Hyaluronan-Loaded Liposomal Dexamethasone-Diclofenac Nanoparticles for Local Osteoarthritis Treatment

Abstract

Osteoarthritis (OA) remains one of the common degenerative joint diseases and a major cause of pain and disability in older adult individuals. Oral administration of non-steroidal anti-inflammatory drugs (NSAIDs) (such as diclofenac, DIC) or intra-articular injected gluco-corticosteroids (such as dexamethasone, DEX) were the conventional treatment strategies for OA to reduce joint pain. Current limitations for both drugs including severe adverse effects with risks of toxicity were noted. The aim of the present study was to generate a novel OA treatment formulation hyaluronic acid (HA)-Liposomal (Lipo)-DIC/DEX to combat joint pain. The formulation was prepared by constructing DIC with DEX-loaded nanostructured lipid carriers Lipo-DIC/DEX mixed with hyaluronic acid (HA) for prolonged OA application. The prepared Lipo-DIC/DEX nanoparticles revealed the size as 103.6 ± 0.3 nm on average, zeta potential as -22.3 ± 4.6 mV, the entrapment efficiency of 90.5 ± 5.6%, and the DIC and DEX content was 22.5 ± 4.1 and 2.5 ± 0.6%, respectively. Evidence indicated that HA-Lipo-DIC/DEX could reach the effective working concentration in 4 h and sustained the drug-releasing time for at least 168 h. No significant toxicities but increased cell numbers were observed when HA-Lipo-DIC/DEX co-cultured with articular chondrocytes cells. Using live-animal In vivo imaging system (IVIS), intra-articular injection of each HA-Lipo-DIC/DEX sufficed to reduce knee joint inflammation in OA mice over a time span of four weeks. Single-dose injection could reduce the inflammation volume down to 77.5 ± 5.1% from initial over that time span. Our results provided the novel drug-releasing formulation with safety and efficiency which could be a promising system for osteoarthritis pain control.

Keywords: dexamethasone; diclofenac; liposomal nanoparticle; osteoarthritis.

Figure 1

Characteristics of various dosages of DIC/DEX-loaded liposomes. (A) Representative figures of DIC/DEX-loaded liposomal nanoparticles captured by transmittance electron microscopy. The left panel depicts an empty liposomal nanoparticle. The right panel depicts a liposomal nanoparticle that was loaded with DIC/DEX. (B) The representative figures of the size distribution and zeta potential in 40 mg/mL DIC combined with 5 mg/mL hydrophilic/hydrophobic DEX-loaded liposomal nanoparticles acquired by DLS analysis (B1: size distribution, B2: zeta potential). (C) Bar figures of size distribution of various dosages of DIC/DEX-loaded liposomal nanoparticles. Note: The average sizes of the DIC/DEX-loaded liposomal nanoparticles were approximately 200-400 nm. Abbreviations: DLS, dynamic light scattering; DIC, diclofenac; DEX, dexamethasone.

Figure 2

The release profiles of various Lipo-DIC/DEX formulations in vitro. (A) DIC release concentrations from Lipo- DIC/DEX at different timepoints. Data were expressed as mean ± SEM. (B) DEX release concentrations from Lipo-DIC/DEX at different timepoints. (C,D) DIC/DEX release concentrations from HA-Lipo-DIC/DEX at different time points were also measured. Data were expressed as mean ± SEM. Abbreviations: Lipo, liposomal; DIC, diclofenac; DEX, dexamethasone; HA, hyaluronic acid; SEM, standard error of the mean.

