IL-1ra delivered from poly(lactic-co-glycolic acid) microspheres attenuates IL-1?-mediated degradation of nucleus pulposus in vitro.
ABSTRACT: INTRODUCTION: Inflammation plays a key role in the progression of intervertebral disc degeneration, a condition strongly implicated as a cause of lower back pain. The objective of this study was to investigate the therapeutic potential of poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with interleukin-1 receptor antagonist (IL-1ra) for sustained attenuation of interleukin-1 beta (IL-1?) mediated degradative changes in the nucleus pulposus (NP), using an in vitro model. METHODS: IL-1ra was encapsulated in PLGA microspheres and release kinetics were determined over 35 days. NP agarose constructs were cultured to functional maturity and treated with combinations of IL-1? and media conditioned with IL-1ra released from microspheres at intervals for up to 20 days. Construct mechanical properties, glycosaminoglycan content, nitrite production and mRNA expression of catabolic mediators were compared to properties for untreated constructs using unpaired Student's t-tests. RESULTS: IL-1ra release kinetics were characterized by an initial burst release reducing to a linear release over the first 10 days. IL-1ra released from microspheres attenuated the degradative effects of IL-1? as defined by mechanical properties, glycosaminoglycans (GAG) content, nitric oxide production and mRNA expression of inflammatory mediators for 7 days, and continued to limit functional degradation for up to 20 days. CONCLUSIONS: In this study, we successfully demonstrated that IL-1ra microspheres can attenuate the degradative effects of IL-1? on the NP for extended periods. This therapeutic strategy may be appropriate for treating early-stage, cytokine-mediated disc degeneration. Ongoing studies are focusing on testing IL-1ra microspheres in an in vivo model of disc degeneration, as a prelude to clinical translation.
Project description:Intervertebral disc degeneration is characterized by a cascade of cellular, biochemical and structural changes that may lead to functional impairment and low back pain. Interleukin-1 beta (IL-1?) is strongly implicated in the etiology of disc degeneration, however there is currently no direct evidence linking IL-1? upregulation to downstream biomechanical changes. The objective of this study was to evaluate long-term agarose culture of nucleus pulposus (NP) cells as a potential in vitro model system to investigate this. Bovine NP cells were cultured in agarose for 49 days in a defined medium containing transforming growth factor-beta 3, after which both mechanical properties and composition were evaluated and compared to native NP. The mRNA levels of NP cell markers were compared to those of freshly isolated NP cells. Glycosaminoglycan (GAG) content, aggregate modulus and hydraulic permeability of mature constructs were similar to native NP, and aggrecan and SOX9 mRNA levels were not significantly different from freshly isolated cells. To investigate direct links between IL-1? and biomechanical changes, mature agarose constructs were treated with IL-1?, and effects on biomechanical properties, extracellular matrix composition and mRNA levels were quantified. IL-1? treatment resulted in upregulation of a disintegrin and metalloproteinase with thrombospondin motifs 4, matrix metalloproteinase-13 and inducible nitric oxide sythase, decreased GAG and modulus, and increased permeability. To evaluate the model as a test platform for therapeutic intervention, co-treatment with IL-1? and IL-1 receptor antagonist (IL-1ra) was evaluated. IL-1ra significantly attenuated degradative changes induced by IL-1?. These results suggest that this in vitro model represents a reliable and cost-effective platform for evaluating new therapies for disc degeneration.
Project description:This paper investigates the protective effect of interleukin-1 receptor antagonist (IL-1Ra) released from hyaluronic acid chitosan (HA-CS) microspheres in a controlled manner on IL-1?-induced inflammation and apoptosis in chondrocytes. The IL-1Ra release kinetics was characterized by an initial burst release, which was reduced to a linear release over eight days. Chondrocytes were stimulated with 10?ng/ml IL-1? and subsequently incubated with HA-CS-IL-1Ra microspheres. The cell viability was decreased by IL-1?, which was attenuated by HA-CS-IL-1Ra microspheres as indicated by an MTT assay. ELISA showed that HA-CS-IL-1Ra microspheres inhibited IL-1?-induced inflammation by attenuating increases in NO2- and prostaglandin E2 levels as well as increase in glycosaminoglycan release. A terminal deoxyribonucleotide transferase deoxyuridine triphosphate nick-end labeling assay revealed that the IL-1?-induced chondrocyte apoptosis was decreased by HA-CS-IL-1Ra microspheres. Moreover, HA-CS-IL-1Ra microspheres blocked IL-1?-induced chondrocyte apoptosis by increasing B-cell lymphoma 2 (Bcl-2) and decreasing Bcl-2-associated X protein and caspase-3 expressions at mRNA and protein levels, as indicated by reverse-transcription quantitative polymerase chain reaction and western blot analysis, respectively. The results of the present study indicated that HA-CS-IL-1Ra microspheres as a controlled release system of IL-1Ra possess potential anti-inflammatory and antiapoptotic properties in rat chondrocytes due to their ability to regulate inflammatory factors and apoptosis associated genes.
