Development of Oromucosal Dosage Forms by Combining

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Article pubs.acs.org/molecularpharmaceutics

Development of Oromucosal Dosage Forms by Combining Electrospinning and Inkjet Printing Mirja Palo,*,†,‡ Karin Kogermann,† Ivo Laidmaë ,† Andres Meos,† Maren Preis,‡ Jyrki Heinam ̈ ak̈ i,† ‡ and Niklas Sandler †

Institute of Pharmacy, Faculty of Medicine, University of Tartu, Nooruse 1, EE-50411 Tartu, Estonia Pharmaceutical Sciences Laboratory, Faculty of Science and Engineering, Åbo Akademi University, Tykistökatu 6A, FI-20520 Turku, Finland

‡

ABSTRACT: Printing technology has been shown to enable flexible fabrication of solid dosage forms for personalized drug therapy. Several methods can be applied for tailoring the properties of the printed pharmaceuticals. In this study, the use of electrospun fibrous substrates in the fabrication of inkjetprinted dosage forms was investigated. A single-drug formulation with lidocaine hydrochloride (LH) and a combination drug system containing LH and piroxicam (PRX) for oromucosal administration were prepared. The LH was deposited on the electrospun and cross-linked gelatin substrates by inkjet printing, whereas PRX was incorporated within the substrate fibers during electrospinning. The solid state analysis of the electrospun substrates showed that PRX was in an amorphous state within the fibers. Furthermore, the results indicated the entrapment and solidification of the dissolved LH within the fibrous gelatin matrix. The printed drug amount (2−3 mg) was in good correlation with the theoretical dose calculated based on the printing parameters. However, a noticeable degradation of the printed LH was detected after a few months. An immediate release (over 85% drug release after 8 min) of both drugs from the printed dosage forms was observed. In conclusion, the prepared electrospun gelatin scaffolds were shown to be suitable substrates for inkjet printing of oromucosal formulations. The combination of electrospinning and inkjet printing allowed the preparation of a dual drug system. KEYWORDS: cross-linked gelatin, electrospinning, inkjet printing, lidocaine hydrochloride, oromucosal drug delivery, piroxicam

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suitable approach.3 Depending on the properties of the carrier material, the drug release from the substrate could be controlled and the risk for drug−drug interactions decreased. Printing technology has been successfully used for printing different APIs on various biocompatible substrates, e.g., commercially available sheets8,11 or solvent-cast films8,12 manufactured in a laboratory scale. Mucoadhesive oral films are typically prepared by hot melt extrusion or solvent casting.13,14 Both these conventional methods are suitable for preparing drug-loaded film formulations. However, it has been shown that the incorporation of an API within the polymer film affects the mechanical properties of the dosage form.15,16 Therefore, other alternative techniques to produce orally administrable carrier systems have been of interest. In addition, the physical, chemical, and mechanical properties of these matrices need to be optimized for the fabrication of inkjetprinted dosage forms.3,8,17

INTRODUCTION In recent decades, the preparation of drug formulations by means of inkjet printing has been recognized to be advantageous in patient-oriented dosage form design.1−4 In principle, the printed formulations consist of a drug-containing ink that is deposited in single or multiple layers on a substrate according to a digitally designed pattern. Excellent reviews have been published on recent studies that have shown the suitability of inkjet printing for the fabrication of dosage forms for oral drug delivery.5−7 Furthermore, the dosing flexibility of printed pharmaceuticals has been examined to meet the individual needs of patients.2,8,9 The preparation of combination drug systems by inkjet printing has not been thoroughly investigated to date,5 whereas Khaled et al.10 demonstrated recently that tablets containing three different drugs with separately defined release mechanisms can be successfully prepared by threedimensional (3D) printing. Printed formulations with multiple components can be prepared by a layer-by-layer or a multicompartmental approach that enables adjustment of the dose of the printed components as well as the physicochemical properties and drug release profiles of the active pharmaceutical ingredient(s) (APIs).4,7,10 Furthermore, in formulations containing two APIs the use of drug-loaded substrates can be a © XXXX American Chemical Society

Received: November 21, 2016 Revised: January 21, 2017 Accepted: January 24, 2017

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DOI: 10.1021/acs.molpharmaceut.6b01054 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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providing additional flexibility for the fabrication of printed dosage forms containing multiple APIs.

Electrospinning is a method for the preparation of fine fibers as well as fibrous mats from polymer solutions or melts using electrical forces.18−20 The main advantages of electrospun nano- and microscale fibers for biomedical and pharmaceutical applications include the applicability of a wide range of biocompatible and nontoxic materials, large surface area to volume ratio, high drug-loading capacity, ease of functionalization, and potential for the optimization of porosity, fiber size, and shape.18−21 The main challenges of electrospinning involve the throughput limitation of production and the randomness of the process that causes challenges in ensuring the uniformity of the produced fiber mats.19−21 Consequently, from the dosage form development point of view, this contributes to the errors caused by the cutting of the final dosage forms. The latter drawback has also been recognized for solvent-cast films.15 Electrospun scaffolds containing APIs in noncrystalline form have been successfully used in intraoral dosage forms.22,23 Furthermore, due to their high surface area and high porosity, electrospun substrate scaffolds are well-suited for printing purposes.3,19 The use of porous substrates can improve the stability of metastable forms of APIs by physical stabilization within the substrate structure.17 The use of porous substrates helps to avoid problems with increased surface roughness, increases the ease of handling, and improves patient compliance. Previously, Planchette et al.11 have shown that both hydrophilic and hydrophobic nonporous orodispersible films exhibit a decrease in surface smoothness after inkjet printing due to the formation of cavities or protuberances, respectively. Despite these issues, they could reduce the surface roughness of the printed dosage forms by adding a hydrophilic coating on the hydrophobic substrate prior to printing. Electrospinning has been shown to be applicable with many natural and synthetic polymers and their combinations.20 The mechanical strength and elasticity of the fiber mats is foremost dependent on the selection of polymers and their aftertreatment.18,24 Gelatin is a natural polymer that is obtained from the alkaline or acidic hydrolysis of collagen. Gelatin is a generally recognized as safe (GRAS) food ingredient, and it is also listed in the United States Food and Drug Administration’s (FDA) Inactive Ingredient Guide. In pharmaceutical dosage forms, it is mainly used for the preparation of hard or soft capsules, but it also has applications in tableting, coating, granulation, and encapsulation processes.25 Gelatin is a hydrogel-type (muco)adhesive polymer, i.e., it requires moisture for adhesion.26 Previously, buccal films with gelatin have been prepared for oromucosal drug delivery.27,28 Despite its good film forming ability, the high viscosity of gelatin during processing limits its applicability in film formulations.13 At the same time, gelatin has been used for electrospinning of fiber mats alone,29−31 in combination32−34 with other polymers and/ or after post-treatment by cross-linking.24,35−39 These kinds of electrospun gelatin fibers have been formulated for tissue engineering and wound healing purposes. The aim of this study was to present a novel approach for the development of printed dosage forms by combining electrospinning and inkjet printing technologies. Single and drug combination dosage forms containing lidocaine hydrochloride (LH) and piroxicam (PRX) as model APIs were prepared for oromucosal drug delivery. In this work, the solid state, mechanical, and drug release properties of the electrospun fibrous substrates and the printed dosage forms were characterized. The presented approach can be beneficial in

