Antioxidant-Enhanced Nanoparticles Delay the Progression of Osteoarthritis

Abstract

Osteoarthritis (OA) is an age-related degenerative disease. Oxidative stress (OS) regulates the pathogenesis of OA by affecting mitochondrial function. Mg²⁺ and citric acid can delay the progression of OA through reducing OS, but integrated therapy. In this study, a mesoporous nanoparticle (PCMg) composed of Mg²⁺, citric acid (CC), and dopamine was developed as a novel delivery platform. SS31, a positively charged peptide, was adsorbed onto the surface of PCMg through the charge interaction to obtain the new nanoparticle platform (PCMS). SS31 endowed PCMS with good biocompatibility and enabled it to be rapidly endocytosed by chondrocytes and simultaneously localized in mitochondria. In vitro, PCMS effectively reduced the reactive oxygen species (ROS) level and restored mitochondrial dysfunction mediated by tert-butyl hydroperoxide (TBHP) through reducing OS in chondrocytes. In addition, both in vivo and in vitro results demonstrated that PCMS had no malignant effect on chondrocyte viability and proliferation and was degradable. Through the destabilization of the medial meniscus (DMM) OA model, hematoxylin–eosin (HE), safranin, fast green (SO), and toluidine blue (TB) staining indicated that PCMS effectively alleviated the progression of OA. Comprehensive analysis indicated that PCMS treatment was an effective strategy for alleviating the progression of OA.

1. Introduction

Osteoarthritis (OA) is a chronic, degenerative cartilage disorder primarily characterized by joint pain, dysfunction, and significant movement impairment, representing a leading cause of disability. Currently, over 300 million individuals worldwide are affected by OA, which places a substantial economic burden on societies. With an aging population, projections indicate that nearly 25% of adults will be afflicted by OA by 2030. , Despite their high prevalence and disability rate, effective treatment options remain scarce. For advanced cases, surgery is often the only viable option, while no therapies exist to reverse or significantly slow the disease in its early stages.

OA pathology is typically characterized by cartilage degeneration, osteophyte formation, and persistent inflammatory responses. − While the precise pathogenic mechanisms remain unclear, oxidative stress (OS)-driven inflammation is recognized as a key factor in the initiation and progression of the disease. Cartilage consists primarily of chondrocytes and extracellular matrix (ECM), with chondrocytes synthesizing ECM components that in turn provide a supportive environment for the cells. This feedback loop maintains cartilage metabolic homeostasis. However, in early OA, altered joint biomechanics and OS-induced inflammation disrupt this equilibrium, reducing the expression of key ECM components, such as collagen and aggrecan. Concurrently, inflammatory mediators, including IL-1, TNF-α, and matrix metalloproteinases such as MMP13, are released, triggering an inflammatory cascade that accelerates OA progression. Consequently, targeting OS offers a promising therapeutic approach to delay OA progression. −

Given the multifaceted factors contributing to arthritis progression and the growing evidence supporting the use of molecules from natural sources in its treatment, leveraging bioactive molecules from natural sources presents a promising therapeutic approach. − Molecules from natural sources serve as invaluable resources for discovering novel pharmacological agents due to their unique structural diversity and the potential for synthetic modification to optimize selectivity and bioavailability. Citric acid (CC), a plant-derived compound found in lemongrass, lemon balm, and ginger, is widely utilized across the food, cosmetic, chemical, and pharmaceutical industries. It is incorporated into various products, including spices, food, and beverages. With documented anti-inflammatory, antioxidant, and antibacterial properties, CC is emerging as a promising candidate for pharmacological intervention. As an essential substrate in energy metabolism, the efficient and targeted delivery of CC remains a challenge in therapeutic applications.

