Alginate/poloxamer hydrogel obtained by thiol-acrylate photopolymerization for the alleviation of the inflammatory response of human keratinocytes

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Abstract

Hydrogel-based wound dressings have been intensively studied as promising materials for wound healing and care. The mixed-mode thiol-acrylate photopolymerization is used in this paper for alginate/poloxamer hydrogels formation. First, the alginate was modified with thiol groups using the esterification reaction with cysteamine, and second, the terminal hydroxyl groups of poloxamer were esterified with acryloyl chloride to introduce polymerizable acrylate groups. Finally, the cross-linking reaction between the two macromers was performed to produce degradable alginate/poloxamer hydrogels. The optimum conditions for the photo-initiated reaction were studied in order to obtain high gel fractions. The resulting hydrogels have high swelling capacity in simulated physiological conditions, good elasticity and strength, and appropriate porosity, some of the physico-chemical properties required for their applications as wound dressings/patches. The biological assays show that the alginate/poloxamer hydrogels induce proliferation of human keratinocyte and have an anti-inflammatory effect on lipopolysaccharides (LPS)-activated keratinocytes by inhibiting the extracellular signal-regulated kinases (ERK)/ nuclear factor (NF)-kB/ tumor necrosis factor (TNF)-α signalling pathway. Taken together, the results showed that the chemical cross-linked alginate/poloxamer hydrogels may function as a dressing/patch applied directly on the skin lesion to heal the wound by reducing the exacerbated inflammation, the main cause of wound healing delay and local infection.

Introduction

Epidermal tissue regeneration that comprises skin wound healing, a process regulated by the controlled functional behavior of keratinocytes and dermal fibroblasts after interaction with cytokines and growth factors, is a challenge for the biomedical field [1]. Based on the healing duration, the skin lesions are classified into acute and chronic wounds, which may result from various dermal conditions or injuries and represent a severe health risk since the skin barrier function is affected and the human body is exposed to exogenous pathogens [2,3]. One of the main causes that exacerbate skin injuries is the inflammatory response of the constituent cells to various external stimuli (e.g. lipopolysaccharides (LPS) from Gram-negative bacterial’ wall) [4,5]. Epidermal cells and keratinocytes in particular have been recognized to release constitutively low levels of soluble pro-inflammatory mediators, including the tumor necrosis factor (TNF-α) [6,7]. Moreover, the increased release of TNF-α by the keratinocytes was associated with skin lesions and is a consequence of the stimulation of the extracellular signal-regulated kinases (ERK) from the mitogen-activated protein kinases (MAPK) pathway and the overexpression of the nuclear factor (NF)-kB [8,9]. For healing acute or chronic skin wounds drugs like dexamethasone, or other materials with anti-inflammatory activity, like alginate, were used [3,10,11]. The wound dressing materials must also fulfill a series of physicochemical, mechanical and biological requirements, such as: high water swelling capacity to maintain a proper moisture environment, mecanical stability, elasticity, biocompatibility, non-cytotoxicity, and capacity to promote cell migration and proliferation [3,10,12]. Preferably, the wound dressings/patches must be biodegradable, with a degradation profile that is concomitant to the wound healing period of time. Hydrogels, with highly hydrated 3D polymeric networks, are the most suitable dressings to cover skin wounds [12].

In-situ formed hydrogels provide the advantage of proper adaptability for wound microstructure and shape [12,13]. Among in-situ fabrication techiques, thiol-acrylate photopolymerization reactions afford a rapid hydrogel formation under mild phisiological conditions and low doses of light, in the absence of toxic monomers and cross-linkers or byproducts [14,15]. Thiol-acrylate photopolymerization proceeds by a combination of the step-grow thiol-ene reaction and acrylate homopolymerization. Since this procedure can enhance the chemical and mechanical properties [14,16,17], it is largelyused for the fabrication of hydrogels with applications in regenerative medicine [15,18] or in tissue engineering [[19], [20], [21]].

Alginate, a biocompatible ionic polysaccharide of biological origin, is known for its numerous applications in biological science and engineering [22], including wound dressings [3,24,25]. Alginate was proved to reduce the concentration of proinflammatory cytokines [25], to increase collagen single bondI expression, and to facilitate re-epithelization [26,27]. The use of alginate in the treatment of chronic wounds is also due to its capacity to absorb high amounts of wound exudate, to maintain a physiologically moist microenvironment, and to minimize bacterial contamination [28]. Various alginate dressings are commercially available as such or in combination with other natural or synthetic polymers [2,3,12,23], to improve its low mechanical properties.