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EXPERIMENTAL SECTION Materials. Lidocaine hydrochloride (LH) monohydrate (≥99%, Sigma-Aldrich, India) and piroxicam (PRX) as an anhydrous form I (Letco Medical Inc., U.S.) were used as model APIs. Gelatin type A from porcine skin (Sigma-Aldrich, U.S.), D-(+)-glucose (≥99.5%, Sigma-Aldrich, U.S.), acetic acid (99.8%, Lach-Ner s.r.o., Czech Republic), dimethylformamide (DMF) (Šostkinskii factory of Chemical Reactives, Ukraine), and purified water were used in the electrospinning process. The inkbase solution for inkjet printing was prepared from propylene glycol (PG) (≥99.5%, Sigma-Aldrich, Germany) and purified water (Milli-Q). Electrospinning of Fibrous Gelatin Substrates. Three different fibrous gelatin substrates were prepared by electrospinning (ESR-200Rseries, eS-robot, NanoNC, South Korea) (Table 1). Glucose was added to the electrospinning solutions Table 1. Composition of Electrospinning Solutions for the Preparation of the Fibrous Gelatin Substratesa G25 gelatin glucose PRX solvent(s) a

G20

25% (w/v) 3.75% (w/v) 10 M acetic acid aq soln

G20-PRX

20% (w/v) 3% (w/v) 16 mg/mL 10 M acetic acid aq soln and DMF in 1:4 (v/v) ratio

PRX, piroxicam; DMF, dimethylformamide.

to enhance the cross-linking capacity of gelatin.37,38 All the solutions for electrospinning were obtained by vigorous stirring at elevated temperature (approximately 40−50 °C). The electrospinning was performed at ambient conditions (temperature of 25 ± 1 °C and relative humidity (RH) of 20 ± 2%). The G25 gelatin substrates (Table 1) were prepared according to a previously published protocol.37 An automatic syringe pump was used with a pumping rate of 8 μL/min and a high voltage of 17−18 kV applied to a metallic syringe needle (23 gauge). The distance between the spinneret and the grounded metal collector plate was approximately 14 cm. The fibrous G25 substrates were cross-linked through the heat treatment in a heating chamber at 170−175 °C for 3 h. For the preparation of G20 and G20-PRX gelatin substrates the composition of the electrospinning solution (Table 1) and the electrospinning conditions were modified. A melt electrospinning setup equipped with an oil circulator (NNC-OCB200, NanoNC, South Korea) was used for keeping the solution temperature at 50 °C to maintain a suitable viscosity for electrospinning. The pumping rate of 25−30 μL/min, high voltage of 14 kV, and an approximate distance of 18 cm between the spinneret and the collector were used. The fibrous G20 and G20-PRX substrates were cross-linked at 130 °C for 3 h. The theoretical concentration of PRX in the final G20-PRX substrate was 6.5% (w/w). After the preparation, the substrates were packed in aluminum foil and stored at ambient conditions (temperature of 25 ± 1 °C and RH of 20 ± 2%). Inkjet Printing. The printed dosage forms were prepared with a PixDro LP50 piezoelectric inkjet printer (Roth&Rau, The Netherlands) equipped with a Spectra SL-128 AA printhead (nozzle diameter of 50 μm). LH ink solution with B

DOI: 10.1021/acs.molpharmaceut.6b01054 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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were recorded during the constant penetration by the probe until the sample broke apart. The tensile test was conducted by fixing the samples with self-tightening roller grips (A/TGT). A constant distance between the grips was 22 mm (initial sample height). The length and width of each sample were measured with a digital caliper (Absolute Digimatic, Mitutoyo Corp., Kawasaki, Japan) to calculate the cross-section area (mm2). During the measurement a tensile speed of 0.1 mm/s with a triggering force of 0.01 N was applied and the tensile strength (N/mm2) and elongation at break (%) were recorded. Furthermore, the elastic modulus (kPa) was calculated as the slope of the initial linear portion of the stress−strain curve. All the measurements were carried out at ambient conditions (temperature of 25 ± 1 °C and RH of 20 ± 2%). A digital caliper (Ironside, France) was used to measure the thickness of the cross-linked substrates prior to printing (n = 60). Differential Scanning Calorimetry. Thermal analysis of the samples was performed by Q2000 differential scanning calorimetry (DSC) (TA Instruments, U.S.). The samples in Tzero aluminum pans covered with crimped Tzero lids were heated from 0 to 300 °C at a rate of 10 °C/min. Additional measurements with modulated temperature DSC (MT-DSC) mode were also performed. In the MT-DSC measurements the samples were heated from −25 to 300 °C at an average rate of 3 °C/min with a modulation amplitude of ±1 °C over a period of 60 s. Prior to MT-DSC analysis, the samples were conditioned at RH of 20 ± 2% and ambient temperature (25 ± 1 °C) for 48 h. Nitrogen was used as purge gas with a flow rate of 50 mL/min. Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy. The infrared spectra of the electrospun gelatin substrates and the printed dosage forms were obtained by attenuated total reflectance Fourier transform infrared (ATRFTIR) spectroscopy (SpectrumTwo, PerkinElmer, U.K.). The spectroscope was equipped with a DiComp crystal composed of a diamond ATR and a ZnSe focusing element. The measurements were performed in a spectral range from 450 to 4000 cm−1 with 4 accumulations and a resolution of 4 cm−1. A force of 140−150 N was applied on the samples during the measurements. Reference materials were measured with a force of 90−100 N. A Spectrum 10.03 software (PerkinElmer, U.K.) was used for data collection and pretreatment with baseline correction and normalization. X-ray Diffraction. X-ray diffraction (XRD) patterns were obtained by a D8 Advance X-ray diffractometer (Bruker AXS GmbH, Germany) equipped with a LynxEye one-dimensional detector (Bruker, Germany). The measurements were carried out in θ/2θ Bragg−Brentano geometry with Cu Kα radiation (λ = 1.54 Å) at 40 mA and 40 kV. The powder samples were measured in a diffraction range from 5 to 40° 2θ with a step size of 0.02° 2θ and a total measuring time of 83 s/step. The fibrous samples were measured from 3 to 55° 2θ angles with a step size of 0.02° 2θ and a total measuring time of 166 s/step. The International Centre for Diffraction Data (ICDD, U.S.) PDF-2 database (2013 edition) was used for the verification of the raw materials of LH monohydrate, PRX anhydrous form I, and D-(+)-glucose. Experimental results of the samples containing PRX were also compared to the theoretical patterns of PRX anhydrous form I (BIYSEH), PRX anhydrous form II (BIYSEH02), PRX monohydrate (CIDYAP01) from the

