A Surface-Engineered Cerium Oxide Nanozyme Functionalized with L-Theanine for Redox and Inflammatory Modulation in Neuropathic Pain

Abstract

Background

Neuropathic pain is associated with interacting oxidative stress-, neuroinflammation-, and apoptosis-related processes, whereas current therapies mainly provide symptomatic relief without fully addressing these pathological components. To overcome this limitation, we developed a surface-engineered nanozyme platform composed of poly(acrylic acid)-coated cerium oxide nanoparticles functionalized with L-theanine (CPT), aiming to combine the redox activity of nanoceria with the bioactivity of a neuroprotective small molecule.

Results

Physicochemical characterization demonstrated that L-theanine functionalization did not alter the crystalline structure or mixed Ce³⁺/Ce⁴⁺ valence state of nanoceria. In H₂O₂-challenged Schwann cells, CPT significantly reduced intracellular reactive oxygen species, improved cell viability, and suppressed the expression of inflammation- and apoptosis-related genes more effectively than free L-theanine or nanoparticle controls. In a rat chronic constriction injury model, repeated administration of CPT improved mechanical and thermal hypersensitivity. These behavioral improvements were accompanied by reduced spinal oxidative and nitrosative stress, partial restoration of endogenous antioxidant defenses, suppression of pro-inflammatory cytokines, and decreased caspase-3 activity.

Conclusion

These findings suggest that CPT attenuates oxidative stress-, inflammatory cytokine-, and caspase-3-associated responses in both H₂O₂-challenged Schwann cells and a rat CCI model of neuropathic pain. Rather than establishing a complete mechanistic pathway, the present study provides preliminary evidence that surface-functionalized nanoceria may serve as a nanozyme-based candidate for redox- and inflammation-associated modulation in neuropathic pain.

Graphical Abstract

Background

Neuropathic pain (NP), caused by injury or dysfunction of the somatosensory nervous system, remains a major clinical challenge due to its chronic nature, limited responsiveness to conventional analgesics, and substantial impact on quality of life.¹ Patients typically experience spontaneous pain, mechanical allodynia, and thermal hyperalgesia, often accompanied by sleep disturbance, emotional distress, and functional impairment.² Current pharmacological treatments, including gabapentinoids, antidepressants, and opioids, provide incomplete symptomatic relief and are frequently associated with adverse effects and tolerance.^3,4 Importantly, these therapies largely fail to target the underlying pathological mechanisms that sustain neuropathic pain, underscoring the need for mechanism-based therapeutic strategies.

Oxidative stress and neuroimmune dysregulation have been increasingly recognized as important contributors to neuropathic pain pathophysiology, although their relative contribution may vary across anatomical sites, disease stages, and experimental models.^5,6 After peripheral nerve injury, redox imbalance and inflammatory signaling can occur locally at the injured peripheral nerve, within dorsal root ganglia, and in spinal cord circuits involved in nociceptive processing. Excessive generation of reactive oxygen and nitrogen species, disruption of redox homeostasis, and mitochondrial dysfunction have been associated with neuronal hyperexcitability, synaptic reorganization, and central sensitization.^7–9 In parallel, activation of immune and glial responses and increased production of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, may further amplify nociceptive signaling and impair functional recovery 5,6. The convergence of oxidative stress, inflammatory signaling, and apoptosis-associated responses therefore represents a relevant therapeutic target in neuropathic pain, although these processes should not be interpreted as the sole determinants of neuropathic pain initiation or maintenance.

L-theanine (γ-glutamylethylamide), a non-proteinogenic amino acid abundant in green tea, has attracted attention for its neuroprotective properties and favorable safety profile.^10,11 L-theanine has been shown to enhance endogenous glutathione synthesis, suppress lipid peroxidation, and inhibit redox-sensitive inflammatory pathways. In experimental models of peripheral nerve injury and neuropathy, L-theanine attenuates behavioral hypersensitivity, reduces oxidative stress markers, and modulates inflammatory responses.^12,13 However, its hydrophilic nature and rapid systemic clearance limit tissue retention and therapeutic efficacy when administered in free form, posing a barrier to clinical translation.

Cerium oxide nanoparticles (CeO₂ NPs, nanoceria) represent a class of regenerative nanozymes with unique and sustained antioxidant activity mediated by reversible Ce³⁺/Ce⁴⁺ redox cycling and oxygen vacancy–dependent ROS scavenging.¹⁴ Nanoceria mimic endogenous antioxidant enzymes and modulate redox-sensitive signaling pathways involved in inflammation and apoptosis.^15,16 In neural injury models, cerium oxide–based nanomaterials have demonstrated neuroprotective effects and functional improvement,^17,18 and recent reviews have highlighted nanozymes as promising therapeutic platforms for neuropathic pain by targeting oxidative stress and neuroinflammation.¹⁹ Nevertheless, challenges related to delivery efficiency, sustained bioactivity, and integration with bioactive molecules remain unresolved. Despite growing interest in nanozyme-based antioxidant strategies, few studies have systematically integrated nanozyme redox buffering with bioactive small molecules to address oxidative stress-, inflammation-, and apoptosis-associated responses in neuropathic pain models.

In this study, we developed a surface-functionalized nanoceria platform in which poly(acrylic acid)-coated cerium oxide nanoparticles were further modified with L-theanine (CPT). The design rationale was to preserve the redox-active properties of nanoceria while introducing a bioactive small molecule with reported antioxidant and anti-inflammatory effects. We hypothesized that this hybrid formulation would provide broader protection against oxidative and inflammation-associated injury than either component alone. To test this hypothesis, we characterized the physicochemical properties of CPT, examined its cytoprotective and gene-modulatory effects in H₂O₂-challenged RSC96 Schwann cells as an in vitro peripheral glial oxidative-injury model, and evaluated its antinociceptive and spinal biochemical effects in a rat chronic constriction injury model. These cellular and in vivo experiments were designed to provide complementary evidence of CPT-associated redox and inflammatory modulation, rather than to establish a complete linear mechanism across all anatomical compartments involved in neuropathic pain.

