Abstract

   Osteoarthritis (OA) is marked by cartilage deterioration, subchondral
   bone changes, and an inflammatory microenvironment. The study
   introduces the Microneedle‐Delivered Polydopamine‐Exosome (PDA@Exo MN),
   a therapeutic that not only preserves cartilage and promotes bone
   regeneration but also improves localized drug delivery through enhanced
   penetration capabilities. PDA@Exo MN shows strong reactive oxygen
   species (ROS) scavenging abilities and high biocompatibility, fostering
   osteogenesis and balancing anabolic and catabolic processes in
   cartilage. It directs macrophage polarization from M0 to the
   anti‐inflammatory M2 phenotype. RNA sequencing of treated chondrocytes
   demonstrates restored cellular function and activated antioxidant
   responses, with modulated inflammatory pathways. The PI3K‐AKT‐mTOR
   pathway's activation, essential for PDA@Exo's effects, is confirmed via
   bioinformatics and Western blot. In vivo assessments robustly validate
   that PDA@Exo MN prevents cartilage degradation and OA progression,
   supported by histological assessments and micro‐CT analysis,
   highlighting its disease‐modifying impact. The excellent
   biocompatibility of PDA@Exo MN, verified through histological (H&E) and
   blood tests showing no organ damage, underscores its safety and
   efficacy for OA therapy, making it a novel and multifunctional
   nanomedical approach in orthopedics, characterized by
   organ‐friendliness and biosecurity.

   Keywords: macrophage polarization, microneedle drug delivery,
   osteoarthritis, polydopamine‐exosome complex, reactive oxygen species
   scavenging
     __________________________________________________________________

   Introducing Microneedle‐Delivered Polydopamine‐Exosome (PDA@Exo MN), a
   novel osteoarthritis therapeutic with enhanced drug delivery via
   improved penetration. Demonstrating strong antioxidative properties and
   high biocompatibility, PDA@Exo MN preserves cartilage, promotes bone
   regeneration, and effectively modulates inflammatory pathways to halt
   disease progression.

   graphic file with name ADVS-11-2406942-g003.jpg

1. Introduction

   OA is a prevalent and debilitating condition that is becoming more
   common due to aging, rising obesity rates, and an increase in joint
   injuries. Worldwide, it is estimated that 250 million people currently
   suffer from osteoarthritis, posing significant health and socioeconomic
   burdens.^[ [48]^1 ^] Currently, there are no non‐surgical interventions
   that can prevent, halt, or slow down the progression of
   osteoarthritis.^[ [49]^2 ^] Pharmaceutical therapies, such as
   nonsteroidal anti‐inflammatory drugs (NSAIDs), primarily focus on pain
   relief and anti‐inflammation. However, they only offer limited control
   over osteoarthritis symptoms and cannot reverse joint damage.
   Furthermore, the effectiveness of these drugs can be compromised by the
   harsh gastrointestinal environment and the liver's first‐pass effect,
   which can result in significantly low drug bioavailability.^[ [50]^3 ^]
   Therefore, it is important to develop new treatments and effective
   anti‐inflammatory drugs for OA to reduce social burden associated with
   OA.^[ [51]^4 ^]

   OA is a chronic disease caused by joint inflammation, resulting from
   damage to the articular cartilage and degradation of extracellular
   matrix components such as collagen II and proteoglycan.^[ [52]^5 ^]
   Activated macrophages are a major contributor to inflammatory responses
   in OA joints, producing significant amounts of ROS which can induce
   extracellular toxicity and degradation of cartilage matrix.^[ [53]^6 ^]
   Prolonged ROS presence can amplify pro‐inflammatory pathways, activate
   macrophages, and worsen inflammation. Targeting the proliferation of
   activated macrophages and reducing existing ROS levels can reduce
   inflammation.^[ [54]^7 ^] Additionally, ROS can be used to induce
   oxidation‐responsive release of anti‐inflammatory drugs, further
   enhancing therapeutic effects in OA.^[ [55]^8 ^] Efforts to decrease
   levels of ROS have focused on creating an effective treatment approach
   that applies multifunctional biomaterials, which can both reduce
   oxidative stress and promote chondrogenesis. Besides, OA is a complex
   disease involving multiple pathways, including inflammation via
   macrophages, although the precise role and mechanisms of macrophages in
   OA remain unknown. Macrophages differentiate into M1 and M2 categories
   in response to microenvironment stimuli, with M1 macrophages secreting
   proinflammatory cytokines and M2 macrophages having anti‐inflammatory
   activity.^[ [56]^8 , [57]^9 ^] The accumulation and polarization of
   macrophages in the synovium and articular cavity during OA development
   suggest a correlation, but recent studies show that macrophage
   depletion, reducing both M1 and M2, increases inflammation and fails to
   alleviate OA severity.^[ [58]^10 ^] However, current biomaterial
   treatments for OA mainly encapsulate growth factors or deliver
   antioxidants, without comprehensively addressing the complex process of
   preventing OA. Therefore, the development of new biomaterial tools with
   multiple functions for OA treatment is still highly anticipated.