Project description:Intervertebral disc degeneration (IDD) is characterized by excessive apoptosis of nucleus pulposus (NP) cells and hyperactive extracellular matrix (ECM) catabolism. Our previous studies revealed the relationship between human islet amyloid polypeptide (hIAPP) and NP cell apoptosis. However, the role of hIAPP aggregates in IDD has not yet been investigated. This study aimed to determine whether the accumulation of hIAPP aggregates promotes IDD progression. The aggregation of hIAPP increased in human NP tissues during IDD. The deposition of hIAPP aggravated the compression-induced IDD that promoted NP cell apoptosis and ECM degradation via IL-1?/IL-1Ra signaling in an ex vivo rat disc model. Moreover, neutralizing IL-1? augmented the protective effects of hIAPP overexpression by decreasing hIAPP aggregation in human NP cells. These results suggest that the aggregation of hIAPP promotes NP cell apoptosis and ECM degradation ex vivo and in vitro by disrupting the balance of IL-1?/IL-1Ra signaling.
Project description:Lumbar disc degeneration is a common cause of chronic low back pain and an important contributor to various degenerative lumbar spinal disorders. However, currently there is currently no effective therapeutic strategy for treating disc degeneration. The pro-inflammatory cytokine interleukin-1? (IL-1?) mediates disc degeneration by inducing apoptotic death of nucleus pulposus (NP) cells and degradation of the NP extracellular matrix. Here, we confirmed that extracellular secretion of IL-1? via secretory autophagy contributes to disc degeneration, and demonstrate that a thermosensitive reactive oxygen species (ROS)-responsive hydrogel loaded with a synthetic growth hormone-releasing hormone analog (MR409) can protect against needle puncture-induced disc degeneration in rats. <b>Methods:</b> The expression levels of proteins related to secretory autophagy such as tripartite motif-containing 16 (TRIM16) and microtubule-associated protein light chain 3B (LC3B) were examined in human and rat disc tissues by histology and immunofluorescence. The effects of TRIM16 expression level on IL-1? secretion were examined in THP-1 cells transfected with TRIM16 plasmid or siRNA using ELISA, immunofluorescence, and immunoblotting. The <i>in vitro</i> effects of MR409 on IL-1? were examined in THP-1 cells and primary rat NP cells using ELISA, immunofluorescence, immunoblotting, and qRT-PCR. Further, MR409 was subcutaneously administered to aged mice to test its efficacy against disc degeneration using immunofluorescence, X-ray, micro-CT, and histology. To achieve controllable MR409 release for intradiscal use, MR409 was encapsulated in an injectable ROS-responsive thermosensitive hydrogel. Viscosity, rheological properties, release profile, and biocompatibility were evaluated. Thereafter, therapeutic efficacy was assessed in a needle puncture-induced rat model of disc degeneration at 8 and 12 weeks post-operation using X-ray, magnetic resonance (MR) imaging, histological analysis, and immunofluorescence. <b>Results:</b> Secretory autophagy-related proteins TRIM16 and LC3B were robustly upregulated in degenerated discs of both human and rat. Moreover, while upregulation of TRIM16 facilitated, and knockdown of TRIM16 suppressed, secretory autophagy-mediated IL-1? secretion from THP-1 cells under oxidative stress, MR409 inhibited ROS-induced secretory autophagy and IL-1? secretion by THP-1 cells as well as IL-1?-induced pro-inflammatory and pro-catabolic effects in rat NP cells. Daily subcutaneous injection of MR409 inhibited secretory autophagy and ameliorated age-related disc degeneration in mice. The newly developed ROS-responsive MR409-encapsulated hydrogel provided a reliable delivery system for controlled MR409 release, and intradiscal application effectively suppressed secretory autophagy and needle puncture-induced disc degeneration in rats. <b>Conclusion:</b> Secretory autophagy and associated IL-1? secretion contribute to the pathogenesis of disc degeneration, and MR409 can effectively inhibit this pathway. The ROS-responsive thermosensitive hydrogel encapsulated with MR409 is a potentially efficacious treatment for disc degeneration.
