rich PLL on DT NP enables effective neutralization of local acidity at the SCI site 13 . However, the aqueous form of these nanoparticles is not well-suited for local administration due to high fluidity and poor tissue adhesion, which limits sustained drug release. Correspondingly, hydrogels—widely used as a slow drug-releasing scaffold material—are often used to accommodate and connect various nanomedicine or drug monomers based on their porous structure and modifiable chemical groups 30 , 31 . The polymeric component of hydrogels offers functional advantages, such as good biocompatibility, biodegradability, biosafety, temperature sensitivity, pH responsiveness, and mucosal adhesion, making them well-suited for local drug delivery in a range of diseases 32 – 35 . In treating neural-related trauma, hydrogels are typically pre-formed into a gel-like state and then applied topically to the injured site. However, this often leads to injection difficulties, excessive gel residue, and air bubble formation due to the high viscosity of the coagulated gel 30 , 36 . To address the above challenges, we selected poloxamer 407 (PLXM407) as a temperature-sensitive phase-change bio-hydrogel to mix antioxidant nanoparticles (DT NP) for local administration in this SCI therapeutic study. PLXM407 solutions at concentrations above 25% remain in a liquid state at 4 ℃ but convert to a gel state at temperatures above 33 ℃ 37 . Additionally, gel-like PLXM407 possesses certain bio-viscous properties and mechanical strength, facilitating adhesion to biological tissues 38 . Therefore, in this study, we successfully designed a ROS-cleared DT NP–Hydrogel for local administration at the injury site in an SCI rat model. In this study, in vitro models of primary spinal cells from 14-day-old fetal rats and female rat SCI models induced by high fall injury were used to evaluate the therapeutic effectiveness and safety of DT NP–Hydrogel. The results indicate that this hydrogel can effectively eliminate ROS at the injury site, neutralize the acidic environment, promote spinal cord repair, and exhibit no toxic side effects in rats—providing a safe and effective strategy for the clinical treatment of SCI. 2. Materials and methods 2.1. Materials and reagents DHA, PLL, SA, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-Hydroxysulfosuccinimide (NHS), 4-dimethylaminopyridine (DMAP), and dimethyl sulfoxide (DMSO) were procured from Macklin, China. 4-Hydroxy-2,2,6,6-tetramethylpiperidine N-oxide (Tempol, TPL), as well as PLXM407, were obtained from Sigma, USA. Dulbecco’s modified eagle medium (DMEM), neuronal growth factor B27, penicillin and streptomycin (PS), fetal bovine serum (FBS), 1× phosphate-buffered saline, normal saline (NS), 3, 3'-Diaminobenzidine (DAB), 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and Avertin were obtained from Dowobio, China. DNaseI and papain were acquired from Solarbio, China. Hydrogen peroxide (H 2 O 2 ) was procured from Macklin, China. Antibodies against 4-hydroxynonenal (4HNE; Bioss, China), class III β-Tubulin III (Tuj1, Immunoway, USA), Caspase-3 (Cap3; Wanleibio, China), and connexin 43 (Cx43; Proteintech, China), were obtained. 2.2. Synthesis and preparation of nanoparticles and hydrogels Hydrogels containing therapeutic nanoparticles were synthesized based on a published article 39 . First, DHA (100 mg), EDC (160 mg), NHS (80 mg), and DMAP (25 mg) were added into DMSO (15 mL) and mixed thoroughly under nitrogen protection at room temperature for 3 h. Subsequently, PLL (900 mg) dissolved in DMSO (10 mL) was added to the reaction system and mixed thoroughly for 24 h to obtain DHA-PLL nanoparticles (DP NP). Second, SA (83 mg), EDC (320 mg), NHS (160 mg), and DMAP (50 mg) were added into DMSO (10 mL) and mixed thoroughly for 3 h. Additionally, TPL (120 mg) dissolved in DMSO (5 mL) was added to the reaction system and mixed thoroughly for 24 h. Next, DP NP (200 mg) dissolved in DMSO (10 mL) was added into the reaction system and mixed thoroughly for another 24 h to obtain DHA-PLL-TPL nanoparticles (DT NP). Third, PLXM407 (3 g) was dissolved in NS (10 mL) at 4 ℃. Then, DP NP (500 mg) or DT NP (500 mg) was added and mixed thoroughly to obtain DP NP–Hydrogel or DT NP–Hydrogel. Fourier transform infrared spectrometer, nuclear magnetic resonance (NMR) spectroscopy, dynamic light scattering (DLS), scanning electron microscope (SEM), transmission electron microscope (TEM), and ultraviolet spectrophotometer (UVS) were used to characterize the nanoparticles and hydrogels. 2.3. Isolation, culture, and identification of primary cells Primary spinal cord neuron (PSCN) cells were obtained from the spinal cord of fetal rats at 14-day gestation. After anesthesia through intraperitoneal injection of Avertin (60 µL/g), fetal rats were obtained and temporarily stored in 75% alcohol at 4 ℃. The spinal cord samples were then carefully separated under sterile conditions, cut into approximately 1 mm tissue blocks, and incubated with DNaseI (100 U/mL) and papain (1 mg/mL) at 37 ℃ for 30 min. The reaction was stopped with FBS, and the suspension was filtered through a 70-mesh filter membrane to obtain PSCN. The spinal cells were then resuspended in DMEM supplemented with 5% FBS and 1% PS and cultured at 37 ℃ with 5% CO 2 for 4 h. The medium was then replaced with a special Neurobasal medium containing 2% B27 and 1% Glu complete medium for 24 h. Subsequently, primary cells were fluorescently stained with Tuj1-antibodies (1:500, anti-rat) to identify PSCN. 2.4. Effects of different reagents on PSCN Well-growing PSCN cells (1 × 10 5 /mL) were seeded into 96-well plates and cultured overnight at 37 ℃ with 5% CO 2 . PSCN cells were then treated with 0–1024 µM H 2 O 2 and 0–1024 µg/mL TPL, DP NP, or DT NP for 24 h. Cell viability was assessed using the CCK-8 assay. Subsequently, a suitable concentration of H 2 O 2 (250 µM) was used to induce oxidative stress injury in PSCN in vitro , as previously described 40 . 2.5. In vitro experiments of PSCN Well-growing PSCN cells (5 × 10 4 /mL) were seeded onto glass slides and cultured overnight at 37 ℃ with 5% CO 2 . In vitro experiment groups were divided into a normal group, a model group (250 µM H 2 O 2 ), a model + TPL group (120 µg/mL), a model + DP NP group (400 µg/mL), and a model + DT NP group (400 µg/mL). After treatment with the respective compounds for 24 h, the neuronal cells on the glass slides were fixed with 4% paraformaldehyde and stained with different antibodies and DAPI. Imaging was performed using an inverted fluorescence electron microscope (IFEM). 2.6. Establishment and hydrogel therapy of SCI rat models The animal models and hydrogel treatment used in this study were ethically approved by the Ethics Committee of Chongqing University Central Hospital (Approval No. 2502001). All Sprague-Dawley (female, 200–220 g, 8 weeks) rats were kept with standard water, diet, and a 12-h circadian rhythm. Before the establishment of SCI models, all rats were kept for a week to acclimate to their new environment. The rats were randomly divided into six groups, each including 6 rats: (1) Sham group (Sham); (2) Model group; (3) TPL group (12 mg/kg); (4) Non–Hydrogel group (100 µL/kg, 30% w/v); (5) DP NP–Hydrogel group (100 µL/kg, 50 mg/mL); (6) DT NP–Hydrogel group (100 µL/kg, 50 mg/mL). All SCI rat models were performed according to the weight-drop method in aseptic conditions 31 . Briefly, after intraperitoneal anesthesia (Avertin, 10 mL/kg), the spinal cord of the 10th thoracic vertebra was carefully exposed and struck with a 15 g weight (2.5 mm in diameter) dropped from a height of 10 cm. Tail spasms and bilateral hindlimb paralysis were observed, revealing the successful establishment of the SCI models. Subsequently, 20 µL Non–Hydrogel, DP NP–Hydrogel, or DT NP–Hydrogel was injected into the damaged spinal cord in the corresponding hydrogel groups, and TPL was injected through the tail vein. After suturing the wound, all operated rats were placed in a 37 ℃ environment for recovery. Artificial urination was performed every two days, and the recovery status of the rats was observed. 2.7. Hematoxylin and eosin (H&E) and immunohistochemical staining (IHC) staining Paraffin sections (4 µm) of spinal cord samples underwent dewaxing, dehydration, membrane permeabilization, and antigen retrieval, then were sealed with 5% bovine serum albumin for 1 h. H&E staining was used to observe SCI in rat models. Subsequently, the tissue sections were incubated overnight at 4 ℃ with the following primary antibodies: 4HNE (anti-rabbit, 1:200), Cap3 (anti-rabbit, 1:200), Tuj1 (anti-mouse, 1:200), and Cx43 (anti-rabbit, 1:1000). Subsequently, tissue sections were stained with DAB agent to observe the degree of brown and yellow color in the tissues. Immunohistochemistry images were obtained from regions of interest in different groups using IFEM. The number of brown-stained cells was quantified using ImageJ software. Six samples from each group were utilized for image acquisition. 