Ionic Polymeric Amphiphiles with Cholesterol Mesogen - American

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Biomacromolecules 2008, 9, 1264–1272

Ionic Polymeric Amphiphiles with Cholesterol Mesogen: Adsorption and Organization Characteristics at the Air/Water Interface from Langmuir Film Balance Studies K. Chandrasekar, R. Vijay, and Geetha Baskar* Industrial Chemistry Laboratory, Central Leather Research Institute, Adyar, Chennai 600 020, India Received November 14, 2007; Revised Manuscript Received January 4, 2008

Ionic polymeric amphiphiles consisting of cholesterol mesogen were investigated for the interfacial adsorption characteristics at the air/water interface using a Langmuir film balance with an aim to understand the influence of ionic segment from 2-acrylamido-2-methyl-1-propane sulfonic acid (AMPS) on the packing behavior of cholesterol at the interface. From surface pressure (π)-area (A) isotherm characteristics, it is demonstrated that the homopolymer and the copolymer C consisting of 0.15 mol fraction CAB segments exhibit the most expanded structures contributing to surface area of about 84 Å2/molecule. It is shown that the copolymer B with 0.1 mol fraction CAB provides optimum hydrophilic liphophilic balance to form the most compact structures contributing to a surface area of 35.75 Å2/molecule. The high surface pressure, >40 mN/m, in contrast to that of PAMPS demonstrates significant adsorption of the copolymers at the interface. An interesting correlation among interfacial packing characteristics, thermal behavior, and solution structures is demonstrated. From molecular models developed for CAB, it is shown that the horizontal orientation of the linker group with respect to cholesterol chain in CAB underlies the expanded structures observed in PCAB and copolymer C.

Introduction Polymeric amphiphiles consisting of segments drawn from biologically related and biocompatible species find tremendous potential in design of modern diagnostic tools like drug delivery systems and biomaterials.1,2 The structural architecture of polymeric amphiphiles and the microenvironment provided in the presence of solvent both dictate formation of self-assembled structures that vary among monolayers, vesicles, and micelles. These structures influence interfacial energy characteristics and control important interfacial processes of wetting, spreading, and adhesion that are significant in biological processes3 such as cell adhesion, protein adsorption, and surface recognition. The micellar assemblies of polymeric amphiphiles have been demonstrated to perform as efficient drug carriers in view of the hydrophobic core that provides an ideal solubilization site for liphophilic drugs. Some, for example, are the diblock polymeric amphiphiles consisting of hydrophilic segments from PEO, PVA, and PVP that are established to form micellar assemblies on a nanometer scale capable of solubilizing drugs and perform as efficient drug carriers.4 The dependence of degradability characteristics on the surface energy of biodegradable polymeric amphiphiles has been studied by Gardella et al.5 Polymeric amphiphiles consisting of hydrophilic and hydrophobic components with blocky or random distribution are preferred over simple surfactants in view of several merits such as robustness of the organized structures, versatility to modulate the hydrophilic liphophilic balance (HLB) through variation in ratio of chain length of different components, and formation of structures with several interesting morphologies at different interfaces.6 Polymeric amphiphiles with optimum HLB tend to form a two-dimensional film at the air/water interface.7,8 The Langmuir film balance (LFB) method provides a simple technique to draw information on the characteristics of thin films * Corresponding author. Phone: +91-44-24911386. Fax: +91-44-24911589. E-mail: [email protected].

adsorbed at the air/water interface. The two-dimensional film of polymeric amphiphiles is considered a suitable model for biomembrane with the advantage of tuning molecular conformation through lateral compression of the film as shown in the recent report on cholesteryl derived cyclen monolayer.9 The influence of structural features on the film characteristics and scope for modulation of these characteristics through changes in chain architecture of the polymer,10 hydrophilic and hydrophobic chain length,11 introduction of spacer group,12 and additives are well demonstrated in the literature.13 Polymeric amphiphiles from homopolymers consisting of comblike structures form monolayer or two-dimensional film wherein the surface energy decreases with an increase in alkyl chain length that is attributed to crystallization of alkyl groups as shown in the case of perfluoroalkyl methacrylate polymers.14 Watersoluble and biocompatible polymers like poly(ethylene oxide) (PEO), which is unique in being amphiphilic, forms a monolayer at the interface, and the surface energy of the film increases with an increase in molecular weight of the polymer that is suggestive of change in conformation of adsorbed structures at the interface. Copolymers consisting of PEO as one of the segments in combination with a nonamphiphilic hydrophobic component like poly(styrene) (PS) is one of the classic systems that have been investigated in detail for the interfacial film characteristics.15 In PEO-derived amphiphiles consisting of amphiphilic copolymer segments, brushlike structures of PEO in subphase with close-packed structures of amphiphilic component at the interface has been demonstrated as in, for example, PEO lipopolymers,16 PEO-caprolactone,17 and PEO surfactants consisting of PVA and a hexanal side chain.18 Different models of pancake-, mushroom-, or brushlike structures have been proposed to explain the interfacial characteristics of PEO surfactants.19 Polymeric amphiphiles consisting of water-soluble ionic segments and an amphiphilic segments as in the case of MAA-silyl compound, form carpet- and brushlike structures at the interface with close-packed structures of long chain segments

