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Tiêu đề Effect of Polymer-Polymer Interactions on the Flow Behavior of Some Polysaccharide-Based Hydrogel Blends
Tác giả Tilen Kopač, Aleš Ručigaj, Matjaž Krajnc
Trường học University of Ljubljana, Faculty of Chemistry and Chemical Technology
Chuyên ngành Chemistry
Thể loại Journal Article
Năm xuất bản 2022
Thành phố Ljubljana
Định dạng
Số trang 12
Dung lượng 6,29 MB

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Nội dung

This study is the continuation of our previous work (Kopaˇc, Abrami, et al., 2021) where the theoretical approach of polymer-polymer interaction to predict the crosslink density of hydrogels was introduced. This theory is further extended to the flow properties of hydrogels that allow the analysis of synergistic effect in hydrogel systems and the understanding of possible anomalous behavior of certain mixtures.

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Available online 15 March 2022

0144-8617/© 2022 The Authors Published by Elsevier Ltd This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Effect of polymer-polymer interactions on the flow behavior of some

polysaccharide-based hydrogel blends

University of Ljubljana, Faculty of Chemistry and Chemical Technology, Veˇcna pot 113, SI-1000 Ljubljana, Slovenia

A R T I C L E I N F O

Keywords:

Zero-shear viscosity

Yield stress

Shear thinning

Hydrogel interactions

Blending effects

Mathematical modeling

A B S T R A C T This study is the continuation of our previous work (Kopaˇc, Abrami, et al., 2021) where the theoretical approach

of polymer-polymer interaction to predict the crosslink density of hydrogels was introduced This theory is further extended to the flow properties of hydrogels that allow the analysis of synergistic effect in hydrogel systems and the understanding of possible anomalous behavior of certain mixtures Various hydrogel structures were prepared accordingly by blending scleroglucan, anionic nanocellulose, Laponite dispersions and alginate solution The relationship between mechanical and flow properties of the hydrogel network was carefully studied and eventually described by mathematical model The linear model equation to predict yield stress of hydrogels

in relation to the crosslink density was designed showing a satisfactory agreement between experimental data and model predictions The correlation was adjusted by defining a proportionality coefficient, representing the energy defined per moles of crosslinks that can be used to restore the deformation

1 Introduction

Rheology plays an essential part in the field of scientific research as

well as in industrial development (Boger, 2009), particularly in the case

of complex systems such as hydrogels whose deformation behavior and

flow properties cannot be described by the classical approaches

belonging to continuum mechanics (the elasticity theory and fluid

dy-namics) (Lei, Li, Xu, & Liu, 2021) The rheological characterization

represents an important tool also to investigate hydrogels' structural

characteristics (Cao, Duan, Zhang, Cao, & Zhang, 2021) in order to

evaluate their potential use and performance in various biomedical

applications (Cal´o & Khutoryanskiy, 2015) Particularly in the case of

carbohydrate polymer systems, which present several advantages over

the synthetic polymers (Farrukh, Mustafa, Hussain, & Ayoub, 2018), the

rheology enables a detailed investigation of the mechanical (crosslink

density ρx and average mesh size) and flow properties (zero shear

vis-cosity η0 and yield stress τ) of hydrogels (Kopaˇc, Abrami, Grassi,

Ruˇcigaj, & Krajnc, 2021; Kopaˇc, Krajnc, & Ruˇcigaj, 2021; Kopaˇc,

Ruˇcigaj, & Krajnc, 2020; Lapasin, Abrami, Grassi, & ˇSebenik, 2017;

ˇ

Sebenik, Krajnc, Aliˇc, & Lapasin, 2018; ˇSebenik, Lapasin, & Krajnc,

2020)

So far, theoretical approaches, that can satisfactorily predict the flow

behavior of complex hydrogels, are undersupplied in literature, except

for very dilute fluids In our previous work (Kopaˇc, Abrami, et al., 2021)

a mathematical model that can satisfactory predict the ρx of hydrogel network on the basis of the number of crosslinks formed due to the polymer-polymer interactions (ionic, hydrogen, electrostatic, and other covalent interactions) was developed and validated for polysaccharide hydrogels Motivated by such ρx prediction, we hypothesize that equa-tion can be leveled up to predict the η0 and τ0 of similar hydrogel sys-tems The literature (Hoti et al., 2021; Lapasin et al., 2017; ˇSebenik

et al., 2020; Zhao, Mayumi, Creton, & Narita, 2017) reports the effect of

ρx on flow behavior of gels based on rheological rotational tests where for the most part, η0 and τ0 increase with the ρx of gels The detailed study of correlations between ρx and η0 or τ0 can be the starting point for further mathematical modeling For this purpose, the comprehensive rheological characterization of (complex) hydrogel systems based on rotational tests is needed to be performed and studied in detail To prove the high applicability of a new mathematical approach for analyzing the

τ0 and η0 data, the hydrogel systems based on TEMPO (2,2,6,6-tetra-methylpiperidine-1-oxyl) oxidized nanocellulose (TOCNF), sodium alginate (ALG), scleroglucan (SCLG) and Laponite (LAP) were prepared The baseline of the model development is associated to the under-standing of polymer-polymer theory in the hydrogel network starting with the careful study of polymers' chemical structures and their possible interactions To fulfil the above orientations, the brief basic

* Corresponding author

E-mail address: matjaz.krajnc@fkkt.uni-lj.si (M Krajnc)

Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

https://doi.org/10.1016/j.carbpol.2022.119352

Received 11 January 2022; Received in revised form 3 March 2022; Accepted 9 March 2022

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structural description of TOCNF, ALG, SCLG and LAP is needed ALG is a

linear copolymer composed of (1 → 4)-linked α-L-guluronic (G) and β-D-

mannuronic (M) residues of varying sequence Anionic carboxyl groups

give the hydrogel pH responsivity (Way, Hsu, Shanmuganathan, Weder,

& Rowan, 2012) in an alkaline release environment and also allowing

control of ρx (Lin, Bruzzese, & Dufresne, 2012), porosity (Pati˜no-Mas´o

et al., 2019) and flow properties (Kopaˇc et al., 2020) by crosslinking via

divalent ions (e.g., Ca2+) (Curvello & Garnier, 2020; Hecht & Srebnik,

2016; Xu et al., 2019)

TOCNF is linear polysaccharide, anionic constituent biopolymer The

physical and chemical properties of cellulose fibers (Lundahl, Berta,

Ago, Stading, & Rojas, 2018) and cellulose-based materials (John &

Thomas, 2008; Liang, Bhagia, Li, Huang, & Ragauskas, 2020) are mostly

influenced by the formation of intra- and intermolecular hydrogen

bonding (Lin & Dufresne, 2014) which are allowed by the presence of

three hydroxyl groups on each β-(1–4)-glucopyranosyl unit (Klemm

et al., 2011; Liang et al., 2020) Significant amounts of C6 carboxylate

groups are selectively formed on each cellulose nanofibril surface by

TEMPO-mediated oxidation that gives the anionic character to

biopolymer (Isogai, Saito, & Fukuzumi, 2011) and the possibility of

ionic crosslinking (Kopaˇc, Krajnc, & Ruˇcigaj, 2021)