Polydopamine nanoparticles (PDA NPs), self-polymerized from the natural neurotransmitter dopamine (DA), are increasingly employed in drug delivery systems due to their biodegradability, biocompatibility, and superior photothermal conversion efficiency. PDA NPs possess abundant reactive groups, including catechol/quinone, amine, and imine, which facilitate molecular interactions via π–π stacking, hydrogen bonding, and electrostatic attraction. However, the drug loading capacity of PDA NPs is constrained by the limited specific surface area. To address this, mesoporous PDA NPs (MPDA NPs) are utilized to enhance the drug loading capacity. Additionally, MPDA NPs exhibit a high density of phenolic groups, which confer excellent reactive oxygen species (ROS) scavenging properties. − The reduction of ROS, in turn, decreases the expression of pro-inflammatory cytokines, potentially slowing disease progression. Thus, PDA-based materials show significant promise as functional drug carriers. Despite the advantages, the conventional approach of drug loading onto the surface of MPDA NPs still faces challenges, such as short-term burst release and limited single-drug loading. ,,− To overcome these limitations, this study proposes to incorporate drugs into MPDA nanoparticles, thereby constructing a multidrug delivery platform.

The traditional delivery strategy relies on the participation of carriers, and drugs adhere to the carriers through electrostatic and hydrophobic interactions. Compared with covalent bond binding, physical loading will cause drugs to be unable to be sustained released for a long time, which is not conducive to maintaining the drug concentration in the body for a long time. Drug-doped delivery platforms have the same advantages as drug self-assembly. The absence of a carrier can produce a longer sustained-release effect, which is conducive to the release and action of the drug. This study takes advantage of the self-polymerization characteristics of dopamine and incorporates CC and Mg²⁺ ions into mesoporous dopamine to synthesize a novel delivery platform.

In this study, Mg²⁺, CC, and DA were chelated and loaded onto the hydrophilic end of Pluronic F127 (PF127), serving as a micelle template with 1,3,5-trimethylbenzene (TMB) as the micelle core, to obtain mesoporous nanoparticles (PCMg) doped with Mg²⁺, CC, and DA. To enhance targeted delivery, the positively charged peptide SS31 was incorporated onto the surface of PCMg via electrostatic interaction. In vitro evaluations were performed to assess the cell viability, ROS elimination, and OS alleviation. Finally, the efficacy of these NPs was tested in an arthritis model to explore their impact on the joint environment in vivo.

2. Method

2.1. Materials

Type II collagenase and tert-butyl hydroperoxide (TBHP) were procured from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies came from Cell Signaling Technology (Danvers, MA), with fluorescent secondary antibodies (Alexa Fluor 488/594-labeled goat antirabbit IgG) being acquired from Abcam. Nuclear staining was performed using DAPI from Beyotime Biotechnology (Shanghai, China). Cell culture media and supplements were primarily obtained from Gibco (Grand Island, NY), unless noted otherwise. DMSO, used as a solvent, was also supplied by Sigma-Aldrich.

2.2. Synthesis of PCMg NPs

Briefly, 50 mg of CC and 50 mg of DA were dissolved in 10 mL of a 1:1 (v/v) mixture of H₂O and ethanol, followed by the addition of 20 mg of MgCl₂, and the mixture was stirred (600 rpm) for 10 min. Subsequently, 0.1 g of PF127 and 150 μL of TMB were added, and the solution was stirred for 30 min. Afterward, 500 μL of NH₄OH was introduced, and the reaction was carried out at room temperature for 2 h. The resulting particles were collected by centrifugation (11,000 rpm) and subjected to ultrasonic treatment with ethanol for 30 min to remove the polymer micelle template. This washing procedure was repeated three times.

2.3. Synthesis and Characterization of PCMS Nanoparticles

The prepared PCMg NPs were stirred (300 rpm) with a 100 μg/mL solution of SS31 at room temperature for 4 h, and the particles were collected by centrifugation (8000 rpm). The washing procedure was repeated three times with PBS under ultrasonic treatment.

The hydrodynamic diameter and surface zeta potential of PDA NPs were characterized by dynamic light scattering using a nanoanalyzer (Malvern, UK). The morphology of the NPs was observed using transmission electron microscopy (TEM) (HT7700, Hitachi, Japan) and scanning electron microscopy (Hitachi SU8010, Japan). For clearly observing the morphology of NPs, the SEM operates at a voltage of 7 kV and a current of 7 μA. To observe the distribution of elements, the EDS mode of TEM was used.

