Annealing temperature influences the cytocompatibility, bactericidal and bioactive properties of green synthesised TiO2 nanocomposites

Annealing is a crucial functional parameter relevant to the green synthesis and bactericidal properties of TiO2 nanocomposites (TiO2-NPs). In this work, the effect of the annealing temperature on the physicochemical, bactericidal and cytocompatibility properties of TiO2-NPs obtained from Calotropis gigantea was comprehensively studied. Results indicated that amorphous-phase TiO2-NPs were transformed into the anatase phase at 500 °C with a crystallite size of 40.9 nm and MIC of 100 mg/mL towards Staphylococcus aureus. Whereas TiO2-NPs annealed at 400 °C demonstrated no bacterial reduction, TiO2-NPs annealed at 500 °C showed a moderate zone of inhibition towards Escherichia coli and Pseudomonas aeruginosa. Findings from this study found that TiO2-500C nanocomposites concentration at 100 mg/mL does not inhibit fibroblast cells proliferation activity after 24 h treatment. The plant-mediated nano-sized cubic and spherical anatase TiO2-NPs encapsulated bioactive green elements, such as carbon, sodium, magnesium, chlorine, potassium, calcium and sulphur, from the C. gigantea extract, ultimately leading to versatile and eco-friendly bactericidal agents with wound-healing properties. Further studies involving in vivo are needed to support this work.


Introduction
Recent progress in research on natural products has resulted in the development of many natural plant-mediated nanoparticles (NPs) with bactericidal properties; amongst these materials, green synthesised titanium dioxide NPs (TiO 2 -NPs) have sparked great interest on account of their potential use in wound healing therapy. Green synthesis method represents an advance over conventional chemical and physical methods (Aravind et al. 2021;Ansari et al. 2022). It has shown multiple benefits such as eco-friendly, simple, economical, sustainable and safe (Ansari et al. 2022). Moreover, green synthesised TiO 2 -NPs produces biocompatible and enhanced antibacterial stable nanomaterials for biomedical applications compared to the chemically synthesized TiO 2 -NPs (Aravind et al. 2021;Ansari et al. 2022). Calotropis gigantea is a traditional medicinal plant with antimicrobial properties that is often used to treat skin diseases (Kumar et al. 2010) and open wounds (Sangeetha et al. 2020). Aqueous solutions of C. gigantea leaf extract function as excellent reducing and capping agents in the formation of green TiO 2 -NPs. Indeed, given their promising bactericidal properties for addressing skin and wound infections due to pathogens, plant-mediated TiO 2 -NPs are amongst the most extensively studied bactericidal agents in the biomedical field (Table 1). TiO 2 -NPs exist in three phases, namely, anatase, rutile and brookite, under different processing conditions (Sugapriya et al. 2013;Tesfaye Jule et al. 2021).
Scientists worldwide have sought to establish methods to control the size and morphology of TiO 2 -NPs as the particle size exerts a massive influence on their bactericidal properties (de Dicastillo et al. 2020). Even slight variations in the annealing conditions could result in prominent effects on the phase and morphology of TiO 2 -NPs (Sugapriya et al. 2013;Długosz et al. 2020). A significant improvement in degree of recrystallization, phase transition and uniform size distribution of TiO 2 -NPs was seen when increasing the annealing temperature (Tesfaye Jule et al. 2021;Muthee and Dejene 2021). Therefore, a slight alteration on size of NPs and transformation of phase could result in dramatic improvement on antibacterial activity (Lin et al. 2014;Senarathna et al. 2017). The present work discusses the formation of bioactive elements of C. gigantea leaf extract and the morphology of the resultant TiO 2 -NPs under the effect of different annealing temperatures (i.e., 400 and 500 °C). The physicochemical properties of the TiO 2 -NPs were characterised using scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDAX), UV-Vis spectrophotometry and Fourier transform infrared (FTIR) spectroscopy. Additionally, bactericidal and cytocompatibility properties of green TiO 2 -NPs were further investigated.