The nonionic biopolymer SCLG is used as a thickener and suspending

agent (Lapasin et al., 2017; Paolicelli et al., 2017) The β-D

-glucopyr-anosyl units (1,3) linked in the main chain create the internal structure

in addition to the three triplex chains which hold the hydrogen bonds

together in the center of the triplex Such a conformation is

character-ized by high stiffness, which is reflected in the special properties of

aqueous solutions of SCLG in a wide pH range and even at relatively high

temperatures (Lapasin et al., 2017) Intra- and intermolecular hydrogen

bonds between the main SCLG chains lead to the formation of stable

rigid triple helices in aqueous solutions (Nazmabadi, Shirdast, Sharif, &

Aalaie, 2020) Furthermore, a single β-D-glucopyranosyl bound to the

unit (1,6) on the main chain prevents polymer precipitation and

inter-molecular aggregation (Coviello et al., 2005; Lapasin et al., 2017)

The synthetic hectorite LAP single layer nanoparticles in a form of

rigid, disk-shape crystals were used as a rheological modifier LAP

hydrogels own a negative charge of faces as a result of Na+ions

disso-ciation Besides, the edges of LAP disks are slightly positively charged

due to the protonation of hydroxyl groups (Park, Dawson, Oreffo, Kim, &

Hong, 2020; ˇSebenik et al., 2020) The face-edge attractions cause a

gelification process, when the system does not contain salt, and depend

on pH, resulting in a gel network also known as a “house of cards”

structure (Becher et al., 2019) Therefore, the LAP addition to weak

polysaccharide-based system could contribute to faster development of

final gel properties (aging), improve the mechanical properties and

shear thinning character of the matrix (ˇSebenik et al., 2020) At last, the

more detailed analysis of the structural formula of TOCNF (Isogai et al.,

2011; Liang et al., 2020), ALG (Homayouni, Ehsani, Azizi, Yarmand, &

Razavi, 2007), SCLG (Coviello et al., 2005), and LAP (Ruzicka &

Zac-carelli, 2011) can be found elsewhere

In this study, neutral (SCLG) and anionic (TOCNF and ALG)

biopolymer-based hydrogels in addition to LAP were prepared in various

concentrations and blended in different weight ratios with the addition

of assorted crosslinking agent concentrations to form complex hydrogel

systems (i.e., ionically crosslinked blends of polysaccharide polymers in

addition to the layered inert component) The effect of polymer and

crosslinking agent concentration, respectively, and the polymer fraction

dependence in the preparation of hydrogels on flow behavior of the

complex hydrogel systems was analyzed Prepared complex hydrogel

systems were subjected to comprehensive rheological rotational tests in

order to broaden the knowledge of the rheological characteristics of

different biopolymer-based systems The study of hydrogel samples

provided the explanation of polymer-polymer interaction effects on the

flow behavior offering the basis for determination of crucial parameters,

such as η0 and τ As a result, the theoretical outlines of the model

development to predict the flow properties were provided following the

polymer-polymer interaction theory correlated to experimental insights

As an upgrade to a wide range of experimental data, a new mathematical approach to analyze η0 and τ0 is eventually given, which can be equiv-alently used to describe the synergistic effect on shear behavior owing to their interrelationship

2 Experimental

2.1 Materials

The TEMPO oxidized nanofibrillated cellulose, a linear poly-saccharide composed of cellobiose repeating units linked by β-(1–4) glycosidic bonds, was purchased in freeze-dried powder from The Pro-cess Development Centre, University of Maine (UMaine PDC), USA The material specifications given by the producer are: (i) chemical formula

of [(C6H10O5)x(C6H9O4COONa)y], (ii) powder density of 1.5 g/cm3, (iii) aqueous gel density of 1.0 g/cm3 (1–3 wt%), (iv) carboxylate level is 0.2–2 mmol/g solids, (v) pKa of substituent group is 3.9, and (vi) the fiber dimensions are classified between 20 and 50 nm in width and lengths of up to several microns

Scleroglucan Actigum CS 11 was purchased from Cargill France SAS, France The supplier provides 99%min purity, and the average molar mass is 1.2⋅106 g/mol

Laponite XLG, synthetic layered silicate with a low heavy metals content was provided by BYK-Chemie GmbH, Germany The supplier specifications provide: (i) disk-shaped crystals with an average diameter

of 30 nm and a thickness of 1 nm, (ii) insolubility in water, (iii) hydrates and swells give clear and colorless colloidal dispersions in water, and (iv) obtains a high surface area (BET) of 800 m2/g

Sodium alginate, a polysaccharide made up of α-L-guluronic acid (G)

content and (1–4)-linked β-d-mannuronic acid (M) monomers, were supplied by Sigma-Aldrich (St Louis, USA) The molar mass of 2⋅104–6⋅104 g/mol and the high (70%) of G content is provided by supplier

Calcium chloride (CaCl2) was as received from Merck (Darmstadt, Germany) and sodium azide (NaN3, an assay of ≥99.5%) was supplied

by Sigma-Aldrich (St Louis, USA)

2.2 Sample preparation

The gels were prepared by slowly added dry TOCNF, ALG, SCLG or LAP in demineralized water with 0.02 wt% of NaN3 (to prevent micro-biological contamination) at constant stirring (500 rpm) by using a four- blade propeller stirrer (pH value of sample was 7) After 2 h, the dispersion was placed in an ultrasonic bath for 1 h (to achieve more homogeneous dispersion - ALG in water form clear solution and so was excluded from this process) The dispersions were stirred overnight at

750 rpm at room temperature

The complex hydrogel systems were prepared by blending single biopolymer dispersion TOCNF or SCLG and LAP or ALG in different weight ratios For homogenization, the same procedure was used as previously reported The samples were then quantitatively transferred in

a 3D printed mold to sample achieve the same characteristics as the rheometer measuring plate (a diameter of 50 mm and thickness of 1 mm)

The crosslinking process including spraying the aqueous solution of calcium ions onto the surface of samples in the mold The mold was designed to have two edges: the first and second corresponding to the equal volume of biopolymer sample and crosslinking agent solution, respectively The crosslinked systems were maintained in a refrigerator

at 4.2 ◦C for 48 h to establish the final hydrogel structure (time required for crosslinking process based on Ca2+ions)

