Amorphization Strategy Affects the Stability and Supersaturation

Mar 26, 2014 - School of Chemical and Biomedical Engineering, Nanyang Technological University, 637459 Singapore. Mol. Pharmaceutics , 2014, 11 (5), ...
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Amorphization Strategy Affects the Stability and Supersaturation Profile of Amorphous Drug Nanoparticles Wean Sin Cheow, Tie Yi Kiew, Yue Yang, and Kunn Hadinoto* School of Chemical and Biomedical Engineering, Nanyang Technological University, 637459 Singapore S Supporting Information *

ABSTRACT: Amorphous drug nanoparticles have recently emerged as a promising bioavailability enhancement strategy of poorly soluble drugs attributed to the high supersaturation solubility generated by the amorphous state and fast dissolution afforded by the nanoparticles. Herein we examine the effects of two amorphization strategies in the nanoscale, i.e., (1) molecular mobility restrictions and (2) high energy surface occupation, both by polymer excipient stabilizers, on the (i) morphology, (ii) colloidal stability, (iii) drug loading, (iv) amorphous state stability after three-month storage, and (v) in vitro supersaturation profiles, using itraconazole (ITZ) as the model drug. Drug−polyelectrolyte complexation is employed in the first strategy to prepare amorphous ITZ nanoparticles using dextran sulfate as the polyelectrolyte (ITZ nanoplex), while the second strategy employs pH-shift precipitation using hydroxypropylmethylcellulose as the surface stabilizer (nano-ITZ), with both strategies resulting in >90% ITZ utilization. Both amorphous ITZ nanoparticles share similar morphology (∼300 nm spheres) with the ITZ nanoplex exhibiting better colloidal stability, albeit at lower ITZ loading (65% versus 94%), due to the larger stabilizer amount used. The ITZ nanoplex also exhibits superior amorphous state stability, attributed to the ITZ molecular mobility restriction by electrostatic complexation with dextran sulfate. The higher stability, however, is obtained at the expense of slower supersaturation generation, which is maintained over a prolonged period, compared to the nano-ITZ. The present results signify the importance of selecting the optimal amorphization strategy, in addition to formulating the excipient stabilizers, to produce amorphous drug nanoparticles having the desired characteristics. KEYWORDS: nanomedicine, nanopharmaceuticals, poorly soluble drug, bioavailability enhancement, drug complexation lumen, higher drug bioavailability is obtained.3 Significantly, in vitro generations of high supersaturation level by amorphous drugs have been shown to translate to enhanced bioavailability in vivo.4−7 Traditionally, amorphous drugs have been formulated as microscale solid dispersions prepared by spray-drying, freezedrying, or hot melt extrusion,2 where the drug is stabilized by polymer excipients having high glass transition temperatures (Tg), such as hydroxypropylmethylcellulose (HPMC) and poly(vinylpyrrolidone) (PVP). The polymer stabilizer reduces the molecular mobility of the dispersed drug, thereby (i)

1. INTRODUCTION A high percentage of promising drug candidates in the development pipeline (∼66%) exhibit low aqueous solubility in their crystalline form,1 resulting in limited drug absorption in the gastrointestinal tract, hence low bioavailability in vivo. Increasing the drug dose in an attempt to increase the amount of bioavailable drug does not represent a viable solution as it simply leads to higher drug wastage, in addition to higher pill burden and cost to the patients. However, amorphization of such sparingly soluble drugs represents a feasible approach to improve the aqueous solubility, by virtue of the high supersaturation generated by the metastable amorphous state, resulting in an apparent drug solubility that is multifolds higher than the saturation solubility of the crystalline counterparts.2 When the high supersaturation level is maintained for a time period sufficient for absorption across the gastrointestinal © 2014 American Chemical Society

Received: Revised: Accepted: Published: 1611

December March 23, March 26, March 26,

31, 2013 2014 2014 2014

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acids or bases.22 Significantly, the development of the amorphous nanoscale drug−PE complex, or drug nanoplex in short, enables an equitable comparison of the two amorphization strategies to be carried out in the same particle length scale. Herein we prepare two amorphous drug nanoparticles by the first (i.e., drug−PE complexation) and second (i.e., pH-shift precipitation) amorphization strategies with the objectives of evaluating their effects on the (i) morphology, (ii) colloidal stability, (iii) drug loading, (iv) amorphous state stability after three-month storage at 25 °C and 30% relative humidity, and (v) in vitro supersaturation profile of the amorphous drug nanoparticles produced. Itraconazole (ITZ), an antifungal drug known for its low aqueous solubility (∼1 ng/mL at neutral pH23), is selected as the model drug. In this regard, various methods have been employed to increase the solubility of ITZ by amorphization, including microscale amorphous solid dispersions prepared by spray drying with hydrophilic polymers24 and melt extrusion,25 as well as amorphous ITZ nanoparticles prepared by antisolvent 9 and pH shift13 precipitations. However, direct comparisons between the different amorphous forms of ITZ have never been performed in the same particle length scale. Central to the utilization of the drug−PE complexation and pH-shift precipitation methods is the realization that although the solubility of ITZ in acid at pH 1 is very low (∼4 μg/mL23), an excess of acid results in the protonation of the triazole ring, in addition to the protonated piperazine nitrogen26 as illustrated in Figure 1A, thus enabling a higher ITZ solubility to be achieved, resulting in a higher production output. Starting from an acidic ITZ solution of up to 30 mg/mL concentration, ITZ is either complexed with the oppositely charged dextran sulfate (DXT) in the first amorphization strategy or

