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Pelagia Research Library

Der Pharmacia Sinica, 2011, 2 (5): 88-97

ISSN: 0976-8688
CODEN (USA): PSHIBD

Isoniazid loaded chitosan microspheres for pulmonary delivery:

Preparation and characterization
Aliasgar J. Kundawala.1*, Vishnu A. Patel2, Harsha V. Patel1 and Dhaglaram Choudhary1.
1
Indukaka Ipcowala College of Pharmacy, New Vallabh Vidyanagar, Dist. Anand (Gujarat)
2
A.R. College of Pharmacy and G H Patel Institute of pharmacy, Vallabh Vidyanagar Dist.
Anand (Gujarat)

ABSTRACT

The objective of the present study was to entrap isoniazid, an Antitubercular drug, in cross
linked chitosan matrix and study in vitro lung deposition of powdered chitosan microspheres
using Andersen cascade impactor. Ionic cross linker Tripolyphospate was used to make cross
linked chitosan microspheres to achieve sustained drug release. Chitosan microspheres were
prepared by spray drying method. The spray dried microspheres were characterized for size,
shape, drug release and in vitro pulmonary drug deposition. Aerosol properties like fine particle
fraction and Mass median aerodynamic diameter were also evaluated. The cross linked
microsphere were prepared with entrapment efficiency of 72 to 88 percentages. Spray dried
microspheres had mean particle size of 3.6 to 5.2 µm and fine particle fraction of 67 %.

Key words: Dry powder inhaler, Isoniazid, Chitosan, tripolyphosphate, dispersing agent.

INTRODUCTION

Microparticles and nanoparticles have been explored as drug delivery carrier to administration of
drug agents used in pulmonary disorders. Microencapsulation offers the advantage of protection
of drug against pulmonary metabolism, sustained and prolong drug release. Dry powder inhalers
have edge over the existing liquid inhaler systems like MDIs, Nebulizer, and Aerosol Sprays etc.
DPI offer better patient compliance as well as overcomes the instability. Controlled release
dosage forms provide advantage over immediate release dosage form: reduction in side effects,
first pass metabolism, maintenance of effective plasma concentration and reduced dosing
frequency [1]. There are many advantages to develop sustained release formulations for
pulmonary delivery of drugs for localized treatment of diseases like asthma, chronic obstructive
pulmonary disease and infectious diseases. Sustained release of drug helps in reducing the
frequency and dose size as well as improves the plasma drug level [2].

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Isoniazid was selected as a model drug for dry powder formulations in treatment of tuberculosis.
INH is less permeated through the stomach and is mainly absorbed through the intestine because
it occurs in the protonated form at acidic pH (pKa = 2) [3]. Therefore, it can be considered as a
good candidate for the development of a site-specific release formulation especially in case of
Tuberculosis to deliver it in lung. Chitosan [Poly (1,4)-b-D-glucopyranosamine], a
polysaccharide produced from crab or shrimp shells, which is a copolymer combined of
glucosamine and an N-acetyl glucosamine unit [4]. It is a bioadhesive, biocompatible and
biodegradable polymer obtained through deacetylation of naturally occurring chitin [5]. The
release modifying and mucoadhesive property of chitosan appears to be a good choice for
preparing sustained release formulation for lung delivery via inhalation. Very handful work has
been done in this directions.[6-9]. A stabilized chitosan based controlled drug delivery system
have been prepared with either chemical cross liking agents such as glutaraldehyde, ascorbic
acid, sodium phosphate and sodium tripolyphosphates, pentapolyphosphates etc.[10 18]. Recent
applications of chitosan are in ophthalmic, nasal, sublingual, buccal, periodontal, gastrointestinal,
colon-specific, vaginal, transdermal drug delivery and as mucosalvaccine and gene carrier
[19].The undesirable side effects like mucosal membrane irritation can be overcome by
ionotropic gelation using multivalent anions. Chitosan microspheres/microparticles can be
prepared by number of techniques such as solvent evaporation, emulsification/ internal gelation,
coacervation, suspension cross linking and spray drying [20-21].

The spray drying technique is advantageous in many ways to prepare microparticles for
pharmaceutical application as it is reproducible, one step, rapid and easy to scale up [20, 22].
Spray drying technique can be used to produce dry powders, granules or agglomerates from
drug-excipient solutions and suspensions [23]. The particle size of the microparticles prepared by
spray drying technique ranged from microns to several tens of microns and had a relatively
narrow size distribution. Recently, spray-drying has been developed to be used as a continuous
process for producing microspheres, especially for the preparation of microspheres for
pharmaceutical applications.

