Building a Safe Future: The Biological Investigation of Doped ZnO Nanocrystals-Based Nanocomposites

Abstract

The chapter presents the outcomes of an extensive investigation of nanosafety concerning various nanocomposites incorporating doped nanoparticles. These findings unveil a diverse range of properties these materials exhibit, including enhanced biocompatibility, unique optical and electronic features, and targeted therapeutic capabilities. Nonetheless, the study underscores the necessity for a cautious approach due to the intricate interactions between doped nanomaterials and biological systems. Optimizing these beneficial properties and mitigating potential risks associated with their utilization necessitate meticulous synthesis, characterization, and evaluation of hybrid nanocomposites. Depending on the dopant used, it has bactericidal advantages but toxic effects, so studying which doping element is essential for developing new efficient and safe products. Therefore, the significance of interdisciplinary collaboration among researchers from diverse fields such as materials science, biology, medicine, and toxicology is emphasized for a comprehensive assessment of the safety and efficacy of these nanomaterials across various applications.

1. Introduction

In the expansive realm of nanotechnology, inorganic nanocomposites have emerged as central players, offering avenues for technological advancement across diverse domains, from electronics to medicine. Within this context, nanoparticles have garnered significant attention as pivotal constituents, showcasing properties ripe for synergistic enhancement and amalgamation within nanocomposites.

Nanotechnology stands at the forefront of addressing contemporary societal challenges [1]. Leveraging the high surface area-to-volume ratio of nanomaterials, this dynamic field bestows distinctive mechanical, magnetic, electrical, and optical attributes upon these materials, facilitating the development of innovative products [2, 3]. Among the array of materials, nanocomposites have garnered considerable interest from academia and industry, owing to the notable enhancements achieved through the synergistic amalgamation of constituent materials [4].

Inorganic nanocomposites epitomize a unique amalgamation of properties stemming from their components. By amalgamating nanoparticles of diverse materials or functionalities, myriad new application avenues emerge. These nanocomposites, spanning optical, electronic, mechanical, and biological realms, can be tailored to meet many needs and applications. Nonetheless, formidable challenges persist. Precise synthesis, stability, toxicity, and scalability demand comprehensive attention to ensure these materials’ safe and practical progression for commercial and biomedical applications.

In this context, inorganic nanocomposites have assumed a prominent role among biomaterials [5], outperforming their microparticle counterparts [6]. These materials find application across diverse sectors, including medicine, textiles, cosmetics, agriculture, food packaging, optoelectronics, semiconductor devices, and catalysis [7, 8]. However, comprehensively addressing synthesis, stability, toxicity, and scalability concerns is imperative to ensure these materials’ safe and practical progression for commercial and biomedical applications [9, 10].

One avenue to advance in the developing frontiers of nanotechnology concerning inorganic nanocomposites involves exploring novel doping strategies with noble metals and examining their properties and applications. Doping presents a viable avenue for developing increasingly sophisticated, versatile, and biocompatible nanomaterials [11]. Characterization tests, luminescence studies, and photocatalysis assessments are indispensable for uncovering potential applications of new nanocomposites (NCPs). In contrast, biocompatibility tests are crucial to ensuring the safe application of NCPs, particularly in nanomedicine [12].

Biocompatibility denotes a material’s ability to elicit an appropriate response in a given model, whether in vivo or in vitro, under specific exposure conditions, such as ingestion, inhalation, or dermal exposure [13]. Evaluating the biocompatibility of new NCPs necessitates rigorous bioassays capable of demonstrating both cell viability rates and the nanotoxic mechanisms of the material [14, 15]. Preceding studies involving mathematical modeling, in vitro cell models encompassing both 2D and 3D models, and in vivo bioassays serve as essential prerequisites for evaluating the biocompatibility of new NCPs [16, 17].

In vivo bioassays for assessing the biocompatibility of nanomaterials may employ traditional murine models; however, alternative models are gaining traction to minimize and refine animal experimentation [18]. The fruit fly Drosophila melanogaster is a promising nanotoxicological model organism due to its well-established characteristics. With its short life cycle and well-defined phases, Drosophila facilitates rapid experimentation, enabling the monitoring of substance effects across all life stages and multiple generations. Despite its relatively simple body organization, Drosophila exhibits homology with the human body’s organizational system [19]. Approximately 70% of genes implicated in human diseases are conserved in Drosophila, rendering it an invaluable tool for studying various pathologies, thus significantly contributing to scientific advancement and societal benefit [20]. Utilizing Drosophila as a tool for studying NCPs accelerates result acquisition, enhances understanding of nanotoxicity, and promotes research accessibility on these emerging substances, thereby fostering the development of safe applications in industries such as medicine and food.

