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J. Phys. Chem. B 2007, 111, 2372-2376
Coil-to-Globule Transition of Poly(N-isopropylacrylamide) Doped with Chiral Amino Acidic Comonomers F. Lebon,† M. Caggioni,‡ F. Bignotti,§ S. Abbate,† F. Gangemi,† G. Longhi,† F. Mantegazza,⊥ and T. Bellini*,‡ Dipartimento di Scienze Biomediche e Biotecnologie, UniVersita` di Brescia, Viale Europa 11, 25123 Brescia, Italy, Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, UniVersita` di Milano, Via CerVi 93, 20090 Segrate (MI), Italy, Dipartimento di Chimica e Fisica per l’Ingegneria e per i Materiali, UniVersita` di Brescia, Via Valotti 9, 25123 Brescia, Italy, Dipartimento di Medicina Sperimentale, UniVersita` di Milano-Bicocca, Via Cadore 48, 20052 Monza (MI), Italy ReceiVed: NoVember 28, 2006; In Final Form: January 12, 2007
By combined light scattering and circular dichroism measurements (CD), we have investigated the coil-toglobule transition of the thermosensitive polymer poly(N-isopropylacrylamide) (pNIPAAm) copolymerized with a 1/10 fraction of valine- or leucine-derived groups randomly positioned along the chains. The comonomers provide the pNIPAAm chains with chirality, electric charge, and increased hydrophobicity. For valine-derived copolymers, the coil-globule transition is basically unmodified with respect to pNIPAAm, whereas doping with leucine-derived groups significantly lowers the transition temperature and makes the transition discontinuous. We find the CD signal of the chiral comonomers to cleanly depend on the local chain density. We interpret this behavior as an effect of the whole chain conformation on the conformations accessible to the chiral groups.
Introduction The dramatic conformational change of the thermosensitive poly(N-isopropylacrylamide) (pNIPAAm) has motivated many investigations aimed at understanding the basic physics of coilglobule transition. Among them, experiments performed on highly diluted solutions of long chains1,2 have enabled measuring the hydrodynamic and gyration radius of the single molecules. A smooth and steep sigmoidal temperature dependence of the molecular size is found, in agreement with the general expectation that globularization should be a second-order type transition.3-5 In parallel, a long line of investigations is devoted to modifying the homopolymer pNIPAAm behavior by synthesizing copolymers in which various kinds of molecular moieties are linked together with NIPAAm monomers, with the intent of adding functionality to the polymer and modifying its thermal response, partly in conjunction with the needs for drug delivery molecular devices. Such a “doping” of pNIPAAm, typically realized with either hydrophobic or ionic groups whose properties compete with pNIPAAm’s, may decrease or enhance the cloud temperature and enable controlling the mutual collapse and aggregation of globules through pH and ionic strength.6-12 Effects of transition smearing and of microphase separation have also been observed in some of these composite polymers. Interesting examples of pNIPAAm doping involve copolymerization with biorelevant molecules such as DNA, which yields globules with a core-shell geometry,13 or with the achiral amino acid glycine, which adds pH sensitivity to the chain,14 or conjugation with proteins for use in diagnostics and biosepa† Dipartimento di Scienze Biomediche e Biotecnologie, Universita ` di Brescia. ‡ Universita ` di Milano. § Dipartimento di Chimica e Fisica per l’Ingegneria e per i Materiali, Universita` di Brescia. ⊥ Universita ` di Milano-Bicocca.
