Download Potential applications of chitosan in veterinary medicine

Advanced Drug Delivery Reviews 56 (2004) 1467 – 1480
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Potential applications of chitosan in veterinary medicine
Sevda Sßenel a,*, Susan J. McClure b
a
Department of Pharmaceutical Technology, Faculty of Pharmacy, Hacettepe University, 06100 Ankara, Turkey
b
CSIRO Livestock Industries, F.D. McMaster Laboratory, Armidale, NSW 2350, Australia
Received 20 June 2003; accepted 18 February 2004
Available online 2 April 2004
Abstract
Chitosan is a partially deacetylated polymer obtained from the alkaline deacetylation of chitin which is a glucose-based
unbranched polysaccharide widely distributed in nature as the principal component of exoskeletons of crustaceans and insects
as well as of cell walls of some bacteria and fungi.
Chitosan exhibits a variety of physicochemical and biological properties resulting in numerous applications in fields such as
waste and water treatment, agriculture, fabric and textiles, cosmetics, nutritional enhancement, and food processing. In addition
to its lack of toxicity and allergenicity, and its biocompatibility, biodegradability and bioactivity make it a very attractive
substance for diverse applications as a biomaterial in pharmaceutical and medical fields, where it has been used for systemic and
local delivery of drugs and vaccines. It also has bioactive properties in its own right.
This paper reviews current veterinary applications for chitosan including wound healing, bone regeneration, analgesic and
antimicrobial effects. It also discusses the potential application of chitosan to drug and vaccine delivery in veterinary species.
Given the restrictions imposed by financial and animal restraint considerations, especially in farming applications, the
veterinary drug delivery areas most likely to benefit from chitosan are the delivery of chemotherapeutics such as antibiotics,
antiparasitics, anaesthetics, painkillers and growth promotants to mucosal epithelium for absorption for local or systemic
activity, and the delivery of immunomodulatory agents to the mucosal associated lymphoid tissue for induction or modulation
of local immune responses. The properties of chitosan expected to enhance these functions are discussed, and the future
research directions in this field are indicated.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Chitosan; Veterinary; Wound healing; Tissue regeneration; Drug delivery; Mucosal immunization
Contents
1.
2.
3.
Introduction . . . . . .
Properties of chitosan . .
2.1.
Physicochemical.
2.2.
Biological. . . .
Veterinary applications .
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* Corresponding author. Tel.: +90-312-305-1241; fax: +90-312-310-0906.
E-mail address: [email protected] (S. Sßenel).
0169-409X/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2004.02.007
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S. Sßenel, S.J. McClure / Advanced Drug Delivery Reviews 56 (2004) 1467–1480
3.1.
Therapy . . . . . . . . . . . . . . . . . . .
3.1.1. Wound healing . . . . . . . . . . .
3.1.2. Antimicrobial activity . . . . . . . .
3.1.3. Tissue regeneration . . . . . . . . .
3.1.4. Analgesia. . . . . . . . . . . . . .
3.2. Drug delivery . . . . . . . . . . . . . . . .
3.2.1. Delivery of chemotherapeutics . . . .
3.2.2. Delivery of immunomodulatory agents
4. Conclusions and future directions . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
Chitin, which is the most abundant biopolymer in
nature after cellulose, is a polysaccharide widely
distributed in nature as the principal component of
exoskeletons of crustaceans and insects as well as of
cell walls of some bacteria and fungi. Like cellulose,
it is a glucose-based unbranched polysaccharide. It
differs from cellulose at the C-2 carbon by having an
acetamido residue in place of a hydroxyl group.
Chitosan is a partially deacetylated polymer of acetyl
glucosamine obtained after alkaline deacetylation of
the chitin. It comprises copolymers of glucosamine
and N-acetyl glucosamine and the term chitosan
embraces a series of polymers which vary in molecular weight (from about 10,000 to 2 million Dalton)
[1]. Chitosan is semi-crystalline and the degree of
crystallinity is a function of the degree of deacetylation. Crystallinity is maximal for both chitin (i.e.
0% deacetylated) and fully deacetylated (i.e. 100%)
chitosan. Minimal crystallinity is achieved at intermediate degrees of deacetylation. Currently, chitin
and chitosan are manufactured commercially in large
scale from the outer shell of shrimps, lobsters and
crabs.
2. Properties of chitosan
2.1. Physicochemical
Chitosan is insoluble at neutral and alkaline pH,
but forms water-soluble salts with inorganic and
organic acids including glutamic, hydrochloric, lactic
and acetic acids. Upon dissolution in acidic media, the
amino groups of the polymer become protonated
rendering the molecule positively charged. Recently,
importance has been attached to chitosan derivatives
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with a defined degree of deacetylation and depolymerization because of their significantly different
physicochemical properties [2]. The degree of acetylation represents the proportion of N-acetyl-D-glucosamine units with respect to the total number of units.
