Abstract

   Wilson's disease (WD), as a typical disease of excessive Cu^2+
   deposition, is characterized by disorders of copper metabolism in the
   brain and thereby damaging the reproductive system. Conventional
   interventions using copper chelators can temporarily reduce
   intracellular copper (in‐copper), but continuous re‐entry of
   extracellular copper (ex‐copper) into cells results in suboptimal
   therapy. Effective therapy requires multipronged copper metabolism
   management. Here, an orally administered, engineered Ganoderma‐derived
   extracellular vesicle (CZGE) is developed for synergistic in/ex‐copper
   regulation. Specifically, CZGE is designed by co‐fusing
   Ganoderma‐derived extracellular vesicle (GEVs) and targeted
   nanomicelles containing zinc‐curcumin (Zn‐Cur) complex (ZCNs). CZGE,
   with β‐glucan‐enriched GEVs and kisspeptin‐10‐modified ZCNs, can
   effectively penetrate intestinal and brain barriers after oral
   administration to target hypothalamic neurons.After internalization,
   Zn‐Cur released from CZGE replaces excess Cu^2+ to form copper‐curcumin
   (Cu‐Cur) and release Zn^2+ via ion exchange. Cu‐Cur reduces in‐copper
   and neuroinflammation via the Nrf2/NLRP3 pathway, while Zn^2+ inhibits
   ex‐copper influx by activating zinc transporter 1. In WD mice, CZGE
   alleviates hypothalamic copper deposition, activates ERK1/2
   phosphorylation, and repairs reproductive dysfunction by modulating the
   hypothalamic‐pituitary‐testicular axis. It first reveals a targeted
   nanomedicine based on ion exchange that multipronged management
   in‐copper and ex‐copper, offering a promising therapeutic strategy for
   addressing WD with reproductive dysfunction.

   Keywords: external copper inhibition, intracellular copper chelation,
   ion exchange strategy, neuroinflammation, oral drug delivery system,
   wilson's disease
     __________________________________________________________________

   This study reports for the first time an oral targeted nanomedicine
   that conducts ion exchange through engineered Ganoderma‐derived
   extracellular vesicle, managing both the in‐copper and ex‐copper in a
   multi‐pronged approach, offering a promising therapeutic strategy for
   addressing WD with reproductive dysfunction.

   graphic file with name ADVS-12-e01689-g004.jpg

1. Introduction

   Wilson's disease (WD) is a severe dysregulation of copper metabolism in
   the brain caused by mutations in the ATP7B gene, characterized by the
   abnormal accumulation of copper ions (Cu^2+) and thereby impairing the
   neuron biofunction.^[ [48]^1 ^] Among them, gonadotropin‐releasing
   hormone (GnRH) neuron, a key of neuron in the hypothalamus that
   controls reproduction by hypothalamic‐pituitary‐gonadal (HPG) axis,
   mediates the secretion of GnRH through the intracellular key protein
   extracellular regulated protein kinases 1/2 (ERK1/2).^[ [49]^2 ^] Once
   GnRH neuron apoptosis/death mediated by Cu^2+ overload in the
   hypothalamus of WD, the secretion of GnRH can be put on brake, leading
   to the inevitable cascade of reproductive dysfunction.^[ [50]^3 ^]
   Therefore, effective removal of excess Cu^2+ in damaged GnRH neurons is
   currently the most routine strategy in preclinical and clinical
   application.^[ [51]^4 ^] However, there is a major limitation in the
   current Cu^2+ chelation strategy that poor intracellular chelation,
   rapid clearance, and high systemic toxicity of small molecule
   chelators, leading to inadequate intracellular chelation effects.^[
   [52]^5 ^] Besides, existing therapies primarily focus on eliminating
   excess intracellular copper (in‐copper), while overlooking the
   continuous accumulation of extracellular copper (ex‐copper), which
   serves as a significant source of in‐copper.^[ [53]^6 ^] It poses a
   substantial challenge to achieving long‐term therapeutic efficacy.
   These limitations put forward higher requirements for the design of
   copper regulation strategy for WD with reproductive dysfunction
   therapy.

   Targeted ion‐regulation therapy based on nanotechnology enables precise
   and efficient reversal of the ion distribution status in target cells,
   thereby circumventing the inherent physicochemical properties of pure
   chelators.^[ [54]^7 ^] The ion exchange strategy, among various
   ion‐regulation approaches, facilitates the separation and replacement
   of distinct ions by leveraging the affinity and selectivity of suitable
   ion exchangers for different ions.^[ [55]^8 ^] It exhibits great
   clinical potential in treating WD complicated with reproductive
   dysfunction as it not only eliminates harmful in‐copper but also
   introduces other functional ions.^[ [56]^9 , [57]^10 ^] Zinc ion
   (Zn^2+), as a bridging link for various proteins, can competitively
   preempt zinc transporter 1 (ZnT1) that mediate ex‐copper entry into
   cells, thereby blocking the continued uptake of ex‐copper.^[ [58]^11 ^]
   Based on this, zinc therapy is widely recognized as a prominent
   adjunctive treatment in the clinical management of WD.^[ [59]^12 ^]
   However, excessive Zn^2+ can potentially induce mitochondrial
   dysfunction and thereby enhance endogenous ROS production, leading to
   certain toxic effects.^[ [60]^13 ^] Therefore, the secure
   implementation of zinc therapeutics via ion‐exchange technology
   constitutes a vital strategy for efficaciously blocking the uptake
   pathway of ex‐copper, but it poses significant challenges.

   Among the various potential ion exchange agents, curcumin (Cur), a
   representative natural antioxidant, has been reported to exhibit
   coordination with manganese ion, Cu^2+, Zn^2+ and other metal ions due
   to its highly conjugated β‐dione group, thereby demonstrating
   selectivity toward specific metal ions.^[ [61]^14 , [62]^15 ^]
   Interestingly, the solubility of Cur is significantly increased upon
   chelation with Zn^2+, while its antioxidant property is markedly
   enhanced following chelation with Cu^2+. Most importantly, the higher
   stability constant of copper‐cur (Cu‐Cur) compared to zinc‐cur (Zn‐Cur)
   (K [Cu‐Cur] = 3.4 × 10^14 mol^−1, K [Zn‐Cur] = 9 × 10^10 mol^−1), makes
   Zn‐Cur as a suitable ion exchange agent for displacing Cu^2+ to form
   the stable Cu‐Cur and free Zn^2+.^[ [63]^16 ^] Based on this, we
   hypothesize that if the ion‐exchange reaction occurs specifically in
   the damaged GnRH neurons of WD, it would enable the in situ synthesis
   of Cu‐Cur for reducing in‐copper and anti‐inflammatory, and releases
   free Zn^2+ to competitively occupy with ZnT1 protein for blocking the
   uptake pathway of ex‐copper.

   Here, an oral engineered Ganoderma‐derived extracellular vesicles
   (CZGE) was proposed based on ion exchange strategy for WD with
   reproductive dysfunction therapy. Specifically, Ganoderma‐derived
   extracellular vesicles enriched with β‐glucan (GEVs) were fused with
   Zn‐Cur nanomicelles modified with targeting peptides of kisspeptin‐10
   (KZCNs). With the help of the penetrating ability of β‐glucan on GEVs,
   GZGE first penetrated the intestinal epithelial barrier (IEB) and blood
   brain barrier (BBB) through specific binding to M cells. After entering
   the brain parenchyma, efficient endocytosis of GnRH neurons is
   facilitated through kisspeptin‐10 (Kiss‐10) on ZCNs specific binding to
   G Protein‐Coupled Receptor 54 (GPR54) protein on the surface of GnRH
   neurons. The Zn‐Cur released in the high in‐copper environment was
   subsequently replaced by Cu‐Cur and free Zn^2+. On one hand, the
   formation of Cu‐Cur effectively decreased in‐copper and augmented
   antioxidant activity. On the other hand, the presence of free Zn^2+
   competitively occupy with ZnT1 protein, thereby blocking the uptake
   pathway of ex‐copper. Consequently, it promoted the hormone secretion
   function of GnRH neurons, thus restoring reproductive dysfunction via
   the HPT axis in WD model mouse. The findings of our study demonstrated
   a novel nanomedicine that employed an ion‐exchange strategy, thereby
   facilitating a dual regulation approach involving in‐copper chelation
   and ex‐copper blockage to effectively enhance the treatment of WD
   accompanied by reproductive dysfunction (Scheme [64]1).

Scheme 1.

   Scheme 1
   [65]Open in a new tab

   Schematic illustration of engineered Ganoderma‐derived extracellular
   vesicle synthesis and its application in the treatment of WD with
   reproductive dysfunction. Fusion of GEVs enriched β‐glucan extracted
   from the ganoderma and KZCNs prepared with self‐assembling technique to
   obtain hybrid CZGE. After oral intragastric administration, GEVs rich
   in β‐glucan specifically penetrated the IEB and BBB. Meanwhile, the
   Kiss‐10 peptide immobilized on the surface of CZGE was recognized and
   captured by the GPR54 receptor of GnRH neurons. The Zn‐Cur released in
   the high intracellular copper environment was subsequently replaced by
   Cu‐Cur and free Zn^2+. The former can effectively reduce in‐copper and
   neuroinflammation through the Nrf2‐NLRP3 pathway, and the latter can
   block ex‐copper influx through activation of ZnT1. Consequently, it
   promoted the hormone secretion function of GnRH neurons, thus restoring
   reproductive dysfunction via the HPT axis in WD model mouse.

2. Results and Discussion

2.1. Clinical Indexes Analysis of WD with Reproductive Dysfunction

   To address the current gap in the clinical application of
   nanotechnology‐based ion‐regulating agents for WD with reproductive
   dysfunction, the screening and identification of effective targets were
   initiated by healthy donors and WD with reproductive dysfunction
   patients in Figure [66]1A. Ceruloplasmin (CP) and magnetic resonance
   imaging (MRI), as a common criterion for clinical diagnosis of WD, were
   used to evaluate the copper deposition in WD patients.^[ [67]^17 ,
   [68]^18 ^] The level of CP in WD with reproductive dysfunction patients
   were markedly reduced compared to those observed in healthy donors
   (Figure [69]1B). Besides, WD with reproductive dysfunction patients
   exhibited distinct MRI imaging characteristics compared to the healthy
   donors. Specifically, the symmetric hypointense on T1‐weighted images
   (T1WI) while the slightly hyperintense on T2‐weighted images (T2WI) and
   fluid‐attenuated inversion recovery (FLAIR) sequences, were clearly
   observed in the brain MRI (Figure [70]1C). This finding was indicative
   of potential copper deposition in the brain.

Figure 1.

   Figure 1
   [71]Open in a new tab

   Clinical indexes analysis from patients WD with reproductive
   dysfunction (n = 6) and healthy donors (n = 6). A) Flowchart of the
   collection and analysis of clinical indicators. B) Relative
   quantitative analysis of CP in serum, in which the expression of normal
   group as denoted as 1. C) Transverse section MRI images of the brain.
   D) Relative expression of GnRH, FSH, LH and T in serum, in which the
   expression of normal group as denoted as 1. E) Sperm motility
   trajectory map, scale bar: 20 µm. F) Relative assessment of semen
   parameters. G,H) Relative quantitative analysis of NLRP3 (G) and PKC
   (H) in serum, in which the expression of normal group as denoted as 1.
   I) 24 h urine copper levels of the patients WD with reproductive
   dysfunction in different course of treatment. All statistics are
   expressed as mean ± standard deviation. Statistical analysis: one‐way
   ANOVA followed by Tukey's HSD post hoc test.

