Academic year 2012-2013

CHARACTERIZATION OF RHEOLOGICAL AND MASS TRANSPORT PROPERTIES OF HYALURONIC ACID FOR

MEDICAL USE

Charlotte COURTENS

First Master of Drug Development

Promoter Prof. Dr. K. Braeckmans

Co-promoters

Prof. Dr. A. Larsson Dr. N. Lorén

Commissioners Dr. K. Remaut

Prof. B. De Geest

GHENT UNIVERSITY FACULTY OF PHARMACEUTICAL SCIENCES Department of Pharmaceutics Laboratory of Genaral Biochemistry and Fysical Pharmacy

Master thesis performed at:

CHALMERS UNIVERSITY OF TECHNOLOGY Department of Chemical and Biochemical

Engineering Laboratory of Pharmaceutical Technology

Academic year 2012-2013

CHARACTERIZATION OF RHEOLOGICAL AND MASS TRANSPORT PROPERTIES OF HYALURONIC ACID FOR

MEDICAL USE

Charlotte COURTENS

First Master of Drug Development

Promoter Prof. Dr. K. Braeckmans

Co-promoters

Prof. Dr. A. Larsson Dr. N. Lorén

Commissioners Dr. K. Remaut

Prof. B. De Geest

GHENT UNIVERSITY FACULTY OF PHARMACEUTICAL SCIENCES Department of Pharmaceutics Laboratory of Genaral Biochemistry and Fysical Pharmacy

Master thesis performed at:

CHALMERS UNIVERSITY OF TECHNOLOGY Department of Chemical and Biochemical

Engineering Laboratory of Pharmaceutical Technology

COPYRIGHT

"The author and the promoters give the authorization to consult and to copy parts of this

thesis for personal use only. Any other use is limited by the laws of copyright, especially

concerning the obligation to refer to the source whenever results from this thesis are cited."

June 3, 2013

Promoter Author

Prof. Dr. Kevin Braeckmans Charlotte Courtens

SUMMARY

In recent years, research in the field of drug delivery has become increasingly important. To

treat diseases more efficiently, there is a growing interest in new ways to deliver drugs in a

more controlled way. Among the most important applications are sustained release

formulations and targeting of drugs. In the search for new drug delivery systems, polymers

play a very important role. Hyaluronic acid (HA) is a natural polysaccharide that can be used

for these applications.

To further research possible drug delivery applications of HA, it is essential to know how long

it takes for the molecules to diffuse out of the HA network. Also, as HA is a natural polymer,

its characteristics potentially depend on the source, isolation and purification method.

Therefore, it is important to look closer into the functional similarities and differences

between HA originating from different sources. We have compared HA from the two mainly

used sources: rooster comb and bacterial or fermented HA.

The bacterial HA used has a molecular weight (MW) of 1,7MDa and a critical overlap

concentration c* of 0,070%. The rooster comb HA used has a MW of 2,7MDa and a c* of

0,046%. The rheology results obtained indicate that the flow properties of HA are

determined by its MW and that there are no intermolecular interactions. As for diffusion, we

have seen that small molecules are not hindered at all. Diffusion rate generally decreases

with increasing MW and/or hydrodynamic radius of the probes. Positively charged molecules

interact with HA and diffuse slowly. Overall, diffusion rate in bacterial and rooster comb HA

is similar. Therefore, there is no reason to prefer the use of rooster comb HA to fermented

HA.

To better understand the implications of the differences between rooster comb and

bacterial HA, future research should be done. Fractionation of HA by size exclusion

chromatography (SEC) should be done in order to further determine the polydispersity. This

way, it would be possible to reduce the impact of polydispersity in MW on the final results.

This would result in a more correct and precise determination of MW and its influence on

rheology. It could also be interesting to calculate the pore size of the network and see if

there is a difference between the rooster comb and fermented HA. As for diffusion, it would

be interesting to make measurements with fluorescently labelled drugs.

SAMENVATTING

De laatste jaren wordt onderzoek op het gebied van drug delivery steeds belangrijker. Om

ziektes efficiënter te behandelen wordt meer en meer aandacht besteed aan hoe we

geneesmiddelen zodanig kunnen afleveren dat we meer controle hebben over waar en

wanneer ze vrijkomen in het lichaam. In dit opzicht zijn vooral vertraagde afgifte en

targeting van geneesmiddelen belangrijke onderzoeksdomeinen. In de zoektocht naar

nieuwe drug delivery systemen spelen polymeren een belangrijke rol. Hyaluronzuur (HA) is

een natuurlijk polysacharide dat voor deze toepassingen gebruikt kan worden.

Bij ontwikkeling van drug delivery systemen op basis van HA is het essentieel te weten

hoelang de diffusie van moleculen in het polymeernetwerk duurt. Aangezien HA een

natuurlijk polymeer is, kunnen diens eigenschappen bovendien afhankelijk zijn van de bron,

isolatie- en opzuiveringsmethode. Daarom is het belangrijk functionele gelijkenissen en

verschillen tussen HA van verschillende bronnen beter te onderzoeken. In deze studie werd

HA van twee frequent gebruikte bronnen vergeleken: hanenkam en bacterieel HA.

Het bacterieel HA heeft een moleculair gewicht (MW) van 1,7MDa en een kritische overlap

concentratie c* van 0,070%. Hanenkam HA heeft een MW van 2,7MDa en c* van 0,046%. De

rheologie resultaten wijzen erop dat de stromingseigenschappen van HA bepaald worden

door diens MW en dat er geen intermoleculaire interacties zijn. Op het vlak van diffusie

hebben we vastgesteld dat kleine moleculen niet gehinderd worden bij eender welke HA

concentratie. Diffusiesnelheid neemt in het algemeen af met toenemend MW en / of Stokes-

Einstein straal van de probes. Positief geladen moleculen interageren met HA en diffunderen

langzaam. In het algemeen is diffusiesnelheid in bacterieel en hanenkam HA vergelijkbaar. Er

is dus geen reden om hanenkam HA te verkiezen voor therapeutische toepassingen.

Om de implicaties van het verschil tussen hanenkam en bacterieel HA beter te begrijpen,

moet verder onderzoek worden gedaan. Fractionering van HA door gelchromatografie (SEC)

moet worden gedaan om de polydispersiteit van HA te bepalen. Dit resulteert in een meer

correcte en nauwkeurige bepaling van het MW en de invloed ervan op de rheologie. Het kan

ook interessant zijn om de poriëngrootte van het netwerk te berekenen en na te gaan of dit

verschilt tussen hanenkam en bacterieel HA. Op vlak van diffusie zou het interessant zijn om

metingen te doen met fluorescent gelabelde geneesmiddelen.

ACKNOWLEDGEMENTS

First of all, I would like to thank Dr. Anna Ström and Dr. Erich Schuster for being the best

supervisors I could’ve asked for. Thank you for your good guidance and positive spirit.

Without your time and energy, I never would have been able to finish this work as well as I

have done now.

I also would like to express my gratitude to Prof. Dr. K. Braeckmans, Prof. Dr. A. Larsson and

Dr. N. Lorén for giving me the opportunity to do my master thesis at Chalmers and SIK.

Thank you to all the kind people at Chalmers and SIK. A special thank you goes to Johanna for

introducing me to Zotero and saving me a lot of precious time.

I also want to thank my family and friends for always supporting me and being there for me

when I need them.

Of course, I would like to thank all the wonderful people I have met in Sweden.

Special thanks go to Peter, Stu, Diego, Ching Chiao, Tone, Toon, Jonathan, Caroline, Victor,

Esther, Nicole, Lucile and Tawan. Thank you for making this Erasmus experience the best

time of my life. “How lucky I am to have something that makes saying goodbye so hard.”

Last but not least, I want to thank Tine. I couldn’t have asked for a better Erasmus

compagnon. I’m really glad I got to know you during our stay in Sweden! We didn’t know

each other before and now you’re one of my best friends Thank you for all the good times!

