Phytocarbon Nanodots Restore Glutathione Homeostasis via γ-Glutamylcysteine Synthetase against Ferroptosis-Driven Renal Fibrosis

Renal fibrosis remains a critical and refractory clinical condition, characterized by a dire lack of safe and effective pharmacological interventions. Its progression is propelled by dysregulated ferroptosis, an iron-dependent cell death pathway that drives pathological extracellular matrix deposition. To bridge this translational gap, we engineered a novel class of intrinsic nanotherapeutics through the atomic-level reengineering of Lycium barbarum polysaccharides (LBP) into phytocarbon nanodots (LBPCDs). This transformation transcends the inherent pharmacokinetic limitations of native LBP, yielding emergent therapeutic properties. The resulting LBPCDs exhibit a defect-rich sp 2 /sp 3 hybrid framework with quantum confinement (~2.5 nm), which facilitates superior renal accumulation and electron-transfer dynamics. Their redox-active surface, dominated by hydroxyl groups, confers potent scavenging capacity against ROS (·OH, ·O 2 - ) and lipid peroxides, effectively alleviating oxidative stress and epithelial-mesenchymal transition. Mechanistic studies indicate that LBPCDs modulate the ferroptosis defense pathway, potentially through interactions with the glutamate-cysteine ligase modifier subunit (GCLM), leading to enhanced γ-glutamylcysteine synthetase activity and restoration of glutathione homeostasis. This dual-action strategy concurrently inhibits lipid peroxidation and ferroptosis, reversing fibrotic pathology in cisplatin-induced fibrosis and diabetic nephropathy models with exceptional biosafety. Our work establishes an innovative “material‑is‑the‑drug” paradigm, synchronizing oxidative homeostasis restoration, ferroptosis interception, and EMT remodeling, thereby presenting a promising next-generation nanotherapeutic platform for fibrotic diseases. Phytocarbon Nanodots Restore Glutathione Homeostasis via γ-Glutamylcysteine Synthetase against Ferroptosis-Driven Renal Fibrosis

Renal fibrosis remains a critical and refractory clinical condition, characterized by a dire lack of safe and effective pharmacological interventions. Its progression is propelled by dysregulated ferroptosis, an iron-dependent cell death pathway that drives pathological extracellular matrix deposition. To bridge this translational gap, we engineered a novel class of intrinsic nanotherapeutics through the atomic-level reengineering of Lycium barbarum polysaccharides (LBP) into phytocarbon nanodots (LBPCDs). This transformation transcends the inherent pharmacokinetic limitations of native LBP, yielding emergent therapeutic properties. The resulting LBPCDs exhibit a defect-rich sp 2 /sp 3 hybrid framework with quantum confinement (~2.5 nm), which facilitates superior renal accumulation and electron-transfer dynamics. Their redox-active surface, dominated by hydroxyl groups, confers potent scavenging capacity against ROS (·OH, ·O 2 ⁻) and lipid peroxides, effectively alleviating oxidative stress and epithelial-mesenchymal transition. Mechanistic studies indicate that LBPCDs modulate the ferroptosis defense pathway, potentially through interactions with the glutamate-cysteine ligase modifier subunit (GCLM), leading to enhanced γ-glutamylcysteine synthetase activity and restoration of glutathione homeostasis. This dual-action strategy concurrently inhibits lipid peroxidation and ferroptosis, reversing fibrotic pathology in cisplatin-induced fibrosis and diabetic nephropathy models with exceptional biosafety. Our work establishes an innovative “material‑is‑the‑drug” paradigm, synchronizing oxidative homeostasis restoration, ferroptosis interception, and EMT remodeling, thereby presenting a promising next-generation nanotherapeutic platform for fibrotic diseases.

Phytocarbon nanodots; γ-glutamylcysteine synthetase; glutathione homeostasis; Ferroptosis; Renal Fibrosis 1. Introduction Chronic kidney disease (CKD) imposes a substantial global health burden, affecting approximately 10% of the population and accounting for 1.2 million annual deaths worldwide [1] . Renal fibrosis, marked by glomerulosclerosis, tubulointerstitial scarring, and pathological extracellular matrix deposition, represents the terminal pathological pathway for most CKD etiologies, ultimately resulting in renal failure and mortality [2, 3] . Central to this pathology is ferroptosis, an iron-dependent cell death mechanism fueled by lipid peroxidation and glutathione (GSH) depletion [4] . Iron overload induces epithelial damage, triggering TGF-β release that activates fibroblasts and accelerates pathological extracellular matrix accumulation [5] . Concurrently, labile Fe²⁺ and ferroptosis-derived lipid hydroperoxides amplify TGF-β/Smad3 signaling, upregulating α-smooth muscle actin (α-SMA) to establish a self-sustaining fibrogenic loop [6-8] . Current anti-fibrotics (e.g., pirfenidone, nintedanib), repurposed from pulmonary fibrosis, suffer from poor renal specificity, dose-limiting systemic toxicities (e.g., hepatotoxicity) [9], and translational barriers including drug resistance and inadequate preclinical models [10] . Thus, innovative therapeutics capable of enhancing renal targeting, bypassing biological barriers, and disrupting ferroptosis-EMT crosstalk represent a promising strategy to halt fibrotic progression [11] . Carbon dots (CDs), as an emerging class of carbon-based nanomaterials, have gained considerable interest in nanomedicine owing to their tunable optical properties, versatile surface chemistry, and exceptional biocompatibility [12-13] . Their ultrasmall size (< 10 nm) facilitates efficient renal accumulation and clearance, rendering them highly promising for theranostic applications in kidney diseases. For example, selenium-doped carbon quantum dots (SeCQDs) have demonstrated broad-spectrum antioxidant activity, effectively mitigating acute kidney injury (AKI) in murine models [14] . Recently, herb-derived CDs have emerged as a novel strategy, leveraging the inherent bioactivity of natural sources to enhance therapeutic efficacy. Ziziphi Spinosae Semen-derived CDs (Z-CDs), for instance, exhibited ROS-scavenging and NF-κB inhibitory effects, protecting against sepsis-associated AKI [15] . However, current