   Theoretically, excessive copper accumulation in the hypothalamus of WD
   patients, specifically within GnRH neurons, may lead to impaired GnRH
   secretion, consequently decreasing the levels of GnRH in blood.^[
   [72]^19 ^] It could in turn affected the synthesis and secretion of
   follicle‐stimulating hormone (FSH), luteinizing hormone (LH), and
   testosterone (T) through the cascade regulation of the HPT axis.^[
   [73]^20 ^] It was evidenced that the levels of GnRH, FSH, LH, and T in
   serum of WD with reproductive dysfunction patients were found to be
   reduced by 3.57, 4.39, 2.19, and 3.73 times than that in healthy
   donors, respectively (Figure [74]1D). In the evaluation of sperm
   function in WD with reproductive dysfunction patients, a comprehensive
   semen analysis was performed to assess sperm motility^[ [75]^21 ^] In
   comparison to sperm from healthy donors, the proportion of grade a + b
   sperm (Grade a > b > c > d) in these WD with reproductive dysfunction
   patients was significantly reduced to 28.95%, whereas the proportion of
   graded sperm was notably elevated Figure [76]1E,F. This observation
   suggested a substantial increase in sperm immotility within these
   patients, which manifests clinically as asthenospermia.

   Previous studies have reported that dysregulation of copper homeostasis
   in the brain may activate the nucleotide‐binding domain and
   leucine‐rich repeat family pyrin domain containing 3 (NLRP3)
   ‐associated pyroptosis pathway, which was implicated in the apoptosis
   of neurons.^[ [77]^22 ^] In light of this, an investigation was
   conducted to ascertain the presence of pyroptosis in WD with
   reproductive dysfunction patients. As shown in Figure [78]1G,H, the
   level of NLRP3 was significantly elevated while the level of protein
   kinase C (PKC) were notably decreased in WD with reproductive
   dysfunction patients compared to those healthy donors. The observed
   outcome supported a hypothesis that excess copper may modulate the
   NLRP3‐PKC pathway, thereby influencing the function of GnRH neurons.
   Zinc agents, as first‐line therapeutics in the clinical management of
   WD, had demonstrated efficacy in inhibiting ex‐copper absorption and
   enhancing its excretion.^[ [79]^23 ^] To further elucidate the therapy
   role of zinc agents in WD with reproductive dysfunction patients, the
   copper excretion in urine significantly increased during the treatment
   period, subsequently declined, and eventually stabilized, which
   confirmed that zinc agents could inhibit the absorption of ex‐copper
   (Figure [80]1I). On the basis of the above clinical experiments
   results, the effectively removal of ex‐copper by zinc agents and the
   reduction of in‐copper by modulating the NLRP3‐PKC pathway can
   significantly reshape copper metabolism for WD with reproductive
   dysfunction therapy. Therefore, an oral engineered extracellular
   vesicle based on ion exchange strategy was designed in the subsequent
   experiment, thereby involving in‐copper chelation and ex‐copper
   blockage.

2.2. Synthesis and Characterization of CZGE

   Based on the aforementioned findings, it is imperative to design a
   targeted nanoformulations capable of dual regulation in‐copper
   chelation and ex‐copper blockade for WD with reproductive dysfunction
   therapy. An ion exchange agents (Zn‐Cur) was first prepared via the
   coordination method in Figure [81]2A.^[ [82]^24 ^] The powders of Cur,
   Zn‐Cur and Cu‐Cur showed yellow, red and dark brown colors, such
   distinct color variations arise from the altered electronic structure
   resulting from metal‐ligand coordination (Figure [83]S1, Supporting
   Information). Compared to that of Cur and Cu‐Cur, Zn‐Cur demonstrated
   superior dispersibility in aqueous solution, which facilitated its
   subsequent loading into nanocarriers (Figure [84]S2, Supporting
   Information). Besides, the results of the ultraviolet‐visible (UV–vis)
   absorption spectrum displayed, a slight absorption peak at ≈445 nm
   emerged in the Zn‐Cur spectrum,^[ [85]^8 ^] while a distinct broad peak
   at 525 nm was observed in the Cu‐Cur spectrum, indicating to a stronger
   coordination between Cur and Cu^2+ than between Cur and Zn^2+
   (Figure [86]2B).^[ [87]^25 , [88]^26 ^] This finding was further
   substantiated by the results obtained from fourier transform infrared
   (FTIR) spectroscopic analysis and ^1H nuclear magnetic resonance
   spectra (^1H NMR) (Figure [89]2C, Figure [90]S3, Supporting
   Information).^[ [91]^27 , [92]^28 , [93]^29 ^] The X‐ray photoelectron
   spectroscopy (XPS) confirmed that the high‐resolution Cu 2p spectrum of
   Cu‐Cur exhibited two characteristic peaks at 934.5 eV (Cu 2p[3/2]) and
   954.2 eV (Cu 2p[1/2]), accompanied by two oscillating satellite peaks,
   indicating that the presence of Cu^2+ in the chelate complex. The
   high‐resolution Zn 2p spectrum of Zn‐Cur displayed two characteristic
   peaks at 1022 eV (Zn 2p[3/2]) and 1045.2 eV (Zn 2p[1/2])
   (Figure [94]2D–F). Besides, an uniform spherical structures with an
   average diameter of 50 nm of Zn‐Cur was observed in transmission
   electron microscopy (TEM) image (Figure [95]S4, Supporting
   Information).

Figure 2.

   Figure 2
   [96]Open in a new tab

   Synthesis and characterization of CZGE. A) Schematic diagram of the
   preparation of Zn‐Cur. B) The UV–vis absorption spectra of Cur, Zn‐Cur,
   and Cu‐Cur. C) The FTIR spectra of Cur, Zn‐Cur, and Cu‐Cur. D) The XPS
   spectrum of Cur, Zn‐Cur, and Cu‐Cur. E,F) XPS spectra of Cu 2p in
   Cu‐Cur (E) and Zn 2p in Zn‐Cur (F). G) TEM image of ZCNs, scale bar:
   200 nm. H) The surface structure of KZCNs. I) Fluorescence images of
   KZCNs with Dil labeled DSPE‐PEG‐Mal (red) and FITC labeled Kiss‐10
   (green) and modification rate of Kiss‐10 on KZCNs (n = 3), scale bar:
   500 nm. J) TEM image of KZCNs, scale bar: 200 nm. K) TEM image of GEVs,
   scale bar: 200 nm. L) The concentration of β‐glucan in GEVs (n = 3). M,
   N) Fluorescence images of CZGE with 1:1 ratio of KZCNs (red) to GEVs
   (green) (M) and fusion rate (N). O) TEM image of CZGE, scale bar: 200
   nm. P,Q) Zeta potential (P) and hydrodynamic diameters (Q) of different
   samples (n = 3). R) The thin layer chromatography analysis of different
   samples (I: Ganoderma; II: GEVs; III: CZGE). S) Drug loading efficiency
   of different samples (n = 3). All statistical data are presented as
   mean ± standard deviation. Statistical analysis: one‐way ANOVA followed
   by Tukey's HSD post hoc test.

   After that, Zn‐Cur nanomicelles (ZCNs) was synthesized by the thin film
   dispersion technique,^[ [97]^30 ^] finding that a homogeneous circular
   dispersed nanosphere by TEM in Figure [98]2G. Kiss‐10 peptide with
   targeting ability to GPR54 protein on GnRH neurons was further
   synthesized and verified to conjugate to maleimide group of ZCNs by
   high‐performance liquid chromatography (HPLC) and time of flight (TOF)
   mass (Figure [99]2H and Figure [100]S5, Supporting Information). It was
   found that 71.28% of Kiss‐10 peptide was grafted into ZCNs, named as
   KZCNs (Figure [101]2I,J). KCZNs exhibited a diameter of ≈125.4 nm
   (Figure [102]S6, Supporting Information). And the zeta potential of
   KCZNs reached −9.13 mV, less negative potential than of ZCNs alone
   (−19.3 mV), which may be due to the modification of the positive
   potential of Kiss‐10 peptide on the ZCNs (Figure [103]S7, Supporting
   Information). Besides, GEVs were extracted from fresh Ganoderma by the
   classic ultracentrifugation method, which was rich in β‐glucan (0.244
   mg·g^−1) (Figure [104]2K,L). Finally, GEVs was incorporated into the
   KZCNs to synthesize CZGE by membrane fusion technology. The successful
   fusion of GEVs (green) with KZCNs (red) was confirmed by fluorescence
   colocalization, resulting the highest fusion rate (78.49%) at a ratio
   of 1:1 and the förster resonance energy transfer (FRET) effect was
   further evaluated to confirm the synthetic CZGE by fusing GEVs and
   KZCNs (Figure [105]2M,N and Figure [106]S8, Supporting Information).
   According to TEM, dynamic light scattering (DLS), nanoparticle tracking
   analysis (NTA) and zeta potentials measurements, the diameter of CZGE
   was ≈143.4 nm and displayed a negative potential of 8.8mV
   (Figure [107]2O–Q and Figure [108]S6, Supporting Information). Next, we
   determined the effects of the fusion process on the surface proteins of
   GEVs and Zn‐Cur loading. It was found that most of the protein was
   retained and the Zn‐Cur loading was still 78.18% (Figure [109]2R,S and
   Figure [110]S9, Supporting Information). Additionally, the stability of
   various samples was evaluated by tracking their particle size changes
   throughout a 7‐day observation period. There was no apparent
   aggregation‐induced precipitation, indicating the satisfactory
   stability for long term application (Figure [111]S10, Supporting
   Information). These findings demonstrated that the modification of
   β‐glucan and encapsulation of Zn‐Cur have been successfully prepared in
   CZGE.

2.3. Penetration and Ion Exchange Efficiency of CZGE In Vitro

   To align with the currently prevalent oral treatment methods for WD in
   clinical practice, a critical prerequisite for the efficacy of the CZGE
   with abundant of β‐glucan proposed in this work is its ability to
   successively penetrate the IEB and BBB.^[ [112]^31 , [113]^32 ,
   [114]^33 ^] Prior to evaluating the penetration, the optimal
   concentration (10^6 particles mL^−1) and incubation time (24 h) for
   CZGE to regulate the activity of GnRH neurons were determine for
   subsequent experiments (Figure [115]S11, Supporting Information).
   First, human colorectal adenocarcinoma cells (Caco‐2) and mouse
   mononuclear macrophage leukemia cells (RAW264.7) were seeded in the
   upper and lower chamber by transwell model to simulate the IEB in vitro
   (Figure [116]3A–C). The IEB penetration efficiency of GEVs and CZGE was
   42.87% and 40.89%, respectively, which was 2.08 and 1.99 times higher
   than that of KZCNs. This difference indicated the important role of
   β‐glucans in penetrating the IEB. To further elucidated the mechanism
   by which β‐glucan penetrates intestinal M cells through specific
   recognition by the Dectin‐1 receptor, laminarin, a specific inhibitor
   of Dectin‐1, was utilized to pre‐treat IEB co‐culture models.
   Subsequently, the penetration rates of different samples were assessed,
   and the penetration rate of CZGE was found to be significantly reduced
   to 21.15%, as anticipated. In addition, the cellular uptake of
   different samples by the lower chamber was further observed (Figure
   [117]S12, Supporting Information). The fluorescence intensity of the
   CZGE group was the strongest compared to that in other group,
   indicating that it had better intracellular internalization.
   Subsequently, a co‐culture model of mouse brain‐derived Endothelial
   cells.3 (bEnd.3) and mouse hypothalamic neuron (GT1‐7) was established
   to simulate the BBB in vitro (Figure [118]3D,E). As shown in
   Figure [119]3F, the penetration efficiency of the BBB by GEVs and CZGE
   was 57.69% and 58.19%, respectively, which was 1.31 and 1.32 times
   higher than that of KZCNs. Additionally, fluorescence images of GTI‐7
   cells in the lower chamber treated by different samples were also
   investigated in Figure [120]S13, Supporting Information. It indicated
   that CZGE successfully penetrated the BBB model and was taken up by
   GTI‐7 cells in the lower chamber.

Figure 3.

