European Journal of Pharmaceutical Sciences 75 (2015) 91–100

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European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier .com/ locate/e jps

Improvement of dissolution rate of indomethacin by inkjet printing

http://dx.doi.org/10.1016/j.ejps.2015.03.0090928-0987/� 2015 Elsevier B.V. All rights reserved.

⇑ Corresponding author. Tel.: +358 2 215 4001.E-mail address: henrika.wickstrom@abo.fi (H. Wickström).

Henrika Wickström a,⇑, Mirja Palo a, Karen Rijckaert a, Ruzica Kolakovic a, Johan O. Nyman a,Anni Määttänen b, Petri Ihalainen b, Jouko Peltonen b, Natalja Genina a, Thomas de Beer c,Korbinian Löbmann d, Thomas Rades d, Niklas Sandler a

a Pharmaceutical Sciences Laboratory, Department of Biosciences, Åbo Akademi University, Tykistökatu 6A, FI-20520 Turku, Finlandb Laboratory of Physical Chemistry, Department of Natural Sciences, Åbo Akademi University, Porthaninkatu 3-5, FI-20500 Turku, Finlandc Laboratory of Pharmaceutical Process Analytical Technology, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgiumd Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 København, Denmark

a r t i c l e i n f o

Article history:Received 26 November 2014Received in revised form 15 March 2015Accepted 17 March 2015Available online 25 March 2015

Keywords:Inkjet printingInk formulationIndomethacinCo-amorphous systemPersonalized medicine

a b s t r a c t

The aim of this study was to prepare printable inks of the poorly water soluble drug indomethacin (IMC),fabricate printed systems with flexible doses and investigate the effect of ink excipients on the printabil-ity, dissolution rate and the solid state properties of the drug. A piezoelectric inkjet printer was used toprint 1 � 1 cm2 squares onto a paper substrate and an impermeable transparency film. L-arginine (ARG)and polyvinylpyrrolidone (PVP) were used as additional formulation excipients. Accurately dosed sam-ples were generated as a result of the ink and droplet formation optimization. Increased dissolution ratewas obtained for all formulations. The formulation with IMC and ARG printed on transparency filmresulted in a co-amorphous system. The solid state characteristics of the printed drug on porous papersubstrates were not possible to determine due to strong interference from the spectra of the carrier sub-strate. Yet, the samples retained their yellow color after 6 months of storage at room temperature andafter drying at elevated temperature in a vacuum oven. This suggests that the samples remained eitherin a dissolved or an amorphous form. Based on the results from this study a formulation guidance forinkjet printing of poorly soluble drugs is also proposed.

� 2015 Elsevier B.V. All rights reserved.

1. Introduction

One of the major challenges in pharmaceutical research anddevelopment is to achieve efficient drug delivery. Today, anincreasing number of the active pharmaceutical ingredients(APIs) being discovered have poor water solubility (Kawabataet al., 2011). Therefore, various formulation strategies have beendeveloped to enhance the solubility and the dissolution rate ofthese Biopharmaceutics Classification System (BCS) class II drugs,with water solubility below 100 lg/ml (Engers et al., 2010;Hörter and Dressman, 2001; Kawabata et al., 2011; Löbmannet al., 2012, 2013a; Vasconcelos et al., 2007). A favorable approachis to form binary amorphous formulations consisting of two smallmolecular weight APIs or to form co-amorphous systems consist-ing of an API and a small molecular weight additive (Löbmannet al., 2012, 2013a). Another approach is to develop stabilizedamorphous solid dispersions by addition of a polymer (Hsu et al.,2013; Matsumoto et al., 1999). The approaches mentioned above

have resulted in an enhanced dissolution rate of poorly solubledrugs.

Personalized medicine is another current trend that sets newdemands on the pharmaceutical research and development as wellas for the health care professionals. Especially the developments inthe diagnostic field and the knowledge obtained thereby highlightsthe need and possibility to offer a more safe and effective treat-ment of patients with different needs in the future (Hamburgand Collins, 2010). The ability to predict and monitor a patient’sresponse to a treatment also sets new demands for flexible dosing.Furthermore, the limited range of doses in commercial medicinestogether with the time-consuming batch-wise manufacturingmethods have also stimulated thinking towards more flexible waysof producing medicines (Buanz et al., 2011; van Melkebeke et al.,2008). Therefore, innovative manufacturing methods are neededto meet potential future requirements of producing pharmaceuti-cals on demand in a tailored and personalized manner (Sandleret al., 2011).

Fabrication of drug delivery systems (DDS) using printing tech-nologies have shown to be a promising approach and could pro-mote the transition to a more individualized treatment of

92 H. Wickström et al. / European Journal of Pharmaceutical Sciences 75 (2015) 91–100

patients. The main printing technologies used for fabrication ofDDS are drop-on-demand (DoD) inkjet – and flexographic tech-nologies (Di Risio and Yan, 2007; Genina et al., 2012, 2013b;Janßen et al., 2013; Lee et al., 2012; Raijada et al., 2013; Sandleret al., 2011). Inkjet technology has shown to allow high dose pre-cision and flexibility (Genina et al., 2012; Raijada et al., 2013;Sandler et al., 2011). Dose flexibility has been obtained either byvarying the drop spacing (resolution), printed area or by applyingmultiple layers on top of each other, for formulations printed bothwith inkjet and flexographic printers (Buanz et al., 2011; Geninaet al., 2013a,b; Janßen et al., 2013). Flexographic printing has beenreported to be a robust and repeatable manufacturing method ofDDS (Janßen et al., 2013). Yet, the printing variability is not as highas for the inkjet printer. DDS with controlled drug release rate havebeen achieved by printing various geometries using inkjet technol-ogy (Lee et al., 2012). Moreover, controlled release rates have alsobeen obtained by combining both technologies. Application of apolymer coating with the flexographic printer onto the inkjetprinted doses was successfully done by Genina et al. (2012). Theprinting technology could optimally allow rapid production ofDDS with optimized dosage and release rates for the patient atthe point of care in the future (Kolakovic et al., 2013).

