Titanium Dioxide/Chromium Oxide/Graphene Oxide Doped into Cellulose Acetate for Medical Applications

Wound dressings have been designed based on cellulose acetate encapsulated with different concentrations of chromium oxide (Cr2O3) and titanium oxide (TiO2) with/without graphene oxide (GO). This study comprises the structural, morphological, optical, thermal, and biological behavior of chromium oxide/titanium dioxide/graphene oxide-integrated cellulose acetate (CA) films. The CA-based film bond formation was introduced by functional group analysis via Fourier transform infrared (FTIR) spectroscopy. The fabricated Cr2O3/TiO2/GO@CA film SEM micrographs demonstrate transition metal oxides Cr2O3 and TiO2 on a nano-scale. The TiO2@CA shows the lowest contact angle with 30°. Optically, the refractive index increases from 1.76 for CA to 2.14 for the TiO2@CA film. Moreover, normal lung cells (A138) growth examination in a function of Cr2O3/TiO2/GO@CA film concentration is conducted, introducing 93.46% with the usage of 4.9 µg/mL. The resulting data showed a promising wound-healing behavior of the CA-based films.


Introduction
Wound healing is a complex process. It occurs in injured skin with progression through various stages, for instance, hemostasis, inflammation, proliferation, and remodeling, according to the intensity and impact of the injury [1]. In recent times, there has been rapid growth in tissue engineering research for medical purposes which is supported by existing traditional transplant techniques. The main goal of tissue engineering is to discover an appropriate strategy for bio-scaffold formation and optimization to perform as a natural extracellular matrix [2]. The anti-inflammatory features of the designed biomaterial are crucial, for instance, biocompatibility and biodegradability. Additionally, a tailored surface for physical and chemical adhesion is considered an important factor [3][4][5][6]. As well, several polymers were tested as basic constituents for bio-scaffold applications due to their biocompatibility.
Among them, cellulose (C 6 H 10 O 5 ) n is considered an accessible polymer that occurs naturally. The three active OH function groups per molecule introduce appropriate physicochemical characteristics to be connected with other substances such as graphene oxide, which can facilitate their reinforcement behavior [7]. These properties might suggest the combination of cellulose as a simple strategy to improve its mechanical properties to be appropriate for medical applications. Biodegradable biopolymers are an eco-friendly alternative; in addition, they originate from renewable and biodegradable sources. Additionally,

Preparation of Scaffold with Different Contents of Oxides
GO is prepared in the laboratory by a modified Hummers' method [18,19]. Moreover, the CA films were formed with the casting methodology. To obtain the fibrogenic solution, CA was dissolved in acetone at the ratio of 1:10 (m/v) = 10.0 wt.%, with the powder ingredients tabulated in Table 1, resting for 24 h in a sealed glass at 25 • C. Each sample containing 2 g of CA was dissolved in 20 mL of acetone (10.0 wt.%), then 0.25 g of the powdered oxides were added through this quantity of dissolved CA solution. Thus, the ratio of the additional oxides into the CA is around 12.5% and it was maintained for all samples, except the first one which represents the pure CA as a control one. Then, the solution was subjected to magnetic stirring for 2 h. The solution was poured onto a glass plate, left to rest until complete solvent evaporation. The ratio of the additional oxides was marinated at 0.25 g for all samples, except the first one which is pure CA. This ratio was selected after experimental trials. In detail, increasing the additional oxide powder in the polymeric solution might reduce the stability of the film and may deteriorate the coherence of the scaffold. However, the low contribution of the additional powder to the polymeric solution might not be effective, or, in some cases, it does not appear in the characterizations. Therefore, the balance between the appropriate stability of the obtained scaffold and the appearance of the additives through characterization is highly required. Thus, several trials can be carried out to select an appropriate ratio to be used for a number of compositions to be examined for medical applications. X-ray diffraction (XRD) analyses were carried out via the X-ray diffractometer model (Panalytical-X'pert pro carried out using Cu k α1 radiation, λ = 1.5404 Å, 45 kV, 40 mA, Malvern, WR14 1XZ, Worcestershire, UK). It was used mainly to investigate the phase composition of the as-synthesized film composite. All XRD curves were scanned in 5 • ≤ 2θ ≤ 70 • scope with a step size of 0.02 • and a step time of 0.5 s.

