Chitosan-coated titanium dioxide nanoparticles: Fabrication, characterisation and toxicological evaluation in Drosophila melanogaster

doi:10.21203/rs.3.rs-4696481/v1

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Chitosan-coated titanium dioxide nanoparticles: Fabrication, characterisation and toxicological evaluation in Drosophila melanogaster

https://doi.org/10.21203/rs.3.rs-4696481/v1

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Titanium dioxide nanoparticle (TiO2 NP) is one the most highly sought-after nanomaterials and are prevalent in many consumer products such as personal care products, paints and coatings, and food colouring. However, their pervasive use and high demand are expected to adversely affect organisms and ecosystems. Several articles suggest that surface modification of TiO2 with appropriate materials could mitigate its negative impacts. To facilitate this, we utilised chitosan (CS), a naturally occurring biopolymer, as a coating material to fabricate a biomaterial-based nanocomposite for consumer applications. TiO2 integration into chitosan was analysed using XRD, FTIR, UV-Vis spectroscopy, and SEM. Drosophila was employed as a model organism to assess the toxicity of the coated nanoparticles, aligning with efforts to prevent animal cruelty. The toxicity was analysed in both larvae and adult flies. Variations in antioxidant enzyme activity were observed, implying activation of nanoparticle clearance pathways. Antioxidant enzyme activation is a normal response to the ingestion of xenobiotics. Nonetheless, the cumulative response did not suggest any severe toxicity despite slight changes in antioxidant mechanisms. Our objective, however, is to employ the nanocomposite for dermal uses. Hence, the nanocomposite can be recommended for consumer applications.

Titanium dioxide

Chitosan

Nanotoxicity

Drosophila

Reactive oxygen species

Titanium dioxide (TiO₂) based nanomaterials have achieved significant usage across various industries due to their unique properties attributed to their heavy metal parameters like density and atomic weight. The nanoparticles when added to food and cosmetic products enhance their quality and product functionality. However, their versatile applications raise concerns regarding their impact on human health and associated environmental risks. TiO₂ particles have distinctive optical and photocatalytic properties, making them an essential compound in various industrial productions. TiO₂ has found its use in sunscreens as ultraviolet A and B radiation blockers, additionally, its unique light scattering properties are highly exploited to create matte-finish cosmetics and personal care products. In the food industry, TiO₂ nanoparticles have been used as white pigment additives (E171) in confectionery, and dairy products. Conversely, the US FDA and EFSA have implemented restrictions on compound usage due to potential genotoxic impacts observed in animal studies [1–3].

The use of any chemical compound in food or personal care products that have direct contact with humans via oral, dermal or inhalation routes has always induced speculations regarding long-term toxic impacts. More so for the usage of heavy metals due to their high systemic retention properties leading to bioaccumulation along the food web. TiO₂, although it shows great benefits and functional applications, its impact on the ecosystem cannot be negated. The environmental risk caused by TiO₂ is prominently due to their release into the environment during production, use and disposal. TiO₂ particles tend to have varied toxic effects based on their size, surface coating and concentration. Their ability to accumulate within the aquatic ecosystem, photocatalytic degradation of pollutants and ROS generation can affect aquatic and soil microbial communities as well as the nutrient cycling processes [4–6].

Toxicological assessments of TiO₂ in humans indicated a significant impact on workers involved in the manufacturing and handling of TiO₂ nanoparticles. Inhalation of these particles led to respiratory issues [1, 7]. However, dermal exposure via cosmetic usage and oral exposure through food additives did not show any prominent impact but their potential accumulation in the gastrointestinal tract has raised serious concerns among the developed countries. In rodent models like rats and mice, TiO₂ particles have demonstrated pulmonary inflammation, altered hepatic gene expressions and liver enzyme levels, decreased sperm quality and impaired fertility [8–11]. Similarly, in cell culture models, TiO₂ particles induced apoptosis and necrosis mechanisms, triggered pro-inflammatory cytokines in addition caused DNA damage, and chromosomal aberrations, raising concerns about their potential genotoxic effects [12, 13]. In the Drosophila melanogaster model, TiO₂ particles demonstrated larval morphological deformities, delayed development, impaired learning and memory in adult flies and significant oxidative stress [14–16].

The conflict over the TiO₂ safety profile calls for a paradigm shift towards biocompatible and non-toxic alternatives. Biocompatible materials, by definition, do not cause an adverse immunological response in the body and are generally not associated with chronic toxicity concerns. The impact of surface modification on titanium oxide nanoparticle toxicity and the influence of biopolymer-coating are critical aspects in understanding their safety and applications in the biomedical field. Surface modification of TiO₂ nanoparticles plays a crucial role in determining their toxicity profiles. Unmodified TiO₂ nanoparticles have been associated with potential adverse health effects due to their ability to generate reactive oxygen species (ROS) upon exposure to ultraviolet (UV) light, leading to oxidative stress and cellular damage. However, surface modifications such as functionalization with biocompatible molecules or polymers can alter their physicochemical properties, reducing ROS generation and mitigating toxicity. These modifications may include coating TiO₂ nanoparticles with materials like silica, polyethylene glycol (PEG), or chitosan, among others [17, 18].

