Nanocrystals embedded in chitosan-based respirable swellable microparticles as dry powder for sustained pulmonary drug delivery

European Journal of Pharmaceutical Sciences 99 (2017) 137–146

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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps

Nanocrystals embedded in chitosan-based respirable swellable microparticles as dry powder for sustained pulmonary drug delivery Rui Ni a, Jing Zhao a, Qiaoyu Liu a, Zhenglin Liang a, Uwe Muenster b, Shirui Mao a,⁎ a b

School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China Chemical and Pharmaceutical Development, Research Center Wuppertal-Aprath, Bayer Pharma AG, Wuppertal 42096, Germany

a r t i c l e

i n f o

Article history: Received 20 August 2016 Received in revised form 13 December 2016 Accepted 13 December 2016 Available online 15 December 2016 Keywords: Chitosan Nanocrystal Spray drying Microparticle Swellable Pulmonary Sustained release

a b s t r a c t In this study, nanocrystals embedded in microparticles were designed to achieve sustained pulmonary drug delivery of hydrophobic drugs. Chitosan based microparticles were engineered to allow sustained drug release via swelling and mucoadhesive properties of the polymer. Taking cinaciguat as a hydrophobic model drug, drug nanocrystals were prepared by high pressure homogenization and then encapsulated in chitosan microparticles via spray drying. Through various in vitro characterizations, it was shown that drug loaded microparticles had a high drug loading with promising aerosolization characteristics (mean volume diameter (Dv50) 3–4 μm, experimental mass mean aerodynamic diameter (MMADe) 4–4.5 μm, fine particle fraction (FPF%) 40–45%, emitted dose (ED%) 94–95%). The microparticles showed high swelling capacity within 5 min, with various sustained drug release rates depending on chitosan concentration and molecular weight. Furthermore, aerosolization performances under various inhalation conditions were investigated. It was found that both inspiratory flow rate and volume had an influence on the aerosolization of developed microparticles, indicating actual inhalation efficiency might be compromised under disease conditions. Taken together, in vitro data indicate that chitosan based swellable microparticles could potentially be useful as nanocrystal carrier to achieve sustained pulmonary delivery. To complete the feasibility assessment of this formulation principle, future in vivo safety and efficacy studies are needed. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The pulmonary route has aroused a great interest during the last few years for the administration of a number of therapeutic molecules, aiming to achieve both local and systemic effects. This attraction of pulmonary drug delivery is due to the numerous advantages over many other delivery routes, including low thickness of the epithelial barrier, extensive vascularization, large alveolar surface area, low enzymatic metabolic activity and the absence of first-pass effect, which are beneficial for drug absorption (Courrier et al., 2002; Lu and Hickey, 2007; Patton and Byron, 2007). Further development of pulmonary sustained or controlled release dosage forms was studied to cut down drug administration frequency, extend drug residence time, maximize drug efficacy and reduce systemic exposure, especially for toxic drugs (Liang et al., 2015; Smyth and Hickey, 2011). However, due to the inherent lung clearance mechanisms of exogenous substances, it is still a challenge to successfully develop respirable carrier systems with adequate aerodynamic properties that can confer ⁎ Corresponding author at: School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, 110016 Shenyang, China. E-mail address: [email protected] (S. Mao).

http://dx.doi.org/10.1016/j.ejps.2016.12.013 0928-0987/© 2016 Elsevier B.V. All rights reserved.

sustained release effect. Generally, particles targeted to the deep lung should be small enough with an aerodynamic diameter of ~ 0.5–5 μm, but should not be too small (b 0.5 μm) otherwise the particles would fail to deposit and be exhaled again (Chow et al., 2007; Grenha et al., 2007; Musante et al., 2002; Telko and Hickey, 2005). The appropriate particle size for inhalation is also the ideal size range for the uptake by alveolar macrophages, therefore, even for hydrophobic drugs, sustained pulmonary drug release can't be achieved despite of their limited dissolution in the lung fluid. Although it was shown that increasing microparticle size could reduce macrophage phagocytosis (Ahsan et al., 2002; Makino et al., 2003), it is unpractical for the objective of pulmonary drug delivery due to the inefficient deposition of fine particle fraction to the targeted region. Therefore, development of particles that can reduce or even escape macrophage phagocytosis and in the meantime have respirable aerodynamic size is the major challenge. Among the several technologies currently under investigation, swellable microparticles are one of the promising strategies to achieve this goal, as this kind of particles is small enough to be inhaled and meanwhile it has swelling capacity of becoming larger in size after deposition upon getting into contact with the lung fluid (El-Sherbiny et al., 2010; El-Sherbiny and Smyth, 2012; Selvam et al., 2011). For the design of swellable microparticles, selection of an appropriate polymer is

