FABRICATION OF MULTIFUNCTIONAL NANO GELATIN/ZINC OXIDE COMPOSITE FIBERS

at concentrations of 0.5%, 1% and 1.5%) was doped with gelatin and the solution was electrospun under specifi c conditions to obtain multifunctional gelatin/ZnO nanofi bers. The morphology of the nanofi bers was studied by fi eld emission scanning electron microscope (FESEM), and energy-dispersive X-ray spectrometry (EDX) analysis indicated the presence of nano Zn on the surface of gelatin fi bers. On the contrary, elemental mapping analysis proved the distribution of nano material along the nano gelatin fi bers. The results show that the produced nano gelatin/ZnO composite increases the ultraviolet (UV) blocking of fabric signifi cantly. It is also observed that electrospun gelatin/ZnO nanofi bers have excellent bactericidal property against both Bacillus cereus (Gram-positive) and Escherichia coli (Gram-negative) bacteria.


Introduction
Gelatus or gelatin is a semitransparent biomaterial which is extracted from the collagen of the skin, bones, and connective tissues (one of the four basic types of animal tissues) [1][2][3]. The hydrolysis of collagen produces a water soluble protein which is breakable in dry conditions and resinous when moist [4,5]. The protein is also soluble in most polar solvents. Gelatin is frequently applied as a gelling material in the food industry, in medications, beauty products, and makeup items, because of its competencies like biocompatibility, biodegradability, and commercial accessibility. Due to the hydrolysis of collagen, gelatin acquires similar amino acids such as proline (∼25%), glycine (∼20%), clutamic acid (∼11%), arginine (∼8%), alanine (∼8%), and other essential/non-essential amino acids. It must be noted that glycine and arginine are the two forerunner amino acids for biosynthesis of creatine [6][7][8][9][10].
In the fi elds of fi ber science, textiles, and polymers, gelatin is simply formed into hydrogels, fi lms, and fi bers, although there are not too many published papers on gelatin nanofi bers and gelatin nanotextiles. Kwak et al. [2] reported that by wet spinning of gelatin fi ber and gelatin/Ag fi ber, its antibacterial property grows. The innovation in our work is using the electrospinning method for fabrication of gelatin fi ber in order to obtain nanofi bers. However, in this work we did not use Ag, so that the fi nal product does not have cellular toxicity (research works have proved the cellular toxicity of Ag particles [11]). One of the simple methods for producing nanofi bers is by electrospinning.
The electrospinning process comprises a polymer solution which is charged, and by overcoming the surface tension, a thin layer of web polymer is formed on the surface by ejection of polymer solution, which gives rise to elongation of the polymer and fi rmed ultrathin fi ber [12][13][14][15][16][17].
Electrospinning nanofi bers have many medical applications such as wound dressing [18], drug delivery, and dental restoration, as well as engineering applications such as fi ltration and tissue. The poor mechanical properties of these fi bers (like Young's modulus) can be enhanced by combining them with carbonaceous nanomaterials [19][20][21][22].
Recently, nanotechnology and nanomaterials are being used in a wide area of fabric formation. Using these nanomaterials gives novel specifi cations to the fi nal produced textile. In the past decade, scientists have focused more on using semiconductors (in the scale of nanometers) such as nano CdS, nano Fe2O3, nano TiO2, nano Ce, and nano ZnO [23 -28].
Nano ZnO is one of the semiconductors which have an energy band gap of 3.3 eV. This semiconductor has many utilizations such as ultraviolet (UV) persistence, bactericide, photocatalytic activity, and low toxicity. Nano ZnO is a nanomaterial that can be sedimented on textile surfaces using ultrasonic devices. The energy of ultrasonic irradiation can have remnant nanoparticles on the surface of fi bers with minimum aggregation [29][30][31][32].
In this study, nano gelatin/ZnO composite has been electrospun and then studied for its antibacterial and UV-blocking properties.

Materials and methods
White to peel yellow gelatin powder from Sigma Aldrich, acetic acid from Merck, and nano ZnO from Sigma Aldrich were purchased. An amount of 1 g gelatin was dissolved in a solution of 30/70 mL acetic acid/distilled water (respectively) and sonicated for 30 min. The reason for using acetic acid is previous works have concluded that using acetic acid can reduce the diameter of electrospun fi bers [33,34]. In addition, Choktaweesap et al. [35] report that acetic acid creates electrical conductivity, and thus the electrospinning is done in a better condition. During this process, nano ZnO solution was prepared in different concentration (0.5%, 1%, and 1.5%). Then, nano ZnO solution was added to gelatin and sonicated again for 60 min in order to distribute the nanomaterial and to prevent aggregation. After obtaining a homogeneous solution, it was loaded into a syringe (3 mL, with blunt needle), which was placed in the device setup. The nanofi bers of gelatin/ZnO were electrospun. The device voltage was 20 kV, pumping rate 0.3 mL/h, collector-needle distance 13 cm, and drum speed 300 rpm. The collector was coated with aluminum sheet.
The method and condition of bactericidal property was done through AATCC 100-2004 against both Bacillus cereus (Grampositive) and Escherichia coli (Lab Assistant Summer Internship Gram-negative) bacteria. The bactericidal property was calculated as follows: (1) where A is the number of bacteria recovered from the inoculated treated test specimen incubated over 24 h while B is the number of bacteria recovered from the inoculated treated test specimen immediately after inoculation.