   Microneedle patches have functional designs that make them highly
   effective in delivering drugs to specific sites in various disease
   microenvironments, such as solid tumors and organs. Their ability to
   penetrate a variety of tissues and organs makes them an ideal
   biomaterial for lesion positioning therapy in medicine. In this paper,
   we present a novel core‐shell MN patch with mesenchymal stem cell
   (MSC)‐derived exosomes (Exo) and polydopamine nanoparticles (PDA NPs)
   encapsulated in the needle tips for OA, as schemed 1. Transdermal drug
   delivery has the advantage of avoiding gastrointestinal digestion
   associated with oral routes and pain sensations caused by injections
   and the ease of self‐administration increases patients’ compliance.^[
   [59]^11 ^] Furthermore, the designed MN patch could be integrally
   degradable and biocompatible, which made the drug delivery and
   absorption more efficient, convenient, and safer.^[ [60]^12 ^] While
   traditional MNs are limited in function, MSC‐derived exosomes offer a
   promising approach for treatment by leveraging their nanoscale size and
   advanced functions inherited from parent stem cells.^[ [61]^13 ^]
   Besides, MSC Exos, created by extruding MSCs through porous membranes,
   offer a 250‐fold increase in yield production and enhanced expression
   of mRNAs and proteins, making them highly valuable for practical
   clinical applications. Despite their successes, the expression amount
   and level of therapeutic growth factors in MSC NVs are still limited,
   requiring further development to meet the demands of more effective
   efficacy.^[ [62]^14 ^]

   To counteract the negative effects of ROS‐derived oxidative stress, PDA
   NPs, which contain reductive functional groups like catechol and imine,
   were encapsulated in the outer methacrylated hyaluronic acid (HAMA)
   shell of MN tips, as PDA has been proven to be an effective antioxidant
   agent.^[ [63]^15 ^] This sustained release of PDA NPs from degrading
   HAMA patch tips effectively suppresses the ROS‐induced inflammation
   reaction at the lesion site. In addition, Exo MN have been shown to
   significantly increase cell proliferation as well as inhibit cell
   apoptosis, while the combination of PDA NPs and Exos can further
   promote M2 macrophage polarization, effectively suppressing
   inflammation. Collectively, the Exo PDA@MN patch has demonstrated
   excellent anti‐inflammatory, antioxidant, and promote osteogenesis,
   making it highly effective for delaying the progression of OA, which
   highlights the potential of the proposed composite core‐shell MN patch
   as a promising candidate for OA treatment.

2. Results and Discussion

2.1. Preparation and Characterization of MSC‐Exos and PDA NPs

   The synthetic route of PDA NPs is shown in Figure [64] 1a. In a typical
   experiment, MSCs were co‐cultured with NPs for 24 h, and the MSCs were
   isolated by ultracentrifugation following established protocol (Figure
   [65]S1, Supporting Information).^[ [66]^16 ^] TEM images reveal that
   PDA nanoparticles exhibit a spherical structure with a diameter of
   ≈100 nm, while Exo microstructure appears as smooth spheres, and the
   morphology of PDA@Exo composite material is consistent with that of PDA
   nanoparticles, also exhibiting a spherical structure (Figure [67]1b).
   Western blot analysis was utilized to detect exosome markers (Alix,
   CD9, TSG101) and endoplasmic reticulum (ER) markers (calnexin) present
   in PDA@Exo exosomes released by cells (Figure [68]1c). Additionally,
   DLS analysis reveals that PDA nanoparticles and PDA@Exo composite
   material both have a narrow size distribution with diameters of ≈108
   and 106 nm respectively, consistent with TEM observations
   (Figure [69]1d) and further supported by Nanoparticle Tracking Analysis
   (NTA) of the exosomes and PDA@Exo composites (Figure [70]S2, Supporting
   Information). Zeta potential and ultraviolet tests were conducted to
   confirm the successful synthesis of PDA@Exo composite material,
   revealing significant changes in its potential value (Figure [71]1e)
   compared to individual PDA and Exo nanoparticles. The UV spectrum of
   the PDA@Exo composite material (Figure [72]1f) also exhibited
   characteristic peaks of Exo extracellular vesicles, distinguishing it
   from the PDA group. Furthermore, the stability of PDA@Exo nanoparticles
   for 48 h (Figure [73]1g) suggests their potential applicability for
   sustained drug delivery and therapeutic applications.

Figure 1.

   Figure 1
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   Synthesis and characterization of PDA@Exo MN. a) Diagrammatic
   representation of the synthesis pathway for PDA@Exo MN. b) Transmission
   electron microscopy (TEM) images depicting PDA, Exo, and PDA@Exo. Scale
   bars: 100 µm. c) Hydrodynamic diameters of Exo, PDA and PDA@Exo
   determined by Dynamic Light Scattering (DLS). d) Western blot analysis
   of exosome markers (Alix, CD9, TSG101) and endoplasmic reticulum (ER)
   markers (calnexin). e) Zeta potential measurements of PDA, Exo, and
   PDA@Exo. f) UV spectra of PDA, Exo, and PDA@Exo. g) Evaluation of the
   size stability of PDA@Exo.

