Compartment-specific Zinc Misallocation in Diabetic Foot Ulcers: Mechanistic Coupling Between Macrophage M1 Locking and MMP-9 Hyperactivation

Abstract

Diabetic foot ulcer (DFU) remains a formidable clinical challenge characterized by a persistent failure of the regenerative process. Affecting approximately 15–25% of diabetic individuals over their lifetime, DFU is associated with a disproportionate risk of lower-limb amputation and mortality. Although zinc is a fundamental requirement for cutaneous repair, conventional supplementation strategies often yield inconsistent therapeutic outcomes. This review addresses this clinical gap by introducing the Zinc Misallocation Hypothesis, which posits that compartment-specific collapse of ionic partitioning represents a mechanistically distinct contributor to DFU chronicity, independent of total zinc deficiency. Under conditions of chronic hyperglycemia and oxidative stress, a pathological spatiotemporal decoupling occurs, leading to functional intracellular Zn²⁺ scarcity alongside a destructive accumulation of labile Zn²⁺ in the extracellular matrix. This ionic misallocation establishes a dual-track inhibitory mechanism that may contribute to sustained repair impairment. Intracellularly, functional Zn²⁺ depletion disrupts the structural integrity of zinc-finger regulatory proteins such as A20 and CYLD, leading to the sustained disinhibition of the NF-κB signaling axis. This biochemical locking prevents the transition of macrophages from a pro-inflammatory M1 state to a pro-reparative M2 phenotype. Simultaneously, the pathological saturation of Zn²⁺ in the extracellular environment facilitates the hyperactivation of matrix metalloproteinases, specifically MMP-9, while concurrently inactivating endogenous inhibitors such as TIMP-1. The markedly elevated proteolytic activity triggers extensive degradation of the basement membrane and induces a state of multi-lineage cellular arrest, where the migratory and proliferative capacity of keratinocytes, fibroblasts, and endothelial cells is substantially attenuated. To resolve this biochemical stalemate, we propose a reciprocal regulatory paradigm utilizing advanced responsive delivery systems, such as ROS-sensitive nanocarriers and metal-organic frameworks. These precision platforms facilitate a dual-action operation designed to sequester pathogenic extracellular zinc while selectively replenishing functional intracellular pools. Ultimately, the restoration of compartment-specific zinc homeostasis represents a rational therapeutic objective for attenuating the self-reinforcing pathological state of the chronic diabetic wound.

Introduction

Diabetic foot ulcer (DFU) represents one of the most severe and clinically intractable complications of diabetes mellitus, contributing significantly to global morbidity and the escalating burden on healthcare systems. Despite advancements in standard wound care, the therapeutic resolution of DFU remains persistently inadequate, often culminating in lower-limb amputations [1]. Traditional wound management has long recognized zinc (Zn) as a fundamental trace element pillar, essential for over 300 enzymatic reactions, protein synthesis, and cellular defense mechanisms [2]. Yet the biological relevance of zinc extends beyond its absolute concentration; its therapeutic efficacy is critically dependent on its precise subcellular distribution. Consequently, clinical outcomes following systemic or non-targeted zinc supplementation in DFU patients have been inconsistent, often failing to reach the threshold of significant regenerative impact [3]. This therapeutic gap suggests that the pathology of the diabetic wound is not merely a consequence of total zinc deficiency, but rather a sophisticated failure of spatial homeostasis [4].

Current evidence indicates that the diabetic microenvironment, characterized by persistent hyperglycemia and oxidative stress, induces a profound collapse of cutaneous zinc partitioning. This review introduces the Zinc Misallocation Hypothesis, a conceptual framework which posits that compartment-specific redistribution of Zn²⁺ constitutes one mechanistically distinct contributor to the failure of DFU healing, operating alongside other established pathological drivers including peripheral neuropathy, microvascular insufficiency, and advanced glycation end-product accumulation. It should be noted that several mechanistic links within this framework are inferred from in vitro studies or indirect clinical observations, and direct in vivo validation of the proposed ionic partitioning remains limited. This hypothesis is therefore intended as a conceptual scaffold to guide future empirical investigation rather than a definitive mechanistic account. In this pathological state, a critical divergence occurs: the intracellular compartment suffers from functional Zn²⁺ scarcity, while the extracellular matrix (ECM) is subjected to pathological Zn²⁺ saturation. This spatiotemporal decoupling creates a dual-track inhibitory mechanism that may maintain the wound in a self-perpetuating cycle of inflammation and structural dissolution.

Intracellularly, the depletion of functional Zn²⁺ serves as a molecular trigger for the disinhibition of the NF-κB signaling pathway. The resulting loss of zinc-dependent coordination in regulatory proteins such as A20 and CYLD is associated with retention of macrophages in a predominant pro-inflammatory M1 state, impairing the transition to the pro-reparative M2 phenotype [5]. Simultaneously, the accumulation of labile Zn²⁺ in the ECM provides the mandatory catalytic requirements for the hyperactivation of matrix metalloproteinases, specifically MMP-9. This elevation in proteolytic activity, often exacerbated by the oxidative inactivation of endogenous inhibitors such as TIMP-1, drives extensive degradation of the basement membrane and the loss of structural prerequisites for re-epithelialization [6].

By interrogating the molecular architecture of zinc-mediated wound failure, this review traces the mechanistic consequences of ionic misallocation from transporter dysfunction to multi-lineage cellular arrest. We further explore the transition from mechanistic elucidation to clinical application, highlighting the potential of pH and ROS-responsive delivery systems to achieve precise spatial redistribution. Ultimately, the successful management of DFU requires a reciprocal regulatory paradigm: a dual-action strategy designed to clear pathogenic extracellular zinc while selectively replenishing functional intracellular pools. By addressing these ionic dynamics, the restoration of spatial zinc homeostasis represents a rational objective for attenuating the self-reinforcing pathological state of the diabetic wound.

Zinc Misallocation and Homeostasis Failure in DFU

The maintenance of cutaneous zinc (Zn) homeostasis represents a strict metabolic and coordinative requirement determined by the precise partitioning of the ion between the cytosol, specialized organelles, and the extracellular matrix (ECM). In the chronic microenvironment of a DFU, this equilibrium undergoes a compartment-specific collapse. This state is characterized by the depletion of intracellular Zn²⁺ alongside its pathological accumulation in the extracellular space. This redistribution serves as a primary biochemical driver of impaired tissue repair, initiating a cascade of enzymatic and transcriptional failures that prevent effective wound closure [7].