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of special importance. Chitosan (CS) is a cationic polysaccharide with many beneficial properties such as biodegradability, low toxicity, low immunogenicity, excellent biocompatibility as well as mucoadhesion and a permeation enhancer (Ding et al., 2012; Hirano et al., 1990; Mao et al., 2010). Chitosan has been used as an absorption enhancer in the nasal mucosa (Mei et al., 2008; Na et al., 2010) and in pulmonary tissues (Jae et al., 2006), attributed to its transient opening of the intercellular tight junctions. A further consideration when delivering particulates for sustained release is the pulmonary mucociliary clearance mechanism which can move particles from the lower lung regions to the throat (Patton and Byron, 2007). Therefore, a suitable mucoadhesive component is essential. Thus, CS is an ideal candidate for sustained pulmonary drug delivery as it can not only act as a drug release modifier but also shows mucoadhesive properties (Dang and Leong, 2006; Harikarnpakdee et al., 2006; Martinac et al., 2005). However, how to incorporate hydrophobic drugs into chitosan microparticles within respirable size is still a big challenge. To guarantee even drug distribution in the microparticles, nanoparticles were prepared and encapsulated (ElSherbiny and Smyth, 2012). Low drug loading and organic solvent remaining are the main shortcomings related to the traditional nanoparticles. With the well-established nanocrystal technology (Jacobs and Müller, 2002; Keck and Müller, 2006; Sun et al., 2011a,b), our hypothesis is that, by encapsulating nanocrystals into chitosan based microparticles, respirable microparticles with sustained pulmonary drug delivery can be obtained. Therefore, in this study, cinaciguat, a nitric oxide (NO)-independent activator of soluble guanylate cyclase for the treatment of pulmonary hypertension, was used as a hydrophobic model drug, and chitosan as matrix, were used to prepare respirable swellable microparticles. The resulting formulations were characterized in terms of particle size, drug loading efficiency, powder crystallinity, water content, morphology, in vitro release, dynamic swelling behavior, powder flowability and in vitro release profile. The in vitro aerodynamic performance was investigated using the next generation impactor (NGI) under various conditions by loading these microparticles in capsules (Vcaps®) and aerosolizing them with a breath-actuated inhaler device (Cyclohaler®). 2. Materials and methods 2.1. Materials Chitosan (Molecular weight 400 kDa, deacetylation degree ≥ 85% and moisture content ≤ 10%) was purchased from Jinan Haidebei Marine Bioengineering Co., Ltd., (China) and degraded as reported previously to get chitosans with molecular weight of ~50 and ~100 kDa (Mao et al., 2004). Cinaciguat (BAY 58-2667) was provided by Bayer Healthcare (Wuppertal, Germany). Polyvinyl pyrrolidone (PVP-k12), Pluronics F68 were donated by International Specialty Products Inc. (USA) and BASF in China respectively. Tweens (20 and 80) of injection grade were from Sigma-Aldrich in China. HPLC grade acetonitrile, dichloromethane (DCM), 1-Methyl-2-pyrrolidone (NMP) and acetic acid were supplied by Shandong Yuwang Co., Ltd. (China). All other reagents, unless otherwise specified, were of analytical grade. 2.2. Preparation of cinaciguat nanosuspension The cinaciguat nanosuspension was prepared by High Pressure Homogenization (HPH) method as described previously (Sun et al., 2011a,b). Briefly, 250 mg of cinaciguat coarse powder were dispersed in 50 mL aqueous solution containing 0.1% (w/v) Pluronic F68. The dispersion was then processed through a high pressure homogenizer AH100D (ATS Engineering Inc., Shanghai, China) at 150 bars for 20 cycles. Thereafter it was homogenized at 300, 600 and 900 bars for 3 cycles respectively followed by 15 cycles at 1200 bars. The exact drug concentration in the nanosuspension was analyzed by HPLC.

2.3. Preparation of cinaciguat nanocrystals embedded in swellable microparticles Cinaciguat loaded swellable microparticles were prepared using spray drying technique. Briefly, 1.5–3.0 g chitosan powder (MW, ~ 50 or ~ 100 kDa) were dissolved in 500 mL diluted acetic acid solution and pH of the final solution was adjusted to 6.0. For drug loading, a specific volume of freshly prepared cinaciguat nanosuspension was added to the chitosan solution. For all the formulations prepared, chitosan concentration and theoretical drug loading (%) were calculated according to the following equation: CS concentration ð%Þ 
 ¼ MCS ðgÞ= Vaqueous solution þ Vdrug nanosuspension ðmLÞ  100%

