An in Situ Anti-Inflammatory Drug Delivery System for Intervertebral Disks
Authors: Pereira I. O. M. , Barbosa M. A. , Gonçalves R. M. , Pereira C. L. , Molinos M. M. 
Published: June 2012

Introduction Intervertebral disk (IVD) degeneration is one of the major causes of low back pain and the exact reason why (and if) it relates with pain symptoms remains to be discovered (Prithvi Raj, 2008). Current conservative treatments include exercise, medications, and physical therapy. Moreover, there is a growing consensus that surgery, towards removing and/or replacing the damaged tissue, may not be effective. This work focuses on the development of a local and minimally invasive anti-inflammatory drug delivery system for IVD. This drug delivery system is based on the incorporation of Chitosan (Ch)/g-Poly(glutamic acid) (PGA) nanocarriers with Diclofenac (Df), a widely used anti-inflammatory drug, in a hyaluronic acid (HA)-based hydrogel. The Df local delivery in IVD would overcome the disadvantages associated with its oral administration such as poor biological half-life, high doses, and side effects such as peptic ulceration. Moreover, the use of a hydrogel envisages the treatment of degenerated IVD, which could be replaced by a novel cell-free or cell-loaded biocompatible gel. Materials and Methods The Ch/PGA nanocarriers with Df were produced by coacervation method. Briefly, PGA and Ch electrostatic interaction at a controlled pH (pH 5) was explored as previously described (Antunes, 2012). The nanoparticles obtained were characterized in terms of size and polydispersion index by dynamic light scattering (DLS) and the Df entrapment was evaluated by UV/Vis absorbance. As a vehicle, a commercially available hydrogel based on HA and Gelatin (HyStem, Glycosan) was used. The gelation of the HA-based gel with the nanoparticles was evaluated at different temperatures and dilutions while the gel net was verified using CryoSEM. The quantity and distribution of the nanoparticles within the gel were characterized by fluorimetry and Confocal Microscopy (using FITC-Ch). The Df release from the nanoparticles within the gel was evaluated at physiological pH by UV/Vis absorbance. In the end, the biological effect of this anti-inflammatory strategy was assessed using Lipopolysaccharide (LPS)-induced macrophages isolated from peripheral blood samples. Cell viability, phenotypic markers, and the levels of diverse pro- and anti-inflammatory cytokines were evaluated (IL-6, TNF-alfa, IL-10, etc.). Results Df entrapment within Ch/PGA nanoparticles was optimized, reaching approximately 93% of drug entrapment with complexes with size of 253 ± 32 nm. These nano complexes remain stable during several days at pH 5.0 and were successfully incorporated into HA-based hydrogels, being distributed along the gel net as observed by CryoSEM and Confocal Microscopy, despite some aggregates. The gelation of the HA-based gel was optimized: the time of gelation range 25 minutes for gels with 10% of nanoparticles (% v/v), at 37 °C. The decrease of temperature (until 4°C) was shown to increase time of gelation (until 45 minutes). The Df delivery from the nanocomplexes entrapped in the HA-based hydrogel was determined at pH 7.4 (PBS) and compared with Df delivery from free nanoparticles. In this case, an immediate release of the drug occurs (∼ 60% after 1 hour incubation), accomplished by particles aggregation. Nevertheless, the Df release rate was hindered by incorporation of the Df-nanoparticles within the HA hydrogel: after 2 h only 26% of the anti-inflammatory drug was released reaching 100% after 24 h. The Df release in a degenerated disk environment (pH 6.5) is currently being evaluated. The cytotoxic effect of this delivery system is being evaluated in LPS-activated human macrophages. Df, in the range of concentrations used, did not cause any toxicity, while cell metabolic activity did not decrease below 80% in the presence of the nanoparticles. The Df seems to promote the expression of CD 86 and MHC II, in CD14+ macrophages, suggesting a polarization towards M1 or M2b phenotype and an increase in IL-10 production. The levels of prostaglandin (PGE2) are also being determined, since PGE2 is inhibited by anti-inflammatory drugs as Df. Conclusion The self-assembly of PGA, Ch, and Df resulted in stable nanosystems which can be used for the controlled release of the drug. Sustained Df release at physiological pH is provided by the incorporation of these NPs into an injectable gel. This work shows the feasibility of an in situ anti-inflammatory drug delivery system, controlled by pH changes, for IVD. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Prithvi Raj P. Intervertebral disc: Anatomy-Physiology-Pathophisiology-Treatment. Pain Practice 2008;8(1):18–44 Antunes JC, et al. Layer-by-layer self-assembly of Chitosan and Poly(γ-glutamic acid) into polyelectrolyte complexes. Biomacromolecules 2012 In press