Magnetic Iron Oxide Nanoparticles

Magnetic FeO NPs mainly consist of iron core (Fe3O<sup>4</sup> or Fe2O3) particles which have been used in several studies as significant antifungals against mycotoxigenic molds [34]. Drug delivery, heat therapy and imaging are other beneficial uses of iron core particles [23,38,39]. Moreover, the iron core can be conjugated with fluorescent shells and drugs or antibodies against targeted cancer cells [24]. Further, surface functionalization of these NPs by polyethylene glycol (PEG) can potentially help to prevent elicitation of the immune response.

#### Semiconductor Quantum Dots

Zinc selenide/telluride/sulphide quantum dots exhibit substantial antifungal potential [39]. QDs are core-shell aqueous materials which exhibit conjugation with drugs or other biological materials including nucleic acids (DNA/RNA), proteins and other biomolecules [17]. The biomolecule conjugated QDs have specific use for detection and diagnosis of diseases or their causative pathogens [23]. Further, QDs find peculiar applications for improved imaging and genetic analysis by observing cell activities under disease conditions, and targeted drug delivery [40].

### Silicate Nanomaterials

These nanomaterials are comparatively biosafe, and do not exhibit high reactivities. Further, the silicate nanomaterials possess diverse morphologies spanning over different particle shapes and sizes which can be easily modified [23]. These silicate nanomaterials are also amenable to functionalization, and other coating treatments. Nanoshells are a specific class of silica nanomaterials which involve a thin metallic coating of the glass core [41]. These nanoparticles have been utilized for the diagnosis of the tumorous tissues [23,42] and simultaneous therapeutics applications [42–45].

#### 2.1.4. Carbon Nanomaterials

Carbon nanomaterials have significant antifungal and antimycotoxin potential [3,5]. The carbon atom contents enable the destruction of pathogen cell walls [40]. These nanomaterials are insoluble in water and do not get digested in the alimentary tract or get excreted on oral administration [26]. The SE nanomaterials pass through the cell membranes of targeted cells to reach to the cytoplasm of the pathogens or cancer cells causing multiplexed

damage resulting in the cell death [46]. Besides, buckyballs can ameliorate pH levels which help in drug delivery to targeted tissues [47]; gene therapy and DNA delivery [48].

#### 2.1.5. Nanobubbles

These are gas core particles suspended in aqueous medium having general size dimensions ranging from 70–120 nm and function as carriers of gas molecules [49]. The nanobubbles are different from the other types of nanoparticles or nanoemulsions as these contain a shell comprised of polymer, phospholipids, proteins or anti-cancer therapeutic agent encasing a gas (generally oxygen) [50–52]. These nanomaterials are finding useful applications in diagnosis and targeted delivery of anticancer drugs [49,53].

#### 2.1.6. Nanovaccines and Nanoadjuvants

Today, there are progressive advances in the application of nanotechnology for the production of vaccines. Nanovaccine formulations effectively activate the humoral immunity by a slow elaboration of antigens and thereby elevating the usefulness of vaccination [17,54]. These can be targeted to lymph tissues which significantly enhances the vaccine activities [55]. Nanomaterials conjugated with antibodies and other biological molecules can be used for the quick detection of pathogens and for effective treatment of the diseases caused by them [39]. However, the nanomaterials possess excellent adjuvant properties as these can bind to a variety of antigens/proteins of pathogenic origin to obtain nano-vaccines thereby replacing the use of the adjuvant material [55]. Different forms of nanomaterials used in animal antifungal nanotherapy was shown in Figure 2.

