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Inhalable resveratrol microparticles produced by vibrational atomization spray drying for treating pulmonary arterial hypertension
Journal of Drug Delivery Science and Technology 29 (2015) 152e158
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Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Research paper
Inhalable resveratrol microparticles produced by vibrational atomization spray drying for treating pulmonary arterial hypertension Frantiescoli A. Dimer a, *, Manoel Ortiz a, Adriana R. Pohlmann a, b, Silvia S. Guterres a ~o em Ci^ cia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil s-Graduaça Programa de Po encias Farmac^ euticas, Faculdade de Farma ^nica and Programa de Po ~o em Química, Instituto de Química, Universidade Federal do Rio Grande do Sul, Porto s-Graduaça Departamento de Química Orga Alegre, RS, Brazil a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 May 2015 Received in revised form 20 June 2015 Accepted 14 July 2015 Available online 15 July 2015
The rare disorder pulmonary arterial hypertension is characterized by elevated pulmonary arterial pressure and right heart failure. Currently, the treatments have improved, but a cure has not yet been discovered. In this study, we developed a new formulation using an innovative controlled delivery system for resveratrol: s dry powder inhaler form designed for pulmonary administration. The particles were composed of resveratrol, as the therapeutic agent; poly(ε-caprolactone), for structuring the controlled-release matrix; sodium deoxycholate, to prevent agglomeration; and trehalose, as drying adjuvant, respectively. The particles were obtained using the piezoelectric atomization technique with a Nano Spray Dryer B-90®. The fine particle fraction was approximately 50% and the theoretical aerodynamic diameter was 2.32 mm. The process afforded excellent yields (approximately 80%) with low powder moisture (less than 2.0%). Due to the low density and the increased flowability of the powder, the spherical shape of the particles and an irregular surface, the microparticles possessed aerodynamic properties suitable for drug deposition on the bronchial and alveolar regions of the lungs. The aerodynamic properties and in vitro sustained release profiles showed a great potential for the inhaled administration of drugs such as resveratrol suitable for the treatment of pulmonary arterial hypertension. © 2015 Elsevier B.V. All rights reserved.
Keywords: Dry powder inhaler Pulmonary arterial hypertension Resveratrol Spray dryer Vibrational atomization
1. Introduction Pulmonary arterial hypertension (PAH) is characterized by the pressure of the lungs exceeding 25 mm Hg at rest or 30 mm Hg during exercise. The major symptoms of PAH are chest pain, fatigue and shortness of breath. The development of this disorder is associated with deregulation of the vasodilator prostaglandin (PGI2) and the vasoconstrictor thromboxane (TXA2) [1,2]. The treatment for PAHuses single or multidrug therapies, such as anticoagulants, diuretics, calcium channel blockers and prostanoids [3]. However, the use of these treatments does not cure of this disease, but these treatments can retard its progression. Additionally, this conventional treatment approach mainly uses mainly the oral and intravenous routes and can induce important systemic side
s-Graduaça ~o em Cie ^ncias Farmace ^uticas, * Corresponding author. Programa de Po cia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga Faculdade de Farma 2752, Porto Alegre CEP 90610-000, RS, Brazil. E-mail addresses: [email protected] (F.A. Dimer), [email protected] (M. Ortiz), [email protected] (A.R. Pohlmann), [email protected] (S.S. Guterres). http://dx.doi.org/10.1016/j.jddst.2015.07.008 1773-2247/© 2015 Elsevier B.V. All rights reserved.
effects, such as breathlessness, liver damage, nausea, diarrhea and pain, leading to limited treatment adherence by patients [2]. Alternatively, the administration of drugs through the inhalation route is quite interesting because it is possible to obtain increased bioavailability compared to oral administration. In addition, it is patient friendly because it is painless and not as invasive as the intravenous route [4]. The conventional treatments for PAH using pulmonary administration have already been approved in some countries with the use of Iloprost [5]. This drug can reduce the mean pulmonary arterial pressure by up to 20% for less than 1 h. Consequently, more than 10 inhalations per day are required to maintain the effect of Iloprost using nebulizers systems [6]. A potential approach to improve the treatment is to administer the drug loaded within a polymeric carrier to control the release of the drug and to reduce the number of daily administrations [7]. These carriers, such as microparticles, can also impart the drugs with great properties, such as increased bioavailability [8] and drug stability [9], and they can deliver the drugs to specific targets [10]. Using a technology recently developed by Buchi Labortechnik, it is possible to obtain dry powders with an aerodynamic diameter between 1 and 5 mm to be efficiently deposited in the lower
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respiratory tract [11,12]. The Nano Spray Dryer B-90® is a new generation of laboratory-scale spray dryers, and it brings improvements compared to conventional spray dryers, such as better yields and powders with reduced particle sizes [12e15]. The achievement of submicrometric and micrometric particles using the Nano Spray Dryer B-90 was facilitated by the piezoelectric atomization technique, which enables the production of small particles directly from solutions or suspensions with high yields when compared with the atomization process in the conventional spray drying technique [12,16]. This piezoelectric system operates by ultrasonically vibrating a membrane that contains tiny holes, forming a mesh, at a frequency of 60 kHz. Moreover, a micro-pump action is created inside the atomizer, due to the vibration of the membrane, which generates millions of droplets with a narrow particle size distribution [17]. Resveratrol (3,5,40-trans-trihydroxystilbene) is also currently used to treat PAH because it can inhibit the production of monocyte chemoattractant protein-1 (MCP-1) and its secretion in vascular endothelial cells, which is a key intermediary factor that stimulates the infiltration of inflammatory cells into the lungs [18,19]. In addition, this drug which is present in grapes and red wine, has attracted considerable interest from pharmaceutical researchers because of ots excellent metabolic properties, such as its powerful antioxidant, anti-inflammatory, lipid metabolism regulator activities and its ability to prevent cancers activities [20e22]. In this context, this work was focused on the development of polymeric microparticles containing resveratrol intended for pulmonary delivery using the Nano Spray Dryer aiming a controlled released formulation with aerodynamic properties suitable for drug deposition on the lungs. 2. Materials and methods 2.1. Materials ~o Paulo, Brazil), Resveratrol (RSV) was purchased from Deg (Sa poly(ε-caprolactone) (Mw 42.500 g mol1) and sodium deoxycholate were acquired from SigmaeAldrich (Steinheim, Germany). Trehalose was kindly donated by Corn Products (Mogi Guaçu, Brazil). The analytical-grade solvent acetone and the highperformance liquid chromatography (HPLC)-grade solvent methanol were purchased from F. Maia (Cotia, Brazil) and Tedia (Rio de Janeiro, Brazil), respectively. 2.2. Preparation of microparticles Initially, 0.1 g of poly(ε-caprolactone) and 0.1 g of RSV were dissolved in 80 mL of acetone in a single flask at 40 ± 2 C with controlled magnetic stirring at 300 rpm. Separately, 0.02 g of sodium deoxycholate and 0.1 g of trehalose were solubilized in 20 mL of ultrapure water. After both phases were completely dissolved, the aqueous phase was added to the organic phase under moderate magnetic stirring. The suspension was loaded in a Nano Spray Dryer B-90 (Büchi Labortechnik AG, Switzerland), using the following drying parameters: pump mode 2 with 100% spray rate, air flow of 110 L/min, inlet temperature of 55 C and the spray mesh with a pore size of 7.0 mm. The spray dryer was maintained in closed-mode configuration with a residual oxygen level of less than 4%. Resveratrol microparticles (RSV-MP) were collected from the particlecollecting electrode using a soft brush. 2.3. Spray-dried powder characterization Particle size distribution of RSV-MP was determined using the laser diffraction (LD) technique without any prior powder
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treatment using Mastersizer 2000 (Malvern Instruments, Germany) and the small volume wet dispersion unit. The particle size of RSV-MP was measured immediately after the addition of the particles with an obscuration level higher than 2%. The particle size parameters were obtained directly from the software, a follows: D4,3; d0,1; d0,5 and d0,9 representing the volume-weighted mean and lower sizes of 10, 50 and 90% of the particles, respectively. The process yield was calculated from the relation of the resulting powder amount obtained by the initial concentration of solids in the solution. The results were expressed in percentage. Moreover, the residual moisture content of the powders was analyzed using Karl Fischer titration (Mettler Toledo 37, Japan). The bulk (rb) and tapped (rt) densities of the spray-dried powders were determined in triplicate using an automatic tapper (J. Engelshmann, Germany). After recording the bulk density, the tapped density was determined by visual inspection after 1250 taps. This number of taps allowed the density to reach a plateau. An indicator of the flowability of the samples, the Carr's index percentage (CI) was determined using the Equation (1). In addition, the theoretical aerodynamic diameter (daero) of the particles was calculated from the particle size determined by laser diffraction (dgeo) and the tapped density (rt) using the Equation (2) [23].
