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Bile-Salt-Induced Aggregation of Poly(N-isopropylacrylamide) and Lowering of the Lower Critical Solution Temperature in Aqueous Solutions Anitha C. Kumar,†,‡ Harikrishna Erothu,‡ Himadri B. Bohidar,*,§ and Ashok K. Mishra*,† Department of Chemistry, Indian Institute of Technology, Madras 600036, India, Laboratorie de Chemie des Polymeres Organiques, ENSBP, 16 AVenue pey Berland, 33607 Pessac cedex, France, and Polymer and Biophysics Laboratory, School of Physical Sciences, Jawaharlal Nehru UniVersity, New Delhi 110067, India ReceiVed: September 22, 2010; ReVised Manuscript ReceiVed: NoVember 30, 2010
The effect of sodium cholate (NaC; concentration 1-16 mM), a biological surfactant, on the aggregation behavior of 1% (w/v, 2.2 × 10-3 M) poly(N-isopropylacrylamide) (PNIPAM) aqueous solutions was studied as a function of temperature. From turbidity, dynamic light scattering, viscosity, and fluorescence measurements, it was observed that (i) there is NaC-induced nanoscale aggregation of PNIPAM in its sol state and (ii) the lower critical solution temperature corresponding to sol-gel transition shifts to a lower temperature by about 2 °C. 1. Introduction Polymeric hydrogels have a wide variety of applications in day-to-day life.1-3 The polymeric hydrogel’s particle size and size distribution have a strong influence on its application.1,2 For example, as a drug delivery carrier, the desired diameter of responsive nanogels is in the range of 20-100 nm.3,4 An interesting subclass of polymeric nanoparticles is thermoresponsive nanogels. Poly(N-isopropylacrylamide) (PNIPAM) is the most widely studied thermoreversible gel and exhibits a lower critical solution temperature (LCST) of 32 °C (values between 31 and 35 °C have been reported) in water.5,6 PNIPAM in water undergoes a coil-to-globule transition upon being heated above its LCST, and such responsiveness to temperature is concentration-dependent. The formation of dense and narrowly size distributed mesoscopic globules (mesoglobules) based on PNIPAM, as a result of the self-assembly of several single chains, is generally stable only under very dilute conditions.7 Dilute solutions of PNIPAM at higher temperature give nanogels.8 In recent literature, Balu et al.9 showed that it was possible to obtain dense stable colloids of nearly pure PNIPAM in a concentration range of 1-6 wt %. PNIPAM nanogels are useful in a wide range of applications because of their stimulusresponsive sol-gel transition behavior, such as drug delivery devices,10 templates for nanoparticles,11 biosensing,12 catalysis,13 etc. They have received increasing attention in the technological and scientific fields. Pelton and Chibante14 first synthesized the PNIPAM microgel particles in 1986. Nearly monodispersed PNIPAM particles are now routinely prepared in a range of colloidal sizes from 50 nm to 1 µm.15,16 Xianglia et al.8 prepared PNIPAM nanoparticles with diameters ranging from 50 to 300 nm by photopolymerization. The LCST is the result of a delicate balance between hydrophobic and hydrophilic interactions. At higher temperatures, i.e., temperatures near the LCST, the entropy contribution to the free energy of mixing will overcome the negative enthalpy * To whom correspondence should be addressed. (A.K.M.) Phone: +91 (44) 2257 4207. Fax: +91 (44) 2257 4202. E-mail:
[email protected]. (H.B.B.) Phone: +91 (11) 2670 4636. E-mail:
[email protected]. † Indian Institute of Technology. ‡ ENSBP. § Jawaharlal Nehru University.
