Release and Formation of Surface-Localized Ionic ... - ACS Publications

Feb 15, 2005 - tales, this study utilizes SDOSS surfactant with a single- tail phospholipid ..... (36) Raven, P.; Johnson, G. B. Biology, 5th ed.; McG...
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Release and Formation of Surface-Localized Ionic Clusters (SLICs) into Phospholipid Rafts from Colloidal Solutions during Coalescence David J. Lestage and Marek W. Urban* Shelby F. Thames Polymer Science Research Center, School of Polymers and High Performance Materials, The University of Southern Mississippi, Hattiesburg, Mississippi 39406 Received October 4, 2004. In Final Form: January 12, 2005 Stimuli-responsive behavior of phospholipids in the presence of ionic surfactants utilized in synthesis of MMA/nBA colloidal particles was investigated. Utilizing 1-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC) phospholipid, and sodium dioctyl sulfosuccinate (SDOSS) surfactant as dispersing media in H2O, narrow unimodal particle size distributions of methyl methacrylate (MMA)/n-butyl acrylate (nBA) copolymers were synthesized. The particle diameters were 154 nm when a SDOSS/MHPC mixture was used and 161 nm using MHPC as the only surface-stabilizing species. When such colloidal dispersions are exposed to 1.7, 3.3, and 6.7 mM aqueous CaCl2 and KCl electrolyte solutions, surface-localized ionic clusters are generated at the film-air interface that may serve as lipid rafts composed of crystalline phases of MHPC deposited on poly(MMA)/nBA films. These studies illustrate that it is possible to control release and morphology developments of surface phospholipid rafts on artificial surfaces.

Introduction Although the use of colloidal dispersions has been known for many decades,1-15 their function as a crystallization medium for self-assembling phospholipids near the surface has only recently been discovered.16 This was accomplished by utilizing a combination of sodium dioctyl sulfosuccinate (SDOSS) surfactant and a dual-tailed hydrogenated soybean phosphocholine (HSPC) phospholipid, which served as stabilizing agents in the synthesis of a random copolymer of methyl methacrylate (MMA) and n-butyl acrylate (nBA) monomers. Although immiscibility of the surfactant and surface-active phospholipid led to a bimodal particle size distribution, the presence of the phospholipid also resulted in the formation of localized ionic clusters (LICs)17 which inhibited SDOSS migration to the filmair (F-A) interface during colloidal particle coalescence. However, exposing the same aqueous dispersions to Ca2+* Author to whom all correspondence should be addressed. (1) Lovell, P. A.; Mohameds, E. A. Emulsion Polymerization and Emulsion Polymers; John Wiley and Sons: New York, 1997. (2) Dreher, W. R.; Urban, M. W. Macromolecules 2003, 36, 1228. (3) Dreher, W. R.; Urban, M. W.; Zhao, C. L.; Porzio, R. S. Langmuir 2003, 19, 10254-10259. (4) Keddie, J. L. Mater. Sci. Eng. 1997, 21, 101-170. (5) Winnik, M. A.; Feng, J. J. Coat. Technol. 1996, 68, 39. (6) Beltran, C. M.; Guillot, S.; Langevin, D. Macromolecules 2003, 36, 8506-8512. (7) Zhao, Y.; Urban, M. W. Polym. Mater. Sci. Eng. 1999, 80, 571572. (8) Evanson, W.; Urban, M. W. J. Appl. Polym. Sci. 1991, 42, 22872296. (9) Evanson, W.; Thorstenson, T. A.; Urban, M. W. J. Appl. Polym. Sci. 1991, 42, 2297-2307. (10) Sethumadhavan, G. N.; Nikolov, A.; Wasan, D. Langmuir 2001, 17, 2059-2062. (11) Shin, J. S.; Lee, D. Y.; Ho, C. C.; Kim, J. H. Langmuir 2000, 16, 1882-1888. (12) Lestage, D. J.; Urban, M. W. Langmuir 2004, 20, 6443. (13) Thorstenson, T. A.; Tebelius, L. K.; Urban, M. W. J. Appl. Polym. Sci. 1993, 50, 1207-1215. (14) Niu, B. J.; Urban, M. W. J. Appl. Polym. Sci. 1996, 60, 371-377. (15) Lovell, P. A.; El-Aasser, M. S. Emulsion Polymerization and Emulsion Polymers; John Wiley and Sons: New York, 1997. (16) Lestage, D. J.; Urban, M. W. Langmuir 2004, 20, 7027-7035. (17) Dreher, W. R.; Urban, M. W.; Zhao, C. L.; Porzio, R. S. Langmuir 2003, 19, 10254-10259.