Thus the aim of the present research was to prepare, chitosan microspheres containing Isoniazid
by a spray drying method. The effects of concentration of tripolyphosphate on microsphere
properties were studied.

MATERIALS AND METHODS

Isoniazid was obtained as a gift sample from Strides Acrolab, Bangaluru (Inida), chitosan was
generously provided by Central institute of fisheries technology, Cochin, India.
Tripolyphosphate was obtained from S D fine Chemicals, Baroda. L Leucine was purchased
from Renkem, India. α, mono lactose was obtained from Meggle, Wasserburg Gmbh and Co.,
Germany as a gift sample. All Other chemicals and solvents used were of analytical grade.

2.1 Determination of Degree of Deacetylation of chitosan

Elemental analysis was used to determine the degree of deacetylation in chitosan with CHNS/O
Elemental Analyzer Perkin Elmer PE 2400 Series II. Using following equation,

Where WC and WN are weight percentage of carbon and nitrogen in chitosan respectively.

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2.2 Preparation of spray dried microspheres

Microspheres were prepared by spray dried technique. 1% w/v Chitosan solution was prepared
by dissolving chitosan in 300ml of 1% v/v acetic acid solution at 50°C by mechanical stirrer.
Isoniazid was dissolved in previously prepared and filtered chitosan solution. The drug chitosan
solution was stirred mechanically at 500 rpm for 5 minutes. 10 ml of Tripolyphosphate (TPP)
solution in concentration (0, 0.3, 0.6, 0.9 1.2, 1.5 w/v) was added drop wise into chitosan-drug
solution and stirred for 30 minutes at 1000 rpm. The Lactose 10 % w/v and L leucine 40%w/v to
polymer concentration were added and stirred for 15 minutes. The chitosan-TPP-Drug
suspension was then spray dried by Lab-ultima spray drier (LU-222 advanced. Mumbai, India)
with standard 0.7 mm nozzle. The spray drying conditions such as inlet temperature, outlet
temperature, pump prate, pressure and aspirator setting were set as 140°C, 70-80°C, 5 ml/min, 3
kg/cm2 and 45 m3/h respectively.

2.3 Spray dried yield and drug content:

The yield of spray dried product was identified as the percentage of anticipated yield.
10 mg of drug loaded chitosan microspheres were dissolved in 50 ml of 0.1 N HCl. The solution
was then passed through 0.22 µm membrane filters (Whatman filters, UK) and then the drug
content was assayed by measuring the absorbance at 264 nm after suitable dilution using UV
spectrophotometer. The experiment was carried out in triplicate (n=3) and loading efficiencies
were calculated according to following

Loading Efficiency = [(total amount of drug/ weight of microspheres) X 100]

x 100 calculated drug content/ theoretical drug content

Entrapment efficiency was determined by extracting (10mg Equivalent drug ) in 7.4 phosphate
buffer. The suspension was centrifugation at 10000rpm for 5 min and supernant collected was
filtered through (0.2µm nylon filters, Whatman, UK). The filtrate was assayed for drug content
by UV spectroscopy (Shimadzu 1700, Japan) at the wavelength of 264 nm.

The percentage encapsulation efficiency for each drug was calculated by the formula

Encapsulation efficiency = [Total amount of drug – free drug/ total amount of drug] x 100

2.4 Morphological characterization of microspheres

Surface morphology of spray dried chitosan microspheres were obtained by using scanning
electron microscope (ESEM TMP with EDAX, Philips, Holland). Spray dried powders were
mounted onto separate, adhesive coated 12.5 mm diameter aluminium stubs. Excess powder was
removed by tapping the stubs sharply and then gently blowing a jet of particle free compressed
gas across each. The specimen was examined using EDAX SEM. The SEM was operated at high
vacuum with accelerate voltage of 5KV and specimen working distance of 12mm. The particle
size was measured directly by optical microscopy on 200 microspheres. for each batch particle
size was measured in triplicate (n=3).

2.5 X ray diffraction (XRD) studies

The physical state of the model drug (INH) and in the spray dried chitosan-TPP microspheres
were assessed by XRD studies. X ray diffraction patterns of pure drug, physical mixture,
chitosan microspheres loaded with drug were obtained using XRD Diffractometer (powder)
Philips Xpert MPD Range (2θ): 3º to 136º.