preventing recrystallization of the amorphous drug during storage and (ii) inhibiting the solution-mediated nucleation and crystal growth of the dissolved drug, resulting in prolonged supersaturation.8 Another amorphous drug formulation is in the form of amorphous drug nanoparticles stabilized by polymer or surfactant excipients, which are physically adsorbed on the nanoparticle surface immediately upon the nanoparticle formation, via hydrophobic or electrostatic interactions. The occupation of the high energy sites on the nanoparticle surface by the excipient stabilizers suppresses the recrystallization propensity of the amorphous form and particle growth due to Ostwald ripening.9,10 Such amorphous drugs have been prepared by various methods, including antisolvent precipitation,9 sonoprecipitation,11 evaporative precipitation,12 and pH-shift precipitation.13 Importantly, the high specific surface area of the nanoparticles ensures a high dissolution velocity, therefore minimizing the time window for solution-mediated crystallization of the remaining solid phase,14 which unfavorably tends to occur in microscale amorphous solid dispersions due to their slower dissolution velocity. This is manifested in the higher supersaturation levels achieved when amorphous drug nanoparticles are used.9 While the effects of different excipient stabilizer formulations on the storage stability and bioavailability of amorphous drugs have been extensively investigated for both the microscale15−17 and nanoscale12,18 formulations, from which the optimal stabilizer formulations are identified, the same cannot be said for the effects of the amorphization strategy. In fact, the selection of the amorphization strategy, where whether to stabilize the amorphous state by adding excipients (1) to reduce the molecular mobility, as pursued in the microscale amorphous solid dispersion, or adding excipients (2) to cover the high energy sites to prevent secondary nuclei formation on the amorphous surface, as pursued in the amorphous drug nanoparticles, is nontrivial. On the one hand, the high excipient content in the first strategy, which typically constitutes more than 50% of the product mass,18 while ensuring the amorphous state stability, may adversely affect the drug dissolution rate as the rate is no longer determined by the amorphous state of the drug but rather is dictated by the type of stabilizer used.19 On the other hand, the smaller amount of stabilizer incorporated in the second strategy (typically less than 10%), while having minimal effects on the dissolution rate, may not be sufficient to fully stabilize the amorphous state. A fair comparison of the two amorphization strategies, however, has not been able to be carried out due to the difference in the length scale of existing amorphous drugs prepared by the two strategies (i.e., microversus nanoscale). This is particularly an issue when the supersaturation profiles of the micro- and nanoscale formulations are compared as the particle size affects dissolution velocity and saturation solubility. In this regard, we demonstrated in an earlier study a novel preparation of amorphous drug nanoparticles via electrostatic complexation of charged drug molecules with oppositely charged polyelectrolytes (PE), where the amorphous state is formed and subsequently maintained by mobility restriction of the drug molecules by the PE;20,21 hence, it exemplifies the first amorphization strategy. The drug−PE complexation method relies on the ability of the sparingly soluble drugs to dissolve in highly acidic or basic medium to form charged molecules, which is achievable considering most drugs are weak organic

Figure 1. (A) Double protonation of ITZ in the presence of excess acid; (B) two different amorphization strategies to prepare amorphous ITZ nanoparticles. 1612