TABLE OF CONTENTS

1 INTRODUCTION .................................................................................................................................................. 1

1.1 HYALURONIC ACID ............................................................................................................................................. 1

1.1.1 Bacterial versus rooster comb HA ............................................................................................................ 3

1.1.2 Biological functions of HA ............................................................................................................................ 4

1.1.3 HA derivatives .................................................................................................................................................... 5

1.1.4 Therapeutic use of HA and its derivatives ............................................................................................. 6

1.1.5 Physical structure and rheology of HA ................................................................................................... 7

1.2 RHEOLOGY ............................................................................................................................................................. 9

1.3 DIFFUSION ............................................................................................................................................................ 10

1.4 CONFOCAL LASER SCANNING MICROSCOPY ......................................................................................... 12

1.5 FLUORESCENCE ................................................................................................................................................. 13

1.6 FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING ................................................................. 14

2 OBJECTIVES .............................................................................................................................................. 17

3 MATERIALS AND METHODS ............................................................................................................... 18

3.1 MATERIALS .......................................................................................................................................................... 18

3.2 METHODS ............................................................................................................................................................. 21

3.2.1 Rheology .............................................................................................................................................................21

3.2.2 FRAP .....................................................................................................................................................................21

4 RESULTS AND DISCUSSION ................................................................................................................. 24

4.1 RHEOLOGY ........................................................................................................................................................... 24

4.2 FRAP ........................................................................................................................................................................ 32

4.2.1 Probe diffusion in PBS solutions ..............................................................................................................32

4.2.2 Probe diffusion in HA solutions ................................................................................................................33

4.2.3 Comparison of the probe diffusion in fermented and rooster comb HA................................38

5 CONCLUSIONS .......................................................................................................................................... 40

6 REFERENCES ............................................................................................................................................ 41

ABBREVIATIONS

CLSM Confocal laser scanning microscopy

CD44 Cluster of differentiation 44

DNA Desoxyribonucleic acid

DGL Dendri-Graft poly-L-lysine

FITC Fluorescein isothiocyanate

FITC-PEG-NH2 Fluorescein isothiocyanate polyethylene glycol amine

FRAP Fluorescence recovery after photobleaching

GAG Glycosaminoglycans

HA Hyaluronic acid / Hyaluronan

HAS HA synthase

MW Molecular weight

NaF Sodium fluorescein

PBS Phosphate buffered saline

RHAMM Receptor for HA-mediated motility

RNA Ribonucleic acid

UDP Uridine diphosphate

1

1 INTRODUCTION

Hyaluronic acid or hyaluronan (HA) is a natural polysaccharide widely spread in eukaryotes.

It is also synthesized by some bacteria. Possibly the most interesting characteristic of HA is

its viscoelasticity. Solutions of HA are viscoelastic due to chain entanglement. HA is a highly

hydrated polymer and the chains start to form an entangled network at relatively low

concentration. This is essential for its lubricating function in joints, muscles and other

tissues.

Commercially available HA is extracted from animal sources, mainly rooster comb, or

isolated from bacteria, mostly Streptococci. Although the chemical structure of HA is

identical in all tissues, HA characteristics and impurities are dependent on the source,

isolation and purification method. This could lead to significant differences, for example in

rheological behaviour, with an important impact on therapeutic activity.

We are interested in HA and its rheological properties because of possible applications of HA

in drug delivery. In recent years, research in the field of drug delivery has become

increasingly important. If we want to develop HA sustained release formulations, it is

essential to know how long it takes for the drug to diffuse out of the HA network. Diffusion

was investigated by fluorescence recovery after photobleaching (FRAP) measurements.

This introduction further discusses HA and gives a brief introduction concerning the

theoretical background of the experiments. The objectives of this study are formulated in

Chapter 2. In Chapter 3 the used materials and methods are described. The results are

written down and discussed in Chapter 4. The conclusions of this study are summarized in

Chapter 5.

1.1 HYALURONIC ACID

Hyaluronic acid (HA) is a linear polysaccharide discovered by Karl Meyer and John Palmer in

1934. The name refers to hyalos, the Greek word for glassy, since it was first found in the

vitreous body of the bovine eye. It consists of alternating disaccharide units of D-glucuronic

acid and N-acetyl-D-glucosamine linked by β–(1–3) and β–(1–4) bonds as illustrated in Figure

2

1.1. Under physiological conditions, it behaves as a salt, hence the term hyaluronate. The

molecule is generally referred to as hyaluronan [1].

Figure 1.1: Chemical structure of HA [2].

HA belongs to the group of glycosaminoglycans (GAG), previously called acid

mucopolysaccharides or connective tissue polysaccharides. Other members of this group are

heparin, heparan sulphate, keratan sulphate, chondroitin sulphate and dermatan sulphate

[3]. These molecules are linear polysaccharides consisting of the amino sugar hexosamine,

mostly in the N-acetylated form, and hexose residues. HA differs from other GAG in several

ways. It doesn’t contain sulphate groups and its molecular weight (MW) is relatively high.

Also, the polysaccharide chain is not covalently linked to protein. Therefore its synthesis

takes place in the plasma membrane rather than in the endoplasmic reticulum and Golgi

apparatus. In contrast to other GAG, sugar residues are added at the reducing end of the

chain [4][5]. The precursors are UDP-glucuronic acid and UDP-N-acetylglucosamine. In

mammals, HA is synthesized by three different types of HA synthases (HAS), HAS1, HAS2 and

HAS3, and degraded by three different types of enzymes. Hyaluronidase, also called hyase,

cleaves HA into oligosaccharide fragments. These fragments are degraded by β-D-

glucuronidase and β-N-acetyl-hexosaminidase by removing non-reducing terminal sugars [1].

HA is widely spread in eukaryotes. Especially high concentrations are found in soft

connective tissues such as skin, synovial fluid, umbilical cord and the vitreous body. Lung,

kidney, brain and muscle contain considerable amounts too [4]. HA serum concentration is

usually low, but high serum levels have been observed in patients with liver cirrhosis, due to

impaired uptake in the liver, and rheumatoid arthritis, due to increased production [6]. HA

can also be synthesized by prokaryotes, namely as a component of the cell coat in some

bacteria [7].

3

1.1.1 Bacterial versus rooster comb HA

Commercially available HA is extracted from animal sources, mainly rooster comb, or

isolated from bacteria, mostly Streptococci. Several bacteria produce HA as a capsule, which

is an important virulence factor [8][9]. Because its chemical structure is source independent,

exogenously applied HA is biocompatible and biodegradable [10]. It is important to note

however that although the chemical structure of HA is identical in all tissues, HA

characteristics and impurities are dependent on the source, isolation and purification

method. This could lead to significant differences, for example in rheological behaviour, with

an important impact on therapeutic activity [7], [11]. The use of bacterial HA will probably

become increasingly important as its production cost is lower than for rooster comb HA

combined with the growing demand of HA for therapeutic applications. Also, the potential

presence of viral agents in animal HA and the use of animals in general make animal derived

HA a less attractive product for biomedical applications [8][10][12][13]. Therefore it is

important to look closer into the functional similarities or differences between the two HA

sources.

In literature, differences between rooster comb and bacterial HA are briefly mentioned,

mostly stating that rooster comb HA is probably contaminated with proteins, whereas

fermented HA is relatively pure [13][14][15]. Also, rooster comb HA is sometimes mentioned

to be of higher molecular weight than bacterial HA [16], [17][18]. However, data originating

from actual comparison between HA from these two sources are rare.

One study comparing human umbilical cord, bovine vitreous, rooster comb and

streptococcal HA has been found. Measurements indicate that bacterially derived and

rooster comb HA are quite similar in MW and its polydispersity. Also, both HA sources have

low total protein, RNA and DNA contaminations. The only significant difference is endotoxin

content, which is a lot higher in rooster comb HA than in bacterially derived HA. However,

this should not lead to differences in physico-chemical properties of the HA solutions [16].

Another study compares several rooster comb and bacterial HA samples rheologically. The

results don't really show a consistent difference between the two sources. Also, the rooster

comb HA used wasn't necessarily of higher MW than the bacterial HA. Rheological behaviour

was generally found to be related to MW, with the exception of two of the bacterial samples,

4

which had lower respectively higher viscosities than expected. No explanation was given for

these exceptions [17].

