Cartilage-Penetrating Framework Nucleic Acid Nanoparticles Ameliorate Osteoarthritis by Promoting Drug Delivery and Chondrocyte Uptake

Abstract

Osteoarthritis (OA) is a chronic joint disease that causes a gradual deterioration of articular cartilage. A major challenge in OA treatment is the limited penetration and delivery efficiency of drugs to cartilage and chondrocytes due to the rapid clearance of drugs through synovial fluid in joints and the osmotic barrier of the cartilage extracellular matrix (ECM). To address this issue, a novel tetrahedral framework nucleic acid (tFNA)-based nanomedicine delivery system (tFNA-2WL) is first synthesized with excellent cartilage permeability and perfect chondrocyte endocytosis properties. After being loaded with ginsenoside Rb1 (Gin), the tFNA-2WL&Gin complex not only penetrates the cartilage but also accumulates in the menisci, ligaments, and joint capsules, thus prolonging the residence time of Gin in OA rat knees. In vitro, tFNA-2WL&Gin effectively promotes chondrogenesis, inhibits cartilage degradation by reducing apoptosis, and scavenges reactive oxygen species (ROS), outperforming free Gin. In OA rats, tFNA-2WL&Gin restores gait, reduces osteophyte formation, inhibits synovial inflammation and hypertrophy, and protects cartilage from further damage more effectively than Gin and other nanomedicines. These results demonstrate the feasibility of tFNA-2WL in improving the pharmacokinetics and efficacy of drugs and highlight the favorable curative effects of tFNA-2WL&Gin for OA, offering a promising paradigm for translational medicine.

Keywords: apoptosis; cartilage‐penetrating; chondrocyte uptake; framework nucleic acid nanoparticles; osteoarthritis; reactive oxygen species.

Figure 1

The synthesis and characterization of tFNA‐2WL and tFNA‐2WL&Gin. A) Characterization of the successful synthesis of tFNA‐2WL and tFNA‐2WL&Gin by PAGE. The results showed that the molecular weights of tFNA‐2WL and tFNA‐2WL&Gin were significantly larger than that of tFNA and the fluorescence of Cy5 in tFNA and Gin had a good colocalization with WL‐FITC (lane V‐VII) [S1 and Gin were labeled with Cy5 (red) and WL were labeled FITC (green)]. B) Spectra of Cy5‐Gin, WL(WL‐FITC), tFNA‐2W[tFNA‐2(WL‐FITC)] and tFNA‐2WL&Gin[tFNA‐2(WL‐FITC)&Cy5‐Gin] between 200 and 800 nm. C and D) Molecular diameters and zeta potential of tFNA, tFNA‐2WL, and tFNA‐2WL&Gin detected by DLS. E) Encapsulation efficiency of Gin loading on tFNA‐2WL. n = 3. F) The unique spatial structure of tFNA‐2WL&Gin was characterized by TEM. Scale bar, 20 nm. G) AFM images of tFNA‐2WL&Gin. Scale bar, 200 and 20 nm. H) Comparison of the stability of tFNA, tFNA‐2WL, and tFNA‐2WL&Gin in 2% and 10% FBS at different times by AGE. I) The release of Gin from tFNA‐2WL&Gin. The data are presented as the mean ± SD. Statistical differences were determined by one‐way ANOVA with Tukey's multiple comparisons test for (E).

