Fabrication and characterization of electrospun cellulose acetate nanofibers derived from rice husk for potential wound healing application

As one of the world’s most abundant biomass, lignocellulosic materials such as rice husk (RH) has been recognized for its various potential usages. Electrospun nanofibrous mats have been fabricated from various natural and synthetic polymers and offers a wide range of promising criteria suitable for wound dressing applications. Attuned to the environmental concerns in current years have led to finding new resources to replace the synthetic polymers. Natural polymers have grabbed considerable attention due to their desirable properties. Therefore, the application of cellulose-derived materials from agricultural waste becomes crucial as a green alternative to produce electrospun wound dressing with excellent wettability, porosity and tunability to promote wound healing at relatively low costs. Interestingly, a specific study on the utilization of cellulose extracted from RH to produce electrospun nanofibrous mat remains unreported. Therefore, this work aimed to investigate the feasibility of using RH as a source of raw materials to create nanofibrous mats for use as prospective wound dressing materials. As researchers currently focus on environmental sustainability, this study focuses on the physicochemical study of the RH derived nanofibers as a biocompatibility drug carrier that are derived from biomass waste. In this paper, cellulose extracted from RH was converted into its derivate, cellulose acetate and electrospun. The nanofibers were then characterized by scanning electron microscope, attenuated total reflectance- fourier transform spectroscopy, water contact angle to evaluate the main properties, and compatibility of the electrospun nano-fibrous mat for wound dressing applications.


Introduction
Wound care is a trendy field of study in biomedicine. In today's wound care, synthetic wound dressings that contain a variety of therapeutic ingredients to promote wound healing, such as antibacterial agents and growth factors, have grown in popularity (Aras et al. 2021). Although there have been significant breakthroughs in wound dressing study, there is still considerable work to be done in finding an ideal wound dressing especially for chronic wounds as it requires better care and more effective wound coverings. According to recent studies, electrospun nanofibers are the ideal material to produce wound dressings. They have ultra-fine fiber diameters, large specific surface areas, high porosity, small hole sizes, and capacity to mimic the extracellular matrix's structure (Bhardwaj et al. 2010). Nanofibrous mats, in comparison to traditional wound dressings, have several crucial features, making them potentially effective for wound healing and drug administration. The desired bioactive compound, natural substances, or other therapeutic substances can be adsorbed on the nanofibers surfaces or contained within the nanofiber's matrix (Sun et al. 2014). A suitable wound dressing should maintain the wound's moisture level, guard against secondary infections, remove wound exudate, and generally encourage tissue regeneration. However, current wound dressing on the market still could not satisfy all therapeutic demands. Thus, to address the problem, more study of new materials that can accelerate wound healing in diabetics especially, is necessary. Selection of suitable dressing materials is crucial, given the various mechanisms involved in wound healing process and the interplay of various external factors (Suarato et al. 2018). Natural biopolymers on the other hand have been widely used as wound dressing due to its characteristics such as versatility, biocompatibility, biodegradability and angiogenic impact (Osmani et al. 2021). Cellulose acetate (CA), a cellulose derivative, is one of the most frequently tested natural biopolymers in nanofibers. CA was chosen for the present study because of its biocompatibility, good water absorption capability, and compatibility with fibroblast cells (Liu et al. 2007). Applications for wound dressing, tissue engineering, and drug delivery systems are among the biomedical uses for CA (Li et al. 2012).
Agricultural waste has been examined as an alternative source of cellulose (Maryana et al. 2021). The utilization of agro waste plays an important role in solving environmental and economic issues (Gao et al. 2018). Rice husk (RH) as an example is a common agricultural by-product of rice milling, accounting 20% of the unhulled paddy in many rice-producing countries (Hossain et al. 2018). According to the Food and Agricultural Organization (FAO), global annual rice production was around 741 million tonnes in 2013, with 148 million tonnes of rice husk (RH) produced as a by-product of rice milling around the world (Chandrasekar et al. 2003). Several studies have been conducted throughout the years to address the conversion RH biomass into high value-added biomaterials. However, the utilization of cellulose derived from RH as wound dressing material remains scarcely available.
RH is made up of lignocellulosic fibers, which contains cellulose (35%), hemicellulose (25%) and lignin (20%) (Yang et al. 2019). Due to its abundance, there is an extensive interest in the extraction and the production of cellulose derivatives (i.e. CA) from RH (Das et al. 2014). Furthermore, studies have shown that CA produced from RH appears to have essential therapeutic properties such as antioxidants, making the consideration of RH as a possible candidate for wound dressing material scientifically and commercially relevant (Gao et al. 2018). The present work aimed at synthesising and investigating the characteristics and the applications of cellulose acetate extracted from RH. The fabricated CA nanofibers was further characterized to understand its physicochemical properties.

