clay physical hydrogels with tailorable structure as potential candidates for wound healing applications

Journal Pre-proof Versatile poly(vinyl alcohol)/clay physical hydrogels with tailorable structure as potential candidates for wound healing applications

Simona Morariu, Maria Bercea, Luiza Madalina Gradinaru, Irina Rosca, Mihaela Avadanei PII:

S0928-4931(19)31847-8

DOI:

https://doi.org/10.1016/j.msec.2019.110395

Reference:

MSC 110395

To appear in:

Materials Science & Engineering C

Received date:

20 May 2019

Revised date:

20 October 2019

Accepted date:

4 November 2019

Please cite this article as: S. Morariu, M. Bercea, L.M. Gradinaru, et al., Versatile poly(vinyl alcohol)/clay physical hydrogels with tailorable structure as potential candidates for wound healing applications, Materials Science & Engineering C (2018), https://doi.org/10.1016/j.msec.2019.110395

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© 2018 Published by Elsevier.

Journal Pre-proof

Versatile poly(vinyl alcohol)/clay physical hydrogels with tailorable structure as potential candidates for wound healing applications Simona Morariu*, Maria Bercea, Luiza Madalina Gradinaru, Irina Rosca, Mihaela Avadanei “Petru Poni” Institute of Macromolecular Chemistry, 41-A Grigore Ghica Voda Alley, 700487 Iasi, Romania

Highlights PVA/Laponite RD hydrogels were obtained by freezing/thawing.



High concentration of clay favors the formation of large size aggregates.



Rheological and mechanical behavior of hydrogels is dependent on the clay content.



Clay does not affect the swelling degree and kinetics of PVA hydrogels in water.



Hydrogels loaded with rifampicin showed an increased antibacterial activity.

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Graphical abstract

*

Corresponding author. E-mail address: [email protected] (S. Morariu).

Journal Pre-proof

Versatile poly(vinyl alcohol)/clay physical hydrogels with tailorable structure as potential candidates for wound healing applications Simona Morariu*, Maria Bercea, Luiza Madalina Gradinaru, Irina Rosca, Mihaela Avadanei “Petru Poni” Institute of Macromolecular Chemistry, 41-A Grigore Ghica Voda Alley, 700487 Iasi, Romania

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ABSTRACT Physical poly(vinyl alcohol) (PVA) hydrogels containing up to 3% Laponite RD (LRD) were

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obtained by freezing/thawing method. The structure and the morphology of PVA/LRD hydrogels were evaluated by ATR-FTIR and SEM measurements, respectively. The

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morphological analysis of the hydrogels revealed the formation of clay agglomerates and

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large size aggregates at LRD concentrations above 2.5%. The rheological and mechanical properties of the PVA hydrogels in presence/absence of clay and their structural regeneration

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capacity after a large deformation were also investigated. The effect of LRD incorporation on the swelling behavior of the PVA hydrogels was discussed. The water vapor sorption capacity

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of PVA hydrogels decreases by clay addition. The analysis of in-vitro rifampicin release data indicated that the presence of a small amount of LRD into hydrogel affects the drug release

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mechanism. The antimicrobial activity of PVA hydrogels in presence/absence of LRD loaded

studied.

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with rifampicin against Staphylococcus aureus, Escherichia coli and Candida albicans was

Keywords: Hydrogel, Poly(vinyl alcohol), Laponite RD, Rifampicin, Nanocomposite

1. Introduction Hydrogels are three dimensional polymer networks able to swell in water or biological fluids. Due to their excellent characteristics, these materials can be successfully used in pharmaceutical [1] and for bone regeneration [2-5] applications, in agriculture to reduce the fertilizer loss in conditions of the excessive rain or irrigation water [6], for cosmetics and *

Corresponding author. E-mail address: [email protected] (S. Morariu).

Journal Pre-proof personal care products [7], as soft actuators [8], for removal of different heavy metals in waste water [9,10], etc. From the multitude of polymers which can be used for the preparation of hydrogels with potential applications in biomedicine, poly(vinyl alcohol) (PVA) is distinguished due to its low toxicity, biodegradability, cytocompatibility, hydrophilicity, good biocompatibility, suitable mechanical properties and low price. In addition, PVA is able to form physical hydrogels with low toxicity (due to the absence of the chemical crosslinker remnants in its structure) by applying several successive freezing/thawing (F/T) cycles to its entangled solutions [11]. The freezing of PVA aqueous solutions causes the formation of ice crystals

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inside the sample while the polymer chains, concentrated in the unfrozen liquid domains, interact with each other forming a physical network structure [12]. PVA hydrogels containing

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chitosan (CS) [13,14] and poly(vinylpyrrolidone) (PVP) [15-18] with possible biomedical

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applications were also obtained by F/T technique. The addition of CS gives to the PVA hydrogels a good antibacterial activity against Escherichia coli.

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Due to their abundance and reduced cost, the clays represent an accessible component for improving the materials properties. Some of the most used clays are those from the smectite

lP

class (for example, montmorillonite, Laponite RD, etc.), which are characterized by a 2:1 layer structure with a sheet of alumina octahedron sandwiched between two sheets of silica

na

tetrahedron. The great interest of researchers for smectite clays is due to their main characteristics: the capacity to swell in water, the ability to form stable dispersions or gel

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structure in aqueous medium, adsorption capacity, large cation exchange capacity. The gelation mechanism of smectite clays in water, which depends on the clay concentration and

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the ionic strength in the system, is still debated in literature. Two main mechanisms for the gelation of smectite clays in water were proposed: i) the electrostatic attractions between negatively charged faces and positively charged rims of plate-like clay particles which give a „card-house structure” [19]; ii) the long-range electrostatic repulsions due to the overlap of double layers of the clay lamellae [20]. The combination of different polymers with clays (low loading, the clay can act as nanofiller) gives materials with unique properties that cannot be achieved by using each component alone: biodegradability, permeability, strength, electrical, magnetic, optical or barrier properties [21,22]. Finding the optimal composition and using a suitable preparation method allow the formulation of materials with targeted properties for specific applications. Due to their properties, polymer/clay nanocomposites have a very high applicative potential in a large variety of domains: automotive (first industry where polymer/clay composites have

