1. Introduction
Cutaneous wound healing is a well-orchestrated process consisting of four overlapping phases: hemostasis, inflammation, proliferation, and remodeling. Most skin wounds heal within 2–3 weeks, however, imbalances in the wound healing phases can lead to non-healing chronic wounds [
1]. Chronic wounds remain in the inflammatory phase, where elevated levels of reactive oxygen species (ROS), pro-inflammatory cytokines, and degradative proteases result in reduced concentrations of growth factors and proteinase inhibitors, and an imbalance in the wound equilibrium [
2]. Diabetes and vascular insufficiency are the main underlying causes of non-healing wounds, with systemic factors such as advanced age or a compromised immune system also contributing to poor wound-healing. The incidence of chronic wounds has dramatically increased in recent years, now reaching epidemic proportions [
3]. Presently, most of the widely used wound dressings only provide an optimal local healing milieu (i.e., they protect the wound from further trauma, provide moisture, and adsorb excess exudate) [
4]. To improve the treatment of chronic wounds, it has been proposed that new types of wound-healing therapies will be required, with the aim of obtaining bioactive dressings that stimulate local cells to migrate and proliferate, stimulate, and guide extracellular matrix deposition, modulate protease activity, and/or neutralize free radicals [
4].
Nanocellulose is emerging as an interesting material for biomedical applications such as tissue engineering, drug delivery, and wound care [
5,
6]. Nanocellulose consists of cellulose fibrils or crystallites with dimensions in the nanoscale and is obtained from a diversity of sources such as wood, algae, bacteria, and tunicates [
7]. Wood-derived cellulose nanofibrils (CNF) comprise individual fibrils that are 2–10 nm in diameter and several micrometers in length, typically forming 20–60 nm thick aggregates [
8]. Characteristics such as being a renewable material, not being of animal origin, having tuneable properties in terms of surface chemistry, aspect ratio and form, and being produced in a process that can easily be scaled-up industrially have contributed to the increased interest in using CNF for biomedical applications [
5,
9]. CNF have been described as a promising material for the treatment of wounds [
5,
10,
11]. Authors have highlighted the physicochemical properties of the material and the absence of cytotoxicity as beneficial characteristics for employing CNF films, aerogels, and gel suspensions as wound dressings [
12,
13,
14]. Moreover, clinical studies have shown encouraging results when using CNF films on skin graft donor sites [
15,
16]. Our group has demonstrated the in vitro and in vivo wound healing properties of an ion-crosslinked CNF hydrogel [
17,
18,
19] together with the evaluation of its antibacterial properties [
20] and the possibility of using the hydrogel as a drug delivery dressing [
21].
In the present work, we selected the amino acid cysteine as an active molecule to endow CNF with bioactivity and for the first time investigate the potential of the functionalized CNF material to modulate the chronic wound environment in vitro. It is believed that addressing the biochemical imbalances typically found in chronic wounds will aid in the resolution of these hard-to-heal wounds. Thus, by targeting the excess of ROS and the high level of metalloprotease activity, it may be possible to restore basal levels of growth factors, promote the restitution of the proper balance between degradation and formation of new tissue, and help overcome the prolonged inflammatory phase observed in chronic wounds [
22,
23]. The radical scavenging properties of cysteine and its metal chelating ability [
24,
25], make this amino acid an excellent candidate molecule to be used in the development of wound dressings capable of interacting with the biochemical environment of chronic wounds.
In this study, cysteine was covalently incorporated into CNF and the functionalized material was characterized in terms of chemical structure and degree of substitution. The radical scavenging properties and the capacity of the material to inhibit metalloprotease activity were investigated. Furthermore, the stability of the thiol groups in cys-CNF and its bioactivity were evaluated over time. Finally, the safety profile of the thiol-functionalized CNF was investigated in an in vitro cytotoxicity study with human dermal fibroblasts.
2. Materials and Methods
2.1. Chemicals and Reagents
Carboxylated-CNF, provided by RISE Bioeconomy (Stockholm, Sweden), was produced from commercial never-dried bleached sulfite softwood dissolving pulp (lignin content < 1.5%, xylose < 1.7%, mannose < 1.8%, Domsjö Fabriker AB, Örnsköldsvik, Sweden) by 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation. L-cysteine methyl ester hydrochloride, N-(3-dimethyolaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), collagenase from Clostridium histolyticum, and calcein-AM were obtained from Sigma Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), penicillinin, streptomycin, 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB), EnzChek™ Gelatinase/Collagenase Assay Kit, Pierce TM Bradford Protein Assay Kit, DMEM:F12 medium, fetal bovine serum, and presto blue cell viability reagent were obtained from ThermoFisher Scientific (Waltham, MA, USA). 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
2.2. Covalent Incorporation of Methyl Cysteine to Cellulose Nanofibrils
L-cysteine methyl ester was covalently incorporated to carboxylated CNF (c-CNF, carboxyl group content 1300 μmol/g dry CNF) via EDC/NHS coupling (
Figure 1). Briefly, 100 g of c-CNF suspension corresponding to 1 g of dried cellulose was dispersed in 50 mL of deionized water and 5 mL of aqueous solution containing 180 mg of NHS and 290 mg of EDC (corresponding to 1.2 mmol of NHS/EDC per mol of carboxyl group in CNF) were added under stirring, followed by the addition of 10 mL of L-cysteine methyl ester aqueous solution. Two different molar ratios cysteine:COOH were used in the synthesis, corresponding to 6 and 20 equivalents of cysteine with respect to the carboxyl groups in c-CNF. The reaction mixture was stirred for 24 h at pH 5 and room temperature. For purification, three cycles of centrifugation (30 min at 3380×
g) were carried out, followed by dialysis against deionized water until the conductivity in water was < 0.005 mS/cm
2.
