2019 - Ahmed, Soliman

Accepted Manuscript
Recent progress in biomedical applications of chitosan and its

nanocomposites in aquaculture: A review
Fatma Ahmed, Faiza M. Soliman, Mohamed A. Adly, Hamdy

A.M. Soliman, Mansour El-Matbouli, Mona Saleh
PII: S0034-5288(19)30163-8
DOI: https://doi.org/10.1016/j.rvsc.2019.08.005
Reference: YRVSC 3850
To appear in: Research in Veterinary Science
Received date: 15 February 2019
Revised date: 3 July 2019
Accepted date: 4 August 2019
Please cite this article as: F. Ahmed, F.M. Soliman, M.A. Adly, et al., Recent progress
in biomedical applications of chitosan and its nanocomposites in aquaculture: A review,
Research in Veterinary Science, https://doi.org/10.1016/j.rvsc.2019.08.005
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Recent progress in biomedical applications of chitosan and its nanocomposites in
aquaculture: A review
Authors: Fatma Ahmed1,2 , Faiza M. Soliman2 , Mohamed A. Adly2 , Hamdy A.M.
Soliman2 , Mansour El-Matbouli1 , Mona Saleh*1
1- Clinical Division of Fish Medicine; University of Veterinary Medicine, Vienna,
Austria.
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2- Department of Zoology, Faculty of Science, Sohag University, Sohag, Egypt.
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*Corresponding Author: Dr. Mona Saleh Clinical Division of Fish Medicine, University
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of Veterinary Medicine Veterinärplatz 1, 1210 Vienna, Austria.
Tel: 0043-1-250774708
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Fax: 0043-1-250775192
E-mail: mona.sale h@vetme duni.ac.at

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Abstract word count: 173
Manuscript word count: 3986

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Number of references: 169
Number of figures: 4
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Tables: 5
The authors declare that they don’t have any commercial associations, current and within
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the past five years, that might pose a potential, perceived or real conflict of interest.
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Abstract
Chitosan nanoparticles (CSNPs) are the nanostructures of chitosan biopolymer which
is derived from chitin polysaccharide, the main component of crustacean shells. Chitosan is a
biocompatible, nontoxic and biodegradable polymer soluble in acidic solutions and easily
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excreted from kidneys. It is widely used in medical and pharmaceutical applications
including artificial matrices for tissue engineering, drug transport, targeted drug delivery and
protein or gene delivery. The antimicrobial activities of chitosan and CSNPS against different
bacterial, fungal and viral pathogens made them valuable for several biological applications
including food preservation purposes. In addition, they have immunomodulatory effects on
fish and crustaceans providing direct positive impact on aquaculture and fish farming
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industry. Sustained release of some bioactive ingredients such as hormones, vitamins,
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nutrients and antioxidants has improved the biological activities of fish. Furthermore, CSNPs
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have recently been employed to diagnose fish diseases. In this review, we present the medical
and biological applications of chitosan and CSNPs on aquatics to provide an update on recent
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advances and the potential for further advanced applications for aquaculture in the future.
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Keywords
Chitosan nanoparticles, Biomedical applications, Antimicrobial agents, Immunomodulation,

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Fish and seafood preservation

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Table of contents:
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1. Introduction
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2. Preparation and characterization of CSNPs

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3. Biomedical applications of chitosan and nano-chitosan complexes on aquatics
3.1 Immunomodulation
3.2 Delivery
3.3 Sustained release of bioactive ingredients
3.4 Potential biomedical fish disease diagnosis
3.5 Fish and seafood preservation
Conclusions
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References
1. Introduction
Chitosan is a natural polysaccharide prepared by alkaline N-de-acetylation of chitin (Zhao
et al., 2011) that is a water insoluble linear polysaccharide consists of repeated N-acetyl-D-
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glucosamine units which are linked by β-(1→4) glycosidic bonds (Gomes et al., 2017). It is a
biocompatible, nontoxic and biodegradable polymer soluble in acidic aqueous solutions (Ji et
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al., 2015), thus it is easily excreted from the kidney. Therefore, chitosan and its derivatives
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are widely used in medical and pharmaceutical applications (Pan et al., 2002) (Kim et al.,
2005). Solubility of chitosan depends mainly on two items; the average degree of acetylation
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and the distribution of acetyl groups along the polymer chains (Brugnerotto et al., 2001).
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Owing to the mucoadhesive properties of chitosan, it could be used as a vehicle improving
penetration of large molecules across mucosal surfaces (Xu and Du, 2003). However, its
cytotoxicity was found to be variable in freshwater and salt water media; the lower the
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salinity, the higher the cytotoxicity (Apetroaei et al., 2018). Therefore, we can expect
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advanced applications of chitosan on marine aquacultures.

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Nanoparticles are small solid particles with a size range of 1–1000 nm providing bigger
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surface areas for the attachment of bioactive ingredients (Jia et al., 2003) and a shorter
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diffusion path for immobilization (Kim and Grate, 2003) (Kim et al., 2005) and slow
sustainable drug release (Lanka and Mittapally, 2016) (Gomes et al., 2017). Recently,
nanoparticles have been reported as novel antimicrobial agents against several fish pathogens
(Shaalan et al., 2016). Gold, silver and zinc oxide nanoparticles were reported by our group
to combat bacterial, fungal and parasitic fish diseases (Saleh et al., 2016) (Shaalan et al.,
2017) and our ongoing research includes the application of chitosan nanoparticles as
antimicrobial and immunostimulatory agent against deleterious fish pathogens. Several