Several lines of indirect clinical and experimental evidence are consistent with the compartment-specific zinc dysregulation proposed here. Serum zinc concentrations have been reported to be significantly reduced in patients with type 2 diabetes mellitus presenting with active foot ulceration compared with those without ulcers, with lower zinc levels independently associated with delayed wound closure [8, 9]. At the wound site level, MMP-9 concentrations in chronic DFU exudate are markedly elevated in non-healing compared with healing ulcers, with wound fluid MMP-9 and the MMP-9-to-TIMP-1 ratio correlating inversely with healing rate at 28 days (P < 0.001) and predicting 12-week healing outcome with an area under the curve of 0.94, underscoring the dysregulated proteolytic milieu of the chronic wound compartment [10, 11]. At the cellular level, high-glucose conditions have been shown to alter the expression and phosphorylation of ZIP and ZnT family transporters, specifically upregulating ZIP7 while downregulating ZnT7, resulting in measurable redistribution of free Zn²⁺ between subcellular compartments as confirmed by real-time FRET-based imaging [12]; analogous transporter dysregulation in wound-resident cells is plausible given shared glucose-sensing machinery, though direct evidence in keratinocytes and fibroblasts remains limited. While direct compartment-specific quantification of labile Zn²⁺ in human DFU tissue remains technically challenging and has not yet been reported, these converging observations provide preliminary empirical support for the ionic partitioning failure central to this hypothesis.

Dysregulation of Zinc Transporters (ZIPs and ZnTs)

The regulation of cytosolic Zn²⁺ bioavailability is contingent upon the reciprocal coordination between the SLC39A (ZIP) and SLC30A (ZnT) transporter families. Persistent hyperglycemia and the associated metabolic strain in the DFU environment are frequently associated with the chronic activation of the Unfolded Protein Response (UPR), involving the PERK/eIF2α and IRE1α/XBP1 signaling axes. These pathways have been implicated in the transcriptional modulation of several zinc transporters, including specific ZIP isoforms such as ZIP7, ZIP10, and ZIP14, in keratinocytes and dermal fibroblasts [13]. The resulting attenuation of Zn²⁺ influx, which may involve the disruption of ZIP7-mediated zinc release from the endoplasmic reticulum, precipitates a state of intracellular Zn²⁺ depletion. Such a deficit alters the structural stability of zinc-finger motifs within transcription factors required for cellular plasticity, including Sox2 and KLF4. A critical biochemical consequence of this depletion is the loss of Zn²⁺ mediated inhibition of Protein Tyrosine Phosphatase 1B (PTP1B). Under Zn²⁺ deficient conditions in the DFU bed, overactive PTP1B facilitates the dephosphorylation of the Insulin Receptor β-subunit at the Tyr1162 and Tyr1163 residues. This catalytic shift extends its inhibitory effect to the downstream PI3K/Akt and MAPK/ERK pathways, reducing the phosphorylation of Akt at Ser473 and Thr308 [14]. This signaling arrest prevents the translocation of GLUT4 to the plasma membrane and inhibits the actin cytoskeleton remodeling required for coordinated cellular mobilization (Fig. 1).

Fig. 1.

Molecular drivers of compartment-specific zinc misallocation in the DFU microenvironment. (A) Dysregulation of ZIP and ZnT zinc transporters. Under conditions of chronic hyperglycemia and oxidative stress, activation of the UPR (PERK/eIF2α and IRE1α/XBP1 axes) suppresses ZIP10/14-mediated Zn²⁺ influx across the plasma membrane and disrupts ZIP7-dependent Zn²⁺ release from the endoplasmic reticulum. Concurrently, AGE-driven overexpression of ZnT1 promotes unscheduled Zn²⁺ efflux into the extracellular space. The resulting intracellular Zn²⁺ depletion disinhibits PTP1B, which dephosphorylates the Insulin Receptor β-subunit at Tyr1162/1163, collapsing downstream PI3K/Akt and MAPK/ERK signaling and arresting GLUT4 translocation and actin cytoskeleton remodeling. (B) ROS-driven metallothionein oxidation and pathological zinc liberation. Mitochondria-derived ROS (H₂O₂, ·OH) and AGEs oxidize the thiolate clusters of MT-1/2, converting Cys-SH groups to disulfide, sulfenic, and sulfinic acid derivatives and triggering the unregulated release of sequestered Zn²⁺ from the α-domain (Zn₄Cys₁₁) and β-domain (Zn₃Cys₉). Oxidative inactivation of MTF-1 prevents MT resynthesis, eliminating intracellular buffering capacity. Liberated Zn²⁺ migrates to the extracellular compartment via damaged membranes and hyperactive ZnT1, where it accumulates as labile ECM Zn²⁺, activates Pro-MMP-9 via the cysteine switch mechanism, and drives the degradation of Type IV collagen, laminin, and elastin within the basement membrane. Intracellular Zn²⁺ depletion simultaneously inactivates Zn-dependent SOD, further exacerbating the oxidative burden. AGEs, advanced glycation end-products; MT, metallothionein; MTF-1, metal-responsive transcription factor-1; ROS, reactive oxygen species; SOD, superoxide dismutase; UPR, unfolded protein response

This influx deficiency is further exacerbated by the aberrant activity and localization of the SLC30A family, notably the plasma membrane exporter ZnT1. In response to the high-glucose milieu and localized elevations of advanced glycation end-products (AGEs), dysregulated ZnT1 promotes the unscheduled efflux of Zn²⁺ into the extracellular environment [15]. This loss of retention prevents the maintenance of the physiological concentration gradients necessary for intracellular signaling and the proper loading of zinc-dependent cofactors into newly synthesized proteins. The synergy between reduced ZIP-mediated intake and increased ZnT-mediated export transforms the cell interior into a zone of functional Zn²⁺ scarcity. This spatiotemporal decoupling undermines the metabolic resilience of resident skin cells and disrupts essential intercellular communication. For instance, the CXCL12/CXCR4 signaling axis, which is vital for the recruitment of bone marrow-derived progenitor cells to the wound site, becomes impaired under these conditions. Consequently, the lack of functional intracellular Zn²⁺ anchors wound cells in a non-migratory and metabolically arrested state. This failure prevents the transition from the inflammatory phase to active tissue synthesis, particularly affecting re-epithelialization and granulation tissue formation through the inhibition of fibroblast differentiation into α-SMA positive myofibroblasts [16].