a concentration of 350 mg/mL was obtained by dissolving LH monohydrate in a 40:60 (v/v) solvent mixture of PG and purified water (inkbase). The printing was performed on the cross-linked G25 or G20-PRX gelatin substrates with a resolution of 500 droplets per inch (dpi) on a printed area of 2 cm2 in two layers. The first printed layer was dried for 2 h at 25 ± 1 °C before applying the second layer. The theoretical dose of printed LH was calculated based on the droplet volume (pL), printing resolution (dpi), printed area (cm2), number of printed layers, and the LH ink concentration (mg/mL). All the printed preparations were stored at ambient conditions (temperature of 25 ± 1 °C and RH of 20 ± 2%) for a short stability study of 4 and/or 8 months. Preparation of Physical Mixtures. Corresponding physical mixtures (PM) of the raw materials were prepared according to the composition of the electrospun substrates and the printed formulations (Table 1). The PM of G25 and G20 contained 87% of gelatin and 13% of glucose. The PM of G20PRX contained 81.3% of gelatin, 12.2% of glucose, and 6.5% of PRX anhydrous form I. The LH monohydrate concentration in the PM of G25-LH and G20-PRX-LH was 32.6% and 28.0%, respectively. Characterization Methods. Scanning Electron Microscopy. Scanning electron microscopy (SEM) (Zeiss EVO MA 15, Germany) was used to visualize the surface topography and morphology of the fibrous gelatin substrates and the printed dosage forms. Before scanning, the samples were sputter-coated with platinum in an argon atmosphere. The SEM images were obtained in high vacuum mode at a high voltage of 20.00 kV using 3000× and 10000× magnifications. An ImageJ 1.49 V software (National Institutes of Health, U.S.) was used to measure the fiber diameters from the SEM images (n = 100). Moisture Uptake. The behavior of the non-cross-linked and cross-linked substrates at high humidity (RH of 70%) in ambient temperature (25 ± 1 °C) was analyzed by a weighing method (APX-200 balance, Denver Instrument, U.S.). Prior to the initial weighing, the substrates were placed on glass slides and kept at RH of 20 ± 2% in a desiccator for 48 h, after which they were transferred to a desiccator with high humidity (RH of 70%). The difference between the initial substrate mass and the substrate mass after 2 and 25 days was recorded (n = 3). Texture Analysis and Substrate Thickness. The mechanical properties of the substrates and the printed dosage forms were measured with TA.XTplus Texture Analyzer (Stable Micro Systems Ltd., Godalming, U.K.) equipped with a 10 kg load cell (sensitivity of 0.001 N). Copy paper (80 g/cm2, Staples Europe B.V., Finland) and 3-ply tissue paper (Kleenex, Kimberly-Clark Worldwide Inc., Kent, U.K.) were used as reference materials. Two methodspuncture and tensile testwere applied to study the physical properties of the fibrous gelatin substrates. Both methods are known to be suitable for measuring the mechanical strength of various film formulations, whereas tensile testing is one of the most widely used methods for determining the strength of electrospun fibers.13,20,40,41 For the puncture test, a film support rig (HDP/FSR) was exploited to fix the samples between two plates with a circular sample section (⌀ 10 mm). A stainless steel cylinder probe with a flat surface (⌀ 5 mm) was used for the measurements of puncture strength (mN/mm2) and elongation at break (%). A test speed of 0.1 mm/s was fixed to move the probe upon contact with the sample surface (triggering force of 0.01 N). The applied force (N) and displacement of the sample (mm) C

DOI: 10.1021/acs.molpharmaceut.6b01054 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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physical properties of the electrospun fibrous substrates and drug content analysis.

Cambridge Structural Database (CSD, U.K.), and PRX anhydrous form III.42 Content Analysis. A high performance liquid chromatograph (HPLC) (Shimadzu Prominence LC20, Shimadzu, Japan) equipped with a photodiode array (PDA) detector was used for the content analysis of LH and PRX. Separations were performed by using a Phenomenex Luna C18(2) column (250 × 4.6 mm) (Phenomenex Inc., U.S.) with basedeactivated end-capped octadecylsilyl gel for chromatography R (5 μm) as stationary phase at 40 °C. For the quantitative analysis, the HPLC method from the European Pharmacopoeia (Ph. Eur. 8) PRX monograph for the test for related substances with minor modifications was applied. A mixture of acetonitrile R1 and 6.81 g/L solution of potassium dihydrogen phosphate R previously adjusted to pH 3.0 with phosphoric acid R in a 30:70 (v/v) ratio was used as the mobile phase with a flow rate of 1.0 mL/min during the analysis. The injection volume of the samples was 10 μL. The compounds were detected spectrophotometrically at a wavelength of 230 nm. The retention times for LH and PRX were approximately at 3.9 and 22.0 min, respectively. The PRX content in the non-cross-linked G20-PRX gelatin substrates was measured in 10 M acetic acid solution. For the content analysis, the dosage forms with LH printed on the cross-linked G25 gelatin substrate were immersed in purified water and measured after 6 h of incubation. The PRX content in the cross-linked G20-PRX fibrous substrates and the dosage forms with LH printed on the cross-linked G20-PRX substrate was determined after enzymatic degradation of the gelatin matrix to ensure the complete drug release from within the cross-linked fibers. In vitro enzymatic degradation43 of cross-linked gelatin fibrous substrates was performed using a Glibco Collagenase Type I isolated from Clostridium histolyticum (Life Technologies Corporation, U.S.) with collagenase activity of 235.00 units/mg. The cross-linked G20-PRX fiber mats (approximately 2 cm2) were immersed in 10 mL of 3 mM CaCl2 aqueous solution containing 0.5 mg/mL of collagenase at 37 °C for 1 h with manual shaking every 5 min. Drug Release Studies. The drug release of PRX from the non-cross-linked and cross-linked fibrous gelatin substrates and the dosage forms with LH printed on G25 or G20-PRX substrates was determined in a simulated saliva solution44 containing sodium chloride (8 g/L), potassium dihydrogen phosphate (0.19 g/L), and disodium hydrogen phosphate (2.38 g/L) with pH 6.8. The cut samples (2 cm2) were inserted into 50 mL Falcon tubes containing 10 mL of simulated saliva. The Falcon tubes were placed into the dissolution vessels of a USP apparatus II (paddle) containing water to ensure a mixing speed of 50 rpm and the temperature of the dissolution media 37 ± 0.5 °C. Each time point was measured separately as an independent sample. The samples were collected by manually withdrawing approximately 2 mL of the solution from the Falcon tubes with a syringe and filtered through 0.45 μm cellulose acetate syringe filters into HPLC vials. The analysis was performed according to the method described in Content Analysis. The drug release measurements were conducted under sink conditions (n = 2). Statistical Data Analysis. Microsoft Office 365 Excel software was used for the data analysis. All experimental results are presented as mean value with ± standard deviation (SD) or relative standard deviation (RSD) in percentage (%). A t test at a confidence level of 95% was used to observe difference in the

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RESULTS AND DISCUSSION Two different dosage forms for oromucosal drug delivery were designed according to the experimental setup demonstrated in Figure 1. The single-drug system (formulation I) contained a

Figure 1. Experimental setup and design of the inkjet-printed dosage forms.

local anesthetic, LH, that was inkjet-printed on electrospun G25 gelatin substrate. In the drug combination system (formulation II) a nonsteroidal anti-inflammatory drug (NSAID), PRX, was incorporated within the electrospun gelatin matrix (G20-PRX) followed by a deposition of LH by inkjet printing. A combination drug therapy with antiinflammatory and anesthetic drugs could increase the efficiency of the treatment of oral ulcers due to the additional relief of pain and discomfort. Preparation and Characterization of the Electrospun Fibrous Substrates. Morphology and Fiber Diameter. The electrospinning parameters (applied voltage, distance between the spinneret and collector, pumping rate) and the properties of the electrospinning solution (viscosity, surface tension, conductivity, polymer concentration) affect the fiber diameter.20,38 In this study, the alteration in the composition of the electrospinning solution influenced significantly the fiber diameter (Figure 2). The increased diameter of G20 and G20-PRX gelatin fibers was predominantly attributed to the higher viscosity of the electrospinning solution. In addition, the process parameters were modified to obtain a continuous production of smooth fibers without any bead formation. Furthermore, the incorporation of PRX increased the average diameter of the electrospun G20-PRX fibers compared to G20 fibers by 13.3% and 9.2% before and after cross-linking, respectively. In this study, the thermal cross-linking had no significant effect on the average diameter of G25 gelatin fibers. However, the effect of cross-linking on the fiber diameter was seen for G20 and G20-PRX gelatin substrates, where the average diameter decreased after cross-linking by 8.6% and 12.7%, respectively. A higher feeding rate of the electrospinning solution has been known to contribute to the higher solvent residue in the fibers.20 The difference between the pumping speed of G20/G20-PRX (25−30 μL/min) and G25 (8 μL/ min) electrospinning solutions could therefore explain this behavior, resulting in an evaporation of residual solvent during cross-linking at elevated temperature. Moisture Uptake and Mechanical Properties. The electrospun gelatin fiber mats were receptive to the increase in the D

DOI: 10.1021/acs.molpharmaceut.6b01054 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 2. Histograms of fiber diameters of the non-cross-linked (top) and cross-linked (bottom) electrospun G25 (A, B), G20 (C, D), and G20-PRX (E, F) gelatin fibers with mean value (n = 100) and relative standard deviation (RSD).

