Article pubs.acs.org/Biomac
Alterations in Mucus Barrier Function and Matrix Structure Induced by Guluronate Oligomers Catherine Taylor Nordgård,*,† Unni Nonstad,‡ Magnus Ø. Olderøy,§,∥ Terje Espevik,‡ and Kurt I. Draget† Departments of †Biotechnology, ‡Cancer Research and Molecular Medicine, and §Physics, The Norwegian University of Science and Technology, NTNU, NO-7491 Trondheim, Norway
ABSTRACT: The effect of guluronate oligomers on the barrier properties of mucous matrices was investigated in terms of the mobility of nanoparticles in mucous matrices by fluorescence recovery after photobleaching (FRAP), cellular uptake of nanoparticles in mucus secreting cells (HT29-MTX), and mucin matrix architecture by scanning electron microscopy (SEM). Guluronate oligomers improved nanoparticle mobility in both native and highly purified mucus matrices and improved cellular uptake of nanoparticles through a mucus layer. Addition of guluronate oligomers to mucin matrices resulted in a decrease in the density of network cross-links and an increase in matrix pore size. Based on these data, we conclude that guluronate oligomers are able to improve nanoparticle mobility in several mucus matrices and alter network architecture in mucin matrices in a manner that suggests a reduction in barrier function. As such, there may be a potential application for guluronate oligomers in mucosal delivery of nanomedicines.
■
INTRODUCTION Mucus is a highly functional, protective secretion that covers the epithelial surfaces of the gastrointestinal, respiratory, and genitourinary tracts, in addition to the surface of the eye and the middle ear. The protective functions of mucus can broadly be considered as the following: (1) Lubrication: mucus is a rheologically reversible gel, which flows under applied shear and regels as the shear abates, thereby absorbing and attenuating shear stress and protecting the underlying epithelium from mechanical damage.1 (2) Barrier: the mucus matrix acts as a steric and interaction barrier, which prevents the penetration of for example digestive enzymes and pathogens to the mucosal cells.2−4 (3) Clearance: the tenacious nature of mucus allows entrapment of particulate material and its viscoelastic properties allow effective removal by the mucociliary clearance system.5,6 These functions are primarily attributable to the mucin matrix of mucus where polymeric mucin molecules interact through noncovalent interactions to form a dynamic 3D meshwork7 that provides the mucus with its viscoelastic properties and acts as a scaffold for other molecular components. While mucus is highly adapted to its physiological function there are some situations where it may be desirable to be able to modify or control mucus properties, for example, to © 2014 American Chemical Society
improve functional properties in disease states with aberrant mucus properties such as cystic fibrosis,8 to modulate fertility,9 or to overcome barrier functions to enable drug/gene delivery.10,11 With this motivation we have previously investigated the ability of guluronate oligomers to modulate the rheology of sputum from cystic fibrosis patients8 and have further studied this phenomenon at a molecular level.12 While mucins are primarily responsible for both the viscoelastic and the barrier properties of mucus, the length scales relevant to these functions are very different,13 with the barrier properties (microrheological properties) having a functional length scale well below that of the viscoelastic (macrorheological) properties. Given this, it cannot be assumed that altering one will alter the other, as has been clearly demonstrated in several studies.14,15 The complex nature of the mucus barrier has been elegantly highlighted in a recent work that demonstrates the influence of pore size distribution (rather than simply pore size) and matrix rigidity on the barrier functions of mucus.16 Here we present a study investigating the effects of guluronate Received: March 28, 2014 Revised: May 13, 2014 Published: May 14, 2014 2294
dx.doi.org/10.1021/bm500464b | Biomacromolecules 2014, 15, 2294−2300
Biomacromolecules
Article
Figure 1. FRAP curves for 200 nm (A, B) and 100 nm (C, D) carboxylate modified fluospheres in native pig gastric mucus (A, C) and with 4.8 mg/ mL guluronate oligomers added (B, D). weight alginates with a high content of guluronic acid residues from Laminaria hyperborea stalk, as previously reported.20,21 Chemical composition and sequence was determined by 1H NMR spectroscopy22 and revealed that the fractions of guluronate containing monad (FG), diad (FGG), and triad (FGGG) were 0.94, 0.83, and 0.80, respectively. The distribution as well as the number-average degree of polymerization (DPn) of this guluronate oligomer sample used was quantified as described earlier,23 applying a HPAEC-PAD system (Dionex BioLC System, Dionex Conrporation, Sunnyvale, Ca). The chromatographic spectrum revealed an average DPn = 12, with 40% of the molecular population in both the Dp range of less than 10, as well as in the range 10−20. No high molecular weight tail was observed. Fluorescence Recovery after Photobleaching (FRAP). The 100 and 200 nm diameter, carboxylate-modified yellow/green fluospheres were purchased from Invitrogen. These nanoparticles are negatively charged with a zeta potential of −43 ± 1.18 mV. Previously frozen native gastric mucus gels were thawed and 0.32 g aliquots were prepared. To each aliquot was added either 64 μL of 30 mg/mL guluronate oligomers in 0.05 M NaCl or 64 μL of ionic strength matched saline and 16 μL of 2% nanosphere suspension to give samples with and without guluronate oligomers at 4.8 mg/mL containing 0.08 wt % nanospheres (giving 1.8 × 1011 nanospheres/mL for 200 nm nanospheres and 2.7 × 1012 nanospheres/mL for 100 nm nanospheres, as calculated from eq 1).24
oligomers on nanoparticle mobility in mucus using three different mucous matrices.
