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Abstract
Objective
IL-1β is involved in the degradation of articular cartilage in various arthritides, including osteoarthritis (OA). Competitive inhibition of IL-1β by IL-1 receptor antagonists (IL-1Ra) may represent a pathogenesis-based strategy for inhibiting degradation of the cartilage matrix. We investigated the hypothesis that controlled release of IL-1Ra using injectable, thermoreversible and complex coacervate combination gels as drug delivery systems might reduce matrix degradation in OA.
Methods
Thermoreversible combination gels that can be injected into joints were formed in aqueous solution by making a complex coacervate with recombinant human IL-1Ra (anakinra) and cationic macromolecules, and this was followed by co-formulation with methylcellulose as a negative thermosensitive polysaccharide. Gels containing anakinra were positioned in the upper insert of a transwell system and human OA chondrocytes were placed in the lower compartment and then they were stimulated with IL-1β. The expression of matrix metalloproteinases (MMPs) was examined by performing real time PCR and ELISA.
Results
Complex coacervation between anakinra and protamine was successfully completed. IL-1Ra was released from the gels in a sustained release pattern for extended periods with minimal initial bursts. IL-1β markedly enhanced the expression of MMP. The IL-1Ra released from the gels significantly inhibited the IL-1β-induced MMP expression in the chondrocytes.
Conclusion
We developed and optimized a novel injectable and thermoreversible gel system for the controlled release of IL-1Ra, and this drug delivery system effectively inhibited the IL-1β-induced MMP expression of chondrocytes in a transwell system. Intraarticular local delivery of injectable and thermoreversible gels containing IL-1Ra into knees has the potential to provide prolonged therapy based on the pathophysiology of knee OA.
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Figure 1.
Experimental protocol. (1) Experiment I (6-days gel, 10 μ g of anakinra, no coacervate): upper arm. Day #0: gel preparation and anakinra loading, Day #1∼#6: daily change of media and measurement of the concentration of anakinra at the indicated days for the release pattern of anakinra, Day #4: transfer of gel into the upper compartment of the new system with plated chondrocytes on the lower compartment, and stimulation of chondrocytes with IL-1β (0.1 ng/ml) for 24 hours, Day #5: preparation of RNA and harvesting the supernatant for real time PCR and ELISA. (2) Experiment II (15-days gel, 10 or 100μ g of anakinra, coacervation with protamine): lower arm. Day #0: gel preparation and anakinra loading, Day #1∼#15: daily change of media and measurement of the concentration of anakinra at the indicated days for the release pattern of anakinra, Day #13: transfer of gel into the new system with plated chondrocytes on the lower compartment, Day #14: stimulation of chondrocytes with IL-1β (0.1 ng/ml) for 24 hours, Day #15: preparation of RNA and harvesting the supernatant for real time PCR and ELISA. IL-1Ra: IL-1 receptor antagonist, RT-PCR: real time polymerase chain reaction, ELISA: Enzyme-Linked ImmunoSorbent Assay.
Figure 2.
Turbidity curves of protamine and the IL-1Ra mixtures. Turbidity curve at various concentration in PBS, pH 7.4, 25 o C (weight ratio of protamine to IL-1Ra: ●: 0.5, ○: 1, ▼: 2). The turbidity results indicated whether or not two macro-molecules formed the coacervate. If the turbidity was increased, then coacervate was formed. At the weight ratio of 2, the turbidity was highly increased within 3 hours. Abs: absorbance.
Figure 3.
In vitro release of anakinra from the thermoreversible, coacervate combination gel. The IL-1Ra released from the gel was measured by ELISA. Without coacervation with protamine, almost 80% of the loaded anakinra was released by Day #5, but coacervation made the release pattern slow down with a minimal initial burst. Only 60% and 40% of the loaded anakinra was released from the gels loaded with 10μ g and 100μ g of anakinra at Day #15, respectively. IL-1Ra: IL-1 receptor antagonist (□: 10μ g IL-1Ra, ●: 10μ g IL-1Ra+20μ g protamine, ○: 100μ g IL-1Ra+200μ g protamine).
Figure 4.
Inhibition of the gene expression of IL-1?-induced MMP-3 (Experiment I) and MMPs (Experiment II) by the anakinra released from the gel. Values are the mean and standard deviation of the indicated gene expression (duplicate measurement) relative to the untreated control. The asterisk indicates statistical significance (∗p<0.05, † p<0.001 vs. the expression level induced by IL-1β, Student's t-test). # indicates the marginal statistical significance (p=0.058 for MMP-1, p=0.059, 0.057, 0.060 respectively for MMP-13). In Experiment I, we examined the effect of the combination gel itself on the gene expression. IL-1β+ Ra indicate the stimulation with IL-1β and Ra300 without the gel in the blank bar, and the stimulation with IL-1β in the presence of the gel loaded with Ra100 in the filled bar. The gel itself (filled bar) did not influence on the result and the 6-days matured gels with anakinra suppressed the IL-1β-induced expression of MMP-3 (Experiment I). In Experiment II, the 15-days matured gels with anakinra significantly inhibited the IL-1β-induced expression of MMP-1, −2, −3 and −13 in a dose-dependent manner. MMP: matrix metalloproteinase, Ra10, 100 and 300: initial loading doses of IL-1 receptor antagonist (anakinra) at 10, 100, and 300μ g, respectively.
Figure 5.
Inhibition of the protein expression of IL-1β-induced MMP-3 in the 6-days (Experiment I) or 15-days (Experiment II) matured gel with anakinra. The values are the mean and standard deviation of the measured concentration (duplicate) of MMP-3 (ng/ml). The asterisk indicates statistical significance (∗p<0.05, † p<0.001 vs. the expression level induced by IL-1β, Student's t-test). # indicates marginal statistical significance (p=0.055 in Exp I). The gel itself (filled bar) did not have a considerable influence on the result (Experiment I). In Experiments I and II, the 6-days and 15-days matured gels with anakinra significantly inhibited the IL-1β (0.1 ng/ml)-induced expression of MMP-3 in a dose-dependent manner. The legend is same as that for Fig. 4.
Table 1.
Primer sets used for real time PCR
Table 2.
Concentration of the released anakinra from the gels into the culture supernatant
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