ROS-Induced Zinc Release from Metallothioneins (MTs)

Intracellular Zn²⁺ buffering and redox sensing are primarily managed by the MT-1 and MT-2 isoforms. These low-molecular-weight proteins utilize twenty conserved cysteine residues to coordinate seven Zn²⁺ ions per molecule across two distinct domains: the α-domain (Zn4Cys11) and the β-domain (Zn3Cys9). These clusters are characterized by a tetrahedral coordination geometry. In the oxidative microenvironment of a diabetic ulcer, the accumulation of mitochondrial-derived reactive oxygen species (ROS) and AGEs targets these high-affinity clusters. The interaction between ROS, such as hydrogen peroxide (H₂O₂) and hydroxyl radicals (·OH), results in the oxidation of the cysteine thiol groups into intramolecular disulfide bonds or higher-order sulfenic and sulfinic acid derivatives. This oxidative modification leads to the sustained and unregulated liberation of sequestered Zn²⁺ ions from their thiolate coordination sites into the cytoplasm [17]. This marks a fundamental transition from redox-sensitive signaling to pathological ion mobilization. In an environment already compromised by transport dysfunction, the sudden surge in free intracellular Zn²⁺ ensures that liberated ions are not effectively re-buffered but instead migrate toward the extracellular compartment.

The potential for cellular recovery in this context is further limited by the inactivation of Metal-responsive Transcription Factor-1 (MTF-1). Under the conditions of chronic oxidative stress prevalent in DFUs, MTF-1 may fail to bind effectively to the Metal-responsive Elements (MREs) located in the promoter regions of MT genes [18]. This binding failure is potentially due to the oxidation of its own zinc-finger DNA-binding domain. The resulting loss of MT resynthesis leaves the cell unable to restore its buffering capacity, allowing liberated Zn²⁺ to migrate into the extracellular matrix through damaged lipid bilayers or hyperactive ZnT1 exporters. This spatial shift establishes a biochemical divergence where functional intracellular depletion undermines antioxidant defenses, including the catalytic activity of zinc-dependent superoxide dismutase (SOD). Simultaneously, the accumulation of labile Zn²⁺ in the extracellular matrix provides the mandatory catalytic requirements for the hyperactivation of matrix metalloproteinases (MMPs), especially MMP-9. This activation proceeds via the “cysteine switch” mechanism. In this process, the displacement of the pro-domain cysteine residue from the catalytic Zn²⁺ ion by the excess extracellular ions converts the inactive pro-enzyme into its proteolytic form [5]. The resulting hyperactivation of MMP-9 leads to the extensive degradation of Type IV collagen, laminin, and elastin within the basement membrane. This compartmental redistribution converts a regulatory ion into a driver of matrix destruction. The persistent breakdown of structural proteins ensures the refractory nature of the diabetic wound bed and prevents the stabilization of the provisional matrix required for effective re-epithelialization (Fig. 1B) [19, 20].

The Immune Stalemate: Macrophage M1 Locking

The resolution of the inflammatory phase in cutaneous wound healing depends on the timely transition of macrophage phenotypes from a pro-inflammatory M1 state to a pro-reparative M2 state. In the microenvironment of a diabetic foot ulcer, this transition is substantially impaired [21]. The compartment-specific depletion of intracellular Zn²⁺ is proposed to function as one molecular constraint on this immune plasticity, where cytosolic Zn²⁺ deficit may impair the timely silencing of pro-inflammatory signaling cascades, contributing to retention of macrophages in a predominant M1 state characterized by sustained secretion of pro-inflammatory cytokines [22].

Intracellular Zinc Depletion and NF-κB Disinhibition

The persistent activation of the Nuclear Factor-κB (NF-κB) signaling pathway represents a primary biochemical driver of the non-healing state in DFU-resident macrophages. Under normophysiological conditions, intracellular Zn²⁺ contributes to the modulation of the IκB kinase (IKK) complex, the gatekeeper of NF-κB activation. This modulation is potentially influenced by zinc-sensitive phosphatases, such as Protein Phosphatase 2 A (PP2A), which are implicated in the dephosphorylation of the IKKβ catalytic subunit at its activation loop. In the Zn²⁺-depleted environment of a diabetic macrophage, the loss of this inhibitory control facilitates the autonomous hyperactivation of IKKβ [23]. The resulting catalytic overactivity leads to the site-specific phosphorylation of IκBα at the Ser32 and Ser36 residues. This phosphorylation serves as a molecular signal for the recruitment of the β-TrCP (beta-transducin repeat-containing protein) E3 ubiquitin ligase complex, initiating the K48-linked polyubiquitination and subsequent proteasomal degradation of IκBα. Consequently, the sustained nuclear translocation and occupancy of p65/p50 heterodimers drive the relentless transcription of pro-inflammatory mediators including TNF-α, IL-1β, and IL-6 [24].

The failure to terminate this inflammatory signaling is further compounded by the functional impairment of zinc-dependent deubiquitinating enzymes (DUBs), specifically the cylindromatosis (CYLD) protein. CYLD is a specialized DUB that restricts NF-κB activation by cleaving K63-linked polyubiquitin chains from signaling adapters such as TRAF2 and NEMO (IKKγ). The structural integrity and catalytic efficiency of the CYLD USP (ubiquitin-specific protease) domain are highly sensitive to local Zn²⁺ concentrations. Zinc coordination is required to maintain the functional conformation of the cysteine-rich pockets within the USP domain, which is essential for substrate recognition and catalytic turnover. In the Zn²⁺-deficient environment of the DFU wound bed, the destabilization of this domain prevents the timely disassembly of the IKK-activating signaling scaffold [25]. This enzymatic failure ensures that upstream signals from Toll-like receptor (TLR) and TNF receptor (TNFR) families are not attenuated but instead continue to feed into the IKK complex. The resulting inability of the macrophage to execute this essential negative feedback loop leads to chronic immune hyper-responsiveness, further obstructing the initiation of the reparative phase.

