ADSC-derived exosomes deliver cerium oxide nanoparticles to promote diabetic wound healing via anti-inflammatory and pro-angiogenic effects

Summary

Diabetic wounds pose a significant clinical challenge due to excessive oxidative stress, chronic inflammation, and impaired angiogenesis. To address these issues, this study developed a novel nanocomposite, CeO₂@Exo, by loading cerium oxide nanoparticles (CeO₂ NPs) into adipose-derived stem cell-derived exosomes. In both in vitro and in vivo diabetic models, CeO₂@Exo effectively scavenged reactive oxygen species, promoted VEGF-mediated angiogenesis, and accelerated wound closure. Importantly, it modulated the inflammatory microenvironment by shifting macrophage polarization from the pro-inflammatory M1 to the pro-regenerative M2 phenotype, thereby enhancing the release of anti-inflammatory cytokines. These multifunctional effects demonstrate that CeO₂@Exo represents a promising, comprehensive therapeutic strategy for diabetic wound healing, overcoming the limitations of conventional single-target treatments.

Graphical abstract

Highlights

• •CeO₂@Exo integrates cerium oxide nanoparticles with ADSC-derived exosomes
• •CeO₂@Exo scavenges ROS and promotes VEGF-mediated angiogenesis
• •CeO₂@Exo reprograms macrophages toward a pro-regenerative M2 phenotype
• •CeO₂@Exo accelerates diabetic wound healing in vitro and in vivo

Nanoparticles; Biological sciences; Materials science; Biomaterials; Composite materials

Introduction

Diabetic wounds (DWs), a severe complication of diabetes with global prevalence, arise from a pathological microenvironment dominated by oxidative stress and sustained inflammation,¹ resulting in delayed healing and increased risk of limb amputation.² Under physiological conditions, reactive oxygen species (ROS) play a critical role in antimicrobial defense and tissue repair³; however, hyperglycemia-driven ROS overproduction disrupts cellular redox balance,⁴ exacerbating oxidative damage, amplifying proinflammatory responses, and skewing macrophage polarization toward a detrimental M1/M2 imbalance.^5,6 It remains a formidable clinical challenge due to the complex pathological microenvironment characterized by excessive oxidative stress,⁷ chronic inflammation,⁸ and impaired angiogenesis.⁹ Conventional therapeutic approaches often fail to simultaneously address these interconnected pathological features, leading to unsatisfactory clinical outcomes.

Adipose-derived stem cell exosomes (ADSC-Exos) have emerged as promising therapeutic agents due to their inherent biological properties that mirror the regenerative potential of parental cells.^10,11 These natural nanovesicles have demonstrated remarkable capabilities in promoting angiogenesis through the transfer of pro-angiogenic miRNAs such as miR-126 and miR-31, which activate endothelial cells and stimulate neovascularization.¹² Concurrently, ADSC-Exos exhibit potent anti-inflammatory effects by modulating macrophage polarization toward the regenerative M2 phenotype and suppressing pro-inflammatory cytokine production through NF-κB pathway inhibition.¹³

While both ADSC-Exos and CeO₂ NPs exhibit distinct therapeutic advantages, their combined application may yield synergistic benefits, potentially addressing multiple pathological facets of diabetic wounds simultaneously. CeO₂ NPs represent another innovative therapeutic approach for diabetic wound healing due to their unique redox properties.¹⁴ The dynamic Ce³⁺/Ce⁴⁺ valence transition enables CeO₂ NPs to function as regenerative antioxidants, effectively scavenging reactive oxygen species (ROS) while maintaining redox homeostasis in the wound microenvironment.^15,16 Beyond their antioxidant capacity, CeO₂ NPs have been shown to modulate inflammatory responses by reducing inflammasome activation and downregulating pro-inflammatory cytokines.¹⁷ However, the clinical translation of free CeO₂ NPs has been hindered by challenges related to targeted delivery and retention at the wound site.

The integration of these two promising therapeutic modalities - ADSC-Exos and CeO₂ NPs - presents a novel strategy to overcome the limitations of individual components while capitalizing on their synergistic effects. This study investigates the therapeutic potential of ADSC-Exos loaded with CeO₂ NPs (CeO₂@Exo) for diabetic wound healing, with particular focus on their combined effects on inflammatory resolution, vascular regeneration, and wound closure kinetics. We hypothesize that the CeO₂@Exo system will not only enhance the stability and targeted delivery of CeO₂ NPs but also synergistically improve the regenerative microenvironment through simultaneous modulation of oxidative stress, inflammation, and angiogenesis, ultimately leading to accelerated wound healing in diabetic conditions. The development of such an integrated therapeutic approach may provide new insights into the treatment of chronic diabetic wounds and other oxidative stress-related disorders.

