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Biodegradable chitosan nanoparticles in drug delivery for infectious disease

Increasing rates of antimicrobial resistance have left a significant gap in the standard antimicrobial armament. Nanotechnology holds promise as a new approach to combating resistant microbes. Chitosan, a form of deacetylated chitin, has been used extensively in medicine, agriculture and industry due to its ease of production, biocompatibility and antimicrobial activity. Chitosan has been studied extensively as a main structural component and additive for nanomaterials. Specifically, numerous studies have demonstrated its potent microbicidal activity and its efficacy as an adjuvant to vaccines, including mucosally administered vaccines. In this review, we present fundamental information about chitosan and chitosan nanoparticles as well as the most recent data about their antimicrobial mechanism and efficacy as a nanotechnology-based drug delivery system. Keywords:  antibacterial • antifungal • antimicrobial • antimicrobial resistance • chitosan • chitosan nanoparticles • vaccine adjuvant

Despite recent advances and efforts to combat infectious disease, microbial drug resistance continues to be one of the greatest challenges for healthcare. Throughout the 20th century, morbidity and mortality from infectious disease had been declining, a phenomenon ascribed to the development of antibiotics, starting with penicillin in the 1940s. However, the pace of antibiotic/antifungal development has fallen behind microbial mutation rates and resistance to these agents has reached a critical level [1–3] . Recently, the dynamic pattern of infectious disease and the emergence of bacterial strains resistant to traditional antimicrobials have caused the biomedical field to turn toward emerging drug delivery platforms. Nanotechnology is one such avenue – a scientifically diverse discipline that encompasses engineering, materials science, physics, chemistry, and the biological sciences. Nanotechnology exploits the complex and remarkably unique properties of matter at the nanoscale, owing largely to a very large ratio of surface area-to-volume. A nanoparticle

10.2217/NNM.15.7 © 2015 Future Medicine Ltd

Angelo Landriscina‡,1, Jamie Rosen‡,1 & Adam J Friedman*,1,2 Department of Medicine (Division of Dermatology), Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA 2 Department of Physiology & Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA *Author for correspondence: adfriedm@ montefiore.org ‡ Authors contributed equally to the production of this work 1

may consist of just a few atoms or may be just a few atoms thick. Given the surface area-tovolume relationship, one can appreciate that a large percentage of the atoms that make up the nanostructure reside on its surface. The behavior of these surface atoms confers many of the unique properties associated with matter at the nanoscale [4,5] . Surface atoms are relatively reactive, having fewer neighbors to share chemical bonds. This facilitates the attachment of a variety of molecules (antibiotics, nucleotides, proteins, antibodies, aptamers) to nanostructured surfaces by chemical and electrostatic means [1] . The optical and electronic properties of the surface atoms behave differently than at the bulk scale. Given these unique properties, antimicrobials at the nanoscale could be designed to have a higher affinity to bacteria and fungi, overcome barriers or resistance mechanisms, and therefore enhance antimicrobial activity. Size alone would confer some benefit as most pathogens measure on the higher end of the nanoscale or even micron scale and therefore the likelihood of interaction with treatment is

