Stereolithographic 3D printing: Formulation design based on percolation thresholds

Journal Pre-proof 1 Stereolitographic 3D printing: Formulation design based on percolation thresholds Vicente Linaresa, Marta Casasa,*, Jörg Huwylerb, Isidoro Caraballoa aDepartment of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, Universidad de Sevilla, C/Profesor García González 2, 41012 Seville, Spain. *m[email protected] bDepartment of Pharmaceutical Sciences, Division of Pharmaceutical Technology, University of Basel, 4056, Switzerland Abstract Stereolitograpy (SLA) is an optical photosolidification technology, which can be used to produce 3D printed dosage forms. In the present work, the model drug 4-Aminosalicylic was combined with the photopolymer poly(ethylene glycol) diacrylate (PEGDA), the plasticizer poly(ethylene glycol) (PEG) and the photoinitiator diphenyl(2,4,6- trimethylbenzoyl) phosphine oxide (DPPO) to form a printable resin. The design of a commercial SLA printer was modified to enable printing of different light-curing mixtures in the same tank. This optimisation allowed to evaluate up to 18 different resins by printing in parallel, thus reducing time and costs. Resulting 3D printed drug delivery systems were analyzed with respect to mechanical parameters and drug dissolution rate. The principles of percolation theory were applied, for the first time, to provide a mechanistic insight into the impact of varying PEGDA-PEG ratios. It could be demonstrated that a critical point is reached at 18 %v/v of PEGDA, representing the minimum amount needed in the resins to produce solid objects. This critical point was related to the site percolation threshold of PEGDA. Additional critical points were identified related to the percolation thresholds of PEGDA and PEG in the formulation. We conclude that percolation theory is a valuable concept to obtain mechanistic insights into 3D printed systems manufactured by SLA and to define the Design Space of these innovative dosage forms. Keywords: 3D printing; stereolithography, percolation theory; printability, critical points. 1. Introduction There is an increasing interest in 3D printing technologies since they can be used for the preparation of solid oral dosage forms offering the possibility to manufacture personalized medicines with complex shapes and modified drug release properties. This is especially relevant in areas such as paediatrics where doses and drug release profiles should be adjusted depending on body weight. Since 1980s, a significant development of 3D printing technology took place, thanks to the previous work of several inventors [1]. In 1986 Charles Hull patented the “apparatus for production of three-dimensional objects of stereolithography” being responsible of the widespread adoption of stereolitography (SLA) technology. SLA is distinguished among other 3D printing techniques as fused deposition modelling (FDM) [2–4], syringe printing systems [5], or binder-jetting, for its high precision. For the printing process, we start initially with a pattern which is first designed and illuminated on the resin surface, and as a result of the exposure to the laser beams, the pattern cures to a certain thickness, joining the previous freshly hardened layer. The platform is then moved so that the new resin is ready to cure [6]. Polymers that are suitable for the SLA technique must be able to solidify (liquid to solid resin) when exposed to laser light. The most commonly used photosensitive polymers are polyethylene glycol diacrylate (PEGDA), diphenyl(2,4,6-thienylbenzoyl) or polyethylene glycol dimethylacrylate 1000 (PEGDMA). These polymers are organic molecules that have the capacity to form chemical bonds with at least two other similar molecules, resulting in very large chains. Polymerization of monomers is initiated through a photoinitiator such as 2- Journal Pre-proof 2 Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2595), 2,2′-azobis[2-methyl-N-(2- hydroxyethyl)promionamide] (VA-086), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (TPO-Li), or diphenyl (2,4,6- trimethylbenzoyl) phosphine oxide (DPPO) that creates a radical monomer [7–11]. PEGDA is very commonly used as biocompatible photo‐crosslinkable monomer for pharmaceutical applications [9,12–14]. The addition of PEG 300 to the formulation improves the printability, as suggested in previous works [8,13]. This