Article pubs.acs.org/molecularpharmaceutics
Controlled Dissolution of Griseofulvin Solid Dispersions from Electrosprayed Enteric Polymer Micromatrix Particles: Physicochemical Characterization and in Vitro Evaluation Jorma Roine,*,† Martti Kaasalainen,† Markus Peurla,‡ Alexandra Correia,§ Francisca Araújo,§,∥,⊥ Hélder A. Santos,§ Matti Murtomaa,† and Jarno Salonen† †
Department of Physics and Astronomy, University of Turku, FI-20014 Turku, Finland Laboratory of Electron Microscopy, University of Turku, FI-20014 Turku, Finland § Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland ∥ INEBInstituto de Engenharia Biomédica, University of Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal ⊥ ICBASInstituto Ciências Biomédicas Abel Salazar, University of Porto, Rua de Jorge Viterbo Ferreira, 4050-313 Porto, Portugal ‡
S Supporting Information *
ABSTRACT: The oral bioavailability of a poorly watersoluble drug is often inadequate for the desired therapeutic effect. The bioavailability can be improved by enhancing the physicochemical properties of the drug (e.g., dissolution rate, permeation across the gastrointestinal tract). Other approach include shielding the drug from the gastric metabolism and targeted drug release to obtain optimal drug absorption. In this study, a poorly water-soluble model drug, griseofulvin, was encapsulated as disordered solid dispersions into Eudragit L 100-55 enteric polymer micromatrix particles, which were produced by electrospraying. Similar micromatrix particles were also produced with griseofulvin-loaded thermally oxidized mesoporous silicon (TOPSi) nanoparticles dispersed to the polymer micromatrices. The in vitro drug dissolution at pH 1.2 and 6.8, and permeation at pH 7.4 across Caco-2/HT29 cell monolayers from the micromatrix particles, were investigated. The micromatrix particles were found to be gastro-resistant, while at pH 6.8 the griseofulvin was released very rapidly in a fastdissolving form. Compared to free griseofulvin, the permeability of encapsulated griseofulvin across the intestinal cell monolayers was greatly improved, particularly for the TOPSi-doped micromatrix particles. The griseofulvin solid dispersions were stable during storage for 6 months at accelerated conditions. Overall, the method developed here could prove to be a useful oral drug delivery solution for improving the bioavailability of poorly water-soluble or otherwise problematic drugs. KEYWORDS: solid dispersion, electrospraying, porous silicon, drug dissolution, drug permeability, enteric release, improved bioavailability
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INTRODUCTION Oral drug administration is often the preferred method for delivering therapeutic molecules to the circulatory system due to the relative comfort, cost effectiveness, and ease of dosage control. Unfortunately, most promising new drug candidates suffer from poor physicochemical and/or pharmacokinetic properties, drastically limiting the drug’s bioavailability when administered orally. Such disadvantageous drug properties include low solubility and slow dissolution rate in the intestinal lumen, poor permeation of the gastrointestinal (GI) tract wall, and high first-pass metabolism.1−4 The principal drug absorption site in the GI tract is drug specific, but most of the absorption generally takes place in the upper and middle parts of the small intestine, the duodenum and the jejunum.5,6 In general, the dissolution rate of an amorphous drug is higher than that of its crystalline forms.7−9 Hence, the drug dissolution rate is frequently improved not only by increasing © 2015 American Chemical Society
the specific surface area but also by producing amorphous drug particles using techniques such as milling, spray-drying, or microemulsifying.10−13 An amorphous drug can also provide a higher apparent solubility through supersaturation.14 For easily crystallizing drugs, a sustainable amorphous state can be achieved, for example, by spatial confinement of the amorphous drug inside the pores of a mesoporous material, or by the formation of stable amorphous co-precipitates with suitable glass-forming materials.7,11,15−20 Griseofulvin is a biopharmaceutics classification system class II drug that has a very high tendency to crystallize, even at room temperature and without the presence of moisture.21 For Received: Revised: Accepted: Published: 2254
November 26, 2014 June 1, 2015 June 2, 2015 June 2, 2015 DOI: 10.1021/mp500787b Mol. Pharmaceutics 2015, 12, 2254−2264
Article