Poloxamer 407 (trade name also as Pluronic 127) is a triblock copolymer with a hydrophobic poly(propylene oxide) block with 54–60 units between two hydrophilic blocks of poly(ethylene oxide) with 95–105 units [29]. Poloxamer is approved by US Food and Drug Administration to be used in cosmetics [30] or as a pharmaceutical ingredient [31]. It was reported to enhance the rate of wound healing process [32,33]. Poloxamer/alginate mixtures were used for in-situ forming hydrogels, where the thermo-gelation property of Poloxamer was exploited alone [34,35] or in combination with the ionic gelation property of alginate [36,37]. Other strategies for in situ forming hydrogels based on Poloxamer and alginate involved the thermo-gelation of alginate-graft-poloxamer [38] or the formation of an inclusion complex between alginate-graft- β-cyclodextrin and Poloxamer [39]. These materials obtained by the physical cross-linking method have limited applications due to the reversibility of the solid-liquid transition and to the low mechanical properties.

In this paper, cross-linked hydrogels based on alginate and Poloxamer with good mechanical properties, high swelling capacity and appropriate porosity were synthesized by thiol-acrylate photopolymerization for potential applications in would healing. Previous to thiol-acrylate photopolymerization, alginate was modified with the introduction of thiol groups, and Poloxamer was modified by introducing two acrylate moieties on terminal hydroxyl groups. For optimization purposes, we studied the influence of the reaction conditions, namely irradiation time, photoinitiator concentration, temperature, and the ratio between thiol and acrylate groups on the hydrogel formation. The influence of the hydrogel composition on its morphology, swelling behavior, degradability, rheological and mechanical properties was also investigated. Biological studies were conducted on the alginate/poloxamer hydrogel, in which the molar ratio of thiol:acrylate was 1:1. The capacity of the hydrogel to induce the proliferation of human keratinocytes (HaCaT cells) and to diminish the inflammatory status of quiescent or LPS-stimulated cells was examined by measuring the released levels of TNF-α. Moreover, the mechanism by which the hydrogel exerts the anti-inflammatory effect was evaluated by the immunoblotting analysis of the proteins involved in the signalling pathway leading to TNF-α expression (i.e. ERK/MAPK and NF-kB).

Section snippets

Materials

Sodium alginate, with Mv = 180,000 Da (determined by viscometric measurements in NaCl 0.1 M, 25 °C, using the Mark-Houwink equation [η] = 0.002 ∙ M0.97 [40], where [η] is the intrinsic viscosity, mL/g) was purchased from Sigma-Aldrich (St. Luis, MO, USA). Poloxamer 407 (Sigma-Aldrich) was dried in vacuum using phosphorus pentoxide, and dichloromethane (Honeywell, Seelze, Germany) was dried over anhydrous sodium sulfate. Triethylamine, acryloyl chloride, 1-ethyl-3-(3-dimethyllaminopropyl)

Synthesis and characterization of macromolecular precursors (PL-DA and Alg-SH)

The esterification of terminal hydroxyl groups of Poloxamer with acryloyl chloride was confirmed by FTIR spectroscopy (Fig. 1A). The spectrum of PL-DA showed a new adsorption band at 1725 cm−1 corresponding to ester Cdouble bondO groups, which was absent in the spectrum of Poloxamer 407 [50]. The peaks at 1632, 1411, and 995 cm−1 are better evidenced in the spectrum of PL-DA because they are also attributed to the double bonds CH2 = CH- [51].

The end group conversion of poloxamer with acrylate was

Conclusions

Poloxamer was successfully modified with acrylate groups on both chain ends (substitution degree 92–97%) and alginate was modified with 20% thiol groups in order to obtain macromers that can be cross-linked by thiol-ene photo-coupling for hydrogel formation. High gel fractions (around 80%) were obtained by using 0.15% Irgacure 2959, an irradiation time of 6 min, 15% concentration of macromolecular precursors, and 1:1 molar ratio between [SH] and [acrylate].

The hydrogels present high swelling

CRediT authorship contribution statement

Irina Popescu and Mihaela Turtoi - Conceptualization, Methodology, Writing - original draft (equal contribution); Dana Mihaela Suflet: Investigation; Maria Valentina Dinu: Mechanical investigation; Raluca Nicoleta Darie-Nita: Rheological investigation; Maria Anghelache: Biological tests investigation; Manuela Calin: Biological experiments designing, Supervision and Writing; Marieta Constantin: Supervision, Writing and Editing.

Declaration of competing interest

None.

Acknowledgments

This work was supported by a grant of Romanian Ministry of Research and Innovation, CNCS - UEFISCDI, project number PN-III-P4-ID-PCCF-2016-0050, within PNCDI III.

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