AGGREGATION BEHAVIOR OF POLYSACCHARIDES IN AQUEOUS …

VYSOKÉ UČENÍ TECHNICKÉ V BRNĚ BRNO UNIVERSITY OF TECHNOLOGY

FAKULTA CHEMICKÁ ÚSTAV FYZIKÁLNÍ A SPOTŘEBNÍ CHEMIE

FACULTY OF CHEMISTRY INSTITUTE OF PHYSICAL AND APPLIED CHEMISTRY

AGGREGATION BEHAVIOR OF POLYSACCHARIDES IN AQUEOUS SOLUTIONS AGREGAČNÍ CHOVÁNÍ POLYSACHARIDŮ VE VODNÝCH ROZTOCÍCH

DISERTAČNÍ PRÁCE PhD THESIS

AUTOR PRÁCE Ing. FILIP MRAVEC AUTHOR

VEDOUCÍ PRÁCE Doc. Ing. MILOSLAV PEKAŘ, CSc. SUPERVISOR BRNO 2008

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ABSTRAKT Tato práce je zaměřena na agregační chování nativního a hydrofobně modifikovaného

hyaluronanu, v různých molekulových hmotnostech a stupních substituce, ve vodném

prostředí. Pro studium bylo vybráno šest fluorescenčních sond s různými vlastnostmi (pyren;

nilská červeň; perylen; akridinová oranž; 6-(p-toluidino)-2-nafthalenesulfonová kyselina;

PRODAN). a výsledky získané pomocí těchto sond byly porovnány s jednoduchým

anionaktivním tenzidem (dodecylsíran sodný). U všech použité sond byly testovány jejich

spektrální vlastnosti v závislosti na polaritě okolí a/nebo na koncentraci. Pro stanovení

vlastností nepolárního jádra hyaluronového agregátu byly vybrány dvě sondy (pyren, nilská

červeň). U domén byly sledovány polarita a viskozita vnitřního prostředí a jejich závislost na

iontové síle a teplotě. Pro modifikované hyaluronany bylo stanoveno, že jejich kritická

agregační koncentrace klesá s rostoucí molekulovou hmotností a stupněm substituce. Pro

vlastní doménu platí, že její kompaktnost roste s rostoucí iontovou silou, ale klesá s rostoucí

teplotou.

ABSTRACT This thesis is focused on aggregation properties of native and hydrophobized hyaluronan,

in different molecular weights and degree of substitution, in aqueous solutions. These samples

were studied using six different fluorescence probes (Pyrene; Nile red; Perylene; Acridine

orange; 6-(p-Toluidino)-2-naphthalenesulfonic acid; PRODAN) and results were compared

with simple anionic surfactant (Sodium dodecyl sulfate). All used probes were tested for their

spectral properties in dependence on polarity and/or concentration. Two of them (Pyrene, Nile

red) were selected to determine apolar core properties of novel hydrophobicaly modified

hyaluronan. Domains were tested for polarity and viscosity, and dependencies of these two

parameters on ionic strength and temperature. For modified hyaluronans were found that

critical aggregation concentration decreasing with increasing degree of substitution and

molecular weight. Domain qualities show their compactness increasing with increasing ionic

strength and decreasing with increasing temperature.

KLÍ ČOVÁ SLOVA Hyaluronan, Polymerní micely, Fluorescenční sonda, Polarita, Mikroviskozita, CAC, CMC,

SDS

KEYWORDS Hyaluronan, Polymeric micelle, Fluorescence probe, Polarity, Microviscosity, CAC, CMC,

SDS

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MRAVEC, F. Aggregation behavior of polysaccharides in aqueous solutions. Brno, 2008.

119 s. Disertační práce na Fakultě chemické Vysokého učení technického v Brně, Ústavu

fyzikální a spotřební chemie. Vedoucí disertační práce Doc. Ing. Miloslav Pekař, CSc.

PROHLÁŠENÍ

Prohlašuji, že jsem disertační práci vypracoval samostatně a že všechny použité literární

zdroje jsem správně a úplně citoval. Disertační práce je z hlediska obsahu majetkem Fakulty

chemické VUT v Brně a může být využita ke komerčním účelům jen se souhlasem vedoucího

disertační práce a děkana FCH VUT.

................................ podpis doktoranda

Poděkování: Mé díky patří především vedoucímu mé disertační práce Doc. Ing. Miloslavu

Pekařovi, CSc. za trpělivost a dlouhodobý systematický přístup. Dále děkuji RNDr.

Vladimírovi Velebnému, CSc., řediteli firmy CPN spol. s.r.o., za poskytnutí vzorků a

inspirativních nápadů. Rovněž nemalé díky patří mým kolegyním Ing. Radce Baarové,

Ing. Radce Lenartové. Ing. Monice Londinové, Ing. Dagmar Slezákové a Bc. Tereze Halasové

a Bc. Petře Kučerové za spolupráci na takto rozsáhlém tématu a dlouhé diskuse nad výsledky.

Samozřejmě chci poděkovat mé ženě Ludmile za neutuchající podporu.

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TABLE OF CONTENTS

1. Introduction ................................................................................................. 7

1.1. Causa Hyaluronate ......................................................................................................... 7

1.2. Modified polysaccharides, sugar-based surfactants, polymers ......................................8

1.3. Interaction between polymers and surfactants ............................................................. 11

2. Theoretical background............................................................................13

2.1. Hyaluronic acid, hyaluronan, hyaluronates and associative systems........................... 13

2.1.1. Brief historical overview.............................................................................................................. 13

2.1.2. Chemical and physical properties ................................................................................................ 13

2.1.3. Hyaluronan Modification............................................................................................................. 14

2.1.4. Micelle formation......................................................................................................................... 16

2.2. Fluorescence spectroscopy........................................................................................... 17

2.2.1. Electronic transition in polyatomic molecule............................................................................... 17

2.2.1.1. Einstein’s coefficients. Transition moment. .......................................................................... 18

2.2.1.2. Selection rule ........................................................................................................................ 19

2.2.1.3. The Franck–Condon principle.............................................................................................. 20

2.2.2. Characteristics of fluorescence emission ..................................................................................... 21

2.2.2.1. Radiative and non-radiative transitions................................................................................ 21

2.2.2.2. Fluorescence......................................................................................................................... 22

2.2.3. Lifetimes ...................................................................................................................................... 23

2.2.4. Quantum yields ............................................................................................................................ 24

2.2.5. Steady-state fluorescence intensity .............................................................................................. 24

2.2.6. Quenching.................................................................................................................................... 25

2.2.6.1. Collisional-quenching mechanism........................................................................................ 25

2.2.7. Polarization and anisotropy.......................................................................................................... 25

2.2.7.1. Characterization of polarization and anisotropy.................................................................. 26

2.2.7.2. Instantaneous anisotropy...................................................................................................... 26

2.2.7.3. Steady-state anisotropy......................................................................................................... 27

2.2.8. Fluorescence Resonance Energy Transfer (FRET) ...................................................................... 27

2.2.8.1. Energy transfer in micelles ................................................................................................... 29

3. Application of the fluorescence spectroscopy .........................................30

3.1. PROBES FOR AGGREGATION BEHAVIOR INVESTIGATION .......................... 30

3.2. Polarity probes.............................................................................................................. 31

3.2.1. Photoinduced charge transfer....................................................................................................... 31

3.2.2. Polarity induced changes in vibronic bands................................................................................. 32

3.3. Determination of microviscosity, fluidity .................................................................... 32

3.3.1. Fluorescence polarization measurements..................................................................................... 33

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3.3.1.1. Isotropic media ..................................................................................................................... 33

3.3.1.2. Ordered systems.................................................................................................................... 34

3.3.2. Intramolecular excimer formation................................................................................................ 34

3.3.3. Molecular rotors........................................................................................................................... 34

3.4. Fluorescence quenching studies ................................................................................... 35

3.4.1. Estimation of average location of fluorophore and quencher ...................................................... 35

3.4.2. Using fluorescence quenching in determination of aggregation number N .................................36

4. Aim of the work .........................................................................................38

5. Materials and methods..............................................................................39

5.1. PROBE’S PROPERTIES............................................................................................. 39

5.1.1. Pyrene .......................................................................................................................................... 39

5.1.2. Nile red ........................................................................................................................................ 39

5.1.3. Perylene ....................................................................................................................................... 39

5.1.4. Acridine orange............................................................................................................................ 39

5.1.5. 6-(p-Toluidino)-2-naphthalenesulfonic acid (TNS) ..................................................................... 40

5.1.6. PRODAN ..................................................................................................................................... 40

5.2. Colloidal systems ......................................................................................................... 40

5.2.1. SDS .............................................................................................................................................. 40

5.2.2. Hyaluronan derivatives - carbamate derivatives .......................................................................... 41

5.3. Determination of critical micelle concentration........................................................... 42

5.3.1. Pyrene 1:3 ratio method as detection of CMC ............................................................................. 42

5.3.2. Perylene-based determination of CAC......................................................................................... 44

5.4. Experimental conditions............................................................................................... 44

6. Results and discussion...............................................................................45

6.1. WATER-INSOLUBLE PROBES IN AGGREGATION............................................. 45

6.1.1. Pyrene .......................................................................................................................................... 46

6.1.1.1. Simple Surfactant System...................................................................................................... 46

6.1.1.2. Native hyaluronan................................................................................................................. 48

6.1.1.3. Hydrophobized hyaluronan .................................................................................................. 49

6.1.1.4. Influence of ionic strength .................................................................................................... 51

6.1.2. Nile red ........................................................................................................................................ 53




6.1.3. Perylene ....................................................................................................................................... 59




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6.2. WATER-SOLUBLE PROBES IN AGGREGATION................................................. 62

6.2.1. Acridine Orange........................................................................................................................... 63




6.2.2. TNS.............................................................................................................................................. 72



6.2.3. PRODAN ..................................................................................................................................... 76




6.3. Fluorescence probes in investigation of core quality ................................................... 82

6.3.1. Domain polarity ........................................................................................................................... 83

6.3.1.1. Pyrene and its polarity scale................................................................................................. 83

6.3.1.2. Nile red and its polarity scale............................................................................................... 87

6.3.2. Domain viscosity ......................................................................................................................... 91

7. Conclusion ..................................................................................................96

8. References...................................................................................................98

9. Abbreviations...........................................................................................106

10. Appendix...................................................................................................108

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1. INTRODUCTION

Biopolymers and their derivatives rise up as very interesting materials in wide area of

medical, pharmacological, and engineering application. They are characteristic for their

quality as biocompatibility and biodegradability. They exhibit fascinating properties and are

inspiration for their role-playing in living organisms. They must prove specifically

interactions with a large variety of different substances.

Hyaluronic acid is a naturally occurring polysaccharide, presents in all vertebrates and in

some Steptococci strains. Native hyaluronan play an important role in the ophthalmology,

viscosurgery, viscosupplementation, and wound healing. Its modification can fortify

hyaluronan’s unique properties and opens a new fields for application this polysaccharide.

Hyaluronan is producing in Czech Republic at high level of quality by Contipro Group from

Dolní Dobrouč. Thus, they are the reasons for introducing this research field to our university.

Native and hydrophobically modified hyaluronan is in front of our research. Progress in

application field necessarily contains investigation of aggregation behavior in solution,

solubilization properties and the others properties following from structure.

One of the most sensitive, progressive, and modern methods successfully used in

investigation of polymer surfactant, is fluorescence spectroscopy. Widely used fluorescence

probe method looks useful in our research; because of it contains additional information about

investigated system – micellar properties, polarity, fluidity, dimensions.

We hope we are able to contribute with our results to clarification of complicated relations

in this interesting system.

We start with brief summary of presence state of works on the field of polymeric micelles

and polysaccharide derivatives with regard to fluorescence spectroscopy utilization.

1.1. Causa Hyaluronate

Pelletier et al. [1] have applied fluorescence probe method for characterization the dilute

solution behavior of modified sodium hyaluronate and sodium alginate. These two polymers

were esterified by long alkyl chains (C12H25 or C18H37). They also used viscometry, 1H and 13C NMR spectroscopy, SEC coupled to a multi-angle laser light scattering detection for

systems characterization. As fluorescence probe was applied “molecular rotor”

1,1-dicyano-(4’-N,N-dimethylaminophenyl)-1,3-butadiene (TICT-probe). They confirmed the

existence of hydrophobic domain in the aqueous solutions by shift fluorescence maximum

and ratio of quantum yields in the presence and absence of polymer.

HA was also used as a tumor-targeting moiety in drug delivery system with N-(2-

hydroxypropyl)methacrylamide (HPMA) polymer [2]. HMPA–HA–DOX bioconjugate with a

lysosomally degradable peptide linkage clearly demonstrated better internalization and

cytotoxicity as compared to non-targeting HMPA-DOX conjugate. The IC50 value of HPMA–

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HA–DOX against HBL-100 (breast) cell line was 0.52 µM for 36 wt % loading of HA, which

is more than one order of magnitude better than that of non-targeting HMPA–DOX (18.7

µM). It was found that the systemic toxicity of HMPA–HA–DOX to the primary cells of

murine fibroblast was very low (IC50 = 21.2 µM).

Graft copolymers have been synthesized [3], using hyaluronic acid (HA) as a hydrophilic

backbone and polylactic acid (PLA) as aliphatic polyester; HA–TBA derivative, the reaction

was performed in dimethylsulfoxide adding as a reagent the N-hydroxysuccinimide derivative

of PLA. Characterization techniques were FT-IR, 1H NMR spectroscopy and gel permeation

chromatography, and rheology.

Amphiphilic hydrogel nanoparticles have been prepared by covalent attachment of

hydrophobic tetradecylamine (TDA) to hyaluronic acid (HA) in the presence of 1-ethyl-3(3-

dimethylaminopropyl)carbodiimide and N-hydroxysulfosuccinimide [4]. This derivative was

investigated by using 1H NMR, dynamic light scattering, FLUORESCENCE

SPECTROSCOPY (Pyrene), and transmission electron microscopy (TEM).

Creuzet et al. [5] prepared alkylated derivatives of hyaluronan. HA derivatives were

selectively modified with adipic dihydrazide (HA-ADH) with a substitution degree of 0.08;

then, alkyl chains with different chain lengths were introduced with a substitution degree

around 0.05. Characterization techniques were NMR, FLUORESCENCE SPECTROSCOPY

(Pyrene).

A novel hyaluronan (HA) derivative was synthesized through silylation [6] reaction to

improve the hydrophobicity of HA, to enhance its solubility in common organic solvents, and

to make it more reactive for further derivatizations (e.g. esterification).

Partially acylated hyaluronates, respectively having a different acyl chain and

polysaccharide chain, were prepared as water-soluble derivatives with substitution degrees of

approximately 1/33. The formation of exciplexes under oil-free conditions suggested that the

partial acyl hyaluronates having a hydrophobic-hydrophilic combination such as HA37-P

might form a micellar polymer in water by themselves [7]. This group also studied

hyaluronates with partial palmitoylation [8, 9].

1.2. Modified polysaccharides, sugar-based surfactants, polymers

Zana and Aoudia [10] have investigated aggregation behavior, critical micelle

concentration (cmc) and micelle aggregation number (N) in aqeous solutions of dodecyl

maltoside (DM), octyl glucoside (OG) and Hecameg (6-O-(N-heptylcarbamoyl)methyl-α-D-

glucopyranoside) with addition of the three water soluble polymers, polyoxyethylene (POE),

polyoxypropylene (POP), and polyvinyl pyrrolidone (PVP) by means of fluorescence probing

a time resolved flurescence quenching. They have reported that the N is nearly invariant with

temperature and concentration for DM and Hecameg. OG micelles showed a rather flat

9

minimum of N at around 30 °C. Hecameg and OG showed intermicellar exchanges of

quenchers becoming significant at T>30 °C. DM and OG did not interact with POE and PVP

based on the invariance of N. Interaction with POP has been occurred, and it resulted in a

decreasing of the micelle aggregation number at low temperature. All N was observed up the

60. They have used cetylpyridinium chloride and tetradecyl-p-cyanopyridinium chloride as

quenchers. The surfactant critical micelle concentrations were determined from the variation

of the ratio 1:3 of the intensities in emission spectra of pyrene solubilized in the micellar

solution.

Nichifor and co-workers [11] used fluorescent techniques to characterize the hydrophobic

domain formed by the side chains of polysaccharides carrying N-alkyl-N,N-dimethyl-N-(2-

hydroxypropyl)ammonium chloride pendant groups in dilute aqeuos solution. They have used

pyrene, N-phenylnaphtylamine, and 1,6-diphenyl-3,5,6-hexatriene. They reported that bellow

a certain concentrations the polymers are in extended form because of the electrostatic

interactions. With increasing concentrations, the increasing of ionic strength causes the

changes in conformation to the less extended form, resulting in the association of hydrophobic

side chain. This early state is sensed by fluorophores with different chemical structures and

hydrophobicities and depends strongly on the polymer’s chemical compositions. By

increasing polymer concentration, more small aggregates are formed along a polymer chain

until almost all hydrophobes are included. An association process continues by an increase in

aggregate size. The characteristic of microdomains depend significantly on the length of the

hydrophobic chain and degree of substitutions.

Duval-Terrié and co-workers [12] have used a set of amphiphilic pullulan derivatives of

high modification ratio. They display surface activity, and from this point, they can behave as

molecular surfactants. Two different critical concentrations were evidenced, related to two

different steps of organization. Amphiphilic pullulans may indeed be involved in hydrophobic

associations in bulk solution while adsorbing at the air – water surface. Their behavior in

dilute solutions was investigated by surface tension mesurment and with polarity probe, the

Coomassie Brilliant Blue dye. Amphiphilic pullulans were used to solubilize integral

membrane proteins.

Simon, Dugast, Le Cerf, Picton, and Muller investigated amphiphilic

carboxymethylpullulans, differing in their degree of octyl-bearing groups, in dilute solutions

[13]. To examine conformation and aggregation states in dilute solution they have used

viscometry and flow field-flow fractionation with multi-angle laser light scattering

(4F/MALLLS). They reported polymer/polymer interactions tendency increases with grafting

degree. At lower grafting degree polymer exists in isolated chain stadium. For greater degree

of substitution, strongly compact structure has been evidenced due to strong intramolecular

hydrophobic association. The critical concentration appears to be largely function of the

content of octyl groups and presents a minimum for about 20 % of C8 groups. In the all cases,

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there is competition between intramolecular and intermolecular interactions depending mainly

on hydrophobic degree and concentration. The transition between dilute and semi-dilute

regime is not classical overlapping phenomenon. For the higher amount of C8, the transition

between intramolecular association and intermolecular associations seems difficult to obtain

in low concentration rage.

Fischer and co-workers [14] used fluorescence spectroscopy to investigate

hydrophobically modified pectin derivatives. These derivatives display an associative

tendency in semidilute aqueous solutions results in the formation of hydrophobic

microdomains. Fluorescent molecular rotors witness the variation of the medium polarity. In

addition, they can also provide further information, particularly about the local cohesion of

the microenvironment of the probe. CAC values derived from polarity changes, using

molecular rotor as the fluorescent probe, are significantly different from those determined

from the cohesion of the microenvironment, accessible only with the molecular rotor.

Hydrophobically modified chitosan derivatives are being studied, with the main focus on

various alkylated chitosan [15, 16], acylated chitosan, e.g. linolenic acid-modified chitosan

[17, 18], palmitoyl chitosan [19-21], and deoxycholic acid-modified chitosan (DAMC) [22-

25]. However, due to the rigidity of the molecular chains in water, it is difficult for

hydrophobically modified chitosan derivatives to form perfect spherical-shaped self-

aggregated nanoparticles [26]. Therefore, we believe that changing the rigidity of chitosan

molecular chains by hydrophilic modification will improve the morphology of this kind of

self-aggregated nanoparticles.

Ortona et al. [27] published way of chitosan derivatization, by applying a very versatile

and quantitative amination reaction. Linear aliphatic chains of length variable from five to 12

carbon atoms have been grafted on the chitosan backbone at 10% in mole of the glycosidic

units. Viscosity and FLUORESCENCE SPECTROSCOPY (Pyrene) have been used to

analyze the intra- and inter-aggregation properties of these modified chitosans, Cn-CHITs,

and to compare them with those of chitosan itself.

Sashiwa and Aiba [28] have published article, in 2004, about chemically modified chitin

and chitosan. Chitin and chitosan are structurally similar to heparin, chondroitin sulfate, and

hyaluronic acid. As well as these mucopolysaccharides (contains carboxyl and sulfuryl

groups – anionic polymers) chitosan is almost the only cationic polysaccharide. Chemical

modification presented in this paper provides derivatives that are soluble at neutral and basic

pH. Modified chitosan is member of polymeric self-assembly systems family (SA’s). His size

could be varied from 130 to 300 nm in diametr. They have reported using deoxycholicacid-

modified chitosan in delivery of adriamycin and using chitosan derivates as physically

crosslinked hydrogel.

About partially modified water soluble polysaccharides pullulan, dextran and mannan

which form by self-assembly hydrogel nanoparticles wrote Akiyoshi and Sunamoto [29].

