Composite material based on pullulan/silane/ZnO-NPs as pH, thermo-sensitive and antibacterial agent for cellulosic fabrics

Pullulan (P) was purchased from natural Lab Ltd (USA). Chitosan medium molecular weight (Fluka), Zinc nitrate hexahydrate (${\rm Zn}{{\left( {\rm N}{{{\rm O}}_{3}} \right)}_{2}}\cdot 6{{{\rm H}}_{2}}{\rm O}$), N-isopropylacrylamide (NIPAAm) and (3-Glycidyloxypropyl)trimethoxysilane (GPTMS) and hexamethylenetetramine (HMT) (Sigma-Aldrich, Germany). Ammonium persulfate (APS), sodium hydroxide (${\rm NaOH}$) and Glutaraldehyde (GA), ${\rm N},{\rm N},{{{\rm N}}^{\prime }},{{{\rm N}}^{\prime }}{{\rm \text -}}$tetramethylethylenediamine (TEMED) and ${\rm N,}{{{\rm N}}^{\prime }}{{\rm \text -}}$methylenebisacrylamide (BIS) were purchase from Fluka, Germany, monochloroacetic acid (MCA) was purchase from Merck, Germany.

2.2.1. Preparation of cross-linked O-carboxymethyl chitosan.

O-carboxymethyl chitosan was prepared as illustrated in scheme 1 [27] it can describe in brief as: 3 g of chitosan was dissolved in 80 ml isopropanol (0.2 M). Then 25 ml ${\rm NaOH}$ $42 \%$ was added dropwise during 30 min and then 3.5 g of MCA in 20 ml isopropanol was added drop by drop to the above mixture in a 250 ml round flask. The final mixture was stirred for 24 h at room temperature. After that, excess ${\rm NaOH}$ was added to precipitate O-carboxymethyl chitosan, the precipitate was washed with ethanol until the pH reached 7. The finally obtained material (O-CMC) was dried in vacuum [28].

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2.2.2. Synthesis of carboxymethyl pullulan (CMP).

2.2.3. Synthesis of ZnO nanoparticles (NPs).

2.2.4. Preparation of the CMC/CMP/ZnO nanogels.

${\rm CMC/CMP/ZnO}$ nanogels have been prepared prepared as illustrated in scheme 2 in three steps as follow: (a) 2 g of O-CMC has been dissolved in 100 ml distilled water. The solution has been stirred at room temperature overnight. GPTMS has been added with half ratio of O-CMC to the solutions under vigorous stirring at $50~\ {}^\circ {\rm C}$ for 3 h to obtain (modified CMC). (b) 2 g of CMP was dissolved in 10 ml water under shaking for 2 h at room temperature. NIPAAm and BIS (2%, w/w, relative to NIPAAm) have been added to the above solution. The whole amount have been transferred to the modified CMC and stirred for 30 min. After that, the solutions have been degassed by passing nitrogen gas through it. Then, TEMED $\left( 30\ \mu {\rm l} \right)$, 0.5 g prepared ${\rm ZnO}$ NPs and APS solution (2.5 %, w/v relative to monomer) have been added. Polymerization has been carried out at room temperature for 48 h. (c) The prepared nanogel was cut into small pieces and immersed in 40 ml of acidified glutaraldehyde (GA) solution for 5 h. Finally, ${\rm CMC/CMP/ZnO}$ nanogel has been washed with water several times each one occurred overnight, then left to dry for 2 d and drying under vacuum for extra 2 d.

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2.2.5. Incorporation of ${{\rm CMC/CMP/ZnO}}$ nanogel to cotton.

2.3.1. Scanning electron microscopy.

2.3.2. Infrared spectrometry.

2.3.3. Shape and particle size determination.

2.3.4. Calculation of degree of substitution (DS).

The degree of substitution (DS) of carboxymethylated chitosan (CMC) was determined according to BeMiller et al [31] by dissolving 0.3 g CMCs in 30 ml ${\rm HCl}$ (1 M) and titrating with 0.1 M aqueous ${\rm NaOH}$. The DS value was calculated as follows:

$$\begin{align} \newcommand{\e}{{\rm e}} \displaystyle {\rm DS}=\frac{161\left( {{V}_{{\rm NaOH}}}\times {{C}_{{\rm NaOH}}} \right)}{{{m}_{{\rm CMCS}}}-58\left( {{V}_{{\rm NaOH}}}\times {{C}_{{\rm NaOH}}} \right)},\nonumber \end{align}$$

where ${{V}_{{\rm NaOH}}}$ and ${{C}_{{\rm NaOH}}}$ are the volume and molarity of ${\rm NaOH}$, respectively, ${{m}_{{\rm CMCS}}}$ was the mass of carboxymethyl chitosan (g). 161 and 58 are the molecular weights of the glucosamine and the carboxymethyl group, respectively.

