Infrared Spectroscopy Reveals the Nonsynchronicity Phenomenon in

Publication Date (Web): June 30, 2010. Copyright © 2010 American ... Jun-Jie Luo , Fu-Gen Wu , Ji-Sheng Yu , Rui Wang , and Zhi-Wu Yu. The Journal of...
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Infrared Spectroscopy Reveals the Nonsynchronicity Phenomenon in the Glassy to Fluid Micellar Transition of DSPE-PEG2000 Aqueous Dispersions Fu-Gen Wu, Jun-Jie Luo, and Zhi-Wu Yu* Key Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China Received April 18, 2010. Revised Manuscript Received June 17, 2010 One challenging question regarding the phase transition mechanism of amphiphiles is to seek the roles individual groups/portions of the amphiphilic molecule play during the transformation. To address this question, we selected a poly(ethylene glycol)-grafted phospholipid, distearoylphosphatidylethanolamine-N-[methoxy(poly(ethylene glycol))2000] (DSPE-PEG2000), to study its glassy to fluid micellar phase transition by using differential scanning calorimetry and Fourier transform infrared (FTIR) spectroscopy. FTIR results revealed that during the glassy to fluid micellar transition, the lipid acyl tails have evident conformational rearrangements but undergo only slight modifications in the packing state. For the lipid interface region, small changes in the hydration state of CdO groups were observed, whereas for the lipid headgroups (NHCO and PO4-), their conformation and hydration states remain unchanged. Thus, the head, the interface, and the tail regions of DSPE-PEG2000 molecules change nonsynchronously during the transition. As to the bulky PEG corona residing at the outer micellar surface, no evident hydration state change was observed upon heating, and its behavior is almost the same as that of the hydrated free PEG2000 molecules. Such a nonsynchronous change in different parts of the self-assembled amphiphilic aggregates undergoing phase transition could be a common phenomenon that needs to be widely recognized.

1. Introduction Polyethylene glycol (PEG) is a kind of biocompatible polymer with properties of nonimmunogenicity, nonantigenicity, and protein rejection.1 When grafted to a lipid molecule, it can modify the properties of the target molecule effectively, and the thus obtained molecule is the so-called PEG-lipid. The PEG-lipid is the most well-known class of lipopolymers (or polymer lipids) which bears the properties of both polymers and amphiphiles. Lipopolymers have been an active research field in recent years as they can serve as simple model molecules for a large variety of different conjugates of lipids and polymers found in nature, such as glucolipids or lipoproteins.2 The neat PEG-lipids can form micelles above the critical micelle concentration (CMC) in aqueous solution and can selfassemble into monolayers at the air-water interface. The formed micelle or monolayer structure is an excellent model for fundamental investigations in colloid and interface science. Moreover, the hydrophobic core of the micelles allows for the solubilization of hydrophobic drugs and the PEG corona residing at the outer micellar surface provides a shield against attacks from enzymes.3 Due to the fundamental interest and practical application, the neat PEG-lipid systems have been studied from various aspects, *To whom correspondence should be addressed. Tel: (þ86)10 6279 2492. Fax: (þ86)10 6277 1149. E-mail: [email protected]. (1) Alcantar, N. A.; Aydil, E. S.; Israelachvili, J. N. J. Biomed. Mater. Res. 2000, 51, 343–351. (2) Baekmark, T. R.; Wiesenthal, T.; Kuhn, P.; Albersd€orfer, A.; Nuyken, O.; Merkel, R. Langmuir 1999, 15, 3616–3626. € uksel, H.; Thiyagarajan, P.; Jacob, J.; Hjelm, R. P. (3) Arleth, L.; Ashok, B.; Ony€ Langmuir 2005, 21, 3279–3290. (4) Kuhl, T. L.; Majewski, J.; Howes, P. B.; Kjaer, K.; von Nahmen, A.; Lee, K. Y. C.; Ocko, B.; Israelachvili, J. N.; Smith, G. S. J. Am. Chem. Soc. 1999, 121, 7682–7688. (5) Naumann, C. A.; Brooks, C. F.; Fuller, G. G.; Knoll, W.; Frank, C. W. Langmuir 1999, 15, 7752–7761. (6) Johnsson, M.; Hansson, P.; Edwards, K. J. Phys. Chem. B 2001, 105, 8420– 8430.