Methods

Materials

Cerium(III) nitrate hexahydrate (Ce(NO₃)₃·6H₂O; Sigma-Aldrich/Merck, Cat. No. 238538), potassium carbonate (K₂CO₃; Sigma-Aldrich/Merck, Cat. No. P5833), poly(acrylic acid) (PAA; Sigma-Aldrich/Merck, Cat. No. 323667), ammonium hydroxide solution (NH₄OH; Sigma-Aldrich/Merck, Cat. No. 221228), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC; Sigma-Aldrich/Merck, Cat. No. E7750), N-hydroxysuccinimide (NHS; Sigma-Aldrich/Merck, Cat. No. 130672), L-theanine (Sigma-Aldrich/Merck, Cat. No. T6576), hydrogen peroxide solution (H₂O₂; Sigma-Aldrich/Merck, Cat. No. H1009), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma-Aldrich/Merck, Cat. No. D6883), and WST-1 cell proliferation reagent (Roche/Sigma-Aldrich/Merck, Cat. No. 11644807001) were used according to the manufacturers’ instructions unless otherwise stated. All aqueous solutions were prepared using deionized water.

Synthesis of Cerium Oxide Nanoparticles (CeO₂ NPs, C)

Cerium oxide nanoparticles were synthesized by a controlled precipitation and thermal treatment method. Briefly, Ce(NO₃)₃·6H₂O (0.02 M) and K₂CO₃ (0.03 M) were dissolved separately in deionized water. The cerium nitrate solution (50 mL) and potassium carbonate solution (20 mL) were added dropwise into 100 mL of distilled water under vigorous stirring while maintaining the pH at approximately 6. The resulting suspension was transferred to a sealed Teflon-lined stainless-steel autoclave and aged hydrothermally at 220 °C for 2.5 h, followed by calcination at 600 °C for 3 h to obtain crystalline CeO₂ nanoparticles. The final product was dried at 65 °C and stored at room temperature.

Synthesis of Poly(Acrylic Acid)-Coated Cerium Oxide Nanoparticles (CeO₂–PAA, CP)

Poly(acrylic acid)-coated CeO₂ nanoparticles were prepared to enhance colloidal stability and provide functional groups for subsequent conjugation. Ce(NO₃)₃·6H₂O and PAA were mixed in a total volume of 5 mL and added dropwise into 36 mL of 25% NH₄OH under continuous stirring at room temperature. The reaction was allowed to proceed for 24 h. The resulting dispersion was centrifuged twice at 4000 rpm for 30 min to remove aggregates, and the supernatant was dialyzed (MWCO 6–8 kDa) against deionized water for 48 h. Purified CeO₂–PAA nanoparticles were lyophilized for further use.

Conjugation of L-Theanine (CeO₂–PAA–Theanine, CPT)

L-theanine was covalently conjugated to CeO₂–PAA nanoparticles via carbodiimide-mediated amidation. CeO₂–PAA nanoparticles (1.5 mg/mL) were dispersed in 0.1 M MES buffer (pH 5.5). Carboxyl groups were activated using EDC and NHS for 1.5 h at room temperature under light-protected conditions. L-theanine (10 mg) was then added, and the reaction mixture was incubated for 20 h in the dark. Unreacted reagents were removed by dialysis (MWCO 6–8 kDa) against distilled water. The pH of the final dispersion was adjusted to 7.2–7.4, and the product was lyophilized and stored at room temperature. Bare CeO₂, PAA-coated CeO₂, and L-theanine–conjugated nanoparticles are referred to as C, CP, and CPT, respectively.

Physicochemical Characterization

Nanoparticle morphology and crystalline structure were examined using transmission electron microscopy (TEM) and selected-area electron diffraction (SAED). Hydrodynamic diameter and zeta potential were measured by dynamic light scattering (DLS) at 25 °C in deionized water, phosphate-buffered saline (PBS), and complete culture medium. Fourier transform infrared (FTIR) spectroscopy was used to confirm surface functionalization and L-theanine conjugation. X-ray diffraction (XRD) analysis was performed to verify crystal phase integrity. X-ray photoelectron spectroscopy (XPS) was used to analyze surface elemental composition and cerium oxidation states (Ce³⁺/Ce⁴⁺).

L-Theanine Incorporation and in vitro Release-Associated Analysis

The apparent L-theanine incorporation content of CPT nanoparticles was quantified using UV–visible spectroscopy based on calibration curves generated with L-theanine standards. For in vitro release-associated analysis, CPT nanoparticles were incubated in phosphate-buffered saline (PBS) at pH 7.4 or pH 5.0. At predetermined time points (0.5, 1, 2, 4, 6, and 8 h), supernatants were collected and replaced with fresh buffer. L-theanine-associated signal in the supernatant was quantified spectrophotometrically, and cumulative release-associated profiles were calculated.

Cell Culture

RSC96 rat Schwann cells were obtained commercially from the American Type Culture Collection (ATCC, Manassas, VA, USA; ATCC^® CRL-2765™). Cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine at 37 °C in a humidified atmosphere containing 5% CO₂. Because this commercially available established cell line was used, no additional institutional ethics committee or institutional review board approval was required for the in vitro cell culture experiments.

Cell Viability Assay

Cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well and allowed to adhere overnight. Oxidative stress was induced by exposure to hydrogen peroxide (H₂O₂, 200 μM) for 30 min. After removal of H₂O₂, cells were washed and incubated with fresh medium containing free L-theanine (50 μM), C, CP, or CPT for 24 h. CPT was applied at 117 μg/mL to provide an equivalent L-theanine dose based on its loading capacity, and C and CP were applied at the same mass concentration. Cell viability was assessed using the WST-1 assay and expressed as a percentage relative to untreated controls.

Intracellular ROS Measurement

Intracellular reactive oxygen species (ROS) levels were measured using the DCFH-DA assay. Following oxidative stress induction and treatment, cells were incubated with DCFH-DA (20 μM) for 45 min at 37 °C in the dark. Fluorescence intensity was measured at excitation/emission wavelengths of 485/535 nm and normalized to untreated controls.

Quantitative Real-Time PCR

RSC96 cells were seeded in 6-well plates at a density of 3×10⁵ cells per well. After oxidative stress induction and treatment with free L-theanine, C, CP, or CPT for 24 h, total RNA was extracted using TRIzol reagent. Complementary DNA was synthesized, and quantitative real-time PCR was performed using SYBR Green chemistry. Gene expression related to oxidative stress (Nrf2, HO-1), inflammation (TNF-α, IL-1β, IL-6), and apoptosis (caspase-3) was normalized to GAPDH and analyzed using the 2⁻ΔΔCt method.

