Effect of hydration on thermodynamic, rheological and structural properties of mucin

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MALMÖ UNIVERSIT Y HEAL TH AND SOCIET Y DOCT OR AL DISSERT A TION 20 1 3:2 Y AN A Z N AMENS k A Y A MALMÖ UNIVERSIT Y 20 1 3 MALMÖ UNIVERSITY 205 06 MALMÖ, SwEDEN www.MAH.SE

YANA ZNAMENSkAYA

EffECT Of HYDRATION

ON THERMODYNAMIC,

RHEOLOgICAL AND

STRUCTURAL pROpERTIES

Of MUCIN

Isbn 978-91-7104-513-3 (print) Isbn 978-91-7104-514-0 (pdf) Issn 1653-5383 Eff ECT O f HYDR A TION ON THERMOD YN AMIC , RHEOL O g IC AL AND S TRUCTUR AL p R O p ERTIES O f MUCIN

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e f f e c t o f h y d r a t i o n o n t h e r m o d y n a m i c , r h e o l o g i c a l a n d s t r u c t u r a l p r o p e r t i e s o f m u c i n

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Malmö University

Health and Society, Doctoral Dissertation 2013:2

Front illustration: image of dehydrated human unpurified saliva in air obtained using polarized light microscopy

Back illustration: 3D image of PGM dumbbell-like structure obtained using AFM ISBN 978-91-7104-513-3 (print)

ISBN 978-91-7104-514-0 (pdf) ISSN 1653-5383

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yana Znamenskaya

effect of hydration on

thermodynamic, rheological

and structural properties

of mucin

Malmö University, 2013

Faculty of Health and Society,

Biomedical Science Department

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abbreviations and symbols

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ABBREVIATIONS AND SYMBOLS

𝑎𝑎𝑤𝑤 – water activity

AFM – atomic force microscopy

BSM – bovine submaxillary gland mucin ∆𝐶𝐶𝑝𝑝 – the difference in heat capacity

DSC – differential scanning calorimetry

𝐻𝐻𝑤𝑤𝑚𝑚 – partial molar enthalpy of mixing of water (enthalpy of hydration)

PEM – polyelectrolyte mutlilayers PGM – pig gastric mucin

PGMC - mucus collected from pig’s stomach Tg – glass transition temperature

QCM-D - quartz crystal microbalance with dissipation monitoring RH – relative humidity

SANS - small angle neutron scattering SAXS - small-angle X-ray scattering

– water activity

BSM – bovine submaxillary gland mucin

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ABBREVIATIONS AND SYMBOLS

– the difference in heat capacity DSC – differential scanning calorimetry

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ABBREVIATIONS AND SYMBOLS

– partial molar enthalpy of mixing of water (enthalpy of hydration) PEM – polyelectrolyte mutlilayers

PGM – pig gastric mucin

SANS - small-angle neutron scattering SAXS - small-angle X-ray scattering

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abstract

Mucus, an adherent hydrated layer covering the epithelium, is found in all internal tracts of the body and plays an important role in animal and human life. One of the essential functions of mucus is protection of tissues against dehydration. A major component of mucus, forming the macromolecular matrix and also being responsible for its viscoelastic properties, is the glycoprotein mucin. Mucin is a complex molecule owing to the high molar mass, its polydispersity and high degree of glycosylation. This thesis is aimed to investigate how thermodynamic, structural and rheological properties of mucin are affected by hydration.

The effect of hydration on thermodynamic properties of pig gastric mucin (PGM) and bovine submaxillary mucin (BSM) has been studied using sorption calorimetry and differential scanning calorimetry (DSC). The analysis of sorption isotherms shows higher water sorption capacity of PGM compared to BSM at relative humidity (RH) levels lower than about 78%. The value of the hydration enthalpy at zero water content at 25ºC for both biopolymers is close to -20 kJ/ mol. RH levels at which glass transition of both mucins occurs are shown to be between 60 and 70%. The RH at which glass transition occurs is weakly dependent on the temperature and mucin type. The phase diagram of PGM demonstrates two different glass transition regions: dependent and independent on hydration levels, respectively. In particular at mucin concentrations from 0 to 67 wt%, the glass transition occurs at a constant temperature of about –15ºC. At higher concentrations of mucin, the glass transition temperature (Tg) increases with increasing mucin concentrations.

It is also demonstrated that the QCM-D technique can be used to obtain the water sorption isotherms of mucin films in particular and in general to monitor glass transitions in biopolymers. Hydration-induced changes of rheological properties of mucin films are investigated using a model-free approach. The ratio

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of G’/G’’ is evaluated as a function of relative humidity. Observed transitions from solid-like behaviour to liquid-like behaviour in mucin system occur in the same humidity range as that found in sorption calorimetric experiments.

The structural properties of mucin have been studied using atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS). AFM indicates the presence of dumbbell as well as fiber-like structures in PGM samples. In the case of BSM samples, only fiber-like structures are observed. SAXS studies show three ranges of scattering vector q corresponding to different fractal dimensions in dilute PGM and BSM solutions. Dehydration-induced structural changes in PGM are demonstrated by a distinct appearance of peaks on scattering curves starting from 20 wt% of mucin. Structural changes at about 80 wt% obtained in SAXS at 25°C, correspond to mucin glass transition, and are in agreement with sorption calorimetry and DSC studies. Temperature-induced phase behaviour changes occur around 60-70°C at intermediate levels of hydration.