ð1Þ

Theoretical drug loading ð%Þ 
 ¼ Mcinaciguat ðgÞ= MCS þ Mcinaciguat ðgÞ  100%

ð2Þ

After adding cinaciguat nanosuspension, the whole suspension was subsequently spray-dried using a spray-dryer equipped with a high performance cyclone and a 0.5 mm two-fluid nozzle (SD-1000, Tokyo Rikakikai Co., Ltd., Japan.). The optimized operation conditions and parameters were: inlet temperature 110 °C, atomizing pressure 190 kPa, drying air flow rate 0.7 m3/min and feeding rate 3.0 mL/min. 2.4. Characterization of cinaciguat nanocrystals loaded swellable microparticles 2.4.1. Particle size analysis Particle size of cinaciguat nanosuspension was measured by Malvern Zetasizer Nano (Malvern, UK) and the Z-average value was used to evaluate the size of the nanosuspension and the size distribution was expressed using Polydispersity index (PDI). Particle size distribution of the drug loaded swellable microparticles was measured using a Beckman-Coulter LS 230 particle size analyzer (Beckman-Coulter LS 230, USA). Approximately 20 mg of the dry powder was re-dispersed in absolute alcohol and then measured. The volume mean diameter (Dv) was used to evaluate the geometric diameter of the microparticles. Each sample was measured in triplicate. 2.4.2. Drug loading efficiency determination Actual drug loading efficiency was determined according to the following procedures. Briefly, 10 mg of the swellable microparticles were weighed precisely and 5.0 mL of 1.0% (w/v) acetic acid solution was used to dissolve the chitosan carrier, then N-methylpyrrolidone was added to 50.0 mL (± 0.05 mL) to dissolve cinaciguat nanocrystals. 10.0 μL of the solution was subjected to RP-HPLC analysis on a Phenomenex Gemini column with pre-column (150 mm × 4.6 mm, particle size: 5 μm) at 40.0 °C. Gradient elution method was used. The detection wavelength was 230 nm and drug retention time was in the range of 14.70–14.80 min. 2.4.3. Powder crystallinity analysis Thermodynamic analysis of the particles was performed using differential scanning calorimeter (DSC-1, Mettler-Toledo, Switzerland). Powder samples (~ 3 mg) were weighed and placed in hermetically sealed aluminum pans. The samples were scanned at a heating rate of 10 °C/min from 25 °C to 350 °C in nitrogen atmosphere. The melting temperature was determined from the endothermic peak of the DSC curve recorded. The solid state form of the drug within swellable microparticles was further validated by X-ray powder diffraction (X-RPD) using an X-ray diffractometer (X'pert PRO, PANalytical B.V., The Netherlands) with Cu-Kα radiation generated at 40 mA and 35 kV. Samples were analyzed in a 2θ range of 4.5° to 40° with a step size of 0.033° and a counting time of 0.6 s per step.

R. Ni et al. / European Journal of Pharmaceutical Sciences 99 (2017) 137–146

2.4.4. Moisture content measurement TGA (TGA-50, Shimadzu Corp. Japan) was performed on 10 mg samples in platinum pans using a nitrogen purge at 20 mL/min. The heating temperature was in the range of 25 °C to 150 °C with a heating rate of 10 °C/min. Water content was calculated as the percentage of mass loss. Measurements were performed in triplicate. 2.4.5. Morphology observation Morphological observation of swellable microparticles was performed using a Hitachi S-3400N (Hitachi Ltd., Japan) and a SUPRA 35 field-emission scanning electron microscope (Zeiss, Jena, Germany). A small amount of powder was sprinkled onto double-sided adhesive tape attached to an aluminum stub and was sputter-coated with gold under vacuum. Photographs were taken at varied magnifications with an accelerating voltage of 5 kV to reveal surface characteristics of the swellable microparticles. 2.4.6. In vitro release study ~5.0 mg of drug loaded microparticles were weighed precisely and dispersed in 5.0 mL phosphate buffer (10 mM PBS, pH = 7.4) containing 2.0% (w/v) Tween-80 (sink condition), then continuously agitated (80 rpm) in an air bath (37 ± 0.5 °C). At predetermined time intervals, the samples were taken out and 1.0 mL medium was withdrawn and centrifuged at 8911 × g for 10 min. Drug content in the supernatant was analyzed by HPLC. Meanwhile, equal volume of fresh medium was added for continuous study. The release profiles were regarded significantly different if f2 b 50 (Li et al. 2013) 2.4.7. Dynamic swelling study 20 mg powder was weighed and dispersed in 2.0 mL release media, then incubated under release conditions. Swelling behavior of the microparticles was studied by determining the increase in volumetric diameter (μm) with time using laser diffractometer (Beckman-Coulter LS 230, USA). 2.4.8. Powder flowability evaluation Powder flowability was estimated through Carr's Index measurement. Carr's Index is defined as the relative percentage between bulk (ρb) and tapped density (ρt) (Eq. (3)) stated by US Pharmacopeia 34. Based on the Carr's Index value, powder flowability can be defined as: excellent (b12%); good (12–18%); fair (18–21%); poor, fluid (21– 25%); poor, cohesive (25–32%); very poor (32–38%) and extremely poor (N40%). Carr’s Index ð%Þ ¼ ½ðρt −ρb Þ=ρ t   100%