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They substituted pullulan by 1.7 cholesterol moieties per 100 glucose units of pullulan. Size-

exclusion column chromatography, light scattering, fluorescence spectroscopy, and electron

micrography were used in investigation solution properties of the hydrophobized

polysaccharides. The particle size (hydrodynamic radius RH) decreases with increase in the

number of cholesterol moieties. A transmission electron micrograph showed that spherical

particles have a narrow size of distribution. The fluorescence quenching study using pyrene –

cetylpyridinium chloride system reveals that one hydrophobic domain consist of

approximately four cholesterol groups. Pullulan with long alkyl chain (ALP) also formed

monodispersive nanoparticles by self-aggregation. One hydrophobic domain of the ALP

selfaggregate consisted of 12-17 alkyl groups. Various water-soluble proteins were

complexed by the CHP self-aggregates; complex showed an excellent colloidal stability.

Sinquin and co-worker presented in 1994 work about behavior of the long-alkyl-chain

derivates of sodium alginate in aqueous solution. They used for evidence aggregation

behavior the fluorescence spectroscopy and rheological measurements. As fluorescence probe

they voted pyrene in concentration ~1 10–6 M. [30].

Work of Benjalloun and co-workers [31] is preliminary work that evidences the

potentialities of molecular rotors as fluorescent probes to detect the formation of hydrophobic

microdomains for amphiphilic polymer, not only on the polarity aspects. Fluorescent rotors

can be advantageously used in the cases where pyrene is excluded due to quenching of its

excited state. They applied fluorescent rotors on homopolymers a copolymers of

3-vinylpyridinium salts. As signal of presentation of the microdomain they used break change

on the wavelength maxima depends on polymer concentration. They have found good

correlation between final plateau value of hypsochromic shift of fluorescence spectra and the

average side-chain length.

1.3. Interaction between polymers and surfactants

Nilsson and Evertsson have measured the microviscosity in dilute solution containing

mixed micelles of the anionic surfactant SDS and non-ionic cellulose ethers of different

hydrophobicity [32]. They have reported that at the critical surfactant concentration where the

clustering process start the microviscosity in the system increases to a maximum, after which

it decreases to an asymptotic level. They used for determination microviscosity by

fluorescence way microviscosity probe 1,3-bis(pyren-1yl)propane.

For determination structural changes of the complex formed by the hyaluronic acid [33],

as its salt, partially modified by a covalently bound fluorescence probe,

4-fluoro-7-nitrobenz-2-oxa-1,3-diazole, and dodecyltrimethylammonium bromide was used

fluorescent depolarization measurement provided by W.F.Reed. He found that conformation

strongly depends on the ionic strength and the surfactant type.

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In their brief note, Winnik , Regismond, and Goddard, have reported using fluorescence

of pyrene in investigation of the interaction between Quatrisoft (polymer based on chloride

salt of a N,N-dimethyl-N-dodecyl derivative of hydroxyethylcellulose) and a homologous

series of alkyltrimethylamonium halides [34]. As the most important tool, in this case they

marked the pyrene 1:3 ratio. From their fluorescence measurements, it has clearly shown that,

despite the unfovorable electrostatic repulsion, alkyltrimethylamonium surfactants associate

with cationic cellulosic polyectrolytes when these polymers contain hydrophobic groups.

Interesting conclusion is observed decreasing the CMC with increasing length of chain in

surfactant.

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2. THEORETICAL BACKGROUND

2.1. Hyaluronic acid, hyaluronan, hyaluronates and associative systems

2.1.1. Brief historical overview

In 1934, Karl Meyer and his assistant, John Palmer, isolated a novel glycosaminoglycan

from the vitreous of bovine eyes. They showed that this substance contained only a uronic

acid and an aminosugar. They named the new substances as hyaluronic acid, from hyaloid

(vitreous) + uronic acid. This macromolecule is most frequently referred to as hyaluronan,

reflecting the fact that it exists in vivo as a polyanion and not in the protonated acid form.

During the 1930s and 1940s, hyaluronan was isolated from many sources such as the vitreous

body, synovial fluids, umbilical cord, skin, and rooster comb and also from Streptococci.

The physical-chemical characterization of hyaluronan was carried out during the 1950s

and 1960s. In 1954, the Meyer’s laboratory finally completed the determination of precise

chemical structure of the basic disaccharide motif forms hyaluronan. Result also showed that

structure is stabilized by virtue of electrostatic interactions.

In 1972, Hardigan and Muir discovered that hyaluronan interacts with cartilage

proteoglycans and serves as the central structural backbone of cartilage.

In 1980s was found the secondary and the tertiary structure which hyaluronan form in

aqueous solution. This was investgated by rotary shadowing-electron microscopy.

After 1980, the research spread in many directions, mainly because until that time it had

been assumed that hyaluronan belonged to the proteoglycans, and that its biosynthesis

proceeded in a similar manner.

2.1.2. Chemical and physical properties

Hyaluronan consists of basic disaccharide units of D-glucoronic acid and D-N-

acetylglucosamine, these being linked together through alternating β-1,4 and β-1,3 glycosidic

bonds (Figure 1.).

The number of repeat disaccharides in a completed hyaluronan molecule can reach 10 000

or more with a molecular mass of ~4 10–6×g mol–1 [35]. At pH 7, the carboxyl groups are

predominantly ionized. In physiological solution, the backbone of a hyaluronan molecule is

stiffened by a combination of the chemical structure of the disaccharide, internal hydrogen

bonds, and interactions with solvent. In addition, the preferred shape in water features

hydrophobic patches on alternating sides of the flat, tape-like secondary structure. The two

sides are identical, so that hyaluronan molecules are ambidextrous, enabling them to

aggregate via specific interactions in water to form meshworks, even at low concentrations.

14

In physiological solution, a hyaluronan molecule assumes an expanded random coil,

which occupies a very large domain. The actual mass of hyaluronan within this domain is

very low. Small molecules such as water, electrolytes and nutrients can diffuse freely through

the solvent.

High molecular weight hyaluronan in high concentration can form entangled molecular

networks through steric interactions and self-association between and within individual

molecules. The latter can occur when a stretch of the hydrophobic face of the ribbon structure

of the backbone interacts reversibly with the hydrophobic face on a comparable stretch of

hyaluronan on another molecule or in a different region of the same molecule. Networks

exhibit different properties than would be isolated hyaluronan molecules. They can resist

rapid, short-duration fluid flow through the network, thereby exhibiting elastic properties,

which can distribute load or shear forces within the network. Slow fluid flow of longer

duration can partially separate and align the molecules, allowing their movement and

exhibiting viscous properties. Procedures for introducing covalent cross-links in hyaluronan

matrices have been developed to create stable networks and semi-solid materials exhibiting

pronounced viscoelastic properties.

2.1.3. Hyaluronan Modification

The derivatization of HA and the synthesis of drug-HA bioconjugates offer a number of

advantages over simple HA-drug mixtures. First chemical modification allows the

physiochemical properties of HA to be tailored according to the desired applications and can

have a significant impact on the natural turnover and clearance of the HA derivative. Ha-drug

conjugated may exhibit improved water solubility relative to parent drug and may be used to

localize a slow-release formulation at specific site in the body.

D-glucuronic acid D-N -acetylglukosamin

repeating unit

Figure 1. Structure of disacharide unit

15

The most commonly used modification procedures use as targets carboxyl group from the

glucuronic sub-unit and hydroxyl group on C6 from N-acetylglukosamine sub-unit. Main

procedures are (according to G. D. Prestwich [35-37]): a) Reaction with the carboxylic group

• Esterification (alkylation of the tetra(n-butyl)ammonium salt of hyaluronan with

an alkyl halide in dimethylformamide(DMF) solution - at higher percentage of

esterification is become insoluble in water).

• Carbodiimide-mediated reactions (generally performed in water at pH 4.75

where the carboxylic group is protonated; the most important derivative is the

HA-Taxol® bioconjugate, which is preparing with adipic dihydrazide and ester

activated Taxol®).

b) Reactions with hydroxyl groups

• Sulfation (for blood-compatible material for medical device coating).

• Esterification (with butyric acid, as the inducing cell differential agent, HA is

coupled to form novel drug-delivery system targeted specifically to tumor cells).

• Isourea coupling (this method contain cyanogens bromide activation of the HA

to produce highly reactive isourea intermediate).

• Periodate oxidation (reactive bisaldehyde functionalities can be generated from

the vicinal secondary alcohol functions on hyaluronan by oxidation with sodium

periodate).

c) Reducing end modification

d) Amide modification

e) Cross-linking strategies

• Bisepoxide and divinylsulfone cross-linking

• Internal esterification

• Photocross-linking

• Glutaraldehyde cross-linking

• Metal cation-mediated cross-linking

• Carbodiimide cross-linking

• Hydrazide cross-linking

• Crosslinking with residual proteins

• Multi-component reactions

• Surface modification

16

2.1.4. Micelle formation

It is well known that surfactants, detergents, or surface-active substances are amphiphilic

molecules. These molecules are consisting of polar group or hydrophilic group (head-group)

and non-polar group or hydrophobic tail (tail-group). To minimize unfavourable solvophobic

interactions, they spontaneously aggregate to form variety of microstructures above a certain

critical concentration (termed CMC or CAC). These molecules associate in aqueous solution

to form large molecular aggregates of colloidal dimensions termed micelles [38]. We can say

that micelles are the simplest of all amphiphilic self-organizing structures. Because they

display rather narrow size distributions, we often can characterize micelles by a single

aggregation number N.

Micelle formation is a cooperative association process. Bellow the critical micelle

concentration the surfactants exist mostly as monomers. Above the CMC, there exists a

dynamic equilibrium between the monomers and micelles. Equilibrium is determined by

equilibrium constant Kn. Three mechanisms; by kinetic point of view, were determined to

describe micelle formation.

First is a stepwise addition of a monomer, S, to the aggregate, Sn-1, as in

nn SSS ↔+ −1 . (2.2.1)

If we neglect additional interactions between aggregates and between monomers, we can

write the equilibrium given in Eq. 2.2.1 as [ ]

[ ][ ]1−

=n

nn SS

SK . (2.2.2)

Previous idea, isodesmic model, is useful in dilute solutions when n is approximately from

0 to 100. In situations that n exceed 100, we must specify a rather intractable number of

equilibrium constants Kn. This model also assumes that Kn is independent of n and

aggregation is a continuous process that does not show the abrupt onset in a narrow

concentration range that typifies micelle formation. Isodesmic model is useful in a description

of the dye’s association in aqueous solutions.

Figure 2. Model of simple surfactant with counter ion (sodium dodecyl sulphate)

17

The second, phase separation, model approximates aggregation as a phase separation

process in which the activity of the monomer remains constant above the CMC. It captures

the start mechanism of aggregation but no stop mechanism.

The third mode is the closed-association model. It assumes that one aggregation number N

dominates. With only monomers and N-aggregates,

NSSN ↔⋅ . (2.2.3)

[ ][ ]N

NN

S

SK = . (2.2.4)

This model describes both start and stop features. The total surfactants concentration in

terms of moles of monomers is

[ ] [ ] [ ] [ ] [ ]SSNKSSNS NNNT +=+= . (2.2.5)

KN relates to the other equilibrium constants of eq. 1.1.2 by

∏= nN

N KK 2 . (2.2.6)

2.2. Fluorescence spectroscopy

2.2.1. Electronic transition in polyatomic molecule

An electronic transition consists of the promotion of an electron from an orbital of a

molecule in the ground state to an unoccupied orbital by absorption of a photon. The molecule

is then said to be in an excited state. A σ orbital can be formed either from two “s” atomic

orbitals, “s” and “p” orbitals, or from two “p” orbitals. The bond formed in this way is called

a σ bond. A π orbital, π bond, is formed from two “p” orbitals overlapping laterally. A

molecule may also possess non-bonding electrons on heteroatoms such as oxygen or nitrogen,

is called n orbital. Absorption of a photon of appropriate energy can promote for example one

of the π electrons to an antibonding orbital π*. he energy of these electronic transitions is

generally in the following order:

n → π* < π → π* < n →σ* < σ → π* < σ → σ*.

In absorption and fluorescence spectroscopy, two important types of orbitals are

considered: HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied

molecular orbital) [40].

When one electron of two electrons of opposite spins is promoted to a molecular orbital of

higher energy, its spin is in principle unchanged – total spin quantum number (S) remains

equal to zero. Because the multiplicities (M = 2S + 1) of both the ground and excited states is

equal to 1, both are called single state, denoted as Sx for general state. Molecule in a singlet

state may undergo conversion into a state where the promoted electron has changed its spin –

there are two electrons with parallel spins, so the multiplicity is equal to three. Such a state is

called a triplet because it corresponds to three states of equal energy. According to Hund’s

18

E1

E2

B12 B21 A21

Figure 3. Scheme for definition Einstein coefficients

rule, the triplet state has lower energy than that of the singlet state of the same configuration

[41, 42].

2.2.1.1. Einstein’s coefficients. Transition moment.

We are considering about a

molecule and two of its energy levels

E1 and E2 (Figure 3.). B12, B21 and A21

are the Einstein coefficients for the

induced absorption, induced

emission, and spontaneous emission

respectively [42].

The rate at which energy is taken

up from the incident light is given by

)(d

d12

12 νρBt

P = . (2.3.1)

where )(νρ is the energy density on the sample at frequency ν . B12 appears as the transition

rate per unit energy density of the radiation. In the quantum mechanical theory 2

2112 M3

2 ΨΨ=h

πB . (2.3.2)

In this expression, τdMM 2121 ∫ ΨΨ=ΨΨ , where Ψ1 and Ψ2 are the wave functions of

states 1 and 2, dτ is over the whole configuration space of the 3N coordinates, M is dipole moment operator ( ∑= jerM , where r j is the vector joining the electron j to the origin of

coordinate system kinked to the molecule)

The emission-induced process E2→E1 occurs at exactly the same rate as the absorption-

induced process E1→E2, so that B12 = B21. The number of molecules in state 1 and 2 is N1 and

N2, respectively. Boltzmann Law gets these together:

19

( )[ ] ( )kThkTEEN

N/exp/exp 21

2

1 ν=−− . (2.3.3)

After several modifications, we get

213

3

21

8B

c

hA

νπ= , (2.3.4)

and note that the ratio A21/B21 is proportional the cube of the frequency. For this reason for

long wavelengths (e.g. radiofrequencies), the spontaneous emission is negligible.

2.2.1.2. Selection rule

a) Spin-forbidden transition. Transitions between states of different multiplicities are

forbidden. The intersystem crossing between S1 to T1 is possible thanks to spin-orbit

coupling. The efficiency of this coupling varies with the fourth power of the atomic

number.

b) Symmetry-forbidden transition. A transition can be forbidden for symmetry reasons.

Symmetry –forbidden transition can nevertheless be observed because the molar

vibrations cause some departure from perfect symmetry (vibronic coupling).

c) Selection rules for the inter-system crossing. El Sayed rules (Figure 4.). The

singlet-triplet interconversion is allowed for

pyrene

S0-S1

S0-S2

anthracene

S0-S1

S0-S2

perylene

S0-S1

S0-S2

NH

indoleS0-S1 (

1La)S0-S2(1Lb)

20

O O

1(n,ππππ*) 3(ππππ,ππππ*)

O O

1(n,ππππ*) 3(n,ππππ*)

X

O

1(ππππ,ππππ*)

O

3(n,ππππ*)

O


O


X

Figure 4. Presentation of the intersystem crossing

with the orbitals. (according to [8]).

( ) ( )*,*, πππ →n transition.

and forbidden for

( ) ( )( ) ( )*,*,

*,*,

ππππππ

→→ nn

transitions.

2.2.1.3. The Franck–Condon principle

According to Born-Oppenheimer

approximation, the motions of electrons are

much more rapid than those of the nuclei.

Promotion of an electron to an antibonding

molecular orbital upon excitation takes

about 10–15 s, which is very quick

compared to the characteristic time for

molecular vibration. An electron is most

likely to occur without changes in the

positions of the nuclei in the molecular

entity and its environment (Figure 5.). The

resulting is called a Franck-Condon state,

and the transition is called vertical

transition.

excited state

ground state0

1

2

0

1

2

0

1

2

0

12

excited state

ground state

E E

nuclear configuration nuclear configuration

λ

0-0

0-10-2

0-3

λ0-0

0-1

0-2

0-3

Figure 5. Potential energy diagrams with vertical transitions. Include the shape of the absorption bands.

21

2.2.2. Characteristics of fluorescence emission

2.2.2.1. Radiative and non-radiative transitions

The Perrin-Jabloński diagram (Figure 6.) is convenient for visualizing in a simple way the

possible processes:

a) photon absorption (10–15 s)

b) internal conversion (IC, 10–11–10–9 s)

c) fluorescence (10–10–10–7 s) – lifetime of the excited state S1

d) phosphorescence (10–6–1 s) – lifetime of the excited state T1

e) intersystem crossing (10–10–10–8 s)

f) delayed fluorescence

g) triplet-triplet transitions

The singlet electronic states are denoted Sx, the triplet states Tx. Vibration levels are

associated with each electronic state. Radiative transitions are representing by the straight

arrows, non-radiative transitions are represent by the wavy arrows.

Internal conversion. It is non-radiative transition between two electronic states of the same

spin multiplicity. In solution, this process is followed by a vibrational relaxation. From S1, IC

to S0 is possible but is less efficient than conversion from S2 to S1, because of the much larger

energy gap between S1 and S0.

S1

S2

T1

S0

ISCIC

ISC

IC

VR

VRABSORPTION

FLUORESCENCE PHOSPHORESCENCE

Figure 6. The Perrin-Jabłoński diagram

22

Fluorescence. See Chapter 2.2.2.2.

Intersystem crossing. It is a non-radiative transition between two iso-energetic vibrational

levels belonging to electronic states of different multiplicities.

Phosphorescence. In solution at room temperature non-radiative de-excitation from the

triplet state T1, is predominant over radiative de-excitation - phosphorescence. The transition

T1 → S0 is forbidden and the radiative rate constant is very low (it can be observed because of

spin-orbit coupling). The phosphorescence can be observed in a rigid medium and/or at low

temperatures. Its lifetime may be in range seconds, minutes or more. The phosphorescence

spectrum is located at wavelengths higher tan fluorescence because of T1 is lower than S1

(Figure 6).

Delayed fluorescence. Reverse intersystem crossing T1 → S1 can occur when the energy

differences between states is small and the lifetime of triplet state is long enough. This results

in emission with the same spectral distribution as normal fluorescence but with a much longer

decay time constant. The fluorescence emission can be activated thermally (E-type of delayed

fluorescence - its efficiency increasing with increasing temperature; first occurs with Eosin),

and by triplet-triplet annihilation (P-type - in concentrated solution where two molecules in

triplet state can collide; observed first time with Pyrene).

2.2.2.2. Fluorescence

It is an emission of photons associated the S1→S0 relaxation. Fluorescence occurs, apart

from a few exceptions, from S1 and therefore its characteristics do not depend on the

excitation wavelength. The 0-0 transition is usually the same for absorption and fluorescence.

The fluorescence spectrum has lower energy values than absorption spectrum because of the

energy loss in the excited states. The wavelength of a fluorescence emission should always be

higher than that of absorption (Stokes Rule). In most cases, the absorption spectrum is partly

overlaps by the fluorescence spectrum, i.e. a fraction of light is emitted at shorter wavelengths

than the absorbed light - this observation it seems to be in contradiction to the principle of

energy conservation. This “energy discomposure” is explaining by the fact that at room

temperature, a small fraction of molecules is in a vibrational level higher then level 0.

Mirror image rule. The differences between the vibrational levels are similar in the

ground and excited states, so that the fluorescence spectrum often resembles the first

absorption band.

Kasha rule. Polyatomic molecular entities luminescence with appreciable yield only from

the lowest excited state of a given multiplicity.

Kasha-Vavilov rule. The quantum yield of luminescence is independent of the wavelength

of exciting radiation.

23

After excitation of a population of molecules by a very short pulse of light, the

fluorescence intensity decreases exponentially with a characteristic time, reflecting the

average lifetime of the molecules in the S1 excited state.

2.2.3. Lifetimes

Figure 7 summarizes rate constants of possible processes in depopulation of the excited

states. Subscripts are:

r…radiative decay ic…internal conversion

nr…for non-radiative decay isc…intersystem crossing

Superscript “S” denotes S1 → S0 processes, and “T” denotes T1 → S0 processes. De-

excitation processes resulting from intermolecular interaction are described in Chapters 2.2.6.

and 2.2.8. After excitation by a very short pulse of light molecules started returns to the

ground state. These processes for molecule “M” can be described by Equation 2.3.5. [ ] ( )[ ]*d

*d Snr

Sr Mkk

t

M +=− . (2.3.5)

Concentration of the excited state at time 0 will be denoted as [M]0, so that after

integration we get

[ ] [ ]

−=

S

tMM

τexp** 0 . (2.3.6)

where τS, the lifetime of excited state S1, is given by

Snr

Sr

1

kkS +

=τ . (2.3.7)

The fluorescence intensity at any time is proportional to the concentration of molecules still excited with the proportionality factorSrk , so we are written

Figure 7. Schematic diagram of depopulation of the excited states

S1

T1

S0

krS kic

S

kisc

krT

kicT

kicS + kisc = knr

S

24

[ ] [ ]

−==

S

tMkMkti

τexp**)( 0

Sr

SrF . (2.3.8)

It’s obvious the δ-pulse response of the system, iF(t), decrease according to a single

exponential.