The composition of the ink depends mainly on the characteris-tics of the API to be printed. Moreover, preferable rheological prop-erties of the ink depend on the printing technology to be utilized(inkjet or flexography). Today, both solutions and suspensions havebeen printed. Water and organic solvents such as ethanol, isobu-tanol, N,N-dimethylacetamide and dimethylsulfoxide (DMSO) havebeen used to form ink formulations in previous studies (Kolakovicet al., 2013; Lee et al., 2012). Ink formulations based on water,ethanol and isopropanol have low viscosities and therefore viscos-ity modifiers and moisturizing agents such as glycerol, propyleneglycol (PG), polyethylene glycol (PEG-200, 2000, 20,000 Da) andsodium carboxymethyl cellulose (CMC) have been added toincrease the viscosity of the inks to ensure better printability (DiRisio and Yan, 2007; Kolakovic et al., 2013). The polymers polyvi-nyl alcohol (PVA), polyvinylpyrrolidone (PVP) and copolymerpoly(lactic-co-glycolic acid) (PLGA) have been included into theink formulations with the aim to modify the viscosity, stabilizethe amorphous form of the drug, control and modify the releaserates of the drug (Di Risio and Yan, 2007; Hsu et al., 2013; Leeet al., 2012; Scoutaris et al., 2011, 2012). A piezoelectric inkjetprinter was used in the studies conducted by Di Risio and Yan(2007) and Lee et al. (2012) where 10–20 pl droplets of the inkwere ejected. Yet, a syringe pump was used to eject 3 ll dropletsof the polymer formulations studied by Hsu et al. (2013). Drug con-taining suspensions and nanosuspensions as ink formulations havebeen printed with flexograpic printers and inkjet microdosing dis-pensers, respectively (Janßen et al., 2013; Pardeike et al., 2011).

The purpose of this work was to formulate printable inks for theBCS class II drug indomethacin (IMC) and to fabricate printed DDSin a flexible and personalized manner using piezoelectric inkjettechnology. The main aim was to investigate the influence ofexcipients on the printability and printing parameters of the inks

Table 1The ink formulations.

Ink IMC_50 IMC⁄a ARG

IMC (mg/ml) 50 50 –ARG (mg/ml) – – 24.3PVP (%) – – –DMSO (%) 70 60 –PG (%) 30 40 30Milli-Q (%) – – 70IMC:ARG (molar ratio) – – –

a Inks used for the ARG_IMC_ARG formulation where IMC⁄ and ARG⁄ were printed in

and, furthermore, the dissolution rate and solid state propertiesof the API in the printed DDS. The choice of excipients was basedon previous research outcomes showing that the excipients wereable to stabilize the amorphous form of IMC (Löbmann et al.,2013a; Matsumoto et al., 1999). Formulation and stabilization ofthe more soluble amorphous form, using printing technology couldpossibly allow production of flexible doses with improved dis-solution properties of the poorly water soluble drug. The aminoacid L-arginine (ARG) and polymer polyvinylpyrrolidone K28(PVP) were used as additives in the inks and the formulations werecompared to inks containing only IMC. Based on these findings, thepaper aims to present a formulation guidance for printing processoptimization of poorly water soluble drugs.

2. Materials and methods

2.1. Materials

A non-steroidal anti-inflammatory agent and poorly water sol-uble drug, indomethacin (IMC) (Sigma–Aldrich, China, P99% TLC),was used as the active pharmaceutical ingredient (API) in this study.Dimethyl sulfoxide (DMSO) (99.5%, Sigma–Aldrich, France), propy-lene glycol (PG) (P99.5%, Sigma–Aldrich, Germany) and purifiedwater (Milli-Q) were used to prepare the ink formulations. An aminoacid L-arginine (ARG) (Sigma–Aldrich, France) and a polymerpolyvinylpyrrolidone (PVP, K28) (BASF, Germany) were includedas excipients into the inks. The ink formulations were printed on afibrous paper substrate (Staples A4 copy paper, code 341 46 56,Staples Europe B.V., Netherlands) and an impermeable transparencyfilm (Dataline™, Transparency film, code 57170, EU).

2.2. Ink formulations

Five formulations with IMC (M = 357.8 g/mol) were preparedand printed using piezoelectric inkjet technology. The compositionof the formulations is displayed in Table 1. Two inks, labeled asIMC_50 and IMC_200, were prepared only with IMC dissolved inthe solvent mixture PG:DMSO. ARG (M = 172.2 g/mol) was usedas an excipient in two of the IMC formulations and both formula-tions were prepared having IMC and ARG in a 1:1 molar ratio.The manufacturing process was the main difference between thesamples containing ARG. IMC_ARG_50 samples were printed withone ink and ARG_IMC_ARG (printing order according to the name)was prepared using two separate inks, namely ARG⁄ and IMC⁄

(Fig. 1). The IMC_PVP_50 ink, consisting of IMC and 0.6% of thepolymer PVP (C6H9NO)n, was prepared in the solvent mixturePG:DMSO.

