Photocurable Bioinks for the 3D Pharming of Combination Therapies

Article Photocurable Bioinks for the 3D Pharming of Combination Therapies

Giovanny F. Acosta-Vélez 1 , Chase S. Linsley 1 , Timothy Z. Zhu 1, Willie Wu 1 and Benjamin M. Wu 1,2,*

1 Department of Bioengineering, University of California, Los Angeles, 420 Westwood Plaza, Room 5121, Engineering V, 951600 Los Angeles, CA 90095, USA; [email protected] (G.F.A.-V.); [email protected] (C.S.L.); [email protected] (T.Z.Z.); [email protected] (W.W.) 2 Division of Advanced Prosthodontics and the Weintraub Center for Reconstructive Biotechnology, University of California, 951600 Los Angeles, CA 90095, USA * Correspondence: [email protected]; Tel.: +1-(310)825-6215

Received: 11 November 2018; Accepted: 10 December 2018; Published: 11 December 2018

Abstract: Combination therapies mediate drug synergy to improve treatment efficacy and convenience, leading to higher levels of compliance. However, there are challenges with their manufacturing as well as reduced flexibility in dosing options. This study reports on the design and characterization of a polypill fabricated through the combination of material jetting and binder jetting for the treatment of hypertension. The drugs lisinopril and spironolactone were loaded into hydrophilic hyaluronic acid and hydrophobic poly(ethylene glycol) (PEG) photocurable bioinks, respectively, and dispensed through a piezoelectric nozzle onto a blank preform tablet composed of two attachable compartments fabricated via binder jetting 3D printing. The bioinks were photopolymerized and their mechanical properties were assessed via Instron testing. Scanning electron microscopy (SEM) was performed to indicate morphological analysis. The polypill was ensembled and drug release analysis was performed. Droplet formation of bioinks loaded with hydrophilic and hydrophobic active pharmaceutical ingredients (APIs) was achieved and subsequently polymerized after a controlled dosage was dispensed onto preform tablet compartments. High-performance liquid chromatography (HPLC) analysis showed sustained release profiles for each of the loaded compounds. This study confirms the potential of material jetting in conjunction with binder jetting techniques (powder-bed 3D printing), for the production of combination therapy oral dosage forms involving both hydrophilic and hydrophobic drugs.

Keywords: 3D printing; pharmaceutical tablets; poly(ethylene glycol); hyaluronic acid; photopolymerization; inkjet printing

1. Introduction Personalized medicine is becoming a reality. For instance, most hearing aids are now custom-ﬁt to each user’s ear canal [1] and the manufacturing process for InvisalignTM aligners and retainers utilizes 3D printing [2]. Additionally, custom bioresorbable tracheal splints fabricated using laser-based 3D printing were successfully used to treat children with tracheobronchomalacia [3]. Recently, interest in applying 3D printing to the manufacturing of pharmaceutical tablets—known as 3D pharming [4]—has been gaining traction since it can allow for on-demand manufacturing of personalized pharmaceutical dosage forms [5,6]. It also allows for the creation of dosage forms with increased complexity, including the manufacturing of oral dosages containing multiple active pharmaceutical ingredients (APIs), which aim to enhance patient compliance by reducing the number of pills required on a daily basis [7].

Polymers 2018, 10, 1372; doi:10.3390/polym10121372 www.mdpi.com/journal/polymers Polymers 2018, 10, 1372 2 of 12

Additionally, several medical conditions utilize combination therapies to improve treatment efficacy, such as hypertension, HIV infection, depression, type 2 diabetes, and cancer [8–17]. There are several challenges to manufacturing dosage forms containing multiple APIs as well as dosing constraints that impact treatment efficacy. Currently, a few fixed dose combinations of varying strength are created for any given combination therapy. However, patients often require dose adjustments to the extent that a tablet with multiple APIs is not a suitable form factor [18]. Additionally, there are challenges to manufacturing multiple-API dosage forms [19]. For instance, a common challenge is designing a single dosage form that combines two chemically incompatible APIs that have to be kept separated. This can result in higher manufacturing costs since a more complex dosage form has to be produced. Three-dimensional pharming is uniquely positioned to overcome these obstacles. In fact, techniques such as fused deposition modeling [20] and extrusion printing at room temperature [21,22] have demonstrated feasibility in the manufacturing of pharmaceutical tablets with multiple APIs. Material jetting technologies, however, allow for compositional control at a voxel level, thereby enabling precise dosage control that is tailorable to each individual patient. Additionally, material can be dispensed at room temperature, which is compatible with thermally labile APIs. The focus of this work was to demonstrate that 3D printing and the development of photocurable formulations can be combined to fabricate pharmaceutical tablets with controlled dosages of both hydrophilic and hydrophobic APIs, and act as a potential carrier for personalized medicine treatments. Previously, this lab engineered a hyaluronic acid-based photocurable bioink for the 3D pharming of hydrophilic compounds via material jetting [23]. Additionally, this lab engineered a poly(ethylene glycol) diacrylate-based bioink capable of loading hydrophobic drugs [24]. In this paper, both bioinks are used to manufacture a tablet with multiple APIs used to treat hypertension. Specifically, the APIs lisinopril and spironolactone were chosen as model drugs for this combination therapy. Lisinopril, an angiotensin-converting enzyme inhibitor (ACEI) for the treatment of hypertension [25,26], was chosen due to its hydrophilicity, and spironolactone, a potassium-sparing diuretic for the treatment of hypertension and congestive heart failure [27], was selected because of its hydrophobicity. The formulated bioinks were dispensed through a piezoelectric nozzle at room temperature into a blank preform tablet featuring two compartments, one for each formulation. Compartmentalizing the preform tablet was required in order to expose each formulation, separately, to the required light exposure times resulting in optimal mechanical properties and release profiles. The preform tablet parts were manufactured by binder jetting process, using calcium sulfate powder as the excipient. The drug-loaded bioinks were subsequently photopolymerized, and the preform tablet parts were assembled to finalize the pharmaceutical dosage form. This tablet produced a sustained drug release profile for both APIs and demonstrated that this 3D pharming approach could successfully fabricate oral combination therapies at room temperature, with quick manufacturing times and controlled dosages.

2. Materials and Methods

2.1. Hydrophilic Photocurable Bioink Preparation Hyaluronic acid norbornene (HANB) was synthesized as previously described [23]. Briefly, hyaluronic acid (HA) (60 kDa·MW) (Genzyme Corporation, Cambridge, MA, USA) was modified with hydrazide groups through a reaction with adipic acid dihydrazide (ADH) in the presence of 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC). The product was dialyzed for 3 days against deionized water (DI) water (Fisherbrand regenerated cellulose, MWCO 12,000–14,000 Da, Houston, TX, USA), frozen, and lyophilized. On a second reaction, the HA functionalized with hydrazide groups (HA–ADH) was reacted cis-5-norbornene-endo-2,3-dicarboxylic anhydride (Sigma–Aldrich, St. Louis, MO, USA), resulting in norbornene-functionalized HA. The product was dialyzed against DI water for 3 days, filtered, lyophilized, and stored at −20 ◦C. Polymers 2018, 10, 1372 3 of 12

Norbornene-functionalized HA was characterized by proton nuclear magnetic resonance spectroscopy (1H NMR) on a Bruker AV300 broad band FT NMR Spectrometer (Billerica, MA, USA). The degree of modiﬁcation obtained was ~50%. Following its synthesis, HANB was dissolved in PBS and mixed with poly(ethylene glycol) dithiol (1500 Da, PEGDT) at a crosslinking ratio (ratio of thiol groups to norbornene groups, rratio) of 0.6. Norbornene-functionalized HA was added at a weight percent (WHANB) of 3%. Eosin Y was added as a photoinitiator and poly(ethylene glycol) (PEG, 200 Da) was added to optimize the viscosity of the formulation for droplet formation. Each of these two components constituted 10% v/v of the bioink. Lisinopril dihydrate (Fisher Scientiﬁc, Pittsburgh, PA, USA) was added at a concentration of 40 mg/mL. All chemicals were purchased from Sigma–Aldrich unless otherwise stated.