   Figure 3
   [121]Open in a new tab

   Penetration and ion exchange efficiency of CZGE in vitro. A) Schematic
   representation of the in vitro construction process for IEB. B)
   Fluorescence images of the upper chamber and lower chambers incubation
   of different Cy5 labeled‐samples using an in vivo imaging system
   (IVIS). C) IEB penetration rate of different Cy5 labeled‐samples (n =
   3). D) Schematic representation of the in vitro construction process
   for BBB. E) Fluorescence images of the upper chamber and lower chambers
   incubation of different Cy5 labeled‐samples using an in vivo imaging
   system (IVIS). F) BBB penetration rate of different Cy5 labeled‐samples
   (n = 3). G) Relative FI of different samples after different cocultured
   time by flow cytometry (n = 3). H) Representative co‐localization CLSM
   images of different samples (red), cell membrane (green) and nucleus
   (blue), scale bar: 20 µm. I,J) The uptake mechanism of CZGE under
   different condition by flow cytometry (n = 3). K) Scheme of Cu‐Cur
   formation and Zn^2+ release after nanomedicine degradation through an
   ion‐exchange reaction. L) The intracellular Cu‐Cur levels investigation
   of GT1‐7 cells after CZGE treatment by UV–vis absorption spectra (n =
   3). M,N) The intracellular Zn^2+ levels investigation of GT1‐7 cells
   under different conditions stained with Zn^2+ fluorescence probe
   detected by flow cytometry, in which the FI of Zn^2+ in the control
   group was denoted as 1 (n = 3). O,P) The intracellular Cu^2+ levels
   investigation of GT1‐7 cells under different conditions stained with
   Cu^2+ fluorescence probe detected by flow cytometry, in which the FI of
   Cu^2+ in the control group was denoted as 1 (n = 3). All statistical
   data are presented as mean ± standard deviation. Statistical analysis:
   one‐way ANOVA (C, F, J, L, N, P); two‐way ANOVA (G).

   Theoretically, the GPR54 protein on the membrane surface of GnRH
   neurons is specifically bound by Kiss‐10 peptide, thereby targeting
   GnRH neurons.^[ [122]^34 ^] Flow cytometry analysis revealed a
   time‐dependent uptake of different Cy5‐labeled samples in GT1‐7 cells,
   and the average fluorescence intensity of KZCNs and CZGE at 4 h was
   found to be significantly higher than that of GEVs (Figure [123]3G).
   The finding was in line with the observed trend of qualitative
   analysis, indicating that the extensive uptake of CZGE by GT1‐7 cells
   can be enhanced through the action of the Kiss‐10 peptide
   (Figure [124]3H and Figure [125]S14, Supporting Information).
   Subsequently, the mechanism of CZGE uptake by GT1‐7 cells was further
   investigated (Figure [126]3I,J). First, low temperature treatment at
   4 °C significantly inhibited the efficiency of cellular uptake of CZGE
   by GT1‐7 cells, indicating that the uptake mechanism was dependent on
   energy endocytosis. Additionally, only in the presence of
   methyl‐β‐cyclodextrin (MβCD, a caveolin‐mediated inhibitor), the uptake
   efficiency of CZGE in GT1‐7 cells was found to be minimally affected,
   indicating that CZGE was dependent on clathrin‐mediated and
   macropinocytosis mediated uptake pathways. The results indicated that
   GT1‐7 cells may consume more energy to uptake CZGE through the three
   energy‐dependent intakes pathways mentioned above.

   We assume that the Zn‐Cur released from CZGE will undergo ion exchange
   reaction with excess intracellular Cu^2+ to displace both Zn^2+ and
   Cu‐Cur (Figure [127]3K). Here, analysis of intracellular Cu‐Cur, Cu^2+
   and Zn^2+ level was performed to verify the effectiveness of ion
   exchange strategy by CZGE using CuSO[4]‐induced WD copper‐overloaded
   cell model in vitro. The level of Cu‐Cur in GT1‐7 cells following CZGE
   treatment was detected using UV–vis absorption spectrum. The
   characteristic peak intensity of Cu‐Cur at 520 nm was observed to
   significantly increase after 24 h, indicating that CZGE can effectively
   generate Cu‐Cur in GT1‐7 cells of the model group (Figure [128]3L).
   Then, the zinquin ethyl ester fluorescence probe was further used to
   evaluate the effects of different samples on the Zn^2+ level in GT1‐7
   cells of the model group. It showed that the Zn^2+ level in the cells
   was significantly increased after KZCNs and CZGE treatment
   (Figure [129]3M,N). Finally, a significant increase in Cu^2+ level was
   observed in the model group compared to the control group. However,
   after KZCNs and CZGE treatment, there was a reduction in Cu^2+ level
   (Figure [130]3O,P and Figure [131]S15, Supporting Information). The
   above results indicated that Zn^2+ was effectively replaced by CZGE by
   chelating intracellular Cu^2+, thereby enhancing the subsequent
   treatment efficacy and minimizing potential side effects.

2.4. The Therapy Mechanism of CZGE In Vitro by Ion Exchange Strategy

   Following confirmation that CZGE entered cells and generated Cu‐Cur and
   Zn^2+ via ion exchange and effectively reduced the intracellular Cu^2+
   level, we further explored its ion regulation mechanism. As a classic
   metal‐polyphenol chelate, Cu‐Cur has been reported to efficiently
   catalyze ROS through phenol groups and was employed in the treatment of
   diseases (sunch as osteoarthritis).^[ [132]^8 ^] Using electron spin
   resonance (ESR) spectroscopy, we observed significant weakened of the
   characteristic signal O[2] ^• − (1:1:1:1) upon Cu‐Cur addition,
   demonstrating its effective O[2] ^• − scavenging ability, consistent
   with SOD activity kit results. Similar ^•OH scavenging effects were
   observed (Figure [133]S16, Supporting Information).^[ [134]^35 ^]
   Subsequently, the ROS clearance effects of different samples on
   copper‐overloaded GT1‐7 cells were further evaluated using
   2,7‐dichlorodihydrofluorescein diacetate (DCFH‐DA) fluorescent probes
   (Figure [135]4A–C). The ROS levels in GT1‐7 cells of the model group
   were found to be significantly elevated, whereas they were
   significantly reduced following intervention with KZCNs and CZGE. The
   above findings suggested that the incorporation of Cu‐Cur in cells
   effectively replaced the loaded Zn‐Cur in KZCNs and CZGE, leading to a
   decrease in intracellular ROS levels. Oxidative stress often induces
   mitochondrial dysfunction, which can directly or indirectly trigger the
   release of inflammatory factors, ultimately leading to cellular damage
   or apoptosis.^[ [136]^36 ^] As anticipated, flow cytometry revealed
   that the ratio of Tetraethyl benzimidazolyl carbocyanine iodide (JC‐1)
   aggregates (indicative of healthy mitochondria) to JC‐1 monomers
   (indicative of mitochondria with impaired membrane potential) was
   significantly increased in the model group following treatment with
   CZGE. This effectively counteracted mitochondrial damage
   (Figure [137]4D,E). Moreover, the results of ELISA kit indicated that
   the levels of intracellular anti‐inflammatory factors (such as
   interleukin‐10 (IL‐10) and interleukin‐4 (IL‐4)) were significantly
   elevated following CZGE treatment, while the levels of pro‐inflammatory
   factors (such as interleukin‐6 (IL‐6) and tumor necrosis factor α
   (TNF‐α)) were significantly decreased (Figure [138]S17, Supporting
   Information). Subsequently, the effects of different samples on the
   polarization phenotype of BV2 cells were assessed using in vitro flow
   cytometry. The results demonstrated that in the model group, the
   expression of the M1 phenotype marker (CD86) was elevated, while the
   expression of the M2 phenotype marker (CD206) was reduced. Following
   treatment with GEVs and KZCNs, the CD86 level in BV2 cells was
   decreased to 12.1% and 8.87%, respectively, and the CD206 level was
   increased to 4.05% and 22.7%, respectively. Upon intervention with
   CZGE, the CD206 level was further elevated to 27.2%. It indicated that
   CZGE can promote the transformation of BV2 cells toward an
   anti‐inflammatory M2 phenotype (Figure [139]S18, Supporting
   Information). These results further substantiated that oxidative stress
   induced by exposure of GnRH neurons to copper could be significantly
   reduced by CZGE, that neuroinflammation could be inhibited, and that
   the brain microenvironment could be improved.

Figure 4.

   Figure 4
   [140]Open in a new tab

   The therapy mechanism of CZGE in vitro by ion exchange strategy. A)
   Representative fluorescent images of GT1‐7 cells treated with different
   samples (green: ROS fluorescence probe, blue: nucleus), scale bar: 50
   µm. B,C) The elimination of ROS in GT1‐7 cells under different
   conditions stained with ROS fluorescent probes by flow cytometry, in
   which the FI of ROS in the control group was denoted as 1 (n = 3). D,E)
   Quantitative determination of mitochondrial membrane potentials after
   different treatments by flow cytometry (n = 3). F) Schematic
   illustration on mechanism of pyroptosis regulation by CZGE throughout
   the process. G–K) Representative western blot image and quantification
   of the relative protein expression of Nrf2, NLRP3, cleaved caspase‐1,
   and GSDMD‐N (n = 3). L,M) Relative quantification of IL‐18 (L) and
   IL‐1β (M) in GT1‐7 cells by different treatments (n = 3). N) Schematic
   illustration on mechanism of inhibiting ex‐copper entry by CZGE
   throughout the process. O) Time‐course fluorescence microscopy images
   and quantification of GT1‐7 cells after incubation with Zn^2+
   fluorescent probe (blue) and addition of CZGE, scale bar: 5 µm. P)
   Time‐course fluorescence microscopy images and quantification of GT1‐7
   cells after incubation with CZGE, Cu^2+ fluorescent probe (red) and
   another addition of CuSO[4], scale bar: 10 µm. Q) Schematic
   illustration on mechanism of GnRH secretion through PKC/ERK pathway by
   CZGE throughout the process. R,S) Representative western blot image and
   quantification of the relative protein expression of PKC, ERK1/2 and
   p‐ERK1/2 (n = 3). T) Schematic illustration of the co‐culture. U)
   Relative quantification of GnRH, FSH, LH, T in GT1‐7 cells by different
   treatments, in which the expression of control group was denoted as 1
   (n = 3). (Samples for H‐U: I: control, II: model, III: GEVs, IV: KZCNs,
   V: CZGE). All statistical data are presented as mean ± standard
   deviation. Statistical analysis: one‐way ANOVA followed by Tukey's HSD
   post hoc test.

   Furthermore, it has been reported that dysregulation of copper
   homeostasis in the brain can facilitate the assembly of the NLRP3
   inflammasome and induce neuronal damage via pyroptosis, which may
   subsequently affect GnRH secretion and contribute to reproductive
   dysfunction.^[ [141]^37 ^] To investigate the correlation between CZGE
   anti‐inflammation and pyroptosis, the expression levels of related
   proteins involved in pyroptosis were further examined by western
   blotting (Figure [142]4G–K). Compared with that in the model group,
   significantly up‐regulated expression of nuclear factor erythroid
   2‐related factor 2 (Nrf2) while down‐regulated expression of NLRP3,
   cleaved Caspase‐1 and gasdermin D N‐terminal domain (GSDMD‐N) were
   observed in the CZGE group. Besides, interleukin‐1β (IL‐1β) and
   interleukin‐18 (IL‐18) play a key role in inflammasome activation and
   cell pyrodeath.^[ [143]^38 , [144]^39 , [145]^40 , [146]^41 ^] The
   expression levels of IL‐1β and IL‐18 was significantly reduced after
   CZGE treatment (Figure [147]4L,M). Then, we subjected the cells to
   treatment with the NLRP3 inhibitor (MCC950) and observed that the level
   of NLRP3 in the model group cells was 2.75 times higher than that in
   the NLRP3 inhibitor group, indicating that excess Cu^2+ can promote the
   expression of NLRP3. Furthermore, the level of NLRP3 in the cells
   treated by CZGE also decreased significantly, suggesting that CZGE can
   inhibit the expression of NLRP3 by reducing copper deposition (Figure
   [148]S19A, Supporting Informing). Conversely, the PKC level in cells of
   the NLRP3 inhibitor group was significantly higher than that in the
   model group, a finding consistent with the PKC trend in the CZGE group
   (Figure [149]S19B, Supporting Informing). These results indicated that
   copper deposition can affect GnRH secretion by regulating the NLRP3‐PKC
   pathway. Based on the above results, it was plausible to infer that
   Cu‐Cur produced through the intracellular ion exchange process in CZGE,
   can effectively attenuate inflammation by inhibiting the
   NLRP3/Caspase‐1/GSDMD pyroptosis signaling pathway (Figure [150]4F).