A comparison between water sorption isotherms of BSM/chitosan multilayers and those of individual biopolymers shows that the water sorption capacity of BSM/chitosan multilayers is similar compared to that of individual biopolymers, although some differences exist. In particular, at low RH levels the water sorption capacity of multilayers is slightly higher compared to each biopolymer, however at high RH the multilayer films are less hydrated than individual biopolymers.

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list of papers

This thesis is based on the following papers, which are cited in the text by their Roman numerals. The papers are appended in the end of the thesis.

I. Znamenskaya, Y.; Sotres, J.; Engblom, J.; Arnebrant, T.; Kocherbitov, V. Effect of Hydration on Structural and Thermodynamic Properties of Pig Gastric and Bovine Submaxillary Gland Mucins. J. Phys. Chem. B. (2012), 116, 5047–5055.

II. Znamenskaya, Y.; Sotres, J.; Gavryushov, S.; Engblom, J.; Arnebrant, T.; Kocherbitov, V. Water Sorption and Glass Transition of Pig Gastric Mucin Studied by QCM-D. J. Phys. Chem. B. (2013), 117, 2554−2563

III. Znamenskaya, Y.; Sotres, J.; Arnebrant, T.; Kocherbitov, V. Hydration of Bovine Submaxillary Gland Mucin/Chitosan Multilayers Studied by QCM-D. Manuscript

IV. Znamenskaya, Y.; Engblom, J.; Arnebrant, T.; Kocherbitov, V. Effects of Dehydration on Structural Properties and Phase Behaviour of Pig Gastric Mucin Evaluated by SAXS. Manuscript

Paper I: I, JE and VK designed the study. I performed sorption calorimetry and DSC experiments and analyzed the data. JS performed AFM experiments, analyzed AFM data and wrote the AFM part of the paper. I wrote the paper with contributions from JS, JE, TA and VK

Paper II: I, JE and VK designed the study. I performed QCM-D experiments and analyzed the data. JS performed AFM experiments, analyzed AFM data and wrote the AFM part of the paper. SG and VK analyzed QCM-D data with model-free approach and extracted the G’/G’’ ratio. I wrote the paper with contributions from JS, SG, JE, TA and VK.

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Paper III: I designed the study with input from TA and VK. I performed sorption calorimetry and QCM-D experiments and analyzed the data. JS performed AFM experiments, analyzed AFM data and wrote the AFM part of the manuscript. I wrote the manuscript with contributions from JS, TA and VK.

Paper IV: I, JE and VK designed the study, performed SAXS experiments and analyzed the data. I wrote the manuscript with contributions from JE, TA and VK.

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chapter 1 introduction

One of the essential functions of the mucous gel is protection of tissues against dehydration (drying). In order to fulfil this function, the mucous gel should be strongly hydrated, i.e. it should contain a large amount of water. The presence of large amounts of water prevents extensive changes of the water activity in the mucous gel when the relative humidity of the surrounding atmosphere changes. Nonetheless, when subjected to dry conditions, mucous gel does dry out. I.e. under 30% RH, the eyes and skin become dry, and under 10% RH the nasal mucous membrane becomes dry. Consequently, dehydration changes the protective properties of the mucous gel and may become a pathogenic factor in many diseases such as cystic fibrosis1-3_{, asthma}1_{, gastric carcinoma}4_{, rhinorrhea}5_.

The focus of this thesis is to investigate effects of mucous gel hydration. In particular, we focus on how macroscopic properties of mucus, such as hydrophilicity and elasticity, are affected by different RH levels. In order to achieve this goal, a further understanding of the components properties in mucous gel is required. The glycoprotein mucin is the main component of mucus, and forms the macromolecular matrix responsible for its viscoelastic properties. Hence, we investigate how thermodynamic, structural and rheological features of mucin are affected by hydration.

The main objectives have been:

• To characterise the hydrophilicity of mucin by determining the equilibrium water vapour sorption at constant temperature as a function of the RH • To obtain RH levels at which glass transition of mucin systems occurs and

show the dependence of the glass transition temperature of mucin on water content

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• To study hydration-induced glass transition behaviour and rheological changes of mucin in thin films

• To indicate temperature and hydration-induced structural changes in mucin systems, using X-ray scattering

 To construct BSM/chitosan multilayers and compare the water sorption isotherms of BSM/chitosan multilayers to those of singular biopolymers

The justification for pursuing this work was to increase the appreciation of the fundamental properties of native tissues, especially how the highly hydrated layer covering mucosal surfaces of our body is affected by changes in ambient conditions; understanding hydration fundamentals can be relevant for areas like biomaterial coatings and transmucosal drug delivery. Moreover, the mucin/ chitosan multilayer system is a mimic of extra-cellular matrices, which is interesting to study with respect to interactions with pharmaceutical components. The “Introduction” part of the thesis contains general aspects on the properties of mucous gel and the glycoprotein mucin. Also the layer-by-layer assembly of oppositely charged polymers is explained. In the “Materials and Methods” section, a detailed description of two major experimental techniques, sorption calorimetry and QCM-D is presented, while others are characterized briefly.

