Water Resistance and Morphology of Electrospun Gelatine Blended with Citric Acid and Coconut Oil Vodoodpornost in morfologija elektropredene želatine , mešane s citronsko kislino in kokosovim oljem

Bio-polymer gelatine can be found in a broad variety of applications, mostly in the food industry. Moreover, it is used in the encapsulation of active pharmaceutical ingredients. In electrospinning, it is used for drug release, but can also strongly infl uence the morphologies of nanofi ber mats when blended with other polymers. In a recent project, we studied the infl uence of adding citric acid and coconut oil to gelatine electrospinning solutions. While the former can be used to modify gelatine nanofi ber diameters and create diverse morphologies between the fi bres and sprayed droplets of diff erent shapes, the latter results in an electrospraying process and additionally increases water resistance, suggesting a possible use of the combination for tailored drug release.


Introduction
Gelatine belongs to bio-polymers which are oft en electrospun to create nanofi ber mats for diverse purposes.Gelatine nanofi bers can be used to mimic an extra-cellular matrix [1,2] and are therefore expected to provide good growth and proliferation conditions for cells.Gelatine is known to bind cell surfaces via fi bronectin binding better than native collagen [3].However, gelatine has a disadvantage since it is water-soluble, which makes crosslinking aft er the electrospinning necessary, e.g. with a heat-treatment in the presence of diff erent other chemicals [4,5], addition of diverse acids [6] or toxic materials, such as glutaraldehyde [8].Another possibility is using gelatine blended with other polymers.Blending silk fi broin with gelatine, for example, resulted in an increased nanofi ber diameter and hydrophobicity, while the mechanical properties decreased [8].Testing diverse PAN/bio-polymer blends, gelatine was found to be the only bio-polymer which signifi cantly increased the PAN fi bre diameters, an eff ect which survived wetting the samples although no cross-linking step

Izvleček
Biopolimerno želatino uporabljajo na številnih področjih, še posebno v živilski industriji, pa tudi pri kapsuliranju aktivnih farmakoloških sestavin.Pri elektropredenju jo uporabljajo za sproščanje zdravil.Ko jo mešajo z drugimi polimeri, pa lahko pomembno vpliva na morfologijo nanovlaknatih kopren.V nedavnem projektu sta bila preučevana vpliva dodatka citronske kisline in kokosovega olja k raztopljeni želatini za potrebe elektropredenja.Medtem ko se citronska kislina lahko uporabi za spreminjanje premera nanovlaken iz želatine in ustvarjanje različne morfologije med vlakni in razpršenimi kapljicami različnih oblik, pa povzroči kokosovo olje elektrorazprševanje in dodatno poveča vodoodpornost, kar kaže na možno uporabo kombinacije za načrtovano sproščanje zdravil.Ključne besede: nanovlaknata koprena, vodoodpornost, elektrorazprševanje was included [9].Similarly, large fi bre diameters could not be gained by modifying spinning and solution parameters of pure PAN [10].Electrospinning PVA/gelatine blends was used to create scaffolds with modifi ed hydrophobicity and morphology [11].Similar results were found in ethyl cellulose/ gelatine blends, which could be tailored between hydrophobic and hydrophilic properties, showing tuneable water stability [12].Zein/gelatine blends were found to show high crystallinity, resulting in the preservation of a porous 3D structure gained by electrospinning, which was not possible in pure gelatine or pure zein nanofi bers [13].Blended with cellulose acetate, gelatine loaded with gabapentin was found to signifi cantly increase injury regeneration in rats [14].Similarly, gelatine-coated poly(butylene succinate) nanofi ber mats could be used to immobilize thrombin, a haemostat, resulting in shorter haemostasis times and less blood loss than commercial gelatine sponges when tested in a rat liver model [15].In the research, apart from some pre-tests with respect to pure gelatine, we examined the infl uence of citric acid on water resistance and morphology of pure gelatine fi bres.Th is acid was shown to increase water stability of collagen/PEO nanofi bers [16], of gelatine scaff olds which were not produced by electrospinning [17], and of electrospun native collagen fi bres [18].It must be mentioned that according to the literature, the bio-polymer which should be crosslinked and the citric acid need to be incubated for several hours and should aft erwards be electrospun from the solution containing a high ratio of ethanol or similar solvents [19].Th e latter was not possible in the electrospinning machine used in this project.Th us, our experiments aim at investigating whether the electrospinning process itself can replace the incubation process due to the large forces and high dynamics working during nanofi ber formation.Electrospinning both materials together without former incubation has, to the best of our knowledge, not been reported before in the scientifi c literature and therefore represents a new approach to create water resistant gelatine nanofi ber mats.If working, it would allow creating waterproof gelatine nanofi ber mats without the necessity of using toxic cross-linkers aft erwards, which could lead to the use of gelatine nanofi ber mats for medical or biotechnological applications.Additionally, the infl uence of coconut oil -which is known to work as a plasticizer [20] -was tested.
Coconut oil also shows good antibacterial, anti-infl ammatory and anti-viral properties [21,22].Electrospinning blends of gelatine and coconut oil were not found in the literature either, and might not only increase the possible application of such electrospun nanofi ber due to combining the intrinsic medical properties of both materials, but may also stabilize gelatine to increase its water resistance.