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including the size, structure, conformation, packing, hydration, and viscoelastic properties.3-10 On the other hand, self-assembled lipid matrixes (i.e., monolayers, bilayers, and vesicles/liposomes) containing PEG-grafted lipids have been frequently investigated, such as the mixed systems of phosphatidylcholine (PC)/PEG-lipid3,7,8,11-17 and phosphatidylethanolamine (PE)/PEG-lipid.18-20 Through the incorporation of PEG-lipids, these systems acquire various unique properties and functions as compared with those without the PEG-lipids. The bulky, highly hydrated PEG segments can form a protective cap on membrane surface and effectively prevent the membrane from fusion or self-closure.14 Moreover, the PEG-lipid can prevent the nonspecific binding of biomolecules in aqueous phase onto the membrane-mimetic surface.15,21-23 It is suggested (7) Xu, Z.; Holland, N. B.; Marchant, R. E. Langmuir 2001, 17, 377–383. € uksel, H. J. Pharm. (8) Ashok, B.; Arleth, L.; Hjelm, R. P.; Rubinstein, I.; Ony€ Sci. 2004, 93, 2476–2487. (9) Tsukanova, V.; Salesse, C. J. Phys. Chem. B 2004, 108, 10754–10764. (10) Sato, T.; Sakai, H.; Sou, K.; Buchner, R.; Tsuchida, E. J. Phys. Chem. B 2007, 111, 1393–1401. (11) Kenworthy, A. K.; Simon, S. A.; McIntosh, T. J. Biophys. J. 1995, 68, 1903– 1920. (12) Chou, T. H.; Chu, I. M. Colloids Surf., A 2002, 211, 267–274. (13) Chou, T. H.; Chu, I. M. Colloids Surf., B 2003, 27, 333–344. (14) Sandstr€om, M. C.; Johansson, E.; Edwards, K. Langmuir 2007, 23, 4192– 4198. (15) Tanwir, K.; Tsoukanova, V. Langmuir 2008, 24, 14078–14087. (16) Lozano, M. M.; Longo, M. L. Soft Matter 2009, 5, 1822–1834. (17) Lozano, M. M.; Longo, M. L. Langmuir 2009, 25, 3705–3712. (18) Baekmark, T. R.; Elender, G.; Lasic, D. D.; Sackmann, E. Langmuir 1995, 11, 3975–3987. (19) Majewski, J.; Kuhl, T. L.; Kjaer, K.; Gerstenberg, M. C.; Als-Nielsen, J.; Israelachvili, J. N.; Smith, G. S. J. Am. Chem. Soc. 1998, 120, 1469–1473. (20) Majewski, J.; Kuhl, T. L.; Gerstenberg, M. C.; Israelachvili, J. N.; Smith, G. S. J. Phys. Chem. B 1997, 101, 3122–3129. (21) Vermette, P.; Meagner, L. Colloids Surf., B 2003, 28, 153–198. (22) Rex, S.; Zuckermann, M. J.; Lafleur, M.; Silvius, J. R. Biophys. J. 1998, 75, 2900–2914. (23) Bianco-Peled, H.; Dori, Y.; Schneider, J.; Sung, L. P.; Satija, S.; Tirrell, M. Langmuir 2001, 17, 6931–6937.

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Figure 1. Molecular structure of DSPE-PEG2000.

that the PEG brushes provide a steric repulsive barrier that prevents the adsorption of proteins onto the membrane surface.21,24-26 When the PEG-lipid was incorporated into the PC lipid to construct liposomes/vesicles, the PEG portion of the PEG-lipid enhances circulatory life in the bloodstream, biocompatibility, and serves as a site for synthetic modification for targeting with monoclonal antibodies or ligands.16 Besides, PEGlipids can be used to create bionon-fouling membrane mimetic surfaces in a lipid matrix for use as tissue-contacting layers on the surfaces of implantable materials and ligand-supporting membranes for biosensors.15 Distearoylphosphatidylethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (DSPE-PEG2000) is a representative PEG-lipid, and its structure is shown in Figure 1. When dispersed in excess water, DSPE-PEG2000 can form oblate spheroidal micelles with a maximum diameter of 18 nm and an aggregation number of ∼90.3 The two saturated, 18-carbon acyl chains endow the DSPEPEG2000 molecules with a strong hydrophobic driving force for self-assembly into micelles, with a very low CMC value of ∼1 μM.8 The extremely low CMC value makes the drug-loaded micelles stable upon the fast dilution that takes place when the drug is injected in blood vessels.3 The thermotropic phase behavior of DSPE-PEG2000 has recently been studied. Kastantin and co-workers reported an endothermic transition of DSPEPEG2000 at 12.8 °C upon heating.27 They attributed it to the lipid chain melting, from glassy to fluid micellar transition, and believed that this phase transition does not perturb the geometry of micelles. In this work, we selected the DSPE-PEG2000 molecule to study its glassy to fluid micellar phase transition process at the submolecular level. We are curious to know the role the individual groups/portions of this polymer-type amphiphile play during the transformation from one phase to another. To answer this question, it is important to consider the cooperativity of the change of the individual groups/portions of the amphiphilic molecule during the phase transformation process. That is, whether the different groups/portions of the amphiphilic molecule change synchronously or nonsynchrously during the phase transitions. The cooperativity issue opens a broad window for us to challenge the important questions including the structure-property-function relationships and the kinetics, polymorphism, metastability, and reversibility of phase transitions. However, not enough attention has been paid to this issue. In a previous study, we observed the nonsynchronicity phenomenon in the lamellar to lamellar transition of a simplestructured double-chained cationic lipid dioctadecyldimethyl(24) Efremova, N. V.; Sheth, S. R.; Leckband, D. E. Langmuir 2001, 17, 7628– 7636. (25) Kuhl, T. L.; Leckband, D. E.; Lasic, D. D.; Israelachvili, J. N. Biophys. J. 1994, 66, 1479–1488. (26) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B 1998, 102, 426–436. (27) Kastantin, M.; Ananthanarayanan, B.; Karmali, P.; Ruoslahti, E.; Tirrell, M. Langmuir 2009, 25, 7279–7286.