2.4. Degradation of PCMS

For degradation assessment, the weight of PCMS was recorded, and the samples were immersed in PBS for 1, 2, and 3 days. After incubation, the freeze-dried NPs were weighed, and the results were recorded at each preset time point. The degradation ratio (%) was calculated using the following formula

$$\mathrm{d}\mathrm{e}\mathrm{g}\mathrm{r}\mathrm{a}\mathrm{d}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}(\%)=(W_{\mathrm{w}}-W_{\mathrm{d}})/W_{\mathrm{d}}\times 100\%$$

where W _d represents the starting weight and W _w represents the weight of the PCMS after immersion.

2.5. Nanoparticle Uptake

PCMS was coincubated with CM-DIL for 12 h, followed by centrifugation (10,000 rpm), to obtain PCMS-DIL. The cells were coincubated with PCMS-DIL for 4 and 12 h, fixed with 4% paraformaldehyde (PFA), permeabilized, and stained with F-actin to visualize the cytoskeleton and DAPI for the nucleus. The localization of PCMS-DIL was examined by using confocal microscopy and analyzed by using ImageJ software.

2.6. Nanoparticle Localization

Following 4- or 12 h coincubation with PCMS-DIL, the cells were washed three times. Subsequently, they were treated with Mitotracker for 30 min, nuclearly stained with Hoechst, and imaged using confocal microscopy.

2.7. Cartilage Extraction

Rats (1 week) were obtained from a specific pathogen-free (SPF) facility. Primary chondrocytes were isolated aseptically from hip joint cartilage, which was dissected into 1 mm³ fragments, washed three times with PBS, and sequentially digested with 0.25% trypsin (1 h) followed by 0.2% collagenase II in DMEM-F12 (4 h, 37 °C, 5% CO₂). The digested suspension was centrifuged (1200 rpm, 5 min), and the pelleted cells were resuspended in complete DMEM-F12 medium supplemented with 10% FBS and 1% penicillin–streptomycin. Cells were maintained at 37 °C in a 5% CO₂ incubator and used within three passages to ensure chondrocyte phenotype stability.

2.8. Live/Dead Assay

Chondrocytes were seeded in a 96-well plate at a density of 10,000 cells per well and cultured in DMEM-F12 containing 10% FBS at 37 °C in a 5% CO₂ incubator. When the cell density reached 70%, the chondrocytes were treated with different formulations for 24 h and divided into four groups: control, tert-butyl hydroperoxide (TBHP), PCMg (10 μL), and PCMS (10 μL). After treatment, the cells were washed with PBS and stained with live/dead dyes for approximately 1 h and were prepared for being observed. Fluorescence images of the treated chondrocytes were captured by using a Nikon fluorescence microscope (ECLIPSE TI-S, Nikon, Japan) (TBHP (30 μM), PCMg (0.1 mg/mL), PCMS (0.1 mg/mL)).

2.9. Cell Proliferation Assay

Chondrocyte viability was evaluated using a cell counting kit-8 (CCK-8) assay following the manufacturer’s protocol. Briefly, 5 × 10³ cells/well were seeded in a 96-well plate and cultured for 24 h (37 °C, 5% CO₂). After treatment with PBS, TBHP, PCMg (10 μL), or PCMS (10 μL) for 24 or 72 h, cells were washed three times with PBS and incubated with 100 μL of DMEM-F12 containing 10 μL of CCK-8 reagent for 1 h. Absorbance at 450 nm was quantified using a Thermo Fisher spectrophotometer (TBHP (30 μM), PCMg (0.1 mg/mL), and PCMS (0.1 mg/mL)).

2.10. Dihydroethidium (DHE) Staining

To assess the ROS levels in chondrocytes under different 24 h treatments, DHE staining was performed. After being cocultured with TBHP, PCMg (10 μL), and PCMS (10 μL) for 24 h, treated chondrocytes were stained with DHE reagent (5 μM) for 50 min. The stained cells were then observed under a confocal microscope, and ROS levels were quantified through cell counting (TBHP (30 μM), PCMg (0.1 mg/mL), and PCMS (0.1 mg/mL)).

2.11. 5-Ethynyl-2′-Deoxyuridine (EDU) Staining

To evaluate chondrocyte proliferation under different treatments, EDU staining was performed. After different treatments (10 μL of PCMg, 10 μL of PCMS), chondrocytes were washed three times with PBS and stained with the EDU reagent for 30 min. The stained cells were subsequently observed under a microscope to assess cell proliferation (TBHP (30 μM), PCMg (0.1 mg/mL), PCMS (0.1 mg/mL)).

