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european j ournal of pharmaceuti cal sci ences 3 5 ( 2 0 0 8 ) 1218

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Micro-particle corrugation, adhesion and
inhalation aerosol efciency
Santoso Adi, Handoko Adi, Patricia Tang, Daniela Traini,
Hak-kim Chan, Paul M. Young

Advanced Drug Delivery Group, Faculty of Pharmacy, University of Sydney,


Sydney, NSW 2006, Australia
a r t i c l e i n f o
Article history:
Received 14 March 2008
Received in revised form 7 May 2008
Accepted 24 May 2008
Published on line 8 June 2008
Keywords:
Dry powder inhaler
Particle corrugation
DPI
AFM
Particle adhesion
a b s t r a c t
Atomic force microscopy (AFM) was used to evaluate the particle adhesion and surface
morphology of engineered particles for dry powder inhaler (DPI) respiratory therapy to gain
a greater understanding of interparticle forces and the aerosolisation process. A series of
spherical model drug particles of bovine serum albumin (BSA) was prepared with differ-
ent degrees of surface corrugation. The particles were evaluated in terms of particle size
(laser diffraction) and microscopic morphology (scanning electron microscopy). Conven-
tional tapping mode AFM was used to evaluate the nanoscopic morphology and derive
specic roughness parameters, while AFM colloid probe microscopy was used to directly
measure the interaction of functionalised probes. The physical characterisation and AFM
measurements were evaluated in terms of in vitro aerosolisation performance, using a con-
ventional Rotahaler

DPI and multistage liquid impinger. A direct relationship between the


root mean square roughness, particle adhesion and in vitro aerosol performance (measured
as ne particle fraction, FPF) was observed suggesting that as the degree of corrugation
increased, particle adhesion was reduced which, resulted in a concomitant increase in FPF.
This study demonstrates that AFM may be used to predict the aerosolisation performance
micron sized particles for inhalation based on their morphological properties.
2008 Elsevier B.V. All rights reserved.
1. Introduction
Dry powder technology has become a popular formulation
approach for the respiratory delivery of both local and sys-
temic drug molecules. In simple terms, a dry powder inhaler
(DPI) formulation contains an active pharmaceutical ingredi-
ent (API) that is engineered to have an aerodynamic diameter
suitable for respiratory delivery; generally considered 5m
(Pritchard, 2001). The API is formulated with, or without
excipients, in an inhalation device that during the inhala-
tion manouveure should ensure efcient aerosolisation of
the API into its primary respiratory sized particulates. These

Corresponding author. Tel.: +61 2 90367035; fax: +61 2 93514391.


E-mail address: py@pharm.usyd.edu.au (P.M. Young).
systems however, tend to be highly cohesive and adhesive,
resulting in relatively poor aerosolisation performance, and
thus respiratory deposition (Smith and Parry-Billings, 2003).
In order to overcome such issues a strong research focus
has emerged concentrating on the particle engineering and
physico-chemical characterisation of these micro-particulate
systems (Chan, 2006a,b).
The force required to aerosolise an adhered API drug par-
ticle will be directly proportional to the sum of the surface
energies of the contiguous surfaces, and inversely propor-
tional to the projected contact area. Thus, the two most
common approaches to improve the aerosolisation efciency
0928-0987/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2008.05.009
european j ournal of pharmaceuti cal sci ences 3 5 ( 2 0 0 8 ) 1218 13
Table 1 Spray-drying conditions for the production of BSA micro-particles with differing degrees of surface corrugation
Spray drying settings Increase in degree of corrugation
Sample no. 1 2 3 4
Feed concentration (mgml
1
) 60 40 25 10
Atomisation rate (l h
1
) 800 550 414 300
Inlet temperature (