containing electrolyte solutions elicited a stimuli-repsonsive behavior in which the mobility of SDOSS and phospholipid can be individually controlled by the strength of the ionic solution. Appareently, disruption of LICs results in controllable release to the F-A interface during coalescence, whereupon phospholipid-rich crystalline domains were formed. During the course of these studies, it became apparent that the structural features of phospholipids significantly affect colloidal particle morphologies and their film formation. In an effort to establish the role of hydrophobic tales, this study utilizes SDOSS surfactant with a singletail phospholipid, 1-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC), in the synthesis of poly(methyl methacrylate)/n-butyl acrylate (p-MMA/nBA) copolymer dispersions. The primary objectives of these studies are to determine stimuli-responsive behaviors elicited in colloidal dispersions as a result of various external stimuli. As shown in the previous studies,16 phospholipid-rich crystalline domains can be generated at the F-A interface that resemble the behavior of naturally occurring membrane lipid rafts, which are semicrystalline microdomains rich in cholesterol and phospholipids. The importance of raft formation and their structural features have been postulated in key functions of biological membranes such as endocytosis and signal transduction.18 Thus, particle coalescence-controlled raft formation on the surface of colloidal films is of particular interest. Because MHPC phospholipid does not self-assemble to form bilayered vesicles19 in which lipid rafts form, the premise behind is to use spherical colloidal dispersion particles as template for assembly of MHPC. As particles coalescence, SDOSS and MHPC become dispersed within the p-MMA/nBA matrix. However, upon proper stimuli, MHPC entities are mobilized to the F-A or film-substrate (F-S) interfaces. Specifically, exposing aqueous colloidal dispersions to electrolytic solutions and coalesced films to (18) Pike, L. J. Biochem. J. 2004, 378, 281-292. (19) Avanti Polar Lipids, I. Polar Lipids Catalog; Avanti: Alabaster, 2000; Vol. VI.

10.1021/la0475526 CCC: $30.25 © 2005 American Chemical Society Published on Web 02/15/2005

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Table 1. Particle Size and Composition of Random Copolymerized p-(MMA/nBA) Colloidal Dispersions with SDOSS/MHPC Mixture and MHPC Only

with a substrate (or F-S interface). For that reason, the F-A and F-S interfaces of each specimen were analyzed using a 2 mm Ge crystal with a 45° angle maintaining constant contact pressure between the crystal and the specimens. The F-S analysis was performed upon the removal of the film from the substrate. All spectra were corrected for spectral distortions using software for the Urban-Huang algorithm.22 Internal reflection infrared imaging (IRIRI) was performed using a Bio-Rad FTS 6000 Stingray focal plane array detector. Using a procedure adapted from previous literature,23 spectra were collected using the following spectral acquisition parameters: step-scan frequency ) 1 Hz, and number of spectrometer steps ) 1777. The spectra were then processed using ENVI software package (Research Systems, Inc.). Surface morphologies of coalesced films were examined using atomic force microscopy (AFM, Nanoscope IIIa, Digital Inst.) in a tapping mode with a Si3N4 tip (Veeco).

composition components

p-(MMA/nBA) SDOSS/MHPC

p-(MMA/nBA) MHPC

DDI (%) methyl methacrylate (%) n-butyl acrylate (%) SDOSS (%) K2S2O8 (%) MHPC (%) solids (%) particle size (nm)