2.8. Quantitative real-time PCR (RT-qPCR) Total RNA was extracted from fresh spinal cord samples through the Trizol method, and RNA concentration was measured at 260 nm with an ultraviolet-visible light spectrophotometer. A FastKing cDNA First Strand Synthesis Kit (Thermo, USA) was used to synthesize cDNA from 2 µg of total RNA per sample. RT-qPCR was performed using SYBR Green PCR Master Mix (Dowobio, China). The expression level of glyceraldehyde 3- phosphate dehydrogenase (GAPDH) was used as the internal control. Each reaction was run in triplicate. All primers, including Glutathione peroxidase algal (GPXH) (5'-GCTCCATGCACGAGTTTTCC-3', 5'-GTTTACTTCGGTCTTGCCTCACT-3'), Cap3 (5'-TACTCTACCGCACCCGGTTA-3', 5'-CGCGTACAGTTTCAGCATGG-3'), Tuj1 (5'-CAAGGTGCGTGAGGAGTATCR-3', 5'-CGGAAGCAGATGTCGTAGAG-3'), and Cx43 (5'-GAGTTTGCCTAAGGCGCTC-3', 5'-AGGAGTTCAATCACTTGGCG3-3'), were customized by Sangon Biotech, China. The experimental results were expressed as 2 −ΔΔCt values, with GAPDH used as the internal reference gene. Each reaction was performed in triplicate. 2.9. Safety studies in vivo Drug distribution of DP NP–Hydrogel and DT NP–Hydrogel was observed in SCI rats, while normal rats served as the negative control group. Fluorescence in the heart, liver, spleen, lung, and kidney was observed using a live animal imaging system. The in vivo safety of the different hydrogels—including Non–Hydrogel, DP NP–Hydrogel, and DT NP–Hydrogel—was assessed through blood component analysis, hemolysis testing, and H&E staining of major organs. Briefly, a 10-fold high dose of DP NP–Hydrogel and DT NP–Hydrogel (500 mg/mL) were injected into the spinal cord of normal rats, and the animals were observed for 28 days after recovery. After anesthesia with Avertin, blood was extracted from the inner canthus using ETDA anticoagulant tubes and analyzed using an animal blood analyzer for blood routine hematological and biochemical indices. Following euthanasia by spinal dislocation, all organs—including heart, liver, spleen, lung, and kidney were harvested and fixed in 4% paraformaldehyde for H&E staining. Subsequently, blood was obtained from untreated normal rats through inner canthus extraction. Then, 50 µL of blood was mixed thoroughly with 50 µL of different solutions—including double distilled water (DDW), NS, DP NP, and DT NP—and centrifugated at 1500 rpm for 10 min at room temperature to assess hemolysis. 2.10. Statistical analysis All statistical analyses were performed using the Statistical Package for the Social Sciences (version 22) and Origin (version 20) software. Data were analyzed using one-way analysis of variance and independent sample t-tests. Results are presented as means ± standard error of the mean. Statistical significance was defined as * p < 0.05, ** p < 0.01 and *** p < 0.001. 3. Results 3.1 Characterization of DP NP and DT NP nanoparticles As biocompatible nanoparticles, the nano framework (DP NP) was first formed by the dehydration condensation of DHA and PLL. Subsequently, DT NP was synthesized through the coupling of SA. DLS analysis displayed that the particle sizes of DP NP and DT NP were 157.42 ± 1.33 and 164.29 ± 1.15 nm, respectively (Figs. 1A-B, Table 1). The surface zeta potential was 44.43 ± 0.42 and -20.67 ± 0.60 mV, respectively (Fig. 1C, Table 1). PDI values were 0.205 ± 0.01 and 0.213 ± 0.02, respectively (Table 1). SEM and TEM results also revealed that DP NP and DT NP were solid, spherical-like nanoparticles (Figs. 1D-E). Chemical binding analysis by NMR revealed that the amine group on PLL and the hydroxyl group were conjugated with the carboxyl group of DHA and SA (Fig. 1F). Size stability analysis by DLS indicated that DP NP and DT NP remained stable in four different liquids after 24 h of incubation (Figs. 1G-H, Table 2). Drug loading measurement by UVS indicated that the drug loading rate of DT NP was approximately 23%. 3.2 Characterization of DP NP and DT NP hydrogels PLXM407 is a temperature-sensitive, biocompatible hydrogel material. The hydrogel samples demonstrated gel formation at temperatures ranging from 4 to 37 ℃ (Fig. 2A). SEM results displayed that the dry hydrogel exhibited a loose and porous structure, with many spherical nanoparticles located on the surface (Fig. 2B). Injection experiments demonstrated that these hydrogels exhibited excellent injectability at 4 ℃ (Fig. 2C). The viscosity experiment revealed that the hydrogels could effectively adhere to a weight of 5 g (Fig. 2D). The swelling experiment displayed that the hydrogels achieved their maximum swelling capacity after 5 min in DDW (Fig. 2E, Table 3). The degradation experiment also indicated that different hydrogels effectively degraded over time in DDW (Fig. 2F, Table 4). 3.3 Identification and cell activity of PSCN To evaluate the cytotoxic effects of DP NP and DT NP in vitro , PSCN cells were obtained from fetal rats at gestation day 14 and cultured for 7 days (Fig. 3A). The morphological result revealed that PSCN developed long synaptic-like extensions (Fig. 3B). The specific surface protein of spinal cord neuron cells, Tuj1, was highly expressed in PSCN and clearly observed by immunofluorescence (IF) staining. These results indicated that the primary cells were successfully obtained and suitable for subsequent experiments. Subsequently, different concentrations of H 2 O 2 , TPL, DP NP, and DT NP were resuspended in a medium and co-cultured with PSCN for 24 h to assess cell viability. As illustrated in Fig. 3D-F and Table 5, PSCN exhibited viability at 2 µM H 2 O 2 , but cell activity decreased as the concentration increased. The result led us to select 250 µM H 2 O 2 as the appropriate concentration for the in vitro oxidative stress model. Additional CCK-8 assays indicated that TPL, DP NP, and DT NP, at concentrations ranging from 0–1024 µg/mL, displayed no significant cytotoxicity after 24 h of co-culture with PSCN, with cell viability remaining at approximately 100%. 3.4 In vitro experiments of PSCN treated with DP NP and DT NP nanoparticles To assess the antioxidant and nerve regeneration effects of DP NP and DT NP on PSCN models, IF staining of 4HNE, Cap3, Tuj1, and Cx43 antibodies was performed and observed using the IFEM. After H 2 O 2 treatment, PSCN indicated a significant increase in oxidative stress and cell apoptosis (Figs. 4A, B, E, Table 6) and a marked decrease in nerve regeneration and junction formation (Figs. 4C–E, Table 6). However, DT NP indicated superior antioxidant and neuroprotective effects compared to TPL and DP NP. These results suggested that TPL reduced ROS levels, oxidative stress, and cell apoptosis induced by H 2 O 2 through scavenging free radicals and that DHA in both DP NP and DT NP also promoted nerve cell growth. 3.5 In vivo experiments of SCI rat models treated with DP NP and DT NP hydrogels To study the effect of DP NP and DT NP on antioxidation and nerve recovery in SCI rat models, hydrogels containing DP NP or DT NP were injected into the site of SCI for local drug release over 28 days (Figs. 5A-B). After 28 days, all spinal cord tissues from the injury site were collected for tissue staining and RT-qPCR tests. H&E staining results revealed that the spinal cord trauma remained evident, and none of the drug treatments effectively restored the normal spinal cord structure (Fig. 5C). However, both IHC staining and RT-qPCR analysis indicated that DT NP–hydrogel could more effectively reduce oxidative stress and cell apoptosis and promoted nerve recovery and connection at the injury site compared to TPL and DP NP–Hydrogel (Figs. 5D–F, Table 7-8). Notably, TPL administered by intravenous injection displayed no significant effect on alleviating the injury site, likely due to the reduced local drug concentration. However, DP NP–Hydrogel and DT NP–Hydrogel, when administered locally, effectively alleviated injury progression and promoted recovery in SCI rat models. These results confirm that local administration of DT NP–Hydrogel has the potential for application in SCI treatment. 