P h o t o - a n d T h e r m o - R e s p o n s i v e P o l y
( E t h y l e n e G l y c o l ) - b a s e d B i o m a t e r i a l s :
S y n t h e s i s , C h a r a c t e r i z a t i o n , P a t t e r n i n g a n d
A p p l i c a t i o n i n B i o l o g i c a l S t u d i e s

Vorgelegt von
Master of Science
Rahima Ra hman
g eb . in Rawalpind i P akistan

Von der Fakul tät II - Mathema tik und Naturwissen schaften
der Technisc hen Univer sität Ber lin
zur Erlangun g des akadem ischen Grade s

Doktor der Na turwiss enschaften
Dr. rer . nat.

genehmig te Dissertat ion

Promotions aussch uss:
Vorsitzen der: Prof. Dr. Ulrike Woggon
Gutachter: P rof. Dr . Ir. Mar ga C. Lensen
Gutachter: Pr of. Dr. Svetlana Santer

Tag der wissen schaf tlichen A ussprache: 4. Apr il 2018

Berlin 2 018

Dedicated to my beautifu l kids, wonderful husband and
ever inspirational woman in my life; my mother

INDEX
List of abbreviations ........................................................................................................................... i
Scope and organization of the thesis .......................................................................................... iii
Chapter 1 Introduction ............................................................................................................... 1
1.1 Materials ................................ ................................................................................................................................... 2
1.2 Stimul i-respons ive materials ........................................................................................................................... 2
1.3 Stimul i-respons ive gels ....................................................................................................................................... 3
1.4 Photo chromic M aterials ..................................................................................................................................... 4
1. 5 Azobenzen e .............................................................................................................................................................. 6
1.6 Azobenzen e Syste ms ............................................................................................................................................ 9
1.7 Bio materials .......................................................................................................................................................... 15
1.8 Idea of motivation and pla n of work ................................................................................................ ........... 20
Chapter 2 Materials and Methods .......................................................................................... 23
2.1 Materials ................................ ................................................................................................................................ . 24
2.2 Synthes is ................................................................................................................................................................ . 25
2.3 An alytical technique s ................................................................................................................................ ........ 30
Ch apt er 3 Effect of Azobenzen e on the Gelation Behavior of PEG -Derivatives ....... 35
3.1 Introdu ction ................................................................................................................................ .......................... 36
3.2 Material an d Method s ................................................................ ........................................................................ 38
3.3 Result s and Discu ssion ...................................................................................................................................... 38
3. 4 Concl usions ............................................................................................................................................................ 47
Chapter 4 Design, Synthesis and Characterization of Chemically Crosslinke d
AZO/PEG Gels ..................................................................................................................................... 49
4.1 Introdu ction ................................................................................................................................ .......................... 50

4.2 Material an d Method s ................................................................ ........................................................................ 51
4.3 An alytical technique s ................................................................................................................................ ........ 51
4.4 Result s and dis cussion ...................................................................................................................................... 51
4.5 Concl usions ............................................................................................................................................................ 66
Chapter 5 Photo- and Thermo- Responsiveness of AZO- 8-PEG-Acr NH 3 Gels ........... 69
5.1 Introdu ction ................................................................................................................................ .......................... 70
5.2 Materials ................................ ................................................................................................................................ . 73
5.3 Experi ments .......................................................................................................................................................... 73
5.4 Result s ...................................................................................................................................................................... 75
5.5 Discus sion ................................................................................................ .............................................................. 84
5.6 Concl usions ............................................................................................................................................................ 88
Chapter 6 Biological Studies of AZO-8- PEG-Acr NH 3 Gels ................................................ 89
6.1 Introdu ction ................................................................................................................................ .......................... 90
6.2 Material and Method s ................................................................ ........................................................................ 91
6.3 Biolog ical Evaluation ......................................................................................................................................... 91
6.4 Result s and Discu ssion ...................................................................................................................................... 93
6. 5 Concl usion .............................................................................................................................................................. 99
Chapter 7 Patterning of AZO -8-PEG-Acr NH 3 Gels ........................................................... 101
7.1 Introdu ction ................................................................................................................................ ....................... 102
7.2 Material an d Method s ................................................................ ..................................................................... 104
7. 3 Result s and Dis cussion ................................................................................................................................... 109
7.4 Concl usions ......................................................................................................................................................... 115
Bibliography ................................ .................................................................................................... 117
Abstract ............................................................................................................................................. 137
Zusammenfassung ......................................................................................................................... 139
Acknowledgements ....................................................................................................................... 143

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List of abbr e viations

θ Contact angle
˚ Degrees
8-PEG-OH 8 Arm star-shaped Poly (ethylene glycol) with OH-end groups
8-PEG- Ac r 8 Arm star-shaped Poly (ethylene gly col) with acrylate -end gr oups
8-PEG- VS 8 Arm star-shaped Poly (ethylene gly col) with vinyl sulfone-end groups
AZO Azobenzene
AFM Atomic Force Microscopy
Au NPs Gold nanoparticles
CL Cross linker
DSC Differential scanning calorimetr y
DCM Dichloromethane
DMF Dimethylformamide
E Young’s Modulu s
FIMIC Fill Molding in Capillaries
FBS Fetal Bovine Serum
G’ Loss Modulus
G‘’ Storage Modulus
h Hour
IC50 Half maximal inhibitory concentration
LC Liquid Crystalline
LCE Liquid Crystalline Elastomer
Mw Molecular weight
NP Nanoparticle
PI Photo- initiator

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PBS Phosphate Buffered Saline solution
PDMS Poly (dimethy lsiloxane)
PEG Poly (ethylene glycol)
PEG- 575 Linear Poly (ethylene glycol) w ith acrylate end groups
PS Penicillin/Streptomycin
Qm Swelling degree
SEM Surface electron Microscopy
Sec Seconds
SRG Surface relief gr ating
t Time
Tg Glass Transition Temperature
TGA Thermogravimetric Analysis
Tm Meltin g temperature
UV Ultraviolet

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Scope and organization of the thesis
There is an increasing interest in ‘acti v e’ materials which can respond or adapt to external
stimuli or changing enviro nmental conditions over traditional non - changing ‘passive’
materials. Multi-responsive gels are parti cularly attractive as p latfor ms for the
development of intelligent devices and comp onents for many practical ap plications lik e
sensing, actuation, permeability control in membranes, drug delivery, tissue engineering
and others.
Light delivers energy to materials and systems at the spee d of li ght, and due to the
possibility of nonconta ct delivery of energy, it acts as an outst anding or thogonal stimulus .
Light-responsive molecules can be used to generate materials that exhibit both
temperature and lig ht-responsiveness; a com mon example is az obenzene. They have been
successfully introduced into several systems in order to mani pulate the matrix p r operties
by reversible trans – cis photochemical iso merization upon exposure to different
wavelengths of light. They have emerged as an effective photo-switch for use in
biomaterials because they ab sor b light in a region that is compatible with many biological
systems (350−550 nm). These properties can be recapitulated in azobenzene -containing
gel networks to control matrix properties.
Most of the today’s research using azobenzene moieties in gel matrix is based on the
synthesis of supramole cular gels which arise due to non-covalent interactions among the
gel matrix an d azobenzene unit s. S upramolecular gels contain ing azobenzene have been
reported to respond to sin g le sti mu li (light). C hemically c rosslinked azobenzene in the gel
matrix can provide a bette r control over t he gel properties (actuation) using both
temperature and light stimuli. Chemical crosslinking of a z obenzenes with gel macromers is
challenging because due to the hyd rophobic nature of azobenzene group, crystallization
and precipitation may occur, which hinders the formation of the gel. Thu s, the successful
design of a chemically c r osslinked, multi-responsive azobenzene-based gels is a significan t
challenge. Despite the known influence of non-covalent inte r actions in supramolecular
gelation, it is still diff icult to rationa lly design and functionalize small azobenzene
molecules to develop a true covalently bonded gel network. With thes e challenges in mind,

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we set about rationall y designing a simple y et effective azobenzene-base d multi-stimuli
responsive gel.
PEG is generally considered biologically in ert and safe therefore; they can be an excellent
candidate f or the pr eparation of biologically safe multi-responsive g els. Hence, we chose
Poly (ethylene glycol) (PEG) as a matrix material for the gel formation.
Chemically in cor porated azobenzene (AZO) can generate multi -responsive materials that
exhibit both te mp erat ure- an d light- r esponsivity; hence act as multi-responsive system.
One of the main aims of the work present ed here was to design no v el multi -responsive gels
having chemically c r osslinked azobenzene moieties incorporated into PE G matrices. Th e
chemically bound azobenzenes in the gel matrix are expect ed to provide a control over the
gel properties using light and te mperatur e stimuli. We expected to cont rol the act uation
and sensing property of synthesized gels using both stimuli. As the PEG used is biologically
inert so in order to inv estigate the cytotoxic effect of AZO in PEG matrix, several biological
evaluations including cell cytotoxicity tests were conducted and documented.
The thesis is organized in following chapters
Chapter 1: Introduction
This chapter gives a general in trod uction to the topic. Details of the azobenzene chemistry
and mechanism of isomerization will b e outlined. Photo -induced micro- and macroscopic
actuation in azobe nzene based mate rials will also be explained in detail. Some intro duction
about Poly (ethy lene g lycol ) (PE G)-based hydrogels will also be presented. At the end,
motivations of the work will be detailed.
Each experimental chapter will contain an introductory section with more specific
information related to its topic.
Chapter 2: Materials and methods
Since several of the reagents, p reparat ion methods and an alytical techniques are common
to many of the experiments, t hey will b e described in this chapter. More specific op erat ing

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conditions or chemical compositions will be st ated at the Mat erials and Met hod s section of
the corresponding chapter.
Chapter 3: Effect of Azobenzen e on the gelation behavior of PEG derivatives
Experimental strategy to obtain a chemically crosslinked azobenzene -based Poly (ethylene
glycol) gel will be outli ned here. Gelation with different type of a zobenzene monomers and
PEG derivatives will be presented using diverse gelation techniques. At the end, merits and
drawbacks of the applied gelation techniques will be discussed in detail.
Chapter 4: Design, synthesis and charact eri zation of chemically crosslin ked AZO/PEG
(AZO-8-PEG-Acr NH 3 ) gels
Depending on the PEG macro-monomer and the gelation techniq ue ap pli ed, the novel
chemically crosslinked azobenzene gels were named as AZO-8-PEG- A cr NH 3 gels. In ord er
to verify the successful chemical incorporation of azobenzene in the gel’s matrix, several
characterization techniques were applied and the results will be detailed in this chapter.
Chapter 5: Photo- and Thermo- Responsiveness of AZO-8-PEG-Acr NH 3 gels
In or d er to evaluate the responsivene ss of the novel gels, they we r e subj ected to light and
thermal stimuli. This c hapter comprises all the results of actuation and also explains the
reason for the response of the novel gels.
Chapter 6: Biological studies of AZ O-8-PEG-Acr NH 3 gels
PEG is a biomaterial known for its inertness. In order to check the feasibility of the novel
gels for biote ch nological application, cell c ytotoxicity measurements were done. Cell
cytotoxicity tests of the novel gels against mouse fibroblast (L -929) were conducted.
Besides, some azobenzenes are known to possess an timicrobial and anti cancer act ivity, in
order to find out the po tential usage of AZO monomer, biological stud ies were carried out.
This chapter details biolog ical evaluations conducted with novel gels.

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Chapter 7: Patterning of AZO-8- P EG-Acr NH 3 gels
As the neat PEG matrix is k nown for its antifoulin g character istics, the inc or poration of AZO
moieties makes the gels less anti -adhesive to cells. In order to pr ovide a p latfor m for the
selective cell adhesion, several patterning te ch niques were ap plied an d compared. A novel
patterning techni que “ Micro - de -Mol ding ” was d esigned to d isplay th e A ZO-8-PEG-Acr NH 3
gels in a lateral micro-patter n at the biomaterial’s surface. This chapter comprises all the
patterning techniques applied for the AZO-8-PEG-Acr NH 3 gels and descr ibes the results of
the comparative cell adhesion studies.

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1 Chapt er 1
Intr oduction

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1.1 Mate rials
Materials technologies have influenced the human civilization so strongly that historians
named ti me p eriods aft er the materials that dominate d in those eras. Ancient artifacts have
been dated and explored to reveal the emer gent sophistication of their manufactur ing
techniques.
A remarkable variety of materials have surrounded the mo dern socie ty. Mankind faces
challenges of previously unknown dimensions; Population growth, scarcity of resources,
increased energy demands and climate chan ge. To cope w ith these ch allenges, mankind
requires creative minds and equally cr eative m aterials to realize essential innovations.
Synthetic materials that can respond to internal or external stimuli represent o ne of the
most emergent areas of scientific interest. Although there are several challenges facing this
field, there are a number of prospects in de sign, synthesis and engineering of stimuli -
responsive systems.
Mother Nature serves as a provider of infinite inspirations for designin g and developing
new mate rials. The truly intelligent stru ctural system lea rns and adapts it s behavior in
response to the external stimulation provided by the environment in which it ope rates [1].
1.2 Stimuli- responsi ve materials
The stimuli- responsive characteristic is commonly defined as “the ability of the system to
undergo obvious responses to enviro nmental changes” [2]. In g eneral, stimuli-responsive
materials can be consid ered as soft materials and besides, they have some significant
features in commo n: th ey respond ei ther to (a ) chemical sti muli, (b) physical stimuli or (c)
biochemical stimuli in solid state, in solution or as gel [3]. Chemical sti muli include changes
in the pH value and in the ionic strength, as well as the addition of chemical agents, e.g.
solvents or gases and redox-reacti ons are the most common exam ples. With regard to
physical stimuli, voltage, te mperature changes, light-irradiation and mechanical stress are
important and promising. In additi on , biochemical stimuli have bee n acknowledged as a

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third category of stim uli that involve responses to enzymes, ligands, an tigens or other
biochemical agents[4].

Figure 1.1: Me thodology fo r material response
A number of stimuli-responsive systems have been established, with the majority of studies
concerning polymeric solu tions [5], gels[6], surfaces[7] – [9], interfaces[ 10], [11]and to some
extent polymer ic solid s[12]. These different state s of matt er impos e a diverse degree of
restrictions on the mo tion of polymeric segments or chains. The challenge in designing
these stimuli-responsive p olymeric systems is to craft networks capab le of inducing minute
molecular, yet coordinated changes that lead to substant ial physicochemical responses
upon internal or external sti muli [7], [13] . Constrained mobility within the network arises
from significant spatial limitat ions, hence imposing restrictions on attainin g stimuli -
responsiveness.
The dimensional r espo nsiveness is simply possib le for the systems wit h a hig her degree of
freedom and least energy inputs. Nov el app roaches to applications inclu ding actuation and
biomimetic sensing require the synthesis an d manipulation of 'soft' materials, respond to
external sti muli with their distincti ve capability. Recently, research on these materials has
increased d ramatically and is appealing eq ual at tention for, physicists , chemists and
engineers. Gels owing both of above ment ioned prerequisites are considered ideal
candidate for fast resp onsiveness. Considerably greater challenges are faced while
designing physically or chemically crosslinked gels networks that require maintaining t heir
mechanical integrity [14].
1.3 Stimuli- responsi ve gels
Owing to their distinctive features such as multifold change of volume in response to small
change in the en viron ment, capacity to function in wet environments, resemblance to soft
biological ti ssues an d chemical adaptability, stimuli-responsive gels r epresent an adaptable

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and a promising class of mate rials for muscle -type actuators, sensors, autonomous
intelligent structures and biomedical applications [13], [15].
The development of res ponsive gels accelerated in the end of the t wentieth cen tury. Tanaka
demonstrated abrupt gel volume change caused b y small change of the environmental
conditions, which was named volume phase t ransition [16 ]. He created gels responsive to
different stimuli, such as solvent composition [16], tempe rature[16] , metal ions [17] ,
electric field [1 8], an d light [19]. In the end twen tieth cen tury, responsive gels became an
important class of functional mate rials. These synthesized materials respond to a variety of
stimuli, e.g., pH[20], temper ature [21], mech a nical force[22], electr ic /magnetic fields[23],
[24] and light[25] .
Among the above mentione d sti muli, light is of particular interest as the use of li ght as the
driving a gent to modul ate properties of mate rials provides many advantages. The use of
light is convenient, and it has noninva sive character. Light with variable wavelength,
polarization, and intensity is readily available. Temporal and spatia l resolution of light with
autonomous, remote, and digital controllab ility renders it an ideal sti mulus to mo dulate the
properties of materials. Light delivers energy to materials and systems at the speed of light
and due to the possibility of noncont act deli very o f energy, light acts as an outstanding
stimulus[26] .
Typically, photochromic molecules are utilize d to alter the mater ial properties in lig ht -
responsive systems. Even one light -responsive unit can be adequate to influence the
properties of the entire matrix. Depending on the chromophore bonded to the polymeric
backbone, the change of behavior can be reversible or irreversible [27].
1.4 Photoch romic M aterials
Photochromism is the “ reversible p hoto-ind uced alterati on of a molecule among two
isomers whose absorption spectra are disting uishably different ” [28]. The shape change
induced by means of light results in in fluential modifications in the behavior of host
material.

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The photochromic units comprise of the molecules that photo-dimerize, such as
anthracenes and coumarins; those that permit intra -molecular photo -induced bond
formation, such as spiro-pyrans, fulgides, an d diarylethenes; and those that display photo -
isomerization, such as crowded alkenes, stilbene s and azobenzenes (shown in Figure
1.2)[29] .

Figure 1.2: Most w idely used photoc hromic groups
The reversible photo-isomerization alters some physicochemical properties of
photochromic compounds, such as f luorescence emission, absorpti on spectra,
electrochemical properties, ele ctron conductivity, dipole interaction, coordinatio n
properties, dielectric constant, refractive index an d geometrical structure tuned by
light[28], [30]. These photochromic compounds are considere d as molecul ar switches.
Figure 1.2 displays some of the most widely us ed photochromic groups.
Generally, molecular switches are used as swi tching unit s in several optoelectronic devices
and functional mat erials to specifically switch the physical properties among two sta tes by
using light [31]. Photochromic molecular switches are promising for the field s su ch as
molecular logic gates[32], [33], data recording and storage[34], [35], multi -photon
devices[36] , surface/ nanop article devices[37] and photo-electronic d evices[38] etc.
Good photochromic mate rials satisfy the prere quisites such as distinct ab sorption p rofiles
for selectivity, high sensitivity, stability of isomers, fast resp onse, high quantum yield s,
fatigue resistance and photo -stationary states predominantly composed of one isomer.

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Light-driven structural changes of photochromic compounds often l ead to modulation of
electronic properties, absorption coefficients , p olarit y, refractive i ndex, electrochemical
redox potentials, conjug ation, conductivity, molecular geometry, physical dimensions,
chirality, solubility, etc. T hese molecular level photo-physical and photo-chemical changes
of the materials can be harnessed to regulate macroscopic properties and functions. As a
consequence, compou nds with p hotochromic fr agments have been invaluable build ing
blocks for the fab rication of light-driven advanced materials and devices with tunable
properties an d performances[26]. Light responsive monomers can also be used to generate
materials that exhibit both tempe rature and light responsiveness; a common example is
azobenzene[3], [4], [39] – [41]
To date, azobenzene (AZO) is considered as one of the smartest chrom ophores amongst all
photochromic molecules ow ing to its thermal stability, a distingui shable absorbance of
trans and cis isomers, and a relatively rapid thermal cis → tra ns back reaction. Photo-
isomerization in AZO results in an evident ch ange in the molecular geometry and dipole
moment [42] .
1.5 Azob enzene
Rhodopsin/retinal protein system that assists vision is the universal natural molecule that
exhibits reversible shape change an d most likely, the inspiration for all artificial bio -mimics.
It is the vital reversible photo-switch for the performance in eyes. The small retinal
molecule implanted in a cage of rhodopsin helices isomerizes (with an absorption of only a
single photon) from a cis → trans geometry around a C=C double bond [43], [44]. The shap e
change of a few angs troms is rapidly amplifie d an d leads to a larger shape and chemical
change, ultimately ending in an electrical signal to the brain for vision.
Possibly the finest ar tificial mimic of this strong photo-switching effect in terms of
simplicity of incorporation, speed and reversibility is azobenzene. Trans an d cis states are
able to switch reversibly in microseconds using light of even low p ower; reversibility of 1 0 5
and 10 6 cycles is obv ious before chemical f atigue. A widespread range of molecular
architectures is access ible to the synthetic materials chemist . C hemically incorporated
azobenzene monomers can generate multi-responsive materials that exhibit both

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temperature an d light responsivity; hence act as multi -responsive responsive system[3],
[4], [39].
1.5.1 Photoch emistry of Azoben zene
Azobenzene ( AZO ), a diazene (HN=NH) derivative with both hydrogens substituted by
phenyl gr oups[4 5].It c an exist in either the cis ( c-AZO ) or tra ns conformation( t- AZO )
(Figure 1.3)[46]. The t ran s → cis isomerization takes p lace after irradi ation with UV-visible
light[45] – [47], mechanical stress[48] or electrostatic stimulation [49]. Thermal cis→trans
isomerization occurs spontaneously due to the thermodynamic stability of the trans
isomer[46]. R represents the attached functional group to the p henyl ring. This absorption
spectrum can be personalized, by means of ri ng substitution, anyplace from the ult raviolet
to the visible-red region.

Figure 1.3: Photo -isomerization i n Azobenzene
The AZO ab sorption spectrum comprises of two well distinguished bands in the UV visi ble
region (Figure 1.4 ). The intense UV band (λmax ∼ 3 20 nm, Ɛ ∼ 22000 L mol -1 cm -1 )
originates from the symmetry allowed π - π * transition. The weaker b and in the visible
region (λmax ∼ 450 nm, Ɛ ∼ 400 L mol -1 cm -1 ) corresponds to the symmetry forbidden n - π*
transition. The π - π* transitions of c- AZO (λ max ∼ 270 nm, Ɛ ∼ 5000 L mol -1 cm -1 ; λmax
∼ 250 nm, Ɛ ∼ 11000 L mol -1 cm -1 ) are weak er, but the n - π* transiti on (λmax ∼ 450 nm,
Ɛ ∼ 1500 L mol - 1 cm -1 ) absorbs more intensely than t -AZO [42] .

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Figure 1.4: Cha nges in the ab sorption spectrum of AZO upon irr adiation w ith 316 nm
light [42] .
The AZO tra ns to cis inte rconversion can be precisely controlled by irradiating at an y
transition or by vary ing the intensity of a mono chromatic source. Uninterrupted irradiation
of t-AZO with either 313 nm or 436 nm radiation results in a photo -stationary state
consisting of ∼ 20% or ∼ 90% of t-AZO , respec tively[42]. On the othe r hand, fluorescence is
enhanced, when t-AZO is embedded in a solid matrix at low temperatures [26], [42].
1.5.2 Isomerizat ion mechan ism
The AZO isomerization mechanism has been the topic of attent ion and controversy since c-
AZO was isolated ab out eighty five years ago. The isomeric form ( cis vs. trans ), irrad iati on
wavelength, excitat ion app roach (thermal vs. radiation), substi tuents on the phenyl rings,
solvent properties, te mperature an d pressur e eff ect the isomerization mechanism an d
quantum yield[42].
Advances in ultrafast time-resolved spectroscopic techniques have enabled scient ists to
discover the complexities in the isomerization process. These techniques ove rcome the
challenges faced by ear lier interpretations of experimental results and well -established
theories concerning the isomerization mechanism.

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Figure 1.5: P roposed mecha nisms for the trans ↔ cis isomeriza tion of AZO
Four mechanisms — rotation, in version, concerted in version, inver sion -assisted rotation –
have been suggested as probable pathways for AZO photo -isomerization ( Figure 1.5)[50] –
[53]The rotational path follo ws rupture of the N=N π -bond to allo w free rotation ab out the
N – N bond. Rotation alters the C – N – N – C dihedral angle, whereas the N – N – C angle remains
fixed at ∼ 120°. In the inversion mechanism, the C – N=N – C dihe dral angle stays fixed at 0°,
while one N=N – C angle rises to 180° which results in a transition sta te with one sp
hybridiz ed azo-nitrogen atom. In concerted in version, b oth N=N – C b ond angles increase to
180° creating a linear transition state. Whereas in case of inversion -assisted rotation,
obvious changes in the C – N=N – C dihedral ang le an d smaller but noteworthy simultaneous
changes in the N=N – C angles occur. The transition state generated in concerted in version
has no net dipole moment, however the other three p aths displays polar transition states.
All four t ransition states can give rise to ei ther the cis or the trans isomer upon relaxation;
thus, all four mechanisms expect photo -stationar y states comprising of both isomers [54].
1.6 Azob enzene Systems
Many AZO’s being high ly colored are utilized in industry as dyes but likewise they have
been widely explored as small molecules, as pen dants on other molecular structures, or

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incorporated (doped o r covalently b ound) into a varied range of a morphous, crystalline, or
liquid crystalline polymeric systems. Significant examples includ e self -assembled
monolayers and super- lattices , sol – gel, silica glasses and biomaterials [55]. Usually, azo
chromophor es are in troduc ed in a solid matrix for studying their behavior or fabrication of
devices. As a conseq uence, matrix effects a re certain; the chromophore behavior can b e
changed due to the matrix and in turn the chromophore alters the matrix [59]. Both of these
effects are in fact useful; the chromophore can be used as a probe of the matrix
(polarizability, fr ee vol ume, mobility etc.) an d when it couples to the chromophore motion,
molecular motions can be transla ted to larger length scales. Therefore, the incorpor ation
approach is important to exploit the AZO’s distinctive behavior.
The azobenzene chromophore was known fro m early on as an ap propr iate photochromic
molecule to alter the polarity within a system in a reversible manner upon light -irradiation
[27]. The polarity change arises due to the change in the dipole moment of tran s → cis from
0-3 Debye. For this reason, the azobenzene chromophor e displays a vital role in
temperature and light responsive polymers[56].
Azobenzene-based photochrom ic syste ms are under kinetic control; after a photochemical
conversion, the spontan eous thermal back reaction occur s. The rate of the thermal
cis→trans back reaction is governed greatly by the chemical architecture of the system. The
response time of the photochr omic switch is an important aspect of its overall
performance[29] .
Keeping in mind the above mentioned versatile properties, AZO’s have been in corpor ated
into several systems i n ord er to control the matrix properties b y reversible trans → cis
photochemical isomerization upon light ir radiation to different wave lengths [2], [3], [48],
[56]. In this regard, worthy contributions are made by Prof. Dr. Svetlana Santer and
coworkers in exploiting the unique behavior of photo-isomerization in azobenzenes , in
various useful applicat ions as well as an effort to explain the mechani sm of photo -induced
motion in azobenzene based systems [57] – [63].

