www.elsevier.com/locate/jconrel

Journal of Controlled Rele

Review

Recent advances on chitosan-based micro- and nanoparticles

in drug deliveryB

Sunil A. Agnihotri, Nadagouda N. Mallikarjuna, Tejraj M. Aminabhavi*

Drug Delivery Division, Center of Excellence in Polymer Science, Karnatak University, Dharwad 580 003, India

Received 15 July 2004; accepted 12 August 2004

Abstract

Considerable research efforts have been directed towards developing safe and efficient chitosan-based particulate drug

delivery systems. The present review outlines the major new findings on the pharmaceutical applications of chitosan-based

micro/nanoparticulate drug delivery systems published over the past decade. Methods of their preparation, drug loading, release

characteristics, and applications are covered. Chemically modified chitosan or its derivatives used in drug delivery research are

discussed critically to evaluate the usefulness of these systems in delivering the bioactive molecules. From a literature survey, it

is realized that research activities on chitosan micro/nanoparticulate systems containing various drugs for different therapeutic

applications have increased at the rapid rate. Hence, the present review is timely.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Microparticles; Nanoparticles; Chitosan; Chemically modified chitosan; Drug delivery

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

2. Methods of preparation of micro/nanoparticles of chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

2.1. Emulsion cross-linking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

2.2. Coacervation/precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2.3. Spray-drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.4. Emulsion-droplet coalescence method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2.5. Ionic gelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2.6. Reverse micellar method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

2.7. Sieving method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

0168-3659/$ - s

doi:10.1016/j.jco

B This pape

* Correspon

E-mail addr

ase 100 (2004) 5–28

ee front matter D 2004 Elsevier B.V. All rights reserved.

nrel.2004.08.010

r is CEPS Communication # 23.

ding author. Tel.: +91 836 2779983; fax: +91 836 2771275.

ess: aminabhavi@yahoo.com (T.M. Aminabhavi).

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–286

3. Drug loading into micro/nanoparticles of chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

4. Drug release and release kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

5. Pharmaceutical applications of chitosan particulate systems . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

5.1. Colon targeted drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

5.2. Mucosal delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

5.3. Cancer therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

5.4. Gene delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

5.5. Topical delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

5.6. Ocular delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

5.7. Chitosan as a coating material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

6. Chemically modified chitosans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

1. Introduction

Chitosan (CS) is a polysaccharide, similar in

structure to cellulose. Both are made by linear h-(1Y4)-linked monosaccharides [see Fig. 1 (a)].

However, an important difference to cellulose is that

CS is composed of 2-amino-2-deoxy-h-d-glucancombined with glycosidic linkages. The primary

amine groups render special properties that make CS

very useful in pharmaceutical applications. Compared

to many other natural polymers, chitosan has a

positive charge and is mucoadhesive [1]. Therefore,

it is used extensively in drug delivery applications [2–

6]. Chitosan is obtained from the deacetylation of

chitin, a naturally occurring and abundantly available

(in marine crustaceans) biocompatible polysaccharide.

However, applications of chitin are limited compared

to CS because chitin is structurally similar to cellulose,

but chemically inert. Acetamide group of chitin can be

converted into amino group to give CS, which is

carried out by treating chitin with concentrated alkali

solution. Chitin and CS represent long-chain polymers

having molecular mass up to several million Daltons.

Chitosan is relatively reactive and can be produced in

various forms such as powder, paste, film, fiber, etc.

[7,8]. Commercially available CS has an average

molecular weight ranging between 3800 and 20,000

Daltons and is 66% to 95% deacetylated.

Chitosan, being a cationic polysaccharide in neutral

or basic pH conditions, contains free amino groups and

hence, is insoluble in water. In acidic pH, amino groups

can undergo protonation thus, making it soluble in

water. Solubility of CS depends upon the distribution of

free amino and N-acetyl groups [9]. Usually 1–3%

aqueous acetic acid solutions are used to solubilize CS.

Chitosan is biocompatible with living tissues since it

does not cause allergic reactions and rejection. It breaks

down slowly to harmless products (amino sugars),

which are completely absorbed by the human body

[10]. Chitosan degrades under the action of ferments, it

is nontoxic and easily removable from the organism

without causing concurrent side reactions. It possesses

antimicrobial property and absorbs toxic metals like

mercury, cadmium, lead, etc. In addition, it has good

adhesion, coagulation ability, and immunostimulating

activity.

If degree of deacetylation and molecular weight of

CS can be controlled, then it would be a material of

choice for developing micro/nanoparticles. Chitosan

has many advantages, particularly for developing

micro/nanoparticles. These include: its ability to

control the release of active agents, it avoids the use

of hazardous organic solvents while fabricating

particles since it is soluble in aqueous acidic solution,

it is a linear polyamine containing a number of free

amine groups that are readily available for cross-

linking, its cationic nature allows for ionic cross-

linking with multivalent anions, it has mucoadhesive

character, which increases residual time at the site of

absorption, and so on. Chitin and CS have very low

toxicity; LD50 of CS in laboratory mice is 16 g/kg

body weight, which is close to sugar or salt. Chitosan

is proven to be safe in rats up 10% in the diet [11].

Various sterilization methods such as ionizing radia-

tion, heat, steam and chemical methods can be

suitably adopted for sterilization of CS in clinical

Fig. 1. (a) Structure of chitosan [poly (h1– 4-d-glucosamine)]. (b)

Structure of cross-linked chitosan.

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–28 7

applications [12]. In view of the above-mentioned

properties, CS is extensively used in developing drug

delivery systems [7,8,13–18]. Particularly, CS has

Table 1

Chitosan-based drug delivery systems prepared by different methods for v

Type of system Method of preparation D

Tablets matrix d

coating p

Capsules capsule shell i

Microspheres/Microparticles emulsion cross-linking t

i

d

m

coacervation/precipitation p

spray-drying c

s

a

ionic gelation f

sieving method c

Nanoparticles emulsion-droplet coalescence g

coacervation/precipitation D

ionic gelation i

reverse micellar method d

Beads coacervation/precipitation a

s

Films solution casting i

g

r

Gel cross-linking c

l

been used in the preparation of mucoadhesive

formulations [19–22], improving the dissolution rate

of the poorly soluble drugs [14,23,24], drug targeting

[25,26] and enhancement of peptide absorption

[20,21,27].

Many reports are available on the preparation of CS

microspheres [23,25,26,28,29]. Many methods used in

the development of microparticulate polymeric drug

delivery devices can also be used to prepare CS

microspheres [30–35]. Dodane and Vilivalam [3]

reviewed new approaches on pharmaceutical applica-

tions of CS and discussed its mechanisms of action in

various in vitro and in vivo models. Recent reviews

[36,37] addressed the issues on biomedical, pharma-

ceutical and biological aspects of chitin, CS and their

derivatives. Chitosan and its derivatives as a non-viral

vector for gene delivery [38] and CS-based gastro-

intestinal delivery systems [39] have been discussed.

The recent review by Sinha et al. [40] covers various

methods of preparation and evaluation of CS micro-

spheres, but no attempt has been made to discuss

nanoparticulate CS systems. Different types of CS-

based drug delivery systems are summarized in Table 1.

arious kinds of drugs

rug

iclofenac sodium, pentoxyphylline, salicylic acid, theophylline

ropranolol HCl

nsulin, 5-amino salicylic acid

heophylline, cisplatin, pentazocine, phenobarbitone, theophylline,

nsulin, 5-fluorouracil, diclofenac sodium, griseofulvin, aspirin,

iphtheria toxoid, pamidronate, suberoylbisphosphonate,

itoxantrone, progesterone

rednisolone, interleukin-2, propranolol-HCl

imetidine, famotidine, nizatidine, vitamin D-2, diclofenac

odium, ketoprofen, metoclopramide-HCl, bovine serum albumin,

mpicillin, cetylpyridinium chloride, oxytetracycline, betamethasone

elodipine

lozapine

adopentetic acid

NA, doxorubicin

nsulin, ricin, bovine serum albumin, cyclosporin A

oxorubicin

driamycin, nifedipine, bovine serum albumin, salbutamol

ulfate, lidocaine–HCl, riboflavin

sosorbide dinitrate, chlorhexidine gluconate, trypsin,

ranulocyte-macrophage colony-stimulating factor, acyclovir,

iboflavine, testosterone, progesterone, beta-oestradiol

hlorpheniramine maleate, aspirin, theophylline, caffeine,

idocaine–HCl, hydrocortisone acetate, 5-fluorouracil

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–288

However, the micro/nanoparticulate drug delivery

systems offer numerous advantages over the conven-

tional dosage forms. These include improved efficacy,

reduced toxicity and improved patient compliance

[35,41–43]. The present review addresses the recent

trends in the area of micro/nanoparticulate CS-based

drug delivery systems. Literature of the past decade has

been covered and results are critically evaluated.

Fig. 2. Schematic representation of preparation of chitosan

particulate systems by emulsion cross-linking method.

2. Methods of preparation of micro/nanoparticles

of chitosan

Different methods have been used to prepare CS

particulate systems. Selection of any of the methods

depends upon factors such as particle size require-

ment, thermal and chemical stability of the active

agent, reproducibility of the release kinetic profiles,

stability of the final product and residual toxicity

associated with the final product. Different methods

used in the preparation of CS micro/nanoparticles are

discussed in this review. However, selection of any of

these methods depends upon the nature of the active

molecule as well as the type of the delivery device.

Since we are concerned only with the micro/nano-

particulate systems of CS and its derivatives, we will

restrict our discussions only on these aspects.

2.1. Emulsion cross-linking

This method utilizes the reactive functional amine

group of CS to cross-link with aldehyde groups of the

cross-linking agent (see Fig. 1b). In this method, a

water-in-oil (w/o) emulsion is prepared by emulsify-

ing the CS aqueous solution in the oil phase. Aqueous

droplets are stabilized using a suitable surfactant. The

stable emulsion is cross-linked by using an appro-

priate cross-linking agent such as glutaraldehyde to

harden the droplets. Microspheres are filtered and

washed repeatedly with n-hexane followed by alcohol

and then dried [44]. By this method, size of the

particles can be controlled by controlling the size of

aqueous droplets. However, the particle size of final

product depends upon the extent of cross-linking

agent used while hardening in addition to speed of

stirring during the formation of emulsion. This

method is schematically represented in Fig. 2. The

emulsion cross-linking method has few drawbacks

since it involves tedious procedures as well as use of

harsh cross-linking agents, which might possibly

induce chemical reactions with the active agent.

However, complete removal of the un-reacted cross-

linking agent may be difficult in this process.

Recently, [33] we have used the emulsion cross-

linking method to prepare chitosan microspheres to

encapsulate diclofenac sodium using three cross-

linking agents viz, glutaraldehyde, sulfuric acid and

heat treatment. Microspheres were spherical with

smooth surfaces as shown in Fig. 3. The size of the

microparticles ranged between 40 and 230 Am.

Among the three cross-linking agents used, glutaral-

dehyde cross-linked microspheres showed the slowest

release rates while a quick release of diclofenac

sodium was observed by the heat cross-linked micro-

spheres. In our continuing study on CS-based

derivatives [34], we have also prepared the nifedi-

pine-loaded microspheres of polyacrylamide-g-chito-

san using three concentrations of glutaraldehyde as

the cross-linking agent. Microspheres were spherical

with the mean particle size of 450 Am.

Glutaraldehyde extracted in toluene was used as a

cross-linking agent by Al-Helw et al. [45] to prepare

CS microspheres encapsulated with phenobarbitone.

Uniform and spherical microspheres with loading

efficiency up to 57.2% were produced. Loading

efficiency was dependent upon the preparation

conditions. Parameters affecting the preparation and

performance of microspheres are molecular weight

Fig. 3. Scanning electron micrograph of chitosan microspheres

produced by emulsion cross-linking method.