The “Results and Discussion” part includes the effect of hydration on the properties of water-mucin system, i.e. Papers I, II, and IV. In Paper III, a study of a more complex system, mucin/chitosan multilayers, is presented, and in particular how the mucin/chitosan system is affected by hydration.

mucous gel (mucus) and mucins

Mucus, an adherent hydrated layer covering the epithelium, is found in all internal tracts of the body and plays an important role in animal and human life1,6_{. The}

properties of mucus are essential for the proper functioning of the digestive, respiratory, and reproductive systems of vertebrates, including humans7,8_.

The function of mucous gel differs depending on its origin, but generally it contributes to protection and transportation. For example, in the gastrointestinal tract mucus protects the epithelial cells from digestive enzymes, bile salts, hydrochloric acid and microorganisms9_{. Mucus is a barrier to pathogens and}

noxious substances and is the first barrier nutrients and delivered drugs will interact with and diffuse through10_{. From an engineering point of view, mucus is}

an outstanding water-based lubricant7_{. However the most important biological}

function of mucus is protection of tissues against dehydration. In order to fulfil this function, the mucous gel needs to be strongly hydrated.

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17 Thus, the highly hydrated gel consists primarily of water (~95%), the glycoprotein mucin, and other components: enzymes, other proteins, electrolytes, lipids10-14_{; the concentration of which depends on the mucus source. The thickness}

of the mucus film also varies with its origin. For example, in the stomach it is reported to be 50-600 µm and in the intestine and colon 15-450 µm 15_{. For the}

respiratory tract mucus the thickness is estimated to be 5-10 µm and for the alveolar regions it is less than or equal to 1 µm16_.

Mucin

The term “mucin” comes from the mucus glycoproteins, secreted by epithelia. However, later it turned out that the epithelia produce transmembrane glycoproteins, which were described as mucins as well. Thus both glycoproteins, membrane bound and secreted, are considered as mucins. Established in 1990, the gene family, with the symbol MUC, followed by a number, represents the mucins17_{. Presently about twenty MUC – genes have been found but not all are}

fully sequenced1,8,10,18_{. Mucus secretion comprises more than one gene product,}

for example, human colon epithelia contains at least three mucin genes: MUC 2, 3, and 419_.

Mucin molecule

Mucin is a major component of mucus, forming the macromolecular matrix responsible for its viscoelastic properties11,12,20_{. Mucins are large glycoproteins}

with high molar masses, varying from 0.5-40 MDa, and are expressed in epithelial and glandular tissues10,21_{. Even though structural and physico-chemical}

properties of mucins vary depending on origin, the glycoproteins share common features. Generally, the mucin molecule consists of hydrophilic and hydrophobic parts: a highly glycosylated central protein core and one or two terminal peptide regions which are either non-glycosylated22_{or carry only a few carbohydrate}

moieties (Figure 1.1).

The protein core, containing domains rich in the amino acids threonine, serine, and proline, represents 10-30% of the total mass of the mucin molecule23_{. These}

domains are composed of tandemly repeated sequences that vary in number, length, and amino acid sequence from one mucin to another24_{. Threonine and}

serine hydroxyl groups are linked to the glycosylated part of the mucin molecule8_.

The carbohydrate content can be up to 80% of the total mass of the molecule, and consists of N-acetylgalactosamine, N-acetylglucosamine, fucose, galactose, and sialic acid25_{. The carbohydrates are present as short chains, often branched,}

and attached to the polypeptide chain by O-glycosidic bonds to the –OH group of serine and threonine residues25_{. The carbohydrate side chains size depends}

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on the mucous secretion source and varies from 2 to 5 sugar units per chain (sheep and pig submaxillary mucus) up to 16 to 19 sugar units per chain (human ovarian cyst and pig gastric mucus)6_.

Non-glycosylated or bare parts of the mucin molecule are rich in cysteine, found in the N- and C-terminal ends of the protein core. Cysteine-rich domains are responsible for dimerization via disulphide bond formation, and afterwards polymerizations of these dimers to form mucin multimers (Figure 1.1). Disulphide bond formation plays an important role in the gel forming properties of mucin10_{. The electrostatic character of mucins is determined by the polypeptide}

backbone, for example, glutamic acid and aspartic acid residues (pK_a=4)26_and

by carbohydrate side chains27,28_{, which are often negatively charged owing to}

the presence of sialic acid residues (pK_a=2.6) and sulphate groups (pK_a=1)29_{. The}

isoelectric point of mucin, caused by these acidic groups, have been determined to 2-327_{. Such negatively charged mucins may form polyelectrolyte complexes}

with positively charged polymers, for example, with chitosan22_.

Models of pig gastric and bovine submaxillary gland mucins

(PGM and BSM)

As previously mentioned, the structural and physico-chemical properties of mucin vary depending on its origin. The high molar mass of the mucin molecule, its polydispersity and high degree of glycosylation10_{make this glycoprotein}

molecule complex and it is difficult to characterise its structure. Hence mucin molecules are described using different models6,7,14,30-32_.

Often PGM and BSM are described with reference to a bottlebrush model (Figure 1.1): a copolymer of relatively rigid (glycosylated) and relatively flexible (bare) moeties6,7,14,30_{. In this mucin model, the carbohydrate side chain length can}

vary from 15 to 50 nm7_.

Figure 1.1 Schematic illustration of a bottlebrush mucin molecule7_.