3.6 Safety studies in vivo The safety of DP NP and DT NP hydrogels was effectively evaluated in SCI rat models using a high dose of DP NP and DT NP (10× treatment dose) administered locally. Subsequently, blood and organs were collected, analyzed with a blood detector, and stained H&E staining, respectively. Blood test results indicated that no significant change in blood composition and blood biochemistry in high-dose groups was found compared to normal groups (Fig. 6A, Table 9). H&E staining results revealed that no abnormal structure was observed in the examined organs (Fig. 6B). The hemolytic test also indicated that the reagent components of different hydrogels did not cause intravascular hemolysis (Fig. 6C). Organ fluorescence imaging also displayed non-specific enrichment in normal organs following local administration (Fig. 6D). These experiments revealed that the local administration of hydrogels was safe. 4. Discussion In recent clinical treatment, it is crucial to alleviate the progression of SCI and avoid secondary injuries 12 . In SCI, excessive oxidative stress and decreased acidic pH values are vital factors that contribute to secondary injuries 10 , 11 , 13 . However, the clinical application of long-lasting local drugs at the SCI site remains insufficient. To achieve sustained drug release targeting ROS and acid in the injured site, this study focused on biocompatible nanoparticles (DT NP) based on the chemical connection between amino groups from PLL, hydroxyl groups from TPL, and carboxylic acids from SA. Subsequently, these DT NP were thoroughly mixed with the thermosensitive hydrogel PLXM407 at 4 ℃ to form a suspension, and at over 33 ℃ to form a gel 37 . Overall, this study aimed to employ a local nanoparticle–hydrogel system for the sustained release of effective drugs to alleviate the injury in SCI rat models. Inevitably, excessive oxidative stress, characterized by elevated ROS and reduced antioxidant defense, is a notably significant pathological process in SCI 41 . In spinal cord tissue with a high metabolic rate and weak antioxidant capacity, biological macromolecules, organelles, and cellular functions are extensively damaged by excessive ROS free radicals 42 . Moreover, these ROS can quickly bind to polyunsaturated fatty acids in biological membrane structure to form highly reactive peroxyl radicals, initiating a chain reaction with other polyunsaturated fatty acids and causing progressive oxidative damage at the injury site. 4HNE is a highly reactive and toxic aldehyde and serve as an important marker of lipid peroxidation 43 . Even more concerning, the myelin sheath of the spinal cord—a type of neural tissue—is particularly rich in lipid components, such as cholesterol, glycosphingolipids, and ceramides, making it particularly susceptible to excessive ROS 44 . Recently, many biological and chemical drugs have been used for direct antioxidant treatment. For example, GSH peroxidase can quickly convert harmful superoxide free radicals into relatively harmless substances, induced by gene regulation for high expression 45 . Similarly, TPL, a strong and low-cost antioxidant molecule, can also effectively capture free radicals through its ring structure, thereby reducing excessive ROS 14 – 16 . In this study, TPL in DT NP demonstrated enhanced antioxidant capacity in both PSCN cell models and SCI rat models, suggesting that the long-acting antioxidant stress property of the DT NP–Hydrogel system may play a potential role in treating neurotraumatic diseases. As a form of programmed cell death, apoptosis is mediated by cysteine aspartate-specific proteases and is prominently observed in SCI 46 . Recently, the biological regulation between ROS and apoptosis has also become a major focus in life sciences. Oxidized low-density lipoprotein and peroxynitrite free radicals induced by H 2 O 2 have been found in different types of apoptosis 47 . Furthermore, during ischemia-reperfusion injury of the spinal cord, a reduction in mitochondrial membrane potential and increased ROS generation lead to mitochondrial dysfunction, excessive release of cytochrome c, and strong activation of Cap3, ultimately causing neural cell apoptosis 48 , 49 . Tuj1 is a neuron-specific marker that plays a significant role in SCI research and treatment 50 . It plays a critical regulatory role in the structural composition of neurons, intracellular substance transport, axonal growth, and neuronal movement 51 – 53 . Cx43 is also a key intercellular communication protein, especially in neural tissues 54 . In SCI, Cx43 participates in inflammation, ferroptosis, and apoptosis of nerve cells 55 . In this study, high expression levels of Cap3 and Cx43, along with reduced levels of Tuj1, were significantly observed and were effectively restored with DT NP–Hydrogel treatment. As three-dimensional polymeric networks, both biological and chemical hydrogels can effectively absorb and retain large amounts of water due to the presence of numerous hydrophilic groups, such as amino, hydroxyl, carboxylic, and ether-oxygen groups. Different biocompatible hydrogels have been commonly prepared for local and long-acting treatment in drug delivery, tissue engineering, and wound healing 56 , 57 . In SCI repair, hydrogels are commonly used to bridge gaps between severed spinal cord tissue, prevent excessive leakage of inflammatory factors, and reduce secondary mechanical injury to the spinal cord 51 , 58 . Moreover, these hydrogels can incorporate various active drugs or therapeutic agents through random distribution, polarity-based encapsulation, electrostatic adsorption, and chemical bonding according to the physicochemical properties of the drug 59 – 61 . Research has revealed that drug-loaded hydrogels can effectively provide a localized therapeutic microenvironment for the removal of harmful substances and the promotion of nerve regeneration. In this study, TPL for ROS clearance, PLL for acid neutralization, and DHA for neurotrophy were all synthesized into biocompatible nanoparticles (DT NP), which were thoroughly mixed with PLXM407 liquid at 4 ℃ to form a DT NP–Hydrogel at 37 ℃. Due to these characteristics, DT NP–Hydrogel can better fill the gaps at the SCI site and enable continuous drug release. These experimental results effectively demonstrated that DT NP–Hydrogel could relieve ROS damage and promote neural recovery under long-term effects in SCI rat models. Additionally, local administration of the DT NP–Hydrogel did not cause abnormal accumulation of nanoparticles in organs, structural damage to organs, changes in blood components, or hemolysis. Consequently, these findings provide new strategies to complement the clinical treatment of SCI. 5. Conclusion In summary, DT NP nanoparticles were demonstrated to play an important role in removing ROS, inhibiting apoptosis, and promoting nerve regeneration in PSCN cell models induced by H 2 O 2 . Moreover, DT NP–Hydrogel could enhance the antioxidant capacity of the injured area in SCI rat models. This therapeutic effect was mainly mediated by the ROS-removing capability of TPL and partly by the acidic neutralization of PLL and the neurotrophic properties of DHA. These results present potential value for developing long-acting sustained-release hydrogels for local therapeutic strategies in SCI. Declarations Acknowledgements We are extremely grateful for the project guidance from Ke Li, PhD, Chongqing Medical University. And we also thank Home for Researchers editorial team (www.home-for-researchers.com) for language editing service. Ethics approval and consent to participate This project was supervised and approved by Laborator Animal Welfare and Ethics Commit of Chongqing University Central Hospital, Chongqing (IACUC Issue No.2502001). Author contributions Gushang Xia, and Renjie Shuai contributed to experiments, data interpretation and writing; Ze Li, Changlin Tang, Yaowen Zhang, Qingli Kong, Wanyou Li and Fangfang Ma contributed to visualization; Xianglin Li and Yan Du contributed with resources, writing - review & editing, project administration; and Yan Du contributed with funding acquisition. All authors reviewed the manuscript. Funding information This study was funded by Project of Chongqing Key Laboratory of Emergency Medicine, Chongqing (No. 2023KFKT02). Declarations of competing interests The authors declare no competing interests. References Courtine G, Sofroniew MV (2019) Spinal cord repair: advances in biology and technology. 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J. (2021) M. C. B. Tissue adhesive hydrogel bioelectronics. 9 https://doi.org/10.1039/d1tb00523e Yongping L, Jiahui H, Baolin GJ (2021) A. N. Functional Hydrogels as Wound Dressing to Enhance Wound Healing. 15. https://doi.org/10.1021/acsnano.1c04206 Ziyuan Y et al (2020) Application of fibrin-based hydrogels for nerve protection and regeneration after spinal cord injury. 14. https://doi.org/10.1186/s13036-020-00244-3 Robert B, S., Yinghui ZJNRR (2020) Hydrogel-based local drug delivery strategies for spinal cord repair. 16. https://doi.org/10.4103/1673-5374.290882 Zengjie F, Xiaozhu L, Yu T, Xie X, Yingying NJA (2019) B. A prevascularized nerve conduit based on a stem cell sheet effectively promotes the repair of transected spinal cord injury. 101. https://doi.org/10.1016/j.actbio.2019.10.042 Hang L et al (2016) A Hydrogel Bridge Incorporating Immobilized Growth Factors and Neural Stem/Progenitor Cells to Treat Spinal Cord Injury. 5. https://doi.org/10.1002/adhm.201500810 Tables Table 1 to 9 are available in the Supplementary Files section. Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Scheme1.tif Scheme 1. Schematic overview of the development and application of DT NP-Hydrogel in SCI rat models. A), Schematic synthesis diagram of DT NP nanoparticles. B),Structure of DT NP nanoparticles and preparation of DT NP-Hydrogel. C), Local administration of DT NP-Hydrogel in SCI rat models. 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(Data are represented as mean ± SD and representative of three independent samples.)\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-6993042/v1/d4a60f8f7c91b66e7e845dc6.png"},{"id":86972403,"identity":"2cdec872-e4fb-4f55-804a-e6149971b5f2","added_by":"auto","created_at":"2025-07-17 19:31:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5910841,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of DP NP and DT NP Hydrogels.\u003c/strong\u003e \u003cstrong\u003eA),\u003c/strong\u003e Coagulation characteristics of thermosensitive hydrogels. \u003cstrong\u003eB), \u003c/strong\u003eMorphology and SEM pictures of dry hydrogels (Scale bar, up 3mm, middle 100um and down 500nm). \u003cstrong\u003eC), \u003c/strong\u003eInjectable pictures. \u003cstrong\u003eD),\u003c/strong\u003e Viscosity pictures (weight, 5g). \u003cstrong\u003eE),\u003c/strong\u003e Swelling ability. \u003cstrong\u003eF),\u003c/strong\u003e Degradation ability. (Data are represented as mean ± SD and representative of three independent samples.)\u003c/p\u003e","description":"","filename":"Figure21.png","url":"https://assets-eu.researchsquare.com/files/rs-6993042/v1/ddbfbb7630600731d675abc0.png"},{"id":86972419,"identity":"147da7f9-9781-45c9-b482-1d607f996a28","added_by":"auto","created_at":"2025-07-17 19:31:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7077966,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell extraction, identification and toxicity experiments of PSCN. A), \u003c/strong\u003eSchematic diagram of primary cell extraction. \u003cstrong\u003eB), \u003c/strong\u003eMorphology pictures (Scale bar, 100X). \u003cstrong\u003eC),\u003c/strong\u003e IF staining of Tuj1 (Scale bar, 100X). \u003cstrong\u003eD-G),\u003c/strong\u003e Cell viability treated with different drugs and detected by CCK-8 assays. (Data are represented as mean ± SD and representative of eight independent samples.)\u003c/p\u003e","description":"","filename":"Figure31.png","url":"https://assets-eu.researchsquare.com/files/rs-6993042/v1/10572b0fa61e3d307d874138.png"},{"id":86972407,"identity":"02dc5279-bb4e-4b47-a9a6-3ce3bf87f59e","added_by":"auto","created_at":"2025-07-17 19:31:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4863596,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of antioxidation and nerve regeneration of DP NP and DT NP nanoparticles in PSCN models.\u003c/strong\u003e \u003cstrong\u003eA), \u003c/strong\u003e4HNE expression. \u003cstrong\u003eB),\u003c/strong\u003e Cap3 expression. \u003cstrong\u003eC), \u003c/strong\u003eTuj1 expression. \u003cstrong\u003eD),\u003c/strong\u003e Cx43 expression. \u003cstrong\u003eE),\u003c/strong\u003e Data analysis. (Scale bar, 100X. Data are represented as mean ± SD and representative of four independent samples.***p＜0.001, ns=no significance.)\u003c/p\u003e","description":"","filename":"Figure41.png","url":"https://assets-eu.researchsquare.com/files/rs-6993042/v1/24ce8323e1efaa074a7b1141.png"},{"id":86972414,"identity":"1c42e411-f09f-4dbe-8bd7-6110f6d163ed","added_by":"auto","created_at":"2025-07-17 19:31:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":17292481,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of antioxidation and nerve regeneration of DP NP and DT NP hydrogels in SCI rat models. A-B), \u003c/strong\u003eSchematic diagram of SCI model and hydrogel administration. \u003cstrong\u003eC),\u003c/strong\u003e HE staining. \u003cstrong\u003eD-E),\u003c/strong\u003e IHC staining and data analysis (Scale bar, 100X). \u003cstrong\u003eF), \u003c/strong\u003eQt-PCR results. (Data are represented as mean ± SD and representative of six independent samples.**p＜0.01, ***p＜0.001, ns=no significance.)\u003c/p\u003e","description":"","filename":"Figure51.png","url":"https://assets-eu.researchsquare.com/files/rs-6993042/v1/5e11d325ca72e6b7748afc76.png"},{"id":86972681,"identity":"a3622ddc-e37d-41ba-9665-f96dc950be11","added_by":"auto","created_at":"2025-07-17 19:39:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":45347236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSafety studies in vivo. A), \u003c/strong\u003eBlood routine and blood biochemical analysis. \u003cstrong\u003eB),\u003c/strong\u003e HE staining of different organs (Scale bar, 100X). \u003cstrong\u003eC),\u003c/strong\u003e Hemolysis test pictures. \u003cstrong\u003eD), \u003c/strong\u003eOrgan distribution. (Data are represented as mean ± SD and representative of six independent samples.)\u003c/p\u003e","description":"","filename":"Figure61.png","url":"https://assets-eu.researchsquare.com/files/rs-6993042/v1/eef62509abae06f5442b50bd.png"},{"id":91800531,"identity":"2bc238d4-c1a6-4389-8e86-1ef4ab247e29","added_by":"auto","created_at":"2025-09-21 20:16:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":78401099,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6993042/v1/99648833-fd44-463d-972c-f4234ded136f.pdf"},{"id":86972756,"identity":"3ab0fe6a-ad1b-4b8a-ba45-8a22bc54b1e1","added_by":"auto","created_at":"2025-07-17 19:47:35","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3401272,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. Schematic overview of the development and application of DT NP-Hydrogel in SCI rat models.\u003c/strong\u003e \u003cstrong\u003eA),\u003c/strong\u003e Schematic synthesis diagram of DT NP nanoparticles. \u003cstrong\u003eB),\u003c/strong\u003eStructure of DT NP nanoparticles and preparation of DT NP-Hydrogel. \u003cstrong\u003eC),\u003c/strong\u003e Local administration of DT NP-Hydrogel in SCI rat models.\u003c/p\u003e","description":"","filename":"Scheme1.tif","url":"https://assets-eu.researchsquare.com/files/rs-6993042/v1/7733bb15dbc188e4cce7b1a3.tif"},{"id":86972401,"identity":"207fd8e8-a8ff-4ed3-92fa-cec9a5d877f3","added_by":"auto","created_at":"2025-07-17 19:31:35","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":70947,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6993042/v1/0772d51e988ddc72e923009f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A reactive oxygen species- and acidic pH-responsive hydrogel loaded with Tempol and poly-lysine enhances spinal cord injury repair in rat models","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSpinal cord injury (SCI) is a potentially fatal condition commonly caused by various traumatic factors, such as falls and traffic accidents, which hinder nerve signal transmission, result in severe spinal cord dysfunction, and place a heavy burden on patients\u0026rsquo; families and society\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Statistically, the annual global incidence of traumatic SCI ranges from 3.3 to 195.4 per million, causing permanent disability in 2\u0026ndash;3\u0026nbsp;million individuals worldwide\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Due to its high mortality and disability rates, the clinical therapy of SCI remains a major global challenge\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Current clinical therapies\u0026mdash;including surgical decompression, methylprednisolone administration, and physical rehabilitation\u0026mdash;aim to reduce secondary injury, repair spinal cord damage, and improve neurological function after SCI\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, the effectiveness of these interventions remains limited due to sustained secondary injury, which results in a series of complex cascade reactions, such as oxidative stress, inflammatory response, and apoptosis in the injured area. Given that existing treatment cannot eradicate the complex microenvironment of the injured site\u0026mdash;which further aggravates ongoing damage\u0026mdash;strategies aimed at improving the local microenvironment present an effective approach to promote SCI recovery\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDuring sustained secondary injury, activated inflammatory factors and cytokines are released into the injured site, leading to intracellular mitochondrial dysfunction. This, in turn, generates a large amount of reactive oxygen species (ROS), which induce overdue oxidative stress, inflammatory response, and various regulatory cell death pathways\u0026mdash;ultimately damaging spinal tissues and hindering regeneration\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Given the complexity of SCI pathophysiology, current clinical therapies cannot effectively restore neurological function\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In addition, oxygen consumption at the injury site increases sharply, resulting in lactic acid accumulation and the formation of a locally acidic microenvironment, which further aggravates spinal cord damage\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Therefore, eradicating ROS and neutralizing acidity at the injury site may be an effective therapy to relieve secondary injury and promote spinal cord recovery. Recently, 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol [TPL]), one of the strongest antioxidants, has been commonly utilized to scavenge ROS by capturing free radicals through its ring structure. Moreover, Tempol can help regulate the local pH by consuming ROS, thereby reducing both acidity and alkalinity \u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. However, the systemic administration of Tempol in SCI therapy is limited by its short half-life and low targeting. Therefore, developing a biocompatible hydrogel incorporating Tempol-coupled lipid nanoparticles for ROS depletion provides a potential therapeutic strategy for SCI.