1.1.2 Biological functions of HA

HA is a molecule with interesting properties essential for its physiological functions. First of

all, HA plays an important role in water homeostasis. The HA network is highly resistant to

water flow as the dynamics of the transient network are relatively slow. As a result, HA is an

efficient barrier against quick changes in water content [6]. Also, HA acts as an osmotic

buffer because of its non-ideal osmotic activity. Osmotic pressure of HA solutions changes

exponentially with increasing concentrations [19]. Secondly, the HA network acts as a sieve

or filter for other molecules. Small molecules are able to diffuse freely in the network, while

larger particles are slowed down or immobilized. This way, HA is able to regulate the

transport of molecules through the interstitial space. Additionally, the HA network takes up

a certain volume of solvent so it is not available for other macromolecules anymore. This

phenomenon is called steric exclusion and plays a major part in influencing chemical

equilibria of reactions with macromolecules and regulating partition of proteins between

tissue compartments [20]. Finally, possibly the most interesting characteristic of HA is its

viscoelasticity. Solutions of HA are viscoelastic due to chain entanglement. Neither viscosity

nor elasticity is constant, but dependent on the shear rate and frequency respectively. This

will be discussed in further detail in 1.1.5. These rheological properties are essential for its

lubricating function in joints, muscles and other tissues. Particularly useful is the shear-

thinning behaviour of HA because this results in a low viscosity during rapid movements and

a high viscosity during slow movements [4][21].

In addition to functions arising from its intrinsic physico-chemical properties, HA also

exhibits functions by specifically binding to proteins, generally called hyaladherins. Different

groups of hyaladherins have different functions. A first group of HA-binding molecules are

matrix components, which bind to HA to form stabilized structures. One example are the

aggrecans, previously called cartilage proteoglycans. A second group of hyaladherins are cell

surface receptors. This group contains among others the CD44 and RHAMM receptors [21].

The combination of physico-chemical properties and specific interactions form the basis for

the role of HA in several biological processes such as inflammation, cell migration,

5

proliferation and aggregation [1].

1.1.3 HA derivatives

HA derivatives are mainly produced to slow down the degradation rate of exogenously

applied HA and to enhance the rheological properties of the HA solutions. As can be seen in

Figure 1.2, two ways of chemically modifying HA can be distinguished [22].

Figure 1.2: Chemical conjugation versus chemical crosslinking [22].

Conjugation is the process in which a compound is connected to HA by a single bond.

Crosslinking is the process in which different HA molecules are linked by two or more bonds.

The main targets for both conjugation and crosslinking are the carboxylic acid group, the

hydroxyl groups and, after deamidation, the N-acetyl group [22]. An example of conjugated

HA derivatives are HA esters. The HA carboxyl group is esterified with therapeutic or

nontherapeutic alcohols. HA esters are insoluble in water [3]. An example of crosslinked HA

derivatives are hylans. These are produced by chemically crosslinking the hydroxyl groups of

the polymer chains [23]. The carboxylic acid and the N-acetyl groups are not affected. The

crosslinking process makes it possible to improve the rheological properties without largely

affecting the biocompatibility [3]. Hylans produced via formaldehyde crosslinking are soluble

in water and are more elastic and viscous at low frequencies and shear rates than HA. Hylans

produced via vinylsulfone crosslinking form infinite molecular networks (gels, membranes,

microparticles) and are more elastic at all frequencies and more viscous at lower shear rates

than HA [24].

6

1.1.4 Therapeutic use of HA and its derivatives

HA and its derivatives can be used therapeutically in several practical applications due to its

unique properties and its biocompatibility, biodegradability and non-immunogenicity. HA

can be used in viscosurgery as a space-filling agent and to protect delicate tissues.

Viscosurgery with HA is mainly used in ophthalmology [25]. In viscoaugmentation, HA is used

as a bulking substance. Possible applications are the correction of leaking sphincters and the

treatment of glottal insufficiency. HA can also be injected into the skin to fill wrinkles [21].

Viscoseparation is the separation of damaged tissue surfaces, often post-surgical, in order to

prevent adhesion, facilitate wound healing and decrease scar formation [25]. Esterified HA

has also been used to prevent bacterial adhesion to biomaterials such as implants, lenses

and catheters [1]. Viscosupplementation is the use of HA solutions to replace or supplement

tissue fluids, usually pathological synovial fluid. Pathological synovial fluid differs from

healthy synovial fluid because HA is degraded by radicals produced during inflammatory

reactions. Viscosupplementation can be applied in arthritis to relieve the pain and improve

the joint mobility [1][3]. In viscoprotection, HA is used to protect tissue surfaces from

dryness or harmful environmental agents, and to facilitate the healing of those surfaces [25].

Gels and solutions of HA and its derivatives are also being used for drug delivery via various

routes of administration. Ophthalmic drug delivery is often problematic because the

majority of the drug is washed away seconds after administration. Viscosity enhancers can

be used to increase the ocular residence time and as a result the bioavailability of the drug.

HA is well suited for this application. Because of its viscoelastic behaviour, blinking reduces

the viscosity resulting in little to no irritation. HA is bioadhesive, which also contributes to

the increase in drug bioavailability. This is applied in nasal delivery as well. Because of the

breakdown and poor absorption of peptides in the digestive tract, this is especially

interesting as an alternative for oral delivery of peptides. In parenteral drug delivery, HA can

be used as a carrier, for example in subcutaneous sustained release formulations of peptides

and proteins [3]. In this application, HA is useful not only as a carrier, but also as an enhancer

of lysosomal entrapment. Topical drug delivery has many advantages over systematic drug

delivery such as avoidance of first-pass metabolism, but is not easily achieved. The stratum

corneum blocks diffusion of most molecules, especially hydrophilic ones, very efficiently.

When this barrier is overcome, the drugs often penetrate all the way into the dermis and are

7

easily washed away from their site of action by the blood stream. This can lead to systematic

side effects. As a result, there is a need of a system that allows the drug to cross the stratum

corneum, but makes sure it is not transported into the bloodstream. One possibility is the

use of HA [7]. Research has been done on lymphatic drug delivery by coupling the drug to

HA. This could be useful because dissemination of tumors often occurs via this way. HA can

also be used in pulmonary delivery and as implant material [3]. In addition to the previous

applications, HA can be used for gene delivery. The polymer matrix releases the DNA slowly,

thereby protecting the DNA from degradation and prolonging the duration of action [26].

Another potential application of HA is tissue engineering. The idea is to create a polymer

network, which acts as a scaffold for cells to grow on [27]. Tissue engineering has received

much interest because it is an interesting alternative for organ transplantation. This is

important because of the lack of donor organs [28].

1.1.5 Physical structure and rheology of HA

HA behaves as an extended, stiffened random coil in solution. This is the result of helix

formation by stabilizing hydrogen bonds parallel to the axis of the chain as can be seen in

Figure 1.3 [29].

Figure 1.3: HA inter-residue hydrogen bonding [29].

The helix also displays relatively hydrophobic regions formed by the axial hydrogen atoms.

This could form the basis for chain-chain associations. The random coil conformation of HA

in solution can be regarded as spherical with a radius of gyration of about 200nm. As can be

seen in Figure 1.4, this indicates the mean distance between the mass center and the outer

layer of the sphere [3][30].

8

Figure 1.4: Radius of gyration.

HA is a highly hydrated and relatively stiff polymer leading to the formation of an entangled

network at relatively low concentration [6]. Increasing the polymer concentration, three

domains can be defined: dilute, semi-dilute and concentrated. In dilute solutions, the single

chains do not interact with each other. In concentrated solutions, coils are overlapping. The

semi-dilute domain is the concentration region where there is only a certain degree of chain

overlapping. c* is the concentration on the border of the dilute and semi-dilute region [15].

HA solutions are viscoelastic and exhibit non-Newtonian behaviour. HA solutions are

pseudoplastic, which means that the viscosity decreases with increasing shear rate [1]. This

is the result of alignment of the chains, which makes the chains flow easier [3]. The elasticity

is frequency-dependent [24]. At low strain frequencies, the viscous behaviour dominates

because the chains have enough time to take up their original conformation while the stress

is being applied. At high strain frequencies, the behaviour of the fluid is mainly elastic, as the

frequency at which the strain is applied is faster than the time scale of topological

interactions [31].

