1. Introduction
Chitin is a natural polysaccharide made of N-acetylglucosamine units bound by covalent β-(1→4) linkages and with a structure similar to that of cellulose, thus able to form fibrils and whiskers. Chitin is primarily found in the shells of crustaceans, cuticles of insects, and cell walls of fungi. It is the second most abundant polymerized carbon-based macromolecular material found in nature and can be converted to innovative high-value bio- and eco-compatible materials [
1].
Chitin consists of both crystalline and amorphous domains, but the elimination of the amorphous phase results in chitin nanofibrils (CNs), nanocrystals characterized by high bioavailability; thus, they have become an appealing source in green cosmetics [
2]. When fibrillated on the nanoscale, chitin loses its pro-inflammatory and allergenic character. Having a backbone structure virtually analogous to that of hyaluronic acid, CN is easily metabolized by the body’s endogenous enzymes, specifically by chitinases and lysozymes present in various human body fluids as well as by some bacterial enzymes present in the colon [
2,
3]. Controlling the crystalline structure and purity of CNs leads to improvement of its antibacterial, anti-inflammatory, cicatrizing, and anti-aging activity. Hence, CNs are suitable for different applications, mainly in pharmaceutical, biomedical, food, textiles and packaging fields [
4,
5,
6]. By all these properties, in addition to non-toxicity toward living organisms and the environment (i.e., air, water, and soil), chitin can play a role as a biopolymer for sustainable industrial development. Moreover, the antibacterial activity and low immunogenicity of chitin have broadened the aspects of research and development on the structure-function relationship toward biological tissues and activities [
1].
Despite the abundance, accessibility, and low cost, the random distribution of acetyl groups in chitin structure, batch-to-batch diversity, and non-accurate characterization of chitin result in a low reproducibility of chitin solubility and eventually in a limitation in product development and access to the market in large volume. Different inorganic acids, bases, and salts have been used for chitin and chitosan dissolution [
7].
The electrohydrodynamic atomization technique, simply called electrospray, accrues when a conductive polymer jet separates into very small droplets under the influence of an electrical field [
8,
9]. The type of solvent is one of the most important parameters in the electrospray technique since, during the flight of droplets to the collector, the primary ones shrink due to the solvent evaporation, which leads to an increase in charge concentration and the breaking of them into smaller offspring [
10]. The surface functionalization materials are possible by using the electrospray method.
This study presents the surface patterning of cellulose tissues via electrospray technique using chitin nanofibrils to decorate the surface with functional (e.g., anti-inflammatory, anti-microbial) nano-metric features, for skincare application. Two types of CNs from different sources (i.e., shrimp (sCN) and mushroom (mCN)) were used for the electrospray technique using two solvent systems, which were used to suspend the CNs in liquid media. The effects of CN type and solvent system (i.e., fully and partially water-based) necessary for electrospraying were investigated. In particular, we assessed the cellulosic tissue surface modification via scanning electron microscopy (SEM) and the skin-compatibility using human dermal keratinocytes (HaCaT cells). The expression of a panel of cytokines involved in inflammation and immune response was studied, including the pro-inflammatory interleukins (ILs) IL-1, IL-6, and IL-8; the tumor necrosis factor α (TNF-α); the transforming growth factor β (TGF-β); and the human beta-defensin 2 (HBD-2).
The successful and uniform surface functionalization of cellulose tissue would enable the development of novel biobased products for potential use for skin-related applications, such as skin contact and skincare.
3. Discussion
The skin is the largest organ of the human body that in a lifetime is directly exposed to the sunlight radiation, environmental agents, and chemical pollutants, as well as injuries and other damages; therefore, an appropriate skincare routine is important to maintain health and wellbeing. Due to a frenetic daily life, many people neglect skincare, thus acquiescing to skin stress and immune reactivity. The application of pure active ingredients and anti-inflammatory biomaterials, possibly under the circular economy approach, endowed with intrinsic preventive or even therapeutic properties for skin disorders and aging, is a key topic in modern cosmetology [
2,
11,
12,
13].
