EXCLI Journal 2021;20:562-584 – ISSN 1611-2156 Received: October 22, 2020, accepted: February 15, 2021, published: March 08, 2021

Review article:

CURRENT APPLICATIONS AND PROSPECTS OF NANOPARTICLES FOR DRUG DELIVERY

Sanam Nami1 , Ali Aghebati-Maleki2 , Leili Aghebati-Maleki3,4*

1 Department of Parasitology and Mycology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran 2 Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran 3 Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 4 Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran

* Corresponding author: Leili Aghebati-Maleki; Ph.D., Assistant Professor of Immunol- ogy, Immunology Research Center, Tabriz University of Medical Sciences Tabriz, Iran. Tel.: 0098- 4113364665, Fax: 0098- 4113364665, E-mail: [email protected]

http://dx.doi.org/10.17179/excli2020-3068

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/).

ABSTRACT Currently, the significance of fungi as human pathogens is not medically concealed in the world. Consequently, suitable recognition and treatment of such are of great importance and necessitate the need for compre- hensive information in this regard. The introduction of new and their use today, especially in the last two decades, have revolutionized the treatment of fungal infections. On the other hand, increasing drug resistance in the world has overshadowed such developments. The use of NPs results in the treatment of fungal infections and owing to their specific properties, these particles, unlike the pure antibiotics, can exert a greater inhibitory power although with less concentration compared with conventional drugs. Important reasons that have led to the use of antifungal drugs in delivery systems include reduced drug efficacy, limited penetration through tissue, poor aqueous solubility, decreased bioavailability, and poor drug pharmacokinetics. It is therefore hoped that unfavor- able properties of antifungal drugs be mitigated via their incorporation into different types of NPs. This review summarizes the different types of NPs as delivery systems of antifungal as well as their advantages over pure drugs.

Keywords: Nanoparticles, drug delivery, antifungal drug

INTRODUCTION fungi can easily endanger the lives of im- Nowadays, the significance of fungi as mune-deficient patients and are now one of human pathogens is medically transparent in the leading causes of death for these patients. the world. Fungi that were not previously in- Therefore, timely recognition and treatment fectious in humans are nowadays among the of such infections are of great importance and opportunistic pathogens which are increasing necessitate the need for comprehensive infor- in number. Thanks to their ability to adapt to mation in this regard. Studies show that more a variety of environmental conditions, these than 300 million people suffer from serious fungal infections, accounting for about 1.4

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million deaths annually (Brown et al., 2012). exerting the maximum effect (Mu and Feng, The introduction of new antifungals and their 2003). The NPs used for drug delivery not use today, especially in the last two decades, only should possess both biocompatibility have revolutionized the treatment of fungal and biodegradability properties but also their infections. On the other hand, unfortunately, timely release, optimum mechanical proper- increasing drug resistance in the world has ties and ease of production need to be taken overshadowed such developments. In devel- into account. NPs get trapped in body through oped countries, antiretroviral therapy (ART) the circulatory system or phagocytosis which has led to a marked decrease in the incidence can be tracked through surface modification of fungal infections among people with hu- and thus, be saved in the circulation system man immunodeficiency viruses (HIV), but (Mahapatro and Singh, 2011). lack of access to such drugs in the developing NPs can be categorized in a variety of countries has brought about a noticeable surge ways in terms of their size, shape and consti- in the incidence of these infections. On the tuting materials (Figure 1). Even the prepara- other hand, the use of invasive therapies in in- tion methods of NPs can lead to the creation tensive care units, use of immunosuppressive of a variety of NPs, with each of them having drugs after organ transplantation, treatment of different loading capacity, delivery, and malignancies, increased use of antifungal shelf-life (Hu et al., 2010). They are divided prophylaxis with derivatives, as well as into dendrimers, nanospheres, nanocapsules, mounting use of for the em- liposomes, micelles, polymersomes, fuller- pirical therapy have led to significant changes enes and nanotubes according to their appear- in terms of heightened incidences of fungal ance. Other studies have classified them into infections with new patterns as well as the de- organic and non-organic groups. The organic velopment of drug resistance, especially in molecules are the main components of the opportunistic fungi (Fisher et al., 2018). NPs within the organic category whereas in Therefore, researchers spare no effort to min- the mineral category, metals (iron, gold, etc.) imize drug resistance and toxicity using effi- and other mineral elements play a pivotal role cient methods. in the structure of the NPs (Erdoğar et al., Today, nanoparticles (NPs) have been 2019). Liposomes, dendrimers, carbon nano- highly appreciated for their wide range of ap- tubes, solid lipid NPs, and polymers belong to plications to various biological, pharmacy and the organic particle category while mineral medical fields. Structurally considered, their NPs contain a central core made of mineral or size barely exceeds the range of 100 nm. A metal elements covered by a coating of or- broad spectrum of drugs, such as small hydro- ganic materials. The cores depict fluores- phobic and hydrophilic drugs, vaccines and cence, magnetic, and electrical properties biological molecules can be controlled by (Frank et al., 2015). In another classification, these NPs (Gupta and Xie 2018). NPs are ex- NPs are made of macromolecular or poly- tensively used in tissue engineering scaffolds, meric materials, natural or synthetic, and are targeted drug delivery, and also the diagnosis classified into two types of nanosphere and of diseases (Mura et al., 2013). NPs have been nanocapsule based on their production meth- widely exploited in forms of nanoliposomes, ods. Nanocapsules are sac-like structures in carbon nanotubes, nanofibers, nanocapsules which the drug is placed in a central chamber as drug carriers, and cellular scaffolds. The surrounded by a polymeric layer. Nanosphere primary purpose of producing NPs as a drug is a matrix system within which the drug and delivery system is to control particle size, sur- polymer are either homogeneously dispersed face properties, and the effective delivery of a or absorbed on the surface. Polymers, being specific drug at a particular time and place for applied as NPs, are accompanied by the drugs

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Figure 1: Different types of nano carriers for drug delivery

with specific therapeutic effects for specific cancers (Langer and Folkman, 1976). Poly- diseases such as cancers. These NPs are mer-based NPs are a proper tool for biomole- bonded to nanocomposites in two ways: 1. cules, drugs, genes and vaccines guidance. The drugs are encapsulated in nanocarriers; 2. The solubility and shelf life of the drugs can The drugs are conjugated over the nanoparti- be honed by being encapsulated within NPs. cle surface (Moritz and Geszke-Moritz, By doing this, the main objectives are targeted 2015). So far, various polymers including pol- drug transfer with a determined concentration yamide, polyamino acid, poly (alkyl α‐cyano- within the range of treatment and ultimately crylates), polyester, polyorthoester, polyure- the patient’s satisfaction (Dinarvand et al., thane, polyacrylamide, and polycaprolactone 2011). Polymer carriers have received a great have been used as drug carriers. Amongst deal of attention in the last few decades thanks them, aliphatic polyester thermoplastics such to their multiple functions and their potential as polylactic acid (PLA), polyglycolic acid to be functionalized. In general, polymer NPs (PGA), and poly lactic-co-glycolic acid are minute colloid systems in which the drug (PLGA) as the copolymer of these two have is physically dispersed or dissolved or chemi- been widely used compared to others due to cally bonded to their main polymer chains. their super biocompatibility and biodegrada- Among the advantages of utilizing polymer bility properties (Cheung et al., 2015). Langer NPs as a pharmaceutical nanocarrier is the en- and Folkman were the pioneers to reveal the hanced drug solubility and stability. Hence, controlled release of macromolecules through polymer NPs are considered to be the most polymers which led to the advancement of the widely administered drug release systems drug release system in anti-angiogenics for

