2
Chitosan-based Drug Delivery Systems
2.1 Introduction
The most important characteristics of chitosans, which make them useful as delivery
vectors for drugs and therapeutic proteins, are their excellent mucoadhesive and
absorption enhancing properties [1-3]. The mucoadhesion is due to the electrostatic
interaction between the positive charge on the chitosan polymer and the negatively
charged sialic acid residues on the mucosal surface [4]. Because of the bioadhesive
characteristics of chitosan, the rate of clearance of drugs from the nasal cavity and
thereby the bioavailability increases [5]. In addition, chitosan is biocompatible with living
tissues and it possesses antimicrobial properties. It absorbs toxic metals such as cadmium,
lead, mercury and so on from the body. Chitosan is biodegradable and it degrades under
the action of human enzymes, especially lysozymes, into harmless products that are
completely absorbed by the human body [6]. Chitosan also has good adhesion,
coagulation ability and immuno-stimulating activity. Other interesting biopharmaceutical
characteristics of chitosan are its ability to control the release of active agents, the
availability of primary amino groups for modification, and the use of aqueous acidic
solvents instead of the use of hazardous organic solvents while fabricating particles.
Chitosan in clinical applications can be sterilised by methods such as ionising radiation,
heat, steam and chemical methods.
Different types of chitosan-based drug delivery systems are currently in use. The drugs
that are delivered as directly compressed tablets are diclofenac sodium, pentoxyphylline,
salicylic acid and theophylline propranolol HCl [7–9]. Drugs such as insulin, 5-amino
salicylic acid, and so on, can be delivered via chitosan capsules. Other delivery systems
include nano/microparticles, beads, films and gels that can deliver DNA, doxorubicin,
riboflavin, isosorbide dinitrate, aspirin, and so on. Micro/nanoparticle-based drug
delivery systems offer numerous advantages over the conventional delivery systems. This
includes improved efficacy, reduced toxicity and improved patient compliance [5, 10].
The following section discusses the various methods of preparation of chitosan-based
micro/nanoparticles.
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Update on Chitosan
2.2 Chitosan Micro/Nanoparticle-based Drug Delivery System
Micro/nanoparticle-based delivery systems can be designed carefully to achieve the
desired result by tailoring the drug encapsulation technologies, varying the copolymer
ratio, molecular weight of polymer and so on. Such a tailor-made delivery system may
increase the life span of active constituents and control the release of bioactive agents.
Chitosan-based particulate delivery systems are used for improving the bioavailability of
biodegradable drugs such as proteins or to enhance the uptake of hydrophilic substances
across the epithelial layers. The large surface-to-volume ratio of the micro/nanospheres is
an advantage for the controlled release of insoluble drugs [11].
2.3 Preparation of Chitosan Micro/Nanoparticles
There are different methods for the preparation of chitosan micro/nanoparticles. The
selection of which one to use is dependent upon the type of delivery device or the active
molecule to be delivered.
2.3.1 Emulsion Crosslinking Method
In this method the reactive amino groups of the chitosan are crosslinked with an aldehyde
group of the crosslinking agent, such as guteraldehyde, formaldehyde or genipin. Briefly,
water in an oil emulsion is prepared by emulsifying an aqueous solution of chitosan in an
oil phase. Aqueous droplets are stabilised using a suitable surfactant. It is further rigged
by crosslinking [12]. The hardened microspheres are filtered, washed with suitable
solvents and dried. It has been reported that the entrapment efficiency of water-insoluble
drugs can be increased by a multiple emulsion method. This method involves the
formation of a primary oil in water emulsion, and the addition of a primary emulsion to
an external oily phase to form an oil/water/oil emulsion followed by crosslinking and
evaporation of an organic solvent [13, 14]. The molecular weight and concentration of
chitosan, concentration of stabilising agent, preparation conditions and so on, will affect
the particle size and performance of the microspheres.
Denkbas and co-workers reported that smaller microspheres with narrow distributions
can be prepared by decreasing the chitosan/solvent ratio and the drug/chitosan ratio [15].
