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Recent progress in applications of nanoparticles in fish medicine: A review
BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 701 – 710
Review Article
nanomedjournal.com
Recent progress in applications of nanoparticles in fish medicine: A review Mohamed Shaalan, MSc a, b , Mona Saleh, PhD a , Magdy El-Mahdy, PhD b , Mansour El-Matbouli, PhD a,⁎ b
a Clinical Division of Fish Medicine; University of Veterinary Medicine, Vienna, Austria Department of Pathology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt Received 21 July 2015; accepted 14 November 2015
Abstract Nanotechnology has become an extensive field of research due to the unique properties of nanoparticles, which enable novel applications. Nanoparticles have found their way into many applications in the field of medicine, including diagnostics, vaccination, drug and gene delivery. In this review, we focused on the antimicrobial effects of nanoparticles, with particular emphasis on the problem of antibiotic resistant bacteria in fisheries. The use of nanoparticle-based vaccines against many viral pathogens is a developing field in fish medicine research. Nanoparticles have gained much interest as a specific and sensitive tool for diagnosis of bacterial, fungal and viral diseases in aquaculture. Nevertheless our review also highlights the many applications of nanotechnology that are still to be explored in fish medicine. From the Clinical Editor: Advance in nanotechnology has enabled the development of nanomedicine, with many ideas being used in clinical diagnosis and therapy. In this review article, the authors described the current use of nanotechnology in fish medicine. The knowledge would also impart important information for our daily living. © 2015 Elsevier Inc. All rights reserved. Key words: Nanoparticles; Fish diseases; Diagnosis; Antimicrobial; Fish vaccines
Nanotechnology is the science of producing and utilizing nanometer-sized particles. Only recently we have developed approaches to understand and control matter at nanoscale dimensions (between approximately 1 and 100 nm). Particles in this size range have unique properties, which pave the way for novel applications. 1 Nanomaterials can be divided into two large groups: ultrafine nano-sized particles that are present normally in nature and not intentionally produced and engineered nanoparticles that are produced in a controlled and intended way. 2 Nanomedicine is the application of nanotechnology in both human and veterinary medicine. This science deals with designing and utilizing nanoparticles and nanodevices for biomedical applications. 3 We acknowledge support from a PhD scholarship offered for Mohamed Shaalan by mission sectors in the Ministry of Higher Education, Egypt. Competing interests: The authors declare that they have no competing interests. The authors declare that they don’t have any commercial associations, current and within the past five years, that might pose a potential, perceived or real conflict of interest. ⁎Corresponding author at: Clinical Division of Fish Medicine, University of Veterinary Medicine, Vienna, Austria. E-mail address: [email protected] (M. El-Matbouli).
Many forms of nanoparticles have been used in medicine 4 (Table 1), for example nanospheres, which are nano-sized spherical particles. 5 Because of their small size and high surface area, a specific drug can be dispersed on the nanosphere's surface to facilitate drug delivery. Nanospheres can also be used for tissue regeneration. 6 Another form of nanoparticle is the nanocapsule, which is composed primarily of an outer nano-scale shell and an inner core. The core may be oil or water, which contains a specific drug, while the shell protects the drug from degradation and hydrolysis. 7 For example, liposomes are lipid bi-layer nano-sized spheres, which are ideal for drug delivery of both lipophilic and hydrophilic medications due to their structure, which is similar to the eukaryotic cell membrane. 3,8 Another form of nanoparticles commonly used in different medical applications is carbon nanotubes (single-walled or multi-walled). These are characterized by having a very large surface area and high ability to penetrate cell membranes, acting as micro-needles, which makes them ideal for drug delivery, including anti-tumor drugs. 9 However, a study has shown that they have high risk of inducing thrombi inside blood vessels. 10 A fourth particle form is dendrimers, which are nanoscale three dimensional structures consisting of tree branch-like units
http://dx.doi.org/10.1016/j.nano.2015.11.005 1549-9634/© 2015 Elsevier Inc. All rights reserved. Please cite this article as: Shaalan M, et al, Recent progress in applications of nanoparticles in fish medicine: A review. Nanomedicine: NBM 2016;12:701-710, http://dx.doi.org/10.1016/j.nano.2015.11.005
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Table 1 Different forms of nanoparticles used in biomedical applications. Type of nanoparticles
Structure
Application in medicine
Nanospheres
Spherical shaped
Nanocapsules
Shell and core combination Cylindrical tubes
Drug delivery, tissue regeneration Controlled drug delivery
Carbon Nanotubes Liposomes
Drug delivery, Anti-cancer
Lipid bi-layer globules
Drug delivery for hydrophobic and hydrophilic drugs Dendrimers Highly branched ends and Delivery system, tissue central core engineering, antimicrobials Polymeric Polymers as Delivey system, nanoparticles chitosan, PLGA tissue regeneration
coming from a central core. These have a highly branched multifunctional surface with low molecular weight, which facilitates their use as a delivery system for genes, vaccines, and drugs, and as a scaffold for tissue regeneration, and as antimicrobial agents. 11–13 The types of nanoparticles applied in fish medicine have to date been limited to mostly nanospheres or polymeric nanoparticles; application of other forms of nanoparticles has not yet been investigated. In this review, we summarize the various demonstrated and potential applications of nanoparticles in the field of fish medicine. These include their deployment as antimicrobials, drug and gene delivery vehicles, vaccination, and for specific diagnosis of fish pathogens.
Synthesis of nanoparticles Generally, there are two main approaches for nanoparticle synthesis: top-down or bottom-up. 14 The top-down approach involves mechanical grinding of bulk metal to convert it from macro or microscale to nanoscale, which is followed by addition of stabilizing agents like colloidal protecting agents to ensure that the nanoparticles do not oxidize, or re-assemble back to the microscale. 15 Bottom-up methods include construction of nanoparticles by various physical and chemical methods, including electrochemical reduction of metals and sonodecomposition. 16 Physical and chemical synthesis Nanoparticle synthesis can be carried out by using thermal decomposition in organic solvents. 17 For metal nanoparticles, cryochemical synthesis yields nanoparticles in the range of 5 to 80 nm in diameter. 18 Physical synthesis using microwaves has been adopted for silver nanoparticles, which involves physical reduction of silver using different microwave radiation frequencies. 19 This method was more rapid and gave a higher concentration of silver nanoparticles when compared with a thermal method, given the same temperature and exposure. Jiang et al 19 also found that the higher the concentration of silver nitrate used, the longer the reaction time and the higher the temperature, the larger the particle that could be obtained. Use of
high poly vinyl pyrrolidone (PVP) concentrations (used for nanoparticle coating) resulted in smaller silver particle sizes, between 15 and 25 nm. Other methods of metal nanoparticle synthesis include: pulsed laser ablation 20; electroreduction of AgNO3 in aqueous solution in the presence of polyethylene glycol 21 ; spark discharge 22 micro-emulsion for preparation of Ag-Fe3O2 nanoparticles 23; chemical reduction of silver nitrate using trisodium citrate combined with sodium borohydride as reducing agent to synthesize PVP coated silver nanoparticles. 24,25 Different methods have also been demonstrated for synthesis of non-metallic nanoparticles, e.g. polymeric chitosan nanoparticles. For example, ionotropic gelation is a simple method involving dissolution of chitosan in acetic acid, with addition of polyanion tripolyphosphate (TPP). This method depends on electrostatic interaction between the chitosan amine group and groups of polyanion polymer. 26,27 The polyelectrolyte complex method depends on electrostatic interaction between cationic groups in chitosan and DNA, which neutralizes the charges with formation of nanoparticles. 26 The microemulsion method utilizes addition of surfactants, but has disadvantages which include use of organic solvents, long preparation time and the complexity of the washing processes. 28 The most common method for synthesis of Poly D, L-lactide-co-glycolic acid (PLGA) nanoparticles is the precipitation method 29 combined with the double emulsion solvent evaporation method. 30,31 Biological synthesis (Green synthesis) There is great interest in finding eco-friendly and economical methods to synthesize nanoparticles. Biological methods are regarded as the key for this approach. 32 Biologically synthesized nanoparticles are derived from three main groups of organisms: bacteria, fungi, and plants. Bio-synthesis of nanoparticles is a bottom-up approach that mostly involves reduction/oxidation reactions. 33 Microbial enzymes or plant phytochemicals with antioxidant or reducing properties act on precursor compounds to produce the desired nanoparticles. The three main components of a biosynthetic nanoparticle system are: a solvent medium for synthesis, an environmentally friendly reducing agent, and a nontoxic stabilizing agent. 33 Silver nanoparticles synthesized using Origanum vulgare leaf extract demonstrated antibacterial activity and cytotoxic effects against a human lung cancer cell line. 34 Cashew nut shell liquid was used for green synthesis of both silver and gold nanoparticles, which showed bactericidal activity against several fish pathogens. 35 Silver nanoparticles synthesized using tea leaf extract (Camellia sinensis) demonstrated bactericidal activity against Vibrio harveyi in juvenile Feneropenaeus indicus, but only at high doses of the nanoparticles. 36 A broth of Aloe leaf extract was used for green synthesis of zinc oxide nanoparticles (ZnO-NPs), which showed higher bactericidal activity than nanoparticles by standard chemistry. 37 A novel method for biological synthesis of zinc oxide nanoparticles uses Aeromonas hydrophila bacteria as the reducing agent. This method is environmentally friendly and economically feasible, and resulted in ZnO nanoparticles with antibacterial and antimycotic effects. 38
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Antibiotic resistant bacteria in fisheries Antibiotics have been used for decades to combat bacterial infections. However unregulated, excessive use of antibiotics can lead to the emergence of resistant bacteria which are no longer sensitive to antibiotics. 39–42 Since there are shared elements between the mobile genomes of both terrestrial and aquatic bacteria, resistance genes can be exchanged between them with major potential impacts on livestock and human health 42,43 Tuševljak et al 44 conducted a survey about usage of antimicrobials in fish farms in 25 countries. They found that tetracycline is the most used antibiotic agent in fish farms. There is a strong relation between emergence of antibiotic resistant bacteria and excessive antibiotics administration in aquaculture. Emergence of many resistant bacteria, including as methicillin resistant Staphyloccus aureus (MRSA) and multi- and super-drug resistant forms has been recorded. 45,46 Some Aeromonas hydrophila isolates from cultured tilapia were resistant to broad spectrum antibiotics like tetracycline, streptomycin and erythromycin. 47 Antibiotic resistance was also observed in Aeromonas salmonicida, Photobacterium damselae subsp. piscicida, Yersinia ruckeri, Vibrio, Listeria, Pseudomonas and Edwardsiella species. 43,48 Di Cesare et al 49,50 isolated antibiotic resistant enterococci from fish farm sediment, which raises the possibility that these bacteria may transfer their resistance to human-infecting strains, making their presence in fish farms an issue of public health importance. Nanoparticles as novel antimicrobials in fish medicine The utilization of nanoparticles as alternative antimicrobials to combat emergence of microbial resistance to antibiotics in aquaculture has been investigated. 37,48 Metal nanoparticles have shown high antimicrobial activity against bacteria, fungi and viruses. 51 In the following sections, we discuss studies on metal nanoparticles that have been applied against fish pathogens. Silver nanoparticles (Ag-NPs) Silver nanoparticles are the most investigated nano-antibacterial agent in research literature. They act against bacteria through multiple mechanisms, and in this way they can evade the bacterial resistance, 52 in contrast to antibiotics that generally act through one mechanism only. 52–54 The release of silver ions (Ag +) is one mechanism 52 : Ag + binds to bacterial cell membrane proteins causing disruption of the membrane, leading to death of the bacterial cells. 55 Intracellularly, Ag + binds to cytochrome and nucleic acids, damaging them and inhibiting cell division. 56 Prakash et al 57 report that silver nanoparticles demonstrate high antibacterial efficacy against multi-drug resistant bacterial isolates. The bactericidal effect of silver nanoparticles has also been reported against methicillin resistant Staphyloccus aureus (MRSA). 58 Silver nanoparticles synthesized using the juice of the Citrus limon as a reducing agent, demonstrated antibacterial activity against Staphylococcus aureus and Edwardesiella tarda, and anti-cyanobacterial activity towards Anabaena and Oscillatoria species. 48 Umashankari et al 59 used leaf bud extract from mangrove Rhizophora mucronata for biological synthesis of Ag-NPs, then demonstrated antimicrobial effects against
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Pseudomonas fluorescens, Proteus species and Flavobacterium species. The efficacy of these ‘green' synthesized silver nanoparticles was the same as commercial antibiotics. In a study on Vibrio harveyi-infected juvenile shrimp Feneropenaeus indicus, long term treatment with silver nanoparticles decreased mortalities by 71% at high doses of Ag-NPs. 36 As an antifungal agent, silver nanoparticles exhibited high inhibitory effects against Candida species, similar to the commercial antifungal Amphotericin B. 60,61 Antifungal activity of silver nanoparticles against dermatophytes has also been recorded. 62 Silver nanoparticles also exhibit anti-viral properties: they can bind to HIV-1 virus proteins in vitro. 63 Both silver nanoparticles and silver nanoparticles-chitosan composite are active against influenza A virus. 64,65 There has been little published work specifically on the antifungal and antiviral effects of silver nanoparticles in fish medicine. Gold nanoparticles (Au-NPs) There is currently great interest toward investigating the antimicrobial effects of gold nanoparticles, due to their low toxicity to eukaryotic cells. 66 Au-NPs can interact with biological proteins and non-proteins, e.g. Lipopolysaccharides (LPS), and have biological functions. 67 Gold nanoparticles supported on zeolite exhibited bactericidal effects against Escherichia coli and Salmonella typhi. 68 Functionalized Au-NPs inhibited the growth of MDR bacterial isolates. 66 Gold nanoparticles made by ‘green' synthesis showed antibacterial activity against fish bacterial isolate. 35 There are three pathways along which gold nanoparticles exert their antibacterial effects. The first way is via interfering with oxidative phosphorylation process with changing the potential of bacterial cell membrane; this leads to decrease in the activity of F-Type ATP synthase with a net decrease in ATP synthesis and metabolism. The second path is interference with binding of tRNA to the two ribosome subunits. The third way is achieved through enhancing chemotaxis. 69 Fungicidal activity against Candida species was reported for gold nanoparticles. Their efficacy was size-dependent, with smaller gold nanoparticles having higher antifungal effects. 70,71 Zinc oxide nanoparticles (ZnO-NPs) Zinc oxide nanoparticles have drawn much attention due to their antibacterial and antifungal effects. 37,48 The antibacterial activity derives from damage the particles do to the bacterial cell membrane, which makes cytoplasmic contents leak from the cell. 72 In the field of fish medicine, ZnO-NPs can inhibit the growth of Aermonas hydrophila, Edwardseilla tarda, Flavobacterium branchiophilum, Citrobacter spp., Staphylococcus aureus, Vibrio species, Bacillus cereus and Pseudomonas aeruginosa. 48 Ramamoorthy et al 73 investigated the antibacterial effects of ZnO nanoparticles against the pathogenic Vibrio harveyi and observed higher bactericidal effects of nanoparticles compared to bulk ZnO. In another interesting study, ZnO nanoparticles were synthesized biologically using Aermonas hydrophila, and these nanoparticles exhibited antibacterial activity against the same bacterium and other species: Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Aspergillus flavus and Candida albicans as shown in Figure 1. 38
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Figure 1. Mueller–Hinton agar plates showing the antifungal effects of ZnO-NPs, and zones of inhibition against both (A) Pseudomonas aeruginosa and (B) Aspergillus flavus. (Adapted with permission from Jayaseelan et al, 2012).