Molecular Pharmaceutics instance, in a previous study where griseofulvin was spray-dried, an improvement in the drug dissolution rate was accomplished by decreasing the drug particle size and adding wetting surfactants, but the produced particles were crystalline.22 Different polymers have been investigated for their capabilities in stabilizing griseofulvin molecular dispersions to prevent drug recrystallization, with varying results.15,23 Enteric polymers are applicable materials for site-specific drug delivery applications that are aimed for drug release targeting in the small intestine. They are insoluble in acidic solutions and dissolve usually above pH 5−6, depending on the polymer.24 These kinds of materials are capable of protecting the carried drug from the harsh conditions of the stomach while also acting as drug state stabilizing binders until the drug release conditions are reached.25 At the same time, the gastric walls are protected from possible drug-induced side effects, such as irritation or erosion.26 In this context, the stabilization of amorphous or molecular solid drug dispersions embedded in the polymer matrix can increase the drug dissolution rate and/ or apparent solubility substantially.7,11,14,15,25 Moreover, enteric polymers can act as wetting enhancers for any embedded hydrophobic drugs, which further improves the drug dissolution rate.15 These properties of the polymer help to achieve maximal drug concentration in the small intestine. Electrospraying, or liquid atomization by electrostatic forces, is considered an effective route to nanotechnology.27,28 The produced droplets can be extremely small and are selfdispersing due to their electric charge.27,29 The droplet size can be controlled by the electrospraying parameters, such as liquid formulation, flow rate, and the voltage used, and the method can be used for the production of monodisperse microand nanoparticles, thin films, and micro- or nanocapsules.27,29−31 Amorphous or crystalline drug particles can be produced with this method, and the crystallinity can be adjusted, to a certain extent, by modifying the electrospraying parameters.32,33 Even novel drug crystal polymorphs have been produced by electrospraying.34 Additionally, in an increasing number of pharmaceutical research papers, single-capillary and parallel or coaxial dual-capillary electrospraying, electroencapsulation (e.g., encapsulation or embedding by electrospraying), and electrospinning systems have been utilized in various novel drug delivery applications, including the production of micro- and nanosized as well as nanostructured drug carrier particles, capsules, fibers, and emulsions for oral, inhalation, and topical drug delivery.27,35−42 Mesoporous silicon (PSi) has been studied extensively as a promising candidate for an orally administrable drug carrier.16−19,43−54 PSi nanoparticles can be made biodegradable, and drugs can be loaded into the pores of PSi at room temperature.16,17,44,47,48 The surface chemistry of PSi can be adjusted to favor adsorption of certain drug molecules or cells, and to control the drug release rate.16,17,43,49,50,54 When a sufficiently small PSi pore size is chosen, the pore walls may spatially prevent the loaded drug molecules from crystallizing, retaining the drug at least partially in an amorphous form, which is advantageous for improving the dissolution rate of many poorly water-soluble drugs, including griseofulvin.7,16−19 Furthermore, the drug dissolution rate can benefit from the wetting properties of PSi.17 The use of drug-loaded PSi nanoparticles has also been found to improve the drug permeation across Caco-2 and Caco-2/HT29 intestinal-derived cell monolayers in certain cases.45,52,53
Electroencapsulation of drug-loaded PSi nanoparticles into enteric polymer micromatrix particles has been previously proposed to be a feasible one-step method to shield the drugloaded PSi nanoparticles from digestive metabolism and to control the dissolution of the loaded drug for oral administration.35 The micromatrix particles, in the order of tens of micrometers in size, also effectively act as packaging units for the PSi nanoparticle solid dispersions.35 Therefore, as an additional benefit, the dry handling and dosing of PSi nanoparticles were possible without any of the problematic agglomeration or formulation issues that generally arise when processing sub-micrometer particles.35,55−57 However, the in vitro drug release and dissolution properties, the in vitro drug permeation properties, and the crystallinity and stability of the drug solid dispersions encapsulated in the micromatrix particles have not been investigated previously, and they remain to be shown. For the present work, griseofulvin was chosen as a model drug for electroencapsulation because of its poor water solubility and high tendency to crystallize. The aim of the work was to study the in vitro release, dissolution, and permeation properties of griseofulvin for the first time from two types of novel micromatrix particles produced by electrospraying: (1) griseofulvin-Eudragit (GE) micromatrix particles, where the griseofulvin was encapsulated as solid dispersions directly into the enteric polymer micromatrices; and (2) griseofulvin-TOPSi-Eudragit (GPE) micromatrix particles, with griseofulvin-loaded thermally oxidized porous silicon (TOPSi) nanoparticles additionally dispersed to the polymer micromatrices. The GE-type micromatrix particles have not been previously produced using the electroencapsulation method. The upper GI tract conditions were simulated for the drug release and dissolution experiments. The permeability experiments were conducted using Caco-2/HT29 cell monolayers at simulated small intestine conditions. The crystallinity and stability of the griseofulvin solid dispersions encapsulated in the micromatrix particles were also studied. Characterization of the micromatrix particles was done by optical microscopy imaging, transmission electron microscopy (TEM) imaging, and X-ray diffraction (XRD).