10.1021/bm701252y CCC: $40.75  2008 American Chemical Society Published on Web 02/29/2008

Ionic Polymeric Amphiphiles with Cholesterol Mesogen

extending into the air.20 Typical control of copolymer composition on the interfacial film characteristics has been recently established in copolymers from poly(acrylic acid) and hexadecyl acylamide wherein, the one consisting of these components at a ratio of 0.1:0.9 mol fraction has been shown to provide higher stability.21 Polymeric amphiphiles consisting of ionic segments as the major component in combination with comblike segments are considered specialty amphiphiles. For this study, we have chosen to investigate the interfacial adsorption characteristics of thin films at the air/water interface of polymers consisting of 2-acrylamido-2-methyl-1-propane sulfonic acid (AMPS), ionic segment, and cholesterol mesogen. We have chosen the AMPS ionic segment in view of a strong acidic functional group capable of withstanding variations in microenvironment. In our series of studies on AMPS-derived copolymers, we observed that the copolymer composition and the nature of the side chain provide control over the solution structures and adsorption at the air/ solution interface. We have chosen cholesterol mesogen in our present study in view of biologically significant unique characteristics of the cholesterol ring. It is shown that cholesterol brings about organization of lipids that is significant in the behavior of the membrane.22 The effect of cholesterol in the ordering of fatty acids with chain length in the range C12-C16 into lamellar structures has been demonstrated.23 Recently, it is shown that cholesterol promotes lamellar structures in mixtures with a phospholipid in water.24 The novel vesicular latex provided by cholesterol attached to maleimide through a spacer group has been established by Menger et al.25 The importance of cholesterol-derived surfactants in view of biological origin and typical liquid crystalline (LC) and chiral behavior has been well brought out in the review of sterol surfactants by Folmer et al.26 Cholesterol mesogen containing amphiphiles are designed with an aim to enhance the biocompatibility and control the membrane-mediated processes especially significant in theraptic systems. Biopolymers like carboxyl methyl chitosan (CMC),27 oligo lactic acid,28 and acryloyl polymer from a phosphoryl choline4 are designed with introduction of cholesterol mesogen through different synthetic strategies. The micellar assemblies from these polymeric amphiphiles are demonstrated to perform as efficient drug carriers. Those polymers from CMC have been shown to form nanomicelles that can perform as an injectable drug carrier. Felder-Flesch et al. have shown that introduction of 10 cholesterol rings to C60 fullerene brings about significant modifications in the interfacial organization behavior controlling aggregation among fullerene rings that are required in enhanced delivery systems.29 It is understood that the spacer group between the mesogen and the main chain influences packing of cholesterol at the interface. In the case of PS-cholesterol polymer, wherein the cholesterol is attached to main chain through the tris-ethyleneoxy group, it is shown that close-packed organized structures of cholesterol are favored at high surface pressure in contrast to related other polymers with poly(maleic acid) backbone that form close packed structures even at low concentrations similar to cholesterol.30 We have designed a new cholesterol-containing monomer with the spacer group of amino butyric acid to promote its incorporation in the copolymer chain and manipulate the steric hindrance due to the cholesterol ring. Three sets of copolymers consisting of different mole fractions of the AMPS and cholesteryl acrylamido butyrate (CAB) have been synthesized and investigated for adsorption characteristics at the air/water interface.