2.2. Hydrophobic Photocurable Bioink Preparation The hydrophobic bioink was formulated as previously described [24], with minor modiﬁcations. Brieﬂy, the bioink was composed of 30% poly(ethylene glycol) diacrylate (PEGDA, 250 Da), 50% PEG200, and 20% ethanol. Eosin Y (1.0 mM) and mPEG-amine (0.05 M) (350 Da, Creative PEG Works, Chapel Hill, NC, USA) were added as photoinitiator and co-initiator, respectively. Spironolactone was added at a concentration of 20 mg/mL. All chemicals were purchased from Sigma–Aldrich unless otherwise stated.

2.3. Bioinks Gelation and Mechanical Properties The tensile strength of polymerized gels was analyzed to characterize the mechanical properties of the bioinks. One-mL syringes (BD & Co., Franklin Lakes, NJ, USA), modified by eliminating their tips, were loaded with 50 µL of bioink. Gels were formed with hydrophilic and hydrophobic bioinks by exposing them to visible light at an intensity of 120 mW/cm2 for a period of 2 min and 1 min, respectively. Additionally, the tensile strength of gels with varied drug concentrations was measured to analyze the impact of drug load on the mechanical properties of the polymerized bioinks. An Instron (5564 model) was used to measure the failure load of the gels fabricated. The tensile strength (σ) was calculated through Equation (1), where D is the gel diameter, H is the thickness, and F represents the failure load [28]. 2F σ = (1) πDH The inverse of the Ohnesorge number (Z value) of the bioinks was calculated to assess the printability of these solutions through inkjet printing piezoelectric nozzles. Equation (2) defines the Z value, where a is the radius of the piezoelectric nozzle printing orifice and ρ, γ, and η represent the density, surface tension, and viscosity of the photocurable formula, respectively [29].

(aργ)1/2 Z = (2) η

The surface tension was measured with a tensiometer (Kimble Chase 14,818 Tensiometer, Cole-Parmer, Vernon Hills, IL, United States) and calculated by using Equation (3), where h is the distance between menisci of the formulation in the test tube and the one in the capillary tube, r is the radius of the capillary, ρ is the density of the formulation, and g is the acceleration due to gravity [30]. One mL of each bioink was weighted and the mass obtained was divided by the pre-determined volume to calculate the density. The viscosity of the bioinks was measured with a rheometer (Discovery HR-2, TA Instruments, New Castle, DE, USA). A cone and plate geometry (using a 40-mm 2.016◦) was utilized for the experiment, with a shear rate ranging from 10 to 100 Hz.

1 γ = hrρg (3) 2 Polymers 2018, 10, 1372 4 of 12

2.4. Scanning Electron Microscopy (SEM) The morphology of the gels was observed with a NOVA 230 NanoSEM scanning electron microscope. Images of lyophilized hydrophilic gels were taken, as well as cross-sectional images of hydrophobic gels.

2.5. Preform Tablet Fabrication and Characterization The drug-containing bioinks were directly printed into tablet preforms that were fabricated by 3D printing (ProJet 660; 3D Systems, Inc.; Rock Hill, SC, USA). The printing materials consisted of calcium sulfate hemihydrate powder (VisiJet PXL Core; 3D Systems, Inc.; Rock Hill, SC, USA), and a liquid inkjet binder comprised of deionized water containing 5% ethanol and 0.25% Tween 80. The polypill preform tablet was designed with two separate chambers that could each hold up to 250 µL of ink and be assembled into a single tablet. The assembled dimensions were kept below the 22-mm maximum tablet size recommended by the Food and Drug Administration (FDA) [31]. Additionally, each chamber was independently modiﬁed to prevent absorption of the respective drug-containing bioink into the preform tablet during printing, as previously reported [23]. Brieﬂy, the chamber holding the hydrophobic formulation was infused with PEG (35 kDa) by submerging the tablet in an acetone solution containing 15% (w/w) PEG for 30 min at 55 ◦C. Next, the well was brush-coated with Eudagrit® E100 (Evonik, Essen, Germany) (polymethacrylate copolymer) dissolved in acetone at 20% (w/w). The chamber holding the hydrophilic formulation was infused with Eudagrit® E100 dissolved in acetone at 10% (w/w). Surface morphology of the preform tablet was characterized by scanning electron microscopy (NOVA NanoSEM 230, FEI Co., Hillsboro, OR, USA).

2.6. Drug Release Kinetics A piezoelectric dispenser with a nozzle diameter of 80 µm (MJ-ABP-01-080, MicroFab, Plano, TX, USA) was used to assess the droplet formation capability of the engineered bioinks. This dispenser was controlled with a microdispensing system (MD-E-3000, Microdrop, Norderstedt, Germany). The dispensing parameters used were 46 V, a 16-µm pulse width, and a frequency of 2000 Hz. The droplet formation process was captured with an analog camera (JAI CV-S3300), equipped with a lens (Edmund Optics, Barrington, NJ, USA). A light-emitting diode (LED) was connected to the microdispensing system in order to control its strobe delay. Lisinopril-loaded hydrophilic bioink was dispensed in the bottom part of the polypill (250 µL) and exposed to visible light (120 mW/cm2) for 2 min, to induce gelation of the hydrogel precursor solution. Spironolactone-loaded hydrophobic bioink was dispensed into the upper piece of the polypill (125 µL) and exposed to light for 1 min. Following the gelation of the bioinks, the pieces of the polypill were assembled to ﬁnalize the pharmaceutical product. The tablets were placed into uni-cassettes (Tissue-Tek) and immersed into beakers containing 500 mL of dissolution medium (monobasic potassium phosphate 1.053 mM, pH of 2.5), conditioned at 37 ◦C and stirred at 60 rpm. Aliquots of 1 mL were taken after 0.5, 2, 4, 6, 8, 12, 18, and 24 h. The volume removed was replenished with fresh dissolution medium conditioned at 37 ◦C. The drug concentration in each aliquot was measured via HPLC (Waters 2690 with a PDA 996 detector). The wavelength used to detect the APIs were 220 nm and 240 nm for lisinopril and spironolactone, respectively.

2.7. Statistical Analysis Statistical analysis was performed with GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA). Statistical signiﬁcance was assessed using single factor ANOVA test with a Tukey post-test and 95% conﬁdence interval. Polymers 2018, 10, 1372 5 of 12

