Confocal Microscopy for the Elucidation of Mass Transport Mechanisms
Involved in Protein Release from Lipid-based Matrices
and Achim Go
Received September 25, 2006; accepted January 31, 2007; published online April 25, 2007
Purpose. It was the aim of this study to identify the governing mechanisms during protein release from
cylindrical lipid matrices by visualizing mass transport and correlating the data with in vitro dissolution
Materials and Methods. Glyceryl trimyristate cylinders of 2 mm diameter, 2.2 mm height and 7 mg
weight were manufactured by compression of a protein–lipid powder mixture prepared by a
polyethylene glycol (PEG) co-lyophilization technique. BSA was fluorescence-labeled and the
distribution visualized and quantified at different stages of the release process by confocal microscopy
in parallel to the quantification in the release buffer. The impact of matrix loading and protein molecular
weight was assessed with the model proteins lysozyme, BSA, alcohol dehydrogenase and thyroglobulin.
Results. Buffer penetration and protein release occurred simultaneously from the outer regions of the
cylinder progressing towards the center. Release from the top and bottom of the matrix was not
negligible but much slower than penetration from the side, probably due to an oriented arrangement of
lipid flakes during compression. The different quantification strategies were found to yield identical
results. At 6% protein loading, buffer penetration was complete after 4 days, while only 60% of the
protein was liberated in that time and release continued up to day 63. Protein release kinetics could be
described using the power law equation M
with an average time exponent n of 0.45 (T0.04) for
loadings varying between 1 and 8%. A percolation threshold at 5% pure protein loading and 3–4%
mixed loading (PEG and protein at a 1:1 mass ratio) could be identified. Release rate was found to
decrease with increasing molecular weight.
Conclusions. Protein release from lipid-based matrices is a purely diffusion controlled mechanism.
Potential protein stabilization approaches should address the time span between complete buffer
penetration of the matrix and 100% release of the remaining loading, which would be exposed to an
aqueous environment before leaving the matrix.
KEY WORDS: confocal microscopy; controlled release of proteins; diffusion; lipid matrices; molecular
weight; release mechanism.
In controlled release science, substantial research efforts are
devoted to the evaluation of suitable matrix materials for the
delivery of protein drugs (1). Triglycerides have gained growing
attention in this context due to their favorable properties: as
physiological substances they have shown good biocompatibility
tested subcutaneously (2) and in the brain (3); they are easily
compactable and display high long-term stability upon incuba-
tion without swelling (4), which qualifies them as a good
alternative to commonly used polymeric matrix materials. A
successful incorporation into microparticles or cylinders and
release thereof has been reported for several peptide and
protein drugs and model substances, amongst them insulin
(5–7), somatostatin (8), interferon a-2a (9), BSA, hyaluroni-
dase (10), lysozyme, brain derived neurotrophic factor (3)and
interleukin-18 (IL-18) (11).
Although stability risks related to the incorporation into
polymers, such as the development of an acidic microclimate
during incubation (12–14) and the formation of detrimental
polymer degradation products (15) can be circumvented by
the use of triglyceride matrices, in parallel studies, we have
encountered problems during release testing, manifesting in a
progressive activity loss (11) or the occurrence of aggregates
(3). These effects were more pronounced at higher incuba-
tion temperatures suggesting an instability mechanism relat-
2007 Springer Science + Business Media, LLC
Pharmaceutical Research, Vol. 24, No. 7, July 2007 (
Department of Pharmaceutical Technology, University of Regensburg,
Universitaetsstr. 31, 93040, Regensburg, Germany.
To whom correspondence should be addressed. (e-mail: achim.
ABBREVIATIONS: ADH, alcohol dehydrogenase; BCA, biscinchoninic
acid; BSA, bovine serum albumin; CLSM, confocal laser scanning
microscopy; DCM, dichloromethane; DIPEA, N-ethyldiisopropylamine;
DMSO, dimethylsulfoxide; FITC, fluorescein isothiocyanate; IL,
, methoxy-PEG-amine; MWCO, molecular
weight cut-off; PEG, polyethylene glycol; TAMRA, carboxy-
tetramethylrhodamine; SRH, sulforhodamine 101 hydrate; THF,