JOURNAL OF AEROSOL MEDICINE AND PULMONARY DRUG DELIVERY

Volume 28, Number 0, 2015

Mary Ann Liebert, Inc.
Pp. 117
DOI: 10.1089/jamp.2014.1187

Development and Evaluation of Chitosan Microparticles

Based Dry Powder Inhalation Formulations
of Rifampicin and Rifabutin
Rohan V. Pai, MPharm,1 Rajesh R. Jain, MPharm1 Anilkumar S. Bannalikar, PhD,2 and Mala D. Menon, PhD1

Abstract

Background: The lung is the primary entry site and target for Mycobacterium tuberculosis; more than 80% of
the cases reported worldwide are of pulmonary tuberculosis. Hence, direct delivery of anti-tubercular drugs to
the lung would be beneficial in reducing both, the dose required, as well as the duration of therapy for
pulmonary tuberculosis. In the present study, microsphere-based dry powder inhalation systems of the antitubercular drugs, rifampicin and rifabutin, were developed and evaluated, with a view to achieve localized and
targeted delivery of these drugs to the lung.
Methods: The drug-loaded chitosan microparticles were prepared by an ionic gelation method, followed by
spray-drying to obtain respirable particles. The microparticles were evaluated for particle size and drug release.
The drug-loaded microparticles were then adsorbed onto an inhalable lactose carrier and characterized for
in vitro lung deposition on an Andersen Cascade Impactor (ACI) followed by in vitro uptake study in U937
human macrophage cell lines. In vivo toxicity of the developed formulations was evaluated using Sprague
Dawley rats.
Results: Both rifampicin and rifabutin-loaded microparticles had MMAD close to 5 lm and FPF values of
21.46% and 29.97%, respectively. In vitro release study in simulated lung fluid pH 7.4 showed sustained release
for 12 hours for rifampicin microparticles and up to 96 hours for rifabutin microparticles, the release being
dependent on both swelling of the polymer and solubility of the drugs in the dissolution medium. In vitro uptake
studies in U937 human macrophage cell line suggested that microparticles were internalized within the macrophages. In vivo acute toxicity study of the microparticles in Sprague Dawley rats revealed no significant
evidence for local adverse effects.
Conclusion: Thus, spray-dried microparticles of the anti-tubercular drugs, rifampicin and rifabutin, could prove
to be an improved, targeted, and efficient system for treatment of tuberculosis.
Key words: chitosan microparticles, dry powder inhaler, Andersen Cascade Impactor (ACI), macrophage uptake

Oral therapy using the currently employed antitubercular

drugs (ATDs) is effective, but associated with several major
drawbacks. More than 80% of TB cases are of pulmonary
TB, which require high drug doses to be administered, because only a small fraction of the total oral dose reaches the
lungs that are cleared in a few hours, making it necessary to
administer multiple ATDs on a regular basis for long periods (6 to 9 months), a regimen which the majority of TB
patients find difficult to adhere to.(1,3,4)

Introduction

uberculosis (TB) is the second most common cause

of death in the world after AIDS.(1) As per World Health
Organization (WHO) estimate, approximately one-third of
the global community is infected with Mycobacterium tuberculosis (M.tb) and India is the country with highest TB
burden, accounting for an estimated one quarter cases (2.0
2.5 million) reported worldwide in the year 2012.(2)

1
2

Department of Pharmaceutics, Bombay College of Pharmacy, Kalina, Santacruz (E), Mumbai, India.
Department of Microbiology, Bombay Veterinary College, Parel, Mumbai, India.

Despite the availability of highly effective drugs for TB,

cure rates have not improved.(57) This is largely due to
problems relating to drugs, formulations, and duration of
therapy. Although the first-line drugs for TB have been
highly effective in curtailing and curing the disease, the daily
dose of these therapeutic agents range from 300600 mg for
rifampicin, rifabutin, and isoniazid, and to 9001500 mg
for ethambutol and pyrazinamide.(3) The World Health Organization (WHO) suggests treatment of tuberculosis with
a standard multi-drug therapy called DOTS (Directly Observed Therapy, Short course) under which a patient is required to take two or more (200300 doses) of the anti-TB
drugs for a period of 612 months under observation.(1)
Such high doses formulated as solid oral formulations
such as tablets and capsules, along with the long duration of
treatment, make it inconvenient for the patients to complete
the course of therapy. Moreover, relatively few ATDs have
been discovered in the past 3 decades because of complex,
lengthy, and expensive process of drug development.(8)
Further to this, the emergence of resistant strains (multidrug
resistant strains) to existing drugs is a growing concern.(6)
Thus, considering the present scenario, due to difficulties in
obtaining new drugs for TB, it is important to look into the
existing drugs and develop novel and targeted delivery
systems of these drugs, which will overcome the drawbacks
of current conventional oral formulations.(912)
Direct delivery to the lungs of antibiotics, anti-infective
agents, and other drugs has been used for treatment of
several diseases of the lungs such as asthma, pneumonia,
and cystic fibrosis. There is potential for administration of
ATDs in a similar way to overcome the drawbacks of oral
delivery.(13) Since lung is the primary entry site and target
for Mycobacterium tuberculosis, direct delivery to the lung
can be expected to reduce the dose required and also significantly reduce the duration of therapy. This can also
overcome the toxicity issue by reducing the amount of drug
required by delivering it directly to the site of action.
Mycobacterium tuberculosis is an intracellular pathogen and
is difficult to treat because infections are localized within
phagocytic cells (alveolar macrophages) where they remain
in a dormant stage. Most antibiotics, although highly active
in vitro, do not actively pass through cellular membranes,
making it difficult to achieve the relatively high concentrations
of the drugs within the infected cells. The main challenge for
intracellular chemotherapy is to design and develop a carrier
system for antibiotics and antifungals that could be efficiently
endocytosed by phagocytic cells and, once inside the cells,
should deliver large concentrations for prolonged periods, so
that the number of doses and associated drug toxicity can be
reduced. It has long been established that macromolecular
drugs and particulate or vesicular drug delivery systems introduced into the deep lung are likely to be taken up by alveolar macrophages (AM).
The particulate carrier systems (micro-particles, liposomes, and nanoparticles) have been found to be effective in
delivering ATDs at the site of infection.(14,15) Among the
first line of drugs used, rifampicin and rifabutin would be
the drug of choice as they act against the intracellular,
nonreplicating dormant organisms, thereby reducing duration of therapy and preventing a relapse of the disease.(1)
The objective of the present study was to develop
microsphere-based dry powder for inhalation (DPI) system