   Ex‐copper serves as a critical source of in‐copper, and its effective
   inhibition is crucial for the long‐term management of WD with
   reproductive dysfunction. Previous research have demonstrated that the
   continuous influx of ex‐copper into cells can be inhibited by Zn^2+
   through the modulation of ZnT1 (Figure [151]4N). Building on this
   foundation, fluorescent probes were employed in this study to monitor
   real‐time changes in Zn^2+ and Cu^2+ levels in the model group treated
   with CZGE, with the aim of elucidating the effects of CZGE on the
   balance of Zn^2+ and Cu^2+ inside and outside the cells and its
   underlying mechanisms. Initially, confocal real‐time imaging using a
   Zn^2+ fluorescent probe revealed a significant enhancement in cellular
   Zn^2+ fluorescence intensity within 10 min of CZGE addition, indicating
   an increase in intracellular Zn^2+ levels (Figure [152]4O). Moreover,
   WB analysis revealed that the expression level of ZnT1 significantly
   increased following CZGE treatment compared with that in the model
   group. This increase might be attributed to the replacement of
   in‐copper by CZGE in a high‐copper environment, thereby causing an
   elevation in the intracellular free Zn^2+ level (Figure [153]S20,
   Supporting Information). Furthermore, following a 4 h pretreatment with
   CZGE and subsequent addition of CuSO[4], no significant change in
   intracellular Cu^2+ fluorescence intensity was observed, suggesting
   that Zn^2+ significantly inhibits the entry of ex‐copper into cells
   (Figure [154]4P). In contrast, the model cells without CZGE
   pretreatment exhibited a gradual increase in Cu^2+ fluorescence
   intensity 10 min after the addition of CuSO[4], further confirming the
   specificity and efficacy of Zn^2+ in blocking copper influx (Figure
   [155]S21, Supporting Information).

   Subsequently, a similar preparation method was used to synthesize CZGE
   excepting for Cur (named CZGE*), to further verify the effect of Zn^2+
   on in‐copper and ex‐copper copper levels (Figure [156]S22, Supporting
   Information). The flow cytometry results showed that the Cu^2+ level of
   cells in the model group with another addition of CuSO[4] (II group)
   was significantly increased compared with that in the model group (I
   group), indicating that the continuous inflow of ex‐copper can increase
   the level of in‐copper. In contrast, no significant increase in the
   Cu^2+ level of cells was observed after pre‐incubation with CZGE* and
   subsequent additional CuSO4 stimulation (III group), indicating that
   Zn^2+ could inhibit the inflow of ex‐copper. Besides, the cells were
   pre‐treated with ZnT1 gene siRNA (si‐ZnT1) to inhibit the expression of
   ZnT1 protein. The quantitative real‐time polymerase chain reaction
   (RT‐qPCR) test confirmed that the expression of ZnT1 was decreased in
   the si‐ZnT1 groups. Therefore, the si‐ZnT1 was selected for subsequent
   experiments (Figure [157]S23, Supporting Information). As illustrated
   in Figure [158]S22, Supporting Information, despite the inhibition of
   ZnT1 expression by si‐ZnT1 in cells, the Cu^2+ levels in cells treated
   with si‐ZnT1 and CZGE* (IV group) were comparable to those of the model
   group (I group). This may be attributed to the minimal impact of ZnT1
   on in‐copper levels. However, upon another addition of CuSO[4] solution
   (V group), the Cu^2+ levels in the cells significantly increased. This
   was attributed to the inhibition of ZnT1, which could not prevent the
   influx of external copper, thereby resulting in a significant elevation
   in cellular Cu^2+ levels. Furthermore, a similar preparation method was
   employed to synthesize CZGE lacking Zn^2+ but with the same other
   factors (named CZGE**). The investigation focused on whether Cur,
   another key component of CZGE, mediates the regulation of ZnT1 on
   in‐copper and ex‐copper levels (Figure [159]S24, Supporting
   Information). Even when the protein expression of ZnT1 was blocked in
   si‐ZnT1 pretreated cells, the Cu^2+ level in the cells treated with
   CZGE** (III group) was still lower than that of the model group (I
   group). Nevertheless, upon re‐addition of CuSO[4] (simulating
   continuous ex‐copper influx), the Cu^2+ level in CZGE**‐treated cells
   was maintained at a high level. This indicated that while the chelation
   of Cu^2+ by Cur effectively reduced in‐copper levels, it had difficulty
   in preventing the influx of ex‐copper. The above results indicated Cur
   and Zn^2+ as the two key components were released from CZGE upon cell
   entry. The former reduced the in‐copper level through chelation, while
   the latter acted on the membrane‐localized ion channel ZnT1 to inhibit
   the continuous influx of ex‐copper.

   As an upstream hormone affecting T secretion, GnRH was regulated by
   PKC/ERK pathway, which influenced GnRH secretion by regulating the
   depolarization of GnRH neurons (Figure [160]4Q).^[ [161]^42 ^] As
   depicted in Figure [162]4R,S, the phosphorylation levels of PKC protein
   and ERK1/2 in the model group were significantly down‐regulated,
   contributing to abnormal GnRH secretion due to oxidative damage induced
   by copper overload. In contrast, the phosphorylation levels of PKC and
   ERK1/2 were significantly up‐regulated in cells treated with CZGE
   compared to the model group, indicating enhanced intracellular ROS
   scavenging. GT1‐7 cells and pituitary cells were co‐cultured in a 3D
   transwell chamber. The GnRH levels in upper chamber solution and FSH
   and LH levels in lower chamber solution were detected treatment with
   different samples. Additionally, the levels of T in TM3 cells was
   measured after 24 h of culture with lower chamber solution, in order to
   evaluate the potential of CZGE to improve reproductive function in
   vitro (Figure [163]4T). It was discovered that the levels of GnRH, FSH,
   LH, and T in the CZGE group were significantly higher than those in the
   model group (Figure [164]4U). This increase could be attributed to the
   abality of CZGE to prevent cell death caused by copper overload and to
   reduce oxidative stress. These actions led to the restoration of GnRH
   neuron endocrine function, regulation of the HPT axis, and promotion of
   reproductive hormone secretion, thereby improving reproductive
   function.

2.5. Evaluation of the Target Ability of CZGE In Vivo

   To ensure the efficacy of CZGE as an oral nanomedicine, maintaining its
   stability in the gastrointestinal (GI) fluid environment is a critical
   prerequisite for therapeutic effectiveness. The stability of GEVs,
   KZCNs, and CZGE was evaluated in simulated gastric fluid (SGF, pH =
   1.8) and simulated intestinal fluid (SIF, pH = 7.4) using DLS and
   subsequent drug release analysis. After 4 h of treatment in both SGF
   and SIF, the particle sizes of GEVs and CZGE remained comparable to
   those treated with KZCNs and PBS, respectively (Figure [165]S25,
   Supporting Information). Additionally, the retention rate of Zn‐Cur in
   CZGE remained largely unchanged under these conditions, suggesting that
   CZGE exhibits stability in the GI tract (Figure [166]S26, Supporting
   Information). It may be attributed to the protective structure of
   plant‐derived EVs with acid resistance.^[ [167]^43 ^] Besides, the
   blood circulation time of CZGE (half‐life of 4.12 h) in healthy
   Sprague‐Dawley rats was significantly prolonged than that (half‐life of
   1.66 h) of free Cy5, which suggested that CZGE exhibits superior in
   vivo stability and biocompatibility, thereby extending its residence
   time within the body (Figure [168]S27, Supporting Information).

   To further investigated the in vivo oral brain‐targeting capability of
   CZGE, the fluorescence distribution of various Cy5‐loaded samples in WD
   mice was monitored following oral gavage using an in vivo imaging
   system (IVIS). The fluorescence images revealed that the brain began to
   exhibit fluorescence signals 2 h post‐administration of different
   samples, with the most intense signals observed at 8 h (Figure
   [169]5A,C). Then the brain and major tissues were collected for ex vivo
   fluorescence imaging and semi‐quantification after 24 h of
   post‐administered. It had been observed that CZGE can successfully
   traverse the intestinal barrier and exhibit significantly higher
   accumulation in the liver and brain compared to that in other groups
   (Figure [170]5B,D). These findings indicated that CZGE was capable of
   overcoming two critical biological barriers (IEB and BBB), thereby
   achieving targeted delivery to the brain following oral administration.
   Additionally, the oral doses of CZGE in various brain regions were
   calculated, which will be critical for the development of future oral
   brain‐targeting nanoparticle systems. The IVIS imaging results revealed
   the distribution of CZGE in the olfactory bulb (OB), cortex (CT),
   hypothalamus (HT), midbrain (MB), hippocampus (HC), and cerebellum
   (CB), with oral doses of 21.98%, 17.08%, 30.24%, 17.04%, 13.50%, and
   9.22%, respectively. Notably, the highest concentration of CZGE was
   observed in the hypothalamus, which was the core region associated with
   reproductive dysfunction in WD and also the primarily distribution
   location of GnRH neurons (Figure [171]S28, Supporting Information).

Figure 5.

   Figure 5
   [172]Open in a new tab

   Evaluation of the target ability of CZGE in vivo. A,C) In vivo
   fluorescence imaging and the corresponding fluorescence quantification
   of WD mice administrated with Cy5‐labeled different samples at
   different time points, scar bar: 1 cm (n = 3). B,D) In vivo
   fluorescence imaging and the corresponding quantitative analysis of the
   ileum, brain and major organs from the normal and WD mice treated by
   different samples after 24 h of oral gavage (n = 3). E–G)
   Representative co‐localization images of different samples in the WD
   ileum after oral with different samples (blue: nucleus, green:
   Dectin‐1for M cells; red: free Cy5 or Cy5‐labled samples). H) Flow
   cytometry analysis and quantitative analysis of intestinal cells
   (Cy5^+, Dectin‐1^+) in the mice in each group (n = 3). J,K)
   Representative co‐localization images of different samples in the WD
   brain after oral with different samples (blue: nucleus, green: GPR54
   for neuron; red: free Cy5 or Cy5‐labled samples). L) Flow cytometry
   analysis and quantitative analysis of hypothalamic cells (Cy5^+,
   GnRH^+) in the mice in each group (n = 3). (Samples for H and L, I:
   Cy5, II: GEVs, III: KZCNs, IV: CZGE). All statistical data are
   presented as mean ± standard deviation. Statistical analysis: two‐way
   ANOVA (C, D); one‐way ANOVA (H, L).

   Previous in vitro studies had demonstrated that CZGE, rich in β‐glucan,
   could penetrate the IEB by targeting the Dectin‐1 receptor on the
   surface of intestinal M cells.^[ [173]^44 , [174]^45 , [175]^46 ^]
   Consequently, histological sections of the isolated tissue were stained
   to further elucidated the delivery pathway from the gut to the brain.
   The numerous CZGE particles (red color) were observed to effectively
   co‐localize with the Dectin‐1 receptor (green color) on intestinal M
   cells (Figure [176]5E–G). Subsequently, the targeting efficiency of
   CZGE in the intestinal was further investigated using in vivo flow
   cytometry. As shown in Figure [177]5H, the proportions of cells
   co‐positive for Cy5 and intestinal M cells in the ileum of mice treated
   with GEVs and CZGE were 58.1% and 68.7%, respectively. These values
   were 3.56 and 4.21 times higher than those in the KZCNs group. It was
   further demonstrated that CZGE can selectively target intestinal M
   cells and pass through IEBs, a process facilitated by the β‐glucan on
   their surface. Furthermore, the co‐localization of CZGE with GnRH
   neurons in the hypothalamus had demonstrated that the red fluorescence
   of the Kiss‐10 peptide effectively overlapped with the green
   fluorescence of GPR54, a receptor on the membrane surface of GnRH
   neurons (Figure [178]5I–K). Subsequently, the targeting efficiency of
   CZGE after penetrating the BBB was further verified in vivo. It was
   found that the number of cells co‐expressing Cy5 and GnRH in the
   hypothalamus of mice treated with GEVs and CZGE were 80.5% and 85.8%,
   respectively, which was 1.97 and 2.10 times higher than that of KZCNs
   (Figure [179]5L and Figure [180]S29, Supporting Information).
   Collectively, these results provided strong evidence that the β‐glucan
   on the surface of CZGE could be utilize to sequentially cross the IEB
   and BBB, successfully delivering to the hypothalamus and targeting GnRH
   neurons with the Kiss‐10 peptide to enhance its accumulation in the WD
   hypothalamus, thereby achieving efficient oral brain targeting.