Within the myriad perspectives for approaching the study of nanocomposite synergism, this chapter endeavors to explore the synergistic marvels of novel inorganic nanocomposites, with a primary focus on those based on nanoparticles and composites doped with transition metals. Subsequent sections will delve into the advantages and challenges of these captivating materials, unveiling their potential applications and biocompatibility studies using the Drosophila model. Thus, contributing to promising pathways shaping the future of nanotechnology, we aim to delineate future directions, offering valuable insights for researchers intrigued by the captivating realm of nanostructured materials.

2. Inorganic nanoparticle-based nanocomposites

2.1 Nanocomposites based in silver-doped nanoparticles

Doping of nanomaterials is advantageous as it offers a range of technological and biotechnological applications, such as the controlled modulation of physical and chemical properties in the nanomaterial according to specific application needs. Furthermore, doping allows for better adjustment of magnetic properties, facilitates surface functionalization, making them more biocompatible, enhances catalytic activation, and increases nanomaterials’ chemical and mechanical stability [21, 22, 23, 24]. While the doping technique in nanomaterials is highly advantageous, it must be executed carefully, especially when the goal is synthesizing nanocomposites for biomedical applications. Depending on the doping concentration, nanoparticle size, morphology, and crystallization, they can potentially make them toxic [22, 23, 25, 26]. Therefore, it is essential to carefully assess the risks associated with using these nanoparticles to ensure their safety in future applications.

Silver (Ag) NCPs have been widely studied and applied in various fields due to their unique properties. Ag, when incorporated into nanocomposites, is well-known for its antimicrobial properties, high electrical conductivity, improvement of thermal and mechanical stability of polymeric materials, and excellent optical properties. These properties ensure applications of Ag in various areas, such as sensors, optoelectronic devices, and catalysts [27]. Ag nanocomposites also find applications in flexible circuit printing and transparent electronic devices, as well as in medical applications such as wound dressings, antibacterial biofilms, and anticancer therapies [28, 29, 30, 31].

The Ag-doped ZnO/AgO/TiO₂ nanocomposite (NCPs) is an example of a nanomaterial enhanced after doping with Ag. In addition to exhibiting enhanced photocatalytic capacity [32], this nanocomposite has demonstrated antioxidant, anti-inflammatory, and wound-healing properties for skin lesions [33]. We synthesized novel NCPs with Ag-ZnO/65%AgO and applied them in vitro against Leishmania braziliensis. It was possible to observe a reduction in intracellular amastigotes of L. braziliensis and low cytotoxicity in human peripheral blood mononuclear cells [34]. However, the toxicity of these NCPs is dependent on the concentration of AgO in the NPs both in in vitro tests [35] and in vivo experiments in animal models of Drosophila melanogaster (unpublished data) and mice [35].

In Drosophila melanogaster, we observed a failure in the eclosion process with a concentration-dependent progression in flies exposed to different Ag-doped ZnO/AgO/TiO₂ (Figure 1). The interaction of these NPs with cells can lead to oxidative stress, inflammation, and even cell death. One of the main toxicity mechanisms of Ag NPs is the generation of reactive oxygen species (ROS), which can cause mitochondrial dysfunction and stress, among other phenomena. Consequently, this may impair essential complexes in the electron transport chain, resulting in an energy deficit for the cells. The imbalance in energy balance can significantly decrease the eclosion rate in fruit flies [36]. In this study, the redox imbalance might be induced explicitly by Ag⁺ ions [26, 37, 38], potentially impairing mitochondrial energy function in excess. Consequently, this may lead to a deficit in energy production, resulting in insufficient energy for the eclosion process in flies.

Figure 1.

