Towards controlled release of BDNF--manufacturing strategies for protein-loaded lipid implants and biocompatibility evaluation in the brain.
It was the aim of this study to establish triglyceride matrices as potential carriers for long-term release of brain-derived neurotrophic factor (BDNF), a potential therapeutic for Huntington's disease. First, four different manufacturing strategies were investigated with lysozyme as a model substance: either lyophilized protein was mixed with lipid powder, or suspended in organic solution thereof (s/o). Or else, an aqueous protein solution was dispersed by w/o emulsion in organic lipid solution. Alternatively, a PEG co-lyophilization was performed prior to dispersing solid protein microparticles in organic lipid solution. After removal of the solvent(s), the resulting powder formulations were compressed at 250 N to form mini-cylinders of 2 mm diameter, 2.2 mm height and 7 mg weight. Protein integrity after formulation and release was evaluated from an enzyme activity assay and SDS-PAGE. Confocal microscopy revealed that the resulting distribution of FITC-lysozyme within the matrices depended strongly on the manufacturing method, which had an important impact on matrix performance: matrices with a very fine and homogeneous protein distribution (PEG co-lyophilization) continually released protein for 2 months. The other methods did not guarantee a homogeneous distribution and either failed in sustaining release for more than 1 week (powder mixture), completely liberating the loading (s/o dispersion) or preserving protein activity during manufacturing (w/o emulsion, formation of aggregates and 25% activity loss). Based on these results, miniature-sized implants of 1 mm diameter, 0.8 mm height and 1 mg weight were successfully loaded by the PEG co-lyophilization method with 2% BDNF and 2% PEG. Release studies in phosphate buffer pH 7.4 at 4 and 37 degrees C revealed a controlled release of either 20 or 60% intact protein over one month as determined by ELISA. SDS-PAGE detected only minor aggregates in the matrix during release at higher temperature. In vivo evaluation of lipid cylinders in the striatum of rat brains revealed a biocompatibility comparable to silicone reference cylinders.
Koennings
S
S
Department of Pharmaceutical Technology, University of Regensburg, Universitaetsstr, 31, 93040 Regensburg, Germany.
Sapin
A
A
Blunk
T
T
Menei
P
P
Goepferich
A
A
D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D019208:Brain-Derived Neurotrophic Factor / Q000008:administration & dosage / Q000493:pharmacokinetics*; D002626:Chemistry, Pharmaceutical / Q000379:methods; D002645:Chickens; D003692:Delayed-Action Preparations / Q000008:administration & dosage / Q000493:pharmacokinetics; D016503:Drug Delivery Systems / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004343:Drug Implants; D005260:Female; D008055:Lipids / Q000008:administration & dosage / Q000493:pharmacokinetics*; D008422:Materials Testing / Q000379:methods; D009113:Muramidase / Q000008:administration & dosage / Q000493:pharmacokinetics*; D051381:Rats; D011916:Rats, Inbred F344
D019208:Brain-Derived Neurotrophic Factor; D003692:Delayed-Action Preparations; D004343:Drug Implants; D008055:Lipids; D009113:Muramidase