Project description:Toll-like receptors (TLR) are activated by endogenous alarmins such as fragmented extracellular matrix compounds found in the degenerating disc. TLRs regulate cytokine, neurotrophin, and protease expression in human disc cells in vitro, and thus control key factors in disc degeneration. However, whether TLR activation leads to degenerative changes in intact human discs is unclear. Nucleus pulposus (NP) cells isolated from non-degenerating discs increase IL-1? and nerve growth factor gene expression following treatment with Pam2CSK4 (TLR2/6 agonist) but not Pam3CSK4 (TLR1/2 agonist). Challenging NP cells with Pam2CSK4 or 30?kDa fibronectin fragments (FN-f, an endogenous TLR2 and TLR4 alarmin) increased secretion of proinflammatory cytokines. We then investigated the effect of TLR activation in intact, non-degenerate, ex vivo human discs. Discs were injected with PBS, Pam2CSK4 and FN-f, and cultured for 28 days. TLR activation increased proteoglycan and ECM protein release into the culture media and decreased proteoglycan content in the NP. Proteases, including MMP3, 13 and HTRA1, are secreted at higher levels following TLR activation. In addition, proinflammatory cytokine levels, including IL-6, TNF? and IFN?, increased following TLR activation. These results indicate that TLR activation induces degeneration in human discs. Therefore, TLRs are potential disease-modifying therapeutic targets to slow disc degeneration.
Project description:<h4>Purpose</h4>To investigate the in vitro release of octreotide acetate, a somatostatin agonist, from microspheres based on a hydrophilic polyester, poly(D,L-lactide-co-hydroxymethyl glycolide) (PLHMGA).<h4>Methods</h4>Spherical and non-porous octreotide-loaded PLHMGA microspheres (12 to 16 ?m) and loading efficiency of 60-70% were prepared by a solvent evaporation. Octreotide release profiles were compared with commercial PLGA formulation (Sandostatin LAR(®)); possible peptide modification with lactic, glycolic and hydroxymethyl glycolic acid units was monitored.<h4>Results</h4>PLHMGA microspheres showed burst release (~20%) followed by sustained release for 20-60 days, depending on the hydrophilicity of the polymer. Percentage of released loaded peptide was high (70-90%); >?60% of released peptide was native octreotide. PLGA microspheres did not show peptide release for the first 10 days, after which it was released in a sustained manner over the next 90 days; >?75% of released peptides were acylated adducts.<h4>Conclusions</h4>PLHMGA microspheres are promising controlled systems for peptides with excellent control over release kinetics. Moreover, substantially less peptide modification occurred in PLHMGA than in PLGA microspheres.
Project description:Abstract Inflammation plays an important role in the pathogenesis of intervertebral disc (IVD) degeneration. The proinflammatory cytokine tumor necrosis factor alpha (TNF??) has shown markedly higher expression in degenerated human disc tissue compared with healthy controls. Anti?inflammatory treatment targeting TNF?? has shown to alleviate discogenic pain in patients with low back pain. Therefore, in vitro and ex vivo inflammatory models utilizing TNF?? provide relevant experimental conditions for drug development in disc degeneration research. The current method article addressed several specific questions related to the model establishment. (a) The effects of bovine and human recombinant TNF?? on bovine nucleus pulposus (NP) cells were compared. (b) The required dose for an inflammatory IVD organ culture model with intradiscal TNF?? injection was studied. (c) The effect of TNF?? blocking at different stages of inflammation was evaluated. Outcomes revealed that bovine and human recombinant TNF?? induced equivalent inflammatory effects in bovine NP cells. A bovine whole IVD inflammatory model was established by intradiscal injection of 100?ng TNF??/ cm3 disc volume, as indicated by increased nitric oxide, glycosaminoglycan, interleukin 6 (IL?6), and interleukin 8 (IL?8) release in culture media, and upregulation of MMP3, ADAMTS4, IL?8, IL?6, and cyclooxygenase (COX)?2 expression in NP tissue. However, results in human NP cells showed that the time point of anti?inflammatory treatment was crucial to achieve significant effects. Furthermore, anticatabolic therapy in conjunction with TNF?? inhibition would be required to slow down the pathologic cascade of disc degeneration. This article describes the methods for establishment of proinflammatory intervertebral disc cell and organ culture models induced by TNF?a.