Green synthesis
TiO 2 -NPs was synthesised via green route by the reaction of C. gigantea leaves extract aqueous solution with titanium (IV) isopropoxide (Sigma-Aldrich). This grounded green synthesis method uses the following protocol (Govindasamy et al. 2021a, b). Green TiO 2 -NPs samples calcined at different low temperatures 400 °C and 500 °C are designated as TiO 2 -400C and TiO 2 -500C, respectively. In this work, commercially available TiO 2 P25, anatase (Sigma-Aldrich, 99.7%) was used as a commercial control.

X-ray diffraction
X-ray diffractometer (XRD; Bruker D8) was used to study crystalline nature and size of green TiO 2 -NPs. The X-ray diffraction peaks were captured via Cu Kα radiation with wavelength of λ = 0.1541 nm and step scan mode with step size of 0.030° in the range of 10° to 90°. The equipment was operated at a voltage of 40 kV and current of 30 mA. Scherrer's equation was applied to determine the average crystallite size of green TiO 2 which is as under where d represents the crystallite size, K = 0.9 is the shape factor, λ stands for the X-ray wavelength of Cu Kα radiation (1.541 Å), θ is used as a Bragg diffraction angle, and β symbolize the full-width at half-maximum (FWHM) of the respective diffraction peak (Govindasamy et al. 2021a).

SEM observation
The morphology of TiO 2 -NPs was further investigated using scanning electron microscopy (SEM Fei Quanta FEG 650). Samples were placed on a stub with carbon tape and coated with Pt for 1-min prior to imaging and elemental analysis. The TiO 2 -NPs and bioderived elements composition and their percentage from green TiO 2 -NPs was further confirmed with EDAX.

TEM analysis
TEM (FEI TECHNAI F20 G2) is used for analysing shape and grain size of TiO 2 -NPs. At first, TiO 2 -NPs was dispersed in absolute ethanol and then sonicated for 30 min. After that, a single drop of TiO 2 -NPs solution was added onto a lacey carbon film-coated copper grid (300 mesh) and then dried at room temperature for 30 min. At last, it was kept in desiccator prior to TEM imaging.

FTIR study
The functional groups of green TiO 2 -NPs were recorded by FTIR spectroscopy (PerkinElmer) within the range of 4000-400 cm −1 through the KBr pellet method. The FTIR samples were prepared by dispersing small dosage of TiO 2 -NPs uniformly in a KBr matrix which was then compressed to thin transparent disc.

UV-visible spectroscopy
The absorption spectrum of TiO 2 -NPs was determined using UV-Vis spectrophotometer (Varian) in wavelength range between 200 and 700 nm.

Antibacterial tests
Kirby-Bauer disc diffusion test and minimum inhibitory concentration (MIC) were determined after 24 h of contact with the prepared green samples according to following methods (Govindasamy et al. 2021a, b, c

Cytocompatibility assay
The cytocompatibility assay was performed on fibroblast cells lines model, L929 obtained from American Type Culture Collection (ATCC, USA) and is maintained in RPMI-1640 media (Gibco, Life technologies) supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) PenStrep (Gibco, USA), sodium bicarbonate, 12.5 g/mL HEPES and 1% (v/v) l-glutamine at 37 °C in a 5% CO 2 humidified atmosphere. 10% (v/v) of dimethyl sulfoxide (DMSO) was adapted as strong cytotoxic material (negative control) while fibroblast cells lines without administration of TiO 2 as blank control. The in vitro cytocompatibility of greensynthesised TiO 2 on fibroblast cells lines was performed by direct contact method according to the protocol recommendations in ISO 10993-5 (2009) (Harun et al. 2021;Chellappa et al. 2015). The cell viability percentage was determined using (1:10) alamarBlue ™ Cell viability reagent DAL1025 (Invitrogen, United Kingdom). At first, cells were subcultured, trypsinized and seeded at a density of 1 × 10 4 cells/well (100 µL/ well) on 96 well plate and grown for 24 h in CO 2 incubator at 37 °C. After that, different concentrated TiO 2 (0, 25, 50 and 100 mg/mL) were added in the culture media and kept overnight. Then, the serially diluted test sample solution (n = 3) was incubated with monolayer cells at 37 °C for 24 h. The proportion of live and healthy cells after treatment were further estimated quantitatively through colour change from blue to pink using Alamar blue assay where the treated cells were incubated as a minimum 20 h before measuring the absorbance at wavelength 570 nm and 600 nm using a microplate reader (Bio-Tek Instruments, USA). The cytocompatibility of test sample was compared with the toxic material (10% (v/v) DMSO) and blank control. The live and dead cells were examined microscopically via Olympus CKX41 optical light microscope at magnification of 10× and 20×.