■
MATERIALS AND METHODS
Mucus Matrices. Pig gastric mucus samples were obtained from the cardia and fundus of recently slaughtered pigs following the previously reported protocol.1,17 The stomachs were rinsed of food debris using cold running tap water and the mucosal surface gently scraped to obtain crude mucus. The purification of the mucins was conducted according to a previously reported protocol.18 The sample was subjected to a brief homogenization (1 min) in 67 mM pH 6.5 phosphate buffer including proteolytic inhibitors (1 mM iodoacetamide, 100 mM aminocaproic acid, 10 mM EDTA, 10 mM Nethylmaleamide, 5 mM benzamidine HC1, and 1 mM phenylmethylsulphonylfluoride) and then centrifuged (8000 g, 4 °C, 1 h) to remove insoluble material. The supernatant was collected and purified by CsCl equilibrium density gradient centrifugation (1.42 g/mL starting density), the mucin-rich fractions (as determined by PAS assay19) were found to have a density between 1.45 and 1.49 g/mL, with DNA (by absorbance at 260/280 nm) appearing in the higher density fractions. The mucin-rich fractions were pooled and exhaustively dialyzed (including dialysis against 0.5 M NaCl to replace any cesium counterions, followed by dialysis against deionized water (resistivity 18.2 MΩ × cm, prepared using a Milli-Q-unit, Millipore)), freezedried, and stored at −20 °C. Guluronate Oligomers. The low molecular weight guluronate oligomer samples were obtained by acid hydrolysis of high molecular
No. of nanospheres/mL = (6C × 1012)/(ρ × π × φ3) 2295
(1)
dx.doi.org/10.1021/bm500464b | Biomacromolecules 2014, 15, 2294−2300
Biomacromolecules
Article
Figure 2. FRAP curves for 200 nm carboxylate modified fluospheres in native pig gastric mucus (A, B) and 20 mg/mL purified pig gastric mucin (C, D), with (B, D) and without (A, C) 4.8 mg/mL guluronate oliomers added. where C = concentration of suspended beads in g/mL (0.02 g/mL for a 2% suspension), φ = diameter of microspheres in μm, and ρ = density of polymer in g/mL (1.05 for polystyrene). These nanosphere concentrations are within the range where a linear relationship exists between concentration and fluorescence.25 Purified mucins were hydrated overnight in 0.05 M NaCl and mixed with guluronate oligomer solution or ionic strength matched saline and nanosphere suspension to give final concentrations 20 mg/mL mucin ± 4.8 mg/mL guluronate oligomers + 0.08 wt % nanospheres (as above). The ionic strength of the guluronate oligomer solution was calculated by considering the monomer weight of sodium guluronate as 200 Da; therefore, 30 mg/mL is equivalent to 15 mM guluronate monomer. Approximately half of these ions will exist in free form (Manning condensation); thus, the guluronate oligomers will contribute an extra 7.5 mM to the ionic strength of the solution. Samples (∼180 μL) were loaded into adhesive imaging chambers (Invitrogen) mounted on microscope slides and allowed to rest overnight at 4 °C to allow for recovery of gel structure. Before imaging samples were allowed to equilibrate to room temperature for 1 h. FRAP experiments were performed on a Zeiss LSM 510 scanning confocal microscope using the 488 nm line of an argon laser and a water immersion objective (×40). Prebleach fluorescence in a defined region of the sample was monitored at 80% laser power/2% transmission, followed by photobleaching of a circular spot at 80% laser power/100% transmission, and fluorescence recovery was monitored as a function of time at 80% laser power/2% transmission.