3. Results and Discussion

Polymers3.1. Bioinks 2018, Characterization10, x FOR PEER REVIEW 5 of 12

Hyaluronic acid is a natural glycosaminoglycan glycosaminoglycan found in connective, neural, and epithelial tissutissueses [[32,33].32,33]. The The biocompatibility biocompatibility of of hyaluronic hyaluronic acid acid hydrogels hydrogels has has been been assessed assessed in in tissue engineering studies, studies, where where diverse diverse cell cell types types have have been been cultured cultured in inhydrogels hydrogels for for the the formation formation of tissuesof tissues and and the the study study of of biological biological processes processes [34 [34––36].36]. Moreover, Moreover, hy hyaluronicaluronic acid photocurable formulations can polymerize under quick gelation times ( (FigureFigure S1). Lisinopril was dissolved in a hyaluronic acid-basedacid-based hydrophilic bioinkbioink atat aa concentrationconcentration ofof 4040 mg/mL.mg/mL. The The l lisinoprilisinopril formulation was dispensed into into a a modified modified syringe syringe (50 (50 µL)µL) and and exposed exposed to tovisible visible light light at an at intensity an intensity of 120 of 120mW/cm mW/cm2 for 22 minfor 2(Figure min (Figure 1). The1). storage The storage modulus modulus of the resulting of the resulting hydrogel hydrogel was quantified was quantified to assess theto assessmechanical the mechanical properties of properties the polymerized of the polymerized bioink, due to bioink, its viscoelastic due to its property. viscoelastic Additionally, property. Additionally,hydrogels with hydrogels different withdrug differentconcentrations drug concentrations (20 and 10 mg/mL) (20 and were 10 polymerized mg/mL) were to polymerizedstudy the effect to ofstudy drug the load effect on of the drug mechanical load on the properties mechanical of this properties hydrophilic of this material hydrophilic (Figure material 2A). (Figure The results2A). Theindicate results that indicate this hydrophilic that this material hydrophilic has materiala low storage has a modulus low storage due modulusto its elevated due towater its elevated content waterand the content low W andHANB the(3%) low utilizedWHANB for(3%) its fabrication. utilized for The its fabrication.elevated water The content elevated of waterthe hydrogel content allows of the forhydrogel the effective allows diffusion for the effective of the hydrophilic diffusion of theAPI hydrophilic out of the oral API dosage out of theform, oral given dosage the form, dissoluti givenon the of thedissolution preform of tablet the preform designed tablet to disintegrate designed to under disintegrate acidic conditions under acidic similar conditions to the similarones found to the in ones the stomach.found in theThe stomach. hydrogels The had hydrogels an average had G’ an of average 1003.86 G’Pa. of Figure 1003.86 2A Pa. indicates Figure2 thatA indicates drug load that had drug no influenceload had noin influence the mechanical in the mechanical properties properties of the hydro ofgel, the hydrogel, where larger where drug larger concentrations drug concentrations had no impacthad no over impact the over G’ of the the G’ gels. of the This gels. result This is result consonant is consonant with the with G’ data the G’previously data previously shown [23], shown where [23], whereropinirole ropinirole-loaded-loaded at a concentration at a concentration of 40 mg/mL of 40 mg/mL had nohad impact noimpact on the onmechanical the mechanical properties properties of the gel,of the comp gel,ared compared to hydrogels to hydrogels with no with drug. no However, drug. However, a decrease a decrease in G’ was in G’noticed was noticedon hydrogels on hydrogels loaded withloaded ropinirole with ropinirole at a concentration at a concentration of 80 mg/mL. of 80 mg/mL. It can be It stated can be that stated the thatG’ remains the G’ remainsstable for stable this lforisinopril this lisinopril formulation formulation at concentrations at concentrations between between 0 and 0 and 40 40 mg/mL, mg/mL, the the maximum maximum lisinoprillisinopril concentration achievable in the hyaluronic acid solution.

Figure 1. 1. PolymerizedPolymerized drug drug-loaded-loaded bioinks. bioinks. The The upper upper row row shows shows images images of the polymerized hyaluronic acid acid-based-based bioink, bioink, loaded loaded with with lisinopril lisinopril atat a concentration a concentration of of40 40mg/mL. mg/mL. This This bioink bioink has W T ahas WHANB a HANBvalue valueof 3% ofand 3% a andTL of a 2 min.L of 2The min. bottom The bottom images imagesshow polymerized show polymerized poly(ethylene poly(ethylene glycol) W W T diacrylateglycol) diacrylate (PEGDA (PEGDA)) gels with gels a W withPEGDA avaluePEGDA of 30%,value a W ofEtOH 30%, value a ofEtOH 20%,value and a ofTL 20%,of 1.0 and min. a L of 1.0 min. Spironolactone was chosen as the second model drug in this study due to its high Spironolactone was chosen as the second model drug in this study due to its high hydrophobicity. hydrophobicity. The model drug was dissolved in a PEG-based bioink specifically engineered for The model drug was dissolved in a PEG-based bioink speciﬁcally engineered for hydrophobic drugs, hydrophobic drugs, at a concentration of 20 mg/mL. Fifty µL of formulation were pipetted into a at a concentration of 20 mg/mL. Fifty µL of formulation were pipetted into a modiﬁed syringe, modified syringe, the solution was exposed to light for 1 min (Figure 1), and the tensile strength of the solution was exposed to light for 1 min (Figure1), and the tensile strength of the resulting gel the resulting gel was measured (Figure 2B). Results show an average tensile strength for this material was measured (Figure2B). Results show an average tensile strength for this material of 176.66 kPa. of 176.66 kPa. The typical tensile strength of pharmaceutical tablets is within the range of 1 and 10 The typical tensile strength of pharmaceutical tablets is within the range of 1 and 10 MPa [37]. MPa [37]. The G’ obtained for this gel is well below this range, and therefore, this material should be The G’ obtained for this gel is well below this range, and therefore, this material should be used used in combination with a preform tablet that provides support and physical stability to the oral dosage form. Furthermore, the effect of drug concentration on the mechanical properties of the gel was studied by measuring the tensile strength of gels containing 100, 80, 40, and 20 mg/mL. Figure 2B shows that drug concentration had no impact on the tensile strength of the material. This contrasts with results previously demonstrated [24], where a decrease in tensile strength was observed with increasing naproxen and ibuprofen concentrations. This divergence is due to the lower WPEGDA value

Polymers 2018, 10, 1372 6 of 12 in combination with a preform tablet that provides support and physical stability to the oral dosage form. Furthermore, the effect of drug concentration on the mechanical properties of the gel was studied Polymersby measuring 2018, 10, thex FOR tensile PEER REVIEW strength of gels containing 100, 80, 40, and 20 mg/mL. Figure2B shows6 thatof 12 drugPolymers concentration 2018, 10, x FOR hadPEER no REVIEW impact on the tensile strength of the material. This contrasts with results6 of 12 usedpreviously in this demonstrated formulation (30%).[24], where These a resultsdecrease indicate in tensile that strength softer materials was observed consisting with increasingof photo- polymerizednaproxenused in this and bioinks, formulation ibuprofen such concentrations. as (30%). the hyaluronic These results This acid divergence indicatehydrogel, that istend due softer to to have the materials lowera steadyW consisting PEGDAmechanicalvalue of stability used photo in- whenthispolymerized formulation exposed bioinks, to (30%).increasing such These as drug the results hyaluronicconcentrations. indicate acid that hydrogel,Stronger softer materials gels tend can to experiencehave consisting a steady substantial of mechanical photo-polymerized differences stability inbioinks,when mechanical exposed such as properties to the increasing hyaluronic (Fig drugure acid S2). concentrations. hydrogel, tend Stronger to have agels steady can mechanicalexperience stabilitysubstantial when differences exposed toin increasingmechanical drug properties concentrations. (Figure S2). Stronger gels can experience substantial differences in mechanical properties (Figure S2).

Figure 2. 2. TensileTensile strength strength of of l lisinoprilisinopril and and s spironolactonepironolactone gels. ( (AA)) Tensile Tensile strength strength of of l lisinoprilisinopril tablets tablets withFigure diverse 2. Tensile drug strength concentrations. of lisinopril Forty and mg/mL spironolactone was was the the maximum maximumgels. (A) Tensile solubi solubility litystrength achieved achieved of lisinopril for for this tabletsactive pharmaceuticalwith diverse drug ingredient concentrations. ( (API)API) in Forty this mg/mL bioink formulation.was the maximum No No statistical solubility difference achieved was for thisobserved active betweenpharmaceutical samples ingredient with varying (API l lisinoprilisinopril) in this concentrations. bioink formulation. ( (BB)) Tensile Tensile No statistical strength strength differenceof of s spironolactonepironolactone was observed tablets withbetween diversediverse samples drugdrug with concentrations.concen varyingtrations. lisinopril One-hundred One-hundred concentrations. mg/mL mg/mL was( Bwas) Tensile the the maximum maximum strength solubility of solubility spironolactone achieved achieved fortablets thisfor thisAPI.with API. Nodiverse statisticalNo statistical drug differenceconcen differencetrations. was was observed One observed-hundred between between mg/mL samples samples was with the with varyingmaximum varying concentrations. solubility concentrations. achieved for this API. No statistical difference was observed between samples with varying concentrations. SScanningcanning electron microscopymicroscopy imagingimaging was was performed performed on on both both gels gels to to observe observe the the morphology morphology of ofthe the polymerizedS canningpolymerized electron structures. structures. microscopy Figure Figure3 imaging shows 3 shows an was irregulara nperformed irregular surface surfaceon both in both ingels both gels to observegels with with small the small crevicesmorphology crevices that thatfurtherof the further polymerized facilitate facilitate the structures. release the release of drugs, Figure of that drugs,3 shows otherwise that an otherwiseirregular diffuse out surface diffuse of the in gelsout both ofthrough gels the with gels their throughsmall polymerized crevices their polymerizedchemicalthat further structures. facilitatechemical The the structures wrinkles release. observed The of drugs, wrinkles in thethat observed hydrophilic otherwise in gel diffuse the (Figure hydrophilic out3A) ofare the gel a consequence gels (Figure through 3A) of are their the a consequencelyophilized/dehydratedpolymerized ofchemical the lyophilized/dehydrated structures nature of. theThe sample wrinkles nature tested. observed of the sample in the tested. hydrophilic gel (Figure 3A) are a consequence of the lyophilized/dehydrated nature of the sample tested.