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Review  Landriscina, Rosen & Friedman much greater [2] . Lastly, nanomaterials can be designed to deliver established or developing drugs in a sustained, controlled and even targeted manner to avoid associated adverse systemic side effects – ­ flexibility, which confers limitless possibilities [6] . Chitosan is a naturally occurring and abundantly available biocompatible polysaccharide derived from chitin, a principal component of the crustacean exoskeleton. Both chitin and chitosan are biocompatible, biodegradable and nontoxic polymers with several applications in wound healing, drug delivery and tissue engineering. Chitosan itself is a widely used antimicrobial agent with activity against bacteria, fungi and viruses  [7] . Chitosan can be structurally and chemically manipulated to generate advantageous properties, functions and applications within the biomedical field. These intriguing properties have been well-known within agriculture, industry and medicine. Chitosan has uses within agriculture as a plant antiviral in fertilizers  [8] , within industry as a strengthening additive in paper [9] and chelating agent for detoxification of hazardous waste [10] . It has also been noted for its anticoagulant properties [11] , application as a biomaterial given its immunostimulatory effects [12] , promoter of wound healing [13,14] and antibacterial and antifungal properties  [15] . Chitosan’s versatility and adaptability allow for unique opportunities in the development of new antimicrobial therapies. These unique properties also make it a prime structural component and additive for nanoparticle platforms in drug delivery. Numerous studies have demonstrated the efficacy of chitosan nanoparticles against a variety of Gram-positive and -negative bacteria, as well as fungi, with chitosan nanoparticles showing increased efficacy compared with chitosan alone [16] . The clinical potential for chitosan nanoparticles to combat the increasing prevalence of microbial resistance may represent a new paradigm for treating infectious disease [1] . The following provides an overview on the antimicrobial properties of chitosan nanoparticles as a nanotechnology-based drug delivery system. Intrinsic properties of Chitosan Chitosan is a linear copolymer of β-(1→4)-linked monosaccharides (2-acetamindo-2-deoxy-β-d-glucopyranose and 2-amino-2-deoxy-β-d-glucopyranose) obtained by the deacetylation of chitin under alkaline conditions or by enzymatic hydrolysis with chitin deacetlyase (Figure 1) . While chitin is defined as a polymer of acetyl-amino-d-glucose units, chitosan’s chemical composition has been more loosely defined. Chitosan is a term applied to a collection of deacetylated chitins; chitosan is a family of polymers with functional properties influenced by the number of sugar units per polymer

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molecule, the molecular weight and degree of deacetlyation [17] . Multiple studies have shown that differences in these parameters impact the expressed properties of chitosan thus altering its biological activity, antimicrobial efficacy and ability to enhance drug delivery [3] . Following cellulose, chitin, the parent molecule of chitosan, is the second most abundant natural polymer. While structurally similar to cellulose, the primary amine group confers chitosan special properties that differentiate and make the polymer extremely useful in drug delivery. Chitosan’s utility as an antimicrobial agent depends significantly on its polycationic structure. Studies have shown that the inhibitory effects of chitosan are altered by the degree of deacetylation, molecular weight, pH, water solubility and type of bacterium  [18–21] . With regard to these parameters, many reports discuss the antimicrobial activity of chitosan with conflicting results. An increase in positive charge, associated with a higher degree of deacetylation (or lower degrees of acetylation), will result in stronger electrostatic interactions. A recent study found that the antibacterial effect of chitosan was strengthened as the degree of acetylation decreased [22] . Chung and Chen similarly found that a low degree of acetlyation confers stronger antibacterial activity against Escherichia coli and Staphylococcus aureus  [23] . An earlier study found that an increased degree of deacetylation leads to a higher positive charge density conferring stronger antibacterial activity against S. aureus than low degrees of deacetylation [24] . Furthermore, Mellegard et al. similarly found that chitosans with a high degree of deacetlyation were more active than the more acetylated chitosans, thus enhancing chitosan’s polycationic structure and conferring enhanced antimicrobial activity [25] . The correlation between chitosan’s molecular weight and bactericidal activity has been less clear. Some studies report low molecular weight chitosan generates a higher inhibition rate against Gramnegative bacteria (i.e., E. coli, Klebsiella pneumoniae, ­Pseudomonas aeruginosa), while the reverse is observed with Gram-positive bacteria – with increased molecular weight generating a greater antibacterial response [26] . This finding was confirmed by another investigation, revealing that that higher molecular weight chitosan had a more pronounced impact against Gram-positive species, Proprionibacterium acnes and S. aureus, than lower molecular weights [27] . This effect was differentiated by comparing chitosan-treated S. aureus (Grampositive) and E. coli (Gram-negative); authors suggest two mechanisms for the differing inhibitory effects: in the case of Gram-positive species, chitosan is able to form a polymeric membrane on the cell surface, preventing nutrients from entering the cell and for Gram-negative species, low molecular weight chitosan