Because of its polyanionic character, the rheological properties of HA are dependent on pH

and ionic strength[7]. Studies show a viscosity optimum at pH 7 with decrease of viscosity

due to breakdown of the hydrogen bonds at lower and higher pH values. This is especially

notable below pH 2,5 to 3,5 and above pH 12,5. Below pH 2,5 to 3,5, the HA carboxyl groups

are protonated and thus not ionized while above pH 12,5, the HA conformation is disturbed

because of ionization of the hydroxyl groups [3][5][32]. At pH 2,5, HA solutions are more

elastic than at other pH values. This could be the result of distinct chain stiffening and

9

increased tendency for chain-chain aggregation. This is the result of a balance between the

attractive and repulsive forces between the chains [3][33][29][32]. An increase in ionic

strength results in a decrease of the viscosity because of intermolecular charge screening

[6][29].

1.2 RHEOLOGY

Rheology studies the relationship between the stress applied on a material and the

deformation or flow of the material. Two main parameters are discussed: viscosity and

elasticity. Viscosity is a measure of energy dissipation. It is also defined as the resistance of a

fluid to flow, as expressed in Newton’s law of viscosity (1.1).

σ = ηγ’ (1.1)

where: σ: stress (Pa)

η: viscosity (Pa.s)

γ’: shear rate (s-)

Elasticity is a measure of reversible deformation or recoverable energy. This is expressed in

Hooke’s law (1.2).

σ = Gγ (1.2)

where: σ: stress (Pa)

G’: elastic modulus (Pa)

γ: strain (1)

Most materials are not purely viscous or elastic, but are viscoelastic. This means that

deformation is partly reversible and partly irreversible, as illustrated in Figure 1.5.

10

Figure 1.5: Strain γ as a funtion of time in response to a period of stress σ for a viscoelastic material.

Most viscoelastic materials are non-Newtonian, which means that the viscosity is dependent

on the shear rate. The viscosity of pseudoplastic or shear-thinning materials decreases with

increasing shear rate, while the viscosity of dilatant or shear-thickening materials increases

with increasing shear rate. This is the result of structure changes in the material in response

to shear rate changes. Sometimes these structural changes take some time, which results in

a time-dependent viscosity. At constant shear rate, thixotropic materials are time-thinning,

while rheopectic materials are time-thickening [34].

1.3 DIFFUSION

Diffusion of suspended pollen is caused by the random thermal motion of the molecules in

the liquid. This was observed by Robert Brown in 1827 and then explained by Albert Einstein

in 1905, and is also called Brownian motion [35][36]. According to Fick’s laws of diffusion

(1.3 - 1.4), particles spontaneously diffuse from an area with high concentration to areas

with lower concentrations until equilibrium is reached. If the particle distribution is

homogenous, diffusive exchange will continue, but there will be no net flux [36]. Fick’s first

law (1.3) describes the transport between two compartments in steady state condition.

11

J=-D.

(1.3)

where: J: diffusion flux (

)

D: diffusion coefficient (m2/s)

: concentration (

)

x: position (m)

Fick’s second law of diffusion (1.4) describes how the concentration changes with time as a

result of diffusion. This equation is also referred to as the diffusion equation.

=D.

(1.4)

where: C: concentration (

)

t: time (s)

D: diffusion coefficient (m2/s)

x: position (m)

The diffusion coefficient depends on temperature, viscosity of the solvent, size and shape of

the molecule and concentration. For spherical particles, this is described by the Stokes-

Einstein equation for infinitely diluted solutions (1.5) [36].

D=

(1.5)

where: D: diffusion coefficient (m2/s)

R: gas constant (8,314m3PaK-1mol-1)

T: temperature (K)

η: viscosity (Pa.s)

r: hydrodynamic radius of the particle (m)

N: Avogadro constant (6,022x1023mol-1)

12

1.4 CONFOCAL LASER SCANNING MICROSCOPY

The principle of confocal fluorescence microscopy was developed by Marvin Minsky in the

1950s. The general idea is to improve the optical imaging resolution by removing the ‘out-of-

focus’ light before the image is formed. This way, the fluorescence emerging from the

regions above and below the focal plane does not disturb the signal from the region of

interest. The basic principle of confocal laser scanning microscopy (CLSM) is illustrated in

Figure 1.6 [37][38].

Figure 1.6: Basic principle of the CLSM

(a) conventional setup – (b) epi-illumination setup [39].

A laser beam passes through the entrance pinhole (a) and is then focused by a lens into a

small spot within the specimen (b), which is usually fluorescent. Scattered and reflected

laser light as well as any fluorescent light from the illuminated spot is then collected by the

objective lens. The light from the focused spot is focused at the exit pinhole (c). The points

(a), (b) and (c) are in conjugate focus or confocal, hence the name CLSM. The light emerging

from the planes below and above the region of interest is out of focus and thus not

transmitted to the detector.

13

Two different setups can be distinguished, the conventional setup and the epi-illumination

setup. In the epi-illumination setup there is only one lens, whereas in the conventional setup

there are two. The light is focused into the region of interest by the condensor lens and the

light is collected by the objective lens. Another difference is the use of a dichroic mirror in

the epi-illumination setup. The dichroic mirror acts as a beam splitter as only a relatively

small range of wavelengths can pass, while the others are reflected. As shown in Figure 1.5,

the excitation wavelength can pass through the dichroic mirror, while the fluorescent

wavelengths are reflected in the direction of the detector [39][38].

1.5 FLUORESCENCE

Molecules can be excited by absorption of the energy of electromagnetic radiation. Shortly

thereafter, the molecules will seek a lower energy state and thus release the absorbed

energy. If this relaxation process happens by the release of photons, it is called

photoluminescence. The process of photoluminescence is illustrated in the Jablonski energy

diagram, as shown in Figure 1.7 [40][41].

Figure 1.7: Jablonski energy diagram [41].

S0 represents the ground state, whereas S1 and S2 represent the first and second singlet

excited states. T1 represents the first triplet excited state. Several vibrational and rotational

energy levels are possible at each electronic energy state. Fluorescence and

phosphorescence are two types of photoluminescence, differing in the nature of the excited

state. Fluorescence is emission of light from the singlet excited state. The electron in the

excited orbital has opposite spin and is paired to the electron in the ground state orbital.

Therefore, transition to the ground state occurs in just 10-9 to 10-7 seconds.

14

Phosphorescence on the other hand is emission of light from the triplet excited state. The

electron in the excited orbital has the same spin orientation as the electron in the ground

state orbital. This results in slower emission rates usually ranging from 10-3 to 102 seconds.

Generally, molecules lose part of the absorbed energy internally, which results in the

emission of electromagnetic radiation with a longer wavelength than originally absorbed.

This difference in wavelength is better known as the Stokes shift. Another consequence of

this phenomenon is that the emission spectrum is usually independent of the excitation

wavelength used. Internal energy loss is caused by internal conversion, vibrational relaxation

and, less likely, intersystem crossing [40][41]. Vibrational relaxation also results in what is

known as the mirror image rule. This means that the lowest energy absorption spectrum is

the mirror image of the emission spectrum. This is because the energy difference between

the vibrational energy levels and the likelihood of vibrational energy transitions in the

excited states is the same as in the ground state [40][41][42].

Most molecules are either not naturally fluorescent or their intrinsic fluorescence is

inadequate for experimental use. As a result, the molecule has to be chemically converted to

a fluorescent molecule or extrinsic fluorescent probes have to be added to visualize the

sample of interest [43]. Fluorophores are typically aromatic compounds [41]. The

fluorescent probes used in this study are described in Chapter 3 (Materials and Methods).

1.6 FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING

Fluorescence recovery after photobleaching (FRAP) is a technique to measure the diffusivity

of fluorescently labeled probes, based on fluorescence intensity measurements as a function

of time and space. The basic principle is illustrated in Figure 1.8. The initial fluorescence Fi in

a specific area of the sample is measured. At t0, this region is illuminated by a high intensity

light beam resulting in partly photobleaching of the available fluorophores and a drop in

fluorescence to F0. This is called the photobleaching phase. Non-bleached fluorescent

molecules from the surrounding areas will diffuse into the bleached area replacing the

bleached molecules. As a result, the fluorescence in this region of the sample will recover to

Fi if all fluorescent molecules in the observed area are mobile or to F∞ (less than Fi) if only a

fraction of the fluorescent molecules in the observed area are mobile.