Figure 1. Structure of sodium cholate.
of solution. Thus, the free energy of mixing takes a positive value, and the phase separation of the polymer solution begins.17 At the LCST temperature there is a coil-to-globule transition, which has been extensively studied experimentally and modeled theoretically.6,18 In this study, we have specifically considered aqueous dispersions of PNIPAM that exhibit a reversible and continuous volume transition in water around 34 °C. Additives such as some salts are known to decrease the LCST of PNIPAM solutions, whereas surfactants tend to increase the LCST.19,20 Only minor studies have been carried out on the interactions of biological surfactants, such as bile salts with PNIPAM. The LCST of PNIPAM in water was modified by small amounts of cholic acid bearing comonomers, since the bile acid residues tend to induce aggregation of the polymers.21 In a recent study, we have examined the effect of sodium cholate (NaC) on the nonflowing macrogels at 12% (w/v) concentration through various techniques such as differential scanning calorimetry (DSC), dynamic light scattering (DLS), turbidity, rheology, and fluorescence using 8-anilino-1-naphthalenesulfonate (ANS) as an external fluorescent probe. We observed that the LCST of PNIPAM slightly decreases in the presence of NaC, accompanied by a broadening of the sol-gel phase transition profile.22 Bile salts are expected to have better biocompatibility when used along with materials such as PNIPAM for biomedical applications. Bile salts (Figure 1) are biological surfactant molecules possessing “facial polarity”. The critical micellar concentration (CMC) of sodium cholate is reported to be in the range of 10-15 mM.23
10.1021/jp109056u 2011 American Chemical Society Published on Web 12/23/2010
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It was reported by Benrebouh et al. that, in the presence of NaC residue, the copolymers of PNIPAM with 1-5% methacrylate derivatives tend to aggregate at very low concentration (1.5 × 10-3 wt %).21 In the present work we examine the interaction of PNIPAM with NaC under dilute PNIPAM concentration so as to form aggregates (nano- and microscale) above the phase transition temperature. Sodium cholate is used as a representative bile salt to study its effect on the aggregation behavior of PNIPAM solutions. Fluorescence studies with an extrinsic fluorescent probe (ANS) and dynamic light scattering, turbidity, and viscosity studies were carried out to understand the PNIPAM-bile salt interactions. The rationale for using ANS as a fluorescent probe has been explained in section 3.4. DSC experiments could not be carried out because of the very low enthalpy change involved. 2. Experimental Section 2.1. Sample Preparation. PNIPAM (nominal MW ≈ 6243) and ANS were purchased from Aldrich and used as such. NaC was purchased from SRL. Triply distilled water having a conductivity 5.53 × 10-6 µS was used for sample preparation. Stock solutions of NaC (20 × 10-3 M) and PNIPAM (2.77 × 10-3 M) were prepared in triply distilled water. The final concentration of PNIPAM in the mixture was 2.2 × 10-3 M (1%, w/v), and the NaC concentration was varied from 0 to 20 mM (0, 1, 5, 10, 16, and 20 mM). A stock solution of 10-3 M ANS was prepared in methanol, and 2 × 10-5 M was used for the fluorescence measurements. The PNIPAM-NaC mixtures were allowed to equilibrate for at least 6 h prior to the experiments. 2.2. Turbidity Measurements. Turbidity measurements were done with a Brinkmann PC 910 colorimeter (Brinkmann Instruments), operating at of 450 nm, details of which are given elsewhere.24 The phase separation temperature is defined in the paper as the temperature where the turbidity observed was the maximum. 2.3. Dynamic Light Scattering. DLS measurements were carried out with a PhotoCor light scattering instrument (PhotoCor Instruments) having a 35 mW He-Ne laser operating at 632.8 nm as the light source. Experiments were carried out with the scattering angle fixed at 90°. This instrument provides temperature regulation in the range of 20-90 °C with an accuracy of (0.1 °C. The whole scattering apparatus was installed on a vibration isolation table. PNIPAM-NaC samples (2 mL) were taken in a 5 mL rectangular quartz scattering cell. The sample was equilibrated for 15 min at the measurement temperature. In the DLS measurements, the intensity correlation function has the analytic form:25-27