They reported the loading of 5-fluorouracil up to a concentration of 10.4 mg/g of
chitosan. However, it was reported that the drug release rate was faster from small size
microspheres compared to large size microspheres. For example Al-Helw reported that
almost 75–95% of the drug was released within three hours depending upon the
molecular weight of chitosan [16]. In order to control the release rate of the drug, Jameela
6
Chitosan-based Drug Delivery Systems
and co-workers prepared smooth, highly spherical, crosslinked chitosan microspheres in
the size range of 45 to 300 µm to release progesterone [17]. They reported that the extent
of crosslinking has a significant influence on drug release characteristics. Highly
crosslinked microspheres released only about 35% of the steroid in 40 days compared to
70% release from the lightly crosslinked microspheres. The in vivo bioavailability of the
drug by intramuscular injection in rabbits showed that a plasma concentration of 1–2
ng/ml was maintained for up to five months without any high burst release effect [17]. It
was also reported that ascorbylpalmitate crosslinked chitosan via the covalent bond with
the amino groups showed microparticles with high loading efficiency and drug release at
a constant rate up to 80 hours [18].
2.3.2 Complex Coacervation
In this method, microparticles are formed by interionic interaction between oppositely
charged polymers. Particles are produced by blowing a chitosan solution into an alkaline
solution through a compressed air nozzle to form coacervate droplets [19]. The
microparticles obtained are hardened in the counter ion solution before washing and
drying. The size and drug release rate of the particles can be controlled by varying the
compressed air pressure or spray-nozzle diameter and the type of crosslinking agent,
respectively. A complex coacervation technique can be used to prepare chitosan-DNA
nanoparticles [20]. Mao and co-workers investigated the properties of the particles by
varying the concentration of DNA, chitosan and sodium sulfate and by changing the
temperature and pH of the buffer and the molecular weight of chitosan, etc. The
nanoparticles of 100–250nm with narrow molecular weight distribution were obtained at
the amino-to-phosphate (N:P) ratio of 3:8 and concentration of 100 µg/ml of chitosan
[20].
2.3.3 Spray Drying
Chitosan microparticles loaded with drugs can also be prepared from a mixture of
chitosan/drug solutions or suspensions by a spray drying method. In this method, chitosan
is first dissolved in an aqueous acetic acid solution; a drug is then dissolved or dispersed
in the solution followed by the addition of a suitable crosslinking agent. The solution of
dispersion is atomised in a stream of hot air. Atomisation leads to the formation of small
droplets, from which the solvent evaporates instantaneously leading to the formation of
free flowing particles [21]. The size of the particles formed depends upon the size of the
nozzle, the spray flow rate, the atomisation pressure, the inlet air temperature and the
extent of crosslinking. He and co-workers prepared both crosslinked and uncrosslinked
chitosan microparticles for the delivery of cimetidine, nizatidine and famotidine by spray
7
Update on Chitosan
drying of multiple emulsions [21]. The particle size and zeta potential of the
microspheres were influenced by the extent of crosslinking. Highly crosslinked particles
showed low particle size. Similarly, particle size can also be controlled by airflow rate.
Large sized particles were obtained by increasing the flow rate by using a large size
nozzle and smaller particles were produced by increasing the airflow rate. The particles
obtained by this method are spherical in shape with smooth surfaces and narrow size
distributions.
The drug release profile, microsphere properties, entrapment efficiency and so on, can be
controlled by using modifiers such as gelatine. For example, Huang and co-workers
prepared chitosan microspheres by the spray drying method using type-A gelatine and
ethylene oxide-propylene oxide block copolymer as modifiers [22]. Betamethasone
disodium phosphate-loaded microspheres demonstrated a good drug stability, high
entrapment efficiency and prolonged release up to 12 hours in the presence of a gelatine
modifier.
2.3.4 Ionic Gelation
This method involves the complexation between oppositely charged macromolecules. It
was first introduced by Bodmeier and co-workers [23]. They reported the preparation of
tripolyphosphate (TPP)-chitosan complex by dropping chitosan droplets into TPP
solution. Because of the physical crosslinking between the oppositely charged polymers
by electrostatic interaction, chemical crosslinking by toxic reagents can be avoided.
However because of the poor mechanical strength, their use is limited in drug delivery.
Urrusuno and co-workers prepared insulin-loaded chitosan nanoparticles by varying the
molecular weight and degree of deacetylation of chitosan [24, 25]. They observed that the
efficiency of the drug loading was dependent on the molecular weight and degree of
deacetylation of chitosan. Xu and co-workers reported that increasing the deacetylation
degree from 75.5% to 92% promoted the encapsulation efficiency [26]. They also found
that as the molecular weight increased from 10 to 210 kDa, the encapsulation efficiency
was almost doubled [26]. The amount of drug released from the chitosan-TPP
nanoparticles depended on the pH of the TPP solution, the molecular weight of chitosan,
the concentration of the materials and also by the crosslinking time. According to Xu and
co-workers, the total release of bovine serum albumin in phosphate buffered saline at pH
7.4 in eight days was reduced from 73.9% to 17.6% when the molecular weight was
increased from 10 to 210 kDa [26]. Ko and co-workers reported that the release of the
drug felodipine decreased as the pH decreased and concentration of the TPP solution
increased [27]. Again, the drug release was increased by decreasing the molecular weight
and concentration of chitosan solution. They also found that the release of the drug from
TPP-chitosan microparticles decreased when the crosslinking time was increased.