61.3 18.7 18.7 0.71 0.47 0.09 38.9 154

61 18.8 18.8

CaCl2/MHPC and KCl/MHPC 0.5:1.0 1.0:1.0 2.0:1.0

conc (mM) 1.7 3.3 6.7

0.47 0.94 38.9 161 conc (mM) 17.0 34.0 68.0

elevated temperatures may elicit stimuli-responsive behaviors of SDOSS and MHPC, thus facilitating the formation of lipid raft domains. Experimental Section MMA, nBA, SDOSS, and potassium persulfate (KPS) were purchased from Aldrich Chemical Co. MHPC phospholipid was purchased from Avanti Polar Lipids, Inc. MMA/nBA copolymer emulsions were synthesized using a semicontinuous process outlined elsewhere20 and adapted for small-scale polymerization. The apparatus was placed in a water bath set at 72 °C and purged using N2 gas. The reaction flask was charged with 10 mL of double deionized water (DDI) water, and while purging for 30 min, the content was stirred at 350 rpm. At this point, 20% w/w KPS solution (DDI and KPS) and 10% w/w of the preemulsion solution (DDI, SDOSS, monomers, and phospholipids, if applicable) were added. The SDOSS levels were in the range of 2.5 wt% of total monomer, thus generating 26.0 mM aqueous solutions that are above the CMC of SDOSS (0.10-0.14 wt% of water,21 or 2.0 mM). After 30 min, preemulsion and initiator solutions were fed at 0.394 and 0.095 g/min into the vessel over a period of 3 and 3.5 h, respectively. Upon completion, the reaction continued for 1 h, after which time, the temperature was raised to 85 °C. Upon cooling, the emulsion was filtered twice and particle size analysis was performed using a Microtrac Nanotrac particle size analyzer. In a typical experiment, the standard deviation for the particle size measurements was (7 nm. CaCl2 and KCl (Aldrich) aqueous solutions were prepared by solubilizing the salts in DDI water at 1.7, 3.3, and 6.7 mM. Table 1 provides details of colloid formulations, results of particle analysis, and electrolyte solution concentrations. Such colloidal dispersions were cast onto a poly(tetrafluoroethylene) (PTFE) mold to achieve free-standing colloidal films with an approximate film thickness of 100 µm. The films were allowed to coalesce for 72 h in a controlled environment at 60% relative humidity (RH) and 23 °C. If applicable, selected films were annealed at 25, 50, 75, 100, 125, and 150 °C for 2 h. It should be noted that SDOSS does not degrade under these conditions, as it melts at 153 °C and decomposes above 450 °C.21 Differential scanning calorimetry (DSC) measurements were performed on a TA Q800 Series DSC instrument and the heating rate was 20 °C/min from -75 to 150 °C, while MHPC decomposition temperature was determined utilizing thermal gravimetric analysis (TGA, TA Q800 Series) in which a ramp of 20 °C/min to 800 °C was performed. Microscopic attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy measurements were conducted on the F-A and F-S interfaces using a Bio-Rad FTS-6000 FT-IR single-beam spectrometer with 4 cm-1 resolution. Due to stratification occurring during film formation, very often the chemical makeup at the surface (or F-A interface) is different from that at the interface in contact (20) Davis, S. D.; Hadgraft, J.; Palin, K. J. Encylopedia of Emulsion Technology; Marcel Dekker: 1985; Vol. 2. (21) Emulsion Polymers and Specialties; Cytec Industries: Stamford, CT, 2002.

Results As indicated in the Introduction, mobility of phospholipids in bio-organism membranes allows formation of surface/interfacial entities such as membrane lipid rafts, which serve functions critical to organism survival. If such lipid entities can be synthetically produced in polymeric water-dispersed particles, control over species involved in raft formation during coalescence may impart stimuliresponsive behaviors of coalesced colloidal films. For that reason, we synthesized aqueous colloidal dispersions of p-MMA/nBA in the presence of SDOSS and MHPC, with structural features shown below.

The use of phosholipids as cosurfactant in colloidal dispersion synthesis presents the opportunity for formation of multiple micellar environments which serve as polymerization loci in emulsion polymerization of uni-, bi-, or even multimodal particle sizes, depending upon the number and size of micelles generated. If both surfacestabilizing species are immiscible within each other, two sizes of micelles were obtained.16 In contrast to the previous studies, an SDOSS/MHPC mixture resulted in unimodal colloidal particles with the diameter of 154 nm. The same p-MMA/nBA dispersions, but prepared in the presence of MHPC only, generated particles with the diameter of 161 nm. Thus, unimodal particle-size distribution was obtained in both cases, and such colloidal dispersions were allowed to coalesce under various stimuli conditions. Molecular-level processes at interfaces are a function of environmental stimuli such as temperature.7,11,24-28 For (22) Urban, M. W. Attenuated Total Reflectance Spectroscopy of Polymers - Theory and Practice; American Chemical Society: Washington, DC, 1989. (23) Otts, D.; Zhang, P.; Urban, M. W. Langmuir 2002, 18, 64736477. (24) Dreher, W. R.; Urban, M. W. Langmuir 2004, 20, 10455-10463. (25) Zhao, Y.; Urban, M. W. Langmuir 2000, 16, 9439-9447. (26) Zhao, Y.; Urban, M. W. Macromolecules 2000, 33, 2184. (27) Zhao, Y.; Urban, M. W. Macromolecules 2000, 33, 7573-7581.

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Figure 1. Polarized ATR-FTIR spectra at the F-A interface depicting 1350-900 cm-1 region of MMA/nBA copolymer films containing SDOSS/MHPC mixture annealed at (A) 100 °C, TE; (A′) 100 °C, TM; (B) 125 °C, TE; (B′) 125 °C, TM; (C) 150 °C, TE; (C′) 150 °C, TM for 2 h.