10 Non-glycosylated or bare parts of the mucin molecule are rich in cysteine, found in the N-

and C-terminal ends of the protein core. Cysteine-rich domains are responsible for

dimerization via disulphide bond formation, and afterwards polymerizations of these dimers

to form mucin multimers (Figure 1.1). Disulphide bond formation plays an important role in

the gel forming properties of mucin

10

_{. The electrostatic character of mucins is determined by}

the polypeptide backbone, for example, glutamic acid and aspartic acid residues (pK

a

=4)

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and by carbohydrate side chains

27,28

_{, which are often negatively charged owing to the}

presence of sialic acid residues (pK

a

=2.6) and sulphate groups (pK

a

=1)

29

. The isoelectric

point of mucin, caused by these acidic groups, have been determined to 2-3

27

_{. Such}

negatively charged mucins may form polyelectrolyte complexes with positively charged

polymers, for example, with chitosan

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_.

Models of pig gastric and bovine submaxillary gland mucins (PGM and BSM)

As previously mentioned, the structural and physico-chemical properties of mucin vary

depending on its origin. The high molar mass of the mucin molecule, its polydispersity and

high degree of glycosylation

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_{make this glycoprotein molecule complex and it is difficult to}

characterise its structure. Hence mucin molecules are described using different models

6,7,14,30-32

_{. Often PGM and BSM are described with reference to a bottlebrush model (Figure 1.1): a}

copolymer of relatively rigid (glycosylated) and relatively flexible (bare) moeties

6,7,14,30

_{. In}

this mucin model, the carbohydrate side chain length can vary from 15 to 50 nm

7

.

Figure1.1 Schematic illustration of a bottlebrush mucin molecule

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_.

The PGM molecule has also been described as having a dumbbell shape (Figure 1.2),

presented as two asymmetric

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_{or symmetric}

32

_{globules per chain separated by a heavily}

Glycosylated (Carbohydrate part)

Bare (Protein part)

Disulphide bonds (S-S)

Glycosylated (Carbohydrate part) Bare (Protein part)

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19 The PGM molecule has also been described as having a dumbbell shape (Figure 1.2), presented as two asymmetric31_{or symmetric}32_{globules per chain separated}

by a heavily glycosylated spacer. The carbohydrate side chain length for a dumbbell shaped molecule is different than that for a bottlebrush, and it is about 2.5 nm32_.

Figure 1.2 Schematic illustration of a dumbbell PGM molecule32_.

The carbohydrate content of BSM is different in comparison with PGM having the higher content; i.e., the carbohydrate content in PGM is about 83-86 wt%23,33_,

and in BSM it is about 61-69 wt%23,33-36_.

Also, the content of sialic acid in PGM and BSM differs. The sialic acid content in PGM is about 2-3 wt%23,33,36_{, therefore PGM is considered as a weakly charged}

molecule28_{. BSM contains much higher amount of sialic acid, i.e. about 32-36}

wt%23,33,36_.

hydration of mucin

Water is the main constituent of our body: cells, tissues and organs primarily consist of water37_{; consequently dehydration of living systems results in}

dysfunction, disease, or ultimately death. In living systems mucins are most often arranged in thin films covering the hydrated tissue. Hydration of mucin films therefore plays a key role in the protection of the tissues against dehydration. Monitoring hydration-induced changes in mucin systems upon addition of water, from the dry to the fully hydrated state, provides information regarding mucin properties in both dry and hydrated states. Thus, sorption isotherms, enthalpies of hydration, and glass transition behaviours need to be considered in the evaluation of mucin hydration.

11 glycosylated spacer. The carbohydrate side chain length for a dumbbell shaped molecule is

different than that for a bottlebrush, and it is about 2.5 nm

32

_.

Figure 1.2 Schematic illustration of a dumbbell PGM molecule

32

_.

The carbohydrate content of BSM is different in comparison with PGM having the higher

content; i.e., the carbohydrate content in PGM is about 83-86 wt%

23,33

_{, and in BSM it is about}

61-69 wt%

23,33-36

_.

Also, the content of sialic acid in PGM and BSM differs. The sialic acid content in PGM is

about 2-3 wt%

23,33,36

_{, therefore PGM is considered as a weakly charged molecule}

28

_{. BSM}

contains much higher amount of sialic acid, i.e. about 32-36 wt%

23,33,36

_.

Hydration of mucin

Water is the main constituent of our body: cells, tissues and organs primarily consist of water

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_{; consequently dehydration of living systems results in dysfunction, disease, or ultimately}

death. In living systems mucins are most often arranged in thin films covering the hydrated

tissue. Hydration of mucin films therefore plays a key role in the protection of the tissues

against dehydration.

Monitoring hydration-induced changes in mucin systems upon addition of water, from the

dry to the fully hydrated state, provides information regarding mucin properties in both dry

and hydrated states. Thus, sorption isotherms, enthalpies of hydration, and glass transition

behaviours need to be considered in the evaluation of mucin hydration.

Sorption isotherm is usually presented in the form of water activity, ��, or RH, as a function

of water content (amount of absorbed water vapour). Water activity is defined as

��=��0

, where �� is the vapour pressure of water in the system studied and ��0 is the

vapour pressure of pure water. Also, the water activity can be expressed in terms of the

40-50 nm

20 nm

2.5 nm (Carbohydrate side chain)

Hydrophobic globule

40-50 nm

20 nm 2.5 nm (Carbohydrate side chain) Hydrophobic globule

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Sorption isotherm is usually presented in the form of water activity, , or RH, as a function of water content (amount of absorbed water vapour). Water activity is defined as

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therms, enthalpies of hydration, and glass transition behaviours need to be considered in the evaluation of mucin hydration.