Data sets in which the focal plane shifted were discarded by visual inspection of image sets. Cellular Uptake. HT29-MTX cells,26 a human colon carcinoma derived, mucin-secreting goblet cell line, were received as a gift from Prof. J. P. Pearson, Newcastle University, U.K. Cells were grown in Dulbecco’s modified Eagles medium (DMEM, Lonza) with 10% fetal calf serum (Gibco), 2 mM L-glutamine (Sigma), and 10 μg/mL ciproflaxin (Cellgro/Mediatech). To enable the production of distinct mucus enhanced and mucus depleted cell populations, cells were grown in 12-well plates (seeding density ∼ 10000 cells/well) either under a filter (Transwell, 12 mm diameter, polycarbonate membrane, pore size 3 μm) and medium changes undertaken through the filter (medium above filter removed, new medium applied), to allow the establishment of an undisturbed layer adjacent to the cells and prevent mechanical removal of the secreted mucins (mucus enhanced), or cells were grown as normal, and cellular secretions were removed with each medium change (mucus depleted). In both cases, uptake studies were performed on day 17 and medium was changed twice weekly. Uptake studies were performed as follows with filters remaining in place for mucus enhanced cells. Medium was removed from wells, as above, and replaced with 750 μL of fresh medium and 250 μL of guluronate oligomer solution (20 mg/mL in physiological saline) or 250 μL of physiological saline. A total of 40 μL of 0.2 wt % suspension of fluospheres (200 nm diameter carboxylate modified yellow/green fluorescent, Invitrogen) was added to wells. Plates were incubated for 1 h. After incubation, medium was removed, cells were washed twice 2296
dx.doi.org/10.1021/bm500464b | Biomacromolecules 2014, 15, 2294−2300
Biomacromolecules
Article
Figure 3. Effect of guluronate oligomers on uptake of 200 nm carboxylate-modified fluospheres in mucus-enhanced and mucus-depleted HT29MTX cells showing autofluorescence in red (A), uptake without guluronate oligomer mucus-depleted cells (grown without filter) (B), uptake with guluronate oligomer mucus-depleted cells (grown without filter) (C), uptake without guluronate oligomer mucus-enhanced cells (grown under filter) (D), uptake with guluronate oligomers mucus enhanced cells (grown under filter) (E), uptake without guluronate oligomer mucus-depleted cells, plus filter (grown without filter, filter added before fluospheres) (F). with cold PBS, and cells were suspended with 0.02% EDTA in PBS (incubate 5 min), transferred to centrifuge tubes, centrifuged (8 min/ 1500 rpm), resuspended in cold PBS, and centrifuged as above (twice), resuspended, and uptake was quantified by flow cytometry using a Beckman−Coulter Flow cytometer model EPICS XL-MCL. Scanning Electron Microscopy (SEM). Mucins were rehydrated in 0.05 M NaCl at a concentration of 20 mg/mL (which produced a material with elastic dominant gel like properties) with and without guluronate oligomers at 4.8 mg/mL as above. A drop of the resultant mucin gel was placed on a coverslip, and the water in the matrix exchanged for acetone by sequential submersion in 50, 90, and 100% acetone. The samples were critical point dried (Emitech K850) and sputter coated (80% Pt, 20% Pd) before imaging in the scanning electron microscope. Imaging was done using an accelerating voltage of 5 kV and the secondary electron detector on a Zeiss Ultra 55 LE microscope.
