Preparation and in vitro evaluation of budesonide spray dried microparticles for pulmonary delivery

Full text

Page 1

Sci Pharm

www.scipharm.at
Research article
Open Access
Preparation and In Vitro Evaluation of
Budesonide Spray Dried Microparticles
for Pulmonary Delivery
Sonali NAIKWADE, Amrita BAJAJ *
C. U. Shah College of Pharmacy, S.N.D.T. Women’s University, Juhu-Tara Road, Santacruz (W), Mumbai-
400049, Maharashtra, India
* Corresponding author. E-mail: bajajamrita@rediffmail.com (A. Bajaj)
Sci Pharm. 2009; 77: 419–441
March 14th 2009
Accepted: March 12th 2009
doi:10.3797/scipharm.0901-11
Published:
Received: January 7th 2009
This article is available from: http://dx.doi.org/10.3797/scipharm.0901-11
© Naikwade and Bajaj; licensee Österreichische Apotheker-Verlagsgesellschaft m. b. H., Vienna, Austria.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly cited.
Abstract
The present study describes development and in vitro evaluation of budesonide
microparticles prepared by spray drying for delivering drug directly to lungs via
dry powder inhaler. This paper introduces new formulations for pharmaceutical
applications which includes conventional formulations and novel spray dried
microparticles viz., pulmosols, microspheres and porous particles. Optimized
spray drying parameters for generation of microparticles were: inlet
temperature, 130 °C; outlet temperature, 80 °C; aspirator rate, 240 mWc (60%);
solution feed rate, 2 ml/min; spraying air flow pressure, 2 bar. Microparticles
appeared to be spherical, low-density particles characterized by smooth
surface. MMAD and GSD ranged from 2.5–4.6 µm and 1.5–2.7 respectively.
Effective index of microspheres (54.48) and porous particle formulations (64.22)
was higher than the conventional formulation (49.21) indicating more effective
deposition of microparticles to the lungs. Carr’s Index (20–30%) and Hausner
ratio (1.2–1.7) for all formulations indicated good powder flow properties.
Formulations emitted a fine particle fraction of 25–47%. Microparticles showed
extended in vitro drug release upto 4 hours with high respirable fractions, thus
use of microparticles potentially offers sustained release profile along with
improved delivery of drug to the pulmonary tract.
Keywords
Spray drying • Budesonide • Microparticles • Dry powder inhaler • Drug deposition

Page 2

420 S. Naikwade and A. Bajaj:
Sci Pharm. 2009; 77; 419–441.
Introduction
Pulmonary route presents several advantages in treatment of respiratory diseases. Drug
inhalation enables a rapid and predictable onset of action and induces fewer side effects
than administration by other routes [1, 2]. Dry powder inhalers (DPIs) are easier to use,
more stable and efficient systems with better lung delivery than nebulizers/ MDIs and are
typically formulated as one-phase, solid particle blends [3]. As a result, preparation of dry
powder formulations for inhalation is an interesting and appreciated proposition [4]. When
preparing a formulation suitable for a DPI, micronization is usually employed to reduce
particle size of the drug powder to less than 5 µm [5], however powders in this size range
exhibit strong interparticulate cohesion leading to poor powder flow properties.
Furthermore, factors known to influence aerosolization properties of dry powders (e.g.
particle morphology, density and surface composition) cannot be controlled effectively
during the micronization process [6]. Researchers have investigated a number of
approaches to improve powder aerosolization, such as mixing the micronized drug with
inert carrier particles or modification of particle morphology, particle surface roughness,
particle porosity or powder density [7-9]. An alternative approach in generation of dry
powders for pulmonary drug delivery is offered by spray drying technology. Spray drying is
one-step constructive process that provides greater control over particle size, particle mor-
phology and powder density whereas micronization is a destructive technique [1, 3, 10].
Spray dried powders that exhibit sustained drug release properties may be generated
through inclusion of drug release modifiers such as chitosan [11]. Chitosan, a
polysaccharide derived from deacetylation of naturally occurring polymer chitin, is a
promising excipient that can be employed in a wide range of applications, including
sustained release preparations [12]. There are many advantages for developing sustained
release formulations for pulmonary drug delivery which includes reduced dosing
frequency, improved patient compliance and reduction in side effects [11, 13, 14].
Chitosan not only acts as a drug release modifier but also has mucoadhesive properties
thus it appear to be a useful excipient while preparing sustained release formulations for
pulmonary drug delivery [10, 11]. Chitosan has shown to be both biocompatible and
biodegradable [15, 16]. The oral LD50 for mice, 16 g/kg, indicates a very low toxicity
potential for this product [17, 18]. It is an approved food additive that has been considered
for pharmaceutical formulation and drug delivery applications, in which attention has been
focused on its absorption-enhancing, controlled release and bioadhesive properties.
Recently, chitosan-based delivery systems have been proposed to increase the
bioavailability of drugs both at the nasal mucosa and in the lungs [19, 20]; also these
systems have been reported as efficient vehicles for pulmonary gene delivery [21–23].
Nevertheless chitosan is not included in the FDA Inactive Ingredient Guide and very
sparse data on its pulmonary toxicity are available.
It is now widely accepted that inhaled corticosteroids (ICSs) are effective in controlling
inflammation, improving lung function and reducing asthma symptoms. As a result, ICSs
are recommended as first-line therapy for all patients with persistent asthma. There is
considerable evidence that treatment with anti-inflammatory ICSs reduces morbidity and
mortality in asthma [24]. ICSs appear to have a place in management of severe COPD,
perhaps by decreasing the frequency of exacerbations and improve quality of life in
patients with COPD [25]. Budesonide (BUD) has high glucocorticoid receptor affinity and
prolonged tissue retention and it inhibits inflammatory symptoms such as edema and

