Microstructure of Pharmaceutical Semicrystalline Dispersions: The

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Microstructure of pharmaceutical semicrystalline dispersions: the significance of polymer conformation Tu Van Duong, Bart Goderis, Jan Van Humbeeck, and Guy Van den Mooter Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01007 • Publication Date (Web): 10 Jan 2018 Downloaded from http://pubs.acs.org on January 11, 2018

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Molecular Pharmaceutics

Microstructure of pharmaceutical semicrystalline dispersions: the significance of polymer conformation Tu Van Duong,†,⊥ Bart Goderis,§ Jan Van Humbeeck,¶ and Guy Van den Mooter*,† †

Drug Delivery and Disposition, Department of Pharmaceutical and Pharmacological

Sciences, KU Leuven, Campus Gasthuisberg O&N2, Herestraat 49 b921, 3000 Leuven, Belgium ⊥

Department of Pharmaceutics, Hanoi University of Pharmacy, 13-15 Le Thanh Tong, Hoan

Kiem, Ha Noi, Vietnam §

Polymer Chemistry and Materials, Department of Chemistry, KU Leuven, Celestijnenlaan

200F b2404, 3001 Heverlee, Belgium ¶

Department of Materials Engineering, KU Leuven, Campus Arenberg, Kasteelpark Arenberg

44 b2450, 3001 Heverlee, Belgium

KEYWORDS solid dispersions, semicrystalline, amorphous, microstructure, crystallization, interlamellar, segregation, conformation, integral folding, non-integral folding, thickening, small angle Xray scattering, indomethacin, polyethylene glycol Table of Contents Graphic

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ABSTRACT The microstructure of pharmaceutical semicrystalline solid dispersions has attracted extensive attention due to its complexity that might result in the diversity in physical stability, dissolution behavior and pharmaceutical performance of the systems. Numerous factors have been reported that dictate the microstructure of semicrystalline dispersions. Nevertheless, the importance of the complicated conformation of the polymer has never been elucidated. In this study, we investigate the microstructure of dispersions of polyethylene glycol and active pharmaceutical ingredients by small angle X-ray scattering and high performance differential scanning calorimetry. Polyethylene glycol with molecular weight of 2000 g/mol (PEG2000) and 6000 g/mol (PEG6000) exhibited remarkable discrepancy in the lamellar periodicity in dispersions with APIs which was attributed to the differences in their folding behavior. The long period of PEG2000 always decreased upon aging-induced exclusion of APIs from interlamellar region of extended chain crystals whereas the periodicity of PEG6000 may decrease or increase during storage as a consequence of the competition between the drug segregation and the lamellar thickening from non-integral folded into integral-folded chain crystals. These processes were in turn significantly influenced by the crystallization tendency of the pharmaceutical compounds, drug-polymer interactions as well as the dispersion composition and crystallization temperature. This study highlights the significance of the


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polymer conformation on the microstructure of semicrystalline systems that is critical for the preparation of solid dispersions with consistent and reproducible quality.



INTRODUCTION Solid dispersion has been widely acknowledged as a potential approach to improve the solubility and dissolution rate of an increasing number of poorly water soluble compounds.1-3 In a system containing a drug and a carrier, the dissolution behavior and pharmaceutical performance of the dispersion is the direct consequence of the physicochemical properties of each component as well as their mutual influence.4, 5 As an illustration, we recently found that in dispersions of polyethylene glycol (PEG) and indomethacin (IMC), the drug inhibits the crystallization of the polymer6 due to the formation of hydrogen bonding between the two components in their molten mixture.7 On the other hand, PEG modified the mobility of IMC molecules in return, thereby inducing the generation of different polymorphic forms of the drug.8 An interesting question that would come to mind is what the microstructure of this system would look like as the result of the mutual impact between the drug and the polymer. As the microstructure of a system strongly affects its macroscopic properties, the structure of PEG in polymer blends has been extensively investigated.9-13 There are however fewer studies on the microstructure of PEG in blends with pharmaceutical compounds. The microstructure of PEG-based dispersions has been found to be complex with a huge discrepancy in the crystallization behavior of both drugs and polymers, in the location and domain size of the active pharmaceutical ingredients (APIs) in the polymer matrices due to the variation in the crystallization tendency of the APIs, the solubility of drug in polymer and the APIs-PEG interactions as well as the crystallization conditions and composition of the dispersions. Furthermore, the microstructure of these systems is likely to change during storage.14-23 It can be anticipated that the difference in the microstructure of solid dispersions can impact their dissolution behavior.20 PEG exhibits a complicated crystallization behavior, starting with the formation of metastable non-integral-folded (NIF) chain crystals, followed by the transformation into integral-folded 4 ACS Paragon Plus Environment

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(IF) chain crystals with the polymer chains either extended or folded an integer number.24-30 The fold number per chain during crystallization is determined by the chain length, the crystallization conditions and time. As the higher folded chain crystals are less stable than the lower folded chain ones, the lamellar thickness of the crystals will increase stepwise according to the quantized reduction in the number of folds per molecule until the polymer chains fully extend.31-35 The structure of PEG is thus dependent on the non-integral to integral folded chains transformation as well as the subsequent unfolding of the integral-folded chains that must play a vital role in the microstructure of solid dispersions of PEG and drug compounds. It is surprising that, to the best of our knowledge, there has been no detailed investigation on the impact of these critical transformation processes during polymer crystallization on the microstructure of pharmaceutical PEG-based dispersions. The purpose of this work is therefore to explore the importance of PEG conformation on the microstructure of pharmaceutical solid dispersions. EXPERIMENTAL SECTION Materials The main model API, γ-IMC (melting point Tm, 161 °C; glass transition temperature Tg, 45 °C)7, was purchased from Fagron (Saint-Denis, France). Other APIs including indoprofen (Tm, 212 °C; Tg, 50 °C) and fenofibrate (Tm, 81 °C; Tg, −19 °C) were obtained from SigmaAldrich (St. Louis, MO, USA). Flurbiprofen (Tm, 115 °C; Tg, −6 °C) was purchased from Acros Organics N.V. (Geel, Belgium). Griseofulvin (Tm, 218°C; Tg, 89°C) and cinnarizine (Tm, 121°C; Tg, 7°C)7 were supplied by Certa N.V. (Braine-l’Alleud, Belgium) and Fagron (Saint-Denis, France), respectively. The IMC methyl ester was synthesized in-house by the method described elsewhere.7 PEG2000 (Tm, 53°C) and PEG6000 (Tm, 60 °C) were obtained from Sigma-Aldrich (Geel, Belgium). The chemical structures of model compounds are shown in Scheme 1. 5 ACS Paragon Plus Environment