suggest the latter is the most probable scenario as it is counterintuitive that smaller beads will be more sterically hindered and the smaller size of the 100 nm beads will allow greater access to matrix sites and thereby greater possibilities for interactions and functional immobilization. This is also compatible with published data, suggesting that beads in this size range are unlikely to be immobilized on steric grounds alone within such a mucus matrix14,29,30 and that such negatively charged nanoparticles have significantly poorer mobility than size matched PEGylated (nonmuocadhesive) particles in mucus.31 It could also be considered that the mucus matrix is functioning in a similar manner to a gel filtration matrix, whereby smaller entities have access to a large volume of the matrix and therefore have retarded mobility when compared to larger entities, but in this case one would expect a slower fluorescence recovery for the smaller nanoparticles but not a reduction in mobile fraction when compared to the larger nanoparticles. In fact, these data indicate the fluorescence recovery is somewhat faster for the smaller nanoparticles, while the mobile fraction is smaller, again supporting matrix− nanoparticle interactions as the dominant retardation mechanism rather than steric effects. Comparing the recovery curves for 200 nm nanospheres in native pig gastric mucus and purified pig gastric mucins there is a higher baseline recovery in the mucin matrix (∼20%) as compared to the mucus (∼10%) (Figure 2A,C). This may reflect either greater nanosphere mobility within the matrix, most likely as a result of reduced steric hindrance since the matrix has fewer macromolecular components, or possibly greater mobility of matrix components (mucins) within the matrix itself leading to a higher mobility of mucin bound nanospheres. In the purified mucin system the addition of guluronate oligomers gives rise to an increase in the mobile fraction, from ∼25 to ∼50% (Figure 2D). This improvement is less than is seen for the native mucus (Figure 2B). The most likely explanation for this is that, since the purification procedure removes nonmucin components, it exposes previously inaccessible interaction sites on the mucins, thus, increasing the density of potential nanosphere entrapment sites, in a similar manner to the 100 nm nanospheres having access to a greater number of interaction sites than the 200 nm nanospheres in native mucus.
■
RESULTS AND DISCUSSION Nanoparticle Diffusion in Mucus. Fluorescence recovery after photobleaching (FRAP) was used to investigate the mobility of negatively charged carboxylate nanoparticles in mucus. The recovery curves for 200 and 100 nm nanospheres in native pig gastric mucus show minimal fluorescence recovery after bleaching (Figure 1A,C). This indicates that these particles are essentially immobile or undergo long time scale transient binding events within the matrix.27 With the addition of guluronate oligomers there is a substantial increase in mobility with the mobile fraction comprising ∼70% and ∼40% for 200 and 100 nm beads respectively (Figure 1 B,D). The nature of the recovery curve, most notably for the 200 nm beads, does not fit a simple one component freely diffusing model but rather indicates multiple populations with differing diffusion rates are present resulting either from short transient binding events 27 or the presence of distinct particle populations.28 Given the monodisperse nature of the nanospheres, the former would appear to be the most likely explanation, but we cannot preclude the possibility that surface accumulation of mucus components may generate distinct mobile bead populations. The immobile fraction may reflect nanospheres that are sterically trapped within pores in the matrix or nanospheres that are bound to the matrix mucins by particularly long time scale interactions or a combination of the two. The higher immobile fraction of the smaller beads would 2297
dx.doi.org/10.1021/bm500464b | Biomacromolecules 2014, 15, 2294−2300
Biomacromolecules
Article
purification procedures. As such, it provides a good complement to the data from the native pig gastric mucus and the purified pig gastric mucin matrices utilized in the FRAP experiments, demonstrating that the effect is independent of the mucous matrix studied and is not related to artifacts or peculiarities of a specific mucous matrix. This is of particular importance when studying mucous systems, as mucus secretions and mucin preparations can vary significantly in their functional properties, for example their viscoelastic and barrier properties, and findings based on one mucous system are not necessarily reproducible in other systems.33,34 The data from these independent systems allow us to conclude with a reasonable degree of certainty that the phenomenon of guluronate oligomer enhanced mucopenetration is both relevant to the in vivo situation, in that it is seen in ex vivo mucus and cellular secretions, and related to the mucin component of mucus, in that it is also seen in purified mucin matrices. This correlates well with previously published data examining the effects of guluronate oligomers on the viscoelastic properties of mucous systems, which again showed that the effects were seen in both native and purified systems.8 Structural Alterations in the Mucin Matrix Induced by Guluronate Oligomers. The mucus barrier has a steric and an interactive component. The interactive component is directly linked to the interaction potential of mucins (and possibly other mucus components), while the steric component is indirectly linked to the interaction potential of mucins (since it is mucin−mucin interactions that form the cross-links of the matrix, and thus, lowering the interaction potential of mucins leads to a lower cross-link density and larger network pores). It has been shown that guluronate oligomers are able to reduce the interactive potential of mucins, at least toward alginate molecules.12 The molecular basis of mucin−mucin interactions is diverse and includes electrostatic interactions, hydrophobic interactions, hydrogen bonding, and van der Waals