The properties of chitosan (e.g. pKa and solubility)
can be modified by changing the degree of deacetylation and formulation properties such as the pH and
ionic strength. At neutral pH, most chitosan molecules
will lose their charge and precipitate from solution.
Chitosan exhibits a variety of physicochemical and
biological properties, therefore it has found numerous
applications in various fields such as waste and water
treatment, agriculture, fabric and textiles, cosmetics,
nutritional enhancement, and food processing. In
addition to its lack of toxicity and allergenicity, its
biocompatibility, biodegradability and bioactivity
make it a very attractive substance for diverse applications as a biomaterial in pharmaceutical and medical
fields [3– 5].
Chemical derivatization of chitosan provides a
powerful means to promote new biological activities
and to modify its mechanical properties. The primary
amino groups on the molecule are reactive and provide a mechanism for side group attachment using a
variety of mild reaction conditions. The general effect
of addition of a side chain is to disrupt the crystal
structure of the material and hence increase the
amorphous fraction. This modification generates a
material with lower stiffness and often altered solubility but the precise nature of changes in chemical
and biological properties depends on the nature of the
side group. In addition, the characteristic features of
chitosan such as being cationic, hemostatic and insoluble at high pH, can be completely reversed by a
sulfation process which can render the molecule
anionic and water-soluble, and also introduce anticoagulant properties [6]. The variety of groups which
S. Sßenel, S.J. McClure / Advanced Drug Delivery Reviews 56 (2004) 1467–1480
can be attached to chitosan is almost unlimited, and
side groups can be chosen to provide specific functionality, alter biological properties or modify physical
properties.
Due to its high molecular weight and a linear
unbranched structure, chitosan is an excellent viscosity-enhancing agent in acidic environments. It
behaves as a pseudoplastic material exhibiting a
decrease in viscosity with increasing rates of shear.
The viscosity of chitosan solution increases with an
increase in chitosan concentration, decrease in temperature and with increasing degree of deacetylation,
which is a structural parameter also influencing
physiochemical properties such as the molecular
weight, the elongation at break and the tensile
strength [7]. Viscosity also influences biological
properties such as wound-healing properties and
osteogenesis enhancement as well as biodegradation
by lysozyme [8].
Chitosan, which is polycationic in acidic environments, possesses an ability to form gels at acidic pH
values because it is hydrophilic and can retain water in
its structure. The acetylation of chitosan in hydroalcoholic media allows the selective modification of
the free amino groups and is responsible for a process
of gelation. It has been shown that the charge density
of the chain segments is an essential parameter for the
formation of gels and all factors that lower this
parameter favor deswelling and reversibility [9]. The
high hydration, the physicochemical and physical
properties, as well as the polyelectrolyte behavior of
this kind of gel allow applications such as bioactive
dressing for wound healing. Gels can also be used as a
slow-release drug-delivery system.
The solubility of chitosan can be sharply reduced
by cross-linking the macromolecules with covalent
bonds using for example glutaraldehyde [10]. Swelling of the films decreases with an increase in the
amount of cross-linking agent added. The swelling of
enzyme-containing films decreases more significantly,
probably because of formation of additional crosslinks due to the participation of the functional groups
of the enzyme in the reaction.
When chitosan is intended for contact with serous
liquids, sterility becomes necessary. Heat is often
employed to facilitate polymer processing and to
sterilize the pharmaceutical and medical products.
However, exposure to high temperatures can change
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the physical properties of chitosan, affecting its
aqueous solubility, rheology, and appearance. It has
been shown that the chitosan acetate membranes are
less aqueous soluble upon heating in an oven for 1
h at 120 jC. One postulation, derived from NMR
and thermal analyses, was a thermal conversion of
the water-soluble chitosan acetate to water-insoluble
chitin. Chitosan films were found to be less hydrophilic when autoclaved at 121 jC for 15– 30 min.
This reduction in solubility was related to the
formation of the anhydrous crystal polymorph observed in chitosan samples heated in the presence of
water. Unlike gamma irradiation, which caused main
chain scissions and a dose-dependent decrease in
viscosity [11], a more complex relationship was
reported between the viscosity of the chitosan and
the amount of heat supplied. The reduction in
viscosity was concomitant with decreased aqueous
solubility and the development of color in the
chitosan samples. The cross-linking of chitosan
chains by glutaraldehyde was characterized by similar coloration and decreased aqueous solubility. The
rate and extent of the changes was accelerated with
increasing temperatures and in the presence of saturated steam [12].