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–28 9

and concentration of CS as well as concentration of

the stabilizing agent. Particle size of the microspheres

varied in the range 274–450 Am. Release rates of

phenobarbitone from different formulations of micro-

spheres showed high initial release (burst effect) of

the drug and about 20–30% of the drug was released

in the first hour. Release was faster from the small

size microspheres, i.e., almost 75–95% of the drug

was released within 3 h depending upon the

molecular weight of CS. Denkbas et al. [46] used

the mixture of mineral oil/petroleum ether in the ratio

of 60/40 (v/v) as the external medium to prepare CS

microspheres using glutaraldehyde as a cross-linking

agent and Tween-80 as an emulsifier. Smaller micro-

spheres with narrow distributions were produced

when CS/solvent ratio and drug/CS ratio were lower.

The 5-fluorouracil was loaded up to a concentration

of 10.4 mg/g of CS.

Thanoo et al. [29] prepared the CS microspheres

by emulsion cross-linking of CS solution in paraffin

oil as an external medium with glutaraldehyde using

dioctyl sulfosuccinate as the stabilizing agent. Addi-

tion of stabilizing agent during particle formation

produced microspheres with spherical geometry and

smooth surfaces. Encapsulation efficiencies up to 80%

were achieved for theophylline, aspirin or griseoful-

vin. These microspheres were used to study the drug

release rates, which were influenced by cross-link

density, particle size and initial drug loading. Sankar

et al. [47] prepared the CS-based pentazocine micro-

spheres for intranasal delivery. Formulation parame-

ters such as drug loading, polymer concentration,

stirring speed during cross-linking and oil phase were

altered to develop microspheres having good in vivo

performance. In vivo studies indicated a significantly

improved bioavailability of pentazocine. Application

of in vitro data to various kinetic models indicated that

these systems followed the diffusion controlled

release kinetics.

Jameela et al. [48] prepared smooth, highly

spherical, cross-linked CS microspheres in the size

range of 45–300 Am for the controlled release (CR) of

progesterone. An aqueous acetic acid dispersion of CS

containing progesterone was emulsified in the dis-

persion medium consisting of liquid paraffin and

petroleum ether stabilized by using sorbitan sesquio-

leate; droplets were hardened by glutaraldehyde cross-

linking. Extent of cross-linking showed a significant

influence on drug release characteristics. Highly

cross-linked microspheres released only about 35%

of steroid in 40 days compared to 70% release from

the lightly cross-linked microspheres. Evaluation of in

vivo bioavailability by intramuscular injection in

rabbits showed that a plasma concentration of 1 to 2

ng/mL was maintained up to 5 months without

showing any high burst release effect. These

data suggest the usefulness of cross-linked CS

microspheres as potential carriers for long-term

delivery of steroids. Bugamelli et al. [49] developed

insulin-loaded microparticles of CS by the interfacial

cross-linking in the presence of ascorbyl palmitate.

Disposition of ascorbyl palmitate at the water– oil

interface allowed the formation of covalent bond with

the amino groups of CS when its oxidation to

dehydroascorbyl palmitate took place during the

formation of microparticles. This method produced

microparticles with high loading efficiency and

released the drug at a constant rate up to 80 h.

2.2. Coacervation/precipitation

This method utilizes the physicochemical property

of CS since it is insoluble in alkaline pH medium, but

precipitates/coacervates when it comes in contact with

alkaline solution. Particles are produced by blowing

CS solution into an alkali solution like sodium

hydroxide, NaOH-methanol or ethanediamine using

a compressed air nozzle to form coacervate droplets

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–2810

[50]. Separation and purification of particles was done

by filtration/centrifugation followed by successive

washing with hot and cold water. The method is

schematically represented in Fig. 4. Varying com-

pressed air pressure or spray-nozzle diameter con-

trolled the size of the particles and then using a cross-

linking agent to harden particles can control the drug

release. In another technique [51], sodium sulfate

solution was added dropwise to an aqueous acidic

solution of CS containing a surfactant under stirring

and ultrasonication for 30 min. Microspheres were

purified by centrifugation and re-suspended in demin-

eralized water. Particles were cross-linked with

glutaraldehyde. Particles produced by this method

have better acid stability than observed by other

methods.

Chitosan microspheres loaded with recombinant

human interleukin-2 (rIL-2) have been prepared by

dropping of rIL-2 with sodium sulfate solution in

acidic CS solution [52]. When protein and sodium

sulfate solutions were added to CS solution and

during the precipitation of CS, the protein was

incorporated into microspheres. This method is

devoid of cross-linking agent. The rIL-2 was released

from microspheres in a sustained manner for up to 3

months. Efficacy of the systems developed was

studied by using two model cells viz., HeLa and L-

strain cell lines. Microspheres were taken up by the

cells and rIL-2 was released from the microspheres.

Fig. 4. Schematic representation of preparation of chitosan

particulate systems by coacervation/precipitation method.

Chitosan–DNA nanoparticles have been prepared

using the complex coacervation technique [53].

Important parameters such as concentrations of

DNA, CS, sodium sulfate, temperature, pH of the

buffer and molecular weights of CS and DNA have

been investigated. At the amino to phosphate group

ratio between 3 and 8 and CS concentration of 100 Ag/mL, the particle size was optimized to 100–250 nm

with a narrow distribution. Surface charge of these

particles was slightly positive with a zeta potential of

112 to 118 mV at pH lower than 6.0, and became

nearly neutral at pH 7.2. The chitosan–DNA nano-

particles could partially protect the encapsulated

plasmid DNA from nuclease degradation.

2.3. Spray-drying

Spray-drying is a well-known technique to pro-

duce powders, granules or agglomerates from the

mixture of drug and excipient solutions as well as

suspensions. The method is based on drying of

atomized droplets in a stream of hot air. In this

method, CS is first dissolved in aqueous acetic acid

solution, drug is then dissolved or dispersed in the

solution and then, a suitable cross-linking agent is

added. This solution or dispersion is then atomized

in a stream of hot air. Atomization leads to the

formation of small droplets, from which solvent

evaporates instantaneously leading to the formation

of free flowing particles [54] as depicted in Fig. 5.

Various process parameters are to be controlled to

get the desired size of particles. Particle size depends

upon the size of nozzle, spray flow rate, atomization

pressure, inlet air temperature and extent of cross-

linking.

He et al. [54] prepared both un-cross-linked and

cross-linked CS microparticles by spray-drying

method for the delivery of cimetidine, famotidine

and nizatidine. Microspheres were spherical with a

smooth and distorted morphology. Particle size of the

un-cross-linked microspheres varied between 4 and 5

Am, while cross-linked microspheres ranged from 2 to

10 Am; they were all positively charged. Particle size

and zeta potential were influenced by the extent of

cross-linking. A decrease in extent of cross-linking

increased both the particle size and the zeta potential.

Particle size was increased when the spray flow rate

was increased using the large size nozzle. Micro-

Fig. 5. Schematic representation of preparation of chitosan particulate systems by spray drying method.

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–28 11

spheres with smaller particle size were produced at

greater airflow rates. However, particle size was less

affected by the inlet air temperature between 140 and

180 8C. Conti et al. [55] produced microparticles by

exposing the spray-dried particles to vapors contain-

ing cross-linking agents. Cetylpyridinium chloride, an

anti-infective agent, was incorporated into CS micro-

spheres produced by spray-drying technique. Extent

of cross-linking was controlled by the time of

exposure to cross-linking agent.

Ganza-Gonzalez et al. [56] have demonstrated that

spray-drying technique is fast, simple and reliable to

obtain microspheres. Microspheres were prepared by

spray drying of aqueous CS dispersions containing

metoclopramide hydrochloride using different

amounts of formaldehyde as a cross-linker. Micro-

spheres released the drug for more than 8 h,

independent of the pH of the medium. In another

study [57], vitamin D2 (VD2), also called as ergo-

calciferol, was efficiently encapsulated into CS micro-

spheres prepared by spray-drying method. The

microencapsulated product was coated with ethyl

cellulose. The sustained release property of VD2

microspheres was used for the treatment of prostatic

disease [58]. Spray-drying method was also used to

prepare ampicillin-loaded methylpyrrolidone CS

microspheres [59] by taking different drug-to-polymer

weight ratios. Spray-dried microparticles were almost

spherical in shape with smooth surfaces and narrow-

size distributions.

Lorenzo-Lamosa et al. [60] prepared the micro-

encapsulated CS microspheres for colonic delivery of

sodium diclofenac. Sodium diclofenac was entrapped

into CS microcores by spray-drying and then, micro-

encapsulated into EudragitR L-100 and EudragitR S-

100 using an oil-in-oil solvent evaporation method.

By spray-drying, CS microspheres of 1.8–2.9 Amsizes were prepared and efficiently microencapsulated

into EudragitR microspheres ranging in size between

152 and 223 Am to form the multireservoir system.

Number of variables such as type and concentration of

chitosan, the core/coat ratio and the type of enteric

polymer have been investigated to optimize the

microsphere properties. Huang et al. [61] prepared

CS microspheres by the spray-drying method using

type-A gelatin and ethylene oxide– propylene oxide

block copolymer as modifiers. Surface morphology

and surface charges of the prepared microspheres

were investigated using SEM and microelectropho-

resis. Shape, size and surface morphology of the

microspheres were significantly influenced by the

concentration of gelatin. Betamethasone disodium

phosphate-loaded microspheres demonstrated a good

drug stability (less 1% hydrolysis product), high

entrapment efficiency (95%) and positive surface

charge (37.5 mV). In vitro drug release from the

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–2812

microspheres was related to gelatin content. Micro-

spheres containing gelatin/CS ratio of 0.4–0.6 (w/w)

showed a prolonged release up to 12 h.

2.4. Emulsion-droplet coalescence method

The novel emulsion-droplet coalescence method

was developed by Tokumitsu et al. [62], which

utilizes the principles of both emulsion cross-linking

and precipitation. However, in this method, instead of

cross-linking the stable droplets, precipitation is

induced by allowing coalescence of CS droplets with

NaOH droplets. First, a stable emulsion containing

aqueous solution of CS along with drug is produced in

liquid paraffin oil and then, another stable emulsion

containing CS aqueous solution of NaOH is produced

in the same manner. When both emulsions are mixed

under high-speed stirring, droplets of each emulsion

would collide at random and coalesce, thereby

precipitating CS droplets to give small size particles.

The method is schematically shown in Fig. 6.

Gadopentetic acid-loaded CS nanoparticles have been

Fig. 6. Schematic representation of preparation of chitosan

particulate systems by emulsion-droplet coalescence method.

prepared by this method for gadolinium neutron-

capture therapy. Particle size depends upon the type of

CS, i.e., as the % deacetylation degree of CS

decreased, particle size increased, but drug content

decreased. Particles produced using 100% deacety-

lated CS had the mean particle size of 452 nm with

45% drug loading. Nanoparticles were obtained

within the emulsion-droplet. Size of the nanoparticle

did not reflect the droplet size. Since gadopentetic

acid is a bivalent anionic compound, it interacts

electrostatically with the amino groups of CS, which

would not have occurred if a cross-linking agent is

used that blocks the free amino groups of CS. Thus, it

was possible to achieve higher gadopentetic acid

loading by using the emulsion-droplet coalescence

method compared to the simple emulsion cross-

linking method.

2.5. Ionic gelation

The use of complexation between oppositely

charged macromolecules to prepare CS microspheres

has attracted much attention because the process is

very simple and mild [63,64]. In addition, reversible

physical cross-linking by electrostatic interaction,

instead of chemical cross-linking, has been applied

to avoid the possible toxicity of reagents and other

undesirable effects. Tripolyphosphate (TPP) is a

polyanion, which can interact with the cationic CS

by electrostatic forces [65,66]. After Bodmeier et al.