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applications in drug and vaccine delivery using CSNPs were summarized previously in
review articles (Agnihotri et al., 2004) (Elgadir et al., 2015). In this review, we aimed to
highlight the potential and significance of using chitosan and CSNPs in various applications
on aquatics encouraging more researchers to further explore the variable characteristics of
CSNPs for more benefits to the aquaculture.
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2. Preparation and characterization of CSNPs
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Chitosan could be prepared via enzymatic or chemical method. The enzymatic methods
are controlled processes form a well-defined chitosan (Younes and Rinaudo, 2015) however
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the chemical methods require lower coasts and then are extensively used in the preparation of
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chitosan for the commercial applications (Ji et al., 2015). A survey on the common
techniques used for the preparation of CSNPs from chitosan biopolymer was reported by
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Grenha (2012) including, ionic gelation (Qi et al., 2004), emulsion (Kataoka et al., 2000),
self-assembling (Liu et al., 2005), reverse micellar (Banerjee et al., 2002) and ultrafine
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milling (Zhang et al., 2012). Ionic gelation (simple ionotropic gelation) is the most widely
used method because it is a simple chemical reaction that needs few organic solvents, could
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be proceeded at room temperature and allows safe and successful encapsulation of sensitive
and fragile molecules such as vaccines and proteins (Zhao et al., 2011). Formation of CSNPs
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starts spontaneously by ionic gelation mechanism via dropwise addition of the gelating
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solution, contains anionic molecules, into chitosan solution, contains positive amino groups
(NH2 +) (Huang and Lapitsky, 2012) and the process could be controlled by changing the pH
to change the charge density of both solutions. The gelating agent, tripolyphosphate (TPP)
has often been used for CSNPs preparation because it is nontoxic, multivalent, negatively
charged and able to form gels through ionic interactions under continuous stirring (Rampino
et al., 2013). Factors influencing the formation of CSNPs, such as pH, concentrations and
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ratios of components, and the method of mixing were previously reported by Nasti et al.
(2009). The Mw of chitosan affects the size of the formed nanoparticles; low Mw generates
smaller nanoparticles with tunable size, morphology, and delivery rate (Xu and Du, 2003).
The higher the Mw, the larger the nanoparticles size, and the lower Mw, the smaller the
nanoparticles size formed (Alishahi et al., 2011b). It is worth mentioning that nanoparticles
formed by the ionic gelation are characterized by limited physical and chemical stability
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when stored for an extended period due to particles aggregation and/or fusion. Larger
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particles may form because single small particles tend to aggregate and fuse together
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generating a larger entity (Rampino et al., 2013). Therefore, the nanoparticles solutions
should be prepared or gently mixed prior to use (Alishahi et al., 2011b).

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Physicochemical properties of the nanoparticles prepared from biopolymers, including
chitosan, have been assessed using different approaches. Size and particles morphology have
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been evaluated by scanning electron microscopy (SEM) and transmission electron
microscopy (TEM) as shown in figure 1 (Parida et al., 2013). In addition, morphology and
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distribution of the nanoparticles in solutions has also been studied by tapping mode atomic
force microscopy after being air dried (Ferosekhan et al., 2014). The size of nanoparticles be
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accurately measured by Fourier transform infrared (FTIR) spectra. The size distribution range
(Zeta Potential) and the average particle size (mean size) can be assessed by laser doppler
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electrophoresis and dynamic light scattering, respectively using Zeta Sizer (Dubey et al.,
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2016).
3. Biomedical applications of chitosan and nano-chitosan complexes on aquatics
The NH2 + and hydroxyl (OH-) groups provide chitosan with many special
characteristics, making it suitable for chemical reactions and applicable in many areas
(Zhao et al., 2011). In addition, chitosan is characterized by its safety and significant
biological and chemical properties such as biodegradability, biocompatibility, bioactivity

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and polycationicity (Muzzarelli, 2010) (Kean and Thanou, 2010) (Plant and LaPatra,
2011). Chitosan and its nanostructures have several antimicrobial properties against the
antibiotic resistant Gram negative and Gram positive bacteria in addition to fungi (Ma et
al., 2016) (Ma et al., 2017) (Table 1). This potential antimicrobial activity needs further
investigations against major bacterial and fungal fish pathogens. Additionally, functional
studies are required to elucidate their mechanisms of action. Owing to their antimicrobial
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activities, chitosan nanocomposites are very important in several applications in the
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textile industry, for water disinfection of fish farms, in food packaging and medicine
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(Yang et al., 2010) (Hajipour et al., 2012) (Gokulalakshmi et al., 2015). CSNPs can bind
with particles that have negative charges on their surfaces (Wisdom et al., 2018) and this
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make them applicable for wide range of purposes. Bioadhesive CSNPs have been also
used as carriers, encapsulators or immobilizers for several bioactive ingredients as

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catechins from green tea (Dudhani and Kosaraju, 2010) (Tang et al., 2013), chlorogenic
acid from fruits and vegetables (Llaiyaraja et al., 2014) and the food preservative
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“Thyme” essential oil (Ghahfarokhi et al., 2016). Fish collagen/chitosan films have been
used for controlled localized drug delivery and chemotherapy (Chen et al., 2015). The
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reported biological and medical applications of chitosan-based complexes on fish and
crustaceans are summarized in this review article to highlight their benefits on the health
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of aquatic organisms. Undoubtedly, the applications related to the immunomodulation

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and antimicrobial activity gained the highest importance since they have high impact on
improving the aquaculture production.
3.1. Immunomodulation
Diets supplemented with chitosan improved growth, survival and meat quality of several
freshwater and seawater fish species including, Rainbow trout (Oncorhynchus mykiss)
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(Meshkini et al., 2012), olive flounder (Paralichthys olivaceus) (Cha et al., 2008), koi
(Cyprinus carpio koi) (Lin et al., 2011), kelp grouper (Epinephelus bruneus) (Harikrishnan et
al., 2012a) (Harikrishnan et al., 2012b), turbot (Scophthalmus maximus L.) (Cui et al., 2013),
gibel carp (Carassius auratus gibelio) (Chen et al., 2014), mrigal carp (Cirrhina mrigala)
(Shanthi Mari et al., 2014), Asian seabass (Lates calcarifer) (Ranjan et al., 2014) and sea bass
(Dicentrarchus labrax) (Zaki et al., 2015). Chitosan as well as CSNPs supplemented diets
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enhanced the survival, growth and meat quality of Nile tilapia (Oreochromis nilotica) (Wang
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and Li, 2011) (Abd El-Naby et al., 2019) (Abdel-Tawwab et al., 2019) and the African catfish
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(Clarius gariepinus) fingerlings (Imefon Udo, 2018). However, CSNPs are more effective
than chitosan in improving growth performance and food quality status. Dietary chitosan and
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chitin supplemented diets can demonstrate immunomodulatory effects (Plant and LaPatra,
2011) and improve the resistance of several fish species against infections and stresses (Ji et
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al., 2015) by enhancing the innate immune response (Vahedi and Ghodratizadeh, 2011). For
the oral immunization against infections, the black tiger shrimp (Penaeus monodon) was
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vaccinated against white spot syndrome virus (WSSV) by the oral feeding on DNA vaccine
encapsulated with CSNPs (Rajeshkumar et al., 2009). Additionally, the crayfish