g(2)(τ) ) 1 + β|g(1)(τ)| 2 ) 1 + βe-2Γτ
(1)
where g(2)(τ) is the normalized second-order intensity correlation function, β is the modulation parameter of the system, g(1)(τ) is the normalized first-order electric field correlation function, τ is the delay time, and Γ is the average characteristic line width. g(1)(τ) can be expressed as
g (τ) ) (1)
∫ G(Γ) e
-Γτ
dΓ
(2)
where G(Γ) is the relaxation time distribution function of Γ purely originating from Brownian motion. The autocorrelation
function was analyzed using the CONTIN method,27 which yields the mean relaxation frequency, Γ, and the variance (polydispersity). For a system that scatters light from moieties under Brownian motion,27 the relaxation frequency is directly proportional to the square of the scattering vector, q. Thus, the z-averaged translational diffusion coefficient, D, was obtained using Γ ) Dq2. The hydrodynamic radius, Rh, was calculated using the Stokes-Einstein equation Rh ) kBT/6πηD, where η, kB, and T are the solvent viscosity, Boltzmann constant, and absolute temperature, respectively. In a typical DLS experiment, the data quality of the correlation function was strictly monitored by insisting that the baseline difference was less than 0.1% and the signal modulation was >85%. Any spectrum that did not meet this stringent requirement was automatically rejected. This ensured that the correlation functions were of very good quality with a smooth baseline. Applying CONTIN analysis to these data yielded reproducible relaxation time distributions independent of the time window provided to CONTIN as input. For convenience, the same is plotted as the particle size distribution. 2.4. Viscosity Measurements. The viscosity of the PNIPAMNaC solutions was measured by using an SV-10 sine wave vibroviscometer (A&D Co. Ltd.) by detecting the driving electric current necessary to resonate the two sensor plates at a constant frequency of 30 Hz and amplitude of less than 1 mm. The solutions were heated to 40 °C, and the viscosity of the sample was measured every 10 s during the period of natural cooling. 2.5. Steady-State Fluorescence Studies. Fluorescence measurements were carried out with a Hitachi F-4500 spectrofluorimeter, with a 150 W xenon lamp as the light source. Excitation and emission spectra were both recorded with a slit width of 5 nm and at the PMT voltage of 700 V. The scan speed was kept at 1200 nm min-1. Temperature-dependent experiments were carried out by circulating water through a jacketed cuvette holder from a thermostat bath (INSREF Ultra Cryostat). The fluorescence anisotropy (rss) values were obtained at the emission maxima using the expression
rss ) (I| - GI⊥)/(I| + 2GI⊥)
(3)
where I| and I⊥ refer to the fluorescence intensities when the emission polarizer is parallel and perpendicular, respectively, to the direction of polarization of the excitation beam and G is the factor that corrects for unequal transmission by the diffraction gratings of vertically and horizontally polarized light. 2.6. Time-Resolved Fluorescence Measurements. Fluorescence lifetime measurements were carried out using an IBH single-photon-counting fluorimeter in a time-correlated singlephoton-counting arrangement, consisting of a picosecond/ femtosecond Ti-sapphire laser system (Tsunami Spectra Physics, Bangalore, India). The pulse repetition rate was 82 MHz, and the full width at half-maximum was less than 2 ps. The emission was collected at magic-angle polarization (54.7°) to avoid any polarization in the emission decay. The instrument response time was approximately 50 ps. The decay data were further analyzed using IBH software. A value of χ2 in the range of 0.99 e χ2 e 1.4 was considered a good fit. 3. Results and Discussion 3.1. Turbidity of the Solutions. Figure 2 shows the turbidity results of PNIPAM-NaC samples as a function of temperature. The turbidimetric titration method as applied to a phaseseparating system is based on the fact that the turbidity is proportional to both the molecular weight and the number
Bile-Salt-Induced Aggregation of PNIPAM
Figure 2. Turbidity data of PNIPAM (1%, w/v) with different concentrations of NaC (0, 1, 5, 10, and 16 mM) as a function of temperature. Notice the sharp increase in turbidity at T ) 34 °C for the PNIPAM solution, which decreases to 32 °C in the presence of NaC, which is indicated by arrows. Solid lines are guides to the eye.