ration.15 In this vein, some of us studied dilute solutions of a copolymer of NIPAAm doped with leucine-derived groups, namely, with the chiral comonomer N-methacryloyl-L-leucine (MALEU), herein called “pNMAL”.16 Circular dichroism (CD) measurements on pNMAL evidenced a remarkable temperature (T) and pH dependence of molar ellipticity ([Θ]) of the positive CD band at ∼220 nm. This behavior was identified as a consequence of the globularization, in turn made pH-sensitive by the specific doping. However, both the details of the molecular origin of the pNMAL CD signal and the cause for its temperature dependence were not explained at the time. Here, we tackle the issue by combined CD and static and dynamic light scattering (LS) measurements on pNIPAAm chains doped with either leucine- or valine-type groups: the aforementioned pNMAL and analogous pNIPAAm doped with N-methacryloylL-valine (MAVA) groups, herein called “pNMAV”, both with composition 10/1 for the NIPAAm/MALEU (MAVA) units ratio. This study ultimately addresses the relevant question of the interplay between optimal molecular packing and induced structural chirality, a key element in modeling the basics of protein folding.17 The behavior of pNMAL and pNMAV here described enables us to draw conclusions about the dependence of the globularization process on the nature of the dopant and about the factors determining the appearance and the features of the CD spectra. We have found that globularization in pNMAL and pNMAV gives rise to electrically stabilized mesoglobules, as described earlier.6,7 We have also found that the slight chemical difference in the comonomers MAVA and MALEU has a remarkable effect. In the case of pNMAV, the coil-globule transition shows progressive globularization, quite analogously to that observed for the pNIPAAm homopolymer. By contrast, the slightly more hydrophobic nature of leucine makes the coil-globule transition sharper, suggesting a discontinuous transition between random
10.1021/jp067877q CCC: $37.00 © 2007 American Chemical Society Published on Web 02/14/2007
Coil-to-Globule Transition of pNIPAAm
J. Phys. Chem. B, Vol. 111, No. 9, 2007 2373
coil and crumpled coil, which further compacts into a tighter globule as T is increased. We describe how this finding could match theoretical predictions for the coil-globule transition. Moreover, in this work, we discuss the origin of the Tdependence of the CD signal. We show that CD signals of pNMAL and pNMAV depend on the local chain density, and we present a collection of clues and evidence unambiguously indicating that the effect of the chain geometry on the measured ellipticity is due to the confinement effect that the chain exerts on the conformational space accessible to the MALEU and MAVA groups. Indeed, as the chain collapses, the peptidic side chains are constrained in a limited, increasingly dehydrated space and, hence, explore a reduced variety of conformations. Accordingly, the overall molecular chirality is affected. To our knowledge, this is the first evidence of a direct dependence of CD on the local packing density of thermosensitive polymers. Data and Discussion (a) Sample Preparation and Characterization. pNMAL in 1:10 molar ratio (MALEU/NIPAAm) was prepared according to ref 18. pNMAV was synthesized as reported in ref 19. Care was taken to conduct the copolymerization under the same conditions for the two systems so that similar random distribution of doping comonomers along the chains may be expected. The number-average molecular mass (Mn) of pNMAL, determined by SEC-MALS (size exclusion chromatography equipped with a multiangle laser light scattering detector) experiments in ref 18, was 577 000 g/mol, corresponding to a mean number of monomer residues per chain (Xn) of 4700. Following a similar procedure, we have evaluated Mn ) 347 000 and Xn ) 2900 for pNMAV. Both CD and LS experiments have been performed on 0.17 mg/mL solutions (pNMAV) and 0.19 mg/mL solutions (pNMAL) at pH4 (1 mM acetate buffer). To test the possible relevance of interactions of contiguous chiral dopants along the chain, we have also studied the homopolymers pMALEU and pMAVA in 0.02 mg/mL solutions. Copolymerization with MALEU and MAVA adds to pNIPAAm a double character, since these monomers are at the same time hydrophobic and become charged upon dissociation of the corresponding carboxylic group. From the modified Henderson-Hasselbalch equation, whose parameters have been previously reported,19 we have estimated the average degree of dissociation of carboxy groups in pNMAV and pNMAL units at pH 4 to be about 50%. (b) Light Scattering. Light scattering is a key technique to study conformational changes of thermosensitive polymers and has enabled determining the temperature dependence of the hydrodynamic radius RH of p-NIPAAm single chains,1,2 microgels,20 and chain aggregates.6,7,9,10,13 We have thus performed quasielastic light scattering (QELS) measurements and determined RH vs T for pNMAL and pNMAV solutions. In the experiment, we used a 30-mW, 633-nm HeNe laser source, and the measurements were performed at the scattering angle of 90°. All the intensity autocorrelation functions G2(t) were acquired with the same accumulation time (15 min) so that the T dependence of the scattered intensity, IS, could be simply determined from the value of G2(t) at large retardation times, since G2(t f ∞) ) IS2. As T was increased in the range explored here (20 °C < T < 50 °C), the scattered intensity grew remarkably while the correlation time τ displayed an overall decrease. We have fitted all correlation functions with exponential decays, obtaining a good (lower T) or excellent (high T) matching to the data. The fits have enabled us to extract
Figure 1. Scattered light intensity, IS, normalized to IS,20, the intensity scattered at 20 °C (open symbols), and hydrodynamic ratio, RH, (full symbols) measured as a function of the temperature, T, for pNMAL (upper panel, diamonds) and for pNMAV (lower panel, dots).