Exposure to Ag-doped ZnO/AgO/TiO₂ NCPs induces a failure in the eclosion process in Drosophila melanogaster. Animals developed in a medium containing ZnAg and its combinations at concentrations of 0.5 and 1 mg/ml failed to undergo eclosion. The emergence failure resulting from treatment with ZnAg is evident in panel A. In panels B, C, and D, it is observed that the combination with TiO₂ (a less toxic phase: rutile-brookite) failed to reverse this effect, with the animals capable of initial puparium rupture but unable to emerge ultimately. The scale bar indicates a size of 2 mm.

In nanomedicine, simonkolleite (SM) nanocrystals possess both antitumor and osteogenic properties [39]. To enhance and broaden their application properties, we synthesized SM doped with different concentrations of Ag. Subsequently, we tested the in vivo biocompatibility of this new nanomaterial in Drosophila. Our results showed toxicity for the 10%Ag-doped SM, as the animals exhibited high larval and pupal lethality rates. We also observed a high rate of depigmentation in adult animals exposed to a concentration of 0.120 mg/mL (Figure 2), which was similarly observed in animals exposed to the Ag-doped ZnO/AgO/TiO₂ NCPs (data not shown). In this regard, we suggest that cuticular depigmentation is a toxic effect mainly induced in fruit flies by the excess of Ag ions from the NPs after undergoing oxidation. We observed this effect in fruit flies exposed to SM:Ag NCPs, Ag-doped ZnO/AgO/TiO₂, and ZnO:Ag (Figure 3) only when exposed to concentrations above 0.1 mg/mL (see Figures 2 and 3).

Figure 2.

Exposure to the high doping of silver-doped zinc oxide (Ag-ZnO) results in cuticular pigmentation loss in Drosophila melanogaster (n = 100). In panels A and B, representative images of animals from the control group and the lowest concentration of ZnAg (0.0125 mg/ml) are shown, respectively, displaying standard cuticular pigmentation of wild-type animals. In panel C, robust cuticular depigmentation is observed due to the high doping of Ag-ZnO exposure. Scale bars A, B, and C = 3 mm.

Figure 3.

Exposure to the high doping of silver-doped simonkolleite (Ag-SM) results in cuticular pigmentation loss in Drosophila melanogaster (n = 100). In panel A, representative images of animals from the control group are shown, displaying standard cuticular pigmentation of wild-type animals. Animals exposed to the lower 0.015 mg/ml concentration show normal cuticular pigmentation (B-B′). However, at the highest concentration, cuticular depigmentation is observed. Scale bars A, B, and C = 3 mm. Scale bars A′, B,′ and C′ = 1 mm.

As previously described, the redox imbalance is considered the primary mechanism of toxicity of ZnOAg and AgO NPs. These undergo oxidation by oxygen (O₂), generating Ag⁺ ions that induce increased formation of ROS, with consequent distress responses and cell death [37, 38, 40, 41]. Here, we demonstrate that fruit fly larvae exposed to SM:Ag NCPs at concentrations above 0.1 mg/mL exhibit an increased hydrogen peroxide (H₂O₂) formation rate, indicating redox imbalance (Figure 4). Thus, our results support the hypothesis that the toxicity observed in the NCPs may be attributed to the presence of Ag. This toxicity may also be concentration- and dose-dependent.

Figure 4.

Exposure to low doping of silver-doped simonkolleite increases mitochondrial H₂O₂ in the larval fat body of Drosophila melanogaster (n = 6 per group). In panel “A,” representative images of the redox homeostasis pattern (405/488 nm) of the roGFP2-Orp1 probe in the larval fat body in different groups are shown. An increase in oxidation is observed in the group exposed to 0.120 mg/ml compared to the control group from the ratiometric image. Scale bar = 150 μm. In panel “B,” the pattern of pro-oxidative redox homeostasis is observed for animals exposed to 0.120 mg/ml of Ag-SM compared to other doping concentrations, indicating an increase in mitochondrial H₂O₂. The graphs present mean ± standard error of the mean, with p-value <0.0001 for the one-way analysis of variance (ANOVA) test.

Therefore, Ag-NPs induce an energy deficit and a phenotype of cuticular melanin loss in Drosophila. These conditions can be explained by the exact competition mechanism between Ag and Cu in CTR transporters (Figure 5), which leads to distress, compromised energy balance, and low myelin synthesis via glutathione depletion [36, 42, 43, 44].

Figure 5.