Hydrogels were prepared in 1–3 wt% concentration of a single polymer since the most important changes in the structure of the hydrogel network occur in this range Furthermore, at polymer con-centration up to 1 wt%, the systems are almost Newtonian and their

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viscoelastic properties are practically undetectable (Lapasin et al., 2017;

ˇ

Sebenik et al., 2020) Ionic crosslinking was performed by adding a

calcium ion solution to a maximum concentration of 2 wt% In this state,

almost all carboxyl groups of TOCNF or ALG participate in the

cross-linking process so that a higher concentration of crosscross-linking agent

so-lution would not (noticeably) affect the rheological properties of the

hydrogels (Kopaˇc et al., 2020; Kopaˇc, Krajnc, & Ruˇcigaj, 2021) The

sample pH value was 7 The prepared hydrogel systems were kept in the

fridge at a temperature of 4.2 ◦C for 15 days to establish the final

hydrogel structure and to avoid the effect of aging which mainly affect

the rheological properties due to the formation of hydrogen bonds

(ac-cording to the literature (Lapasin et al., 2017; ˇSebenik et al., 2018))

2.3 Rheological characterization

Rheological rotational tests were performed on rheometer Anton

Paar Physica MCR 301 equipped with a crosshatched plate with a

diameter of 50 mm (PP50/P2) The samples were prepared to

corre-spond to a thickness of 1 ± 0.1 mm between the plates and were

measured at a temperature of 25 ◦C

Flow curve – viscosity (η) was measured over a range of shear stresses

(σ) from 0.01 to 3000 Pa The flow curves were presented in a log

vis-cosity–log shear stress plot The experimental data were fitted by the

Roberts-Barnes-Carew (RBC) model The RBC is the modified original

Ellis equation (Roberts, Barnes, & Carew, 2001):

η=η∞[1 + (σ / σ2)s] +

η0

1+(σ / σ1 )pη∞[1 + (σ / σ2)s]

1 +

(

σ

c

where η is dynamic viscosity, η0 and η∞ are the asymptotic values of the

viscosity at zero and infinite shear stress, respectively, σ is shear stress,

σc is critical shear stress, which locates the transition region between

two moderate shear thinning regions, m describes the rate of viscosity

decrease in the transition region, σ1 and σ2 are critical shear stresses in

the composite Ellis model, p and s are adjustable parameters (Roberts

et al., 2001)

3 Results and discussion

3.1 Flow behavior

3.1.1 Single polymer hydrogel and yield stress determination

Single polymer hydrogel based on constituent biopolymers (TOCNF

and SCLG) and LAP (clay) exhibits pronounced shear thinning character (Fig 1) The η0 increases from TOCNF, over SCLG to LAP dispersions Especially in the case of SCLG and LAP, the behavior is plastic with a significant viscosity drop in a narrow stress interval, while TOCNF hydrogels present a longer stress interval of viscosity decrease which appears at lower τ The 1st Newtonian plateau of TOCNF (η0) is the consequence of the existence of invisibly small clusters formed during aging which are enabled by entanglements of nanofibers and non-covalent and non-covalent interactions between them (not only hydrogen bonding, also van der Waals interactions and electrostatic repulsion due

to negatively charged carboxyl groups (Sebenik et al., 2018ˇ )) The clusters arrangement of the TOCNF internal structure in the shear flow direction is therefore shown in a longer stress interval of viscosity decrease (effect of clusters concentration, shape, and distribution) Accordingly, the TOCNF has the most pronounced 2nd Newtonian plateau and the network-of-forces of the TOCNF internal structure are weaker than in the case of stable rigid triple helices in SCLG structure and LAP crystals (η0 values) As opposed to η0, the τ0 occurs in the lower shear stress in the LAP in comparison with SCLG The reason may be attributed to different interactions that predominantly form a structure

in both samples The intra- and inter-molecular hydrogen bonds among the main chains of SCLG lead to the formation of a stable structure until the increased shear stress predominates over hydrogen interactions in the SCLG structure (τ) On the other hand, the LAP forms rigid, disk- shaped crystals whose random distribution determines the internal structure (the crystals are folded and occupy the most energy-efficient state) The viscosity is constant until the crystals start to steer in the direction of shear (τ) Fig 1 shows that the SCLG structure based on hydrogen interactions between polymer-polymer chains and LAP disk- shaped crystals structure (predominantly electrostatic interactions) are more solid than the TOCNF dispersion The internal structure in TOCNF

is determined by intra- and intermolecular hydrogen bonding (Lin & Dufresne, 2014), which are allowed by the presence of three hydroxyl groups on each β-(1–4)-glucopyranosyl unit The crosslink density of TOCNF is significantly lower than in the case of SCLG, due to lower hydroxyl group content which results in lower number of polymer- polymer hydrogen interactions, which also results in lower η0 and τ0

Differently, the ALG is soluble in water and does not form gels structure

in the absence of crosslinking agent and therefore exhibits fluid-like viscoelastic behavior (Fig 1)

The values of η0 and τ0 are experimentally difficult to determine uniformly, as they generally depend on the type and experiment per-formance rate The η0 values can be determined by extrapolating the experimental points of the flow curve on the log viscosity–log shear stress (or shear rate) diagram The measurements in Fig 1 show a flow behavior where even at very low shear stresses the samples “creep” and become plastically deformed This is the main reason for the difficult determination of η0 and τ All samples with plastic flow behavior (SCLG, LAP and TOCNF) are characterized by a very high viscosity due

to a strong internal structure (based on polymer-polymer interactions) that resists the shear The η0 values for 2% SCLG, TOCNF and LAP single dispersion were determined at 101000, 7327 and 520,600 [Pa⋅s], respectively (Table S1)

Furthermore, yield points are not material constants as they depend

as well on the measuring method as well as on the analysis method used The value of τ0 is often determined as a rough approximation by extrapolating the experimental points of the flow curve on log shear stress–log shear rate or log shear strain– log shear stress diagram The

τ0 determination via the “tangent crossover method” is illustrated in

Fig 2 The τ0 determination via the best straight fitting line (“tangent”)

in the linear–elastic deformation range is also realizable Shear stress ramp experiments from 0.1 to 200 Pa were performed in variable measuring point duration logarithmic profile (initial point duration was set at 300 s and final point duration was set at 20 s, along with ramp log +9.63 points/decade) From Figs 2A–C, the τ0 values for 2% SCLG, TOCNF and LAP single dispersion were determined at 32, 1.9 and 22

Fig 1 Flow behavior of single polymer systems based on LAP (squares), SCLG

(circles), TOCNF (triangles) and ALG (diamonds) The lines represent RBC

model fit

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[Pa], respectively Fig 2D illustrates obvious fluid behavior of ALG