from G2(t) the long time asymptote and the correlation time, from which IS(T) and RH(T), respectively, were extracted. The T dependence of IS and RH is shown in Figure 1 for pNMAL (upper panel) and for pNMAV (lower panel). For both polymers, IS(T) markedly increases while RH(T) displays, instead, a more complicated nonmonotonic behavior, eventually leveling off at high T. Since RH is always shorter than the wavelength (i.e., RHq < 1, where q is the scattering vector), IS is sensitive to the mass of the scatterers but not to their specific structure. Hence, in this regime, the growth of IS reflects mutual chain aggregation: IS ∝ n, where n is the mean number of chains coagulated in each pNMAV (or pNMAL) cluster. At the lowest T, data refer to isolated chains. This notion is supported by the saturation of IS as T decreases and by the measured RH ∼ 80 nm for pNMAL, and RH ∼ 70 nm for pNMAV, in agreement with the hydrodynamic radius measured for p-NIPAAm random coils, once properly scaled by the contour length.2 Hence, n ) IS/IS,20, where IS,20 is IS measured at T ) 20 °C. Accordingly, in Figure 1 (right axis) we have scaled the scattered intensity to enable directly determining n. The behavior of pNMAV and pNMAL in Figure 1 indicates that upon growing T, as the solubility of the pNIPAAm segments decreases, the chains collapse and, at the same time, aggregate. The indication of this double mechanism phase transition was hypothesized also in ref 16 on the basis of CD and UV measurements; herewith, though, a more satisfactory proof and a quantitative evaluation of the phenomenon is provided. The decrease of RH, conveying the shrinking of the volume taken by each chain, dominates at the lower temperatures, where the aggregation is less substantial. As T is increased, the cluster radius is instead dominated by an increase of RH, due to the increasing number, n, of chains collapsing in the same cluster. At T > 40 °C, the solution is composed of polymer nanoclusters, analogous to the charged, stabilized “mesoglobules” observed for pNIPAAm with various in-chain dopants.6,7 Here, we carry the analysis further, and by combining RH(T) and n(T), we extract the local chain density, F(T) ∝ n/RH3, across the coilto-globule transition, which we plot in Figure 2A. Although RH(T) and n(T) are necessarily concentration-dependent, F(T) represents a property intrinsic to the polymer under study. We find that for both pNMAV and pNMAL, F(T) ranges from
2374 J. Phys. Chem. B, Vol. 111, No. 9, 2007
Figure 2. Panel A: Temperature dependence of the local polymer density, F, for pNMAL (open diamonds) and for pNMAV (full dots). For comparison, we overplot literature data of F vs T obtained for a highly diluted pNIPAAm solution (thick gray dashed line, data from ref 1). Vertical lines indicate the value of TCG for pNMAL (dotted line), pNMAV (dashed line), and pNIPAAm (dot-dashed line). Panel B: Temperature dependence of the mean distance between chiral units in pNMAL (open diamonds) and pNMAV (full dots).