2.2. Biological
Chitosan has been used as a safe excipient in drug
formulations over the last two decades [13]. This
polymer also attracted the attention of pharmaceutical
scientists as a mucoadhesive polymer. Chitosan in the
swollen state has been shown to be an excellent
mucoadhesive and as a natural bioadhesive polymer
that can adhere to hard and soft tissues, it has been
used in dentistry, orthopaedics, ophthalmology and in
surgical procedures. It adheres to epithelial tissues and
to the mucus coat present on the surface of the tissues.
A variety of chitosan-based colloidal delivery systems
have been described in the literature for the mucosal
delivery of polar drugs, peptides, proteins, vaccines
and DNA. Clinical tests carried out in order to
promote chitosan-based biomaterials do not report
any inflammatory or allergic reactions following implantation, injection, topical application or ingestion
in the human body [14]. Biomedical applications and
some bioactive properties of chitosan are summarized
in Table 1. Chitosan is easily hydrolyzed by various
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chitosanases, which are completely absent in mammals, and depending on degree of deacetylation, by
lysozyme. Its biodegradation leads to release of aminosugars, which can be incorporated into glycosaminoglycan and glycoprotein metabolic pathways, or
excreted.
Recently, chitosan has shown promise as a carrier
for delivery to the colon, results suggesting that
degradation of chitosan by colonic bacterial enzymes
might be an important factor in its successful targeting
of the colon [15]. The degradation by the bacterial
enzymes was shown to be dependent on both molecular weight and degree of deacetylation of the chitosan sample. Samples with a lower molecular weight
and lower degree of deacetylation were found to be
more susceptible substrates. On the other hand, chitosan was shown not to be affected by the environment of the small intestine [16].
It has been demonstrated that degree of deacetylation has no significant influence on the in vitro and
in vivo cytocompatibility of chitosan films with
keratinocytes and fibroblasts. The chitosan films with
a low degree of deacetylation are very good biomaterials for superficial wound-healing [14]. Once
placed on the wound, they adhere to fibroblasts and
favor the proliferation of keratinocytes and thereby
epidermal regeneration. The higher the degree of
Table 1
Biomedical applications and bioactive properties of chitosan
Artificial skin
Surgical sutures
Artificial blood vessels
Controlled drug release
Contact lens
Eye humor fluid
Bandages, sponges
Burn dressings
Blood cholesterol control
Anti-inflammatory
Tumor inhibition
Anti-viral
Dental plaque inhibition
Bone healing treatment
Wound healing accelerator
Hemostatic
Antibacterial
Antifungal
Weight loss effect
deacetylation of chitosan films, the lower was the
adhesion to cells and the cell proliferation. It has
been reported that the degree of deacetylation influences the strength as well as the elongation of films
[17]. The chitosan films with a high degree of
deacetylation were found to be more brittle and
difficult to handle than those with a lower degree
of deacetylation. Tissue responses to chitin and its
derivatives were studied by implanting the films in
the subcutaneous tissue of rats for different periods
of time [17]. A relatively severe inflammatory reaction was observed in the tissue surrounding the chitin
and chitosan films implanted for 1 week. However,
this tissue response subsided 2 weeks after implantation. When films were implanted for 7 weeks, an
encapsulation around the implanted films by a collagenous tissue was observed. Implanted films were
more strongly encapsulated 12 weeks after implantation, as are conventional, non-biodegradable biomaterials. The surface properties of implanted
biomaterials influence the tissue response. The film
surface becomes less hydrophobic upon deacetylation of chitin to chitosan but the extent of hydration
does not increase in proportion to the degree of
deacetylation. In contrast to the hydration, the biodegradation of the films of chitin and its deacetylated
derivatives does not seem to be governed by the
physical microstructure but by the chemical structure
of the films. The degradation kinetics of chitosan
appear to be inversely related to the degree of
crystallinity, which is controlled mainly by the degree of deacetylation. Highly deacetylated forms
(e.g. >85%) exhibit the lowest degradation rates
and may last several months in vivo, whereas samples with a lower level of deacetylation degrade
more rapidly.
3. Veterinary applications
Recently, much attention has been given to the use
of chitosan in veterinary applications, as a wound
healing agent, antimicrobial agent, bandage material,
skin grafting template, hemostatic agent and drug
delivery vehicle. In this section, various investigations
carried out recently on veterinary applications of
chitosan are discussed as well as their potential uses
in veterinary medicine.