Sorption isotherm is usually presented in the form of water activity, 𝑎𝑎𝑤𝑤, or

RH, as a function of water content (amount of absorbed water vapour). Wa-ter activity is defined as 𝑎𝑎𝑤𝑤 = 𝑝𝑝𝑤𝑤⁄ , where 𝑝𝑝𝑝𝑝𝑤𝑤0 𝑤𝑤 is the vapour pressure of

wa-ter in the system studied and 𝑝𝑝𝑤𝑤0 is the vapour pressure of pure water. Also,

the water activity can be expressed in terms of the relative humidity (RH) of air 𝑎𝑎𝑤𝑤 = 𝑝𝑝𝑤𝑤⁄𝑝𝑝𝑤𝑤0 = 𝑅𝑅𝑅𝑅 100⁄ . The chemical potential can also be expressed in

terms of RH 𝜇𝜇𝑤𝑤 = 𝜇𝜇𝑤𝑤𝑜𝑜 + 𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅(𝑎𝑎𝑤𝑤) = 𝜇𝜇𝑤𝑤𝑜𝑜 + 𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅(𝑝𝑝𝑤𝑤⁄ ) = 𝜇𝜇𝑝𝑝𝑤𝑤0 𝑤𝑤𝑜𝑜 + 𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅(𝑅𝑅𝑅𝑅 100⁄ ), where 𝜇𝜇𝑤𝑤

is the chemical potential of water in the system; 𝜇𝜇𝑤𝑤𝑜𝑜 is the chemical potential

of pure water at the absolute temperature 𝑅𝑅; 𝑅𝑅 is the gas constant (8.314 J/molK). For a condensed phase, the chemical potential of water is equal to that of the vapour in equilibrium with the condensed phase.

The enthalpy 𝑅𝑅 is the measure of heat at constant pressure. The heat required for a temperature change 𝑑𝑑𝑅𝑅 at constant pressure 𝑃𝑃 is defined as heat capacity 𝐶𝐶𝑝𝑝= (𝑑𝑑𝑅𝑅 𝑑𝑑𝑅𝑅⁄ )𝑃𝑃. In sorption calorimetry, it is possible to measure the

enthal-py of hydration or partial molar enthalenthal-py of mixing of water, 𝑅𝑅𝑤𝑤𝑚𝑚. This is a

measure of water material interactions as it represents the change in partial molar enthalpy of water upon mixing, when the sorption process occurs at equilibrium conditions. Enthalpy of hydration is defined as the heat produced when one mole of the condensed vapour is mixed with an infinite amount of a material (at constant values of vapour content and total pressure)38_.

Measuring the water activity and the enthalpy of hydration simultaneously, using sorption calorimetry, allows for detecting phase transitions and the iso-thermal glass transition. A more common method of determination of glass transition is differential scanning calorimetry (DSC). A solid phase is defined as a glass if it is noncrystalline and exhibits what appears to be a

second-is the vapour pressure of water in the system studied and

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is the vapour pressure of pure water. Also, the water activity can be expressed in terms of the relative humidity (RH) of air

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the water activity can be expressed in terms of the relative humidity (RH) of air 𝑎𝑎𝑤𝑤= 𝑝𝑝𝑤𝑤⁄𝑝𝑝𝑤𝑤0 = 𝑅𝑅𝑅𝑅 100⁄ . The chemical potential can also be expressed in

second-. The chemical potential can also be expressed in terms of RH

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RH, as a function of water content (amount of absorbed water vapour). Wa-ter activity is defined as 𝑎𝑎𝑤𝑤= 𝑝𝑝𝑤𝑤⁄ , where 𝑝𝑝𝑝𝑝𝑤𝑤0 𝑤𝑤 is the vapour pressure of

terms of RH 𝜇𝜇𝑤𝑤= 𝜇𝜇𝑤𝑤𝑜𝑜 + 𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅(𝑎𝑎𝑤𝑤) = 𝜇𝜇𝑤𝑤𝑜𝑜 + 𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅(𝑝𝑝𝑤𝑤⁄ ) = 𝜇𝜇𝑝𝑝𝑤𝑤0 𝑤𝑤𝑜𝑜 + 𝑅𝑅𝑅𝑅𝑅𝑅𝑅𝑅(𝑅𝑅𝑅𝑅 100⁄ ), where 𝜇𝜇𝑤𝑤

second-where

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the water activity can be expressed in terms of the relative humidity (RH) of air 𝑎𝑎𝑤𝑤= 𝑝𝑝𝑤𝑤⁄𝑝𝑝𝑤𝑤0 = 𝑅𝑅𝑅𝑅 100⁄ . The chemical potential can also be expressed in

is the chemical potential of water in the system;

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is the chemical potential of pure water at the absolute temperature T; R is the gas constant (8.314 J/mol/K). For a condensed phase, the chemical potential of water is equal to that of the vapour in equilibrium with the condensed phase.

The enthalpy H is the measure of heat at constant pressure. The heat required for a temperature change dT at constant pressure P is defined as heat capacity

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second-. In sorption calorimetry, it is possible to measure the enthalpy of hydration or partial molar enthalpy of mixing of water,

22

second-. This is a measure of water material interactions as it represents the change in partial molar enthalpy of water upon mixing, when the sorption process occurs at equilibrium conditions. Enthalpy of hydration is defined as the heat produced when one mole of the condensed vapour is mixed with an infinite amount of a material (at constant values of vapour content and total pressure)38_.

