Introduction The recent advances in regenerative medicine have led to the development of novel experimental therapies, at both the cellular and molecular levels, which aim at reversing the devastating consequences of acute spinal cord injury [1-2]. Several bioactive molecules able to promote regeneration of the injured nervous system show therapeutic potential only when delivered in a controlled and sustained fashion, via appropriate microcarriers. The aim of this work was to evaluate the suitability of poly(lactic-co-glycolic) acid (PLGA)-based microspheres (MS) as a means to deliver therapeutic amounts of either neurotrophic factors (for axonal protection), or inhibitors of bone morphogenetic proteins (for inhibition of the glial scar), following acute spinal cord injury in mice. Several MS formulations were prepared, in order to achieve proper release kinetics of the selected molecule(s), and the effect of the encapsulation technique on the structure and stability of the loaded molecule was assessed. The encapsulation of environmental regulators (ERs) in bioresorbable PLGA-based MS might be a promising approach to promote the regeneration of the injured nervous system. Materials and Methods Microsphere synthesis: PLGA-based MS were synthesized by means of a double emulsion/solvent evaporation technique (water/oil/water), as previously described . The ER (e.g., recombinant mouse chordin) was loaded in the presence of bovine serum albumin (BSA) as stabilizing molecule, and MS were synthesized starting from different PLGA concentrations. Microsphere characterization: The size distribution of MS was evaluated through dynamic laser light scattering, whereas MS morphology was assessed through scanning electron microscopy (SEM). The encapsulation efficiency was evaluated by UV spectrophotometry. Release study: In vitro release of the ER was monitored in PBS at 37°C with gentle shaking, over a period ranging from 72 hours to 30 days, depending on the given MS formulation tested. In order to assess any effects of the presence of BSA on the ER release kinetics, both the total amount of protein (BSA+ER) released at selected time points and the released amount of ER were measured, by UV spectrophotometry (with a BCA-assay protein kit) and ER-specific ELISA assays respectively. Protein/ER stability and bioactivity: The effect of the encapsulation technique on the structure of the loaded molecule(s) was assessed through sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectrofocalization (IEF), performed at selected time points along the sustained release. Specific bioassays, to assess the biological activity of the released ER, are currently in progress. Results and Discussion The average MS size was found to be significantly affected by the PLGA amount initially dissolved in the organic solvent used to prepare the double emulsion, with lower diameters attained for lower PLGA concentrations. As expected, release kinetics was greatly dependent on the mean MS size. Indeed, for the MS formulations tested, it was possible to modulate the time length of the sustained release of the ER from 3-4 days (for an average MS size of 15 μm, attained for a 10% w/v PLGA concentration) to 35 days (average MS size of 30 μm, attained for a 20% w/v PLGA concentration). The encapsulation efficiency was about 80-90% for all the MS formulations tested. The structural integrity of both BSA and ER was not affected by the encapsulation technique used, as demonstrated by the results of SDS-PAGE and IEF. However, the released fraction of ER as measured by the ELISA assays was found to be significantly lower than the amount expected from the total release study (BSA+ER). Along with in vitro bioactivity assays, specific release studies to understand this discrepancy between the total release of BSA and ER and the release of the only ER are currently being performed. Conclusions ER-loaded PLGA-based MS, possessing different mean diameters, were synthesized by a double emulsion/solvent evaporation technique. This technique yielded MS batches with a small size distribution and a high encapsulation efficiency, and preserved the structural integrity of the loaded molecules. The release kinetics could be tuned by varying the mean MS diameters. Based on these results, PLGA-based MS show potential for the local delivery of ERs to the injured nervous system. However, the suitability of the proposed MS to this purpose will be proved only following in vitro and in vivo bioactivity assays, scheduled in the next future.
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