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1.6.1 Photo - in duced Motions
The geometrical change is the fundamental molecular photo -motion in azobenzenes that
arises upon absorption of light. I n c -AZO, the p henyl rings are twisted at 90 ° relative to the
C-N=N-C plane[64], [65] . Photo-isomerization decreases the distance from 0.9 9 nm in the
trans state to 0.55 nm for the cis state between th e 4 and 4’ positions ( Figure 1.3 ) [66]. This
geometric change alters the dip ole moment from 0-3D from trans to cis isomer [67]. The
free volume required for the photo -isomerization in case of the c-AZO is more than the t -
AZO [68]. The minimum free volu me pocket necessary to allow photo -isomerization to
proceed through the inversion pathway[64] is 0.12 nm 3 and is approximately 0.38 nm 3 via
the rotation pathway[69]. The effects of matrix free volume limitations on photochemical
reactions are considerable [70]. The geometrical changes in azobenzene modifies a varied
wealth of material properties[55] .
Natansohn and Roch on explained all possible photo -induced motions in azobenzenes in
detail in or der of increasing size scale [71]. H ere in this thesi s, we a re mainly concerned
with two types of them mentioned below.
1.6.2 Ma croscopic Motion
The azobenzene molecular conformation can result in modification of the bulk properties or
even to macroscopic motion. Various examples of this photomechanical conversion are
displayed in many azob enzene base d systems. Polymers remain an ideal candidate for
expressing this photomechanical conversion because of the flexible network structure,
which allows a higher degree of freedom and displays qu icker response on exposure to light
stimuli[2], [27], [56], [72], [73] .
The most considerable macroscopic motion demonstration in azobenzenes due to photo-
isomerization is the mechanical bending a nd unbending of an azo polymer film. The
bending direction can either be selected with polarized light or by aligning the
chromophor es with rubbing. The inter-relationship between photo-isomerization and
geometry changes of azobenzene gr oups with photo -induced macroscopic ben ding an d
stress hav e been investigated and analyzed in polymer films containing azobenzene -based
cross-linkers. Light -driven changes in the azobe nzene moieti es cause volume contraction at

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the polymer film surface leading to ben ding phenomenon. In this regard, an interestin g
article is contributed by Prof. Dr. Svetlana Santer and cow orkers explaini ng the
comparative study of photoinduced d efo rmation in azobenzene containing polymer
films[58].
Ikeda group fabricated a LC polymer film by using mono - and di-acrylates containing
azobenzene group. This film was shown to ben d precisely along controlled directions by
irradiation with linearl y polarized light ( Figure 1.6). Light-induced bending studies on such
films were conducted by varying the crosslinking density. It was observed that the
magnitude and rate of bending is different. The light -driven bending p henomenon has been
explained by taking into account the large ab sorption extinction coefficient of the
azobenzene chromophor es. It is suggested t hat changes in the molecul ar size of the
azobenzene moieties cause volume contraction which give rise to bending
phenomenon[74].

Figure 1.6: Lig ht-control led bending of a po lymer film containing azobe nzene groups.
Reproduced fr om ref [74] .
Ikeda et al. reported the fabrication as well as light -driven anisotropic bending and
unbending of liquid crystalline gels formed through azobenzene derivatives [75]. Liquid
crystalline gels formed by the cross-linked ne twork of azo monomers in tolue ne show
anisotropic swelling behavior. Upon UV light irradiation, they exhibit anisotropic bending

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toward the light direction, whereas they retrieve b ack to original st ate upon visible light
irradiations. Figure 1.7 displays the reversible bending and unbending phenomenon in a
liquid crystalline gel f ilm. The bending behavior of the film has been attributed to the
absorption gradient between the surface and bulk of the film.

Figure 1.7: P hoto-ind uced bendi ng and unbending in the liquid crystalline gel fi l m.
Reproduced fr om refere nce [75] .
The same group reported the photo-mechanics in Ferroelectric liquid crystalline elastomers
(LCE) films conta ining azobenzene moieties, fabricated by photo -polymerization under the
applied electric field. Light - driven bending phenomenon in these fil ms was in vestigated.
Interestingly, it was observed that photo - generated mechanical force in these films is
comparable to the contraction of human muscles. This attr ib ute qualifies these materials for
potential application in artificial muscles and light - driven mechanical devices. A li ght -
driven p lastic motor was demonstrated using a laminated azobenzene - containing LCE film
(Figure 1.8)[76] .
The film was fabricated from the polymerizable acrylates. A cyclic belt made from the LCE
film was able to drive a pai r of pulleys when irradiated simultaneously at different positio ns

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with UV/ Visible light. This is a clear demonstration of the transformation of light energy
into mechanica l work. Interesting 3D movements have been demonstrated in cross -linked
LC polymer films by light illumination.

Figure 1.8: Light-driven plas tic motor. Reprodu ced from referenc e [76]
1.6.3 Micrometer- Scale Motion (Hologra phic lithography)
The azobenzene based matrices demonstrate a significan t surface -mass transport under the
light illumination. This surface- ma ss transport results in op tical patterning of a micrometer
and sub -micrometer le ngth scale. In 1995, an unexpected and uniq ue optical effect was
revealed in poly mer thin f ilms having the azo chromophore Disp erse Red 1. The
Natansohn/Rochon [77 ] research team and the Tripathy/Kumar collaboration [78 ]
simultaneously and in dividually revealed a large-scale surf ace-mas s transport when the
films were irradiated with a light interference pat tern. In this experiment, two coherent
laser b eams (having a wavelength lying in the azo absorption ban d) are intersected at the
sample surface (Figure 7.5) . This technique is called “ holographic litho graphy”. The samp le
typically consists of a thin sp in-cast azo-polymer film (10 – 1000 nm) on a transpar ent
substrate. The sinusoidal light interference pattern irradiated at the sa mple surface leads to
a sinusoidal surface pattern, referred as a surface relief grating (SRG) ( Figure 1.9). On the
other hand, the surface mass transp ort in azo is not limited to j ust gratings and can yield
arbitrary structures, de fined by the polarization pattern and spatial intensity of the incident
light. Therefore, the phenomenon more accurately can be called photo-patterning.

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Figure 1.9: A ty pical surface r elief grating (SRG)
The SRGs diffracts efficiently, and it was revealed that various reports of large diffraction
efficiency before 1995, ascribed to birefringence were actually due to surface gratings. The
phenomenon arises at room temperature (below the Tg of the amorphous polymers) with
moderate irradiation (1 – 100 mW/cm2) completed from seconds to minutes. As the original
thickness is recovered upon heating above Tg, the p rocess is a reversible mass transport .
Analytically; it involves the presence and photo-isomerization of azobenzene
chromophor es. Other absorbing but non -isomerizing chr o mophores do not yield SRGs.
Several other s ystems can show opti cal surface pat terning, but does not involve mass
transport; the amp litude of the modification is much smaller an d generally involves
additional processing steps. Since its discovery , t his unique optical patterning of
azobenzenes has been exploite d in tensively, yet there re mains controversy regarding the
mechanism. Many reviews on this effect with experimental results are available [78] – [81].
1.7 Biomaterials
Just 60 years ago the word “biomaterial” was not known. There w ere no medical device
manufacturers (except for external prosthetics such as limbs, fracture fixation devices ,
dental fillings and glass eyes), no appreciative of biocompatibility, no formal regulatory
approval processes and indeed no academic courses on b iomaterials. A definition of
“biomaterial” certified b y consent of experts in the field is “A biomaterial is a nonviabl e
material used in a medical device, in tended to interact with biological systems”. Now in
twenty-first century, biomaterials are extensively used in various fields[82].

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Figure 1. 10 : The pa th from the bas ic science of biomaterials, to a medical device, to clinical
applicatio n . Reproduced fr om reference [83]
To design successful biomaterials for clinical application (Figure 1. 10 ), one must consider
the interaction between the targeted cells/tissues and the environmen tal cues. Essential
factors of ten consist of soluble growth factors, cell – cell and cell – material interacti ons and
mechanical properties of the microenv ironme nt [15], [84], [85 ].
Polymer mate rials hold a range of distinctive properties which make them suitab le into a
wide selection of b iomaterial applications such as dental, orthopedics, hard and soft tissue
replacements an d cardi ovascular devices. Actually, polymers symbolize the largest class of
materials used in the medical industry [6], [86].
1.7.1 Gels
Gels are usually design ed by physical and/or chemical crosslinking o r by supramolecu lar
interaction of mo lecular chains distr ibuted in a solv ent . The d riving forces for the gel
networking are covale nt bonds[87] and non-covalent interactions, such as hydrogen-
bonding, stacking, hydrophobic or van der Waals interactions [88 ] . For in stance, if a
polymer matrix is tied by crosslinked points or entanglements, sta ble hydrogels an d/or
organogels (depending on the solvents) with retained bulk structures are achieved.
Hydrog els have been found in nature since li fe on earth evolved. Bacterial biofilms hydrated
extracellular matrix components an d plant stru ctures are ever -pre sent, water- swollen
motifs in nature. Gelation and agar were used for various ap plications since early human
history. On the other hand, the modern hist ory of hydrogels as a material designed for
medical ap plications can b e outlined precisely. They have received s ubsta ntial attention for
their high water contents and related p ote nt ial biomedical applications. Hydrog els are
polymeric structu res held together as water -swollen gels by: (1) pri mary covalent cross -

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links; (2) ionic forces; ( 3) hydrogen bonds; (4) affinity or “bio - recognit ion” interactions ; (5)
hydr ophobic interactions; (6) polymer crystallit es; (7) p hysical entanglements of individual
polymer chains; or ( 8) a combination of two or more of the above interactions [89].
1.7.1.1 Structural evaluation of gels
Structural evaluation of the gels discloses that ideal networks are seldom obse rved. F igure
1. 11 ) ill ustrates an ideal macromolecular net work (gel ) rep resenting the te tra -functional
cross-links (junctions) resulting from covalent bonds[89].

Figure 1. 11 a) Ideal macr omolecular network o f a gel; (b) N etwork with multifu nctional
junctions; (c) Physical entangle ments in a gel; (d) Unreacted functio nality in a ge l;(e ) Chain
loops in a gel [89] .
Though in real netwo rks it is likel y to en counter multi -functional jun ctions (Figure 1.1 1b)
or physical molecular en tanglements (Figure 1.11c) playing the rol e of semi -permanent
intersections. There always exists a possi bility of molecular defects in gel network. Figure
1.11d an d e indicate two such effects: unreacted functionalities with partial entanglements
(Figure 1 .11 d) and ch ain loops (Figure 1.11e).The terms “ cross - link,” “j unction” or “tie -
point” (shown by an open circle symbol in Figure 1.11d show the covalent or secondary

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connection points of several chains. In the example of covalent linkages, these j unctions
may be carbon atoms, but usually they are small chemical bridges (e .g., an acetal bridge in
crosslinked poly (vinyl alcohol), or an ethylene glycol diester brid ge in the polyHEMA
contact lens gel) with molecular weights much smaller than thos e of the cross -linked
polymer chains..
1.7.2 Poly (Et hylene Glycol)
Ever since from the start of twenty first century , p oly(ethylene glycol) (PEG) hydr ogels
have been widely used for practical solutions as scaffolds and have been used for cell
culture; for controlled release of therap eutics; as matrices for contro lling drug delivery, as
well as cell delivery vehicles for promoting tissue regeneration [15], [90]. The versatility of
the PE G macromer chemistry[91] an d its ex cellent biocompatibility has prompted the
development of several intelligently-designed hydrogel systems for r egenerative medicine
applications.
Polyethylene glycol s (PEGs) are hydrophilic oligomers or pol ymers co nsisting of a repeating
unit of – (O – CH 2 – CH 2 ) – synthesized from ethylene oxide. Poly (ethylene glycol) (PEG) is
also called poly (ethylene oxide) (PEO). PEG in its high molecular weight form when the
chains are crosslinked, can b e categorized as a gel [92] – [94]. Depending on the solvent
retained, they are categ orized as hydrogel (water) or organogel ( organic solv ent). The
capacity to at tach a range of reactive funct ional groups to the terminal sites of PE G
polymers has significantly extended their utilization. Gels of poly (ethylene oxide) (PEO)
and poly (ethylene glycol) (PEG) have drawn growing attention recently for biomedical
applications beca use of their non -toxic behavior [15], [95], [96]. The inertne ss of PEG with
living organisms has been acknowledged from the time in 1944, when it was inspected as
a possible vehicle for intravenously administering fat-soluble hormones [97].
Branched PEG-based macromonomers have taken substantial in terest as they combin e
multifunctional architectures with small hydrodynamic radii so that the molecules have
relatively low viscosities in contrast to the linear an alogues. M oreover, the molecular
architecture of such molecules can be adapted as star, H -shaped, or dend ritic. Star polymers

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are an exclusive class of b ranched polymers consisting of a single bran ch point and attached
linear chains to the central core.
They have drawn signi ficant in terests due to their substant ial properties that differ than
their linear analogues with a small hydrody namic radius, low viscosity and high
functionality. They may present variations in chemical composition, molecular weight o r
different peripheral functiona lities, dep ending on the ir synthesis ro ute. A mong sta r-type
polymers, 8-PEG possesses higher number of branches and provides higher functiona lities.
Here in this work, we mainly deal with multifunctional, 8 -arm PE G macromonomers [98],
[99].
1.7.2.1 Major techniques for the preparation of PEG networks
Three main techniques exist for the p reparation of PE G networks [89]; (1) chemical cross-
linking between PEG chains, such as reaction of di -functional PEGs and multi -functional
cross-linking agents; (2) radiation cross-linking of PEG chains to each other and (3) physical
interactions of hydrophobic blocks of triblock copolymers having bot h hydrop hobic blocks
and PEG blocks. The benefit of using radiati on -cross-linked PE G net works is that no toxic
cross-linking agents are required. Though, it is challenging to control t he ne twork str ucture
of these materials, Lowman et al. in 199 9 described a metho d for the preparation of PEG
gels with controlled geo metry [100]. In this work, highly cross -linked a nd tethered PE G gels
were synthesized from PEG -dimethacrylates and PE G mono-methacr ylates. The tech nique
explained in this work has bee n used for the development of a new c lass of functionalized
PEG containing gels used for a wide range of drug release applications.
Dendrimers an d star p olymers are sensational new materials b ecause of the large number
of functional groups of fered in a very small molecular volume. These systems could have
great p otential in biomedical applications. Recently Zhang et al. from our group introduced
a new method of gelation “ amin e Michael-type ” addition for the star shaped 8-PE G
derivatives using ammonia linker [93]. He determined that by varying amounts of am monia
cross linker use d in the polymerization, provides a contr ol over the cross - linking density,
surface and bulk elasticity and morphologies. Therefore, amount of the cross linker is an

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important factor in the polymeriz ation of PEG-based gels formed by amine Michael-type
addition.
1.8 Idea of motiv ati on and plan o f wor k
Seeing believes. We are able to see and applau d the vivid and sparkling natural world that
surrounds us due to the natural process of cis→t rans photo-isomerization of the retinal [43],
[44]. At the outset, light actuate s this na tural phenomenon followed by a cascad e of
photomechanical events, eventually leading t o “ sight ” otherwise r ef erred to as “ vision ” .
Therefore, it is suitably sta ted that “ there is no sight without light ” . This single example is
adequate to ap preciate the role of light -d riven processes in the animal world. As far as the
plant kingdom is concerned, there functions another similar light -driven cis→trans photo-
isomerization process in the photo -receptor phytochrom e, a linear tetrapyrrole. This light -
driven photochromic phenomenon governs p hysiological, growth, and developmental
processes in plants cul minating in certain heliotropic and ph ototropic motions in addition
to stem mo vements. The above-mentioned two representative examples in the na tural
world convincingly demonstrate the importance of light -triggered processes and
phenomena in nature.
The elegance of nature exhibit ing such light-driven phenomena has been a great b asis of
inspiration for scientists in the research and development of light -dr iven materials and
systems not only fo r f undamental scientific studies but also for device app lications.
Subsequently, they have investigated into the dominion of design, synthesis, and evaluation
of properties of dynamic and r econfigurable light driven materials and systems.
Multi-responsive gels are particularly attractive as platforms for the development of
intelligent devices and components for many p ractical applications like sensin g, actuation,
drug delivery, tissue engineering and others.
There is an increasing interest in ‘active’ materials which can adap t or respond to external
stimuli or changing environmental conditions over traditi onal non- changing ‘passive’
materials. A variety of different stimuli for responsive systems have been reported, namely,
ionic electrochemical, pH and light etc. Among these external stimuli, light has attracte d

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much attention as it offers a b road range of tunab le parameters, e.g., intensity, wavelength ,
and duration, to manipulate the rheological properties of the gel. Photochromic unit s within
these systems experience structural transformations such as isomerization (e.g;
azobenzene tautomeriz ation (e. g; spiro-pyran) and electro-cycling ring closures and
openings(e.g; fulgimide) to control gel's properties.
Azobenzenes have been successfully introduced into several systems in order to manipulate
the matrix p roperties by reversible tran s → cis photochemical isomerization upon exposure
to different wavelengths of light. In small molecu le systems, azobenzene converts from the
trans isomer to the cis isomer upon irradiation with 360 nm light in a remarkably efficient
reaction ( ∼ 80%−95%). Removal of the light or irradiation in the visi ble range regenerates
the Tr ans isomer almost completely (>9 9%) because it is the mor e thermody namically
stable conformation. T hey have been used to control properties of polymeric networks,
such as the gel −sol tran sition an d the s welling ratio. Az obenzenes hav e emerg ed as an
effective photo switch for use in biomaterials because they absorb light in a region that is
compatible with many biological systems (350−5 50 nm). These properties can b e
recapitulated in azobenzene-containing gel networks to control matrix properties.
Most of today’s research using azobenzene moiety in g el matrix is based on the synthes is of
supramolecular gels which arise d ue to non-covalent inte ractions among the gel mat rix and
azobenzene. Most supr amolecular gels contain ing azobenzene have been stated to respond
to limited stimuli. Th is is due to the challenges of incor porating additional stimuli
responsive functionalities into the g el matrix stru cture in addition to the hy drophobic
azobenzene group. Due to its hy drophobicity, crystallization and prec ipitation of
azobenzene molecules occurs, which hinders t he formation of the gel.
Thus, the successful de sign of a chemically crosslinked multi -responsive azobenzene based
gel remains a signifi cant challenge. Despite the known influence of non - covalent
in teractions in supramolecular gelation, it is still difficult to r ationally design and
functionalize small azobenzene molecules to develop a true covalently bonded g el network.

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With this challenge in mind, we set about rationa lly designing a simp le yet e ff ective
azobenzene based multi-stimuli responsive gel.
Application of gels a s actuators was historically considered promising because gel
properties resemble properties of muscles. Thanks to this r esemblan ce, such systems
became known as “a rtificial muscles” . The y are particularly at tractive for making
biologically inspired de vices and robots that mimic the movements of humans, animals, and
insects. For many appli cations, the requirement of keeping the gel wet represents a hur dle.
Control of actuation with environmental stimuli such as temperature, pH, or light is also
relatively uncommon in current actuator designs. Among the others, light and temperature
could be esp ecially interesting due to ease of remote control. The most important
shortcoming of gel a ctuators, however, is their response rate. Bulk macroscopic gels are
unacceptably slow for most act uators ap plications. Therefore methods of improvement of
response rate are hig hly relevant f or g el act uators. Nonet heless, gel actuators possess other
unique pr operties, suc h as no power requirements, and no moving parts. Therefore, they
are ideally suited for sp ecialized app lications, such as autonomous medical p umps for long -
term drug release and autonomous v alves for power-f ree field irrigation.
PEG is gene rally considered biologically inert an d safe so they can be an excellent candidate
for the prep aration of biol ogically safe multi- responsive gel. PE G is a suitable mate rial for
biological applications because it does not generally elicit an immune response.
Chemically in corporated azobenzene monome rs can generate multi - responsive mate rials
that exhibit both temperature and light responsivity; hence act as multi responsive
system[40], [41], [56]. One of the main aims of the work presented here was to design
novel multi-responsive gels having chemically crosslinked azobenzene moiety in corporated
into PEG matrix.
The chemically bonded azobenzenes in the gel matrix are e xpected to provide a control
over the gel properties using light and temp erature sti muli. We ex pected to control the
actuation and sensin g property of synthesized gels using b oth stimuli. As the PE G matrix
used is cytocompatible, the gels are also expected to possess cytocompatibility. In order to
evaluate the b iocompatibility, the in teractions of ge ls with cells will also be monitored.

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2 Chapt er 2
Materials and Meth ods

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As man y of the e mplo y e d reagents, pr eparation methods a nd an al y tical chara cterizations re levant
to this thesis are comm on to man y o f the ex periments, the y will be described in this chapter.
Concrete op erating conditions and composition of the materials are outlined in the Materials a nd
Methods section of the corresponding c h apter.
2.1 Mate rials
Reagents and chemicals of high p urity were u sed fo r all the exp eriments. Solvents were at
least analytical grade quality. Poly (ethylene glycol) di -acrylate ( P EG- 575 , M w 575 Da),
Photo init iator, 2 -hyd roxy- 4’ - (2 -hydroxyethoxy)-2-methylpropiophenone ( PI , M w 224.26
gmol -1 ), Cr oss lin ker, Pentaerythritol triacrylate ( CL , Mw 298 gmol -1 ), Acryloyl chloride (Mw
90.51 gmol -1 ), Divinyl sulfone(Mw 118.15 gmol -1 ) and Ammonia(Mw 35.05 gmol -1 ) 30 %
solution was purchased from S igma-Aldrich. 8-Arm Poly (ethylene glycol)- OH ( 8-PEG- OH
Mw 15 kDa) was purchased from Jenke m Technology USA.
Cross-linker ( CL ) , (7.5%) was a dded to the pre -cur ing mi xture to en hance the cross -linking
density and photo initiator ( PI ) was added ( 1-5 %) to achieve the UV -curing. Figure 2.1 and
Figure 2.2 present the AZO monomers and the PEG derivatives used throughtout the thesis.

