Interactions of DPPC with Semitelechelic Poly(glycerol methacrylate)s

Article pubs.acs.org/Langmuir

Interactions of DPPC with Semitelechelic Poly(glycerol methacrylate)s with Perfluoroalkyl End Groups Peggy Scholtysek, Zheng Li, Jörg Kressler, and Alfred Blume* Institute of Chemistry, Martin-Luther University Halle-Wittenberg, D-06120 Halle (Saale), Germany S Supporting Information *

ABSTRACT: Semitelechelic poly(glycerol methacrylate)s having a perfluoroalkyl end group (PGMAn-F 9) were synthesized by ATRP. The interactions of these polymers with different degrees of polymerization with chiral or racemic dipalmitoylphosphatidylcholine (L-DPPC, D-DPPC, or racDPPC) monolayers at the air/water interface were studied. Langmuir trough measurements coupled with epifluorescence microscopy allowed for the observation of domain formation within the coexistence region of liquid-expanded (LE) and liquid-condensed (LC) states of DPPC in mixed DPPC−polymer films prepared by spreading a solution of both compounds in the same organic solvent (cospread films). Because of the incorporation of PGMAn-F9 polymers into the LE phase and their lineactive behavior, a formation of novel types of domains could be observed. During compression, a thinning out of the tips of twoto six-lobed flowerlike domain structures and consecutive spiral formation appeared for L- and D-DPPC within the two-phase coexistence region (LE/LC) of the monolayer. When rac-DPPC was used, symmetrical stripe formation was induced at the vertices of the domains and fingerprint-like structures were created by convection-inducing movements of the domains at the air/ water interface. Additional investigations of the interaction of PGMAn-F9 with DPPC vesicles using differential scanning calorimetry (DSC) supported the finding on the monolayer system that the incorporation of the polymers into the lipid monolayers is not solely driven by the perfluoroalkyl chain but significantly by the hydrophilic polymer part. Apparently, interactions of the PGMA chain with the lipid headgroups are important as the interactions increase with the elongation of the polymer chain, indicating that the polymer also has hydrophobic character.

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INTRODUCTION Monomolecular films of phospholipids at the air/water interface can be used as simple models for half of a lipid bilayer. Because of the large planar area of the monolayers, they are ideally suited for using various microscopic and scattering methods.1,2 In addition, the monolayer composition and the molecular area of amphiphilic molecules spread on the air/ water interface can be controlled easily by compressing the film to the desired surface pressure. For our investigations of the interaction of PGMAn-F9 polymers consisting of a polymer with different degrees of polymerization end-capped with a perfluoroalkyl chain with nine carbon atoms (Scheme 1c) with lipid monolayers, we used L-enantiomer 1,2-dipalmitoylsn-glycero-3-phosphocholine (L-DPPC) and D-enantiomer 2,3dipalmitoyl-sn-glycero-1-phosphocholine (D-DPPC)3 and also a 1:1 racemic mixture of the two enantiomers (rac-DPPC). DPPC has a glycerol backbone with two hydrophobic fatty acid chains connected to the sn-1 and sn-2 positions of glycerol for L-DPPC and to the sn-2 and sn-3 positions for D-DPPC (Scheme 1a,b). When a DPPC monolayer at the air/water interface is compressed at constant temperature, a phase transition is induced from a liquid-expanded (LE) to a liquid-condensed (LC) phase. The LC domains in the LE/LC coexistence region have typical triskelion structures. This propeller domain shape © 2012 American Chemical Society