that reason, coalesced p-MMA/nBA colloidal films were annealed and analyzed at the F-A interface using ATRFTIR spectroscopy. Figure 1, Traces A-C′, illustrate ATRFTIR spectra of p-MMA/nBA colloidal films synthesized using an SDOSS/MHPC (2.5:0.3% w/w) mixture and annealed at temperatures ranging from 100 to 150 °C for 2 h after coalescence. In an effort to determine preferential orientation of a given species at the film surfaces,7,28 these spectra were recorded using transverse magnetic (TM; 90°) and transverse electric (TE; 0°) polarizations.28 Films were annealed at 25, 50, and 75 °C (spectra not shown), and small increases of the band at 1046 cm-1 corresponding to the symmetric S-O stretching of SO3-Na+- - -H2O interactions of SDOSS were detected at elevated temperatures.29 However, significant surface changes are detected for films annealed at 100 °C (Traces A/A′) and show the bands at 1310, 1300, and 1265 cm-1. Interestingly enough, these bands are not detected for individual components of this colloidal system, suggesting that other molecular entities are produced as a result of coalescence and annealing. Postponing temporarily the origin of these bands, another observation is that the presence of the bands at 1250 and 1061 cm-1, which exhibit enhanced intensities in TE and TM polarizations, respectively, is detected, which indicates that the PO4- entities30 represented by the 1250 cm-1 band of MHPC are preferentially aligned parallel with respect to the F-A interface, while the P-O-C segmental motions at 1061 cm-1 are normal. Although it is not surprising that surfactants and phospholipids may be driven to the F-A interface with water flux during colloidal particle coalescence7,10,11,31-34 and these processes have been enhanced with elevated tem(28) Zhao, Y.; Urban, M. W. Langmuir 2001, 17, 6961-6967. (29) Niu, B. J.; Urban, M. W. J. Appl. Polym. Sci. 1996, 62, 19031911. (30) Lambert, J. B.; Shurvell, H. F.; Lightner, D.; Cooks, R. G. Introduction to Organic Spectroscopy; Macmillan Publishing Co.: New York, 1987. (31) Manev, E. D.; Sazdanova, S. V.; Wasan, D. T. J. Disp. Sci. Technol. 1984, 5, 111-117. (32) Niu, B. J.; Urban, M. W. J. Appl. Polym. Sci. 1998, 70, 13211348.

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Figure 2. Polarized ATR-FTIR spectra at the F-S interface depicting the 1350-900 cm-1 region of MMA/nBA copolymer films containing SDOSS/MHPC mixture annealed at (A) 25 °C, TE; (A′) 25 °C, TM; (B) 50 °C, TE; (B′) 50 °C, TM; (C) 75 °C, TE; (C′) 75 °C, TM for 2 h.

peratures, another surprising observation is that these bands are not detected when the films were annealed at 125 °C (Traces B/B′). Considering the fact that the evaporation/decomposition temperature is 450 °C21 for SDOSS and 250 °C for MHPC, as determined by TGA analysis, these data indicate that, above 100°C, SDOSS and MHPC are not decomposed, but displaced, and above 125°C, MHPC and SDOSS are again detected (Figure 1, Traces C/C′), as demonstrated by the bands due to P-O, O-CH2, and P-O-C vibrations8,30,35 at 1082, 1025, and 1061 cm-1, respectively. As shown above, annealing coalesced films at elevated temperatures facilitates migration of SDOSS and MHPC to the F-A interface. Let us examine if similar processes occur at the F-S interface. Figure 2, Traces A-C′, show ATR-FTIR spectra of p-MMA/nBA films containing an SDOSS/MHPC mixture and annealed from 25 to 75 °C. As seen, upon annealing, the band intensities at 1300, 1310, and 1061 cm-1 decrease, and above 100 °C (not shown), they are not detected. Subsequently, these data indicate that there are significant differences, especially at elevated temperatures, in stratification behavior between the F-A and F-S interfaces, where mobility toward the F-A interface appears to be dominant. In view of the above experimental data and previous studies,16,34 the presence of ionic species has a significant effect not only on the mobility of individual components, but also on the orientation of the surface groups. For that reason, we prepared a series of CaCl2 and KCl solutions (Table 1) that were added to p-(MMA/nBA) colloidal dispersions. While the choice of Ca2+ as a counterion was dictated by its ability to collapse or extend polymeric chains,34 as well as act as a stimulus in coalescing colloidal dispersions containing phospholipids,16 K+ was chosen because of its importance as a primary counterion in (33) Urban, M. W.; Provder, T. Film Formation in Coatings: Mechanisms, Properties, and Morphology; Oxford University Press: Washington, DC, 2001. (34) Yacoub, A.; Urban, M. W. Biomacromolecules 2003, 4, 52-56. (35) Lin-Vien, D.; Colthup, N. B.; Fateley, W. G.; Grasselli, J. G. The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules; Academic Press: New York, 1991.

Release and Formation of SLICs into Phospholipid Rafts

Figure 3. Polarized ATR-FTIR spectra at the F-A interface depicting the 1350-900 cm-1 region of MMA/nBA copolymer films containing SDOSS/MHPC mixture: (A) TE; (A′) TM, and colloidal dispersions treated with KCl molar ratios: (B) 0.5: 1.0, TE; (B′) 0.5:1.0, TM; (C) 1.0:1.0, TE; (C′) 1.0:1.0, TM; (D) 2.0:1.0, TE; (D′) 2.0:1.0, TM.