2.12. Histological Assessment

Mouse knee joints were fixed overnight in 4% PFA, followed by decalcification in 10% EDTA (pH 7.4) for one month with weekly solution changes. After dehydration and paraffin embedding, sagittal sections (4 μm) were prepared for histological analysis. Tissue morphology was evaluated using hematoxylin and eosin (H&E), toluidine blue (TB), and Safranin O-fast green (SO) staining. Two blinded investigators independently assessed cartilage degeneration according to OARSI guidelines. Stained sections were imaged by light microscopy and were quantified using ImageJ software.

2.13. OA Model

All operations involving animal experiments were conducted in accordance with the Animal Care and Use Committee of Zhejiang University (Ethics Number: Year 2025 No. 94). Sixty 10 week old male C57BL/6 wild-type mice were obtained from the Chinese Academy of Sciences Animal Center (Shanghai, China). The mice were randomly assigned to three groups: sham, DMM, and PCMS (n = 5 per group). The OA model was established through surgical destabilization of the medial meniscus (DMM) under 2% pentobarbital anesthesia. In the DMM group, the right knee joint was exposed via medial parapatellar arthrotomy. Following identification of the patellar tendon, the medial collateral ligament was transected to allow for complete excision of the medial meniscus under aseptic conditions. This procedure induces mechanical instability, leading to progressive cartilage degeneration. Sham-operated controls underwent identical surgical exposure without meniscal resection. Postoperatively, DMM mice received daily intraperitoneal injections of PCMS (5 mg/kg every 3 days) for 8 weeks. Terminal procedures were performed under 10% chloral hydrate euthanasia, with subsequent knee joint harvesting for histological and molecular analyses.

2.14. Degradation of Nanoparticles In Vivo

The biodistribution of the materials was studied by using 15 DMM mouse models. A 10 μL injection of Cy5-labeled PCMS was administered into the knee joint, and the biodistribution was monitored on days 1, 3, 5, 7, and 14 using an intrabody imaging device.

2.15. Statistical Analysis

All experiments were repeated at least three times. Data are expressed as the mean ± standard deviation. One-way analysis of variance (ANOVA) followed by Tukey’s test was performed using SPSS 23.0 to compare different treatments in cells and tissues. Nonparametric data (OARSI scores) were analyzed using the Kruskal–Wallis H test.

3. Results

3.1. Characterization of PCMg and PCMS NPs

The preparation process of PCMS is presented in Figure A. Initially, Mg²⁺ was chelated with PDA and CC, which were then loaded into the hydrophilic end of PF127. Following this, TMB and NH₄OH were added, and the mixture was washed with ethanol to obtain PCMg. SS31 was subsequently loaded onto the surface and within the mesopores of PCMg through electrostatic interactions, resulting in the formation of PCMS. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed that, similar to mesopore polydopamine (MPDA), PCMg exhibited a complete spherical shape while retaining mesopores (Figure B,C). Results demonstrated that PCMg had a size of 141 ± 20.11 nm, while its hydrated particle size increased to 185 ± 2.78 nm, with a polydispersity index (PDI) of 0.25 ± 0.32 (Figure D–F). Upon the PCMg was immersed in PBS, the release profile of CC was assessed, showing a fast release in the first 30 h, after which the release rate decreased but continued over time. This controlled release is particularly beneficial for the absorption of CC by chondrocytes in the pathological environment of OA (Figure G). In addition, the encapsulation efficiency and drug loading were also measured. Encapsulation efficiency was 85.12%, and drug loading capacity was 15.55%. To enhance the targeted release of CC from PCMg, SS31 was incorporated onto the surface of PCMg via electrostatic interactions. TEM results confirmed the successful encapsulation of SS31 on PCMg, with Mg²⁺ remaining stably present in PCMS (Figure H). Zeta potential measurements further validated this finding, as the potential of PCMS was increased compared to that of PCMg (Figure I). Additionally, the degradation of PCMS was examined. TEM images on Day 1 showed that PCMS retained its spherical shape, whereas by Day 3, it had disintegrated into irregular aggregates, some of which remained encapsulated by SS31, confirming the stabilizing effect of SS31 on PCMS. On Day 1, the weight of PCMS had degraded to 62.12%, and by Day 3, it had further degraded to 49.42% (Figure J,K). These indicated that Mg²⁺ and citric acid-doped PCMg were successfully constructed and attached to the surface of PCMg through the electrostatic interaction.