PAI ET AL.

of ATDs, using methods free from organic solvents and

toxic cross-linking agents, with a view to achieve localized
and targeted delivery of these drugs to the alveolar macrophages within the lungs. Chitosan is a high molecular
weight polysaccharide linked by b-1, 4 glycoside. It is
composed of N-acetyl-glucosamine and glucosamine. It is
considered to be the most widely distributed biopolymer,
having huge resources.(16) It is a cationic polyelectrolyte
which is nontoxic, biocompatible, biodegradable, and has
been shown to be enzymatically degraded by the body, including in organs like the lungs.(16,17) The nontoxicity of
chitosan has been previously established by many authors in
lung epithelial cell lines as well as in vivo.(18,19)
The primary amine groups in chitosan renders it a positive
charge and contributes to mucoadhesive properties that
make chitosan very useful in drug delivery applications.(20)
Chitosan has attracted a great deal of attention in pharmaceutical drug delivery applications, including colon-targeted
drug delivery, mucosal delivery, cancer therapy, vaccine
delivery, gene delivery, and nasal and pulmonary delivery.(21,22) Today chitosan-based inhalation delivery systems
have become the focus of researchers for developing treatment strategies for asthma, COPD, and even diabetes.(13,23)
Thus, there is potential for administration of ATDs in a
similar way to overcome the drawbacks of oral delivery.
Materials and Methods

Rifampicin (RIF) and rifabutin (RFB) were obtained as

gift samples from Lupin Ltd, India. Chitosan (CHT) having
degree of deacetylation value of 90% was obtained as gift
sample from Esvee Agro and Feeds, Pune, India. Tripolyphosphate (TPP) was procured from Sigma Aldrich, India.
Lactohale 100 and Lactohale 300 were obtained as gift
samples from DFE Pharma, Germany. Hard gelatin capsules
were obtained as gift samples from Associated Capsules
Ltd, India. All other chemicals and reagents used were of
analytical grade.
Preparation of drug-loaded chitosan-TPP
microparticles

The chitosan microparticles were prepared by ionic gelation technique described by Ko et al.(24) and further isolated by spray drying. Briefly, accurately weighed quantities
of the drugs were dissolved in 1% (w/v) chitosan solution
prepared in 0.1 N acetic acid. Different concentrations
(0.5%2% w/v) of TPP solution were added dropwise to the
chitosan-drug solution under high-speed homogenization
(Silverson homogenizer, Sri Ram Industries, India) at
2000 rpm for 15 min. The suspension of chitosanTPPdrug
microparticles was then spray dried (LSD-48 spray dryer,
JISL, India) to obtain TPP cross-linked drug loaded microparticles. The parameters for spray drying are given in
Table 1. Batches with different ratios of chitosan and TPP
were prepared and characterized in order to evaluate its
effect on properties of microparticles.
Preparation of microparticle-based dry powder
inhalation (DPI) system

The microparticles obtained after spray drying were adsorbed onto an inhalable lactose carrier. A blend of a coarser

CHITOSAN MICROPARTICLES FOR PULMONARY DELIVERY

Table 1. Spray Drying Parameters

for Preparation of Chitosan Microparticles
Parameter

Values
150C 20C
65C 10C
10 rpm 4 rpm
40% 2%
1.5 kg/m3
23C 1C
and 40% 5% RH

Inlet temperature
Outlet temperature
Feed rate
Aspirator
Atomization pressure
Temperature and humidity

RH, Relative humidity; All values are expressed as mean SD

grade lactose (Lactohale 100) and fine grade lactose (Lactohale 300) was prepared in the ratio of 70:30 and used as the
carrier system for the microparticles. Briefly, 400 mg of
drug-loaded microparticles were mixed well with 2000 mg of
pre-formed Lactohale blend. The admixture was then passed
through ASTM (American Society for Testing and Materials) 60 sieve (250 micron) and mixed well again. Samples
were taken from three different locations from the bulk blend
and analyzed for drug content in order to ascertain content
uniformity. The remaining blend was filled into size 3 hard
gelatin capsules (average fill weight: 25 mg/capsule).
Characterization of drug-loaded
chitosan-TPP microparticles
Moisture content. The moisture content of the microparticles was determined by the Karl-Fisher titration method. Accurately weighed quantities of microspheres (100 mg)
were titrated on a Mettler DL40GP Memotitrator using Karl
Fisher reagent. The percent moisture content was determined using the titer reading by the following formula:

Moisture Content (%)

Burette reading Factor

100
Weight of sample (mg)

Particle size and zeta potential. Particle size and zeta

potential were determined using a Malvern ZetaSizer
(Nano-ZS, U.S.A). For determination of particle size, the
microparticles were suspended in methanol containing 0.1%
Tween 80 to prevent aggregation, and the mean particle size
and polydispersity index were determined. For determination of zeta potential, the microparticles were suspended in
de-ionized water and placed in disposable polystyrene
electrophoretic cells. The total zeta runs were 100 and the
count rate was 250 particles/sec. The experiments were
performed in triplicate.
X ray diffraction (XRD). XRD patterns of the free drug,
excipients, and drug-loaded microparticles were recorded to
assess their solid state characteristics. The samples were
prepared and analyzed using a Phillips X Pert MPD diffractometer (TIFR, Mumbai) with a copper target, operated at
a voltage of 40 kV and 20 mA current, at a scanning speed of
2 per minute. The experiment was performed in triplicate.
Differential scanning calorimetry (DSC). The DSC thermograms of RIF, RFB, and their microparticles were re-

corded on a Diamond DSC (Perkin Elmer) thermal analyzer.