**Figure 2.** Various types of nano-based materials employed in antifungal nanotherapy in veterinary medicine.

### **3. Applications of Nanoantifungals in Veterinary Medicine**

*3.1. Therapeutic and Preventive Aspects of Nanomaterials*

3.1.1. Metal/Metal Oxide/Non-Metal Oxide NPs and their Hybrids as Nanoantifungal Agents

The use of nanomaterials as antifungal agents is an established attribute. The nanomaterials that exhibit antifungal potentials have been evaluated in several studies with

primary inhibitory impact on the vegetative growth of the fungal mycelia. The noble metal nanoparticles including the silver and gold nanoparticles possess potent antifungal properties. Nasar et al. [56] have evaluated the broad antimicrobial activity of AgNPs against human pathogenic bacteria (*Escherichia coli*, *Klebsiella pneumonia*, and *Bacillus subtilis*), and common fungal pathogen *Aspergillus niger.* The AgNPs have been found to be effective antifungals against dermal infections [57] Moreover, AgNPs can remove the human oral microbial infections caused by *S. aureus* and *C. albicans* [58], and *C. albicans*, and *Trichophyton mentagrophytes* infections in buffaloes [59]. The nanosized silver can inhibit the growth of *Fusaium* sp. at very low concentrations (<100 ppm) [16,60] and led to decreased mycotoxin production [1,61]. Also, Kischkel et al. [37] observed the antifungal activity of the AgNPs against *C. albicans*, *F. oxysporum* and *M. canis*.

Abd-Elsalam et al. [62] have discussed the fungal growth inhibitory potential of a variety of metal oxide NPs. Among the metal oxide NPs, the most promising candidates are zinc oxide NPs which inhibited the *Candida albicans* growth at very low concentrations of 1.013–296.0 µg/mL [63]. The shape and size of ZnO NPs has been an important characteristic that decides for the extent of the antifungal activity. Flower-shaped ZnO nanostructures inhibited the development of *Aspergillus flavus* and aflatoxin production at concentrations below 5 mM [64]. The next metal oxide NPs showing considerable antimicrobial potential are the iron oxide NPs. A study on magnetic NPs (Fe2O<sup>3</sup> NPs) described the antifungal activity against *A. flavus* and prevention of the aflatoxin production [38]. While, Mouhamed et al. [65] documented the inhibitory effect of iron oxide NPs on ochratoxigenic *Aspergillus* sp. Moreover, Abd El-Tawab et al. [66] have detected the growth inhibitory properties of Fe2O<sup>3</sup> NPs against causative pathogens of bovine skin diseases (*Trichophyton verrucosum*, *T. mentagrophytes*, and *Dermatophilus* sp.).

The coating or surface functionalization of the metal/metal oxide nanoparticles can further improve their antimicrobial properties. The chitosan NPs derived from deacetylated derivative of chitin can prevent growth of *Fusarium* sp., *Rhizopus* sp. and *Aspergillus niger* and thus can be used as an alternative to chemical pesticides [67]. Further, chitosan NPs have also been observed to inhibit fish pathogens under in vitro conditions [68]. Chitosan polymers can also be utilized to develop surface coatings on metal oxide NPs to improve their interactions and passage through the biological membranes. Recently, Abd-Elsalam et al. [69] have detected significant antifungal activity of CuNPs singly and in combination with chitosan against mycotoxigenic fungi, which also led to the prevention of aflatoxin production. The use of an acrylic resin reinforced with ZnONPs and Ag NPs can inhibit the growth of *Candida albicans* [70].

Nowadays, combinations of nanomaterials with beneficial biological active compounds are used to produce nanocomposites of significant use for animal health [1]. The conjugation and overlay of nanomaterials by other biological molecules are related to their chemical properties and used in detection of pathogens inside the body [71]. In this respect, Hassan et al. [1,5] have reported that the conjugation of metals nanomaterials with natural oils significantly improved the antifungal activity. They have detected that the composites of AgNPs, ZnONPs, and essential oils can effectively prevent the growth of fungal and bacterial pathogens. Hybrids of Ag NPs/essential oil were employed in therapy of bovine skin and udder infections [5,72] and carbon NPs [73]. Hassan et al. [5,16,74] have reported the efficient conjugation of ZnNPs and AgNPs with cinnamon and olive oils for use at low safe doses for inhibition of growth of toxigenic *A. flavus* and *E. coli* and production of respective toxins, whereas, Wang et al. [75] successfully detected that the hybrid of Au NPs with antibodies help in immune-chromatographic exploration and diagnosis of toxic AFM<sup>1</sup> in milk. Similar activities were obtained for QDs to observe events and activities of body cells that were found to be better than the use of traditional dyes and this helped for release of drug to the required site of infection [9,76].