2.3. Viscosity and surface tension

Viscosity measurements of the solvent mixtures and inks wereperformed with a controlled stress rheometer (Bohlin-CS, MalvernInstruments, Worcestershire, UK) using a 15 mm cone-plategeometry with a 4� angle. The viscosity measurements were

⁄a IMC_ARG_50 IMC_PVP_50 IMC_200

50 50 20024.3 – –– 0.6 –50.5 70 7030 30 3019.5 – –1:1 – –

a 1:1 molar ratio.

Fig. 1. Visualization of the formulations and samples IMC_50, IMC_ARG_50 and ARG_IMC_ARG. (a) IMC_50 formulation (only IMC) and the 3, 6, 9 layer samples, (b)IMC_ARG_50 formulation and the 3, 6, 9 layer samples made from one ink designed to have IMC and ARG in a 1:1 molar ratio, and (c) ARG_IMC_ARG formulation printed withtwo separate inks and designed to have IMC and ARG in a 1:1 molar ratio.

H. Wickström et al. / European Journal of Pharmaceutical Sciences 75 (2015) 91–100 93

conducted using 1 ml of the 25–26 �C ink formulations. The surfacetension was measured using a bubble tensiometer (Sensadyne PC900, M&H Technologies Inc., Flagstaff, USA) and 20 ml of 25–26 �C tempered ink formulation was analyzed. The viscosity andsurface tension measurements were conducted once per inkformulation. The IMC⁄ and ARG⁄ inks used to fabricated theARG_IMC_ARG sample were not analyzed.

2.4. Solubility in solvents and solvent mixture

The solubility of IMC was determined spectrophotometrically inDMSO at 265 nm and in PG at 321 nm using an UV/VIS-spectropho-tometer (Ultrospect 2100 pro, Biochrom Ltd., Cambridge, UK). Thesolubility of IMC was also determined in the solvent mixturePG:DMSO, which was prepared in a 30:70 vol% ratio. All measure-ments were done in triplicate.

2.5. Piezoelectric inkjet printing

The printing was performed using a drop-on-demand (DoD)piezoelectric inkjet printer (PIJ) PixDro LP 50 (Roth & Rau,Eindhoven, the Netherlands) equipped with a printhead SpectraSE-128 AA with 128 nozzles. The nozzle diameter was 35 lmand the nozzle spacing was 508 lm. The doses were printedaccording to a customized template (Fig. 2).

Before printing, the ink formulations were filtered into the inkreservoir using a 0.2 lm polypropylene membrane filter (VWEInternational, USA). The ink pressure (�19 mbar), firing frequency(1400 Hz), quality factor (8) and resolution (500 � 500 dpi) wereheld constant during the printing task for all ink formulations.The appropriate jetting voltage applied on the piezoelectric ele-ments, in order to achieve droplet ejection, and the pulse shape

Fig. 2. Template of the 1 � 1 cm2 printed doses.

for each ink formulations were determined after systematic stud-ies, where different values were applied.

The printed samples IMC_50, IMC_ARG_50 and IMC_PVP_50were obtained by printing 1 � 1 cm2 squares onto a paper sub-strate applying 3, 6 and 9 layers to fabricate different doses forthe 50 mg/ml inks. The flexible doses for IMC_200 were manufac-tured by applying 1, 3, 6 and 9 layers of the 200 mg/ml ink onto thesubstrate (1 � 1 cm2). Each formulation was printed on the sameday.

The ARG_IMC_ARG samples were printed on a transparencyfilm and the printing task was performed during 3 days; 2 layersof ARG were applied the first day, 4 layers of IMC the second dayand 2 layers of ARG ink the third day. The printing ofARG_IMC_ARG was carried out during three days due to the clean-ing procedure that was required when changing the ink and theoptimization of the printing parameters for the separate inks.

2.6. Attenuated total reflectance infrared spectroscopy (ATR–IR)

Infrared spectra of the samples were obtained using a universalattenuated total reflectance infrared spectroscope (ATR–IR)Spectrum Two (PerkinElmer, UK) to study the solid state propertiesof the printed DDS. All measurements were done in triplicate and aforce gauge of 140 N was applied on the samples. Spectra of theprinted samples were captured both before and after drying in avacuum oven. The solvent drying in the vacuum oven (BinderGmbh, Tuttingen, Germany) was done at 60 �C for 1 h. The spectraof the printed samples were compared with the spectra of raw andamorphous IMC, the excipients and the substrates. The spectralregion used for the analysis was 1000–1800 cm�1. Baseline correc-tion and normalization were applied to the spectra for bettervisualization.

2.7. Scanning electron microscopy (SEM)

The morphology of the printed samples was studied with scan-ning electron microscopy (SEM) (LEO Gemini 1530, Oberkochen,Germany) supplied with a thermo scientific ultradry silicon driftdetector. An ultrathin layer of carbon coating was deposited onthe samples using a vacuum evaporator. This was done beforethe scanning took place at a working voltage of 2.7 kV with themagnification of 25,000�.

The energy dispersive X-ray spectroscopy (EDX) (ThermoScientific, Madison, Wisconsin, USA) SEM extension was used toidentify the chemical elements on the surface of the printedIMC_200 samples. The analysis was done at an accelerated voltageof 15 kV and a magnification of 250�.

Fig. 3. IMC doses (1 � 1 cm2) printed on fibrous paper substrate with the IMC_50ink formulation. The color contrast has been modified for better visualization. (Forinterpretation of the references to color in this figure legend, the reader is referredto the web version of this article.)

Table 2Solubility of IMC in solvents and a solvent mixture.