Measuring the water activity and the enthalpy of hydration simultaneously, using sorption calorimetry, allows for detecting phase transitions and the isothermal glass transition. A more common method of determination of glass transition is differential scanning calorimetry (DSC). A solid phase is defined as a glass if it is noncrystalline and exhibits what appears to be a second-order transition in heat capacity measurements. Glass transition is a change of the system from a solid-like to a liquid-like behaviour or a transition from glassy to elastic states. At this type of transition structural changes of the system do not take place, only changes of the chain mobility occur39_.

Only a few reports on water sorption isotherms of mucin can be found in the literature; in particular, pedal mucus trails40_{, porcine mucin}41_{, bovine and porcine}

submaxillary mucins34_{, using gravimetric methods. In this work we present}

sorption isotherms of PGM and BSM at 25, 40 and 50°C using the sorption calorimetry method, which as far as we are aware was not applied in studies of mucin systems. Since mucin is a carbohydrate polymer, it is expected that at low RH, water molecules easily and primarily interact with charged groups, and therefore the energy of interaction of water with charged polymers might be expected to be higher than with non-charged polymers. However reported data

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21 on sorption energies of charged and non-charged biopolymers show that water-ion interactwater-ions do not affect hydratwater-ion enthalpies42_{, and that the enthalpy of}

hydration values of charged and non-charged biopolymers and proteins at zero water content at 25°C are about -18 kJ/mol42-44_.

A number of studies have been focused on phase behaviour of mucins using different techniques7,14,45-47_{including polarized light microscopy}7,46,47_{, small angle}

neutron scattering (SANS)29_{and DSC}7_{. Some studies reported the mucin ability}

to form liquid crystals. For example, Viney et al. observed formation of liquid crystalline domains in slug pedal mucin46_{and partially dried giraffe saliva}47_by

polarized light microscopy. Waigh et al.29_{observed a polydomain nematic phase}

in porcine stomach mucin using light scattering measurements, small angle neutron scattering (SANS) and rheological experiments and suggested that the mucin phase behaviour is a function of concentration.

Table 1.1 Literature data of glass transition temperature (Tg) of mucins

Type of mucin Tg, ºC Concentration, wt%

Pig gastric mucin7 _24.8 _5-50

Porcine gastric mucin41 40.4 (onset)

68.6 (endset) 100

Snail mucin45 ₁₁₁ ₁₀₀

The glass transition temperature as a function of water content is also an important parameter for polymers since their properties undergo dramatic changes at the glass transition42_{. According to several DSC studies mucins can undergo glass}

transitions, and the transition temperature vary for different types of mucins, and can depend on experimental conditions, in particular water content (Table 1.1). Because of variety of discrepancies in reported data on Tg, the exact phase behaviour of mucin as a function of temperature and hydration levels is not completely understood. In an effort to reconcile the literature data we undertook a study of the phase behaviour of PGM and BSM and tried to determine the glass transition temperature using different techniques.

polyelectrolyte multilayers

Formation of polyelectrolyte multilayers (PEM) has gained significant interest since Decher et al.48_{for the first time introduced the construction of PEM based}

on adsorption of oppositely charged polyelectrolytes, using a layer-by-layer deposition technique. PEM present a wide range of potential applications in

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22

different fields such as coating for biomaterials49,50_{, drug delivery}51,52_{, biosensors}53

etc. The mechanism of PEM construction is based on electrostatic interactions and charge overcompensation54,55_{. The thickness and physico - chemical properties}

of PEM films can be controlled by changing the experimental conditions of deposition such as number of adsorption cycles, pH, salt concentration, polyelectrolyte concentration, adsorption time and type of polymer used56-58_{. In}

the latter case, both synthetic and natural polymers can be used. Although many studies focus on synthetic multilayers, in recent years the use of natural polymers, such as polysaccharides, has attracted significant attention because of their low toxicity, biocompatibility and biodegradability. For example, biodegradation in

vitro and in vivo of a chitosan/hyaluronic acid multilayer film was reported by

Picart et al. 59_{. Moreover, polysaccharide-based PEM is a mimics of extra-cellular}

matrices from plant and animal tissues which is interesting to study as a model of native tissues60_.

Mucin/chitosan multilayers

Chitosan, one of the most abundant polysaccharides in the world61_{, is}

biodegradable, biocompatible and non-toxic to living tissues62_{, which makes}

it suitable for mucosal delivery. Studying mucin/chitosan multilayer films can help to understand the fundamental properties of native tissues. In particular, it is interesting to study the properties of mucin-chitosan based multilayers films, such as hydration and water sorption capacity, compared to the properties of the individual biopolymers.

Hydration and swelling behaviour of PEM are often studied in liquids (high water activity), using different techniques, and the estimated water content in the multilayers is quite high, ranging from 60 to 90% for different systems60_{. For}

example, Svensson et al.63_{studied BSM/chitosan multilayers using ellipsometry}

and reported a water content of more than 90% at high water activity. Hydration of PEM at different RH levels has been studied by several authors using different techniques64-67_{. However most of them are focused on hydration of synthetic}

polymers and only a few reports present the water sorption isotherms of PEM and compare them to those of the individual polyelectrolytes64-66_{. For example,}

swelling dynamics of the poly(sodium-4 styrene sulfonate)/poly[(allylamine) hydrochloride] multilayers was studied by Kugler et al. and reported to be slow compared to individual synthetic polymers66_{. All mentioned above show the}

water sorption capacity of polysaccharide-based multilayers at different RH is yet to be determined.