Additionally, η0 and τ0 can also be defined as a model parameter

when a rheological model is used to describe plastic flow behavior Such

generally well-known rheological models are the Bingham model,

Her-schel–Bulkley model and Casson model Both methods for η0 and

τ0 values determination were used The results of all samples are

sum-marized in Table S1

3.1.2 Polymer concentration and fraction dependence hydrogel systems

Fig 3A presents flow curve of SCLG/TOCNF hydrogels systems The

samples were prepared by blending various (1–3 wt%) concentration of

SCLG and TOCNF dispersions in equal weight ratios The concentration

of biopolymer plays a crucial role in determining final rheological

properties of SCLG/TOCNF hydrogels, which can be confirmed by a very

wide interval of yield stresses from 1.28 < τ0 <43.7 [Pa] and also zero-

shear viscosity of 4.15⋅102 < η0 <1.05⋅105 [Pa⋅s] (more data can be

found in Table S1) Shear thinning character increases with increasing

biopolymer concentration which suggests the existence of invisibly

small clusters with significant swell-ability in water Clusters are formed

during the process of storage (aging) of prepared hydrogels when

pre-dominantly hydrogen, van der Waals and electrostatic repulsive forces

begin to intertwine in SCLG and TOCNF network structure In this case,

the clusters increase, which reduces their distance from each other and

further enhances the noncovalent interactions among them After two

weeks, the growth of the clusters slows down reaching the final

prop-erties of the hydrogel structure (ˇSebenik et al., 2018) In the case of

TOCNF polymers, having modified carboxyl groups on the surface, the

clusters are also formed during the ionic or covalent crosslinking

pro-cess The experimental data show higher η0 when using a higher

con-centration of SCLG (the effective volume fraction of the dispersed phase

is increased) A less important effect on shear thinning character has the

concentration of TOCNF, which significantly affects especially on τ0 in

low concentration biopolymer systems The flow curve shape of the

sample with a 1 wt% concentration of both biopolymers is extremely

similar to the single 2 wt% TOCNF hydrogel It may be concluded that

hydrogel with SCLG below 1 wt% does not have plastic behavior

(Lapasin et al., 2017)

The effect of the mass fraction of different biopolymers on the

rheological properties of hydrogel mixtures was studied in the case of

blending 2 wt% of SCLG and TOCNF (Fig 3B), LAP and ALG (Fig 3C) as

well as SCLG and ALG (Fig 3D) (see single polymer hydrogel properties

from Section 3.1.1 as reference) The mixtures were prepared in mass

fractions of both biopolymers from 0.9 to 0.1 In Fig 3B, flow curves

show almost negligible change in η0 between 0.1 and 0.25 mass fraction

of SCLG and also 0.5–0.90 On the other hand, τ0 increased with an

increase in the mass fraction of SCLG The shape of flow curves is typical for shear thinning behavior of hydrogel blends All the 2 wt% SCLG/ TOCNF systems exhibit an evident plastic behavior (the 1st Newtonian plateau is distinctive) with a significant viscosity drop more typical for a single SCLG hydrogel Furthermore, the 2nd Newtonian plateau is also detected, as the main contribution of the TOCNF biopolymer (see the shape of the flow curve for single TOCNF hydrogel - triangle) to the SCLG/TOCNF hydrogel blends It may be concluded that 2 wt% SCLG/ TOCNF systems form pronounced viscoelastic properties of hydrogels, where the addition of SCLG mainly affects the elastic properties of the hydrogel (hydrogen interactions contributed to stronger network-of- forces of internal structure), while the addition of TOCNF gives the hydrogel a viscous character at high shear stresses (due to the presence

of the small invisible clusters or flocs which are dispersed in a gel-like matrix (ˇSebenik et al., 2018)) (also discussed after in Figs 7A and B) Based on the calculated data from Table S1, the zero-shear viscosity interval is between 9.94⋅103 < η0 <3.19⋅104 [Pa⋅s], the yield stress

interval is determined in the range of 7.84 < τ0 <30.7 [Pa]

In Figs 3C–D, the effect of ALG in the hydrogel blend system is presented The ALG in water forms a clear solution which is the main reason that it does not participate in forming polymer-polymer inter-action in addition to various biopolymer (SCLG) or rheological modifier (LAP) In Fig 3C, it is clear that the flow curves of blends containing LAP

in 1.0–0.25 mass fractions have a pronounced plastic behavior evident

in 1st Newtonian plateau with a significant viscosity drop in a narrow stress interval (same as for single LAP dispersion presented in Fig 3C – square symbols), in addition to the decreasing effect of η0 and τ This may be the consequence of diluting effect of ALG in the 2% LAP/2% ALG systems (see also discussion of Figs 7A and B) In addition, the ALG addition gives the more viscous character of systems in high shear stresses where the 2nd Newtonian plateau is clearer (Fig 3C) than in single LAP dispersions (Fig 1) At the lower mass fractions of LAP in the mixture, the plastic behavior is not expressed According to the litera-ture (Lapasin et al., 2017), the LAP dispersion of less than 0.75 wt% has properties of fluid or does not show shear thinning and viscoelastic character, therefore the blending would have only a diluting effect and not synergistic interaction between LAP nanoparticles and biopolymer network The same conclusions (diluting effect) may be given for 2% SCLG/2% ALG systems (Fig 3D) where the flow behavior is a function of SCLG concentration dependence changed by the addition of ALG solu-tion While dilution effect of ALG can be observed in non-crosslinked samples, the drastic change in ALG participation in hydrogel mixtures

on the flow behavior is presented in ionically crosslinked systems (Section 3.1.3)

3.1.3 Fraction dependence of biopolymer blends on crosslinked hydrogel systems

Biopolymer blends from Section 3.1.2 were additionally crosslinked with 2 wt% CaCl2 solution The rheological properties of such complex hydrogel systems are illustrated in Fig 4 Compared to the non- crosslinked systems a η0 increased by up to two decades (see Ta-ble S1), demonstrating even more plastic behavior of hydrogel systems

On the opposite, the η0 increases with increasing TOCNF or ALG mass fraction in the case of crosslinked systems as a result of crosslinking polymer chains by calcium ions through the carboxylic functional groups on the polymer surface As anticipated, the higher it is concen-tration of the carboxylate level, the more plastic behavior could be observed More than that, the non-ionic SCLG cannot contribute to ionic interaction with TOCNF or ALG (ˇSebenik et al., 2020) (see also discus-sion of Figs 7C and D) Furthermore, the 2nd Newtonian plateau established in non-crosslinked systems (Fig 3) was no longer existed in crosslinked hydrogels (Fig 4) The effect of increasing the τ0 of cross-linked systems with increasing mass fraction of TOCNF or ALG biopolymer is also evident (see Table S1)

