Temperature-Induced Surface Effects on Drug

Pharm Res (2018)35:69 https://doi.org/10.1007/s11095-017-2300-6

RESEARCH PAPER

Temperature-Induced Surface Effects on Drug Nanosuspensions Simone Aleandri 1 & Monica Schönenberger 2 & Andres Niederquell 1 & Martin Kuentz 1

Received: 30 August 2017 / Accepted: 2 November 2017 # Springer Science+Business Media, LLC, part of Springer Nature 2018

ABSTRACT Purpose The trial-and-error approach is still predominantly used in pharmaceutical development of nanosuspensions. Physicochemical dispersion stability is a primary focus and therefore, various analytical bulk methods are commonly employed. Clearly less attention is directed to surface changes of nanoparticles even though such interface effects can be of pharmaceutical relevance. Such potential effects in drug nanosuspensions were to be studied for temperatures of 25 and 37°C by using complementary surface analytical methods. Methods Atomic force microscopy, inverse gas chromatography and UV surface dissolution imaging were used together for the first time to assess pharmaceutical nanosuspensions that were obtained by wet milling. Fenofibrate and bezafibrate were selected as model drugs in presence of sodium dodecyl sulfate and hydroxypropyl cellulose as anionic and steric stabilizer, respectively. Results It was demonstrated that in case of bezafibrate nanosuspension, a surface modification occurred at 37°C compared to 25°C, which notably affected dissolution rate. By contrast, no similar effect was observed in case of fenofibrate nanoparticles. Conclusions The combined usage of analytical surface methods provides the basis for a better understanding of phenomena that take place on drug surfaces. Such understanding Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11095-017-2300-6) contains supplementary material, which is available to authorized users. * Martin Kuentz [email protected] 1

Institute of Pharma Technology, University of Applied Sciences Northwest, Switzerland, Gründenstrasse 40, Basel, Switzerland

2

Swiss Nanoscience Institute, Nano Imaging Lab, University of Basel, Klingelbergstrasse 82, Basel, Switzerland

is of importance for pharmaceutical development to achieve desirable quality attributes of nanosuspensions.

KEY WORDS atomic force microscopy . nanosuspension . surface characterization . surface dissolution . wet-milling

ABBREVIATIONS AFM BF BFns DLS DSC DWS FF FFns HPC iGC ORD PXRD SDS SEM WAXS

Atomic force microscopy Bezafibrate Bezafibrate nanosuspension Dynamic light scattering Differential scanning calorimetry Diffusing wave spectroscopy Fenofibrate Fenofibrate nanosuspension Hydroxypropyl cellulose Inverse gas chromatography Optical rotatory dispersion Powder X-ray diffraction Sodium dodecyl sulfate Scanning electron microscopy Wide angle X-ray scattering

INTRODUCTION Nanosuspensions provide an efficient way to increase dissolution rate and absorption of poorly soluble drugs (1). Rapamune® (sirolimus, Wyeth), Emend® (aprepitant, MSD), TriCor® (fenofibrate, Abbott Laboratories), and Invega® (paliperidone, J&J) are examples of pharmaceutical products based on nanosuspension technology, which are already on the market. Nanosuspensions can be formed by breaking larger micron-sized particles down (top-down approach) and by stabilizing them with a mixture of polymer/surfactant, as applied in the wet milling technique (2).

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Aleandri, Schönenberger, Niederquell and Kuentz96:53 )8102(