Titanium dioxide nanoparticles (TiO2-NPs) TiO2-NPs, when doped with magnetic Fe3O4-NPs had a bactericidal effect against Streptococcus iniae, Edwardsiella tarda and Photobacterium damselae, after activation by light. 74,75 These particles can be used to disinfect water, as the fish pathogens bind with the nanoparticles, which can then be easily extracted from water using a magnet. 74,75 However, Jovanovic et al 76,77 concluded that TiO2-NPs affect the fish immune system by inhibiting the antibacterial activity of fish neutrophils, making the fish liable to infection and hence increased mortality especially in disease outbreaks.
hormone releasing hormone (LHRH) was bound to chitosan nanoparticles in one group and compared with LHRH conjugated with chitosan-gold nanoparticles. Both groups showed an increase in blood hormone levels with sustained release of hormones groups, compared to the group injected with hormone alone. Egg fertilization rates were elevated after one injection dose of hormone-chitosan nanoparticle conjugate and hormone-chitosan-gold nanoparticles, respectively 87% and 83%, compared with multiple injections of LHRH alone which gave a fertilization rate of 74%. 82 PLGA nanoparticles for drug delivery
Nanoparticles as vehicles for drug and gene delivery An ideal drug delivery system has several key properties, which include safety, biocompatibility and biodegradability of the delivery system, plus stability of the drug, specificity of delivery, and few or no side effects 4 As drug delivery systems, nanoparticles have gained much interest because of their small sizes and because they can cross biological barriers like the blood brain barrier (BBB). Nanoparticles also have a high surface area to volume ratio, which allows increased reactivity with other conjugates and compounds. 4,78,79 In fish medicine specifically, chitosan nanoparticles and PLGA nanoparticles have been investigated the most as nanoparticles for drug delivery.
PLGA is a copolymer consisting of poly lactic acid and poly glycolic acid. It is biocompatible, biodegradable and non-toxic. It has been approved by the FDA. Hence many researchers have investigated the feasibility of using PLGA as a drug carrier. 83,84 In a recent study on zebra fish embryos, PLGA nanoparticles were loaded with the anti-mycobacterial agent rifampicin then injected. As zebra fish embryos are transparent, the treatment impact on Mycobacterium marinum-infected could be measured using non-invasive imaging as shown in Figure 2. The rifampicin-PLGA nanoparticles showed an increased therapeutic effect against M. marinum and higher embryo survival, compared to rifampicin alone. 85
Chitosan nanoparticles for drug delivery
Nanotechnology-based fish vaccines
Chitosan nanoparticles have excellent properties as drug delivery vehicles. They are comprised of a biocompatible, non-toxic and biodegradable polymer, which is easily excreted from the kidney. 4 Their mucoadhesive properties mean that they can be adapted for slow and sustainable drug release. 80 For example, vitamin C was conjugated with chitosan nanoparticles in a study on rainbow trout (Oncorhynchus mykiss). The vitamin was released up to 48 h after oral administration, and stimulation of the fish innate immune system was observed due to potent synergism between chitosan and vitamin C. 81 In another study, chitosan nanoparticles were used as a hormone delivery system in Cyprinus carpio. Luteinizing
Incorporation of nanoparticles in vaccine formulations is an exciting avenue of medical research. Polymeric nanoparticles have been most widely investigated, as they have several advantages as vaccine releasing vehicles, including their ability to ensure antigen stability against degradation by enzymes, preserving immunogenicity and for sustained release of the vaccine. 86,87 Nanovaccines have been used as immunostimulant adjuvants or as a delivery system for targeted antigen delivery, with slow antigen release a feature of both purposes. 87 Other nanoparticle forms that have been used as a vaccine delivery system include: virus-like particles, liposomes, immunostimulant complex, metal nanoparticles and polymeric nanoparticles. 87,88 Concerns about nanovaccines have
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Figure 2. (A, B, C, D) Lateral view showing formation of Mycobacterium marinum granulomas in zebra fish embryos. After injection of nanoparticles in the posterior caudal vein, panels (E, F, G, H) show rifampicin-loaded PLGA-nanoparticles targeting the granulomas. (Adapted with permission from Fenaroli et al, 2014. Copyright (2014) American Chemical Society).
been directed mainly towards technical difficulties in producing them with stable properties, their potential toxicity, and a lack of information about their distribution within biological systems. 88 The most investigated nanoparticles in fish vaccine research up to now are polymeric chitosan and PLGA nanoparticles. Chitosan nanoparticles have been used for the development of fish vaccines, for example the inactivated virus vaccine against infectious salmon anemia virus (ISAV), which incorporates the DNA coding for ISAV replicase as an adjuvant. This vaccine showed protection rates that reach up to 77% against ISAV. 89 An oral DNA vaccine against Vibrio anguillarum in Asian sea bass (Lates calcarifer) was developed using chitosan and chitosan/tripoly phosphate nanoparticles. The nanovaccine conferred only moderate protection against the pathogen. 27,90
Another oral DNA vaccine was developed by loading the outer membrane protein K (ompK) gene of Vibrio parahemolyticus onto chitosan nanoparticles. This recombinant nanovaccine elicited a protective immune response in black seabream (Acanthopagrus schlegelii) against Vibrio parahemolyticus. 91 The effectiveness of recombinant DNA-chitosan nanoparticles in providing protection against white spot syndrome virus (WSSV) in shrimps was investigated. It was found that when administered orally, the vaccine enhanced shrimp immunity, providing a protective response against WSSV. 92,93 The efficacy of PLGA nanoparticles as a DNA vaccine carrier and adjuvant has been reported. 30,94,95 PLGA nanoparticles have had limited use in fisheries so far. An oral DNA vaccine in Japanese flounder (Paralichthys olivaceus) against lymphocystis disease virus (LCDV) was developed. 96 In a different study, an
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Figure 3. The principle of direct detection of SVCV RNA using unmodified gold nanoparticles (Adapted with permission from Saleh et al, 2012).
orally administered DNA vaccine was used to immunize rainbow trout against infectious hematopoietic necrosis virus, and an immune response was observed. 31 Nanoparticles in the diagnosis of fish pathogens Nanoparticles have been adopted for rapid and sensitive diagnosis of diseases, and these nanoparticle-based detection methods are called nanodiagnostics. 97 One of the most widely used nanoparticles in diagnostics is gold nanoparticles, which are appropriate for use in different methods. 98,99 Diagnosis of bacterial and mycotic fish diseases The first report of using gold nanoparticles for detection of fish pathogen was with A. salmonicida antibody-gold nanopar-
ticles conjugated for the specific immunodiagnosis of furunculosis in fish tissues. 100 Kuan et al 101 designed an electrochemical DNA biosensor for detection of Aphanomyces invadans in fish, based on conjugation of Au-NPs with a DNA reporter probe. This assay could detect fungi at a lower level than PCR. Diagnosis of viral fish diseases Jaroenram et al 102 combined a AuNPs colorimetric assay with loop-mediated isothermal amplification (LAMP) for visual detection of yellow head virus in shrimp. This method was rapid, was specific, and showed high sensitivity. A similar combination of DNA-functionalized AuNPs with LAMP was developed for the detection of white spot syndrome virus (WSSV) in shrimp. 103 This method was specific, sensitive and suitable for field applications.
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Figure 4. Results of a gold nanoparticle-based assay for detection of SVCV: in a positive test, the color of the solution changes from red to blue (Adapted with permission from Saleh et al, 2012).
In another study, Toubanaki et al 104 developed a method for NNV detection using a gold nanoparticles-based biosensor for detection of viral nucleic acids after amplification by RT-PCR. This method was quite cost effective, as it did not require antibody conjugation. Yang et al 105 report an immunomagnetic reduction assay for nervous necrosis virus (NNV) in grouper fish using magnetic nanoparticles coated with rabbit anti-NNV antibody. When an external magnetic field was applied, immunodiagnosis was based on the motility of magnetic nanoparticles: if the antibody coated-nanoparticles bound to the viral antigen, they form clusters which decrease their motility. The virus titer was calculated using a magnetic immunoassay analyzer. A colorimetric assay for detection of spring viremia of carp virus (SVCV) was developed using unmodified Au-NPs. The probe, which was complementary for SVCV, was added first, followed by gold nanoparticles. If target viral RNA was present, it hybridized to the probe, thereby preventing the probe from stabilizing the gold nanoparticles. The Au-NPs could aggregate, with a resultant change in the solution, from red to blue. If no viral nucleic acid was present, the probe could freely adsorb onto the surface of gold nanoparticles and prevent their aggregation, and the solution stayed red (Figures 3-4). 106 This method was highly specificity and rapid, without the need for prior amplification of viral nucleic acids. The same principle was adopted for the development of a rapid, specific and sensitive assay for the detection of the DNA virus, cyprinid herpes virus-3 (CyHV-3). 107
Concluding summary and future directions In this review article, we have summarized the current applications of nanoparticles for diagnosis, and treatment of fish pathogens. A range of different methods has been employed for nanoparticle synthesis, and the emerging use of ‘green' synthetic methods appears safer and more environmentally friendly while
retaining the efficacy of the formed nanoparticles. 32–38 Naked metal nanoparticles exhibit antimicrobial effects, and have been applied to combat microbial resistance in aquaculture. 36–38,48,59,66,73–75 Many nanoparticles are reported to be excellent vehicles for drug, gene and vaccine delivery due to their unique properties. The most investigated nanoparticles in fish medicine for these applications are polymeric chitosan nanoparticles and Poly D, L-lactide-co-glycolic acid (PLGA) nanoparticles. 3,4,78–80,84,86 Both unmodified and conjugated nanoparticles have been shown to facilitate rapid, cost effective and specific detection of fish pathogens. There are, however, many research gaps in the field of nanotechnology applications in fish medicine. Different forms of nanoparticles like nanocapsules, liposomes, dendrimers and nanotubes could theoretically have applicability in fish diseases research. The antifungal and antiviral effects of nanoparticles against fish diseases have yet to be explored. More studies for vaccine development in fish are essential if nanotechnological approaches are to be applied widely. Few studies have examined nanoparticle applications for diagnosis of bacterial and mycotic diseases in aquaculture. Given the demonstrated potential of nanoparticles, there are needs for more targeted investigations of their application in many fish medicine research topics, to promote more efficient fish disease diagnostics and therapy, to meet the ever-growing aquatic animal health demand.
