Rheological Study and Molecular Dynamics Simulation of Biopolymer

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Rheological Study and Molecular Dynamics Simulation of Biopolymer Blend Thermogels of Tunable Strength Erfan Dashtimoghadam, Ghasem Bahlakeh, Hamed Salimi-Kenari, Mohammad Mahdi Hasani-Sadrabadi, Hamid Mirzadeh, and Bo Nyström Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00846 • Publication Date (Web): 21 Oct 2016 Downloaded from http://pubs.acs.org on October 21, 2016

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Biomacromolecules

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Rheological Study and Molecular Dynamics

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Simulation of Biopolymer Blend Thermogels of

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Tunable Strength

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Erfan Dashtimoghadam a,b,c, Ghasem Bahlakeh d, Hamed Salimi-Kenari e,

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Mohammad Mahdi Hasani-Sadrabadi f,g, Hamid Mirzadeh c,*, Bo Nyström b,*

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a

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Department of Developmental Sciences, Marquette University School of Dentistry, Milwaukee, WI, USA.

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b

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c

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Department of Chemistry, University of Oslo, Oslo, Norway. Department of Polymer Engineering and Color Technology, Amirkabir University of Technology, Tehran, Iran

11

d

Department of Engineering and Technology, Golestan University, Aliabad Katool, Iran.

12

e

Faculty of Engineering & Technology, University of Mazandaran, Bābolsar, Iran.

13

f

14 15 16

Laboratoire de Microsystemes (LMIS4), Institute of Microengineering, Institute of Bioengineering, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

g

Parker H. Petit Institute for Bioengineering and Bioscience, and G.W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, USA.

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ACS Paragon Plus Environment

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ABSTRACT:

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The temperature-induced gelation of chitosan/glycerophosphate (Chs/GP) systems through

3

physical interactions has shown great potential for various biomedical applications. In the

4

present work, hydroxyethyl cellulose (HEC) was added to the thermosensitive Chs/GP solution

5

to improve the mechanical strength and gel properties of the incipient Chs/HEC/GP gel in

6

comparison with the Chs/GP hydrogel at body temperature. The physical features of the

7

macromolecular complexes formed by the synergistic interaction between chitosan and

8

hydroxyethyl cellulose in the presence of β-glycerophosphate disodium salt solution have been

9

studied essentially from a rheological point of view. The temperature and time sweep rheological

10

characterizations of the thermogelling systems revealed that the sol-gel transition temperature of

11

the Chs/HEC/GP blends is equal to 37 °C at neutral pH; with increasing HEC content in the

12

solutions, more compact networks with considerably improved gel strength are formed without

13

influencing the gelation time. The formed hydrogel matrix has enough mechanical integrity and

14

adequate strength for using it as injectable in-situ forming matrices for biomedical applications.

15

The classical Winter–Chambon (W-C) and Fredrickson– Larson (F–L) theories were applied to

16

determine the gel point. In view of the obtained results, it is shown that the F–L theory can be

17

employed as a robust and less tedious method than the W-C approach to precisely determine the

18

gel point in these systems. At the end, molecular simulation studies were conducted by using ab

19

initio quantum mechanics (QM) calculations carried out on Chs and HEC models, and molecular

20

dynamics (MD) simulations of solvated Chs/HEC blend systems showed the binding behavior of

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Chs/HEC polymers. Analyses of interaction energy, radial distribution function, and hydrogen

22

bonding from simulation studies strongly supported the experimental results; they all disclosed


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that hydrogen-bond formation between Chs moieties with regards to HEC chains plays an

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important role for the stabilization of the complexes.

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Keywords: Thermoresponsive hydrogel, chitosan; hydroxyethyl cellulose, rheology, ab initio

5

quantum mechanics, molecular dynamics simulation.

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23


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1. INTRODUCTION

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In the past few years, thermosensitive biopolymer-based hydrogels that are biodegradable

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and biocompatible and with adjustable gel characteristics have attracted a great deal of interest

4

because of their unlimited potential as therapeutic delivery systems and for tissue engineering as,

5

e.g., injectable depot systems.