Figure 2

tFNA‐2WL and tFNA‐2WL&Gin can penetrate the normal and OA cartilage matrix deeply, in vitro and in vivo. A) Schematic illustration of porcine cartilage explant harvesting and detection of the penetration and distribution of fluorescent nanoparticles in cartilage via an IVIS and a confocal fluorescence microscope imaging. B and C) Fluorescence images and quantitative analysis of cartilage explants incubated with Control (PBS), tFNA (Cy5‐tFNA), WL(WL‐FITC), and tFNA‐2WL[Cy5‐tFNA‐2(WL‐FITC)] for 12 h using IVIS in FITC and Cy5 fluorescence channels, respectively. n = 4, *p < 0.05, **p < 0.01, ***p < 0.001. D) Horizontal fluorescence scanning images and quantitative analysis of these cartilage samples from (B) and (C) at different depths using a confocal microscope. The black arrow indicates the direction of nanoparticle penetration. n = 4, Scale bars, 200µm. E) Representative confocal microscopy images of cryosections harvested from normal rats 2 h after intra‐articular injection of tFNA (Cy5‐tFNA) and tFNA‐2WL [Cy5‐tFNA‐2(WL‐FITC)], respectively. n = 3. F) Schematic illustration of OA rats with surgery on the right hind knee (top) and timeline of intra‐articular injection of different nanomedicines. G and H) Representative IVIS images and quantitative analysis of OA rat knee joints over 7 h after injection of Gin (Cy5‐Gin), tFNA&Gin (tFNA&Cy5‐Gin), and tFNA‐2WL&Gin [tFNA‐2(WL‐FITC)&Cy5‐Gin] in vivo. n = 3. I) Representative confocal microscopy images of OA rat knee cryosections from (G). Scale bars, 250µm. J) Light‐sheet fluorescence microscopy three‐dimensional (3D) reconstruction images of the rat's OA joint with intra‐articular injection of tFNA‐2WL&Cy5‐Gin, after tissue clarity, and 3D fluorescence imaging by light‐sheet microscopy, showed that Cy5‐Gin fluorescence signals retained in cartilage, meniscus, ligament, and articular capsule (shown by white arrows). Note: yellow, Cy5‐Gin; green, tissue autofluorescence. AC: articular capsule, Lig: ligaments, Men: meniscus, Car: cartilage. Scale bars, 1 mm. All data are presented as the mean ± SD. Statistical differences were determined by one‐way ANOVA with Tukey's multiple comparisons test for (B), and (C).

Figure 3

Schematic illustration. We designed and fabricated tFNA‐2WL and tFNA‐2WL&Gin via self‐assembly of bases and click chemistry. tFNA‐2WL&Gin was administrated into the knee joints of OA rats by intra‐articular injection. The tFNA‐2WL&Gin penetrated deeply into the cartilage matrix, was effectively uptake by chondrocytes, and sustainably released Gin to protect cartilage from osteoarthritic damage by rebalancing anabolism and catabolism in OA chondrocytes.

Figure 4

tFNA‐2WL&Gin can be efficiently uptake by chondrocytes, effectively reduce ROS, and significantly inhibit apoptosis of chondrocytes in vitro. A) Schematic illustration of extraction of chondrocytes, construction of OA chondrocyte model, characterization of chondrocyte uptake, and detection of the ability of nanomedicines to intervene in ROS and apoptosis of OA chondrocytes. B) Cytotoxicity of nanomedicine systems of tFNA‐2WL and tFNA‐2WL&Gin, characterized by CCK8 kit after co‐incubation of chondrocytes with different concentrations of tFNA‐2WL (0, 100, 200, 300, 400, 500 nm) and tFNA‐2WL&Gin (Gin: 0, 2.5, 5, 10, 20, 40 µm) for 12 h. C and D) Evaluation and quantitative analysis of uptake efficiency of Cy5‐Gin, tFNA&Gin (tFNA&Cy5‐Gin), and tFNA‐2WL&Gin (tFNA‐2WL&Cy5‐Gin) measured by flow cytometry. n = 3. E) Representative confocal fluorescence images of chondrocytes co‐cultured with nanomedicines of Cy5‐Gin, tFNA&Gin (tFNA&Cy5‐Gin) and tFNA‐2WL&Gin [tFNA‐2(WL‐FITC)&Cy5‐Gin] for 6 h. Scale bars, 250 µm. F and G) Representative fluorescence and quantitative analysis of the apoptosis in OA chondrocytes treated with IL‐1β, IL‐1β + Gin, IL‐1β + tFNA‐2WL, IL‐1β + tFNA&Gin, and IL‐1β + tFNA‐2WL&Gin was measured by flow cytometry. H and I) Quantitative analysis and fluorescence images of ROS in OA chondrocytes of the above groups (H) were detected by fluorescence imaging. Scale bars, 100µm. All data are presented as the mean ± SD. Statistical differences were determined by one‐way ANOVA with Tukey's multiple comparisons test for (B), (D), (G), and (H). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5