∼0.0004 g/cm3, when random coils, to 0.1 g/cm3, when globulized. For comparison, in Figure 2A, we also report the density from the experiments on pNIPAAm reported in ref 2. We understand the smaller density of pNMAV and pNMAL with respect to pNIPAAm in both coil and globule form as due to the electric charges of the doping group, which make the polymer chain stiffer and selfrepulsive. Another parameter of interest for the discussion is the mean distance between MAVA (MALEU) moieties in pNMAV (pNMAL). This value can be extracted from RH(T) and n(T) under the assumption of uniform distribution of dopants within the globules. The result is plotted in Figure 2B. Finally, as cross-checks, we have performed a few experiments on pNIPAAm and on homopolymers pMAVA and pMALEU. We have found the cloud temperature for pNIPAAm to be around 32 °C, indicating that the comparison between our data and the literature results is on a coherent T scale. We have found no T dependence for RH and n in the case of pMAVA and pMALEU. As clearly visible in Figures 1 and 2, despite the similarity in the chemical structure of MALEU and MAVA, the behavior of pNMAL and pNMAV shows remarkable differences. Approaching from below the coil-to-globule transition temperature, TCG (TCG,pNMAL ∼ 24 °C and TCG,pNMAV ∼ 33.5 °C, vertical lines in Figure 2), pNMAV displays a continuous pretransitional shrinking in the wide T range 25-33 °C, in close analogy with the pNIPAAm behavior. The slightly higher TCG of pNMAV with respect to pNIPAAm has to be understood as a consequence of the stabilizing effect of the ionic dopants, in analogy to previous experiments in the literature.6-8,11,14 Overall, despite the difference in TCG and in packing density, pNIPAAm and pNMAV behave similarly. On the contrary, the behavior of pNMAL appears to be different: the TCG is ∼8 °C lower than in the pNIPAAm homopolymer, and most important, there is no pretransitional behavior for T < TCG. After an abrupt decrease in radius (increase in density), the pNMAL globules progressively compact in the whole T interval between TCG,pNMAL and TCG,pNIPAAm, with a milder T dependence of n and F than for pNMAV and pNIPAAm. We regard these differences as quite
Lebon et al. interesting and wish to offer a possible line of explanations, to be further investigated in the future. Doping with MAVA and MALEU adds to the pNIPAAm chain properties a stabilizing electrostatic contribution and, at the same time, a destabilizing hydrophobicity. Our results indicate that in the case of pNMAV, at our working conditions, the two contributions compensate, yielding only a small increase in TCG. The slightly larger hydrophobicity of MALEU drives, instead, the pNMAL chains toward a more discontinuous behavior. The lowering of TCG is certainly due to the cohesive interactions of MALEU, which, at 8 °C below the pNIPAAm TCG, overcome the still favorable solvation energy of the NIPAAm monomers. Accordingly, we surmise that the globularization is promoted by micellelike intrachain clusters of MALEU moieties, interspersed with the (more) soluble NIPAAm sequences. Chain conformations displaying this type of clustering, sometimes referred to as crumpled coil, have been found by computer simulation studies of hydrophobic/hydrophilic random copolymers.21 The formation of crumpled coils as an intermediate state in the globularization of pNMAL could explain (i) the progressive compacting of the pNMAL globules for T > TCG, as the NIPAAm sequences along the chains become insoluble, and (ii) the discontinuous character of the pNMAL transition. Indeed, the presence of cooperative hydrophobic interactions is considered to be at the origin of the discontinuous two-state behavior of proteins.22 Moreover, computer simulations of homopolymers with three-body attractive interactions are found to produce rich phase diagrams featuring both first- and second-order coil-globule transitions.23 Additionally, this description helps us to understand why at high T the more hydrophobic pNMAL forms smaller mesoglobules (RH ∼ 47 nm) than pNMAV (RH ∼ 65 nm). The burying of the MALEU groups into the clusters driving the pNMAL transition leaves exposed, in the T range where the globularization occurs, the rather soluble NIPAAm groups, thus disfavoring aggregation. The formation of mesoglobules at high T can be explained as the result of competing short-range attraction (e.g., solvation and Van der Waals forces) and long-range electrostatic repulsion, in analogy to phenomena such as the pearling of hydrophobic polyelectrolytes24 and the formation of stable clusters (“cluster phase”) in dispersions of electrically stabilized colloids experiencing depletion attractions.25 This behavior is different from that observed in analogous experiments performed on pNIPAAm under the same conditions of concentration, pH, and ionic strength, where the low solubility of the chains at high T induces unrestrained chain aggregation and sedimentation. The formation of stable nanoglobules is a consequence of the electric charges provided by the carboxyl group in the MAVA/MALEU monomers, which at our working pH are partly dissociated. Quantitatively, the stability of pNMAL and pNMAV aggregates can be evaluated in the frame of the DLVO theory,26 which allows one to extract the surface electric charge, Q, needed to prevent aggregation of clusters having RH ) 65 nm and F ) 0.10 g/cm3 (pNMAV) or RH ) 47 nm and F ) 0.09 g/cm3 (pNMAL), that is, the stable nanoglobules we observe for T > 40 °C. We model the globules as charged spheres interacting by van der Waals potential, with an effective Hamaker constant of ∼1 kBT, compatible with the real density and chemical nature of the globules. We further assume a volume fraction of 4 × 10-4 and an ionic strength of 1 mM monovalent electrolyte. By requiring that such a dispersion be stable for periods on the order of months, we derive a value of Q ∼ 500 electron charges for pNMAV and Q ∼ 300 electron charges for pNMAL. These figures are much smaller than the number of MAVA/MALEU groups in the aggregate, and hence,
Coil-to-Globule Transition of pNIPAAm
Figure 3. Upper panel: CD spectra in molecular ellipticity ([Θ]) units for pNMAL measured below (open diamonds) and above (full diamonds) the coil-to-globule transition temperature. Dashed curve: difference of the two pNMAL CD spectra below and above TCG. Continuous curve: CD spectra of the chiral homopolymers pMALEU. Lower panel: Analogous set of measurements performed on pNMAV and pMAVA solutions.