density of particles present in the dispersion. Turbidity appears in the samples when the aggregates assume the size comparable to that of the wavelength of visible light. It is routinely used in determining the cloud point because it gives a clear first-hand indication of phase separation. However, as shown in Figure 2, this methodology has limited resolution, which requires that the information obtained is supported by independent measurements. The data in Figure 2 unambiguously imply the existence of phase separation close to 32 °C, though they could not be used to distinguish the same from sample to sample. Thus, we resorted to other measurements, such as light scattering, to deduce the phase separation temperatures pertaining to each sample. Moreover, the turbidity profile indicates a steplike change close to 32 °C. The data reported in Figure 2 were a result of several independent measurements, and the turbidity profiles were highly reproducible. The temperature at which the maximum turbidity is observed is considered as the phase separation temperature. In our previous work,22 we reported that the PNIPAM samples become turbid at the onset of the phase transition temperature. In a macrogel with an increased concentration of NaC, the onset of the PNIPAM phase transition temperature decreased.22 In the current study for all samples of PNIPAM (the NaC concentration was varied from 0 to 16 mM), the turbidity begins to abruptly increase at 32 °C. In contrast, for PNIPAM solutions the same was observed at 34 °C. Thus, the presence of NaC in solution reduced the phase transition temperature by 2 °C. This is indicative of the fact that large soluble aggregates are formed of PNIPAM molecules induced by NaC. However, it is clearly established that PNIPAM molecules undergo intermolecular aggregation, mediated by bile salt. The quantification of the aggregate size for temperatures lower and higher than the phase transition temperature necessitated the use of a particle sizing study. This was achieved through light scattering measurements. 3.2. Dynamic Light Scattering Studies. DLS measurements are useful to study the structure of macromolecules and molecular assemblies. It was felt imperative to characterize the PNIPAM samples first. The particle size (Stokes radius) of PNIPAM molecules as a function of temperature is shown in Figure 3, and the inset depicts the particle size distribution at room temperature, which indicates the existence of polydispersity in this sample (limited to 20%). The nominal molecular
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Figure 3. Variation of the PNIPAM size as a function of temperature. The particle size distribution is shown in the inset pertaining to measurements taken at room temperature (20 °C). The arrow points to the phase transition temperature (LCST), which is 34 °C.
Figure 4. Particle size distribution of the aggregates in a PNIPAM-16 mM NaC solution recorded at room temperature. Notice the bimodal nature of the distribution.
weight of PNIPAM is ∼6243, which translates to a mean radius of ∼1.3 nm (assuming the partial specific volume is ∼0.75 cm3/ g). Thus, the data shown in Figure 3 imply that even in the absence of the bile salt PNIPAM is capable of exhibiting heatinduced aggregation. The formation of dense and narrowly size distributed mesoscopic globules based on PNIPAM molecules, as a result of the self-assembly of several single chains, is generally stable only under dilute conditions as has been discussed earlier.7 At a characteristic temperature of T ) 34 °C, the aggregates abruptly become large, implying approach to the LCST. In the presence of NaC, the PNIPAM chains start to aggregate substantially in the solutions. The particle size distribution for a typical sample is shown in Figure 4 (16 mM NaC solution) and clearly implies the existence of two sets of aggregates: one in the nanorange and another in the range of 100-300 nm. Both of these fractions revealed an increase in size with temperature and NaC concentration. However, both indicated the presence of a characteristic temperature (32 °C) where aggregation was abruptly high (Figures 5 and 6). Invariably, for all the samples, at 32 °C the size of the aggregates increased from their room temperature values typically by 2-fold or more. The variation of the Rh of PNIPAM aggregates as function of temperature and NaC concentration is shown in Figure 5. With an increase in the concentration of sodium cholate, there was an increase
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Figure 5. Variation of the hydrodynamic radii of the aggregates as a function of temperature for PNIPAM (1%, w/v) with different concentrations of NaC. The data pertain to a smaller size fraction of aggregates. The arrow points to the LCST, which is 32 °C. Solid lines are guides to the eye.