The fluorescence decay time τS is one of the most important characteristic of a fluorescent

molecule because it defines the time window of observation of dynamic phenomena. No

accurate information on the rate of phenomena occurring at time-scale shorter than about

τ/100 or longer than about 10τ can be obtained.

2.2.4. Quantum yields

The fluorescence quantum yield ΦF is the fraction of excited molecules that return to the

ground state S0 with emission of fluorescence photons:

Skkk

k τSrS

nrSr

Sr

FΦ =+

= . (2.3.9)

According to Eq. 2.3.8 and integration, we get

[ ]∫∞

Φ==0 FS

SrF d)(

*

1 τkttiM

. (2.3.10)

Using radiative lifetime τr as Sr1 k , the fluorescence quantum yield can be also written as

r

S

ττ

=FΦ . (2.3.11)

2.2.5. Steady-state fluorescence intensity

In steady-state measurements, we illuminate the sample, with molecule’s concentration

[M], by the photons with the constant amount N0. Expression αN0 represents the amount of

absorbed photons per unit volume involved in excitation process - pseudo-first rate constant for absorption of the photon is very large ( 15

a s10 −≈k ). Rate constants for radiative and

non-radiative decay have values between 107 and 1010 s–1.

Under continuous illumination, the concentration [M*] remains constant. This means M*

is in steady-state. The rate of change [M*] is equal to zero, and [ ] ( )[ ]*α0d

*d Snr

Sr0a MkkNk

t

M +−== . (2.3.12)

Expression 0aαNk represents the amount of absorbed photon per unit volume and per unit

time. It can be written as 0αI where 0I represents intensity of incident light (moles of photon

per litre and second). So, the steady concentration of the M* can be written as

[ ]Snr

Sr

0α*kk

IM

+= . (2.3.13)

25

According to Equation 2.3.8 for steady-state fluorescence intensity we obtain

[ ] F0Snr

Sr

Sr

0SrF αα* Φ=

+== I

kk

kIMki . (2.3.14)

2.2.6. Quenching

We introduce to the system, solvent – probe, another apolar component – fluorescent

quencher. If the excited molecule of fluorescence comes close to the quencher, a rapid non-

radiative decay of the fluorescent state occurs and the probe molecules do not produce any

fluorescence.

2.2.6.1. Collisional-quenching mechanism

When an excited state M* is quenched by collision with a quencher Q, the simplest form of

the rate equation is [ ] [ ]( )[ ]** d/d MQkkItM qMSex +−= , (2.3.15)

which under steady-state condition leads to the Stern-Volmer equation [ ] [ ] ( )[ ]QkkMM SqMq /1/ *

0* += . (2.3.16)

There are SSk τ=−1 as lifetime of excited state, qMk as the collisional quenching rate

constant and ratio SqM kk / as the Stern-Volmer quenching constantSVK . Variations in

fluorescence intensities II /0 versus [Q] (in which I0 and I are intensities in the absence and

presence of Q respectively) fit by a least square’s method to the best straight-line yields to the

experimental parameter Kobs.

2.2.7. Polarization and anisotropy

Upon excitation with polarized light, the emission from many samples is also polarized.

The extent of polarization of the emission is described in terms of the anisotropy (r). Samples

exhibiting non-zero anisotropies are said to display polarized emission.

A number of phenomena can cause depolarization of the emission: rotational diffusion,

energy transfer, angle between absorption and emission dipole, scattering the incident light,

etc.

Fields of application of fluorescence polarization contain: a) spectroscopy - difference between excited states

b) polymers - chain dynamics; local viscosity in polymer environments

c) micellar system - internal microviscosity of micelles; fluidity

d) biological membranes - fluidity; phase transition temperature

e) molecular biology - DNA-protein interaction

f) immunology - immunoassays

26

2.2.7.1. Characterization of polarization and anisotropy

When the incident light is vertically

polarized, the vertical axis Oz is an axis

of symmetry for the emission of

fluorescence according to the Curie

principle - Ix = Iy. For the vertically

polarized light the components of

fluorescence intensity are denoted as Iz =

III, Ix = Iy = I⊥. The fluorescence intensity

observed along the Oz axis is

Ix + Iy = 2I⊥. Commonly fluorescence is

observed in a horizontal plane at 90° to

the propagation direction of the incident

beam (Figure 8).

The polarization ratio is given by:

⊥

⊥

+−

=II

IIp

II

II

. (2.3.17)

The emission anisotropy is given by:

⊥

⊥

+−

=II

IIr

2II

II . (2.3.18)

In the expression of the polarization ratio, the denominator represents the fluorescence

intensity in the direction of observation, whereas in the formulation of the anisotropy, the

denominator represents the total fluorescence intensity.

The relationship between r and p follows from Eqs. 2.3.17 and 2.3.18:

p

pr

−=

3

2. (2.3.18)

2.2.7.2. Instantaneous anisotropy

After the infinitely short pulse of excited light, at the time t, the total fluorescence

intensity is I(t) = III(t) + 2I⊥(t), and the instantaneous emission is given by

)(

)()()( II

tI

tItItr ⊥−

= . (2.3.19)

Figure 8. Usual configuration for measuring fluorescence anisotropy

excitationy

z

I II

I ⊥⊥⊥⊥

emission

27

Each polarized component evolves according to

[ ] [ ])(13

)()( ; )(21

3

)()(II tr

tItItr

tItI −=+= ⊥ . (2.3.20)

2.2.7.3. Steady-state anisotropy

Steady state anisotropy r is measured under the continuous illumination [40, 41, 42].

With the general definition of an averaged quantity, with the total normalized fluorescence

intensity as the probability law, we obtain

∫

∫∞

∞

=

0

0

d)(

d)()(

ttI

ttItrr . (2.3.21)

So, in the case of a single exponential decay with the excite-state lifetime τ, the steady-

state anisotropy is given by

∫∞

−=0

d)/exp()(1

tttrr ττ

. (2.3.22)

In the case samples contains a mixture of fluorophores, the important relation is that total

emission anisotropy is the weighted sum of the individual anisotropies:

∑=i

ii rfr . (2.3.23)

2.2.8. Fluorescence Resonance Energy Transfer (FRET)

Fluorescence energy transfer has been widely use to determination dimension either

within or between molecules over distances of 10-100 Å, a range is well suited to probing for

example protein structure. Absolute distances measured by FRET spectroscopy remain

problematical because of what appears to be an unjustified assumption, namely that

calculation of FRET distances assume that the probes are able to undergo free , isotropic

D A

S1

S0

S1

S0 radiative transitionradiationless transition

RET

abso

rptio

n fluorescence

Figure 9. Simplified Jabłoński diagram

28

motion. Common sense suggests that a large (300–500 g mol–1) hydrophobic FRET probe

covalently bound to polymer chain can not undergo true rotational freedom [43].

Simplified mechanism of radiationless (resonance) energy transfer from donor to acceptor

is displayed in Figure 9 Condition for FRET: a) a donor has a high quantum yield (φD)

b) existence overlap of donor emission spectrum and acceptor absorption spectrum (JDA)

c) there is an appropriate alignment of the absorption and emission transition moments

and their separation vector ( parameter κ2)

d) the donor-acceptor distances are within (1 ± 0.5)R0 of each other.

ad a) The quantum yield of the donor is defined as the ratio of the number of photons

emitted to the number absorbed. It is parameter which depends on the immediate

environment of the probe.

ad b) The overlap integral (Figure 10) of the donor emission spectrum with the acceptor

absorption spectrum is defined as

∫∫= λλλλλελ d)(/d)()( 4DA DAD FFJ . (2.3.24)

ad c) and d) The Förster distance R0 was originally defined as the distance between the donor

and acceptor at which energy transfer is 50% efficient. The relationship is given by

( ) 10875.8 422560 DAD JnR φκ −−×= . (2.3.25)

where:

� 8.79x10-25 is derived from 9000(ln10)/128π5 x Avogadro’s number � κ2 …is the Förster’s orientation factor. This factor is indeterminate. Theoretically,

values of κ2 may range from 0 to 4. Its value depends on the relative orientations of the donor and acceptor dipoles.

� n…refractive index of the medium between the donor and acceptor

Figure 10. Overlap integral for perylene-fluorescein pair [18]

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50 60 70 80 90 100 110 120 130

distance

tran

sfer

eff

icie

ncy

R0

Figure 11. Transfer (E) efficiency as the function of the D-A distance (R0 = 40 Å)

29

Transfer efficiency E can be obtained by measuring either the fluorescence intensities or

the fluorescence lifetimes [43] of the donor with and without an acceptor

00/1/1 DDDD FFE ττ−=−= . (2.3.26)

where FD (τD) a FD0 (τD0) are the intensities (lifetimes) in the presence and the absence of

acceptor. The relationship between donor and acceptor is illustrated in Figure 11. Efficiency

is defined by ( )66

060 / RRRE += . (2.3.27)

2.2.8.1. Energy transfer in micelles

Assumptions of the model: a) all donors and acceptors are associated with micelles

b) the distribution of donor and acceptor molecules follows the Poisson statistic, given by

,!n

exP

xnS

n

−

= . (2.3.28)

where SnP is the probability that the micelles contains n solutes S, and x is the mean

number of solutes per micelles, that is

[ ][ ]micelle

solute=x . (2.3.29)

c) quenching of the donor fluorescence can be ascribed entirely to intramicellar Förster-

type energy transfer.

For the Försters’s orientation factor, κ2, Dos Remeidos and Moens [43] predicted value

2 / 3. Sánchez and Ruiz [44] have used for micellar media value equal to 0.475 M·cm3.

According to these conditions, it is possible to determine the micellar radius directly from

attenuation of the fluorescence emission after addition of donor.

30

3. APPLICATION OF THE FLUORESCENCE SPECTROSCOPY

3.1. PROBES FOR AGGREGATION BEHAVIOR INVESTIGATION

There are many ways how to sort a CAC probe type into separate sections. A selection

was made according to their behavior in aqueous environment. Contrary to a “standard

sorting” which was used in Theoretical background chapter probes were not sorted to polar or

non-polar but to water-soluble and water-insoluble ones. For description, TNS probe is water

soluble because of sulfonyl group but in aqueous environment has a weak fluorescence. The

fluorescence quantum yield rapidly increases in non-polar environment like enzyme’s non-

polar active centers.

Table 1. Summary of the fluorescence probes used in this thesis for CAC determination

probe observed parameter additional information

changes fine vibrational structure

of emission spectra polarity

position of excitation maxima polarity;

quotient of bounded Pyrene PYRENE

changes in excimer emission

efficiency viscosity

quantum yield - PERYLENE

anisotropy viscosity

quantum yield - NILE RED

position of emission maxima polarity

position of emission maxima polarity PRODAN

emission ratio multi-domain anylysis

quantum yield - TNS

position of emission maxima polarity

ACRIDINE

ORANGE quantum yield -

These probes show a variation of their spectral properties in presence of supra-molecular

aggregate (mainly negatively charged) or hydrophobic domain. In adequate concentration

range, the significant change of their properties is expected.

31

3.2. Polarity probes

3.2.1. Photoinduced charge transfer

The dipole moment of an aromatics molecule in the excited state eµ differs from that in

ground state gµ . Most of polarity probes undergo intramolecular charge transfer upon

excitation so that eµ > gµ . Following excitation, the solvent cage undergoes a relaxation

leading to a relaxed state of minimum free energy. The higher the polarity of the solvent, the

lower energy of the relaxed state and the larger red-shift of the emission spectrum. It is

important to note that the rate of solvent relaxation depends on the solvent viscosity.

Figure 12. Structures of PCT probes

One of the most important probes is ANS (1-anilino-8-naphtalene sulfonate) and its

derivative TNS (p-toluidinyl-6-naphtalene sulfonate). ANS is non-fluorescent in aqueous

solutions and highly fluorescent in solvent of low polarity. ANS and TNS have been

extensively used for probing biological membrane, micellar systems and proteins [45-54].

Valeur suggests parameters of an ideal PCT probe: • large change in dipole moment value but without change in direction

• bear no permanent charge (avoid contribution from ionic interactions)

• be soluble in solvents of various polarity

PRODAN, 6-propionyl-2-(dimethylamino)naphthalene, designed by Weber and Farris

(1979) fulfills most of requirements described above. Difference in dipole moment of ground

and excited state reach to the 5-7 D, but this value cannot explain the outstanding

solvatochromism of PRODAN. In fact, PRODAN contains hydrogen bond acceptor groups so

care must be taken when interpreting data from solvatochromic shift in microenvironments

containing hydrogen donor groups.

NHSO O

O-

CH3

NH

S

O

OO

-

CH3

CH3

N

CH3

O

ANS TNS

PRODAN

32

3.2.2. Polarity induced changes in vibronic bands

In some aromatic molecules with a high degree of symmetry, the first singlet absorption ( )10 SS → may be symmetry forbidden. The intensities of the various forbidden vibronic

bands are highly sensitive to solvent polarity (Ham effect). In polar solvents, the intensity of

the 0-0 band increases at the expense of the others.

The relative changes in intensity of the vibronic bands in the Pyrene fluorescence

spectrum has its origin in the extent of vibronic coupling between the weakly allowed first

excited state and the strongly allowed second excited state; dipole-induced dipole interaction

between the solvent and Pyrene play a major role.

Reconstructed polarity scale based on ratio of the first and third vibronic band of the

pyrene emission spectrum, according to data from Dong and Winnik (1984), is showed in the

Figure 13.

Figure 13. Pyrene polarity scale (DMF = N,N-Dimethylformamide; ACN = Acetonitrile;

DMSO = Dimethylsulfoxide; THF = Tetrahydrofuran)

3.3. Determination of microviscosity, fluidity

There are some several techniques how to determine microviscosity or fluidity. According

to Valeur, the microviscosity term should be used with caution, and fluidity can be

alternatively used to characterize the effects of viscous drag and cohesion of the probe

microenvironment.

Main techniques are summarized and described below: • Molecular rotors - influence of internal torsional motion on fluorescence quantum

yield and lifetime [55-60]

• Excimer formation - translational diffusion (intermolecular) or internal rotational

diffusion (intramolecular) → ratio of excimer and monomer bands [61-63]

DMSO

ACNDMF

MeOH

EtOHBenzyl alkohol

THFEthyl acetate

Chloroform

Toluene

Cyklohexan0.50

0.75

1.00

1.25

1.50

1.75

2.00

0 10 20 30 40 50

Dielectric constant

Py

1:3

Cyclohexane

33

• Fluorescence quenching - translational diffusion → fluorescence quantum yield

• Fluorescence polarization (steady-state, time-resolved) - rotational diffusion of the

whole probe → emission anisotropy [64-72]

3.3.1. Fluorescence polarization measurements Requirements for polarization probe:

• minimum disturbance of the medium to be probed

• symmetry and directions of the transition moments - isotropic rotations

• minimum specific interactions with the surrounding molecules

• minimum sensitivity of the excited state lifetime to the microenvironment when only

steady-state anisotropy is measured

On the topic of disturbance of the medium, small molecules are to be preferred, but the

measurement is then often sensitive to specific interaction with the surrounding molecules.

3.3.1.1. Isotropic media

Rotations are isotropic when the probe has a spherical shape, but most fluorescent probes

are aromatics and less or more planar. When the direction of the absorption and emission

transition moments coincide with the long molecular axis (rod-like molecules - DPB, DPH),

the rotation can be considered as isotropic because any rotation about this long axis has no

effect on the emission anisotropy.

For isotropic motion in isotropic medium, we obtain for instantaneous and steady state

emission anisotropies: )6exp()( 0 tDrtr r−= , (3.3.1)

( )τrDrr

6111

0

+= , (3.3.2)

where rD means the rotational diffusion coefficients and τ means the lifetime of the probe.

In principle, the value of the viscosity η could be calculated from Stokes-Einstein relation:

V6

T

r8

T3 ηπη

kkDr == , (3.3.3)

where r is the hydrodynamics radius of the sphere and V its hydrodynamic volume. Introducing the rotational correlation time as 1)6( −= rc Dτ , equation 3.3.2 can be rewritten as

+=

crr ττ

111

0

, (3.3.4)

34

3.3.1.2. Ordered systems

In anisotropic media such as bilayers and liquid crystals, the hindered rotation is observed. In this media anisotropy does not decay to zero, but to a steady value∞r , and instantaneous

emission anisotropy can written as ( ) ∞∞ +−−= rtrrtr c )/exp()( 0 τ . (3.3.5)

3.3.2. Intramolecular excimer formation

Bifluorophoric molecules consisting of two identical fluorophores linked by a short

flexible chain may form an excimer (e.g. see Figure 3.1.1 c). Intramolecular excimer

formation requires close approach of the two moieties through internal rotations the lifetime

of excited state. Diffusion processes or microheterogenity of the medium does not affect this

process. The efficiency of excimer formation does not depend on the concentration of

fluorophores. Efficiency can be denoted as ratio of the fluorescence intensities of monomer ( MI ) and excimer (EI ). Its relationship to monomer concentration is described as

1E

/

M

E kk

k

I

I

r

r τ= , (3.3.6)

where Eτ is the excimer lifetime, rk and /rk is radiative rate constants of the monomer

and excimer, respectively. 1k is the diffusional rate constant, and if assumed to be this

constant independent of time, it can be expressed by the following simplified form -

Smoluchowski relation: DNRk cπ41 = , (3.3.7)

where cR is the distance of closest approach, D is the mutual diffusion coefficient, N is equal

to 1000/AN , AN being Avogadro’s number.

It is important that ratio is proportional to the excited-state lifetime of the excimer, which

is reduced by oxygen quenching. The rate of oxygen quenching decrease with increasing

viscosity and its effect on excimer formation becomes negligible at viscosities higher than

0.1 N.s.m-2.

3.3.3. Molecular rotors

Molecular rotors are molecules, which undergo

internal rotation resulting in viscosity dependent

changes in its emissive properties. The example is

shown in Figure 14. In solvents of medium and high

viscosity, an empirical relation has been proposed to

link non-radiative rate constant to the ratio of the van der Waals volume (0V ) to the free volume (fV ):

NN CC

N

Figure 14. Molecular rotor

35

−=

fnrnr V

Vxkk 00 exp , (3.3.8)

whrere 0nrk is the free-rotor reorientation rate and x is a constant for a particular probe.

Fluorescence quantum yield is related to the radiative and non-radiative rate constant (Eq.

2.3.9) and so we obtain

=

Φ−Φ

fnr

nr

F

F

V

Vx

k

k 00

exp1

. (3.3.9)

Combination with Doolittle equation, which compare relationship between viscosity and free volume (“ ( )fVV00 expηη = ”) yields

x

F

F aη=Φ−

Φ1

. (3.3.10)

For small quantum yields and when changes in viscosity are achieved by variations of

temperature, Eq. 3.3.10 should be rewritten as ( )x

F b Tη≅Φ . (3.3.11)

It is important denote, that the effective viscosity probed by a molecular rotor appears to be less than the bulk viscosity η because of free volume effects.

3.4. Fluorescence quenching studies

The kinetics of the quenching of probes incorporated into microaggregates depends on a

variety of factors: solubility and partition of the quencher between aqueous medium and

micellar phase, relative location of probe and quencher, and their mobility inside the

aggregates. These characteristic depend on the chemical and spatial structures of the probe,

the quencher, and the microheterogenous system [73]. Determination of fraction of the probe partition between the pseudophase and medium is useful. Ratio ( )sAA IIIE += / , where IA is

the intensity fluorescence measured from aggregate, or membrane, and Is is the intensity in

supernatant solution. M. Deumié and co-workers described that fluorescence of Py, PDA and

BaP in the supernatant was least hundred times less than fluorescence from the solution of

erythrocyte membranes [74].

3.4.1. Estimation of average location of fluorophore and quencher

Fluorescence measurement using quenchers can also be employed to estimate the location

of probe/fluorophore and quencher [73, 74]. It is necessary to use various quenchers located at

different depths in the aggregates and a totally incorporated probe in the same phase as the

quenchers or the quenching of various probe at different depths by one quencher. In order to

evaluate the relative proximity of the quencher-fluorophore from the steady-state quenching

rate constant, both species should be totally incorporated into aggregates or else the intra-

aggregate concentration must be known and employed in calculations. In aggregates, where

36

diffusion-controlled quenching process is allowed, the quenching rate constant is not directly

related to the local quencher concentration around the fluorophore; it is related to its rate of

accessing the probe locus [72].

In the presence of long-lifetime family of pyrene and its derivatives, we can verify the

incorporation of the probe in compact aggregate (e.g. membrane) by the absence of excimer

[73].