Interestingly, the same or even slightly higher τ0 of non-crosslinked SCLG/TOCNF mixtures with the SCLG mass fraction of 0.9 (29 Pa)

Fig 2 Yield point determination via the tangent crossover method for 2%

SCLG (A), TOCNF (B), LAP (C) and ALG (D) single systems

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than their analogous crosslinked samples (28 Pa) is measured In other

words, SCLG forms many hydrogen bonds between polymer chains and

water molecules, which diffuse into the SCLG network in the process of

swelling Also, due to the lower mass fraction of TOCNF, a smaller

number of ionic bonds during crosslinking are formed because of the

lower concentration of carboxyl functional groups Hydrogen bonds are

predominant over a few ionic interactions, in this case, reflecting no

effect of increasing τ0 in flow curves In a previous study (Kopaˇc,

Abrami, et al., 2021) was shown that the hydrogen bonding interaction

arising among SCLG triple helices and TOCNF nanofibrils reduces the

tendency to prone to establish Ca2+mediated ionic bonds The opposite

could be clearly seen at higher TOCNF mass fractions (Fig 4A) This

anomalous behavior of SCLG/TOCNF gels is further studied in detail in

Section 3.4

Additionally, to prove the predominant effect of electrostatic

inter-action over hydrogen bonds, the crosslinked LAP/TOCNF systems

(Fig 4B) were prepared The rheological properties of non-crosslinked

LAP/TOCNF are presented in the literature (ˇSebenik et al., 2020),

therefore the η0 and τ0 values are shown only in Table S1 and not also in

a graphical form The effect of hydrogen bonds is lower in exchange for

electrostatic interactions between LAP nanodisks (positively charged

edges of particles) and TOCNF fibrils (anionic side groups) The clay

particles of LAP and its aggregated forms are deposited between the

polymer chains of TOCNF (Lapasin et al., 2017; ˇSebenik et al., 2020),

which additionally connects the polymer nanofibers and strengthens the

internal structure of hydrogel systems For that reason, the higher η0 and

τ0 values of crosslinked LAP/TOCNF hydrogels in comparison to SCLG/

TOCNF systems, especially in low TOCNF mass fractions, may possibly

be explained by stronger electrostatic interactions over hydrogen bonds

3.1.4 Effect of crosslinking agent concentration on complex hydrogel systems

To study the effect of crosslinking agent concentration on the rheo-logical properties of hydrogel systems, 2% SCLG/2% TOCNF and 2% LAP/2% TOCNF samples were blended in the 0.5/0.5 weight ratio and additionally crosslinked with different concentrations of calcium ion solution (0.17–2 wt%) Fig 5 shows almost negligible effect of cross-linking agent concentration on flow curves of ionically crosslinked hydrogels Crosslinked hydrogels have a rigid and stable structure even

at higher shear stresses (over 100 Pa) The ionic bonds between the TOCNF polymer chains formed during the crosslinking process with calcium ions (Curvello & Garnier, 2020) are obviously predominant over other interactions (explained in Section 3.1.3.) Flow curves are mostly plastic with a drastic drop in viscosity when the structure is destroyed (see the τ0 data from Table S1) The LAP-containing hydrogel systems possess higher values of η0 and τ0 (Table S1) As already dis-cussed in Section 3.1.3, the LAP mainly contributes to electrostatic interaction in the mixture unlike SCLG-containing systems where the hydrogen bonds are strengthened In the crosslinked systems, the polymer chains are closer to each other which allows the electrostatic interaction to play a more important role in a strong hydrogel structure (Fig 5) In the light of high η0 and τ0 illustrated in Fig 5 for TOCNF- containing systems, for ALG-containing systems with SCLG and LAP only a few measurements at different concentration of crosslinker were assessed for verification purposes (data not shown) Accordingly, due to the higher concentration of carboxyl groups in ALG than in TOCNF (more ionic interactions), the presence of ALG in the crosslinked mixture exhibit an even stronger internal structure (higher η0 and τ) compared

to TOCNF gels

Fig 3 Flow behavior of biopolymer concentration and fraction dependence hydrogel systems In the panel A, the different shape (and color) of symbols represents

the various concentrations of biopolymer (blends of SCLG and TOCNF in equal weight ratio) in single polymer hydrogel preparation (the legend inside panel A) Panel B (blends of 2% SCLG and TOCNF biopolymers dispersion), C (blends of 2% LAP and ALG) and D (blends of 2% SCLG and ALG) illustrate the flow curve of biopolymers different weight ratio as shown in the legend below figure The lines represent RBC model fit The open symbols correspond to single polymer systems presented in Fig 1 (circles – SCLG, triangles – TOCNF, squares – LAP, and diamonds - ALG)

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3.2 Model development

The previous sections present the results of rheological rotational

tests and detailed characterization of hydrogel flow properties The

shear thinning behavior was observed since the gels' structure reveals

almost constant viscosity at very small shear stresses (η0) followed by the

structure deformation at the τ Accordingly, mathematical model to

predict these flow characteristics of hydrogels is designed based on the

following assumptions The literature (Buscall, Goodwin, Hawkins, &

Ottewill, 1982; Chen & Zukoski, 1990) already reports that plateau

modulus (Gp) is proportional to the yield stress τ:

Further on, the average molecular weight between crosslinks Mc can

be derived from the following equation, including Gp as is clearly pre-sented in recent paper (Yan et al., 2018):

M c=c ρ RT

where c is the polymer concentration, ρ is the density of water, T is the absolute temperature and R is the ideal gas constant which represent the product of Avogadro (NA) and Boltzmann (kB) constant The Eq (3)

leads to the crosslink density determination:

ρ x= ρ g

where ρg is hydrogel density Accordingly, the Eqs (2)–(4) allow to find the relation between τ0 and ρx It is followed that a proportionality exists between τ0 and ρx:

In the previous work (Kopaˇc, Abrami, et al., 2021) we presented an equation (Eq (6)) which may be used to predict the crosslink density ρx

of hydrogels and analyze data of shear moduli adopted by the rheo-logical oscillatory tests The equation is based on the polymer-polymer interaction as well as properties of polymers and crosslinking agents and avoids difficult-to-determine parameters:

ρ x=∑

n

i=0

N ion,i m p,i X ion,i

aion

n

i=0

N hyd,i

(

m p,im p0,i

)

X hyd,i

Fig 4 Flow behavior of crosslinked (constant – 2 wt% addition of crosslinking agent solution) systems, presented in Fig 3 The lines represent RBC model fit The open symbols correspond to single polymer systems presented in Fig 1 (squares - LAP and circles – SCLG)