polymer, interspersed within the PEGDA chains, reduces the degree of crosslinking between the PEGDA chains, modifying the mechanical properties of printed materials [8]. However, an excessive amount of PEG 300 causes poor strength and system deformation. Therefore, different PEGDA-PEG 300 ratios have been tested without yielding a clear definition of the optimal range of these polymers. In view of these shortcomings, the questions arises on how the composition of SLA resins can be tested and optimized. Here, we propose that percolation theory, applied in pharmaceutical systems, could be used as a tool to predict the changes in the proportions of components of mixed systems that can induce abrupt phase transitions. These transitions are associated with socalled critical points. Percolation theory has been widely applied to the estimation of the critical points of pharmaceutical systems obtained by direct compression, ultrasound-assisted compresion, and, more recently, extrusion [15–17]. According to the above, the objective of this work is to apply the percolation theory, for the first time, to the 3D printed systems obtained by SLA to better describe and predict their properties. For this purpose, resins with varying PEGDA-PEG 300 ratios were prepared and used to manufacture 3D printed systems by SLA. These systems were evaluated from a mechanical and biopharmaceutical point of view. Critical points, associated to geometrical phase transitions of the components were estimated and the design space of the formulation was explored. Parallelly, an optimisation of the SLA equipment was performed to enable the economic printing of drug delivery systems. 2. Materials and Methods 2.1 Materials 4-Aminosalicylic acid (4-ASA, MW 153.14) was used as a model drug, poly(ethylene glycol) diacrylate (PEGDA, average MW 700), was used as a photopolymer, poly(ethylene glycol) (PEG 300, average MW 300) was used as a plasticizer and diphenyl(2,4,6- trimethylbenzoyl) phosphine oxide (DPPO) was added to the formulation as a photoinitiator. All materials were purchased from Sigma–Aldrich, UK. 2.2 Methods 2.2.1. Preparation of the photopolymer solution PEGDA and PEG 300 were mixed in varying ratios from 10-90% w/w, to a total volume of 120 mL, which means 93% of the whole formulation. After 1 hour, DPPO was added to the mixture at a concentration of 1% (w/v) and mixed for at least 8 h, until DPPO was completely dissolved. Finally, 4-ASA was added to the mixture at a concentration of 6% (w/w). The photoreactive solution was mixed until the drug was dissolved. The final solution was loaded into the printer. 2.2.2. Printing process Journal Pre-proof 3 The SolidWorks Corp. software version 2016 (Dassault Systèmes, Suresnes, France) was used to design a cylinder measuring 11 mm long by 4 mm wide. The design was then loaded into the printer software (Preform Software v.1.9.1, Formlabs, UK) for the configuration of the printing parameters, which were as follows: "open mode" configuration, layer thickness 0.1 mm, and no support structures. 2.2.3. Modification of the platform and the 3D printing tank In order to optimise the process of producing drug delivery systems with different light-curing mixtures, modifications were made to both the printing platform and the resin tank, thanks to the Zentrale Mechanische Werkstatt Department (Biozentrum, University of Basel). Thus, 18 structures were printed at the same time and six replicates per resin were carried out. 2.2.4. Physical tests of printed systems 3D printed structures were weighted on a precision balance Scaltec type SBC 31 (Gram Precision S.L., Barcelona, Spain) and the thickness and diameter of all the 3D printed structures were measured using a digital micrometer (Comecta, SA, Barcelona, Spain). Photographs were taken at different zooms of the cross sections of the 3D systems with a stereo microscope SMZ800N (Nikon Instruments Inc., Melville, NY, USA). 2.2.5. Scanning electron microscopy (SEM) The 3D printed systems were evaluated using scanning electron microscopy (SEM) with a FEI TENEO electronic microscope (FEI Company, Hillsboro, OR, USA), operating at 5 kV. Systems were coated with a 12 nm thin Pt/Pd layer with a Leica EM SCD500 high vacuum sputter coater. 