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polymers with a lower critical solution temperature (LCST) can often be manipulated to undergo

7

gelation near body temperature when they are introduced into the target tissue in an easily

8

injectable way6. Certain thermogelling systems are able to remain in solution at low temperature,

9

where growth factors, cells and other biologically active elements can be incorporated, and

10

1-5

Thermosensitive systems, especially those based on natural

became in situ solid at body temperature.

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There are several studies in the literature on block copolymers of the type ABA, which form

12

gels at elevated temperatures. Classical examples are block copolymers based on poly(ethylene

13

oxide), such as poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (trade name

14

Pluronics)

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biomedical applications, these poloxamers may have limited areas of use because of inherent

16

features such as high injection temperature, weak mechanical strength, the need for high polymer

17

concentrations, and rapid dissolution in aqueous media due to the low hydrophobicity of the PPO

18

spacer block.12-14 The shortcomings of these gelling systems are usually that high polymer

19

concentrations are needed to form gels and the gels exhibit fairly weak mechanical strength.

7-10

, which have been studied extensively as in situ gelling polymeric systems.11 For

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To circumvent these defects, several research groups have considered the use of various

21

stimuli-responsive hydrogels, based on aqueous polysaccharide systems 15, 16. Among the various

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gelling polysaccharide systems, stimuli-responsive chitosan (Chs)/glycerophosphate (GP) is an

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attractive system that exhibits an interesting temperature-induced sol–gel with transition under


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physiological conditions. The polysaccharide chitosan has been used in a wide range of medical

2

and pharmaceutical applications because this biopolymer is biocompatible, biodegradable, it

3

displays an anti-bacterial nature, and it is bio-adhesive with permeation enhancement ability.5, 17-

4

19

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In view of the special physicochemical features of the thermoresponsive Chs/GP system, a

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wide range of biomedical applications is possible, such as local drug and gene delivery, suitable

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microenvironment for living cells to maintain functional characteristics after injection for tissue

8

engineering. 5, 20

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In the past, different stimuli-responsive Chs/GP formulations have been developed. Several

10

studies

have

focused

on

the

understanding

of

11

chitosan/glycerophosphate (Chs/GP) solutions.

12

chitosan solution, the pH of the solution increases because phosphate groups neutralize chitosan

13

amine groups and enhanced interactions occur. If a semidilute chitosan solution is heated,

14

gelation occurs.23 In this process, many types of interactions between Chs and GP come into play

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21

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heating. ii) Increased structuring of free water by the glycerol groups of GP and this leads to

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enhanced interchain hydrophobic attraction. iii) Temperature-induced transfer of protons from

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chitosan amine groups to the phosphate moieties of GP, and this reduces both chain charge

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density and chitosan attraction to GP; this promotes attractive interchain and hydrogen-bonding

20

between chains. It can generally be argued21, 24-27 that presence of GP in chitosan solutions leads

21

to screening of electrostatic repulsion and enhanced hydrophobic effect emerges, resulting in

22

favorable conditions for gel formation.24 In other words, hydrophobic interactions between

17, 18, 21, 22

the

thermogelling

mechanism

of

. The idea is that when GP is added to

including: i) reduced polarity of the chitosan chains and augmented hydrophobicity upon


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chitosan chains and reduced solubility are the main driving force for this thermally induced sol–

2

gel transition.

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However, several research groups22, 25, 26 have established that the mechanical properties of

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the thermogelling Chs/GP systems may not be sufficiently durable for biomedical applications.

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To overcome this limitation, composites consisting of the Chs/GP system and another

6

biocompatible polymer have been proposed.

7

that the gel strength of the Chs/GP system can be increased by adding 2% gelatin due to

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enhanced molecular interactions between gelatin and chitosan. These types of thermosensitive

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hydrogels, based on Chs/gelatin/GP compositions, have been successfully developed for nucleus

22, 27, 28

For instance, Roughley et al. suggested

22, 27

29

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pulposus regeneration, as a controlled release system for ferulic acid delivery.