The drug sample was heated in an aluminum crimped pan,
sealed with a platinum lid at a heating rate of 20C/min in
the 50250C temperature range under a nitrogen atmosphere purged at a flow of 50 mL/min. The experiment was
performed in triplicate.
Surface morphology. Surface topography of the free drug,
drug-loaded microparticles and the DPI system was visualized
using a scanning electron microscope (Zeiss EVO LS10,
Germany). The samples were prepared by plasma deposition
method using a gold coating unit to make the surface of the
microparticles conductive to the scanning electron beam.
About 40100 A thick coat of gold was applied on the microparticle surfaces. The gold coating was done under argon
atmosphere. The gold-coated microparticles were scanned
using secondary electron beam of 10 KV intensity.
Drug content/loading efficiency. Drug content was determined by UV spectrophotometry. The drug from 10 mg of
accurately weighed microparticles was extracted in 5 mL of
methanol by sonication in a bath sonicator (Ultrasonic
Systems, India) for 15 min, followed by centrifugation
(Remi, India) at 1000 g for 10 min. The sediment was given
two washings with 5 mL of filtered distilled water and the
above steps of sonication and centrifugation were repeated.
The supernatant and washings were combined and the absorbance was recorded on a UV-Vis spectrophotometer
( Jasco 530V, Japan) at 239 nm for RIF and 277 nm for RFB,
using appropriate blanks as the reference. The experiment
was performed in triplicate.
Swelling index. The swelling properties of the chitosan
microparticles was evaluated by the difference in weight of
the microparticles before and after incubation in simulated
lung fluid (SLF), pH 7.4 for a period of 24 h, at 37 0.5C in
an incubator (Tempo Instruments and Equipments, India).
The composition of simulated lung fluid, pH 7.4 is given in
Table 2.(25) The experiment was performed in triplicate.
In vitro drug release. The in vitro drug release from the
microparticles was evaluated by a static method in simulated lung fluid (SLF), pH 7.4 at 37 0.5 C and compared
to that of free drug under the same conditions. An accurately
weighed quantity (10 mg) of drug-loaded microparticles was
taken in a dialysis bag (Himedia; MWCO: 1200014000
Daltons) containing 5 mL of SLF, pH 7.4 and tied at both
ends. The dialysis bag was suspended in a 100 mL stoppered
test tube containing 50 mL of SLF, pH 7.4, and the assembly
was placed in an incubator (Tempo Instruments and
Equipments, India) maintained at 37 0.5C.
At specific intervals of time, the whole dissolution medium was replaced with 50 mL of fresh pre-warmed medium
(37 0.5C) and the dialysis bag was inverted each time to
prevent the microparticles from adhering to the sides of the
bag or aggregating. The aliquots were evaluated for drug
content on a UV-Vis spectrophotometer ( Jasco 530V, Japan)
using SLF, pH 7.4, as the blank.
In order to further understand drug release mechanism,
the drug release data was plotted into the Korsmeyer-Peppas
equation as log cumulative percentage of drug released
versus log time, and the value of diffusion exponent (n) was

PAI ET AL.

Table 2. Composition of Simulated

Lung Fluid, pH 7.4
Ingredients

Quantity

Magnesium chloride hexahydrate

Sodium chloride
Potassium chloride
Disodium hydrogen orthophosphate
(anhydrous)
Sodium sulfate (anhydrous)
Sodium bicarbonate
Sodium acetate trihydrate
Sodium citrate dihydrate
Calcium chloride dihydrate
0.1 M hydrochloric acid; q.s to adjust
Distilled water

0.102 gm
5.727 gm
0.298 gm
0.142 gm
0.142 gm
2.604 gm
0.826 gm
0.098 gm
0.368 gm
pH 7.4
q.s 1000 mL

calculated using the slope of the straight line. The experiment was performed in triplicate.
In vitro lung deposition. In vitro lung deposition of drugloaded microparticles, plain drug, and microparticles with
lactose blends was evaluated on an Andersen Cascade Impactor(ACI) (Copley Scientific, U.K) at a flow rate of
28.3 L/min using a Lupihaler device (Lupin, Ltd.). Fine
particle dose (FPD), fine particle fraction (FPF), mass median aerodynamic diameter (MMAD), and geometric standard deviation (GSD) were calculated as per USP using
CITDAS software application (Copley Scientific, U.K). The
experiment was performed in triplicate.
Stability study

The stability study of selected batch (RIF2) of RIF-loaded

microparticles, formulated as the dry powder inhaler blend
in hard gelatin capsule (size 3) and sealed in tri-laminate
pouches, was carried out at room temperature and at
40C 2C/75% RH 5% RH for a period of 3 months. At
intervals of 1 month, the samples were evaluated to detect
any changes with respect to visual appearance, drug content,
moisture content, and in vitro lung deposition. The analysis
was performed in triplicate
In vitro macrophage uptake study

In vitro uptake by macrophages was assessed on U937 human macrophage cell lines by the method mentioned by Evora
et al.(26) The RIF2 and RFB3 batches, which showed highest
in vitro lung deposition, were selected for the study. Different

concentrations of the drug-loaded microparticles were added

to a cell culture suspension of U937 human macrophage cells
equivalent to 2 106 cells per well and incubated in a 5% CO2
environment at 37C for a period of 90 min in a CO2 incubator
(Thermo Scientific, India). The macrophages were isolated by
centrifugation (Remi, India) at 1000 g for 15 min and the supernatant was analyzed spectrophotometrically for drug content. The experiment was performed in triplicate.
In vivo acute toxicity study

Acute inhalation toxicity evaluation of drug-loaded microparticles of RIF and RFB was carried out by intratracheal
instillation in female Sprague Dawley rats. The protocol followed for the study was approved by an independent animal
ethics committee of Bombay College of Pharmacy (30/04/
2010/CPCSEA/BCP/2010/04). The animals were divided into
seven groups of three animals each as shown in Table 3.
The animals were anesthetized with an intra-peritoneal
injection of 0.2 mL of ketamine hydrochloride. The animal
was harnessed onto the wooden board with the help of
stretch bands. A ventral incision of 0.5 cm was made over
the tracheal region superior to the supraclavicular notch.
The trachea was exposed through the longitudinal incision
along the ventral aspect of the neck. 200 lL of suspension of
microparticles (containing equivalent amount of 1 mg/mL of
drug) or drug solutions (1 mg/mL) in phosphate buffered
saline (PBS), pH 7.4, were administered intratracheally with
a tuberculin syringe having 27-gauge needles.
Similarly, 200 lL of suspension of blank chitosan microparticles (0.5 % w/v) in PBS, pH 7.4, was administered
to the respective control groups. The skin was then sutured
with a sterile braided surgical silk thread and an antiseptic
ointment (Betadine) was applied over the stitches. The animals were kept upright against a wooden board such that
they remained upright till they regained consciousness.
The animals were then administered 200 lL of amoxicillin
subcutaneously to prevent infection. The dosed animals were
observed daily for any abnormal reactions or mortality for a
period of 14 days. At the end of this period, the animals were
humanely sacrificed by ketamine injection and their lung
tissues were excised and subjected to histopathological examination to assess any bronchiolar epithelial hyperplasia,
wall thickening, edema or accumulation of eosinophils,
neutrophils, or mononuclear inflammatory cells.
Results and Discussion