2.3. Material Characterization
2.3.1. Characterization of the Molecular Structure
The chemical structure of cys-CNF was characterized by solid-state nuclear magnetic resonance (NMR). The NMR spectra were obtained on a Bruker Avance III 600 MHz spectrometer (Bruker, Billerica, MA, USA) using a double-resonance 4 mm (1H&19F)/(15N-31P) CP-MAS probe and 4 mm ZrO2 rotors. The 13C cross-polarization (CP) magic angle spinning (MAS) NMR spectra were recorded at a spinning frequency of 12 KHz, a contact time of 1–2 ms and a repetition delay of 3–5 s. The experiments were performed at 298 K.
2.3.2. Degree of Substitution
The cysteine content of cys-CNF was determined by elemental analysis of total sulfur content. For this, cys-CNF freeze-dried samples were submitted to MEDAC Ltd., analytical and chemical consultancy services (Cobham, UK), where a Thermo FlashEA
R 1112 instrument was used for sulfur quantification. The weight percentage of sulfur in cys-CNF was converted to mmol cysteine/g CNF [
26] and the degree of substitution (DS) was calculated as the cysteine content (mmol/g CNF) per number of carboxyl groups in the starting material c-CNF (1.3 mmol/g CNF).
2.3.3. Thiol Group Content
The thiol group content was quantified by the Ellman assay. Briefly, 2.5 mL of reaction buffer (0.1 M sodium phosphate, pH 8.0, containing 1 mM EDTA) was mixed with 250 µL of cys-CNF suspension (0.05 wt%) and 50 µL of DTNB solution (4 mg/mL in reaction buffer). After 15 min, the absorbance at 412 nm was measured using a spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan). The number of thiol groups in the CNF samples was calculated using a L-cysteine methyl ester standard curve and expressed as mmol SH/g CNF.
2.4. Radical Scavenging Capacity
Free radical scavenging capacity was evaluated with DPPH by measuring the decrease in absorbance of the radical at 517 nm after incubation with the CNF materials. In brief, 1.2 mL of CNF suspension in water (containing 0.5 mg, 2 mg, and 5 mg of dry CNF materials) was mixed with 10.8 mL of DPPH (0.2 mM in methanol). After 90 min incubation time protected from direct light, reaction tubes were centrifuged to spin down the CNF materials and the absorbance of the supernatant was measured at 517 nm with a plate reader (TECAN infinite M200, Männedorf, Switzerland). Radical scavenging capacity was expressed as percentage of DPPH inhibition according to Equation (1):
where A
S is the absorbance at 517 nm of the DPPH solution after incubation with the CNF samples and A
R is the absorbance at 517 nm of the DPPH reference solution (i.e., incubated alone).
2.5. Evaluation of the Material Interactions with the Metalloprotease Collagenase
Collagenase from Clostridium histolyticum was selected as a model protein to investigate the interaction of cys-CNF with metalloproteases. Aqueous suspensions (0.7 mL) of the CNF materials containing 0.1 mg, 0.2 mg, and 3.5 mg of dry cellulose were mixed with collagenase in reaction buffer (0.5 M Tris-HCl, 1.5 M NaCl, 50 mM CaCl2, and 2 mM sodium azide, pH 7.6) at a final protein concentration of 10 μg/mL in a final volume of 1.5 mL. The mixture was incubated for 24 h at 37 °C under agitation (400 rpm). Afterward, reaction tubes were centrifuged to spin down the fibers and the supernatant was collected to determine the collagenase activity and concentration.
2.5.1. Evaluation of Protease Inhibition
The EnzChek
TM Collagenase Assay Kit was used to quantify the activity of collagenase after incubation with the materials. The assay was performed as indicated by the manufacturer. Briefly, 20 µL of fluorescein conjugate-gelatin (DQ
TM Gelatin, 0.5 mg/mL) was added to 100 µL of reaction buffer (0.5 M Tris-HCl, 1.5 M NaCl, 50 mM CaCl
2, and 2 mM sodium azide, pH 7.6) mixed with 80 µL of CNF-incubated collagenase solution. The increase in fluorescence signal (excitation/emission at 485/530 nm), as a result of substrate cleavage by the collagenase, was measured from minute 0 to 120 with a plate reader (TECAN infinite M200, Männedorf, Switzerland). To calculate the inhibition of collagenase activity, the 30 min time point was selected and the fluorescence intensity of the CNF-incubated collagenase samples was compared to the fluorescence intensity of the collagenase control according to Equation (2):
where F is fluorescence intensity (a.u.) at the 30 min time point.