1.

Preparation and characterization of MPDA, PCMg, and PMCS. (A) Preparation process of PCMS. (B) SEM and TEM images of MPDA. (C) TEM image of PCMg. (D) Size of MPDA and PCMg. (E) Hydrodynamic size of MPDA and PCMg. (F) PDI of MPDA and PCMg. (G) Cumulative release of CC. (H) TEM image of PCMS. (I) Zeta potential of SS31, MPDA, PCMg, and PCMS. (J) TEM image of PCMS immersed in PBS on day 1, day 2, and day 3. (K) Degraded ratio of PCMS. All values are presented as mean ± standard deviation (n = 3).

3.2. Biosafety and Biocompatibility of PCMS NPs

Chondrocyte viability following treatment with PCMg and PCMS was assessed by using the CCK-8 assay. The results indicated that after 1 and 3 days of treatment, the PCMg-treated and PCMS-treated groups did not affect cell viability compared to the control group (Figure A). CA/PI staining results showed that after 1 day of PCMg and PCMS treatment, chondrocytes predominantly exhibited green fluorescence, indicating viable cells, with only a small number of red fluorescence signals present, suggesting minimal cell death. Besides, there was no statistically significant difference between the PCMg and PCMS groups compared with the control group. These results suggest that PCMg and PCMS treatments did not significantly induce chondrocyte death (Figure B).

2.

Biosafety and biocompatibility of PCMS NPs. (A) CCK8 assay results of chondrocytes treated with PBS, PCMg, and PCMS for 1 day and 3 days. (B) Live/dead assay of PC12 cells treated with PCMg and PCMS. (C,D) Live/dead assay, EDU stain, and quantification of TBHP-stimulated chondrocytes treated with PCMg and PMCS. All values are presented as mean ± standard deviation (n = 3). *P < 0.05 and **P < 0.01. One-way analysis of variance (ANOVA) followed by Tukey’s test was used as the statistical test.

To assess the effects of PCMg and PCMS on cell viability under OS conditions, tert-butyl hydroperoxide (TBHP) was used to induce OS in chondrocytes. The results demonstrated a significant increase in dead cells following TBHP treatment, while both PCMg and PCMS effectively protected chondrocytes from TBHP-induced cell death. To further examine the impact of PCMg and PCMS on chondrocyte proliferation under OS, we performed EDU staining. TBHP treatment significantly reduced chondrocyte proliferation, and both PCMg and PCMS mitigated this inhibitory effect, enhancing the proliferation of TBHP-treated chondrocytes. Notably, PCMS showed a statistically more significant effect than PCMg (Figure C,D). Overall, these results indicated that PCMg and PCMS had no adverse impact on chondrocyte activity and could alleviate oxidative stress-induced chondrocyte death while promoting chondrocyte proliferation.

3.3. Antioxidant Capacity and Intracellular Localization of PCMS NPs In Vitro

To investigate the intracellular localization of PCMS, we evaluated its uptake efficiency by chondrocytes. PCMS-DIL was prepared by coincubating CM-DIL with PCMS, followed by coculturing with chondrocytes for 4 and 12 h. The cells were then fixed, stained with F-actin, and observed under a confocal microscope. At 4 h, red fluorescence signals appeared within the green fluorescence range, indicating that chondrocytes had internalized some PCMS. After 12 h, the red fluorescence signals significantly increased, suggesting an enhanced uptake of PCMS by chondrocytes. These results confirmed that PCMS was successfully taken up by chondrocytes and localized within them (Figure A,B). To further investigate PCMS’s ability to target mitochondria, PCMS-DIL was cocultured with chondrocytes for 4 h, followed by labeling with Mitotracker to identify mitochondria. Confocal imaging revealed colocalization of the green fluorescence signal (Mitotracker) with the red fluorescence signal, resulting in yellow fluorescence. Co-localization analysis confirmed that PCMS was present within the mitochondria, demonstrating its ability to target these organelles (Figure C,D).