Figure 3. CrossCross-sectional-sectional SEM SEM micrographs micrographs of of polymerized polymerized bioinks bioinks loaded loaded with with l lisinoprilisinopril and sspironolactone.Figurepironolactone. 3. Cross ( (A-Asectional)) Lyophilized Lyophilized SEM lisinopril lisinopril-loadedmicrographs-loaded of h polymerized hydrogelydrogel (40 (40 mg/mL) mg/mL) bioinks with loaded with a W a HANBW withHANB of l 3%isinoprilof and 3% anda r andratio a

ofrsratiopironolactone. 0.6.of This 0.6. This bioink (A bioink) wasLyophilized was exposed exposed l isinopril to a to T aL T-ofloadedL of 2 min. 2 min. hydrogel (B ()B Polymerized) Polymerized (40 mg/mL) hydrophobic hydrophobicwith a WHANB ink inkof 3% loaded loaded and a with rratio sspironolactoneofpironolactone 0.6. This bioink (20 mg/mL). was exposed This This formulation formulation to a TL of 2contained contained min. (B) a Polymerizeda WWPEGDAPEGDA of of20% 20% hydrophobic and and a T aLT ofL 1of ink min. 1 min. loaded with spironolactone (20 mg/mL). This formulation contained a WPEGDA of 20% and a TL of 1 min. 3.2. Droplet Formation 3.2. Droplet Formation To study the droplet formation ability of these bioinks, the inverse of the Ohnesorge number, denominatedTo study asthe Z droplet value, formation was calculated ability (Equation of these bioinks, 3). This the dimensionless inverse of the number Ohnesorge considers number, the inertiadenominated and surface as Z tension value, wasforces calculated of a fluid (Equation over its viscosity 3). This for dimensionlessces to define number its droplet considers formation the ability.inertia andThe orificesurface radius tension of forcesthe nozzle of a fluidused overfor the its inkjet viscosity printing forces of tothe define fluid itsis alsodroplet a factor formation taken intoability. consideration The orifice within radius this of the number. nozzle Z used values for between the inkjet 4 and printing 14 are of considered the fluid isprintable also a factor fluids taken [29]. into consideration within this number. Z values between 4 and 14 are considered printable fluids [29].

Polymers 2018, 10, 1372 7 of 12

3.2. Droplet Formation To study the droplet formation ability of these bioinks, the inverse of the Ohnesorge number, denominated as Z value, was calculated (Equation (3)). This dimensionless number considers the inertia and surface tension forces of a fluid over its viscosity forces to define its droplet formation Polymers 2018, 10, x FOR PEER REVIEW 7 of 12 ability.Polymers 2018 The, 10 orifice, x FOR radius PEER REVIEW of the nozzle used for the inkjet printing of the fluid is also a factor taken7 of 12 into consideration within this number. Z values between 4 and 14 are considered printable fluids [29]. Values above 14 typically exhibit the formation of satellite droplets, whereas values below four Values above above 14 14 typically typically exhibit exhibit the the formation formation of satellite of satellite droplets, droplets, whereas whereas values values below four below present four present strong viscous forces. The viscosity, surface tension, and density of the bioinks was strongpresent viscous strong forces. viscous The forces. viscosity, The surface viscosity, tension, surface and density tension, of the and bioinks density was of quantified, the bioinks in order was quantified, in order to determine their Z value. Table 1 shows the results obtained for these toquantified, determine in their orderZ value. to determine Table1 shows their the Z results value. obtained Table 1 for shows these parameters the results atobtained room temperature for these parameters at room temperature and the Z value for the two bioinks utilized in this study. The andparameters the Z value at room for the temperature two bioinks and utilized the inZ thisvalue study. for the The two hydrophilic bioinks bioink utilized experienced in this study. a higher The hydrophilic bioink experienced a higher viscosity value (9.83 cP), resulting in a lower Z value than viscosityhydrophilic value bioink (9.83 experienced cP), resulting a inhigher a lower viscosityZ value value than (9.83 the hydrophobic cP), resulting formulation. in a lower Z The value later than one the hydrophobic formulation. The later one had lower viscosity and surface tension parameters (4.88 hadthe hydrophobic lower viscosity formulation. and surface The tension later parametersone had lower (4.88 viscosity cP and 31.41and surface mN/m, tension respectively), parameters resulting (4.88 cP and 31.41 mN/m, respectively), resulting in a higher Z value of 10.52. However, both formulations incP aand higher 31.41Z mN/m,value of respectively), 10.52. However, resulting both in formulations a higher Z value fell within of 10.52. the However, defined range both formulations for printable fell within the defined range for printable fluids as depicted in Figure 4 and Figure 5, where the fluidsfell within as depicted the defined in Figures range4 for and printable5, where fluids the droplet as depicted formation in Figure sequence 4 and of Figure these bioinks5, where can the droplet formation sequence of these bioinks can be observed. bedroplet observed. formation sequence of these bioinks can be observed.

Table 1. Physical properties and Z value of formulated bioinks. Table 1. Physical properties and Z value of formulated bioinks. Bioink 풓 (mm) Ρ (kg/m3 3) 휸 (mN/m) 휼 (mPa·s) Z Bioink Bioink r 풓 (mm)P (kg/mΡ (kg/m) 3) 휸 (mN/m)γ 휼 (mPa η (mPa·s)· s) Z Z Hydrophilic 0.08 1022.27 57.76 9.83 6.99 HydrophilicHydrophilic 0.08 0.08 1022.271022.27 57.76 9.83 9.83 6.99 6.99 Hydrophobic 0.08 1048.00 31.41 4.88 10.52 HydrophobicHydrophobic 0.08 0.08 1048.001048.00 31.41 4.88 4.88 10.52 10.52

Figure 4. Droplet formation sequence for hyaluronic acid-based bioink with a WPEGDA of 3%, a rratio of Figure 4. Droplet formation sequence for hyaluronic acid-basedacid-based bioink with a WPEGDA ofof 3%, 3%, a arratiorratio of 0.6, and loaded with lisinopril at a concentration of 40 mg/mL. This formulation had a Z value of 6.99, of0.6, 0.6, and and loaded loaded with with lisinopril lisinopril at a at concentration a concentration of 40 of mg/mL. 40 mg/mL. This Thisformulation formulation had a had Z value a Z value of 6.99, of falling within the printable range (4–14). 6.99,falling falling within within the printable the printable range range (4–14). (4–14).