Figure 2.1: Typ es of Azobenze ne monomers

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Figure 2.2: Struct ure of differe nt PEG derivatives: P oly (ethylene glyco l) di-acrylate, 8-arm
PEG a crylate (8-PEG-Acr) a nd 8-arm PEG V inyl sulfone (8-PEG -VS).The u nit s a t the end of
chain repres ent acrylat e and vinyl sulfon e groups.
2.2 Synthesis
2.2.1 8-PEG Acryla te and Azo Acrylate (8-PEG /AZ-Acr)
8-Arm Poly (ethylene glycol)-acrylate ( 8-PEG-Acr) was s ynthesized by Dr. Zhenfang Zhang,
a fellow in Lensen Lab and the acry lation p rocedur e is explai ned well in his PhD thesis.
Azo monomers (AZ 1 -A cr /AZ 2 -A cr /AZ 3 -A cr ) and 8- PEG-Acr were sy n thesized by follo wing
procedure. The reagents ( AZ - OH / 8-PEG- OH ) an d the catalyst K 2 CO 3 (3 g), were dr ied in a
vacuum oven at 100°C f or 4 hours. A pre -back e d 2 necked r o und bottom flask was fitted in
the vacuum line to avoid the moisture in the reaction. The reagents were added in 50 ml
CH 2 Cl 2 (DCM) under N 2 -atomsphere. Acryloyl chloride (1 mL) was added dropwise in a ice
bath. The mixture was stirred at 50 ° C for 4 days in absence of light to ensure the maximal
conversion. The solu tion was filtered and p oured into the cold p etroleum ether. The
products were filtered and the crude product was dissolved in 50mL of DCM an d extracted
with saturated NaCl solution for 3 times. The or ganic layer was collected. The solution was
dried overnight by MgSO 4 , filtered and finally the flask was p laced on a rotary evaporator
(Heidolph Instruments GmbH & Co. KG, Germany) to remove the solvent . The polymer was

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stored in dark . 1 H NMR analysis showed complete conversion of hydr o xyl-groups into
acrylate. 1 H NMR (400 MHz, CDCl 3 ): (O CH 2 CH 2 O 3.64ppm (1 496H), (C=O) OCH 2 4.31ppm
(16H), =C- H trans 5.83ppm (8H), C H =C 6.15ppm (8H), =C- H cis 6.42ppm (8H).
2.2.2 Sulf onation of 8-PEG-OH (8-PEG – VS)
8-PEG-VS was synthesized by Dr . Zhen fang Zhang, a fellow in Lensen Lab.
8-PEG-VS was synthesized by coupling 8-PEG- OH with an excess of Div inyl sulfone. 8 -PEG -
OH (5g) was dissolved in 300 mL of dry dichloromethane (DCM). S odium hydride (NaH)
was added in 8-PEG- OH solution under nitrogen, at 5-fold molar excess over OH groups.
When hydrogen gas evolved, Divinyl sulfone was added quickly at 50-100-fold molar
excess over OH groups. The reaction was carried out for 3 days at room te mperature under
N 2 atmosphere with const ant sti rring. On com pletion, the reaction solution was ne utralized
with acetic acid, filtered and reduced to a small volume (10mL) by rotary evaporation. 8 -
PEG-VS was precip itated in ice-cold diethyl ether. The polyme r was filtered, washed with
diethyl ether and dried under vacuum. The cru de polymer was then dissolved in 200mL of
deionized water containing 5g of NaCl an d extracted three times with 200mL of DCM. This
solution was dried with Na 2 CO 3 . To end, the product was precipitated and washed many
times with diethyl ether to remove all remaining Divinyl sulfone. F inal product was dried
under vacuum and stor ed under argon at -20 ° C. Conversion was confirmed with 1 H NMR
(CDCl 3 ): 3.6 pp m (PEG b ackbone), 6.1 ppm (d, 1H, dCH 2 ), 6.4 ppm (d, 1H, dCH 2 ), and 6.8
ppm (dd, 1H, -dSO 2 CH).
2.2.3 AZO -PEG Co -G els
The prep aration of AZO-PEG Co -Gels was done at room-temperature by add ing the 5% of
photo- initiator (PI) into the precursor soluti on of AZ 1 -Acr along with 7.5% cross-linker ( CL)
and poly (ethylene glycol) di -acrylate (PEG-5 75) w ith 100% DMF content. Mixture was
vigorous l y stirred ma gneti cally to achieve a viscous liquid. Compositions were set to obtain
0 wt.%, 5 wt.%, 15 wt.%, 25 wt .%, 35 wt.% ,50 wt. %, 75 wt.%, an d 100 wt.%, AZ 1 -Acr to
PEG- 575 by weight. Then, the resulting liquid (50 μL ) was ispensed on a glass sli de and
covered with a cover slip and UV cured (λ = 365 nm Vilber Lourmat GmbH) for 1 -2h using a
working distance of 10 cm, in glovebox. The cured gels were peeled off using tweezers and

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the samples were placed on the clean glass slide. The solvent was ev aporated and further
dried until constant weight.
2.2.4 AZO -PEG NH 3 Co -Gels
The preparati on of AZO-PEG NH 3 Co -Gels w as achie ved by adding the 40% ammonia
solution into the precursor solution of AZ 1 -Acr along with 7.5% cross-linker (CL) and p oly
(ethylene glycol) di -acrylate (PEG-575) with 100% DMF content at room tempe rature.
Solution was magnetically stirred to achieve a v iscous liquid. Compositions were set to
prepare 0 wt.%, 5 wt.%, 15 wt.%, 25 wt.%, 35 wt.% ,50 wt.%, 75 wt.%, and 100 wt.%, AZ -
Ac to PEG by weight. T he resulting liquid ( 50 μL) was deposited on the clean glass slide,
capped with a cover g lass (18 mm × 18 m m; Carl Roth GmbH & Co. KG) and left to gelate.
The required time for gelation was found to be of different for different ratio of AZ 1 -Acr
precursor.
2.2.5 AZO -8-PEG-Acr NH 3 Gels
The preparation of 8-PEG-Acr NH 3 gels was conducted at room temperature. 40% ammonia
solution was added in to the precursor s olution of precursor solution of AZ 1 -Acr along with
8-arm poly (ethylene glycol) ( 8-PEG-Acr) with 100% DMF. Magnet ic stirring was done until
the solution turned into a viscous liquid. Compositions were set to get 0 wt.%, 5 wt.%, 15
wt.%, 25 wt.%, 35 wt.% ,50 wt.%, 75 wt.%, and 100 wt.%, AZ 1 -Acr to PE G b y weight. Then,
the resulting liq uid (50 μ L) was casted on a glass slide and shielded with a glass cover slip.
The required gelation time was found to be of different for different ratios of AZ1-Acr
precursor.
2.2.6 AZO -8-PEG-Acr NH 3 P hysical Gels
The preparation of AZ O-8-PEG NH 3 Physical Gels was accomplished by ad ding the 40 %
ammonia solution into the precursor solution of AZ 1 -OH along with poly (ethylene gl ycol)
acrylate (8-PEG-Acr) wit h 100% DMF solvent at room temperature. Solution was turned
into a v iscous liquid by vigor o us magnetic stirring. Compositions were set to receive 0
wt.%, 5 wt.%, 15 wt.%, 25 wt.%, 35 wt.% ,an d 50 wt.% of AZ 1 -OH to 8-PEG-Acr by weight.
Then, the resulting liquid (50 μL) was d ispen sed on a glass slide an d covered with a glass

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cover slip . After formation gels were p eeled off with tweezers. The samples were p ut on the
clean glass slide. The solvent was evap orated and further dried until gels obtained a
constant weight.
2.2.7 AZO -8-PEG-Acr Co -Gels
The prep aration of AZO-8-PEG Co -Gels was done by add ing the 5% of photo - initiator (PI)
into the precursor solution of AZ 1- Ac r along with 7.5% cross-linker (CL) and 8 -arm poly
(ethylene glycol) acryl ate ( 8-P EG -Acr ) with 100% DMF content at room-tempe rature.
Mixture was vigorously stirred magnetically to achieve a viscous liquid. Compositions were
set to have 0 wt.%, 5 wt.%, 15 wt.%, 25 wt. %, 35 wt.% ,50 wt.%, 75 wt.%, and 100 wt.%,
AZ1-Acr to 8-PEG-Acr by we ight. Then, the resulting liquid ( 50 μL) was casted on a glass
slide and UV cured for 1-2h in a glovebox. The cured gels were pee led off and the samples
were p laced on clean gl ass slide. The solvent was evap orated to acquire constant weight of
gels.
2.2.8 AZO -8-PEG-Acr NH 3 Co -Gels
The preparati on of AZO -8-PEG NH 3 Co Gels was accomplished by addi ng the 40% ammonia
solution into the precursor solution of AZ 1 - Ac r along with 7.5% cross-linker (CL) an d poly
(ethylene glycol) acr ylate (8-PEG-Acr) with 100% DMF content at room temperature under
vigorous magnetic stirring until the solution turned into a viscous liquid. Compositions
were set in order to rec eive 0 wt.%, 5 wt.%, 15 wt.%, 25 wt.%, 35 wt. % , 50 wt.%, 75 wt.%,
and 100 wt. %, AZ1-Acr to 8-PEG-Acr by we ight. The n, the resulting liq uid (50 μL ) was
dropped on a glass slide and protected with a glass cover slip. After t he gelation, gels were
peeled off with tweeze rs. And then the samp les were put on the clean g lass slid e. The
solvent was evaporated and fur ther dried until constant weight was achieved by the gels.
2.2.9 UV st abilized AZO-8-PEG-Acr NH 3 Gels
In order to obtain UV stabilizing AZO -8-PEG NH 3 Co -Gel, 5% of photo- initiator (PI) was
added in same pr ocess of AZO- 8-PEG NH 3 Co -G els formation. After gel formation with NH 3 ,
the gels were exposed to UV light (λ = 365 nm Vilber Lourmat GmbH) for 1.5 min using a
working distance of 10 cm, in a nitrogen -filled glovebox. The cured gel s were peeled off an d

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the samples were placed on clean glass slide. The solvent was evap orated and further dried
until constant weight.
2.2.10 AZO-8-PEG-VS NH 3 Gels
The preparation of AZO-8-PEG-VS NH 3 Gels was achieved by adding the 4 0% ammonia
solution into the precursor solution of AZ 1 - Acr along with poly (ethylene glycol ) vinyl
sulfone (8-PEG-VS) with 100% DMF content at room temperature under vigorous magnetic
stirring until the soluti on turned into a viscou s liquid. Compositions were set to receive 0
wt.%, 5 wt.%, 15 wt.%, 25 wt.%, 35 wt .% ,50 wt.%, 75 wt.%, and 10 0 wt.%, of AZ1-A cr to
8-PEG- VS by weight. The resulting liquid ( 50 μ L) was depo sited on glass slide. The resulting
gel was peeled off and dried to obtain constant weight.
2.2.11 AZO-8-PEG-VS NH 3 Physical Gels
The preparation of AZO-8-PEG-VS NH 3 Physical Gels was accomplish ed by adding the 40%
ammonia solution into the precursor solution of AZ 1 -OH along with poly (ethylene gl ycol)
vinyl sulfone (8 -PEG-VS) with 100% DMF solvent at room temperature. Solution was
turned into a viscous liquid by magnetic stirring. Compositions were set to collect 0 wt.%, 5
wt.%, 15 wt.%, 25 wt.%, 35 wt.% ,and 50 wt.% of AZ 1 -OH to 8-PEG-VS by weight. Then, the
resulting liquid (50 μL) was dispensed on a glass slide and covered with a glass cover slip .
After formation gels were peeled off with tweezers. The samples were put on the clean glass
slide. The solvent was evap orated and fur th er dried until constant weight of gels was
obtained.
2.2.12 AZO-8-PEG-VS Co -Gels
The p reparation of AZO -8-PEG-VS Co -Gels was done by adding the 5% of photo - initiator
(PI) into the precursor solution of AZ - Ac r (AZ 1 -A cr /AZ 2 -A cr / AZ 3 -A cr ) , along with 7.5%
cross-linker (C L) and 8-arm poly (ethylene glycol) vinyl sulfone (8 -PEG-VS) with 100%
DMF was magnetically stirred till the solution turned in to a viscou s liquid. Mixture was
stirred magnetically to achie ve a viscous liquid. Compositions were set in order to receive 0
wt.%, 5 wt.%, 15 wt.%, 25 wt.%, 35 wt.% , 50 wt.%, 75 wt.%, and 100 wt.%, AZ -Acr to 8-
PEG-VS by weight. Then, the resulting liquid (50 μ L) was casted on a glass slide, covered

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with a glass cover slip and UV cured for 1-2h in a nitrogen-filled glovebox. The cured gels
were peeled off with tweezers. And then the samples were put on the clean glass slide. The
solvent was evaporated to obtain constant weight of gels.
2.2.13 AZO-8-PEG-VS NH 3 Co -Gels
The preparati on of AZO-8- PEG-VS NH 3 Co Gels was accomplished by add ing the 40%
ammonia solution into the precursor solution of AZ 1 -Acr along with 7.5% cross-linker (CL)
and poly (ethylene glycol) vinyl sulfone (8-PEG -VS) with 100% D MF was magnetically
stirred till the solution turned into a viscous liquid. Compositions were set to receive 0
wt.%, 5 wt.%, 15 wt.%, 25 wt.%, 35 wt.% ,50 wt.%, 75 wt.%, and 100 wt.%, AZ1-Acr to 8-
PE G- VS by weight. Then, the resulting liquid (50 μL) was dr opped on a glass slide and
protected with a glass cover slip. After the gelation, gels were peeled and the sol vent was
evaporated until constant weight was achieved.
2.2.14 UV stabilized AZO -8-PEG-VS NH 3 Co -Gels
In order to obtain UV s tabilizing AZO-8-PEG-VS NH 3 Co -Gel, 5% of photo - initiator (PI) was
added in same p rocess of AZO -8-PEG-VS NH 3 Co -Gels formation. After gel formation with
NH 3 , the gels were exposed to UV light (λ = 365 nm Vilber Lourmat GmbH) fo r 1.5 min using
in glovebox. The cured gels were put on the clean glass slide. The solvent was evaporated
and further dried until constant weight.
2.3 Analytica l techniq ues
2.3.1 Spectrosco pic analysis
2.3.1.1 FTIR
FTIR spectra were collected on a Perkin Elmer FTIR spec trometer (UATR Two) equipped
with an ATR cell. Measurements were done between“450 -4000 cm -1 ”at room temperature.
2.3.1.2 Raman
Raman Measurements were done with the help of M.Sc. Taravat Saeb Gilani (AG
Woggon/Eichler) at Institute for Optic and Atomic Physic TU Berlin. I nstruments used was

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Ventana 785 Raman spectrometer, Ocean Optics having Wavelength ran ge of 800 -940 nm
and Raman shifts were measured between 20 0 -2000 cm -1 .
2.3.1.3 UV -Vi sible spectroscopy
UV -Visible spectroscopy was analyzed on Car y 4000 UV -Vis spectrophotometer (Agilent
technologies) at operati ng wavelength of 200 - 800 nm. Solutions wer e measured in DMSO
solvent. Gels were measured directly mounted on glass slides.
2.3.1.4 1 H NMR spectroscopy
1 H NMR studies for solution were carried out in in DMSO solvent by using of Bruker 400
MHz Advance II d igital NMR instrument usin g TMS as internal reference.
2.3.1.5 Fluorescence measuremen ts prot ocol
Absorption spectra were obtained with F -4500 FL S pectrophotometer spect rophotometer s
equipped with constant- temperature cell holders. The measurements were done at 25 ˚C.
One-millimeter quartz cells were used in most cases in ord er to avo id self -absorption. The
excitation wavelength of 360 nm was used to see t he fluorescence emission spectrum.
2.3.2 Microscop ic analysis
2.3.2.1 Surface electron microscopy
SEM was performed by Dip l. Phys. Christoph Fahrenson at the ZELM I institute, using a
DSM982 F E-SEM with Gemini optics from ZEISS (1 5 kV ) using an accelerat ion voltage
between 10-2 0kv and a working distance of 12 mm . Instrument is coupled with EDAX
Apollo detector and TE AM software for EDX analysis. Samples were mounted on silica
slides and sputtered wi th carbon p rior to the measurements. An acceleration volt age of 8
keV was ap plied yieldi ng EDX sp ectra with a penet ration depth of ap proximately 2.0 μm
and work ing distance of 14mm.
2.3.2.2 Atomic Force microscopy
An Atomic Force M icroscope (JPK instrume nts, Nano wizard II) was used in or der to
measure the topography of gels with increase in Azo content in PEG.

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2.3.2.3 Topographical Imaging
Imaging was done in inte rmittent contact using silicon nitride cantilevers (PNP TR k ≈ 0 .0 8
N/m f0 ≈ 17 kHz; Nano wor ld Innovative technologies) w ith a chromium -gold coating.
Images were edited with Nano wizard IP Versi on 3.3a (JPK instruments).
2.3.3 Rheology
Rheological measurements were conducted in order to access the gelation time in
dependence of the Azo content. Measureme nts were conducted wit h the help of Sarah
Schatte in Prof. Dr. M. G radzielski lab oratory TU Berlin, using a Gemini 200 HR Rh eometer
(Malvern Instruments), with a 4cm cone and plate, having geometry of 4° cone an gle and
0.15-mm g ap. The rheometer w as used to test gels in thr ee distinct method s: time sw eep,
frequency sweep, an d tempe rature sweep. Oscillatory experiments were performed with
5% constant strain , applying the strain -controlled mode for all measurements. Prior to all
experiments, the linear elastic range of the sam ples was ascertained by an amplitude sweep
with a frequency of 1 Hz. It was observed, when Storage Modulus (G’) (indicating the elastic
property of the system) and the Loss Modulus (G”) (indicating viscous p roperties of the
fluid) were achieving a constant plateau.
2.3.3.1 Gelation time measurement
Gelation time of the gels was determined with Gemini 200 HR Rheometer (Malvern
Instruments), with a 4cm cone and plat e, having geometry of 4° con e angle and 0.15 -mm
gap. All measurements were started at room tempe rature. Around 200 μL of the mixture of
precursor was added on t he plate and a sol vent trap was used throughout the measurement
to avoid loss of sol vent. A ti me sweep involve d a constant applied freq uency over a time
range to record the gelation time at the crossover of G` and G''. Measurements were
repeated at least 3 times for each set of samples.
2.3.3.2 Rheological measurements with frequency and temperature sweep
Frequency sweep mode involves an applied low strain (5%) over a range of frequencies
determining the gel’s viscoelastic properties as a function of freq uency. A 4cm plate was
used and measurements were taken at room t emperature. First, the linear elastic r ange of

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the samples w as determined w ith the help of the amplitude sw eep .This is observed when
Storage Modulus (G’) (i ndicating the elastic property of the network) and the Loss Modulus
(G”) (indicating viscous properties of the fluid) reach a constant platea u. The value obtaine d
was transferred to the frequency sweep, where the suitable values were determined within
a range of 0.01 to 10 H z. Frequency of 1Hz an d 0.01 – 0.1 as deformat ion value (γ) were
chosen as ap propriate parameters for all measured samples. The value of the observed
plateau was record ed and the bulk elasti city was calculated by the following equation as
described by Flory ,
E = 3 G’
Where E is the Young’s Modulus and G’ is the Storage Modulus. Each measurement was
recorded at least 3 time s.
The final test which was conducted on the g el samples was a temperature sweep. This
method in volved a constant strain over a range of increasing t emperature and was used to
determine the effect of temperature increase on Storage Modulus (G’) of the gels. These
tests were run at a constant strain and over a temperature range of 2 5 - 60°C.
2.3.4 Swelling behavior and observation of gel degradation
Swelling be havior of AZO-8-PEG-Acr NH 3 g els was monitored as a part of physiochemical
characterization. For this, at regular time intervals, the swelling ratio (Qm) was calculated
by dividing the weight of the swollen gel (after in cubation at 37˚C in deionized water) by
the ini tia l weight of the gel.

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3 Chapt er 3
Effect of Azobenzene on the Gelation
Beha vior of PEG - Deri v ati v es
In this chapter, we h ave investigated the effect of a zobenzene monomers on gelation
behavior of different PE G-derivatives. For these studies, different azobenzene monomers
synthesized and charac terized. Further, they were subjected to gelation with different PE G
derivatives (PE G-575, 8-PEG-Acr, 8-PE G-VS) using two gelation methods. One of them was
conventional photo-polymerization using a photo - initiator, while the other employ ed
strategy was amine Michael -type addition usi ng ammonia linker, designed by Len sen Lab.
Photo-polymerization failed with Az obenzene and PEG derivatives while the successful
gelation of Azobenzene(AZO) with 8-PEG-Acr, 8-PEG-VS was achieved using amine Michael -
type addition. Possible reasons for the failure and success of gelation were investigated.

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3.1 Introductio n
Chemical modification of a hydrogel with photo -responsive moieties is the most straight-
forward method to obtain a photo-responsive gel. They usually consist of a polymeri c
network an d a photo-reacti ve moiety, typically a chromophore as the functional unit [101] .
The optical signal is first captured by the ph otochromic molecules and converted into a
chemical signal through a photo-reaction such as isomerization, cleavage or dimerization.
The signal is then transferred to the f unctional part of gel an d controls it s properties. The
change of the chromophores upon photo irra diation strongly depen ds on thei r molecular
structures and correspondingly , the req uired irradiation also varies[102] .
Gels based on poly (et hylene glycol) (PEG) are wid ely used because of the renow ned bio -
inertness and biocompatibilit y[1 03] . They also provide the fascinating versatility of PE G -
macromonomer chemistry, facilitate the incorporation of bioche mical units , which
promotes cell adhesion and can control cell behavior[104].
One of the most ap pealing platforms for a reversible, photo -based reaction to control gel
matrix mechanics is the use of a photo-isomer, esp ecially azobenzene[105]. They ha ve
emerged as an effective photo switch for use in biomaterials because they absorb light in a
region that is compa tible with many biological systems (350−550 nm) [105], [106].They can
be isomerized from the E -form ( t rans ) to the Z-for m ( ci s ) b y UV -irradiation and back to the
original form by visible light irradiation or heating [107]. When it is i n cis configuration, it
shows higher polarity than in trans, which can be used to cont rol the hydrophobic
interactions [108], [109].
This phenomenon was already used to const ruct a photo -responsive hydrogel system in
1967[110]. Besides a change in the polarity, a change in conformation of the azobenzene
can induce ste ric hindrance for sta cking or complex formation. Thus Azobenzenes h ave
been used to control properties of polymeric networks, such as the gel − sol transi tion and
the swelling ratio[39], [111], [112].

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One of the main advanta ges of controlling gel mechanics with azobenzene is the lack of
initiator required to complete photo -isomerization. Because this photo-reaction p roceeds
with light alone, no free radicals are created from the ma terial during irradiation.
Furthermore, light is a noninvasive stimulus that allows for changes without altering the
chemical environment of the cells. A zobenzene undergoes a struct ural change, and it is
therefore anti cipated that gel mechanics will b e controlled without affecting the overall
network connectivity. Although extensi ve studies hav e been conducted on azobenzene
based polymers,[113] only limited attention has been pai d to p hot o -responsive polymer
gels[39], presumably because of the dif ficulties associated with their synthesis.
Keeping in view the above ment ioned interests, the main aim of the research was to design
new AZO/ PE G based g els that were expected to possess versatile optical properties. The
idea was the chemical modification of the PEG matrix using a zobenzene monomer to obtain
a chemically crosslinke d AZO/PEG gel. The choice of the gel matrix and the design of
compatible AZO monomers were the crucial steps for achieving the desired g oal.
Thorough literature stu dy had revealed that di- azobenzene monomers are exp ected to have
faster photo-response becau se of the p resenc e of two azobenzene groups. PEG derivatives,
bearing acry late groups are normally crosslin ked with photo- initiator into gel s using UV
light; moreover “ amin e Michael-type addition ” is p racticed to crosslink the acrylate bearing
PEG derivatives.
The first ste p toward the experiment al plan was to synthesize a zobenzene monomer
bearing di -acrylate groups , which could be crosslinked w ith a crylate bearing PEG
derivatives either by photo crosslinking or amin e Michael-type addition reaction. For this
purpose three different type of azobenzene monomers were prepared . Di -acrylate
azobenzene monomers were synthesized and characterized using different spectroscopic
techniques like FTIR, Raman, 1 H NMR and UV-V isible sp ectroscopy.
The d esigned monomers were subjected to gelation with three different PE G d erivatives
(PEG-575, 8-PEG-Acr, 8-PE G-VS) using two different gelation te chniqu es. Several strategies
were employed to improve the gelation method to obtai n tunable gels. The succes sful
gelation technique which give rise to homogenous dispersion of the AZO within the gel

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matrix and p rovide a control o ver the gel properties will be dis cussed. This chapter
presents the characteriz ation of the AZO monomers and the summary of the results
obtained through d iffe rent gelation techniques.
3.2 Mate rial and Met hods
The synthesis and details for the preparation of azobenzene monomers and PEG derivatives
was explai ned in detail in Chapter 2. Characterization techniq ues and op timal measuring
conditions are explained in the relevant section briefly.
3.3 Results and Discussion
3.3.1 Monomer prepa ration
For choosing the correct monomer for our system we prepared three diff erent monomers
namely AZ 1 - Ac r, AZ 2 - Ac r, AZ 3 - Ac r (Figure 2.1). These di -acrylate azobenzene monomers
were prepared from azobenzene diols which were prepared through d ia zotization of
aromatic diamines with three different couplin g agents(phenol, biphenyl, nap hthol) using a
previously explai ned method [114]. The azo benzene diols were then converted into di-
acrylate azobenzene monomers.
The conversion of azo benzene diols to azobenzene di -acrylate is exp lained in Chapter 2
(2.2.1). The physical data of the monomers are shown in Table 3.1.
Table 3.1: Phys ical Prope rties of Azob enzene Monom ers
Azo
Monomer

Mol. W t.
gmol -1

Color

%
Yield

M.P
˚C

AZ 1- Ac r

C 30 H 22 N 4 O 5
Mw=518.16

Yellowish
brown

85

224

AZ 2- Ac r

C 42 H 30 N 4 O 5
Mw=670.22

Yellow

47

244

AZ 3- Ac r

C 38 H 26 N 4 O 5
Mw=618.64

Maroon

70

231

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All the azobenzene di-acr ylate monomers were highly colored owing to the presence of
azobenzene chromophore[115]. These monomers were then characterized using different
spectroscopic techniques such as FTIR, UV-Vis and 1 H NMR.
For the confirm ation of the functional groups, FTI R spectroscopic studies of the samples
were done in powd ere d state. The FTIR data is tabula ted in Table 3.2. The spectra showed
stretching vibration of Azo band (N=N ) between 1562-1586 cm -1 , while the C – H alip hatic
stretching of the acr ylate groups was observed between 2945-2969 cm -1 . Characteristic
bands of C=O stretching vibrations was dis played between 1751-1757 cm -1 . The peaks
between 829-841 cm - 1 were assigned to the b ending vibration of C -H in the phenyl rings
[116] . Figure 3.1 depicts clear conversion evidence from d iol to acryla te group.
Table 3.2 : FTIR (cm - 1) Analysis for Azob enzene Monomers
Band
assignmen t

-N = N -

C=O

Aromatic
C- H

Aliphatic
C- H

Phenyl
C-H bend

AZ 1- Ac r

1562

1756

3040

2969

829

AZ 2- Ac r

1586

1751

3053

2945

841

AZ 3- Ac r

1574

1757

3017

2958

833

Figure 3.1: Compar ison of FTIR spec tra of a zobenzene diol to di-acryla te

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UV -Visible spectroscopic analysis of the azobenzene monomers was done in DMSO solvent
at operating wavelength of 200-800 nm. These studies w ere done in order to evaluate the
absorption behavior of azobenzene monomers.
Dilute solutions of 10 -6 M concentration wer e prepared for the measurement becaus e
azobenzene monomers solution was highly c olored.
Table 3.3 : UV-Visible Anal ysis of the Azobe nzene Monom ers
Azobenzene
Monomer

AZ 1- Ac r

AZ 2- Ac r

AZ 3- Ac r

 max (nm)

355

369

401

The  m ax values of azobenzene monomers are summarized in Table 3.1. The azobenzene
monomers showe d the ir first and second ab sorption bands at λ = 280 – 420 and 404 – 630
nm, respectively. Broad ab sorption around 350 nm mainly due to t he tran s form of the
azobenzene while the less energetic 404-630 is because of cis form of the azobenzene.