[67] reported the preparation of TPP–CS complex by

dropping CS droplets into a TPP solution, many

researchers have explored its potential pharmaceutical

usage [68–73]. In the ionic gelation method, CS is

dissolved in aqueous acidic solution to obtain the

cation of CS. This solution is then added dropwise

under constant stirring to polyanionic TPP solution.

Due to the complexation between oppositely charged

species, CS undergoes ionic gelation and precipitates

to form spherical particles. The method is schemati-

cally represented in Fig. 7. However, TPP/CS micro-

particles formed have poor mechanical strength thus,

limiting their usage in drug delivery.

Insulin-loaded CS nanoparticles have been pre-

pared by mixing insulin with TPP solution and then

adding this to CS solution under constant stirring [74].

Two types of CS in the form of hydrochloride salt

(SeacureR 210 Cl and ProtasanR 110 Cl), varying in

Fig. 7. Schematic representation of preparation of chitosan particulate systems by ionic gelation method.

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–28 13

their molecular weight and degree of deacetylation,

were utilized for nanoparticle preparation. For both

types of CS, TPP concentration was adjusted to get a

CS/TPP ratio of 6:1. Chitosan nanoparticles thus

obtained were in the size range of 300–400 nm with a

positive surface charge ranging from +54 to +25 mV.

Using this method, insulin loading was modulated

reaching the values up to 55%. Efficiency of the

method was dependent upon the deacetylation of CS,

since it involves the gelation of protonated amino

groups of CS.

There are many ongoing investigations, which

demonstrate the improved oral bioavailability of

peptide and protein formulations. Bioadhesive poly-

saccharide CS nanoparticles would seem to further

enhance their intestinal absorption. Pan et al. [75]

prepared the insulin-loaded CS nanoparticles by

ionotropic gelation of CS with TPP anions. Particle

size distribution and zeta potential were determined by

photon correlation spectroscopy. The ability of CS

nanoparticles to enhance the intestinal absorption of

insulin and the relative pharmacological bioavailability

of insulin was investigated by monitoring the plasma

glucose level of alloxan-induced diabetic rats after the

oral administration of various doses of insulin-loaded

CS nanoparticles. The positively charged, stable CS

nanoparticles showed particle size in the range of 250–

400 nm. Insulin association was up to 80%. The in vitro

release experiments indicated initial burst effect, which

is pH-sensitive. The CS nanoparticles enhanced the

intestinal absorption of insulin to a greater extent than

the aqueous solution of CS in vivo. After adminis-

tration of 21 I.U./kg insulin in the CS nanoparticles,

hypoglycemia was prolonged over 15 h. The average

pharmacological bioavailability relative to s.c. injec-

tion of insulin solution was up to 14.9%.

Xu and Du [76] have studied different formulations

of CS nanoparticles produced by the ionic gelation of

TPP and CS. TEM indicated their diameter ranging

between 20 and 200 nm with spherical shape. FTIR

confirmed tripolyphosphoric groups of TPP linked

with ammonium groups of CS in the nanoparticles.

Factors that affect the delivery of bovine serum

albumin (BSA) as a model protein have been studied.

These include molecular weight and deacetylation

degree of CS, concentrations of CS and BSA, as well

as the presence of polyethylene glycol (PEG) in the

encapsulation medium. Increasing molecular weight

of CS from 10 to 210 kDa, BSA encapsulation

efficiency was enhanced nearly twice. The total

release of BSA in phosphate buffered saline pH 7.4

in 8 days was reduced from 73.9% to 17.6%.

Increasing deacetylation degree from 75.5% to 92%

promoted the encapsulation efficiency with a decrease

in release rate. Encapsulation efficiency decreased

greatly by increasing the initial concentration of BSA

and CS. Higher loading capacity of BSA enhanced the

BSA release from nanoparticles. However, adding

PEG hindered the BSA encapsulation and increased

the release rate.

Ko et al. [77] prepared CS microparticles with TPP

by the ionic cross-linking method. Particle sizes of

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–2814

TPP-CS microparticles varied from 500 to 710 Amwith drug encapsulation efficiencies more than 90%.

Morphologies of TPP-CS microparticles have been

examined by SEM. As the pH of TPP solution

decreased and molecular weight of CS increased,

microparticles acquired better spherical shape having

smooth surface. Release of felodipine as a model drug

was affected by the preparation method. Chitosan

microparticles prepared at lower pH or higher

concentration of TPP solution resulted in a slower

release of felodipine. With a decreasing molecular

weight and concentration of CS solution, the drug

release increased. The release of drug from TPP-CS

microparticles decreased when the cross-linking time

was increased.

2.6. Reverse micellar method

Reverse micelles are thermodynamically stable

liquid mixtures of water, oil and surfactant. Macro-

scopically, they are homogeneous and isotropic,

structured on a microscopic scale into aqueous and

oil microdomains separated by surfactant-rich films.

One of the most important aspects of reverse micelle

hosted systems is their dynamic behavior. Nano-

particles prepared by conventional emulsion poly-

merization methods are not only large (N200 nm),

but also have a broad size range. Preparation of

ultrafine polymeric nanoparticles with narrow size

distribution could be achieved by using reverse

micellar medium [78]. Aqueous core of the reverse

micellar droplets can be used as a nanoreactor to

prepare such particles. Since the size of the reverse

micellar droplets usually lies between 1 and 10 nm

Fig. 8. Schematic representation of preparation of chitos

[79], and these droplets are highly monodispersed,

preparation of drug-loaded nanoparticles in reverse

micelles will produce extremely fine particles with a

narrow size distribution. Since micellar droplets are

in Brownian motion, they undergo continuous

coalescence followed by re-separation on a time-

scale that varies between millisecond and micro-

second [80]. The size, polydispersity and thermody-

namic stability of these droplets are maintained in the

system by a rapid dynamic equilibrium.

In this method, the surfactant is dissolved in a

organic solvent to prepare reverse micelles. To this,

aqueous solutions of CS and drug are added with

constant vortexing to avoid any turbidity. The aqueous

phase is regulated in such a way as to keep the entire

mixture in an optically transparent microemulsion

phase. Additional amount of water may be added to

obtain nanoparticles of larger size. To this transparent

solution, a cross-linking agent is added with constant

stirring, and cross-linking is achieved by stirring

overnight. The maximum amount of drug that can

be dissolved in reverse micelles varies from drug to

drug and has to be determined by gradually increasing

the amount of drug until the clear microemulsion is

transformed into a translucent solution. The organic

solvent is then evaporated to obtain the transparent

dry mass. The material is dispersed in water and then

adding a suitable salt precipitates the surfactant out.

The mixture is then subjected to centrifugation. The

supernatant solution is decanted, which contains the

drug-loaded nanoparticles. The aqueous dispersion is

immediately dialyzed through dialysis membrane for

about 1 h and the liquid is lyophilized to dry powder.

The method is schematically represented in Fig. 8.

an particulate systems by reverse micellar method.

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–28 15

Mitra et al. [81] have encapsulated doxorubicin–

dextran conjugate in CS nanoparticles prepared by

reverse micellar method. The surfactant sodium

bis(ethyl hexyl) sulfosuccinate (AOT), was dissolved

in n-hexane. To 40 mL of AOT solution (0.03 M), 100

AL of 0.1% CS solution in acetic acid, 200 ALdoxorubicin–dextran conjugate (6.6 mg/mL), 10 ALliquor ammonia and 10 AL of 0.01% glutaraldehyde

solution were added with continuous stirring at room

temperature. This procedure produced CS nanopar-

ticles encapsulating doxorubicin–dextran conjugate.

Solvent was removed by rotary evaporator and the dry

mass was resuspended in 5 mL of pH 7.4 Tris–Cl

buffer by sonication. To this, 1 mL of 30% CaCl2solution was added dropwise to precipitate the

surfactant as calcium salt of diethylhexyl sulfosucci-

nate. The precipitate was pelleted by centrifugation at

5,000 rpm for 30 min at 4 8C. The pellet was

discarded and the supernatant containing nanopar-

ticles was centrifuged at 60,000 rpm for 2 h to pellet

the nanoparticles. The pellet was dispersed in 5 mL of

pH 7.4 Tris–HCl buffer.

2.7. Sieving method

Recently, Agnihotri and Aminabhavi [82] have

developed a simple, yet novel method to produce CS

microparticles. In this method, microparticles were

prepared by cross-linking CS to obtain a non-sticky

glassy hydrogel followed by passing through a sieve

as shown in Fig. 9. A suitable quantity of CS was

dissolved in 4% acetic acid solution to form a thick

Fig. 9. Schematic representation of preparation of ch

jelly mass that was cross-linked by adding glutaralde-

hyde. The non-sticky cross-linked mass was passed

through a sieve with a suitable mesh size to get

microparticles. The microparticles were washed with

0.1 N NaOH solution to remove the un-reacted excess

glutaraldehyde and dried overnight in an oven at 40

8C. Clozapine was incorporated into CS before cross-

linking with an entrapment efficiency up to 98.9%.

This method is devoid of tedious procedures, and can

be scaled up easily. Microparticles were irregular in

shape, with the average particle sizes in the range

543–698 Am. The in vitro release was extended up to

12 h, while the in vivo studies indicated a slow release

of clozapine.

3. Drug loading into micro/nanoparticles of

chitosan

Drug loading in micro/nanoparticulate systems can

be done by two methods, i.e., during the preparation

of particles (incorporation) and after the formation of

particles (incubation). In these systems, drug is

physically embedded into the matrix or adsorbed onto

the surface. Various methods of loading have been

developed to improve the efficiency of loading, which

largely depends upon the method of preparation as

well as physicochemical properties of the drug.

Maximum drug loading can be achieved by incorpo-

rating the drug during the formation of particles, but it

may get affected by the process parameters such as

method of preparation, presence of additives, etc.

itosan particulate systems by sieving method.

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–2816

Both water-soluble and water-insoluble drugs can

be loaded into CS-based particulate systems. Water-

soluble drugs are mixed with CS solution to form a

homogeneous mixture, and then, particles can be

produced by any of the methods discussed before.

For instance, cisplatin was loaded [83] during the

formation of particles with encapsulation efficiency

as high as 99%. The initial concentration of

cisplatin and volume of glutaraldehyde had no

effect on the encapsulation efficiency. Drug encap-

sulation increased as the concentration of CS

increased. Water-insoluble drugs and drugs that

can precipitate in acidic pH solutions can be loaded

after the formation of particles by soaking the

preformed particles with the saturated solution of

drug.

Diclofenac sodium, which precipitates in acidic pH

conditions, has been loaded by the soaking method

[33]. In this method, loading depends upon the

swelling of particles in water. Percentage loading of

drug decreased with increasing cross-linking due to

decreased swelling. Water-insoluble drugs can also be

loaded using the multiple emulsion technique. In this

method, drug is dissolved in a suitable solvent and

then emulsified in CS solution to form an oil-in-water

(o/w) type emulsion. Sometimes, drug can be dis-

persed into CS solution by using a surfactant to get the

suspension. Thus, prepared o/w emulsion or suspen-

sion can be further emulsified into liquid paraffin to

get the oil-water-oil (o/w/o) multiple emulsion. The

resulting droplets can be hardened by using a suitable

cross-linking agent.

In a study by Jameela et al. [84], bovine serum

albumin (BSA) and diphtheria toxoid were loaded

into preformed glutaraldehyde cross-linked CS

microspheres by passive absorption from aqueous

solutions. This method is an alternative to loading

biological macromolecules that are sensitive to

organic solvents, pH, temperature, ultrasound, etc.

In vitro release of BSA showed a high burst effect.

