Phospholipid Stabilized Colloidal

Biomacromolecules 2005, 6, 1561-1572

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Stimuli-Responsive Surfactant/Phospholipid Stabilized Colloidal Dispersions and Their Film Formation David J. Lestage, Min Yu, 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 December 20, 2004; Revised Manuscript Received March 21, 2005

Methyl methacrylate (MMA) and n-butyl acrylate (nBA) were copolymerized into stable colloidal particles in the presence of micelle forming sodium dioctyl sulfosuccinate (SDOSS) and liposome forming 1,2dilauroyl-sn-glycero-3-phosphocholine (DLPC) in aqueous media that serve as thermodynamically stable loci for lipophilic monomers and nanostructured templates. These studies show for the first time that hollow colloidal particles may coalesce to form polymeric films and the combination of SDOSS and DLPC dispersing agents provides a stimuli-responsive environment during film formation through which individual surface stabilizing components can be driven to the film-air (F-A) or film-substrate (F-S) interface. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) of p-MMA/nBA colloidal dispersions revealed preferential and enhanced mobility of SDOSS and DLPC lipid rafts to the F-A and F-S interfaces in response to thermal, ionic, and enzymatic stimuli. Introduction Although colloidal particles have been of interest for a number of years, their chemical complexity derived from the presence of ionic or nonionic surfactants, hydrophilic/ hydrophobic monomers, their components, and their interactions make the understanding of these polymeric species challenging and not trivial.1-12 Thus, continuous quests for advancing our knowledge continue by exploring new avenues. For example, it has not been realized until recently that their synthesis may be facilitated by amphiphilic bioactive molecules, such as phospholipids,13,14 and this approach opened up new scientific and application avenues of polymeric colloidal dispersions. Although one function of these species is to serve as dispersion stabilizing agents, they may also be deliberately mobilized to designated areas within polymer matrixes during or after coalescence. As a result, and depending on concentrations levels of ionic environments, their structural features, as well as other solution properties, phospholipids may form unique entities. Such stimuli-responsive behaviors of these and other bio-active entities offer numerous opportunities for preparing polymeric films for biomedical and biotechnological applications. For example, under certain conditions, self-assembled bilayered liposomes may serve as lipophilic drug carriers to target sites in aqueous media.15-19 Such systems, derived from physiological structures, are expected to have increased biocompatibility due to chemical and compositional similarities to their biological counterparts.20 Although it is well established that phospholipids may form liposomes, the liposome architecture is inherently fragile which limits encapsulation properties, thus affecting loads and shelf life stability. In an attempt to reinforce liposomes by polymerization of hydro* To whom all correspondence should be addressed.

phobic monomers such as styrenes, methacrylates, isodecyl acrylate, and oleic acid solubilized within the vesicle bilayer, several studies were conducted.21-27 Induced either thermally or photochemically, polymerization reactions were initiated by either hydrophilic or lipophilic initiators resulting in hollow polymer capsules,27,28 although hollow capsules may exhibit parachute-like morphologies with a polymer bead attached to the vesicle exterior.25,26 To advance how liposomes may be utilized in polymerization of unique shape colloidal particles, the roles of self-assembled liposomes in colloidal film formation processes need to be further understood. For that reason, these studies not only focus on the formation of liposomes in the presence of monomer/ initiator species which are thermally polymerized but also coalescence processes during film formation from colloidal dispersions prepared in the presence of liposomes. Of particular interest are stimuli-responsive behaviors which may be tailored for specific applications. For example, Figure 1 illustrates how temperature, ionic strength, and pH may mobilize colloidal particle surface stabilizing entities to designated areas of coalesced films which may serve as signal/receptor species in bio-processes.29-32 Although further details concerning these surfaces will be explored in the Results and Discussion, the observed crystalline structures shown in Figure 1 exhibit significant differences in size and morphology and are elucidated preferentially at the filmair (F-A) and film-substrate (F-S) interfaces as a result of changes in temperature, ionic strength, pH, and concentration of active enzyme in coalescing colloidal dispersions. Experimental Section Methyl methacrylate (MMA), n-butyl acrylate (nBA), styrene (Sty), sodium dioctylsulfosuccinate (SDOSS), and potassium persulfate (KPS) were purchased from Aldrich

10.1021/bm049195j CCC: $30.25 © 2005 American Chemical Society Published on Web 04/13/2005

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Lestage et al. Table 1. Particle Size and Composition of Random Copolymerized p-(MMA/nBA) Colloidal Dispersions with SDOSS/ DLPC Mixture and DLPC Only composition components: DDI (%) methyl methacrylate (%) n-butyl acrylate (%) SDOSS (%) K2S2O8 (%) DLPC (%) Solids (%) particle size (nm) CaCl2/DLPC: 0.5/1.0 1.0/1.0 2.0/1.0

Figure 1. Cross-polarized optical micrographs of SDOSS/DLPC stabilized p-MMA/nBA films recorded from F-A and F-S interfaces at (A1) F-A, 50 °C; (A2) F-S, 50 °C (A3) F-A, 0.5/1.0 CaCl2/DLPC; (A4) F-S, 0.5/1.0 CaCl2/DLPC; (A5) F-A, pH ) 5.0; (A6) F-S, pH ) 5.0 and DLPC stabilized films at (B1) F-A, 50 °C; (B2) F-S, 50 °C; (B3) F-A, 0.5/1.0 CaCl2/DLPC; (B4) F-S, 0.5/1.0 CaCl2/DLPC; (B5) F-A, pH ) 5.0; (B6) F-S, pH ) 5.0.