Carr 0 s Indexð%Þ ¼
daero ¼ dgeo
rt rb 100 rb
sffiffiffiffiffiffiffiffiffiffiffiffi rt r0
(1)
(2)
where, r0 ¼ 1 g cm3 (spheric shape). Morphological analyses of the polymeric microparticles were performed using a scanning electron microscope (SEM) (Zeiss EVO HD15, Germany). Samples of raw resveratrol and RSV-MP were added in aluminum stubs with carbon conductive double-sided tape and sputter-coated with a 15e20 nm layer of gold. The analyses were conducted using an accelerating voltage of 5 kV at magnifications of 2000 and 20,000. The resveratrol content was assayed using high-performance liquid chromatography (HPLC) according to a previously validated method [9]. The chromatographic system consisted of a Discovery C18, 5 mm, 4.6 150 mm column (Supelco, USA) and a PerkineElmer series 200 chromatograph (PerkineElmer, USA). The isocratic mobile phase was composed of methanol:water (50:50, v/ v) at pH 3.0 adjusted with acetic acid, which was pumped at a flow rate of 0.6 mL/min. The sample volume injected was 50 mL, and RSV was detected using a wavelength of 305 nm. The HPLC showed linearity in the range of 0.5e20 mg/mL (R2 ¼ 0.9845). The total amount of RSV from the DPI was extracted from 20 mg of RSV-MP with 10 mL of mobile phase of methanol:water (50:50, v/v) after 10 min of ultrasonic extraction and subsequent centrifugation at 1.500 g. In addition, 250 mL of the samples was diluted using the mobile phase. The samples were filtered (Millipore 0.45 mm, USA) and injected into the HPLC. The crystallinity and the compatibility of resveratrol with other components of the particles were characterized using a differential scanning calorimeter (DSC e TA Instruments, model Q100, USA) equipped with a refrigerated cooling accessory. Nitrogen was used as the purge gas was used at a rate of 10 mL/min. The specimens (3e7 mg) were packed in hermetic aluminum pans and heated from 20 to 300 C at a heating rate of 10 ºC/min under a dry nitrogen atmosphere. The DSC heating curves were analyzed using Universal Analysis 2000 software (TA Instruments, USA).
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2.4. In vitro aerosol performance The in vitro aerosol performance of RSV-MP was investigated using the USP pharmacopeia apparatus 1, called the Andersen cascade impactor (ACI, Type ACI-MDI 1000 Erweka, Germany) [24]. Initially, the inhalation rate generated by the vacuum pump (VP 1000, Erweka, Germany) was adjusted and calibrated to 28.3 ± 1 L/ min during 4 s after five inhalations of empty capsules. The analyses (n ¼ 3) were performed using a gelatin capsule (size n 3) filled with 20 mg of the RSV-MP powder. ACI consists of eight stages, and it is composed of a first part called the throat and eight subsequent stages (0e7). After inhalation using the Aerolizer® (Novartis, Switzerland) as the inhaler device in the mouthpiece adaptor, the powders were impacted into the different stages, according to their aerodynamic diameters [24]. The particles that were retained in the inhalation device, mouthpiece adaptor, throat device and each stage from zero to seven were rinsed into volumetric flasks using the HPLC mobile phase. The amount of RSV in each step was determined using the previously validated HPLC method. The fine particle fraction (FPF; less than 5.8 mm) was calculated from the sum of the stages 2e7 of the ACI. In addition, the mass median aerodynamic diameter (MMAD) was determined. MMAD is defined as the diameter above and below where 50% of the mass of the particles lie. 2.5. In vitro dissolution profile The dissolution of free RSV and the drug release of RSV from RSV-MP formulations (n ¼ 4) were performed using in a Vankel VK 7010 automatic sampling dissolution system (Vankel, United Kingdom). Approximately 30 mg of the RSV-MP powder (containing approximately 9 mg of RSV) or the same amount (9 mg) of bulk Resveratrol (RSV-FREE) was loaded in a gelatin capsule (size n 3). Sink conditions were maintained following the conditions employed: baskets, speed of 75 rpm, 900 mL of an aqueous medium containing 1% of polysorbate 80 and controlled temperature of 37 ± 1 C. Samples (5 mL) were automatically withdrawn at predetermined time points of 5, 10, 15, 20, 25, 30, 45, 60, 120, 240, 720 and 1440 min and filtered through a 0.45 mm filter. Prior to HPLC analysis, the samples were diluted with 5 mL of mobile phase. The apparent kinetic rate constants for both formulations were obtained from the release profiles fitted according to the first-order (C ¼ C0$e kt) mathematical model using the software MicroMath Scientist® 2.0 (Missouri, USA), where k and C0 are the apparent kinetic rate constant and the initial drug concentration at time t (h). Additionally, we used a model-independent model by using the statistical moment of the mean dissolution time (MDT), which is commonly used to describe the in vitro drug release profile for the dissolution of controlled release products [25]. Statistical analysis of the in vitro data was performed via t-test using GraphPad Prism 5 software (GraphPad Software, USA) and a value of p < 0.05 was considered significant. 3. Results and discussion The delivery of drugs to the lungs using the inhalation therapy for the local treatment of diseases could decrease the side effects because lower systemic drug concentrations are reached compared to oral delivery. In this work, microparticles were developed as dry powder inhalers (DPI) systems intended for pulmonary application, which were composed of PCL, sodium deoxycholate and trehalose and loaded with Resveratrol using a Nano Spray Dryer B-90®. Using this new formulation, the aim was to target RSV to the lungs, leading to an increased bioavailability in the tissue, yield and FPF of the particles. Thus, inhalable RSV-MP was prepared by means of
spray drying using the vibrational atomization technique. One of the disadvantages of using Nano Spray Dryer B-90® is the presence of incrustations at the nozzle, and thus, an optimization process is always required [12]. Initially, the drying process used in this work also presented this problem. In order to produce RSV-MP powder formulation without incrustations, the process was optimized by decreasing the inlet temperature from 70 C to 55 C, reducing the solids contents of the feeding solution from 1.0% to 0.3%, and modifying the proportions acetone:water from 50:50 to 80:20 (v/v). After all changes, the RSV-MP powder formulation was prepared without incrustations. For the purposes of this study, a poly(ε-caprolactone) polymer with a medium molecular weight (42.5 kDa) was used as a shell structure to control the drug release rate. This water-insoluble polymer is well known as a matrix for nano- or microparticle formulations because of its biodegradability and biocompatibility properties [26]. Acetone was selected as a solvent due to its high capacity to dissolve both the polymer and the drug. The solvent is a very important as process parameter because it can control the process temperature, thereby influencing the drug loading. Sodium deoxycholate was used as a surfactant to avoid particle agglomeration and to increase the redispersion of the microparticles in the airstream, which also increased the flowability because the liquid/ air interface of the droplets formed during the atomization process prior to drying should preferably be occupied by a surfactant rather than drug or polymers [27]. Trehalose was used as a drying adjuvant. This carbohydrate is readily soluble in water and is widely used in inhalation preparations [28,29]. In addition to the materials used, the integrity of the polymeric particles is dependent of the different steps of the spray drying process: formulation feed and atomization, spray mixture with air drying, solvent evaporation, and dry product separation [30]. One of the mainly problems in the spray drying process is the high aggregation and fusion of the particles resulting from the temperature