Mucus as an Arrested Phase Separation Gel - Macromolecules (ACS

Oct 2, 2017 - We investigate the spontaneous restructuring and aging behavior of pig gastric mucus gels by combining rheology and nonconventional stat...
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Mucus as an Arrested Phase Separation Gel Adrian-Marie Philippe,† Luca Cipelletti,† and Domenico Larobina*,‡ †

Laboratoire Charles Coulomb, UMR 5221, Université de Montpellier and CNRS, 34095 Montpellier, France Institute for Polymers, Composites and Biomaterials, National Research Council of Italy, P.le E. Fermi 1, Naples, 80055 Portici, Italy

‡

ABSTRACT: We investigate the spontaneous restructuring and aging behavior of pig gastric mucus gels by combining rheology and nonconventional static and dynamic light scattering. We find that the mucus elasticity weakens over time, concomitantly to structural evolution toward a locally more compact configuration and the onset of large scale density fluctuations. We interpret this behavior as stemming from an arrested phase separation, as observed in many gel-forming polymer systems. This scenario is confirmed by spaceand time-resolved dynamic light scattering data, showing that the gel restructuring is due to intermittent bursts of spatially correlated rearrangements, leading to ballistic dynamics.

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INTRODUCTION Mucus protects epithelial cells (the lining of the surfaces) in the respiratory, gastrointestinal, and urogenital tracts in mammals. In a healthy person, mucus is composed by 90−95% w/w of water, 1% salts, and a mix of macromolecules responsible for the physical properties of mucus: 2−5% of mucins, 1−2% lipids, and 500 min), although minor events are occasionally seen also in the exponential regime, e.g., around tw = 500 min. These analogies suggest that the mucus gel network initially builds up, following the fluidization imposed when preparing the sample and loading it in the scattering cell. During this phase, the gel becomes increasingly compact, as in phaseseparating systems (see the increase of df, inset of Figure 6), and the dynamics drastically slow down as the network strengthens. After a few hundred minutes, internal stresses become so large that the network develops large-scale dense regions (upturn of I(q) in the low q limit, see Figure 6) through large scale rearrangement event (drops of τa, see Figure 8a). To further substantiate this picture, we analyze in more detail the dynamics, since stress-driven dynamics in gels have distinctive features that affect the shape of the correlation functions, the q dependence of τ a , and the spatial correlation of the dynamics.29−31,42,55,64 The KWW fits to the intensity correlation functions shown in Figure 7 have exponent β = 1.6 ± 0.2. Similar values are also found for the other SALS data and for the larger angles probed by DLS: over the full set of measured q and tw, we find 1.1 ≤ β ≤ 1.9. Values of β larger than unity correspond to a decay of g2 − 1 steeper than exponential, which has been termed “compressed” exponential,55 in contrast to the stretched exponential decay of correlators typically observed in glassy systems39 and reported previously for gelling mucin solutions.65 We attribute the different shape of the correlation functions measured in this work and those reported in ref 65 to the different regimes explored in the two studies. In our work, we measure the aging dynamics of well-formed gels, on time scales from tens to thousands of seconds. Cao et al., by contrast, investigated the sol−gel transition, measuring correlation functions in the microseconds−seconds range. A compressed exponential shape of g2 − 1 is the signature of microscopic dynamics stemming from the relaxation of internal stress.55,66,29,30 It is usually associated with an anomalous dependence of the dynamics on the probed length scale, resulting in the scaling τa ∝ q−1 (“ballistic” dynamics), rather than the τa ∝ q−2 scaling indicative of the more common diffusive motion. Figure 9 shows the q dependence of τa for the SALS measurements (solid symbols). Data taken at various representative ages, both in the exponential aging regime and in the plateau regime discussed previously, are collapsed on a single curve by multiplying τa by a scaling factor k. A power law fit to the data yields τa ∝ q−0.95±0.04, consistent with ballistic dynamics. Thus, both the shape of g2 − 1 and the q dependence of the relaxation time exhibit the distinctive signatures of stressinduced dynamics. Figure 9 also shows data collected at larger q with the DLS apparatus (open symbols). These data were collected for a physically distinct sample, for which the preparation protocol was not identical. Thus, a scaling factor k′ different from k was determined independently from that of the SALS measurements; k′ was chosen such that the DLS data point at the smallest q follows the trend of the SALS data. A slight upturn of τa vs q is observed at the larger scattering

Figure 9. q dependence of the relaxation time τa measured by SALS (solid symbols) and DLS (open symbols). Data taken at various tw are rescaled by factors k and k′ for SALS and DLS, respectively, as shown in the label. The line is a power law fit to the SALS data, yielding an exponent −0.95 ± 0.04, indicative of ballistic motion.

