IL-1ra delivered from poly(lactic-co-glycolic acid) microspheres attenuates IL-1β-mediated degradation of nucleus pulposus in vitro

Intervertebral disc degeneration is strongly implicated as a cause of chronic low back pain, a condition that will affect up to 85% of people at some point during their lives [1, 2]. Disc degeneration is a cascade that includes early changes to the cellular microenvironment and progresses over time to structural breakdown and functional impairment [3–5]. Anatomically, the intervertebral disc is composed of three distinct regions: the central gelatinous nucleus pulposus (NP), the surrounding fibrocartilaginous annulus fibrosus, and superiorly and inferiorly thin endplates of hyaline cartilage that interface with the vertebral bodies [5]. Changes to the NP are implicated in the initiation of the degenerative cascade. Decreasing proteoglycan content in the NP and the associated reduction in hydrostatic pressure impair the ability of the disc to evenly distribute and transfer compressive loads between the vertebrae [6, 7].

Inflammation plays a key role in the progression of disc degeneration [8–11]. NP cells express the inflammatory cytokines interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), in particular, more highly in degenerate discs than in healthy discs [10]. The matrix composition of the NP, principally collagens and proteoglycans, is key to its mechanical function. Increasing levels of inflammatory cytokines in the degenerate disc have been associated with increased activity of catabolic proteases - including matrix metalloproteinases (MMPs) 1, 2, 3, 7, and 13 and a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4) and others - which degrade these constituents [11–14]. Current therapies for disc degeneration, including conservative techniques such as physical therapy and surgical techniques such as spinal fusion [15], treat the symptoms of low back pain without addressing the underlying biological mechanisms and, as a result, fail to sustain or restore disc structure and function.

We recently described a three-dimensional (3D) agarose culture model of the NP that recapitulates key molecular, compositional, and biomechanical properties of the native tissue and is responsive to inflammatory stimulus [16]. In that study, NP constructs treated with IL-1β exhibited increased mRNA expression of inducible nitric oxide synthase (INOS), ADAMTS-4, and MMP-13 and decreased glycosaminoglycan (GAG) content and aggregate modulus [16]. These findings highlighted the importance of IL-1β as a mediator of intervertebral disc degeneration by linking upregulation of key catabolic molecules with compositional and biomechanical changes. Attenuating the catabolic effects of IL-1β in the NP may be an essential therapeutic step in slowing or reversing disc degeneration. Interleukin-1 receptor antagonist (IL-1ra) is an endogenous inhibitor of IL-1β in vivo, where it acts by competitively blocking the binding of IL-1β to IL-1 receptors on IL-1-responsive cells [17, 18]. Whereas IL-1β is upregulated with disc degeneration, IL-1ra is not [9], resulting in an imbalance between catabolic and anabolic signaling.

Given the central role of IL-1 signaling in inflammatory diseases, considerable effort has been devoted to the development of IL-1ra for clinical applications [17, 19–24]. IL-1ra has successfully been used clinically for the treatment of rheumatoid arthritis for a number of years [22–24]. IL-1ra has also been investigated as a potential treatment for osteoarthritis in animal and human clinical trials, and the results have been encouraging [19, 20, 25]. There are pathophysiological similarities between osteoarthritis and intervertebral disc degeneration, including chronic cytokine-mediated extracellular matrix degradation [26]. There are also phenotypic similarities between articular cartilage chondrocytes, which mediate the inflammatory cascade in osteoarthritis, and NP cells, which mediate the cascade in the disc [27]. As such, it has been suggested that IL-1ra may have potential for the treatment of disc degeneration [26]. This potential has been demonstrated by recent in vitro studies [16, 28]. One study showed that IL-1ra delivered both directly in solution and by gene therapy to disc tissue explants was able to inhibit mRNA expression and activity of catabolic enzymes upregulated by IL-1β [28]. In addition, our recent study used an agarose 3D culture model of the NP to show that co-delivery of soluble IL-1ra could effectively inhibit IL-1β-mediated degradation at the molecular, compositional, and functional levels [16].