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Table 1. Synthesis and Characterization of the Copolymers from AMPS and CAB

polymer

mole fraction of the comonomers in copolymer (AMPS:CAB)

segmental mol wt

viscosity ava mol wt (Mv × 105)

A B C

0.95:0.05 0.90:0.10 0.85:0.15

223 239 255

1.0 1.4 2.9

a

K ) 3.65 × 10-5 mL/g; R ) 0.77 of AMPS segment.

Scheme 1. Structural Representation of the Monomer CAB

In view of the influence of microstructures of organized assemblies in many biologically related interfacial phenomenon and material design, a fundamental insight into the interfacial organization characteristics of amphiphiles that would focus on structure–property correlation becomes highly important. We aim to address and understand how the composition of the copolymer influences the packing characteristics at the interface from the LFB method. We have carried out these investigations in comparison to the homopolymer from cholesterol in order to understand the effect of ionic AMPS segments on the organization characteristics. Molecular modeling studies have been performed to rationalize the experimental results. The thermal behavior of these polymeric amphiphiles has been investigated in order to get information on the crystalline behavior of these copolymers that would serve useful in rationalizing the results on the interfacial packing characteristics.

Experimental Section Materials. 2-Acrylamido-2-methyl-1-propanesulfonic acid (AMPS) (99%), cholesterol (99%), aminobutyric acid (99%), and acryloyl chloride from Aldrich, were used as received without further purification. Dimethylformamide and dichloromethane solvents were of HPLC grade from s.d. fine chemicals, India. Azo-bis(isobutyronitrile) (AIBN) was recrystallized twice from methanol and used. Synthesis and Characterization of Cholesteryl Acrylamido Butyrate (CAB) and AMPS-CAB Copolymers. The monomer CAB and copolymers were synthesized as reported in our previous work.31 Copolymers of AMPS-CAB were synthesized by a solution polymerization technique in dimethylformamide using AIBN initiator and employing different feed compositions of the monomers, wherein the composition of CAB was varied in range 0.05-0.15 mol fraction (Table 1). Structural representation of the copolymer is presented in Scheme 1. The composition of the copolymers has been estimated from 1H NMR spectroscopy using the characteristic integral values of the respective monomers. Molecular weight of the polymers was estimated from a single point viscosity method. This was calculated from intrinsic viscosity, fitting K and R values of AMPS derived polymers in the Mark–Houwink relation.32

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Thermal Characteristics. Differential Scanning Calorimetry (DSC). DSC analysis of the polymers was performed using a NETZSCHGeratebauv GmbH instrument with a scanning speed of 10 °C/min under nitrogen atmosphere using aluminum pans. The samples were characterized from -50 to 200 °C. Liquid N2 was used as a purge gas for measurements below 25 °C and dry N2 for those above 25 °C. The stability of the baseline was checked before each measurement. The heating-cooling cycle was repeated two or three times until reproducible results were obtained. The second heating cycle was used in most cases for measurement of the glass transition temperature (Tg). Adsorption Characteristics of the Copolymers at the Air/Water Interface Using the Langmuir-Blodget Technique. The surface pressure (π)-area (A) isotherm measurements were performed at the air/water interface with LFB on a Langmuir trough coated with Teflon (model no. 6015) supplied by Nima technology, U.K., on the subphase of double distilled water showing a surface tension of 71.5 ( 0.5 mN/m. In view of poor solubility of the copolymers in nonaqueous waterimmiscible solvents like dichloromethane, the mixed solvent of methanol and chloroform was used in the preparation of polymer solution. The stock solution of the polymer at a concentration of 1 mg/mL was employed. The polymer solution was slowly spread on the water subphase using a microsyringe. Different volumes of polymer solution, 25 µL for PCAB, 50–200 µL for copolymer A, 50–100 µL for copolymer B, and 25 µL for copolymer C were spread on water subphase. Sufficient time was allowed for the complete evaporation of the solvent after spreading the polymer solution on the subphase of water. The surface pressure of the monolayer was measured during compression of the sample at a barrier speed of 2.5 × 10-1 nm2/ (molecule/min) with the Wilhelmy balance (accuracy 0.01 mN/m). Each measurement was repeated at least three times to check the reproducibility of the isotherms. All measurements were performed at a temperature of 25 ( 0.1 °C. The molar contribution from the cholesterol mesogen component is taken in surface area calculations. The minimum area of closest packing (A0) and maximum surface pressure (πmax) were estimated from π-A isotherms. The packing characteristics of the monolayer were further evaluated from compressibility coefficient (k) that is calculated from the extrapolated area A0 and the slope of the π-A isotherm by applying eq 1.15

k)-

1 (δA ⁄ δπ) A0

(1)

