Hyaluronic Acid Molecular Weight-Dependent Modulation of Mucin

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Hyaluronic Acid Molecular Weight-Dependent Modulation of Mucin Nanostructure for Potential Mucosal Therapeutic Applications Irene Maria Hansen, Morten Frendø Ebbesen, Liselotte Kaspersen, Troels Thomsen, Konrad Bienk, Yunpeng Cai, Birgitte Mølholm Malle, and Kenneth Alan Howard Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 12 May 2017 Downloaded from http://pubs.acs.org on May 14, 2017

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Molecular Pharmaceutics

Hyaluronic Acid Molecular Weight-Dependent Modulation of Mucin Nanostructure for Potential Mucosal Therapeutic Applications Irene Maria Hansen1†, Morten Frendø Ebbesen1†, Liselotte Kaspersen1, Troels Thomsen1, Konrad Bienk1, Yunpeng Cai1, Birgitte Mølholm Malle2 and Kenneth Alan Howard1* 1

Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics,

Aarhus University, 8000 Aarhus C, Denmark.

2

Novozymes Biopharma, Novozymes A/S,

Brudelysvej 32, 2880 Bagsværd, Denmark †These authors contributed equally to this work. *

Corresponding author at: Interdisciplinary Nanoscience Center (iNANO), 8000 Aarhus C,

Denmark. E-mail address: [email protected] (K.A. Howard). TOC:

ACS Paragon Plus Environment


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ABSTRACT This study investigates the effects of different molecular weight hyaluronic acid (HA) on the mucosal nanostructure using a pig stomach mucin hydrogel as a mucosal barrier model. Micro- (1.0 µm) and nanoparticles (200 nm) were used as probes and their movement in mucin was studied by a 3D confocal microscopy-based particle tracking technique and by nanoparticle tracking analysis (NTA) after addition of high (900 kDa) and low (33 kDa) molecular weight HA. This demonstrated a molecular weight-dependent HA modulation of the mucin nanostructure with a 2.5-fold mobility reduction of 200 nm nanoparticles. To further investigate these mechanisms and to verify that the natural viscoelastic properties of mucus is not undesirably altered, rheological measurements were performed on mucin hydrogels with or without HA. This suggested the observed particle mobility restriction not to be attributed to alterations of the natural mucin cohesive and viscoelastic properties but, instead, indicate that the added high molecular weight HA primarily modulates the mucin nanostructure and mesh size. This study, hereby, demonstrates how mucus nanostructure can be modulated by the addition of high molecular HA that offers an opportunity to control mucosal pathogenesis and drug delivery.

Keywords: Hyaluronic Acid, Mucin, Mucus, Nanoparticles, Drug Delivery


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INTRODUCTION The mucosal barrier lining the respiratory, gastrointestinal and genitourinary tracts provides protection against environmental and pathogenic particulates such as viruses and bacteria, but conversely limits the therapeutic potential of particle-based drug delivery.1 The mucus layer is a viscoelastic gel that contains mucin glycoproteins forming a heterogeneous network of fibers, through which, micro- and nanoparticles need to diffuse in order to reach the underlying epithelial cells.2 Mucin polymers comprised of disulfide linked monomers contain repeated hydrophilic glycosylated amino acid sequences and hydrophobic cysteine-rich domains that can interact by electrostatic, hydrogen and hydrophobic interactions with particles,3 bacteria4 and viruses in a sizedependent manner.5 The mucin mesh pore size varies according to the bulk elasticity and region, it is, for example, ~340 nm in human cervical mucus and 400-650 nm in purified gastric mucin.6 The conformational state of the mucin fibers, that determines both pore size and binding domain availability, is pH-dependent and supports a structure-function relationship at specific anatomical sites.6,

7

Mucin conformation, therefore, need to be addressed in order to understand mucosal

pathogenesis and optimize particle mucosal drug delivery. Pathogenic mechanisms have evolved to circumvent the mucus barrier and allow contact with the epithelial cells. These include pathogenic adhesion to mucin followed by degradation of mucin fibers,8 reduced adhesion to mucin fibers,5 or by directed bacterial movement by chemotaxis.9 These mucoadhesive10 and penetrative2 approaches have been adopted as strategies for particle-based drug delivery using mucin-interacting polymers such as chitosan11,

12

, stealth poly(ethylene glycol)

(PEG)-ylated coatings13, 14 or poly(2-ethyl-2-oxazoline).15 Altering the mucin fibrous nanostructure is likewise an attractive strategy for controlling pathogenic migration or improved particle drug delivery. Wang et al.16 reported an increased mucin pore size