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2.6 Swelling index determination

The water uptake capacities of uncross linked and cross linked chitosan microspheres were
determined by swelling microspheres in phosphate buffer pH 7.4 at, 37º ± 0.5° C and measuring
their changes in weight during swelling. 100mg of microspheres were taken and placed in
dialysis membrane with continuous stirring at 50 rpm for 2 hours. The samples were removed
periodically (0.25, 0.5, 0.75, 1.0, 1.5 and 2 hrs) and their net weight was determined by first
blotting their surface with a filter paper. Each experiment was repeated thrice and average values
were taken as the degree of swelling. The degree of swelling is calculated by following formula

Degree of Swelling = [(Wt- W0) / W0] x 100

Where Wt is weight of the microspheres at time t and W0 is initial weight of the microspheres.

2.7 In vitro drug release studies

Dissolution testing was performed as described by Amit misra et al with slight modification [24].
briefly, Dissolution test was performed on a USP Type II tablet dissolution test apparatus
(VEEGO) at a stirring speed of 150 rpm. A dialysis membrane (Himedia, LA 401) was cut into
equal pieces of about 5 cm x 3 cm and pre-treated. Microspheres (50 mg) were accurately
weighed out on the pre-treated dialysis membrane and sealed with clips. The pouch thus formed
was attached to the paddles of the apparatus using cotton threads over the clips. 900 ml of
phosphate-buffered at pH of 7.4 was used as a dissolution medium to ensure sink conditions.
Samples were withdrawn for analysis at specified time points, and assessed for Isoniazid content
by UV spectroscopy (Shimazdu UV-1700, Japan) at 264 nm. Each dissolution experiment was
performed in triplicate.

2.8 Characterization of aerodynamic properties of dry powder

An eight stage Andersen cascade impactor was used to determine the fine particle fraction of the
INH formulation. Eight metal plates were installed on the impactor before use. A throat piece
(non-USP type was connected on the top of the device. A dose equivalent to 10 mg of free drug
was loaded directly into the chamber with the help of the respirable device, cyclohaler (cipla Ltd)
and dispersed at an aspiration rate of 28.3L/min for an inhalation time of 10 sec. Powder
deposited on each plate and throat piece was extracted with 0.1 N HCl and analysed by UV
spectroscopy at the wavelength of 265nm after suitable dilution. The Emitted dose (ED), Fine
Particle Fraction (FPF) and Mass Median Aerodynamic Diameter (MMADae) were determined.
The emitted dose was calculated as the percentage of total loaded powder mass exiting the
capsule. The % FPF was determined as the total percentage deposition at stages 2–7 of the
cascade impactor. MMADae of the powders was also derived, defined as the particle size at the
50% mark of a plot of cumulative fraction vs. effective cut-off diameter.

RESULTS AND DISCUSSION

3.1 Degree of deacetylation:

The degree of deacetylation [DDA] influences its interaction with cross linkers and media. The
DDA is measure of free amino group in chitosan molecule and on increasing its value in the
chitosan. DDA also influence the degree of crosslinking extent and hydrophobic interactions
with encapsulated drugs. The DDA of received chitosan was determined by elemental analysis as
described by [25]. In elemental analysis the weight percent of carbon [WC] and weight
percentage of Nitrogen [WN] was used in equation to calculate the DDA of Chitosan. The DDA
was found to be 89 %.

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3.2 Characterization of chitosan TPP cross linked microspheres:

Chitosan microspheres were prepared by spray drying method. Tripolyphosphate (TPP) was
employed as a physical cross linker to cross link chitosan and to impart sustained drug release.
TPP is a polyion and can interact with cationic chitosan in acidic medium. The chitosan- TPP
microspheres have been prepared by emulsion or syringe methods. However, these methods are
tedious and prepare larger sized microparticles; and are unsuitable for bulk production of
microspheres/microparticles. To control the size and reproducibility, spray drying method was
employed [26].

The powder characteristics of the five different formulations of chitosan microspheres are shown
in table 1. In the process of spray drying the chitosan-TPP- Drug suspension was sprayed in the
chamber, circulated with hot air, and collected as dry powder which was collected in cyclone
separator. Here, TPP interacted with chitosan before formation of particles on spray drying.