S. Sßenel, S.J. McClure / Advanced Drug Delivery Reviews 56 (2004) 1467–1480
3.1. Therapy
3.1.1. Wound healing
The chemical structure of chitosan, which is similar to that of hyaluronic acid, suggested that it would
act as a wound healing agent [18]. Mechanisms of
acceleration of wound healing have been investigated
in vitro and in vivo by several groups [19 – 21]. Since
1985, Minami and his group have been developing
chitin and chitosan products for veterinary use and
they have reported their clinical effects on wound
healing and their biological activities [22]. They found
these polymers and products to accelerate wound
healing, decrease treatment frequency, and give comfortable and painless wound surface protection. In
addition to accelerating wound healing, chitosan is
also capable of activating host defenses to prevent
infection, thereby offering an alternative to the use of
antibiotics.
Chitosan enhances the functions of inflammatory
cells such as polymorphonuclear leukocytes (PMN)
(phagocytosis, production of osteopontin and leukotriene B4), macrophages (phagocytosis, production
of interleukin (IL-1), transforming growth factor h1
and platelet-derived growth factor). As a result,
chitosan promotes granulation and organization.
Therefore, it is beneficial for healing of large open
wounds in animals [23]. One of the most interesting effects of chitin and chitosan on wound healing
is formation of granulation tissue with angiogenesis.
Chitin and chitosan induce fibroblasts to release
interleukin-8, which is involved in migration and
proliferation of fibroblasts and vascular endothelial
cells.
There have been many studies carried out by
different groups showing that chitin and chitosan
accelerated wound healing in clinical veterinary
cases (Table 2) and many remedies using chitin
and chitosan for treatment of wounds have already
been marketed. In most of these studies, the chitin
and chitosans have been used as filaments, powders,
granules, sponges, or as a composite with cotton or
polyester. The effects of chitin and chitosan on
wound healing were demonstrated in small animals
(dogs and cats), livestock (cows) and zoo animals
using the chitin products (Chitipack S and Chitipack
P), and a chitosan product (Chitopack C), which are
commercialised in Japan [24]. In comparison with
1471
conventional therapy with irrigation and antibiotic
administration to a wound, new treatments with
chitin and chitosan products permitted a substantial
decrease in treatment frequency with minimal scar
formation.
The application of chitosan hydrogels has also
been demonstrated to effectively interact with and
protect the wound, ensuring a good, moist healing
environment. An insoluble flexible hydrogel-like soft
rubber was prepared following application of ultraviolet light irradiation to a photo-crosslinkable chitosan aqueous solution. The resulting gel was
evaluated as a dressing for wound occlusion and
an accelerator in the healing process, using a mouse
model with full-thickness skin incisions. Application
of the gel onto an open wound induced significant
wound contraction and accelerated wound closure
and healing. Furthermore, in cell culture studies, it
was shown that the immobilized chitosan hydrogel in
the presence of fetal bovine serum had a chemoattractive influence on dermal fibroblasts and stimulated cell growth [25].
In addition to effects on superficial wound repair,
the influence of chitin on the healing of sheep flexor
tendons after repair with non-woven polyester fiber
has been investigated. Chitin was found to have a
beneficial effect on collagen differentiation around
non-woven fabric tendon implants [26].
For more detailed information on the accelerating
effects of chitin and chitosan on wound healing,
readers are referred to the studies published by Minami and his group [27].
3.1.2. Antimicrobial activity
In addition to the requirements for wound healing,
it is important to control any infection of a wound
under dressing. Infectious organisms preferentially
target wounds beneath the dressing materials and
elicit serious infections that frequently require removal of the wound dressing. For these reasons, the
treatment of wounds requires the suppression of
bacterial growth. Chitosan has been shown to provide inhibition of bacterial proliferation in the treatment of infected wounds [28]. The antimicrobial
activity of chitosan has been recognized against
several bacteria and fungi, and is influenced by a
number of factors that include the type of chitosan,
the degree of polymerization and some of its other
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Table 2
Veterinary applications of chitosan and chitin for therapy
Chitin/chitosan type
Form
Animal model