References
1. The Royal Society and Royal Academy of Engineering. Nanoscience and nanotechnologies: opportunities and uncertainties; 2004 [London, UK]. 2. Oberdörster G, Oberdörster E, Oberdörster J. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect 2005;113:823-39. 3. Cavalieri F, Tortora M, Stringaro A, Colone M, Baldassarri L. Nanomedicines for antimicrobial interventions. J Hosp Infect 2014;88(4):183-90.
708
M. Shaalan et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 701–710
4. De Jong WH, Borm PJ. Drug delivery and nanoparticles: applications and hazards. Int J Nanomedicine 2008;3(2):133-49. 5. Donbrow M. Microcapsules and nanoparticles in medicine and pharmacy. CRC Press; 1991. 6. Ramalingam M, Jabbari E, Ramakrishna S, Khademhosseini A, editors. Micro and nanotechnologies in engineering stem cells and tissues, vol. 39. John Wiley & Sons; 2013. 7. Torchilin VP. Nanoparticulates as drug carriers. Imperial college Press; 2006. 8. Lasic DD. Novel applications of liposomes. Trends Biotechnol 1998;16(7):307-21. 9. Reilly RM. Carbon nanotubes: potential benefits and risks of nanotechnology in nuclear medicine. J Nucl Med 2007;48(7):1039-42. 10. Gaffney AM, Santos-Martinez MJ, Satti A, Major TC, Wynne KJ, Gun'ko YK, et al. Blood biocompatibility of surface-bound multiwalled carbon nanotubes. Nanomedicine 2015;11(1):39-46. 11. Aulenta F, Hayes W, Rannard S. Dendrimers: a new class of nanoscopic containers and delivery devices. Eur Polym J 2003;39(9):1741-71. 12. Gillies ER, Frechet JM. Dendrimers and dendritic polymers in drug delivery. Drug Discov Today 2005;10(1):35-43. 13. Wu LP, Ficker M, Christensen JB, Trohopoulos PN, Moghimi SM. Dendrimers in medicine: therapeutic concepts and pharmaceutical challenges. Bioconjug Chem 2015;26(7):1198-211. 14. Holmes JD, Lyons DM, Ziegler KJ. Supercritical fluid synthesis of metal and semiconductor nanomaterials. Chemistry 2003;9(10):2144-50. 15. Gaffet E, Tachikart M, El Kedim O, Rahouadj R. Nanostructural materials formation by mechanical alloying: morphologic analysis based on transmission and scanning electron microscopic observations. Mater Charact 1996;36:185-90. 16. Thirumalai Arasu V, Prabhu D, Soniya M. Stable silver nanoparticle synthesizing methods and its applications. J Biol Sci Res 2010;1:259-70. 17. Esumi K, Tano T, Torigue K, Meguro K. Preparation and characterization of bimetallic Pd-Cu colloids by thermal decomposition of their acetate compounds in organic solvents. Chem Mater 1990;2:564-7. 18. Sergeev MB, Kasaikin AV, Litmanovich AE. Cryochemical synthesis and properties of silver nanoparticle dispersions stabilised by poly(2dimethylaminoethyl methacrylate). Mendeleev Commun 1999;9:130-2. 19. Jiang H, Moon K, Zhang Z, Pothukuchi S, Wong CP. Variable frequency microwave synthesis of silver nanoparticles. J Nanopart Res 2006;8:117-24. 20. Jang D, Kim D. Synthesis of nanoparticles by pulsed laser ablation of consolidated metal microparticles. Appl Phys 2004;A79:1985-8. 21. Zhu J, Liao X, Chen HY. Electrochemical preparation of silver dendrites in the presence of DNA. Mater Res Bull 2001;36:1687-92. 22. Tien DC, Tseng KH, Liao CY, Tsung TT. Colloidal silver fabrication using the spark discharge system and its antimicrobial effect on Staphylococcus aureus. Med Eng Phys 2007;30:948-52. 23. Gong P, Li H, He X, Wang K, Hu J, Tan W. Preparation and antibacterial activity of Fe3O4 Ag nanoparticles. Nanotechnology 2007;18:604-11. 24. Van Dong P, Ha CH, Kasbohm J. Chemical synthesis and antibacterial activity of novel-shaped silver nanoparticles. Int Nano Lett 2012;2:1-9. 25. El Mahdy MM, Eldin TAS, Aly HS, Mohammed FF, Shaalan MI. Evaluation of hepatotoxic and genotoxic potential of silver nanoparticles in albino rats. Exp Toxicol Pathol 2015;67(1):21-9. 26. Kumari A, Yadav SK, Yadav SC. Biodegradable polymeric nanoparticles based drug delivery systems. Colloids Surf B: Biointerfaces 2010;75(1):1-18. 27. Rajesh Kumar S, Ishaq Ahmed VP, Parameswaran V, Sudhakaran R, Sarath Babu V, Sahul Hameed AS. Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in Asian sea bass (Lates calcarifer) to protect from Vibrio (Listonella) anguillarum. Fish Shellfish Immunol 2008;25(1-2):47-56.
28. Nagpal K, Singh SK, Mishra DN. Chitosan nanoparticles: a promising system in novel drug delivery. Chem Pharm Bull 2010;58(11):1423-30. 29. Barichello JM, Morishita M, Takayama K, Nagai T. Encapsulation of hydrophilic and lipophilic drugs in PLGA nanoparticles by the nanoprecipitation method. Drug Dev Ind Pharm 1999;25(4):471-6. 30. Tinsley-Bown AM, Fretwell R, Dowsett AB, Davis SL, Farrar GH. Formulation of poly (D, L-lactic-co-glycolic acid) microparticles for rapid plasmid DNA delivery. J Control Release 2000;66(2-3):229-41. 31. Adomako M, St-Hilaire S, Zheng Y, Eley J, Marcum RD, Sealey W, et al. Oral DNA vaccination of rainbow trout, Oncorhynchus mykiss (Walbaum), against infectious haematopoietic necrosis virus using PLGA [poly(D, L-lactic-co-glycolic acid)] nanoparticles. J Fish Dis 2012;35(3):203-14. 32. Kalishwaralal K, Deepak V, Ramkumarpandian S, Nellaiah H, Sangiliyandi G. Extracellular biosynthesis of silver nanoparticles by the culture supernatant of Bacillus licheniformis. Mater Lett 2008;62:4411-3. 33. Prabhu S, Poulose EK. Silver nanoparticles: mechanism of antimicrobial action, synthesis, medical applications, and toxicity effects. Int Nano Lett 2012;2:32. 34. Sankar R, Karthik A, Prabu A, Karthik S, Shivashangari KS, Ravikumar V. Origanum vulgare mediated biosynthesis of silver nanoparticles for its antibacterial and anticancer activity. Colloids Surf B: Biointerfaces 2013;108:80-4. 35. Velmurugan P, Iydroose M, Lee SM, Cho M, Park JH, Balachandar V, et al. Synthesis of silver and gold nanoparticles using cashew nut shell liquid and its antibacterial activity against fish pathogens. Indian J Microbiol 2014;54(2):196-202. 36. Vaseeharan B, Ramasamy P, Chen JC. Antibacterial activity of silver nanoparticles (AgNps) synthesized by tea leaf extracts against pathogenic Vibrio harveyi and its protective efficacy on juvenile Feneropenaeus indicus. Lett Appl Microbiol 2010;50(4):352-6. 37. Gunalan S, Sivaraj R, Rajendran V. Green synthesized ZnO nanoparticles against bacterial and fungal pathogens. Prog Nat Sci Mater Int 2012;22(6):693-700. 38. Jayaseelan C, Rahuman AA, Kirthi AV, Marimuthu S, Santhoshkumar T, Bagavan A, et al. Novel microbial route to synthesize ZnO nanoparticles using Aeromonas hydrophila and their activity against pathogenic bacteria and fungi. Spectrochim Acta A 2012;90:78-84. 39. Aarestrup FM. Veterinary drug usage and antimicrobial resistance in bacteria of animal origin. Basic Clin Pharmacol Toxicol 2005;96(4):271-81. 40. Hajipour MJ, Fromm KM, Ashkarran AA, de Aberasturi DJ, de Larramendi IR, Rojo T, et al. Antibacterial properties of nanoparticles. Trends Biotechnol 2012;30(10):499-511. 41. Pelgrift RY, Friedman AJ. Nanotechnology as a therapeutic tool to combat microbial resistance. Adv Drug Deliv Rev 2013;65(13):1803-15. 42. Cabello FC, Godfrey HP, Tomova A, Ivanova L, Dölz H, Millanao A, et al. Antimicrobial use in aquaculture re‐examined: its relevance to antimicrobial resistance and to animal and human health. Environ Microbiol 2013;15(7):1917-42. 43. Sørum H, Lie Ø. Antibiotic resistance associated with veterinary drug use in fish farms. Improving farmed fish quality and safety; 2008157-82. 44. Tuševljak N, Dutil L, Rajić A, Uhland FC, McClure C, St‐Hilaire S, et al. Antimicrobial use and resistance in aquaculture: findings of a globally administered survey of aquaculture‐allied professionals. Zoonoses Public Health 2013;60(6):426-36. 45. Atyah MAS, Zamri-Saad M, Siti-Zahrah A. First report of methicillinresistant Staphylococcus aureus from cage-cultured tilapia (Oreochromis niloticus). Vet Microbiol 2010;144(3):502-4. 46. Davies J, Davies D. Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 2010;74(3):417-33.