Currently, where environmental sustainability is a paramount ideology, the utilization of waste materials to create valuable resources has become a critical venture. Among these waste materials, crustacean shells stand out as a significant contributor to environmental pollution if not properly managed. With the global demand for seafood continuing to rise, so does the accumulation of discarded shells. The conversion of crustacean shell waste into chitosan represents a pivotal step towards sustainable resource utilization, mitigating environmental harm while creating valuable commercial opportunities. By repurposing discarded shells through advanced extraction processes, chitosan—a versatile biopolymer renowned for its biocompatibility and antimicrobial properties, is produced. This transformation not only diverts waste from landfills but also fuels innovation across industries [19]. Furthermore, chitosan finds applications in the food industry as a natural preservative, reducing food waste and enhancing food safety [20].

Our study aims to develop a chitosan-coated TiO₂ nanoparticle formulation that can be substituted in cosmetics like sunscreen and lotions instead of conventional UV blockers. The importance of animal testing in cosmetic toxicology is a crucial and inevitable step to assess the safety profile of the cosmetic product before its intended human use. Toward this, we aimed to fabricate a versatile, non-toxic cosmetic substituent and analyse its safety profile using cruelty-free alternative testing methods followed by many developed nations. The utilization of the Drosophila melanogaster (fruit fly) model for toxicity testing in cosmetics holds significant importance within the framework of regulations banning animal testing [21, 22]. The fruit fly model offers several advantages, including rapid reproduction, genetic tractability, and physiological similarity to humans in many aspects of biological processes. By utilizing Drosophila models, researchers can assess the potential toxicity of cosmetic ingredients and formulations in a manner that is both ethically sound and scientifically rigorous, ultimately contributing to the development of safer and more sustainable cosmetic products within the confines of regulatory guidelines [23].

While there have been limited studies regarding the use of chitosan-coated TiO₂ nanoparticles as cosmetic substitutes in sunscreens, lotions, etc, our study addresses this gap by successfully synthesizing these nanoparticles via the ionic gelation method. We further characterized the synthesized particles using XRD, FTIR, UV-Vis, and SEM techniques, providing a comprehensive understanding of their physicochemical properties. Additionally, we evaluated their safety profile utilizing the fruit fly model, shedding light on their potential applicability in cosmetic formulations.

2.1. Chemicals

D-glucose, agar-agar type 1 (Himedia), yeast extract, methylparaben, ortho-phosphoric acid, propionic acid, NADH, tetrasodium pyrophosphate (TSPP), phenazonium methosulphate (PMS), nitroblue tetrazolium (NBT), sodium phosphate monobasic (Himedia), sodium phosphate dibasic (Himedia), potassium chloride (KCl), 2’-7’dichlorofluorescin diacetate (DCFH-DA) (Sigma Aldrich), tris-HCl, glutathione reduced (GSH), 1-chloro-2,4-dinitrobenzene (CDNB), sodium carbonate, sodium hydroxide (NaOH), copper sulphate, Folin-Ciocalteau reagent, phosphorus pentoxide (Spectrochem), titanium dioxide ultrapure nanopowder, medium molecular weight chitosan (MMWC), acetic acid, sodium tripolyphosphate (STPP), bovine serum albumin (BSA), ethanol. Unless mentioned otherwise, all chemicals were purchased from SRL and were of analytical grade.

2.2. Fabrication of CS-TiO₂ nanocomposite

Chitosan was coated on titanium dioxide nanoparticles using an established method [24]. In a nutshell, 1% (w/v) MMWC was dissolved in acetic acid, and the contents were stirred until the chitosan was completely dissolved. 0.1% (w/v) sodium tripolyphosphate was used as a cross-linking agent, in which 1% (w/v) titanium dioxide nanoparticles were suspended and mixed until a milky white colloid was formed. The TiO₂ suspension was added dropwise to the chitosan solution under shaking conditions. The mixture was then placed on ice and sonicated for 30–45 minutes and subsequently centrifuged for 15 min at 4℃. The pellet was retained and washed twice to purge contaminants with distilled water. The sample was dried and used for further experiments.

2.3. Structural characterisation of the nanocomposite

2.3.1. X-ray diffraction

XRD provides information on the crystalline nature of the nanoparticle. The analysis was performed using a PANalytical make X-ray diffractometer (Malvern Panalytical, Worcestershire, UK), copper-K-alpha X-rays (λ) = 1.5406 Å, and the crystalline peaks were analysed in 2θ range of 20° to 80°.

2.3.2. Fourier transform infrared spectroscopy

The structural changes in CS by the incorporation of TiO₂ nanoparticles were determined with a Shimadzu, IRTRACER 100 spectrophotometer between 4000 − 400 cm^− 1 (Shimadzu Corporation, Kyoto, Japan).