Journal Pre-proof been used), aeronautics, packaging industry, electronics (digital optical discs), biomedical applications (drug vehicles, tissue scaffolds, wound dressings and biosensors) [23]. Clay addition improves optical, thermal and barrier properties. Besides clays, other inorganic fillers are used for enhancing the polymer properties: carbon nanotubes, graphene, nanocellulose. Carbon and metal nanoparticles add electrical conductivity, catalytic activity or plasmotic properties. Synergistic combination of different nanofillers determines a considerable enhancement of intrinsic and extrinsic properties for hydrogels used in nanomedicine [22]. The combination of two nanofillers can shift the dispersion limit of one of them, making possible the inclusion of a higher amount of filler into nanocomposite (for example, hybrid

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nanocellulose/carbon nanostructures) [24]. Simultaneous incorporation of different 1D, 2D or 3D nanofillers significantly improves the properties of nanocomposites [25]. Nanocomposites

develop

covalent

and

graphene/polymer composites) [26].

noncovalent

interactions

(such

as

nacre

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simultaneously

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with high toughness and tensile strength were obtained by using the fillers which

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A large number of works refers to the effect of non-ionic polymers presence, for example, poly(ethylene oxide) (PEO), into Laponite RD (LRD) aqueous dispersions on their structure

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and properties [27-35]. By addition of CS into PEO/LRD hydrogels, injectable hydrogels used as articular cartilage scaffolds (with increased fibroblast cell adhesion, biocompatibility,

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biodegradability) were obtained [36]. CS presence improves the rheological properties of LRD/PEO hydrogels due to the new interactions CS-CS, CS-PEO or/and CS-clay in addition

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to those established between PEO chains and clay particles [37]. PVA hydrogel films containing LRD with good transparency and flexibility, high surface

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roughness, high water retention capacity and with viscoelastic properties appropriate for wound dressing applications were obtained by crosslinking PVA chains in the presence of glutaraldehyde and clay [38]. The PVA/LRD aerogels, prepared by directional freezing from the bottom-up, showed fragility at high amount of LRD and more rigidity at high PVA fraction [39]. The quantity of clay added into the PVA hydrogels represents the main factor to obtain the properties appropriate to their use as wound dressing [40]. The presence of clay particles improves the hemolysis ratio and the blood coagulation activity [41]. Hydrogels based on PVA and LRD, applicable in various fields, can be tailored by changing the polymer/clay ratio or the obtaining method, by the addition of a second polymer into the PVA/LRD mixture, etc. Thereby, good mechanical properties and fast thermal response of the PVA/poly(N-isopropylacrylamide) hydrogel, physically crosslinked with Laponite XLG, recommend it for applications in controlled drug release [42].

Journal Pre-proof In the present study, the freezing/thawing method was applied to PVA/LRD/water mixtures in order to design hybrid hydrogels and to investigate the influence of the experimental conditions on the final properties of materials which can be potential candidates for wound healing applications. The effect of LRD concentration on the morphology, viscoelastic and mechanical properties and swelling degree of PVA hydrogels was studied and discussed. The rifampicin (RI) release and the antimicrobial activity of these hydrogels were also evaluated.

2. Experimental

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2.1. Materials

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Poly(vinyl alcohol) (PVA) with a hydrolysis degree of 98–99% was purchased from LOBA Feinchemie AG (Austria Chemical Companies). The synthetic hectorite clay, Laponite

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RD (LRD), has been donated by Rockwood Additives Limited U.K. (now BYK Additives Ltd). Rifampicin (RI) was offered by S.C. Antibiotice SA Iasi. PVA, LRD and RI were used

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without any further purification.

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The viscometric molecular weight ( M v ) of PVA was established as being 6.263  104 gmol-1 by applying the following Mark–Houwink equation [43]: (1)

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[𝜂] = 4.28 ∙ 10−4 ∙ 𝑀𝑣0.64 (dLg-1)

where   represents the intrinsic viscosity determined in water at 30 C by applying the

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classical Huggins equation.

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2.2. Preparation of hydrogels

4% PVA solution was prepared by dissolving the polymer in deionized water at 80 C under vigorous stirring and kept overnight at room temperature to reach the equilibrium state. Different amounts of clay were added into the PVA homogeneous solution in order to obtain samples with various compositions (Table 1). Then, the samples were slowly mixed for 1 h at room temperature to achieve a good dispersal of clay particles. The PVA/LRD hydrogels were obtained by subjecting the polymer/clay aqueous dispersions to five F/T cycles (freezing in liquid nitrogen for about 5 min and thawing at room temperature for 4 h) followed by their freezing-drying in a Martin Christ ALPHA 1-2LD lyophilizer for 48 h. Polymer/clay aqueous dispersions containing different quantities of LRD and 0.1% RI were prepared as mentioned above, they were submitted to five F/T cycles and lyophilized in order to obtain hydrogels for drug release and antibacterial activity measurements. The concentrations of the hydrogel

Journal Pre-proof components are expressed as weight percentage. The hydrogels were denoted differently depending on the measurements to which they were subjected: i) P-CX, where X represents the weight percentage of LRD for ATR-FTIR, SEM, rheology and swelling measurements and ii) H, for RI release and antibacterial activity investigations. For ATR-FTIR and SEM measurements, lyophilized hydrogels were used and rheological measurements were performed on hydrogels in swollen state.

2.3. Characterization of hydrogels 2.3.1. ATR-FTIR spectroscopy

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The Fourier transform infrared (FTIR) spectra were registered in the range 4000–600 cm-1

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on a Bruker Vertex 70 spectrometer (Bruker Optics, Germany) in the attenuated total reflection (ATR) configuration. The spectra were recorded at room temperature with a

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2.3.2. Scanning electron microscopy

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resolution of 2 cm-1 and by accumulation of 128 scans.

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The hydrogels morphology was investigated with a Quanta 200 Scanning Electron Microscope (SEM) (FEI, Brno, Czech Republic), operating at 20 kV in low vacuum mode

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using a large field secondary electron detector. The SEM measurements were performed on the cross-section of the hydrogels dried by lyophilization and cut with a sharp razor blade.