there is no problem in justifying the stability of the cluster phase on the basis of structural charged groups. Among the pNMAV/ pNMAL ionizable groups, only a small fraction contribute to the electric stabilization of the globule because of many factors: partial dissociation because of the acidic conditions of our study, charge renormalization,27 recondensation of ions buried inside the globule where the effective dielectric constant is smaller, and ionic screening (in our working conditions, the screening length is much shorter than RH). (c) Circular Dichroism (CD) Measurements. CD spectra were measured for buffered solutions of pNMAV, pNMAL, pMAVA, and pMALEU, prepared as described in paragraph (a) of this section, in 1-mm path length quartz thermostated cuvettes. A JASCO J500 instrument was used, and for each spectrum, up to 30 scans were taken. All the CD spectra of copolymers pNMAL and pNMAV and of the homopolymers pMAVA and pMALEU have an intense negative band at ∼195 nm and a positive band at ∼215 nm. As expected, homopolymers’ CD spectra are not influenced by temperature, and only the presence of NIPAAm introduces a temperature sensitivity. In Figure 3, we report the CD spectra for pNMAL and pNMAV, below and above the coil-to-globule transition temperatures. In the same figure, we overlay the CD spectra of pMAVA and pMALEU (continuous lines), the superposition being possible since the CD is here expressed in [Θ] per chiral unit (deg cm2 dmol-1). All CD spectra of Figure 3 have the typical shape attributed to the so-called random coil CD spectrum, which is dominated by the CD of the polyproline-II (ppII) type helix: the negative band at ∼195 nm can be attributed to π f π* transitions; the positive band involves also contributions from n f π* transitions. Contributions can be also given by the coupling of the amide group transitions with those of the carboxylic group. In the case of MALEU, the pNMAL CD spectra at high and low T are similar in shape, except for a slight red shift and an evident decrease in intensity of the positive band going toward low T. The homopolymer pMALEU exhibits a high positive [Θ], similarly to the
J. Phys. Chem. B, Vol. 111, No. 9, 2007 2375 copolymer CD spectrum at high T; however, the peak wavelength is also red-shifted, concurrently with the broadening of the negative band. In the case of MAVA, the shape of the CD spectrum of the homopolymer pMAVA is similar to the spectrum of pNMAV at high T, except that both positive and negative peaks have a much lower [Θ]. The spectrum of pNMAV at low T, instead, has a weak, broad, and evidently red-shifted positive band; the negative band dominates the spectrum, since it is considerably broader. The effects of globulization on the chirality of the copolymers here investigated could in principle be explained in two basic ways: either we are dealing with a coupled oscillator effect of interacting chiral groups whose coupling strength is modulated by the chain behavior, or the observed behavior is due to the effect of the chain on the conformation of the MAVA and MALEU groups, in turn determining the CD spectrum. From the elements gathered here, the latter appears as the correct explanation for the following reasons: (1) The signal per chiral group is quite similar for pMAVA and pNMAV, and for pMALEU and pNMAL, suggesting that the signal is basically proportional to the total concentration of chiral dopants. (2) The quite large inter-MAVA and inter-MALEU distance (see Figure 2B) is a negative factor for oscillator coupling to take place and determine the aspect of the CD spectra. Indeed, to our knowledge, the maximum interaction distance has been measured as 5 nm for porphyrinic systems, bearing much higher oscillator strengths for πfπ* transitions.28 Given the resemblance of the spectra of the coil states with those of the pMAVA (pMALEU) homopolymers, the transition dipoles in contiguous groups should interact with the same strength as those a few nanometers apart. (3) Crucial additional information is provided by the recent experiments by Gokce et al.,29 who studied the CD spectra of a variety of single peptides. In that paper, it is shown that molecules extremely similar to the chiral dopants used in our experiments, such as the acetylated aminoacid L-valine (N-Ac-VAL), may provide by themselves spectra as those in Figure 3. (4) Ref 29 also shows that the intensity of the positive band and the location of the negative maxima generally depend on T. For example, a red shift has been observed in N-Ac-VAL going from low to high temperature. In other cases, it was found that upon increasing T, not only does the negative band shift to the red, but also the positive