A major molecular mechanism contributing to this sustained disinhibition involves the functional and structural compromise of the zinc-finger protein A20, also known as Tumor Necrosis Factor α-Induced Protein 3 (TNFAIP3). A20 serves as a critical dual-function regulator of the NF-κB pathway, exhibiting both K63-deubiquitinase and K48-E3 ubiquitin ligase activities. The structural integrity of A20 is defined by its seven C-terminal zinc-finger (ZnF) motifs. Notably, ZnF4 is required for the specific recognition and binding of K63-linked polyubiquitin chains on RIP1, while ZnF7 facilitates the direct interaction with the NEMO subunit. These domains require Zn²⁺ ions to coordinate their cysteine-rich scaffolds and maintain the functional conformation necessary for signal termination. In the Zn²⁺-deficient milieu of a DFU, the structural destabilization of these ZnF domains leads to the functional inactivation of A20 [4]. This loss of ubiquitin-editing control prevents the displacement of RIP1 from the signaling complex and the removal of K63-linked chains from TRAF6. Effectively, the macrophage is trapped in a constitutively active configuration, where the biochemical machinery required for signal resolution is structurally compromised by the lack of Zn²⁺-dependent coordination. This molecular locking of the M1 phenotype prevents the restoration of immune homeostasis and ensures the persistent degradative state of the diabetic wound (Fig. 2A) [26].

Fig. 2.

Intracellular zinc depletion as the molecular constraint on macrophage immune plasticity in DFU. (A) NF-κB disinhibition and M1 phenotype locking. Intracellular Zn²⁺ depletion abolishes PP2A-mediated inhibition of IKKβ, driving its autonomous hyperactivation. Phosphorylation of IκBα at Ser32/Ser36 recruits the β-TrCP E3 ligase, triggering K48-linked polyubiquitination and proteasomal degradation of IκBα, thereby enabling sustained p65/p50 nuclear translocation and transcription of TNF-α, IL-1β, and IL-6. Negative feedback is simultaneously disabled through two parallel mechanisms: Zn²⁺ deficiency destabilizes the CYLD USP domain, preventing K63-chain disassembly from TRAF2 and NEMO and perpetuating IKK scaffold assembly; and structural collapse of the A20/TNFAIP3 zinc-finger motifs (ZnF4, ZnF7) abolishes ubiquitin editing of RIP1 and TRAF6. The convergence of unattenuated TLR/TNFR upstream input and failed negative feedback locks the macrophage in a state of constitutively active NF-κB and chronic inflammatory stasis. (B) Dual-axis blockade of the M1-to-M2 transition. Even in the presence of M2-polarizing cytokines (IL-4/IL-10), intracellular Zn²⁺ depletion erects two parallel barriers to phenotypic transition. On the JAK-STAT6 axis, Zn²⁺-disinhibited PTP1B prematurely dephosphorylates JAK1/JAK3 and STAT6 at Tyr641, blocking STAT6 dimerization and nuclear entry and rendering the macrophage unresponsive to anti-inflammatory stimuli. On the transcription factor axis, insufficient Zn²⁺ coordination destabilizes the C4-type zinc-finger DNA-binding domains of PPARγ and KLF4, suppressing transcription of M2 effector genes including Arg1, Mrc1, and Chil3. The downstream consequence is iNOS dominance over Arginase-1, amplifying NO and peroxynitrite production, while concurrent suppression of TGF-β and VEGF secretion blocks myofibroblast differentiation and angiogenesis. The macrophage is thus anchored in a persistent M1 state, ensuring permanent inflammatory stasis and failed re-epithelialization. DUB, deubiquitinase; iNOS, inducible nitric oxide synthase; IKK, IκB kinase; JAK, Janus kinase; NEMO, NF-κB essential modulator; PP2A, protein phosphatase 2A; PTP1B, protein tyrosine phosphatase 1B; STAT6, signal transducer and activator of transcription 6; TRAF, TNF receptor-associated factor; USP, ubiquitin-specific protease

M1-to-M2 Transition Barriers: Blockage of Pro-reparative Signaling

The phenotypic shift from M1 to M2 macrophages represents a transcriptionally intensive process coordinated by the activation of specific pro-reparative signaling axes, primarily the IL-4/STAT6 and IL-10/STAT3 pathways. In the Zn²⁺-depleted intracellular environment of a DFU, this transition is significantly obstructed by a compartment-specific failure in signal transduction. This attenuated responsiveness to anti-inflammatory stimuli is potentially exacerbated by the loss of Zn²⁺-mediated inhibition of PTP1B. Overactive PTP1B has been implicated in the premature dephosphorylation of the IL-4 receptor-associated JAK1 and JAK3 kinases, as well as the STAT6 protein at its critical Tyr641 residue [14]. This enzymatic interference prevents the dimerization and subsequent nuclear translocation of STAT6, effectively silencing the primary transducer of M2-polarizing stimuli. Consequently, even in the presence of exogenous reparative cytokines, DFU-resident macrophages exhibit a blunted signaling capacity, maintaining their pro-inflammatory profile due to the inability to activate the primary cytoplasmic transducers of the M2 program. It should be noted that PTP1B overactivation is not selective for anti-inflammatory JAK-STAT signaling; pro-inflammatory JAK-STAT pathways, including IFNγ/STAT1 and IL-6/STAT3, are subject to parallel dephosphorylation under the same conditions [27, 28], which could theoretically attenuate pro-inflammatory JAK-STAT-dependent outputs and partially offset the inhibitory effects on anti-inflammatory signaling. However, in the DFU microenvironment, the net inflammatory outcome is unlikely to be neutral for two reasons. First, the sustained activation of NF-κB driven by the functional loss of A20 and CYLD operates independently of JAK-STAT inputs, and its transcriptional output of TNF-α, IL-1β, and IL-6 is sufficient to maintain M1 macrophage retention regardless of concurrent JAK-STAT modulation [29, 30]. Second, the magnitude of NF-κB-driven cytokine secretion in the zinc-depleted DFU bed is proposed to substantially exceed any anti-inflammatory benefit that might theoretically arise from PTP1B-mediated attenuation of IFNγ/STAT1 signaling. The net effect of PTP1B overactivation in this context is therefore determined primarily by its blockade of M2-polarizing signal transduction rather than by any balancing influence on pro-inflammatory JAK-STAT pathways [31, 32].

Beyond the failure of cytoplasmic signaling, the terminal execution of the M2 transcriptional program is fundamentally compromised by the functional impairment of zinc-dependent transcription factors. The master regulators of the M2 phenotype, including PPARγ and KLF4, are characterized by highly conserved zinc-finger motifs within their DBDs. Specifically, PPARγ utilizes two C4-type zinc fingers to coordinate with specific PPREs in the promoter regions of M2-specific genes. In the state of functional Zn²⁺ scarcity within the DFU bed, the cysteine residues of these zinc fingers may undergo conformational instability due to insufficient metal coordination [33]. This structural compromise prevents the effective binding of PPARγ and KLF4 to their genomic targets, thereby blocking the transcription of essential reparative genes such as Arg1 (Arginase-1), Mrc1 (CD206), and Chil3 [34]. The loss of Zn²⁺ coordination thus acts as a molecular constraint that prevents the transcription of the M2 toolkit, ensuring the cell remains functionally anchored in an M1 configuration despite local reparative cues.