Statistical analysis
The different calcination temperature treated group of samples were analysed using analysis of two-way ANOVA implemented in the GraphPad Prism software package. Results were considered statistically significant if p value is less than 0.05. Data are presented as mean ± standard deviation (SD).

Physicochemical characterisation of the TiO 2 -NPs
The SEM images in Fig. 1a, b show that the TiO 2 -400C and TiO 2 -500C NPs are spherical in shape with a nano-sized and agglomerated morphology (Fig. 1a, b, insets). The EDAX images of the TiO 2 -NPs confirm the presence of Ti and O, which make up approximately 29.28 wt% and 36.72 wt%, respectively, of TiO 2 -400C and 33.55 wt% and 41.91 wt%, respectively, of TiO 2 -500C (Table 2, Fig. 1c, d). The TEM images in Fig. 1e, f reveal that the NPs produced at a low calcination temperature are nearly spherical with a large, agglomerated morphology whilst those calcined at a high temperature are spherical and cuboidal in shape with a partly agglomerated morphology.
EDAX analysis successfully identified an abundance of bioactive elements in C. gigantea, such as carbon (C), calcium, chlorine, sodium, magnesium, potassium and sulphur, in agreement with a previous research (Govindasamy et al. 2021a, b) (Table 2). These elements are by-products of the green synthesis of the NPs. The combination of these bioderived elements with high concentrations of C and antimicrobial species, such as hydroxyl radicals, hydrogen peroxide, superoxide anions and titanium (IV), may arrest the growth and development of skin pathogens (Cheng et al. 2009).
The functional groups responsible for the formation of green TiO 2 -NPs were determined by FTIR analysis. The FTIR spectrum of the green nanocomposites shown in Fig. 2b reveals a broad peak at 3438 cm −1 , which could be assigned to the O-H stretching vibrations of flavonoids from C. gigantea (Govindasamy et al. 2021a, b). The peak at 1633 cm −1 could be attributed to C=C (carbonyl group), and the peaks at 1361 and 1119 cm −1 could respectively be attributed to the O-C-O stretching vibrations of esters and the C-O stretching vibrations of bio-derived elements from the C. gigantea leaf extract (Govindasamy et al. 2021a, b). The peak at 595 cm −1 confirms the presence of TiO 2 -NPs with Ti-O-Ti framework bonds (Nasrollahzadeh et al. 2016;Zhou et al. 2021).
UV-Vis spectrophotometry was applied to measure the bandgap of the green synthesised TiO 2 -NPs, and the results obtained are shown in Fig. 2c. The absorbance peak at 228 nm is due to the dispersion of natural C in deionised water (Govindasamy et al. 2021a, b), and the small peak at 327 nm confirms the formation of green synthesised TiO 2 -NPs (Zhou et al. 2014). The UV-Vis spectrum of the control C. gigantea leaf extract showed two prominent absorbance peaks; the sharp peak at 206 nm corresponds to the carbonyl group, whilst the low broad peak at 269 nm represents phenolic compounds (Govindasamy et al. 2021a, b).  aureus because staph skin infections are most commonly observed in open wounds. The MIC of TiO 2 -500C for S. aureus was 50.0 mg/mL. TiO 2 -500C exhibited strong bactericidal activity at a high concentration of 100 mg/mL (Fig. 3e). However, the results for TiO 2 -400C showed no bacterial reduction despite these NPs having a low MIC of approximately 25.0 mg/mL. A sharp decrease in colony count from 4.3 log 10 (control S. aureus) to 1.01 log 10 was observed for TiO 2 -500C at a concentration of 100 mg/mL (Fig. 3). Here, * indicates statistically significant differences (****p ≤ 0.0001) between the different calcination groups for each measurement. There is significant difference between control strain group and anatase TiO 2 -500C group at concentration of 100 mg/mL. The stronger bactericidal effect of TiO 2 -500C compared with that of TiO 2 -400C may be attributed to the anatase crystalline structure of the former, as confirmed by the XRD pattern shown in Fig. 2a (Fu et al. 2005;Senarathna et al. 2017). Inhibition of bacterial colonies was observed only at a high dosage of the NPs. Thus, green TiO 2 -500C nanocomposites may be considered an excellent bactericidal agent against S. aureus, a gram-positive bacterium (Behera et al. 2017). Table 3 and Fig. 3a-e illustrates the MICs of the green TiO 2 -NPs for S. aureus.