2.2. Preparation and Characterization of Core–Shell MN Patch

   The successful coating with the PDA@Exo composite, evident from the
   increased surface roughness in Figure [75] 2a, is highlighted through
   SEM imaging, which also shows the maintained uniform conical geometry
   of the microneedle arrays. Notably, the microneedles manifest a
   distinctive conical shape, characterized by uniform dimensions and
   remarkably sharp tips, which collectively meet the essential geometric
   criteria required for optimal and efficient skin penetration. These
   microneedles exhibit a remarkable height of 1000 ± 10 micrometers, a
   base width of 900 ± 10 micrometers and a diameter of 904.06 µm in blank
   MN and 879.36 µm, respectively (Figures [76]S3 and [77]S4, Supporting
   Information). The analysis of the force‐displacement curve demonstrates
   that the fracture force of the microneedles significantly exceeds the
   skin's minimum penetration threshold of 58 mN, confirming their
   effectiveness for reliable skin penetration. Moreover, the
   incorporation of the PDA@Exo composite material leads to a noticeable
   increase in the microneedles' mechanical strength, as evidenced by the
   enhanced slope in the force‐displacement curve within the 20–40% range,
   indicative of improved mechanical properties (Figure [78]2b).
   Additionally, Figure [79]2c displays a micrograph of the microneedle
   array with 49 needles organized in a precise 7 × 7 arrangement,
   highlighted by uniform FITC dye fluorescence, signifying even coating
   distribution. A 3D reconstructed image from laser confocal microscopy
   offers a detailed perspective of the microneedles’ topography and
   precise architecture, as shown in Figure [80]2d. Upon application to
   rat skin, water‐soluble microneedles demonstrate a swift dissolution
   and delivery of compounds, with the skin exhibiting rapid healing
   within minutes, as the puncture marks nearly vanish by the 15 min
   observation, underscoring a quick recovery and the non‐invasive nature
   of the delivery system (Figure [81]2e). Histological analyses confirm
   the patches’ ability to penetrate various epidermal layers, reaching up
   to 159 µm into the dermis and thereby reducing discomfort by avoiding
   deeper, more sensitive tissue areas (Figure [82]2f). To further assess
   transdermal penetration, PKH26‐labeled EXOs/MN were applied to fresh
   porcine skin; fluorescence imaging of frozen skin sections demonstrated
   that EXOs could penetrate deeply, up to 800 µm into the skin (Figure
   [83]S5, Supporting Information). The in vitro release of EXOs from
   microneedles was evaluated using a BCA protein assay in a 24‐well
   Transwell setup, where the cumulative release rate reached 58.85% by 12
   h and then stabilized, indicating effective and controlled delivery
   (Figure [84]S6, Supporting Information). Additionally, methylene
   blue‐labeled microneedles release their payload rapidly upon insertion
   into agar gel and achieve a stable release over 7 days, suggesting
   their suitability for sustained drug delivery and thus, this could
   enhance compliance by reducing the frequency of dosing, making them
   promising for chronic condition management (Figure [85]S7, Supporting
   Information).

Figure 2.

   Figure 2
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   Microstructural Analysis and Mechanical Validation of Microneedles. a)
   SEM Micrographs of MN and PDA@Exo MN. b) Mechanical Characterization of
   Microneedles: MN versus PDA@Exo MN. c) Photomicrograph of a fluorescent
   MN. d) 3D reconstruction image of Laser Confocal Microscopy Image of a
   Microneedle. e) Sequential evaluation of skin recovery at various time
   points post‐microneedle insertion. Scale bars: 1 cm. f)
   Immunofluorescent and histological staining of MN penetration in rat
   skin. Scale bars: 50 and 200 µm, respectively.