Results

Synthesis and characterization of ceo₂@exo

The successful fabrication and characterization of CeO₂@Exo nanocomposites were systematically investigated, as shown in Figure 1. Transmission electron microscopy (TEM) analysis revealed well-dispersed CeO₂ NPs with uniform morphology (Figure 1A), while high-resolution TEM imaging clearly demonstrated the crystalline lattice structure of CeO₂, as evidenced by the distinct lattice fringes marked in the circular region (Figure 1B). The result of XRD clearly showed that the synthesized CeO₂ NPs exhibit a typical fluorite structure with good crystallinity, matching the standard reference (JCPDS No. 34–0394) well. This confirms the successful preparation of CeO₂ NPs with the correct crystal structure (Figure 1C). The efficient loading of CeO₂ Np into exosomes was confirmed by TEM observation (Figure 1D), which showed the typical vesicular structure of exosomes incorporating the Nps. The SAED pattern has been successfully obtained and is now included as an inset. The diffraction rings observed in the pattern can be clearly indexed to the (111), (200), and (220) crystal planes of the fluorite structure of CeO₂ (JCPDS No. 34–0394). This result provides direct experimental evidence that the crystalline phase of the CeO₂ nanoparticles is well preserved after their integration with the exosomes to form the CeO₂@Exo complex. The EDS elemental mapping data clearly show the distribution of the Ce signal (representing the CeO₂ nanoparticles) in the CeO₂@Exo (Figure 1E). This provides direct spatial evidence confirming the successful association of the CeO₂ nanoparticles with the exosomal structure. The exosomal origin of the carrier was verified by Western blot analysis, which confirmed the presence of characteristic ADSC-Exo markers (Figure 1F). The Zeta potential showed that CeO₂ NP, ADSC-Exo, and CeO₂@Exo were relatively stable (Figure 1G). CeO₂ NP has a particle size of ∼4.8 nm, while ADSC-Exo and CeO₂@Exo have similar particle sizes of ∼156.1 nm (Figure 1H). The low PDI value confirms a relatively narrow and monodisperse size distribution of the nanocomposite, which aligns well with the homogeneous morphology observed in our TEM images. This comprehensive physicochemical evaluation confirmed the successful synthesis of CeO₂@Exo nanocomposite system and laid a solid foundation for subsequent biological applications.

Figure 1.

Characterization of CeO₂@Exo

(A) TEM image of CeO₂ Np.

(B) High-resolution transmission electron microscopy (TEM) image of CeO₂ Np, with the CeO₂ lattice marked in the circle.

(C) XRD of CeO₂ Np.

(D) TEM image and SAED pattern of CeO₂@Exo; The yellow arrow indicates cerium dioxide nanoparticles modified on the surface of exosomes.

(E) The corresponding energy-dispersive X-ray spectroscopy (EDS) elemental maps for Ce (white), N (pink), and O (yellow).

(F) Western blot analysis confirms ADSC-Exo. (G) Zeta potential of CeO₂ Np.

(H) DLS for particle sizes. Scale bars: (A) 50 nm, (B) 10 nm, (D) 100 nm, and (E) 10 nm.

Biosafety evaluation of CeO2@Exo

The potential accumulation of NPs in vivo has raised significant concerns and posed challenges for clinical translation. Prior to investigating the therapeutic potential of CeO₂@Exo for diabetic wound healing, we conducted comprehensive biosafety evaluations to ensure the biocompatibility and clinical applicability of this novel nanocomposite. As shown in Figures 2A and 2B, confocal laser scanning microscopy (CLSM) confirmed efficient cellular uptake of Rho-labeled CeO₂@Exo by human umbilical vein endothelial cells (HUVECs). CCK-8 assays demonstrated excellent cytocompatibility and established 100 μg/mL as the maximum safe concentration, with viability maintained above 90% for both HUVECs and HaCaT cells across a wide concentration range (Figure 2C), indicating minimal cytotoxicity toward critical cell types involved in wound repair. Hemocompatibility testing (Figure 2D) revealed negligible hemolytic activity (<5%) at 100 μg/mL, meeting international standards for biomedical materials. Histopathological examination of major organs (Figure 2E) showed normal tissue architecture in kidney and liver sections, confirming the absence of systemic toxicity or significant organ accumulation following topical administration. These thorough biosafety assessments provide compelling evidence that CeO₂@Exo meets fundamental safety requirements for clinical translation while maintaining biological functionality, thereby supporting its potential application in diabetic wound therapy research.