used being considerably higher than the melting point (Tm). However, the Nano Spray Dryer B-90 uses lower temperatures compared to conventional spray dryers, facilitating the drying process of particles [17]. The inlet temperature used was 55 C and below the Tm of the polymer poly(ε-caprolactone) (60 C) [26]. During the few seconds of the drying step, the temperatures of the atomized droplets and obtained powder remained lower than the temperature of the circulating air. Therefore, it is possible to obtain powders without directly exposing the materials to high temperatures, thereby allowing for the production of particles containing sensitive materials without causing degradation [31]. Moreover, formulation components self-assembled to form the structure of the particles at this stage [32]. Most particles were obtained in the designed inhaled formulation, with particle sizes ranging from 1 to 5 mm for their maximum efficiency deposition in the deepest lung [11,33]. Although particles larger than 5 mm would be deposited in the throat and easily cleared, the smaller particles above 0.5 mm may not be effective due to the Brownian motion in the airstream and can be exhaled during breathing [2,24]. Using the laser diffraction technique, the volumeweighted mean (D4,3) of RSV-MP was determined to be approximately 3.8 mm; additionally 10, 50 and 90% of the particles presented particle sizes of less than 0.7, 2.0 and 9.5 mm, respectively, based on the volume distribution (Fig. 1A). When the number distribution was used, 10, 50 and 90% of the particles presented particle sizes lower than 0.4, 0.6 and 1.1 mm, respectively (Fig. 1B). Therefore, the vast majority of the particles were in the respirable range. The bulk density, tapped density and CI of the RSV-MP spraydried powders are listed in Table 1. The obtained bulk and tapped densities were 0.32 g/cm3 and 0.37 g/cm3, respectively. The
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Fig. 1. Particle size distribution of RSV-MP as measured by laser diffractometry using volume (A) and number distributions (B).
Table 1 Technological parameters of spray-dried powder of resveratrol submicrometric particles. Technological parameter
Value (±SD)
rb (g/cm3) rt (g/cm3)
0.32 0.37 17.29 2.32 79.5 1.84 31.01 46.48 5.22
CI (%) daero (mm) Yield (%) Residual moisture (%) Drug content (%) FPF (%) MMAD (mm)
(0.01) (0.02) (5.41) (0.17) (8.3) (0.25) (0.12) (1.25) (0.3)
rb ¼ bulk density; rt ¼ tap density; CI ¼ Carr Index; daero ¼ aerodynamic diameter; FPF ¼ fine particle fraction; MMAD ¼ mass median aerodynamic diameter.
calculated aerodynamic diameter was approximately 2.3 mm. As previously reported, spray-dried particles with densities of less than 0.4 g/cm3 and with a mean volume diameter of less than 5 mm are suitable for pulmonary administration and would be capable of delivering drugs to the deep lung for providing a prolonged residence time in the alveolar region [34]. The powder flowability directly affects directly the dry powder inhalation performance and can be obtained from the CI value. CI values less than 25% indicates a powder with good flowability, and values greater than 25% indicates cohesive powder characteristics, corresponding to poor flowability [13]. The CI value of RSV-MP was approximately 17%, indicating a fluidity powder. Yields of approximately 50% are typically achieved in spray drying processes; however, a higher yield (approximately 80%) was obtained for RSV-MP (Table 1). The increase in the yield with the Nano Spray Dryer B-90 is due to the electrostatic particle collector rather than to the cyclone collector in the conventional spray drying technique [12]. The electrostatic collection separation method consist of a stainless steel outer cylinder (anode) and a starshaped inner part (cathode). After a high voltage is passed between the electrodes, an electrostatic potential difference is created, which attracts the produced particles. In addition, our research group previously demonstrated that the addition of surfactant facilitates the passage of liquid through the membrane pores and that the use of ionic surfactants promotes a greater attraction of the particles to the collector cylinder, and consequently increasing the process yield [17]. The residual moisture content in RSV-MP was less than 2%, indicating that the drying process was efficient [35], even at the low drying temperature used (55 C). It is well-known that a high moisture content or increased moisture sorption by powders can affect the chemical, physico-chemical and microbial stability of powders [36]. Note that these biodegradable microparticles must
be stored under lower moisture conditions to avoid potential polymer degradation through hydrolysis and agglomeration during storage [36]. The drug content determined by HPLC in the obtained powder was 31.01 ± 0.12%. This value corresponds to 99.24% of the theoretical drug content (312.5 mg/g). Morphological analyses of the dry powders were performed using SEM to verify their morphology and particle size. The inhalable microparticles consisted of mainly spherical particles with an irregular surfaces (Fig. 2A and B), characteristic of amorphous substances obtained by spray drying [37]. The surface asperities can reduce the van der Waals forces between the particles, thereby improving powder flow, fluidization, and dispersibility [38]. Regarding the irregular surface of the particles, this is an important characteristic because it results in an increased superficial area between the airstream and the particles surface, and it can also improve the flowability as well. In addition, submicrometric particles sizes can be observed, supporting the data obtained from the laser diffraction analysis. From the SEM micrograph, It is also possible to observe that the particles are partially agglomerated into a single structure, but these submicrometric particles can readily redisperse in aqueous media. The thermal properties of the submicrometric particles determined via DSC analysis are useful for verifying interactions between components within a range of temperatures. As observed (Fig. 3), the resveratrol melting temperature (268.9 C) is similar to that reported in a previous study (265.9 C) [39]. The DSC curve of RSV-MP did not show characteristic peaks of the drug, polymer or drying adjuvant, suggesting a complex interaction between the components. The aerodynamic behavior of the particles is the main parameter that influences their deposition in the lungs. Whereas the larger particles are deposited mainly in the bronchial region, the smaller particles can reach the deepest parts of the lung (the alveolar region). The size of the microparticles is determined by the formulation and the spray-drying process parameters, such as the solvents used, the concentration of solids and mainly by the aperture size of the spray mesh [14]. In the study of the aerosol properties, about 85% of the RSV in the RSV-MP was emitted from the capsules (Fig. 4). This emitted dose met the European pharmacopoeia specification, which should be greater than 75% of the loaded dose. In addition, the fraction of powder that can be deposited in the lowers parts of the lungs (equivalent to the stages 2e7, FPF) represented by the particle size of the RSV-MP less than 5.8 mm in size (in these experimental parameters) was 46.48 ± 1.25. This FPF value is in accordance with conventional dry powder inhaler formulations [2,33]. Moreover, the MMAD of the RSV-MP obtained using the ACI was 5.22 ± 0.3 mm. This value is slightly greater than the daero possibly due to some individual particles behaving as aggregates after the aerosolization. This specific MMAD is in the ideal range for PAH treatment using the inhalation therapy because
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Fig. 2. Scanning electron microscopy images of resveratrol submicrometric particles at 2000 (A) and 20,000 (B).
