Early Stage Phase Separation in Pharmaceutical Solid Dispersion

Article pubs.acs.org/molecularpharmaceutics

Early Stage Phase Separation in Pharmaceutical Solid Dispersion Thin Films under High Humidity: Improved Spatial Understanding Using Probe-Based Thermal and Spectroscopic Nanocharacterization Methods Sheng Qi,* Jonathan G. Moffat, and Ziyi Yang School of Pharmacy, University of East Anglia, Norwich, Norfolk, NR4 7TJ, United Kingdom ABSTRACT: Phase separation in pharmaceutical solid dispersion thin films under high humidity is still poorly understood on the submicrometer scale. This study investigated the phase separation of a model solid dispersion thin film, felodipine-PVP K29/32, prepared by spin-coating and analyzed using probe-based methods including atomic force microscopy, nanothermal analysis, and photothermal infrared microspectroscopy. The combined use of these techniques revealed that the phase separation process occurring in the thin films under high humidity is different from that in dry conditions reported previously. The initial stage of phase separation is primarily initiated in the bulk of the films as amorphous drug domains. Drug migration toward the surface of the solid dispersion film was then observed to occur under exposure to increased humidity. PVP cannot prevent phase separation of felodipine under high humidity but can minimize the crystallization of amorphous felodipine domains in the solid dispersion thin films. This study demonstrates the unique abilities of these nanocharacterization methods for studying, in three dimensions, the phase separation of thin films for pharmaceutical applications. KEYWORDS: phase separation, solid dispersion, thin films, crystallization, nanocharacterization

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INTRODUCTION Solid dispersions have been widely used for the purpose of enhancing the dissolution of poorly water-soluble drugs via the involvement of polymers that can rapidly dissolve in biological fluids.1−3 However, the physical instability of many solid dispersion formulations has resulted in few successes in terms of marketed products. In particular, for systems containing hygroscopic polymers, physical instability upon exposure to a humid environment can be a major drawback and compromise the anticipated advantage in drug release.4,5 Phase separation is one of the direct results of the physical instability of these formulations. Although a high number of publications have described the physical instability of solid dispersions as well as the relationship between phase separation and miscibility between drug and polymer, few studies have focused on the formulation-processing effect on phase separation behavior.6−8 The lack of knowledge of process-specific phase separation behavior of formulations can lead to the generalization of solid dispersion formulation strategies and possibly result in the unforeseen physical instability of products being processed using different methods. This study focuses on understanding the phase separation behavior of solid dispersion thin films with a thickness of 2−10 μm, prepared by rapid solvent evaporation (spin coating), under high humidity. The involvement of mechanical stress in a formulation process (such as hot melt extrusion) often can complicate the driving force of phase © 2013 American Chemical Society

separation. Therefore, spin coating is a good sample preparation method for conducting studies into the fundamental understanding of the phase behavior of a drug in polymer base solid dispersions. However, the limitation is that the knowledge gained only applies to polymer−drug thin films prepared by rapid solvent evaporation. Nevertheless, polymer−drug thin films have been increasingly used in medical and biomedical applications.9 Drug-loaded polymeric thin films (within a few micrometer thicknesses) have attracted increased attention in the field particularly for coating medical implants and stents for drug delivery purposes as well as applications in bioadhesive formulations, skin delivery, and wound management.9−11 Extensive knowledge of the phase behavior of thin films produced from polymeric blends and polymeric systems containing small molecular additives has been accumulated in the field of polymer sciences and organic semiconductor thin films.12−17 The phase separation behavior is significantly affected by physical properties, such as the thickness of the film, the external environment, and the chemical compatibility, such as miscibility of the blend.12−17 For example, a recently published study has shown that phase separation is minimized if the Received: Revised: Accepted: Published: 918

June 7, 2012 January 7, 2013 January 15, 2013 January 15, 2013 dx.doi.org/10.1021/mp300557q | Mol. Pharmaceutics 2013, 10, 918−930