94 H. Wickström et al. / European Journal of Pharmaceutical Sciences 75 (2015) 91–100

2.8. Content analysis

The IMC content analysis of the printed 1 � 1 cm2 squares wasdetermined in phosphate buffer pH 5.0 (Ph.Eur.) at 37 �C in tripli-cate. The printed doses were put in 100 ml of the buffer and wereheld on a shaker for 3 h. The absorbance was measured at 265 nmusing an UV/VIS-spectrophotometer (Ultrospect 2100 pro,Biochrom Ltd., Cambridge, UK). Reference measurements weredone with a blank substrate. The method was linear for IMC con-centrations between 0.2 and 50 lg/ml (R2 = 0.99).

Solvents/solvent mixture Solubility (mg/ml)

DMSO 487.8 ± 17.5PG 11.0 ± 0.5PG:DMSOa 236.0 ± 17.5

a Prepared in a 30:70 vol% ratio.

2.9. Drug release

The release of the poorly soluble drug IMC from the printedpaper substrates before and after oven drying was monitored dur-ing 60 min. The drug release of IMC from the samples printed ontransparency film was also analyzed. The dissolution testing ofthe printed samples (n = 3) was performed using an in-housedeveloped experimental setup with a rotation speed of 150 rpm.The samples were placed in metal sinkers to prevent floating andintroduced to 100 ml of phosphate buffer pH 5.0 at the tempera-ture of 37 �C. Manual sampling without medium replacement atpre-determined time points was conducted. Concentration of thesamples was determined using an UV/VIS-spectrophotometer(Ultrospect 2100 pro, Biochrom Ltd., Cambridge, UK) at 265 nm.The dissolution rates of the printed samples were compared toraw crystalline IMC. The dissolution rate of the reference crys-talline IMC was measured under sink conditions (m = 2.0 mg ofIMC in 500 ml phosphate buffer pH 5.0 at the temperature of37 �C). Due to the sensitivity limits the volume of the dissolutionmedia was scaled down because of the lower API amount in theprinted samples. The dissolution rates were expressed as acumulative amount of drug (in %) released over time. Percentageswere calculated based on the average dose gained from the contentmeasurements.

The effect of sample drying at elevated temperature in the vac-uum oven on the dissolution rate was also studied. The samplesdried in room temperature (hereafter called untreated samples)were compared with the samples dried in the vacuum oven (here-after called treated samples). The dissolution rate time point valuesfor the treated and untreated samples were statistically analyzedusing Students unpaired t-test with a significance level ofp-value > 0.05 using Microsoft Excel software.

3. Results and discussion

3.1. Characteristics of the API and the excipients

IMC was chosen as a model compound for this study, due to itsphysicochemical properties such as poor water solubility, acidicnature (pKa = 4.5) and existence of the polymorphic forms.Furthermore, the yellow color of the drug when dissolved or inamorphous form enables a visual characterization of the printeddoses on the substrates (Fig. 3). The drug solubility was the limit-ing factor. The highest solubility was seen for IMC in DMSO, whichis a solvent with low viscosity and high surface tension (Table 2).Since the rheological properties of the ink could be improved withregard to printability, the solubility of IMC was also determined inthe viscosity increasing agent PG. Furthermore, the solubility ofIMC was determined in the solvent mixture PG:DMSO(30:70 vol% ratio). Consequently, the ink concentrations chosenfor the ink formulations, 50 mg/ml and 200 mg/ml, were basedon the IMC solubility.

Only a limited number of ink excipients have been studied withregard to their influence on the characteristics of the printed DDS.

In this study, the differences gained from the addition ofexcipients into the DDS were studied. The small molecular weightadditive ARG was chosen as a stabilizing excipient to form theIMC_ARG_50 and the ARG_IMC_ARG formulations (Table 1). Thechoice was based on the results from a previous study, whichconcluded that amino acids could be favorable excipients whendealing with the stability and dissolution rate challenges of a drug(Löbmann et al., 2013a,b). The addition of the stabilizing polymerPVP to the ink formulation (IMC_PVP_50) was also studied. PVPwas chosen because it has shown to inhibit the recrystallizationbehavior of amorphous IMC through hydrogen bonding betweenthe drug and the PVP molecules in the molecular dispersions pre-pared by Matsumoto and Zografi (1999). PVP has previously beenused as an ink excipient when the ink distribution was done usingsyringe pumps and piezoelectric dispensers (Hsu et al., 2013;Scoutaris et al., 2011).

Porous cellulose was chosen as a substrate because a previousstudy showed that it serves as a good reference with adequateabsorptive properties when flexible doses were printed (Geninaet al., 2013a). Furthermore, printability tests of pharmaceuticalinks have also shown to be possible on theses substrates. In thisstudy, substrates such as sugar and rice paper were also initiallystudied. However, these substrates were seen to disintegrate asthe ink was printed onto the substrate. Transparency films werechosen as an impermeable reference substrate.

3.2. Properties of the ink formulations

To be able to deposit the inks in a controlled way, the inkformulations should possess good physical properties to achievea good fluid flow, droplet formation and, consequently, a robustprinting result (Jang et al., 2009). Viscosity values ranging from 1to 30 mPa s and surface tension values from 25 to 50 mN/m havebeen reported in various studies to give a uniform droplet forma-tion (Genina et al., 2013b; Huang et al., 2012; Jang et al., 2009;Kamyshmy and Magdassi, 2012; Sandler et al., 2011). Yet, the opti-mal range of viscosity and surface tension values are usually men-tioned in the manual of the printer and it is still important toremember that the values are device dependent (Kolakovic et al.,2013). An appropriate viscosity range for the PixDro LP 50equipped with the Spectra SE-128 AA printhead is 8–20 mPa saccording to the Spectra SE-128 AA manual (Dimatix, 2010). Theoptimal surface tension range was, however, not mentioned. Thephysical properties of the inks and the solvent mixtures measuredare displayed in Table 3. The ink properties were initially measuredfor the solvent mixtures without API and excipients in order to getan understanding about the printability of the solvent systems. Themeasured properties of the ink formulations as well as the ejected

Table 3Properties of the solvent mixtures and the IMC ink formulations measured at25–26 �C.