ð3Þ

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volume diameter in this study; ρ is the mass mean density of the particles, which was provided by tapped density here; ρ0 is a reference density (1 g/mL) and χ is the dynamic shape factor, which is assumed as 1 (for a sphere) (Vanbever et al., 1999). The in vitro aerodynamic particle size distribution of the microparticles based powders was determined using the Next Generation Impactor (NGI) with a stainless steel induction port (i.e. USP throat) and pre-separator attachments according to the procedures described in US Pharmacopeia 34 Chapter b601N. The impactor was equipped with a Copley TPK 2000 critical flow controller, a Copley DFM 2000 flow meter and a Copley HCP5 vacuum pump (Copley Scientific, UK). Prior to measurement, the NGI plates were coated with a thin film to decrease the particle entrainment. Specifically, each plate was coated with 10% Tween-20 ethanol (w/v) solution and ethanol was evaporated under a fuming hood for 1 h. Vcaps® (No.3 HPMC capsule, Capsugel®, China) were used to load the powder samples with the inhaler device Cyclohaler® (Pharbita BV, The Netherlands), which was tightly connected to the NGI equipment. 20.0 mg of powder was weighed precisely and loaded in one capsule and 10 capsules were used for one measurement. The standard flow rate for Cyclohaler® is 100 L/min and the actuation time lasted for 2.4 s per capsule. After actuations, particles intercepted in each of the NGI stages were collected into separate volumetric flasks and dissolved, then analyzed by HPLC. The emitted dose (ED%) is defined as the percent of particles exiting the capsules versus the total loaded powder mass (Eq. (5)). The fine particle fraction (FPF%) is defined as the percent of particles intercepted in inhalable stages versus the total mass of particles intercepted in each component of NGI (Eq. (6)). The experimental mass median aerodynamic diameter (MMADe) is defined as the mass median aerodynamic diameter tested by NGI. According to USP, deriving a plot of cumulative mass percentage of powder intercepted in each stage versus aerodynamic diameter (log scale) of the respective stage, and MMADe of the particles was determined from the graph as the particle size at which the line crosses the 50% mark. The FPF% and MMADe were also directly provided by the bundled software of NGI provided by the Copley Scientific Inc.
ED% ¼ M powder exited from the capsules =Mtotal loaded powder  100%

ð5Þ

  FPF% ¼ M stage 1 through 5 =MtotalðInduction portþPre−separatorþall stagesÞ  100%

ð6Þ

Moreover, powder aerosolization was tested at various flow rates and lower simulated respiratory volumes. For the NGI used in this study, the nominal cut-off aerodynamic diameter of each stage is flow rate dependent, as outlined in the literature (Marple et al., 2004) as

Tapped density was measured by a modified method according to US Pharmacopeia 34 and literature report (Ungaro et al., 2009). Briefly, a known amount of swellable microparticles (approximately 100 mg) was placed in a purpose-made 10 mL (±0.05 mL) graduated cylinder and the initial volume (mL) was recorded. The cylinder was then tapped 500 times onto the work station until a consistent height (volume plateau) was achieved and the final volume (mL) was recorded. Tapped density was expressed as the ratio between sample weight (g) and the volume occupied after tapping (mL). 2.4.9. In vitro aerodynamic performance Theoretical mass mean diameter (MMADt) is defined as the mass mean diameter of spheres of geometric diameter d and density ρ that reaches the same settling velocity as non-spherical particles. And MMADt of the developed microparticles can be calculated using the measured tapped density according to the following equation: MMADt ¼ d  ðρ=ρ0 χÞ1=2 ρ0 ¼ 1g=cm3 ; χ ¼ 1




ð4Þ

where d is the geometric mean diameter, which is expressed as mean

Fig. 1. Particle size and polydispersity index of cinaciguat nanosuspension stabilized by PVP-k12 and Pluronic F68.

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Table 1 The parameters of cinaciguat loaded swellable microparticles. Chitosan MW (kDa)

Concentration (%, w/v)

Inlet temperature (°C)

Dv (μm) (span)

50 50 50 50 100 100 100

0.3 0.4 0.5 0.6 0.5 0.5 0.5

110 110 110 110 90 110 130

3.66 2.71 3.30 3.45 4.02 4.00 4.28

well as described in US Pharmacopeia 34. At a given flow rate Q (30– 100 L/min), the effective cut-off aerodynamic diameters of the various stages can be calculated from reference calibration cut-off diameters (Eq. (7)) at reference flow (Qref) rate 60 L/min. In the case of Pre-separator and Micro-Orifice Collector (MOC), the effective aerodynamic diameters are calibrated using Eqs. (8) and (9). Here, D50 is mass median diameter of the particle size distribution, it is the value of the particle diameter at 50% in the cumulative distribution. And D80 describes the diameter where 80% of the distribution has a smaller particle size and 10% has a larger particle size in the cumulative distribution. D50 ¼ D50;ref  ðQ ref =Q Þx