2.2.6. Determination of drug loading The printed system was placed in a volumetric flask with 1 L of deionized water under magnetic stirring until complete dissolution. Samples (5 ml) were withdrawn and filtered through 0.45mm filters (Millipore Ltd., Ireland). Drug concentration was determined using an Agilent 8453 UV - Vis spectrophotometer (California, USA) at 302 nm, based on the validated calibration curve (A = 0.0554 C – 0.0317, R² = 0.9986). The drug load reported is the average of 3 samples from each batch. 2.2.7 Mechanical property testing of 3D printed systems The mechanical properties of the SLA systems were evaluated using a Texture Analyser TA.XTPlus (Stable Micro Systems, Godalming, UK) at room temperature. A stiffness test was applied to measure the toughness of the filaments following the method previously described by Linares et al. [2]. Briefly, systems were placed on top of the Texture Analyzer platform and were cut using a Warner Bratzler blade. The area under curve (AUC) and maximum stress were calculated using the Macro program in Texture Analyzer software. Three anchors are inserted into the plot: at the starting point of the strain, at the maximum strain, and at the end of the test, when the stress approached zero. Toughness is calculated by the sum of AUC between the first and third anchors and is expressed in kg/mm2. The test was carried out in triplicate for each system. 2.2.8. Dissolution testing and modelling The dissolution studies were carried out using a USP Apparatus II (Erweka DT 600 HH, Heusenstamm, Germany) with a rotation speed of 50 rpm, in 900 ml of distilled water at 37 ± 0.5 ◦C. 3D printed systems were analysed by triplicate. Filtered samples were withdrawn at specific interval times (30, 60, 90, 120, 180, 240, 300, 360, 420, 480, 1440 min). The percentage Journal Pre-proof 4 of drug released was measured on an Agilent 8453 UV-vis spectrophotometer (Agilent Technologies, California, CA, USA) at 302 nm. Sink conditions were guaranteed throughout the study. Drug release data (Mt/M∞≤ 0.6) were analysed according to zero order, Higuchi (1963) [18], Korsmeyer et al. (1983)[19] and Peppas and Sahlin (1989)[20] equations (1-3): 𝑀𝑡 𝑀∞=𝑏𝑡12 ⁄ (1) 𝑀𝑡 𝑀∞=𝑘´𝑡𝑛 (2) 𝑀𝑡 𝑀∞=𝑘𝑑𝑡𝑚+ 𝑘𝑟𝑡2𝑚 (3) where Mt/M∞ is the drug released fraction at time t (the drug loading was considered as Mt), b and 𝑘´ are kinetic constants characteristic of the drug/polymer system, t is the release time, n is the release exponent that depends on the release mechanism and the shape of the matrix tested [21], 𝑘𝑑 and 𝑘𝑟 are the diffusion and relaxation rate constants, respectively, m is the purely Fickian diffusion exponent for a device of any geometrical shape which exhibits controlled release. The determination coefficient (r2) was used to test the applicability of the release models. Furthermore, drug release profiles were compared using the similarity factor, f2, calculated by the following equation: 𝑓2=50 ∙ log{[1 + (1 𝑛)∑ (𝑅t − 𝑇t)2 𝑛 𝑡=1 ]−0.5 ∙100} (4) where Rt and Tt are the percentages released at each time point. A value of f2 between 50 and 100 indicates similarity between two release profiles [22]. 2.2.9. Study of critical points according to percolation theory According to the percolation theory, if one property of the system behaves as a critical one, we can expect the following relation for this property X near to the percolation threshold pc (Eq. 5): 𝑋 ∝𝑆 (𝑝 − 𝑝𝑐)𝑞 (5) where X is the property studied, S is a proportional constant or scaling factor; p is the occupation probability -in our case can be assimilated to the concentration in volume of the studied component-, pc is the percolation threshold, and q is a critical exponent, which only depends on the property studied and the dimensionality of the system -in our case three dimensions-. Therefore, at polymer p close to pc the excpient spans the whole system, and at p > pc it percolates the system changing the characteristics of the blend with respect to the studied property. Thus, the mechanical and kinetic parameters were analysed versus the % v/v of PEGDA to observe possible discontinuities related to geometrical phase transitions of the components. 