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SEM images were analysed by using an ImageJ software.

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2.3.3. Rheological measurements

The rheological measurements were performed at 25 C, by using a MCR 302 Anton-Paar rheometer, equipped with plane-plane geometry (diameter of 25 mm). The temperature control was provided with a Peltier device and the water evaporation was limited by using an anti-evaporation device. Amplitude sweep tests were performed at 10 rad/s in order to establish the linear viscoelastic region (LVR) of the investigated samples, where the storage (G′) and loss (G′′) moduli are not dependent on the shear stress () and strain (). The frequency sweep tests were performed in the frequency range of 0.1–100 rads-1, at a constant , chosen from LVR. Finally, oscillatory step tests, with three strain intervals, were carried out at 5 rads-1. The hydrogels were firstly subjected to a low strain (1%) for 200 s, followed by a

Journal Pre-proof very high strain (1000%) for 100 s and, then, the structure recovery was monitored for 400 s at a strain of 1%.

2.3.4. Tensile tests Tensile testing was carried out on an Instron 3365 machine at ambient temperature at a constant speed of 200 mm/min. The measurements were made on dried hydrogel samples, which were swollen in distilled water for about 3 min in order to decrease their rigidity and to prevent slipping from the device clips. The tests were performed on dumbbell-shaped specimens, having thickness of about 0.3 mm, width of 4 mm and length of 50 mm. Two

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independent tests were carried out for each sample, the average values were taken into

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account and the stress-strain curves were recorded.

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2.3.5. Swelling experiments

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The swelling of PVA/LRD hydrogels in water was investigated at room temperature. The hydrogels were weighed in lyophilized state (before swelling) and after different times of

hydrogel with an absorbent paper.

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swelling. Prior each weighing, the water excess was removed by gently wipe out the swollen

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The swelling degree, S, was calculated with the following relationship: 𝑆 = (𝑚𝑡 − 𝑚0 )⁄𝑚0 ∙ 100 (%)

(2)

immersion time t.

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where mo is the weight of the dried hydrogel and mt represents the hydrogel weight at the

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The swelling degree was calculated as an average value from three swelling measurements and the standard deviations were lower than 8% of the average value. The porosity of the hydrogels in swollen state was calculated with the following relationship proposed by Okay and coworkers [44]: 𝑑

𝑃𝑠 = (1 − 𝑞𝑣 ∙ (1 + (𝑞𝑤 − 1) (𝑑2 ))−1 × 100 (%) 1

(3)

The equilibrium volume (qv) and the equilibrium weight (qw) swelling ratios of the investigated gels were calculated as: 𝑞𝑣 = 𝑉𝑒𝑞 /𝑉𝑑𝑟𝑦

𝑞𝑣 = 𝑚𝑒𝑞 /𝑚𝑑𝑟𝑦

(4)

where Vdry and mdry represent the volume and weight of dried sample after swelling and Veq and meq are the volume and weight of the sample which reached the equilibrium swelling.

Journal Pre-proof The diameter (D) and the length (L) of the samples swollen and dried after swelling measurements were measured using a digital calliper, and the volume values (Veq and Vdry) were calculated with the following relationship: 𝑉 = (𝜋 ∙ 𝐷2 ∙ 𝐿)⁄4

(5)

2.3.6. Dynamic water vapor sorption Dynamic water vapor sorption capacity of the PVA hydrogel in the absence/presence of LRD was measured at 25 C, in the relative humidity range of 0–90%, by using a fully automated gravimetric analyzer IGAsorp supplied by Hiden Analytical, Warrington (UK).

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The samples were dried before sorption measurements at 25 C in flowing nitrogen (250

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mL/min) until the weight of the sample was in equilibrium at relative humidity lower than 1 %. The weight change by the modification of the sample chamber humidity was determined

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by using an ultrasensitive microbalance. The vapors pressure was increased in humidity steps

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of 10% with a pre-established equilibrium time between 40 and 60 min. The weight gained after each step was measured by electromagnetic compensation between tare and sample

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when equilibrium was reached. By decreasing the vapor pressure in steps, the desorption isotherms were also obtained. The measurement system is controlled by a user-friendly

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software package.

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2.4. In-vitro drug release studies

In order to investigate the drug release, a known amount of lyophilized PVA hydrogels in

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absence/presence of LRD containing RI was placed in 10 mL MilliQ water at 37 C, in a shaker incubator (100 rpm). At different time intervals, 1 mL of solution was withdrawn from the medium and filtered through a 0.20-μm cellulose acetate filter (Advantec, Japan). The absorbance of the release medium was determined at a wavelength of 475 nm, by using a JENWAY-650 UV-VIS Spectrophotometer (Jenway, UK). The recorded absorbance values were used to calculate the released drug concentration in mgmL-1, considering the slope and the intercept established from the calibration curve obtained previously under the same conditions. The experiments were performed in duplicate and the average value was considered for the evaluation of RI release. The mechanism of RI release from the PVA/LRD hydrogels was determined by fitting the kinetics of drug released into mathematical models including Korsmeyer–Peppas and Peppas-Sahlin models.

Journal Pre-proof 2.5. Antimicrobial activity The antimicrobial activity was assessed on two different bacterial reference strains, i.e. Escherichia coli ATCC 25922 (E. coli) and Staphylococcus aureus ATCC25923 (S. aureus) and against one fungal strain represented by Candida albicans ATCC10231 (C. albicans). To assess the antimicrobial activity, the PVA/LRD hydrogels containing RI were dried under vacuum, at 70 C for 72 h and stored in a cool dry place until their use. All microorganisms were stored at -80 C in 10% glycerol. The bacteria were refreshed in Mueller–Hinton broth (Merck) at 36±1 C, and afterward were inoculated on Plate Count Agar (Merk) for purity

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checking. Fungi were refreshed on Sabouraud dextrose agar (Merk) and were grown at 36±1 °C, pH = 7.