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(Procambarus clarkii) was protected against WSSV infection via the dietary CSNPs with
significantly higher survival rate (Sun et al., 2016). The reported studies on the
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immunomodulation of fish against infections and stresses are summarized in table 2.

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However, fish immunomodulation effects of CSNPs need further studies to improve our
knowledge on how and to which extent they can enhance fish health.
3.2. Delivery
Since they have been used for longer release, CSNPs are promising carriers for drug oral
administration (Zhao et al., 2014). CSNPs have the ability to penetrate deeply into tissues
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through fine capillaries allowing efficient delivery into body organs (Vimal et al., 2014).
Chitosan and CSNPs are bioactive ingredient carriers, encapsulators and immobilizers used in
oral delivery of drugs, vitamins, nutrients, genes (DNA) and vaccines into fish
gastrointestinal tract. CSNPs enhance the mucoadhesive potential improving intestinal
absorption and oral bioavailability. Therefore, they can be incorporated with further natural
ingredients and antioxidants to enhance fish immunity (Ferosekhan et al., 2014). FTIR
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spectroscopy can be used to confirm cross linking of loaded bioactive ingredients (Bhat et al.,
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2018).
CSNPs are used as stabilizers for bio-molecules and dietary supplements as RNA and α-
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tocopherol with limited bioavailability, due to unsuitable pH and intestinal cell repulsion, and
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short half life in blood which significantly improve growth, immunity and disease resistance
of fish (Ferosekhan et al., 2014).

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In vitro, Zein/chitosan complexes were used for food supplementation and considered
promising for delivery of hydrophobic molecules (Luo and Wang, 2014) (Luo et al., 2011).
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Currently, gene (DNA) delivery is considered the most effective strategy for fish vaccination
(Mandal, 2010). Delivery systems improve the solubility, bioavailability and sustained
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release of hydrophobic drugs and protect therapeutic agents from degradation, thereby,
facilitating cell and tissue targeting (Shi et al., 2010). CSNPs can be used as gene vectors for
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the rapidly degrading molecules such as DNA, plasmids (pDNA), siRNA (Vimal et al.,
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2014). DNA immunization of fish is usually applied either by direct intramuscular injection
(Kumar et al., 2007) or by immersion (Fernandez-Alonso et al., 2001). However, oral
vaccination is the ideal way for fish immunization as it can effectively induce the mucosal
immunity of the gastrointestinal tract avoiding the exhausting injection of large numbers of
fish in aquacultures (Ramos et al., 2005) (Tian et al., 2008). Because chitosan is resistant to
degradation, it can protect the activity of encapsulated compounds from destruction by the
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critical conditions inside gastrointestinal tract enhancing their absorption (Aranaz et al.,
2009). Therefore, CSNPs are one of the most widely used drug and gene delivery vehicles in
aquaculture (Ji et al., 2015). Immune genes expression depends on CSNPs/plasmid ratio, pH
and the combination of deacetylation degree (DD) and M W of chitosan (Lavertu et al., 2006).
Oral plasmid DNA (pDNA) delivery into gastrointestinal tract of fish can be achieved via
encapsulation with chitosan or its micro or nanoparticles. OMPs of bacteria act as antigenic
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sites with high immunogenic properties on the outer surfaces of bacterial cell membranes
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(Dubey et al., 2016). Antigen vaccines constructed using recombinant OMPs of several
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pathogens have been used for fish vaccination via intramuscular injection (Kumar et al.,
2007). Currently, CSNPs provide oral vaccines administration system that enables higher
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tissue availability enhancing fish immunity. Figure 2 showing size distribution intensity and
TEM of CSNPs encapsulated with Edwardsiella tarda OmpA for the oral vaccination of
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fringed-Lipped peninsula carp (Dubey et al., 2016). Moreover, the combination between
microspheres of chitosan and other biodegradable composites has been used as antigen
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carriers for both parenteral and oral immunization of fish. Chitosan and gantrez has been
effective in incorporating the surface antigens of P. dicentrarchi for the stimulation of innate
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immune response against scuticociliatosis (León-Rodríguez et al., 2013). Reports on the
delivery of bio-molecules and vaccines into fish tissues via encapsulation with chitosan, its
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microspheres and nanocomplexes are summarized in table 3.