8
Chitosan-based Drug Delivery Systems
2.3.5 Reverse Micellar Method
In the reverse micellar method, the surfactant is dissolved in an organic solvent and the
drug is solubilised in an aqueous solution of chitosan. The aqueous solution is added to
organic solution and mixed by vortexing until the entire mixture is in an optically
transparent micro emulsion phase. The mixture is crosslinked by adding a crosslinking
agent and stirring overnight. The particle size can be increased by adding more water in
the aqueous phase. The organic solvent is then evaporated and the obtained dry mass is
dispersed in water. The surfactant is removed by adding a suitable salt to precipitate it
out. The mixture is then centrifuged and re-suspended in water, dialysed through a
membrane for one hour and then lyophilised to dry powder.
2.4 Drug Loading in Chitosan Micro/Nanoparticles
The loading efficiency of drugs in the chitosan micro/nanoparticles may be affected by
various factors such as chitosan concentration, physico-chemical properties of the drug,
methods of preparation, drug polymer ratio, presence of additives, stirring speed and so
on. Drug loading into the nano/microparticles can be achieved in two ways, the
incorporation method or the incubation method. In the incorporation method, the drug is
physically incorporated into the matrix during the preparation of the particles. In the
incubation method, the drug is adsorbed onto the surface after the formation of particles.
It has been observed that the incorporation method is more efficient than the incubation
method with respect to loading [28, 29]. Water-soluble drugs can be loaded into the
particle by the incorporation method, i.e., the drug can be made water soluble and
homogenised with a chitosan solution. From the mixture, particles can be prepared by any
of the methods. In this case, the entrapment efficiency increases with increase in chitosan
concentration. For example, Nishioka and co-workers have reported that loading
efficiency of cisplatin is increased with concentration of chitosan and that it had no effect
on the concentration of cisplatin or the volume of crosslinking agent glutaraldehyde [30].
This is due to the increase in the viscosity of the chitosan solution, which prevents drug
crystals from leaving the droplet. Water insoluble drugs and the drugs which precipitate
at an acidic pH can be loaded by the incubation method by soaking the preformed
particles with the saturated solution of the drug and also by the multiple emulsion
method. Kumbar and co-workers reported that diclofenac sodium, which is precipitated in
acidic pH conditions, has been loaded by the socking method [31]. In this method,
loading efficiency depends upon the swelling of the particles in water. Increase in
crosslinking decreases the swelling and therefore the drug loading efficiency. In the
multiple emulsion method, an aqueous solution of chitosan is emulsified with the solution
of the drug in suitable solvent to form an oil-in-water type emulsion. A drug can be
entrapped by a multiple emulsion method like oil-water-oil. In this method, the drug is
9
Update on Chitosan
first dispersed into a chitosan solution by using a surfactant to get the suspension of oil in
water. This emulsion can further be emulsified into liquid paraffin to get an oil-water-oil
emulsion. The resulting droplets can be hardened by a suitable crosslinking agent [31].
2.5 Pharmaceutical Applications of Chitosan
In addition to its distinctive physicochemical properties chitosan is also a bioactive agent,
which makes it attractive in pharmaceutical and biomedical applications. Depending on
their size, surface charge, hydrophilic/lipophilic balance and specific transmembrane
ability, a polymeric drug carrier can change pharmacological and immunological activity
of drugs and their delivery. The following section illustrates the unique features of
chitosan as a pharmacologically active delivery system.
2.5.1 Antibacterial Activity of Chitosan
At an acidic pH, chitosan possesses antimicrobial activity against many bacteria and
fungi. One of the suggested mechanisms behind this property is that the positive amino
group of the glucosamine units interacts with negative charged components in microbial
cell membranes and alters their barrier properties, thereby preventing the entry of
nutrients or causing the leakage of intracellular contents [32]. This eventually leads to the
cellular breakup of the bacteria or fungi [33, 34]. Another mechanism suggested is that
chitosan penetrates into the cell and binds to the DNA of the bacteria and subsequently
inhibits protein synthesis [35]. Factors such as molecular weight, degree of deacetylation,
pH of the chitosan solution, position of glucosamine unit and so on, all influence the
biological activity of chitosan [36]. It has been reported that high molecular weight
chitosan inhibits Escherichia coli’s activity almost completely by stacking outside the
cell and inhibits the permeation of nutrition in comparison to low molecular weight
chitosan.