research has predominantly focused on acute kidney injury or nephrotoxicity models, while the more complex and chronic fibrotic processes remain largely unexplored. Furthermore, most CDs exert their therapeutic effects through generic antioxidant and anti-inflammatory mechanisms, with no reported instances of directly modulating glutathione metabolism to combat renal fibrosis. Herein, we report the rational design of phytocarbon nanodots (LBPCDs) derived from Lycium barbarum polysaccharides (LBP), a selection justified by well-established nephroprotective profile and unique ability of LBP to activate the Nrf2-mediated antioxidative response [16-17] . Through controlled low-temperature hydrothermal synthesis, we transmuted these polysaccharides into uniform architectures and highly hydroxyl-functionalized surfaces. These phytocarbon nanodots exhibit excellent renal accumulation, superior electron transfer kinetics, mitochondrial targeting, and enhanced anti-fibrosis activity than LBP. Mechanistically, LBPCDs function as dual-pathway nanoinhibitors of fibrogenesis: (1) surface hydroxyl-dominated redox activity enables potent elimination of ·OH, ·O₂⁻, and lipid peroxides, directly mitigating oxidative stress-triggered epithelial-mesenchymal transition (EMT); (2) high-affinity targeting of glutamate-cysteine ligase modifier subunit (GCLM) reprograms glutathione metabolism, boosting γ-glutamylcysteine synthetase (γ-GCS) activity and elevating GSH/GSSG ratios to suppress ferroptotic cell death. In cisplatin-induced renal interstitial fibrosis and diabetic nephropathy models, LBPCDs potently ameliorated parenchymal damage and fibrotic pathologies by synchronously restoring redox homeostasis and blocking iron-dependent cell death, with exceptional biosafety. In summary, this work pioneers phytocarbon nanodrugs as first-in-class therapeutics that reprogram glutathione metabolism through interaction with GCLM, effectively addressing longstanding challenges in bioavailability and targeting precision via innovative nano-bio interface engineering. By concurrently facilitating ROS scavenging, ferroptosis inhibition, and extracellular matrix remodeling, this study establishes a sustainable Herb-Nano platform that offers a transformative strategy for the treatment of fibrotic diseases. 2. Results 2.1 Synthesis and Structural Characterization of LBPCDs Lycium barbarum polysaccharides (LBP) were extracted as previously reported and underwent controlled hydrothermal carbonization (220 °C, 8 h) to yield uniform carbon dots (LBPCDs) (Figure 1A). To elucidate the morphological features, transmission electron microscopy (TEM) analysis was performed, and High-resolution TEM (inset top right, Figure 1B) showed distinct 0.21-nm lattice fringes corresponding to graphitic (100) planes, thereby confirming partial graphitization, revealing monodisperse quasi-spherical nanoparticles with an average diameter of 2.48 nm (Figure 1C). The aqueous dispersion of LBPCDs exhibited intense blue photoluminescence under 365-nm UV excitation and a clear Tyndall effect (inset bottom, Figure 1B), confirming excellent colloidal stability. Photoluminescence characterization indicated promising bioimaging potential of LBPCDs, with excitation/emission maxima at 450/520 nm (red/blue) and a characteristic UV-vis absorption band at 290 nm (black) (Figure 1D), indicating π-π* transitions. To assess practical utility under physiological conditions, environmental stability tests were conducted. Remarkably, LBPCDs maintained 98.6% fluorescence intensity after 30 days (ambient), withstood pH extremes (3-10), and resisted high ionic strength (1.0 M NaCl) (Figure S1, Supporting Information), demonstrating exceptional robustness. Complementarily, X-ray diffraction (XRD) analysis further confirmed an amorphous carbon structure, evidenced by broad peaks centered at To probe chemical composition and functional group evolution, comparative FTIR spectroscopy was employed. The results confirmed retention of key hydrophilic groups from LBP (Figure 1F), including O-H (3387 cm⁻¹), C-H (2926 cm⁻¹), C=O (1669 cm⁻¹), and C=C (1403 cm⁻¹) stretching vibrations, preserving aqueous solubility while inheriting bioactive motifs. To quantify elemental restructuring during carbonization, X-ray photoelectron spectroscopy (XPS) was utilized. The data showed significant oxygen decrease (68.04% to 49.21%) and carbon increase (31.96% to 50.71%) (Figure 1G). Deconvolution of C1s spectra (Figure 1H) identified four carbon states in LBPCDs (C-C: 284.4 eV; C-O: 286.0 eV; C-O-C: 287.1 eV; C=O: 288.5 eV), while O1s analysis showed dominant C=O (531.5 eV) and C-O (532.6 eV) environments (Figure 1I; LBP spectra in Figure S2, Supporting Information). To resolve molecular-level reorganization, the 13 C NMR in C 2 D 6 SO was performed. This technique uncovered emergent sp²-hybridized carbon network (100-150 ppm) and oxygenated domains (140-180 ppm), sharply contrasting with polysaccharide signatures of LBP (Figure 1J). Corroborating these findings, ¹H NMR detected aromatic hydrogens (7-10 ppm, Figure S3, Supporting Information), conclusively establishing glycosidic bond cleavage and reconstruction into oxygen-functionalized sp² frameworks. In summary, the integrated characterization spanning morphology, optoelectronics, surface chemistry, and molecular structure, collectively establishes that LBPCDs possess a well-defined quasi-spherical nanostructure, retained bioactivity, and uncompromised stability. Consequently, these properties position LBPCDs as versatile theranostic platforms for targeted biomedical applications. FIGURE 1 | Synthesis and Structural Characterization of LBPCDs. (A) Schematic of LBPCDs fabrication. (B) TEM image of LBPCDs (inset: HRTEM showing lattice fringes; lower inset: photoluminescence under UV illumination and Tyndall effect in aqueous dispersion). (C) Size distribution histogram. (D) UV-vis absorption (black), fluorescence excitation (red), and emission spectra (blue; λ ex = 450 nm). (E) XRD pattern. (F) Comparative FTIR spectra of LBP and LBPCDs. (G) XPS survey spectra. High-resolution XPS spectra: (H) C 1s and (I) O 1s. (J) Comparative 13 C NMR spectra of LBP and LBPCDs. 