Fig 5 Flow curve of crosslinked 2 wt% SCLG/TOCNF (A) and 2 wt% LAP/

TOCNF (B) hydrogel systems blended in the equal weight ratio The symbols

represent the concentration of the added crosslinking agent solution The lines

represent RBC model fit

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where N is substituent groups content on the polymer surface (OH,

COOH, NH2, etc.) defined per gram of dry polymer [mmol/g], mp and

mp0 are the mass concentration of dry polymer in the solvent and a

minimum mass concentration of polymer with the ability of hydrogel

formation [g/m3], ahyd is the hydrogen bonds functionality affinity [/],

the aion is the functionality affinity [wt%] of the ionic crosslinking agent,

x is the crosslinker concentration [wt%], i and n is the particular

poly-mer and the number of polypoly-mers in the hydrogel system, respectively,

and Xion,i is the mass fraction of polymer i in the hydrogel system The

development of Eq (6) for particular hydrogel system and the detail

explanation of symbols as well as its determination can be observed in

(Kopaˇc, Abrami, et al., 2021)

According to the relation between τ0 and ρx presented in Eq (5), the

mathematical model to predict yield stress of complex hydrogel systems

can be proposed by analogy to Eq (6):

τ0=

n

i=0

S ion

N ion,i m p,i X ion,i

aion

n i=0

S hyd

N hyd,i

(

m p,im p0,i

)

X hyd,i

where S is the coefficient of proportionality in units J/mol and may

represents the energy defined per moles of crosslinks that can be used to

restore the deformation It is defined as a function of polymer-polymer

interaction type (predominant effect of ionic (Sion) and hydrogen

(Shyd) interactions) and shapes of building blocks (depends on consistent

polymer or clay, etc.) in the gel system In polymer dispersions or

so-lutions, the internal structure is formed by polymer molecules that are

randomly distributed and occupy the most energy-efficient

conforma-tion Chains can be intertwined, there may be electrostatic repulsive or

attractive forces between them, and parts of the chains can be

inter-connected (crosslinked) These effects significantly affect the S value

(more detailed discussion of this parameter is further provided in

Sec-tion 3.3.)

Eq (7) can be further simplified for single: (i) polymer systems giving

origin to hydrogels relaying on inter-chains hydrogen bons (Eq (8)), and

(ii) ionically crosslinked hydrogel systems (Eq (9)), and can be written

as analogy to model development in (Kopaˇc, Abrami, et al., 2021):

τ0=S hyd

N hyd

(

m pm p0

)

τ0=S ion

N ion m p

aion

As it was possible to deduce from experimental observations, other

phenomena can be introduced and discussed in the development of

mathematical model, namely dilution effect In samples where one of

the polymers does not participate in ionic, hydrogen, or any other

in-teractions process (e.g., ALG solution without crosslinking agent), the

dilution effect described by the Sdil is introduced in Eq (10) It is

spec-ulated that the internal structure of the polymer solutions plays an

important role in the formation of the hydrogel network

τ0=N hyd

(

m pm p0

)

(

ShydXhyd− Sdil

(

1 − Xhyd ) )

(10) The Eq (10) is written for two-component non-crosslinked hydrogel

systems (e.g., SCLG/ALG) where the first term represents the effect of

hydrogen bonding interactions between SCLG chains on τ0 and the

second term is the dilution effect contribution wherein the ALG polymer

molecules inhibit hydrogen interactions between SCLG chains and

reduce the Shyd of SCLG due to the steric hindrances

3.3 Analysis of zero–shear viscosity and yield stress (model verification)

As an upgrade to a wide range of experimental data, the proposed

mathematical approach to analyze τ0 in a form of Eq (7) was used to

describe the synergistic effect on shear behavior owing to their

inter-relationship For this evaluation, the experimentally determined ρx data

presented in Fig 6 are adopted from our previous paper (Kopaˇc, Abrami,

et al., 2021) (samples preparation protocol in this research is identical)

On the other hand, the τ0 data were determined based on flow curve measurements (illustrated in Figs 3-5) via the tangent crossover method

as presented in Fig 2 During the action of shear force, hydrogels can store part of the energy entering the system, and accordingly, after removing the stress, they can restore part of the deformation In this

context, parameter S as a coefficient of the energy that can be restored

defined per mole of crosslinks was introduced Below the τ, a hydrogel deforms elastically and returns to its original shape when the applied stress is removed due to energy stored in the network (described by

parameter S) The Shyd for hydrogen interactions and Sion for ionic in-teractions were determined by fitting the Eqs (8) and (9) to single non- crosslinked and crosslinked polymer hydrogels (SCLG, LAP, TOCNF,

ALG), respectively The predetermined biopolymer individual S

co-efficients values were further endorsed in mixtures as proposed in Eq

(7) to calculate the yield stress of complex hydrogel of (crosslinked)

polymer mixtures Furthermore, Sdil was determined in fitting the Eq

(10) of non-crosslinked ALG-containing mixtures The results of the model fitting parameters are presented in Table 1

The Eq (7) proved to be a satisfactory model prediction of τ0 (see red lines in Fig 6) in most of the samples except for ionically crosslinked SCLG-containing systems whose unforeseen behavior is detailly dis-cussed in Section 3.4 It may be observed that the SCLG internal

struc-ture, predominantly formed based on the hydrogen interactions (Shyd), are able to restore more energy than LAP and TOCNF per moles of crosslinks On the other hand, in the case of crosslinked TOCNF and ALG

hydrogels, where the ionic interactions are predominant, the Sion is

significantly lower than Shyd This means that internal structures based

on ionic interactions have less potential to restore after deformation compared to hydrogen interactions-based structures On the other hand,

it has to be noted that ionic interactions are much stronger than hydrogen interactions along with the usually higher ρx of ionically crosslinked hydrogels (the affinity to create crosslinks are higher) While the shear stress should be significantly higher to deform the gels' structure in crosslinked hydrogels than in non-crosslinked (where the

hydrogen interactions are predominant), the parameter S defined here

only refers to the energy that can be used to restore the deformation (elastic character) The magnitude of shear stress is therefore hidden in ionic and hydrogen polymer-polymer interactions (see Eqs (6) and (7)) Moreover, not only polymer-polymer interaction but also the (differ-ently shaped) building blocks that form a hydrogel significantly affects

the flow properties Therefore, a wide difference between the Shyd of SCLG and TOCNF appears although the hydrogen interactions are pre-dominant in both systems (see Table 1) The higher values of Shyd of SCLG than LAP and TOCNF may indicate that by the additional forma-tion of the network structure SCLG helices contribute to higher resis-tance to flow than LAP nanodisks and TOCNF clusters, respectively

In non-crosslinked ALG solutions the hydrogels' structure is not formed since ALG is water-soluble and as such non-swellable (see Fig 1)