C) 55 45 45 45
Outlet temperature (

C) 36 33 33 36
in these systems is to reduce the surface free energy of the
contacting surfaces or modify the particle shape to limit con-
tact area. Alteration of the surface energy may be achieved by
the addition of excipients during manufacturing (Ahfat et al.,
1997; Begat et al., 2005; Hickeyet al., 1990; Iidaet al., 2004; Lucas
et al., 1999; Young et al., 2002) or by controlled crystallisation
(ChanandGonda, 1989, 1995; Zeng et al., 2001) (promoting spe-
cic faces, andthus, surface chemistry); however, the former is
usually a more popular approach due the physical challenges
in producing high quality crystalline material at the micron
range. Modication of the surface topology of the API parti-
cles, is alsofraught withchallenges, specically whentrying to
modify crystalline geometry. However, the use of spray drying
to prepare physically stable API powders with modied sur-
face morphology has been shown to be a successful method
of improving aerosolisation efciency (Chew and Chan, 2001;
Chew et al., 2005; Dellamary et al., 2000; Duddu et al., 2002;
Edwards et al., 1997).
Previous work by Chew & Chan, has demonstrated that
the aerosolisation performance of particles of bovine serum
albumin (BSA) could be controlled by altering the spray drying
conditions during preparation (Chew and Chan, 2001). These
primarily spherical particles had different degrees of sur-
face corrugation (presumably due to the differences in initial
feed concentration and solvent evaporation rate) and dif-
ferent aerosolisation performance characteristics. In general,
the corrugated particles produced in this study had signi-
cantly improved aerosol performance when delivered from a
Rotahaler

DPI (Chewand Chan, 2001). In a more recent study,


Chan et al., demonstrated that the degree of surface corruga-
tionof BSAparticles could be successfully controlled using the
previous technique, and the aerosolisationefciency modied
(Chew et al., 2005). However, the relationship between degree
of particle corrugationandaerosolisationefciency couldonly
be based on secondary measurements and derived fractural
dimensions.
The invention of the scanning tunnelling microscope in
1985 and subsequent atomic force microscope (AFM) in 1986
(Binnig et al., 1986), has allowed surface scientists unprece-
dented views of structure, morphology and forces at the
nanoscopic and atomic level. Although a very powerful tool,
the AFMwas not truly utilised for probing particle interactions
until the end of the twentieth century, when the appara-
tus was modied to directly measure the force of interaction
between individual colloidal particles and any given substrate
(Ducker et al., 1991). Simply, a micro-fabricated cantilever,
with a known spring constant, can be functionalised with an
individual drug particle, using a micromanipulation process.
The functionalised drug probe can be subsequently ramped
towards, in contact with and away from a sample using a
precision piezo. Monitoring the deection of the cantilever,
as it travels through the Z-axis, and applying Hooks law, the
forces acting on a particular drug probe may be measured
with extreme accuracy. Furthermore, multiple forcedistance
curves may be conducted as a function of a samples X and
Y coordinates, so to form a spatial adhesion map (referred to
as force volume imaging). The force volume technique, cou-
pled with the high resolution imaging capabilities of the AFM
(using conventional cantilevers and imaging modes), allows
unprecedented insight into the particle interactions and mor-
phology of APIs used in inhalation therapy.
Here, the relationship between API morphology, adhesion
and aerosolisation efciency was investigated. Specically,
model API particles of BSA, were prepared with different
degrees of surface corrugation. As in the previous study, the
particles were investigated in terms of particle size distribu-
tion and in vitro aerosolisation efciency (Chew et al., 2005).
However, in this study the relationship between morphology,
adhesion and efciency was investigated quantitatively using
AFM imaging and colloid probe measurements.
2. Materials and methods
2.1. Materials
Bovine serum albumin (Lot: 42K1578, Fraction V, minimum
98%) was supplied by Sigma Chemical Co. (MO, USA). Water
was puried by reverse osmosis (MilliQ, Millipore Australia Pty
Ltd., Sydney, Australia). All organic solvents were supplied by
Biolab Ltd. (Victoria, Australia) and were of analytical grade.
2.2. Preparation of BSA micro-particles with different
degrees of surface corrugation
Micro-particles of BSA, with different degrees of surface cor-
rugation were prepared by spray-drying aqueous solutions in
a Buchi B-290 mini spray dryer (Flawil, Switzerland). Settings
and feed concentrations are shown in Table 1. In general, a
decrease in feed concentration resulted in increased corru-
gation. Subsequently the feed concentration (10, 25, 40 and
60mgml
1
) were used as the identier for each BSA powder.
All powders were stored and tested at 25

C and 45% RH.