Animal Model of Peripheral Neuropathic Pain

All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University (approval no. 20210438) and were conducted in accordance with the institutional guidelines for the care and use of laboratory animals. No separate animal permit or animal license number was issued by the institution. Adult male Wistar rats (body weight: approximately 250 g) were randomly assigned to experimental groups (n = 6 per group). The sample size (n = 6/group) was selected based on prior CCI studies reporting robust effect sizes in behavioral and biochemical endpoints and was considered sufficient to detect group differences with the present design. Chronic constriction injury (CCI) was established to induce peripheral neuropathic pain. For the surgical procedure, anesthesia was induced with isoflurane (4–5% in oxygen) and maintained at 1.5–2.5% via inhalation throughout surgery. Adequate depth of anesthesia was confirmed by the absence of the pedal withdrawal reflex before surgical manipulation. The right sciatic nerve was exposed at the mid-thigh level, and four loose ligatures (4–0 chromic gut) were placed around the nerve with approximately 1 mm spacing to induce partial nerve injury without transection. Sham-operated rats underwent identical surgical exposure without nerve ligation. To minimize postoperative pain, meloxicam (1–2 mg/kg, subcutaneously) was administered perioperatively and continued for up to 48–72 h in accordance with the approved animal protocol. Animals were monitored daily for general health, pain-related behavior, wound condition, and postoperative recovery. Drug administration began on postoperative day (POD) 1. CeO₂–PAA–Theanine nanoparticles (CPT) were administered intraperitoneally once daily from POD 1 to POD 14 at a dose of 135 mg/kg. Based on the measured drug loading capacity (DLC = 7.42%), this dose corresponded to a theanine-equivalent dose of 10 mg/kg. For a 250 g rat, the injected CPT mass was approximately 33.8 mg per animal. To ensure fair comparison among formulations, cerium oxide nanoparticles (C) and CeO₂–PAA nanoparticles (CP) were administered at the same nanoparticle mass dose (135 mg/kg). The free L-theanine (Th) group received L-theanine at a dose of 10 mg/kg, matched to the theanine-equivalent dose of CPT. Vehicle-treated animals received phosphate-buffered saline (PBS) at an equivalent injection volume (1 mL/kg). Animals were randomized prior to surgery, and all subsequent assessments were performed by investigators blinded to group allocation.

Behavioral Assessment of Neuropathic Pain

Mechanical allodynia and thermal hyperalgesia were assessed by investigators blinded to treatment allocation. Mechanical sensitivity was evaluated using a dynamic plantar aesthesiometer. Rats were placed in individual transparent chambers on an elevated wire mesh platform and allowed to acclimate prior to testing. A calibrated filament was applied to the plantar surface of the ipsilateral hind paw with an automatically increasing force until paw withdrawal occurred, and the withdrawal threshold was recorded. Thermal hyperalgesia was assessed using the Hargreaves test. Rats were placed on a glass surface and acclimated before testing. A radiant heat source was focused on the plantar surface of the ipsilateral hind paw, and paw withdrawal latency was recorded with a predefined cutoff time to prevent tissue damage. Behavioral assessments were conducted at baseline (pre-surgery) and on postoperative days 3, 7, 10, and 14.

Biochemical Analysis

At postoperative day 14, immediately after completion of behavioral assessments, rats were euthanized using compressed carbon dioxide (CO₂) delivered by a gradual-fill method at a displacement rate of 30–70% of the chamber volume per minute, in accordance with the approved institutional animal protocol and the AVMA Guidelines for the Euthanasia of Animals: 2020. The euthanasia chamber was not prefilled with CO₂. Death was confirmed by cessation of respiration and heartbeat before rapid collection of the lumbar spinal cord tissues corresponding to sciatic nerve innervation. Tissues were homogenized and analyzed for oxidative stress markers, including malondialdehyde (MDA), nitric oxide (NO), reduced glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT), using commercial assay kits according to the manufacturers’ instructions. Levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and caspase-3 activity were quantified as inflammation-associated cytokine and apoptosis-associated biochemical endpoints. These in vivo endpoints were selected to evaluate whether CPT-associated behavioral effects were accompanied by changes in spinal redox status, inflammation-associated cytokines, and caspase-3 activity. They were not intended to provide definitive pathway-level validation or to identify cell-type-specific mechanisms. All biochemical analyses were performed with n = 6 animals per group.

Statistical Analysis

Physicochemical characterization data are presented as mean ± SD. Biological data, including in vitro assays, behavioral outcomes, and spinal biochemical endpoints, are presented as mean ± SEM. In vitro comparisons were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Behavioral data were analyzed using two-way repeated-measures ANOVA (group × time), followed by Sidak’s multiple-comparisons test. Biochemical data collected at postoperative day 14 were analyzed using one-way ANOVA followed by Tukey’s post hoc test. Statistical significance was set at p < 0.05.

Results

Synthesis and Physicochemical Characterization of CeO₂–PAA–Theanine Nanoparticles

The microstructure and crystalline characteristics of CeO₂–PAA (CP) and CeO₂–PAA–Theanine (CPT) nanoparticles were examined by transmission electron microscopy (TEM). As shown in Figure 1A and B, both CP and CPT exhibited nanoscale morphology with well-defined lattice fringes, indicating high crystallinity. High-resolution TEM analysis revealed an interplanar spacing of approximately 3.12 Å for both CP and CPT, which corresponds to the (111) crystallographic plane of cubic cerium oxide, consistent with the ICDD standard (PDF No. 81–0792).

Figure 1.

Transmission electron microscopy (TEM) characterization of CeO₂–PAA (CP) and CeO₂–PAA–Theanine (CPT) nanoparticles. (A) High-resolution TEM (HRTEM) image of CP nanoparticles showing clear lattice fringes with an interplanar spacing of approximately 3.12 Å, corresponding to the (111) plane of cubic fluorite CeO₂. The white parallel lines are visual guides used to indicate the representative lattice fringes from which the d-spacing was measured. (B) HRTEM image of CPT nanoparticles showing a comparable lattice spacing, indicating that the crystalline structure of CeO₂ was preserved after L-theanine conjugation. The white parallel lines similarly mark representative lattice fringes for d-spacing annotation. (C) Selected-area electron diffraction (SAED) pattern of CPT nanoparticles showing diffraction rings consistent with the cubic fluorite structure of CeO₂.

Selected-area electron diffraction (SAED) analysis further confirmed the crystalline structure of CPT nanoparticles (Figure 1C). The observed diffraction rings matched well with the characteristic reflections of fluorite-type CeO₂, indicating that the conjugation of L-theanine did not alter the intrinsic crystal phase of cerium oxide nanoparticles. These results indicate that both CP and CPT retained a highly crystalline fluorite structure following surface modification and bioactive molecule conjugation.