2.3.3. UV-Vis spectroscopy

The UV absorption spectra were examined using a Shimadzu-make UV-vis spectrophotometer (Shimadzu Corporation, Kyoto, Japan). The spectrum was recorded between 200–800 nm.

2.3.4. Scanning electron microscopy

The structure and morphology of the nanocomposite were analysed using a High-resolution scanning electron microscope (HE-SEM), Thermoscientif Apreo S (Thermo Fisher Scientific, Waltham, USA) and the elemental analysis was carried out using an energy-dispersive spectrometer.

2.4. Moisture absorption

Moisture absorption (Ra) studies were performed based on a previously described method by Ta Q. et al with minor modifications [25]. The following equation was used to calculate moisture absorption, where Wo and Wn denote the initial and the final weight of the sample respectively. The assay was performed in duplicates.

$$\:Ra\:\left(\%\right)=100\:x\:(Wn-Wo)/W0$$

2.5. Moisture retention

The moisture retention (Rh) assay was performed using phosphorus pentoxide as a desiccant following the protocol established by Ta Q. et al with minor modifications [25].

$$\:Rh\:\left(\%\right)=100\:x\:\left(Hn\right)/\left(Ho\right)$$

The equation mentioned above was utilised to quantify the rate of moisture retention. Hn and Ho represent the final and initial weight of the sample respectively.

2.6. D. melanogaster strain and rearing conditions

Wild-type D. melanogaster (Oregon-K) was used in the study. The flies were raised and maintained in polystyrene flasks on a standard cornmeal agar at 25 ± 1℃ and approximately 60% humidity. The media was autoclaved, following which antifungals such as propionic acid, orthophosphoric acid and Tego (methylparaben in ethanol) were added to the corn-flour agar when the temperature of the media reached between 55 ℃ to 60 ℃.

2.7. Treatment of Drosophila with CS-TiO₂ nanoparticles

The CS-TiO2 nanoparticles suspended in water were formulated in the diet for toxicity assays. A stock suspension of 1% (w/v) CS-TiO₂ was sonicated for 30 minutes to get a homogenous dispersion. The experimental setup consisted of triplicates of control, and seven concentrations (10-, 50-, 100, 200-, 400-, 600-, and 800 µg/ml) of CS-TiO₂ dispersed in food for analysing the behavioural parameters of the flies and larvae and the survivability of the flies. An appropriate volume of CS-TiO₂ suspension from the stock was aliquoted to achieve the treatment concentrations, which were then added to each partially cooled treatment food. 5 ml of the food was transferred into a 25 ml polystyrene vial and left undisturbed overnight. For biochemical assays, three experimental concentrations (8-, 80-, and 800 µg/ml) were chosen based on the cosmetic purpose of the nanoparticle.

2.8. Lifespan experiment

The assay was carried out to evaluate the outcome of long-term exposure to different doses of CS-TiO₂ nanoparticles, following a previously described method [26]. Briefly, 30 male flies (n = 3) were maintained in control and the seven previously mentioned treatment doses of CS-TiO₂ until all the flies died, excluding the flies that escaped during transfer. To find out how many dead flies were inside, the vials were examined daily. GraphPad Prism 8 was used to create a survival curve and the statistical significance between the treated and control groups using the log-rank Mantel-Cox test.

2.9. Behavioural assays

2.9.1. Crawling assay

Following a previously defined protocol, the crawling ability of the larvae was determined [27]. Three 3rd instar larvae (n = 3) were collected from all the treatment groups. The larvae were allowed to crawl on the surface of a 2% agarose gel. The larval movements were captured in a video using a graph sheet for reference. The distance travelled by the larvae was determined by counting the number of grids they crossed in a minute.

2.9.2. Negative geotaxis assay

The climbing assay was performed by maintaining 30 adult male flies (n = 3) in the control and treatment groups. The food was changed once every three days and the flies were treated with the particles for two weeks. The difference in behaviour was compared with the control group. The assay was performed in triplicates and the number of flies that could climb beyond the 3 cm mark was noted and the values were represented in percentage [26].

2.10. Biochemical assays

For biochemical assays, flies and larvae were exposed to three different concentrations of CS-TiO₂ (8-, 80-, and 800 µg/ml) for 2 weeks. The concentrations were finalised based on the results obtained from behavioural assays and previous literature. Additionally, the cosmetic use of the particle had a factor in the concentration range selection [28]. The assays were performed in both larvae and adult flies, for which 20 adult male flies/third instar larvae were collected. The samples were homogenised with 0.1M sodium phosphate buffer for SOD, GST, and protein analysis while Tris buffer was used for ROS estimation. After centrifuging the homogenate for 10 minutes at 6000 rpm, the supernatant was stored and used for further experiments.

2.10.1. Estimation of protein content

With minor adjustments, a previously reported procedure was used to carry out the assay [29]. The protein content was measured in 3rd instar larvae and adult flies using BSA as a standard with 1mg/ml as the stock concentration. Lowry’s reagent A was added to 1ml of the homogenate and incubated in the dark for 10 mins. Following this reagent B was added and incubated in dark conditions for half an hour and the absorbance was read at 660 nm.