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3.3. Sustained release of bioactive ingredients
Nano-encapsulation is one of the most promising strategies for controlled release of
bioactive compounds at the right time and the right site with efficient cell absorption
(Ezhilarasi et al., 2013). CSNPs have specific physical properties (size, surface charge, or
loading capacity), which allow controlled delivery, improve targeting and stimulation of the
immune system (Ji et al., 2015) and provide protection against gastrointestinal conditions
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(Luo et al., 2011). In fish, CSNPs have been reported in the controlled release of some
bioactive components including vitamins (Alishahi et al., 2011a) and proteins such as
enzymes and hormones (Wisdom et al., 2018). Figure 3 shows particle size distribution of
leutinizing hormone releasing hormone (LHRH) encapsulated CSNPs and the effect of their
injection on the ovarian histology (Rather et al., 2013).
CSNPs can persist inside cells up to two weeks and are considered as good carriers
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for sustained release of various biological substances. Moreover, they can diffuse into the
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cytoplasm and nucleus, therefore they are able to pass from mother to daughter cells through
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several mitotic cycles (Malatesta et al., 2015). They are enriching additives for freshwater
and marine organisms enhancing their immunity (Sun et al., 2016) (Abd El-Naby et al.,
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2019). When encapsulated with antioxidants, they can penetrate intestinal epithelium
providing controlled oral delivery of bioactive ingredients (Rojas et al., 2015). The
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functionality, the thermal stability at 45°C and the shelf life of vitamin C has been enhanced
by encapsulation with CSNPs (Rojas et al., 2015) in vitro as well as in vivo (Alishahi et al.,
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2011a). In vitro, the stability and control release of vitamin C has been improved through
encapsulation with double layer coating from Zein/CS nanoparticle complex (Dong and
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Wang, 2016). Similarly, the chemical stability of vitamin D3 was enhanced when loaded on
zein nanoparticles coated with carboxymethyl chitosan (Luo et al., 2012). Additionally,
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significant increase in the total antioxidant capacity of vitamin C has been seen in zebrafish
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liver cell line when loaded on CSNPs without affecting cell viability (Jiménez-Fernández et
al., 2014). In vivo, vitamin C has been control released from CSNPs into the gastrointestinal
tract and serum of some fish species inducing their nonspecific immunity. In addition,
releasing of vitamin C from CSNPs was pH dependent and its shelf life increased when
compared with the non encapsulated form (Alishahi et al., 2011b).

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CSNPs are sufficiently stable to carry and protect proteins (Katas et al., 2013). The
higher Mw and degree of deacetylation (DD) of chitosan, the more the encapsulation
efficiency (EE) and the slower the protein release rate obtained. However, increasing in
loading capacity of proteins decreases the EE and accelerates its release rate from
nanocomplexes (Xu and Du, 2003). EE of bovine serum albumin (BSA) as a protein model
loaded on CSNPs reached up to 80% provided continued release for up to 1 week (Calvo et
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al., 1997). Recently, BSA/siRNA was controlled released for up to 48 h in vitro when loaded
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on CSNPs (Katas et al., 2013). Additionally, CSNPs improved mucoadhesive properties and
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permeability when adsorbed in vitro onto the excised carp intestinal mucosa (Liu et al.,
2012b) without toxicity or pathological damage in carp tissues (Liu et al., 2012a) (Liu et al.,
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2013).
Oral administration of protein derivatives including, hormones and enzymes in diets is

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limited because of its thermal nature, pH specificity and intestinal damage hazard.
Conjugation and encapsulation with CSNPs provides oral controlled delivery systems to
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overcome the problem of their short life in blood and enhance the efficacy and safety of their
oral administration. Figure 4 shows particle size distribution of trypsin enzyme encapsulated
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with CSNPs and the effect of their oral administration on the intestinal histology (Kumari et
al., 2013). Recently, nanoconjugation with chitosan was reported for the oral administration
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of various gonadal enzymes avoiding multiple injections of fish (Wisdom et al., 2018).
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Studies using chitosan and CSNPs for vitamin and protein delivery into fish are presented in
table 4.
3.4. Potential biomedical fish disease diagnosis (fish pathogen detection)
Fernandes et al. (2015) have used chitosan-based nanoparticles in the formation of a
highly sensitive electrochemical genosensor for the detection of fish pathogenic Aeromonas
spp. in spiked tap water. This genosensor is a promising potential biomedical tool for fish
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diseases diagnosis. It has been constructed from multi-walled carbon nanotubes chitosan–
bismuth complex (MWCNT–Chi–Bi) and lead sulphide nanoparticles. Thiol-modified
oligonucleotides have been used as fixing probe DNA. Lead sulphide NPs have been capped
via amide bond with 5′- (NH2) oligonucleotides and used as signalizing probe DNA (sz-
DNA). The two probes and the DNA of Aeromonas sequence are forming a hybrid complex.
Lead nanoparticles released from sz-DNA have been electrodeposited on the surface of glass
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carbon electrode provided with the MWCNT–Chi–Bi complex for improvement of lead
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deposition and electrical signal transmission. Finally, the differential pulse voltammetry is
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used in detecting the hybridization signal.
3.5. Fish and Seafood preservation

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In spite of the reported toxicity of CSNPs on zebrafish embryos (Hu et al., 2011)
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(Wang et al., 2016), there is a good number of investigations considering the inorganic
biodegradable nanoparticles, including CSNPs safe. For seawater aquaculture, chitosan,

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mostly from marine sources, was reported as the most promising stabilizer for seafood
products that are highly susceptible to deterioration because of their high water activity,
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neutral pH and their high contents of autolytic enzymes, polyunsaturated fatty acids, and free
amino acids (Alishahi and Aïder, 2012). Several in vitro and in vivo studies have considered
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chitosan safe, nontoxic (Kean and Thanou, 2010) and effective antibacterial agent for food
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preservation (Islam et al., 2011) and fish additive (Aref et al., 2018) improving their quality
(Gomes et al., 2017). CSNPs are non genotoxic and can enhance food protection by reducing
the permeation of gases, minimizing odor loss and increasing mechanical strength and
thermal stability (de Lima et al., 2010). Moreover, CSNPs have been incorporated in
biosensors for detection of xanthine in fish meat as an indicator for meat freshness (Devi et
al., 2012) (Devi et al., 2013). Owing to its antioxidative and antimicrobial properties,
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chitosan and its nanocomplexes as N-stearoyl O-butylglyceryl/chitosan have been used for
encapsulation and sustained release of fish oil for protection against oxidative degradation by
decreasing heat transfer and increasing the thermal stability of the encapsulated fish oil
(Chatterjee and Judeh, 2015). The quality of the unsaturated fatty acids (ω-3) content
obtained from carp viscera has been preserved against oxidative degradation when
encapsulated with CSNPs (Esquerdo et al., 2015).
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Nanocapsules constructed from the encapsulation of the liquid smoke of coconut shell
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have been obtained by the condensation of coconut wood smoke under limited oxygen
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conditions, with chitosan and maltodextrin and used for tuna fish preservation at ambient
temperature (Saloko et al., 2014). During refrigeration of fish meat at 4°C, chitosan
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composites have been used for the inhibition of the bacterial growth and reduction of the
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formed volatile bases and oxidation products (Mohan et al., 2012). Additionally, CSNPs have
been used in bio-nanocomposite technology applications for fabrication and strengthening