The hydrodynamic particle size and surface charge of CP and CPT nanoparticles are summarized in Table 1. CP nanoparticles exhibited an average particle size of 63.45 ± 23.47 nm, whereas CPT nanoparticles showed an increased average size of 90.31 ± 28.76 nm. This increase in hydrodynamic size is consistent with surface functionalization by L-theanine. Both CP and CPT displayed low polydispersity indices (PDI ≈ 0.28), indicating a relatively narrow size distribution.

Table 1.

Particle Size, Polydispersity Index (PDI), and Zeta Potential of CeO₂–PAA (CP) and CeO₂–PAA–Theanine (CPT) Nanoparticles

	Particle Size (nm)	PDI	Zeta Potential (mV)
CP	63.45 ± 23.47	0.28 ± 0.03	−46.10 ± 2.86
CPT	90.31 ± 28.76	0.28 ± 0.02	−20.50 ± 1.45

Zeta potential measurements revealed that CP nanoparticles possessed a highly negative surface charge (−46.10 ± 2.86 mV), resulting from the abundant carboxyl groups of the PAA coating. In contrast, CPT nanoparticles exhibited a reduced negative zeta potential (−20.50 ± 1.45 mV), which can be attributed to the formation of amide bonds between the carboxyl groups of PAA and the amine groups of L-theanine via EDC/NHS-mediated conjugation. The less negative zeta potential is consistent with partial consumption or shielding of surface carboxyl groups after L-theanine functionalization.

Surface Functionalization and Redox Properties of CPT Nanoparticles

Fourier transform infrared (FTIR) spectroscopy was employed to analyze the surface chemical functionalities of CP and CPT nanoparticles (Figure 2A). The characteristic absorption band of poly(acrylic acid) (PAA) corresponding to C=O stretching was observed at approximately 1702 cm⁻¹ in both CP and CPT spectra, confirming successful PAA coating on the nanoparticle surface. Free L-theanine exhibited a distinct N–H stretching vibration at approximately 3324 cm⁻¹. Notably, CPT nanoparticles displayed an additional absorption band at approximately 1631 cm⁻¹, which is attributed to the C=O stretching vibration of an amide bond. This result indicates that the amine group of L-theanine successfully reacted with the carboxyl groups of PAA via EDC/NHS-mediated amidation, confirming covalent conjugation of L-theanine onto the CP nanoparticle surface.

Figure 2.

Chemical characterization and crystal structure analysis of CeO₂–PAA (CP) and CeO₂–PAA–Theanine (CPT) nanoparticles. (A) Fourier transform infrared (FTIR) spectra of free L-theanine, CP, and CPT nanoparticles. The characteristic N–H stretching vibration of L-theanine (~3324 cm⁻¹) and the appearance of an amide C=O stretching band at ~1631 cm⁻¹ in CPT confirm successful conjugation of L-theanine to PAA via amide bond formation. (B) X-ray diffraction (XRD) patterns of CP and CPT nanoparticles, showing diffraction peaks corresponding to the (111), (200), (220), (311), (331), (420), and (422) planes of cubic fluorite CeO₂ (ICDD PDF No. 81–0792), indicating preservation of the crystalline structure after surface modification. (C) X-ray photoelectron spectroscopy (XPS) survey spectra of CP and CPT nanoparticles. Characteristic Ce 3d peaks corresponding to both Ce³⁺ and Ce⁴⁺ oxidation states are observed, indicating the coexistence of mixed valence states associated with oxygen vacancies and redox activity.

To assess whether surface modification affected the crystalline structure of cerium oxide, X-ray diffraction (XRD) analysis was performed (Figure 2B). Both CP and CPT nanoparticles exhibited distinct diffraction peaks at 2θ values of approximately 28.5°, 33.1°, 47.5°, 56.3°, 76.7°, 79.1°, and 88.4°, corresponding to the (111), (200), (220), (311), (331), (420), and (422) crystallographic planes of cubic fluorite CeO₂, respectively. These diffraction patterns closely matched the ICDD reference (PDF No. 81–0792), indicating that neither PAA coating nor L-theanine conjugation altered the intrinsic crystal structure of cerium oxide nanoparticles.

Given that the antioxidant activity of cerium oxide nanoparticles is closely associated with the coexistence of Ce³⁺ and Ce⁴⁺ oxidation states and the presence of oxygen vacancies, X-ray photoelectron spectroscopy (XPS) was employed to analyze the surface elemental composition and cerium valence states of CP and CPT nanoparticles (Figure 2C). Survey spectra revealed characteristic peaks corresponding to carbon (C 1s, ~284.4 eV), oxygen (O 1s, ~531.3 eV), and cerium (Ce 3d, 870–920 eV). Deconvolution of the Ce 3d region identified multiple peaks corresponding to Ce³⁺ (875.1, 900.6, 904.8, and 912.0 eV) and Ce⁴⁺ (881.2, 890.9, 897.3, and 915.5 eV) oxidation states in both CP and CPT nanoparticles. The coexistence of mixed cerium valence states suggests the presence of oxygen vacancies and supports the redox cycling capability of these nanoparticles, which is essential for their sustained reactive oxygen species (ROS)-scavenging activity. Importantly, mixed Ce³⁺/Ce⁴⁺ states remained detectable after L-theanine functionalization, suggesting retention of the redox-active character of nanoceria in the CPT formulation.

Drug Loading Capacity and pH-Dependent Release Profile of CPT

The apparent L-theanine incorporation metrics of CeO₂–PAA–Theanine (CPT) nanoparticles are summarized in Table 2. CPT nanoparticles exhibited an apparent incorporation efficiency of 16.52 ± 4.26% and an apparent L-theanine content of 7.42 ± 2.66% (w/w), supporting successful introduction of L-theanine into the nanoplatform. To assess pH-associated release behavior, CPT nanoparticles were incubated in phosphate-buffered saline (PBS) at pH 7.4 and pH 5.0, and cumulative L-theanine-associated signal in the supernatant was monitored over time (Figure 3). A more rapid increase was observed under acidic conditions than under neutral conditions. Because the present assay does not distinguish between weakly associated and covalently linked L-theanine species, these data are interpreted as apparent release-associated behavior rather than direct evidence of selective amide bond cleavage. The findings nevertheless suggest that the CPT interface is responsive to environmental pH and may facilitate faster L-theanine availability under acidic conditions.