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approach, the effect of adding hydroxyethylcellulose (HEC) ,which is a nonionic hydrophilic and

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biodegradable biopolymer with a typical polysaccharide structure, on the physico-chemical

13

properties of aqueous mixtures of thermosensitive Chs/GP solutions have been tested as a cell

14

carrier for cartilage repair by Hoemann et al.

15

these studies on HEC/Chs/GP blends showed the formation of hydrogels with high mechanical

16

strength and uniform microporous network structure.

30

and Naderi‐Meshkin et al.

28

In a similar

. The results from

17

In spite of several studies on hydrogels of Chs/HEC/GP blends, there is still a serious lack of

18

detailed understanding of the gelation mechanism of these temperature-sensitive and tunable

19

blends that are controlled through intricate multiple interactions. In this work, critical quantities

20

such as gelation time, gelation temperature, gel strength of the Chs/HEC/GP thermosensitive

21

hydrogels are characterized by using experimental techniques such as rheology, turbidity, and

22

small angle light scattering. To gain a better insight into the gelation process of Chs/HEC/GP

23

blends with a deeper understanding of the sol-gel transition and incipient gel features, the focus


6

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of this study is on the analysis of the rheological results in combination with theoretical

2

techniques, based upon ab initio quantum mechanics (QM) and molecular dynamics (MD)

3

simulations. The computational QM and MD investigations were carried out to obtain a detailed

4

electronic/atomic scale insight into the experimentally-found observations; in particular the

5

chain-chain interactions governing the gel behavior of Chs/HEC systems. These techniques

6

allow us to attain microscopic view of gel characteristics and to elucidate new information, such

7

as inter/intra-molecular hydrogen bonds, interaction energy and correlation between Chs and

8

HEC, which cannot be achieved through experimental efforts. Computational methods have

9

recently been applied to investigate the behavior of thermoresponsive polymer chains in water.31-

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33

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2. EXPERIMENTAL

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2.1. Materials and Preparation of Solutions

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Chitosan (Chs, deacetylation degree of 84% and a weight-average molecular weight of ~

14

4×l05) was acquired from Pronova Biopolymers. Hydroxyethylcellulose (HEC) was obtained

15

from Hercules, Aqualon Division. The weight-average molecular weight of HEC was found to

16

be 4×l05 by using intensity light scattering.

17

supplied by Merck. To prepare chitosan/β-glycerophosphate disodium salt (Chs/GP) solution, as

18

well as the Chs/HEC/GP blends, Chs was first dissolved and mixed with various amounts of

19

HEC solutions. The prepared solutions were then cooled down to 4 ºC, and subsequently cold GP

20

solution in water was added drop-wise under magnetic stirring to adjust the pH of the polymeric

21

solutions to the physiological range (pH 7.4). In all formulations, the concentration of Chs was

22

fixed (2 wt %) and also the GP (0.78 M) concentration, but the HEC was varied (0, 0.5, and 1

34

Glycerol 2-phosphate disodium salt hydrate was


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wt%). The samples were assigned as Chs2.0/HECx/GP, where x is the weight percentage of the

2

HEC in the blend formulations.

3 4

2.2. Rheological Measurements

5

Oscillatory sweep experiments were carried out in a Paar-Physica MCR 301 rheometer using

6

a cone-and-plate geometry, with a cone angle of 1o and a diameter of 75 mm. This geometry was

7

employed in all measurements. To prevent evaporation of the solvent, the free surface of the

8

sample was always covered with a thin layer of low-viscosity silicone oil (the value of the

9

viscosity is practically not affected by this layer). The measuring device is equipped with a

10

temperature unit (Peltier plate) that gives an effective temperature control (± 0.05 oC) over an

11

extended time in the temperature range considered in this work. In the oscillatory shear

12

experiments, the values of the strain amplitude were checked to ensure that all measurements

13

were carried out within the linear viscoelastic regime, where the dynamic storage modulus (G‫)׳‬

14

and loss modulus (G‫ )״‬are independent of the strain amplitude. To determine gelation

15

temperature, the measurements were carried out over the angular frequency (ω) interval 1–10

16

rad/s, while the temperature was increased with a heating rate of 1 K min-1 and small strain

17

amplitude (γ) of 0.03 to minimize the perturbation of the network during the gel evolution. The

18

gelation time was monitored during time sweep measurements at 37◦C and at fixed angular

19

frequency of 1.0 rad/s. The sample was inserted between the cone and plate at ambient

20

temperature, and thereafter the sample was heated to 37 oC. When the temperature reached 37

21

o

22

the rheometer over an extended period of time.