Scheme 1. Chemical structures of model compounds. Sample Preparation Dispersions of APIs and polymers were prepared by heating the mixture of the two components to 5 °C above the melting point of the APIs under stirring for 3 minutes to ensure complete melting, followed by cooling the melt in ambient atmosphere to room temperature and storage in a desiccator containing phosphorus pentoxide. Small-Angle X-ray Scattering (SAXS) Samples were wrapped in aluminum foil and cooled at 20 °C/min from 5°C above the melting points of the APIs to a certain crystallization temperature using a Linkam HFS 91 hot stage and a TP-93 temperature programmer (Linkam Scientific, Surrey, UK). The SAXS measurements were performed with a XeuSS X-ray camera (Xenocs, Sassenage, France), comprising a GeniX 3D molybdenum ultralow divergence X-ray beam delivery system 6 ACS Paragon Plus Environment

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(wavelength λ = 0.71 Å) at a power of 50 kV–1 mA, a collimating assembly based on scatterless slits, a sample stage, a He flushed flight tube, and a Mar345 image plate detector (Marresearch, Norderstedt, Germany). The 2D-patterns were processed by Fit2D software.13 The scattering angles were calibrated using silver behenate and high density polyethylene standards. Data were azimuthally averaged using the program CONEX36 and corrected for the empty sample holder signal. The Lorentz corrected intensities I(q)q2 are expressed versus the magnitude of scattering vector q = (4π/λ)·sin(θ/2), where λ is the wavelength of incident radiation and θ the scattering angle. The angular range in these experiments covered 0.16° < θ < 15.3°. This range includes the scattering at wide angles, which was not considered for analysis. Each scattering pattern was recorded with an exposure time of 60 min. The SAXS data were processed using Origin 8.5 (OriginLab Corp., Northampton, USA) High Performance Differential Scanning Calorimetry (HPer DSC) HPer DSC analyses were performed using a Perkin-Elmer DSC 8500 equipped with an Intracooler 3 cooling accessory (Perkin-Elmer, Massachusetts, USA). Measurements were conducted under an inert dry nitrogen gas purge at a flow rate of 20 ml/min. The calibration was performed as described earlier.37 A 15 µm aluminum foil of approximately 0.5 x 1 cm2 was used to wrap the sample of ca. 1.0 mg instead of using an aluminum pan as sample container. The aluminum foil has a much lower thickness and provides a drastically improved heat transfer. An aluminum foil of the same dimensions was placed on the reference holder. The samples were kept isothermally at ca. 5 °C above the melting temperatures of APIs for 3 minutes before cooling to 20°C at a cooling rate of 20 °C/min. The samples were stored at 20°C inside the DSC cell for different time periods and were then subjected to heating from 20 °C to ca. 5 °C above the melting points of the APIs using various heating rates.



Gel Permeation Chromatography 10 mg PEG2000 or PEG6000 was dissolved in 10 ml mobile phase (0.01 M Na2HPO4, adjusted to pH 7.0 with 3 M H3PO4). The injection volume amounted to 100 µl and the column was a TSK G3000 PW, 60 cm × 7.5 mm (Tosoh Corp., Tokyo, Japan). The experiments were carried out at room temperature at a flow rate of 1 ml/min with a refractive index detector. PEG-standards with molecular weights of 620, 1080, 1900, 4120, 6450, 11800, 22800 g/mol were supplied by Polymer Labs (Amherst, MA, USA). The average molecular weight of PEG2000 and PEG6000 were determined to be 1980 and 6775 g/mol, respectively. RESULTS PEG2000 and PEG6000 were selected to investigate the influence of polymer conformation on the microstructure of semicrystalline dispersions as the two polymers exhibited different folding behaviors: PEG2000 has been reported to only exist as extended chain crystals while PEG6000 might crystallize into once-folded or twice folded chain crystals.32 Polymers that can form crystals with higher fold numbers such as PEG8000 (three-times folded) or PEG10000 (four-times folded) showed relatively weak SAXS signals in dispersions with APIs, likely as a result of being poorly crystalline, and were thus ineligible for the measurements. Microstructure of pure PEGs Both pure polymers have been found to already fully crystallize after cooling to 20oC from the melt and the SAXS diffractograms remained unchanged thereafter. Thus, the long periods extracted from the maxima in I(q)q2 were assumed to be representative of the crystal lamellar thickness. 8 ACS Paragon Plus Environment

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PEG2000 showed the most intense peak at the scattering vector q ≈ 0.051 Å-1 and weaker but well resolved signals at scattering vectors of 0.102, 0.153 and 0.204 Å-1 which were due to the second, third and fourth order diffraction, respectively, of the first peak (Figure 1). The corresponding long period L of PEG2000 lamellar stacks was 123.6 Å, calculated from Bragg’s law L=2nπ/q (where n is the diffraction order and q is the scattering vector). This value is quite consistent with the theoretical chain length (l) of the polymer (125.2 Å) that was obtained from the formula l=m/v where m is the molecular weight and V being the molar mass per unit length (158.2 g/nm) along the crystal c axis.38 These data suggest that PEG2000 exists as extended chain crystals.