Empirical studies on excipients for stabilization have shown that often combinations of surfactants and polymers are needed to achieve stable nanosuspensions (3). Interestingly, drugs with a high enthalpy of fusion (or melting point i.e. high hydrophobicity) as well as high lipophilicity (logP), may be stabilized by various types of surfactants, whereas compounds of either high hydrophobicity or high lipophilicity were typically stabilized with ionic surfactants (4). The interactions between drug, polymer, and surfactant on the nanosuspension surface play an important role in the formation of nanosuspensions (5). Specifically, the interactions between the functional groups of stabilizers and drug surfaces have been demonstrated (6), indicating that the more stable nanosuspensions are obtained if the drug surface energy is comparable to that of the stabilizer (7). Recently, the amount of polymer and surfactant attached on the drug surface was also quantified to better evaluate excipient/drug interactions (8). The literature offers at least a starting point for rational nanosuspension development and a more thorough investigation of drug-stabilizer interactions would be desirable to better understand physical changes. However, trial-and-error is still the prevailing approach to develop nanosuspensions with long-term stability. Most published works evaluated particle size effects like those due to Ostwald ripening (9). Further, solid-state changes were studied in the bulk, such as drug amorphization, crystal defects and/or formation of an amorphous phase induced by the preparation method (such as media milling) (10), or by the drying technique (11). There is generally a focus on different techniques of bulk analytics, whereas potential surface changes have only been studied by few authors (12). It seems that research on surface effects in pharmaceutical nanosuspensions needs to be much better explored to bring this rather neglected domain to a technical focus in developing nanomedicines. The current study emphasizes temperature effects between 25 and 37°C, which have obvious relevance for storage and oral administration. Especially lipophilic compounds with comparatively low crystal energy may exhibit surface changes in such a temperature interval, which can be of technological or biopharmaceutical relevance. The current approach with usage of the different surface analytical tools is to the best of our knowledge new in the field of pharmaceutical nanosuspensions. Fenofibrate (FF) and bezafibrate (BF) (two cholesterol- reducing drugs) served as models for poorly water-soluble drugs in presence of commonly used excipients, i.e., sodium dodecyl sulfate (SDS) and hydroxypropyl cellulose (HPC) as anionic and steric stabilizer, respectively. FF and BF nanosuspensions (FFns and BFns, respectively) were fully characterized, using different technics, with particular attention to temperature induced-phenomena on the surface by atomic force microscopy (AFM), inverse gas chromatography (iGC) and UV imaging of drug dissolution. AFM has been employed previously to study the crystallization behavior of a pharmaceutical drug

(probucol) in nanoparticles (13). However, within this study AFM was used for the first time in combination with a UV imaging system of drug dissolution to evaluate the changes on the nanosuspension surface induced by temperature.

MATERIALS AND METHODS Materials Chemical structures of drug and excipients are depicted in Fig. 1. Fenofibrate (FF) and bezafibrate (BF) were purchased from AK scientific, Inc. (USA), hydroxypropyl cellulose (HPC) was obtained from Nisso Chemical Europe and Sephadex G25 and sodium dodecyl sulfate (SDS) were purchased from Sigma Aldrich. All solutions were prepared using Mill-Q water (18.2 MΩ cm−1). Methods Wet Milling The milling mixture was produced by dispersing FF or BF 10 wt.% in a HPC/SDS (2.5 and 0.5 wt.%, respectively) aqueous solutions under vigorous stirring for 2 h. The obtained suspension was transferred into a small agitator bead mill (Dyno-mill research Lab., Willy A Bachofen Ltd., Muttenz, Switzerland) that was previously filled (60%, v/v) with 300– 400 μm yttrium-stabilized zirconia beads. The dispersion was then wet milled at 25°C for 2 h using a controlled agitator speed of 3000 rpm. During the process, aliquots were taken from the milling chamber after 10, 30, 60, 90, and 120 min for size analysis. Dynamic Light Scattering (DLS) for Particle Sizing. The size of the nanosuspensions was measured during the milling process, immediately after preparation, and 2 weeks later with a Zeta Sizer Nano ZS (Malvern Instruments, Malvern, UK. Operating at 173°scattering angle) at 37 ± 0.1°C. The samples were filled in disposable polystyrene cuvettes of 1 cm optical path length. At least three independent samples were taken, each of which was measured between three to five times. The width of the DLS hydrodynamic diameter distribution was indicated by the polydispersity index (PDI). The intensity size distribution of the nanosuspension was typically unimodal; therefore, the autocorrelation function was analyzed according to the cumulant method (14). Optical Rotatory Dispersion (ORD) The amount of HPC attached on the nanosuspension surface was measured with ORD at 400 nm. ORD can be generated by chiral elements in a macromolecular backbone or by the

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Fig. 1 Structures of drugs, stabilizers employed and schematic drawing of a wet milling process used to form nanosuspensions

helical conformation of a polymer (15). HPC does not contain any chiral center in the main chain or side chain; however, if a right-handed or left-handed helical conformation is generated in excess, the polymer exhibits optical activity. In particular, HPC exhibits an ORD curve with monotonic increase in negative rotation with decreasing wavelength (16). The variation in optical rotation with wavelength for dilute solutions of HPC was evaluated using a Jasco J-815-150S CD spectrometer (JASCO Inc., Easton, MD, U.S.A.) equipped with a Peltier accessory and an ORD measuring unit as described in literature (16). The samples were measured in a 5 cm glass cuvette in triplicates at room temperature. Size Exclusion Chromatography Following the milling process, the nanosuspension was filtered at 25°C trough a Sephadex G-25 packed column to remove the free HPC, which is not attached on the nanoparticle surface. The nanosuspension elution profile was retrieved plotting the value of the mean count rate (Kcps), obtained by DLS measurements (Zeta Sizer Nano ZS from Malvern Instruments, Malvern, UK.) of the fractions eluted from the column vs the elution volumes (uL). The HPC elution profile was obtained plotting the percentage of HPC present in the fractions eluted from the column vs the elution volumes. The percentage of HPC in the fractions (% HPC) was evaluated according with Eq. 1. %HPC ¼ ðORDFRs :=ORDB Þ 100