2.2 Surface Hydroxyl-Dominated ROS Scavenging Capacity of LBPCDs Lycium barbarum polysaccharides (LBP) known for their inherent antioxidant properties, which are critical for nephroprotection [18] . To investigate whether hydrothermal carbonization preserves this key bioactivity, we systematically evaluated and compared the reactive oxygen species (ROS) scavenging capacities of LBPCDs and native LBP. We first employed the ABTS assay to quantify the general antioxidant potential of LBP and LBPCDs. In this assay, ABTS is oxidized by an oxidant to form the green radical cation ABTS·⁺, which exhibits maximum absorbance at ~420 nm (Figure 2A). Analysis of the UV-vis absorption spectra revealed that incubation of ABTS·⁺ solutions with increasing concentrations of LBPCDs (Figure 2B) or LBP (Figure S4, Supporting Information) resulted in significant suppression of ABTS·⁺ formation. Crucially, LBPCDs demonstrated a markedly greater antioxidant capacity compared to native LBP (Figure 2C). Given that hydroxyl radicals (·OH) and superoxide anions (·O₂⁻) consist of majority of reactive oxygen species, we next specifically evaluated the ·OH and ·O₂⁻ scavenging capacity of LBPCDs. To generate ·OH, we employed a Fenton reaction system (Fe²⁺/H₂O₂) using 3,3’,5,5’-tetramethylbenzidine (TMB) as a chromogenic probe (Figure 2D). Oxidation of TMB produces green oxTMB, detectable at 652 nm. UV-vis spectra of oxTMB solutions treated with escalating concentrations of LBPCDs (Figure 2E) and LBP (Figure S5, Supporting Information) clearly showed that LBPCDs exhibited a > 2-fold greater ·OH scavenging capacity than native LBP (Figure 2F). Subsequently, we assessed the superoxide anion (·O₂⁻) scavenging capacity of LBPCDs using a riboflavin/methionine photochemical system. In this system, photoexcited riboflavin transfers electrons to methionine, undergoing self-oxidation to form a semiquinone radical that reacts with O₂ to generate ·O₂⁻ [19] . The resulting ·O₂⁻ reduces nitroblue tetrazolium chloride (NBT) to blue-violet formazan, monitored at 560 nm (Figure 2G). Notably, at a concentration of 100 µg/mL, LBPCDs achieved near-complete ·O₂⁻ elimination (> 90% scavenging activity), significantly surpassing the scavenging activity of LBP (62.95%) at the same concentration (Figure 2H). In summary, our comparative study demonstrates that hydrothermal carbonization not only preserves but significantly enhances the inherent antioxidant properties of LBP. The resulting LBPCDs exhibit robust and superior scavenging capacities against both hydroxyl radicals (·OH) and superoxide anions (·O₂⁻). This potent dual ROS-neutralizing activity, as illustrated in Figure 2I, highlights LBPCDs as effective antioxidants. To elucidate the key functional groups governing the ROS scavenging capacity of LBPCDs, we conducted selective surface modification of their functional moieties [20] . The strategy involved a two-step modification process: first, carbonyl groups were reduced to hydroxyl groups using NaBH₄, which served to create reactive sites for subsequent functionalization; then, propane sultone (PS) was conjugated to these hydroxyl groups, followed by hydrolysis to yield two distinct derivatives: LBPCDs-A (with -OH groups masked) and LBPCDs-B (with both -OH and C=O groups masked) (Figure 2J). FTIR analysis confirmed successful modification, evidenced by signatures of sulfonic acid groups (-SO₃⁻ at 528, 610, 1047 cm⁻¹) and the emergence of aliphatic C-H stretches (2840–3000 cm⁻¹), consistent with ether linkage formation (Figure 2K). Notably, functional group passivation significantly attenuated the ROS scavenging activity of both derivatives compared to the unmodified LBPCDs. Specifically, the capacities to scavenge ·OH (Figure 2L, M) and O₂⁻ (Figure 2N) were markedly reduced following modification. Critically, a comparative analysis revealed that suppressing hydroxyl groups (as in LBPCDs-A) led to a more profound impairment of ROS scavenging than merely masking carbonyl groups (as in LBPCDs-B). This differential effect underscores the pivotal role of surface -OH groups as the primary active sites responsible for ROS neutralization. FIGURE 2 | Surface Hydroxyl-Dominated ROS Scavenging Capacity of LBPCDs. (A) ABTS ·+ scavenging assay schematic. (B) Concentration-dependent UV-vis spectra of ABTS ·+ solutions treated with LBPCDs. (C) Comparative total antioxidant capacity of LBP vs. LBPCDs. (D) Hydroxyl radical (·OH) detection system schematic (Fe 2+ /H 2 O 2 /TMB). (E) oxTMB absorption spectra after LBPCDs treatment. (F) ·OH elimination capacity of LBP vs. LBPCDs. (G) Superoxide radical (·O 2 - ) generation schematic (riboflavin/NBT). (H) ·O 2 - scavenging capacity of LBP vs. LBPCDs. (I) Proposed ROS scavenging mechanisms by LBPCDs. (J) Surface modification strategy for functional group masking. (K) Comparative FTIR spectra of modified LBPCDs-A/B. (L) oxTMB absorption after treatment with modified nanoparticles. (M) ·OH scavenging capacity of surface-modified variants. (N) ·O 2 - neutralization by modified LBPCDs. For statistical analysis, data represent mean ± SD (n = 3). Statistical significance was calculated by using an unpaired two-tailed Student’s t-test. ** p < 0.01, *** p < 0.001. 2.3 LBPCDs Reverse EMT through Mitochondrial-Targeted ROS Scavenging The transition of renal tubular epithelial cells into myofibroblasts through epithelial-mesenchymal transition (EMT) is a key mechanism driving renal interstitial fibrosis in chronic kidney disease [21] . To explore potential therapeutic strategies, we established an in vitro model of renal fibrosis using TGF-β-stimulated human kidney proximal tubular epithelial (HK-2) cells, a well-established system for studying TGF-β-driven EMT which is a core process in fibrosis development [22] . Comparative evaluation revealed that LBPCDs exhibited superior anti-fibrotic efficacy over native LBP, as evidenced by more pronounced inhibition of fibrosis-related phenotypes (Figure S6, S7, Supporting Information). Building on this preliminary observation, we further investigated the dose-dependent effects of LBPCDs (100, 200, and 400 µg/mL) in reversing TGF-β-induced EMT. Notably, treatment with 400 µg/mL LBPCDs fully preserved the characteristic cobblestone morphology of epithelial cells, whereas control cells displayed the spindle-shaped fibroblastic phenotype typical