2.10.2. Estimation of reactive oxygen species production

ROS generation upon exposure to CS-TiO₂ was evaluated with the help of 2’,7’-dichlorofluorescein diacetate (DCHF-DA). It is a non-polar compound that upon interaction with ROS becomes a highly fluorescent compound. The assay was performed using a previously defined method with minor changes in both flies and 3rd instar larvae. Briefly, 20 male flies / 3rd instar larvae exposed to CS-TiO₂ were homogenised in 20 mM Tris buffer (pH7). The samples were placed on ice while homogenising and were centrifuged immediately. The supernatant was added to a 96-well plate in duplicates (n = 3). 0.025 ml of DCHF-DA was added and incubated for 1 hour. The fluorescence product was measured at the excitation wavelength of 488 nm and emission wavelength of 525 nm. The results' mean value was given in arbitrary units [30].

2.10.3. Estimation of superoxide dismutase (SOD) activity

A modified version of Ahmed et al.'s procedure was used to measure SOD activity [31]. The following were the contents of the reactions: 1 ml of TSPP (pH8), 0.1 ml of PMS, 0.3 ml of NBT, 0.2 ml of distilled water, 0.2 ml of sample and 0.2 ml of NADH which initiates the reaction. 1 ml of glacial acetic acid was used to stop the reaction after 1 min and absorbance was read at 560 nm. A unit of enzyme activity is denoted in terms of the concentration of the enzyme to inhibit 50 % cromogen production. The results are expressed as units/min.

2.10.4. Estimation of glutathione S-transferase (GST) activity

The technique developed by Siddique Y et al. was used to calculate the GST activity [32] with CDNB as a substrate. Briefly, 0.5 ml of sodium phosphate buffer (pH7), 0.15 ml of CDNB, 0.2 ml of reduced GSH and 0.05 ml of the homogenate were combined and the absorbance was recorded at 340 nm. The results were expressed as µmols/min/mg protein.

2.11. Statistical analysis

Statistical analyses were performed using GraphPad Prism 8. The mean values between the treatment and control groups were compared using ANOVA and Dunnett's multiple comparison test. A significance threshold of P < 0.05 was established, and all results are represented as mean ± standard error of the mean (SEM).

Chitosan-coated TiO₂ NPs (Fig. 1) exhibit enhanced biocompatibility compared to their uncoated counterparts, rendering them suitable for diverse biomedical applications. Chitosan’s presence improves TiO₂ NP dispersibility in biological fluids, diminishes cytotoxicity, and mitigates immune responses. Moreover, these nanoparticles can be tailored by targeting ligands or biomolecules, enhancing specificity and biocompatibility for applications such as targeted drug delivery or imaging. With precise control over particle properties during synthesis and assured compatibility with biological systems, chitosan-coated titanium dioxide nanoparticles emerge as versatile nanomaterial with promising prospects in various fields [33–39]. Several in vitro studies have assessed the potency of chitosan-coated titanium dioxide nanoparticles however, further investigation is warranted to evaluate their efficacy in vivo. Drosophila offers a valuable alternative to traditional animal testing methods, aligning with the principles of cruelty-free testing while still providing relevant and reliable toxicity data. Regulatory bodies such as the European Union's Cosmetics Regulation recognize and endorse the use of alternative testing methods, including Drosophila models, as part of efforts to phase out animal testing for cosmetics [21, 22].

3.1. Characterisation of CS-TiO₂ nanoparticles

3.1.1. X-ray diffraction

The XRD profile of the commercially procured TiO₂, pure chitosan and CS-TiO₂ nanoparticles are depicted in Fig. 2a, 2b and 2c. The pattern in Fig. 2a represents diffraction peaks at 2θ values of 27.36°, 36.01°, 39.19°, 41.17°, 43.96°, 54.26°, 56.56°, 62.69°, 64.18°, 68.77° corresponding to Miller indices (110), (101), (200), (111), (210), (211), (220), (002), (310) and (301) planes, pointing to the crystalline rutile phase of TiO₂ which is consistent with previous reports [40, 41]. The characteristic and crystalline diffraction peaks in the figure between 27° and 80° correlate with the tetragonal structure of pure rutile TiO₂ and do not indicate the presence of other diffraction peaks arising from impurities. The XRD pattern of chitosan, as shown in Fig. 2b, revealed a peak at 2θ position at 11.06° and another distinct peak at position 19.7° which are associated with the hydrated crystalline structure and the amorphous nature of chitosan. The diffraction pattern of CS-TiO₂ is exhibited in Fig. 2c and upon addition of chitosan to TiO₂ all the peaks corresponding to TiO₂ appeared with a very minute shift in the angle with 2 theta values of 27.58°, 36.63, 39.47°, 41.44°, 43.97°, 54.64° 56.93°, 63.6°, 64.1°, 69.29° while both the diffraction peaks of chitosan at positions 11.06° and 19.7° appeared rather fused [42]. The findings from the analysis demonstrate that the formation of the CS-TiO₂ nanoparticle is facilitated by the formation of intermolecular hydrogen bonding between the polymer and metal oxides. The crystallite size of the CS-TiO₂ nanoparticles was calculated using the Debye-Scherrer equation: $\:D=\:k\lambda\:/\beta\:\text{cos}\theta\:$ From the equation, D is the crystallite size measured in nm. k is the Scherrer’s constant and the value is 0.9, λ indicates the wavelength of X-rays. β indicates the full-width half maximum of the peaks and θ indicates the Bragg’s diffraction angle. The average crystallite size of the CS-TiO₂ nanoparticles calculated from the above equation is 106.3nm. It can be seen from the graph that the Braggs peak values are more intense at 2θ = 27.36°, 36.01° and 54.26° denoting the presence of larger crystallites while the peaks with lower intensity signify the presence of smaller crystallite sizes.