Coating of particles with paraffin or polylactic acid

modulated the drug release. Diphtheria toxoid loaded

CS microspheres showed constant antibody titres for

5 months.

Hejazi and Amiji [85] have prepared CS micro-

spheres by ionic cross-linking and precipitation with

sodium sulfate. Two different methods were used

for drug loading. In method I, tetracycline was

mixed with CS solution before simultaneous cross–

linking and precipitation. In method II, drug was

incubated with the pre-formed microspheres for 48

h. Cumulative amount of tetracycline that was

released from CS microspheres and stability of drug

was examined in different pH media at 37 8C.Microspheres with a spherical shape having an

average diameter of 2– 3 Am were formed. When

drug was added to CS solution before cross-linking

and precipitation, only 8% (w/w) was optimally

incorporated in the final microsphere formulation.

When drug was incubated with the pre-formed

microspheres, a maximum of 69% (w/w) could be

loaded. About 30% of tetracycline either in solution

or when released from the microspheres was found

to degrade at pH 1.2 in 12 h. Preliminary results of

this study suggested that CS microspheres can be

used to incorporate antibiotic drugs, which may be

effective when administered locally in the stomach

against H. pylori.

4. Drug release and release kinetics

Drug release from CS-based particulate systems

depends upon the extent of cross– linking, morphol-

ogy, size and density of the particulate system,

physicochemical properties of the drug as well as

the presence of adjuvants. In vitro release also

depends upon pH, polarity and presence of enzymes

in the dissolution media. The release of drug from CS

particulate systems involves three different mecha-

nisms: (a) release from the surface of particles, (b)

diffusion through the swollen rubbery matrix and (c)

release due to polymer erosion. These mechanisms are

shown schematically in Fig. 10.

In majority of cases, drug release follows more

than one type of mechanism. In case of release

from the surface, adsorbed drug instantaneously

dissolves when it comes in contact with the release

medium. Drug entrapped in the surface layer of

particles also follows this mechanism. This type of

drug release leads to burst effect. He et al. [54]

observed that cemetidine-loaded CS microspheres

have shown burst effect in the early stages of

dissolution. Most of the drug was released within

few minutes when particles were prepared by spray

drying technique. Increasing the cross-linking den-

Fig. 10. Mechanism of drug release from particulate systems.

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–28 17

sity can prevent the burst release. This effect can

also be avoided by washing microparticles with a

proper solvent, but it may lead to low encapsulation

efficiency.

Drug release by diffusion involves three steps.

First, water penetrates into particulate system, which

causes swelling of the matrix; secondly, the

conversion of glassy polymer into rubbery matrix

takes place, while the third step is the diffusion of

drug from the swollen rubbery matrix. Hence, the

release is slow initially and later, it becomes fast.

This type of release is more prominent in case of

hydrogels. Al-Helw et al. [45] observed a high

initial release of the drug in all the prepared

formulations. Nearly, 20– 30% of the incorporated

drug was released in the first hour. Release was

dependent on the molecular weight of CS and

particle size of the microspheres. The release rate

from microspheres prepared from high molecular

weight CS was slow compared to those prepared

from medium and low molecular weight CS. This

could be attributed to both lower solubility of high

molecular weight CS and higher viscosity of the gel

layer formed around the drug particles upon contact

with the dissolution medium. The release within the

first 3 h was fast (75– 95%) from microspheres

within the size range of 250– 500 Am, but for

particles in the size range of 500– 1,000 Am, drug

release was 56– 90% in 5 h. This is attributed to

large surface area available for dissolution with a

small particle size, thus favoring rapid release of the

drug compared to larger microspheres.

Kweon and Kang [86] prepared the CS-g–

poly(vinyl alcohol) matrix to study the release of

prednisolone under various conditions. Relationship

between the amount of drug release and square root

of time was linear indicating the diffusion-controlled

release. Drug release was controlled by the extent of

PVA grafting, heat treatment or cross-link density,

but it was less affected by the pH when compared to

plain chitosan. Ganza-Gonzalez et al. [56] analyzed

the drug release data using Higuchi equation [87].

Higuchi equation was used to describe the release of

a solute from a flat surface, but not from a sphere

[88], but the good fit obtained suggests that the

release rate depends upon the rate of diffusion

through the cross-linked matrix. Authors have also

fitted the release data to equations developed by Guy

et al. [89] to describe the diffusion from a sphere.

The most commonly used equation for diffusion-

controlled matrix system is an empirical equation used

by Ritger and Peppas [90], in which the early time

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–2818

release data can be fitted to obtain the diffusion

parameters,

Mt

Ml¼ ktn ð1Þ

Here, Mt/Ml is the fractional drug release at time t, k

is a constant characteristic of the drug-polymer

interaction and n is an empirical parameter character-

izing the release mechanism. Based on the diffusional

exponent [91], drug transport is classified as Fickian

(n=0.5), Case II transport (n=1), non-Fickian or

anomalous (0.5bnb1) and super Case II (nN1). Drug

release from the CS microspheres cross– linked with

glutaraldehyde, sulfuric acid and heat have shown

[33] different n values varying from 0.47 to 0.61. The

n values increase with increasing loading of diclofe-

nac sodium in different cross-linked formulations.

Recently, Agnihotri and Aminabhavi [82] analyzed

the dynamic swelling data of CS microparticles using

Eq. (1) to predict drug release from the water uptake

data of the microparticles cross-linked with (5.0, 7.5

and 10.0)�10– 4 mL of glutaraldehyde/mg of CS. It

was observed that as the cross-linking increases,

swelling of CS microparticles decreases. Values of n

obtained in the range of 0.160 to 0.249 indicating that

the release mechanism deviates from the Fickian

trend. The values of n are b0.5 due to the irregular

shaped particles and these decrease systematically

with increasing cross-linking.

In the swelling controlled release systems, drug is

dispersed within a glassy polymer. Upon contact with

biological fluid, the polymer swells, but no drug

diffusion occurs through the polymer phase. As the

penetrant enters the glassy polymer, glass transition

temperature of the polymer is lowered due to

relaxation of the polymer chains. Drug could diffuse

out of the swollen rubbery polymer. This type of

system is characterized by two moving boundaries:

the front separating the swollen rubbery portion and

the glassy region, which moves with a front velocity

and the polymer fluid interface. The rate of drug

release is controlled by the velocity and position of the

front dividing the glassy and rubbery portions of the

polymer.

Jameela et al. [48] have obtained a good correlation

fit for the cumulative drug released vs. square root of

time, demonstrating that the release from the micro-

sphere matrix is diffusion-controlled and obeys

Higuchi equation [87]. It was demonstrated that the

rate of release depends upon the size of microspheres.

Release from smaller size microspheres was faster

than those from the large size microspheres due to

smaller diffusional path length for the drug and the

larger surface area of contact of smaller particles with

the dissolution medium. Orienti et al. [92] studied the

correlation between matrix erosion and release

kinetics of indomethacin-loaded CS microspheres.

Release kinetics was correlated with the concentration

of CS in the microsphere and pH of the release

medium. At high concentrations of CS and at pH 7.4,

deviations from Fickian to zero order kinetics have

been observed. Variations induced by these parame-

ters on drug diffusion and solubility in the matrix

undergoing erosion have been analyzed.

5. Pharmaceutical applications of chitosan

particulate systems

Chitosan-based particulate systems are attracting

pharmaceutical and biomedical applications as poten-

tial drug delivery devices. Some important applica-

tions are discussed below.

5.1. Colon targeted drug delivery

Chitosan is a promising polymer for colon drug

delivery since it can be biodegraded by the colonic

bacterial flora [93,94] and it has mucoadhesive

character [1]. The pH-sensitive multicore microparti-

culate system containing CS microcores entrapped

into enteric acrylic microspheres was reported [60].

Sodium diclofenac was efficiently entrapped within

these CS microcores and then microencapsulated into

Eudragit L-100 and Eudragit S-100 to form a multi-

reservoir system. In vitro release study revealed no

release of the drug in gastric pH for 3 h and after the

lag-time, a continuous release for 8– 12 h was

observed in the basic pH.

5.2. Mucosal delivery

Nowadays, mucosal surfaces such as nasal, peroral

and pulmonary are receiving a great deal of attention

as alternative routes of systemic administration.

Chitosan has mucoadhesive properties and therefore,

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–28 19

it seems particularly useful to formulate the bioadhe-

sive dosage forms for mucosal administration (ocular,

nasal, buccal, gastro-enteric and vaginal-uterine ther-

apy) [95]. Nasal mucosa has high permeability and

easy access of drug to the absorption site. The

particulate delivery to peroral mucosa is easily taken

up by the Peyer’s patches of the gut associated

lymphoid tissue. Chitosan has been found to enhance

the drug absorption through mucosae without damag-

ing the biological system. Here, the mechanism of

action of CS was suggested to be a combination of

bioadhesion and a transient widening of the tight

junctions between epithelial cells [27].

Genta et al. [95] studied the influence of gluta-

raldehyde on drug release and mucoadhesive proper-

ties of CS microspheres. A new in vitro technique was

developed based on electron microscopy to study the

effect of polymer cross-link density on the mucoad-

hesive properties of CS microspheres modulating the

rate of theophylline release. The ability of insulin-

loaded CS nanoparticles to enhance the nasal absorp-

tion of insulin was investigated in a conscious rabbit

model. Chitosan nanoparticles enhanced the nasal

absorption of insulin to a greater extent than the

aqueous solution of CS [74]. van der Lubben et al.

[96] incorporated the model protein ovalbumin into

CS microparticles and the uptake of ovalbumin

associated with CS microparticles in murine Peyer’s

patches was demonstrated using confocal laser scan-

ning microscopy. In a further study, van der Lubben et

al. [97] investigated the ability of CS microparticles to

enhance both systemic and local immune responses

against diphtheria toxoid (DT) vaccine after the oral

and nasal administration in mice. Systemic and local

IgG and IgA immune responses against DT associated

to CS microparticles were strongly enhanced after the

oral delivery in mice.

Even though oral vaccination has numerous

advantages over the parenteral injection, degradation

of the vaccine in the gut and low uptake in the

lymphoid tissue of the gastrointestinal tract still

complicate the development of oral vaccines. In this

direction, van der Lubben et al. [98] prepared the CS

microparticles and characterized them for size, zeta

potential, morphology- and ovalbumin-loading as

well as release characteristics. The in vivo uptake of

CS microparticles by murine Peyer’s patches was

studied by using confocal laser scanning microscopy

(CLSM). Chitosan microparticles were prepared using

a precipitation/coacervation method. The size of CS

microparticles was 4.3±±0.7 Am and were positively

charged (20±1 mV). Since only microparticles smaller

than 10 Am can be taken up by M-cells of Peyer’s

patches, these microparticles were used as vaccination

systems. The CLSM studies showed that the model

antigen ovalbumin was entrapped within the CS

microparticles. Field emission scanning electron

microscopy demonstrated the porous structure of CS

microparticles, thus facilitating the entrapment of

ovalbumin. Ovalbumin loading in CS microparticles

was about 40%. Release studies have shown the low

release of ovalbumin within 4 h, but most of

ovalbumin (about 90%) remained entrapped in the

microparticles. Since CS microparticles are biode-

gradable, the entrapped ovalbumin was released after

intracellular digestion in Peyer’s patches. Initial in

vivo studies demonstrated that fluorescently labeled

CS microparticles can be taken up by the epithelium

of the murine Peyer’s patches. Since the uptake by

Peyer’s patches is an essential step in oral vaccination,

these results have shown that the porous CS micro-

particles developed are most promising vaccine

delivery systems.