Figure 1. SAXS diffractograms of PEG after cooling to 20°C from the melt at 20 °C/min. PEG6000 exhibited the first strong reflection at q ≈ 0.045 Å-1, another peak at 0.090 Å-1 which has been assigned to the second order as well as two weak and poorly resolved peaks at wider angles, most likely due to the third and fourth order diffraction, respectively, of the first signal (Figure 1). The calculated long period of PEG6000 lamellae was 139.7 Å, corresponding to the twice-folded conformation of the polymer chains with the theoretical length of 428.3 Å. DSC thermograms of PEG2000 (Figure 2A) displayed a single melting peak of extended polymer chains at any heating rate. The melting peak shifted from 52.5 °C at the heating rate 9 ACS Paragon Plus Environment


of 1 °C/min to 58.3 °C and became broader when heating at 100 °C/min due to the thermal lag. For PEG6000, fast heating at not less than 60 °C/min showed a single melting peak whereas slower heating led to the appearance of two distinct endothermic events with the intensity of the lower temperature endotherm increasing relative to the higher temperature one and becoming dominant as the heating rate increased. The two melting peaks occurring at low heating rates normally represents the presence of two distinct lamellae with different long periods in the PEG6000 sample. As the heating rate increased, the two endotherms might merge together because their widths increased with the heating rate due to the thermal inertia effect.

Figure 2. DSC thermograms of (A) PEG2000 and (B) PEG6000 as a function of heating rate during heating immediately after cooling to 20°C at 20 °C/min from the melt. From bottom to top: 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 °C/min. However, SAXS diffractograms revealed the existence of only twice-folded chain lamellae of PEG6000 (Figure 1). Accordingly, the higher endotherm on the DSC thermograms of the polymer should be the result of the unfolding of twice-folded chains during heating, forming a thicker lamellae with higher melting point. The unfolding phenomenon can only be observed at low heating rates when the polymer has enough time to stretch out. The heating as rapid as 60 °C/min might be sufficient to prevent the unfolding. 10 ACS Paragon Plus Environment

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Figure 3 provides more evidence of the reorganization of the twice-fold lamellae of PEG6000 during heating. The sample stored for different periods up to 50 hours showed identical thermograms, suggesting that the unfolding must take place during the course of DSC experiments rather than during isothermal annealing at 20°C.

Figure 3. DSC thermograms of PEG6000 as a function of storage time at 20°C during heating with the rate of 25 °C/min (A) and 100 °C/min (B).

Figure 4. SAXS diffractograms of PEG6000 measured at different temperatures. In order to explore the nature of the unfolded product, SAXS diffractograms of PEG6000 were recorded at different temperatures (Figure 4). The polymer exhibited the twice-folded lamellar structure with a characteristic peak at q ≈ 0.045 Å-1 below 40°C. At higher temperatures, this peak decreased in intensity concurrently with the evolution of two new 11 ACS Paragon Plus Environment


reflections, one at smaller and the other at larger scattering vector. At 57°C, PEG6000 showed well resolved peaks at the scattering vector of 0.027, 0.054 and 0.081 Å-1 which corresponded to the first, second and third diffraction order, respectively, of stacks from the once-folded lamellae. These findings confirm the unfolding of PEG6000 lamellae from twice-folded into once-folded upon heating during the DSC measurements.

Figure 5. SAXS diffractograms of PEG PEG6000 as a function of storage time at 20°C. The unfolding behavior of PEG6000 was also detected during long-term storage of weeks. The PEG6000 sample prepared by cooling the melt at the cooling rate of 1 °C/min to 20°C initially showed diffraction peaks at 0.030, 0.060 and 0.090 Å-1 (Figure 5) which corresponds to the first, second and third diffraction order of stacks from the once-folded lamellae with the calculated long period of ca. 215.2 Å. A shift of reflections of PEG6000 measured at 20°C to lower scattering vectors compared to those at 57°C (Figure 4) is the result of lamellar crystal distortion which is presumably a monotonic increasing function of temperature. An extra signal at q ≈ 0.045 Å-1 also occurred, revealing the presence of the twice-folded lamellae. The atypically strongest peak at 0.090 Å-1 is therefore the accumulation of the signal from the third diffraction order of stacks from the once-folded lamellae and the second diffraction order of stacks from the twice-folded ones. Upon storage at 20°C, the reflection at 0.045 Å-1 weakened before it almost disappeared after 10 weeks albeit its visible contribution to the strong peak at 12 ACS Paragon Plus Environment

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0.090 Å-1. This suggests the unfolding of PEG6000 from the twice-folded to once-folded lamellae during storage at 20°C, similarly to the behavior of the polymer at elevated temperatures (Figure 4) although the first process requires much longer time. Microstructure of PEG-IMC solid dispersions Although the crystallization of PEG2000 and PEG6000 had virtually finished after the first hour in dispersions containing 20% IMC, the SAXS diffractograms displayed remarkable changes (Figure 6). For PEG2000, the polymer remained in the conformation of the extended chains and both reflections corresponding to the first and second order diffraction initially occurred at lower scattering vectors than for the pure polymer, which corresponded to a larger periodicity. Subsequently, the diffraction peaks continuously shifted to wider angles, indicating the decrease in the long period of the lamellae over time at 20°C (Figure 6A).

Figure 6. SAXS diffractograms of solid dispersions containing 20 weight percent of IMC in (A) PEG2000 and (B) PEG6000 as a function of storage time at 20°C. The opposite was true for PEG6000: all diffraction peaks appeared at higher scattering vectors than for the pure polymer, corresponding to smaller periodicity (Figure 6B). The peaks constantly shifted to lower angles upon storage, suggesting that the long period of the lamellae was increasing. It should be noted that adding IMC resulted in the formation of the once-folded lamellae of PEG6000 rather than the twice-folded lamellae that was observed for 13 ACS Paragon Plus Environment


the pure polymer (Figure 1 and 4). For both dispersions, the SAXS diffractograms changed until the diffraction peaks appeared at approximately the same positions as for the pure PEGs.

