Exploring the translational potential of PLGA nanoparticles for intra-articular rapamycin delivery in osteoarthritis therapy

Abstract

Osteoarthritis (OA) is a prevalent joint disease that affects all the tissues within the joint and currently lacks disease-modifying treatments in clinical practice. Despite the potential of rapamycin for OA disease alleviation, its clinical application is hindered by the challenge of achieving therapeutic concentrations, which necessitates multiple injections per week. To address this issue, rapamycin was loaded into poly(lactic-co-glycolic acid) nanoparticles (RNPs), which are nontoxic, have a high encapsulation efficiency and exhibit sustained release properties for OA treatment. The RNPs were found to promote chondrogenic differentiation of ATDC5 cells and prevent senescence caused by oxidative stress in primary mouse articular chondrocytes. Moreover, RNPs were capable to alleviate metabolism homeostatic imbalance of primary mouse articular chondrocytes in both monolayer and 3D cultures under inflammatory or oxidative stress. In the mouse destabilization of the medial meniscus (DMM) model, intra-articular injection of RNPs effectively mitigated joint cartilage destruction, osteophyte formation, chondrocytes hypertrophy, synovial inflammation, and pain. Our study demonstrates the feasibility of using RNPs as a potential clinically translational therapy to prevent the progression of post-traumatic OA.

Keywords: Intra-articular injection; Osteoarthritis therapy; PLGA nanoparticles; Rapamycin; mTORC1.

Fig. 1

Preparation and characterization of RNPs and RMPs. (A) Average diameter of RNPs with different PVA concentrations measured by dynamic light scattering. (B) PDI of RNPs with different PVA concentrations. (C) Zeta potential of RNPs with different PVA concentrations. (D) Encapsulation efficiency of RNPs with different PVA concentrations measured by HPLC. (E) Encapsulation efficiency of RNPs with different initial drug-to-material ratios measured by HPLC. (F) Average diameter of RNPs with different initial drug-to-material ratios measured by dynamic light scattering. (G) PDI of RNPs with different initial drug-to-material ratios. (H) Scanning electron micrograph of RNPs, Scale Bar 1 μm. (I-K) Average diameter, encapsulation efficiency, PDI of RNPs and RMPs (0.5% PVA concentration, drug-to-material ratio of 1:10). (L) Quantification of in vitro release profiles of Rapa, RMPs-12 μm, RMPs-2 μm and RNPs groups over 9 days. n = 3. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc analysis. Data are presented as means ± SD. ns, no significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 2

In vitro cellular uptake and biocompatibility. (A, B) Cellular uptake of RNPs and RMPs by primary articular chondrocytes measured by flow cytometry (n = 3). (C) Cellular uptake of RNPs and RMPs visualized by CLSM, Scale Bar 20 μm. (D) Cell viability of primary articular chondrocytes cultured with different concentrations of NPs measured by CCK8 assay (n = 4). (E) Cell viability of primary articular chondrocytes cultured with different concentrations of free rapamycin and RNPs measured by CCK8 assay at 48 h (n = 4). (F) Fluorescence images of Live/Dead staining on primary articular chondrocytes cultured with different concentrations of free rapamycin and RNPs, Scale Bar 500 μm. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc analysis. Data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

RNPs promote chondrogenic differentiation of ATDC5 cells. (A) Safranin O and Toluidine blue staining of ATDC5 cells cultured in chondrogenic medium for 7 days. (B-D) The expression of chondrogenic genes Col2a1, Acan and Sox9 in ATDC5 cells after 96 h of chondrogenic differentiation. (E-H) The expression of relative proteins (Col II, SOX9 and p-S6) of ATDC5 cells cultured in chondrogenic medium for 7 days. (I) Alcian blue staining of ATDC5 cells encapsulated in hydrogels for chondrogenic differentiation and cultured in chondrogenic medium for 14 days, Scale Bar 200 μm. n = 3 per group. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc analysis. Data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 4

RNPs alleviate IL-1β-induced metabolic homeostatic imbalance in chondrocytes. The expression levels of the catabolic marker Mmp13 (A) and the cartilage-specific marker Col2a1 (B) in primary articular chondrocytes, which were induced with IL-1β, except the Ctrl group, the other groups were treated with IL-1β for 24 h. (C-G) The expression of proteins (Col II, SOX9, MMP13 and p-S6) in primary articular chondrocytes, which were induced with IL-1β for 24 h. Toluidine blue (H) and Alcian blue (I) staining of primary articular chondrocytes, encapsulated in hydrogels and induced with IL-1β for 24 h, Scale Bar 200 μm. n = 3. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc analysis. Data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 5

RNPs prevent H₂O₂-induced metabolic homeostatic imbalance and prevent senescence in chondrocytes. (A) Alcian blue staining of primary articular chondrocytes, treated with 200 µM H₂O₂ for 24 h and maintained in 50 µM H₂O₂ for an additional week in the absence or presence of NPs, Rapamycin and RNPs, Scale Bar 500 μm. Except the Ctrl group, the other groups were treated with H₂O₂. (B, C) The expression of the catabolic marker Mmp13 (n = 3) and the cartilage-specific marker Col2a1 (n = 3) in primary articular chondrocytes induced with 200 µM H₂O₂. (D-H) The expression of relative proteins (Col II, SOX9, MMP13 and p-S6) in primary articular chondrocytes induced with 200 µM H₂O₂. (I) SA-β-gal staining of primary articular chondrocytes after H₂O₂-induction, Scale Bar 100 μm. (J) Quantification of SA-β-gal positive primary articular chondrocytes (n = 3). (K) The expression of the senescence marker P21 in primary articular chondrocytes, induced with 200 µM H₂O₂ (n = 3). Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc analysis. Data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 6

Joint retention of RNPs. (A) Representative fluorescence images of knee joints from OA mice over 21 days after intra-articular injection of free DID (DID), DID-labeled MPs (DID-MPs) and DID-labeled NPs (DID-NPs), respectively. (B) Quantitative analysis of the time course of relative fluorescence intensity within knee joints after intra-articular injection of free DID (DID), DID-labeled MPs (DID-MPs) and DID-labeled NPs (DID-NPs), respectively. (C) Quantitative analysis of the area under the curve (AUC) based on the relative fluorescence intensity profile in (B). n = 5 per group. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc analysis. Data are presented as means ± SD. ns, non-significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 7

RNPs alleviate knee osteoarthritis in vivo. (A) Micro-CT imaging of the morphological structure of the knee in mice at week 8 after surgery. Scale bar = 10 mm. (B) Osteophyte maturity (ROI zone) in the different groups of mice at week 8 after surgery, n = 8 mice per group. (C) Safranin-O/fast green staining of the mouse knee joint cartilage (hypertrophic chondrocytes were marked with black arrows), hematoxylin and eosin staining of cartilage and synovium at week 8 after surgery, Scale Bar 50 μm. (D, E) OA severity of knee joints at week 8 after surgery evaluated by OARSI score (D) and Mankin score (E), n = 8 mice per group. (F) Synovitis score, n = 8 mice per group. (G) Von Frey assay was performed at week 8 after surgery, n = 8 mice per group. (H) Immunohistochemical staining to detect the expression of cartilage specific indicators Col II (scale bar 50 μm) and the catabolic marker MMP13 (scale bar 20 μm) in mouse knee cartilage after sham or DMM surgery at week 8. Immunofluorescence staining to detect the expression of the cartilage specific indicator SOX9 (scale bar 20 μm). (I-K) Quantitative analysis of the staining images in (H), n = 3. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc analysis. Data are presented as means ± SD. ns, non-significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001