Table 1: Physical Characterization of chitosan microspheres (n=3)

w/v % TPP Mean particle Drug loading % Encapsulation

Formulations (%)Yield
concentration /10 ml size (µm) Efficiency efficiency
28.31 ±
F1 0 5.2 ± 0.8 49.12 ± 2.03 87.5 ± 1.6
2.12
30.54 ±
F2 0.3 4.9 ± 0.4 48.67 ± 2.58 81.9 ± 1.2
1.59
30.89 ±
F3 0.6 4.3 ± 0.5 46.91 ± 1.51 78.4 ± 1.4
0.97
32.62 ±
F4 0.9 3.7 ± 0.2 46.20 ± 1.44 79.1 ± 1.1
1.04
32.46 ±
F5 1.2 3.6 ± 0.6 45.70 ± 2.61 76.3 ± 2.7
1.32
31.01 ±
F6 1.5 3.6 ± 0.3 45.56 ± 1.86 72.1 ± 2.1
1.95

The % yield was in the range of 28.31 to 32.62 %. SEM micrographs revealed that most of the
spray dried microspheres were spherical in shape. No significant difference was observed when
different concentration of cross linking agent was employed. The size of microspheres was found
in the range of 3.6 to 5.2 µm. The particle size decreased slightly on increase in TPP
concentration from 0.3 to 1.5 % w/v. However, as the cross linking extent was increased the
encapsulation efficiency was found to be decreased from 87.5 to 72.1 %. In contrast the drug
loading was found to be more than 45 percentile in each formulation. The uniform size of the
chitosan-TPP cross linked microspheres was due to the entrapment of TPP in the microsphere
core [27].

3.3 Surface morphology by SEM

The surface morphology of spray dried non crosslinked and chitosan-TPP cross linked
microspheres are shown in figure 1. Microspheres produced by spray drying method showed
spherical shape and smooth surface. However, slight roughness was produced when the TPP
concentration was increased from 0.3 to 1.5 % w/v.

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Figure 1: Scanning electron microscope pictures of non cross linked (a), non-cross-linked drug loaded (b) and
cross linked drug loaded (c) chitosan microspheres.

The study revealed that the SEM micrographs of non cross linked chitosan microspheres have
smooth surface compared to cross linked chitosan microspheres. The SEM micrograph also
indicates the aggregation of chitosan microspheres due to existing weak interparticle bonding.

3.4 X-ray diffraction studies

Physical state of the isoniazid in chitosan matrix was assessed by XRD studies. X ray
diffractograms of pure drug has shown in figure 2 its characteristic crystal peaks between 2θ of
14.56, 16.88, 26.76, 27.52, 29.33, 32.39, 35.10, 40.37 and 51.18 ° indicating the presence of
crystalline isoniazid, shown in figure 2 [X-1].

Under the present spray drying conditions, chitosan TPP microspheres did not show any
characteristic peaks, although peaks with reduced intensity were seen. Where as the
characteristic us nature of peaks were absent in the spray dried formulations indicating the
amorphous nature and entrapment and complexation with polymer. Encapsulation of drug
showed unidentified, broad and diffused peaks with very low intensities. This is indicative of
amorphous nature of drug.

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FIGURE 2: X-ray diffractograms of pure drug Isoniazid [X-1], uncrosslinked drug loaded chitosan
microspheres [X-2] and cross linked drug loaded chitosan microspheres [X-3].

400

350

300

250 0.3 % TPP

% swelling

0.6 % TPP
200
0.9 % TPP

150 1.2 % TPP

1.5 % TPP
100

50

0
1 2 3 4 5 6
time point

Figure 3: Swelling behavior of chitosan-TPP-microspheres with different concentration of Tripolyphosphate

3.5 Swelling behavior

It is known that micromolecular chain of polymer can be fixed by ionic cross linking or chemical
cross linking so as to get the swelling ability decreases. However, the swelling of spray dried
chitosan is reported to be higher compared to pure chitosan. Understanding swelling behavior of
chitosan microsphere is helpful in loading and drug release characteristics of the chitosan
microspheres. The degree of swelling of chitosan-TPP microspheres was studied in Phosphate
buffer pH 7.4 and is shown in figure 3. It was observed that swelling capacity of the TPP

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crosslinked chitosan microspheres was increased with time. However at the higher concentration
of TPP the capacity of chitosan microsphere decreases. It may be the result of tightly packing of
matrix in presence of TPP. The present study confirms the rigid chitosan –TPP network.