Influence of drug loading Figure 7 shows the change in the long period of PEG lamellar stacks as a function of drug loading and storage time at 20°C. In PEG2000 dispersions, increasing the drug loading led to a faster drop in the long period of extended chains (Figure 7A). At 20% IMC, the long period remained unchanged at 139.7 Å until 100 hours before dropping and it took 40 hours for the periodicity to decrease to the final value of 129.6 Å. As the drug content increased to 30%, the decline in the long period started earlier from 60 hours and finished only 20 hours afterwards. The 40% drug sample exhibited the fastest decline in the long period of extended polymer chains: the periodicity already decreased from 40 hours to the ultimate value at 50 hours.

Figure 7. The long period of lamellar stacks of the polymer in dispersions of IMC with PEG2000 (A) and PEG6000 (B) as a function of drug loading and time at 20°C. For dispersions of PEG6000-IMC, the change in the long period of stacks from the oncefolded lamellae showed the opposite trend: it increased more slowly with the increasing drug loading (Figure 7B). It took 60, 140 and 190 hours for the long period of the polymer to 14 ACS Paragon Plus Environment

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increase from 181.2 Å to 215.2 Å for the sample containing 20, 30 and 40% drug, respectively. If the long period of PEG lamellar stacks is proportional to the crystal thickness of the polymer, Figure 7 should have implied that the crystals became thinner for PEG2000 while thicker for PEG6000. However, the thinning of PEG2000 crystals is unrealistic because that would lead to the formation of less stable crystals with time, revealed by lower melting temperature as expressed by the Tamman equation31, 39, 40 1

σ

∆×

(1)

where Tm is the melting point of a lamellar crystal of thickness L, is the melting point of the infinite chain length crystals, σ is the surface free energy and ∆H is the enthalpy of fusion. In order to verify this supposition, the evolution of stability for PEG crystals was examined via the melting points of PEG2000 and PEG6000 in dispersions with IMC measured by HPer DSC. Due to the fact that heating might induce reorganization of the lamellae conformation, a heating rate of 100 °C/min was selected as this would be sufficiently rapid to avoid transformation of the lamellae during heating as previously shown in Figure 2.

Figure 8. DSC thermograms of PEG2000 and PEG6000 as well as 20% IMC dispersions with the polymers measured at 100 °C/min. 15 ACS Paragon Plus Environment


Figure 8 exhibits the thermograms of PEG2000 and PEG6000 as well as 20% IMC dispersions with the polymers measured at 100 °C/min. All samples displayed a single melting peak, indicating the absence of unfolding upon heating. The significant melting point depression of the polymers was observed in the presence of the drug. The melting temperature of pure PEG2000 is 58.2°C, reducing by 7.1°C to 51.1 °C in the dispersion whereas the melting point depression of PEG6000 was 7.3 °C (decreasing from 64.2 °C for the pure polymer to 56.9°C for 20% drug dispersion), which was comparable to that of PEG2000based dispersions. Figure 9 presents the melting peak of 40% IMC dispersion in PEG2000 as a function of storage time at 20°C. Evidently, the peak continuously shifted from 44.9°C after 1 hour to 46.7°C when the sample was stored as long as 50 hours. The increase in the melting temperature of PEG2000 in this dispersion did not follow the Tamman equation since the long period of the extended chain crytals decreased from 139.7 to 129.6 Å during that storage period (Figure 7). This suggested that the decrease in the periodicity of the polymer lamellar stacks was not related to the thinning process. It would rather be attributed to the segregation of IMC from the interlamellar region of PEG2000 crystals that resulted in the increasing of and thus Tm in the Tamman equation.


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Figure 9. DSC thermograms (A) and melting point (B) of 40% IMC dispersion in PEG2000 as a function of storage time at 20°C. In contrast, the melting behavior of PEG6000 seemed to be consistent with the Tamman equation as the melting peak of the polymer rose from 57.4°C to 58.8°C during storage (Figure 10) as the long period of stacks from the once-folded lamellae also increased from 181.2 to 215.2 Å (Figure 7).

Figure 10. DSC thermograms (A) and melting point (B) of 20% IMC dispersion in PEG6000 as a function of storage time at 20°C. Influence of storage temperature



Figure 11. The long period of lamellar stacks of the polymer in (A) PEG2000-IMC 60-40 and (B) PEG6000-IMC 80-20 dispersions as a function of storage temperature and time. Figure 11 displays the influence of the storage temperature on the long period of lamellar stacks of PEG2000 and PEG6000 in dispersions with IMC. For PEG2000, increasing the storage temperature led to faster reduction of the long period (Figure 11A). The long period started decreasing from 139.7 Å after 110 hours to the final value of 129.6 Å after 140 hours of storage at 20°C. Increasing the temperature to 30°C resulted in the decline of the long period as early as 20 hours to reach 129.6 Å after 60 hours. At 40°C, the long period decreased from 136.2 Å rather than 139.7 Å and reached the final value after only 30 hours. For PEG6000, the long period of the stacks containing once-folded lamellae increased faster as the temperature rose from 20°C to 30° (Figure 11B). At 20°C, the periodicity rose from 181.2 Å to 215.2 Å over 60 hours. Increasing the storage temperature to 30°C led to a slight increase in the initial period to 185.4 Å and it took only 24 hours for the long period to develop to the final value of 215.2 Å. The long period of lamellar stacks surprisingly showed a downward trend at 40°C: the periodicity decreased from 250.7 Å to the final value of 229.3 Å within only 14 hours, indicating the strong segregation of the drug from the interlamellar region of PEG6000 crystals. The final value of the long period at 40°C was higher than that at lower temperatures. Influence of crystallization tendency of APIs and drug-polymer interactions Being a good glass former, IMC belongs to class III in the classification scheme of crystallization tendency of organic molecules proposed by Baird et al41 and this API can form hydrogen bonds with PEG. We hypothesized that both crystallization propensity of the APIs and the drug-polymer interactions might influence the microstructure of semicrystalline dispersions. In order to test this hypothesis, dispersions of various APIs in PEG2000 and PEG6000 were prepared. The selected compounds were fenofibrate (class III – slow 18 ACS Paragon Plus Environment

