Reversible Condensation of Mucins into Nanoparticles - Langmuir

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Reversible condensation of mucins into nanoparticles Hongji Yan, Cristina Chircov, Xueying Zhong, Benjamin Winkeljann, Illia Dobryden, Harriet Elisabeth Nilsson, Oliver Lieleg, Per Martin Claesson, Yolanda Susanne Hedberg, and Thomas Crouzier Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02190 • Publication Date (Web): 16 Oct 2018 Downloaded from http://pubs.acs.org on October 18, 2018

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Reversible condensation of mucins into nanoparticles

Hongji Yan1, Cristina Chircov1, Xueying Zhong2, Benjamin Winkeljann3, Illia Dobryden4, Harriet Elisabeth Nilsson2,5, Oliver Lieleg3, Per Martin Claesson4, Yolanda Hedberg4, Thomas Crouzier1* 1

Division of Glycoscience, Department of Chemistry, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, AlbaNova University Center, 106 91 Stockholm, Sweden 2

Department of Biomedical Engineering and Health Systems, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, 141 83 Huddinge, Sweden. 3

Division of Surface and Corrosion Science, Department of Chemistry, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Drottning Kristinas väg 51, SE-10044 Stockholm, Sweden 4

Department of Mechanical Engineering and Munich School of Bioengineering, Technical 10 University of Munich, Boltzmannstrasse 11, 85748, Garching, Germany 5 Department

of Biosciences and Nutrition, Karolinska Institutet, 141 83 Huddinge, Sweden

*Email: [email protected]

KEYWORDS. Mucin nanoparticles, Nanoparticle Tracking Analysis, In situ atomic force microscopy, Shear thinning

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ABSTRACT. Mucins are high molar mass glycoproteins that assume an extended conformation and can assemble into mucus hydrogels that protect our mucosal epithelium. In Nature, the challenging task of generating a mucus layer several hundreds of micrometers in thickness by micrometer-sized cells, is elegantly solved by the condensation of mucins inside vesicles and their on-demand release from the cells where they suddenly expand to form the extracellular mucus hydrogel. We aimed to recreate and control the process of compaction for mucins, the first step towards a better understanding of the process and creating biomimetic in vivo delivery strategies of macromolecules. We found that by adding glycerol to the aqueous solvent, we could induce a drastic condensation of purified mucin molecules, reducing their size by an order of magnitude down to tens of nanometers in diameter. The condensation effect of glycerol was fully reversible and could be further enhanced and partially stabilized by cationic crosslinkers such as calcium and polylysine. The change of structure of mucins from extended molecules to nanosized particles in the presence of glycerol translated into macroscopic rheological changes as illustrated by a dampened shear thinning effect with increasing glycerol concentration. This work provides new insight into mucin condensation, which could lead to new delivery strategies mimicking cell release of macromolecules condensed in vesicles such as mucins and heparin.


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Introduction. The extracellular matrix in which mammalian cells are embedded is composed of a highly

hydrated network of polymeric proteins, polysaccharides, and glycoproteins. The structural components of the extracellular matrix have sizeable steric footprint owing to their high hydration, high molar mass, and extended conformation. For instance, collagen molecules can span over the length of several cells. The secretion of such voluminous molecules represents a delivery challenge for cells. This problem has been elegantly solved by Nature in several ways. Some molecules such as collagen have their extracellular auto-assembly encoded in their structure1. Others like hyaluronic acid, can be directly synthesized and secreted at the cell membrane thanks to membrane-anchored enzymes2. For several macromolecules that do not need to be constantly secreted, the molecules are first synthesized in an extremely compacted form, then encapsulated in vesicles. The vesicles are stored in the cell and their content can be released within seconds given an environmental trigger. Examples of such vesicular secretion include heparin secretion from mast cells that require rapid response to inflammatory signals3 and mucin secretion from goblet epithelial cells that respond to mucosal irritants4.

In contrast, bioengineers are still challenged by the delivery of high molar mass and highly hydrated molecules. The injection of highly viscous solutions leads to large shear stresses when extruded through a needle5. A current solution to this problem consists in the in situ crosslinking of macromolecules6. However, these often require a chemical functionalization of the molecule and could require a two-step injection process. With a better understanding of the driving forces behind the compaction and decompaction of macromolecules, one can envision creating new delivery methods that would mimic the efficient delivery of extracellular structural biopolymers


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by cells. The molecules would be condensed hundredfold, thereby lowering the solution viscosity and perhaps increasing resistance to enzymatic degradation. A simple change in environmental conditions would revert the condensation and create a hydrated matrix in situ.

In this work, we explore this concept focusing on the mucin molecules; the main structural component of mucus. Mucins are multifunctional molecules which hydrate, lubricate, assemble into hydrogels with selective filter capacity, and are able to provide biochemical signals to both bacteria and mammalian cells7. Several studies have shown the potential use of mucins, for instance, to treat mucosal related conditions such as ulcers8, dry mouth, mucosal infections9, and microbiome imbalances. Mucins are also of particular interest to us because the mechanisms behind their compaction, their encapsulation and their release are not fully understood. In several studies, mucin vesicles were isolated, their membrane removed, and the dramatic swelling of their content upon the change in environmental pH and ionic composition shown by video microscopy 10–12.