15

Figure 1.8: Basic principle of FRAP measurements [44].

Two parameters can be derived from FRAP measurements: the mobile fraction and the

apparent translational diffusion coefficient. The mobile fraction is derived from the amount

of fluorescence recovered at the end of the experiment. The translational diffusion

coefficient can be estimated by the characteristic diffusion time τd, which is the time

required for the fluorescence to recover to

[44][45][46]. More recently, quantitative

models have been developed, ready to use on commercial CLSM [47].

FRAP was originally developed to study molecular mobility in biological samples such as cell

membranes, cellular compartments and tissues. Nowadays, it is used to study diffusion in all

kinds of soft materials [45][46][48]. FRAP has become a valuable technique especially in

pharmaceutical research. Knowledge about diffusion of molecules is important in drug

delivery. Both release of the drug from its matrix as uptake of the drug at its target location

in the body are determined by diffusion. Especially polymer solutions and gels have gained a

lot of interest [45][46]. FRAP can also be used in binding studies by determining the fraction

of molecules that are immobile resulting in only partial recovery of fluorescence. Other

applications are immunological assays by coupling fluorescent labels to antibodies and flow

velocity measurements if movement by flow is significantly faster than diffusion. FRAP can

16

also be used in binding studies of antitumor drugs in tumor tissue, research on intracellular

mobility of gene complexes and mobility of drugs in membranes before transmembrane

uptake [45].

17

2 OBJECTIVES

We are interested in HA and its rheological properties because of possible applications of HA

in drug delivery. The polymer matrix releases the molecules slowly, protecting them from

degradation and prolonging the duration of action. If we want to develop HA sustained

release formulations, it is essential to know how long it takes for the drug to diffuse out of

the HA network.

Commercially available HA is extracted from animal sources, mainly rooster comb, or

isolated from bacteria, mostly Streptococci. Although the chemical structure of HA is

identical in all tissues, HA characteristics and impurities are dependent on the source,

isolation and purification method. This could lead to significant differences, for example in

rheological behaviour, with an important impact on therapeutic activity. The use of bacterial

HA will probably become increasingly important as its production cost is lower than for

rooster comb HA combined with the growing demand of HA for therapeutic applications.

Also, the potential presence of viral agents in animal HA and the use of animals in general

make animal derived HA a less attractive product for biomedical applications. Therefore it is

important to look closer into the functional similarities or differences between rooster comb

and fermented HA.

The first aim of my research is to investigate the diffusion of several probes of different size,

shape and charge. The probes used are sodium fluorescein, FITC-PEG-NH2, DGL, FITC-

Albumin and 10, 70 and 500kDa FITC-dextran. The second aim is to compare rooster comb

and bacterial HA regarding MW, rheological and mass transport properties. MW is

determined with the capillary viscometer. Flow properties are investigated by rotational

rheology measurements. Diffusion is investigated by fluorescence recovery after

photobleaching (FRAP) measurements.

18

3 MATERIALS AND METHODS

3.1 MATERIALS

Rooster comb HA was kindly provided by Bohus Biotech, Sweden. The water content as

reported by the supplier was 12,6%. HA from bacterial fermentation was also provided by

Bohus Biotech, Sweden. The bacterial HA was dissolved in phosphate buffered saline (PBS).

The concentrations of the two different batches (A and B) were 2,5% and 1% respectively.

The solvent used for all samples was PBS. The PBS was purchased from Sigma-Aldrich, USA,

yielding a 0,01M phosphate buffer, 0,0027M potassium chloride and 0,137M sodium

chloride solution of pH 7,4 at 25°C when dissolved in Milli-Q-water. Different lots of PBS

were used for rheology measurements (P4417-50TAB, 021M8221) and FRAP measurements

(P4417-50TAB, Lot SLBD8390V, PCode 1001449151).

Seven fluorescent probes of different MW and/or charge were used: NaF, 10kDa, 70kDa and

500kDa FITC-dextran, FITC-Albumin, DGL and FITC-PEG-NH2.

Sodium fluorescein (NaF) (Sigma-Aldrich, Standard Fluka, USA, 46960, Lot 456103/1, Filling

code 41606065) has a MW of 376,3Da and is the smallest probe that has been used [49].

Figure 3.1 shows the chemical structure of NaF, the sodium salt of fluorescein. It is

negatively charged in aqueous solutions.

Figure 3.1: Chemical structure of NaF [50].

Fluorescein is a fluorophore commonly used for FRAP applications. As can be seen in Figure

3.2, fluorescein has absorption maxima at 460 and 493,5nm. It is important to note however

19

that these spectra are strongly pH-dependent and that fluorescein is relatively sensitive to

photobleaching [49].

Figure 3.2: Absorption and fluorescence emission spectra of fluorescein in pH 9,0 buffer [51].

FITC-dextran of different MW have been used: 10kDa FITC-labeled dextran (D1821, Lot

682637), 70kDa FITC-labeled dextran (D1823, Lot 1231886) and 500kDa FITC-labeled dextran

(D7136, Lot 408193). All were purchased from Invitrogen Molecular Probes, USA. As shown

in Figure 3.3, dextrans are branched glucans consisting of α-1,6 linkages and α-1,3 branches.

Dextrans are hydrophilic and neutral. FITC-dextrans are slightly anionic due to the

fluorophore. The chain length of dextrans is variable so their MW is not fixed. Consequently,

dextrans are polydisperse. Dextran is fixed to fluorescein by succinimidyl coupling [52].

Figure 3.3: Chemical structure of FITC-dextran [52].

20

FITC-labeled albumin (Sigma-Aldrich, USA, A9771-50MG, 080M7400) is a carbohydrate-free

serum protein with a MW of 66kDa, which makes up 55 to 62% of the serum protein. The

albumin used was bovine albumin coupled to FITC through the ε-amino group of the lysine

residues of the albumin. The degree of substitution is 7 to 12 moles of FITC per mole of

albumin. The probe is negatively charged in aqueous solutions at pH 7,4 [53].

As can be seen in Figure 3.4, Dendri-Graft poly-L-lysine (DGL) is a dendrimer – a tree-like

polymer of L-lysine. The amino group of L-lysine is positively charged at pH 7,4. Therefore,

DGL is polycationic at physiological pH. The kind of DGL used in this experiment (Colcom,

France, DGL G3, Batch FI109-03) is made up of 123 lysine groups. With a MW of 22 kDa, DGL

is a relatively small probe [54].

Figure 3.4: Chemical structure of DGL [54].

The last probe used was fluorescein polyethylene glycol amine (FITC-PEG-NH2) (NANOCS,

USA, Lot 091010). Its chemical structure can be seen in Figure 3.5. It is a fluorescent PEG

derivative with a maximum absorption at 495nm and emission around 515-520nm. The

amino group is positively charged at pH 7,4. The probe used in this experiment had a MW of

10kDa [55].

Figure 3.5: Chemical structure of FITC-PEG-NH2 [55].

21

3.2 METHODS

3.2.1 Rheology

HA was weighed on a Shimadzu AUW220D scale and put into cups. The rooster comb HA

solutions were obtained by dissolving the polymer fibers in PBS. The bacterial HA solutions

were obtained by diluting the supplied solutions with PBS. After stirring, the solutions were

stored in the refrigerator to facilitate dissolving and to avoid degradation. All solutions were

prepared at least one day prior to the measurements in order to obtain full dissolution of

the sample.

Viscometric measurements were performed at 25°C with an automated Ubbelohde

viscometer (Schott-Geräte, Germany) equipped with capillary Type No. 531 0a. Each sample

was measured five times.

Rotational rheology measurements were carried out using a MCR300 Rheometer (Anton

Paar Physica, Germany, SN405539), using a CP50-1 cone-plate geometry (Anton Paar

Physica, Germany, diameter: 50mm, truncation: 52m and an angle: 1°) and a TEK150P-C

temperature control unit (Anton Paar Physica, Germany). The temperature during the

measurements was set at 25°C. The rotational rheology measurements were carried out at

shear rates of 0,01-1000s-1 and 1000 to 0,01 s-1 in approximately 30 minutes preceded by 30

seconds of pre-shear at 100s-1 and a resting period of 5 minutes.