ð1Þ

Where ORDFRs is the ORD value of the fractions eluted from Sephadex filtration and ORDB is the ORD value before Sephadex filtration. Scanning Electron Microscopy (SEM) Size and morphology of the particles constituting the formulation were analyzed by Supra M-40 scanning electron microscopy (Zeiss AG, Jena GmbH). A small drop of the suspension was first air- dried followed by drying in an oven. Samples were then fixed on an SEM stub using double-sided adhesive tape and coated with Au at 20 mA through a sputter-coater (Ion sputter JFC 1100). A scanning electron microscope with a

secondary electron detector was used to obtain digital images of the samples at an accelerating voltage of 15 kV. Diffusing Wave Spectroscopy (DWS) DWS RheoLab (LS Instruments AG, Fribourg, Switzerland) was used as optical technique for microrheological measurements as reported previously (17). The theory of DWS-based microrheology was already explained in detail in our previous work (18). The DWS was calibrated prior to each measurement with a suspension of polystyrene particles, PS, (Polysciences Europe Ltd. Germany) in purified water (50 wt.%). The PS particles (NIST-standard) have a mean size of 250 ± 0.30 nm with a solid content of 1 wt.% in dispersion. This suspension was filled in cuvettes with a thickness L of 1 and 2 mm prior to measuring for 60 s at 25°C. The value of the transport mean free path, l* (microns) was determined experimentally as reported previously (19). The transmission count rate was measured several times until a constant value was reached and the cuvette length, L, was considerably larger than the obtained values for l* (L » l) ensuring diffusive transport of light. The transport mean free path of the sample l* is needed for the determination of the correlation intensity function and thus for the microrheological characterization. Nanosuspensions were stirred, kept overnight for equilibration and afterwards analyzed using quartz cuvettes with L of 1 and 2 mm. All samples were equilibrated at 25°C in the measuring chamber prior to measurement. The measurement time was set to 60 s and each sample was measured 5 times to confirm reproducibility. Viscosity measurements were also performed for HPC solutions. Thus, 1 wt.% polystyrene (PS) nanoparticles were added to the clear samples to ensure the correct regime (guarantee a L/l* ratio larger than 7) (17). 5 mm quartz cuvettes were employed and data acquired for 60 s. Each sample was measured 5 times as previous measurements. Spray Drying Spray drying was performed using a Büchi Mini Spray Dryer B-290 (Büchi Labortechnik AG, Switzerland). Suspensions were properly diluted with deionized water if necessary. In

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order to maintain its homogeneity, the suspension was stirred slowly using a magnetic stirrer during the spray drying process. The spray dryer was fitted with a 0.7 mm pneumatic nozzle and operated at 6 bar air pressure, 11 ml/min pump speed, 600 l/h air flow rate, 80% aspirator level (pump 33%) and 150°C inlet temperature. Powder X-Ray Diffraction (XRPD) Powder X-ray diffraction was used to assess crystallinity of spray-dried nanosuspension at ambient temperature using a Bruker D2 PHASER (Bruker AXS GmbH, Germany) with a PSD-50 M detector and EVA Application Software version 6. Samples were prepared by spreading powder samples (obtained by spray dry) on PMMA specimen holder rings from Bruker. Measurements were performed with a Co Kα radiation source at 30 kV voltage, 10 mA current and were scanned from 10 to 40 2θ, with 2θ being the scattering angle at a scanning speed of 2θ/min. Differential Scanning Calorimetry (DSC) A DSC 4000 System, from PerkinElmer (Baesweiler, Germany) was calibrated for temperature and enthalpy using indium. Nitrogen was used as the protective gas (20 mL/min). Samples (3 mg) were placed in 40 μL aluminium pans with pierced aluminium lids. The melting onset temperatures (Tm) and the enthalpy of fusion (ΔH) were determined by a singlesegment heating ramp of 10°C/min from 25°C to a maximum temperature of 200°C. For drug classification and to determine midpoint glass transition temperatures (Tg), the samples were heated in separate experiments from 25 to 5°C above the melting temperature and held isothermally for 5 min to ensure complete melting of the sample. The samples were then cooled to −50°C and held for 5 min. Finally, samples were heated again to 20°C above the melting temperature. A constant heating and cooling rate of 10°C/ min was used. All DSC measurements were carried out in triplicate. Wide Angle X-Ray Scattering (WAXS). WAXS measurements were used to confirm the crystallinity of nanosuspensions at 37°C and to categorize the drugs in glass formers (GFs) and non-glass formers (nGFs), which was in line with a classical DSC approach (20,21). Briefly, the milled samples were heated 10°C above the Tm and let equilibrate for 30 min before acquiring a spectra. Then, the samples were cooled at 37°C and 25°C, using the same heating and cooling cycle as with DSC. The intensity profile was acquired after 30 min equilibration time. Experiments were performed on a Bruker AXS Micro (Bruker AXS GmbH, Germany), with a microfocused X-ray source, operating at voltage and filament current of 50 kV and 1000 μA, respectively. The Cu Kα radiation