Molecular Modeling. The molecular model for the monomer and the copolymers was built using Cerius2 package and minimized in different arrangements using the Discover package33 by employing the COMPASS (condensed phase optimized molecular potentials for atomistic simulation studies) force field.34,35

Results and Discussion Synthesis and Characterization of Copolymers. The new monomer CAB with cholesterol mesogen consisting of a novel spacer group from aminobutyric acid has been designed. This spacer group with medium polar characteristics has been shown to provide copolymers with CAB as high as 15 mol %. Table 1 presents the characteristics of copolymers. It is significant to observe that the number of cholesterol chains for 20 AMPS chains varies roughly as 1, 2, and 3 in copolymers A, B, and C, respectively. This signifies different HLB of the designed polymers. These copolymers exhibit maximum solubility to an extent of 5 mg/mL in water under acidic pH conditions. Thermal Characteristics of Copolymers from Differential Scanning Calorimetric (DSC) Analysis. The DSC traces for the homopolymer PCAB and the copolymers A, B, and C are given in Figure 1. PCAB shows transitions at three different temperatures. The exothermic transition observed at about 27 °C must be arising due to transition in crystalline phase

Figure 1. DSC curves of PCAB and AMPS/CAB copolymers: a, PCAB; b-d, copolymers A, B, and C.

structures of the cholesterol side chain of the CAB. The sharp endotherm at 75 °C corresponds to melting (Tm) of the cholesterol side chain. With introduction of the PAMPS segment, considerable changes in the crystalline phase transition and melting temperatures could be observed. We have attributed the transformations occurring below 75 °C in the copolymers due to the cholesterol segment in view of absence of transitions in PAMPS in this temperature range.36 In the copolymer C consisting of 15 mol % CAB, the transitions due to transformation in crystalline phase and melting are distinctly observed similar to the homopolymer PCAB. However, such transitions occur at slightly lower temperatures of about 5 and 50 °C. This is suggestive of higher mobility of the segmental chain or, in other words, flexibility of the cholesteryl side chain in the copolymer C in comparison to the homopolymer PCAB. On the other hand, the copolymers A and B show different thermal behavior. The transitions at lower temperature corresponding to crystalline phase structure transformation and melting of cholesterol chain are not very sharp. However, a broad and small endotherm over the temperature range of about 40–75 °C could be observed. These results suggest that rigidity of copolymers increase in the order C < B < A. The different thermal behavior of the copolymers is suggestive of different packing characteristics of cholesteryl side chain of the copolymers. Surface Pressure (π)-Area (A) Isotherm Characteristics of the Homopolymer and Copolymers at the Air/Water Interface. The two-dimensional organization behavior at the air/water interface has been investigated for the copolymers in comparison with the homopolymer PCAB using the wellestablished LFB method. The π-A isotherm of PCAB is presented in Figure 2a. From the shape of the π-A isotherm, it could be observed that PCAB forms an expanded monolayer at the interface especially at low π. The monolayer consists of two phases, liquid condensed (LC) at π e 10 mN/m and solid condensed (SC) at high π. This is demonstrated in compressibility coefficient (k) values estimated at different π that are discussed in the subsequent section. The area (Ao) of closepacked structures obtained from the extrapolation of the steep portion of upper part of the curve is estimated as 83.44 Å2/ molecule (Table 2). From the structure of PCAB (Scheme 1), it could be expected that the methylene spacer group along with linker amido group organize predominantly at the interface with the amido group acting as an anchor to the aqueous subphase and the cholesterol side chain projecting toward air. This could account for a higher Ao of PCAB that is measured as 83.44 Å2/molecule. The organization of close-packed structures of cholesterol chain of PCAB at the interface is expected to contribute to much lower Ao at about 47.4 Å2/molecule as measured by us on simple cholesterol and agreeing with the



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Figure 2. Surface pressure (π)-surface area (A) isotherms of polymers on an aqueous subphase: (a) PCAB; (b-d) copolymer A from 50, 100, and 200 µL; (e and f) copolymer B from 50 and 100 µL; (g) copolymer C from 25 µL of spreading solution. Temperature 25 °C. Table 2. Surface Pressure (π) and Surface Area (A) Characteristics of Polymers on an Aqueous Subphase at Different Volumes of Spreading Solution, 25 °C system

volume (µL)