vectors, reminiscent of what reported for gels formed by attractive polystyrene particles.67 In ref 67 the q dependence of the dynamics was interpreted in the framework of a simple model where the decorrelation of g2 − 1 is due to intermittent rearrangement events. Each event entails a typical particle displacement δ such that τa exhibits a transition from a q−1 scaling to a regime independent of q, for qδ > 1. Although the range of scattering vectors covered in our experiments does not allow this transition to be precisely located, it is worth noting that the data set an upper bound δ < 1/q* ≈ 0.1 μm, where q* ≈ 10 μm−1 is the q vector where τa starts to depart significantly from the q−1 scaling. Figure 10 shows G4, the spatial correlation of the dynamics, which quantifies the similarity of the temporal fluctuations of the relaxation time measured in sample points separated by a distance Δr (see eq 5). For all ages, the dynamics are correlated over macroscopic distances: following the literature,32 we define a correlation length ξ4 such that G4(ξ4) = exp(−1) and find that

Figure 10. Spatial correlation of the dynamics, as calculated following eq 5, over time intervals ending as shown by the arrows in the inset. The correlation length ξ4 is 270, 460, 1050 and ≥2500 μm for tw = 0, 80, 200, and 400 min, respectively. G

DOI: 10.1021/acs.macromol.7b00842 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules ξ4 increases from 270 μm at tw = 80 min to more than 2.5 mm at tw = 1200 min. The existence of ultralong spatial correlations of the dynamics is a distinctive feature of gels,42,62,29,31,30 as opposed to suspensions of repulsive hard particles, where ξ4 is at most of the order of a few particle sizes.32 In gels, spatial correlations of the dynamics stem from the propagation of the strain field due to a rearrangement event throughout the highly connected elastic network.42,31,30 In mucus, the range ξ4 of the spatial correlation of the dynamics increases significantly during the aging (see Figure 10), a behavior never investigated in previous experimental studies. Interestingly, a similar trend has been reported very recently in simulations,31 thus reinforcing the analogies between the gel mucus dynamics and that of model systems undergoing gelation as a result of arrested phase separation.

biological role of mucus: the gel should be strong enough not to be disrupted by thermal energy alone but weak enough to fulfill its physiological functions, e.g., by yielding under the action of beating cilia in mucociliary transport.17 Future work coupling simultaneous rheological, structural, and dynamical measurements will be needed to fully elucidate the subtle interplay between structure, dynamics, and mechanical properties endowing mucus gels with the required biological functions.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected], Fax +39 081 7758850; Tel +39 081 7758810 (D.L.).

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ORCID

CONCLUSIONS We have investigated the spontaneous restructuring of pig gastric mucus gels by combining rheology and static and dynamic light scattering, thus probing the sample structure and dynamics on a wide range of length scales. The overall picture emerging from our experiments indicates that the behavior of mucus gels can be rationalized in the framework of gel formation resulting from arrested phase separation, in analogy to many colloidal and polymer systems undergoing a phase separation. Following the initial disruption of the original network due to sample preparation, a gel is rapidly re-formed. The SALS data show that larger, more compact domains tend to form over time, consistent with the notion of an underlying phase separation. This evolution implies a decrease of network connectivity, which provides a microscopic scenario for the observed decrease of the elastic modulus. The dynamics are characterized by slow relaxations that exhibit peculiar features, not seen in repulsive glassy systems, but similar to those reported in gels undergoing phase separation:29−31 compressed exponential decay of the correlators, ballistic motion, temporal intermittency, and long-ranged spatial correlations. A key parameter controlling the behavior and the ultimate fate of phase-separating systems is the depth of the quench in the two-phase region. Given the complexity of mucus, it is very difficult to make quantitative estimates. Nonetheless, it is interesting to compare the results presented here to the two limiting cases highlighted by simulations and experimental work on model colloidal systems. For shallow quenches, the thermal energy is sufficient to continuously remodel the gel network via bond breaking and re-formation. In this case, phase separation can proceed to very late stages; the dynamics are driven by the minimization of surface tension and exhibit nonintermittent behavior, with stretched exponential relaxations and very shortrange spatial correlations.19,29,30,68 This is clearly not the case of our mucus gels, which must be deeply quenched in the twophase region. Indeed, in deeply quenched colloidal gels, the interaction energy is much higher than the thermal energy: full phase separation is effectively prevented and rearrangements are due to the buildup of internal stresses, leading to intermittency, anomalous compressed exponential relaxations, and extended spatial correlations. Interestingly, we find that both the relaxation time and the range of spatial correlations are significantly smaller for the mucus gels than for “strong” gels made of polystyrene colloids.42,55 This suggests that in mucus the interaction strength is somehow smaller than in strong colloidal gels but larger than in gels easily remodeled by thermal fluctuations. We speculate that this is dictated by the

Domenico Larobina: 0000-0002-5543-0054 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank M. Tassieri for kindly providing us with the i-Rheo software and for discussions. We thank L. Berthier for discussions, F. Docimo for technical assistance on the SALS setup, and G. Peres from the I.C.S. Factory for donating the material on which this paper is based.

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REFERENCES

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DOI: 10.1021/acs.macromol.7b00842 Macromolecules XXXX, XXX, XXX−XXX