Figure 2.

Biosafety evaluation

(A) Confocal laser scanning microscope (CLSM) shows the uptake of Rho-labeled CeO₂@Exo by HUVECs.

(B) Quantitative analysis of cellular uptake efficiency.

(C) Cell viability of HaCaT and HUVEC cells after coculture with different concentrations of CeO₂@Exo, as determined by CCK-8 assay.

(D) Hemolysis assay.

(E) H&E staining of kidney and liver tissues. Scale bars: (A) 20 μm and (E) 200 μm. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Data are presented as mean ± SD. For in vitro experiments, n represents the number of independent biological replicates (n = 5). Statistical significance is indicated as ∗∗∗p < 0.001.

Dual mechanism of action: reactive oxygen species scavenging and VEGF upregulation by CeO2@Exo

The detrimental impact of intracellular and mitochondrial reactive oxygen species (ROS/mtROS) on endothelial cell function represents a pivotal pathological mechanism underlying impaired diabetic wound healing. Excessive ROS accumulation, particularly within mitochondria, disrupts endothelial homeostasis through multiple pathways, including oxidative damage to macromolecules (DNA, lipids, and proteins), activation of pro-inflammatory signaling cascades (e.g., NF-κB pathway), and impairment of mitochondrial membrane potential and energy metabolism. Our systematic investigation of CeO₂@Exo’s antioxidant capacity and its beneficial effects on endothelial function, as illustrated in Figure 3, revealed that CeO₂@Exo treatment significantly reduced both intracellular and mitochondrial ROS levels in HUVECs compared to control groups (Figure 3A), with quantitative analysis demonstrating dose-dependent ROS scavenging efficiency (Figures 3B and 3C). This potent antioxidant activity translated into functional improvements, evidenced by enhanced cell migration in scratch wound assays (Figure 3D) and accelerated wound closure rates at both 12 h and 24 h time points (Figure 3E). Importantly, the antioxidant effects were accompanied by pro-angiogenic activation, with qRT-PCR analysis showing significant upregulation of VEGF transcription in CeO₂@Exo-treated cells (Figure 3F). These findings collectively demonstrate that mitochondrial-targeted ROS scavenging plays a crucial role in endothelial functional recovery, as mtROS directly influences cellular migration and angiogenic signaling. The ability of CeO₂@Exo to not only effectively mitigate endothelial oxidative stress but also promote functional recovery and angiogenic potential suggests a dual therapeutic mechanism for improving vascular function in diabetic wounds. This comprehensive ROS modulation capability likely explains the superior performance of CeO₂@Exo compared to conventional antioxidants that typically target specific cellular compartments. The coordinated antioxidant and pro-angiogenic effects of CeO₂@Exo are particularly significant for disrupting the vicious cycle of oxidative damage and endothelial dysfunction that characterizes diabetic wound pathophysiology, offering a promising therapeutic strategy for this challenging clinical condition.

Figure 3.

Antioxidant effects of CeO₂@Exo on endothelial cells

(A) CLSM (confocal laser scanning microscopy) images show intracellular ROS and mitochondrial ROS (mtROS) levels in HUVECs before and after different treatments.

(B and C) Corresponding statistical analyses of ROS/mtROS levels.

(D) Scratch wound healing assay of HUVECs.

(E) Quantitative analysis of wound closure at different time points (from Figure 3D).

(F) qRT-PCR analysis of VEGF transcriptional levels across treatment groups. Scale bars: (A) 100 μm; (D) 200 μm. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Data are presented as mean ± SD. For in vitro experiments, n represents the number of independent biological replicates (n = 3). Statistical significance is indicated as n.s., ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Enhancement of vascularization and healing in diabetic wounds through CeO2@Exo treatment