Direct delivery of antibiotics, other anti-infective agents,

and therapeutic agents to the lungs has been advocated for

Table 3. Grouping of Animals for in Vivo Toxicity Study

Group
Vehicle control
Blank chitosan
microparticles (0.5 % w/v)
RIF solution*
RFB solution#
RIF microparticles*
RFB microparticles#

Description

No. of animals

PBS, pH 7.4 administered

Suspension of chitosan microparticles in PBS, pH 7.4

3
3

Free RIF suspension in PBS, pH 7.4 administered

Free RFB suspension in PBS, pH 7.4 administered
Suspension of RIF microparticles in PBS, pH 7.4 administered
Suspension of RFB microparticles in pH, 7.4 administered

3
3
3
3

PBS, phosphate buffered saline, pH 7.4; RFB, rifabutin; RIF, rifampicin; *Equivalent to 200 lg of RIF; #Equivalent to 200 lg of RFB.

CHITOSAN MICROPARTICLES FOR PULMONARY DELIVERY

treatment of several locally occurring diseases of the lungs

such as asthma, pneumonia, and cystic fibrosis. Thus there is
a potential for administration of ATDs in a similar way to
overcome the drawbacks of oral delivery.(15) Since the lung
is the primary entry site and target for Mycobacterium tuberculosis, direct delivery to the lung would not only reduce
the dose required, but also significantly reduce the duration
of therapy.
Mycobacterium tuberculosis is known to reside in alveolar
macrophages, thus delivery to the lung in the form of particulate or vesicular delivery systems such as microspheres,
nanoparticles, and liposomes can also help in targeting the
bacilli that remain dormant within these macrophages. Many
researchers have reported delivery systems for targeting alveolar macrophages via the pulmonary route with promising
results.(27) Inhalable microparticles containing ATDs fabricated as dry powder for inhalation (DPI) have great potential
in overcoming the drawbacks of current therapy.(13)
Preparation of drug-loaded chitosan-TPP
microparticles

Chitosan microparticles are prepared by several methods

involving use of cross-linking agents such as formaldehyde,
glutaraldehyde, sodium hydroxide, and ethylene glycol,
di-glycidil ether. However, these chemical cross-linking
agents are capable of imparting undesirable effects to the
formulation, the active ingredient, and also to the mucosal
membranes.(28,29) To overcome these disadvantages of
chemical cross-linking, ionic cross-linking has been widely
proposed.(3032) Tripolyposphate is nontoxic in nature and
thus a good alternative to the harsh cross-linking agents like
formaldehyde.
Chitosan is insoluble in aqueous solutions; however it is
soluble in aqueous acids. In acidic solutions, the -NH2
group of chitosan is protonated (  NH3 ), rendering the
polymer a cationic charge.(16,20,31) The cationic nature of
chitosan provides mucoadhesive properties, which enable it
to adhere to the mucosa of the respiratory tract and prolong
drug release. The cationic nature also enables it to be a good
substrate for cross-linking with negatively charged molecules.(33) TPP is a multivalent anion and thus it can ionically
interact with the positively-charged amino groups of chitosan to form a matrix structure.(24)
This type of interaction between chitosan and TPP is
reported to affect the characteristics of the formed micro-

particles like surface morphology, particle size, and release

profile. Moreover the particle size and the release pattern of
the microparticles can be easily controlled by varying the
concentration of either chitosan or TPP solution.(34,35) The
microparticles have to be isolated from the suspension by
using suitable methods. Filtration and freeze drying techniques have been unsuccessful in isolating the formed microparticles owing to their gel like characteristics, and hence
other methods like spray drying need to be explored.(35)
Spray drying is a one-stage continuous process widely used
in pharmaceutical industries to produce powders for inhalation
delivery. The microparticles obtained after spray drying are
fine, light, and can be aerosolized easily, enabling efficient
delivery to the lungs. The particle size obtained by spray
drying is near to 1 lm and thus can reach the alveolar regions
of the lungs.(10,36,37) In the present study, microparticles prepared using different ratios of chitosan and TPP were spray
dried to assess the effect of concentration of TPP on particle
size, process yield, and drug entrapment (Table 4).
The yield of the microparticles obtained after spray drying was found to be between 9%30% for RIF-loaded microparticles (Table 4) and decreased with an increase in the
amount of TPP. However, no significant difference was
observed in the yield obtained for RFB-loaded microparticles for different concentrations of TPP used. The differences in the yield for different batches of RIF and RFB
loaded microparticles were due to the different amounts
of TPP used for cross-linking as seen from ratios in the
Table 4. Higher concentration of TPP resulted in formation
of a viscous dispersion prior to spray drying, which in turn
resulted in low yield as compared to batches with less viscous dispersions. For RIF loaded microparticles, higher
amount of TPP was used as compared to RFB loaded microparticles, resulting in viscous dispersion prior to spray
drying and hence differences in the yield.
Characterization of microparticles
Moisture content. Moisture content is a critical parameter for microparticles to be administered by inhalation as it
affects the deposition of particles in lungs. High residual
moisture content can lead to aggregation of particles causing
an increase in particle size, thus resulting in lesser fraction
of inhaled drug reaching the alveolar region of lung.(38,39)
Selected batches of microparticles (RIF2 and RFB3) were
characterized for moisture content. RIF and RFB

Table 4. Composition and Characterization of Different Batches of Microparticles

Batch

TPP:
CHT ratio

Yield (%)

Particle size
(mm)

P.I.

Loading
efficiency (%)

Zeta potential
(mv)

Swelling
index (%)

RIF1
RIF2
RIF3
RIF4
RFB1
RFB2
RFB3
RFB4

1:4
1:2
1:1.5
1:1
1:8
1:6
1:4
1:2

30.17
29.52
18.16
9.15
15.61
17.25
20.17
14.37

1.904 0.623
2.407 0.536
2.471 0.481
3.403 0.935
1.146 0.832
1.187 0.393
1.270 0.609
1.769 0.588

0.672 0.063
0.531 0.052
0.368 0.022
0.751 0.083
0.258 0.073
0.283 0.065
0.623 0.086
0.950 0.045

59.35 3.63
56.36 1.93
50.65 2.47
45.51 1.48
89.41 1.83
83.83 3.63
70.77 2.42
69.47 1.57

19.5 1.2
18.6 2.3
24.7 1.9
22.8 1.6
18.1 0.9
21.7 2.6
29.4 3.2
23.2 1.7

1064.7 69.3
758.2 52.6
684.6 47.2
576.3 32.4
1659.1 67.1
1682.9 60.9
1576.5 60.5
1259.5 55.8

CHT, chitosan; P.I., polydispersity index; TPP, tripolyphosphate.