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crystallizer without interactions with PEG), flurbiprofen and cinnarizine (class II – intermediate crystallizer with and without interactions, respectively), indoprofen and griseofulvin (class I – fast crystallizer, with and without interactions, respectively) as well as the IMC methyl ester (class III without interactions). For comparison to IMC, we maintained the same molar ratio of APIs to PEG monomer as in 40% and 20% IMC dispersions in PEG2000 and PEG6000, respectively.

Figure 12. The long period of lamellar stacks of the polymer in dispersions of APIs in (A) PEG2000 and (B) PEG6000 as a function of time at 20°C. The amounts of APIs were calculated to maintain the same molar ratio of API to PEG monomer as in 40% and 20% IMC dispersions in PEG2000 and PEG6000, respectively. For PEG2000, the long period of lamellar stacks almost immediately reached the final value of 129.6 Å in dispersions with the IMC methyl ester, fenofibrate, cinnarizine, indoprofen and griseofulvin (Figure 12A). These are APIs that are either fast crystallizer (indoprofen and griseofulvin) or not able to interact with PEG2000 (IMC methyl ester, fenofibrate and cinnarizine). Flurbiprofen, an intermediate crystallizer that can form hydrogen bonds with the polymer, was able to slow down the decrease of the long period of lamellar stacks for several hours whereas IMC hampered this process further.



In dispersions with PEG6000, the fast crystallizer indoprofen or APIs without proton donating groups including the IMC methyl ester, fenofibrate and cinnarizine had practically no influence on the microstructure of the polymer: the long period of lamellar stacks reached the final value of 215.2 Å after the first measurements. In contrast, IMC was able to impede the thickening of PEG6000 lamellae until 60 hours (Figure 12B). The long period of lamellar stacks showed the opposite trend in dispersions with flurbiprofen: the periodicity decreased from 256.3 Å to the common final value after 17 hours. The periodicity of lamellar stacks of PEG6000 exhibited an unusual behavior in the sample with griseofulvin: it increased marginally from 199.2 Å to 204.2 Å after 8 hours and remained unchanged thereafter. DISCUSSION Folding behavior of PEG The melting and crystallization behaviors of PEG have been systemically investigated by Spegt et al.42-45 and Kovacs et al.31-35, 39 Low molecular weight PEG (2,000 – 10,000 g/mol) fractions melt-crystallize with polymer chains either folded an integral number (n ≥ 1) of times or fully extended (n = 0), forming plate-like structures called lamellae with the hydroxyl end groups rejected from the crystal interior and located at the surface layers of the crystalline lamellae. For a polymer with a given molecular weight, the fold number n per chain depends not only on the crystallization temperature but also the crystallization time. The n-times folded chain crystals are metastable with respect to the (n-1)-times folded ones and even more so to the extended chain crystals. During crystallization or annealing, the lamellae thicken stepwise with increasing crystallization temperature and time due to the quantized reduction of the fold number until full chain extension. As can be seen in Figure 2A, PEG2000 always crystallized as extended chain crystals which concurs with the conformation of this polymer fraction reported by Buckley and Kovacs.32 In


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fact, the folding behavior of PEG was observed only in polymers with a molecular weight from 3000 g/mol that was considered as the borderline fraction. Lower molecular weight fractions showed only one endothermic peak on the DSC thermograms corresponding to the melting of extended chain crystals. Due to the fact that unfolding proceeds more rapidly with decreasing molecular weight, folded chain crystals in these samples, if any, unfold too fast to be detected.32 For PEG6000, the pure polymer crystallized as the twice-folded crystals when cooling the melt to 20°C at the rate of 20 °C/min (Figure 1) whereas lower cooling rate of 1 °C/min led to the formation of a portion of once-folded lamellae along with the twice-folded chain crystals (Figure 5). At higher cooling rates, the polymer crystallized at lower temperatures i.e. higher degrees of supercooling. According to the Ostwald’s step rule,46, 47 the crystallization of the melt into the metastable twice-folded lamellae which grow faster at high extent of supercooling will be kinetically preferred. In contrast, slow cooling thermodynamically favors the formation of more stable once-folded crystals. The presence of a mixture of once-folded and twice-folded lamellar crystals at the cooling rate of 1 °C/min reflects the delicate balance between the thermodynamic and kinetic factors during polymer crystallization. Upon heating, the twice-folded lamellae of PEG6000 transformed into the once-folded chain crystals at low heating rates (Figure 2B), but further unfolding to the extended chains did not occur, as confirmed by SAXS data measured at temperatures adjacent to the melting point of the polymer (Figure 4). The absence of any separate exothermic event during the unfolding process should be noted, indicating that chain extension does not involve large-scale melting followed by recrystallization. Instead, these two processes occur simultaneously.33 Generally, unfolding of folded lamellae is slower as the fold number decreases. For instance, the three-times folded chain crystals of PEG 10,0000 transform into the twice-folded ones within a few minutes while the subsequent conversion into the once-folded chain 21 ACS Paragon Plus Environment