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is able to penetrate the microbial cell and disturb its metabolism [28] . Lastly, pH is an important parameter that extends chitosan’s antimicrobial activity. Its free amino groups render chitosan a cationic polysaccharide in neutral and alkaline pH conditions. The use of chitosan is limited given its insolubility in water and its tendency to coagulate with proteins at high pH. Chitosan’s loss of bactericidal activity in alkaline pH environments may be explained by the lack of positively charged amine groups [29] . However, chitosan does show antibacterial activity in acidic mediums, where the degree of protonation is largest [19,22] . When the pH is below the respective pKa, the amine groups of the polymer are protonated, making chitosan soluble in water. Chitosan as an antimicrobial agent Chitosan’s antimicrobial properties have gained considerable attention in recent years. Chitosan, being a polycationic polysaccharide, demonstrates a broad spectrum of antimicrobial activity against a variety of microorganisms including bacteria, fungi and viruses with differing inhibitory efficiency [15] . Many studies report chitosan’s stronger bactericidal effect against Gram-positive bacteria than Gram-negative bacteria. While chitosan markedly inhibited growth of most bacteria, No et al. found chitosan to have stronger bactericidal activity against Gram-positive bacteria (Listeria monocytogenes, Bacillus megaterium, B. cereus, S. aureus, Lactobacillus plantarum, L. brevis, L. bulgaricus) than Gram-negative bacteria (E. coli, Pseduomonas fluorescens, Salmonella typhimurium, Vibrio parahaemolyticus)  [19] . As previously discussed, chitosan’s antimicrobial efficacy is enhanced in sufficiently low pH environments; the acidity of the solution exposes a larger positive charge density, enabling chitosan’s amine groups to interact with negatively charged microbial cell walls and cytoplasmic membranes [13,18,19,30] . The inhibitory activity of chitosan against Gramnegative bacteria is closely correlated with cell surface characteristics. Chitosan’s polymeric macromolecular structure prevents its passage through the hydrophobic outer membranes of Gram-negative bacteria. Since direct access is unlikely, chitosan’s positively charged amine groups allow its interaction with anionic components such as lipopolysaccharides and surface proteins, disrupting the integrity of the outer membrane  [31] . The positive charge may also compete with the positively charged cations (Ca 2+, Mg2+) that are typically present in the cell wall, causing the destabilization of lipopolysaccharide and disrupting the barrier function of the cell wall [3,32] . Je et al. further elucidated this mode of action via 1-N-phenyl-naphthylamine assays [33] ; authors found that that the addition

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CH2OH

CH2OH

O

O O

OH NH2

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OH NH2

Figure 1. Structure of chitosan [poly(β1→4- d glucosamine)]. Reprinted from [73] with permission from Elsevier.