All values are expressed as mean SD; n = 3.

PAI ET AL.

microparticles had moisture content of 1.43% and 1.58%,

respectively, which were found to be relatively lower than
previously reported for spray-dried formulations.(38,40,41)
Particle size and zeta potential. The particle sizes of the
spray-dried microparticles with different concentrations of
TPP are as shown in Table 4. The particle size increased
with an increase in the concentration of TPP, for both RIF
and RFB loaded microparticles. Many authors have reported
increase in particle size of microparticles with an increase in
the amount of cross-linking agent added.(24,31,35) This may
be due to increase in the viscosity of the solution to be spray
dried, resulting in a larger droplet size during spray drying

and also due to increasing amount of cross-linking agent

getting incorporated in the same volume of droplets which
are produced during atomization before drying.(35)
The particle size showed a unimodal distribution that is
also evident from low polydispersity indices. The particle
sizes of all the batches were below 5 lm, which is critical
for inhalation delivery. Particles below 5 lm are regarded as
respirable fraction and defined as the fraction of the inhaled
particles which can reach the alveolar regions of the lungs.
All spray-dried batches of microparticles showed positive
zeta potential values, ranging from 18.129.4 mV (Table 4).
No particular trend was observed in the zeta potential of
spray-dried microparticles with respect to concentration of

FIG. 1. X-ray diffractograms of (a) RIF, RFB, and their microparticles (RIF1; RFB1);
(b) chitosan (CHT), tripolyphosphate (TPP), and lactohale (LH).

CHITOSAN MICROPARTICLES FOR PULMONARY DELIVERY

TPP. The results were as expected, since chitosan is a cationic polymer owing to the protonated  NH3 groups
present in an acidic medium and thus shows an inherent
positive charge.(16) However, even after interaction with
anionic TPP, chitosan retained its cationic nature.
X-ray diffraction (XRD). The X-ray diffractograms of
free drugs and drug loaded microparticles are shown in
Figure 1a. No sharp peaks, prominent for RIF, were observed in XRD spectra of RIF-loaded microparticles (RIF1),
indicating that the drug was completely embedded within
the polymer matrix and not adhering to the surface of the
particles. RFB was obtained as an amorphous powder, thus
the results for free RFB and RFB-loaded microparticles
(RFB2) are consistent with the nature of RFB.
Two diffused peaks were observed at 2h values of 10 and
20 in both RIF and RFB-loaded microparticles. This pattern was similar to the XRD pattern of chitosan.(16) Thus,
chitosan was found to retain its partial crystalline nature
even after the spray drying process. The sharp peaks observed in the XRD pattern of TPP (Fig. 1b) were not seen in
the XRD patterns of RIF as well as RFB-loaded microparticles, indicating absence of any residual TPP.
Differential scanning calorimetry (DSC). The thermogram of RIF showed an endotherm having an onset at
186.49C and reaching a peak value at 197.75C, corresponding to its melting point, immediately followed by an
exotherm corresponding to the recrystallization of the melt.
The thermogram of RFB showed an endotherm having an
onset at 137.63C and reaching a peak at 143.06C. The
peaks corresponding to the endotherms of RIF and RFB
were not observed in the thermograms of the drug-loaded

FIG. 2.

microparticles, indicating that the drugs were embedded

within the amorphous polymeric matrix (Fig. 2).
Drug content/loading efficiency. The drug loading of
microparticles with different concentrations of TPP was
between 45%60% for RIF loaded microparticles and 70%
89% for RFB loaded microparticles (Table 4). The increase
in the concentration of TPP was found to affect the drug
loading of the microparticles. The amount of drug loaded
onto the microparticles decreased with an increase in the
concentration of TPP added, which may be attributed to the
increase in the extent of cross-linking, resulting in less accommodation of drug within the polymer matrix.(35) As
discussed earlier, the interaction of chitosan with TPP results in the formation of a polymeric matrix. At lower
concentrations of TPP, the chitosan network is loose and has
high hydrodynamic free volume to accommodate more of
the solvent molecules, whereas at higher concentrations of
TPP, the chitosan network is dense, thus having low free
volume for accommodation of solvent molecules. RIF has
been reported to interact with the binding sites on chitosan.
Thus the decrease in drug loading with an increase in
amount of TPP may also be due to availability of lesser
binding sites on chitosan polymer network, due to its interaction with TPP ions.(24,31,35)
The aliquots sampled from three different locations of the
bulk blend demonstrated acceptable blend content uniformity with an RSD of less than 5%, indicating formation of a
homogenous blend of microparticles and the carrier system
prior to filling in hard gelatin capsules.
Surface morphology. The surface morphologies of the

free

drugs

and

selected

batches

DSC thermograms of RIF, RFB, and their drug loaded microparticles.

of

drug

loaded

PAI ET AL.

FIG. 3. SEM images of (a) RIF and (b) RFB in free form showing irregularly shaped and
non-uniform particles.
microparticles (RIF2 and RFB3) were visualized using an
EVO LS10 (Ziess, Germany) scanning electron microscope.
The surface morphology of the spray-dried microparticles is
important as it gives valuable information about the particulate nature of spray dried products. The properties of
materials may change after the spray drying, leading to
formation of agglomerates, skin-forming particles, crystalline structures, hollow or porous particles.(36)

Also, flow properties, release profile, and swelling behavior can depend on the nature of matrix or surface of the
polymeric microparticles that can be ascertained using
SEM.(40,42) The surface morphology of particles can also
affect their deposition into the lungs. Particles that are
spherical and have smooth surfaces have good aerodynamic
properties and thus deposit deep into the lungs. However,
irregularly shaped particles with rough surfaces deposit in

CHITOSAN MICROPARTICLES FOR PULMONARY DELIVERY

FIG. 4. SEM images of (a) RIF microparticles and (b) RFB microparticles showing
differences in surface characteristics.
the upper regions of the respiratory tract by impaction due to
their poor aerodynamic properties.(43)
The SEM images of the free drugs are shown in Figure 3.
RIF appears as distinct rod shaped, non-uniform crystals of
size ranging from 10100 lm, whereas RFB appears as fine
agglomerated particles with un-uniform particle sizes. Such
a morphology and particle size is not suitable for the lung
delivery. This further justifies the need to incorporate these

drugs into a more aerodynamically suitable system such as

the microparticles for delivery to the lungs.
The SEM images for RIF and RFB loaded microparticles
are shown in Figure 4. The sizes of the microparticles of
both RIF and RFB were observed to be 12 lm, which was
within the required size range of 15 lm, and hence suitable
for lung delivery. The particle size was also in conformation
with the particle size obtained from the zeta sizer.