Molecular Pharmaceutics

Article

monomer of the polymer and the small molecular additives are highly chemically similar.16 It has also been highlighted in another recent work that the film thickness and polymer-additive interactions can have a significant impact on the crystal growth.17 Polymer−drug films may share some similarities in phase separation behavior with polymer films containing small molecular additives. An increasing number of publications have indicated that there are a number of dominating factors that can play critical roles in the phase separation of polymer−drug dispersions. 19−22,25,26,28 These include the formation of intermolecular interactions between the polymer and the drug, the physical stability of the amorphous drug and polymer, and the hygroscobicity of the polymer (particularly concerning the stability under high humidities).19−22,25,26,28 An important aspect of understanding phase separation/drug crystallization in polymer−drug thin films is that it can directly affect the in vivo functionality of the films used for drug delivery. In a solid dispersion film, the growth of phase-separated drug crystals on the surface or in the bulk of the film can have a significant impact on the release of the drug if the drug is poorly water-soluble. It has been shown that drug crystals embedded in the bulk of a solid dispersion still can have significantly increased dissolution over that of drug crystals alone;18 in contrast, it is easy to understand that a high level of crystal growth on the surface of a solid dispersion can dramatically reduce the wettability of the formulation and result in decreased dissolution (as compared to a corresponding solid solution or drug embedded in polymeric material). Therefore, the key goal of this study is to use combined macroscopic and nanocharacterization methods to develop a fuller understanding of the phase separation process of solid dispersion thin films containing drugs, which is essential for development and to maximize the potential of using thin films for drug delivery. There is little reported literature on understanding the phase separation behavior of polymer−-drug thin films under high humidity in a three-dimensional fashion.4,5,19−22 Previously, this may have been directly associated with the limitations of available characterization methods to study a micrometer to submicrometer thick film. Many conventional analytical methods have been used for film characterization. However, these conventional macroscopic characterization methods often have low spatial resolution with respect to the distribution of different components within the thin films. The lack of detailed and localized characterization capacity leads to the difficulties of characterizing and understanding the phase separation process at a submicrometer scale.23−25 Recently developed nanocharacterization tools using scanning probe-based localized characterization methods including pulsed force atomic force microscopy (AFM), nanothermal analysis (nanoTA), and photothermal infrared microspectroscopy (PT-MS) allow detailed and highly localized physicochemical characterization of formulations.23−25 These analytical techniques are highly suitable for studying polymeric thin films. In this study, we propose the combined use of macroscopic and scanning probe-based localized characterization methods, as illustrated in Figure 1, to thoroughly understand the evolution of the phase separation in drug-loaded polymer thin films with three different drug-loading architectures. Importantly, the uses of the localized micro- and nanocharacterization methods allow us to study the phase separation process in a three-dimensional fashion, which cannot be achieved using conventional macroscopic methods.

Figure 1. Illustration of the combined macroscopic and scanning probebased microscopic characterization methods used to study the polymer−drug thin films.

The model system used in this article is the well-studied felodipine-PVP solid dispersion system.4,5,19−22,26−28 Moistureinduced phase separation is of particular importance to hydrophilic and hygroscopic polymer-based solid dispersions since it is a more likely environmental stress to cause stability concerns during product storage than high temperature. Therefore, this study used high humidity as the main stress condition to induce phase separation. It has been reported that PVP-based solid dispersions often exhibit a strong tendency to phase separate on exposure to moisture.4,20,26 This observation has been suggested to be a result of the disruption of drug− polymer interactions (if they exist) in the presence of moisture.4,20 In the case of felodipine-PVP solid dispersions, amorphous−amorphous phase separation was reported after the exposure to high humidity.4,20 However, there is no specific knowledge of the phase separation in three dimensions, such as surface phase separation and bulk phase separation in a thin film formulation. This study reports for the first time the bulk phase separation dominating mechanism of felodipine in PVP solid dispersion thin films under high humidity and demonstrates the unique abilities of nanoTA and PT-MS for studying the phase separation of polymer−drug thin films.

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EXPERIMENTAL SECTION Materials. Felodipine was provided as a gift from Evonik (Darmstadt, Germany). PVP K29/32 was a gift from BASF. Dichloromethane (ChromAR grade) and ethanol were obtained from Sigma-Aldrich (United Kingdom). Spin-Coated Films. Films with a thickness of 5−10 μm were prepared using a SCS G3P-8 lab scale spin coater (Cookson Electronics, RI) with a spinning speed of 3000 rpm. Amorphous felodipine films were prepared by dissolving the crystalline drug in 1:1 dichloromethane/ethanol and spin coated with a spinning speed of 3000 rpm for 2 min onto thin 18 mm × 18 mm glass microscopic cover slides (Menzel-Gläser, Braunschweig, Germany). Felodipine and PVP K29/32 with mass ratio of 1:1 were dissolved in 1:1 dichloromethane/ethanol and spin coated onto glass slides. Fresh films were analyzed within 2 h of preparation. The humidity chamber containing tissues overly saturated with water was equilibrated at room temperature overnight to create the saturated humidity. Films were exposed to saturated humidity at room temperature and were examined after 24 h of exposure. For the production of layered films, the solution of 15% (w/w) solid content of either the drug or the polymer was first spin coated on the slides. Once the first layer was completely dry, the second layer was spin coated on top using the same conditions. All humidity-treated films were dried overnight under 0% RH (P2O5) before measurements. 919

dx.doi.org/10.1021/mp300557q | Mol. Pharmaceutics 2013, 10, 918−930


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Figure 2. Representative PLM images of the humidity-treated (a) amorphous felodipine films after 24 h, (b) felodpine-PVP solid dispersion films after 24 h, and (c) the double-layered film with the felodipine layer on the top and the PVP layer underneath after 24 h and (d) after 7 days of exposure to saturated humidity.