(λCu Kα = 1.5418 Å) was collimated by a 2D Kratky collimator, and the data were collected by a 1D VÅNTEC-1 detector. The scattering vector q = (4π/λ) sinθ, with 2θ being the scattering angle, was calibrated using silver behenate. Data were collected in a q range from 13 to 20 nm−1. The samples (dry powder, obtained after spray dry) were placed inside a stainless steel cell between two thin replaceable mica sheets and sealed by an O-ring, with a thickness of ∼1 mm. Measurements were performed at different temperatures, and samples were equilibrated for 30 min before measurement, whereas scattered intensity was collected over 60 min. UV Imaging of Drug Dissolution. The intrinsic dissolution rates were determined using a real-time UV imaging dissolution system (Actipix®SDI300) from Sirius Ltd. (York, UK) as reported in our previously work (22). The images were acquired using UV filter at 300 nm for FF and 250 nm for BF. In line with maximum absorption, a wavelength was selected with 310 and 230 nm for FF and BF, respectively. The light source was a pulsed xenon lamp and the total imaging area was 9 × 7 mm (4 mm light path). Downstream from the specimen centre, a designated quantification region was automatically selected by the software (approx. 0.56 mm × 3.14 mm) to determine the dissolution rate. All experiments were carried out consecutively (n = 6) at 25 and 37°C. The dissolution medium (PBS pH 7.5) was added via syringe pump at 0.2 ml/min over 18 min (zero turbulence flow) through the flow cell to contact the sample surface. The drug was dissolved from the surface, flowing downstream. The absolute absorbance was obtained by comparing the intensity at each camera spot against the corresponding background value (dark images and blank dissolution media). The intrinsic dissolution rate was calculated at defined time points (60 s intervals) by averaging 207 frames per interval using the Actipix®SDI300 software from Paraytec Ltd. (V.1.7.2210). Using the molecular weight, molar extinction coefficient at the wavelength used, flow rate and surface area of the drug compact at the centre (3.14 mm2), the absorbance was converted to the drug concentration. The molar extinction coefficient was evaluated by a Jasco V630 spectrophotometer (JASCO Inc., Easton, MD, U.S.A.). The data from the first minute of dissolution were disregarded and an interval was selected arbitrarily at 960– 1200 s to determine the mean intrinsic dissolution rates for each sample. Inverse Gas Chromatography (iGC) A commercially available inverse gas chromatograph, iGCSEA (Surface Measurement Systems, London, UK) was used to determinate the Tg of BF and BFns (stationary phase) using 1-Butanol as gas probe. For BF, approximately 170–200 mg of sample was packed in a silanized glass column using a standardized packing method. In the case of BFns, due to


the high surface area and column pressure, we used a physical mixture of 50 mg BFns and 1 g of 250 μm sized silanized glass beads (Supelco Analytical, Pennsylvania, USA). Samples were equilibrated with dry helium (10 sccm at 303 K for 1 h) prior to measurements. This treatment allowed for removal of any sorbed water or impurities from the system. Each sample was then subjected to a temperature program composed of 13 single injections of the probe molecules at infinite dilution conditions (5% surface coverage), starting at 300 K and increasing by 2° steps to 324 K. Prior to each injection, methane was used for dead volume correction and a flame ionization detector was used for detection. All experiments were performed in triplicate, using three different sample columns. Prior to replicate measurements, the samples were preequilibrated (dry helium with 10 sccm at 303 K for 1 h). Surface phase transformations (Tg values in Table I as mean ± SD) were determined by plotting of the natural log of the quotient between the specific retention volume and the temperature vs. the reciprocal of the absolute temperature (ln(VSp /T)) using a method reported in literature (23). Surface area values of BF and BFns, used herein, were determined by physical absorption of octane molecules. Multiple injections of the probe were done with different surface coverages (0.5–100%) at 303 K and a flow rate of 10 sccm. iGC analysis software v. 1.4 was used in the Brunauer–Emmett–Teller (BET) range (p/p0 of 0.05–0.35) to calculate the surface area of a samples. All measurements were done in triplicate using individual sample columns and the same equilibration conditions as for the Tg measurements were used. Atomic Force Microscopy (AFM) A NanoWizard4 AFM (JPK Instruments AG, Berlin, Germany) was used to visualize the surface of the nanosuspension particles. Quantitative imaging (QI™) maps of FFns and BFns were acquired in air, at 25 and 37°C, using the JPK high temperature heating stage. One microliter of a nanosuspension (0.03 wt.%), was dropped on a glass coverslip and let dry. QI™ height images were acquired with a silicon nitride tip, MLCT-A (Bruker Nano Inc., USA) used for soft contact imaging modes and liquid operation, with a nominal spring constant of 0.07 N/m.