The cumulative result of these signaling and transcriptional blocks is a marked deficiency in the anti-inflammatory and pro-angiogenic output of the macrophage, contributing to a sustained inflammatory state within the wound bed. Under physiological conditions, the activation of the STAT6/PPARγ axis drives the synthesis of Arginase-1, which competes with iNOS for the substrate L-arginine, effectively reducing the production of reactive nitrogen species. In Zn²⁺-deficient DFU macrophages, the persistent dominance of the M1 program ensures that L-arginine is preferentially converted into NO and peroxynitrite, further exacerbating local tissue oxidative stress [35]. In parallel, the failure to activate the M2-associated TGF-β and VEGF secretion pathways prevents the initiation of myofibroblast differentiation and the organization of the vascular niche. This state creates a refractory inflammatory microenvironment where the biochemical machinery required for resolution of inflammation and stabilization of the ECM is substantially compromised. This state of chronic immune arrest represents a sustained pathological equilibrium, where the structural inactivation of essential reparative nodes prevents the re-establishment of tissue homeostasis and ensures the self-perpetuating nature of the diabetic wound (Fig. 2B) [36, 37].

Extracellular Matrix Degradation and Pathological Proteolysis

The transition from a dormant wound to a chronic non-healing state is architecturally defined by the destabilization of the provisional wound scaffold prior to its organized replacement. Building on the compartment-specific zinc misallocation described in preceding sections, this phase represents a functional shift from intracellular signaling arrest to active extracellular structural dissolution, driven by the pathological accumulation of labile Zn²⁺ in the extracellular space and its role as a catalytic cofactor for matrix-degrading enzymes [5].

Zinc-dependent MMP Catalysis

Matrix metalloproteinases constitute a family of Zn²⁺-dependent endopeptidases defined by a highly conserved catalytic domain architecture. At the atomic level, the catalytic efficacy of these enzymes, particularly the gelatinase MMP-9, is contingent upon the precise coordination of a catalytic Zn²⁺ ion. This ion is structurally anchored by a conserved histidine triad localized within the HEXXHXXGXXH consensus sequence motif, where it functions as a strong Lewis acid. In the latent pro-enzyme state, the catalytic Zn²⁺ remains biologically silent due to its coordination with a conserved cysteine residue within the N-terminal pro-domain. This cysteine switch mechanism physically prevents water molecules from accessing the catalytic center [19], which effectively suppresses proteolytic activity under normophysiological conditions.

The transition from a latent zymogen to a functional protease requires the disruption of this cysteine–zinc coordinate bond, a process primarily initiated by the prevailing oxidative burden in the DFU. ROS-mediated oxidation of the pro-domain thiol groups facilitates the displacement of the cysteine residue. While the precise relative contributions of autolytic cleavage versus intermolecular trans-activation mediated by plasmin or other MMPs remain to be fully delineated in vivo, both pathways converge on the exposure of the catalytic cleft. Once this cleft is accessible, the substrate selectivity of MMP-9 is dictated by its unique domain architecture. This structural specificity is not incidental; as a member of the gelatinase subfamily, MMP-9 possesses three fibronectin type II-like modules inserted within its catalytic domain. These modules function as an auxiliary collagen-binding interface, providing the high-affinity anchoring required to engage the triple-helical structure of Type IV collagen. This proteolytic capacity is directly reflected in clinical measurements: MMP-9 activity in chronic DFU exudate has been reported at levels orders of magnitude above those recorded in acute healing wounds [38], providing one of the more robust empirical indicators of the extracellular zinc-dependent enzymatic dysregulation proposed in this framework.

The catalytic cycle of the activated enzyme is driven by the polarization of a coordinated water molecule by the Zn²⁺ ion, a process further stabilized by the conserved glutamate residue within the HEXXH motif acting as a general base and proton shuttle. The subsequent nucleophilic attack on the carbonyl carbon of the scissile peptide bond initiates the sustained fragmentation of ECM substrates. Critically, this proteolysis targets the principal structural and adhesive constituents of the basement membrane, specifically Type IV collagen and laminin-332. The structural consequences of this proteolysis are architecturally irreversible. The fragmentation of laminin-332 disrupts both the hemidesmosomal anchorage mediated by α6β4 integrins and the migratory adhesion contacts governed by α3β1 integrins. This dual failure effectively silences the FAK/Src signaling axis, which serves as the primary intracellular engine for directional cell migration. Consequently, the disruption of these matrix-bound migratory cues arrests keratinocytes in a non-migratory, repair-incompetent state. Within the DFU microenvironment, the continuous availability of labile Zn²⁺ may sustain this catalytic cycle without adequate attenuation, maintaining the wound in a state of impaired repair (Fig. 3) [39].

Fig. 3.

Zinc-dependent MMP-9 catalytic activation and downstream signaling silencing. ROS-mediated oxidation of the pro-domain thiol disrupts the cysteine–zinc coordinate bond, converting latent Pro-MMP-9 to its active form. The continuous availability of labile extracellular Zn²⁺ ensures sustained catalytic turnover: within the active cleft, Zn²⁺ polarizes a coordinated water molecule while the conserved glutamate acts as a general base, driving nucleophilic attack on the scissile peptide bond. Active MMP-9 cleaves Type IV collagen and laminin-332, fragmenting the basement membrane and disrupting α6β4- and α3β1-mediated integrin signaling. This dual integrin failure silences the FAK/Src axis, arresting keratinocytes in a repair-incompetent state. The self-sustaining labile Zn²⁺ pool ensures this destructive cycle persists without attenuation. Cys, cysteine; FAK, focal adhesion kinase; Glu, glutamate; Hid, histidine; ROS, reactive oxygen species

The Self-reinforcing Biochemical Loop

The chronic persistence of the DFU wound environment is proposed to be governed by a self-reinforcing pathological cycle integrating transcriptional, ionic, and post-translational mechanisms. The sustained nuclear occupancy of NF-κB p65/p50 heterodimers drives transcription of the MMP9 gene through binding to the conserved 9-base pair NF-κB response element in its proximal promoter region, maintaining an elevated supply of pro-MMP-9 zymogen within the cellular compartment [20].