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(Hickey et al., 2015). To date, several poly- 2015). Relatively low drug loading is proba- mers have been investigated based on their bly the major obstacle to the use of drug- characteristics, some of which are as follows: loaded PLGA NPs in clinical trials. As a re- Chitosan, a carrier of NPs, is a plentiful sult, further progress is needed to transform natural polysaccharide known for its benefi- the concept of drug-loaded PLGA NPs into a cial properties. Compared to the synthetic realistic action plan as a new generation of polymers, chitosan obtained by chitin acety- drug delivery systems with the primary goal lation is suitable for the formation of nanogels of enhancing the therapeutic effects and min- due to its biocompatibility, better biodegrada- imizing side effects (Le Garrec et al., 2004). bility, and stability as well as low toxicity Proteins are a bountiful source of natural (Onyebuchi and Kavaz, 2019; Saito et al., materials for making nanoparticle systems. 2019). However, the low solubility of chi- Various proteins including gelatin, albumin, tosan in the alkaline and neutral medium due and curcumin are widely applied for drug de- to the large number of amino groups in its livery (Verma et al., 2018). The use of protein chain on the one hand, and the formation of NPs for treatment is yielding astonishing re- strong hydrogen bonds and stable crystal sults and promises greater efficacy in the fu- structure on the other hand limits its use in ture. With respect to functional comparisons pharmaceutical and biomedical applications and therapeutic efficacy, proteins and other (Rodrigues et al., 2012; Bruinsmann et al., existing delivery systems still suffer from 2019). As a result, some different chitosan some defects, necessitating further investiga- amendments have been implemented, but tions in this area (Tosi et al., 2013). these may include toxicity issues. To over- Many studies are being conducted world- come such restrictions, polyethylene glycol wide today concentrating on the improvement methyl ether is an excellent hydrophilic poly- of therapeutic methods for corneal fungal in- mer whose bond with chitosan chain not only fections and invasive mycoses in particular, improves the compatibility of chitosan but due to the poor bioavailability of antifungal also prevents protein absorption and escape drugs that arise from various protective mech- from the reticuloendothelial system (Szy- anisms. Numerous in vivo investigations indi- mańska et al., 2016; Antoniraj et al., 2018). cated that the accumulation of drug-loaded PLGA is one of the most successful poly- nanoparticles in lungs was significantly mers used for drug release applications, sev- higher than that of a pure drug; in addition to eral outstanding benefits of which include bi- this, the prolonged retention of particles in ocompatibility, drug compatibility, mechani- lungs was observed (Balabathula et al., 2020; cal properties, easy production process; be- Faustino and Pinheiro, 2020). Sometimes oral sides, its hydrolysis results in the formation of therapy is not adequate especially for topical monomers of lactic acid and glycolic acid me- fungal infections; thus, topical application of tabolites. Since these two monomers are an- this drug formulation can yield better results drogens and are easily metabolized by body such as improved skin penetration due to en- through the Krebs cycle or TCA cycle, PLGA hanced contact between antifungal and skin. induces poor systemic toxicity (Richard and One of the preferred mechanisms in advanc- Margaritis, 2001; Yang, 2019). PLGA NPs ing drugs for better penetration is the use of protect the drug against degradation and im- these nanosystems (Dhamoon et al., 2019). So prove its stability (Le Garrec et al., 2004; Tian in general, due to the limited antifungals, we et al., 2017; Yang, 2019). These systems also are facing a number of problems such as pro- have some downsides; namely, rather low longed treatment times, high costs, complex drug loading for many drugs, costly produc- route of administration, side effects, drug re- tion and the issues pertaining to expanding the sistance, toxicity, and drug interactions scale of use (Moritz and Geszke-Moritz, (Souza and Amaral, 2017). The benefits of us- ing these NPs in combination with antifungals

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entail increased solubility, improved permea- ability of these nanostructures in encapsulat- bility, bioavailability, enhanced storage sta- ing large quantities of drugs, minimizing un- bility, prolonged half-life, reduced therapeu- wanted side effects, and having high efficacy tic cost, and adequate dose of drugs (Faustino and low toxicity has attracted researchers' in- and Pinheiro, 2020). In this review, we will terest. Also, other benefits of nanoliposomes summarize the major findings related to nano- are the ease of production in industrial vol- particles and some of the strategies using nan- umes, excellent fabrication quality, variety in otechnology to improve the conventional particle size, chemical composition, and elec- therapy. trical charge. Liposomes have a broad particle size range, spanning from micron-sized mac- roliposomes to nano-sized liposomes. Nano- TYPES OF SMART DRUG DELIVERY liposomes (liposomes of less than 200 nm) are Recent clinical and pre-clinical studies frequently used in the pharmaceutical indus- have revealed that targeted drug delivery sys- try. These liposomes can easily pass through tems are a great way to treat various life- a variety of obstacles they face such as blood threatening diseases. Smart drug delivery is barriers (Moghimipour et al., 2012). divided into the following categories: Polymer nanoparticles Polymeric NPs as pharmaceutical carriers Nano-scale carriers Drug delivery using nanocarriers is abso- are made up of both biodegradable polymers lutely desirable due to their very small diam- and non-biodegradable types. In recent years, eters (10 to 1000 nm) which greatly contrib- their biodegradable formulation has attracted utes to improved encapsulated drug treatment considerable attention as potentially suitable efficacy (Kumar Giri et al., 2016). In recent drug delivery systems due to their ability to years, many nanometer structures have been release drugs modestly, to load large amounts investigated and developed for drug delivery of pharmaceuticals and prevent drug degrada- purposes including nanoliposome, polymer tion (Faraji and Wipf, 2009). In this system, NPs, solid lipid NPs, and dendrimers. the drugs are either trapped or bound by cova- lent bonding to the polymer matrix. Besides, Nanoliposome polymer NPs are also used to improve the sur- Nanoliposomes are self-forming nano- face quality that can in turn, enhance drug ab- structures that come together in an aqueous sorption efficiency. Polyethylene glycols solution of lipid molecules. Phospholipid lip- have been widely used in polymer NPs to ophilic molecules are grouped in such a way hone biodegradation (Cho et al., 2008; Pérez- that their hydrophobic groups are directed in- Herrero and Fernández-Medarde, 2015). In ward whereas their hydrophilic groups are general, polymers used in the preparation of aligned in the outer layer of the sphere, thus these structures fall into two categories: natu- forming a spherical bilayer membrane. This ral polymers such as chitosan, albumin and mode of orientation allows the loading of hy- heparin, and synthetic polymers such as drophilic drugs in the nucleus, and hydropho- (HPMA) N- (2-hydroxypropyl) methacryla- bic agents in the liposomes shell (Figure 2) mide PLA, PGA, and PLGA. However, given (Gunasekaran et al., 2014). Today, these the importance of biodegradability and safety, nanostructures are used as a drug or gene car- natural polymers are preferably used to de- riers, and for modeling purposes of cell mem- liver a wide range of drugs from macromole- branes, both in animals and in humans. The cules to small molecules.

