Surface Analytical Techniques in Solid-State Particle Characterization for Predicting Performance in Dry Powder Inhalers

The pulmonary route is of interest for both ef fective local therapy for respiratory and lung diseases, such as asthma, chronic obstructive pulmonary disease and cystic fibrosis, and systemic administration of drugs, such as proteins and peptides. Dry powder inhalers (DPIs) are devices through which a dry powder formulation of drug is delivered via the pulmonary route. The DPIs are highly ef ficient but complicated systems, the performance of which relies on many aspects, including aerodynamic diameter of the powder formulation, particle density, bulk density, surface morphology and composition, particle shape, interparticulate cohesive forces between drug particles and interparticulate adhesive forces between drug and carrier particles. Among them, surface morphology of both drug particles and carrier particles within the formulation is a very important factor in determining the interparticulate contact area and forces, aerosolization ef ficiency and subsequent lung deposition. Techniques that have been applied to study surface properties of solidstate particles in DPIs include atomic force microscopy, microand nanothermal analysis, inverse gas chromatography and X-ray photoelectron spectroscopy. This paper reviews dif ferent aspects of DPIs, with emphasis on their surface properties and influence on aerosol performance, and the techniques that are utilized to examine their surface properties.

and compressability 21) . Typically, the thermodynamically stable cr ystalline form is preferred since the risk of solid-state transformations (ie. solid-state phase transitions) during process and storage is minimized.
Thermodynamically over time, the less stable solid form possesses a higher Gibbs free energy, and hence, will convert to the more stable form which has a lower Gibbs free energy. Hence, solid-state phase transitions are of great importance in pharmaceutical material science. A phase transition can arise through melting, in solution, and solution-mediated, solid-solid transition. Molten transition takes place when a compound is heated and subsequently cooled after melting. A new solid phase can be obtained through solvent evaporation after being suspended or dissolved in a solvent. Solution-mediated phase transition has three steps: 1). dissolution of the metastable form; 2). nucleation as a more stable form; and 3). crystal growth of the stable phase. The solid-solid phase transition circumvents the transient liquid or vapor phase. The solid-solid transition can be categorized into monotropic and enantiotropic systems, in the case of polymorphic pairs. In a monotropic system, one polymorph is stable and the transition is irreversible. However, in an enantiotropic system, the transition is reversible and each polymorph is stable within a certain temperature range. Solid-state phase transformations [22][23][24][25] include polymorphic interconversions, dehydration or desolvation processes, and order-disorder transformations 26) . Furthermore, solid-phase transitions can be either thermodynamically or kinetically controlled processes.
In crystalline pharmaceutical powders, a crystalline particle has a number of cr ystal faces 27) . Each cr ystal face possesses unique surface properties. Typical cr ystal faces and Miller indices are shown in Fig. 1. Various faces of the L-L ysine monohydrochloride dihydrate (LH) cr ystals have different surface free energies which relates to crystal habit and orientation 28) . Therefore, understanding the surface energetics 29) of a particular crystalline face is of importance in pharmaceutical formulation, processing, and therapeutic delivery. For instance, surfaces energetics of a particular crystal face influences the nature of chemical interactions with excipients in the formulation, as well as with other particles.