Chemical Co. 1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC) phospholipid was purchased from Avanti Polar Lipids, Inc. MMA/nBA copolymer emulsions were synthesized using a semicontinuous process outlined elsewhere33 and adapted for small-scale polymerization. The reaction flask 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 dionized (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) was added while 10 w/w% preemulsion solution (DDI, SDOSS, DLPC, and monomers), mixed under vortex followed by 15 min sonication using a G112SP1 ultrasonic cleaner (Lab. Supp. Co.), was added to the reaction flask. The SDOSS levels were in the range of 2.7 wt. % of total monomer, thus generating 26.0 mM aqueous solutions that are above the CMC of SDOSS (0.100.14 wt. % of water,34 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. The reaction continued for 1 additional hour after which time

p-(MMA/nBA) SDOSS/DLPC

p-(MMA/nBA) DLPC

76.7 10.9 10.9 0.58 0.45 0.47 21.9 132 concentrated (mM) 4.6 9.2 18.4

76.8 11.0 11.0 0.44 0.76 22 69 concentrated (mM) 7.6 15.3 30.6

the temperature was raised to 85 °C. Upon cooling, the emulsion was filtered twice and particle size analysis was performed using a Microtrac Nanotrac 250 particle size analyzer. In a typical experiment, the standard deviation was (10 nm. CaCl2 aqueous solutions were prepared by solubilizing CaCl2 (Aldrich) in DDI water at 6.9, 11.5, 13.8, 23.0, 27.6, and 45.0 mM. When applicable, 1.0 mg (1,283 units/ mg) of phospholipase A2 (PLA2) enzyme (Aldrich) was added to 1.0 mL of aqueous dispersions adjusted to pH ) 2.0, 5.0, and 8.0 before film casting. Table 1 summarizes details of colloid formulations, results of the particle size analysis, and electrolyte and enzyme 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 its melting point is 150 °C and the decomposition temperature is above 450 °C.34 Molecular weight was determined using gel permeation chromatography (GPC; Waters Inc.), and each sample was precipitated in tetrahydrofuran (THF) and eluted through a divinylbenzene (DVB) column. Sample elution times were analyzed with polystyrene standards. Colloidal dispersion particle morphologies were analyzed using a Zeiss EM 109-T transmission electron microscope (TEM) in which colloidal dispersions were diluted at a 20:1 vol. ratio (DDI H2O:dispersion) and deposited on Formvar coated copper TEM grids. Optical micrographs of film surfaces were acquired using a Nikon Optiphot biological microscope equipped with cross-polarizers. 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. The surfaces were analyzed using a 2 mm Ge crystal with a 45° angle maintaining constant contact pressure between the

Stabilized Colloidal Dispersions

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Figure 2. a. TEM images of p-MMA/n-BA particles prepared in the DLPC/SDOSS and DLPC. b. TEM micrographs of a p-MMA/nBA particle exposed to the electron beam for 0 s (A′) and 240 s (B′). TEM micrographs A through E are images of with 69 nm diameter prepared in the presence of DLPC liposomes illustrating decomposition of polymer in time.

crystal and the specimens. All spectra were corrected for spectral distortions using software for the Urban-Huang algorithm.35 Results and Discussion Aside from applications, mobility of phospholipids and their ability to form unique architectures within a polymer matrix is of importance as the same species may serve as polymerization loci in colloidal dispersion synthesis.13,14,36,37 For example, phospholipids may function as surfactants and stabilize colloidal particles while providing a self-assembled bilayer scaffold in which hydrophobic polymers may localize. Since the DLPC phospholipid may play such a role, we synthesized colloidal dispersions of MMA/nBA in the presence of SDOSS and DLPC in an aqueous phase, and their chemical structures are shown below. Utilizing an

aqueous environment containing micelle forming SDOSS and liposome forming DLPC, a p-MMA/nBA particle size of 132 ( 9 nm was generated with a mol wt. of 350 000 g/mol,

whereas the presence of only DLPC resulted in 69 ( 11 nm colloidal particles with a mol. wt. of 630 000 g/mol. The particle diameter of 69 nm in p-MMA/nBA colloidal particles synthesized in the presence of DLPC is mostly due to the formation of liposomes in the aqueous phase. Aqueous solutions of DLPC form large multilamellar vesicles (LMVs), which upon sonication, disassociate to assemble small unilamellar vesicles (SUVs), or liposomes, ranging in size from 50 to 75 nm.38 As a matter of fact, the particle size of DLPC dispersed in DDI H2O before polymerization was about 50 nm, and the increase to 69 nm likely results from polymer partitioning in the hydrophobic bilayer of liposomes during dispersion synthesis. TEM images of particles prepared in the presence of DLPC/SDOSS and DLPC are shown in Figure 2a, respectively. Interestingly enough, the TEM micrograph series shown in Figure 2b illustrate 69 nm p-MMA/nBA particles synthesized in the presence of DLPC and appear to decompose under the electron beam with time. At beam exposure time t ) 0 in Figure 2b, A, spherical particles with nonuniform distribution of electron (e-) densities are detected at 30 000× magnification, but upon longer exposure times to the electron beam, the darker regions due to e- rich polymer appear to decrease in quantity and contrast, as depicted in Figure 2b, B and C, and at extended exposure times up to 240 s (Figure 2b, E), e- rich regions are not detected. These data indicate that, due to