Ao (Å2/molecule)

πmax (mN/m)

PCAB polymer A

25 50 100 200 50 100 25

83.44 43.68 25.15 17.34 35.75 33.67 79.91

49.98 44.67 44.99 51.82 50.88 52.87 52.06

polymer B polymer C

reported value within limits of structural variation and experimental error.37 The methylene spacer and the amido linker group and also the steric effect due to rigid cholesterol side chain all must account for the observed expanded conformation of the PCAB. The significant adsorption of PCAB at the interface is demonstrated from the high πmax at about 50 mN/m. Although PCAB forms expanded monolayer film at the interface, the film exhibits good stability. This could be seen from the good reproducibility of three compression–expansion cycles and almost negligible change in area at different π in one compression–expansion cycle (Table 3). For example in the SC phase,

at π ) 30 mN/m, the loss in area is about 8.5%. At both the low and high π regions, it could be seen that the loss in area in one cycle is in the range of 8.5–11.85%. This trend is suggestive of the viscoelastic nature of the film. In view of limited solubility of copolymers A, B, and C in water as against PCAB, first of all an optimum level of copolymer concentration that can provide significant adsorption at the interface is to be identified. This is performed by spreading at least two or three different volumes of copolymer solution on the water subphase. In these experiments, πmax > 35 mN/m is taken to be indicative of significant adsorption of copolymer film at the interface on the basis of adsorption characteristics of PCAB as described above. We have chosen those concentrations of the copolymer solution that yield π g 35 mN/m and also initial π (πi) at zero. At a very high concentration, it is known that πi will be much greater than zero and, therefore, here compression of film from the gaseous phase into other phases is ruled out. Accordingly, from several preliminary experiments, the volume of spreading solution was chosen. The π-A isotherms of the copolymer A formed from spreading different volumes of the solution are presented in panels b-d of Figure 2. The results from π-A isotherms on Ao and πmax at different volumes of the spreading solutions of polymers are

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Table 3. Surface Area (A) and Area Difference (%) at Different Surface Pressures and Concentrations of Polymers in One Cycle of Compression and Expansion, 25 °C

polymer

surface area (A), Å2/molecule surface volume pressure % area (µL) (π), mN/m compression expansion difference

PCAB

25

polymer A

50 100

polymer B

50 100

polymer C

25

10 30 40 10 30 40 10 30 40 10 30 40 10 30 40 10 30 40

87.55 61.17 55.44 45.84 33.35 30.29 24.29 19.97 18.69 34.76 26.64 23.83 32.16 25.02 22.47 75.72 61.85 56.55

77.23 56.01 50.28 42.52 31.31 28.50 22.77 18.69 17.42 32.52 25.23 22.43 30.12 23.24 20.69 72.05 56.96 50.03

11.79 8.44 9.31 7.23 6.11 5.89 6.29 6.39 6.82 6.44 5.26 5.87 6.34 7.13 7.93 4.85 7.91 11.54

presented in Table 2. It is significant to note that copolymer A forms a more compressed film at all concentrations in comparison to the homopolymer PCAB. Ao is measured as 43.68 Å2/molecule on spreading 50 µL of the copolymer A solution on the water subphase (Figure 2b; Table 2). We observed that PAMPS spread on a water surface does not contribute to increase in π and this suggests that the interface is predominantly covered with the polar groups from the side chain of AMPS. On the contrary, with the introduction of 5 mol % CAB in the PAMPS as in copolymer A, formation of film exhibiting πmax > 35 mN/m could be observed. This demonstrates that in the copolymer A, CAB accounts for the predominant adsorption at the interface. A considerable lower Ao of the copolymer A in comparison to that of PCAB suggests that the conformation of the adsorbed group must be different. It is possible that the orientation of spacer, linker, and cholesterol ring of CAB underlies such change in the conformation of adsorbing group of the polymer at the interface. This is supported from molecular modeling studies in the subsequent section. On spreading 100 µL of copolymer A, Ao is reduced to 25.15 Å2/molecule, i.e., by about 44% with retention of πmax at about 45 mN/m. This reduction is suggestive of bilayer formation at the interface especially at high π. With further increase in volume to 200 µL, Ao is still reduced to 17.34 Å2/molecule, which is about 60% reduction in comparison to the first one or 32% of the second one. These results suggest that the copolymer A has a tendency to form bilayer at higher concentrations, with more compact structures. It could be argued that the reduction in area is indicative of dissolution of the material into the subphase. But, this is expected to result in considerable decrease in πmax. However a significant πmax at >45 mN/m at all concentrations with concomitant reduction in Ao supports bilayer formation. In copolymer B, Ao from the isotherm (50 µL) is estimated as 35.75 Å2/molecule. Interestingly, Ao remains almost constant and measured as 33.67 Å2/molecule with increase in volume of spreading solution from 50 to 100 µL. This is suggestive of tendency of the copolymer to form monolayer under these conditions. The Ao value of copolymer B is indicative of compact packing arising from considerable change in the conformation of adsorbing group, i.e., the spacer and the linker group of CAB. Both isotherms show high π of >45 mN/m.