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Figure 2: Liposomes are bilayered vesicles having an aqueous core and one or more concentric phos- pholipid membranes. This structure allows liposomes to act as effective delivery systems for both hy- drophilic and hydrophobic drugs.

Solid lipid NPs delivery by SLN depends on various factors Solid lipid NPs (SLN) are colloidal struc- such as the way the samples are administered, tures that can be prepared by emulsification the type of lipid and active ingredient used, as and by means of mechanical forces such as ul- well as the type of interaction of the body with trasound and homogenizer, they get reduced the particles (Qu et al., 2016). The most im- to below micrometers in size. These systems portant enzyme that affects these structures in are formed by replacing the w/o emulsion oil the body is lipase. The rate of degradation of phase with a solid oil or a mixture of solid different lipids varies with this enzyme. For oils; that is, a mixture of lipid matrix particles example, the longer the length of the lipid that are solid at room temperature and body. chain, the slower the effect of the enzyme on SLN consist of 0.1 to 30 % solid fat dispersed its degradation. In the presence of some emul- in the liquid phase and if necessary, 0.5 to 5 sifiers, the degradation rate also decreases and percent of the surfactant is also used in their the emulsifier acts as a lipid shield (Cunha et preparation. The average SLN particles range al., 2017). from 40 to 1000 nm. Studies have shown that Dendrimers the physicochemical and stability properties Dendrimers are a family of three-dimen- of drugs loaded in the SLN depend on the sional, nano-sized polymers characterized by properties of the drugs and the components a compact spherical structure in solution. Alt- used. These structures are capable of carrying hough the origin of dendrimers is linear poly- drugs and active substances in their lipid por- mers and then branched polymers, the re- tion, thereby protecting the target material markable structural properties of high from environmental damages. As a result, this branched dendrimers and macromolecules are spectrum of NPs can be used to deliver drugs quite different from those of traditional poly- and prolong their effectiveness (Li et al., mers (Bugno et al., 2015a). Despite the use of 2017; Rodriguez-Torres et al., 2020). Drug

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polymers in drug delivery systems, den- cosmetics, perfumes or pharmaceutical indus- drimers are more beneficial than them. They tries. The nanocapsule is used in various have finite polydispersity and nanometer di- fields: for drug delivery in case of tumors, or mensions that facilitate passing through bio- as a nanocapsule bandage to fight , logical barriers. Dendrimers can encapsulate as a liposomal nanocapsule in food science guest molecules by receptors at their surface and agriculture, in delivering radio therapy or within the cavities between branches and as a self-healing material. Nanocapsules (Fréchet, 2003). Unlike linear polymers, den- can be used as smart drugs that have specific drimers are macromolecules that branch out chemical receptors and only bind to specific of a single nucleus, with all the branches cells. It is this receptor that makes the drug eventually reaching a central nucleus. In mak- ‘smart, allowing it to target cancer or disease. ing dendrimers, their molecular size and mass The advantages of nano-encapsulation tech- can be precisely controlled. The presence of a nologies for pharmaceutical applications in- large number of terminal branches increases clude: higher dose loading with smaller dose the solubility and reactivity of the dendrimers. volumes, longer site-specific dose retention, The solubility of dendrimers is strongly influ- more rapid absorption of active drug sub- enced by the nature of the surface groups. For stances, increased bioavailability of the drug, example, the presence of hydrophilic groups higher safety and efficacy, and improved pa- makes the dendrimers soluble in polar sol- tient compliance (Radhika and Sivakumar, vents whereas the hydrophobic end groups 2011). make the dendrimers more soluble in nonpo- lar solvents. The importance of dendrimers is Targeted pro-drug highlighted since the therapeutic effect of any Pro-drugs are inactive biological com- drug depends on its optimal solubility in the pounds that become active after encountering aquatic environment. There are numerous a specific physiological barrier (Hoste et al., substances with strong therapeutic properties 2004). The pro-drugs are designed to improve but are not used for therapeutic purposes due parameters such as undesired side effects, sol- to their insolubility. Water-soluble den- ubility, stability, biodegradability, toxicity drimers can bind to hydrophobic molecules and systemic metabolism. In recent years, a with antifungal or antibacterial properties. group of pro-drugs called targeted precursors The release of the bound drug is plausible have received much attention (Greco and upon contact with the target organisms and Vicent, 2008). In general, a targeted drug con- thus, these complexes are considered as drug tains a parent drug or its derivatives, a bond delivery systems (Bugno et al., 2015b). breakable by a chemical or enzymatic activity Nanocapsules such as amide and ester, an enzymatically or chemically breakable spacer, and a transient Nanocapsules consist of a thin membrane surrounding a core (Liquid, solid) with their target structure (Mahato et al., 2011). Precise size ranging from 10 nm to 1000 nm. selection of the breakable bond and the target structure of the transition play a critical role Nanocapsules are submicroscopic colloidal drug carrier systems composed of an oily or in the construction of the targeted drug. The an aqueous core surrounded by a thin polymer breakable bonds commonly used in the man- membrane. The membrane may be composed ufacture of pro-drugs include amides, esters, of natural or synthetic polymers (Mora- disulfide bonds, and phosphate esters, among Huertas et al., 2010). Two technologies can be which ester and amide are more widely used. used to obtain such nanocapsules: the interfa- The ester bond is usually broken by the ester- cial polymerization of a monomer and the in- ase enzyme which is highly dispersed in the terfacial nano deposition of a preformed pol- body. This stability problem can be tackled by ymer. The nanometric size of nanocapsule replacing carboxyl esters or phosphate esters can be interesting for many applications like with carbamate esters (D’Souza and Topp,