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by bundling of mucin fibers mediated by mucoadhesive nanoparticles, and the mucolytic agent Nacetylcysteine has also been reported to enhance particle uptake across mucus, by disruption of the mucus barrier after cleavage of disulfide bonds.17 Here, we investigate a novel strategy to reduce penetration of pathogens or prolong the residence time of drug delivery particles by addition of hyaluronic acid (HA) to alter the nanostructure of the mucin hydrogel by polymer-mucin entanglement with consequent pore size reduction to control the movement of particulates. Hyaluronic acid is a natural linear glycosaminoglycan composed of alternating D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine (GlcNAc) disaccharide units18 widely distributed in the extracellular matrix of the connective tissue, synovial fluid and vitreous body. HA coated planar or particle surfaces have been shown to exhibit mucoadhesive properties when applied to mucosal epithelium or onto mucus gels as demonstrated by tensile stress, rheologic or rotating cylinder methods19-21 or by mucociliary transport studies across mucosal tissue.22, 23 These properties are attributed to the presence of carboxyl and hydroxyl groups that can hydrogen bond with glycosyl groups on the mucin polymers;23 interactions, facilitated by the flexible conformation of HA.18 Other relevant parameters for the mucus pore size and particulate movement are the cohesive interactions between mucin fibers that control the interlinked fibrous nanostructure and forms part of the basis for the natural viscoelastic properties of mucus.16 Despite many investigations on HA mucoadhesiveness, the impact of pure HA polymer on bulk mucus natural cohesive and viscoelastic properties has, to the best of our knowledge, not been evaluated directly and the effects of free HA polymer within the mucin nanostructure remain unclear. In this work, the effect of free HA polymers of 33 or 900 kDa on the mucin nanostructure was evaluated by two in vitro methods both based on the diffusion of micro- and nanoparticles in mucin hydrogels; 1) particle tracking analysis to determine the particle diffusion coefficient within a HAmucin hydrogel (Figure 1, A) and 2) a confocal-based technique to analyze the transit of particles


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across HA-mucin hydrogels (Figure 1, C). The aim was to investigate if addition of free HA polymers would modulate the capability for micro- and nanoparticles to diffuse within the hydrogels, thereby revealing if addition of free HA could alter the nanostructure of mucin hydrogels (Figure 1, B). Carboxyl-modified polystyrene model particles of size 200 nm and 1.0 µm were selected to investigate appropriate size scales associated with viruses and bacteria respectively, in order to evaluate the potential application of HA to control pathogenic movement across mucus. This work identifies the selective ability of high molecular weight HA (900 kDa) modulate the mucin nanostructure and inhibit particle movement across a mucin hydrogel that presents a potential new therapeutic strategy to reduce pathogen invasion and increase the residence and consequent local drug release of particle-based drug delivery systems at the mucosal-epithelial interface without interfering with the natural mucus viscoelastic properties.

Figure 1. Experimental particle tracking methods. A) Diffusion analysis using Nanoparticle Tracking Analysis (NTA). The particle suspension in the mucin/HA matrix is laser illuminated and the point scattering and horizontal positions of the



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particles are recorded for calculation of the particle diffusion coefficient. (B) The tracking methods are used here for investigating the nanostructure of the mucin and HA mesh and any possible interactions between the mesh and particles. (C) Particle tracking using confocal laser scanning microscopy (CLSM) performed in small wells, in which porcine stomach mucin is placed in the bottom. The particle migration through the hydrogel is visualized in 3D by rapid confocal imaging of the well volume and the particle depth distribution in the mucin hydrogel presented as histograms at selected time points.

EXPERIMENTAL SECTION Materials Porcine stomach mucin (Type III) (M1778) was purchased from Sigma-Aldrich. Hyaluronic acid with a mass average molecular weight of 33 and 900 kDa were supplied by Novozymes Biopharma DK A/S (Denmark). The experimental sample of 33 kDa was prepared by well-controlled hydrolysis of 900 kDa HA.24 Weight averaged molar mass (Mw) and the molecular mass distribution (Mw/Mn) were determined by size-exclusion chromatography with online multiangle laser light scattering and refractive index detection (SEC-MALLS-RI). Mw/Mn for HA ranged from 1.3 to 1.7 for the 33 kDa and 1.4 for the 900 kDa. Simulated Intestinal Fluid (SIF) without pancreatin was prepared according to the International Pharmacopedia vol. 5, 3rd edition. 200 nm and 1.0 µm yellow-green

fluorescent

(505/515)

and

carboxyl-modified

polystyrene

microspheres

(FluoSpheres®, F8811 and F8823) were purchased from Invitrogen, Oregon, USA and here termed 200 nm and 1.0 µm PS-COOH. For particle tracking analysis, µ-slides Angiogenesis (81506, ibidi, Germany) were used.

