Near Infrared Responsive Gold Nanorods Attenuate Osteoarthritis Progression by Targeting TRPV1

Abstract

Osteoarthritis (OA) is the most common degenerative joint disease worldwide, with the main pathological manifestation of articular cartilage degeneration. It have been investigated that pharmacological activation of transient receptor potential vanilloid 1 (TRPV1) significantly alleviated cartilage degeneration by abolishing chondrocyte ferroptosis. In this work, in view of the thermal activated feature of TRPV1, Citrate-stabilized gold nanorods (Cit-AuNRs) is conjugated to TRPV1 monoclonal antibody (Cit-AuNRs@Anti-TRPV1) as a photothermal switch for TRPV1 activation in chondrocytes under near infrared (NIR) irradiation. The conjugation of TRPV1 monoclonal antibody barely affect the morphology and physicochemical properties of Cit-AuNRs. Under NIR irradiation, Cit-AuNRs@Anti-TRPV1 exhibited good biocompatibility and flexible photothermal responsiveness. Intra-articular injection of Cit-AuNRs@Anti-TRPV1 followed by NIR irradiation significantly activated TRPV1 and attenuated cartilage degradation by suppressing chondrocytes ferroptosis. The osteophyte formation and subchondral bone sclerosis are remarkably alleviated by NIR-inspired Cit-AuNRs@Anti-TRPV1. Furthermore, the activation of TRPV1 by Cit-AuNRs@Anti-TRPV1 evidently improved physical activities and alleviated pain of destabilization of the medial meniscus (DMM)-induced OA mice. The study reveals Cit-AuNRs@Anti-TRPV1 under NIR irradiation protects chondrocytes from ferroptosis and attenuates OA progression, providing a potential therapeutic strategy for the treatment of OA.

Keywords: TRPV1; ferroptosis; near infrared‐inspired nanoparticle; osteoarthritis; photothermal therapy.

Scheme 1

Illustration of Cit‐AuNRs@Anti‐TRPV1 switch for photothermal activation of TRPV1 signaling to attenuate OA.

Figure 1

Preparation and characterization of Cit‐AuNRs@Anti‐TRPV1. a) Schematic of preparation of Cit‐AuNRs@Anti‐TRPV1. b,c,d) TEM images, of as‐prepared CTAB‐AuNRs, Cit‐AuNRs and Cit‐AuNRs@Anti‐TRPV1, respectively. e) Statistical average size, f) UV‐vis spectra and g) the zeta (ζ) potential of as‐prepared CTAB‐AuNRs, Cit‐AuNRs and Cit‐AuNRs@Anti‐TRPV1. h) FTIR spectra of Cit‐AuNRs and Cit‐AuNRs@Anti‐TRPV1.

Figure 2

In vitro photothermal effect of Cit‐AuNRs@Anti‐TRPV1. a) Real‐time infrared thermography of Cit‐AuNRs@Anti‐TRPV1 with NIR irradiation at different power levels (1.0 mg ml⁻¹, 880 nm for 20 s). b) In vitro temperature change curves of Cit‐AuNRs@Anti‐TRPV1 at different NIR irradiation power levels (1.0 mg ml⁻¹, 880 nm for 20 s). c) In vitro temperature change curves of PBS, CTAB‐AuNRs (1.0 mg ml⁻¹), Cit‐AuNRs (1.0 mg ml⁻¹), Cit‐AuNRs@Anti‐TRPV1 (1.0 mg ml⁻¹) with NIR irradiation for 20 s (880 nm, 0.75 W cm⁻²). d) Real‐time infrared thermography of PBS, CTAB‐AuNRs (1.0 mg ml⁻¹), Cit‐AuNRs (1.0 mg ml⁻¹), Cit‐AuNRs@Anti‐TRPV1 (1.0 mg ml⁻¹) with NIR irradiation for 20 s (880 nm, 0.75 W cm⁻²). e) Real‐time infrared thermography of Cit‐AuNRs@Anti‐TRPV1 with NIR irradiation at different concentrations (880 nm, 0.75 W cm⁻² for 20 s). f) In vitro temperature variation curves of Cit‐AuNRs@Anti‐TRPV1 at different concentrations of NIR irradiation (880 nm, 0.75 W cm⁻² for 20 s). g) Transient thermal measurements of Cit‐AuNRs@Anti‐TRPV1 (1.0 mg ml⁻¹) under repeated cycles of NIR irradiation (880 nm, 0.75 W cm⁻²). Each cycle consisted of 15 s irradiation followed by a 20 s cooling phase. For all graphs, data are expressed as mean ± S.D. of three independent tests.