3.6 In vitro drug release

Concentration and molecular weight of chitosan, concentration of cross linking agent employed
and drug to chitosan ratio determines the drug release from the chitosan matrix. Spray dried
chitosan microspheres are having a faster swelling capacity and dissolve quickly in dissolution
media [22]. Again chitosan microsphere dissolves faster in acidic media as compared to alkaline
media. Therefore the release studies were carried out in phosphate buffer pH 7.4. The drug
release profile of different formulation is shown in figure 4. A burst release of drug was observed
in this study indicating approximately 50-60% of drug release within first 30 minutes.

120
0.3 % TPP

100
cumulative % drug release

0.6 % TPP
80
0.9 % TPP
60
1.2 % TPP
40

1.5 % TPP
20

0
0 2 4 6 8 10
Tim e (in hrs)

Figure 4: in vitro drug release profile of chitosan microspheres loaded with isoniazid

The water solubility and faster swelling of spray dried chitosan microspheres was responsible for
burst drug release. The faster swelling as well as low diffusion path due to small particle size
resulted in faster drug release from the chitosan-TPP microspheres. The higher drug loading in
microspheres also contributed towards the initial burst release. It was found that addition of TPP
into chitosan microspheres influenced the retardation of drug from the chitosan matrix. Increase
in TPP concentration from 0.3 to 1.5 % w/v decreases the release of isoniazid from the chitosan
microspheres. The high crosslinking density and rigid structure due to cross linking with TPP
might have resulted in less swelling ability. The sustained isoniazid release was seen after two
hours which followed zero order release pattern, more than 90 % of isoniazid released in 8 hours.

3.7 Characterization of aerodynamic properties of formulation

The Isoniazid loaded chitosan-TPP microspheres were prepared by spray drying technique with
dispersing agent L-Leucine and a bulking agent lactose. For aerodynamic properties, a powder of
noncrosslinked chitosan microspheres and powder of TPP crosslinked chitosan microspheres
[0.3% w/v] was taken. The HPMC capsules containing 10 mg equivalent of INH was loaded to
Andersen cascade impactor. The data obtained for various aerodynamic properties are shown in
the table 2. No significant difference was observed in both the formulations. The results
demonstrated that 10-12 % drug was retained in the capsule and device. That has given

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indication of good dispersibility of prepared powder. 88.34 ± 2.16 % and 89.86 ± 1.95 % of drug
was found to be emitted. However, the role of leucine is not discussed in detail in this research
work.

Table 2: Summary of aerosol deposition data of spray dried chitosan microsphere formulations (n=3).

Dry powder formulation (%) Emitted dose (%) FPF MMADae

F1 88.34 ± 2.16 63.2 ± 3.2 2.79 ± 0.22
F4 89.86 ± 1.95 66.8 ± 2.7 2.33 ± 0.41

The hydrophobic and surfactant like properties of Leucine might have influence the surface
characteristics of dry powder formed in spray dying resulting in a good dispersibility of dry
powder. FPF of drug loaded chitosan-TPP microsphere was found to be 68.8 ± 2.7 and for
noncrosslinked chitosan microspheres was 63.2 ± 3.2 indicative of no significant difference. The
FPF obtained in this range are considered to be satisfactory [28-30]. A MMADae of less than
5µm is prerequisite to have an inhalation of powders into the lower region of the lung. The SEM
micrograph suggested agglomeration of particles but obtained MMADae values indicative of
particles behaving as individual particles on aspiration. This may be due to the week cohesive
forces between the particles and the higher drag force applied.

CONCLUSION

The recent studies on spray dried chitosan microspheres demonstrated possibility of generating
highly respirable powders that exhibits good aerosolization properties. Effective
microencapsulation and loading of Isoniazid with controlled size was feasible with spray drying
technique. It is possible to produce good spherical particles with size less than 10 µm with spray
drying technique. The applied processing conditions resulted in drug loaded chitosan-TPP
micropsheres having good surface properties and spherical shape suitable for inhalation.
Sustained drug release was achieved with addition of tripolyphosphate TPP as a crosslinking
agent. The chitosan microspheres loaded with Isoniazid was found to possess effective fine
particle fraction as well as lower impaction loss and device retention for effective aerosolization.
These spray dried microspheres would be predicted to deposit predominately in the central and
peripheral regions of the lung following inhalation. Chitosan, natural polysaccharide with
biodegradable properties can be used as a carrier for inhalation delivery.

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