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MATERIALS AND METHODS Materials. Enteric methacrylate copolymer Eudragit L 10055, with a dissolution threshold pH of 5.5, was received from Evonik Industries (Germany). Griseofulvin was purchased from Sigma-Aldrich (USA), and talc was received from Ciba Specialty Chemicals Oy (Finland). Analytical-grade glycerol (99.9% purity) was obtained from MP Biomedicals (Solon, OH). Dichloromethane (DCM) was obtained from Merck KGaA (Germany) and ethanol (99.5 vol %) from Altia (Finland). Phosphate-buffered saline (PBS) was prepared by dissolving 1.063 g (7.81 mmol) of KH2PO4, 2.789 g (12.21 mmol) of K2HPO4·3H2O, and 8.506 g (0.146 mol) of NaCl in 1000 mL of deionized water. Prior to finalizing the buffer volume, the pH was adjusted to 6.80 ± 0.02 by adding a small amount of 0.10 M HCl. The final phosphate molar concentration was 0.02 M. The KH2PO4, NaCl, and KI salts were obtained from Merck KGaA (Germany), and the deionized water was produced using a Direct-Q 5 UV system (Merck Millipore, USA). HCl (2 M) was obtained from Oy FFChemicals Ab (Finland) and diluted using deionized water. The HCl used for the dissolution experiments was adjusted to pH 1.19 ± 0.01 by diluting to a concentration of 0.043 M. 2255
DOI: 10.1021/mp500787b Mol. Pharmaceutics 2015, 12, 2254−2264
Article
Molecular Pharmaceutics
talc, except for the smallest particles, was sedimented to the bottom of the liquid dispensers before the electrospraying. Glycerol was used as a plasticizer to control charging during the collection. The electrosprayed liquid formulations are presented in Table S1 in the Supporting Information. The produced micromatrix particle batches were consistent, and the electrospraying parameter variations within the applied ranges were considered to have no significant effect on the drug dissolution or permeation properties of the micromatrix particles, as the form and ordering of the drug particles and TOPSi particles in the polymer matrices were not dependent on the morphology or size of individual micromatrix particles. Particle Size Distribution Measurements. The micromatrix particles were imaged using an Olympus BH-2 (Japan) optical microscope and a mounted Canon Powershot A630 (Japan) camera. The micromatrix particle size distributions were determined by measuring the projection area A of individual particles from the microscope images, which was done using ImageJ 1.47v software (Wayne Rasband, NIH, USA). Particle size d was then calculated for each particle from d = 2(A/π)1/2, as the diameter of a circle with an area equal to A. Transmission Electron Microscopy (TEM) Imaging. A JEM-1400Plus (Jeol, Japan) microscope was used for imaging the PE type micromatrix particles with no griseofulvin. The sample particles were embedded directly in Epon resin, which was sliced into 70 nm thick sheets and imaged. X-ray Diffraction (XRD). A PANalytical (Netherlands) X’Pert Pro MPD diffractometer, equipped with a PW3050/60 goniometer and a PIXcel multichannel detector, was used in the XRD measurements. Cu Kα radiation with a wavelength λ = 1.541874 Å was used. The primary beam was collimated with a 0.04 rad soller slit, a 0.25° divergence slit, and a 10 mm mask. A 7.5 mm receiving slit and a 0.04 rad soller slit were used in the diffracted beam side. The measured 2θ angle range was 6.3°− 34.0°, with a scanning speed of 0.026°/s per channel. The powder samples were prepared on Cu sample holders. In Vitro Drug Dissolution Experiments. The dissolution media corresponding to the gastric and small intestine pH conditions (HCl, pH 1.2; and PBS, pH 6.8) and three samples (free griseofulvin, GE micromatrix particles, and GPE micromatrix particles) were used to study the dissolution of griseofulvin in all six possible combinations. The dissolution experiments were conducted using 50 mL glass containers as dissolution vessels, each containing 25.0 mL of dissolution medium. Magnetic stirring bars (35 mm) were placed inside the vessels, and a constant stirring rate of 100 rpm and T = 37 ± 2 °C were maintained during the experiments. The sample griseofulvin masses mgr were 25 ± 1 μg, with a larger mass variation in the free griseofulvin samples. This corresponds to a concentration