Intact vesicles are characterized by a low internal pH and a high calcium concentration. It has been shown that calcium solutions can stabilize de-membraned vesicles, possibly by shielding repulsive negative charges on the mucin glycans, while acting as ionic crosslinkers that maintain the molecules in close proximity11–13. The presence of calcium and low pH were also shown to be necessary to crosslink and organize recombinantly produced N-terminal VWD1-D2-D′D3 domains of the MUC2 mucins14. In addition to the mucin crosslinkers, solvent quality was also shown to effectively mediate mucin vesicle condensation. Using glycerol as a stabilizing reagent, giant mucin vesicles extracted from the slug Ariolimax columbianus could expand up to 600


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folds within seconds when glycerol was removed13. Studies on polyanionic gels like polyacrylamide gels have shown that such gels can undergo discontinuous15 or more continuous phase16 transition between condensed and a swollen state. The transitions are triggered by environmental changes such as pH, temperature, and ionic compositions and are characterized by reversible and often large volume changes (up to 1000 folds)17. It is hypothesized that in mucin vesicles, crosslinkers such as calcium ions and other environmental conditions contribute to locking the mucin in a condensed state. Once the vesicle is opened to the extracellular environment the increase in pH and the entry of sodium ions displace calcium ions and triggers a rapid unfolding of the molecules driven by electrostatic and steric repulsions18,19.

Mucin condensation and decondensation have thus mostly been studied in the context of mucin vesicles, and little is known about the response of individual mucins and mucin oligomer molecules to these environmental changes. Varma et al. showed by dynamic light scattering measurements that calcium ions could condense porcine submaxillary mucins20. Later, Bastardo et al. showed a reduction in hydrodynamic radius of bovine submaxillary mucin molecules when mixed with sodium dodecyl sulfate, which was interpreted as mucin disaggregation21. Herein we show that glycerol and cationic crosslinkers can condense mucin molecules from micrometer into nano-sized particles in a fully reversible manner. We investigate the combined effect of ionic crosslinkers and solvent quality on mucin nanoparticle formation and demonstrate an effect of nano-scale structural changes on the macroscopic rheological properties of mucin solutions. These results constitute an important step towards a better understanding of the mucin condensation process, and the potential use of such a process in the delivery of macromolecules.


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Results and discussion. Glycerol induces the condensation of PGM molecules. In this study, we used pig gastric mucin (PGM) that we purified from the mucosa of porcine stomachs. The resulting PGM contains MUC5AC and MUC5B22, which share sequence similarity with their human counterparts23 and can be found in many mucosal surfaces including the tear film24, respiratory tract25, stomach, vagina, and cervix26,27. As opposed to commercial sources of mucins, the in-lab purified mucins are close to the native conformation and maintain a high molecular weight and the ability to undergo a pH-dependent sol-gel transition28,29. In this study, we have not varied the pH of the mucin solution, which was measured to be around pH 6 when dissolved in water and the glycerol solutions. At that pH, PGM does not form gels, which allows us to focus our understanding of the other environmental factors we have varied.

Nanoparticle Tracking Analysis (NTA), which estimates the size of nano-sized objects based on the tracking of dynamic light scattering signals, in-situ atomic force microscopy (AFM), and negative stain transmission electron microscopy (TEM) were used to investigate the size of PGM molecules at low concentration (0.2 mg/mL for NTA, 20 µg/mL for AFM and TEM) and in different glycerol/H2O mixtures. When increasing the glycerol concentration from 0% to 90% (v/v) , the average diameter of the PGM molecules estimated by NTA decreases from a micrometer to about 20 nm (Figure 1A). PGM in H2O exceeded the upper detection range of NTA, and thus the estimate of about 1 µm is to be taken cautiously. Similarly, sizes below 20 nm are below the lower detection range. The representative particle size distribution graphs measured by NTA measurement revealed a broad distribution in all conditions, but that tended to narrow with increasing glycerol concentration (Figure 1B, 1C, and 1D).


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Both AFM and TEM image supported these findings. In water or the 30% glycerol/H2O, AFM images recorded in quantitative Imaging (QI) mode (Figure 1B and 1C inserts) and TEM images using a negative stain (Figure 1E, 1F) confirmed the presence of extended and condensed molecules, which reflected the broad size distribution measured by NTA at lower glycerol/H2O ratio solutions. AFM measurements in 60% glycerol/H2O(Figure 1D inserts), performed in a contact imaging mode due to the high viscosity also revealed the presence of highly condensed particles and some extended mucins. However, measurements done with the contact mode may disrupt or unfold the condensed mucin particles. Indeed, as shown in Figure SI 1, the adsorbed mucins were disrupted by the application of a higher imaging force. However, when imaged by TEM, PGM in 60% glycerol/H2O solutions (Figure 1G) exhibited only condensed particles homogeneous in size.