Application
Effect
Result
Reference
Chitosan
DD: 82%
MW: 80,000 Da
Source:
crab shells
Cotton type
(Chitopack C)
Small animals
Large animals
Wild and zoo
animals
Topical
Wound/
infection
healing
Disappearance
of purulence,
formation of
granulation tissue,
re-epithelization,
decrease in
treatment frequency
[24]
Chitin
DD: 9%
MW>100,000
Source:
squid pen
Sponge-like chitin
(Chitipack S)
Small animals
Topical
Wound
healing
Composite chitin
sheet
(Chitipack P)
Small animals
Large animals
Topical
Implantation
Wound
healing
Decrease in
treatment frequency
with minimal scar
formation
Good wound
contraction,
re-epithelization,
granulation
tissue formation
Chitin
MW: 370,000
Source:
squid pen
Non-woven fiber
and chitin complex
(Chitipack P)
Sheep
Implantation
Tendon
healing
Increased immature
and intermediate
fibroplasia
[26]
Methylpyrrolidone
chitosan
Freeze-dried
soft sponge
Rabbit
Topical
Bone
healing
Induced bone
repair
[35]
Phosphorylated
chitosan
(P-chitosan)
DD: 63%
MW: 17,000
P-chitosan
containing
calcium
phosphate
cements
Rabbit
Implantation
Bone
healing
Osteoinduction
with relatively
low doses of
inducing signals
[36]
N,Ndicarboxymethyl
chitosan
Soft spongy
and hydrophilic
material
Sheep
Topical
Bone
healing
Accelerated
regeneration
of bone tissue
[37]
Chitosan
DD: 72%
MW:
Source: cuttlefish
Powder
Dog
Topical
Bone
healing
Enhancement of
bone healing
[38]
Chitosan
MW: 70,000
Source:
prawn shells
Solution
Rat
Injection
into knee
Tissue
regeneration
(chondrogenic)
Intra-articular
fibrous tissue
formation, articular
chondrocyte
proliferation,
reduced decrease
in the epiphyseal
cartilage thickness
[39]
Injection
into pleural
cavity
S. Sßenel, S.J. McClure / Advanced Drug Delivery Reviews 56 (2004) 1467–1480
1473
Table 2 (continued )
Chitin/chitosan type
Form
Animal model
Application
Effect
Result
Reference
Chitin
DD: < 10%
MW: 300,000
Chitosan
DD: >80%
MW: 80,000
Chitin
DD: < 30%
Chitosan
DD: >80%
Suspension
Mouse
Intraperitoneal
injection
Analgesic
A dose-dependent
decrease of the
abnormal behaviors
due to pain
[40]
Granule
Dog
Topical
Wound
healing
Re-epithelization
in chitin and
chitosan groups
(no statistical
difference
between chitin
and chitosan groups)
[42]
Chitosan
DD: 82%
MW: 30 – 80,000
Cotton type
(Chitopack C)
Dog
Topical
Wound
healing
Accelerated
infiltration of PMN
cells at the early
stage, followed by
the production of
collagen by fibroblasts
[43]
Chitosan
DD: 82%
MW: 80,000
Source:
crab shells
Cotton type
(Chitopack C)
Dog
Implantation
Immunopotentiator
Enhanced activity of
polymorphonuclear
cells, increase in
number of white
blood cells
[44]
Phosphated
chitin
Solution
Mouse
Intravenous
infusion
Anti-inflammatory
Potentially useful
in chitosaninduced acute
respiratory
distress syndrome
[45]
DD, degree of deacetylation; MW, molecular weight (Da); PMN, polymorphonuclear cells.
chemical and physical properties [29,30]. Chitosan
more effectively inhibits growth of bacteria than
chitosan oligomers but the inhibitory effects differ
with regard to the molecular weight of chitosan and
the type of bacterium. Chitosan generally exerts
stronger bactericidal effects against gram-positive
than gram-negative bacteria. The antibacterial activity of chitosan is affected by pH (pH 4.5– 5.9 range
tested), with greater activity being found at the lower
pH values. In dilute acid solutions, the positive
charges of chitosan interfere with the negatively
charged residues of macromolecules at the bacterial
cell surface, presumably by competing with Ca+ for
electronegative sites on the membrane without conferring dimensional stability, rendering the membrane leaky [31].
3.1.3. Tissue regeneration
Cellular interactions of chitosan with mammalian
tissues have been positive from the tissue repair and
regeneration standpoint. One of chitosan’s most promising features is its excellent ability to be processed
into porous structures for use in cell transplantation
and tissue regeneration [32,33]. Porous chitosan structures can be formed by freezing and lyophilizing
chitosan solutions in suitable molds. The mechanical
properties of chitosan scaffolds depend largely on the
pore sizes and pore orientations [34]. Such scaffolds
can enhance bone repair by supporting the proliferation of osteoblastic cells as well as their differentiation. Chitosan and its derivatives have been reported
to have osteogenic activity and promote bone mineralization on artificially made bone defects in animal
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models [35 –37]. In a study investigating the bone
healing property of chitosan in dogs, the time required
for bone repair was shorter by 1 week when compared
to controls without chitosan, resulting in reduced
animal stress and medical cost [38].