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DLS values and were 178.5 ± 1.3 and 200.2 ± 2.3 nm for FFns and BFns, respectively. A short-term stability study was carried out as shown in Fig. 2b. The nanosuspension was kept at 4°C for 2 weeks after preparation and DLS was used to monitor the nanosuspension particle size at 37°C. As a result, the data indicated at least acceptable short-term stability since no marked size changes were noted. The final nanosuspensions were spray dried and the product was analyzed by powder X-ray diffraction (PXRD) at 25°C. Processes like milling and spray drying can induce a change in the crystal form of FF or BF (24,25). More importantly, the solid state form can influence physical stability, dissolution behavior, and possibly also in vivo performance of the drug. Therefore, PXRD experiments were conducted to identify the crystalline state of FF and BF after milling and drying treatments. As shown in Fig. 3, even though a slight decrease in intensity of peaks was observed, the characteristic peaks for milled FF or BF were observed at same 2θ value as those of the unmilled drug. Since an increase in temperature could lead to change the crystallinity, wide angle X-ray scattering (WAXS) was used to confirm the crystallinity of nanosuspensions at 37°C. As shown in Fig. 4, the nanosuspensions are crystalline also at 37°C. A DSC analysis was performed to investigate the thermal properties of nanosuspensions and to categorize the molecules in glass formers (GFs) and non-glass formers (nGFs) (21). nGFs compounds (class I) crystallize directly in the first cooling cycle, whereas the stable GFs (class III) remain amorphous upon cooling and display a glass transition in a subsequent heating cycle. Some compounds alternatively crystallize in the second heat and were assigned to a category II (26). As summarized in Table I (the thermograms are shown in SI; Fig. S1), the endothermic peaks of FF and BF are the same as those observed for FFns and BFns. Moreover, both the raw drugs and their version formulated as nanosuspensions belong to stable GF (class III). The DSC results were also confirmed by WAXS experiments that employed a corresponding temperature cycle (See SI, Fig. S2). While below the Tm the nanosuspension showed the characteristic peaks of the crystalline drugs, above the Tm, the peaks disappeared, indicating that the nanosuspensions become amorphous, and remained amorphous even upon cooling. Surface Characterization of the Nanosuspensions

RESULTS Bulk Characterization of the Nanosuspensions The nanosuspensions were composed of 10 wt.%. FF or BF, 2.5 wt.% HPC and 0.5 wt.% SDS (chemical structures are depicted in Fig. 1). The particle size reduction was monitored during the milling process as shown in Fig. 2a. Nanoparticle size after 2 h of milling agreed with SEM (Fig. 2c, d, e, f) and

Following the milling process, the nanosuspensions were filtered at 25°C through a Sephadex G-25 packed column (as described above) to quantify the amount of HPC that effectively covered the nanoparticle surface. Analogous to our recent findings of nanosuspensions using HPC as stabilizer (19), only 41 or 53% of the total amount of HPC used in the milling medium (2.5 wt.%) covered the FFns or BFns surface (see SI Fig. S3). Samples with these reference amounts of polymer and SDS (without drugs) and the nanosuspension before and

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Table I Thermal Properties (i.e. Melting Point, Tm, Enthalpy of Fusion ΔHTm, Enthalpy Change Associated with Glass Formation ΔHTg, and Glass Transition Temperature, Tg) of the Model Drugs and Their Nanosuspensions with Assigned Class of Glass Forming Ability

Tm (°C)

ΔHTm (J/g)

Tg (°C)

ΔHTg(J/g)

Tg (°C) a

Classb

BF

178.6 ± 0.1

52.3 ± 3.0

42.2 ± 0.4

4.2 ± 0.6

36.2 ± 2.7

GF(Class III)

