Human skin plays an essential role in preventing water loss in the body as it provides the outermost layer of protection from the external environment. In skincare applications, bio-polymers, synthetic polymers, and organic polymers are used to control formulation viscosities, transfer moisture to the skin, increase the stability of the formulation and active ingredient, and protect the skin by forming a coating on its surface [1
]. In particular, bio-polymers, such as cellulose, chitosan, and polysaccharide, are known to be skin-friendly substances as they are biocompatible and biodegradable. Among these, cellulose is the most abundant bio-polymer in plants and microorganisms and possesses a number of unique properties that, depending on its origin and the extraction process, allow it to be used in various applications.
Recently, cellulose nanofibers (CNFs) have attracted wide interest due to their nanoscopic size, ease of preparation, low cost, tunable surface properties, and enhanced mechanical properties, which makes them well suited for use as drug carriers, tissue regenerating scaffolds, water purifying membranes, electrodes, supercapacitors, fluorescent probes, and flexible electronics [2
]. In the field of skincare, CNFs have attracted attention as a new potential bio-material with thixotropic properties that allow it to be used for emulsion stabilization, water retention, and rheology modification applications [3
Bacterial cellulose (BC), which is referred to as bio-cellulose in the skincare field, is a bio-based polymer that is synthesized directly from microorganisms, such as Acetobacter xylinum (A. xylinum
). BC nanofibers (BCNFs) have a number of advantages over plant-derived cellulose, including a high physical strength, water absorption and retention properties, and a uniform fiber network structure [4
]. They are also available in a variety of structural forms, including spheres, gels, sheets, membranes, mats, etc., all of which can be produced by introducing simple modifications into the production strategy. However, a limitation is that it is challenging to use conventional CNF preparation methods to create BCNFs that can be dispersed in water.
CNFs obtained from plant sources can be prepared using chemical, physical, or oxidation methods [8
]. Chemical methods typically involve preparing cellulosic nanofibers via the acid digestion of an amorphous area of fibers that, when destroyed, yield nanocrystalline nanofibers. Some physical methods produce CNFs by the mechanical nanofibrillation of chemically purified cellulose pulps using a grinder, high-pressure homogenizer, blender, and high intensity ultrasonicator [8
]. Alternatively, aqueous cellulose dispersions of nano-sized structures have been prepared by aqueous counter collision (ACC) using paired water jets without any chemical modification and were then used to convert naturally occurring cellulose fibers into nanofibers [9
In contrast, a higher yield of CNFs can be obtained through the oxidation of cellulose mediated via 2,2,6,6–tetramethyl–1–piperidine–N–oxy radical (TEMPO), which is an oxidation catalyst capable of replacing alcoholic groups of cellulose with aldehyde, ketone, and carboxy groups under mild conditions at room temperature and normal pressure. TEMPO-oxidized cellulose can be dispersed in the aqueous phase by the repulsion force caused by the anionic charge on the surface of the carboxyl groups in the modified cellulose [10
]. As the resulting material has the advantage of maintaining its physical fiber structure when dispersed in water, its use has been studied in a variety of fields, such as papermaking, membrane filters, heavy metal removal, and cell transfer.
A number of researchers have studied TEMPO immobilized on solid supports, such as polystyrene-bound TEMPO (PS-TEMPO) [12
], FibreCat TEMPO [13
], TurboBeads-TEMPO (TEMPO immobilized on the outer surface of Fe3
magnetic nanoparticles) [14
], and organosilica xerogel (SiliaCat TEMPO) [15
]. While these types of heterogeneous TEMPO catalyst clouds have been applied to the conversion of molecular chemicals, their use in the form of structured polymers, such as cellulose nanofibers, has been limited due to the steric hindrance between the solid supports and the nanofibers. Recently, Patanker et al. studied nanofibrillated cellulose that had been synthesized via the magnetically separable TEMPO-mediated oxidation and mechanical disintegration of wood pulp [17
]. However, to the best of our knowledge, no researchers have yet studied the TEMPO oxidation of bacterial cellulose using immobilized TEMPO as a catalyst.
Previously, we reported the synthesis of TEMPO-oxidized bacterial cellulose nanofibers suitable for use as bio-based polymers for engineered green materials without sodium bromide, which were confirmed to exhibit unique properties on the skin surface [7
]. In this case, the TEMPO-oxidized cellulose nanofibers were obtained conventionally using a centrifugation method for washing or drying. However, while these results are promising, it is difficult to implement centrifugation at an industrial scale. In addition, the chemicals used in the drying process, which contain N–oxyl compounds, bromide or iodide, and an oxidizing agent, all remain after drying; thus, this drying method is unsuitable for use in skincare applications.
In contrast, the one-pot synthesis method proposed in this study for producing TOCNs, using TEMPO immobilized on silica beads, is simple, efficient, and easy to use, and the synthesis process can use a simple filtration instead of washing after the cellulose nanofiber dispersion has been prepared. When the TOCNs produced via the one-pot synthesis were applied to the skin surface, they were found to exhibit unique properties similar to those of conventional TOCNs. Thus, they are not only a viable alternative for industrial production for use in the real field, they are also potentially suitable for use in skincare and biomedical research applications.