Table 2.

Apparent L-Theanine Incorporation Efficiency and Apparent L-Theanine Content of CeO₂–PAA–Theanine (CPT) Nanoparticles

	C-TAA (mg)	Theanine (mg)	EE (%)	DLC (%)
CPT	75	20	16.52 ± 4.26	7.42 ± 2.66

Figure 3.

Apparent L-theanine incorporation and pH-associated release behavior of CeO₂–PAA–Theanine (CPT) nanoparticles. (A) Cumulative release-associated profile of L-theanine from CPT nanoparticles in phosphate-buffered saline (PBS) at pH 7.4. (B) Cumulative release-associated profile of L-theanine from CPT nanoparticles in PBS at pH 5.0. L-theanine-associated signal in the supernatant was quantified at predetermined time points (0.5, 1, 2, 4, 6, and 8 h) using UV–visible spectroscopy based on a calibration curve. CPT nanoparticles showed slower release-associated behavior at pH 7.4 and a faster response at pH 5.0, indicating pH-dependent interfacial responsiveness.

CPT Attenuates Oxidative Stress and Enhances Cell Viability Under in vitro Stress Conditions

Following a 30-min oxidative insult with H₂O₂ (200 μM), RSC96 Schwann cells exhibited a marked reduction in cell viability compared with untreated controls (Figure 4B). Treatment with CPT for 24 h substantially restored cell viability, outperforming free L-theanine and nanoparticle controls. Consistently, intracellular ROS levels were markedly elevated after H₂O₂ exposure, whereas CPT treatment reduced ROS to near-baseline levels comparable to untreated controls (Figure 4C). In addition, both CP and CPT demonstrated excellent cytocompatibility in RSC96 cells in the absence of oxidative stress, with viability remaining above the ISO 10993–5 threshold (Figure 4A). Collectively, these results indicate that CPT attenuates H₂O₂-induced oxidative stress-associated cytotoxicity while maintaining favorable cytocompatibility in vitro.

Figure 4.

In vitro cytocompatibility and protection against oxidative stress. (A) Cytocompatibility of C, CP, and CPT nanoparticles in RSC96 Schwann cells assessed by WST-1 assay according to ISO 10993–5. The dashed line indicates the 70% viability threshold. (B) Cell viability following oxidative insult induced by H₂O₂ (200 μM, 30 min) and subsequent treatment with free L-theanine (Th), C, CP, or CPT for 24 h. (C) Intracellular reactive oxygen species (ROS) levels measured by DCFH-DA assay after H₂O₂ challenge and 24 h treatment. ROS signals were normalized to the H₂O₂-treated group. Data are presented as mean ± SEM (n = 6). *p < 0.05 vs H₂O₂-treated group; ^#p < 0.05 vs untreated control.

CPT Attenuates Oxidative Stress-Associated Inflammatory and Apoptotic Gene Responses in vitro

Quantitative real-time PCR analysis was performed to investigate the effects of CPT on oxidative stress-, inflammation-, and apoptosis-related gene expression in H₂O₂-challenged RSC96 cells (Figure 5). Exposure to H₂O₂ markedly upregulated the expression of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, as well as the apoptotic marker caspase-3, indicating oxidative stress-associated inflammatory and apoptotic responses. Treatment with free L-theanine partially reduced the expression of these genes, whereas cerium oxide-based nanoparticles (C and CP) exerted more pronounced suppressive effects. CPT treatment produced the greatest attenuation of inflammatory and apoptotic gene expression among the tested groups. Furthermore, H₂O₂ exposure induced compensatory upregulation of the antioxidant-related genes Nrf2 and HO-1, reflecting activation of endogenous stress-response pathways. CPT reduced this stress-associated transcriptional response, consistent with the observed reduction in intracellular ROS levels. Collectively, these results indicate that CPT attenuates oxidative stress-associated inflammatory and apoptosis-related gene responses in vitro.

Figure 5.

CPT modulates oxidative, inflammatory, and apoptotic gene expression in H₂O₂-challenged RSC96 cells. RSC96 cells were exposed to H₂O₂ (200 μM, 30 min) to induce oxidative stress, followed by treatment with free L-theanine (Th), cerium oxide nanoparticles (C), CeO₂–PAA nanoparticles (CP), or CeO₂–PAA–Theanine nanoparticles (CPT) for 24 h. Quantitative real-time PCR analysis was performed to assess the expression of oxidative stress-related genes (Nrf2, HO-1), pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), and the apoptotic marker caspase-3. Gene expression levels were normalized to GAPDH and expressed as fold change relative to untreated control cells. Data are presented as mean ± SEM (n = 6). *p < 0.05 vs H₂O₂-treated group; ^#p < 0.05 vs untreated control.

CPT Improves Mechanical and Thermal Hypersensitivity in a Rat Model of Traumatic Neuropathic Pain

Mechanical allodynia was rapidly established following CCI surgery, as evidenced by a marked reduction in paw withdrawal thresholds from day 3 onward in vehicle-treated rats (Figure 6A). CPT treatment significantly attenuated mechanical hypersensitivity beginning at day 7, with progressive improvement observed through days 10 and 14. Rats receiving CPT exhibited significantly higher withdrawal thresholds compared with vehicle-treated CCI animals, and the effect was more pronounced than that observed with free L-theanine, cerium oxide nanoparticles, or CeO₂–PAA nanoparticles alone.

Figure 6.

CPT alleviates mechanical allodynia and thermal hyperalgesia in a rat model of traumatic neuropathic pain. (A) Mechanical sensitivity was evaluated using a dynamic plantar aesthesiometer and expressed as paw withdrawal threshold. Chronic constriction injury (CCI) induced a marked reduction in mechanical withdrawal thresholds beginning at day 3 post-surgery in vehicle-treated rats. Treatment with CPT significantly attenuated mechanical hypersensitivity from day 7 onward, with sustained improvement observed through days 10 and 14 compared with vehicle-treated CCI rats. (B) Thermal hyperalgesia was assessed using the Hargreaves test and expressed as paw withdrawal latency. CCI resulted in a persistent decrease in thermal withdrawal latency, whereas CPT treatment progressively increased latency at later time points, indicating attenuation of thermal hyperalgesia. Data are presented as mean ± SEM (n = 6 per group). Vehicle-treated CCI rats served as the injury control group.