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limited volume inside the hydrophobic zone available for monomer diffusion during polymerization, the resulting molecular weight of the polymer is low, and its distribution is likely nonuniform. As a consequence, high powered electron beam exposure during TEM analysis either decomposes or depolymerizes a relatively weak polymer that reinforces the hydrophobic zone. However, regardless of the mechanism of disintegration of these unstable particles, the particles prepared in the presence SDOSS/DLPC are stable upon exposure to the electron beam. This is illustrated in Figure 2b, A′ and B′, which are TEM images for the p-MMA/nBA particles polymerized in the presence of SDOSS/DLPC and exposed for 0 and 240 s or longer to the same intensity electron beam. Although the primary purpose for these experiments was to controllably produce particles with relatively narrow particle sizes, our ultimate goal is to elucidate the effect of different micellar environments on film formation in response to various stimuli. As shown in Figure 1, A1-A6 and B1B6, which illustrate optical images obtained from the F-A and F-S interfaces, temperature, ionic strength, and pH effectively alter interfacial morphologies. This initial observation is important because, under specific conditions, selfassembled phospholipid rafts may be deliberately generated in designated areas of coalesced films when phospholipid is utilized in the synthesis, and such entities may potentially serve as protein docks, cell-cell signaling receptors,29,39,40 or other applications. In an effort to illustrate how temperature, ionic strength, pH, and the presence of enzymes may generate stimuli-responsive behaviors, the remaining part of this paper is divided into the following sections: Temperature Responses, The Effects of Ionic Strength, Responses to pH Changes, and Enzyme Effects. Temperature Responses. In an effort to establish the effect of temperature on response of these entities, we conducted a series of annealing experiments on SDOSS/ DLPC stabilized p-MMA/nBA films as well as p-MMA/nBA films containing only DLPC (Table 1) and spectroscopically analyzed the F-A and F-S interfaces. Since formation of rafts lead to preferentially aligned crystalline morphologies, transverse electric (TE - 0°) and transverse magnetic (TM - 90°) polarizations were utilized from which dichroic ratios (R)41 were determined and are listed in Table 2. When R < 1.0, molecular segments are preferentially aligned perpendicular to the film interface, whereas for R > 1.0, parallel orientation to that interface is anticipated. These values will be used to assess approximate orientation of surface/ interfacial entities. The F-A interfaces of p-MMA/nBA colloidal films synthesized in the presence of a SDOSS/DLPC mixture exhibited no spectral changes as a result of elevated temperature. In contrast, as shown in Figure 3, traces A-D′, spectroscopic analysis of the F-S interface illustrates the presence of low mol. wt. species is manifested by the bands at 1310, 1300, 1265, 1250, and 1061 cm-1 upon annealing from 50 to 100 °C. As seen in traces B-D′, enhanced intensity of the band at 1061 cm-1 in TM polarization which is reflected in the R values in Table 2 indicates preferential perpendicular orientation of DLPC P-O-C42 segments at

Lestage et al.

the F-S interface, whereas R ) 1.3 for PdO43 linkages at 1250 cm-1 indicates mostly random orientation. At the same time, the band at 1300 cm-1 due to crystalline phospholipid entities14 (Figure 1, A2) indicates that these segments are aligned preferentially parallel to the F-S interface as R > 1.0. Furthermore, upon annealing at 75 °C, the 1310 and 1300 cm-1 bands in trace C exhibit enhanced magnitude for TE polarization with R values of 2.2 and 2.8, respectively, indicating an increase in population of preferentially parallel oriented species at the F-S interface as a result of elevated temperature. Upon annealing at 100 °C (traces D/D′), the bands at 1310, 1300, 1265, and 1250 cm-1 exhibit reduced intensity and, above this temperature, are not detected. Previous studies37 showed that in p-MMA/nBA colloidal dispersions synthesized in the presence of a SDOSS and 1-myristoyl-2-hydroxy phosphocholine (MHPC) mixture the same species are present at the F-A interface. However, when DLPC is utilized in the same colloidal environment, these features are detected at the F-S interface, thus indicating that, depending upon the phospholipid, surface localized ionic clusters (SLICs) generated as a result of thermal stimuli can be created at either interface. Although previous studies21-27 have attempted polymerization within liposome bilayers or incorporation of liposomes in colloidal dispersion synthesis and their film formation,13 stimuli-responsive behaviors of pMMA/nBA were not addressed. For that reason, coalesced colloidal films synthesized with DLPC were analyzed, and Figure 4, traces A-D′, illustrates ATR-FTIR spectra recorded at the F-A interface using TE and TM polarizations (Table 2). As seen in traces A/A′, upon annealing at 25 °C, the bands at 1310, 1300, 1265, 1250, and 1061 cm-1 are detected at the F-A interface. However, when annealed at 50 and 75 °C (not shown), these bands are not present. Furthermore, as seen in traces A/A′ the bands at 1265 and 1250 cm-1 exhibit enhanced magnitude in TE polarization with R values of 4.3 and 7.2, respectively, whereas the bands at 1310, 1300, and 1061 cm-1 display strongest intensity for TM polarization with R < 0.25. These data indicate that ordered, crystalline morphologies are present at the F-A interface after particle coalescence at 25 °C (Figure 1, B1) and contain primarily parallel aligned DLPC PO4- segments represented by the PdO band at 1250 cm-1. However, as shown in traces B/B′ for films annealed at 100 °C, the bands at 1310, 1300, 1265, and 1250 cm-1 exhibit reduced intensity and broader bandwidths, thus indicating a decrease in population and more random orientation which is reflected in the R values, and at 125 °C (traces C/C′) are not detected. Interestingly enough, upon annealing at 150 °C, the bands at 1082 and 1061 cm-1 are detected, thus revealing the presence of DLPC C-OH43,44 and P-O-C species with R ) 1.0, and for TM polarization (trace D′), the band at 1025 cm-1 is detected indicating an alignment of O-CH245 entities normal to the F-A interface. The above data illustrate that crystalline phospholipid species are generated at the F-A interface as a result of coalescence and temperature response, and cross-polarized optical micrographs of the F-A and F-S interface of SDOSS/DLPC and DLPC films shown in Figure 1, A1-A2