3. Results and discussion In a first set of experiments, the printing platform and printing tank of the SLA printer were modified to allow for tablet prototyping using small amounts (10 mL) of liquid resin per tablet. This modification allows for the use of more than one type of resin to be printed in parallel. It was thus possible to print up to 18 3D objects at the same time (Figure S1). As a result of Journal Pre-proof 5 the modification of the 3D printer, eighteen wells were obtained (Figure 1A). It should be noted that a similar platform design was recently introduced to enhance efficiency of pharmaceutical formulation printing [23]. For each printing process, wells were filled with three different light-curing mixtures (six replicates per mixture). Three printing processes were performed for a total of 54 structures. This technique allows more than one structure to be printed at the same time, so that in 35 minutes, eighteen drug delivery systems could be manufactured. The resulting structures are shown in Figure 1B. Figure 1. (A) Modified tank and parallel printing platform. The 18 tank compartments can be filled with different resins allowing for a parallel printing of different 3D structures. (B) 3D cylinders printed with different PEGDA-PEG300 ratios on the new printing platform. With this methodology, photopolymer solutions with varying PEGDA-PEG300 ratios were tested in order to evaluate the ability of these blends to be printed by SLA. The resins were successfully printed obtaining systems with the designed structure, with the exception of the mixtures with ratio 10-90% of PEGDA-PEG300. Taking into account the volume occupied by PEGDA in the whole formulation, this result indicates the existence of a first critical point between 9.8-18.8% v/v of PEGDA. This critical point corresponds to the minimum amount of this photopolymer in the resin to produce enough crosslinking to obtain solid systems. Based on percolation theory, this range could be related to a mechanical threshold according to a bond percolation model. Bond percolation models study the probability at which, a certain number of randomly formed bridges, is able to stablish a connection through the whole system. This is called a percolating cluster of bond elements. In our case, this bond percolating cluster would develop the sufficient strength to keep the integrity of the solid system. In other words, to obtain a solid system instead of a liquid one. To the best of our knowledge, this proportion of PEGDA was the lowest that has been reported to allow the fabrication of 3D printed systems using the SLA technique [7,8]. The cylinders were printed according to the designed structure. Their physical characteristics are shown in Table 1. Based on these results, we can confirm that SLA technology is particularly accurate, which makes it a technique of interest when high resolution structures are desired. Furthermore, according to the results of the weight of the printed systems, there is a general trend showing that the higher the amount of PEGDA, the higher the weight and the more transparent is the printed system, as shown in the photographs of the cross sections taken with a stereo microscope SMZ800N (Fig. 2). Journal Pre-proof 6 Table 1. Physical characteristics of 3D printed systems. Values represent the mean ± standard deviation (n=6) Figure 2. Images of the cross section of 3D printed systems taken by the stereo microscope. SEM microphotographs of 3D printed systems were taken for all batches under study (Figure 3). The continuum medium obtained by the complete dissolution of all components of the mixture can be appreciated in the images. Figure 3. SEM microphotographs of the SLA printed system with 90 % PEGDA. Front view (A) and cross section (B). %PEGDA (w/w) Weight (mg) Diameter (mm) Height (mm) 20 406.63 ± 32.60 11.03 ± 0.06 3.92 ± 0.02 30 492.90 ± 12.14 11.00 ± 0.02 3.85 ± 0.10 40 480.80 ± 31.06 11.01 ± 0.03 4.10 ± 0.09 50 474.83 ± 21.04 10.95 ± 0.07 4.05 ± 0.05 60 464.93 ± 11.47 10.90 ± 0.06 3.90 ± 0.04 70 507.93 ± 15.46 11.10 ± 0.02 4.08 ± 0.06 80 524.93 ± 12.49 11.00 ± 0.05 3.94 ± 0.04 90 551.47 ± 18.36 11.01 ± 0.00 4.01 ± 0.08 Journal Pre-proof 7 The drug loading of the SLA systems was determined using the methodology described in Section 2.2.6. The quantity of 4- ASA in the systems was confirmed to be 5.00 ± 0.46 mg. A stiffness test was performed to study the mechanical properties of 3D printed systems. The toughness values obtained for the different batches studied is shown in Figure 4. As expected, the toughness of the systems increases with the higher amount of PEGDA. Figure 4. Toughness versus the percentage w/w of PEGDA in the systems prepared by SLA printing (n=3). Drug dissolution studies from SLA systems are shown in Figure 5A. As