10

PAI ET AL.

FIG. 5. SEM images of DPI blend of (a) RIF and (b) RFB-loaded microparticles adsorbed onto the lactohale carrier particles.
The concentration of TPP was seen to have an effect on
the surface morphology of spray-dried chitosan microparticles. Lower concentrations of TPP resulted in formation
of irregularly shaped microparticles with smooth surfaces,
whereas higher concentrations of TPP resulted in formation of distinctly spherical particles having rough surfaces.(24) This can be seen from the SEM images of the

drug-loaded microparticles of both the drugs. RIF-loaded

microparticles have a rough surface and look more
spherical than RFB-loaded microparticles, which have a
wrinkled or corrugated surface. This is due to the difference in the amount of TPP used in their preparation (Table
4). The batch RIF2 of RIF loaded microparticles had more
than twice the amount of TPP than batch RFB3 of RFB

CHITOSAN MICROPARTICLES FOR PULMONARY DELIVERY

11

FIG. 6. Release profile of (a) RIF and (b) RFB-loaded microparticles in SLF, pH 7.4,
showing sustained release as compared to free RIF and RFB. (All values are expressed as
mean SD; n = 3)
loaded microparticles, which may have resulted in formation of denser structure as observed from the images
(Fig. 4).
The SEM images of DPI blends of drug loaded microparticles are as shown in Figure 5. The drug-loaded microparticles are seen adhering to the larger particles of the
carrier system indicating formation of a homogenous blend
for DPI.

Swelling studies. Cross-linked chitosan has a matrix-like

structure and thus has the ability to absorb water and swell.
The extent of swelling is directly proportional to crosslinking density, which in turn depends on the amount of
cross-linking agent used.(31) At higher concentrations of
cross-linking agent, the polymer network is densely packed
and thus can accommodate less volume of solvent molecules, whereas at lower concentrations of cross-linking

12

agent, the polymer network is loosely held, resulting in

voids and is able to accommodate higher volume of solvent
molecules.(35)
All the batches of microparticles underwent swelling in
aqueous medium as seen in Table 4. The swelling index decreased with an increase in concentration of TPP for both RIF
as well as RFB-loaded microparticles. This difference in
swelling indices between RIF and RFB-loaded microspheres is
also evident from the differences in the surface morphology of
the two systems, as seen in the SEM images (Fig. 4). The RIF
loaded microparticles appear dense, spherical, and highly
cross-linked, owing to higher concentration of TPP and thus
would accommodate lesser volume of medium. RFB-loaded
microparticles show a less rough surface, which is corrugated,
and loosely cross-linked due to lesser amounts of TPP used and
thus accommodates greater amount of the media in the study.
In vitro drug release. The release profiles of different
batches of RIF-loaded microparticles are shown in Figure 6a.
The RIF-loaded microparticles released 90% of the drug at
the end of 12 hours. The release profile of RIF-loaded microparticles was found to be sustained as compared to the
release of free RIF and no burst release of the drug was
observed. The release of drug from the microparticles was
found to increase with an increase in the amount of TPP.
This was in contradiction to the findings reported by
previous authors,(35) who found that release of drug from the
chitosan-TPP microparticles decreased as the amount of
cross-linking agent is increased as a result of formation of
dense polymeric network, thus slowing drug release. However, for polymers such as chitosan that exhibit varied
swelling behavior with varying amounts of cross-linking
agent, the release pattern is different.
The swelling of microparticles affects the release profile by
changing the path length followed by the drug, embedded in
the microparticles, to diffuse into the surrounding medium.
Greater extent of swelling results in longer path length,
whereas lesser extent of swelling keeps the path length short.
At higher concentrations of cross-linking agent, the polymer
network is densely packed and hence enables swelling to a
lesser extent, whereas at lower concentrations of cross-linking
agent, the polymer network is loosely held resulting in swelling
to a greater extent.(31,44) Hence, the RIF microparticles crosslinked with less amount of TPP (RIF1) showed slower release
than those cross-linked with higher amounts of TPP (RIF4).
In order to further understand drug release mechanism,
the drug release data was plotted into the Korsmeyer-Peppas
equation as log cumulative percentage of drug released
versus log time, and the value of diffusion exponent (n) was
calculated using the slope of the straight line. If the exponent n is less than 0.45, then the drug release mechanism is
by Fickian diffusion, and if n is 0.45 < n <0.89, then it is by
non-Fickian or anomalous diffusion. An exponent value of
0.89 is indicative of Case-II Transport or typical zero-order
release, while value higher than 0.89 is indicative of Super
case-II Transport.(44,45) The values of n obtained from the
slope of the equations, for different batches of Rifampicin
loaded microparticles are shown in Table 5. All batches of
rifampicin-loaded microparticles had n values less than 0.45
indicating Fickian diffusion.
The release profiles of different batches of RFB loaded
microparticles are shown in Figure 6b. The RFB-loaded

PAI ET AL.