3.4.2. Using fluorescence quenching in determination of aggregation number N

• steady state point of view

If the concentration of micelles equals [M], the average number of quenchers per micelle,

q, is defined by

[ ][ ]M

Qq = . (3.4.1)

A random placing of m objects (quenchers) in n boxes (micelles) results in a Poisson

distribution for large m and n. This distribution implies that the probability P(p) of having p

objects in an arbitrary box is

( ) ( )!

exp

p

qqpP p −= . (3.4.2)

where ]/[][/ MQnmq == . In particular, P(0) signifies the probe ability of finding an empty

micelle box

( ) )exp(0 qP −= (3.4.3)

since 0! = 1. The steady state fluorescence at a quencher concentration [Q] is thus

[ ][ ]

−=M

QII exp0 (3.4.4)

The slope, [ ]QII q vs.ln

0

, gives the concentration of micelles[ ]M . From this value we can

determine the aggregation number N (eq. 3.4.4)

[ ]( )[ ]M

CMCSN tot −= , (3.4.5)

where [ ]totS is total surfactant concentration.

• time resolved point of view

Steady-state fluorescence quenching works only for small N. Time-resolved fluorescence

is method that is more sophisticated. In systems, where is only fluorescence probe, the

fluorescence intensity I decay exponentially (Equation 3.1.8)

37

−=0

exp)0()(τ

tItI , (3.4.6)

where 0τ is the fluorescence lifetime.

The addition of quencher to system leads to fact, that fluorescence intensity of probe

decay more rapidly. With quencher, fluorescence shows nonexponential decay, this can be

described by Equation 3.1.9.

[ ]1)exp()0(

)(ln

0

−−+−=

tkq

t

I

tIQτ

. (3.4.7)

38

4. AIM OF THE WORK

Aim of this work is to investigate aggregation behavior of native and hydrophobicaly

modified Hyaluronan in aqueous solution by fluorescence probe method.

1) From literary sources, select most suitable group of fluorescence probes for investigation

of the aggregation process and qualities of a hydrophobic core.

2) Verify or/and specify spectral behavior of these probes in model system – solvents and

simple surfactant systems.

3) Collect information about aggregation process of native and hydrophobicaly modified

hyaluronan from fluorescence probes point of view.

4) With selected probe(s), investigate hydrophobic core qualities in modified hyaluronan

solution. Determine influence of the ionic strength and temperature on core qualities.

39

5. MATERIALS AND METHODS

5.1. PROBE’S PROPERTIES

5.1.1. Pyrene

For details, see Chapter 4.2.1

5.1.2. Nile red

Nile red (CAS # 7385-67-3) is one of the most useful probes. Nile red (NR) is a

hydrophobic, highly fluorescent, solvatochromic probe that has been extensively applied to

examine the structure, dynamics and environment in biological and microheterogeneous

systems. The remarkable sensitivity of its absorption and fluorescence characteristics to the

microenvironment has been exploited in many studies included polarity in zeolites, lipid

bilayer, ionic liquids, and liquid crystals [75].

Large shifts in the absorption and emission bands are due to large changes in the excited

state dipole moment of the molecule. This corresponds to nearly complete charge separation

between the diethylamino group which acts as an electron donor and the quinoid part of the

molecule which is the electron acceptor. The charge separation may be explained by the

formation of the twisted intramolecular charge transfer state (TICT state) of the molecule in

polar solvents due to the rotation of the flexible diethylamino group attached to the rigid

structure of the molecule [76].

Fluorescence intensity and position of emission maxima were selected as observed

parameters for Nile red. Nile red has very low fluorescence quantum yield in the presence of

water molecules. This made NR an ideal probe for aggregation investigation in aqueous

solution [77-83].

5.1.3. Perylene

Perylene (CAS # 198-55-0) is thermally and chemically stable fluorescent probe. It has

high fluorescence quantum yield, ~ 0.98 in CHCl3, and short fluorescent lifetime, ~ 5.5 ns. In

ground state S0, has planar configuration. Efficiency of ISC process is too low but is

compensated for long phosphorescence lifetime (~ 0.3 ms).

5.1.4. Acridine orange

Acridine orange (as hydrochloride hydrate, CAS # 65-61-2) is a cationic dye mainly used

in nucleic acid staining. It is cationic dye, which is protonated at a pH lower than 10-10.5.

Acridine orange (AO) is a much-studied member of a class of cationic dyes whose planar

molecules aggregate in aqueous solution to cause concentration-dependent spectral changes

(metachromasy). Direct evidence for the aggregation of AO has been obtained from proton

40

chemical shifts. The concentration dependence of changes in the visible absorption spectrum

of dilute AO solutions is consistent with dimerization equilibrium. The simplest such

equilibrium for the AO monomer, D+, is 2D+ = D22+ [84]. The AO-dimer formation is

ascribed to “H-type aggregation”. This aggregation is caused by transition-dipole moments

interactions between two, or more, chromophores. In this case, the dipole interaction leads to

the excitation energy level splitting and to new energy levels. Absorption is allowed only to

higher energy level (caused hypsochromic shift of the absorption band), but radiation

transition from lower level is forbidden and no fluorescence is occurs.

The absorption spectrum of dilute AO, having a maximum at 492 nm (α band), is

gradually substituted by a new spectrum with a maximum at 465 nm (β band). In the

concentration range below (4-5) 10-5 mol L-1 an isosbestic point is observed at 471.5 nm. At

higher concentrations a third band (γ band) with a maximum at 450 nm substitutes the

“465 nm band” and the isosbestic point disappears [84-95].

The emission spectrum of AO in aqueous solution has a maximum at 532 nm. In the

highest concentration range a new weak emission band at 630 nm is occurs. This band is

related to the forbidden H-type aggregate transition. Figure 36 summarizes excitation and

emission spectra.

5.1.5. 6-(p-Toluidino)-2-naphthalenesulfonic acid (TNS)

TNS (CAS # 7724-15-4) is common and well-known fluorescence probes for peptides and

proteins. It is non-fluorescent in aqueous solutions and highly fluorescent in solvents of low

polarity. This allows us to visualize only hydrophobic regions of biological systems without

interference from non-fluorescent TNS molecules localized in surrounding aqueous

environment.

5.1.6. PRODAN

PRODAN (N,N-Dimethyl-6-propionyl-2-naphthylamine, CAS # 70504-01-7) was

designed by Weber and Farris at 1979. PRODAN has non-specific solubility and successfully

probing different chemical and biological systems. It shows a large Stokes shift from

4300 cm–1 in cyclohexane to 8640 cm–1 in water [40].

5.2. Colloidal systems

5.2.1. SDS

For the model of anionic amphiphilic system, a sodium dodecylsulphate (SDS) was

selected. This simple surfactant is well known and its CAC concentration has been

determined in 1966 by Elworthy. In aqueous solution at 298.2 K, the CAC value was

determined to 8.2 10–3 mol L–1 by a surface tension measurement [96].

41

Figure 15. 3D structure of Sodium Dodecyl Sulfate.

5.2.2. Hyaluronan derivatives - carbamate derivatives

These derivatives were prepared by Czech company CPN spol. s.r.o. Dolni Dobrouc [97].

In fact, the modification targets were hydroxyl groups on the C2 on glucuronic sub-units (hot

hydroxyl). These groups were activate by CNBr and result react with alkyamine compound to

form alkyamine-HA conjugate (see Figures 16 and 17).

These derivatives can be described by three parameters:

a) Molecular weight of unmodified hyaluronan (det. by SEC-MALS)

b) Alkyl-chain type – C10, Decyl-derivative

Figure 16. Schematic modification HA on the glucuronic sub-unit leads to carbamate

derivates. Hot hydroxyl is red-marked.

Br N

N

O

OH

+ NH

O

OOH

-

NH

OH

O-

O

Na+

NH2

O

O-

O

Na+

NH2R

R

OH

NH

O

O

NH2Na

O

O

OH

O

OH

OH

42

c) Substitution degree (SD). The substitution degree is defined as the ratio of the

number of the monomer with and without the alkyl chain per polymer chain, and it

was determined from 1H NMR spectra.

5.3. Determination of critical micelle concentration

5.3.1. Pyrene 1:3 ratio method as detection of CMC

Figure 18. Pyrene derivatives: a) pyrenealdehyde; b) 1-alkyl-pyrene; c) 1,3-bis(pyrene-1-yl)propane)

Widely used fluorescence probes are pyrene and its derivates (Figure 18) [98-109],

common use as polarity-probe. Pyrene is a strongly hydrophobic polyaromatic hydrocarbon

with low solubility in water. Its fluorescence spectrum presents a fine structure, where the

relative peak intensities are highly influenced by the polarity of the solvent molecules by

which this probe is surrounded. The ratio of the fluorescence intensity of the highest energy

n

O

H

R

O

NH

O

OHNH

OOH

OH

O

O

OHO

OH

O

CH3

Figure 17. Hyaluronan derivative

43

vibrational band (I1) to that of the third highest energy band (I3) correlates with the solvent

polarity: 1:3 is around 0.6 in hydrocarbon solvents, 1.1 in ethanol and around 1.6 in water.

This unique property is based on fact that dipole moment of pyrene (3.24 ± 0.1 D) predict in

situation when some nonpolar domain exists in aqueous solution that pyrene is situated in less

polar area and indicate local environment by changes ratio of I (372 nm) to III (382 nm).

This method has been widely used not only in puremicellar solutions, but also in mixed-

surfactant systems, to investigate polymer–surfactant interactions, and in studies on the effect

of additives on the micellar properties of ionic and non-ionic surfactants. The plots of the

pyrene 1:3 ratio vs. logarithm of total surfactant concentration show sigmoid decrease around

critical micelle concentration.

The pyrene 1:3 ratio plots can adequately described by a decreasing Boltzmann sigmoidal

curve, which is given by

( ) be

bay

xxx+

+−= ∆− 01

, (3.1.1)

where the variable y corresponds to the pyrene 1:3 ratio value, independent variable x is the

logarithm of the total surfactant concentration, a and b are the upper and lower limits of the

sigmoid, respectively. x0 is the center of the sigmoid (inflection point), and ∆x is directly

related t to the independent variable range where abrupt change of dependent variable occurs.

Figure 19. Decreasing sigmoidal curve.

Figure 19 illustrates two singular points, labeled as CMC1 and CMC2. CMC1 should the

CMC values when the surfactant has a very low CMC value and CMC2 the corresponding

values in the remaining cases.CMC1 coincide with the inflection point x0. The “x” coordinates

of the CMC values are in the relationship described by xxx ∆+= 2)CMC()CMC( 12 . (3.1.2)

Log c

Py1

:3

a

b

CMC2

CMC1

44

5.3.2. Perylene-based determination of CAC

Figure 20. Perylene in determination of the CMC

This method is based on the fact, that some fluorescent probes, such as Perylene, have a

tendency to associate with micelles rather than with water phase and their fluorescence

changes depend on surroundings. Below the CMC, the probe (Perylene) is solubilized in a

polar solvent (water) to a very small extent. In the case, when micelles begin to appear in the

system and the probe becomes associated with the micelle core, fluorescence intensity is

strongly increasing. Figure 20 presents typical curve describing fluorescence changes of probe

in the presence of varying concentrations of a micelle-forming amphiphile and demonstrate

how CMC value is calculated.

5.4. Experimental conditions

The fluorescence emission spectra were monitored with a luminiscence spectrophotometer

(AMINCO-Bowman, Series 2) at 293.15 ± 0.1 K (Thermostat Grant), except experiments

with increasing temperature. In most of all cases, the excitation and emission slit widths were

set to 4 nm. Scan rate was set to 5 nm s–1, and obtained spectra were corrected.

Measurements were carried out in 1 cm quartz cuvette with stopper. In case of Acridine

orange experiments, samples were removed immediately after measurement, and cuvette was

washed in Ethanol and Water, to avoid sorption on cuvette wall.

After some measurement series, cuvette were placed into acid hydrogen peroxide and

stored through the night to remove all organic impurities. Next day cuvette was washed in

organic solvents and at last in deionized water.

0

2

4

6

8

10

12

14

16

18

-8 -6 -4 -2 0 2

Log c

Flu

ores

cenc

e (a

.u.)

log CMC

45

6. RESULTS AND DISCUSSION

This part is divided into three sections:

1) aggregation behavior monitored by water insoluble,

2) aggregation behavior monitored by water soluble probe,

3) fluorescence probes in monitoring of core quality.

All fluorescence probes were tested in simple surfactant system - aqueous solution of the

Sodium dodecyl sulfate.

6.1. WATER-INSOLUBLE PROBES IN AGGREGATION

Water insoluble probes have an extremely low solubility in water and from theirs aqueous

solutions no fluorescence signal is observed. In self-aggregate systems, the water insoluble

probes are predominantly located in the nonpolar core.

In colloidal system, presence of probes in solution is not instantaneous after addition to a

solution. In case, when probe is introduced to a flask via evaporating of volatile solvent, we

obtained thin layer of probe on the flask wall. This layer has manifold larger surface, than

compact probe crystals. This larger surface makes probe’s molecules more accessible for

surfactant molecule and can reduce time needed to full solubilization of probe in colloidal

system. In most of all cases, samples were equilibrated at least for 24 hours. Samples were

shaking on overhead or vibrating shaker and subsequently storeed at dark at laboratory

temperature.

Form a large group of water insoluble probes, three probes was selected – Pyrene, Nile red

and Perylene.

46

6.1.1. Pyrene

6.1.1.1. Simple Surfactant System

As the CAC probe for bio-polyanions Pyren’s response to the aggregation behavior was

tested with anionactive surfactant SDS. Figure 21 shows emission properties of the Pyrene in

different area of polarity environment - water and n-Heptane. The Pyrene polarity ratio varies

from 1.8 in aqueous solution to 0.5 in n-alkane environment. For this reason, Pyrene is useful

because its emission band is not shifting according to polarity of a microenvironment. Only

emission band at 373 nm is polarity-sensitive and changes probability of transition. This

transition probability is increasing in environment with high polarity (high dielectric

constant).

Figure 21. Emission properties of the Pyrene in aqueous and n-heptane solution.

The plot of the polarity index on SDS concentration is shown in Figure 22. Raw data were

fitted by Boltzmann S-type curve. Results from non-linear curve fitting with OriginPro75 are

displayed as inset table in the Figure 22. According to the Equation 3.1.2, we obtained two

possible critical aggregation values:

CAC1 = (2.68 ± 0.15) mol dm-3, and CAC2 = (7.43 ± 0.45) mol dm-3.

Statistical parameters of this fit was R2 = 0.9977, and χ2/DoF = 0.0002.

0

0.2

0.4

0.6

0.8

1

360 400 440 480 520

Wavelength (nm)

No

rmal

ize

d flu

ore

sce

nce

inte

nsi

ty (

-)

n-HeptaneWater

47

Figure 22. Typical dependency of the polarity index on the SDS concentration. Inset table displays fit

parameters obtained from non-linear curve fit with OriginPro75.

Both values are slightly below the Elsworthy’s CAC value (8.2 10–3 mol L–1[96]). This

could be caused by an induction effect on aggregation process caused by the presence of non-

polar species, fluorescence probes, in solution. Pyrene’s molecules can organize surfactant

molecules to form probe-surfactant aggregates. These aggregates can be formed before the

micelles are presented in solution. In this aggregate type, the Pyrene molecule is covered with

not only surfactant but also the water molecules are presented in immediate surroundings.

This aggregate induction leads to the first moderate decrease of the polarity index. In all

cases, the Pyrene as the CAC probe was confirmed.

In the simple surfactant system, the influence of the Pyrene’s concentration was studied.

Seven concentrations of SDS were selected in order to Elsworthy’s CAC value.

Concentrations of the Pyrene were selected as range from 10–7 to 10–5 mol L–1. Figure 23

summarizes influence of Pyrene’s concentration on the polarity index in different SDS

concentration. Stronger concentration influence shows concentrations below the CAC value,

above the CAC the probe concentration is irrelevant. Shifting of the polarity index to higher

values in concentrations below the CAC can be explained as increasing of the part Pyrene in

aqueous environment. This caused the environment polarity looks like more polar. For

following experiments, the Pyrene concentration was used in range 1-6 10–6 mol L–1.

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

0.001 0.01 0.1 1 10 100 1000

Concentration of SDS (10-3 mol L-1)

Po

larit

y in

de

x (-

)

Raw dataFit

parameter value errora 1.504 ± 0.006b 0.841 ± 0.009

x 0 2.680 ± 0.147∆∆∆∆x 2.375 ± 0.153

Fit parameters

48

Figure 23. Dependency of the polarity index on the probe’s concentration in different concentration of

SDS. Three lowest concentrations show significant changes, from 8 mM the polarity index is not

influenced.

6.1.1.2. Native hyaluronan

Figure 24. Polarity index in native hyaluronan in aqueous solution. Different molecular weights were

selected to demonstrate almost constant character of the polarity index value in wide concentration

range. Fluctuation of the polarity index value is insignificant considering to range of the polarity

index.

0.60

0.80

1.00

1.20

1.40

0.1 1.0 10.0

Pyrene concentration (10-6 mol L-1)

Pol

arit

y in

dex

(-)

3 mM 4 mM 6 mM 8m M 12 mM 16 mM 20 mM

Below CAC

Above CAC

49

First measurements have been focused on possible aggregation behavior of native

hyaluronan in aqueous solution. Four molecular weights (30, 97, 560, and 1,630 kg mol-1)

were selected to investigate their concentration dependencies in the presence of Pyrene.

Figure 24 shows that the polarity index, ranges from 1.40 to 1.42 through a wide

concentration range. Value of the polarity index in this concentration range can be taken as

constant and invariant. So, from the hydrophobic polarity probe point of view no aggregation

behavior was observed in these solutions and in this concentration range.

6.1.1.3. Hydrophobized hyaluronan

Typical results with hydrophobized hyaluronans, obtained for D30/30 a 1,470/30, are

presented in Figure 25. For good lucidity, concentration values are plotted in dimension g L–1.

The CAC value for lower molecular weight looks to be lower than for higher molecular

weight. After the conversion of concentration dimension from g L–1 to 10–6 mol L–1 the CAC

value for 1,470 kg mol–1 (1,667 10–6 mol L–1) is 25-times smaller than for 30 kg mol–1

(0,068 10–6 mol L–1).

Aguiar and co-workers [110] suggested a condition to select the CAC value from the

pyrene polarity index. If the x0/∆x (cf. Eq. (1)) is less than 10, the CAC point is determined by

the x-coordinate of the inflection point x0 - CAC1 in our case. Table 2 summarizes determined

values of the x0/∆x ratio of hyaluronate derivatives. All of them passed the “less than 10-

condition”.

Table 2. The representative samples of hydrophobized hyaluronan with the x0, ∆x, and criterion value

obtained from curve-fitting. All samples have passed the “less than 10-condition” criterion.

samples x0 ∆∆∆∆x x0/∆∆∆∆x R2

- g L–1 g L–1 - -

D134/10 0.61 1.51 0.40 0.99

D183/30 0.15 1.55 0.10 0.98

D360/50 0.08 1.48 0.05 0.98

D1470/70 0.20 1.03 0.19 0.97

For investigation of the influence of molecular weight and degree of substitution, mixed

group of derivatives were selected. In this group, molecular weights range from

44 kg mol–1 to 1,470 kg mol–1. Substitution degrees are preferably 10, 30, 50, and 70 %.

Unfortunately, it was not possible to obtain all substitution degrees for all selected molecular

weights.

50

Figure 25. Pyrene polarity index in hydrophobized hyaluronan (with the same degree of substitution

and different molecular weight) aqueous solution. For transparency, concentration values are plotted

in dimension g L–1. CAC points, determined from inflection points, are marked with arrows.

Figure 26. Dependency of CAC values on substitution degree for different molecular weights

(complete sets of molecular weights were disposable only for 30 and 50 % degree of substitution).

0.5

0.7

0.9

1.1

1.3

1.5

1.7

0.01 0.1 1 10Concentration (g L-1)

Pol

arit

y in

dex

(-)

30/301,470/30

M = 30 kg mol-1, SD = 30 %

M = 1,470 kg mol-1, SD = 30 %

0.01

0.1

1

10

100

0 10 20 30 40 50 60 70 80

Substitution degree (%)

CA

C (

10-6 m

ol L

-1)

441341833601,470

51

The CAC values of various derivatives show two types of trends. First, the CAC values

decrease when the substitution degree increases, with a small exception for samples D 1470

that in general have very small CAC. Second, the CAC values decrease with increasing M.

These trends are obvious from Figure 26 and summarized in Table 3. The greatest decrease of

CAC values with increasing substitution degree show samples D 44. It is possible to relate

this behavior to the fact that D 44 samples have the shortest chains and new types of

interactions – hydrophobic – can much easily change chains orientation and conformation

leading to their aggregation. Results are compared with Perylene measurement and they are

briefly discussed in Appendix V.

Table 3. Summary of the CAC values for hHA samples determined by the Pyrene polarity index

method. Difference between the highest CAC value 12.27 10-6 mol L–1 (D44/10) and the lowest

CAC value 0.06 10-6 mol L–1 (D44/50; and D1470/50) is three orders. These results clearly

show that CAC value can be modified not only by the substitution degree but also by the

molecular weight of hyaluronan’s derivative.

M SD CAC

kg mol-1 % x 10-6 mol L–1

10 12.27 30 1.02 44

50 0.06 10 4.35 30 1.21 50 0.76

134

70 0.20 10 0.99 30 0.73 183

50 0.47 10 0.51 30 0.19 360

50 0.18 30 0.15 50 0.06 1470

70 0.10

6.1.1.4. Influence of ionic strength

Because of presence of carboxylic groups, low molecular salts can influence CAC. For

this experiment were selected group of modified hyaluronans. Molecular weight was

100 kg mol–1 a three different substitution degrees – 10, 30, and 50 %. Sodium chloride was

select for modification of ionic strength of a hyaluronan solution. CAC were determined in

three salt concentrations – 0, 0.1, and 0.4 mol L–1. Figure 27 shows dependencies for

derivative with substitution degree 10 %. Table 4 summarizes results from measurements.