Page 3

Budesonide Micropartciles for Pulmonary Delivery 421
Sci Pharm. 2009; 77; 419–441.
vascular hyperpermeability [25]. Budesonide has low molecular weight of 430.53 Da
having oral bioavailability of 6–11% with half life of 2–3 hr [26]. Budesonide forms esters in
all tissues, PK modeling has shown that ester formation occurs primarily in the large
airways and lungs, sustaining local anti-inflammatory activity. High doses of ICSs are
recommended in the treatment of moderate to severe persistent asthma. It is well
accepted that ICSs have fewer systemic side effects than oral or parenteral
glucocorticosteroids [27, 28], but there is still some concern about the long-term safety of
high doses of ICSs [29, 30]. Long-term systemic side effects of high doses of ICSs include
thinning of the skin and easy bruising. The aim of inhaled administration of corticosteroids
in respiratory disease is to achieve high local concentrations of active drug in the lungs
while limiting systemic exposure. A dose-response relationship has been demonstrated for
budesonide [31, 32] and an increase in the dose and frequency of budesonide
administration has been shown to be beneficial in quickly reducing inflammation and
broncho-constriction in patients with unstable asthma [27]. Thus, inhaled corticosteroids
should preferentially combine a high fraction of the dose that reaches the airways with a
low swallowed fraction [33, 34]. Respirable fraction of currently available DPI formulations
is not more than 30% [35–37] which means that only 30% of total dose reaches at the site
of action thus increasing frequency and dose of drug administration. This also increases
the systemic side effects mentioned above. Conventional DPI formulations of this drug are
available in the market but not as controlled release formulations. This necessitates the
development of novel formulations. Pulmonary targeting can be achieved by prolongation
of pulmonary residence time either by reducing dissolution rate of drug particle (drug
lipophilicity or crystal structure), reducing release from drug delivery system (liposomes or
microparticles) or by initiation of biological interaction resulting in prolonged pulmonary
residence time (ester formation or capturing in membrane structures). Thus, development
of useful controlled-release formulations for use in the respiratory tract presents additional
challenges because apart from controlling drug release in the lung environment, drug
particles need to avoid removal by the lung clearance mechanisms for the period of drug
delivery.
No report is available comparing effect of various carriers on respirable fraction of drug
and particle engineering technology in order to generate sustained release microparticles
using chitosan as polymer with improved deposition profiles of BUD. The objective of
current work was to develop and characterize conventional formulations (using various
grades of inhalable lactose) and novel spray dried microparticles viz., pulmosols (prepared
with various sugars), microspheres and porous particles (generated using natural
polymers viz. gelatin and chitosan) in order to achieve sustained release profile and to
improve respirable fraction of formulation to the lungs. Microparticles could be used in DPI
after being blended with standard excipient such as lactose.
Results and Discussion
Optimization of spray drying process parameters
Effect of aspirator rate, spraying air flow pressure and inlet temperature on moisture
content of product, % yield and % drug entrapment was studied (Table 1) [38] by plotting
surface response curves (Figure 1) and was interpretated in terms of % contribution of
each factor and from equations obtained from Stat-Ease Design-Expert v.7 software.
Effect of aspirator rate and spraying air flow pressure on % yield was graphically shown in

