Insulin Particle Formation in Supersaturated Aqueous Solutions of Poly(Ethylene Glycol)

Abstract

Protein microspheres are of particular utility in the field of drug delivery. A novel, completely aqueous, process of microsphere fabrication has been devised based on controlled phase separation of protein from water-soluble polymers such as polyethylene glycols. The fabrication process results in the formation of spherical microparticles with narrow particle size distributions. Cooling of preheated human insulin-poly(ethylene glycol)-water solutions results in the facile formation of insulin particles. To map out the supersaturation conditions conducive to particle nucleation and growth, we determined the temperature- and concentration-dependent boundaries of an equilibrium liquid-solid phase separation. The kinetics of formation of microspheres were followed by dynamic and continuous-angle static light scattering techniques. The presence of PEG at a pH that was close to the protein's isoelectric point resulted in rapid nucleation and growth. The time elapsed from the moment of creation of a supersaturated solution and the detection of a solid phase in the system (the induction period, t(ind)) ranged from tens to several hundreds of seconds. The dependence of t(ind) on supersaturation could be described within the framework of classical nucleation theory, with the time needed for the formation of a critical nucleus (size <10 nm) being much longer than the time of the onset of particle growth. The growth was limited by cluster diffusion kinetics. The interfacial energies of the insulin particles were determined to be 3.2-3.4 and 2.2 mJ/m(2) at equilibrium temperatures of 25 and 37 degrees C, respectively. The insulin particles formed as a result of the process were monodisperse and uniformly spherical, in clear distinction to previously reported processes of microcrystalline insulin particle formation.

Comparison between Protein−Polyethylene Glycol (PEG) Interactions and the Effect of PEG on Protein−Protein Interactions Using the Liquid−Liquid Phase Transition

“…1,2 In pharmaceutical science, it is important for the preparation of protein microspheres relevant to drug delivery. 3,4 In enzymology, it is important for the preparation of novel cross-linked enzyme particles for catalysis in aqueous and nonaqueous media. 5,6 However, a sound scientific basis of the thermodynamic behavior of protein aqueous solutions is still missing.…”

Surface functional modification of self-assembled insulin nanospheres for improving intestinal absorption

“…The insulin nanospheres were spontaneously formed by a modified temperature induced phase separation [15]. Briefly, a solution buffered at pH 5.3-5.5 containing 12% PEG 2000 and 0.5% PVA was prepared and pre-heated to 75 • C. Bulk porcine insulin powder was directly added while stirring to allow complete dissolution within 3-5 min.…”

Formulations for delivery of therapeutic proteins

“…To achieve a high protein loading, an alternative to the classical microencapsulation protocols is production of a formulation from therapeutic protein itself, such as a preparation of protein particles. The formulations in a form of protein microspheres have been produced by different methods, including a gradual dialysis of protein solution against precipitating agents (Yakovlevski 2004), or by cooling of a pre-heated supersaturated solution (Bromerg et al 2005). Under the latter conditions, the presence of polyethylene glycol at a pH close to the protein isoelectric point results in a rapid nucleation and a particle growth, forming approximately 1-2 lm spherical particles.…”