of EMT (Figure 4A, quantitative analysis in Figure 4B) [23, 24] . At the molecular level, TGF-β stimulation significantly upregulated the expression of fibrotic markers, including α-smooth muscle actin (α-SMA), Collagen type I (Col 1), and Vimentin, as measured by qPCR (Figure 4C-E). LBPCDs effectively counteracted this upregulation in a dose-dependent manner, with all tested concentrations inducing significant suppression of these markers. Western blot analysis (Figure S8, Supporting Information) corroborated these transcriptional changes at the protein level, confirming that LBPCDs dose-dependently attenuated TGF-β-induced overexpression of α-SMA, Col 1, and Vimentin (Figure 4F). Collectively, these data demonstrate that LBPCDs mitigate TGF-β-driven renal fibrosis in HK-2 cells through dual regulation of fibrotic marker transcription and translation. Given our prior demonstration of the ROS-scavenging capacity of LBPCDs, we next explored their role in counteracting oxidative damage within the context of renal fibrogenesis, specifically targeting the ROS-EMT axis [25, 26] . Consistent with this role, LBPCDs potently suppressed intracellular ROS accumulation in TGF-β-stimulated HK-2 cells (Figure 4G, H), directly mitigating partial key fibrosis trigger. Mechanistically, LBPCDs dose-dependently rescued catalase (CAT) activity, an essential enzyme for H₂O₂ detoxification, from TGF-β-induced impairment (Figure S9, Supporting Information). By restoring endogenous antioxidant defenses, LBPCDs disrupted the ROS-EMT-fibrosis cascade, linking their free radical scavenging activity to the observed anti-fibrotic effects. Since mitochondria are the primary intracellular source of ROS and particularly vulnerable to oxidative damage in fibrosis [27], we hypothesized that the efficacy of LBPCDs in scavenging ROS and mitigating fibrosis might depend on their ability to target these organelles. To test this hypothesis and elucidate the subcellular mechanism antioxidant action of LBPCDs, we first investigated their intracellular distribution. Cellular uptake studies using CY5-fluorophore-labeled LBPCDs (LBPCDs@CY5) revealed progressive intracellular accumulation, predominantly localized in the cytoplasm (Figure S10, Supporting Information). Critically, colocalization analysis with MitoTracker demonstrated strong mitochondrial targeting (Pearson’s coefficient 0.91), indicating preferential accumulation at the primary sites of ROS generation (Figure 4I). Notably, lysosomal colocalization was significantly lower (Pearson’s coefficient 0.62 vs. mitochondrial 0.91), confirming organelle-selective targeting (Figures S11, Supporting Information). Based on this observed mitochondrial localization, we next assessed the impact of LBPCDs on mitochondrial oxidative stress and function. MitoSOX™ Red assays, specifically detecting mitochondrial superoxide (mtROS), showed that LBPCDs significantly attenuated TGF-β-induced production in HK-2 cells (Figure 4J; Figure S12, Supporting Information). Furthermore, parallel JC-1 assays revealed that LBPCDs dose-dependently restored the TGF-β-compromised mitochondrial membrane potential (Δψm), as indicated by increased red/green fluorescence ratios (Figure 4K; Figure S13, Supporting Information). Collectively, these results suggest that LBPCDs alleviate renal fibrosis through mitochondrial targeting, enabling efficient clearance of mtROS at its source, while simultaneously protecting mitochondrial integrity and function to maintain intracellular redox homeostasis. FIGURE 3 | LBPCDs Inhibit Renal Fibrosis via Mitochondrial Targeting of ROS Scavenging. ( A) Representative micrographs of HK-2 cells after TGF-β induced and co-cultured with or without LBPCDs (scale bar = 20 μm). ( B) Quantitative analysis of fibrotic cell proportions. (C-E) Expression of pro- fibrotic factors (α-SMA, Col 1 and Vimentin) in HK-2 cells in gene levels. ( F) Protein levels of α-SMA, Col 1, and Vimentin by Western blot. ( G) Intracellular ROS detection using DCFH-DA fluorescence probe (scale bar = 20 μm). ( H) Quantified ROS fluorescence intensity. ( I) Mitochondrial colocalization of LBPCDs@CY5 (red) with MitoTracker (green); inset panels: intensity correlation curves. ( J) JC-1 staining showing mitochondrial membrane potential (red: J-aggregates; green: monomers; scale bar = 20 μm). ( K) Mitochondrial superoxide detection by MitoSOX™ Red (scale bar = 20 μm). For statistical analysis, data represent mean ± SD (n = 3). Statistical significance was calculated by using an unpaired two-tailed Student’s t-test. # p < 0.05, ### p < 0.001, vs Ctrl group; ns p ≥ 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, vs TGF-β group. 2.4 LBPCDs Ameliorate Renal Fibrosis via γ-Glutamylcysteine Synthetase-Mediated Glutathione Homeostasis and Ferroptosis Suppression To further elucidate the molecular mechanisms underlying the anti-fibrotic effects of LBPCDs we performed RNA sequencing (RNA-seq) analysis in TGF-β-induced HK-2 cells with or without LBPCDs treatment. Principal component analysis (PCA) revealed distinct intergroup separation with high intra-group reproducibility (Figure 4A), indicating that LBPCDs induce transcriptional reprogramming in TGF-β-stimulated HK-2 cells. A total of 264 differentially expressed genes (DEGs) were identified between LBPCDs-treated and model cells (95 upregulated and 169 downregulated; Figure 4B). KEGG enrichment highlighted glutathione metabolism and ferroptosis pathways as primary targets (Figure 4C), with hierarchical clustering visualizing key DEG expression patterns (Figure 4D). To validate the biological relevance of these transcriptomic findings, GO functional enrichment analysis revealed ”chromosome segregation” as the most significantly upregulated biological process (Figure S14, Supporting Information), suggesting that LBPCDs may enhance the precision of chromosomal segregation, thereby reducing profibrotic factor secretion. Conversely, ”extracellular organization” was markedly downregulated, aligning with the anti-fibrotic effects of LBPCDs. GSEA further confirmed the enrichment of glutathione metabolism- and ferroptosis-related gene sets following LBPCDs treatment (Figure S15, S16, Supporting Information), supporting the hypothesis that LBPCDs mitigate renal fibrosis