Page 4

422 S. Naikwade and A. Bajaj:
Sci Pharm. 2009; 77; 419–441.
Figure 1a and their individual and combined effects on yield of product is found to be
98.32%, 1.05% and 0.63% respectively; which was calculated from Eq. 6. This indicated
that aspirator rate was the major parameter affecting product yield.
Eq. 6. Log10(% yield) = 1.0803 + 6.3538 ×Aspirator rate − 0.0246 × Spraying air flow
pressure + 6.6669 Aspirator rate × Spraying air flow pressure
Effect of aspirator rate and spraying air flow pressure on % drug entrapment was
graphically shown in Figure 1b and their individual and combined effects on drug
entrapment is found to be 51.44%, 1.62% and 46.94% respectively; which was calculated
from Eq. 7. This proved that aspirator rate alone and aspirator rate- spraying air flow
pressure together were major parameters affecting the drug entrapment.
Eq. 7. Log10(% Drug entrapment)= −0.6103 + 0.0445 × Aspirator rate + 0.5706 ×
Spraying air flow pressure − 0.011 × Aspirator rate × Spraying air flow pressure
Effect of aspirator rate and inlet temperature on % moisture content was graphically shown
in Figure 1c. Their individual (7.39% and 15.55%) and combined (77.05%) effects on
moisture content were calculated from Eq. 8. This indicated that aspirator rate- inlet
temperature together was major parameter affecting moisture content.
Eq. 8. Log10(Moisture content) = −4.5851 + 0.0874 × Aspirator rate + 0.0332 ×
Inlet temp. −7.3062 × Aspirator rate × Inlet temp.
From all this data, it was concluded that aspirator rate and inlet temperature were the
major parameters affecting yield, drug entrapment and moisture content of product.
Tab. 1. Combinations of factors and their effect on various responses in optimization of
spray drying
Response noted*
F1 (LL)
Effect on % yield
21.8 + 3.7
Sr.
No.
1.
Combination
of factors
Aspirator rate
and feed spray
pressure
Aspirator rate
and feed spray
pressure
Aspirator rate
and inlet
temperature
* Mean ± SD; n = 3; LL, Both factors at low limit; HH, Both factors at high limit; HL, First
factor at high limit and another at low limit; LH, First factor at low limit and another at high
limit
F2 (HL)
F3 (LH) F4 (HH)
22 + 6.7 31.06 + 2.4 33.33 + 5.7
Effect on % drug entrapment
27.0392 +
0.07 0.09
Effect on % moisture content
0.206 ±
0.14 0.28
2.
49.3174 + 76.2938 +
0.32
50.5038 +
0.18
3.
0.267 ± 0.272 ±
0.38
0.186 ±
0.17

Page 5

Budesonide Micropartciles for Pulmonary Delivery 423
Sci Pharm. 2009; 77; 419–441.

Fig. 1. Factorial design for optimization of process parameters
Development of HPLC method for estimation of Budesonide
A review of literature revealed that all HPLC methods published so far for budesonide are
based on separation of two epimers even though both epimers are similar in their potency
with respect to their anti-inflammatory activity [39–41]. Since our work involved design and
development of pulmonary drug delivery systems for budesonide, our objective was to
develop a simple, rapid and stability indicating HPLC method of analysis. Thus, we
developed a method, which elutes drug quickly and also separates all degradation
products and other impurities from the main peak. This method was successfully used to
analyze the developed budesonide formulations (polymeric microspheres and porous
particles prepared by spray drying technology).
Proportions of the organic and aqueous phases were adjusted to obtain a rapid and simple
assay method for budesonide with a reasonable run time, suitable retention time and
sharpness of the peak. Under experimental conditions, the chromatogram of budesonide
showed a single peak around 4 min. Our method has several advantages over earlier
reported methods viz. [1] Cheaper mobile phase (methanol and water) in comparison with
earlier methods involving acetonitrile and phosphate buffers; [2] Routinely used C18
column; [3] Short run time (5 min). The mobile phase was easily prepared and gave
reproducible results. With the use of non-buffered mobile phases, problems associated
with buffers viz. time required in its preparation, pH adjustments, chocking of tubings, and

Page 6

424 S. Naikwade and A. Bajaj:
Sci Pharm. 2009; 77; 419–441.
proper washing of the system after its use has been avoided. The method was linear over
a concentration range 10 ng-100 μg/mL (R2 = 0.9990). LOD of budesonide was 25 ng/mL
and LOQ was 100 ng/mL, values which are lower than other HPLC methods. The method
has been proven to be stability indicating. Peaks of degradation products, as well as those
of excipients in the budesonide loaded microparticles did not interfere with the analysis.
Recovery of budesonide from the developed dry powder inhaler formulations was
essentially quantitative. The structure of major degradation product formed under basic
aqueous conditions was also identified. Furthermore, degradation products formed under
alkaline condition are very well resolved using newly developed HPLC method [42].
Preparation of microparticles
There are no literature reports which compare effects of various carriers on respirable
fraction of budesonide and particle engineering technology in order to generate sustained
release microparticles using chitosan as polymer with improved deposition profiles of BUD.
Therefore we developed and characterized conventional formulations (using various
grades of inhalable lactose) and novel spray dried microparticles viz., pulmosols (prepared
with various sugars), microspheres and porous particles (generated using natural
polymers viz. gelatin and chitosan) in order to achieve sustained release profile and to
improve drug-targeting to lungs. Microparticles could be used in DPI after being blended
with standard excipient such as lactose. Formulation of BUD dry powder inhalers was
designed based on two different approaches-
Strategies in formulation developemnt of BUD
Conventional DPI
Novel and engineered DPI
Mixing of drug and
carrier lactose and
thus delivery to lung
Have disadvantage of high
and repeated drug dosing
and low pulmonary
bioavailability
Pulmosols- Altering
physical shape, length,
density of drug particles
after spray drying them
with different carriers
Use of Microspheres
as drug carriers-
generated using natural
polymers gelatin and
chitosan
Use of Porous particles as
drug carriers- generated
using natural polymers
gelatin and chitosan by
inclusion of blowing agent
Low density partciles