Physicochemical characterization of particles The particle surface charge was measured with a Zetasizer Nano ZS (Malvern, UK) after dilution 1:500 in SIF at 25 °C and ~4 min sonication. The hydrodynamic diameter of the particles was measured by nanoparticle tracking analysis (NTA software version 2.3) of 60 s particle trajectories


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recorded using a NanoSight LM10-HS (NanoSight Limited, Amesbury, UK). For this, particles were diluted in SIF to an appropriate concentration of 2.5·108 particles/mL, sonicated ~4 min prior to use, and measured in triplicates. Data was presented as mean of the modes of size distributions with standard deviations.

Studies on the influence of hyaluronic acid on particle mobility in mucin hydrogels The influence of HA on particle mobility in porcine stomach mucin was investigated using two complimentary methods for tracking particles within the mucin hydrogels. Nanoparticle Tracking Analysis (NTA) (Figure 1, A-B) and 3D tracking of particles using confocal laser scanning microscopy (CLSM) (Figure 1, C).

NTA diffusion studies after addition of free 33 kDa hyaluronic acid and 900 kDa hyaluronic acid to a porcine stomach mucin hydrogel 200 nm PS-COOH particles were diluted 1:100 in SIF pH 7.5, and sonicated for ~4 min to disperse potential particle flocculates and the suspension was analyzed by NTA prior to each experiment to ensure a uniform size and no particle aggregation. Prior to each experiment a stock of mucin hydrogel was prepared by dissolving porcine stomach mucin in SIF buffer by gentle stirring for 4 hours at 4 degrees. Samples were prepared by mixing mucin and HA to a final mucin concentration of 10 mg mL-1 containing 0, 2.5, 7.5 and 12.5% w/w HA (33 kDa or 900 kDa) in a final volume of 400 µl. Two µl of particles were added to all solutions giving a final particle dilution of 1:20,000. Samples were incubated at 37 °C for 2 h and cooled to room temperature prior to the experimental procedure. 60 s videos were recorded in triplicates for nanoparticle tracking analysis using NanoSight LM10HS fitted with a 405 nm-laser. Pentaplicates of the particle diffusion coefficient was measured using



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the NTA software and used for evaluating the influence of HA on the diffusion of particles in the mucin hydrogel. The presented Dm/Dm+HA ratio denotes the particle diffusion coefficient in pure mucin divided by that for mucin and HA mixtures and was calculated from the mean of the mode of diffusion for each measurement, and presented with the corresponding (added) standard deviation of the means. Statistical analysis was performed by two-tailed Student’s t-test with unequal variance.

Particle tracking using CLSM after addition of free 900 kDa hyaluronic acid to a porcine stomach mucin hydrogel The particle tracking method is based on the CLSM 3D-visualization of fluorescent particles (200 nm and 1.0 µm) translocation through a porcine stomach mucin layer in small µ-slide wells (Figure 1, C). The 200 nm PS-COOH and 1.0 µm PS-COOH particles were diluted 1:1000 in SIF and sonicated for ~4 min prior to use. Stocks of porcine stomach mucin (33.3 mg mL-1) and 900 kDa HA (10.0 mg mL-1) were prepared in SIF. Samples with a final concentration of 20 mg mL-1 mucin and a 0% and 10% weight ratio (w/w) of 900 kDa HA were mixed and incubated 2 h at 37 °C just before the experiment. 20 µL of mucin hydrogel was placed in µ-slide microscopy wells of an approximate height of 800 µm and fluorescently labelled particles (in a volume of 5 µl) loaded gently on top. The translocation of 200 nm and 1.0 µm PS-COOH particles across the mucin layer with or without 900 kDa HA was followed over time and captured by CLSM using a LSM700 confocal microscope (Zeiss, Switzerland) with a 488 nm laser for capturing particle positions inside the mucin layer. The wellplate was placed under the microscope with 10x magnification and presets found and installed prior to particle loading (gain and pinhole for the individual particle types). Focus was adjusted by focusing on a mark on the well bottom and used as the lower limit in the recorded stack of images


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each covering an area of 804x600x600 µm with a vertical spacing of 12 µm. Each well was then automatically imaged consecutively with 30 min intervals for 9 h with the capture of each individual well taking ~1.5 min. The particle number for each captured image was extracted by ImageJ ver. 1.47 (NIH, USA) at 8 bits resolution and with intensity threshold of 60-255. The particle numbers in each image were normalized to the total number of particles counted in one well and averages of triplicates plotted for visualizing the particle distribution throughout the mucin layer. Additionally, the numberweighted mean particle count at each well depth was calculated and plotted for further quantifying the particle penetration effects in the presence and absence of added HA. Multiple t tests on the particle distribution data were performed using GraphPad Prism version 6.01 for Windows (La Jolla, USA) under the assumption of similar standard deviations and with correction for multiple comparisons (Holm-Šídák).