ð7Þ

± ± ± ± ± ± ±

0.07 (1.24 0.16 (1.02 0.01 (1.13 0.02 (1.22 0.04 (1.42 0.07 (1.41 0.05 (1.51

± ± ± ± ± ± ±

0.03) 0.05) 0.01) 0.01) 0.01) 0.11) 0.02)

Drug loading (%)

Water content (%)

5.15 4.89 5.07 4.94 4.57 4.81 4.90

9.01 ± 0.55 10.62 ± 0.43 9.95 ± 0.46 9.72 ± 0.52 10.20 ± 0.35 9.11 ± 0.27 7.53 ± 0.15

± ± ± ± ± ± ±

0.10 0.01 0.02 0.03 0.02 0.05 0.05

D50 ¼ 12:8−0:07  ðQ −Q ref Þ

ð8Þ

D80 ¼ 0:14  ðQ ref =Q Þ1:36

ð9Þ

2.5. Statistical analysis All the experimental results were depicted as the mean value ± standard deviation (SD) from at least three measurements (unless otherwise specified). Significance of difference was evaluated using oneway ANOVA at a probability level of 0.05.

Fig. 2. The DSC thermograms of cinaciguat loaded swellable microparticles.

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PVP-k12 concentration was increased from 0.1% to 0.3%. In contrast, when Pluronic F68 was used as the stabilizer, the particle size decreased significantly with a narrow size distribution. The particle size did not change significantly within an F68 concentration range of 0.1–0.5% (w/v). Particle size ranged from 424.8 ± 4.66 to 473.2 ± 4.33 nm (Fig. 1). Therefore, a concentration of 0.1% (w/v) F68 was selected as the stabilizer to prepare cinaciguat nanosuspension in the following studies. 3.2. Influence of chitosan concentration

Fig. 3. The X-RPD patterns of cinaciguat loaded swellable microparticles.

3. Results and discussion 3.1. Preparation and characterization of cinaciguat nanosuspension Cinaciguat is practically insoluble in water. To guarantee homogeneous distribution of drug in the microparticles, first of all, nanonization of coarse cinaciguat was conducted by high pressure homogenization. To get a stable nanosuspension, stabilizers are essential (Sun et al., 2011a,b). Taking both safety and system simplicity into consideration, Pluronic F68 and PVP-k12 were selected as the potential stabilizers and the appropriate concentrations were investigated. The results are shown in Fig. 1. When PVP-k12 was used as the stabilizer, the particle size was above 700 nm with poly-distribution (PDI ~ 0.8) even when

By keeping theoretical drug loading at 5%, freshly prepared drug nanosuspension was added to chitosan solution and swellable microparticles were prepared by spray-drying. First of all, taking chitosan 50 kDa as an example, by keeping the inlet temperature at 110 °C, influence of chitosan concentration (0.3, 0.4, 0.5 and 0.6%, w/v) on the properties of the microparticles were investigated and listed in Table 1. No statistical change in geometric particle size, drug loading and water content was found when chitosan 50 kDa concentration increased from 0.3% to 0.6%. The solid state of cinaciguat in the spray dried microparticles was characterized using DSC studies. As shown in Fig. 2, small endothermic peaks were observed at around 160 °C, which were lower than the melting point (170 °C) of cinaciguat alone. This melting point depression can probably be explained by presence of the polymer chitosan. Meanwhile, the microparticles were further analyzed by X-RPD (Fig. 3), it was shown that cinaciguat existed in the crystalline state within the spray dried microparticles. Morphologies of the spray dried microspheres were observed by SEM and shown in Fig. 4(A–D). The powder blend (micro-cinaciguat and inhalable lactose) was presented for comparison (Fig. 4H). No influence of chitosan concentration on the particles appearance was found, with all the particles presenting wrinkled shape. This particle morphology was a typical case that fell into the category of high Peclet number particle formation (Chew et al., 2005; Kim et al., 2002;

Fig. 4. The morphology of swellable microparticles prepared with 50 kDa chitosan and 110 °C (A 0.3%; B 0.4%; C 0.5%; D 0.6%, w/v), 0.5% 100 kDa chitosan (w/v) (E 90 °C; F 110 °C; G 130 °C) and cinaciguat-lactohale powder blend (H).

