Conclusions and perspectives
In this work, we have produced and characterised biodegradable microparticles for the local treatment of arthritis. These microparticles contain two active ingredients: the first one, dexamethasone acetate (DXM), is dissolved in the polymer matrix and provides the anti-inflammatory effect, and the second one, the superparamagnetic nanoparticles (SPIONs), is incorporated as a suspension in the polymer matrix and has the role of keeping the whole system in place under the action of an external magnetic field. The formulation of these microparticles has been optimised by an experimental design approach, which allowed us to find the best technological conditions that lead to biodegradable microparticles of 5 to 10 μm in diameter that encapsulate both DXM and SPIONs with drug loadings suitable for future clinical administration. This study outlined the significance of parameters, such as drug/PLGA ratio, stirring speed during solvent extraction, and extractive volume, on the particle characteristics.
The microparticles formulated according to the optimised process were subsequently characterised from a physicochemical point of view, focusing on aspects such as the inner structure of the microparticles, including electron-energy loss spectroscopy, zeta potential and magnetisation. In this respect, we could demonstrate that the encapsulation process did not affect the magnetic properties of the SPIONs or the iron oxidation state, ensuring a superparamagnetic behaviour of the final microparticles. Moreover, the zeta potential measurements showed that both DXM and SPIONs were completely embedded into the microparticles, thus validating the results obtained by transmission electron microscopy, from which we could see that the SPIONs were homogeneously dispersed into the microparticle matrix. The DXM loading in the microparticle complied with the therapeutically effective dose that has to be administered into the articulation, which is around 1.2 mg/kg in rats or mice, according to El Hakim et al. [1]. The in vitro and in vivo release patterns showed
Conclusions and Perspectives 72
similarity in terms of DXM release in the case of batches prepared with RG 502S polymer.
An important aspect is that the in vivo release was performed in an inflammatory environment (air pouch model), such as in the case of arthritis, giving us an insight into the microparticle behaviour in the joint.
The next step of our work focused on the interaction between microparticles and synovial fibroblasts, a key issue considering that the microparticles are intended for intra-articular administration. This study revealed that neither the microparticles nor their individual components were toxic for synoviocyte cell cultures. Moreover, it has been clearly demonstrated that the microparticles are taken up by the synovial fibroblasts by an actin-dependent process, namely, phagocytosis. In summary, DXM-containing superparamagnetic microparticles seem to be promising drug delivery systems for the local treatment of arthropathies as they present the double advantage of internalisation by the synoviocytes and a prolonged drug action due to magnetically increased microparticle residence time in the joint.
Finally, the biological action of the microparticles was studied in an antigen-induced arthritis model in mice in the presence and in absence of a subcutaneously implanted magnet near the knee of the mice. Following i-a. injection of DXM- and SPION-containing microparticles in arthritic joints, a diminution in the synovial inflammation was observed 4 days after the injection. Furthermore, magnetic microparticles were still detectable in healthy joints up to 3 months after i-a. injection, proving that this type of versatile system has the potential of delivering locally/in a targeted manner not only corticosteroids, such as DXM, but also other substances, such as TNF- or p38 mitogen-activated protein kinase inhibitors.
Nevertheless, a pronounced influence of the subcutaneously implanted magnets was not observed. The reasons for this lack of effect could be the limited magnetic retention in the joint or the high animal response variability in the studied model. In this respect, experiments using an osteoarthritis model over extended time periods will be more appropriate to evidence the benefit of SPION incorporation. In any case, our results represent a proof of concept on which future work could be based.
Conclusions and Perspectives 73
This research project opens numerous perspectives for future work in at least two main directions. First of all, due to the drug delivery system high versatility, it would be possible to encapsulate other substances into the same microparticle type, while their release rate could be tailored by changing the material of the microparticle matrix. In this respect, new formulation strategies could be found for very active compounds, which actually could not be used otherwise due to high systemic toxicity. As extensively discussed in the first chapter, numerous p38 MAPK or IL-1 inhibitors represent only a few examples of such compounds.
This is substantiated by the fact that we have recently been able to embed VX 745, a potent p38 MAPK inhibitor, with high loading efficacies in the same type of microparticles (data not shown). Based on this encouraging preliminary result, future work should be directed towards the study of the biological activity of this particle type in animal models of arthritis, after intra-articular administration. The second research direction opened by this thesis consists of chemically or physically modifying the microparticles to permit them to reach specific target sites in the inflamed joint [2]. For instance, we suggest the use of the v3 integrin, which is a dimeric transmembrane molecule that is up-regulated in neoangiogenic vessels; the folate receptor FR, which is up-regulated on activated synovial macrophages; and E-selectin, which is an adhesion molecule that is up-regulated on the vascular endothelium of inflamed tissue.
[1] I. E. El Hakim, I. S. Abdel-Hamid, A. Bader, Tempromandibular joint (TMJ) response to intra-articular dexamethasone injection following mechanical arthropathy: a histological study in rats, Int. J. Oral Maxillofac. Surg. 34 (2005) 305-310.
[2] T. Garrood,C. Pitzalis, Targeting the inflamed synovium: the quest for selectivity, Arthritis Rheum. 54 (2006) 1055-1060.