e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 5 ( 2 0 0 8 ) 12–18
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Micro-particle corrugation, adhesion and inhalation aerosol efficiency 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
a b s t r a c t
Article history:
Atomic force microscopy (AFM) was used to evaluate the particle adhesion and surface
Received 14 March 2008
morphology of engineered particles for dry powder inhaler (DPI) respiratory therapy to gain
Received in revised form 7 May 2008
a greater understanding of interparticle forces and the aerosolisation process. A series of
Accepted 24 May 2008
spherical model drug particles of bovine serum albumin (BSA) was prepared with differ-
Published on line 8 June 2008
ent degrees of surface corrugation. The particles were evaluated in terms of particle size (laser diffraction) and microscopic morphology (scanning electron microscopy). Conven-
Keywords:
tional tapping mode AFM was used to evaluate the nanoscopic morphology and derive
Dry powder inhaler
specific roughness parameters, while AFM colloid probe microscopy was used to directly
Particle corrugation
measure the interaction of functionalised probes. The physical characterisation and AFM
DPI
measurements were evaluated in terms of in vitro aerosolisation performance, using a con-
AFM
ventional Rotahaler® DPI and multistage liquid impinger. A direct relationship between the
Particle adhesion
root mean square roughness, particle adhesion and in vitro aerosol performance (measured as fine 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 systemic drug molecules. In simple terms, a dry powder inhaler (DPI) formulation contains an active pharmaceutical ingredient (API) that is engineered to have an aerodynamic diameter suitable for respiratory delivery; generally considered ≤5 m (Pritchard, 2001). The API is formulated with, or without excipients, in an inhalation device that during the inhalation manouveure should ensure efficient aerosolisation of the API into its primary respiratory sized particulates. These
∗
Corresponding author. Tel.: +61 2 90367035; fax: +61 2 93514391. E-mail address:
[email protected] (P.M. Young). 0928-0987/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2008.05.009
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 particle will be directly proportional to the sum of the surface energies of the contiguous surfaces, and inversely proportional to the projected contact area. Thus, the two most common approaches to improve the aerosolisation efficiency
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Table 1 – Spray-drying conditions for the production of BSA micro-particles with differing degrees of surface corrugation Increase in degree of corrugation ⇒
Spray drying settings Sample no. −1
Feed concentration (mg ml Atomisation rate (l h−1 ) Inlet temperature (◦ C) Outlet temperature (◦ C)
)
1
2
3
4
60 800 55 36
40 550 45 33
25 414 45 33
10 300 45 36
in these systems is to reduce the surface free energy of the contacting surfaces or modify the particle shape to limit contact area. Alteration of the surface energy may be achieved by the addition of excipients during manufacturing (Ahfat et al., 1997; Begat et al., 2005; Hickey et al., 1990; Iida et al., 2004; Lucas et al., 1999; Young et al., 2002) or by controlled crystallisation (Chan and Gonda, 1989, 1995; Zeng et al., 2001) (promoting specific faces, and thus, 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. Modification of the surface topology of the API particles, is also fraught with challenges, specifically when trying to modify crystalline geometry. However, the use of spray drying to prepare physically stable API powders with modified surface morphology has been shown to be a successful method of improving aerosolisation efficiency (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 surface corrugation (presumably due to the differences in initial feed concentration and solvent evaporation rate) and different aerosolisation performance characteristics. In general, the corrugated particles produced in this study had significantly improved aerosol performance when delivered from a Rotahaler® DPI (Chew and Chan, 2001). In a more recent study, Chan et al., demonstrated that the degree of surface corrugation of BSA particles could be successfully controlled using the previous technique, and the aerosolisation efficiency modified (Chew et al., 2005). However, the relationship between degree of particle corrugation and aerosolisation efficiency could only 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 unprecedented views of structure, morphology and forces at the nanoscopic and atomic level. Although a very powerful tool, the AFM was not truly utilised for probing particle interactions until the end of the twentieth century, when the apparatus was modified 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 deflection of the cantilever, as it travels through the Z-axis, and applying Hook’s law, the forces acting on a particular drug probe may be measured with extreme accuracy. Furthermore, multiple force–distance 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, coupled with the high resolution imaging capabilities of the AFM (using conventional cantilevers and imaging modes), allows unprecedented insight into the particle interactions and morphology of APIs used in inhalation therapy. Here, the relationship between API morphology, adhesion and aerosolisation efficiency was investigated. Specifically, 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 distribution and in vitro aerosolisation efficiency (Chew et al., 2005). However, in this study the relationship between morphology, adhesion and efficiency 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 purified 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 corrugation 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 corrugation. Subsequently the feed concentration (10, 25, 40 and 60 mg ml−1 ) were used as the identifier 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 Mastersizer 2000, Malvern, UK). Approximately 10 mg of sample was dispersed in chloroform and sonicated for 5 min in a
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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 obscuration of 5–25% in triplicate.
taken diagonally across each particle, were recorded. A representative adhesion/topography matrix is shown in Fig. 1. A minimum of five particles were analysed for each drug probe; three drug probes for each BSA type were studied.