FTIR Measurements
Fourier-transformed infrared (FTIR) spectra were scanned via an FTIR spectrometer (Perkin-Elmer 2000 Akron, OH, USA) in the range of 4000-400 cm −1 . The samples were prepared for the FTIR test by combining the films with KBr and a ratio of 20:1. The samples then were fixed on the holder. Finally, the sample was placed in the front of the beam and scanned within the range of 400 to 4000 cm −1 .

Examination of Films Morphology
FESEM (QUANTA-FEG250, Kolkata, WB, India) was used to investigate the morphology of MG-63 cells on the films-based NC. For this issue, the nano-film was sanitized using a UV lamp for 30 min of exposure. Each sample was cropped into two pieces of 0.5 × 0.5 cm 2 , then they were added to 12-well plates. An amount of 1.5 mL of MG-63 cells was added to each well. The plate was then incubated at 37 • C for 3 days. After this time, the films were washed with phosphate-buffered saline (PBS) (Merck, Beijing, China). To keep the cells fixed on the film surface, the scaffolds were submerged in a glutaraldehyde

Thermogravimetric Analysis
TGA was carried out from room temperature up to 600 • C in a thermal analyzer (DTG-60H SHIMADZU, Kyoto, Japan) using the airflow rate of 100 mL/min. The heating rate was 10 • C/min.

UV Measurements
Double beam UV-visible (UV-Vis) (Shanghai Metash instruments Co., Shanghai, China) spectroscopy was used to investigate the optical properties of polymeric samples. Each sample was cropped with a dimension of 2 cm × 2 cm. The thickness of these films was maintained at 0.185 mm.

Contact Angle
A piece of 1 cm × 1 cm was obtained for each sample. Then, it was placed in front of a small camera on a plate. A drop of deionized water was dropped on the horizontal film, while the camera is working, to take one image for each drop on the film. The camera used was a "1600× 8 LED, Ningbo, China". The images were taken manually after 1 s of the drop.
The noticeable resultant peaks are sharp because of the high sintering temperature during merged oxides preparation. Additionally, the cellulose acetate peak decreased in intensity upon binary NC formation that indicated the successful incorporation of transition metal oxides [1]. GO peak is hidden in Cr 2 O 3 /TiO 2 /GO@CA film owing to the interruption of its 2D crystal lattice by inserting the rest of the composition. Thus, the XRD data verified the chemical structure of the studied films.
FTIR analysis is tested in the wave number scope (400 to 4000 cm −1 ). FT-IR analysis of CA, Cr 2 O 3 @CA, TiO 2 @CA, Cr 2 O 3 /TiO 2 @CA, and Cr 2 O 3 /TiO 2 /GO@CA films are given in Figure 2. The CA spectra exhibit bands at 1030, 1230, 1372, and 1745 cm −1 equivalent to C-O-C, C-O-C, C-CH 3 , and C-O, respectively. In the binary compositions (transition metal oxide@CA) film, the intensity of CA peaks from 3000 cm −1 to 3400 cm −1 declines because of Cr 2 O 3 /TiO 2 incorporations. This peak is attributed to the stretching mode of the OH group, and changes in its appearance reflect the H-bond between CA and metal oxides, which indicates the film's compatibility [20]. The GO characteristic peaks at 1150 and 1520 cm −1 are assigned to the C-O stretching mode [1]. Regarding Cr 2 O 3 bands , the weak band at 580 cm −1 refers to Cr-O stretching [21]. The FTIR peaks also verified the chemical composition of the prepared films. The noticeable resultant peaks are sharp because of the high sintering temperature during merged oxides preparation. Additionally, the cellulose acetate peak decreased in intensity upon binary NC formation that indicated the successful incorporation of transi tion metal oxides [1]. GO peak is hidden in Cr2O3/TiO2/GO@CA film owing to the inter ruption of its 2D crystal lattice by inserting the rest of the composition. Thus, the XRD data verified the chemical structure of the studied films. sities [10]. The data in Figure 3 (inset) confirm the existence of C, N, O, Ti, and Cr elemen with 58.89, 4.71, 36, 0.17, and 0.22%. Undeniably, oxygen represents the second uppermo atomic percent after carbon because of its appearance with almost all individual elemen at 0.5 keV, as shown in Figure 3 [22]. Lately, technologies assembly of EDX with SEM executed for giving the quantification picture of materials' chemical composition [10 EDX analysis affirmed the successful synthesis and the existence of the correspondin elements ( Figure 3). The quantitative and qualitative data of the Cr 2 O 3 /TiO 2 /GO@CA film are obtained via the EDX technique. The quantitative detection of elements is obtained by the signal intensities [10]. The data in Figure 3 (inset) confirm the existence of C, N, O, Ti, and Cr elements with 58.89, 4.71, 36, 0.17, and 0.22%. Undeniably, oxygen represents the second uppermost atomic percent after carbon because of its appearance with almost all individual elements at 0.5 keV, as shown in Figure 3 [22]. Lately, technologies assembly of EDX with SEM is executed for giving the quantification picture of materials' chemical composition [10]. EDX analysis affirmed the successful synthesis and the existence of the corresponding elements ( Figure 3).