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2004). Other breakable bonds, such as amines ( (, and ), ben- bond / oxime bond along with thioether non- zylamine ()), breakable bond are used in the manufacture of () (Campoy and Adrio 2017; targeted precursors. The choice of the linker Nami et al., 2019). Given all the advances in depends on the use of the pro-drug. Disulfide the optimal development of such classes of bonds, for example, are commonly used to antifungal medicine, the use of NPs drug de- target cancerous tissues because the glutathi- livery systems has been proposed for their ex- one in these types of tissues breaks the bond. ceptional efficiency as well as their maxim- Similarly, hydrazone acid-sensitive bonds re- ized activity. The use of NPs results in the lease drugs into endosomal structures at low treatment of fungal infections and owing to pH (D’Souza and Topp, 2004; Tai et al., their specific properties, these particles, un- 2011). like the pure antibiotics, can exert a greater inhibitory power although with less concen- NPS FOR ANTIFUNGAL DRUG tration compared with drugs. Important and DELIVERY prominent reasons that have led to the use of The main classes of antifungal medica- antifungal drugs in delivery systems include tions used in mycoses treatment include poly- reduced drug efficacy, limited penetration enes (amphotericin B (deoxycholate, and li- through tissue, poor aqueous solubility, de- pid-based), , , ), creased bioavailability, reduced drug stabil- (imidiazoles (, , ity, side effects, and poor drug pharmacoki- , , …), (flu- netics (Soliman, 2017; Hassanpour et al., conazole, , isavuconazole, 2020). It is therefore hoped that encapsulating , , , drugs in NPs (≤ 100 nm) can partially over- …), and (abafungin)), pyrimidine an- come such problems of antifungals (Figure 3). alogue (), (caspo- In the following, we will discuss the different fungin, , , cilofun- types of NPs as delivery systems of antifungal gin, and biafungin), as well as their advantages over pure drugs.

Figure 3: NPs can over- come many of the unfa- vorable drug properties through virtue of their ver- satility, multifunctionality and wide range of proper- ties.

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Solid lipid nanoparticle pure drug (Butani et al., 2016). Another anti- Today, the delivery of antifungal drugs to fungal cream in the treatment of cutaneous the target skin region is one of the major prob- disease is clotrimazole (CLT) and in several lems when using systemically. studies, SLNs were employed for the encap- The use of systemic drugs in the treatment of sulation of CLT. In a study by Souto et al., in cutaneous diseases, in addition to having var- 2004 on azole drug, CLT-loaded SLN was de- ious side effects, can cause poor drug availa- veloped by hot high-pressure homogenization bility at the infection site. Therefore, one of to demonstrate the physical stability of these the main goals of accessing an effective novel lipid particles in their study (Souto et al., drug delivery route is to increase drug locali- 2004). Cassano et al. (2016) investigated the zation in the targeted organ. In recent years, vaginal infections sustained by Candida albi- SLNs have drawn more attention in topical cans. The researchers used SLNs based on applications due to their chemical affinity for polyoxyethylene-40 stearate for the admin- the skin. Several prominent advantages that istration of CLT, all yielding satisfactory re- make SLNs apt for drug delivery systems in- sults in the treatment of vulvovaginal candid- clude their nanometer range, sustained re- iasis infection. Nowadays, recurrent vulvo- lease, biocompatibility, and reduced side ef- vaginal candidiasis (≥ 4 episodes within 1 fects, with lipid matrix being the leading year) is a common cause of morbidity affect- cause of a reduction in the risk of toxicity ing millions of women worldwide; however, (Pople and Singh, 2006; Trombino et al., it is hoped that by further studies on this new 2016). Studies have shown that the physico- formulation and clinical trials, this drug can chemical and stability properties of drugs be used to treat the so-called patients loaded in the SLNs depend on the properties (Cassano et al., 2016). Another topical medi- of the drugs and the components used. These cation is econazole (ECN), whose poor water structures are capable of carrying drugs and solubility has limited its bioavailability and active substances in their lipid portion, antifungal effects. To this end, Sanna et al. thereby protecting the target material from en- (2007) worked on the econazole nitrate drug vironmental damage. As a result, this spec- using the o/w high-shear homogenization trum of NPs can be used to deliver drugs and technique, by which SLN was designed for prolong their effectiveness (Rodriguez-Torres the topical administration of ECN to improve et al., 2020). SLNs are also used in the deliv- ECN penetration through skin. In this in vivo ery of antifungal drugs for topical applica- study, they showed that SLN promoted an im- tions such as amphotericin B (AmB), azoles, mediate penetration of ECN through the stra- griseofulvin (GF), etc, some of which will be tum corneum compared to conventional gel discussed later on. In a study by Butani et al., (Sanna et al., 2007). A study by Bhalekar in in 2016 on AmB, it was suggested that SLNs 2009 on miconazole nitrate (MN), who pro- were effective for the topical delivery of duced MN loaded SLN by hot homogeniza- AmB. He noted impressive advantages of us- tion, revealed that MN-SLN formulations in- ing AmB loaded SLNs compared to drugs creased the accumulation of MN in the skin as only, including higher skin deposition, lower opposed to the conventional gel and improved skin irritation, and better antifungal activity. skin targeting effect (Bhalekar et al., 2009). In The researcher used several SLNs formula- 2010, Jain et al., conducted a similar study tions of AmB in his studies, asserting that with MN-loaded SLNs, produced by a modi- SLN5 (lipid ratio 1:10 and Pluronic F 127 fied solvent injection method. The researchers 0.25 % as a surfactant) have the best formula- used an animal model of rats infected with tion. He also showed that the SLN5 formula- Candida species and examined them for cuta- tion on the Trichophyton rubrum fungus has a neous candidiasis. They reported that MN- higher zone of inhibition compared to the loaded SLN-bearing hydrogel was more ef- fective than the free drug in the treatment of