Figure 6. Variation of the hydrodynamic radii of the aggregates as a function of temperature for PNIPAM (1%, w/v) with different concentrations of NaC. The data pertain to a larger size fraction of aggregates. The LCST is shown by the arrow. Solid lines are guides to the eye.
in the size of the aggregates. The Rh of these aggregates increased with temperature too, which is indicative of a bilesalt-induced aggregation phenomenon. Due to the significant gain in turbidity of the PNIPAM solutions, DLS measurements could not be performed above 34 °C. Below the LCST, in the absence of sodium cholate, the Rh of individual PNIPAM chains increased albeit by a small amount due to thermal aggregation (Figure 3). Above the transition temperature, a further increase in temperature had no effect on the Rh. It was observed that the presence of NaC resulted in generation of two differently sized aggregates, and both fractions exhibited a qualitatively similar aggregation trend. 3.3. Viscosity Measurements. The variation in the specific viscosity of PNIPAM-NaC samples as function of temperature is shown in Figure 7. The specific viscosity decreases with increasing temperature, but increases with increasing bile salt concentration for a given temperature. It is known that the specific viscosity is related to the particle size and geometrical shape; i.e., the specific viscosity increases with particle size and particle asymmetry. During the aggregation process, the larger aggregates coalesce to form microdroplets that undergo Ostwald ripening and eventually sediment out of the solution. Thus, the data presented in Figure 7 mostly owe their origin to the smaller
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Figure 7. Variation of the specific viscosity of PNIPAM-NaC solutions as a function of temperature. [PNIPAM] ) 1% (w/v), and [NaC] ) 1-16 mM. The arrow points to the LCST. Solid lines are guides to the eye.
aggregates. However, these aggregates grow in size with an increase in temperature, and beyond 32 °C, these aggregates become sufficiently large to remain in a stable suspension. Loss of these big aggregates to sedimentation may manifest itself in the reduction of the viscosity of the solution beyond 32 °C. However, it must be noted that, though the viscosity studies were carried out on a poorly sedimenting solution, the data were devoid of noise and exhibited consistency that allowed identification of the phase separation temperature without difficulty. Figure 2 reveals that, beyond the transition temperature, there is a plateau region where the solution is turbid but poorly sedimenting. This allowed the viscosity to be measured over an extended range of temperature. From Figures 3 and 7 it is clear that the LCST point of pure PNIPAM solution is at 34 °C, and for the NaC-added samples the same shifts to 32 °C. 3.4. Fluorescence Studies. 3.4.1. Fluorescence Emission Characteristics. From our previous work,22 it was identified that ANS is one of the most suitable probes for the study of the interaction of PNIPAM-NaC interactions. This is because it has a very low fluorescence in water and it shows a significant increase in fluorescence intensity, lifetime, emission energy (blue shift), and steady-state fluorescence anisotropy on partitioning to the hydrophilic-hydrophobic interface. The increase of these fluorescence parameters is due to the combined effect of the decrease in the local polarity at the interface and restricted mobility of the probe molecule. ANS being anionic, it preferentially partitions to the PNIPAM domain and not to the anionic bile salt micellar domain. Thus, as a fluorescent molecular probe, it reports on the structural changes of PNIPAM gel. We have continued to use ANS as an extrinsic fluorescent probe in this work too. The emission spectra of NaC-added PNIPAM samples at 20 °C (sol state of PNIPAM) are shown in Figure 8. The PNIPAM concentration was 1% (w/v, 2.2 mM), and the NaC concentration was varied from 0 to 20 mM. The ANS concentration was maintained at a low level (2 × 10-5 M) so that there would be negligible perturbation due to the probe on the PNIPAM aggregation process, if any. In water, ANS gives a very weak fluorescence at 510 nm. The fluorescence intensity in the 1% PNIPAM solution at 20 °C (sol state) was slightly higher than that in water. With a progressive increase in the concentration of NaC in the medium, there was a progressive increase in the ANS fluorescence intensity and blue shift. At 20 mM NaC, the
Bile-Salt-Induced Aggregation of PNIPAM
Figure 8. Emission spectra of ANS in PNIPAM solutions having varying concentrations of NaC at 20 °C. [PNIPAM] ) 1% (w/v), [NaC] ) 0-20 mM, and [ANS] ) 2 × 10-5 M.