52

Table 4. Summary of the CAC values (10–6 mol L–1) for selected hHA samples in solutions containing

different concentration of sodium chloride.

M SD 0 0.1 0.4

kg mol-1 % mol L–1 mol L–1 mol L–1

10 3.83 3.5 3.05

30 1.36 1.11 0.97 100

50 1.22 0.95 0.81

Results clearly show decreasing of the critical aggregation value with increasing value of

ionic strength for all degrees of substitution. The highest decrease shows sample with

substitution degree 50 %. The decrease is about 40 %.

Figure 27. Dependency of polarity index values on concentration of modified hyaluronans in aqueous

solution, with different concentrations of NaCl in different concentration. Molecular weights are

100 kg mol–1 and degrees of substitution 10, 30, 50 %.

Presences of low molecular salts in solution suppress repulsive forces between dissociated

carboxylic groups of the modified hyaluronan. This enables polymer’s chain approach to each

other and form polymeric micelles at lower concentration.

0.9

1.1

1.3

1.5

0.0 0.0 0.1 1.0 10.0

Concentration (g L-1)

Pol

arit

y in

dex

(-)

0 M0.1 M0.4 M

CAC

ionic strength

53

6.1.2. Nile red

Emission properties of the Nile red in chloroform and methanol solution are shown in

Figure 28. Inset figure displays structure of the Nile red probe. Visible positive shifts of the

excitation and emission maxima confirmed the Nile red as solvatochromic probe.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

400 450 500 550 600 650 700 750 800 850

Wavelength (nm)

No

rma

lize

d flu

ore

scen

ce in

ten

sity

(-)

EM chloroformEX chloroformEM methanolEX methanol

O

N

O

N

CH3

CH3

Figure 28. Emission properties of the Nile red in chloroform and methanol solution. Inset figure

displays structure of the Nile red probe. Visible positive shifts of the excitation and emission maxima

confirmed the Nile red as solvatochromic probe.


For checking of spectral properties of NR in associative system, the SDS was selected as

well as for Pyrene probe.

Nile red fluorescence in effectively quenched in aqueous microenvironment, so, a sharp

increase of fluorescence intensity above the CAC concentration were expected.

In high polar media, NR has emission maximum around 655 nm. In non-polar

environment, the emission maximum wavelength decreases to 590 nm. According to these

conditions above the CAC a significant shift were expected.

54

Table 5. Summary of the CAC values for SDS in aqueous solution obtained from Nile red

measurements. Data were collected using the sigmoidal fit.

CAC Intensity Wavelength

x 103 mol L–1 - -

CAC1 6.96 ± 1.01 3.07 ± 0.03

CAC2 9.11 ± 2.12 6.09 ± 0.09

STAT

R2 0.9958 0.9889 χ2/DoF 0.0012 0.6753

Figure 29 shows evolution of both studied parameters. Results from non-linear curve

fitting with Boltzman sigmoidal curve are displayed as inset tables in this figure. Form both

dependencies, we obtained again two possible critical aggregation values, showed in Table 5.

For accuracy holding three measurements were performed. Results are collected in Table 6.

Table 6. Summary of the CAC values for SDS in aqueous solution obtained from Nile red

measurements. CAC Intensity Intensity Wavelength

x 103 mol L–1 1 2 3 av. Wavelength

1 2 3 av.

CAC1 6.10 6.23 6.80 6.38 ± 0.30 0.10 1.22 0.52 0.61 ± 0.46

CAC2 6.95 7.08 7.14 7.06 ± 1.02 0.11 1.78 1.16 1.02 ± 0.69

In Table 5 statistical parameters of the fit are also displayed, as same as in Table 6 a

standard errors are noted. From these statistics is obvious that the CAC values from intensity

measurement are less accurate. For that reason, another method was used to solve the CAC

value from experimental data. We had been looked for first breakpoint in data’s dependence,

so, two lines method was selected. Evaluation of the Nile red data is showed in Figure 30.

Point of intersection has independent coordinate after the conversion equal

3.93 10–3 mol L–1. This is the lowest value obtained by Nile red. Correlation coefficients of

the linear fits are on the margin of linearity. This should be related again to the induction

effect on the low-molecular surfactant SDS.

55

Figure 29. Fluorescence intensity and position of emission maxima dependencies on the SDS

concentration. Both dependencies have sigmoidal character Data were fitted by Boltzman S-type

curve and inset tables display parameters of these fits. Solved CAC values are described in text.

Figure 30. Alternative data evaluation method from fluorescence intensity dependency on logarithm of

the SDS concentration. CAC as the point of intersection is 3.93 10-3 mol L–1.

635

640

645

650

655

660

0.0001 0.001 0.01 0.1 1 10 100 1000

Concentration SDD (10-3 mol L-1)

Em

issi

on m

axim

um (

nm)

0.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

flu

ores

cenc

e in

tens

ity (

-)

EM maximumMaximum fitFluorescenceFluorescence fit

wavelength dataparameter value error

a 655.30 ± 0.41b 640.10 ± 0.38x 0 3.07 ± 0.03∆∆∆∆x 1.51 ± 0.03

-0.2

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

-5 -4 -3 -2 -1 0 1 2 3

Log(concentration) (10-3 mol L-1)

Nor

ma

lize

d fl

uo

resc

ence

inte

nsity

(-)

y = 0.0013x + 0.0088

R2 = 0.9525

y = 1.8359x - 1.0829

R2 = 0.9511

56


Similarly to pyrene’s measurements, the Nile red was firstly tested in native hyaluronan’s

aqueous solution. Two samples with different molecular weights, 310 and 1,109 kg mol-1,

were selected. Concentration dependencies of both studied parameters are displayed in

Figure 31. Polarity parameter, emission maximum, displayed almost linear character. Mean

wavelength value displays polar microenvironment and response to the values obtained from

measurements with SDS bellow its CAC value. On the other hand, fluorescence intensity

looks to be strongly influenced by the concentration of the native hyaluronan in aqueous

solution (displays as inset Graph in Figure 31). This is caused by a relativity of displayed

normalized data. In fact, obtained intensities were too low and they were obtained with the

highest sensitivity of detector. This is confirmation of the fact native hyaluronan domains are

completely hydrated a present water molecules effectively quench the Nile red emission.

Figure 31. Concentration dependencies of the Nile red parameters, emission maximum wavelength

and fluorescence intensity (inset Figure), on concentration of two hyaluronan’s samples in aqueous

solution. Emission maximum response to polar environment and looks to be not affected by

hyaluronan’s concentration. Normalized fluorescence intensities were extremely low to display them

in common scale and show no clear effect of the hyaluronan concentration too.

640

645

650

655

660

0.01 0.1 1 10


Em

issi

on m

axim

um

(n

m)

310-EM max1,109-EM max

0.00

0.40

0.80

1.20

0.01 0.1 1 10


No

ram

lize

d flu

ore

sce

nce

inte

nsity

(-)

310-fluorescence

1109-fluorescence

57


A group of hydrophobized hyaluronans was selected for the study of influence of the

molecular weight and degree of substitution on CAC: 1470/30, 50, and 70, 183/50, 500/50.

All samples were prepared in common way in wide concentration range in aqueous solution.

Analogously to measurements with Pyrene, emission properties were affected by varying

concentration of modified hyaluronan. Selected parameters, the position of emission

maximum and the intensity of fluorescence, show an abrupt decrease in their concentration

dependencies. In Figure 32, two samples are showed to demonstrate aggregation behavior

Figure 32. Concentration dependencies of the Nile red emission maximum wavelength on

concentration of two hyaluronan’s samples in aqueous solution.

Obtained CAC values are listed in Table 7. This table included CAC from both parameters

dependencies. All dependencies had sigmoidal character. Listed CAC values are inflection

points from Boltzman sigmoidal fit of raw data.

CAC values obtained from intensity parameter are 4 times higher than values obtained

from wavelength measurements. This can be related to the fact, that quantum yield of the Nile

red is related not only to amount of the probe dissolved in solution but also to the

presence/absence of water molecules in the probe microenvironment. In fact, the wavelength-

polarity parameter can be collected even if the fluorescence intensity is very low. In aqueous

environment the Nile red is effectively quenched, so non-solubilized probes have no effect on

0.001 0.01 0.1 1605

610

615

620

625

630

635

640

645

650

655

1,470 kg mol-1; 70 %

183 kg mol-1; 50 %

em

issi

on

ma

xim

m w

ave

leng

th (

nm

)

concentration (g dm-3)Concentration (g L–1)

E

mis

sio

n m

axim

um

(nm

)

58

polarity parameter. This means the polarity-wavelength decrease sooner than the fluorescence

intensity.

Table 7. Summary of the CAC values for selected hHA samples determined by Nile red parameters.

These results clearly show that CAC value can be modified not only by the substitution degree

but also by the molecular weight of hyaluronan’s derivative.

M SD CAC intensity CAC wavelength

kg mol-1 % x 106 mol L–1 x 106 mol L–1

183 50 2,514 0.437

360 50 0.778 0.194

30 0.313 0.075

50 0.116 0.048 1470

70 0.129 0.034

59

6.1.3. Perylene

This probe has extremely low solubility in water and it is predicted Perylene is presented

only in micellar core. Spectral properties are shown in Figure 33. As the excitation

wavelength the second maximum at 410 nm were selected. Emission intensity was monitored

at 443 nm.

Figure 33. Spectral properties of Perylene in chloroform. Dashed line displays excitation spectrum,

full-body line displays emission spectrum. Both spectra were normalized.


Tests in simple surfactant system clearly show Perylene is able to determine aggregation

processes’ in aqueous solutions. Dependency of the fluorescence intensity on a sample

concentration and data evaluation method is similar to Nile red fluorescence intensity

measurements and they are displayed in Figure 34.

Obtained the CAC value for SDS is (7.5 ± 0.2) 10-3 mol L–1. This value is smaller than

values obtained with surface tension measurements and confirm the theory that probe

presented in solution slightly below the CAC can induced forming of the micelles which

result in decreasing of the CAC value.

0

0.2

0.4

0.6

0.8

1

320 370 420 470 520 570Wavelength (nm)

No

ram

lize

d f

luor

esc

enc

e (

-)

ExcitationEmission

410 nm

443 nm

Absorption

Emission

60

Figure 34. Perylene’s intensity of fluorescence dependency on concentration of the SDS. Raw data are

fitted with linear curves. Their point of intersection determines the CAC value.


Situation in native hyaluronan is similar to the situation with other hydrophobic probes.

Low solubility of the Perylene was not enhanced in presence of native hyaluronan in different

molecular weights.

From above mentioned reasons, Perylene was found as unsuitable for detection of the

aggregation of the native hyaluronan.


Samples with hydrophobized hyaluronan were tested in the same way as were used for

Pyrene and Nile red.

Three samples were selected with same degree of substitution (30 %) and with different

molecular weights (134, 183, and 360 kg mol–1). Evaluation of experimental data was selected

according to degree of probe saturation at high concentration of hyaluronan. Measurements

where probe is unsaturated were evaluated with the point of intersection method,

measurements which have a constant value of fluorescence intensity at high concentration

were evaluated with sigmoidal fitting. Experimental data with evaluation are shown in

Figure 35.

R2 = 0.9939

R2 = 0.9950

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

4 6 8 10 12

Concentration (10-3 mol L-1)

No

rma

lized

flu

ores

cenc

e in

ten

sity

(-)

CAC = 7.05 mM

61

Figure 35. Perylene’s intensity of fluorescence dependency on concentration of the hydrophobized

hyaluronan. In Figure are displayed different types of data evaluation. The sample with the lowest

molecular weight did not reach the probe saturation level and were evaluated with the point of

intersection. The other samples were evaluated with sigmoidal fit.

Form sigmoidal fit were obtained two possible points again, which can determine the

CAC value. Values are collected in Table 8. CAC1 means point of intersection or point with

independent coordinate (defined as “x0 – 2 ∆x”) from sigmoidal fit. CAC2 means the

independent coordinate of the inflection points obtained from sigmoidal fit.

These results, were briefly discussed and published – see Appendix V.

Table 8. Summary of the CAC values for selected hHA samples determined by Perylene.

M SD CAC1 CAC2

kg mol-1 % x 10-6 mol L–1 x 10-6 mol L–1

134 30 0.66 ND

183 30 0.54 0.96

360 30 0.09 0.12

If CAC1 values are plotted against the molecular weight, we will obtain strictly linear

dependency of determined values. Correlation coefficient of this dependency is equal to “1”.

0

0.2

0.4

0.6

0.8

1

0.01 0.1 1 10 100

Concentration (10-6 mol L-1)

No

rma

lized

flu

ore

sce

nce

flu

ore

sce

nce

(-)

134/30183/30360/30

62

6.2. WATER-SOLUBLE PROBES IN AGGREGATION

Water-soluble probes are wide and varied group of fluorescent probes. We were interested

if the changes in the hydrophobic core, investigated by water-insoluble probes, have an image

at the hyaluronan’s charged surface. These probes allowed to us investigate possible

aggregation behavior of the native hyaluronan.

We involved in this group probes Acridine Orange, TNS and PRODAN. If the probe and

surface are charged oppositely (Acridine Orange) and there is no steric protect, the probe and

the surface will form an ionic pair with different photo-physical behavior from free probe in

solution. This change can be expressed as new absorption or emission band, shift of existing

bands, fluorescence quenching, etc. On the other hand, this helps us to localize probe in

solution - inner part of Stern layer.

When the probe and the surface are charged identically or non-charged, electrostatic

interactions are suppressed or have repulse character. In this system are attracted forces based

only on hydrophobic (disperse) interactions. Photo-physical properties of the probe are not

influenced. Identically charged probe also interact charged surface. It can be localized in

Gouy-Chapman layer where the surface’s charge is compensate by counter ions or by addition

of electrolyte.

TNS probe has extremely weak fluorescence in water and in fact, fluorescence grow up

when TNS is adhered on non-polar supra-molecular surface, but its structure contains sulfonyl

group and to the system it can added as aqueous solution.

PRODAN probe has observable fluorescence from alkanes to the water. Other probes can

be charged positively or negatively or, in fact, non-charged. This quality is deterministic for

probe’s position around the charged surface.

63

6.2.1. Acridine Orange

Figure 36. Excitation (Ex) and emission (Em) spectra of the Acridine Orange in different

concentration in aqueous solution (10 and 250 10–6 mol L-1). Bathochromic shift with increase

concentration is evident. Inset shows chemical structure of the Acridine Orange as hydrochloride

hydrate.


Acridine orange was firstly tested similarly to hydrophobic probes in aqueous solution of

the SDS. In solution above the CAC, negatively charged micellar surface is presents. For this

reason is the SDS an ideal model for aggregation studies on the hyaluronan.

Figure 37 shows fluorescence intensity data dependency on the SDS concentration in its

aqueous solution. Data were related to the zero SDS concentration. From zero to

approximately 5 mM of the SDS, fluorescence intensity decrease. Around the expected CMC

(8.2 mM), fluorescence intensity sharply increases to a constant value. This value is slightly

higher (~ 20 %) than value obtained from the sample without the SDS.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

450 500 550 600 650 700 750

Wavelength (nm)

Flu

ores

cenc

e (a

.u.)

250 uM Em

250 uM Ex

10 uM Em

10 uM Ex

Cl-

H

N+

N N+

64

Data around the CMC and higher were evaluated using Boltzman’s sigmoidal curve. From

fit parameters, CAC1 a CAC2 are evaluated. Obtained values 9.80, and 10.18 respectively, are

slightly higher than expected Elworthy’s value [96]. When we take account of the first point

of change, realizes as “x0 – 2∆x”, we obtained value 9.42. This value reflects mathematical

representation of the first change in decreasing trend and it is the nearest value to the

tabulated CMC. On the other hand, these quite different CAC values are expectable. Probe’s

cation may shield repulsive interaction between sulfate groups and allows more surfactant

molecules may occupy air/water interface. This phenomenon should shifts the CMC value to

slightly higher value.

Figure 37. Fluorescence intensity, related to the zero SDS concentration, dependency on the SDS

concentration in aqueous solution. Data were fitted with the Boltzman’s sigmoidal curve.

Next addition of the surfactant has no observable influence on the fluorescence intensity.

This corresponds to a new equilibrium between bound dimers (or oligomers) and bound

monomers.


Contrary to measurements with hydrophobic fluorescence probes, experiments with

cationic fluorescent dye brought many interesting results. Predominantly dissociated carboxyl

groups in aqueous solution at pH ~7 interact with the acridine orange cation. This interaction

leads to formation of probe’s dimers and to decreasing of the fluorescence intensity of the

monomer.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0.01 0.10 1.00 10.00 100.00

Concentration SDS (x 10-3 mol L-1)

Nor

mal

ized

flu

ores

cenc

e (-

)

parameter value errora 0,01841 ± 0,00753b 1,15127 ± 0,00616x 0 9,80225 ± --∆∆∆∆x 0,18982 ± --

65

Shape of the dependency of the AO’s fluorescence intensity on the concentration of the

native hyaluronan should be affected by the initial concentration of the probe. This influence

should be checked off if the fluorescence intensity is plotted against a ratio of a polymer

binding sites to the concentration of the probe – P/D, polymer-to-dye ratio. For the selected

hyaluronan’s molecular weights, typical dependencies are shown in Figure 38. Displayed

range is equal to a hyaluronan concentration range from 0 to 0.05 g L-1. In this dilute region, a

dependency can be separate to two linear curves. Theirs point of intersection is end-point of

titration, (P/D)e, a point of equivalence where all free carboxyl groups are occupied with at

least one dimer of acridine orange.

Figure 38. Dependency of the relative fluorescence intensity of AO on P/D for native hyaluronan in

aqueous solution with two different molecular weights – 106 and 253.9 kg mol–1. There is also shown

end-point of titration, (P/D)e, a point of equivalence where all free carboxyl groups are occupied with

at least one dimer of acridine orange.

Because of electrostatic origin of AO dimer formation on polyelectrolytes, influence of the

low molecular weight electrolytes addition was also studied. First experiment was focused on

a change of end-point of titration. Figure 39 shows dependency of the AO fluorescence

intensity in dilute region of HA aqueous solution in the presence of NaCl in two different

concentrations – 0.1 and 0.5 mol L–1. Due to an electrostatic shielding of carboxylic groups by

low-molecular weight salt, end-point of titration is moved to lower values. These values are in

the same order of magnitude.

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 1 2 3 4 5 6 7 8

P/D (-)

Re

lativ

e flu

ores

cenc

e in

tens

ity (

-)

106 kDa253,9 kDa

(P/D

) e

○ 106 kDa ■ 253.9 kDa

66

Figure 39. Dependency of the relative fluorescence intensity of AO on P/D for native hyaluronan in

aqueous solution with a different presence of NaCl (0, 0.1, 0.5 mol L-1).

Next experiments handled with different types of chlorides – Li+, Na+, K+, Ca2+ in the

constant concentration of the native hyaluronan. Hyaluronan with the molecular weight

106 kg mol−1 was selected in the concentration 5 g L−1. Concentration of the probe was

2 10−5 mol L−1. These relatively high concentrations of the probe and hyaluronan were due to

absorption measurements, which has lower sensitivity and higher amount of the probe in the

solution is necessary. Because of relatively high concentration of the probe in solution, we

increased of hyaluronan concentration to final P/D ~2000. This value is compromise between

concentration of the hyaluronan, solution become a quite viscous at this concentration, and

concentration of the probe – value of the ratio is much more higher than end-point of titration

in pure water.

Obtained results from absorption measurements are shown in Figure T. In this graph is

shown dependency of the Monomer:Dimer ratio on the ionic strength. This ratio is realized as

ratio of absorbance values at 492 nm and 465 nm respectively. Its value has information about

changes in quantities of monomers and dimers in solution. Influences of ionic strength are

rather same for all cations in the region above 0.05 mol L–1. Below this value increasing ions

concentration, lead to higher values of ratio. Dimers, which are presented in the solution, are

depolymerized in the presence of the low molecular salts. This process is shown in Figure 40.

Unfortunately, we are not able to differentiate bound and free AO monomer, so we can

predict according to high P/D value, that AO monomers from dimers are bound to polymer in

equilibrium.

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

0 1 2 3 4 5 6 7 8

P/D (-)

Rel

ativ

e f

luo

resc

ence

inte

nsi

ty (

-)

0 M0.1 M0.5 M

0.5

M

0.1

M

0 M

67

Figure 40. Dependency of the Monomer:Dimer ratio from absorption spectra of AO on the ionic

strength in the presence of HA (5 g L–1) for different types of chlorides.

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

0.01 0.10 1.00

Ionic strength (mol L-1)

Flu

ores

cenc

e (a

.u.)

LiClNaClKClCaCl2

Figure 41. Dependency of the fluorescence intensities of AO on the ionic strength in the presence of

hyaluronan (5 g L–1) for different types of chlorides.