Table 2. Physical Properties of Non-Cross-Linked (NonCL) and Cross-Linked (CL) G25, G20, and G20-PRX Gelatin Substrates, Printed Dosage Forms with Lidocaine Hydrochloride (LH) (Formulations I and II), and Reference Materials (n = 3−5) moisture uptake (%) thickness of the substrate (mm) G25 Substrate G25 nonCLa G25 CLa formulation I G20 Substrate G20 nonCL G20 CL G20-PRX Substrate G20-PRX nonCL G20-PRX CL

puncture test burst strength (mN/mm2)

elongation at break (%)

tensile strength (N/mm2)

elongation at break (%)

elastic modulus (kPa)

2 days

25 days

0.05 ± 0.02

6.7 ± 1.5 8.5 ± 2.9

11.4 ± 2.0 12.8 ± 1.9

32.3 ± 3.6 203.7 ± 6.4 188.9 ± 13.5

1.1 ± 0.1 6.6 ± 0.8 21.9 ± 6.5

4.4 ± 0.4 6.1 ± 0.7

1.2 ± 0.4 8.1 ± 2.3

48.6 ± 7.7 9.8 ± 0.7

0.06 ± 0.02

2.8 ± 0.7 5.5 ± 0.6

6.1 ± 1.7 6.0 ± 1.0

79.0 ± 16.2 95.8 ± 22.2

1.4 ± 0.2 2.3 ± 0.5

2.3 ± 0.7 2.8 ± 0.6

1.1 ± 0.4 2.7 ± 0.3

24.7 ± 3.1 6.5 ± 1.1

0.06 ± 0.02

5.6 ± 1.2 6.9 ± 0.3

5.7 ± 2.2 8.2 ± 1.2

37.8 ± 8.8 71.7 ± 12.9

0.6 ± 0.1 1.7 ± 0.1

1.6 ± 0.4 2.5 ± 0.03

1.3 ± 0.2 2.1 ± 0.4

16.2 ± 1.4 9.0 ± 2.9

56.5 ± 17.6b 63.8 ± 9.0

5.8 ± 0.6b 120.3 ± 24.1 5.6 ± 0.8 1.2 ± 0.1

86.5 ± 6.6 1.1 ± 0.1

formulation II Reference Materials copy paper 3-ply tissue a

tensile test

0.09 ± 0.01 0.12 ± 0.01

1685.8 ± 75.0 137.5 ± 11.2

6.3 ± 0.4 23.4 ± 6.9

50.0 ± 5.4 2.5 ± 0.1

NonCL: non-cross-linked gelatin substrate. CL: cross-linked gelatin substrate. bValues for the top layer(s) of the printed surface.

into unfavorably hard, brittle, and more transparent film-like structures at high humidity. The physical properties of the electrospun gelatin substrates before and after cross-linking are presented in Table 2. Previously, various cross-linking methods have been reported to enhance the mechanical properties of the electrospun gelatin fibers and films.35,36,38 Here the cross-linking improved the strength of the fibrous G25 and G20-PRX gelatin substrates. For example, the burst strength of the G25 gelatin fiber mats showed an approximately 5-fold increase after the heat treatment. In addition, the elongation at break upon puncture/tension was increased for all the fibrous substrates after cross-linking. The results showed that according to the calculated elastic modulus the flexibility of the drug-free G25/ G20 gelatin substrates increased significantly after cross-linking. It was noted that the addition of PRX into the fibers had no remarkable influence on the tensile strength and the elastic

humidity (Table 2). The extent of moisture uptake is relevant for predicting the stability of the substrates during storage, but it also gives an indication about their behavior upon contact with the printing solution and the oral mucosa. A noticeable difference in the moisture uptake was seen between G25 and G20/G20-PRX gelatin substrates, due to the changes in the electrospinning process that influenced the fiber diameter and the packing density of fibers within the substrate. In the crosslinked substrates the absorptivity seemed not to be timedependent at the time points observed, suggesting that posttreatment of fiber mats by cross-linking could potentially give smaller variations in stability between the drug-free and drugloaded fibrous gelatin. Visual inspection showed that the crosslinked G25 gelatin substrate was able to maintain its original appearance, whereas others were prone to contraction. Furthermore, the non-cross-linked gelatin fiber mats turned E

DOI: 10.1021/acs.molpharmaceut.6b01054 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics properties of the gelatin fiber matrix, whereas the elongation at break was slightly lower for the cross-linked G20-PRX fibers compared to the drug-free G20 gelatin substrate. Due to the small size of the printed area (2 cm2), only puncture strength and elongation at break were measured for the printed formulations I and II. The results showed that the deposition of two layers of the printed LH ink did not affect the burst strength of the preparations compared to the electrospun gelatin substrates. However, the elongation at break of the final preparations was enhanced after inkjet printing. The main drawback was the randomness of the fibrous alignment within the electrospun substrates that has been known to cause higher deviations and errors in the physical properties.38 Due to the extensive variability in the used apparatuses, measurement parameters, and sample preparation, the comparison of results from different studies is very difficult and out of the scope of the current study. Nevertheless, the mechanical properties of the electrospun gelatin substrates seemed to be comparable to those of many oromucosal film formulations.41 Thermal Properties. The interpretation of conventional DSC measurements was difficult due to the presence of residual water in the samples of pure gelatin and fibrous gelatin substrates. Nevertheless, the characteristic endotherm that was attributed to the water evaporation with an overlapping denaturation of gelatin decreased from 107.2 to 73.0 °C for G25, 79.1 °C for G20, and 77.1 °C for G20-PRX fibers. Similar decrease has been shown for gelatin nanofibers prepared from aqueous and organic solutions by electrospinning. 24,31 Previously, it has been reported that the thermal stability of gelatin nanofibers was enhanced by cross-linking with glutaraldehyde.24,35 In this study, the characteristic endotherm of gelatin that has been related to its thermal stability was not affected by cross-linking. The melting endotherm of glucose was observed at 149−160 °C in the PMs of G25, G20, and G20-PRX and in the respective electrospun gelatin substrates before cross-linking. The disappearance of the melting endotherm of glucose was seen for all the substrates after cross-linking (Figure 3A). This suggested that the use of high temperature and/or the extent of Maillard reaction between glucose and gelatin stabilized the thermal behavior of these fibrous substrates. The MT-DSC results showed that the helix− coil conformational transition was presented as a step change with low intensity at approximately 197 and 210 °C before and after cross-linking of G20 gelatin fibers, respectively. This irreversible supercontraction temperature for raw gelatin was at about 222.6 °C (onset at 213.9 °C) and remained unchanged in the PMs (data not shown). Previous studies have reported that the amorphous state of PRX can be stabilized by incorporation of drug into the electrospun polymer fibers.45,46 Paaver et al.45 presented a successful preparation of electrospun Soluplus nanofibers with amorphous PRX at 7.5% (w/w). Additionally, drug-loaded polyvinylpyrrolidone fibers containing indomethacin or griseofluvin with concentrations up to 33% (w/w) have been published.47 In this study, no melting endotherm for PRX in the electrospun fibers was seen, indicating the presence of an amorphous PRX (Figure 3B). However, this could have been superposed by the melting endotherm of glucose in the noncross-linked G20-PRX substrate at 149.5 °C (onset at 125.8 °C). Furthermore, the possible detection of a glass transition and crystallization seemed to be impeded due to the PRX incorporation within the polymer matrix. Therefore, no characteristic thermal behavior for PRX (crystalline or