2.3. Particle size analysis
The particle size distribution of each of the spray dried BSA
samples was evaluated using laser diffraction (Malvern Mas-
tersizer 2000, Malvern, UK). Approximately 10mg of sample
was dispersed in chloroform and sonicated for 5 min in a
14 european j ournal of pharmaceuti cal sci ences 3 5 ( 2 0 0 8 ) 1218
water bath (Model FXT8; Unisonics Pty Ltd., Australia). The
dispersed samples were transferred rapidly to the Malvern
sample dispersion unit (Hydro SM, Malvern, UK) for analysis.
Particle size distributions were measured between an obscu-
ration of 525% in triplicate.
2.4. Scanning electron microscopy
The morphology of each of the spray dried BSA samples was
qualitatively evaluated using a eld emission scanning elec-
tron microscopy (SEM) at 5keV (JEOL JSM 600F, Jeol, Japan).
Five images for each formulation were taken at random loca-
tions, at three magnications (1000, 5000 and 10,000).
Prior to imaging samples were dispersed onto carbon sticky
tabs and coated withplatinumto a thickness of approximately
1520nm.
2.5. Atomic force microscopy
The degree of corrugation and surface morphology of each
sample was quantied using atomic force microscopy (AFM)
(Multimode AFM, Nanoscope IIIa controller, Veeco Inc., Cali-
fornia, USA). Samples of each BSA powder were mounted in
Tempx
TM
(SPI, Pennsylvania, USA) using methods described
previously (Young et al., 2004), and were imaged at a scan
rate of 1Hz using ultra-sharp intermittent contact tips (Micro-
Masch tips, Group Scientic Ltd., Adelaide, Australia) in Tap-
ping Mode

. The variation in surface corrugation was quan-


tied by post image analysis of the sample roughness. Indi-
vidual particles (n5) were analysed over 1m1m areas
and the root mean square roughness calculated using Eq.
(1).
R
RMS
=

1
n
n

i=1
y
2
i
(1)
where n is the number of data points in a topographical prole
and y
i
is the distance of asperities (i) from the centre line.
2.6. Colloid probe microscopy
Particleparticle adhesion in each BSA system was evaluated
using the colloid probe microscopy technique. Briey, indi-
vidual particles were mounted onto the apex of V-shaped
tipless AFM cantilevers (NP-0 wafer, nominal spring constant
0.58nN, Veeco Inc., California, USA) using a micromanipula-
tion technique described elsewhere (Young et al., 2002). The
force of adhesion between each drug probe and Tempx