\u003c/p\u003e\u003cp\u003eRecently, nanoparticle drug delivery technology has been widely used in various diseases, such as cancer, cardiovascular disease, and chronic inflammation\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The nanoparticle-based drug-loading platform can effectively encapsulate or link therapeutic molecules, including chemical drugs, proteins or peptides, and nucleic acids\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. This closed spherical structure greatly enhances the solubility of lipophilic drugs, the stability of easily degraded drugs, and the targeting efficiency of otherwise non-specific drugs\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Consequently, antioxidant drugs such as curcumin\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, metallic nanozymes\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, glutathione (GSH)\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, and microRNA\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e have been used for ROS clearance in nervous system injury. These active ingredients are administered through nanomedicine at the local injury site, enabling effective treatment through slow release\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, ROS elimination\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, and antioxidant enzyme enhancement\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In this therapeutic study of SCI, we attached Tempol to the poly-lysine (PLL)-docosahexaenoic acid (DHA) nanoparticle framework using strong succinic acid (SA) linkages to synthesize antioxidant nanoparticles (DT NP), leveraging Tempol\u0026rsquo;s strong ROS-scavenging ability and the presence of hydroxyl groups\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Additionally, the ammonium-rich PLL on DT NP enables effective neutralization of local acidity at the SCI site\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, the aqueous form of these nanoparticles is not well-suited for local administration due to high fluidity and poor tissue adhesion, which limits sustained drug release.\u003c/p\u003e\u003cp\u003eCorrespondingly, hydrogels\u0026mdash;widely used as a slow drug-releasing scaffold material\u0026mdash;are often used to accommodate and connect various nanomedicine or drug monomers based on their porous structure and modifiable chemical groups\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The polymeric component of hydrogels offers functional advantages, such as good biocompatibility, biodegradability, biosafety, temperature sensitivity, pH responsiveness, and mucosal adhesion, making them well-suited for local drug delivery in a range of diseases\u003csup\u003e\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In treating neural-related trauma, hydrogels are typically pre-formed into a gel-like state and then applied topically to the injured site. However, this often leads to injection difficulties, excessive gel residue, and air bubble formation due to the high viscosity of the coagulated gel\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. To address the above challenges, we selected poloxamer 407 (PLXM407) as a temperature-sensitive phase-change bio-hydrogel to mix antioxidant nanoparticles (DT NP) for local administration in this SCI therapeutic study. PLXM407 solutions at concentrations above 25% remain in a liquid state at 4 ℃ but convert to a gel state at temperatures above 33 ℃\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Additionally, gel-like PLXM407 possesses certain bio-viscous properties and mechanical strength, facilitating adhesion to biological tissues\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Therefore, in this study, we successfully designed a ROS-cleared DT NP\u0026ndash;Hydrogel for local administration at the injury site in an SCI rat model.\u003c/p\u003e\u003cp\u003eIn this study, \u003cem\u003ein vitro\u003c/em\u003e models of primary spinal cells from 14-day-old fetal rats and female rat SCI models induced by high fall injury were used to evaluate the therapeutic effectiveness and safety of DT NP\u0026ndash;Hydrogel. The results indicate that this hydrogel can effectively eliminate ROS at the injury site, neutralize the acidic environment, promote spinal cord repair, and exhibit no toxic side effects in rats\u0026mdash;providing a safe and effective strategy for the clinical treatment of SCI.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials and reagents\u003c/h2\u003e\u003cp\u003eDHA, PLL, SA, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-Hydroxysulfosuccinimide (NHS), 4-dimethylaminopyridine (DMAP), and dimethyl sulfoxide (DMSO) were procured from Macklin, China. 4-Hydroxy-2,2,6,6-tetramethylpiperidine N-oxide (Tempol, TPL), as well as PLXM407, were obtained from Sigma, USA. Dulbecco\u0026rsquo;s modified eagle medium (DMEM), neuronal growth factor B27, penicillin and streptomycin (PS), fetal bovine serum (FBS), 1\u0026times; phosphate-buffered saline, normal saline (NS), 3, 3'-Diaminobenzidine (DAB), 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and Avertin were obtained from Dowobio, China. DNaseI and papain were acquired from Solarbio, China. Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) was procured from Macklin, China. Antibodies against 4-hydroxynonenal (4HNE; Bioss, China), class III β-Tubulin III (Tuj1, Immunoway, USA), Caspase-3 (Cap3; Wanleibio, China), and connexin 43 (Cx43; Proteintech, China), were obtained.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Synthesis and preparation of nanoparticles and hydrogels\u003c/h2\u003e\u003cp\u003eHydrogels containing therapeutic nanoparticles were synthesized based on a published article\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. First, DHA (100 mg), EDC (160 mg), NHS (80 mg), and DMAP (25 mg) were added into DMSO (15 mL) and mixed thoroughly under nitrogen protection at room temperature for 3 h. Subsequently, PLL (900 mg) dissolved in DMSO (10 mL) was added to the reaction system and mixed thoroughly for 24 h to obtain DHA-PLL nanoparticles (DP NP). Second, SA (83 mg), EDC (320 mg), NHS (160 mg), and DMAP (50 mg) were added into DMSO (10 mL) and mixed thoroughly for 3 h. Additionally, TPL (120 mg) dissolved in DMSO (5 mL) was added to the reaction system and mixed thoroughly for 24 h. Next, DP NP (200 mg) dissolved in DMSO (10 mL) was added into the reaction system and mixed thoroughly for another 24 h to obtain DHA-PLL-TPL nanoparticles (DT NP). Third, PLXM407 (3 g) was dissolved in NS (10 mL) at 4 ℃. Then, DP NP (500 mg) or DT NP (500 mg) was added and mixed thoroughly to obtain DP NP\u0026ndash;Hydrogel or DT NP\u0026ndash;Hydrogel. Fourier transform infrared spectrometer, nuclear magnetic resonance (NMR) spectroscopy, dynamic light scattering (DLS), scanning electron microscope (SEM), transmission electron microscope (TEM), and ultraviolet spectrophotometer (UVS) were used to characterize the nanoparticles and hydrogels.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Isolation, culture, and identification of primary cells\u003c/h2\u003e\u003cp\u003ePrimary spinal cord neuron (PSCN) cells were obtained from the spinal cord of fetal rats at 14-day gestation. After anesthesia through intraperitoneal injection of Avertin (60 \u0026micro;L/g), fetal rats were obtained and temporarily stored in 75% alcohol at 4 ℃. The spinal cord samples were then carefully separated under sterile conditions, cut into approximately 1 mm tissue blocks, and incubated with DNaseI (100 U/mL) and papain (1 mg/mL) at 37 ℃ for 30 min. The reaction was stopped with FBS, and the suspension was filtered through a 70-mesh filter membrane to obtain PSCN. The spinal cells were then resuspended in DMEM supplemented with 5% FBS and 1% PS and cultured at 37 ℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e for 4 h. The medium was then replaced with a special Neurobasal medium containing 2% B27 and 1% Glu complete medium for 24 h. Subsequently, primary cells were fluorescently stained with Tuj1-antibodies (1:500, anti-rat) to identify PSCN.