1.3

1.4

1.5

1.6

1.7

0.01 0.10 1.00Ionic strength (mol L-1)

Mo

nom

er:

Dim

er

ratio

(-)

LiClNaClKClCaCl2

68

Evaluation of the fluorescence measurements in the presence of different types of chloride

is shown in Figure 41. This measurement confirms absorption measurements. In the

increasing concentration of low molecular salts increases amount of monomer in system.

Increasing amount of monomers is observable by fluorescence intensity increasing. In the

case of Ca2+ fluorescence intensity increases sharply (more than 50% increase) to ionic

strength value 0.025 mol L–1. Above this value, fluorescence remains constant. Monovalent

cations increasing slowly and reach maximum ~0.1 mol L–1 Next addition of the salts lead to

the decreasing of the fluorescence intensity in all cases. In this, relatively high, concentration

is possible to observe not only changes of formation/destruction of the dimers but this

decrease can be explained by dynamic fluorescence quenching of AO molecules by present

Cl– anions.

If we summarize effects of the low molecular salt on system AO-HA, we obtained these

conclusions:

• in the presence of the low molecular salt, end-point of titration is moved to lower

values

• in the increasing presence of ionic strength, formed dimers are destructed which lead

to the increasing of the fluorescence intensity and ratio between amounts of monomers

and dimers in solution

• in comparable values of ionic strength, Ca2+ has more intensive effect on system than

monovalent ions


Motivation for this experiment was question if the aggregation process, observable with

hydrophobic probes, has its own image in outer part of formed aggregate, and if it is able to

determine CAC.

Derivative with MW 360 kg mol–1 and 50% substitution degree was selected. As the

reference probe, Nile red was selected because of its zero fluorescence in aqueous

environment. Nile red was found suitable for aggregation measurements because it carrying

two different types of information, fluorescence intensity and position of the emission

maxima, respectively.

Concentration range was kept wide for both probes. Evaluation was focused on abrupt

changes or break points in emission spectra, which can be found for both probes.

Results are shown in Figures 42 and 43. In Figure 42, fluorescence of both probes are

compared. In Figure 43, we focused on second parameter – emission maximum, which can be

used for determination of the critical aggregation concentration, and is compared with

fluorescence intensity of AO.

69

Figure 42. Fluorescence intensities of the Acridine Orange (AO) and Nile red in different

concentration of the hydrophobized hyaluronan with MW 360 kg mol–1 an SD 50 %. Dashed line

represents CAC value.

Figure 43. Fluorescence intensity of the Acridine Orange (AO) and position of emission maxima of the

Nile red (fitted) in different concentration of the hydrophobized hyaluronan with molecular weight

360 kg mol–1 and SD 50 %. “X” represents inflection point, and dashed line represents CAC value.

0.0

0.2

0.4

0.6

0.8

1.0

0.0001 0.001 0.01 0.1 1 10


Flu

ores

cenc

e (

a.u

.)

0.60

0.70

0.80

0.90

1.00

Flu

ores

cenc

e (

a.u

.)

NRAO

CAC

610

620

630

640

650

660

0.0001 0.001 0.01 0.1 1 10


Em

issi

on m

axi

mum

(nm

)

0.60

0.70

0.80

0.90

1.00

No

rma

lize

d fl

uor

esc

ence

(a.

u.)

NR

AO

inflex point

CAC

70

Fluorescence intensity dependency for the Nile red has three different regions. First

region, below the CAC value, has zero fluorescence. Second region, around and above the

CAC, with exponential (linear in logarithmic scale) increasing. Third region, in high

concentration of the hydrophobized hyaluronan, has constant value of the fluorescence

intensity. Second and third regions are separate with leap increasing of fluorescence intensity.

Likewise, for dependency of the AO, three regions can be found. First decrease is followed by

a region with constant value of the fluorescence intensity. Fluorescence dependency changes

in the point, which corresponds to the first break point from the Nile red dependency - this is

common CAC point. Constant region is followed by a decreasing region (!), which agrees

with constant region from the Nile red measurements – compare with simple surfactant

measurements, chapter 5.2.1.1.

Figure 44. Graphical illustration of potential aggregation behavior of hydrophobized hyaluronan in

aqueous solution. This is a case, where distribution of alkyl chains is unequal. Two possible aggregate

types can be found in solution – intermolecular (a) and intramolecular (b). Black represents polymer,

wavy red line represents side alkyl chain.

Dependency of the position of the Nile red maximum wavelength has typical S-type

shape. Inflection point from its fit corresponds not only with first break point from

measurements of the Nile red fluorescence intensity but also with first break point from AO

measurements.

This experiment confirmed aggregation process has its image in the outer part of formed

aggregate. If we analyze AO measurements we can assign first decrease, below the critical

concentration, to the fact addition of carboxylic groups induce dimer/oligomer formation.

Followed region remain at constant value through the one order of magnitude of

concentration. If we convert concentration into P/D ratio, margins of constant region are 100

and 800, respectively. Both these values are too high in comparison with determined end-

accesible COO- group

inaccesible COO- group

a) b)

71

points of titration from the native hyaluronan. Constant region represents a state, where

formation and destruction of the dimers/oligomers are in dynamic equilibrium. Surprisingly,

this region is not followed by increasing region (theoretically hundreds of COO– groups are

presented in solution per one molecule AO), but next decreasing is expected. Because

dependency has not reached increasing phase, is hardly to say if we not cross the end-point of

titration yet. Continuous formation of dimers may mean structure of hydrophilic region of

aggregate is “multilayer” and make almost all COO– group inaccessible for AO molecule,

however these molecules ale quite small and planar (Figure 44). It is a question if these layers

are intra- or intermolecular origin.

72

6.2.2. TNS

Figure 45 shows spectral properties of TNS in different polarity environments. Polarity

was adjusted by methanol in aqueous solution. With increasing amounts of methanol in

aqueous solution (from 0 to 80 % vol.), quantum yield of TNS fluorescence sharply increases

(Figure 46).

Figure 45. Excitation and emission spectra of TNS in solutions with different polarity (aqueous

solution of methanol).

Figure 46. Dependency of the fluorescence intensity on increasing volume fraction of MeOH in

aqueous solution

0

2

4

6

8

10

250 300 350 400 450 500 550

Wavelength (nm)

Flu

ores

cenc

e in

tens

ity (

a.u.

)

EX 20 % MeOH aq.

EX 80 % MeOH aq.EM 80 % MeOH aq.

EM 20 % MeOH aq.

0

1

2

3

4

5

6

7

8

9

0 10 20 30 40 50 60 70 80 90

Volume fraction of MeOH in aq. (%)

Flu

ore

sce

nce

(a.u

.)

73

If we focused on dependency of position of emission maximum on polarity of

microenvironment, we obtain non-significant results (Figure 47). Dependency is polynomic-

like (best-fit results were obtained for 4th-order polynom).

Figure 47. dependency of the emission maximum of TNS on the relative permittivity in pure alcohols

and alcohol/water mixtures.


Experiments in aqueous solution of SDS and theirs evaluation were made with respect to

the nature of this probe. TNS is hydrophobic polarity probe but it has pretty good solubility in

aqueous environment due to its sulfonic group. These facts made this probe amphiphilic-like.

It can be predicted TNS will not penetrate into non-polar core, but it stay in the position

between aqueous and non-polar environments. Penetration of TNS into the SDS core makes

difficult repulsive electrostatic forces between sulfonic groups. Next difficulties rise from

non-monotonous dependency of the emission maximum on polarity of its microenvironment.

This fact can disable using of second parameter from emission measurement for evaluation of

aggregation process.

Figure 48 shows dependency of the fluorescence maximum and fluorescence intensity at

420 nm on concentration of aqueous solution of SDS. Standard value of SDS’ CMC is also

displayed. Emission maximum has from global point of view increasing character. This

monotonous trend is abrupt by decreasing with local minimum at 9 10–3 mol L–1. This value is

really close to the values obtained by other probes and Elworthy’s value from surface tension

measurements.

Methanol

Ethanol

1-Propanol1-Oktanol

400

420

440

460

480

0 20 40 60 80 100

Relative permittivity (-)

Em

issi

on m

axim

um

(n

m)

"pure alcohols"

"alcohol/water mixture"

"wa

ter"

1-Octanol

74

Figure 48. Dependency of the emission maximum and fluorescence intensity at 420 nm on

concentration of SDS in aqueous solution. Bold dashed line represents CMC of SDS at 8.2 mmol L–1.

Fluorescence intensity shows typical dependency for apolar probes (e.g. Perylene, see

Figure 4.2.3), but abrupt point, and so possible CMC point, is moved to a lower value -

around 1.5 mM. Above this value, another break point can be found nearby 10.3 mM.

Both measurements brought non-significant results. Decreasing in emission maximum

trend is hard to explain because of its complicated dependency on polarity in non-colloidal

solutions. Data from intensity measurements can be affected by possible amphiphilic

character of TNS. TNS as water-soluble probe can induce pre-micellar aggregates in its

microenvironment.

We must conclude TNS is not suitable for determination of the CMC in SDS solution.


Experiments in native have point to successfully probing the hydrophobic patches in

native hyaluronan. For experiments three molecular weights of HA were selected – 100, 253,

and 740 kg mol–1, respectively. Because of TNS is negatively charged in solution with pH ~7,

to suppress charge repulsion low molecular salts were added into a solution. Concentration of

HA was kept constant at 2.5 g L–1.

Due to a non-specific reaction of the emission maximum on polarity, only fluorescence

intensity was take in data evaluation. Table 9 summarizes results of dependency of

fluorescence intensity of TNS on ionic strength for different cations in HA solution. For

comparison, fluorescence intensities of TNS from aqueous samples were added. From

400

405

410

415

420

425

430

435

0.01 0.1 1 10 100 1000

Concetration of SDS (mmol L-1)

Em

issi

on m

axim

um

(nm

)

1

1.1

1.2

1.3

1.4

1.5

1.6

Flu

ore

sce

nce

inte

nsity

(a

.u.)Wavelength

Emission at 420 nm CMC

75

obtained results is obvious, that ionic strength influence was insignificant. In all cases,

samples with molecular weight 253 kg mol–1 have generally two times lower intensity than

other samples. These values are equal to fluorescence intensities obtain from aqueous

samples. On the other hand, detector in luminiscence spectrometer was set to the highest

values for all samples, so this made these differences insignificant.

Table 9. Summary of the TNS fluorescence intensity values for selected HA samples in different

concentration of NaCl, KCl, and CaCl2, respectively. For comparison, fluorescence intensities

of TNS from aqueous samples were added. In brackets are standard deviations.

M I NaCl KCl I CaCl 2

kg mol-1 mol L–1 Fluorescence (a.u.) Fluorescence (a.u.) mol L–1 Fluorescence (a.u.) 0.01 4.50 (0.82) 4.39 (0.66) 0.03 4.32 (0.72)

0.1 4.40 (0.15) 4.50 (0.35) 0.3 4.48 (0.32)

0.2 4.41 (0.14) 4.48 (0.62) 0.6 4.44 (0.91) 100

0.5 4.64 (0.13) 4.57 (0.15) 1.5 4.45 (0.54)

0.01 2.12 (0.15) 2.11 (0.16) 0.03 1.12 (0.14)

0.1 2.23 (0.53) 1.97 (0.75) 0.3 2.66 (0.23)

0.2 2.41 (0.85) 2.32 (0.71) 0.6 2.39 (0.72) 253

0.5 2.41 (1.15) 2.30 (0.85) 1.5 2.49 (0.47)

0.01 4.60 (0.84) 4.64 (0.15) 0.03 4.69 (0.20)

0.1 4.60 (0.53) 4.72 (0.61) 0.3 4.73 (0.77)

0.2 4.59 (0.45) 4.70 (0.12) 0.6 4.59 (0.38) 740

0.5 4.55 (0.19) 4.42 (0.15) 1.5 4.64 (0.15)

0.01 2.40 (0.15) 2.46 (0.71) 0.03 2.40 (0.45)

0.1 2.60 (0.64) 2.20 (1.10) 0.3 2.13 (0.78)

0.2 2.39 (0.4) 2.70 (0.92) 0.6 2.19 (0.91) water

0.5 2.50 (1.15) 2.42 (0.181) 1.5 2.54 (0.95)

Under these conditions, we were not able to successfully probing hydrophobic patches in

hyaluronan. Samples were measured with the highest sensitivity of luminiscence detector.

Difference s between intensities obtained from these samples a for example from the

methanol is minimum two orders of magnitude (under same conditions – concentration,

temperature)

76

6.2.3. PRODAN

PRODAN spectral properties are strongly dependent on polarity of its microenvironment.

Fluorescence intensities from chloroform to alcoholic aqueous solutions are comparable. The

lowest fluorescence intensities come from pure hydrocarbons (n-Heptane, Cyclohexane) and

from pure water. Difference can reached one order of magnitude. Figure 49 represents

excitation spectrum, which is roughly the same for most of environments, and emission

spectra of n-Heptane and Methanol. It is obvious decreasing polarity causes hypsochromic

shift of emission wavelength.

Figure 49. Typical excitation spectrum and emission spectra of PRODAN in n-Heptane and Methanol.


Due to a non-specific solubility of PRODAN, we obtained fluorescence signal as

composite signal from various PRODAN’s molecules in polarity-different environments. In

aqueous solutions, a “water-peak” with maximum at 530 nm is always presents. The fact

enables use this “aqueous signal” as reference and evaluates polarity changes in system

during for example aggregation processes.

Figure 50 shows emission spectra in different concentration of SDS in aqueous solution.

Two groups of peaks are differentiated by relative intensity and position of central maximum.

Below the critical micellar concentration of SDS emission maximum can be found at 517 nm.

Around and above CMC is emission wavelength ~502 nm. Because of differences between

Excitation; 360 Emission n-Heptane; 396 Emission Methanol; 496

0

0.2

0.4

0.6

0.8

1

250 300 350 400 450 500 550 600 650

Wavelength (nm)

Nor

ma

lize

d flu

ore

sce

nce

(-) Excitation

Emission n-HeptaneEmission Methanol

77

intensity and position of emission maximum with respect to existence reference wavelength at

530 nm, three possible data evaluation can be found. First, is fluorescence intensity

dependency, second is emission wavelength dependency, and third is dependency of the ratio

of intensities at 502 and 530 nm, respectively.

Figure 50. Emission spectra of PRODAN in different concentration of aqueous solution of SDS.

Figure 51 and Figure 52 summarizes all three approaches to data evaluation. All

parameters show sigmoidal dependencies on surfactant concentration and can be evaluated by

Boltzman’s S-type fitting procedure. Form general point of view we obtained three possible

points, and contrary of the Pyrene-type data evaluation, first intersection point is added in

analysis. Table 10 shows all possible points for all measurements obtained from non-linear

curve fitting.

Table 10. Summary of points from non-linear curve fitting of PRODAN’s data in aqueous solution of

SDS. Points represent possible CMC points and should be compared with literature value

8.2 10–3 mol L–1.

DATA type 1. Intersection

point 103 mol L–1

Inflection point

103 mol L–1

2. Intersection point

103 mol L–1 R2

Intensity 5.1 7.3 9.4 0.990

Ratio 4.7 6.9 9.0 0.998

Wavelength 3.1 5.4 7.7 0.998

15 mM

10 mM

8 mM

6 mM

0

2

4

6

8

400 450 500 550 600

Wavelength (nm)

Flu

ore

scen

ce in

ten

sity

(a.

u.)

Concentrationof SDS

78

Figure 51. Dependency of the PRODAN’s fluorescence intensity and emission wavelength on SDS

concentration. Raw data are fitted by Boltzman’s S-type curve.

Figure 52. Dependency of the PRODAN’s fluorescence intensity and intensity ratio of hydrophobic

(502 nm) and hydrophilic peak (530 nm) on SDS concentration. Raw data are fitted by Boltzman’s S-

type curve.

0

1

2

3

4

5

6

7

8

0.0001 0.001 0.01 0.1 1 10 100 1000

Cocentration od SDS (mmol L-1)

Flu

ores

cen

ce in

tens

ity (

a.u

.)

500

504

508

512

516

520

Em

issi

on w

avel

engt

h (n

m)

IntensityEmission wavelength

0

1

2

3

4

5

6

7

8

0.0001 0.001 0.01 0.1 1 10 100 1000

Cocentration od SDS (mmol L-1)

Flu

ore

scen

ce in

tens

ity (

a.u

.)

0.20

0.60

1.00

1.40

1.80

502

:53

0 r

atio

(n

m)

IntensityRatio

79

Obtained data seem to be in good agreement with Elworthy’s value 8.2 mM. Literature

value corresponds to interval between inflection and second intersection point from intensity

and ratio measurements. Values obtained form emission wavelength dependency is generally

lower than intensity values. As well as in other cases with hydrophobic probes, wavelength

reacts more sensitively on polarity of its microenvironment than fluorescence intensity.

Molecules of PRODAN can interact with pre-micellar aggregate or induce its forming, and

subsequently undergo hypsochromic shift before “literature CMC”.


Experiments in native hyaluronan (polymer with MW = 1 109 kg mol–1 was selected) came

off according to assumption. Non-specific solubility led to the conclusion emission properties

of PRODAN are not affected by increasing hyaluronan concentration (Figure 53). However, it

must be noted emission maximum was hypsochromicaly shifted to ~520 nm against signal

from pure water, 530 nm. It is possible PRODAN interacts with hyaluronan’s chain

hydrophilic interface (Karukstsis 2005, Langm).

Figure 53. dependenycy of the emission wavelength a fluorescence intensity on concentration of native

hyaluronan (MW = 1 109 kg mol–1).

Molecules of this have probably too small hydrophobicity, that successful penetration to

hyaluronan’s hydrophobic patches needed.

490

500

510

520

530

540

0.01 0.1 1 10

Concentration of HA (g L-1)

Em

issi

on

wa

vele

ng

th (

nm

)

0

1

2

3

4

5

Flu

ore

sce

nce

inte

nsi

ty (

a.u

.)

WavelengthIntensity

80


For experiments with PRODAN in hydrophobized hyaluronan solution were selected

same samples as for experiments with Nile red. For obtained data, two types of evaluation

were selected. First was based on emission wavelength, second was ratio of fluorescence

intensities in different wavelengths. Wavelength selection demonstrates Figure 54. Increasing

concentration causes at first increasing of fluorescence at “green” side (side with a high

polarity). Around CAC, the “green side” increasing is stopped and “blue” wavelengths

increasing in theirs intensity. At the highest concentration is center of the blue region at least

two times higher than from the “green” region. For evaluation, handled with intensity ratio,

were selected centers of these two regions – 420 nm for the blue region and 520 nm for the

green region, respectively. The question why was selected 520 nm as “green” wavelength

against reference value 530 nm from free water environment, can be solved according to

measurements in native hyaluronan. Signal from these samples was constant and remain at

420 nm.

Figure 54. Emission spectra recorded from modified hyaluronan solution at different concentration.

0

2

4

6

8

10

380 400 420 440 460 480 500 520 540 560 580 600

Wavelength (nm)

Flu

ore

sce

nce

inte

nsity

(a

.u.)

above CACcenter at 420 nm

concentration

below CACcenter at 520 nm

81

Table 11. Summary of the CAC values for selected hHA samples determined by PRODAN

experiments. Two parameters were selected to determine these values. First was dependency

of the emission maximum, and second was dependency of the intensity ratio, 420 nm : 520 nm,

on the concentration of hHA samples. Both dependencies were fitted by sigmoidal curve.

M SD Wavelength Ratio

kg mol-1 % x 106 mol L–1 x 106 mol L–1

183 50 7.760 1.639

360 50 3.250 1.500

30 0.891 0.095

50 0.381 0.197 1 470

70 0.313 0.122

Both parameters dependency were evaluated using S-type fitting procedure. As final CAC

point, the inflection point was selected. Table 11 summarizes obtained CAC values. CAC

values are decreasing with increasing molecular weight and substitution degree. Differences

between wavelength and ratio values are major, especially for sample with 1 470 kg mol–1 and

substitution degree 30 %.

In comparison with same samples, analyzed with Nile red, CAC values are different in the

low molecular weight samples. For samples with molecular weight 1 470 kg mol–1, obtained

values are comparable. This relatively good agreement can be ascribed to fact PRODAN can

be taken as hydrophilic probe as well as hydrophobic probe. Contrary to the acridine orange,

PRODAN can penetrate inner parts, the hydrophobic core, of aggregates, but fluorescence

from the lowest polarity region is very weak, contrary to signal from free molecules in

aqueous environment. That is the reason, why PRODAN is embodied in water-soluble probes

chapter.

If we will be interested only in formation of the hydrophobic core, or its concentration

dependency, PRODAN can bring slightly misleading results. On the other hand, emission

spectrum is compact of signals from different PRODAN’s molecules, allow us to recognize

nature of hyaluronan’s inner microenvironment, because aggregate structure, formed in

aqueous environment, cannot be seen as simple surfactant’s micelle. According experiments

wit the acridine orange intra-chain or chain-chain interactions can lead to formation of

instable mesophasic environments (Figure 44). These “meso-environments” can entrap

fluorescence probes but is for discussion stability of them. This can be dangerous when

fluorescence probe is replaced by active drug, which must be protecting from e.g. blood or

stomach aggressive environment. Interactions with these environments can lead to drug

deactivating or unwished early interaction with its neighborhood.