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in charge densities of 2.8 × 10−6 and 4.8 × 10−6 mol·charge/mg for ITZ and DXT, respectively. 2.3. Preparation of Amorphous Nano-ITZ. The amorphous ITZ nanoparticles prepared by the pH-shift precipitation method are termed nano-ITZ, to differentiate it from the ITZ nanoplex prepared in the previous section. To prepare nano-ITZ, an acidic ITZ solution is added dropwise into an alkaline solution containing the stabilizer upon which ITZ immediately precipitates due to a sudden pH shift. Owing to the rapid precipitation, formation of organized crystalline structures is prevented. In addition, physical adsorption of the stabilizer (i.e., HPMC) present in the basic solution on the nano-ITZ surface suppresses secondary nucleation and subsequent particle growth. Briefly, ITZ is first dissolved in 25% (w/v) HCl at a concentration of 30 mg/mL, while HPMC is dissolved in 0.5 M KOH at a concentration of 0.75 mg/mL. One milliliter of the acidic ITZ solution is then added dropwise into 20 mL of the alkaline solution under vigorous stirring after which a milky suspension is immediately formed. After equilibrating for 60 s to allow the adsorption of HPMC, the suspension is washed to remove visibly large particles and excess HPMC, HCl, and KOH by three cycles of centrifugation (13000 × g for 10 min), followed by resuspending the nanoparticles in DI water. 2.4. Physical Characterizations of Amorphous ITZ Nanoparticles. The size and zeta potential of the amorphous ITZ nanoparticles (i.e., ITZ nanoplex or nano-ITZ) in their aqueous suspension form are measured by photon correlation spectroscopy (PCS) using Brookhaven 90Plus Nanoparticle Size Analyzer (Brookhaven Instruments Corporation, USA). To measure the nanoparticle size in their dry powder form, the nanoparticles are freeze-dried and sputter coated with platinum and imaged using scanning electron microscope (SEM) model JSM-6700F (JEOL, USA), followed by particle size analysis using ImageJ software (NIH, USA) with a minimum of 200 particle counts. The ITZ loading, which is defined as the mass percentage of ITZ making up the nanoparticles, is determined by measuring the amount of ITZ released in PBS when a known mass of the nanoparticles is completely dissolved. ITZ concentrations in the solution are measured by isocratic reverse-phase high performance liquid chromatography (HPLC) (Agilent 1100, Agilent Technologies, USA) equipped with a vacuum degasser, binary pump, autosampler, and temperature-controlled column oven. A Vydac C18 column (250 × 4.6 mm, 5 μm particle size) is used for the chromatographic separation, while detection is performed at 260 nm. The mobile phase consists of acetonitrile−water (70:30 v/v) with a flow rate of 0.75 mL/ min. Under these conditions, the retention time of ITZ in 90% methanol (10% PBS) is 6.3 min. The efficiency of the nanoparticle formation, which is defined as the mass percentage of the ITZ initially added that forms amorphous ITZ nanoparticles, by either drug−PE complexation or pH-shift precipitation, is characterized by % ITZ utilization. Specifically, the % ITZ utilization is determined from the mass percentage of ITZ that is not recovered in the supernatant of the nanoparticle suspension after the first centrifugation relative to the initial amount of ITZ added. The efficiency of the nanoparticle recovery is characterized by % yield to take into account the particle loss incurred during the recovery and purification (i.e., washing and centrifugation). The % yield is defined as the ratio of the nanoparticle mass recovered to the total solid mass initially added (i.e., ITZ, DXT,

precipitated upon addition into a basic solution containing HPMC as the stabilizer in the second strategy, to form amorphous ITZ nanoparticles (see Figure 1B). As the ITZDXT complex formation is demonstrated for the first time here, the investigations leading to the optimal ITZ nanoplex formulation, using low and high molecular weight DXT as the PE, are presented first.

2. MATERIALS AND METHODS 2.1. Materials. Sodium chloride (NaCl), potassium hydroxide (KOH), hydrogen chloride (HCl), HPMC, glacial acetic acid, methanol, and phosphate buffer saline (PBS, pH = 7.4) were purchased from Sigma−Aldrich (USA). ITZ was purchased from Alfa Aesar (USA). DXT (MW 5000 kDa and 500 000 kDa) was purchased from Wako Pure Chemical (Japan). 2.2. Preparation of Amorphous ITZ Nanoplex. To prepare the amorphous drug nanoplex, the drug is first dissolved in an aqueous acid solution to form an ionized drug solution, which is subsequently added to the oppositely charged aqueous PE solution, upon which drug−PE electrostatic interaction takes place to form soluble drug−PE complex in the presence of salt. Upon reaching a certain critical concentration, whose value depends on the drug hydrophobicity, the drug−PE complexes form agglomerates by means of interdrug hydrophobic interactions to finally form insoluble drug nanoplex. The role of salt is to provide a charge screening effect to minimize repulsions of the like-charged PE chains, which can inhibit the drug−PE complex formation. The amorphous form is obtained because the strong drug−PE electrostatic interaction prevents the drug molecules from assembling into ordered crystalline structures upon precipitation. As ITZ forms cations upon dissolution in aqueous acetic acid solution, DXT, a polyanion, is used as the PE. A ternary phase diagram of the ITZ-DXT complex is constructed by varying mass compositions of the drug, PE, and salt, while keeping the ITZ concentration at 5 mg/mL. Briefly, 1.5 mL of 5 mg/mL of ITZ in 28% (v/v) acetic acid is added to 0.6 mL of DXT solutions at various DXT and NaCl concentrations. The mixture is left for 1 h in ambient conditions to allow the complexation to equilibrate. To remove the excess drug and PE (i.e., those that do not form nanoplex), as well as NaCl and acetic acid, the nanoplex suspension is washed by three cycles of centrifugation and resuspension in deionized water. The nanoplex suspension is then transformed into dry powders by freeze-drying. The complexation products are categorized as either nanoplex or soluble complex. In order to identify nanoplex suspensions, removal of the excess salt and resuspension in DI water is necessary as the charge-stabilized nanoplex flocculates in the presence of salt. To categorize the complexation products, the samples are centrifuged for 15 min at 13 000 × g. The sediments obtained at the bottom of the centrifuge tubes are redispersible suspensions when nanoplex is obtained. Drug−PE mixtures are categorized as soluble complex when a clear solution is observed after centrifugation. The drug to PE charge ratios (RITZ/DXT) are calculated from the molecular weight (MW) and number of charge per molecule of the ITZ and DXT. For example, ITZ (MW 705.6 g/mol) has two charges when dissolved in excess acid, while there are 24 −OSO3− groups per DXT molecule (MW 5000 kDa), resulting 1613