Macroscopic Characterization Methods. Polarized Light Microscopy (PLM). PLM studies were conducted using a Leica DM LS2 polarized light microscope (Wetzlar GmbH, Germany) connected to a video capture system. The phase separation and crystallization behavior of the edges of the films were not considered representative of the entire films as there is over flow of the coating solution on the edges. This can lead to changes in the thickness of the film and variation in the crystallization behavior. Only data from the central regions of the films are reported in this study. The growth of the phase-separated domains was followed by taking a series of images of the thin films after exposure to the humidity for different time periods (from 0 min to 24 h). Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy. The IR spectra of the spin-coated films were collected using an FTIR spectrometer (IFS66/S model from Bruker Optics Limited, Coventry, United Kingdom) with a mercury/cadmium/telluride detector coupled with a GoldenGate single-reflection diamond ATR accessory (SPECAC, Orpington, United Kingdom). Sixty-four scans were acquired for each film sample with a resolution of 2 cm−1. Powder X-ray Diffraction (PXRD). PXRD studies on the spincoated thin films were carried out at room temperature on Thermol-ARL Xtra diffractormeter (Thermo Scientific, United Kingdom). The X-ray source used was Cu Kα1 with a voltage of 45 kV and current of 40 mA. It was mounted with the wavelength of 1.54 Å. The scanning angular range was from 3 to 80° (2θ) with a step size of 0.01°. The scan time per step was 4 s to minimize the background noise. Dynamic Vapor Sorption (DVS). The DVS studies were conducted using a TGA Q5000 (TA Instruments, New Castel, United States). A small fraction of the film-coated slides was cut and used for the measurements. The isohumidity experiments were performed at 25 °C/75% RH, and a per-drying step at 25 °C/0% RH for 4 h (to ensure reaching the complete drying) followed by a minimum of 6 h of exposure to 75% RH were allowed to reach equilibrium. A postdrying was performed after

the 75% isohum experiments to investigate the dehydration of the films. Microscopic Characterization Methods. AFM and nanoTA. AFM imaging and nanoTA were performed using a Veeco diCaliber scanning probe microscope head (Veeco, CA) equipped with a thermal nanoprobe (AN nanoprobes) (Anasys Instruments, Santa Barbara, CA). For imaging, a scanning speed of 0.5 Hz was used at a resolution of 200 pixels per line. Each image was obtained over an approximately 20 min period. Localized nanoTAs were performed using a nanoTA2 controller also from Anasys Instruments, which controls the voltage supplied to the tip. The probe was calibrated for temperature by supplying a scanning voltage profile while in contact with polymeric materials with known melting points. A thermal event is defined as penetration of the probe into the surface due to softening of the material. Spin-coated slides were fixed to a magnetic stub using double-sided tape and mounted onto an X-Y translating microscope stage. A contact mode AFM image was generated in the first instance. The thermal nanoprobe was brought to selected locations according to the AFM image, and nanoTA measurements were performed at each point. Three− four locations were selected on the surface of each sample and tested. An extension of the nanoTA technique is a method known as transition temperature microscopy (TTM). With this technique, a series of nanoTA measurements were performed over a selected area on a sample surface with the same probe described previously. The sample surface was imaged with an optical microscope instead of an AFM, however. Each measurement stopped when a thermal event was detected. The detected transition temperature was assigned a color based on a particular palette; hence, an image was assembled based on transition temperatures. These measurements were carried out on a specialized system known as the VESTA (Anasys Instruments). Transition temperature maps were measured over a 50 μm × 50 μm area, and each nanoTA measurement was carried out from room temperature until the transition temperature was detected. After each measurement, the probe was retracted and moved to 920



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Figure 3. Physical characterizations of the thin films of amorphous felodipine before and after exposure to high humidity. The top panel contains the ATR-FTIR spectra of the (a) N−H stretching region and the (b) CO region of the pure drug films before and after aging. The bottom panel contains the nano-TA profiles of the tested regions on the left-hand side and the optical images on the right showing the areas on the film that were analyzed and the corresponding transition temperature maps that were generated.

instance. Ten different locations on the film within the image were selected, and the thermal probe was approached onto the selected sampling point for spectrum collection. After the tip was placed in contact with the sample, 200 scans of each tested point were acquired with a resolution of 8 cm−1. The PT-MS spectra at different locations were then compared.

the next location. The distance between locations for measurements can be controlled and was set at 5 mm for single component systems and 1 mm for multicomponent systems. All nanoTA measurements were carried out at a heating rate of 10 °C s−1. Photothermal Infrared Microspectroscopy (PT-MS). PT-MS analysis was implemented by integrating an Explorer AFM (Veeco) equipped with a Wollaston wire thermal probe (Veeco) with a FTIR spectrometer (IFS66/S model from Bruker Optics Limited) via a dedicated optical interface. More detail on the technique can be found in other articles,29,30 but in short, the thermal probe detects the vibrational-induced temperature changes of a material as it absorbs the IR radiation. The resultant output is a spectrum similar to that found in conventional IR spectroscopy. Topography images were acquired in the first

