Development of Triamcinolone Acetonide-Loaded Nanoparticles for Inflammatory Knee Arthritis Treatment

Abstract

Introduction

Intra-articular corticosteroid injections (IACIs) provide an alternative treatment for inflammatory arthritis. Nevertheless, IACIs are limited by adverse effects, including injection-related pain, corticosteroid-induced chondrocyte toxicity, and progressive cartilage degeneration following prolonged use.

Methods

In this study, human serum albumin (HSA) was employed as a biocompatible nanocarrier for the encapsulation of triamcinolone acetonide (TA) to develop TA/HSA nanoparticles (TA/HSA NPs). The physicochemical characteristics and drug-release profile of the TA/HSA NPs were characterized. The in vitro cytocompatibility was evaluated in Hs68 fibroblasts, chondrocytes, and MG-63 osteoblast-like cells. The therapeutic efficacy of TA/HSA NPs was further evaluated in an inflammatory knee arthritis model following intra-articular administration.

Results

This system could significantly decrease the direct cytotoxicity of TA and achieve rapid and sustained release of TA in the joint cavity by reducing the crystallization properties of TA. Due to the sustained release of TA from TA/HSA NPs, the intra-articular injection of TA/HSA NPs could reduce inflammation-induced knee swelling, extend its anti-inflammatory effects, and thereby protect against cartilage erosion and condyle damage.

Conclusion

TA/HSA NPs represent a promising intra-articular drug delivery strategy for inflammatory arthritis. The formulation reduced the cytotoxicity of TA and provided sustained drug release in vitro, while demonstrating enhanced anti-inflammatory and cartilage-protective effects in an MIA-induced arthritis model.

Graphical Abstract

Introduction

Arthritis commonly refers to a group of inflammatory diseases that affect various joints in the body. Symptoms include joint redness, swelling, heat, pain, or stiffness, and in some cases, a reduced range of motion in the affected joints. Based on its onset and symptoms, arthritis can be classified into acute and chronic forms. During the acute phase, affected joints exhibit redness, swelling, heat, and pain, while the chronic phase is characterized by joint pain and stiffness. Clinically, there are over 100 types of arthritis, the most common of which are osteoarthritis (OA), rheumatoid arthritis, ankylosing spondylitis, and gouty arthritis.¹

OA is the most prevalent form of arthritis, impacting millions of individuals globally.^2,3 It is characterized by a gradual and progressive degradation of articular cartilage, which serves as a protective layer at the ends of bones within synovial joints. As the cartilage deteriorates over time, joint function becomes compromised, with the hands, knees, hips, and spine being the most frequently affected anatomical sites. OA is a chronic disease characterized by the progressive degeneration of articular cartilage accompanied by synovial inflammation. The main pathological changes include subchondral bone thinning, formation of periarticular bone outgrowths, meniscal alterations, ligament injuries, and joint capsule hypertrophy.^4,5 OA frequently occurs in the hip and knee joints, as these joints are most sensitive to mechanical stress. In such cases, the available treatment options are primarily symptomatic. Patients can take oral and topical pain relievers or nonsteroidal anti-inflammatory drugs to alleviate early OA symptoms.⁶ As the disease progresses to the intermediate stage, intra-articular corticosteroid injections (IACIs) and viscosupplementation with hyaluronic acid become the only treatment options for relieving pain and inflammation. IACIs can effectively and readily relieve OA-induced pain. The pain relief duration typically lasts around 2–4 weeks, depending on the corticosteroid type used. Although IACI is considered a relatively safe procedure, common side effects include post-injection flare pain (2–10%), skin atrophy at the injection site (1%), fat atrophy (1%), and facial flushing (<1%). Animal studies suggest that long-term IACI use may lead to cartilage loss. Therefore, current clinical guidelines recommend an injection interval of at least three months.^7–9