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or HPMC), where the nanoparticle mass recovered is determined by freeze-drying of an aliquot of the nanoparticle suspension after the three washing and centrifugation cycles. The reported ITZ loading, % ITZ utilization, and % yield are obtained from at least two independent experiments on different days with two measurement replicates each. The stability of the ITZ nanoparticle powders is examined in which the powder X-ray diffraction (PXRD) patterns of the powders are characterized after three-month storage at 25 °C and 30% relative humidity, using a D8 Advance X-ray diffractometer equipped with Cu Kα radiation (Bruker, Germany) from 10° to 60 (2θ) with a step size of 0.02°/s. The PXRD pattern of the ITZ nanoparticle powders is compared with that of the native crystalline ITZ powders. Thermal stability of the powders is examined by differential scanning calorimetry (DSC) using DSC 822e (Mettler Toledo, USA) in which 2 mg of powders are filled into a sealed aluminum crucible and heated from 25 to 180 °C at 10 °C/ min. In addition, thermogravimetric analysis (TGA) of the powders are performed to determine the moisture content, using SDT Q600 (TA Instruments, USA) where about 5 mg of powder is filled into an alumina pan and heated from 30 to 400 °C at 10 °C/min. 2.5. Generation and Quantification of ITZ Supersaturation. The ITZ saturation solubility (Ceq) is determined first by adding native crystalline ITZ in excess (∼100 mg) into 20 mL of PBS supplemented with 1 mg/mL of HPMC. After 24 h incubation in a shaking incubator at 37 °C, a 2 mL aliquot of the incubated sample is centrifuged and filtered through 0.1 μm filters (Puradisc, Whatman, USA), after which the dissolved ITZ concentration is measured by HPLC as described earlier. Next, the supersaturated ITZ solution is generated by adding ITZ nanoparticles (suspension and dry powder) at concentrations equal to 20× and 80× the saturation solubility into 20 mL of PBS containing 1 mg/mL of HPMC at 37 °C under constant stirring. The role of HPMC is to suppress the ITZ precipitation from the supersaturated solution, thereby the supersaturation level can be accurately determined. At specified time points, 0.5 mL of the release medium is withdrawn and filtered after which 50 μL of the 0.5 mL filtered ITZ solution is immediately diluted 10-fold with methanol to prevent ITZ precipitation from the supersaturated solution. Afterward, the ITZ concentration (C) is measured by HPLC to obtain the supersaturation profile, defined as the ratio of Ceq to C, as a function of time.

Figure 2. (A) Ternary diagram showing the compositions of ITZ, LMW DXT, and NaCl that result in the formation of ITZ nanoplex (closed circles) or soluble ITZ−DXT complex (open circles); (B) % ITZ utilization as a function of RITZ/DXT at different NaCl concentrations (i.e., 0.03 to 0.42 M) (lines are provided as guide to the eyes only).

increasing salt concentration at all RITZ/DXT, where the % ITZ utilization decreases from ∼70−90% at 0.03 M NaCl to ∼50% at 0.42 M NaCl. This is because higher salt concentrations intensify the screening of the PE charge that in turn inhibits the drug−PE complex formation. Second, for the effects of RITZ/DXT on the % ITZ utilization, at salt concentrations ≤0.14 M, the % ITZ utilizations remain relatively constant for RITZ/DXT ≤ 1.0−1.2 above which the % ITZ utilization starts to decrease. The decrease in the % ITZ utilization as RITZ/DXT increases above 1.0 is because there are more ITZ than DXT to interact electrostatically at RITZ/DXT > 1.0, resulting in an increased amount of noncomplexed ITZ. At higher salt concentrations (i.e., 0.24 and 0.42 M), however, the decrease in the % ITZ utilization at RITZ/DXT > 1.0 is not observed in the range of RITZ/DXT investigated. The highest % ITZ utilization (∼90%) is obtained at 0.03 M NaCl and RITZ/DXT = 1.2, therefore suggesting that excess DXT (i.e., RITZ/DXT < 1.0) does not necessarily lead to higher % ITZ utilizations. In fact, at 0.24 M NaCl, lower % ITZ utilizations are observed as RITZ/DXT is lowered to below 0.2. The % yield of the run having the highest % ITZ utilization, however, is found to be only ∼28%, denoting inefficient nanoparticle recovery. In fact, the % yields of all the LMW DXT runs are low at ∼6−40% as shown in Figure 3A. The low % yields are postulated caused by the loss of ITZ and/or LMW DXT from the ITZ nanoplex during washing and centrifugation caused by their insufficiently strong electrostatic interactions. The % yields, unlike the % ITZ utilization, are found to be minimally affected by the salt concentration, thus the yields in

3. RESULTS 3.1. Optimal Preparation of ITZ Nanoplex. The feasibility of preparing ITZ nanoplex is first investigated using low molecular weight DXT (i.e., LMW DXT) as the PE. The drug−PE−salt ternary diagram in Figure 2A shows the compositions at which ITZ nanoplex (closed circles), or soluble ITZ-DXT complex are formed (open circles). The ternary diagram shows that ITZ nanoplex is not formed at % ITZ ≤ 20% and (i) % NaCl ≥ 40% and % LMW DXT ≤ 40% or (ii) % NaCl ≤ 30% and % LMW DXT ≥ 60%. All other ITZ:DXT:NaCl compositions result in the ITZ nanoplex formation. To determine the optimal ITZ:DXT:NaCl composition, we evaluate the nanoplex produced in terms of the % ITZ utilization in Figure 2B, where the data are grouped based on the salt concentration used in the preparation. First, the % ITZ utilizations plotted against RITZ/DXT in Figure 2B suggest a decrease in the % ITZ utilization with 1614