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RESULTS AND DISCUSSION Amorphous Felodipine Thin Films. The amorphous drug thin films were prepared to allow comparison with the phase separation/recrystallization tendency of the amorphous drug from the solid dispersion films stored at high humidity. The freshly prepared drug thin films by spin coating were a completely transparent glassy film where no features can be observed using PLM (data not shown). After exposure to 921



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Figure 4. Optical images, TTM images, and typical nano-TA measurements of the fresh and aged felodipine-PVP (50:50) solid dispersion films before and after saturated humidity treatment.

nanoTA results of the freshly prepared and humidity-treated amorphous felodipine films. The freshly prepared films were completely amorphous within the detection resolution of the TTM image, which is 1 μm × 1 μm. As seen in Figure 3, the TTM imaging of the freshly prepared felodipine film only showed a blue color code with a transition temperature of approximately 40 °C. This temperature correlates well with the glass transition temperature of the amorphous felodipine. The TTM image of the humidity-treated felodipine film (after 24 h of treatment) shows clear phase separation of blue and red dominated region. The red region has a transition temperature (∼85 °C) higher than the glass transition but lower than the melting of the crystalline drug (141 °C). This may indicate that in the red region there is mixed amorphous and crystalline felodipine. The nanoTA results of the interface between the clear amorphous and the crystalline regions show double transitions indicating the double layer (vertically) configuration of the aged film. The double transitions are at a lower temperature (around the glass

saturated humidity at room temperature for 24 h, largely transparent glassy felodipine with a few crystalline spherulites with radii of 1−5 mm were observed using PLM (Figure 2). The ATR-FTIR spectra of the freshly prepared and aged felodipine films are shown in the top panel of Figure 3. It can be seen that after saturated humidity treatment, the N−H peak shifts from 3342 cm−1 in the freshly prepared amorphous drug film to 3372 cm−1, which is close to the N−H peak of crystalline felodipine. However, it is also noted that the shape of the N−H peak is asymmetrical with a shoulder peak in the region of 3340−3330 cm−1. This suggests that a small amount of amorphous felodipine is still present in 24 h humidity-treated drug films. Therefore, the ATR-FTIR spectroscopy results indicate the coexistence of crystalline and amorphous felodipine in the humidity-treated drug film. However, no spatial information of the crystal growth can be obtained from ATR-FTIR spectroscopy study. The bottom panel of the Figure 3 displays the representative nanoTA profiles and the TTM image constructed based on the 922



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transition temperature of the amorphous felodipine) and a higher one at around 100 °C. The latter one is lower than the melting transition of any polymorphs of crystalline felodipine. However, as the top layer is confirmed to be the amorphous layer according to the nanoTA results, it is possible that the glassy felodipine may dissolve crystalline felodipine to some extent with an increase in the temperature and depress the melting point. Another possibility is that the sizes of the crystals are extremely small (within the nanometer range). It is well-known that many nanocrystals have lower melting temperatures than their corresponding bulk crystalline materials.31−33 This possible double layer configuration of the aged felodipine indicated that the growth of felodipine crystals is likely to be largely initiated from the bulk (close to the bottom of the thin film) under high humidity. Once the crystals start to approach and penetrate the surface, the red regions are created. These results demonstrated the capacity of nanoTA on detecting the in-depth profile of the composition of the film. This is extremely helpful in confirming whether the main mechanism of phase separation is dominated by surface or bulk crystallization. With the understanding of the crystallization behavior of the drug film alone, the solid dispersion films were studied to evaluate the effect of polymer on the crystallization of the drug. Solid Dispersion Thin Films. The AFM images of the freshly prepared felodipine-PVP K29/32 films showed a smooth featureless surface indicating a homogeneous molecular dispersion of the drug in the polymer (data not shown). The nanoTA of the different locations on the fresh film revealed a highly reproducible single glass transition (Tg) at 93 ± 2 °C, as seen in Figure 4. This single transition correlates well with the calculated glass transition of dry molecular dispersion of felodipine in PVP with a ratio of 50:50 (w/w). This indicated the absence of phase separation in the freshly prepared film. The TTM image of the fresh film also reflected the homogeneous nature of the molecular dispersion as indicated by the uniform green color of the map (the red dot on the image is an artifact created by the detachment of the probe to the sample surface temperately). After exposure to saturated humidity, the solid dispersion films were first observed using PLM. As seen in Figure 2b, many fractal-like patches that have distinct boundaries in comparison to the rest of transparent glassy molecular dispersion film can be clearly observed. These fractal-like structures are typically formed by a diffusion-limited aggregation (DLA) process.34 In most cases of the growth of crystalline materials, this type of branch-structured dendritic crystallization is believed to be a result of crystallization from supersaturated states.36 However, in this case, unlike crystalline material, these fractal-like patches showed no birefringence under polarized light. Previously, the system has been reported to form amorphous−amorphous phase separation.4 Therefore, PXRD was used to further confirm the nature of the phase separations in the solid dispersion films. It can be seen in Figure 5 that after saturated humidity treatment, the XRD spectra are nearly identical to the freshly prepared solid dispersion films, which have the characteristic halo amorphous pattern. Thus, these patches are confirmed as phase-separated amorphous felodipine domains. Contact mode AFM results showed an increase in surface roughness from ∼50 nm (data not shown) to ∼0.36 μm (Figure 6a) after 1 day of humidity treatment. The TTM image of the aged film was taken at the edge of the amorphous patterns observed using PLM (Figure 4). Although the resolution of the TTM image is only 1 μm × 1 μm, the pattern of phase separation