Fig. 3. DSC heating curves of: Resveratrol, PCL (poly(ε-caprolactone)), trehalose and RSV-MP (resveratrol microparticles).
the small diameter of the particles ensure the alveolar deposition [3]. Therefore, the microparticles loaded in hard gelatin capsules are suitable for use in commercially available DPIs, such as Aerolizer®, allowing targeted pulmonary administration of resveratrol, and they could represent an effective treatment for PAH. The spherical shape of these inhalable particles is appropriate for
Fig. 4. In vitro inhalation performance of resveratrol microparticles using Andersen cascade impactor. C, T and FPF represent the capsule stage, throat stage and the sum of stages 2e7, respectively. Each point represents the mean ± S.D. (n ¼ 3).
pulmonary delivery because it can improve the impaction and diffusion phenomena's in the respiratory tract. In addition to the particle morphology, the irregular surface of RSV-MP is responsible for the improved aerosol performance of the particles when compared to particles with a smooth surface. With all of these important characteristics associated with the very small and controlled aerodynamic particle size (3 mm) and high FPF, these particles can reach in the deepest part of the lungs, thereby improving the PAH treatment [38]. To evaluate the changes in the solubility and dissolution rate of RSV-MP obtained through the use of the Nano Spray Dryer B-90® compared to the RSV raw material in bulk form, dissolution in vitro studies were conducted using a dissolution system with a basket apparatus (Fig. 5). After 30 min, more than 80% of RSV-FREE had already solubilized in the medium. Moreover, RSV-MP released only 25% in the same time period. The release was controlled up to 720 min, and more than 85% of RSV was released in this period. This drug release of RSV from the microparticles is consistent with other spray-dryed powders for different drugs [21,26]. An empirical firstorder model was used to fit the drug release data to compare both formulations. In this model, the amounts of drug released are directly proportional to the amount remaining in the dosage form following a concentration gradient pattern based on Fick's law [40].
Fig. 5. The in vitro cumulative release profiles of resveratrol microparticles (RSV-MP) and the free drug (RSV-FREE) (n ¼ 4).
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By applying this model, the first-order rate constant (K) and the time at which 50% of the drug is dissolved (T50) were obtained. For RSV-FREE, the values of K and T50 were 3.67 ± 0.67 h1 and 0.19 ± 0.04 h, respectively, whereas for RSV-MP, the values of K and T50 were 0.30 ± 0.04 h1 and 2.34 ± 0.35 h, respectively. Additionally, the MDT values were 0.214 ± 0.03 h and 3.41 ± 0.4 h for RSV-FREE and RSV-MP, respectively. By assessing the release through both dependent models as with the independent model, a significant controlled release of Resveratrol from the microparticles was obtained. The controlled release of RSV-MP represented a suitable dose of the drug at the site of action because the first times with the slight burst released resveratrol, and a sustained dose was released during the evaluated period. Despite the need for further studies on the drug release, it could be possible to administer RSVMP using inhalation once to three times per day for the treatment of PAH, which is considerably much less than the actual treatment. Although the formulation presented many qualities and advantages in vitro, further in vivo studies should be performed to confirm the potential of the developed formulation. 4. Conclusions In this paper, we produced a microtechnology-based powder by vibrational atomization spray-drying capable to control the resveratrol release from the microparticles. This new dry inhalable formulation containing resveratrol was produced with aerodynamic particle properties suitable to deposition in the deep lung. The results open the perspective to further investigation towards the biological evaluation of the new formulation, as well as to apply this microtechnology as a platform to produce other drug-loaded powders intended for pulmonary administration. Conflict of interest The authors declare no competing financial interest. Acknowledgments These studies were supported by the following Brazilian agencies: Conselho Nacional de Desenvolvimento Científico e Tec~o de Aperfeiçoamento de Pessoal de Nível gico (CNPq), Coordenaça nolo ~o de Amparo a Pesquisa do Estado do Superior (Capes) and Fundaça Rio Grande do Sul (FAPERGS). References [1] E. Kassem, T. Humpl, M.K. Friedberg, Prognostic significance of 2-dimensional, M-mode, and Doppler echo indices of right ventricular function in children with pulmonary arterial hypertension, Am. Heart J. 165 (2013) 1024e1031. [2] A. Saigal, W.K. Ng, R.B. Tan, S.Y. Chan, Development of controlled release inhalable polymeric microspheres for treatment of pulmonary hypertension, Int. J. Pharm. 450 (2013) 114e122. , A. Torbicki, R. Barst, P. Dartevelle, S. Haworth, T. Higenbottam, [3] N. Galie H. Olschewski, A. Peacock, G. Pietra, L.J. Rubin, G. Simonneau, Guidelines on diagnosis and treatment of pulmonary arterial hypertension. The task force on diagnosis and treatment of pulmonary arterial hypertension of the European Society of Cardiology, Eur. Heart J. 25 (2004) 2243e2278. [4] M. Smola, T. Vandamme, A. Sokolowski, Nanocarriers as pulmonary drug delivery systems to treat and to diagnose respiratory and non respiratory diseases, Int. J. Nanomed. 3 (2008) 1e19. [5] S.E. Baker, R.H. Hockman, Inhaled iloprost in pulmonary arterial hypertension, Ann. Pharmacother. 39 (2005) 1265e1274. [6] L.J. Rose-Jones, V.V. McLaughlin, Pulmonary hypertension: types and treatments, Curr. Cardiol. Rev. 11 (2015) 73e79. [7] M.B. Chougule, B.K. Padhi, A. Misra, Nano-liposomal dry powder inhaler of amiloride hydrochloride, J. Nanosci. Nanotechnol. 6 (2006) 3001e3009. [8] S. Naikwade, A. Bajaj, P. Gurav, M. Gatne, P. Singh Soni, Development of budesonide microparticles using spray-drying technology for pulmonary administration: design, characterization, in vitro evaluation, and in vivo efficacy study, AAPS PharmSciTech 10 (2009) 993e1012.