2.3. Antioxidant Effect of PDA@Exo Nanoparticles against ROS

   To ensure safe use for bio‐applications, the cytotoxicity of PDA@Exo
   was evaluated using a cell counting kit‐8 (CCK‐8) assay on primary rat
   chondrocytes. Results showed no significant decrease in cell viability
   after 48 h of incubation with PDA@Exo at concentrations up to
   400 µg mL^−1, indicating minimal cytotoxicity (Figure [87]S8,
   Supporting Information). Subsequently, chondrocytes were pre‐treated
   with H[2]O[2] (800 µm) for 24 h to confirm the protective effect of
   PDA@Exo against cellular ROS damage by Live/dead cell double staining
   using calcein‐acetoxymethyl ester/propidium iodide (Calcein‐AM/PI) kit.
   The results showed that PDA@Exo efficiently protected chondrocytes from
   H[2]O[2]‐induced cell death, as evidenced by the significant increase
   in live cells after PDA@Exo addition compared to the almost complete
   cell death in the control group (Figure [88] 3a,b). Studies have
   demonstrated excessive production of ROS and oxidative stress in
   chondrocytes are major contributors to the pathogenesis of OA.^[
   [89]^17 ^] Under pathological conditions in OA, excessive ROS act as
   secondary messengers, promoting cartilage degradation by inducing
   matrix‐degrading proteases, reducing ECM synthesis, and inducing
   chondrocyte apoptosis.^[ [90]^18 ^] Therefore, leveraging PDA NPs,
   renowned for their ROS scavenging capabilities in conditions such as
   tumors, infections, and inflammatory diseases including OA, may offer
   therapeutic potential by diminishing ROS production and impeding
   cartilage matrix catabolism.^[ [91]^19 ^] Additionally, numerous
   studies have demonstrated that MSCs‐derived exosomes possess the
   ability to repair damaged tissues, protect cells, regulate immune
   responses, and inhibit inflammation.^[ [92]^20 ^] Intracellular ROS
   levels were quantitatively assessed using the ROS‐sensitive fluorescent
   probe DCFH‐DA, which diffuses into cells and fluoresces upon oxidation,
   with flow cytometry providing quantitative measurement of oxidation
   levels. Enhanced green fluorescence indicative of increased ROS was
   observed in chondrocytes subjected to oxidative stress by H[2]O[2].
   Notably, the application of PDA@Exos markedly attenuated the
   H[2]O[2]‐induced ROS levels, as evidenced by reduced fluorescence
   intensity in the treated samples (Figure [93]3c,d). FITC results
   corroborated the observed decrease in ROS, quantitatively confirming a
   significant reduction in median fluorescence intensity, which
   illustrates the effective suppression of ROS by PDA@Exos in treated
   cells (Figure [94]3e,f). Superoxide dismutase (SOD) serves as a crucial
   defensive enzyme against oxidative stress by neutralizing superoxide
   anions, while malondialdehyde (MDA) levels indicate the extent of lipid
   peroxidation by ROS. In this study, PDA@Exos was found to significantly
   restore SOD activity diminished by H[2]O[2] exposure in chondrocytes,
   suggesting a recovery of the cells' antioxidative mechanisms.
   Additionally, PDA@Exos effectively reduced the elevated MDA content,
   indicating a decrease in lipid peroxidation and an overall reduction of
   intracellular ROS levels (Figure [95]3g,h). Enzymes like SOD3, GPX, and
   CAT play essential roles in cellular defenses against oxidative stress
   by regulating the balance of ROS. PDA@Exos treatment was observed to
   significantly enhance the expression of these enzymes, suggesting its
   supportive role in reinforcing the cell's antioxidative capacity.
   Concurrently, PDA@Exos also reduced the mRNA expression of iNOS and
   COX‐2, inflammatory markers linked to cartilage damage, highlighting
   its dual protective effect against both oxidative stress and
   inflammation in chondrocytes (Figure [96]S9, Supporting Information).
   These results collectively highlight PDA@Exo as a potent ROS scavenger,
   capable of preventing cartilage deterioration and thereby emerging as a
   promising candidate for OA therapy.

Figure 3.

   Figure 3
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   Protective effect of PDA@Exo MN on chondrocytes against ROS in vitro.
   a) Live/Dead staining of chondrocytes treated with Control, PDA, Exo,
   and PDA@Exo under H[2]O[2] stimulation, showing nuclei in blue (33258),
   live cells in green (AM) and dead cells in red (PI). Scale bars: 20 µm.
   b) Ratio of live/dead cells measured by MFI in chondrocytes
   post‐treatment with Control, PDA, Exo, and PDA@Exo under H[2]O[2]
   stimulation. c) ROS detection in chondrocytes treated with Control,
   PDA, Exo, and PDA@Exo under H[2]O[2] stimulation, where green
   fluorescence (DCF) indicates ROS levels, with DAPI staining for nuclei.
   Scale bars: 50 µm. d) Quantitative analysis of intracellular ROS levels
   as indicated by mean fluorescence intensity (MFI) in chondrocytes
   treated with Control, PDA, Exo, and PDA@Exo following H[2]O[2]
   stimulation. e) ROS detection using FITC in chondrocytes treated with
   Control, PDA, Exo, and PDA@Exo under H[2]O[2] stimulation. f) MFI in
   chondrocytes treated with Control, PDA, Exo, and PDA@Exo following
   H[2]O[2] stimulation. g) Relative SOD activity restored by PDA@Exo in
   H[2]O[2]‐induced chondrocytes. h) Cellular MDA content of chondrocytes
   treated with Control, PDA, Exo, and PDA@Exo under H[2]O[2] stimulation
   All data are shown as the mean ± standard deviation (SD). *p < 0.05,
   **p < 0.01, ***p < 0.001.