Viscosity(mPa s)

Surfacetension(mN/m)

Density(kg/l)

Z-value Dropletvolume (pl)

PG:DMSOa 4.7/5.6 42.6/42.2 1.07/1.07 8.5/7.1 –/–IMC_50 5.4 42.6 1.05 7.3 13.1 ± 1.0IMC⁄ – – – – 13.2 ± 1.6IMC_ARG_50 7.8 48.3 1.04 5.3 12.1IMC_PVP_50 5.8 43.0 1.06 6.9 16.0 ± 3.0IMC_200 7.8 44.9 1.09 5.3 8.9 ± 1.0

a Prepared in a 30:70/40:60 vol% ratio.

Fig. 4. The average pulse shape set for the printing tasks. (a) The time to reach theset voltage (4 ls), (b) the time period when voltage is applied on the piezoelectricelement (10 ls), and (c) the time period as the voltage decreases to the initial setpoint (3 ls). The pulse shape plays an important role in the droplet formation andejection.

H. Wickström et al. / European Journal of Pharmaceutical Sciences 75 (2015) 91–100 95

droplet volumes are displayed in Table 3. Different rheologicalproperties of the inks have been reported to influence the dropletformation and the droplet volume (Dong et al., 2006).Furthermore, in this study the printing parameters showed to havea minor influence on the droplet volume.

3.3. Printability prediction and printing parameters

The printability of an ink can be predicted based on the Z-valueof the inverse Ohnesorge number and it is calculated according toEq. (1). The value is based on the viscosity, surface tension anddensity values of the inks as well as the nozzle diameter(Kamyshmy and Magdassi, 2012). Unfavorable physical parame-ters of inks give rise to unfavorable droplet formation which ischaracterized as droplets with long tails or droplets having a satel-lite droplet traveling alongside the actual droplet (Genina et al.,2013b). Originally, Fromm (1984) estimated that uniform dropletswould be ejected from an inkjet printer if the Z-value of the inkwas more than 2 (Kamyshmy and Magdassi, 2012). Reis et al.(2005) reported that the most optimal droplets would be ejectedwith inks having a Z-value in the range of 1–10. Some years laterJang et al. (2009) conducted a study ending up with a slightlyshifted Z-value range of 4–14. Again few years later Kuscer et al.(2012) narrowed the range to 5–9 for optimal droplet formation.Yet, inks with both lower and higher values have been reportedto be printable (Kuscer et al., 2012). Inks with lower Z-values havebeen reported to form droplets with tails more easily, while inkswith high values have contributed to the formation of satellitedroplets traveling alongside the real droplet (Jang et al., 2009).The Z-values of the inks prepared in this study were 5.3–7.3 andconsequently uniform droplets were formed, when suitable print-ing parameters were applied. All in all, droplet formation and vol-ume depend to a high extent on fluid properties of the ink and onthe choice of printhead (nozzle size) attached to the printer.

Z ¼ ðdqcÞ1=2

gð1Þ

The diameter of the orifice (d), density (q), surface tension (c)and viscosity (g) are used to calculate the Z-value (Kamyshmyand Magdassi, 2012).

In order for a droplet to be formed and ejected, a current has tobe applied on the piezoelectric element of the PIJ. This causes theelement to deform, which forces a droplet to be ejected throughthe nozzle (Scoutaris et al., 2011). Consequently, the droplet for-mation and volume are also influenced by the printing parametersapplied. The voltage and the pulse shape, also called the piezoelec-tric activation voltage wave form, are key parameters to beadjusted and optimized with regard to droplet formation and size(Mogalicherla et al., 2014). Furthermore, other parameters to be setbefore printing are ink pressure and firing frequency. The voltagesapplied on the piezoelectric elements in this study varied between

80 and 92 V and were systematically determined for each ink.Some small changes on the pulse shapes were also done for the dif-ferent ink formulations (Fig. 4). Resolution and firing frequencywere further parameters to be set before the printing task.

3.4. Characterization of the printed DDS

The yellow colored ink formulations printed on fibrous papersubstrates and transparency film were characterized using ATR–IR spectroscopy and analyzed with regard to peak shifts of thefunctional groups of the components, e.g. ink formulations andsubstrate. The solid state properties of IMC could not be deter-mined for the formulations printed on fibrous paper substrates,neither before nor after drying in vacuum oven, due to stronginterference from the substrate. Even though the solid statecharacterization was not conclusive, the yellow color indicatedthat the API was either in a dissolved or an amorphous form. Thesamples remained yellow after 6 months of storage in room tem-perature and after drying at elevated temperature in vacuum oven.

Solid state characterization was successfully conducted for theARG_IMC_ARG formulation printed with two separate inks (IMC⁄

and ARG⁄) on transparency film. The spectra of the printed DDSwere compared with the spectra of amorphous IMC, crystallineARG and the transparency film (Fig. 5). The vibrational modes at1500–1750 cm�1 were seen for amorphous IMC and crystallineARG corresponding to carbonyl and carboxylic acid group vibra-tions alongside with amino and guanidyl group vibrations forARG (Löbmann et al., 2013b). These absorbance bands were seenas a plateau with a small peak at 1590 cm�1 for theARG_IMC_ARG sample referring to a co-amorphous system asreported earlier by Löbmann et al. (2013b). Peak shifts were foundupon co-amorphization by ball-milling (Löbmann et al., 2013b).These changes were also observed on the spectrum forARG_IMC_ARG prepared by PIJ. The typical vibration modes of car-boxylic acid for amorphous IMC at 1707 and 1734 cm�1 were notseen in the spectra of the ARG_IMC_ARG sample. The formationof a salt was seen, since the vibrational mode of the amide II incrystalline ARG at 1551 cm�1 was seen as a shoulder at1554 cm�1 in the co-amorphous sample. Similar peak shifts havepreviously been reported (Löbmann et al., 2013b).