biological fluids36 and in cellular processes such as cell membrane homeostasis. From the chemical point of view, both cations are similar: K+ atomic number and weight are 19 and 39.1, whereas Ca2+ has 20 and 40.1, but the latter is divalent. Previous work37 has demonstrated that due to divalency, when placed in aqueous media containing phospholipids, Ca2+ ions may be predominantly bound to phosphate moieties of two lipids. Subsequently, molar ratios of CaCl2 and KCl to MHPC were adjusted to 0.5: 1.0, 1.0:1.0, and 2.0:1.0, and such colloidal dispersions were allowed to coalesce, followed by the same F-A interfacial analysis of the coalesced films using polarized ATR-FTIR spectroscopy. Polarized ATR-FTIR spectra (not shown) of p-MMA/ nBA films were recorded from the F-A interface of colloidal dispersions containing CaCl2 with the CaCl2/ MHPC molar ratio of 0:1.0, 0.5:1.0, 1.0:1.0, and 2.0:1.0. The results of these experiments have shown that upon CaCl2 addition, relatively weak bands at 1310, 1300, and 1061 cm-1 initially detected in dispersions not containing CaCl2 solution are now absent, revealing that the species responsible for these vibrational modes have been disrupted by the presence of Ca2+. Furthermore, films containing a 0.5:1.0 molar ratio of CaCl2/MHPC exhibit MHPC P-O stretching vibrations16,34 at 1089 cm-1, indicating that Ca2+ also facilitates the migration of MHPC to the F-A interface, but this band is not detected for higher CaCl2/MHPC ratios. When KCl with the molar ratios of KCl/MHPC equal to 0:1.0, 0.5:1.0, 1.0:1.0, and 2.0:1.0 were added to p-MMA/ nBA solutions and polarized ATR-FTIR spectra were recorded from the F-A interface of coalesced films, Figure 3, Traces A-D′, resulted. In the same manner as films containing a 0.5:1.0 CaCl2/MHPC ratio, specimens con(36) Raven, P.; Johnson, G. B. Biology, 5th ed.; McGraw-Hill Co.: New York, 1999. (37) Bockmann, R. A.; Grubmuller, H. Angew. Chem., Int. Ed. 2004, 43, 1021-1024.

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taining the KCl/MHPC ratio (Traces B/B′) exhibit no IR bands at 1310 and 1300 cm-1, but the P-O stretching at 1089 cm-1 is detected. These spectroscopic changes accompanied by the intensity differences of the bands at 1123 and 1135 cm-1 in TE and TM polarizations indicate the presence of SDOSS and MHPC at the F-A interface. Similarly to the previous studies,16 the presence of these bands at the F-A interface reveal the presence of surfactant and phospholipid at the F-A interface which is facilitated by concentration changes of counterions inducing migration. However, as shown in Traces C/C′, this process is controlled by the concentration ratio of KCl/ MHPC, as these bands are not detected for a 1.0:1.0 KCl/ MHPC ratio. At this point, it is important to note that, in contrast to previous studies16 in which electrolyte solutions were added to aqueous dispersions and resulted in precipitation, no precipitates were formed when CaCl2/ MHPC or KCl/MHPC ratios were utilized. Thus, the absence of the bands at 1123 and 1135 cm-1 in Traces C/C′ indicates that ionic components do not precipitate out of the solution, but are composed of LICs suspended within the p-MMA/nBA matrix. Interestingly enough, higher KCl/MHPC ratios of 2.0:1.0 (Traces D/D′) induce the same behavior observed for films with 0.5:1.0 KCl/ MHPC ratios, and the only differences are the magnitude of TE and TM band intensities, which are greater for higher KCl concentration levels. The analysis of TE and TM intensities for the PO4- vibrations at 1089 cm-1 and the band at 1123 cm-1 shown in Figure 3, Trace D, indicate that these species have preferential parallel (TE) orientation with respect to the F-A interface. However, the bands at 1135 and 1200 cm-1 reveal a preferential perpendicular (TM) alignment of C-O-C stretching entities. In contrast, spectral data recorded from the F-S interface (not shown) exhibited no presence of MHPC at this interface. Since simultaneous presence of SDOSS and MHPC in the presence of or without ionic environments appears to have a significant effect on stimuli-responsiveness of these species stabilizing p-MMA/nBA particles, let us examine p-MMA/nBA films prepared in the presence of MHPC only. For that reason, colloidal dispersions were synthesized using only MHPC, and Figure 4, Traces A-F′, illustrates ATR-FTIR spectra recorded from the F-A interface of p-MMA/nBA colloidal films annealed at temperatures ranging from 25 to 150 °C after coalescence. As shown in Traces A/A′ of films annealed at 25 °C, the bands at 1310 and 1300 cm-1 exhibit enhanced intensities in TE polarization, whereas 1061 cm-1 due to P-O stretching vibrations are enhanced in TM. As the annealing temperature is increased to 50 °C (Traces B/B′), these bands are further enhanced, and furthermore, the bands at 1265 and 1250 cm-1 due to PO4- entities are observed only for TM polarization. However, at 75 °C (Traces C/C′), these bands are not detected but are present again at 100 and 125 °C (Traces D/D′ and E/E′). Again, spectra recorded from the F-S interface at different annealing temperatures (not shown) revealed no spectral changes. In the same manner, CaCl2 and KCl electrolyte solutions were added to MHPC-stabilized p-MMA/nBA colloidal dispersions, and ATR-FTIR spectra were recorded from the F-A interface using TE and TM polarizations. As shown in Figure 5A, Traces A-D′ of films containing 0:1.0, 0.5:1.0, 1.0:1.0, and 2.0:1.0 CaCl2/MHPC solutions and varying ratios of Ca2+ elicit numerous spectral changes. As seen in Traces B/B′, which represent the F-A interface of p-MMA/nBA films containing 0.5:1.0 CaCl2/MHPC, the bands at 1089 (P-O), 1105 (sec -OH),30 1123, 1135 (CO-C),38 and 1200 cm-1 (C-O-C)38 are detected. At the same time, PO4- entities exhibit preferential parallel