We also investigated the effect of glycerol on the ζ-potential and electrophoretic mobility (EM) of the PGM particles in glycerol/H2O mixtures between 0 and 30%, the measurement being impossible in mixtures higher than 30% because of the high viscosity of glycerol (Table 1). The glycerol-mediated condensation of the mucins decreased the negative ζ-potential of the particles, from -49,7 ± 8,83 mV in H2O to -22 ± 16 and -22 ± 23 in 25% and 30% glycerol/H2O, respectively. The trend suggests that the interactions between PGM and glycerol affect the protein electrostatic charge distribution, perhaps through conformational changes. When comparing the ζ-potential in different glycerol concentrations one should consider a couple of aspects. First, the ζ-potential is calculated based on Stokes-Einstein relation and Henry’s law which assume spherical particles. Due to their extended nature, the mucins molecules may be


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more rod shaped, in particular at lowest glycerol concentration. Second, although the dielectric constant was entered as an input value30, errors in the values could somewhat affect the ζpotential. Indeed, decreasing dielectric constant with increasing glycerol concentration leads to stronger electrostatic forces, a shorter Debye screening length and a shift in all acid-base equilibria (e.g., sialic acid) towards the uncharged state31. Still, the measurements could be useful for comparison with other studies and between conditions with similar glycerol concentrations. PGM exhibited similar EM in all the solutions with a slight decrease (towards more positive values) in 25% and 30% glycerol (Table 1), which also suggests PGM is more condensed in a higher viscous solution with increasing glycerol concentration.


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Figure 1. Mucin condensation by glycerol/H2O solutions. (A) NTA examined the average size of condensed mucins in different glycerol/H2O solutions. The red line indicates the approximate lower detection limit of NTA (20 nm in diameter). (B, C, D) Representative size distribution curves for PGM in H2O and glycerol/H2O solutions measured by NTA. Corresponding AFM images of mucins on untreated mica surfaces in water and glycerol/H2O mixtures are shown in the insets. (E, F, G) Negative staining TEM images of mucins in H2O, 30%, and 60% glycerol/H2O. The dark circle in the mucin in 60% glycerol/H2O may be due to the interaction of PGM with glycerol and uranyl acetate since control images with 60% glycerol/H2O and uranyl acetate only show no artifacts (H).

Table 1 ζ-potential and electrophoretic mobility (EM) of PGM compacted by glycerol/H2O mix Glycerol/H2O

0%

5%

15 %

20 %

25 %

30 %

ζ-potential [mV] -49.7 ± 8.83

-41.3 ± 7.36

-37.7 ± 11.5

-48.4 ± 8.8

-22.0 ± 15.9

-22.2 ± 22.8

EM [µmcm/Vs] -3.62 ± 1.13

-2.75 ± 1.01

-1.58 ± 0.53

-2.02 ± 0.95

-0.75 ± 0.54

-0.69 ± 0.81

We and others take cautiously the absolute values of sizes provided by NTA measurements. Errors can originate from the viscosity values we have used for glycerol/water. In addition, mixed solvents can give rise to fluctuations and light scattering, and affect the absolute values of NTA measurements32,33. For instance, we observed a systematic underestimation of the size of gold nanoparticles in glycerol-water mixtures above 60% (Figure SI 2). Such results emphasize the need to take caution when analyzing the absolute values provided by NTA. But they also do


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not perfectly reflect the results obtained for mucins since local heating of gold nanoparticles34 can significantly affect the viscosity of the surrounding solution. There is little doubt, however, that a large compaction is occurring with increasing glycerol concentration, as supported by AFM and TEM imaging and by the diffusion coefficient data directly measured by NTA that showed little variation up to 60% glycerol/H2O, which can only be explained by significant concomitant compaction of the mucin molecules (Figure SI 3).

The glycerol-driven condensation is reversible. A hallmark of the phase-transition observed during in vitro experiments on mucin vesicles in

cells is its reversibility17. A fully reversible compaction of mucin molecules would also prove useful if such a strategy is to be used in biomimetic macromolecule delivery systems. We thus tested the reversibility of the compaction by adding PGM molecules to 90% or to 10% glycerol/H2O solutions, followed by either decreasing the glycerol concentration from 90% down to 10% or increasing it from 10% up to 90%. The diameter of the particles measured by NTA are shown in Figure 2. The mucin diameter was affected by increasing or decreasing the solution glycerol concentration, suggesting the transformation is not unidirectional but indeed reversible. Thus, irreversible phenomena such as protein degradation can be excluded. The results also show the compaction depends on the continued presence of glycerol, and the changes in particle size occur rather rapidly as seen by the measurements taken only a few minutes after addition or dilution of glycerol.


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Figure 2. The reversibility of condensed mucins by glycerol/H2O solutions recorded by NTA. PGM was introduced to 90% glycerol/H2O then diluted down to 50% then to 10%. In a separate experiment, PGM was introduced to 10% glycerol/H2O, and the ratio brought to 50% then to 90%. The red line indicates the approximate lower detection limit of NTA (20 nm in diameter). Three independent repeats of the experiments are shown.

As opposed to globular proteins that have well-defined tertiary structures due to the action of intramolecular interactions, mucins are considered self-avoiding molecules that adopt extended conformations (persistence length of the order of tens of nm7). AFM images of de-glycosylated mucin showed they had re-folded into globular structures35 suggesting the dense O-glycosylation of mucin counteracts protein-protein interactions36. These unique properties make mucins exceptional good candidates for extreme compaction of the molecule as observed in mucin granules in vivo. While mucin reduced it size by roughly 60% when transferred from 0% to 30% glycerol/H2O, a globular protein such as lysozyme showed only a 10 % reduction at similar test conditions (Figure SI 4).