3.1.2. Fourier transform infrared spectroscopy

Figure 3a shows the FTIR spectra of pure TiO₂, pure chitosan and the CS-TiO₂ nanocomposite respectively. The analysis is useful to identify the functional groups present in the composite. The absorption peak that can be seen at 480 cm^− 1 is consistent with O-Ti-O and Ti-O-Ti bonding vibrations [43].

The FTIR spectrum of pure chitosan revealed vibrations around 3350 cm^− 1 and 3450 cm^− 1 which correspond to O-H and N-H bonds. The characteristic absorption band of chitosan emerged at 1656 cm^− 1 which indicates the presence of the N-acetyl group and the C-O group was present as a functional group around 1026 cm^− 1. The stretching vibration at 2934 cm^− 1 denotes the survival of the C-H bond in the chitosan spectrum [24, 44]. Between the regions of 1300 cm^− 1 and 1650 cm^− 1, the amide bands I, II, and III were found to be present. The successful incorporation of chitosan into the TiO₂ can be seen from the spectrum depicted in Fig. 3c. The hydrogen bonds around 3300 cm^− 1 can be attributed to the binding of TiO₂ in the amorphous region of chitosan also strengthening the hydrogen bonds around the region. The peaks around 1029 cm^− 1 can be correlated to the bending vibrations of Ti-O-C [45], and the stretching vibrations of C-O, NH₂, and OH groups around 3450 cm^− 1 and 2970 cm^− 1 are related to Ti-O-Ti bonds indicating that chitosan has attached strongly to TiO₂ [44]. Furthermore, a change in the vibration patterns of chitosan between 1406 cm^− 1 and 1576 cm^− 1 was observed, possibly due to the formation of hydrogen bonds between the amine group of chitosan and titanium [45].

3.1.3. UV-Vis spectroscopy

The UV-Vis spectra of pure chitosan, pure TiO₂, and CS-TiO₂ are depicted in Fig. 3b. The analysis was performed to find the optical properties of pure TiO₂, chitosan and CS-TiO₂. TiO₂ has a broad absorption range in the UV region of the electromagnetic spectrum between 200–400 nm because of its low photocatalytic ability under the visible light spectrum. In Fig. 3b, it can be seen that the absorption of TiO₂ peaked at about 360 nm and had a band gap value of 3.4 eV. In the absorption spectrum of chitosan, there was a wide absorption band in the UV region but the intensity of the absorption decreased as the wavelength progressed into the visible spectrum. The CS-TiO₂ band as illustrated in the figure shows that there were two sharp peaks, at 270 nm and 370 nm and another small peak at around 330 nm exhibiting a calculated bandgap value of 4.5eV, 3.3eV and 3.7eV respectively. The peaks around these wavelengths indicate that the composite can absorb all three types of UV light. The bandgap value of 4.5eV is surprisingly higher than the energy value of TiO₂ in bulk which is usually between 3.0 eV and 3.4eV which could be due to the spatial confinement of the electron pairs of both chitosan and TiO₂ and as a consequence, there is a widening of the bandgap [41]. The intensity of these absorption peaks, however, decreases in the visible light regions [46, 47].

3.1.4. Scanning electron microscopy

The SEM and EDX images are represented in Fig. 4. TiO₂ nanoparticles appeared as macroreticular spherical agglomerates while EDX analysis of the same confirms the presence of TiO₂ with an atomic ratio of titanium to oxygen of around 1:1 and a weight percentage of 74.77 and 24.68 per cent respectively. The morphology of chitosan appeared as layers and the elemental analysis revealed a composition of carbon, nitrogen, and oxygen with a weight percentage of 32.08, 14.73, and 49.26 per cent respectively. The SEM images of CS-TiO₂ showed larger agglomerates of TiO₂ nanoparticles on the chitosan matrix with only mild changes to the particle size. These agglomerations could lead to a reduction in the surface area of the nanoparticle. However, the downside to large cluster-size agglomerates is that it could affect the mechanical properties of the composite. The EDX analysis of CS-TiO₂ showed the presence of both the elemental molecules of chitosan and titanium dioxide with a composition of 5.88% carbon, 4.81% nitrogen, 36.66% oxygen, and 51.64% titanium. The weight percentage of carbon, nitrogen, and oxygen in chitosan was decreased in CS-TiO₂ suggesting that the reactions between chitosan and titanium dioxide lead to the formation of new functional groups [45, 48, 49].