of 1.0 μg/mL at 100% dissolution, satisfying the sink conditions, as the aqueous solubility of griseofulvin is at least 8.9 μg/mL.58,59 At predetermined time points (15, 30, 60, 90, 120, 180, and 240 min) after the sample was introduced, a 500 μL aliquot of the release medium solution was withdrawn from the dissolution vessel and compensated with an equal volume of fresh medium. All of the experiments were performed in triplicate. The aliquots were centrifuged for 5 min at 17000g using a Thermo (Germany) MicroCL 17 centrifuge. The griseofulvin concentration of each aliquot was measured from 400 μL of the supernatant, using a Labrox (Finland) UV spectrophotometer and Greiner UV-Star (Germany) microplates. The aliquot
Thermally oxidized porous silicon (TOPSi) nanoparticles, with a hydrodynamic diameter (Z-average) of 163.0 nm and polydispersity index (PDI) of 0.094, were produced using a previously reported method.51 The TOPSi nanoparticle size distribution is shown in Figure S1 in the Supporting Information. The Si wafers used for the TOPSi nanoparticle production were obtained from Siegert Wafer GmbH (Germany). Drug Loading into TOPSi Nanoparticles. Griseofulvin was loaded into the pores of the TOPSi nanoparticles by dispersing the particles in a DCM loading solution, with a griseofulvin concentration of 150 mg/mL. The loading solution was kept at a temperature T = 30 °C and stirred gently for 40− 96 h. After loading, the solution was centrifuged, and the supernatant was removed as completely as possible. The griseofulvin loading degree to the TOPSi nanoparticles was defined as the ratio of loaded drug mass to the total mass of loaded particles. The loading degree was estimated from the griseofulvin dissolution in ethanol from dried TOPSi nanoparticles after 24 h of vigorous stirring. The loading degree varied between 3.2% and 7.2%, as measured by ultraviolet (UV) spectrophotometry using a wavelength of λ = 292 nm. Micromatrix Particle Production by Electrospraying. The utilized electrospraying setup consisted of a dual-capillary electrospraying device, integrated to the lid of the electrospraying chamber. The chamber height was 1.04 m, and the inner diameter was 0.21 m. For the production of each micromatrix particle batch, identical liquid was electrosprayed from the oppositely charged capillaries in the stable cone-jet mode, using equal liquid flow rates of 0.8−1.2 mL/h, and oppositely signed atomization voltages with equal absolute magnitudes in the range of 3.4−3.9 kV. The jets dispersed due to the repulsive Coulomb forces between the droplets within the same jet, while the droplets of neighboring jets were mutually attracted. Whenever contact occurred with an oppositely charged droplet, the droplets were neutralized partially or completely due to charge transfer. Sufficiently neutralized droplets fell freely onto the collection dish at the open bottom of the chamber. The chamber was heated to promote the evaporation of excess solvents from the droplets during the fall. The collection efficiency, i.e., the ratio of the collected micromatrix particle mass to the total dry mass introduced to the system, was on average 15%. The electrospraying setup and process have been described in more detail in a previous work.35 Two types of micromatrix particles were produced by electrospraying for the dissolution and permeation experiments: GE micromatrix particles, with the griseofulvin solid dispersions embedded in the enteric polymer matrices, and GPE micromatrix particles, with griseofulvin-loaded TOPSi nanoparticles additionally dispersed to the polymer matrices. The griseofulvin mass proportions were confirmed to be 1.0% and 2.0% for these GE and GPE micromatrix particles, respectively, as measured by UV spectrophotometry after the dissolution of the micromatrix particles to ethanol. In addition, a single GE micromatrix sample with a griseofulvin content of 5.0% by mass was produced for XRD measurements only. Finally, TOPSi-Eudragit (PE) micromatrix particles, with no griseofulvin, were produced exclusively for TEM imaging. Ethanol was used as the base solvent for all of the electrosprayed liquids, with an Eudragit L 100-55 concentration of 30.0 mg/mL. Ground talc (particle size