Thermal hyperalgesia showed a similar but delayed pattern of recovery (Figure 6B). CCI induced a sustained reduction in paw withdrawal latency, whereas CPT treatment progressively increased thermal withdrawal latency over time. Significant improvement was observed at later time points, with CPT-treated rats exhibiting the greatest recovery among all treatment groups. Collectively, these results indicate that CPT improves mechanical and thermal hypersensitivity in this rat model of traumatic neuropathic pain.

CPT Attenuates Spinal Oxidative Stress and Inflammation-Associated Biochemical Markers in vivo

To evaluate whether the behavioral effects of CPT were accompanied by spinal biochemical changes, oxidative stress markers, antioxidant defenses, pro-inflammatory cytokines, and caspase-3 activity were assessed in lumbar spinal cord tissues at postoperative day 14 following chronic constriction injury (CCI). As shown in Figure 7, CCI induction resulted in a marked elevation of lipid peroxidation and nitrosative stress, as evidenced by significantly increased levels of malondialdehyde (MDA) and nitric oxide (NO) compared with the sham control group. Concurrently, endogenous antioxidant defenses were substantially compromised in CCI animals, reflected by pronounced reductions in reduced glutathione (GSH) content as well as superoxide dismutase (SOD) and catalase (CAT) activities.

Figure 7.

CPT attenuates spinal oxidative stress, inflammation-associated cytokine changes, and caspase-3 activity following chronic constriction injury (CCI). At postoperative day 14, lumbar spinal cord tissues corresponding to sciatic nerve innervation were collected and analyzed for oxidative and nitrosative stress markers, including malondialdehyde (MDA) and nitric oxide (NO); antioxidant defenses, including reduced glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT); pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6; and caspase-3 activity. CCI increased spinal oxidative and nitrosative stress markers, elevated inflammation-associated cytokines, increased caspase-3 activity, and reduced endogenous antioxidant capacity compared with the sham control group. CPT treatment attenuated these CCI-associated biochemical abnormalities and shifted redox- and inflammation-associated markers toward sham levels, whereas Th, C, and CP exerted partial effects. Data are presented as mean ± SEM (n = 6 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 vs CCI group; ^#p < 0.05 vs sham control group.

Treatment with theanine (Th), nanoceria (C), or PAA-coated nanoceria (CP) partially mitigated CCI-induced oxidative stress, as indicated by moderate decreases in MDA and NO levels accompanied by partial restoration of GSH content and antioxidant enzyme activities. Notably, CPT treatment produced the most pronounced improvement in oxidative stress-associated biochemical markers among all treatment groups, significantly suppressing MDA and NO accumulation while partially restoring GSH content and SOD and CAT activities toward sham levels. In parallel with the changes in oxidative stress markers, levels of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were significantly elevated in the CCI group compared with sham controls. Individual component treatments (Th, C, or CP) partially reduced these cytokine levels, whereas CPT produced the most pronounced reduction among the treatment groups and shifted cytokine levels toward those observed in sham-operated rats. Caspase-3 activity was also markedly increased following CCI, consistent with an apoptosis-associated biochemical response under neuropathic conditions. CPT treatment significantly reduced caspase-3 activity compared with the CCI group, whereas Th, C, and CP exerted partial effects. Collectively, these biochemical findings indicate that CPT-associated behavioral improvement was accompanied by attenuation of spinal oxidative and nitrosative stress markers, inflammation-associated cytokines, and caspase-3 activity.

Based on the in vitro and in vivo findings, a schematic summary of the proposed working model of CPT in CCI-induced neuropathic pain is presented in Figure 8. The model summarizes the measured cellular, behavioral, and spinal biochemical endpoints and illustrates a possible association among reduced oxidative/nitrosative stress, lower inflammation-associated cytokine levels, decreased caspase-3 activity, and improved pain-related behaviors. This schematic should be interpreted as a working model based on the current endpoints rather than definitive evidence of a complete causal pathway.

Figure 8.

Proposed endpoint-based working model of CPT-associated effects in CCI-induced neuropathic pain. Chronic constriction injury (CCI) of the sciatic nerve is associated with pain-related behavioral changes and spinal biochemical alterations, including oxidative/nitrosative stress markers, inflammation-associated cytokines, and caspase-3 activity. CPT treatment was associated with attenuation of measured ROS/RNS-related endpoints, reduced inflammation-associated cytokines, decreased caspase-3 activity, and improved mechanical and thermal hypersensitivity. Upward and downward arrows indicate the direction of measured or summarized endpoint changes only. This schematic represents a proposed working model based on the measured cellular, behavioral, and spinal biochemical endpoints and should not be interpreted as evidence of direct spinal biodistribution, a complete causal pathway, or cell-type-specific neuroimmune mechanisms.

Discussion

Neuropathic pain remains difficult to treat because available therapies often provide incomplete symptom control while failing to fully address biological processes associated with persistent nociception.^2,3 In this study, we developed a surface-functionalized nanoceria platform, CeO₂–PAA–Theanine (CPT), designed to combine the redox-active properties of nanoceria with the reported anti-inflammatory and cytoprotective potential of L-theanine.^10–16 Across complementary in vitro and in vivo readouts, CPT showed the strongest protective profile among the tested formulations. In H₂O₂-challenged RSC96 Schwann cells, CPT reduced intracellular ROS accumulation, improved cell viability, and attenuated inflammation- and apoptosis-associated gene responses. In the rat CCI model, CPT improved mechanical and thermal hypersensitivity, accompanied by reduced spinal oxidative and nitrosative stress markers, partial restoration of antioxidant defenses, lower pro-inflammatory cytokine levels, and decreased caspase-3 activity. These findings are consistent with previous evidence implicating oxidative stress, inflammatory signaling, and apoptosis-associated responses in neuropathic pain pathophysiology.^5–7 Nevertheless, the present data should be interpreted as evidence of CPT-associated biochemical modulation rather than definitive proof of a complete causal mechanism or cell-type-specific neuroimmune pathway.