Figure 3.2: Co mparison of U V-Vis spectra of a zobenzene diol to di-ac rylate

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Figure 3.2 shows the comparison UV Visible spectra of monomer’s initial precursor to the
final product. The two absorption bands of azobenzene monomers are attributed to
electronic excitations taking place within the molecule. λ max between 280 – 420 nm is
assigned to π - π* electroni c transitions while the b and between 404 – 630 nm arise because
of n- π* transitions [117].
Fo r further structural analysis, 1 H NM R study of the monomers was also carried out usi ng
DMSO as a solvent (Table 3.1). In this study, disappearance of the signals for the OH group
between 9.55-10.1 ppm and ap pearance of signal for aliph atic C – H between 5.64-6.22 p pm
was observed showing conversion of diols in to the azobenzene di -acrylate[118]. The
mutiplets in the spectra confirm ed the presence of aromatic groups in the compound.

Table 3. 4 : 1 H NMR data of Azob enzene Monome rs (DMSO -d6 ) in  (ppm)
Azo
Monomer

AZ 1- Ac r

AZ 2- Ac r

AZ 3- Ac r

Aromatic-H

7.32-8.01(m)

7.40-8.15(m)

7.30-8.03(m)

Ethylene- Ha (CH)

5.90-5.97(1H,d)

5.93-5.99(1H, d )

5.89-5.95(1H,d)

Methylene- Hb (CH 2 )

5.64-5.69(1H, d )

5.66-5.70(1H, d )

5.61-5.66(1H, d )

Methylene- Hc ( CH 2 )

6.19-6.22(1H, d )

6.15-6.21 (1 H, d)

6.14-6.17(1H, d )

The ab sorptions manifest in the FTIR and 1 H NMR spectra are in full agreement with the
expected constitution of the synthesized products, which indicates that all the three
compounds were sy nth esized successfully.
The azobenzene monomers p repared had different aromatic chain length attached to
azobenzene unit; each had a different effect on the conversion yield and phys ical propertie s
as shown in Table 3.5. Monomer AZ 1 -Acr was p repared in good yield (85%), w hile the rest

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of two monomers could be synthesized in compara ble yield. These monomers were
processed further for g elation studies with PEG deriv atives.
Table 3. 5: Monomers interactio n with PEG
Azo
Monomer

AZ 1- Ac r

AZ 2- Ac r

AZ 3- Ac r

Conversion
Yield(%)

85

47

70

Observation

Good yield and
homogenous

Poor Yield and
Viscous

Intense color and
viscous

AZ 1 -Acr was able to dev elop a homogeneous p hase with PE G derivatives while the rest of
two monomers were hard to process to get smooth gels b ecause of formation of viscous
phase. This b ehavior of the azobenzene monomers could b e attributed to the bulky
biphenyl and naphthol groups attached to azobenzene groups.
Owing to the better yield and processability of AZ 1 - Ac r with PEG deri vatives, this monomer
will be subjected for the further stud ies to make gels.
3.3.2 Gela tion behavior of different PEG derivate
In order to eval uate the feasibility of AZO for gel formation with Pol y (ethylene) glycol, a
systematic experimental plan was outlined. After the successful synthesis of desired
azobenzene monomers (Figure 2.1), they were subjected to gelation with PE G derivatives
(Figure 2.2 ) using different techniques an d str ategies.
Gelation studies were done in DMF solvent at room temperature. Othe r solvents were tried
but the AZO monomer was not completely dissolved in solvents lik e H 2 O, et hanol and
acetone. The weight ratios kept between solvent and reage nts was 1:1. Different weight
ratios were chosen to study the effect of monomer on gelation behavior. Different gelation
techniques were used to achieve the AZO-PEG gels.

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The detail of the gels formation is briefly explained in Chapter 2 ( 2.2). Gelation behavior
was monitored against three PEG derivatives shown in Figure 2. 2 . We started with simpler
linear PEG-575 and then 8-PEG-Acr and 8-PEG-VS were also studied .
i. PEG - 575
The first simpler PEG d erivative used was the poly (ethylene glycol) d i -acrylate (PE G-575).
PEG-575 is a linear PEG di -acrylate with low molecular weight. The synthesized azobenzene
monomers possess di- acrylate groups as well. This PEG derivative is normally photo
crosslinked into a gel using photo- initiator.
Initially, azobenzene monomer was subj ected to gelation with PEG -575 using photo-
crosslinking method. T he results are tabulated in Table 3. 6. The detailed conditions and
procedure for t he formation of these gels is well explained in Chapter 2. The neat PEG 575
can form the hydrog el with 30 mins exposur e to UV light using 1% photo- initiator [119] .
Table 3.6 : Gelation of PEG-575 with different content of Azobenz ene monomer: Gel s
crossl inked by Am ine Michael-type addition (NH 3 40%) and by UV-curing PI (5%). Gelatio n
(+), partial gelat ion (o) and no gelation (- ).
Content of
AZ 1 -Acr in
PEG-5 75(%)

NH 3 (40%)

PI (5%)

0%CL

7.5%CL

0%CL

7.5%CL

0

+

+

+

+

5

-

+

-

+

15

-

o

-

o

25

-

-

-

-

35

-

-

-

-

50

-

-

-

-

75

-

-

-

-

100

-

-

-

-

Photo-polymerization of PE G 575 with azobenz ene monomers was started from 1% photo -
initiator. The polymeriz ation failed even when the exposure time w as increased up to 2
hours and photo- initiator (PI) ratio was increased up to 5%. In order to maximiz e the

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chance of crosslinking, 7.5% Cross linker (CL) was used. The 7.5% CL could onl y
accommodate the gel structure up to 5% weight ratio of azo monomer.
The second strategy for the gelation of PEG 575 used was the amine Michael -type addition
method to make the AZO-PE G NH 3 Co -Gels. This methods was designed in the Lensen Lab
( LL ) and was well studied [120]. This strategy was applied with 7.5% CL with 40%
ammonia ratio but again it could not accommodate azobenzene monomer mor e than 5%.
ii. 8-PEG - Ac r
The star shaped PEG de rivatives are very versa tile , because they offer high er number of end
groups per molecule that allow interconnectivity and functionalization . After the failure of
gelation with L inear PE G, the star shaped 8-PE G-Acr was subjected to gelation with
azobenzene monomers as it can offer more connectivity.
Table 3.7: Gelation of 8-PEG-Arc with different content of Azobenzene monomer: Gels
crossl inked by Am ine Michael-type addition (NH 3 40%) and by UV-curing PI (5%). Gelatio n
(+), partial gelat ion (o) and no gelation (- ).
Content of
AZ 1 -Acr in
8- PEG -Acr (%)

NH 3 (4 0%)

PI (5%)

0%CL

7.5%CL

0%CL

7.5%CL

0

+

+

+

+

5

+

+

+

+

15

+

+

O

+

25

+

+

-

o

35

+

+

-

-

50

+

+

-

-

75

-

-

-

-

100

-

-

-

-

Both p hoto-polymerization as well as amine Michae l-type addition method was used. AZO -
8-PEG Co-Gels were prepared by using the photo-polymerization crosslinking method. The
ratios of PI an d CL were kept at 5% an d 7.5%, respectively. By using the photo -
polymerization, the 8-P EG-Acr can accommodate 5% azobenzene mon omer while using the

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CL the percentage increase up to 15 % b y weig ht. Lat er on, the amin e Michael -type addition
method was employed t o prepare AZO-8-PEG-Acr NH 3 Gels.
The gelation was successful even without CL. 8-PEG-Acr could accommodate up to 50% by
weight ratio of azobenzene monomers as shown in Table 3.7. When we increased the ratio
further, it could accommodate 57% of the azoben zene but later on adding mo re AZO, the
phase separation between AZO and PEG derivative was observ ed.
iii. 8-PEG- VS
8-PEG-VS have reactive Sulfone functionality and enhance d connectivity. In or der to
compare the reactivity of 8 -PEG-VS with 8-PE G-Acr, the former was also subj ected to
gelation using the same strategies. Differe nt AZO: 8-PEG-VS ratios were employed to
completely get an in sight of how much AZO could b e accommodated in 8 -PEG-VS matrix.
The results of g elati on of 8-PEG-VS are quite simila r to 8-PEG-Acr; the only difference is that
the gelation was achieved earlier. The results are show n in Table 3.8.
Table 3.8 : Gelation of 8-PEG-VS with different content of Azoben zene monomer: Gels
crossl inked by Am ine Michael-type addition (NH 3 40%) and by UV -curing PI ( 5% ). Gelatio n
(+), partial gelat ion (o) and no gelation (- ).
Content of
AZ 1 -Acr in
8-PEG -VS (%)

NH 3 (40%)

PI (5%)

0%CL

7.5%CL

0%CL

7.5%CL

0

+

+

+

+

5

+

+

+

+

15

+

+

o

+

25

+

+

-

o

35

+

+

-

-

50

+

+

-

-

75

-

-

-

-

100

-

-

-

-

The PEG derivatives were subjected to gelation and sho wed different behavior.

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Photo polymerization of azobenzene di-acrylate monomer failed in linear PEG because of
two possible reasons. The first possible reason could be that PEG 575 is a line ar molecule
and azobenzene di-ac rylate monomers are also having smaller chain so the direct
polymerization of these molecules is difficult t hrough photo-pol ymerization. The proposed
mechanism is explained in Figure 3.3. For instance, the line ar molecule does not provide
sufficient connectivity to hold the gel matrix to gether which result in partial or no gelation.
As we add the CL, the p robability for connectivity increase which can l ead to better gelation.
But again the photo-polymerization failed with 8-PEG-Acr and 8-PEG-VS where one can
observe sufficient connectivity.
The second p ossible and more convincing reason for the failure of photo -polymerization
method is that the azob enzene monomers themselves absorb in the UV region as shown in
Figure 3.2 and undergoes photo -isomerization which create kind of blanket on the g el
matrix, hence doesn’t allow the sufficient radical formation for the gelation and fails the
polymerization .
Amine Michael-type addition method is well practiced w ith 8-PEG-Acr and 8-PEG-VS gels
and it also successfully formed the gels with az obenzene monomers up to 50% weight ratio.
The mechanism of gelation is well explained by Dr. Zhenfang Zhang [120]. The same
mechanism worked well with azobenzene monomers as they also have acrylate functional
groups. The acrylate end -gr oups of 8-PEG and azobenzene monomers bin d together
through ammonia by using amine Michael-type addition method as proposed in Figure 3.3.

Figure 3.3: P roposed mechanism o f gelatio n in AZO-8 -PEG- Ac r NH 3 Gels

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In order to confirm the chemical bonding of AZO with PE G derivatives, the AZO-8-PEG-Acr
NH 3 physical gels were also prepared and compared with AZO-8-PEG- Ac r NH 3 Gels. A
marked difference in th e texture of the gels was observed. The AZO-8- PEG - Ac r NH 3 gels had
a better elasticity as compared to the physical gels.
3.4 Conclusio ns
Di -acrylate based azobenzene monomers ( AZO ) were synthesized an d their suitab ility for
incorporation in different PEG derivatives was monitored.
Three selected AZO monomers were s ynt hesized. The structur al elucidation using
characterization techniques confirmed the successf ul synthesis. AZO monomer with less
steric hindr ance (AZ 1 - Ac r) was found to be pre pared with ease in g ood yield and have good
processability with PEG derivative s while th e sterically hindered bip henyl (AZ 2 - Ac r) and
naphthalol (AZ 3 - Ac r) based AZO had poor yield and reduced processabilit y with PEG
derivatives. Therefore ow ing to better processab ility of AZ 1 -Ac, it w as selected for further
gelation studies with PEG derivatives (PEG-575, 8-PEG-Acr and 8-PEG-VS).
After the successf ul syn thesis of desired a zobenzene monomers ( AZO ), they were subjected
to gelation with PEG derivatives using different techniques an d strategies. First simpler PEG
derivative used was the po ly (ethylene glycol) di-acrylate ( PEG- 575 ). This PEG derivative is
normally photo-crosslinked in to a gel usin g photo - initiator. So initially, azobenz ene
monomer was subjected to gelati on with PE G -575 using photo crosslinking method but it
did not work even when the amount of photo- initiator was increased and the cross linker
( CL ) was added to enhance the crosslinking density.
Failure of photo crosslinking with PEG -575 give rise to an idea that may be p hoto -
crosslinking of short chai ned PE G -575 is difficult with AZO so the long chaine d 8-PEG-Acr
and 8-PEG-VS were introduced with the same experimental conditio ns. Nevertheless, the
results were not so promising and even with the addition of cross linker (CL) the
mechanical strength of the gel was poor also it c ould not accommodate AZO more than 5%.
Now, there was a nee d to find the logical reason for the failure of photo -crosslinking

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method with AZO/PEG gels and to find a better alternat ive method to successfully gelate
the AZO and PEG derivatives.
Different gelation techniques with varying p recursor r atio were ap plied. UV curing was
found not to work with all of three tested PE G derivatives even with high ratio of PI an d CL.
At this p oint, “ amine Michael-type addition” method using ammonia linker (d esigned by a
coworker in Lensen Lab ( LL ) Dr. Zhenfang Zhang) was ap plied to crosslink the AZO and
PEG derivatives. This method was employed to obtain gels with PEG -5 75, 8-PEG-Acr an d 8 -
PEG-VS.
Am ine Michael-type addition applied as second alternative did not work well with PE G - 575
but 8-PEG-Acr and 8-PEG-VS made excellent gel s with using this technique. These gels were
prepared with varying AZO: PEG ratio and could accom modate up to 50% wt. ratio of both
precursors.
So the main target of the experimental plan was achiev ed. We successfully synthesized
Novel chemically cr ossl inked A ZO/PEG ( AZO -8-PEG-Acr NH 3 ) g els using “ amine Michael-
type addition”.

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4 Chapt er 4
Design, S ynthesis and Char act e rization of
Chemicall y Cr ossli nk e d AZ O/PEG Gels
As mentione d in chapter 3, the gelation of the azobenzene monomers was achie ved with
8-PEG-Acr/V S using amine Michael-type addition method. In this chapter, the
characterization of chemically crosslinke d AZO/PE G ( AZO-8-PEG- Ac r NH 3 ) gels using
different techniques will be exp lained in detail. Structural studies were carried out using
FTIR, Raman, and UV -Visible spectroscopic studies. Rheological measurements were
performed to evaluate the gelation ti me and mechanical strength of gels using time,
frequency and temperature sweep. Surface characterizations of AZO-8-PEG- Ac r NH 3 gels
were done using atomic force (AFM) and sur face ele ctron microscopy (SEM).

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4.1 Introductio n
Photo-responsive materials have attracted considerable attention because of their potential
us e in various optical applications, such as nonlinear optics, erasab le memory storage and
processing and electro optical displays [121]. P hoto-responsive gels ha ve been of increasing
interest because of rev ersible; photo induced physical and chemical p roperties that can b e
transferred to the micro-environment by a photochromic molecule incorporated in such
systems. For this reason, numerous efforts have been devoted to polymeric gels an d/or
hydr o gels [122]. Their swelling and shrinking behavior has many potential uses in
applications such as controlled release of d rugs, separations, and construction of
actuators[123], [124].
Azobenzenes are the most commonly us ed photochromic unit for photo induced
transitions. A lthough extensive studies have been conduc ted on azobenzene based
polymers, only limited atte ntion has bee n paid to photo-r espo nsive polymer gels,
presumably b ecause of the difficulties associated with their synthesis. More often,
Azobenzenes are used in supramolecular assemblies to trigger reversible environmental
changes in a wide variety of hydrogels [125][126]. But few studies are available on the
synthesis of chemically crosslinked azo ben zene base d poly (ethylene) glycol g els [ 127] .
As explained in chapter 2, AZO -8-PEG-Acr/VS NH 3 gels were synthesized using “ amine
Michael-type add ition” method. The AZO- 8-PEG- Ac r NH 3 gels show e d a smooth morphology
and flexible texture, while the AZO-8-PEG-VS NH 3 gels showed rigid and stiffer morphology.
As we aimed to design the responsive materials, stiffer gels because of higher crosslinking
density may not respond q uickly to the external stimulus. AZO-8-P EG-VS NH 3 g els being
stiffer don’t seem to be a promising candidate for our desi red project. Keeping in mind the
softer flexible morphology of AZO-8-PEG- Ac r NH 3 gels, w e decided t o continue w ith them
for further stud ies.

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The next important step was the structural an d physicochemical anal ysis of the AZO-8-PEG-
Acr NH 3 gels for the co mplete investigation of the gels. In order to insp ect the gel network
and morphology, we applied several techniques.
4.2 Mate rial and Met hods
In this chapter, the br ief characterization of AZO-8-PEG-Acr NH 3 gels is presented. The
synthetic method of the respective gels was explained in chapte r 2.
4.3 Analytical techniq ues
Several an alytical techniq ues were employed to characterize the AZO-8-PEG- Ac r NH 3 gels.
The details can be found in chapter 2.
4.4 Results and discussion
4.4.1 Spectr oscopic characterization
In order to perform the structural studies of AZO-8-PEG- Ac r NH 3 gels characterization was
carried out by using FTIR, Raman, and UV -Visible spectroscopy.

Figure 4.1: FTI R spectra o f AZO-8-PEG- Ac r NH 3 gels
The results of FTI R an d Raman analysis are shown in Figure 4.1 and Figur e 4.2.

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FTIR and Raman spectral data of synthesi zed gels with differe nt weight ratios of
Azobenzene monomer was record ed to co nfirm the chemical binding of azobenzene
monomers into the PE G matrix. In case of successful incorporati on of the AZO in th e PEG
matrix, a small peak is expected around 158 0 cm -1 [116].

Figure 4.2: Rama n spectr a of AZO-8-PEG -Acr NH 3 gels
It is evident in both the analysis that the increase in the % ratio of azobenzene monomers
causes an increase in the intensity of N=N (Azo) p eak, an d aromatic/ aliphatic CH stretch.
Appearance of the azo peak around 1585 cm - 1 confirms the successful incorpor ation of the
azo functionalities into the PEG matrix. Raman studies were performed to complement the
FTIR studies. The anticipated azo peaks (N=N) were detected as shown in Figure 4.2 .
The azo band showed a slight shift from the FTIR analysis. It was o bserved at 1440 cm - 1
[128]. It can be eluci dated from the Figu re 4. 2 that increase in the AZO % increase the
Raman intensity f or the azo (N =N) group. Likewise, it is clear that not all the acr ylat e
groups from the AZO and 8-PEG-Acr a re utilized for making the hydrogel ne twork. The

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concentration of the unreacted acrylate group have shown an increase with in crease in AZO
%, which means the unreacted AZO in the ge l matrix is also present. Both IR and Raman
studies confirm the incorporation of azo ban d into PEG matrix. Noticeable fluorescence was
observed during the Raman measurements.
Fluorescence and photo-isomeriz ation both are d isplayed in photo excited state. Individual
azobenzene units are less fluorescent b ecause the princip le photo phenomenon i n them is
the isomerization which suppresses the fluorescence. This ph enomenon becomes
prominent an d pronounced when azobenzene unit is incorporated into the gel matrix [26],
[42].The possible reason for this change is that the photo -isomerization becomes
suppressed w hen the az obenzene unit is chemically crosslinked in the long chains of star
shaped PEG uni ts. The crosslinked ne twork does not allow the structural conformation in
the azobenzene unit that’s why fluorescence becomes the major photo active p henomena
[129].

Figure 4.3: Fluor escenc e emission spectra o f monomers a nd AZO-8-P EG- Ac r NH 3 gels

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To prove this principle, fluorescence emission sp ectroscopy was done with the monomer
and AZO-8-PEG-Acr N H 3 gels. The results a re shown in Figure 4.3 . The fluo rescence
emission spectrum of AZO monomer showed no fluorescence as the unbound AZ O mainly
display the photo-isomeriz ation, which ov errules the fluor escence emission. The pure 8-
PEG-Acr showed so me fluorescence but the emission pat tern is not clear . As we kept on
increasing the AZO % in the P EG matrix, the f luorescence became m ore pronounced. This
can be observed from the emission spectrum of 25% AZO-8-PE G-Acr NH 3 gels. When the
concentration is further increased up to 50% AZO: 8 -PEG-Acr r atio the fluorescence
becomes the most prominent phenomena as could be seen in the Figu re 4.3 with 50% AZO-
8-PEG-Acr NH 3 gel.
In order to observe the absorption behavior of AZO-8-PEG- Ac r NH 3 gels UV visible spectral
analysis was performed in the dried state. The results are summarized in Figur e 4.4. Pure
PEG does not show any ab sorption pattern in visible region due t o the abse nce of an y
photo-chrome. Addition of azobenzene moiety into the PEG matrix introduces the
photochromic unit ; lea ds to absorption in UV and visible region. UV -Visible sp ectra o f
synthesized gels with different weight ratios of Azobenzene mono mer and 8-PEG-Acr
showing broad enin g of absorption peak with increase in AZO content.

Figure 4.4: UV -Visible spectra of AZO -8-PEG-Acr NH 3 gels

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The two prominent absorption bands of azobenzene monomer observ ed at 355nm and
472nm are overlapped in gel matrix. 5% AZO-8-PEG-Acr NH 3 gels exhibited the distinct π -
π*(388nm) and n - π* (45 0nm) electronic ex citations which could be seen from the U V -
Vi sible sp ectrum of the corresponding gel [49]. As the AZO percentage was in creased a
marked chan ge in the absorption behavior was observ ed. The pea ks become broader and
50% AZO-8-PEG- Ac r NH 3 gel show ed the maxi mum broadening.
The possible reason for the broadening of ab sorption spectrum could be that amine
Michael-type addition provides various possibilit ies of crosslinking using ammonia linker
as sho wn in Figure 3. 3. As the concentrati on of azobenzene monomers increases, the
possible combinations of cr osslinking between P EG and azobenzene unit also increase.
Presence of several different type of AZO -PEG crosslinked units give different absorption
peak, which overlap to give broad absorption band as shown in F igure 4.4. This fact was
also strengthened by the photo-isomerization studies.
Photo-isomerization studies were done by exposing the gels to alternating UV an d visible
light exposures and measuring their absorption behavior. The azobenzenes switch from
trans to cis confo rmation upon exposure to UV light and reverse t he conformation upon
exposure to the visible light. The AZO-8-PEG- Ac r NH 3 gel does not ex hibit clear maximum
absorption pea k, rather they show a b roadening of the band due to several combinations of
azo an d PE G crosslinking so no p rominent switching could be obse rved in the gels with
higher AZ O concentration. The second reason for an unclear photo -is omerization behavior
in AZO-8-PEG- Ac r NH 3 gels could be the bondi ng of azobenzene units with PEG chains lim it
the freedom of mo vement in them which is necessary for the p hoto -isomerization. This
phenomenon is supported fr om the fl uorescence emission spect roscopy of the AZO-8-PEG-
Ac r NH 3 gels where the increase of AZO % in creases the emission behavior of the gels as
shown in Figure 4 . 3. Isom erization is the main photo phenomenon in the 5% AZO-8-PEG-
Ac r NH 3 gels in the excited state so it displays a clear isomerization.