All sixteen conventional batches (Table 2) developed using inhalable lactose in
combination of fine lactose: coarse lactose, 60: 40 and 70: 30 were evaluated for various
parameters but the optimized batch was selected based on the % FPF. Batch L2
(Lactohale 300M: Pharmatose 150M, 60: 40) was selected as it gave maximum FPF
(34.50%) compared to other batches.

Page 7

Budesonide Micropartciles for Pulmonary Delivery 425
Sci Pharm. 2009; 77; 419–441.
Tab. 2. Development of BUD DPI formulations
Formulation
type
Batch
code
Excip./ drug:
polymer ratio
Excip.
(w/w)
% drug
content/
loading
105.6875
% FPF EI % CI Hausner
ratio
L1
L2
L3
L4
L5
L6
L7
L8
K1
K2
K3
K4
K5
K6
K7
K8
PS1
PS2
PS3
PS4
G1
G2
G3
G4
G5
A + B
C + B 109.6011
D + B 115.6885
F + B 113.0335
A + E 101.0742
C + E 103.4362
D + E 107.0000
F + E 103.5188
A + B 109.3114
C + B 112.5635
D + B 109.9085
F + B 106.0086
A + E 101.7149
C + E 104.6747
D + E 99.1107
F + E 108.5848
10% 31.2680
20% 10.0160
20% 49.6030
20% 87.7118
56.8719
88.5608
38.8170
24.1402
92.4756
31.7606
34.5082
26.1204
22.1722
12.8226
27.2027
24.5655
31.6768
31.2728
30.9298
30.7926
31.8673
18.4171
33.3384
31.4507
30.4080
–
–
–
29.3275
–
18.2420
–
–
–
46.3303 28.57 1.40
49.2185 25.0
44.8396 42.85 1.75
43.9040 28.57 1.40
31.8021 22.22 1.28
36.4843 20.0
43.0648 18.18 1.22
51.7964 37.50 1.60
49.6251 22.22 1.28
43.6226 18.18 1.22
48.2394 25.0
48.8284 33.33 1.50
37.7805 25.0
45.5773 22.22 1.28
48.5436 25.0
48.3153 27.27 1.37
– –
– –
– –
48.5685 12.50 1.14
– –
38.2699 30.0
– –
– –
– –
1.33
1.25
Conventional
Fine Lactose:
Coarse
Lactose
60: 40
1.33
1.33
1.33
Conventional
Fine Lactose:
Coarse
Lactose
70: 30
1: 50
1: 200
1: 100
1: 50
1: 1
1: 2
1 : 5
1 : 7
1: 0.5: 1.5
–
–
–
Pulmosols
Drug: mannitol
–
1.42
–
–
–
Microsph. D.
gelatin
Microsph. D.
gelatin:
HPβ-CD
Microsph. D.
chit.
1%
CH1
CH2
CH3
CH4
CH5
CH6
1: 2
1: 5
1: 2
1: 4
1: 7
1: 1: 1
1: 0.5: 1.5
1: 2: 2
1: 2: 5
1: 1: 1
1: 1
1: 2
1: 5
1: 2
7.3474
106.7436
85.9734
90.6250
101.3991
101.4084
102.8516
83.0364
109.8440
55.7134
44.7347
49.2864
91.1380
98.3962
–
–
35.6785
–
–
–
–
–
–
–
–
–
–
12.1883
–
–
54.4813 30.76 1.44
– –
– –
– –
– –
– –
– –
– –
– –
– –
– –
31.7858 25.0
–
–
–
–
1%
–
–
–
–
–
–
–
–
–
–
1.33
0.5%
Microsph. D.
chit.: HPβ-CD CH7
Microsph. D.
gelatin: chit.
0.5%
GC1
GC2
GC3
CM1
CM2
CM3
P1
1%
Microsph. D.
compritol 888
ATO
Porous. Part.
Drug: gelatin
Porous. Part.
Drug: chit.
A, Pharmatose 125M; B, Pharmatose 150M; C, Lactohale 300; D, Lactohale 200; E, Lactohale 100;
F, Inhalac; FPF, fine particle fraction; EI, Effective index; Chit., Chitosan; CI, Carr’s index; Excip.,
Excipients; Microsph. D., Microspheres Drug; Porous Part., Porous Particles.
1%
1%
PC1 1: 2 0.5% 95.9390 46.8199 64.2257 26.66 1.36