Triamcinolone acetonide (TA) is a long-acting synthetic glucocorticoid widely used for the treatment of inflammatory and autoimmune disorders because of its potent anti-inflammatory and immunosuppressive activities. Owing to its prolonged pharmacological action, TA is frequently administered by intra-articular injection for the management of osteoarthritis and other inflammatory joint diseases. The anti-inflammatory activity of TA is mediated, in general, through binding to cytosolic glucocorticoid receptors (GRs), followed by translocation of the TA–GR complex into the nucleus, where it can modulate the transcription of inflammation-related genes through interactions with glucocorticoid response elements (GREs). These well-established glucocorticoid-mediated mechanisms have been associated with suppression of pro-inflammatory cytokine production and modulation of inflammatory mediator pathways, thereby contributing to the therapeutic effects of TA.^10,11 Despite its established therapeutic efficacy, TA possesses extremely low aqueous solubility and is therefore commonly formulated as a crystalline suspension for intra-articular administration. These suspended drug crystals may induce post-injection pain and discomfort, while the therapeutic benefit is typically limited to approximately 2–4 weeks after a single injection. Moreover, repeated or prolonged administration of TA has been associated with both local and systemic adverse effects, including chondrocyte toxicity, progressive cartilage degeneration, adrenal insufficiency, hyperglycemia, hypertension, immunosuppression, muscle wasting, and an increased risk of infection.¹² These limitations restrict the long-term clinical application of TA and highlight the need for improved delivery strategies. Accordingly, the development of an optimized intra-articular TA delivery system capable of improving drug solubility, prolonging intra-articular retention, minimizing injection-associated pain, and reducing treatment-related toxicity represents a promising strategy to enhance the safety and therapeutic efficacy of corticosteroid therapy for inflammatory arthritis.

Nanomedicine is the medical application of nanotechnology and has garnered widespread attention due to its notable advantages over conventional drugs.¹³ These benefits include reduced dosage and administration frequency, enhanced drug bioavailability and therapeutic efficacy for targeted tissues, extended shelf-life, and enhanced patient compliance and convenience in clinical applications.¹⁴ Several nanomedicine and biomaterial-based platforms have been explored to prolong the intra-articular availability of TA. For example, dextran sulfate–TA conjugate nanoparticles (NPs) have been developed to target scavenger receptor A-expressing activated macrophages and attenuate inflammatory responses and cartilage damage in osteoarthritis.¹⁵ In addition, polymeric microsphere-based systems, including PLGA- and polyesteramide-based formulations, have been investigated to achieve sustained intra-articular TA release and prolonged therapeutic effects.^11,16–18 Extended-release TA formulations have also demonstrated prolonged synovial exposure and reduced systemic drug exposure compared with conventional TA suspensions.¹⁹ Despite these advances, most reported TA delivery systems rely on synthetic polymeric microspheres, polymeric NPs, or hydrogel-based depots, whereas the use of human serum albumin (HSA) as a protein-based nanocarrier for TA delivery remains comparatively less explored.

HSA, the predominant plasma protein in humans, has garnered significant attention as a drug delivery vehicle due to its favorable biological properties, including non-toxicity, non-immunogenicity, biocompatibility, and biodegradability.^20–22 HSA exhibits a high binding affinity for hydrophobic drugs, satisfactory tolerance for various pharmaceuticals, and no severe side effects.²¹ In recent years, multiple methodologies have been established for the fabrication of HSA-based NPs, encompassing approaches such as emulsification, desolvation, thermal gelation, nanospray drying, nanoprecipitation, and self-assembly techniques.²¹ Nonetheless, many of these approaches necessitate the use of hazardous organic solvents and high-cost instrumentation, posing limitations for large-scale and clinical applications.

This study utilized the previously developed lyophilization–hydration method to prepare a HSA-based nanocarrier as a TA delivery system.^23,24 Rather than introducing a new targeting mechanism, our strategy focuses on the combined advantages of HSA-based encapsulation, reduced direct cellular toxicity, and sustained drug release. Accordingly, TA/HSA NPs were developed and characterized, and their cytocompatibility, in vitro drug-release behavior, and therapeutic effects were evaluated in an MIA-induced rat knee arthritis model (Scheme 1).

Scheme 1.

Schematic illustration of the sustained release and therapeutic effects of TA/HSA NPs following intra-articular administration. Compared with free TA, which exhibits rapid burst release and higher cytotoxicity, TA/HSA NPs provide sustained TA release, reduce inflammation-induced knee swelling, prolong anti-inflammatory effects, and protect against cartilage erosion and condylar damage.

Experimental Section

Materials

TA, HSA (purity ≥ 96%), monosodium iodoacetate (MIA), dimethyl sulfoxide (DMSO), methanol, collagenase, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were supplied by Sigma-Aldrich (St. Louis, MO, USA).

Preparation of TA-Loaded NPs (TA/HSA NPs)

TA-loaded NPs were prepared as follows: 0.5 mg TA powder was dissolved in 1 mL DMSO solution and then added to 100 μL of 5 or 10 mg/mL HSA solution at the TA/HSA weight ratio of 1/1 or 1/2, respectively. The TA/HSA mixtures with different weight ratios were dispersed in 1 mL ddH₂O by ultrasonication after lyophilization to form TA/HSA(1/1) and TA/HSA(1/2) NPs. Finally, the prepared TA/HSA NPs were re-lyophilized for the following studies.