2.4. Therapeutic Modulation of Chondrocyte Function and Macrophage
Polarization by PDA@Exo In Vitro

   Given that mitochondria constitute a principal source of intracellular
   ROS and serve as the epicenter of energy metabolism, they inherently
   become susceptible targets for ROS‐induced cellular impairment. To
   evaluate the influence of PDA@Exo on the mitochondrial function of
   chondrocytes, we employed a JC‐1 mitochondrial membrane potential
   assay, wherein well‐functioning mitochondria with high membrane
   potential emit red fluorescence due to JC‐1 aggregation, while
   dysfunctional mitochondria with low membrane potential emit green
   fluorescence due to JC‐1 monomers. Healthy chondrocytes exhibited
   pronounced red fluorescence, which was significantly diminished upon
   exposure to H[2]O[2], leading to an increase in green fluorescence; the
   presence of PDA@Exo significantly elevated the aggregate to A/M
   fluorescence ratio, which had been sharply reduced by H[2]O[2]
   exposure, indicating a substantial mitigation of mitochondrial
   dysfunction compared to groups treated with PDA or Exo alone (Figure
   [98]4a; Figure [99]S10, Supporting Information). Furthermore, the ATP
   content within chondrocytes, a direct measure of mitochondrial
   function, was notably compromised by H[2]O[2], with PDA@Exo leaching
   solution effectively restoring ATP production, indicating the potential
   of PDA@Exo to support cellular energy requirements under oxidative
   stress (Figure [100]S11, Supporting Information). Furthermore, to
   investigate the protective effect of PDA@Exo on chondrocyte apoptosis,
   the expression levels of Bax, Bcl‐2, Caspase 3, cleaved Caspase 3,
   Caspase 9 and cleaved Caspase 9, were examined using western blot
   analysis.^[ [101]^21 ^] The results showed that IL‐1β treatment
   significantly upregulated the expression of Bax, cleaved Caspase 3 and
   cleaved caspase 9 and while downregulating the expression of Bcl‐2,
   suggesting an induction of chondrocyte apoptosis.^[ [102]^22 ^]
   However, pretreatment with PDA@Exo resulted in a significant decrease
   in the expression of Bax, cleaved caspase 3 and cleaved caspase 9 while
   increasing the expression of Bcl‐2 comparing to control group,
   indicating a suppression of chondrocyte apoptosis (Figure [103]4b;
   Figure [104]S12, Supporting Information). RT‐PCR analysis of critical
   cartilage matrix anabolic genes—including type II collagen (COL2A1),
   aggrecan (ACAN), and SRY‐box 9 (SOX9)—revealed that their suppressed
   expression in IL‐1β‐treated chondrocytes was significantly reversed by
   treatments with PDA@Exo and PDA@Exo, indicating a protective role
   against chondrocyte dysfunction in OA. This therapeutic potential was
   further highlighted by the inhibition of the upregulation of Adamts5,
   Adamts1, and MMP13, genes pivotal to cartilage matrix degradation,
   suggesting an inhibitory effect of PDA@Exo on catabolic processes
   within OA chondrocytes (Figure [105]S13, Supporting Information). To
   investigate the protective effect of PDA@Exo on chondrocytes in an
   IL‐1β‐induced model, the expression levels of MMP9, MMP13, SOX9, COL1A2
   and RUNX2 were examined using western blot analysis (Figure [106]4c;
   Figure [107]S14, Supporting Information).^[ [108]^23 ^] IL‐1β induced
   model remarkably increased the levels of MMP9 and MMP13 while
   chondrocytes co‐treated with PDA@Exo resulted in decreased levels of
   MMP9 and MMP13, which are known to be involved in cartilage
   degradation. Furthermore, RUNX2 expression was downregulated by
   PDA@Exo, indicating a protective effect against hypertrophy.^[ [109]^24
   ^] PDA@Exo increased the expression levels of SOX9 and COL1A2, which
   are important markers of chondrogenic differentiation.^[ [110]^25 ^] In
   addition, the osteogenic potential of BMSCs, assessed via Alizarin red
   staining after 21 days of culture in osteogenic induction medium with
   or without PDA@Exo, demonstrated an enhanced formation of calcium
   nodules in the PDA@Exo groups (Figure [111]4d). This was indicative of
   a bolstered osteogenic capability, further corroborated by elevated
   protein levels of osteogenesis‐associated genes (OPN, RUNX2, and BMP2),
   underscoring the contribution of PDA@Exo to subchondral bone repair in
   vitro (Figure [112]4e; Figure [113]S15, Supporting Information).
   Overall, PDA@Exo can protect chondrocytes by inhibiting apoptosis and
   promoting extracellular matrix synthesis, suggesting its potential as a
   therapeutic strategy for the management of osteoarthritis.

Figure 4.