Crucial to the propagation of this cycle is the pathological ionic redistribution that facilitates the transition of the pathology from the intracellular to the extracellular space. As established in the preceding sections, oxidative stress initiates the liberation of Zn²⁺ from MT-1 and MT-2 clusters. The resulting surge in labile cytosolic zinc is actively redirected through dysregulated ZnT1 exporters, which are overexpressed under hyperglycemic conditions. This spatiotemporal misallocation is the critical juncture of the loop [40]. It simultaneously maintains the intracellular depletion required to prevent the silencing of pro-inflammatory signals and saturates the extracellular space with the catalytic cofactor necessary for enzyme activation. The kinetics of this Zn²⁺ efflux effectively bridge the gap between nuclear induction and extracellular execution.

The external activation ring involves the functional collapse of the endogenous proteolytic balance, completing the self-reinforcing circuit. The pathological accumulation of labile Zn²⁺ in the extracellular space triggers the cysteine switch of the latent MMP-9 pool while concurrently rendering TIMPs functionally inadequate. Specifically, the oxidative milieu of the DFU promotes the oxidative modification of critical methionine and cysteine residues within TIMP-1, compromising its structural integrity and inhibitory capacity [41]. This dual mechanism ensures that once MMP-9 is activated, its catalytic turnover remains unchecked by stoichiometric inhibitors. Critically, the resulting ECM degradation products, such as collagen-derived matrikines, function as DAMPs that engage TLR4 on the macrophage surface. This interaction re-activates the NF-κB signaling axis, thereby re-initiating the internal ring of the loop and sustaining the recalcitrant inflammatory state (Fig. 4) [42, 43].

Fig. 4.

The self-reinforcing biochemical loop and multi-lineage cellular arrest in DFU. Three interconnected zones perpetuate DFU chronicity. In the internal ring, Zn²⁺ depletion drives constitutive IKKβ activation and sustained NF-κB-dependent pro-MMP-9 transcription. The ionic redistribution hub links both compartments: ROS-driven MT-1/2 oxidation liberates labile Zn²⁺, which ZnT1 exports to the ECM while MTF-1 inactivation eliminates intracellular buffering. In the external activation ring, accumulated ECM Zn²⁺ triggers the MMP-9 cysteine switch while oxidative TIMP-1 inactivation abolishes stoichiometric inhibition. Unchecked MMP-9 generates anti-angiogenic matricryptins and DAMPs that engage TLR4, re-activating NF-κB and closing the loop. Convergent cleavage of TGF-β and VEGF, compounded by Smad3 blockade, arrests fibroblast and endothelial repair programs. The resulting multi-lineage cellular arrest ensures permanent inflammatory stasis. DAMPs, damage-associated molecular patterns; MT, metallothionein; MTF-1, metal-responsive transcription factor-1; TIMP-1, tissue inhibitor of metalloproteinases-1

Structural Fragmentation and Multi-lineage Cellular Arrest

The cumulative consequence of this proteolytic loop is extensive degradation of the basement membrane and subsequent disruption of the vascular niche. Beyond physical fragmentation, the hyperactivation of MMP-9 targets the structural and adhesive integrity of the basement membrane, specifically Type IV collagen and laminin-332. The fragmentation of Type IV collagen is particularly deleterious, as it generates specific matricryptins including arresten, canstatin, and tumstatin. These bioactive fragments exhibit potent anti-angiogenic properties, inhibiting endothelial cell proliferation and migration by interfering with α1β1 and α2β1 integrin signaling. This biochemical shift transforms the ECM from a supportive scaffold into an actively inhibitory microenvironment that thwarts revascularization and perpetuates the vascular insufficiency characteristic of the DFU bed [44].

Simultaneously, the excessive proteolytic activity of MMP-9 targets the regenerative signaling capacity of the wound bed through the direct cleavage of growth factors and their receptors. MMP-9 does not merely displace factors such as TGF-β and VEGF from their matrix anchors; it directly cleaves their bioactive domains, leading to functional inactivation prior to receptor engagement. This signaling deficit is further compounded by the loss of Smad-dependent transcriptional activity in fibroblasts [45]. Intracellular Zn²⁺ depletion, as described in previous sections, disrupts the structural integrity of zinc-finger domains within the Smad transcriptional complex, thereby preventing Smad3 phosphorylation and subsequent nuclear translocation. The resulting failure in Smad signaling inhibits the synthesis of Type I collagen and α-SMA, effectively halting the differentiation of fibroblasts into contractile myofibroblasts. Furthermore, the MMP-mediated shedding of cell-surface receptors, such as the VEGFR-1 extracellular domain, creates decoy receptors that sequester circulating VEGF. This multi-level disruption effectively silences the biochemical signaling required for both mesenchymal activation and endothelial organization [46].

The cumulative failure of these matrix-dependent pathways leads to the arrest of keratinocytes, fibroblasts, and endothelial cells in a non-migratory, repair-incompetent state, initiated by attenuation of the FAK/Src axis and disruption of the growth factor niche [47]. DAMPs generated during progressive matrix dissolution, including fibronectin fragments and other ECM-derived danger signals, continuously feed back into the TLR4/NF-κB axis, sustaining the self-reinforcing pathological cycle [48]. This convergence of structural collapse and sustained inflammatory signaling represents a state of chronic homeostasis where the molecular and structural prerequisites for tissue repair have been systematically eliminated. The DFU thus remains in a persistent inflammatory state, where destructive feedback from the extracellular environment impairs the re-establishment of a pro-reparative intracellular program (Fig 0.4).

Zinc-based Precision Interventional Strategies

The elucidation of compartment-specific zinc misallocation as a central driver of DFU chronicity necessitates a fundamental transition in therapeutic approach. Conventional, systemic zinc supplementation often proves inadequate or even deleterious in the DFU microenvironment, as it risks fueling extracellular MMP activity without addressing the metabolic arrest within the intracellular compartment [49]. Consequently, effective restoration of tissue homeostasis requires a reciprocal regulatory paradigm: a dual-action strategy designed to sequester pathogenic labile Zn²⁺ from the extracellular matrix while selectively replenishing functional Zn²⁺ within the cytoplasm [50]. This reciprocal approach aims to neutralize the proteolytic environment while simultaneously re-activating the dormant reparative programs of resident wound cells. By shifting the focus from systemic supplementation to spatial redistribution, these precision interventions may offer a more targeted approach to attenuating the pathological ionic imbalance inherent in chronic diabetic wounds.