BFns FF

176.4 ± 0.4 81.4 ± 0.6

52.2 ± 4.1 46.6 ± 3.7

40.5 ± 0.2 −14.5 ± 0.7

5.2 ± 0.9 1.6 ± 3.3

36.7 ± 0.6

GF(Class III) GF(Class III)

FFns

79.8 ± 1.1

41.0 ± 5.2

−14.3 ± 0.3

1.7 ± 4.2

GF(Class III)

Mean values ± standard deviations were obtained from DSC as well as iGC measurements (in case of Tga ) a

Glass transition temperature measured by iGC

The solidification behavior of the samples during the heat − cool − heat treatment was obtained by DSC and confirmed by WAXS experiments. Details about the classification as glass forming drugs (GF) are given in the text

b

after filtration were measured by DWS. This approach hence compares filtration results with DWS. After filtration, the polymer concentration was reduced and the viscosity decreased accordingly. Moreover, the viscosity values for the reference amounts of HPC (1.025 or 1.35 wt.% for FFns and BFns, respectively) and filtered nanosuspensions were indeed the same. Since the main purpose of this study was to use the surface characterization techniques to investigate surface Fig. 2 Kinetic evaluation of the wet milling process of BFns (filled circles) and FFns (opened circles) (a). Size analysis of the nanosuspensions obtained by dynamic light scattering (DLS) after 1 and 2 weeks storage at 4°C, white and gray bars, respectively (b) and SEM pictures of FFns (c and d) and BFns (e and f) after 2 h of wet-milling

changes of nanosuspension as a function of temperature, inverse gas chromatography (iGC) was further used to confirm the Tg of BFns. The Tg value was determined by plotting the natural log of the retention volume vs. the reciprocal of the absolute temperature (23). As shown in Table I, either for the BF or for the BFns the Tg value obtained by DSC is slightly higher than the values obtained by iGC. However, it has to be noted, that the onset of glass transition in the DSC


Fig. 3 Powder X-ray diffraction (XRPD) plots of FF (a), FFns (b), BF (c) and BFns (d) at 25°C

thermograms was around 34°C (see SI, Fig. S1), and this value was close to the Tg values given by iGC (36.2 ± 2.7 and 35.7 ± 0.6, for BF and BFns, respectively) and to the value reported in literature (20). Afterwards, the surface dissolution of nanosuspensions was evaluated at 25 and 37°C respectively. Since the system required several seconds to achieve constant laminar flow and temperature control, we disregarded the first two minutes of the measurements (as reported in a previous publication (22)) and the dissolution was monitored from 2 up to 18 min (1200 s) to ensure that only a small amount of the compacted drug was dissolved. Figure 5a shows that at either 25 or 37°C the FFns exhibit the same dissolution profile without any appreciable difference. By contrast, BFns revealed a different dissolution profile at 25 and 37°C. As depicted in Fig. 5b, the intrinsic dissolution rate increases with an increase of temperature. The values towards the end of the experiment (960–1200 s) were nearly constant. This time interval provided a mean intrinsic dissolution rate from this pseudo-

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equilibrium (see Fig. 5c) and the t-test revealed the significant differences in the dissolution rates between the BFns at 25 or 37°C (p < 0.005), whereas no statistical difference is present in the case of FFns. Finally, since atomic force microscopy (AFM) can provide information on the 3D topography of the samples at the nanoscale level, quantitative advanced imaging mode (QI™) was used to visualize the FFns and BFns surface at 25 and 37°C. QI™ mode was developed by JPK Instruments to get quantitative mechanical information such as stiffness, adhesion and dissipation. However, it is also ideal for high-resolution imaging, particularly for challenging samples that are soft and sticky, loosely attached or that have steep edges. Since in both nanosuspensions (FFns and BFns) the sample became sticky after heating to 37°C, it was difficult to measure normal AC mode under these circumstances. In AC mode (the signal driving the feedback is the amplitude of an oscillating cantilever, rather than the static deflection, as used in contact mode), the oscillating tip remains close to the adhesive surface, thereby resulting in unstable and blurred topography images. Therefore, QI™ mode was applied, where the tip is brought close to the surface, indenting by a few nN of vertical deflection and again fully withdrawn from the sample at every cycle and pixel by at least 300 nm off the surface. First, as shown in Fig. 6, the AFM images at 25°C confirmed the particle size value obtained by DLS and the shape of the nanosuspension particles obtained by SEM. In the case of FFns, there were no appreciable differences on the nanosuspension surface at 25 or 37°C (Fig. 6a, b, c, d respectively). By contrast, the case of BFns revealed surface changes with increasing temperature. As shown in Fig. 6i at 37°C, the shape of the particle became more elongated with respect to the same spot at 25°C (Fig. 6g) and thus, the total surface area of this particular particle increased. The surface change was confirmed by the AFM height profile of a selected line through BFns at 25 and 37°C (blue and red lines, respectively in Fig. 6j).