Nanoparticles can also be conjugated with known standard antifungal agents or other molecules where these NPs function as nanovehicles for better delivery of the antifungal therapeutic agents at the targeted site. Therefore, common antifungal agents can also

be conjugated on the metal or metal oxide NPs to enhance their antifungal activities. Kischkel et al. [29] have illustrated the potentials of different types of nanoantifungals for the treatment of mycosis caused by *Candida* sp. and *Aspergillus* sp. Hamad et al. [77] have developed a gold nanorod-fluconazole nanoconjugate which exhibited significantly high antifungal activity (9 to 12-fold) against *C. albicans* compared to either component alone, whereas, Huang et al. [36] reported the possibility of using AgNPs as antifungals singly or in conjugation with epoxiconazole (8:2 and 9:1), respectively. The concentration of AgNPs required to suppress the growth of 50% of the fungal colony was 170.20 µg/mL. The combination of AgNPs with fluconazole and florfenicol produced more antimicrobial potential against the causes of animal diseases than their single forms [5].

Inorganic mesoporous silica nanoparticles (MSNs) can also act as nanocarriers for drug delivery to target affected cells inside the body [78]. Functionalized silica NPs can be tethered to drug molecules or they can also adsorb or sequester the drug compound on the surface or inside the nanopores thereby elevating their delivery to the target organs [79,80]. Kanugala et al. [81] have developed phenazine-1-carboxamide-functionalized MSN-based antimicrobial biomaterial surfaces to prevent the formation of bioflms on medical implants. The developed MSNs exhibited superior anti-Candidal activity besides polymicrobial antibiofilm potential. Silica NPs can also be used for the development of topical cream formulations to treat skin fungal infections. Montazeri et al. [82] have synthesized and evaluated an aminopropyl functionalized MSN-econazole topical cream formulation against *Candida albicans* skin infections and observed improved antifungal activity at lower concentrations of the loaded drug.

### 3.1.2. Polymer Nanoparticles for Antifungal Drug Delivery

Recent drug and vaccine delivery strategies in biomedical research advocate the use of nanomaterials for successful delivery of drugs to targeted cells and tissues [10]. These strategies are beneficial as they can ensure the delivery of drugs to target tissues resulting in a decrease in the amount and required doses for the treatment of diseases. The most promising nanodelivery agents for drugs can be the polymer nanoparticles encapsulating antifungal drugs. In these respects, chitosan (CS) NPs which themselves possess considerable antifungal potential can be used for the delivery of the antibiotic drugs [68]. The encapsulation of antifungal drugs or development of their formulation as nanoemulsions can improve their action potential. Deaguero et al. [83] observed that nanoencapsulation of miconazole in cholesterol/sodium oleate vesicles have significant antifungal activity against several fungal pathogens. Siopi et al. [84] have reported that the liposome-encapsulated amphotericin B possess significant therapeutic potential against mycotic respiratory infections in animals caused by *A. fumigatus*.

Drug molecules can also be nanoformulated as nanomicelles comprised of a hydrophobic core and hydrophilic shell which improves the water solubility and therefore bioavailability of the hydrophobic drugs [85]. Further, these nanosystems can be used for the targeted delivery of the drug [86], treatment of cancer in animals [87] and to ensure drug delivery without stimulation of immunity [88].

#### 3.1.3. Carbon Nanomaterials as Nano-Antifungals

Different forms of carbon-based nanomaterials also exhibit antimicrobial activity against bacterial and fungal pathogens causing diarrhoea [3]. These nanomaterials can inhibit the growth of *E. coli* and mycotoxigenic fungi [89]. Furthermore, conjugation of sugars with CNTs improve the ability to affect the viability of *C. albicans*, *A. flavus* [5]. Several benefits of nanoantifungal applications were detected, as illustrated in Figure 3.

**Figure 3.** Theragnostic applications of nanoantifungals in animal science. **Figure 3.** Theragnostic applications of nanoantifungals in animal science.
