A Review of Bioactive Release from Nerve Conduits as a Neurotherapeutic Strategy for Neuronal Growth in Peripheral Nerve Injury
Typical W/O/W double emulsion method to prepare microspheres containing protein drug (upper panel) and microscopic events during fabrication process (lower panel). The sequence of fabrication is primary emulsion, secondary emulsion, solvent extraction/evaporation (not shown), freeze-drying, and drug release test. With negligible partition of protein into oil phase (A), the organic solvent-water interface during W1/O emulsion results in protein denaturation (B). During generation of secondary emulsion, water channels connecting internal (W1) and external (W2) aqueous phases (E) allow proteins to escape from droplets (C) and provide more chances of protein denaturation by increased surface area of the oil-water interface (D). The water channels become pores (F) of microspheres hardened by freeze-drying. Ice crystal (G) is known to provide a hazardous condition inducing protein denaturation (I). Irreversible aggregation (H) between protein molecules can be formed if stabilizer or cryoprotectant is not added. Normally, microspheres made by double emulsion have a broad range of particle size distribution as well as different protein amount in each microparticle. In a release test, a burst release of protein at the initial period (<24 h) is mostly due to the protein release (K) from the proteinaceous film on the particle surface (D). With time, proteins are release from particles (J) by diffusion and degradation (L) of polymer (e.g., PLGA). Microparticle degradation cumulates acidic products inside particles (M), which further facilitates protein denaturation (N). Protein adsorption on hydrophobic polymer surface (O) often leads to incomplete release of protein drugs  (reproduced with permission from Elsevier B.V. Ltd., 2010).