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Figure 4.5: P hoto-isomerization ob served in 5% AZO-8-PEG- Ac r NH 3 gel
As it can be seen from the Figure 4.4 , π - π* and n - π* electronic transitions are only
prominent in 5 % AZO-8- PEG- Ac r NH 3 gel, so this gel showed clear p hoto-isomerization
behavior. Alternate UV/Visible light generated the photo -isomerization phenomenon in 5%
AZO-8-PEG- Ac r NH 3 gel as shown in Figure 4.5. The tra ns azobenzene was converted to cis
isomer with 10 min exp osure to UV light and this phenomena was reversible.
4.4.2 Mech anical Characterization of AZO -8-PEG- Ac r NH 3 gels
Rheological measurements of AZO-8-PEG-Acr NH 3 gels were carried out to determine the
gelation time and mec hanical properties. Time, frequency and temperature Sweep modes
were used to measure in situ gelation time and the mechanical strength of gels respectively.
Gelation times was determined by measuring the storage (G’) and loss modulus (G’’ ) in the
course of 250 mins. The storage modulus (G’) refers to the elastic while the loss modulu s
(G”) refers to the viscous propertie s of the gel network. Both the sto rage and loss moduli
increased w ith time, a pprov ing that the g elation network is progressively stabilized b y
chemical crosslinking. I n the rheological curves, the gel point (crossover of storage modules
and loss modules) was used to determine the gelati on time.

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Figure 4.6: Tim e sweep o f in situ gel formation of AZO-8-PEG- Ac r NH 3 gels
Moreover, in the plateau region after the cr ossover p oint, the storage modulus became
higher than the loss mo dulus. Table 4.1 enlists the determined gelati on ti mes of the various
investigated AZO-8-PEG- Ac r NH 3 gels. These studies revealed d if ferent gelation time
depending on the concentration of azobenzene unit into PEG matrix.

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Table 4.1 : Gelation time studies of AZO -8-PEG- Ac r NH 3 gels
Content of AZ 1 - Ac r
in 8-PEG-Acr (%)

0%

5%

15

25

35

50

100

Gelation time
(mins)

79 ± 0.7

44 ± 1.2

59 ± 1.5

82 ± 2.2

94 ± 3.5

113 ±5.5

-

5% AZO-8 -PEG- Ac r NH 3 gel showed the shortest gelation time while the 50% AZO-8-PEG-
Acr NH 3 gel showed the longest gelation time. The gelation times were dramatically affected
by the added azo mono mers (AZO) %. Greate r was the concentrati on of AZO, more was the
gelation time, p robably beca use in creased amount of AZO in PE G matrix delays the gelation
as compared to the pure PEG.
Frequency sweeps mode characterizations of AZO-8-PEG-Acr NH 3 gels with different weight
ratios of AZO and PEG was carried out to determine the b ulk elasticity. These
measurements provide information about the mechanical strength of the gels. The data
obtai ned for all gels was characterized by G’ exhibiting an almost consta nt value in the
lower frequency range (0.10-10 Hz) with a slight increase when applied the higher
frequencies. The reason for this observed in crease in modulus with increasing frequency
ca n be attributed to the partial fluidic viscoelastic nature of the gels. The increased
frequency allows less time for polymer r elaxat ion and thus the incomplete relaxation may
lead to a pre- stressed state of the gels with a corresponding hig her You ng’s Modu lus.
The storage modulus is proportional to the gel crosslinking density .M ore is the
concentration of aromatic azobenzene units in the gel matrix, the lower will be the
crosslinking density. This leads to more flui dic nature of the gels resulting in a mo re
pronounced deviation from the i deal b ehavior, as observed in Figure 4.7 (G’’ not shown fo r
the sake of clarity).
The larger amount of AZO added le ads to lower storage moduli; the G’ values of 5% AZO-8-
PEG-Acr NH 3 gel are more than 10 times than that of 50% AZO-8-PEG-Acr NH 3 gel ,
suggesting that the 50% AZO-8-PEG-Acr NH 3 gel has quite a low crosslinking density and
hence the most softest gel.

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Figure 4.7: Fr equency sweep of in situ gel fo rmation of AZO-8-PEG -Acr NH 3 gels

Figure 4.8: Te mp sweep o f in situ gel formation of 0 and 5 0% AZO-8-PEG-Acr NH 3 gels

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Similarly, this behavior is ap proved from the Raman results of 50% A ZO-8-PEG-Acr NH 3 gel
as shown in Figure 4.2 where most of the acrylate groups are shown unreacte d.
Temperature sweep mod e of rheological characterization of AZO-8-PEG-Acr NH 3 gels with
0% and 50% AZO content was done to observe the effect of te mperature on storage
modulus shown in F igure 4.8. Temperature ramp was done from 25- 60˚C with a frequency
range of 0.1-1 0Hz. The 0% AZO-8-PEG-Acr NH 3 gels which is pure 8-PEG-Acr NH 3 gel
possess highest storage modulus among the gels. This gel is when subj ected to tempe rature
ramp from 25- 60˚C, showed a gradual decrease in elasticity until the melting is achie ved.
Decrease in storage modulus was obse rved with increase in temperature. Likewise, the
50% AZO-8-PEG-Acr NH 3 was subjected to temperature ramp at the same temperature
range. The same behavior was observed in this case. The elasticity decreased with increase
in te mperature. The reason for this change is attributed to the increase in temperature
increase the degree of freedom of the pol ymer chains and hence the gel becomes softer.
4.4.3 Surfa ce morphology of AZO -8-PEG-Acr NH 3 gels
In order to investigate the surface morphology of the AZO-8-PEG-Acr NH 3 gels surface
electron microscopy (SEM) and atomic force microsco py (AFM) were conducted.
Surface electron microscopy of the AZO-8-PE G-Acr NH 3 gels was conducted at room
temperature an d results are shown in Figure 4.9. In case of 0% AZO-8- PE G-Acr NH 3 gels
which have 0% of AZO monomer and only possess p ure PEG in the gel matrix, regular
spherulites, ranging from around 40 -1 00 µm with radial st ripes were detected; presentin g
that 0% AZO-8-PEG-Acr NH 3 gels possess crystallinity. Moreover, patterns propagated from
the center, appear ed primarily in diagonal d ire ctions.
However, on 5% AZO- 8-PEG-Acr NH 3 gels showed smaller spherulites ranging from 4 µm to
10 µm. For 5% AZO-8-PEG -Acr NH 3 gels spheru lites structures with clear boundaries can b e
observed, but the sp herulites surfaces are smal ler than the those of 0 % AZO-8-PEG-Acr NH 3
and many dots distribute on the surface. There was observed marked decrease in
crystallinity when the ratio of AZO was further increased as in case of 25% AZO-8-PEG-Acr
NH 3 gels (Figure 4.9) no microscale structure of crystalline can be found via SEM, indicating
only nano-scale crystals are formed and there is obs erved more wrinkling on the surface

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because of less cr ystal linity. SEM imagining of the AZO-8-PEG-Acr NH 3 gels was quite
difficult because the AZO chrom ophore itself absorb radiations and the gels surface was
getting destroyed due to breakdown of pol ymer c hains. The SEM images of the gels with
higher AZO content (more than 25%) w ere n ot achie ved due to this reason.

Figure 4.9: SEM -ima ges of 0- 25% AZO-8-PEG -Acr NH 3 gels
AFM is a good techniqu e that provides structural d etails of crystallization at a considerably
higher resolution than optical and electron microsco py. In order to verify the observations
made b y S EM, atomic force micr oscopy (AFM) was used. Imaging was done at ambien t
conditions of a d ried AZO-8-PEG-Acr NH 3 gels film by using intermittent contact mode. The
SEM results coincide with the appear ance of AZO-8-PEG-Acr NH 3 gel films under AFM.
Figure 4. 10 displays the AFM results of t he AZO-8-PEG-Acr NH 3 with 0 and 25%
respectively. Both phase and height images display the difference of the crystal structure
among the two kinds of gels.

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Figure 4. 10 : AFM-heig ht profile (right) a nd 3D image (le ft) of 0% AZO-8 -PEG-Acr NH3gels

Figure 4. 11 : AFM-heig ht profile (left ) and 3D im age (right) of 2 5% AZO-8-PEG-Acr NH 3 gels

Figure 4. 10 show that 0%AZO-8-PEG NH 3 gel forms large and regular spherulites. In
comparison, the crystals formed in 25%AZO-8-PE G NH 3 gel are small, irregular , and possess
more defects, as show n in Figure 4. 11 . Moreover, compared with Figure 4. 10 , we can see
that the surface top ography of 25%AZO-8-PEG NH 3 gel varies greater in F igure 4. 11 which
is caused b y the increase in flexibility of polymer chains with increase in AZO content. This
results in smooth morphology of the gel surface due to addition of smaller units, which

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break the stiffer PEG network. Both AFM and SEM images of s ynthesized gel showing the
increase of N=N (AZO) unit decrease the crystallinity in gel surface.
The surface roughness of the gels d ecreased with increase in the AZO%. In case of 0% AZO-
8-PEG-Acr NH 3 gel 50nm roughness was calculated while in case of 25% AZO- 8-PEG-Acr
NH 3 gel, this value dropped to 35nm.
4.4.4 Swelling Ratio and degra dation studies of AZO-8-PEG-Acr NH 3 gels
Swelling behavior of AZO-8-PEG-Acr NH 3 gels was investigated as a part of the
physicochemical characterizations. T he swelling ratio (Qm) was d eter mined by d ividing the
weight of the swollen gel (after incubation at 37 ˚C in deionized water) to the ini tia l weight
of the gel at regular time interval.
The swelling tests ( Figure 4 . 12 ) sho wed that the sof test gels i.e. those w ith the highest
amount of AZO added; 50% AZO-8-PEG-Acr N H 3 swelled twice as much as the other gels.
This also leads to a quicker d eg radation.

Figure 4. 12 : Swell ing Ratio of AZO-8-PEG -Acr NH 3 gels
The 35% AZO-8-PEG-Acr NH 3 showed the same behavior. Actually, these loosely
crosslinked gels had disinte grated after 3 h. As a result, could not be studied any further in
the swelling tests. The other AZO-8-PEG-Acr NH 3 gels were stab le for at least 24 h .
Subsequently, 25% AZ O-8-PEG-Acr NH 3 , 15% AZO-8 -PEG-Acr NH 3 and 5% AZO-8-PEG-Acr
0 200 400 600 800 1000 1200 1400 1600
2
4
6
8
10
12
14
16
18
20
Swelling ratio
Time(mins)
0%
5%
15%
25%
35%
50%

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NH 3 gels degr aded ; after 2 days, 3d ays and 5days respec tively, while and 0% AZO-8-PEG-
Acr NH 3 gels r emain ed stable up until 7days.
These results can be exp lained on the b asis of the observation that higher AZO a ddition
leads in less cr osslink ing of the gel that p ermit faster hy drolysis of the ester groups, lead ing
to quicker degradation. It is significan t to note that the swelling behavior was proportional
to crosslinking density. More is the AZO c ontent higher would be the swelling ratio. 5%
AZO-8-PEG-Acr NH 3 gels did not swell like the 50% AZO-8-PEG-Acr NH 3 gels.
Presence of ester moieties in the gel matrix, c an leads to hydrolysis an d aminolysis which
causes the observed degradation of the gels[130].

Figure 4. 13 : Reactio n mechanism o f gelation in AZO-8-PEG -Acr NH 3 gels
The degradation p roducts are helpful in e xplaining the gel ne twork st ructure and possible
reaction mechanisms. Amine Michael-type add ition w ith 8 -PEG-Acr using ammonia is well
established and studied b y the Dr. Zhenfang Zhang [9 3] . F igure 4. 13 shows the possible
reaction chemistry of AZO-8-PEG-Acr NH 3 ge ls. Analysis of the d egradation p roducts of
AZO-8-PEG-Acr NH 3 was carried out through U V visible spectroscopy. Degraded gels were
dried in vacuum overnight and then the dried powdered degradation product was dissolved
in DMSO to measure the UV-Visible absorption spectrum.
Figure 4. 14 presents the absorption behavior of degraded gels in DMSO. It can be seen tha t
50% AZO-8-PEG-Acr NH 3 exhibited more than one absorption maxima which strengthen the
possibility of s everal crosslinking combinat ion of AZO and PEG as p red icted in Figure 3 .3.
The different crosslinki ng combina tions on hydr olysis gi ve different degr adation prod ucts
which show s different absorption b ehavior in UV v isible sp ectroscopic measur ements.

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Figure 4. 14 : UV visib le spectra of degrade d AZO-8-PEG -Acr NH 3 gels in DMSO

Leaching studies of 25% AZO -8-PEG-Acr NH 3 gels were done to determine the chemistry of
gels. For lea ching studies, the gel was washed several times in DMSO so that the unreacted
monomer, azobenzene di-acrylate and small c hain AZO -PEG derivatives could be leached
out.

Figure 4. 15 : UV visible spectra of monomer and the leached 25% AZO-8- PEG-Acr NH 3
gels in DMSO

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The absorption behavior of the monomer and the leached solution wa s expected to display
difference. It can be seen from the Figure 4. 15 that absorption band of the leached gel is
exhibiting add itiona l a bsorption bands because of the formation of several crosslinking
combinations. This behavior is complimented with the UV - Visible absorption behavior of
the dried gels where peak broadening was observed due to the overlapp ing of these
individual bands.
4.5 Conclusio ns
In this chapter, detailed spectral, mechanical and surface characterization of the AZO-8-
PEG-Acr NH 3 gels was presented.
The spectral characterization using FTIR and Raman spectr oscopy revealed th e
confirmation of the incorporation of the desired functional group (N=N) in the gel matrix.
UV -Visible absorbance and fluorescence emission spectroscopic sh owed the absorbance
and emissi on behavior of gels. AZO bonded in gel matrix showed prominent emissi on while
exhibited the supp ressed photo isomerization. While the gels with lesser amount of AZO in
gel matrix i.e. 5% exhibited a noticeable photo isomerization.
Mechanical characterizations of the AZO-8-PEG-Acr NH 3 gels were carried out to find t he
effect of AZO % on gelation time in PEG matrix. It was observed that with in crease in AZO %
a gradual increase in the gelation time was observed. Likewise, there was observed a
marked decrease in storage mo dulus of the gels with addition of AZO in PEG matrix.
Te mperature increase predicted the decrease in storage modulus p robable due to softening
of gels with increase in the degr ee of freedom of polymer chains. Surface characterization
using SEM and AFM d isplayed a decrease in crystallinity due with increase in A ZO %
ascribed to low crosslinking density with additive concentration.
The degrad ation and swellings tests revealed that higher AZO addition leads to less
crosslinks in the bulk of the gel, which al lows f astest hydrolysis o f the ester groups, lea d ing
to quicker degradation. It is significan t to note that the swelling behavior was proportional
to crosslinking density. More is the AZO content higher would be the swelling ratio. The
different crosslinking combinations on hydroly sis give dif ferent d egradation products

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which shows different absorption b ehavior in UV visible spectroscopi c measurements. The
absorption band of the lea ched gel is exhibiting additional absorption bands beca use of the
formation of seve ral cr osslinking combinations. This behavior is c omplime nted with the
UV -Visible absorption behavior of the dried gels where broadening of the p eak was
observed due to overlapping of these individual bands.

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5 Chapt er 5
Phot o - and Thermo - R esponsi v eness of
AZ O - 8 - PE G - A cr N H 3 Gels
Light irradiation generates geometric changes in azobenzenes. Under appropriate
conditions, these changes can be translated into larger -scale motions, even in macroscopic
movements of the material system. AZO-8-PEG-Acr NH 3 polymeric gels showed mechanical
actuation under the sunlight. Also, these gels showed response to body heat and proved to
be thermal responsive as well. In or der to separate the thermal and p hoto -response of AZO-
8-PEG-Acr NH 3 gels, experiments were done with solar simulator and effective actuati on
was achieved. Thermal characterization was done by using differential scann ing
calorimetry (DSC) and thermogravimetric analysis (TGA). These stu dies were performed to
evaluate the effect of azobenzene concentration on thermal stability and melti ng enthalpy
of synthesized gels. These characterizations also provided an in sight of noticeable thermal
response of novel gels.

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5.1 Introductio n
The world nowadays is mainly relying on limited fossil fuels for the production of en ergy
[131] . Hence, we are now challenged with a diff icult problem: generating energy without
burning f ossil fuels. Us e of unlimite d resources like sun, wind and w ater for produc tion of
energy is a great need of ou r time [132].
Sun is the unlimited so urce of light energ y [ 133]. Radiant heat and light energy from sun is
being harnessed by humans since ancient times using a range of ever-evolving technologies.
Solar energy technologies includ e solar photovoltaics , solar heating , and solar architecture
make significant contributions toward solving some of the most cruc ial energy problems
faced by the wo rld [134], [135]. The conversion of solar energy into electricity is a suita ble
way of utilizing this unlimited resource. H owever, it is necessary to convert the electricity
into mechanical work with devices, such as batteries, motors an d gear s [136]. F urthermore,
those devices are usually macroscopic an d made up of metallic co mponents. If polymer
materials convert solar energy directl y into mechanical work, a new system could be
developed which involves no power machinery and possibly will b e applied in a light -
weight working d e vice of any size and shape[137].
Identifying materials th at can convert an input sti mulus to the mecha nical work have b een
of long standing interest. Photo-control of molecular alignment is an intelligent and useful
strategy to convert a signal (e.g. sunlight) into a r esponse in the polymer network, i.e.
deformation[138]. Polymer actuators ha ve prospective for energ y conversion due to
presence of formability[139]. Poly mer gels comprise of flexible polymer ic chains cross-
linked to ea ch other. Their distin ct ab ility to deform easily generate s stimuli-sensitive
properties. Numerous types of gels have b een developed for a range of app lications, such as
biological sensors, actuators, op tically tunable lenses and self -oscillating devices[6], [75],
[139], [140].
Photo-responsive polymer gels usually consist of a polymeric network an d a photo-reactive
group i. e. a photochromic molecule (chromophore) as the functional part. The op tical signal

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is captured by the chr omophore and the resulting photo -reaction such as isomeriz ation or
bond cleavage convert s it into a chemical or mechanical s ignal [141]. This change on the
molecular level thus triggers a microscopic or macrosco pic change in the gels
properties[142].
Azobenzene is a uniq ue molecular switch, whose function can be amplified to alter the
larger-scale mate rial properties in response to light. To date, azobenzene is considered one
of the smartest chromophores among all photochrom ic molecules because of its thermally
stability, a distinguishable absorbance of tran s and cis isomers, a nd a relatively rapid
thermal cis→trans b ack reaction. Isomerization lea ds to changes in molecular geometry an d
polarity [42] . The geom etric change arises in azo benzenes upon absorption of light; results
in bending of the molecule. The characteristic banan a -shape is shorter and slightly broader
while it is polar; it has a dipole moment of 3.1D[143].
Polymer mate rials bearin g azobenzene monomers are widely tested for actuation using UV
and visible light [29], [76], [144], [145] but very few systems are explo red where the p hoto -
actuation could be achieved using sunlight, also the actuati on is of micro scale [26], [107],
[146] .
Sunlight at the earth's surface is around 52-5 5% infrared (IR), 4 2-43% visible (Vis) and 3 -
5% ultraviolet (U V) [26] . The change in the chromophore’s shape upon photo -irradiation
depends on their molecular structures. If we develop a mo lecular structure using
azobenzene that might make use of ultraviolet and visible light from s u n for isomerization,
we can produce the photo-mechanics directly by using sunlight. Such an achievement could
be a milestone for producing solar motors and actuators [147].
Keeping in view the above mentioned interests, we aimed to synthesize photo -responsive
azobenzene base d gels which can p ossibly utilize solar energ y for actu ation. M any synthetic
polymers can form gels by chemical crosslinking, poly (ethylene glycol) (PE G) remains one
of the most extensively studied systems [148], [149] . Moreover, the star shaped 8-PEG is
considered as an interesting class of materia ls because of its flexibilit y and multiple
crosslinking p oints. So we chose the 8-PEG-A cr as matrix and chemically incorporated
azobenzene (AZO) thr ough our recently discovered “ amine Michael -type addition”

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method[120]. Incorporat ing the azobenzen e units covalently in the newly synthesized star
shaped 8-PEG acrylates, made the system so flexible that it could use the UV an d Visible
light from the sun for cycling b etween the two isomerization states. These star shap ed po ly
(ethylene glycol) gels are suitab le candidate s for providing the f re e volume for cycling
between two states of isomerization, because of the flexibility of the polymer chains;
capable of mechanical actuation powered directly by sunlight energy.
The main aim of project was to design and prepare a mate rial which could directly convert
solar energy into motion as explained in Figure 5.1 .
The synthetic a pproach for AZO-8-PEG-Acr NH 3 gels is different from com monly used
photo-polymerized gelation to fabricate PEG -based gels (as explain ed in chapter 3). The
complete characterization of the gel was explained in Chapter 4.

Figure 5.1: Photo -mechanics in A ZO using su nlight
In this chapter, the detailed experiment s were conducted for pro ving t he responsiveness of
AZO-8-PEG-Acr NH 3 gels. The photo-response was inspected in sunlig ht and with solar
simulator while the thermal response was monitored using the thermal effect of body heat
from hands.

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Photo an d Thermal response was also studi ed separately in order to understand the
mechanism of actuation in AZO-8-PEG-Acr NH 3 gels. This chapte r p rovides all the details of
the responsiveness of the synthesized g els.
5.2 Mate rials
The synthetic method a nd exact ratio of AZO- 8-PEG-Acr NH 3 gels was explai ned in detail in
Chapter 2. The actuation studies were done on polymer gel film. Figure 5.2 presents the
methodology adopted to prep are the p olymer film. The film was then peeled off from the
glass surface and was used for experiments.

Figure 5.2: Me thodology a chieved to prepare AZ O-8-PEG-Acr NH 3 gels
5.3 Experi ments
5.3.1 Responsiveness
5.3.1.1 Photo
Photo-responsiveness of the azo based polyethylene glycol gels was monitored under two
conditions. One aspect of the photo -response was measur ed in sunlight and other was
evaluated with solar simulator, to remove the p ossible effect of the thermal radiation of real
sunlight.
5.3.1.1.1 Sunlight
The solar response of AZO-8-PEG-Acr NH 3 gels was monitored in the presence of sunlight.
In order to avoid the eff ect of wind an d te mperature, the experiments were conducted
indoor. The effect of sea sonal temperature was monitored in both summer and winter .
Results were collected in form of visual imaging and videos.

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5.3.1.1.2 Solar LED simulator
Solar LED simulator (shown in F igure 5. 3) was assembled an d p rovided by D r.
Michael Schwarze and Maxim ilian Neumann (Prof. Dr. Reinha rd Schomäcker group TU
Berlin).

Figure 5.3: So lar simulator used for monitori ng photo-response o f synthesized ma terials
In order to evaluate the effect of the solar frequency on the p hoto-responsiveness of novel
synthesized materials, experiments were conducted with solar LED simulator having the
radiations of 200-1000 nm wavelength . Distance between the sample and beam was 15 cm.
Intensity of the current app lied for LED source was 0.14 A (i.e. the lowest possib le intensity
to avoid the temperature effect).
The results were recor ded with web camera connected to a lap top. Atten uators were used
to cut off the exposure of camera.
5.3.1.2 Thermal
Thermal response of the novel synthesized gels was monitored against body heat (37˚C).
Degree of response was measured by bringing the gels films near finger an d hand as well as
placing the gels on palm. Results were recorded with a camera in the form of visual imaging
and videos.

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5.3.1.2.1 TGA
Thermogravimetric an alyses (TGA) were carried out on a TGA 1 instrument from Mettler
Toledo under O 2 atmos phere. Alumina powder was used as standard. The temperature was
raised from room temperature to 10 00 °C at a heating rate of 10 °C min − 1 . Weight of the
measuring samp les was 100 mg each. Dried gel films were cut into small pieces. The
reproducibility of the i nstrument reading was determined by repeating ea ch experiment
more than twice.
5.3.1.2.2 DSC
Differential scannin g calorimetry (DSC) scans were p erformed on a M ulti -Cell Differential
Scanning Calorimeter MC -DSC (TA Instruments) with the help of Dr. Leonardo Chiappisi in
Prof. Dr. M . Gradzielski lab oratory TU Berlin. Measurements were done with a scan rate of
1°C/min from -30 to 70 °C with an equilibration time of 600 Sec prior to each scan.
5.4 Results
In order to demonstrate the responsive behavior of the AZO-8-PEG-Acr NH 3 gels, they were
subjected to p hoto an d thermal sti muli. These gels with different AZ O % were e xposed to
light and heat, their response was recorded with camera in the form of visual imaging and
videos. In this chapter, pictures are taken from the videos t o explain the phenomena.
5.4.1 Photo -responsive studies
Photo-responsiveness of the AZO-8-PEG-Acr NH 3 gels was examined under two conditions.
One aspect of the photo -response was measured in sunlight and other was evaluate d with
solar simulator.
5.4.1.1 In Sunli ght
Al l the AZO-8-PE G-Acr NH 3 gels showed some response to sunlight, but the 25% AZO-8-
PEG-Acr NH 3 gel showed prominent actuation in response to sunlight in winter when the
room was recorded ab out 18˚C. The film thickness was record ed arou nd 100µm. Figure 5.4
displays the series of e vents taken place during the solar response of 25% AZO -8-PEG-Acr

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NH 3 gel. It can b e seen that the gel shows movement over the period of 5 Sec. Interesting is
to note that this response was periodic.
The same experiments for the photo -actuation were conducted in summer with room
temperature of about 30˚C. It was expected th at if the AZO- 8-PEG-Acr NH 3 gels are
responding to sunlight, they might b ehave differently with high solar reflux in summe r.
Experiments were d one with all ratios of AZO-8- PEG-Acr NH 3 gels and no actuation was
observed with 0%, 25 %, 35% an d 50% AZO-8-PEG-Acr NH 3 gels. However, 5%, 15% AZO-
8-PEG-Acr NH 3 gels showed w eak actuation.