Characterization of TA/HSA NPs

The effect of different TA/HSA weight ratios on the particle size distribution of TA/HSA NPs in aqueous suspension was investigated using a Zetasizer Nano-ZS90 (Malvern Instruments Ltd., UK). The surface morphology of TA/HSA NPs was examined under a transmission electron microscope (TEM, Hitachi H-7500, Tokyo, Japan) after precipitation and drying of TA/HSA NPs with different TA/HSA weight ratios on carbon-coated copper grids. The loading efficiency of TA in NPs was investigated as follows: The re-lyophilized TA/HSA(1/1) or TA/HSA(1/2) NPs were suspended in methanol and subsequently filtered using a 0.22-μm Millex GP syringe filter unit (Merck Millipore Ltd., Tullagreen, Ireland) to separate particulate matter. An aliquot of 50 μL of the filtrate was subsequently analyzed on a high-performance liquid chromatography (HPLC) system (ACQUITY Arc System, Waters, USA). An acetonitrile/water solution (30:70, v/v) as the mobile phase and a Waters Symmetry^® C18 reversed-phase column (XBridge™) were used to perform the chromatographic separation. The flow rate was maintained at 1 mL/min for the quantitative determination of unencapsulated (free) TA. TA was detected at 254 nm using a UV detector. Herein, a calibration curve was constructed by analyzing standard TA solutions prepared in methanol at concentrations ranging from 0.1 to 10.0 μg/mL. The TA loading efficacy (LE) in the prepared NPs was calculated using the following equations:

The absorbance spectra of FITC-HSA conjugates (0.5 mg and 1.0 mg) in DMSO aqueous solution were obtained using an ELISA microplate reader (SpectraMax M2 Multi-Mode Microplate Reader, Molecular Devices, USA). The crystalline structures of the four samples (TA, HSA, TA/HSA(1/1) NPs, and TA/HSA(1/2) NPs) were characterized by powder X-ray diffraction (XRD) using a D2 PHASER X-ray diffractometer (Bruker AXS Inc., WI, USA) equipped with Cu Kα radiation (λ=1.5418 Å). The FTIR spectra of the TA, HSA, TA/HSA(1/1) NPs, and TA/HSA(1/2) NPs were acquired using a Nicolet™ Summit FTIR Spectrometer (Thermo Fisher Scientific, MA, USA) equipped with MicromATR. The storage stability of prepared TA/HSA(1/1) NPs or TA/HSA(1/2) NPs was evaluated by determining the particle size change of re-suspended TA/HSA(1/1) NPs or TA/HSA(1/2) NPs in aqueous solution at 0, 3, 5, 7, 10, 14, 21, and 28 days after lyophilization.²⁵

In vitro TA Release from TA/HSA NPs

The release profiles of TA from TA/HSA NPs were analyzed employing the sample and separate method.¹⁹ In brief, 2 mL of either TA/HSA nanoparticle suspension or free TA solution was mixed with 2 mL of phosphate-buffered saline (PBS, pH = 7.4), and then introduced into an Amicon Ultra centrifugal filter device (Millipore, Billerica, MA, USA). The assembly was maintained at 37°C under gentle agitation at 100 rpm to simulate physiological conditions. At predetermined time points (0, 1, 2, 3, 4, 6, 8, 10, 12, and 24 hours), the device was centrifuged at 2,500 rpm for 5 minutes, and a 0.5 mL aliquot of the filtrate was collected for analysis. Subsequently, an equal volume (0.5 mL) of fresh PBS was added to keep the total volume at 4 mL. The concentration of TA in each collected sample was quantified via HPLC.

In vitro Cytotoxicity of TA/HSA NPs

The Hs-68 human foreskin fibroblasts from ATCC (CRL-1635™) was cultured in 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin–amphotericin (PSA) supplemented Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, USA). The MG-63 human osteosarcoma cell line was obtained from ATCC (CRL-1427™) and maintained in 20% FBS and 1% PSA supplemented Minimum Essential Medium (MEM; Gibco, USA). Primary porcine chondrocytes were isolated from full-thickness articular cartilage obtained from adult porcine knee joints within 12 h after slaughter. The chondrocytes were isolated by enzymatically digestion of cartilage specimens in DMEM/F12 medium (Gibco, USA) containing 0.2% collagenase.²⁶ On the second day, the isolated porcine chondrocytes were pooled and still cultivated in DMEM/F12 medium supplemented with 10% FBS and 1% PSA. All cell types were maintained at 37°C in a humidified incubator with 5% CO₂, and the culture medium was refreshed every other day until confluence was reached.