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The size decrease can be explained by at least two phenomena. Disaggregation of mucins driven by changes in intermolecular interactions, and the compaction of the mucin molecules driven by favorable intramolecular interactions. In the disaggregation model, we assume the PGM solution is composed of multimeric structures held together in part by hydrophobic interactions37. Glycerol in water was shown to stabilize proteins and prevent aggregation by interacting as an amphiphilic interface between the hydrophobic domains of the protein and the polar solvent38. Thus, it could be that in highly concentrated glycerol solutions, the condensed mucin particles limit their intermolecular hydrophobic interactions, which would decrease mucin aggregation and result in single molecules that can be condensed to nano-scale sizes.

In the compaction model, we hypothesize that the strong compaction observed is driven by poor solubility of mucins in glycerol. Indeed, mucins are typically insoluble in poorly polar solvents such as hexane39 and simple sugars40, which are essential for the solubility of the mucin protein36,41, are also less soluble in glycerol than in water40. Increasing the glycerol concentration would then favor mucin intramolecular interactions over mucin-solvent interactions, leading to a compaction of the molecular structure. This reasoning is supported by previous work showing that glycerol can reduce the specific volume of even already folded globular protein by up to 8%42. The effect here is much more pronounced because of the largely extended conformation of mucins in water, and perhaps also because of the intrinsic capacity of mucins to condense and unfold as exemplified by the compaction and release of mucins by goblet cells.

As we decrease glycerol concentration of a PGM solution, the size increase seen in Figure 2 could also be due to (re)aggregation of the molecules. However, the fact that the size increase is


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achieved nearly instantaneous, that it is stable over time (PGM particles retained their size over 4 days storage at 4ºC, Figure SI 5), and that size is independent of the protein concentration (Figure SI 6) weigh against the possibility of intramolecular aggregates. We thus hypothesize that decompaction of previously condensed molecules is the driving mechanism here. The underlying mechanisms of mucin compaction and mucin de-compaction could very well be different. However, we would then not expect to find a similar size distribution when reaching a particular glycerol concentration by glycerol concentration or by dilution (Figure 2).

Positively charged ionic crosslinkers further compact glycerol compacted mucin particles. We showed that PGM could be condensed many folds and reverted to its original conformation by changing glycerol concentration alone. In vivo, mucin granules most likely do not contain glycerol, but a combination of low pH and positively charged calcium ions (Ca2+) that possibly decrease electrostatic repulsion of mucins43, and that together with macromolecular crowding contribute to mucin entanglement and to maintaining mucins in a compacted state. We thus wished to investigate whether the combined effect of glycerol and ionic crosslinking could further compact the mucins and perhaps contribute to stabilizing the condensed form of mucin in the absence of glycerol. Cationic molecules are expected to interact with mucin molecules since mucins bear an overall negative charge over a large range of pHs owing to carboxylic groups of sialic acid sugar residues and sulfated glycans.

We first performed a two-step mucin condensation, which consisted of compacting the mucins with glycerol followed by exposure to either Ca2+ or positively charged polylysine (PLL)


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polymers while maintaining the glycerol concentration constant. In our hands, we saw no effect of Ca2+ on the size of the particles measured by NTA when added directly to PGM dissolved in water. However, the addition of Ca2+ further decreased the size of the mucin particles previously condensed by glycerol/H2O (Figure 3A). Increasing the Ca2+ concentration from 1 mM to 50 mM did not affect the particle size. Both low molar mass (1.6 kDa) and high molar mass (66 kDa) PLL further condensed the mucins in glycerol. The condensation effect of PLL was significantly stronger than for Ca2+ only when the mucin was first condensed in 30% glycerol/H2O, and when comparing the effect of 1 mM Ca2+ with 1.6 kDa PLL in 60% glycerol/H2O. (Figure 3A). At high glycerol content (90% glycerol/H2O), ionic crosslinkers had less effect on the size, probably because there were fewer interactions sites accessible to the ionic crosslinkers due to steric hindrances.

We next investigated the interactions between the ionic crosslinkers and the glycerolcondensed mucin particles by measuring their effect on the ζ-potential. The addition of 1 mM Ca2+ and 0.1 mg/ml PLL increased the ζ-potential of the glycerol-condensed mucin particles (Table 1, S-1). The addition of 50 mM Ca2+ resulted in higher ζ-potential than 1 mM Ca2+ (Figure 3B) which suggests more Ca2+ binding to the mucins even though the particle diameter was similar. It has previously been noted that the presence of calcium ions affect adsorbed mucin layers towards a more compact state, which is consistent with the role of calcium ions as a physical cross-linker44,45. The addition of 1.6 kD and 66 kD PLL significantly rose the ζ-potential to + 11.6 ± 9.3 mV and + 32.4 ± 9.6 mV, respectively (Figure 3C). This large increase in ζpotential is due to the high number and high density of positive charges on the PLL molecules. The ionic cross-linkers interact with the glycerol-condensed particles, most likely via


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electrostatic interaction with the negative charges of mucin-associated glycans. The interaction resulted in further condensation of the particle due to these electrostatic interactions. The 66 kDa PLL molecules, owing to their high charge density and high molar mass, increased the surface charge to a greater extent compared to 1.6 kD PLL. It is also possible that along with this penetration into the mucin particles, the cationic species disrupt mucin-mucin interactions, which would lead to disaggregation of mucins.

Figure 3. Further condensation of glycerol/H2O-compacted mucins by addition of positively charged species. (A) The average diameter of mucins condensed by 30%, 60% or 90% glycerol/H2O followed by addition of different positively charged species without changing the


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glycerol/H2O concentration. (B, C) ζ-potential of mucins condensed by 30% glycerol/H2O, or by 30% glycerol/H2O followed by addition of Ca2+ or PLL. The red line indicates the approximate lower detection limit of NTA (20 nm in diameter).