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centrifugation. A similar dependence of the % yield on RITZ/DXT, where the highest yield is obtained at RITZ/DXT ≈ 1.0, and a minimal influence of the salt concentration on the % yield are also observed for the HMW DXT runs. In terms of the % ITZ utilization, the HMW DXT runs result in % utilizations (Figure 4) that are comparable in magnitude

Figure 4. Percent ITZ utilization of ITZ nanoplex formation as a function of RITZ/DXT at different NaCl concentrations when HMW DXT is used.

to the values reported earlier for the LMW DXT runs (Figure 2B), except for the % utilizations at the highest salt concentration investigated (i.e., 0.42 M), where the HMW DXT runs result in ∼10−20% lower ITZ utilizations. Similarly, the % utilizations of the HMW DXT runs also decrease with increasing salt concentration from ∼80−90% at 0.03 M NaCl to ∼20−50% at 0.42 M NaCl. Importantly, the ITZ:DXT:NaCl composition that produces the highest % utilization at 93% (i.e., 0.03 M NaCl and RITZ/DXT = 1.0 at ITZ = 5 mg/mL) corresponds to the composition that also produces the highest % yield. This optimal composition is subsequently used to prepare the ITZ nanoplex used for comparison with the nanoITZ. 3.2. ITZ Nanoplex versus Nano-ITZ Comparisons. 3.2.1. Physical Characteristics. Comparisons between the amorphous ITZ nanoplex and nano-ITZ in terms of their physical characteristics are presented in Table 1. The two nanoparticles in their aqueous suspension forms are similar in size at ∼400 ± 120 and 480 ± 250 nm for the ITZ nanoplex and nano-ITZ, respectively, with the ITZ nanoplex exhibiting a considerably narrower size distribution in their wet state as shown in Figure 5A. This is due to the lower zeta potential of the nano-ITZ at nearly neutral ∼−7 mV compared to ∼−47 mV for the ITZ nanoplex, denoting lower colloidal stability of the former. The lower zeta potential renders the nano-ITZ more prone to agglomeration, hence a broader size distribution, which is reflected in Figure 5B by the significant presence of particles in the ∼700−1000 nm range. The higher zeta potential for the ITZ nanoplex is not unexpected considering that DXT is a polyanion capable of having multiple negative charges, whereas HPMC is nonionic in nature. SEM images of the ITZ nanoplex and nano-ITZ in Figure 6A,B, respectively, show that both nanoparticles are spherical in shape, as expected from amorphous particles, having sizes ∼300 nm after drying. The smaller size for the nanoparticle powders

Figure 3. Percent yield of ITZ nanoplex formation as a function of RITZ/DXT when (A) LMW DXT or (B) HMW DXT is used (lines are provided as guide to the eyes only).

Figure 3A are not grouped based on the salt concentration used in the preparation. Despite their low values, the variations in the % yields as a function of RITZ/DXT of the LMW DXT runs do exhibit the trend expected from the theory, where the yield increases with increasing RITZ/DXT up to RITZ/DXT ≈ 1.0 at which the highest yield (∼40%) is obtained, followed by its gradual decrease at RITZ/DXT > 1.0. At RITZ/DXT < 1.0 (i.e., excess DXT), there are insufficient ITZ to complex with DXT, resulting in a large amount of noncomplexed DXT, hence the low yield, which is gradually improved as RITZ/DXT approaches 1.0. The opposite is true at RITZ/DXT > 1.0 (i.e., excess ITZ), resulting in the lower % ITZ utilization, hence the gradually decreasing yield. To improve the % yield, the study is repeated using high molecular weight DXT (HMW DXT) as an increase in the PE’s molecular weight has been found to result in a lower critical aggregation concentration in surfactant−PE complexation, denoting stronger intrinsic binding between the two oppositely charged molecules.27 The results in Figure 3B indicate that the highest % yield is indeed increased from ∼40% to 80% when the HMW DXT is used in place of the LMW DXT. Moreover, the average yield of all the HMW DXT runs is calculated to be equal to ∼43%, which is substantially higher than the average yield of all the LMW DXT runs at ∼19%. The improved % yield when HMW DXT is used therefore aligns with our postulate of the stronger interactions between ITZ and HMW DXT, such that the nanoplex can stay intact upon washing and 1615

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Table 1. Physical and Preparation Characteristics of ITZ Nanoplex and Nano-ITZ sample

wet size (nm)

zeta potential (mV)

ITZ loading (wt %)

ITZ utilization (wt %)

yield (wt %)