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Microbial suspensions were prepared with these cultures in sterile saline solution to obtain a turbidity optically comparable to that of the 0.5 McFarland standards (yielding a suspension

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containing approximately 1  108 CFU mL−1 for all microorganisms). Volumes of 0.3 mL

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from each inoculum were spread onto Mueller–Hinton Agar and Saboraud Dextrose Agar in Petri dishes and the biopolymer films were added after drying of the medium surface.

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To evaluate the antimicrobial properties, the inhibition of growth was measured under standard conditions, after 24 h of incubation at 36±1 °C. All tests were carried out in triplicate

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to verify the results. The diameter of the inhibition zone around the films was measured using

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a ImageJ software.

3. Results and discussion

(Table 1).

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For the present study, PVA hydrogels with different concentrations of LRD were prepared

3.1. ATR-FTIR analysis ATR-FTIR spectroscopy analysis was performed in order to check the incorporation of clay into PVA/LRD hydrogels. Fig. 1 shows comparatively the spectra of dried PVA hydrogels in absence (P-C0 sample), presence of LRD (P-C3 sample) and LRD powder. The ATR-FTIR spectrum of P-C3 sample is dominated by two intense and wide bands, whose positions are characteristic to the stretching vibration of hydroxyl groups in PVA and those of silicate, silanol, and magnesium hydroxide groups in LRD. The (OH) band in PVA/LRD sample (centered on 3338 cm-1) is broader than in pure PVA (3295 cm-1). Its

Journal Pre-proof greater spectral width is due to the combined signals of the hydroxyl groups from PVA and that of physically adsorbed water in LRD. The spectral shift is mainly arising from the change in the strength of inter- and intramolecular hydrogen bonding between C-OH groups of PVA and –OH and Si-O-Si groups of LRD. The (OH) vibrations from Si-OH and Mg-OH of LRD, which are usually observed as well-defined shoulders at 3636 and 3690 cm-1 [45], are now hardly seen as a weak tail.

Table 1. Composition, rheological parameters and porosity (Ps) of PVA/LRD hydrogels in swollen Samplea

LRD Water LRD/PVA

lb

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state. G′c

G′′c

Elastic

d

(%)

g/g

(Pa)

0

96

0

7.9

P-C0.5

0.5

95.5

0.12

5.9

P-C1.5

1.5

94.5

0.38

18.7

P-C2.1

2

94

0.53

P-C2.2

2.2

93.8

0.57

44

P-C2.5

2.5

93.5

P-C2.7

2.7

93.3

3

93

(%)

lossf

(%)

(%)

74.90

86.9

8.3

174

21.6

90.16

75.9

23.46

676

79.4

93.75

78.4

13.13

47.0 2290 272

92.69

74.8

5.71

1680 232

61.78

76.7

7.76

0.64

107 4120 444

79.10

-

-

0.68

92.3 5140 731

86.56

81.4

7.17

51.77

81.3

6.99

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12.2

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P-C3

(Pa)

mass

142

0.78

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P-C0

(Pa)

-p

(%)

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recovery

Pse

52

3430 484

PVA concentration for all hydrogels subjected to rheological measurements was 4%;

b

established by amplitude sweep tests at 10 rads-1;

c

determined from frequency sweep tests at 1 rads-1;

d

calculated as recovery percent of G′ after applying a strain of 1000% at 5 rads-1;

e

calculated with equation 3;

f

mass loss during the swelling measurements.

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a

The intense band with a maximum at 993 cm-1 from PVA/LRD spectrum is due to the contribution of (Si-OH) + (Si-O-Si) + (Mg-OH) in LRD, which overlaps with the contribution of (C-OH) of PVA. Its profile and bandwidth are different from that of LRD. Thereby, this maximum observed at 993 cm-1 in PVA/LRD sample is blueshifted to 997 cm-1

Journal Pre-proof and it decreased very much in intensity in LRD spectrum. The shoulder at 970 cm-1 in LRD is assigned to the deformation vibration of Mg-OH in the inter-sheets space [46]. The characteristic (C-OH) of PVA at 1089 cm-1 is deeply buried in the 993 cm-1 band, but the crystalline (C-OH) peak at 1142 cm-1 shows that the crystalline fraction is not totally broken by insertion into LRD platelets. Therefore, the hydroxyl groups of PVA have a strong interaction not only with the Si-OH groups located on the edge surface of the LRD platelets, but also with the Mg-OH groups from the space between the sheets. The vibration bands of

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na

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-p

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the groups identified in our spectra are in agreement with literature data [16,38,47].

Fig. 1. ATR-FTIR spectra of PVA (P-C0 sample), PVA/LRD (P-C3 sample) hydrogels in dried state and of LRD powder.

3.2. Hydrogels morphology All hydrogels are macroporous with large and interconnected pores. The pores average size (determined with the ImageJ software) ranged from about 77 m for P-C0 to 63.4 m for P-C1.5 and 36.9 m for P-C3. 4% PVA solution submitted to five F/T cycles develops a hydrogel with sponge-like structure (Fig. 2a). The addition of different LRD concentrations leads to macroporous

Journal Pre-proof structures with a morphology similar to PVA hydrogel (Figs. 2b,c,d), possessing some not

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very well-defined interconnected pores.

(b)

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(a)

(c)

(d)

Fig. 2. SEM micrographs of (a) P-C0, (b) P-C1.5, (c) P-C2.5 and (d) P-C3 samples.

The distribution of clay particles into the PVA hydrogel structure differs as a function of LRD concentration. Thereby, in the P-C1.5 hydrogel, the LRD particles are uniformly distributed amongst the polymer chains, due to the adsorption of PVA chains on the clay surface, and the network structure is homogeneous (Fig. 2b). The clay plays the role of inorganic filler in PVA hydrogels containing low amount of LRD. By increasing the LRD concentration at 2.5%, in the hydrogel structure starts to appear some clay aggregates with an

Journal Pre-proof average diameter of about 30 m (some of these agglomerations are exemplified by the arrows in the Fig. 2c). The further increase of the LRD concentration to 3% determines the formation of large size aggregates through association of the clay agglomerates with smaller size (see Fig. 2d). The rich-polymer and rich-clay zones appear, and the hydrogel structure becomes inhomogeneous. The addition of LRD does not significantly change the porosity of the PVA hydrogel (Table 1). During the F/T process, the formation of the ice crystals in the PVA solution determines the development of PVA-rich and solvent-rich phases. In the polymer-enriched phase, the interactions between PVA chains by hydrogen bonds are favored and the crystalline zones,

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which act as connection points between polymer chains, are envolved. (Fig. 3a).