Proposed mechanism of cuticular depigmentation in Drosophila mediated by exposure to silver-doped nanoparticles. Silver-doped nanoparticles, upon reaching the extracellular space, undergo different processes of ionic dissociation, resulting in the formation of Ag⁺ ions. The increased concentration of Ag⁺ ions leads to competition with Cu²⁺ ions for intracellular copper transport channels (CTR) in the plasma membrane. The decrease in intracellular influx of Cu²⁺ ions due to competition with Ag⁺ ions prevents the assembly of copper-dependent enzymes such as tyrosinase, which converts tyrosine into dihydroxyphenylalanine (Dopa). Subsequently, Dopa is converted into Dopa melanin, which is responsible for cuticular black pigmentation. Thus, the high concentration of Ag⁺ ions inhibits the cuticular pigmentation process.

Copper (Cu) is a chemical element essential for cellular homeostasis, serving as a cofactor for cellular metabolism processes and the synthesis of biological compounds. It is no wonder that the CTR transporter family is present in various groups, including mammals, playing an essential role in Cu homeostasis [45]. The redox potential of this element is utilized for enzymatic activity, such as Cytochrome c oxidase (COX), mainly in mitochondrial inner membrane complex VI. Additionally, it participates as a cofactor in cellular protection against oxidative stress via the activity of superoxide dismutase 1 (Cu/Zn SOD1). It has a crucial role in adequately absorbing iron in the small intestine and its incorporation into hemoglobin molecules [46, 47, 48]. This transition metal also acts in enzymatic catalysis for the synthesis of compounds such as dopamine-beta-hydroxylase (DBH) and lysyl oxidase (LOX), thus playing a crucial role in the nervous system and collagen synthesis [48, 49]. Therefore, the high concentration of Ag⁺ ions in nanoparticles may inhibit the cuticular pigmentation process in Drosophila, as seen here, disrupt intracellular copper homeostasis, and interfere with crucial cellular processes [49]. Therefore, we recommend the cautious use of Ag-doped NCPs.

2.2 Nanocomposites of the doped ZnO nanocrystals

Nanocomposites based on zinc oxide (ZnO), whether pure or doped, offer a wide range of functionalities and hold promise for various applications in electronics, photonics, energy, environmental remediation, and biomedicine, driven by their unique properties and synthesis versatility. Additionally, zinc oxide nanoparticles (ZnO NPs) are widely used in the production of creams and sunscreens due to their whitening capacity and high absorption of ultraviolet (UV) light [50, 51, 52]. Doping with transition metals has been extensively employed in ZnO NPs. This technique enables the incorporation of elements into the crystal lattice of ZnO, reducing undesired effects and adjusting the advantageous properties of the nanomaterial.

Due to their biodegradability capability, ZnO nanoparticles’ biological applications extend to fluorescent probes, biosensors, and drug delivery systems. Although they are generally considered biocompatible [53], the most proposed toxicity mechanisms of ZnO nanoparticles involve pH-dependent release [54] of Zn²⁺ ions and the production of reactive oxygen species (ROS). Therefore, the biocompatibility of this substance is deemed uncertain, underscoring the importance of studies regarding the biological impacts of these nanoparticles [55, 56, 57].

In this perspective, we synthesized and characterized various ZnO nanoparticles doped with different transition metals, such as gold (Au), iron (Fe), nickel (Ni), magnesium (Mg), copper (Cu), and calcium (Ca) [58]. Our findings from the optical absorption spectra of doped NCs demonstrated the successful incorporation of metal ions into ZnO NCs. The dopant element was selected considering the expected new functionality and with a focus on increased biocompatibility. For example, Sajjad [59] demonstrated that doping ZnO NPs with copper (Cu) positively influenced the band gap of ZnO. This modification favored the photocatalytic activity of the nanomaterial, resulting in improved bactericidal activity of the substance.

Practical improvements in the properties of ZnO nanoparticles were also observed when doped with iron (Fe). The presence of iron delays the release of Zn²⁺, contributing to the increased nanosafety of the substance [60]. In this sense, doping favors enhancing NPs properties and improved biocompatibility. Therefore, continuous research efforts in materials synthesis, characterization, device integration, and doping methodologies can further unlock their applications in future technologies with higher biocompatibility.