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edible films of food packaging and preservative coatings to increase shelf life and decrease
the deterioration of fish meat during refrigerating and freezing storage (De Moura et al.,
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2008). Chitosan coatings reduce the off-flavour compounds and improve flavour retention of
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chilled fish (Yu et al., 2018). Combination of chitosan with natural preservatives such as
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plant extracts, essential oils or antioxidants significantly improves the preservation quality
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(Zarei et al., 2015) (Qiu et al., 2014) (Li et al., 2012) (T. Li et al., 2013).
Recently, the physicochemical properties of the food edible films constructed from
fish gelatine (FG) fillers have been enhanced via incorporation with chitosan (Hosseini et al.,
2013). Strong, applicable, eco-friendly and bio-nanocomposite films have been produced via
reinforcing the FG matrix by incorporation with CSNPs (Hosseini et al., 2015) (Hosseini et
al., 2016). These films are safe to eat, hence, can be used in manufacturing the packaging
materials of food and helps in decreasing the environmental pollution resulting from plastic
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materials usually used for food packaging (Feng et al., 2016). FG/chitosan coating and film
can control the bacterial contamination, provide good quality characteristics and extend the
meat shelf life, however, the coating is better than the film in reducing the lipid oxidation of
fish fillets during chilled storage (Nowzari et al., 2013). Additionally, FG/chitosan edible
films with the plasticizer hydrophobic D-limonene have provided excellent mechanical
resistance against light and water penetration and enhanced the antibacterial properties of the
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films (Yao et al., 2017). Further, combination with natural preservatives significantly
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enhances the preservative characters of FG/chitosan-based edibles (Gómez-Estaca et al.,
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2010). During freezing, the aqueous chitosan solution has been used to form coating for
frozen storage of fish meat (Fan et al., 2009). Supplying the edible coatings with chitosan and
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CSNPs introduces effective barriers for protection of fish meat against deterioration for long
frozen storage periods of fish fingers (Abdou et al., 2012). It provides considerable
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improvements in chemical, microbial, sensory and textural properties even at -18°C frozen
storage (Berizi et al., 2018). The applications for fish meat preservation during chilled and
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frozen storage are presented in table 5.
On crustaceans, chitosan has been used as an antimicrobial agent to reduce the microbial
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loads on the surface of fresh shrimp and for preservation of shrimp meat (Alfaro et al., 2018).
Chitosan combined with shrimp protein/lipid concentrate has been used for coating and
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improving shrimp quality during cold storage (Arancibia et al., 2015). Chitosan coatings
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incorporated with some bioactive factors such as oregano and thyme essential oil preserved
ready-to-eat peeled shrimp and controlled the growth of microorganisms during refrigeration
storage (Carrión-Granda et al., 2016). In addition, chitosan coating combined with
pomegranate peel extract significantly lowered the melanosis and increased the quality of
Pacific white shrimp (Litopenaeus vannamei) during iced storage up to 10 days (Yuan et al.,
2016). Moreover, gelatin coating supplemented with chitosan improved the quality of shrimp
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under refrigerated storage (Farajzadeh et al., 2016). Recently, a novel packaging film had
been produced from chitosan coatings containing the essential oil of (Ziziphora
clinopodioides), pomegranate peel extract and cellulose nanoparticles, either separately or in
combination. High antibacterial activity had been observed during refrigerating storage of
peeled shrimp for up to 11 days (Mohebi and Shahbazi, 2017). More recently,
chitosan/carvacrol coating has been used for active packaging and extending the shelf life of
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L. vannamei during iced storage for 10 days (Wang et al., 2018). CSNPs have been used as
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glazing material for controlling lipid oxidation and reducing aerobic counts, yeasts and molds
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during 30 days frozen storage of shrimp at -21°C (Solval et al., 2014). Chitosan applied by
vacuum tumbling was helpful in the preservation of shrimp meat quality frozen at -20°C
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(Chouljenko et al., 2016). Similarly, vacuum tumbling with chitosan and CSNPs reduced the
aerobic plate counts and lipid oxidation besides maintaining the desired physicochemical
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properties of shrimp during frozen storage at -20°C (Chouljenko et al., 2017).

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Conclusions
There is a potential need for the development of novel encapsulation or immobilization

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carriers of several bioactive ingredients for several applications in aquaculture. This review
highlights the reported applications of chitosan and its nanostructure complexes on fish and
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crustaceans. CSNPs have many favorable biological properties such as safety,

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biocompatibility, biodegradability and antibacterial ability that make them promising
candidates for several applications in fish medicine. They are promising carriers for
biological ingredients including DNA vaccines, dietary supplements and nutrients such as
RNA nucleotides, antioxidants like vitamin C and even proteins including hormones and
enzymes. Dietary CSNPs exhibit bioactive immune-inducing activity and antimicrobial
properties on fish and crustaceans. However, additional studies are required particularly in
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the field of fish disease diagnosis and the oral delivery of DNA vaccines, siRNA, and
bioactive ingredients including vitamins, proteins and nutrients aiming at inducing of fish
immunity for the protection against infections.
Acknowledgement
We acknowledge support for a PhD scholarship offered for Fatma El Zahraa Ahmed by
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mission sectors in the ministry of higher education, Egypt.
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Figures and table legends
Figure 1. (A) SEM and (B) TEM of chitosan nanoparticles (Parida et al., 2013).
Figure 2. (A) Size distribution intensity and (B) TEM of freeze-dried CSNPs, encapsulating
OmpA of E. tarda (Dubey et al., 2016).
Figure 3. (Rather et al., 2013)