Figure 5. PXRD spectra of the freshly prepared (bottom spectra) and 24 h saturated humidity-treated (top spectra) solid dispersion films of felodipine and PVP.

shows similarity to the PLM images. Localized nanoTA of the humidity-treated films (followed by drying in P2O5 overnight) show domains with different transition temperatures within the regions of 40−45, 52−57, and 60−65 °C, indicating phase separation at the surface of the solid dispersion film. The glass transitions of the amorphous felodipine are at ca. 45 and 168 °C for PVP K29/32. It is unlikely that the differences in Tg of these domains are solely caused by the water content differences between them as the films were dried prior to the tests. It is possible that these domains also have different drug concentrations, which further leads to the different water content in these domains as a result of different moisture uptake capacity. This hypothesis was further tested using DVS measurements to compare the moisture interaction affinity difference between solid dispersion films and pure PVP K29/32 films. The DVS results of the solid dispersion films indicate slightly lower moisture uptake capacity than the pure PVP films during the exposure to humidity, but the difference is not significant (Figure 7). To calculate the drug concentrations in the domains with different Tg, the moisture content of the film was estimated using DVS. The films were exposed to ambient environment (as the nanothermal measurements were conducted under ambient conditions) and dried at 0% RH using DVS to assess the moisture loss. The moisture loss is within the range of 5−7% (w/w) (the inserted DVS drying stage results in Figure 7). On the basis of the assumption that all domains contain relatively similar amounts of moisture, the Fox equation was then employed to roughly estimate the drug and polymer contents in the domains,35 where the Tg of the drug−polymer blend can be calculated as follows: x 1 − x1 1 = 1 + Tgmix Tg1 Tg2 (1) In the Fox equation, Tgmix is the Tg of the molecular dispersion of the drug in the polymer, Tg1 is the Tg of the amorphous drug, Tg2 is the Tg of the pure polymer, x1 is the mass fraction of the drug, and 1 − x1) is the mass fraction of the polymer. The calculated drug contents are approximately on the order of 75 (w/w), 60 (w/w), and 50% (w/w) for the domains with Tg values of 40−45, 52−57, and 60−65 °C, respectively. 923



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Figure 6. Contact mode AFM images of the humidity-treated (a) solid dispersion films and (b) the drug-top-polymer-bottom films.

Figure 7. DVS results of the solid dispersion and pure PVP films during drying and moisture uptake under saturated humidity.

humidity treatment, two peaks at 868 and 849 cm−1 belonging to the drug and the polymer, respectively, within the fingerprint regions were chosen as they did not overlap. The changes in the ratio of the intensities of these two peaks can quantitatively represent the changes in the drug concentration of the film. As seen in Figure 8, the ATR-FTIR results of the films show a higher drug concentration in the top layer of the treated films as the relative peak ratio between 868 and 849 cm−1 peak decreases,

ATR-FTIR spectroscopy was used to study the surface chemical compositions of the solid dispersion film before and after the saturated humidity treatment. It can be seen in Figure 7 that the N−H peak of the humidity treated solid dispersion film shifts to the higher wavenumber of 3347 cm−1, which is close to the NH peak of pure amorphous felodipine. This again confirms the separate phases being amorphous felodipine domains. To estimate the changes in drug concentration before and after 924



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Figure 8. Partial ATR-FTIR spectra of (a) the solid dispersion films and (b and c) the double-layered films before (top spectra) and after (bottom spectra) saturated humidity treatment.

indicating the increase of drug concentration. This agrees well with the nanothermal results and suggests the migration of drug to the surface of the solid dispersion films after exposure to saturated humidity. For ATR spectroscopy, the IR beam within the midinfrared region can penetrate approximately 1−3 μm for most polymeric materials.36 Therefore, the ATR-FTIR results of a solid dispersion thin film can provide the average spectroscopic information of the top layer of a film. The disadvantage of this method is that it is not able to obtain localized information within the top layer, such as drug concentration variation/distribution (horizontally), which can be important for understanding the uniformity of the drug across the surface of the formulation. For this reason, PT-MS was also used in this study to provide complementary information on the local distribution of drug at the surface of the films.