Stabilized Colloidal Dispersions Table 2. Dichroic Ratios (R) of Selected IR Bandsa

dichroic ratio (R) of specific molecular segments SDOSS/DLPC F-S annealing T (°C)

SLIC

SLIC

SLIC

PdO

P-O-C

1310 cm-1

1300 cm-1

1265 cm-1

1250 cm-1

1061 cm-1

25 50 75 100 125 150

n/a 1.848 2.186 2.163 n/a n/a

n/a 1.640 2.845 1.989 n/a n/a

n/a 1.202 1.073 2.229 n/a n/a

n/a 1.295 0.999 2.012 n/a n/a

1.000 0.043 0.051 0.335 0.056 0.895

DLPC F-A annealing T (°C)

1310 cm-1

1300 cm-1

1265 cm-1

1250 cm-1

1061 cm-1

25 50 75 100 125 150

0.210 n/a n/a 0.001 n/a n/a

0.249 n/a n/a 2.082 n/a n/a

4.343 n/a n/a 2.074 n/a n/a

7.156 n/a n/a 1.670 n/a n/a

0.007 n/a n/a 0.045 1.000 0.943

DLPC F-A CaCl2/DPLC

1310 cm-1

1300 cm-1

1265 cm-1

1250 cm-1

1061 cm-1

0/1.0 0.5/1.0 1.0/1.0

0.500 n/a n/a

0.476 n/a n/a

3.650 n/a n/a

5.100 n/a n/a

0.238 1.000 1.000

SDOSS/DLPC F-S CaCl2/DPLC

1310 cm-1

1300 cm-1

1265 cm-1

1250 cm-1

1061 cm-1

0/1.0 0.5/1.0 1.0/1.0

1.125 1.200 1.270

1.353 1.850 1.780

0.663 1.000 1.000

1.097 1.300 1.180

0.513 0.370 0.333

DLPC F-S CaCl2/DLPC

1310 cm-1

1300 cm-1

1265 cm-1

1250 cm-1

1061 cm-1

0/1.0 0.5/1.0 1.0/1.0

1.000 n/a n/a

1.300 n/a n/a

1.300 n/a n/a

1.173 n/a n/a

0.813 n/a n/a

SDOSS/DLPC F-A pH

1310 cm-1

1300 cm-1

1265 cm-1

1250 cm-1

1061 cm-1

2.0 5.0 8.0

n/a 2.500 3.200

n/a 3.000 4.170

n/a 0.769 0.909

n/a 0.769 0.909

n/a 0.125 0.192

DLPC F-A

1310 cm-1

1300 cm-1

1265 cm-1

1250 cm-1

1061 cm-1

2.0 5.0 8.0

0.500 n/a n/a

0.476 n/a n/a

3.650 0.870 n/a

5.100 1.000 n/a

0.238 0.400 1.000

SDOSS/DLPC F-S pH

1310 cm-1

1300 cm-1

1265 cm-1

1250 cm-1

1061 cm-1

2.0 5.0 8.0

n/a 1.000 n/a

n/a 1.000 n/a

n/a 1.000 n/a

n/a 1.000 n/a

1.000 1.000 1.000

a R ) A /A , where A and A are band areas for parallel (TE) and perpendicular (TM) polarizations. R values were obtained by ratioing the baseline| ⊥ | ⊥ corrected IR bands of interest. For each band, the same wavenumber range was used to determine the band area for TE and TM polarizations.

and B1-B2, support these observations. As seen, the F-S interface of SDOSS/DLPC stabilized p-MMA/nBA films (Figure 1, A2) exhibits birefringent crystalline clusters with an average domain size of 30 µm. Upon annealing at desired temperatures, the crystalline entities exist and reveal no morphology changes. However, above 100 °C, no birefringence is observed at the F-S interface. Under the same conditions, the micrograph in Figure 1, B1, reveals crystalline

entities present at the F-A interface of films stabilized with DLPC only and exhibit domain sizes of approximately 160 µm. These domains, however, are observed only at the F-A interface of the films annealed at 25 °C, clearly indicating that the simultaneous presence of SDOSS and DLPC forms SLICs at the F-S interface which differ in morphology compared to SLICs formed at the F-A interface of films prepared with DLPC only. These data are summarized in

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Figure 3. Polarized ATR-FTIR spectra at the F-S interface depicting 1350-950 cm-1 region of MMA/nBA copolymer films containing SDOSS/DLPC mixture annealed at (A) 25, TE; (A′) 25, TM; (B) 50, TE; (B′) 50, TM; (C) 75, TE; (C′) 75, TM; (D) 100, TE; (D′) 100 °C, TM for 2 h.