The Amorphous state & other metastable
states Unlike crystalline compounds, which have orientational and positional long-range molecular order in all three dimensions of space, the amorphous state shows no long-range molecular packing ie. lacks longrange molecular order. Since, the amorphous state is a mesophase/metastable phase (ie. not a thermodynamically stable phase), physicochemical properties such as higher solubility, higher bioavailability, higher molecular mobility (ie. more disordered with higher entropy), higher chemical and physical degradation kinetics, and less stability over pharmaceutically relevant time scales are obser ved. Pharmaceutical development of amorphous form is being conducted in recent years to gain higher solubility 30) because of a higher molecular mobility, although lower physical and chemical stability is associated with it. The strategy of rationally designing the amorphous state into a pharmaceutical product can be utilized to overcome low bioavailability of poorly water-soluble crystalline drug candidates 6,9,31,32) which is increasingly more common with newer and promising drug candidates. The amorphous form can be generated intentionally or unintentionally by many common pharmaceutical processing methods, including milling [33][34][35] , quenchcooling 36) , spray-drying 37-42) , freeze-drying 9, 43, 44) and compression 9, 32) .
The amorphous phase often originates on the surfaces of particles during various types of pharmaceutical processing procedures. The oxygen gas and water vapor adsorption on these disordered regions (having a higher molecular mobility) occurs initially on the surface of particles (ie. adsorption) resulting in acceleration in oxidation and hydrolysis reactions Fig. 1 Illustration of the miller indices of crystal faces of a cubic crystal. The short dash ( ) plane (001) meets the x, y and z axes at infinity, infinity and a, respectively. The long dash dotted ( . . ) plane (110) meets the x, y and z axes at a, a and infinity, respectively. that then propagates below the surface into the bulk region (ie. absorption). Furthermore, the relatively higher molecular mobility of compound is further increased, where absorbed water acts as a plasticizer. Consequently, the physical and chemical stability 45) over pharmaceutical timescales for the powder becomes compromised.
The thermodynamic tendency for metastable phases existing on the surface of particles in the solidstate to readily take up small amounts of water vapor molecules in the environment is the basis on which quantification of very small amounts (1% or less) of these metastable phases can be carried out using gravimetric water vapor sorption. Gravimetric vapor sorption (GVS), also sometimes called dynamic vapor sorption (DVS), is a powerful and highly quantitative analytical interfacial technique used in pharmaceutical solid-state development, including inhalation aerosols. Moreover, vapor-induced solid-state phase transitions (eg. disorder-to-order phase transition, recr ystallization, liquid cr ystallization, etc.) can be systematically examined and quantified. It is of particular utility in the characterization and performance prediction of particles intended for inhalation aerosol delivery, as often particle size reduction methods (eg. micronization) induce minute but significant amounts of metastable surface phases that can impact aerosol powder dispersion and particle behavior in the high relative humidity of the lungs (> 90%), as well as overall stability of the aerosol formulation product.
In addition to the amorphous form, other mesophases/metastable phases 46,47) are important in the solid-state phase behavior of pharmaceuticals and phase transitions induced by pharmaceutical processing techniques. Based on the type of molecular mobility and directional molecular order along one or two dimensions, a mesophase can be classified into liquid crystal 48) (with positional and if applicable conformational mobility), plastic cr ystal (with orientational mobility) [49][50][51] , and condis crystal (with conformational mobility) 52) .
Therefore, it is important to be able to determine that the desired form chosen for a pharmaceutical formulation is not contaminated with other solid-state forms that have varying bioavailability and stability. Based on this, it is essential to determine the effects of processing and storage conditions on the API, which may induce solid-state phase transitions and polymorphic transformations caused by mechanical (milling, compression) and thermal stresses or interactions with formulation ingredients. Hence, solidstate transformations have to be closely monitored and trace amount of contaminating polymorphs or non-cr ystalline metastable forms (eg. amorphous) should be detected to ensure safety, quality and efficacy of the pharmaceutical product 53) .
Finally, the presence of partially ordered/disordered phases are influential in affecting performance of particles constituting powders intended for aerosol inhalation, both in vitro and in vivo 23,26,54,55) . These partially ordered/disordered phases may processinduced or rationally designed into the particle for improving aerosol performance [56][57][58] and therapeutic properties (ie. enhanced solubility and bioavailability). These phases include nanocr ystals [59][60][61] (nonamorphous state lacking long-range order), liquid cr ystals, plastic cr ystals, and amorphous phases. There is much interest in dry powder inhaler (DPI) performance prediction based on the static and dynamic properties of the powder, as a manifestation of the solid-state phases described above. Recently, novel insight and predictive correlations between DPI particle surface properties and DPI aerosol performance have been reported [62][63][64][65] .