M. Shaalan et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 701–710 47. Son R, Rusul G, Sahilah AM, Zainuri A, Raha AR, Salmah I. Antibiotic resistance and plasmid profile of Aeromonas hydrophila isolates from cultured fish, Telapia (Telapia mossambica). Lett Appl Microbiol 1997;24(6):479-82. 48. Swain P, Nayak SK, Sasmal A, Behera T, Barik SK, Swain SK, et al. Antimicrobial activity of metal based nanoparticles against microbes associated with diseases in aquaculture. World J Microbiol Biotechnol 2014;30(9):2491-502. 49. Di Cesare A, Vignaroli C, Luna GM, Pasquaroli S, Biavasco F. Antibiotic-resistant enterococci in seawater and sediments from a coastal fish farm. Microb Drug Resist 2012;18(5):502-9. 50. Di Cesare A, Luna GM, Vignaroli C, Pasquaroli S, Tota S, Paroncini P, et al. Aquaculture can promote the presence and spread of antibioticresistant Enterococci in marine sediments. PLoS One 2013;8(4):e62838. 51. Seil JT, Webster TJ. Antimicrobial applications of nanotechnology: methods and literature. Int J Nanomedicine 2012;7:2767-81. 52. Knetsch ML, Koole LH. New strategies in the development of antimicrobial coatings: the example of increasing usage of silver and silver nanoparticles. Polymers 2011;3(1):340-66. 53. Hindi KM, Ditto AJ, Panzner MJ, Medvetz DA, Han DS, Hovis CE, et al. The antimicrobial efficacy of sustained release silver–carbene complex-loaded L-tyrosine polyphosphate nanoparticles: characterization, in vitro and in vivo studies. Biomaterials 2009;30(22):3771-9. 54. Antony JJ, Nivedheetha M, Siva D, Pradeepha G, Kokilavani P, Kalaiselvi S, et al. Antimicrobial activity of Leucas aspera engineered silver nanoparticles against Aeromonas hydrophila in linfected Catla catla. Colloids Surf B: Biointerfaces 2013;109:20-4. 55. Lara HH, Ayala-Núnez NV, Turrent LDCI, Padilla CR. Bactericidal effect of silver nanoparticles against multidrug-resistant bacteria. World J Microbiol Biotechnol 2010;26(4):615-21. 56. Huang CM, Chen CH, Pornpattananangkul D, Zhang L, Chan M, Hsieh MF, et al. Eradication of drug resistant Staphylococcus aureus by liposomal oleic acids. Biomaterials 2011;32(1):214-21. 57. Prakash P, Gnanaprakasam P, Emmanuel R, Arokiyaraj S, Saravanan M. Green synthesis of silver nanoparticles from leaf extract of Mimusops elengi, Linn. for enhanced antibacterial activity against multi drug resistant clinical isolates. Colloids Surf B: Biointerfaces 2013;108:255-9. 58. Ayala-Núñez NV, Lara HH, Turrent LDCI, Padilla CR. Silver nanoparticles toxicity and bactericidal effect against methicillinresistant Staphylococcus aureus: nanoscale does matter. Nanobiotechnology 2009;5:2-9. 59. Umashankari J, Inbakandan D, Ajithkumar TT, Balasubramanian T. Mangrove plant, Rhizophora mucronata (Lamk, 1804) mediated one pot green synthesis of silver nanoparticles and its antibacterial activity against aquatic pathogens. Aquat Biosyst 2012;8(1):11. 60. Sanjenbam P, Gopal JV, Kannabiran K. Anticandidal activity of silver nanoparticles synthesized using Streptomyces sp. VITPK1. J Mycol Med 2014;24(3):211-9. 61. Mallmann EJJ, Cunha FA, Castro BN, Maciel AM, Menezes EA, Fechine PBA. Antifungal activity of silver nanoparticles obtained by green synthesis. Rev Inst Med Trop Sao Paulo 2015;57(2):165-7. 62. Kim KJ, Sung WS, Moon SK, Choi JS, Kim JG, Lee DG. Antifungal effect of silver nanoparticles on dermatophytes. J Microbiol Biotechnol 2008;18:1482-4. 63. Elechiguerra JL, Burt JL, Morones JR, Camacho-Bragado A, Gao X, Lara HH, et al. Interaction of silver nanoparticles with HIV-1. J Nanobiotechnol 2005;3:6. 64. Mehrbod P, Motamed N, Tabatabaian M, Soleimani R, Amini E, Shahidi M, et al. In vitro anti viral effect of "nanosilver" on influenza virus. DARU 2009;17:88-93. 65. Mori Y, Ono T, Miyahira Y, Nguyen VQ, Matsui T, Ishihara M. Antiviral activity of silver nanoparticle/chitosan composites against H1N1 influenza A virus. Nanoscale Res Lett 2013;8(1):1-6. 66. Li X, Robinson SM, Gupta A, Saha K, Jiang Z, Moyano DF, Sahar A, et al. Functional gold nanoparticles as potent antimicrobial agents against multi-drug-resistant bacteria. ACS Nano 2014;8(10):10682-6.
709
67. Sumbayev VV, Yasinska IM, Gibbs BF. Biomedical applications of gold nanoparticles. Recent advances in circuits, communications and signal processing. Athens: WSEAS Press; 2013342-8. 68. Lima E, Guerra R, Lara V, Guzmán A. Gold nanoparticles as efficient antimicrobial agents for Escherichia coli and Salmonella typhi. Chem Cent J 2013;7(1):11. 69. Cui Y, Zhao Y, Tian Y, Zhang W, Lü X, Jiang X. The molecular mechanism of action of bactericidal gold nanoparticles on Escherichia coli. Biomaterials 2012;33(7):2327-33. 70. Ahmad T, Wani IA, Lone IH, Ganguly A, Manzoor N, Ahmad A, et al. Antifungal activity of gold nanoparticles prepared by solvothermal method. Mater Res Bull 2013;48(1):12-20. 71. Wani IA, Ahmad T. Size and shape dependant antifungal activity of gold nanoparticles: a case study of Candida. Colloid Surf B: Biointerfaces 2013;101:162-70. 72. Liu Y, He L, Mustapha A, Li H, Hu ZQ, Lin M. Antibacterial activities of zinc oxide nanoparticles against Escherichia coli O157: H7. J Appl Microbiol 2009;107(4):1193-201. 73. Ramamoorthy S, Kannaiyan P, Moturi M, Devadas T, Muthuramalingam J, Natarajan L, et al. Antibacterial activity of zinc oxide nanoparticles against Vibrio harveyi. Indian J Fish 2013;107–112. 74. Cheng TC, Yao KS, Yeh N, Chang CI, Hsu HC, Chien YT, et al. Visible light activated bactericidal effect of TiO2/Fe3O4 magnetic particles on fish pathogens. Surf Coat Technol 2009;204(6):1141-4. 75. Cheng TC, Yao KS, Yeh N, Chang CI, Hsu HC, Gonzalez F, et al. Bactericidal effect of blue LED light irradiated TiO2/Fe3O4 particles on fish pathogen in seawater. Thin Solid Films 2011;519(15):5002-6. 76. Jovanović B, Anastasova L, Rowe EW, Zhang Y, Clapp AR, Palić D. Effects of nanosized titanium dioxide on innate immune system of fathead minnow (Pimephales promelas Rafinesque, 1820). Ecotoxicol Environ Saf 2011;74(4):675-83. 77. Jovanović B, Whitley EM, Kimura K, Crumpton A, Palić D. Titanium dioxide nanoparticles enhance mortality of fish exposed to bacterial pathogens. Environ Pollut 2015;203:153-64. 78. Lockman PR, Mumper RJ, Khan MA, Allen DD. Nanoparticle technology for drug delivery across the blood-brain barrier. Drug Dev Ind Pharm 2002;28(1):1-13. 79. Wang JJ, Zeng ZW, Xiao RZ, Xie T, Zhou GL, Zhan XR, et al. Recent advances of chitosan nanoparticles as drug carriers. Int J Nanomedicine 2011;6:765-74. 80. Costa ADS, Brandão HM, Silva SR, Bentes‐Sousa AR, Diniz JAP, Viana Pinheiro JDJ, et al. Mucoadhesive nanoparticles: a new perspective for fish drug application. J Fish Dis 2015, http:// dx.doi.org/10.1111/jfd.12373. 81. Alishahi A, Mirvaghefi A, Tehrani MR, Farahmand H, Koshio S, Dorkoosh FA, et al. Chitosan nanoparticle to carry vitamin C through the gastrointestinal tract and induce the non-specific immunity system of rainbow trout (Oncorhynchus mykiss). Carbohydr Polym 2011;86(1):142-6. 82. Rather MA, Sharma R, Gupta S, Ferosekhan S, Ramya VL, Jadhao SB. Chitosan-nanoconjugated hormone nanoparticles for sustained surge of gonadotropins and enhanced reproductive output in female fish. PLoS One 2013;8(2):e57094. 83. Lü JM, Wang X, Marin-Muller C, Wang H, Lin PH, Yao Q, et al. Current advances in research and clinical applications of PLGA-based nanotechnology. Expert Rev Mol Diagn 2009;9(4):325-41. 84. Makadia HK, Siegel SJ. Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier. Polymers 2011;3(3):1377-97. 85. Fenaroli F, Westmoreland D, Benjaminsen J, Kolstad T, Skjeldal FM, Meijer AH, et al. Nanoparticles as drug delivery system against tuberculosis in zebrafish embryos: direct visualization and treatment. ACS Nano 2014;8(7):7014-26. 86. Myhr AI, Myskja BK. Precaution or integrated responsibility approach to nanovaccines in fish farming? A critical appraisal of the UNESCO precautionary principle. Nanoethics 2011;5(1):73-86.
710
M. Shaalan et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 701–710
87. Zhao L, Seth A, Wibowo N, Zhao CX, Mitter N, Yu C, et al. Nanoparticle vaccines. Vaccine 2014;32(3):327-37. 88. Gregory AE, Titball R, Williamson D. Vaccine delivery using nanoparticles. Front Cell Infect Microbiol 2013;3:13, http:// dx.doi.org/10.3389/fcimb.2013.00013. 89. Rivas-Aravena A, Fuentes Y, Cartagena J, Brito T, Poggio V, La Torre J, et al. Development of a nanoparticle-based oral vaccine for Atlantic salmon against ISAV using an alphavirus replicon as adjuvant. Fish Shellfish Immunol 2015;45(1):157-66. 90. Vimal S, Taju G, Nambi KSN, Abdul Majeed S, Sarath Babu V, Ravi M, et al. Synthesis and characterization of CS/TPP nanoparticles for oral delivery of gene in fish. Aquaculture 2012;358–359:14-22. 91. Li L, Lin SL, Deng L, Liu ZG. Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in black seabream Acanthopagrus schlegelii Bleeker to protect from Vibrio parahaemolyticus. J Fish Dis 2013;36(12):987-95. 92. Rajeshkumar S, Venkatesan C, Sarathi M, Sarathbabu V, Thomas J, Anver Basha K, et al. Oral delivery of DNA construct using chitosan nanoparticles to protect the shrimp from white spot syndrome virus (WSSV). Fish Shellfish Immunol 2009;26(3):429-37. 93. Vimal S, Abdul Majeed S, Taju G, Nambi KS, Sundar Raj N, Madan N, et al. Chitosan tripolyphosphate (CS/TPP) nanoparticles: preparation, characterization and application for gene delivery in shrimp. Acta Trop 2013;128(3):486-93. 94. Wang D, Robinson DR, Kwon GS, Samuel J. Encapsulation of plasmid DNA in biodegradable poly(D, L-lactic-co-glycolic acid) microspheres as a novel approach for immunogene delivery. J Control Release 1999;57(1):9-18. 95. Hølvold LB, Myhr AI, Dalmo RA. Strategies and hurdles using DNA vaccines to fish. Vet Res 2014;45(1):21-31. 96. Tian J, Sun X, Chen X, Yu J, Qu L, Wang L. The formulation and immunisation of oral poly(DL-lactide-co-glycolide) microcapsules containing a plasmid vaccine against lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus). Int Immunopharmacol 2008;8(6):900-8.
97. Jain KK. Nanodiagnostics: application of nanotechnology in molecular diagnostics. Expert Rev Mol Diagn 2003;3(2):153-61. 98. Baptista P, Pereira E, Eaton P, Doria G, Miranda A, Gomes I, et al. Gold nanoparticles for the development of clinical diagnosis methods. Anal Bioanal Chem 2008;391(3):943-50. 99. Saleh M, Soliman H, El-Matbouli M. Gold nanoparticles as a potential tool for diagnosis of fish diseases. Methods Mol Biol 2015;1247:245-52, http://dx.doi.org/10.1007/978-1-4939-2004-4_19. 100. Saleh M, Soliman H, Haenen O, El-Matbouli M. Antibody-coated gold nanoparticles immunoassay for direct detection of Aeromonas salmonicida in fish tissues. J Fish Dis 2011;34(11):845-52. 101. Kuan GC, Sheng LP, Rijiravanich P, Marimuthu K, Ravichandran M, Yin LS, et al. Gold-nanoparticle based electrochemical DNA sensor for the detection of fish pathogen Aphanomyces invadans. Talanta 2013;117:312-7. 102. Jaroenram W, Arunrut N, Kiatpathomchai W. Rapid and sensitive detection of shrimp yellow head virus using loop-mediated isothermal amplification and a colorogenic nanogold hybridization probe. J Virol Methods 2012;186(1):36-42. 103. Seetang-Nun Y, Jaroenram W, Sriurairatana S, Suebsing R, Kiatpathomchai W. Visual detection of white spot syndrome virus using DNA-functionalized gold nanoparticles as probes combined with loop-mediated isothermal amplification. Mol Cell Probes 2013;27(2):71-9. 104. Toubanaki DK, Margaroni M, Karagouni E. Nanoparticle-based lateral flow biosensor for visual detection of fish nervous necrosis virus amplification products. Mol Cell Probes 2015;3:158-66. 105. Yang SY, Wu JL, Tso CH, Ngou FH, Chou HY, Nan FH, et al. A novel quantitative immunomagnetic reduction assay for nervous necrosis virus. J Vet Diagn Invest 2012;24(5):911-7. 106. Saleh M, Soliman H, Schachner O, El-Matbouli M. Direct detection of unamplified spring viraemia of carp virus RNA using unmodified gold nanoparticles. Dis Aquat Organ 2012;100(1):3-10. 107. Saleh M, El-Matbouli M. Rapid detection of cyprinid herpesvirus-3 (CyHV-3) using a gold nanoparticle-based hybridization assay. J Virol Methods 2015;217:50-4.
