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Effect of a PEGylated Lipid on the Dispersion Stability and Dynamic Surface Tension of Aqueous DPPC and on the Interactions with Albumin Yoonjee Park and Elias I. Franses* School of Chemical Engineering, Purdue University, 480 Stadium Mall Drive, West Lafayette, Indiana 47907-2100 Received November 3, 2009. Revised Manuscript Received January 18, 2010 Dispersions of dipalmitoylphosphatidylcholine (DPPC) vesicles at 0.1 wt % (1000 ppm) in aqueous isotonic buffer solutions produced by extensive sonication were found to be colloidally stable for hours and days. They also had very low (20 mN/m. Only at X=0.119 was the DSTM lower than 10 mN/m. Similar results (68) Kim, S. H.; Franses, E. I. J. Colloid Interface Sci. 2006, 295, 84. (69) Kim, S. H.; Franses, E. I. Colloids Surf., B 2005, 43, 256.
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were observed with the S-PEG/DPPC mixed vesicles in the HBS buffer solution. Evidently, the mixed lipid-protein interaction, first in the dispersion and then in the mixed monolayer, is quite complex and sensitive to the value of X. One would expect that the more stable mixed vesicles at X = 0.188 will diffuse and possibly adsorb faster than those at lower values of X. Nevertheless, the different lipid composition may change the thermodynamic surface tension/surface density isotherm in such a way as to produce larger DSTs. Moreover, as the PEGylated lipid mole fraction increases, the DPPC concentration decreases, and this may affect the surface tension to some extent, as has been reported in ref 49. The exact molecular basis of the dependence of the equilibrium or dynamic surface tension on the lipid composition X or the lipid-protein composition of the dispersed particles and of the mixed surface layer remains unclear. Moreover, it is possible that there are also other mole fractions X for which favorable DSTM behavior may exist. Future work may focus on discovering such systems and elucidating their behavior. To obtain some information on the state of the interfacial layer, certain ellipsometry data were obtained (Figure 11). For pure DPPC, where a lipid monolayer of thickness 2-3 nm is expected, the values of |δΔ| were 0.3-0.5. Spread DPPC lipid monolayers with surface densities Γ ranging from 410-6 to 210-6 mol/m2 (surface area a ranging from 0.4 to 0.8 nm2/molecule, respectively) have |δΔ| values of about 0.5 ( 0.1.11,70 Since δΔ for this system is close to that of monolayers, it cannot be determined whether there are any vesicles attached to the interfacial monolayer, as has been found before for DPPC vesicles in water.63 The |δΔ| value of the mixed lipid at X = 0.119 (line 2 in Figure 11) was somewhat higher, probably because of the presence of the longer PEG moiety, which may increase the overall monolayer thickness. The |δΔ| value for albumin alone, 0.7-0.8, reflects the larger size of the molecule (4 4 14 nm3), as has been reported previously.71 The interfacial layer of DPPC mixed with BSA had much higher values of |δΔ|, 2 after 30 min. These values indicate a much thicker lipid/protein layer on the surface than an albumin monolayer or a DPPC monolayer, or the presence of aggregated particles. A schematic diagram is shown in Figure 12B. When a mixed lipid with X = 0.119 was used, the value of |δΔ| was about the same as that with the mixed lipid alone. Hence, the (70) Wen, X. Y.; Franses, E. I. Langmuir 2001, 17, 3194. (71) McClellan, S. J.; Franses, E. I. Colloids Surf., B 2005, 260, 265.
DOI: 10.1021/la904183e
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Park and Franses
Figure 12C). We speculate that this is the reason why the surface tension lowering ability of the mixed lipid is not inhibited. More detailed studies are needed to probe the compositions and surface densities of the interfacial monolayers at conditions which yield low DSTM.
Figure 12. Schematic diagrams of postulated adsorption mechanisms of DPPC or DPPC þ BSA vesicles: (A) a DPPC vesicle adsorbs and DPPC molecules spread at the surface, in a vesicle “unzipping” type mechanism; (B) DPPC þ BSA mixture; BSA molecules adsorb first and remain adsorbed; some vesicles may adsorb and open to form a partial interfacial lipid film; the vesicles remain attached to the monolayer; big aggregates in the bulk phase due to hetoerocoagulation with BSA may cause the formation of a thick layer at the surface; the aggregates may absorb and open up to form a partial interfacial lipid film; (C) S-PEG/DPPC þ BSA mixture; aggregation does not occur in the bulk phase due to the steric repulsive effect of the PEG; the modified vesicles adsorb and form a lipid film; some adsorbed S-PEG molecules may play a role of repelling BSA molecules and help maintain the low dynamic surface tension minima at the surface.
presence of a lipid monolayer is inferred. Any protein or particle adsorption appears to be insignificant (a schematic is shown in
6942 DOI: 10.1021/la904183e
Conclusions The stability of mixed vesicles of dipalmitoylphosphatidylcholine (DPPC) with N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)750]} (S-PEG) in two physiological buffer solutions and at 25 or 37 C was evaluated with dynamic light scattering (DLS) at a concentration of 1000 ppm (1 g/L) of total lipid. For mole fractions X of S-PEG in the total lipid ranging from 0.028 to 0.188, the vesicles become more stable, for days and weeks. At mole fractions of 0.028 and 0.119, the dynamic surface tension minima (DSTM) behavior is quite similar to that of DPPC alone. At X = 0.057, the behavior is slightly different. When mixed with 1000 ppm bovine serum albumin (BSA), the pure DPPC lipid surface tension lowering ability is significantly impaired. The albumin induces fast and massive lipid vesiclealbumin heterocoagulation. Also a substantial inhibition in the surface tension lowering ability of the lipid by the introduction of the albumin is observed. Protein and lipid-protein aggregates may adsorb at the air/water interface, creating a layer with less lipid and more protein, and thus lead to higher DSTM. When the DPPC lipid is mixed with S-PEG, such lipid-protein heterocoagulation is mostly prevented or slows down considerably. A simple molecular model indicates that there are substantial electrostatic attractive interactions between the positive ionic groups in the DPPC zwitterionic headgroup and the negatively charged protein molecules. These interactions become much weaker with the mixed lipid vesicles, probably because of a steric PEG barrier. The DSTM behavior also improves considerably. At a narrow range of X-values, the DSTM are lower than 10 mN/m within 5-20 min, similar to DSTM before the introduction of the albumin in the solution. These results may have implications for understanding the shelf life of lipid formulations, lipid-protein inhibition phenomena, and perhaps future lipid formulations for lung-surfactant replacement therapies. Acknowledgment. This research was supported by NSF Grant # CBET 0651942. We thank Mrs. Debbie Sherman for advice and help with the electron microscopy images. We thank Professor You-Yeon Won for allowing us to use the DSL ZetaPALS instrument. We thank Professor Nien-Hwa Linda Wang for helpful discussions about short-range ionic interations.
Langmuir 2010, 26(10), 6932–6942