conformation requires hours.44 Chain extension of the once-folded lamellae proceeds even more slowly: the once-folded chain of PEG6000 crystals do not unfold appreciably during heating but remain essentially in their native form until completion of melting at least for heating rates above 0.5 °C/min.32 The unfolding of the once-folded chain crystals of PEG6000 was observed only at crystallization temperatures close to the melting point at which the crystals grow relatively slowly in the once-folded chain conformation39 and subsequently unfold to extended chains after hours.32 The presence of IMC in PEG6000 depressed the melting point and lowered the degree of supercooling of the molten polymer, thereby favoring the crystallization into the thermodynamically more stable once-folded chain crystals (Figure 6B) rather than metastable twice-folded lamellae as found in the pure polymer (Figure 1 and 4). Microstructure of PEG2000-APIs dispersions PEG2000 exists as extended chain crystals with the long period of 123.6 Å (calculated from the second order diffraction peak at 0.153 Å-1) showing a single melting peak at any heating rates (Figure 2A). In dispersions with IMC, the diffraction peak initially appeared at lower scattering vector of 0.090 Å-1 (Figure 6A) corresponding to the longer period of 139.7 Å, which is 15.1 Å larger than the lamellar periodicity of the pure polymer. The long period is the sum of the thickness of the crystalline lamellae and amorphous layers. When the API resides in the amorphous layer between lamellae, i.e. interlamellar incorporation, it would result in the increase of the thickness of the amorphous layer and hence the long period. Since crystals exist in the extended form, the increase in the long period of PEG2000 in dispersions with IMC cannot be attributed to a lower degree of supercooling due to the melting point depression caused by the presence of IMC. Consequently, the 15.1 Å increase in the long period of the polymer in the PEG2000-IMC


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dispersion must be the result of the incorporation of IMC in the interlamellar region of the polymer. Upon storage, the diffraction peak of PEG2000 extended chain lamellae continuously shifted to higher scattering vectors (Figure 6A), indicating the decrease in the long period of the polymer. Evidently, the shift in the diffraction peak of PEG2000 appeared concurrently with the evolution of a new diffraction peak at q ≈ 0.605 Å-1 due to the crystallization of IMC (Figure 13). Therefore, the decrease of the long period of the polymer should be the consequence of the segregation of the drug from the interlamellar region of the PEG matrix and its crystallization outside of the lamellar stacks. The microstructure of PEG2000-IMC dispersions as well as the exclusion of the drug from the interlamellar domain of the polymer are illustrated in Figure 14.

Figure 13. SAXS diffractograms of 20% IMC dispersion in PEG2000 during storage at 20°C.



Figure 14. Illustration of the microstructure evolution of PEG2000-API dispersion. Initially, the API resides in the amorphous layer between lamellae of PEG2000. Upon storage, the drug crystallizes and is segregated from the interlamellar domain of the polymer matrix. PEG2000 exhibited the same initial lamellar periodicity of 139.7 Å in dispersions containing 20, 30 and 40% IMC as shown in Figure 7A, suggesting that the interlamellar incorporation of the drug into PEG matrix was already saturated at 20% drug loading. As the drug content increased, the crystallization of IMC was also accelerated due to the larger driving force for crystallization. Consequently, the drug was expelled more rapidly from the interlamellar region, leading to the faster decrease in the long period. The segregation of IMC from the interlamellar domain resulted in the higher purity of the PEG phase and reduced the melting point depression effect, thereby increasing the melting temperature of the polymer as presented in Figure 9 albeit the decline in the long period. Similarly, when the storage temperature increased, IMC crystallized more rapidly due to higher mobility of the drug molecules, leading to the faster reduction of the long period (Figure 11A). 24 ACS Paragon Plus Environment

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The aforementioned data imply that the decrease in the long period of PEG2000 lamellar stacks depends on how fast the drug is expelled from the interlamellar region which in turn correlates to the crystallization rate of the API. This explains why the periodicity of lamellar stacks of the polymer almost instantly reached the final value (Figure 12A) in solid dispersions with indoprofen and griseofulvin, the two fast crystallizers that belong to class I in the classification scheme of crystallization tendency of organic molecules. However, the crystallization propensity of the APIs is not the only factor that contributes to the change in the long period of PEG2000. Good glass formers including fenofibrate41 and the IMC methyl ester (crystallization tendency was determined by the method proposed by Baird et al.41) with low crystallization propensity can be expected to lead to a slow decrease of the periodicity of the polymer lamellar stacks. However, the periodicity reaches the final value immediately (Figure 12A) without evidence of crystallization of the two APIs, suggesting the absence of interlamellar incorporation of the APIs into interlamellar region of the polymer matrix. The most likely justification for the non-incorporation of these APIs into the interlamellar domain is the lack of drug-polymer specific interactions. Indeed, both fenofibrate and the IMC methyl ester do not contain any proton donating groups and thus are not able to interact with the polymer. For the same reason, cinnarizine is immiscible with the amorphous layers of PEG2000 and hence does not affect the long period of the polymer (Figure 12A). The absence of interlamellar incorporation of non-interacting APIs into the PEG matrix has also been reported for loratadine,17 ketoconazole18 and haloperidol.19 Both flurbiprofen and IMC contain a carboxylic hydroxyl that is able to form hydrogen bonds with the ether oxygen of PEG.7 The long period of the polymer in the presence of these APIs starts decreasing only after certain storage periods when the APIs crystallize and are segregated from the interlamellar regions of the polymer. Since flurbiprofen is a faster



crystallizer than IMC, the long period of PEG in the dispersion with this API decreases more rapidly than in the PEG/IMC dispersion (Figure 12A). Mircrostructure of PEG6000-APIs dispersions PEG6000 exists as twice-folded lamellae in the pure polymer (Figure 1) and once-folded conformation in dispersions with IMC (Figure 6B). The long periods of stacks from the twicefolded and once-folded lamellae of pure PEG6000 are 139.7 Å (Figure 1) and 209.6 Å (Figure 5), respectively. In dispersions with 20% IMC, PEG6000 shows the initial lamellar periodicity of 181.2 Å (Figure 6B) which does not correspond to either twice-folded or once-folded fraction of the polymer. It would be possible that the interlamellar incorporation of IMC into PEG6000 matrix leads to the expansion of the long period of stacks from the twice-folded lamellae. If this would be the case, the periodicity of PEG6000 is expected to reduce upon crystallization of IMC that leads to the separation of the drug from the interlamellar region. Nevertheless, the diffraction peaks of PEG6000 constantly shift to lower scattering vectors over time at 20°C, indicating the continuous increase of the long period despite the crystallization of IMC from 30 hours characterized by the occurrence of a new scattering peak of the drug at 0.61 Å-1 as shown in Figure 15. For those reasons, the microstructure evolution of PEG6000-IMC dispersions cannot be attributed simply to the segregation of the drug from interlamellar region of the polymer as for dispersions of the drug in PEG2000.