In this case, ALG participates in the diluting process in the mixture of SCLG or LAP dispersion with ALG solution In ALG polymer solutions, the internal structure of the liquid is formed by polymer molecules that are randomly distributed and occupy the most energy-efficient confor-mation (Lee & Mooney, 2012) Therefore, dilution with such solutions (η

of 2 wt% ALG solution at 25 ◦C is 29 mPa⋅s) is significantly different from dilution with water (η of water at 25 ◦C is 0.89 mPa⋅s) The shear thinning character of SCLG or LAP dispersions decreases linearly with increasing the water content, due to the decreasing polymer

concen-tration (as formulated, Sdil for water should be equal to zero) Compared

to water dispersions of SCLG or LAP, the material becomes less elastic by introducing ALG solution since ALG molecules dispersed in the SCLG network or LAP hydrogel cannot contribute to the crosslinking process and may present barriers in the formation of a hydrogel network

Consequently, Shyd, having an elastic character, is here reduced by the

value of Sdil

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Additionally, to analyze in detail the relationship between

mechan-ical and flow properties of hydrogels, the effect of ρx on η0 was also

identified and illustrated in Fig 7 In absence of (ionic) crosslinking

agent, the predominant effect on the formation of hydrogel network and

consequently on rheological properties have the polymers which in pure

dispersion state forms strong gels' structure due to many interactions

(hydrogen, electrostatic, covalent, other van der Waals bonds…) present

between polymer chains (see Fig 1 for reference) Fig 7A reports the

effect of SCLG addition on ρx and η0 of non-crosslinked hydrogel

sys-tems Due to the previously exposed dilution effect of ALG it can be

presumed that ALG solution has no contribution to hydrogel network

formation in a mixture with SCLG Therefore, the presence of ALG in SCLG systems has only a diluting effect, to the point, where gel's for-mation is no longer possible (see also Fig 3D for reference) which is for fractions lower than 0.25 of 2 wt% SCLG or 0.5 wt% SCLG (according to the literature (Lapasin et al., 2017), SCLG water dispersions under 0.75 wt% are almost Newtonian or slightly shear thinning with undetectable viscoelastic properties) Differently, in SCLG/TOCNF samples, due to TOCNF nanofibers hydrogen interaction contribution, the hydrogel structure is observed for all fractions (from 0.9 to 0.1) of 2 wt% SCLG Interestingly, although the different effects of ALG and TOCNF on ρx of SCLG based systems, the same linear trend on log η0 vs ρx plot can be observed in both cases It can be figured out that the fraction (concen-tration) of polymers contributes to the size of invisibly small clusters which are enabled by entanglements of nanofibers and (non)covalent interactions between them and the size of beads which are formed due to triple helical tertiary structure (triplex) in TOCNF and SCLG systems, respectively

Similar hydrogel systems as in previous case were prepared by changing SCLG with LAP (Fig 7B) The presence of ALG has only diluting effect also in the non-crosslinked mixture with LAP This phe-nomenon is obvious at 50/50 mixture of LAP/ALG (see circle 0.5 in

Fig 7B), where the trend is out of linearity on log η0 vs ρx diagram in comparison to mixtures with higher LAP content On the other hand, the TOCNF reduces the effect of the edge-face, edge-edge, and face-face interactions between LAP particles on the viscoelastic properties, through the steric hindrance (Lapasin et al., 2017; ˇSebenik et al., 2020)

It can be observed that LAP/TOCNF system (black circles) possess higher

Fig 6 Correlation between crosslink density and yield stress The τ0 data were determined by the same procedure as shown in Fig 2 and refers to hydrogel samples presented in Figs 3 and 4 The ρx data were adopted from our publication (Kopaˇc, Abrami, et al., 2021) where the same samples (the same procedure of preparation) were prepared The red lines represent the Eq (7) model prediction and numbers represent a mass fraction of first written polymer or clay in the mixture (in panel C the numbers represent the concentration of both polymers in 0.5/0.5 mass fractions) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 1

The values of proportionality coefficient S (Eq (7)) for a specific hydrogel

constituent material The Shyd was adopted by fitting the Eq (8) of non-

crosslinked single polymer (SCLG, LAP, TOCNF) hydrogels, Sion was adopted

by fitting the Eq (9) of crosslinked single polymer (ALG, TOCNF) hydrogels, and

Sdil was adopted by fitting the Eq (10) of non-crosslinked ALG-containing

mixtures The predetermined values for SCLG, LAP, and TOCNF were fixed in

further model calculations

Shyd [J/mol] S[J/mol] ion S[J/mol] dil

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η0 values at the same ρx of the network than in LAP/ALG system (red

circles) This is contributed to building blocks that form a hydrogel in

different shapes The LAP/TOCNF system is made of LAP disks which are

smaller and more homogeneous than TOCNF fibers which form

ag-glomerates (the meshes of invisibly small clusters) between which LAP

disks are distributed (ˇSebenik et al., 2020) It may be also assumed that

LAP contributes to the rheological response of the system in the same

way as other particulate fillers, although the individual clay

nano-particles are capable of aggregation (squares in Fig 1) The contact

between the particles is also different due to the various external

sur-faces and therefore the complex LAP/TOCNF building blocks are

accordingly the main reason for higher η0 values

Figs 7C and D illustrate the effect of TOCNF and ALG presence on ρx

and η0 in ionically crosslinked hydrogel systems, respectively It may be

figured out that the ρx and η0 are much higher than in the same non-

crosslinked samples (Figs 7A and B) At this point, the hydrogel

for-mation of ALG systems is stimulated by ionic interactions, omitting the

previously reported dilution effect of ALG in non-crosslinked samples

This leads to the different dynamics of hydrogel properties, which is why

the ALG-containing crosslinked systems have higher ρx due to more

carboxyl groups on the surface of ALG than in the case of TOCNF

Generally, the linear (exponential on a normal scale) growth in log

η0 versus ρx can be observed with some deviations marked at lower mass

fractions of ionic polymer A very similar trend can be observed in LAP/

TOCNF (red circles in Fig 7C) and LAP/ALG (red circles in Fig 7D)

samples which may be explained by the noncompetition of LAP disks

with TOCNF or ALG mediated ionic interactions via calcium ions

Furthermore, LAP nanodisks are distributed (interpenetrate) within the

TOCNF matrix due to electrostatic interactions between positively

charged edges of LAP disks and negatively charged carboxyl groups of

TOCNF nanofibers The LAP nanodisks act as bridging agents between

polymer nanofibers (ˇSebenik et al., 2020) which may be the reason for higher η0 values for crosslinked TOCNF or ALG systems containing LAP (unlike nonionic SCLG-containing systems) (see Figs 7C and D) Differently, TOCNF–SCLG hydrogen bonds lead to the scarce effect of calcium ions due to the hydrogen bonding interaction arising among SCLG triple helices and TOCNF nanofibrils that are no longer so prone to establish Ca2+mediated ionic bonds (see Fig 7C) In the SCLG/ALG systems (Fig 7D), the presence of SCLG reduces ρx as a result of steric hindrances, especially evident in the intermediate region of SCLG frac-tions (0.25–0.75) The effect of SCLG in complex hydrogel systems on ρx

and τ0 which leads to anomalous rheological behavior is profoundly discussed in the next section Here, it may be concluded, that the pres-ence of SCLG in ionically crosslinked hydrogels reduces the ρx because it complicates the ionic interactions between ALG or TOCNF polymer chains (Figs 7C and D) On the other hand, the addition of SCLG con-tributes to high values of η0 even at lower ρx (under 2 mol/m3), indi-cating that by the additional formation of the network structure SCLG helices contribute to higher resistance to flow