Figure 4. Polarized ATR-FTIR spectra at the F-A interface depicting 1350-950 cm-1 region of MMA/nBA copolymer films containing DLPC annealed at (A) 25, TE; (A′) 25, TM; (B) 100, TE; (B′) 100, TM; (C) 125, TE; (C′) 125, TM; (D) 150, TE; (D′) 150 °C, TM for 2 h.

Figure 5, which depicts SLIC formation with preferential orientation at alternate interfaces as a result of the surfactants present in annealed p-MMA/nBA colloidal films. As seen for a specimen annealed at 25 °C, a SDOSS/DLPC mixture inhibits the preferential migration of these species to the F-A and F-S interface. The presence of DLPC allows the formation of LICs with SDOSS which at elevated temperatures exhibit enhanced mobility to the F-S interface when films are annealed up to 100 °C. However, at higher temperatures, these species are not observed. To further support these behaviors resulting from the presence of DLPC, films only stabilized with DLPC exhibit SLICs at the F-A interface, and higher temperatures elicit their disappearance. However, SLICs reappear at 100 °C but at 150 °C, their molecular order has decreased and DLPC entities are detected as an amorphous phase. These data indicate that interactions between SDOSS and DLPC form entities with directionality to the F-S interface at elevated temperatures. However, annealing at 150 °C disrupts these associations resulting in the release of DLPC to the F-A interface.

Lestage et al.

Effect of Ionic Strength. In view of the above experimental data, previous studies2,13 determined that utilization of phospholipids in colloidal dispersion synthesis imparted responsive behaviors to coalescing films as a result of various stimuli. Although the effects of elevated temperatures have been addressed above, of particular importance is the ionic strength of a colloidal dispersion. For that reason, a series of CaCl2 solutions were prepared and added to p-MMA/nBA colloidal dispersions and the choice of Ca2+ as a counterion was dictated by its ability to collapse or extend polymeric chains,13 bind multiple low mol. wt. molecules,46 and disrupt ionic interactions, thus facilitating the formation of SLICs.14,46,47 Although details of experimental conditions are provided in the Experimental Section, such colloidal dispersions were allowed to coalesce, followed by the F-A and F-S interfacial analysis of the coalesced films using polarized ATR-FTIR spectroscopy to determine the presence of species of interest at the F-A and F-S interfaces, as well as to identify their preferential orientations. Although CaCl2/DLPC molar ratios added to SDOSS/ DLPC stabilized p-MMA/nBA dispersions showed no responsiveness at the F-A interface as determined by ATRFTIR spectra (not shown), significant spectral changes were observed in polarized spectra collected from the F-S interface of SDOSS/DLPC stabilized dispersions exposed to 0.5/1.0 and 1.0/1.0 CaCl2/DLPC ratios. As seen in Figure 1, A4, minute crystalline domains are observed, and in Figure 6, traces B/B′ and C/C′, the bands at 1310, 1300, and 1265 cm-1 attributed to SLIC formation, as well as the bands at 1250 and 1061 cm-1 due to DLPC PO4- entities, are detected as a result of 0.5/1.0 and 1.0/1.0 CaCl2/DLPC addition. As a result, P-O-C entities (1061 cm-1) are preferentially oriented perpendicular to the F-S interface. As shown in Table 2, whereas R values for 1250 and 1061 cm-1 bands remain steady despite Ca2+ ion concentration changes, the R ) 1 for SLIC segments at 1310, 1300, and 1265 cm-1, and increase in response to higher Ca2+ levels indicates a more parallel ordered species present at the F-A interface. ATR-FTIR spectra recorded from the F-A interface of p-MMA/nBA dispersions prepared in the presence of DLPC and exposed to CaCl2 ionic solutions are shown in Figure 7. As seen in traces B/B′ at 0.5/1.0 CaCl2/DLPC ratios, IR bands at 1310, 1300, and 1265 cm-1 due to crystalline phospholipid moieties, and 1250 and 1061 cm-1 bands of PO4- segmental motions, are suppressed at the F-A interface compared to untreated films (traces A/A′), and at a 1.0/1.0 CaCl2/DLPC ratio (traces C/C′), are not detected. In addition, dispersions treated with 0.5/1.0 ratios exhibit new bands at 1135 and 1123 cm-1, indicating the formation of alternate LICs14 resulting from higher concentrations of Ca2+ ions which appear as crystalline domains at the F-A interface but are not detected at the F-S interface (Figure 1, B3B4). It should be noted that model experiments in which all components were mixed together and examined under the same conditions revealed no formation of the F-A and F-S interfacial entities. This observation is consistent with the previous studies.14 For example, when p-MMA/nBA disper-



Figure 5. Schematic diagram depicting preferentially oriented manifestations at the F-A and F-S interfaces of SDOSS/DLPC and DLPC stabilized p-MMA/nBA dispersions annealed at various temperatures.

