Combination of anti-inflammatory therapy and RNA interference by light-inducible hybrid nanomedicine for osteoarthritis treatment

Abstract

Osteoarthritis (OA) is a type of highly prevalent heterogeneous degenerative disease that leads to joint pain, deformity, the destruction of articular cartilage, and eventual disability. The current treatment strategies for OA often suffer from systemic side effects, poor anti-inflammatory efficacy, and persistent pain. To address these issues, we develop light-inducible nanomedicine that enables the co-delivery of anti-inflammatory drug (diacerein, DIA) and small interfering RNA (siRNA) targeting nerve growth factor (NGF) for pain relief to enhance the therapeutic efficacy of OA. The nanomedicine is based on poly(β-amino-ester)-coated gold nanocages (AuNCs), which is further incorporated with the phase-change material (lauric acid/stearic acid, LA/SA). Following intra-articular (IA) injection in vivo, the nanomedicine displays high degree of drug accumulation and retention in the joint lesion of OA mouse models. The photothermal effect, induced by AuNCs, not only promotes DIA and siRNA release, but also upregulates the expression of heat shock protein 70 (HSP-70) to resist the apoptosis of chondrocytes in the inflammatory condition. The internalization of both DIA and siRNA results in strong anti-inflammatory and pain-relieving effects, which greatly contribute to the joint repair of OA mice. This study offers a promising combination strategy for OA treatment.

Keywords: Combinational therapy; Diacerein; Gold nanocages; Osteoarthritis; Pain relief; Phase-change material; Photothermal effect; Poly(β-amino-ester).

Graphical abstract

Scheme 1

Schematic illustration of the preparation of AuNCs-based nanodrug system (AuDPNAs) for the co-delivery of DIA and siRNA, and its therapeutic mechanism for OA in vitro and in vivo. (A) Schematic illustration of the preparation procedures of AuDPNAs nanosystem. (B) The application process and (C) Therapeutic mechanism of photothermally controlled multi-therapeutic nanosystems.

Figure 1

Preparation, characterization, and in vitro release behavior of nanodrug. (A) Schematic illustration for the formations of AuDPNAs. (B–E) TEM image and size distribution of AuDs and AuDPNs. Scale bar: 100, 500 nm. (F) The zeta potentials of AuNCs, AuDs, AuDPNs and AuDPNAs were determined by DLS. (G) The absorbance spectra of AuNCs, AuDs, AuDPNs and AuDPNAs were determined by UV–Vis spectrometer. (H) TEM image of AuDPNAs. (I) Size distribution of AuDPNAs. Scale bar: 100, 500 nm. (J) Photothermic stability of AuDPNAs upon 808 nm laser irradiation of 0.2 W/cm² for five on/off cycles at room temperature. (K) The laser thermography of PBS, AuNCs and AuDPNAs with 2.0 mg/mL after 808 nm laser irradiation of 0.2 W/cm² for 5 min. (L) In vitro release profiles of DIA from AuDs and AuDPNAs with or without laser irradiation/37 °C water bath for 5 h determined by UV–Vis.

Figure 2

In vitro transfection, photothermal-responsiveness and biocompatibility of the nanodrug with chondrocytes. (A) Illustration for fluorescence intensity of the FAM-labeled siRNA in chondrocytes after transfection by siNGF@PEI, siNGF@PBAE, and AuDPNs at various mass ratios of AuNCs/PBAE (1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, w/w). Green, siRNA. Scale bar: 100 μm. (B) The fluorescence intensity of chondrocytes was quantitatively assessed by flow cytometry after transfection. (n = 4). (C) The cellular uptake and endosomal escape of AuDPNs in chondrocytes were observed via confocal laser scanning microscopy (CLSM). Green, siNGF; Blue, nucleus; red, LysoTracker red; yellow, colocalization of siNGF and LysoTracker red. Scale bar: 20 μm. (D) Relative NGF expression of chondrocytes treated with IL-1β (10 ng/mL) at negative control siRNA (siNC) and three siNGFs transfection, respectively (n = 3). (E, F) NGF expression (E) and secretion (F) in chondrocytes stimulated with IL-1β and co-treated with PBS, siNC@PBAE, AuNCs, diacerein (DIA) and AuDPNs for 24 h by RT-qPCR and ELISA, respectively (n = 3). (G) Distribution of siNGF in chondrocytes after transfection by AuDPNs, AuDPNAs and AuDPNAs + L by a CLSM. Green, siRNA; Blue, nucleus. Scale bar: 20 μm. (H, I) NGF expression (H) and secretion (I) in chondrocytes stimulated by IL-1β, and co-treated with AuDPNs, AuDPNAs and AuDPNAs + L for 24 h (n = 3). (J) The quantity of viable cells obtained from the Live/Dead assay of chondrocytes co-cultured with PBS, DIA, AuDs, siNGF@PBAE, AuDPNs, AuDPNAs, and AuDPNAs + L (n = 3 biologically independent samples). Data are presented as mean ± S.D. Significance was calculated using one-way ANOVA with Tukey's post-hoc test. ∗∗∗P < 0.001. ns, no significant difference.