Definition of DPIs
In medical therapy, inhaled and aerosolized medications have been used for the treatment respiratory tract diseases, such as asthma and chronic obstructive pulmonary disease, for many decades 66) .
Based on the physical states of dispersed-phase and continuous medium, inhaled drug delivery systems can be divided into three principle categories 67) : pressurized metered-dose inhalers (pMDIs), dr y powder inhalers (DPIs), and nebulizers 68) . Within each class further differentiation is based on metering, means of dispersion, or design 69) . In nebulizers, the drug is dissolved or suspended in a polar liquid, usually water. Because of their large dimensions (including pump, tubing and possibly ventilating air supply), nebulizers are not convenient to use. They are usually used in hospital or ambulatory care settings, and the aerosol is delivered continuously over an extended period of time. pMDIs are bolus drug delivery devices that contain solid drug, suspended or dissolved in a nonpolar volatile propellant. Dry powder inhalers are devices through which a dry powder formulation of drug is delivered for local or systemic ef fect via the pulmonar y route. Lactose carrierbased DPI formulations are comprised of coarse nonrespirable carrier particles of -lactose monohydrate (FDA-approved) physically mixed with jet-milled drug that is in the respirable size range 62,63,[70][71][72] . Advanced particle engineering design techniques have enabled the formulation of lactose carrier-free DPI formulations 73) .

Advantages of a dr y powder inhaler
The development of DPIs has been motivated by the desire for alternatives to pMDIs, In 1987, the Montreal protocol called for signatory countries to phase out the production of CFC propellants in medical products, in order to stop depletion of the ozone layer 74) .
In addition to being propellant free, DPIs are very portable, patient friendly, easy to use and do not require spacers 73) . Because a pMDI is pressurized, it emits the dose at high velocity, probably causing premature deposition in the oropharynx 75,76) . Despite use of spacers, incorrect use of pMDIs is still a prevalent problem 77) . Thus, pMDIs require careful coordination of actuation and inhalation. Contrastingly, DPIs with passive devices are activated by the patient s respiratory airflow. They require little or no coordination of actuation and inhalation and can achieve better lung delivery 78,79) .
Since DPIs are comprised of particles in the solidstate (ie. powders) 73) , they possess higher stability relative to the liquid state 80) and compared with pMDI formulations containing propellant and cosolvents 81) . Summary of the advantages and disadvantages of dry powder inhalers is shown in Table 1.

Drugs incorporated in DPIs
There are several factors which should be taken into consideration in the development of a dry powder aerosol delivery system, including drug powder production, aerosol performance and delivery device.
Among them, the desired site of deposition should be the starting point for ever y DPI development. When local effects are desired, receptor densities may be indicative for the preferred site of drug deposition, while when systemic absorption is desired, differences in membrane permeability and clearing mechanisms may be decisive. The majority of pulmonary drugs on the market are short-acting and longacting ß agonists, corticosteroids, and anti-cholinergic agents. Bronchodilators (ß2-agonists) in asthma and COPD, such as salbutamol and formoterol, interact with ß2-adrenoceptors, which are located on a variety of cells, including smooth muscle and epithelial cells. The concentration of ß-receptors throughout the lungs varies. Most of them are located in the alveoli [82][83][84][85] . Inhalation steroids are the cornerstone in asthma therapy and their molecular action occurs at intracellular glucocorticoid receptors, which are located through out the airways and alveoli 86,87) . Anti-cholinergic agents target muscarinic receptors, which are moderately distributed throughout the airways and periphery 82,88) .