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ammonium bromide (DODAB).28 We attributed the origin of the nonsynchronous change of the lipid head and tail to the lack of special intermolecular attractive forces (hydrogen bonding and electrostatic interactions) between the neighboring polar headgroups of DODAB molecules. In another study concerning the lamellar to nonlamellar transition of a single-chained phospholipid 1-stearoyllysophosphatidylcholine (SLPC) dispersed in excess water, we also found the nonsynchronous change of the different portions of the molecule during the lamellar-micellar phase transitions.29 We suggested that the lack of change in the intermolecular attractive electrostatic interactions between the neighboring polar headgroups of SLPC molecules is the origin of the occurrence of the nonsynchronicity phenomenon. Despite these two studies, questions like whether this nonsynchronicity phenomenon exists in other self-assembled systems or in other types of phase transitions still remain open. Moreover, the origin of the nonsynchronicity phenomenon also needs to be exploited. In this work, by using differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy, we will identify the presence of such a nonsynchronicity phenomenon in a nonlamellar to nonlamellar transition, namely the glassy micellar to fluid micellar transition, of the polymer-type amphiphilic molecule, DSPE-PEG2000. Specifically, we are curious to know whether different parts of DSPE-PEG2000 take identical pace in response to temperature changes during the transition. The previous two studies28,29 have uncovered the existence of the nonsynchronicity phenomenon in two typical amphiphiles (or surfactants) with medium molecular weights. The present studied molecule is a polymer, which possesses the self-assembly property of an amphiphile. While the previous systems involved the lamellar-lamellar and lamellar-nonlamellar transitions, the present work deals with a new transition type, the nonlamellarnonlamellar (micellar-micellar) transition. Besides, we have found the special role the PEG2000 part plays in regulating the intermolecular interactions between the neighboring headgroups of the DSPE moiety, which eventually leads to the occurrence of nonsynchronicity. To the best of our knowledge, the present work is the first observation of the nonsynchronicity phenomenon in a polymer-type amphiphile undergoing a nonlamellarnonlamellar transition process with a new origin of nonsynchronicity.

2. Experimental Section 2.1. Sample Preparation. DSPE-PEG2000 was obtained from Avanti Polar Lipids (Birmingham, AL). PEG2000 was from Beijing Yili Fine Chemicals Co., Ltd. Double deionized water with a resistivity of 18.2 MΩ cm or D2O (99.9% of deuterium, from Cambridge Isotopes) was used for the aqueous hydration and suspension of the lipid samples. The lipid/water ratio was 1/3 (w/w). Homogeneous lipid dispersion was prepared by repeated (28) Wu, F. G.; Wang, N. N.; Yu, Z. W. Langmuir 2009, 25, 13394–13401. (29) Wu, F. G.; Wang, N. N.; Yu, J. S.; Luo, J. J.; Yu, Z. W. J. Phys. Chem. B 2010, 114, 2158–2164.

