Enhanced photocatalytic and photodynamic activity of chitosan and garlic loaded CdO–TiO2 hybrid bionanomaterials

Herein, the work addresses the synthesis of biomaterials (chitosan and garlic) loaded CdO–TiO2 hybrid nanocomposites for photocatalytic water treatment and photodynamic cancer therapeutic applications that were reported the first time. CdO–TiO2 (CT) nanocomposites were synthesized and loaded with the biomaterials such as chitosan and garlic by simple sol–gel method. The nanomaterials were characterized and the photodegradation of three model pollutants, Methylene blue (MB), Methyl orange (MO) and Rhodamine B (Rh-B) was opted to investigate the efficiency of the synthesized photocatalyst under the solar light. From the results, the garlic-loaded CdO–TiO2 (AS-CT) hybrid nanocomposites exhibit a superior photocatalytic activity than the chitosan-loaded CdO–TiO2 (CS-CT) and CdO–TiO2 (CT) nanocomposites under the irradiation of solar light. Additionally, the cell viability of the synthesized nanocomposites was carried out in HeLa cell lines under different concentrations, light doses and incubation periods using an LED light source. Compared to the CS-CT and CT nanocomposites, an efficient photodynamic activity was achieved in the case of AS-CT hybrid nanocomposites. Actually, the end-use properties required for both processes in AS-CT nanocomposites appear similar due to the presence of organo sulphurus compounds.


Result and discussion
Characterization. The  The effect of the catalyst relies on the morphological behaviour of the sample. The surface morphology of the synthesized nanocomposites was determined from FE-SEM analysis. The micrographs of CT, CS-CT and AS-CT hybrid nanocomposites were displayed in Fig. 2a-c. Compared with FE-SEM images of CT, the images of CS-CT and AS-CT (Fig. 2b,c) displays that the NPs are uniform and spherical in shape. The results highlight that the NPs appear spherically shaped with some irregularities. The particle size varies from 16 to 45 nm with an average crystal size of 8-15 nm.
The TEM image of nanocomposites was presented in Fig. 3a-c. The present TEM images were undoubtedly exposed to FE-SEM reflection. Transmission electron microscope (TEM) images in Fig. 3a-c give close view of nanocomposites; there is no significant difference in the morphologies, which is almost spherical in shapes. However, there is a slight difference in particle size for all three synthesized nanomaterials but fall within the range of . This is about twice to thrice that of the average crystal size obtained from XRD and the nanocomposites are polycrystalline in nature. This result indicates that different sizes of nanocomposites might be obtained by using biomaterials.
The elemental composition was detected by using the EDX technique. Figure 4a-c displays the EDX analysis of CT, CS-CT, and AS-CT nanocomposites. The results proved the existence of all the elements including, Ti, O, and Cd from TiO 2 and CdO, in the synthesized nanocomposites. Similarly, presence of N and S along with Ti, O and Cd in CS-CT and AS-CT nanocomposites confirms the existence of chitosan and garlic in the bionanomaterials. In addition, the presence of O is associated with the oxygen in TiO 2 lattice as well as in the surface -OH groups, and the Ti and O values are not even with the actual elemental composition of TiO 2. The absolute amount of the elements present in the materials was not determined by using the EDX analysis, but the presence of specific elements can be determined 47,48 .
The visible light activity and the band gap of the synthesized CT, CS-CT, and AS-CT nanocomposites were studied by using UV-DRS. As shown in Fig. 5a-c, the light absorption characteristics of CT were also modified by loading of biomaterials. Figure 5a-c indicate that loading of biomaterial with CT nanocomposites had increased the absorbance from UV to the visible-light region, improving the photocatalytic and photodynamic behaviour of the nanocomposites. DRS of CdO-TiO 2 and garlic-loaded CdO-TiO 2 nanocomposites show absorption in the visible region of 500-600 nm. Chitosan-loaded CdO-TiO 2 nanocomposites show absorption in the UV region. In the case of garlic-loaded CdO-TiO 2 calcined at 450 and 700 °C, the absorption edge was observed in the visible region of the solar spectrum, that represents the catalyst excitation efficiently exploits more photons.  www.nature.com/scientificreports/ This kind of absorption explained the substitution of titanium lattice by S 6+ , a newly isolated band formed above the valence band of TiO 2 VB and the band gap narrowed consequently 49 . Furthermore, the results suggest that loading significantly promotes the band gap to red-shift, which eases the electron excitation from the VB to the CB that results in higher photocatalytic and photodynamic activity.