5.3. Cancer therapy

Gadopentetic acid-loaded CS nanoparticles have

been prepared for gadolinium neutron-capture therapy

[62]. Their releasing properties and ability for long-

term retention of gadopentetic acid in the tumor

indicated that these nanoparticles are useful as intra-

tumoral injectable devices for gadolinium neutron-

capture therapy. The accumulation of gadolinium

loaded as gadopentetic acid (Gd-DTPA) in CS nano-

particles designed for gadolinium neutron-capture

therapy (Gd-NCT) for cancer have been evaluated in

vitro in cultured cells [99]. Using L929 fibroblast

cells, Gd accumulation for 12 h at 37 8C was

investigated at Gd concentrations lower than 40

ppm. The accumulation leveled above 20 ppm and

reached 18.0±2.7 (mean±S.D.) Ag Gd/106 cells at 40

ppm. Furthermore, the corresponding accumulations

in B16F10 melanoma cells and SCC-VII squamous

cell carcinoma, which were used in the previous Gd-

NCT trials in vivo were 27.1±2.9 and 59.8±9.8 Ag Gd/106 cells, respectively. This explains the superior

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–2820

growth-suppression in the in vivo trials using SCC-

VII cells. The accumulation of nanoparticles in these

cells was 100– 200 times higher in comparison to

dimeglumine gadopentetate aqueous solution (Mag-

nevistw), a magnetic resonance imaging contrast

agent. The endocytic uptake of nanoparticles was

suggested from TEM. These findings indicated that

nanoparticles had a high affinity to cells, thus

contributing to the long retention of Gd in tumor

tissue leading to significant suppression of tumor

growth in in vivo studies.

Tokumitsu et al. [100] demonstrated the potential

usefulness of Gd-NCT using gadolinium-loaded nano-

particles. The potential of gadolinium neutron-capture

therapy (Gd-NCT) for cancer was evaluated using CS

nanoparticles as a novel gadolinium device. The

nanoparticles incorporated with 1200 mg of natural

gadolinium were administered intratumorally twice in

mice-bearing subcutaneous B16F10 melanoma. The

thermal neutron irradiation was performed for the

tumor site, with the fluence of 6.32�1012 neutrons/

cm2, 8 h after the second gadolinium administration.

After irradiation, the tumor growth in the nano-

particle-administered group was significantly sup-

pressed compared to that in the gadopentetate

solution-administered group, despite radioresistance

of melanoma and the smaller Gd dose than that

administered in past Gd-NCT trials.

Jameela et al. [101] have prepared glutaraldehyde

cross-linked CS microspheres containing mitoxan-

trone. The antitumor activity was evaluated against

Ehrlich ascites carcinoma in mice by intraperitoneal

injections. The tumor inhibitory effect was followed

by monitoring the survival time and change in the

body weight of the animal for 60 days. Mean survival

time of animals which received free mitoxantrone was

2.1 days and this was increased to 50 days when

mitoxantrone was given via microspheres. In another

study [102], the in vitro release of mitoxantrone was

controlled for 4 weeks in phosphate buffer at 27 8C.Mitra et al. [81] have encapsulated doxorubicin–

dextran conjugate into long circulating CS nano-

particles. In an attempt to minimize cardiotoxicity of

doxorubicin, a conjugate with dextran was prepared

and encapsulated in CS nanoparticles. Size of the

nanoparticle was 100±10 nm, which favors enhanced

permeability and retention effect. Antitumor effect of

these doxorubicin– dextran-loaded nanoparticles was

evaluated in J774A.1 macrophage tumor cells

implanted in Balb/c mice. The in vivo efficacy of

these nanoparticles was determined by tumor regres-

sion and increased survival time compared to doxor-

ubicin– dextran conjugate and the free drug. These

results suggest that the system not only reduced the

side effects, but also improved its therapeutic efficacy

in the treatment of solid tumors.

Janes et al. [103] evaluated the potential of CS

nanoparticles as carriers for doxorubicin (DOX). The

challenge was to entrap a cationic, hydrophilic

molecule into nanoparticles formed by ionic gelation

of the positively charged CS. To achieve this

objective, the authors have masked the positive charge

of DOX by complexing it with dextran sulfate. This

modification doubled the DOX encapsulation effi-

ciency relative to controls and enabled real loadings

up to 4.0 wt.% of DOX. Authors also investigated the

possibility of forming a complex between CS and

DOX prior to the formation of particles. Despite low

complexation efficiency, no dissociation of the com-

plex was observed upon the formation of nano-

particles. Fluorimetric analysis of the in vitro drug

released showed the initial release phase, the intensity

of which was dependent upon the association mode,

followed by a very slow release. Evaluation of the

activity of DOX-loaded nanoparticles in cell cultures

indicated that those containing dextran sulfate were

able to maintain cytostatic activity relative to free

DOX, while DOX complexed with CS before the

nanoparticle formation showed a slightly decreased

activity. Additionally, confocal studies showed that

DOX was not released in the cell culture medium, but

entered the cells while being associated to nano-

particles. These studies have shown the feasibility of

CS nanoparticles to entrap DOX and to deliver it to

the cells in its active form.

5.4. Gene delivery

Gene therapy is a challenging task in the treatment

of genetic disorders. In case of gene delivery, the

plasmid DNA has to be introduced into the target

cells, which should get transcribed and the genetic

information should ultimately be translated into the

corresponding protein. To achieve this goal, number

of hurdles are to be overcome by the gene delivery

system. Transfection is affected by: (a) targeting the

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–28 21

delivery system to target cell, (b) transport through the

cell membrane, (c) uptake and degradation in the

endolysosomes and (d) intracellular trafficking of

plasmid DNA to the nucleus. Chitosan could interact

ionically with the negatively charged DNA and forms

polyelectrolyte complexes. In these complexes, DNA

becomes better protected against nuclease degradation

leading to better transfection efficiency.

DNA–CS nanoparticles have been prepared [53]

to examine the influence of several parameters on

their preparation. The transfection efficiency of CS-

DNA nanoparticles was cell-type dependent. Typi-

cally, it was 3 to 4 orders of magnitude, in relative

light units, higher than the background level in

HEK293 cells, and 2 to 10 times lower than that

achieved by LipofectAMINE–A–DNA complexes.

The presence of 10% fetal bovine serum did not

interfere with their transfection ability. The study

also developed three different schemes to conjugate

transferrin or KNOB protein to the nanoparticle

surface. The transferrin conjugation only yielded a

maximum of 4-fold increase in their transfection

efficiency in HEK293 cells and HeLa cells, whereas

KNOB conjugated nanoparticles could improve the

gene expression level in HeLa cells by 130-fold.

Conjugation of PEG on nanoparticles allowed

lyophilization without aggregation, and without loss

of bioactivity for at least 1 month in storage. The

clearance of PEGylated nanoparticles in mice follow-

ing i.v. administration was slower than the unmodi-

fied nanoparticles at 15 min, and with higher

depositions in kidney and liver. However, no differ-

ence was observed during the first hour.

Self-aggregates were prepared [104] by hydro-

phobic modification of CS with deoxycholic acid in

aqueous media. Self-aggregates have a small size

(mean diameter of 160 nm) with an unimodal size

distribution. Self-aggregates can form charge com-

plexes when mixed with plasmid DNA. The useful-

ness of self-aggregates/DNA complex for transfer of

genes into mammalian cells in vitro has been

suggested. Several transfection studies using chemi-

cally modified CS have been reported. Trimethyl CS

oligomers were examined for their potency as DNA

carriers [105]. Chitosan and lactosylated CS carriers

were investigated for their transfection efficiencies in

vitro [106]. Recently, galactosylated CS-g– dextran–

DNA complexes have been prepared [107]. Galactose

groups were chemically bound to CS for liver

specificity and dextran was grafted to increase the

stability of the complex in water. It was shown that this

system could efficiently transfect liver cells.

Chew et al. [108] studied the i.m. immunization

with full-length Der p 1 cDNA induced significant

humoral response to the left domain (approximately

corresponding to amino acids 1– 116), but not to the

right domain (approximately corresponding to amino

acids 117– 222) of Der p 1 allergen. Authors explored

the use of CS–DNA nanoparticles for oral immuniza-

tion to induce the immune responses specific to both

left and right domains of Der p 1. DNA constructs

pDer p 1 (1– 222) and pDer p 1 (114– 222), which

were complexed with CS and delivered orally

followed by an i.m. injection of pDer p 1 (1– 222)

after 13 weeks. Such an approach has successfully

primed Th1-skewed immune responses against both

domains of Der p 1. It was suggested that such a

strategy could be further optimized for more effica-

cious gene vaccination for full-length Der p 1.

Numerous studies have been reported on prophy-

lactic and therapeutic use of genetic vaccines for

combating a variety of infectious diseases in animal

models. Recent human clinical studies with the gene

gun have validated the concept of direct targeting of

dendritic cells (Langerhan’s cells) in the viable

epidermis of the skin. However, it is unclear whether

the gene gun technology or other needle-free devices

will become commercially viable. Cui and Mumper

[109] investigated the topical application of CS-

based nanoparticles containing plasmid DNA

(pDNA) as a potential approach to genetic immuni-

zation. Two types of nanoparticles were investigated:

(i) pDNA-condensed CS nanoparticles and (ii)

pDNA-coated on pre-formed cationic CS/carboxy-

methylcellulose (CMC) nanoparticles. These studies

have shown that both CS and a CS oligomer can

complex CMC to form stable cationic nanoparticles

for subsequent pDNA coating. Selected pDNA-

coated nanoparticles (with pDNA up to 400 mg/

mL) were stable to challenge with the serum.

Several different CS-based nanoparticles containing

pDNA resulted in both detectable and quantifiable

levels of luciferase expression in mouse skin 24 h

after topical application and significant antigen-

specific IgG titer to expressed h-galactosidase at

28 days.

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–2822

Borchard [110] has recently published a review on

the efficient non-viral gene delivery using cationic

polymers as DNA-condensing agents. The gene

delivery is dependent on several factors such as

complex size, complex stability, toxicity, immunoge-

nicity, protection against DNase degradation, intra-

cellular trafficking and processing of the DNA. The

review also examined the advances made in the

application of CS and CS derivatives to non-viral

gene delivery. It gives an overview of the transfection

studies performed by using CS as a transfection agent.

5.5. Topical delivery

Due to good bioadhesive property and ability to

sustain the release of the active constituents, CS has

been used in topical delivery systems. Bioadhesive CS

microspheres for topical sustained release of cetyl

pyridinium chloride have been evaluated [55].

Improved microbiological activity was shown by

these microparticulate systems. Conti et al. [111]

prepared microparticles composed of CS and designed

as powders for topical wound-healing properties.

Blank and ampicillin-loaded microspheres were pre-

pared by spray-drying technique. In vivo evaluation in

albino rats showed that both drug-loaded and blank

microspheres have shown good wound healing

properties.

5.6. Ocular delivery

De Campos et al. [112] investigated the potential of

CS nanoparticles as a new vehicle to improve the

delivery of drugs to ocular mucosa. Cyclosporin A

(CyA) was chosen as a model drug. A modified ionic

gelation technique was used to produce CyA-loaded

CS nanoparticles. These nanoparticles with a mean

size of 293 nm, a zeta potential of +37 mV, high CyA

association efficiency and loading of 73% and 9%,

respectively were obtained. The in vitro release

studies, performed under sink conditions, revealed

the fast release during the first hour followed by a

more gradual drug release during the 24-h period. The

in vivo experiments showed that after topical instilla-

tion of CyA-loaded CS nanoparticles to rabbits,

therapeutic concentrations were achieved in the

external ocular tissues (i.e., cornea and conjunctiva)

within 48 h while maintaining negligible or undetect-

able CyA levels in the inner ocular structures (i.e., iris/

ciliary body and aqueous humour), blood and plasma.