Fig. 3. Schematic structure of PVA hydrogels (a) free of LRD, (b) with low LRD - PVA,

- LRD,

- crystalline

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concentration and (b) with high LRD amount (

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zones developed during the F/T cycles).

Thereby, both crystalline and amorphous zones are formed in the PVA-rich domain during the F/T cycles. By the addition of a low amount of LRD, the clay particles adsorb the PVA chain segments on their surface and new connection points (besides PVA crystallites) appear (Fig. 3b). The addition of a large amount of clay causes the association of the clay particles and the formation of large size aggregates through the PVA chains from amorphous domain (Fig. 3c). The aggregation mechanism of LRD platelets in water depends on the dispersion pH. Thereby, at acidic pH, the faces and edges of LRD platelets are oppositely charged and a cards-house structure forms due to the face-to-edge interactions. At basic pH, when both edge and face of platelets are negative, edge-to-edge or/and face-to-face interactions are responsible for clay platelets agreggation [48]. The pH values of the investigated PVA/LRD

Journal Pre-proof dispersions were between 6.5, for 0.5% LRD, and 9, for 3% LRD, indicating a basic medium favorable for edge-to-edge or/and face-to-face interactions between clay platelets.

3.3. Rheological properties The rheological measurements at a constant oscillation frequency of 10 rads-1 revealed gellike properties (G > G) for all investigated hydrogels (Fig. 4a). Viscoelastic moduli are constant up to a limiting stress (l) or strain (l) value, whereupon G and G decrease and the hydrogel network structure starts to change irreversibly [49]. As the hydrogel network is stronger, the shear stress necessary to destruct the hydrogel structure increases and l shifts to

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higher values. By further increasing of shear stress above l, a critical shear stress (c) is

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reached when G becomes lower than G and the sample acquires liquid-like properties. In Fig. 4b is shown the LRD concentration dependence of the maximum deformation

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permissible for structure preservation, l, for PVA hydrogel containing different concentration

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lP

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of clay.

Fig. 4. Evolution of (a) the viscoelastic moduli, G and G, as a function of shear stress, , at 10 rads-1 and (b) limiting strain value, l, as a function of LRD concentration at 25 C.

The addition of a small amount of clay (0.5%) into the PVA hydrogel determines a slight increase of the limiting strain value due to the additional interactions between clay particles and PVA chains, along with the polymer-polymer interactions, conferring a higher flexibility to the hydrogel. The small amount of clay, which is well dispersed into the PVA solution, reduces the possibility to develop interactions between clay particles. Further increase of LRD

Journal Pre-proof concentration leads to the development of clay aggregates which determine the decrease of network flexibility and the l value starts to decrease. From a clay concentration of about 1.6%, l magnitude remains approximately constant. In Table 1, it can be seen that in the LRD concentration range of 1.6–3% there is a maximum value of l at 2.5%. For clay concentration higher than 2.5%, the linear viscoelastic region becomes narrower as a result of the weakening of the gel network. The frequency sweep tests on the investigated hydrogels have evidenced the increase of viscoelastic moduli by increasing of the clay content up to 2.7% (Fig. 5a). All investigated samples have revealed gel-like properties with G > G, regardless the LRD concentration

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lP

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(Table 1).

Fig. 5. (a) Evolution of G versus oscillation frequency, , in linear viscoelastic domain; variation of (b) viscoelastic moduli and (c) G during the step strain measurements when strain is alternating from 1% to 1000% and again to 1% at a constant frequency of 5 rads-1 s for PVA/LRD hydrogels with different content of clay.

Journal Pre-proof The structural regeneration of PVA/LRD hydrogels after they were submitted to large deformation (1000%) was investigated by step strain measurements at 5 rads-1. Fig. 5b exemplifies the evolution of G and G in time when a stepwise sequence of 1%–1000%–1% was applied. At low deformation (within LVR domain), the viscoelastic moduli are constant and G values are larger than values of G for all investigated hydrogels. By increasing the strain to 1000%, G and G decrease abruptly and the systems become predominately viscous with G > G. For PVA/LRD hydrogels with 2%, 2.2% and 2.5% LRD, it was observed the increase of G values during the large strain step (Fig. 5c). The interactions established in a given range of compositions favor the “shake gel” behavior. It was known that PEO/LRD

of

aqueous dispersions can have a “shake gel” behavior under a vigorous shaking near the

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saturation threshold of clay particles surface with polymer (which depends on the polymer molecular weight, nature and concentration as well as clay amount) [27,50,51].

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The “shake gel” phase is characterized by bulk elasticity and its capacity to return to the initial state after shaking stops. Pozzo et al. [50] explained the “shake gel” behavior of PEO

re

(Mw = 3  105 gmol-1)/LRD mixture, in water, at a composition close to surface saturation

lP

limit, by obtaining of a new surface area as a result of deformation of clay particles aggregates under high shear stress and the establishment of unstable polymer bridges which determine

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the increase of macroscopic elasticity. After stopping the shear, the bridges break up, the desorption on the clay particles of the weakly bound segments occurs and the system returns at initial state. In our case, the hydrogels with clay concentration between 2% and 2.5% LRD

ur

exhibit “shake gel” behavior and G increases if a high deformation is applied. The highest

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increase of G (from 7.96 Pa to 22.36 Pa) under high shear stress was observed for the hydrogel with 2.5% clay. After application of the high stress (above LVR domain), the ability to recover the initial gel structure was about 90% for the hydrogels with clay concentration lower than 2% and around 80% for concentration near 2.5%.

3.4. Mechanical properties Three hydrogels, with low and high amounts of LRD and free of clay were chosen for mechanical measurements in order to observe the effect of clay on the tensile strength, the elongation and the tensile modulus. The stress-strain curves are shown in Fig. 6.