Cationic crosslinkers can stabilize the glycerol condensed mucin particles We hypothesized that the interactions of the cationic crosslinkers after glycerol condensation could have a stabilizing effect on the mucin particles, which could then remain condensed after the removal of the glycerol. We thus studied the reversibility of compaction of mucin by 90% glycerol/H2O followed by 50 mM Ca2+, and 90% glycerol/H2O followed by 1.6 kD or 66 kD PLL. We decreased the glycerol concentration to 60% and 30% sequentially without changing the concentrations of cationic crosslinkers. The presence of positively charged species (Ca2+, 1.6 kD and 66 kD PLL, Figure 4A, B, C) could stabilize the condensed PGM particles when reducing the glycerol concentration from 90% to 30% glycerol/H2O. Interestingly, a monovalent positive ion such as sodium could not fully replicate the stabilizing effect of Ca2+ since the PGM in 90% glycerol/H2O and 50 mM Na+ reverted to larger sizes when the glycerol concentration was decreased (Figure 4A, B, C). The small, but a statistically significant stabilizing effect of Na+ ions could be due to electrostatic shielding of mucin charges. The cationic crosslinkers tested here were thus able to counteract the unfolding of the mucin that occurs with decreasing glycerol concentration.

Notably, the size of PGM particles condensed by 30% glycerol/H2O followed by 1.6 kD PLL was smaller than mucin condensed by 90% glycerol/H2O followed by 1.6 kD PLL and then diluted down to 30% glycerol/H2O without changing PLL concentration (100 ± 14 nm vs 54 ± 3


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nm) (Figure 4D). These discrepancies could point to differences in the amount and arrangement of PLL around mucins condensed with 30% or 90% glycerol/H2O. One can speculate that in 90% glycerol/H2O, the well-condensed PGM limited the interactions between PLL and PGM particles due to a steric effect. In this scenario, the PLL molecules are poorly entangled into the mucin particle and have a limited stabilizing effect on the mucin particles when decreasing the glycerol/H2O ratio.


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Figure 4. The reversibility of the mucin condensation triggered by glycerol/H2O solution followed exposure to a positively charged ionic crosslinker. (A) The average diameter of condensed mucins as measured by NTA, in decreasing glycerol/H2O ratios (90%, 60% 30%) with constant cationic crosslinker concentration. (B, C) The average diameter of condensed mucins by 90% glycerol/H2O and 90% glycerol/H2O followed by a cationic crosslinker after decreasing the concentration of glycerol/H2O ratio from 90% to 60% (C) and 30% (B) without changing the concentration of the positively charged ionic crosslinker. (D) The average diameter of condensed mucins directly exposed to 30% glycerol/H2O followed by exposure to 1.6 kDa PLL, compared to mucins exposed to 90% glycerol/H2O followed by 1.6 kDa PLL, then diluted to 30% glycerol/H2O. The red line indicates the approximate lower detection limit of NTA (20 nm in diameter).

PLL but not Ca2+ can stabilize the glycerol condensed particles dialyzed against a saline solution. Although the ionic crosslinkers stabilized the complex when present in solution, it is unknown whether the particles would remain compacted when removing both glycerol and ionic crosslinkers. After dialysing the mucin particles against a saline (140 mM NaCl, pH 7.4) for 48 hours, we performed NTA to measure the mucin particle size. Mucin particles condensed by 30% glycerol, followed by Ca2+ or 1.6 kDa PLL, and 60%, and 90% glycerol/H2O followed by Ca2+ showed full reversibility when compared to the size of particles in 140 mM NaCl (Figure 5A). We hypothesize that the Ca2+ ions could be displaced by Na+ ions from the saline solution, leading to the size increase of the mucin particles. This is consistent with previous quartz crystal


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microbalance work showing a reversible effect of calcium ions on adsorbed mucin layers45. In contrast, the mucin particles condensed by 90% and 60% glycerol/H2O followed by 1.6 kDa PLL remained partially compacted. It is likely due to the multivalent nature of the PLL which enhances its affinity to the mucin molecules, leading to more stable condensation. Interestingly, mucin particles condensed by 30% glycerol/H2O followed by PLL also showed full reversibility, which might be due to the limited compaction of PGM in 30% glycerol/H2O.

To investigate the fate of the cationic crosslinkers after dialysis, ζ-potential measurements were performed before and after dialysis of mucin particles condensed by 30% glycerol/H2O followed by exposure to Ca2+ or PLL. Under all conditions, the mucin particles exhibited negative ζpotentials, but they were less negative than for mucins never exposed to the cationic crosslinkers (-22.2 ± 22.8 mV), suggesting that the bound Ca2+ and PLL did not fully dissociate from the condensed PGM molecules (Figure 5B, Table S-3). Na+ displaced only a fraction of the bound Ca2+ and PLL, affecting the mucin condensation while leaving the surface charge relatively unchanged. This may indicate a limited role of the surface-bound ions in the compaction effect.


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Figure 5. Reversibility of the two-step condensation of mucins after dialysis against a saline solution. (A) The reversibility of the size of mucin particles first condensed by 30%, 60% and 90% glycerol/H2O followed by exposure to Ca2+ or PLL and finally dialysed against saline solution. The sizes are normalized to the sizes in saline solution, a value of 1 corresponding to full reversibility. (B) ζ-potential of condensed mucins in 30% glycerol/H2O followed by Ca2+ or PLL, before and after dialysis.