Figure 5. DropletDroplet formation formation sequence sequence for for PEGDA PEGDA-based-based bioink bioink with with a W a PEGDAWPEGDA of 30%of 30%and loaded and loaded with Figure 5. Droplet formation sequence for PEGDA-based bioink with a WPEGDA of 30% and loaded with withspironolactone spironolactone at a concentration at a concentration of 20 mg/mL. of 20 mg/mL. This formulation This formulation had a Z had value a Z ofvalue 10.52, of falling 10.52, within falling spironolactone at a concentration of 20 mg/mL. This formulation had a Z value of 10.52, falling within withinthe printable the printable range (4 range–14). (4–14). the printable range (4–14). 3.3. Preform Tablet Characterization 3.3. Preform Tablet Characterization 3.3. PreformThe role Tablet of the C preformharacterization tablet was to serve as a vessel for the formulations dispensed prior to their The role of the preform tablet was to serve as a vessel for the formulations dispensed prior to their curing,The role given of the that preform their polymerizationtablet was to serve occurred as a vessel after afor desired the formulations amount of formulation dispensed prior was allotted.to their curing, given that their polymerization occurred after a desired amount of formulation was allotted. Therecuring, was given a need that totheir compartmentalize polymerization theoccurred tablet forafter each a desired formulation amount added, of formulation since each was formulation allotted. There was a need to compartmentalize the tablet for each formulation added, since each formulation wasThere cured was a for need different to compartmentalize light exposure the times tablet to obtain for each the formulation desired mechanical added, since properties each formulation and drug was cured for different light exposure times to obtain the desired mechanical properties and drug releasewas cured proﬁles. for different Calcium light sulfate exposure hemihydrate times wasto obtain clinically the useddesired in themechanical preparation properties of plaster and of Paris,drug release profiles. Calcium sulfate hemihydrate was clinically used in the preparation of plaster of Paris, whichrelease is profiles. used for Calcium casts that sulfate immobilize hemihydrate fractures, was and clinically is not used in in the tablet preparation formulations of plaster [38]. Calciumof Paris, which is used for casts that immobilize fractures, and is not used in tablet formulations [38]. Calcium sulfatewhich is dihydrate, used for casts however, that immobilize has been commonly fractures, and used is innot pharmaceutical used in tablet formulations applications [38]. [39], Calcium and the sulfate dihydrate, however, has been commonly used in pharmaceutical applications [39], and the dihydratesulfate dihydrate, is formed however, when hemihydrate has been commonly is mixed with used water in pharmaceutical [40]. In this study, applications water was [39], used and as the dihydrate is formed when hemihydrate is mixed with water [40]. In this study, water was used as the liquiddihydrate binder is formed during when the fabrication hemihydrate of the is perform mixed with tablets water via 3D[40]. printing In this (Figurestudy, water6). Upon was contact used as with the liquid binder during the fabrication of the perform tablets via 3D printing (Figure 6). Upon contact liquid binder during the fabrication of the perform tablets via 3D printing (Figure 6). Upon contact with the powder, water causes the dissolution of the calcium sulfate hemihydrate and with the powder, water causes the dissolution of the calcium sulfate hemihydrate and recrystallization of the dihydrate form [41]. The SEM micrographs (Figure 7A) show that pores exist recrystallization of the dihydrate form [41]. The SEM micrographs (Figure 7A) show that pores exist within the uncoated preform tablet wall. These pores negatively impacted the performance of tablets within the uncoated preform tablet wall. These pores negatively impacted the performance of tablets during printing of the bioink. Specifically, the porosity in the preform tablet allowed both the during printing of the bioink. Specifically, the porosity in the preform tablet allowed both the hydrophilic and hydrophobic bioinks to soak into the preform tablet during printing, which not only hydrophilic and hydrophobic bioinks to soak into the preform tablet during printing, which not only weakened the preform tablet, but also negates the advantages of using 3D printing for weakened the preform tablet, but also negates the advantages of using 3D printing for pharmaceutical applications, such as control over drug positioning. To fill the pores, two different pharmaceutical applications, such as control over drug positioning. To fill the pores, two different

Polymers 2018, 10, 1372 8 of 12 the powder, water causes the dissolution of the calcium sulfate hemihydrate and recrystallization of the dihydrate form [41]. The SEM micrographs (Figure7A) show that pores exist within the uncoated preform tablet wall. These pores negatively impacted the performance of tablets during printing of the bioink. Specifically, the porosity in the preform tablet allowed both the hydrophilic and hydrophobic bioinks to soak into the preform tablet during printing, which not only weakened the preform tablet, butPolymersPolymers also 20182018 negates,, 1010,, xx FORFOR the PEERadvantagesPEER REVIEWREVIEWof using 3D printing for pharmaceutical applications, such as control88 ofof 1212 over drug positioning. To fill the pores, two different coatings were used depending on the ink being usedcoatingscoatings for were printing.were usedused For dependingdepending the hydrophilic onon thethe inkink bioink, beingbeing the usedused preform forfor printing.printing. tablets wereForFor thethe soaked hydrophilichydrophilic in Eudagrit bioink,bioink,®E100 thethe (Figurepreformpreform7 B). tabletstablets For were thewere hydrophobic soakedsoaked inin Eudagrit®Eudagrit® bioink, the E100E100 preform ((FigureFigure tablets 7B).7B). ForFor were thethe first hydrophobichydrophobic soaked in bioink,bioink, a high-molecular thethe preformpreform weighttabletstablets werewere PEG first (35first kDa) soakedsoaked solution. inin aa highhigh High-molecular--molecularmolecular weightweight weight PEGPEG PEG (35(35 kDa)kDa) was selectedsolution.solution. because HighHigh--molecularmolecular it is immiscible weightweight withPEGPEG thewas was low-molecular selected selected because because weight it it is is PEGDAimmiscible immiscible in the with with bioink the the low low (Figure--molecularmolecular7C). Additionally, weight weight PEGDA PEGDA a thin in in coating the the bioink bioink of Eudagrit((FigureFigure 7C).7C).®E100 Additionally,Additionally, was added aa to thinthin the coatingcoating preform ofof tablet Eudagrit®Eudagrit® well to E100E100 further waswas inhibitaddedadded to theto thethe absorption preformpreform tablet oftablet the wellwell bioink toto duringfurtherfurther printing.inhibitinhibit thethe The absorptionabsorption SEM micrographs ofof thethe bioinkbioink show duringduring that the printing.printing. polymeric TheThe coating SEMSEM fillsmicrographsmicrographs the pores ofshowshow the preformthatthat thethe tabletpolymericpolymeric (Figure coatingcoating7D). fillsfills thethe porespores ofof thethe preformpreform tablettablet ((FigureFigure 7D).7D).

FigureFigure 6. 6. 6. Multi-compartment MultiMulti--compartmentcompartment preform preform preform tablet. tablet. tablet. (A ) (( SideAA)) Side Side view view view of the of of three the the piecesthreethree constitutingpiecespieces constituting constituting the preform the the tablet.preformpreform From tablet.tablet. left FromFrom to right, leftleft toto top right,right, cap, toptop top cap,cap, compartment, toptop compartment,compartment, and bottom andand bottombottom compartment. compartment.compartment. These TheseThese pieces piecespieces were manufacturedwerewere manufacturedmanufactured by powder-bed byby powderpowder 3D--bedbed printing 3D3D printingprinting using asusingusing binder asas binderbinder DI water DIDI with waterwater 5% withwith ethanol 5%5% ethanolethanol and 0.25% andand Tween 0.25%0.25% 80.TweenTween The 80.80. powder TheThe powderpowder utilized utilizedutilized for their forfor construction theirtheir constructionconstruction was calcium waswas calcium sulfate.calcium Each sulfate.sulfate. of the EachEach two ofof wells thethe twotwo have wellswells a 250 havehaveµL capacity.aa 250 250 µL µL ( capacity.B capacity.) Front view ( (BB)) Front Front of the view view preform of of the the tablet preform preform fabricated. tablet tablet fabricated. fabricated.(C) Comparison ( (CC)) Comparison Comparison between the between between assembled the the versionassembledassembled of the versionversion preform ofof thethe tablet preformpreform and commercially tablettablet andand commerciallycommercially available gel availableavailable capsules. gelgel capsules.capsules.