DISCUSSION

Fig. 4 Wide angle X-ray scattering (WAXS) plots of FFns (a and b) and BFns (c and d) at 25 (black lines) and 37°C (red lines)

In line with the objective to better explore the mostly neglected surface domain of nanosuspensions, we studied different fibrates as model compounds. The nanosuspensions were prepared by wet-milling of FF and BF in presence of 2.5 wt.% HPC and 0.5 wt.% SDS. These stabilizing excipients as a charged lipophilic surfactant and a polymer represent typical choices for nanosuspensions of lipophilic compounds. The milling was stopped after 2 h, since the size of drug crystals was sufficiently reduced and an increase of milling time could lead to particles agglomerations (12,27). The nanosuspensions exhibited an acceptable short-term stability, confirming suitable selection of the stabilizing excipients.

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Fig. 5 Initial phase of intrinsic surface dissolution from 120 to 720 s at 25°C (filled circles) and 37°C (opened circles) for FFns (a) and BFns (b). Summary of intrinsic dissolution rates in a pseudo-equilibrium (960–1200 s) for FFns and BFns at 25°C (blue bars) and 37°C (red bars) (c). Data are expressed as mean ± SD from six independent experiments. Statistically significant differences are indicated by * (p < 0.005) via the t test vs BFns at 25 and 37°C

Longer periods were not analyzed because this was beyond the current study objective and the formulation did not contain any preservatives. Wet-milling, spray drying and temperature can alter the solid state form of drugs (24,25). Such

changes can affect physical stability as well as dissolution behavior and finally the absorption step in vivo (28). Therefore, XRPD and WAXS were conducted to identify the crystalline state of FF and BF after milling and drying treatments at 25 and 37°C. Although both FFns and BFns retained their crystallinity from the results of the DSC (Table I), PXRD (Fig. 3), and WAXS (Fig. 4) analyses, the diffraction intensity of spraydried nanosuspensions was slightly decreased in X-ray experiments. This can be attributed to some process-induced disorder and a decreased particle size of FF and BF crystals (29). The present study considered glass forming ability (GFA) of the compounds, which is the ability of a material to vitrify on cooling from the supercooled melt. This inherent compound property is likely to be of relevance for also other processinduced glass formation for example upon spray-drying or wet-milling (30,31). Both compounds were identified as glass formers that were stable (category III), whereas FF has previously also been assigned to less stable glass formers (i.e. class II) (20). This ability of the model fibrates to be easily converted into a glass may be accompanied by fast loss of crystallinity during processing. However, the milling and the drying process did not lead to a very pronounced loss of bulk crystallinity as suggested by DSC and X-ray. In line with the study objectives, there was a primary interest in surface effects. In most pharmaceutical studies, the amount of polymer adsorbed onto drug nanoparticles is unknown. Here, the amount of HPC that effectively covers the nanoparticles surface was estimated to be 41 and 53% for FFns and BFns, respectively. Differences in the absorbed surface concentration of excipients can be a factor with respect to temperature induced drug changes on the surface. Nanosuspension drug surfaces are always accompanied by stabilizing excipients so their influence is essentially compounded with any observed drug effect on the surface. Particularly interesting were the results of surface dissolution imaging. These studies revealed significant differences in the dissolution rates between BFns at 25 vs. at 37°C (p < 0.005) and AFM images evidenced a surface change when increasing the temperature. At 37°C the surface became more elongated with respect to the same spot at 25°C (Fig. 6g). On the other hand, there were no appreciable differences observed on the nanosuspension surface of FF at 25 vs. 37°C (Fig. 6a, b, c, d respectively), which was also reflected by an intrinsic dissolution rate that was barely affected by the temperature difference. Differences in the glass transition temperature (Tg) of the drugs can provide a possible explanation. FFns exhibited a Tg at −14°C, so that at the working conditions (25 and 37°C) the materials were all above the Tg. On the other hand, BFns showed a Tg close to 40°C. This is an interesting value for a thermal phase transition of a drug given that it is close to the body temperature of 37°C. In the dissolution experiment at 25°C, the BFns was below its Tg. The surface was also rigid