82

6.3. Fluorescence probes in investigation of core quality

For potential drug delivery systems is basic to know at which concentration or under

which conditions a supramolecular domain is formed. But it is always necessary to know, if

formed domain is able to solubilize a different kind of hydrophobic/amphiphilic species. For

this prediction is useful to know “how much hydrophobic” formed domain is. Because

different probes can react differently in same environment, it is need to estimate polarity

scales for each probe. These polarity scales allow us to compare information form inner core

with pure solvents. For many reasons, as were mentioned above, two probes were selected –

Pyrene and Nile red.

Next information, which can bring using of fluorescence probes, is local viscosity or

microviscosity. Microviscosity contains information if domain is compact or if domain is soft.

It can be measured by anisotropy probes, intra-/intermolecular excimer formation, or via

molecular rotors. Information about viscosity will depends on geometry of a system too. For

small cavities is useful using molecular rotors, because of only small segment of molecule is

moving. Large (long) anisotropy probes like DPH may have long axis equal to cavity

diameter and cannot move freely in inner environment. Then obtained anisotropy is too high

and depolarization is equal to ration of the whole aggregate.

83

6.3.1. Domain polarity

Through aggregation experiments, four polarity probes were used. According to

experiments with native hyaluronan, TNS was found unsuitable for experiments with

identically charged polysaccharide. PRODAN as fluorescence probe has composite spectrum

and results without sophisticated mathematic interpretation are unclear.

Probes can show different reaction in different environments. For each probe, a specific

polarity scale must be estimated. These polarity scales have only relevance for specific probe.

Because we use polarity indexes as physical quantities in determination of CAC (CMC), is

obvious that polarity index varying with amphiphile concentration. So, if we are interested in

system polarity a choice of concentration or ratio of probe’s molecules to aggregates is

decisive. Principle is to use the lowest detectable amount of probe for specific concentration.

This is important especially for probes with non-zero signal from water – Pyrene and

PRODAN. Emission intensity of the Nile red from aqueous environment

6.3.1.1. Pyrene and its polarity scale

Pyrene is the most common polarity probe. Its polarity ratio is often using to determinate the critical aggregation concentration. Polarity-sensitive is 0

001 SS → transition. Probability of

this transition, and subsequently fluorescence intensity, is relative to polarity of its

microenvironment (see Figure A). Dong and Winnik estimated first Pyrene-based polarity

scale at 1984.

Figure 55. Measured polarity scale in different pure solvents (DMSO dimethylsulfoxide).

0.40

0.60

0.80

1.00

1.20

1.40

1.60

0 10 20 30 40 50 60 70 80 90

Dielectric constant (-)

pola

rity

ind

ex (

-)

Cyclohexane

n-Heptane

1-Octanol

1-Propanol

Ethanol

Methanol

Water

DMSOAcetonitrile

Acetone

Chloroform

Toluen

84

From Figure 55 is resulting that solvent with the highest polarity is DMSO. Scale ranges

between 0.54 for Cyclohexane and 1.63 for DMSO. Overall dependence of the polarity index

on dielectric constant is non-linear and can be separate in two different dependencies. First

dependency is consisting of solvents with OH group, except Toluene (A scale). Toluene

belongs to second dependency. There are solvents with unsaturated bond with small exception

– Chloroform (U scale). Ability to form hydrogen bond plays an important role for polarity

index value. Pyrene, from solvents with comparable value of dielectric constant, exhibits

lower index from solvents with H-bond forming ability. In comparison with original data, our

scale is shifted to lower values, approximately by factor 0.2.

For determination of the core-polarity is necessary to use the highest concentration of

amphiphile and the lowest concentration of the probe. As the indicator of optimal

concentration, an emission of Pyrene excimer can be used. Emission intensity of excimer

depends on concentration of Pyrene. Center of excimer emission is at 470 nm. Although,

formation of excited dimer also depends on diffusion coefficient in inner core, it can be used

as indicator of local concentration. Example dependencies show Figures 56 and 57. These

Figures summarize dependencies of polarity index and ratio of excimer to monomer

intensities on probe concentration in modified hyaluronan solution. Hyaluronan molecular

weight was 100 kg mol–1 and three different substitution degrees (10, 30, and 50 %) were

used.

From dependencies resulting, that currently used concentration of the Pyrene not affected

polarity index. Increasing core-concentration of Pyrene is expressed as non-zero value of

excimer to monomer ratio. This value is decreasing to a constant value below concentration

1 10–6 mol L–1. Ratio remains non-zero. This mean, from statistic point of view, that in

solution are present polymeric micelle with one or more Pyrene molecules and micelles

without any probe. Probes’ parting between particular micelles is describing by Poisson

statistic. Selected samples were in molecular weights 100, 134, 183, and 360 kg mol–1 and in

three different degrees of substitution - 10, 30, and 50 %. Polarity indexes were obtained as

minimum values from the sigmoidal fit

Table 12. Summary of the polarity index values for selected hHA samples determined by Pyrene.

M Substitution Degree

kg mol-1 10 % 30 % 50 %

100 0.91 0.85 0.84

134 0.88 0.86 0.79

183 0.89 0.80 0.82

360 0.96 0.83 0.80

85

Figure 56. Concentration dependencies of polarity index on Pyrene concentration in solution of

modified hyaluronan, with different degrees of substitution and molecular weight 100 kg mol–1.

Figure 57. Concentration dependencies of excimer to monomer ratio on Pyrene concentration in

solution of modified hyaluronan, with different degrees of substitution and molecular weight

100 kg mol–1.

0.5

0.6

0.7

0.8

0.9

1.0

1.1

0 0.5 1 1.5 2 2.5


Po

lari

ty in

dex

(-)

100/10100/30100/50

0.00

0.10

0.20

0 0.5 1 1.5 2 2.5


Exc

ime

r :

Mo

nom

er

(-)

100/10100/30100/50

86

Results are summarized in Table 12. Obtained polarity indexes varied from 0.79 to 0.96.

In solvent polarity scale these values correspond to Toluene, 1-Propanol, and 1-Octanol. In

modified hyaluronan solution, there are no aromatic groups. Only alkyl chains and OH groups

are presented. From different dependencies, the “A scale” was selected. From this

dependency, it follows that dielectric constant of inner polymeric micelle core ranges between

10 and 20. Without a taking account of sterical effects, this environment can

solubilize/dissolve all species soluble in alcohols form 1-Propanol to 1-Octanol, but this

statement needs to be tested.

Influence of the ionic strength on inner core polarity brings interesting results. Prediction

is low molecular salt molecules, shielding repulsive interaction, make domain more compact

and more apolar, Table 13 shows opposite results. In tested system with molecular weight

100 kg mol–1 and substitution degrees 10, 30, and 50 %, increasing ionic strength caused

increasing of the polarity index. Mean increase is 0.07 points of the polarity scale. This value

amounts around 6 % from whole polarity scale.

Increasing can be caused by changes in organization of water molecules around carboxylic

groups. Because Pyrene exhibits non-zero fluorescence from pure water, it can be also found

in region nearby polymeric micelle surface, which is directly affected by low molecular salt.

Table 13. Summary of the polarity index values for selected hHA samples determined by Pyrene in

presence of sodium chloride in different concentration.

SD Ionic strength (mol L–1)

% 0 0.1 0.4

10 0.91 0.91 0.98

30 0.85 0.90 0.90

50 0.84 0.90 0.91

87

6.3.1.2. Nile red and its polarity scale

Nile red (or Nile Blue oxazone) exhibits blue shift of its emission maximum with

decreasing polarity of its microenvironment. This quality was successfully used in

determination of the critical aggregation concentration of simple surfactant system and

modified hyaluronans.

Figure 58. Excitation spectra of Nile red in pure solvents with different polarity.

Nile red excitation and emission spectra are shown in Figure 58 and Figure 59,

respectively. From these measurements is obvious abnormal behavior of Nile red in pure

hydrocarbons. The best example is shown in Figure 59. Shape and position of emission

spectrum is different, than can be expected. Maximum in n-Heptane and Cyclohexane is

moved against Chloroform of about 75 nm to lower wavelengths and band has two local

maximums. This considerable shift to higher energies mean, that system lost part of its

aromaticity.

If position of emission maxima against dielectric constant is plotted, two dependencies are

obtained (Figure 60). Accordingly to Pyrene measurements, groups of solvents are

differentiate to alcohols and to unsaturated solvents. If we not take into account n-Heptane

and Cyclohexane, we obtained two linear dependencies with correlation coefficient above

0.95. First group is consisting of alcohols and water; members of second group are

Chloroform, Acetone, Acetonitrile, DMSO, and Water.

0

2

4

6

8

10

400 450 500 550 600 650 700

Wavelength (nm)

Flu

resc

en

ce in

ten

sity

(a

.u.)

n-Heptane

Cyclohexane

Chloroform

1-Octanol

Acetone

Ethanol

Methanol

Acetonitrile

DMSO

88

Figure 59. Emission spectra of Nile red in pure solvents with different polarity.

Figure 60. Dependency of the Nile red emission maximum on the dielectric constant. Two possible

linear dependencies are fitted.

0

2

4

6

8

10

500 550 600 650 700 750

Wavelength (nm)

Flu

ore

sce

nce

inte

nsi

ty (

a.u

.)

n-Heptane EMCyclohexane EMChloroform EMOctanol EMAcetone EMEthanol EMMethanol EMAcetonitrile EMDMSO EM

520

545

570

595

620

645

670

0 20 40 60 80 100

Dielectric constant (-)

Em

issi

on

ma

xim

um

(nm

)

n-HeptaneCyclohexane

Chloform

1-Octanol

Acetone

EthanolMethanol

Acetonitrile

DMSO

Water

R2 = 0.96; Slope = 0.41

R2 = 0.97; Slope = 0.71

89

For determination of the core polarity is necessary to know, if the Nile red emits from

non-polar core like from pure hydrocarbons (two maximums, emission band below 600 nm),

or if emits like rest of the solvents from polarity scale in Figure 60. Emission spectra of the

Nile red in three different concentration of modified hyaluronan with molecular weight

360 kg mol–1 and substitution degree 50 % is shown in Figure 61. From this Figure is

obvious, that emission of the Nile red from hyaluronan core has only one maximum localized

nearby 610 nm.

Figure 61. Emission spectra of the Nile red in modified hyaluronan in different concentration.

Molecular weight is 360 kg mol–1 and substitution degree 50 %.

With obtained results we again must select, which dependency, from two possible

dependencies, is relevant for determination of the core polarity. With respect to the fact, only

saturated chains form that core environment with possible presence of OH groups or water

molecules, “the alcohol scale” is selected. Wavelength value 610 nm refers to dielectric

constants around 1-Octanol. This is in good agreement with the Pyrene polarity scale.

Obtained polarity indexes belong to region between 1-Octanol and 1-Propanol.

Table 14 summarizes results for selected samples of modified hyaluronans. Unfortunately,

in comparison of Pyrene measurements, only few of identical samples were in sufficient

amount. From results is not obvious any possible dependency on the substitution degree or

molecular weight. Polarity of formed core in different samples ranges from 608 to 611 nm.

This difference means only 2% change of whole polarity scale, and it is not significant.

0

1

2

3

4

580 600 620 640 660 680 700 720 740

Wavelength (nm)

Flu

ore

scen

ce in

ten

sity

(a.

u.) 2.5 g L-1

1.0 g L-1

0.2 g L-1

90

Table 14. Summary of the emission maximum (in 10–9 m) of the Nile red for selected hHA samples.

ND = Not determined (because of insufficient amount of sample)

M Substitution Degree

kg mol-1 10 % 30 % 50 %

100 608 611 611

183 611 ND ND

360 611 ND ND

The same way as pyrene measurements, influence of the ionic strength was studied. Same

samples, molecular weight 100 kg mol–1 and 10, 30, and 50 % substitution degrees, were

placed into solutions with different concentration of sodium chloride. Results are showed in

Table 15.

Table 15. Summary of the emission maximum wavelengths for selected hHA samples determined by

Nile red, in presence of sodium chloride in different concentration.

SD Ionic strength (mol L–1)

% 0 0.1 0.4

10 608 607 611

30 611 600 604

50 611 607 606

The most interesting result came from measurements of 30 % substitution degree in solution

with ionic strength 0.1 mol L–1. Emission maximum was found at 600 nm and in polarity

scale, its value is lower than Chloroform. The second-lowest value shows same sample in

solution with 0.4 mol L–1 of Sodium chloride. Nevertheless, from global point of view, there

is not remarkable trend of emission maximums.

91

6.3.2. Domain viscosity

Different approaches to determination of a domain viscosity can be used. One of them is

based on intermolecular aggregate formation. Due to a long lifetime Pyrene formed excited

dimer, the excimer, if its local concentration increases. For monitoring of the excimer

formation, a ratio between intensity of excimer (470 nm) and monomer (373 nm), was used

(Figure 62). It is hardly to assign excimer-to-monomer ratio to concrete value of viscosity.

Excimer formation is a type of bimolecular reaction, quenching. Probability of excimer

formation, and subsequently its emission intensity, depends on, among others, diffusion

coefficient. With increasing of the diffusion coefficient increases probability, that Pyrene

molecule in excited state meets another Pyrene molecule in ground state, and subsequently

form excimer. Diffusion coefficient is inversely proportional to the viscosity. This means with

increasing of the core viscosity the diffusion coefficient and probability of excimer formation

decreases and excimer-to-monomer ratio decreases too. Ratio can be used especially for

monitoring of changes in viscosity of the inner micellar core.

Figure 62. Emission spectra of Pyrene in micellar system with Excimer emission band at 470 nm.

For experiments were selected samples of modified hyaluronan with molecular weight

25 kg mol–1 and two degrees of substitution 30 and 50 %. At first, the critical aggregation

concentrations of derivatives were determined using Pyrene polarity index. Determined

aggregation concentration was 2 10–6 mol L–1 (0.05 g L–1) for 30 % and 0.1 10–6 mol L–1

(0.003 g L–1) for 50 %, respectively. According to CAC values, for each derivative a specific

0

1

2

3

4

5

6

360 390 420 450 480 510 540

Wavelength (nm)

Flu

ores

cenc

e in

tens

ity (

a.u

.)

Monomer373 nm

Excimer470 nm

92

measurement concentration was selected. Concentration was set to 0.5 g L–1 for 30 % and

0.1 g L–1 for 50 % degree of substitution, respectively. This study comprises influence of the

temperature and ionic strength on monitored ratio.

Results for substitution degree 30 % are showed in Figure 63. In this Figure is also marked

range of human body temperature (from 35 to 40 °C), encloses by dashed rectangle. Ratio is

increasing (and viscosity is decreasing!) in all cases when sample is heated from laboratory

temperature to human body region temperature. Above 36 °C, ratio decreases and in all cases

is at 50 °C higher than for 15 °C. This behavior may show process of domain expanding and

subsequent collapsing. Domain collapsing is also noted in analysis of the influence of the

ionic strength. For all temperatures, increasing of the ionic strength is leading to the

increasing of the inner viscosity.

Figure 63. Dependency of the Excimer:Monomer ratio on the temperature in solution with different

ionic strength. Sample was in molecular weight was 25 kg mol–1 and substitution degree 30 %. In

Figure, dashed rectangle encloses range of the human body temperature.

Another sample shows different behavior (Figure 64). Dependency of the sample with no

addition of sodium chloride is at constant value and decreasing above 36 °C. Sample with

ionic strength 0.01 mol L–1 is showed only monotonous decreasing. In physiological ionic

strength, 0.1 mol L–1, ratio below 36 °C remains constant, around physiological temperature

slightly increases and above 45 °C follow next decreasing. The last dependency showed in

0.4

0.5

0.6

0.7

0.8

0.9

10 20 30 40 50 60

Temperature (°C)

Exc

ime

r:M

ono

me

r (-

)

0 M0.01 M 0.1 M 1 M

Ionic Strenght

Human body temperature range

93

Figure AS at ionic strength 1 mol L–1 remains constant from 15 to 25 °C and follows with

monotonous decreasing.

Figure 64. Dependency of the Excimer:Monomer ratio on the temperature in solution with different

ionic strength. Sample was in molecular weight was 25 kg mol–1 and substitution degree 50 %. In

Figure, dashed rectangle encloses range of the human body temperature.

If these measurements are compared, it can be said, that sample with substitution degree

50 % has globally higher inner viscosity at all temperatures and ionic strengths than sample

with substitution degree 30 %. In most of all cases (except sample with SD 50 % and I.S.

0.1 mol L–1), increasing temperature caused at first increasing of the ratios, and above

physiological temperature caused theirs decreasing. This mean that in polymeric micelles,

formed by modified hyaluronan, heating firstly make core fluid and subsequently, above

physiological concentration, domains are collapsing and going to be more viscous than at

laboratory temperature.

Results of the influence of the temperature on the ratio can be resolved by another way.

Excimer formation is depending on local concentration of the Pyrene. Excimer formation can

be hindering not only by viscosity, but sterical effects must be taken into account. Decreasing

of the ratio above 36 °C can be caused not only by viscosity increasing, but can be caused by

extreme expanding of hyaluronan domain. This domain diluting can shielded excimer

concentration, because Pyrene lifetime can be short than time, which is needed to interaction

of the Pyrene in the excited state with another one in ground state.

0.0

0.1

0.2

0.3

0.4

0.5

0.6

10 20 30 40 50 60

Temperature (°C)

Exc

ime

r:M

ono

me

r (-

)

0 M0.01 M 0.1 M 1 M

Ionic Strenght

Human body temperature range

94

For making decision if hyaluronan domain is collapsing or expanding, is necessary to use

another point of view. On e of the point of view is resulting form Pyrene measurements. It is

polarity index, which can give an answer to expending/collapsing question. Measurements for

both samples are showed in Figure 65 and Figure 66, respectively.

Figure 65. Dependency of the polarity index on the temperature in solution with different ionic

strength. Sample was in molecular weight was 25 kg mol–1 and substitution degree 50 %. In Figure,

dashed rectangle encloses range of the human body temperature.

In both cases, increasing temperature caused increasing of the polarity index. This can be

indicative of domain expanding. Domain is diluting and water molecules are penetrating into

inner parts of micellar core. These water molecules are increasing polarity of the Pyrene

environment. For sample with substitution degree 50 % is index saturated above 35 °C.

Next important result, consequent upon these measurements, is that ionic strength no

affected or affected in positive way polarity index. Polarity index remain the same or is

decreasing wit increasing of the ionic strength. This mean, that domain is no only more

apolar, but that observed decreasing of the Excimer:Monomer ratio is caused by increasing in

viscosity of the micellar core.

0.8

0.9

1.0

1.1

1.2

1.3

10 15 20 25 30 35 40 45 50 55

Temperature (°C)

po

lari

ty in

de

x (-

)

0 M0.01 M 0.1 M 1 M

95

Figure 66. Dependency of the polarity index on the temperature in solution with different ionic

strength. Sample was in molecular weight was 25 kg mol–1 and substitution degree 50 %. In Figure,

dashed rectangle encloses range of the human body temperature.

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

10 15 20 25 30 35 40 45 50 55

Temperature (°C)

po

lari

ty in

de

x (-

)

0 M0.01 M 0.1 M 1 M

96

7. CONCLUSION

Aim of this doctoral thesis was to investigate, using of fluorescence probes, colloidal

properties in aqueous solutions of native and modified sodium hyaluronates. From a large

family of fluorescence probes, six of them were selected. These probes have different and

interesting spectral and physiochemical properties which are sensitive to specific parts of

colloidal behaviour.

As a first step, probe spectral properties in various pure solvents were checked or

determined. Obtained values were sometime slightly different in comparison with literature

values, but from the global point of view, no basic differences were found.

Next step focused on the behavior of probes in simple and well-known colloidal media.

From the surfactants family, the sodium dodecylsulfate was selected. This surfactant has well-

known cirtical micellar concetration and aggregation number. All of the probes determined its

CMC. It is hardly to compare CMCs obtained by fluorescence probes with literature value,

which resulted from surface tension measurement. In fluorescence probe case, additional

specie is added into surfactant-solvent system, and this addition can affect the system via

inducing pre-micellar aggregation. This influence is resulting into wide range of determined

CMC: from 2.1 to 10.3×10–3 mol L–1. TNS was found to be the least suitable probe for the

SDS-water system.

All probes were used in investigation of aggregation properties of the native hyaluronan.

Unfortunately, probing of the hydrophobic patches was not successful. Only two probes

changed spectral properties in presence of native hyaluronan. Acridine orange interacted by

expected way, and the end-points of titration were determined. Also was determined influence

of the ionic strength on the end-point of titration. Interaction of the native hyaluronan with the

PRODAN was confirmed by hypsochromic shift of its emission band.

All probes, except TNS, were used in study of aggregation properties of C10 derivatives of

hyaluronan. Due to vast amount of measurements and small amounts of experimental

samples, not all experiments could be performed with all probes and with all derivatives. But

from the global point of view it can be said that hyaluronan aggregation concentration is

decreasing with increasing molecular weight and degree of substitution. It depends on probe

selection if differences will be significant or not. Choice of the probe shall take into account

additional information, which probes can afford. If we are interesting in polarity of its

microenvironment, we select the Pyrene or the Nile red, as probes. In case of monitoring

viscosity of inner core, Perylene or Pyrene shall be used.