2.5.2. Evaluation of Protease Entrapment
The Bradford assay was used for protein quantification. Simply, 100 µL of CNF-incubated collagenase was mixed with 100 µL of Bradford reagent in a transparent 96 well plate. Following the protocol from the manufacturer, the plate was shacked for 30 s and, after 10 min incubation at room temperature, absorbance was measured at 595 nm with a plate reader (TECAN infinite M200, Männedorf, Switzerland). Protein entrapment was expressed as percentage of collagenase in solution after incubation with CNF over the content of the protein in the control solution, as expressed in Equation (3):
where A
S and A
R are the absorbance values at 595 nm of the sample and of the control solution, respectively.
2.5.3. Zn2+ Binding Assay
With similar conditions as in the collagenase inhibition experiment, water suspension of the CNF materials (5 mg dry content) were mixed and incubated with 0.1 mM zinc acetate in a final volume of 3 mL. The mixture was left in a shaking plate at 37 °C for 24 h. Then, fibers were spun down and the zincon assay was performed to quantify the remaining zinc in the supernatant. Briefly, 25 µL of sample (supernatant) was mixed with 950 µL of 53 mM borate buffer, pH 9; and 25 µL of 1.6 mM zincon (2-carboxy-2′-hydroxy-5′-sulfoformazyl-benzene) solution were added to the mixture. The absorbance at 620 nm was measured 5 min later. Zinc content was expressed as a percentage of the reference solution (i.e., where the CNF suspension was replaced by water).
2.6. Material Stability over Time
The thiol content and the radical scavenging properties of the cys-CNF suspension were measured by the Ellman assay and the DPPH assay, respectively, over a time period of 30 days to evaluate the stability of the thiol group in the cys-CNF suspension and the correlation to its radical scavenging capacity. The material was kept at 4 °C in three different storage conditions: suspensions in a closed container, aerogels prepared by freeze-drying, and suspensions stored under an inert atmosphere. The Ellman assay and the DPPH assay were conducted following the protocols described in
Section 2.3 and
Section 2.4, respectively.
2.7. Cell Studies
2.7.1. Cell Culture
Adult human dermal fibroblasts (hDF) (primary cells from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, England) were cultured in DMEM-F12 medium supplemented with 10% v/v fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were cultured at 37 °C and 5% CO2 in a humidified atmosphere and passaged at 80% confluency.
2.7.2. Cytotoxicity Assessment
The cytotoxicity of cys-CNF was assessed by exposing hDF monolayers to suspensions of cys-CNF (1:20). Concentrations from 0.1 to 1 mg/mL in cell culture medium were prepared from 10 mg/mL stock suspension in water the day of the experiment. A control experiment showed that the dilution of cell culture medium due to the water content of the CNF stock suspension did not significantly affect the cell viability.
hDF cells (passage numbers 10–16) were seeded in a 96 well-plate at a density of 4.8 × 103 cells/well and after 24 h of culture, they were exposed to the cys-CNF suspensions (200 µL/well) and cultured for another 24 h. Non-treated cells were used as the negative control and cells exposed to 5% DMSO in cell culture medium were the positive control. c-CNF was also included in the toxicity assessment in the same concentration range as cys-CNF.
Cellular metabolic activity was measured with the presto blue (PB) assay after 24 h of exposure to the CNF materials. Cell culture media were removed from the cell culture wells and cells were carefully washed with warm PBS. Thereafter, 200 µL of PB reagent diluted 1:10 in cell culture media were added to each well and incubated for 90 min at 37 °C, and 5% CO2 in a humidified atmosphere. Aliquots of 100 µL were transferred to a black 96-well plate and fluorescence was measured at 560 nm excitation and 590 nm emission wavelengths using a plate reader (Tecan infinite M200, Männedorf, Switzerland). Cell metabolic activity was used as an indicator of cell viability and the results were expressed as the percentage of the negative control. The interaction of the CNF samples with the PB reagent was evaluated by applying the same protocol as above, but in the absence of cells and the results showed no interference of the samples with the PB assay.
Cell viability and morphology were also assessed by microscopic observations of calcein-AM stained cells. Briefly, cell media were removed from the wells, and 100 µL of calcein-AM (0.1% v/v in PBS) were added to each well. After 15 min incubation time in the cell incubator, cells were imaged with a fluorescence microscope (Nikon Eclipse TE2000-U, Tokyo, Japan).
2.8. Statistical Analysis
All data are presented as mean ± standard error of the mean from at least three independent experiments, each with triplicate sampling. Statistical analyses were performed using GraphPad Prism software. Differences between groups were evaluated by one-way analysis of variance (ANOVA), followed by Dunnett’s test for pairwise comparisons. A t-test was used for paired comparison of the two treatment groups. The differences were considered statistically significant when the p value was < 0.05.