ITZ nanoplex nano-ITZ

400 ± 120 480 ± 250

(−) 47 ± 0.8 (−) 7 ± 0.6

65 ± 6 94 ± 7

93 ± 2 99 ± 1

84 ± 5 35 ± 4

the frequency size distributions of the dry powders plotted in Figure 5. The nano-ITZ powders, nevertheless, remain to exhibit a broader size distribution than the ITZ nanoplex powders due to the presence of agglomerated particles with sizes larger than 600 nm. Despite their comparable size, the nano-ITZ has significantly higher ITZ loading at ∼94 ± 7% (w/w) than that of the ITZ nanoplex (∼65 ± 6%), which is well anticipated because the HPMC is only present on the nano-ITZ surface via adsorption, whereas the HMW DXT is present throughout the ITZ nanoplex’s volume via electrostatic complexation. On this note, the ITZ loading of the ITZ nanoplex remains relatively high compared to the typical drug loading of amorphous drugs prepared by the first amorphization strategy. In terms of the preparation, both amorphization strategies result in high % ITZ utilizations at ∼93 and 99% for the ITZ nanoplex and nanoITZ, respectively. The % yield, however, is significantly lower for the nano-ITZ at 35% compared to 84% for the ITZ nanoplex. The low % yield of the nano-ITZ is because visibly large particles formed in the pH shift precipitation method, likely due to Ostwald ripening, are not recovered. 3.2.2. Stability of the Amorphous State. The amorphous states of the ITZ nanoplex and nano-ITZ after three-month storage at 25 °C and 30% relative humidity are demonstrated by the PXRD spectra presented in Figure 7, where broad diffuse Figure 5. Volume-averaged size distributions of the (A) ITZ nanoplex and (B) nano-ITZ.

Figure 7. PXRD spectra of the amorphous ITZ nanoparticles compared to the native crystalline ITZ.

peaks are observed for the ITZ nanoparticles, in contrast to sharp crystalline peaks for the native ITZ powders. Next, thermal behaviors of the two stabilizers (i.e., HMW DXT and HPMC) are examined from which the HMW DXT and HPMC are found to exhibit Tg at 135 and 158 °C, respectively (Figure 8A). In comparison to pure amorphous ITZ (i.e., without stabilizers), which was found in a separate study to exhibit Tg at ∼59 °C,28 the significantly higher Tg for the HMW DXT is expected to lead to high Tg for the ITZ nanoplex, lending the ITZ-DXT complex a high amorphous state stability, whereas for the nano-ITZ significant Tg change is not expected because

Figure 6. SEM images of the (A) ITZ nanoplex and (B) nano-ITZ showing spherical particles with relatively monodispersed size.

is due to the swelling of the polysaccharide stabilizers (i.e., DXT and HPMC) in water. The SEM images also suggest that the size monodispersity of the nanoparticles is improved upon their transformation into dry powders, which is also reflected in 1616

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observed elsewhere at a similar recrystallization temperature.28 The recrystallized nano-ITZ later melts at 158 °C, close to the melting point of the native ITZ crystals at 168 °C, where the difference in their melting points is likely due to the presence of HPMC as contaminants. In addition to temperature, another factor that affects the amorphous state stability is the moisture content of the nanoparticle powders after the three-month storage as the absorbed moisture can lower the Tg due to the plasticizing effect of water.29 The TGA results presented in Figure 8B show that the nano-ITZ has only ∼1% (w/w) moisture as indicated by ∼1% mass loss upon heating below 100 °C, whereas the ITZ nanoplex has ∼5% (w/w) moisture. Despite its higher moisture content, the ITZ nanoplex remains more stable than the nanoITZ as the former does not recrystallize upon the thermal treatment. The large mass losses observed in the TGA thermograms at 175 and 250 °C for the ITZ nanoplex and nano-ITZ, respectively, are due to their thermal degradation. 3.2.3. Supersaturation Profiles. Supersaturation profile comparisons between the ITZ nanoplex and nano-ITZ are presented in Figure 9. When the nanoparticles in their aqueous suspension form are added at a concentration equal to 20× the ITZ saturation solubility, the nano-ITZ exhibits a faster dissolution rate, where the maximum ITZ concentration of ∼10.5× the saturation solubility is reached in 30 min, compared to the 45 min taken for the ITZ nanoplex to reach its maximum ITZ concentration, which is also lower at 8.5× supersaturation (Figure 9A). The higher supersaturation level delivered by the nano-ITZ, however, is accompanied by extensive ITZ recrystallization from the supersaturated solution, where the ITZ concentration decreases to 2.5× supersaturation at t = 150 min. In contrast, the ITZ concentration delivered by the ITZ nanoplex only decreases to ∼6.5× supersaturation at t = 150 min. The ITZ concentrations of both nanoparticles remain at these values until the end of the experiment (t = 180 min). When the initial nanoparticle concentration is increased to 80× the ITZ saturation solubility, similar supersaturation profiles are observed, where the nano-ITZ exhibit a faster dissolution rate and more extensive recrystallization than the ITZ nanoplex (Figure 9B). The supersaturation level of the nano-ITZ also peaks after 30 min, albeit at a slightly lower supersaturation level of ∼8.9× compared to that obtained at 20× the saturation solubility. The ITZ concentration then rapidly decreases to ∼4× the supersaturation level only 15 min after the peak supersaturation level is reached and remains at that value until the end of the experiment. In contrast, the ITZ nanoplex delivers its maximum supersaturation level (∼8.7×) at a slower rate in 60 min, after which a gradual decrease of the supersaturation level to ∼4× in 60 min is observed, and it remains at that value until the end of experiment. The supersaturation profiles at 20× and 80× the saturation solubility suggest that an increase in the initial nanoparticle concentration does not necessarily lead to an increase in the maximum ITZ supersaturation level achieved, which is due to the increased recrystallization propensity of the more highly supersaturated solution. Next, the supersaturation profiles of the nanoparticles in their dry powder form are investigated at an initial concentration of 20× of the saturation solubility (Figure 9C). The same trend in which the nano-ITZ dissolves at a faster rate than the ITZ nanoplex is observed, where the maximum supersaturation levels are reached in 30 and 150 min for the nano-ITZ and ITZ nanoplex, respectively. However, in contrast to the high