tFNA‐2W&Gin can effectively reverse the impaired chondrogenesis and reduce the cartilage degradation of OA chondrocytes. A) Schematic illustration of detection of chondrogenic and cartilage degradation abilities of OA chondrocytes after treatment with different nanomedicines. B) Schematic diagram of key processes getting involved in promoting impaired chondrogenesis and reducing the catabolism of OA chondrocytes of tFNA‐2W&Gin. C) RT‐PCR analysis of the gene expression of ACAN, Col II, ADAMTS5, and MMP13 of the chondrocytes in the above six groups. D and E) Western blotting analysis of the protein expression of Col II, ADAMTS5, and MMP13 of the chondrocytes in each group. F–I) Immunofluorescence images and quantitative analysis of ACAN, Col II, ADAMTS5, and MMP13 expression in chondrocytes after multiple drug treatments. n = 3, Scale bars: 100 µm. All data are presented as the mean ± SD. Statistical differences were determined by two‐way ANOVA for (E) and by one‐way ANOVA with Tukey's multiple comparisons test for (C), and (F–I). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 6

tFNA‐2W&Gin can attenuate the development of OA via relieving pain and gait, alleviating collagen tissue damage, and reducing osteophyte formation and subchondral bone resorption in OA rats. A) Schematic of the timeline and protocol of multiple treatments for OA rats induced by the ACLT + pMMx surgery. B) Representative photographs of the rats’ gaits after different treatments for 4 weeks. Left front foot, sky blue; Right front foot, purple; Left hind foot, yellow; Right hind foot, red. C) Quantitative analysis of average walking speed, average front/rear print length, average stride length, and relative contact area of the right hind limb of the rats after different treatments for 4 weeks. n = 6. D) Schematic of detection of joint collagen tissue damage for all rats after conditional treatment for 4 weeks by the intra‐articular injection of Cy5‐CHP in vivo. E and F) Representative IVIS images and quantitative analysis of rat knee joints with different treatments for 4 weeks after over 1.5 h by injection of Cy5‐CHP. n = 6. G) Representative 2D micro‐CT images (first row), 3D reconstruction images of knee joints (second row), and trabeculae of the tibial plateau (third row) showing the occurrence of osteophytes, cartilage and bone defects and bone resorption (shown by red arrows). n = 6, Scale bar, 2 mm. H–M) Quantitative analysis results of the parameters of subchondral bone microarchitecture (BV/TV, BS/BV, Tb.Th, Tb.Sp, Tb.N, and Tb.Pf) in the tibial epiphysis. All data are presented as the mean ± SD. Statistical differences were determined by one‐way ANOVA with Tukey's multiple comparisons test for (C), and (H–M). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

tFNA‐2W&Gin not only can significantly reduce cartilage degradation, and promote cartilage regeneration, but also alleviate synovial inflammation and Collagen degradation in OA rats. A–C) Representative images of SO&FG, Col II, and MMP13 staining of cartilage from the rats after 4 weeks of different treatments. Scale bars, A) 500 µm B,C) and 50 µm. D) Representative fluorescence images of CHP and Tunel staining of cartilage from the rats with conditional treatments. Scale bars, 100 µm. E and F) Representative images of H&E and MMP13 staining of the articular capsule tissue after 4 weeks of treatments. Scale bar, 100 µm. G) Representative fluorescence images of CHP and Tunel staining of articular capsule tissue from the rats after 4 weeks of treatments. Scale bars, 100µm. H) Quantitative analysis of cartilage degeneration was evaluated by OARSI score, Col II⁺ area, the rate of MMP13⁺ chondrocytes and fluorescence intensity of CHP, and quantitative of the intensity of Tunel fluorescence staining to explore the apoptosis in chondrocytes of each group. n = 6. I) Quantitative analysis of the inflammation, the expression of MMP13, collagen destruction, and apoptosis in synovium. n = 6. All data are presented as the mean ± SD, and statistical differences were determined by one‐way ANOVA with Tukey's multiple comparisons test for (H) and (I). *p < 0.05, **p < 0.01, ***p < 0.001.