Review Article
nanomedjournal.com
Recent progress in applications of nanoparticles in fish medicine: A review Mohamed Shaalan, MSc a, b , Mona Saleh, PhD a , Magdy El-Mahdy, PhD b , Mansour El-Matbouli, PhD a,⁎ b
a Clinical Division of Fish Medicine; University of Veterinary Medicine, Vienna, Austria Department of Pathology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt Received 21 July 2015; accepted 14 November 2015
Abstract Nanotechnology has become an extensive field of research due to the unique properties of nanoparticles, which enable novel applications. Nanoparticles have found their way into many applications in the field of medicine, including diagnostics, vaccination, drug and gene delivery. In this review, we focused on the antimicrobial effects of nanoparticles, with particular emphasis on the problem of antibiotic resistant bacteria in fisheries. The use of nanoparticle-based vaccines against many viral pathogens is a developing field in fish medicine research. Nanoparticles have gained much interest as a specific and sensitive tool for diagnosis of bacterial, fungal and viral diseases in aquaculture. Nevertheless our review also highlights the many applications of nanotechnology that are still to be explored in fish medicine. From the Clinical Editor: Advance in nanotechnology has enabled the development of nanomedicine, with many ideas being used in clinical diagnosis and therapy. In this review article, the authors described the current use of nanotechnology in fish medicine. The knowledge would also impart important information for our daily living. © 2015 Elsevier Inc. All rights reserved. Key words: Nanoparticles; Fish diseases; Diagnosis; Antimicrobial; Fish vaccines
Nanotechnology is the science of producing and utilizing nanometer-sized particles. Only recently we have developed approaches to understand and control matter at nanoscale dimensions (between approximately 1 and 100 nm). Particles in this size range have unique properties, which pave the way for novel applications. 1 Nanomaterials can be divided into two large groups: ultrafine nano-sized particles that are present normally in nature and not intentionally produced and engineered nanoparticles that are produced in a controlled and intended way. 2 Nanomedicine is the application of nanotechnology in both human and veterinary medicine. This science deals with designing and utilizing nanoparticles and nanodevices for biomedical applications. 3 We acknowledge support from a PhD scholarship offered for Mohamed Shaalan by mission sectors in the Ministry of Higher Education, Egypt. Competing interests: The authors declare that they have no competing interests. The authors declare that they don’t have any commercial associations, current and within the past five years, that might pose a potential, perceived or real conflict of interest. ⁎Corresponding author at: Clinical Division of Fish Medicine, University of Veterinary Medicine, Vienna, Austria. E-mail address: [email protected] (M. El-Matbouli).
Many forms of nanoparticles have been used in medicine 4 (Table 1), for example nanospheres, which are nano-sized spherical particles. 5 Because of their small size and high surface area, a specific drug can be dispersed on the nanosphere's surface to facilitate drug delivery. Nanospheres can also be used for tissue regeneration. 6 Another form of nanoparticle is the nanocapsule, which is composed primarily of an outer nano-scale shell and an inner core. The core may be oil or water, which contains a specific drug, while the shell protects the drug from degradation and hydrolysis. 7 For example, liposomes are lipid bi-layer nano-sized spheres, which are ideal for drug delivery of both lipophilic and hydrophilic medications due to their structure, which is similar to the eukaryotic cell membrane. 3,8 Another form of nanoparticles commonly used in different medical applications is carbon nanotubes (single-walled or multi-walled). These are characterized by having a very large surface area and high ability to penetrate cell membranes, acting as micro-needles, which makes them ideal for drug delivery, including anti-tumor drugs. 9 However, a study has shown that they have high risk of inducing thrombi inside blood vessels. 10 A fourth particle form is dendrimers, which are nanoscale three dimensional structures consisting of tree branch-like units
http://dx.doi.org/10.1016/j.nano.2015.11.005 1549-9634/© 2015 Elsevier Inc. All rights reserved. Please cite this article as: Shaalan M, et al, Recent progress in applications of nanoparticles in fish medicine: A review. Nanomedicine: NBM 2016;12:701-710, http://dx.doi.org/10.1016/j.nano.2015.11.005
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Table 1 Different forms of nanoparticles used in biomedical applications. Type of nanoparticles
Structure
Application in medicine
Nanospheres
Spherical shaped
Nanocapsules
Shell and core combination Cylindrical tubes
Drug delivery, tissue regeneration Controlled drug delivery
Carbon Nanotubes Liposomes
Drug delivery, Anti-cancer
Lipid bi-layer globules
Drug delivery for hydrophobic and hydrophilic drugs Dendrimers Highly branched ends and Delivery system, tissue central core engineering, antimicrobials Polymeric Polymers as Delivey system, nanoparticles chitosan, PLGA tissue regeneration
coming from a central core. These have a highly branched multifunctional surface with low molecular weight, which facilitates their use as a delivery system for genes, vaccines, and drugs, and as a scaffold for tissue regeneration, and as antimicrobial agents. 11–13 The types of nanoparticles applied in fish medicine have to date been limited to mostly nanospheres or polymeric nanoparticles; application of other forms of nanoparticles has not yet been investigated. In this review, we summarize the various demonstrated and potential applications of nanoparticles in the field of fish medicine. These include their deployment as antimicrobials, drug and gene delivery vehicles, vaccination, and for specific diagnosis of fish pathogens.
Synthesis of nanoparticles Generally, there are two main approaches for nanoparticle synthesis: top-down or bottom-up. 14 The top-down approach involves mechanical grinding of bulk metal to convert it from macro or microscale to nanoscale, which is followed by addition of stabilizing agents like colloidal protecting agents to ensure that the nanoparticles do not oxidize, or re-assemble back to the microscale. 15 Bottom-up methods include construction of nanoparticles by various physical and chemical methods, including electrochemical reduction of metals and sonodecomposition. 16 Physical and chemical synthesis Nanoparticle synthesis can be carried out by using thermal decomposition in organic solvents. 17 For metal nanoparticles, cryochemical synthesis yields nanoparticles in the range of 5 to 80 nm in diameter. 18 Physical synthesis using microwaves has been adopted for silver nanoparticles, which involves physical reduction of silver using different microwave radiation frequencies. 19 This method was more rapid and gave a higher concentration of silver nanoparticles when compared with a thermal method, given the same temperature and exposure. Jiang et al 19 also found that the higher the concentration of silver nitrate used, the longer the reaction time and the higher the temperature, the larger the particle that could be obtained. Use of
high poly vinyl pyrrolidone (PVP) concentrations (used for nanoparticle coating) resulted in smaller silver particle sizes, between 15 and 25 nm. Other methods of metal nanoparticle synthesis include: pulsed laser ablation 20; electroreduction of AgNO3 in aqueous solution in the presence of polyethylene glycol 21 ; spark discharge 22 micro-emulsion for preparation of Ag-Fe3O2 nanoparticles 23; chemical reduction of silver nitrate using trisodium citrate combined with sodium borohydride as reducing agent to synthesize PVP coated silver nanoparticles. 24,25 Different methods have also been demonstrated for synthesis of non-metallic nanoparticles, e.g. polymeric chitosan nanoparticles. For example, ionotropic gelation is a simple method involving dissolution of chitosan in acetic acid, with addition of polyanion tripolyphosphate (TPP). This method depends on electrostatic interaction between the chitosan amine group and groups of polyanion polymer. 26,27 The polyelectrolyte complex method depends on electrostatic interaction between cationic groups in chitosan and DNA, which neutralizes the charges with formation of nanoparticles. 26 The microemulsion method utilizes addition of surfactants, but has disadvantages which include use of organic solvents, long preparation time and the complexity of the washing processes. 28 The most common method for synthesis of Poly D, L-lactide-co-glycolic acid (PLGA) nanoparticles is the precipitation method 29 combined with the double emulsion solvent evaporation method. 30,31 Biological synthesis (Green synthesis) There is great interest in finding eco-friendly and economical methods to synthesize nanoparticles. Biological methods are regarded as the key for this approach. 32 Biologically synthesized nanoparticles are derived from three main groups of organisms: bacteria, fungi, and plants. Bio-synthesis of nanoparticles is a bottom-up approach that mostly involves reduction/oxidation reactions. 33 Microbial enzymes or plant phytochemicals with antioxidant or reducing properties act on precursor compounds to produce the desired nanoparticles. The three main components of a biosynthetic nanoparticle system are: a solvent medium for synthesis, an environmentally friendly reducing agent, and a nontoxic stabilizing agent. 33 Silver nanoparticles synthesized using Origanum vulgare leaf extract demonstrated antibacterial activity and cytotoxic effects against a human lung cancer cell line. 34 Cashew nut shell liquid was used for green synthesis of both silver and gold nanoparticles, which showed bactericidal activity against several fish pathogens. 35 Silver nanoparticles synthesized using tea leaf extract (Camellia sinensis) demonstrated bactericidal activity against Vibrio harveyi in juvenile Feneropenaeus indicus, but only at high doses of the nanoparticles. 36 A broth of Aloe leaf extract was used for green synthesis of zinc oxide nanoparticles (ZnO-NPs), which showed higher bactericidal activity than nanoparticles by standard chemistry. 37 A novel method for biological synthesis of zinc oxide nanoparticles uses Aeromonas hydrophila bacteria as the reducing agent. This method is environmentally friendly and economically feasible, and resulted in ZnO nanoparticles with antibacterial and antimycotic effects. 38
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Antibiotic resistant bacteria in fisheries Antibiotics have been used for decades to combat bacterial infections. However unregulated, excessive use of antibiotics can lead to the emergence of resistant bacteria which are no longer sensitive to antibiotics. 39–42 Since there are shared elements between the mobile genomes of both terrestrial and aquatic bacteria, resistance genes can be exchanged between them with major potential impacts on livestock and human health 42,43 Tuševljak et al 44 conducted a survey about usage of antimicrobials in fish farms in 25 countries. They found that tetracycline is the most used antibiotic agent in fish farms. There is a strong relation between emergence of antibiotic resistant bacteria and excessive antibiotics administration in aquaculture. Emergence of many resistant bacteria, including as methicillin resistant Staphyloccus aureus (MRSA) and multi- and super-drug resistant forms has been recorded. 45,46 Some Aeromonas hydrophila isolates from cultured tilapia were resistant to broad spectrum antibiotics like tetracycline, streptomycin and erythromycin. 47 Antibiotic resistance was also observed in Aeromonas salmonicida, Photobacterium damselae subsp. piscicida, Yersinia ruckeri, Vibrio, Listeria, Pseudomonas and Edwardsiella species. 43,48 Di Cesare et al 49,50 isolated antibiotic resistant enterococci from fish farm sediment, which raises the possibility that these bacteria may transfer their resistance to human-infecting strains, making their presence in fish farms an issue of public health importance. Nanoparticles as novel antimicrobials in fish medicine The utilization of nanoparticles as alternative antimicrobials to combat emergence of microbial resistance to antibiotics in aquaculture has been investigated. 37,48 Metal nanoparticles have shown high antimicrobial activity against bacteria, fungi and viruses. 51 In the following sections, we discuss studies on metal nanoparticles that have been applied against fish pathogens. Silver nanoparticles (Ag-NPs) Silver nanoparticles are the most investigated nano-antibacterial agent in research literature. They act against bacteria through multiple mechanisms, and in this way they can evade the bacterial resistance, 52 in contrast to antibiotics that generally act through one mechanism only. 52–54 The release of silver ions (Ag +) is one mechanism 52 : Ag + binds to bacterial cell membrane proteins causing disruption of the membrane, leading to death of the bacterial cells. 55 Intracellularly, Ag + binds to cytochrome and nucleic acids, damaging them and inhibiting cell division. 56 Prakash et al 57 report that silver nanoparticles demonstrate high antibacterial efficacy against multi-drug resistant bacterial isolates. The bactericidal effect of silver nanoparticles has also been reported against methicillin resistant Staphyloccus aureus (MRSA). 58 Silver nanoparticles synthesized using the juice of the Citrus limon as a reducing agent, demonstrated antibacterial activity against Staphylococcus aureus and Edwardesiella tarda, and anti-cyanobacterial activity towards Anabaena and Oscillatoria species. 48 Umashankari et al 59 used leaf bud extract from mangrove Rhizophora mucronata for biological synthesis of Ag-NPs, then demonstrated antimicrobial effects against
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Pseudomonas fluorescens, Proteus species and Flavobacterium species. The efficacy of these ‘green' synthesized silver nanoparticles was the same as commercial antibiotics. In a study on Vibrio harveyi-infected juvenile shrimp Feneropenaeus indicus, long term treatment with silver nanoparticles decreased mortalities by 71% at high doses of Ag-NPs. 36 As an antifungal agent, silver nanoparticles exhibited high inhibitory effects against Candida species, similar to the commercial antifungal Amphotericin B. 60,61 Antifungal activity of silver nanoparticles against dermatophytes has also been recorded. 62 Silver nanoparticles also exhibit anti-viral properties: they can bind to HIV-1 virus proteins in vitro. 63 Both silver nanoparticles and silver nanoparticles-chitosan composite are active against influenza A virus. 64,65 There has been little published work specifically on the antifungal and antiviral effects of silver nanoparticles in fish medicine. Gold nanoparticles (Au-NPs) There is currently great interest toward investigating the antimicrobial effects of gold nanoparticles, due to their low toxicity to eukaryotic cells. 66 Au-NPs can interact with biological proteins and non-proteins, e.g. Lipopolysaccharides (LPS), and have biological functions. 67 Gold nanoparticles supported on zeolite exhibited bactericidal effects against Escherichia coli and Salmonella typhi. 68 Functionalized Au-NPs inhibited the growth of MDR bacterial isolates. 66 Gold nanoparticles made by ‘green' synthesis showed antibacterial activity against fish bacterial isolate. 35 There are three pathways along which gold nanoparticles exert their antibacterial effects. The first way is via interfering with oxidative phosphorylation process with changing the potential of bacterial cell membrane; this leads to decrease in the activity of F-Type ATP synthase with a net decrease in ATP synthesis and metabolism. The second path is interference with binding of tRNA to the two ribosome subunits. The third way is achieved through enhancing chemotaxis. 69 Fungicidal activity against Candida species was reported for gold nanoparticles. Their efficacy was size-dependent, with smaller gold nanoparticles having higher antifungal effects. 70,71 Zinc oxide nanoparticles (ZnO-NPs) Zinc oxide nanoparticles have drawn much attention due to their antibacterial and antifungal effects. 37,48 The antibacterial activity derives from damage the particles do to the bacterial cell membrane, which makes cytoplasmic contents leak from the cell. 72 In the field of fish medicine, ZnO-NPs can inhibit the growth of Aermonas hydrophila, Edwardseilla tarda, Flavobacterium branchiophilum, Citrobacter spp., Staphylococcus aureus, Vibrio species, Bacillus cereus and Pseudomonas aeruginosa. 48 Ramamoorthy et al 73 investigated the antibacterial effects of ZnO nanoparticles against the pathogenic Vibrio harveyi and observed higher bactericidal effects of nanoparticles compared to bulk ZnO. In another interesting study, ZnO nanoparticles were synthesized biologically using Aermonas hydrophila, and these nanoparticles exhibited antibacterial activity against the same bacterium and other species: Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Aspergillus flavus and Candida albicans as shown in Figure 1. 38
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Figure 1. Mueller–Hinton agar plates showing the antifungal effects of ZnO-NPs, and zones of inhibition against both (A) Pseudomonas aeruginosa and (B) Aspergillus flavus. (Adapted with permission from Jayaseelan et al, 2012).