of dimethylaminoethyl-chitosan to E. coli resulted in increased fluorescence, an indication of cell membrane damage. This same study also found that dimethylaminoethyl-chitosan increased the permeabilization of the inner membrane of E. coli as evaluated by the release of cytoplasmic B-galactosidase. Several reports support this hypothesis, showing the leakage of cell constituents (glucose and lactate dehydrogenase) from E. coli cells following exposure to chitosan [34] . On the other hand, the cell wall of Gram-positive bacteria is comprised of peptidoglycan and teichoic acid. Data has suggested that contact between chitosan and the negatively charged cell wall is driven by electrostatic forces. Some report chitosan’s antibacterial mechanism on Gram-positive bacteria is driven by its molecular weight. High-molecular-weight chitosan is able to form a polymeric membrane, depriving the cell of nutrients and resulting in cell death [28] . Alternatively, low molecular weight chitosan is able to permeate the cell wall and inhibit mRNA and protein synthesis via its interaction with negatively charged DNA [30] . Teichoic acid also clearly plays an important role in this interaction as seen with the teichoic acid deletion mutant, found to be the most resistant strain to the antimicrobial activity of chitosan [35] . The lack of teichoic acid results in a less negatively charged cell wall, substantiating the hypothesis that the polycationic structure of chitosan is the major antibacterial factor against Grampositive bacteria. Regardless of its target on Gram-positive or -negative cell walls, chitosan’s binding to cell wall polymers disrupts bacterial membrane functions. This enables chitosan to gain entry into microbial cell membranes resulting in osmotic instability, membrane disruption and ­ eventually l­eakage of intracellular ­elements  [13,18,19,30] . Despite its virulence against bacteria, many reports have found chitosan to be a more efficacious antifungal agent. Chitosan’s antifungal activity has been reported in a variety of fungi [22,36–40] . The literature does not reveal general trends in fungal inhibition with regard to chitosan’s characteristics. The effect of molecular weight and degree of deacetylation has been investigated on Rhizopus stolonifer  [37] , Aspergillus niger  [41] ,

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Review  Landriscina, Rosen & Friedman Fusarium oxysporum and Alternaria solani  [22] , finding no unique patterns. Thus we can conclude that the differences in antifungal activity are influenced by chitosan’s distinct inhibition activity on each microorganism rather than chitosan’s intrinsic characteristics. However, through scanning and transmission electron microscopy, chitosan was found to exert its antifungal activity by suppressing mycelial growth, sporulation and spore germination via in vitro germination with R. stolonifer [37] . Fungi are also capable of forming biofilms on foreign body materials, prolonging infection and making treatment challenging. In vitro and in vivo experiments found that chitosan significantly inhibited biofilm formation on Candida albicans-infected catheters [42,43] . The antibiofilm properties of chitosan are attributed to chitosan’s interference with surface colonization/adhesion by microbes with chitosan demonstrating a profound effect on the negative charge of the cellular membrane [15,42] . A similar study revealed that chitosan significantly reduced the metabolic activity of the biofilms (as seen by XTT reduction assays) and cell viability (as seen by CFU determinations) of chitosan treated Cryptococcus neoformans  [44] . By immunofluorescence authors concluded that chitosan damages melanin, a component of the polysaccharide capsule responsible for the negative charge of C. neoformans, thereby strengthening chitosan’s antifungal activity [44] . Lastly, while chitosan’s intrinsic properties enhance its antimicrobial efficacy, when used prophylactically, chitosan can prevent infection from occurring altogether. Chitosan’s immunostimulatory properties include macrophage-induced nitric oxide production and chemotaxis, polymorphonuclear cell activation and potentiation of IL-12 mediated Th1 immune responses [45–47] . Taken together, chitosan is capable of stimulating both a phagocytic and cytotoxic immune response, both preventing and fighting infection. Chitosan is also a potent modulator of inflammatory cytokines, thereby promoting healing within the wound bed by recruiting fibroblasts and enhancing collagen III deposition [3,48,49] . It acts as an accelerant of wound healing and thus inhibits infection from occurring. Given chitosan’s multiple mechanisms to simultaneously combat microbial activity, development of resistance to chitosan is unlikely – as this would require multiple mutations within the same microbial cell [2,32] . Given chitosan’s poor solubility in vivo, utilizing chitosan at the nanoscale in the form of a nanoparticle, for example, could increase its translatability through improved delivery. Even more importantly, chitosan’s inherent antimicrobial activity would be remarkably enhanced due to the increasing density of positively charged amino groups on its surface at the nanoscale,