5 × 10³ cells per well of Hs-68 cells, chondrocytes, or MG-63 cells were individually seeded into 96-well culture plates. Following a 24 h incubation period, the culture media was replaced with fresh media containing different TA concentrations of free TA or TA/HSA NPs (0, 0.125, 0.25, 0.5, and 1.0 mg/mL), respectively. Following an additional 24-h exposure period, cell viability was assessed utilizing the MTT assay. The viability of cells in each treatment group was quantified and presented as a percentage relative to the viability of the untreated control group.²⁷

In vivo OA Treatment

Establishment and Treatment of the OA Model

Twenty-four male SD rats (160–180 g) were obtained from BioLASCO Shortcourse (Taipei, Taiwan), and received care in accordance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals (8th edition). The experimental procedures were performed in accordance with protocols approved by the National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee (IACUC number: 20220045) and conducted in accordance with established ethical guidelines.

OA was induced in SD rats by intra-articular injection of MIA (2 mg in 100 μL of sterile normal saline) into the left knee joint under isoflurane anesthesia. Rats were anesthetized by induction with 5% isoflurane, followed by maintenance of anesthesia with 1–3% isoflurane during the procedure. The day of MIA injection was designated as day 0 of the experimental timeline. Four days post-induction (day 4), the SD rats were randomly assigned into four groups (six in each group): (a) normal group (no MIA, no treatment), (b) saline group (100 μL, normal saline), (c) TA group (0.6 mg TA/100 μL saline), and (d) TA/HSA NP group (0.6 mg TA/100 μL saline). Treatments were administered intra-articularly on days 4, 7, and 11 following OA induction. On day 25, blood samples were collected from the tail vein under isoflurane anesthesia. At the end of the experimental period, all animals were euthanized in a transparent chamber by gradual exposure to CO₂ at a displacement rate of approximately 50% of the chamber volume per minute for at least 5 min. Following CO₂ administration, animals were observed for an additional 2 min to confirm death before tissue collection and subsequent analyses.

Measurement of Knee Width and Body Weight

Knee swelling was evaluated by measuring the knee width adjacent to the tibial plateau using a digital vernier caliper on days 0, 4, 7, 11, and 25. The percentage change in knee width (Δ Width), relative to the baseline measurement (day 0), was calculated using the following equation:

where d0 and dt represent the knee widths measured at baseline (day 0) and at each designated time point, respectively.²⁸

Body weight was also recorded throughout the experimental period to evaluate the systemic safety and potential treatment-related toxicity. The percentage change in body weight (Δ Weight) was calculated according to the following equation:

where W0 and Wt denote the body weights measured at baseline (day 0) and at each corresponding time point, respectively.

Micro-CT and Histopathological Analysis

At day 25, the rats were sacrificed, the whole leg was fixed in 10% paraformaldehyde solution for 48 h, and images of the knee were acquired using a Bruker SkyScan 1276 micro-CT scanner system (Bruker, Kontich, Belgium). Three-dimensional (3D) image reconstruction was performed using Instarecon reconstruction software (Bruker-micro-CT, Kontich, Belgium) on the acquired image data to assess structural alterations in the subchondral bone. Moreover, hematoxylin and eosin (H&E) staining of the whole knee joint was performed to evaluate histopathological changes in the femur, tibia, and synovium. The severity of joint lesions was assessed using the Mankin scoring system, which evaluates key histopathological features, including cartilage structure, cellularity, and tidemark integrity, as well as chondrocyte abnormalities.²⁹

Determination of Inflammatory Factors in Serum

On the 25th day of the experiment, the blood samples were collected using BD Vacutainer^® Serum Tubes (BD, USA). The collected blood was centrifuged at 1500 rpm for 15 minutes to separate the serum. Serum concentrations of IL-1β, IL-6, and TNF-α were subsequently quantified using ELISA kits specific to each cytokine (Invitrogen, Thermo Fisher Scientific Inc., USA), in accordance with the manufacturer’s instructions.

Statistical Analysis

All data were expressed as the mean ± standard deviation (SD). A one-way ANOVA was performed separately at each time point to compare the different treatment groups, and a p-value less than 0.05 was considered indicative of statistical significance.