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Figure 4. Polarized ATR-FTIR spectra at the F-A interface depicting the 1350-900 cm-1 region of MMA/nBA copolymer films containing MHPC annealed at (A) 25 °C, TE; (A′) 25 °C, TM; (B) 50 °C, TE; (B′) 50 °C, TM; (C) 75 °C, TE; (C′) 75 °C, TM; (D) 100 °C, TE; (D′) 100 °C, TM; (E) 125 °C, TE; (E′) 125 °C, TM; (F) 150 °C, TE; (F′) 150 °C, TM for 2 h.

Lestage and Urban

species, to our surprise, coalesced films of dispersions containing 2.0:1.0 CaCl2/MHPC molar ratios (Traces D/D′) exhibited no presence of the bands at 1089, 1105, 1123, 1135, and 1200 cm-1. To compare divalent Ca2+ with monovalent K+, Figure 5B, Traces A-D′, illustrates TE- and TM-polarized ATRFTIR spectra recorded from the F-A interface of dispersions treated with KCl. As seen in Figure 5B, Traces B/B′, and similar to Figure 5A, Traces B/B′, IR bands are present at 1089, 1105, 1123, 1135, and 1200 cm-1, which result from the presence of K+ (0.5:1.0 KCl/MHPC ratio) and indicate that the species responsible for the 1089 (P-O) and 1123 cm-1 bands exhibit preferential parallel orientation, while the band at 1200 cm-1 reveals that C-O-C entities are perpendicular to the F-A interface. Interestingly enough, Figure 5A and B, Traces B/B′ and C/C′, reveals that films containing 0.5:1.0 and 1.0:1.0 molar ratios of both CaCl2 and KCl exhibit the same molecular species at the F-A interface. Subsequently, these spectral changes depend on ionic concentration rather than counterion valency. However, as seen in Traces D/D′ for films containing 2.0:1.0 KCl/MHPC, the bands are present at 1089, 1105, 1123, 1135, and 1200 cm-1 (same as 1.0:1.0 KCl/MHPC) with increased intensity are detected but not for 2.0:1.0 ratio of CaCl2/MHPC (Figure 5A, Traces D/D′), thus indicating that ionic interactions present in the p-MMA/nBA matrix are a function of ion valency. Specifically, the number of (+) charges in 2.0:1.0 KCl/MHPC ionic solution is the same as that in 1.0:1.0 CaCl2/MHPC. Again, the F-S interface is not affected. Discussion

Figure 5. (A) Polarized ATR-FTIR spectra at the F-A interface depicting 1350-900 cm-1 region of MMA/nBA copolymer films containing MHPC: (A) TE; (A′) TM, and colloidal dispersions treated with CaCl2 molar ratios: (B) 0.5:1.0, TE; (B′) 0.5:1.0, TM; (C) 1.0:1.0, TE; (C′) 1.0:1.0, TM; (D) 2.0:1.0, TE; (D′) 2.0: 1.0, TM. (B) Polarized ATR-FTIR spectra at the F-A interface depicting the 1350-900 cm-1 region of MMA/nBA copolymer films containing MHPC: (A) TE; (A′) TM, and colloidal dispersions treated with KCl molar ratios: (B) 0.5:1.0, TE; (B′) 0.5:1.0, TM; (C) 1.0:1.0, TE; (C′) 1.0:1.0, TM; (D) 2.0:1.0, TE; (D′) 2.0:1.0, TM.

orientation, while C-O-C stretching entities (1135 and 1200 cm-1) are primarily perpendicular to the F-A interface. Higher ratios of 1.0:1.0 CaCl2/MHPC (Traces C/C′) induce the same behavior as films containing 0.5: 1.0 ratios; however, the magnitude of TE and TM band intensities is greater. While intensity increases are expected as a result of increased population of absorbing (38) Silverstein, R. M.; Webster, F. X. Spectrometric Identification of Organic Compounds, 6th ed.; John Wiley and Sons: New York, 1998.