Rheological measurements on concentrated mucin-hyaluronic acid hydrogels Porcine stomach mucin was dissolved at a concentration of 100 mg mL-1 in SIF pH 7.5, under continuous mixing for 4 h at room temperature. Samples of 4.5 mL were distributed in individual tubes and HA powder of 33 kDa or 900 kDa was added in 10% ratio (w/w) to mucin and mixed gently overnight at 4 °C. Mucin without added HA was used as control. Samples containing 33 kDa HA and 900 kDa HA alone were prepared by dissolving HA in SIF pH 7.5 to a concentration of 10 mg mL-1. All samples were prepared in triplicates and any air bubbles formed in the samples during mixing was removed by mild sonication prior to rheological measurements. Rheological measurements were performed using a MCR501 rheometer (Anton Paar GmbH) with a concentric cylinder (CC DG26.7) at 37 °C and instrument control and data collection was carried out using



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Rheoplus V3.21. Measurements were initiated by 45 min rest of the sample, ensuring a temperature of 37°C throughout the entire sample and relaxation of the hydrogel components. Initial amplitude sweeps were performed to assure that subsequent measurements were carried out within the linear viscoelastic (LVE) region. Frequency sweeps in the angular frequency range 0.01-500 rad/s with 0.2% deformation were then performed with 6 measuring points per angular frequency decade and the duration of each time point following a logarithmic scale from 200-1 s, resulting in determination of the elastic and loss modulus (G’ and G’’) as a function of angular frequency (ω).

RESULTS AND DISCUSSION This work demonstrates the mucin nanostructure-modulating effect of HA probed via tracking the movement of micro- and nanoparticles through a porcine stomach mucin hydrogel using a confocal microscopy-based- and particle tracking analysis techniques.

Hyaluronic acid-mediated nanoparticle diffusion restriction in hyaluronic acid-mucin hydrogels Particle movement within, and diffusion across, mucus barriers has been utilized for exploring the mucin nanostructure and interactions between particles and mucus.25, 26 There has, however, been no reports of the effects on the mucus barrier or the nanostructure of mucin hydrogels with HA in its free form as addressed in this work. The ability of particles to cross mucus membranes has been studied by side-on-three compartment diffusion,27 diffusion chambers,28 modified Franz diffusion cells26 and modified Transwell-Snapwell® diffusion chambers29. Li et al. developed a micro fluidic system to investigate the diffusion of ions, peptides or nanoparticles into a mucin hydrogel in vitro.30,

31

The study of particle tracking within mucus has, for example, been evaluated using

multiple particle tracking (MPT)32 and fluorescence recovery after photobleaching (FRAP).33


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Figure 2. Diffusion analysis of 200 nm PS-COOH in 10 mg mL-1 porcine stomach mucin (10 mg mL-1) mixed with 33 or 900 kDa HA in ratios of 0 – 12.5% w/w. The diffusion restriction caused by the addition of HA is presented as the diffusion in mucin divided by the diffusion in the mucin+HA mixture Dm/Dm+HA. The mode of the diffusion coefficients is presented as averages of pentaplicates with standard deviation. *denotes p < 0.05.

In this work, in order to investigate and compare structural effects of 33 or 900 kDa HA addition to mucin hydrogels, the movement of 200 nm carboxyl-modified particles within the mucin mesh was analyzed using NTA (Figure 1, B) following incubation for 2 h at 37°C in mucin hydrogels containing 0 - 12.5% w/w HA to identify any concentrations effects (Figure 2). Diffusion analysis by NTA resembles MPT which, however, use elaborate custom made set-ups composed of several individual components; microscope, camera, image acquisition and processing software. Conversely, NTA uses a commercially available instrument (NanoSight LM10-HS), which represent a more consistent and convenient way of studying the particle diffusion within mucin hydrogels as was utilized by Mansfield et al. for assessing the influence of surface coatings on particle diffusion in mucin.15, 34 Table 1. Diameter and zeta potential of PS-COOH particles in SIF (pH 7.5)



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Size

Diameter [nm]a ζ potential [mV]a

1 µm

1096 (±51)

200 nm 202 (±5) a

-33.5 (±1)

-54.0 (±2)

Size and ζ potential average of three measurements.