Powder production process
The first step of DPIs manufacture is to produce bulk drug preparations by crystallization from solution, filtration and dr ying. Subsequently, to create particles in the respirable size range (< 5 m in diameter), the drug particle size must be reduced. The conventional size-reduction technique is milling [89][90][91][92] , which is simple, cheap and easy to scale up. There are many different mills, three main types of which used in active pharmaceutical ingredient manufacture are fluid-energy mills, high-peripheral-speed Spacers not required and dose counters incorporated in most newer designs mills, and the ball mill. The shortcoming of milling is its poor control over the particle crystallinity, shape, size and size distribution.
Other techniques for production of stable micronsized particles involve spray dr ying [93][94][95] and supercritical fluid technology [96][97][98] . In spray dr ying, the feed solution is supplied at room temperature and pumped to the nozzle where it is atomized by the nozzle gas. The atomized solution is then dried by preheated drying gas in a special chamber to remove water moisture from the system, thus forming dr y particles. These prepared particles are collected with a cyclone separation device 99,100) . Compared with milling, spray-drying offers more flexibility and the possibility of morphology control in addition to size control 101,102) ; however, spray dried particles are mostly amorphous 103) .
The basic feature of the supercritical fluid process is the controlled cr ystallization of drugs from dispersion in supercritical fluids, carbon dioxide. This method has demonstrated a wide range of application in producing pulmonary inhalable formulations [104][105][106] . Supercritical fluid technology can be divided into several classes. The most important two are supercritical anti-solvent precipitation (SAS) 107,108) and supercritical fluid extraction of emulsions (SFEE) 109,110) . The fundamental mechanism of SAS is based on rapid precipitation when a drug solution is brought into contact with a supercritical CO2. SFEE is based on extraction of the organic phase in oil-in-water or multiple emulsions using supercritical CO2 109,110) . Because most of the drugs (e.g., asthma drugs) are not soluble in CO2, SAS processes provide an easy and excellent way to produce dry powder inhalation formulations. SFEE can provide uniform crystalline drug particles, composite particles containing polymeric materials and the drugs 109,110) . For the formulator, it is important to consider the effect of each technique on the drug when choosing a suitable one to produce the desired results.

Interparticulate forces and strategies to
improve aerosol performance Micron-sized particle (< 5 µm) produced by highenergy operations such as milling usually have large sur face areas and sur face energies, which cause poor flow and a high tendency to aggregate 111) . These interpar ticulate cohesive forces include mechanical interlocking due to surface asperities, capillary forces from the presence of water, electrostatics arising from the insulating nature of the material, and van der Waals forces from the fundamental elec-tromagnetic nature of matter 92,112) . One way to add in particle separation is to agglomerate micron-sized drug particles with larger "carrier" particles of the excipients 113) . Excipients can reduce drug cohesiveness by occupying the high-energy sites of the drug particles, consequently improving handling, dispensing, and metering of the drug. According to the FDA guidance for dry powder inhaler drug products, -lactose monohydrate is the only approved sugar that can be used as a larger carrier particle in dry powder inhalation aerosol products to fluidize and disperse the respiratory drug while itself not being delivered to the lung. Lactose is highly crystalline and has the smooth surfaces and satisfactory flow properties desirable for a DPI carrier particle. The disadvantage of lactose is that it is a reducing sugar, which may lead to chemical instability particularly in formulations containing protein and peptides 114) . Other sugars, such as mannitol and glucose have been shown to be feasible alternatives to lactose 115) .
Except for blends using fine carriers and ternary components, several other methods can be used to improve the performance of dry powder aerosol delivery systems: Low aerodynamic diameters (e.g., small physical size) Low particle density (e.g., porous particles) 116) Low bulk density (loose particle packing to reduce particle contacts) 117) Rough surface (to increase air drag force and reduce particle interaction) [118][119][120] Surface composition (to reduce surface energy) Shape (e.g., elongated particles) The basic principle of the above methods is to overcome the cohesive forces between drug particles. Moreover, to generate an aerosol containing drug particles in the aerodynamic size range of 1 5 m and deliver the drug particles to the target area for deposition, the drug particles must be detached from the carrier crystals in adhesive mixtures or nucleus agglomerates by shear forces during an adequate inhalation. Coates et al. analyzed computationally and experimentally the influence of airflow rate of a DPI on particle dispersion and throat deposition. An optimal inhaler performance was found at a respiratory airflow rate of 65 liters/min 121) .

Device design of a dr y powder inhaler
From the design viewpoint, the primar y components of a dr y powder inhaler are the same for all types of devices on the market as well as those in development. They consist of a powder formula-tion, a dose mechanism, a powder de-agglomeration principle, and the inhaler s mouthpiece. The dose mechanism is applied to measure a single drug dose. The powder de-agglomeration principle is used to dispense the powder into the inhaled air stream. In some devices, secondary inhaler parts may be applied for different purposes, such as safety, ease of handling, signaling to the patient and moisture protection of the drug formulation. The de-agglomeration principle is one of the most important parts of the inhaler, as to a large extent it determines the de-agglomeration ef ficiency and thereby the lung deposition of the drug. The dry powder formulation is also a critical component of a DPI 122,123) . Most formulations are sensitive to moisture during manufacturing, storage, and usage, which may affect the ultimate delivery of 1 to 5 micron-sized, stable, and reproducible drug aerosols. Therefore, new packaging technologies, including multidose blister packs, the use of nonirritating excipients, and improved device designs have made DPIs more acceptable 124) .