The use of RSC96 Schwann cells should be interpreted within a defined experimental scope. Schwann cells are peripheral glial cells involved in peripheral nerve homeostasis, injury responses, inflammatory signaling, and axonal support after nerve damage. In the present study, RSC96 cells were used as an in vitro peripheral glial oxidative-injury model to determine whether CPT could protect glial-lineage cells from H₂O₂-induced oxidative stress and suppress stress-associated inflammatory and apoptotic gene responses. This model was not intended to reproduce the full multicellular and multi-compartment pathology of neuropathic pain. In contrast, lumbar spinal cord tissue was selected for in vivo biochemical analysis because spinal redox imbalance, inflammatory cytokine production, and apoptosis-associated signaling are closely associated with nociceptive processing and central sensitization after CCI. Therefore, the in vitro and in vivo experiments should be interpreted as complementary evidence supporting CPT-associated redox and inflammatory modulation, rather than as a single linear mechanistic pathway from Schwann cells to spinal cord responses.

A central finding of this study is that CPT outperformed free L-theanine and cerium oxide nanoparticle controls. Nanoparticle mass-matched dosing was intentionally employed across the C, CP, and CPT groups to isolate the contribution of L-theanine functionalization rather than differences in nanoparticle exposure. Free L-theanine has recognized neuroprotective potential but is limited by rapid systemic clearance and restricted tissue retention.²⁰ In contrast, nanoceria provide persistent redox activity through reversible Ce³⁺/Ce⁴⁺ cycling but may not sufficiently suppress downstream inflammatory and apoptosis-associated responses when used alone.²¹ Combining these features within a single surface-engineered platform may therefore provide broader biological coverage than either component alone.²² The inclusion of both C and CP groups further indicates that PAA coating alone does not account for the full activity observed with CPT.

Our findings are consistent with the concept that oxidative stress and neuroinflammation form a tightly coupled pathological network in neuropathic pain.^23–25 In Schwann cells, oxidative challenge triggered upregulation of pro-inflammatory cytokines and caspase-3, consistent with ROS-associated inflammatory and apoptotic signaling.^26–28 CPT markedly suppressed these responses while reducing the stress-associated transcriptional elevation of Nrf2 and HO-1. In the context of the concurrent reduction in intracellular ROS, these data are consistent with a lower oxidative burden rather than activation of a stronger compensatory stress response. Together, the in vitro findings suggest that CPT is associated with concurrent attenuation of redox-, inflammation-, and apoptosis-associated cellular responses under oxidative stress conditions.²⁵

A broadly consistent pattern of biochemical improvement was also observed in vivo following chronic constriction injury. CPT did not uniformly return all parameters to sham values, but it consistently shifted oxidative, inflammatory, and apoptosis-associated markers toward a less pathological state. In neuropathic pain, therapeutic benefit does not necessarily require full normalization to a naïve baseline; rather, partial correction of feed-forward oxidative and inflammatory signaling may be sufficient to reduce pain maintenance.^23,29 CPT significantly suppressed lipid peroxidation and nitrosative stress while restoring endogenous antioxidant capacity, and these effects were accompanied by reductions in pro-inflammatory cytokines and caspase-3 activity. Given the established contribution of oxidative stress and inflammatory mediators to spinal pain sensitization,^30–33 these biochemical changes are consistent with reduced spinal pathological burden after CPT treatment.

Several anatomical and cell-type-specific limitations should be considered when interpreting these findings. Neuropathic pain involves coordinated responses across multiple compartments, including the injured peripheral nerve, dorsal root ganglia, spinal cord, and supraspinal pain-processing regions. Peripheral nerve injury can engage Schwann cells and macrophages at the injury site, satellite glial cells and sensory neurons in the dorsal root ganglia, and microglia and astrocytes within the spinal dorsal horn.^34–36 Therefore, the present spinal cord biochemical endpoints cannot determine whether CPT acts primarily at the injured nerve, dorsal root ganglia, spinal cord, peripheral immune compartment, or through systemic redox and inflammatory modulation. In addition, although reduced spinal TNF-α, IL-1β, IL-6, and caspase-3 activity support attenuation of inflammation- and apoptosis-associated biochemical responses, we did not directly assess microglial activation, astrocytic activation, neuronal injury, or cell-type-specific cytokine production. Future studies should incorporate Iba1 and CD68 for microglial/macrophage responses, GFAP for astrocytic activation, NeuN or ATF3 for neuronal injury responses, and compartment-specific analyses of injured sciatic nerve, dorsal root ganglia, lumbar spinal cord, and relevant supraspinal regions.

The exclusive use of male rats is another important limitation. Sex-dependent neuroimmune mechanisms have been reported in neuropathic pain, particularly with respect to microglia-dependent versus microglia-independent pathways after peripheral nerve injury.^37,38 Because the present study used only male animals, the findings should not be generalized to female subjects or interpreted as evidence of sex-independent efficacy. Future studies should include both sexes and evaluate whether CPT differentially affects microglial, astrocytic, peripheral immune, and sensory neuronal responses in male and female animals.

The temporal profile of behavioral improvement is also noteworthy. CPT-mediated improvement in mechanical thresholds emerged around postoperative day 7 and progressed through day 14, whereas recovery of thermal latency became evident later. This delayed pattern is compatible with gradual modification of biochemical processes associated with neuropathic pain rather than immediate analgesic masking.^23,39 Biochemical analyses at postoperative day 14 showed that CPT-treated animals exhibited reduced oxidative and inflammatory abnormalities together with lower caspase-3 activity. Although microglial and astrocytic activation markers were not directly assessed, the broad suppression of spinal cytokines together with restoration of antioxidant defenses is consistent with reduced inflammation-associated biochemical burden that may contribute to neuropathic pain behaviors.^24,32,39

From a translational perspective, CPT represents an exploratory nanozyme-based formulation designed to integrate redox buffering with L-theanine functionalization. The PAA coating provides colloidal stabilization and introduces functional groups for subsequent L-theanine conjugation, thereby supporting rational surface engineering of the nanoceria platform 41. In vitro, CP and CPT exhibited favorable cytocompatibility under the tested conditions, while CPT attenuated H₂O₂-induced oxidative stress-associated cytotoxicity. In vivo, repeated intraperitoneal dosing improved behavioral and spinal biochemical endpoints in the CCI model. However, these findings should be interpreted as short-term efficacy-associated evidence, and they do not establish long-term systemic safety, tissue distribution, or clinical translatability.