Recently, it has been shown that the chitosan
solution injected into the knee articular cavity of rats
causes a significant increase in articular cartilage
chondrocyte density, suggesting that chitosan could
act on the growth of epiphyseal cartilage and wound
healing of articular cartilage [39]. These biochemical
and morphological results coupled with favorable
biological properties of chitosan indicate that chitosan
is a promising scaffold for cartilage tissue engineering. It is also very important that the chitosan scaffold
possesses the necessary mechanical properties in order
to provide seeded chondrocytes with a proper biomechanical environment. Development of a chitosanbased material that can support chondrogenesis may
be significant not only in terms of the quality of
neocartilage produced, but also in terms of the ability
of that tissue to integrate with the host matrix.
Combination of chitosan-based cartilage scaffolds
with gene therapy and the use of growth factors
may improve the long-term outcomes of cartilage
repair in clinical settings [6].
3.1.4. Analgesia
The analgesic effects of chitin and chitosan on
inflammatory pain were evaluated using the acetic
acid-induced writhing test in mice [40]. Following
intraperitoneal administration of chitin and chitosan
solutions prepared in acetic acid solution, a dosedependent decrease in the number of the abnormal
behaviors (writhing) due to pain, including extension
of the hind legs, abdominal rigidity, and abdominal
torsion was observed. This effect was greater in the
animals administered the chitosan than those administered the chitin. The level of bradykinin in the
peritoneal lavage fluid was lower in the animals
administered chitin than those receiving chitosan.
The chitin particles absorbed bradykinin more extensively than chitosan. The main analgesic effect of
chitosan was suggested to be due to the absorption of
protons released in the inflammatory site, while that of
chitin was due to the absorption of bradykinin.
Chitosan and N-O-carboxymethyl chitosan incorporated with morphine have been used to develop
a product to provide 8– 12 h of effective analgesia
and which is inexpensive, non-irritating at the site
of injection and devoid of side effects other than
those normally associated with morphine. The use
of opioids for the provision of pain relief in
animals is common in both veterinary practice
and laboratory animal medicine. Most opioid drugs
have a relatively short half-life and duration of
action in most companion animal species, necessitating multiple injections at 4 – 6-h intervals if
adequate analgesia is to be maintained. Although
not being directly used for its analgesic effect,
chitosan as a gel matrix was demonstrated to
provide more prolonged absorption, lower maximal
serum concentrations and more sustained serum
morphine concentrations than conventional morphine sulfate solutions [41].
In Table 2, the recent studies carried out on
applications of chitosan for therapy in veterinary
medicine are summarized [24,26,35 – 40,42 – 45].
However, there are some reported complications of
chitosan applications. It is reported to cause lethal
hemorrhagic pneumonia in dogs, following a subcutaneous high dose of chitosan [46]. In spite of
application of chitosan to various species, this finding was observed only in dogs. Secondly, intratumor
injection of chitosan on mice bearing tumors was
found to increase the rate of metastasis and tumor
growth. The development of excessive granulation
tissue and accumulation of exudates was also encountered when large amounts of chitin and chitosan
were administered in subcutaneous tissue [47]. Suitable levels of chitin and chitosan for organization of
the implants in the rat were found to be 1 –10 and
0.1 –1 mg/ml, respectively. Therefore, it is important
to consider these effects of chitosan prior to drug
delivery.
3.2. Drug delivery
Chitosan is not currently used as a drug delivery
system for veterinary species but its characteristics
briefly described above suggest that it has considerable potential for such an application, although there
will be restrictions imposed by animal restraint considerations, especially in farming applications. The
veterinary areas most likely to benefit from chitosan
are discussed in this section.
S. Sßenel, S.J. McClure / Advanced Drug Delivery Reviews 56 (2004) 1467–1480
3.2.1. Delivery of chemotherapeutics
There is a need for delivery of chemotherapeutics
such as antibiotics, antiparasitics, anaesthetics, painkillers and growth promotants to mucosal epithelium
for absorption for local or systemic activity. Potential
sites for application are buccal, nasal, conjunctival,
mammary, vaginal and large intestinal mucosae. Oral
administration intended for intestinal absorption may
be possible, but would require formulation to cope
with dilution and degradation in the stomach(s); this
is particularly complex for ruminants where the physiological conditions of each of the stomachs are
different, and the system is designed for microbial
degradation of ingested substances [48]. Should chitosan be shown to enhance permeability of skin
stratum corneum and epithelium, there would also be
potential application to the transdermal delivery of
drugs by backline application. A potential role has
been identified in companion animals for delivery of
chemotherapeutics to oral mucosal epithelium for
local effect [49] and in ruminants for systemic regulation of reproduction [50] and to mammary mucosal
epithelium for local effect [51]. In the future, when
interfering RNA (RNAi) becomes a useful tool, chitosan may permit its mucosal delivery for local
activity.