These levels were significantly higher than those

obtained following the instillation of CS solution

containing CyA and an aqueous CyA suspension. The

study indicated that CS nanoparticles could be used as

a vehicle to enhance the therapeutic index of the

clinically challenging drugs with potential application

at the extraocular level.

5.7. Chitosan as a coating material

Chitosan has good film forming properties and

hence, it is used as a coating material in drug delivery

applications. Chitosan-coated microparticles have

many advantages such as improvement of drug

payloads, bioadhesive property and prolonged drug

release properties over the uncoated particles. Chito-

san-coated microspheres composed of poly(lactic

acid)– poly(caprolactone) blends have been prepared

[113]. These microspheres showed good potential for

the targeted delivery of antiproliferative agents to treat

restenosis. Shu and Zhu [73] have prepared the

alginate beads coated with CS by three different

methods. The release of brilliant blue was not only

affected by CS density on the particle surface, but also

on the preparation method and other factors. Chiou et

al. [114] have used different molecular weight

chitosans for coating the microspheres. The initial

burst release was observed in the first hour with 50%

release of lidocaine. But, 19.2% release occurred at

25th hour for the un-coated particles and 14.6% at the

90th hour for the CS-coated microspheres.

6. Chemically modified chitosans

Various chemical modifications of CS have been

studied to alter its properties. N-Trimethyl chitosan

chloride (TMC), a quaternized CS derivative, has

been proven to effectively increase the permeation of

hydrophilic macromolecular drugs across- the

mucosal epithelia by opening the tight junctions

[115]. The study investigated the intestinal absorption

of octreotide when it is co-administered with a

polycationic absorption enhancer, TMC. Chitosan

succinate and CS phthalate were synthesized and

assessed as potential matrices for colon-specific orally

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–28 23

administered drug delivery applications. The prepared

matrices resisted the dissolution under acidic con-

ditions. On the other hand, improved drug release

profiles were observed in basic conditions. These

results suggested the suitability of the prepared

matrices in colon specific and orally administered

drug delivery applications [116]. In order to overcome

the low solubility of CS in neutral pH, which is the

major drawback to use this type of polymer as a

transfection agent, N-trimethylated and N-triethylated

oligosaccharides have been synthesized [105].

Lee et al. [104] synthesized the hydrophobically

modified CS containing 5.1 deoxycholic acid groups

per 100 anhydroglucose units by 1-ethyl-3-(3-dime-

thylaminopropyl) carbodiimide (EDC)-mediated cou-

pling reaction as shown in Fig. 11. Since deoxycholic

acid can form self-assemblies in aqueous media, it

was found that the modified CS also formed the self-

aggregates. The self-aggregates were characterized by

Fig. 11. A scheme of the coupling mechanism between chitosan and deox

(EDC) through amide linkage formation [taken from Ref. 104].

fluorescence spectroscopy and dynamic light scatter-

ing method. A charge complex was produced between

the cationically charged self-aggregates and the

negatively charged plasmid DNA. The feasibility of

self-aggregates as an in vitro delivery vehicle was

investigated for the transfection of genetic material in

mammalian cells.

Microcrystalline CS has been investigated as a gel

forming excipient [117]. Matrix granules of CS of

differing physicochemical properties loaded with

either ibuprofen or paracetamol as model drugs have

been prepared. Varying the amount or molecular

weight of the microcrystalline CS and to a lesser

extent by the degree of deacetylation controlled

release rate. Giunchedi et al. [59] prepared and

characterized a new derivative of CS: methyl

pyrrolidone CS. It randomly carries pyrrolidinone

groups covalently attached to the polysaccharide

backbone. This CS derivative combines the biocom-

ycholic acid using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–2824

patibility of CS [118] and hydrophilic characteristics

of the pyrrolidinone moiety [119], being particularly

susceptible to the hydrolytic action of lysozyme

[120]. The microparticles were characterized by

S.E.M., particle size analyzer, DSC and in vitro

ampicillin release. Drug release characteristics

depend upon the nature of CS used.

Chen et al. [121] studied the modification of CS by

coupling with linoleic acid (LA) through 1-ethyl-3-(3-

dimethylaminopropyl) carbodiimide-mediated reac-

tion to increase its amphipathicity for improved

emulsification. The micelle formation of linoleic

acid-modified CS in 0.1 M acetic acid solution was

enhanced by O/W emulsification with methylene

chloride, an oil phase. Fluorescence spectra indicated

that without emulsification, the self-aggregation of

LA-CS occurred at the concentration of 1.0 g/L or

above, and with emulsification, self-aggregation was

greatly enhanced followed by a stable micelle

formation at 2.0 g/L. Addition of 1 M NaCl solution

promoted the self-aggregation of LA-CS particles

both with and without emulsification. The nanosize

micelles of LA-CS were formed ranging in size

between 200 and 600 nm. The LA-CS nanoparticles

were used to encapsulate the lipid soluble model

compound, retinal acetate, with 50% efficiency.

Chitosan was chemically modified [122] by graft

copolymerization of poly(ethylene glycol) diacrylate

macromonomer onto CS backbone. Microspheres

based on chitosan and polymer grafted chitosan were

prepared by a polymer dispersion technique. A

comparative study in relation to structural deviation

among CS and modified CS microspheres was

evaluated. These chemically modified CS micropar-

ticles were hydrophilic in nature and formed aggre-

gates. Chitosan derivative with galactose groups was

synthesized by introducing galactose group into the

amine group of CS [123]. The results indicated that

although acyl reaction on the part of amino groups of

CS took place, the degree of galactosylated substitu-

tion was 20%. Crystallinity, solubility, stability and

other physical properties were different from CS.

Microspheres of CS and galactosylated CS were

prepared by the physical precipitation and coacerva-

tion techniques, respectively. Microspheres of CS and

galactosylated CS were spherical in nature with an

average diameter of 0.54 and 1.05 Am and an average

zeta potential of +17 and +15 mV, respectively. It was

suggested that galactosylated CS microspheres could

be used for passive and active hepatic targeting.

7. Conclusions

Chitosan has the desired properties for safe use as a

pharmaceutical excipient. This has prompted accel-

erated research activities worldwide on chitosan micro

and nanoparticles as drug delivery vehicles. These

systems have great utility in controlled release and

targeting studies of almost all class of bioactive

molecules as discussed in this review. Recently,

chitosan is also extensively explored in gene delivery.

However, studies toward optimization of process

parameters and scale up from the laboratory to pilot

plant and then, to production level are yet to be

undertaken. Majority of studies carried out so far are

only in in vitro conditions. More in vivo studies need

to be carried out. Chemical modifications of chitosan

are important to get the desired physicochemical

properties such as solubility, hydrophilicity, etc. The

published literature indicates that in the near future,

chitosan-based particulate systems will have more

commercial status in the market than in the past.

Acknowledgements

Authors thank the University Grants Commission

(UGC), New Delhi, India for a major grant (F1-41/

2001/CPP-II) sanctioned to Karnatak University to

establish Center of Excellence in Polymer Science.

References

[1] P.C. Berscht, B. Nies, A. Liebendorfer, J. Kreuter, Incorpo-

ration of basic fibroblast growth factor into methylpyrrolidi-

none chitosan fleeces and determination of the in vitro release

characteristics, Biomaterials 15 (1994) 593–600.

[2] L. Illum, Chitosan and its use as a pharmaceutical excipient,

Pharm. Res. 15 (1998) 1326–1331.

[3] V. Dodane, V.D. Vilivalam, Pharmaceutical applications of

chitosan, Pharm. Sci. Technol. Today 1 (1998) 246–253.

[4] O. Felt, P. Buri, R. Gurny, Chitosan: a unique polysaccharide

for drug delivery, Drug Dev. Ind. Pharm. 24 (1998) 979–993.

[5] K.D. Yao, T. Peng, Y.J. Yin, M.X. Xu, Microcapsules/

microspheres related to chitosan, J. Macromol. Sci., Rev.

Macromol. Chem. Phys. C35 (1995) 155–180.

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–28 25

[6] H.S. Kas, Chitosan: properties, preparations and application

to microparticulate systems, J. Microencapsulation 14 (1997)

689–711.

[7] R.A.A. Muzzarelli, C. Jeuniauk, G.W. Gooday, Chitin in

Nature and Technology, Plenum, New York, 1986.

[8] G. Sjak-Braek, T. Anthonsen, P. Sandford, Chitin and

Chitosan, Elsevier, New York, 1992.

[9] T. Sannan, K. Kurita, Y. Iwakura, Studies on chitin, 2. Effect

of deacetylation on solubility, Makromol. Chem. 177 (1976)

3589–3600.

[10] S. Nicol, Life after death for empty shells, New Sci. 129

(1991) 46–48.

[11] K. Arai, T. Kinumaki, T. Fujita, Toxicity of chitosan, Bull.

Tokai Reg. Fish Lab. 43 (1968) 89–94.

[12] T. Chandy, C.P. Sharma, Chitosan as a biomaterial, Biomater.

Artif. Org. 18 (1990) 1–24.

[13] W.M. Hou, S. Miyazaki, M. Takada, T. Komai, Pharmaceut-

ical application of biomedical polymers. Part XVI. Sustained

release of indomethacin from chitosan, Chem. Pharm. Bull.

33 (1985) 3986–3992.

[14] S. Miyazaki, K. Ishii, T. Nadai, Pharmaceutical application of

biomedical polymers. Part IV. The use of chitin and chitosan

as drug carriers, Chem. Pharm. Bull. 29 (1981) 3067–3069.

[15] T. Handa, A. Kasai, H. Takenaka, S.Y. Lin, Y. Ando, Novel

method for the preparation of controlled-release theophylline

granules coated with a polyelectrolyte complex of sodium

polyphosphate-chitosan, J. Pharm. Sci. 74 (1985) 264–268.

[16] S. Miyazaki, H. Yamaguchi, C. Yokouchi, M. Takada, W.-M.

Hou, Sustained-release and intragastric-floating granules of

indomethacin using chitosan in rabbits, Chem. Pharm. Bull.

36 (1988) 4033–4038.

[17] Y. Sawayanagi, N. Nambu, T. Nagai, Use of chitosan for

sustained-release preparations of water-soluble drugs, Chem.

Pharm. Bull. 30 (1982) 4213–4215.

[18] S. Shiraishi, T. Imai, M. Otagiri, Controlled release of

indomethacin by chitosan– polyelectrolyte complex: optimi-

zation and in vivo/in vitro evaluation, J. Control. Release 25

(1993) 217–225.

[19] C.M. Lehr, J.A. Bouwstra, E.H. Schacht, H.E. Junginger, In

vitro evaluation of mucoadhesive properties of chitosan and

some other natural polymers, Int. J. Pharm. 78 (1992) 43–48.

[20] H.L. Luehen, C.M. Lehr, C.O. Rentel, A.B.J. Noach, A.G.

Boer, J.C. Verhoef, H.E. Junginger, Bioadhesive polymers for

the peroral delivery of peptide drugs, J. Control. Release 29

(1994) 329–338.

[21] L. Illum, N.F. Farraj, S.S. Davis, Chitosan as a novel nasal

delivery system for peptide drugs, Pharm. Res. 11 (1994)

1186–1189.

[22] T. Imai, S. Shiraishi, H. Saito, M. Otagiri, Interaction of

indomethacin with low molecular weight chitosan, and

improvements of some pharmaceutical properties of indome-

thacin by low molecular weight chitosans, Int. J. Pharm. 67

(1991) 11–20.

[23] I. Genta, F. Pavanetto, B. Conti, P. Giunchedi, U. Conte,

Spray-drying for the preparation of chitosan microspheres,

Proc. Int. Symp. Control. Release Bioact. Mater. 21 (1994)

616–617.