band around 220 nm decreases in intensity. In those experiments, both the red shift and the vanishing of the positive band were attributed to a transition between two states; namely, from PPII to β form. The increased thermal energy increases the population of the less likely conformation. In our case, we interpret the red shift and the reduction of the intensity of the positive band observed as pNMAV and pNMAL expand from the globular state (high T) to the coil state (low T) as a consequence of the increased variety of conformations made accessible by the looser packing. This notion is supported by systematic ab initio calculations based on time dependent density functional theory (TDDFT) using B3LYP functionals and 6-31G* basis set of the CD spectra on a MAVA “monomer” in vacuo. By use of the Gaussian03 suite of programs,30 we found ∼20 possible conformations, and we obtained two principal patterns of calculated CD spectra with bands of opposite sign. On that basis, we argue that the presence of many conformations gives rise to a low CD signal. This computational analysis shows that by acting on the conformational space, it is, indeed, possible to significantly affect the molecular CD. Specifically, in the globular state and in the case of homopolymer, the packing of the pendent groups introduces steric interactions that limit the
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Lebon et al. eliminating the pretransitional coil shrinking at T < TCG and making the onset of the globularization more discontinuous. 2. We explain why the globularization of the achiral pNIPAAm affects the chirality of the amino acid-based copolymers. We rule out the possible contributions from interacting transition dipoles in distinct MAVA or MALEU groups and show by comparisons with literature data and ab initio calculations that the dependence of the CD spectra on the local chain density is due to the constraints exerted by the collapsing chains on the conformational freedom of the chiral groups. References and Notes
Figure 4. Comparison of the temperature dependence of the local polymer density, F, as derived by DLS experiments (open symbols, left axis) and of the [Θ] value of the positive band at 215 nm in the CD spectra ([Θ]215, full symbols, right axis) for pNMAL (top panel) and for pNMAV (bottom panel).
number of possible conformations. It may be worth noting that a similar conclusion had also been reached by Oku et al.31 on a similar system by comparing polymer and specially prepared monomer CD spectra. A possible additional contribution to the observed spectra that we cannot rule out and that will be worth considering in future experiments is an involvement of the NIPAAm achiral monomers. In the globular state, some chirality could, indeed, “propagate” to neighboring achiral NIPAAM units in a way reminiscent of the sergeant-soldier model32 and, thus, increase the CD signal. This could help in the understanding of the large value of the positive CD band of pNMAV and pNMAL. The overall effect of globularization on the CD spectra can be more clearly evidenced by taking the difference between coil and globule CD spectra, analogously to what is typically done to separate the CD contributions when a two-state unfolding is expected.29 The difference CD is a negative signal, shown as dashed lines in Figure 3. The fact that the area of the CD difference spectrum for pNMAL is smaller suggests that the conformational mobility of the bulkier and more hydrophobic MALEU group is less than that of MAVA. Following the analysis here presented, the peak intensity of the positive CD band can be thus taken as a quantifier of the effects of globularization on the conformational freedom of the chiral moieties. As previously shown for the case of pNMAL,16 a good marker for the intrachain phase transition is the positive value of [Θ] at 215 nm ([Θ]215). With the aim to compare and combine the information gained from LS and from CD, in Figure 4, we plot the T dependence of [Θ]215 (right axis, full symbols) and the local chain density F (left axis, open symbols). The ranges of the vertical axes have been adjusted to produce the best overlap. The agreement is evidently remarkable, thus supporting the notion that the local density is, indeed, the relevant parameter controlling the CD signal. Summary and Conclusions The work here presented delivers two main achievements: 1. We compare two thermosensitive copolymers that are quite similar to each other and show that the inclusion along the chain of sufficiently hydrophobic moieties not only lowers TCG but also modifies the nature of the coil-to-globule phase transition,
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