3.

Antioxidant capacity and intracellular localization of PCMS NPs in vitro. (A) Distribution of PCMS after 4 h and 12 h (green = F-actin, red = PCMS). (B) Quantification of red fluorescence. (C,D) Quantification and co-localization image of mitochondrial and PCMS (green = mitochondrial, red = PCMS). (E) Effects of TBHP, PCMg, SS31, and PCMS on regulating the level of ROS in vitro. (F) Quantification of DCFH-DA levels. (G) Quantification of DHE levels. All values are presented as mean ± standard deviation (n = 3). *P < 0.05 and **P < 0.01. One-way analysis of variance (ANOVA) followed by Tukey’s test was used as the statistical test.

Additionally, DHE and DCFH-DA staining were used to assess PCMS’s capacity to clear ROS in vitro. After TBHP treatment, the green fluorescence signal in the cells increased, indicating elevated intracellular ROS levels. However, following treatment with PCMg, SS31, and PCMS, the green fluorescence signal decreased. Among these, the most significant reduction in green fluorescence was observed in TBHP-treated chondrocytes after PCMS treatment, indicating that PCMS was more effective than PCMg and SS31 in mitigating TBHP-induced ROS accumulation (Figure E–G).

3.4. Effect of PCMS NPs on Mitochondrial Function

JC-1, a fluorescent probe, is commonly used to assess the mitochondrial membrane potential (MMP). At high MMP, JC-1 forms aggregates (J-aggregates) that emit red fluorescence, whereas at lower membrane potentials, JC-1 exists as monomers and emits green fluorescence. Chondrocytes treated with TBHP and stained with JC-1 showed a significant decrease in red fluorescence and an increase in green fluorescence compared to those in the control group, indicating a reduction in mitochondrial membrane potential (MMP). The decreased red-to-green fluorescence ratio suggested that TBHP may induce mitochondrial dysfunction or early apoptosis. However, when chondrocytes were cotreated with TBHP and PCMg, SS31, or PCMS, the red fluorescence signal was enhanced and the green fluorescence signal weakened, indicating that these treatments helped rescue mitochondrial function disrupted by TBHP. Notably, PCMS demonstrated the most significant effect in mitigating TBHP-induced mitochondrial damage compared to PCMg and SS31 (Figure A,B). Intracellular calcium ion levels serve as another key indicator of mitochondrial function. After cells were treated with TBHP, PCMg, SS31, and PCMS, the treated cells were coincubated with the Fluo-4 AM probe. Results showed that TBHP increased the intracellular Ca²⁺ concentration. However, compared to the other groups, PCMS significantly reduced Ca²⁺ levels, suggesting an improvement in mitochondrial function (Figure C,D). Further assessments of ATP levels and mitochondrial ROS, using Mitosox, revealed that TBHP reduced ATP synthesis and increased mitochondrial ROS, while treatments with PCMg, SS31, and PCMS enhanced ATP production and reduced mitochondrial ROS levels. Among these, PCMS exhibited the most significant effect. These results demonstrate that PCMS alleviated the impact of oxidative stress on mitochondrial function, with its stronger effect compared to PCMg highlighting the critical role of targeting mitochondria in mitigating mitochondrial damage (Figure E,F).

4.

Effect of PCMS NPs on mitochondrial function in vitro. (A,B) Quantification and the image of JC-1 of different groups. (C,D) The levels of Ca²⁺ of different groups. (E) ATP levels of various drug-treated groups. (F) MitoSox levels of various drug-treated groups. All values are presented as mean ± standard deviation (n = 3). *P < 0.05 and **P < 0.01. One-way analysis of variance (ANOVA) followed by Tukey’s test was used as the statistical test.