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Effects of different reagents on PSCN\u003c/h2\u003e\u003cp\u003eWell-growing PSCN cells (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e/mL) were seeded into 96-well plates and cultured overnight at 37 ℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e. PSCN cells were then treated with 0\u0026ndash;1024 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 0\u0026ndash;1024 \u0026micro;g/mL TPL, DP NP, or DT NP for 24 h. Cell viability was assessed using the CCK-8 assay. Subsequently, a suitable concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (250 \u0026micro;M) was used to induce oxidative stress injury in PSCN \u003cem\u003ein vitro\u003c/em\u003e, as previously described\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. \u003cem\u003eIn vitro\u003c/em\u003e experiments of PSCN\u003c/h2\u003e\u003cp\u003eWell-growing PSCN cells (5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e/mL) were seeded onto glass slides and cultured overnight at 37 ℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e. \u003cem\u003eIn vitro\u003c/em\u003e experiment groups were divided into a normal group, a model group (250 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), a model\u0026thinsp;+\u0026thinsp;TPL group (120 \u0026micro;g/mL), a model\u0026thinsp;+\u0026thinsp;DP NP group (400 \u0026micro;g/mL), and a model\u0026thinsp;+\u0026thinsp;DT NP group (400 \u0026micro;g/mL). After treatment with the respective compounds for 24 h, the neuronal cells on the glass slides were fixed with 4% paraformaldehyde and stained with different antibodies and DAPI. Imaging was performed using an inverted fluorescence electron microscope (IFEM).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Establishment and hydrogel therapy of SCI rat models\u003c/h2\u003e\u003cp\u003eThe animal models and hydrogel treatment used in this study were ethically approved by the Ethics Committee of Chongqing University Central Hospital (Approval No. 2502001). All Sprague-Dawley (female, 200\u0026ndash;220 g, 8 weeks) rats were kept with standard water, diet, and a 12-h circadian rhythm. Before the establishment of SCI models, all rats were kept for a week to acclimate to their new environment. The rats were randomly divided into six groups, each including 6 rats: (1) Sham group (Sham); (2) Model group; (3) TPL group (12 mg/kg); (4) Non\u0026ndash;Hydrogel group (100 \u0026micro;L/kg, 30% w/v); (5) DP NP\u0026ndash;Hydrogel group (100 \u0026micro;L/kg, 50 mg/mL); (6) DT NP\u0026ndash;Hydrogel group (100 \u0026micro;L/kg, 50 mg/mL). All SCI rat models were performed according to the weight-drop method in aseptic conditions\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Briefly, after intraperitoneal anesthesia (Avertin, 10 mL/kg), the spinal cord of the 10th thoracic vertebra was carefully exposed and struck with a 15 g weight (2.5 mm in diameter) dropped from a height of 10 cm. Tail spasms and bilateral hindlimb paralysis were observed, revealing the successful establishment of the SCI models. Subsequently, 20 \u0026micro;L Non\u0026ndash;Hydrogel, DP NP\u0026ndash;Hydrogel, or DT NP\u0026ndash;Hydrogel was injected into the damaged spinal cord in the corresponding hydrogel groups, and TPL was injected through the tail vein. After suturing the wound, all operated rats were placed in a 37 ℃ environment for recovery. Artificial urination was performed every two days, and the recovery status of the rats was observed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Hematoxylin and eosin (H\u0026amp;E) and immunohistochemical staining (IHC) staining\u003c/h2\u003e\u003cp\u003eParaffin sections (4 \u0026micro;m) of spinal cord samples underwent dewaxing, dehydration, membrane permeabilization, and antigen retrieval, then were sealed with 5% bovine serum albumin for 1 h. H\u0026amp;E staining was used to observe SCI in rat models. Subsequently, the tissue sections were incubated overnight at 4 ℃ with the following primary antibodies: 4HNE (anti-rabbit, 1:200), Cap3 (anti-rabbit, 1:200), Tuj1 (anti-mouse, 1:200), and Cx43 (anti-rabbit, 1:1000). Subsequently, tissue sections were stained with DAB agent to observe the degree of brown and yellow color in the tissues. Immunohistochemistry images were obtained from regions of interest in different groups using IFEM. The number of brown-stained cells was quantified using ImageJ software. Six samples from each group were utilized for image acquisition.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Quantitative real-time PCR (RT-qPCR)\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from fresh spinal cord samples through the Trizol method, and RNA concentration was measured at 260 nm with an ultraviolet-visible light spectrophotometer. A FastKing cDNA First Strand Synthesis Kit (Thermo, USA) was used to synthesize cDNA from 2 \u0026micro;g of total RNA per sample. RT-qPCR was performed using SYBR Green PCR Master Mix (Dowobio, China). The expression level of glyceraldehyde 3- phosphate dehydrogenase (GAPDH) was used as the internal control. Each reaction was run in triplicate. All primers, including Glutathione peroxidase algal (GPXH) (5'-GCTCCATGCACGAGTTTTCC-3', 5'-GTTTACTTCGGTCTTGCCTCACT-3'), Cap3 (5'-TACTCTACCGCACCCGGTTA-3', 5'-CGCGTACAGTTTCAGCATGG-3'), Tuj1 (5'-CAAGGTGCGTGAGGAGTATCR-3', 5'-CGGAAGCAGATGTCGTAGAG-3'), and Cx43 (5'-GAGTTTGCCTAAGGCGCTC-3', 5'-AGGAGTTCAATCACTTGGCG3-3'), were customized by Sangon Biotech, China. The experimental results were expressed as 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e values, with GAPDH used as the internal reference gene. Each reaction was performed in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Safety studies \u003cem\u003ein vivo\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eDrug distribution of DP NP\u0026ndash;Hydrogel and DT NP\u0026ndash;Hydrogel was observed in SCI rats, while normal rats served as the negative control group. Fluorescence in the heart, liver, spleen, lung, and kidney was observed using a live animal imaging system. The \u003cem\u003ein vivo\u003c/em\u003e safety of the different hydrogels\u0026mdash;including Non\u0026ndash;Hydrogel, DP NP\u0026ndash;Hydrogel, and DT NP\u0026ndash;Hydrogel\u0026mdash;was assessed through blood component analysis, hemolysis testing, and H\u0026amp;E staining of major organs. Briefly, a 10-fold high dose of DP NP\u0026ndash;Hydrogel and DT NP\u0026ndash;Hydrogel (500 mg/mL) were injected into the spinal cord of normal rats, and the animals were observed for 28 days after recovery. After anesthesia with Avertin, blood was extracted from the inner canthus using ETDA anticoagulant tubes and analyzed using an animal blood analyzer for blood routine hematological and biochemical indices. Following euthanasia by spinal dislocation, all organs\u0026mdash;including heart, liver, spleen, lung, and kidney were harvested and fixed in 4% paraformaldehyde for H\u0026amp;E staining. Subsequently, blood was obtained from untreated normal rats through inner canthus extraction. Then, 50 \u0026micro;L of blood was mixed thoroughly with 50 \u0026micro;L of different solutions\u0026mdash;including double distilled water (DDW), NS, DP NP, and DT NP\u0026mdash;and centrifugated at 1500 rpm for 10 min at room temperature to assess hemolysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were performed using the Statistical Package for the Social Sciences (version 22) and Origin (version 20) software. Data were analyzed using one-way analysis of variance and independent sample t-tests. Results are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean. Statistical significance was defined as *\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05, **\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01 and ***\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Characterization of DP NP and DT NP nanoparticles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;As biocompatible nanoparticles, the nano framework (DP NP) was first formed by the dehydration condensation of DHA and PLL. Subsequently, DT NP was synthesized through the coupling of SA. DLS analysis displayed that the particle sizes of DP NP and DT NP were 157.42 \u0026plusmn; 1.33 and 164.29 \u0026plusmn; 1.15 nm, respectively (Figs. 1A-B, Table 1). The surface zeta potential was 44.43 \u0026plusmn; 0.42 and -20.67 \u0026plusmn; 0.60 mV, respectively (Fig. 1C, Table 1). PDI values were 0.205 \u0026plusmn; 0.01 and 0.213 \u0026plusmn; 0.02, respectively (Table 1). SEM and TEM results also revealed that DP NP and DT NP were solid, spherical-like nanoparticles (Figs. 1D-E). Chemical binding analysis by NMR revealed that the amine group on PLL and the hydroxyl group were conjugated with the carboxyl group of DHA and SA (Fig. 1F). Size stability analysis by DLS indicated that DP NP and