The sequential combination of two ionic crosslinkers following glycerol further condenses the mucin particles to a limited extent. The Ca2+ and PLL cationic crosslinkers tested here have different effects on mucin condensation and stabilization of compacted mucin form, which most likely stems from differences in cross-linking mechanisms. We thus wanted to test whether the combination of the two crosslinking strategies could provide additional compaction to the mucin particles. Since mucin condensed by glycerol/H2O followed by 1 mM Ca2+ results in negatively charged particles (Figure 3C), we hypothesized the particles could interact further with other cationic molecules. For this three-step mucin compaction protocol, we added PLL to mucin particles previously condensed by sequential addition of glycerol/H2O and 1 mM Ca2+. The addition of PLL to mucin condensed by 30%, 60% or 90% glycerol/H2O followed by 1 mM Ca2+ significantly decreased the size of particles as measured by NTA (Figure 6A). The reduction in size was even more pronounced when adding high molar mass PLL (66 kD) to mucin compacted by 30% glycerol/H2O followed by 1 mM Ca2+. While the same PLL gave rise to a small increase in size of mucin compacted by 60% glycerol/H2O followed by 1 mM Ca2+. The mucin particles condensed with 30% glycerol followed by 1mM Ca2+ then by PLL increased their ζ-potentials


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suggesting that there is indeed PLL binding to the particles. Here again, the highest ζ-potentials were measured for mucin particles complexed to 66 kD PLL (Figure 6B, Table S-2).

Figure 6. Three steps mucin condensation by using 30%, 60% or 90% glycerol/H2O, Ca2+, and then PLL. (A) The average diameter of the condensed mucins was evaluated by NTA. (B) ζpotential of mucins in 30% glycerol/H2O, 30% glycerol/H2O followed by addition of Ca2+ and 30% glycerol/H2O followed by addition of Ca2+ and then PLL. The red line indicates the approximate lower detection limit of NTA (20 nm in diameter). The upper limit is 1000 nm.

The compaction of the mucin by glycerol affects the rheological properties of mucin solutions. The compaction of mucin molecules into nano-size objects could have a direct impact on their utilization in various biomedical applications. We hypothesized that the conformational change of mucins from linear to condensed could affect the shear viscosity of mucin solutions. However, the use of highly viscous glycerol as a compaction reagent masked any effect of the mucin conformational changes on the absolute viscosity of the solutions. Indeed, at constant mucin


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concentration, the viscosity of the mucin solution increased with the increase of glycerol concentration (Figure SI 7). However, some conformational changes of mucin induced by glycerol, even though occurring at the molecular level, could still be detected at macro-scale by comparing the dynamic viscosity of condensed and native mucin solutions. Indeed, the classical drop of viscosity with increasing shear (i.e shear thinning) rate seen for many polymer solutions is gradually reduced as the glycerol concentration is increased (Figure 7). In contrast, the glycerol solutions are Newtonian fluids and thus showed a shear rate independent viscosity as expected (Figure SI 7). The drop in shear thinning with increasing glycerol concentration could be explained by a transition of elongated mucin molecules to globular mucin particles as the glycerol/H2O ratio increases. Another possible cause could be the disruption of a mucin aggregates or network with increasing shear. However, considering that the concentration of PGM is low (0.25 % w/v in H2O or glycerol/H2O solutions) and that the pH at which we performed in the experiment was above the critical gelling pH of 4, we do not expect the formation of a significant mucin network to occur. Notably, the mucin in water solution exhibited a weaker shear thinning behavior compared to mucin in 10% or 30% glycerol/H2O solutions (Figure 7). This could be due to various effects induced by low glycerol concentrations, such as the disassembly of mucin complexes into more linear molecules or the intramolecular unfolding of mucin domains. It is worth noting that in 60% and 90% glycerol/H2O solution we observed a reminiscent shear thinning effect even though the majority of mucin molecules are supposedly in condensed colloidal form. Several scenarios should be considered here. Large particles left undetected by NTA might contribute to such a background shear thinning effect. The mucin nanoparticles could interact under shear, leading to shear thinning effects. Indeed, a similar phenomenon was shown for aluminum oxide nanoparticle


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solutions.46 Finally, it is also possible that the shear exerted on the sample leads to a partial or complete unraveling of the mucins from their condensed form. The shear-extended molecules could then align and contribute to the shear thinning behavior.

Figure 7. Shear thinning properties of mucin solutions. The viscosity of 2.5 mg/ml PGM in different glycerol/H2O solutions (0%, 10%, 30%, 60%, and 90%) were evaluated by shear rheology. The relative viscosity values shown denote the viscosity of each solution normalized to the value obtained at the highest applied shear rate, which is set to zero afterwards. The error bars denote the standard error of the mean as obtained from three independent measurements. If error bars are not shown, they were smaller than the symbol size.