mounted particulates of the same type was investigated using


forcevolume imaging

. Individual force curves were con-


ducted over 10m10m areas using the following settings:
approach retraction cycle 3m, cycle rate 8Hz and constant
compliance distance 60nm. The use of forcevolume mapping
to measure the adhesion force resulted in a spatial adhesion
map and limited topographical data relating to the slope of
constant compliance. Data was exported and analysed using a
custom-built software package to produce a force of adhesion
and topography matrix. Topographical data was used to iden-
tify individual particles and individual force curves (n25),
taken diagonally across each particle, were recorded. A rep-
resentative adhesion/topography matrix is shown in Fig. 1. A
minimum of ve particles were analysed for each drug probe;
three drug probes for each BSA type were studied.
2.7. In vitro aerosol particle characterisation
The relationship between particle corrugation, adhesion and
aerosol performance was assessed using a multi-stage liquid
impinger (MSLI) (BritishPharmacopoeia). The MSLI is designed
to evaluate the regional lung deposition of particles for res-
piratory medicine. The design of the MSLI is such that at a
ow rate of 60l min
1
, estimated as equivalent to an inhala-
tion manoeuvre when using a DPI, the aerodynamic cut-off
diameters of stages 1, 2, 3 and 4 are 13, 6.8, 3.1 and 1.7m
respectively. Stage 5 contains a lter housing for capturing
particles less than 1.7m. Cumulative drug concentrations
collected in each stage of the MSLI can be plotted against the
log of effective cut-off diameter to calculate the concentration
of drug below a chosen aerodynamic diameter. In this case
an effective cut-off diameter of 5m was chosen as a suit-
able descriptor of particles with an appropriate diameter for
respiratory delivery (Pritchard, 2001).
Prior to testing, 20ml of water was added to stages 1
through 4 and the ow rate through the MSLI set to 60l min
1
using a GAST Rotary vein pump (Erweka GmbH, Germany) and
calibrated owmeter (TSI 3063, TSI instruments Ltd., Bucking-
hamshire, UK).
Approximately 20mg of BSA powder was accurately
weighed into a size 3 hydroxy-proyl-methyl-celulose (HPMC)
capsule (Capsugel, Sydney, Australia), which was placed into
the sample compartment of a Rotahaler
TM
DPI device (GSK,
UK). The device was activated, connected to a mouthpiece
adapter, inserted into a United States pharmacopoeia (USP)
throat (connected to the MSLI), and tested for 4s at 60l min
1
.
The procedure was repeated using a second capsule con-
taining 20mg of the same powder. After actuation of both
capsules, the device, capsules, throat, and all sample stages
were washed into separate volumetrics using water.
Recovered aqueous samples from the MSLI were quanti-
ed using high performance liquid chromatography (HPLC)
with the following components: Waters 717+ autosampler,
515 pump, 2487 detector, 600 controller with Millennium V.32
software (all Waters Ltd., Sydney, Australia), BioSep-SEC-S
3000 column, 3007.8mm (Phenomenex, USA). Standards
and samples were prepared in MillQ water. Mobile phase con-
sisted of 0.05M potassium dihydrogen orthophosphate which
was adjusted to pH 7.4 with sodium hydroxide. The HPLC
settings were as follows: detection wavelength 214nm, ow
rate 1.0ml min
1
, injection volume 100l and retention time
of 13min. Linearity was obtained with BSA concentration
ranging between 20200gml
1
(R
2
=0.999). All BSA powder
samples were tested using the MSLI procedure in triplicate.
3. Results
3.1. Particle size analysis
In general all BSA samples had similar particle size dis-
tributions with median volume diameters of 2.47m
european j ournal of pharmaceuti cal sci ences 3 5 ( 2 0 0 8 ) 1218 15
Fig. 1 Force volume (A) greyscale height image, (B) grey scale force of adhesion image and (C) single force curve taken at
the intersection point of (B). Lighter grey scales indicate increased height or adhesion.
0.01m (60mgml
1
), 2.67m0.02m (40mgml
1
),
2.96m0.01m (25mgml
1
) and 3.17m0.02m
(10mgml
1
). These diameters were in good agreement with
the previous study by Chewet al. (2005), and were chosen such
that the aerodynamic diameter would remain constant. Parti-
cle size distributions of each of the BSA powder are shown in
Fig. 2.
Fig. 2 Particle size distributions of BSA particles prepared
from different stock solutions.
3.2. Scanning electron microscopy
Scanning electron microscope images of the spray dried BSA
micro-particles (Fig. 3) corroborated previous ndings where
a reduction in the feed concentration (from 60mgml
1
to
10mgml
1
) with a concurrent reduction in the atomisation
rate (from 800l h
1
to 300l h
1
) resulted in an increase in
surface corrugation. It is speculated that a decrease in shell
density and increased drying time, observed in the lower feed
concentrations, would result in increased particle corrugation
due to particle collapse.
3.3. Atomic force microscopy
Particle roughness by AFM indicated R
RMS
values of
14.07nm1.46nm, 41.53nm7.08nm, 66.71nm7.32nm
and 94.6nm11.49nm for the BSA particles prepared from
60mgml
1
, 40mgml
1
, 25mgml
1
, and 10mgml
1
feed con-
centrations, respectively. Statistical analysis of the roughness
data for particles produced using each spray drying condition
suggested statistical signicance in R
RMS
with respect to feed
concentration (ANOVA p<0.05).
3.4. Colloid probe microscopy
The forces of adhesion between each drug probe and a mini-
mum of ve particles (n125 force curves) was processed as
cumulative adhesion distributions. The cumulative adhesion
16 european j ournal of pharmaceuti cal sci ences 3 5 ( 2 0 0 8 ) 1218
Fig. 3 Scanning electron microscopy images of BSA
particles spray dried from stocks solutions containing (A)
60mgml
1
(B) 40mgml
1
(C) 25mgml
1
and (D) 10mgml
1
BSA, respectively.
Fig. 4 Cumulative separation force distributions for
interactions between BSA probes prepared using the
different spray drying conditions and mounted particles of
the same type.
distributions for four drug probes, prepared using the four
spray drying conditions, and corresponding mounted parti-
cles (Fig. 4) show that as the feed concentration is reduced
(corresponding to an increased particle roughness), the force
of adhesion is concurrently reduced. As with previous stud-
ies, the median force of adhesion (f
0.5
) was taken as the best
descriptor for particle adhesion (Young et al., 2003, 2004). The
particle measurement procedure was repeated using a further
two drug probes from each spray drying condition and data
processed, as described above, to produce adhesion distribu-
tions andf
0.5
values. The meanf
0.5
standarddeviationfor the
interaction of three drug probes, from each formulation, with
their respective mounted particles were 217.8nN16.9nN,
112.3nN12.7nN, 50.3nN8.3nN and 19.9nN1.0nN for
the 60mgml
1
, 40mgml
1
, 25mgml
1
, and 10mgml
1
feed
concentrations, respectively. Statistical analysis of the f
0.5
for
particles produced using each spray drying condition (n=3),
indicated statistical signicance (ANOVA p<0.05).
3.5. In vitro aerosol particle characterisation
Total, emitted and ne particle dose values, as well as ne
particle fractions (FPF), were calculated and are shown in
Table 2. In general, as the spray drying feed concentration was
decreased, a signicant increase in ne particle fraction was
observed (ANOVA p<0.05).
4. Discussion
The BSA particulate systems were produced with similar
aerodynamic diameters but different surface roughness and
corrugation. As the projected contact area between contigu-
ous particle faces was reduced a concomitant reduction in the
net adhesion force is also appartment. Subsequently, exclud-
ing particle interlocking, an increase in particle corrugation
would result inincreased particle de-agglomeration, and thus,
aerosol performance. Indeed, comparison of the R
RMS
of the
european j ournal of pharmaceuti cal sci ences 3 5 ( 2 0 0 8 ) 1218 17
Table 2 In vitro aerosolisation data
Increase in degree of corrugation
60
a
40
a
25
a
10
a
Total loaded dose (TD)
b
(mg) 39.2 1.3 38.3 0.7 39.6 0.6 40.00.0
Emitted dose (ED)
c
(mg) 24.3 0.6 23.8 0.4 32.2 1.1 32.02.4
Fine particle dose (FPD)
d, *
(mg) 6.2 0.3 7.7 0.3 12.1 0.1 14.30.0
Fine particle fraction (FPF)
e, *
(%) 25.7 0.7 32.5 0.6 37.4 1.4 44.71.8
a
Feed concentration (mgml
1
).
b
Sum of BSA concentration recovered from all stages of the device and MSLI.
c
Sum of BSA concentration recovered from all stages of the MSLI (excluding the device).
d
Sum of BSA concentration recovered from stage 3-lter of the MSLI.
e
FPD/ED100.