through dual regulation of glutathione homeostasis and ferroptosis. To identify molecular targets through which LBPCDs influence glutathione metabolism and inhibit ferroptosis, we integrated cellular thermal shift assays (CETSA) (Figures 4E, Figure S17, Supporting Information) with data-independent acquisition proteomics (Figure S18, Supporting Information). Target enrichment analysis from this approach highlighted the glutamate-cysteine ligase modifier subunit (GCLM) as a potential high-affinity target of LBPCDs (Figure 4F). Molecular docking further confirmed a strong interaction between LBPCDs and GCLM (binding energy: -9.7 kcal/mol; Figure S19, Supporting Information). Clinically, analysis of the GEO database revealed significant downregulation of GCLM in patients with AKI/DKD compared to healthy controls (Figure S20, Supporting Information), suggesting its deficiency contributes to renal pathogenesis and thus supporting its relevance as a therapeutic target. Mechanistically, GCLM serves as a critical regulatory component of glutathione (GSH) biosynthesis. As a modulatory subunit of glutamate-cysteine ligase (GCL) complex, GCLM associates with the catalytic subunit GCLC to form γ-glutamylcysteine synthetase (γ-GCS), enhancing catalytic efficiency and promoting de novo GSH synthesis, which is essential for maintaining redox homeostasis [28] . Disruption of GSH synthesis impairs GPX4 function, resulting in toxic lipid peroxide accumulation and ferroptosis. Profibrotic mediators such as TGF-β are known to exacerbate renal fibrosis by promoting redox imbalance, iron overload, and lipid peroxidation, collectively accelerating ferroptotic cell death [29, 30] . Of note, LBPCDs counteracted TGF-β-induced suppression of GPX4 activity (Figure S21, Supporting Information). In line with possible GCLM engagement, CETSA results indicated that LBPCDs stabilized GCLM under thermal challenge (Figure 4G). LBPCDs were also observed to promote GCLM–GCLC complex formation (Figures 4H, I), thereby enhancing γ-GCS activity (Figure 4J) and increasing the GSH/GSSG ratio (Figure 4K). As this ratio is a central indicator of cellular antioxidant capacity, its elevation helps maintain redox balance and suppresses ferroptosis [31], identifying LBPCDs as a potent modulator of this cell death pathway. Multi-dimensional validation further supported these findings. Western blot analysis demonstrated that LBPCDs dose-dependently upregulated key ferroptosis regulators, including γ-GCS catalytic subunit (GCLC), modulatory subunit (GCLM), cystine/glutamate antiporter SLC7A11, ferritin heavy chain (FTH1), and Glutathione Peroxidase 4 (GPX4) (Figures 4L, Figure S22, Supporting Information). This multi-target upregulation demonstrates concurrent activation of both glutathione metabolism and ferroptosis suppression pathways [32] . Specifically, LBPCDs dose-dependently attenuated TGF-β-induced accumulation of Fe²⁺ (Figure 4M), a core driver of ferroptotic cell death. By reducing the labile iron pool, LBPCDs directly suppress iron-dependent lipid peroxidation [33, 34] . Moreover, direct inhibition of lipid peroxidation represents another key anti-ferroptotic mechanism of LBPCDs. Live-cell imaging with BODIPY™ 581/591 C11 revealed that LBPCDs dose-dependently reversed TGF-β-induced lipid peroxidation, as indicated by restoration of the 581/591 nm to 488/510 nm fluorescence ratio (Figure 4N, Figure S23, Supporting Information). Biochemical assays further confirmed concentration-dependent suppression peroxidation markers, including reduced 4-HNE protein adducts (Figure S24, Supporting Information) and attenuated malondialdehyde (MDA) accumulation (Figure 4O). Critically, LBPCDs rescued HK-2 cells from diverse ferroptosis inducers, potently countering the effects of the GPX4 inhibitor RSL3 and the system Xc⁻ blocker Erastin (Figure S25, S26, Supporting Information) [35], achieving efficacy comparable to the ferroptosis inhibitor Ferrostatin-1 (Fer-1). This broad protection against multiple inducers establishes LBPCDs as a robust nano-inhibitor of ferroptotic cascades. Building on integrated omics and experimental validation, LBPCDs effectively inhibited ferroptosis. Treatment of HK-2 cells with the ferroptosis inducer RSL3, and with or without LBPCDs, showed that LBPCDs significantly attenuated ferroptosis-induced activation of fibrotic factors (Figure S27, Supporting Information). In summary, LBPCDs alleviate renal fibrosis largely through a GCLM-mediated protective cascade. By stabilizing the GCLM–GCLC complex and enhancing γ-GCS function, LBPCDs promote GSH biosynthesis, attenuate lipid peroxidation, and inhibit ferroptosis in renal tubular epithelial cells. Together, these actions reduce extracellular matrix accumulation and fibrotic remodeling (Figure 4P). These results not only propose GCLM as a promising target for LBPCD-based antifibrotic strategies but also provide important insights into the therapeutic potential of modulating the glutathione–ferroptosis axis in renal fibrosis. FIGURE 4 | LBPCDs Ameliorate Renal Fibrosis via γ-Glutamylcysteine Synthetase-Mediated Glutathione Homeostasis and Ferroptosis Suppression. ( A) Principal component analysis (PCA) of transcriptomic profiles. ( B) Volcano plot of differentially expressed genes (DEGs) in LBPCDs vs. Model groups. ( C) KEGG pathway enrichment of DEGs. ( D) Hierarchical clustering heatmap of selected DEGs. ( E) Cellular thermal shift assay (CETSA) workflow. ( F) LBPCDs-protein binding affinity scores. ( G) Thermal stabilization of GCLC and GCLM by LBPCDs in CETSA. Quantification of ( H) GCLC and ( I) GCLM protein thermal resistance. ( J) γ-glutamylcysteine synthetase (γ-GCS) activity. ( K) GSH/GSSG redox ratio. ( L) Western blot of ferroptosis regulators (GCLC, GCLM, SLC7A11, FTH1, and GPX4). ( M) Intracellular Fe 2+ levels. ( N) Lipid peroxidation imaging via BODIPY™ 581/591 C11 (red: non-oxidized; green: oxidized; scale bar = 20 μm). ( O) Malondialdehyde (MDA) quantification. (P) Proposed mechanism of LBPCDs-mediated fibrosis suppression. Statistical plot of relative fibrotic protein expression levels. For statistical analysis, data represent mean ± SD (n = 3). Statistical significance was calculated by using an unpaired two-tailed Student’s t-test. # p < 0.05, ## p < 0.01, ### p < 0.001, vs Ctrl group; * p < 0.05, ** p < 0.01, *** p < 0.001, vs TGF-β group. 