Figure 8. (A) DSC and (B) TGA thermograms of the amorphous ITZ nanoparticles showing higher amorphous state stability for the ITZ nanoplex.

of the low HPMC content (∼6%) and the fact that HPMC is only present on the nano-ITZ surface. The DSC thermograms in Figure 8A, however, indicate that the Tg of the ITZ nanoplex (∼72 °C) is not raised to a significantly higher temperature compared to the Tg of pure amorphous ITZ, and it is considerably lower than the Tg of the native DXT despite the high DXT content (>30%). Nevertheless, the Tg of the ITZ nanoplex is higher than that of the nano-ITZ (∼55 °C), which is very similar to the Tg of pure amorphous ITZ as anticipated. Despite not exhibiting a significantly higher Tg, the ITZ nanoplex does not exhibit recrystallization and melting point peaks, which indicates that its amorphous form remains stable upon the thermal treatment attributed to the mobility restriction of the ITZ molecules by the HMW DXT. In contrast, the nano-ITZ displays broad exothermic events above its Tg, denoting molecular rearrangements of the ITZ, which is not unexpected as the second amorphization strategy does not rely on molecular mobility restriction in its stabilization mechanism. On this note, the slight endothermic peak above Tg at ∼70 °C in the nano-ITZ’s thermograms has been attributed to a thermal phenomenon related to rotational restrictions of the ITZ molecules above its Tg.28 The molecular rearrangement in the nano-ITZ is capped by an exothermic recrystallization peak at 120 °C. Recrystallization of amorphous ITZ upon being subjected to thermal treatment has been 1617

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4. DISCUSSION In the preceding sections, the ITZ nanoplex preparation has been optimized in terms of the % ITZ utilization and % yield by varying the drug:PE:salt compositions using the HMW DXT as the PE. Subsequently, the optimal ITZ nanoplex and the nanoITZ have been compared in terms of their physical characteristics (i.e., morphology, colloidal stability, and ITZ loading), amorphous state stability, and their in vitro supersaturation profiles. While both amorphization strategies result in >90% ITZ utilization, the % yield of the nano-ITZ production is significantly lower due to the nonrecovery of visibly large ITZ particles formed. The % yield of the nano-ITZ preparation can nevertheless be improved by suppressing the particle growth, for example, by increasing the mixing intensity of the acid and base to enhance the degree of supersaturation, resulting in nucleation-dominated precipitations, or by increasing the amount of stabilizer used. Solely on the basis of the physical characteristics, the nanoITZ can be deemed superior by virtue of its higher ITZ loading, despite its lower colloidal stability. A high ITZ loading is much desirable as it translates to a smaller dosage needed to achieve the same level of bioavailability. As these amorphous ITZ nanoparticles are intended for oral solid dosage form delivery, the lower colloidal stability of the nano-ITZ does not represent a significant liability as the nanoparticle suspension can be immediately transformed into dry powders. The higher amorphous state stability of the ITZ nanoplex, however, overrides the higher ITZ loading of the nano-ITZ as more stabilizers would inevitably be needed to stabilize the nano-ITZ in its solid dosage preparation, resulting in its lower ITZ loading. When the in vitro supersaturation profiles of the ITZ nanoplex and nano-ITZ are compared, there is not a distinct advantage of one over the other, despite their different supersaturation profiles. The nano-ITZ achieves its maximum supersaturation 20−30 min and 2 h earlier than the ITZ nanoplex for their aqueous suspension and dry powder forms, respectively, where the maximum supersaturation levels are in general higher for the nano-ITZ. The role of specific surface areas in causing their varying dissolution rates is likely to be minimal as the nanoparticles are of similar size and shape. Therefore, the slower dissolution rate for the ITZ nanoplex is more likely caused by (i) its lower ITZ loading and (ii) the fact that the ITZ must decomplex from the HMW DXT before the ITZ can be released, as opposed to the nano-ITZ, which only requires the dissolution of a small amount of HPMC on the surface before the ITZ can be released. At the same time, the supersaturation level of the nano-ITZ decreases more rapidly than that of the ITZ nanoplex. The slower decrease of the supersaturation level for the ITZ nanoplex is postulated owing to the presence of decomplexed DXT in the solution that helps the HPMC in inhibiting the ITZ recrystallization from the supersaturated solution, resulting in more prolonged supersaturation for the ITZ nanoplex. As ITZ is known to exhibit a high permeability in the gastrointestinal mucosa,30 the maximum achievable supersaturation level could be more important than the duration that the supersaturation is sustained above a certain level. An in vivo study will better demonstrate the superiority of one nanoparticle over the other, if any, in terms of the supersaturation profiles, which ultimately impacts the bioavailability.