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candidiasis (Jain et al., 2010). Also, Aljaeid enhanced corneal drug permeation as well as and Hosny developed MN-SLNs produced by revealing the non-irritating property of SLN hot homogenization /ultrasonication, to over- compared to the pure drug (Kumar and Sinha, come the problem of poor aqueous solubility 2016). Another study on VRZ-SLN was per- of this drug. They investigated antifungal ac- formed by Füredi and his colleagues in 2017 tivity against Candida albicans during the in to develop a novel formulation of the ophthal- vitro tests and used rabbits in vivo to ulti- mic dosage form. They worked on Candida mately observe the increased oral bioavaila- glabrata and Aspergillus flavus in this study bility of MN-SLN compared to the commer- and finally reported that antifungal study us- cially available capsules (Aljaeid and Hosny, ing this formulation could inhibit the repro- 2016). Other studies on MN include the 2017 duction of fungus (Füredi et al., 2017). An- study by Kenechukwu et al., by which solidi- other commonly used in either treat- fied reverse micellar solution-based mucoad- ment or prophylaxis is (FLZ), the hesive nano lipid gels encapsulating SLNs focus of Gupta and colleagues for investiga- were developed and evaluated for improved tion in 2011 as a result of which FLZ-loaded localized oromucosal delivery of MN for ef- SLNs were prepared by the aqueous diffusion fective treatment of oropharyngeal candidia- method against cutaneous candidiasis which sis (Kenechukwu et al., 2017). One of the tri- yielded satisfactory outcomes when com- azoles, widely used in the treatment of fungal pared with the pure drug (Gupta et al., 2013). infections today, is a case of itraconazole EL-Housiny et al. (2018) generated FLZ- (ITZ) developed by Mohanty et al., in 2015 as loaded SLNs topical gel to treat Pityriasis ver- a result of which ITZ loaded SLN was de- sicolor (the most common cutaneous derma- ployed for topical ocular delivery through tologic conditions worldwide) and observed melt-emulsion sonication and low tempera- impressive outcomes than candistan (clotri- ture-solidification technique. Many studies mazole) cream. The researchers used modi- are being conducted in the world today con- fied high shear homogenization and ultrason- cerning the improvement of therapeutic meth- ication method to prepare SLN loaded with ods for corneal fungal infections due to the FLZ and then used Carbopol (CP 934) as a poor bioavailability of antifungal drugs aris- gelling agent to prepare FLZ-SLN topical gel. ing from various protective mechanisms of It has been reported that one of the advantages eyes. One of the preferred mechanisms in ad- of using FLZ -loaded SLNs topical gel is that vancing drugs for better penetration in the eye the treatment of this fungal infection via pure is the use of these nanosystems. The research- FLZ is feasible solely through oral therapy; ers reported the antimicrobial efficacy of thus, the topical application of this drug for- these formulations by investigating Aspergil- mulation can produce better results such as lus flavus (Mohanty et al., 2015). Other im- improved skin penetration due to enhanced portant triazoles are voriconazole (VRZ) that contact between FLZ and skin. The research- has been used in the treatment of candidiasis ers performed a clinical study of over 30 pa- and aspergillosis infections in skin together tients with Pityriasis versicolor to evaluate with keratitis following topical administration FLZ-SLNs topical gels whose findings were (Marangon et al., 2004; Al-Badriyeh et al., contrasted against Candistan cream. In this 2010). Studies have suggested that nano- study, each patient was followed up by a clin- based VRZ formulation can be used to en- ical and mycological examination every week hance VRZ permeation (Nassiri-Kashani et during therapy to assess the clinical efficacy al., 2016). One of those studies was the paper and safety of different treatment regimens. of Kumar and Sinha (2016) that comprehen- The researchers eventually reported that the sively examined SLNs for improved ocular formulation appeared to be faster and more delivery of VRZ, with satisfactory results ob- effective than topical Candistane (El-Housiny tained both in vitro and in vivo. They reported et al., 2018). All of these studies on various

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azoles have indicated that the use of SLNs as tration capacity over pure drugs. Based on re- an antifungal drug delivery system, especially search on rabbits’ eyes, it was confirmed that for topical medicinal applications, has several AmB-CH-NLC could successfully penetrate important merits including good skin penetra- the cornea (Fu et al., 2017). On the basis of tion, reduced side effects, biocompatibility, the findings of this study, the AmB-CH-NLC and enhanced therapeutic efficacy. system may also be suggested as a treatment Another SLN study was carried out on for fungal keratitis in the future given the dif- GF, a drug frequently used to treat dermato- ficulty of choosing the right drug for keratitis phytosis. However, today, considering the today. several shortcomings of this drug such as its poor oral bioavailability and some other side Cubic liquid crystalline nanoparticle effects, the topical application of drug deliv- (CUBOSOMES) ery system of GF has been proposed. For this Cubosomes are extremely firm NPs purpose, Anurak and his colleagues in 2011 formed from the lipid cubic phase and stabi- developed a GF-loaded SLN by microemul- lized by a polymer-based outer corona sion method. The researchers showed that us- (Barriga et al., 2019). The cubosomes as drug ing this formulation makes GF release pro- carriers have a small size, low viscosity, large files be a delayed-release and also makes interfacial areas, and a hydrophobic core. The large scale production of SLNs quite viable cavern-like structure of these NPs lead to ef- (Anurak et al., 2011). ficient drug loading (Yang et al., 2012; Zhang et al., 2020). These NPs have properties such Nanostructured lipid carrier as high biocompatibility and bioadhesive that Another drug delivery system is led Yang and his colleagues in 2014 to pre- nanostructured lipid carriers (NLCs), being pare AmB which is loaded in glyceryl composed of both solid and liquid lipids as a monoolein (GMO) cubosomes to hone the core matrix (Aghebati‐Maleki et al., 2020). oral delivery of AmB. They used rats in this The utilization of these NPs in combination in vivo study and reported that using these with antifungals yielded increased solubility. NPs could facilitate the oral delivery of AmB The diameter of NLCs ranges from 10 to 1000 (Xu et al., 2014). nm so their minute particle size affects their solubility, biocompatibility, and rate of drug Silver nanoparticle release. In addition, the shape of NLCs betters Various studies on silver-containing com- encapsulation efficiency, affects cellular up- pounds have shown that silver is a broad- take and receptor binding. NLCs as drug car- spectrum antimicrobial agent, containing dis- riers improve permeability and bioavailability ruptive microbial cell membrane as well as an and lead to enhanced storage stability and interfering power with DNA replication prolonged half-life. Eventually, using NLCs (Bhabra et al., 2009; Vazquez-Muñoz et al., as drug carriers protect the drugs from degra- 2014). Silver NPs (AgNPs) also have unique dation in the body (Fang et al., 2013; Haider physical and chemical properties such as elec- et al., 2020). In 2017, Fu et al. investigated the trical, thermal, high electrical conductivity, efficacy of AmB loaded, chitosan-modified, and biological properties that lead these NPs nanostructured lipid carriers (AmB-CH- to be widely used in medicine (Nozari et al., NLC) for the effective ocular delivery of 2012; Zhang et al., 2016). Ahmad’s study in AmB for fungal keratitis using solvent emul- 2016 involved Candida albicans and Candida sification and low temperature-solidification tropicalis using AmB-conjugated biogenic technology. It was reported that due to the cat- silver NPs (AmB-bAgNPs). What they found ionic and mucoadhesive properties of CH, in this study was that AmB-bAgNPs have a AmB-CH-NLC obtains better corneal pene- significant effect on enhancing their antifun- gal activities against Candida spp. compared