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allowing for more frequent interaction with microbial cell walls and membranes. Chitosan nanoparticle platforms as antimicrobial therapy Chitosan nanoparticles can be synthesized through a variety of methods that depend on either the cationic character or chemical structure of the molecule and its ability to polymerize into long chains (Figure 2) . These methods include ionotropic gelation, microemulsion, emulsification solvent diffusion and polyelectrolyte complex methods, as well as others [47] . Drugs can be added to these nanoparticle preparations during polymerization (incorporation) or after formation of nanoparticles by soaking the preformed nanoparticles in a saturated solution (incubation). Furthermore, chitosan can be used as an additive to other nanoparticle platforms in order to exploit its unique properties while using another structural molecule. When incorporated into nanomaterials, chitosan has a higher surface-to-volume ratio. This higher surface area results in a higher charge density, allowing for increased interaction with microbial elements [16] . These nanoparticle preparations are more effective antimicrobial agents than chitosan itself, and also work by the same method of cell membrane rupture and attachment to nucleic acids and other intracellular components, theoretically impeding essential processes like DNA transcription and translation and cellular respiration (Figure 3)  [16,50] . In vitro studies have not demonstrated an increase in toxicity. Chitosan nanoparticles have shown biocompatibility with human monocytes, endothelial cells and respiratory epithelial cells as well as mouse fibroblasts and chick embryos  [3,51–54] . These data suggest that the intrinsic effects of chitosan on microbes are augmented at the nano scale with little or no increase in adverse effects (Table 1) . Chitosan nanoparticles as an antibacterial agent

Chitosan nanoparticles have been proven effective against a broad range of bacteria. Qi et al. showed that chitosan nanoparticles had a significant antimicrobial effect against E. coli and several species of Staphylococcus including S. aureus. These chitosan nanoparticles were superior to both chitosan alone and doxycycline in inhibiting the growth of these species [16] . Xing et al. demonstrated that Oleoyl-chitosan nanoparticles exerted their antimicrobial effect on E. coli and S. aureus by both direct cell membrane damage on electron microscopy and through attachment to nucleic acids by DNA/RNA gel retardation assays, having both intracellular and extracellular effects [50] .

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These studies highlight that chitosan nanoparticles and chitosan alone exert their antimicrobial effect via the same mechanism. Given the concern of microbial resistance due to the chronic use of traditional antibiotic based acne therapeutics, Friedman et al. investigated the antimicrobial and immunological properties of chitosan-alginate nanoparticles, demonstrating potent inhibition of growth of P. acnes  [3] . While its exact mechanism of action is unclear, scanning electron microscopy and transmission electron microscopy demonstrated loss of the well-demarcated outer and inner lipophilic layers of P. acnes’s cell membrane. As previously described, polycationic chitosan interacts with the electronegative charges of microbial cell membranes, inducing perturbations in membrane integrity. Furthermore, targeting these highly conserved membrane structures reduces the probability of antimicrobial resistance. Chitosan nanomaterials have shown promise as a prophylactic agent against biofilm formation. Biofilm formation is a critical virulence factor for many bacteria that cause nosocomial infections. These bacteria form a film on catheters and other instrumentation that may act as fomites for infection. Chavez de Paz  et al. showed that chitosan nanoparticles, especially those formed with low molecular weight chitosan, were able to inhibit Streptococcus mutans biofilm formation in vitro  [55] . Furthermore, they showed that chitosan nanoparticles were able to disperse evenly throughout the sample, and induce significant cell membrane damage [55] . Holban et al. showed similar results on S. aureus and P. aeruginosa biofilm formation when incubated on a chitosan nanosphere-based coating  [53] . These findings show promise for chitosan nanoparticle-based technologies as prophylaxis for hospital-associated infections. Recent investigations have shown a synergistic effect of chitosan and metals against S. aureus [56] and E. coli  [39] . Compared to silver nanoparticles and chitosan alone, chitosan-coated silver nanoparticles were found to be highly active against S. aureus, suggesting a synergistic effect. This effect may be explained by the altered surface chemistry and biological properties when covered with chitosan; the addition of chitosan may decrease the aggregation potential, increasing the surface-area-to-volume ratio, thereby allowing more particles to interact with cell surfaces. Chitosan has also been used as a carrier system for a variety of nanoparticles  [3,57] . For example, encapsulating benzoyl peroxide into the chitosan-alginate nanoparticle enhanced its antimicrobial activity against P. acnes compared with benzoyl peroxide alone [3] . Chitosan nanoparticles can also provide a controlled environment for antibacterial substances that were not