Determination of zone of inhibition
The bactericidal activity of the TiO 2 -NPs was further evaluated against S. aureus, the gram-negative bacterium Escherichia coli and the antibiotic-resistant bacteria Klebsiella pneumoniae and Pseudomonas aeruginosa via the Kirby-Bauer disc diffusion method. In this study, TiO 2 -500C showed a moderate zone of inhibition (ZOI) for E. coli and P. aeruginosa (Table 4 and Fig. 4). By comparison, commercially available anatase TiO 2 (P25) and TiO 2 -400C showed poorer ability to interfere with the cell wall of multi-drug resistant (MDR) and non-MDR strains and disrupt their biochemical processes at a concentration of 100 mg/mL. Cefoxitin antimicrobial discs showed a large ZOI against all four bacterial strains. Researchers found that green TiO 2 -NPs synthesised from Trigonella foenum-graecum extract have a ZOI of approximately 11.2 mm against S. aureus (Subhapriya and Gomathipriya 2018).
Previous studies demonstrated enhancements in the photocatalytic properties of C-decorated TiO 2 -NPs for water purification (Nasrollahzadeh et al. 2016;Sharma et al. 2018;Shah et al. 2012;Atchudan et al. 2017) and solid rocket propellants (Dey et al. 2013). None of these works, FT-IR spectrum of TiO 2 -NPs and c UV-Vis spectrum of TiO 2 -NPs ▸ however, have highlighted the bactericidal activity of natural C-encapsulated TiO 2 -NPs. The present study is the first to report that TiO 2 -500C nanocomposites could encapsulate the bioactive elements of C. gigantea leaf extracts and possess strong inhibitory effects against the tested organisms. This plant-mediated anatase TiO 2 -based bactericidal agent may be a promising eco-friendly and non-hazardous biomaterial for future pharmaceutical applications.