FigureFigure 7.7. TheThe SEMSEM micrographsmicrographs micrographs ofof of thethe the untreateduntreated untreated andand and coatedcoated coated preformpreform preform tablettablet tablet surfaces.surfaces. surfaces. ((AA ())A SurfaceSurface) Surface ofof ofuntreateduntreated untreated preformpreform preform tablet.tablet. tablet. ((BB)) ( BottomBBottom) Bottom compartmentcompartment compartment infusedinfused infused withwith with aa 10%10% a 10% EE--100100 E-100 inin acetoneacetone in acetone solution.solution. solution. ((CC)) (TopTopC) Top compartmentcompartment compartment infusedinfused infused withwith with aa 15%15% a 15% PEGPEG PEG (35(35 (35 kDa)kDa) kDa) inin in acetoneacetone acetone solution.solution. solution. ((D (DD)) ) InnerInner Inner sectionsection section ofof the the top toptop compartmentcompartment preformpreform tablet,tablet, brushedbrushed withwith aaa 20%20%20% E-100EE--100100 ininin acetoneacetoneacetone solution.solution.solution.

3.4.3.4. PolypillPolypill DissolutionDissolution TestTest TheThe hyaluronichyaluronic acidacid--basedbased hydrophilichydrophilic bioinkbioink waswas loadedloaded withwith llisinoprilisinopril atat aa concentrationconcentration ofof 4040 mg/mL.mg/mL. TwoTwo--hundredhundred--andand--fiftyfifty µLµL of of this this formulation formulation were were dispensed dispensed into into the the bottom bottom compartmentcompartment ofof thethe preformpreform tablettablet andand furtherfurther exposedexposed toto visiblevisible lightlight forfor 22 min,min, toto induceinduce gelationgelation ofof the the photocurable photocurable bioink. bioink. Likewise, Likewise, the the PEGPEG--basedbased hydrophobic hydrophobic bioink bioink waswas loaded loaded with with sspironolactonepironolactone atat aa concentrationconcentration ofof 2020 mg/mL.mg/mL. OneOne--hundredhundred--andand--twentytwenty--fivefive µLµL ofof thisthis formulationformulation werewere loadedloaded intointo thethe toptop compartmentcompartment ofof thethe preformpreform tablettablet andand exposedexposed toto visiblevisible lightlight forfor aa periodperiod ofof 11 minmin ((FigureFigure 8).8). OnceOnce thethe bioinksbioinks werweree polymerized,polymerized, thethe twotwo compartmentscompartments werewere attached,attached, andand

Polymers 2018, 10, 1372 9 of 12

3.4. Polypill Dissolution Test The hyaluronic acid-based hydrophilic bioink was loaded with lisinopril at a concentration of 40 mg/mL. Two-hundred-and-fifty µL of this formulation were dispensed into the bottom compartment of the preform tablet and further exposed to visible light for 2 min, to induce gelation of the photocurable bioink. Likewise, the PEG-based hydrophobic bioink was loaded with spironolactone atPolymers a concentration 2018, 10, x FOR of PEER 20 mg/mL. REVIEW One-hundred-and-twenty-five µL of this formulation were loaded9 of 12 into the top compartment of the preform tablet and exposed to visible light for a period of 1 min (Figurethe small8). cap Once was the placed bioinks to were seal polymerized,the top compartment, the two compartmentscompleting the were oral attached,dosage form. and The the smalltablet capwas wasimmersed placed in toa beaker seal the containing top compartment, 500 mL of dissolution completing medium, the oral conditioned dosage form. at 37 The C and tablet stirred was immersedat 60 rpm. inAliquots a beaker were containing taken after 500 mL0.5, of2, dissolution4, 6, 8, 12, 18, medium, and 24 conditionedh of dissolution at 37 and◦C andtheir stirred drug atconcentration 60 rpm. Aliquots was assessed were taken through after HPLC 0.5, 2, analysis. 4, 6, 8, 12, The 18, results and 24 obtained h of dissolution show a and dual their sustained drug concentrationrelease of lisinopril wasassessed and spironolactone through HPLC in a period analysis. of 24 The h results(Figure obtained9). The preform show a tablet dual sustaineddissolved releasealmost of in lisinopril its entirety and with spironolactone the exception in a of period the lower of 24 hpart (Figure of the9). top The compartment, preform tablet completely dissolved almostexposing in itsthe entirety gels to withthe dissolution the exception medium of the lower (Figure part S3). of theLisinopril top compartment, experienced completely faster drug exposing release thekinetics, gels to when the dissolution compared medium to spironolactone. (Figure S3). LisinoprilThis result experienced can be explained faster drug by the release differences kinetics, in when the comparedmicroarchitecture to spironolactone. and composition This result of the can gels.be explained The hydrophilic by the differences formula in has the over microarchitecture 90% of water andcontent, composition facilitating of the diffusion gels. The of hydrophilic lisinopril, formulaa hydrophilic has over compound, 90% of water into the content, dissolution facilitating medium. the diffusionMoreover, of the lisinopril, hydrogel a hydrophilic has a WHANB compound, of only 3%, into allowing the dissolution small molecules medium. to Moreover, easily diffuse the hydrogel through hasthe polymerize a WHANB of matrix. only 3%, The allowing hydrophobic small moleculesformulation to has easily a higher diffuse polymeric through thecontent polymerize (WPEGDA matrix.= 30%) Theresulting hydrophobic in a mesh formulation size of ~ 11 hasÅ [24]. a higher Naprox polymericen and ibuprofen content ( WhavePEGDA a hydrodynamic= 30%) resulting radius in a of mesh 3.77 sizeÅ and of ~11 6.80 Å Å [24 [42],]. Naproxen respectively, and ibuprofen and molecular have a weights hydrodynamic of 230.26 radius Da and of 3.77 206.29 Å and Da. 6.80 It can Å [42 be], respectively,hypothesized and that molecular the hydrodynamic weights of radius 230.26 ofDa s andpironolactone 206.29 Da. is It close can be to hypothesized the mesh size that of the the hydrodynamicpolymerized hydrophobic radius of spironolactone gel (11 Å ), is since close it to has the a mesh significantly size of the higher polymerized molecular hydrophobic weight than gel (11naproxen Å), since and it hasibuprofen, a significantly model higherdrugs previously molecular weightutilized than under naproxen similar andexperiment ibuprofen, conditions model drugs [24]. previouslyThis would utilized explain under the slower similar release experiment profile conditions observed [with24]. Thisspironolactone, would explain since the diffusion slower release of the profilemolecule observed through with the spironolactone, gel matrix would since be diffusionimpeded of. The the use molecule of a higher through PEGDA the gel molecular matrix would weight be impeded.for the fabrication The use of of a the higher gel PEGDA could result molecular in larger weight mesh for sizes the fabrication and consequently of the gel, enhanced could result drug in largerrelease mesh kinetics. sizes and consequently, enhanced drug release kinetics.

FigureFigure 8. MultiMulti-compartment-compartment preform preform tablet tablet loaded loaded with with hydrophilic hydrophilic (bottom) (bottom) and hydrophobic and hydrophobic (top) (top)bioinks. bioinks.

The dual release of these APIs for the treatment of hypertension demonstrates the use of inkjet The dual release of these APIs for the treatment of hypertension demonstrates the use of inkjet printing for the fabrication of combination therapies. Moreover, it shows that the therapy could contain printing for the fabrication of combination therapies. Moreover, it shows that the therapy could both hydrophilic and hydrophobic compounds. This technology would be especially applicable contain both hydrophilic and hydrophobic compounds. This technology would be especially towards drugs that achieve their pharmacological effect at low dosages and could be targeted towards applicable towards drugs that achieve their pharmacological effect at low dosages and could be targeted towards the development of oral dosage forms for children who require small dosages not always commercially available. Moreover, children experience drastic changes in metabolism that affect the dosage needed to achieve a given target pharmacological effect.