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Fig. 6. Stress-strain curves for the hydrogels containing different amounts of LRD.

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The tensile strength decreases by addition of LRD from 10.82 MPa, for PVA hydrogels free of clay, to 2.05 MPa, for 2.7% LRD due to the aggregates formed inside the voids

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generated by the F/T process which determines the formation of PVA crystalline zones. Tensile strength of P-C0.5 was observed at 3.89 MPa. The clay aggregates reduce the

lP

interfacial area between PVA chains and clay nanoparticles and create domains that can be broken under mechanical stress [52]. PVA hydrogel shows the highest elongation at break

na

(446.6%), while the hydrogels P-C0.5 and P-C2.7 show elongations up to 339.7% and 381.9%, respectively. The tensile moduli were determined as slopes of the linear part in the

ur

stress-strain curves. Tensile modulus of sample P-C0 (1.63 MPa) increased by addition of LRD at 1.75 MPa, for P-C0.5, and 2.56 MPa, for P-C2.7, proving an improvement of the

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stiffness level of the hydrogels.

3.5. Swelling behavior

The addition of clay does not significantly influence the swelling degree of physical PVA hydrogels in water. S values are close to 900%, indicating the ability of these hydrogels to adsorb the wound fluids and exudates. In Fig. 7, it is exemplified the variation of S as a function of time for PVA (P-C0 sample) and PVA/LRD (P-C2.7 sample) hydrogels in water at room temperature. PVA hydrogel showed the overshoot phenomena explained by the water diffusion rate into the gel pores higher than the relaxation rate of polymer chains from the network [53]. The fast diffusion of water into the gel determines the increase of S in the first hours and then the rearrangement of the PVA chains causes the water exclusion and an equilibrium S value is reached. The curve corresponding to the hydrogel with clay (sample

Journal Pre-proof P-C2.7) did not shown overshoot. The mass loss was 8.31% for PVA hydrogel free of LRD

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and 7.17% for the hydrogel with clay.

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Fig. 7. Swelling degree versus immersion time in water of P-C0 (Δ) and P-C2.7 (□) hydrogels at room temperature. The inset figure shows the plots of ln(Mt/M) as a function of ln t for

lP

PVA hydrogels in absence/presence of LRD.

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The diffusion mechanism of the water molecules into the hydrogel network during its

Mt  k tn M

(6)

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Peppas model) [54]:

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swelling can be establish with the relationship proposed by Korsmeyer et al. (Korsmeyer–

where Mt and M are the mass of water adsorbed at time t and at equilibrium, respectively. k (h-n) represents the kinetic constant and n is the power law coefficient which gives the indication about the type of diffusion mechanism. Thereby, n for cylindrical samples can have the following values: n = 0.45 for Fickian diffusion, 0.45 < n < 0.89 for non-Fickian or anomalous diffusion, and n = 0.89 for relaxation-controlled transport [55]. k and n values, determined from the representation of ln(Mt/M) as a function of ln t for Mt/M < 0.60 (inset Fig. 7), were 0.76 h-n and 0.18, for PVA hydrogel free LRD, and 0.61 h-n and 0.21, for the hydrogel containing 2.7% clay. The n values lower than 0.45 show a pseudo-Fickian diffusion, characterized by a rapid initial diffusion of water into the hydrogel followed by a slow reaching of the equilibrium value. The swelling process of macroporous hydrogels

Journal Pre-proof occurs mainly through water-filled pores and the deviations from Fickian behavior are due to the interactions established between diffusing water and hydrogel components. These interactions cause the diffusion through both the network formed by clay particles and PVA chains and the water-filled pores [56]. The diffusion rate (k) is slightly higher for the PVA hydrogel. The pseudo-Fickian diffusion mechanism, insufficiently explained in literature, was attributed to the heterogeneous structure of the gel given by the existence of the pores of different sizes and to the polydispersity of the sample [55,57]. n values lower than 0.45 can be also a consequence of the finite rate of polymer network structure changing as a result of the

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stress caused by the water sorption during the diffusion process [58]. The amorphous and the crystalline domains generated by the PVA chains and the LRD aggregates from network

ro

structure give polydispersity to hydrogels. The heterogeneity of hydrogels structure

-p

determines a rapid transport of the water molecules inside of the samples during the first stage of swelling but the equilibrium is reached very slowly as a result of the transport delay over a

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long period of time. n values lower than 0.45 were also previously reported for PVA/PVP

lP

hydrogels [17,18].

3.6. Water vapors sorption capacity

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Water sorption-desorption isotherms registered at 25 C, in the relative humidity range of 0–90%, for P-C0, P-C0.5 and P-C2.7, are shown in Fig. 8. It can be observed that all

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isotherms present similar shape with hysteresis due to the interactions established between

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water and hydrogel components. A slight decrease of the sorption capacity has been observed by addition of LRD into PVA hydrogel, due to the morphological changes. According to IUPAC classification, the sorption-desorption curves can be associated to type III behavior, indicating relatively weak adsorbent-adsorbate interactions. The data obtained from adsorption experiments were fitted with Brunauer-Emmett-Teller (BET) equation: W

Wm  C  RH 1  RH  1  RH  C  RH 

(7)

where W is the weight of sorbed water, Wm is the weight of water forming a monolayer, C is the sorption constant, and RH represents relative humidity. BET model is applicable with good results up to a relative humidity of 40%. The values of specific surface area and water monolayer adsorbed content are presented in Table 2. One can

Journal Pre-proof observe that water vapor sorption capacity decreases from 23.336%, for PVA hydrogels free

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ro

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of clay (P-C0), to 17.115%, for PVA hydrogels with 2.7% LRD (P-C2.7).

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Fig. 8. Water sorption-desorption isotherms of the PVA hydrogels in the absence/presence of

lP

LRD.

Table 2.

Samples

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BET data evaluated from sorption isotherms up to 40%. Total water vapors

BET analysis

ur

sorption capacity, weight (% d.b.)