Surface Properties of Powder Formulations and Surface Analytical Techniques
For pharmaceutical solid systems, the surface of the primary active pharmaceutical ingredient and associated excipient particles is crucial during manufacturing, as it affects not only the handling characteristics such as powder flowability and compressibility but also the product performance such as wettability, hardness, and dissolution rates [125][126][127] . For dry powder inhalation aerosols, surface morphology (corrugated or smooth surface) of both drug particles and carrier particles within the formulation directly influence the interparticulate contact area, interparticulate forces, aerosolization efficiency and subsequent lung deposition 120,128,129) . Surface properties of dry powders can be exploited for DPI formulation design by creating or selecting drug particles and carrier particles with specific morphology 130,131) . Drug particles, with a higher degree of surface rugosity are reported in lower powder cohesion and consequently increased dispersibility of powder particles 120) . Conversely, high degrees of surface roughness may also promote mechanical interlocking between particles, influencing the powder flow and dispersion 132) . Hence, it is important to choose an optimal value considering both advantages and disadvantages. Surface morphology, together with particle size and shape, determines surface area, which in turn determines the contact area and interparticulate forces between particles. Corrugated particles have more surface area than smooth par ticles that occupy the same volume. Therefore, ideally, surface properties, including surface morphology, contact area and interparticulate forces, should be adjusted to a level that provides enough adhesion between drug and carrier to obtain a stable formulation, and also allows easy separation upon inhalation. Techniques that have been applied to study surface properties of solid-state particles in dry powder inhalers include atomic force microscopy, micro-and nanothermal analysis, inverse gas chromatography, and X-ray photoelectron spectroscopy. They are summarized in Table 2 and described in the following sections.

Surface analytical techniques Applications
Atomic force microscopy (AFM) Microscopic analytical technique. Characterizes surface structure, morphology, adhesive forces, interparticulate interaction between drug and drug particles, and drug and carrier particles.
Micro-and nanothermal Analysis (MTA/NanoTA) Highly localized (micrometer or sub-micrometer scale) thermal analytical technique. Measures the composition, morphology, and transition of inhalation powder particles. Distinguishes between the drug substance and excipients in solid dispersion. Evaluates heterogeneity of multicomponent systems and provides three-dimensional information.
Inverse gas chromatography (IGC) Chromatographic analytical technique. Elucidates a wide range of physicochemical properties of the solid, including surface energy, heats of sorption, adsorption isotherms, glass transition temperatures, solubility parameters, energetic heterogeneity profiles, diffusion coefficients and particular functional groups on the surface of solid materials.

X-ray photoelectron spectroscopy (XPS)
Quantitative spectroscopic technique. Measures surface elemental composition of a dry powder inhalation aerosol, empirical formula, chemical state and electronic state of the elements that exist within a material.