3.2. Moisture absorption and retention

The moisture absorption ability was analysed for pure chitosan and CS-TiO₂ at a relative humidity of 43%. One of the appreciable physical properties of chitosan is that it is highly hygroscopic and capable of forming hydrogen bonds. The results represented in Fig. 5a, show that both chitosan and CS-TiO₂ were able to quickly absorb moisture in the first two hours which can be attributed to the high partial pressure of saturated vapour. The rate of moisture absorption neither increased nor decreased between the 6th and 24th hours after which there was a decline in the moisture sorption rate due to the saturation of moisture content within the chitosan molecules. While it is evident from other studies that chitosan has the inherent property to absorb moisture and act as a hydrant, titanium dioxide, being a heavy metal cannot absorb moisture. Furthermore, it has been previously reported that TiO₂ can impede the absorption rate of the polymer used for its coating. However, this study's results suggested no marked difference between the moisture sorption rate of pure chitosan and CS-TiO₂ and that TiO₂ nanoparticles did not limit chitosan’s ability to absorb moisture [25, 50, 51]. Similarly, water retention indicates the rate of evaporation of water and the results of the moisture retention assay, as depicted in Fig. 5b, revealed that both pure chitosan and CS-TiO₂ were able to retain the water in their matrix. Additionally, humidity and temperature also play a major role in the amount of water initially absorbed as well as the amount of water that can be retained.

3.3. Lifespan experiment

Drosophila melanogaster has been established as a well-suited model for toxicological investigations. The initial screening for the chronic oral toxicity of CS-TiO₂ and optimisation of the exposure period was investigated by performing a survival assay. The lifespan curve for the treated population is represented in Fig. 6. The survivability of the flies upon exposure to different concentrations of CS-TiO₂ was monitored and compared against the control group. The control flies showed a half-life of approximately one and a half but a decline in the survivability of the flies exposed to CS-TiO₂ particles with concentrations ranging between 200 µg/ml and 800 µg/ml was noted after 25 days of exposure. To better understand the effects of CS-TiO₂ on the life cycle of Drosophila, the behavioural and biochemical parameters of both the larvae and flies were investigated. Based on the findings of this assay, the exposure period for the adult flies to CS-TiO₂ was fixed as two weeks for both behavioural and biochemical assays.

3.4. Behavioural assays

3.4.1. Crawling assay

In the larval stages of development, Drosophila progresses through the contraction of the body wall muscles, synchronised by a central pattern generator and is responsible for the rhythmic firing of motor neurons in the larvae’s ventral ganglion. These firings generate the peristaltic contractions of the body wall muscles, resulting in larval movement [52]. The larval crawling assay has been a well-grounded analysis for studying any deviations in the crawling abilities and also to comprehend the effects of the xenobiotics on their behaviour. From the larval crawling assay results depicted in Fig. 7, it can be seen that there was a mild decrease in activity of the larvae treated with 200 µg/ml of CS-TiO₂. At the same time, other treatment groups did not exhibit any noticeable changes in the crawling activity. Sood K., et al [53] investigated the toxicity of graphene oxide (GO) and zinc oxide (ZnO) nanoparticles on Drosophila. The larvae treated with GO demonstrated a non-monotonic trend in their crawling ability and a similar pattern of results was observed in this study.

3.4.2. Negative geotaxis assay

Negative geotaxis is one of the robust methods to assess the behaviour of adult male flies to assess any variations arising in the fly upon exposure to any xenobiotics or alterations in the organism's genetic makeup. The presence of any dysfunction in the locomotion post-treatment with CS-TiO₂ was analysed using this assay. Adult male flies were exposed to seven different concentrations (10-, 50-, 100-, 200-, 400-, 600-, 800 µg/ml) of CS-TiO₂ nanoparticles for two weeks and the results are represented in Fig. 8. The assay revealed that in the control group, 92.59% of flies were able to traverse the 3 cm mark. Further, a marginal decline in the activity was observed between the control group and the flies treated with concentrations of 50-, 100-, and 600 µg/ml of CS-TiO₂. The treatment group with 50 µg/ml of CS-TiO₂ showed that 85.93% of flies were able to cross the 3cm mark, while the treatment with 100- and 600 µg/ml of CS-TiO₂ 87.04% and 85.56% of flies were able to cross the demarcation. Interestingly, the highest concentration, 800 µg/ml, did not exhibit any significant difference when compared against the control group [54–56]. Although an ebb in the activity was discernible, there was only a minor disparity in the percentage of flies displaying decreased activity. This can be rationalised using the hormesis theory based on a non-linear dose-response relationship. It stated that an organism can exhibit stimulatory reactions at low dosages and inhibitory responses at greater doses [57, 58].

