Microcapsules As A Delivery Strategy For Inflammation-Modulating Proteins

D Federici Canova2,1, LV Norling1, A Pavlov3, GB Sukhorukov3, M Perretti1. 1William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, London, UK, 2Dept. Healthy Sciences, University of Eastern Piedmont, Novara, Italy, 3School of Engineering & Materials Science Queen Mary University of London, London, UK

Introduction. Microparticles are small membrane-bound vesicles released from different cell types that may serve as important signalling elements between cells. Depending on their cargo of distinctive mediators, microparticles can exert pro- or anti-inflammatory effects [1]. A new technology for construction of Microcapsules made of dextran, poly-arginine and poly-lysine, with defined size, composition and content of encapsulated materials has been described [2]. We hypothesised that this technique could be exploited to i) mimic the biology of endogenous microparticles and ii) study single encapsulated mediators. The focus here is on alpha-2-macroglobulin (α2M), a protein abundant in a subset of neutrophil-derived microparticles and endowed with protective functions in experimental sepsis (unpublished).

Aim. Evaluate the biological properties of α2M-enriched microcapsules.

Methods. α2M-microcapsules were produced by chemical synthesis using 0.8 mg of α2M (BioMac) and verified by flow cytometry, microscopy and Western blotting analyses. In functional assays, α2M-microcapsules were tested in an in vitro flow chamber assay of neutrophil-endothelial cell interactions (human umbilical vein endothelial cells- HUVEC- stimulated 4 h with 10 ng/ml TNF-α prior to 8-min flow of freshly prepared neutrophils) [1] and in phagocytosis assay. For the latter, human monocyte-derived macrophages (MDM) were prepared by 8-days culture in RPMI 1640 medium containing 20% foetal bovine serum. Cells were controlled by flow cytometry (monocytes CD14+vehicle; MDM, CD68+vehicle) and incubated with fluorescent particles (Zymosan for 20 min and E. Coli for 1 h); α2M-microcapsules were added 24 h before. As a control, a blank preparation of microcapsules was used, prepared and handled as the α2M-microcapsules but devoid of any protein. Data are mean ± SEM; *p<0.05 (t-test).

Results and Conclusions. Microcapsules enriched with active α2M replicated the biological properties that we observed with neutrophil-derived microparticles. Incubation of endothelial cells with α2M-microcapsules (10-100x103; 4 h) enhanced recruitment of neutrophils to the endothelial monolayer (p<0.05 vs. un-stimulated cells). In phagocytosis assays, α2M-microcapsules promoted uptake of Zymosan and E.Coli particles by MDM with optimal responses between 10-100x103 microcapsules (p<0.05 vs. un-stimulated cells). In either assay, α2M-microcapsules mimicked the effect of soluble α2M (10nM). In summary, α2M-microcapsules are a new tool to harness the biological properties of active α2M and other immune-active proteins, affected by short half-life, with potential therapeutic/delivery applications that now must be tested in in vivo. Further, microcapsule technology can be used to replicate specific biological properties of microparticles, selecting one or two of their components, to elicit distinct cellular responses to ultimately modulate inflammation.

References .

[1] Dalli J, Norling LV, Renshaw D, Cooper D, Leung KY, Perretti M. Blood 112: 2512-2519 (2008).

[2] Sukhorukov GB, MÖhwald H. Trends in Biotecnology 25: 93-98 (2007).