P-C0.5 P-C2.7

23.336

302.057

0.086

18.898

115.795

0.033

17.115

142.686

0.041

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P-C0

ABET (m2g-1) Monolayer (gg-1)

The addition of LRD into PVA hydrogels determines the decrease of the specific surface area and water monolayer adsorbed content, due to the decreasing of the hydrophilicity, by including of clay particles into the hydrogel voids.

3.7. In-vitro rifampicin release Rifampicin (RI) is an antimicrobial drug mainly used in tuberculosis and leprosy treatment, although some positive results have also been obtained for leishmaniasis, rhinoscleroma, and

Journal Pre-proof psoriasis [59]. The addition of LRD into the RI-PVA hydrogel can have as effect, on the one hand, the improvement of the drug solubility in water (RI is slightly soluble in water) and, on the other hand, the change of the drug release time. Knowing that the solubility of RI in water at 25 °C and pH 7.3 is 2.5 mgmL-1 (according to supplier) and at 37 °C and pH values of 7.4 and 8 are 3.35 mgmL-1 and 5.44 mgmL-1, respectively [60], the concentration of drug in initial LRD/PVA aqueous solution (submitted subsequently to five F/T cycles) has been selected 1 mgmL-1 (0.1 g %). The released amount of RI was calculated with the following relationship: Nt  100 Nl

(%)

(8)

of

DR 

ro

where Nt and Nl represent the amount of RI released from the hydrogel at time t and the initial quantity of the RI loaded in the hydrogel.

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Fig. 9a illustrates the experimental release of RI from investigated hydrogels in MilliQ water, at 37 °C. The PVA hydrogel free of LRD releases the entire amount of loaded RI, due

re

to the weak interactions established between the polymer chains and drug molecules (Table 3,

Jo

ur

na

lP

Fig. 9a).

Fig. 9. (a) RI release profile from PVA hydrogels with different LRD contents, and (b) Korsmeyer-Peppas model kinetic release plots of RI for H1 (■), H2 (●), H3 (▲) and H4 (♦) samples. The inset figure shows the plot of drug release as a function of t for PVA hydrogels in absence of LRD.

Journal Pre-proof Moreover, a large amount of polymers (PVA) from hydrogel (32.94% compared with the mass loss for unloaded PVA hydrogel of 8.31%) dissolves, as a result of the negative influence of the drug incorporation on the formation of PVA crystallites during F/T cycles, leading to the weakening of interactions between the polymer chains. The addition of LRD into the hydrogel determines the decrease of the drug release percent to values in the range 79.50–87.12%, due to the additional interactions occurred between RI molecules and LRD particles. The presence of LRD into the PVA network causes an insignificant decrease of the time required to reach the maximum drug amount released from 160 min for PVA hydrogel free of LRD to about 130 min for the hydrogels containing clay.

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The drug release plot of the PVA hydrogel free of LRD (H1 sample) exhibits a high burst release (45%) in the first 10 min (inset Fig. 9a). The presence of burst could be attributed to

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small fraction of RI which is adsorbed or weakly bound to the hydrogel surface [61].

Table 3.

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Composition of hydrogels loaded with RI and the parameters which characterize the release processes. DRma

/PVA

H1d

0/1/38

H2

g/g

(%)

n

k

Peppas-Sahlin

b

R2

(min-n)

model parameters k1

k2

lossc R2

(%)

(min-m) (min-2m)

0.996

0.169 0.0017 0.43 0.999 32.94

14/1/38

0.38 79.50 0.12 0.612

0.963

0.437

H3

21/1/38

0.56 87.12 0.50 0.140

0.993

0.123 -0.0034 0.69 0.995 26.39

H4

25/1/38

0.66 82.81 0.44 0.220

0.992

0.218 -0.0054 0.48 0.996 31.44

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0.176 0.09 0.998 17.69

a

represents the maximum percent of released RI;

b

the models were applied for the experimental data up to 75% released drug;

c

m

mass b

100 0.44 0.168

ur

0

model parameters

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(g/g/g)

Korsmeyer-Peppas

lP

Sample LRD/RI/PVA LRD

PVA and LRD mass from hydrogel released in solution during the same time with the drug;

d

for H1 sample, the models were applied for the experimental data after burst effect and up to

75% released drug.

This fast initial release, on the one hand, can be an advantage if it is desired to attain rapidly a shock dose of drug and, on the other hand, it can be a therapeutic drawback leading to a toxic level for the body.

Journal Pre-proof The first model, used to establish RI release kinetics, was the Korsmeyer–Peppas model (Eq. (6)), where Mt - the mass of RI released at time t; M - the mass of RI released over a long time period; k - the kinetic constant (min-n); n - the release exponent. The ln(Mt/M) - ln t dependences during RI release in water are shown in Fig. 9b. The drug release from H1 (free of LRD) and H3, H4 (high amount of LRD) hydrogels shows a Fickian mechanism (n is close to 0.45), characterized by the water diffusion rate higher than the one of polymer chain relaxation. The presence of a small amount of LRD into hydrogel affects the RI release mechanism and n value becomes lower than 0.45 (pseudo-Fickian diffusion). RI incorporation into the PVA hydrogel (H1 sample) prevents the formation of crystalline

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polymer domains during the F/T cycles, due to the hydrogen bonds established between

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hydroxyl groups of PVA and RI. On the one hand, these interactions prevent the rapid relaxation of polymer chains and, on the other hand, the absence of crystalline zone in the

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network structure favors the diffusion of water into the hydrogel. RI release from the PVA hydrogel involves the following steps: i) the diffusion of water

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into the hydrogel; ii) the swelling of polymer network (polymer chain relaxation), and the

lP

formation of a gel layer, which expands as the water permeates into the polymer network; iii) the dissolution and the diffusion of the drug through the formed gel layer, for soluble drug, or by exposure through erosion, for poorly soluble drug; iv) the drug is released in water;

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v) the erodation of the polymer matrix at the hydrogel surface, due to the dissolution of polymer. The RI release in water from H1 sample is mainly due to the erosion mechanism of

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very small.