Cellulose is the most widely available and renewable material on Earth; it is also present as naturally nanofibrous substrates, i.e., bacterial cellulose, imbued with remarkable properties in wound repair, including eardrum perforations [
14,
15,
16,
17]. Surface decoration of cellulose tissues with proper functional coatings based on natural biopolymers will allow their cosmetic performance to be improved, thus enabling simple but effective biobased products to effectively replace petrochemical counterparts in the personal care sectors [
2]. As an example, CNs, alone and complexed with nanolignin in the form of microcarriers, have shown bioactive features in skin contact and cosmetics, being recently proposed in a novel soluble nonwoven beauty mask based on pullulan [
1,
6,
18]. Electrospray, which is analogous to electrospinning, has aroused interest as an eco-friendly technology: it usually works at room temperature and there are no post-process residues, with a virtual yield of 100% for the electrosprayed active ingredients. In the case of water-based solvent systems, this technique is safe and green, having only electricity as an energy supply [
6,
19].
In our study, we used the electrospray technique to decorate the surface of cellulose tissue with CNs aimed at improving its antibacterial and anti-inflammatory properties to propose a novel skincare product. We thus investigated shrimp-based and mushroom-based CNs as active ingredients for electrospray and focused on water-based solvent systems. It is well known that a solvent, upon interaction with an electric field, plays a decisive role in the size and the homogeneity of the particles being collected downstream of the electrospray process [
20]. Other techniques for surface functionalization in cosmetic and packaging applications, such as spray [
21], casting [
22], dry powder impregnation [
6], multilayer coatings assembled via dipping and spraying [
23], or spinning methods [
24], have been proposed for modification of different surfaces, including fibrous nonwovens and delicate films using CNs or CN complexes [
6,
23,
24].
CNs are biobased nano compounds that can be considered useful bioactive agents for functionalizing skin contact substrates. In fact, on the nanoscale, chitin is able to modulate the pro- and anti-inflammatory reaction of dermal keratinocytes [
1,
25]. The application of an easy and efficient method for decorating the biomaterial surfaces with such components and for quality control of the outcomes has been the subject of recent studies [
26]. Water-based spray of CNs may affect the cellulose substrate surface by wetting. Searching for environmentally friendly solvents, we thus considered that electrospray could be a valuable method for CN deposition on cellulose tissue surfaces, as it can uniformly functionalize the surface of the substrate without a wetting effect due to the nanodrops being generated in presence of a high voltage field. Different parameters can affect the production of nanoparticles with uniform size and morphology during the electrospray process [
27,
28,
29]. Solution parameters include the solvent type and solution viscosity, and electrospinning parameters include applied voltage, flow rate, and needle tip to collector distance. In fact, a polymer solution used for the electrospray technique should be enough diluted so that a low sufficient viscosity allows the solution to break up into droplets at the same time so as to not be too viscous to form the continuous fibers [
8,
10]. The volatility of solvents is also important since the electrospray principle is based on solvent evaporation.
Usually, aggregated three-dimensional (3D) chitin networks resulting from high crystallinity, hydrogen bonds, and strong cohesive forces cause the lack of solubility in conventional solvents, which has limited its applications [
7]. In our study, the so-called “solvent systems” were selected to electrospray finely dispersed CNs in a liquid means. In this way, the CN properties were better preserved, as chitin was neither dissolved to form a solution (rather CN was in a colloidal suspension state with the solvent systems) nor deacetylated. Therefore, in our intent, the primary role of the solvent system was to allow separation among the CNs, along with an efficient interaction between the CNs and the electric field, which in turn results in smaller size droplets and larger deposited areas. We investigated the effect of CN source and solvent system types on the decoration of cellulose tissues and their bioactive properties for skin contact applications. Using distilled water as a dispersion agent resulted in the formation of large size droplets containing aggregated CNs for both shrimp and mushroom types. This phenomenon occurred when electrospray was performed using both aluminum and cellulose tissue as collecting surfaces. Using water/acetic acid as a solvent led to the deposition of CNs with uniform size and morphology, in particular when mCNs were employed as a chitin source. The observed differences can be due to the improved nano-suspension of CNs in an acidic solution [
21]. In the acidic system, the protonation of -NH
2 groups present on the surface of CNs occurs, leading to the formation of positive charges at the surface. The repulsions between nanofibrils counteract coalescence phenomena, thus favoring a homogenous deposition by electrospray.