3.2.2. Delivery of immunomodulatory agents
Chitosan may provide for delivery of immunomodulatory agents such as vaccines, adjuvants and
anti-inflammatory agents in veterinary medicine.
There is a growing need, particularly in livestock
industries, for effective mucosal immunomodulation
as a sustainable disease control alternative to chemotherapeutics which are becoming less effective because of pathogen resistance or less acceptable
because of environmental concerns. The target sites
would include follicular associated epithelium and M
cells of Peyer’s patches, and possibly also the
absorptive epithelium, not associated with organized
lymphoid tissue, of the small and large intestines and
epithelium of respiratory tract, mouth, eye and vagina. The aim would be to deliver the agents to the
mucosal associated lymphoid tissue for induction or
modulation of local immune responses. The targets
of such vaccines would include bacterial, viral and
parasitic pathogens, reproductive hormones and
ingested plant toxins, for the induction of either
1475
immune responsiveness or tolerance. Bowersock
and Martin [52] identified the following issues as
needing to be addressed by future veterinary vaccine
formulation and delivery:
Stimulation of immunity at mucosal surfaces. This
is highly desirable, as most pathogens enter by this
route, but the practical difficulties involved mean
that it is yet to be achieved. One difficulty is that
systemic immune responses are not necessarily
effective at mucosal sites, and systemic vaccination
usually produces poor mucosal immune responses.
Local delivery may therefore be required, however,
oral delivery for intestinal uptake has inherent
problems such as enzymic and bacterial degradation and dilution in the stomach(s) and intestine,
and interference by pH and mucus. There is thus a
need for practical and effective delivery of
inactivated vaccines so as to induce protective
mucosal immune responses.
Oral alternatives to systemic vaccines, in order to
avoid hide and carcass damage, and restraint and
handling difficulties. Oral vaccines for systemic
effect would also be useful for feral animal control,
in terms of reproduction, zoonotic disease, and
disease transmissible to domestic livestock and
companion animals. They may also permit priming
of the neonate immune system in the presence of
maternal antibodies [53]. In order to generate
systemic immunity, the target pathway of antigen
uptake and processing may differ from that desired
for local immunity.
Controlled release of inactivated vaccines. Given
the cost of individual vaccination, especially in
livestock species, there would be potential application for single administration biocompatible
implants or devices, which were long-acting or
self-boosting.
The desired destination of the drug or vaccine
will influence the mechanism of uptake required and
therefore the formulation of the delivery system.
Drugs for local or systemic effect are likely to be
less specific in their transport requirements, and
where drug uptake depends on permeability of the
intercellular tight junctions site and type of epithelium may be unimportant. In laboratory species,
chitosan particles have been shown to be an effective
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delivery system for the transport of a range of
macromolecules (including peptides and DNA)
across nasal, tracheal, oral and ocular epithelium
[54,55]. Chitosan solutions, powder and nanoparticles have enhanced absorption across nasal mucosa,
enhanced efficacy and prolonged action time of
drugs [56 – 59]. However, vaccines will need to be
delivered to appropriate lymphoid tissues.
may therefore differ with the solubility of the antigen
[60].
Thus, a mucosal delivery system for drugs or
vaccines should provide protected delivery, delivery
to a specific target site, optimal uptake by the target
route, and for vaccines, appropriate immunomodulation. Chitosan has a number of properties which can
address these requirements.
Mucosal associated lymphoid tissues (MALT)
The anatomy and physiology of the mucosal immune system of the veterinary species has been
comprehensively reviewed [60]. These species have
a common mucosal immune system in that cells
primed at one mucosal site recirculate to other mucosal sites. In general, these species have both diffuse
lymphoid cells, especially in the lamina propria of the
small intestine, and organized lymphoid tissue associated with conjunctiva, bronchi, small and large
intestines. This organized tissue may be solitary
nodules, often reactive rather than constitutive, or
complex constitutive tissues such as tonsils, Peyer’s
patches and lymph nodes. Immune responses to
foreign antigens are initiated in these organized tissues. Peyer’s patches have specialized lympho-epithelium which includes M (microfold or membranous)
cells with a capacity for active particle uptake, the
subsequent processing of which differs between ileal
and jejunal patches [61]. Fish are an exception in that
they lack Peyer’s patches, the skin functions as a
mucosal barrier and skin, gill and hindgut are all
capable of macromolecular uptake.