Table 5. Values of Diffusion Exponent (n)

for RIFand RFB Loaded Microparticles
Batch

Diffusion exponent (n)

Interpretation

RIF1
RIF2
RIF3
RIF4
RFB2
RFB3
RFB4

0.16
0.36
0.25
0.1
0.52
0.67
0.89

Fickian diffusion
Fickian diffusion
Fickian diffusion
Fickian diffusion
Non-Fickian diffusion
Non-Fickian diffusion
Non-Fickian diffusion

microparticles released 90% of the drug at the end of 96

hours. The release profile of RFB-loaded microparticles was
more sustained as compared to the release of free RFB and
no burst release of the drug was observed. The release of
RFB-loaded microparticles decreased, with an increase in
the amount of TPP added. This was unlike RIF-loaded microparticles, for which the drug release increased with an
increase in the amount of TPP.
Although both RIF and RFB-loaded microparticles exhibited swelling in the media, the difference in the release
profiles of the microparticles may be due to the differences in
the drug solubility. RIF is more readily soluble in aqueous
media than RFB, which is only slightly soluble. Thus the rate
limiting step for release of RIF from the microparticles is
diffusion through the chitosan matrix. In the case of RFBloaded microparticles, the solubility of RFB being very low,
the rate limiting step for release of drug from the microparticles is dissolution into the surrounding medium. Thus, release
of RFB from the microparticles is a function of the drug
present within the microparticles, hence slower release of RFB
from microparticle as compared to RIF microparticles. This
can also be seen also be seen from the values of diffusion
exponent, obtained after plotting Korsmeyer-Peppas equation
were greater than 0.45 but less than 0.89, indicating nonFickian diffusion (Table 5).
In vitro lung deposition
Deposition on ACI. Particle size distribution is one of the
important parameters for in vitro testing of DPI systems and
is usually obtained with the help of cascade impactors. The
ACI is comprised of different plates or stages corresponding
to the different regions of the respiratory system, from the
oral cavity to alveoli. The particulate matter is drawn at a predetermined air-flow rate through the apparatus, resulting in
deposition of the particles on different plates of ACI as a
function of their particle size and aerodynamics. The ACI is
similar to a twin stage impinger (TSI) in function. However
in addition to particle size, it gives a detailed lung deposition
data with additional parameters like MMAD, GSD, and FPF.
MMAD can be used as the predictor of the particle distribution in the lung. GSD is a measure of the variability of the
particle diameters within the system. A DPI system with a GSD
less than 1.2 is described as monodisperse. The FPF is generally
regarded as the fraction of the total inhaled dose that reaches the
stages corresponding to the cut-off diameter of 5 lm.
The batches of microparticles (RIF2, RIF3, RFB2, and
RFB3), which showed promising results (data not shown) on
TSI, were selected for a detailed study on an 8-stage Andersen

CHITOSAN MICROPARTICLES FOR PULMONARY DELIVERY

13

FIG. 7. Parameters of lung deposition study of free drugs and their microparticles on
ACI with respect to MMAD, GSD, and FPF. All values are expressed as mean SD; n = 3,
FPF, fine particle fraction; GSD, geometric standard deviation; MMAD, mass median
aerodynamic diameter.

cascade impactor. It was observed that free RIF and RFB,

along with the carrier blend, did not show any appreciable
results on the ACI as seen from the low FPF values (Fig. 7).
A major portion of the emitted dose was found deposited on
the pre-separator stage (oral cavity). This is due to the large
particle size of the drug crystals, rod shaped morphology of RIF
and agglomerated nature of RFB as observed in SEM images
(Fig. 3). Among the two batches of RIF-loaded microparticles,
no significant differences were observed with respect to
MMAD, GSD, and FPF. However, the values of MMAD and
GSD near to 5 lm and 1.2, respectively, are indicative of
monodisperse DPI system suitable for deep lung delivery.
The results obtained for RFB-loaded microparticles were
similar to those obtained for RIF-loaded microparticles (Fig.
7). However, as compared with RIF-loaded microparticles,
RFB-loaded microparticles showed better deposition in the
later stages of the ACI, indicating a greater FPF. This can
again be attributed to the surface characteristics of the microparticles as seen in Figure 4. RFB microparticles appear

smooth and have a corrugated surface as compared to RIF

microparticles, which exhibited a rough surface. These
differences in the surface morphology of the microparticles
result in a change in their aerodynamic pattern, thus affecting in vitro deposition.(36.40)
Stability study

Spray-dried particles are prone to moisture uptake and

aggregation during storage, which may further result in an
increase in the particle size distribution, and thereby alter the
lung deposition profile of the DPI.(41) Thus it is important to
evaluate stability of spray-dried particles with moisture
content and particle size and in vitro lung deposition.
The selected batch of RIF-loaded microparticles (RIF2),
packed in trilaminate aluminum pouches, was subjected to storage at room temperature (ambient temperature) and at 40C
2C/75% RH 5% RH in order to evaluate stability. Different
parameters were evaluated, such as color, appearance, particle

Table 6. Results of Stability Studies of RIF2 Microparticles at RT and 40C/75% RH

Time

Condition

Appearance

Moisture content (%w/w)

Particle size (mm)

Drug content (%w/w)

0M
1M

R.T
40 C/75% RH
R.T
40 C/75% RH
R.T
40 C/75% RH

No change
No change
No change
Darkening
No change
Darkening

1.43 0.90
2.37 0.97
3.63 1.41
6.49 1.92
7.00 1.82
6.68 1.91
7.95 2.18

2.41 0.88
2.47 0.67
2.42 0.95
2.35 0.87
3.64 1.26
2.43 0.91
4.11 1.05

50.65 2.47
47.76 2.66
46.42 1.71
49.21 1.56
48.95 2.79
46.14 2.22
45.12 2.03

2M
3M

FPF, fine particle fraction; GSD, geometric standard deviation; M, month; MMAD, mass median aerodynamic diameter; RH, relative
humidity; RT, room temperature.
All values are expressed as mean SD; n = 3.

14

PAI ET AL.

Table 7. Results of ACI Deposition Study of RIF2 Microparticles at RT and 40 C/75% RH
Time

Condition

MMAD (mm)

GSD (%)

FPF (%)

0M
1M

R.T
40 C/75% RH
R.T
40 C/75% RH
R.T
40 C/75% RH

5.73 0.41
5.45 0.26
5.98 0.45
5.83 0.29
6.87 0.48
5.99 0.18
7.37 0.36

1.6 0.04
1.51 0.04
1.64 0.08
1.73 0.04
1.83 0.05
1.76 0.05
1.96 0.03

21.46 1.36
24.24 1.77
20.01 1.84
20.58 1.51
17.49 1.18
20.10 1.08
15.78 1.02

2M
3M

FPF, fine particle fraction; GSD, geometric standard deviation; M, month; MMAD, mass median aerodynamic diameter; RT, room
temperature; RH, relative humidity.
All values are expressed as mean SD; n = 3.