Materials and methods
For electrospinning, a needleless electrospinning machine "Nanospider Lab" from Elmarco (Czech Republic) was used.Th e spinning parameters were as follows: temperature in the spinning chamber (21 ± 1) °C, relative humidity 33%, carriage speed 75-150 mm/s, substrate speed 0 mm/min, electrode distance 175-220 mm, electrode-substrate distance 50 mm, high voltage 70-75 kV between the lower electrode and the grounded upper electrode, and nozzle diameter 0.6-0.9mm, depending on the viscosity of the spinning solutions.A polypropylene (PP) substrate (from Elmarco) was used as the substrate during electrospinning.A sketch of the electrospinning machine is shown in Figure 1.

Results and discussion
Th e fi rst experiments aimed at investigating the infl uence of boiling the gelatine solution on the nanofi ber mat morphology, as compared to its working at room temperature.Figure 2 depicts nanofi ber mats, prepared from the solutions G1 and G1b, spun at the voltage of 70 kV.Th e images show that, on the one hand, the fi bres became thinner aft er boiling the solution, and on the other hand, spherical agglomerates were formed.Th inner fi bres can be explained by the reduction of gel-building abilities of gelatine when it is boiled.Both changes in the nanofi ber mat morphology are not desired; the relatively thick gelatine nanofi bers are oft en preferable for the application in cell growth, compared to the thinner ones.Consequently, in the next test, a slightly higher concentration of gelatine was used to prepare a solution which had to be boiled again before the spinning to slightly reduce the viscosity and thus support the spinning process.Higher concentrations oft en result in thicker fi bres, as investigations of other polymers have shown [10,23].Since former experiments in our group revealed a similar trend to signifi cantly higher spinnability for slightly lower gelatine concentrations, the experiments with gelatine/citric acid blends were performed with reduced gelatine concentrations and without boiling.Th e results are depicted in Figure 4.While sample GCA1, containing the highest gelatine and the lowest citric acid concentration, resulted in a mat of nanofi bers with reduced diameters, increasing the citric acid concentration and at the same time decreasing the amount of gelatine resulted in electrospraying instead of electrospinning, with strongly changed droplet morphologies between spiky arrangements of very short nanofi bers (GCA2) and smooth drops in a broad variety of diameters (GCA4).Th is fi nding can be explained by the well-known suppression of the gelling properties of gelatine in the presence of acids.Th e most interesting structure, however, is visible in sample GCA3.Here, a relatively thick coating on substrate fi bres can be recognized, on top of which a labyrinth-like structure is revealed, looking like a strongly cross-linked net of thick gelatine fi bres.It should be mentioned that the width of these features is approx.1.5-2 μm, i.e. thicker than the nanofi bers gained for sample GCA1 with the diameters in the range of 600-900 nm.
Unfortunately, the tests of water resistance of these nanofi ber mats showed that all electrosprayed or electrospun structures were completely washed off of the substrate.Th e same occurred at all tests to increase water resistance by spraying citric acid onto pure gelatine fi bre mats or vapour coating the electrospun gelatine mats with citric acid.It does not seem to be possible to increase water resistance of gelatine nanofi ber mats in this way.Apparently, the incubation step mentioned in the literature cannot be replaced by the electrospinning process itself.A similar structure, however, can be created by electrospraying poloxamer/dextran blends [24].Opposite to the tests with gelatine/citric acid, which aimed at increasing water resistance compared to pure gelatine, adding coconut oil could indeed reduce the water solubility of the gelatine spray-coating.As depicted in Figure 5 (right panel), the surface morphology clearly changed aft er wetting the sample with a drop of water which dried on the fabric at room temperature, not washing off the coating.Th is fi nding is consistent with former experiments on casein, for which water stability could be signifi cantly increased by adding wax or paraffi n oil to the spinning solution [25].We assume that this reduction of water solubility can be attributed to closing small pores in the coating, thus reducing the contact area between gelatine and water [26].Th is suggests further experiments with diff erent gelatine : coconut oil ratios to create nanofi bers which may also show increased water resistance, making them suitable for a slow release of medical drugs etc.

Conclusion and outlook
To conclude, this study shows that citric acid can be used to modify the nanofi ber mat morphology and create not only thinner fi bres than with pure gelatine, but also diff erent sprayed structures, including a labyrinth-like surface.Coconut oil, on the other hand, has indeed increased the water resistance of the resulting electrosprayed coatings.Future research will concentrate on combining both features, i.e. creation of nanofi ber mats or labyrinthlike coatings with increased water resistance, possibly also using the combination of coconut oil and citric acid to enable the use of such materials for drug delivery and similar applications where a large surface : volume ratio of nanofi bers should be combined with slow dissolving in aqueous environments.

Figure 4 :Figure 5 :Figure 5
Figure 4: Nanofi ber mats and electrosprayed samples GS1-GS4, prepared combining gelatine and citric acid, using carriage speed of 75 mm/s, electrode distance of 200 mm and high voltage of 75 kV