In the interest of predicting the type of band-to-band transitions in the synthesized nanocomposites, the absorbance data of the DRS was plotted into the direct band gap transitions equation 50 . Figure 6a-c gives [F(R) hν] 2 Vs. photon energy plot for direct transitions. From the recorded reflectance (R), the F(R) data were deduced by Kubelka-Munk algorithm application [F(R) = (1-R 2 )/2R]. F(R) depicts the sample's absorptivity at a specific wavelength. From the modified Kubelka-Munk algorithm plot, the corresponding wavelengths (λ g ) and the absorption edges (E g ) of the nanocomposites were determined.
The interaction in metal oxides and biomaterials doped TiO 2 NPs was studied using FT-IR. The FT-IR analysis of the CT, CS-CT, AS-CT nanocomposites, chitosan and garlic extract were displayed in Fig. 7a-e. From the observance of Fig. 7a, a broad and strong transmittance band at 3400 cm −1 which is attributed to the O-H stretching vibration of TiO 2 NPs. The peak at 2300 cm −1 corresponds to atmospheric CO 2 vibrations, and a peak at 1630 cm −1 represents water deformation (δH-OH). A band in the range of 650 and 800 cm −1 corresponds to TiO 2 different vibrational modes. In CS-CT hybrid nanocomposites (Fig. 7b) show a peak around 3400 cm −1 and 1630 cm −1 that indicates hydroxyl (-OH) and amine (-NH 2 ) groups which behave as reactive and coordination sites for the organic species adsorption. A band at 700 cm −1 and 2300 cm −1 attributes TiO 2 and the atmospheric CO 2 vibrations. The presence of amide or amine and OH groups along with metal oxides favoring the confirmation of the efficient dye removal by adapting the process of photodegradation-adsorption 51 . AS-CT hybrid nanocomposites (Fig. 7c)   www.nature.com/scientificreports/ the broadening of the peak occurs when loaded with garlic. For comparison, the FT-IR spectra of chitosan and garlic also displayed in the Fig. 7d,e. The presence of a peak at 1630 cm −1 corresponds to amine (-NH 2 ) groups in chitosan and the observance of a peak at 1636 cm −1 corresponds to S = O functional groups in garlic along with the other peaks supports and matches with the synthesized bionanomaterials. Figure 8a-e gives the TG-DTA studies of the nanocomposites. From TG-DTA, the thermal behaviour of the nanomaterials was determined in the range of 50 and 800 °C at nitrogen atmosphere. The thermogram of CT nanocomposite ( Fig. 8a) gives weight loss around 345 °C that represents the decomposition of the residual -OH groups. After that, there was no observance of peak since the compound remained intact. DTA graph shows a convexity appearance centered at 375 °C indicating the residual -OH decomposition. The thermo-gravimetric analysis of CS-CT (Fig. 8b) shows weight loss with two decompositions. The first weight loss that occurred at 115 °C is owing to the degradation of the chitosan polymer chain and the second weight loss at 354 °C is because of the crystallization of TiO 2 . The DTA of CS-CT nanocomposite reveals two prominent peaks. The first peak at 118 °C indicates the degradation of the chitosan polymer chain. The second peak at 354 °C may be corresponding to the template removal and TiO 2 crystallization. Thermogravimetric analysis of AS-CT nanocomposite (Fig. 8c) shows two-weight losses with two decomposition steps. The weight loss below 300 °C is attributed to the decomposition of the residual -OH groups and the second weight loss at 620 °C is due to the phase transformation of TiO 2 . In DTA of AS-CT shows two convex appearances centered at 300 °C and 610 °C. The first exothermic peak is due to the removal of the residual OH group and the second weight loss is attributed to the phase transformation of TiO 2 . The TG-DTA of chitosan and garlic also displayed in the Fig. 8d,e, respectively. The TGA of chitosan shows two weight losses at 340 °C and 400 °C, first stage of weight loss was due to decomposition of the residual OH groups and the second stage of weight loss was attributed to the degradation of the polymer chain. In TGA of garlic extract, the major weight loss was observed at 400 °C that represents the main loss of natural extract. The noteworthy characteristic is that almost similar trends in mass losses were obtained up to 800 °C for all three synthesized nanocomposites.   www.nature.com/scientificreports/ The exhibited higher photocatalytic efficacy of AS-CT nanocomposite may be due to organic sulphurous compounds that possess a superior property of degradation 46 .