In view of the above data, it appears that SDOSS/MHPCand MHPC-stabilized p-MMA/nBA dispersions are sensitive to temperature and ionic strength stimuli. Furthermore, as a result of interactions of individual components at the F-A interface, formation of new entities resulting from complexation is apparent. For example, when annealing at 100 °C (Figure 2, Traces D/D′), the bands at 1310 and 1300 cm-1, as well as 1265 and 1250 cm-1, are detected simultaneously and annealing at 150 °C (Traces F/F′) reveals the presence of the bands at 1082, 1061, and 1025 cm-1. Since the bands at 1310, 1300, and 1265 cm-1 are not present in neat SDOSS and MHPC spectra (not shown), formation of unique molecular entities which are stratified at the F-A interface in the presence of p-MMA/ nBA matrix is evident, especially at elevated temperatures. Furthermore, as shown in Figure 5B, Traces D/D′ of films stabilized by MHPC, the bands at 1135, 1123, and 1089 cm-1 are detected at the same time when 2.0:1.0 KCl/MHPC ionic solution is utilized. As shown in the Results, analysis of spectra recorded from about 0.18 µm from the F-A interface revealed stimuli-responsive behaviors of SDOSS/MHPC. However, an immediate question to be addressed is the formation of the localized F-A interface entities and what morphological features are present. In an effort to correlate chemical morphologies at the F-A interface resulting from temperature and ionic strength of electrolyte solutions with ionic interactions manifested in the solutions, we collected a series of IRIR images from the F-A interface of 2.0:1.0 KCl/MHPC p-MMA/nBA colloidal solutions that were subjected to 100 and 150 °C annealing temperatures for 2 h after coalescence. As seen in Figure 6 for p-MMA/ nBA films stabilized by a SDOSS/MHPC mixture and annealed at 100 °C, AFM amplitude and phase images correspond to IRIR images when tuned to 1310, 1300, 1265, and 1250 cm-1 bands. These data illustrate that

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Figure 6. AFM amplitude and phase images at the F-A interface of SDOSS/MHPC-stabilized p-MMA/nBA films annealed at 100 °C and IRIR images tuned to 1265, 1250, 1310, and 1300 cm-1 with regions labeled 1, 2, and 3 corresponding to IR Traces 1, 2, and 3.

surface morphologies resulting from annealing at 100°C are due to surface LICs (SLICs). Specifically, IRIR image regions labeled 1 and 2 (Traces 1 and 2) indicate that the heterogeneities present at the F-A interface in AFM images are primarily due to species responsible for 1265 and 1250 cm-1 bands due to SLIC components and MHPC. This is manifested by darker regions generated by higher intensities of these bands, thus higher SLIC concentrations, whereas the region labeled 3 and Trace 3 reveal the surrounding p-MMA/nBA matrix. It should be noted that the AFM images are provided to illustrate surface morphological features, whereas IR images obtained from the same areas illustrate the chemical makeup of the analyzed area. Thus, the scales and the color palette in both imaging experiments do not necessary correspond to each other. The same applies to Figures 7 and 8.Since thermal history appears to be a significant stimulus, specimens annealed at 25, 50, 75, 100, 125, and 150 °C were analyzed utilizing DSC (not shown) to determine the presence of temperature-induced molecular phases at the F-A interface. Polymeric films stabilized by SDOSS/ MHPC and MHPC exhibit endothermic transitions indicating Tg’s at 15 °C for SDOSS/MHPC films and -7 °C in MHPC-stabilized films. In addition, a number of exothermic transitions are detected well above annealing temperatures which indicate crystallization processes, and within these transitions, sharp endotherms are present which reveal melting of crystals that form at the F-A interface. Figure 7 illustrates AFM and IRIR images recorded from the F-A interface of SDOSS/MHPC-stabilized pMMA/nBA films annealed at 150°C. As shown in the AFM images, temperature effectively stimulates the formation of SLICs which exhibit ordered crystalline geometries with 5 µm × 10 µm dimensions at the F-A interface. Areas labeled 1-3 in the IRIR images, from which multiple images were collected, illustrate that when tuned to 1082 (-OH) and 1061 (P-O-C) cm-1, IRIR images exhibit the structures observed with AFM. These data indicate that