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thermal cycling between 0 and 50 °C. The PEG2000 aqueous solution containing the same weight ratio of PEG2000 as that of the DSPE-PEG2000 aqueous sample was also prepared by dissolving PEG2000 in H2O. 2.2. DSC. Calorimetric data were obtained with a differential scanning calorimeter DSC821e equipped with the high-sensitivity sensor HSS7 (Mettler-Toledo Co., Switzerland). 2.3. FTIR Spectroscopy. FTIR spectra were recorded using a Nicolet 5700 Fourier transform infrared spectrometer with a DTGS detector in the range of 4000-1000 cm-1 with a spectral resolution of 2 cm-1 and a zero filling factor of 2. The precision of the frequency is better than 0.1 cm-1. Samples were coated onto the inner surfaces of a pair of CaF2 windows between a 6 μm Teflon spacer, which were then mounted on a Linkam heating-cooling stage for temperature control ((0.1 °C). Spectra were recorded every ∼30 s, and each spectrum consisted of 16 scans. For the IR difference spectroscopy, each IR spectrum of the DSPE-PEG2000 aqueous dispersion was subtracted by that of the PEG2000 aqueous solution (with the same PEG2000 weight ratio as that of the DSPE-PEG2000-H2O system) at the same temperature using the Nicolet OMNIC 7.2 software (Thermo Electron Co.). A subtraction coefficient (which is always close to 1) was applied and adjusted to achieve the complete remove of the characteristic IR absorption bands of PEG2000 based on the following rational. As the CdO group is only present in DSPEPEG2000, we used this group as a probe to examine if the subtraction is optimal. Specifically, the coefficient was such adjusted that the resultant spectral contour of the CdO group obtained by subtracting PEG2000 from DSPE-PEG2000 in H2O was identical with that in the DSPE-PEG2000-D2O system at the same temperature. The thus obtained spectra were then smoothed using the 11-point Adjacent-Averaging method (Origin 7.0, OriginLab Co.). The peak fitting procedures of the IR band of CdO groups were carried out using the PeakFit v4.05 software (AISN Software Inc.). The baseline was created by the two-point linear method and the peak type was GaussianþLorentz for all the peak fitting treatments.

3. Results and Discussion 3.1. DSC. Thermotropic phase transition of DSPE-PEG2000 dispersed in excess H2O and D2O was monitored by DSC and the results are shown in Figure 2. For the dispersion in H2O, a single endothermic peak is seen, with the onset and peak temperatures of 11.0 and 13.6 °C, respectively. The transition enthalpy is 37.3 kJ/mol. These values are in agreement with the literature data.27 This transition has been ascribed to the glassy micelle to fluid micelle phase transition.27 The thermal event in the figure

Figure 2. DSC results of DSPE-PEG2000 in H2O and D2O. The heating rate is 0.5 °C/min. Langmuir 2010, 26(15), 12777–12784

reflects mainly the melting of DSPE lipid chains in the core of the DSPE-PEG2000 micelles, as the enthalpy is close to the main transition enthalpy of hydrated DSPE (43.9 kJ/mol).30 For the dispersion in D2O, the results are very similar. The onset and peak temperatures are 11.5 and 14.1 °C, respectively. The transition enthalpy is 39.1 kJ/mol. Although the phase transition enthalpy in DSPE-PEG2000 is slightly smaller than that of DSPE, the glassy to fluid micellar transition temperature is much lower than that of hydrated DSPE (74 °C).30 This significant decrease (∼60 °C) of the phase transition temperature when PEG2000 is grafted onto DSPE is related with the drastic structural alteration from bilayer in DSPE to micelle in DSPE-PEG2000. This drastic structural alteration is caused by the change in the molecular geometry from rod-like DSPE to cone-shaped DSPE-PEG2000. The lowered phase transition enthalpy and temperature in DSPE-PEG2000 may suggest that the PEG-PEG interactions on the highly curved micellar surface are weak, and the resultant bulky PEG brushes may affect the packing state of the lipid acyl chains, leading to an earlier chain melting process. On the other hand, the broad feature of the DSC peak (∼20 °C) suggests that the glassy micelle changes into the fluid micelle in a noncooperative way. Experimentally it has been found that the DSC profiles of the lipid melting process are broadened for curved membranes,31-33 which also support our present DSC results for the highly curved micellar-micellar transition. Upon cooling from the fluid micellar state, the glassy micelle can form readily, indicating that the transformation of these two micellar states is reversible. 3.2. FTIR. As shown in Figure 1, the DSPE-PEG2000 molecule can be divided into two segments: the PEG (OCH2CH2) part and the lipid DSPE moiety. The latter can further be divided into three regions: the lipid head region including mainly NHCO and PO4- groups, the lipid interface region containing mainly the ester COO group, and the lipid tail region consisting of two acyl chains (CH2s) (the micellar core). These functional groups were selected as the IR probes for monitoring the changes of the head, interface, and tail regions of DSPE-PEG2000 molecules, respectively, to see how these groups change during the glassy to fluid micellar transition. Shown in Figure 3 are the time-resolved FTIR spectra in the selected wavenumber ranges of a DSPE-PEG2000-H2O dispersion recorded during the glassy (5 °C) to fluid (35 °C) micellar transition at a heating rate of 0.5 °C/min. To know the origins of these IR bands in the two selected wavenumber regions (3000-2800 and 1600-1000 cm-1), we also gave the IR results of PEG2000-H2O system with the same PEG2000 weight ratio as that of the DSPE-PEG2000-H2O system at two temperatures (5 and 35 °C), and compared their IR bands with those of the DSPE-PEG2000 molecules. The corresponding results are shown in Figure 4. By comparing the IR spectra of DSPE-PEG2000 and PEG2000 in the 3000-2800 cm-1 region at 5 and 35 °C in Figure 4, panels A and C, we can see that in this region, the DSPEPEG2000 contains the asymmetric and symmetric stretching vibrations of CH2 and CH3 groups from both the DSPE moiety and the PEG part. By comparing those in the 16001000 cm-1 region at 5 and 35 °C in Figure 4B and 4D, we can see that the spectra of DSPE-PEG2000 and PEG2000 are very similar both in band shape and band intensity. This indicates that in (30) Seddon, J. M.; Cevc, G.; Marsh, D. Biochemistry 1983, 22, 1280–1289. (31) Schneider, M. F.; Marsh, D.; Jahn, W.; Kloesgen, B.; Heimburg, T. Proc. Natl. Acad. Sci., U.S.A. 1999, 96, 14312–14317. (32) Heimburg, T. Biochim. Biophys. Acta 1998, 1415, 147–162. (33) Brumm, T.; Jørgensen, K.; Mouritsen, O. G.; Bayerl, T. M. Biophys. J. 1996, 70, 1373–1379.