Building upon the promising antioxidant results obtained in vitro, we further systematically evaluated the therapeutic efficacy of CeO₂@Exo in diabetic wound healing through comprehensive in vivo experiments. Photographic documentation and quantitative analysis revealed significantly accelerated wound closure in the CeO₂@Exo-treated group. Notably, the treatment group exhibited a ∼42% improvement in wound closure rate by day 7 and achieved complete healing within just 14 ± 0.32 days, compared to only ∼59% closure in the control group at the same time point (Figures 4A and 4B). This enhanced healing correlated with an improved systemic inflammatory profile, as evidenced by a significant reduction in serum CRP and PCT levels (by approximately 33.41% and 76.86%, respectively, Figure 4C). Histological examination provided deeper mechanistic insights. H&E-stained sections showed more pronounced re-epithelialization and granulation tissue formation in the CeO₂@Exo-treated wounds at days 7 and 14 (Figure 4D). Furthermore, Masson’s trichrome staining at day 21 revealed more mature and well-organized collagen deposition. This was quantitatively confirmed by a ∼13.29-fold increase in epidermal thickness and a ∼15.59-fold higher collagen content in the treatment group compared to the control (Figures 4F and 4G). These findings collectively demonstrate that CeO₂@Exo promotes diabetic wound healing through multiple synergistic mechanisms: markedly accelerating wound closure kinetics, modulating systemic inflammation, enhancing re-epithelialization capacity, and facilitating superior extracellular matrix remodeling. The robust therapeutic outcomes observed in this study, from macroscopic wound closure to histological and molecular improvements, suggest that CeO₂@Exo effectively addresses the complex pathophysiology of diabetic wounds through its multifaceted action.

Figure 4.

CeO₂@Exo treatment promotes diabetic wound healing

(A) Photographic documentation of diabetic wounds at different time points. The schematic diagram above illustrates the wound margins of each group at days 0, 7, and 14 post-injury.

(B) Quantitative analysis of wound healing kinetics.

(C) Heatmap representation of systemic inflammatory markers (CRP and PCT) in blood samples.

(D) Representative H&E-stained sections at days 7 and 14 post-treatment.

(E) Representative Masson’s trichrome-stained sections at day 21.

(F) Statistical analysis of epidermal thickness.

(G) Collagen index (CI) quantification based on Masson’s trichrome staining. Scale bars: (D) 100 μm and (E) 100 μm. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Data are presented as mean ± SD. n represents the number of biological replicates used for the corresponding analyses (n = 3 per group). Statistical significance is indicated as n.s., ∗∗p < 0.01, and ∗∗∗p < 0.001.

Implantation of CeO2@Exo in vivo Reshapes the inflammation microenvironment and promotes angiogenesis

Diabetic wounds are characterized by dysregulated inflammation and impaired angiogenesis, both of which contribute to delayed healing. Insufficient vascularization, driven by diminished VEGF signaling and endothelial dysfunction, restricts oxygen and nutrient supply to the wound bed, perpetuating a non-healing state. To elucidate the therapeutic mechanisms of CeO₂@Exo in diabetic wound repair, we investigated its pro-angiogenic and immunomodulatory effects. As shown in Figure 5, immunohistochemical analysis revealed a significant increase in CD31⁺ microvessel density in CeO₂@Exo-treated wounds at day 21 post-treatment (Figures 5A and 5C), accompanied by the substantial upregulation of VEGF transcription (Figure 5B).

Figure 5.

CeO₂@Exo enhances angiogenesis via VEGF upregulation and modulates the inflammatory microenvironment

(A) Representative immunohistochemical (IHC) staining images of CD31 (a marker for angiogenesis) in wound tissues at day 21 post-treatment; CD31-positive vascular endothelial cells are stained in brown, while nuclei are counterstained in blue with hematoxylin. (C) Quantitative analysis of CD31⁺ microvessel density.

(B and C) qRT-PCR analysis of VEGF transcriptional levels across treatment groups.

(D and E) qRT-PCR analysis of macrophage polarization in wound tissues (CD86⁺: M1; CD206⁺: M2).

