Understanding the interactions of lipid membranes with various cations, which can be found in the body, is of great importance considering their role in processes occurring at the molecular level in the cell membrane. Emphasis is given in literature on the importance of calcium based processes happening at the membrane interface, which include protein binding, cell signaling and cell activation.7 In this work, the effect of
calcium on the size and electrostatic stability of PC/PS liposome was studied. PC/PS liposome suspensions were prepared in a PBS buffer with 10 mM CaCl2 at a constant
ionic strength of ~164 mM.
Figure 4.5 displays the average hydrodynamic sizes and corresponding PDIs of PC/PS liposomes suspended in PBS solution with and without Ca2+ added. The introduction of Ca2+ di-cation, at a 10 mM concentration, results in a slight decrease in the
hydrodynamic size of PC/PS liposomes from 148 ± 2 nm (PDI 0.4) to 128 ± 3 nm (PDI 0.3). Liposomes size was stable over a range of 1 h for both PBS and PBS.CaCl2 buffers. The
slight decrease in liposome diameter is consistent with reported effects of divalent cations on the curvature of phospholipid vesicles and membranes.20, 22, 23, 45-47 It is well stablished that a stronger interaction occurs between lipid molecules and Ca2+ ions when compared to Na+ ions and lipid molecules. The stronger interaction for lipid/Ca2+ ion combination results in a significant change in the lipid membrane conformation.7, 21,
48 It is also well known that the introduction of Ca2+ results in a lower degree of
94 Figure 4.5.Hydrodynamic size (blue) of PC/PS liposomes in the absence and presence of 10 mM CaCl2 and its respective polidispersity index (PDI-red). Liposomes were extruded
through a nylon membrane (450 nm pore diameter). The hydrodynamic size was measured immediately after extrusion, for t=30 min and for t=60 min.
To understand the effect of Ca2+ on the electrostatic stability of the liposomes, ζ- potential measurements were performed on PC and PC/PS liposomes, Figure 4.6. As previously observed, the PC liposomes suspended in a PBS solution exhibit a ζ-potential of ~ 0 mV which is dramatically decreased to - 85 ± 1 mV when PS is added to the liposome, Table 4.2. The introduction of Ca2+ in the PBS buffer does not seem to interfere with the ζ-potential of PC liposomes that remain close to 0 mV, Figure 4.6. Interestingly, PC/PS liposomes have their ζ-potential increased to – 44 ± 1 mV, suggesting that the ζ-potential of the negatively charged liposome is significantly affected by the di-cation.
95 Figure 4.6.Apparent ζ-potential values of PC (red) and PC/PS (blue) liposomes in the
absence (solid line) and presence (dot line) of CaCl2 in the PBS solution.
Recently, Melcrova et al.7 reported on the interaction of Ca2+ with PC/PS membranes and suggested three possible binding sites for Ca2+ on the phospholipid membrane: (i) carboxyl groups of PS, (ii) phosphate groups of PC and PS, and (iii) carbonyl groups of PC and PS.7 Literature studies on the Ca2+ and phospholipid membranes have shown that Ca2+ binds primarily to the phosphate group of all phospholipids, including PC.47 Also, the binding of Ca2+ to the carboxyl group of PS have been studied by molecular dynamics simulation.47-48 It was shown that in the presence of PS, the Ca2+ binds both at the phosphate groups of PC and PS but also to the carboxyl group of PS.48
3. Conclusions
In this chapter, the physico-chemical properties of phospholipids liposomes were studied. Firstly, the FT-IR spectra of PC and PS phospholipids was analysed. Subsequently, PC and PS were combined in a liposome and their electrostatic stability in solution was examined regarding liposome preparation method, type and nature of phospholipids present in the liposome and buffer pH and ionic composition.
PC-containing liposomes prepared through the Morrisey protocol exhibit similar diameter when comparing with the Freeze-thaw procedure. The stability of the PC liposomes in solution was observed for both preparation protocols; the Morrisey
96 protocol was used due to its easy and simple protocol when compared with the Freeze- thaw.
The influence of PS on PC-based liposomes was analysed and it was found that the introduction of PS in a PC-containing liposome results in a smaller hydrodynamic size. Additionally, the PC/PS liposomes exhibit a significantly higher electrostatic stability in solution when compared with PC liposomes, displaying ζ-potential values in the stability range (< -20 mV), while PC liposomes display ζ-potential values close to 0 mV.
The influence of pH on PC/PS electrostatic stability was also studied. ζ-potential measurements on the PC/PS liposomes suspended in PBS buffer in a pH range of 4.5-9.5 showed that buffer pH does not affect significantly the ζ-potential of PC/PS liposomes in suspension. With regards to the hydrodynamic size, PC/PS liposomes hydrodynamic diameter reaches a minimum at pH 8.5 and was found to increase when PC/PS liposomes were suspended in more acidic and basic conditions.
Finally, the influence of buffer ionic composition was examined and showed that the introduction of Ca2+ in the PBS solution has a great influence on the ζ-potential of PC/PS
liposomes. Ca2+ is known to bind to the PC and PS phosphate groups and also to the PS
carboxylic group, resulting in an increase of ζ-potential values for PC/PS liposomes. The study presented in this chapter clarifies the physico-chemical properties of PC/PS liposomes in suspension. The results in this chapter on the PC/PS liposomes will be utilised in the following chapter. In subsequent chapters, the PC/PS liposomes will be incubated with carbon surfaces and their interaction will be examined with regards to buffer ionic composition and pH and amorphous carbon physico-chemical properties.
4. Experimental
4.1. Chemicals and Materials
Phosphatidylcholine from egg yolk 99.9% (PC) and 3-sn-Phosphatidyl-L-serine from bovine brain 95% (PS) were purchased from Sigma Aldrich as a lyophilized powder. Phosphate buffer saline solution (PBS 0.01 M, 0.0027 M KCl and 0.137 M NaCl pH 7.4)
97 was prepared by dilution of ready to use tablets (Sigma). Chloroform (HPLC grade) and calcium chloride were purchased from Sigma Aldrich, and were used without further purification. All water used for experiments was obtained from a Millipore purification system.