Development and evaluation of a beta-glucan biopolymer formulation of lactoferrin produced using a novel cryomilling technique

Abstract

Transformation of therapeutic proteins from the new compound stage to a marketed product for clinical use depends on the development of an appropriate formulation, using technologies that avoid the underlying causes of protein degradation. Although synthetic biodegradable polymers have been extensively studied in the search for better protein delivery formulation development, the use of natural biodegradable polymers in drug delivery continues to be an area of active research. In the present study, a natural polymer, β-glucan, was investigated as a protein carrier. A novel methodology was developed to produce microparticles encapsulating bovine lactoferrin (bLF), a whey protein from milk. β-glucan was investigated as a protein delivery candidate for the first time in this study, the formulation demonstrated a significant improvement in in vivo activity, which suggested improved bioavailability of bLF when formulated using β-glucan. β-glucan is a polysaccharide obtained from a natural source, barley, it is extensively used as a food additive and is well known for its potential nutritional and biological applications. The physicochemical properties (rheological behaviour, mechanical strength, stress studies, molecular weight and density) of the β-glucan were investigated. Simulated gastric pH condition was found to have no effect on molecular weight and viscosity of β-glucan at all tested concentrations. The bLF was characterised for its mass, purity and amino acid sequence, bLFcin was generated by pepsin digestion, purified by ion exchange adsorptive membrane technique and characterised. Biological activity of bLF and bLFcin was evaluated. β-glucan films containing bLF (1 and 10% w/w) were cast with or without hydrophobic and hydrophilic excipients. Films were milled and optimised at different milling conditions and particle size analysis was conducted. The entrapment efficiency of bLF in films with hydrophobic excipients was found to be higher than films without excipients. A High pressure liquid chromatography (HPLC) method was developed and validated to measure the concentrations of bLF in the formulations. Complete recovery of bLF from films and particles could not be achieved and further studies were conducted to investigate the stability of bLF and interactions between bLF and β-glucan. The release of bLF from films was higher than from particles. Particles without excipients showed lower release. Solid state characterisation showed that there was no change in the structure when bLF was milled alone but minor changes were observed when bLF in β-glucan films were milled to form particles. These changes were due to interactions between β-glucan and bLF, which were investigated by surface plasmon resonance (SPR) studies. The stability evaluation of bLF and β-glucan was carried out using liquid chromatography-mass spectrometry (LC-MS/MS) and size exclusion chromatography-multi-angular laser light scattering (SEC-MALLS) respectively. In vitro evaluation of biological activity of bLF showed that there was no change in osteoblast proliferation activity after milling but reduction in activity was seen when bLF from the particles was tested. In vivo evaluation using a mouse model demonstrated a considerable improvement in bioavailability with particles encapsulating bLF and bone mineral density gain increased significantly. This study demonstrated the potential of β-glucan as an encapsulating agent for the oral delivery of therapeutic proteins.