The shape of the isotherm is indicative of compressed film similar to copolymer A. On the contrary in copolymer C, consisting of CAB at 15 mol %, a different behavior is observed. A stable and slightly more expanded monolayer is formed from spreading much lower volume at 25 µL on the water subphase. Ao from the π-A isotherm is estimated as 79.91 Å2/molecule, which is close to that obtained with PCAB (83.44 Å2/molecule). This is suggestive of similarity in conformation of the CAB component that is adsorbed at the interface with respect to PCAB and copolymer C. Copolymer C forms a more expanded film in comparison to the other two copolymers; the πmax was high at >45 mN/m. With increase of the volume of the spreading solution, initial π was observed to be greater than zero with a tendency to form more expanded film. From these results it could be concluded that the composition of copolymer B provides optimum HLB to form monolayer film at the interface with the most compact structures. Stability Characteristics of Adsorbed Films of Copolymers. The stability characteristics of the copolymer film at the air/water interface have been investigated measuring surface area at different surface pressures after repeated compression–expansion cycles. The change in area in one compression–expansion cycle is calculated and expressed as percent change in Table 3. Though, by virtue of high molecular weight, these polymers are expected to be stable, in view of water solubility characteristics considerable changes in interfacial adsorption characteristics on repeated compression–expansion cycles could be anticipated due to dissolution of the material into the subphase. Such a desorption process would result in a progressive decrease in surface concentration or in other words increase in surface area at the corresponding surface pressures. It is to be mentioned that the hysteresis curves are highly reproducible in three cycles. The hysteresis curves at selected concentration of the copolymers are presented in Figure 3. The data on change in area at different π in one compression–expansion cycle of the copolymer films are presented in Table 3. From the experimental results, it could be observed that the average percent loss over the wide π region is 5.96, 6.00, and 8.30 for the copolymers A, B, and C, respectively. Such small changes demonstrate the viscoelastic characteristics of the copolymer film and also the excellent stability. It is significant to observe that at high surface pressure of 40 mN/m, both PCAB and copolymer C show higher percent loss of 9 and 11 in surface area, respectively, than copolymers A and B. The expanded structures of cholesteryl chain favor reorganization especially in the SC phase, i.e., at high π when allowed to relax through repeated compression– expansion cycles. Packing Characteristics in Copolymer Films from Compressibility Coefficient (k) Estimations. The packing characteristic of the homopolymer (PCAB) and the copolymer film at the interface that is suggestive of phase structure of the film generally described in monolayer is investigated from the value of the compressibility coefficient (k) calculated from the respective π-A isotherms at different π. The results are presented in Table 4. For PCAB, k is estimated as 2.9 × 10-2 m/mN at π ) 5 and 10 mN/m which decreases significantly to 0.84 × 10-2 m/mN with further increase in π above 10 mN/m. This is suggestive of phase transition in the monolayer wherein the structures can be considered to transform from the liquidcondensed (LC) to solid-condensed (SC) phase. For copolymer A, k was estimated to be in range of 1.99 × 10-2 m/mN at π of 5 and 10 mN/m. At high π, for example, at 30 mN/m, k decreases significantly to about 0.80 × 10-2 m/mN. In the first place, the k value over the wide range of π suggests that the



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Figure 3. Hysteresis π-A isotherm on an aqueous subphase of (a) PCAB, (b, c) copolymer A from 50 and 100 µL, (d, e) copolymer B from 50 and 100 µL, and (f) copolymer C from 25 µL of spreading solution. Temperature 25 °C. Table 4. Compressibility Coefficients (k, m/mN) of Polymers on an Aqueous Subphase, at Different Pressures (π), Temperature 25 °C k × 10-2 (m/mN) π (mN/m)