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Fig. 6 QI™ mode (by atomic force microscopy, AFM) of FFns at 25 and 37°C (upper panel; from A to D) and of BFns at 25 and 37°C (bottom panel F to I). The white squares (500 × 500 nm) in figure A, C, F and H shows the area zoomed in figures B, D, G and I. Panel E show the AFM height profile of a selected line through FFns. Panel J show the AFM height profile of a selected line through BFns. 25°C (blue lines) and 37°C (red lines)

and less elongated as evidenced by AFM (Fig. 6f, g). An increase to 37°C means for BF that the temperature comes into the range of the Tg (transition onset at 34°C) and it was shown by AFM that the surface softened and became more elongated (see Fig. 6h, i). These surface modifications must have contributed to an increase of intrinsic dissolution rate as shown in Fig. 5b, c. It is important to specify that Tg was determined by DSC from undercooled melts and the drug Tg was evaluated during the second heating ramp (21). However, when approaching the Tg from room temperature as in WAXS experiments (Fig. 4), no changes were shown in the bulk phases. BFns was crystalline at 37°C, and remained crystalline even when the sample was heated up to 75°C (see SI, Fig. S4). This supports the importance of analyzing surface properties apart from the bulk. While DSC and WAXS evaluate the physical changes in the bulk, iGC is another powerful technique to obtain nanosuspension surface information and to determine a surface glass transition (12). IGC has been already used to study the surface properties of pharmaceutical systems and observed structural relaxation was previously shown to be comparable to the bulk relaxation obtained from DSC method (32). IGC can detect the fraction of amorphous surface of BFns and it can localize the surface fraction in crystalline substances that transforms into amorphous state upon intensive milling (33). Plotting the natural log of retention volume versus the reciprocal of temperature, is a common way to obtain a glass transition. It is hence a characteristic of the confined surface and does not have to correspond to a bulk Tg. A continued straight line of the transformed retention volume (see methods section) is indicating no transition, whereas, a first deviation from linearity is attributed to the onset of a thermal transition that was here a Tg. An increase in retention volume is due to both, surface adsorption and bulk sorption because of the increased molecular mobility due to the Tg (23). As showed in Table I,

either for the BF or for BFns the Tg values obtained by DSC (42.2 and 40.5°C, respectively) were slightly higher than the values obtained by iGC (36.2 and 36.7 for BF and BFns, respectively). However, the onset of glass transition in the DSC thermograms was around 34°C (see SI, Fig. S1), and this value was close to the Tg values obtained by iGC and to the values reported in literature (20). Slight differences between Tg of drug and nanosuspensions can be caused by the excipients, the process or by the fact that a surface is confined compared to a bulk. When BFns was heated from 25°C onwards, an increase in gas retention time was visible due to both surface adsorption and bulk sorption because of the increased molecular mobility caused by the Tg. This result confirmed the interesting evidences obtained by the AFM and intrinsic dissolution findings. BFns revealed some surface amorphization induced by temperature as shown by the complementary methods of surface analytics, whereas no such a clear effect was seen in the bulk methods. Since the intrinsic dissolution rate is typically a critical quality attribute, these surface changes are of biopharmaceutical relevance and the surface changes can also be relevant for manufacturing processes high pharmaceutical relevance.

CONCLUSIONS Nanosuspensions of the model fibrates FFns and BFns were studied by different methods of bulk as well as surface analytics. It was observed that the preparation method did not greatly impact on the bulk properties of the drugs. On the other hand, while FFns was barely affected by the temperature difference of 25 and 37°C, BFns clearly altered surface properties. AFM demonstrated a BFns surface modification induced by temperature. These changes were likely causing the higher intrinsic dissolution rate of this formulation at 37°. However,

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even though both drugs share similar lipophilicity, they also exhibit different molecular structures, for example, FF is neutral and BF is an acidic compound. This could have further contributed to the observed temperature effect on intrinsic dissolution rate. Regarding a surface influence, the iGC results confirmed that the observed changes were linked to the onset of a phase transition, since the BFns surface Tg was close to the temperature 37°C used in the intrinsic dissolution experiments. These findings underpin the importance to study surface properties of nanosuspensions by complementary modern methods because the bulk analytics alone would lead to an incomplete physical characterization. The observed changes induced by the body temperature (i.e. 37°C) are not only relevant for a better understanding but may be also technologically harnessed to for example target a specifically increased dissolution rate. However, to this end more knowledge is needed about how adsorbed excipients and the surface confinement itself may lead to different thermal changes than observed in the bulk. All of such future research would have to make use of complementary surface analytics that seems to be indispensable for today’s advancement of pharmaceutical nanosuspensions.

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ACKNOWLEDGMENTS AND DISCLOSURES Prof. Raffaele Mezzenga is acknowledged for his support to conduct the ORD and WAXS experiments at the ETH in Zurich Switzerland. Author contributions: The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

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