Polymers 2018, 10, 1372 10 of 12 the development of oral dosage forms for children who require small dosages not always commercially Polymersavailable. 2018 Moreover,, 10, x FOR PEER children REVIEW experience drastic changes in metabolism that affect the dosage needed10 of 12 to achieve a given target pharmacological effect.

Figure 9. Dissolution test of polypill. A sustained release profile was achieved for both APIs. Around Figure90% of 9. the Dissolution lisinopril loaded test of waspolypill. release A sustained in a period release of 24 h,profile releasing was achieved most of the for drug both withinAPIs. Around the first 90%8 h. Aboveof the l 60%isinopril of the loaded spironolactone was release loaded in a period was released of 24 h, inreleasing a period most of 24 of h. the drug within the first 8 h. Above 60% of the spironolactone loaded was released in a period of 24 h. 4. Conclusions 5. ConclusionsIn this study, a combination therapy oral dosage form for the treatment of hypertension was designed.In this The study, polypill a combination featured the therapy use of a hyaluronicoral dosage acid form photocurable for the treatment hydrophilic of hypertension bioink and a PEGwas photocurabledesigned. The hydrophobic polypill featured bioink, the loaded use of with a hyaluronic lisinopril acid and spironolactone,photocurable hydrophilic respectively. bioink A preform and a PEGtablet photocurable with two compartments hydrophobic able bioink, to hold loaded these with bioinks lisinopril was designed and spironolactone, and manufactured respectively. through A preformbinder jetting tablet 3D with printing. two compartments The formulations able wereto hold dispensed these bioinks through was a piezoelectricdesigned and nozzle manufactured designed throughfor material binder jetting jetting and 3D subsequently printing. The polymerized formulations through were exposure dispensed to visiblethrough light. a piezoelectric The preform nozzle parts weredesigned assembled for material and a jetting dissolution and subsequently study was carried polymerized out, where through a dual exposure sustained to release visible of light. the drugs The preformlisinopril parts and spironolactone were assembled was and observed a dissolution over a period study ofwas 24 carried h. This studyout, where shows a the dual feasibility sustained of release3D printing of the photocurable drugs lisinopril formulations and spironolactone to manufacture was combinationobserved over therapy a period oral of dosage 24 h. This forms study that showsincorporate the feasibility both hydrophilic of 3D printing and hydrophobic photocurable APIs, formulations with a special to manufacture application combination towards drugs therapy that oralachieve dosage their pharmacological forms that incorporat effect ate low both dosages. hydrophilic and hydrophobic APIs, with a special application towards drugs that achieve their pharmacological effect at low dosages. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4360/10/12/1372/ s1, Figure S1: Gelation kinetics obtained through in situ photorheology of the hydrophilic bioink, Figure S2: Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: Gelation Tensile Strength of PEGDA-based (WPEGDA = 100%) gels loaded with varying concentrations of spironolactone, kineticsFigure S3: obtained Exposed through gels after in situ 24 hphotorheology of dissolution of time. the hydrophilic bioink, Figure S2: Tensile Strength of PEGDA- based (WPEGDA = 100%) gels loaded with varying concentrations of spironolactone, Figure S3: Exposed gels Author Contributions: Conceptualization, B.W., G.A., and C.L.; Investigation, G.A., C.L., T.Z., and W.W.; afterResources, 24 h of B.W.;dissolution Data curation,time. G.A., C.L.; Writing—original draft preparation, G.A.; Writing—review and editing, C.L.; Funding acquisition, B.W. Author Contributions: Conceptualization, B.W., G.A., and C.L.; Investigation, G.A., C.L., T.Z., and W.W.; RFunding:esources,This B.W research.; Data curation,was funded G.A., by GlaxoSmithKline, C.L.; Writing—original grant number draft preparation, PO No. 36,257,529 G.A.; W OS.riting—review and editing,Conflicts C.L of.; Interest: FundingThe acquisition authors, declareB.W. no conflict of interest. Funding: This research was funded by GlaxoSmithKline, grant number PO No. 36,257,529 OS. References Conflicts of Interest: The authors declare no conflict of interest. 1. Banks, J. Adding value in additive manufacturing: Researchers in the United Kingdom and Europe look to References3D printing for customization. IEEE Pulse 2013, 4, 22–26. [CrossRef][PubMed] 2. Kaye, R.; Goldstein, T.; Zeltsman, D.; Grande, D.A.; Smith, L.P. Three dimensional printing: A review on the 1. Banks, J. Adding value in additive manufacturing: Researchers in the United Kingdom and Europe look to utility within medicine and otolaryngology. Int. J. Pediatr. Otorhinolaryngol. 2016, 89, 145–148. [CrossRef] 3D printing for customization. IEEE Pulse 2013, 4, 22–26. [PubMed] 2. Kaye, R.; Goldstein, T.; Zeltsman, D.; Grande, D.A.; Smith, L.P. Three dimensional printing: A review on 3. Zopf, D.A.; Hollister, S.J.; Nelson, M.E.; Ohye, R.G.; Green, G.E. Bioresorbable Airway Splint Created with a the utility within medicine and otolaryngology. Int. J. Pediatr. Otorhinolaryngol. 2016, 89, 145–148. Three-Dimensional Printer. N. Engl. J. Med. 2013, 368, 2043–2045. [CrossRef][PubMed] 3. Zopf, D.A.; Hollister, S.J.; Nelson, M.E.; Ohye, R.G.; Green, G.E. Bioresorbable Airway Splint Created with 4. Acosta-Vélez, G.F.; Wu, B.M. 3D Pharming: Direct Printing of Personalized Pharmaceutical Tablets Abstract a Three-Dimensional Printer. N. Engl. J. Med. 2013, 368, 2043–2045. Powder Bed Inkjet 3D Printing. Polym. Sci. 2016, 1, 1–10. 4. Acosta-Vélez, G.F.; Wu, B.M. 3D Pharming: Direct Printing of Personalized Pharmaceutical Tablets Abstract Powder Bed Inkjet 3D Printing. Polym. Sci. 2016, 1, 1–10. 5. Norman, J.; Madurawe, R.D.; Moore, C.M.V.; Khan, M.A.; Khairuzzaman, A. A new chapter in pharmaceutical manufacturing: 3D-printed drug products. Adv. Drug Deliv. Rev. 2017, 108, 39–50.