2.6. Assessment of the Therapeutic Mechanisms of CZGE in WD Mice

   As illustrated in Figure [181]6A, the efficacy of CZGE in treating WD
   with reproductive dysfunction was further evaluated using TX mice, a
   recognized animal model for WD. Homozygous male toxic milk (TX) mice
   (6–8 weeks old) were randomly divided into model, GEVs, KZCNs, and CZGE
   groups, with homologous Dunn‐Lewis (DL) mice serving as the control
   group. For therapeutic intervention, oral gavage administration of 8
   mg·kg^−1 was performed daily for 28 days, with the control group
   receiving an equivalent dose of PBS. Post‐treatment evaluation of
   therapeutic efficacy was conducted using susceptibility‐weighted
   imaging (SWI) imaging and histopathological analysis.

Figure 6.

   Figure 6
   [182]Open in a new tab

   Assessment of the therapeutic mechanisms of CZGE in WD mice. A) The
   treatment schedule of the experiment. B) Representative SWI images and
   phase signal value analysis in the hypothalamus. C) Immunofluorescence
   staining and quantitative analysis of CD206 (red) in the hypothalamus
   of mice by different treatment (n = 3). D) Immunofluorescence staining
   and quantitative analysis of iNOS (red) in the hypothalamus of mice by
   different treatment (n = 3). E) Immunofluorescence staining and
   quantitative analysis of NLRP3 (red) in the hypothalamus of mice by
   different treatment (n = 3). F) Immunofluorescence staining and
   quantitative analysis of GSDMD‐N (red) in the hypothalamus of mice by
   different treatment (n = 3). G) Relative quantification of Nrf2,
   Caspase‐1 in the hypothalamus of mice by different treatments (n = 3).
   H) Immunohistochemical staining and quantitative analysis of GnRH in
   the hypothalamus of mice by different treatment (n = 3). I) Relative
   quantification of GnRH, FSH, LH and T in the serum of mice by different
   treatments (n = 3). J) The sperm survival rate for the mice after
   different treatments by an optical microscope (n = 3). K) The sperm
   plasma‐membrane and acrosome integrity for the mice after different
   treatments by flow cytometry (n = 3). (Samples for B‐K: I: Cy5, II:
   GEVs, III: KZCNs, IV: CZGE, VI: cetrorelix). All statistical data are
   presented as mean ± standard deviation. Statistical analysis: one‐way
   ANOVA.

   First, SWI was used to dynamically assess the therapeutic effects of
   CZGE on the brains of WD mice (Figure [183]6B). It significantly
   low‐signal dark areas in the hypothalamic region of WD mice compared to
   that in the control group. Quantitative analysis showed that treatment
   with CZGE prevented the sharp decline of phase values in the
   hypothalamus of WD mice, restoring them to almost the same level as
   that of the mice in the control group. It suggested that secondary
   damage caused by copper deposition in the WD hypothalamus can be
   effectively mitigated by CZGE. As anticipated, CZGE treatment reduced
   copper levels in hypothalamic tissue of WD mice using inductively
   coupled plasma mass spectrometry (ICP‐MS) (Figure [184]S30, Supporting
   Information). Besides, the expression levels of pro‐inflammatory
   cytokines (TNF‐α and IL‐6) were significantly reduced, whereas the
   levels of anti‐inflammatory cytokines (IL‐10 and IL‐4) were elevated in
   the CZGE‐treated group compared to that in the model group, which
   elucidated the reduction of copper levels to effectively mitigate the
   cascading neural effects by CZGE (Figure [185]S31, Supporting
   Information). Furthermore, microglia play a crucial role in brain
   inflammation in WD. Among these, Iba‐1, serving as an important
   indicator of neuroinflammation, was significantly upregulated in the WD
   group but was significantly downregulated following CZCG treatment
   (Figure [186]S32, Supporting Information). And the inflammatory
   microenvironment shifted from a pro‐inflammatory state (the expression
   of iNOS) to an anti‐inflammatory state (the expression of CD206) in the
   hypothalamus of WD mice following CZGE processing (Figure [187]6C,D;
   Figures [188]S33 and [189]S34, Supporting Information).

   To explore the anti‐inflammatory mechanism in vivo, the expression
   levels of key pyroptosis‐related proteins in the hypothalamus of WD
   mice were examined. The expression levels of NLRP3 and GSDMD‐N were
   markedly elevated in WD mice compared to the control group, whereas
   these levels were significantly reduced in mice treated with CZGE
   (Figure [190]6E,F; Figures [191]S35 and [192]S36, Supporting
   Information). Additionally, the expression levels of Caspase‐1, IL‐18,
   and IL‐1β in the hypothalamus of the CZGE‐treated mice were notably
   decreased, while Nrf2 expression was significantly increased
   (Figure [193]6G and Figure [194]S37, Supporting Information). This
   result corresponds to the ability of CZGE to effectively inhibit
   pyroptosis of hypothalamic neurons in vitro by upregulating Nrf2.
   Besides, the co‐expression of GnRH and NLRP3, as well as GnRH and PKC,
   in the hypothalamus of WD mice was further examined. It was found that
   in the WD model group, the expression of GnRH in the hypothalamus was
   significantly decreased, the expression of NLRP3 was significantly
   increased, and the expression of PKC was significantly decreased.
   Following treatment with CZGE, the expression of GnRH increased, the
   expression of NLRP3 decreased significantly, and the expression of PKC
   increased significantly (Figure [195]S38, Supporting informing). These
   findings further confirm the hypothesis that copper deposition can
   affect the function of GnRH neurons by regulating the NLRP3‐PKC
   pathway. In addition, the zinc levels in hypothalamic tissue of the
   KZCNs and CZGE groups were found to be significantly higher than those
   in the model group, indicating that the nanomaterial successfully
   facilitated ion exchange and released Zn^2+ in the brain within a
   high‐copper environment (Figure [196]S39, Supporting Information).
   Urinary and fecal copper levels in WD mice were measured, and it was
   found that, compared with the model group, both urinary and fecal
   copper levels were significantly elevated in the KZCNs and CZGE groups,
   with the most pronounced increase observed in the CZGE group (Figure
   [197]S40, Supporting Information). This further confirmed that the
   synergistic therapeutic effect of CZGE is achieved by inhibiting copper
   absorption and promoting copper excretion through the release of Zn^2+,
   in addition to chelating intracellular copper.

   The HPT axis plays a crucial role in regulating reproductive function.
   Specifically, the most important secretion GnRH by GnRH neurons can
   promote the production of secretions that promote sperm or testicular
   function (such as LH and FSH) by specifically binding to the specific
   protein (GnRHR I) of pituitary cells, thereby affecting reproductive
   function (Figure [198]S41, Supporting Information). Therefore, the
   hormone secretion capability of the hypothalamus was initially
   evaluated using immunohistochemical staining. Compared to that in the
   control group, GnRH expression in the hypothalamus was significantly
   reduced in the model group but markedly increased in the CZGE‐treated
   group (Figure [199]6H). Subsequently, the causal relationship between
   copper deposition and GnRH neuronal injury was further investigated.
   The results demonstrated that, compared with that in the control group,
   red fluorescence (Cu^2+) in the model group was markedly enhanced,
   whereas green fluorescence (GnRH) was significantly reduced. This
   suggested that copper deposition in the hypothalamic tissue of WD mice
   was substantially increased, leading to the inhibition of GnRH
   expression. However, following CZGE treatment, the red fluorescence
   intensity of Cu^2+ in the hypothalamic neurons of WD mice decreased to
   a level comparable to that of the control group, while the green
   fluorescence intensity of GnRH was significantly elevated to 1.57 times
   that of the model group (Figure [200]S42, Supporting Information).
   These findings indicated that excessive Cu^2+ can markedly suppress the
   expression and secretion of GnRH, whereas CZGE can restore GnRH
   secretion by reducing Cu^2+ levels. Additionally, the serum levels of
   GnRH, FSH, LH, and T in the mice of the model group were found to be
   significantly decreased compared with that in the control group.
   However, following treatment with CZGE, these levels were observed to
   have increased significantly in Figure [201]6I. Furthermore, the
   potential of CZGE to enhance testicular prognosis and function through
   the regulation of GnRH secretion and the utilization of the HPT axis
   was further explored using the GnRH secretion inhibitor (cetrorelix).
   It was observed that, compared to that in the CZGE group, the serum
   level of T in mice treated with cetrorelix was significantly reduced
   (Figure [202]6I). As the primary organ responsible for sperm production
   and testosterone secretion, the structure and function of the testis
   play a crucial role in reproductive function.^[ [203]^47 , [204]^48 ,
   [205]^49 ^] Through hematoxylin and eosin (H&E) staining, structural
   changes in testicular tissue were observed, with damage to seminiferous
   tubules and necrosis of spermatogenic cells at all stages noted in the
   model group. In contrast, after CZGE treatment, spermatogenic cells
   were found to be more densely arranged. Meanwhile, spermatogenic
   tubules in the cetrorelix group exhibited varying degrees of damage and
   necrosis (Figure [206]S43, Supporting Information). These findings
   suggested that enhancing GnRH secretion in the hypothalamus can
   influence T secretion and improve testicular morphology through the HPT
   axis. High‐quality sperm is essential for reproductive function.^[
   [207]^50 ^] Sperm function was further assessed to confirm reproductive
   recovery. Observations of sperm smears revealed a significant increase
   in sperm survival in the CZGE group compared to the model group
   (Figure [208]6J). Additionally, flow cytometry analysis revealed that,
   compared with the model group, treatment with CZGE significantly
   enhanced the integrity of the plasma membrane and acrosome of mouse
   sperm. In contrast, the integrity of the plasma membrane and acrosome
   of mouse sperm in the cetrorelix group was significantly compromised
   (Figure [209]6K and Figure [210]S44, Supporting Information). These
   results indicated that spermatogenesis was promoted and sperm quality
   improved in WD mice by CZGE.

   Furthermore, to evaluate the long‐term efficacy of CZGE, the
   observation period was extended for 4 weeks after the completion of
   treatment, and changes in reproductive function were re‐evaluated. It
   was found that, compared to that in the model group, the level of T,
   sperm plasma membrane and acrosome integrity, and spermatogenic tubule
   structure of mice in the CZGE group were all improved to varying
   degrees. These findings indicated that CZGE has a beneficial long‐term
   therapeutic effect (Figure [211]S45, Supporting Information). In
   conclusion, CZGE was found to reduce hypothalamic copper deposition,
   inhibit neuroinflammation, regulate HPT axis function, and enhance
   sperm quality, thereby maintaining reproductive function. Additionally,
   CZGE demonstrated a beneficial long‐term therapeutic effect.

   Finally, in‐depth biocompatibility and safety analyses in vivo to
   understand the potential systemic toxicity of CZGE. Weight changes in
   WD mice were monitored over 28 days post‐treatment, revealing no
   significant differences among groups (Figure [212]S46, Supporting
   Information). Blood routine analysis and H&E staining of brain and
   major organs (heart, liver, spleen, lung, kidney and intestinal) were
   also examined in each group. The results showed that no significant
   changes were observed in routine blood levels, no abnormal pathological
   changes were found in major organs or tissues, and a significant
   improvement was observed in the structure of hypothalamic tissue
   post‐treatment (Figure [213]S47, Supporting Information and
   Table [214]S1, Supporting Information). These findings demonstrated
   that CZGE treatment was biocompatible and non‐toxic. Taken together,
   the above experimental findings revealed that CZGE served as a highly
   efficient and safe gut‐to‐brain drug delivery system for the oral
   treatment of WD with reproductive dysfunction.