2.7. 2.4.
The morphology of each of the spray dried BSA samples was qualitatively evaluated using a field emission scanning electron microscopy (SEM) at 5 keV (JEOL JSM 600F, Jeol, Japan). Five images for each formulation were taken at random locations, at three magnifications (1000×, 5000× and 10,000×). Prior to imaging samples were dispersed onto carbon sticky tabs and coated with platinum to a thickness of approximately 15–20 nm.
2.5.
Atomic force microscopy
The degree of corrugation and surface morphology of each sample was quantified using atomic force microscopy (AFM) (Multimode AFM, Nanoscope IIIa controller, Veeco Inc., California, USA). Samples of each BSA powder were mounted in TempfixTM (SPI, Pennsylvania, USA) using methods described previously (Young et al., 2004), and were imaged at a scan rate of 1 Hz using ultra-sharp intermittent contact tips (MicroMasch tips, Group Scientific Ltd., Adelaide, Australia) in Tapping Mode® . The variation in surface corrugation was quantified by post image analysis of the sample roughness. Individual particles (n ≥ 5) were analysed over 1 m × 1 m areas and the root mean square roughness calculated using Eq. (1).
n 1 RRMS = y2 n
i
(1)
i=1
where n is the number of data points in a topographical profile and yi is the distance of asperities (i) from the centre line.
2.6.
In vitro aerosol particle characterisation
Scanning electron microscopy
Colloid probe microscopy
Particle–particle adhesion in each BSA system was evaluated using the colloid probe microscopy technique. Briefly, individual particles were mounted onto the apex of V-shaped tipless AFM cantilevers (NP-0 wafer, nominal spring constant 0.58 nN, Veeco Inc., California, USA) using a micromanipulation technique described elsewhere (Young et al., 2002). The force of adhesion between each drug probe and Tempfix® mounted particulates of the same type was investigated using force–volume imaging® . Individual force curves were conducted over 10 m × 10 m areas using the following settings: approach retraction cycle 3 m, cycle rate 8 Hz and constant compliance distance 60 nm. The use of force–volume 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 identify individual particles and individual force curves (n ≥ 25),
The relationship between particle corrugation, adhesion and aerosol performance was assessed using a multi-stage liquid impinger (MSLI) (British Pharmacopoeia). The MSLI is designed to evaluate the regional lung deposition of particles for respiratory medicine. The design of the MSLI is such that at a flow rate of 60 l min−1 , estimated as equivalent to an inhalation manoeuvre when using a DPI, the aerodynamic cut-off diameters of stages 1, 2, 3 and 4 are 13, 6.8, 3.1 and 1.7 m respectively. Stage 5 contains a filter housing for capturing particles less than 1.7 m. 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 ≤5 m was chosen as a suitable descriptor of particles with an appropriate diameter for respiratory delivery (Pritchard, 2001). Prior to testing, 20 ml of water was added to stages 1 through 4 and the flow rate through the MSLI set to 60 l min−1 using a GAST Rotary vein pump (Erweka GmbH, Germany) and calibrated flow meter (TSI 3063, TSI instruments Ltd., Buckinghamshire, UK). Approximately 20 mg 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 RotahalerTM 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 4 s at 60 l min−1 . The procedure was repeated using a second capsule containing 20 mg 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 quantified 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, 300 × 7.8 mm (Phenomenex, USA). Standards and samples were prepared in MillQ water. Mobile phase consisted of 0.05 M potassium dihydrogen orthophosphate which was adjusted to pH 7.4 with sodium hydroxide. The HPLC settings were as follows: detection wavelength 214 nm, flow rate 1.0 ml min−1 , injection volume 100 l and retention time of 13 min. Linearity was obtained with BSA concentration ranging between 20–200 g ml−1 (R2 = 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 distributions with median volume diameters of 2.47 m ±
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 5 ( 2 0 0 8 ) 12–18
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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.
0.01 m (60 mg ml−1 ), 2.67 m ± 0.02 m (40 mg ml−1 ), −1 and 3.17 m ± 0.02 m 2.96 m ± 0.01 m (25 mg ml ) (10 mg ml−1 ). These diameters were in good agreement with the previous study by Chew et al. (2005), and were chosen such that the aerodynamic diameter would remain constant. Particle size distributions of each of the BSA powder are shown in Fig. 2.
3.2.