After synthesizing and characterizing pure ZnO and nickel-doped transition metal nanoparticles (ZnO:Ni) (Figure 6A), we investigated their biocompatibility in Drosophila melanogaster. From the first larval stage (L1), treatments were administered to the animals orally by adding them to the standard culture medium at final concentrations of 0.05, 0.10, 0.25, and 0.50 mg/mL, in addition to the control group. By closely monitoring the developmental stages of the animals from the larval stage to adulthood, we identified the impacts of the ZnO:Ni at each life cycle phase (Figure 6B). Remarkably, animals exposed to pure ZnO exhibited high larvae and pupal mortality (Figure 7A and B, respectively), developmental delays, and incomplete post-embryonic development. The ZnO nanoparticles showed high toxicity in a concentration-dependent manner. Moreover, based on our experiments, we hypothesize that any ZnO-based NCPs presents some toxicity at concentrations equal to or greater than 1 mg/mL, regardless of the dopant element.

Figure 6.

In vivo evaluation of the effects of exposure to pure ZnO and nickel-doped ZnO (ZnO:Ni) NCs. In A, the nanoparticle’s structure with and without doping is observed. In B, the life cycle of Drosophila melanogaster is observed, which serves as the basis for the exposure method and subsequent monitoring of the development stages. In C, it is noticeable that only animals exposed to 0.25 mg/mL present several pupae formed per day, similar to the control; exposure to other concentrations causes delays during the larval stage. These concentrations also result in a reduction in the larval lethality rate (D) and, consequently, in a reduction in pupal viability (E). Furthermore, they decrease the hatching rate (F) after the pupal stage.

Figure 7.

In vivo assessment of the effects of exposure to ZnO, ZnO:Au, ZnO:Cu, ZnO:Ni, ZnO:Fe, and ZnO:Mg NCPs. In A, it is notable that both investigated concentrations of ZnO cause larval lethality and, consequently, a decrease in pupal viability (B). However, this scenario can be modified through doping. Among the different dopings evaluated, ZnO:Au and ZnO:Fe proved to be the most promising, being biocompatible not only in the control group but also compared to the pure ZnO group.

However, the adverse effects were significantly reduced when the animals were exposed to 0.25 mg/mL of ZnO:Ni, as those treated with concentrations equal to or greater than 0.50 mg/mL demonstrated a delay of up to 6 days in post-embryonic development (Figure 6C) and larval and pupal mortality rates exceeding 50% when exposed to a concentration of 1.0 mg/mL (Figure 6D), characterizing high toxicity compared to the control.

Additionally, the pupal mortality and emergence rates were nearly zero when exposed to 2 mg/mL of ZnO:Ni. Thus, the data obtained so far indicate that the presence of Ni in ZnO nanoparticles may even increase in vivo biocompatibility, but only at low concentrations.

Among the studies conducted to evaluate the biological activity of ZnO:Ni, tests were implemented to assess antimicrobial activity using the broth microdilution technique against Staphylococcus aureus (ATCC 25923) and Staphylococcus epidermidis (ATCC 31488) species. The initial and final Ni doping concentrations showed the highest minimum inhibitory concentration (MIC) obtained in the tests (500 μg/mL). In contrast, nanoproducts with intermediate doping concentrations of Ni-doped ZnO exhibited improved performance (250 μg/mL). The antimicrobial activity of Ni-derived nanoproducts against pathogenic microorganisms has been reported in the literature, which has even pointed out synergism with increased antimicrobial activity when associated with ZnO.

Contrary to the in vivo results found for ZnO:Ni, animals fed a culture medium containing ZnO nanoparticles doped with Cu (ZnO:Cu) showed better performance in post-embryonic development [22]. This scenario was also observed when conducting bioassays in Drosophila exposed to ZnO doped with Fe (ZnO:Fe) or ZnO doped with Au (ZnO:Au) at concentrations of 0.25 and 0.50 mg/mL (Figure 7).

Animals exposed to 0.25 and 0.50 mg/mL of ZnO:Au and ZnO:Fe showed larval lethality rates similar to those of the control and demonstrated higher pupal viability than those exposed to pure ZnO NPs. Additionally, we found that ZnO:Au and ZnO:Fe NCPs exhibited better biocompatibility when compared to ZnO:Ni and ZnO:Cu at the concentration of 0.50 mg/mL (Figure 7). On the other hand, ZnO:Mg NCPs demonstrated toxicity, resulting in a high mortality rate at both tested concentrations (0.25 and 0.50 mg/mL). Thus, even at the highest concentration, ZnO:Au and ZnO:Fe demonstrate biocompatibility in both the control and pure ZnO. The doping of Au and Fe can neutralize the effects of pure ZnO, increasing its biocompatibility and providing stability and improved physicochemical performance to the sample, such as luminescence. These new properties can be utilized for bioimaging and in sensors, as Martins et al. [24] proposed in an immunosensor based on ZnOCu.