Page 8

426 S. Naikwade and A. Bajaj:
Sci Pharm. 2009; 77; 419–441.
For optimization of pulmosols, initially blank batches of lactose, sucrose, mannitol,
fructose, dextrose, albumin and PEG 4000 were prepared. Except mannitol, all other
carriers viz. lactose, sucrose, fructose, dextrose, albumin and PEG 4000 yielded cohesive
product which could not be removed from the cyclone, so mannitol was chosen for drug
loading as it gave free flowing powder. Different drug: carrier ratios, 1:50, 1:100 and 1:200
at concentrations of 10% and 20% w/w solution were tried (Table 2). Batch PS4 was
selected to determine % FPF (10 mg of formulation ≈ 200 µg of BUD) as drug: carrier ratio
was minimum (1: 50) with optimum drug loading of about 88.0% w/w as compared with
other batches of pulmosols.
Microspheres of gelatin, chitosan (alone and in combination) and compritol 888 ATO were
generated with drug: polymer ratios as shown in Table 1. Batch G2 (1% w/w solution, 1:2
drug: gelatin) and CH3 (0.5% w/w solution, 1:2 drug: chitosan) were selected for which %
drug entrapment was about 89.0 and 86.0% w/w, respectively. During preparation of
chitosan microspheres, initially 1% w/w solution was used for batches CH1 and CH2 for
spray drying. Although batch CH2 showed maximum entrapment efficiency, this batch was
not further continued because blocking of feed pipe was noticed sometimes during spray
drying process due to viscosity of the solution. Further batches of chitosan microspheres
were prepared with 0.5% w/w solution and drug: chitosan ratio of 1:2, 1:4 and 1:7; batch
CH3 (1:2, drug: chitosan) was selected as the amount of chitosan was less with 86.0%
w/w drug content as compared with other batches. Effect of HPβ-CD on entrapment
efficiency and drug release was studied and its addition increased entrapment efficiency of
BUD without changing the release profile. Combinations of gelatin-chitosan were
evaluated but not continued as the quantity of polymer required for these batches was
larger than batches prepared with gelatin and chitosan alone. Similar observations were
noted in case of batches prepared using compritol 888 ATO.
Porous particles of gelatin and chitosan were prepared (Table 2) with % drug entrapment
of about 98% and 96% w/w, respectively. Microspheres and porous particles were
formulated with inhalable lactose (lactohale 300M: pharmatose 150M, 60: 40) based on
the entrapment efficiency such that 25 mg of formulation ≈ 200 µg of BUD.
In vitro assessment of developed aerosol formulations
Selected formulations were characterized for in vitro deposition by twin stage impinger and
Anderson cascade impactor [43]. Pulmosols, formulations of microspheres and porous
particles prepared using gelatin showed FPF of 29.32%, 18.24% and 12.18%, so these
were not selected for further characterization. FPF for conventional (L2), microspheres
(CH3) and porous particles formulation (PC1) was about 34%, 36% and 47% respectively
with SD of ≤ 0.5 was very promising. Values were compared using normality (p=
0.159>0.05) and paired t test (p= 0.727>0.05). The difference is considered to be
statistically significant (p<0.001) (One Way Analysis of Variance Test). MMAD, for
conventional, microspheres and porous particles formulation were 2.75, 4.60, and 4.30
respectively. GSD for above batches was 2.56, 1.75 and 2.54.
Drug content, content uniformity and in vitro release studies
Drug content and content uniformity for all conventional formulations was in the range of
90-110% w/w. Drug entrapment for developed novel formulations varied from 10-110%
w/w as given in Table 2. In vitro release profile is shown in Figure 2a. Mechanism of drug

Page 9

Budesonide Micropartciles for Pulmonary Delivery 427
Sci Pharm. 2009; 77; 419–441.
release was determined using various kinetic models. Coefficient of correlation were
calculated from plots of Q vs t (cumulative % drug release vs time), log Q vs log t and Q vs
square root of t [44]. Regression coefficients (near to 1) for zero order, matrix and
korsmeyer-peppas kinetic equations confirmed the release of drug by slow zero order
kinetics through diffusion matrix (Table 3, Figure 2b and 2c). Korsmeyer-peppas plot
indicated good linearity (r2 = 0.9877).