Morphological Investigation
Nano-composites' topographical and elemental information was displayed in SEM micrographs which appear in Figure 4, where Figure 4a displays micrographs for Cr 2 O 3 @CA, Figure 4b for TiO 2 /Cr 2 O 3 @CA, and Figure 4c for Cr 2 O 3 /TiO 2 /GO@CA. The binary polymeric film Cr 2 O 3 @CA micrograph confirms the good distribution of chromium (III) oxide grains upon the CA matrix. The grains appeared with semi-spherical shaping, recording an average grain size of 0.32 µm. Additionally, TiO 2 /Cr 2 O 3 @CA ternary film shows a significant reduction in the size of Cr 2 O 3 grains. Moreover, TiO 2 merging is accompanied by originating of narrow grooves in between metal oxide grains with better distribution over the CA matrix. Further, the Cr 2 O 3 /TiO 2 /GO@CA film demonstrates with resolution scopes in Figure 4c. The oxides grains show less aggregation tendency with better grain distribution over the polymeric matrix. The close scope displays GO sheets with a decent spread of transition metal oxides. Both Cr 2 O 3 and TiO 2 appear in the nano-scale (<50 nm). This surface alteration points to expected variations in biological responses. Indeed, surface grooves are vital in biomaterials' biological potential.

Wettability Study via Contact Angle Detection
Contact angle measuring is performed to appraise the wettability of polymeric films, as shown in Figure 5. Undeniably, the narrower the contact angle, the greater the adhesion potential, and also the higher the bio-applicability. The wettability angles for CA,

Wettability Study via Contact Angle Detection
Contact angle measuring is performed to appraise the wettability of polymeric films, as shown in Figure 5. Undeniably, the narrower the contact angle, the greater the adhesion potential, and also the higher the bio-applicability. The wettability angles for CA, Cr 2 O 3 @CA, TiO 2 @CA, Cr 2 O 3 /TiO 2 @CA, and Cr 2 O 3 /TiO 2 /GO@CA films are 38, 39, 30, 37, and 41.5, respectively. In brief, the chemical composition directly influences contact angle and adhesion capability.
x FOR PEER REVIEW 9 of 15  [23]. The CA resultant contact angle matches our study's experimental data. Based on the previous experimental values, the films' wettability properties tended to have more hydrophilic behavior with the insertion of TiO2. The wettability potential recommends the usage of such NC films biologically.