SLICs generated at the F-A interface with annealing at 150 °C contain higher concentration levels of the tuned entities, thus revealing that the primary component of these surface structures is MHPC. Interestingly enough, spectral Traces 1 and 2, corresponding to Regions 1 and 2, exhibit elevated intensities at 1082 and 1061 cm-1, which indicate the presence of -OH and P-O-C segments of MHPC. The spectral map generated when tuned to 1025 cm-1 (O-CH2) exhibits the same SLIC heterogeneities; however, the concentration of O-CH2 species present in areas 1-3 is lower, thus revealing a minor contribution of SDOSS to SLIC formation at 150 °C. Figure 8 illustrates the formation of SLICs generated as a result of 2.0:1.0 KCl/MHPC electrolyte solution added to MHPC-stabilized p-MMA/nBA colloidal dispersions. As seen in the AFM amplitude and phase images, structures present at the F-A interface of MHPC-stabilized films exhibit clustered architectures which have been chemically mapped by IRIR images tuned to 1089 (P-O), 1123, and 1135 (C-O-C) cm-1. When tuning IRIR images to these bands, spectral maps illustrate similar features, thus indicating the structures detected in the AFM images are SLICs composed of at least three molecular segments. While IRIR spectral Traces 1-3, corresponding to image regions labeled 1-3, exhibit bands at 1089, 1123, and 1135 cm-1, Trace 3 reveals that its corresponding region is higher in p-MMA/nBA matrix content with enhanced bands at 1148 and 1162 cm-1. This, however, is expected as IRIRI Traces 1 and 2 were collected directly from the surface of the domains obtained in by AFM images. When treated with 2.0:1.0 KCl/MHPC ratio, a band at 1123 cm-1 is generated in the spectrum of MHPC. Furthermore, the magnitude of the 1123 cm-1 band is diminished at lower KCl/MHPC ratios and is not detected in model studies in which SDOSS is present. However, when combined with p-MMA/nBA matrix, the 1123 cm-1 band is detected in both SDOSS/MHPC and MHPCstabilized dispersions treated with CaCl2 and KCl ionic solutions, but crystalline domains are not formed at the

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Figure 7. AFM amplitude and phase images at the F-A interface of SDOSS/MHPC-stabilized p-MMA/nBA films annealed at 100 °C and IRIR images tuned to 1082, 1061, and 1025 cm-1 with regions labeled 1, 2, and 3 corresponding to IR Traces 1, 2, and 3.

Figure 8. AFM amplitude and phase images at the F-A interface of SDOSS/MHPC-stabilized p-MMA/nBA films annealed at 100 °C and IRIR images tuned to 1089, 1123, and 1135 cm-1 with regions labeled 1, 2, and 3 corresponding to IR Traces 1, 2, and 3.

F-A interface in the presence of CaCl2/MHPC ratios. These data indicate that the band at 1123 cm-1 is due to molecular vibrations of MHPC in the presence of Ca2+ and K+ ions and not Na+ (SDOSS counterion). Furthermore, while crystalline domains are not detected at the F-A interface of CaCl2-treated dispersions, organized structures at the surface of KCl-containing colloidal dispersions with corresponding IRIR images and spectra

reveal that segmental motions at 1123 cm-1 are present in SLICs of MHPC containing p-MMA/nBA colloidal dispersions. On the basis of these experiments and model studies, the following model of SLICs is proposed. Figure 9 illustrates that the effect of annealing SDOSS/MHPCstabilized films is responsive formation of crystalline SLICs at the F-A interface, whereas at elevated tem-

Release and Formation of SLICs into Phospholipid Rafts

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Figure 9. Effect of temperature and ionic strength on mobility and crystallization at the F-A interface.

peratures not exceeding 150°C, SDOSS/MHPC ionic interactions are disrupted due to SDOSS melting, thus allowing highly ordered MHPC crystalline domains to form at the film surface with a SDOSS subphase beneath. In a similar manner, MHPC crystalline domains are formed at the F-A interface in MHPC-stabilized dispersions when exposed to 2.0:1.0 KCl/MHPC ratios; however, the domains are less ordered likely due to lack of co-surfactant (SDOSS) assisting in MHPC diffusion and stratification at the F-A interface, thus facilitating the formation of SLICs or rafts containing phospholipid. Conclusions These studies illustrate that the responsive mobility of LICs to the F-A interface may be induced by external stimuli, which include environmental temperature and concentration of ionic species. Furthermore, crystallization of bio-active lipid domains occurs at the F-A interface which is affected by annealing temperature and concentration of electrolyte solutions. When MHPC is used as a

cosurfactant in colloidal synthesis, the CMC of the colloidal dispersion remains unchanged, thus resulting in unimodal particle size, which facilitates migration of surfactants with water flux. Subsequently, phospholipid-rich crystalline domains are formed that resemble membrane lipid rafts that occur in nature. These studies indicate that it is possible to create liposomes reinforced via a phospholipid-stabilized emulsion polymer partitioned in the hydrophobic region of bilayered vesicles and capable of stimuli-responsive behavior. Upon exposure to appropriate stimuli, stable synthetic hollow vesicles39 may undergo lipid raft self-assembly at interfaces, thus mimicking processes inherent to natural cellular membranes. Acknowledgment. This work was supported primarily by the MRSEC Program of the National Science Foundation under Award No. DMR 0213883. LA0475526 (39) Lestage, D. J.; Urban, M. W. submitted for publication.