Figure 3. A: Conventional DSC thermograms of untreated gelatin and non-cross-linked (···) and cross-linked (−) G25 (black) and G20 (blue) gelatin substrates. B: Modulated DSC (MT-DSC) (total heat flow signal) thermograms of G20-PRX gelatin substrates before (G20PRX nonCL) and after (G20-PRX CL) cross-linking. C: MT-DSC thermograms (reversing heat flow signal) of non-cross-linked (···) and cross-linked (−) G20 (blue) and G20-PRX (green) gelatin substrates.

amorphous) in the non-cross-linked fibers was seen. The cross-linked G20-PRX gelatin substrate, however, exhibited a melting endotherm at 173.1 °C (onset at 150.2 °C) (Figure 3B). This was not characteristic for any previously reported solid state forms of PRX, but most likely could be PRX form III, which is known to be the least stable crystalline form of PRX obtained from amorphous PRX.42,48−50 The thermal analysis showed a melting endotherm for the raw PRX anhydrous form I at 201.4 °C (onset at 200.5 °C) and in the PM of G20-PRX at 196.6 °C (onset at 194.2 °C) (data not shown). The decrease of the melting endotherm could give some indication on physical drug−polymer interactions already during mixing.51 The glass transition and/or crystallization of PRX were not detected for cross-linked G20-PRX substrate with MT-DSC. In addition, the thermal behavior of gelatin was influenced by the addition of PRX into the electrospun fibers as seen by the differences between the fibrous G20 and G20-PRX gelatin substrates. The results showed that the helix−coil transition temperature of gelatin was decreased approximately 20 °C after the addition of PRX, presented in Figure 3C for non-crosslinked fibers at 178.4 °C (onset at 168.9 °C) and for crosslinked fibers at 176.9 °C (onset at 166.8 °C). F

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Molecular Pharmaceutics Solid State Analysis. ATR-FTIR Spectroscopy. Several characteristic peaks for gelatin and glucose were identified on the ATR-FTIR spectra of raw materials and their PMs (Figure 4).52,36−38 The vibrational modes of amide I (1634 cm−1) and

acid was reduced noticeably, however, the formation of a helical structure was still hindered. The less-ordered coil formation was promoted by the use of a higher electrospinning temperature (50 °C) and the addition of DMF into the solvent mixture.54 No interactions between gelatin and DMF were detected in the G20 and G20-PRX fibers due to the rapid solvent evaporation during electrospinning.29 Furthermore, the molecular interactions attributing to the secondary structure of gelatin seemed not to be affected by the thermal cross-linking. The changes on the ATR-FTIR spectra due to the crosslinking are presented in Table 3. Permanent changes in the fibrous structure of gelatin substrates during cross-linking were due to Maillard reaction between the carbonyl group of glucose and amino group of gelatin as also reported in the literature.37,38,53 A nonenzymatic Maillard reaction was also seen between lactose and gelatin during the heat-promoted glycation of solvent-cast films.53 The glycation between reducing sugars and gelatin is induced by high temperature, and its extent is dependent on the amount of the crosslinker.37,38,53 Siimon et al.37 speculated that, if the ratio between gelatin and glucose ratio is above 5:1 (w/w), the caramelization increases significantly due to the limited extent of the crosslinking reaction. ATR-FTIR spectra of the non-cross-linked and cross-linked G20-PRX substrate were analyzed to determine the solid state of the drug within the fibers after electrospinning and consequent cross-linking. Comparison was made with several PRX crystalline forms (I, II, III, and monohydrate) as well as with a recently proposed new polymorphic form of PRX obtained by electrospinning.50 The PRX-specific vibration modes at 1436, 875, and 773 cm−1 were detected in the spectra of G20-PRX substrates (Figure 4). However, these absorbance bands were overlapping between the different polymorphic forms, thus, the solid state of the API could not be determined based on the analysis of the “fingerprint” region of the spectra. Clear difference between the spectra of pure PRX anhydrous form I, G20-PRX substrate, and the corresponding PM was observed at higher wavenumbers above 3000 cm−1 (Figure 4). A sharp peak at 3337 cm−1 due to the N−H stretching vibration of PRX was not detected in the G20-PRX substrate spectrum, which might be due to the intermolecular interactions between PRX and gelatin. The spectral regions from 3400 to 3300 cm−1 and from 600 to 700 cm−1 are known to describe the inter- and intramolecular hydrogen bonding of PRX molecules enabling to differentiate between its different

Figure 4. ATR-FTIR spectra of piroxicam (PRX) anhydrous form I (a), physical mixture (PM) of G20-PRX (b), non-cross-linked (c) and cross-linked (d) G20 substrate, and non-cross-linked (e) and crosslinked (f) G20-PRX substrate in the ranges of 500−1700 cm−1 (A) and 2600−3500 cm−1 (B). The dashed lines (---) represent the characteristic absorbance bands for PRX anhydrous form I. Spectra are offset in absorbance for clarity.

amide II (1524 cm−1), as well as the CO vibrations (1033 and 1080 cm−1) of both components, were considerably affected by the electrospinning and cross-linking processes (Table 3). The electrospinning process caused a slight shift of the amide I and amide II absorbance bands of gelatin to higher wavenumbers. The amide I vibrations give an indication about the secondary structure of the proteins.53 Fibers of gelatin electrospun from nonacidic solutions have been shown to exhibit a helical structure, whereas the ones derived from acidic solutions have the triple helix structure destabilized already during the solution preparation.30,36,37 During the electrospinning of nonacidic gelatin solutions, a conversion from a less-ordered structure into α-helix can be seen from the peak shift of the amide I absorbance band to a lower wavenumber.36 No formation of a helical structure has been identified when an acetic acid aqueous solution was used for the electrospinning of gelatin fibers.37 The spectral analysis showed a significant difference in the amide I absorbance band between the raw gelatin powder and the fibers, indicating formation of a less-ordered amorphous structure after electrospinning. In the G25 fibers this phenomenon was due to the presence of an acetic acid that influenced the molecular reorientation of gelatin.29 In the G20 and G20-PRX electrospinning solutions the amount of acetic