3.2.2 FRAP

HA and the fluorescent probes were weighed on a Mettler Toledo XS204 scale and put into

cups. The fluorescent probes were added to PBS to obtain a concentration of 200ppm. This

concentration was chosen in order to get enough fluorescence intensity while staying in the

linear range of the relationship between fluorescence intensity and fluorochrome

concentration [47]. Rooster comb HA was dissolved in the probe solution and the bacterial

HA solutions were obtained by diluting the supplied solutions with the probe solutions. After

stirring, the cups were wrapped in aluminum foil to avoid photobleaching. The solutions

were stored in the refrigerator to facilitate dissolving and to avoid degradation. All solutions

were prepared at least one day prior to the measurements in order to obtain a homogenous

sample.

22

Bacterial HA measurements were performed on batch (A) only. To ensure that the results

are not batch dependent, the diffusion of each probe was measured at one concentration in

batch (B). Comparing these results to those obtained with the same concentration of batch

(A), we have established that indeed, diffusion in the two batches of fermented HA is not

significantly differing.

The microscope used was a Leica SP2 AOBS (Germany). The 488nm line of an argon laser was

used. Wavelengths between 500 and 550nm were detected. FRAP measurements were

performed at room temperature with a water based objective with 20 x magnification and a

numerical aperture of 0,5 and the following settings: 256 x 256 pixels, zoom factor 4 and

800Hz, yielding a pixel size of 0,732422μm and every 0,5 seconds images acquired. The

beam expander was set to 1. The FRAP images were stored as 12 bit images. The areas

bleached were 30μm large discs and measurements were made approximately in the middle

of the sample. The analysis was carried out in MATLAB utilizing a pixel-based likelihood

framework for analysis of the data [56]. An example of the FRAP measurement of 10kDa

FITC-dextran in 0,1% fermented HA is shown in Figure 3.6.

Figure 3.6: FRAP measurement of 10kDa FITC-dextran in 0,1% fermented HA, as observed under the microscope.

23

To avoid bleaching during non-bleaching periods, the laser intensity is set as low as possible

with the acousto-optical tunable filter. To ensure that there is no bleaching, the baseline

should be stable during 10 to 30 frames. During bleaching, the laser intensity is set at

maximum intensity. The amount of bleaching should be between 20 and 40%. If the amount

of bleaching is too low, the recovery curves are difficult to analyse. To boost bleaching, the

476 and 496 lines are used as well. This is preferred over increasing the bleach time since

FRAP theory assumes that there is no diffusion during the bleaching. If the amount of

bleaching is too high, the recovery dynamics are not described by the used analytical models

[47]. This can be solved experimentally by decreasing the laser intensity during bleaching.

The fluorescence recovery is followed for 30 to 50 frames.

24

4 RESULTS AND DISCUSSION

4.1 RHEOLOGY

As we are interested in the difference in MW between rooster comb and fermented HA, the

intrinsic viscosity of the polymer is measured. This value is related to the MW via the Mark-

Houwink-Sakurada equation (4.1) [57].

[η] =KMwa (4.1)

= 3,39 x 10-4 MW0,778 (105 < MW < 106Da)

= 3,95 x 10-3 MW0,604 (MW > 106Da)

where: K: proportional constant (dL/g)

a: Staudinger index

Mw: molecular weight (Da))

[η]: intrinsic viscosity (dL/g)

The Mark-Houwink-Sakurada equation is characterized by two constants. K is a proportional

constant depending on solvent conditions. The Staudinger index (a) on the other hand

depends on polymer flexibility. The value of (a) increases as flexibility decreases. Typically,

values between 0,5 and 0,8 are found for flexible polymer chains in a ‘good’ solvent while

values over 0,8 are found for stiff chains. Two different values have been used to calculate

the MW of HA. A value of 0,79 has been used extensively for HA and is found to be applicable

to low MW HA. A value of 0,6 is found to be applicable to high MW HA [57]. These values

indicate that low MW HA is relatively stiff, whereas high MW HA is more flexible.

The intrinsic viscosity is determined graphically after applying Hagenbach corrections. This is

done by combining the Huggins (4.2) and Kraemer (4.3) plots, as can be seen in Figure 4.1

and 4.2. The mean value of the y-intercept of the resulting plots is the intrinsic viscosity.

25

y =

(4.2)

where:

: relative viscosity (1)

c: HA concentration (g/dL)

y: reduced viscosity (dL/g)

y =

(4.3)

where:

= relative viscosity (1)

c: HA concentration (g/dL)

y: inherent viscosity (dL/g)

Figure 4.1: Derivatization of intrinsic viscosity by Huggins (nrel-1)/c and Kraemer ln(nrel)/c plots for bacterial HA.

y = -26,43x + 28,141

y = 2,0972x + 28,661

0

5

10

15

20

25

30

35

0 0,05 0,1 0,15 0,2 0,25

(nre

l-1

)/c

- ln

(nre

l)/

c (d

L/

g)

HA Concentration (%)

26

Figure 4.2: Derivatization of intrinsic viscosity by Huggins (nrel-1)/c and Kraemer ln(nrel)/c plots for rooster comb HA.

Although R2 values are rather low, the standard deviation within each point is relatively

small and the two values of the y-intercept are very close to each other. Initially, more

measurements were done at higher concentrations. However, these data are not taken into

account since it is probable that these concentrations are close to or exceeding c* while the

Huggins and Kraemer equations are applicable in the dilute region only.

The intrinsic viscosity of fermented HA was found to be 28,4dL/g. The intrinsic viscosity of

rooster comb HA was found to be 41,5dL/g. This corresponds to a MW of 1,70 (with (a)

0,778) – 2,43MDa (with (a) 0,604) respectively 2,75 (with (a) 0,778) – 4,55MDa (with (a)

0,604). Comparing to prior literature, the MW of both the fermented and rooster comb HA

agree well with reported data when calculated with the (a)-value of 0,778, while the number

obtained using the (a)-value of 0,604, appears relatively high compared to literature data

[58][59][60]. The Mw trend is consistent with what is generally stated in literature, namely,

the MW of rooster comb HA is higher than the MW of fermented HA [17][18]. Literature data

originating from actual comparison between HA from these two sources however are rare.

Two studies comparing rooster comb and bacterial HA have been found and both show that

rooster comb HA is not necessarily of higher MW than bacterial HA [16][17]. One study

comparing human umbilical cord, bovine vitreous, rooster comb and streptococcal HA has

y = -440,32x + 42,633

y = -746,03x + 40,401

0

5

10

15

20

25

30

35

40

45

50

0 0,005 0,01 0,015 0,02 0,025

(nre

l-1

)/c

- ln

(nre

l)/

c (d

L/

g)

HA concentration (%)

27

determined that bacterially derived and rooster comb HA are quite similar in MW and its

polydispersity. Also, both HA sources have low total protein, RNA and DNA contaminations.

The only significant difference is endotoxin content, which is a lot higher in rooster comb HA

than in bacterially derived HA. However, this should not lead to differences in rheological

behaviour [16]. Another study compares several rooster comb and bacterial HA samples

rheologically. The results don't really show a consistent difference between the two sources.

Also, the rooster comb HA used was not necessarily of higher MW than the bacterial HA.

Rheological behaviour was generally found to be related to MW, with the exception of two of

the bacterial samples, which had lower respectively higher viscosities than expected. No

explanation was given for these exceptions [17].

To determine c*, the viscosity of HA solutions of different concentrations is measured as a

function of shear rate. The viscosity is measured as the shear rate increases and decreases

so two measurements are taken at each shear rate. Plotting both results in two curves,

which superpose very well. This means that the HA network aggregates and disaggregates

very quickly in response to changing shear rate. This characteristic is useful for certain

applications, for example delivering HA solutions with a syringe. While injecting, the HA

solution is under stress and will behave as a liquid. Once HA is injected, the stress disappears

and the solution will immediately regain its gel-like behaviour. In Figure 4.3 and 4.4, only the

values found with the decrease of shear rate are plotted since these are expected to be less

subject to variation as the solutions have experienced an identical shear rate gradient for a

relatively long time.