Aerodynamic size 5m.


four BSA samples, with the in vitro aerosolisation efciency
(FPF), indicated that as the BSA particle roughness increased
an increase in FPF was observed (Fig. 5). In both cases the
observed increase was statistically signicant with respect to
feed concentration (ANOVA p<0.05).
To further analyse the relationship betweenparticle rough-
ness and aerosolisation efciency, the in vitro FPF values for
each feed concentration were plotted as a function of R
RMS
(Fig. 6A). Alinear correlation between FPF and R
RMS
exists with
regression analysis indicating an R
2
value of 0.96. It may be
concluded that an increase in R
RMS
leads to a reduction in
particle adhesion and thus results in a linear increase in par-
ticle de-agglomeration. To further evaluate this observation,
the particle adhesion, measured using colloidal probe AFM
was plotted as function of FPF (Fig. 6B). An inverse relationship
between FPF and mean particle separation force was observed
(where linear regression analysis indicated R
2
values of
0.94).
Interestingly, the mean separation force for the for-
mulation with the highest degree of roughness (feed
concentration=10mgml
1
) appeared to have an elevated FPF
(when compared to the regression t). It is possible that such
observations are due to a degree of mechanical interlocking in
the force volume data set that would not occur so routinely in
a free powder.
Fig. 5 AFM derived particle roughness data (n=5StdDev)
and ne particle fractions (n=3StdDev) for BSA particles
produced from different feed concentrations.
Fig. 6 Relationship between (A) particle roughness and
ne particle fraction and (B) separation force and ne
particle fraction.
5. Conclusions
The inuence of particle morphology on the surface rough-
ness and interparticulate adhesion has been investigated
using AFM in conventional and colloid probe modes of oper-
ation, respectively. Signicant differences in micro-particle
18 european j ournal of pharmaceuti cal sci ences 3 5 ( 2 0 0 8 ) 1218
roughness and particle cohesion were observed between BSA
particles with different roughness parameters. A correla-
tion between particle roughness, adhesion and aerosolisation
efciency was reported. Clearly understanding the inu-
ence of micro-particle structure and interparticle adhesion
mechanisms is of extreme importance when considering dry
powder inhaler formulations as such critical parameters will
ultimately affect the aerosol efciency and respiratory pene-
tration.
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