Intra-articular interleukin-1 receptor antagonist (IL1-ra) microspheres for posttraumatic osteoarthritis: in vitro biological activity and in vivo disease modifying effect

Abstract

Background: Interleukin-1 receptor antagonist (IL-1 ra) can be disease-modifying in posttraumatic osteoarthritis (PTOA). One limitation is its short joint residence time. We hypothesized that IL-1 ra encapsulation in poly (lactide-co-glycolide) (PLGA) microspheres reduces IL-1 ra systemic absorption and provides an enhanced anti-PTOA effect.

Methods: IL-1 ra release kinetics and biological activity: IL-1 ra encapsulation into PLGA microsphere was performed using double emulsion solvent extraction. Lyophilized PLGA IL-1 ra microspheres were resuspended in PBS and supernatant IL-1 ra concentrations were assayed. The biological activity of IL-1 ra from PLGA IL-1 ra microspheres was performed using IL-1 induced lymphocyte proliferation and bovine articular cartilage degradation assays. Systemic absorption of IL-1 ra following intra-articular (IA) injection of PLGA IL-1 ra or IL-1 ra: At 1, 3, 6, 12 and 24 h following injection of 50 μl PLGA IL-1 ra (n = 6) or IL-1 ra (n = 6), serum samples were collected and IL-1 ra concentrations were determined. Anterior cruciate ligament transection (ACLT) and IA dosing: ACLT was performed in 8-10 week old male Lewis rats (n = 42). PBS (50 μl; n = 9), IL-1 ra (50 μl; 5 mg/ml; n = 13), PLGA IL-1 ra (50 μl; equivalent to 5 mg/ml IL-1 ra; n = 14) or PLGA particles (50 μl; n = 6) treatments were performed on days 7, 14, 21 and 28 following ACLT. Cartilage and synovial histopathology: On day 35, animal ACLT joints were harvested and tibial cartilage and synovial histopathology scoring was performed.

Results: Percent IL-1 ra content in the supernatant at 6 h was 13.44 ± 9.27 % compared to 34.16 ± 12.04 %, 47.89 ± 12.71 %, 57.14 ± 11.71 %, and 93.90 ± 8.50 % at 12, 24, 48 and 72 h, respectively. PLGA IL-1 ra inhibited lymphocyte proliferation and cartilage degradation similar to IL-1 ra. Serum IL-1 ra levels were significantly lower at 1, 3, and 6 h following PLGA IL-1 ra injection compared to IL-1 ra. Cartilage and synovial histopathology scores were significantly lower in the PLGA IL-1 ra group compared to PBS and PLGA groups (p < 0.001).

Conclusions: IL-1 ra encapsulation in PLGA microspheres is feasible with no alteration to IL-1 ra biological activity. PLGA IL-1 ra exhibited an enhanced disease-modifying effect in a PTOA model compared to similarly dosed IL-1 ra.

Keywords: Bioactivity of IL1ra; IL1 ra microspheres; Interleukin-1 receptor antagonist; Posttraumatic osteoarthritis.

Fig. 1

IL-1 ra containing PLGA microspheres. a, b Representative scanning electron micrographs of PLGA IL-1 ra microspheres at different magnifications. c Release of IL-1ra from PLGA-IL1 ra microspheres over time. Cumulative IL-1 ra release at 72 h was significantly higher than cumulative release at 6, 12, 24 and 48 h. Similarly, cumulative IL-1 ra release at 24 and 48 h was significantly higher than cumulative release at 6 and 12 h, respectively. *p < 0.001. Data represents the average ± SD of 4 different preparations