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Figure 3. Time-resolved FTIR absorbance spectra of DSPE-PEG2000-H2O during the glassy to fluid micellar transition in the 30002800 cm-1 region (A) and 1600-1000 cm-1 region (B) at 0.5 °C/min. The spectra are 1 °C apart.

Figure 4. Subtraction of PEG2000 from DSPE-PEG2000 to obtain the IR spectra of DSPE moiety at 5 °C (A and B) and 35 °C (C and D).

DSPE-PEG2000 there are several IR bands mainly originating from the absorption of the PEG part in this wavenumber region: the CH2 wagging vibration (ωCH2, ∼1348 cm-1), the CH2 twisting vibrations (τCH2, ∼1289 cm-1 and ∼1253 cm-1), and the C-O-C stretching vibration (νC-O-C, ∼1091 cm-1). Besides, we have also compared the IR bands of DSPE-PEG2000 with those of DSPE at the two temperatures (5 and 35 °C) in the 12780 DOI: 10.1021/la101539z

1600-1000 cm-1 region, and found that for the same content of the DSPE moiety, the IR absorption at ∼1348 cm-1 for DSPE is almost neglectable (data not shown), which is consistent with the above assignments. The results in Figure 3B show that during the glassy to fluid micellar transition, the CH2 groups in PEG part do not produce significant changes in the band shapes and band positions of Langmuir 2010, 26(15), 12777–12784

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Figure 5. Time-resolved FTIR absorbance spectra of DSPE moiety-H2O during the glassy to fluid micellar transition in the 30002800 cm-1 region (A) and 1600-1000 cm-1 region (B) at 0.5 °C/min. The spectra are 1 °C apart.

ωCH2 and τCH2, indicating that their chemical environment or conformation do not change upon heating. While for νC-O-C, almost no band shift occurs, indicating that the hydration of the PEG segments is almost conserved over the temperature range (5-35 °C). Moreover, by careful comparison of the ωCH2, τCH2, and νC-O-C bands of DSPE-PEG2000 at 5 and 35 °C with those of the two corresponding spectra of PEG2000 in Figure 4, panels B and D, we can see that the change of the PEG part during heating from glassy micelle (5 °C) to fluid micelle (35 °C) is almost identical as that of the hydrated free PEG2000 molecules in water. This in turn confirms our hypothesis that the PEG part of DSPEPEG2000 has the same behavior (and IR band absorptions) as that of the hydrated free PEG2000 molecules. In order to examine in detail how the lipid part, the DSPE moiety, changes during the phase transition, the IR spectra of PEG2000 were subtracted from those of DSPE-PEG2000. Figure 4A-D illustrates the spectra of DSPE-PEG2000, PEG2000, and the subtracted results at the two typical temperatures, 5 and 35 °C. From this figure, it can be seen that the subtracted spectra contain mainly the IR bands of the DSPE moiety. Spectra of the DSPE moiety at other temperatures could be obtained in the same way. Shown in Figure 5 are the timeresolved FTIR spectra of the DSPE moiety during the glassy to fluid micellar transition. In Figure 5A, we can see that the glassy to fluid micellar transition is characterized by a shift toward high wavenumbers and an increase of the width of the bands of the CH2 stretching vibrations. At the glassy micellar state (5 °C), the CH2 asymmetric and symmetric stretching bands (νasCH2 and νsCH2) center at 2919.1 and 2850.8 cm-1, which shift to 2923.3 and 2853.2 cm-1 in the fluid micellar state at 35 °C, respectively. These special properties have been used frequently to follow the conformational order of the lipid methylene chains and the trans-gauche isomerization of the CH2 groups in lipid tail regions.34,35 The increase in wavenumbers is partly due to the increase in the gauche conformers of methylene chains, and partly (34) Wu, F. G.; Chen, L.; Yu, Z. W. J. Phys. Chem. B 2009, 113, 869–872. (35) Lewis, R. N. A. H.; McElhaney, R. N. In Methods in Molecular Biology; Dopico, A. M., Ed.; Humana Press: Totowa, NJ, 2007; Vol. 400, pp 207-226. (36) Umemura, J.; Cameron, D. G.; Mantsch, H. H. Biochim. Biophys. Acta 1980, 602, 32–44.