At low shear rates, the viscosity is shear-independent. The zero shear viscosity is taken by

extrapolation of the viscosity plateau value and gives an idea of the extent of chain

entanglement. High zero shear viscosity values indicate abundant chain interactions. The

decrease in viscosity with increasing shear can be attributed to alignment of the polymer

chains, which makes the chains flow easier.

28

Figure 4.3: Shear rate dependence of the viscosity of bacterial HA solutions for a range of different concentrations.

1% 0,6% 0,3% 0,2% 0,15% 0,1% 0,05% 0,02% 0,01% 0,005%

Figure 4.4: Shear rate dependence of the viscosity of rooster comb HA solutions for a range of different concentrations.

1% 0,6% 0,4% 0,3% 0,2% 0,15% 0,1% 0,05% 0,02% 0,01% 0,005%

0,001

0,01

0,1

1

10

100

1000

0,01 0,1 1 10 100 1000

Vis

cosi

ty (

Pa

.s)

Shear rate (1/s)

0,001

0,01

0,1

1

10

100

0,01 0,1 1 10 100 1000

Vis

cosi

ty (

Pa

.s)

Shear rate (1/s)

29

Data points at low shear rates are omitted for the less concentrated solutions as the

viscosity values in this region are below the specifications of the rheometer. This results in

less accurate values of the zero shear viscosity of the less concentrated solutions. The zero

shear viscosity values at different concentrations are shown in Table 4.1.

Table 4.1: Zero shear viscosities of a concentration series of fermented and rooster comb HA solutions.

HA Concentration (%) Fermented HA Zero shear viscosity (Pa.s)

Rooster Comb HA Zero shear viscosity (Pa.s)

0 0,0010 0,0010

0,005 0,0012 0,0012

0,010 0,0017 0,0013

0,020 0,0022 0,0014

0,050 0,0036 0,0025

0,100 0,013 0,020

0,150 0,047 0,040

0,200 0,26 0,30

0,300 1,4 1,5

0,400 no measurement 8,0

0,600 40 60

1,000 2,2 x 1O2

30

To determine c*, the specific viscosity is plotted as a function of HA concentration. The

dilute and entangled concentration regions can be distinguished by a difference in slope. As

c* is the concentration on the border of the dilute and semi-dilute region, it can be

estimated from the intercept between the two lines. This is illustrated in Figure 4.5 and 4.6.

30

Figure 4.5: Determination of c* of bacterial HA by plotting specific viscosity as a function of HA concentration.

Figure 4.6: Determination of c* of rooster comb HA by plotting specific viscosity as a function of HA concentration.

c* was determined to be 0,070% for bacterial HA and 0,046% for rooster comb HA. As

expected, c* is higher as the MW is lower. These values are consistent with what is found in

the literature [2][15].

y = 73,617x1,0721 R² = 0,9557

y = 276361x4,4079 R² = 0,9959

0,001

0,01

0,1

1

10

100

1000

10000

100000

1000000

10000000

0,001 0,01 0,1 1 10

ηsp

HA concentration (%)

y = 2,8845x0,5 R² = 0,9905

y = 337145x4,2919 R² = 0,9818

0,001

0,01

0,1

1

10

100

1000

10000

100000

1000000

0,001 0,01 0,1 1 10

ηsp

HA concentration (%)

31

Plotting the specific viscosity as a function of the coil overlap parameter, which is the

concentration multiplied by the intrinsic viscosity, generates a universal curve for all random

coil polymers without specific intermolecular interactions. Therefore, data from bacterial

and rooster comb HA should superpose if we don’t have intermolecular interactions. The

data superpose quite well. Typical values for random coil polymers are a slope of 1,4 and 3,3

and the intercept of the two lines on a log-scale at a coil overlap parameter of 0,6 with a

specific viscosity of 1,0. As can be seen in Figure 4.7, the slope of both lines is slightly higher

than expected and specific viscosity and coil overlap parameter at the intercept are slightly

lower. This indicates that HA is a relatively stiff polymer.

Figure 4.7 The log-value of the specific viscosity is plotted as a function of the log-value of the coil overlap parameter.

Data from rooster comb and bacterial HA should superpose and generate a mastercurve if HA is a random coil

without intermolecular interactions.

Data at low concentrations are a bit scattered due to less accurate measurements of the

rheometer at such low viscosity values. We can see that data from the rooster comb HA are

systematically higher than those from the bacterial HA. This is probably due to minor errors

y = 1,1107x + 0,1599 R² = 0,7427

y = 4,6897x - 1,48 R² = 0,9644

-2

-1

0

1

2

3

4

5

6

-1 -0,5 0 0,5 1 1,5 2

logη

sp

logc[η]

32

in the value of the intrinsic viscosity as determined with the capillary viscometer. The overall

trend however is nicely followed.

4.2 FRAP

FRAP was used to measure the diffusion of probes of different size, shape and charge in HA

solutions. The probes used are NaF, FITC-PEG, DGL, FITC-Albumin and 10, 70 and 500kDa

FITC-dextran.

4.2.1 Probe diffusion in PBS solutions

To compare the FRAP measurements on different probes better, reference measurements of

the free diffusion coefficients of the probes in PBS, in the absence of HA, were performed.

These measurements allow us to calculate the hydrodynamic radius of the probes with the

Stokes –Einstein equation – see Equation (1.5). In Figure 4.8, the hydrodynamic radius of the

probes is plotted.

Figure 4.8: Hydrodynamic radius of the probes in PBS at 25°C, calculated by Stokes-Einstein equation.

Generally, we can see that the hydrodynamic radius of the probes increases with increasing

MW. However, comparing 10kDa FITC-dextran and FITC-PEG-NH2, two probes of identical

MW, we can see that the size of the probes does not only depend on the MW, but also on the

shape of the polymer. 10kDa FITC-dextran is a branched polymer whereas FITC-PEG-NH2 is a

flexible, linear polymer. Such polymers generally occupy a smaller volume in solution since

0

2

4

6

8

10

12

Hyd

rod

yn

am

ic r

ad

ius

rH (

nm

)

33

the chains are able to fold back on themselves. Also, the hydrodynamic radius depends on

the solvent and how the polymer interacts with it. Reference measurements rule out such

effects in the following analysis. We can now test the HA with this selection of probes with

sizes ranging from 1-30nm.

4.2.2 Probe diffusion in HA solutions

In the following part, we study the probe diffusion of the fluorescent tracers discussed

above, in a concentration series of HA. In Figure 4.9 we can see a quick overview of the

results. The diffusion coefficient of the probes in a 1% fermented HA solution is plotted as a

function of the MW of the probes.

Figure 4.9: Diffusion coefficient in 1% fermented HA solutions as a function of molecular weight of the probes.

NaF FITC-PEG-NH2 10kDa FITC-dextran DGL FITC-Albumin 70KDa FITC-dextran 500kDa FITC-dextran

Again, as expected, the overall trend is a decrease in diffusion coefficient as the MW

increases. However, 10kDa FITC-dextran and FITC-PEG-NH2, two probes of identical MW,

have different diffusion coefficients. As mentioned above, 10kDa FITC-dextran is a branched

polymer, while FITC-PEG-NH2 is a linear, flexible polymer. As a result, the diffusion of FITC-

PEG-NH2 is less hindered since the polymer occupies a smaller volume in solution and has

the flexibility to diffuse through the network with relative ease.

0

50

100

150

200

250

300

350

0,1 1 10 100

1%

HA

D (μ

m2/

s)

MW (kDa)

34

We will now analyse the data of the individual probes in closer detail and compare the

diffusion rate of different probes. This way it is possible to get an idea of how the size, shape

and charge affect the diffusion through the HA network. At first, the diffusion in the

fermented HA will be examined, followed by the diffusion in the rooster comb HA. Finally,

we will look at any differences in diffusion between fermented and rooster comb HA.

For three of the probes, 10kDa FITC-dextran, 70kDa FITC-dextran and DGL, we observed a

higher diffusion coefficient at 0,01% HA than the free diffusion coefficient. This is possibly

due to diluting the probe solutions with HA when preparing the samples, resulting in less

concentrated probe solutions and less hindrance. These data were normalised relative to the

diffusion coefficient found at 0,01% HA.