Conclusions. In this study, we show that by changing the solvent quality by addition of glycerol to aqueous mucin solutions we can control the mucin conformation from large and extended molecules,


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down to highly condensed nanometer-sized particles. We also show that cationic crosslinkers can both contribute to the compaction of mucins in conjunction with glycerol, but also help to stabilize the particles in the absence of glycerol. The scale of the compaction and the reversibility of the mechanism match the properties of mucin vesicles in cells. Thus, one could hypothesize that in addition to Ca2+ and low pH, other unidentified small molecules modulate the mucinmucin interaction and mucin solubility inside the vesicles. These would dilute with the fusion of the vesicle with the cell membrane and lead to a “jack in the box” effect driven by rapid phase transition of the mucins. This work is also an important first step toward the biomimetic delivery of mucin for therapeutic applications. Although the glycerol used in this study is poorly biocompatible and highly viscous, other strategies to stabilize the condensed form of the mucin, which could be removed once triggered by specific environmental conditions, would open the door to instant gelation from low viscosity solutions.

Materials and methods. All ratios given in this manuscript are volume ratios, if not denoted differently. All chemical reagents were purchased from Sigma-Aldrich. PGM was purified and characterized in our laboratory as described below. Purification of PGM. PGM was purified as described before47 with the difference, that the cesium chloride gradient centrifugation was omitted. Briefly, the mucus from the mucosa of porcine stomachs epithelium was scraped off gently and diluted at the ratio of 1 to 5 (v/v) in water containing 200 mM NaCl, 5 mM benzamidine HCl, 1 mM 2,4’-dibromoacetophenone, 1 mM phenylmethylsulfonylfluoride and 5 mM EDTA. The pH of the solution was adjusted to 7.4 with NaOH and gently


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stirred overnight at 4 °C. The PGM solution was centrifuged at 20 000 rpm for 1 hour followed by ultracentrifugation at 40 000 rpm for 1 hour at 4 °C to remove the cellular and food debris and the being fractionated by preparative size exclusion chromatography. The fraction of PGM was identified by periodic acid Schiff assay as described in the previous publication48. The PGM fraction was pooled together, and then concentrated and desalted by reversed osmosis using a 100 kDa molar mass cutoff. The desalted PGM solution was then flash frozen in liquid nitrogen and lyophilized. Samples were stored at -20 °C.

Condensation of PGM. The PGM was pre-dissolved in water at 5 mg/ml overnight at 4 °C. For one-step PGM condensation, 100 µl of condensation buffer (different glycerol/H2O mixtures) was added into 1.5 ml bottom of Eppendorf tube, followed by the addition of 25 µl of pre-dissolved PGM onto the tube wall. The solutions were then mixed on a vortex mixer at high speed for 30 seconds. For two-step condensation, the same glycerol/H2O mixture having different positively charged species was added after one-step condensation, to reach final concentrations of 1 mM, 50 mM Ca2+ (CaCl2), 0.1 mg/ml 1.6 kD and 66 kD PLL individually. For three steps sequential condensation procedure, PGM was firstly mixed with the glycerol/H2O, followed by addition of same glycerol/H2O containing Ca2+, and then the same glycerol/H2O containing Ca2+ (to maintain the Ca2+ concentration constant) and PLL, to make the final concentration of 1 mM Ca2+ and 0.1 mg/ml of 1.6 kD or 66 kD PLL.

Particle size determination.


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Nanoparticle Tracking Analysis (NTA, Nanosight NS300 instrument, Malvern, Uppsala, Sweden) with a 405 nm laser was used to evaluate the size of condensed PGM (diameters) in-situ according to the instrument manual. Latex beads having a size of 100 nm (Thermo Fisher) in ultrapure water were used to confirm correct size measurements by NTA and showed satisfying accuracy (within 5% error). Each sample was firstly diluted by the same buffer which was used to condense the PGM to a final PGM concentration of 0.2 mg/ml (to minimize the contribution of PGM to solution viscosity), and a triplicate measurement on triplicate independent condensed PGM particles with 60 s captures was performed at 20 °C. Between each sample, the sample chambers were washed with 16.4 mM (double critical micelle concentration) sodium dodecyl sulfate adjusted to a pH of 2.9 with HNO3, followed by 5% ethanol in ultrapure water. This cleaning procedure should both remove adsorbed proteins and calcium ions. This was verified by measurements in ultrapure water showing acceptable particle contamination (camera level 15-16, 2 particles/frame). Chambers were dried by nitrogen flow. The data of average size and diffusion coefficient was processed by using NTA 3.2 software.

The diameters estimated by NTA measurements are calculated based on the Stoke Einstein equation describing particle motions, in which viscosity is an input parameter. We thus set the correct viscosity values as input values, based on the solvent (water/glycerol) viscosity, to compensate for the increase in viscosity30 with increasing glycerol composition.

Control NTA measurements with gold nanoparticles (50 nm) and lysozyme in H2O in different glycerol/H2O ratios were also conducted. Citrate-coated 50-nm gold nanoparticles were obtained as stock suspensions of approximate 1 mg/mL in 2 mM sodium citrate from NanoComposix


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(BioPure, batch no MGM2244). 2 µl of this stock solution was added to 1 mL of water or waterglycerol mixtures, and vortexed until a homogenous color was obtained in the solution (after about 3-10 seconds). For lysozyme, 5 mg/mL stock solution was prepared, 25 µl of the lysozyme stock solution was added to 100 µl of the desired glycerol/H2O mixtures following the same compaction method as performed for PGM. The solutions were further diluted by the same glycerol/H2O to obtain the concentration of 0.2 mg/mL before NTA measurements.