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formation occurred earlier in the evaporation process. And if the shell did not become rigid quickly, it was then buckled and folded as the inner water evaporated to the outside, with wrinkle surface formed. For inhalation purpose, the particle corrugation is advantageous to improve delivery efficiency by lowering the aerodynamic diameter of the particles caused by decreased particle density (Eq. (4)). Moreover, a corrugated surface could further assist in dispersibility by minimizing contact areas (Vehring, 2008). Influence of chitosan concentration on the in vitro drug release was further studied. As illustrated in Fig. 5, the release rate of cinaciguat nanosuspension was very fast and all the nanocrystals were dissolved in b10 min. Compared to drug suspension, remarkable sustained release was achieved with all the chitosan based microparticles, and the drug release rate decreased with increasing chitosan concentration. It was noted that higher burst release was found at chitosan concentration 0.3% (~60%) and 0.4% (~50%), and no statistical difference in burst release was found between 0.5% and 0.6% chitosan group (~40%). Thereafter, drug release decreased with the increase of polymer concentration. Taking both burst release and system viscosity into consideration, a chitosan concentration of 0.5% was selected for the following studies. Fig. 5. The in vitro release profiles of nano-cinaciguat and cinaciguat loaded swellable microparticles.

3.3. Influence of chitosan molecular weight

Lechuga-Ballesteros et al., 2008). During the spray drying process, for droplets with increasing Peclet numbers, the time to reach the critical concentration for shell formation at the surface decreased, i.e. shell

Keeping chitosan concentration at 0.5% (w/v) and the inlet temperature at 110 °C, the influence of chitosan molecular weight (MW 50 and 100 kDa) on the properties of the spray dried microparticles was

Fig. 6. The in vitro dynamic swelling behavior of cinaciguat nanocrystal loaded chitosan microparticles.

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investigated. The particle size increased slightly with the increase of chitosan MW from 3.30 ± 0.01 μm (50 kDa) to 4.00 ± 0.07 μm (100 kDa, Table 1), which was most likely due to the increased viscosity of the feeding suspension at higher chitosan molecular weight (Rabbani and Seville, 2005). No significant influence of chitosan MW on drug loading, solid state form and microparticle morphology was found (Figs. 2–4). The influence of chitosan MW on drug release profile is shown in Fig. 5. When higher molecular weight chitosan was applied, the retarding effect was significant (f2 b 50). Overall, decreased drug release was found by increasing the chitosan MW. When comparing microparticles containing 50 kDa chitosan and 100 kDa Chitosan, the burst release decreased from approx. 40% (50 kDa) to 15% (100 kDa).

3.4. Influence of inlet temperature As reported, inlet temperature could be an important factor to influence the properties of particles during the spray drying process (Mohajel et al., 2012). Therefore, keeping the chitosan concentration at 0.5% (w/v) and the MW at 100 kDa, the influence of inlet temperature (90, 110 and 130 °C) on the properties of spray dried microparticles was investigated. It was found that the particles prepared at 110 °C and 130 °C had no obvious difference in properties. As shown in Table 1, the particle size increased slightly when the inlet temperature increased from 110 °C to 130 °C, no statistical change in drug loading was found. Under all three conditions, the drug substance stayed in the crystalline state (Figs. 2, 3). Slight particle agglomeration was observed at 90 °C (Fig. 4). The moisture content decreased slightly with the increase of inlet temperature from 90 °C and 110 °C, but the moisture change was significant when the inlet temperature was further increased to 130 °C (from 9.11% to 7.53%). However, this moisture content appeared to be more dependent on polymer itself because chitosan had a water content of no N 10% (w/w). Taking the above properties into consideration, inlet temperature 110 °C was selected for the microparticles preparation.

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3.5. Dynamic swelling behavior Formulations prepared with two different chitosan MW (50 kDa and 100 kDa) at a concentration of 0.5% and an inlet temperature of 110 °C were selected for the swelling study. The evolution of particle size and size distribution along with medium contact time of the microparticles are shown in Fig. 6. It was noted that upon immersion in the release media, swelling of the microparticles was very fast and reflected by the significant change of volume mean diameter values. For chitosan 50 kDa microparticles, the particle size increased from 3.3 μm to 20 μm in 5 min, almost constant particle size of approximately 70 μm was observed in 30 min. As to chitosan 100 kDa microparticles, swelling of the particles was much faster, and the particle size increased from 4.0 μm to 30 μm in 5 min, but after 10 min the particle size kept more or less constant at appro. 50 μm. However, it should be noted that during particle size analysis, the particles for testing were probably contacting and adhering to each other. All these contributed to the errors of the final calculated mean value. Considering the error bars and the broad particle size distribution, there was no significant difference in particle size between the two MW chitosan samples. The swelling results can only qualitatively show the swelling property of chitosan microparticles. Taking chitosan 50 kDa as an example, this change of particle size was also observed by light microscope to provide direct evidence, as presented in Fig. 7. The swelling properties of chitosan microparticles would be beneficial to the objective of sustained release in the lung, because it has been proven that the larger particle size can reduce, or even escape the uptake of lung macrophages (Ahsan et al., 2002; El-Sherbiny and Smyth, 2012; Makino et al., 2003). It was also noted that after long time in vitro incubation (1 h), the particles observed were adhesive and clusters were formed. As reported, the diameter of human bronchi was in the range of 3–5 mm and decreased to 250 μm in average at alveoli region (Patton and Byron, 2007). Therefore, it would not necessarily implicate a clogging risk of human bronchi and alveoli despite the swelling

Fig. 7. The micrographs of chitosan 50 kDa based swellable microparticles versus time (10 × 40).