The avascular nature of the disc [29], in addition to the short terminal (serum) half-life and high therapeutic concentrations of IL-1ra [30], suggest that, in vivo, systemic delivery for treatment of disc degeneration is unlikely to be successful. Clinically, repeated intradiscal injections of IL-1ra necessary to sustain high local concentrations are impractical and may induce further degenerative changes via structural damage to the annulus fibrosus. Biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres have been used clinically for both local and systemic delivery of a number of small-molecule and protein-based therapies [31]. Given that these microspheres could be delivered directly to the disc space, they represent a novel mechanism for local, sustained, and controlled delivery of anti-inflammatory agents, including IL-1ra, to the disc. Therefore, the objective of this study was to advance this technology toward clinical application by investigating the therapeutic potential of IL-1ra-loaded PLGA microspheres for sustained attenuation of IL-1β-mediated NP degradation by using a 3D culture model.

In this study, we present evidence that IL-1ra delivered from PLGA microspheres can effectively attenuate IL-1β-mediated degradation of engineered NP constructs. IL-1ra is currently prescribed for the treatment of rheumatoid arthritis, and the treatment regime normally consists of daily self-administered subcutaneous injections [43]. This dosing frequency is necessary because of the short terminal half-life of the drug (approximately 4 to 6 hours) [30]. The predominantly avascular nature of the intervertebral disc [29] suggests that systemic delivery of IL-1ra is unlikely to be effective for treating inflammation in this tissue. Cells in the NP acquire nutrition via solute diffusion, primarily through the vertebral endplates [44]. It is possible that some drug may reach the NP via this route. However, given the short terminal half-life, the amount would most likely be small and lower than the therapeutic concentration. Thus, local delivery of IL-1ra into the NP would likely be necessary for effective treatment of NP inflammation. However, the tissues of the NP are subjected to continuous fluid flow because of mechanical loading and osmotic swelling, suggesting that the drug would rapidly diffuse away from the delivery site. Given that high-frequency injections to the disc are impractical, a sustained delivery vehicle would be required to make such a therapy clinically viable.

A key goal of sustained delivery techniques is achieving therapeutic levels of released factors over biologically relevant timescales. In one recent study, it was shown that IL-1ra released from PLGA microspheres could inhibit the stimulatory effects of IL-1β on tumor cell progression for at least 7 days [45]. Similarly, in the present study, we demonstrated that released IL-1ra comprehensively inhibited the degradative effects of IL-1β on NP constructs for up to 7 days in terms of composition, nitric oxide production, mRNA expression of catabolic factors and biomechanical properties. Beyond 7 days, the inhibitory effects of IL-1ra were limited to attenuation of construct GAG loss (up to 10 days) and of loss of biomechanical function (up to 20 days). Decreased overall efficacy at later time intervals may reflect both the decreasing concentration of released IL-1ra with time and potentially decreased bioactivity of the encapsulated protein. In a previous study, we demonstrated that 100 ng/mL of IL-1ra was sufficient to completely inhibit the degradative effects of 10 ng/mL of IL-1β on NP constructs [16]. As shown in Table 2, cumulative IL-1ra release was greater than 100 ng/mL for all intervals, suggesting that the decreased efficacy may be due to decreased bioactivity rather than insufficient delivery dose. We selected PLGA microspheres as the delivery vehicle for this study since they are biocompatible and easily optimized for different applications. The water-oil-water double-emulsion fabrication technique is an effective and well-characterized method for producing PLGA microspheres [31]. One limitation of this technique, however, is maintaining protein stability and activity during the fabrication process. The organic-aqueous interface can interfere with the hydrostatic mediated tertiary structure of proteins, and the mechanical cavitation forces present during emulsification can denature proteins [46]. After encapsulation, acid stress from the lactic and glycolic acid monomers from PLGA degradation can interfere with protein hydrogen bonding, leading to loss of protein activity [31]. For this study, we did not add additional stabilizing agents beyond those already present in the drug-buffering solution. In the future, additional protein stabilizers such as bovine serum albumin or additional acid buffers could be used to improve and maintain the bioactivity of IL-1ra after release. Another important consideration when evaluating the translational potential of this treatment is the fact that the concentration of IL-1β of 10 ng/mL used in this study likely far exceeds that which would likely be present in situ in the microenvironment of the degenerate disc, simulating an artificially harsh inflammatory environment.