200 nm and 1.0 µm PS-COOH particles of well-defined size distributions (Table 1) were chosen for the study due to their strongly negative surface charge in SIF buffer (pH 7.5) that leads to good colloidal charge stabilization of the particles and ensures non-adherence to negatively charged mucin fibers35 or HA; a prerequisite for using the particles as probes in this system1. Dm/Dm+HA denotes the HA-mediated diffusion restriction (Figure 2). Mixtures containing 900 kDa HA from 7.5% w/w significantly limited particle diffusion compared to mucin alone with 1.5 and 2.5-fold increased restriction for 7.5% and 12.5% w/w added 900 kDa HA (p < 0.05). In contrast, the addition of 33 kDa HA to 10 mg mL-1 porcine stomach mucin did not alter the diffusion coefficients of 200 nm PS-COOH. The NTA data, thus, suggests an HA- and molecular weight-dependent mobility restriction that can be explained by the increased molecular motion required for relaxation and untangling of larger 900 kDa HA polymers within mucin nanostructures for allowing particle passage.1


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CLSM tracking of nano- and microparticles in a hyaluronic acid-mucin hydrogel

Figure 3. Selected histograms of particle tracking after 1, 3, 5, 7 and 9 h of 200 nm and 1.0 µm PS-COOH particles in 20 mg mL-1 mucin hydrogel incubated with or without 10% w/w 900 kDa HA for 2h prior to particle addition and tracking.



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The y-axis is aligned with the walls of the µ-slide wells and scaled so to indicate the particle mucin penetration depth with the bottom of the well at ~700 µm.

Figure 4. Effect of 10% w/w 900 kDa HA addition on particle penetration through mucin of 20 mg mL-1. The graphs display the number-weighted means of the particle height distributions from the CLSM particle tracking of which some are displayed in Figure 3. A) 200 nm PS-COOH particles and B) 1.0 µm PS-COOH particles. Data is presented as averages of triplicates w. 95% confidence intervals.

Based on the ability of 900 kDa HA to alter the diffusion coefficient of 200 nm carboxyl-modified particles in porcine stomach mucin, further investigations on the mobility restriction of 200 nm and 1.0 µm particles in a 10% w/w 900 kDa HA-containing mucin hydrogel was performed using a CLSM method for 3D-visualization of fluorescent particles mucin penetration (Figure 1, C). This introduces an improvement to previously used diffusion chambers28, 29 as the visualization of the mucin penetration provides additional information e.g. if the particle movement is characterized by particle distribution throughout the entire mucus layer or by a defined particle front moving through


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the layer. The effects of HA on the mucin penetration of both 200 nm and 1 µm carboxyl-modified particles were analyzed within 20 mg mL-1 mucin to identify any capability of HA to modulate movement of different sized particles under in vivo gastric mucus concentrations of 20-50 mg mL1 1, 25

.

Histograms of the particle distribution along the height of the mucin hydrogel at selected time points are presented for visualizing the particle mucin penetration (Figure 3) with the numberweighted mean penetration of the individual distributions given to further quantify and analyze the effect of HA addition (Figure 4). All particle distributions penetrated gradually through the mucin layer and accumulated at the bottom of the µ-slide wells at 650 – 700 µm (Figure 3, A-D). 200 nm particles quickly penetrated the pure mucin layer and reached the well bottom ~3 h after start, whereas the addition of 10% w/w 900 kDa HA to the mucin hydrogel delayed mucin penetration to ~5 h after start of the experiment with a high number of particles still in the remaining mucin bulk (depth < 600 µm, Figure 3, A-B). Further analyzing the particle penetration by plotting the number-weighted means of the particle height distributions (Figure 4, A), the same effect was evident, showing a significant (p < 0.05) HAmediated reduced particle penetration up to 5 h, where the particles started to approach the well bottom and slow down. Increasing the amount of added 900 kDa HA to 20% w/w further decreased the penetration of 200 nm PS-COOH particles (Figure S1 – Supporting Information). The larger particles of 1.0 µm showed an overall slower penetration through pure mucin compared to those of 200 nm despite the particle mass and gravity force differences with e.g. a penetration of 300 µm and 500 µm after 3 h for particles of 1.0 µm and 200 nm, respectively (Figure 4). No major influence of HA could be inferred from the 1.0 µm particles data with only a single time-point (6.5 h, Figure 4, B) showing a real difference with and without added HA.