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Figure 2. Transmission electron microscopy micrograph of chitosan nanoparticles. Reproduced from [51] with permission from Elsevier (2013).

previously usable. Enzymes represent one such class of macromolecules that are difficult to use due to instability when exposed to various conditions encountered in different routes of administration. Nanoencapsulation surrounds each enzyme with a lattice network resulting in stabilization of enzyme activity without a disruption of its intrinsic properties [58] . Piras et al. formulated lysosyme-loaded chitosan nanoparticles that show augmented antimicrobial activity against S. epidermidis when compared with either lysozyme or chitosan nanoparticles alone [54] . To the authors’ knowledge, this is the first investigation of its kind; loading antimicrobial peptides into polymeric nanoparticles may represent a promising strategy for combating infectious disease.

3 µm Mag = 2.61 KX EHT = 15.00 kV Signal A = SE1 WD = 13 mm Figure 3. Scanning electron microscopy micrograph of Escherichia coli cells after 60 min of treatment with chitosan–AgNPs composite material. Reproduced from [74] with permission from Elsevier.

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Table 1. Summary of antimicrobial activity of chitosan nanoparticle platforms. Study (year)

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Organisms studied

Results

Qi et al. (2004)  Chitosan nanoparticles

Nanoparticle platform

Staphylococcus aureus

Inhibited growth of both organisms more effectively than chitosan and doxycycline

 

 

Escherichia coli

 

Xing et al. (2009)

Oleoyl-chitosan nanoparticles

S. aureus

Nanoparticles showed antimicrobial activity, and attachment to nucleic acids

 

 

E. coli

 

Friedman et al. (2013)

Chitosan-alginate nanoparticles, Benzoyl peroxide releasing chitosanalginate nanoparticles

Propionibacterium acnes

Inhibition of growth, and cell membrane disruption. Greater antimicrobial activity than benzoyl peroxide alone

Chavez de Chitosan nanoparticles Paz et al. (2011)

Streptococcus mutans

Inhibited biofilm formation

[55]

Holban et al. (2014) 

Chitosan nanosphere-based coating

S. aureus

Inhibited biofilm formation

[53]

 

 

Pseudomonas aeruginosa

 

Potara et al. (2011)

Chitosan-silver nanocomposites

S. aureus

These studies showed a synergistic effect between chitosan and silver nanomaterials of these two organisms, respectively

[56]

El-Sharif et al. (2011) 

 

E. coli

 

[39]

Piras et al. (2014) 

Lysozyme loaded chitosan nanoparticles

Staphylococcus epidermidis

Greater antimicrobial activity than chitosan nanoparticles or lysozyme alone

[54]

Ing et al. (2012) Chitosan nanoparticles

Candida albicans

Lower MIC90 than chitosan for C. albicans and F. solani. Resistance of A. niger to treatment

 [60]

 

 

Fusarium solani

 

 

 

Aspergillus niger

 

Saharan et al. (2013)

Chitosan nanoparticles

Aspergillus alternata

Inhibition of mycelial growth and germination

 

 

Macrophomina phaseolina

 

 

 

 

Rhizoctonia solani

 

 

Zhou et al. (2013)

Amphotericin-B-loaded chitosan nanoparticles

C. albicans

Similar activity to amphotericin-B with increased corneal penetration

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Ref.  [16]

  [50]

  [3]

 

    [63]

 [65]

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Chitosan nanoparticles for infectious disease 