Table 3. Changes in the ATR-FTIR Spectra of G25, G20, and G20-PRX Gelatin Substrates in Comparison with the Raw Materials vibrational assignment

raw material

G25 and G20

G20-PRX

refs

CO stretching CO stretching CO and CC stretching CN stretching NH bending

gelatin, 1029 cm−1

1032 cm−1, intensity decreased after cross-linking

glucose, 1080 cm−1

1081 cm−1, intensity decreased after cross-linking

gelatin, 1161 cm−1; glucose −1147 cm−1 gelatin (amide III), 1234 cm−1 gelatin (amide II), 1524 cm−1

peak shift from 1157 to 1167 cm−1 after cross-linking 1167 cm−1 due to the decreased content of unbound glucose peak shift from 1243 to 1239 cm−1 after cross-linking; weak to no change in the intensity

Maillard reaction37,38,53 Maillard reaction37,38,53 intensity the same36 or decreased39

peak shift from 1539 to 1532 cm−1 after cross-linking; weak to no change in the intensity

change in intensity35,37

CO stretching

gelatin (amide I), 1634 cm−1

1644 cm−1; weak to no change in the intensity

G

peak shift from 1533 to 1528 cm−1; overlapping with PRX anhydrous form I (1528 cm−1)

29, 37, 53

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Molecular Pharmaceutics solid state forms.48,55 The disappearance of the peak at 3400− 3300 cm−1 (shown in Figure 4) has been described as a proof for hydrogen bonding between the NH group of PRX and polymers with >N− and CO functional groups in amorphous solid dispersions.56,57 In addition, the disappearance of characteristic bands for PRX anhydrous forms (I, II, III) and monohydrate strongly suggests that PRX is in a noncrystalline state within the gelatin fibers. X-ray Diffraction. The results from the XRD analysis of gelatin supported the DSC and ATR-FTIR spectroscopy findings. A wide reflection at 4.5 Å (2θ ≈ 19.9°) demonstrated the amorphous nature of gelatin in the electrospun fibers, and the reflections at 11.3 Å (2θ ≈ 7.8°) and 2.9 Å (2θ ≈ 31.3°) indicated the presence of a triple helix in the raw material (Figure 5). Previously, wide-angle X-ray scattering was used to

layers of ink were deposited on top of each other with an intermediate drying step. Similarly, in the production of pharmaceutical film formulations by solvent casting, the drying process plays a crucial role. The solvent-cast polymer films reach the dried equilibrium state after several hours depending on the drying environment (relative humidity and temperature) and the amount of plasticizer.59 The physical stability of the produced films can be ensured by an adequately fast and optimized drying process in one or several steps to avoid the sedimentation of the drug and/or premature solidification of the film surface.60 The drying of the printed dosage forms has been shown to be dependent on the absorption capacity of the substrate, the volume of the ink droplets, and the evaporation rate of the ink.3,9,12 Genina et al.12 have presented that up to 9 layers of ink could be printed consecutively on porous copy paper without any drying between the layers, whereas the drying of water-based inks on substrates with limited absorptivity was performed successfully at ambient conditions for 1 h.12 Furthermore, the drying at elevated temperature of 60 °C for 1 h in a vacuum oven was applied for the removal of dimethyl sulfoxide from the printed samples on copy paper without any influence on the stability of the printed drug.9 In this study, it was observed that the applied drying time (2 h) could have been reduced significantly due to the rapid absorption of the solution into the substrate. The photographic images of the electrospun gelatin substrates before and after cross-linking and the printed dosage forms on G25 and G20-PRX matrices are presented on Figure 6. The PRX in an amorphous state can be visually observed

Figure 5. X-ray diffractograms of gelatin (a), glucose (b), piroxicam (PRX) anhydrous form I (c), physical mixture (PM) of G20-PRX (d), non-cross-linked G20 (e) and G20-PRX (f) gelatin substrates, and cross-linked G20 (g) and G20-PRX (h) gelatin substrates. Diffractograms are offset in the y-axis for clarity.

demonstrate that the gelatin helicity decreased in aqueous fibers electrospun at elevated temperatures and fibers from an acidic solution showed no triple helix structure.30 Electrospinning has been known to stabilize the amorphous state of various APIs by incorporating the drug molecules within the polymer nanofibers.22,47 Polymers, such as hydroxypropyl methylcellulose (HPMC), Soluplus, or chitosan-based biodegradable complexes, have been studied for the stabilization of the amorphous PRX by electrospinning.45,46,58 According to the XRD patterns, PRX presented no crystallinity in the electrospun G20-PRX fibers (Figure 5). Thus, all different solid state analysis techniques (DSC, ATR-FTIR, and XRD) confirmed the presence of amorphous PRX within the gelatin fibers. PRX Content in Electrospun Fibrous Substrates. The drug loading of PRX in the non-cross-linked and cross-linked G20PRX fibers was 6.3 ± 0.2% (n = 3) and 6.2 ± 0.2% (n = 3), respectively. The measured drug content correlated well with the theoretical drug loading showing no significant loss (below 5%) in the drug content after electrospinning. Furthermore, the thermal cross-linking (at 130 °C) did not affect the chemical stability of the PRX within the gelatin fibers, although PRX has been shown to be sensitive to higher temperatures and degrades upon quench cooling of the melt.49 Preparation and Characterization of the Printed Dosage Forms. The printed dosage forms were obtained by depositing the ink solution in picoliter-size droplets at a predetermined spatial resolution on the cross-linked fiber mats by inkjet printing. For obtaining the desired drug content, two

Figure 6. Images of the G25 (A), G20 (B), and G20-PRX (C) gelatin substrates before (nonCL) and after (CL) cross-linking and the formulations with printed lidocaine hydrochloride (LH) on G25 and G20-PRX substrates (formulations I and II). The unprinted edges of the final dosage forms are left to illustrate the borders of the printed area.

within the non-cross-linked G20-PRX fiber mats due to the color change compared to drug-free G25/G20 fibers. After printing, changes in the appearance of the substrate can be observed, and thus the printed surface can be clearly distinguished from the unprinted area of the substrate. Morphology. Individual fibers were easily distinguished on the surface of the substrate despite the extensive cross-linking (Figure 7). The SEM imaging demonstrated that the printed ink penetrated into the surface layer of the substrate and altered H

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Molecular Pharmaceutics the structure of the fibrous G25 and G20-PRX substrates. Smoother surface was achieved due to increased moisture that caused the swelling of the gelatin fibers and adhesion to each other. The substrates remained intact after the printing with identifiable contours of the fibers. Furthermore, the SEM did not reveal any crystallization of the drug(s).

Figure 8. A: Modulated DSC (MT-DSC) thermograms (reversing heat flow signal) of cross-linked (−) G25 (black) and G20-PRX (green) gelatin substrates and formulations I (−·−) and II (−·−) with printed lidocaine hydrochloride (LH). B: X-ray diffractograms of LH monohydrate (a), physical mixtures (PMs) of G25-LH (b) and G20PRX-LH (c), cross-linked G25 substrate (d), LH printed on crosslinked G25 substrate (formulation I) (e), cross-linked G20-PRX substrate (f), and formulations with inkbase (g) and LH (h) printed on cross-linked G20-PRX substrate (formulation II). The characteristic reflections for piroxicam (PRX) in the printed formulation with inkbase (g) are marked (*).