forces,7 and interfering with any of these interaction types has the potential to alter the barrier properties of mucus. It is also important to note that barrier properties may be indirectly affected by altering intramolecular interactions within the mucin molecules if this then alters the accessibility of interaction sites. Nevertheless, the multiplicity of interaction types leads to a rather robust gel that is able to withstand the various disruptive agents, such as bile salts, low pH, and surfactants it is exposed to in vivo.2,35−37 The polyelectrolyte nature of guluronate oligomers would suggest they inhibit electrostatic interactions,8 however, the alteration in barrier properties could simply be due to inhibition of interactions between matrix mucins and the mobile component or may additionally involve inhibition of matrix cross-links and thereby a reduction in the steric barrier. Rheological data is compatible with a guluronate oligomerinduced reduction in network cross-link density;8 however, the latter is an indirect measurement and cannot be taken as evidence for an altered network structure. For this reason, scanning electron microscopy was used to directly investigate mucin network structure in the absence and presence of guluronate oligomers (Figure 4A,B). The images obtained are similar to those that have previously been published for cervical mucus.9 It should be noted that the scanning electron microscopy data cannot be used to directly quantify the pore size of the mucin matrix, as the sample preparation is likely to affect the observed structures. However, a qualitative comparison of the samples can be made, which clearly shows that the presence of guluronate oligomers alters the matrix
The complex nature of the recovery curves, which indicate multiple diffusing populations,27 preclude simple quantitative estimates of diffusion coefficients based on the half-life of the recovery,32 however, qualitatively, these data clearly indicate that the addition of guluronate oligomers reduce the barrier to diffusion of nanospheres to a considerable degree. While Kirch et al.16 have concluded that matrix rigidity is relevant to mucus barrier function and rheological studies have shown that guluronate oligomer treated mucous matrices are less rigid (they are less resistant to applied deformation),8 it is unlikely that alterations in matrix rigidity are central to the changes seen here. First, the FRAP studies are passive, involving no deformation applied to the matrix, which will limit the impact of rigidity (or deformability) on the results. Second, if changes in rigidity and resultant molecular rearrangement were responsible for the increase in particle mobility one would expect the results to be somewhat independent of nanoparticle size. Given that the results here vary considerably with nanoparticle size then it is reasonable to assume that a change in matrix rigidity is not the dominant mechanism underlying these results. It can therefore be considered that the guluronate oligomers alter either the interactions between the nanoparticles and the mucus matrix, such as they have been shown to inhibit binding of negatively charged polymers to mucins by AFM,12 or the structure of the matrix and, thus, the steric barrier, or both. Cellular Uptake of Nanospheres in Mucus Enhanced and Mucus Depleted Cells. While both mucus enhanced (Figure 3D) and mucus depleted cells (Figure 3B) showed uptake of 200 nm nanospheres compared to cell auto fluorescence (Figure 3A), the presence of an unstirred mucin containing layer adjacent to the cells (mucus enhanced cells) led to a substantial decrease in uptake compared to mucus depleted cells, with the fluorescence intensity shift relative to cell autofluorescence being substantially reduced. Addition of guluronate oligomers concurrently with the fluorescent nanospheres brought about an increase in nanosphere uptake for the mucus enhanced cells (Figure 3E; as seen by the increased shift in fluorescence intensity compared to cell autofluorescence) but no changes in uptake for the mucus depleted cells (Figure 3C). This indicates that the site of action of the guluronate oligomers is the extracellular mucus barrier rather than the cell membrane of the HT29-MTX cells. Independent investigations showed that the presence of a filter over mucus depleted cells did not impede nanosphere uptake (Figure 3F), and this is also strongly supported by the data showing a level of uptake for the guluronate oligomer treated mucus enhanced cells approaching that of the mucus depleted cells (Figure 3B,E). It should be noted that flow cytometry will not distinguish between nanoparticles with have been internalized by the cell and any nanoparticles which may have remained adherent to the cell surface despite the washing procedure. However, even in the event of nanoparticles adhering to the cell surface, the increase in fluorescence intensity comes about as a result of nanoparticles that have traversed the mucus/unstirred layer adjacent to the cells and, as such, can be used to consider the barrier modifying effects of guluronate oligomers. These data support the FRAP data, indicating that guluronate oligomers increase the mobility of nanospheres in mucus, allowing access to the cell surface. The mucus matrix in these experiments is a sterile cellular secretion, which, while it is likely to contain cellular debris, as would be the case in vivo, contains no luminal debris and has not been subject to 2298
dx.doi.org/10.1021/bm500464b | Biomacromolecules 2014, 15, 2294−2300
Biomacromolecules
Article
reflects the in vivo situation. It has also been shown that guluronate oligomers alter mucin matrix architecture and thereby the steric barrier component of mucous matrices. This gives important insights into the relevance of intermucin interactions to the architecture of the mucus barrier in vivo. The ability of guluronate oligomers to modify the barrier properties of mucus may make them an interesting proposition within the field of mucosal drug delivery, particularly for nanomedicines where mucus can provide a significant barrier to effective delivery.