3.5. Biocompatibility of PCMS NPs

PCMS was coincubated with Cy5 to produce PCMS-Cy5, which was subsequently injected into the joint cavity. In vivo imaging was performed at 1, 3, 5, 7, and 14 days postinjection. The results revealed that on day 5, PCMS remained predominantly within the joint cavity, with a noticeable decrease in signal intensity by day 7 and a near-complete disappearance of the signal by day 14. These results suggest that PCMS-Cy5 undergoes degradation in vivo (Figure A). Hemolysis assays were conducted to evaluate the biocompatibility of PCMS, and the results demonstrated that PCMS, PMCg, and SS31 all exhibited hemolysis rates below 5%, in line with internationally recognized biocompatibility safety standards (Figure B). To further assess the biocompatibility of PCMS, blood samples were collected from PCMS-treated mice after 8 weeks for analysis. The results showed no significant impact on liver and kidney function markers, including ALT, BUN, and creatine kinase, indicating that PCMS had no adverse effect on these parameters. These findings collectively confirm that PCMS is biocompatible and undergoes degradation in vivo (Figure C–I).

5.

Biocompatibility of PCMS NPs. (A) Image and quantification of PCMS in vivo. (B) Hemolysis test of PCMg, PCMS, SS31, and PBS. (C–I) Blood biochemical test of PCMS. All values are presented as mean ± standard deviation (n = 3). *P < 0.05 and **P < 0.01. t-test was used as the statistical test.

3.6. Effect of PCMS NPs on Alleviating OA Progression

The therapeutic effect of PCMS in delaying OA progression was assessed using histological staining combined with the OARSI scoring system. In H&E staining, the cartilage structure in the normal group appeared intact, while the injury group exhibited typical fissures and cell clusters. In contrast, the PCMS treatment group exhibited near-normal cartilage structure, indicating restoration of tissue integrity. Safranin O staining revealed uniform and intense proteoglycan staining in the normal group, whereas the injury group showed significant matrix loss. The treatment group significantly restored the proteoglycan content (Figure A–C). Quantitative analysis of toluidine blue staining confirmed that chondroitin sulfate levels were significantly lower in the injury group compared to the normal group, while PCMS treatment notably restored these levels. These results, consistent with the OARSI scoring criteria, confirm that PCMS effectively reverses the key pathological features of OA, restoring the histological appearance to near-normal levels (Figure D). The ROS levels in the joint fluid of the OA mice were detected with the ROS kit. The results showed that after DMM surgery, the ROS levels significantly increased, while PCMS effectively reduced the ROS levels (Figure E). These results collectively indicate that PCMS significantly delays the progression of OA.

6.

Effect of PCMS NPs on alleviating OA progression. (A–C) HE, S–O, and TB stains of the knee joint of different groups. (D) OARSI scores of different groups. (E) Relative fluorescence intensity of ROS in mice OA models. All values are presented as mean ± standard deviation (n = 5). *P < 0.05 and **P < 0.01. The Kruskal–Wallis H test was used as the statistical test.

4. Discussion

This study successfully synthesized a mesoporous NP capable of loading magnesium, CC, and DA. By encapsulation of SS31 into PCMg, a targeted delivery system (PCMS) was created, which enhanced the antioxidant and mitochondrial function-regulating properties of PCMg, demonstrating excellent biocompatibility and biosafety. In vivo experiments confirmed that PCMS had good biocompatibility and effectively delayed the progression of OA. In conclusion, this study developed a mesoporous drug delivery platform (PCMg) and demonstrated the therapeutic effect of PCMS, offering a promising strategy for OA treatment and a potential platform for drug delivery.

DA and CC were successfully loaded onto the hydrophilic end of PF127 via magnesium chelation, while the hydrophobic end was occupied by TMB. Under alkaline conditions, DA polymerized, facilitating the self-assembly of PF127 micelles into NPs. After the TMB was washed away, the mesoporous NP, PCMg, was obtained. This platform can accommodate multiple drugs, providing versatile strategies for drug delivery. Compared with direct injection of hydrophilic drugs, loading them onto PCMg enables better control of drug release and enhances delivery efficiency. Furthermore, magnesium as a chelating agent can be replaced for other disease models, facilitating the creation of mixed multidrug mesoporous NP delivery platforms, offering a promising strategy for drug delivery synthesis.

SS31 is a synthetic tetrapeptide with an outstanding mitochondrial targeting ability. Its core advantage lies in stabilizing the inner mitochondrial membrane by specifically binding cardiolipin, thereby reducing the generation of ROS from the source rather than simply eliminating them, improving ATP synthesis, and inhibiting apoptosis. However, as a bioactive factor, the impact of repeated injections in vivo on immunogenicity is lacking in research, which is crucial for the expansion of SS31’s application.