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Collectively, this suggests a particle size-dependent effect of the mucin mesh sterically limiting the mobility of the 1.0 µm particles more and indicating the mucin mesh size to lie within this size range from 200 nm – 1.0 um. This is in agreement with other studies were particles >0.5 µm show a limited diffusion ability in mucus.36, 37 Comparing the penetration of 200 nm particles in mucin with and without 900 kDa HA, a similar mobility-limiting effect was observed (Figure 3 and 4), however, now induced by the presence of HA within the mucin hydrogel. This suggests an HA-mediated modulation of the mucin nanostructure with reduction of the effective mucin mesh size to below a limit of 200 nm. The further strengthening of the effect by increasing the HA ratio to 20% w/w (Figure S1) indicates an opportunity for adjusting the effect by the added HA amount. The CLSM tracking analysis, thereby, supports the NTA data and the hypothesis that HA-mediated alteration of the mucin nanostructure is an important part of the explanation for the restriction of particle mobility. This concept has also been proposed as the reason for the reduction of mucin mesh spacing as observed in sputum from cystic fibrosis patients due to the presence of DNA and actin components altering the mucin nanostructure38 or for mucus with added nonionic detergents that unbundle the mucin fibers to create a finer elastic mucin mesh.39 Even though the 1.0 µm particles showed a slower mucin penetration, the different particle masses are, in general, a factor to take into account when performing 3D CLSM tracking in the directionand under the influence of gravitational force. Furthermore, the NTA technique is, under some circumstances, limited to the detection of nanoscale particles < 1.0 µm. This, however, also highlights the advantages of the synergistic combination of these complementary methods as is presented in this paper for studying both nanoparticle and microparticle systems.


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Rheometric analysis of mucin and hyaluronic acid mixtures The suggested mucoadhesiveness of HA40, 41 could, in theory, create additional crosslinks between mucin and HA fibers that could be detrimental for therapeutic use as it is essential that the viscoelastic properties of the mucus lining are not undesirably altered by addition of HA.39 Such adhesive interactions between HA and the mucin fibers would lead to a stronger and less flexible HA-mucin mesh that would be less prone to reptation and untangling events for allowing passage of nanoparticulate probes, providing additional reasons for the observed mobility restrictions. While anionic polymers in general are reported to be mucoadhesive due to hydrogen bond formation between carboxyl groups and hydroxyl groups on mucin glycoproteins,41 the case has been less clear for HA with conflicting reports on its mucoadhesiveness. This is likely due to the great variation of analysis techniques and setups applied, such as measurements on surfaces or in bulk, but also due to differences in ionization and hydration states during measurement.40, 41 Therefore, the effect of HA addition was analyzed by probing the rheological properties of mucin and HA mixtures to elucidate if the natural properties of mucin was left intact or if additional network links, entanglements or secondary bonding was formed upon addition of HA. A mucin concentration of 100 mg mL-1 was used that is slightly higher than physiological levels (in vivo gastric mucus concentrations range from 20 to 50 mg mL-1

24

) for better sensitivity towards any possible

interactions. Dynamic oscillatory rheology is as a robust method for evaluation of mucoadhesive properties in bulk, and is considered optimal for the purpose of completely characterizing both elastic and viscous components of a sample. Operated within the LVE region, it is a non-destructive technique that does not disturb the sample as it is the case for constant shear and flow measurements.40, 42 Initial amplitude sweeps determined the strain limits of the LVE region with the strain amplitude limit, γL, to be ~ 0.5% (data not shown) and a deformation of 0.2% was therefore well within the



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LVE region and was used throughout. This is a typical LVE limiting value γL for materials showing a lightly crosslinked network such as soft gels.40

Figure 5. Rheological behavior of the mucin hydrogels with 10% ratio (10 mg mL-1) HA of either 33 or 900 kDa showing the elastic and loss moduli (G’ and G’’) as a function of the angular frequency, ω. The elastic moduli, G’, shows no significant differences upon adding a 10% ratio of HA to the mucin hydrogel. All data are presented as averages of triplicates with standard deviations. G’ measured for 33 and 900 kDa pure HA, was much lower (~1%) than G’ for the mucin HA mixtures (data not shown).

The elastic and loss (viscous) moduli (G’ and G’’) as a function of the angular frequency, ω, were recorded in triplicates for mucin hydrogels alone or containing 33 or 900 kDa HA at 37 °C within a concentric cylinder system (Figure 5). All samples maintained G’ > G’’ within the measured ω range with a phase angle (δ = tan-1(G’’/G’) at ω = 0.9 s-1) of 15.5 ± 0.8° indicating a weak primarily elastic gel-like behavior32,

40

that is consistent with present literature values (11 - 22°) on pig

gastrointestinal mucin.43 G’ and G’’ slowly increased with ω that is typical for polymers with wide molecular mass distributions and widely distributed and transient physical linkages or entanglements.44, 45