The temporal dynamics of these pathological events are relevant to the rational design of interventional strategies. In the acute inflammatory phase, occurring within approximately the first three to seven days following injury, transient ROS elevation and early NF-κB activation represent physiologically necessary responses to tissue damage and microbial challenge [51–53]. Zinc transporter dysregulation and measurable intracellular Zn²⁺ depletion are proposed to emerge as secondary consequences of sustained hyperglycemia and oxidative burden [54, 55], becoming clinically relevant in wounds that fail to transition out of the inflammatory phase beyond two to four weeks. The accumulation of labile extracellular Zn²⁺ and the associated hyperactivation of MMP-9 are temporally downstream of transporter dysfunction [56], and are therefore most pronounced in wounds that have entered a chronic non-healing state, typically defined clinically as persisting beyond four to twelve weeks without measurable closure progress [57, 58]. This temporal sequence suggests that interventions targeting zinc redistribution are most likely to be effective during the transition window between early inflammatory arrest and established chronicity, rather than in the acute phase where transient zinc flux may serve physiological functions. Once the self-reinforcing proteolytic cycle is fully established, the structural loss of basement membrane components may limit the extent of recoverable regenerative capacity [59], underscoring the importance of early identification of patients at risk of chronic wound progression (Fig. 5).

Fig. 5.

Proposed temporal sequence of compartment-specific zinc misallocation events in DFU and the corresponding therapeutic intervention window. The horizontal axis represents disease duration across four operationally defined stages: the acute inflammatory phase (Day 0–7), the delayed resolution phase (Week 1–4), the chronic lock establishment phase (Week 4–12), and established chronicity (Week 12+). Curves depict the schematic trajectories of six key pathological parameters: ROS level, NF-κB activation intensity, intracellular Zn²⁺ level, extracellular labile Zn²⁺ level, MMP-9 activity, and M1/M2 macrophage ratio. The gray dashed curve represents the expected trajectory of ROS in normally resolving acute wounds, provided as a reference. The schematic icon at the base of the Phase 1/2 boundary illustrates the proposed directional shift in ZIP and ZnT1 transporter activity under sustained hyperglycemic and oxidative conditions. The blue shaded region spanning Week 2 to approximately Week 8 indicates the proposed therapeutic window during which zinc redistribution strategies are most likely to achieve consistent benefit, prior to full establishment of the self-reinforcing proteolytic cycle. All curves are schematic representations based on existing literature regarding wound healing phase transitions and clinical definitions of chronic wound chronicity and are not drawn to scale

Advanced Responsive Delivery Systems

Achieving spatial precision in zinc redistribution requires the utilization of responsive materials capable of sensing the unique biochemical signatures of the DFU bed. Unlike acute wounds that exhibit a transient acidic shift, chronic DFUs are characterized by a persistent alkaline environment (pH 7.2–8.9) and an overwhelming oxidative burden. These parameters provide specific triggers for controlled ion release. ROS-responsive nanocarriers, typically incorporating aryl boronic ester or thioether linkages, represent a primary strategy for targeted delivery [6]. These systems remain stable under physiological conditions but undergo rapid degradation upon exposure to high concentrations of H₂O₂. This mechanism ensures that Zn²⁺ liberation is synchronized with the intensity of local oxidative stress, providing functional ions precisely when the cellular antioxidant machinery is most compromised [60].

Beyond traditional polymers, Metal-Organic Frameworks (MOFs), particularly zeolitic imidazolate frameworks such as ZIF-8, have emerged as sophisticated reservoirs for spatial correction [61]. ZIF-8 consists of Zn²⁺ ions coordinated with imidazolate ligands, providing an inherent source of functional zinc with high loading capacity. However, the utilization of ZIF-8 in DFUs presents unique kinetic challenges; while its structure is highly sensitive to acidic environments, it remains relatively stable under the alkaline conditions typical of chronic ulcers. To overcome this stability barrier, recent strategies have focused on functionalizing MOF surfaces with enzyme-responsive coatings or integrating them into composite scaffolds [62]. These modifications allow for a complementary, dual-targeted delivery of Zn²⁺. Specifically, the surface-triggered release of ions mediated by the high local concentrations of MMP-9 targets the extracellular matrix requirements, while subsequent cellular uptake through endocytosis ensures the cytosolic delivery of ions required to quench pro-inflammatory signaling [63].

Reciprocal Regulation and Biological Restoration

The therapeutic efficacy of these precision systems is defined by their ability to execute a simultaneous “quench and rescue” operation within the DFU microenvironment. In the extracellular compartment, the incorporation of specific chelating moieties or ion-exchange interfaces within hydrogel scaffolds facilitates the sequestration of excess labile Zn²⁺ [64]. This reduction in extracellular zinc availability deprives the latent MMP-9 zymogen pool of the cofactor required for activation, while directly attenuating the catalytic turnover of already-active enzymes. By suppressing this proteolytic tide without requiring the reversal of the irreversible pro-domain cleavage, these interventions prevent the further generation of anti-angiogenic matricryptins. This architectural preservation is a prerequisite for the restoration of the FAK/Src signaling engine required for keratinocyte mobilization [5].

In parallel, the selective replenishment of the intracellular zinc pool addresses the root cause of the M1-to-M2 transition arrest. Upon cellular internalization, responsive nanocarriers release functional Zn²⁺ that is postulated to restore the structural integrity of zinc-finger proteins, including A20 and CYLD [4]. The re-activation of these deubiquitinating enzymes facilitates the disassembly of the IKK complex, thereby terminating the persistent NF-κB signaling that drives the pro-inflammatory phenotype. This intracellular restoration is not merely a silencing of inflammation; it provides the coordinative environment required for the phosphorylation of Smad3 and the dimerization of STAT6. By resolving the internal zinc hunger of resident macrophages and fibroblasts, these precision strategies unlock the transcriptional toolkit necessary for collagen synthesis and granulation tissue formation. This dual-compartment correction facilitates the restoration of tissue homeostasis by systematically re-establishing the molecular and physical prerequisites required for wound resolution (Fig. 6).

Fig. 6.