Results and Discussion

Characterization of TA/HSA NPs

TA, a synthetic glucocorticoid, binds to specific cytosolic glucocorticoid receptors, which then translocate to the nucleus. There, the receptor-ligand complex interacts with glucocorticoid response elements on DNA, leading to the modulation of gene expression. This interaction promotes the synthesis of anti-inflammatory proteins while suppressing the production of pro-inflammatory mediators.¹¹ TA can be used topically to treat various skin diseases, arthritis, and allergic rhinitis. Owing to its low solubility in water, the direct intra-articular injection of TA can prolong its action time, but it can also easily cause pain in the affected area. Herein, the hydrophobic TA molecules could interact with the hydrophobic functional groups of HSA molecules and be incorporated into HSA-based NPs. The average particle sizes of TA/HSA(1/1) and TA/HSA(1/2) NPs were 378 ± 11.0 nm and 384 ± 11.2 nm, respectively. The surface charges of TA/HSA(1/1) and TA/HSA(1/2) NPs were ‒29.9 ± 0.54 mV and ‒31.0 ± 2.57 mV, respectively. Although the TA/HSA(1/2) NPs exhibited a larger particle size than TA/HSA(1/1) NPs, the TA/HSA(1/2) NPs displayed a narrower size distribution than TA/HSA(1/1) NPs (Figure 1a). TEM images show that the TA/HSA(1/1) NPs displayed an irregular morphology, but the TA/HSA(1/2) NPs exhibited a solid morphology with well-defined margins (Figure 1b). Specifically, the characteristic FTIR bands of HSA at 1618 and 1662 cm⁻¹ are mainly associated with intermolecular β-sheet and β-turn structures, respectively.³⁰ The higher intensities of these two bands observed for TA/HSA(1/1) NPs compared with TA/HSA(1/2) NPs suggest greater alterations in the secondary structure of HSA in the 1/1 formulation (Figure 1c). In addition, 0.5 mg HSA exhibited an approximately 10-nm red shift in the FITC-HSA spectrum compared with 1.0 mg HSA (Figure 1d), suggesting a greater tendency toward HSA intermolecular association and aggregation under the tested conditions. This observation might be related to the preferential solvation behavior of HSA in the 90.9% DMSO aqueous environment, in which lower HSA concentrations may allow greater solvent–protein interactions and conformational perturbation, potentially exposing hydrophobic domains and promoting intermolecular association. At higher HSA concentrations, increased molecular crowding may restrict protein diffusion and reduce the formation of large aggregates.³¹ These structural effects might contribute to the broader particle size distribution and less uniform morphology observed for TA/HSA(1/1) NPs. In contrast, TA/HSA(1/2) NPs exhibited a narrower size distribution and more uniform morphology.

Figure 1.

(a) Particle size distributions of TA/HSA NPs with different TA/HSA ratios. (b) TEM images of TA/HSA(1/1) and TA/HSA(1/2) NPs. Samples were negatively stained with 2% uranyl acetate prior to imaging. Scale bar = 200 nm. (c) FTIR spectra of HSA, TA/HSA(1/1), and TA/HSA(1/2) NPs. (d) Absorption spectra of FITC-HSA in DMSO aqueous solution (90.9%, v/v).

TA is a hydrophobic and white crystalline powder, and its state in the prepared NPs could be investigated by comparing the difference in XRD patterns of TA, HSA, and prepared NPs. As illustrated in Figure 2a, the diffraction pattern of TA exhibited distinct and sharp peaks within the 2θ range of 10–30°, indicating its strong crystallization. But the intensity of peaks decreased dramatically when TA was incorporated into the prepared NPs, especially in the TA/HSA(1/2) NPs, suggesting a significant decrease in the crystalline nature of TA within the nanoparticle matrix. These observations indicate that the reduced aggregation of HSA molecules during the preparation of TA/HSA(1/2) NPs led to more TA molecules interacting with the hydrophobic groups of HSA, resulting in their successful incorporation into the TA/HSA(1/2) NPs. The LEs of TA in TA/HSA(1/1) NPs and TA/HSA(1/2) NPs were 50.9 ± 0.60% and 53.4 ± 0.79%, respectively. Because crystalline drugs in an amorphous state or as small crystals uniformly distributing in the prepared NPs may enhance their dissolution rate, thereby potentially improving bioavailability in subsequent clinical applications.³² Therefore, the presence of TA molecules in an amorphous state or as finely dispersed microcrystals within the TA/HSA(1/2) NPs should reduce the pain caused by insoluble suspended TA particles after intra-articular injection.

Figure 2.

(a) X-ray diffraction (XRD) diagrams of TA, HSA, TA/HSA(1/1), and TA/HSA(1/2) NPs. (b) FTIR spectra of TA, HSA, TA/HSA(1/1), and TA/HSA(1/2) NPs. (c) The storage stability of TA/HSA(1/1) or TA/HSA(1/2) NPs was determined by dissolving the re-lyophilized TA/HSA(1/1) or TA/HSA(1/2) NPs in 1 mL H₂O at 0, 1, 5, 7, 10, 14, 21, and 28 days.