2. Materials and Methods
A key material in the proposed one-pot process is SiliaCat TEMPO, which is a heterogeneous catalyst/reagent fabricated from a proprietary class of organosilica-entrapped radicals, and which is suitable for the selective oxidation of delicate substrates into higher valued carbonyl derivatives [15
]. The SiliaCat TEMPO material used in this study was purchased from SiliCycle Inc. (Quebec, QC, Canada). With regard to the other materials used in the study, sheets of BC were purchased from EZ Costec Co. Ltd. (Gyeonggi, Korea); TEMPO, sodium bromide, and sodium hypochlorite were purchased from Sigma-Aldrich (St Louis, MO, USA), and ethanol was purchased from Daejung Chemical & Metal Co. Ltd. (Gyeonggi, Korea). All other reagents were purchased from Sigma-Aldrich in the highest available commercial grade.
2.2. TEMPO Oxidation of BC
The synthesis of TOCNs via the conventional process (C-TOCNs): 20 g of a bacterial cellulose sheet that had been cut into small pieces was suspended in 500 mL of distilled (DI) water containing dissolved sodium hypochlorite and 20 mg of TEMPO catalyst. The oxidation reaction was maintained at a pH of 10 with 0.5 M NaOH. The mixture was vigorously agitated overnight using a magnetic stirrer at 25 °C. The oxidation reaction was then quenched by adding ethanol to the suspension. The products were washed with DI by centrifugation at 10,000× g several times, until all of the reactants were completely removed. The final product was stored at room temperature for later use.
The synthesis of TOCNs via the proposed one-pot process (O-TOCNs): the same amounts of bacterial cellulose and sodium hypochlorite were used for the chemical treatment of the process-cellulose. Before SiliaCat-TEMPO was used in the oxidation reaction, it was pretreated to activate the catalyst (Figure 1
) by stirring a mixture containing SiliaCat-TEMPO (1 eq.), 4 M HCl (6 eq.) in dioxane, and a 0.5 M solution of N-chlorosuccinimide (5 eq.) in dichloromethane (DCM) was stirred for 15 min. The activated SiliaCat TEMPO was then washed with ethanol and dried completely under vacuum to ensure that all of the reactant had evaporated [15
]. An oxidation reaction consisting of bacterial cellulose, sodium hypochlorite, and the activated SiliaCat-TEMPO was facilitated by stirring while the pH was maintained at 10.
A pure bacterial cellulose aqueous solution was obtained by preparing the aqueous dispersion of cellulose as follows. Ascorbic acid was added to the reacted cellulose solution to neutralize the hypochlorite acid. Then, the SiliaCat-TEMPO was removed by filtering the solution through a vacuum decompression device using a nylon mesh through which only the cellulose nanofiber solution could pass. As a result, the SiliaCat-TEMPO beads remained on the nylon mesh, while the cellulose nanofiber solution passed through to produce a pure nanofiber solution.
2.3. Detection of the Sodium Hypochlorite Content
To neutralize the hypochlorous acid remaining in the reacted cellulose solution, ascorbic acid with a different concentration was added, and the mixture was stirred. Then, a quantitative analysis was conducted using a phosphate buffer solution and N,N–diethyl–p–phenylenediamine (DPD) reagent to determine the amount of free chlorine content. In this test, if residual hypochlorous acid is present, then the color of the reactant will turn red, and its value can be obtained at a wavelength of 510 nm on a spectrophotometer.
2.4. Characterization by Scanning Electron Microscopy
Oil in water (o/w) emulsions containing TOCNs were prepared, consisting of TOCNs, 1% dimethicone, 3% pentaerythrityl tetraethylhexanoate, 1% hydrogenated polydecene, 7% dipropylene glycol, 7% glycerin, 0.4% methyl glucose sesquistearate, 3% cyclopentasiloxane, 4% xanthan gum, 12% carbomer, 1.5% sorbitol, 0.4% chelating agent, 2% trisodium ethylenediaminetetraacetic acid (EDTA), 2% pH adjusting agent, and water up to 100%. When investigating the surface characteristics, 0.05% of O-TOCNs and C-TOCNs were prepared by casting the solution on porcine skin (1 × 1 cm) and drying at room temperature overnight. These samples were then observed using a field-emission-type scanning electron microscope (FE-SEM; Hitachi S-4000, Tokyo, Japan) after platinum sputtering at 20 mA for 120 s.
2.5. Fourier Transform Infrared Spectroscopy (FT-IR) Analysis
The chemical structures of three types of BC samples (pure BC, C-TOCN, and O-TOCN) were analyzed via Fourier transform infrared spectroscopy (PerkinElmer, FT-IR microscope spotlight 200i, Courtaboeuf, France) over a frequency range of 4000 to 450 cm−1 and a resolution of 8 cm−1.
2.6. X-ray Diffractometer (XRD) Analysis
The XRD patterns of the BC samples were obtained with an X-ray diffractometer (Rigaku, MiniFlex 300/600, Tokyo, Japan) using copper Cu Kα radiation (λ = 1.5406 Å) at 40 kV and 15 mA. The samples were examined with a scanning angle of 2θ from 10° to 80° at a rate of 5°/min, the crystallinity index (CrI) was calculated as a function of the maximum intensity of the diffraction peak from the crystalline region (I200
) at a 2θ of about 22.5°, and the minimum intensity from the amorphous region (Iam
) at a 2θ angle of about 18° [18
2.7. Contact Angle Measurements
The surface wettability of the TOCNs and o/w emulsions containing various concentrations of TOCNs were evaluated by contact angle measurements using a Contact Angle System (OCA, Dataphysics, Filderstadt, Germany) and a high-speed camera. During the measurements, water droplets were deposited directly on the surface of the dried cellulose solution on the porcine skin, and the water contact angles were measured. Three measurements were performed per sample, and the results were averaged. The volume of each water droplet was 10 μL, and each drop was placed using a precision stainless steel tip (Gauge 32, EFD).