is due to long-range orientational order in the domains leading to optical anisotropy along the triskelion arms.4,5 A competition between line tension and electrostatic dipole−dipole repulsion between the domains with different dipole densities as well as the chirality of the phospholipid (intermolecular chiral forces) determines the domain size and shape.3,4,6,7 The chirality of the molecules is a prerequisite for the chiral domain shapes provided the stereogenic center is situated in a larger functional group (hydrophilic headgroup) compared to the size of residual functional groups of the molecules (cross sections of the hydrophobic fatty acid chains). This is the case for phosphatidylcholines with their larger headgroups compared to those of other phospholipids such as phosphatidylethanolamines (PE) and phosphatidic acids (PA) that have smaller cross sections of their headgroups, and LC domains formed during compression are nonchiral.7 The perfluoroalkyl moieties in amphiphilic molecules possess special properties, namely, hydrophobicity and lipophobicity, high thermal and chemical stability and low polarizibility of the C−F bonds, high electron density, stability against biological decomposition, and a strong tendency to self-assemble.8−18 Received: July 12, 2012 Revised: October 9, 2012 Published: October 9, 2012 15651

dx.doi.org/10.1021/la3028226 | Langmuir 2012, 28, 15651−15662

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micellar concentration (cmc) of approximately 10−5 mol L−1. We will show that they are also surface-active and interact strongly with preformed lipid monolayers at the air/water interface. Using epifluorescence microscopy, it could be shown that these polymers act as line-active compounds leading to marked changes in the shape of the LC domains of DPPC in the LE/LC phase coexistence region of DPPC. Additional measurements of mixed lipid/polymer vesicles suspended in water were performed using differential scanning calorimetry (DSC) to test whether these polymers also incorporate into lipid bilayers and influence the thermotropic properties of the liposomes.

Scheme 1. Chemical Structures of (a) 1,2-Dipalmitoyl-snglycero-3-phosphocholine (L-DPPC), (b) 2,3-Dipalmitoylsn-glycero-1-phosphocholine (D-DPPC), and (c) Semitelechelic PGMA of Type PGMAn-F9, Where n = 14, 40, or 90

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EXPERIMENTAL SECTION

Materials. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (L-DPPC) was purchased from Genzyme Pharmaceuticals (Liestal, Switzerland) or Sigma-Aldrich (Schnelldorf, Germany) with a purity of >99% and was used without further purification. The 2,3-dipalmitoyl-sn-glycero1-phosphocholine (D-DPPC) enantiomer was purchased from SigmaAldrich (Schnelldorf, Germany) with a purity of >99% and used without further purification. For the preparation of the racemic mixture (rac-DPPC), D-DPPC was mixed with L-DPPC in a 1:1 molar ratio. The semitelechelic poly(glycerol monomethacrylate) (PGMAnF9) polymers were synthesized as described below. N-(Lissamine rhodamine B sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (rhodamine-DHPE) was purchased from Invitrogen (Karlsruhe, Germany) and used without further purification. The water used for the experiments was ultrapure Millipore quality (conductivity Lβ′ => Pβ′ => Lα, where the last transition denotes the main phase transition of DPPC from a tilted ripple phase (Pβ′) to a liquid-crystalline bilayer phase (Lα) of the phospholipid. The main phase transition occurs between an

Figure 8. DSC thermograms of lipid bilayer vesicles of (a) L-DPPC/ PGMA14-F9, (b) L-DPPC/PGMA40-F9, and (c) L-DPPC/PGMA90-F9. 15659

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The observed pressure increase depends on the polymer length, increasing with a higher molar mass of the polymer. This shows that the hydrophilic polymer moiety interacts with the lipids, probably by polar interactions with and/or hydrogen bonds to the lipid headgroups. The compression isotherms and the epifluorescence images prove that the polymer is mainly located in the LE phase of the lipid monolayer but accumulates at the edges of the LC domains, decreasing the line tension. This leads to the formation of extended spiral LC domains for LDPPC or D-DPPC with a curling direction dependent on the chirality of the lipid but very long domain stripes for the racemic DPPC mixture. The DSC measurements of DPPC−polymer mixtures as vesicle systems support the findings obtained with the lipid monolayers. The incorporation of the polymer with the longest chain is the most effective, leading to a significant shift of the main transition to lower temperature and a considerable broadening of the transition. For the shorter-chain compounds, the effects are smaller, as observed before in the monolayer experiments. The results show that a single fluorinated chain with a length of nine fluorinated carbon atoms leads only to weak surface activity and a minor incorporation into lipid bilayers. In contrast to expectations, the hydrophilic polymer part plays an important role in the interactions with lipid monolayers as well as bilayers because of polar interactions in the lipid headgroup region.