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to pure AmB. It was also reported that the an- NPs could damage Candida cells by generat- tifungal stability of AmB-bAgNPs remained ing reactive oxygen species (ROS) (Thakur et higher than the pure AmB for several days al., 2019). Szerencsés showed the same re- and this formulation could reduce the thera- sults by using citrate-coated AgNPs to inhibit peutic cost and dose of AmB as well as its tox- the biofilm formation of Candida (Szerencsés icity (Ahmad et al., 2016). Another study in et al., 2020). this area belongs to Tutaj et al., (2016) who worked on hybrid-AmB silver NPs (AmB-Ag Poly-lactic-co-glycolic acid nanoparticle NPs). By investigating the antifungal activity Many studies have suggested the use of of these NPs in Aspergillus niger, Candida al- PLGA NPs in the drug delivery system to op- bicans and Fusarium culmorum strains com- timize the therapeutic efficacy of many clas- pared to Fungizone (AmB), the researchers sical drugs and their toxicity as a result of the reported that the formulation had a higher an- decreased dose of the drug. Several notewor- tifungal trait. They, therefore, suggested that thy benefits of PLGA NPs include biocompat- using these NPs could open up a new avenue ibility, drug compatibility, and mechanical in the treatment of pathogenic fungi responsi- properties; besides, its hydrolysis results in ble for severe mycotic infections (Tutaj et al., the formation of monomers of lactic acid and 2016). An additional research in this area was glycolic acid metabolites. Since these two that of Hussain et al. (2019) who investigated monomers are androgens and are easily me- the enhanced antifungal efficacy of nystatin tabolized by body through the Krebs cycle or (NYS) and FLZ after conjugation with Ag; to TCA cycle, PLGA is associated with poor this end, they designed NYS- and FLZ-coated systemic toxicity. PLGA NPs protect the drug Ag. The researchers employed strains of Can- against degradation and improve its stability dida albicans ATCC 10231 and Aspergillus (Yang, 2019). Among these studies, a study brasiliensis ATCC 16404 using the agar tube by Amaral et al. (2010) can be pointed out, in dilution method to perform in vitro studies. which immunoprotective peptide P10 (the What they discovered was that the combina- major diagnostic antigen secreted by Para- tion of Ag with NYS and FLZ may have clin- coccidioides brasiliensis) loaded on PLGA ical implications in the treatment of fungal in- was utilized to treat paracoccidioidomycosis. fections after observing the enhanced antifun- Male BALB/c mice infected with Para- gal effects of NYS-Ag and FLZ-Ag (Hussain coccidioides brasiliensis (inoculated intratra- et al., 2019). Mussin ’s study in 2019 visual- cheally) were used in this study. They also de- ized the interaction between AgNP- Malas- ployed animal models for which a combina- sezia furfur and evaluated the synergism with tion of sulfamethoxazole/trimethoprim with KTZ to produce an antimicrobial gel based on the P10 peptide entrapped within PLGA was carbopol formulated with AgNP–KTZ. Inves- utilized and observed fulfilling results in tigating the antifungal activity of this gel al- some treatments due to P10 elicits, a Th1-like lowed the improvement of the topical therapy immune response which can control fungal of superficial mycoses, reduction in the num- infection (Amaral et al., 2010). In 2014, Liu ber of applications, and prevention of relapse and his colleagues managed to prepare AmB- (Mussin et al., 2019). Thakur’s study in 2019 loaded diblock copolymer D-α-tocopheryl indicated that the zinc ferrite nanoparticles polyethylene glycol 1000 succinate-b-poly (ε- and silver nanowires (ZnFe2O4@AgNWs) caprolactone-ran-glycolide) (PLGA-TPGS) manifest both biofilm inhibition capacity for by a modified nanoprecipitation method. To Candida albicans cells and potentially novel investigate the effect of these NPs, they first antifungal activities. They showed that performed antifungal activity in vitro on a AgNWs penetrated the biofilm matrix and strain of Candida albicans using a paper-plate transported ZnFe2O4 NPs into it, so ZnFe2O4 technique and reported that AmB-NPs have an antifungal activity similar to that of free

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AmB. The in vivo therapeutic efficacy of production of NPs. Several noticeable ad- AmB-NPs was then tested using an animal vantages of gelatin as NPs are their biode- model of BALB/c mice which showed high gradable, non-toxic, non-immunogenic bio- stability of AmB-NPs in this study compared compatible nature with human tissues as well to free AmB (Liu et al., 2014). Other studies as their easy crosslinking and potential to include Das’ study on pulmonary delivery of modify chemically. Gelatin NPs are delivered VRZ as a result of which PLGA NPs contain- to special tissues without compromising drug ing antifungal drug VRZ was developed. The stability and concentration which is consid- in vivo investigations indicated that the accu- ered a plus point (Singh and Mishra, 2014; mulation of VRZ-loaded PLGA in lungs was Sabet et al., 2017). In a study by Ahsan and significantly higher than that of a pure drug; Rao in 2017 on controlling infection and in- in addition to this, the prolonged retention of flammation in keratitis, they prepared double- particles in lungs was observed. They finally desolvation KTZ loaded gelatin nanostruc- showed that VRZ-loaded PLGA is a proper tures. In their study on rats’ corneas infected system for delivering a drug in deep lung tis- with Aspergillus flavus, the researchers found sue in high concentrations for a prolonged pe- that these NPs can enhance drug residence riod (Das et al., 2015). Working on AmB en- time, down-regulate inflammation, and re- trapped within PLGA and incorporated with lease antifungal drugs (Ahsan and Rao, 2017). dimercaptosuccinic acid (NANO-D-AMB), affirmed by the in vitro and in vivo findings, Alginate nanoparticle helped confirm that NANO-D-AMB im- Alginic acids or alginates are natural pol- proves AmB delivery (Souza et al., 2015). ymers consisting of linear copolymers and Radwan et al. (2017) prepared AmB loaded to varying amounts of (1→4′)-linked β-d-man- PEGylated polylactic-polyglycolic acid co- nuronic acid and α-l-guluronic acid residues polymer (PLGA-PEG) NPs and observed that (Rehm and Valla, 1997). Alginate is often ob- the MIC of AmB loaded to PLGA-PEG NPs tained from brown algae and certain bacteria against Candida albicans was reduced com- and is used for drug delivery applications pared with fungizone (free AmB). On the nowadays (Spadari et al., 2017). Alginate is a other hand, studies on rats disclosed that the natural, non-toxic, biocompatible, biode- oral delivery system of the NPs manifested gradable, water-soluble, and non-immuno- minimal toxicity and better efficacy com- genic polymer. One of the noticeable benefits pared to fungizone (Radwan et al., 2017). In of alginate as NPs is the sustained release of another study, Ahmed and Aljaeid (2017) de- drugs so that they can be constantly released veloped optimized ketoconazole (KTZ) into the tissue. The different drug release PLGA NPs with in situ gel (ISG) formulation mechanisms of these NPs are diffusion and for ophthalmic drug delivery in an attempt to matrix erosion for water-soluble drugs and streamline ocular KTZ delivery. Using this poorly water-soluble drugs, respectively formulation, the researchers observed the en- (Spadari et al., 2019). In this regard, Pandey hanced antifungal activity against the stand- and his colleagues worked on two CLT and ard strain of Candida albicans ATCC 76615 ECN drugs in 2005 and managed to produce and also reported that these NPs could be used cation-induced controlled gelification of algi- in the treatment of both keratitis and endoph- nate, CLT-loaded alginate nanoparticles, and thalmitis owing to their ability for ECN-loaded alginate nanoparticles. These transcorneal permeation (Ahmed and Aljaeid, two NPs were orally administered to mice and 2017). thus improved the oral bioavailability of the drugs. Other benefits of this formulation are Gelatin nanoparticle their simplicity of preparation and increased Gelatin (a natural polymer derived from duration of sustained drug release (Pandey et collagen) is one of the substances used in the al., 2005). Sangeetha et al. (2007) scrutinized