FT-IR analyses have been running to provide the structure of O-CMC. FT-IR spectra of chitosan and O- are shown in figure 1(a). The spectra of chitosan show the peaks at 1653 and $1595~\,{\rm c}{{{\rm m}}^{-1}}$ for ${\rm N{\text {--}}H}$ bending and at 1078 and ${\rm 1032}\,{\rm c}{{{\rm m}}^{-1}}$ for ${\rm C{\text {--}}O}$ stretching [32]. It is noticed that, a shoulder peak at $3063\,{\rm c}{{{\rm m}}^{-1}}$ for both chitosan and O-CMC is due to ${\rm N}{{{\rm H}}_{2}}$ [33]. In addition, a stretching vibration for ${\rm OH}$ groups is presenting at ${\rm 3400}\,{\rm c}{{{\rm m}}^{-1}}$. A peak at ${\rm 1325}\,{\rm c}{{{\rm m}}^{-1}}$ is corresponding to the ${\rm CH}$ rocking of the ring and the band for amide I at ${\rm 1655}\,{\rm c}{{{\rm m}}^{-1}}$. In the spectra of O-CMC, a new peaks at ${\rm 1414}\,{\rm c}{{{\rm m}}^{-1}}$ was appeared characteristic to carboxylate group asymmetrical and symmetrical stretch $\left( {{\rm \text {--}}CO}{{{\rm O}}^{-}} \right)$ [34].

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FT-IR analyses have been running to provide the structure of CMP. Figure 2 shows the FT-IR spectra of pullulan and CMP, and explain that, the characteristic absorption bands for pullulan are at 3432, 2929, 1654, 1125, 1032 1642 and ${\rm 860}\,{\rm c}{{{\rm m}}^{-1}}$, which they relative to ${\rm O{\text {--}}H}$, ${\rm C{\text {--}}H}$, ${\rm O{\text {--}}C{\text {--}}O}$, ${\rm C{\text {--}}O{\text {--}}C}$ and ${\rm C{\text {--}}O}$ stretching, ${\rm C{\text {--}}O{\text {--}}H}$ bending and D-glucosidic bands respectively [29]. Furthermore, the characteristic absorption bands of CMP appears at ${\rm 3350 c}{{{\rm m}}^{-1}}$ stretching for ${{\rm \text {--}}OH}$ of anhydroglucose units [17], ${\rm 2920}\,{\rm c}{{{\rm m}}^{-1}}$ for ${\rm C{\text {--}}H}$ groups, ${\rm 1590}\,\,{\rm c}{{{\rm m}}^{-1}}$ [17] for asymmetric carboxylate group (${\rm CO}{{{\rm O}}^{-}}$) and at ${\rm 1416}\,{\rm c}{{{\rm m}}^{-1}}$(symmetric carboxylate group (${\rm CO}{{{\rm O}}^{-}}$) [17]. At ${\rm 1087}\,{\rm c}{{{\rm m}}^{-1}}$, a characteristic stretching peak attributed to ether bond in pullulan have been observed [29]. In addition, there are three peaks for D-glucosidic which they appear at 857, 757 and ${\rm 932}\,{\rm c}{{{\rm m}}^{-1}}$ [35].

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The particle size of ZnO NPs has been illustrated in figure 3. Mean particle size diameter and PDI index were all measured in solutions using dynamic light scattering (DLS). The size of the colloidal ZnO NPs and their granule metric distribution are disclosed in figure 3. As is evident the size of the particles is around 9 nm. Particle size analysis displays the presence of ZnO NPs with low ${\rm PDI}=0.45$, which mean a good- quality of the colloidal suspensions [36]. Figure 3 shows TEM image which indicate that, the structure of ${\rm ZnO}$ NPs are in spherical or slightly irregular shape.

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There are a several possible interactions between CMP, CMC, poly-NiPAAm and GPTMS when using dispersion copolymerization. Theses interactions could be covalent bonding, electrostatic and/or hydrogen interactions.

Hydrogen interactions and/or electrostatic were presence between hydroxyl groups of CMP and amine groups of poly-NiPAAm. In addition, covalent bonding is presence between hydroxyl groups in C6 of CMP and poly-NiPAAm in polymerisation process [37].