Properties of domain formed upon hyaluronan aggregation are the last topic of this thesis.

Domains were investigated from polarity and viscosity point of view. As polarity probes,

Pyrene and Nile red were selected. Solvent-based polarity scales were determined with

different results. Both probes showed different results in interaction with alcohols and with

unsaturated solvents. This make harder to assign specific value of dielectric constant or

97

another polarity factor to measured polarity indexes or wavelengths. Likewise in polarity

determination, viscosity measurements, based on intermolecular fluorescence quenching, are

not suitable for determination of the process by itself. These measurements must be compared

with e.g. polarity measurements. This is clearly shown in influence of the temperature on the

Excimer:Monomer ratio. Most of all dependencies of the ratio on the temperature have

inverse quadratic character with maximum nearby human body temperature. Nevertheless,

from Pyrene polarity index point of view, only monotonous increasing was observed. This

increasing mean domain is expanding and excited Pyrene molecules cannot interact with the

other molecule during its lifetime. On the other hand, polarity measurements confirm

influence of the salt as agent for increasing core viscosity. Through the 2 orders of magnitude

of the ionic strength values, index remain same or it was moved to the lower values, which

mean, that domain was more compact.

Results obtained from measurements with native hyaluronan show no interaction between

hydrophobic fluorescence probes and hyaluronan. Alkylation enhanced interaction between

hydrophobic probe and polymer and aggregation behavior of the modified hyaluronan is

monitored. Obtained results are comparable with simple surfactants. It can be said

aggregation in modified hyaluronan solution lead to the formation of hydrophobic cavity.

This cavity can be influenced (and qualities can be modified) by temperature and ionic

strength. Presence of the hydrophobic cavity makes this system suitable for drug delivery

application. Next advantage is hyaluronan is biocompatible and biodegradable polymer and it

can specifically interact with cell receptors (CD44, RHAMM). All of these qualities make

hydrophobized hyaluronan as one of the most promising system in drug carriers.

98

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106

9. ABBREVIATIONS

Å Ångström [1 10–10 m]

ACN Acetonitrile

ANS 1-anilino-8-naphthalene sulfonate

AO Aridine orange

CAC Critical aggregation concentration

CMC Critical micellar concentration

DMF Dimethylformamide

DMSO Dimethylsulfoxide

DOX Doxorubicin

F Fluorescence intensity

FT-IR Fourier transform infrared spectroscopy

HA Hyaluronic acid, Sodium hyaluronate

hHA hydrophobicaly modified sodium hyaluronate

IC50 Median Inhibition Concentration

I.C. Ionic strength [mol L–1]

I⊥ Intensity, when polarizators are perpendicularly oriented

I || Intensity, when polarizators are in a parallel way

M Molecule

[M] Molecule concentration (ground state)

[M* ] Molecule in excited state concentration

M [mol L–1]

mM [10–3 mol L–1]

µM [10–6 mol L–1]

MW Molecular weight (weight averaged)

N Aggregation number (number of surfactant molecules per micelle)

NMR Nuclear magnetic resonance

p polarization

P/D ration of the mole of polymer binding sites per mole of dye

Q Quencher molecule

r fluorescence anisotropy

S Singlet state

SEC Size exclusion chromatography

SD Substitution degree

SDS Sodium dodecyl sulfate

THF Tetrahydrofuran

TICT Twisted intramolecular charge transfer

κ Orientation factor

107

π, σ, n, π*, σ* Molecular orbitals

ΦF Fluorescence quantum yield

108

10. APPENDIX

I. AMINCO-Bowman Series 2, Luminiscence spectrophotometer

II. Experimental apparature

Cooler

AB 2

Thermostat

109

III. Cuvette in the cell-holder with the PRODAN in polar environment (Water, 530 nm)

IV. Cuvette in the cell-holder with the PRODAN in non-polar environment (Choroform, 405 nm)

110

V. Mravec, F., Pekař, M., Velebný, V. Aggregation behavior of novel hyaluronan derivatives – a

fluorescence probe study. Colloid and Polymer Science, In Press. DOI: 10.1007/s00396-008-1941-8

(Accepted 30 Sep 2008)

Aggregation behavior of novel hyaluronan

derivatives – a fluorescence probe study

Filip MRAVEC1, 2, Miloslav PEKAŘ1, Vladimír VELEBNÝ2

(1) Institute of Physical and Applied Chemistry, Faculty of Chemistry, Brno University of Technology, Purkyňova 118, 612 00 Brno, CZ

(2) CPN spol. s.r.o., Dolní Dobrouč 401, 561 02 Dolní Dobrouč, CZ

tel.: +420 541 149 483

fax.: +420 541 149 389

[email protected]

www.fch.vutbr.cz/ipac ; www.hyaluronan.cz

Abstract: Aggregation properties of hydrophobized hyaluronan (hHA) with different molecular weights and

degrees of substitution were studied using pyrene and Perylene as fluorescence probes. Both probes in contrast to

native biopolymer confirmed aggregation of modified hyaluronan. The critical aggregation concentration (cac)

was determined by the pyrene I1/I3 and Perylene fluorescence intensity method. The cac value varied both with

the molecular weight and the degree of substitution and was between 0.610-0.003 g⋅L−1. Pyrene Polarity Scale

confirmed formation of hydrophobic domains.

hyaluronate derivatives - fluorescence probes - critical aggregation concentration - core

hydrophobicity

Introduction

Polysaccharides and their derivatives have become major components in the development of biocompatible

and biodegradable materials with many areas of applications (e.g. tissue engineering, drug delivery). Chemical

modification, which does not affect the biodegradability and does not suppress biological activity, can lead to

further expansion of medicine and engineering applications [1, 2].

Hyaluronan (HA) is a major component of pericellular and extracellular matrices [3]. It is a linear polymer

formed by repeating disaccharide units composed of the disaccharide unit formed by D-glucuronic acid-1-β-3-N-

acetylglukosamine. It plays an important role in stabilizing the extracellular matrix in many tissues by binding to

specific proteins called hyaladherines. The main hyaluronan fraction is localized in the skin tissue [4].

111

Synthesis of hyaluronan derivatives is generally based on the esterification on the D-glucuronic subunit [5].

Recently, preparation of new type derivatives that are subject of this study was reported [6]. Modification was

made on the secondary hydroxyls of the glucuronic subunit. This way of modification leaves all carboxylic

groups free in contrast to common derivatives prepared via esterification on the COOH group and enables to

achieve high substitution degrees while maintaining polyelectrolytic character. Amphiphilic hyaluronan

polyelectrolyte is thus obtained (hydrophobized hyaluronan, hHA) which is supposed to be still water-soluble

and to aggregate in aqueous solution forming micelle-like structures with a non-polar core that will be able to

entrap hydrophobic species, e.g. drugs.

In this work, first results on studying their aggregation capabilities are reported. Aggregation behavior of

amphiphiles can be studied by non-polar fluorescence probes, which can be solubilized into this core.

Fluorescence probe techniques have been used successfully in the study of a wide range of surfactants [7-9].

They are able to determine not only the critical micellar or aggregation concentration (cac) but also the polarity

index of probe’s microenvironment [10-12] and effective viscosity of the micellar core [13, 14].

Pyrene I1/I3 ratio method is a widely used method to determine the cac value for many surfactant-based systems.

Its unique response to the microenvironment polarity is well known and described [15]. In this method, the ratio

of the fluorescence intensity at 373 nm (I1) and at 383 nm (I3) is plotted against the logarithm of the aggregating

molecule concentration. Below the cac the pyrene I1/I3 ratio does not change in a wide range of concentration.

Near the cac value, this ratio sharply decreases with increasing concentration up to a final, nearly constant, value.

To confirm the results obtained with pyrene we used also the Perylene fluorescence method [16]. Using

different probes in aggregation studies is not common. Perylene is non-fluorescent in aqueous environment.

Fluorescence intensity of the Perylene increases with the number of non-polar domains formed in the solution.

No fluorescence is observed until these domains are present in solution. When the domains are formed, a sharp

increase in the fluorescence is observed. Straight lines can fit these two trends and the concentration-coordinate

of their point of intersection define the cac value directly.

Materials and Methods

Sodium hyaluronate and its alkyl derivatives (Figure 1) were obtained from CPN Ltd. (Dolní Dobrouč,

Czech Republic). Details on the synthesis of derivatives and their molecular characteristics have been published

elsewhere [6]. Hyaluronans were of the following weight-average molecular weights: 97, 560, and 1 630

kg·mol−1.

Figure 1 Schematic structure of the sodium hyaluronate (a) and its C10 alkyl-derivative

112

Derivatives were of the weight-average molecular weights of 44, 134, 183, 360, and 1 470 kg·mol−1 and

their substitution degrees were in the range from 10 to 70 %. Substitution degree is defined as the ratio of the

mol of substituents per mol of the disaccharide unit [6], e.g. SD 100 % means one alkyl chain per each

disaccharide unit in the hyaluronate chain. Molecular weights were determined by SEC-MALLS and the

substitution degree is defined from the 1H NMR spectra, for details, see [6]. All the molecular parameters were

determined and provided by the producer. The hyaluronate samples were dissolved in doubly distilled water to

the concentration 2 g·L−1. This stock solution was stabilized by addition of sodium azide (p.a., Lachema) in final

concentration 10−3 mol·L−1. The alkyl-type abbreviation comes as the first followed by the original molecular

weight (before the derivatization) and the substitution degree. For example D 134/10 means C10-derivate of the

molecular weight 134 kg·mol-1 and with the substitution degree 10 %.

Sodium dodecylsulfate (p.a., Lachema) was dissolved in water to obtain concentration of 2⋅10−2 mol·L−1.

Pyrene and Perylene (fluorescence grade) were obtained from Fluka, acetone p.a. from Lachema.

The hyaluronate samples were listed in correspondence to their characteristics. Stock solutions of pyrene

and Perylene were prepared in acetone in final concentration 10–4 mol·L−1. Probe stock solution was introduced

into a vial and acetone was evaporated. The concentration of both probes in final samples was set to

5·10−6 mol·L−1. The stock solution of HA or hHA was introduced into the vial with the probe, diluted to the

desired concentration, and the resulting solution was sonicated for 4 hours and stored during next 20 hours. The

fluorescence emission spectra were monitored with a luminiscence spectrophotometer (AMINCO-Bowman,

Series 2) at 293.15 ± 0.1 K. The excitation and emission slit widths were set to 4 nm, and the excitation

wavelength was 335 nm and 408 nm for pyrene and Perylene, respectively.

The experimental data, i.e. the pyrene I1/I3 ratio (y) dependency on concentration (x), were evaluated using

non-linear fitting with Boltzman’s curve containing four parameters – the maximum (a), the minimum (b), the

inflex point (x0), and the width of the step change (∆x) (Equation 1).

b

e

bay

x

xx+

+

−=∆− )( 0

1 (1)

The data were fitted using the nonlinear curve fitting with Origin 75. The cac was obtained from the inflex

point of the non-linear fitting. This is denoted as the cac1-point. Alternatively, we determined also the cac2-point

defined as (cf. Figure 2)

xx ∆+= 2cac 02 (2)

Figure 2 shows the typical dependency of the I1/I3 on the logarithm of the concentration in a model

surfactant system, SDS, with two possible points, cac1 and cac2, which can determine the critical aggregation

concentration. Its critical aggregation concentration, 8.2 10-3 mol⋅L-1 in aqueous solution at 293.15 K, via surface

tension experiments, was reported elsewhere [17]. Obtained cac values from the fit are 8.3 and 10.2 10-3 mol⋅L-1

for cac1 and cac2, respectively. It is obvious the cac1 value is closer to surface tension value and it seems to be

more relevant as cac point.

113

Figure 2 Typical dependency of the Pyrene Polarity Ratio on the concentration of an amphiphile (Sodium

dodecyl sulphate).

Perylene data evaluation was based on a fit of two linear trends. From equations of these straight lines “x-

coordinate” of the point of intersection was evaluated as the cac.

Results and Discussion

Aggregation Properties

First measurements were focused on possible aggregation behavior of native hyaluronan in aqueous

solution. Three molecular weights (97, 560, and 1,630 kg⋅mol−1) were selected to investigate possible

aggregation of native hyaluronan using the pyrene fluorescence method. Figure 3 shows that the I1/I3 ratio, called

also the polarity index, ranges from 1.37 to 1.45 through a wide concentration range. Value of the polarity index

in this concentration range can be taken as constant and invariant. So, from the hydrophobic polarity probe point

of view no aggregation behavior was observed in these solutions. Hydrophobic patches supposed to exist along

HA chain [18] do not form domains able to solubilize non-polar substances like pyrene.

Figure 3. Plot of the I1/I3 vs. Log CHA for native hyaluronan in aqueous solution of different molecular weights.

Table 1. The representative samples of hydrophobized hyaluronan with the x0, ∆x, and criterion value obtained

from curve-fitting.

114

samples x0 ∆x x0/∆x R2

- g·L−1 g·L−1 - -

D44/10 0.54 1.42 0.38 0.99

D134/10 0.61 1.51 0.40 0.99

D183/30 0.15 1.55 0.10 0.98

D360/50 0.08 1.48 0.05 0.98

D1470/70 0.20 1.03 0.19 0.97

On the contrary, hyaluronate derivatives manifested clear aggregation behavior. Example of results obtained

for D134/10 is presented in Figure 4. As explained above there are two possible cac points on the concentration

dependency of the pyrene I1/I3 ratio. Aguiar and co-workers [15] suggested a condition to select the cac value

from the pyrene I1/I3 ratio. If the x0/∆x (cf. Eq. (1)) is less than 10, the cac point is determined by the x-

coordinate of the inflex point x0 - cac1 in our case. Table 1 examples determined values of the x0/∆x ratio of

hyaluronate derivatives. All of them passed the “less than 10-condition”.

Figure 4. Plot of the normalized integral fluorescence (IF) and the I1/I3 vs. the Log CHA for the D134/30 sample.

The Perylene data are fitted with two lines. The pyrene data are fitted by sigmoid curve with marked cac1 (X).

The point of intersection (↓) from Perylene dependence (x-coordinate value = −0.747, which is equal to

0.179 g·L−1) is identical with the pyrene cac1 point (x-coordinate value = −0.750, which is equal to 0.178 g·L−1).

Perylene results confirmed the selection of cac1 as the proper critical concentration. In Figure 4, the integral

Perylene fluorescence intensity is plotted against the logarithm of hyaluronate concentration. The Perylene data,

resolved by two straight lines, lead to the cac value 0.179 g⋅L−1, which corresponds to the cac1 value determined

by the pyrene method (0.178 g·L−1). Accordingly, we used cac1 for the evaluation of the pyrene data.

The cac values of various derivatives decrease when the substitution degree increases, with a small

exception for samples D 1470 (Table 2). At constant substitution degree there is no clear trend of cac change

with molecular weight (Table 2). The greatest decrease of cac values with increasing substitution degree was

observed for samples D 44. These samples have the shortest biopolymer chain. Aggregation is driven by

hydrophobic interactions introduced by the alkylation of native hyaluronan backbone. Formation of aggregates is

accompanied by reorientation of hyaluronan chains and also by changing its conformation. Short chains can be

115

reoriented and deformed more easily than long ones, therefore, their cac values are more sensitive to the degree

of substitution. But not only cac values are decisive for the final selection of the derivative, which can be use in

future for an application in drug delivery system.

Table 2 Overview of the cac values for hHA samples from pyrene and Perylene, in brackets, experiments.

SD (%) MW

(kg·mol−1) 10 30 50 70

cac

(g·L−1)

44

134

183

360

1470

0.540 (0.520)

0.610 (0.600)

0.190 (0.210)

0.190 (0.200)

ND (ND)

0.045 (0.050)

0.179 (0.088)

0.150 (0.099)

0.080 (0.032)

0.260 (0.280)

0.003 (0.003)

0.130 (0.140)

0.110 (ND)

0.110 (0.140)

0.110 (0.130)

NDa (ND)

0.030 (0.025)

ND (ND)

ND (ND)

0.200 (0.160)

a...ND – not determined

In comparison of cac values with another alkyl-derivative of HA [19], pyrene as the cac probe was used and

the cac value was realized as the first decreasing of the pyrene polarity index dependency on the derivative

concentration, our derivatives with high SD or MW have same or lower cac values. Similarly, in the same orders

of magnitude, range cac values of the alkyl-chitosan [20, 21], cholesteryl-chitosan derivatives [22] or heparin-

deoxycholic acid derivative [23], where decreasing tendency of the cac on increasing SD was observed.

Domain Hydrophobicity

As the base for determination of the hydrophobicity of the non-polar core, the pyrene polarity scale (PPS)

was selected [24]. The limiting values of the I1/I3 ratio from the concentration dependencies are shown in Table

3. Data indicate a general trend of increasing hydrophobicity (decreasing pyrene polarity parameter) with

increase in the substitution degree, which is easily understandable and expectable. Closer inspection of data

obtained for samples of the same molecular weight of the native hyaluronan does not always reveal simple trend

(Table 3). This can indicate different distribution of alkyl substituents along the polysaccharide backbone of

varying substitution degree, non-uniform alkyl distribution on highly substituted hyaluronan and also different

changes in conformational behavior of chains of various molecular weights modified by alkyls to different

degrees.

Table 3 Overview of the hydrophobicity indexes for hHA samples.

SD (%) M W

(kg·mol−1) 10 30 50 70

Pyrene

polarity

parameter

(-)

44

134

183

360

1470

0.96

0.88

0.89

0.96

-

0.83

0.86

0.80

0.83

0.84

0.89

0.79

0.82

0.8

0.81

-

0.82

-

-

0.81

116

Conclusion

The novel hyaluronan derivatives show surfactant-like aggregation behavior in aqueous solutions. Their

critical aggregation concentrations can be modified by the molecular weight and substitution degree and ranges

between 0.610-0.003 g⋅L−1. Hydrophobic domains are formed with the relative hydrophobic index (PPS) nearby

0.85. This value is comparable to that of simple surfactants. Novel hyaluronate hydrophobized derivatives with

preserved free carboxyl groups are thus potential candidates for preparing systems for targeted delivery of

hydrophobic active substances.

Acknowledgement.

We thank CPN Ltd Dolní Dobrouč (Czech Republic) for material and financial support. The research was

supported also by the Ministry of Education of the Czech Republic, project. No. MSM 00216305001.

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VI. Mravec. F., Pekař, M., Velebný, V. Fluorescence Probes In Investigation Of Hydrophobized

Hyaluronan Aggregation. Luminiscence, 2008, vol. 23, pp. DOI: 10.1002/bio.1074

FLUORESCENCE PROBES IN INVESTIGATION OF HYDROPHOBIZ ED HYALURONAN

AGGREGATION

F. Mravec1,2 , M Pekař2, V. Velebný1 1Laboratory of Transport Systems, CPN Ltd., Dolni Dobrouc, Czech Republic,

E-mail: [email protected] 2Institute of Physical and Applied Chemistry, Faculty of Chemistry, Brno University of Technology, Brno,

Czech Republic

Aggregation properties of hydrophobized hyaluronan (hHA) in different molecular weights and degree of

substitution were studied by pyrene fluorescence method. The critical aggregation concentration (cac) was

determined from the emission spectra by the pyrene polarity ratio I1/I3 (1). The value of the cac for the hHA in

aqueous solution was measured at different molecular weights and at 298.2 K. They showed variation of the cac

value with the molecular weight. The I1/I3 value of the pyrene fluorescence spectrum in the highest concentration

of the hHA varied with the molecular weight too.

Figure 1 Compare of the molecular weight influence on cac value.

0.5

0.7

0.9

1.1

1.3

1.5

1.7

0.01 0.1 1 10

concentration (g dm-3)

pyre

ne p

olar

ity in

dex

30/30

1,470/30

MW = 30 kg mol-1, SD = 30 %

MW = 1,470 kg mol-1, SD = 30 %

119

Formed micelle-like aggregates were studied with the fluorescence probe called Prodan. This probe exhibit

large Stokes shift depends on a polarity of its microenvironment. The complex fluorescence spectra of the

Prodan, recorded form the hydrophobized hyaluronan solution, contain signals from many polarity-different

environments (2, 3). For the determination how many microenvironments are in the hyaluronan solution and

nature of these, we used deconvolution method (to Guassian curves) based on the steady-state anisotropy

measurements.

Figure 2 Deconvoluted Prodan emission spectrum in the hydrophozided hyaluronate solution above the cac.

(1) J. Aguiar , P. Carpena, J.A. Molina-Bolívar, C.C. Ruiz, J. Colloid Interface Sci. 2003, 258, 116.

(2) K. Karukstis, J. McDonough, Langmuir 2005, 21, 5716.

(3) E. Krasnowska, E. Gratton, T. Parasassi, Biophys. J. 1998, 74, 1984.

0.0

0.2

0.4

0.6

0.8

1.0

380 430 480 530 580 630

wavelenght (nm)

fluor

esce

nec

inte

nsity

(a.u

.)

Raw data

Gauss 1

Gauss 2

Gauss 3

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