3.4 Analysis of synergistic effects in hydrogel systems and anomalous behavior of gels based on scleroglucan and TEMPO nanofibrillated cellulose mixture

In Fig 8 the black and red lines represent the systems where the hydrogen and ionic (electrostatic) interactions, respectively, play a pivotal role in the formation of hydrogel network In Figs 8B-D, the predominant effect of ionic interactions over hydrogen bonds (and other interactions, explained in the introduction) are evident even at low ρx

(the intersection of the black and red curves is at the point where there is the lowest concentration (fraction) of the polymer with carboxyl groups

- i.e., ALG or TOCNF) Meanwhile, in Fig 8A, the linear trendline (black)

Fig 7 Zero-shear viscosity (η0) obtained by RBC model against crosslink density (ρx) of various hydrogel systems (different colors) Panel A and B reports the effect

of SCLG and LAP, respectively, on rheological properties of non-crosslinked hydrogel system Panel C and D show the predominant effect of ionic polymer (TOCNF and ALG, respectively) on crosslinked samples The numbers represent a mass fraction of first written polymer or clay in the mixture The double color circles correspond to the single biopolymer or clay dispersion (2% SCLG, 2% LAP, crosslinked 2% TOCNF, and crosslinked 2% ALG in panel A, B, C, and D, respectively) summarized from Fig 1

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of the SCLG/TOCNF system without a crosslinking agent can be

forwardly forecast into the “red region” where the SCLG/TOCNF

sam-ples were ionically crosslinked It was proven in Fig 8A, where the black

circles/lines present the samples without addition of ionic crosslinker

(without ionic interactions) Differently, the red circles/lines present the

same samples which were ionically crosslinked However, it was

spec-ulated that the ionic interactions are predominant over hydrogen which

results especially in drastic increase in crosslink densities (see different

slopes of red and black lines on panels B, C and D) In panel A, the slope

of black line (predominant hydrogen interactions) is almost the same as

the slope of red line This clearly shows that in the case of SCLG/TOCNF

systems the hydrogen bonds play a predominant role even at low

con-centrations of ionic interaction (Kopaˇc, Abrami, et al., 2021) As a

matter of fact, in Fig 6, the (only) nonlinear behavior between τ0 and ρx

is revealed in crosslinked 2% SCLG/2% TOCNF gels in different weight

ratios of both constituent biopolymers (Fig 6B) A small deviation can

be also observed in crosslinked 2% SCLG/2% ALG system (Fig 6H)

Apparently, the SCLG biopolymer in mixture with the polymer that

could be ionically crosslinked often leads to anomalous behavior of gel's

rheological properties Interestingly, a similar deviation was observed in

our previous study (Kopaˇc, Abrami, et al., 2021), where the SCLG/

TOCNF hydrogel systems clearly differ from the ρx prediction of the

developed model equation (Eq (6)) According to this model prediction,

it can be speculated that ρx is underestimated, since the experimentally

determined values of ρx are lower than the Eq (6) prediction To support

this assumption, the model prediction of τ0 by Eq (7) is added in Fig 8A

for reference (gray circles) The observed deviations can be explained by

the predominance of hydrogen interactions that probably hinder the

formation of ionic bonds in obtaining the hydrogel network Therefore,

the competition between TOCNF – Ca2+− TOCNF mediated ionic

in-teractions and TOCNF–SCLG hydrogen bonds lead to the scarce effect of

calcium ions due to the hydrogen bonding interaction arising among

SCLG triple helices and TOCNF nanofibrils that are no longer so prone to establish Ca2+ mediated ionic bonds It is also not negligible that, theoretically, it is possible that more than twice more hydrogen bonds (2.8 mmol/g of hydroxyl group content) can be formed between TOCNF polymer chains than ionic ones (1.2 mmol/g of carboxyl group content) The SCLG has even more hydroxyl pendant groups (4.2 mmol/g of hy-droxyl group content) on the surface than TOCNF that are accessible for hydrogen bond formation (Kopaˇc, Abrami, et al., 2021) Moreover, as illustrated in Fig 8C, the presence of SCLG due to the steric hindrance also reduces the effect of the ionic interactions in the SCLG/ALG system

on the ρx (viscoelastic properties)

On the other hand, except for SCLG, Fig 8 reports that the addition

of TOCNF, ALG, and LAP to the polymer mixture produces only a quantitative effect on its viscous behavior, which does not change qualitatively In the LAP-containing systems, the synergistic effects of polymer-clay blending are crucial The linear response shown in

Figs 6D, F, G, and I and the apparent switch in the slope of the linear curve (black to red lines) when the crosslinking agent is added (see

Figs 8 B and D) may be evidence that the synergistic effect of polymer- clay interaction gradually decreases when, despite high concentrations, nanoparticles, distributed within the polymer network, are no longer able to develop an extended aggregation structure (Lapasin et al., 2017) Therefore, an increase of LAP fraction implies an increasing number and size of nanoparticle aggregates, but polymer-clay interactions do not disturb possible ionic interactions between crosslinked ALG or TOCNF chains and thus no anomalous flow behavior occurs as in the case of SCLG-containing systems Moreover, addition of LAP in a mixture with anionic polymer (ALG or TOCNF) contributes to the interpenetration and electrostatic interaction between TOCNF fibrils or crosslinked ALG particles and LAP nanodisks

Fig 8 Correlation between crosslink density and yield stress presented for four different types of polymer mixtures in addition of Laponite The values in panel: (i) A

refers to Figs 6A and B, (ii) B refers to Figs 6F and I, (iii) C refers to Figs 6 E and H, and (iv) D refers to Figs 6 D and G The black and red circles represent the non- crosslinked and crosslinked samples, respectively The lines correspond to linear fit where black and red color refers to predominant effect of hydrogen and ionic interaction in hydrogel systems, respectively In panel A, the gray color represents the linear correlation between τ0 and theoretically calculated ρx by Eq (6) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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