The PT-MS spectra of the freshly prepared and humiditytreated films are shown in Figure 9a,b. In the PT-MS spectra of the fresh films, the N−H stretching band at 3290 cm−1 indicated drug−polymer hydrogen bonding, confirming the molecular dispersion nature of the drug and the polymer in the fresh film.4,20 The PT-MS spectra of the humidity-treated films showed significant changes in the N−H stretching region. The peak representing hydrogen bonding between drug and polymer at 3290 cm−1 disappeared, indicating that the drug−polymer interaction was disrupted. This was replaced with the absorption peak at 3340 cm−1, indicating the presence of cohesive hydrogen bonding between the felodipine molecules in the amorphous felodipine.4,20 This replacement of drug−polymer hydrogen bonding by the drug−drug hydrogen bonding is caused by the moisture-induced phase separation. This finding is in good agreement with the results reported using macroscopic analytical 925



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Figure 9. (a) 3700−3000 cm−1 and (b) 1000−500 cm−1 partial PT-MS spectra of PVP, crystalline felodipine, and thin films before and after humidity treatment and (c) PT-MS spectra of eight treated points on the surface of the film after humidity treatment and the distribution of the 868/849 cm−1 peak ratios of these points before and after humidity treatment.

prepared and humidity-treated films were examined using PTMS. At each sampling point, the thermal diffusion length of the transmitted IR beam detected by the thermal tip is approximately 10−20 μm radii for this method.29,30 This allows the comparison of the uniformity of drug distribution across the film before and

methods, such as ATR-FTIR spectroscopy.4,20,26 In addition to the detection of phase separation, the nanocharacterization techniques introduced in this study can also semiquantitatively analyze the distribution of drug at the surface of the film. Ten random sampling points across the surface of the freshly 926



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dispersion films disrupted the drug−polymer interactions19−21 and lead to the nucleation of phase separated amorphous drug domains. This is then followed by drug migration toward the surface of the solid dispersion film, and nucleation and crystallization of the drug can be promoted. Overall, the solid dispersion films demonstrated nonsurface crystallization (not including surface crystallization induced by introducing heterogeneous nucleus) in the early stages of crystallization similar to the drug film alone. This implies that the presence of PVP K29/ 32 did not fundamentally alter the crystallization process. To further confirm that the surface crystallization is not the dominating crystallization process of felodipine films, doublelayered films were prepared and analyzed. These consisted of a PVP K29/32 layer on top of an amorphous felodipine layer and a felodipine layer on top of a PVP layer. The main purpose of the PVP-top-drug-bottom configuration is to test the surface crystallization pathway, and for the drug-top-PVP-bottom configuration, it is to evaluate the effect of substrate on the drug crystallization. Double-Layered Thin Films. After exposure to humidity, double-layered films with separated PVP and felodipine layers were analyzed using the same methods as described previously. If surface crystallization is the dominating mechanism of the pure drug film,37−40 no difference on drug crystallization tendency in the samples with the drug layer as the top layer should be observed. Also, no drug crystallization should be seen in the samples with polymer top layers, as the polymer layer should act as a protection layer for the drug from the humidity unless the polymer layer is highly moisture permeable. As predicted, no drug crystals were detected with PLM in the humidity-treated layered polymer-top-drug-bottom films (images not shown). Even after 1 week of exposure, no crystal growth was observed in these films. The ATR-FTIR spectra of the freshly prepared, layered, polymer-top-drug-bottom films (Figure 8c) show largely PVP-related peaks. However, the spectra of the humidity-treated film show the clear presence of the N−H peak at 3340 cm−1. This indicates the presence of phase-separated amorphous felodipine domains in the polymer layer. It was also noted that more drugrelated peaks could be observed in the fresh films, particularly in the 1200−1000 cm−1 region. This indicates the vertical migration of drug molecules into the polymer layer (Figure 8c). The nanothermal results of the humidity-treated polymer-top-drugbottom films show no obvious transition temperature but continuous deflection from room temperature (Figure 11). This indicates the soft nature of the films, which may be associated with a relatively high amount of absorbed moisture in the top layer of PVP K29/32 after exposure to high humidity. This demonstrates one of the limitations of thermal probe-based techniques, as it is extremely challenging to analyze soft samples. It is noted that the drug-top-polymer-bottom films show amorphous domains with clear boundaries to the glassy film under PLM (Figure 2c,d), which are similar to the ones observed in the solid dispersion films. No crystalline patch was observed using PLM on the films (except the edges, which was discarded from the discussion as the reason stated earlier). ATR-FTIR spectra of the humidity-treated films (Figure 8b) reveal that the top felodipine layer remains amorphous with a characteristic N− H peak around 3345 cm−1. The AFM images of the aged films reveal higher surface roughness than the solid dispersion films, indicating the more aggressive surface morphology changes (Figure 6b). The nanothermal results of the drug-top-polymerbottom films show reproducible transitions in the temperature range of 60−70 °C as seen in Figure 9. This transition

after the humidity treatment. The peak intensity ratios of the chosen peaks of the eight tested points on the surface of the film are plotted in Figure 9c. It was found that freshly prepared felodipine-PVP K29/32 films have little variation in the drug− polymer peak intensity ratio, whereas the humidity-treated films show significantly greater variation in the peak ratios in comparison to the fresh films. This indicates that after humidity treatment, the drug distribution is highly uneven across the film. Also, it appeared that half of the tested locations contained a higher drug concentration than before the humidity exposure. As the material close to the surface contributes most to the PT-MS spectra (the contribution has exponential decay with the depth of the samples), it is valid to suggest that this result indicates migration of the drug toward the surface of the thin film after humidity exposure. The change in local drug concentration (that exceeds the saturation of drug in polymer) may further initiate/ accelerate the nucleation and surface crystallization of the drug. This is in good agreement with the nano-TA measurements discussed previously. The kinetic process of the phase separation within the studied 24 h period is presented in Figure 10. No phase separation was