The improved behavior obtained by adding 50%
w/
w glacial acetic acid can be explained by the change in surface tension, which enables better interaction with the electric field. It has been reported that to electrospinning the chitosan-a partially deacetylated form of chitin, the use of acetic acid/water solvents at higher ratios (10% up to 90%) improved the fiber formation by decreasing the surface tension, which is considered a key parameter to switch from particles into beads or fibers. In this study, 50% acetic acid still was in the particle formation window [
30].
As a further example of organic solvent miscible in water, we tested HFIP, a polar fluoroalcohol with strong hydrogen bonding properties, which has an acidic behavior. HFIP is able to solubilize mostly insoluble polymers, peptides, and β-sheet protein aggregates. By using the mixture of distilled water/HFIP (60/40
w/
w%), continuous chitin nanofiber webs with a homogeneous morphology and a quite uniform diameter were formed on the surface of the cellulose tissue. This phenomenon has been described by dissolving chitin in pure HFIP, as an in vitro self-assembly (i.e., bottom-up) of biogenic chitin nanofibers initiated by solvent evaporation [
31]. The authors did not observe deacetylation under the mild conditions applied, and the crystalline structure of chitin was maintained. Apparently, the application of HFIP as a co-solvent in an equal concentration to water led to the formation of a CN solution with a higher viscosity than the solution produced with distilled water or diluted acetic acid. Such a viscous solution led to the formation of CN nanofibers instead of CNs. Min et al. investigated the variation in viscosity with the concentration of chitin in HFIP [
32]. They demonstrated that at lower concentrations, fibers with a large number of beads were formed while increasing concentration led to the significant chain entanglements and subsequent increase of the viscosity, which ultimately formed the continuous fibers of chitin.
We showed that electrospray is a safe technology to decorate cellulose tissue since no significant difference was observed in the chemical structure of cellulose tissue after its surface modifications and in the main characteristic bands of CN observed on the spectrum of cellulose tissue functionalized with CN from different sources. Chitins may have different acetylation depending on the source type, such as fungi, insects, crustaceans, mollusks, and the extraction processes, which may determine variations in their biological activity [
7]. The search for animal-free cosmetic and skincare products is looking specifically at vegetable sources of chitins, such as mushrooms, whereas the biobased industries are particularly interested in chitin as a biowaste, such as crustaceans. Therefore, the diverse chitin source can comply with the specific needs of the market, and an investigation about the specific biological properties of different CNs could enable the best choice for each final application. After both sCNs and mCNs were electrosprayed via diverse water-based solvent systems upon cellulosic tissues, the functionalized paper was cytocompatible with HaCaT cells chosen as an epidermal skin model. Previous studies performed on free sCNs and electrosprayed sCNs onto polyhydroxyalkanoate fiber meshes corroborated the good interaction of sCNs with HaCaT cells [
1,
9]. There are many recent studies that investigated cellulose as a scaffold, also reporting excellent properties of paper in cell cultures in vitro and cell migration in vivo [
14,
33,
34,
35,
36]. In our results, the CN functionalized cellulosic tissue was highly cytocompatible in vitro using a model of human dermal keratinocytes under indirect and direct testing modes.
Independent of the solvent systems applied for CN dispersion, viable HaCaT cells were detected in all the samples and conditions used. Apparently, a few dead cells were observed in the indirect cytotoxicity tests in both samples processed using water/acetic acid to electrospray the CNs, in which also the metabolic activity was slightly lower than in the other samples. Even though this fact could be a consequence of some acidic residue of acetic acid, the direct cytocompatibility tests corroborated the very good cytocompatibility of these samples. Acetic acid is a weak organic acid of natural origin; thus, its environmental impact is lower than that of the other acids tested, i.e., HFIP. In the electrospinning process, the solvents are trapped in a filter, which must be disposed or possibly regenerated with solvent recovery [
37]. In the case of a solvent mixture, such as water/solvent, the separation process and cost must be evaluated. Moreover, the use of HFIP led to the obtainment of self-assembled chitin nanofibers instead of separate CNs, so the mixture of water/acetic acid could be better suitable for a circular economy approach [
2].