Figure 9. Supersaturation profiles of the amorphous ITZ nanoparticles in their aqueous suspension form at initial ITZ concentration equal to (A) 20× and (B) 80× the saturation solubility, and (C) the supersaturation profiles of the dry powder form at 20× the saturation solubility.

supersaturation level achieved by the nanoparticle suspensions, the nanoparticle powders result in significantly lower maximum supersaturation levels of ∼4× and 2.3× for the nano-ITZ and ITZ nanoplex, respectively. Despite the lower maximum supersaturation level, where the ITZ recrystallization from the supersaturation solution is anticipated to be less extensive, the ITZ concentrations rapidly decrease to 1× supersaturation, denoting the absence of supersaturation, for both the nano-ITZ and ITZ nanoplex. 1618

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The lower maximum supersaturation level achieved by both nanoparticles in their dry powder form is caused by the smaller surface areas available for dissolution as the nanoparticles now exist in the form of loose agglomerates in the micrometer range, thus not all nanoparticles are in contact with the dissolution medium. The slower dissolution rate in turn leads to the increased propensity of the remaining solid phase to recrystallize during dissolution, which is commonly observed in microscale amorphous solid dispersions, hence further inhibiting the supersaturation generation. The presence of recrystallized solid phase in the solution then promotes Ostwald ripening, resulting in the further decrease in the supersaturation level, despite the low supersaturated solution being generated. These sequential events are believed to be responsible for the less than desirable supersaturation profiles for the nano-ITZ and ITZ nanoplex powders. In order to fully utilize the supersaturation generation potentials of the amorphous nanoparticles, it is crucial that their dry powder transformation, which is required for their solid dosage form preparation, ensures complete and rapid reconstitutions of the nanoparticles from their agglomerated form upon contact with the dissolution medium. Methods, such as salt-induced flocculation18 and electrostatic adsorption31 of the nanoparticles followed by drying, or freeze-drying of nanoparticles in the presence of highly water-soluble excipients,32 have been proposed for this purpose. The feasibility of employing these different granulation-and-drying methods for the ITZ nanoparticles is currently being evaluated in our laboratory. For example, the salt-induced flocculation is likely not suitable for the ITZ nanoplex as the presence of salt would induce decomplexation of the ITZ from DXT during drying, hence altering the ITZ release profile, while the electrostatic adsorption is likely not feasible for the nano-ITZ due to its nearly neutral charge.

Article

ASSOCIATED CONTENT

S Supporting Information *

DSC and SEM images. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(K.H.) Tel: (65) 6514 8381. Fax: (65) 6794 7553. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Sabrina Nian Zi Tay of Dunman High School Singapore for her contribution in the ITZ nanoplex preparation. A financial support from GlaxoSmithKline Singapore for their Green and Sustainable Manufacturing research partnership is gratefully acknowledged.

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ABBREVIATIONS Ceq, saturation solubility of itraconazole; C, supersaturated concentration of itraconazole; DI, deionized; DSC, differential scanning calorimetry; DXT, dextran sulfate; HMW DXT, high molecular weight dextran sulfate; LMW DXT, low molecular weight dextran sulfate; HPLC, high performance liquid chromatography; HPMC, hydroxypropylmethylcellulose; ITZ, itraconazole; ITZ nanoplex, amorphous ITZ nanoparticles prepared by the first amorphization strategy; MW, molecular weight; nano-ITZ, amorphous ITZ nanoparticles prepared by the second amorphization strategy; PBS, phosphate buffer saline; PCS, photon correlation spectroscopy; PE, polyelectrolyte; PVP, poly(vinylpyrrolidone); PXRD, powder X-ray diffraction; SEM, scanning electron microscope; t, time; Tg, glass transition temperature; TGA, thermogravimetric analysis

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5. CONCLUSIONS Two amorphous forms of ITZ nanoparticles, i.e., nano-ITZ and ITZ nanoplex, have been prepared by two amorphization strategies, i.e., pH-shift precipitation and drug−PE complexation, respectively, which are distinct in their stabilizing mechanisms of the amorphous state. Even though they share a similar morphology, the ITZ nanoplex, which relies on molecular mobility restriction of the ITZ by the PE for the stabilization, has been shown to exhibit superior amorphous state stability in the dry powder form after three-month storage than the nano-ITZ, whose stability is derived from the occupation of the amorphous particle surfaces by polymer stabilizers. The higher stability of the ITZ nanoplex, however, comes with a lower ITZ loading due to the higher amount of stabilizer used and slower supersaturation generations in vitro, and also lower maximum supersaturation levels achieved in general, compared to the nano-ITZ. Nevertheless, the supersaturation condition is prolonged for the ITZ nanoplex owed to the lower solution-mediated recrystallization propensity postulated due to the presence of decomplexed PE in the solution. An in vivo study needs to be carried out in the future to determine which supersaturation profile is more desirable for ITZ. Lastly, the supersaturation profiles of both nanoparticles have been shown to be greatly affected by their transformation into dry powders, therefore determining the optimal drying methods is crucial for their oral solid dosage form preparation.

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