Zeta potential measurements. Zeta potential (ζ-potential) of the condensed particles were evaluated by using a Zetasizer instrument (Nano ZS, Malvern instrument, Ltd., UK). Briefly, electrophoretic mobility was first determined and then converted to ζ-potential by the instrument software using Henry’s equation. For each measurement, the condensed PGM particles were first diluted by using the same condensation buffer to the final concentration of 0.2 mg/ml and agitated by a vortex shaker. The solution was transferred into a 1 ml syringe and then loaded gently into folded capillary zeta cells (Malvern) without generating air bubbles. For each sample, the duration of 10 s equilibrium time was allowed for samples to reach the temperature of 25 °C. Triplicate measurements were performed for each sample having 10-100 runs.

Atomic force microscopy imaging. Adsorption of PGM particles onto a freshly cleaved mica surface was studied with a JPK NanoWizard® 3 Atomic force microscopy (AFM, JPK Instruments AG, Berlin, Germany). The measurements were conducted in a droplet of MilliQ water, 30% and 60% glycerol/H2O mixtures. Quantitative Imaging mode (QI) was utilized to obtain topography images of mucin


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particles in water and 30% glycerol/H2O mixture. The QI mode allows an accurate control of the peak imaging force and thus is preferable for such soft sample. The maximum applied normal force during QI imaging in water and in 30% glycerol/H2O mixture was about 0.3-0.4 nN and no particle damage was observed. The measurements in water were conducted using the MLCT probe (Bruker) with a nominal spring constant of 0.1 N/m and a nominal outer tip radius of 20 nm. However, to perform measurements in 30% glycerol/H2O mixture, a cantilever with a stiffer spring constant was needed due to higher mixture viscosity. An SNL-10 probe (Bruker) with a nominal spring constant of 0.12 N/m and an outer tip radius of 2 nm was thus used to image mucins in the 30% glycerol/H2O mixture. The QI imaging became unreliable with an increase of glycerol concentration to 60% due to high viscosity. Instead reliable imaging of mucins in 60% glycerol/H2O was possible using contact mode employing a soft MLCT probe (Bruker) with a nominal spring constant of 0.03 N/m. However, the contact imaging mode can be destructive for soft materials due to constant contact of the cantilever tip with the surface and higher lateral forces. It was found that the lowest normal force needed to obtain a good image of mucins without inducing their destruction was 2-3 nN (Figure SI 1). The imaging speed using QI mode was about 40 μm/s and the tip velocity in contact mode was around 1.9 μm/s.

Preparation and TEM imaging of PGM in water and 60% glycerol/H2O. Stock solution (5mg/ml PGM in milli-Q water) were diluted to desired concentration by either milli-Q water or 60% glycerol/H2O for negative-stain sample preparation. Aliquots (4 μL) were adsorbed onto glow-discharged continuous carbon-coated copper grids (400 mesh, Analytical Standards) for 2 min. The grids were subsequently blotted with filter paper, washed with two drops of milli-Q water and negatively stained with one drop of uranyl acetate (2% (w/v) for 60%


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glycerol alone and 0.75% (w/v) for PGM samples) for 45 s before final blotting and air-drying. The samples were imaged using a Jeol JEM2100F field emission gun transmission electron microscope (Jeol, Japan) operating at 200 kV. Single micrographs of the sample were recorded on a Tietz 4k × 4k CCD camera, TVIPS (Tietz Video and Image Processing Systems, GmbH, Gauting, Germany) at the nominal magnification of × 50,000 and 1.5–3.5 μm defocus.

Dialysis against saline solution. The condensed PGM solutions by different conditions were transferred into a Slide-A-Lyzer™ Dialysis Cassettes with a molecule weight cut-off of 10 kDa. The dialysis cassettes were then transferred to a beaker containing 300 mL of saline buffer, pH 7.4 for 2 days at 4 °C.

Rheological characterization. Rheological measurements were conducted on a commercial shear rheometer (MCR302, Anton Paar, Graz, Austria) using a cone/plate measuring setup (CP50, Anton Paar, Graz, Austria). For each measurement, a sample volume of 570 µL was required. The solutions were prepared as follows: First, porcine gastric mucin (MUC5AC) was solubilized at a concentration of 12.5 mg/mL in Milli-Q water. This concentrated mucin solution was then further compacted with different glycerol/H2O solutions (10%, 30%, 60% or 90%, respectively) in a ratio of 1:4 (v/v) to obtain a final mucin concentration of 2.5 mg/mL. Viscosities were determined for shear rates between dγ/dt = 10s-1 and dγ/dt = 4000s-1 at 25 °C. A solvent trap was installed to avoid dehydration of the samples during the measurement. To compare the degree of shear thinning between the different samples, the measured viscosities were normalized to the values obtained


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for the highest shear rate, and this value obtained at the highest shear rate was set to zero after normalization.

Statistical analysis. To test for statistical differences between conditions, ordinary one-way ANOVA tests were performed among at least three independent data sets by using Graphpad Prism 7.0. ‘*‘, ‘**’, ‘***’, and ‘****’ indicates the p value < 0.05, 0.001, 0.0005 and 0.0001, respectively.

Supporting Information. Supporting information attached with seven (7) figures and (3) tables.

Acknowledgments. The authors would like to thank Prof. Hans Hebert for fruitfull discussions regarding electron microscopy observation of mucins. Also thank you to Zheng Wei for assitance with experimental supervision.

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