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I. (A) Particle size distributions and (B) TEM of chitosan-LHRH and chitosan-gold-LHRH
nanoparticles.
II. Ovarian histology of Cyprinus carpio:
A] Control: 1. Ovigerous lamellae with immature oocyte; 2. Mature oocyte; 3. Maturing
oocyte; 4. Atretic oocyte.
B] After injection with bare LHRH: 1. Mature/ripe oocyte; 2. Partially spent oocyte; 3.
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Cortical alveoli stage oocyte; 4. Spent follicle; 5. Nest of immature oocytes.
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C] After injection with CS/ LHRH NPs: 1. Lamellae containing nests of Oocytes 2. Spent
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follicle 3. Spent oocytes 4. Immature oocytes in ovigerous lamellae
D] After injection with CS–gold/LHRH NPs: 1. Mature oocytes 2. Spent oocytes 3. Immature
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oocytes 4. Discharged follicle.
Figure 4. (Kumari et al., 2013)

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a) Particle size distribution and three-dimensional images of CSNPs: A] 0.4% free
CSNPs, B&D] 0.4% CSNPs/0.01% trypsin and C&E] 0.4% CSNPs/0.02% trypsin.
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b) Intestinal histology (H&E, 40X) after feeding of Cyprinus carpio with:
(A): Control fish fed: Regular villi with continuous basement membrane lined the
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intestinal mucosa.
(B): 0.4% free CSNPs: 1. Villi with mildly swollen apical surfaces.
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(C1 & C2) 0.02% bare trypsin: 2. Broadened villi, marked foamy cells with lipid
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vacuoles. 3. Atrophied submucosal layer and muscularis.
(D) 0.4% CSNPs/0.01% trypsin: 4. longer villi with healthy apical surfaces. 5.
Improved morphological appearance of villi with continuous basement membrane. 7.
Distributed goblet cells along the villi for mucin production indicating better gastro-
intestinal health.
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(E) 0.4% CSNPs/0.02% trypsin: 6. Less crypt depth. 7. Villi containing goblet cells
and are similar to control group.
Table 1. Collection of the reported In vitro studies on the antimicrobial (antibacterial and
antifungal) activities of chitosan and its nanocomposites.
Table 2. Immunomodulatory effects of chitosan-based complexes on several fish species
against infections and stresses.
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Table 3. Published studies on bio-molecules and vaccine delivery via loading on chitosan, its
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microspheres and nanocomplexes.
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Table 4. Published studies on control and sustain release of bioactive ingredients via loading
on chitosan nanocomposites.
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Table 5. The reported applications in fish meat preservation during chilled and frozen
storage.
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Table 1
microorganism Reagent Reference
Gram negative bacteria Gram positive bacteria Fungi
Salmonella paratyphi, Staphylococcus aureus ___________ Chitosan (Islam et al.,
Escherchia coli 2011)
Human colonic anaerobic, nonpathogenic bacteria ___________ Chitosan (Šimůnek et
al., 2012)
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Escherchia coli __________________ ____________ N-diethylmethyl (Avadi et al.,
chitosan 2004)
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SC
__________________________________ filamentous fungi & N,O-(chloracetyl)-N- (Li and Guo,
yeasts (trimethyl) chitosan 2010)

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chloride
Klebsiella pneumonia, Staphylococcus aureus ______ CSNPs (Divya et al.,

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Escherchia coli, 2017)
Pseudomonas aeruginosa
________________________________ Yeast Chitosan & trimethyl (Ing et al.,

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(Candida albicans) CSNPs 2012)

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& Fungi
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(Aspergillus niger)
& Fusarium solani

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Escherchia coli, Staphylococcus aureus __________ CSNPs & copper-loaded (Qi et al.,
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Salmonella choleraesuis, CSNPs 2004) (Du et
Salmonella typhimurium al., 2008) (Du
et al., 2009)
Escherchia coli ______________ ______________ Chitosan, carboxy- (Liu et al.,
methylated chitosan 2001)
______________ ______________ Chitosan (Liu et al.,
2006)
Staphylococcus aureus Candida albicans chitosan-g– (Hebeish et

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Aspergillus niger acrylonitrile/silver al., 2015)
nanoparticles
__________ ___________________ Colletotrichum Chitosan/silver (Chowdappa
gloeosporioides nanoparticles composite et al., 2014)
Escherchia coli, Pseudomonas Listeria monocytogenes, ______________ Chitosan & chitosan (No et al.,
fluorescens, Salmonella Bacillus megaterium, oligomers 2002)
typhimurium, Vibrio Bacillus cereus,
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parahaemolyticus, Staphylococcus aureus,
Lactobacillus plantarum,
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Lactobacillus brevis,
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Lactobacillus bulgaricus
Escherchia coli, Salmonella Staphylococcus aureus, ______________ Chitosan (Chung et al.,

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typhimurium, Pseudomonas Streptococcus faecalis 2004)
aeruginosa
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______________ Streptococcus mutans ______________ CSNPs (de Paz et al.,
biofilms 2011)
Aeromonas hydrophila, ______________ ______________ Chitosan (Yildirim-

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Edwardsiella ictaluri, Aksoy and
Flavobacterium columnare Beck, 2017)

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Escherchia coli, Vibrio Streptococcus uberis, ______________ Chitosan microparticles (Jeon et al.,
cholerae, Staphylococcus aureus, 2014)

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Salmonella enterica, Enterococcus

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Klebsiella pneumonia
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Table 2
Immunomodulati on against infections Immunomodulati on against stresses
Fish species Pathogen Diets Reference Fish species Stress Diets Reference
Kelp grouper Philasterides (Harikrishnan Olive flounder Oxidative (Samarakoon
(Epinephelus dicentrarchi et al., 2012a) (Paralichthys cellular and et al., 2013)
bruneus) olivaceus) DNA damage
induced by
Chitin and Chitosan
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H2 O2 .
Chitosan
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Kelp grouper Vibrio (Harikrishnan Caspian kutum Environmental (Najafabad
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(Epinephelus alginolyticus et al., 2012b) fingerlings stresses et al., 2016)
bruneus) (Rutilus frisii

NU
kutum)
Indian major carp Aeromonas (Aathi et al., Common carp Oxidative stress (Mehrpak et
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(Labeo rohita) hydrophila 2013) (Cyprinus and toxicity of al., 2015)
Carpio L.) CdCl2 on liver
Chitosan and Vitamin C

tissues
T ED
Common carp Aeromonas (Alishahi et Common carp Toxicity of (Sharifinasab