   Figure 4
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   PDA@Exo restore the cellular function of chondrocytes under oxidative
   stress and M2 polarization. a) JC‐1 staining of chondrocytes treated
   with Control, PDA, Exo, and PDA@Exo under H[2]O[2] stimulation to
   monitor mitochondrial membrane potential. Scale bars: 50 µm. b) Western
   blot analysis of the expression of apoptosis markers in chondrocytes
   treated with Control, PDA, Exo, and PDA@Exo under IL‐1β stimulation. c)
   Western blot analysis of the expression of MMP9, MMP13, SOX9, COL1A2,
   and RUNX2 in chondrocytes treated with Control, PDA, Exo, and PDA@Exo
   under IL‐1β stimulation. d) Alizarin red S staining of BMSCs treated
   with Control, PDA, Exo, and PDA@Exo. e) Western blot analysis of the
   expression of osteogenesis‐related genes (RUNX2, OPN, and BMP2) in
   BMSCs treated with Control, PDA, Exo, and PDA@Exo. f)
   Immunofluorescence staining of the RAW 264.7 cells after being
   cocultured with control, PDA, Exos and PDA@Exo under LPS stimulation,
   iNOS (green); CD206 (red); Blue: nuclei. Scale bars: 50 µm. g) Western
   Blot analysis of RAW 264.7 cells after being cocultured with control,
   PDA, Exos and PDA@Exo under LPS stimulation. (Scale bars: 50 µm) All
   data are shown as the mean ± standard deviation (SD). *p < 0.05, **p <
   0.01, ***p < 0.001.

   Macrophage polarization is a crucial factor in the development and
   progression of osteoarthritis. An imbalance toward M1 macrophages leads
   to inflammation and cartilage degradation, while M2 macrophages promote
   cartilage repair through the secretion of anti‐inflammatory cytokines
   and growth factors, making M2 polarization a promising therapeutic
   strategy for osteoarthritis.^[ [115]^26 ^] To investigate the potential
   antioxidative and anti‐inflammatory effects of PDA@Exo on macrophage
   polarization in osteoarthritis, we incubated PDA, Exo and PDA@Exo with
   50 ng mL^−1 lipopolysaccharide (LPS)‐stimulated RAW264.7 cells.
   Additionally, immunofluorescence staining of Vihecle, PDA, Exo and
   PDA@Exo with LPS‐stimulated RAW264.7 cells revealed that iNOS
   expression was significantly decreased in PDA@Exo groups and CD206 was
   increased compared to the levels in the control group (Figure [116]4f;
   Figure [117]S16, Supporting Information). Post‐incubation with PDA@Exo,
   western blot analysis of RAW264.7 cells revealed a notable upregulation
   in M2 macrophage markers, Arg1 and CD206, and a concurrent
   downregulation of the M1 marker iNOS, indicating a shift toward
   anti‐inflammatory phenotype (Figure [118]4g; Figure [119]S17,
   Supporting Information). ELISA results revealed that PDA@Exos
   effectively attenuates LPS‐induced increases in pro‐inflammatory
   cytokines IL‐6, TNF‐α, and IL‐1β, highlighting its potential to
   counteract M1 macrophage‐mediated inflammation (Figure [120]S18,
   Supporting Information). These findings suggest that PDA@Exo treatment
   can induce M2 polarization of macrophages, consistent with the observed
   anti‐inflammatory effects. Since the M1/M2 macrophage balance plays a
   critical role in the progression of various diseases, including
   osteoarthritis, our study demonstrated that PDA@Exo treatment can
   effectively shift the balance toward the M2 phenotype, indicating its
   potential as a therapeutic strategy for osteoarthritis.