(F–G) ELISA quantification of local inflammatory cytokines: (F) IFN-γ and (G) IL-10. Scale bars: (A) 100 μm. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Data are presented as mean ± SD. n represents the number of biological replicates used for the corresponding analyses (n = 3 per group). Statistical significance is indicated as n.s., ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Concurrently, macrophages play a critical role in wound repair by transitioning from a pro-inflammatory (M1) phenotype during early stages to an anti-inflammatory, pro-reparative (M2) phenotype during tissue remodeling. However, under diabetic conditions, this polarization is frequently disrupted, resulting in persistent M1 dominance and excessive inflammation that exacerbates vascular dysfunction. In Figures 5D and 5E, it was demonstrated that CeO₂@Exo treatment promoted macrophage polarization toward the regenerative M2 phenotype (CD206⁺) while suppressing the pro-inflammatory M1 subset (CD86⁺). This favorable immunomodulation was further confirmed at the cytokine level, with ELISA quantification showing significant reductions in pro-inflammatory mediators (IFN-γ; Figure 5F) alongside elevated anti-inflammatory IL-10 (Figure 5G). Our findings demonstrate that CeO₂@Exo effectively modulates macrophage polarization toward the M2 phenotype while enhancing VEGF-driven angiogenesis, thereby addressing two fundamental pathological features of diabetic wounds. The coordinated enhancement of vascularization and resolution of inflammation not only mitigates chronic inflammation but also restores functional vascular networks, creating a regenerative microenvironment conducive to healing. The dynamic interplay between macrophage reprogramming and angiogenic activation highlights the potential of CeO₂@Exo as a multifaceted therapeutic strategy for diabetic wound repair.

This integrated approach, targeting both the inflammatory and vascular components of diabetic wound pathophysiology, represents a significant advance over conventional therapies that typically focus on single pathological aspects. The ability of CeO₂@Exo to simultaneously promote tissue repair while modulating the immune response underscores its potential as a next-generation treatment for chronic diabetic wounds.

Discussion

This study successfully developed CeO₂@Exo, an innovative nanocomposite system engineered through the conjugation of CeO₂ NPs with adipose-derived stem cell-derived exosomes (ADSC-Exos), to address the multifactorial pathology of diabetic wounds. Our experimental results demonstrate that CeO₂@Exo exhibits remarkable therapeutic efficacy by synergistically scavenging reactive oxygen species (ROS) and modulating the inflammatory microenvironment. The nanocomposite significantly enhanced diabetic wound healing by promoting VEGF-mediated angiogenesis, accelerating re-epithelialization, and improving extracellular matrix remodeling. A particularly noteworthy finding is that CeO₂@Exo effectively reprogrammed macrophage polarization from the pro-inflammatory M1 phenotype to the pro-reparative M2 phenotype, thereby resolving chronic inflammation. This synergistic combination of ADSC-Exos and CeO₂ NPs overcomes the limitations of conventional therapies by simultaneously targeting three key pathological features of diabetic wounds: oxidative stress, inflammatory dysregulation, and impaired angiogenesis. The exosomal delivery system not only improves the stability and bioavailability of CeO₂ NPs but also enhances their targeted delivery to wound sites. Our findings position CeO₂@Exo as a next-generation therapeutic strategy with significant potential for chronic diabetic wound treatment, offering a solution that addresses both cellular dysfunction and molecular imbalances underlying impaired healing. Future studies should focus on optimizing dosage regimens and investigating the long-term safety profile of CeO₂@Exo to facilitate its clinical translation. This work establishes a strong foundation for developing advanced combinatorial therapies for diabetic wound management and other oxidative stress-related disorders.

Limitations of the study

This study has several inherent limitations. First, only male C57BL/6 mice were used in the in vivo experiments. Although this approach reduces biological variability, it may limit the generalizability of the findings to female subjects. Second, the present work primarily focuses on demonstrating the therapeutic efficacy of CeO₂@Exo in promoting wound healing through anti-inflammatory and pro-angiogenic effects; however, deeper mechanistic investigations were beyond the scope of this study. Finally, the in vivo assessments were conducted over a relatively short experimental period, and long-term outcomes beyond the wound-healing phase were not evaluated. These limitations should be taken into consideration when interpreting the results.

Resource availability

Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Leilei Cao (caoleilei@sysush.com).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •Data reported in this article will be shared by the lead contact upon request.
• •This article does not report original code.
• •Any additional information required to reanalyze the data reported in this article is available from the lead contact upon request.

Acknowledgments

The care and use of all animals were conducted in accordance with the guiding principles of the Animal Research Ethics Committee of Institutional Animal Care and Use Committee (IACUC), the Sun Yat-Sen University (experimental Program no. SYSU-IACUC-2025-B2075), and was strictly compliant with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. This research was sponsored by Shenzhen Key Medical Discipline Construction Program Matching Funds (Seventh Affiliated Hospital of Sun Yat-sen University; Qingyan Li, No.00302600004).