Figure 3

Biocompatibility analysis of HA-Lipo-DIC/DEX. Bar figure of the viability of human chondrocytes SW 1353 co-cultured with HA-Lipo-DIC/DEX. (A) SW 1353 chondrocytes co-cultured with various drug concentration ratio for 24 h. (B) SW 1353 chondrocytes co-cultured with various drug concentration ratio for 48 h. (C) SW 1353 chondrocytes co-cultured with various drug concentration ratio for 72 h. SW 1353 chondrocytes viability were significantly higher in co-culturing with liposome only, and HA-Lipo-DIC/DEX in compared with free drugs. (^★ indicates p < 0.05, ^★★ indicates p < 0.01, one-way ANOVA). Abbreviations: HA, hyaluronic acid; Lipo, liposomal; DIC, diclofenac; DEX, dexamethasone.

Figure 4

Comparative efficacy of HA-Lipo-DIC/DEX and free drugs for arthritis therapy. Arthritis was induced in C57BL/6 mice by subcutaneous injection of FCA as described in Materials and Methods. At the time of appearance of signs of arthritis (disease onset), mice were randomized (n = 5 per group) and given an intraarticular injection single dose from day eight of the following preparations: FCA only, free DIC, free DEX, or HA-Lipo-DIC/DEX. The opposite paw was served as the reference for comparison with other groups. (^★ indicates p < 0.05, ^★★ indicates p < 0.01, one-way ANOVA). Abbreviations: FCA, Freund’s Complete Adjuvant; HA, hyaluronic acid; Lipo, liposomal; DIC, diclofenac; DEX, dexamethasone.

Figure 5

In vivo imaging of systemically administered liposomes. Arthritis was induced in C57BL/6 mice by subcutaneous injection of FCA as described in Materials and Methods. At the time of appearance of signs of arthritis (disease onset), mice were randomized (n = 5 per group) and given intravenously NE680 (1 nmol/25 μL) two injections every 14 days starting from day 7 (indicated by arrows) to monitor the inflammation status. Mice then receiving single-dose treatment from day 8 of the following preparations: FCA only, free DIC, free DEX, or HA-Lipo-DIC/DEX. (A) The fluorescence imaging of arthritic mice receiving various treatments including FCA only, free DIC, free DEX, or HA-Lipo-DIC/DEX after NE680 injection and their distribution in the paw was assessed by Near-infrared fluorescence-imaging. (B) Fluorescence activities of arthritic mice in various treated groups (mean ± SEM). Mice receiving free DEX or HA-Lipo-DIC/DEX exhibited the least fluorescence in the first 2 weeks. Four weeks after mice receiving FCA induced arthritic, the HA-Lipo-DIC/DEX treated group exhibited the least fluorescence imaging (^★ indicates p < 0.05, ^★★ indicates p < 0.001, one-way ANOVA).

Figure 6

HA-Lipo-DIC/DEX inhibited leukocyte infiltration and cartilage destruction in OA-induced mice. (A) Hematoxylin and eosin staining revealed the histological changes in the various anti-inflammation formulation treatments including control (A1 and A6), phosphate-buffered saline (PBS) (A2 and A7), DIC (A3 and A8), DEX (A4 and A9), and HA-Lipo-DIC/DEX (A5 and A10) on day 28, respectively. (Scale bar (A1~A5) 40×: 200 μm; (A6~A10) 200×: 50 μm). (B) Arthritic changes, including inflammatory cell infiltration, synovial proliferation/enlargement, articular cartilage breakdown, and bone destruction, were evaluated 28 days after administration of Freund’s Complete Adjuvant (CFA), graded from 0 to 3, and summed. Results are presented as the mean ± SD of 3 animals/group.

Figure 7

The schematic diagram of HA-Lipo-DIC/DEX structure. Liposomal nanoparticle Lipo-DIC/DEX containing both hydrophilic/hydrophobic DEX and DIC was first prepared. The preparation procedure was as described previously. HA-Lipo-DIC/DEX was prepared by mixing equal volume of liquefied HA (20 mg/mL) and Lipo-DIC/DEX as described in Materials and Methods Section. Liposomal nanoparticle structure was originally obtained from the website: https://www.slideshare.net/SunealSaini/liposomes-47868772 and modified by author. The final liquefied HA concentration was determined as 10 mg/mL.