Chitosan
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(Cyprinus Carpio hydrophila al., 2014) (Cyprinus paraquat on et al., 2016)
L.) (Mustafa et Carpio L.) muscle tissues

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al., 2014)
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Nile tilapia Aeromonas (Abu-Elala et
(Oreochromis hydrophila al., 2015)
nilotica)
Zebrafish larvae Aeromonas (Nikapitiya et
(Danio rerio) hydrophila CSNPs al., 2018)

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Table 3
Bio-molecules delivery
Bio-molecules Carrier Fish species Delivery Reference
The plant extract Indian freshwater catfish Parenteral (Bhat et al.,
(Eurycomanone) (Clarias magur) administration 2018)
(induces the secretion of
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CSNPs
testosterone hormone)
The exogenous nucleotide Indian major carp (Ferosekhan
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RNA extracted from yeast fingerlings et al., 2014)
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(important nutrients) (Labeo rohita)
Oral administration
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“Ulvan” polysaccharide CSNPs Solea senegalensis (Fernández-
extracted from Ulva ohnoi Díaz et al.,

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macroalgae 2017)
(immunomodulation)
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Vaccines delivery
Vaccine Antigen carrier Fish species Delivery Reference

T
EP
The exogenous reporter Nile tilapia (Ramos et al.,
gene, encoding for the (Oreochromis nilotica) 2005)

C
bacterial enzyme (β-

Chitosan
AC
galactosidase)
Oral administration
Nodavirus DNA vaccine European sea bass juveniles (Valero et al.,
(Dicentrarchus labrax) 2016)
The aerolysin antigen of Oleoyl- Common carp (Liu et al.,
Aeromonas hydrophila carboxymethy- (Cyprinus carpio) 2016)
chitosan/hyaluronic
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acid
pDNA containing major Chitosan Japanese flounder (Tian et al.,
capsid protein gene of microspheres (Paralichthys olivaceus) 2008)
lymphocystis disease virus
The surface antigens of Chitosan/gantrez Turbot Parenteral (León-
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Philasterides dicentrarchi microparticles (Scophthalmus maximus) administration Rodríguez et
parasite causing al., 2012)
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scuticociliatosis
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The OMP antigen of Indian major carp juveniles Parenteral (Behera and
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Aeromonas hydrophila (Labeo rohita) administration Swain, 2013)
chitosan composite microspheres
Oral (Behera and

Alginate-coated
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administration Swain, 2014)
Model pDNA vaccine Gilthead sea bream juveniles Oral & parenteral (Sáez et al.,
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(Sparus aurata) administration 2018)

T
EP
pDNA constructed from the Asian sea bass (Vimal et al.,
RNA2 capsid protein gene (Lates calcarifer) 2014)

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of nodavirus
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Oral administration
pDNA vaccine of Vibrio Asian sea bass (Vimal et al.,

CSNPs
anguillarum Juveniles 2012)
(Lates calcarifer)
pDNA encoding OMP38 of Asian sea bass (Rajesh
Vibrio anguillarum (Lates calcarifer) Kumar et al.,
2008)
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A combination of OMP K black seabream (Acanthopagrus (L. Li et al.,
gene of Vibrio schlegelii) Bleeker 2013)
parahaemolyticus strain and
the eukaryotic expression
vector against infection.
DNA coding for the replicas Atlantic salmon (Rivas-
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of alphavirus of infectious (Salmo salar) Aravena et
salmon anemia virus al., 2015)
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SC
The recombinant OMP fringed-lipped peninsula carp (Dubey et al.,
A of Edwardsiella tarda (Labeo fimbriatus) 2016)

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Table 4
Bioactive ingredients Fish species Delivery Carrier Reference
Vitamins Rainbow trout (Alishahi
(Oncorhynchus mykiss) et al.,
2011a)
Vitamin C
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Brachionus plicatilis & the (Jiménez-
post-metamorphic larvae of Fernández
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Solea senegalensis et al.,
SC
2014)
Oral delivery
CSNPs
Enzymes The exogenous Indian major carp (Kumari et
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proteolyses enzyme (Labeo rohita) al., 2013)
“trypsin”
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Aromatase inhibitors Indian freshwater catfish (Wisdom
“letrozole” for the fingerlings et al.,

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gonadal development (Clarias magur) 2018)
of fish
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Hormones Salmon gonadotropic Female grass carp Intramuscular Chitosan & (Rather et
hormone "leutinizing (Ctenopharyngodon idellus) injection chitosan gold al., 2013)

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hormone releasing nanoconjugates

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hormone" (LHRH)
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Table 5
Fish meat preservation during chilled storage
Fish species Coating/Film Duration/°C Reference
Indian oil sardine Edible chitosan coatings up to 8 days for 1% (Mohan et

(Sardinella longiceps) chitosan and 10 days for al., 2012)
2% chitosan /(4°C)
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Large yellow croaker fillets (Li et al.,
(Pseudosciaena crocea) Chitosan combined with tea polyphenol 20 days/(4°C) 2012)
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& rosemary & grape seed extract (T. Li et al.,
Red drum fillets extracts 2013)
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(Sciaenops ocellatus)
Large yellow croaker Chitosan combined with different 8 days/(4°C) (Guohua et

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(Pseudosciaena crocea) concentrations of nisin peptide al., 2016)
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Japanese sea bass fillets Chitosan incorporated with citric acid & 12 days/(4°C) (Qiu et al.,
(Lateo labrax japonicas) licorice extract 2014)
Raw grass carp fillets Chitosan combined with glycerol 7 days/(4°C) (Yu et al.,
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(Ctenopharyngodon idellus) monolaurate & clove bud essential oil 2018)

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Rainbow trout fillet FG/chitosan coating & film 16 days/(4±1°C) (Nowzari et

(Oncorhynchus mykiss) al., 2013)
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Golden pomfret fillet FG/chitosan up to 17 days/(4°C) (Feng et al.,