Fig. 2.
In vitro release profile and release kinetics of formulations (a) In vitro release
profile of microspheres and porous particles prepared by spray drying method;
(b) Release kinetics of chitosan microspheres; (c) Release kinetics of porous
particles of chitosan
Tab. 3. Regression coefficients for formulations
r2
Formulations
Zero order
Korsmeyer-
peppas
Matrix
T50%
(min)
Log Q vs
Log t
Q vs
SQRT
Gelatin
microspheres
Chitosan
micropsheres
Gelatin porous
particles
Chitosan porous
particles
0.4272 0.9920 0.9166 4 0.6749 0.7637
0.8338 0.9722 0.9898 27 0.8132 0.9680
1.0000 1.0000 1.0000 2 0.6684 0.7953
0.8315 0.9877 0.9906 56 0.8595 0.9619

Page 10

428 S. Naikwade and A. Bajaj:
Sci Pharm. 2009; 77; 419–441.
Fourier transform infrared spectroscopy
IR spectrum of BUD showed peaks at 3378 cm-1 (O-H stretch), 2935 (C-H stretch) and
1720, 1659 cm-1 (C=O stretch). Chitosan showed typical peaks at 3446 and 1633 cm-1 for
N-H stretch and C=O stretch for free amine and amide carbonyl functionalities
respectively. In the IR spectrum of BUD chitosan microparticles, typical peaks for the N-H
stretch of free NH2 of chitosan disappeared due to possible crosslinking of chitosan with
drug via amine and hydroxyl functionalities while the broad peak ranging from 2800–2600
cm-1 was observed as well as intensity of peaks in the range of 1650–1720 cm-1 was
dramatically reduced which confirmed entrapment of BUD in chitosan.
Characterization of particle shape by scanning electron microscopy
Morphology of microparticles was investigated and SEM micrographs are illustrated in
Figure 3. Figure 3a of pulmosols showed spherical particles with wide particle size
distribution (1–20 µm) but uniform spherical microspheres and porous particles (Figure 3b
and 3c) were obtained with diameter ranging from 1 to 4 µm, with similar particle
morphology and size.

Fig. 3. SEM micrographs (a) Pulmosols; (b) Chitosan microspheres; (c) Chitosan
porous particles

Page 11

Budesonide Micropartciles for Pulmonary Delivery 429
Sci Pharm. 2009; 77; 419–441.
Characterization of microparticles by differential scanning calorimetry and
crystalline state by X-ray powder diffraction
DSC scans are shown in Figure 4. Chitosan showed broad endotherm (Figure 4d) at
109.13 °C while BUD showed sharp endotherm (Figure 4c) at 264.14 °C. DSC spectra of
microspheres revealed 2 endotherms (Figure 4b) for chitosan and BUD at 105.18 °C and
235.38 °C respectively. In case of porous particles 2 endotherms (Figure 4a) were
observed for chitosan and BUD at 106.50 °C and 222.98 °C respectively. BUD
conventional formulation showed 2 sharp endotherms (Figure 4e) for inhalable lactose and
BUD at 148.44 °C and 221.06 °C respectively. This confirmed no interaction between BUD
and excipients occurred after spray drying process [45].

Fig. 4. DSC spectra of developed formulations (a) BUD porous particles; (b) BUD
microspheres; (c) BUD pure; (d) Chitosan pure; (e) BUD conventional
formulation
On the other hand, X-ray powder diffraction patterns (Figure 5) showed that spray-drying
process did not completely affect the crystalline form of BUD (Figure 5a). Peaks that
represent the spray dried samples (both microspheres and porous particles) (Figure 5b
and 5c) correspond to those of chitosan (Figure 5d) but differ in intensity, indicating that
the major component (in formulations) is partly amorphous.

Page 12

430 S. Naikwade and A. Bajaj:
Sci Pharm. 2009; 77; 419–441.

Fig. 5. X-ray powder diffraction patterns of (a) BUD; (b) Microspheres; (c) Porous
particles; (d) Chitosan
Evaluation of other physicochemical characteristics
Effective index (EI) of microspheres (54.48, Table 2) and porous particle formulations
(64.22) was higher than the conventional formulation (49.21) suggesting more effective
microparticles drug deposition to the lungs might be possible [37]. Carr’s Index and
Hausner ratio, which are considered as appropriate methods of evaluating flow properties
of solids, were also determined from tapped and bulk density values [38]. Carr’s Index
values of less than 25 are usually taken to indicate good flow characteristics; values
beyond 40 indicate poor powder flowability. Carr’s Index values (Table 2) for all
formulations were found to be in the range of 20-30% which indicated good powder flow
properties. Hausner ratio is measure of flowability of powder. A low Hausner ratio means
that the powder has a high flowability (but it should be >2.0). For all formulations this ratio
was in the range of 1.2–1.7 (Table 2) indicating good flowability. Extent of porosity for
chitosan microspheres and porous particles formulation was 30.76 and 26.66 respectively
which was good as compared to developed conventional formulations (6–20%). Moisture
content for all formulations was <1% w/w.
Discussion
Chitosan, a polysaccharide derived from deacetylation of naturally occurring polymer chitin
is a promising excipient that can be employed in a wide range of applications, including
sustained release preparations. Deacetylation value of chitosan was determined using IR
spectroscopy in order to know quality of chitosan employed in formulation development.