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equals to 1 only when the particle is a sphere. However, the developed microparticles in this study, as observed from SEM, were particles with many irregular voids and wrinkles on the surface rather than spheres. So the application of “χ = 1” during MMADt calculation might contribute to errors. On the other hand, the particles vibrated and sprayed out from capsules may more or less form aggregates during actuation of the inhaler. Therefore, the actual aerodynamic diameter value was larger, which was more close to the experimental results. Powder flowability was characterized using Carr's index. As shown in Table 2, both swellable microparticles exhibited very poor flow property with Carr's Index 38.82 ± 1.30% and 34.18 ± 2.46% for chitosan 50 kDa and chitosan 100 kDa based microparticles respectively, while the powder blend presented fair flowability as a whole (20.17 ± 3.55%). This result is common for the powders with respirable particle size (0.5–5 μm) when no flow agents or excipients with larger particle size (e.g. inhalable lactose) are added. 3.7. Influence of flow rate and simulated respiratory volume Fig. 8. In vitro aerodynamic diameter distribution of cinaciguat loaded chitosan based swellable microparticles and cinaciguat-lactohale powder blend.

and increased particle size of the microparticles, as indicated in the in vitro simulated conditions. In addition, the liquid lining layer at the cell surface decreased from 8 mm (bronchi) to approximately 70 nm (alveoli) (Patton and Byron, 2007). Moreover, when inhaled, the limited number of particles would be more sufficiently aerosolized and dispersedly deposited in the fluid layer with limited volume, particle inter-adhesion and clusters-forming might not happen in vivo. 3.6. Powder aerosolization property and flowability evaluation The in vitro aerodynamic properties of chitosan 50 kDa and 100 kDa based microparticles were tested by NGI and the results are shown in Fig. 8. For comparison, conventional DPI composed of micro-cinaciguat and inhalable lactose powder blend (micro-cinaciguat/LH300/ LH200 = 5/20/75) was also prepared and tested. As listed in Table 2, both the microparticles and the powder blend showed high emitted dose (~ 93–95%). Compared with the powder blend (MMADe, 4.82 ± 0.08 μm; FPF%, 36.00 ± 1.40%), chitosan-based microparticles presented smaller experimental mass mean aerodynamic diameter (MMADe, 3.91 ± 0.03 and 4.48 ± 0.12 μm for chitosan 50 kDa and 100 kDa, respectively) and higher fine particle fraction (FPF%, 44.90 ± 1.35% and 39.25 ± 2.09% for chitosan 50 kDa and 100 kDa, respectively). As to the influence of chitosan MW, chitosan 50 kDa based microparticles presented smaller MMADe and higher FPF% than the one with chitosan100 kDa, which might be contributed to the smaller Dv value due to its lower viscosity. In addition, tapped density (ρt) and the theoretical mass mean aerodynamic diameter (MMADt) were tested and calculated. Chitosan based swellable microparticles had apparently much lower density than that of the powder blend. This low density was mainly due to its wrinkled morphology as observed in SEM, which is beneficial for particles aerosolization and deposition via dry powder inhalation. It was worth noting that the calculated theoretical mass mean aerodynamic diameter (MMADt), was obviously smaller than the one measured by NGI. This can probably be explained by the fact that, for MMADt calculation, there is a dynamic shape factor (χ) in the theoretical formula, and it