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23 In this work assembly and hydration of BSM/chitosan multilayers was studied using the QCM-D technique; water sorption isotherms of multilayers were determined and compared to those of the singular polymers BSM and chitosan.

studied systems

In the present work commercially available, partially purified PGM (Sigma, cat. no M1778) type III, containing 0.5-1.5% bound sialic acid and BSM (Sigma, cat. no M3895), type I-S, containing 9-17% bound sialic acids were used as received without further purification. PGM from the same batch was used for all experiments. However in Papers I, III, and IV, the BSM used was from different batches.

Mucus, collected from a pig’s stomach (PGMC), was used in Paper I without purification, and calculation of masses before and after drying indicated that the mucus contained 95% of water and 5% of solids.

Mucins were dried in vacuum at room temperature in contact with 3 Å molecular sieves for 24 hours directly prior to the experiments. Samples with mucin concentrations below 50 wt% were prepared by adding the appropriate amount of solvent to a weighed amount of dry PGM.

After mixing mucin with solvent, samples were left for equilibration for several days. Preparation of the samples with mucin concentrations above 50 wt% was done, using desiccators with saturated salt solutions which give different relative humidity levels68_{, where samples were equilibrated for 7-10 days at 25°C. Water}

vapour was absorbed by dry mucin, the mucin-water mixtures were weighed to obtain the water content, and then samples were examined in experiments (Paper I and IV).

Chitosan, used in Paper III, was obtained from Fluka BioChemika (low-viscous, Prod. No. 50494) and the degree of acetylation, determined from titrimetric analysis of the amino groups69_{, was 19%.}

The water used in all experiments was of ultrahigh quality (UHQ), purified at 25ºC by Elgastat UHQ II Model UHQ-PS-MK3 (Elga Ltd., High Wycombe, Bucks, UK).

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25

chapter 2 experimental techniques

In the present work a combination of several structural and thermodynamic methods was used to characterise hydration-induced changes in mucin systems. The major measuring techniques in this thesis, sorption calorimetry and QCM-D, are described in detail. Other techniques, DSC, SAXS and AFM are explained briefly.

isothermal sorption calorimetry

The word “calorimetry” comes from “calor”, meaning “heat” and “metron” meaning “measure”. Isothermal calorimetry is a method where heat and thermal power are measured at constant temperature. All physical, chemical or biological processes are accompanied by release or absorption of heat and therefore calorimetry is a general measurement technique70_.

Isothermal sorption calorimetry is a method for studying hydration of surfactant71_{, phospholipid}72_{, protein}43,73_{, and polymer}42,44_{systems. This method}

allows simultaneous measurement of the water activity, and the partial molar enthalpy of mixing of water, ,38_{providing a complete thermodynamic}

characterisation of sorption process at a given temperature.

The sorption calorimetric experiments are performed in a two-chamber calorimetric cell inserted in a double-twin microcalorimeter74_{. In the sorption}

calorimetric measurements completely dry samples are required, i.e. the initial water activity and water content in the samples should be zero. Therefore, the sample drying process usually takes place in vacuum at room temperature in contact with 3 Å molecular sieves for 24–48 hours directly prior to the experiment. The initial dry mass of the sample is usually in the range of 50-150 mg. The dry sample is placed in the upper (sorption) chamber and pure water is injected in the lower (vaporisation) chamber. Evaporated water diffuses through the tube that

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26

connects the upper and lower chambers of the calorimetric cell and is absorbed by the sample (Figure 2.1).

The thermal power P released or absorbed in the two chambers are calculated using calorimetric signal U and the corresponding calibration coefficient

29 The sorption calorimetric experiments are performed in a two-chamber calo-rimetric cell inserted in a double-twin microcalorimeter74_{. In the sorption}

cal-orimetric measurements completely dry samples are required, i.e. the initial water activity and water content in the samples should be zero. Therefore, the sample drying process usually takes place in vacuum at room temperature in contact with 3Å molecular sieves for 24 – 48 hours directly prior to the ex-periment. The initial dry mass of the sample is usually in the range of 50-150 mg. The dry sample is placed in the upper (sorption) chamber and pure water is injected in the lower (vaporisation) chamber. Evaporated water diffuses through the tube that connects the upper and lower chambers of the calori-metric cell and is absorbed by the sample (Figure 2.1).

The thermal power P released or absorbed in the two chambers are calculated using calorimetric signal 𝑈𝑈 and the corresponding calibration coefficient 𝜀𝜀:

𝑃𝑃 = 𝑈𝑈𝜀𝜀 (2.1)

The calibration coefficient 𝜀𝜀 is determined by electrical calibration. Since the thermal power is changing with time, Tian’s equation is applied for calcula-tion of the thermal power75_:

𝑃𝑃 = 𝜀𝜀 �𝑈𝑈 + 𝜏𝜏𝑑𝑑𝑑𝑑_{𝑑𝑑𝑑𝑑}� (2.2) where the time constant 𝜏𝜏 is determined in calibration experiments.