Finally, small differences in biological activity were detected between sCNs and mCNs. Specifically, the cellulose substrate decorated with sCNs showed strong immunomodulatory activity; conversely, using mCNs had an overall anti-inflammatory activity. The different reactions of HaCaT cells to the substrates covered by different CNs could be explained by the diverse immune recognition, which is also affected by size and shape, as well as source and purification methods [
38]. It has been demonstrated that chitin lower than 0.2 µm loses immunogenicity; however, aggregated CNs may be perceived differently from the cells. Other possible concurring factors are physico-chemical treatments used to purify chitin, which can alter the chitin structure, thus its recognition by the immune cells [
38]. Therefore, all these aspects must be taken into account when developing a cosmetic product using CNs.
4. Materials and Methods
4.1. Materials
Both sCNs (water solution, 1.5%) and mCNs (Glentham, Mushroom-based) (water solution, 1.5%) were supplied by Celabor (Mouscron, Belgium). Cellulose tissue used as a substrate was kindly provided by LUCENSE (Lucca, Italy). Acetic acid (code: 33209) and hexafluoro-2-propanol (HFIP) (code: 105228) were bought from Sigma-Aldrich (Milan, Italy). HaCaT cell line was obtained from CLS–Cell Lines Service, Eppelheim, Germany. Absolute ethanol was supplied by VWR (by Avantor, Radnor, PA, USA). Formalin was purchased from BioOptica (Milan, Italy). Fetal bovine serum (FBS) and Live/Dead kit (propidium iodide and calcein, AM, USA) were bought from Gibco (by Life Technologies, ThermoFisher Scientific, Waltham, MA, USA). Dulbecco’s Modified Essential Medium (D-MEM), l-Glutamine, penicillin, streptomycin, MgCl2, LiCl, LC Fast Start DNA Master SYBR Green Kit, and Tri Reagent® were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Phosphate buffered saline (PBS) was supplied by Lonza (Basel, Switzerland).
4.2. Preparation of CN Solutions and Electrospray Protocols
CN was used at 0.52 w% in all applied solvents. Distilled water, distilled water/acetic acid (50:50 w/w), and distilled water/HFIP (60:40) w/w) were used as solvents. Each solution was magnetically stirred for 3 h until it appeared uniform. Each solution was electrosprayed using an electrospinning bench apparatus (Linari Engineering s.r.l., Pisa, Italy) with a distance of 7 cm from the positively charged needle tip to the grounded aluminum static collector at 17 kV at a flow rate of 0.136 mL/h for 30 min.
In the following step, cellulose tissue was connected to the static aluminum collector, and for each CN solution, the same electrospray parameters were used for 3 h to electrospray CN on the surface of cellulose tissues.
4.3. Morphological Characterization of CNs
Morphological analysis of the samples was performed using field emission electron scanning microscopy (FE-SEM) with FEI FEG-Quanta 450 instrument (Field Electron and Ion Company, Hillsboro, OR, USA) and inverted optical microscope (Nikon Ti, Nikon Instruments, Amsterdam, The Netherlands). The samples were sputtered with gold or platinum for analysis. ImageJ software (version 1.52 t) was used to evaluate the size of nanofibrils and fibers. An average of 50 measurements was reported for each sample.
4.4. Chemical Structure Characterization
Fourier transform infrared spectroscopy (FTIR) was pursued using a Nicolet T380 instrument (Thermo Scientific, Waltham, MA, USA) equipped with a Smart ITX ATR attachment with a diamond plate, which was employed for chemical structure characterization of both solid chitin-based substances and cellulose tissue.
4.5. Cytotoxicity Evaluation with Epidermal Cells
Human keratinocytes HaCaT cells were employed to investigate the cytocompatibility of the different coatings of the cellulosic substrates using both a direct and an indirect cytotoxicity evaluation method. The cells were expanded in 25 cm2 tissue culture flasks using DMEM supplemented with 10% FBS, 1% l-glutamine, and 1% penicillin-streptomycin until semi-confluence was reached. Briefly, 4 coated samples + 4 uncoated controls were cut into 1 cm2 square, sterilized by immersion in absolute ethanol 12 h, washed in PBS, and seeded with 2.5 × 104 cells/sample.