C, the measurements started and the time evolution of the dynamic moduli was monitored with

23


8

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2.3. Small Angle Light Scattering

2

Small-angle light scattering (SALS) experiments have been carried out using polarized light

3

scattering. In all measurements, a 10 mW diode laser operating at a wavelength of 658 nm was

4

used as the light source. The laser beam was deflected and passed through the sample confined

5

between the parallel glass plates. Plate diameter was 43 mm and the gap between the plates was

6

small (0.25 mm) so that the effect of multiple scattering is less pronounced when the sample

7

becomes turbid at elevated temperatures. The CCD camera (driver LuCam V. 3.8), placed in a

8

parallel position to the screen, was utilized to capture two-dimensional images from the

9

scattering patterns of the samples with an exposure time of 200 ms. The temperature was

10

gradually changed from 10 to 45°C. Subsequently, the pictures were analyzed using the SALS

11

software program (version 1.1) developed by the Laboratory of Applied Rheology and Polymer

12

Processing, Katholieke Universite in Leuven, Belgium. The experimental details of this

13

technique have been provided in an earlier publication. 35

14

2.4. Turbidimetry

15

The turbidity values of Chs/GP solution and Chs/HEC/GP blends with different compositions

16

of HEC (0.5, 1 wt.%) were determined using an NK60-CPA cloud point analyzer from Phase

17

Technology, Richmond, BC, Canada. The scanning diffusive technique was utilized to

18

characterize phase changes of the solutions over a temperature range from 25 to 60 °C at heating

19

rate of 1°C min-1.36 The light beam from an AlGaAs light source, operating at 654 nm, is focused

20

on the measuring solution applied onto a glass plate coated with a thin metallic layer of very high

21

reflectivity. Directly above the sample, an optical system with a light-scattering detector

22

continuously monitors the scattered intensity signal (S) of the solution as it is subjected to

23

prescribed temperature alterations. The relation between the signal and the turbidity (τ) is given


9

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by the following empirical equation, τ(cm-1) = 9×10-9S3.751.37 The method of turbidity

2

determination and specification of the instrument have been described elsewhere.38

3 4

3. Computational Method and Simulation Details

5

3.1. Quantum Mechanics Modeling

6 7

In order to gain a fundamental understanding of interactions between Chs and HEC

8

polymers, ab initio QM calculations were applied for clusters of Chs-HEC and Chs-Chs. Due to

9

the computational time of such calculations, only one monomeric unit of each polymer (see

10

Supplementary Figure S1) was modeled. First, all the starting structures (i.e., Chs and HEC as

11

well as Chs-Chs and Chs-HEC clusters) were optimized using Hartree-Fock (HF) theory

12

employing 6-31G** basis set.39 The resulting geometries were further relaxed by density

13

functional theory (DFT) 40, 41 using B3LYP 42, 43 first with 6-31G** basis set and then with larger

14

basis function of 6-311G**.44 All these calculations were carried out with Gaussian 09

15

software.45 Finally, to determine the magnitude of interactions for Chs/HEC complex, the

16

interaction energy ( ∆E ) for structures extracted from DFT calculations was calculated.46

17

3.2. Molecular Dynamics Simulation

18

Besides QM examinations, MD simulations were performed to shed further light on

19

interactions controlling the gel characteristics of Chs/HEC systems. Unlike QM tools, which are

20

limited to molecular systems with small length scale, the use of MD simulations allows to

21

examine interacting systems at larger and more realistic length scales even under solvation

22

conditions, whereby mimicking experiments.