Figure 6. Polarized ATR-FTIR spectra at the F-S interface depicting 1350-950 cm-1 region of MMA/nBA copolymer films containing SDOSS/DLPC mixture: (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.

sions were prepared in the presence of SDOSS and hydrogenated soybean phosphocholine (HSPC) surface stabilizing species and exposed to CaCl2 ionic solutions, as a result of increasing ionic strength, a precipitate was formed in the aqueous dispersions. For DLPC, no precipitate was formed at 1.0/1.0 CaCl2/DLPC ratios, but increased CaCl2/DLPC ratios (2.0/1.0 CaCl2/DLPC) resulted in precipitation where specimens gelled within 2 h. IR analysis recorded from the gel showed the presence of the 1046 cm-1 band due to S-O stretching vibrations of SDOSS, thus indicating the release of SDOSS from particle surfaces. Based on these data, Figure 8, A, summarizes the direction of mobility during coalescence as a function of concentration levels of Ca2+ as well as dispersion stabilizing agents. As shown in Figure 8, B1, the F-A and F-S interfaces of

Figure 7. Polarized ATR-FTIR spectra at the F-A interface depicting 1350-950 cm-1 region of MMA/nBA copolymer films containing DLPC: (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.

SDOSS/DLPC and DLPC stabilized p-MMA/nBA dispersions respond to ionic environments, and when SDOSS and DLPC are present in solution, low ionic strength Ca2+ ions may bind to the DLPC species, thus causing their stratification at the F-S interface during coalescence. In contrast, when only DLPC is present in a low ionic strength environment (Figure 8, B2), a fraction of DLPC molecules not complexed with Ca2+ ions are released from the particle interfaces thus are able to migrate to the F-A interface and form SLICs. However, as shown in Figure 8, B3 and B4, at high ionic strength, solutions containing SDOSS/DLPC and DLPC coagulate as the electrical double layer surrounding colloidal particles is disrupted by Ca2+ ions and there is no longer a substantial energy barrier separating negatively charged neighboring particles. This behavior is consistent with the DLVO theory.48,49

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Figure 8. (A) Schematic diagram depicting preferentially oriented manifestations at the F-A and F-S interfaces of SDOSS/DLPC and DLPC stabilized p-MMA/nBA dispersions prepared in the presence of CaCl2/DLPC molar ratios. (B) Molecular-level interactions between SDOSS and DLPC species in the presence of Ca2+.

Figure 9. Polarized ATR-FTIR spectra at the F-A interface depicting 1350-950 cm-1 region of MMA/nBA copolymer films containing SDOSS/DLPC and adjusted to pH: (A) 2.0, TE; (A′) 2.0, TM,: (B) 5.0, TE; (B′) 5.0, TM; (C) 8.0, TE; (C′) 8.0, TM.

Response to pH Changes. It is well know that the formation of liposomes and their stability depends on properties of the aqueous media and one of the important parameters is pH. For that reason p-MMA/nBA colloidal dispersions were adjusted to pH values of 2.0, 5.0, and 8.0. Using these solutions, films were cast and allowed to coalesce for 72 h, followed by interfacial analysis using polarized ATR-FTIR. As seen in Figure 9, traces A-C′, SDOSS/DLPC stabilized films formed from dispersions at pH ) 2.0, 5.0, and 8.0 exhibit bands due to crystalline DLPC moieties at 1310, 1300, and 1265 cm-1 along with the PO4-

Figure 10. Polarized ATR-FTIR spectra at the F-A interface depicting 1350-950 cm-1 region of MMA/nBA copolymer films containing DLPC and adjusted to pH: (A) 2.0, TE; (A′) 2.0, TM,: (B) 5.0, TE; (B′) 5.0, TM; (C) 8.0, TE; (C′) 8.0, TM.

segmental motions at 1250 and 1061 cm-1 at the F-A interface, and as shown in Figure 1, A5, such crystalline domains are detected in cross-polarized microscopy. Interestingly enough, in addition to the band at 1061 cm-1, the 1069 cm-1 band is present which exhibits greater intensity in TM polarization for pH ) 5.0 (traces B/B′) and 8.0 (traces C/C′). However, only the 1061 cm-1 band is detected in TE mode. These data indicate that with the increased dispersion alkalinity, basic ions generate another environment around PO4- which forces P-O-C entities to orient preferentially perpendicular to the F-A interface. The R values in Table 2 reflect these orientation changes as a result of solution



Figure 11. (A) Schematic diagram depicting preferentially oriented manifestations at the F-A and F-S interfaces of SDOSS/DLPC and DLPC stabilized p-MMA/nBA dispersions as a result of adjusted pH values. (B) Molecular-level interactions between SDOSS and DLPC species in the presence of low and high pH.