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Figure 15. SAXS diffractograms of 20% IMC dispersion in PEG 6000 during storage at 20°C. The initial lamellar periodicity of 181.2 Å of PEG6000 in 20% dispersion with IMC is intermediate between that of the long periods of the two integral folding conformations. This could be the result of the intercalation of the drug into the interlamellar region of the polymer matrix or might correspond to the periodicity of NIF chain crystals. It has been found by using real-time synchrotron SAXS that during crystallization of low molecular weight PEG fractions, the NIF chain crystals will form at the beginning, then transform into IF chain crystals with polymer chains either folded an integral number of times or fully extended.24-30 The existence of the NIF chain crystals of PEG as well as the transformation into IF chain crystals have also been independently observed by longitudinal acoustic mode Raman spectroscopy.48-51 Figure 16 illustrates the primary NIF chain crystallization of PEG6000 and the evolution of nearest IF chain crystals through thickening or thinning. The scheme is adapted to the model originally proposed by Ungar et al.52 then modified by Cheng et al.24 with the hydrogen bonding formation between hydroxyl end groups of polymer chains.53 It is known that the lamellar thickness is proportional to the crystal thermodynamic stability with the melting



point obeying the Tamman equation. Therefore, the thickening process is thermodynamically favored as the crystals convert to a more stable form whereas lamellar thinning is not preferred due to the decrease of the lamellar thickess.

Figure 16. Schematic representation of non-integral folded chain crystallization and the transformation into integral-folded chain crystals. Nevertheless, the inclusion of chain-end defects within the crystal interior in addition to dangling chain ends causing rough and irregular folded surfaces are the destabilizing factors that make NIF crystals the least stable with respect to both once-folded and twice-folded crystals despite the fact that they possess a thicker fold length than the twice-folded crystals. In other words, NIF crystals are thermodynamically metastable to IF crystals and the free energy barrier to form NIF crystals must be the lowest among three states. The initial formation of NIF crystals from melt-crystallization are kinetically favored as they grow fastest, a behavior that follows the well-established Ostwald’s rule of stages.46, 47


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Upon storage, NIF crystals will relax to a lower free energy state via thickening or thinning. The Gibbs energy change during the thickening or thinning of NIF crystals can be described as: ∆G = ∆GNIFIF + ∆G*

(2)

where ∆GNIFIF represents the Gibbs energy difference between initial NIF crystals and final IF ones and ∆G* is the activation energy of the diffusional motion required for thickening and thinning. ∆GNIFIF represents the thermodynamic driving force for NIF to IF transformation and is always negative in contrast with the positive activation energy ∆G*. Which process will occur, either thickening or thinning, is determined by the competition between the thermodynamic driving force for NIF to IF transformation and the kinetic factor. While the overall crystallization of PEG completes within minutes, the transformation of NIF to IF crystals might continue to occur long afterwards through annealing and perfection. The conversion rate is inversely proportional to the molecular weight because shorter polymer chains are associated with the decrease in the barrier to polymer chain motion, and with greater difference in the fold lengths between the initial NIF and the final IF crystals and thus higher thermodynamic driving force for transformation. For these reasons, the NIF chain crystals of the low molecular weight PEG2000, if any, would be extremely unstable and immediately convert to extended chain ones. Due to the formation of NIF chain crystals of PEG6000, it is now unambiguous why the initial lamellar thickness of the polymer in dispersions with IMC is intermediate between the long periods of stacks from the twice-folded and once-folded lamellae. Moreover, the thickening of NIF to once-folded chain crystals will result in the continuous increase in the long period of lamellar stacks during storage.



The evolution of the microstructure of PEG6000-IMC dispersions as illustrated in Figure 17 involves two distinct phenomena with opposite effects on the long period of stacks from the lamellae: the lamellar thickening increases the long period while the segregation of the drug from the interlamellar region of the polymer matrix leads to the reduction of the lamellar thickness. Both events synergistically increase the melting temperature of the polymer during storage as shown in Figure 10. Depending on the crystallization tendency of the API, the drug segregation might occur during or after the thickening period. The overall trend in the change of the long period of PEG6000 depends on the interplay between the two processes.

Figure 17. Illustration of the microstructure evolution of PEG6000-API dispersion. Lamellar thickening is apparently the dominant process as the long period only exhibits the upward trend in dispersions containing various IMC contents (Figure 7B). For example, in 20% IMC dispersions, even though the drug is removed from the interlamellar region of the polymer matrix from 30 hours due to the IMC crystallization as shown in Figure 15, the long 30 ACS Paragon Plus Environment