Page 13

Budesonide Micropartciles for Pulmonary Delivery 431
Sci Pharm. 2009; 77; 419–441.
Spray drying process was optimized for aspirator rate, spraying air flow pressure and inlet
temperature and its effect on moisture content of product, % yield and % drug entrapment
was studied. Above mentioned optimized parameters were selected as it was observed
that aspirator rate alone affected yield and drug entrapment, aspirator rate along with
spraying air flow pressure was also affecting drug entrapment and aspirator rate- inlet
temperature together was major parameter affecting moisture content of product. Initially
conventional BUD formulations were developed using novel inhalable lactose and effect of
those on respirable fraction of BUD was assessed. It was observed that for all formulations
FPF was in the range of 12-34%. Microparticles viz. pulmosol, microspheres and porous
particles were developed using spray drying technology at optimized process parameters
in order to further improve FPF and thus delivery of BUD into deep lungs. These
developed microparticles were also assessed for in vitro deposition studies using TSI and
ACI. Data was statistically analysed. Drug content, content uniformity and in vitro release
profile was monitored and it was found that developed microparticles showed drug release
by slow zero order kinetics through diffusion matrix. From SEM micrographs, it was
observed that spray drying process yielded hallow, porous and spherical micropsheres
with uniform particle size distribution. To prove compatibility of drug with excipients, DSC
studies confirmed that there was no interaction between drug and polymer as endotherms
of drug and polymers were separate in formulation even after spray drying. From XRPD
studies it was observed that spray drying did not affect crystalline form of BUD. From other
physicochemical parameters like EI, Carr’s index, Hausner ratio and % porosity it was
clear that spray drying process generated particles for inhalation with uniform particle size
distribution and good flow properties.
Experimental
Materials
Budesonide was obtained from Lupin Ltd., Mumbai; gelatin and chitosan were procured
from S.D. Fine Chemicals, Mumbai and different grades of inhalable lactose were obtained
as gift sample from DMV Int., The Netherlands. Methanol and chloroform were of
analytical grade and were procured from S.D. Fine Chemicals, Mumbai.
Assay for degree of deacetylation of chitosan
Degree of deacetylation of chitosan affects overall charge density. An increasing presence
of ammonium groups results in decrease in the crosslinking density related to hydrogen
bonding and hydrophobic interactions [12, 46]. Increase in degree of deacetylation results
in increased swelling due to an increase in number of ionic sites and their counter-ions.
Degree of deacetylation dictates the reactivity, solubility and viscosity of chitosan solutions
which was determined using FTIR spectroscopy (Nicolet, USA) by Eq. 1.
Eg. 1.
115
)(
NH
%
2
3×=
freeofstretchNH ofAbsorbance
NHCOCHamideofstretchcarbonylofAbsorbance
ionDeacetylat

Page 14

432 S. Naikwade and A. Bajaj:
Sci Pharm. 2009; 77; 419–441.
IR spectra of chitosan showed the characteristic peaks for N-H stretch (νmax 3346 cm-1) for
free amine and C=O stretch (νmax 1633 cm-1) for amide carbonyl. From the Eq. 1, %
deacetylation was calculated as 45% which was in the acceptable limit.
Optimization of spray drying process parameters
Microparticles were prepared at laboratory scale by spray drying using Labultima Mini
Spray Dryer (Mumbai, India). Spray-drying is a one-step process that converts liquid feed
(solution, coarse suspension, colloidal dispersion) to a dried particulate form. Principle
advantages of spray drying with respect to pulmonary drug delivery are ability to
manipulate and control particle size and size distribution, particle shape and density in
addition to macroscopic powder properties such as bulk density, flowability and
dispersibility [47]. Various process parameters were optimized by 22 factorial design.
Surface response curves were plotted using Stat-Ease Design-Expert v.7 software. Effect
of aspirator rate (varied form 40–60%), spraying air flow pressure (2-4 bar) and inlet
temperature (varied from 100-130 °C) on moisture content of product, yield and
entrapment efficiency of drug was studied (Table 1). Following were the optimal conditions
of spray-drying: inlet temperature, 130 °C; outlet temperature, 80 °C; aspirator rate, 240
mWc (60%); solution feed rate, 2 ml/min; spraying air flow pressure, 2 bar [38, 48]. These
process parameters were optimized for batch CH3 with concentration of chitosan solution
as 0.5% w/v.
HPLC method development for estimation of Budesonide in formulations
HPLC apparatus
The HPLC analysis was carried out on Phenomenex C18 analytical column (5 µ, 250 mm
× 4.6 mm) connected to a HPLC (model SPD-M2OA 230V) system (Shimadzu corporation,
Japan) consisting a LC-8A pump, SPD- M2OA PDA detector, C3M-20A flow cell, a
degasser unit and 20 μL injection loop. LC Solution software was used for data collection.
HPLC conditions
The mobile phase consisted of methanol: water (80:20 v/v) with the flow rate of 1.5
mL/min. The wavelength of detection was 244 nm (λmax for BUD) and the injection volume
was 20 µL [42].
Preparation of microparticles
Conventional DPI formulations
BUD DPI formulations were developed on laboratory scale (100 g) using various grades of
inhalable lactose like pharmatose, lactohale, inhalac and mannitol in various combinations
(fine lactose: coarse lactose, 60: 40 and fine lactose: coarse lactose, 70: 30). The
development of DPI formulations of salbutamol sulphate using various carriers by sieving
process was reported in literature [43, 49] therefore; we employed sieving process in
development of BUD DPI formulations on laboratory scale. BUD was initially mixed with
fine lactose using mesh #100 and this premix was blended with coarse lactose to
homogeneity in geometric proportions using mesh #80. In all these 16 developed batches
(Table 2), 25 mg of formulation was equivalent to 200 µg of BUD. Effect of particle size of
excipients (fine or coarse) on fine particle fraction (FPF) of drug was assessed using TSI
study [44].