Figure 5.4 : Series of events duri ng solar r esponse of 2 5% AZO-8-PEG-Acr NH 3 gel
The durability of the films was monitored over th e period of two years and it still retained
the actuation property. It means, the gels don’ t degrade in normal atmosphere and sustain
the shape an d property over the longer peri od of time. The 0% AZO-8-PEG-Acr NH 3 gel,
having the pure PEG matrix didn’t sho w any actuation with sunlight.
5.4.1.2 Solar LED simulator
In order to evaluate the effect of the solar frequency on the photo -responsiveness of AZO-8-
PEG-Acr NH 3 gels, exp eriments were conducted with solar L ED. Figure 5.5 presents the
response of 25 % AZO-8-PEG-Acr NH 3 gel to solar LED simulator. It c an be c seen that the
film was curlin g on exposure to light. This curled film could be relaxed back to original form
with exposure to heat s timuli of hand. The response was also monitored with other AZO %

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but the 25% AZO-8-PEG-Acr NH 3 gel s howed f astest actuation. As comparison, experiments
were done with 0% AZO-8-PEG-Acr NH 3 gel. But it showed no response to light as seen in
figure 5.6. This clearly depicts that the photo-responsiveness of AZO-8-PEG-Acr NH 3 gels is
because of the AZO unit. As the 0% AZO-8-PEG-Acr NH 3 gel is lacking the AZO unit so it
showed no response to light.

Figure 5.5 : Series of events duri ng solar r esponse of 2 5% AZO-8-PEG-Acr NH 3 gel

Figure 5.6: Compar ison of pho to-response in 0 and 2 5% AZO-8-PEG-Acr NH 3 gel
5.4.2 Thermo- responsive studies
In order to investigate the effect of temperature on the responsiveness in AZO-8-PEG-Acr
NH 3 gels , response was monitored against body heat ( 37˚C). Degree of response was

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measured b y bringing the gel films ne ar finger and hand as well as placing the gels o n palm.
Results were recorded with a camera in the form of v isual imaging and videos.
5.4.2.1 Thermal Characterization of AZO- 8-PEG-Acr NH 3 gels
Thermal characteri zation of the AZO- 8-PEG-Acr NH 3 gels was done to measure the thermal
stability as well as the meltin g enthalpy. The thermal stab ility was measured b y using TGA
and DSC instruments.
Thermal studies were conducted from room t emperature to 1000˚C with a heating rate of
10˚C min − 1 in O 2 atmosphere. Figure 5.7 shows the analyzed thermogram for the respective
AZO-8-PEG-Acr NH 3 gels. It can be seen from t he thermogram that th e thermal stability is
maintained almost till 280. The recorde d TGA thermogram proposes multistage
degrad ation [150]. T he first degradation up to 100 ˚ C was due to l oss of moisture. The
second degradation curve from 200 ˚− 400 ˚ C arises beca use of the eli mination of nitrogen by
the degradation of AZO linkage.

Figure 5.7: TGA th ermogra m of AZO-8-PEG-Acr NH 3 gels
The third degradation step occurring between 400 ˚C − 550 ˚ C was attributed to the breakage
of ester linkage present within the gel networ k. The aromatic backbone present in the gel
matrix degr a ded in the end.
200 400 600 800 1000
0
20
40
60
80
100
Temperature (  C)
% Wt.loss
0%
5%
15%
25%
35%
50%

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To investigate the melting en thalpy of the AZO-8-PEG-Acr NH 3 gels, DSC was conducted.
Measurements were ac complished with a scan rate of 1°C/min from -30 to 70 °C, with an
equilibration time of 600 sec prior to each scan.

Figure 5.8 : DSC thermogram of AZO-8-PEG-Acr NH 3 gel s
Figure 5.8 shows the DSC thermogr am for the AZO-8-PEG-Acr NH 3 ge ls. Sharp exothermic
peaks were detected and attributed to the melting temperatures (T m).
Tm of the 0 % A ZO-8-PE G-Acr NH 3 gels poly mer is 45 ± 0.2 °C as shown in Figure 5.8 .
Noteworthy is that the melting te mperatures decrease with in crease in the concentration of
AZO content. The low values of crystallinity and corresponding melting temperatures can
be at tributed to the non-perfect crystal formed in the gels and the irregular chain folding
(De tailed mechanism is discussed by Dr. Zhenfang Zhang L L member (unpublished results).

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Figure 5.9: Cha nge in melting e nthalpy of AZO-8 -PEG-Acr NH 3 gels with additive
concentratio n
During the gel formation processes, the PEG chains are restricted in a confined space as
they form the gel ne twor k. The 8-PEG network structure using different gelators an d effect
of the structure on the crystallinity of the gel is w ell explained b y Dr. Zhenfang Zhang
(unpublished results under review). The network formation generate s lower crystallinity,
resulting in decreased meltin g te mperatures. This behavior is also complimented with AFM
and SEM studies where decrease in crystallinity was observed with increase in AZO %,
resulted in lower melting enthalp y. 0% AZO -8-PEG-Acr NH 3 gels showed the highest
melting enthalp y and maximum crystallinity while the 50% AZO-8-PEG-Acr NH 3 gels
possessed the lowest melting enthalpy and lowest crystallinity as shown in Figure 5.9.
5.4.2.2 Body heat
Thermal response of the AZO-8-PEG-Acr NH 3 gels was monitored against body heat (37˚C)
with all AZO %. The AZO-8-PEG-Acr NH 3 gels showed quick response to body heat.

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Figure 5. 10 : Series o f events durin g thermal respo nse of 25% AZO-8-PE G-Acr NH 3 gel
As soon as the film was placed on the palm whose recorded tempe r ature was 3 7 ˚C, the film
showed continuous movement of cur ling and bending as shown in Figure 5. 10 . The film was
moving like a diving f ish. Interesting is to note that this phenomena of bending and
response was monitored with all AZO % and it was r emarked that even the 0% AZO-8-PEG-
Acr NH 3 gel which was having pure PEG matrix also showed actuation and movement to
thermal stimuli as shown in Figure 5. 11 .

Figure 5. 11 : Series o f events durin g thermal respo nse of 0% AZO-8-PEG -Acr NH 3 gel
Thus, it seems that the therm al response of the AZO-8-PEG-Acr NH 3 gels is to some extent
because of the gel matrix not exclusively because of the AZO.
5.4.2.3 Bendin g
In order to see the range of body heat on t he thermo -responsiveness of AZO-8-PEG-Acr NH 3
gels, the effect was monitored f rom a distance as well. All the A ZO-8-PEG-Acr NH 3 gels

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showed clear ben d ing from the heat and the thermal field for this response was calculated
be around 2.5cm. The results are presented visually in Figure 5. 12 and Figure 5. 13 .

Figure 5. 12 : Thermal b ending of 2 5% AZO-8-PEG -Acr NH 3 gel

Thermal bending is observed by all the AZO -8-PEG-Acr NH 3 gels. The 0% AZO-8-PEG-Acr
NH 3 gel which is based on pure PEG matrix also exhibited the ben d ing fr om the heat sti mu li
produced by the warm t h of hand s as shown in Figure 5. 13 .

Figure 5. 13 : Thermal b ending of 0 % AZO-8-PEG-Acr NH 3 gel

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Thermal response of the AZO-8-PEG-Acr NH 3 gels seems irrespective of the AZO
concentration. In order to rule out the evap or ation effect, thermal r esponse of 0 % A ZO -8-
PEG-Acr NH 3 gels prep ared w ith diff e rent solvents ranging from mo r e volatile(acetone ) to
less volatile (DMF), was monitored. Therm al re sponse was evident in each case.
This response was attributed to the gel network that arises because of the “ Amine Michael-
type addition”. Because when 8-PEG-Acr gels were prepared by photo -crosslinking method,
showed no thermal response.
5.4.3 Photo a nd thermal re sponse
In order to distinguish between thermal and photo -r esponse of AZO-8 -PE G-A cr NH 3 gel,
experiments were conducted in sunlight. In normal day light, experiments were carried out
with AZO-8-PEG-Acr NH 3 gels films. T he result s were collected with a camera in the form of
videos. Here in this sec tion, images from various in terv als are presented in Figure 5. 14 to
show the photo and thermal response of 25% A ZO -8-PEG-Acr NH 3 gels films. The normal
film exposed to ord inary light at T=0 Sec. has a slight ly bent appearance. When this film is
exposed to hand even from a distan ce of 2 cm, it showed response with in a period of
microseconds.

Figure 5. 14 : Series o f events durin g photo and t hermal response of 2 5% AZO-8-PEG-Acr NH 3
gel

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Later on, this film was exposed to sunshine having the concomitant temperature of around
25˚C and it was noted that the thermal response w as also of same intensity. Actuation of the
polymer film was seen as presented in Figu re 5. 15 . The film was dr a wn back from the palm
and curl ed inward.

Figure 5. 15 : Series o f events durin g photo (Suns hin e ) and thermal response of
25% AZO-8 -PEG-Acr NH 3 gel
5.5 Discussio n
The above mentioned results evidently demonstrate that AZO-8 -PEG-Acr NH 3 gels are
photo an d thermo-responsive. The experiments were performed with all AZO: PE G ratios
and several striking observations were collected.
For the photo-responsive studies with sunlight and solar simulator, it was revealed that in
case of 25% AZO-8-PEG-Acr NH 3 gel the response was more pronounced while the rest of
the gels showed weak response. Also the second interesting observation was that photo -
actuation was seen in winter when the room temperature was aroun d 18˚C, but when same
response was tried to b e rec orded in summer (30˚C) it was not pronounced but weak. It
was expected that if the gels are responsive to sunlight then may be in summer high solar
reflux produces better actuati on. The reason for this difference co uld be c omprehended

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from the DSC thermogram of AZO-8-PEG-Acr NH 3 gels shown in Figur e 5.8 that gives a clear
reason for the more pronounced photo-response in winter rather than summer.
It can be seen from the results that the melting enthalpy and Tm both are inversely directly
proportional to AZO%. It means, when we keep on increasing the AZO%, decrease in
melting en thalpy is observ ed, as a result the gel melt at lower tempe r ature. So 50 % AZO-8-
PEG-Acr NH 3 gels melt at lower temperature than 0%. It can be seen t hat at the tempe r ature
above 20˚C, the melting of all the AZO-8-PEG-Acr NH 3 g els starts. A ctually the gels don’t
melt truly like solids but they become softer at their melting poin t.
Solar radiations possess infrared (IR), visible (Vis), and ultraviolet (UV) light. The AZO- 8 -
PEG-Acr NH 3 gel, if they are responsive to the UV and Vis radiation com ing from sun should
behave like as shown in Figure 5. 16 (a)[151] an d it can be observed f rom the Figure 5. 16 (b)
that the 25% AZO-8-PEG -Acr NH 3 gel possess es the required shape in the sunlight. It
clarifies that AZO-8-PEG-Acr NH 3 gels can respond to the solar radiations.

Figure 5. 16 : No rmal state of expected AZO-8 -PEG-Acr NH 3 gel films in differen t radiations
In winter, when the temperature(18˚C) is below Tm of AZO-8-PEG-Acr NH 3 , AZO present in
the gel matrix act as p hotochrom ic unit that absorbs light and co verts it to heat energy
which is transfe rred to the polymer chains. This in cr eases the degree of freedom in the
polymer chains an d mov ement in the polymer chains causes act uation. This actuati on is
more pronounced with 25% AZO- 8- PE G-A cr NH 3 g els because it has moderate melting
enthalpy and melting temperature is around 30˚C.

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Normally, the gel network is intact and polymer chains have lower d egree of freedom but
when exposed to sunlight, the AZO ab sorb su nlight, which have both UV and visible light
along with IR radiations, isomerization takes place within the AZO unit as well as the energy
absorbed is transferred to the polymer chai ns. This energy cause the movement and
softening of the gel which give rise to p hoto- mechanics. While in the AZO-8-PEG-Acr NH 3
gels which have even lower melting enthalpy like 35% and 50%, probably the polymer
chains are already in a relaxed state at this te mperature and could not produce pronounced
actuation. Film d imensions are another imp ortant factor to keep in mind. Thinner the film
is, better was the response.
In summer, for the AZO -8-PEG-Acr NH 3 gels w hich possess high melting enthalpy and high
Tm, they are expected to p erform good because the 2 5% AZO-8-PEG-Acr NH 3 gel hav e
melting te mperature of around 30˚C w hich is similar to the room temperature. The gel is
already in a melted state, so no further flexibility can be brought into the p olymer chains as
they are in the most relaxed state. Same is the case with 25% and 50% AZO-8-PEG-Acr NH 3
gels which are already melted at this temperature. The ab sence of noticeable act uation in
5% and 15% AZO-8-PEG-Acr NH 3 gels can be the conc. of AZO is not enough and the en ergy
required for the melting of poly mer chains is not prov ided with this small concentrati on of
AZO.
0% AZO-8-PEG-Acr NH 3 gels sho w no response to sunlight b ecause they have 0% of AZO
which is necessary for c apturing the light energy and cou l d translate it to photo -mechanics.
In order to completely eliminate the thermal effect of the solar radiation on AZO-8 -PEG-Acr
NH 3 gels, experiments with solar LED simulator were recor ded whic h revealed that 25%
AZO-8-PEG-Acr NH 3 gels show actuation even by using the lower possib le intensity. The
temperature rise with 20min exposure o f ligh t having intensi ty of 0.14A was observed only
0.2˚C. Therefore, the thermal effect is almost negligible. It mean s th e AZO-8-PEG-Acr NH 3
gels are responsive to light stimuli.
The thermal response of the AZO-8 -PEG-Acr NH 3 gels is directly attributed to the Tm of the
gels. The body temperature is around 37˚C. So when the finger is brought near the film or
when the gel is placed on the palm, the heat radiated by the body cause the melting of the

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polymer gel and results in t he movement. This melting of the gels is mainly because of the
crosslinked network and it is only dependent on the crosslinking densit y. Consequently,
highly c rosslinked gels show slower melting as observed in 0% AZO-8-PEG-Acr NH 3 gels,
while the 50% AZO-8-PEG -Acr NH 3 gels exhibits lowest melting enthalpy and Tm. At 37˚C ,
0%, 5% and 15% are exp ected to show better response because 25%, 35% and 50% AZO-8-
PEG-Acr NH 3 gels are already in the meltin g state. It means the thermal response is
independent to AZO.As a proof of principle that this thermal responsiveness of AZO-8-PEG-
Acr NH 3 is not because of solvent, 0% AZO-8-PEG-Acr NH 3 gels were p repared in different
solvents like DMF, Water Ethanol, and Acetone an d their response was monitored. Gels
were active with all the solvents.
In order to evaluate if the difference in crosslinking chemistry plays a role, response was
monitored with 0% AZO-8 -PEG-Ac r NH 3 , 0% AZO-8-PEG-VS gels and 0% AZO-PEG gels.
The gels prepared using photo-crosslinking did not show response. The 0% AZO-8-PEG-VS
NH 3 gels were prepare d using amine Michael-type addition also showed no response to
thermal stimuli .
All these experiments revealed that PEG gels prepared using p hoto - initiator consum e
almost all the active sites; as a result it s crosslinking densit y becomes higher. The melting
enthalpy is expected to in crease so they show no response to thermal stimu li of 37˚ C. It is
expected that these gel mig ht respond to high temperature gradient closer to the T m. Same
is the case with 0% AZO-8-PEG-VS NH 3 gels because due to the high reactivity of VS group
these gels are formed very quickly and the c rosslinking den sity is expected to be higher.
The only responsive 0% AZO-8-PEG-Acr NH 3 gels exhibit free active units which could move
in the gel matrix and possess higher degree of freedom s o it shows response to thermal
stimuli.
Thermal response was monitored both by pl acing the film on palm and b y bringing the
finger near and the re sponse was pronounced in both cases which shows that the heat
radiated by the body ca n also cause movement in gel film.
Thermal and p hoto-response was brought tog ether in the presence of sun shine and it was
revealed that the gel is responding to both stimuli simultaneously and also independently.

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Gel films always curl inward when are exposed to thermal or photo -response because the
crosslinking density of the upper part and lower p art of the gel is diff erent. The solvent
used fo r gelation was DMF (0.95 g/cm3 at 20 °C) has higher density than the ammonia
(0.88 g/cm3 at 18˚C) which is used as cr osslinking agent. So at the surface, higher
concentration of ammonia will be present so more crosslink in g points than the b ottom. So
when a gel film is exposed to any stimuli, the upper mor e crosslinked surface exerts force
on lower p art and the film curl inward. Also the top of the film is non -smooth because of the
bubbles formed while the lower part is shiny and smooth.
5.6 Conclusio ns
As per target of the p roject, we aimed to build a photo -sensitive system which could
respond to the solar radiation. From the results ment ioned above, it can be seen AZO-8 -
PEG-Acr NH 3 gels are multi-responsive.
The AZO-8- PE G- Ac r NH 3 gels are good can didates for photo -mechanical actuation usi ng
sunlight. Such polymer gels could be used as li ght -weight solar motors and sensors instead
of traditional heavy batteries and gears. This would be a useful achie vement in the field of
renewable energy utilizing the sun as an unlimited light source in to practical usage. The
AZO-8-PEG-Acr NH 3 were proved to be p hoto and thermo-responsive gels.
These gels show actuation t o both photo and th ermal stimuli. The photo -response of AZO- 8-
PEG-Acr NH 3 gel is solely because of the AZO group present in the PE G matrix. This
photochromic unit absorbs the solar radiation and translates it into heat , results in the
actuation of gels. 25% AZO-8-PEG-Acr NH 3 gels exhibit actuation with solar radiation in
winter because of the moderate melting enthalpy. Concentration of the AZO in PEG matrix is
also an important factor to keep in mind for determining the photo -response of AZO-8-PEG-
Acr NH 3 gels.
The thermal response of the AZO-8-PEG-Acr NH 3 gels monitored demonstrates that all the
AZO-8-PEG-Acr NH 3 gel s are thermo- responsive. The therm al stud ies showed a d ecrease in
melting enthalpy d ue to increase in AZO%. Du e to the low e r crosslinking density, the
polymer chains have more f reedom of movement and he nce they melt quickly.

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6 Chapt er 6
Biological Studies of AZ O - 8 - PE G - A cr NH 3
Gels
Growth of Microbes at the implant surf ace leads to infections that are major cause of
implantation failur e in medical field. Controlling infections can in cre ase the success rate of
implants in biomedical field. Chemical induction of the antimicrobial units in biocompatible
materials can limit the grow th of microbes on the implant surfaces. This might be an
interesting a nd log ical strategy to solve t his problem. Azobenzenes ex hibit some
antimicrobial properties against bacteria and fungi. PEG is a well - known biocom pat ible
substrate in biomedical industry. Therefore PE G matrix with ch emically crosslinked
antimicrobial azobenzene moiety dr aws an att ention for biological evaluatio n.
Keeping in mind these captivating ideas of medical importance, chemically cr osslinked
AZO/PEG gels (AZO-8- PEG -Acr NH 3 gels) were synthesized, characte rized and studied for
various ap plications. Biological evaluation of AZO-8-PEG-Acr NH 3 gels will be detailed in
this chapter. Cell studies against M ouse F ibroblast (L -929) will be conducted to investigate
the effect of AZO-8-PEG-Acr NH 3 gels on cell cytotoxicity and cell adhe sion behavior. In the
end, antibacterial testing of azobenzene monomer against E.c oli bacterium and H ep -G2 will
be presented.

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6.1 Introductio n
Infections by pathogenic microorg anisms a re of great concern in medical science
predominantly in surgery equipment, medical devices, hospital surfaces and health care
products. Infections are normally combatted with an timicrobial agents[152]. Many times,
infections caused by re sistant microorg anisms fail to respond to conventional treatment,
which result in prolonged illness and higher risk of death. The emergence of bacterial
strains resistant to the most common classes of anti biotics is prompt ing a dr amatic quest
for the development of new antimicrobial drugs[153] .
In b iomedical field, use of the materials with antimicrobia l p roperties stretches the serv ice
life of these materials, and avoids damage cau sed by growth of infection causing microbes.
The synthesis of biologically active materials can be carried ou t eit her by impregnation with
antimicrobial compoun ds, or by chemi cal reaction (adding antimicrobial compounds by
means of chemical bonding to functional groups).
Some stilbenes, like resveratrol, are well -known natural antibioti cs. Unfortunately,
stilbenes derivatives display only moderate anti microbial effects [154],and they are usually
toxic compounds, a major drawback when developing new medications. Therefore we
considered the azobenzenes, a class of mole cules having high structural similar ity with
stilbenes. Though azoben zenes have been widely studied as dyes or p hoto responsive
materials [155], little is reported about their potential antimicrobial activity.
Recent advances in biological studies using azobenzenes opened the door for biomedical
applications of these materials. They have shown some antimicrobial properties [156] –
[15 8] . It would be a great idea to combine the biomaterials with antimicrobial azobenzene
compounds, this may possibly increase the success rate of cell growth in b iomedical
applications. P EG is known for long for its biocompatibility and inertne ss. Thus, if th e
antimicrobial units are chemically embedded in the PE G matrix, the chances of infections
generated with microbes can be minimized.

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In this work, we have desi g ned a series of azob enz ene based PEG gels ( AZO-8-PEG-Acr NH 3
gels) and studied their poten tial toxicity by means of a Live/Dead assay. The first step
toward incorporating AZO for biomedical ap pl ications would be to check its
cytocompatibility w ith cells. For this purpose, the monomer solution was tested for the
cytotoxicity test again st Mouse F ibroblast (L-929 ) using Live/Dead assay. No remarkable
change in morphology of the cells was detected. H ence in order to investigate the
cytocompatibility of AZO-8-PEG-Acr NH 3 gels, cell cytotoxicity of the gels against M ous e
Fibroblast (L -9 29) was conducted. For using th e AZO-8-PEG-Acr NH 3 g els for anti micr obial
properties, ant ibact eria l testi ng of AZO against E .coli and anti cancer ac tivity again st Hep -G2
was accompanied.
6.2 Mate rial a nd Methods
The synthetic methods adopted for the preparation of azobenzene monomer (AZO) and
azobenzene based PEG gels (AZO-8-PEG-Acr NH 3 gels) w ere e xplained in Chapter 2.
Different ratios of AZO-8-PEG-Acr NH 3 gels were prepared and tested for the biological
evaluation. The details of the b iolog ical proced ures adopte d for activ ity measurements are
explained in the section below.
6.2.1 Wett ability ch anges
Wettability changes of gels were examin ed by measuring the change in water conta ct angle
of the gels mounte d o n coated on glass sub strate. Dimensions of gel film were foun d
18x18mm a nd ≈200µm thickness. Water contact angle was measured using OCA15 Contact
angle measurement pendent drop instrument.
6.3 Biological Evaluation
Biological evaluation includes the cell viabilit y , cell adhesion, an tibac terial and anticancer
studies. The cells used for the cell viability and cell adhesion were Mouse Fibroblast (L -
929). The activity was d one with different AZO-8-PEG-Acr NH 3 gels. Antibacterial studie s
were accomplished against E .coli bacterial strain and anticancer activity was monitored
against H ep - G2. Cell studies were conducte d w ith the help o f Ci gd e m Yeşildağ L ensen L ab

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member. Wh ile the anti bacterial and anticancer activity was done a t Leibniz insti tut e of
molecular pharmacolog y Berlin, Germany, assi stance provided by Dr. Jens Peter von Kries
and Dr. Martin Neuenschwender.
6.3.1 Cell cult ure
Mouse fibroblasts L -9 29 (provided by Dr. Lehmann, Fraunhofer Instit ute for Cell Therapy
and Immunology, IZ I , Leipzig, Germany) were cultured in RPM I 1640 medium with addition
of 10% Fetal Bovine S eru m (FBS) and 1 % P enicillin/Streptomycin (PS) in an incubator
CB150 Series (Binder GmbH, Germany) at controlled temperature (37°C) and CO 2
atmosphere (5%) and 100 % humidity. Medium and reagents were provided by P AA
Laboratories GmbH, Ger many.
6.3.2 Cell viability
Mouse Fibroblast (L-929) viability on AZO-8-PE G-Acr NH 3 gels substrates was monitored
using a Live/Dead assa y. The gels substrates ( 1 cm x 1 cm) were washed with 7 0% ethanol
rinsed in PBS and kept in a μ - slide. 30 0 μ L of a cell suspension containing 50000 L-929 cells
were seeded ont o each substrate and incubated at 3 7 °C, 5 % CO 2 at mosphere and 100 %
humidity. The viability of cells on AZO-8-PEG-Acr NH 3 gels substrates was assessed afte r 24
h incubation period. Later on, cells were stained with 100 μL of a vit ality staining solution
containing fluorescein diacetat e (stock solution 0.5 m g/ml in aceton e, Sigma Aldrich) and
Propidium iodide (stock solution 0.5 mg/ml in PBS, Fluka). Detection of Viable and dead
cells was accomplished by fluorescence micr osco py. Cell viabilit y against the monomers
solution (AZ 1 - Ac r ) was monitored by using the same protocol .
6.3.3 Cell adh esion
Cell adhesion of Mouse Fibroblast (L -929 ) was studied both on neat and patterned AZO-8-
PEG-Acr NH 3 gels substrates. Pro cedure f or cell seeding is similar as explained for cell
viability studies. Neat and patte r ned gel samples were in cu bated at 37°C and 5% CO 2 an d
100 % humidity f o r 24 h.
Prior to microscope observation; the medium was removed an d the s amples were gently
washed with PBS two times. After incubati on, the cells were fix ed. For this purpose ,

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Formaldehyde 4% (Carl Roth GmbH & Co, KG ) was added and left for 30 minutes. At the
end, samples were washed with PBS before observation.
6.3.4 Antibacterial studies
Antibacterial studies of the azo monomers were done against E.coli bacterium. Following
protocol was used f o r c arr ying out experiments.
6.3.4.1 Protocol for antibacteri al t esting with E.coli
Testing protocol for the an tibact erial activity comprised of 5 days. On day 1, bacteria were
cultured overnight at 37˚C. On Day 2, 0.086D o f bacterial culture was put in lysogeny broth
(LB) medium with d ilut ion factor of 1:50 with monomer. Solution was centrifug ed for 15sec
at 1300 rpm and left for next 2 d ays. Bacte ria were counted by using Teca reader on day 5.
6.3.5 Anticancer activity
Anticancer activity of monomers was evaluat ed against Hep -G2 cells. Following protocol
was conducted for measurements.
6.3.5.1 Protocol for anticancer testing with Hep- G2
Testing p r otocol for t he ant icancer activity comprised of 5 days. On d ay 1, Cell seeding was
carried out by placing 2000 cells/ well in 40l RPMI/ 10% FBS in 384 well plat e. After that
cells were incubated for 24 h at 37˚ C. Monomer solutions were transferred to cell plates on
2nd day .The cells were seeded for the next two days. F ixation of Hep -G2 cells were done on
day 5 by a dd ing 40μl/well 4% PFA (Paraformaldehyde) by dispenser. They were further
incubated for 1h at room te mper ature and then cell number was determined by using Teca
reader.
6.4 Results and Discussion
In order to accom plish t he biolog ical stud ies of AZO-8-PEG-Acr NH 3 , cell cytotoxicity tests of
mouse fibroblasts (L-929) were conducted usin g L ive/Dead assay. Later on, cell adhesion
was monitored both on AZO-8-PEG-Acr NH 3 g el substrates. M or eover, an tibacterial studies
against E.coli bacterium and anticancer testing against Hep -G2 cells were cond ucted.