Titanium dioxide nanoparticles (TiO2-NPs) TiO2-NPs, when doped with magnetic Fe3O4-NPs had a bactericidal effect against Streptococcus iniae, Edwardsiella tarda and Photobacterium damselae, after activation by light. 74,75 These particles can be used to disinfect water, as the fish pathogens bind with the nanoparticles, which can then be easily extracted from water using a magnet. 74,75 However, Jovanovic et al 76,77 concluded that TiO2-NPs affect the fish immune system by inhibiting the antibacterial activity of fish neutrophils, making the fish liable to infection and hence increased mortality especially in disease outbreaks.
hormone releasing hormone (LHRH) was bound to chitosan nanoparticles in one group and compared with LHRH conjugated with chitosan-gold nanoparticles. Both groups showed an increase in blood hormone levels with sustained release of hormones groups, compared to the group injected with hormone alone. Egg fertilization rates were elevated after one injection dose of hormone-chitosan nanoparticle conjugate and hormone-chitosan-gold nanoparticles, respectively 87% and 83%, compared with multiple injections of LHRH alone which gave a fertilization rate of 74%. 82 PLGA nanoparticles for drug delivery
Nanoparticles as vehicles for drug and gene delivery An ideal drug delivery system has several key properties, which include safety, biocompatibility and biodegradability of the delivery system, plus stability of the drug, specificity of delivery, and few or no side effects 4 As drug delivery systems, nanoparticles have gained much interest because of their small sizes and because they can cross biological barriers like the blood brain barrier (BBB). Nanoparticles also have a high surface area to volume ratio, which allows increased reactivity with other conjugates and compounds. 4,78,79 In fish medicine specifically, chitosan nanoparticles and PLGA nanoparticles have been investigated the most as nanoparticles for drug delivery.
PLGA is a copolymer consisting of poly lactic acid and poly glycolic acid. It is biocompatible, biodegradable and non-toxic. It has been approved by the FDA. Hence many researchers have investigated the feasibility of using PLGA as a drug carrier. 83,84 In a recent study on zebra fish embryos, PLGA nanoparticles were loaded with the anti-mycobacterial agent rifampicin then injected. As zebra fish embryos are transparent, the treatment impact on Mycobacterium marinum-infected could be measured using non-invasive imaging as shown in Figure 2. The rifampicin-PLGA nanoparticles showed an increased therapeutic effect against M. marinum and higher embryo survival, compared to rifampicin alone. 85
Chitosan nanoparticles for drug delivery
Nanotechnology-based fish vaccines
Chitosan nanoparticles have excellent properties as drug delivery vehicles. They are comprised of a biocompatible, non-toxic and biodegradable polymer, which is easily excreted from the kidney. 4 Their mucoadhesive properties mean that they can be adapted for slow and sustainable drug release. 80 For example, vitamin C was conjugated with chitosan nanoparticles in a study on rainbow trout (Oncorhynchus mykiss). The vitamin was released up to 48 h after oral administration, and stimulation of the fish innate immune system was observed due to potent synergism between chitosan and vitamin C. 81 In another study, chitosan nanoparticles were used as a hormone delivery system in Cyprinus carpio. Luteinizing
Incorporation of nanoparticles in vaccine formulations is an exciting avenue of medical research. Polymeric nanoparticles have been most widely investigated, as they have several advantages as vaccine releasing vehicles, including their ability to ensure antigen stability against degradation by enzymes, preserving immunogenicity and for sustained release of the vaccine. 86,87 Nanovaccines have been used as immunostimulant adjuvants or as a delivery system for targeted antigen delivery, with slow antigen release a feature of both purposes. 87 Other nanoparticle forms that have been used as a vaccine delivery system include: virus-like particles, liposomes, immunostimulant complex, metal nanoparticles and polymeric nanoparticles. 87,88 Concerns about nanovaccines have
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Figure 2. (A, B, C, D) Lateral view showing formation of Mycobacterium marinum granulomas in zebra fish embryos. After injection of nanoparticles in the posterior caudal vein, panels (E, F, G, H) show rifampicin-loaded PLGA-nanoparticles targeting the granulomas. (Adapted with permission from Fenaroli et al, 2014. Copyright (2014) American Chemical Society).
been directed mainly towards technical difficulties in producing them with stable properties, their potential toxicity, and a lack of information about their distribution within biological systems. 88 The most investigated nanoparticles in fish vaccine research up to now are polymeric chitosan and PLGA nanoparticles. Chitosan nanoparticles have been used for the development of fish vaccines, for example the inactivated virus vaccine against infectious salmon anemia virus (ISAV), which incorporates the DNA coding for ISAV replicase as an adjuvant. This vaccine showed protection rates that reach up to 77% against ISAV. 89 An oral DNA vaccine against Vibrio anguillarum in Asian sea bass (Lates calcarifer) was developed using chitosan and chitosan/tripoly phosphate nanoparticles. The nanovaccine conferred only moderate protection against the pathogen. 27,90
Another oral DNA vaccine was developed by loading the outer membrane protein K (ompK) gene of Vibrio parahemolyticus onto chitosan nanoparticles. This recombinant nanovaccine elicited a protective immune response in black seabream (Acanthopagrus schlegelii) against Vibrio parahemolyticus. 91 The effectiveness of recombinant DNA-chitosan nanoparticles in providing protection against white spot syndrome virus (WSSV) in shrimps was investigated. It was found that when administered orally, the vaccine enhanced shrimp immunity, providing a protective response against WSSV. 92,93 The efficacy of PLGA nanoparticles as a DNA vaccine carrier and adjuvant has been reported. 30,94,95 PLGA nanoparticles have had limited use in fisheries so far. An oral DNA vaccine in Japanese flounder (Paralichthys olivaceus) against lymphocystis disease virus (LCDV) was developed. 96 In a different study, an
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Figure 3. The principle of direct detection of SVCV RNA using unmodified gold nanoparticles (Adapted with permission from Saleh et al, 2012).
orally administered DNA vaccine was used to immunize rainbow trout against infectious hematopoietic necrosis virus, and an immune response was observed. 31 Nanoparticles in the diagnosis of fish pathogens Nanoparticles have been adopted for rapid and sensitive diagnosis of diseases, and these nanoparticle-based detection methods are called nanodiagnostics. 97 One of the most widely used nanoparticles in diagnostics is gold nanoparticles, which are appropriate for use in different methods. 98,99 Diagnosis of bacterial and mycotic fish diseases The first report of using gold nanoparticles for detection of fish pathogen was with A. salmonicida antibody-gold nanopar-
ticles conjugated for the specific immunodiagnosis of furunculosis in fish tissues. 100 Kuan et al 101 designed an electrochemical DNA biosensor for detection of Aphanomyces invadans in fish, based on conjugation of Au-NPs with a DNA reporter probe. This assay could detect fungi at a lower level than PCR. Diagnosis of viral fish diseases Jaroenram et al 102 combined a AuNPs colorimetric assay with loop-mediated isothermal amplification (LAMP) for visual detection of yellow head virus in shrimp. This method was rapid, was specific, and showed high sensitivity. A similar combination of DNA-functionalized AuNPs with LAMP was developed for the detection of white spot syndrome virus (WSSV) in shrimp. 103 This method was specific, sensitive and suitable for field applications.
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Figure 4. Results of a gold nanoparticle-based assay for detection of SVCV: in a positive test, the color of the solution changes from red to blue (Adapted with permission from Saleh et al, 2012).
In another study, Toubanaki et al 104 developed a method for NNV detection using a gold nanoparticles-based biosensor for detection of viral nucleic acids after amplification by RT-PCR. This method was quite cost effective, as it did not require antibody conjugation. Yang et al 105 report an immunomagnetic reduction assay for nervous necrosis virus (NNV) in grouper fish using magnetic nanoparticles coated with rabbit anti-NNV antibody. When an external magnetic field was applied, immunodiagnosis was based on the motility of magnetic nanoparticles: if the antibody coated-nanoparticles bound to the viral antigen, they form clusters which decrease their motility. The virus titer was calculated using a magnetic immunoassay analyzer. A colorimetric assay for detection of spring viremia of carp virus (SVCV) was developed using unmodified Au-NPs. The probe, which was complementary for SVCV, was added first, followed by gold nanoparticles. If target viral RNA was present, it hybridized to the probe, thereby preventing the probe from stabilizing the gold nanoparticles. The Au-NPs could aggregate, with a resultant change in the solution, from red to blue. If no viral nucleic acid was present, the probe could freely adsorb onto the surface of gold nanoparticles and prevent their aggregation, and the solution stayed red (Figures 3-4). 106 This method was highly specificity and rapid, without the need for prior amplification of viral nucleic acids. The same principle was adopted for the development of a rapid, specific and sensitive assay for the detection of the DNA virus, cyprinid herpes virus-3 (CyHV-3). 107
Concluding summary and future directions In this review article, we have summarized the current applications of nanoparticles for diagnosis, and treatment of fish pathogens. A range of different methods has been employed for nanoparticle synthesis, and the emerging use of ‘green' synthetic methods appears safer and more environmentally friendly while
retaining the efficacy of the formed nanoparticles. 32–38 Naked metal nanoparticles exhibit antimicrobial effects, and have been applied to combat microbial resistance in aquaculture. 36–38,48,59,66,73–75 Many nanoparticles are reported to be excellent vehicles for drug, gene and vaccine delivery due to their unique properties. The most investigated nanoparticles in fish medicine for these applications are polymeric chitosan nanoparticles and Poly D, L-lactide-co-glycolic acid (PLGA) nanoparticles. 3,4,78–80,84,86 Both unmodified and conjugated nanoparticles have been shown to facilitate rapid, cost effective and specific detection of fish pathogens. There are, however, many research gaps in the field of nanotechnology applications in fish medicine. Different forms of nanoparticles like nanocapsules, liposomes, dendrimers and nanotubes could theoretically have applicability in fish diseases research. The antifungal and antiviral effects of nanoparticles against fish diseases have yet to be explored. More studies for vaccine development in fish are essential if nanotechnological approaches are to be applied widely. Few studies have examined nanoparticle applications for diagnosis of bacterial and mycotic diseases in aquaculture. Given the demonstrated potential of nanoparticles, there are needs for more targeted investigations of their application in many fish medicine research topics, to promote more efficient fish disease diagnostics and therapy, to meet the ever-growing aquatic animal health demand.