Figure 10. Time profile of the growth of the amorphous felodipine patches based on the PLM images of the saturated humidity-treated films taken over 24 h (with inserted representative PLM images taken at 2, 12, and 24 h).

observed in the first 30 min of the humidity treatment. Phaseseparated fractal-like patches could be observed after 1 h of exposure to saturated humidity, and the phase separation coverage area increased with exposure time. This suggests that the nucleation of amorphous phases occurs within the first 1 h of the exposure. However, the individual domains show a slow increase in size within the first 16 h but had increased dramatically after 24 h. Only a few crystalline felodipine spherulites could be observed after 24 h. As this study is focused on the early phase separation in the humidity-treated solid dispersion film instead of the crystallization of the model drug, no further data on the crystallization of felodipine were collected after 24 h. Nevertheless, there is a clear indication that the solid dispersion films studied in this paper undergo phase separation stages in the order of nucleation of amorphous drug, continuous formation of amorphous domains, intensive growth and fusion of existing amorphous drug domains, and nucleation and crystallization of the model drug. The solid dispersion film results appear to suggest that the early stage of phase separation was initiated within the bulk film upon exposure to moisture. The moisture absorbed by the solid 927



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study explored the unique ability of the probe-based nanothermal and spectroscopic techniques for studying phase separation in these thin films. The focus of this study is to understand, on the nanoscale, the phase/separation/crystallization process of the system under high humidity. The key question to be addressed is whether the surface or bulk crystallization of the amorphous felodipine in a thin film is the dominating mechanism of phase separation from the solid dispersion films with PVP K29/32. This is because a fuller understanding of the phase separation will facilitate the development of formulation strategies to prevent/minimize drug crystallization. Crystallization of Amorphous Felodipine Thin Films under High Humidity. The crystallization of amorphous felodipine under dry (in some studies vacuum) conditions has been reported previously from melt-quenched solids or powders.26−28 Below the glass transition temperature of amorphous felodipine, surface crystallization (into polymorph I) of felodipine has been reported to take place at a faster rate than the bulk crystallization, but the difference between the bulk and the surface crystallization rates is smaller than other systems such as amorphous indomethacin, nifedipine, and griseofulvin.37−40 In this study, amorphous felodipine prepared by spin coating was treated under saturated humidity to maximize the external physical stress of the samples. The PLM and nanoTA results confirmed that the majority of the nucleation and growth of amorphous felodipine thin films initiated in the bulk. In contrast to the previously reported crystallization of amorphous felodipine under dry conditions,27 bulk crystallization may be the dominating process for crystallization of amorphous felodipine thin films prepared by spin coating under high humidity. This was further confirmed by the results of the double-layered films with a drug layer on the top and a polymer layer at the bottom. The amorphous felodipine patches were found to only grow at the interface where the drug and polymer can interdiffuse and form solid dispersion locally. Phase Separation of Felodipine in Solid Dispersion Films under High Humidity. Various polymers including PVP have been used to reduce the nucleation and crystal growth of amorphous felodipine from solid dispersions.41−43 Under dry conditions (such as over P2O5), Kestur and co-workers suggested that the presence of 3% PVP can have a major effect on the nucleation process and a minor contribution to reducing the rate of the felodipine surface as well as bulk crystallization.27 Under humidity conditions of up to 75% RH, felodipine-PVP solid dispersions undergo amorphous−amorphous drug−-polymer phase separation and crystallization of felodipine.4,20,26 However, a detailed understanding of the separation process on a nanoscale has not been demonstrated experimentally. The combined nanocharacterization and PLM analysis allowed the visualization of phase separation process and gained three-dimensional information of the phase separation. It was found that in the PVP K29/32-based solid dispersion thin films, felodipine show rapid phase separation as amorphous domains with the absence of crystallization within 24 h of exposure to saturated humidity. These amorphous domains can be stable for very prolonged period of time (weeks) prior to crystallization. Although this may mean that PVP K29/32 is not effective for stabilizing the drug as molecular dispersions under high humidity, this also indicates that PVP can protect amorphous felodipine domains from crystallization. More importantly, this study revealed for the first time that the phase separation in thin films goes through a process of drug migration that leads to a gradual build-up of high drug concentration toward the surface of the film after high

Figure 11. Nano-TA profiles of the humidity-treated double-layered films in comparison to the humidity-treated pure amorphous drug and the solid dispersion films.