Atomic force microscopy (AFM)
Atomic force microscopy is a technique employed to analyze and characterize the surface structure, morphology and forces of materials at the nanoscopic and atomic level [133][134][135][136] .
One of the important components of AFM is a micro-fabricated cantilever whose deflection is usually recorded by laser reflection when it interacts with a moving substrate. Direct measurement of force can be achieved by ramping the tip vertically in z axis direction, towards and away from a sample. When the tip approaches, makes contact with, and is removed from a surface, the relative deflection of the cantilever produces a force-distance curve. The change in hysteresis in cantilever deflection during approach and retraction is related to the magnitude and type of forces acting between the two surfaces 137,138) . A schematic representation of principle of AFM is shown in Fig. 2.
Based on its capability to measure morphological properties of materials, AFM is used to predict the aerosolization performance of micron-sized particles for inhalation. As stated above, the force required to aerosolize an adhered drug particle is directly proportional to the sum of the surface energies of the contiguous surfaces, and inversely proportional to the projected contact area. The two most common approaches to improve the aerosolization efficiency in these systems are to reduce the surface free energy of the contacting surfaces or modify the particle shape to limit contact area. Producing drug particles with rough surfaces can reduce surface free energy and particle adhesive forces. Spray drying process has demonstrated the potential to prepare physically stable drug powders with modified surface morphology, presumably due to the differences in initial feed concentration and solvent evaporation rate 118,120,[139][140][141] . To clarify this, in one study, a series of spherical model drug particles of bovine serum albumin (BSA) was prepared with different degrees of surface corrugation by spray drying process 142) . The relationship between the morphology of the prepared particles, adhesion and aerosolization efficiency was investigated quantitatively using AFM imaging and colloid probe measurements. The results confirmed that as the degree of corrugation increased, particle adhesion was reduced which, resulted in a concomitant increase in fine particle fraction FPF and better aerosolization performance.
AFM has also been identified as a tool capable of measuring the interactions between individual particulates and substrates at specific environmental conditions, such as temperature and relative humidity (RH). Capillary interaction due to the water vapor is one of the primary interparticulate forces in dry powder delivery systems. Young et al. investigated into the effect of humidity on drug-drug interactions using the AFM with an attached custom-built perfusion apparatus 143) . Salbutamol sulfate drug particulates were chosen as a model system and mounted on V-shaped AFM cantilevers using a novel micromanipulation technique. Force-distance curves obtained from the measurements between cantilever drug probes and model compacts of salbutamol sulfate were integrated to determine separation energies as a function of RH (15-75%). Significant increases in the median separation energies were observed as humidity was increased, most likely attributed to stronger capillary interaction at higher humidity. This study also shows the potential of AFM as a rapid preformulation tool to quantify the separation energies between micronized drug particles.
In addition to the characterization of the surface morphology and interpar ticulate interactions between drug and carrier particles, the AFM provides opportunity to assess the interaction between inhaled drug particles and the internal surfaces of the deep lung, that is to say the interaction between micronized drug par ticles and pulmonar y sur factant. In one study, the force of adhesion between micronized budesonide particles and simulated pulmonary surfactant (PS) monolayers (Survanta monolayers) were visualized and quantified by the combination of AFM with a Langumir-Blodgett (LB) approach 144) . The results indicated that budesonide is hydrophobic and Survanta films at increasing surface pressure exhibit a rising hydrophobic character. AFM revealed that PS properties were governed by applied surface pressure and that the degree of interaction of budesonide was greater at higher surface pressure, where packing of the lipid film was increased; consistent with the point of exhalation. The increasing hydrophobicity of the PS film, on increased pressure, was believed to be the primary reason for increased interaction with the hydrophobic budesonide, which correlates well with the accepted inhaler technique. Besides, AFM has been used to observe and monitor the cr ystallization of lactose and the ef fect of mechanical processing on the powder surface. The cohesive-adhesive balance (CAB) approach to colloid probe AFM has been employed to measure the interfacial force balance between an active pharmaceutical ingredient and substrate surfaces 145,146) and determine the influence of micronized budesonide crystal habit on the prediction of dry powder inhalation formulation performance 147) . Tapping mode AFM effectively images crystals of various organic compounds, including drugs (cimetidine 148) and felodipine 149) ), and the adhesional properties of carrierparticle 130,145,[150][151][152] .