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due to the decrease in density or change in the packing state of methylene chains.36 The 1600-1000 cm-1 region (Figure 5B) contains several important IR bands. The first is the amide II (NHCO) band at 1548 cm-1, which mainly contains the NH bending band (δNH). It should be noted that the NH4þ ions in the dispersion reside preferentially around the PO4- groups. The asymmetric bending vibration of this ammonium species is at ∼1377 cm-1 and its symmetric counterpart is above 1600 cm-1,37 both depart clearly from the amide II band. The amide II band is known sensitive to hydration and hydrogen-bonding changes and can serve as an important probe to detect the environment of the lipid head region. It is interesting to find that, during the transition, the IR band shape and band position remain unchanged, indicating that no hydration state or hydrogen-bonding network change occurs upon heating. Another important band is the CH2 scissoring band (δCH2) at 1490-1430 cm-1. This band is sensitive to the intermolecular forces and can serve as a key band for examining the state of packing of the methylene chains in various phases.35 For both of the two micellar phases at 5 and 35 °C, this band is broad and very slight changes in band contour occur during the transition. The “broad” band shape indicates that the lipid tails of the two micellar phases are in a fused state, without regular acyl chain packing. The slight modification of the packing state of lipid tails can be explained as the geometrical and energetical difficulty in the reorganization from the glassy micelle to the fluid micelle. The asymmetric and symmetric stretching vibrations of the PO2- group (νasPO2- and νsPO2-) are centered at around 1215 and 1072 cm-1, respectively. These two bands are also sensitive to hydration and hydrogen-bonding changes. We can see that the PO4- groups tend to retain comparable levels of hydration irrespective of the phase state. Figure 6 shows the temperature-dependences of νasCH2 and νsCH2 of the DSPE moiety during the glassy to fluid micellar transition process. The change behavior of the band positions correlates well with the sluggish DSC trace (Figure 2). Such a slow and gradual increase in the wavenumbers of the two vibrations (37) Marinova, D.; Georgiev, M.; Stoilova, D. J. Mol. Struct. 2009, 938, 179– 184.

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Figure 6. Dependency of the IR band positions (νasCH2 and νsCH2) on temperature of the DSPE moiety in the glassy to fluid micellar transition process.

implies that the transition is much less cooperative than the main transitions of common phospholipids (such as the saturated diacyl PEs38) dispersed in water, and the trans-to-gauche transformation of the lipid acyl chains is not easy and is temperature dependent during the glassy to fluid micellar transition. In DSPE-PEG2000-H2O system, the absorption band of the CdO group is overlapped by the neighboring H2O band. To avoid this problem, we investigated the change of the CdO stretching band in the DSPE-PEG2000-D2O system, and the results are presented in Figure 7. Shown in Figure 7A is the dynamic evolution of the CdO absorbance band upon heating from 5 to 35 °C during the glassy to fluid micellar transition. We can see that the overall CdO absorbance band is roughly composed of two bands. Figure 7B shows that the CdO contours of the initial glassy micelle (5 °C) and the final fluid micelle (35 °C) exhibit only a slight difference in the band contour of the higher wavenumber band, and almost no band contour change occurs in the lower wavenumber band. To further investigate the subcomponents of the CdO stretching vibrations of the two phases, peak fitting procedures were carried out and the results are depicted in Figure 7, panels C and D, for the glassy micellar (5 °C) and fluid micellar phase (35 °C), respectively. For both of these two phases, the CdO stretching band can be fitted by four subcomponents centering at 1742, 1725, 1701, and 1681 cm-1. The bands at 1742 and 1725 cm-1 are assigned to the stretching vibrations of the interfacial CdO groups in the absence and presence of hydrogen bonding interactions, respectively.38,39 The slight increase in the peak area of the band at 1725 cm-1 (from 28% to 31%, as indicated in the figure) shows that the interfacial CdO groups are slightly more hydrated in the fluid micellar state than those in the glassy micellar state. The other two CdO components centered at 1701 and 1681 cm-1 are attributed to the CdO stretching vibrations of the NHCO moiety in the lipid head region, which may be associated with two different hydration or conformation states of the CdO groups. No changes on the peak area of these two CdO components were found. Together with the two stacked IR bands shown in Figure 7B, we conclude that only the CdO groups in the lipid interface region undergo an increase in the hydration degree during the phase transition. In summary, from these FTIR results, we can see that, although there are evident conformational rearrangements of the lipid acyl tails during the glassy to fluid micellar transition, (38) Lewis, R. N. A. H.; McElhaney, R. N. Biophys. J. 1993, 64, 1081–1096. (39) Lewis, R. N. A. H.; McElhaney, R. N. Chem. Phys. Lipids 1998, 96, 9–21.