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6.4.1 Cell cytot o xicity
In order to measure the compatibility of AZO-8-PEG-Acr NH 3 gels for biomedical studies,
the first step was to measure the cell cytotoxicity of synthesized materials .For this purpose
Mouse Fib r oblast (L-9 29) were c hosen. Cell cytotoxicity was meas ur ed using Live/Dead
assay under fluorescence microscope. I n this assay, under the fluorescence microscope, the
live cells shows green color beca use they con vert the non -f luorescent FDA into the green
fluorescent metabolite fluorescein, and the dead cells with a non -integer cell membrane
display a red fluorescence because of the incorpor ation of a second dye PI at DNA.

Figure 6.1: C ell cytotoxicity t ests of AZO-8-PEG- Acr NH 3 gels after 24 h with Mouse
Fibroblast (L-929)

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Figure 6 .1 displays th e micrograms ima ges of AZO-8-PEG-Acr NH 3 gels cell cytotoxicity
results with Mouse Fibroblast (L -9 29) after 24 h. It can be see n that no evident change in
the morphology of cells is observ ed. Alm ost 99 % cells showed the green color in microgr am
images which depicts that these gels are not toxic to Mouse Fibroblast (L-929) cell lines.
6.4.2 Cell adh esion
During the cell cytotoxicity measurements of Mouse Fibroblast (L -9 29) with AZO-8-PEG-
Acr NH 3 gels, it was observed that cells adhe r e to the gels substrate . Normally the neat PEG
matrix is non adherent to Mouse Fibroblast (L -9 29) cells. I n order to evaluate the cell
adhesion behavior of Mouse Fibroblast (L -9 29) on AZO-8-PEG-Acr NH 3 gels experiments
were conducted . T he re sul ts are displayed in F igu re 6.2 .

Figure 6.2: Cell adhesion mon itored in AZO-8-P EG-Acr NH 3 gels after 24 h with L - 929
(fibroblas t mouse cells)
It can be seen from the figure that the cells like the AZO-8-PEG-Acr NH 3 gels surface. The
number of cells adherin g on the gel surface increased with the increase ratio of A ZO.

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Control presents the 0 % AZO-8-PEG-Acr NH 3 gel which is having only PE G in the matrix. It
can be seen that there are not many cells adhering on the pure PEG substrate.
There was obse rved a marked in crease in the number of cells adhering on the gel substrate
as we move form 0-50% AZO-8-PEG-Acr NH 3 gels. The 50% ratio depict s the gel which has
50% AZO: PEG ratio. It can b e seen that number of cells a dhering on this surface increased
significantly.
These substrates proved to be an interesting platform with enhanced cell adhesion
properties. The nor mal PEG is non adherent to cells and possesses antifouling properties.
Therefore it was interesting to investigate a n ontoxic cell adhesive surface and explore the
reason for this striking change in PEG properties by adding azo benzen es.
Literature review revealed that the possible reason for the cell adhesive property of AZO-8-
PEG-Acr NH 3 gels might be the change in the surface chemistry of the substrate [159 ] .

Figure 6.3: Cell-sub strate interactio ns [159]
Figure 6.3 explains the cell substrate in teractions. It can be understood from the figure that
the cell adherence is subjected to the surface chemistry. The surfaces which exhibit the
moderate wettability are preferred by the cells. The pure PEG sur face is non -fouling
because it is too hydrophilic; as a result the re is almost no adherence of cells on this
substrate. The same behavior is observe d b y t he hydrophobic substrate s. It is evident fr om

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the figure that cells like the surf ace o f moderate wettab ility. They don’ t adhere to either too
hydr ophilic o r too hydrophobic surfaces.
PEG exhibit s water contact angle (WCA) of ar ound 40° and cell adhesion is minimum on
such surfaces. Cells needs moderate surface wettab ility from around 45 to 81° to a dhere on.
Therefore, in order to investigate the reason for cell adhesion on AZO-8-PEG-Acr NH 3 gels, it
was interesting to figure out the WCA of AZO-8-PE G-Acr NH 3 g els.
Table 6.1 : WCA values of AZO-8 -PEG-Acr NH 3 gel s
AZO %

0

5

15

25

35

50

WCA

43±1.7

49±2.1

55±2.7

67±3.4

74±4.7

77 ±5.8

Table 6.1 shows the WCA values obtained for AZO-8-PEG-Acr NH 3 gels. The surface
chemistry of the AZO-8-PEG-Acr NH 3 gels shows that they exhibit WCA values ranging from
42 -77°.
This data explains the rea son for cell adhesion on AZO-8-PE G-Acr NH 3 gels with increase in
AZO content. It means a s we keep on increasing the organic a romatic unit in the PE G matrix,
the hydrophilicity of the PEG decreases and it moves to more h ydrophobic surface. As cells
like the surface of moderate wettabilit y so they adhere on AZO-8 -PE G-Acr NH 3 gels. It is
noticeable that 50% AZO-8-PEG-Acr NH 3 gel shows maximu m adhes ion because the WCA
value is around 77° which is the most liked surface by the cell as shown in figure 6.3.
6.4.3 Antibacterial a nd anticancer activity
The cell cytotoxicity and cell adhesion results gave us a hope that these materials can be a
used for cell testing. It means these mate rials can be used to promot e cell adhesion and if
these materials also exh ibit some antimicrobial p roperties then i t would be a good
advancement in their studies.
Subsequently, in ord er to evaluate the antimicrobial activity, monomer which was
crosslinked with PEG to form gel was tested for the an tibacterial tests against E.coli an d

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anticancer activity against H ep -G2. For these studies, IC50 val ues, activity difference an d
Hill coefficient were determined show n in Table 6.2 .
Table 6. 2: Antibacterial a nd anticancer data o f AZO monom er with E.col i and Hep - G2
AZ 1 - Ac r

IC50
(mg/mL)

Activity differe nce

Hill co efficient

E.coli

0.8085

33

0.97

HepG2

0.0005

57

2.0

The half maximal inhibitory concentration (I C50) is “a measure of the eff ectiveness of a
substance in in hi bitin g a specific biological or b iochemical function” [160]. S maller is the
IC50 value m ore effective will be the compound. Activity difference disp lays that how much
difference in the activity of host cell can be caused by the minimal concentration of the
tested compound.
The Hill coefficient offers a way to q uantify the degree of interaction as well as binding
mode of the te sted co mpounds with host cel l. A coefficient of 1 specifies non -cooperative
binding. Value higher than one shows positive cooperativity. Whereas, the value less than
one displays negative cooperativity is indicated. Positively cooperative binding means when
one guest molecule is b ound to the host bind ing site, its affinity for other guest molecules
in creases. Negatively cooperative b inding means when one guest molecule is bound to the
host, its affinity for other guest molecules decreases. Non -cooperative binding shows that
the affinity of the host for a gu est molecule is i ndependent of guest concentra tion. [161].
The IC 50 value achieve d for E.coli was 0.8085 mg/mL while the activity difference of 33%
shows that AZO causes 33% activity diff eren ce in E.coli bacterium. The hill coefficient value
of 0.97 which is close to 1 sugg ests in dependent cooperativity.
For Hep -G2, IC50 value was 0.0005 mg/mL while the activity differen ce of 57% shows that
AZO causes 5 7% activi ty difference in Hep -G2. The hill coefficient v alue of 2.0 suggests
positive cooperativity.

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The above mentioned te sts shows that the synthesized and tested AZO e xhibits the
antibacterial and anticancer activity. The AZO monomer seems to exh ibit more anti cancer
activity. Thus these tests provide interesting preli minary evid ence of the potential test ing of
AZO-8-PEG-Acr NH 3 gels for further biological evaluation for diff erent microbial stra ins.
6.5 Conclusio n
In this chapter, the biological evaluations of th e AZO/PEG hybrid gels ( AZO-8-PEG-Acr NH 3 )
were detailed. The cyto compat ibility of AZO-8 -PEG-Acr NH 3 gels for cell applications was
tested against M ouse Fib roblast (L -929 ). Tests were conducted wit h different AZO: PEG
ratios. All AZO-8-PEG-Acr NH 3 gels showed good compatibility to Mouse Fibroblast (L -929 ).
It was observed that AZO-8-PEG-Acr NH 3 gels substrate promotes the cell adhesion of
Mouse Fibroblast (L -929 ) the refore, cell adhesion beh avior was monitored. Noticeable
increase in the cells adherence was observed on the AZO -8-PEG-Acr NH 3 gels substrate
exhibiting higher ratio of AZO. In order to eval uate the dependence of the cell adherence on
the surface wetta bility, WCA values o f AZO-8-P EG -Acr NH 3 gels were determined. It was
observed that the substrate with more AZO content p romotes cell adhesion. The reason for
this marked increase is the change in the W CA values of AZO-8-PEG-Acr NH 3 g els. The
addition of aromatic unit in the PE G backbo ne causes this modification. This addition
changes the hydrophilic PEG surface to hydr o phobic one. The WCA values of AZO-8-PEG-
Acr NH 3 gels were found between 42- 77°. More cells adhere to the 50% AZO-8-PEG-Acr NH 3
gels because the exhibit the optimal WCA values for cell adhesion.
Antibacterial and anticancer activity of AZO monomer was monitored in solution state . AZO
monomer showed moderate antibacterial activity against E. coli bacterium and good
anticancer activity against Hep -G2 cell line.

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7 Chapt er 7
P att erni ng of AZ O - 8 - PE G - A cr NH 3 Gels
AZO-8-PEG-Acr NH 3 gels substrates are capable of supporting the adhesion of mouse
fibroblast (L-929) cells, while pure PEG is anti-adhesive to cells. Patte rned AZO-8-PEG-Acr
NH 3 gels cou ld act as a p latform for the selective adhesion of cells. The focus of this chapte r
would be the patternin g techniques using AZO- 8-PEG-Acr NH 3 gels. In this contribution, we
present a ne w and vers atile technique to p attern the AZO-8-PEG-Acr NH 3 ge ls on hyd rogel
surface. In this work, the AZO-8-PEG-Acr NH 3 gels were patterned on PEG -575 hydrogel
through a novel “M icro - de -Molding (µ-dM) patterning method. The patterns were
transferred to PE G-575 hydrogel films. Afterwards, these p atterned AZO-8-PEG-Acr NH 3
gels on PEG-575 hy drogels were investigated in cell culture with mouse fibroblasts (L - 929)
to evalu ate the feasibility of using these patterned surfaces for guiding cell adhesion.
Selective and guided cells adhesion was observed on those patterned h ydrogels. Gold
nanoparticles (Au NPs) pat terned on hydrogel substrate are a suitable can didates for
selective cell adhesion. Additionally, the micrometer -sized (50 µm) Au NPs strip es were
patterned by means of our recently develope d “micro -contact depri nti ng method”. Those
patterned Au NPs stripes were transferred to AZO- 8-PEG-Acr NH 3 gels. In the end,
Holographic lithography of AZO-8-PEG-Acr NH 3 gels was cond ucted. Patterns (SRGs) w ere
fabricated by mean s of a holographic set up. They were analyzed through atomic fo rce
microscopy (A FM ).

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7.1 Introductio n
Surface patterning acts as a vital tool in b iomedical research. The abilit y to sp ecifically
define the spatial locati on of biomolecules and/or cells on surfaces or in three dimensions
offers an influential tool to researche rs to insp ect the interaction between b iomolecules,
cells and artificial materials in a controlled sp a tial environment. First and the critical step in
the pathogenesis of implant infection is the bacterial adhesion to the surface. Structured
and pat terned surfaces inhibit bacterial growth because of reduction in conta ct area
between cells and surf ace [85]. Topographies generate a substantial reduction in bacterial
adhesion (30 – 45%) co mparative to the smooth control samples irrespective of surface
hydr ophobicity/h ydrophilicity[162].
Patterning of the functional polymer s at dif ferent scale length plays a vital role in several
research areas including medicinal science, cell biology, ti ssue engineering an d the
development of optics and electronics [163 ], [164]. The interest in th e polymer patterning
have coined in from the ab undance of fun cti onalities of polymers and a variety of
applications of the patterns [165], [1 66] . Topography of the material surfaces is
acknowledged to affect the cell behavior at different levels: from adhesion up to
differentiation. To investigate the v arious aspects of cell behavior , different micro - and
nano -patterning techniques have b een employed to create patterned surfaces [119], [162],
[163], [1 67] – [171]. Hyd rogels are particularly considerable for this purpose as they fulfill
numerous characteristics of the mechanics and architecture of most soft tissues [172].The
presence of water in gel matrix pr o vides softness, a high porosity, flexibility, larg e surface
area, and biocompatibility [173] – [176]. PEG hydrogels are among the most extensively
studied and widely used polymers for cell studies[177], [178]. The gel network p roperties,
swelling an d the elasti city can be controlled by tuning the chain length functionalities of
polymers[92 ] .
PEG-based substrate s are non-permissive to bact erial adhesion, protein adsorption, and
eukaryotic cell adhesion[174], [179], [18 0]. M oreover, optical transparence of the PEG
hydr o gels allows effective opti cal detection with minimal background signals [178], [181 ] .

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PEG being resistant to non -specific protein adsorptions and undesired cell at tachments,
serve as a perfect cell-resistant substrate for those biomedical investigations , where specific
and controlled bio-interactions are targeted [182].
Cell micro-patterning is mainly focused on mi cro-fabrication techniques ba sed on glass or
silicon or substrate s, which limit applicat ions to tissue engineering [183 ]. PE G hydrogels,
being inert and protei n -repellent surface have established to be useful as a background
platform for the in vitr o investigation of cell behavior ap plied in tissue enginee ring and
biosensor systems [174], [178].
Thus, in this work, PE G matrices have been chosen as the b asic material to template the
immobilization of AZO unit. As explained in chapter 6, AZO monomer exhibits an tibacteria l
properties and the AZO- 8-PEG-Acr NH 3 gels are proved to promote cell adhesion . Therefore,
it would be interesting to develop the p atterne d surfaces of these materials for selective cell
adhesion.
Patterned surfaces of AZO-8-PEG-Acr NH 3 gels were fabricated by using different patterning
techniques. New patterning techniques were dev eloped to obtain the patterns. Here a new
and versatile technique to pattern the AZO-8-PEG-Acr NH 3 gels on hydrog el su rface is
presented. In this work, the regularly arranged (5 0 µm) AZO-8-PEG-Acr NH 3 gels were
patterned on PEG-575 hydrogel through newly designed “Micro - de -Molding (µ- dM)”
method. The pat terns were transferred convenient ly and accurately to PEG -575 hydrogel
films. Subseq uently these pat terned AZO-8 -PEG -Acr NH 3 gels on PEG -575 hydrogels were
examined in cell culture with mouse fibroblasts (L - 929) to e valuate the viability of using
these patterned surfaces for guided cell adhesion.
Recently, it was revealed that the presence of non -functionalized Au NPs on cell - anti -
adhesive PE G hydrogels assi sted cell adhesion [11 9], [184 ], [185]. Au NPs can be
immobilized onto hydrogels by chemical adsorption, physical adsorption or by entrapment
method [18 6]. Physical adsorption method is o ne of the widely use d m ethods for modifyin g
Au NPs on the hydrogel surface [187]. As the development of nan otechnology and bio -
conjugate chemistry pr ogresses, immobilization of Au NPs can be do ne by highly specific

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biomolecular interactions [188 ] . In this study, our recent ly developed “ Wet M icro-Contact
Deprinting Method” was implemented to immobilize Au NPs onto gel surfaces[189].
Last patterning method employed to AZO-8-PEG-Acr NH 3 gels was Holographic lithography.
This is a three dimensi onal techniq ue est ablished by the projection of three dimensional
images via interference patterns from in tersecting laser bea ms. With the app ropriat e
number of beams and alignment, we can p attern the photoactive mater ial [190 ] .
When an azobenzene -c ontaining material is illuminated by a superposition of two coherent
laser beams, in add ition to the formation of a volume gr ating modulations of the initially
flat surface of the material can occur g enerating surface relief grating (SRG) [72] .
Azobenzene-based polymers are known well for this effect [ 191 ] (for details see chapter 1).
Hardly any studies have been focused on azobenzene containing polymer gels because the
phase sep aration in polymer gel can scatter the visible lig ht and reduce the d iffraction
efficiency[192 ] .
In this study azobenzene c ontaining AZO-8-PEG-Acr NH 3 gels were subjected to holographic
lithography and patterns (SRG) were fabricated by means of a holographic setup. A key
advantage of holographic patterning to the typically used patterning method is that
patterning is controlled remotely using light. Moreover, this is rather a simple techniq ue to
generate 3D pattern q uickly without the use of mask, masters and serial scanning . The
patterning is an all-optical process com pleted in single ste p and needs no post -treatment.
Likewise, the grating period and the height can be adjusted in a wide range [193 ] .
These three dif ferent patterning methods of AZO-8-PEG-Acr NH 3 gels may find use in the
elucidation of fundamen tal structure – function relationships, tissue engineering and the
formation of immobilized cell and protein arrays for biotech nology.
7.2 Mate rial and Methods
7.2.1 Prepara tion of Hydro gels
For all the pat t erning methods, AZO/PEG gels ( AZO-8-PEG-Acr NH 3 gels) and PEG-575 was
used.

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7.2.1.1 AZO-8-PEG-Acr NH 3 gels
The synthetic method u sed for the preparation of AZO-8-PEG-Acr NH 3 gels was exp lained in
Chapter 2.
7.2.1.2 PEG-575 liquid precursor
PEG-575 liquid precursors having 1% of PI ( 1 wt. % with respect to the amount of the
precursor) were mixed in a v ial. Later it w as placed into oven at 6 0 °C for 5 min until the
mixture became cle ar. Afterwards, for patterning experiments, 80 μL of the mixture was
dispensed on a clean g lass slide, covered with a cover glass (18 m m × 18 mm Carl Roth
GmbH & Co KG). The glass slide was placed un der the UV lamp (λ = 366 nm Vilber Lourmat
GmbH) for 30 min at a working distance of 10 cm in a nitrogen-filled glovebox.
7.2.2 Pat ternin g methods used for AZO- 8 -PEG- Ac r NH 3 gels
Patterning of the gels was done to study the cell adhesion behavior of mouse fibroblast (L -
929) cells. All the patterning studies were done in col laboration w ith Cigdem Yeşilda ğ (LL
member).
7.2.2.1 Fill-Molding in Capillaries (FIMIC) Method
In the p ast years, a new soft lithographic method has b een established in our group: the
Fi ll - M olding I n C apillaries ( FIMIC ) method. T his method has enabled us to fabricate sub-
micrometer precise pa tterns of elasticity. Th ose are surface patter ns, ideally horizontal
perfectly smooth in hydrated sta te an d possess an alternating elasti city. In addition, thes e
fabricated FIMIC platforms may incorporat e chemical funct ionalities, which can be
introduced in a sp atially controlle d manner. T he detailed descripti on of these methods can
be found in LL publications[194] .
The FIMIC method was ap plied to make the p attern of AZO-8-PEG-Acr NH 3 gels as shown in
Figure 7.2. The PE G-575 mold was prepared by the replication from the silicon master of
(width × distance × height = 10 × 50 × 10) as shown in Figure 7.1, which conta ins patte rned
stripes fabricated into microscale lines.

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Figure 7.1: Schema tic view o f a patterned silico n master
Silicon wafers were cleaned with water, acetone and isopropa nol befor e use and dried
using stream of nitrogen . Proceeding to the replicat ion, the cleaned silicon masters were
fluorinated with 97% trichloro (1H, 1H, 2 H, 2H-perfluorooctyl) silane (Si gma-Aldrich,
Steinheim, Germany). The viscous PEG -575 liquid was dispen sed on the silicon master
covered with a thin glass coverslip and exposed to UV light (λ = 366 nm ) for 1 5 min at a
working d istan ce of 1 0 cm in a g lovebox. Following the crosslinking, mold was pee led off
from the silicon master with the help of tweezers.
The patterned PEG-575 mold was then inverte d on the glass slide and liquid precursor of
AZO-8-PEG-Acr NH 3 gels was filled in the channe ls. The AZO-8-PE G-Acr NH 3 gels form
within the voids of PEG-575 molds.

Figure 7.2: FIMI C patter ning technique appli ed on AZO-8-PEG -Acr NH 3 gels
7.2.2.2 Micro- de -Molding Method (µ-dM)
In addition to FIMIC, a new method of pat terning “Micro - de - Molding” ( µ - dM ) was
designed. The PDMS mold was obtaine d from the silicon master of 10- 50 - 10 size. The mold
was casted by the replic ation from the silicon m aster of ( width × distance × height = 10 × 50

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× 10) as displayed in Figure 7.1, which contains patterned stripes fabricated into microscale
lines.
The mold was inverted on the silicon wafer and the gaps were filled with AZO- 8-PEG-Acr
NH 3 gels precursor solution. The patterned lines of AZO-8-PEG-Acr NH 3 gels we re obtained
by peeling off the mold. The patterned strips were characterized by using optical and
surface electron microscopy. Later on, 80µ L PEG -575 p recursor solution was dispensed on
the pat terned of AZO-8-PEG-Acr NH 3 gels substrate an d UV cured to obtain a hybrid gel as
shown in Figure 7.3.

Figure 7.3: Micr o- de -Mo lding (µ-dM) patterni ng technique ap plied on AZO-8 -PEG-Acr NH 3
gels
These hybrid patterned surfaces are interesting candidate to study the cell adhesion on
mouse fib roblast (L-929) cells. 25% AZO-8-PEG -Acr NH 3 gels were selected because they
exhibit moderate wettab ility and good consisten cy. Later on, these patterned surfaces were
subjected to cell studies with mouse fibroblast (L -929) cells.

7.2.3 Pat terning of AZO-8-PEG-Acr NH 3 gels with Au NPs
The pat terning of AZO- 8-PEG-Acr NH 3 gels w as also done with Au NPs p rovided by Cigd em
Yeşildağ. The spherical Au NPs of almost 80nm with citrate capping agent were synthesized
by seed mediated grow th methods. The “wet micro contact deprinting” patterning method
designed by Cigdem Yeşildağ et al. was used[189] .
Firstly the silicon mas ter was patterned with gold nanoparticles ( Au -NPs) and then the
pattern was transferred to AZO- 8-PEG-Acr NH 3 gels. Au NPs were initially deposited on (3-

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Aminopropyl) triethoxysilane (APTES) -modified silicon wafers t hrough the ele ctrostatic
interaction between the positively charged amin o groups of APTES and negative charges on
the citrate-stab ilized Au NPs. Next, 80µL p recu rsor solution of AZO-8-PE G-Acr NH 3 gels was
dispensed on the Au NPs-decorated silicon wafers.
Lastly, the immobilization of Au NPs on the gels was accomplished by peeling off the gels
from silicon wafer s. U sing this p rocedure, Au NPs were efficiently transferred f rom the
silicon wafers to the AZO-8-PEG-Acr NH 3 gels surface.
Figure 7.4 presents the schematic view of p atterning of AZO-8-PEG-Acr NH 3 gels with Au
NPs. Transfer of the Patt erned Au NPs fro m S ilicon Wafer to AZO-8- P EG-Acr NH 3 gels was
achieved by dispensing the 80 µL precursor solutions of 25% AZO-8-PEG-Acr NH 3 gels. The
substrate was allowed t o gelate for almost 2 h. Then, it was p eeled off carefully to t ransport
all Au NPs from silicon t o the gel s urface. The final samples were kept i n Petri dish and were
subjected to further analysis by SEM for the evaluation of successful tr a nsfer.