Micro-and nanothermal analysis
Micro-and nanothermal analysis is an emerging localized thermal analysis technique which in essence involves the replacement of the tip of a conventional atomic force microscope with a thermal probe, thereby allowing site-specific characterization [153][154][155][156][157][158] . It overcomes the disadvantage of conventional bulk thermal measurement techniques, such as dif ferential scanning calorimetr y (DSC) and modulated temperature DSC (MTDSC). DSC and MTDSC only provide results representing the sum of all the constituents in the specimen and no information on the size, shape or spatial distribution of constituents within a multicomponent system. The principle of microand nanothermal analysis is based on performing highly localized material property characterization on a micrometer to a sub-micrometer scale on a sample subjected to a controlled temperature programme using a heated tip.
Like an AFM, the major component of a micro-and nanothermal system is a cantilever with an integrated tip that is used to scan the surface, its position being controlled in the x, y and z directions by means of a piezoelectric scanner. Laser light is used to measure the bending degree of the cantilever, and thereby the normal force acting between the tip and sample. Instead of standard AFM tips, the instrument uses a thermal probe. Two popular thermal probes are Wollaston wire based probes (platinum/rhodium core surrounded by a silver sheath) and silicon-based nanoprobe. Wollaston wire based probes are approxi-mately 1 m, although the use of thinner wires 159) or the addition of diamond tips 160) has achieved a resolution around 100 nm. Silicon-based nanoprobe with a miniature heater has a topographic spatial resolution of around 5 nm and thermal property measurement resolution of up to 20 nm 161) .
Micro-and nanothermal techniques allow the investigation of the composition, morphology and transition of inhalation powder particles 162,163) . It is able to distinguish between the drug substance and excipients in solid dispersions. This technique also allows the study of sample surface as well as analysis of the distribution of materials below the surface of the material by having the probe penetrate the sample surface. Consequently, materials can be characterized in a spatially resolved manner, especially in terms of providing three-dimensional information.
The manufacturing of dry powder inhalers involves the micronization of the drug and mixing with an inert carrier such as lactose in order to facilitate adequate drug penetration into the lungs. The surface properties of both components are crucial to subsequent performance. Since the amorphous material is thermodynamically unstable in terms of recrystallization and may also be more susceptible to chemical degradation, the generation of even small quantities of amorphous material may have a profound effect if the material is located extensively on the powder surface. Therefore, the quantification and mapping of amorphous materials in other wise cr ystalline samples has become a major concern. Dai et al. has described a case study using nanothermal analysis for a reliable identification of partially amorphous surface 153) . The authors prepared compressed tablets of amorphous and crystalline lactose and used thermomechanical analysis (L-TMA) performed by using micro-and nanothermal analysis in addition to single point variable temperature pull-off force measurements for mapping amorphous and cr ystalline lactose at a nano-scale. L-TMA was shown to be able to differentiate the amorphous and crystalline forms of lactose via measurement of the thermal events associated with them. Pull-off force measurement showed that amorphous lactose has higher adhesion than cr ystalline, even at temperatures below glass transition tomperature (Tg), and this kind of adhesion increased on approaching the Tg.
Nanothermal analysis (nano-TA) has also been utilized to characterize nano-and micro-scale heterogeneity in the solid-state properties of drug-polymer formulations 164) . Zhang et al. demonstrated that the morphology of a nano-dispersed pharmaceutical sys-tem containing carbamazepine (CBZ) and hydroxypropyl methyl cellulose (HPMC) at a 50/50 polymer/ drug ratio was mapped and determined by using nano-TA probes 164) . High resolution image obtained from AFM tapping mode showed a structure with an occluded phase, and the continuous matrix corresponded to HPMC while the bright occluded phase was attributed to the presence of CBZ nano-crystals, approximately 50 nm in size. The phase image contrast observed confirmed the presence of heterogeneity.