Indeed, beyond their essential roles in vital metabolic processes for all living organisms [61, 62], zinc oxide (ZnO) and copper (Cu) on the nano-scale exhibit significant bactericidal and antifungal activity. They also find application in cancer treatment and are used in bioimaging, serving as visualizers and sensors [63]. Fe is also essential for various biological processes, including enzymatic reactions, DNA synthesis, mitochondrial function, and oxygen transport through hemoglobin. Thus, NCPs or NPs containing Fe can be valuable tools in nanomedicine. Fe-containing NCPs are promising for the treatment of iron deficiencies and anemia. Depending on the synthesis method, they can be used for targeted drug delivery and encapsulated in liposomes to increase bioavailability and prevent immune system-mediated hemolysis [64].

Furthermore, they can act in the theranostic area as diagnostic agents and in cancer treatment [65]. An important characteristic is that ultra-small iron oxide nanoparticles (USIONPs) have demonstrated facilitated cellular penetration [66]. Their applications include thrombolysis, vascular grafts, atherosclerosis treatment, cardiovascular regeneration, and antibacterial activity [67]. Moreover, nanoparticles based on ZnO, Cu/CuO, or Fe have promising applications in agriculture to assist in growth and control fungal growth [68].

Gold (Au), although not an essential element for life, stands out for its high stability, high biocompatibility, and ease of functionalization. These characteristics make it ideal for applications in diagnosis, including various biosensors, cancer therapy, drug delivery, and enhancement of medical bioimaging acquisition [69, 70, 71]. The incorporation of gold, iron, silver, or copper nanoparticles with another nanomaterial to build hybrid nanoparticles can lead to synergistic effects, improving their biocompatibility, as shown in this chapter, and their possibilities of application in biosensors [72], biomedicine, catalysis, and magnetism [71, 73, 74].

Therefore, nanoparticles (NPs) and nanocrystals (NCs) can alter the levels of these ions in the body or exploit homeostatic pathways in the human body. To achieve this, new elements and different physicochemical processes can be explored to synthesize novel nanomaterials, resulting in various shapes and sizes with specific properties.

3. Conclusion

Therefore, although doped nanomaterials offer numerous opportunities for technological and biomedical advancements, ongoing research is essential to fully understand their effects and ensure their safety in future applications. The diverse range of properties exhibited by doped nanoparticles and nanocomposites, including enhanced biocompatibility, unique optical and electronic properties, and targeted therapeutic capabilities, holds great promise for various applications, from biomedicine to environmental remediation.

However, the complex interactions between doped nanomaterials and biological systems necessitate a cautious approach to their development and deployment. The synthesis, characterization, and evaluation of hybrid nanocomposites must be conducted meticulously, ensuring the optimization of their beneficial properties and mitigating potential risks associated with their use. Furthermore, interdisciplinary collaboration between researchers from various fields, including materials science, biology, medicine, and toxicology, is paramount to comprehensively assess the safety and efficacy of doped nanomaterials across different applications.

Moreover, considering the dynamic nature of nanotoxicology and the evolving regulatory landscape surrounding nanomaterials, continuous monitoring and risk assessment strategies are imperative to address emerging concerns and ensure the responsible advancement of nanotechnology. By adopting a proactive approach to research and regulation, we can harness the transformative potential of doped nanomaterials while safeguarding human health and the environment.

In conclusion, while doped nanomaterials represent a paradigm shift in materials science with profound implications for diverse fields, their safe and sustainable integration into real-world applications requires concerted efforts from the scientific community, industry stakeholders, and regulatory bodies. Through collaborative research, rigorous testing, and informed decision-making, we can realize the transformative benefits of doped nanomaterials while minimizing potential risks, thus paving the way for a brighter and more sustainable future.

Acknowledgments

This work was supported by grants from CNPq, CAPES, FAPEAL, and RENORBIO.

Conflict of interest

The authors declare no conflict of interest.