DT NP remained stable in four different liquids after 24 h of incubation (Figs. 1G-H, Table 2). Drug loading measurement by UVS indicated that the drug loading rate of DT NP was approximately 23%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Characterization of DP NP and DT NP hydrogels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;PLXM407 is a temperature-sensitive, biocompatible hydrogel material. The hydrogel samples demonstrated gel formation at temperatures ranging from 4 to 37 ℃ (Fig. 2A). SEM results displayed that the dry hydrogel exhibited a loose and porous structure, with many spherical nanoparticles located on the surface (Fig. 2B). Injection experiments demonstrated that these hydrogels exhibited excellent injectability at 4 ℃ (Fig. 2C). The viscosity experiment revealed that the hydrogels could effectively adhere to a weight of 5 g (Fig. 2D). The swelling experiment displayed that the hydrogels achieved their maximum swelling capacity after 5 min in DDW (Fig. 2E, Table 3). The degradation experiment also indicated that different hydrogels effectively degraded over time in DDW (Fig. 2F, Table 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Identification and cell activity of PSCN\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;To evaluate the cytotoxic effects of DP NP and DT NP \u003cem\u003ein vitro\u003c/em\u003e, PSCN cells were obtained from fetal rats at gestation day 14 and cultured for 7 days (Fig. 3A). The morphological result revealed that PSCN developed long synaptic-like extensions (Fig. 3B). The specific surface protein of spinal cord neuron cells, Tuj1, was highly expressed in PSCN and clearly observed by immunofluorescence (IF) staining. These results indicated that the primary cells were successfully obtained and suitable for subsequent experiments. Subsequently, different concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, TPL, DP NP, and DT NP were resuspended in a medium and co-cultured with PSCN for 24 h to assess cell viability. As illustrated in Fig. 3D-F and Table 5, PSCN exhibited viability at 2 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, but cell activity decreased as the concentration increased. The result led us to select 250 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as the appropriate concentration for the \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eoxidative\u003cem\u003e\u0026nbsp;\u003c/em\u003estress model. Additional CCK-8 assays indicated that TPL, DP NP, and DT NP, at concentrations ranging from 0\u0026ndash;1024 \u0026micro;g/mL, displayed no significant cytotoxicity after 24 h of co-culture with PSCN, with cell viability remaining at approximately 100%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 \u003cem\u003eIn vitro\u003c/em\u003e experiments of PSCN treated with DP NP and DT NP nanoparticles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the antioxidant and nerve regeneration effects of DP NP and DT NP on PSCN models, IF staining of 4HNE, Cap3, Tuj1, and Cx43 antibodies was performed and observed using the IFEM. After H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment, PSCN indicated a significant increase in oxidative stress and cell apoptosis (Figs. 4A, B, E, Table 6) and a marked decrease in nerve regeneration and junction formation (Figs. 4C\u0026ndash;E, Table 6). However, DT NP indicated superior antioxidant and neuroprotective effects compared to TPL and DP NP. These results suggested that TPL reduced ROS levels, oxidative stress, and cell apoptosis induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e through scavenging free radicals and that DHA in both DP NP and DT NP also promoted nerve cell growth.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 \u003cem\u003eIn vivo\u003c/em\u003e experiments of SCI rat models treated with DP NP and DT NP hydrogels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo study the effect of DP NP and DT NP on antioxidation and nerve recovery in SCI rat models, hydrogels containing DP NP or DT NP were injected into the site of SCI for local drug release over 28 days (Figs. 5A-B). After 28 days, all spinal cord tissues from the injury site were collected for tissue staining and RT-qPCR tests. H\u0026amp;E staining results revealed that the spinal cord trauma remained evident, and none of the drug treatments effectively restored the normal spinal cord structure (Fig. 5C). However, both IHC staining and RT-qPCR analysis indicated that DT NP\u0026ndash;hydrogel could more effectively reduce oxidative stress and cell apoptosis and promoted nerve recovery and connection at the injury site compared to TPL and DP NP\u0026ndash;Hydrogel (Figs. 5D\u0026ndash;F, Table 7-8). Notably, TPL administered by intravenous injection displayed no significant effect on alleviating the injury site, likely due to the reduced local drug concentration. However, DP NP\u0026ndash;Hydrogel and DT NP\u0026ndash;Hydrogel, when administered locally, effectively alleviated injury progression and promoted recovery in SCI rat models. These results confirm that local administration of DT NP\u0026ndash;Hydrogel has the potential for application in SCI treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Safety studies \u003cem\u003ein vivo\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The safety of DP NP and DT NP hydrogels was effectively evaluated in SCI rat models using a high dose of DP NP and DT NP (10\u0026times; treatment dose) administered locally. Subsequently, blood and organs were collected, analyzed with a blood detector, and stained H\u0026amp;E staining, respectively. Blood test results indicated that no significant change in blood composition and blood biochemistry in high-dose groups was found compared to normal groups (Fig. 6A, Table 9). H\u0026amp;E staining results revealed that no abnormal structure was observed in the examined organs (Fig. 6B). The hemolytic test also indicated that the reagent components of different hydrogels did not cause intravascular hemolysis (Fig. 6C). Organ fluorescence imaging also displayed non-specific enrichment in normal organs following local administration (Fig. 6D). These experiments revealed that the local administration of hydrogels was safe.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn recent clinical treatment, it is crucial to alleviate the progression of SCI and avoid secondary injuries\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In SCI, excessive oxidative stress and decreased acidic pH values are vital factors that contribute to secondary injuries\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, the clinical application of long-lasting local drugs at the SCI site remains insufficient. To achieve sustained drug release targeting ROS and acid in the injured site, this study focused on biocompatible nanoparticles (DT NP) based on the chemical connection between amino groups from PLL, hydroxyl groups from TPL, and carboxylic acids from SA. Subsequently, these DT NP were thoroughly mixed with the thermosensitive hydrogel PLXM407 at 4 ℃ to form a suspension, and at over 33 ℃ to form a gel\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Overall, this study aimed to employ a local nanoparticle\u0026ndash;hydrogel system for the sustained release of effective drugs to alleviate the injury in SCI rat models.\u003c/p\u003e\u003cp\u003eInevitably, excessive oxidative stress, characterized by elevated ROS and reduced antioxidant defense, is a notably significant pathological process in SCI\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In spinal cord tissue with a high metabolic rate and weak antioxidant capacity, biological macromolecules, organelles, and cellular functions are extensively damaged by excessive ROS free radicals\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Moreover, these ROS can quickly bind to polyunsaturated fatty acids in biological membrane structure to form highly reactive peroxyl radicals, initiating a chain reaction with other polyunsaturated fatty acids and causing progressive oxidative damage at the injury site. 4HNE is a highly reactive and toxic aldehyde and serve as an important marker of lipid peroxidation\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Even more concerning, the myelin sheath of the spinal cord\u0026mdash;a type of neural tissue\u0026mdash;is particularly rich in lipid components, such as cholesterol, glycosphingolipids, and ceramides, making it particularly susceptible to excessive ROS\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Recently, many biological and chemical drugs have been used for direct antioxidant treatment. For example, GSH peroxidase can quickly convert harmful superoxide free radicals into relatively harmless substances, induced by gene regulation for high expression\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Similarly, TPL, a strong and low-cost antioxidant molecule, can also effectively capture free radicals through its ring structure, thereby reducing excessive ROS\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In this study, TPL in DT NP demonstrated enhanced antioxidant capacity in both PSCN cell models and SCI rat models, suggesting that the long-acting antioxidant stress property of the DT NP\u0026ndash;Hydrogel system may play a potential role in treating neurotraumatic diseases.