References
1. The Royal Society and Royal Academy of Engineering. Nanoscience and nanotechnologies: opportunities and uncertainties; 2004 [London, UK]. 2. Oberdörster G, Oberdörster E, Oberdörster J. Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect 2005;113:823-39. 3. Cavalieri F, Tortora M, Stringaro A, Colone M, Baldassarri L. Nanomedicines for antimicrobial interventions. J Hosp Infect 2014;88(4):183-90.
708
M. Shaalan et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 701–710
4. De Jong WH, Borm PJ. Drug delivery and nanoparticles: applications and hazards. Int J Nanomedicine 2008;3(2):133-49. 5. Donbrow M. Microcapsules and nanoparticles in medicine and pharmacy. CRC Press; 1991. 6. Ramalingam M, Jabbari E, Ramakrishna S, Khademhosseini A, editors. Micro and nanotechnologies in engineering stem cells and tissues, vol. 39. John Wiley & Sons; 2013. 7. Torchilin VP. Nanoparticulates as drug carriers. Imperial college Press; 2006. 8. Lasic DD. Novel applications of liposomes. Trends Biotechnol 1998;16(7):307-21. 9. Reilly RM. Carbon nanotubes: potential benefits and risks of nanotechnology in nuclear medicine. J Nucl Med 2007;48(7):1039-42. 10. Gaffney AM, Santos-Martinez MJ, Satti A, Major TC, Wynne KJ, Gun'ko YK, et al. Blood biocompatibility of surface-bound multiwalled carbon nanotubes. Nanomedicine 2015;11(1):39-46. 11. Aulenta F, Hayes W, Rannard S. Dendrimers: a new class of nanoscopic containers and delivery devices. Eur Polym J 2003;39(9):1741-71. 12. Gillies ER, Frechet JM. Dendrimers and dendritic polymers in drug delivery. Drug Discov Today 2005;10(1):35-43. 13. Wu LP, Ficker M, Christensen JB, Trohopoulos PN, Moghimi SM. Dendrimers in medicine: therapeutic concepts and pharmaceutical challenges. Bioconjug Chem 2015;26(7):1198-211. 14. Holmes JD, Lyons DM, Ziegler KJ. Supercritical fluid synthesis of metal and semiconductor nanomaterials. Chemistry 2003;9(10):2144-50. 15. Gaffet E, Tachikart M, El Kedim O, Rahouadj R. Nanostructural materials formation by mechanical alloying: morphologic analysis based on transmission and scanning electron microscopic observations. Mater Charact 1996;36:185-90. 16. Thirumalai Arasu V, Prabhu D, Soniya M. Stable silver nanoparticle synthesizing methods and its applications. J Biol Sci Res 2010;1:259-70. 17. Esumi K, Tano T, Torigue K, Meguro K. Preparation and characterization of bimetallic Pd-Cu colloids by thermal decomposition of their acetate compounds in organic solvents. Chem Mater 1990;2:564-7. 18. Sergeev MB, Kasaikin AV, Litmanovich AE. Cryochemical synthesis and properties of silver nanoparticle dispersions stabilised by poly(2dimethylaminoethyl methacrylate). Mendeleev Commun 1999;9:130-2. 19. Jiang H, Moon K, Zhang Z, Pothukuchi S, Wong CP. Variable frequency microwave synthesis of silver nanoparticles. J Nanopart Res 2006;8:117-24. 20. Jang D, Kim D. Synthesis of nanoparticles by pulsed laser ablation of consolidated metal microparticles. Appl Phys 2004;A79:1985-8. 21. Zhu J, Liao X, Chen HY. Electrochemical preparation of silver dendrites in the presence of DNA. Mater Res Bull 2001;36:1687-92. 22. Tien DC, Tseng KH, Liao CY, Tsung TT. Colloidal silver fabrication using the spark discharge system and its antimicrobial effect on Staphylococcus aureus. Med Eng Phys 2007;30:948-52. 23. Gong P, Li H, He X, Wang K, Hu J, Tan W. Preparation and antibacterial activity of Fe3O4 Ag nanoparticles. Nanotechnology 2007;18:604-11. 24. Van Dong P, Ha CH, Kasbohm J. Chemical synthesis and antibacterial activity of novel-shaped silver nanoparticles. Int Nano Lett 2012;2:1-9. 25. El Mahdy MM, Eldin TAS, Aly HS, Mohammed FF, Shaalan MI. Evaluation of hepatotoxic and genotoxic potential of silver nanoparticles in albino rats. Exp Toxicol Pathol 2015;67(1):21-9. 26. Kumari A, Yadav SK, Yadav SC. Biodegradable polymeric nanoparticles based drug delivery systems. Colloids Surf B: Biointerfaces 2010;75(1):1-18. 27. Rajesh Kumar S, Ishaq Ahmed VP, Parameswaran V, Sudhakaran R, Sarath Babu V, Sahul Hameed AS. Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in Asian sea bass (Lates calcarifer) to protect from Vibrio (Listonella) anguillarum. Fish Shellfish Immunol 2008;25(1-2):47-56.
28. Nagpal K, Singh SK, Mishra DN. Chitosan nanoparticles: a promising system in novel drug delivery. Chem Pharm Bull 2010;58(11):1423-30. 29. Barichello JM, Morishita M, Takayama K, Nagai T. Encapsulation of hydrophilic and lipophilic drugs in PLGA nanoparticles by the nanoprecipitation method. Drug Dev Ind Pharm 1999;25(4):471-6. 30. Tinsley-Bown AM, Fretwell R, Dowsett AB, Davis SL, Farrar GH. Formulation of poly (D, L-lactic-co-glycolic acid) microparticles for rapid plasmid DNA delivery. J Control Release 2000;66(2-3):229-41. 31. Adomako M, St-Hilaire S, Zheng Y, Eley J, Marcum RD, Sealey W, et al. Oral DNA vaccination of rainbow trout, Oncorhynchus mykiss (Walbaum), against infectious haematopoietic necrosis virus using PLGA [poly(D, L-lactic-co-glycolic acid)] nanoparticles. J Fish Dis 2012;35(3):203-14. 32. Kalishwaralal K, Deepak V, Ramkumarpandian S, Nellaiah H, Sangiliyandi G. Extracellular biosynthesis of silver nanoparticles by the culture supernatant of Bacillus licheniformis. Mater Lett 2008;62:4411-3. 33. Prabhu S, Poulose EK. Silver nanoparticles: mechanism of antimicrobial action, synthesis, medical applications, and toxicity effects. Int Nano Lett 2012;2:32. 34. Sankar R, Karthik A, Prabu A, Karthik S, Shivashangari KS, Ravikumar V. Origanum vulgare mediated biosynthesis of silver nanoparticles for its antibacterial and anticancer activity. Colloids Surf B: Biointerfaces 2013;108:80-4. 35. Velmurugan P, Iydroose M, Lee SM, Cho M, Park JH, Balachandar V, et al. Synthesis of silver and gold nanoparticles using cashew nut shell liquid and its antibacterial activity against fish pathogens. Indian J Microbiol 2014;54(2):196-202. 36. Vaseeharan B, Ramasamy P, Chen JC. Antibacterial activity of silver nanoparticles (AgNps) synthesized by tea leaf extracts against pathogenic Vibrio harveyi and its protective efficacy on juvenile Feneropenaeus indicus. Lett Appl Microbiol 2010;50(4):352-6. 37. Gunalan S, Sivaraj R, Rajendran V. Green synthesized ZnO nanoparticles against bacterial and fungal pathogens. Prog Nat Sci Mater Int 2012;22(6):693-700. 38. Jayaseelan C, Rahuman AA, Kirthi AV, Marimuthu S, Santhoshkumar T, Bagavan A, et al. Novel microbial route to synthesize ZnO nanoparticles using Aeromonas hydrophila and their activity against pathogenic bacteria and fungi. Spectrochim Acta A 2012;90:78-84. 39. Aarestrup FM. Veterinary drug usage and antimicrobial resistance in bacteria of animal origin. Basic Clin Pharmacol Toxicol 2005;96(4):271-81. 40. Hajipour MJ, Fromm KM, Ashkarran AA, de Aberasturi DJ, de Larramendi IR, Rojo T, et al. Antibacterial properties of nanoparticles. Trends Biotechnol 2012;30(10):499-511. 41. Pelgrift RY, Friedman AJ. Nanotechnology as a therapeutic tool to combat microbial resistance. Adv Drug Deliv Rev 2013;65(13):1803-15. 42. Cabello FC, Godfrey HP, Tomova A, Ivanova L, Dölz H, Millanao A, et al. Antimicrobial use in aquaculture re‐examined: its relevance to antimicrobial resistance and to animal and human health. Environ Microbiol 2013;15(7):1917-42. 43. Sørum H, Lie Ø. Antibiotic resistance associated with veterinary drug use in fish farms. Improving farmed fish quality and safety; 2008157-82. 44. Tuševljak N, Dutil L, Rajić A, Uhland FC, McClure C, St‐Hilaire S, et al. Antimicrobial use and resistance in aquaculture: findings of a globally administered survey of aquaculture‐allied professionals. Zoonoses Public Health 2013;60(6):426-36. 45. Atyah MAS, Zamri-Saad M, Siti-Zahrah A. First report of methicillinresistant Staphylococcus aureus from cage-cultured tilapia (Oreochromis niloticus). Vet Microbiol 2010;144(3):502-4. 46. Davies J, Davies D. Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 2010;74(3):417-33.