■
AUTHOR INFORMATION
Corresponding Author
*Fax: +47 735 91283. Tel.: +47 735 94069. E-mail: catherine.t.
[email protected]. Present Address ∥
The Norwegian Center for Stem Cell Research, Oslo University Hospital, Oslo, Norway (M.Ø.O.). Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work is supported by Innovation Norway, Sør Trøndelag County Pre-Seed fund and the Research Council of Norway FORNY fund. We gratefully acknowledge Astrid Vik Bjørkøy of the Norwegian Molecular Imaging Consortium (NorMIC) at the Department of Physics, NTNU, for assistance with the FRAP studies.
■
REFERENCES
(1) Taylor, C.; Draget, K. I.; Pearson, J. P.; Smidsrod, O. Biomacromolecules 2005, 6, 1524−1530. (2) Allen, A.; Flemstrom, G. Am. J. Physiol.: Cell Physiol. 2005, 288, C1−C19. (3) Perez-Vilar, J. Gastrointest. Mucus Gel Barrier 2009, 21−48. (4) Swidsinski, A.; Sydora, B. C.; Doerffel, Y.; Loening-Baucke, V.; Vaneechoutte, M.; Lupicki, M.; Scholze, J.; Lochs, H.; Dieleman, L. A. Inflamm. Bowel Dis. 2007, 13, 963−970. (5) Randell, S. H.; Boucher, R. C.; Univ, N. C. V. L. G. Am. J. Respir. Cell Mol. Biol. 2006, 35, 20−28. (6) Wanner, A.; Salathe, M.; Oriordan, T. G. Am. J. Respir. Crit. Care Med. 1996, 154, 1868−1902. (7) Taylor, C.; Allen, A.; Dettmar, P. W.; Pearson, J. P. Biomacromolecules 2003, 4, 922−927. (8) Nordgard, C. T.; Draget, K. I. Biomacromolecules 2011, 12, 3084− 3090. (9) Willits, R. K.; Saltzman, W. M. Biomaterials 2001, 22, 445−452. (10) Cone, R. A. Adv. Drug Delivery Rev. 2009, 61, 75−85. (11) Lai, S. K.; Wang, Y. Y.; Hanes, J. Adv. Drug Delivery Rev. 2009, 61, 158−171. (12) Sletmoen, M.; Maurstad, G.; Nordgard, C. T.; Draget, K. I.; Stokke, B. T. Soft Matter 2012, 8, 8413−8421. (13) Lai, S. K.; Wang, Y. Y.; Wirtz, D.; Hanes, J. Adv. Drug Delivery Rev. 2009, 61, 86−100. (14) Dawson, M.; Wirtz, D.; Hanes, J. J. Biol. Chem. 2003, 278, 50393−50401. (15) Lai, S. K.; Wang, Y. Y.; Cone, R.; Wirtz, D.; Hanes, J. Plos One 2009, 4, e4294. (16) Kirch, J.; Schneider, A.; Abou, B.; Hopf, A.; Schaefer, U. F.; Schneider, M.; Schall, C.; Wagner, C.; Lehr, C.-M. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 18355−18360. (17) Taylor, C.; Pearson, J. P.; Draget, K. I.; Dettmar, P. W.; Smidsrod, O. Carbohydr. Polym. 2005, 59, 189−195. (18) Fogg, F. J. J.; Hutton, D. A.; Jumel, K.; Pearson, J. P.; Harding, S. E.; Allen, A. Biochem. J. 1996, 316, 937−942.