Figure 2.1 A sorption calorimetric cell (figure from www.mah.se)

The activity of water is calculated from the thermal power of evaporated wa-ter regiswa-tered in vaporization chamber and a simplified equation can be pre-sented as 76_:

𝑎𝑎𝑤𝑤 = 1 −_𝑃𝑃𝑚𝑚𝑚𝑚𝑚𝑚𝑃𝑃 (2.3)

:

(2.1) The calibration coefficient

𝑎𝑎𝑤𝑤= 1 −_𝑃𝑃𝑚𝑚𝑚𝑚𝑚𝑚𝑃𝑃 (2.3)

is determined by electrical calibration. Since the thermal power is changing with time, Tian’s equation is applied for calculation of the thermal power75_:

𝑎𝑎𝑤𝑤= 1 −_𝑃𝑃𝑚𝑚𝑚𝑚𝑚𝑚𝑃𝑃 (2.3)

(2.2) where the time constant

is determined in calibration experiments.

Figure 2.1 A sorption calorimetric cell (left figure from www.mah.se)

The activity of water is calculated from the thermal power of evaporated water registered in vaporization chamber and a simplified equation can be presented as76_:

Equations for Chapter2. Capital letters are more pronounced than in template file.

max

1 P

P

a

_w

=

−

(2.3) vap sorp vap w vap w mix w

H

P

_P

H

=

+

(2.4) (2.3)

where P is the thermal power of vaporisation and Pmax_{is its maximum value. In} practice, a more complicated equation is used for calculations76_.

The partial molar enthalpy of the mixing of water (often referred to as the enthalpy of hydration) is calculated using the following equation:

P

sorp

Effect of hydration on thermodynamic, rheological and structural properties of mucin

YANA ZNAMENSkAYA

EffECT Of HYDRATION

ON THERMODYNAMIC,

RHEOLOgICAL AND

STRUCTURAL pROpERTIES

Of MUCIN

Malmö University

Health and Society, Doctoral Dissertation 2013:2

yana Znamenskaya

effect of hydration on

thermodynamic, rheological

and structural properties

of mucin

Malmö University, 2013

Faculty of Health and Society,

Biomedical Science Department

contents

abbreviations and symbols

ABBREVIATIONS AND SYMBOLS

ABBREVIATIONS AND SYMBOLS

ABBREVIATIONS AND SYMBOLS

abstract

list of papers

chapter 1 introduction

mucous gel (mucus) and mucins

Mucin

Mucin molecule

Models of pig gastric and bovine submaxillary gland mucins

(PGM and BSM)

10

Non-glycosylated or bare parts of the mucin molecule are rich in cysteine, found in the N-

and C-terminal ends of the protein core. Cysteine-rich domains are responsible for

dimerization via disulphide bond formation, and afterwards polymerizations of these dimers

to form mucin multimers (Figure 1.1). Disulphide bond formation plays an important role in

the gel forming properties of mucin

. The electrostatic character of mucins is determined by

the polypeptide backbone, for example, glutamic acid and aspartic acid residues (pK

=4)

and by carbohydrate side chains

, which are often negatively charged owing to the

presence of sialic acid residues (pK

=2.6) and sulphate groups (pK

=1)

. The isoelectric

point of mucin, caused by these acidic groups, have been determined to 2-3

. Such

negatively charged mucins may form polyelectrolyte complexes with positively charged

polymers, for example, with chitosan

.

Models of pig gastric and bovine submaxillary gland mucins (PGM and BSM)

As previously mentioned, the structural and physico-chemical properties of mucin vary

depending on its origin. The high molar mass of the mucin molecule, its polydispersity and

high degree of glycosylation

make this glycoprotein molecule complex and it is difficult to

characterise its structure. Hence mucin molecules are described using different models

. Often PGM and BSM are described with reference to a bottlebrush model (Figure 1.1): a

copolymer of relatively rigid (glycosylated) and relatively flexible (bare) moeties

. In

this mucin model, the carbohydrate side chain length can vary from 15 to 50 nm

.

Figure1.1 Schematic illustration of a bottlebrush mucin molecule

.

The PGM molecule has also been described as having a dumbbell shape (Figure 1.2),

presented as two asymmetric

or symmetric

globules per chain separated by a heavily

Glycosylated (Carbohydrate part)

Bare (Protein part)

Disulphide bonds (S-S)

hydration of mucin

11

glycosylated spacer. The carbohydrate side chain length for a dumbbell shaped molecule is

different than that for a bottlebrush, and it is about 2.5 nm

.

Figure 1.2 Schematic illustration of a dumbbell PGM molecule

.

The carbohydrate content of BSM is different in comparison with PGM having the higher

content; i.e., the carbohydrate content in PGM is about 83-86 wt%

, and in BSM it is about

_{. The electrostatic character of mucins is determined by}

_{, which are often negatively charged owing to the}

_{. Such}

_.

_{make this glycoprotein molecule complex and it is difficult to}

_{. Often PGM and BSM are described with reference to a bottlebrush model (Figure 1.1): a}

_{. In}

_.

_{or symmetric}

_{globules per chain separated by a heavily}

_.

_.

_{, and in BSM it is about}

_.

_{, therefore PGM is considered as a weakly charged molecule}

_{. BSM}

_.

_{; consequently dehydration of living systems results in dysfunction, disease, or ultimately}

��=��0

_P