2.5 LBPCDs Attenuate Cisplatin-Induced Renal Fibrosis via Ferroptosis Suppression Having established the renal fibrosis improvement potential of LBPCDs in vivo, we next sought to determine whether these effects translate to a physiological context by evaluating their therapeutic potential in a cisplatin (CDDP)-induced renal fibrosis mouse model. Mice were treated with CDDP (7 mg/kg, once weekly for four weeks), followed by administration of varying doses of LBPCDs (10, 20, 40 mg/kg, once every two days, i.p.) or the positive control drug Roxadustat [36] (10 mg/kg/d, oral gavage) with the experimental timeline and treatment regimen detailed in Figure 5A [37] . Compared to controls, CDDP-treated animals exhibited significant reductions in body weight and kidney weight. Notably, both Roxadustat and LBPCDs effectively restored these weight parameters to near-baseline levels (Figure 5B, C; Figure S28, Supporting Information). Consistent with these findings, biochemical analyses confirmed that LBPCDs attenuated CDDP-induced renal dysfunction. Specifically, LBPCDs significantly lowered blood urea nitrogen (BUN) and serum creatinine levels [38], which are key biomarkers of kidney dysfunction (Figure 5D, E). Furthermore, LBPCDs suppressed fibrotic progression, as evidenced by reduced expression of the key fibrosis markers α-smooth muscle actin (α-SMA) and collagen type I (Col 1) (Figure 5F, G). Comprehensive histological assessments revealed robust protective effects of LBPCDs across multiple renal compartments. H&E staining demonstrated preserved renal architecture in LBPCDs treated mice, with attenuated glomerular basement membrane thickening, diminished extracellular matrix deposition, and normalized tubular structure compared to the CDDP-only group (Figure 5H). Corroborating the reduction in fibrosis markers, Masson trichrome staining demonstrated diminished collagen deposition in tubules and glomeruli. Immunohistochemistry further confirmed reduced α-SMA-positive areas and peritubular fibrosis (Figure 5H). Critically, these structural improvements coincided with direct targeting of our previously identified molecular axis: immunohistochemical staining revealed significantly enhanced GCLM-positive signals in renal tissues following LBPCD treatment, confirming GCLM as a direct in vivo molecular target. Concurrently, LBPCDs markedly suppressed accumulation of 4-hydroxynonenal (4-HNE), a key lipid peroxidation byproduct mechanistically linked to ferroptosis, providing histological evidence of ferroptosis inhibition in CDDP-injured kidneys (Figure 5H). Collectively, these results establish that LBPCDs exert potent therapeutic effects in vivo by alleviating CDDP-induced renal injury and fibrosis progression, mediated through their established mechanism of GCLM-dependent ferroptosis suppression. FIGURE 5 | LBPCDs Attenuate Cisplatin-Induced Renal Fibrosis via Ferroptosis Suppression. ( A) Schematic diagram of the construction and treatment of CDDP-induced renal fibrosis mice. ( B) Weight and ( C) Kidney weight changes after different treatments. ( D) Serum creatinine and ( E) urea nitrogen levels after different treatments. (F, G) Expression of pro- fibrotic factors (α-SMA and Col 1) in the kidneys of renal fibrosis model mice after different treatments. ( H) Representative immunohistochemical images showing H&E and Masson staining, α-SMA (fibrosis marker), 4-HNE (ferroptosis marker), and GCLM (LBPCDs target) expression in kidney tissues across experimental groups (scale bar = 50 μm). For statistical analysis, data represent mean ± SD (n ≥ 5). Statistical significance was calculated by using an unpaired two-tailed Student’s t-test. ## p< 0.01, ### p < 0.001, vs Ctrl group; ns p≥0.05, * p < 0.05, ** p< 0.01, *** p < 0.001, vs Model group. 2.6 LBPCDs Attenuate Renal Fibrosis in Diabetic Kidney Disease Mice by Inhibiting Ferroptosis To further evaluate the renoprotective effects of LBPCDs, we employed a well-established diabetic kidney disease (DKD) mouse model induced by a high-fat diet (HFD) and streptozotocin (STZ) administration (Figure 6A) [39, 40] . DKD, a major microvascular complication of diabetes, induces renal fibrosis and ultimately progresses to end-stage renal disease. Sustained hyperglycemia drives metabolic dysregulation and cellular damage, prompting injured cells to release profibrotic signals. Critically, LBPCDs treatment not only improved renal function but also exhibited a glucose-independent renoprotective effect, highlighting its unique therapeutic advantage (Figure 6B). Notably, this glucose independence aligns with a similar observation reported by Sourris et al [41] . Compared with untreated DKD mice, LBPCDs intervention also increased both body weight and kidney weight (Figure 6C, Figure S29, S30, Supporting Information). Biochemical analysis confirmed the characteristic renal impairment in DKD mice, as evidenced by significantly elevated levels of urinary protein, serum creatinine, and blood urea nitrogen compared to controls (Figure 6D-F). There is a strong pathological association between renal injury indicators and renal fibrosis, where these biomarkers act as both drivers of fibrogenesis and indicators of fibrotic severity. Both metformin and LBPCDs dose-dependently attenuate these renal injury markers, with LBPCDs demonstrating potent renoprotection. At the molecular level, LBPCDs effectively suppressed DKD-induced upregulation of fibrotic markers α-SMA and Col 1 (Figure 6G, H), matching the anti-fibrotic efficacy of metformin. In addition, serum iron and triglycerides synergistically accelerate renal fibrosis through ferroptosis. Diabetic mice exhibited significant hypertriglyceridemia and elevated serum iron which are the key drivers of ferroptosis , while LBPCDs treatment normalized (Figure 6I, J) [42, 43] . Complementary histological analysis (H&E/PAS staining) demonstrated that LBPCDs ameliorated characteristic diabetic nephropathological features, including glomerular hypertrophy, mesangial expansion, and glycogen deposition. Consistently, Masson’s trichrome staining revealed that LBPCDs dose-dependently reduced the pathognomonic mesangial collagen deposition and