Thermal Study Using a Thermo-Gravimetric Analysis Technique
The thermal behavior of Cr2O3/TiO2/GO@CA has been investigated by following the weight loss % of NC film by raising the temperature, using the thermo-gravimetric analysis technique (TGA). The oxy-nano-composite is exposed to temperature scope (20-600 °C) at a rate of 20 °C/min under nitrogen gas. Figure 6 shows the thermal behavior with the temperature peak of tested NC weight loss. The decomposition exhibits two overlapped steps with temperature peaks of 354.7 and 452 °C. The two steps reveal temperature scopes of (300-380 °C) and (380 to 460 °C). Regarding the TGA thermogram obtained for the FAC-based NC, originally, there is an insignificant mass loss of 7% up to 250 °C, corresponding to the H2O attach to the hydrophilic hydroxyl groups of the CA chains and, The contact angle value of the CA was detected to be lower when compared to the CA/polyurethane films [23]. The CA resultant contact angle matches our study's experimental data. Based on the previous experimental values, the films' wettability properties tended to have more hydrophilic behavior with the insertion of TiO 2 . The wettability potential recommends the usage of such NC films biologically.

Thermal Study Using a Thermo-Gravimetric Analysis Technique
The thermal behavior of Cr 2 O 3 /TiO 2 /GO@CA has been investigated by following the weight loss % of NC film by raising the temperature, using the thermo-gravimetric analysis technique (TGA). The oxy-nano-composite is exposed to temperature scope (20-600 • C) at a rate of 20 • C/min under nitrogen gas. Figure 6 shows the thermal behavior with the temperature peak of tested NC weight loss. The decomposition exhibits two overlapped steps with temperature peaks of 354.7 and 452 • C. The two steps reveal temperature scopes of (300-380 • C) and (380 to 460 • C). Regarding the TGA thermogram obtained for the FACbased NC, originally, there is an insignificant mass loss of 7% up to 250 • C, corresponding to the H 2 O attach to the hydrophilic hydroxyl groups of the CA chains and, afterward, CA chains deacetylation. There were two clear stages of thermal degradation for this material, as previously mentioned: The first step (300-380 • C/Ts 354.7 • C) with a mass loss of 63.3% agrees with the main thermal decomposition stage and could be assigned to CA chain decomposition owing to the interruption of glycosidic bonds [8]. The second (380 to 460 • C/Ts 452 • C) with a mass loss of 20.7% was assigned to CA carbonization, resulting in full degradation of the organic part of the film [8]. Thus, the residual hits 9% owing to the slight contribution of oxides in the FAC-based composition. Additionally, Kuila et al. presented that annealing of GO at 450 °C or above is equivalent to a chemical reduction process by hydrazine monohydrate at 80 °C shadowed by heating at 200 °C [24]. The previous studies state the thermal decomposition of the TiO2 nanoparticles that culminated in water evaporation, then the evaporation of inorganic material within a sample, and the evaporation of unreacted organic impurities. It reported a total weight loss of 3.9% from room temperature to 800 °C [8]. This points to the slight loss owing to mineral oxides, especially with a minor contribution. In addition, the attractive interactions between the film ingredients such as hydrogen bonding might play a stabilization role [25]. This behavior is different from that detected for pure CA films [8]. On the other hand, the more compact structure (films) offers a larger number of polymeric chains and their electrostatic interactions with inserted oxides [23]. Figure 7 shows the optical behavior of CA, Cr2O3@CA, TiO2@CA, Cr2O3/TiO2@CA, and Cr2O3/TiO2/GO@CA films. The absorption behavior demonstrates the fact the oxides insertion causes clear deviations in the absorption pattern. These tightly studied insertions lead to clear increases in CA peak intensity with the appearance of new peaks related to the inserted oxides. Thus, it is significant that all oxy composites show a higher position than CA. The peak positioned at 239 nm is a point to n → π* transitions. There is a minor shift with oxides merging, which reflects the good dispersion of oxygenated ingredients Thus, the residual hits 9% owing to the slight contribution of oxides in the FACbased composition. Additionally, Kuila et al. presented that annealing of GO at 450 • C or above is equivalent to a chemical reduction process by hydrazine monohydrate at 80 • C shadowed by heating at 200 • C [24]. The previous studies state the thermal decomposition of the TiO 2 nanoparticles that culminated in water evaporation, then the evaporation of inorganic material within a sample, and the evaporation of unreacted organic impurities. It reported a total weight loss of 3.9% from room temperature to 800 • C [8]. This points to the slight loss owing to mineral oxides, especially with a minor contribution. In addition, the attractive interactions between the film ingredients such as hydrogen bonding might play a stabilization role [25]. This behavior is different from that detected for pure CA films [8].