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Rheological synergism covers the ability of a test material, when incorporated in e.g. mucin, to synergistically increase the overall modulus of the mixture to more than the sum of the individual moduli. This has been suggested as a method for evaluating mucoadhesive properties of polymers42 and was here carried out by comparing the elastic moduli, G’, of mucin, HA and mucin-HA mixtures. Despite the high mucin and HA concentration (100 mg mL-1 and 10 mg mL-1, respectively) no significant change of the elastic modulus, G’, was observed upon addition of 10% w/w 33 and 900 kDa HA over the measured frequency range and no indications for additional bond formation or other interactions were found for the mucin and HA mixtures (Figure 5). G’ measured for 33 and 900 kDa pure HA, was much lower (~1%) than G’ for the mucin HA mixtures (data not shown). This indicates that HA does not restrict the particle movement by the formation of an interconnected HA/mucin network, but rather exists as a non-interacting polymer freely dispersed in the mucin hydrogel. In summary, these investigations support our hypothesis that the particle mobility restriction mediated by 900 kDa HA mucin is due to alterations in the nanostructure of the mucin hydrogel by entanglement of HA, resulting in a smaller network pore size and inhibition of particle movement as depicted in Figure 6. The discrepancy between the restrictive effect of 33 and 900 kDa HA is ascribed to the requirement for increased reptation and untangling events of larger 900 kDa HA polymers within the mucin mesh for allowing passage of the nanoparticulate probes.



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Figure 6. Schematic illustration of the suggested nanostructure of mucin mesh after the addition of 33 kDa HA or 900 kDa HA. A) Mucin hydrogel with a certain average mesh spacing. B) Mucin hydrogel with 33 kDa HA. Mesh spacing is only slightly reduced and 200 nm particle mobility is unaffected. C) Mucin hydrogel with 900 kDa HA. The mesh pore size is altered due to entanglement of 900 kDa HA and 200 nm particle mobility is significantly reduced.

This work focuses on interactions with mucin that is the major component of mucus. Mucus, however, also contains lipids, salts, free protein, DNA and cellular debris1, but the findings remain relevant to particle movement studies due to the prominent role of the mucin fiber network. Viruses and bacteria can vary in shape and surface characteristics that can influence their movement in mucin.3 For example, the higher aspect ratios of some pathogens could enhance their mucus penetration. Furthermore, the combination of positive and negative surface charge has been suggested as the reason why surfaces of capsid virus proteins is neither repelled nor attracted to mucin glycan domains and, therefore, do not stick readily to mucin.5 Decreasing the mucin mesh size by HA addition is, therefore, a generic and exciting approach for limiting pathogen penetration. Whilst negatively charged model spherical particles of two different sizes have been used in this work to exemplify the approach, future studies could be directed towards investigations in whole mucus with viruses and bacteria. The mucus layer provides protection against pathogens, but also restricts entry of particle-based drug delivery systems. This work identifies the selective ability of 900 kDa HA to inhibit particle movement across mucin hydrogels that presents a potential new therapeutic strategy to reduce pathogen invasion and increase the residence and consequent local drug release of particle-based drug delivery systems at the mucosal-epithelial interface (Figure 7). Importantly, the added material did not alter the natural viscoelastic properties of mucin that is an important prerequisite for safe use and for maintaining normal function of the mucus layer. This is important for potential clinical applications that could be based on direct application of the HA to the mucosal lining. Preliminary data in our group on the diffusion of fluorescent labelled HA when applied directly onto a mucin


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matrix, without prior mixing, indicates that HA is able to diffuse and distribute evenly into a mucin hydrogel. Moreover, dependent on the site of application, blinking, swallowing and copulation could potentiate mixing in situ. The results in this work could, therefore, be extended to in vivo applications in which direct topical application of the HA allows incorporation into the mucus layer. HA can be easily formulated into a gel and applied to mucosal surfaces such as in the vagina that has a maximal ambient fluid volume of 2 mL,46 and would require an easy manageable amount of 4 – 10 mg HA to obtain a final concentration of 10% w/w HA.

Figure 7. Schematic illustration of the proposed therapeutic applications of 900 kDa HA for blocking pathogen transfer and controlling local drug delivery. Epithelial cell layer covered by mucus under normal conditions, where some pathogens are able to penetrate the mucus layer and reach the epithelial cells (A). Application of 900 kDa hyaluronic acid restricts the diffusion of pathogens, limiting their penetration to epithelial cells and reducing possible infection (B). Diffusion restriction of nanoparticle drug carriers with concomitant prolonged retention could facilitate a controlled and continuous local release of drug (C).