alkalinity, and as observed for the bands at 1265 and 1250 cm-1, R ) 0.8 indicates primarily random orientation of specific SLIC and PdO molecular segments at pH ) 5.0. At the same time, the bands at 1310 and 1300 cm-1 have R values of 2.5 and 3.0, respectively, revealing preferential parallel orientation. Furthermore, upon adjusting pH to 8.0, the R values increase to 3.2 and 4.2, thus suggesting that a more basic environment is conducive to parallel alignment of SLIC entities. Figure 10, traces A-C′ are the F-A interface spectra of p-MMA/nBA colloidal films stabilized by DLPC not containing Ca2+ ions and adjusted to pH of 2.0, 5.0, and 8.0. As seen in traces A/A′ for pH ) 2.0, and in contrast to films stabilized by a SDOSS/DLPC mixture (Figure 9, traces A/A′), the bands at 1310, 1300, 1265, and 1250 cm-1 are detected, revealing the presence of crystalline domains at the F-A interface which are also present in the crosspolarized micrographs of Figure 1, B1. The R values for the

bands at 1310 and 1300 cm-1 (SLICs) are 0.5, indicating a preferential perpendicular orientation of these species, whereas for the 1265 and 1250 cm-1 bands R > 1.0, and thus SLIC segments (1265 cm-1) and DLPC PO4- entities are preferentially parallel to the F-A interface. It should be noted that dispersions synthesized in this study have an initial pH of 2.0. Thus, dispersions stabilized with a SDOSS/DLPC mixture require an increase of the pH to stimulate formation of crystals at the F-A interface, whereas dispersions containing only DLPC do not. However, similarly to SDOSS/ DLPC stabilized films, p-MMA/nBA films containing only DLPC exhibit highly oriented P-O-C segments normal to the F-A interface at 1061 cm-1 with R < 1.0, but structural rearrangements occur when pH ) 5.0 (traces B/B′). Specifically, the bands at 1310 and 1300 cm-1 are not detected, whereas the bands at 1292 and 1284 cm-1 have lower magnitudes and broader absorbances with the R < 1.0, thus indicating a chemical disruption of crystalline moieties,

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which were previously detected at 1310 and 1300 cm-1 with preferential perpendicular orientation, and as a result of higher pH, the bands are shifted to 1292 and 1284 cm-1 and their intensity changes indicate orientation primarily parallel to the F-A interface. For the films coalesced at pH ) 8.0, no significant spectral features are observed. Based on the analysis of spectroscopic data shown in Figures 9 and 10, the following physical and chemical processes resulting from pH changes are depicted. As shown in Figure 11, A, when pH ) 2.0, SLICs are formed with preferential perpendicular orientation (R < 1.0) at the F-A interface of DLPC stabilized films but increased pH to 5.0 randomizes orientation (R ) 1.0) of SLICs and induces an alternate crystalline phase as seen in Figure 1, B5. At the same time, SLICs are present at the F-A interface in SDOSS/DLPC films and are preferentially parallel (R > 1.0). At higher pH values of 8.0, these entities maintain their order but are no longer detected in DLPC stabilized films. Spectroscopic data also allows us to identify chemical interactions resulting from pH changes. As shown in Figure 11, B1, chemical interactions for low pH are depicted where S-O- and P-O- entities are protonated, thus causing a decrease of ionic repulsion forces between like charges.50 As a consequence, stratification at the F-A and F-S interface does not occur. Similary for DLPC (Figure 11, B2), a low pH value of the solution protonates P-O- species, but due to increased packing they remain intact and form SLICs at the F-A interface. In contrast, for high pH values, as shown in Figure 11, B3, the ionic interactions between SDOSS/DLPC species are disrupted due to the presence of excess Na+ and OH- ionic species, thus facilitating their mobility with water flux during particle coalescence. Similarly, for higher pHs, DLPC interactions (B4) are no longer present due to deprotonation of P-O-H+ entities, thus allowing neighboring DLPC molecules to migrate to the F-A interface and assemble (Figure 1, B5). It should be noted that pH values of 10.0 and higher cause excessive coagulation of SDOSS/DLPC and DLPC dispersions. This is again attributed to the release of DLPC and SDOSS from the surface of colloidal particles. Enzyme Effects. As indicated above, DLPC forms liposomes, thus facilitating hollow particle morphologies. Therefore, to intentionally inhibit the ability of DLPC to form liposomes and compare the resulting stimuli-responsive behaviors of p-MMA/nBA colloidal particles, the active enzyme phospholipase A2 (PLA2) was added to aqueous dispersions before film coalescence. PLA2 is an active enzyme consisting of a single polypeptide chain of 123 amino acids which reacts with glycerophospholipids (DLPC). This process is pH dependent with the highest activity achieved at pH ) 8.9, and upon the addition of H2O, DLPC is cleaved at its C2 position to form a single tailed lyosophospholipid and lauric acid.51-54 Considering the above discussion, one would expect that dispersions prepared in the presence of DLPC only and exposed to PLA2 would elicit drastic spectral changes due to the concentration of DLPC present in the dispersion, however the bands indicative of SLICs at 1310, 1300, 1265, 1250, and 1061 cm-1 are detected at pH)2.0, which is

Lestage et al.