23

MD simulations were conducted on three-dimensional cells consisting of Chs and HEC


10

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polymers solvated by water molecules. In this regard, polymeric Chs and HEC 10-mer were

2

initially built (shown in Supplementary Figure S1). The Chs and HEC chains were then

3

minimized. Afterwards, we used of the 5-nm cubic simulation cell where is included of random

4

orientation and arrangement of ten optimized Chs chains together with five optimized HEC

5

molecules. The constructed simulation box was then solvated, as illustrated in Supplementary

6

Figure S1. Furthermore, to acquire a better understanding of interaction characteristics of

7

Chs/HEC blends, and influence of interactions upon the morphology of polymers, another

8

solvated cell contacting only Chs chains was built and simulated. The MD simulation details

9

have been summarized in Table 1.

10 11 12

Table 1. The MD simulation details considered for pure Chs and Chs-HEC blend simulation systems. System Pure Chs Blend Chs–HEC Chs chain length

10

10

HEC chain length

–

10

No. of Chs chains

10

10

No. of HEC chains

–

5

No. of water molecules

3210

3210

Total No. of atoms

11850

13260

13 14 15

For MD simulations, the starting solvated Chs and Chs-HEC blend simulation boxes were

16

initially subjected to NVT simulation of 50 ps at a temperature of 300 K, followed by 100 ps

17

NPT simulations (at 300 K and 1 atm). Then, simulations were run for 40 ns in NPT ensemble,

18

from which the last 10 ns were used for subsequent analyses. MD simulations were done by

19

GROMACS code47,

48

using OPLS-AA force field49,

50

for Chs and HEC chains, and SPC


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potential51 for water. All bond lengths were kept fixed via the LINCS algorithm.52 Non-bonded

2

interactions were truncated at 1.2 nm cutoff and long-range electrostatic interactions were

3

computed by Particle Mesh Ewald (PME) method.53 Leapfrog algorithm54 was employed to

4

solve equation of motion with a time step of 2 fs. The Nose-Hoover thermostat55;56 and

5

Parrinello-Rahman barostat 57 were used to control the temperature and pressure, respectively.

6 7

4. RESULTS AND DISCUSSION

8

4.1. Analysis of Rheological Data and Investigation of Gel Properties

9

Oscillatory sweep experiments were used to investigate the gelation temperature and the

10

viscoelastic features of temperature-induced gelling of the Chs/HEC/GP blends at different

11

mixture compositions. According to the method of Winter and Chambon58, the gelation

12

temperature can be determined by observation of a frequency-independent value of tan δ (=

13

G''/G') obtained from a multi-frequency plot of tan δ versus temperature. Alternatively, the

14

gelation temperature can also be obtained by plotting the “apparent” viscoelastic exponents n'

15

and n'' (G'~ ωn', G''~ ωn") calculated from the frequency dependence of G' and G'' at different

16

temperature and observing a crossover where n'=n''=n (see the inset graphs in Figure 1(a-c)).59, 60

17

Figure 1 shows a decrease in the damping factor during the gel formation of Chs/HEC/GP

18

solutions and this trend indicates enhanced elasticity of the thermogelling network with raising

19

temperature. Furthermore, the effect of different HEC concentrations on the gelation temperature

20

of Chs/HEG/GP blends is illustrated in Figure 1. Both methods yield the same gelation

21

temperature (37 °C) for all the considered polymer systems, and the addition of 0.5 and 1 wt %

22

HEC to the Chs/GP solution did not significantly alter the incipient gelation temperature from 37

23

°C as shown in Figure 1(b, c). This is an interesting finding for biomedical applications that the


12

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gel temperature is virtually not affected by the addition of HEC. However, as will be discussed

2

below, the gel strength of the gels will be enhanced as the HEC content in the blends increases.

(a)

(b)

(c)

3

Figure 1. Illustration of methods for the determination of the gel point: viscoelastic loss tangent

4

as a function of temperature at the indicated frequencies for (a) Chs2.0/HEC0.0/GP, (b)

5

Chs2.0/HEC0.5/GP, and (c) Chs2.0/HEC1.0/GP. The inset plots show changes of the apparent

6

exponents (n' for the storage and n'' for the loss moduli) at various temperatures.