M. Shaalan et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 701–710 47. Son R, Rusul G, Sahilah AM, Zainuri A, Raha AR, Salmah I. Antibiotic resistance and plasmid profile of Aeromonas hydrophila isolates from cultured fish, Telapia (Telapia mossambica). Lett Appl Microbiol 1997;24(6):479-82. 48. Swain P, Nayak SK, Sasmal A, Behera T, Barik SK, Swain SK, et al. Antimicrobial activity of metal based nanoparticles against microbes associated with diseases in aquaculture. World J Microbiol Biotechnol 2014;30(9):2491-502. 49. Di Cesare A, Vignaroli C, Luna GM, Pasquaroli S, Biavasco F. Antibiotic-resistant enterococci in seawater and sediments from a coastal fish farm. Microb Drug Resist 2012;18(5):502-9. 50. Di Cesare A, Luna GM, Vignaroli C, Pasquaroli S, Tota S, Paroncini P, et al. Aquaculture can promote the presence and spread of antibioticresistant Enterococci in marine sediments. PLoS One 2013;8(4):e62838. 51. Seil JT, Webster TJ. Antimicrobial applications of nanotechnology: methods and literature. Int J Nanomedicine 2012;7:2767-81. 52. Knetsch ML, Koole LH. New strategies in the development of antimicrobial coatings: the example of increasing usage of silver and silver nanoparticles. Polymers 2011;3(1):340-66. 53. Hindi KM, Ditto AJ, Panzner MJ, Medvetz DA, Han DS, Hovis CE, et al. The antimicrobial efficacy of sustained release silver–carbene complex-loaded L-tyrosine polyphosphate nanoparticles: characterization, in vitro and in vivo studies. Biomaterials 2009;30(22):3771-9. 54. Antony JJ, Nivedheetha M, Siva D, Pradeepha G, Kokilavani P, Kalaiselvi S, et al. Antimicrobial activity of Leucas aspera engineered silver nanoparticles against Aeromonas hydrophila in linfected Catla catla. Colloids Surf B: Biointerfaces 2013;109:20-4. 55. Lara HH, Ayala-Núnez NV, Turrent LDCI, Padilla CR. Bactericidal effect of silver nanoparticles against multidrug-resistant bacteria. World J Microbiol Biotechnol 2010;26(4):615-21. 56. Huang CM, Chen CH, Pornpattananangkul D, Zhang L, Chan M, Hsieh MF, et al. Eradication of drug resistant Staphylococcus aureus by liposomal oleic acids. Biomaterials 2011;32(1):214-21. 57. Prakash P, Gnanaprakasam P, Emmanuel R, Arokiyaraj S, Saravanan M. Green synthesis of silver nanoparticles from leaf extract of Mimusops elengi, Linn. for enhanced antibacterial activity against multi drug resistant clinical isolates. Colloids Surf B: Biointerfaces 2013;108:255-9. 58. Ayala-Núñez NV, Lara HH, Turrent LDCI, Padilla CR. Silver nanoparticles toxicity and bactericidal effect against methicillinresistant Staphylococcus aureus: nanoscale does matter. Nanobiotechnology 2009;5:2-9. 59. Umashankari J, Inbakandan D, Ajithkumar TT, Balasubramanian T. Mangrove plant, Rhizophora mucronata (Lamk, 1804) mediated one pot green synthesis of silver nanoparticles and its antibacterial activity against aquatic pathogens. Aquat Biosyst 2012;8(1):11. 60. Sanjenbam P, Gopal JV, Kannabiran K. Anticandidal activity of silver nanoparticles synthesized using Streptomyces sp. VITPK1. J Mycol Med 2014;24(3):211-9. 61. Mallmann EJJ, Cunha FA, Castro BN, Maciel AM, Menezes EA, Fechine PBA. Antifungal activity of silver nanoparticles obtained by green synthesis. Rev Inst Med Trop Sao Paulo 2015;57(2):165-7. 62. Kim KJ, Sung WS, Moon SK, Choi JS, Kim JG, Lee DG. Antifungal effect of silver nanoparticles on dermatophytes. J Microbiol Biotechnol 2008;18:1482-4. 63. Elechiguerra JL, Burt JL, Morones JR, Camacho-Bragado A, Gao X, Lara HH, et al. Interaction of silver nanoparticles with HIV-1. J Nanobiotechnol 2005;3:6. 64. Mehrbod P, Motamed N, Tabatabaian M, Soleimani R, Amini E, Shahidi M, et al. In vitro anti viral effect of "nanosilver" on influenza virus. DARU 2009;17:88-93. 65. Mori Y, Ono T, Miyahira Y, Nguyen VQ, Matsui T, Ishihara M. Antiviral activity of silver nanoparticle/chitosan composites against H1N1 influenza A virus. Nanoscale Res Lett 2013;8(1):1-6. 66. Li X, Robinson SM, Gupta A, Saha K, Jiang Z, Moyano DF, Sahar A, et al. Functional gold nanoparticles as potent antimicrobial agents against multi-drug-resistant bacteria. ACS Nano 2014;8(10):10682-6.
709
67. Sumbayev VV, Yasinska IM, Gibbs BF. Biomedical applications of gold nanoparticles. Recent advances in circuits, communications and signal processing. Athens: WSEAS Press; 2013342-8. 68. Lima E, Guerra R, Lara V, Guzmán A. Gold nanoparticles as efficient antimicrobial agents for Escherichia coli and Salmonella typhi. Chem Cent J 2013;7(1):11. 69. Cui Y, Zhao Y, Tian Y, Zhang W, Lü X, Jiang X. The molecular mechanism of action of bactericidal gold nanoparticles on Escherichia coli. Biomaterials 2012;33(7):2327-33. 70. Ahmad T, Wani IA, Lone IH, Ganguly A, Manzoor N, Ahmad A, et al. Antifungal activity of gold nanoparticles prepared by solvothermal method. Mater Res Bull 2013;48(1):12-20. 71. Wani IA, Ahmad T. Size and shape dependant antifungal activity of gold nanoparticles: a case study of Candida. Colloid Surf B: Biointerfaces 2013;101:162-70. 72. Liu Y, He L, Mustapha A, Li H, Hu ZQ, Lin M. Antibacterial activities of zinc oxide nanoparticles against Escherichia coli O157: H7. J Appl Microbiol 2009;107(4):1193-201. 73. Ramamoorthy S, Kannaiyan P, Moturi M, Devadas T, Muthuramalingam J, Natarajan L, et al. Antibacterial activity of zinc oxide nanoparticles against Vibrio harveyi. Indian J Fish 2013;107–112. 74. Cheng TC, Yao KS, Yeh N, Chang CI, Hsu HC, Chien YT, et al. Visible light activated bactericidal effect of TiO2/Fe3O4 magnetic particles on fish pathogens. Surf Coat Technol 2009;204(6):1141-4. 75. Cheng TC, Yao KS, Yeh N, Chang CI, Hsu HC, Gonzalez F, et al. Bactericidal effect of blue LED light irradiated TiO2/Fe3O4 particles on fish pathogen in seawater. Thin Solid Films 2011;519(15):5002-6. 76. Jovanović B, Anastasova L, Rowe EW, Zhang Y, Clapp AR, Palić D. Effects of nanosized titanium dioxide on innate immune system of fathead minnow (Pimephales promelas Rafinesque, 1820). Ecotoxicol Environ Saf 2011;74(4):675-83. 77. Jovanović B, Whitley EM, Kimura K, Crumpton A, Palić D. Titanium dioxide nanoparticles enhance mortality of fish exposed to bacterial pathogens. Environ Pollut 2015;203:153-64. 78. Lockman PR, Mumper RJ, Khan MA, Allen DD. Nanoparticle technology for drug delivery across the blood-brain barrier. Drug Dev Ind Pharm 2002;28(1):1-13. 79. Wang JJ, Zeng ZW, Xiao RZ, Xie T, Zhou GL, Zhan XR, et al. Recent advances of chitosan nanoparticles as drug carriers. Int J Nanomedicine 2011;6:765-74. 80. Costa ADS, Brandão HM, Silva SR, Bentes‐Sousa AR, Diniz JAP, Viana Pinheiro JDJ, et al. Mucoadhesive nanoparticles: a new perspective for fish drug application. J Fish Dis 2015, http:// dx.doi.org/10.1111/jfd.12373. 81. Alishahi A, Mirvaghefi A, Tehrani MR, Farahmand H, Koshio S, Dorkoosh FA, et al. Chitosan nanoparticle to carry vitamin C through the gastrointestinal tract and induce the non-specific immunity system of rainbow trout (Oncorhynchus mykiss). Carbohydr Polym 2011;86(1):142-6. 82. Rather MA, Sharma R, Gupta S, Ferosekhan S, Ramya VL, Jadhao SB. Chitosan-nanoconjugated hormone nanoparticles for sustained surge of gonadotropins and enhanced reproductive output in female fish. PLoS One 2013;8(2):e57094. 83. Lü JM, Wang X, Marin-Muller C, Wang H, Lin PH, Yao Q, et al. Current advances in research and clinical applications of PLGA-based nanotechnology. Expert Rev Mol Diagn 2009;9(4):325-41. 84. Makadia HK, Siegel SJ. Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier. Polymers 2011;3(3):1377-97. 85. Fenaroli F, Westmoreland D, Benjaminsen J, Kolstad T, Skjeldal FM, Meijer AH, et al. Nanoparticles as drug delivery system against tuberculosis in zebrafish embryos: direct visualization and treatment. ACS Nano 2014;8(7):7014-26. 86. Myhr AI, Myskja BK. Precaution or integrated responsibility approach to nanovaccines in fish farming? A critical appraisal of the UNESCO precautionary principle. Nanoethics 2011;5(1):73-86.
710
M. Shaalan et al / Nanomedicine: Nanotechnology, Biology, and Medicine 12 (2016) 701–710
87. Zhao L, Seth A, Wibowo N, Zhao CX, Mitter N, Yu C, et al. Nanoparticle vaccines. Vaccine 2014;32(3):327-37. 88. Gregory AE, Titball R, Williamson D. Vaccine delivery using nanoparticles. Front Cell Infect Microbiol 2013;3:13, http:// dx.doi.org/10.3389/fcimb.2013.00013. 89. Rivas-Aravena A, Fuentes Y, Cartagena J, Brito T, Poggio V, La Torre J, et al. Development of a nanoparticle-based oral vaccine for Atlantic salmon against ISAV using an alphavirus replicon as adjuvant. Fish Shellfish Immunol 2015;45(1):157-66. 90. Vimal S, Taju G, Nambi KSN, Abdul Majeed S, Sarath Babu V, Ravi M, et al. Synthesis and characterization of CS/TPP nanoparticles for oral delivery of gene in fish. Aquaculture 2012;358–359:14-22. 91. Li L, Lin SL, Deng L, Liu ZG. Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in black seabream Acanthopagrus schlegelii Bleeker to protect from Vibrio parahaemolyticus. J Fish Dis 2013;36(12):987-95. 92. Rajeshkumar S, Venkatesan C, Sarathi M, Sarathbabu V, Thomas J, Anver Basha K, et al. Oral delivery of DNA construct using chitosan nanoparticles to protect the shrimp from white spot syndrome virus (WSSV). Fish Shellfish Immunol 2009;26(3):429-37. 93. Vimal S, Abdul Majeed S, Taju G, Nambi KS, Sundar Raj N, Madan N, et al. Chitosan tripolyphosphate (CS/TPP) nanoparticles: preparation, characterization and application for gene delivery in shrimp. Acta Trop 2013;128(3):486-93. 94. Wang D, Robinson DR, Kwon GS, Samuel J. Encapsulation of plasmid DNA in biodegradable poly(D, L-lactic-co-glycolic acid) microspheres as a novel approach for immunogene delivery. J Control Release 1999;57(1):9-18. 95. Hølvold LB, Myhr AI, Dalmo RA. Strategies and hurdles using DNA vaccines to fish. Vet Res 2014;45(1):21-31. 96. Tian J, Sun X, Chen X, Yu J, Qu L, Wang L. The formulation and immunisation of oral poly(DL-lactide-co-glycolide) microcapsules containing a plasmid vaccine against lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus). Int Immunopharmacol 2008;8(6):900-8.
97. Jain KK. Nanodiagnostics: application of nanotechnology in molecular diagnostics. Expert Rev Mol Diagn 2003;3(2):153-61. 98. Baptista P, Pereira E, Eaton P, Doria G, Miranda A, Gomes I, et al. Gold nanoparticles for the development of clinical diagnosis methods. Anal Bioanal Chem 2008;391(3):943-50. 99. Saleh M, Soliman H, El-Matbouli M. Gold nanoparticles as a potential tool for diagnosis of fish diseases. Methods Mol Biol 2015;1247:245-52, http://dx.doi.org/10.1007/978-1-4939-2004-4_19. 100. Saleh M, Soliman H, Haenen O, El-Matbouli M. Antibody-coated gold nanoparticles immunoassay for direct detection of Aeromonas salmonicida in fish tissues. J Fish Dis 2011;34(11):845-52. 101. Kuan GC, Sheng LP, Rijiravanich P, Marimuthu K, Ravichandran M, Yin LS, et al. Gold-nanoparticle based electrochemical DNA sensor for the detection of fish pathogen Aphanomyces invadans. Talanta 2013;117:312-7. 102. Jaroenram W, Arunrut N, Kiatpathomchai W. Rapid and sensitive detection of shrimp yellow head virus using loop-mediated isothermal amplification and a colorogenic nanogold hybridization probe. J Virol Methods 2012;186(1):36-42. 103. Seetang-Nun Y, Jaroenram W, Sriurairatana S, Suebsing R, Kiatpathomchai W. Visual detection of white spot syndrome virus using DNA-functionalized gold nanoparticles as probes combined with loop-mediated isothermal amplification. Mol Cell Probes 2013;27(2):71-9. 104. Toubanaki DK, Margaroni M, Karagouni E. Nanoparticle-based lateral flow biosensor for visual detection of fish nervous necrosis virus amplification products. Mol Cell Probes 2015;3:158-66. 105. Yang SY, Wu JL, Tso CH, Ngou FH, Chou HY, Nan FH, et al. A novel quantitative immunomagnetic reduction assay for nervous necrosis virus. J Vet Diagn Invest 2012;24(5):911-7. 106. Saleh M, Soliman H, Schachner O, El-Matbouli M. Direct detection of unamplified spring viraemia of carp virus RNA using unmodified gold nanoparticles. Dis Aquat Organ 2012;100(1):3-10. 107. Saleh M, El-Matbouli M. Rapid detection of cyprinid herpesvirus-3 (CyHV-3) using a gold nanoparticle-based hybridization assay. J Virol Methods 2015;217:50-4.