2.5. Transcriptomic Analysis of PDA@Exo against IL‐1β Induced OA Chondrocytes

   Building on our previous promising results, we extended our research to
   perform RNA sequencing analysis, aiming to understand the effects of
   PDA@Exo on chondrocytes in osteoarthritis (OA). After RNA sequencing,
   Figure [121] 5a shows a volcano plot identifying differentially
   expressed genes, with significant upregulation and downregulation noted
   byFDR < 0.05, log2FC > 1, or log2FC < −1. A Venn diagram in
   Figure [122]5b illustrates the overlap of gene expression changes
   between conditions, highlighting a core set of affected genes among
   control, OA group and OA with PDA@Exo MN treatment group. Additionally,
   the heatmap details gene expression levels across samples, with
   hierarchical clustering indicating distinct expression profiles, as
   decoded by the legend (Figure [123]5c). To delve deeper into the
   affected biological functions and pathways, we carried out Gene
   Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and
   Genomes (KEGG) pathway enrichment analysis. Our GO analysis highlights
   PDA@Exo's anti‐inflammatory role in IL‐1β‐stimulated chondrocytes,
   showing an upregulation of antioxidative responses, modulation of lipid
   biosynthesis, and enhancement of extracellular matrix integrity.
   Notably, the upregulation of “response to hydrogen peroxide” and
   “response to reactive oxygen species” underscores an enhanced
   antioxidative defense mechanism. Simultaneously, the activation of
   “cholesterol biosynthetic process” and “sterol biosynthetic process”
   signals a reparative shift in lipid metabolism. Additionally, the
   enrichment of terms like “extracellular matrix” and
   “collagen‐containing extracellular matrix” indicates an improvement in
   tissue repair and resilience to inflammation. These findings highlight
   PDA@Exo's role in dampening IL‐1β‐mediated inflammatory pathways,
   showcasing its potential for osteoarthritis therapy (Figure [124]5d,e).
   The KEGG pathway enrichment analysis further revealed a significant
   modulation of the PI3K‐Akt signaling pathway in chondrocytes treated
   with PDA@Exo post IL‐1β stimulation (Figure [125]5g). This pathway,
   crucial for various cellular functions such as growth, survival, and
   inflammation, showed significant gene expression changes, suggesting
   PDA@Exo's ability to mitigate osteoarthritis pathophysiology by
   modulating the PI3K‐Akt pathway. Moreover, related inflammatory
   pathways like TNF and IL‐17 signaling were also modulated, with a
   notable emphasis on the PI3K‐Akt pathway's role.The downstream effects
   of this modulation hint at a potential reduction in pro‐inflammatory
   mediators alongside an improvement in cellular homeostasis. These
   insights reveal the complex impact of PDA@Exo, especially highlighting
   the significant role of the PI3K‐Akt pathway in managing chondrocyte
   pathophysiology amidst inflammatory stress. The extensive changes in
   gene expression related to this pathway highlight its therapeutic
   promise for mitigating inflammation in osteoarthritis.

Figure 5.

   Figure 5
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   Changes in transcriptome profile of PDA@Exo ‐treated OA chondrocytes.a)
   The volcano plot of differentially expressed genes (DEGs) and b) Venn
   that are essential for chondrocyte bio‐function and provide cellular
   protection against inflammation induced by IL‐1β are identified and
   highlighted. c) Heatmap representing the gene expression profiles of OA
   chondrocytes stimulated with IL‐1β and OA chondrocytes treated with
   IL‐1β + PDA@Exo. d–f) The results of Gene Ontology (GO) enrichment
   analysis in both up‐regulated and down‐regulated genes. g) Enrichment
   analysis of the KEGG pathways for the differentially expressed genes of
   interest. h) The expression of p‐PI3K, PI3K, p‐Akt, Akt, p‐mTOR, and
   mTOR protein in chondrocyte treated with control, PDA, Exos and PDA@Exo
   under IL‐1β stimulation.

   To validate the anti‐inflammatory mechanisms of PDA@Exo in chondrocytes
   and its capacity to decelerate osteoarthritis progression, as inferred
   from RNA sequencing and KEGG GO analysis, Western blot assays were
   executed, targeting the phosphorylation profiles of essential proteins
   in the PI3K‐AKT‐mTOR signaling pathway. Figures [127]5h and Figure
   [128]S19 (Supporting Information) demonstrate that PI3K, AKT, and mTOR
   phosphorylation levels are significantly elevated in the PDA, Exo, and
   PDA@Exo treatment groups. The PI3K‐AKT‐mTOR pathway, crucial for cell
   growth, survival, and metabolic regulation, showed an upregulation in
   response to IL‐1β stimulation, with marked increases in the
   phosphorylation of PI3K, AKT, and mTOR. PDA@Exo treatment moderated
   phosphorylation levels, indicating its regulatory impact on the pivotal
   PI3K‐AKT‐mTOR pathway and related cellular functions in chondrocytes,
   aligning with observed enhancements in chondrocyte proliferation,
   matrix synthesis, and cellular defense mechanisms. n conclusion, our
   research reveals that PDA@Exo adjusts the chondrocyte transcriptome to
   facilitate functional recovery, activate antioxidant enzymes, and
   reduce inflammation. These outcomes corroborate the theory that PDA@Exo
   reduces intracellular ROS and inflammation in cartilage, thus
   maintaining chondrocyte structural and functional integrity, and
   presenting a viable strategy for osteoarthritis treatment.

2.6. In Vivo Therapeutic Effect and Efficacy of PDA@Exo MN Patch on OA

   To assess the efficacy of PDA@Exo MN in treating rat knee
   osteoarthritis (OA), a model was established via anterior cruciate
   ligament transection (ACLT) and followed by intra‐articular
   administrations of treatments including control, Blank MN, PDA MN, Exo
   MN, and PDA@Exo MN, starting four weeks post‐surgery, every three days
   for a duration of four weeks. At eight weeks post‐treatment, the rats
   were euthanized for their knee joints to be analyzed with hematoxylin
   and eosin (H&E), safranin‐O/fast green (SO‐FG) staining and toluidine
   blue (TB), providing insight into the therapeutic impacts of the
   various treatments. Consequently, OA knees exhibited pronounced
   cartilage degradation and subchondral bone lesions, whereas PDA@Exo MN
   treatment preserved the integrity of both cartilage surfaces and
   subchondral bone. This protective effect on OA was further validated by
   OA Research Society International (OARSI) scores (Figure [129]6a,b).
   Micro‐computed tomography (micro‐CT) analyses, including 3D
   reconstructions of the medial tibial plateau, illustrated typical OA
   manifestations like osteophyte formation and joint deformity in control
   OA knees of ACLT rats, which were absent in PDA@Exo MN‐treated groups
   (Figure [130]6c, upper row). Sagital and coronal micro‐CT images showed
   severe bone resorption and sparse trabeculae in control OA rats,
   whereas PDA@Exo MN treatment effectively mitigated these changes,
   improving trabecular bone volume and reducing trabecular separation as
   confirmed by micro‐CT quantitative analysis (Figure [131]6d–f).