marginally incorporated into lipid bilayers in the liquidcrystalline phase. Very slight shifts of Tm can be seen for the 50:1 and 10:1 mixtures, which, however, can also be preparation effects. For the other mixtures, Tm does not change. For the 5:1 and 2:1 mixtures, the pretransition disappears, which is clearly an effect caused by the greater amount of polymer now incorporated into the bilayer. As a consequence, the ripple phase cannot be formed. For mixtures of DPPC with PGMA40-F9 where the polymer has a longer chain length, the influence on the phase transition is more pronounced. For the 20:1 mixture of L-DPPC/ PGMA40-F9, the transition suddenly becomes very broad, but a sharp peak with a shoulder on the high-temperature side develops at higher polymer concentration (Figure 8b). An indication of this effect, but less pronounced, can also be found for shorter polymer PGMA14-F9 mixed with the phospholipid. Also, the regaining of the peak intensity for the 10:1, 5:1, and 2:1 mixtures appears for both polymers. An explanation is difficult to find. There are possibly two counteracting effects, namely, the incorporation of the fluorinated chain into the bilayer and the interaction of the polymer with the lipid headgroups and the chains. Although the stiff fluorinated chain probably increases the order of the bilayer (increases in Tm), the polymer chain perturbs the chain packing (decrease in Tm). The total effect observed then depends on the length of the polymer chain and on the partition coefficient of the molecules into the bilayer. Thus, it could be possible that for L-DPPC/ PGMA40-F9 mixtures the perturbation effect of the polymer part is overcompensated for by the fluorinated chain at a higher polymer concentration. For mixtures with PGMA90-F9, the polymer with the longest chain, the largest effects are seen. The transition is shifted slightly to lower temperature, and in particular, the width of the transition increases strongly (Figure 8c), which indicates a destabilization of the ordered lipid bilayer phase due to the incorporation of the polymer into the liquid-crystalline phase. The DSC thermograms of DPPC/PGMAn-F9 mixtures show again that the interaction of the polymers with the lipid depends not only on the presence of the perfluorinated alkyl chain but also on the length of the PGMA chain. The polymer with the highest molar mass shows the largest effects. This agrees with the observations presented above on the interactions of the polymers with lipid monolayers where we found that the incorporation of PGMA90-F9 leads to a higher surface pressure increase compared to that for the shorter-chain analogues. Also, the DSC curves indicate that the hydrophilic part of the molecules influences the packing of the lipid molecules in the bilayers by interacting with the headgroups and possibly partially inserting into the headgroup region and thus leading to a perturbation of the packing in the gel phase.

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ASSOCIATED CONTENT

S Supporting Information *

Epifluorescence microscopy images of monolayers of D-DPPC/ PGMA90-F9 and D-DPPC/PGMA40-F9. Isotherms for PGMAnF9 monolayers after spreading from a solution in methanol. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49-345-5525850. Fax: +49-345-5527157. E-mail: alfred. [email protected]. Website: http://phys.chemie.unihalle.de/groups/blume. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft (Forschergruppe FOR 1145, TP 3, and TP 4).

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REFERENCES

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CONCLUSIONS The interaction of semitelechelic PGMAs end-capped with a perfluorinated alkyl chain with monolayers and bilayers of DPPC was investigated using different methods. The polymers are surface-active because of the fluorinated chain. However, the surface activity increases with increasing length of the PGMA block. This indicates that the PGMA chain also has hydrophobic character. When polymer is injected underneath a lipid monolayer at the air/water interface, a significantly higher pressure increase is observed as expected from the surface activity of the polymer, indicating that the polymers have a higher affinity for the monolayer than for the air/water surface. 15660

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