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PVA hydrogel, in which the number of crystalline domains formed during the F/T process, is

Effect of LRD incorporation on the water diffusion into PVA hydrogels loaded with RI depends on the clay concentration. The water diffusion into PVA/LRD/RI hydrogels is influenced by the various interactions formed between the polymer chains, clay particles and drug molecules (polymer-polymer, polymer-clay, clay-clay, clay-drug molecule, drug-drug, etc.). The RI release rate from H2 sample (low LRD amount) is higher than those corresponding to H3 and H4 samples (high LRD amount). The mass loss value increases from 17.69% for H2 sample to 31.44% for H4 sample proving that the addition of LRD favors the erodation of the hydrogel network during the drug release process. The preparation of the RI loaded hydrogels consisted in mixing of PVA and LRD, in the first step, and the addition of RI, in the second step. Thereby, by addition of a small amount of LRD into PVA solution, the clay particles are uniformly embedded through the polymer chains, leading to a strong network. The further addition of RI into the PVA/LRD dispersion does not change the

Journal Pre-proof network strength and the drug is released faster, with a smaller mass loss. The addition of a higher amount of LRD in PVA solution causes the formation of clay aggregates through the polymer chains, weakening network strength. The release of RI from these hydrogels occurs mainly by the bulk erosion which implies the fast penetration of water into polymer and clay particles matrix causing its partial destruction and the release of polymer chains or/and clay particles along with rifampicin release. This mechanism is supported by the values of the mass loss after the release experiments. The second approach used to fit the experimental drug release data was Peppas-Sahlin model, which takes into account both diffusional phenomena and polymer chains relaxation

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for the drug release process [62]:

(9)

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Mt  k1  t m  k2  t 2 m M

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where the first term represents the Fickian contribution and the second term is Case-II relaxational contribution. m is the Fickian diffusion exponent and it is related to the n

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coefficient from Korsmeyer–Peppas equation. For all samples, the Fickian contribution to the

lP

drug release is predominant, relative to the PVA chains relaxation (k1 > k2) (Table 3). 3.8. Antimicrobial activity

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Rifampicin (RI) is a broad-spectrum antibiotic, which was approved by Food and Drug Administration in 1971 for treatment of patients with tuberculosis [63], and being a low

ur

toxicity drug, it was used afterwards for the treatment of various infections produced by

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various types of bacteria, especially of S. aureus [64], various combined therapies such for catheter infections [65], osteomyelitis [66] and prosthetic joint infections [67], but also in other resistant Gram-negative bacteria [68]. The antibacterial activity was quantified on dried films, by using two types of different strains: S. aureus (Gram-positive) and E. coli (Gram-negative). These are very common and virulent pathogens that are usually met in care-associated disease. Data on the average inhibition diameters and images of inhibition zones are presented in Fig. 10 and Table 4. The control samples, H5 and H7, did not present any inhibition zone for the tested bacterial strains. The samples H6 and H8 show high activity against Gram-positive bacteria, represented by S. aureus. The sample H8 exhibits the highest antibacterial activity (4.15±0.45 cm).

Journal Pre-proof

Fig. 10. Antibacterial activity of samples: (a) H5–H6 and (b) H7–H8 against S. aureus; (c) H5–H6 and (d) H7–H8 against E. coli.

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Moreover, the samples H6 and H8 also presented antibacterial activity against Gram-

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negative bacteria, represented by E. coli, but at a smaller scale. The sample H8 shows again better antibacterial activity (3.05±0.07 cm), and it is more efficient than sample H6 (2.7±0.28

-p

cm).

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Table 4.

PVA

RI

%

%

%

H5

100

0

H6

98.1

1.9

na

lP

Antibacterial activity of the samples H5-H8 against the references strains (cm).

H7

61

0

H8

60

1.9

LRD LRD/PVA

S. aureus

E. coli

C. albicans

0

0

0

0

0

0

0

3.9±0.14

2.7±0.28

0

ur

g/g

Inhibition zone (cm)

39

0.64

0

0

0

38.1

0.64

4.15±0.45

3.05±0.07

0

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Samples

The compounds did not present any effect against the fungal strain represented by C. albicans, even if RI proved to have in-vitro activity, in combination with other drugs, against fungal diseases produced by Candida species, Rhizopus and Cryptococcus [69-71].

4.

Conclusions Physical crosslinked (PVA)/LRD hydrogels, obtained by freezing/thawing method,

revealed macroporous structure, with similar morphology, but with different distribution of clay particles into the polymer network, as a function of the clay concentration. The addition of a small amount of LRD leads to homogeneous PVA hydrogel, with a uniform distribution of the clay particles into the hydrogel structure. The increase of LRD concentration above

Journal Pre-proof 2.5% determines the appearance of the agglomerates and large size aggregates by association of the clay particles. At high clay concentration, the hydrogel structure becomes inhomogeneous. The rheological measurements evidenced the gel-like properties of all prepared samples. Moreover, the development of clay particles aggregates into the PVA hydrogels, at high clay concentration, determines the weakening of the network. PVA/LRD hydrogels containing 2%, 2.2% and 2.5% LRD showed a “shake gel” behavior characterized by increasing of storage modulus, by applying a high deformation. The recovery capacity of the initial gel structure, after application of a stress above linear viscoelastic range, was about 90% for the hydrogels with low clay concentration and around 80% for concentrations near

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2.5%. The equilibrium swelling degree was of about 900% regardless the composition. The rifampicin release mechanism from hydrogels is dependent of the clay concentration. The

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LRD incorporation into the PVA hydrogel loaded with rifampicin determines the increase of

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the antibacterial activity against gram positive (S. aureus) and negative (E. coli) bacteria. The incorporation of a low LRD quantity into the physical PVA hydrogels leads to materials

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which show the enhanced rheological and mechanical properties, high capacity of structural regeneration (about 90%) after applying a large deformation, high water absorption capacity

lP

(900%) and improved antibacterial activity, making them as potential wound healing

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dressings.

Acknowledgments

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The authors thank to Dr. Maria Cazacu, Dr. Alexandra Nistor and Dr. Codrin Tugui from

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„Petru Poni” Institute of Macromolecular Chemistry for dynamic water vapor sorption and mechanical measurements.

Conflicts of interest The authors declare no conflict of interest.

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