Cytocompatibility profiles
The cytocompatibility of TiO 2 -400C and TiO 2 -500C with different concentration (0, 25, 50 and 100 mg/mL) were investigated by fibroblast cells lines model as described in Fig. 5 with the blank control group and 10% (v/v) DMSO. Blank control group set as 100% viability. Based on ISO 10993-5 (2009) recommendation it can be concluded that all different-concentrated green TiO 2 nanocomposites is considered as cytocompatible since the cell viability is higher than 70%. Comparatively, DMSO exhibited significant cytotoxicity to the tested cell lines with cell viability percentage of approximately 31%. It strongly indicates statistically significant differences (****p ≤ 0.0001) between differentconcentrated green TiO 2 group and DMSO treated group. In addition, more healthy live cells with elongated filopodia (the leg like of the cell) between 95 and 129% were seen in all TiO 2 -400C (Fig. 6b, c, d, f, g, h; Supplementary material) and TiO 2 -500C (Fig. 6j, k, l, n, o, p; Supplementary material) treated nanocomposites, respectively. The outcome showed green TiO 2 had ability in promoting proliferation and viability of fibroblast cells lines.
However, TiO 2 -500C caught much more attention in this study due to its strong bactericidal activity against Grampositive S. aureus (Fig. 3e) and high proliferation of fibroblast cells lines at concentration level of 100 mg/mL (Fig. 5). The sedimentation of agglomerated green TiO 2 on bottom plate was picturized through yellow circle ( Fig. 6; Supplementary material). Previously, researchers have investigated the effect of TiO 2 nanoparticles concentration related to cytotoxicity and cytocompatibility against MG63 cell lines Fig. 3 Number of colony-forming units per millilitre (CFU/ mL) of S. aureus remained after treatment of different concentration of different calcined TiO 2 -NPs; a control strain, b TiO 2 -400C at 50 mg/ mL, c TiO 2 -500C at 50 mg/ mL, d TiO 2 -400C at 100 mg/ mL and e TiO 2 -500C at 100 mg/ mL. Petri dish with more than 300 colonies cannot be counted and are designated too many to count (TMTC). Data shown are mean value ± SD (n = 3, ****p value ≤ 0.0001)   S. aureus 6 ± 0.00 6 ± 0.00 6 ± 0.00 NA 10 ± 0.00 E. coli 6 ± 0.00 6.83 ± 0.29 6 ± 0.00 NA 10 ± 0.00 K. pneumoniae 6 ± 0.00 6 ± 0.00 6 ± 0.00 NA 9 ± 0.00 P. aeruginosa 6 ± 0.00 6.33 ± 0.14 6 ± 0.00 NA 9 ± 0.00 and discovered that rapid cell proliferation and enhanced viability were seen at different concentrations without any adverse toxicity (Chellappa et al. 2015). Although, proliferation and survival of fibroblast cells lines slightly decreased when the concentration of green TiO 2 is further increased. It is ascribed to the higher level of ROS generation at high concentration level of TiO 2 (Behera et al. 2017). Future works on ROS release quantification and long-term cytocompatibility properties of green TiO 2 on fibroblast cells lines model is needed for further understanding of the molecular level.

Conclusions
Nano-sized cubic and spherical anatase TiO 2 -500C decorated bioactive green elements nanocomposites with cytocompatible behaviour was successfully synthesised from C. gigantea leaf extract solution. Differences in annealing temperature exerted remarkable impacts on the crystalline phases, morphology, cytocompatibility and bactericidal activities of the green TiO 2 -NPs towards S. aureus, E. coli and P. aeruginosa. Green TiO 2 -NPs was found to be cytocompatible on fibroblast cells lines with increased cell viability (≥ 116%). Thus, the TiO 2 -NPs developed in this work can address current limitations related to pathogen-induced open wound skin infections and wound healing characteristic. However, further investigation is needed to determine the detailed bactericidal mechanism of bioderived anatase TiO 2 -NPs.
Author's contribution GAG mainly contributes in writing this manuscript and carried out all experimental works. NHH, WNFWEE, and SS assist in the procedures. RBSMNM is the principal investigator contributes in the concept idea, experimental design, writing process and gave final approval of this paper for publication. All authors have given approval to the final version of the manuscript.  Fig. 4 Zone of inhibition (mm) exhibited by 100 mg/mL of TiO 2 -NPs against non-MDR and MDR skin pathogens. a TiO 2 -400C against S. aureus, b TiO 2 -500C against S. aureus, c commercial P25 against S. aureus, d positive control (cefoxitin-30 µg) against S. aureus, e TiO 2 -400C against E. coli, f TiO 2 -500C against E. coli, g commercial P25 against E. coli, h positive control (cefoxitin-30 µg) against E. coli, i TiO 2 -400C against P. aeruginosa, j TiO 2 -500C against P. aeruginosa, k commercial P25 against P. aeruginosa, l positive control (cefoxitin-30 µg) against P. aeruginosa, m TiO 2 -400C against K. pneumonia, n TiO 2 -500C against K. pneumonia, o commercial P25 against K. pneumonia, and p positive control (cefoxitin-30 µg) against K. pneumonia. 10% DMSO as negative control sample did not show any antibacterial activity towards tested pathogens. These data statistically analysed by two-way ANOVA test using GraphPad Prism software. Data shown are mean value ± SD (n = 3, ****p value ≤ 0.0001)