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only very slight modifications in the packing state of the lipid acyl tails and small hydration state change of the lipid interfacial CdO groups were observed during the phase transition. For the lipid headgroups (including the NHCO and PO4- groups), their conformation and hydration states remain unchanged upon phase transition. Thus, the lipid head, interface, and tail regions do not change synchronously during the glassy to fluid micellar transition upon heating. As to the bulky PEG corona residing at the outer micellar surface, no evident hydration state change was observed upon heating, and its behavior is almost the same as that of the hydrated free PEG2000 molecules. These IR data also provide a detailed submolecular evidence to understand why DSPE-PEG2000 maintains the integrity of its micellar structure undergoing the glassy to fluid micellar transition. 3.3. Nonsynchronicity Phenomenon. The wide-angle X-ray scattering (WAXS) technique can provide information on the structure of the DSPE-PEG2000 micellar core consisting of the lipid acyl tails. This has been done by Kastantin et al. at 2 and 25 °C to examine the glassy and fluid micellar phases.27 It was found that both phases displayed a broad peak near q = 1.5 A˚-1, indicating the lack of regular packing in the lipid acyl chains at both temperatures. The peak position shifts to a slightly lower q value at 25 °C as compared with that at 2 °C, indicating a larger average carbon-carbon spacing in the fluid micellar state. The lack of regular packing observed in WAXS data correlates well with our IR data analyses (δCH2 in Figure 5). The rearrangement of the packing state is relatively small, and the observed broad DSC peak in Figure 2 suggests that the glassy micelle changes to the fluid micelle in a less cooperative way, which may be related with the geometrical constraint in the lipid tail region, induced by the high spontaneous curvature of the micelle structure. The geometrical constraint will, in turn, set obstacles to the rearrangement of the lipid acyl chains. Shown in Figure 8 is the schematic model showing the glassy to fluid micellar transition of DSPE-PEG2000 in excess water. With the bulky PEG corona linking to the lipid head, the DSPEPEG2000 molecule possesses positive conical chape, and it can form micelles (oblate spherical shape) when dispersed in water. Dynamic light scattering (DLS) measurements on DSPEPEG2000 micelles indicate that the phase transition seen in DSC does not cause a dramatic shape transition.27 Besides, the DLS measurements revealed that the hydrodynamic diameter of DSPE-PEG2000 micelles increase gradually from 11 (2 °C) to 16 nm (35 °C).27 It was thus concluded that the continuous increase in micelle size upon heating is likely due to the increasing aggregation number and subsequent effects on the axial ratio of the spheroidal micelle as slight shape changes are required to pack more PEG-lipids into the micelle.27 In a micellar structure that is lacking in regular acyl chain packing, when it approaches the onset temperature of the glassy to fluid micellar phase transition, the van der Waals forces between the interchains of the lipid tails will be markedly weakened, and the packing of the tail region of the glassy micellar structures is to some extent perturbed. This facilitates the transto-gauche transformation, and the new formed gauche conformers may act as the nuclei of the fluid micellar state. The loosening of the tail portion induces more space of the interface region and more water molecules are allowed to penetrate in, resulting in the increase in the hydration of the CdO group. As the hydration of the interfacial CdO groups is mostly likely induced by the disordering of the tail region, the change in lipid tails is the factor that controls the glassy to fluid micellar transition. There are similar reports that the interfacial glycerol backbone conformation is principally governed by the vicinally Langmuir 2010, 26(15), 12777–12784

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Figure 7. Time-resolved FTIR absorbance spectra showing the change of the CdO stretching band in the 1800-1600 cm-1 region at 0.5 °C/ min in DSPE-PEG2000-D2O system (A). The spectra are 1 °C apart. The two stacked absorbance spectra of the glassy micelle at 5 °C and the fluid micelle at 35 °C (B). The peak fitting results of the CdO stretching bands at 5 °C (C) and 35 °C (D).

Figure 8. Schematic model showing the glassy to fluid micellar transition of the DSPE-PEG2000 aqueous dispersion. During the transition, the lipid head, interface, and tail regions of DSPEPEG2000 molecules change nonsynchronously. Note that only the hydration change of the lipid interface is indicated.

arranged acyl chains in phospholipid aggregates such as bilayers or micelles.40,41 Although there are possibilities for DSPE-PEG2000 molecules to form intermolecular hydrogen bonding networks between the (40) Hauser, H.; Pascher, I.; Sundell, S. Biochemistry 1988, 27, 9166–9174. (41) Arora, A.; Gupta, C. M. Biochim. Biophys. Acta 1997, 1324, 47–60.