In Figure 4.10, the relative diffusion coefficients D/D0 of 10kDa, 70kDa and 500kDa FITC-

Dextrans are plotted as a function of HA concentration. Comparing the diffusion of these

probes is interesting as the shape and characteristics of these probes are identical. This way,

different results are the consequence of differences in MW only.

As discussed above, the rheological experiments yielded the following overlap

concentrations: for fermented HA c*=0,070%, and for rooster comb HA c*=0,046%. Below c*,

the FITC-dextran diffusion is not or only slightly hindered by the polymer chains. Above c*,

HA chains form an entangled network which hinders the probes from diffusing. As expected,

this effect is more important with increasing MW of the probe. However, we can see that

70kDa FITC-dextran diffuses more slowly than 500kDa FITC-dextran. This is probably the

result of different procedures of sample preparation. The 70kDa FITC-dextran probes were

dissolved in PBS and afterwards, HA was diluted with this solution. The 500kDa FITC-dextran

probes on the other hand were dispersed in a HA solution of the desired concentration. This

way, they may be less enclosed in the network and thus less hindered in motion. It would be

useful to repeat the measurements of 500kDa FITC-dextran to draw concise conclusions.

35

Figure 4.10: Relative diffusion coefficients of 10kDa, 70KDa and 500kDa FITC-dextrans as a function of HA

concentration in bacterial HA solutions.

Figure 4.11 shows the relative diffusion coefficients of NaF, DGL and FITC-PEG-NH2 as a

function of HA concentration. Although all of these probes are rather small, they strongly

differ in diffusivity.

Figure 4.11: Relative diffusion coefficients of NaF FITC-PEG-NH2 and DGL as a function of HA concentration in

bacterial HA solutions.

0

0,2

0,4

0,6

0,8

1

1,2

0 0,2 0,4 0,6 0,8 1 1,2

D/

D0

HA Concentration (%)

0

0,2

0,4

0,6

0,8

1

1,2

0 0,2 0,4 0,6 0,8 1 1,2

D/

D0

HA Concentration (%)

36

NaF is so small that diffusion is not hindered over the full concentration range. FITC-PEG-NH2

shows a slight decrease in diffusion rate with increasing HA concentration. This decrease is

rather limited as this polymer is very flexible and thus able to diffuse through the network

quite fluently. DGL on the other hand shows a large decrease in diffusion speed despite its

rather small size. This can be explained by the chemical structure of the probe. DGL is a

dendrimer with amine-modified ends, all of which are positively charged at physiological pH.

This leads to electrostatic interactions with the negatively charged HA. In Figure 4.12, we can

see the fluorescence recovery curve of DGL in a 0,1% fermented HA solution.

Figure 4.12: Fluorescence recovery curve of DGL in a 1% fermented HA solution.

The fluorescence does not fully recover, which also indicates that the molecules are

interacting with each other. It might be interesting to further analyse these interactions with

the FRAP and binding framework [61].

Figure 4.13 shows the relative diffusion coefficients of FITC-Albumin and 70kDa FITC-dextran

as a function of HA concentration. 70kDa FITC-dextran and FITC-Albumin are very similar in

MW. Comparing these probes indicates how diffusion is affected by the shape of the

molecule. FITC-Albumin is a spherical polymer, whereas FITC-dextran is a branched polymer.

FITC-Albumin is slightly less slowed down with increasing HA concentration than 70kDa FITC-

dextran. This is consistent with the fact that FITC-Albumin takes up less volume than 70kDa

FITC-dextran, as can be seen looking back at the hydrodynamic radius in Figure 4.8.

0,6

0,65

0,7

0,75

0,8

0,85

0,9

0,95

1

1,05

1,1

0 10 20 30 40 50 60

Flu

ore

sce

nce

in

ten

sity

Time (s)

37

Figure 4.13: Relative diffusion coefficients of FITC-Albumin and 70KDa FITC-dextran as a function of HA

concentration in bacterial HA solutions.

Looking at the rooster comb HA plots, we can see that the trends discussed above are

followed. This is shown in Figures 4.14 and 4.15.

Figure 4.14: Relative diffusion coefficients of NaF, FITC-PEG-NH2 and DGL as a function of HA concentration in

rooster comb HA solutions.

0

0,2

0,4

0,6

0,8

1

1,2

0 0,2 0,4 0,6 0,8 1 1,2

D/

D0

HA Concentration (%)

0

0,2

0,4

0,6

0,8

1

1,2

0 0,2 0,4 0,6 0,8 1 1,2

D/

D0

HA Concentration (%)

38

Figure 4.15: Relative diffusion coefficients of FITC-Albumin and 70KDa FITC-dextran as a function of HA

concentration in rooster comb HA solutions.

4.2.3 Comparison of the probe diffusion in fermented and rooster comb HA

Comparing diffusion of the probes in fermented versus rooster comb HA solutions, diffusion

rate is comparable. This is certainly the case at low HA concentrations. At higher HA

concentrations, we can see that there are some differences. FITC-PEG-NH2 and DGL are

slowed down more in rooster comb HA than in bacterial HA, while 70kDa FITC-dextran

diffuses faster in the rooster comb HA. A possible explanation for these observations is that

as the MW of rooster comb HA is higher, the network structure will consist of longer chains

at the same HA concentration. This suggests that the charged probes feel more attraction or

repulsion, depending on their charge, resulting in slower or faster diffusion. FITC-Albumin is

unaffected although it is negatively charged. This could be because the probe has

hydrophobic parts as well, thus compensating for the charge effect.

These results are useful as they provide us with a toolbox to estimate diffusion in the HA

network. As the probes used cover different sizes, shapes and charges, they can be related

to molecules that we want to deliver such as drugs and proteins. Small probes diffuse out of

the network as if dissolved in water, whereas large probes are hindered. Especially positively

0

0,2

0,4

0,6

0,8

1

1,2

1,4

0 0,2 0,4 0,6 0,8 1 1,2

D/

D0

HA Concentration (%)

39

charged probes diffuse very slowly. The time over which delivery occurs depends on the

molecule delivered and the HA concentration and can be calculated (4.4).

t =

(4.4)

where: x: mean square displacement (m)

D: diffusion coefficient (m2/s)

t: time (s)

Assuming HA systems with low concentrations of the drugs/active substances, in the range

of hundreds of ppm as investigated here via FRAP, this means that we can get extended

release over hours to days to weeks as necessary for the application. The diffusive release

times of the probes used in 1% fermented HA have been calculated and is shown in Table 4.2

for a mean square displacement of 1cm.

Table 4.2: Diffusive release time of probes in 1% fermented HA for a mean square displacement of 1cm.

Probe Time (hours) Time (days)

NaF 15 <1

10kDa FITC-dextran 86 4

FITC-PEG-NH2 32 >1

DGL 443 18

FITC-Albumin 171 7

70kDa FITC-dextran 723 30

It would be interesting to do future research on diffusion of drugs by attaching a fluorescent

label to the drug and making FRAP-measurements. However, we have to keep in mind that

these measurements do not take into account degradation and dilution of HA in the body.

40

5 CONCLUSIONS

The fermented HA has a MW of 1,7MDa and a c* of 0,070%. The rooster comb HA used has a

MW of 2,7MDa and a c* of 0,046%. We can conclude that the rooster comb HA used has a

higher MW and lower c* than the bacterial HA used. The rheology results obtained indicate

that the flow properties of HA are determined by its MW and that there are no

intermolecular interactions.

As for diffusion, we can conclude that small molecules are not hindered at all. Diffusion rate

generally decreases with increasing MW and/or hydrodynamic radius of the probes.

Positively charged molecules interact with HA and diffuse slowly. Overall, diffusion rate in

bacterial and rooster comb HA is similar. Therefore, there is no reason to prefer the use of

rooster comb HA to fermented HA.

The value of the Staudinger index to use is not obvious due to separation of the high and low

MW HA. Therefore, fractionation of HA by size exclusion chromatography (SEC) should be

done in order to further determine the polydispersity. This way, it would be possible to

reduce the impact of polydispersity in MW on the final results. This would result in a more

correct and precise determination of MW and its influence on rheology. It could also be

interesting to calculate the pore size of the network and see if there is a difference between

the rooster comb and fermented HA. As for diffusion, as mentioned above, it would be

interesting to make measurements with fluorescently labelled drugs.

41

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