intensity increase was about 10-fold and the blue shift of λmax was 450 nm. The general features observed upon incorporation of ANS in an organized medium are a blue shift in λmax arising out of the decreased local polarity and an increase in the fluorescence lifetime as well as quantum yield arising out of restricted mobility of the fluorophore.28,29 A previous study indicates that, in NaC micelles, the ANS fluorescence spectral changes are not very significant, as both ANS and NaC are anionic.22 Figure 8 shows that there is an increase in ANS fluorescence intensity even after addition of 1 mM NaC to the PNIPAM solution, which is well below the micellation concentration of NaC, which is 10-15 mM.23 This indicates that NaC does play a significant role in inducing nanoscale aggregation of PNIPAM even in the sol state of PNIPAM and that micellation of NaC is not a prerequisite for this NaC-induced aggregation of PNIPAM. Thus, the fluorescence of ANS corroborates the results obtained from DLS, turbidimetry, and viscosity presented in the previous sections. The facial polarity of NaC molecules and their aggregates could be a reason for the sol-state aggregation of PNIPAM in the presence of NaC.22 The hydrophilic surface of the NaC aggregates brings the PNIPAM chains closer in the sol state. Although the magnitudes are different, these results are similar to the behavior of 12% PNIPAM-NaC systems.22 Experiments involving the temperature dependence of the fluorescence response of ANS at different [NaC] values were carried out in the temperature range of 20-40 °C, and the results are shown in Figure 9. In the temperature range of 20-32 °C PNIPAM exists in its sol state. Increasing the concentration of NaC causes the ANS fluorescence to increase in this temperature range as discussed in the previous paragraph. For all the samples there is a sudden increase in fluorescence intensity at the onset of the sol-to-gel transition at 34 °C. At higher temperatures, significant scattering of the turbid solution tends to cause a small drop in the fluorescence intensity. These results closely follow the trend of DLS and turbidity results. Figure 10 shows the emission frequency of ANS in different solvents and PNIPAM in the presence and absence of NaC as a function of the z-scale solvent polarity. A 1% (w/v) PNIPAM solution without NaC shows a polarity between those of water and ethylene glycol. The addition of 20 mM NaC to 1% (w/v) PNIPAM decreases the z-scale polarity and gives the medium a methanol-like polarity. The z-scale polarity of the 1% (w/v) PNIPAM-NaC sample is higher than that of 12% (w/v) PNIPAM. The 12% (w/v) PNIPAM sample shows ethylene glycol-like polarity, which is shifted close to ethanol-like polarity by the addition of 16 mM NaC.22
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Figure 9. Variation of the fluorescence intensity of ANS in PNIPAM solutions having varying concentrations of NaC as a function of temperature. [PNIPAM] ) 1% (w/v), [NaC] ) 0-20 mM, and [ANS] ) 2 × 10-5 M. Solid lines are guides to the eye.
Figure 10. Change in the emission frequency of ANS as a function of the z-scale solvent polarity of ANS in different solvents and the PNIPAM-NaC system.
TABLE 1: Fluorescence Lifetime Data of ANS in PNIPAM-NaC Samples at 15 °C (below the Phase Transition)a
τ1 (R1) τ2 (R2) τ3 (R3) χ2 τav
[NaC] ) 0 mM
[NaC] ) 1 mM
[NaC] ) 5 mM
[NaC] ) 10 mM
[NaC] ) 16 mM
0.24 (68) 1.11 (17) 4.11 (15) 1.183 0.97
0.23 (61) 0.96 (20) 4.70 (19) 1.170 1.22
0.25 (56) 1.44 (21) 5.98 (23)
0.27 (41) 1.50 (25) 5.63 (24) 1.227 2.04
0.27 (22) 1.75 (34) 5.50 (44) 1.183 3.07
2.36
a
The τ values are given in nanoseconds. The amplitude values are given in percent. [PNIPAM] ) 1% (w/v), [NaC] ) 0-16 mM, [ANS] ) 2 × 10-5 M, λex ) 360 nm, and λem ) 500 nm.