Figure 2b shows the FTIR spectra of the TA, HSA, and prepared NPs. The TA, TA/HSA(1/1), and TA/HSA(1/2) NPs presented two typical infrared absorbance bands around 1700 cm⁻¹ attributed to the C–O aliphatic ketone and at 1056 cm⁻¹ corresponded to the C–F stretching vibration of the halogenated ring present in TA molecules.^33,34 In addition, the intensity of the absorption bands displayed a negative correlation with the addition of HSA content in the prepared NPs. This reconfirmed that more HSA content could interact with TA molecules, causing them to distribute uniformly in the prepared NPs in an amorphous state or as small crystals, resulting in a reduced intensity of absorption bands due to the covering by HSA. As shown in Figure 2c, the prepared TA/HSA(1/2) NPs exhibited more storage stability than the TA/HSA(1/1) NPs. As TA solubility in water seemed low, the content of un-incorporated TA molecules may affect NP suspension stability in water. The TA/HSA(1/1) NPs displayed more unloaded TA than TA/HSA(1/2) NPs, causing low storage stability. The prepared TA/HSA(1/2) NPs exhibited better characteristics than TA/HSA(1/1) NPs and were thus used for further study.

In vitro TA Release from TA/HSA NPs

TA is a hydrophobic molecule with a low water solubility (approximately 0.1 mg/mL).³⁵ Its low water solubility affects its pharmacokinetics, pharmacodynamics, and several other parameters, such as body distribution, protein binding, and absorption.³⁶ Herein, the preparation process of the TA/HSA NPs resulted in TA molecules existing in amorphous or small crystalline states within the NPs, potentially enhancing their release rate and efficacy. As shown in Figure 3, the release of TA was slow and continuous. The in vitro accumulated release of TA from TA/HSA NPs was found to be 18.4% after a 24-h incubation period in PBS, which significantly exceeded that of the free TA crystal powder (8.9%). Owing to the amorphous or nanocrystalline state of TA within the nanoparticle matrix, TA/HSA NPs might facilitate a more rapid and sustained release of TA to prolong the action time of the anti-inflammatory response on arthritis and reduce the pain in the affected area.

Figure 3.

The release profile of TA from free TA or TA/HSA NPs in PBS solution.

In vitro Cytotoxicity of TA/HSA NPs

Several reports have shown TA with chemical toxicity on retinal pigment epithelial cells,^37,38 phototoxicity on erythrocytes in vitro, and weak photosensitizing properties in vivo,³⁹ and its toxicity is dose- and time-dependent. As shown in Figure 4, the cell viability of Hs-68, chondrocyte, or MG-63 cells remained at 67%, 60%, and 70%, respectively, after 24-h incubation with free TA. This outcome was independent of the concentration of free TA and differed from the results reported in the literature.^37,38 Because TA-induced cytotoxicity occurs when TA crystals contact closely with cellular membrane and correlates with the number and size of TA crystal aggregates,⁴⁰ the present study introduced no statistically significant differences in the number or size of TA crystals at concentrations ranging from 0.125 to 1 mg/mL, resulting in consistent toxicity levels on Hs-68, chondrocyte, or MG-63 cells. Although the cytotoxic effect of TA/HSA NPs toward Hs-68, chondrocyte, and MG-63 cells was found to be dose-dependent, cellular viability remained above 80%, suggesting that the prepared TA/HSA NPs had high biocompatibility. This indicates their potential suitability as a nanomedicine for intra-articular injections in OA treatment.

Figure 4.

In vitro cytotoxic effects of free TA and TA/HSA NPs on (a) Hs-68, (b) chondrocyte, and (c) MG-63 cells. *: p < 0.05.

In vivo OA Treatment

Because the pathological and pharmacological characteristics of the MIA-induced rat OA model are similar to those of human OA, it is one of the most established and widely utilized preclinical models.⁴¹ The OA model could be successfully established on the 4th day following intra-articular injection of MIA. The width of the knee joint, articular cartilage destruction, body weight, and serum levels of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) were assessed after three intra-articular injections of free TA and TA/HSA NPs to explore their treatment efficacy on OA (Figure 5a).

Figure 5.

In vivo therapeutic effects of free TA and TA/HSA NPs on OA induced by MIA. (a) Schematic representation of the MIA-induced rat model establishment and treatment protocol. (b) Changes in knee joint width across groups during treatment (n = 6). (c) Changes in body weight across groups during treatment (n = 6). *: p < 0.05.