Figure 4. Scanning electron microscopy of 20 mg/mL purified pig gastric mucin with (B) and without (A) 4.8 mg/mL guluronate oligomers added.
structure. The addition of guluronate oligomers leads to a substantial increase in the average pore size within the mucin matrix, confirming that guluronate oligomers affect the steric components of the mucus barrier. While even in the guluronate-free mucin matrix, the pore size is well in excess of the 200 nm diameter of the nanospheres (Figure 4A), there is still a substantial steric component to the barrier since the matrix fibers (mucins) have such a high interaction potential they are essentially “sticky” to traversing particles, so the higher the density of network cross-links, the more statistically likely it is that a particle will become entrapped through interactions with mucins. This is compatible with the FRAP data analysis presented here, which would indicate that the interactive component is the dominant element of the mucus barrier, something that is in agreement with other published data.11 Additionally, AFM experiments have demonstrated the ability of guluronate oligomers to reduce the interaction potential of mucins,12 which also supports this interpretation.
■
CONCLUSIONS The data presented here demonstrate that guluronate oligomers are able to alter the barrier properties of mucous matrices, that this effect is linked to the mucin component of mucus, and that it is relevant to native mucus matrices. This is particularly important since purification procedures may significantly alter the behavior of mucin-based systems and potentially induce artifacts so that the material no longer 2299
dx.doi.org/10.1021/bm500464b | Biomacromolecules 2014, 15, 2294−2300
Biomacromolecules
Article
(19) Mantle, M.; Allen, A. Biochem. Soc. Trans. 1978, 6, 607−609. (20) Haug, A.; Larsen, B.; Smidsrod, O. Acta Chem. Scand. 1966, 20, 183−&. (21) Haug, A.; Larsen, B.; Smidsrod, O. Acta Chem. Scand. 1967, 21, 691−&. (22) Grasdalen, H. Carbohydr. Res. 1983, 118, 255−260. (23) Gimmestad, M.; Ertesvag, H.; Heggeset, T. M. B.; Aarstad, O.; Svanem, B. I. G.; Valla, S. J. Bacteriol. 2009, 191, 4845−4853. (24) Invitrogen Product Information: FluoSpheres, Fluorescent Microspheres. Revised 25 October 2005. (25) Peeters, L.; Sanders, N. N.; Braeckmans, K.; Boussery, K.; de Voorde, J. V.; De Smedt, S. C.; Demeester, J. Invest. Ophthalmol. Visual Sci. 2005, 46, 3553−3561. (26) Smirnova, M. G.; Guo, L.; Birchall, J. P.; Pearson, J. P. Cell. Immunol. 2003, 221, 42−49. (27) Phair, R. D.; Misteli, T. Nat. Rev. Mol. Cell Biol. 2001, 2, 898− 907. (28) Lippincott-Schwartz, J.; Altan-Bonnet, N.; Patterson, G. H. Nat. Rev. Mol. Cell Biol. 2003, S7−S14. (29) Lai, S. K.; O’Hanlon, D. E.; Harrold, S.; Man, S. T.; Wang, Y. Y.; Cone, R.; Hanes, J. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 1482−1487. (30) Lai, S. K.; Wang, Y. Y.; Hida, K.; Cone, R.; Hanes, J. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 598−603. (31) Yang, X. Y.; Forier, K.; Steukers, L.; Van Vlierberghe, S.; Dubruel, P.; Braeckmans, K.; Glorieux, S.; Nauwynck, H. J. Plos One 2012, 7, e51054. (32) Axelrod, D.; Koppel, D. E.; Schlessinger, J.; Elson, E.; Webb, W. W. Biophys. J. 1976, 16, 1055−1069. (33) Crater, J. S.; Carrier, R. L. Macromol. Biosci. 2010, 10, 1473− 1483. (34) KocevarNared, J.; Kristl, J.; SmidKorbar, J. Biomaterials 1997, 18, 677−681. (35) Bansil, R.; Turner, B. S. Curr. Opin. Colloid Interface Sci. 2006, 11, 164−170. (36) Bernhard, W.; Postle, A. D.; Rau, G. A.; Freihorst, J. Comp. Biochem. Physiol., Part A: Mol. Integr. Physiol. 2001, 129, 173−182. (37) Macierzanka, A.; Rigby, N. M.; Corfield, A. P.; Wellner, N.; Bottger, F.; Mills, E. N. C.; Mackie, A. R. Soft Matter 2011, 7, 8077− 8084.
2300
dx.doi.org/10.1021/bm500464b | Biomacromolecules 2014, 15, 2294−2300