2.7. The Therapy Mechanisms by Transcriptomics Analyses

   To further our understanding of the therapeutic process of CZGE, a
   comprehensive transcriptomic analysis of the hypothalamus was conducted
   in both the CZGE‐treated and model groups of mice, revealing potential
   therapeutic mechanisms at the genetic level. The Venn diagram
   illustrated that 24 959 genes were co‐expressed in the model and CZGE
   groups, while 2965 genes were exclusive in the CZGE group (Figure
   [215]7A). Furthermore, volcano plot analysis of differentially
   expressed genes (DEGs) indicated that 1780 genes were up‐regulated and
   1456 genes were down‐regulated in the CZCG treatment group
   (Figure [216]7B).

Figure 7.

   Figure 7
   [217]Open in a new tab

   Transcriptional sequencing and biological analysis results. A) Venn
   diagram of differentially expressed genes in model and CZGE groups. B)
   Volcano plots showing the down‐regulated and up‐regulated genes between
   model and CZGE groups. C) Heatmap of expression levels of the refined
   gene classes (copper channel, inflammasome and oxidative stress) after
   the indicated treatments. D,E) Regulation of neuroinflammatory response
   and MAPK signaling pathway GSEA enrichment analysis. F) Dot plots of
   KEGG pathway enrichment analysis of the DEGs between the model and CZGE
   groups. G) GO enrichment analysis of DEGs in cellular biological
   process showing the most significantly enriched categories. n  =  3
   biologically independent cell samples per group.

   To delve deeper into the underlying mechanisms, heat maps were
   constructed to analyze gene expression patterns associated with copper
   channels, inflammasomes, and oxidative stress. The classification of
   DEGs revealed downregulation in genes associated with copper channels,
   inflammasomes, and oxidative stress, confirming that CZGE can inhibit
   pyroptosis and reduce inflammation at the genetic level
   (Figure [218]7C). Additionally, Gene Set Enrichment Analysis (GSEA)
   demonstrated that the neuroinflammatory response was actively regulated
   in the CZGE treatment group, with significant up‐regulation of the MAPK
   signaling pathway (Figure [219]7D,E). It aligned with previous studies
   and provided further evidence that CZGE plays a crucial role in GnRH
   secretion by inhibiting inflammation and modulating the MAPK‐PKC‐ERK
   pathway. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment
   analysis indicated significant enrichment of the NOD‐like receptor
   signaling pathway and the TNF signaling pathway among DEGs following
   CZGE treatment. Previous experimental studies had shown these pathways
   to be closely associated with the anti‐inflammatory therapeutic
   mechanisms of CZGE. Specifically, the NOD‐like receptor signaling
   pathway had been linked to pyroptosis, while ROS could induce systemic
   inflammatory responses through the activation of the TNF signaling
   pathway. Therefore, normalizing the expression levels of these genes
   could inhibit pyroptosis, thereby mitigating damage caused by copper
   deposition. Moreover, DEGs were significantly enriched in pathways
   regulating GnRH secretion, including the MAPK signaling pathway, GnRH
   signaling pathway, and Ras signaling pathway (Figure [220]7F).^[
   [221]^51 , [222]^52 , [223]^53 ^] Genetic ontological (GO) enrichment
   analyses confirmed changes in copper ion import and activation of
   pathways involved in the regulation of neuroinflammatory responses, as
   well as alterations in biological functions related to reproductive
   system development before and after treatment (Figure [224]7G). The
   results of the transcriptomic analysis were also validated by qPCR
   revealed significant downregulation of hypothalamic
   inflammasome‐related gene (NLRP3), alongside significant upregulation
   of GnRH secretion‐regulating gene (MAPK14 and GnRH), consistent with
   the transcriptomic data (Figure [225]S48, Supporting Information).
   Collectively, these findings suggested that hypothalamic neuronal
   pyroptosis was alleviated by CZGE through the reduction of hypothalamic
   copper deposition, thereby inhibiting inflammation, promoting GnRH
   secretion, and improving reproductive function to a certain extent.

3. Conclusion

   In summary, an oral brain‐targeted drug delivery system via ion
   exchange strategy was developed and proposed for the treatment of WD
   with reproductive dysfunction. Specifically, the therapeutic system of
   CZGE, which incorporated the targeting functions of β‐glucan and
   Kiss‐10, can effectively surmount both the IEB and BBB, thereby
   accurately targeting the damaged neurons associated with WD lesions.
   Followed by internalization, the ion exchange replacement between
   excess Cu^2+ and CZGE was rapidly occurred and released Zn‐Cur to form
   Cu‐Cur and the subsequent release of free Zn^2+. The former exhibited
   anti‐inflammatory properties, effectively reducing in‐copper and
   neuroinflammation by promoting the Nrf2‐NLRP3 pathway, while Zn^2+
   blocks ex‐copper influx by activating ZnT1. Furthermore, it was
   demonstrated that oral administration of CZGE can significantly reduce
   WD‐related copper deposition, alleviate neuroinflammation, and markedly
   improve reproductive function. Given its high efficacy and favorable
   biosafety profile, CZGE was considered to hold significant potential
   for future clinical applications in treating WD with reproductive
   dysfunction.

4. Experimental Section

Ethics Statement

   All animal experiments were approved by the Laboratory Animal
   Management Committee of Institute of health and medicine, Hefei
   comprehensive national science center (Permit Number: IHM‐AP‐2024‐051).
   The use of human clinical indexes was approved by the Ethics Committee
   of the First Affiliated Hospital of Anhui University of Chinese
   Medicine (Permit Number: 2023AH‐56).

Materials

   All the Fmoc‐protected amino acids, Rink amide resins were purchased
   from Bide Pharmatech Co., Ltd. (Shanghai, China).
   3,3′‐dioctadecyloxacarbocyanine perchlorate (DiO), DCFH‐DA,
   5,5′,6,6‐Tetrachloro‐1,1′,3,3′‐tetraethyl‐imidacarbocyanineiodide
   (JC‐1) and cetrorelix were all purchased from Shanghai Beyotime
   Biotechnology Co., Ltd. (Shanghai, China). Zinquin ethyl ester was
   purchased from Maokang Biochemical Technology Co., Ltd. (Shanghai,
   China). Cu^2+ fluorescence probe was purchased from Fount Biochemical
   Technology Co., Ltd. (Beijing, China). Curcumin, CuSO[4]·5H[2]O, Zinc
   acetate dihydrate, 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐
   [methoxy (polyethylene glycol)]‐Cy5 (DSPE‐PEG‐Cy5),
   1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate
   (Dil) were all provided from Bide Pharmatech Co., Ltd. (Shanghai,
   China). Methyl‐β‐cyclodextrin (MβCD), amiloride hydrochloride were
   purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai,
   China). Chlorpromazine hydrochloride (CPZ) was purchased from
   Altascientific Co., Ltd. (Tianjin, China). • OH and SOD assay kit was
   purchased from Nanjing Success Biotechnology Research Institute Co.,
   Ltd. (Nanjing, China). The Cell Counting Kit‐8 (CCK‐8) was purchased
   from Dojindo Laboratories (Kumamoto, Japan). NLRP3 inhibitor (MCC950)
   was purchased from Shandong Sparkred Biotechnology Co., LTD. Rabbit
   Anti‐Cleaved Caspase‐1, Rabbit Anti‐GSDMD, Rabbit Anti‐PKC, Rabbit
   Anti‐ERK1/2, Rabbit Anti‐p‐ERK1/2 were purchased from Jiangsu Kinko
   Biological Research Center Co., LTD. Rabbit Anti‐Nrf2, Mouse
   Anti‐NLRP3, Mouse Anti‐GnRH, Coralite488, FITC‐CD86 and PE‐CD206 were
   purchased from Wuhan Saneagle Biotechnology Co., LTD. DMEM/F12 (high
   glucose) and Pen‐Strep were purchased from Dalian MeilunBio Co., Ltd.
   Trypsin‐EDTA (0.25%) was purchased from Anhui KETU Biotechnology Co.,
   Ltd. Mouse Tumor Necrosis Factor Alpha (TNF‐a), Mouse Interleukin‐4
   (IL‐4), Mouse Interleukin‐10 (IL‐10) and Mouse Interleukin‐6 (IL‐6)
   ELISA Kits was purchased from Jianglai Industrial Co., Ltd. (Shanghai,
   China). Mouse Interleukin‐18 (IL‐18), Mouse Interleukin‐1β (IL‐1β),
   Mouse NLRP3 and Mouse PKC ELISA Kits was purchased from Meimian
   Industrial Co., Ltd. (Jiangsu, China).

Instrumentation

   The exosomes are obtained by ultra‐high centrifuge (OptimaXPN‐100,
   Beckman, USA). The Zeta potential was recorded by Zetasizer (ZEN3690,
   Malvern, USA). The Fluorescence resonance energy transfer (FRET) was
   detected by Fluorescence spectrophotometer (F4600, Japan). The ^1H‐NMR
   spectrum was recorded on the Bruker ADVANCE III 600 MHz spectrometer.
   Transmission electron microscopy (TEM) images was obtained using
   Hitachi HT7800 (80 kV). The particle concentration and particle size of
   GEVs, KZCNs, and CZGE were detected by nanoparticle tracking analysis
   (NTA, Malvern, UK). All fluorescence images of cells were captured by
   CLSM (Zeiss LSM900, Germany) and Inverted fluorescence microscope
   (LEICA DMI8, Germany). Cell viability was detected by Microplate reader
   (SpectraMax i3X, USA). The clearance of intracellular ROS, and
   dysfunction of mitochondria were assessed using flow cytometry. (BD
   FACS Celesta, BD, USA). Biological distribution in vivo and in vitro
   was performed on the IVIS imaging system (PerkinElmer). In vivo
   magnetic resonance imaging of mice was performed on a 9.4 T MRI scanner
   (uMR 9.4T, Shanghai, China).

Cell Lines and Animals

   GT1‐7 cell lines were purchased from Meilun Biotechnology Co., LTD.
   (Dalian, China). Caco‐2 and bEnd.3 cell lines were purchased from
   Shanghai Xinyu Biotechnology Co., LTD. (Shanghai, China). The complete
   medium was prepared by adding 10% FBS and 1% Pen/Strep/Amp solution to
   basic medium DMEM/F12 or DMEM, and cultured in a 5% CO[2] incubator at
   37 °C. TX mice (C3He‐ATP7B^tx‐j) carrying an ATP7B genemutation were
   purchased from the Jackson Laboratory Animal Center (Maine, USA).

Isolation and Purification of GEVs

   GEVs were isolated from ganoderma using differential
   ultracentrifugation. 50 g of dried ganoderma were soaked in 200 mL of
   ice‐cold phosphate‐buffered saline (PBS, 10 mM, pH 7.4) and processed
   in a blender to obtain the juice. The juice underwent centrifugation at
   500 g for 10 min, 2000 g for 20 min and 10 000 g for 30 min to remove
   large ganoderma fibers. The supernatant was then subjected to further
   ultracentrifugation at 150 000 g for 2 h, followed by resuspension of
   the pellet in PBS. The GEVs was stored at −80 °C for future use, and
   the particle size and zeta potential of GEVs were measured by dynamic
   light scattering (DLS) and TEM.

Preparation of Zn‐Cur and Cu‐Cur

   The Zn‐Cur chelate was synthesized by mixing zinc acetate dihydrate
   with methanolic solution of curcumin at a molar ratio of 1:1.
   Specifically, 50 mL of methanolic solution of curcumin (2 mM) was
   prepared and then heated to 60 °C for 0.5 h. Concurrently, 2 mM of zinc
   acetate dehydrate was dispersed in 100 mL of methanol by magnetic
   stirring for 0.5 h. The prepared solution was then added to the
   curcumin solution under magnetic stirring. The product was subsequently
   filtered, rinsed three times with cold methanol, and freeze‐dried
   overnight for further use. The Cu‐Cur chelate was prepared similarly,
   substituting CuSO[4]·5H[2]O for zinc acetate dihydrate. It was
   subsequently characterized using ^1H‐NMR spectrum, ultraviolet‐visible
   (UV–vis) absorption spectrum, and FTIR spectra.