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period constantly increases up to 60 hours until it reaches the value of the pure polymer. It is obvious that IMC slows down the thickening of PEG6000 as the long period of the pure polymer already reaches the final value after the first hour while the process is much longer in the dispersion with 20% IMC. The hindering effect of IMC on the polymer thickening should be ascribed to the drug-carrier specific interactions via hydrogen bonding and the decreased mobility of the polymer chains. The same phenomenon of polymer thickening hindrance was reported by Chen et al.21 that the non-integral to integral folding of PEG3350 could be observed in dispersions with acetaminophen but was absent in the pure polymer because the transformation was too rapid to be recorded. After 60 hours, the long period remains unchanged despite further crystallization of the drug, suggesting that the expulsion of IMC from the PEG matrix has already completed before that point. As the drug loading increases, the thickening process is further hampered by the decrease in the mobility or diffusional motion of polymer chains along the crystallographic c axis. Additionally, due to the inhibition effect of the drug on the polymer crystallization,6 the crystallinity of PEG6000 increases more slowly at higher drug content. As a consequence, it takes more time for the long period of lamellar stacks of PEG6000 to rise to the level of the pure polymer (Figure 7B). The influence of drug segregation on the long period only becomes noticeable at elevated storage temperature. In Figure 11B, the long period increases faster as the temperature rises from 20°C to 30°C because the chain mobility is higher and thus the thickening is accelerated. Interestingly, when the temperature further increases to 40°C, the long period shows a downward trend instead of commonly observed upward trend. This can be explained by the fact that the thickening process finishes rapidly at 40°C due to the rather high diffusional motion of the polymer chains. The change in the long period afterwards is solely determined by the drug segregation from the PEG6000 matrix that leads to the descending trend. It should 31 ACS Paragon Plus Environment


be noted that the final periodicity at 40°C is somewhat longer than that at lower temperatures, possibly due to the distortion of the lamellae of the polymer. As can be seen for PEG2000, interlamellar incorporation of APIs into the polymer matrix is favored for molecules that can form hydrogen bonding with PEG whereas non-interacting compounds show no interlamellar intercalation. Likewise, the presence of APIs that cannot interact with the polymer does not affect the long period of PEG6000, regardless of the crystallization tendency of the drug compounds. This is the case for the IMC methyl ester, fenofibrate and cinnarizine but surprisingly not for griseofulvin (Figure 12B). The periodicity of PEG6000 in the dispersion with griseofulvin slightly increases and then remains constant. Generally, in non-interacting dispersions, the thickening of PEG6000 is very rapid, comparable to the thickening of the pure polymer. In these systems, the factor that influences the thickening of the polymer is the diffusional motion of the polymer chains. In fact, griseofulvin possesses a quite high glass transition temperature (Tg) of 89°C which is much larger than other APIs in this study. This high Tg will reduce the mobility of the polymer chains in the dispersion with griseofulvin, thereby suspending the thickening of PEG6000. Flurbiprofen, an intermediate crystallizer that can form hydrogen bonds with PEG, crystallizes quickly after 5 hours of storage and hence exhibits almost no effect on the rapid thickening of the polymer. Therefore, the long period of PEG6000 in the dispersion with this API only shows the downtrend (Figure 12B) due to the interlamellar segregation of the drug, which is similar to what is observed for PEG2000-flurbiprofen dispersions (Figure 12A). The periodicity of the polymer decreases upon storage concurrently with the crystallization of flurbiprofen (data not shown). Notably, the incorporation of flurbiprofen into the interlamellar region of PEG6000 increases the initial long period of the polymer by ca. 40 Å to around 255 Å which is comparable to that found in the PEG6000-IMC 80-20 sample at 40°C (Figure 11B) and is around 3 times larger 32 ACS Paragon Plus Environment

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than the rise in the periodicity of PEG2000 in the dispersion with the drug (Figure 12A). The greater degree of intercalation of IMC into the interlamellar region of PEG6000 than PEG2000 indicates the more disordered structure of PEG6000 as a result of longer polymer chains that entangle with each other and impede the formation of well-defined crystals. This can be seen on the SAXS diffractograms in Figure 1: the third and fourth order diffraction peaks are weak for PEG6000 but well-resolved for PEG2000. The increase of ca. 40 Å in the long period of PEG6000 lamellar stacks due to the intercarlation of IMC and flurbiprofen is within the range of lamellar expansion of 30-50 Å of stacks from the once-folded lamellae of PEG3350 in dispersions with chlopropamide, aceclofenac,17 benzocaine, ibuprofen and nilutamide.18 CONCLUSIONS This study highlights the significance of polymer conformation on the microstructure of semicrystalline solid dispersions. PEG2000, a polymer that exists as extended chain crystals, shows a commonly observed and consistent decreasing trend in the long period of lamellar stacks upon interlamellar segregation of APIs from the PEG matrix. PEG 6000 that exhibits the non-integral folded to integral-folded chain transformation as well as the subsequent unfolding of integral-folded chains shows a more complicated behavior: the long period of PEG6000 might increase or decrease during storage as the result of the interplay between the lamellar thickening that expands the thickness of the crystals and the exclusion of the APIs from the interlamellar region of the polymer that reduces the periodicity of lamellar stacks. Numerous factors such as drug loading, storage temperature, crystallization tendency of the APIs as well as the drug-polymer interactions might affect the long period of the two polymers as these factors can slow down or accelerate both lamellar thickening and APIs segregation. All of these differences might translate into the diversity in the physical stability, dissolution behavior and pharmaceutical performance of the semicrystalline dispersions. 33 ACS Paragon Plus Environment


These mechanistic findings further expand our knowledge about the complex nature of semicrystalline dispersions and are of importance for the preparation of solid dispersions with reproducible and consistent properties and pharmaceutical performance. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel.: +32 16 330 304. Fax: +32 16 330 305. ORCID Tu Van Duong: 0000-0002-8918-0268 Guy Van den Mooter: 0000-0001-9166-6075 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS

The authors would like to acknowledge the financial support from Research Foundation Flanders (FWO-Vlaanderen). Tu Van Duong gratefully acknowledges the scholarship (G.0764.13) and travel grant (K1H0416N) awarded by FWO-Vlaanderen. We thank Olivier Verkinderen, Maarten Colaers and Dorien Baeten for technical assistance.

ABBREVIATIONS


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API, active pharmaceutical ingredient; IF, integral-folded; IMC, indomethacin; DSC, differential scanning calorimetry; NIF, non-integral-folded; PEG, polyethylene glycol; SAXS, small-angle X-ray scattering

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