**4. Nanoantifungals: Can These Be the Future Innovations in Veterinary Biomedicine?** *4.1. Mechanism of Action of Nanomaterials as Antifungal Agents*

Nanomaterial possess a range of activities to inhibit the growth and multiplication of fungal-pathogens resulting in cell damage and loss of functions [3,5,35,38]. Nanomaterials exhibit a large surface area compared to the corresponding bulk materials [147]. These materials interact with the various biomolecules in the biological milieu eliciting formation of reactive oxygen species. The action of several nanoantifungals leads to an augmentation of intracellular ROS, an important mediator for exerting antifungal effects. The antifungal activity of nanosilver has been associated with the induction of mitochondrial dysfunctional apoptosis through an increase in oxidative stress via ROS generation especially hydroxyl radicals [148]. The ROS generation is initiated as a response to attachment of antifungal nanomaterial with targeted cells leading to elaboration of O<sup>2</sup> atom and metal ions [149], whereas, the elaborated O<sup>2</sup> increases the oxidative stress causing damage of the mitochondria proteins, leading to denaturation and loss of their functions. These potentials of ROS production have been observed on supplementation of C<sup>60</sup> fullerenes, SWNTs, and QDs [1,5,150].

#### *4.2. Cytoxicity Risks of the Use of Nanoantifungal Agents*

The continuous awareness about the toxicity risk of nanomaterials to animal and the environment have led to refusals of applications of nanomaterials in animal science by several international authorities [151]. The toxicity of nanomaterials can be affected by a variety of factors such as particle size, dose level, type of animal species and the period of exposure [94] and the physico-chemical characters of the nanomaterials used [152]. Chronic exposure of buffalo sperm to ZnNPs and TiONPs (100 µg/mL) caused several abnormalities resulting in suppression of viability and diminished fertility [143], while, sperm exposure to 100–500 µg/mL of Zn NPs caused their damage and rapid death [153]. Hence, the estimation of safe doses of the used nanomaterial should be investigated in laboratory animals before application to field animals [35,72,94].

Furthermore, upon ingestion of nanomaterials by humans and animals they enter the alimentary tract, reach the circulatory system and are carried over via the liver and spleen [35,154,155], whereas, inhalation and skin exposure to nanomaterials allows for their penetration through the skin tissues and nerve cells [152,155]. Inhalation of TiO<sup>2</sup> NPs was identified to have an effect in the development of lung cancer [154]. When NPs reach blood vessels, pathological effects occur such as blood clots and disorders in the cardiovascular system functions [156]. Inhalation of low doses of TiO<sup>2</sup> NPs can cause vascular disorders in rats [157], besides inhalation of single wall and multiwall CNTs [158,159]. We have little knowledge about toxicity and the journey of the nanoparticles in the animal body from the site of administration, passage through absorption, blood vessels, distribution in body tissues and their further journey. Hence, broad toxicological studies are needed before launching commercially viable nanotechnology applications in biomedicine and animal health.

#### *4.3. Safety Concerns of Nanoantifungals*

There are many challenges related to the potentially toxic effects of nanomaterials. Incorporation of nanomaterials into polymeric hydrogel matrices may reduce the toxicity and improve its efficacy because of sustained and controlled release of the incorporated NPs. The effective delivery of the nanomaterials can be ensured by their functionalization with polymers at low doses to avoid elicitation of the cellular toxicity [1,3,5,160]. Moreover, several benefits of nanomaterials use for improvement in biomedical applications have also been realized. Although, information related to their harmful impacts is not sufficient and special attention is required for identification of their toxicity risk before practical biomedical applications can be approved for use.

#### **5. Conclusions and Future Perspectives**

Over the past decades, nanotechnology has offered progressive novel advances to improve animal health and production. Today, several nanomaterials are used as nanoantifungals, besides having other benefits such as disease detection, diagnosis and therapy, use of additives to animal feed and their products, and finally food safety. The essential therapeutic and preventive activities of nanoantifungals, particularly the zinc and copper nanomaterials, have been evaluated against a variety of fungal diseases and mycotoxicosis in animals. Also, super paramagnetic iron, semiconductor quantum dots and gold nanoparticles are finding applications for early and sensitive detection followed by detailed prognosis and therapy of cancer. Both inorganic and organic polymeric nanomaterials have also been utilized for targeted delivery of various vaccines, quick and on-site detection of pathogens or their signature protein and other biological molecules. The mechanisms of nanoantifungal activity are related to their ability to penetrate the cell membrane, damage the cytoplasmic contents, leading to loss of function and death of the cells. Therefore, further studies illustrating the cellular toxicity mechanisms that result in oxidative stress and leading to genotoxicity and cancers need a detailed evaluation to manipulate the roles of nanomaterial in animal health. Moreover, the toxicity risks of nanomaterials must be determined before application of nanomaterials in veterinary medicine for safeguarding the health of the animals and their role in animal production.

**Author Contributions:** Conceptualization, A.A.H., M.A.A.; resources, A.A.H. writing—original draft preparation, A.A.M.E.H., A.K.; writing—review and editing, R.M.H.S.E.A., K.K., K.A.A.-E.; supervision, K.A.A.-E.; funding acquisition, K.K., P.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the project UHK VT2019-2021.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors wish to express their grateful appreciation and heartfelt thanks to M.K. Refai, Professor of Microbiology, Cairo University, Egypt for his continuous assistance and advice for initiating this work.

**Conflicts of Interest:** The authors declare no conflict of interest.