Optical Study
On the other hand, the more compact structure (films) offers a larger number of polymeric chains and their electrostatic interactions with inserted oxides [23]. Figure 7 shows the optical behavior of CA, Cr 2 O 3 @CA, TiO 2 @CA, Cr 2 O 3 /TiO 2 @CA, and Cr 2 O 3 /TiO 2 /GO@CA films. The absorption behavior demonstrates the fact the oxides insertion causes clear deviations in the absorption pattern. These tightly studied insertions lead to clear increases in CA peak intensity with the appearance of new peaks related to the inserted oxides. Thus, it is significant that all oxy composites show a higher position than CA. The peak positioned at 239 nm is a point to n → π* transitions. There is a minor shift with oxides merging, which reflects the good dispersion of oxygenated ingredients via the polymeric medium. Additionally, the CA polymer spectrum was almost flat after 300 nm [26]. The TiO 2 NPs showed a near-UV absorbance peak at 345 nm, while aggregated TiO 2 NPs were shown at 395 nm. The band gap energy of the TiO 2 NPs was valued above 3.503 eV. Chromium III oxide existence is confirmed by a rounded peak at 405 nm (introducing a band gap of 3.08, and 3.38 eV in Cr2O3 NP, and Cr2O3, respectively). Regarding these fact and tabulated data, NC film formation causes a significant reduction in band gaps in com parison with pure mineral oxides band gaps.

Optical Study
The absorption coefficients (α) are detected via Beer-Lambert's formula: where A is absorbance and d is the film's thickness. As displayed in Figure 7, α is meas ured as a function of (photon energy hν) and it is clear that the absorption edge is reposi tioned along the x-axis with varying mixed oxides amount and types. It started from 4.9 eV for CA, and then it minimized to 2.2 eV for TiO2@CA. Additionally, the band gap could be calculated using the next equation: where hν is the photon energy, Eg is the band gap, and A refers to the band tailing param Chromium III oxide existence is confirmed by a rounded peak at 405 nm (introducing a band gap of 3.08, and 3.38 eV in Cr 2 O 3 NP, and Cr 2 O 3 , respectively). Regarding these facts and tabulated data, NC film formation causes a significant reduction in band gaps in comparison with pure mineral oxides band gaps.
The absorption coefficients (α) are detected via Beer-Lambert's formula: where A is absorbance and d is the film's thickness. As displayed in Figure 7, α is measured as a function of (photon energy hν) and it is clear that the absorption edge is repositioned along the x-axis with varying mixed oxides amount and types. It started from 4.9 eV for CA, and then it minimized to 2.2 eV for TiO 2 @CA. Additionally, the band gap could be calculated using the next equation: where hν is the photon energy, Eg is the band gap, and A refers to the band tailing parameter. The power (m) refers to the type of transition; where (direct if m = 0.5, and indirect if m = 2). The diminishing of band gap is accompanied by the raising in crystallographic ordering, this is explained by the effect of mixed oxides insertions through the CA metrics. The refractive index (n) could be found via Dimitrov and Sakka's equation as a function of indirect energy band gap as follows: The refractive index parameter increases from 1.76 for CA to 2.14 for TiO 2 @CA film. As a result, the composite formation merging tactic is crucial in refractive index alteration, as demonstrated in Table 2. The refractive index does not provide distinctive values for each component because its measurement depends strongly on the thickness of the sample and also the preparation conditions of the materials. It was reported that CA might possess a refractive index of 1.5. Thus, the increase in the refractive index could reflect the increase in polymeric density due to the additional metal oxides.