API inks have been applied onto different substrates such asuncoated paper, coated paper, edible icing sheets, polyethyleneterephthalate (PET) films, hydroxypropylcellulose (HPC) films,hydroxypropylmethylcellulose (HPMC) films and chitosan films(Genina et al., 2012, 2013b; Hsu et al., 2013; Janßen et al., 2013;Raijada et al., 2013). Routine solid state analysis has been men-tioned to be a challenge when API has been applied onto sub-strates, mainly because of the small amounts of printed dosesand/or the interference from the substrate (Genina et al., 2013b;Raijada et al., 2013). The solid state properties of samples in

Fig. 5. ATR–IR spectra of crystalline ARG, amorphous IMC, printed ARG_IMC_ARGformulation and transparency film. The spectra are offset in absorbance for clarity.

96 H. Wickström et al. / European Journal of Pharmaceutical Sciences 75 (2015) 91–100

crystalline form have been possible to detect on HPC, HPMC andPET films using X-ray diffraction (XRD) and differential scanningcalorimetry (DSC) (Genina et al., 2013b; Janßen et al., 2013). Yet,

Fig. 6. SEM images of the pure paper substrate and of a printed sample before drying in v(a) Cellulose fiber as seen in the pure substrate, (b) crystals of filler as seen in the pure pap(d) IMC_ARG_50, 6 layers before drying seen in the area of crystalline paper filler.

characterization using XRD and DSC has not been successfully uti-lized on edible icing sheets and similar porous paper substrates(Genina et al., 2013a; Raijada et al., 2013). Near-infrared (NIR)hyperspectral imaging has successfully been utilized as a quan-tification of the printed amount of API on uncoated copy paper(Vakili et al., 2014). However, this would not have helped for thesolid state characterization. In this study, ATR-IR analysis, micro-scopy and visual inspections were considered to provide adequateinformation at this time for the copy paper substrates as detectionlimits prevented us from gaining more data.

3.5. SEM and EDX

SEM was used to study the morphology and the distribution ofIMC on the printed samples both before and after drying in the vac-uum oven. Images of the printed ink formulation on the porouspaper substrates were compared with the pure paper substratereference consisting of cellulose fibers and crystals of filler mate-rial. A partially coated surface was distinguished on the printedsamples (Fig. 6). No IMC crystals could be detected from theuntreated and treated samples 6 months after printing. No signifi-cant morphological changes could be distinguished between thetreated and the untreated samples. SEM-EDX confirmed the pres-ence of IMC in the DDS.

3.6. Theoretical dose

The theoretical dose for each formulation was calculated basedon the concentration of the ink, resolution, printed area and dro-plet volume. In this study, the resolution was set on 500 � 500dpi and the inks were printed on an area of 1 � 1 cm2. With these

acuum oven. Images were taken with a 25,000�magnification (scale bar of 200 nm).er substrate, (c) IMC_ARG_50, 6 layers before drying seen on the fiber structure, and

Table 4Calculated theoretical dose (lg) of IMC in the printed formulations.

Layers IMC_50 IMC_ARG_50 IMC_PVP_50 IMC_200

1 24.5 23.0 30.4 67.73 76.4 69.0 91.3 203.16 152.9 138.0 182.5 406.19 229.3 207.0 273.8 609.2

H. Wickström et al. / European Journal of Pharmaceutical Sciences 75 (2015) 91–100 97

settings a total number of 38,025 droplets were printed per layer.The inks were prepared to have IMC concentrations of 50 mg/mland 200 mg/ml. Since the resolution, area and concentrations werefixed, the varying droplet volumes were the reason for the differentdoses. The rheological properties of the ink formulations affectedthe droplet volumes and the average volumes displayed inTable 3 were based on snapshots (n = 20) of the droplets usingthe DropAnalysis function in the PixDro software prior to the print-ing task. The doses of IMC_50 and IMC_ARG_50 inks with smalleraverage droplet volumes (12–14 pl) contributed to slightly smallerdoses compared to IMC_PVP_50 (13–19 pl) (Table 4).

3.7. Content analysis

Accurately printed doses, within the samples of the sameamount of layers, were produced (Table 5). API doses from 0.19to 1.13 mg were manufactured when one to nine layers wereapplied onto the porous paper substrates. RSD% below 3% wereobtained for the 50 mg/ml doses and slightly higher RSD% wereseen for the 200 mg/ml IMC ink formulation printed on the papersubstrates. The formulation ARG_IMC_ARG, where four layers ofIMC ink was printed on transparency film, was seen to have a smal-ler dose (165.9 lg) with a higher SD value (±25.2) and RSD%(15.2%). Doses with low SD% and RSD% have previously beenprinted using the PIJ Dimatix DMO-2800 (Genina et al., 2012;Raijada et al., 2013; Sandler et al., 2011). The printed API doseswere only prepared using one nozzle in the above mentioned stud-ies. In this study the doses were printed using 73–128 nozzles andno correlation regarding the content uniformity could be seenwhen comparing the samples printed with a lower or higher num-ber of nozzles. A favorable advantage of the increased number ofnozzles in use was the reduction of the manufacturing time forthe printing task. It took 2 min per layer to print 15 doses usingall 128 nozzles.