Author contributions

Conceptualization, Y.Z. and L.C.; methodology, Y.H., Q.L., and Y.N.; investigation, Y.H., Q.L., Y.N., Y.Z., T.Y., and T.P.; writing – original draft, Y.Z. and L.C.; writing – review and editing, Y.Z., Y.H., Y.N., and L.C.; funding acquisition, Q.L.; resources, Y.H., Q.L., Y.N., Y.Z., T.Y., T.P., and Y.N.; supervision, Y.N. and L.C.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-CD63 antibody	Abcam	Cat# ab134045; RRID: AB_2800495
Anti-CD81 antibody	Abcam	Cat# ab79559; RRID: AB_1603682
Anti-CD31 antibody	Invitrogen	Cat# MA5-37858; RRID: AB_2897778
Chemicals, peptides, and recombinant proteins
Cerium(III) nitrate hexahydrate	Sigma-Aldrich	238538-5G
Ammonium hydroxide	Sigma-Aldrich	221228-500 ML
Ethanol	Sigma-Aldrich	459836-1L
Uranyl acetate (2%)	Electron Microscopy Sciences	22400–2
Rhodamine B (Rho)	Thermo Fisher Scientific	R6626
DCFH-DA probe	Sigma-Aldrich	R6626
DAPI (4′,6-diamidino-2-phenylindole)	Thermo Fisher Scientific	D1306
Streptozotocin	Sigma-Aldrich	S0130-50 MG
Paraformaldehyde (4%)	Sigma-Aldrich	158127-500G
CCK-8 assay kit	Beyotime	C0037
ELISA kits(IFN-γ)	R&D Systems	DY485B-05
ELISA kits(IL-10)	R&D Systems	DY417-05
Dulbecco’s Modified Eagle Medium (DMEM)	Gibco	11965092
Endothelial Cell Medium (ECM)	ScienCell	1001
Fetal bovine serum (FBS)	Gibco	10099141
Exosome-depleted FBS	Gibco	A2720803
Primers	Sangon Biotech	N/A
Experimental models
C57BL/6 mice	Gempharmatech Co., Ltd	RRID:IMSR_JAX:000664
Human Umbilical Vein Endothelial Cells	Cellverse Co., Ltd	RRID:CVCL_2959
HaCaT Keratinocytes	Cellverse Co., Ltd	RRID:CVCL_0038
Adipose-Derived Stem Cells	SUNNCELL	N/A
Software and algorithms
Malvern ZS Xplorer	Malvern Panalytical Ltd.	https://www.malvernpanalytical.com
ImageJ	National Institutes of Health	RRID:SCR_003070
GraphPad Prism	GraphPad Software	RRID:SCR_002798

Experimental model and study participant details

Animal model

Species/Strain: C57BL/6 mice.

Sex and Age: Male, 8 weeks old.

Developmental Stage: Adult.

Maintenance and Care: Mice were housed under standard laboratory conditions with ad libitum access to food and water. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-Sen University (Approval No. SYSU-IACUC-2025-B2075). All procedures were conducted in strict compliance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Consideration of Sex Influence: This study utilized only male mice to minimize variability in wound healing responses that can be influenced by estrous cycle in females. While this provides internal consistency, it may limit the generalizability of the findings to female subjects, which should be considered as a limitation of the study.

Cell lines

Human Umbilical Vein Endothelial Cells (HUVECs): Primary endothelial cells derived from human umbilical veins. Cells were cultured in Endothelial Cell Medium (ECM) supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified 5% CO2 atmosphere.

HaCaT Keratinocytes: Immortalized human keratinocyte cell line. Cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified 5% CO2 atmosphere.

Cell line authentication and mycoplasma testing: The HUVECs and HaCaT cell lines used in this study were authenticated by the suppliers prior to use. Cell line identity was further confirmed based on consistent morphological characteristics and growth behavior during routine cell culture. All cell lines were tested for mycoplasma contamination using a commercial detection kit, and no contamination was detected.

Primary cell culture

·Adipose-Derived Stem Cells (ADSCs): Primary human adipose-derived stem cells were cultured in DMEM supplemented with 10% exosome-depleted FBS. Cells were maintained at 37°C in a humidified 5% CO₂ atmosphere. For exosome isolation, cells at 80% confluency were switched to serum-free medium for 48 h to collect conditioned medium. Cells were tested for mycoplasma contamination using a commercial detection kit prior to experimental use, and no contamination was detected.