Project description:A biomimetic approach to organic solvent-free microencapsulation of proteins based on the self-healing capacity of poly (DL)-lactic-co-glycolic acid (PLGA) microspheres containing glycosaminoglycan-like biopolymers (BPs), was examined. To screen BPs, aqueous solutions of BP [high molecular weight dextran sulfate (HDS), low molecular weight dextran sulfate (LDS), chondroitin sulfate (CS), heparin (HP), hyaluronic acid (HA), chitosan (CH)] and model protein lysozyme (LYZ) were combined in different molar and mass ratios, at 37 °C and pH7. The BP-PLGA microspheres (20-63 ?m) were prepared by a double water-oil-water emulsion method with a range of BP content, and trehalose and MgCO3 to control microclimate pH and to create percolating pores for protein. Biomimetic active self-encapsulation (ASE) of proteins [LYZ, vascular endothelial growth factor165 (VEGF) and fibroblast growth factor (FgF-20)] was accomplished by incubating blank BP-PLGA microspheres in low concentration protein solutions at ~24 °C, for 48 h. Pore closure was induced at 42.5 °C under mild agitation for 42h. Formulation parameters of BP-PLGA microspheres and loading conditions were studied to optimize protein loading and subsequent release. LDS and HP were found to bind >95% LYZ at BP:LYZ>0.125 w/w, whereas HDS and CS bound >80% LYZ at BP:LYZ of 0.25-1 and <0.33, respectively. HA-PLGA microspheres were found to be not ideal for obtaining high protein loading (>2% w/w of LYZ). Sulfated BP-PLGA microspheres were capable of loading LYZ (~2-7% w/w), VEGF (~4% w/w), and FgF-20 (~2% w/w) with high efficiency. Protein loading was found to be dependent on the loading solution concentration, with higher protein loading obtained at higher loading solution concentration within the range investigated. Loading also increased with content of sulfated BP in microspheres. Release kinetics of proteins was evaluated in-vitro with complete release media replacement. Rate and extent of release were found to depend upon volume of release (with non-sink conditions observed <5 ml release volume for ~18 mg loaded BP-PLGA microspheres), ionic strength of release media and loading solution concentration. HDS-PLGA formulations were identified as having ideal loading and release characteristics. These optimal microspheres released ~73-80% of the encapsulated LYZ over 60 days, with >90% of protein being enzymatically active. Nearly 72% of immunoreactive VEGF was similarly released over 42 days, without significant losses in heparin binding affinity in the release medium.
Project description:PURPOSE:The purpose of this study was to investigate the feasibility of poly lactic/glycolic acid (PLGA) as a drug delivery carrier of Rho kinase (ROCK) inhibitor for the treatment of corneal endothelial disease. METHOD:ROCK inhibitor Y-27632 and PLGA were dissolved in water with or without gelatin (W1), and a double emulsion [(W1/O)/W2] was formed with dichloromethane (O) and polyvinyl alcohol (W2). Drug release curve was obtained by evaluating the released Y-27632 by using high performance liquid chromatography. PLGA was injected into the anterior chamber or subconjunctiva in rabbit eyes, and ocular complication was evaluated by slitlamp microscope and histological analysis. RESULTS:Y-27632 incorporated PLGA microspheres with different molecular weights, and different composition ratios of lactic acid and glycolic acid were fabricated. A high molecular weight and low content of glycolic acid produced a slower and longer release. The Y-27632 released from PLGA microspheres significantly promoted the cell proliferation of cultured corneal endothelial cells. The injection of PLGA did not induce any evident eye complication. CONCLUSIONS:ROCK inhibitor-incorporated PLGA microspheres were fabricated, and the microspheres achieved the sustained release of ROCK inhibitor over 7-10 days in vitro. Our data should encourage researchers to use PLGA microspheres for treating corneal endothelial diseases.