3.8. Dose escalation

In this study, dose escalation was achieved by varying the num-ber of printed layers. Interestingly, the measured dose deviatedfrom a linear dose escalation based on the theoretical values. Thedrug amount per layer decreased as the layers increased. Theamount of drug applied per layer was 50–80 lg for the samplesprinted with 50 mg/ml inks and 125–191 lg for the samplesprinted with IMC_200. Dose escalation studies by applyingvarious number of layers have been done earlier using thermalinkjet printers (TIJ) with the face-down (Deskjet, D1660,

Table 5The content analysis of the printed IMC samples with the relative standard deviation (%).

IMC_50 IMC_ARG_50

Layers Amounta RSD% Amounta RSD%

1 – – – –3 194.8 ± 5.3 2.7 194.6 ± 2.4 1.36 318.9 ± 2.8 0.9 324.0 ± 2.2 0.79 451.8 ± 2.9 0.6 452.7 ± 3.9 0.9

a Data are presented as mean (lg) ± standard deviation (n = 3).

Hewlett-Packard Inc.) and face-up loaded paper designs (PixmaMP495, Canon Inc.) (Buanz et al., 2011; Genina et al., 2013a).Dose wear off seen for the printed doses manufactured with theTIJ printers mentioned above, has been explained by the shearingforce caused by the feeding rollers during substrate feeding. Onlyone dose escalation study has previously been performed using anon-contact PIJ printer where the substrate was situated on a sub-strate plate (Genina et al., 2013b). That study obtained flexibledoses by varying either the drop spacing (resolution) or the areaof the printed sample.

In this study, the printed doses of IMC (Table 5) were roughly2–3 times higher compared to the calculated theoretical values.Similar variations have also been reported in previous studies per-formed using PIJ (Genina et al., 2013b; Raijada et al., 2013; Sandleret al., 2011). Sandler et al. (2011) have reported both 25% higherand 31% lower amounts compared to the theoretical values, whileGenina et al. (2013b) have reported only higher values. However,Raijada et al. (2013) managed to print a dose that was equal tothe theoretical dose. Both differences in the ink properties andthe parameters set during the printing task affect the droplet vol-ume (Dong et al., 2006; Genina et al., 2013b; Jang et al., 2009). Yet,the reason for the increasing mismatch between the theoreticaland actually printed dose as the layers increases is somewhatunclear. Dose drifting is likely to happen during the printing pro-cess and could be affected by changes in different printing parame-ters (ink pressure, voltage, pulse shape, printhead temperature). Tobe able to control operating parameters, such as pressure levelsand vacuum, has been reported to be important to ensure ejectionof expected droplet volumes (Lyman, 2015). If the pressure is toohigh, droplets meant to be printed are sucked into the ink con-tainer and if the pressure is too low a larger droplet is ejected. Ifthe operating parameters could sensitively be monitored and keptin a predefined acceptable range, accurate dose escalation wouldbe possible. Based on the high printing accuracy, the fast manufac-turing and the possibility to control operating parameters, PIJ couldbe viewed as a potential method of fabricating personalized doses.

3.9. Drug release studies

Increased dissolution rates were obtained for all printed IMCformulations compared to the raw crystalline reference (Fig. 7).After 20 min over 90% of IMC was released from the untreatedIMC_50, IMC_ARG_50 and IMC_PVP_50 samples and the drugwas entirely released from treated samples. Within the same time,a release of over 80% and 72% was seen for the 200 mg/mluntreated and treated samples, respectively. A burst effect wasseen for the untreated and treated IMC_50, IMC_ARG_50 andIMC_PVP_50 samples during the first 10 min of dissolution. Aslower dissolution behavior was observed for the IMC_200, forwhich the release rate decreased as a function of applied layers.In general, the even and accurate spatial distribution of the inksresulting in an increased surface area of the API that is in contactwith the dissolution media, is one of the reasons for the increaseddissolution rates obtained in this study. An increased dissolutionrate was also seen for the ARG_IMC_ARG samples printed on an

IMC_PVP_50 IMC_200

Amounta RSD% Amounta RSD%

– – 191.4 ± 12.4 6.5238.8 ± 6.8 2.9 430.7 ± 22.7 5.3393.1 ± 3.9 1.0 797.3 ± 26.8 3.4557.3 ± 13.3 2.4 1130.7 ± 34.1 3.0

Fig. 7. Release profiles after drying the samples at elevated temperature in vacuum oven (treated samples). Reference and the 3, 6 and 9 layer samples of (a) IMC_50, (b)IMC_ARG_50, (c) IMC_PVP_50 and 1, 3, 6 and 9 layer samples of (d) IMC_200 and 4 layer IMC sample of (e) ARG_IMC_ARG. The reference was crystalline IMC. Data arepresented as mean ± standard deviation (n = 3). 1 layer, 3 layers, 4 layers, 6 layers, 9 layers, and IMC.

98 H. Wickström et al. / European Journal of Pharmaceutical Sciences 75 (2015) 91–100

impermeable transparency film. The release rate was slightly fasterdue to the properties of the substrate. More than 120% of IMC wasreleased and this can be explained by the high SD and RSD% of theprinted API content that was used for the calculations in therelease studies.

The addition of ARG and PVP as excipients did not show to haveany effect on the dissolution rate of IMC_ARG_50 or IMC_PVP_50compared to the formulation containing only IMC (IMC_50).Furthermore, no significant differences were seen when comparingthe release rates values at specific time points for the untreatedand treated samples (p > 0.05). The only statistically significanttime point was seen at 15 min when the untreated and treatedIMC_200 samples were compared. Stability studies should be con-ducted in the future, since then the dissolution rates might be dif-ferent for the formulations with and without excipients.