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CONCLUSION This work demonstrates the modulation of the mucin nanostructure by the introduction of high molecular weight HA that restricts the movement of nanoparticles within a mucin-HA hydrogel in a molecular weight dependent manner. The presence of 900 kDa HA did not affect the natural viscoelastic properties of mucin supporting its application as a safe therapeutic. This, therefore, potentially offers a convenient and simple therapeutic approach to control mucosal pathogenesis and drug delivery.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXX. CLSM nanoparticle tracking in mucin containing 20 % w/w HA.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; phone: +45 51272573

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors are grateful to Senior Technician Martin Mellergaard (Novozymes Biopharma) for help with rheology measurements.

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TOC 30x10mm (300 x 300 DPI)


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Figure 1. Experimental particle tracking methods. A) Diffusion analysis using Nanoparticle Tracking Analysis (NTA). The particle suspension in the mucin/HA matrix is laser illuminated and the point scattering and horizontal positions of the particles are recorded for calculation of the particle diffusion coefficient. (B) The tracking methods are used here for investigating the nanostructure of the mucin and HA mesh and any possible interactions between the mesh and particles. (C) Particle tracking using confocal laser scanning microscopy (CLSM) performed in small wells, in which porcine stomach mucin is placed in the bottom. The particle migration through the hydrogel is visualized in 3D by rapid confocal imaging of the well volume and the particle depth distribution in the mucin hydrogel presented as histograms at selected time points. 81x62mm (300 x 300 DPI)



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Figure 2. Diffusion analysis of 200 nm PS-COOH in 10 mg mL-1 porcine stomach mucin (10 mg mL-1) mixed with 33 or 900 kDa HA in ratios of 0 – 12.5% w/w. The diffusion restriction caused by the addition of HA is presented as the diffusion in mucin divided by the diffusion in the mucin+HA mixture Dm/Dm+HA. The mode of the diffusion coefficients is presented as averages of pentaplicates with standard deviation. *denotes p < 0.05. 62x51mm (600 x 600 DPI)


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Figure 3. Selected histograms of particle tracking after 1, 3, 5, 7 and 9 h of 200 nm and 1.0 µm PS-COOH particles in 20 mg mL-1 mucin hydrogel incubated with or without 10% w/w 900 kDa HA for 2h prior to particle addition and tracking. The y-axis is aligned with the walls of the µ-slide wells and scaled so to indicate the particle mucin penetration depth with the bottom of the well at ~700 µm. 180x200mm (600 x 600 DPI)



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Figure 4. Effect of 10% w/w 900 kDa HA addition on particle penetration through mucin of 20 mg mL-1. The graphs display the number-weighted means of the particle height distributions from the CLSM particle tracking of which some are displayed in Figure 3. A) 200 nm PS-COOH particles and B) 1.0 µm PS-COOH particles. Data is presented as averages of triplicates w. 95% confidence intervals. 95x118mm (600 x 600 DPI)


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Figure 5. Rheological behavior of the mucin hydrogels with 10% ratio (10 mg mL-1) HA of either 33 or 900 kDa showing the elastic and loss moduli (G’ and G’’) as a function of the angular frequency, ω. The elastic moduli, G’, shows no significant differences upon adding a 10% ratio of HA to the mucin hydrogel. All data are presented as averages of triplicates with standard deviations. G’ measured for 33 and 900 kDa pure HA, was much lower (~1%) than G’ for the mucin HA mixtures (data not shown). 64x51mm (600 x 600 DPI)



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Figure 6. Schematic illustration of the suggested nanostructure of mucin mesh after the addition of 33 kDa HA or 900 kDa HA. A) Mucin hydrogel with a certain average mesh spacing. B) Mucin hydrogel with 33 kDa HA. Mesh spacing is only slightly reduced and 200 nm particle mobility is unaffected. C) Mucin hydrogel with 900 kDa HA. The mesh pore size is altered due to entanglement of 900 kDa HA and 200 nm particle mobility is significantly reduced. 52x17mm (300 x 300 DPI)


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Figure 7. Schematic illustration of the proposed therapeutic applications of 900 kDa HA for blocking pathogen transfer and controlling local drug delivery. Epithelial cell layer covered by mucus under normal conditions, where some pathogens are able to penetrate the mucus layer and reach the epithelial cells (A). Application of 900 kDa hyaluronic acid restricts the diffusion of pathogens, limiting their penetration to epithelial cells and reducing possible infection (B). Diffusion restriction of nanoparticle drug carriers with concomitant prolonged retention could facilitate a controlled and continuous local release of drug (C). 72x35mm (300 x 300 DPI)


Hyaluronic Acid Molecular Weight-Dependent Modulation of Mucin

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