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systemic candidiasis both in vitro and in vivo Chitin nanoparticle (the in vivo study was carried out on candidi- Other NPs used today are chitin nanogels asis induced mice models) conditions. So- (CNGs) which are natural polymeric NPs dium alginate nanospheres of AmB were pre- with great potential in the field of nanothera- pared by the controlled gellification method peutics. Chitin is an excellent material for the in this study. Having compared the nano- synthesis of nanoparticles as a natural renew- sphere-bound drug with the free drug, the re- able resource, demonstrating biodegradable, searchers reported that upon using this formu- biocompatible, and non-toxicity traits (Zhang lation, a significant reduction in colony-form- et al., 2018; López et al., 2020). Mohammed ing units (CFU) in the liver and lungs was ob- and colleagues used FLZ-loaded chitin nano- served. This finding, in turn, contributes to a gels (FLZ-CNGs) in the treatment of corneal reduction in the total dose required for ther- fungal infections in 2013 to overcome barriers apy and, consequently, the degree of drug tox- such as minimal dose absorption due to icity (Sangeetha et al., 2007). unique anatomy and physiology of eyes. By producing these NPs, they managed to detect Magnetic nanoparticle the good antifungal activity of this formula- Magnetic NPs (MNPs) (such as iron oxide tion in comparison to the pure drug against NPs) are a class of NPs that can be manipu- Candida tropicalis, which has a determining lated using magnetic fields and due to mag- role in higher uptake by fungal cells. The re- netic properties, antibiotics included in such searchers used the cornea obtained from the complexes can be delivered to specific areas porcine eye to investigate ex vivo corneal per- using a magnetic field (Akbarzadeh et al., meation which ultimately demonstrated the 2012; Niemirowicz et al., 2016). Magnetic effective penetration of FLZ-CNGs into the nanoparticles possess multimodal targeting deeper sections of the cornea (Mohammed et potential and have thermal therapy applica- al., 2013). tions due to their ability to convert energy from an alternating magnetic field to thermal Chitosan nanoparticle energy (Hauser et al., 2015). In 2016, Niemi- Chitosan obtained by chitin acetylation is rowicz and his colleagues studied two poly- suitable for the formation of nanogels. Chi- ene antifungals, AmB and NYS, using Mas- tosan is a non-toxic and biocompatible poly- sart's procedure; namely, MNP-AmB and mer with strong mucoadhesive properties, MNP-NYS. After comparing MNP with pure which is used now either as a matrix or coat- drugs on Candida strains, they reported that ing for NPs (Onyebuchi and Kavaz, 2019). In these formulations were able to prevent Can- 2013, Song et al. succeeded in preparing dida biofilm formation and improve their bi- AmB-loaded NPs based on poly (lactic acid)- ocompatibility (Niemirowicz et al., 2016). grafted-chitosan (AmB/PLA-g-CS) for the Muñoz-Escobar and Reyes-López investi- ocular delivery of AmB by dialysis. In vitro gated the antifungal activity of polycaprolac- antifungal activity was performed by re- tone-copper fibers (PCL-CuONPs) in Can- searchers against Candida albicans using the dida albicans, Candida glabrata, and Can- M27-A3 (2008) guidelines of clinical and la- dida tropicalis in 2020. They showed that the boratory standards institute indicating that formation of mycelia was inhibited in the AmB-loaded NPs have an antifungal potential presence of synthesized NPs, a pre-requisite similar to that of the free drug. After investi- in the dimorphic transition from yeast to my- gating ocular pharmacokinetics, they reported celial form in superficial fungal infections the prolonged residence time on the ocular caused by Candida (Muñoz-Escobar and surface. On the other hand, in a corneal pene- Reyes-López 2020). tration study, they observed the penetration of NPs into the cornea (Song et al., 2013). In an- other study, Vasquez-Marcano et al., in 2018

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used AmB-loaded chitosan-coated poly (ε-ca- NPs not only evinced higher intracellular kill- prolactone) NPs (AmB-loaded CS-coated ing of Candida albicans in infected macro- PCL NPs) produced by the nanoprecipitation phages of female BALB/C mice, but also in- method for the oral delivery of AmB and a re- hibited yeast to hyphae transition, and en- duction in its toxicity. They also revealed that hanced in vivo efficacy and reduced toxicity by using NPs, the antifungal activity against (Khan et al., 2020). Candida parapsilosis strain intensifies as against the pure AmB. It was also divulged Polycaprolactone nanoparticle that these NPs could safely release AmB via Polycaprolactone (PCL) is one of the syn- the oral route (Vásquez Marcano et al., 2018). thetic biodegradable polymers for drug deliv- Paul and his colleagues also achieved similar ery and biomedical applications. PCL NPs de- results in 2018 by preparing Chitosan-coated pict low particle size and high flexibility. poly lactic-co-glycolic acid NPs of VRZ PCL-based NP formulations have slow re- (VChNP), affirming that using this formula- lease due to high crystalline nature. Also, tion, compared with free VRZ, depicts effec- these NPs have less water permeability and tive pulmonary delivery and boosted bioavail- consequently a very slow degradation rate ability (Paul et al., 2018). Sandhya et al. (Ashour et al., 2019; Muñoz-Escobar and (2018) also observed that by preparing AmB Reyes-López, 2020). Chandasana et al. inves- encapsulated sulfated CS NPs (AmpB- tigated natamycin for the treatment of my- SCNPs), synthesized NPs manifested higher cotic keratitis (natamycin 5 %, FDA approved intracellular killing of Candida glabrata in in- and commercially available for topical ocular fected RAW 264.7 macrophage cell lines. use) and benefited from the formulation of na- Studies have shown that both lectin and CS tamycin encapsulated poly-D-glucosamine are both biocompatible and biodegradable functionalized polycaprolactone NPs (PDG- polymers, which is why Lecithin/chitosan na- PCL NPs) and finally succeeded in devising a noparticles (L/CS NPs) are highly regarded in targeted corneal nanoformulation to reduce the drug delivery system these days. To this the dose and dosing frequency of natamycin. end, Chhonker et al. made use of AmB loaded Using this method hand in hand with the in L/CS NPs prepared by ionic gelation and vitro studies, the prolonged release of na- achieved the prolonged release of AmB with tamycin was reported and subsequently, the in a reduction in the dosage of pure drug and the vivo conditions yielded highly satisfactory consequent side effects. In the in vitro study outcomes as well (Chandasana et al., 2014). of Candida albicans and Aspergillus fumiga- In the same vein, Lucena et al. (2018) also tus, the researchers perceived the potential an- achieved impressive results by preparing ITZ tifungal efficacy by preparing AmB loaded loaded PCL nanocapsules (ITZ-NC) for the L/CS NPs and similarly, based on the in vivo treatment of vulvovaginal candidiasis after study conducted in New Zealand on albino topical application in the vagina. Having con- rabbit eyes, improved bioavailability com- ducted the in vivo study of female BALB/c pared to the free drug was noted. Thus, they mice, the researchers observed the effect of reported that this formulation holds a higher ITZ-NC formulation on reducing Candida al- ocular tolerance than the marketed formula- bicans and the arising inflammation. Based on tion, however, further studies are necessary their studies, it was reported that their for commercial applications (Chhonker et al., nanocapsules have the potential to increase 2015). Khan and his colleagues (2020) syn- drug contact with the mucosa and open new thesized Methylglyoxal Chitosan Nanoparti- perspectives for the treatment of this disease cles (MGCN) against fluconazole-resistant (Lucena et al., 2018). All studies are summa- Candida albicans and investigated them both rized in Table 1. in vitro and in a mouse model. Synthesized