Figure 3

The chondroprotective effect of Cit‐AuNRs@Anti‐TRPV1+NIR in OA. a,b) Immunofluorescence (IF) staining analysis for p‐Camk II expression in the cartilage of mouse by Sham or DMM surgery treated as indicated. AC, articular cavity; C, cartilage; SB, subchondral bone (a) and its quantification (b) (n=6). c) The OARSI score shows the extent of cartilage injury in DMM mice receiving different treatments (n=6). d) The quantification of Safranin‐O/fast green (S.O.) staining (n=6). e) Safranin‐O/fast green (S.O.) staining. f) Hematoxylin & Eosin (H&E) staining of mouse knee. g) Alcian blue staining of mouse knee. h) The quantification of Safranin‐O/fast green (S.O.) staining (n=6). i) The quantification of immunohistochemical (IHC) staining of Col II in the articular cartilage of mouse treated as in (a) (n=6). j) The quantification of IF staining of Mmp13 in the articular cartilage of mouse treated as in (a) (n=6). k) Immunohistochemical (IHC) staining of Col II. (i) Immunofluorescence (IF) staining of Mmp13. Scale bars, 50 µm (a, e (lower panel), f, g, k (lower panel), l), 100 µm (e (upper panel), k (upper panel)). One‐way ANOVA with Tukey's post‐hoc test. Data are shown as mean ± SD. *p< 0.05; **p< 0.01; ***p< 0.001.

Figure 4

Cit‐AuNRs@Anti‐TRPV1+NIR plays an anti‐ferroptotic role in the mice OA model. a,b,c,d) Representative images of immunofluorescence (IF) staining for Gpx4 (a), Ncoa4 (b), Cox2 (c), and immunohistochemical (IHC) staining for 4‐HNE (d) of mice articular cartilage treated as indicated. AC, articular cavity; C, cartilage; SB, subchondral bone. Scale bars, 50 µm (a, b, c, d (lower panel)), 100 µm (d (upper panel)). e,f,g,h) The quantification of a, b, c, d, respectively (n=6). One‐way ANOVA with Tukey's post‐hoc test. Data are shown as mean ± SD. **p< 0.01; ***p< 0.001.

Figure 5

Cit‐AuNRs@Anti‐TRPV1+NIR attenuates bone remodeling in DMM mice. a) 3D images of the mouse knee joint were reconstructed by micro‐computed tomography (micro‐CT) to highlight changes in the femoral and tibial surfaces. The sagittal images of the medial joint compartment show changes in the thickness of the subchondral bone plate (SBP). A red line marks the thickness of SBP. Quantified changes in b) subchondral bone volume (SB‐BV), c) number of osteophytes, d) SBP thickness, e) trabecular Number (Tb.N), f) trabecular separation (Tb.Sp) (n=6). Scale bars, 1mm (a). One‐way ANOVA with Tukey's post‐hoc test. Data are shown as mean ± SD. *p< 0.05; **p< 0.01; ***p< 0.001.

Figure 6

Cit‐AuNRs@Anti‐TRPV1+NIR treatment group improves physical activities and reduces pain in DMM mice. a,b) The weight (a) and the knee diameter (b) of mice treated as indicated (n=6). c) Thresholds of paw contraction were tested using von Frey fibers to reflect mechanical sensitivity (n=6). d) Representative trajectory plots show that the spontaneous activity of mice after DMM surgery decreases in the open field test. Changes in spontaneous activity, including relative activity, active time, distance, and mean speed (e) (n=6). The footprints of the two front paws of the manipulated mice were marked with red ink and the footprints of the two hind paws were marked with blue ink. f) Representative pictures of the footprints of each group. g) Cit‐AuNRs@Anti‐TRPV1+NIR treatment group increased relative stride length, relative step length, and shortened relative front/rear print length in DMM mice (n=6). One‐way ANOVA with Tukey's post‐hoc test. Data are shown as mean ± SD. *p< 0.05; **p< 0.01; ***p< 0.001.

Figure 7

Cit‐AuNRs@Anti‐TRPV1+NIR toxicity evaluation. The serum biochemical analysis for a) alanine aminotransferase (ALT), b) aspartate aminotransferase (AST), c) albumin (ALB), d) cholesterol (CHO), and e) lactate dehydrogenase (LDH) of mice treated as indicated (n=6). f) Hematoxylin & Eosin (H&E) staining of mice heart, liver, spleen, lung, kidney. Scale bars, 50 µm. One‐way ANOVA with Tukey's post‐hoc test. Data are shown as mean ± SD.