Page 15

Budesonide Micropartciles for Pulmonary Delivery 433
Sci Pharm. 2009; 77; 419–441.
Pulmosol formulations
Based on report of spray dried composites of drug bendroflumethiazide with polyethylene
glycol by Corrogan et al. [50] we attempted development of composites of BUD with
different sugars. Different sugars like lactose, sucrose, mannitol, fructose, dextrose,
albumin and PEG 4000 were used as carriers in formulation development of pulmosols.
Initially, blank batches were spray dried to find out optimum concentration of sugar as well
as to determine powder flow properties. Mannitol was chosen as carrier in formulations as
it yielded free flowing powder with maximum drug loading. Different ratios of drug: mannitol
(Table 2) was spray dried at optimized process conditions.
Microspheres formulation
BUD and natural polymers viz. chitosan, gelatin was spray dried at optimized process
parameters. Salbutamol chitosan co-spray dried multiparticulates were developed and
reported by Corrigan et al. with varying concentration of 0.5–2% chitosan solution and
there coworkers observed that 0.5% w/v was the optimum concentration of polymer based
on entrapment efficiency and release profile from developed composites [44]. Hence
gelatin (1% wt/ml solution in water), chitosan (0.5% wt/ml solution in 0.1M HCl) were spray
dried and drug: polymer ratio was optimized based on the % drug entrapment and release
profile (Table 2). Final concentration of solution to be spray dried was adjusted to 1% wt/ml
and 0.5% wt/ml for gelatin and chitosan, respectively. BUD and polymer were dissolved in
equal parts of methanol and water. Polymeric phase was mixed using Ultra-turrax at
13000 rpm to which methanolic phase was slowly added and solution was stirred to
homogeneity. This solution was spray dried to get microspheres.
Porous particle formulations
BUD and natural polymers viz. chitosan, gelatin was spray dried in water: methanol (1:1)
as 1.0% and 0.5% w/v, respectively. Solution containing BUD, polymer and a blowing
agent was atomized into the drying chamber and brought in contact with a hot air stream.
Blowing agent which is trapped in droplets decomposes at higher temperatures creating a
void in the center of particle [51, 52]. Since air stream temperature is greater than that of
the droplet, the droplet temperature increases until the evaporation temperature of solvent
is reached. Solvent at the surface (blowing agent) begins to evaporate causing solvent
below the surface of droplet to diffuse to the surface. The droplet, as it passed through the
spray chamber forms a hollow particle [45]. Hence porous particles were generated by
adding chloroform (5% v/v) as a blowing agent. Drug: polymer ratio was optimized based
on the % drug entrapment and release profile.
Effect of HPβ-CD (HPβ-cyclodextrin) on entrapment efficiency and drug release of
microspheres and porous particles was assessed [53]. Microspheres and porous particles
obtained by spray drying were formulated with inhalable lactoses.
In vitro assessment of developed aerosol formulations
In vitro deposition of dry powders for inhalation was determined using a twin impinger
[Copley Instruments (Nottingham) Ltd]. A 25 mg formulation was weighed and loaded into
size 3 hard gelatin HPMC capsules (Associated Capsules Pvt. Ltd., India), which were
individually installed in a rotahaler device. The rotahaler was attached to the impinger
which contained 7 and 30 ml of collecting solvent [acetonitrile: buffer (disodium hydrogen