The toxicity of TiO₂ nanoparticles relating to developmental toxicity and DNA damage in Drosophila was previously tested by Sario S., et al [28] who reported that TiO₂ nanoparticles, at concentrations 8-, 40-, 80-, and 800 µg/ml did not cause any adverse effects in the organism. These concentrations were fixed based on the cosmetic applications of TiO₂. Although reported as non-toxic, in the current study we decided to test a panel of concentrations for the CS-TiO₂ nanoparticles, using survival and behavioural assays, to analyse if the particles caused any untoward effects in the model organism. While there were considerable changes in the behavioural analysis for certain concentrations, the variations were minimal and did not drastically alter the behaviour or cause any adverse toxicity in either larvae or adult flies. Therefore, the established concentrations of 8-, 80-, and 800 µg/ml of CS-TiO₂ were chosen to study the biochemical parameters to check if the nanoparticles caused any modifications at the molecular level.

3.5. Biochemical assays

3.5.1. Estimation of protein content

The protein content in adult male flies and third-instar larvae was estimated using Lowry’s method. The results for the protein content in both adult flies and larvae are depicted in Fig. 9a and 9b. From the results, it is evident that there is no statistical significance in the protein content present in adult flies and larvae when examined against the control group. Nonetheless, there was a trend of increase in the protein content in the larvae treated with dietary 8- and 80 µg/ml CS-TiO₂ upon scrutinising with the control. The protein content in flies also did not show any statistical significance, however, there was a slight decrease in the total protein content in flies treated with 80-, and 800 µg/ml CS-TiO₂ when compared to the control group. The results, consequently suggest that CS-TiO₂ nanoparticles did not elicit any untoward reaction in both flies and larvae implying the non-toxic nature of the particle.

3.5.2. Estimation of ROS production

ROS is one of the primal factors that influence the toxic effects of the nanoparticles. In the toxicological studies, the estimation of ROS generation triggered by the xenobiotics is a well-valued assay. With that established, the intracellular ROS levels in fly whole body homogenate and in the larval homogenate were estimated using DCFH-DA, a widely utilised fluorogenic probe to monitor the intracellular redox process. The oxidation of DCFH-DA to 2’- 7’ dichlorofluorescein (DCF) is a result of an enzymatic activity, where the cellular esterase cleaves the acetyl groups. The levels of intracellular ROS measured in both the larvae and the flies are represented in Fig. 10a and 10b. The larval samples exhibited a distinct diminution in the basal level of ROS in all three tested concentrations. In contrast, the levels of ROS in the adult flies did not alter considerably. Chitosan is notable for its antioxidant properties among the other versatile biological properties. The rationale behind the reduced ROS levels in the larvae could be accredited to the inherent antioxidant nature of chitosan. Similar results were reported by Kumar, P. P., et al. who studied the role of low molecular weight chitosan in subduing the toxicity of acrylamide [59]. Concurrently, there was no significant increase or decrease in ROS levels in the adult flies. This variation between the larval and adult ROS levels can be associated with the larval-to-adult transition as metamorphosis activates unique cellular events and initiates specific molecular pathways contributing to this heterogeneity in ROS generation [60, 61].

3.5.3. Estimation of SOD activity

Nanoparticle-mediated toxicity has become a subject of interest as it is being used daily in food, medicine and other consumer products. Oxidative stress is a major concern, as various studies have reported that nanoparticles trigger the production of ROS. SOD is the first antioxidant that gets activated to neutralise the superoxides among the other antioxidant enzymes that are activated in response to an increase in ROS levels. It converts high-reactive superoxides to less-reactive hydrogen peroxide which again is reduced to water by catalase (CAT). The present study evaluated the levels of SOD in 3rd -instar larvae and adult male flies post-treatment with CS-TiO₂ and the results are presented in Fig. 11a and 11b. The enzyme activity was inconsequential in both larvae and the adults supplemented with dietary CS-TiO₂ suggesting that the particle did not induce oxidative stress. There have been reports from previous studies that TiO₂ nanoparticles have had a negative impact on the survival rate, developmental abnormalities and induction of oxidative stress in Drosophila [62, 63]. However, the results from this study show that chitosan outplays its antioxidant role and, in some way, alleviates the effects of TiO₂.

3.5.4. Estimation of glutathione S-transferase (GST) activity

GSTs are a highly diverse superfamily of enzymes whose primary function is detoxifying chemical compounds of both endobiotic and xenobiotic origin. They are also an important stress biomarker and their activity is triggered when the organism comes in contact with xenobiotics, environmental pollutants etc. Flies and larvae were tested to check if the activity of the GST enzyme was triggered after dietary exposure to CS-TiO₂. The results are presented in Fig. 12a and 12b corresponding to larvae and flies respectively. It was noted from the results that there was an increase in the level of the enzyme in the larvae fed with 800 µg/ml of dietary CS-TiO₂ when compared to control. When flies' enzyme activity was examined, there was no noticeable distinction between the treatment and control groups.