Reciprocal zinc redistribution via responsive nanocarrier platforms as a dual-compartment therapeutic strategy. A ZIF-8/MOF-based nanoparticle system executes a simultaneous "quench and rescue" operation to resolve compartment-specific zinc misallocation. In the extracellular space, chelating hydrogel scaffolds sequester excess labile Zn²⁺, depriving the latent Pro-MMP-9 zymogen pool of its essential cofactor and suppressing active MMP-9-mediated ECM cleavage, thereby preserving collagen fiber architecture. ROS-triggered degradation of the nanocarrier surface initiates the quench loop, capturing pathogenic extracellular Zn²⁺ in proportion to local oxidative burden. Concurrently, cellular endocytosis of the nanoparticle system delivers functional Zn²⁺ to the intracellular compartment, initiating the rescue loop. Replenished intracellular Zn²⁺ restores the structural integrity of zinc-finger DUBs A20 and CYLD, facilitating IKK complex disassembly and terminating NF-κB-driven M1 polarization. Downstream restoration of Smad3 phosphorylation and STAT6 activity unlocks the M2 transcriptional program, upregulating Arginase-1, CD206, collagen synthesis, TGF-β, and VEGF. This dual-compartment correction simultaneously preserves ECM integrity, activates wound healing programs, and terminates the chronic inflammatory cycle. DUB, deubiquitinase; ECM, extracellular matrix; IKK, IκB kinase; MOF, metal-organic framework; ROS, reactive oxygen species; ZIF-8, zeolitic imidazolate framework-8

Conclusion and Future Perspectives

The Zinc Misallocation Hypothesis

The conceptual framework of compartment-specific zinc misallocation offers one mechanistic perspective to partially account for the recalcitrant nature of diabetic foot ulcers. While zinc misallocation is unlikely to represent the sole determinant of DFU chronicity, its mechanistic coupling of intracellular signaling arrest with extracellular matrix degradation identifies a specific pathological axis that may be amenable to targeted intervention. By bridging the gap between intracellular signaling failure and extracellular structural dissolution, this spatiotemporal decoupling identifies a specific pathological state in the wound healing process. The persistent M1 macrophage locking and the hyperactivation of matrix metalloproteinases are not independent pathological events; rather, they are proposed to be functionally coupled through the dysregulated partitioning of Zn²⁺, among other contributing mechanisms. This ionic imbalance may constitute one factor sustaining chronic inflammation, operating in conjunction with neuropathic, vascular, and glycation-related pathological processes. Consequently, the resolution of the DFU necessitates a departure from traditional, systemic supplementation toward precision strategies that can restore the delicate spatial balance of this essential trace element.

Several limitations of the present framework merit explicit acknowledgment. The mechanistic links described in this review, particularly those involving ZIP7-mediated ER zinc release, MTF-1 oxidative inactivation, and PTP1B-dependent JAK-STAT attenuation, are largely inferred from in vitro models or studies conducted in non-DFU contexts. Direct quantification of compartment-specific labile Zn²⁺ concentrations in human DFU tissue remains technically challenging and is currently absent from the literature. Furthermore, the relative contribution of zinc misallocation to DFU chronicity, compared with other established pathological drivers including advanced glycation end-product accumulation, peripheral neuropathy, and microvascular insufficiency, has not been experimentally delineated. The proposed hypothesis should therefore be interpreted as a mechanistic framework requiring prospective validation through spatially resolved zinc quantification, such as synchrotron X-ray fluorescence microscopy or fluorescent Zn²⁺ biosensors applied to clinical biopsy material.

Towards Spatial Metallomics and Precision Repair

To translate these mechanistic insights into clinical success, the next phase of DFU research must prioritize high-resolution mapping of the wound metal-niche. The integration of spatial metallomics with single-cell transcriptomics offers a promising avenue to delineate the precise ionic boundaries between the inflammatory and reparative zones of the wound bed. Such multi-omic profiling would allow for the identification of cell-specific “zinc signatures,” potentially revealing novel therapeutic targets within the SLC30A and SLC39A transporter families. The translation of these fundamental multi-omic maps into point-of-care diagnostic tools represents a critical step for clinical management. For instance, the development of smart biosensors for the real-time monitoring of labile Zn²⁺ levels in wound exudate could serve as a personalized diagnostic tool to assess proteolytic risk and guide the timing of interventional strategies.

Beyond diagnostics, the clinical translation of responsive delivery systems, including MOFs and smart hydrogels, remains a critical frontier. While the application of pH and ROS-responsive nanocarriers demonstrates significant potential in preclinical models, further investigation into their long-term biocompatibility and metabolic clearance is essential. Future interventional research should also explore the synergistic potential of combining zinc-redistribution platforms with NF-κB pathway inhibitors or TIMP-1 stabilizing agents. Ultimately, the clinical resolution of DFU chronicity will require interventions capable of operating simultaneously at both the ionic and transcriptional levels, dismantling the self-reinforcing proteolytic loop while restoring the intracellular coordinative environment upon which reparative gene expression depends.

Author Contributions

Conceptualization, A.Y.F.W. and T.H.Y.Q.; methodology, A.Y.F.W. and T.H.Y.Q.; investigation, A.Y.F.W., A.A., H.L.T. and X.W.L.; data curation, A.Y.F.W., A.A., H.L.T. and X.W.L.; validation, A.Y.F.W., A.A. and H.L.T.; visualization, T.H.Y.Q.; writing – original draft preparation, A.Y.F.W. and T.H.Y.Q.; writing – review and editing, all authors; supervision, T.H.Y.Q.; project administration, T.H.Y.Q.; funding acquisition, T.H.Y.Q. and H.L.T. All authors have read and agreed to the published version of the manuscript.

Funding

Open access funding provided by The Ministry of Higher Education Malaysia and Universiti Malaya. This publication was supported by the Universiti Malaya Bantuan Khas Penyelidikan Early Career Research Grant (BKP-ECRG, Project No. BKP031-2025-ECRG) and the Universiti Malaya Specialist Centre C.A.R.E. Fund Research Grant (UMSC-RG, Project No. UMG004C-2025).

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Ethics Approval and Consent to Participate

Not applicable. As a narrative review, this article does not contain any new studies involving human participants or animals performed by any of the authors.

Declaration of Generative AI

The authors acknowledge the use of AI tools strictly for language editing and grammatical refinement during the preparation of this manuscript. The authors have critically reviewed all outputs and take full responsibility for the final content, originality, and academic rigor of the publication.

Competing interests

The authors declare no competing interests.