Chitosan nanoparticles as an antifungal agent

The antifungal activity of chitosan is also augmented when incorporated into nanomaterials. Previous investigations suggest that these nanomaterials exert their antifungal activity in much the same way as chitosan itself. It has been proposed that chitosan nanoparticles have tighter membrane binding with fungal cells, and diffuse into fungal cells at a higher rate based on in vitro studies [16,59] . These findings have been attributed to their higher surface charge density and small size. Ing et al. showed that the MIC90 (minimum concentration needed to inhibit 90% of growth) of chitosan nanoparticles was significantly less than chitosan alone when used against C. albicans and F. solani  [60] . Interestingly, they showed that C. albicans was more easily inhibited by nanoparticle preparations made from lower molecular weight chitosans, while F. Solani was more susceptible to nanoparticles made with higher molecular weight chitosans. This relationship was not seen in treatments made from chitosan alone. It was also found that nanoparticles of smaller sizes more easily inhibited the growth of both species, supporting the hypothesis that nanoparticle size itself imparts more antifungal activity. However, the same study showed resistance of A. niger to chitosan nanoparticles. This resistance is explained by the fact that A. niger contains 10% of chitin in its cell wall, a factor that has been associated with resistance to chitosan by other fungi  [61,62] . Chitosan nanoparticles have also been shown to inhibit mycelial growth and germination of Alternaria alternata, Macrophomina phaseolina and Rhizoctonia solani in vitro  [63] . These results taken together suggest that the versatility of chitosan, and the ease of its manipulation allow for specific formulations to address different pathogens. However, these preparations do not show universal antifungal activity. Chitosan nanoparticles can also be used as a vehicle to optimize other antifungal treatments. Modi et al. have developed a ketoconazole-loaded chitosan nanoparticle that overcomes poor gastric absorption of the drug [64] . They showed high binding of the nanoparticles to pig mucin. An ex vivo mucosal diffusion assay using mouse stomachs showed sustained diffusion of the nanoparticle drug after gastric emptying. However, when treated with ketoconazole alone, diffusion was halted after gastric emptying. The nanoparticle formulation also showed sustained release over time. A similar study done by Zhou et al. studied amphotericin-Bloaded poly(lactic acid)-grafted-chitosan nanoparticles and their ocular use [65] . They similarly found high interaction between their nanoparticles and mucin, showing a decrease in mucin’s negative charge when exposed to nanoparticles. This supports the notion that the positive surface charge of the nanoparticles

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is a primary mechanism of their mucosal adherence. They also found similar in vitro drug release and antifungal activity against C. albicans between amphotericin B and the nanoparticle preparation. When studied in vivo with New Zealand white rabbits, the nanoparticle preparation showed higher corneal penetration than amphotericin B alone, with no increase in corneal irritation. These data are encouraging for the prospect of using chitosan-based nanoparticle platforms in order to improve peroral and ocular formulations of antifungals by providing more efficient penetration, higher bioavailability and sustained release of already widely used drugs. Chitosan nanoparticle-based vaccines As discussed above, chitosan’s intrinsic properties including nonreactivity, its polycationic nature and high affinity for metals and other substances allow it to be used as a carrier system. Nanoparticles are a useful adjuvant in vaccines, since they are easily taken up by antigen presenting cells, due to their small size [66] . It has been proposed that since the size of nanoparticle carriers is comparable to the size of pathogens encountered during the maturation of the immune system, they are more easily internalized by antigen presenting cells [67] . Additionally, nanoparticle preparations may enhance the mucosal uptake of vaccines, initiating a mucosal protective immune response. By mucosal administration, both IgA-mediated mucosal immunity and systemic immunity may be achieved. As mentioned above, chitosan nanoparticle platforms are ideal for mucosal administration, due to tight binding with mucin, small size and the ability to open tight junctions between epithelial cells. Previous studies have shown that chitosan nanoparticles adhere to dendritic cells, and induce their maturation as shown by IL-6 and IL-12 release [68] . Additionally, administration of chitosan itself exerts an adjuvant effect by activation of macrophages and polymorphonuclear cells [47] . These data together show that chitosan is not only an ideal vehicle for antigens, but also a significant immune potentiator. Chitosan nanoparticle-encapsulated mucosal vaccines have been shown to produce significant IgG and IgA responses in mice after intranasal administration of vaccines against influenza, diphtheria, pertussis and hepatitis B virus [69,70] . It has been shown that these vaccines have a delayed clearance time from the nasal mucosa, which activates both mucosal and humoral immunity [70] . Similar results have been seen with oral chitosan nanoparticle vaccines loaded with tetanus toxoid  [71,72] . Taken together, these data substantiate claims that chitosan’s negative zeta potential, mucoadhesive and immunostimulatory properties and ease