Materials
RH was bought from Kubang Rotan Rice Factory Sdn. Bhd in Kedah, Malaysia. Acetic acid, Sulphuric acid (H 2 SO 4 ), sodium hypochlorite (NaClO), acetone, and N, N-dimethylacetamide (DMAC) were purchased from Sigma-Aldrich and used as received without further purification.

Acetylation of cellulose
Cellulose was extracted from RH using chemical pretreatments with some modifications from previous study (Johar et al. 2012). The cellulose fiber was acetylated to form cellulose acetate (CA) following methods from previous studies (Das et al. 2014, Achtel andHeinze 2016). 2.5 g of the extracted cellulose microfibrils was dissolved in 10 mL of glacial acetic acid and heated at 47.5 °C for an hour (Bello et al. 2016). Then, 5 mL of acetic anhydride was added with 5.5 wt% H 2 SO 4 as a catalyst for the reaction. The mixture was heated for an hour at the same temperature. Then, the precipitate was filtered and washed thoroughly with distilled water and dried in an oven overnight (Bello et al. 2016;Jassem et al. 2020).

Attenuated total reflectance-fourier transform infrared (FTIR) spectroscopy
The spectra for samples were analyzed to determine the changes in the functional groups after successive chemical treatments. The spectra were collected using a one-bounce ATR mode of the FTIR Spectrometer (Perkin Elmer, USA). The samples were analyzed with the resolution of 4 cm −1 within the range of 400-4000 cm −1 .

Scanning electron microscopy (SEM)
Microscopic examinations of the morphology of electrospun nanofiber samples were performed by SEM (Hitachi TM3030) at the accelerating voltage of 15 kV. Prior to analysis, the surface of the sample was coated with a thin gold layer and observed at magnifications of 1000×.
Fabrication of the extracted CA by electrospinning process

Preparation of CA solution for electrospinning
For the RH-derived CA solution, CA at concentrations of 17%, 18%, 19% and 20% (w/v) were dissolved and continuously stirred by a magnetic stirrer for 6 h at room temperature in acetone: N, N-dimethylacetamide (DMAC) (2:1) solution for 5 h (Liu et al. 2007;Huan et al. 2015). The solution was stirred vigorously until a homogenous solution formed (Huan et al. 2015).

Electrospinning of the CA nanofibers
Electrospinning of the CA solutions was performed using 5 mL plastic syringes (Becton Dickinson, UK), equipped with 18G gauge needles (ID: 0.84 mm). The syringe was placed in the syringe pump and operated at a flow rate of 1 mL/min. After that, the solution was electrospun at 11 kV at 15 cm onto a static collector wrapped with aluminum (Table 1).

Morphological characterization
Microscopic examinations of the nanofibrous mats were observed using a Scanning Electron Microscope (Hitachi TM3030) at the accelerating voltage of 15 kV. Prior to analysis, the surface of the sample was coated with gold and observed at magnifications of 1000× . Water contact-angle measurement Using a deionized water contact angle measurement equipment, the sessile drop method was used to determine the nanofibrous mat's wettability. (VCA, AST Product Inc, USA) (Unnithan et al. 2014). The contact angle measurements were performed by dropping 2.0 µL of deionized water onto the nanofibrous mat (Unnithan et al. 2014).