Polymers 2018, 10, 1372 11 of 12

5. Norman, J.; Madurawe, R.D.; Moore, C.M.V.; Khan, M.A.; Khairuzzaman, A. A new chapter in pharmaceutical manufacturing: 3D-printed drug products. Adv. Drug Deliv. Rev. 2017, 108, 39–50. [CrossRef] [PubMed] 6. Boudriau, S.; Hanzel, C.; Massicotte, J.; Sayegh, L.; Wang, J.; Lefebvre, M. Randomized Comparative Bioavailability of a Novel Three-Dimensional Printed Fast-Melt Formulation of Levetiracetam Following the Administration of a Single 1000-mg Dose to Healthy Human Volunteers Under Fasting and Fed Conditions. Drugs R D 2016, 16, 229–238. [CrossRef][PubMed] 7. Preis, M.; Öblom, H. 3D-Printed Drugs for Children—Are We Ready Yet? AAPS PharmSciTech 2017, 18, 303–308. [CrossRef][PubMed] 8. Chow, C.K.; Thakkar, J.; Bennett, A.; Hillis, G.; Burke, M.; Usherwood, T.; Vo, K.; Rogers, K.; Atkins, E.; Webster, R.; et al. Quarter-dose quadruple combination therapy for initial treatment of hypertension: Placebo-controlled, crossover, randomised trial and systematic review. Lancet 2017, 389, 1035–1042. [CrossRef] 9. Weichselbaum, R.R.; Liang, H.; Deng, L.; Fu, Y.-X. Radiotherapy and immunotherapy: A beneficial liaison? Nat. Rev. Clin. Oncol. 2017, 14, 365–379. [CrossRef] 10. Gradman, A.H.; Basile, J.N.; Carter, B.L.; Bakris, G.L. Combination therapy in hypertension. J. Am. Soc. Hypertens. 2016, 4, 42–50. [CrossRef] 11. Cahn, A.; Cefalu, W.T. Clinical considerations for use of initial combination therapy in type 2 diabetes. Diabetes Care 2016, 39, S137–S145. [CrossRef][PubMed] 12. Melero, I.; Berman, D.M.; Aznar, M.A.; Korman, A.J.; Gracia, J.L.P.; Haanen, J. Evolving synergistic combinations of targeted immunotherapies to combat cancer. Nat. Rev. Cancer 2015, 15, 457–472. [CrossRef] [PubMed] 13. Cicero, A.F. Combination Therapy with Olmesartan/Hydrochlorothiazide to Improve Blood Pressure Control. J. Clin. Exp. Cardiol. 2015, 6, 384. [CrossRef] 14. Sun, W.; Sanderson, P.E.; Zheng, W. Drug combination therapy increases successful drug repositioning. Drug Discov. Today 2016, 21, 1189–1195. [CrossRef][PubMed] 15. Terrie, Y.C. Monitoring combination drug therapy. Pharm. Times 2010, 76, 1–6. 16. Follath, F. Challenging the dogma of high target doses in the treatment of heart failure: Is more always better? Arch. Cardiovasc. Dis. 2009, 102, 785–789. [CrossRef][PubMed] 17. Farr, M.; Bacon, P.A. How and when should combination therapy be used? The role of an anchor drug. Br. J. Rheumatol. (UK) 1995, 34, 100–103. [CrossRef] 18. Desai, D.; Wang, J.; Wen, H.; Li, X.; Timmins, P. Formulation design, challenges, and development considerations for fixed dose combination (FDC) of oral solid dosage forms. Pharm. Dev. Technol. 2013, 18, 1265–1276. [CrossRef] 19. Pourkavoos, N. Unique Risks, Benefits, and Challenges of Developing Drug-Drug Combination Products in a Pharmaceutical Industrial Setting. Comb. Prod. Ther. 2012, 2, 1–31. [CrossRef] 20. Goyanes, A.; Wang, J.; Buanz, A.; Martinez-Pacheco, R.; Telford, R.; Gaisford, S.; Basit, A.W. 3D printing of medicines: Engineering novel oral devices with unique design and drug release characteristics. Mol. Pharm. 2015, 12, 4077–4084. [CrossRef] 21. Khaled, S.A.; Burley, J.C.; Alexander, M.R.; Yang, J.; Roberts, C.J. 3D printing of five-in-one dose combination polypill with defined immediate and sustained release profiles. J. Control. Release 2015, 217, 308–314. [CrossRef][PubMed] 22. 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. [CrossRef][PubMed] 23. Acosta-Vélez, G.F.; Linsley, C.S.; Craig, M.C.; Wu, B.M. Photocurable Bioink for the Inkjet 3D Pharming of Hydrophilic Drugs. Bioengineering 2017, 4, 11. [CrossRef][PubMed] 24. Acosta-Vélez, G.F.; Zhu, T.Z.; Linsley, C.S.; Wu, B.M. Photocurable poly(ethylene glycol) as a bioink for the inkjet 3D pharming of hydrophobic drugs. Int. J. Pharm. 2018, 546, 145–153. [CrossRef][PubMed] 25. Trachtman, H.; Frymoyer, A.; Lewandowski, A.; Greenbaum, L.A.; Feig, D.I.; Gipson, D.S.; Warady, B.A.; Goebel, J.W.; Schwartz, G.J.; Lewis, K.; et al. Pharmacokinetics, pharmacodynamics, and safety of lisinopril in pediatric kidney transplant patients: Implications for starting dose selection. Clin. Pharmacol. Ther. 2015, 98, 25–33. [CrossRef][PubMed] Polymers 2018, 10, 1372 12 of 12

26. Snauwaert, E.; Vande Walle, J.; De Bruyne, P. Therapeutic efficacy and safety of ACE inhibitors in the hypertensive paediatric population: A review. Arch. Dis. Child. 2017, 102, 63–71. [CrossRef][PubMed] 27. Larik, F.A.; Saeed, A.; Shahzad, D.; Faisal, M.; El-Seedi, H.; Mehfooz, H.; Channar, P.A. Synthetic approaches towards the multi target drug spironolactone and its potent analogues/derivatives. Steroids 2017, 118, 76–92. [CrossRef][PubMed] 28. United States Pharmacopoeia Convention. <1217> Tablet Breaking Force; USP 37-NF 32; United States Pharmacopeia and the National Formulary; The United States Pharmacopeial Convention: Rockville, MD, USA, 2014. 29. Jang, D.; Kim, D.; Moon, J.; Jang, D.; Kim, D.; Moon, J. Influence of Fluid Physical Properties on Ink-Jet Printability. Langmuir 2009, 25, 2629–2635. [CrossRef][PubMed] 30. Cole-Parmer Instrument Company. Available online: https://pim-resources.coleparmer.com/ instructionmanual/surface-tension-apparatus-instruction-manual.pdf (accessed on 12 January 2017). 31. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER). Size, Shape, and Other Physical Attributes of Generic Tablets and Capsules; Guidance for Industry; Center for Drug Evaluation and Research (CDER): Silver Spring, MD, USA, 2015. 32. Fraser, J.R.; Laurent, T.C.; Laurent, U.B. Hyaluronan: Its nature, distribution, functions and turnover. J. Intern. Med. 1997, 242, 27–33. [CrossRef][PubMed] 33. Allison, D.D.; Grande-Allen, K.J. Review. Hyaluronan: A Powerful Tissue Engineering Tool. Tissue Eng. Part A 2006, 12, 2131–2140. [CrossRef][PubMed] 34. Highley, C.B.; Prestwich, G.D.; Burdick, J.A. Recent advances in hyaluronic acid hydrogels for biomedical applications. Curr. Opin. Biotechnol. 2016, 40, 35–40. [CrossRef][PubMed] 35. Owen, S.C.; Fisher, S.A.; Tam, R.Y.; Nimmo, C.M.; Shoichet, M.S. Hyaluronic acid click hydrogels emulate the extracellular matrix. Langmuir 2013, 29, 7393–7400. [CrossRef][PubMed] 36. Kim, I.L.; Mauck, R.L.; Burdick, J.A. Hydrogel design for cartilage tissue engineering: A case study with hyaluronic acid. Biomaterials 2011, 32, 8771–8782. [CrossRef][PubMed] 37. Juban, A.; Nouguier-Lehon, C.; Briancon, S.; Hoc, T.; Puel, F. Predictive model for tensile strength of pharmaceutical tablets based on local hardness measurements. Int. J. Pharm. 2015, 490, 438–445. [CrossRef] [PubMed] 38. Sheskey, P.J.; Cook, W.G.; Cable, C.G. Handbook Pharmaceutical Excipients, 6th ed.; Pharm Press: London, UK, 2009. 39. Williams, R.O.; Reynolds, T.D.; Cabelka, T.D.; Sykora, M.A.; Mahaguna, V. Investigation of excipient type and level on drug release from controlled release tablets containing HPMC. Pharm. Dev. Technol. 2002, 7, 181–193. [CrossRef][PubMed] 40. Thomas, M.V.; Puleo, D.A. Calcium sulfate: Properties and clinical applications. J. Biomed. Mater. Res. Part B Appl. Biomater. 2009, 88, 597–610. [CrossRef][PubMed] 41. Singh, N.B.; Middendorf, B. Calcium sulphate hemihydrate hydration leading to gypsum crystallization. Prog. Cryst. Growth Charact. Mater. 2007, 53, 57–77. [CrossRef] 42. Bešter-Rogaˇc,M. Nonsteroidal Anti-Inflammatory Drugs Ion Mobility: A Conductometric Study of Salicylate, Naproxen, Diclofenac and Ibuprofen Dilute Aqueous Solutions. Acta Chim. Slov. 2009, 56, 70–77.

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).