7


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The gelation process is reminiscent of a phase separation phenomenon. In view of this, as the

2

formation of spatially bicontinuous percolated networks is considered as the critical point for

3

gelation, the Fredrickson–Larson (F–L) theory was also implemented as an alternative approach

4

to determine the bicontinuous percolation structuring point of thermosensitive Chs/HEC/GP

5

blends. Generally, it can be assumed that the gelation temperature, the temperature where the

6

spatially bicontinuous percolation structure forms, can be interpreted as the spinodal

7

temperature, which was introduced by the F-L theory as the temperature where the phase

8

separation mechanism switches from nucleation and growth to spinodal decomposition. In this

9

framework, thermoresponsive gelation and phase separation are phenomena that are closely

10

linked to each other, and this coexistence has been recognized in a number of studies.61-63

11 12 13 14

(b)

(a)

15 16 17 18 19 20

(c)

21 22 23


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1 2 3

Figure 2. Determination of the sol-gel transition temperature based on Fredrickson-Larson

4

theory for (a) Chs2.0/HEC0.0/GP, (b) Chs2.0/HEC0.5/GP, and (c) Chs2.0/HEC1.0/GP.

5 6

Fredrickson et. al

64

developed a mean-field theory to derive the contribution to the shear

7

stress caused by critical fluctuations near the order–disorder transition. They demonstrated that

8

extra stress induced by critical fluctuations can be estimated from the total free energy

9

differential change through integration over the whole wave vector space. Based on the derived 18,

10

expression (the details of derived equations based upon F-L theory were reported previously

11

65

12

shows a divergence behavior near the sol-gel transition temperature. In the framework of the F-L

13

approach, the peak positions of the curves depicted in Figure 2 determine the gel temperatures

14

(37 ⁰C) for the Chs/HEC/GP blends with different HEC content. The gel-temperatures of 37 oC

15

are in perfect agreement with those obtained from the Winter–Chambon model.

) for the storage and loss modulus, according to F-L theory, the resulted ratio of (G' T/G''2)2

16

The thermal evolution of the dynamic moduli (G' and G'') of Chs2.0/HEC0.0/GP and

17

Chs2.0/HEC1.0/GP solutions are shown in Figure 3 (a). In the initial stage of the gelation process

18

up to about 33 oC, G''>G' and the viscous response dominates, but at higher temperatures G'>G''

19

and the elastic modulus dictates the viscoelastic response and an incipient gel is formed (37 oC).

20

At higher temperatures, the post-gel region is evolved; G' rises strongly and a solid-like behavior

21

emerges with an enhancement of the mechanical properties of the gel. The inset plot reveals that

22

a very similar behavior is observed for the system with added HEC.

23

This observation indicates that the raise in temperature leads to breaking of intramolecular

24

hydrogen bonds stabilizing chitosan molecules and the water clouds around chitosan chains are


15

Biomacromolecules

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1

perturbed. This may lead to neutralization of chitosan chains by heat-induced proton transfer

2

from protonated amino groups from GP.

3

This suggests that hydrophobic interactions between Chs-Chs chains and disruption of

4

hydrogen bonding of Chs-HEC chains are responsible for the formation of the incipient gel

5

network. This type of thermosensitive gelation has previously been reported and the same

6

explanation of the gelation mechanism was proposed by other researchers. Cho and co-workers

7

66

8

weaker electrostatic repulsive interactions between chitosan chains. Therefore, the synergic

9

effect of Chs-Chs hydrophobic interactions and hydrogen bonding between chitosan chains are

10

assumed as the main molecular forces in the temperature-induced gelation process. Chenite et al

11

17, 21

12

and this plays a key role in the gelation process. Li et al

13

CHs/HEC/GP solutions. They hypothesized that multi-site hydrogen bonding between hydroxyl

14

groups at each side chain of HEC with the hydroxyacetyl group on the glucose ring in chitosan

15

plays a predominant role in hydrogel formation and the control of the gel strength. 29, 68-70 Cheng

16

et al.