Porosity
Equation 3 was used to determine the porosity of the fabricated mat (Razak et al. 2016). By submerging the nanofibrous mat in cyclohexane, which has a density of 0.778 g/cm 3 , the pore volume, or Vp, was determined. Meanwhile, the volume of the nanofibrous mat, or Vd, was measured by measuring the volume of the cyclohexane that was absorbed (Razak et al. 2016).
Equation 1 shows the porosity measurement:

Swelling behavior study
Prior to submerging the dried nanofibrous mat (W0) in a wound-like solution, the swelling of the material was measured, in accordance with YYT0471.1-2004, contact wound dressing test technique (Cheng et al. 2021). The solution was made by mixing 8.298 g of NaCl, 0.368 g of CaCl 2 .2H 2 O in 1 L of distilled water (Xie et al. 2018). The nanofibrous mat was cut into a size of 1 cm × 1 cm and the initial weight was recorded. The samples then were immersed into the wound solution and incubated at the room temperature for 14 days (Franco et al. 2012). The fiber was dried using filter paper and weighed daily until it reached constant weight (Ravikumar et al. 2017). The swelling of the nanofibrous mat was calculated by using Eq. 2 and referred as swelling rate (%) (Yousefi et al. 2017). (1)

Biodegradation study
After being submerged in a wound exudate-like solution for 14 days, the weight loss of the nanofibers was measured to analyse the biodegradation activity of the CA nanofibrous mats. (Adeli et al. 2019). The fibre was dried in a vacuum oven at 50 °C for 14 days and weighed until a constant weight was achieved. The biodegradation rate was calculated by using Eq. 3 (Adeli et al. 2019) as follows: W0 and W1 are the initial weight and after immersion in wound exudate like solution, respectively.

Tensile properties
The tensile properties (tensile strength, Young's modulus, and elongation at break) of the prepared samples were measured according to ASTM D3822 using a universal testing machine (Zwick/Roell Z020, Zwick, Germany). For this test, five electrospun nanofibers samples with dimensions of 2 cm × 10 cm (length) of the optimized nanofibers were prepared and tested in dry and wet conditions. The tensile testing machine (Zwick/Roell Z020, Zwick, Germany) mounts a load of 500 N, with gauge length of 3 cm, and a crosshead speed of 1 mm/ min. Each sample's diameter was measured using a micrometer and was repeated three times. Figure 1a-e show images of raw RH, alkali-treated RH, bleached RH, acetylated cellulose, and CA dissolved in electrospinning solutions. The results observed that the alkali treatment caused colour changes in raw RH in Fig. 1a from brownish to brownish yellow in Fig. 1b which could be associated with the partial removal of the lignin fraction

Acetylation of cellulose
of the RH (Luduena et al. 2011). Following the bleaching treatment, the colour change observed in Fig. 1d was more obvious. The CA was then dissolved in the electrospinning solutions of acetone: N, N-dimethylacetamide (DMAC) (2:1) solution for 5 h (Liu et al. 2007;Huan et al. 2015). The solution was stirred vigorously until a homogenous solution formed as shown in Fig. 1e (Huan et al. 2015).

FTIR spectrum
To confirm the acetylation of cellulose from RH, the FTIR spectrum of CA derived from RH was compared with the commercial CA (Sigma Aldrich, US) and the results are shown in Fig. 2. Absorption bands of CA derived from RH displayed typical absorption bands of the commercial CA. Absorption band at wavenumber 3323 cm −1 was attributed to OH vibration (Zhang et al. 2016). Absorption band around 1600 cm −1 represented a strong bond between cellulose and water (Mendoza et al. 2016). Moreover, the absorption bands observed  (Harvey et al. 2012). These findings confirmed the acetylation process of cellulose derived from RH was successfully performed.