size, moisture content, drug content, release profile, and in vitro

lung deposition as shown in Table 6 and Table 7.
The microparticles were found to be stable at room
temperature (ambient temperature), however at 40C
2C/75% RH 5% RH, an increase in moisture content
and physical appearance was observed, indicating requirement of more robust packaging systems. The effect of
moisture content on in vitro lung deposition of the microparticles is evident from the ACI deposition data. The
RIF microparticles stored at 40C 2C/75% RH 5% RH
for all time periods showed a decrease in the FPF as
compared to RIF microparticles stored at room temperature. The increase in the particle size is confirmed by
particle sizes and MMAD obtained on Malvern ZetaSizer
and ACI, respectively (Table 7).
In vivo toxicity

Administration of free RIF and RFB to the rats by intratracheal instillation produced severe peribronchiolar infiltration of the inflammatory cells accompanied with hyperplasia
of BALT (bronchus associated lymphoid tissue) and interalveolar septal thickening, evident of severe toxicity. The inflammatory cells were also present in bronchiolar and alveolar
lumen (Fig. 8c and 8d).

n comparison to free drugs, the RIF and RFB-loaded

microparticles produced only mild changes in the lung pathology, indicating no significant toxicity of the prepared
microparticles of RIF and RFB to the lungs (Fig. 8e and 8f).
The blank chitosan microparticles were found to be nontoxic
to the lungs as previously reported.(19) The blank microparticles produced only mild septal thickening and infiltration, thus indicating no toxicity of chitosan microparticles
produced by ionic interaction process (Fig. 8a and 8b).
In vitro macrophage uptake study

The causative agent for tuberculosis, Mycobacterium tuberculosis, is known to reside in the alveolar macrophages
and remain dormant for years. Alveolar macrophages are
reported to internalize microparticles when administered
to the lungs. Particles within the size range of 13 lm are
efficiently internalized, whereas particles less than 1 lm and
larger than 10 lm escape internalization by alveolar macrophages.(46) Thus, size is an important parameter for microparticles meant for targeting Mycobacterium tuberculosis
within alveolar macrophages. Many authors have reported
internalization of microparticles by alveolar macrophages(4749)
and their visualization by fluorescence microscopy or staining
techniques.(50)

FIG. 8. Photomicrographs of lungs of rats showing histopathological changes after administration of (a) PBS, pH 7.4; (b) blank chitosan microparticles; (c) RIF; (d) RFB; (e)
RIF microparticles; (f) RFB microparticles. B, bronchiole; BL, bronchiolar lumen; HPB,
hyperplasia of BALT; PBI, peribronchiolar infiltration; STI: interalveolar septal thickening
and infiltration;

CHITOSAN MICROPARTICLES FOR PULMONARY DELIVERY

Table 8. Concentration of RIF and RFB

Microparticles Internalized by U937 Macrophages

Batch
RIF2

RFB3

Conc.
(ppm)

Conc.
internalized
(ppm)

Estimated number
of particles
internalized

1
5
10
20
1
5
10
20

0.10 0.02
0.24 0.13
1.37 0.31
9.45 0.96
0.40 0.06
1.83 0.14
3.46 0.26
13.43 0.37

1.45 105
3.65 105
2.08 106
1.43 107
4.75 106
2.16 107
4.08 107
1.58 108

15

differences in particle size between RIF and RFB-loaded

microparticles. RFB microparticles being smaller in size
than RFB microparticles (Table 4), would incorporate a
larger number of particles at the same given concentration
and volume than RIF-loaded microparticles.
Within the tested levels, the internalization of microparticles was maximal at 20 lg/mL for both RIF and RFBloaded microparticles, and no saturation in uptake was
observed. Similar observations have been reported elsewhere in the literature for RIF.(27) The internalization of
drug-loaded microparticles was found to be a volumedependent process based on the observation that the internalization of the particles increased with the number of
available particles.

All values are expressed as mean SD; n = 3.

Conclusion

In the present study, an indirect method was used, wherein

the drug content in the supernatant was analyzed by UV
spectrophotometry and the amount of drug entrapped within
the macrophages was estimated (Table 8). The number of
microparticles corresponding to the estimated concentration
was determined based on the true density (0.453 gm/mL) and
diameter of the microparticles (Table 4).
The percent internalization of the drug loaded microparticles is shown in Figure 9. It was observed that all concentrations of RIF and RFB-loaded microparticles were
internalized to some extent at the end of 90 minutes. The
internalization was found to increase with an increase
in the number of particles for both RIF and RFB-loaded
microparticles. However, RFB-loaded microparticles were
internalized to a greater extent than RIF-loaded microparticles at all concentrations tried. This can be attributed to the

The spray-drying method was efficient in producing microparticles suitable for lung delivery with good aerodynamic characteristics that were evident by their deposition on
the later stages of the ACI. The chitosan-based microparticles containing anti-tubercular drugs are apparently nontoxic
to the lung tissues within the scope of studies described;
however, repeated-dose inhalation toxicology studies of
these formulations will be needed to better assess their longterm safety. The microparticles are also taken up by alveolar
macrophages, thus enabling targeting of the Mycobacterium
tuberculosis residing within the macrophages.
Microparticles of the anti-tubercular drugs, RIF and RFB,
prepared by a spray-drying technique, have a good potential
for direct delivery to the lungs, when formulated as dry
powder for inhalation (DPI) and can possibly provide the advantages of lower doses and shorter treatment times compared

FIG. 9. In vitro macrophage uptake of RIF and RFB microparticles. All values are
expressed as mean SD; n = 3.

16

to the conventional oral delivery approach. However, further

detailed studies are required to evaluate the uptake in alveolar
macrophages and efficacy of these microparticles on Mycobacterium tuberculosis.

PAI ET AL.

16.
17.

Acknowledgments

The authors thank Lupin Ltd, India for providing gift

samples of rifampicin and rifabutin, and Mumbai University
for providing financial assistance (Project No.314 & Ref.
no.: APD/237/172 of 2011).
Author Disclosure Statement

18.
19.

20.

The authors report no conflicts of interest. The authors alone

are responsible for the content and writing of the publication.
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Received on October 9, 2014

in final form, July 22, 2015
Reviewed by:
James Blanchard
Anthony Hickey
Address correspondence to:
Dr. Mala D. Menon
Department of Pharmaceutics
Bombay College of Pharmacy
Kalina, Santacruz (E)
Mumbai 400098, Maharashtra
India
E-mail: maladmbcp@yahoo.co.in