The knee width and body weight of the rats on days 0, 4, 7, 11, and 25 were recorded and shown in Figure 5b. The knee width of normal rats continued to increase with rat growth during the experiment, while that of saline-treated rats first increased to the highest at 4–7 days of post-MIA induction, then decreased, and then increased as the rat grew. The initial increase in knee width was mainly due to joint swelling caused by MIA-induced arthritis. Later, as the autologous repair decreased the inflammation, the joint swelling disappeared, and the knee width increased as the rat grew. In contrast, intra-articular administration of either free TA or TA/HSA NPs markedly alleviated arthritis progression by suppressing the inflammatory response, resulting in a significantly greater reduction in knee swelling than that observed in the saline-treated group on days 7, 11, and 25 (Figure 5b and Figure S1a). Furthermore, changes in body weight were monitored to evaluate the systemic effects of treatment (Figure 5c and Figure S1b). Compared with the saline-treated group, rats receiving free TA or TA/HSA NPs exhibited an initial decrease in body weight during the treatment period, followed by gradual recovery after treatment cessation. Notably, the reduction in body weight was significantly greater in the free TA-treated group than in the TA/HSA NP-treated group. Because skeletal muscle makes up approximately 45% of total body mass in rats, the observed reduction in body weight may primarily result from muscle tissue loss. Glucocorticoid administration, such as TA, has been reported to reduce amino acid uptake by muscle cells, inhibit the incorporation of amino acids into proteins, and enhance proteolytic activity within muscle tissue.⁴² Therefore, TA administration might lead to significant atrophy of the gastrocnemius muscle and overall weight loss by lowering intracellular levels of free amino acids, decreasing the ability to incorporate amino acids into protein, activating myofibrillar proteases in skeletal muscle, and binding to two distinct cytoplasmic proteins in muscle to induce muscle wasting.^43,44 And HSA nanoparticle encapsulation alleviated the systemic adverse effects associated with TA administration while maintaining its therapeutic efficacy.

Through micro-CT scanning images (Figure 6a), irregular joint surfaces, severe cartilage erosion, subchondral bone sclerosis, and extensive destruction of the femoral condyle were observed in the saline-treated group. However, in the intra-articular injection of the free TA group, only irregular small cartilage erosions and mild femoral condyle damage were noted. Compared to the intra-articular injection of free TA, the intra-articular injection of TA/HSA NPs provided better protective effects. Histopathological assessment (Figure 6b) was performed on the knee joints to determine the extent of cartilage degeneration and synovial hyperplasia using the Mankin scoring system (Table 1).²⁹ In the normal group, the femur and tibia revealed surface irregularities of articular cartilage and minimal chondrocyte necrosis due to the intra-articular injection of saline that induced mild inflammation, but no significant histopathological changes were noted in the synovium. Regardless of intra-articular saline or TA injections, histopathological results revealed complete destruction of the femur, severe chondrocyte necrosis, and blood vessel penetration through the tidemark. Similarly, the tibia showed complete destruction, moderate chondrocyte necrosis, and blood vessel penetration through the tidemark. In addition, the knee joint presented slight synovial hyperplasia, slight inflammation, and slight granulation tissue formation in the synovium and periarticular soft tissues. For the TA/HSA NP-treated group, the articular cartilage of the femur and tibia only revealed clefts to the transitional zone and severe chondrocyte necrosis, and the knee joint presented slight synovial hyperplasia, slight inflammation, and slight granulation tissue formation in the synovium and periarticular soft tissues. Therefore, the intra-articular injection of TA/HSA NPs might effectively reduce joint tissue damage caused by inflammation by lowering the direct cytotoxicity of TA and achieving rapid and sustained TA release.

Figure 6.

(a) Micro-CT images of the knees in OA rats after treatment with saline, free TA, and TA/HSA NPs. (b) H&E staining of rat femur, tibia, and synovium in each treated group. Scale bar = 200 μm in the femur and tibia images. Scale bar = 50 μm in synovium images.

Table 1.

Mankin’s Score Results for the Cartilage Structure of the Rats After Intra-Articular Administration of Saline, Free TA, and TA/HSA NPs

	Normal	Saline	TA	TA/HSA NPs
Femur structurea	2.2	5.6	6.0	3.2
Femur lesion severity of structure b	1	1.0	1.0	1.0
Tibia structurea	1.8	4.4	4.2	3.2
Tibia lesion severity of structure b	1	1.6	1.0	1.4
Pannus (granulation tissue) formation b	1	1.6	1.8	1.6
Mononuclear cell and neutrophil infiltration b	1	1.6	1.8	1.8
Synovial hyperplasia b	1	1.6	1.6	1.8

Notes: ^a0: normal; 1: surface irregularities; 2: pannus (granulation tissue) and surface irregularities; 3: clefts to transitional zone; 4: clefts to radial zone; 5: clefts to calcified cartilage; 6: complete disorganization.^b0: normal; 1: minimal (≤25%); 2: slight (26–50%); 3: moderate (51–75%); 4: severe (76–100%).