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Research paper
Inhalable resveratrol microparticles produced by vibrational atomization spray drying for treating pulmonary arterial hypertension Frantiescoli A. Dimer a, *, Manoel Ortiz a, Adriana R. Pohlmann a, b, Silvia S. Guterres a ~o em Ci^ cia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil s-Graduaça Programa de Po encias Farmac^ euticas, Faculdade de Farma ^nica and Programa de Po ~o em Química, Instituto de Química, Universidade Federal do Rio Grande do Sul, Porto s-Graduaça Departamento de Química Orga Alegre, RS, Brazil a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 May 2015 Received in revised form 20 June 2015 Accepted 14 July 2015 Available online 15 July 2015
The rare disorder pulmonary arterial hypertension is characterized by elevated pulmonary arterial pressure and right heart failure. Currently, the treatments have improved, but a cure has not yet been discovered. In this study, we developed a new formulation using an innovative controlled delivery system for resveratrol: s dry powder inhaler form designed for pulmonary administration. The particles were composed of resveratrol, as the therapeutic agent; poly(ε-caprolactone), for structuring the controlled-release matrix; sodium deoxycholate, to prevent agglomeration; and trehalose, as drying adjuvant, respectively. The particles were obtained using the piezoelectric atomization technique with a Nano Spray Dryer B-90®. The fine particle fraction was approximately 50% and the theoretical aerodynamic diameter was 2.32 mm. The process afforded excellent yields (approximately 80%) with low powder moisture (less than 2.0%). Due to the low density and the increased flowability of the powder, the spherical shape of the particles and an irregular surface, the microparticles possessed aerodynamic properties suitable for drug deposition on the bronchial and alveolar regions of the lungs. The aerodynamic properties and in vitro sustained release profiles showed a great potential for the inhaled administration of drugs such as resveratrol suitable for the treatment of pulmonary arterial hypertension. © 2015 Elsevier B.V. All rights reserved.
Keywords: Dry powder inhaler Pulmonary arterial hypertension Resveratrol Spray dryer Vibrational atomization
1. Introduction Pulmonary arterial hypertension (PAH) is characterized by the pressure of the lungs exceeding 25 mm Hg at rest or 30 mm Hg during exercise. The major symptoms of PAH are chest pain, fatigue and shortness of breath. The development of this disorder is associated with deregulation of the vasodilator prostaglandin (PGI2) and the vasoconstrictor thromboxane (TXA2) [1,2]. The treatment for PAHuses single or multidrug therapies, such as anticoagulants, diuretics, calcium channel blockers and prostanoids [3]. However, the use of these treatments does not cure of this disease, but these treatments can retard its progression. Additionally, this conventional treatment approach mainly uses mainly the oral and intravenous routes and can induce important systemic side
s-Graduaça ~o em Cie ^ncias Farmace ^uticas, * Corresponding author. Programa de Po cia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga Faculdade de Farma 2752, Porto Alegre CEP 90610-000, RS, Brazil. E-mail addresses: [email protected] (F.A. Dimer), [email protected] (M. Ortiz), [email protected] (A.R. Pohlmann), [email protected] (S.S. Guterres). http://dx.doi.org/10.1016/j.jddst.2015.07.008 1773-2247/© 2015 Elsevier B.V. All rights reserved.
effects, such as breathlessness, liver damage, nausea, diarrhea and pain, leading to limited treatment adherence by patients [2]. Alternatively, the administration of drugs through the inhalation route is quite interesting because it is possible to obtain increased bioavailability compared to oral administration. In addition, it is patient friendly because it is painless and not as invasive as the intravenous route [4]. The conventional treatments for PAH using pulmonary administration have already been approved in some countries with the use of Iloprost [5]. This drug can reduce the mean pulmonary arterial pressure by up to 20% for less than 1 h. Consequently, more than 10 inhalations per day are required to maintain the effect of Iloprost using nebulizers systems [6]. A potential approach to improve the treatment is to administer the drug loaded within a polymeric carrier to control the release of the drug and to reduce the number of daily administrations [7]. These carriers, such as microparticles, can also impart the drugs with great properties, such as increased bioavailability [8] and drug stability [9], and they can deliver the drugs to specific targets [10]. Using a technology recently developed by Buchi Labortechnik, it is possible to obtain dry powders with an aerodynamic diameter between 1 and 5 mm to be efficiently deposited in the lower
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respiratory tract [11,12]. The Nano Spray Dryer B-90® is a new generation of laboratory-scale spray dryers, and it brings improvements compared to conventional spray dryers, such as better yields and powders with reduced particle sizes [12e15]. The achievement of submicrometric and micrometric particles using the Nano Spray Dryer B-90 was facilitated by the piezoelectric atomization technique, which enables the production of small particles directly from solutions or suspensions with high yields when compared with the atomization process in the conventional spray drying technique [12,16]. This piezoelectric system operates by ultrasonically vibrating a membrane that contains tiny holes, forming a mesh, at a frequency of 60 kHz. Moreover, a micro-pump action is created inside the atomizer, due to the vibration of the membrane, which generates millions of droplets with a narrow particle size distribution [17]. Resveratrol (3,5,40-trans-trihydroxystilbene) is also currently used to treat PAH because it can inhibit the production of monocyte chemoattractant protein-1 (MCP-1) and its secretion in vascular endothelial cells, which is a key intermediary factor that stimulates the infiltration of inflammatory cells into the lungs [18,19]. In addition, this drug which is present in grapes and red wine, has attracted considerable interest from pharmaceutical researchers because of ots excellent metabolic properties, such as its powerful antioxidant, anti-inflammatory, lipid metabolism regulator activities and its ability to prevent cancers activities [20e22]. In this context, this work was focused on the development of polymeric microparticles containing resveratrol intended for pulmonary delivery using the Nano Spray Dryer aiming a controlled released formulation with aerodynamic properties suitable for drug deposition on the lungs. 2. Materials and methods 2.1. Materials ~o Paulo, Brazil), Resveratrol (RSV) was purchased from Deg (Sa poly(ε-caprolactone) (Mw 42.500 g mol1) and sodium deoxycholate were acquired from SigmaeAldrich (Steinheim, Germany). Trehalose was kindly donated by Corn Products (Mogi Guaçu, Brazil). The analytical-grade solvent acetone and the highperformance liquid chromatography (HPLC)-grade solvent methanol were purchased from F. Maia (Cotia, Brazil) and Tedia (Rio de Janeiro, Brazil), respectively. 2.2. Preparation of microparticles Initially, 0.1 g of poly(ε-caprolactone) and 0.1 g of RSV were dissolved in 80 mL of acetone in a single flask at 40 ± 2 C with controlled magnetic stirring at 300 rpm. Separately, 0.02 g of sodium deoxycholate and 0.1 g of trehalose were solubilized in 20 mL of ultrapure water. After both phases were completely dissolved, the aqueous phase was added to the organic phase under moderate magnetic stirring. The suspension was loaded in a Nano Spray Dryer B-90 (Büchi Labortechnik AG, Switzerland), using the following drying parameters: pump mode 2 with 100% spray rate, air flow of 110 L/min, inlet temperature of 55 C and the spray mesh with a pore size of 7.0 mm. The spray dryer was maintained in closed-mode configuration with a residual oxygen level of less than 4%. Resveratrol microparticles (RSV-MP) were collected from the particlecollecting electrode using a soft brush. 2.3. Spray-dried powder characterization Particle size distribution of RSV-MP was determined using the laser diffraction (LD) technique without any prior powder
153
treatment using Mastersizer 2000 (Malvern Instruments, Germany) and the small volume wet dispersion unit. The particle size of RSV-MP was measured immediately after the addition of the particles with an obscuration level higher than 2%. The particle size parameters were obtained directly from the software, a follows: D4,3; d0,1; d0,5 and d0,9 representing the volume-weighted mean and lower sizes of 10, 50 and 90% of the particles, respectively. The process yield was calculated from the relation of the resulting powder amount obtained by the initial concentration of solids in the solution. The results were expressed in percentage. Moreover, the residual moisture content of the powders was analyzed using Karl Fischer titration (Mettler Toledo 37, Japan). The bulk (rb) and tapped (rt) densities of the spray-dried powders were determined in triplicate using an automatic tapper (J. Engelshmann, Germany). After recording the bulk density, the tapped density was determined by visual inspection after 1250 taps. This number of taps allowed the density to reach a plateau. An indicator of the flowability of the samples, the Carr's index percentage (CI) was determined using the Equation (1). In addition, the theoretical aerodynamic diameter (daero) of the particles was calculated from the particle size determined by laser diffraction (dgeo) and the tapped density (rt) using the Equation (2) [23].