\u003c/p\u003e\u003cp\u003eAs a form of programmed cell death, apoptosis is mediated by cysteine aspartate-specific proteases and is prominently observed in SCI\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Recently, the biological regulation between ROS and apoptosis has also become a major focus in life sciences. Oxidized low-density lipoprotein and peroxynitrite free radicals induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e have been found in different types of apoptosis\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Furthermore, during ischemia-reperfusion injury of the spinal cord, a reduction in mitochondrial membrane potential and increased ROS generation lead to mitochondrial dysfunction, excessive release of cytochrome c, and strong activation of Cap3, ultimately causing neural cell apoptosis\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Tuj1 is a neuron-specific marker that plays a significant role in SCI research and treatment\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. It plays a critical regulatory role in the structural composition of neurons, intracellular substance transport, axonal growth, and neuronal movement\u003csup\u003e\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Cx43 is also a key intercellular communication protein, especially in neural tissues\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. In SCI, Cx43 participates in inflammation, ferroptosis, and apoptosis of nerve cells\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. In this study, high expression levels of Cap3 and Cx43, along with reduced levels of Tuj1, were significantly observed and were effectively restored with DT NP\u0026ndash;Hydrogel treatment.\u003c/p\u003e\u003cp\u003eAs three-dimensional polymeric networks, both biological and chemical hydrogels can effectively absorb and retain large amounts of water due to the presence of numerous hydrophilic groups, such as amino, hydroxyl, carboxylic, and ether-oxygen groups. Different biocompatible hydrogels have been commonly prepared for local and long-acting treatment in drug delivery, tissue engineering, and wound healing\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. In SCI repair, hydrogels are commonly used to bridge gaps between severed spinal cord tissue, prevent excessive leakage of inflammatory factors, and reduce secondary mechanical injury to the spinal cord\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Moreover, these hydrogels can incorporate various active drugs or therapeutic agents through random distribution, polarity-based encapsulation, electrostatic adsorption, and chemical bonding according to the physicochemical properties of the drug\u003csup\u003e\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Research has revealed that drug-loaded hydrogels can effectively provide a localized therapeutic microenvironment for the removal of harmful substances and the promotion of nerve regeneration. In this study, TPL for ROS clearance, PLL for acid neutralization, and DHA for neurotrophy were all synthesized into biocompatible nanoparticles (DT NP), which were thoroughly mixed with PLXM407 liquid at 4 ℃ to form a DT NP\u0026ndash;Hydrogel at 37 ℃. Due to these characteristics, DT NP\u0026ndash;Hydrogel can better fill the gaps at the SCI site and enable continuous drug release.\u003c/p\u003e\u003cp\u003eThese experimental results effectively demonstrated that DT NP\u0026ndash;Hydrogel could relieve ROS damage and promote neural recovery under long-term effects in SCI rat models. Additionally, local administration of the DT NP\u0026ndash;Hydrogel did not cause abnormal accumulation of nanoparticles in organs, structural damage to organs, changes in blood components, or hemolysis. Consequently, these findings provide new strategies to complement the clinical treatment of SCI.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, DT NP nanoparticles were demonstrated to play an important role in removing ROS, inhibiting apoptosis, and promoting nerve regeneration in PSCN cell models induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Moreover, DT NP\u0026ndash;Hydrogel could enhance the antioxidant capacity of the injured area in SCI rat models. This therapeutic effect was mainly mediated by the ROS-removing capability of TPL and partly by the acidic neutralization of PLL and the neurotrophic properties of DHA. These results present potential value for developing long-acting sustained-release hydrogels for local therapeutic strategies in SCI.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are extremely grateful for the project guidance from Ke Li, PhD, Chongqing Medical University. And we also thank Home for Researchers editorial team (www.home-for-researchers.com) for language editing service.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was supervised and approved by Laborator Animal Welfare and Ethics Commit of Chongqing University Central Hospital, Chongqing (IACUC Issue No.2502001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGushang Xia, and Renjie Shuai contributed to experiments, data interpretation and writing; Ze Li, Changlin Tang, Yaowen Zhang, Qingli Kong, Wanyou Li and Fangfang Ma contributed to visualization; Xianglin Li and Yan Du contributed with resources, writing - review \u0026amp; editing, project administration; and Yan Du contributed with funding acquisition. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by Project of Chongqing Key Laboratory of Emergency Medicine, Chongqing (No. 2023KFKT02).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations of competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCourtine G, Sofroniew MV (2019) Spinal cord repair: advances in biology and technology. 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A prevascularized nerve conduit based on a stem cell sheet effectively promotes the repair of transected spinal cord injury. 101. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.actbio.2019.10.042\u003c/span\u003e\u003cspan address=\"10.1016/j.actbio.2019.10.042\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHang L et al (2016) A Hydrogel Bridge Incorporating Immobilized Growth Factors and Neural Stem/Progenitor Cells to Treat Spinal Cord Injury. 5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/adhm.201500810\u003c/span\u003e\u003cspan address=\"10.1002/adhm.201500810\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 9 are available in the Supplementary Files section.\u003c/p\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Spinal cord injury, Oxidative stress, Hydrogel, Tempol","lastPublishedDoi":"10.21203/rs.3.rs-6993042/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6993042/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSpinal cord injury (SCI) is characterized by excessive reactive oxygen species (ROS) and an acidic pH, which hinder repair. This study aimed to develop and evaluate a local therapeutic strategy using a temperature-sensitive hydrogel loaded with antioxidant and acid-neutralizing nanoparticles to form a DT NP\u0026ndash;Hydrogel designed to clear ROS, neutralize acidity, and promote neurological recovery after SCI. Both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, DT NP and the local administration of DT NP\u0026ndash;Hydrogel significantly reduced ROS levels (4-hydroxynonenal), cell apoptosis (caspase-3), and connexin 43 expressions in primary spinal cord neuron models induced by hydrogen peroxide, and in SCI rat models caused by fall injury, while increasing class III β-tubulin expression. Moreover, our findings also revealed that the locally administered DT NP\u0026ndash;Hydrogel exhibited no side effects, making it a promising therapeutic alternative for SCI.\u003c/p\u003e","manuscriptTitle":"A reactive oxygen species- and acidic pH-responsive hydrogel loaded with Tempol and poly-lysine enhances spinal cord injury repair in rat models","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-17 19:31:30","doi":"10.21203/rs.3.rs-6993042/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"999a43ac-1a1b-4bc8-8ab4-812e6e774fba","owner":[],"postedDate":"July 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-21T20:08:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-17 19:31:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6993042","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6993042","identity":"rs-6993042","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}