Fig. 2

Impact of PLGA incorporation on IL-1 ra biological activity. a Inhibition of interleukin-1 beta (IL-1β)-induced human lymphocyte proliferation by IL-1 ra or PLGA-encapsulated IL-1 ra (PLGA IL-1 ra). The percent inhibition of lymphocyte proliferation following treatment with 25 ng/ml of IL-1 ra or PLGA IL-1 ra was significantly higher than the percent inhibition of lymphocyte proliferation following treatment with 1 ng/ml of IL-1 ra or PLGA IL-1 ra. There was no significant difference in percent inhibition of lymphocyte proliferation between IL-1 ra and PLGA IL-1 ra treatments at 1 ng/ml or 25 ng/ml. *p < 0.001. Data represents the average ± SD of three independent experiments, each with duplicate wells per group. PLGA IL-1 ra was pooled from 4 different preparations. b cumulative sulfated glycosaminoglycan (sGAG) release, over 7 days, from control bovine articular cartilage explants (control), interleukin-1 α-stimulated (IL-1α), IL-1α stimulated + treatment with IL-1 ra at 20 ng/ml (IL-1 + IL-1 ra (20 ng/ml)), IL-1α stimulated + treatment with PLGA IL-1 ra at 20 ng/ml (IL-1 + PLGA IL-1 ra (20 ng/ml)), IL-1α stimulated + treatment with IL-1 ra at 100 ng/ml (IL-1 + IL-1 ra (100 ng/ml)) and IL-1α stimulated + treatment with PLGA IL-1 ra at 100 ng/ml (IL-1 + PLGA IL-1 ra (100 ng/ml)). (n = 6 in each group). IL-1α treatment resulted in a significantly higher sGAG cartilage release compared to control. Treatment with IL-1 ra or PLGA IL-1 ra at 20 ng/ml resulted in a significant reduction in sGAG release compared to IL-1α alone. Similarly, treatment with IL-1 ra or PLGA Il-1 ra at 100 ng/ml resulted in a significant reduction in sGAG release compared to IL-1 ra alone. There was no significant difference in sGAG release between IL-1 ra and PLGA IL-1 ra at 20 ng/ml or 100 ng/ml. *p < 0.001. Data represents the average ± SD of 6 explants per group. PLGA IL-1 ra was pooled from 4 different preparations

Fig. 3

Serum IL-1 ra concentrations following intra-articular administration of IL-1 ra or PLGA IL-1 ra. *p < 0.001. Data represents the average ± SD of 6 animals in each experimental group

Fig. 4

The disease-modifying activity of IL-1 ra or PLGA IL-1 ra in the rat ACLT model. a Cartilage histopathology scores of control joints and joints undergoing ACLT and receiving weekly intra-articular injections of PBS (n = 9), IL-1 ra (n = 13), PLGA-encapsulated IL-1 ra (PLGA IL-1 ra; n = 14) or PLGA microspheres (PLGA; n = 6) for 4 weeks starting one week following ACLT. PBS treated ACLT joints exhibited a significantly higher mean cartilage histopathology score compared to control. Similarly, PLGA-treated ACLT joints exhibited a significantly higher mean cartilage histopathology score compared to control. PLGA IL-1 ra treated ACLT joints demonstrated a significantly lower mean cartilage histopathology score compared to PBS or PLGA-treated ACLT joints. *p < 0.001. Data represents average ± SD. b Representative Safranin O/Fast Green stained tibial plateau cartilage specimens from control, ACLT animals receiving PBS, IL-1 ra, PLGA-IL1 ra or PLGA. ACLT joints exhibited loss of Safranin O staining indicative of glycosaminoglycan loss, clefts extending into the middle zone and hypocellularity. PLGA IL-1ra treatment reduced glycosaminoglycan loss and the number and depth of cartilage tissue fissures. Scale = 100 μm. c Synovial histopathology scores of control joints and joints undergoing ACLT and receiving weekly intra-articular injections of PBS (n = 9), IL-1 ra (n = 13), PLGA IL-1 ra (n = 14) or PLGA (n = 6) for 4 weeks starting one week following ACLT. PBS treated ACLT joints exhibited a significantly higher mean synovial histopathology score compared to control. Similarly, PLGA-treated ACLT joints exhibited a significantly higher mean synovial histopathology score compared to control. IL-1 ra treated ACLT joints exhibited a significantly lower mean synovial histopathology score compared to PBS or PLGA-treated ACLT joints. Similarly, PLGA Il-1 ra treated ACLT joints exhibited a significantly lower mean synovial histopathology score compared to PBS or PLGA-treated ACLT joints. *p < 0.001. Data represents average ± SD. d Representative H&E-stained synovia specimens from control, ACLT animals receiving PBS, IL-1 ra, PLGA IL-1 ra or PLGA. Arrows point to synovial hyperplasia evident in ACLT animals treated with PBS, IL-1 ra and PLGA

Fig. 5

Urinary CTXII (uCTXII) concentrations, normalized to urinary creatinine level, collected over 24 h in control animals and animals undergoing ACLT and receiving weekly intra-articular injections of PBS (n = 7), IL-1 ra (n = 9), PLGA IL-1 ra (n = 9) or PLGA (n = 6) for 4 weeks starting one week following ACLT. PBS treated ACLT animals exhibited a significantly higher mean uCTXII concentration compared to control. Similarly, PLGA-treated ACLT animals exhibited a significantly higher mean uCTXII concentration compared to control. PLGA IL-1 ra treated ACLT animals demonstrated a significantly lower mean uCTXII concentration compared to PBS or PLGA-treated ACLT animals. *p < 0.001. Data represents average ± SD