[24] Y. Sawayanagi, N. Nambu, T. Nagai, Enhancement of

dissolution properties of prednisolone from ground mix-

tures with chitin or chitosan, Chem. Pharm. Bull. 31

(1983) 2507–2509.

[25] J.M. Gallo, E.E. Hassan, Receptor-mediated magnetic

carriers: basis for targeting, Pharm. Res. 5 (1988) 300–304.

[26] E.E. Hassan, R.C. Parish, J.M. Gallo, Optimized formulation

of magnetic chitosan microspheres containing the anticancer

agent, oxantrazole, Pharm. Res. 9 (1992) 390–397.

[27] P. Artursson, T. Lindmark, S.S. Davis, L. lllum, Effect

of chitosan on the permeability of monolayers of

intestinal epithelial cells (CACO-2), Pharm. Res. 11

(1994) 1358–1361.

[28] J. Akbuga, G. Durmaz, Preparation and evaluation of cross-

linked chitosan microspheres containing furosemide, Int. J.

Pharm. 111 (1994) 217–222.

[29] B.C. Thanoo, M.C. Sunny, A. Jayakrishnan, Cross-linked

chitosan microspheres: preparation and evaluation as a matrix

for the controlled release of pharmaceuticals, J. Pharm.

Pharmacol. 44 (1992) 283–286.

[30] K.S. Soppimath, A.R. Kulkarni, T.M. Aminabhavi, Con-

trolled release of antihypertensive drug from the interpene-

trating network poly(vinyl alcohol) guar gum hydrogel

microspheres, J. Biomater. Sci., Polym. Ed. 11 (2000) 27–43.

[31] K.S. Soppimath, A.R. Kulkarni, T.M. Aminabhavi, Water

transport and drug release study of cross-linked guar gum

grafted polyacrylamide hydrogel microspheres for the con-

trolled release application, Eur. J. Pharm. Biopharm. 53

(2002) 87–98.

[32] A.R. Kulkarni, K.S. Soppimath, T.M. Aminabhavi, Con-

trolled release of cefadroxil using sodium alginate inter-

penetrating network with gelatin/egg albumin, Eur. J. Pharm.

Biopharm. 51 (2001) 127–133.

[33] S.G. Kumbar, A.R. Kulkarni, T.M. Aminabhavi, Cross-

linked chitosan microspheres for encapsulation of diclofenac

sodium: effect of cross-linking agent, J. Microencapsulation

19 (2002) 173–180.

[34] S.G. Kumbar, T.M. Aminabhavi, Synthesis and character-

ization of modified chitosan microspheres: effect of the

grafting ratio on the controlled release of nifedipine through

microspheres, J. Appl. Polym. Sci. 89 (2003) 2940–2949.

[35] K.S. Soppimath, T.M. Aminabhavi, A.R. Kulkarni, W.E.

Rudzinski, Biodegradable polymeric nanoparticles as drug

delivery devices, J. Control. Release 70 (2001) 1–20.

[36] D.K. Singh, A.R. Ray, Biomedical applications of chitin,

chitosan and their derivatives, J. Macromol. Sci., Rev.

Macromol. Chem. Phys. C40 (2000) 69–83.

[37] A.K. Singla, M. Chawla, Chitosan: some pharmaceutical and

biological aspects an update, J. Pharm. Pharmacol. 53 (2001)

1047–1067.

[38] W.G. Liu, K.D. Yao, Chitosan and its derivatives– a promis-

ing non-viral vector for gene transfection, J. Control. Release

83 (2002) 1–11.

[39] R. Hejazi, M. Amiji, Chitosan-based gastrointestinal delivery

systems, J. Control. Release 89 (2003) 151–165.

[40] V.R. Sinha, A.K. Singla, S. Wadhawan, R. Kaushik, R.

Kumria, K. Bansal, S. Dhawan, Chitosan microspheres as

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–2826

a potential carrier for drugs, Int. J. Pharm. 274 (2004)

1–33.

[41] J. Kreuter, Nanoparticles, in: J. Kreuter (Ed.), Colloidal Drug

Delivery Systems, Marcel Dekker, New York, 1994, pp.

219–342.

[42] L. Brannon Peppas, Recent advances on the use of

biodegradable microparticles and nanoparticles in the con-

trolled drug delivery, Int. J. Pharm. 116 (1995) 1–9.

[43] P. Couvreur, L. Grislain, V. Lenaerts, F. Brasseur, P. Guiot,

in: P. Guiot, P. Couvreur (Eds.), Polymeric Nanoparticles and

Microspheres, CRC Press, Boca Raton, FL, 1986.

[44] J. Akbuga, G. Durmaz, Preparation and evaluation of cross-

linked chitosan microspheres containing furosemide, Int. J.

Pharm. 11 (1994) 217–222.

[45] A.A. Al-Helw, A.A. Al-Angary, G.M. Mahrous, M.M. Al-

Dardari, Preparation and evaluation of sustained release

cross-linked chitosan microspheres containing phenobarbi-

tone, J. Microencapsulation 15 (1998) 373–382.

[46] E.B. Denkbas, M. Seyyal, E. Piskin, 5-Fluorouracil loaded

chitosan microspheres for chemoembolization, J. Micro-

encapsulation 16 (1998) 741–749.

[47] C. Sankar, M. Rani, A.K. Srivastava, B. Mishra, Chitosan

based pentazocine microspheres for intranasal systemic

delivery: development and biopharmaceutical evaluation,

Pharmazie 56 (2001) 223–226.

[48] S.R. Jameela, T.V. Kumary, A.V. Lal, A. Jayakrishnan,

Progesterone-loaded chitosan microspheres: a long acting

biodegradable controlled delivery system, J. Control. Release

52 (1998) 17–24.

[49] F. Bugamelli, M.A. Raggi, I. Orienti, V. Zecchi, Controlled

insulin release from chitosan microparticles, Arch. Pharm.

331 (1998) 133–138.

[50] K. Nishimura, S. Nishimura, H. Seo, N. Nishi, S. Tokura, I.

Azuma, Macrophage activation with multiporous beads

prepared from partially deacetylated chitin, J. Biomed. Mater.

Res. 20 (1986) 1359–1372.

[51] A. Berthod, J. Kreuter, Chitosan microspheres– improved

acid stability and change in physicochemical properties by

cross-linking, Proc. Int. Symp. Control. Release Bioact.

Mater. 23 (1996) 369–370.

[52] S. Ozbas-Turan, J. Akbuga, C. Aral, Controlled release of

interleukin-2 from chitosan microspheres, J. Pharm. Sci. 91

(2002) 124–125.

[53] H.Q. Mao, K. Roy, V.L. Troung-Le, K.A. Janes, K.Y. Lim, Y.

Wang, J.T. August, K.W. Leong, Chitosan DNA nano-

particles as gene delivery carriers: synthesis, characterization

and transfection efficiency, J. Control. Release 70 (2001)

399–421.

[54] P. He, S.S. Davis, L. Illum, Chitosan microspheres prepared

by spray drying, Int. J. Pharm. 187 (1999) 53–65.

[55] B. Conti, T. Modena, I. Genta, P. Perugini, C. Decarro, F.

Pavanetto, Microencapsulation of cetylpyridinium chloride

with a bioadhesive polymer, Proc. Int. Symp. Control.

Release Bioact. Mater. 25 (1998) 822–823.

[56] A. Ganza-Gonzalez, S. Anguiano-Igea, F.J. Otero-Espinar,

J.B. Mendez, Chitosan and chondroitin microspheres for

oral-administration controlled release of metoclopramide,

Eur. J. Pharm. Biopharm. 48 (1999) 149–155.

[57] X.Y. Shi, T.W. Tan, Preparation of chitosan/ethylcellulose

complex microcapsule and its application in controlled

release of vitamin D-2, Biomaterials 23 (2002) 4469–4473.

[58] C.W. Bishop, J.C. Knutson, C.R. Valliere, Method of treating

prostatic disease using delayed and/or sustained release

vitamin D formulation, US Pat. 5 795 882, 1998.

[59] P. Giunchedi, I. Genta, B. Conti, R.A.A. Muzzarelli, U.

Conte, Preparation and characterization of ampicillin loaded

methylpyrrolidinone chitosan and chitosan microspheres,

Biomaterials 19 (1998) 157–161.

[60] M.L. Lorenzo-Lamosa, C. Remunan-Lopez, J.L. Vila-Jato,

M.J. Alonso, Design of microencapsulated chitosan micro-

spheres for colonic drug delivery, J. Control. Release 52

(1998) 109–118.

[61] Y.C. Huang, M.K. Yeh, C.H. Chiang, Formulation factors in

preparing BTM-chitosan microspheres by spray drying

method, Int. J. Pharm. 242 (2002) 239–242.

[62] H. Tokumitsu, H. Ichikawa, Y. Fukumori, Chitosan– gado-

pentetic acid complex nanoparticles for gadolinium neutron

capture therapy of cancer: preparation by novel emulsion-

droplet coalescence technique and characterization, Pharm.

Res. 16 (1999) 1830–1835.

[63] A. Polk, B. Amsden, K.D. Yao, T. Peng, M.F.A. Goosen,

Controlled release of albumin from chitosan-alginate micro-

capsules, J. Pharm. Sci. 83 (1994) 178–185.

[64] L.S. Liu, S.Q. Liu, S.Y. Ng, M. Froix, T. Ohno, J. Heller,

Controlled release of interleukin-2 for tumor immunotherapy

using alginate:chitosan porous microspheres, J. Control.

Release 43 (1997) 65–74.

[65] Y. Kawashima, T. Handa, H. Takenaka, S.Y. Lin, Y. Ando,

Novel method for the preparation of controlled-release

theophylline granules coated with a polyelectrolyte complex

of sodium polyphosphate-chitosan, J. Pharm. Sci. 74 (1985)

264–268.

[66] Y. Kawashima, T. Handa, A. Kasai, H. Takenaka, S.Y. Lin,

The effect of thickness and hardness of the coating film on

the drug release of theophylline granules, Chem. Pharm.

Bull. 33 (1985) 2469–2474.

[67] R. Bodmeier, K.H. Oh, Y. Pramar, Preparation and evaluation

of drug-containing chitosan beads, Drug Dev. Ind. Pharm. 15

(1989) 1475–1494.

[68] S. Shirashi, T. Imai, M. Otagiri, Controlled release of

indomethacin by chitosan– polyelectrolyte complex: optimi-

zation and in vivo: in vitro evaluation, J. Control. Release 25

(1993) 217–225.

[69] A.D. Sezer, J. Akbuga, Controlled release of piroxicam from

chitosan beads, Int. J. Pharm. 121 (1995) 113–116.

[70] Z. Aydin, J. Akbuga, Chitosan beads for the delivery of

salmon calcitonin: preparation and characteristics, Int. J.

Pharm. 131 (1996) 101–103.

[71] P. Calvo, C. Remunan-Lopez, J.L. Vila-Jata, M.J. Alonso,

Chitosan and chitosan: ethylene oxide-propylene oxide block

copolymer nanoparticles as novel carriers for protein and

vaccines, Pharm. Res. 14 (1997) 1431–1436.

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–28 27

[72] P. Calvo, C. Remunan-Lopez, J.L. Vila-Jata, M.J. Alonso,

Novel hydrophilic chitosan-polyethylene oxide nanopar-

ticles as protein carriers, J. Appl. Polym. Sci. 63 (1997)

125–132.

[73] X.Z. Shu, K.J. Zhu, A novel approach to prepare tripoly-

phosphate/chitosan complex beads for controlled release drug

delivery, Int. J. Pharm. 201 (2000) 51–58.

[74] R. Fernandez-Urrusuno, P. Cavlo, C. Remunan-Lopez, J.L.

Vila-Jato, M.J. Alonso, Enhancement of nasal absorption of

insulin using chitosan nanoparticles, Pharm. Res. 16 (1999)

1576–1581.