Carr 0 s Indexð%Þ ¼
daero ¼ dgeo
rt rb 100 rb
sffiffiffiffiffiffiffiffiffiffiffiffi rt r0
(1)
(2)
where, r0 ¼ 1 g cm3 (spheric shape). Morphological analyses of the polymeric microparticles were performed using a scanning electron microscope (SEM) (Zeiss EVO HD15, Germany). Samples of raw resveratrol and RSV-MP were added in aluminum stubs with carbon conductive double-sided tape and sputter-coated with a 15e20 nm layer of gold. The analyses were conducted using an accelerating voltage of 5 kV at magnifications of 2000 and 20,000. The resveratrol content was assayed using high-performance liquid chromatography (HPLC) according to a previously validated method [9]. The chromatographic system consisted of a Discovery C18, 5 mm, 4.6 150 mm column (Supelco, USA) and a PerkineElmer series 200 chromatograph (PerkineElmer, USA). The isocratic mobile phase was composed of methanol:water (50:50, v/ v) at pH 3.0 adjusted with acetic acid, which was pumped at a flow rate of 0.6 mL/min. The sample volume injected was 50 mL, and RSV was detected using a wavelength of 305 nm. The HPLC showed linearity in the range of 0.5e20 mg/mL (R2 ¼ 0.9845). The total amount of RSV from the DPI was extracted from 20 mg of RSV-MP with 10 mL of mobile phase of methanol:water (50:50, v/v) after 10 min of ultrasonic extraction and subsequent centrifugation at 1.500 g. In addition, 250 mL of the samples was diluted using the mobile phase. The samples were filtered (Millipore 0.45 mm, USA) and injected into the HPLC. The crystallinity and the compatibility of resveratrol with other components of the particles were characterized using a differential scanning calorimeter (DSC e TA Instruments, model Q100, USA) equipped with a refrigerated cooling accessory. Nitrogen was used as the purge gas was used at a rate of 10 mL/min. The specimens (3e7 mg) were packed in hermetic aluminum pans and heated from 20 to 300 C at a heating rate of 10 ºC/min under a dry nitrogen atmosphere. The DSC heating curves were analyzed using Universal Analysis 2000 software (TA Instruments, USA).
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2.4. In vitro aerosol performance The in vitro aerosol performance of RSV-MP was investigated using the USP pharmacopeia apparatus 1, called the Andersen cascade impactor (ACI, Type ACI-MDI 1000 Erweka, Germany) [24]. Initially, the inhalation rate generated by the vacuum pump (VP 1000, Erweka, Germany) was adjusted and calibrated to 28.3 ± 1 L/ min during 4 s after five inhalations of empty capsules. The analyses (n ¼ 3) were performed using a gelatin capsule (size n 3) filled with 20 mg of the RSV-MP powder. ACI consists of eight stages, and it is composed of a first part called the throat and eight subsequent stages (0e7). After inhalation using the Aerolizer® (Novartis, Switzerland) as the inhaler device in the mouthpiece adaptor, the powders were impacted into the different stages, according to their aerodynamic diameters [24]. The particles that were retained in the inhalation device, mouthpiece adaptor, throat device and each stage from zero to seven were rinsed into volumetric flasks using the HPLC mobile phase. The amount of RSV in each step was determined using the previously validated HPLC method. The fine particle fraction (FPF; less than 5.8 mm) was calculated from the sum of the stages 2e7 of the ACI. In addition, the mass median aerodynamic diameter (MMAD) was determined. MMAD is defined as the diameter above and below where 50% of the mass of the particles lie. 2.5. In vitro dissolution profile The dissolution of free RSV and the drug release of RSV from RSV-MP formulations (n ¼ 4) were performed using in a Vankel VK 7010 automatic sampling dissolution system (Vankel, United Kingdom). Approximately 30 mg of the RSV-MP powder (containing approximately 9 mg of RSV) or the same amount (9 mg) of bulk Resveratrol (RSV-FREE) was loaded in a gelatin capsule (size n 3). Sink conditions were maintained following the conditions employed: baskets, speed of 75 rpm, 900 mL of an aqueous medium containing 1% of polysorbate 80 and controlled temperature of 37 ± 1 C. Samples (5 mL) were automatically withdrawn at predetermined time points of 5, 10, 15, 20, 25, 30, 45, 60, 120, 240, 720 and 1440 min and filtered through a 0.45 mm filter. Prior to HPLC analysis, the samples were diluted with 5 mL of mobile phase. The apparent kinetic rate constants for both formulations were obtained from the release profiles fitted according to the first-order (C ¼ C0$e kt) mathematical model using the software MicroMath Scientist® 2.0 (Missouri, USA), where k and C0 are the apparent kinetic rate constant and the initial drug concentration at time t (h). Additionally, we used a model-independent model by using the statistical moment of the mean dissolution time (MDT), which is commonly used to describe the in vitro drug release profile for the dissolution of controlled release products [25]. Statistical analysis of the in vitro data was performed via t-test using GraphPad Prism 5 software (GraphPad Software, USA) and a value of p < 0.05 was considered significant. 3. Results and discussion The delivery of drugs to the lungs using the inhalation therapy for the local treatment of diseases could decrease the side effects because lower systemic drug concentrations are reached compared to oral delivery. In this work, microparticles were developed as dry powder inhalers (DPI) systems intended for pulmonary application, which were composed of PCL, sodium deoxycholate and trehalose and loaded with Resveratrol using a Nano Spray Dryer B-90®. Using this new formulation, the aim was to target RSV to the lungs, leading to an increased bioavailability in the tissue, yield and FPF of the particles. Thus, inhalable RSV-MP was prepared by means of
spray drying using the vibrational atomization technique. One of the disadvantages of using Nano Spray Dryer B-90® is the presence of incrustations at the nozzle, and thus, an optimization process is always required [12]. Initially, the drying process used in this work also presented this problem. In order to produce RSV-MP powder formulation without incrustations, the process was optimized by decreasing the inlet temperature from 70 C to 55 C, reducing the solids contents of the feeding solution from 1.0% to 0.3%, and modifying the proportions acetone:water from 50:50 to 80:20 (v/v). After all changes, the RSV-MP powder formulation was prepared without incrustations. For the purposes of this study, a poly(ε-caprolactone) polymer with a medium molecular weight (42.5 kDa) was used as a shell structure to control the drug release rate. This water-insoluble polymer is well known as a matrix for nano- or microparticle formulations because of its biodegradability and biocompatibility properties [26]. Acetone was selected as a solvent due to its high capacity to dissolve both the polymer and the drug. The solvent is a very important as process parameter because it can control the process temperature, thereby influencing the drug loading. Sodium deoxycholate was used as a surfactant to avoid particle agglomeration and to increase the redispersion of the microparticles in the airstream, which also increased the flowability because the liquid/ air interface of the droplets formed during the atomization process prior to drying should preferably be occupied by a surfactant rather than drug or polymers [27]. Trehalose was used as a drying adjuvant. This carbohydrate is readily soluble in water and is widely used in inhalation preparations [28,29]. In addition to the materials used, the integrity of the polymeric particles is dependent of the different steps of the spray drying process: formulation feed and atomization, spray mixture with air drying, solvent evaporation, and dry product separation [30]. One of the mainly problems in the spray drying process is the high aggregation and fusion of the particles resulting from the temperature