temperature is much higher than the amorphous drug alone, indicating the possibility of PVP penetrating into the drug layer and elevating the transition temperature of the layer. The observed phase separation is likely to be developed at the interface of the drug layer and the underneath polymer layer. It is reasonable to speculate that the interdiffusion/penetration and mixing of PVP into drug layer promote the separation of amorphous drug patches. On the basis of the assumption of PVP mixing into the top drug layer, similar calculations were conducted with the rough estimation of the presence of 5−8% moisture. The calculated ratio of drug to polymer is around 50:50 (w/w %). The fact that the ATR-FTIR spectra of the drug-toppolymer-bottom films after humidity treatment show many similarities to the treated solid dispersion films (Figure 8b,c) indicates that the presence of the PVP bottom surface can have a similar effect on phase separation of the drug to the molecular dispersion of felodipine and PVP. To further confirm the effect of the solid substrate on the phase separation of felodipine, two more model polymers, Eudragit E OP and Soluplus, were tested. For both polymers, the films with the polymer layer as the top layer showed no crystalline of felodipine after saturated humidity treatment (data not shown). This indicates that surface protection can greatly contribute to the prevention of drug crystallization. When the polymer layers were placed as the bottom layers, for both Eudragit and Soluplus, neither crystallization nor amorphous phase separation was observed after 24 h of saturated humidity treatment. The detailed behavior of the films containing Eudragit and Soluplus and the comparison with the PVP-containing films are out of the scope of this study and will be presented in a separated paper. This confirmed that the dominating nucleation and crystallization mechanism of felodipine is not surface but mainly bulk crystallization, and the solid substrate for laying the amorphous drug film can significantly affect the crystallization process. Discussion. The phase separation behavior of molecular dispersions prepared by spray drying, fusion, and hot melt extrusion has been extensively studied.6,19−23,25−28 However, there is little known on the phase separation in molecular drug dispersion thin films with thicknesses within the range of 5−10 μm, which often are used for pharmaceutical applications. This 928



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dispersion films under high humidity. Three key findings of this study are that (1) our data suggest that nucleation and growth of amorphous drug domains in the bulk of the film are the dominating process of the early stage of phase separation in the solid dispersion films, (2) drug migration occurs toward the surface of the solid dispersion film during the exposure to high humidity, and (3) under high humidity PVP K29/32 cannot prevent phase separation of felodipine in the solid dispersions but can effectively protect amorphous felodipine domains from crystallization. This study demonstrated that these nanocharacterization tools can allow the localized analyses of the films, which are often difficult to achieve by conventional analytical methods. In addition to identification of the size and chemical makeup of the phase separation, the combined used of these novel techniques also allows the measurements of drug/ phase distribution across the solid dispersion film. This can not only provide detailed understanding of the physical structure of pharmaceutical films but bring insights into the mechanism of phase separation in solid dispersion formulations.

humidity treatment, as illustrated in Figure 12. Similar behavior has been reported in ultrathin polymer blend films.44 The demixing of the blend polymers was induced by the presence of humidity, and the surface tension effect is believed to be the dominating driving force of the phase separation and the crystallization of the more hydrophobic material at the surface of the film.44 Although the surface tension effect has a more profound effect on the crystallization of ultrathin films (within few nanometer thick),38,39 we speculate that this may also contribute to the phase separation observed in this study. By concentrating the more hydrophobic components toward the surface of the thin film, the total surface energy of the thin drug− polymer film can be reduced. Because of the hygroscopic nature of the PVP film, after exposure to the high humidity, the film is much more mobile, allowing the migration of the drug molecules to the surface within a relatively short period of time. The high chain mobility is also contributed to the similar layered phase separation of PEO/PMMA blend films under humidity.43 To further confirm our hypothesis of overall surface tension reduction as the driving force of the phase separation and drug migration, the double-layered thin films with the PVP layer as the top layer were studied after exposure to high humidity. The migration of drug into the top PVP layer was observed in the absence of drug crystallization. This suggests that the interaction of drug and PVP at the interface of the two layers is likely to be promoted by the presence of high humidity, which further progresses into the diffusion/migration of drug into the top PVP layer.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +44 1603592925. Fax: +44 1603592015. E-mail: sheng. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the contribution of the Interreg project funded by the European Union. Z.Y. thanks Evonik for the financial support for his Ph.D.

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REFERENCES

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Figure 12. Schematic illustration of the phase separation and drug migration process of the thin films containing felodipine and PVP K29/ 32.

The finding of this study may not be generalized into the behavior of solid dispersion films exposed to other lower humidity environments and other amorphous drugs. Previous studies have suggested that the crystallization can vary with the degree of relative humidity and the nature of the drug molecules. However, this study has demonstrated the capabilities of probebased nanocharacterization methods to provide extremely important information about the phase separation process of the thin films that other characterization techniques cannot achieve.

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CONCLUSION Nanocharacterization methods including AFM, nanoTA, and PT-MS revealed detailed three-dimensional information of the early stage phase separation process in felodipine-PVP solid 929



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Early Stage Phase Separation in Pharmaceutical Solid Dispersion

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