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Review  Landriscina, Rosen & Friedman of use in nanoparticulate platforms make it an efficacious adjuvant for vaccines. Compared with traditional vaccine delivery, mucosal nanoparticulate vaccines have the potential to increase patient compliance and decrease costs of manufacturing, though clinical data are needed to solidify their efficacy. Conclusion With antimicrobial resistance reaching a critical level, chitosan nanoparticle platforms represent and new and innovative treatment strategy for infectious disease. However, there are still numerous questions that remain unanswered. While many studies demonstrate the efficacy of chitosan nanoparticles against infection in vitro, there remains paucity of in vivo data to support them. Similarly, chitosan nanoparticles have shown promise as an adjuvant in vaccines, though there is no clinical data to support their use. With chitin being one of the most abundant polymers, second only to cellulose, chitosan has to potential to be manufactured on a large scale. While nanotherapeutics hold the potential to combat the continued rise of resistant microorganisms, its successful translation from bench to bedside depends on overcoming numerous challenges and obstacles in terms of scalability, reproducibility and toxicity. Nevertheless, chitosan-based nanoparticles

represent a promising new strategy for the treatment of resistant microorganisms. Future perspective As mentioned above, there is a lack of in vivo data about the efficacy and safety of chitosan nanoparticle platforms. In the future, we expect to see more in vivo studies to evaluate the antimicrobial activity and biocompatibility of these platforms. Additionally, their activity against a variety of bacteria, fungi and viruses has yet to be elucidated, though these data are likely to emerge in the coming years due to the promising studies mentioned previously. While the current data are encouraging, there have been no studies about use in humans – a necessary bridge to investigate the ­possibility of obtaining true clinical data. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Executive summary Chitosan as an antimicrobial • Chitosan is formed by the deacetylation of chitin, making it highly accessible and easily produced. • Numerous studies have shown the antimicrobial efficacy of chitosan, owing to its polycationic structure. • Chitosan is able to disrupt the integrity of cell membranes resulting in osmotic instability, attachment to microbial elements (i.e., mRNA, DNA) and leakage of intracellular elements.

Chitosan nanoparticle platforms as antimicrobial therapy • When incorporated into nanoparticles, chitosan has a higher surface area-to-volume ratio, resulting in a higher charge density and allowing for an increased interaction with microbial elements. • Many studies have shown increased efficacy against bacteria and fungi compared with chitosan alone. • Chitosan nanoparticles are capable of carrying other substances such as metals, traditional antimicrobials and enzymes, yielding a synergistic effect.

Chitosan nanoparticle-based vaccines • Chitosan nanoparticles have been investigated as a vaccine adjuvant due to their small size, mucosal adhesion properties and immunostimulatory effect. • In vivo studies have demonstrated chitosan nanoparticle based vaccines’ ability to activate both mucosal and humoral immune pathways.

Future directions • Chitosan nanoparticles may represent a promising avenue for overcoming antimicrobial resistance. • The paucity of in vivo and clinical data challenges its translatability to clinical practice, highlighting the need for further investigation.

References Papers of special note have been highlighted as: • of interest; •• of considerable interest. 1

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Biodegradable chitosan nanoparticles in drug delivery for infectious disease.

Increasing rates of antimicrobial resistance have left a significant gap in the standard antimicrobial armament. Nanotechnology holds promise as a new...
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