used being considerably higher than the melting point (Tm). However, the Nano Spray Dryer B-90 uses lower temperatures compared to conventional spray dryers, facilitating the drying process of particles [17]. The inlet temperature used was 55 C and below the Tm of the polymer poly(ε-caprolactone) (60 C) [26]. During the few seconds of the drying step, the temperatures of the atomized droplets and obtained powder remained lower than the temperature of the circulating air. Therefore, it is possible to obtain powders without directly exposing the materials to high temperatures, thereby allowing for the production of particles containing sensitive materials without causing degradation [31]. Moreover, formulation components self-assembled to form the structure of the particles at this stage [32]. Most particles were obtained in the designed inhaled formulation, with particle sizes ranging from 1 to 5 mm for their maximum efficiency deposition in the deepest lung [11,33]. Although particles larger than 5 mm would be deposited in the throat and easily cleared, the smaller particles above 0.5 mm may not be effective due to the Brownian motion in the airstream and can be exhaled during breathing [2,24]. Using the laser diffraction technique, the volumeweighted mean (D4,3) of RSV-MP was determined to be approximately 3.8 mm; additionally 10, 50 and 90% of the particles presented particle sizes of less than 0.7, 2.0 and 9.5 mm, respectively, based on the volume distribution (Fig. 1A). When the number distribution was used, 10, 50 and 90% of the particles presented particle sizes lower than 0.4, 0.6 and 1.1 mm, respectively (Fig. 1B). Therefore, the vast majority of the particles were in the respirable range. The bulk density, tapped density and CI of the RSV-MP spraydried powders are listed in Table 1. The obtained bulk and tapped densities were 0.32 g/cm3 and 0.37 g/cm3, respectively. The
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Fig. 1. Particle size distribution of RSV-MP as measured by laser diffractometry using volume (A) and number distributions (B).
Table 1 Technological parameters of spray-dried powder of resveratrol submicrometric particles. Technological parameter
Value (±SD)
rb (g/cm3) rt (g/cm3)
0.32 0.37 17.29 2.32 79.5 1.84 31.01 46.48 5.22
CI (%) daero (mm) Yield (%) Residual moisture (%) Drug content (%) FPF (%) MMAD (mm)
(0.01) (0.02) (5.41) (0.17) (8.3) (0.25) (0.12) (1.25) (0.3)
rb ¼ bulk density; rt ¼ tap density; CI ¼ Carr Index; daero ¼ aerodynamic diameter; FPF ¼ fine particle fraction; MMAD ¼ mass median aerodynamic diameter.
calculated aerodynamic diameter was approximately 2.3 mm. As previously reported, spray-dried particles with densities of less than 0.4 g/cm3 and with a mean volume diameter of less than 5 mm are suitable for pulmonary administration and would be capable of delivering drugs to the deep lung for providing a prolonged residence time in the alveolar region [34]. The powder flowability directly affects directly the dry powder inhalation performance and can be obtained from the CI value. CI values less than 25% indicates a powder with good flowability, and values greater than 25% indicates cohesive powder characteristics, corresponding to poor flowability [13]. The CI value of RSV-MP was approximately 17%, indicating a fluidity powder. Yields of approximately 50% are typically achieved in spray drying processes; however, a higher yield (approximately 80%) was obtained for RSV-MP (Table 1). The increase in the yield with the Nano Spray Dryer B-90 is due to the electrostatic particle collector rather than to the cyclone collector in the conventional spray drying technique [12]. The electrostatic collection separation method consist of a stainless steel outer cylinder (anode) and a starshaped inner part (cathode). After a high voltage is passed between the electrodes, an electrostatic potential difference is created, which attracts the produced particles. In addition, our research group previously demonstrated that the addition of surfactant facilitates the passage of liquid through the membrane pores and that the use of ionic surfactants promotes a greater attraction of the particles to the collector cylinder, and consequently increasing the process yield [17]. The residual moisture content in RSV-MP was less than 2%, indicating that the drying process was efficient [35], even at the low drying temperature used (55 C). It is well-known that a high moisture content or increased moisture sorption by powders can affect the chemical, physico-chemical and microbial stability of powders [36]. Note that these biodegradable microparticles must
be stored under lower moisture conditions to avoid potential polymer degradation through hydrolysis and agglomeration during storage [36]. The drug content determined by HPLC in the obtained powder was 31.01 ± 0.12%. This value corresponds to 99.24% of the theoretical drug content (312.5 mg/g). Morphological analyses of the dry powders were performed using SEM to verify their morphology and particle size. The inhalable microparticles consisted of mainly spherical particles with an irregular surfaces (Fig. 2A and B), characteristic of amorphous substances obtained by spray drying [37]. The surface asperities can reduce the van der Waals forces between the particles, thereby improving powder flow, fluidization, and dispersibility [38]. Regarding the irregular surface of the particles, this is an important characteristic because it results in an increased superficial area between the airstream and the particles surface, and it can also improve the flowability as well. In addition, submicrometric particles sizes can be observed, supporting the data obtained from the laser diffraction analysis. From the SEM micrograph, It is also possible to observe that the particles are partially agglomerated into a single structure, but these submicrometric particles can readily redisperse in aqueous media. The thermal properties of the submicrometric particles determined via DSC analysis are useful for verifying interactions between components within a range of temperatures. As observed (Fig. 3), the resveratrol melting temperature (268.9 C) is similar to that reported in a previous study (265.9 C) [39]. The DSC curve of RSV-MP did not show characteristic peaks of the drug, polymer or drying adjuvant, suggesting a complex interaction between the components. The aerodynamic behavior of the particles is the main parameter that influences their deposition in the lungs. Whereas the larger particles are deposited mainly in the bronchial region, the smaller particles can reach the deepest parts of the lung (the alveolar region). The size of the microparticles is determined by the formulation and the spray-drying process parameters, such as the solvents used, the concentration of solids and mainly by the aperture size of the spray mesh [14]. In the study of the aerosol properties, about 85% of the RSV in the RSV-MP was emitted from the capsules (Fig. 4). This emitted dose met the European pharmacopoeia specification, which should be greater than 75% of the loaded dose. In addition, the fraction of powder that can be deposited in the lowers parts of the lungs (equivalent to the stages 2e7, FPF) represented by the particle size of the RSV-MP less than 5.8 mm in size (in these experimental parameters) was 46.48 ± 1.25. This FPF value is in accordance with conventional dry powder inhaler formulations [2,33]. Moreover, the MMAD of the RSV-MP obtained using the ACI was 5.22 ± 0.3 mm. This value is slightly greater than the daero possibly due to some individual particles behaving as aggregates after the aerosolization. This specific MMAD is in the ideal range for PAH treatment using the inhalation therapy because
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Fig. 2. Scanning electron microscopy images of resveratrol submicrometric particles at 2000 (A) and 20,000 (B).