Since solubility of chitosan depends on pH, the antimicrobial activity is also influenced
by pH. However, modification of chitosan such as quarternisation and hydrophilic
substitution improves the solubility. N,N,N-trimethyl chitosan prepared by methylating
the primary amino group of chitosan using methyl iodide in the presence of sodium
hydroxide as a base and N-methyl-2-pyrrolidone as a solvent is the most promising
chitosan derivative [37]. Since this derivative is soluble in lower sections of the
gastrointestinal tract, it can act as an absorption enhancer. The quaternary ammonium
chitosan prepared by the introduction of a quaternary ammonium group on the
dissociative hydroxyl group and amino group also shows good solubility and inhibition
effects against E. coli. [38].
10
Chitosan-based Drug Delivery Systems
Chitosan derivatives such as poly(N-vinylimidazole) grafted chitosan [39], guanidylated
chitosan [40], galactosylated chitosan [41] and disaccharide conjugated chitosan [42]
were found to show better antibacterial activity against bacteria such as Gram positive
and Gram negative, Staphylococcus aureus and Bacillus subtilis, E. coli and Pseudomanas aeruginosa. All these derivatives exhibit higher activity than native unmodified
chitosan at pH 7.0 [42]. Silver is a well-known antimicrobial agent but the complex of
chitosan with Ag+ is unstable. Therefore silver ion derivatives of chitosan were prepared
by the reaction of chitosan with ammonium thiocynate in ethanol. Thiourea chitosan-Ag+
complex improves the instability of silver as well, as it shows better antibacterial activity
[43]. Chitosan/alginate sponges can be used as wound healing agents for severe burns,
trauma, diabetic and venous stasis ulcers, and similar tissue damage. Chitosan accelerates
the healing process by improving fibroblast growth and affects macrophage activity [44].
The wound healing process can be further improved by loading antibacterial agents such
as ciprofloxacin [44], norfloxacin [45] and gatifloxacin [46] into the sponge.
2.5.2 Antioxidant Activity of Chitosan
Antioxidants are compounds that protect the body against oxidative damage by inhibiting
or delaying the oxidation of cellular oxidisable substrates caused by reactive oxygen
species (ROS) and degenerative diseases. Superoxide anion (-O2-), hydroxyl radical (OH) and hydrogen peroxide are the ROS produced by sunlight, ultraviolet light, ionising
radiation, chemical reactions and metabolic processes which have a wide variety of
pathological effects such as stroke, cancer, diabetes, atherosclerosis and cardiovascular
disease. Low molecular weight, partly deacetylated chitosan exhibits antioxidant
properties and can be considered as a natural antioxidant [36]. Peng and co-workers
reported that chitosan inhibits the activity of linolenic acid peroxidation by 83.7% against
hydroxyl radicals [47]. It was observed that sulfated and sulfanilamide derivatives of
chitosan, having various molecular weights, increase the antioxidant and scavenging
activities [48, 49]. From the range of derivatives of chitosan, it was found that the major
factor for free radical scavenging activity is the amino group [50]. Among the four forms
of amino groups, the primary amino group, imino group, secondary amino group and the
quaternised amino group, the latter showed the highest antioxidant activity against
hydroxyl radicals. Therefore it can be concluded that high positive charge density
increases antioxidant activity [51].
2.5.3 Antitumour Activity of Chitosan
Decreasing the molecular weight of chitosan causes an increase in water solubility,
transformation of crystal structure and alteration of thermostability without change in
11
Update on Chitosan
chemical structure. This improves the antitumour activity of chitosan. In order to
elucidate the structural features of low molecular weight chitosan and its effect in
inhibiting angiogenesis, Prashanth and co-workers injected chitooligosaccharides intraperitoneally into mice [51]. They observed apoptosis in Ehrlich ascites tumour and over
90% decrease in the ascites volume. Copper complexes of chitosan at the ratio of 0.11
mol copper per one chitosan residue, induces cleavage of DNA and arrested the cell cycle
progression in tumour cell lines such as 293 and HeLa. But normal human lung fibroblast
cell lines were not affected by the complex [52]. Water soluble chitosan derivatives were
also used as antitumour drug or gene delivery vectors [53-55]. This will be discussed in
later chapters.
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