matrix expansion (Figure 6K). Immunohistochemical analysis corroborated anti-fibrotic effects of LBPCDs by showing reduced α-SMA-positive areas and diminished extracellular matrix deposition in treated kidneys versus untreated DKD controls (Figure 6K). Of mechanistic significance, LBPCDs enhanced renal expression of GCLM while attenuating key ferroptosis markers. This confirms the target-specific activation of the glutamate-cysteine ligase modifier subunit. Concurrently, LBPCDs suppressed the accumulation of 4-HNE, a lipid peroxidation end-product (Figure 6K). The coordinated suppression of multiple ferroptotic drivers underscores multi-mechanistic renoprotection of LBPCDs in DKD. Taken together, these results establish that GCLM-targeted ferroptosis suppression by LBPCDs underpins their potent renoprotective efficacy in diabetic kidney disease. FIGURE 6 | LBPCDs Attenuate Renal Fibrosis in Diabetic Kidney Disease Mice by Inhibiting Ferroptosis. ( A) Experimental timeline for high-fat diet/streptozotocin (HFD/STZ)-induced diabetic kidney disease (DKD) model and therapeutic regimen. (B) Fasting blood glucose levels. (C) Kidney weight and (D) urinary albumin excretion. Renal function biomarkers: (E) Serum creatinine and (F) blood urea nitrogen (BUN). (G, H) Expression of pro-fibrosis factors (α-SMA and Col 1) in the kidneys of DKD model mice after different treatments. (I) Serum triglycerides (TG) and, (J) iron (Fe) concentrations. (K) Representative renal histology: H&E (structural integrity), PAS (glycoprotein accumulation), Masson trichrome (collagen deposition). Immunohistochemistry for α-SMA (fibrosis), 4-HNE (lipid peroxidation), and GCLM (target engagement). Scale bar = 50 μm. For statistical analysis, data represent mean ± SD (n ≥ 5). Statistical significance was calculated by using an unpaired two-tailed Students t-test. ## p< 0.01, ### p < 0.001, vs Ctrl group; ns p ≥ 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, vs Model group. 2.7 LBPCDs Exhibit Favorable Biocompatibility The biosafety of ultrasmall carbon nanoparticles remains a key research focus. Consistent with this priority, our in vitro cytotoxicity assessment in NCM460 (colon), HUVEC (endothelium), and HK-2 (renal) cell lines revealed > 100% viability after 24 h exposure to 400 μg/mL LBPCDs (Figure 7A, B, Figure S31, Supporting Information), indicating excellent cytocompatibility and even suggesting potential proliferative effects. Hemocompatibility, a key prerequisite for nanotherapeutics, was further evaluated. Spectrophotometric analysis of hemolysis showed that LBPCDs (≤ 800 μg/mL) preserved erythrocyte integrity after 2 h incubation, supported by visual inspection confirming the absence of hemolysis (Figure 7C). The biocompatibility profile was further corroborated by outstanding systemic biosafety observed in vivo . A 7-day high-dose administration (100 or 500 mg/kg) induced no significant changes in hematological parameters, including white blood cell count, lymphocyte levels, platelet count, and red blood cell count, relative to control groups (Figure S32, Supporting Information ). Serum biomarkers of organ function, specifically liver enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST]) and renal indicators (urea nitrogen, creatinine), remained stable throughout the treatment period, with no treatment-related fluctuations (Figure 7D-G). Histopathological examination of major organs (heart, liver, spleen, lungs, and kidneys) via H&E staining further revealed intact tissue architecture without evidence of structural abnormalities or inflammatory infiltration (Figure 7H, Figure S33, Supporting Information ). These comprehensive in vitro and in vivo analyses unequivocally confirm the exceptional biocompatibility of this plant-derived nanotherapeutic platform. Additionally, to characterize the tissue targeting and pharmacokinetic profile of LBPCDs, we conducted near-infrared fluorescence imaging after intravenous administration of CY5-labeled LBPCDs (LBPCDs@CY5) via tail vein injection. Real-time tracking over 24 h revealed peak systemic distribution at 6 h (Figure 7I, J). Subsequent organ-specific quantification demonstrated predominant accumulation in renal tissue, with significantly higher fluorescence intensity compared to other major organs (heart, liver, spleen, and lungs) (Figure 7K, L). Notably, hepatic retention remained low (Figure S34, Supporting Information), as further quantified by the kidney-to-liver fluorescence ratio (Figure S35, Supporting Information). These data collectively establish LBPCDs as a promising novel nanoplatform for precision therapeutic intervention in kidney diseases. FIGURE 7 | LBPCDs Exhibit Favorable Biocompatibility and Renal-Targeting Specificity. (A) In vivo time-course fluorescence imaging and (B) quantified renal fluorescence intensity of LBPCDs@CY5 (2.5 mg/kg, i.v.) over 24 h. (C) Ex vivo organ biodistribution (heart, liver, spleen, lungs, kidneys) and (D) renal accumulation efficiency. In vitro cytotoxicity of LBPCDs (0-400 μg/mL, 24 h) in (E) NCM460 and (F) HUVEC cells. (G) Hemolysis assay using mice erythrocytes (0-800 μg/mL). Serum biochemistry after high-dose LBPCDs (100/500 mg/kg): Hepatic enzymes of (H) ALT and (I) AST, (J) Blood urea nitrogen (BUN), (K) Creatinine. (L) Major H&E-stained organs showing structural integrity (scale bar = 100 μm). 