Swelling and degradation test
One of the most significant qualities of a wound dressing is to evaluate its swelling capacity, or how much fluid that can be absorbed from the wound site. Exudates from the wound site should be able to be absorbed by a suitable wound dressing. A good wound dressing should be able to absorb fluid at a rate of 100-900%. Additionally, correct hydrophilicity and absorption permeates the intake of nutrients, cells and bioactive compounds (Morgado et al. 2015). The swelling and degradation properties of the electrospun nanofibers (CA17, CA18, CA19, and CA20) were subsequently analyzed by immersing at 37 °C in wound-like solution (according to YYT0471.1-2004, contact wound dressing test method) (Cheng et al. 2021), mimicking the physiological conditions. Swelling of the dry nanofibrous mat occurred rapidly, with the maximum swelling ratio of 446.45% achieved by CA17 nanofibers. High water uptake property of all samples is related to the water penetration rate into the porous structure of the nanofibers. The number of water molecules bonded to the fiber surface, between polymer chains, and

Weight loss (%)
contained inside a porous structure are all factors that can affect water absorption (Lalani et al. 2012). Figure 3b shows the percentage weight loss of the CA17, CA18, CA19, and CA20 samples immersed in a wound-like solutions (pH = 7.4, 37 °C) for 14 days. Of all samples, CA20 had the highest percentage of weight loss over 15-days, which was 24.19%. As compared to CA17, the percentage of weight loss in CA18, CA19 and CA20 was also lower by differences of 1.09%, 0.13%, and 1.29%, respectively. According to a study, the high percentage of degradation rate is related to increased water accumulation, indicating that the fiber is swollen and surrounded by the solvent (Wutticharoenmongkol et al. 2019) and consequently causing the fiber to degrade.
Wettability study of the nanofibers Nanofiber's wettability characteristics are crucial for drug release and cellular growth (Chen et al. 2020). The wettability of the CA nanofibers was assessed using the water contact angle, and the results are displayed in Fig. 4. At 0 s, the contact angles of the CA17, CA18, CA19 and CA20 nanofibers mat were 122.80°, 121.40°, 118.90° and 108.60°, respectively. The water contact angle results proved the hydrophobic nature (> 90) of the nanofibers (Prietto et al. 2017). Nanofibers' physicomechanical properties, such as the diameter of the fiber, pore diameter, and stiffness, have impacts on wound healing process. Recent research has found that fiber diameter (200-400 nm) and scaffold pore diameter (6-20 μm), which are similar to the native ECM, improve human skin fibroblast adhesion, proliferation and infiltration while reducing bacterial infiltration (Hodgkinson et al. 2014).

Morphological studies
The average fiber diameter for CA17, CA18, CA19, and CA20, respectively, is 300.94 57.19 nm, 318.56 80.96 nm, 335 58.24, and 352.25 50.86 nm as shown in Table 2, Each electrospun nanofiber mat represents a smooth morphological surface with bead-free forms. The processing parameters (flow rate, voltage, distance between the syringe and collector) influenced the morphology of the CA nanofibers, while the concentration of CA had a greater impact on the fiber diameter. By increasing the polymer concentration, the uniformity decreased, but not necessarily result in statistical difference (

Fiber distribution
The average diameters of the nanofibers produced ranged from 149.6 to 473.6 nm. Large fiber diameter variation is a common problem in electrospinning, especially when utilizing volatile solvents, although few studies have provided a workable solution (Huang et al. 2003;Angel et al. 2020). The diameters of the nanofibers were found to increase with the increase in solution concentration as shown in Fig. 6. The average electrospun nanofibers diameters were of 300.94 ± 57.19 nm, 318.56 ± 80.96 nm, 335 ± 58.24, and nm for CA17, CA18, CA19 and CA20, respectively. The findings are in agreement with the study conducted by Chumpol and Siri (2016), in which the electrospun CA nanofibers produced were in the diameter range of 367.6−458.0 nm. Previous research has revealed that fiber diameter increases with increase polymer concentration (Ryu et al. 2003;Kong and Ziegler 2014). Consequently, larger solid content and viscosity would hinder flow and elongation, resulting in less stretched jet, explaining why fiber diameter and diameter uniformity increase with increasing solution concentration (Tarus et al. 2016).