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NH groups and the hydrogen bonding acceptors (such as PO4- or CdO) in the lipid polar region, the bulky PEG2000 portion in the molecules (which produces a positive micellar curvature) may enlarge the donor-acceptor distance and thus suppress the formation of hydrogen bonds. The fact that the CdO stretching band at 1714 cm-1 due to the hydrogen-bonding interaction between the headgroup amine protons and the sn-2 ester carbonyl38 is absent (see Figure 7C and 7D) also partially supports our above conclusion. As a result, the polar lipid headgroups (NHCO and PO4-) tend to form hydrogen bonds only with the water molecules, and there may be no particularly strong intermolecular attractive forces (such as hydrogen bonding and attractive electrostatic interactions) between the neighboring lipid headgroups of DSPE-PEG2000 molecules. The small modifications in the packing state of lipid acyl tails and the small hydration changes of the lipid interface region, together with the enlarged distance between the neighboring lipid headgroups caused by the bulky PEG2000 portion, eventually cause the nonsynchronous change of the head, the interface, and the tail of the DSPE-PEG2000 during the glassy to fluid micellar transition. It has been noticed that the nonsynchronicity phenomena have been reported in the protein folding/unfolding processes and polymer phase transitions. When a protein undergoes folding/unfolding DOI: 10.1021/la101539z

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processes, one domain may change prior to other domains.42-44 In the polymer phase transition process, the side chains may change earlier than the main chain.45,46 These nonsynchronicity phenomena are very important for the maintenance/regulation of the properties and functions of proteins and polymers. While our present work is, to the best of our knowledge, the first attempt to reveal the cooperativity or synchronicity phenomenon of the phase transition mechanism of a polymer-type amphiphilic molecule (with properties of both polymers and amphiphiles), DSPE-PEG2000, at the submolecular level. The observed nonsynchronicity phenomenon in the nonlamellar to nonlamellar transition of this PEG-grafted lipid, together with our previous findings of the nonsynchronicity phenomena in the lamellar to lamellar and lamellar to nonlamellar transitions of the amphiphilic lipid systems,28,29 strongly suggest that such a nonsynchronous change in different parts of the selfassembled amphiphilic aggregates undergoing phase transition could be a common phenomenon that needs to be widely studied. It was previously presumed that for PEG brushes to be effective in reducing the adsorption of proteins they must be highly stretched and hydrated.24,26 Our work provides further information that the glassy to fluid micellar transition is associated with (42) Zhou, P.; Xie, X.; Knight, D. P.; Zong, X. H.; Deng, F.; Yao, W. H. Biochemistry 2004, 43, 11302–11311. (43) Shashilov, V. A.; Lednev, I. K. J. Am. Chem. Soc. 2008, 130, 309–317. (44) Ashton, L.; Barron, L. D.; Czarnik-Matusewicz, B.; Hecht, L.; Hyde, J.; Blanch, E. W. Mol. Phys. 2006, 104, 1429–1445. (45) Sun, B. J; Lin, Y. N; Wu, P. Y. Appl. Spectrosc. 2007, 61, 765–771. (46) Curtis, M. D.; Nanos, J. I.; Moon, H.; Jahng, W. S. J. Am. Chem. Soc. 2007, 129, 15072–15084.

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almost no change in the hydration state of the micellar surface dominated by the bulky PEG corona, which is related to the flexibility, permeability, fusion ability, and environment and activity of the functionized biomimetic and biocompatible surface, especially when the micelles are used as drug carriers encountering proteins or DNA.

4. Conclusions To summarize, we have studied the thermotropic phase behavior of DSPE-PEG2000 aqueous dispersions by DSC and FTIR techniques. The FTIR combined with the IR difference spectroscopic method make it possible to reveal the nonsynchronous change in the organization of the lipid head, interface, and tail regions during the conversion from glassy micelle to fluid micelle. Such a nonsynchronicity phenomenon in the self-assembled aggregates composed of the medium-sized lipid molecules reflects the regional (head, interface, and tail) imbalance in molecular interactions and may have profound significance in understanding the nature of the phase transition processes of amphiphiles. The understanding of the mechanism as represented in this work will, we hope, provide a rationale for the behavior of other amphiphilic molecules used for model membrane studies. Acknowledgment. This work was supported by grants from the Natural Science Foundation of China (NSFC: 20633080 and 20973100) and a “973” National Key Basic Research Program of China (Grant No. 2006CB806203).

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