[75] Y. Pan, Y. Li, H. Zhao, J. Zheng, H. Xu, G. Wei, J. Hao, F.

Cui, Chitosan nanoparticles improve the intestinal absorption

of insulin in vivo, Int. J. Pharm. 249 (2002) 139–147.

[76] Y. Xu, Y. Du, Effect of molecular structure of chitosan on

protein delivery properties of chitosan nanoparticles, Int. J.

Pharm. 250 (2003) 215–226.

[77] J.A. Ko, H.J. Park, S.J. Hwang, J.B. Park, J.S. Lee,

Preparation and characterization of chitosan microparticles

intended for controlled drug delivery, Int. J. Pharm. 249

(2002) 165–174.

[78] Y.S. Leong, F. Candau, Inverse microemulsion polymer-

ization, J. Phys. Chem. 86 (1982) 2269–2271.

[79] A. Maitra, Determination of size parameters of water–

Aerosol OT– oil reverse micelles from their nuclear magnetic

resonance data, J. Phys. Chem. 88 (1984) 5122–5125.

[80] P.L. Luisi, M. Giomini, M.P. Pileni, B.H. Robinson, Reverse

micelles as hosts for proteins and small molecules, Biochim.

Biophys. Acta 947 (1988) 209–246.

[81] S. Mitra, U. Gaur, P.C. Ghosh, A.N. Maitra, Tumor targeted

delivery of encapsulated dextran– doxorubicin conjugate

using chitosan nanoparticles as carrier, J. Control. Release

74 (2001) 317–323.

[82] S.A. Agnihotri, T.M. Aminabhavi, Controlled release of

clozapine through chitosan microparticles prepared by a

novel method, J. Control. Release 96 (2004) 245–259.

[83] J. Akbuga, N. Bergisadi, Effect of formulation variables on

cis-platin loaded chitosan microsphere properties, J. Micro-

encapsulation 16 (1999) 697–703.

[84] S.R. Jameela, A. Misra, A. Jayakrishnan, Cross-linked

chitosan microspheres as carriers for prolonged delivery of

macromolecular drugs, J. Biomater. Sci., Polym. Ed. 6 (1994)

621–632.

[85] R. Hejazi, M. Amiji, Stomach-specific anti-H. pylori therapy.

I: preparation and characterization of tetracyline-loaded

chitosan microspheres, Int. J. Pharm. 235 (2002) 87–94.

[86] D.K. Kweon, D.W. Kang, Drug-release behavior of chitosan-

g-poly(vinyl alcohol) copolymer matrix, J. Appl. Polym. Sci.

74 (1999) 458–464.

[87] T. Higuchi, Mechanism of sustained action medication, J.

Pharm. Sci. 52 (1963) 1145–1149.

[88] C. Washington, Drug release from microdisperse systems: a

critical review, Int. J. Pharm. 58 (1990) 1–12.

[89] R.H. Guy, J. Hadgraft, I.W. Kellaway, M.J. Taylor, Calcu-

lations of drug release rates from spherical particles, Int. J.

Pharm. 11 (1982) 199–207.

[90] P.L. Ritger, N.A. Peppas, A simple equation for description

of solute release. II. Fickian and anomalous release from

swellable devices, J. Control. Release 5 (1987) 37–42.

[91] N.A. Peppas, R.W. Korsmeyer, in: N.A. Peppas (Ed.),

Hydrogels in Medicine and Pharmacy, vol. 3, CRC Press,

Boca Raton, FL, 1987, p. 103.

[92] I. Orienti, K. Aiedeh, E. Gianasi, V. Bertasi, V. Zecchi,

Indomethacin loaded chitosan microspheres. Correlation

between the erosion process and release kinetics, J. Micro-

encapsulation 13 (1996) 463–472.

[93] D. Pantaleone, M. Yalpani, M. Scollar, Advances in chitin

and chitosan, in: C.J. Brine, P.A. Sandford, J.P. Zizakis,

Proceed. 5th Int. Conf. Chitin and Chitosan, Princeton, New

York, 1991.

[94] H. Zhang, I.A. Alsarra, S.H. Neau, An in vitro evaluation of a

chitosan-containing multiparticulate system for macromole-

cule delivery to the colon, Int. J. Pharm. 239 (2002) 197–205.

[95] I. Genta, M. Costantini, A. Asti, B. Conti, L. Montanari,

Influence of glutaraldehyde on drug release and mucoadhe-

sive properties of chitosan microspheres, Carbohydr. Polym.

36 (1998) 81–88.

[96] I.M. van der Lubben, F.A.J. Konings, G. Borchard, J.C.

Verhoef, H.E. Junginger, In vivo uptake of chitosan micro-

particles by murine Peyer’s patches: visualization studies

using confocal laser scanning microscopy and immuno-

histochemistry, J. Drug Target. 9 (2001) 39–47.

[97] I.M. van der Lubben, G. Kersten, M.M. Fretz, C. Beuvery,

J.C. Verhoef, H.E. Junginger, Chitosan microparticles for

mucosal vaccination against diphtheria: oral and nasal

efficacy studies in mice, Vaccine 21 (2003) 1400–1408.

[98] I.M. van der Lubben, J.C. Verhoef, A.C. van Aelst, G.

Borchard, H.E. Junginger, Chitosan microparticles for oral

vaccination: preparation, characterization and preliminary in

vivo uptake studies in murine Peyer’s patches, Biomaterials

22 (2001) 687–694.

[99] F. Shikata, H. Tokumitsu, H. Ichikawa, Y. Fukumori, In vitro

cellular accumulation of gadolinium incorporated into chito-

san nanoparticles designed for neutron-capture therapy of

cancer, Eur. J. Pharm. Biopharm. 53 (2002) 57–63.

[100] H. Tokumitsu, J. Hiratsuka, Y. Sakurai, T. Kobayashi, H.

Ichikawa, Y. Fukumori, Gadolinium neutron-capture therapy

using novel gadopentetic acid-chitosan complex nanopar-

ticles: in vivo growth suppression of experimental melanoma

solid tumor, Cancer Lett. 150 (2000) 177–182.

[101] S.R. Jameela, P.G. Latha, A. Subramoniam, A. Jayakrishnan,

Antitumor activity of mitoxantrone-loaded chitosan micro-

spheres against Ehrlich ascites carcinoma, J. Pharm. Phar-

macol. 48 (1996) 685–688.

[102] S.R. Jameela, A. Jayakrishnan, Glutaraldehyde cross-linked

chitosan microspheres as a long-acting biodegradable drug-

delivery vehicle-studies on the in-vitro release of mitoxan-

trone and in-vivo degradation of microspheres in rat muscle,

Biomaterials 16 (1995) 769–775.

[103] K.A. Janes, M.P. Fresneau, A. Marazuela, A. Fabra, M.J.

Alonso, Chitosan nanoparticles as delivery systems for

doxorubicin, J. Control. Release 73 (2001) 255–267.

S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 5–2828

[104] K.Y. Lee, I.C. Kwon, Y.H. Kim, W.H. Jo, S.Y. Jeong,

Preparation of chitosan self-aggregates as a gene delivery

system, J. Control. Release 51 (1998) 213–220.

[105] B.I. Florea, M. Thanou, M. Geldof, C. Meaney, H.E.

Junginger, G. Borchard, Modified chitosan oligosaccharides

as transfection agents for gene therapy of cystic fibrosis,

Proc. Int. Symp. Control. Release Bioact. Mater. 27 (2000)

846–847.

[106] P. Erbacher, S. Zou, T. Beuinger, A.M. Steffan, J.S. Remy,

Chitosan-based vector/DNA complexes for gene delivery:

biophysical characteristics and transfection ability, Pharm.

Res. 15 (1998) 1332–1339.

[107] Y.K. Park, Y.H. Park, B.A. Shin, E.S. Choi, Y.R. Park, T.

Akaike, C.S. Cho, Galactosylated chitosan graft-dextran as

hepatocyte-targeting DNA carrier, J. Control. Release 69

(2000) 97–108.

[108] J.L. Chew, C.B. Wolfowicz, H.Q. Mao, K.W. Leong, K.Y.

Chua, Chitosan nanoparticles containing plasmid DNA

encoding house dust mite allergen, Der p 1 for oral

vaccination in mice, Vaccine 21 (2003) 2720–2729.

[109] Z. Cui, R.J. Mumper, Chitosan-based nanoparticles for

topical genetic immunization, J. Control. Release 75 (2001)

409–419.

[110] G. Borchard, Chitosans for gene delivery, Adv. Drug Deliv.

Rev. 52 (2001) 145–150.

[111] B. Conti, P. Giunchedi, I. Genta, U. Conte, The preparation

and in vivo evaluation of the wound-healing properties of

chitosan microspheres, STP Pharma. Sci. 10 (2000) 101–104.

[112] A.M. De Campos, A. Sanchez, M.J. Alonso, Chitosan

nanoparticles: a new vehicle for the improvement of the

delivery of drugs to the ocular surface. Application to

cyclosporin A, Int. J. Pharm. 224 (2001) 159–168.

[113] T. Chandy, R.F. Wilson, G.H.R. Rao, G.S. Das, Changes in

cisplatin delivery due to surface-coated poly(lactic acid)-

poly(q-caprolactone) microspheres, J. Biomater. Appl. 16

(2002) 275–291.

[114] S.H. Chiou, W.T. Wu, Y.Y. Huang, T.W. Chung, Effects of

the characteristics of chitosan on controlling drug release of

chitosan coated PLLA microspheres, J. Microencapsulation

18 (2001) 613–625.

[115] M. Thanou, J.C. Verhoef, P. Marbach, H.E. Junginger,

Intestinal absorption of octreotide: N-trimethyl chitosan

chloride (TMC) ameliorates the permeability and absorption

properties of the somatostatin analogue in vitro and in vivo, J.

Pharm. Sci. 89 (2000) 951–957.

[116] K. Aiedeh, M.O. Taha, Synthesis of chitosan succinate and

chitosan phthalate and their evaluation as suggested matrices

in orally administered, colon-specific drug delivery systems,

Archiv.der Pharmazie 332 (1999) 103–107.

[117] M. Sakkinen, U. Seppala, P. Heinanen, M. Marvola, In vitro

evaluation of microcrystalline chitosan (MCCh) as gel-

forming excipient in matrix granules, Eur. J. Pharm.

Biopharm. 54 (2002) 33–40.

[118] R.A.A. Muzzarelli, V. Baldassarre, F. Conti, G. Gazzanelli, V.

Vasi, P. Ferrara, G. Biagini, The biological activity of chitosan:

ultrastructural study, Biomaterials 8 (1988) 247–252.

[119] R.A.A. Muzzarelli, Depolymerization of methylpyrrolidinone

chitosan by lysozyme, Carbohydr. Polym. 19 (1992) 29–34.

[120] R.A.A. Muzzarelli, In vivo biochemical significance of

chitin-based medical items, in: S. Dumitriu, M. Szycher

(Eds.), Polymeric Materials for Biomedical Applications,

Marcel Dekker, New York, 1992, pp. 179–198.

[121] X.G. Chen, C.M. Lee, H.J. Park, O/W emulsification for the

self-aggregation and nanoparticle formation of linoleic acids

modified chitosan in the aqueous system, J. Agric. Food

Chem. 51 (2003) 3135–3139.

[122] K.L. Shanta, D.R.K. Harding, Synthesis and characterization

of chemically modified chitosan microspheres, Carbohydr.

Polym. 48 (2002) 247–253.

[123] C. Zhang, Q. Ping, Y. Ding, Y. Cheng, J. Shen, Synthesis,

characterization and microsphere formation of galactosylated

chitosan, J. Appl. Polym. Sci. 91 (2004) 659–665.