Cell Viability (Normal)
Normal lung cell (A138) growth examination as a function of Cr 2 O 3 /TiO 2 /GO@CA NC film concentration is displayed in Figure 8. In other words, the size and distribution of the cells through and over the films were observed. The usage of 4.9 µg/mL shows a cell viability percentage of 93.46%, while 156.25 µg/mL hits 86.35%. On the subject of the usage of the highest concentrations of 5000 and 10,000 µg/mL, the cell viability % declined to 61.35 and 55.19%, respectively. The viability results point to the minor toxicity of Cr 2 O 3 /TiO 2 /GO@CA with a definite threshold. The cell viability was higher than 50% at a very high concentration of (10,000 µg/mL), which indicates the high biocompatibility of these scaffolds to be used for dressing applications. Khalid et al., 2017, have reported the successful merging between TiO 2 NPs into cellulose [16]. The NC bandage displayed good healing with 71% wound contraction. Histopathological evidence such as the reepithelization refers to the healing progress [16]. Prakasha et al., 2020, have stated GO/TiO 2 NC has clear antibacterial activity in the presence of light. This is explained by the photocatalytic activity of NCs, besides the release of ROS in light [17]. Additionally, the GO/TiO 2 NC displays significant biocompatible and hemocompatibility in this study, also displaying potential in wound healing applications [17]. Accordingly, NC films based on these ingredients have various biomedical potentials. Regarding the previous studies [27][28][29][30][31][32], as shown in Table 3, it was reported that A. Ullah studied the modification of CA for wound healing utilization. The results showed that the incorporation of manuka honey into CA nanofiber led to the formation of nano-composites that were able to inhibit the colonization of bacterial cells over the surface of wounds. Furthermore, the high porosity of the fabricated scaffolds might introduce a transfer of moisture through the wound [31].

Conclusions
Herein, cellulose acetate-based films showed biocompatibility behavior. The chromium oxide/titanium dioxide/graphene oxide integrated cellulose acetate (CA) films were fabricated. SEM micrographs of Cr2O3/TiO2/GO@CA film revealed Cr2O3 and TiO2 ingredients on a nano-scale. The EDX analysis resultant data confirmed the existence of C, N, O, Ti, and Cr elements. The TiO2@CA showed the lowest contact angle with 30°, besides introducing the highest refractive index with 2.14. Likewise, normal lung cells (A138) growth examinations were introduced at 93.46% with exposure of 4.9 µg/mL. The resulting data promoted the usage of the tested NCs for wound healing hastening.  High effectiveness against both tumor cells and bacterial invasion. [28] CA nanofiber/GO/TiO 2 /Curcumin Wound healing High hemocompatibility and promoted cell proliferation and migration. [29] Lignin/CuO/CA nanofiber Wound healing The Cu/CA showed faster release (80%) of copper ions within 24 h. [30] Manuka honey/CA nanofiber Wound healing High efficacy to prevent bacterial growth, high porosity suitable for wound breathability, and high cytocompatibility. [31] Ag-sulfadiazine/CA nanofiber Wound healing Appreciable antimicrobial activity against E. Coli and Bacillus Subtilis. [32]