Figure 3

In vitro protective effects of the nanodrug on chondrocytes against ROS and apoptosis. IL-1β (10 ng/mL) was applied in chondrocytes to mimic the pathological process of OA, and was co-cutreated with PBS (Ⅱ), DIA (Ⅲ), AuDs (Ⅳ), siNGF@PBAE (Ⅴ), AuDPNs (Ⅵ), AuDPNAs (Ⅶ), and AuDPNAs + L (Ⅷ), along with a blank control group (Ⅰ). (A) Intracellular ROS levels were assessed in chondrocytes using DCFH-DA. Scale bar: 100 μm. (B) SOD activity assay of chondrocytes (n = 3). (C) CAT activity assay of chondrocytes (n = 3). (D) Chondrocytes apoptosis was assessed by flow cytometry after labeling with annexin V–fluorescein isothiocyanate (FITC) conjugate and propidium iodide (PI) (n = 4). (E) The Western blot analysis of chondrocytes, showed the protein expression of HSP-70, Bax, Bcl-2, caspase-3, and cleaved caspase-3 (n = 3). (F) The protein expression of COL2A1, ACAN, MMP-13, and ADAMTS-5 expression in chondrocytes (n = 3). (G–J) Quantitation of COL2A1 (G), ACAN (H), MMP-13 (I), and ADAMTS-5 (J) expression in chondrocytes tested by immunofluorescent staining (n = 3). Data are presented as mean ± S.D. Significance was calculated using one-way ANOVA with Tukey's post hoc test. ∗∗∗P < 0.001. ns, no significant difference.

Figure 4

Intra-articular residence and photothermal conversion property of nanodrug. (A) The photothermal performance of AuNCs and AuDPNAs in knee joints upon laser irradiation (808 nm, 0.2 W/cm²) over 5 min (n = 5). (B) In vivo joint temperature variations with and without AuNCs at specific intervals (n = 5). (C) IVIS images displaying mice knee joints across 28 days post-injection with Cy5, AuDs, AuDPNAs, and AuDPNAs + L (n = 5). (D) Temporal progression of fluorescent radiant efficiency within the joints at indicated time points (n = 5). Data are presented as mean ± S.D. Significance was calculated using two-way ANOVA with Bonferroni post-hoc test. ∗∗P < 0.01, ∗∗∗P < 0.001, ns, no significant difference.

Figure 5

The nanodrug under laser irradiation exhibited cartilage protection in mice. (A) Regimen illustration for the DMM-induced OA model. (B) Exemplary micro-CT scans of each group (n = 5). (C) Histological staining analysis was conducted on the cartilage sections using the H&E and Safranin O-Fast Green methods to evaluate the treatment of DMM-induced OA mice after being injected with PBS (G2), DIA (G3), siNGF@PBAE (G4), AuDPNAs (G5), and AuDPNAs + L (G6) at 4 weeks post-surgery (n = 5). Scale bar: 50, 100 μm. (D, E) Histological (D) and OARSI (E) scores of the articular cartilage for each group were assessed after the 4-week treatment period (n = 5). (F) Representative fluorescence images displaying the expression of COL2A1 and MMP-13 proteins in the articular cartilage, 4 weeks post intra-articular injection of G2‒G6 (n = 5). Scale bar: 50 μm. (G, H) COL2A1 protein expression level (G) and percentage of MMP-13-positive chondrocytes (H) obtained from the fluorescence intensity (n = 5). Data are presented as mean ± S.D. Significance was calculated using one-way ANOVA with Tukey's post-hoc test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. ns, no significant difference.

Figure 6

In vivo NGF knockdown and modulation of pain-related symptoms with nanodrug in OA model. (A) Illustration of the mitigation process of DMM-induced OA pain-related behaviors. (B) Representative fluorescent images of NGF protein expression levels in articular cartilage following intra-articular injection of PBS (G2), DIA (G3), siNGF@PBAE (G4), AuDPNAs (G5), and AuDPNAs + L (G6) into the knee joints of mice at 4 weeks postoperatively (n = 5 biologically independent mice). Scale bar: 50 μm. (C) Assessment of NGF-positive cells proportion in cartilage (n = 5 biologically independent mice). (D, E) The weight (D) and diameter (E) of mice were measured at 3-day intervals until their sacrifice (n = 5 biologically independent mice). (F–H) Assessment of pain sensitivity and mobility by paw withdrawal threshold (F), average speed (G) and distance (H) test at 1, 2, and 4 weeks post-surgery (n = 5 biologically independent mice). Data are presented as mean ± S.D. Significance was calculated using one-way ANOVA with Tukey's post-hoc test (C, F–H) or two-way ANOVA with Bonferroni post-hoc test (D and E). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns, no significant difference.