FESEM, map images, and EDX analysis
In order to study the morphology of electrospun nanofi bers, FESEM images were obtained. FESEM was done at 15 kV and 100 kx magnifi cation. Figure 1(B) clearly demonstrates the presence of nano ZnO and its distribution. As shown, the average diameter of nano gelatin fi bers is 40 nm and the average particle size of nano ZnO is 25 nm. The fi gure also shows a good distribution of nanoparticles without any aggregation or agglomeration. This proves to be a suitable method and condition for preparing the solution and electrospinning. Figure 1(A) indicates that nanofi bers of gelatin (without nano ZnO) were electrospun correctly with a minimum diameter of approximately 11 nm for these fi bers. Figure 1(B) shows the distribution of nano ZnO particles, and as shown in red, the particle size ranges between 21 and 31 nm.
For the chemical characterization or elemental analysis of materials of the fi nished samples, energy-dispersive X-ray spectrometry (EDX) was done. The energy peaks correspond to the various elements in the sample. The main element detected through EDX on the treated sample is Zn, which is related to the use of nano ZnO.
On the basis of EDX analysis (Figure 2), it is observed that the treated sample contains a signifi cant amount of nano ZnO, which indicates the presence of Zn on the fi ber surface.
In order to demonstrate and prove the distribution of nano Zn on electrospun gelatin nanofi bers, elemental mapping analysis was done. Figure 1(C) indicates that nano Zn particles have great distribution along the fi bers. 1% nano ZnO is about 100% and 98.2% for E. coli and Bacillus cereus, respectively, and for the sample containing 0.5% nano ZnO, it is 97.4% and 96.1% respectively. Comparing the antibacterial property of samples, it is determined that the antibacterial property of samples against E.coli is more than that of Bacillus cereus, because of the difference in the thicknesses of the cell walls. Bacillus cereus has a thicker cell wall.

Conclusion
This study explains the successful fabrication of gelatin/ZnO nanofi ber through electrospinning. Four gelatin samples (0.0%, 0.5%, 1%, and 1.5% ZnO) were electrospun in specifi ed conditions. Elemental mapping analysis of samples from FESEM shows the excellent distribution of nano Zn on electrospun gelatin nanofi bers. However, while the transmission spectrophotometer outcome shows good UV blocking of the nano gelatin/ZnO composite, the blank sample does not

UV blocking analysis
The UV transmission diagram of electrospun gelatin nanofi ber containing different amounts of nano ZnO is illustrated in Figure 3. The irradiation wavelength was 200-400 nm. As it indicates, the electrospun gelatin nanofi ber without any nano ZnO (blank sample) has a higher transmittance compared with those containing nano ZnO. That is, UV protection of the blank sample is low. Moreover, the UV-blocking activity of the electrospun fi ber is improved in the presence of nano ZnO, which is owing to the synergetic UV absorption of nano ZnO. Also, the spectrum shows that the sample containing 1.5% nano ZnO has greater protection against UV radiation, so by increasing nano ZnO in gelatin nanofi ber, the UV blocking of samples is enhanced.

Bactericidal properties detection
Bacillus cereus and E.coli are two common Gram-positive and Gram-negative bacteria. Bacillus cereus is a rod-shaped bacteria which is found in abundance in food and soil and can cause nausea, diarrhea, and vomiting syndrome [36]. While the Gram-negative bacteria E.coli can cause gastroenteritis, urinary tract infections, neonatal meningitis, hemorrhagic colitis, and Crohn's diseases [37]. Nanoparticles of ZnO have unparalleled specifi cations such as photocatalytic property and bacteriostatic effects [38]. Furthermore, nano ZnO is biocompatible for pharmaceutical textile usages [39]. Nano ZnO can prevent the increase of bacterial cells by penetrating the hydrogen peroxide into the cell membranes and causeing destruction of the bacteria. In addition, the positive ions of zinc(Zn 2+ ) can react with the negative particles of bacteria cell and thus lead to the death of bacteria. In this study, these two kinds of bacteria have been used to investigate the antibacterial effi ciency of electrospun nano gelatin/ZnO. The results of the antibacterial experiment are presented in Figure 4. As it is demonstrated, there is no antibacterial property on the blank sample against both Gram negative and positive bacteria, but the samples that contain nano ZnO have antibacterial property. As shown in Figure 4, the sample containing 1.5% nano ZnO has 100% antibacterial property against both the bacteria. As it is shown, the antibacterial activity of the sample treated with    have suitable UV blocking, but by doping nano ZnO, its UV blocking property enhances tremendously. This is because of the UV-blocking property of nano ZnO and its synergetic UV adsorption. Also, the bactericidal property of samples was investigated with both Gram negative and Gram positive bacteria, E.coli and Bacillus cereus. The results indicate that nanofibers of gelatin/ZnO have more than 96% antibacterial property against both bacteria.