To create microspheres for this study, we chose a PLGA with a high lactic acid ratio. The 75:25 PL/GA copolymer ratio used in this study is less hydrophilic than the more commonly used 50:50 ratio, leading to slower degradation [42, 47]. This results in extended release and slower accumulation of acidic degradation by-products. Nevertheless, these degradation products, specifically lactic and glycolic acids, could be expected to lower the pH at the delivery site, placing stress on the local cell population. In their native environment, NP cells exist in relatively acidic conditions in part due to anaerobic glycolysis, a product of which is lactic acid [48], suggesting that they may be relatively resistant to acidic PLGA degradation products. There is also evidence that acidity in the NP is higher in symptomatic degenerate discs, which are the most likely candidates for therapeutic intervention [49]. Nevertheless, to evaluate these effects in vitro, we co-cultured our NP constructs with blank microspheres. While there was no significant effect on GAG content or release, there were increases in the mRNA levels of catabolic mediators, suggesting that the microsphere degradation products did have some effect. This highlights the importance of appropriate control groups to assess these effects when microspheres are tested in vivo.

Maintenance of biomechanical properties at later release intervals, despite increases in inflammatory mediators and decreased GAG content, was encouraging but unexpected and likely points to the importance of extracellular constituents other than GAG in contributing to mechanical function. Indeed, a previous study examining functionally mature chondrocyte-seeded agarose constructs cultured under free-swelling conditions found only a moderate correlation between GAG content and aggregate modulus (r² = 0.37) [50].

In this study, we found that treatment with IL-1β resulted in upregulation of TLR-4 mRNA expression. To our knowledge, this is the first time this finding has been reported for NP cells. While there are similarities between the cytoplasmic domains of IL-1 receptor and TLR-4, their extracellular domains are structurally distinct [51], suggesting that IL-1β does not signal directly through TLR-4. It is possible, however, that TLR-4 contributes to the inflammatory response indirectly by responding to endogenous ligands that are produced, for example, as a consequence of matrix catabolism.

A limitation of this study is our use of an engineered NP model, which lacks an exogenous biological response, mechanical loading, and other physiological factors. To address this limitation, ongoing work in our laboratory will seek to test this therapy in an in vivo model system. An additional limitation is our use of bovine cells. The adult bovine disc is considered to be an effective surrogate for the adult human disc for a number of reasons, including the absence of notochord-like NP cells and the large size leading to similar nutritional limitations for the cellular microenvironment. In this study, we specifically targeted IL-1β because this cytokine has been implicated as a primary mediator of matrix catabolism in disc degeneration. The inflammatory cascade is, however, a complex process mediated by a large variety of other cytokines, including IL-6 and TNF-α. Ongoing work will investigate therapies that specifically target these additional factors as well as the associated nuclear factor-kappa-B and mitogen-activated protein kinase signaling pathways [52].

In this study, we provide evidence that IL-1ra delivered from PLGA microspheres can effectively attenuate IL-1β-mediated inflammatory changes in an engineered NP construct in vitro. Sustained delivery of anti-inflammatory agents may be very effective in preventing catabolic changes during early-stage disc degeneration but is unlikely to be sufficient to regenerate a painful and severely degenerate NP. A strength of this therapy may lie in a synergistic approach, in which sustained release of anti-inflammatory molecules is combined with complementary therapies for disc degeneration, such as those that augment and replace degenerate NP tissue, thereby halting the inflammatory cascade while simultaneously restoring disc height and mechanical function. Additional future work in our laboratory will focus on developing such synergistic approaches. More broadly, the techniques described in this study may have application for a wide variety of other musculoskeletal conditions characterized by acute or chronic inflammation.