Method details

Preparation and characterization of CeO2@Exo nanocomposites

Synthesis of CeO₂ NPs

CeO₂ NPs were synthesized via a hydrothermal method. Briefly, cerium(III) nitrate hexahydrate (Ce(NO₃)₃·6H₂O) was dissolved in deionized water, followed by the addition of ammonium hydroxide (NH₄OH) under vigorous stirring. The mixture was transferred to a Teflon-lined autoclave and heated at 180°C for 12 h. The resulting precipitate was centrifuged, washed with ethanol and deionized water, and dried at 60°C overnight. The obtained CeO₂ NPs were characterized using transmission electron microscopy (TEM) to confirm their size, morphology, and crystalline structure.

Isolation and purification of ADSC-Exosomes (ADSC-Exos)

Adipose-derived stem cells (ADSCs) were cultured in DMEM supplemented with 10% exosome-depleted FBS. Upon reaching 80% confluency, the culture medium was replaced with serum-free medium for 48 h.¹⁸ The conditioned medium was collected and subjected to sequential centrifugation at 300 × g for 10 min, 2000 × g for 20 min, and 10,000 × g for 30 min to remove cellular debris. Exosomes were then isolated by ultracentrifugation at 100,000 × g for 70 min at 4°C. The exosome pellet was resuspended in PBS and further purified using a 0.22 μm filter. Exosome identity was confirmed by Western blot analysis for markers CD63, CD81, and TSG101.

Fabrication of CeO2@Exo nanocomposites

CeO₂ NPs were loaded into ADSC-Exos using an electroporation method.¹⁹ Briefly, 100 μg of CeO₂ NPs were mixed with 200 μg of exosomes in 200 μL of electroporation buffer (provided by the kit/invited in PBS). The mixture was transferred to a 4-mm electroporation cuvette and subjected to five electrical pulses at 500 V with a pulse duration of 5 ms each. Subsequently, the electroporated mixture was incubated at 37°C for 1 h to allow for vesicle membrane recovery. To remove unencapsulated or loosely attached CeO₂ NPs, the solution was centrifuged at 10,000 × g for 30 min. The pellet containing the CeO₂@Exo nanocomposite was resuspended in 1× PBS for further use.

The successful loading of CeO₂ NPs into exosomes was confirmed by Transmission Electron Microscopy (TEM). Imaging was performed using a JEOL JEM-1400Flash (JEOL Ltd., Japan) microscope operating at an acceleration voltage of 100 kV. Sample preparation involved negative staining with 2% uranyl acetate. The hydrodynamic size, size distribution, and Zeta potential of the bare exosomes and the CeO₂@Exo nanocomposite were characterized using Dynamic Light Scattering (DLS) and Laser Doppler Micro-electrophoresis, respectively. Measurements were performed on a Malvern Zetasizer Nano ZS (Malvern Panalytical Ltd., UK) at a fixed temperature of 25 °C. For DLS analysis, samples were appropriately diluted in 1× PBS to achieve an optimal scattering intensity. Each measurement was performed in triplicate with an equilibration time of 60 s. The hydrodynamic diameter was calculated from the intensity-based distribution using the instrument’s built-in software (Malvern ZS Xplorer), which is based on the Non-Invasive Back-Scatter (NIBS) technology and utilizes a non-negative least squares (NNLS) algorithm for size distribution analysis. The polydispersity index (PDI) was provided as a measure of the size distribution breadth. For Zeta potential measurements, samples were diluted in 1× PBS and loaded into a disposable folded capillary cell (DTS1070, Malvern Panalytical). The measurement was conducted using the Smoluchowski model. The reported Zeta potential value represents the average of at least three independent measurements.

In vitro experiments

Cell culture

Human umbilical vein endothelial cells (HUVECs) and HaCaT keratinocytes were cultured in endothelial cell medium (ECM) and DMEM, respectively, supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were maintained at 37°C in a humidified 5% CO₂ atmosphere.

Cellular uptake and biosafety evaluation

For cellular uptake studies, CeO₂@Exo was labeled with Rhodamine B (Rho) and incubated with HUVECs for 4 h. Cells were fixed, and uptake was visualized using confocal laser scanning microscopy (CLSM). Cytocompatibility was assessed using the CCK-8 assay. HUVECs and HaCaT cells were treated with varying concentrations of CeO₂@Exo (0–200 μg/mL) for 24 h, and cell viability was measured. Hemocompatibility was evaluated by incubating CeO₂@Exo with red blood cells (RBCs) and measuring hemolysis spectrophotometrically at 540 nm.

