Hydration Potential of Lysozyme: Protein Dehydration Using a Single Microparticle Technique

Abstract

For biological molecules in aqueous solution, the hydration pressure as a function of distance from the molecular surface represents a very short-range repulsive pressure that limits atom-atom contact, opposing the attractive van der Waals pressure. Whereas the separation distance for molecules that easily arrange into ordered arrays (e.g., lipids, DNA, collagen fibers) can be determined from x-ray diffraction, many globular proteins are not as easily structured. Using a new micropipette technique, spherical, glassified protein microbeads can be made that allow determination of protein hydration as a function of the water activity (a(w)) in a surrounding medium (decanol). By adjusting a(w) of the dehydration medium, the final protein concentration of the solid microbead is controlled, and ranges from 700 to 1150 mg/mL. By controlling a(w) (and thus the osmotic pressure) around lysozyme, the repulsive pressure was determined as a function of distance between each globular, ellipsoid protein. For separation distances, d, between 2.5 and 9 A, the repulsive decay length was 1.7 A and the pressure extrapolated to d = 0 was 2.2 x 10(8) N/m(2), indicating that the hydration pressure for lysozyme is similar to other biological interfaces such as phospholipid bilayers.

From Single Microparticles to Microfluidic Emulsification: Fundamental Properties (Solubility, Density, Phase Separation) from Micropipette Manipulation of Solvent, Drug and Polymer Microspheres

“…First used in a non-mechanical way in 1998 to simply position micro-hydrogel beads with and without a loaded-drug (doxorubicin) in a controlled flow field of different pH [23,24], the micropipette technique was then developed by Needham et al [1][2][3][4][25][26][27][28][29][30][31][32][33][34][35][36][37], Tony Yeung et al [38], and others, in a series of papers on adsorption at interfaces and two-phase oil-aqueous systems. It has now become a highly versatile experimental setup that allows a wide variety of studies at microscopic interfaces and with single-and pairs-of microparticles.…”

“…It has now become a highly versatile experimental setup that allows a wide variety of studies at microscopic interfaces and with single-and pairs-of microparticles. Among other applications, the technique has been established for studying solvent dissolution, measuring fundamental properties such as diffusion coefficients and solubilities of the dissolving liquids [4,27,30], and for evaluating the phase separation, precipitation of droplets containing different solutes upon solvent loss, such as the micro-glassification of proteins by fast removal of water into water-imbibing solvents like octanol [3,31], and drug-containing polymer microspheres by removal of the organic solvent into water [28,29]. It is this last application that has motivated and guided the current studies with poly(lactic-co-glycolic acid) (PLGA) microparticles containing Ibuprofen [34].…”

“…For the particular droplet shown here in Figure 6a, it can be clearly observed that it rapidly dissolved and reached the size of the holding pipette in 9 s (dotted line in Figure 6b-i), at which point the holding suction pressure in the pipette aspirated the small droplet far into the pipette. A re-plot of the data is presented in Figure 6b-ii, and fitted to the linear form of the EpsteinPlesset (EP) model, Equation (3). This representation collapses the data onto a single line, independent to the droplet initial size.…”

“…Con sid erin g t dissolution into water: CS = 17.22 mg/mL and ρ = 1325 mg/mL (both at 25 °C), the EP model provided an experimental value for the diffusion coefficient of DCM in water of D = 1.77 ± 0.01 × 10 −5 cm −2 /s. The estimated D value of DCM from the Wilke and Lee model was 1.22 × 10 −5 cm 2 /s [71], which was A re-plot of the data is presented in Figure 6b-ii, and fitted to the linear form of the Epstein-Plesset (EP) model, Equation (3). This representation collapses the data onto a single line, independent to the droplet initial size.…”

“…In this respect, we introduce here our micropipette manipulation technique [1][2][3][4], capable of making fundamental single particle measurements and analyses, and how this information is critical to processing parameters such as in microfluidic processing, and also homogenization. Thus, in this paper, where we start to combine the two techniques, we simply wanted to introduce and show that the micropipette manipulation technique can guide certain aspects of scale-up study associated with parameters especially the use of microfluidic devices.…”