C
(Trachinotus blochii) 2016)

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Dolphinfish FG/chitosan incorporated with clove 10 days/2°C (Gómez-

(Coryphaena hippurus) essential oil with edible films Estaca et al.,
2010)
Minced trout fillet FG/chitosan films incorporated with (1 11 days /(4 ± 1 °C) (Kakaei and
& 2%) grape seed extract & (1 & 2%) Shahbazi,
Ziziphora clinopodioides essential oil, 2016)
either separately or in combination.
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Silver carp fillets (Ramezani et

(Hypophthalmicthys molitrix) al., 2015)
Chitosan & CSNPs coating
Carangoides coeruleopinnatus (Alboghbeish
12 days/(4°C)
fillets and
Khodanazary,
2018)
Silver carp fillets CSNPs incorporated with orange & (Zarei et al.,
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(Hypophthalmicthys molitrix) pomegranate peel extracts 2015)
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Fish meat preservation during frozen storage
Fish species Coating Duration / °C Reference
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Silver carp fingers Chitosan & CSNPs up to 6 months/ -18°C (Fan et al.,
(Hypophthalmicthys molitrix) 2009)
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Silver carp (Hypophthalmicthys 2% aqueous chitosan solution 30 days/ -3°C (Fan et al.,
molitrix) 2009)
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Salmon 0.5% and 0.75% chitosan solutions 14 weeks/ -5°C (Soares et al.,
directly on the surface of frozen fish 2013)
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Rainbow trout Chitosan & Pomegranate peel extract up to 6 months/ -18°C (Berizi et al.,
(Oncorhynchus mykiss) 2018)
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C EP
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Highlights
Chitosan-based complexes are effective immunomodulators of fish and crustaceans.
Chitosan nanoparticles utilization for controlled drug delivery and sustained release.
Chitosan improves fish meat and oil preservation.
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Figure 1
Figure 2
Figure 3
Figure 4

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Accepted Manuscript

Recent progress in biomedical applications of chitosan and its

Fatma Ahmed, Faiza M. Soliman, Mohamed A. Adly, Hamdy

Recent progress in biomedical applications of chitosan and its nanocomposites in

Authors: Fatma Ahmed1,2 , Faiza M. Soliman2 , Mohamed A. Adly2 , Hamdy A.M.

Soliman2 , Mansour El-Matbouli1 , Mona Saleh*1

1- Clinical Division of Fish Medicine; University of Veterinary Medicine, Vienna,

E-mail: mona.sale h@vetme duni.ac.at

Abstract word count: 173

Manuscript word count: 3986

Number of references: 169

Chitosan nanoparticles (CSNPs) are the nanostructures of chitosan biopolymer which

excreted from kidneys. It is widely used in medical and pharmaceutical applications

including food preservation purposes. In addition, they have immunomodulatory effects on

Chitosan nanoparticles, Biomedical applications, Antimicrobial agents, Immunomodulation,

Fish and seafood preservation

2. Preparation and characterization of CSNPs

3. Biomedical applications of chitosan and nano-chitosan complexes on aquatics

3.3 Sustained release of bioactive ingredients

3.4 Potential biomedical fish disease diagnosis

3.5 Fish and seafood preservation

Chitosan is a natural polysaccharide prepared by alkaline N-de-acetylation of chitin (Zhao

Owing to the mucoadhesive properties of chitosan, it could be used as a vehicle improving

advanced applications of chitosan on marine aquacultures.

antimicrobial and immunostimulatory agent against deleterious fish pathogens. Several

on aquatics encouraging more researchers to further explore the variable characteristics of

CSNPs for more benefits to the aquaculture.

should be prepared or gently mixed prior to use (Alishahi et al., 2011b).

been evaluated by scanning electron microscopy (SEM) and transmission electron

3. Biomedical applications of chitosan and nano-chitosan complexes on aquatics

biological and chemical properties such as biodegradability, biocompatibility, bioactivity

used as carriers, encapsulators or immobilizers for several bioactive ingredients as

reported biological and medical applications of chitosan-based complexes on fish and

of aquatic organisms. Undoubtedly, the applications related to the immunomodulation

improving the aquaculture production.

encapsulated with CSNPs (Rajeshkumar et al., 2009). Additionally, the crayfish

immunomodulation of fish against infections and stresses are summarized in table 2.

gastrointestinal tract. CSNPs enhance the mucoadhesive potential improving intestinal

of fish (Ferosekhan et al., 2014).

(Kumar et al., 2007) or by immersion (Fernandez-Alonso et al., 2001). However, oral

immune response against scuticociliatosis (León-Rodríguez et al., 2013). Reports on the

microspheres and nanocomplexes are summarized in table 3.

3.3. Sustained release of bioactive ingredients

Nano-encapsulation is one of the most promising strategies for controlled release of

injection on the ovarian histology (Rather et al., 2013).

compared with the non encapsulated form (Alishahi et al., 2011b).

Oral administration of protein derivatives including, hormones and enzymes in diets is

3.4. Potential biomedical fish disease diagnosis (fish pathogen detection)

Fernandes et al. (2015) have used chitosan-based nanoparticles in the formation of a

bismuth complex (MWCNT–Chi–Bi) and lead sulphide nanoparticles. Thiol-modified

3.5. Fish and Seafood preservation

biodegradable nanoparticles, including CSNPs safe. For seawater aquaculture, chitosan,

encapsulated with CSNPs (Esquerdo et al., 2015).

been used in bio-nanocomposite technology applications for fabrication and strengthening

frozen storage are presented in table 5.

storage (Carrión-Granda et al., 2016). In addition, chitosan coating combined with

clinopodioides), pomegranate peel extract and cellulose nanoparticles, either separately or in

properties of shrimp during frozen storage at -20°C (Chouljenko et al., 2017).

There is a potential need for the development of novel encapsulation or immobilization

crustaceans. CSNPs have many favorable biological properties such as safety,

biocompatibility, biodegradability and antibacterial ability that make them promising

enzymes. Dietary CSNPs exhibit bioactive immune-inducing activity and antimicrobial

immunity for the protection against infections.