3.4.1. FT-IR, TEM and particle size analyser.

FT-IR spectra of CMP and ${\rm CMC/CMP/ZnO}$ nanogel have been illustrated in figure 4. Sharp peaks from hydroxyl and amine groups of both CMP and CMC have been detected at $3350{{\rm \text {--}}}3440\ {\rm c}{{{\rm m}}^{-1}}$ [17, 29]. The absorption peaks at 1460 and ${\rm 1651}\,\,{\rm c}{{{\rm m}}^{-1}}$ are resulted from the overlapping of the carboxylate groups from CMP and the amide I from PNIPAAm [38]. The sharp peak at ${\rm 1533}\,\,{\rm c}{{{\rm m}}^{-1}}$ in the NIPAAm spectrum (amide II) becomes wider with lower intensity in the spectra of the nanogel which provide the presence of BIS and PNIPAAm [38]. Figure 4 shows TEM image of the ${\rm CMC/CMP/ZnO}$ nanogel, which indicates that, the particles shape is spherical or slightly irregular. In addition, the nanogel particle size can be easily determined and provide from the TEM figure. Figure 4 shows also the particle and distribution size of ${\rm CMC/CMP/ZnO}$. It is clear that, the size of ${\rm CMC/CMP/ZnO}$ particles is around 50 nm. In addition there are some particles in the range of 7 nm. These differences are may be due to the different surface properties of both CMC and CMP.

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3.4.2. Effect of temperature and pH on ${{\rm CMC/CMP/ZnO}}$ NPs.

Particles diameter of the prepared nanogel have been investigated at pH 6.5 (figure 5), and it provides that the nanogel particles are swollen at $25\ {}^\circ {\rm C}$ with 50 nm average diameter. While, at $40\ {}^\circ {\rm C}$, the nanogel having 7 nm in particles size. Furthermore, the temperature was increased gradually from 27 to $33\ {}^\circ {\rm C}$ lead to reduce in the particle size. Above $35\ {}^\circ {\rm C}$, size of nanoparticles does not vary significantly with increase in temperature. A sharp collapse at $32\ {}^\circ {\rm C}$ was observed which is corresponding normally to poly-NiPAAm homo-polymer. Observation of broad transition temperature in range of $27\ {}^\circ {\rm C{\text {--}}}33\ {}^\circ {\rm C}$ is due to presence of CMP. Ionization of CMP groups at pH 6.5 caused increasing of the hydrophilicity and imparts charge repulsion between particles.

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Dynamic light scattering analyser was used to investigate the effect of pH on changing in the particle size of the prepared nanogel at $20\ {}^\circ {\rm C}$ (figure 5). ${\rm CMC/CMP/ZnO}$ NPs are in swollen state at high pH (pH 10) with size of 118 nm. In addition, decreasing the pH value led to decreasing the particle size of the prepared nanogel. Swelling/de-swelling effect may be attributed to presence of CMP and/or CMC. Therefore, at high pH, nanogel particles are similar to cellulosic material (swelling behaviour). At low pH, CMP and/or CMC become soluble. Therefore, one can conclude that ${\rm CMC/CMP/ZnO}$ nanogel swell (basic medium) and deswell (acid medium).

Cotton fabrics have been treated with the prepared composite gel of ${\rm CMC/CMP/ZnO}$ mixture using pad-dry-cure technique; at first—${\rm CMC/CMP/ZnO}$ nanogel were deposited onto the surface, then, at $160\ {}^\circ {\rm C}$ forms ester, between carboxylic groups on CMP and hydroxyl groups on cellulose [39]. Two types of carboxyl groups are available for reaction from both CMC and CMP from ${\rm CMC/CMP/ZnO}$ nanogel. The crosslinking was occurs via esterification with these carboxylic groups and hydroxyl group of cotton fabric.

Surface morphology of both untreated and treated cotton fabrics have been illustrated in figure 6. SEM micrograph show that, Incorporation of nanogel onto surface of cotton fibre have been observed and the nanoparticles seem embedded onto the fibre surface. In addition, a thin layer of the nanogel was covers the surface of cotton fibre which have some irregular moieties observed as nanoparticles clusters.

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Two bacteria, namely Escherichia coli as Gram-negative bacteria and Staphylococcus aureus as Gram-positive bacteria have been used to investigate the antibacterial activity of the prepared composite gel against bacterial growth. As expected, from table 1 untreated fabric did not show any reduction in bacterial growth for both bacteria. In addition, treated fabric with CMP only shows poorly activity of fabric against both bacteria. That is because CMP didn't have functional group which can attack the bacteria cell wall. Presences of amino group in CMC give the fabric antibacterial activity against both bacteria under investigation.

Table 1. Antibacterial activities, mechanical and physical properties of cotton fabrics.