While the results from ROS analysis did not indicate any untoward effect in flies and larvae, the increase in GST activity in the larvae can again be correlated to the difference in the physiology of larvae and adult flies. Further, GST is a sensitive enzyme that can get triggered easily and several mechanisms were proposed to unravel the effect of nanomaterials and metal on GST activity but there is no perspicuous explanation for the increase or decrease in GST activity [64–66]. Owing to the particle’s nature as a heavy metal, it will be perceived as a xenobiotic by the organism, triggering a set of stress mechanisms to remove the particle from its system. Consequently, the variations shown in the biochemical and behavioural tests are the outcome of a series of responses set off by the imposed stress and do not purportedly represent particle toxicity.

TiO₂ nanoparticles are one of the five leading nanoparticles utilized in multiple industries for their diverse functionalities. Developments in various societal domains have elevated the potential for exposure to TiO₂ NPs. Sourced from the exoskeletons of crustaceans and insects, chitosan is prized for its natural composition and exceptional biocompatibility, while also being cost-effective thereby reducing the financial burden on the users. Since surface properties could influence the inherent behaviour of a material, we utilised chitosan as a surface modifier for TiO₂ NPs. Therefore, to determine the effects of the coated particle and for the obvious reasons outlined in the preceding section, Drosophila was used to evaluate the toxicological characteristics of the particle, even though the planned application of this study is to employ CS-TiO₂ for cutaneous applications. To the best of the authors’ knowledge, this is the first study to evaluate the toxicity of CS-TiO₂ nanoparticles on the Drosophila model. The lifespan of the flies, behavioural patterns and biochemical changes upon exposure to the nanoparticles were assessed. To summarise, no gross changes were observed in the crawling and climbing activities of the larvae and adult fly respectively. Though there were mild fluctuations detected in biochemical parameters, they did not affect the flies or the larvae adversely and this can be supported by the results from survival and behavioural analysis. Though Drosophila is considered an ideal model for toxicological studies, investigation of the CS-TiO₂ nanoparticle toxicity on higher-order organisms is called to validate the toxicity of the particle to be utilised as a replacement in consumer products.

Conflict of interest – The authors declare no competing interest

Ethical approval - Not applicable

Consent to participate - Not applicable

Consent to publish - Not applicable

Data availability statement – All data sets used and/or generated in this work are obtainable from the corresponding author upon reasonable request.

Funding – The authors did not receive any funding for carrying out the study.

CRediT authorship contribution statement

Sharine Priscilla: Conceptualization, Methodology, Data curation, Writing - original draft, Writing - review & editing, Software, Validation. G. Devanand Venkatasubbu: Conceptualization, Methodology & Validation. Sahabudeen Sheik Mohideen: Conceptualization, Methodology, Validation, Writing - review & editing, Supervision.

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgement

The authors thank SRM Central Instrumentation Facility (SCIF) for the SEM facility and SRM Nanotechnology Research Centre (NRC) for providing XRD, FTIR, and UV-vis spectroscopy facilities.

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Chitosan-coated titanium dioxide nanoparticles: Fabrication, characterisation and toxicological evaluation in Drosophila melanogaster

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Chemicals

2.2. Fabrication of CS-TiO2 nanocomposite

2.3. Structural characterisation of the nanocomposite

2.3.1. X-ray diffraction

2.3.2. Fourier transform infrared spectroscopy

2.3.3. UV-Vis spectroscopy

2.3.4. Scanning electron microscopy

2.4. Moisture absorption

2.5. Moisture retention

2.6. D. melanogaster strain and rearing conditions

2.7. Treatment of Drosophila with CS-TiO2 nanoparticles

2.8. Lifespan experiment

2.9. Behavioural assays

2.9.1. Crawling assay

2.9.2. Negative geotaxis assay

2.10. Biochemical assays

2.10.1. Estimation of protein content

2.10.2. Estimation of reactive oxygen species production

2.10.3. Estimation of superoxide dismutase (SOD) activity

2.10.4. Estimation of glutathione S-transferase (GST) activity

2.11. Statistical analysis

3. Results and discussion

3.1. Characterisation of CS-TiO2 nanoparticles

3.1.1. X-ray diffraction

3.1.2. Fourier transform infrared spectroscopy

3.1.3. UV-Vis spectroscopy

3.1.4. Scanning electron microscopy

3.2. Moisture absorption and retention

3.3. Lifespan experiment

3.4. Behavioural assays

3.4.1. Crawling assay

3.4.2. Negative geotaxis assay

3.5. Biochemical assays

3.5.1. Estimation of protein content

3.5.2. Estimation of ROS production

3.5.3. Estimation of SOD activity

3.5.4. Estimation of glutathione S-transferase (GST) activity

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

2.2. Fabrication of CS-TiO₂ nanocomposite

2.7. Treatment of Drosophila with CS-TiO₂ nanoparticles

3.1. Characterisation of CS-TiO₂ nanoparticles