Mg²⁺ and CC play significant roles in the treatment of OA. As an essential intracellular cation, Mg²⁺ exerts anti-inflammatory effects mainly by inhibiting the NF-κB inflammatory pathway, thereby reducing the production of pro-inflammatory cytokines such as IL-1β and TNF-α. They are also crucial for maintaining the normal function of chondrocytes and delaying cartilage degradation. CC, as an alkaline buffer salt, primarily acts on the extracellular environment. It neutralizes the acidic substances produced by inflammation and metabolism in the joint cavity and around chondrocytes, improving the acidic microenvironment of the joint. This helps reduce the damage to cartilage caused by acidic conditions and indirectly suppresses inflammatory responses. CC creates a healthier external environment for cells, while Mg²⁺ enhances the physiological functions within cells, jointly alleviating the pathological process of OA through multiple pathways.

The cell membrane surface is generally negatively charged, which has a negative impact on the endocytosis of negatively charged nanoparticles. However, the charge of nanoparticles is not constant during in vitro and in vivo cultivation. The formation of the protein corona has both advantages and disadvantages for the endocytosis of nanoparticles. Research shows that specific proteins in serum (such as apolipoproteins A–I) will adsorb onto the negatively charged surface of silica nanoparticles to form a protein corona. This protein corona not only does not inhibit uptake but also significantly promotes the endocytosis of nanoparticles in hepatocytes (expressing SR-BI) by being recognized by the scavenger receptor SR-BI. In addition, under conditions with serum (with a protein corona), the intracellular uptake of nanoparticles is significantly higher than that in serum-free conditions. They further confirmed that the formation of the protein corona alters the mechanism of the interaction between nanoparticles and cells. These indicate that the formation of the protein corona helps to promote the interaction between nanoparticles and cells, thereby facilitating the endocytosis of the nanoparticles. Therefore, the formation of the protein corona is a potential mechanism for the endocytosis of PCMS by the cells. However, the endocytosis mechanism of negatively charged nanoparticles is related not only to the protein corona but also to the specific ligand–receptor binding. In conclusion, the endocytosis mechanism of PCMS by cells remains to be further studied.

Dopamine has the function of antioxidation and removal of reactive oxygen species (ROS). By doping CC and Mg into mesoporous dopamine, we obtained PCMg with a stronger antioxidation and ROS removal function. In addition, PCMS combined with PCMg through electrostatic interaction further enhances the ROS removal function of PCMg and endows PCMg with the function of targeting mitochondria. This ROS removal enhanced delivery platform shows strong effects both in vivo and in vitro and effectively alleviates the progression of OA, which is a potential and feasible treatment strategy.

Additionally, the targeting properties of PCMS confirmed the importance of targeting in drug delivery systems. However, the study did not address the in vivo metabolism of PCMS NPs. While biosafety was assessed in animal samples, multiple time points were not considered, which would be critical for a thorough evaluation. Moreover, although combined treatment with multiple drugs is a common clinical approach for complex pathological conditions, the study primarily demonstrated the therapeutic effect of the synthesized NPs with DA and CC as cotherapeutic agents, without exploring potential interactions between them. Drug interactions are an essential aspect of biosafety. While biocompatibility and biosafety tests indicated no significant effects on chondrocytes or mice in vitro and in vivo, the study did not examine potential impacts on mental health, such as anxiety or sleep disorders. In addition, this study lacks long-term safety verification, and the safety impact on female mice is unclear. More importantly, the potential mechanism of action of PCMS is not clear. What’s more, the lack of data on the long-term stability of PCMS in physiological conditions will restrict the application of PCMS. Therefore, further research into the safety of these NPs is necessary to support their potential use as a clinical treatment for patients.

Despite limitations in biosafety and drug interaction research, this study demonstrated that PCMS effectively delayed OA progression by reducing ROS and regulating mitochondrial function while providing a feasible strategy for developing drug delivery platforms.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c07948.

• Effect of PCMS on chondrocytes (PDF)

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C.S. and J.Y. have contributed equally to this work. C.S. and J.Y. finished experiment plans and manuscript. H.X., J.L., and K.J. finished the data processing. All author revised the manuscript. All authors have approved publishing the final manuscript.

The authors declare no competing financial interest.