Sample	Antibacterial activities						Mechanical and physical properties
	Zone of inhibition diameter in mm						Tensile strength (kg)	Elongation at a break (%)	Roughness $(\mu {\rm m})$	CRA
	E. coli			S. aureus						Warp	Weft
	24 h	48 h	72 h	24 h	48 h	72 h
Untreated	0	0	0	0	0	0	41	15	11.65	50	45
Treated with modified CMP	1.1	1.1	1.1	1	1	1	37.5	13.8	12.36	85	82
Treated with modified CMC	6.1	6.2	6.3	5.5	5.7	5.8	39.1	14.1	17.43	77	70
Treated with modified nanogel	14.2	14.8	14.9	12.1	12.5	12.6	39.3	14.5	14.12	81	73

On the other hand, the treated fabric with produced gel showed highly reactivity against both bacteria comparing with untreated and treated fabric with CMP or CMC only. As expected, presence of amine group and ${\rm ZnO}$ NPs in the nanogel structure increasing the inhibition zone, demonstrating the antimicrobial activity of the tested treated fabric with nanogel.

The positive charge of the amine groups results in a nanogel structure and presence of ${\rm ZnO}$ NPs were cooperate with the transcendently anionic lipopolysaccharides of the Gram-negative cell surface advancing hindering the vehicle of supplements into the cells or the spillage of intracellular segments [40, 41].

Mechanical and physical properties of untreated and treated cotton fabrics have been investigated and the parameters have been illustrated in table 1. The results provide that, the finishing treatment show a significant decreasing in both tensile strength and elongation at a break of the treated fabrics comparing to untreated one. In addition, roughness properties show a slight increase in its values, which due to the deposition of nanogel and ${\rm ZnO}$ NPs onto the surface of cotton fabrics. The produced film onto the cotton surface led to improve the crease recover angel (CRA) of all treated fabrics, and treated fabrics with nanogel shows highly value comparing with the other treated fabric with CMC or CMP only.

Effect of both temperature and pH on the treated and untreated cotton fabrics have been investigated by measuring water uptake at varies temperatures range from 24 to $40~\ {}^\circ {\rm C}$ and different pH (3, 7 and 10) (figure 7). Water uptake for untreated fabrics was almost constant (95% to 100%) for all measured pH at ranged temperature.

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The water uptake at pH 7 was constant while increasing temperature from 24 to $26\ {}^\circ {\rm C}$ led to decreasing of water uptake from 111 to 97% whenever increasing temperature from 26 till $30\ {}^\circ {\rm C}$. In addition, water uptake remained constant with further temperature increase. In acidic medium (pH 3), a significant weaker response compared to values at pH 7, but it shows same behaviour. On the other hand, at alkaline medium (pH 10), water uptake was in higher values than those values at pH 3 or 7. Furthermore, when the nanogel was in collapsed state, water uptake was always lower comparing to untreated fabric. This phenomenon confirmed that, in collapsed state, the nanogel surface becomes hydrophobic and increased hydrophobicity of treated fabric. Raising the temperature cause that, PNIPAAm undergoes collapse transitions [16, 42].

At the point when the chain is warmed up, all the hydrogen bonds were broken, subsequent the chain in the collapse sharp. The captured water molecules through hydrogen bonds in nanogel are released. Moreover, in light of its hydrophilic nature influenced by pH, incorporation of both CMP and CMC expected that, bearing many hydroxyl groups (hydrophilic nature) into the PNIPAAm network.

At lower pH and temperature ($24\ {}^\circ {\rm C}$), the particles of nanogels were in marginally collapsed state. Thus, the primary water uptake was investigated at $24\ {}^\circ {\rm C}$ for pH 3 is lower than its value at pH 7. Notwithstanding, above $30\ {}^\circ {\rm C}$ for all measured pH, the water uptake was almost same. Nevertheless, this procedure is being backed off because of CMP, CMC and PNIPAAm chains present in the nanogel, which keeps water molecules inside the nanogel network.

Furthermore, from another point of view, in the figure 5(a), which indicates the effect of the temperature on the size of ${\rm CMC/CMP/ZnO}$ nanogel particles, it is obvious that the nanogel start decomposing (collapse) at $26\ {}^\circ {\rm C}$ and completely decomposing at $36\ {}^\circ {\rm C}$ with particle size around 9 nm (particle size of 9 nm is the size of only ${\rm ZnO}$). So, when then the fabric coated with the nanogel and applying high temperature for drying and curing processes until $160\ {}^\circ {\rm C}$ the nanogel around ${\rm ZnO}$ NPs is completely decomposed and make a thin film around the fibre surface. This coated film covered the whole surface and have the nanoparticle embedded through its network. In addition, presence of the crosslinker during these processes allows the nanogel to be chemically bonded with cellulose chains and the nanoparticle was rented in the gaps between cellulose chains and nanogel network.