A direct cytotoxicity test was performed by adding 20 µL of a HaCaT suspension in a culture medium containing 1∙105 cells on the top surface of cellulosic tissues previously layered onto 24-well plates (n = 2). The samples were placed to rest in a humidified incubator (95% air/5% CO2) at 37 °C for 30 min to allow cell adhesion. Then, 1.5 mL of culture medium was added to each sample and the culture was continued for 36 h. To perform a Live/Dead assay, the cellularized samples were added with sterile PBS, containing the fluorescent dyes according to the manufacturer’s protocol, and were observed under an inverted fluorescence microscope equipped with a camera (Nikon-Ti, Tokyo, Japan).
At the endpoint, the samples were fixed in 10% formalin for 10 min, rinsed in distilled water, and dried in a vacuumed oven set at 37 °C. The specimens were mounted on aluminum stumps, sputter-coated via an argon gas carrier for 15 s (Coater SC7620, Quorum Technologies Ltd., West Sussex, UK), and observed by SEM (Phenom Pro, Thermo Scientific).
The indirect cytotoxicity assay was carried out by plating HaCaT cells into 24-well plates at 1∙105 cells/well, including wells for untreated controls, and let to adhere overnight (n = 2). Samples of cellulosic tissues with the coatings, including uncoated controls, previously sterilized as above, were placed in tubes containing culture medium (2 cm2/tube/2.5 mL culture medium) for 18 h. The subsequent day, the culture media in the cell samples were replaced with the conditioned media. To perform Live/Dead assay, 36 h after seeding, the media were removed from the wells, and the cells were added with sterile PBS containing the fluorescent dyes according to the manufacturer’s protocol and were observed under an inverted fluorescence microscope equipped with a camera (Nikon-Ti, Tokyo, Japan).
4.6. Cell Metabolic Activity Evaluation
The HaCat cells, cultured as described above and seeded in 12-well plates until 80% of confluence, were incubated for 24 h with the sterile CN/cellulose tissues. At the end of this time, resazurine was added to the concentration of 0.5 mg/mL and incubated for 4 h. At the endpoint, the AlamarBlue test was performed following the manufacturer’s protocol. Briefly, AlamarBlue incorporates a redox indicator that changes color according to cell metabolic activity. The supernatants were read with a spectrophotometer using a double wavelength reading at 570 nm and 600 nm. Finally, the reduced percentage of the dye (% AB
RED) was calculated by correlating the absorbance values and the molar extinction coefficients of the dye at the selected wavelength following the protocol provided by the manufacturer. The equation applied is shown below, in which: λ = absorbance,
s = sample, and
c = control. The results obtained are expressed as % AB
red, which is related to metabolically active cells.
4.7. Immune Response by Epidermal Cells
The immunomodulatory properties of decorated cellulose substrates were assayed using HaCaT cells. The films, sterilized overnight in EtOH and rinsed 3 times with PBS, were placed on the bottom of 6-well plates, then HaCaT cells were plated on them and incubated for 6 h and 24 h with the cellulosic tissues (
n = 3). At the endpoint, the mRNA was extracted from the cells, and the levels of expression of the proinflammatory cytokines IL-8, IL-6, IL-1 β, IL-1 α, and TNF-α anti-inflammatory cytokine, TGF-β, and antimicrobial peptide HBD-2 were evaluated by real-time PCR. Briefly, the total RNA was isolated with TRizol, and 1 µm of RNA was reverse-transcribed into complementary DNA (cDNA) using random hexamer primers at 42 °C for 45 min according to the manufacturer’s instructions. Real-time polymerase chain reaction (PCR) was carried out with the LC Fast Start DNA Master SYBR Green kit using 2 µL of cDNA, corresponding to 10 ng of total RNA in a 20 µL final volume, 3 mM MgCl
2, and 0.5 µM sense and antisense primers (
Table 2).
4.8. Statistical Analysis
Each marker expression level was compared against its untreated control using Student’s t-test. Probability (p) values < 0.05 were considered as statistically significant differences.