Figure 7.4: Patterni ng of AZO-8- PEG-Acr NH 3 gels with Au NPs
7.2.4 Hologra phic Lithography
Holographic studies w ere done in collaboration with Dr. Tina Sabel (LL member) at
institute of optics and atomic physics TU Berlin, Germany. A major advantage of
holographic patte rning to the typically used patterning method is that it can be controlled
remotely using light and is relatively simple to generate 3D pattern quickly without the use
of mask, masters and serial scann ing. Surf ace relief gratings were an alyzed th rough atomic
force microsco py (AFM).

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Figure 7.5: Experimen tal s etup for holograph ic pat terning
Figure 7.5 presents the experimental setup used for holographic lith ography use d for the
holographic patte rning. Green laser of 532 nm was used to produce the in terference
pattern. Laser output power was 17.9mW and power per exposure beam was 6mW. Beam
diameter was kept 6mm.
7.3 Results and Disc ussion
Patterned surfaces with three different techniques were obtained.
7.3.1 Pat terning of AZO-8-PEG-Acr NH 3 gels
7.3.1.1 FIMIC Method
In order to make the p atte rned AZO-8-PEG-Acr NH 3 gels, the first patterning method used
was FIMIC[194]. The PEG-575 mold was filled with AZO-8-PEG-Acr NH 3 gels precursor. NH 3
used for gelation of AZ O-8-PEG-Acr NH 3 gels contains water; hence it swells the PE G 575
mold. That’s wh y filling was not achieved complete ly. The filling of the swollen mold fails
the patterning. Thus FIMIC pat terning technique did not wor k well with A ZO-8-PEG-Acr
NH 3 gels.
7.3.1.2 Micro- de -molding Method (µ-dM)
To overcome the failure of FIMIC with AZO-8-PEG-Acr NH 3 gels, a novel and versatile
patterning method “Micr o - de -Molding ( µ- dM )” was d esigned with the help of Cigdem
Yeşildağ (a L L member). Patterned lines having width of 50 µm 25% AZO-8-PEG-Acr NH 3

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gels were prepared b y using 10- 50 - 10 PDMS master. Later on, PEG 575 was dispended on
the patterned surface and UV cured to get a h ybrid surface exhibiting AZO-8 -PEG-Acr NH 3
gels.
7.3.1.2.1 Characterization of AZO -8-PEG-Acr NH 3 gels pattern
The 50 µm lines of AZO-8-PEG-Acr NH 3 g els were achieved by fillin g the PDMS mold on
glass substrate. The patterned gels line s wer e formed by “ amine M ichael -type addition ”
method. The mold was peeled off and the p atter ned substrate an alyzed through op tical and
surface electron microscopy as shown in Figure 7.6 and Figure 7.7

Figure 7.6: Opt ical image o f 50µm lines of 2 5%AZO-8-PEG-Acr NH 3 gels obtained on glass

Straight line s with 50 μm in width (distance between stripes is 10 μm) can be recognized
from Figure 7.6. Optical image displays that the patterned AZO-8-PEG-Acr NH 3 gels stripes
of any dimensions can be fabricated by using the mold.
The SEM imaging of the patte rn obtained from 50 - 50 -10 PDMS master is shown in Figure
7.7 . Straight lines with 50 μm in width (distance bet ween stripes is 50 μm) can be
recognized. The image at higher resolution indicates that the p atterne d 25% A ZO -8-PEG-Acr
NH 3 gels stripes are not smooth; rather they possess an uneven surface morphology.
The optical and SEM images show that the µm lines a re obtained positiv ely.

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Figure 7.7: SE M images of 50µm lines of AZO-8- PEG-Acr NH 3 gels o btained on glass

7.3.1.2.2 Characterization of AZO -8-PEG-Acr NH 3 gels pattern obtained by “Micro - de -
Molding” by atomic force microscopy
To investigate the success of our novel “ M icro- de -Molding ” (µ -dM) patterning method , the
micro patterned lines were then transferred on PEG -575 hydrogel. The final patterned
substrate was characterized by AFM.
The micrograph showed in Figure 7.8 displays that a hybr id PE G-575 and 2 5% AZO-8-PEG-
Acr NH 3 gels patterned surface was formed positively. The 50µm lines corr espond to 25%
AZO-8-PEG-Acr NH 3 gels lines while the 10 µm lines relate to PEG -575 lines.
The morphology of the patte rned surface is not smooth r ather it exhibit s some topography.
Also it can be seen that that PEG -575 lies are a bit higher in the dried patterned surface .
While in the swollen st ate, the AZO- 8-PEG-Acr NH 3 gels lines are exp ected to sho w a high
topography because of swelling in the medium.

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Figure 7.8: AF M image of Micr o- de - Molded (µ-d M) patterned AZ O-8-PEG-Acr NH 3 gels

7.3.1.2.3 Cell Adhesion tests
In order to monitor the selective adhesion of mo use fibroblast (L -929) cells, adhesion tests
were carried ou t on pat terned AZO-8-PEG-Acr NH 3 gels
Although the PEG-based hy drogel background is supposed to be a nti -ad hesive, and the
AZO-8-PEG-Acr NH 3 gels ar e not (bi o) function alized to assist cell adhesion they exhibit a
moderate surface wett abilit y which assi st the cell adhesion as explained in Chapter 6.
Therefore, selective cell ad hesion on AZO-8-PEG-Acr NH 3 gels micro-stripes was expected
(see Figure 7.9)

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Figure 7.9: Cell adhesion stud ies on Micro- de -Mo lded (µ-dM) pattern ed surface
The cellular behavior of mouse fibroblast L-929 cells on the surface of PEG-575 hydrogel
with 25% AZ O-8-PEG-Acr NH 3 gels (50 µ m in width and 1 0 µm in distance) was further
investigated. It is evident from the F igure 7.9 that the cells adhere to the pat terned AZO-8-
PEG-Acr NH 3 gel stripes after 24 h of in cubation. The cells grow with random distribution.
Hardly any cells can be seen on the non -ad hesive PEG-575 (10 µm ) lines. The patterned
AZO-8-PEG-Acr NH 3 gels stripes indeed induce cell adhesion. T hus thes e patterned surfaces
can be used for selective adhesion of cells on a particular substrate.
7.3.2 Pat terning of AZO-8-PEG-Acr NH 3 gels with Au NPs
The patterning of A ZO-8-PEG-Acr NH 3 gels wa s done with spherical Au NPs of 80 nm. These
Au NPs possessing citrate capping agent were syn thesized by see d mediated growth
methods. “ Wet micro contact deprinti ng” p atterning method was used to pattern the AZO-
8-PEG-Acr NH 3 gels. The patte rning methods was designed by Cigde m Yeşildağ et al. [189] .
Initially, the silicon m aster was patterned with gold NPs and th en the pat tern was
transferred to AZO-8-PEG-Acr NH 3 gels.

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7.3.2.1 Characterization of Immobilized Au NPs on the Surface of AZO-8-PEG-Acr NH 3
gels by SEM
To investigate the transfer efficiency of Au NPs from silicon wafers to AZO-8-PEG-Acr NH 3
gels the via “wet micro contac t deprinting pat terning method” SEM was utilized to
characterize the patterned AZO-8-PEG-Acr NH 3 gel with Au NPs after the transferring
procedure.

Figure 7. 10 : SEM ima ges o f Au- NPs transferred o n 25% AZO-8-PEG -Acr NH 3 gel
The size of Au NPs is 80 nm, the width of stripe is 50 μm and t he distance between
patterned stripes is 1 0 μm. Resulted patterned AZO-8-PEG-Acr NH 3 gels are shown in
Figure 7. 10 . S traight g rey lines with lines of 50 μm in width (distance bet ween strip es is 10
μm) can be recognized indicating that patterned Au NPs stripes are formed on the AZO-8-
PEG-Acr NH 3 gels. At higher magnification, it can be seen how the Au NPs are distributed on
the gels; not perfectly homogeneously, but large agglomerations are not present either.
The result demonstrates that the pat terned Au NPs stripes can be transferred to the AZO-8-
PEG-Acr NH 3 gel in an ordered way. Aft er in corporation of Au NPs on to the surface of gels,
an increase in the stiffness and roughness of c omposite gels may induce cell adhesion. The
present study shows that the patterned Au NPs stripes can be used for controlling a cellular
response affect the morphology and adhesion of the cells. Importantly, patterning cells on
the substrate is useful for the development of tissue engineering and fundamental studies in
cell biology.

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7.3.3 Hologra phic patterning of AZO- 8-PEG-Acr NH3 gels
In order to investigate the r esponse of azobenzene (AZO) containing polymer gels toward
holographic lithography, experiments were c onducted. In this study 5% AZO-8-PEG-Acr
NH 3 gel films was subjected to holographic exposure to observe the diff raction pattern and
obtain the SRG. We aimed to form the 2D gratings with interfering tw o beams of Green laser
of 532 nm with laser o utput power of 17.9mW an d power per expo sure b eam was 6 mW.
Beam diameter used was 6mm. Expected grating period was approximately 2µm.
Figure 7. 11 represent s the AFM image of the 2D grating of 5% AZO-8-PE G-Acr NH 3 gel film
obtained through holographic exposures. The original 5% AZO-8-PEG-Acr NH 3 gel film was
not smooth rather it e xhibits crystal spheruli tes as explained in Chapter 4. Therefore the
grating obtained cannot b e smooth either. \A gr ating of around 2 µm w as obtained with 5%
AZO-8-PEG-Acr NH 3 gel film which provide s the basic evidence to study further the
holographic prospects of these materials.
Holographic exposures of AZO-8- PEG-Acr NH 3 gel film with higher AZO ratio did not
achieve clear patterning which might be attributed to diffraction of these gels.

Figure 7. 11 : AFM imag e of the 2D grati ng of 5% AZO-8-P EG-Acr NH 3 gel film
7.4 Conclusio ns
In this work, several p atterning techniques were employed to de velop pat terned A ZO-8-
PEG-Acr NH 3 gel. Different patterning techniques were used to achieve the d esired goal.

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The first patterning techniq ue employed was FIMIC but it did not work because the gelator
(NH 3 ) used fo r the preparation of AZO-8-PEG-Acr NH 3 gels has water which swells the PEG-
575 mold so f il ling was not achieved.
In order to overcome the above mentioned problem a ne w versatile “Micro - de -Molding ( µ -
dM )” patterning technique w as developed. This method enabled us to obtain patterned
AZO-8-PEG-Acr NH 3 gels stripes on the surface of PEG-575 hydrogel.
These patterned surfaces were characterized through optical microsco py using SEM and
AFM. T hese measurements provided a pr oof for the positive develo pment of the desired
pattern. The patterned sur faces were subjected to cell studies to monitor the selective
adhesion of mouse fibroblast (L-929) cells. The L-929 showed a selecti ve adhesion on the
AZO-8-PEG-Acr NH 3 gels stripes. The cells were distributed randomly.
Keeping in mind the importance of Au -NPs, patterning of AZO-8-PEG-Acr NH 3 gels w as also
carried out with Au-NPs of 80 nm size. S EM ch aracterization of the pattern p rovided the
clue for eff ective p atterning.
At the end, holographic lithography w as used to pattern the AZO-8-PEG-Acr NH 3 gels. SRG
of 2 µm spacing w e re expected and the results were reasonably promising.
These patterned surfaces can act as a te mplate for studying the selective cell adhesions;
serve as a tool for various biomedical application

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A bstr act
Chemically incorporated azobenzenes (AZO) with in a matrix can gene rate multi -responsive
materials that exhibit both temperature and light responsivity; hence act as multi-
responsive system. The main aim of the work present ed here was to design novel multi -
responsive gels having chemically crosslinked azobenzene moiety in corporated into Poly
(Ethylene Glycol) (PEG) matrix. The chemically bonded azobenzenes in the gel matrix are
expected to provide a control over the gel properties using light and temperatur e stimuli.
We expected to control the actuation an d sensing property of synthesized gels using both
stimuli. As the PEG matrix used is biocompatible, the gels are also exp ected to possess
biocompatibility.
Di -acrylate b ased azobenzene monomers (A ZO) were synthesized and w ere subjected to
gelation with PEG derivatives (PEG -575 , 8-PEG-Acr and 8-PEG-VS) using different
techniques and strategies. UV curing was found not to work with all of three te sted PE G
derivatives even with high ratio of P I and C L. Am ine Michael-type addition app lied as
second alternative d id not work w ell with P EG -575 but 8-PEG-Acr and 8-PEG-VS mad e
tunable gels by using this technique. These gels were p repared with varying AZO: PEG ratio
an d could accommoda te up to 50% wt. ratio of both p recursor s. Hence, we positi vel y
synthesized Novel ch emically crosslinked A ZO/PE G g els using “ amine M ichael -type
addition”.
The characterization of chemically crosslinked AZO/PEG gels was done using different
techniques. Structural studies were done using FTIR, Raman, and UV -Visible spect roscopic
studies. Rheological measurements were done to evaluate the gelation time and mechanical
strength of gels using ti me, frequency and temperature sweep. Surface charact erization gels
were done using atomic force (AFM) and surface electron microscopy ( SEM).
Light irradiation produces geometric changes in azobenzenes and under appropriate
conditions, these changes can be translated into larger-scale motions, even in macroscopic
movements of the material system. In order to test the responsiveness of AZO/PEG gels
with light and temperature, they were subject ed to many experiments. AZO/PEG polymeric

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gels showed mechanical actuation under the sunlig ht. Also, these gels showed response to
body heat and proved t o be thermal responsive as well.
Also, azobenzenes exhibits some antimicrobial properties against bacteria and fungi. PEG is
a well-known biocomp atible substrate in biomedical in dustry . Therefore PE G matrix with
chemically crosslinked anti microbial azobenzene moiety draws an attention for biological
evaluation. The cytoco mpatibility of AZO/PEG gels for cell app lications was tested against
Mouse Fib roblast (L-929). Gels showe d goo d compatibilit y to Mouse Fibroblast (L -929)
cells. It was observed that AZO/PEG gels substrate promotes the cell adhesion of Mouse
Fibroblast (L-929) therefore, cell adhesion beh avior was monitored. Noticeable increase in
the cells adherence was observed on the AZO/PEG gels substrate exhibitin g highe r ratio of
AZO. In order to evalua te the dependence of t he cell adherence on the surface wettability,
WCA values of AZO/P EG gels were determined. It was observed that the substrate with
more AZO content promotes cell adhesion. The reason for this marked in crease is the
change in the WCA values of AZO/PEG gels. The addition of aromat ic unit in the PEG
backbone causes this modification. This addition changes the hydrophilic PEG surface to
hydr ophobic one. Antibacterial and anticancer activity of AZO monomer w as monitored in
solution state . AZO monomer showed moderate antibacterial activ ity against E. col i
bacterium and good anticancer activity against Hep -G2 cell line.
In order to provide a platform for the selective cell adhesion, several patterning technique s
were app lied and co mpared. A novel p atterning te chnique “Micro - de - Molding” was
designed to display the AZO/PE G gels in a micro - pat tern at the biomaterial’s surface. These
patterned surfaces were characterized thro ugh opti cal microscopy using S EM and AFM.
These measurements provided a proof for the p ositive development of the desired patte rn.
The pat terned surfaces were subjected to cell studies to monitor the selective adhesion of
mouse fibroblast (L-929) cells. The L -929 sho wed a select ive adhesion on the AZO/PEG gels
stripes. The cells were distributed randomly. Keeping in mind the importance of Au -NPs,
patterning of AZO/PEG gels was also carri ed out with Au -NPs of 80 nm size. SEM
characterization of the pattern prov ided the clue for effective pat terning . At the end ,
holographic litho graphy was used to pat tern the AZO/PEG gels. S RG s of 2 µm spacing were
expected and the results were reasonably promising.

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Z usammenfassung
Chemisch inkor poriert e Azobenzole (A ZO) in einer Matrix können multires ponsive
Materialien generieren, die sowohl Temperatur - als auch Lichtresponsivität aufweisen; nun
als multiresponsives System agieren. Das Haup tziel der Arbeit war es neuartige
multiresponsive Gele zu designen, die chemisch vernetzte Azobenzoleinheiten in
Poly(Ethylenglycol) (PEG) -Matrix enthalten. Es wird erwartet, dass die chemisch
gebundenen Azobenzole in der Gel-M atrix die Eigenschaften vom Gel mittels Licht- und
Temperatureinfluss kontrolliert. Wir erwarteten die Betätigungs - und Sen soreigenschafte n
der synthetisie rten Gele durch beide E inflüsse zu kontrollieren. Da die verwen dete PEG -
Matrix biokompatibel ist, wird das azobenz ol -gebundene Gel auch als biokompatibel
erwartet.
Di -acrylat basierte Azobenzole (AZO) wurd e n synthetisiert und mit PE G -Derivaten zur
Gelbildung unterworfen (PE G-575, 8-PEG-Acr und 8-PEG-VS), wobei verschiedene
Techniken und S trategien benutzt wurden. Bei der UV - Vernetzung wurde festgestellt, dass
es nicht bei allen der drei verschiedenen PEG -Derivaten funktioniert, sogar b ei ho hen PI
und CL-Anteilen. Ami ne Michael-Type Addition Reaktion wurde als zweite alternative
Vernetzungsmethod e e ingesetzt, welche bei PEG 575 nicht sehr gut funktionierte aber b ei
8-PEG-Acr un d 8-PE G-VS abstimmbare Gele hervorbrachte. Diese Gele wurden durch die
Variation von AZO: PE G-Anteilen hergestellt und es konnten 50 gew.% d er beiden
Prekursor en untergebr acht werden. Infolgedessen haben wir ne uartige chemisch ve rnetzte
AZO/PEG- Gele durch „Amin - Michael Typ Additionsreaktion“ erfolgreich sy ntheti siert.
Die Charakterisierung der chemisch vernetzten AZO/PEG-Gele wurd e mittels verschiedener
Techniken vollzogen. Strukturelle Studien wurden mittels FTIR, Raman, und UV-Vis-
spektroskopischen S tud ien untersucht. Rheologische M essungen wurd en vollzogen, um die
Vernetzungsz eit un d die mechanische Festigk eit der Gel e mittels Ze it-, Frequenz- und
Temperaturabtastung zu best immen. Oberflächencharakterisierungen der Gele wurden
mittels Atomkraftmikroskopie (AFM) und Raste relektronenmikr o skopie (REM/SEM)
durchgeführt.

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Lichteinflüsse führten zu geometrischen Veränderunge n der Az obenzole und unte r
bestimmten Voraussetzung en konnten diese Veränderungen auch in größere Skale n
übertragen werden, sogar als makroskopische Bewegungen des Materials. Um die
Responsivität d es AZO/PEG-Gels mit Licht und Temperatur zu bestimmen, wurd en
verschiedene Experimente durchgeführt. AZO/PEG polymere Gele wiesen mechanische
Bewegungen unter S onnenlicht auf. Diese Gele zeigten auch Responsivität auf
Körpertemperatur auf und wurden bewiese n, dass sie auch auf thermische Anregungen
reagieren.
Ebenfalls haben die A zobenzole antimikrobielle E igenschaften gegenüber Bakterien und
Pilze. PEG ist ein weit b ekannter, biokompatibler Stoff in der biomedizinischen Industrie.
Demzufolge weist die PEG -Matrix mit chemisch vernetzten, antimikrobiellen
Azobenzoleinheiten eine hohe Aufmerksamkeit in der biologischen Auswertung auf. Die
Zytokompatibilität der AZO/PEG -Gele für zelluläre Anwendungen wurden mittels Maus
Fibroblasten (L-929) getestet. Die Gele zeigten eine gute Kompatibilität gegenüber Maus
Fibroblast (L -929) Zellen. Es wurd e beob achtet, dass AZO/PEG -Gel Substrate
Zelladhäsionen von Maus Fibroblasten (L-929) fördern, diese wurd en
Zelladhäsionsexperimente gezeigt. Einen auffälligen Anstieg der Ze lladhäsion auf AZO/PEG -
Gel Substrate mit hohen AZO-Anteilen wurde beobachtet. Um den Einfluss der
Oberflächenbenetzungsverhalten auf die Zelladhäsion zu überprüfen, wurden
Wasserkontaktwinkelmessungen (WCA) der AZO/PEG-Gele untersucht. Eine hohe
Zelladhäsion bei hohen Anteilen an AZO wu rde festgestellt; der Grund für dieses Verhalten
wurde durch die verschiedenen WCA-Werte der AZO/PEG -Gele festgestellt. Das
Herbeiführen der aromatischen Einheiten ins PEG -Rückgrat war der Grund für diese
Modifikation. Diese Addition änderte die hydrophile PEG -Oberfläche zu einer hyd rophoben
Oberfläche. Antib akterielle- und Antikrebsaktivitäten von AZO -Monomeren wurden in der
Lösung gezeigt. AZO-Monomere zeigten eine mittelmäßige anti bakterielle Aktivität
gegenüber E-coli Bakterien und eine gute Antikrebsaktivität gegen Hep -G2 Zell- Li nien.
Um eine Plattform für d ie selektive Zelladhäsion zu ermöglichen, wurden versch iedene
Strukturierung stechnik en angewendet und verglichen. Eine neuartige
Strukturierung smetho de „Micro - de - Molding“ wurde entworfen um die AZO/PE G -Gele als

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Mikrostruktur en auf Biomaterialoberflächen zu designen. Diese strukturierten Oberflächen
wurden mittels optischer Mikroskopie, SEM und AFM charakterisiert. Diese Methoden
bestätigten die erf olgreichen Stru kturierungen der g eplant en Oberflächen. An den
strukturierten Oberfl ächen wurden Zelladhäsion der Maus Fibroblasten (L -92 9)
untersucht. Die Maus Fibroblasten (L -9 29) zeigten eine selektive Adhäsion auf den
AZO/PEG-Gel Streifen. Die Ze llen verteilten sich zuf ällig au f den Linien. Aufgrund der
Wichtigkeit der Au NPs wurden AZO/PEG-Gele mit 80 nm gr oßen Au NPs strukturiert. SEM
Charakterisierung bes tätigte die erfolgreiche Strukturierung. Sc hließlich wurde die
holographische Lithographie für die Strukturierung der AZO/PEG-Gel e benutzt. SRGs von 2
µm Abständen wurden erwartet und die Resultaten waren vielversprechend.

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A ckno w ledgements
First and foremost I e xpress my sincerest gratitude to my supervisor, Prof. Dr. Marga C.
Lensen, who has suppor ted me throughout my thesis with her patience, motivation, and
im mense knowledg e w hilst allowing me the room to work in my own way. Her guidance
helped me in all the time of research and writing of this thesis.
I would also like to thank Prof. Dr. Svetlana Santer for being the part of thesis reviewing
co mmittee and reading my thesis.
Special and sincere thanks also go to Prof. Dr. Michael Gradzielski for his collaboration in
monitoring wettability changes, thermal studies and Rheological m easurements. I owe a
special thanks to Dr. Jens Pe ter von Kries and Dr. Martin Neu enschwender (Leibniz institute
of molecul ar pharmacology, Berlin, Germany) for providing all the facilit ies an d assistance
for the biological studies. Thanks to Dr. Tina Sabel (LL member) for p roviding equipment
and help for the holographic studies. I am a lso in debted to Dr. Michael S chwarz e and
Maximilian Neumann for p roviding the solar simulator equip ment for photoresponsive
studies.
I also want to than k the membe rs of the Lensen Lab for the help they offered me when I
joined the group. I owe heartiest gratitude to LL members, Dr. Tina Sabel, Dr. Gonzalo de
Vicente Lucas, Dr. Zhen fang Zhang and especially to m y friend Cigdem Yesildag for their
encouragement, insightful comments and valuable guidance to carry out my project
objectively. I am thankful to all my friends in Germany and Pa kistan, who always supp orted
and encouraged me during my difficult times , ga ve me good advice an d made my life
colorful .
The completion of this work wo uld not have b een p ossible without the funding which was
ki ndly provided by Higher Education Commission of Pakistan (HEC) and PAS TU Berlin. I
am highl y obliged to b oth agencies for providing the all the faciliti es for research and my
stay in Berlin .
I highly ackno wledge and appreciat e the endless support , encouragement and sacrifices
offered by my parent s for educating and p reparing me for my f uture. I t is hard using wor ds

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to express my gratitude to them. I am very much thankful to my husband Rahim Gul for his
love, understanding and continuous support to reach my goal and realize my dreams.
Special an d heartiest thanks to my daughter Hadia Gul and son M uhammad Ahmad who
remained patie nt an d cooperative during this whole tenure, which he lped me to complete
this goal successfully.
I am in debted to my brothers and sisters specially Nab ila Rahman and Riffat Rahman who
put all their lo ve and support for b ringing up Hadia Gul an d Muhammad Ahmad during the
times when I was not around.

Rahima Rahman

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