PCAB

polymer A

polymer B

polymer C

5 10 20 30 40

2.90 2.90 0.84 0.84 0.84

1.99 1.99 0.80 0.80 0.80

1.66 1.66 0.83 0.83 0.83

2.00 2.00 0.82 0.82 0.82

copolymer A forms more compressed film in comparison to PCAB in both the LC and SC phases. The plot of k vs π for PCAB and the copolymers are presented in Figure 4. It is observed that a sudden drop in k from 1.99 × 10-2 to 0.80 × 10-2 m/mN occurs with an increase in π from 10 to 20 mN/m in copolymer A after which k remains almost constant even at a high π of 40 mN/m. From this, it is inferred that copolymer

A forms an LC phase structure at π e 10 mN/m, which gets transformed at high π into SC structures. The π corresponding to transformation of phase structure here is measured at 10 mN/ m, similar to PCAB. A similar trend of significant drop in k occurs at π >10 mN/m for the other copolymers B and C. However from the comparative evaluation, it could be seen that the copolymer B exhibits more compressed packing especially in the LC phase, i.e., at π e 10 mN/m, as observed from the least k at 1.66 × 10-2 m/mN. The more expanded packing in copolymer C almost similar to PCAB in LC phase could be inferred from comparatively higher k at 2.00 × 10-2 m/mN at π < 10 mN /m. It is significant to note that at high π the packing characteristics of PCAB and all copolymers are almost the same. Discussion on the Adsorption Characteristics of Copolymers. Surface concentration (Γ) in mg/m2 is calculated from the respective π-A isotherms at different surface pressures with an aim to understand the packing characteristics. The results

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Figure 4. Compressibility coefficient (k) vs surface pressure plots of (a) PCAB and (b-d) copolymers A, B, and C at 25 °C. Table 5. Surface Concentration (Γ) in Terms of mg/m2 of Polymers on an Aqueous Subphase, at Different Pressures (π) with Temperature at 25 °C polymer

volume (µL)

PCAB

25

polymer A

50 100 200

polymer B

50 100

polymer C

25

surface pressure (π), mN /m

surface concn (Γ), mg/m2

10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30

1.002 1.249 1.425 1.930 2.319 2.620 3.540 4.154 4.384 5.152 5.913 6.283 2.531 2.926 3.333 2.642 3.026 3.611 1.168 1.295 1.423

are presented in Table 5. It is observed that in all copolymers and the homopolymer, surface concentration increases with an increase in surface pressure from 10 to 30 mN/m. In the case of copolymers A and B, the increase in surface concentration at a fixed surface pressure with an increase in volume of the spreading solution showed interesting results. For example, at π of 30 mN/m, surface concentration increases by about 73 and 140% on increasing the volume of spreading solution from 50 to 100 and 200 µL. On the contrary, in copolymer B the surface concentration increases to a small extent of about 7% with an increase in volume of spreading solution from 50 to 100 µL. The increase in surface concentration can be considered to be indicative of enhanced surface adsorption, but this is expected to result in high surface pressure. The increase can also arise from change in conformation of the adsorbed structures at the interface, and this is expected to bring about changes in compressibility coefficient. The fact that such an enormous increase in Γ brings about almost a nil change in surface pressure and k support the formation of bilayer assemblies at the interface. The small change in Γ in copolymer B substantiates the formation of close-packed monolayer structures. It is interesting to note that PCAB and copolymer C show lower Γ of 1.423 and 1.425 mg/m2 at 30 mN/m. Such a trend is suggestive of a more expanded conformation of these polymers. It is interesting