A further translational limitation is the absence of direct biodistribution, pharmacokinetic, clearance, and tissue accumulation data. The present study therefore cannot determine whether intraperitoneally administered CPT directly reaches the injured sciatic nerve, dorsal root ganglia, lumbar spinal cord, or supraspinal structures at pharmacologically meaningful levels. Nanoparticle biodistribution is strongly influenced by particle size, surface chemistry, aggregation state, protein corona formation, route of administration, and mononuclear phagocyte system uptake. Studies of cerium oxide nanoparticles have shown that tissue distribution and long-term retention can vary substantially depending on formulation and exposure conditions, with liver, spleen, lung, kidney, brain, lymph nodes, urine, and feces being relevant compartments for biodistribution and excretion assessment.⁴⁰ Accordingly, the behavioral and spinal biochemical effects observed here may reflect direct neural tissue exposure, indirect modulation of peripheral immune or redox responses, systemic anti-inflammatory effects, or a combination of these mechanisms. Future studies should use fluorescently labeled, radiolabeled, or elemental cerium-quantified CPT to define time-dependent distribution in injured nerve, dorsal root ganglia, spinal cord, brain, liver, spleen, kidney, and excretion routes.

In addition, nanoparticle behavior under biologically complex conditions was not fully resolved. Although hydrodynamic size and zeta potential were measured in aqueous and culture-related media, we did not perform time-course colloidal stability testing in serum-containing medium or plasma. This is important because adsorbed proteins and other biomolecules can form a corona around nanoparticles, altering their apparent size, zeta potential, colloidal stability, cellular uptake, biodistribution, immune recognition, and biological identity.^41,42 Future work should therefore assess CPT stability in serum-containing medium and plasma over time, including hydrodynamic size, polydispersity index, zeta potential, aggregation behavior, and protein corona composition.

The in vivo CPT dose also requires careful interpretation. The selected CPT dose of 135 mg/kg was calculated to provide a 10 mg/kg L-theanine-equivalent dose based on the measured apparent L-theanine content of 7.42%. The C and CP groups were administered at the same nanoparticle mass dose to allow mass-matched comparison among nanoceria-based formulations and to isolate the contribution of L-theanine functionalization. However, daily systemic administration of an inorganic nanoceria-containing material for 14 days represents a relatively high cumulative nanoparticle exposure. Although no mortality or gross treatment intolerance was observed during the experimental period, the present study did not include comprehensive systemic toxicity assessment, hematology, serum biochemistry, organ histopathology, immunotoxicity profiling, dose de-escalation, long-term retention analysis, or post-treatment recovery follow-up. Therefore, the current data should be interpreted as short-term efficacy-associated evidence rather than a complete safety evaluation. Future studies should define the minimum effective dose, no-observed-adverse-effect level, dose-response relationship, organ distribution, long-term retention, and clearance profile before translational development.

Several limitations should be acknowledged. First, the study used a single traumatic neuropathic pain model and one dosing regimen. Evaluation in additional neuropathic pain models, such as chemotherapy-induced, diabetic, inflammatory, or nerve transection-associated neuropathy, together with dose-response and delayed-treatment designs, would strengthen generalizability. Second, only male rats were included; therefore, sex-dependent neuroimmune responses and possible sex-specific treatment effects remain unresolved. Third, although the study provides cellular, behavioral, and spinal biochemical evidence, it does not define the anatomical site or cellular target of CPT action. Analyses of injured sciatic nerve, dorsal root ganglia, spinal cord, and supraspinal regions, together with cell-type-specific markers of microglia, astrocytes, Schwann cells, macrophages, satellite glial cells, and neurons, will be needed to clarify the mechanism. Fourth, pathway-level signaling was not directly assessed. Measurements of NF-κB activation, Nrf2 nuclear translocation, MAPK signaling, mitochondrial function, and neuronal injury markers would be required to establish a causal mechanistic pathway. Fifth, biodistribution, pharmacokinetic, clearance, serum stability, protein corona, and long-term tissue retention analyses were not performed. These studies are essential to determine whether CPT reaches relevant neural compartments and to evaluate the translational safety of repeated systemic nanoceria administration. Finally, although FTIR and zeta potential analyses support successful L-theanine functionalization, the current release-associated assay does not distinguish covalently linked from weakly associated L-theanine species. Accordingly, the release data should be interpreted as cumulative L-theanine-associated signal rather than definitive evidence of selective amide bond cleavage. Despite these limitations, the present findings support CPT as a promising candidate nanoplatform for further investigation of redox- and inflammation-associated modulation in neuropathic pain.

Conclusions

In conclusion, this study developed a surface-functionalized CeO₂–PAA–Theanine nanozyme platform and evaluated its redox- and inflammation-associated effects in cellular and CCI-based neuropathic pain models. CPT retained key crystalline and mixed-valence characteristics of nanoceria, showed evidence of L-theanine functionalization, and attenuated H₂O₂-induced oxidative injury, inflammatory gene responses, and caspase-3-associated gene expression in RSC96 Schwann cells. In the rat CCI model, CPT improved mechanical and thermal hypersensitivity and was associated with reduced spinal oxidative and nitrosative stress markers, partial restoration of antioxidant defenses, lower pro-inflammatory cytokine levels, and decreased caspase-3 activity. These findings support CPT as a candidate nanozyme platform for further investigation in redox- and inflammation-associated modulation of neuropathic pain. However, the present study does not establish a complete causal mechanism, anatomical site of action, biodistribution profile, or long-term safety profile. Future studies incorporating cell-type-specific neuroimmune markers, pathway-level validation, biodistribution analysis, serum stability testing, dose optimization, and extended toxicological assessment are warranted.

Funding Statement

This work was supported by the National Science and Technology Council, Taiwan (Grant No. NSTC 112-2221-E-195-001-).

Data Sharing Statement

All data generated or analyzed during this study are included in this article. Additional raw data supporting the findings are available from the corresponding author, Dr. Cheng-Chia Tsai, upon reasonable request.

Ethics Approval and Consent to Participate

All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan University (approval no. 20210438) and were conducted in accordance with the institutional guidelines for the care and use of laboratory animals. Surgical procedures were performed under isoflurane anesthesia. Animals were euthanized using compressed carbon dioxide (CO₂) delivered by a gradual-fill method at a displacement rate of 30–70% of the chamber volume per minute, in accordance with the approved institutional protocol and the AVMA Guidelines for the Euthanasia of Animals: 2020. No separate animal permit or animal license number was issued by the institution. The RSC96 rat Schwann cell line used in this study was obtained commercially from the American Type Culture Collection (ATCC, Manassas, VA, USA; ATCC^® CRL-2765™). Because this commercially available established cell line was used, no additional institutional ethics committee or institutional review board approval was required for the in vitro cell culture experiments.

Author Contributions

All authors made a significant contribution to the work reported, whether in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare that they have no competing interests.