Inverse gas chromatography (IGC)
IGC is a physical characterization technique that has been used for the measurement of the surface properties of a wide range of solid materials, including pharmaceutical powders, nanomaterials, polymers and coatings.
The concept of IGC is rather simple; taking a standard gas chromatography (GC) experiment and inverting the roles of the stationary (solid) and mobile (gas or vapor) phases. In GC, a standard column is used to separate and characterize several gases and/ or vapors. With IGC, the unknown surface is the powder that is packed into a column and the known materials are the vapor probes that are injected into the column (Fig. 3). The retention time of the probe molecule is then measured by traditional GC detectors, such as flame ionization detector and thermal conductivity detector. Measuring how the retention time changes as a function of probe molecule chemistry, probe molecule size, probe molecule concentration, column temperature, or carrier gas flow rate can elucidate a wide range of physicochemical properties of the solid.
Parameters accessible by IGC include heats of sorption, adsorption isotherms, glass transition temperatures 165) , solubility parameters 166) , and particularly surface energy of solids [167][168][169] . Many publications have shown potential for IGC use in batch to batch variability 170) , the influence of milling on the surface free energy 171,172) , differences of surface free energy for two isomers 28) , the influence of humidity on surface free energy of different powders and drug-carrier interactions for dry powder formulations 173,174) .
The surface energetics of two supercritical CO2processed and two commercial reference samples of salmeterol xinafoate (SX), a selective long-acting ß2adrenergic bronchodilator used clinically for asthma prophylaxis, have been characterized by IGC at infinite dilution 175,176) . The results showed that the metastable SX-II polymorph possessed a higher surface free energy, higher surface entropy, and a more polar surface than the stable SX-I polymorph.
IGC can give information on the relative exposure of par ticular functional groups on the sur face of solid materials, due to electron acceptor and donor properties 177) . Grimsey et al. have demonstrated that the acidic and basic parameters of saccharides determined by IGC reflected the concentration of hydroxyl groups exposed on their surface 178) . Furthermore, IGC can be used to determine the functional groups on the surface of solid materials at various relative humidities, to explore the extent of the interaction between the surface and water 179,180) . Sunkersett et al. have demonstrated that the specific energies of adsorption of polar probes for paracetamol and carbamazepine changed as RH was raised and the particular interaction sites of water molecules on the surface have been identified using a combination of IGC and molecular modeling 173) .
Summarily, IGC appears to be of particular utility for DPI formulation work, as it requires only small samples for analysis, and it is nondestructive, fast and information rich. A more comprehensive review of this technique has been published elsewhere 169,[181][182][183] .

X-ray photoelectron spectroscopy (XPS)
As a quantitative spectroscopic technique, X-ray photoelectron spectroscopy measures the elemental composition, empirical formula, chemical state and electronic state of the elements within a material. Briefly, powder samples are irradiated by an X-ray beam, which induces the ejection of electrons from the atoms. The kinetic energies of the photoelectrons are analyzed and their binding energies are determined. Since the binding energies of electrons in the atom of origin are characteristic for the elements in a certain chemical environment, XPS provides an Gas probes injected into a column as mobile phase elemental analysis and further information on functional groups, including the atomic compositions of a sample, the chemical state of a certain element, the electronic structure and band structure. In many cases, chemical shifts can be used to draw direct conclusions on the local coordination in a system and the electronic change upon adsorption. One example of the application of this technology in the characterization of surface composition of a dry powder inhalation aerosol is given here. The surface composition of a dry powder aerosols prepared by spray-drying was analyzed by XPS 184) . The powder aerosols were made of albumin, dipalmitoylphosphatidylcholine (DPPC) and a protein stabilizer (lactose, trehalose or mannitol). The powders exited a Spin-halerTM inhaler as particle aggregates. XPS results revealed a large surface excess with DPPC relative to albumin under certain spray-drying conditions and self-organized in a gel phase in the particle. Furthermore, no sugar or mannitol crystals were detected by X-ray diffraction and water sorption isotherms showed that albumin/sugar/DPPC combinations were not prone to crystallization upon exposure to moisture. The surface enrichment of DPPC and the avoidance of crystallization offer a physical environment favorable to protein stability.

Conclusion
The fundamental understanding of rational DPI formulation and aerosol dispersion performance is rooted in fundamental principles in the basic science disciplines of solid-state physics, physical chemistry, surface chemistr y, micromeritics, powder technology, and aerosol science. This paper describes the important features of DPIs and the techniques used to examine the surface properties of the particles comprising a DPI formulation. Surface analytical techniques such as AFM, MTA/NanoTA, IGC, XPS, and GVS can provide very useful and unique surface parameters, including surface composition, morphology, structure, energetics, phase behavior, and interparticulate forces. The fundamental data acquired from these surface analytical techniques can greatly facilitate an in-depth and rigorous understanding of the powder formulation at the level of particle interfaces (ie. the solid-solid interface, the solid-liquid interface, and/or the solid-air interface) existing in dry powder inhalation aerosol formulations. These interfaces give rise to the interfacial interactions acting between particles and consequently greatly influence the aerosol dispersion performance of particles in the solid-state. A fundamental understanding, as such, can provide unique predictive insight into rationally designing particles in the solid-state that are intended for use in DPIs having optimal surface properties to provide optimal aerosol dispersion performance for a given DPI formulation. Furthermore, a given DPI formulation can be rationally tailored at the particle surface level to increase its aerosol dispersion performance.