ROS scavenging and angiogenic effects

Intracellular and mitochondrial ROS levels in HUVECs were measured using DCFH-DA and MitoSOX Red probes, respectively, followed by CLSM imaging and quantitative analysis. A scratch wound assay was performed to evaluate cell migration.

In vivo experiments

Diabetic wound model

The animal use protocol listed below has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC), Sun Yat-Sen University (No. SYSU-IACUC-2025-B2075). Male C57BL/6 mice (8 weeks old) were rendered diabetic by intraperitoneal injection of streptozotocin (STZ, 50 mg/kg for 5 consecutive days). Full-thickness excisional wounds (8 mm diameter) were created on the dorsal skin after blood glucose levels stabilized at >300 mg/dL. Following the creation of wounds, each wound was covered with a sterile, transparent film dressing (Product Code: 1626W; 3M Company, USA) to protect the site and simulate a moist wound healing environment. At the end of the experiment, all mice were euthanized by CO₂ inhalation followed by cervical dislocation.

Treatment groups and wound healing assessment

Mice were randomly divided into four groups (n = 9 per group): (1) Control (PBS), (2) CeO₂ NPs, (3) ADSC-Exos, and (4) CeO₂@Exo. Treatments (100 μL containing 100 μg/mL of the respective material) were topically applied every other day. Wound closure was monitored by digital photography on days 0, 3, 7, 10, and 14, and the wound area was quantified using ImageJ software.

Histological and immunohistochemical analysis

On days 7, 14, and 21, mice were euthanized, and wound tissues were harvested for histological analysis. Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Hematoxylin and eosin (H&E) staining was performed to evaluate re-epithelialization and granulation tissue formation. Masson’s trichrome staining was used to assess collagen deposition. For immunohistochemistry (IHC), sections were stained with antibodies against CD31 (angiogenesis marker).

ELISA for cytokine analysis

Wound tissues were homogenized, and the levels of inflammatory cytokines (IFN-γ and IL-10) were quantified using commercial ELISA kits according to the manufacturer’s instructions.

Qt-PCR assay

To quantify gene expression levels (e.g., VEGF, CD86, CD206), total RNA was extracted from cells or wound tissues using TRIzol reagent according to the manufacturer’s protocol. RNA concentration and purity were measured spectrophotometrically (NanoDrop) at 260/280 nm. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using a reverse transcription kit (e.g., PrimeScript RT Reagent Kit) with oligo(dT) primers.

qRT-PCR was performed in triplicate using SYBR Green Master Mix on a real-time PCR system (e.g., Applied Biosystems 7500). The reaction mixture (20 μL) contained 10 μL SYBR Green Mix, 1 μL cDNA, 0.8 μL each of forward and reverse primers (10 μM), and 8.4 μL nuclease-free water. The thermal cycling conditions were: 95°C for 30 s (initial denaturation), followed by 40 cycles of 95°C for 5 s and 60°C for 30 s (annealing/extension). Melt curve analysis was conducted to confirm primer specificity. Primer sequences:

 VEGF

 (Forward) 5′-AGGGCAGAATCATCACGAAGT-3′ (Reverse) 5′-AGGGTCTCGATTGGATGGCA-3′

 CD86 (M1 marker)

 (Forward) 5′-CTCTGGAAAGGGGACTCAGC-3′ (Reverse) 5′-CAGGTGAAATGGGGATGACA-3′

 CD206 (M2 marker)

 (Forward) 5′-CTCTGTTCAGCTATTGGACGC-3′ (Reverse) 5′-CGGAATTTCTGGGATTCAGCTTC-3′

 GAPDH

 (Forward) 5′-GGAGCGAGATCCCTCCAAAAT-3′ (Reverse) 5′-GGCTGTTGTCATACTTCTCATGG-3′

Relative gene expression was calculated using the 2−ΔΔCt method, normalized to GAPDH, and presented as fold changes compared to control groups.

Quantification and statistical analysis

All quantitative data are presented as mean ± standard deviation (SD). The number of biological replicates (n) for each experiment is indicated in the corresponding figure legends. Statistical analyses were performed using GraphPad Prism software. Comparisons between two groups were conducted using unpaired two-tailed Student’s t test. Comparisons among multiple groups were performed using one-way ANOVA followed by Tukey’s post hoc test. Statistical significance was defined as ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Published: December 31, 2025