Preparation of ZCNs, KZCNs, and CZGE

   ZCNs were prepared by a thin film hydration method according to
   previously reports. In brief, DSPE‐PEG[2000]‐Mal (2 mg) was dissolved
   in 4.5 mL of acetonitrile, while curcumin (1 mg) was dissolved in 4.5
   mL of the mixture solvent of ethanol and methanol (1:2, v: v). The two
   solutions were combined in a round flask, and the organic solvent was
   removed by rotary evaporation to obtain a thin dried film. The dried
   was then hydrated with 20 mL of PBS at 60 °C for 30 min. After
   centrifugation at 10 000 rpm for 3 min to remove free curcumin to
   obtain ZCNs. Subsequently, the targeted peptide of Kiss‐10 was prepared
   by standard Fmoc solid‐phase peptide synthesis (SPPS) method, and 0.62
   mg of Kiss‐10 was further added to the above ZCNs solution (10 mL), and
   the mixture was incubated under N[2] protection for 12 h to obtain
   KZCNs. To investigate if Kiss‐10 were successfully grafted onto ZCNs
   surface, Dil‐labeled ZCNs were combined with FITC‐labeled Kiss‐10
   according to the described procedure. The resulting mixtures were
   imaged using the inverted fluorescence microscope, and the fluorescence
   intensity was measured at 490 nm excitation wavelength and 520 nm
   emission wavelength using fluorescence spectroscopy. The same number of
   KZCNs and GEVs, quantified and extruded through 0.2 µm polycarbonate
   membrane filter 10 times and further merged for 12 h to obtain uniform
   CZGE. The particle size and zeta potential were characterized by DLS
   and TEM. The particle concentration and particle size of GEVs, KZCNs,
   and CZGE were detected by NTA.

Vesicle Fusion Studies

   GEVs and KZCNs were labeled with 20 µM DiO and Dil, respectively. The
   labeled GEVs and KZCNs were then incubated at room temperature for 12 h
   and extruded through a 0.2 µm polycarbonate membrane at a 1:1 ratio,
   followed by a further incubation for 12 h. Finally, the mixture was
   imaged using an inverted fluorescence microscope.

   FRET assay was used to verify membrane fusion to check whether GEVs
   were incorporated into KZCNs. GEVs were labeled with Dil
   (excitation/emission = 549/565 nm) and DiD (excitation/emission =
   644/663 nm). KZCNs were then added to the dye‐labeled GEVs at a 1:1
   ratio for fusion. The fluorescence spectrum of KZCNs were measured
   within the 550 to 750 nm range, using an excitation wavelength of 525
   nm.

Antioxidation Performance and Mechanism of Cur, Zn‐Cur, and Cu‐Cur

   The inhibition rates of •OH and O[2] ^•‐ for Cur, Zn‐Cur and Cu‐Cur at
   concentrations ranging from 20 to 120 µg mL^−1 were determined by •OH
   and SOD detection kit. In addition, the ESR spectroscopy of Cur, Zn‐Cur
   and Cu‐Cur was performed using DMPO/BMPO as the spin trapping reagent.
   For O[2] ^•‐ detection assay, 1 mM of xanthine, 0.2 unit mL^−1 of XO, 5
   mM of BMPO and Cur, Zn‐Cur or Cu‐Cur (120 µg mL^−1) were added into the
   PBS solutions. Subsequently, an aliquot of the solution (100 µL) was
   transferred into the quartz tube for ESR measurements. For •OH
   detection assay, 2 mM of H[2]O[2], 10 µM of FeSO[4], 1.5 of mM DMPO,
   and Cur, Zn‐Cur or Cu‐Cur (120 µg mL^−1) were added into the PBS
   solutions.

Evaluation of the Stability of GEVs, KZCNs, and CZGE

   GEVs, KZCNs and CZGE were stored at 37 °C in 10% FBS to mimic blood
   conditions. Their size distribution was examined by DLS from 1 day to 7
   days at a set time.

Cell Viability Assay

   About 1 × 10^4 cells mL^−1 GT1‐7 cells were inoculated in 96‐well
   plates and cultured for 24 h. In the stimulation group, CuSO[4] with a
   final concentration of 300 µM was added and cultured at 37 °C for 24 h.
   Different concentrations of CZGE were added. After incubation for 24 h,
   CCK‐8 assay was utilized to evaluate the viability of GT1‐7 cells after
   different treatments. In addition, the experimental procedures for cell
   viability in different samples and at different times were consistent
   with the above.

Experiment of a Simulated IEB and BBB—In the IEB Mimetic Model

   The Caco‐2 cells and RAW264.7 cells were mixed in a 9:1 ratio were
   seeded with a density of 1 × 10^5 cells mL^−1 in the upper chambers (12
   well, pore size: 0.4 µm). After a 7‐day incubation period, the IEB was
   successfully formed. RAW264.7 cells were then seeded in the lower
   chambers. The IEB was then stimulated with CuSO[4]​ (300 µM) for 24 h.
   Subsequently, different Cy5‐labeled samples (10^6 particle mL^−1) were
   introduced into the upper chamber and incubated for an additional 8 h.
   In the Dectin‐1 inhibitor group, Laminarin (1 mg mL^−1) was
   pre‐incubated for 3 h prior to the addition of CZGE labeled with Cy5
   for further incubation.

In the BBB Mimetic Model

   The bEnd.3 cells were seeded with a density of 5 × 10^4 cells mL^−1 in
   the upper chambers (12 well, pore size: 0.4 µm) for 10 days to form the
   blood brain barrier layer. GT1‐7 cells were then seeded in the lower
   chambers, treated with 300 µM CuSO[4] for 24 h. Following this,
   different Cy5‐labeled samples (10^6 particle mL^−1) were added to upper
   chamber and incubated for 24 h. After discarding the culture medium,
   the cells in the lower and upper chambers were collected and were
   stained with DAPI (5 µg mL^−1) for 10 min, respectively. The stained
   GT1‐7 cells were recorded by CLSM and IVIS system.

   Penetration rate (%) = Fluorescence intensity of Cy5 from the lower
   chambers /Fluorescence intensity of Cy5 from the lower chambers and the
   upper chamber × 100%.

Cellular Uptake

   GT1‐7 cells with a density of 5 × 10^4 cells mL−^1 were inoculated in
   confocal dishes. A final concentration of 300 µM of CuSO[4] was
   stimulated for 24 h. Different samples were added to confocal dishes at
   different times (1, 2, and 4 h) for co‐culture. Then, the cells were
   cleaned with PBS and stained with DiO (10 µM) and DAPI (5 µg mL^−1) for
   15 min. After washing with PBS, fluorescence images of GT1‐7 cells were
   detected by CLSM. To quantitatively measure the level of cell uptake,
   GT1‐7 cells were grafted as described above, collected and cleaned for
   flow cytometry analysis.

Endocytosis Mechanism Assay

   To explore the endocytic pathways in GT1‐7 cells, various endocytic
   inhibitors were introduced. GT1‐7 cells (5 × 10^4 cells mL^−1) were
   seeded in 24‐well plates. For stimulation, CuSO[4] was administered to
   the designated group at a final concentration of 300 µM and incubated
   at 37 °C for 24 h. To assess the impact of endocytosis inhibitors,
   specific inhibitors were added: chlorpromazine (CPZ, 10 µg mL^−1),
   amiloride (1 mM) and methyl‐β‐cyclodextrin (MβCD, 500 µM) for
   caveolin‐mediated endocytosis. These inhibitors were incubated for 4 h
   at 4 °C. Following this, DiO‐labeled samples (10^6 particle mL^−1) were
   introduced and incubated for an additional 4 h. Subsequently, the cells
   were collected, and the DiO fluorescence signal was measured using flow
   cytometry.

Analysis of Intracellular Zn^2+ and Cu^2+ Level

   GT1‐7 cells were seeded into culture dishes at a density of 5 × 10^4
   cells mL^−1 and cultured for 24 h. Subsequently, the cells were
   stimulated with a 300 µM CuSO[4] solution and incubated at 37 °C for 24
   h. Various samples, at a concentration of 10^6 particle mL^−1, were
   then added and incubated for an additional 24 h. Then, 200µL of zinquin
   ethyl ester (20 µM) or 200 µL of Cu^2+ fluorescence probe (1 µM) was
   added in the dark at 37 °C for 30 min. After washing with PBS three
   times, the cells were collected to observe and detect using
   fluorescence microscopy and flow cytometry analysis.

   The GT1‐7 cells were plated in the same experimental manner. The cells
   were incubated with CuSO[4] (300 µM) for 24 h, followed by the addition
   of the CZGE* (10^6 particle mL^−1) for 4 h, and then CuSO[4] (300 µM)
   was added for stimulation. A Cu^2+ fluorescent probe (1 µM) was used
   for incubation at 37 °C in the dark for 30 min and subsequent flow
   cytometry detection.

   The GT1‐7 cells were plated in the same experimental manner and
   incubated with CuSO[4] (300 µM) for 24 h. Si‐ZnT1 (Huzhou Hippobio Co.,
   LTD.) was then transfected, followed by incubation with CZGE* (10^6
   particle mL^−1) for 4 h, after which CuSO[4] was added for stimulation.
   Subsequently, 200 µL of a Cu^2+ fluorescent probe (1 µM) was added to
   the cells, which were then incubated at 37 °C in the dark for 30 min,
   followed by flow cytometry detection. Following the same experimental
   procedure, CZGE** (10^6 particle mL^−1) was added for 4 h, and a Cu^2+
   fluorescent probe was used for incubation and subsequent flow cytometry
   detection.

Analysis of Intracellular ROS Level

   GT1‐7 cells, at a density of 5 × 10^4 cells mL^−1, were inoculated into
   confocal dishes and cultured overnight. Following induction with 300 µM
   CuSO[4​] for 24 h, various samples were introduced and incubated for an
   additional 24 h. The cells were then washed three times with PBS and
   stained with 2 µM DCFH‐DA for 30 min. Subsequently, the cells were
   observed under an inverted fluorescence microscope. and prepared for
   flow cytometry analysis.

Determination of Mitochondrial Membrane Potential

   The mitochondrial membrane potential was evaluated using a JC‐1
   fluorescence probe assay kit. GT1‐7 cells were seeded into culture
   dishes at a density of 5 × 10^4 cells mL^−1 per well and cultured for
   24 h. Subsequently, the groups designated for stimulation were treated
   with CuSO[4​] at a final concentration of 300 µM and incubated at 37 °C
   for 24 h. After that, different samples (10^6 particle mL^−1) were
   added to the culture dish and incubated for 24 h. The cells were then
   washed and incubated with JC‐1 probe (5 µg mL^−1) for 30 min. Finally,
   the cells were washed three times with PBS, and the fluorescence signal
   was analyzed using flow cytometry.

Evaluation of BV2 Cell Polarization

   The polarization of M1 microglia was evaluated by immunofluorescence
   staining (CD86 representing M1 phenotype and CD206 representing M2
   phenotype). A density of 5 × 10^4 BV2 cells were inoculated in a 6‐well
   plate. After CuSO[4] (300 µM) induction for 24 h, media containing
   GEVs, KZCNs and CZGE (10^6 particle mL^−1) were added, respectively.
   After 24 h, the cells were washed with PBS and incubated with
   fixation/osmotic buffer (BD Biosciences, 554 722) for 20 min, and
   incubated with perm/wash buffer (BD Biosciences, 554 723) for 15 min.
   Subsequently, BV2 cells were collected and labeled with antibodies
   FITC‐CD86 (PTG, 65 068) and PE‐CD206 (PTG, 141 705). After washing with
   flow cytometry staining buffer, the suspended cells were detected by
   flow cytometry immediately.