The arthritic joint microenvironment is characterized by the presence of numerous pro-inflammatory cytokines, chemokines, alarmins, and other inflammatory mediators, including TNF-α, IL-1β, IL-6, IL-17, and CCL19, which collectively contribute to synovial inflammation and cartilage degradation.^45–47 Among these mediators, TNF-α and IL-1β are recognized as important contributors to OA-associated inflammation and cartilage destruction by promoting inflammatory signaling and the expression of matrix-degrading enzymes.^48–52 IL-6 is also involved in the inflammatory and catabolic responses associated with OA and can be induced downstream of IL-1β and TNF-α signaling.^53,54 Therefore, changes in these circulating cytokines may provide an indication of the overall inflammatory status associated with experimental arthritis. As shown in Figure 7, the content of IL-1β, IL- 6, and TNF-α were elevated in saline-treated rats compared to normal rats, indicating an inflammatory response associated with OA. Intra-articular administration of free TA resulted in a reduction of the levels of pro-inflammatory cytokines IL-1β and IL-6. Notably, treatment with TA/HSA NPs led to a more pronounced decrease in IL-1β and IL-6 levels relative to the saline-treated group. Because IL-6 is an important mediator of the acute-phase inflammatory response,⁵⁵ the significant reduction in circulating IL-1β and IL-6 following TA/HSA NP treatment indicates that TA remained pharmacologically active after encapsulation and was released in vivo to exert its anti-inflammatory activity. These findings are consistent with the histopathological observations, which showed improved joint tissue morphology following treatment with TA/HSA NPs, further supporting their therapeutic efficacy against MIA-induced joint inflammation and tissue damage.

Figure 7.

The levels of (a) IL-1β (b) IL-6 and (c) TNF-α in serum. (n = 6; *p < 0.05).

Several TA nanocarrier systems have previously been reported;^11,15–19 therefore, the novelty of the present study does not rely solely on the use of nanoparticles for TA delivery. Rather, this study demonstrates an HSA-based formulation strategy designed to improve the biocompatibility of TA and modulate its release following intra-articular administration. In the present study, TA/HSA NPs reduced TA-associated cytotoxicity, exhibited sustained TA release in vitro, and improved anti-inflammatory and cartilage-protective outcomes in the MIA-induced arthritis model. These findings support the potential of HSA-based encapsulation as an alternative strategy for intra-articular TA delivery. Although the inflammatory cytokines measured in this study were determined from serum samples and may not fully reflect the local inflammatory microenvironment within the joint cavity. And the cartilage-degrading mediators, such as MMP-13 and ADAMTS-5, were not directly evaluated in joint tissues. Thus, the reductions in circulating IL-1β, IL-6, and TNF-α, together with the observed histopathological improvements, provide supportive but not definitive evidence for the local anti-inflammatory and chondroprotective effects of TA/HSA NPs. Future studies incorporating synovial fluid cytokine analysis, joint-tissue assessment of cartilage-degrading enzymes, and direct evaluation of intra-articular drug retention may be important to further elucidate the local molecular mechanisms and pharmacokinetic behavior underlying the therapeutic effects of TA/HSA NPs.

Conclusion

Intra-articular corticosteroid injection remains an effective treatment option for relieving inflammation associated with OA; however, its therapeutic benefits may be limited by drug-associated adverse effects and insufficient intra-articular retention. In this study, TA was encapsulated within HSA nanoparticles (TA/HSA NPs) to improve the formulation properties and provide sustained drug release. Compared with free TA, TA/HSA NPs demonstrated reduced cytotoxicity in vitro and favorable therapeutic effects in an MIA-induced rat OA model, as evidenced by reduced joint swelling, decreased serum IL-1β and IL-6 levels, and improved histopathological and micro-CT outcomes. These findings suggest that HSA-based encapsulation may improve the therapeutic profile of intra-articularly administered TA. However, functional outcomes and local molecular mechanisms were not directly evaluated in the present study. Further studies incorporating behavioral assessments and local joint biomarkers are therefore warranted to validate the functional benefits and chondroprotective mechanisms of TA/HSA NPs. Overall, TA/HSA NPs represent a promising platform for sustained intra-articular TA delivery and warrant further investigation as a potential strategy for improving the treatment of OA.

Funding Statement

This work was supported by the National Science and Technology Council (113-2314-B-002 −221- and 114-2314-B-002-132-).

Data Sharing Statement

The data that support the findings of this study are available from the corresponding author, C.H. Chang, upon reasonable request.

Disclosure

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.