Scanning electron microscopy
Scanning electron microscope images of the spray dried BSA micro-particles (Fig. 3) corroborated previous findings where a reduction in the feed concentration (from 60 mg ml−1 to 10 mg ml−1 ) with a concurrent reduction in the atomisation rate (from 800 l h−1 to 300 l 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 RRMS values of 14.07 nm ± 1.46 nm, 41.53 nm ± 7.08 nm, 66.71 nm ± 7.32 nm and 94.6 nm ± 11.49 nm for the BSA particles prepared from 60 mg ml−1 , 40 mg ml−1 , 25 mg ml−1 , and 10 mg ml−1 feed concentrations, respectively. Statistical analysis of the roughness data for particles produced using each spray drying condition suggested statistical significance in RRMS with respect to feed concentration (ANOVA p < 0.05).
3.4.
Fig. 2 – Particle size distributions of BSA particles prepared from different stock solutions.
Colloid probe microscopy
The forces of adhesion between each drug probe and a minimum of five particles (n ≥ 125 force curves) was processed as cumulative adhesion distributions. The cumulative adhesion
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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 particles (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 studies, the median force of adhesion (f0.5 ) 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 distributions and f0.5 values. The mean f0.5 ± standard deviation for the interaction of three drug probes, from each formulation, with their respective mounted particles were 217.8 nN ± 16.9 nN, 112.3 nN ± 12.7 nN, 50.3 nN ± 8.3 nN and 19.9 nN ± 1.0 nN for the 60 mg ml−1 , 40 mg ml−1 , 25 mg ml−1 , and 10 mg ml−1 feed concentrations, respectively. Statistical analysis of the f0.5 for particles produced using each spray drying condition (n = 3), indicated statistical significance (ANOVA p < 0.05).
3.5.
In vitro aerosol particle characterisation
Total, emitted and fine particle dose values, as well as fine particle fractions (FPF), were calculated and are shown in Table 2. In general, as the spray drying feed concentration was decreased, a significant increase in fine particle fraction was observed (ANOVA p < 0.05).
4.
Fig. 3 – Scanning electron microscopy images of BSA particles spray dried from stocks solutions containing (A) 60 mg ml−1 (B) 40 mg ml−1 (C) 25 mg ml−1 and (D) 10 mg ml−1 BSA, respectively.
Discussion
The BSA particulate systems were produced with similar aerodynamic diameters but different surface roughness and corrugation. As the projected contact area between contiguous particle faces was reduced a concomitant reduction in the net adhesion force is also appartment. Subsequently, excluding particle interlocking, an increase in particle corrugation would result in increased particle de-agglomeration, and thus, aerosol performance. Indeed, comparison of the RRMS of the
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Table 2 – In vitro aerosolisation data Increase in degree of corrugation ⇒ 60a Total loaded dose (TD)b (mg) Emitted dose (ED)c (mg) Fine particle dose (FPD)d , * (mg) Fine particle fraction (FPF)e , * (%) a b c d e ∗
39.2 24.3 6.2 25.7
± ± ± ±
40a 1.3 0.6 0.3 0.7
38.3 23.8 7.7 32.5
± ± ± ±
25a 0.7 0.4 0.3 0.6
39.6 32.2 12.1 37.4
± ± ± ±
10a 0.6 1.1 0.1 1.4
40.0 ± 0.0 32.0 ± 2.4 14.3 ± 0.0 44.7 ± 1.8
Feed concentration (mg ml−1 ). Sum of BSA concentration recovered from all stages of the device and MSLI. Sum of BSA concentration recovered from all stages of the MSLI (excluding the device). Sum of BSA concentration recovered from stage 3-filter of the MSLI. FPD/ED × 100. Aerodynamic size ≤5 m.
four BSA samples, with the in vitro aerosolisation efficiency (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 significant with respect to feed concentration (ANOVA p < 0.05). To further analyse the relationship between particle roughness and aerosolisation efficiency, the in vitro FPF values for each feed concentration were plotted as a function of RRMS (Fig. 6A). A linear correlation between FPF and RRMS exists with regression analysis indicating an R2 value of 0.96. It may be concluded that an increase in RRMS leads to a reduction in particle adhesion and thus results in a linear increase in particle 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 R2 values of 0.94). Interestingly, the mean separation force for the formulation with the highest degree of roughness (feed concentration = 10 mg ml−1 ) appeared to have an elevated FPF (when compared to the regression fit). 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. 6 – Relationship between (A) particle roughness and fine particle fraction and (B) separation force and fine particle fraction.
5.
Fig. 5 – AFM derived particle roughness data (n = 5 ± StdDev) and fine particle fractions (n = 3 ± StdDev) for BSA particles produced from different feed concentrations.
Conclusions
The influence of particle morphology on the surface roughness and interparticulate adhesion has been investigated using AFM in conventional and colloid probe modes of operation, respectively. Significant differences in micro-particle
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roughness and particle cohesion were observed between BSA particles with different roughness parameters. A correlation between particle roughness, adhesion and aerosolisation efficiency was reported. Clearly understanding the influence 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 efficiency and respiratory penetration.
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