Promoting chondrogenesis by targeted delivery to the degenerating cartilage in early treatment of osteoarthritis

Abstract

Osteoarthritis (OA) is a highly incident total joint degenerative disease with cartilage degeneration as the primary pathogenesis. The cartilage matrix is mainly composed of collagen, a matrix protein with a hallmark triple-helix structure, which unfolds with collagen degradation on the cartilage surface. A collagen hybridizing peptide (CHP) is a synthetic peptide that binds the denatured collagen triple helix, conferring a potential disease-targeting possibility for early-stage OA. Here, we constructed an albumin nanoparticle (An) conjugated with CHP, loaded with a chondrogenesis-promoting small molecule drug, kartogenin (KGN). The CHP-KGN-An particle exhibited sustained release of KGN in vitro and prolonged in vivo retention selectively within the degenerated cartilage in the knee joints of model mice with early-stage OA. Compared to treatment with KGN alone, CHP-KGN-An robustly attenuated cartilage degradation, synovitis, osteophyte formation, and subchondral bone sclerosis in OA model mice and exhibited a more prominent effect on physical activity improvement and pain alleviation. Our study showcases that targeting the degenerated cartilage by collagen hybridization can remarkably promote the efficacy of small molecule drugs and may provide a novel delivery strategy for early-stage OA therapeutics.

Keywords: Albumin nanoparticle; Collagen; Early-stage osteoarthritis; Targeting therapy; Triple helix.

Graphical abstract

Fig. 1

Preparation and characterization of the CHP-functionalized and KGN-encapsulated albumin particles (CHP-KGN-An) as a drug-delivery vehicle. (a) Schematic of the conjugation of CHP to KGN-An using a copper-free click reaction. (b) Size distribution of nanoparticles CHP-An, KGN-An, and CHP-KGN-An measured by DLS. (c) SEM images of nanoparticles CHP-An, KGN-An, and CHP-KGN-An. Scale bars, 1000 nm. (d) Zeta potentials of nanoparticles CHP-An, KGN-An, and CHP-KGN-An. Data are shown as mean ± SD. (e) The release profile of KGN from KGN-An and CHP-KGN-An in PBS over 7 days (n = 3 independent experiments; data shown as mean ± SD).

Fig. 2

In vivo targeted retention of CHP-KGN-An in the degenerated cartilage. (a) Representative ex vivo fluorescence images of knee joints 5 h post-tail-vein injection of Cy5-CHP-KGN-An in mice 4 weeks post-DMM-operation (n = 5 mice). (b) Representative fluorescent images of the cryosections of knee joint cartilage taken 5 h following intra-articular injection in mice. (c) Daily in vivo fluorescence images showing Cy5-CHP-KGN-An's retention in the knee joints of the mice following an intra-articular injection on day 0 (1 h p.i., n = 5 mice). The ratio between the fluorescence signals quantified from each mouse's DMM- and sham-operated knee area is displayed for each image with the only ratio lower than 1 shown in brown. (d) Post-mortal measurements of the paired fluorescence signals from the isolated knee joint specimens on day 4 post-injection with a representative fluorescence image (n = 5 mice). Different imaging parameters were used for the ex vivo and in vivo fluorescence images. Data are analyzed with a paired t-test. **P < 0.01.

Fig. 3

CHP-KGN-An improved the therapeutic effect of KGN on the motion function of the DMM mice. (a) The weight of the mice in this study (n = 5 mice). (b) The knee diameter of mice treated as indicated. (c) The quantification of paw contract thresholds was measured with the Von Frey Fiber test. (d) Representative mice movement trajectories in the open field test. (e) The quantification of the relative activity, distance, mean speed, and active time in the open field test (n = 5). (f) Representative mouse footmarks in the footprint assay. Red footprints: forelimbs; black footprints: hind limbs. Scale bars, 1 cm (left), 5 mm (right). (g) The CHP-KGN-An treatment further increased the relative stride and step length and reduced the relative front/rear print length in the DMM mice compared to the KGN treatment group (n = 5). Data are shown as mean ± SD and analyzed using one-way ANOVA with Tukey's post-hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4

CHP-KGN-An increased the KGN's protection against cartilage degeneration in the DMM mice (n = 5). (a) The representative Safranin-O/fast green staining of the mouse cartilage. (b) The representative toluidine blue staining of the mouse cartilage. (c) The quantification of the Safranin-O/fast green staining in terms of the uncalcified cartilage thickness. (d) The OARSI scores showing the extent of cartilage degradation in each treatment group. (e, f) The number of chondrocytes and full cartilage thickness quantified from the Safranin-O/fast green staining. (g) The representative IHC staining for Col II, Aggrecan, Sox9, and Mmp13 in the cartilage of mice by DMM or sham surgery. (h) The quantification of the Col II⁺ area in the cartilage surface as shown by IHC staining. (i) The quantification of Aggrecan⁺ chondrocyte population in the cartilage surface from the IHC staining. (j) The quantification of Sox 9⁺ chondrocyte percentage in the cartilage surface from the IHC staining. (k) The quantification of Mmp13⁺ chondrocyte percentage in the cartilage surface from the IHC staining. Scale bars, 100 μm (a, b), 50 μm (g). Data are shown as mean ± SD and analyzed using One-way ANOVA with Tukey's post-hoc test (c, e, f, h, i, j, k) or Kruska-Wallis test (d). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5

CHP-KGN-An further attenuated bone remodeling and synovitis in the DMM mice compared with KGN (n = 5). (a) Representative reconstructed 3D micro-CT images of the mouse knee joints showed the osteophytes around the knee joints. The sagittal images of the medial tibial plateau demonstrated subchondral osteosclerosis. The red line marked the subchondral bone thickness. (b) Representative H&E staining of the mouse knee joints. (c) The quantification of the synovial score. (d) The quantification of subchondral bone thickness. (e) The quantification of the osteophyte number. Scale bars, 1 mm (a), 100 μm (b). Data are shown as mean ± SD and analyzed using one-way ANOVA with Tukey's post-hoc test (d, e) or Kruska-Wallis test (c). **P < 0.01; ***P < 0.001.

Fig. 6

In vivo and in vitro biocompatibility evaluation of CHP-KGN-An. (a) Representative Calcein-AM/PI staining of chondrocytes after incubating with 5 μM KGN or CHP-KGN-An capable of releasing equivalent dose of KGN within one day (n = 6 independent experiments). (b) The quantification of cell survival rate based on Calcein-AM/PI staining. (c) The representative phalloidin showing the chondrocyte morphology after incubating with 5 μM KGN or CHP-KGN-An capable of releasing the same dose of KGN within one day (n = 6 independent experiments). (d,e) CCK-8 assay for the viability of chondrocytes treated for with 100 nM to 5 μM KGN (d, 200 μL) or CHP-KGN-An capable of releasing 100 nM to 5 μM (200 μL) KGN (e) for 72 h. (f) Representative H&E staining of DMM mice's spleen, heart, lung, liver, and kidney. Scale bars, 100 μm (a,c), 50 μm (f). Data are shown as mean ± SD and analyzed using one-way ANOVA with Tukey's post-hoc test.