3.4.2. Fluorescence Lifetime Studies on the Sol State. Table 1 shows the fluorescence lifetime values of PNIPAM-NaC samples at 15 °C, at which PNIPAM exists in its sol state. A similar study for the gel state was not possible due to extensive scattering. The fluorescence decay profiles, obtained by excitation at 360 nm, shows a good fit for three exponential decay components for all the samples. An average lifetime was calculated by taking the weighted average lifetime (τav) of the three-component decay given by
τav )
τ1(R1) + τ2(R2) + τ3(R3) R1 + R2 + R3
(4)
where Ri are the amplitudes of each component in percent. The calculated τav of ANS increases with increasing concentrations
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Figure 11. Average fluorescence lifetime of ANS against the fluorescence intensity of ANS in PNIPAM-NaC samples at 15 °C (below the LCST). [PNIPAM] ) 1% (w/v), [NaC] ) 0-16 mM, [ANS] ) 2 × 10-5 M, λex ) 360 nm, and λem ) 490 nm.
of NaC, and it was shifted from 0.97 ns in pure PNIPAM (1%) solution to 3.07 ns in PNIPAM solution with 16 mM NaC. For comparison, the average lifetime of ANS in pure water is reported to be 0.25 ns.30 Figure 11 shows the plot of the average lifetime of ANS against the fluorescence intensity in PNIPAMNaC samples in the sol state (15 °C). The enhancement of the average fluorescence lifetime roughly corresponds to the increase in the fluorescence intensity. This suggests that the intensity enhancement is primarily due to the decrease of ∑knr, the sum of the rate constants of all nonradiative decay processes. Since such a behavior is expected from the presence of ANS in a hydrophobic microenvironment, the conclusion of NaC-induced aggregation of PNIPAM in the sol state is further substantiated. These observations related to 1% (w/v) PNIPAM can be contrasted with those of 12% (w/v) PNIPAM.22 For 12% PNIPAM, τav was 2.28 ns, and it increased to 7.02 ns in the presence of 16 mM NaC.22 3.4.3. Fluorescence Anisotropy Studies. Fluorescence anisotropy (rss) is a measure of the average angular displacement of the excited-state probe molecule in a microenvironment, which reveals the degree of rotational diffusion of the fluorescent probe during its excited-state lifetime. The rotational diffusive motion of the molecule depends on the viscosity of the medium. Thus rss is expected to be a useful parameter in obtaining information on the efficiency of the rotational motion of ANS. Generally, the value of rss varies between the maximum of 0.4 for a completely restricted fluorophore (limiting rss) and the minimum of 0.0 for a completely free molecule.31 The reported steady-state fluorescence anisotropy of ANS in water is 0.03, and in 95% glycerol at 0 °C, its limiting rss is 0.32 as per the literature.32 The variation of fluorescence anisotropy values of PNIPAMNaC samples is shown in Figure 12. The rotational diffusive motion of the molecule depends on the viscosity of the medium. The rss values in PNIPAM solutions in its sol state (20 °C) in the presence and absence of NaC are close to 0.1. At 32 °C there is a sudden increase in the rss of NaC-containing PNIPAM samples. This temperature is lower than the temperature of 34 °C observed for pure (1%) PNIPAM. It is already known that the maximum value in the rss vs T plot corresponds closely to the onset of the sol-gel transition of the samples.22 Thus, as was inferred from turbidity, DLS, and viscosity measurements, the onset of the sol-gel transition appears to occur at a lower LCST in the presence of NaC. As PNIPAM formed a turbid solution above the LCST, there was a sudden drop in the fluorescence anisotropy due to massive light scattering.31 It is to be noted that, although there appears to be a good cor-
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Figure 12. Change in the fluorescence anisotropy of ANS in PNIPAM-NaC samples as a function of temperature. [PNIPAM] ) 1% (w/v), [NaC] ) 0-16 mM, [ANS] ) 2 × 10-5 M, λex ) 360 nm, and λem 490 nm. The arrow points to the LCST. Solid lines are guides to the eye.
