Effect of buffer composition and preparation protocol on the dispersion stability and interfacial behavior of aqueous DPPC dispersions

Abstract

The effect of the buffer composition and the preparation protocol on the dynamic surface tension (DST) and vesicle sizes of aqueous dipalmitoylphosphatidylcholine (DPPC) dispersions was studied. Four isotonic buffers were used in preparing DPPC dispersions at physiological conditions for possible biological applications: (1) a standard PBS solution; (2) the above PBS with 1 mM CaCl₂; (3) PBS with one tenth the previous standard phosphate salt concentrations and 2.5 mM CaCl₂; and (4) 150 mM NaCl with 2.5 mM CaCl₂ and 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). Two protocols, with a new method and an old method (Bangham method), were used in preparing the DPPC dispersions. The DPPC dispersions prepared with the new method contained mostly vesicles and were quite stable at 25 or 37 °C. Dynamic light scattering (DLS) and spectroturbidimetry (ST) results showed that the DPPC vesicle sizes in buffer (4) were much smaller than those in the other buffers. When the DPPC dispersions were prepared with the new method, the diameter of the DPPC particles was smaller than those with the old method. The DPPC vesicles with the new method were more stable than those with the other method. The DPPC dispersions of 1000 ppm at 37 °C with the new method produced, at pulsating area conditions at 20 cycles per minute, low tension minima (γ_min), lower than 10 mN/m, in buffers (1), (2), and (4). With buffer (4) the DSTs were lower and were achieved faster than with the other buffers. A minimum concentration of 1000 or 250 ppm DPPC was needed to produce DSTs lower than 10 mN/m within 10 min or less, with buffer (2) or (4), respectively. IRRAS results suggest that DPPC in buffer (2) or (4) forms a close-packed monolayer at the interface. These results have implications for designing efficient protocols of lipid dispersion preparation and lung surfactant replacement formulations in treating respiratory disease.

Introduction

Dipalmitoylphosphatidylcholine (DPPC) is the major lipid ingredient of natural lung surfactant, which controls the dynamic surface tension (DST) and helps maintain the lung alveoli healthy [1]. Above 41 °C, the DPPC chain melting transition temperature, DPPC forms fluid “liposomes,” which are multilamellar liquid crystal droplets or “onions,” and fluid “vesicles,” which are unilamellar, with one lipid bimolecular layer, or bilayer, surrounding an aqueous core [2], [3], [4], [5]. As these dispersions are cooled to 25 or 37 °C, they form “frozen” liposomes or “frozen” vesicles, in which the chains are in a solid-like state. DPPC lipid is also the major component of some commercial synthetic lung surfactant replacement formulations [1], [5].

At conditions of large size particulates dispersed in aqueous solution, DPPC is reported to adsorb at the air/water surface, and reduce DSTs at pulsating area conditions quite slowly [5]. Our hypothesis has been that the slow rate of adsorption is linked to slow diffusion of particles to the surface [6]. In order to increase the DPPC particles diffusion rate to the interface, DPPC particles should be made as small as possible. The sizes of DPPC particles, liposomes or vesicles, vary from about 20 nm to over 1 μm, depending on the protocol of the dispersion preparation and composition [5], [6], [7], [8], [9], [10], [11]. One method for producing vesicles is by sonication following extensive stirring. For DPPC, we have explored and developed new protocols for preparing dispersions with very low minimum DST’s, lower than 5 mN/m [12], [13]. In one protocol we produced colloidally stable dispersions of DPPC “frozen” vesicles in both water and non-isotonic phosphate saline buffer. The protocol involves several key steps. The first is stirring extensively DPPC liposomes (multilamellar liquid microcrystalline droplets, or “onions”) above the chain melting transition temperature, T_c = 41 °C. The second step is extensive sonication of the small liposomes to produce small vesicles, which are fluid at a temperature T = 55 °C. Cooling the dispersion to 37 or at 25 °C, produces frozen vesicles. The present research aims at finding out how general this behavior of DST lowering is at physiologically relevant conditions, and evaluating the possibility of the lipid to be used in practice by producing low DSTs when the surface area is compressed by about 50% at pulsating area conditions.

This article aims also to clarify the issue on whether DPPC can have such behavior with no supporting lipids, lung protein, or spreading agents. Another goal is to compare our protocol to the one described by Bangham [14], [15]. More importantly, the effect of using physiologically relevant, or “biocompatible,” aqueous solutions is examined.

If one needs to add some calcium ion concentration, in the range of 1–2.5 mM, then some phosphate salt may precipitate. To use 2.5 mM CaCl₂, one has to either reduce the phosphate concentration, or eliminate it, or use another method for pH buffering, such as an organic molecule called HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). The question arises then as to the effect of the buffer composition on the DPPC vesicle formation and stability, and on the level of DSTs at 37 °C. In this article, the two protocols are compared.

To find the proper concentration range of DPPC in producing adequately low DSTs, the effect of concentration on the DST of DPPC is studied. The pulsating bubble surfactometer (PBS) was primarily used. The interfacial layer was also probed directly with infrared reflection absorption spectroscopy (IRRAS), which can detect adsorbed components qualitatively and determine quantitatively the surface density. In addition, dynamic light scattering (DLS), and UV–vis spectroturbidimetry were used to characterize particles sizes and microstructure in detail.