3. Discussion Our study pioneers the development of Lycium barbarum polysaccharide-derived carbon dots (LBPCDs) as a multifunctional nanotherapeutic that concurrently targets mitochondrial oxidative stress and ferroptosis to combat renal fibrosis. By reorganizing LBP into sub-5 nm phytocarbon dots with defect-rich sp²/sp³ hybrid frameworks, we have overcome the inherent pharmacokinetic limitations of native polysaccharides, including rapid clearance and poor bioavailability. Critically, surface functionalization of these nanocarriers confers dual advantages: exceptional biocompatibility (as validated by in vitro cytotoxicity and in vivo systemic safety assays) and renal accumulation (driven by size-dependent accumulation and charge interactions), enabling precise delivery of therapeutic payloads to renal tissues. Through this structural and functional engineering, LBPCDs effectively neutralize reactive oxygen species (e.g., ·OH, ·O₂⁻), chelate free iron ions, and suppress lipid peroxidation-driven ferroptosis [44, 45] . Furthermore, LBPCDs represent the first-in-class agent targeting the glutamate-cysteine ligase modifier subunit (GCLM) to reprogram glutathione metabolism. This action enhances γ-glutamylcysteine synthetase (γ-GCS) activity and elevates the GSH/GSSG ratio, further fortifying cellular defenses against ferroptosis. Importantly, LBPCDs achieve high renal accumulation without compromising systemic safety, exhibiting no detectable off-target toxicity. These findings position LBPCDs as a transformative and safe nanotherapeutic strategy for the treatment of kidney fibrosis and related disorders. Renal fibrosis arises from a complex interplay of oxidative stress [46, 47] . Excessive ROS generation and compromised antioxidant defenses drive cellular damage and directly activate pro-fibrotic TGF-β pathways [48, 49] . Significantly, the spatial targeting capability of LBPCDs disrupts the ROS-fibrosis vicious cycle. Their hydroxyl groups facilitate electron transfer for ROS neutralization, while their sub-5 nm size and negative charge enable mitochondrial enrichment (Pearson’s coefficient: 0.91). By neutralizing mtROS at the primary generation site, LBPCDs prevent TGF-β-induced overexpression of fibrosis factors (e.g., α-SMA, Col 1), thereby interrupting collagen deposition and extracellular matrix expansion observed in Masson staining. This organelle-precise intervention represents a paradigm shift from broad-spectrum antioxidants. Ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation, serves as a critical pathogenic nexus linking metabolic dysfunction and renal fibrosis. Following renal injury (e.g., ischemia, toxins, diabetes), tubular epithelial cells (TECs) are particularly susceptible to ferroptosis due to their high metabolic rate, polyunsaturated fatty acid-rich membranes, and iron handling mechanisms. Ferroptotic TEC death releases damage-associated molecular patterns (DAMPs), redox-active iron, and lipid peroxidation products [50, 51] . These molecules potently activate adjacent fibroblasts and recruit/polarize macrophages towards a pro-fibrotic M2 phenotype. Activated fibroblasts proliferate and differentiate into collagen-secreting myofibroblasts, ultimately leading to excessive extracellular matrix deposition and renal fibrosis. Our multi-model investigations spanning TGF-β-stimulated HK-2 cells, cisplatin-induced renal fibrosis mice, and HFD/STZ-induced diabetic nephropathy mice, consistently demonstrated LBPCDs potently significantly reducing lipid peroxidation markers (MDA, 4-HNE) and pathological iron accumulation to relieve renal fibrosis. We identify GCLM as the linchpin target, with LBPCDs binding GCLM at -9.7 kcal/mol to stabilize the GCLC-GCLM heterodimer (γ-GCS, GSH synthesis rate-limiting enzyme), boosting glutathione biosynthesis and GPX4-mediated lipid peroxide reduction. Mechanistically, LBPCDs counteract ferroptosis drivers concurrent lipid peroxide accumulation and glutathione (GSH) depletion [52] through dual-pathway modulation: targeting glutamate-cysteine ligase modifier subunit (GCLM) to restore GSH biosynthesis while enhancing glutathione peroxidase 4 (GPX4) activity. This coordinated action reduced tubular epithelial cell death and extracellular matrix deposition, attenuating fibrosis across acute and chronic injury models. Compared to conventional renal therapies, LBPCDs offer distinct therapeutic advantages rooted in their natural LBP origin, which ensures inherent biocompatibility and reduces the risk of adverse effects [53] . Notably, their nanoscale dimensions (< 5 nm) synergize with intrinsic mitochondrial and renal tropism to maximize therapeutic delivery to target tissues while minimizing off-target distribution. This platform’s multimodal mechanism, simultaneously addressing mtROS overload, GSH metabolism dysregulation, and ferroptosis, positions it as a versatile therapeutic candidate for fibrosis-related kidney diseases. Despite these advancements, several challenges remain for clinical translation. First, long-term biodistribution, clearance pathways, and cumulative toxicity require comprehensive preclinical characterization. Second, rigorous pharmacokinetic (PK) and pharmacodynamic (PD) profiling is needed to optimize dosing regimens. Third, evaluating synergies with standard-of-care therapies (e.g., ACE inhibitors, immunosuppressants) will be critical for real-world applicability. Finally, developing standardized clinical-grade manufacturing protocols and conducting large-animal safety studies will be essential to bridge the gap from bench to bedside. 4. Conclusion This work pioneers atomically precise carbonization of Lycium barbarum polysaccharides into therapeutic phytocarbon nanodots (LBPCDs), successfully overcoming the pharmacokinetic limitations of natural polysaccharides and endowing them with excellent renal accumulation capabilities. This nanodrug operates via multiple mechanisms, including scavenging reactive oxygen species, inhibiting ferroptosis, and regulating glutathione metabolism (enhancing γ-glutamylcysteine synthetase activity through interaction with GCLM), synergistically ameliorating oxidative stress, reversing epithelial-mesenchymal transition, and extracellular matrix remodeling. It demonstrated significant anti-fibrotic effects and favorable biosafety in both cisplatin-induced and diabetic renal fibrosis models. This work pioneers a novel “material-is-the-drug” therapeutic paradigm using phytocarbon dots, offering a sustainable and translatable Herb-Nano platform strategy for the treatment of fibrotic diseases. 5. Experimental Section Experimental details are provided in the Supporting Information.

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Authors Metrics & Citations Metrics Article Usage 227views 139downloads Citations Download citation Haojia Li, Nannan Song, Ping Huang, et al. Phytocarbon Nanodots Restore Glutathione Homeostasis via γ-Glutamylcysteine Synthetase against Ferroptosis-Driven Renal Fibrosis. Authorea. 24 September 2025. DOI: https://doi.org/10.22541/au.175874423.33548868/v1 DOI: https://doi.org/10.22541/au.175874423.33548868/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.