Figure 6.

   Figure 6
   [132]Open in a new tab

   PDA@Exo attenuated OA progression in vivo. a) Representative images of
   H&E, TB, and SO‐FG staining in rat knee joints, and b) the
   corresponding OARSI scores after different treatments 8 weeks
   post‐surgery. Scale bars: 500 µm c) Representative 3D reconstruction
   images of differently treated rat knees at week 8 (Scale bars: 2 µm)
   and coronal micro‐CT images of the rat medial tibial plateau in
   different groups. (Scale bars: 1 µm); d–f) Quantitative analysis of
   BV/TV and increased Tb. Sp conducted by micro‐CT in 8 weeks
   post‐surgery. All data are shown as the mean ± standard deviation (SD).
   *p < 0.05, **p < 0.01, ***p < 0.001.

2.7. In Vitro and In Vivo Biocompatibility Tests

   After demonstrating the effects of PDA@Exo in vitro, further
   investigation in OA animal models is crucial to understand its
   potential in vivo. Animal models allow for evaluation of
   pharmacokinetics, biodistribution, efficacy, safety, and mechanisms of
   action of PDA@Exo in a more physiologically relevant context. HA has
   good degradation characteristics and HAMA has been proved to degrade in
   the presence of hyaluronidase and the result showed the MN dissolving
   progressively from 0 to 10 min, confirming their quick biodegradability
   and biosafety profile (Figure [133]S20, Supporting Information).^[
   [134]^27 ^] Histopathological evaluations of key organs, including
   heart, liver, spleen, lung, and kidney, in rats treated with PDA@Exo MN
   revealed no morphological or pathological changes, highlighting the
   treatment's non‐toxic nature (Figure [135]S21, Supporting Information).
   Furthermore, histological assessments, including HE staining at
   microneedle application sites, confirmed an absence of inflammatory or
   pathological responses, with no erythema, edema, or cellular
   infiltration observed. Routine blood tests from the PDA@Exo MN‐treated
   animals revealed stable hematological parameters, with serum
   biochemical assays corroborating these findings. Liver and kidney
   function indicators, such as ALT, AST, and creatinine, remained within
   normal limits, indicating the absence of hepatic or renal toxicity due
   to the PDA@Exo MN treatment (Figure [136]S22, Supporting Information).
   In conclusion, PDA@Exo MNs displayed exceptional biocompatibility and
   safety, as evidenced by their lack of toxicological impact on major
   organ functions, confirmed through histopathological and biochemical
   analyses. This foundational safety, coupled with their precise drug
   delivery, establishes these microneedles as a promising and effective
   method for osteoarthritis treatment.

3. Conclusion

   In conclusion, our comprehensive studies affirm that PDA@Exo MNs serve
   as a potent intervention for osteoarthritis, providing dual benefits by
   reducing intracellular ROS and promoting osteogenesis while
   simultaneously preserving cartilage and promoting an M2 reparative
   macrophage phenotype. This therapeutic efficacy is supported by
   histological and micro‐CT analyses that show significant reduction of
   cartilage and bone damage in osteoarthritic models. Further
   pharmacokinetic assessments underscore the biocompatibility and safety
   profile of PDA@Exo MNs, positioning them as a promising,
   multifunctional approach to osteoarthritis treatment that concurrently
   addresses the disease's inflammatory and structural deteriorations, as
   depicted in Scheme [137]1 .

Scheme 1.

   Scheme 1
   [138]Open in a new tab

   Schematic depicting PDA@Exo MN‐mediated modulation of cartilage
   degradation inhibition, osteogenesis enhancement, and macrophage
   polarization via the PI3K‐AKT‐mTOR signaling pathway.

4. Experimental Section

Statistical Analysis

   Data were presented as mean ± SD and analyzed using GraphPad Prism
   software. Multiple comparisons among more than two groups were analyzed
   using multiple‐factorial ANOVA to determine statistical significance,
   indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Student's t‐test or
   Welch's t‐test was used for two‐group comparisons, based on variance
   homogeneity.

Conflict of Interest

   The authors declare no conflict of interest.

Supporting information

   Supporting Information
   [139]ADVS-11-2406942-s001.pdf^ (8.6MB, pdf)

Acknowledgements