Lipid Corralling and Poloxamer Squeeze-out in Membranes
Guohui Wu 1, Jaroslaw Majewski 2, Canay Ege 1,
Kristian Kjaer 3, Markus Weygand 3 & Ka Yee C. Lee 1
1 Department of Chemistry, the University of Chicago, Chicago, IL 60637
2 Manuel Lujan Jr. Neutron Scattering Center, Los Alamos National Laboratory, Los Alamos, NM 87545
3 Materials Research Department, Risø National Laboratory, DK-4000 Roskilde, Denmark
Poloxamers are a family of non-ionic surface-active block copolymers constituted by a hydrophobic poly-propylene oxide (PPO) moiety capped with hydrophilic poly-ethylene oxide (PEO) moieties on the two ends, rendering this class of molecules amphiphilic. Poloxamers have been demonstrated to be effective in sealing permeabilized cell membranes. It turns out that traumas and diseases such as electrical shocks, radiation injuries, thermal burns, frostbites, ischemia-reperfusion injuries and sickle cell disease can all lead to a loss of the integrity of the cell membrane [1]. Such a break in structural integrity can immediately compromise the essential role of the cell membrane as a barrier, affecting its control over the transport of materials into and out of the cell, and can eventually kill the affected cell [2, 3]. In spite of the importance of poloxamers’ ability to help restore structurally compromised membranes, the mechanisms involved are not well understood.
Poloxamer 188 (P188, MW = 8400 g/mol, with 80 wt% PEO) was the first among this family of copolymers to be tested as a candidate for membrane sealant, primarily because it had been widely used in medical, pharmaceutical and cosmetic systems as a solubilizing, wetting and emulsifying agent with low toxicity. P188 was found to be effective in reducing the leakage of dye from loaded cells after electroporation [1].
To gain insight into the molecular mechanisms of interaction between P188 and damaged membranes, we have used lipid monolayers composed of dipalmitoylphosphatidylcholine (DPPC), which is abundant in human erythrocytes [18], as a model for the outer leaflet of the membrane. We have performed Langmuir isotherm, X-ray reflectivity (XR) and grazing incidence X-ray diffraction (GIXD) experiments on the lipid/poloxamer system to address the following questions: Does P188 seal by inserting into portions of the membrane whose structural integrity has been compromised? What is the effect on lipid packing in the damaged membrane when P188 is present? If P188 really helps seal the membrane, what is the fate of the poloxamer when the membrane regains its integrity? To the best of our knowledge, this combination of techniques is used for the first time to investigate the lipid/P188 system, allowing us to assess both macroscopic information and molecular details of the lipid/poloxamer interactions, and providing answers to the questions posed.
Using concurrent Langmuir isotherm and fluorescence microscopy measurements, we have found that P188 changes the phase behavior and morphology of the monolayers. P188 inserts into both dipalmitoylphosphatidlycholine and dipalmitoylphosphatidylglycerol monolayers at surface pressures equal to and lower than ~22 mN/m at 30°C; this pressure corresponds to the maximal surface pressure attained by P188 on a pure water subphase. Similar results for the two phospholipids indicate that P188 insertion is not influenced by headgroup electrostatics. Because the equivalent surface pressure of a normal bilayer is on the order of 30 mN/m, the lack of P188 insertion above 22 mN/m further suggests the poloxamer selectively adsorbs into damaged portions of electroporated membranes, thereby localizing its effect [4, 5].
Our X-ray results indicate that the selective insertion of P188 into low lipid-density regions of the membrane “corrals” lipid molecules to pack tightly, giving rise to unexpected Bragg peaks at low nominal lipid density and inducing the film to separate into P188-rich and P188–poor phases. This corralling of the lipid molecules helps the membrane to regain its barrier function. As the cell heals and reestablishes its normal lipid packing density, the once inserted P188 is squeezed out from the lipid film, thus providing a route for the poloxamer to gracefully exit when the membrane integrity is restored [6].
1. Lee, R. C., River, L. P., Pan, F. S., Ji, L. & Wollmann, R. L. (1992) Proc. Natl. Acad. Sci. U. S. A.89, 4524-4528.
2. Lee, R. C., Zhang, D. & Hannig, J. (2000) Annu. Rev. Biomed. Eng.02, 477-509.
3. McNeil, P. L. & Steinhardt, R. A. (1997) J. Cell Biol.137, 1.
4. Maskarinec, S. A., Hannig, J., Lee, R. C. & Lee, K Y. C. (2002) Biophysical Journal82 1453-1459.
5. Maskarinec, S. A. & Lee, K Y. C. (2003) Langmuir19, 1809-1815.
6. Wu, G., Majewski, J., Ege, C., Kjaer, K., Weygand, M. & Lee K. Y. C., Physical Review Letters 93 (2004) 028101.