Enzyme‐Linked Immunosorbent Assay

   GT1‐7 cells were plate and treated according to the above method, and
   cells treated with different samples were collected. Among them, the
   NLRP3 inhibitor group was treated with MCC950 (10 µM) for 24 h. The
   cell suspension was diluted with PBS, and the cell was broken by
   ultrasonic wave to destroy the cell and release the intracellular
   components, and the supernatant was carefully collected after
   centrifugation at 2–8 °C for 20 min (2000–3000 r min^−1). The levels of
   IL‐1β, IL‐18, IL‐6, TNF‐α, IL‐4, IL‐10, NLRP3 and PKC were then
   measured by ELISA kit.

Western Blotting Experiment

   GT1‐7 cells with a density of 5 × 10^5 were inoculated in 6‐well plates
   overnight and induced by CuSO[4] (300 µM) for 24 h. PBS was washed
   twice, and 1mL medium containing PBS, GEVs, KZCNs, CZGE (10^6 particle
   mL^−1) was treated for 24 h, respectively. After collection, the medium
   was suspended with 100 µL lysis buffer, store on ice for a few minutes
   to use, and ultrasonic cracking, 10 000 rpm centrifuge for 20 min to
   take supernatant for use. After the protein concentration was
   determined with the BCA protein detection kit (Jiangsu KeyGEN BioTECH
   Corp., Ltd, KGA902), appropriate samples were absorbed by SDS‐PAGE
   electrophoresis, and then transferred to PVDF membrane. Closed with
   skim milk powder and Rabbit Anti‐ZnT1 (1:2000; Bioss, bs‐6440R) was
   incubated at 4 °C overnight, cleaned by TBST, and incubated at room
   temperature for 1 h (1:2000). Finally, the protein signal was detected
   by ECL reagent and analyzed by GAPDH normalized Gel‐Pro32.

Confocal Microscopy Experiments

   Real‐time confocal fluorescence imaging of Zn^2+: GT1‐7 cells were
   seeded in a confocal dish at a density of 5 × 10^4 cells mL^−1 and
   stimulated with 300 µM CuSO[4] for 24 h. Subsequently, 200 µL of
   Zinquin ethyl ester (20 µM) was added, and the cells were incubated at
   37 °C for 30 min in the dark. After recording was started, CZGE (10^6
   particle mL^−1) was introduced into the dish, and fluorescence was
   recorded over a period of 10 min. The fluorescence intensity of Zn^2+
   was quantified on a per‐minute basis.

   Real‐time confocal fluorescence imaging of Cu^2+: GT1‐7 cells were
   seeded in a confocal dish with a density of 5 × 10^4 cells mL^−1,
   supplemented with 300 µM CuSO4 for 24h, rinsed with PBS, CZGE (10^6
   particle mL^−1) was added to the dish and incubated for 4 h, and then
   200 µL Cu^2+ fluorescence probe (1 µM) was added, and the cells were
   incubated at 37 °C for 30 min in the dark. After recording was started,
   CuSO4 (300 µM) was added to the dish, the fluorescence was recorded for
   10 min, and the fluorescence intensity of Cu^2+ was quantified per
   minute. In addition, GT1‐7 cells without CZGE (10^6 particle mL^−1)
   were incubated with the same method as above to observe the changes of
   Cu^2+ fluorescence intensity within 10 min.

In Vitro Co‐Culture Experiment Modeling the Hypothalamic‐Pituitary‐Testicular
Axis

   GT1‐7 cells were inoculated at a density of 5 × 10^4 cells mL^−1 in the
   upper chamber (12 well, pore size: 0.4 µm), and the primary pituitary
   cells of TX mice were extracted and inoculated in the lower chamber.
   After treatment with 300 µM CuSO[4] for 24 h, different samples (10^6
   particle mL^−1) were added to the upper chamber and incubated for 24 h.
   The supernatant of the upper and lower chambers was collected and
   centrifuged, and the hormone levels in the supernatant were detected
   according to the instructions of GnRH, FSH and LH ELISA kits. In
   addition, TM3 cells were inoculated into 12‐well plates at a density of
   5 × 10^4 cells mL^−1, and cultured with the collected supernatant in
   the lower chamber for 24 h. After the supernatant was collected and
   centrifuged, T levels in the supernatant were detected according to the
   instructions of the T ELISA kit.

Animals and Administration

   Male homozygous TX mice aged 6 to 8 weeks were randomly allocated into
   four experimental groups: model group, GEVs group, KZCNs group, and
   CZGE group. Homologous DL mice served as the control group (n = 8 per
   group). Mice in the experimental groups received daily administrations
   of respective samples (200 µL, 8 mg·kg^−1), whereas those in the
   control group received an equivalent volume of PBS for a total duration
   of 28 days. Among that, the GnRH inhibitor group received daily
   intraperitoneal injections of cetrorelix (5 µg kg^−1) during the last 7
   days of treatment with CZGE.

In Vivo Bio‐Distribution Assay and Pharmacokinetic Study

   To evaluate the in vivo distribution of nanoparticles, WD mice were
   administered 200 µL of free Cy5 and various Cy5‐labeled samples (8
   mg·kg^−1) via oral gavage. Subsequently, imaging was conducted using
   the IVIS system at 2, 4, 6, 8, 12, and 24 h post‐administration. For ex
   vivo imaging, the mice were sacrificed after in vivo imaging, and
   brains, iluem, hearts, livers, spleens, lungs, and kidneys were
   collected. The brain tissue of mice was dissected into the olfactory
   bulb, cortex, hypothalamus, midbrain, hippocampus, and cerebellum, and
   subsequently homogenized. The Cy5 fluorescence signal in each organ was
   visualized using IVIS.

   To examine the pharmacokinetic properties of the nanoparticles, WD mice
   were given 200 µL of free Cy5 and Cy5‐labeled CZGE (8 mg·kg^−1) by oral
   gavage. Blood samples (1 mL) were collected from the mice at 0.5, 2, 4,
   6, 8, 12, and 24 h post‐administration, centrifuged at 4 °C for 10 min
   (4000 rpm), and the fluorescence intensity of Cy5 in the supernatant
   was measured using a fluorescence spectrometer.

Stability of CZGE under GI Conditions

   To assess the stability of various samples under gastrointestinal
   conditions, in vitro studies were conducted. Samples were incubated in
   simulated gastric fluid (SGF, pH 1.8, prepared with 10% hydrochloric
   acid) and simulated intestinal fluid (SIF, pH 7.4, phosphate‐buffered
   saline) at 37 °C. Specifically, samples were exposed to SGF for 2 h
   followed by SIF for 4 h. Aliquots were collected at 0, 1, 2, and 4 h,
   and the drug content was subsequently analyzed.

Flow Cytometry Analysis In Vivo

   To evaluate the in vivo targeting efficiency of nanoparticles after
   penetrating the IEB, WD mice were randomly assigned to different groups
   administered 200 µL of free Cy5 and various Cy5‐labeled samples (8
   mg·kg⁻¹) via intragastric gavage. Then, the mice were euthanized, and
   the ileum tissue was promptly excised and finely minced after 8 h. The
   tissue was then completely immersed in 2 mL of digestion solution
   (RPMI‐1640 containing 1 mg mL^−1 of collagenase IV). The above mixture
   was digested at 37 °C for 30 min and then added to complete medium to
   terminate the digestion reaction. A 70 µM cell filter was used to
   further remove inadequately digested tissue and cell masses, yielding
   an ileal tissue suspension. Following fixation and permeabilization,
   the cells were incubated with rabbit anti‐Dectin‐1 (1:300, PTG,
   22809‐1‐AP) overnight at 4 °C. Subsequently, CoraLite488 (1:500, PTG,
   SA00013‐2) was applied for 1 h in the dark. Flow cytometry was employed
   for analysis.

   To evaluate the in vivo targeting efficiency of nanoparticles after
   penetrating the BBB, WD mice were administered 200 µL of free Cy5 and
   various Cy5‐labeled samples (8 mg·kg⁻¹) via intragastric gavage. After
   24 h, the mice were sacrificed, and the hypothalamic tissues from each
   group were immediately isolated and fully homogenized in PBS at 4 °C.
   Hypothalamic cells were subsequently extracted through filtration and
   centrifugation. Following fixation and permeabilization, the cells were
   incubated with rabbit anti‐GnRH (1:200, PTG, 26950‐1‐AP) overnight at 4
   °C. Subsequently, CoraLite488 (1:500, PTG, SA00013‐2) was applied for 1
   h in the dark. Flow cytometry was employed for analysis.

SWI Imaging

   In vivo magnetic resonance imaging was performed on a 9.4 T MRI scanner
   (uMR 9.4T, Shanghai, China). During the scanning, the mice were
   anesthetized by inhaling isoflurane, and the mice's heart rate and
   respiration were monitored in real time. For analysis, the hypothalamic
   arcuate nucleus was manually defined as a region of interest (ROI) in
   imaging slices of each mouse. The phase value was measured on the phase
   diagram.

Immunofluorescence Staining and Measurement of Brain Cytokines

   After the treatment, the major organs (heart, liver, spleen, lungs,
   kidneys, brain, iluem, testicle) of mice in each experimental group
   were collected and fixed in 4% paraformaldehyde. Then, the brain was
   paraffin embedded to cut into sections of 3 µm in thickness.
   Immunofluorescence staining was performed to detect Iba‐1, CD206, iNOS,
   GSDMD‐N and NLRP3 in the hypothalamus region of the brain.
   Additionally, immunofluorescence staining was employed to detect the
   co‐expression of Cu^2+ with GnRH, GnRH with NLRP3, and GnRH with PKC in
   the hypothalamus region of the brain. GnRH expression in hypothalamus
   was detected by immunohistochemical staining. Subsequently, the organs
   were embedded in paraffin and 5 µm sections were prepared for
   hematoxylin and eosin (H&E) staining. Furthermore, the levels of
   cytokines (Nrf2, Caspase‐1, IL‐1β, IL‐18, TNF‐α, IL‐10, IL‐6, IL‐4) in
   the brain hypothalamus of each group were quantified using specific
   assay kits.

Measurement of Sperm Survival Rate

   A 4 µL aliquot of eosin Y stain was applied to a glass slide and
   combined with an equal volume (4 µL) of sperm suspension. After 3 min,
   the slide was covered with a coverslip and examined under an optical
   microscope. Normal sperm appeared unstained, whereas dead sperm
   exhibited pink staining due to cell membrane rupture. Then, 200 sperms
   were randomly counted, and the ratio of unstained live sperm to total
   sperm was calculated.

Measurements of Sperm Plasma Membrane and Acrosome Integrity

   A 100 µL volume of sperm suspension was introduced into a 1.5 mL
   centrifuge tube and sequentially stained with
   isothiocyanate‐fluoresceinated peanut agglutinin (PNA‐FITC) and
   propidium iodide (PI). The integrity of the sperm plasma membrane and
   acrosome was assessed using flow cytometry. PI‐ indicated the integrity
   of the sperm plasma membrane, while PNA‐ indicated the integrity of the
   sperm acrosome.

Quantitative Real‐Time PCR

   Total RNA was extracted from hypothalamus tissue using TRIzol reagent
   (15 596 018, Life Technologies, New York, USA) and processed with
   chloroform, isopropanol, and 75% ethanol following homogenization. The
   integrity and purity of the RNA were assessed using a
   microspectrophotometer. RNA was reverse‐transcribed into cDNA and
   subsequently amplified by PCR. The amplification conditions were as
   follows: pre‐denaturation at 95 °C for 1 min, denaturation at 95 °C for
   20 s, annealing and extension at 60 °C for 1 min, for a total of 40
   cycles. The relative expression of the target genes was calculated
   using the 2^‐ΔΔCt method. Primer sequences are listed in Table [226]S2,
   Supporting Information.

Statistical Analysis

   All experiments were conducted a minimum of three times. Data are
   presented as means ± standard errors. Statistical significance was
   assessed using Student's t‐test for pairwise comparisons and one‐way or
   two‐way ANOVA with Tukey's post hoc test for multiple comparisons.

Conflict of Interest

   The authors declare no conflict of interest.

Supporting information

   Supporting Information
   [227]ADVS-12-e01689-s001.docx^ (13.1MB, docx)

Acknowledgements