to observe that there is a correlation between solution structures and interfacial adsorption characteristics from LFB experiments. From fluorescence quenching experiments in aqueous solutions, it was established that copolymer A forms intermolecular aggregated structures with an increase in concentration in contrast to copolymer B that forms a unimolecular structure.31 Copolymer C tends to form intramolecular aggregated structures. By virtue of close coil structures, copolymer A promotes intermolecular aggregated structures in solution and bilayer or multilayer kind of assemblies at the air/water interface. On the contrary, the typical coil structures in copolymer B favor unimolecular aggregated structure in solution and monolayer film at the air/water interface. The presence of a higher CAB component estimated as 1 for every 6 AMPS chain as in copolymer C favors intramolecular aggregated structures in solution and more expanded film structures at the air/water interface for steric reasons. Thus, it is understood that the conformation of the copolymer chain as influenced by the HLB or the copolymer composition underlies the interfacial adsorption characteristics that has a direct correlation on its aggregation behavior in solution. The close coil structures in copolymer A account for rigidity, and this favors formation of bilayers at higher concentrations. It is significant to mention here that the thermal behavior of copolymer A is also indicative of rigid structures. The expanded conformation of copolymer C similar to PCAB provides more flexibility, and this corroborates well with that from thermal analysis that shows transitions at lower temperature. Overall, copolymer B exhibits optimum HLB to form the most close-packed structures of monolayer film at the interface. It could be visualized that in these sets of copolymers, AMPS segments form brushlike structures wherein the hydrophilic sulfonic acid groups are oriented toward the subphase and the CAB segment organizes at the interface with different conformations as dictated by the coil structures of polymeric chain. Molecular Modeling Studies. In order to understand different conformations of the copolymers at the interface, molecular models have been developed using the Cerius2 package with minimization of energy. Here, the models for the three copolymers have been developed wherein the distribution of CAB chain for about 20 AMPS chains is varied as 1, 2, and 3, more or less matching with the copolymer composition. The model for PAMPS with 20 AMPS chain is also developed for comparison. The models are shown in Figure 5. It could be seen that AMPS homopolymer exhibits coiled structure. The introduction of CAB brings about significant changes in the conformation of the chain. The most significant observation is that copolymer B provides more stretched chain conformation. Also, the orientation of spacer, linker, and the cholesterol ring of CAB chain changes significantly in these copolymers, and this demonstrates that the ratio of hydrophilic to hydrophobic component plays a significant role in influencing the conformation and the packing characteristics at the interface. With an aim to understand the area contribution from different stable conformations of CAB, molecular models were developed individually for CAB. The two different models that basically differ in the orientation of spacer and linker group with respect to cholesterol side chain are presented (Figure 6). It is observed that the orientation of spacer group with respect to cholesterol ring could bring about modifications in area from about 84 to 45 Å2/molecule. Thus a horizontal orientation of the spacer group with respect to the cholesterol ring contributes to a spacer group area of 84 Å2/molecule and a tilted vertical



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Figure 5. Molecular modeling images of (a) PAMPS and (b-d) copolymers A, B, and C.

Figure 6. Molecular modeling images of CAB segments at the interface with contact area shaded: (a) 46.47 Å2/molecule and (b) 84 Å2/molecule.

orientation of the spacer group shows an area of about 46 Å2/molecule. From these models, one can understand that in the homopolymer PCAB and the copolymer C, the horizontal orientation of the spacer group with respect to cholesterol ring at the interface accounts for the measured surface area of about 84 Å2/molecule. With an increase in AMPS comonomer segment, the typical conformation of polymer chain favors tendency for vertical orientation of spacer group, wherein the contact surface area could vary between 35 and 43 Å2/molecule. The projection of two molecular models wherein the contact surface area is marked by shaded portion is shown in Figure 6. From these models it could be visualized that the orientation of spacer and linker group with respect to cholesterol side chain mainly underlies the packing characteristics.

Conclusion Three sets of ionic copolymers consisting of different mole ratios of AMPS and cholesterol mesogen have been investigated for adsorption characteristics at the air/water interface using the Langmuir film balance method. The presence of CAB even at a level of 0.05 mol fraction as in copolymer A is able to bring about significant adsorption of the copolymer at the interface in contrast to homopolymer PAMPS. The interfacial adsorption characteristics are dependent on the copolymer composition. It is observed that the copolymers and homopolymer, PCAB form compressed stable film at the interface consisting of two different phases of liquid condensed phases at π < 10 mN/m and solid condensed (SC) phase at higher π as demonstrated from compressibility coefficient (k). The tendency to form a bilayer

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at the interface as a function of volume of spreading solution is demonstrated in copolymer A from progressive decrease in surface area and increase in surface concentration with negligible changes in π. This is explained on the basis of rigid and close coil structures of copolymer A that are supported from thermal and solution structures. Copolymer B is identified to provide optimum HLB to form a monolayer with the most compact structures with surface area at 35.75 Å2/molecule that varies to a very small extent with volume of spreading solution. It is shown that due to steric reasons PCAB and the copolymer C promote expanded structures at the interface. The transitions at temperatures

Ionic Polymeric Amphiphiles with Cholesterol Mesogen - American

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