Photocatalytic activity.
The second higher photocatalytic degradation of the dyes by CS-CT catalyst because of the anionic dye adsorption increased by a positively charged chitosan matrix surface. The amine groups present in the chitosanbased nanomaterials undergo protonation (formation of protonated amine), which could adsorb the dye molecules using various types of interaction mechanisms like chelation, electrostatic attraction, etc. It could have a higher adsorbent capacity to remove pollutants from the wastewater. It produces high active sites for complex formation with the attracted molecules, result in enhancement of the solar light photocatalytic efficiency 52 .
The CT catalyst also shows some favourable activity but not comparable with CS-CT and AS-CT nanocomposites. The significant photocatalytic effect of CT nanocomposite may be due to the dye-sensitized reaction. However, the catalytic deterioration of the dyes by TiO 2 NPs is sluggish and there are no remarkable changes that could modify the degradation process.
The reason for enhanced photocatalytic activity of the biomaterials based hybrid nanomaterials is owing to (i) the high crystallization degree of doped/loaded anatase and stability that feeble the transfer of electron and subsequent reduction in the recombination of photo-generated holes, and/or (ii) the increase of vacancies of oxygen result in the doping or deformity lattice defects that attract the photoinduced electrons thereby suppress the e − − h + recombination [53][54][55] . Generally, the doped materials may deform the lattice of TiO 2 and the substitution possible either Ti 4+ or O 2− . Thus, h + in the valence band trapped by OH − or the H 2 O adsorption produces the radical on the catalyst surface, whereas the photo-generated e − in the conduction band reduces the adsorbed oxygen into ˙O 2 that enhances the catalytic activity 56 . In addition to that, the hole itself achieves the oxidation of the target pollutants effectively, which is adsorbed over the catalyst surface 57 .
The degradation behavior of the pollutants (Rh-B, MB, and MO) improved over the surface of the catalyst 58 by the synergistic effect of generated radicals and holes but not processing in the bulk solution; the reason is the lifetime of the photo-generated radicals was short and inclined to the recombination 59 . Additionally, the occurrence of the enhancement of the dye degradation in the liquid phase has been made by the adding dopants of the slightly distorted lattice and presence of anatase phase with a high degree of crystallinity 60,61 . www.nature.com/scientificreports/ Recyclability and reusability. The recyclability experiment was also carried out for the synthesized photocatalysts (Fig. 10). After each experiment, the photocatalysts were isolated from the reaction mixture, washed thrice by using absolute alcohol, oven-dried at 80 °C. The photocatalysts exhibit favorable reusability after 3 times of recycling. There was an observance of some extent of loss in catalytic activity after each reporting period. The fall in the photocatalytic activity/rate of photodegradation may be the result of loss in the amount of catalyst during the catalyst collection or weakening of the photocatalyst absorbing capacity.
Cell viability. Figure  The CT nanocomposites had no significant impact on the HeLa cells with increasing concentration, light dose and also incubation time (Fig. 11a-d). Considering the CS-CT effect (Fig. 12a-d), LC 50 was found to be 184, 174, 171 and 167 µg/ml at the light dose 53.65, 71.54, 89.43 and 107.31 J/cm 2 respectively after 24 h of incubation (Fig. 12d). Also, the clear observation reveals that further increase in the concentration of the dose above the LC 50 level also had produced 60% of cell death at the different tested light doses except for the lowest dose at 36.77 J/cm 2 . Hence this is highly significant in considering the individual effect of CS-CT . Figure 13a-d shows cell viability of AS-CT nanocomposite on the Hela cells. Remarkably, AS-CT had shown increased anticancer activity when compared to the CS-CT. LC 50 was found to be 189, 175, 159, 149 and 124 µg/ ml for 53.65, 71.54, 89.43, 107.31 J/cm 2 respectively for the AS-CT after 24 h of incubation (Fig. 13d). At the increased intensity of light dose tested at 89.43, 107.31 J/cm 2 , the concentration of anticancer activity slightly decreased to 159, 149 µg/ml for the AS-CT nanocomposite when compared to CS-CT nanocomposite, which was at 171 and 167 µg/ml, respectively. After 24 h of incubation, AS-CT nanocomposite shows a remarkable decrease in cell viability concerning the light dose and it was found to be 75% of cancer cells died for 107.31 J/ cm 2 of light dose at 200 µg/ml. It justifies that the garlic loading improve the anticancer effects by inhibiting Hela cells' growth. The efficiency of cell viability was progressively reduced with extending the incubation time. The times of incubation were optimized in order to confirm the time required for the maximum amount of cellular

Synthesis of CdO-TiO 2 (CT).