expected, the higher amount of PEGDA in the composition, results in a lower drug release rate. In all cases, the systems showed prolonged drug release, even those with 20% of PEGDA. Three different groups of profiles can be distinguished in the Figure, which have been confirmed by the similarity factor f2 (71-75 for 20-40%, 53-83 for 50-70%, and 58 for 80-90% of PEGDA). So, while systems with 20-40% ratio of PEGDA showed similar values, a change in the values of the release rates can be appreciated between 40% and 50 %. In a similar way, from 70 to 80% of PEGDA a trend change is observed. Figure 5B represents the mean of Higuchi kinetic constant of each group to highlight these differences of drug release rate. The three groups with similar release rates, separated by two discontinuities, could be correlated with the geometrical phase transitions of the two polymers, according to a site percolation model. In a system with two components, two site percolation thresholds can be expected. The first discontinuity observed in the drug release profiles may correspond to the site percolation threshold of PEGDA, (between 38-47% v/v) where this polymer starts to exert strong influence in the system. Above this point, drug release decreases because the volume occupied by PEGDA is hindering the transport through the system. Therefore, with respect to the PEGDA concentration, two percolation thresholds have been observed. The bond percolation threshold, related to the mechanical properties of the system, between 9.8-18.8% v/v, as the minimum amount of PEGDA needed to develop links enough to obtain a solid object; and the site percolation threshold, between 38-47 % v/v, related to the transport properties of the system, which leads to a slowdown of the drug release rate. 0.00 5.00 10.00 15.00 20.00 25.00 010 20 30 40 50 60 70 80 90 100 Toughness (kg/mm2) % w/w PEGDA Journal Pre-proof 8 Figure 5. (A) Dissolution profiles from 3D printed systems obtained by the SLA technique with different ratios of PEGDA. (B) Three groups of drug release profiles represented according to the Higuchi kinetic constant. Values are means ± SD, n=3. Between the bond and site percolation thresholds of PEGDA, the polymer skeleton has a structure developing sufficient mechanical strength but containing “holes” enough to exert low influence in the transport of the drug through the system. Since the development of percolation theory, the bond percolation model has been usually related to the mechanical properties of the system [24] whereas the site percolation model, whose thresholds are obtained at higher concentrations have been more frequently associated to kinetic properties [25–28]. The second discontinuity in the release rates, observed between 66-75% v/v of PEGDA (i.e. between 25-34% v/v of PEG) corresponds to the site percolation threshold of PEG. That means that below 25% v/v of PEG the volume occupied by this polymer is insufficient to form an infinite cluster of neighbour molecules of this hydrophilic filler. This results in a strong decrease in the transport properties of the system. The relationship between particle size and percolation threshold was discovered and extensively studied by our research group in the past [29–34]. In the present study, this concept can be applied to the size of the molecules of the two employed polymers. Using an analogy with the particle size, a higher molecular size should result in a higher percolation threshold, i.e., this component would need a higher concentration to constitute a cluster able to connect every side of the system. In other words, the component with the higher molecular size should need a higher concentration to reach its critical point. Despite new studies are needed to confirm this hypothesis, the site percolation thresholds, and therefore the critical points governing the release properties of the SLA 3D printed systems, fit quite well with this proposal: the component whose molecules have a bigger size, i.e., the PEGDA, exhibit a critical point between 38-47 % v/v, whereas the geometrical phase transition, and the corresponding critical point of PEG has been found between 25-34% v/v. 0 10 20 30 40 50 60 70 80 90 100 0246810 % drug release Time (h) Figure 5A 90% 80% 70% 60% 50% 40% 30% 20% 0 0.01 0.02 0.03 0.04 0.05 0.06 20-40 % PEGDA 50-70 % PEGDA 80-90 % PEGDA Higuchi kinetic constant Figure 5B Journal Pre-proof