Uptake of antigen by M cells is dependant on
particle size, surface hydrophobicity and targeting
ligands [62,63] and on the physiological characteristics of the M cells [64]. Thus microparticles may
enhance the uptake of vaccines by M cells and
therefore target them more effectively to lymphoid
sites of immune induction [56,57,65]. Although
microparticles are not commonly used in veterinary
vaccines, a number have been investigated for veterinary applications [52]. Soluble antigens are thought
to be transported through and between the absorptive
epithelia of the gastrointestinal tract to local dendritic
antigen presenting cells thus reaching the draining
lymph nodes, or to liver. The site and type of immune
response generated by mucosally delivered antigens
Mucoadhesion. Adherence of macromolecules to
mucosal surfaces has been shown for various
mucosae and may permit enhanced or prolonged
drug absorption [66,67]. This would address the
problems of washout and dilution effects, and
permit maximal availability to the mucosal epithelium. In the case of chitosan, this adherence is
generally the result of physicochemical interactions, and is thus not limited to specific ligands
[68 –70], but specific glycosylation can result in
binding to specific lectins on epithelial cells [71].
Rapid turnover of gastrointestinal mucus may still
be a problem.
Enhanced mucosal epithelial permeability. Chitosan has been shown to enhance permeability of
small polar molecules and peptide/protein drugs
through nasal mucosa in animal models and human
volunteers [72]. Other studies have shown an
enhancing effect on penetration of compounds
across the intestinal mucosa, buccal mucosa and
cultured Caco-2 cells [73 – 76]. Several mechanisms have been proposed such as increasing cell
permeability by opening of intercellular tight
junctions, and favoring paracellular and intracellular transport.
Capacity to customize particle size, protection,
release rates, administration formats and biodegradability. A number of microparticular delivery
systems have been used to administer drugs and
vaccines via mucosal routes in order to make use of
the characteristics of the macromolecular uptake by
epithelial cells of MALT [62]. The size of particles
influences susceptibility to gut washout and transepithelial uptake, with nanospheres more readily
taken up than microspheres [70]. There is currently
a great interest in the use of biodegradable
polymers for the delivery of vaccines, both
parenterally and to mucosal surfaces. Entrapment
of antigens in particles prepared from biodegrad-
S. Sßenel, S.J. McClure / Advanced Drug Delivery Reviews 56 (2004) 1467–1480
able polymers such as copolymers of polylactide
and polyglycolide esters, and alginates for mucosal
immunization, have given successful outcomes in
animals [52]. Chitosan can be formulated as microor nano-particles and chitosan microparticles have
been shown to be taken up by epithelium of murine
Peyer’s patches [77]. Chitosan can also be
protected from degradation by glutaraldehyde or
formaldehyde cross-linking, although this reduces
its mucoadhesive properties [78,79]. Release rates
from chitosan formulations can be varied by
altering the chitosan properties and the initial drug
concentration [80].
Adjuvant effects. Chitosan for mucosal vaccination
has been reported to enhance local and systemic
immune responses. Possible mechanisms suggested have been induction of cytokine release by
epithelial cells, shifting of the Th1/Th2 balance,
activation of macrophages, or simply by increased
absorption of antigen [56,57,81]. Chitosan has
been shown to enhance local and systemic immune
responses to influenza, tetanus toxoid, diphtheria
and pertussis vaccines when delivered intranasally
[82 – 85], and to DNA when delivered orally [86] to
laboratory species. Chitosan given in an emulsion
also enhanced both T and B lymphocyte responses
to the poorly immunogenic hhCG delivered by
intraperitoneal injection [87]. Subcutaneous implantation of chitosan alone in dogs increased the
number of white blood cells, particularly neutrophils and activated both polymorphonuclear cells
and macrophages, suggesting that chitosan may be
useful as an immunopotentiator for prevention of
immunosuppression as well as enhancement of
vaccination [44].
4. Conclusions and future directions
Chitosan, which is a biodegradable, non-toxic
biopolymer, is currently being used for therapy in
veterinary medicine due to its various biological
properties. It is capable of activating host defenses
to prevent infection and accelerate wound healing as
well as repairing the tissues. Studies are continuing on
new derivatives of chitosan, which would not only
exert new biological activities but also provide a
better form for application to the site of effect. Little
1477
work has been published with chitosan for veterinary
drug or vaccine delivery systems. Given the potential
usefulness of this system, studies in livestock and
companion animals are required. The most rewarding
applications would appear to be for mucosally effective vaccines, oral vaccines for control of disease and
reproduction in feral animals and possibly for sustained delivery of low doses of drugs such as hormones. Future studies need to include the stability of
drugs and vaccines in chitosan formulations, and
stability of the complex in the presence of gut bacteria; the optimal epithelial site of uptake for a given
purpose; the optimal particle size for uptake by the
desired cell type; and the requirement for bolus versus
sustained delivery of mucosal vaccines.
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