Figure 7. SEM images of the cross-linked G25 (A) and G20-PRX (B) gelatin substrates before and after printing of lidocaine hydrochloride (LH) (formulations I and II) at 3000× and 10 000× magnification (top to bottom).

systems, further XRD measurements were conducted to confirm the solid state of the drugs. Solid State Analysis. X-ray Diffraction. The XRD analysis did not detect any crystallinity in the printed single-drug system (Figure 8B). Most likely the LH was present in a molecularly dispersed state. The solvent system is known to affect the solid state form that is present in the printed formulations.1 The presence of PG has been shown to inhibit the crystallization of APIs through the nucleation inhibition or its solubilizing effect.8,62 Furthermore, the homogeneous distribution of the LH after the printing stabilized the noncrystalline nature of the printed drug. When solely the inkbase was deposited on the cross-linked G20-PRX substrate, the crystallization of PRX anhydrous form I was observed by XRD. The reflections with low intensity were observed approximately at 14.6° and 17.7° on the diffractograms (Figure 8B). This was attributed to the extensive contact between the inkbase and G20-PRX fibers that promoted the crystallization of the drug from the surface of the fibers. Interestingly, in the drug combination system with printed LH on the cross-linked G20-PRX substrate (formulation II) no crystallization of PRX was observed (Figure 8B). It was suggested that the water amount in the drug-loaded ink and the evaporation rate were not sufficient to promote the crystallization of PRX. In addition, a molecularly dispersed layer of LH on the surface of the substrate prevented the penetration of the inkbase into the fibrous structure of the substrate and therefore contributed to the stabilization of the amorphous PRX within the fibers.

Thermal Properties. The DSC analysis showed that the melting endotherm of LH monohydrate was at 79.3 °C (onset at 73.7 °C). The melting of LH monohydrate was easily observable in the corresponding PMs of G25-LH and G20PRX-LH (data not shown). However, the characteristic melting endotherm of LH monohydrate was superposed by the water evaporation of gelatin in the electrospun fibers. The MT-DSC measurements were conducted to reveal the changes in the heat capacity (Cp) caused by the addition of LH. The results demonstrated a clear increase in the Cp of the printed dosage forms with LH (presented as a decrease in the heat flow on the reversing heat flow curve) (Figure 8A). Thus, the MT-DSC allowed distinguishing between the different overlapping thermal events. No melting endotherm for the LH anhydrous form at approximately 130 °C was detected in the printed dosage forms.61 Previous publications have shown that inkjet printing can result in a solidification of the ink solution on the substrate.8,11,16 Therefore, based on these findings, the printed preparations exhibited endothermic events characteristic for LH in monohydrate form or molecularly dispersed state. According to the thermal analysis, the printing did not affect the solid state of PRX. Despite the overlapping with the thermal events of LH monohydrate, a characteristic endothermic event for PRX was identified at 175.7 °C on the DSC thermograms of the printed dosage forms on the cross-linked G20-PRX substrate. This showed similarity to the endotherm of the cross-linked G20-PRX gelatin substrate presented previously (Figure 3B). Due to the complexity of the printed I

DOI: 10.1021/acs.molpharmaceut.6b01054 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics ATR-FTIR Spectroscopy. The spectral analysis of the printed dosage forms was conducted to support the XRD results. The ATR-FTIR spectra of formulations I and II were compared with spectra from LH monohydrate, PG, and the LH ink solution. The characteristic absorbance bands for the LH salt were detected in the printed samples (Figure 9). Moreover, the

Even though LH is considered as a fairly stable API at room temperature, a degradation of the printed LH was observed over time probably due to the hydrolysis of the amide functional group (Table 4).64,65 Interestingly, the LH hydrolysis was more pronounced in formulation II, where the LH was printed on cross-linked G20-PRX gelatin substrates. These samples contained 0.35 ± 0.01 mg of PRX per printed area of 2 cm2. The PRX drug loading (6.5% (w/w)) in the fibers was constant over the short-term stability study of 4 months at ambient conditions. Drug Release from Printed Dosage Forms. The PRX release from the G20-PRX substrate was determined before and after cross-linking (Figure 10). Direct electrospinning of the

Figure 9. ATR-FTIR spectra of lidocaine hydrochloride (LH) monohydrate (a), physical mixtures (PMs) of G25-LH (b) and G20-PRX-LH (c), LH printed on cross-linked G25 substrate (formulation I) (d), LH printed on cross-linked G20-PRX substrate (formulation II) (e), and LH ink solution (f) in the ranges of 500− 1700 cm−1 (A) and 2200−3700 cm−1 (B). The dotted (···) and dashed (---) lines represent the characteristic absorbance bands for LH monohydrate and the printed LH, respectively. Spectra are offset in absorbance for clarity.

spectral changes (broadening, shifting, merging, change of intensity) in the LH monohydrate absorbance bands at 1477, 984, 952, 773, 716, and 691 cm−1 supported the potential formation of a solidified ink layer on the fibrous gelatin substrates. The interactions between PG and LH were unclear from the ATR-FTIR analysis due to spectral overlapping with the substrate. There are major spectral differences between LH anhydrate and LH monohydrate on the ATR-FTIR spectra in the range from 2200 to 3000 cm−1.63 In that region, the NH stretching vibrations of LH monohydrate were not identified for the printed samples, thus supporting the fact that after the printing the API did not recrystallize into its original form as a LH monohydrate. Drug Content Analysis and the Chemical Stability during Short-Time Storage. The drug content in the printed dosage forms was analyzed. In both of the formulations (I and II) a low therapeutically relevant dose of LH was deposited on the electrospun substrates (Table 4). The measured content showed no significant difference from the calculated values.

Figure 10. A: Drug release (%) of piroxicam (PRX) from the noncross-linked (◇) and cross-linked (◆) G20-PRX gelatin substrate and formulation II with printed lidocaine hydrochloride (LH) on G20PRX substrate (■). B: Drug release (%) of LH from formulation I (△) and formulation II (▲) with LH printed on cross-linked G25 and G20-PRX substrates, respectively. Drug release is presented as average with standard deviation bars (n = 2).

drug-loaded fibers has been known to result in a drug dissolution profile with a noticeable initial burst release. The results showed a burst release of approximately 80% from the non-cross-linked G20-PRX fibers that decreased after a few minutes. This kind of behavior has been attributed to the fast formation of PRX monohydrate that has a lower solubility compared to PRX anhydrous forms in aqueous media in a wide pH range.66,67 The burst release of PRX was caused by the drug present on the surface of the fibers.19,21,35 On the other hand, cross-linking of polymer fibers has been shown to decrease the drug release rate in the later phases.32,34 The release of PRX from the cross-linked G20-PRX fibers showed a noticeably decreased burst release (approximately 40%) within the first 30 s followed by a continuous drug release at a slower rate. Further studies are needed in order to reveal the solid state of PRX during the dissolution from the cross-linked fibrous substrates. As expected the LH dissolution from the printed formulations was immediate, releasing over 95% of the drug

Table 4. Drug Content of Lidocaine Hydrochloride (LH) in the Formulations Printed on the Cross-Linked G25 and G20-PRX Gelatin Fibrous Substrates with the Relative Standard Deviation (RSD) and the Difference (%) from the Initial Content after Storage (n = 4−6) lidocaine hydrochloride (LH) drug content (mg) per 2 cm2 G25 Substrate initial (theoretical) after 8 months G20-PRX Substrate initial (theoretical) after 4 months

RSD (%)

difference from the initial content (%)