17

chitosan/gelatin/ glycerol-phosphate blends as a cell carrier. As a result, they assumed that the

18

formation of physical entanglements between chitosan and gelatin chains at higher temperature is

19

a main driving force to build-up the gel-network structure.

proposed that heating the solution may lead to a progressive neutralization of chitosan and

reported that hydrophobic interactions between Chs-Chs chains are enhanced upon heating

22

67

reported a rheological study of

reported the preparation and rheological characterization of thermosensitive


16

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Biomacromolecules

(b) (a) 1 2

Figure 3. Temperature evolution (a) and time dependence at 37 ⁰C (b) of the storage (G') and

3

loss (G'') modulus for the Chs2.0/HEC0.0/GP sample. Insets show the corresponding graphs for

4

the Chs2.0/HEC1.0/GP sample.

5

The results above show that the addition of nonionic HEC polymer to the Chs/GP solution

6

leads to augmented gel strength at elevated temperatures because of formation of boosted intra-

7

and inter-molecular physical interactions (i.e., hydrogen bonding and hydrophobic interactions)

8

into the Chs/HEC/GP system. In addition, the HEC chains may contribute to more entanglements

9

and thereby a stronger gel network.

10

Furthermore, the gelation time is considered as one of the most important features for in situ

11

gel-forming systems. Figure 3 (b) shows the time evolution of the gel for the Chs2.0/HEC0.0/GP

12

and Chs2.0/HEC1.0/GP systems by measuring the storage modulus (G') and loss modulus (G'') at

13

37 ⁰C. In this type of experiment, the sample is rapidly heated in the rheometer to 37 oC and the

14

time evolution storage and loss modulus of the gel are monitored immediately after the sample is

15

37 oC. After an induction time of about 90 s, G' raises very fast while G'' is virtually constant.

16

This feature suggests that the elastic response prevails and the gel-network is formed. This can

17

be rationalized in the following scenario. The chitosan concentration (2 wt%) considered in this


17

Biomacromolecules

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Page 18 of 35

1

work is well located in the semidilute concentration regime and we have a transient network at

2

shorter times than 90 s. However, at the gel temperature (37 oC) and after that 90 s, strong

3

hydrophobic interactions come into play and the network becomes physically crosslinked. The

4

rheological results clearly show that the reorganization of the network and the development of

5

temperature-induced intermolecular interactions take a few minutes. In the presence of HEC

6

chains (inset plot in Figure 3b), the general rheological behavior is very similar, but in this case

7

the growth of G' is stronger due to Chs-HEC interactions. Furthermore, the results show that

8

addition of HEC to the systems leads to higher values of G’ than without HEC. For instance,

9

after 240 s from start of the gelation process, it is clear that in the presence of HEC chains

10

(Chs2.0/HEC1.0/GP) the value of G’ is 88.5 Pa, which is significantly larger than the value (64

11

Pa) of G’ in the absence of HEC (Chs2.0/HEC0.0/GP) (cf. Figure 3b). The higher values of G' in

12

the presence of HEC indicate that HEC addition strengthened the gel network. The details of

13

molecular interactions for Chs and HEC association are further discussed in the section about

14

computational modeling.

15 16

17

18 19

In this work, the theoretical model of Winter and Chambon has been utilized to describe the gel strength for an incipient gel, according to the following relationship 71,

‫ܩ‬ᇱ =

ீ ᇲᇲ ௧௔௡ఋ

= ܵ߱௡ Γሺ1 − ݊ሻ cos ߜ

(1)

where Γ(1-n) is the gamma function, n is the relaxation exponent, δ is phase angle between stress and strain, and S is the gel strength parameter.

20

Muthukumar elaborated a theoretical model72, based on the assumption that variations in the

21

strand length between cross-linking points of the incipient gel network give rise to changes of the

22

excluded volume interactions, to rationalize values of n in the whole physically accessible range


18

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Biomacromolecules

1

(0

Rheological Study and Molecular Dynamics Simulation of Biopolymer

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