Fig. 3. DSC heating curves of: Resveratrol, PCL (poly(ε-caprolactone)), trehalose and RSV-MP (resveratrol microparticles).
the small diameter of the particles ensure the alveolar deposition [3]. Therefore, the microparticles loaded in hard gelatin capsules are suitable for use in commercially available DPIs, such as Aerolizer®, allowing targeted pulmonary administration of resveratrol, and they could represent an effective treatment for PAH. The spherical shape of these inhalable particles is appropriate for
Fig. 4. In vitro inhalation performance of resveratrol microparticles using Andersen cascade impactor. C, T and FPF represent the capsule stage, throat stage and the sum of stages 2e7, respectively. Each point represents the mean ± S.D. (n ¼ 3).
pulmonary delivery because it can improve the impaction and diffusion phenomena's in the respiratory tract. In addition to the particle morphology, the irregular surface of RSV-MP is responsible for the improved aerosol performance of the particles when compared to particles with a smooth surface. With all of these important characteristics associated with the very small and controlled aerodynamic particle size (3 mm) and high FPF, these particles can reach in the deepest part of the lungs, thereby improving the PAH treatment [38]. To evaluate the changes in the solubility and dissolution rate of RSV-MP obtained through the use of the Nano Spray Dryer B-90® compared to the RSV raw material in bulk form, dissolution in vitro studies were conducted using a dissolution system with a basket apparatus (Fig. 5). After 30 min, more than 80% of RSV-FREE had already solubilized in the medium. Moreover, RSV-MP released only 25% in the same time period. The release was controlled up to 720 min, and more than 85% of RSV was released in this period. This drug release of RSV from the microparticles is consistent with other spray-dryed powders for different drugs [21,26]. An empirical firstorder model was used to fit the drug release data to compare both formulations. In this model, the amounts of drug released are directly proportional to the amount remaining in the dosage form following a concentration gradient pattern based on Fick's law [40].
Fig. 5. The in vitro cumulative release profiles of resveratrol microparticles (RSV-MP) and the free drug (RSV-FREE) (n ¼ 4).
F.A. Dimer et al. / Journal of Drug Delivery Science and Technology 29 (2015) 152e158
By applying this model, the first-order rate constant (K) and the time at which 50% of the drug is dissolved (T50) were obtained. For RSV-FREE, the values of K and T50 were 3.67 ± 0.67 h1 and 0.19 ± 0.04 h, respectively, whereas for RSV-MP, the values of K and T50 were 0.30 ± 0.04 h1 and 2.34 ± 0.35 h, respectively. Additionally, the MDT values were 0.214 ± 0.03 h and 3.41 ± 0.4 h for RSV-FREE and RSV-MP, respectively. By assessing the release through both dependent models as with the independent model, a significant controlled release of Resveratrol from the microparticles was obtained. The controlled release of RSV-MP represented a suitable dose of the drug at the site of action because the first times with the slight burst released resveratrol, and a sustained dose was released during the evaluated period. Despite the need for further studies on the drug release, it could be possible to administer RSVMP using inhalation once to three times per day for the treatment of PAH, which is considerably much less than the actual treatment. Although the formulation presented many qualities and advantages in vitro, further in vivo studies should be performed to confirm the potential of the developed formulation. 4. Conclusions In this paper, we produced a microtechnology-based powder by vibrational atomization spray-drying capable to control the resveratrol release from the microparticles. This new dry inhalable formulation containing resveratrol was produced with aerodynamic particle properties suitable to deposition in the deep lung. The results open the perspective to further investigation towards the biological evaluation of the new formulation, as well as to apply this microtechnology as a platform to produce other drug-loaded powders intended for pulmonary administration. Conflict of interest The authors declare no competing financial interest. Acknowledgments These studies were supported by the following Brazilian agencies: Conselho Nacional de Desenvolvimento Científico e Tec~o de Aperfeiçoamento de Pessoal de Nível gico (CNPq), Coordenaça nolo ~o de Amparo a Pesquisa do Estado do Superior (Capes) and Fundaça Rio Grande do Sul (FAPERGS). References [1] E. Kassem, T. Humpl, M.K. Friedberg, Prognostic significance of 2-dimensional, M-mode, and Doppler echo indices of right ventricular function in children with pulmonary arterial hypertension, Am. Heart J. 165 (2013) 1024e1031. [2] A. Saigal, W.K. Ng, R.B. Tan, S.Y. Chan, Development of controlled release inhalable polymeric microspheres for treatment of pulmonary hypertension, Int. J. Pharm. 450 (2013) 114e122. , A. Torbicki, R. Barst, P. Dartevelle, S. Haworth, T. Higenbottam, [3] N. Galie H. Olschewski, A. Peacock, G. Pietra, L.J. Rubin, G. Simonneau, Guidelines on diagnosis and treatment of pulmonary arterial hypertension. The task force on diagnosis and treatment of pulmonary arterial hypertension of the European Society of Cardiology, Eur. Heart J. 25 (2004) 2243e2278. [4] M. Smola, T. Vandamme, A. Sokolowski, Nanocarriers as pulmonary drug delivery systems to treat and to diagnose respiratory and non respiratory diseases, Int. J. Nanomed. 3 (2008) 1e19. [5] S.E. Baker, R.H. Hockman, Inhaled iloprost in pulmonary arterial hypertension, Ann. Pharmacother. 39 (2005) 1265e1274. [6] L.J. Rose-Jones, V.V. McLaughlin, Pulmonary hypertension: types and treatments, Curr. Cardiol. Rev. 11 (2015) 73e79. [7] M.B. Chougule, B.K. Padhi, A. Misra, Nano-liposomal dry powder inhaler of amiloride hydrochloride, J. Nanosci. Nanotechnol. 6 (2006) 3001e3009. [8] S. Naikwade, A. Bajaj, P. Gurav, M. Gatne, P. Singh Soni, Development of budesonide microparticles using spray-drying technology for pulmonary administration: design, characterization, in vitro evaluation, and in vivo efficacy study, AAPS PharmSciTech 10 (2009) 993e1012.
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