A suspension of 0.038 g of CdO and 20 mL of distilled ethanol was allowed to stir for 1 h for a homogeneous suspension. To the homogenous solution, 3 ml of tween-80 was added dropwise and continued stirring for another 30 min. Followed by 3 mL of TTIP with 10 mL IPA was added drop-wise to the above mixture and allowed to stir for 2 h for obtaining gel. The gel obtained was separated and washed completely by using 1:1 aqueous ethanol and dried for 6 h at 120 ºC. The resultant material was calcined at 500 ºC for 3 h. www.nature.com/scientificreports/

Synthesis of chitosan loaded CdO-TiO 2 (CS-CT).
The same procedure has been adopted up to the formation of a gel. The procedure for the CS synthesis from crab shells has been followed from the reported literature 62 . To the gel, CS solution (1 g of CS in 100 mL of 1% (v/v) acetic acid) was added and stirred for 1 h. The resultant product was filtered off and washed completely with 1:1 aqueous ethanol and dried for 6 h at 120 ºC. The samples were calcined at 500 ºC for 3 h by a Muffle furnace.

Synthesis of garlic loaded CdO-TiO 2 (AS-CT).
The same procedure has been adopted up to the formation of a gel. Freshly crushed garlic cloves were made into tiny pieces and grounded finely with a required amount of water for getting the garlic extract. To the gel, 10 ml of as-prepared garlic extract was mixed on continuous stirring and the resultant solution was allowed for ageing (24 h) and then filtered, dried for 6 h at 120 °C and calcinated at 500 °C for 3 h to get the corresponding AS-CT nanocomposites. The experiments done on plants are in accordance with international, national and/or institutional guidelines.
Instrumentation. Bruker D2 Phaser Desktop X-ray Diffractometer (Cu Kα radiation (λ = 1.542 Å)) was used to analyze the XRD. FE-SEM was investigated using a DXS-10 ACKT scanning electron microscope with EDAX. TEM images were examined from the JEOL JEM-3010 microscope with 600 and 800 k times magnification operated at 300 keV. Shimadzu 2100 UV-Visible spectrophotometer was used to study the DRS-UV Visible spectra between 200 and 800 nm. FT-IR spectrum was investigated by using a Perkin Elmer RX1 using solid To the as-prepared dye solution (50 mL), a required quantity of photocatalyst was added and kept under an air atmosphere at a constant rate. The photocatalyst was isolated after the illumination and the dye was measured spectrophotometrically.
The solar light intensities were evaluated by using a New 200,000 Lux Digital Meter Light Luxmeter Meter Photometer with Footcandle FC and the intensity was 1200 X 100 ± 100 lx and almost identical during the experiments.
All the photocatalytic analysis was conducted under the same environment on sunny days between 12.00-2.30 p.m. and the reaction mixture (  The measurement of cell viability plays a fundamental role in all forms of cell culture. Cell-based assays are used to study the direct cytotoxic effects of drugs. Among the cell viability assays, the MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) is one of the most prominent methods for studying the mitochondrial dehydrogenase activity in living cells for safety and easy to use. In this method, viable cells convert MTT into a purple-colored formazan crystal having an absorbance maximum of around 570 nm. Thus, the color formation provides a convenient and effective marker of only the viable cells. 10 μl of MTT was added to each well after 8, 12, 16 and 24 h of incubation. The obtained formazan crystals were dissolved in dimethyl sulfoxide (200 μl) and the absorbance intensity at 570 nm was determined [63][64][65] . www.nature.com/scientificreports/