Figure 12. Polarized ATR-FTIR spectra at the F-A interface depicting 1350-950 cm-1 region of MMA/nBA copolymer films containing SDOSS/DLPC and adjusted to pH: (A) 2.0, TE; (A′) 2.0, TM,: (B) 5.0, TE; (B′) 5.0, TM; (C) 8.0, TE; (C′) 8.0, TM in the presence of PLA2 enzyme.

attributed to low enzyme activity in acidic environments.52 At higher pH values, the spectra recorded from the F-A and F-S interfaces of DLPC stabilized films prepared in the presence of PLA2 (not shown) revealed no bands at 1310, 1300, 1292, 1284, 1265, 1250, and 1061 cm-1, thus indicating that DLPC cleaves as a result of PLA2 reactions, and SLICs previously observed in DLPC stabilized films (Figure 1, B1) are no longer capable of forming at the F-A interface. In contrast, Figure 12, traces A-C′, shows the F-A interface spectra of p-MMA/nBA colloidal films stabilized by SDOSS/ DLPC and exposed to PLA2 in an aqueous phase before coalescence. As seen in traces A/A′, at pH ) 2.0, p-MMA/ nBA spectra resemble untreated SDOSS/DLPC stabilized films (Figure 9, traces A/A′). However, when the dispersion is at pH ) 5.0, the bands at 1310, 1300, 1265, and 1250 cm-1 are detected (traces B/B′), thus indicating the formation of crystalline entities, as illustrated in the optical micrographs as well as the presence of DLPC PdO segments at the F-A interface. When comparing the R values in Table 2 to those determined from the same bands of films adjusted to pH ) 5.0 but not treated with PLA2 in Figure 9, traces B/B′, their magnitudes are opposite. The R values of the 1310 and 1300 cm-1 bands are 2.5 and 3.0, respectively, thus indicating the crystalline components are oriented parallel to the F-A interface (Figure 9) whereas, upon exposure to PLA2, they are oriented perpendicular with the R < 1.0. Similarly, the R values of crystalline segments at 1265 cm-1 and PdO entities at 1250 cm-1 increase from 0.8 to 4.2 and 6.0 respectively, thus revealing a preferential parallel orientation



Figure 13. Schematic diagram depicting preferentially oriented manifestations at the F-A and F-S interfaces of SDOSS/DLPC and DLPC stabilized p-MMA/nBA dispersions prepared in the presence of PLA2 at adjusted pH values.

stabilized films are summarized in Figure 13, which schematically illustrates the decrease in preferentially oriented SLIC formations detected at the F-A interface as a result of enzymatic degradation. Conclusions

Figure 14. Schematic diagram illustrating the mobility of SDOSS/ DLPC and DLPC surface stabilizing species to the F-A and F-S interfaces in response to changes in temperature, ionic strength, pH, and PLA2. Open and closed circles illustrate preferential location of DLPC and SDOSS/DLPC, respectfully.

at the F-A interface in the presence of PLA2. Furthermore, at pH ) 5.0 and 8.0 in dispersions not containing PLA2 (Figure 9, traces B/B′ and C/C′), separate bands with equal magnitudes were detected at 1069 and 1061 cm-1 (P-O-C entities), whereas PLA2 exposed samples in Figure 12, traces B/B′ and C/C′, this is not the case. Thus, the alternate P-O-C environments created by the presence of basic ions were also dependent on the molecular structure of DLPC. The F-S interface spectra (not shown) of the films discussed above reveal only minute spectral changes; however, bands at 1056 and 1046 cm-1 are exhibited with R values greater than 1.0 in dispersions adjusted to pH ) 5.0 and 8.0, and these band intensities reveal the presence of SO3-- - -HOOC8 and SO3-- - -H2O interactions8,55 between SDOSS and lauric acid species with preferential parallel orientation at the F-S interface formed as a result of PLA2 cleaving DLPC molecules. These data, along with that of SDOSS/DLPC

These studies show that stimuli-responsive behaviors of p-MMA/nBA colloidal films prepared in the presence of liposome forming phospholipids and ionic surfactants can be elicited as a result of altering thermal, ionic, pH, and enzymatic environments of colloidal particles. In an effort to summarize responses of SDOSS and DLPC to temperature, pH, ionic strength, and enzyme changes, Figure 14 was constructed which illustrates the direction of migration or lack of it for these stimuli. As seen, particles stabilized by a SDOSS/DLPC mixture upon coalescence may release surface stabilizing species to the F-S interface with elevated temperature and low ionic strength stimuli or to the F-A interface in response to solution alkalinity and enzymatic degradation of DLPC. Acknowledgment. This work was supported primarily by the MRSEC Program of the National Science Foundation under Award Number DMR 0213883. The authors are also thankful to the National Science Foundation Major Research Instrumentation Program under the Award 0315637 for partial financial support of these studies. References and Notes (1) Dreher, W. R.; Urban, M. W.; Zhao, C. L.; Porzio, R. S. Langmuir 2003, 19, 10254-10259. (2) Dreher, W. R.; Urban, M. W. Macromolecules 2003, 36, 1228. (3) Lestage, D. J.; Urban, M. W. Langmuir 2004, 20, 6443. (4) Zhao, Y.; Urban, M. W. Langmuir 2001, 17, 6961-6967. (5) Zhao, Y.; Urban, M. W. Langmuir 2000, 16, 9439. (6) Zhao, Y.; Urban, M. W. Macromolecules 2000, 33, 8426-8434. (7) Zhao, Y.; Urban, M. W. Macromolecules 2000, 33, 7573-7581.

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Phospholipid Stabilized Colloidal

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