Abstract
Monoolein-based cubic and hexagonal mesophases were investigated as matrices for insulin loading, at low pH, as a function of temperature and in the presence of increasing amounts of oleic acid, as a structural stabilizer for the hexagonal phase. Synchrotron small angle X-ray diffraction, rheological measurements, and attenuated total reflection-Fourier transform infrared spectroscopy were used to study the effects of insulin loading on the lipid mesophases and of the effect of protein confinement in the 2D- and 3D-lipid matrix water channels on its stability and unfolding behavior. We found that insulin encapsulation has only little effects both on the mesophase structures and on the viscoelastic properties of lipid systems, whereas protein confinement affects the response of the secondary structure of insulin to thermal changes in a different manner according to the specific mesophase: in the cubic structure, the unfolding toward an unordered structure is favored, while the prevalence of parallel β-sheets, and nuclei for fibril formation, is observed in hexagonal structures.
Highlights
Lyotropic liquid crystalline (LLC) mesophases, cubic and hexagonal ones, have been extensively studied as vehicles for molecules of different size and polarity such as small drugs, vitamins, peptides, proteins, and nucleic acids.[1−6] encapsulation permits the control of the release of the enclosed molecule in space and time, possibly allowing targeted therapies, and represents an efficient strategy for biomolecule stabilization
The use of complementary techniques including small angle X-ray scattering (SAXS), rheological measurements, and ATR-FTIR spectroscopy provided a useful approach for studying the effect of encapsulated insulin on the cubic and hexagonal mesophases and of the confinement in nanostructured water channels on the secondary structure of the protein and on its stability
Upon heating with the concomitant increase of the random coils, Data collected at room temperature showed that insulin whereas just a small increase in the β-sheets was observed. encapsulation in GMO cubic mesophase did not affect the According to these results, GMO cubic system seems to favor structure since the aqueous channels are large enough to load the heat-induced unfolding toward an unordered structure, the protein even in the dimeric form
Summary
Lyotropic liquid crystalline (LLC) mesophases, cubic and hexagonal ones, have been extensively studied as vehicles for molecules of different size and polarity such as small drugs, vitamins, peptides, proteins, and nucleic acids.[1−6] encapsulation permits the control of the release of the enclosed molecule in space and time, possibly allowing targeted therapies, and represents an efficient strategy for biomolecule stabilization. Different hydrophilic proteins, ranging from small cytochrome c and lysozyme to large fibrinogen and apo-ferritin, have been incorporated within the lipidic cubic[12] or hexagonal mesophases,[23] sometimes inducing structural transitions. Among these proteins is insulin, a protein hormone important for the regulation of glucose metabolism and used worldwide in the treatment of diabetes. GMO cubic structures were used to protect insulin from aggregation induced by either agitation,[28] temperature,[26] or enzymatic degradation.[29] On the other hand, different modified reverse hexagonal systems based on GMO and co-surfactants were used by Garti and co-workers to explore the effect of confinement on the stability, morphology, and unfolding behavior of the protein upon heating or pH changes.[30−33]. These spectra were curve-fitted in the 1800−1400 cm−1 spectral range; the number and the position of the underlying bands were identified by second derivative minima analysis and fixed during the peak-fitting procedure with Gaussian functions; the integrated areas of all the underlying bands were obtained (GRAMS/AI 9.1, Galactic Industries, Inc., Salem, New Hampshire)
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