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Table 1: Studies conducted in nanoparticles for antifungal drugs delivery Target(s) Antifungal Nanoparticle type Admin- References agent istra- tion Trichophyton rubrum AmB SLN Topical Butani et al., 2016 N/A CLT SLN Topical Souto et al., 2004 Candida albicans CLT SLNs based on polyoxy- N/A Cassano et al., ethylene-40 stearate 2016 N/A ECN SLN Topical Sanna et al., 2007 N/A MN SLN Topical Bhalekar et al., 2009 Candida species MN SLN-bearing hydrogel Topical Jain et al., 2010 Candida albicans MN SLN Oral Aljaeid and Hosny, 2016 Candida species MN SLN Oro- Kenechukwu et al., mucosal 2017 Aspergillus flavus ITZ SLN Ocular Mohanty et al., 2015 N/A VRZ SLN Ocular Kumar and Sinha, 2016 Candida glabrata & VRZ SLN Ocular Füredi et al., 2017 Aspergillus flavus Candida species FLZ SLN Topical Gupta et al., 2013 Malassezia species FLZ SLN Topical El-Housiny et al., 2018 Dermatophyte GF SLN N/A Anurak et al., 2011 N/A AmB CH-NLC Ocular Fu et al., 2017 N/A AmB (GMO) cubosomes Oral Xu et al., 2014 Candida albicans & AmB Ag N/A Ahmad et al., 2016 Candida tropicalis Aspergillus niger, AmB Ag N/A Tutaj et al., 2016 Candida albicans & Fusarium culmorum Candida albicans & NYS & FLZ Ag N/A Hussain et al., Aspergillus brasili- 2019 ensis Malassezia furfur KTZ Ag Topical Mussin et al., 2019 Candida albicans N/A ZnFe2O4@AgNWs N/A Thakur et al., 2019 Candida species N/A citrate-coated Ag N/A Szerencsés et al., 2020 Paracoccidioides Sulfameth- PLGA N/A Amaral et al., 2010 brasiliensis oxazole/tri- methoprim Candida albicans AmB PLGA-TPGS N/A Liu et al., 2014 N/A VRZ PLGA Pulmo- Das et al., 2015 nary Paracoccidioides Amb PLGA N/A Souza et al., 2015 brasiliensis Candida albicans AmB PLGA-PEG Oral Radwan et al., 2017 Candida albicans KTZ PLGA Ocular Ahmed and Aljaeid, 2017 Aspergillus flavus KTZ Gelatin Ocular Ahsan and Rao, 2017 N/A CLT & ECN Alginate Oral Pandey et al., 2005

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Candida species AmB Sodium alginate N/A Sangeetha et al., 2007 Candida species AmB & NYS Magnetic N/A Niemirowicz et al., 2016 Candida albicans, N/A PCL- CuO N/A Muñoz-Escobar Candida glabrata & and Reyes-López Candida tropicalis 2020 Candida tropicalis FLZ CNG Ocular Mohammed et al., 2013 Candida albicans AmB PLA-g-CS Ocular Song et al., 2013 Candida parapsilosis AmB CS-coated PCL Oral Vásquez Marcano et al., 2018 N/A VRZ CS Pulmo- Paul et al., 2018 nary Candida glabrata AmB Sulfated CS N/A Sandhya et al., 2018 Candida albicans & AmB L/CS N/A Chhonker et al., Aspergillus fumigatus 2015 Candida albicans N/A MGCN N/A Khan et al., 2020 N/A Natamycin PDG-PCL Ocular Chandasana et al., 2014 Candida albicans ITZ PCL Topical Lucena et al., 2018

TOXICITY OF NANOPARTICLES and inconsistent (Badman et al., 2020). Toxi- NPs are being increasingly employed in a cological studies provide a sound grounding variety of sectors. NPs have been studied for for the protection of both humans and envi- cell toxicity, immunotoxicity, and genotoxi- ronment. Therefore, on the basis of available city. Tetrazolium-based assays such as MTT, experimental models, it may be inaccurate to MTS, and WST-1 are used to determine cell list some of the more valuable NPs as being viability. Cell inflammatory response induced toxic to biological systems and vice versa. by NPs is checked by measuring inflamma- Considering the potential applications of NPs tory biomarkers, such as IL-8, IL-6, and tu- in many fields and to address the knowledge mor necrosis factor, using ELISA. Lactate de- gap, the relevant toxic effects of NPs should hydrogenase assay is used for cell membrane be assessed by utilizing internationally agreed integrity (Bahadar et al., 2016). Different and unbiased in vivo toxicological models, types of cell cultures, including cancer cell targeting the vital systems (Pacheco-Blandino lines have been employed as in vitro toxicity et al., 2012). However, we are of the opinion models. It has been generally agreed that NPs that designing, adapting, and validating such interfere with either assay materials or detec- new models in the future for toxicity testing, tion systems. Based on multiple applications route of exposure, coating material and steril- ity of NPs, and type of cell cultures need to be in multiple areas, human exposure to NPs, both intentionally and unintentionally, is in- carefully examined (Crisponi et al., 2017). evitable. Before being considered for human Moreover, the US FDA as a public health application, all nanoproducts are subjected to agency has also recently taken the important toxicological studies and for this purpose, issue of toxic effects associated with products several experimental studies have been car- containing NPs into account and does not ried out. To meet this regulatory requirement, consider them either totally safe or harmful some toxic effects of nanomaterials have been for human use, maintaining that each product evaluated, but according to reports, the toxi- must be subjected to regulation (Bahadar et cological data derived so far are conflicting al., 2016).

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