4.5

−18.2

4.5

−30.2

2.84 2.33 ± 0.10 2.18 1.52 ± 0.07

J

DOI: 10.1021/acs.molpharmaceut.6b01054 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics Notes

after 4 min. This is beneficial for decreasing the discomfort and pain caused by oral ulcers. In oromucosal dosage forms unidirectional drug release toward the mucosal tissue is preferred to decrease the loss of drug through gastrointestinal pathways and to improve the drug permeation through the layers of oral mucosa.7,68 It has been recognized that this type of drug delivery system could be produced by inkjet printing.7 The direction of the drug release from the substrate/ electrospun fibers or from the surface of films/patches (e.g., printed dosage forms) could be controlled by designing multilayered systems with an impermeable backing layer.7,68 Furthermore, the development of layered systems allows also producing delivery systems with modified drug release kinetics.69 A study by Thakur at al.70 showed the fabrication of a combination drug release system of LH and an antibiotic mupirocin by means of dual spinneret electrospinning setup. It was found that the presence of two drugs in one polymer matrix (single spinneret electrospinning) altered the release kinetics of at least one of the drugs, whereas in the dual spinneret approach a scaffold with dual drug release was obtained. In the formulation II with LH printed on the crosslinked G20-PRX fibrous substrate, the release kinetics of both drugs remained the same compared to the single drug systems (Figure 10). This indicated that the use of two different techniques could allow controlling the physicochemical properties and the release kinetics of both drugs separately.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the NordForsk, the PUT1088 project, and IUT-34-18 project. M. Palo is thankful to the Finnish Cultural Foundation for financial support. Jaan Aruväli (Institute of Ecology and Earth Sciences, University of Tartu) is acknowledged for carrying out the XRD experiments. PhD Urve Paaver, MSc Kristian Semjonov (Institute of Pharmacy, University of Tartu), and Prof. Kalle Kirsimäe (Institute of Ecology and Earth Sciences, University of Tartu) are thanked for conducting the SEM imaging.

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(1) Meléndez, P. A.; Kane, K. M.; Ashvar, C. S.; Albrecht, M.; Smith, P. A. Thermal Inkjet Application in the Preparation of Oral Dosage Forms: Dispensing of Prednisolone Solutions and Polymorphic Characterization by Solid-State Spectroscopic Techniques. J. Pharm. Sci. 2008, 97 (7), 2619−2636. (2) Buanz, A. B. M.; Saunders, M. H.; Basit, A. W.; Gaisford, S. Preparation of Personalized-Dose Salbutamol Sulphate Oral Films with Thermal Ink-Jet Printing. Pharm. Res. 2011, 28 (10), 2386−2392. (3) Sandler, N.; Mäaẗ tänen, A.; Ihalainen, P.; Kronberg, L.; Meierjohann, A.; Viitala, T.; Peltonen, J. Inkjet Printing of Drug Substances and Use of Porous Substrates − Towards Individualized Dosing. J. Pharm. Sci. 2011, 100 (8), 3386−3395. (4) Scoutaris, N.; Alexander, M. R.; Gellert, P. R.; Roberts, C. J. Inkjet Printing as a Novel Medicine Formulation Technique. J. Controlled Release 2011, 156, 179−185. (5) Kolakovic, R.; Viitala, T.; Ihalainen, P.; Genina, N.; Peltonen, J.; Sandler, N. Printing Technologies in Fabrication of Drug Delivery Systems. Expert Opin. Drug Delivery 2013, 10 (12), 1711−1723. (6) Alomari, M.; Mohamed, F. H.; Basit, A. W.; Gaisford, S. Personalised Dosing: Printing a Dose of One’s Own Medicine. Int. J. Pharm. 2015, 494 (2), 568−577. (7) Preis, M.; Breitkreutz, J.; Sandler, N. Perspective: Concepts of Printing Technologies for Oral Film Formulations. Int. J. Pharm. 2015, 494, 578−584. (8) Genina, N.; Fors, D.; Palo, M.; Peltonen, J.; Sandler, N. Behavior of Printable Formulations of Loperamide and Caffeine on Different Substrates − Effect of Print Density in Inkjet Printing. Int. J. Pharm. 2013, 453 (2), 488−497. (9) Wickström, H.; Palo, M.; Rijckaert, K.; Kolakovic, R.; Nyman, J. O.; Mäaẗ tänen, A.; Ihalainen, P.; Peltonen, J.; Genina, N.; de Beer, T.; Löbmann, K.; Rades, T.; Sandler, N. Improvement of Dissolution Rate of Indomethacin by Inkjet Printing. Eur. J. Pharm. Sci. 2015, 75, 91− 100. (10) Khaled, S. A.; Burley, J. C.; Alexander, M. R.; Yang, J.; Roberts, C. J. 3D printing of tablets containing multiple drugs with defined release profiles. Int. J. Pharm. 2015, 494, 643−650. (11) Planchette, C.; Pichler, H.; Wimmer-Teubenbacher, M.; Gruber, M.; Gruber-Woelfler, H.; Mohr, S.; Tetyczka, C.; Hsiao, W.-K.; Paudel, A.; Roblegg, E.; Khinast, J. Printing Medicines as Orodispersible Dosage Forms: Effect of Substrate on the Printed Micro-Structure. Int. J. Pharm. 2016, 509 (1−2), 518−527. (12) Genina, N.; Janßen, E. M.; Breitenbach, A.; Breitkreutz, J.; Sandler, N. Evaluation of Different Substrates for Inkjet Printing of Rasagiline Mesylate. Eur. J. Pharm. Biopharm. 2013, 85, 1075−1083. (13) Dixit, R. P.; Puthli, S. P. Oral Strip Technology: Overview and Future Potential. J. Controlled Release 2009, 139, 94−107. (14) Morales, J. O.; McConville, J. T. Manufacture and Characterization of Mucoadhesive Buccal Films. Eur. J. Pharm. Biopharm. 2011, 77, 187−199. (15) Janßen, E. M.; Schliephacke, R.; Breitenbach, A.; Breitkreutz, J. Drug-Printing by Flexographic Printing Technology − A New

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CONCLUSIONS For the first time, printed oromucosal formulations were successfully prepared by combining electrospinning and inkjet printing technologies. For the printed solid dosage forms, fibrous gelatin substrates with high surface area to volume ratio were obtained by direct electrospinning and consequent thermal cross-linking. The mechanical properties of the electrospun gelatin substrates were improved after the crosslinking. A dual drug release system was developed by incorporating an anti-inflammatory drug, piroxicam, within the fibrous gelatin substrates and depositing a local anesthetic, lidocaine hydrochloride, on the thermally cross-linked fiber matrix by piezoelectric inkjet printing. The amorphous state of piroxicam was stabilized within the gelatin fibers, and the printed lidocaine hydrochloride remained in a molecularly dispersed state. An immediate release of lidocaine hydrochloride suitable for fast pain relief was obtained irrespective of the substrate used. This study demonstrated an alternative method for the preparation of printed dosage forms containing multiple drugs by combining electrospinning and inkjet printing techniques. The results indicated that the presented approach has a high potential for obtaining solid dosage forms for local drug therapy. Further studies will be directed toward investigating the flexibility and overall limits of dosing as well as the applicability of various other substrate materials.

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*Pharmaceutical Sciences Laboratory, Faculty of Science and Engineering, Åbo Akademi University, Tykistökatu 6A, FI20520 Turku, Finland. Tel:+358 2 215 4001. E-mail: mirja. palo@abo.fi. ORCID

Mirja Palo: 0000-0003-3108-6049 Jyrki Heinämäki: 0000-0002-5996-5144 K

DOI: 10.1021/acs.molpharmaceut.6b01054 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

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DOI: 10.1021/acs.molpharmaceut.6b01054 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.molpharmaceut.6b01054 Mol. Pharmaceutics XXXX, XXX, XXX−XXX