Porosity of the nanofibers
For improved wound respiration and oxygen gas permeability, the ideal wound dressing should have high porosity. One of the major benefits of the electrospun nanofibrous mats is their highly interconnected porosity, which closely resembles the natural ECM's structural design (Adeli et al. 2019). The average porosity of the nanofibers is shown in Table 2. The porosity of the nanofibers decreased as the concentration (w/v) of the CA solution increased. CA17, CA18, CA19 and CA20 nanofibers had nanofibers porosity of 80.25, 80.67, 79.67 and 76.09, respectively. Nonwoven fibers were more generated and denser packed as the amount of CA solution increased, resulting in smaller pore size and lower porosity (Naseri et al. 2016). There was no significant difference between the porosity of the electrospun nanofibers, however, by increasing the concentration of CA, the porosity of and d CA20 collected on the aluminum foil at 500 × magnifications the fiber slightly decreased. This could be attributed to the increase in the average fiber diameter of the CA nanofibers due to higher CA % (Fig. 7).

Tensile properties
Tensile testing is required to assess the nanofiber's strength as the wound dressing will be placed directly to the skin. This is critical since the material must be both robust and flexible in order to tolerate body movement (Boateng and Catanzano 2015). The tensile test was performed to measure the mechanical properties of the CA nanofibers in dry and wet conditions, mimicking the physiological condition of a chronic wound. The nanofibrous mats should be able to give acceptable mechanical qualities as well as flexibility in both dry and wet conditions, as they are designed for the application of wound dressings (Adeli et al. 2019). Five trials were made for each sample, and stress-strain curves were extracted from the data as shown in Fig. 7. The tensile strength of the CA19 nanofiber in dry condition was higher (3.30 ± 1.45 MPa), compared to 2.98 ± 0.78 MPa in wet condition. The fibers lose rigidity and become more malleable as the moisture level rises (Zukowski et al. 2020). Their strength decreased as compared to dry fibers, and might attain larger ultimate strains, but their elasticity modulus fell. However, the tensile strength was within the acceptable range for wound dressing application. It is reported that the tensile strength ranging from 0.7 to 18.0 MPa is 6 ( Table 3). To conclude, the mechanical properties of the electrospun CA19 nanofibers were in the acceptable range as wound dressing. It was reported in the literature that the elongation of the human skin varies in the range of 1-32 MPa for tensile strength, and the range for Young's modulus is from 0.008 Mpa (Mattei et al. 2008) up to 70 Mpa (Shevchenko et al. 2010), suggesting the potential application of the CA nanofibers as wound dressing.

Conclusions
In conclusion, the optimization of the CA nanofibers to be fabricated with the active compound was investigated in this study. The morphology, the wettability, the porosity, the swelling, and the degradation as well as the tensile strength were investigated. The mean fiber diameter in this study ranged from 318.55 to 352.25 nm. The diameters of the CA nanofibers increased with increasing CA concentration. This study found that the best quality fibers, i.e., continuous fibers with little to no beading, were produced by the solutions with 17-19% (w/v) CA concentration. The results obtained herein suggest that the electrospun nanofibers can be utilized as wound dressing since they possess the advantages of being porous (76.09-80.67%), good water absorption (maximum swelling ratio of 446.45%) as well as mechanical stability with tensile strength of 3.30 ± 1.45 MPa, based on the physicochemical tests conducted. However, to assess the biocompatibility of the nanofibers produced, bioassay tests should be done to understand the biological properties of the fabricated mat for wound healing application.