ATR-IR spectra did not reveal any differences between theuntreated and the treated samples printed on porous cellulose.Moreover, the method did not allow identification of the solid stateproperties of the drug, due to the strong interference from the sub-strate. Evaporation of the solvents used in the inks was initially

checked by pipetting 10 ll of the IMC_200 ink onto the paper sub-strate. Amorphous IMC (1680 cm�1) was detected with ATR-IRimmediately after pipetting ink onto the porous substrate and alsoafter drying at elevated temperature in vacuum oven. DMSO andPG were detected for the wet samples and evaporation of DMSOwas confirmed after drying treatment (data not shown). Thedetected amount of IMC applied by pipetting corresponded toalmost double of the highest amount printed with the IMC_200ink.

Increased dissolution rates for the printed systems compared tocrystalline reference could be attributed in this study for the sam-ples prepared on porous substrates by the two following reasons:(1) IMC was present in an amorphous form and (2) IMC was stilldissolved in the viscosity modifier PG. The deposition of small dro-plet volumes, fast drying and absorption of the ink into the poroussubstrate could contribute to having the drug in an amorphousform. Increased dissolution rate has earlier been reported for APIdoses printed with inkjet technology (Raijada et al., 2013).Viscosity increasing agents such as PG and glycerol has been sug-gested to contribute to fast release rates by keeping the API in a

H. Wickström et al. / European Journal of Pharmaceutical Sciences 75 (2015) 91–100 99

dissolved form. Furthermore, these viscosity modifying agents incombination with thermal treatment have also been suggested tobe the reason for the existence of two polymorphs in the samples(Meléndez et al., 2008). Since there was no difference in the dis-solution rates for the untreated and treated samples after 6 monthsof storage one could assume that the amorphous form was par-tially stabilized by the substrate. Moreover, the samples remainedyellow for at least 6 months which would indicate that IMC was inan amorphous form. The increased dissolution rate for theARG_IMC_ARG printed on transparency film was achieved by theformation of co-amorphous systems.

3.10. Formulation guidance

Based on the results from this study the formulation guidancefor poorly soluble drugs is proposed below. The main steps to con-sider when developing printed DDS using inkjet technology arepresented in Fig. 8. Herein, the factors to consider with regard toprintability are discussed.

The poorly water soluble drugs need to be dissolved in non-toxic organic solvent approved for pharmaceutical applicationand the solubility sets the base for the possible concentration ofthe ink. Favorable physical properties with regard to printabilityset the base for the ink formulation design. Furthermore, calcula-tion of the Z-value during the development phase is a favorabletool to ease the estimation of ink printability. The droplet forma-tion and volume are dependent on the physical properties of theink formulations alongside with the printing parameters applied.Furthermore, printheads with different nozzle sizes also affectthe droplet volume and the printability. These factors in turn affectthe dose and the content uniformity of the printed DDS.

Characterization of the solid state properties of the printed DDShave so far shown to be a challenge mainly because of the smallamounts of printed doses and/or the interference from the poroussubstrate (Raijada et al., 2013). Yet, the detection of the solid stateproperties is important to be able to control the stability and releaserate of the API in the DDS. The characteristics of the substrate mayalso set limitations with regard to dose escalation. Linear dose esca-lation might be possible if the operating parameters could

Fig. 8. Formulation guidance

sensitively be monitored and kept in a predefined acceptable rangeand if real time monitoring of the droplet volume could be con-ducted. This would result in better understanding of the productionprocess and also of the droplet volumes applied throughout theprinting task. Accurately printed doses and desirable dissolutionbehavior have also previously been reported for printed DDS(Genina et al., 2012; Raijada et al., 2013). The dissolution rate isaffected by several factors as (1) the even and spatially accuratelydistributed ink, (2) the printed drug amount, (3) the remaining sol-vents on the substrate, (4) the drying/coating and (5) the substratecharacteristics. The knowledge above sets a base for further designand development of more sophisticated DDS.

4. Conclusions

Ink formulations with favorable physical properties containingIMC were successfully prepared. As a consequence, accuratelyprinted DDS and flexible doses were fabricated using piezoelectricinkjet technology. The ink formulations with ARG and PVP showedsimilar release properties as the inks without excipients. Furtherstudies are needed to investigate the effect of these excipients.Also systematic studies on the effect of the solvents remaining inthe carrier substrate structure and solvent evaporation are neededto fully understand the behavior of the printed systems. The evenand accurate spatial distribution of the deposited ink was shownto make a relevant contribution to the increased dissolution ratefor all printed DDS. The solid state of the formulation was success-fully determined and co-amorphous systems were formed on thetransparency film. Although the solid state properties of IMC couldnot be determined for the samples on fibrous paper substrate, theyellow color of the printed formulations suggest that IMC remainedin a dissolved or an amorphous form after solvent drying and up to6 months storage at room temperature. Previously stated findingsare believed to lead to improved dissolution rates of the printedsamples compared to the crystalline reference. Overall, it is clearthat printing is a complex interplay of many aspects, ranging fromthe printer and printhead technology used to the ink and substrateproperties. The challenges open up the need to further explore and

for poorly soluble drugs.

100 H. Wickström et al. / European Journal of Pharmaceutical Sciences 75 (2015) 91–100

study the true potential of inkjet technology in personalization ofdosing and fabricating tailored drug delivery systems.

Acknowledgements

Kristian Gunnelius is thanked for conducting the rheologicalmeasurements. Henrika Wickström would like to acknowledgefunding from Waldemar von Frenckell Foundation.

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