According to the description in US pharmacopeia, the standard testing flow rate for Cyclohaler® is 100 L/min and lasts for 2.4 s. Clinically, patients who suffer from pulmonary hypertension are characterized by increasing dyspnea (short of breath) and difficulties to reach such high flow rate or keep for a few seconds (Giaid and Saleh, 1995). Furthermore, the respiratory volume specified in pharmacopeia is 4.0 L per simulated inhalation. And this value is considered to be the normal forced inhalation capacity of an average sized male weighing approximately 70 k (http://www.copleyscientific.com/files/ww/brochures/ Inhaler%20Testing%20Brochure%202015_Rev4_Low%20Res.pdf, 201608-15). In practice, it is necessary to widen the scope of the test parameters to cover a broader target patient population, such as geriatrics and pediatrics, as well as those already suffering from pulmonary problems, including typical use and unintentional misuse conditions. As specified in the FDA guidelines (http://www.fda.gov/downloads/drugs/ guidancecomplianceregulatoryinformation/guidances/ucm070573.pdf, 2016-08-15), the effect of varying the flow rate at a constant volume and lower simulated respiratory air volume (2.0 L) should be studied for breath-activated drug products or those that are intended to be marketed with an expansion or holding chamber, spacer, or similar component. And these studies can assess the sensitivity of the device to widely varying flow rates generated by patients of different age, gender and with different severity of disease. To keep the specified simulated respiratory volume (4 L) constant, the aerodynamic performance of the spray-dried microparticles was conducted at 100 L/min, 90 L/min, 75 L/min, 60 L/min, 45 L/min, and 30 L/min respectively. Then keeping the flow rate at 100 L/min, lower simulated respiratory volume (2 L) was applied to investigate the aerodynamic particle size distribution of the particles. As shown in Fig. 9A, the different flow rates did have effects on the powder aerosolization when the inspiratory volume was constant. With the flow rate decreasing, a downward trend on the FPF% value was observed, from 39.25 ± 2.09% at 100 L/min to 16.64 ± 2.30% at 30 L/min. This implied that the lower flow rate could not provide sufficient strength to vibrate the capsules loaded in the inhaler, leading to the incomplete dispersion of the particles. Owing to the insufficient powder aerosolization, the emitted dose (ED, %), as presented in Fig. 9B, also showed a downward trend with the decrease of flow rate, from 95.65 ± 0.74% at 100 L/min to 84.09 ± 1.33% at 30 L/min (Fig. 9B). Furthermore, the application of lower simulated

Table 2 Aerodynamic and flow properties of swellable microparticles compared to powder blend. Formulation

ED (%)

MMADe (μm)

FPF%

ρt (g/cm3)

MMADt (μm)

Carr's index (%)

50 kDa CS 100 kDa CS Powder blend

94.15 ± 0.07 95.65 ± 0.74 93.61 ± 0.50

3.91 ± 0.03 4.48 ± 0.12 4.82 ± 0.08

44.90 ± 1.35 39.25 ± 2.09 36.00 ± 1.40

0.278 ± 0.006 0.255 ± 0.010 1.103 ± 0.048

1.82 ± 0.02 2.02 ± 0.04 –

38.82 ± 1.30 34.18 ± 2.46 20.17 ± 3.55

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Fig. 9. Fine particle faction and the emitted dose of chitosan based swellable microparticles (100 kDa) tested under different inspiratory flow rate and volume.

inspiratory volume (2 L) resulted in a reduced fine particle fraction (FPF%, 26.0 ± 43.47%). The aforementioned results indicate that once this dry powder inhalation formulation type was used in practice by patients, actual inhalation efficiency might be compromised. For Cyclohaler®, reduced aerosolization efficiency caused by lower flow rate and simulated respiratory volume was also reported in the literatures (Mohammed et al., 2014; Srichana et al., 1998), however, for a lactose-drug physical mixture formulation type. So far, safety of chitosan itself in nasal and oral formulations has been demonstrated (Ding et al., 2012; Zhang et al., 2015), however, in vivo safety evaluation of the swellable microparticles upon lung delivery requires further in-depth investigations. Especially for the in vivo swelling of these types of particles it needs to be observed if the swelling might lead to clogging of alveoli and/or inflammatory processes within the lung. 4. Conclusions In this study, cinaciguat nanocrystals encapsulated in chitosanbased respirable swellable microparticles were developed and evaluated as a potential carrier system for sustained pulmonary delivery of hydrophobic drugs using DPIs. This newly designed system combines the advantages of nanocrystals and the respirable swellable microparticles while avoiding the shortcomings of the traditional nanoparticles including the complicated preparation process and the risk of remaining organic solvents. A full panel of in-vitro characterization studies showed promising properties of this type of nanocrystal-in-microparticle formulation for sustained pulmonary delivery of hydrophobic drugs. Furthermore, it was found that the formulation principle of swellable microparticles together with the Cyclohaler® was sensitive to the various flow rates and the inspiratory air volumes. However, even though in this work we have shown highly promising in vitro properties of the formulation principle, the potential usefulness for therapy in humans remains to be proven by in vivo efficacy studies as well as by in vivo safety evaluation studies. Acknowledgements This project is financially supported by Bayer Pharma AG (Germany) and the Construction of Innovative Drug Incubation Platform of Liaoning Province (No.8). References Ahsan, F., Rivas, I.P., Khan, M.A., Torres Suarez, A.I., 2002. Targeting to macrophages: role of physicochemical properties of particulate carriers–liposomes and microspheres– on the phagocytosis by macrophages. J. Control. Release 79, 29–40. Chew, N.Y.K., Tang, P., Chan, H.K., Raper, J.A., 2005. How much particle surface corrugation is sufficient to improve aerosol performance powders? Pharm. Res. 22, 148–152. Chow, A., Tong, H.P., Shekunov, B., 2007. Particle engineering for pulmonary drug delivery. Pharm. Res. 24, 411–437.

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