respondence of the increase of rss with the onset of the sol-gel transition, the actual value of the rss maximum does not correlate with [NaC]. As turbidity sets in, possibly the light-scatteringinduced rss loss does not permit such a correlation. Thus, all three fluorescence parameters, fluorescence spectral shift, intensity enhancement, and increase in fluorescence anisotropy, consistently report similar LCST changes from 34 °C in pure PNIPAM (1%, w/v) to a slightly lower temperature, i.e., 32 °C, in PNIPAM-NaC samples. These fluorescence parameters also consistently indicate NaC induced (i) an increase in aggregation and (ii) a decrease in the motional freedom of a substrate molecule such as ANS in the PNIPAM-NaC aggregates even in the sol state. 4. Conclusions The incorporation of natural compounds such as sodium cholate is expected to improve the biocompatibility of polymers. The effect of added sodium cholate on the aggregation behavior of PNIPAM was studied by different techniques. The change in different fluorescence parameters (intensity, emission wavelength, anisotropy) and data from other techniques such as DLS as well as turbidity indicate that even a small amount of sodium cholate can influence the aggregation of the polymer significantly in the sol state. It was observed from the fluorescence, DLS, and viscosity measurements that the onset of the LCST of PNIPAM solution changes slightly from 34 to 32 °C in the presence of NaC. On the other hand, in PNIPAM gelation the onset of the sol-gel transition is shown to shift from 32 to 29 °C in the presence of 16 mM NaC.22 The facial polarity of the NaC bile salt appears to be the factor that induces PNIPAM aggregation in the sol state, which results in lowering of the LCST. The results show that even a small fraction of the cholic acid can influence the properties of the polymer significantly. Acknowledgment. A.C.K. thanks the University Grants Commissions, Government of India, for a research fellowship. H.B.B. acknowledges receiving a research grant from the Department of Science and Technology, Government of India. The fluorescence lifetime data were measured at the National Centre for Ultrafast Processes, Madras University, India. References and Notes (1) Vinogradov, S.; Batrakova, E.; Kabanov, A. Colloid Surf., B 1999, 6, 291. (2) Esfand, R.; Tomalia, D. A. Drug DiscoVery Today 2001, 6, 427.
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J. Phys. Chem. B, Vol. 115, No. 3, 2011 439 (19) Yoshioka, H.; Mikami, M.; Mori, Y. J. Macromol. Sci., Pure Appl. Chem. 1994, A31 (1), 113. (20) Wu, C.; Zhou, S. J. Polym. Sci., Part B: Polym. Phys. 1996, 34, 1597. (21) Benrebouh, A.; Avoce, D.; Zhu, X. X. Polymer 2001, 42, 4031. (22) Kumar, A. C.; Bohidar, H. B.; Mishra, A. K. Colloids Surf., B 2009, 70, 60. (23) Danielsson, H. In The Bile Acids: Chemistry, Physiology and Metabolism; Nair, P. P., Kritchevsky, D., Eds.; Plenum Press: New York, 1973; Vol. 2, p 1. (24) Mohanty, B.; Bohidar, H. B. Biomacromolecules 2003, 4, 1080. (25) Bohidar, H. B. In Handbook of Polyelectrolytes; Tripathy, S. K., Kumar, J., Nalwa, H. S., Eds.; American Scientific Publishers: Stevenson Ranch, CA, 2002; Vol. 2, p 117. (26) Schmitz, K. S. An Introduction to Dynamic Light Scattering by Macromolecules; Academic Press: New York, 1990. (27) Provencher, S. W. Comput. Phys. Commun. 1982, 27, 213. (28) Chakrabarti, S. K.; Ware, W. R. J. Chem. Phys. 1971, 55, 5494. (29) Kosower, E. M.; Dodiuk, H.; Kanety, H. J. Am. Chem. Soc. 1978, 100, 4179. (30) Pal, S. K.; Peon, J.; Zewail, H. A. Proc. Natl. Acad. Sci. U.S.A. 2002, 24, 15297. (31) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer Academic, Plenum Publishers: New York, 1999; Vol. 314, p 295. (32) Churchich, J. E. Protein Sci. 1998, 7, 2587.
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